Proteins 9780128172865, 012817286X, 9780128166956

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PROTEINS: SUSTAINABLE SOURCE, PROCESSING, AND APPLICATIONS

PROTEINS: SUSTAINABLE SOURCE, PROCESSING, AND APPLICATIONS Edited by

CHARIS M. GALANAKIS Department of Research & Innovation, Galanakis Laboratories, Chania, Greece; Food Waste Recovery Group, ISEKI Food Association, Vienna, Austria

Academic Press is an imprint of Elsevier 125 London Wall, London EC2Y 5AS, United Kingdom 525 B Street, Suite 1650, San Diego, CA 92101, United States 50 Hampshire Street, 5th Floor, Cambridge, MA 02139, United States The Boulevard, Langford Lane, Kidlington, Oxford OX5 1GB, United Kingdom Copyright © 2019 Elsevier Inc. All rights reserved. No part of this publication may be reproduced or transmitted in any form or by any means, electronic or mechanical, including photocopying, recording, or any information storage and retrieval system, without permission in writing from the publisher. Details on how to seek permission, further information about the Publisher’s permissions policies and our arrangements with organizations such as the Copyright Clearance Center and the Copyright Licensing Agency, can be found at our website: www.elsevier.com/permissions. This book and the individual contributions contained in it are protected under copyright by the Publisher (other than as may be noted herein). Notices Knowledge and best practice in this field are constantly changing. As new research and experience broaden our understanding, changes in research methods, professional practices, or medical treatment may become necessary. Practitioners and researchers must always rely on their own experience and knowledge in evaluating and using any information, methods, compounds, or experiments described herein. In using such information or methods they should be mindful of their own safety and the safety of others, including parties for whom they have a professional responsibility. To the fullest extent of the law, neither the Publisher nor the authors, contributors, or editors, assume any liability for any injury and/or damage to persons or property as a matter of products liability, negligence or otherwise, or from any use or operation of any methods, products, instructions, or ideas contained in the material herein. British Library Cataloguing-in-Publication Data A catalogue record for this book is available from the British Library Library of Congress Cataloging-in-Publication Data A catalog record for this book is available from the Library of Congress ISBN: 978-0-12-816695-6 For Information on all Academic Press publications visit our website at https://www.elsevier.com/books-and-journals

Publisher: Charlotte Cockle Acquisition Editor: Megan R. Ball Editorial Project Manager: Katerina Zaliva Production Project Manager: Omer Mukthar Cover Designer: Miles Hitchen Typeset by MPS Limited, Chennai, India

List of Contributors Nils K. Afseth Nofima AS - Norwegian Institute of Food, Fisheries and Aquaculture Research, Norway Tatiana Q. Aguiar CEB Centre of Biological Engineering, University of Minho, Braga, Portugal Peter Alexander School of Geosciences, University of Edinburgh, Edinburgh, United Kingdom; Global Academy of Agriculture and Food Security, The Royal (Dick) School of Veterinary Studies, University of Edinburgh, Easter Bush Campus, Midlothian, United Kingdom Daniel Ananey-Obiri Food and Nutritional Sciences Program, North Carolina Agricultural and Technical State University, Greensboro, NC, United States Rene´ Renato Balandra´n-Quintana Center for Research in Food and Development, A.C. Coordination of Technology of Foods from Vegetal Origin, Hermosillo, Sonora, Mexico Ulrike Bo¨cker Nofima AS - Norwegian Institute of Food, Fisheries and Aquaculture Research, Norway Calum Brown Karlsruhe Institute of Technology, Institute of Meteorology and Climate Research, Atmospheric Environmental Research (IMK-IFU), Garmisch-Partenkirchen, Germany Cristina Chuck-Herna´ndez Tecnologico de Monterrey, School of Engineering and Sciences, Monterrey, Mexico Clare Dias Land Economy and Environment Research Group, SRUC, Edinburgh, United Kingdom Lucı´lia Domingues CEB Centre of Biological Engineering, University of Minho, Braga, Portugal Parag R. Gogate Chemical Engineering Department, Institute of Chemical Technology, Mumbai, India ´ ngel Huerta-Ocampo CONACYT-Center for Research in Food and Development, Jose´ A A.C. Coordination of Food Science, Hermosillo, Sonora, Mexico Rajeshree A. Khaire Chemical Engineering Department, Institute of Chemical Technology, Mumbai, India Kenneth A. Kristoffersen Nofima AS - Norwegian Institute of Food, Fisheries and Aquaculture Research, Norway Diana Lindberg Nofima AS - Norwegian Institute of Food, Fisheries and Aquaculture Research, Norway Ivone M. Martins CEB Centre of Biological Engineering, University of Minho, Braga, Portugal Lovie G. Matthews Food and Nutritional Sciences Program, North Carolina Agricultural and Technical State University, Greensboro, NC, United States

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LIST OF CONTRIBUTORS

ˆ ngelo Paggi Matos Federal University of Santa Catarina, Floriano´polis, Brazil A Ana Marı´a Mendoza-Wilson Center for Research in Food and Development, A.C. Coordination of Technology of Foods from Vegetal Origin, Hermosillo, Sonora, Mexico Dominic Moran Global Academy of Agriculture and Food Security, The Royal (Dick) School of Veterinary Studies, University of Edinburgh, Easter Bush Campus, Midlothian, United Kingdom Carla Oliveira CEB Centre of Biological Engineering, University of Minho, Braga, Portugal Ce´sar Ozuna Food Department, Bioscience Graduate Program, Division of Life Sciences, Campus Irapuato-Salamanca, University of Guanajuato, Guanajuato, Mexico Puneet Parmar Teagasc, Moorepark, Fermoy, Co. Cork, Ireland Seema Patel Bioinformatics and Medical Informatics Research Center, San Diego State University, San Diego, CA, United States Shivani Pathania Teagasc, Ashtown, Dublin, Ireland Gabriela Ramos-Clamont Montfort Center for Research in Food and Development, A.C. Coordination of Food Science, Hermosillo, Sonora, Mexico Mark D.A. Rounsevell School of Geosciences, University of Edinburgh, Edinburgh, United Kingdom; Karlsruhe Institute of Technology, Institute of Meteorology and Climate Research, Atmospheric Environmental Research (IMK-IFU), GarmischPartenkirchen, Germany Sı´lvio B. Santos CEB Centre of Biological Engineering, University of Minho, Braga, Portugal Reza Tahergorabi Food and Nutritional Sciences Program, North Carolina Agricultural and Technical State University, Greensboro, NC, United States Brijesh K Tiwari Teagasc, Ashtown, Dublin, Ireland Eva Veiseth-Kent Nofima AS - Norwegian Institute of Food, Fisheries and Aquaculture Research, Norway Kathryn E. Washburn Nofima AS - Norwegian Institute of Food, Fisheries and Aquaculture Research, Norway Sileshi G. Wubshet Nofima AS - Norwegian Institute of Food, Fisheries and Aquaculture Research, Norway

Preface Proteins are essential for life and provide many food systems with functional properties. As the quality of proteins of animal origin is higher than plant proteins, the human population demands to consume more meat. In addition, the global use of protein ingredients in formulated foods, beverages, and dietary supplements is estimated to be at 5.5 million metric tons by 2018 and exceed US$24.5 billion by 2015. The increasing demands for proteins, the increasing population, and the depletion of resources has led researchers to investigate more sustainable sources for these valuable compounds in order to feed the world and meet the market’s needs. In addition, the production of meat has a much larger impact compared with the production of vegetable-based proteins or proteins from other sources. Thus, the academic and industrial sectors have two important challenges ahead: (1) to identify alternative sources of proteins that meet the nutritional expectations; and (2) to innovate to offer acceptable and economically sustainable products. Among other activities (webinars, workshops, e-courses etc.), the Food Waste Recovery Group (www.foodwasterecovery.group) of ISEKI Food Association has published books over the last 5 years that deal with different issues of sustainable food systems, such as innovations in the food industry and traditional foods, food waste recovery, and nonthermal processing. Following the needs of our current time, this book covers proteins’ properties and health effects in view of the new trends in sustainable sources, recovery procedures, stability aspects, and application trends. The ultimate goal is to support the scientific community, professionals, and enterprises that aspire to develop the industrial and commercialized applications of proteins. The book consists of 10 Chapters. Chapter 1, Sustainable Proteins Production, provides an introduction to the book by discussing alternatives to conventional animal products as well as exploring the potential change in global agricultural land requirements associated with each alternative. It compares the agricultural land area given shifts from conventional animal product consumption to one of the alternative products. The analysis uses stylized transformative consumption scenarios where half of current conventional animal products are substituted to provide at least equal protein and calories. Chapter 2, Insects as a Source of Sustainable Proteins, reviews the available literature on edible insects, presents the latest developments in this field, discusses their scope for food security, and proposes the advantages and disadvantages of this emerging food trend. Chapter 3, Microalgae as a Potential Source of Proteins, assesses the properties of some microalgae—microscopic photosynthetic organisms present in both marine and freshwater environments. The term microalgae, in applied phycology, usually includes the microscopic algae and the photosynthetic bacteria (i.e., cyanobacteria), formerly known as Cyanophyceae. Chapter 4, PlantBased Proteins, condenses the basic concepts of protein sustainability and protein quality, then provides an overview of the characteristics of proteins from common plant sources as

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PREFACE

well as other unconventional ones. Emphasis is given to the aspects of greatest interest, such as protein quality, extraction methods, functional properties, and bioactivity. Chapter 5, Protein Isolates From Meat Processing By-Products, discusses the recovery of proteins from meat processing by-products and covers all the important aspects of achieving optimal utilization of proteins in such residual raw materials, identifying those eligible for human consumption as coproducts and for feed applications as by-products. In Chapter 6, Proteins From Fish Processing By-Products, different methods of fish protein recovery, including hydrolysis and isoelectric precipitation, and their impact on the protein functional properties are succinctly reviewed. Fish protein hydrolysates can be produced through exogenous means, using either acids, base, enzymes, or microorganisms. In addition to their bioactivity, structural and functional properties such as solubility, foaming, emulsification, water holding, and fat holding capacities of fish are also enhanced during fish protein hydrolysis. Chapter 7, Whey Proteins, presents an overview of the processing steps for the recovery of proteins from whey, along with guidelines on the operating conditions and recent advances for improved processing. A brief overview of the proven and upcoming applications has also been presented with information on the global markets. Chapter 8, discusses the bioanalytical aspects of the enzymatic protein hydrolysis of byproducts. It covers the classical analytical methodologies used to measure these parameters, as well as emerging approaches of using rapid spectroscopic techniques as process control and optimization tools. Moreover, key factors and process considerations in relation to the use of proteases for recovery of peptides will be discussed. Food proteins, depending on the food matrix and (multiple) processing conditions, undergo physical and chemical changes, affecting their quality and functionality in the food system. Therefore, Chapter 10, denotes the stability of proteins during processing and storage. Finally, Chapter 9, Production and Bioengineering of Recombinant Pharmaceuticals, discusses the production and bioengineering of recombinant approaches used for obtaining phage display-derived proteins/peptides, antimicrobial peptides, phage-encoded endolysin enzymes, and lectins. Conclusively, the book fills the existing gap in the current literature by providing information in different critical dimensions, namely properties, sustainable sources, recovery processes, and food applications. It is a guide for all scientists, technologists, engineers, chemists, and new product developers working in the whole food science field. It could be a helpful reference book for researchers, academics, and professionals dealing with food applications and food processing as well as those who are interested in the development of innovative products and functional foods. It could be used by University libraries and Institutes all around the world as a textbook and/or ancillary reading in undergraduates and postgraduate level multidiscipline courses dealing with nutritional chemistry, and food science and technology. During this project I was fortunate to have the opportunity of collaborating with so many experts from Brazil, Germany, India, Ireland, Mexico, Norway, Portugal, the United Kingdom, and the United States. Hereby, I would like to honor and thank all the authors of the book for accepting my invitation, and for bringing together several topics of sustainable proteins into one comprehensive textbook. Their alignment with the editorial guidelines and timeline is much appreciated. I would also like to thank the acquisition editor

PREFACE

xi

Megan Ball, the book manager Katerina Zaliva, and all colleagues of Elsevier’s production team for their help during the publishing process. Last but not least, I have a message for all the readers of this book. Collaborative book projects may contain errors as they are developed via scientific discussions by different experts. Therefore, any comments, notifications, or even criticism are and always will be welcome. In that case, please do not hesitate to contact me to discuss these issues in detail. Charis M. Galanakis1,2 1 Food Waste Recovery Group, ISEKI Food Association, 2 Vienna, Austria Research & Innovation Department, Galanakis Laboratories, Chania, Greece Email: [email protected]

C H A P T E R

1

Sustainable Proteins Production Peter Alexander1,2, Calum Brown3, Clare Dias4, Dominic Moran2 and Mark D.A. Rounsevell1,3 1

School of Geosciences, University of Edinburgh, Edinburgh, United Kingdom 2Global Academy of Agriculture and Food Security, The Royal (Dick) School of Veterinary Studies, University of Edinburgh, Easter Bush Campus, Midlothian, United Kingdom 3Karlsruhe Institute of Technology, Institute of Meteorology and Climate Research, Atmospheric Environmental Research (IMK-IFU), Garmisch-Partenkirchen, Germany 4Land Economy and Environment Research Group, SRUC, Edinburgh, United Kingdom O U T L I N E 1.1 Introduction

2

1.4.4 Aquaculture

1.5 Comparison of Land Requirements 15 1.5.1 Alternative Animal Product Scenarios 15 1.5.2 Insect Consumption 17 1.5.3 Cultured (In Vitro) Meat 17 1.5.4 Imitation Meat 17 1.5.5 Aquaculture 18 1.5.6 Conventional Livestock Consumption Changes 18 1.5.7 Waste and Other Dietary Change Scenarios 18 1.5.8 Uncertainty Quantification 19 1.5.9 Yields of Alternatives to Animal Product 20 1.5.10 Land Requirements of Scenarios 20 1.5.11 Discussion 24

1.2 Human Appropriation of Land for Food 4 1.2.1 Allocating Areas for Food Commodities 5 1.2.2 Allocating Areas for Animal Feed and Pasture 6 1.2.3 Assessing the Land Use Impact of Different Diets 7 1.3 Global and Country-Level Consumption Patterns and Trends 7 1.3.1 Intercountry Variation in Appropriation of Land for Food 7 1.3.2 Changing Dietary Patterns 9 1.4 Alternatives to Current Animal Products 1.4.1 Insects 1.4.2 Cultured Meat 1.4.3 Imitation Meat Proteins: Sustainable Source, Processing and Applications DOI: https://doi.org/10.1016/B978-0-12-816695-6.00001-5

14

12 13 13 14

1

1.6 Conclusions

32

References

33

© 2019 Elsevier Inc. All rights reserved.

2

1. SUSTAINABLE PROTEINS PRODUCTION

1.1 INTRODUCTION Livestock provides a quarter of all the protein (and 15% of energy) consumed in food, but also creates substantial environmental impacts (FAO, 2012; Herrero et al., 2016). The area of global pasture is more than twice that of cropland, with livestock animals additionally consuming around one-third of the crops harvested as feed (FAO, 2006). Despite rises in crop yields and in the efficiency of livestock production, global agricultural land area has been expanding, increasing by 464 Mha between 1961 and 2011 (Alexander et al., 2015). Land use change in recent decades has accounted for 10% 12% of total anthropogenic carbon dioxide emissions, and one-third since 1850 (Houghton et al., 2012; Le Que´re´ et al., 2015). Livestock production also contributes to atmospheric greenhouse gas (GHG) emissions, due to methane from enteric fermentation (presently 2.1 Gt CO2 eq/year; Gerber et al., 2013), and nitrous oxide emissions from fertilizer use on pasture and croplands in fodder production (Smith et al., 2014). In total, livestock is responsible for 12% of global anthropogenic GHG emissions (Havlı´k et al., 2014). A larger global population consuming a diet richer in meat, eggs, and dairy (Popkin et al., 1999; Keyzer et al., 2005; Kearney, 2010; Tilman et al., 2011; Bodirsky et al., 2015) has meant that agricultural land use change in the past 50 years has been dominated by the expansion of livestock production (Alexander et al., 2015). Besides the direct GHG emissions, agriculture also has large indirect emissions (e.g., from agrochemicals production and fossil fuel usage) (Smith and Gregory, 2013). The combination of land use change and other emissions increases the share of agriculture in all global anthropogenic GHG emissions to between 17% and 32% (Smith and Gregory, 2013). Therefore changing demands on agricultural production, and in particular for animal products (i.e., meat, milk, and eggs), has the potential to substantially alter GHG emissions (Bustamante et al., 2014; Havlı´k et al., 2014). Additionally, the sparing of agricultural land would provide options for further climate change mitigation measures, including afforestation or bioenergy (Humpeno¨der et al., 2014; Henry et al., 2018). The projected rise in global population and higher per capita rates of animal product consumption, arising from higher incomes and urbanization, suggests that livestock production will continue to increase (Tilman et al., 2011). Changes in production practices and animal genetics that increase efficiencies may help to offset some of the potential land use and associated environmental impacts (Le Cotty and Dorin, 2012; Havlı´k et al., 2014). Substantial research attention has been given to supply-side responses, including expanding land in agricultural use and increasing food yields, especially crops (e.g., closing the “yield gap” or “sustainable intensification”) (Foley et al., 2011; Mueller et al., 2012; Kastner et al., 2014; West et al., 2014); or the potential benefits and trade-offs associated with increasing livestock intensities (Davis et al., 2015; Herrero et al., 2016; de Oliveira Silva et al., 2017). Nevertheless, demand-side measures to reduce animal product consumption may be necessary to meet climate change targets (UNFCC, 2015; van Vuuren et al., 2018), while helping to achieve food security (Smil, 2013; Bajˇzelj et al., 2014; Meadu et al., 2015; Lamb et al., 2016). High levels of meat consumption are also detrimental to human health, with links to obesity, cardiovascular diseases, and cancer (Popkin and Gordon-Larsen, 2004; Hu, 2011; Bouvard et al., 2015; Cesare et al., 2016). Despite both the health and

PROTEINS: SUSTAINABLE SOURCE, PROCESSING AND APPLICATIONS

1.1 INTRODUCTION

3

environmental benefits, changing consumer preferences toward a low meat diet is difficult because of cultural, social, and personal associations with meat consumption (Grac¸a et al., 2015; Macdiarmid et al., 2016). Although there is some evidence for increasing rates of vegetarianism and reduced meat diets in western countries (Vinnari et al., 2010; Leahy et al., 2011), the global average per capita rate of animal product consumption has continued to increase (FAOSTAT, 2015a). Analyses of the sustainability of the food system that do consider dietary changes tend to do so through an exogenous wealth-based factor, and anticipate continuations of current dietary trends (Schmitz et al., 2014; Engstro¨m et al., 2016a). However, diets and the food preferences that shape them do not necessarily follow fixed trends. Instead, they alter over time, influenced by technology, policies, and changes in social norms (e.g., Hollands et al., 2015). Modeling work has been done to project the impact of alternative assumptions regarding future diets (Stehfest et al., 2009; Popp et al., 2010; Haberl et al., 2011; Bajˇzelj et al., 2014), and the ability of the agricultural system to supply the global population with a diet containing adequate calories has also been considered (Cassidy et al., 2013; Davis et al., 2014). Further studies in this area have taken a life cycle analysis (LCA) approach that typically consider either GHG emissions, energy or water requirements for individual commodities (Carlsson-Kanyama and Gonza´lez, 2009; Marlow et al., 2009; Gonza´lez et al., 2011; Pelletier et al., 2011). Studies of the food system that include the impact of dietary change typically assume the continuation of existing consumption patterns and income and price elasticity relationships (e.g., Engstro¨m et al., 2016a,b; Schmitz et al., 2014; Tilman et al., 2011), implicitly discounting the possibility of major shocks or transformative changes in diets. There has also been an increasing number of studies considering the impact of alternative assumptions regarding future diets, such as lower animal product consumption, healthy diets, vegetarianism, or veganism (e.g., Stehfest et al., 2009; Popp et al., 2010; Haberl et al., 2011; Bajˇzelj et al., 2014; Erb et al., 2016; Mora et al., 2016). However, technology changes or radical alteration of consumer preferences, which could be transformative for the food system, remain unexplored. New technologies raise the possibility of supplying high-quality food from novel sources, for example, cultured meat, also known as in vitro meat (Thornton, 2010). Also, behavior, preferences, and social norms change over time, such that food previously considered unacceptable or undesirable (e.g., insects, in western countries) could become a more common part of future diets (van Huis, 2013). There are historical precedents for foods becoming acceptable after long periods of rejection; for example, tomatoes in Britain were widely viewed with suspicion and dismissed for over 200 years (Smith, 2013a; Bir, 2014). Similarly, lobster in America was initially a poverty food eaten by slaves and prisoners, and used as fertilizer and fish bait, due to their abundance (Dembosky, 2006). It was not until the late 19th century that lobster developed a status as a luxury food, supported by the expansion of the US railway network giving access to new markets (Townsend, 2012). But while alternative food sources may become technologically feasible or publically acceptable in the future, their potential contribution to sustainability remains unclear. This chapter aims to review and compare the potentially transformative alternatives to conventional animal products, including cultured meat, imitation meat and insects, and consider the implications for global agricultural land use requirements given widespread

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1. SUSTAINABLE PROTEINS PRODUCTION

adoption, based on Alexander et al. (2016, 2017a). The Human Appropriation of Land for Food (HALF) index is presented and used to provide a relative measure of the scale of the impacts of alternative diets on land use. The approach is explorative, rather than predictive, and assumes half of existing animal products are substituted by each alternative food, to provide at least equal energy and protein. The objective is to compare the alternatives on an equal basis and to assess their potential to reduce agricultural land requirements, and contribute to food system sustainability. To allow comparison with other current diets and potential dietary change, other scenarios were included using the same methodology. These scenarios include shifts in conventional animal product consumption, changes to high and low animal product diets (based on average consumption in India and the United States), and reductions in consumer waste. The focus is on animal products due to their dominance in the food system for land use and environmental impacts (Herrero et al., 2016), and because of their relative inefficiency in converting inputs into human-edible food (FAO, 2006; Mottet et al., 2017). The premise is that due to the cultural and personal associations with animal product consumption (Grac¸a et al., 2015; Macdiarmid et al., 2016), consumers with higher incomes continue to eat large quantities of animal products and consumers currently eating at lower rates will increase their consumption as incomes increase. Therefore alternatives that mimic aspects of these products in a manner that is acceptable to consumers need to be explored for environmental sustainability.

1.2 HUMAN APPROPRIATION OF LAND FOR FOOD A metric of the land use impact of alternative diets that is not impacted by the effect of local or domestic production or international trade is needed to compare diets. In this chapter the HALF index (Alexander et al., 2016) is presented and used for this purpose. The HALF index expresses the land area required for the global population to consume a particular diet, as a percentage of the world land surface. HALF therefore provides a relative measure of the scale of the impacts of alternative diets on land use. Diet here is assumed to include the quantities of commodities lost and wasted after reaching the consumer. The index is calculated from global average production intensities and yields from a baseline year, primarily 2011. HALF is accordingly not predictive, as adaptive responses in production systems that may result from changes in demand are excluded. Rather, the HALF index is a metric that characterizes the land use impact of alternative scenarios of dietary patterns. The HALF index does not provide a land use footprint for particular countries or regions, but addresses questions such as “how much land would be used if the global population adopted diet X.” The inclusion of local production systems within a land footprint would tend to obscure the understanding of the role of diet in the global food system. The results can be interpreted in terms of both methods and areas of production, with a given increase in the HALF index implying the same increase in agricultural areas, an equivalent increase in productive efficiency, or some combination of the two. FAO country-level panel data for crop areas, production quantities, commodity uses, and nutrient values were used to construct the HALF index (FAOSTAT, 2015a,b,c,d,e,f).

PROTEINS: SUSTAINABLE SOURCE, PROCESSING AND APPLICATIONS

1.2 HUMAN APPROPRIATION OF LAND FOR FOOD

5

Global average production values and efficiencies for primary crops, processed commodities, and livestock products were used to calculate the agricultural areas needed to meet per capita consumption for each country. Assessments of country average diet do not use production or international trade associated with that country, except as they contribute to the world average. The calculations and assumptions are described in Alexander et al. (2016).

1.2.1 Allocating Areas for Food Commodities The areas associated with the production of 90 commodities, representing 99.4% of global food consumption by calorific value, were each allocated between three categories of use: food for human consumption, animal feed, and nonfood related uses (primarily biofuels and fiber). The commodities comprise 50 primary crops that are directly grown, 32 processed commodities derived from them, and 8 livestock products. The FAO commodity balance data (FAOSTAT, 2015c) identifies the quantities used for food, feed, processing, other nonfood related uses (primarily, bioenergy and fiber), seed, and waste. To provide an assessment of the embedded areas required for delivering the consumed commodities two adjustments were made. Firstly, for each primary crop, the quantities used as seed and wasted (e.g., in storage and transport) were distributed across the remaining categories of use (i.e., food, feed, processing, and nonfood). The second adjustment deals with the difference between the total cropland area and the harvested areas (e.g., in 2011, respectively, 1556 and 1378 Mha; FAOSTAT, 2015b,f) due to set-aside, multiple-cropping, and failed or unharvested crops. To account for these differences, the cropland area for each primary crop was adjusted by the ratio of these areas (e.g., in 2011 areas they are increased by a factor of 1.129). After applying both the adjustments, the cropland area for each primary crop was then allocated pro rata between the categories of use (i.e., food, feed, processing, and nonfood), by the mass used for each category. This approach removes the areas used to produce commodities for bioenergy, fiber, or other nonfood uses (see supplementary material of Alexander et al. (2016), e.g., calculations). The areas used to grow the primary crops for processing were further mapped to the commodities output from the processing. Where multiple commodities are produced from a single crop, the areas used to grow the primary crop were allocated on an approximate economic value basis (see Table S4; Alexander et al., 2016). For example, processed oil crop areas were divided equally between the resulting oil (used primarily for food and biofuel), and the seed meals or cakes (used primarily for livestock feed). In 2011, 224.1 Mt of soybeans, which represent the single biggest vegetable oil crop (48% of the total), were processed globally into 41.6 Mt of oil and 174.7 Mt of meal (7.8 Mt is assumed lost during processing). This gives a similar total market value for the oil and meal (45% of value is in the oil and 55% in meal), at 2011 market prices of US$1103 and 321 per ton, respectively (Index Mundi, 2016), suggesting that an equal division of input area is a reasonable approximation. Alternative allocations would introduce additional biases. For example, calculations on the basis of mass would be biased toward associating the area with the seed meals, while conversely, accounting for them as a by-product with no area allocated would implicitly and incorrectly assume they can be freely produced and have no value.

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1. SUSTAINABLE PROTEINS PRODUCTION

1.2.2 Allocating Areas for Animal Feed and Pasture Animal nutrition derives from grassland and feed crops including forage crops. Data are available to quantify the area of pasture and quantities of crops used as feed (FAOSTAT, 2015c,f). However, there are no empirical data to describe directly how these sources of nutrition are divided between livestock species, and hence between commodity types such as meat, milk, and eggs. Instead, feed conversion ratios, describing the efficiency of converting inputs into edible animal products, were used to estimate animal feed requirements (Table 1.1). Commonly, feed conversion ratios are expressed in terms of dry matter (DM) of feed per animal live weight (LW). To represent the production efficiency of meat consumed by humans, these ratios were adjusted to express feeding requirements per unit edible weight (EW), and also to account for the need to raise sire and dam animals (Smil, 2002). The nutritional requirements of monogastric livestock (i.e., poultry and pigs) were assumed to be met solely from feed, while nutrients for ruminant species (e.g., cattle and sheep) come from feed and grazed pasture. Firstly, the produced masses from monogastric animals were multiplied by the feed conversion factors (Table 1.1) to give estimates of the feed requirements. These feed amounts, and the cropland areas needed to provide them, TABLE 1.1 Global Average Feed Conversion Ratios and Efficiencies for Animal Products Feed Conversion Animal Ratio (kg DM Product Feed/kg EW)

Percentage Edible (% EW of LW)

Energy Feed Conversion Efficiency (%)

Protein Feed Conversion Efficiency (%)

Direct Energy for Housing or Processing (MJ/kg EW)

Poultry

3.3

70

13

19.6

4.5

Macleod et al. (2013), Smil (2013)

Pork

6.4

55

8.6

8.5

1.8

Macleod et al. (2013), Smil (2013)

Data Source

Beef

25

40

1.9

3.8

0.08

Opio et al. (2013), Smil (2013)

Other meata

15

55

4.4

6.3

0.09

Opio et al. (2013), Smil (2013)

Eggs

2.3

19

25

1.3

Macleod et al. (2013), Smil (2013)

Whole milk

0.7

24

24

0.22

Opio et al. (2013), Little (2014)

a

The “other meat” category, which forms 6.6% of all meats produced in 2011, is based on sheep and goat meat (65% by mass of “other meat” in 2011), but includes other sources of meat, for example, horse, rabbit, and camelids. The feed conversion efficiencies and direct energy for housing are given for reference, and are not used in the analysis. Source: Alexander, P., Brown, C., Rounsevell, M., Finnigan, J., Arneth, A., 2016. Human appropriation of land for food: the role of diet. Global Environ. Change 41, 88 98.

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1.3 GLOBAL AND COUNTRY-LEVEL CONSUMPTION PATTERNS AND TRENDS

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were allocated to the monogastric livestock products. Secondly, the remaining feed (23% in 2011 using feed DM content; INRA, CIRAD, AFZ, FAO, 2016), and associated cropland areas were allocated pro rata by the estimated feed requirements across the ruminant products. The same pro rata allocation was used to associate the pasture area with products derived from ruminant animals.

1.2.3 Assessing the Land Use Impact of Different Diets The average consumption per capita and per commodity were calculated globally and nationally (FAOSTAT, 2015a,c). The area required to produce each commodity was determined from the global production system land use allocations (described earlier). The area needed to provide all the commodities for each country’s diet if it were adopted by the global population could then be calculated (FAOSTAT, 2015g). This was expressed as a proportion of total global land area to obtain the HALF value. HALF values were also calculated to quantify the land use impacts of changes in country-level diets over time. The values primarily used here were calculated with variable diet only, and a constant baseline population and production system (2011 was chosen as the most recent year with available values; FAOSTAT, 2015c). National land footprints for food, that is, an estimate of the actual agricultural land area used to supply each country’s food, were also calculated based on domestic production and the land displaced through international trade. This used the same data as the HALF calculation, and accounted for imports and exports following the approach of previous studies (Jalava et al., 2014; Alexander et al., 2015). For each commodity, net exports were included using the domestic production yields, and net imports using the global mean yields of net exports (weighed by net export quantities). The country footprints were expressed as an area per capita using country populations (FAOSTAT, 2015g).

1.3 GLOBAL AND COUNTRY-LEVEL CONSUMPTION PATTERNS AND TRENDS 1.3.1 Intercountry Variation in Appropriation of Land for Food The total agricultural area used for human food production was 4484 Mha in 2011, of which 871 Mha was used for cropland for human consumption, and 3700 Mha for animal products (497 Mha of cropland for feed and 3203 Mha of pasture). The remaining cropland was used for biofuels (140 Mha), fiber (33 Mha), feed for nonfood uses of animal products (9 Mha), and net variations in stock levels (7 Mha). Expressed as a percentage of the global land surface (13,009 Mha; FAOSTAT, 2015f) the HALF index is 35.1, or an average area per person of 0.65 ha. Expressing HALF as a percentage of global land surface includes land that is unlikely to be suitable for agriculture, for example, ice-covered or desert areas. However, the use of an estimate of suitable land suffers from difficulty in definition and measurement, and also would vary with climate change. Consequently, the clarity of comparing to the global land surface was preferred. There are large differences in HALF values between country-level average diets. For example, the global adoption of the diet in the United States would require over six times

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the agricultural area that adoption of the diet in India, with a HALF index of 97.7 compared to India’s 15.8. Fig. 1.1 shows the HALF index at 2011 for the average diets of 170 countries for which sufficient data were available. The highest HALF values are for diets in New Zealand, Argentina, and Australia at 135.8, 114.9, and 112.2, respectively, due to the high levels of animal products—particularly beef—consumed. At the other extreme are Mozambique, Liberia, Bangladesh, and Sri Lanka all with a HALF index below 11.5, that is, less than a third of the global average. The HALF results use global mean production efficiencies, and so no specific account is taken of domestic (national) production except as it contributes to the world average. The national food footprints include aspects of diet and production within them, whereas HALF (Fig. 1.1) only includes variations in diet. The distribution of these national footprints differ from the distribution of HALF values as a result (e.g., Mongolia has a per capita footprint three times greater than any other country (39 ha per person), due to the use of extensive grazing). Many developed countries have a lower land use footprint than implied by the HALF index, due to the high agricultural yields in these countries. For example, the United States was found to have a national food footprint of 1.0 ha per person, but a HALF of 1.8 ha per person. The first value addresses, “how much land is used to produce the food consumed in the United States?” and the second “how much land would be used if the global population adopted the average diet in the United States?” Using a global average production system is reasonable because of the global scale of the analysis (considering global adoption of alternative diets), and also because of the levels of

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FIGURE 1.1 Map of Human Appropriation of Land for Food index for average country-level diets in 2011. Countries where the index could not be calculated due to no commodity consumption data being available (FAOSTAT, 2015c), for example, Libya, Somalia, and Greenland, are shown in light gray. Source: Alexander, P., Brown, C., Rounsevell, M., Finnigan, J., Arneth, A., 2016. Human appropriation of land for food: the role of diet. Global Environ. Change 41, 88 98.

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1.3 GLOBAL AND COUNTRY-LEVEL CONSUMPTION PATTERNS AND TRENDS

international trade in agricultural commodities and the associated globalized markets (Fader et al., 2013; Meyfroidt et al., 2013; D’Odorico et al., 2014). The inclusion of production systems within the land footprint to some degree obscures the understanding of the role of diet in the global food system. HALF, therefore, provides both a clearer comparative metric between countries of the land requirements of different diets, and also a way to consider the impacts from changes in dietary patterns.

1.3.2 Changing Dietary Patterns Calculating the time-dependent HALF index for dietary variations only, that is, assuming a constant 2011 population and production systems, demonstrates the impacts of changes in food consumption patterns (solid lines in Fig. 1.2). The global agricultural land required has increased by 8.7% due to dietary changes, from a HALF value of 32.3 in 1961 to 35.1 in 2011. For country-level average diets, results for Brazil and China show particularly substantial increases, due to the transitions in diets that are associated with increasing per capita wealth (Godfray et al., 2010), as well as the influence of urbanization (Huang and David, 1993; Popkin et al., 1999; Dong and Fuller, 2010; Seto and Ramankutty, 2016)

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20

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China

India

Nigeria

Diet only variable (2011 population and production)

UK

USA

World

Diet, population, and production variable

FIGURE 1.2 Human Appropriation of Land for Food (HALF) index from 1961 to 2011, globally and for selected counties. Solid lines show variable diets, but constant population and agricultural production systems (at 2011 values). Dashed lines show variable diet, population, and agricultural production systems over time. Source: Alexander, P., Brown, C., Rounsevell, M., Finnigan, J., Arneth, A., 2016. Human appropriation of land for food: the role of diet. Global Environ. Change 41, 88 98.

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and globalization of food markets (Popkin, 2006; Meyfroidt et al., 2013). The land required for the diet in Brazil more than doubled between 1961 and 2011, from 43.5 to 88.2, making it the 11th highest ranked country globally in 2011. However, the Chinese diet’s HALF increased nearly five times, from 6.0 in 1961 (the lowest at that period), to 28.6 (but still below the global average). The gap between the United States and Indian diets has reduced slightly, from the United States value being 7.5 times the Indian value in 1961 to 6.2 times in 2011, with an 8% reduction in the United States and a 11% increase for the Indian diet. When the time-dependent HALF indices are recalculated to take account of changing production efficiencies and population sizes (Fig. 1.2, dashed lines), they show a high degree of similarity to the diet-only case (Fig. 1.2, solid lines). This is because increasing agricultural efficiencies and population growth in the past have acted in opposite directions on land requirements, largely offsetting one another. If production efficiencies from 2011 had been available and used in 1961, less than half of the agricultural land used at the time would have been required to feed the population at the time. However, populations have more than doubled since 1961, and therefore the 2011 population would have required more than twice the land for food production based on 1961 production systems. The net effect is that if the mean global diet of 1961 had been consumed by the 2011 population, using 2011 production systems, agricultural land area would have remained largely unchanged from 1961 (just 5 Mha less land is estimated to have been needed than was used in 1961). When HALF values including variation in the production system and population (dashed lines in Fig. 1.2) are lower than the HALF values for dietary changes only (solid lines), then cumulative improvements in agricultural efficiencies achieved by 2011 have not fully offset the rise in population. However, diets have also been changing. Dietary changes alone between 1961 and 2011 has caused the agricultural area for food to increase by 368 Mha or 2.8% of the land surface. HALF has increased less than the 464 Mha expansion of global agricultural land since 1961 (FAOSTAT, 2015f), as an increasing proportion of land is used for nonfood uses of agricultural commodities, that is, feedstocks for biofuels. The central role of the types of foods consumed in determining the agricultural land requirements of different diets, compared to the overall quantity of nutrients consumed, can be seen from the calculated energy intake and the percentage derived from animal products (Fig. 1.3). Variation in total food energy consumed between countries and over time is substantially smaller than the variations in the land needed (Fig. 1.3). In 2011 the per capita land required to sustain a US diet was 635% of that required for an Indian diet, even though the energy content of the food was only 65% greater (or 99% greater in terms of protein). This disparity stems from the profile of commodities consumed, with 30% of energy derived from animal products in the United States and 9% in India (65% and 19%, respectively, for protein). This greater proportion of animal products increases the land requirements in comparison to a predominantly vegetarian diet, for example, as in India. In developed countries such as the United States and the United Kingdom, per capita dietary land requirements have been falling (Fig. 1.2) even while energy and protein consumption continue to rise (Fig. 1.3A). This apparent discrepancy is explained by the fall in the proportion of nutrients from animal products (Fig. 1.3B), and a shift in the mix of animal products consumed (Fig. 1.4). The drop in the proportion of nutrients from animal products is in large part due to the increased consumption of vegetal products, particularly vegetal

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1.3 GLOBAL AND COUNTRY-LEVEL CONSUMPTION PATTERNS AND TRENDS

(i) Energy

(A)

(B) 40

% From animal products

Energy (MJ per person per day)

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FIGURE 1.3 Per capita mean consumption (A), and percentage derived from animal products (B), of energy (i), and protein (ii), in foods consumed from 1961 to 2011 globally, and for selected countries, using global average nutritional values (FAOSTAT, 2015d,e). This includes commodities wasted after reaching the consumer, but not in the food supply chain. Source: Alexander, P., Brown, C., Rounsevell, M., Finnigan, J., Arneth, A., 2016. Human appropriation of land for food: the role of diet. Global Environ. Change 41, 88 98.

oil, for example, soybean oil. For example, in the United States vegetal oils provided 9.6% of calories in 1961, but this expanded to 19.2% by 2011 (14.5% from soya bean oil alone). Consumption of these oils accounts for over half (55%) of the 3.2 MJ (765 kcal) per person per day increase in energy consumed in the United States, with other sweeteners (i.e., corn syrup) and poultry meat respectively accounting for 26% and 18% of the rise.

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Consumption per capita (g per person per day)

Bovine meat

Pig meat

Poultry meat

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FIGURE 1.4 Per capita daily rates of bovine, pig, and poultry meat consumption from 1961 to 2011. Data Source: FAOSTAT, 2015d. Food Supply—Livestock and Fish Primary Equivalent (2015-12-16). Food and Agriculture Organization of the United Nations, Rome. Source: Alexander, P., Brown, C., Rounsevell, M., Finnigan, J., Arneth, A., 2016. Human appropriation of land for food: the role of diet. Global Environ. Change 41, 88 98.

The relative quantities of different animal products consumed changes over time, influencing the HALF results. The effects of this are evident in the results for China, where since 1961 the proportion of nutrients derived from animal products has increased toward that found in developed countries (Fig. 1.3), but the HALF values have converged more slowly (Fig. 1.2). The energy and protein intake and the percentages derived from animals are all higher than the global averages in China in 2011 (Fig. 1.3). Nonetheless, the HALF is lower in China compared to its global value (Fig. 1.2). This is due to the high rates of consumption of the commodities derived from monogastric animals (Fig. 1.4), which have lower feed conversion ratios and lower land requirements in comparison to ruminants, although direct energy inputs are higher (Table 1.1). For example, the average diet in China contained around half the global average amount of beef (53%), but more than twice that of pork (239%). The rise in global HALF (8.5%) is also modest (Fig. 1.2), given the rise in nutrients (28% rise in energy and protein) and the proportions derived from animals (increased by 11% for energy and 25% for protein). Again, this can be understood by reference to the changes in the relative quantities of meats consumed (Fig. 1.4). Global consumption per capita of bovine meat has been broadly constant, while poultry and pig meat have seen substantial rises, with 399% and 91% increases respectively from 1961 to 2011. Global average per capita consumption of beef is now less then pork and poultry in mass, energy, and protein.

1.4 ALTERNATIVES TO CURRENT ANIMAL PRODUCTS The proceeding work underlines the dominance of animal products in the food system for land use. This section introduces several potential alternatives to existing animal

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products as food protein and energy sources, that is, insects, cultured meat, imitation meat, and aquaculture. Subsequently we consider scenarios where these alternatives are used in substitution to part of the current mix of animal products using the HALF index methodology presented earlier.

1.4.1 Insects Edible insects have the potential to become a major source of human nutrition, and can be produced more efficiently than conventional livestock, that is, in terms of converting biomass into protein or calories (van Huis, 2013; Tabassum-Abbasi and Abbasi, 2016). They are high in fat, protein, and micronutrients (Rumpold and Schlu¨ter, 2013; Persijn and Charrondiere, 2014), and can be produced with lower levels of GHG emissions and water consumption (van Huis, 2013). The efficiency of insects to convert feed into edible food is in part due to the higher fraction of insect consumed (up to 100%), compared to conventional meat (e.g., 40% of live animal weight is consumed with cattle). Insects are poikilothermic, so they do not use their metabolism to heat or cool themselves, reducing energy usage. They tend to have higher fecundity than conventional livestock, potentially producing thousands of offspring (Premalatha et al., 2011). Efficiency is also increased by rapid growth rates and the ability of insects to reach maturity in days rather than months or years. Isotope analysis of bones indicates that insectivorous diets are entrenched in human evolution (Ramos-Elorduy, 2009; De-Magistris et al., 2015), and a variety of species are currently consumed ( . 2000 species; Rumpold and Schlu¨ter, 2013) across many regions of the world (119 countries; Rumpold and Schlu¨ter, 2013). But the issue of limited consumer acceptability is prevalent particularly in Western countries. These are also the countries with high animal product consumption rates per capita, and are therefore where a switch from animal product to insect consumption would have the greatest impact. There are already signs that consumer attitudes in developed countries such as the United States and the United Kingdom may be starting to change (Jamieson, 2015), and there may be less of a barrier to including insect-derived materials in other products, for example in powdered form (Little, 2015). However, in some jurisdictions, there are legal barriers (De-Magistris et al., 2015).

1.4.2 Cultured Meat Cultured meat, also termed in vitro, “lab-based,” or synthetic meat, refers to meat produced outside of a living animal. The meat is produced by culturing animal stem cells in a medium that contains nutrients and energy sources required for the division and differentiation of the cells into muscle cells that form into tissue (Bhat et al., 2015), with commercial-scale production anticipated by 2021 (Verstrate, 2016). The tissue produced can be separated for further processing and packaging. The amount of nutrients and energy needed may be relatively small, as only muscle tissue develops, without the need for biological structures such as respiratory, digestive, or nervous systems, bones, or skin (Bhat et al., 2014). Rapid growth rates mean that tissue is maintained for a shorter time than for animal rearing, further reducing required inputs.

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Cell and tissue culture are currently not efficient processes in terms of energy, water, and feedstock expenditure, and have been primarily employed in scientific and medical applications (Moritz et al., 2015). The financial and sustainability advantages are also unclear as the reductions in some inputs may be offset by the extra costs of a stricter hygiene regime and other energy inputs (Bhat et al., 2014). The cell culture medium can be produced from materials of animal origin (e.g., bovine serum), but this defeats many of the sustainability benefits of cultured meat (Bhat et al., 2014). Although suitable culture medium can be produced from nonanimal sources (e.g., hydrolyzed cyanobacteria, sometimes known as blue-green algae (Tuomisto and de Mattos, 2011) and Maitake mushroom extract (Bhat et al., 2014)), an efficient process to manufacture animal-free media is still viewed as a major challenge, and a barrier to cultured meat adoption (Mattick et al., 2015a). Consumer perceptions are also a potential barrier (Hocquette, 2016). The product needs to be of sufficiently similar taste, texture, and appearance to livestock meat for wide acceptance, and this is currently difficult to achieve (Moritz et al., 2015).

1.4.3 Imitation Meat Imitation meat or meat analogues attempt to mimic specific types of meat, including the aesthetic qualities (e.g., texture, flavor, and appearance) and the nutrient qualities, without using meat products. Soy-based products, such as tofu or tempeh, are perhaps the most widely known imitation meats (Malav et al., 2015). Tofu is soybean curd, made from coagulated soy milk, and has been prepared and consumed in Asia for centuries. It can be further prepared to approximate meat products in flavor and texture, for example, with flavoring added to make it taste like chicken, beef, lamb, ham, or sausage (Malav et al., 2015). Soy and tofu contain high levels of protein, while being low in fat (Sahirman and Ardiansyah, 2014). Beef and soy have a similar Protein Digestibility Corrected Amino Acid Score (PDCAAS), indicating that they have similar protein values in human nutrition (Schaafsma, 2000). More recent imitation meats include mycoprotein-based Quorn (Finnigan et al., 2010), and textured vegetable protein, again often made from soy.

1.4.4 Aquaculture Global aquaculture is already a major source of food, and has grown substantially over the past 50 years to produce around 61.9 Mt in 2011 (FAO, 2016), which is similar to the quantity of bovine meat (FAOSTAT, 2015h). As a global per capita average, protein from fish contribute 10% (2.72 g per capita per day) of that from meat, milk and eggs (27.69 g per capita per day; FAOSTAT, 2015h), around half of which is from aquaculture. Asia dominates aquaculture production (accounting for 89% by mass), with 62.4% produced in China alone, due to preexisting aquaculture practices and a relaxed regulatory framework (Bostock et al., 2010). Carnivorous fish, such as salmon, can consume up to five times the quantity of fish (as feed) than they ultimately provide (Naylor et al., 2009). Therefore limitations on the sustainable sourcing of feed represents a barrier to increases in farmed carnivorous fish (Diana, 2009), making substantial substitution with existing animal products less likely. This issue is less acute for herbivorous and omnivorous species, as they have much lower

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“fish-to-fish” conversion ratios, for example, carp currently has a ratio of 0.1, with further reductions predicted (Tacon and Metian, 2008) as fish-derived feed consumption is not essential for their nutrition (Bostock et al., 2010). Freshwater aquacultural systems dominate production, accounting for around two-thirds of all outputs from aquaculture. The main species are herbivorous or omnivorous, with the largest production from carp, although tilapia and catfish production have increased more recently (Bostock et al., 2010).

1.5 COMPARISON OF LAND REQUIREMENTS To provide an assessment of the consequences of adoption of the above alternative protein sources on agricultural land requirements, separate scenarios for each were considered, assuming replacement of 50% of current animal products. These scenarios assume that perceptions and diets alter over time, such that current animal product (i.e., meat, milk, and eggs) consumption declines and is substituted by a replacement food. The reduction in current animal products is evenly distributed across existing sources, and is replaced by one commodity so that the new diet contains at least the same quantities of energy and protein to the existing diet. The 50% replacement assumption is largely arbitrary, but is simply used as a reference point against which to compare alternative diets. It would have been equally accurate to select an alternative value, and the relative changes between these substitution scenarios would not have been impacted, that is, the changes would scale proportionately. However, the assumptions followed could also be considered to reflect a “demitarian” diet, where half the “normal” meat is consumed (Barsac Declaration Group, 2009; Lang, 2017; Weindl et al., 2017a,b). Further scenarios considered conventional animal products in the same manner (i.e., a 50% replacement), to provide a basis for comparison with the transformative scenarios. The scales of animal product substitution tested is not highly relevant, but rather the comparative outcomes between the substitution scenarios. The scenarios of reduced consumer waste (including both food waste and consumption in excess of nutritional requirements) and global adoption of the current average per capita diets in India and the United States were also constructed. These scenarios are not chosen to be equally probable or desirable, but rather to provide a broad comparison between the impacts of potential transformations in consumer behavior.

1.5.1 Alternative Animal Product Scenarios The scenarios considering alternative animal product also make the same assumption of a 50% replacement from current animal product consumption. Nutrient contents and feed conversion ratios were estimated for the substitute commodities (Table 1.2). The protein and energy contents were used to calculate the mass of the commodity required to replace the conventional foods removed. Feed conversion ratios were applied to evaluate the feed requirements to produce the substitute product. The feed was assumed to be provided from the current mix and yields of animal feeds, except for imitation meat, which was calculated using soybean production. The net changes in cropland and pasture areas were then calculated assuming the conventional livestock area reduces by 50% (assuming constant production practices) plus the requirements from the replacement commodity.

PROTEINS: SUSTAINABLE SOURCE, PROCESSING AND APPLICATIONS

TABLE 1.2 Feed Conversion Efficiencies, in Dry Matter (DM) Weight of Feed Required per Unit Edible Weight (EW), for Alternatives to Convention Animal Products Considered Feed Conversion by Mass (kg DM Percentage feed/kg EW) Edible [uncertainty (% EW of LW) range]

Energy Content (MJ/kg EW)

Protein Content (g/kg EW)

Energy Feed Conversion Efficiencya (%)

Protein Feed Conversion Efficiencya (%)

Direct Energy for Housing and Processing (MJ/kg EW)

1.8 [1.6 2.1]

8.9

179

33

50

7.3

Oonincx and de Boer (2012), Spang (2013), Persijn and Charrondiere (2014)

Crickets: 80 adults (Acheta domesticus)

2.1 [1.9 2.4]

5.9

205

19

49

No data

Finke (2002), van Huis (2013)

Cultured meat

4 [2 8]

8.3

190

17

24

18 25c

Tuomisto and de Mattos (2011)

0.29 [0.27 0.35]

3.2

81

47

72

11.4

Wang and Cavins (1989), Sahirman and Ardiansyah (2014), USDA (2015)

Commodity Mealworm: larvae (Tenebrio molitor)

100

100

Imitation meat (based on soybean curd)d

b

Data Sources

Tilapia

37

4.6 [3.7 5.5]

4.0

201

5.8

21.8

5.4

Pelletier and Tyedmers (2010), USDA (2015)

Chinese Carp

37

4.9 [3.9 5.9]

5.3

178

7.3

18.3

5.4e

Tacon and Metian (2008), Bauer and Schlott (2009), USDA (2015)

Notes. a Energy and protein conversion efficiency based on feed content of 15 MJ/kg DM and 200 g/kg protein. b Mealworm feed efficiency adjusted from Spang (2013), assuming 62% moisture content (Persijn and Charrondiere, 2014). c Excluding production of biomass feedstock. d Feed columns relates to inputs of soy to tofu production process. e Based on Tilapia production. Source: Alexander, P., Brown, C., Arneth, A., Dias, C., Finnigan, J., Moran, D., et al., 2017a. Could consumption of insects, cultured meat or imitation meat reduce global agricultural land use? Global Food Security 15, 22 32.

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1.5.2 Insect Consumption Mealworm larvae and adult crickets were selected to assess the impact of insect consumption, based on the availability of data for these species (Table 1.2). Protein from conventional livestock and insects was considered substitutable on an equal mass basis, as all essential amino acids for humans are available from insects, although profiles differ between species (van Huis, 2013; Persijn and Charrondiere, 2014). Insects are also high in a variety of micronutrients such as the minerals copper, iron, magnesium, manganese, phosphorous, selenium, and zinc and the vitamins riboflavin, pantothenic acid, biotin, and in some cases folic acid (Rumpold and Schlu¨ter, 2013; Persijn and Charrondiere, 2014). However, the analysis is limited to considering equivalence of protein and energy only. Although insects can be produced from organic wastes, given the high levels of production required under this scenario, it is assumed that production is from purpose-grown feed, rather than waste sources.

1.5.3 Cultured (In Vitro) Meat Process efficiency values from Tuomisto and de Mattos (2011) were used as feed conversion ratios, but assuming that the raw materials for the production of the culture medium is from conventional livestock feeds (Table 1.2). Tuomisto and de Mattos (2011) suggest 99% less land is required to produce cultured meat rather than livestock meat, but this assumes production of biomass for the culture medium using an algae-based system. This increases direct energy requirements while reducing land requirements, but depends upon a conflation of two novel technologies; production of algae biomass and cell culturing of meat. Producing feed from algae is likely to reduce the land required for conventional livestock production, while increasing other inputs, and therefore we consider only the cultured meat aspect. Production of the nutrient “broth” in which the cells are cultured (Mattick et al., 2015a; Verbeke et al., 2015) is possible from different inputs. However, as commercial-scale processes for cultured meat are not yet available (Mattick et al., 2015a), the assessment of which feedstock would be selected to produce the culture media in the required quantities, and the associated efficiency are both uncertain. To represent this uncertainty the conversion efficiency range tested is large (Table 1.2).

1.5.4 Imitation Meat The calculation was based on the use of soybean curd, that is, tofu, for imitation meat. Manufacturing soybean curd from soybeans creates some losses in protein and energy content (Wang and Cavins, 1989), for example, during the washing, grinding, boiling, and pressing involved (Sahirman and Ardiansyah, 2014), and also requires direct input of energy to these operations (Table 1.2). The production of the soybean curd was considered analogously to livestock production, with soy being used to produce soybean curd, rather than livestock inputs producing animal products. The losses in preparation of imitation meat from the soybean curd are expected to be low, and given the relatively simple processes, such as extrusion (Malav et al., 2015), have substantially lower direct energy inputs in comparison to cultured meat.

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1.5.5 Aquaculture Production of Chinese carp and tilapia were taken as examples in the analysis, due to their high contribution to current aquaculture and, compared to carnivorous fish (e.g., salmon), their low requirements for fishmeal or fish oil as feeds, and more advantageous feed conversion ratios. The feed conversion ratios to LW for tilapia and carp are 1.7 and 1.8, respectively (Tacon and Metian, 2008), but given that only 37% of the fish by weight is fillet (Bauer and Schlott, 2009; Pelletier and Tyedmers, 2010), this leads to a feed conversion ratios to EW of 4.6 4.9 (Table 1.2). Although some fishmeal and fish oil are currently used as feed for these species, these are not essential for nutrition in herbivorous and omnivorous species (e.g., carp and tilapia) (Bostock et al., 2010). Therefore the assumption is that all feed is provided from land-based production (e.g., soybeans and cereals). Any contribution from fishmeal and fish oil, that could be provided sustainably from fish processing by-products is neglected (Tacon and Metian, 2008; Bostock et al., 2010). The 50% replacement scenario would imply an approximately 10-fold increase in protein terms.

1.5.6 Conventional Livestock Consumption Changes Each of the conventional animal products was also considered as replacements for 50% of the current mix. Thus more than half of the calories or protein were assumed to be provided by the commodity being considered in each of these scenarios. For example, poultry meat currently provides 24% of all animal proteins, which reduces to 12% under all the other protein meat substitution scenarios except the poultry meat scenario. Under this scenario 62% of animal product consumption is from poultry, that is, the 12% of unchanged poultry consumption plus the 50% substituted for the current animal product mix. The feed and pasture area requirements were calculated using the results derived from the FAO data (FAOSTAT, 2015a,b,c,d,e,f), as described earlier.

1.5.7 Waste and Other Dietary Change Scenarios 1.5.7.1 Waste Reduction The waste reduction scenario uses losses from Alexander et al. (2017b). The scenario assumes that the combination of food discarded by consumers and due to overconsumption halves from the 2011 rates to 11% of energy and 26% of protein (assuming requirements of 9.8 MJ per person per day of energy and 52 g/day of protein; Institute of Medicine, 2005; SACN, 2011). The reduction in this waste was applied equally across all commodities. Losses during production, processing, and distribution were not changed, as the focus here is on the impact of consumer behavior on the food system. 1.5.7.2 High and Low Animal Product Diets To assess the impact of diets with high and low rates of animal products consumption, the average per capita consumption in the United States and India were chosen, respectively. These diets lie toward but not at global consumption extremes, and therefore provide a framework for understanding land use impacts arising from different food

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consumption habits. Additionally, the difference between the global average diet and the diets in each of the countries was decomposed into two parts. The first part represents a shift in the total quantity of nutrients consumed while holding the proportional contribution of each commodity constant. The second part represents a shift in the ratio or profile of commodities consumed, while holding the total nutrient level constant. These two parts were expressed both in protein and energy terms, with nutritional values by mass for each commodity derived from global FAO food supply data (FAOSTAT, 2015d,e). For example, the average energy consumed per capita globally is 11.9 MJ per person per day, while in the United States the average is 16.6 MJ per person per day, that is, 40% more. Therefore if the current global profile commodities remained unchanged, but the energy consumed increased to that of the United States, 40% more land would be required for production, in the absence of production intensification. This is reflected in a 40% increase in HALF. However, consumption in the United States also differs in the relative profile of the different commodities consumed. These differences also have an effect on the land required, evaluated without the influence of the quantity differences in the “profile” type.

1.5.8 Uncertainty Quantification A number of the parameter values used are uncertain, with perhaps the most influential ones being the livestock feed conversion ratios and the food nutrient contents. Feed conversion ratios are difficult to estimate, and have been the subject of misrepresentation by both sides of the sustainability meat consumption debate (Fairlie, 2010). The feed conversion ratios used here are for the global average production, derived in FAO studies (Macleod et al., 2013; Opio et al., 2013). While some uncertainty in feed conversion ratios remains, changes in the ratios only affect the disaggregation of the global pasture and feed areas between animal products. Biases introduced by inaccurate feed conversion ratios will cancel out in the baseline case. When alternative consumption profiles are considered they may not perfectly cancel out and result in a residual bias in the required land areas calculated. This is likely to be small relative to the scale of the overall effects shown, due in part to the offsetting between animal products. As a check on the accuracy of the feed conversion ratios used, the allocation of feed between monogastric animal and ruminants was compared against the results of a survey of the feed use from 134 countries (Alltech, 2013). This survey showed that 26% of total feed use was for ruminants in 2012, while 23% of feed was calculated as used for ruminants in 2011 in the results presented here. The level of agreement between these values gives additional confidence in the feed conversion ratios rates used. To assess the impact of uncertainties parameters for feed conversation ratios as well as energy and protein contents were randomly sampled from assigned uncertainty ranges (i.e., a Monte Carlo uncertainty method was used). The range of feed conversion ratios for conventional livestock was taken as 20% to 120% of the assumed value (Table 1.1), and for the alternative commodities the ranges are given in Table 1.2. The ranges for protein and energy contents were 10% to 110% for the 90 agricultural commodities, carp, tilapia, soybean curd, and cultured meat. However, the nutrient content of the insect species appears to be less certain, so a 30% to 130% range was used. All of these uncertainty

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ranges are indicative of qualitative levels of confidence in the default values used in the absence of relevant quantitative data. Uniform distributions were used for all parameter uncertainties, sampled 500 times.

1.5.9 Yields of Alternatives to Animal Product The energy and protein produced per unit of agricultural area were found to vary by more than 100-fold across conventional animal products and the alternatives considered (Fig. 1.5). Soybean curd had the highest energy and protein yields (2.2 MJ/m2 and 57 g/m2) and beef the lowest (0.02 MJ/m2 and 0.4 g/m2). After soybean curd, the two insect species gave the next highest yields. The yields for cultured meat were similar to eggs, and also relatively close to those for poultry. The order of commodities by yield differed between protein and energy, due to the differences in nutrient contents. For example, tilapia has a higher protein, but a lower energy yield, than carp. The areas for the ruminant derived products (i.e., mutton and goat meat, milk, and beef) include both cropland to produce feed and pasture area for grazing, while the other products use only feeds from cropland.

1.5.10 Land Requirements of Scenarios 1.5.10.1 Alternative Animal Product Scenarios Global cropland and pasture areas vary substantially under the scenarios (Fig. 1.6). The animal product substitute scenarios suggest that the HALF index (i.e., the percentage of land area required for food production), is 21.8 for soybean curd, and 112.2 for beef, compared to a baseline of 35.1 in 2011. There is also considerable variability in the cropland areas. The highest cropland requirement occurs in the tilapia scenario, where an additional 709 Mha of cropland is needed for feed, a 46% increase in the total cropland area. However, total agricultural land area reduces by 18% or 892 Mha, as cropland increases are more than offset by a 1601 Mha drop in pasture area. For the animal product replacement scenarios, the lowest cropland area is for milk with the cropland reducing by 217 Mha (14%) of cropland and 590 Mha (18%) of pasture, due to higher feed conversion ratios than the current mix of animal products, and because nutrients are also derived from pasture. Pasture changes dominate the results, with the cropland changes for most of the other scenarios being more modest. For example, the results with the largest agricultural area change have only a 7% 9% change in cropland, with soybean curd decreasing by 137 Mha and beef increasing by 110 Mha, while the pasture areas decrease by 1601 Mha and increase by 9916 Mha, respectively. The animal production replacement scenarios all provide at least the same amount of both energy and protein. The binding constraints were by energy for all scenarios except pork. In these scenarios the replacement food provides an equal amount of energy, but a greater quantity of protein. Conversely, for pork the binding constraint was on protein, due to the relatively low ratio of protein to energy in pork compared to the other animal products (FAOSTAT, 2015d).

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2 Calorific yield (MJ/m per year)

(A)

2

1

0 Soy bean curd

Mealworm larvae

Adult crickets

Eggs

Cultured meat

Pork

Poultry meat

Carp

Tilapia

Milk and products

Mutton and goat meat

Beef

Eggs

Poultry meat

Carp

Pork

Milk and products

Mutton and goat meat

Beef

Protein yield (g protein/m2 per year)

(B)

60

40

20

0 Soy bean curd

Mealworm larvae

Adult crickets

Cultured meat

Tilapia

FIGURE 1.5 Energy and protein per unit area of agricultural land for conventional and alternatives to animal production. Error bars show the yield range from uncertainty in feed conversion ratios and nutrient contents. Source: Alexander, P., Brown, C., Arneth, A., Dias, C., Finnigan, J., Moran, D., et al., 2017a. Could consumption of insects, cultured meat or imitation meat reduce global agricultural land use? Global Food Security 15, 22 32.

22

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120

HALF index

100

80

60

40

20

0 Soy bean curd

Mealworm larvae

Adult crickets

Eggs

Cultured meat

Cropland and pasture area in 2011

Poultry meat

Pork

Cropland area in 2011

Carp

Cropland area

Tilapia

Milk and products

Mutton and goat meat

Beef

Pasture area

FIGURE 1.6 Total cropland and pasture areas for food production under scenarios assuming 50% of current nutrients from animal productions are substituted with the indicated food, to provide at least equal energy and protein. The results are expressed as the percentage of global land required, or HALF index, based on 2011 population and food production systems. Error bars show the HALF range from uncertainty in feed conversion ratios and nutrient contents. Source: Alexander, P., Brown, C., Arneth, A., Dias, C., Finnigan, J., Moran, D., et al., 2017a. Could consumption of insects, cultured meat or imitation meat reduce global agricultural land use? Global Food Security 15, 22 32.

The range of agricultural land areas required based on uncertainty in feed conversion ratios and food nutrients (Fig. 1.6, error bars) are small for the animal product scenarios with low HALF indices (e.g., soybean curd and insects). This is because the uncertainty from new food commodities, for example, for soybean curd, is only a small proportion of the total agricultural area, therefore a large percentage uncertainty (Fig. 1.5) only produces a small absolute uncertainty in land area (Fig. 1.6). The opposite is the case for the results with higher HALF (e.g., beef), where the areas for replacement production are large and so, therefore, are the associated uncertainties. Fig. 1.5 shows uncertainty for each scenario per unit of energy or protein. The similarity in land requirements between the commodities with low HALF indices (Fig. 1.6) suggests that substantial land use and associated environmental benefits could be achieved from the adoption of any of them individually or in combination. Land requirements are always reduced by further increases in efficiencies of production per unit area. For example, a doubling of efficiency between two alternative scenarios always produces a halving of land use requirements. However, as the land use requirements decrease, the differences in the absolute areas also decrease, creating diminishing returns from increasing efficiency. The selection of the most appropriate mix of the more efficient products (Fig. 1.6) may therefore be more greatly influenced by other production externalities, for example, biodiversity or water usage, rather than the land requirements.

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1.5.10.2 Alternative Diet Scenarios Changes in diets and dietary impacts on land use are uncertain and are influenced by multiple factors, both economic and environmental. Two contrasting alternative scenario were used as exemplars to analyze the impacts of diet on global agricultural land use; the global adoption of the current diets of India and the United States. Although these countries are not the most extreme cases, they are major economies, with large populations, in which diets lie close to the lowest and highest land use requirements, respectively (of the 170 countries included, India has the 13th lowest HALF value and the United States has the 6th highest). Consideration of the adoption of these diets by the global population therefore provides a broad envelope within which human appropriation of land for food is likely to vary, but these are intended to be illustrative rather than represent equally plausible alternative futures. The net change in land use from a shift in global diet was decomposed into two parts; one considering a change in the quantity of nutrients consumed, and a second the profile of commodities consumed. The profile of commodities (i.e., the sources from which nutrients are derived) was found to have a greater impact on land use than the quantities of nutrients consumed, in the dietary transitions considered (Table 1.3). For both dietary scenarios, changes in quantities and profiles act in the same direction, intensifying the overall impact. The impact of contrasting diets is much larger for the livestock area compared to cropland area used for food for human consumption. A more than threefold increase is required in livestock area (pasture and cropland for feed) under the US diet scenario, increasing HALF by 178%. This area is needed both to support the increased quantities of nutrients consumed and the changes in dietary profile toward a greater proportion of animal products. Conversely, the lower overall consumption and the lower proportion from animal products in India suggests the livestock area would drop to less than a third of the current area and reduce the overall HALF by 55%. The changes in cropland required to produce food for human consumption are comparatively modest with both the Indian and US diets, with a 4% fall and a 21% rise, respectively. The profile of the Indian diet is weighted toward vegetal crops, but the impact of this is offset by the lower level of nutrient intake overall. The opposite is the case for the average diet in the United States, with a lower emphasis on crops, but higher overall consumption. Fig. 1.6 shows the 2011 HALF index values for these scenarios, with cropland (for food and feed) and pasture identified separately. 1.5.10.3 Results Summary The results from the scenarios presented are summarized in Table 1.4. The table includes HALF results from meat substitution scenarios, and the adoption of high and low animal product diets scenarios (based on average consumption in India and the United States). Additionally, consumer waste scenarios from Alexander et al. (2017b) are included to provide a further comparison. This consumer waste scenario, assuming foods discarded and lost due to overconsumption are halved, was found to spare 9% of agricultural land. As the high and low animal product diets and waste scenarios involve different assumptions, that is, they do not consider a 50% substitute of animal products, direct comparisons between these two scenario groups must be limited. However, the high and low

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TABLE 1.3 Changes in HALF From Transitions of Average Global Diet to That of India or the United States in 2011, Divided Into the Impact From Quantity of Consumption (Quantity) and the Types of Commodities Consumed (Profile) Dietary Scenario Country

Type and Nutrient Basis

India

Profile: energy

113

22

61

47

India

Profile: protein

127

12

56

40

India

Quantity: energy

16

India

Quantity: protein

25

India

Overall

5

34

67

55

United States

Profile: energy

11

121

1122

197

United States

Profile: protein

17

113

1109

185

United States

Quantity: energy

1 41

United States

Quantity: protein

1 50

United States

Overall

1 25

1 71

1 214

1 178

Cropland Area for Food Change (%)

Total Cropland Area Change (%)

Livestock (Feed and Pasture) Area Change (%)

Agricultural Area Change (%)

For the quantity and profile cases, the change in areas are calculated based on providing the same energy and protein as current consumption. The overall type includes changes in quantities and profile of foods consumed, and by definition (1 1 overall change rate) 5 (1 1 profile change rate) 3 (1 1 quantity change rate), in terms of energy or protein. A single “overall” row is given for each dietary scenario, as this is equal in both nutrient terms. Source: Alexander, P., Brown, C., Rounsevell, M., Finnigan, J., Arneth, A., 2016. Human appropriation of land for food: the role of diet. Global Environ. Change 41, 88 98.

animal product diets (based on United States and India), respectively, were found to have higher and lower land impacts than the meat alternatives, with the exception of beef (Table 1.4). This is because the diets include both a shift in the amounts of food consumed and, more importantly, in the types of food consumed (Table 1.3 and Fig. 1.7). These diets involve different rates of meat consumption, and therefore are not restricted to maintain 50% of the current animal products as in the other scenarios.

1.5.11 Discussion 1.5.11.1 Limitations of the Analysis A stylized and exploratory approach is used to better understand and ensure comparison on a like-for-like basis of potential land use outcomes across a range of scenarios, from

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TABLE 1.4 Summary of Results Across All Scenarios, Ordered by Increasing Agricultural Land Use

Scenario

Description

Low animal product diet

Average diet globally becomes that of the average diet in India

Percentage Change in Required Agricultural Area for Food

HALF Index

Comments

55

15.7

Influenced by lower overall consumption, and lower rates of meat in the diet. In both these aspects global diets are changing in the opposite direction of current trends, making this scenario of low plausibility.

Soybean curd Soybean curd replaces 50% of current animal products

35

21.7

Increase in direct energy inputs in comparison to animal products, but less substantial than for cultured meat. 50% uptake seems unlikely to be acceptable to consumers.

Insects

34

22.2

Consumer acceptability barriers in some regions. A lower level of uptake in combination, perhaps as an ingredient, for example, in prepackaged foods, seems more likely.

Mealworm larvae replaces 50% of current animal products

Most efficient Eggs or chicken replaces conventional 50% of current animal animal products products

30 to

Cultured meat

29

24.0

Technology still rather uncertain (Bhat et al., 2014), and benefits compared to other sources of nutrients currently are not well demonstrated. The high direct energy used in production also a concern.

Most efficient Carp replaces 50% of aquacultural current animal products product

22

26.8

Potential for environmental pollution issues with large-scale production, although this is also the case with other intensive animal production.

Milk and products

Milk and products replaces 50% of current animal products

16

28.9

Associated with the largest reduction of cropland, while still providing material reduction in overall agricultural area.

Reduction in waste

Consumer waste, including food discard and due to overconsumption is halved

9

32.0

Feasible, but opposite to current direction of change, particularly with respect to overconsumption. Health, as well as environmental, benefits for policies or social changes to reverse these changes.

Cultured meat replaces 50% of current animal products

28

23.7 24.4 The direction of recent changes, with rapid growth in the consumption rates for chicken in particular, supported by intensification in production.

(Continued)

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TABLE 1.4 (Continued) Percentage Change in Required Agricultural Area for Food

Scenario

Description

High animal product diet

Average diet globally becomes that of the average diet in the United States

Least efficient Beef replaces 50% of conventional current animal products animal product

HALF Index

Comments

1178

97.7

Not possible given production systems currently used. Direction of recent changes for overall nutrients and rates of animal products consumption. Approaching this consumption globally would be expected to increase food price, suppress demand and intensify production practices.

1204

112.2

Physically impossible with production systems currently used, and contrary to current trends of average per capita consumption falling since 1970s.

Source: Alexander, P., Brown, C., Arneth, A., Dias, C., Finnigan, J., Moran, D., et al., 2017a. Could consumption of insects, cultured meat or imitation meat reduce global agricultural land use? Global Food Security 15, 22 32.

100

HALF index

80

60

40

20

0 Indian diet: overall

Indian diet: profile

Indian diet: quantity

Cropland and pasture area in 2011

2011 baseline

Cropland area in 2011

USA diet: quantity

Cropland area

USA diet: profile

USA diet: overall

Pasture area

FIGURE 1.7 Cropland and pasture required to produce food under alternative dietary scenarios, expressed as required percentage of world land, or Human Appropriation of Land for Food (HALF) index, using global 2011 population and production systems. For each scenario (from Table 1.3) the case is shown that provides at least equal amounts of both energy and protein, for example, the protein case is shown for the Indian diet profile, as the energy case provides insufficient protein. Source: Alexander, P., Brown, C., Rounsevell, M., Finnigan, J., Arneth, A., 2016. Human appropriation of land for food: the role of diet. Global Environ. Change 41, 88 98.

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the more unusual and transformational (e.g., insects and cultured meat), to the more conventional (e.g., changes in proportions of livestock demand). The replacement of at least equal quantities of protein and calories has been considered, leaving the potential for reductions in micronutrients between the scenarios. The results are not intended as predictive, nor are they presented to suggest equal plausibility, but rather to allow comparisons in land use requirements between the scenarios. Fixed global average production figures based on 2011 were used and no spatial variation in production practices are taken into account. These production practices would be expected to respond to the substantial changes considered in these scenarios, mediated by international trade in agricultural commodities. For example, increased agricultural land requirement would tend to intensify production, with higher rates of inputs used to achieve greater yields. Conversely, if less agricultural land is needed for food, this may cause a lowering of the production intensity. In both cases, such adaptation in production moderates the land use consequences, but alters the resource requirements for other inputs, for example, fertilizer or pesticide use (Smith, 2013b; Hertel et al., 2016). However, the results do characterize the demands placed on agricultural production, which can be interpreted as implying an increase in agricultural areas, an equivalent increase in productive efficiency (perhaps through greater inputs, i.e., higher intensity), or some combination of the two. Nonetheless, comparison with a previous more complex model results suggests that the outcomes here are broadly equivalent. For example the vegan and vegetarian diets in Erb et al. (2016) have a central value for cropland area of approximately 1200 and 1000 Mha, respectively, compared to the low meat diet used here (based on the average diet in India) of 1022 Mha. As expected, for the reasons given earlier, the intensity changes considered in Erb et al. (2016) (which are not a direct parallel of the scenarios used here) appear to moderate the land use outcomes, with less agricultural land relinquished, but coupled with a decrease in intensity of production. Therefore although the adopted approach neglects aspects that would allow robust spatial or temporal predictions of land use, it does provide a consistent methodology across scenarios allowing comparisons between them, a primary aim of the chapter. The results demonstrate that milk production is more efficient than the current animal product mix, with the milk scenario showing a decrease in land requirements (Table 1.4). Cull dairy cows and male dairy calves could also be used to produce beef, which is not accounted for in these results. If the additional beef production from an expanded dairy sector were considered, the land requirements in the milk scenario would be further reduced, as less land would be required to produce the remaining beef consumed. The magnitude of this bias is perhaps moderate, as the fraction of emissions from the dairy herd currently assigned to milk rather than meat production is between 90% and 96% (Opio et al., 2013). 1.5.11.2 Imitation Meat and Soybean Production The imitation meat scenario, based on soybean curd, implies that more cropland is used for growing soybeans, while the other meat replacement scenarios use a more diverse mix of feeds. The additional soybean areas may be less suited to the crop and so would have lower yields than existing production, potentially leading to an underestimate of the area needed when using average yields. An additional 111 Mha of soybean area was calculated

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as being needed (i.e., a doubling of 2013 area; FAOSTAT, 2015b), while 248 Mha of cropland currently used for animal feed is spared. Therefore the net cropland area decreases in this scenario suggest that suitable land may be available, although this would also be constrained by climatic suitability. However, higher soybean yields would be anticipated to have only a small impact on the results as the net percentage agricultural area change is dominated by the change in pasture area. The expansion of soybean area may have substantial local impacts, for example, on biodiversity and soil quality, due to the intensity of production. However, the land spared from agricultural production by the transition could be potentially used to offset such negative outcomes. This would be a form of “land sparing,” that is, separation of land for conservation and food production, in contrast to “land sharing” with integration of conservation and production (Phalan et al., 2011). However, attempting to account for the associated trade-offs and scale effects, as well as the challenges and controversy involved (Fischer et al., 2014), are out of scope for consideration here. 1.5.11.3 Cultured Meat and Energy The results suggest that the benefits claimed for cultured meat (Tuomisto and de Mattos, 2011) may not be justified. Although cultured meat was found to have a lower land footprint than beef, it had a similar efficiency to poultry meat (Figs. 1.2 and 1.5), but with substantially higher direct energy requirements (Table 1.2). Direct energy inputs are needed for cultured meat to process raw biomass material into the cell medium, to then culture the cells and process them into a consumable product, including sterilization and hydrolysis (Tuomisto and de Mattos, 2011). Conventional livestock use direct energy primarily in housing, for example, lighting, heating, and cooling (Macleod et al., 2013). Direct energy inputs for cultured meat (18 25 GJ/t; Tuomisto and de Mattos, 2011; Table 1.2) are higher than any of the other foods considered here (at least four times the highest conventional animal product, poultry meat (4.5 GJ/t; Macleod et al., 2013)). This suggests that a low-cost and low-carbon source of energy may be a prerequisite for cultured meat to be economically and environmentally viable. Furthermore, the provision of growth factors, vitamins, and trace elements, for example, B12, will also have an impact on the resources used for cultured meat, although the scale of this is unclear. However, the overall primary energy used in the production of cultured meat production was shown to be 46% lower than for beef production (e.g., including energy in fertilizer production and machinery), but 38% higher than for poultry meat. Given the relative novelty of this technology, further development and optimization may be able to reduce these energy and cost requirements and increase the efficiency of production (Bhat et al., 2017). These improvements would potentially involve development of improved methods for producing the cell culture medium beyond that assumed here. The types of feed used may not match the current animal feed mix, although the land use consequences of such differences are likely to be lower than that associated with the uncertainty in efficiency of cultured meat production and would not be expected to alter our conclusions. Overall, currently cultured meat could provide some benefits (e.g., land use savings compared to beef), but result in higher direct energy requirements and also potentially primary energy (e.g., in comparison to poultry meat). This conclusion concurs with a more recent anticipatory LCA of culture meat production (Mattick et al., 2015b).

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1.5.11.4 Insects, Promising but More Research Needed Insects are the most efficient animal production system considered, although less so than soybean curd. However, insects have the additional advantage that they are able to use a wide variety of feeds, including by-products and waste (Ocio and Vinaras, 1979; van Broekhoven et al., 2015). The results here assume that insect feed uses the same mix of feeds currently used for conventional livestock. However, if half of food discarded by consumers (from Alexander et al. 2017b) could be used as feed for mealworms, this would replace 8.1% of current animal production. Where the total feed is reduced there is potential for this to occur primarily for food commodities (e.g., cereals), and thereby increase the proportion of by-products. Although by-products are ascribed some value when considering their impacts (Elferink et al., 2008), the system efficiency increases by replacing lower yielding conventional livestock with insects (Fig. 1.5). For instance, soybeans could be used to produce soybean curd, and then feed insects from the residues. More research is needed to understand how the large-scale production of insects could be achieved, the inputs required, the suitability of feeds, and other constraints (e.g., location) (van Huis, 2013). There is little published data on the feed efficiency of insect productions. However, direct energy inputs for intensive insect production appears comparable to intensive conventional livestock production (Oonincx and de Boer, 2012). Perhaps the biggest barrier to the large-scale global adoption of insects as a food source is consumer acceptability (Looy et al., 2013; Shelomi, 2015), where again further research is required to understand how best to increase adoption and what rate and levels of consumption might be possible. 1.5.11.5 Existing Diets With High and Low Animal Products The two contrasting examples of current average consumption in the United States and India were used to examine how changes in food consumption preferences and behaviors might affect agricultural commodity demand and land use. Although the average global diet transitioning to the current average US diet scenario is unlikely in the short term, consumption patterns have been shifting in this direction, due to increases in per capita incomes in developing countries (e.g., China and Brazil), rural urban migration, and globalization, leading to more overall per capita food consumption, and a greater percentage consumption of animal products (Lambin and Meyfroidt, 2011; Tilman et al., 2011; Seto and Ramankutty, 2016). However, a substantial gap in consumption patterns remains between countries, with the US diet requiring 2.8 times the land area of the global average diet, and 3.4 times that of the Chinese diet. Consequently, given current yields and production systems, it would clearly not be possible for the world’s population to consume food as in the United States; indeed, this would require 98% of all land, including snowcover and deserts. Apart from being physically impossible, changes to approach this level of consumption would also generate strong market signals that would act to increase the price of food, suppress demand, and intensify production practices (additional inputs, e.g., irrigation water, fertilizer, or labor, leading to higher yield). Conversely, if more land were to be used for agriculture, suitable land would become more scarce, and the additional land would tend to be of lower quality and produce lower yields, leading to a greater area requirements (Lambin and Meyfroidt, 2011). Price signals may be particularly

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large for the less efficient and potentially costlier commodities, for example, beef. Arguably, these impacts are already evident, with a shift toward chicken and away from beef (Fig. 1.4) supported by intensification of chicken production and the associated efficiency increases (Havenstein, 2006). The contrasting scenario considers the global diet becoming equivalent to the average diet of India. This is more plausible from an environmental and agricultural system viewpoint. However, it implies shifts in consumption that are the opposite of the global consumption trends that have occurred over previous decades, as per capita incomes have increased in developing countries. A reversal of these trends would either require a substantial shift in consumer preferences (toward the consumption of vegetal crops, for example, higher rates of vegetarianism), or a catastrophic global economic collapse reducing per capita incomes, particularly in wealthier countries. Changes in food preferences may be achievable through either behavioral or economic approaches. For example, less food is consumed when people are offered smaller-sized portions, packages, or tableware than when offered larger-sized versions, leading to the possibility of policies to reduce consumption (Hollands et al., 2015). Economic approaches such as taxes (e.g., a fat tax or a tax on sugar-sweetened beverages) and subsidies (e.g., on fruit and vegetables) could be used to provide fiscal incentives to change behaviors (Thow et al., 2010; Wang et al., 2012). However, the effectiveness of taxation and subsidies alone to alter diets, without other policies that target a number of different levels within society, has been questioned (Tiffin and Arnoult, 2011). 1.5.11.6 Obesity, Malnutrition, and Waste The results presented here are based on the average food reaching consumers rather than human nutritional requirements, and it is important to consider the extent to which these differ within a population. Distinctions arise due to overeating and, conversely, malnutrition, through waste of food by consumers (Eshel and Martin, 2006), and also inequalities in distribution (Porkka et al., 2013). Losses and waste occur at each stage of the food supply chain, with overall food waste, accounting for losses in production and at the consumer, estimated to be around 25% 40% of total food production (Godfray et al., 2010; Kummu et al., 2012). HALF values include losses both in the production system (e.g., unharvested crops and losses in storage, transportation, and processing) and at the consumer. Production system losses are derived from the global production efficiencies, and therefore are considered only as a global average. By contrast, food waste by consumers is included at a country-specific level, as this is included in the FAO commodity balance data used (FAOSTAT, 2015c). Consequentially, the HALF index includes (but does not separately identify) the variations in the rates of per capita food waste by consumers. 95 115 kg/year of food has been estimated to be wasted per capita after reaching the consumer in Europe and North America, while in sub-Saharan Africa and South/Southeast Asia this is only 6 11 kg/year (Gustavsson et al., 2011), which equates to 9% 12% and 1% 3% of food delivered to consumers respectively. Applying the mean values of these rates for United States and India suggests that the HALF value for consumer waste alone is 10.3 and 0.3, respectively. The protein requirement of adult men and women depends on body weight. For an average body weight of 60 kg, 50 g/day of protein is the minimum safe limit (WHO, FAO, UNU, 2007).

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No country with a population of more than 20 million currently falls below this limit, although several smaller countries consume 40 50 g per person per day, that is, Guinea, Guinea-Bissau, Haiti, Liberia, Madagascar, Mozambique, Zambia, and Zimbabwe. The energy requirements also vary by sex, weight, and the level of physical activity. For instance, average energy requirements for the population of UK adult females and males, are respectively 8.7 MJ/day (2079 kcal/day) and 10.9 MJ/day (2605 kcal/day) (SACN, 2011). To compare with the calculated energy intakes, we assume the mean energy requirement value is 9.8 MJ per person per day (2342 kcal per person per day). This value is somewhat higher than the 2100 kcal per person per day energy intake used in some previous studies (Eshel and Martin, 2006; Kummu et al., 2012), and likely to exceed the intake needed to avoid hunger or malnutrition (WFP, 2016). The average Indian consumption appears close to the population’s energy requirements, given the relatively low levels of consumer waste in South and Southeast Asia (Gustavsson et al., 2011), just 1% more, assuming 2% food is discarded. Even if there is sufficient food to avoid malnutrition within a country or region, this does not mean that these foods are distributed equitably. Globally, 37% of men and 38% of women were overweight in 2014 (Ng et al., 2014), while approximately 12% of people were undernourished between 2010 and 2012 (FAO, IFAD, WFP, 2015). The populations living in countries with critically low food supply (,2000 kcal per capita per day) have also been dropping over time, from 52% in 1965 to 3% in 2005 (Porkka et al., 2013). In India (ranked 25th worst in the 2015 Global Hunger Index Report; von Grebmer et al., 2015) 20% of the population are overweight (including nearly 5% obese) and 15% undernourished (Ng et al., 2014; FAO, IFAD, WFP 2015), while for adults in the United States 66% are overweight, including 33% obese (Ng et al., 2014). Given there are three times more overweight people than undernourished, and that levels of malnutrition have been declining over recent years, better national and international distribution of food is more relevant to achieving global food security than additional production. The US per capita energy consumption is 16.6 MJ/day, which suggests that 41% of food (in energy terms) is either due to overeating or consumer waste (34% of energy intake is in excess of requirements, assuming 10.5% food waste; Gustavsson et al., 2011). This is in line with a previous finding, showing that in the United States, overeating and food discarded by consumers accounted for 44% of food distributed to consumers (Eshel and Martin, 2006). The results suggest that under the global adoption of US consumer behaviors the land required to produce the food wasted by consumers (including overconsumption), would be sufficient to provide more than twice the entire food requirements assuming adoption of Indian consumption patterns. 1.5.11.7 Comparisons to Previous Studies The results show that either substitution of source of current animal protein sources or adoption of diets already consumed by hundreds of millions of people could lead to a magnitude of change around a doubling or halving of current agricultural land area. There have been few previous studies that have quantified the impact of such substantial shifts in diets on agricultural land areas and none that consider the impact alternative protein sources. Stehfest et al. (2009) is one example, where dietary scenarios for 2050 are

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considered, including a “healthy diet” (low rates of ruminant meat and pork and moderate poultry and consumption) and a no-meat diet. The current diet in India falls between these scenarios (i.e., rates of animal product consumption are lower than the Stehfest et al. “healthy diet,” but higher than the no-meat diet), and likewise the land use results found here lie between those of Stehfest et al. (2009). The impact of a “healthy diet” was also considered in Bajˇzelj et al. (2014), and showed a somewhat lower drop of 32% in pasture areas in 2050 compared to the authors’ business-as-usual scenario. The few studies published to date have shown that shifts in dietary preferences have a substantial impact not only on agricultural land use, but also on externalities such as GHG emissions and bioenergy potential (Popp et al., 2010; Haberl et al., 2011). Further studies that do not include land use change have also shown substantial GHG emissions implications from alternative diets, for example, a 55% reduction from a vegetarian diet (Tilman and Clark, 2014). Considering the trade-offs between land for bioenergy production or afforestation (Williamson, 2016), reducing agricultural GHG emissions and meeting the food requirements of a growing population, a greater focus is justified in examining demand-side measures, including waste reduction (Smith and Gregory, 2013). 1.5.11.8 A Future for Ruminants? The land use footprint of ruminant meat production is high, and therefore consuming more beef and sheep meat requires large increases in land areas (Fig. 1.6). Although ruminants are less efficient converters of feed to edible foods than monogastrics (Table 1.2), their high reliance on forage that is inedible to humans from nonarable land reduces their claim for feeds produced on cropland (Smil, 2013). Livestock production can also provide a range of other benefits, for example, recycling plant nutrients, maintaining ecosystems, and providing social benefit (Oltjen and Beckett, 1996; Janzen, 2011). Therefore ruminants that are mainly grass-fed from land that is unsuitable for the production of other crops may provide substantial benefits, but this implies a move away from intensive production practices, that is, that use large quantities of feed produced from cropland. Such extensive grazing-based systems are likely to produce a reduced quantity of livestock, and therefore per capita consumption rates of ruminant meat would have to continue to fall to avoid unsustainable land use change. Additionally, changes toward consumption of diets with lower land use requirements also provide the prospect of reduced competition for land between food production and climate change mitigation measures, for example, bioenergy or afforestation (Smith et al., 2014).

1.6 CONCLUSIONS Dramatically different requirements of land for food production could arise depending on future dietary change, and in particular the combination of animal products. The results presented in this chapter suggest that alternatives to the current mix of livestock production systems could substitute current animal products and substantially reduce the current agricultural land use footprint from food production. Reducing meat consumption overall is likely to have the greatest effect on the land use footprint, but replacing beef or lamb with any of the foods considered here has the potential for substantial sustainability

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benefits. Although the two most efficient products considered, that is, imitation meat and insects, both come with consumer perception barriers, a shift toward poultry meat, eggs, and milk was also found to offer land use and associated environmental benefits, of only slightly smaller magnitudes. Reductions in consumer waste have potentially important but smaller impacts on resource requirement than the other scenarios considered. A wide range of land requirements for food was found based on global adoption of current country-level average diets, far wider than the divergence in energy or protein intakes, with the difference due to the types of commodities in each diet, and in particular the level of ruminant animal products. For example, if the diets of India or the United States were adopted globally the impact from the change in the mix of commodities would be about twice that from the quantities consumed. What we individually eat (or even waste), rather than how much, appears to be more important for agricultural land requirements. However, waste and overeating are still important issues, with the results suggesting that the land required to produce the food wasted by consumers (including overconsumption) given US consumption, could provide more than twice the food required under adoption of Indian consumption patterns. We conclude that a diet which reduces agricultural land requirements may best be achieved through a combination of approaches, includes both shifts toward more efficient conventional animal products (e.g., chicken and eggs), and greater use of alternatives such as insect and imitation meat, as well as waste reduction. A more balanced approach than those in the stylized scenarios considered here would also require less extreme shifts in diets and therefore need less dramatic changes in consumer consumption habits. This chapter focuses principally on the land requirements, although out of scope here, a similar consistent GHG LCA across all options is warranted, as well as consideration of consequences for biodiversity, water requirements, and other ecosystem services. Further research is also required into the technologies and production systems for the large-scale production of insects, including what feeds are most appropriate and the potential use of food waste and by-products, and to better understand how consumer behavior and preferences can be influenced toward a healthier and more sustainable diet.

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Schaafsma, G., 2000. Criteria and significance of dietary protein sources in humans The Protein Digestibility Corrected Amino Acid Score 1, 1865 1867. Schmitz, C., van Meijl, H., Kyle, P., et al., 2014. Land-use change trajectories up to 2050: insights from a global agro-economic model comparison. Agric. Econ. 45, 69 84. Seto, K.C., Ramankutty, N., 2016. Hidden linkages between urbanization and food systems. Science 352, 943 945. Shelomi, M., 2015. Why we still don’t eat insects: assessing entomophagy promotion through a diffusion of innovations framework. Trends Food Sci. Technol. 45, 1 8. Smil, V., 2002. Worldwide transformation of diets, burdens of meat production and opportunities for novel food proteins. Enzyme Microb. Technol. 30, 305 311. Smil, V., 2013. Should We Eat Meat? Evolution and Consequences of Modern Carnivory. Wiley, New York. Smith, K.A., 2013a. Why the Tomato Was Feared in Europe for More Than 200 Years: How the Fruit Got a Bad Rap from the Beginning. Smithsonian. Smith, P., 2013b. Delivering food security without increasing pressure on land. Global Food Security 2, 18 23. Smith, P., Gregory, P.J., 2013. Climate change and sustainable food production. Proc. Nutr. Soc. 72, 21 28. Smith, P., Bustamante, M., Ahammad, H., et al., 2014. Agriculture, Forestry and Other Land Use (AFOLU). In: Edenhofer, O.R., Pichs-Madruga, Sokona, Y., Farahani, E., Kadner, S., Seyboth, K., et al.,Climate Change 2014: Mitigation of Climate Change. Contribution of Working Group III to the Fifth Assessment Report of the Intergovernmental Panel on Climate Change. Cambridge University Press, Cambridge and New York, pp. 811 922. Spang, B., 2013. Insects as Food: Assessing the Food Conversion Efficiency of the Mealworm (Tenebrio molitor). Evergreen State College, WA. Stehfest, E., Bouwman, L., Van Vuuren, D.P., Den Elzen, M.G.J., Eickhout, B., Kabat, P., 2009. Climate benefits of changing diet. Clim. Change 95, 83 102. Tabassum-Abbasi, A.T., Abbasi, S.A., 2016. Reducing the global environmental impact of livestock production: the minilivestock option. J. Clean. Prod. 112, 1754 1766. Tacon, A.G.J., Metian, M., 2008. Global overview on the use of fish meal and fish oil in industrially compounded aquafeeds: trends and future prospects. Aquaculture 285, 146 158. Thornton, P.K., 2010. Livestock production: recent trends, future prospects. Philos. Trans. R. Soc. Lond. Ser. B Biol. Sci. 365, 2853 2867. Thow, A.M., Jan, S., Leeder, S., Swinburn, B., 2010. The effect of fiscal policy on diet, obesity and chronic disease: a systematic review. Bull. World Health Organization 88, 609 614. Tiffin, R., Arnoult, M., 2011. The public health impacts of a fat tax. Eur. J. Clin. Nutr. 65, 427 433. Tilman, D., Clark, M., 2014. Global diets link environmental sustainability and human health. Nature 515, 518 522. Tilman, D., Balzer, C., Hill, J., Befort, B.L., 2011. Global food demand and the sustainable intensification of agriculture. Proc. Natl Acad. Sci. U.S.A. 108, 20260 20264. Townsend, E., 2012. Lobster: A Global History. University of Chicago Press, Chicago, IL. Tuomisto, H.L., de Mattos, M.J.T., 2011. Environmental impacts of cultured meat production. Environ. Sci. Technol. 45, 6117 6123. UNFCC, 2015. COP21: Adoption of the Paris Agreement. United Nations Framework Convention on Climate Change. USDA, 2015. National Nutrient Database for Standard Reference Release 28. United States Department of Agriculture, Agricultural Research Service. van Huis, A., 2013. Potential of insects as food and feed in assuring food security. Annu. Rev. Entomol. 58, 563 583. Verbeke, W., Marcu, A., Rutsaert, P., Gaspar, R., Seibt, B., Fletcher, D., et al., 2015. “Would you eat cultured meat?”: Consumers’ reactions and attitude formation in Belgium, Portugal and the United Kingdom. Meat Sci. 102, 49 58. Vinnari, M., Mustonen, P., Ra¨sa¨nen, P., 2010. Tracking down trends in non-meat consumption in Finnish households, 1966 2006. Brit. Food J. 112, 836 852. van Broekhoven, S., Oonincx, D.Ga.B., van Huis, A., van Loon, J.Ja, 2015. Growth performance and feed conversion efficiency of three edible mealworm species (Coleoptera: Tenebrionidae) on diets composed of organic by-products. J. Insect Physiol. 73, 1 10.

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von Grebmer, K. Bernstein, J., Prasai, N., Yin, S., Yohannes, Y., 2015. 2015 Global Hunger Index. International Food Policy Research Institute, Bonn, Washington, DC, and Dublin. van Vuuren, D.P., Stehfest, E., Gernaat, D.E.H.J., et al., 2018. Alternative pathways to the 1.5  C target reduce the need for negative emission technologies. Nat. Climate Change 8, 1 7. Verstrate, P., 2016. Feeding the 7 Billion: Cultured Meat. In: Edinburgh International Science Festival, Summerhall, Edinburgh. WFP, 2016. What Is Hunger? World Food Programme (WFP), Rome. WHO, FAO, UNU, 2007. Protein and Amino Acid Requirements in Human Nutrition. World Health Organization Technical Report Series, p. 935. Wang, H.L., Cavins, J.F., 1989. Yield and amino acid composition of fractions obtained during tofu production. Cereal Chem. (USA) 66, 359 361. Wang, Y.C., Coxson, P., Shen, Y., Goldman, L., 2012. A penny-per-ounce tax on sugar-sweetened beverages would cut health and cost burdens of diabetes. Health Affairs 31, 199 207. Weindl, I., Popp, A., Bodirsky, B.L., et al., 2017a. Livestock and human use of land: productivity trends and dietary choices as drivers of future land and carbon dynamics. Global Planet. Change 159, 1 10. Weindl, I., Bodirsky, B.L., Rolinski, S., et al., 2017b. Livestock production and the water challenge of future food supply: implications of agricultural management and dietary choices. Global Environ. Change 47, 121 132. West, P.C., Gerber, J.S., Engstrom, P.M., et al., 2014. Leverage points for improving global food security and the environment. Science 345, 325 328. Williamson, P., 2016. Scrutinize CO2 removal methods. Nature 530, 5 7.

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Insects as a Source of Sustainable Proteins Seema Patel Bioinformatics and Medical Informatics Research Center, San Diego State University, San Diego, CA, United States O U T L I N E 2.1 Introduction

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2.1 INTRODUCTION The importance of dietary proteins cannot be underestimated (Pihlanto and Korhonen, 2003). Proteins form the enzymes, transcription factors, antibodies, neurotransmitters, hormones, and a whole lot of other vital body components. Protein deficiency can lead to a gamut of illnesses (Bhutta and Sadiq, 2012), and undernutrition is killing millions of people annually. So far, most of the starvation and famine conditions are confined to developing countries. But, as climate change is causing erratic weather patterns, and the human population is poised to exceed 9 billion by 2050, global food insecurity looms on the horizon. At this rate of human population growth, conventional protein production from livestock, poultry, and fish cannot keep up (van Huis, 2013), so novel and affordable sources of proteins are being searched for. As sustainable meat substitutes, options like soybean, algae, insects, mycoproteins, among others, have been explored (Smetana et al., 2015). Each of these alternatives has their pros and cons. Particularly, insects, generally considered pests for agriculture and vectors of zoonotic diseases, have emerged as a prospective solution (Ramaswamy, 2015;

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Nadeau et al., 2015; van Huis et al., 2015). In the past half-decade, insects have drawn immense interest from diversified hierarchies. Humankind consuming certain arthropods is not new. Especially crustaceans, which encompass crabs, lobsters, prawns, and shrimps, are considered as delicacies (Hadley, 2006). Krill (Euphausia superba), a marine crustacean, is being harvested for its oil, rich in omega-3 fatty acids, choline, and antioxidant astaxanthin (Maki et al., 2009; Barros et al., 2014). Aquaculture along with fishery, is a billion dollar sector. Bee (Apis sp.) products as honey, pollen, and propolis are dietary supplements (Al-Hariri, 2011; Rossano et al., 2012; Silva-Carvalho et al., 2014; Patel, 2016a). The cochineal insects (Dactylopius coccus)-derived dye carmine (the pigment carminic acid) is used in food processing (Voltolini et al., 2014). Apiculture (Verde, 2014) and cochineal insect farming (De Leo´n-Rodrı´guez et al., 2006; Ramos-Elorduy et al., 2011) are popular traditional practices. Also, crickets and mealworms are reared to feed zoo animals and birds (McClements et al., 2003). Silkworm (Bombyx mori, Antheraea assamensis, etc.) culture or sericulture, to harvest silk, is an age-old practice, and is a major part of some economies (Takeda, 2009; Tikader et al., 2013). Cantharidin, a topical vesicant from blister beetles (Cantharis vesicatoria), has been traditionally used to treat warts, calluses, and other skin conditions. Also, it is currently being used as a sexual stimulant (Torbeck et al., 2014). The horseshoe crab (Limulus polyphemus) hemolymph is harvested for multiple biomedical applications (Hurton et al., 2005). In certain ethnic cultures and some Oriental countries, insects have been integral part of diet. But overall global consumption is minimal and often considered a taboo, insects being regarded as filthy and disease carriers (Sidali et al., 2018). But with changing times and the escalating need for additional protein sources, insects are being viewed in a new light. Their merits are outnumbering the demerits. Insects are rich in protein (40 75 g/100 g dry weight), and other micronutrients (Verkerk et al., 2007). Their “feed conversion ratio” is high, and turnover time is low (Premalatha et al., 2011; Nowak et al., 2016). Insect husbandry releases less greenhouse gases, while requiring less water and agricultural land (Van Huis and Dunkel, 2016). Black soldier fly (Hermetia illucens) can grow on organic materials, which is an added advantage as wastes are accumulating and their disposal is an additional problem (Wang and Shelomi, 2017). Insect farming is energy-efficient compared to livestock farming or aquaculture (Grau et al., 2017). Edible insects can be reared on organic byproducts (Lang and Barling, 2013). So, insect farming and entomophagy practices by humans are being promoted (Gahukar, 2011; Nadeau et al., 2015). Several countries are allocating funds and resources for insect-based protein food developments. While insects as the sources of protein and their relevance as human food are indisputable, some strong hiccups lie in the path of their popularization. The most important hurdles are “repulsion towards arthropods,” which is psychological (Caparros Megido et al., 2016), and “allergenicity,” which is of clinical significance. This chapter delves into the present status of insect-based protein foods, enumerates the roadblocks in the path of insects emerging as “sustainable protein source,” and discusses the prospects.

2.2 ETHNIC AND MODERN ENTOMOPHAGY PRACTICES The class Insecta includes the orders Coleoptera, Orthoptera, Hemiptera, Hymenoptera, Lepidoptera, Isoptera, Ephemeroptera, Odonata, and Mantodea (Chakravorty et al., 2011, 2013).

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Humans have consumed insects since the early days of evolution and before as their other primate ancestors. During the hunter-gatherer stage of mankind, insects were a major source of nutrition (Morris, 2008). Insects have been part of subsistence diets for millennia (Pal and Roy, 2014). They have served as an emergency food, as a staple, and as delicacies (Johnson, 2010). Until now, entomophagy, the practice of consuming insects, has been followed across the world (Raubenheimer and Rothman, 2013). Several ethnic food habits encompass insects (Bodenheimer, 1951; Costa-Neto and Dunkel, 2016). As per one report, about 1700 insects species are consumed globally (Chakravarthy et al., 2016), while as per another report, 2086 species are consumed by 3071 ethnic groups (Ramos-Elorduy, 2009). As per another report, 527 different insects are consumed across 36 countries in Africa, 29 insects in Asia, and 23 in the America. The eggs, larvae, pupae, and adults of several insects are consumed by frying, stewing, drying, smoking, steaming, blanching, and roasting (Chen et al., 2009). Snacks, beverage, and condiments are popular modes of insect consumption. Fried honey bees (Apis sp.) are deemed a delicacy in parts of China (Hartmann et al., 2015). The black chafer beetle (Holotrichia parallela Motschulsky) and black ant (Polyrhachis vicina Roger) are traditionally consumed in China. In Cambodia, roasted crickets are a popular delicacy (Walia et al., 2018). The Japanese consume hachinoko (boiled wasp larvae), sangi (fried silk moth pupae), zazamushi (aquatic insect larvae), semi (fried cicada), and inago (fried grasshopper) (Nonaka, 2010; Ce´sard et al., 2015). Dried Tenebrio molitor, Oxya chinensis sinuosa, B. mori, Protaetia brevitarsis seulensis, and Verlarifictorus asperses are consumed in Korea (Kim et al., 2017). About 164 insect species are consumed in Thailand (Boulidam, 2010; Hanboonsong, 2014). Rural Filipinos consume migratory locust, field crickets, mole crickets, carpenter ant eggs, coconut beetles grubs, June beetles, and katydids (Adalla and Cervancia, 2010). In Laos, weaver ant eggs, bamboo worms, crickets, and wasps are consumed (Barennes et al., 2015). In Borneo, more than 80 species of insects, including honey bee brood, grasshoppers and sago grubs, crickets, rice bugs, cicadas, termites, ants, and beetles, are consumed by the Kadazandusun, Murut, and Rungus people (Chung, 2008). The Vedda tribal people of Sri Lanka consume bee brood and larvae of Apis dorsata, Apis cerana, and Apis florea (Nandasena et al., 2010). Tribal people inhabiting the North Eastern part of India, as in Assam and Manipur, consume several insects to supplement their diet (Nath et al., 2005; Shantibala et al., 2014). In Manipur, they ingest Lethocerus indicus, Laccotrephes maculatus, Hydrophilus olivaceous, Cybister tripunctatus, and Crocothemis servilia (Shantibala et al., 2014). Green weaver ant (Oecophylla smaragdina) is consumed as a condiment. The edibility of 18 species of insects in the Kolhapur region of India has been reported (Sathe, 2015). Emperor moth (Gonimbrasia belina) caterpillar (mopane worm) is consumed in Southern Africa (Okezie et al., 2010). African palm weevil (Rhychophorus phoenicis) larvae are consumed by some tribes (Elemo et al., 2011). The Azande and Mangbetu people of Congo (van Huis, 2017) and inhabitants of Limpopo Province, South Africa (Netshifhefhe et al., 2018) consume termites. In Nigeria, the termite (Macrotermes natalensis), African cricket (Brachytrupes membranaceus), and pallid emperor moth (Cirina forda) are popular insect foods (Agbidye et al., 2009). The Pangwe people in Guinea, Gabon, and Cameroon gather aquatic larvae of dragonflies. People living around Lake Victoria consume black ants (Carebara vidua Smith) (Ayieko et al., 2012).

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Native American and Latin American tribes consume a large variety of insects (Navarro et al., 2010). Tukanoan Indians in the Northwest Amazon consumed over 20 species of insects which included beetle larvae (Rhynchophorus), ants (Atta), termites (Syntermes), and caterpillars (from the families Noctuidae and Saturniidae) (Dufour and Dufour, 1987). In Mexico, roasted ants, lime and chile cricket, grasshoppers (chapulines), mescal worms (gusanos de maguey), and insect eggs (escamoles) are traditional snacks. The leafcutter ant (Atta laevigata, Atta cephalotes, and Atta sexdens) is eaten in parts of Colombia and Brazil. Brazil, Colombia, Ecuador, Mexico, Peru and Venezuela are countries where insect consumption is prevalent. Regional differences in insect-eating practices have been seen in the United States (Schrader et al., 2016). Australian Aborigines have been consuming Bogong moth (Agrotis infusa), larvae of cossid moth (Xyleutes leucomochla), honeypot ant (Melophorus bagoti Lubbock), and carpenter ant (Camponotus spp.) (Yen, 2010; Warrant et al., 2016). Tribes in the New Guinea island, collect oviposits of the beetle Rhynchophorus ferrugineus papuanus from the sago palm (Ponzetta and Paoletti, 1997). In the Carnia region of Italy, Zygaena moths are eaten as a seasonal delicacy. In the putrid cheese Casu Marzu, a traditional Sardinian sheep milk cheese, live larvae of cheese fly (Piophila casei) are present (Manca et al., 2015). A review on termites records 43 species being used in the human diet (Figueireˆdo et al., 2015). People consume locally-available insects to supplement their diet for hunger and taste, but insects are rich in nutrients as well. Insects are dense sources of carbohydrates, proteins, fats, minerals, and vitamins (Gahukar, 2011). They are particularly abundant in protein, containing 40 75 g/100 g dry weight (Verkerk et al., 2007). Also, insect meat has more polyunsaturated fatty acid than conventional meat (Van Huis, 2016). The minerals obtained from eating insects include K, Ca, P, Mg, Fe, Mn, and Zn. Organic compounds include 9-octadecenoic acid, ethyl oleate, cholesterol and n-hexadecanoic acid. Some other fatty compounds include hexadecanoic acid, ethyl ester, linoleic acid, ethyl oleate, oleic acid, and cholesta-3, 5-diene (Shen et al., 2006). A study found that the lipids from three Orthopterans, Acheta domesticus, Conocephalus discolor, and Chorthippus parallelus, contain much higher amounts of essential fatty acids than those of T. molitor larvae. A. domesticus and C. discolor contain linoleic acid in major quantities, while C. parallelus contains α-linolenic acid in major quantities (Paul et al., 2017). Fig. 2.1 shows some of the edible insect preparations. Not only interspecies biochemical compositions differ, but differences exist in the various stages of a species’ life cycle, as the egg, larvae, pupae, and adult forms contain very different biochemical profiles.

2.3 ISSUES WITH INSECTS-BASED PROTEIN AND THE SOLUTIONS FOR THEM Food neophobia prevents people from ingesting new things (Dematte` et al., 2014). Consuming a new plant-based food is easier than that of a fauna-based food. When it comes to insects, disgust is a major factor (Hamerman, 2016; Menozzi et al., 2017), and neophobia is high (Caparros Megido et al., 2016). People in Western countries have had

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FIGURE 2.1 Edible insect preparations.

access to other source of proteins, so they have not had to ingest insects. As a result, they are not conditioned to consume insects with ease, and consider insect eating as culturally inappropriate (Tan et al., 2016). Experts predict that controlled exposure to insect-based foods can help overcome the repulsion. Chopping insects into ready-to-eat preparations obtained a better response than the whole forms (Caparros Megido et al., 2016). Whole forms evoke rejection because of the characteristic cuticle, antennae, wings, strong odor, crawling, filthy habitats, etc. Insects use their pheromones to communicate and for defense, so emit unpleasant smell (Suwannapong and Benbow, 2011). Ground insect powder was used as an additive in cookies, crackers, and protein bars (Smetana et al., 2016). Based on a study involving response towards cricket flour based chips, it was derived that the exposure to processed insect products can increase consumer willingness to taste, compared to unprocessed insects (Hartmann and Siegrist, 2016). In a study conducted in Australia, incorporating insects into familiar products like biscuits or cookies gained better appeal (Wilkinson et al., 2018). In a survey among the Belgian population, the participants mostly showed neophobia, but also partial willingness for insect preparations with flavor and crisp texture (Caparros Megido et al., 2014). People who do not consume pork or beef can consume their derivative gelatin. Similarly, while blended in a known food, insect extract can obtain better acceptance. Japanese relish sushi and sashimi and the Hawaiians consume ahi-poke where fish is largely raw. Western consumers are showing interest towards them, though infections of parasitic helminth (roundworm, tapeworm, and fluke) from the raw fish consumption have been reported (Kaneko and Medina, 2009). Sea urchin roe is considered a gourmet food, though it has been associated with IgE-mediated allergy (Rodriguez et al., 2007). Palm civet coffee or kopi luwak is an expensive coffee, even though the bean is collected from the droppings of civet cat (Onishi, 2010). Such examples of seemingly gross yet popular food abound. Approval of insects as food is an acquired taste, so transitioning into insectivore requires time and exposure (Hartmann and Siegrist, 2016). Furthermore, food habits are culturally-ingrained,

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reflected by halal (Islam), kosher (Jewish), and vegetarian (Hindus and Jainism). So, acceptance of insects is sure to be sluggish. So far, insect farming and marketing as food, is highly unregulated. Legislatives supporting their safety and nutrition can make them popular (Van Huis and Dunkel, 2016). Anaphylaxis is a life-threatening allergic condition, which can be triggered by insects (Kemp and Lockey, 2002). Case studies of anaphylaxis after the consumption of chapuline have been documented (Sokol et al., 2017). Hymenoptera (bees, wasps, hornets, yellowjackets, and ants) sting-caused allergy, has been observed (Przybilla and Rue¨ff, 2010). House dust mites (Dermatophagoides pteronyssinus), cockroaches (Blatella germanica, Periplaneta americana), and moths have allergens that can provoke IgE-mediated hypersensitivity in atopic individuals (Arlian, 2002; Kim and Hong, 2007; Okezie et al., 2010). These allergens can be serine proteases (trypsin, chymotrypsin, collagenase) (Wan et al., 2001; Sudha et al., 2008; Dumez et al., 2014), aspartic proteases, chitinases, calycin, troponin, tropomyosin, arylophorin, glutathione-S-transferases, and chitin (Arlian, 2002; Hindley et al., 2006; Jeong et al., 2006; Kim and Hong, 2007; Reese et al., 1999), among others. Mosquitoes have salivary gland proteins such as D7 protein family, adenosine deaminase, serpin, and apyrase (Doucoure et al., 2013). The saliva and secretions of flea, grain weevil, black fly, bluebottle fly, horse fly, bedbugs, cockroach, head louse, butterfly caterpillars, or silkworm have immunogenic components as well (Buczylko et al., 2015). The medical potential of antimicrobial peptides (AMP) from insects have been explored (Patel and Akhtar, 2017). The α-helical AMPs melittin, cecropin, abaecin, moricin, formaecin, and ponericin have been studied regarding their pathological and antibacterial roles (Chen and Lin-Shiau, 1985; Sousa et al., 2013). The peptides may have potential to inhibit microbes, but also have human cytotoxicity as well (Tonk and Vilcinskas, 2017). Also, insect protease inhibitors can inhibit human chymotrypsin, elastase, and plasmin (Wan et al., 2013; Negulescu et al., 2015). Some of these insects are not edible, but they share the same protein repertoire, which indicates the immunogenicity of the edible insects. Insects have storage proteins which are important for their metamorphosis and egg production (Telang et al., 2002). Storage proteins are synthesized in fat bodies and then secreted into the hemolymph. These vital proteins are hexameric glycoproteins which include hemocyanin, arylphorin, etc. Arylphorin is a major cockroach allergen (Arruda et al., 2001; Kim et al., 2003) and it possesses mitogenic properties as well (Hakim et al., 2007). Hexamerin1B, another insect storage protein in Gryllus bimaculatus (field cricket), can induce allergy (Srinroch et al., 2015; Pener, 2016). Not only insects, but plants also contain storage proteins which have exerted allergenicity towards humans. Peanut vicilin allergen Ara h 1, cashew vicilin-like Ana o 1 (Wang et al., 2002), conglutin beta in Lupinus sp. (Goggin et al., 2008), 7S and 11S globulins of soybean are all some of the allergenic storage proteins in plant-based foods (Shewry and Jones, 2006). However, it seems plant-origin storage proteins are easier to inactivate by cooking, compared to those of insects. Lipocalin family of β-barrel proteins are transport proteins with allergenicity (Chudzinski-Tavassi et al., 2010). Insect lipocalins induce IgE production in atopic individuals. Nitrophorins are salivary heme proteins, from a lipocalin homology family, that are present in blood-sucking insects, which transfer nitric oxide (NO) to the victim, causing vasodilatation, sequestering histamine, and inhibiting blood coagulation (Ascenzi et al., 2002).

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Insect exoskeleton chitin is an immunotoxin and a neurotoxin which, if regularly exposed to, can cause profound illnesses (Patel and Goyal, 2017; Patel et al., 2017). Even the inhalation of chitin particles can provoke the immune, neural, and endocrine systems. Chitin is the substrate of host chitinase, which is crucial for inhibiting infectious agents, but can damage host tissues as well. Enzymes work in cascade, so it can unleash a range of other aberrant enzyme activity. It would not be wrong to regard chitin as an endocrine disruptor or estrogenic agent. It is a top-grade inflammatory agent, and likely to induce excess cytochrome oxidase production. Aromatase is a cytochrome P450 enzyme that converts androgens (C19) into estrogens (C18) (Patel, 2017a). Estrogen is mitogenic and proliferative in nature and may cause breast cancer, prostate cancer, ovarian cancer, gastric cancer, pituitary cancer, polycystic ovary syndrome, diabetes, endometriosis, osteoporosis, Alzheimer’s disease, and schizophrenia, among others (Patel et al., 2018). Some moths and butterflies, such as Burnet moths (Zygaena filipendulae), contain toxic hydrogen cyanide in their tissues (Zagrobelny and Møller, 2011), and monarch butterfly (Danaus plexippus) larvae contain the cardiac glycoside cardenolide (Petschenka and Agrawal, 2015). Cardenolide manipulates the Na(1)/K(1)-ATPases, which can affect blood pressure and electrolyte balance (Patel, 2016b). These allergens and venoms can provoke the human immune system in a myriad of ways. They can disrupt cell membranes, cleave tight junction proteins between the epithelial cells, manipulate the cytoskeleton, and induce cytokine proliferation (Chapman et al., 2007; Navarro-Garcia et al., 2010; Kempkes et al., 2014; Zhang et al., 2014). The immune activation can cause asthma, dermatitis, urticaria, sinusitis, rhinitis, otitis, etc. (Arshad, 2010), or even anaphylaxis (Asokananthan et al., 2002; Macan et al., 2003; Ichikawa et al., 2009; Ahmed et al., 2010). Insect venom causing mastocytosis (Bonadonna et al., 2010) has been documented. Females seem to be particularly hypersensitive to insect allergens due to their higher level of estrogen and better immune defense (Patel et al., 2018). Also, individuals with inflammatory diseases are more likely to show sensitivity toward the allergens as the host proteins are highly glycosylated and they recognize the insect proteins. It is an evolutionary adaptation to protect the stressed body from further threats by escalating immune surveillance. Like methylation of proteins is akin to an off switch, glycosylation, due to the presence of N- and O-linked oligosaccharides, alters protein properties. Reports are emerging that hyperglycosylation prolongs the circulation of coagulation factor IX (Bolt et al., 2012). In silico analyses have revealed that all living organisms, viruses to humans and in between all, possess some evolutionarily conserved protein domains (Patel, 2017b). These motifs are pivotal in “offense and defense,” and so are strictly conserved. Some of such virulent protein domains in insects include chitin-binding domain (ChtBD3) (Patel et al., 2017). The allergens often cause cross-reactivity, so the sensitive individuals should not consume insects (Pier and Lomas, 2017). Incidences of allergic reactions on consumption of silkworm pupae, cicadas, and crickets have been reported in China (Feng et al., 2018). Caterpillars, sago worm, locust, grasshopper, bee, etc., have also been associated with insect allergy (de Gier and Verhoeckx, 2018). The allergen proteins are so stable that even thermal processing and digestion cannot eliminate all of them (de Gier and Verhoeckx, 2018). So, food processing of the insects ought to be rigorous and the products should have warning labels like other allergy-inducing foods. Insect allergy is also a cause of

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occupational health problems. Systemic allergic reactions in beekeepers has been documented (Ludman and Boyle, 2015). Post-hire asthma instances among insect-rearing workers have been observed (Suarthana et al., 2012). Those rearing the insects ought to be aware of the risks (Pener, 2016). Both potential consumers and breeders ought to be cognizant of the risks of embracing insects, as the immune sensitization may not manifest immediately, but after a certain titer of IgE antibodies are formed. Once the antibodies are formed, and inflammasomes are activated, it takes years to restore the immune system homeostasis (Patel and Meher, 2016). Even after exposure to the allergens is discontinued, the health hazards continue to occur Also, insects are vectors of human pathogens such as viruses (Campos et al., 2015), bacteria (Hager et al., 2006), protozoa (Takeo et al., 2009), fungi, and nematodes. Food pathogens such as Staphylococcus aureus, Bacillus cereus, Clostridium perfringens, enterohemorrhagic Escherichia coli, Listeria monocytogenes, and Salmonella spp. have been found associated with crickets (Walia et al., 2018). Insects being protein dense often serve as substrates for fungal growth. Dried insects in Zambia have been detected with aflatoxin-producing fungi. Aflatoxins elaborated by Aspergillus flavus are carcinogenic (Barrett, 2005). In the study, above 10 μg/kg aflatoxin was detected in Gynanisa maja, Gonimbrasia zambesina, and Macrotermes falciger (Kachapulula et al., 2018). The insect families Muscidae, Glossinidae, Culicidae, and Phlebotominae are vectors for diarrhea, myiasis, sleeping sickness, filariasis, malaria, leishmaniases, and bartonellosis, among others (Burgess, 2010). So, a complete risk assessment of the edible insects-based food candidates must be carried out, before recommending them for mass consumption (Belluco et al., 2015; Grabowski and Klein, 2016). In fact, the European Union (EU) has published an edible insect food safety guide (Robinson, 2015). Other publications also have discussed the risk and safety aspects (Marshall et al., 2016). Fig. 2.2 presents the merits and demerits of insect-based foods. FIGURE 2.2 The merits and demerits based food.

PROTEINS: SUSTAINABLE SOURCE, PROCESSING AND APPLICATIONS

of

insect-

2.4 LOOKING AHEAD AND DISCUSSION

49

2.4 LOOKING AHEAD AND DISCUSSION Despite the hurdles in the path, insects as a sustainable food source are an optimistic prospect (Sun-Waterhouse et al., 2016). Food futurists believe that insects as an alternative protein source will claim a major niche in the next few years. The UN FAO is recommending the consumption of insects to mitigate food insecurity (Costa-Neto and Dunkel, 2016; Nowak et al., 2016). The US Department of Agriculture (USDA) is willing to fund edible insect research (LIGMAN, 2015). Entomoculturing and entomophagy is part of the “One World One Health” (OWOH) movement as well (Yates-Doerr, 2015). United Nations (UN) and NGOs are promoting insect-based food availability in regions with food insecurity. Undernutrition-afflicted countries like Congo can benefit from insect-based foods. In a pilot study, school children were provided with biscuits containing 10% cricket powder (Homann et al., 2017). Another study in Nigeria determined the protein quality of common edible insects such as moth caterpillar, termite, cricket, and grasshopper, finding them to be substitutes for dietary protein (Oibiokpa et al., 2018). China and Thailand have numerous insect farms, both small and medium enterprises, and industrial scale farms. Thailand is a leader at insect farming, with an estimated 20,000 food insect farms, mostly rearing crickets and palm weevils (Hanboonsong et al., 2013). Cricket farming in Thailand, Cambodia, Lao People’s Democratic Republic (Lao PDR), the Democratic Republic of the Congo (DRC), and Kenya was studied, and in most countries their infancy status was reported (Halloran et al., 2018). In Latin America, chapuline farming is common, given its popularity as a food. The European Commission (EC) is also promoting the inclusion of insects in food (Finke et al., 2015). The Netherlands is open to the integration of insects in food (House, 2016) and insect farming practices are rising (De Goede et al., 2013). The Dutch populace are being motivated to include crickets, worms, and caterpillars as nutritious protein sources (Tagliabue, 2011). Canada-based Entomo Farms raises crickets and mealworms for food. The North American Coalition for Insect Agriculture (NACIA), comprises stakeholders who arrange conferences to promote insect agriculture. Many startups in California (United States) are selling insect-based food products. In fact, insect farming has been present in almost every continent, as animals in captivity and pets are fed with them. Now, with the new wave of insect-based food consumption by humans, the safety and hygiene dimensions are being emphasized, and human-grade insect farms are being set up. The edible insect business is now a more than $20 million industry (Hoffman, 2014). Entrepreneurs across the world are attempting to rear edible insects and to take them mainstream (Costa-Neto and Dunkel, 2016). Insects in the mass rearing facilities are prone to viral diseases, which requires adequate knowledge to avert the problem (Maciel-Vergara and Ros, 2017). To make insects into edible products, they are subjected to sun drying, freeze-drying, grinding, defatting, and acid hydrolysis (Kim et al., 2016). Each processing technique is likely to affect the biochemical composition of the insect biomass differently. In a study of sun-dried edible black ant, out of the 28 organic components, five were lost while four were formed. Fatty acids, aldehyde, and alkanes appeared during the sun drying (Li et al., 2009). For the economic viability of insects in the food sector, better rearing, harvesting, and processing tools and techniques ought to be developed (Rumpold and Schlu¨ter, 2013). Diet optimization for the insects can enhance their biomass. As genetic engineering has

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2. INSECTS AS A SOURCE OF SUSTAINABLE PROTEINS

improved crop yield (Khan et al., 2013), the development of less immunogenic and more meat-containing edible insects by genetic manipulation can be very economical. Nowadays, interested consumers are purchasing edible insects online. Mixed bag of grasshoppers, crickets, silk worms, and sago worms are marketed as energy snacks like granola bars. Cricket powder is sold and recommended to be mixed with flour for baking purposes. Chocolate-coated roasted crickets or mealworms, and insect lollipops are some of the innovative products being marketed. Even global leaders like Amazon are selling these products. Although at present there are only a handful of insect-based food traders, such as ecoEat, Don Bugito Prehispanic Snackeria, Mercado Mio, Newport Jerky Company, Hotlix, Merci Mercado, Candy Crate, Thailand Unique, EntoVida, and Meat Maniac, soon new establishments are likely to emerge. Renowned chefs are serving insect-laced recipes like cricket-sprinkled salads, mealworm lettuce wraps, cricket fried rice, mescal worm tacos, fried dragonflies, ant egg tostada, mealworm-peppered noodles, silkworm powder-flavored broth, etc. Cookbooks, featuring the culinary uses of insects are being published (Linn, 2014). Experimenting with insects has just begun and possibilities abound. In a study, sausage fortified with mealworm larvae and silkworm pupae was evaluated (Kim et al., 2016). In an insect-eating festival held in the Philippines, various agricultural pest insects were processed into chayote bars. rice cakes, burgers, sandwich fillers, etc., and presented to people (Sabado and Aguanta, 2014). In a festival in Switzerland, pizzas were served topped with mealworms and beetle larvae (Wallace, 2010). Even countries like the Czech Republic are showing interest in including insects in the diet (Bednarova et al., 2010). Human societies are appearance-conscious. The same insect product that will be normally dismissed as repulsive might attract attention if it is claimed that it prevents obesity, skin wrinkles, hair loss, or restores fertility. Similarly, anticancer, antidiabetic, and immunity building claims can raise the acceptance of insect-based foods. It will not be surprising if traders adopt this path for profits. Silkworm moth (B. mori L.) larvae was being evaluated as an animal protein source for astronauts on space missions (Tong et al., 2011). Apart from silkworm, the hawkmoth, the drugstore beetle, and the termite are considered candidates for space agriculture (Katayama et al., 2008). It appears that compared to the whole insects or their crushed forms, there is lower aversion and better prospects for acceptance for bioactives extracted from them. Insects elaborate proteins and AMP for immune defense (Slocinska et al., 2008; Ezzati-Tabrizi et al., 2013), which might be exploited as dietary supplements. Lipid extracted from meadow grasshopper (C. parallelus) was found to have an interesting fatty acid composition (Paul et al. 2015). However, the immunogenicity or immunosuppressive aspect of the extracted components must be addressed as not all proteins are benign and healthpromoting, but can be perceived as antigens. Astaxanthin from krill is known to be an antioxidant. However, recent studies have revealed that astaxanthin by virtue of its oxidative stressor elimination might resolve immunopathology, but can exert an immune depressive effect (Dhinaut et al., 2017). Other studies have shown how astaxanthin feeding suppresses the expression of inflammatory cytokines, including nuclear factor (NF)-κB, tumor necrosis factor (TNF)-α, and interleukin (IL)-1β. While insects no doubt are proteinrich, the ingestion of inadequately prepared insects can cause immune activation and inflammatory diseases. The FDA and other regulatory bodies ought to keep a tab on unscrupulous merchandise and marketing.

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2.4 LOOKING AHEAD AND DISCUSSION

Every new application of an object requires imparting education on the merits and demerits. In every element, there is a trade-off. If the risks are too high, the element is not worth applying for the new use. Some of the merits and demerits have been discussed earlier. Host factors are different, so the response to a substance can vary. Insects are allergenic as discussed previously. Therefore not all human beings are expected to fare well after insect consumption. The way a lot of people cannot consume seafood, shrimps, and crabs for hypersensitivity reasons (Lopata et al., 2010), some will not be fit to consume insects. However, different processing techniques might neutralize the allergens and make them amenable for mass consumption. In fact, if the allergenicity issue can be taken care of, insects can be a superfood, a way better substitute to canned, frozen, food additivelaced packaged foods. Ethnic people have been consuming them for a long time, and are thriving. To urban people in developed countries, insects are a novelty. Those who can savor shrimps or clams should not have a psychological issue with the consumption of insects. Apart from a sustainable protein source, insect farming can lower the burden on the environment, and can provide livelihood opportunities to many (Halloran et al., 2017). By choice, few people would have consumed insects, but protein requirements can lead to their consumption. Food insecurity looms, but the situation is still under control. Maybe in the next few decades, the availability of nutritious foods will be challenging, and insects will have to be coerced onto the human dietary platter. So, this time period ought to be directed towards the safety evaluation of insects, developing facilities, and processing the edible insects into human consumption-worthy food objects. In fact, future meat options are likely to include cultured meat and imitation meat, apart from insect meat (Alexander et al., 2017). Table 2.1 presents a list of insects that are or have been consumed in different regions of the world. This is a fraction of the actual list, as the exact picture is elusive and exhaustive. TABLE 2.1 Some Common Edible Insects and Their Common Names No.

Scientific Name

Common Name

1.

Acheta domesticus

House cricket

2.

Agrotis infusa

Bogong moth

3.

Anabrus simplex

Mormon cricket

4.

Apis spp.

Honey bees

5.

Arsenura armada

Giant silk moth

6.

Atractomorpha psittacina

Slant-faced grasshopper

7.

Bombyx mori

Silkworm moth

8.

Brachytrupes membranaceus

Tobacco cricket

9.

Brontispa longissimi

Coconut leaf beetles

10.

Camponotus spp.

Jet-black carpenter ants

11.

Carebara vidua Smith

Black ant

12.

Cirina forda

Pallid emperor moth (Continued)

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2. INSECTS AS A SOURCE OF SUSTAINABLE PROTEINS

TABLE 2.1 (Continued) No.

Scientific Name

Common Name

13.

Chorthippus parallelus

Meadow grasshopper

14.

Coloradia pandora

Pandora moth

15.

Corcyra cephalonica

Rice moth

16.

Crocidolomia pavonana

Cabbage worm

17.

Crocothemis servilia

Scarlet skimmer

18.

Cybister tripunctatus

Three-punctured diving beetle

19.

Ephydra hians

Alkali fly

20.

Gonimbrasia zambesina

Bulls eye silk moth

Gonimbrasia belina

Emperor moth

21.

Gryllotalpa africana

Mole crickets

22.

Gynanisa maja

Speckled emperor

23.

Hermetia illucens

Black soldier fly

24.

Holotrichia parallela Motschulsky

Black chafer beetle

25.

Hydrophilus olivaceous

Water scavenger beetle

26.

Hypopta agavis

Tequila worm

27.

Laniifera cyclades

Nopal worm

28.

Latebraria amphipyroides

29.

Leptocorisa oratorius

Rice bug

30.

Lethocerus indicus

Giant water bugs

31.

Liometopum apiculatum

32.

Locusta migratoria

Locusts

33.

Macrotermes falciger

Termites

Macrotermes natalensis Macrotermes michaelseni 34.

Melophorus bagoti

Honeypot ant

35.

Odontotermes sp.

Termites

36.

Oecophylla smaragdina

Red weaver ant

37.

Omphisa fuscidentalis

Bamboo borers

38.

Oxya chinensis

Chinese grasshopper

39.

Patanga succincta

Bombay locusts

40.

Piophila casei

Cheese fly (Continued)

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TABLE 2.1 (Continued) No.

Scientific Name

Common Name

41.

Polyrhachis vicina Roger

Black ant

42.

Protaetia brevitarsis seulensis

White-spotted flower chafer

43.

Rhynchophorus ferrugineus

Asiatic palm weevil

Rhynchophorus phoenicis

African palm weevil

44.

Schistocerca gregaria

Desert locust

45.

Scotinophara coarctata

Malaysian black bug

46.

Sphenarium sp.

Grasshoppers

47.

Tribolium castaneum

Red flour beetle

48.

Verlarifictorus aspersus

49.

Xyleutes leucomochla

Cossid moth

50.

Zygaena sp.

Burnet moth

CONCLUSION Food trends keep evolving, and if certain impediments in the path of mass adoption of insect diet can be addressed, insects can become a substantial part of the human diet. Going by the significant spike in the number of publications on the potential of insects as human food, the rising interest and fund allocations, can be gauged. Research projects and partnerships are being forged and entrepreneurs are establishing insect farms and facilities. The interest in insects as human food has been stirred in academia, the food sector, as well as among the general public (Evans et al., 2015). It can be anticipated that insects can be a regular part of the food production chain in the next few years. Surveys, food fests, and symposia are being held to create consumer awareness and to familiarize people to the emerging novel edible protein source (Liu and Zhao, 2018). Chefs, insect farmers, and nutrition experts are collaborating to formulate novel insect-based food preparations. Despite the hurdles and genuine concerns, opinions are cohesive that insects have the potential to be used as a functional food source. If the above-voiced concerns can be resolved, and nutritional analyses optimized, insects can be a sustainable protein food source, and can generate an alternative food industry with less pressure on environmental resources.

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Verkerk, M.C., Tramper, J., van Trijp, J.C.M., Martens, D.E., 2007. Insect cells for human food. Biotechnol. Adv. 25, 198 202. Voltolini, S., Pellegrini, S., Contatore, M., et al., 2014. New risks from ancient food dyes: cochineal red allergy. Eur. Ann. Allergy Clin. Immunol. 46, 232 233. Walia, K., Kapoor, A., Farber, J.M., 2018. Qualitative risk assessment of cricket powder to be used to treat undernutrition in infants and children in Cambodia. Food Control 92, 169 182. Wallace, A., 2010. Insect toppings. Sci. World 67, 22. Wan, H., Winton, H.L., Soeller, C., et al., 2001. The transmembrane protein occludin of epithelial tight junctions is a functional target for serine peptidases from faecal pellets of Dermatophagoides pteronyssinus. Clin. Exp. Allergy 31, 279 294. Wan, H., Lee, K.S., Kim, B.Y., et al., 2013. A spider-derived Kunitz-type serine protease inhibitor that acts as a plasmin inhibitor and an elastase inhibitor. PLoS One 8, e53343. Available from: https://doi.org/10.1371/journal.pone.0053343. Wang, F., Robotham, J.M., Teuber, S.S., et al., 2002. Ana o 1, a cashew (Anacardium occidental) allergen of the vicilin seed storage protein family. J. Allergy Clin. Immunol. 110, 160 166. Available from: https://doi.org/ 10.1067/mai.2002.125208. Wang, Y.-S., Shelomi, M., 2017. Review of black soldier fly (Hermetia illucens) as animal feed and human food. Foods 6, 91. Available from: https://doi.org/10.3390/foods6100091. Warrant, E., Frost, B., Green, K., et al., 2016. The Australian Bogong moth Agrotis infusa: a long-distance nocturnal navigator. Front. Behav. Neurosci 10, 77. Available from: https://doi.org/10.3389/fnbeh.2016.00077. Wilkinson, K., Muhlhausler, B., Motley, C., et al., 2018. Australian consumers’ awareness and acceptance of insects as food. Insects. Available from: https://doi.org/10.3390/insects9020044. Yates-Doerr, E., 2015. The world in a box? Food security, edible insects, and “One World, One Health” collaboration. Soc. Sci. Med. 129, 106 112. Available from: https://doi.org/10.1016/j.socscimed.2014.06.020. Yen, A.L., 2010. Edible insects and other invertebrates in Australia: future prospects. For insects as food humans bite back 65. In: Proceedings of a Workshop on Asia-Pacific Resources and Their Potential for Development, Chiang Mai, Thailand. ISBN: 978-92-5-106488-7. Zagrobelny, M., Møller, B.L., 2011. Cyanogenic glucosides in the biological warfare between plants and insects: the Burnet moth-Birds foot trefoil model system. Phytochemistry 72, 1585 1592. Available from: https://doi. org/10.1016/j.phytochem.2011.02.023. Zhang, H., Zeng, X., He, S., 2014. Evaluation on potential contributions of protease activated receptors related mediators in allergic inflammation. Mediators Inflamm. 2014, 829068. Available from: https://doi.org/ 10.1155/2014/829068.

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3.4 Chemical Composition of Microalgae 3.4.1 Protein 3.4.2 Amino Acids 3.4.3 Peptides 3.4.4 Enzymes

69 70 72 74 75

3.5 Protein Extraction Methods 3.5.1 Bead Milling 3.5.2 Ultrasound-Assisted Extraction 3.5.3 Pulsed Electric Field-Assisted Extraction 3.5.4 Microwave-Assisted Extraction 3.5.5 Ionic Liquids

76 77 77 77 78 78

3.6 Extraction of Proteins and Active Substances From Arthrospira platensis and Porphyridium cruentum 78

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3.8 Application of Algal Protein for Human Consumption 3.8.1 Spirulina as a Superfood

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3.10 Toxicological Aspects 3.10.1 Nucleic Acids 3.10.2 Algal Toxins 3.10.3 Heavy Metals

86 87 87 88

3.11 Challenges Surrounding Algal Usage 3.11.1 Food Safety 3.11.2 Genetically Modified Algae 3.11.3 Scalability 3.11.4 Price

89 89 89 90 90

3.12 Conclusion

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3.1 INTRODUCTION Microalgae are a group of eukaryotic organisms and photosynthetic cyanobacteria able to accumulate sugars, carbohydrates, proteins, lipids, and other valuable organic substances by the efficient use of solar energy, CO2, and nutrients. These microorganisms convert inorganic substances such as carbon, nitrogen, phosphorus, sulfur, iron, and trace elements into organic matter (green, blue-green, red, brown, and other colored biomass) (Batista et al., 2013). It is unknown how many species of algae exist, with estimates ranging between several hundred thousand and several million different species—with new types identified all of the time (Lourenc¸o, 2006; Moheimani et al., 2015). Only a small proportion of microalgal species can be kept alive in culture, and only a handful of them have been successfully grown commercially (Mata et al., 2010). The ideal microalga must be able to grow very well even under high biomass concentration and varying environmental conditions. It must also be able to produce high concentrations of products of interest (i.e., high-value products, lipids, and proteins) (Borowitzka, 2013). Commercial large-scale production of microalgae for bioproducts began in the 1960s with Chlorella vulgaris and in the 1970s with Arthrospira platensis (Spirulina), followed in the 1980s with production of β-carotene from Dunaliella salina (Varshney et al., 2015). All three species were successfully grown in open ponds (Craggs et al., 2013). The ability to grow in highly selective environments is the main reason for the successful growth of these species (Spirulina 5 high pH and high HCO32, D. salina 5 high salinity, and Chlorella 5 high nutrients) (Borowitzka and Borowitzka, 1990; Henrikson, 2009; Lourenc¸o, 2006). In the late 1990s, commercial production of the freshwater green alga Haematococcus pluvialis as a source of the carotenoid astaxanthin started at Cyanotech in Hawaii (Moheimani et al., 2015). The culture system is a combination of “closed” tower reactors and raceway ponds. Haematococcus production by several other small producers commenced in Hawaii in subsequent years using a wide range of, mainly, “closed” culture systems (Olaizola, 2000). The astaxanthin from Haematococcus is mainly sold as a nutraceutical for human uses and a coloring agent for farming of salmonids fish (Haque et al., 2016; Panis and Carreon, 2016). In the past five decades, there have been numerous attempts by researchers and profitseeking companies to commercialize the production of microalgae primarily as a food or food ingredient (Pulz and Gross, 2004). The impetus for large-scale algae cultivation was provided by the “Third World Food Survey” published by the FAO in 1963, drawing attention to the problems of protein supply, foreseeing a general scarcity of food in the world by the year 2000. NASA considered it an excellent, compact space food for astronauts. The WHO has called it one of the greatest superfoods on earth. And New Agers all over the world are rediscovering the wonders of Spirulina as a superfood (Becker, 2013). Indigenous populations have consumed microalgae for centuries. Aztecs used the cyanobacterium Spirulina (A. platensis, Arthrospira maxima) from Lake Toxcoco (Mexico) CE c.1300 as complement food. Spanish chroniclers described local fishermen collecting bluegreen masses from the lakes that were prepared as a dry cake, known as “tecuitlatl.” For centuries, the population in Chad has been harvesting Spirulina (known as “dihe´”) from

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3.1 INTRODUCTION

Lake Kossorom at the north-east fringe of Lake Chad and using it for food on a daily basis. Nostoc, filamentous cyanobacteria, has been also widely used as food. The species Nostoc commune, Nostoc flagelliforme, and Nostoc punctiforme are traditionally consumed in China, Mongolia, and South America (known as “fa cai” and “lakeplum”). In Japan, another edible cyanobacterium Aphanotheca sacrum (formerly Phyloderma sacrum) is considered a special delicacy known as “suizenji-nori.” The filamentous green algae Spirogyra and Oedogonium are also used as a dietary component in Burma, Thailand, Vietnam, and India (Ga´rcia et al., 2017). Nowadays, Chlorella and Spirulina are widely commercialized in health food stores, gaining worldwide popularity because they are one of the most nutritious foods known to man. These microalgae are also used to feed many types of animals (e.g., cats, dogs, aquarium fish, ornamental fish, birds, horses, poultry, cows, and breeding bulls). Moreover, other microalgae such as Tetraselmis, Isochrysis, Pavlova, Phaeodactylum, Chaetococeros, Nannochloropsis, Skeletonema, and Thalassiosira are also used as feeds in aquaculture (Ga´rcia et al., 2017). In addition to aquaculture application, microalgae are considered as perfect candidates for contemporary “nutraceutical” or “functional food” due to their ability to synthesize valuable products like carotenoids, long-chain fatty acids, essential and nonessential amino acids, enzymes, vitamins, and minerals useful for human nutrition (Matos, 2017). A conventional microalgae production system consists of growth and cultivation of microalgae, biomass harvesting and dewatering, cell disruption for algae that have rigid cell walls, and extraction/conversion of the biomass to the product of interest. A microalgal biorefinery plant concept is shown in Fig. 3.1.

Microalgae biomass production

FIGURE 3.1

Microalgal biorefinery

plant concept.

Cell disruption (e.g., pulsed eletric field)

Extraction (e.g., polymers, ionic liquids)

Hydrophilic compounds

Hydrophobic compounds

(e.g., proteins, carbohydrates)

(e.g., lipids, isoprenes)

Specialized industry

Further fractionation

(e.g., food, feed, fine chemicals)

(e.g., food, feed, chemicals, fuel)

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3.2 MARKETS AND PRODUCTS FROM MICROALGAE Market prices for algal biomass and its valued components are strongly fluctuating globally depending upon where the production site is located, the actual market situation, and especially the production purity (Koller et al., 2014). In 1999, global production volumes of microalgae were estimated at only 1000 tons dry weight. This had increased to 5000 tons dry weight representing US$ 1 billion by 2004, which represented a fivefold increase in 5 years (Miledge, 2012). In 2011, the total production volume had risen to 9000 tons dry weight. The value of the global marine biotechnology market in 2011, with microalgae as its main component, was estimated at US$ 2.4 billion, with an expected yearly growth of 10% (Guedes et al., 2011). According to Transparency Market Research (accessed on www.algaeindustrymagazine), in a new market report entitled “Algae market, by application, by cultivation technology, and geography global industry analysis, size, share, growth, trends, and forecast 2016 2024,” the global algae market was value at US$ 608.0 million in 2015 and is projected to reach US$ 1.1 billion with a volume of 27,552 tons by 2024 (Algae Industry Magazine, 2016). Note that this volume is still small compared to other food commodities, for instance global wheat production is around 700 million tons annually (Enzing et al., 2014). Traditionally, microalgae such as Spirulina and Chlorella are directly sold as dietary supplements, without any kind of processing except drying. The development of these products is relatively mature and they are produced by a relatively large number of producers. More than 12,000 tons of Spirulina biomass are produced every year (at about 20 US$/kg); nearly 70% is produced in China, India, and Taiwan. Worldwide, Chlorella producers cultivate an estimated 5000 tons per year (Draaisma et al., 2013). Besides the sales of the whole dried algae, nowadays also specific high-value components from microalgae are being produced. In general, microalgae-based molecules are less competitive than standard synthetic and traditional alternatives. However, some microalgae-based molecules have specific advantages over their conventional alternatives which make their use commercially viable. In Fig. 3.2, for example, it is possible to visualize the main features of some commercial traditional microalgae species. Astaxanthin (a carotenoid used as pigment) from dried H. pluvialis is the most developed product in this domain. Astaxanthin is either available as a dietary supplement (mostly US-produced), or as food additive. A relatively large group of companies produces β-carotene, a food additive and ingredient, from D. salina. Although most producers are located in Asia or Australia, European multinationals such as BASF and DSM have acquired a number of leading producers in Australia and the United States. More recently, a selected number of producers in Europe and the United States started producing omega-3 eicosapentaenoic and docosahexaenoic acids from microalgae, to be used for dietary supplements or food ingredients. The pigment phycocyanin is produced from Spirulina by a small number of companies (Draaisma et al., 2013; Enzing et al., 2014).

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3.3 MICROALGAL PRODUCTION SYSTEMS

Arthrospira platensis

Phorphyridium cruentum

Chlorella vulgaris

Haematococcus pluvialis

Cyanobacteria (blue-green algae)

Rodophyta (red algae)

Chlorophyta (green algae)

Chlorophyta (green algae)

Phycocyanin Amino acids Vitamin complex B γ-Linolenic acid

Phycoerythrin Sulfated polysaccharides Arachidonic acid

Chlorophyll Lutein Protein α-Linolenic acid

Astaxanthin Lipids Palmitic acid Oleic acid

Canthaxanthin Lipids/PUFAs Eicosapentaenoic acid

Fucoxanthin Lipids/PUFAs Silicates Ceramides

Fragile cell wall

Very fragile cell wall

Very rigid cell wall

Very rigid cell wall

Rigid cell wall

Rigid cell wall

Nannochloropsis oculata

Phaeodactylum tricornutum

Heterokontophyta Heterokontophyta (Eustigmatophyceae) (diatom)

FIGURE 3.2 Commercial traditionally microalgae (free-access photos assembled from Culture Collection of Algae (SAG) at Goettingen University). Source: Adapted from Idakiev, H., Baecker, S., 2018. Extraction of proteins and active substances from microalgae. INFORM (International News on Fats, Oils, and Related Materials) 29, 22 25.

3.3 MICROALGAL PRODUCTION SYSTEMS The three most important classes of microalgae in terms of abundance are the diatoms (Bacillariophyceae), the green algae (Chlorophyceae), and the golden algae (Chrysophyceae). All these microalgae are eukaryotes distinguished by the presence of a nucleus and separate organelles for photosynthesis (chloroplasts) and respiration (mitochondria). It is important to note that cyanobacteria (Cyanophyceae or blue-green algae) are also referred to as microalgae, for example, the species Spirulina (A. platensis and A. maxima). The cyanobacteria are part of the eubacteria and are prokaryotes lacking a membrane-bounded nucleus (Enzing et al., 2014). Diatoms are the dominant life form in phytoplankton and probably represent the largest group of biomass producers on earth. It is estimated that more than 100,000 species exist. The cell walls of diatoms contain polymerized silica, and they often accumulate oils and chrysolaminarin (a storage polysaccharide) (Lourenc¸o, 2006). Green algae are especially abundant in freshwater. The main storage compound of green algae is starch, although oils can also be synthesized. The freshwater green algae H. pluvialis is commercially important as a source of astaxanthin, C. vulgaris as a supplementary food product or food ingredient, and the halophilic algae species Dunaliella as a source of β-carotene (Borowitzka, 2013). The golden algae also synthesize oils and carbohydrates and are in this respect similar to diatoms. The blue-green algae (Cyanobacteria) are found in a variety of habitats and several of them are known for their water polluting effect due to their production of toxins (Enzing et al., 2014).

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Photobioreactors cultivation (4 L–6 L)

Cylinder photobioreactors cultivation (200 L)

Open raceway ponds cultivation (5000 L)

FIGURE 3.3 Cultivation of different types of algae (e.g., Chlorella vulgaris, Nannochloropsis gaditana, Nannochloropsis oculata, Phaeodactylum tricornutum, and Porphyridium cruentum) in photobioreactors, e.g., cylindrical inverted photobioreactor (4 L), balloon photobioreactor (6 L), Erlenmeyer photobioreactor (5 L) and cylinder photobioreactor (200 L), and raceway open ponds (B5000 L, Chlorella sp. and Scenedesmus sp.). Source: Photographs courtesy of Matos, A.P., Feller, R. Laboratory of Food Biotechnology and Laboratory of Algae Cultivation, Federal University of Santa Catarina, Floriano´polis, Santa Catarina, Brazil.

Microalgae are cultivated in a wide range of different cultivation systems that can be placed outdoors or indoors. Cultivation systems range from open shallow raceway ponds to closed photobioreactors (Fig. 3.3). The systems mostly used on a large scale and on a commercial basis are open systems. For small-scale algae production, mostly for research purposes, a wide range of systems is used. Closed systems (photobioreactors) of algal cultures are not exposed to the atmosphere and are covered with a transparent material or contained within transparent tubing. Photobioreactors have the distinct advantage of preventing evaporation. Culturing microalgae in these kinds of systems has the added benefit of reducing the contaminants risks, limiting the CO2 losses, creating reproducible cultivation conditions, and flexibility in technical design (Moheimani et al., 2015). Closed and semiclosed photobioreactors are mainly used for production of high-value products such as pigments, protein, and singlecell oils (EPA 1 DHA 1 AA) for human consumption (Borowitzka, 2013). The main disadvantages of closed systems are the high costs of construction and operation, in terms of both energy (pumping and cooling) and maintenance such as cleaning and sterilization (Kumar et al., 2015). However, if these difficulties can be overcome, the controlled closed systems may allow commercial mass production of an increased number of microalgal species at a wider number of locations. Open ponds are the most usual setting for large-scale outdoor microalgae cultivation. The major commercial production of algae is today based on open channels (raceway) which are less expensive, and easier to build and operate compared with closed photobioreactors (Moheimani et al., 2015). Furthermore, the growth of microalgae meets is less challenging in open than closed cultivation systems. However, just a few species of

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69

microalgae (e.g., Chlorella, D. salina, Spirulina platensis, and Pleurochrysis carterae) have been successfully grown in open ponds (Lourenc¸o, 2006; Borowitzka, 2013). Profitable production of microalgae, at present, are limited to a comparatively few smallscale (,10 ha) plants producing high-value compounds, most located in south-east Asia, Australia, and the United States(Chisti, 2007). Relatively low cost of construction and operation are the main reasons for culturing algae in open ponds. However, the high contamination risks and low productivity, induced mainly by poor mixing regime and light penetration, are the main disadvantages of open systems (Rogers et al., 2014).

3.4 CHEMICAL COMPOSITION OF MICROALGAE The chemical composition of algae is frequently determined with the objective of providing the necessary nutritional information for the consumer, and also of determining variations with respect to the conditions under which the microalgae are being cultivated. Chemical composition of algae varies from species to species, from strain to strain, and from batch to batch. To obtain an algal biomass with a desired pattern of constituents, their proportion can be modified to a certain extent by varying the culture conditions. Many analyses of gross chemical composition have been published in the literature. In order to give a general overview on these estimations, selected data have been compiled in Fig. 3.4. Although there are marked differences in the compositions of the microalgal classes and species and strains, protein is generally the major organic constituent, followed by lipid and then by carbohydrate. Due to the varying culture conditions, as Arthrospira platensis

Porphyridium cruentum

35%

55%

Haematococcus pluvialis

Nannochloropsis oculata

25%

Protein

Fiber

42%

Ash

Moisture

FIGURE 3.4 Proximate composition (protein content highlighted) of some microalgae traditionally utilized in the food industry. Source: Data from Matos, A. P., Feller, R., Moecke, E.H.S., 42% Oliveira, J.V., Junior, A.F., Derner, R.B., et al., 2016. Chemical characterization of six microalgae with potential utility for food application. J. Phaeodactylum tricornutum Am. Oil Chem. Soc. 93, 963 972 (Matos et al., 2016). Chlorella vulgaris

39%

Carbohydrate

Lipid

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mentioned above, it is not surprising that even for the same strain different compositions have been reported. In almost all applications, where the harvested and concentrated algal biomass is to be utilized further, a product with a water content of less than 10% is required. Moisture affects spoilage of the dried algal product by supporting the growth of bacteria, mold, and fungi. In this context, a distinction is made between water bound to the biological material that is not freely available to maintain biological processes (,2% moisture) and moisture content above this level at which the water becomes available for harmful microorganisms. A moisture content of ,7%, which technically is realistic, is the level that still protects the algae from prolonged microbial growth (Becker, 2013).

3.4.1 Protein The high protein content of several microalgal species (e.g., 55% 70% for S. platensis and 42% 55% for C. vulgaris per dry matter) has been one of the main reasons for considering these organisms as a source of food. It is important to note that protein content in algae is normally obtained by measuring the total nitrogen and multiplying this value by the factor N 3 6.25. N-Protein factors for specific microalgal species have been determined experimentally and they range from N 3 3.06 to 5.95 (Gonza´lez Lo´pez et al., 2010; Lourenc¸o et al., 2004). This different N-protein factor is attributed to the fact that about 10% of the nitrogen found in microalgae consists of nonprotein nitrogen, including amines, glucosamides, nucleic acids, and cell-wall materials. Since protein is one of the most valuable algal components, four important parameters of its quality are used to determine the appropriate nutritive value of algal protein, that is, protein efficiency ratio (PER), biological value (BV), digestibility coefficient (DC), or true digestibility and net protein utilization (NPU) (Becker, 2013). PER is the simplest method to evaluate protein quality and is performed by 3 4 weeks of animal feeding trials with weanling rats. The response to the diets fed is expressed in terms of weight gain per unit of protein consumed, that is PER 5 weight gain (g)/protein intake (g). The PER values are normally compared with a reference protein such as casein. To eliminate fluctuations, the PER values for casein are customarily adjusted to a figure of 2.50, which requires a corresponding correction of the experimental data. As examples, Scenedesmus obliquus has PER values in the range of 1.12 2.00 (Becker et al., 1976; Pabst, 1974), Spirulina sp. PER are between 1.78 and 2.20 (Becker, 1994; Bourges et al., 1971), while Chlorella sp. PER values range from 0.84 to 2.00 (Cheeke et al., 1977; Saleh et al., 1985). The selected results of PER studies in microalgae depend on the context of the drying process, which is vital to determine the utilization of algal biomass as a proteinaceous matter. Thus, the nutritive value of the alga protein depends on the type of postharvesting process. With the exception of the cyanobacteria Spirulina and Aphanizomenon flos-aquae, most of the other types of microalgae have relatively thick cell walls, which makes improperly treated algal biomass indigestible for humans. This cellulosic cell wall poses a serious problem in digesting/utilizing the algal biomass. Hence, to make the algal protein nutritionally accessible, various disruption methods (mechanical or nonmechanical) have been currently applied for algal cell wall disruption. For instance, mechanical forces

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such as solid-shear forces (e.g., high speed homogenization, bead mill), liquid-shear forces (e.g., microfluidization, high-pressure homogenization), energy transfer through waves (e.g., microwave, ultrasonication), currents (e.g., pulsed electric field, PEF), or heat (e.g., autoclaving, thermolysis), and nonmechanical methods such as enzymatic cell lysis and chemical cell disruption (Gunerken et al., 2015). BV is one of the principles based on the quotient between nitrogen retained and nitrogen absorbed. The absorbed nitrogen is defined as the difference between ingested and excreted fecal and urinary nitrogen: BV 5 [I (F F0) (U U0)]/[I (F F0)], where I 5 nitrogen intake, F 5 fecal nitrogen, U 5 urinary nitrogen, and F0 and U0 are fecal and urinary nitrogen excreted when the animals are maintained on a diet free of or low in nitrogen (Becker, 2013). DC—sometimes called true digestibility—is another parameter that reflects the quality of a protein. It expresses the proportion of food nitrogen that is absorbed by the animal, calculated by using the parameters already used for the calculation of the BV, that is, DC 5 [I (F F0)]/I. NPU can be calculated by the expression: nitrogen retained divided by nitrogen intake. It is equivalent to the calculation BV 3 DC and is a measure of both the digestible BV 3 DC 5 (B Bk)/I, where B 5 body nitrogen, measured at the end of the test period on animals fed with the test diet, and Bk 5 body nitrogen, measured on another group of animals fed with a protein-free/low diet. As mentioned early, the importance of an adequate drying process is vital to obtain nutritive protein from algae. Since the results of the PER studies already showed the importance of the drying step on the nutritive value of the algae, additional studies (BV, DC, NPU) demonstrate the relevance between processing and nutritive quality (Table 3.1). In general, the drum-dried algae had a nutritional quality that is about 85% that of casein. For Scenedesmus, for instance, at 10% level all parameters of the drum-dried material were TABLE 3.1 Comparative Data on Biological Value (BV), Digestibility Coefficient (DC), and Net Protein Utilization (NPU) of Various Algae Processed by Different Methods Alga

Processing

Casein

BV

DC

NPU

References

87.8

95.1

83.4

Becker et al. (1976)

Chlorella sp.

Drum-dried

77.9

89.3

69.6

Saleh et al. (1985)

Scenedesmus obliquus

Drum-dried

81.3

82.3

67.3

Pabst (1974)

S. obliquus

Sun-dried

72.1

72.5

52.0

Becker et al. (1976)

Spirulina sp.

Raw

63.0

76.0

48.0

Clement et al. (1967)

Spirulina sp.

Sun-dried

77.6

83.9

65.0

Becker (1994)

Spirulina sp.

Drum-dried

68.0

75.5

52.7

Narasimha et al. (1982)

Chlorella sp.

Protein extract

79.9

83.4

66.2

Yamaguchi et al. (1973)

Chlorella sp.

Drum-dried

71.6

79.9

57.1

Thananunkul et al. (1977)

Data from Becker, E.W., 2013. Microalgae for human and animal nutrition. In: Richmond, A., Hu, Q. (Eds.), Handbook of Microalgal Culture: Applied Phycology and Biotechnology. Wiley Blackwell, Oxford, pp. 461 503.

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better than those of the samples dried by other methods. The NPU values were significantly lower than for casein, indicating that the algal protein is limited by at least one of the essential amino acids, most probably methionine. The data on the nitrogen balance studies with Spirulina confirm that this alga with its thin cell membrane does not present serious problems in protein utilization, even with simple sun-drying (Becker, 2013). 3.4.1.1 Protein Digestibility of Microalgae Bioavailability can be described as the fraction of ingested food components that is available at the target site of action for utilization in various physiological functions. Bioavailability entails the entire process following consumption of the food element, including digestibility and solubility of the food element in the gastrointestinal tract, absorption/ assimilation of the food element across the intestinal epithelial cells and into the circulatory system, and finally, incorporation into the target site of utilization (Ekmekcioglu, 2002). The digestibility of algal protein has been examined within the literature, including in vitro digestion of microalgal biomass from freshwater species for monogastric and ruminant animal feed applications, and results showed that protein solubility (PS) was statistically highest and the same for C. vulgaris at 84% and Micractinium reisseri at 78%, lowest for Nannochloropsis bacillaris at 64%, and intermediate for Tetracystis sp. at 73% (Tibbetts et al., 2016). Generally, microalgae such as S. obliquus, Spirulina sp., and Chlorella sp. have DC values of 88.0%, 77.6%, and 76.6%, respectively (Becker, 2007). This is in comparison to protein digestibility of other commonly consumed plants, including grains (69% 84%), legumes (72% 92%), fruits (72% 92%), and vegetables (68% 80%) (Bleakley and Hayes, 2017). Some differences might be expected between various conventional plant proteins and algal proteins based on the relative hydrophobicity of their amino acids. Very recently, Wild et al. (2018) evaluated in vitro crude protein digestibility of four noncell-disrupted microalgae, and algal protein digestibility decreased in the order of C. vulgaris (79%) . Phaeodactylum tricornutum (77%) . A. platensis (74%) . Nannochloropsis oceanica (54%), while in the same algal cell that was disrupted the values were as follows: C. vulgaris (84%) . P. tricornutum (83%) . N. oceanica (79%) . A. platensis (78%). Thus, cell disruption increased in vitro protein digestibility in all algal species, but the extent of the increase was variable. This is primarily attributed to the wide range to methodical differences in the enzyme mixtures, assay conditions, sample processing, and cell disruption (cell wall structure and composition). In sum, bioavailability, bioaccessibility, and bioactivity are assessed by different in vitro and in vivo studies that need to be harmonized to obtain univocal data. Considering the protein digestibility, a harmonizing attempt is the INFOGEST, a static in vitro digestion model (Infogest, 2014).

3.4.2 Amino Acids Amino acids are subunits that make up proteins and therefore the nutritional quality of a protein is determined by the content, proportion, and availability of these organic compounds. Nine proteinogenic amino acids are called “essential” for humans and must be acquired through food. Because of their biological importance, amino acids are important

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in nutrition and are commonly used in nutritional supplements, fertilizers, and food technology. Industrial uses include the production of drugs, biodegradable plastics, and chiral catalysis (Becker, 2013; Tibbetts et al., 2016). Selected data on the amino acid profile of some algae are compiled in Table 3.2 and compared with some basic conventional food items. According to Becker (2013) the amino acids pattern of almost all algae are comparable with that of the FAO (Food and Agriculture Organization) requirement, with minor deficiencies among the sulfur-containing amino acids methionine and cystein, a fact that is characteristic for many plant proteins. TABLE 3.2 Amino Acid Profile of Different Algae as Compared with Conventional Protein Sources and the WHO/FAO Reference Pattern (g/100 g protein) Amino Acid

Recommended Compositiona

Chlorella Eggb Soybeanb vulgarisc

Spirulina platensisb

Nannochloris bacillarisd

Tetracystis sp.d

ESSENTIAL AMINO ACIDS Arginine

6.2

6.2

5.4

7.3

9.2

9.3

Histidine

1.9

2.4

2.4

1.8

2.2

2.3

2.3

Isoleucine

2.8

6.6

5.3

3.7

6.7

7.8

7.0

Leucine

6.6

8.8

7.7

7.7

9.8

13.8

12.6

Lysine

5.8

5.3

6.4

5.7

4.8

8.9

9.1

Methionine

2.5

3.2

1.3

16.0

2.5

2.8

2.9

Phenylalanine 6.3

5.8

5.0

7.7

5.3

8.8

9.1

Threonine

3.4

5.0

4.0

5.1

6.2

7.1

7.4

Tryptophan

1.1

1.7

3.7

8.5

5.3

6.6

7.3

Valine

3.5

7.2

5.3

5.2

7.1

9.7

8.8

11.0

1.3

8.1

11.8

15.3

12.9

5.0

7.5

9.5

10.7

10.6

1.0

1.8

2.4

NONESSENTIAL AMINO ACIDS Aspartic acid Alanine Cystine

2.3

1.9

12.6

19.0

13.7

10.3

15.1

14.4

Glycine

4.2

4.5

6.1

5.7

7.8

8.5

Proline

4.2

5.3

7.7

4.2

6.8

7.3

Serine

6.9

5.8

4.6

5.1

6.5

7.0

Tyrosine

4.2

3.7

5.3

5.7

5.5

Glutamic acid

a

Recommendation for children of 2 5 years old, according to FAO/WHO (1973). b Becker (2004). c Wei et al. (2011). d Tibbetts et al. (2015). Based on FAO/WHO (1973), Becker (2004), We et al. (2011) and Tibbetts et al. (2015).

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Special attention is given during prolonged storage or excessive heat treatment (140 C 165 C) of algal biomass that can induce the so-called Maillard reaction between amino acids and reducing carbohydrates, resulting in the nonavailability of essential amino acids. In the case of Spirulina, for instance, a considerable reduction of the digestibility was found due to the interaction of heat, pH, and lysine (Adrian, 1975). Two methods are common for estimating the quality of a given protein by its amino acid composition, that is, the chemical score (CS) and the essential amino acid index (EAAI). The estimation of the CS involves hydrolysis of the protein into its amino acids and their comparison with that of a high-quality protein such as egg, milk, or a reference pattern. The lowest score for any of the essential amino acids designates the limiting amino acid and gives a rough estimate of the quality of the protein tested. In practice, it is preferable to test for lysine, methionine plus cystine, and tryptophan, because one of these amino acids is usually present in a limiting amount in most conventional and unconventional protein sources (Matos, 2017). The EAAI index is based on the assumption that the BV of a protein is a function of the levels of the ten essential amino acids lysine, tryptophan, valine, isolecine, leucine, threonine, phenylalanine, methionine and cystine (as one), arginine, and histidine in comparison with their content in a reference protein (egg). The EAAI results in figures close to the value determined in feeding tests. Deficiencies in certain amino acids can be compensated for by supplementing with proteins from other sources or by consuming the limiting amino acid directly (Becker, 2013). As microalgae are able to synthesize a wide range of amino acids, an interesting cosmetic active product based on algal-derived amino acid is “Exsy-Algine” (Exsymol, Monaco) that has been marketed with the claim of having skin care properties due to a better polar alga-peptide (derivative of arginine) (www.exsymol.com).

3.4.3 Peptides Bioactive peptides are usually composed of 2 20 amino acid residues and have received much attention due to their biological activities and health benefits. Algae organisms produce a variety of bioactive molecules, which can be developed as nutraceutical and pharmaceuticals for human nutrition supplementation and disease therapy (Korhonen and Pihlanto, 2006). As many microalgae have high protein content, there is an enormous interest in using marine alga protein as a source of bioactive peptides (Samarakoon and Jeon, 2012). Depending on the amino acid sequence, marine algae-derived biopeptides may be involved in various biological functions, including antioxidant, anticancer, antihypertensive, antiatherosclerotic, and immunomodulatory effects (Fan et al., 2014; Kim and Kim, 2013; Sheih et al., 2010). Some of these molecules retain their natural functions when used for anthropogenic purposes; for example, mycosporine-like amino acids (MAAs) are effective UV-absorbing pigments and have been identified in taxonomically diverse organisms, including cyanobacteria. Two novel MAAs were discovered in N. commune as protective agents against UV damage and as antioxidants relevant to anhydrobiosis. In this case, MAAs have been incorporated in a number of human sunscreen products, with the claim of having UV

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protective properties. A protein peptide cyanovirin-N (CV-N), also called Nostopeptins, extracted from Nostoc ellipsosporum was observed to inactivate diverse strains of HIV-1, HIV-2, and simian immunodeficiency viruses, while noscomin extracted from N. commune is a promising antibacterial compound (Coates et al., 2013). There are three ways to release bioactive peptides: solvent extraction, enzymatic hydrolysis, and microbial fermentation (Fan et al., 2014). To obtain bioactive peptides from algae, the first step is to effectively extract the proteins. Many algae have cell walls made of sulfated and branched polysaccharides, such as lignin, cellulose, and hemicellulose. In this case, it is essential to break down cell walls for extracting algal proteins. For the pharmaceutical and food industries, enzymatic hydrolysis is preferred, due to the lack of residual chemicals in the final peptide products. After enzymatic hydrolysis, the most widely used peptide fractionation methods include ultrafiltration with different pore sizes (3, 5, 10, and 30 kDa), sequential chromatography (i.e., gel filtration chromatography and ion-exchange chromatography), and reverse-phase liquid chromatography. Then, the molecular structures of bioactive peptides can be characterized by mass spectrometry, such as liquid chromatography-mass spectrometry and mass-mass spectrometry (Chabeaud et al., 2009). The enzymatic hydrolysis or enzyme-assisted extraction (EAE) technology is a highly sophisticated technique and can also be applied for large-scale operations. Furthermore, EAE is an ecofriendly, nontoxic, high-bioactivity, and relatively low-cost technology (Wijesinghe and Jeon, 2012). The commonly used food-grade enzymes include cellulose, α-amylase, and pepsin. Compared to extracts obtained using conventional extraction methods, enzyme-assisted extracts have higher antioxidant activity. Similarly, enzyme proteolysis was found to enhance extraction of ACE inhibitory and antioxidant peptides from Porphyra columbina residual cake (Cian et al., 2013).

3.4.4 Enzymes Enzymes from microorganisms could be used as catalysts in diverse industrial processes. The search for new sources of microbial enzymes is ongoing and requires sustainable solutions (Adrio and Demain, 2014). To date, there is no industrial production of enzymes from microalgae. However, several reports have shown the great capacity of microalgae cells to synthesize enzymes, including hydrolases, oxidoreductases, and lyases (Brasil et al., 2017). 3.4.4.1 Proteases Proteases constitute a wide ranging group of enzymes that catalyze peptide-bond cleavage in proteins and peptides (Zhu et al., 2011). Proteases have several metabolic functions and have been used in many industrial applications, primarily in the detergent, pharmaceutical, and food industries (Rao et al., 1998). In microalgae metabolism, protease activity increases during environmental stress such as light or nutrient limitation and cell apoptosis. Proteolysis also plays a role in the control of organelle senescence and heteorocyst formation in cyanobacteria (Pe´rez-Llore´ns et al., 2003). In the cyanobacteria Anabaena variabilis, a calcium-dependent serine protease (trypsin-like protease)

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has been described (Lockau et al., 1988). Using A. variabilis mutant IM 141, which lacks this calcium-dependent protease, a second soluble trypsin-like protease, namely propylendopeptidase, has been studied (Strohmeier et al., 1994). A novel serine protease associated with the selective proteolysis of phycobiliproteins was isolated, purified, and partially characterized from A. platensis (Nanni et al., 2001). In another study, a novel arginine-specific protease (Sp-protease) that hydrolyzed gelatin and fibrin has been described. Both enzymes from A. platensis had a molecular weight of 80 kDa and act independently of Ca21 (Yada et al., 2004). Since some studies have reported the ability of microalgae for protease production and because synthesis of protease is directly associated with the availability and nature of the nitrogen source, future research should evaluate how changes of the nitrogen source in the medium affect the induction of protease activity in microalgae.

3.5 PROTEIN EXTRACTION METHODS Microalgae have low protein digestibility in their raw (unprocessed) form and it is for this reason that great emphasis has been placed on developing improved methods for algal protein extraction in order to improve their bioavailability. Algal proteins are conventionally extracted by means of aqueous, acidic, and alkaline methods, followed by several rounds of centrifugation and recovery using techniques such as ultrafiltration, precipitation, or chromatography (Bleakley and Hayes, 2017). Current extraction methods used on algae to date are limited for commercial use due to concerns with upscaling. But, in general, algal proteins are extracted by using the Kjeldahl method, which is recognized as a universal, precision, and reproductive method. Conventional mechanical and enzymatic methods for protein extraction may affect the integrity of extracted algal proteins due to the release of proteases from cytosolic vacuoles (Ganeva et al., 2003). In addition, these techniques are also laborious and time-consuming. As many microalgae have rigid cell walls composed of lignin, cellulose, and hemicellulose, improved extraction methods coupled with cell wall disruption are therefore required. Pretreatment with cell-disruption techniques aid the breakdown of the tough algal cell wall, increasing the availability of proteins and other high-value components for later protein extraction. There are several factors that collectively determine the suitability of a cell disruption process for the release of cell contents from microalgae. In general, a good cell disruption technique should be characterized by easy handling, low energy demand, and high disruption yields, by using economic and less toxic disruption reactants. The final product application should be taken into consideration when selecting a suitable disruption technique. To extract the biofunctional proteins from microalgae while preserving their bioactivity and functionality, it is necessary to use a mild pretreatment process to facilitate the recovery of the fragile intracellular compounds. Exposure of the microalgal cells to harsh conditions, such as high pressures, high shear levels, or high temperatures that might change the structure and subsequently cause the loss of specific functionality or activity, could thus be avoided (Phong et al., 2018).

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Some examples of existing cell disruption technologies prior to protein extraction include bead milling, ultrasonication, ionic liquids, microfluidization, enzymatic disruption, PEF, cationic polymer coated membranes, and microwave-assisted extraction (MAE; Kadam et al., 2013).

3.5.1 Bead Milling Bead milling is a mechanical cell disruption method that causes direct mechanical damage to cells. Advantages such as high disruption efficiency and high biomass loading are the primary factors that make bead milling an attractive cell disintegration method. Although bead milling has been found to be effective in breaking the cell wall of microalgae, most studies have mainly focused on the disruption of microalgae for lipid extraction. To date, the investigation on the mild release of fragile components such as microalgal proteins using bead milling is still limited (Phong et al., 2018). But, Postma et al. (2015) demonstrated that mild disintegration of C. vulgaris using bead milling for the release of water-soluble proteins below 35 C is possible, with yields ranging between 32% and 42%.

3.5.2 Ultrasound-Assisted Extraction Ultrasound-assisted extraction (UAE) is recognized as an efficient extraction technique that dramatically reduces working times, increasing yields and often the quality of the extract. UAE comprises mechanical waves that need an elastic medium to spread, creating regions of alternating compression and rarefaction waves induced on the molecules of the medium. UAE can be applied in analytical chemistry in two ways: directly to the sample, or indirectly through the walls of the sample container using a water bath (Pico´, 2013). After interaction with subjected biological or nonbiological material, ultrasound waves alter their physical and chemical properties releasing the extractable compounds. Ultrasound-aided extraction was evaluated in microalgae for a number of compounds, including algal proteins. For example, ultrasound treatment of C. vulgaris significantly increased (56.7%) crude protein digestibility in rats compared to electroporated (44.3%) and untreated spray-dried (46.9%) biomass (Janczyk et al., 2005). Keris-Sen et al. (2014) reported that ultrasound at a power intensity of 0.4 kWh/L yielded the optimum concentrations of proteins from Scenedesmus sp. microalgae. Due to its characteristics, UAE can be also used as a pretreatment prior to more sophisticated extraction. For example, a UAE/solid-phase extraction (SPE)/supercritical fluid extraction (SFE) method was developed for trace concentrations of isoflavones in algae and cyanobacteria (Klejdus et al., 2010). During the sonication pretreatment, certain parts of the matrix (e.g., walls of the cells or organelles in algal material) are damaged, and the SFE mass transfer takes place much more easily.

3.5.3 Pulsed Electric Field-Assisted Extraction PEF has been used as a cell disruption technique in microalgae, although its primary use has thus far been for the extraction of lipids for conversion to biofuel. PEF involves

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applying high electric currents in order to perforate a cell wall or cell membrane, causing reversible electroporation. Electroporation enables the introduction of various foreign components to cells, including DNA, proteins, and drugs (Vanthoor-Koopmans et al., 2013). Goettel et al. (2013) were among the first to report the use of PEF as a means of extracting multiple intracellular components from algae. Since then, PEF has been demonstrated to increase the yield of several high-value microalgae components, including lipids, carbohydrates, carotenoids, and chlorophyll (Postma et al., 2016). Protein yield from Chlorella sp. and Spirulina sp. was reported to increase by 27% and 13%, respectively, following PEF-treatment at 15 kV/cm and 100 kJ/kg (Bleakley and Hayes, 2017). In another study, the enhancement of the extraction efficiency of pigments such as C-phycocyanin from PEF electroporated microalgae (S. platensis) has been observed (Martı´nez et al., 2017).

3.5.4 Microwave-Assisted Extraction MAE involves heating a material, causing moisture to evaporate, thus creating bubbles under high pressure rupturing the algal cell wall. Increased levels of soluble proteins were extracted from green microalgae (Stigeoclonium sp. and Monoraphidium sp.) and diatoms (Nitzschia sp. and Navicula sp.) using microwave pretreatment compared to ultrasound (Passos et al., 2015).

3.5.5 Ionic Liquids Ionic liquids can be an ecofriendly alternative to traditional solvents and their applications in the processing of microalgae appears to be promising. Ionic liquids are organic salts in the liquid state, consisting of a large asymmetric organic cation and an organic or inorganic anion, and they have excellent properties for use in cellulosic biomass treatment due to their high hydrogen bond accepting ability, which could disrupt the extensive hydrogen bonding network of polymers, leading to the breakdown of complex networks of lignin, cellulose, and hemicelluloses (Phong et al., 2018). Ionic liquids exhibited better microalgal disruption efficiency under mild conditions compared to organic solvents (Orr and Rehmann, 2016). Synechocystis sp. and Scenedesmus sp. have successfully been dissolved in ionic liquids during the extraction process (Phong et al., 2018). Furthermore, a few studies revealed that ionic liquids are suitable for use in the extraction of fragile and sensitive compounds from microalgae, such as phycocyanin from Spirulina (Zhang et al., 2014) and astaxanthin from H. pluvialis (Desai et al., 2016).

3.6 EXTRACTION OF PROTEINS AND ACTIVE SUBSTANCES FROM ARTHROSPIRA PLATENSIS AND PORPHYRIDIUM CRUENTUM Two important active substances in the microalgae A. platensis and Porphyridium cruentum are phycocyanin and phycoerythrin, respectively. These are accessory pigments in photosynthesis and belong to the phycobiliproteins (pigment protein complexes). Phycocyanin (blue pigment) and phycoerythrin (red pigment) have fluorescent and

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3.6 EXTRACTION OF PROTEINS AND ACTIVE SUBSTANCES FROM ARTHROSPIRA PLATENSIS

Microalgae

Cell disruption

Extraction

Separation

·High pressure (homogenization– 3 passes)

·pH value pH 3–11

·“Salting-out” Precipitation at different ammonium sulfate concentrations

·Ultrasound Continuous (3 passes and 1 batch)

·Ionic:strengh NaCl: 0.1; 0.5; 1.0 mol/L

79

·Ultra-turrax 20,500 ppm ·Cell lysis pH = 10 and 12

FIGURE 3.5 Optimal conditions steps for pigment and protein extraction from Arthrospira platensis and Porphyridium cruentum. Source: Based on Idakiev, H., Baecker, S., 2018. Extraction of proteins and active substances from microalgae. INFORM (International News on Fats, Oils, and Related Materials) 29, 22 25.

antioxidant properties, which are marketed as high-fluorescent dyes in biological and biomedical research, and as food supplements and natural dyes in the food and cosmetics sectors (Ariede et al., 2017; Matos, 2017). The process for extracting functional proteins in conjunction with phycocyanin and phycoerythrin extraction from A. platensis and P. cruentum has been shown in Fig. 3.5. Various methods of cell disruption, such as high-pressure homogenization, ultrasound treatment, ultra-turrax homogenization, and cell lysis have been applied. In addition, the solubility behavior of proteins and pigments depends primarily on the variation of pH values and ionic strengths (Pereira et al., 2018). The extracted proteins and pigments, however, can be separated by precipitation at different ammonium sulfate concentrations using the salting-out method (Idakiev and Baecker, 2018). Fig. 3.5 provides an overview of the methods and testing parameters applied for these compounds. Generally, the optimal cell disruption method for A. platensis turned out to be highpressure homogenization. Phycocyanin has maximum solubility at pH 6, and is unstable within the alkaline pH range, where the majority of other proteins are most soluble. The proteins of A. platensis are also highly soluble at pH 6 and maximally soluble at pH 10. Therefore, the optimum pH for extracting both substrates together is 6. In general, ion concentration does not increase phycocyanin and PS. Therefore, to solubilize them more selectively, cell disruption can be conducted at pH 6 without adding salts. The phycocyanin and proteins in the obtained extract can be separated from each other—for example, by precipitation at different ammonium sulfate concentrations (Benelhadj et al., 2016). Additionally, phycocyanin is separated from proteins at higher ammonium sulfate concentrations (25% and 35%), as the phycocyanin fractions are distinguished by their typical blue color. The main disadvantages of this process, however, is the fact that the addition of ammonium sulfate introduces obstacles such as the high cost of adding and recovering ammonium sulfate and the possible impairment of solubility in water. Another possibility would be to use ultrafiltration. In this case, using membranes with different

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3. MICROALGAE AS A POTENTIAL SOURCE OF PROTEINS

Arthrospira platensis

Porphyridium cruentum

Cell disruption pH 6

Cell disruption pH 7

Centrifugation

Supernatant

Residue

Centrifugation

Alcaline extraction

Phycoerythrin COOH COOH

Separation

Centrifugation

Phycocyanin (linked to protein) “Phycocyanobilin” Cys

Supernatant

H3C

O

CH3 H3C

H3C

H3C N H

N H

N

H2C

N H

O

Purification

COOH COOH

S H O

H N H

Protein N H

N

N H

O

FIGURE 3.6 Process design for obtaining the pigments (phycocyanin and phycoerythrin) and proteins from Arthrospira platensis and Porphyridium cruentum. Source: Redrawn from Idakiev, H., Baecker, S., 2018. Extraction of proteins and active substances from microalgae. INFORM (International News on Fats, Oils, and Related Materials) 29, 22 25.

molecular weight cut-offs achieves effective separation, as the pigment and protein have different molecular weights (Idakiev and Baecker, 2018; Kannaujiya and Sinha, 2016). The residue from cell disruption still contains a large portion of the proteins, which can be subsequently extracted under basic conditions. The protein solution recovered by centrifugation can also be purified by precipitation at the isoelectric point (pH 4) or, alternatively, by ultrafiltration. The process diagram for extraction of proteins and phycocyanin from A. platensis is shown in Fig. 3.6. In the case of P. cruentum microalga, most of the cells can be disrupted using ultrasound. Phycoerythrin is maximally soluble at pH 7, unstable at the alkaline pH range, and less soluble in saline solution. In contrast, P. cruentum proteins are poorly soluble at a neutral pH range. They are most soluble at pH 11 and less soluble in saline solution. Therefore, these two products can be extracted simultaneously. By applying successive extraction, both products can be obtained with high yields and quality. In this case the P. cruentum cells should be disrupted first at pH 7 to solubilize phycoerythrin more selectively. Since only a small fraction of P. cruentum proteins will be recovered, separation can be omitted if high purity is not desired. The extract obtained by centrifugation can be marketed as a phycoerythrin-rich product. The residue can be subsequently extracted under basic conditions to obtain the proteins. The protein solution recovered by centrifugation can be further purified by precipitation at the isoelectric point (pH 3) or by ultrafiltration (Idakiev and Baecker, 2018; Safi et al., 2013). The process scheme for extracting proteins and phycoerythrin from P. cruentum is presented in Fig. 3.6.

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Using the process shown in Fig. 3.6, proteins are obtained as coproducts of phycocyanin and phycoerythrin extraction. Moreover, the technofunctional properties of the proteins obtained can be studied, and their application possibilities determined based on their functionality. The extraction of other valuable compounds such as sulfated polysaccharides and polyunsaturated fatty acids remaining in the residues should also be considered. These steps are necessary to use the full potential of the A. platensis and P. cruentum. Maintenance and stability of phycocyanin and phycoerythrin for long-lasting applications in the biotechnology, biomedical, and food industries are challenging tasks. Purity ratios of around 0.7, 3.9, and .4.0 are used to classify phycocyanin and phycoerythrin as food grade, reactive grade, and analytical grade, respectively (Chaiklahan et al., 2012). Kannaujiya and Sinha (2016) have investigated the thermokinetic stability of phycocyanin and phycoerythrin from Nostoc sp. strain HKAR-2 with edible preservatives such as benzoic acid, citric acid, sucrose, ascorbic acid, and calcium chloride over 30 days of incubation at 4 C, 25 C, and 40 C. Overall, benzoic acid at 4 C was found to be the best preservative as colorant for food application for both phycocyanin and phycoerythrin, and thus can be similarly applied to protect the phycobiliproteins from A. platensis and P. cruentum.

3.7 PHYSICOCHEMICAL AND TECHNOFUNCTIONAL OF ALGAL PROTEINS The term “technofunctional” has been suggested to describe the properties of proteins and other nutrients that, in addition to their nutritional function, are capable of playing other roles in food formulation. The technofunctional properties of proteins include all the physicochemical properties that would affect their technological applications during processing, handling, and consumer use. These properties are strongly involved in enhancing the organoleptic characteristics of processed food, including texture, viscosity, palatability, and mouthfeel. In general, the technofunctional properties of food proteins involve two molecular aspects, that is, protein surface-related properties (solubility, wettability, dispersibility, foaming, emulsification, flavor, and fat binding) and hydrodynamic properties (viscosity, thickening, gelation, and texturization) (Barka and Blecker, 2016). Today, the technofunctionality of proteins of only a few microalgae species (Spirulina sp., Tetraselmis sp., and C. vulgaris) is known. Examples of such properties are highlighted below. Very recently, Pereira et al. (2018) studied the technofunctional properties of Spirulina sp. LEB 18 biomass. Results have shown that the proteins are solubilized at pH 11.0 and precipitated at pH 4.2 (protein isoelectric point determined by potentiometric titration). In addition, it was possible to obtain a protein concentrate with B84.0%wt. of protein. Furthermore, protein extraction from this microalgae allowed a significant increase in PS and foam stability. Microalgae proteins usually have an isoelectric point at acidic pH due to amino acids composition. In the case of Spirulina sp. LEB 18, biomass presents high amounts of acidic amino acids, aspartic acid (11.8%), and glutamic acid (10.3%), in relation to the total amino acids. These amino acids are classified as acids because they get negatively charged at pH above 3.0 (isoelectric point). Basic amino acids, histidine (2.2%), lysine (4.8%), and arginine

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(7.3%) also constitute the amino acids of the biomass and they contribute to the isoelectric point above pH 3.0 (Pereira et al., 2018). Protein extracted from the microalga Tetraselmis sp. is completely soluble at pH values of 5.5 and higher, independent of ionic strength. Such insensitivity to ionic strength opens the door to applications in a wide range of salt concentrations. In contrast, oil seed proteins are usually minimally soluble at pH values of 5.5 6.5 and low ionic strengths (I # 0.3). Due to the strong influence of solubility behavior of the functional properties of a protein, this difference in the solubility behavior of microalgal protein extracts compared to the plant proteins can lead to unique technofunctional properties. For example, Tetraselmis proteins exhibit stronger emulsified behavior compared to the sunflower protein helianthinin (Hel), and the emulsifier-stabilizing abilities of the protease inhibitor protein isolated from potato juice is comparable to that of whey protein isolate, the most important protein-containing emulsifier in food applications (Schwenzfeier et al., 2011). Protein extracts from C. vulgaris also show an excellent emulsifying capacity that is comparable to or higher than that of commercial proteins such as sodium caseinate and soy proteins (Ursu et al., 2014). The protein isolate extracted from A. platensis (API) has relatively high oil- and water-absorption capacities. Moreover, API is able to form films when sorbitol (30% w/w) is used as a plasticizer, and forms gels when the API concentration exceeds 12% (w/w) (Benelhadj et al., 2016). Another interesting technofunctionality from algal protein is the production of type II plywood adhesive using total proteins extracted from S. platensis and Chlamydomonas reinhardtii (Roy et al., 2014). According to the authors, of the two algae sources tested, C. reinhardtii proteins had better adhesive strength and water resistance than S. platensis proteins and showed comparable adhesive properties to soy proteins. Notably, bioadhesives made from both algal proteins had lower viscosity than soy proteins. This feature should allow easier spreading of adhesive on wood surfaces and deeper penetration into veneers.

3.8 APPLICATION OF ALGAL PROTEIN FOR HUMAN CONSUMPTION The growing world population and consequential deficiency in protein supply for human nutrition has led to increased activity in exploring novel and alternative protein sources like single-cell proteins (SCPs). Many microorganisms (algae, bacteria, fungi yeast/filamentous) can be used as a source of SCP, but due to their low nucleic acid content and high level of essential amino acids, algae are preferred over fungi and bacteria as a source of SCP for human consumption (Anupama and Ravindra, 2000). It has been reported that Yury Romanenko, a Soviet Union cosmonaut, was able to perform a series of important experiments on the Chlorella genus, in consideration of its future utilization as an SCP in long space journeys. The flavor of this microalga is not enjoyable in comparison with ordinary dishes. However, evidence of protein, nitrogen, and amino acid positive balance has been documented (Nicoletti, 2016). An interesting algal manufacturer, that is, TerraVia, has recently announced that Health Canada has issued regulatory approval, and classified as Generally Recognized as Safe (GRAS) in compliance with US FDA, to its portfolio of AlgaVia Whole Algae Ingredients

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from Axenochlorella protothecoides. These include AlgaVia Whole Algal Protein that contains approximately 65% vegan protein plus amino acids, fiber, and microelements and AlgaVia Whole Algal Flour making it an option for fortifying food due to its neutral flavor profile and texture (Algae Industry Magazine, 2017). With regard to a protein-cosmetic product, a relevant cosmeceutical derived from a protein-rich Spirulina extracts is “Protulines” by Exsymol S.A.M (Monaco) that has been used in the cosmetic industry for its skin properties, such as antiaging, performing face and body benefits (Enzing et al., 2014). The functional properties of the proteins in the microalgae species such as A. platensis and P. cruentum suggest that such proteins have a high potential as additives in numerous food as well as nonfood products, such as cosmetics. For example, microalgae proteins and their derivatives have moisture-retention properties and provide nutrients to skin and hair, making them good candidates for functional cosmetics (Idakiev and Baecker, 2018). They can also serve as biological substitutes for commercial chemical emulsifiers and foaming agents in such personal care products as moisturizing creams, lotions, shampoos, soaps, hair foam, shaving foam, and many others. Furthermore, many biological activities in microalgae (e.g., antioxidant, hypotensive, hepatoprotective, immunomodulatory, anticancer, and anticoagulant) are associated with their proteins, protein hydrolyzates, or peptides (Brasil et al., 2017; Fan et al., 2014). Thus, extracting such proteins and other active compounds in tandem with phycocyanin, phycoerythrin, astaxanthin, lipids, and other primary active substances could open up additional revenue streams and add value to microalgae processing.

3.8.1 Spirulina as a Superfood Spirulina sp. has been used for a long time by people as consumable food in several parts of the world. Many food products can be produced by using Spirulina or their compounds, for instance, isotonic beverages, cereal bars, instant soups, pudding, cake powder mix, and biscuits (Andrade et al., 2018; Santos et al., 2016). In this sense, Spirulina sp. can be used as a source of protein and amino acids for malnourished people. Furthermore, Spirulina biomass can be used in foods because it has a GRAS certificate granted by FDA. The protein content in Spirulina can vary from 50% to 70%wt., since its synthesis can be manipulated or influenced by cultivation or environmental conditions, such as concentrations of nitrogen and minerals, light intensity, and climatic conditions (Lourenc¸o, 2006). Some foods with added Spirulina biomass have been developed, including bakery Spirulina products with 2% of microalga, for example, rice pasta, sfogliatine, and grissini are produced by Microlife Nutrition in Italy. Novel yoghurt products containing high protein content, supplemented with 6% of Spirulina biomass and 10% of papaya pulp have been developed, too. Spirulina powder was used as ingredient for an orange-flavored chewable wafer by Microlife Nutrition to produce chocolate bars and biscuits (Fradique et al., 2010). Elaborated fresh spaghetti enriched with Spirulina maxima has been compared to standard semolina spaghetti (chemical composition, optimal cooking, coking losses, swelling index, water absorption, etc.) (Fradique et al., 2010). Authors concluded that the presence of microalgae enhanced the nutritional and sensorial quality of pasta. In addition, no changes in cooking and textural properties were observed.

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Different Italian companies produce pasta with Spirulina biomass, such as tagliolini, rice fusilli, corn penne (2% of microalga) (Microlife Nutrition), corn flour conchiglie (10% of microalga) (Nutracentis), wheat fusilli, kamut penne, spelt spaghetti (3% of microalga) (La finestra sul cielo). In addition, French companies produce novel pasta including tagliatelle duo curry and Spirulina (Cornand), and tagliatellines with Spirulina (GlopeXplore) (Fradique et al., 2010). The use of protein concentrates from Spirulina sp. LEB 18 as an alternative protein ingredient for athletes’ supplements in food formulation has been proposed by Pereira et al. (2018). This protein concentrate has higher solubility, foam stability, and thermal stability than Spirulina biomass, presenting a high protein content and good functional properties as benchmark proteins. Most cyanobacteria, including Spirulina, are able to synthesize vitamin B12, also called cobalamin, a water-soluble vitamin that is involved in the metabolism of every cell function in human body; it is a cofactor in DNA synthesis and amino acid metabolism. People who have a vegan diet, such as vegans and vegetarians, are highly advised to consume Spirulina biomass as source of B12. In addition to the vitamin B complex, other vitamins such as vitamin K, vitamin A, isomers of tocopherols (vitamin E), as well as metabolic intermediates can be found in Spirulina biomass. Among microalgal pigments, phycobiliproteins (phycocyanin and phycoerythrin) have an important role in the commercial and biotechnological sectors, with wide applications as natural dyes, food supplements, and antioxidants, and in biomedical and pharmaceutical areas (Borowitzka, 2013). Phycocyanin mainly produced by A. platensis is used in the food industry as a colorant, especially in dairy and milky products (fermented milk, milk shakes, and ice cream), jellies, coated soft candies, soft drinks, health drinks, sour milk and green tea, confectionaries (blue smarties), desserts, and sweet cake decoration (Mishra et al., 2010), a good commercial example of phycocyanin product is the “Lina Blue” developed by Dainippon Ink & Chemicals of Japan. The product is an odorless nontoxic blue powder with a slight sweetness. When dissolved in water it is brilliant with a faint reddish fluorescence. Its color (absorbance maxima 618 nm) is between those of blue colors No. 1 (brilliant blue) and No. 2 (indigo carmine). It is stable from pH 4.5 to 8.0, and thermostable up to 60 C, but exhibits poor light stability (Vonshak, 2002). Beyond the food product and nutrition value, both phycocyanin and phycoerythrin have medical properties (e.g., anti-inflammatory, antiviral, anticancer, neuroprotective, and hepatoprotective) and have recently been used in photodynamic therapy for the treatment of certain cancers (Richa et al., 2011; Zheng et al., 2013). In addition, the fluorescence properties of phycobiliproteins have led to their use in many molecular and pharmaceutical diagnostic applications (Matos, 2017). The worldwide market of phycocyanin has been reported to be about US$10 50 million per annum, and the food-grade price for phycocyanin is around US$450 kg21 (Borowitzka, 2013).

3.9 NUTRITIONAL STANDARD AND REGULATIONS As many of the high-value products from microalgae are intended for human and animal nutrition or related use, these compounds are subject to a range of regulations and

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standards. There exist internationally testing programs for unconventional foodstuffs, such as single-cell proteins, which have to be or should be performed. There are only a few countries that have so far stipulated legislative standards for Spirulina. It is not known whether official regulations also exist for other types of microalgae, that is, Chlorella and Dunaliella. These official requirements as well as suggested quality criteria found in the literature and elsewhere are summarized in Table 3.3. According to Becker (2013) several steps need to be evaluated for the approval of algal biomass for human and animal consumption. It seems useful to have the following specifications to become available: • • • • • •

proximate chemical composition; evaluation of protein quality and quantity of essential amino acids; biogenic toxic substances (phycotoxins, nucleic acids, other toxicants); nonbiogenic toxic compounds (heavy metals, residues from harvesting and processing); sanitary analyses (microbial analyses for contamination); toxicological and safety evaluations (feeding trials with experimental animals).

TABLE 3.3 Some Quality Standards for Microalgae

IUPAC (1974)

WHO (1972)

Earthrise Algal Farm (United States)

Indian Japan France Brasil, (2001, Standard (1990) (Jassby, 1988) (Becker, 1994) 2013, 2014)

Total protein (%)

.55.0

Total ash (%)

,9.0 ,9.0

.45.0

Moisture (%)

,7.0

,7.0

Standard plate count ( 3 106 g21)

,0.2

,0.005

Mold (number/g)

,100

,100

Coliformes (number/g)

Absent

Absent

Salmonella sp.

.50.0

,10.0 ,0.1

,10

,10

Absent

Absent

Absent

Staphylococcus sp. (number/g)

Absent

,100

,500

Filth (insect fragment) (number/g)

,10

Absent

,0.2

,0.30

Pb (ppm)

,5.0

,0.1

Hg (ppm)

,0.1

,0.001 ,0.025

,0.50

Cd (ppm)

,1.0

,0.01

,0.10

As (ppm)

,2.0

,0.05

,0.2

,1.00

Adapted from Becker, E.W., 2013. Microalgae for human and animal nutrition. In: Richmond, A., Hu, Q. (Eds.), Handbook of Microalgal Culture: Applied Phycology and Biotechnology. Wiley Blackwell, Oxford, pp. 461 503 and Matos, A.P., 2017. The impact of microalgae in food science and technology. J. Am. Oil Chem. Soc. 94, 1333 1350.

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The issues of food safety at the international level are considered by the Codex Alimentarius Commission, which was created by the Food and Agriculture Organization and the World Health Organization with the intent to develop food standards and guidelines. Laws and regulations relating to food additives and novel foods (including nutraceuticals and functional foods) vary from country to country. For example, in the United States the FDA has the primary responsibility for regulating new food ingredients, whereas the EU through the EFSA (European Food Safety Authority) has regulations for food additives, novels foods, and genetically modified (GM) organisms (Borowitzka, 2013). In January 2011, the FDA Food Safety Modernization Act was signed into law in the United States. It means that the manufacturer may seek approval for a new ingredient by filing a food additive petition with the FDA to (1) make a GRAS determination, or (2) request a formal pre-market review. This petition needs to be supported by clinical and nonclinical studies (Borowitzka, 2013; Enzing et al., 2014). In Australia and New Zealand, the products are most likely to come under the novel foods and novel food ingredients category regulated under “Standard 1.5.1 Novel Foods” of the Australian and New Zealand Food Standards Code administered by Food Standards Australia New Zealand (Borowitzka, 2013). In Brazil, the ANVISA (Ageˆncia Nacional de Vigilaˆncia Sanita´ria) has estimated some limits for microbial and heavy metal contaminants in tablets/capsules of Spirulina sold as dietary supplement (BRASIL, 2001, 2013, 2014; see Table 3.3). In Japan, the Ministry of Health, Labor, and Welfare defined the specifications and standards for food additives where the approved food additives are reported. Chlorella powder is generally provided as food and food additives—color, flavor, and taste enhancer. Furthermore, in the list of food additives appear the phycocyanin from Arthrospira sp., carotene from D. salina, and the microalga H. pluvialis as colorant (Japan External Trade Organization (JETRO), 2011). To improve algal safety and quality standards in China, the National Health and Family Planning Commission of the People’s Republic of China (NHFPC) announced the “Algae and their products food safety national standard,” applying to all algae products brought to market. This algal standard was developed in collaboration to the Chinese Microalgae Industry Alliance (CMIA) that submitted several advisories, which included quality testing data and current market statutes (Chen et al., 2016). For many markets and certifying or regulatory authorities, Good Manufacturing Practice certification and ISO 9001 2000 are essential. For food and related industries, an HACCP (Hazard Analysis & Critical Control Points) methodology is highly recommended (Borowitzka, 2013). Whereas, specific markets may require additional certifications, for example, organic certification by a recognized certifying authority, for example, USDA Organic in the United States and IBD Accreditation in Brazil (Matos, 2017).

3.10 TOXICOLOGICAL ASPECTS Microalgae, which are considered as unconventional food, have to undergo a series of toxicological tests to prove their harmlessness. Several recommendations and toxicological evaluations have been published by different international organizations, but it has to be

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assumed that additional national regulations exist from country to country, which specify the recommended analyses (Becker, 2013). As part of the toxicological characterization, the algal material has to be analyzed for the presence of toxic compounds, either synthesized by the alga itself (biogenic toxins) or accumulated from the environment (nonbiogenic toxins). The biogenic toxins include nucleic acids and algal toxins, whereas nonbiogenic products comprise environmental contaminants, such as heavy metals (Matos, 2017).

3.10.1 Nucleic Acids Nucleic acids are biopolymers essential to all known forms of life, and these include RNA and DNA, which are sources of purines. The association between a purine-rich diet and an increased plasma urate concentration and risk of gout has long been recognized (Kelley and Andersson, 2014; Liu et al., 2017). Uric acid is the end-product of purine degradation so the avoidance of purine-rich foods is commonly recommended to gout patients (Liu et al., 2017). As microorganisms are sources of single-cell protein that may contain purines, their daily consumption is limited for humans. In general, the nucleic acid content in algae varies between 4% and 6% (8% 12% for yeast and up to 20% for bacteria) in dry matter (Becker, 2013). Because of a possible health hazard, the Protein Advisory Group of the United Nations (Nutrition Bulletin) has recommended a maximum daily intake of 4.0 g/day nucleic acid for unconventional food source. As mentioned early, single-cell protein from algae sources are preferred over fungi and bacteria sources due to their low nucleic acid content (Matos, 2017). However, the safe level should be at about 20 g of algae per day or 0.3 g of algae per kg of body weight (Becker, 2013).

3.10.2 Algal Toxins Algal toxins are produced by various algae and are found both in seawater and freshwater and they have no taste or smell, and are not eliminated by cooking or freezing. Poisoning of humans and animals by algal toxins, resulting from blooms of algae, occurs with considerable frequency—sometimes unpredictably—in several regions of the globe and can lead to fish die-offs, cities cutting off water to residents, or states having to close fisheries. Certain marine algae (Anabaena sp., Mycrocystis sp., Dynophysis sp. and Pseudonitzschia) produce potent toxins (saxitoxins, brevetoxin, domoic, and okadaic acids) that have an impact on human health through consumption of contaminated fish, oysters, scallops, clams, shellfish, and mussels, which have filtered these toxins from the water and accumulated them to levels that can be lethal to consumers, including humans (Proenc¸a et al., 2011). Algal toxins can give rise to a number of different poisoning syndromes (Enzing et al., 2014): • • • • •

NSP—neurotoxic shellfish poisoning; PSP—paralytic shellfish poisoning; ASP—amnesic shellfish poisoning; DSP—diarrheic shellfish poisoning; Ciguatera fish poisoning.

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TABLE 3.4 Safety Aspect of Relevant Microalgae for Food Application Organism

Species

Safety Aspect Organism

Species

Safety Aspect

Chlorophyta

Chlamydomonas reinhardtii

NT

Isochrysis galbana

NT

Chlorella vulgaris

GRAS

Pavlova sp.

NT

Dunaliella salina

NT

Heterokontophyta Nitzschia dissipata

NT

Haematococcus pluvialis NT

Nannochloropsis sp.

NT

Scenedesmus sp.

NT

Phaedactylum tricornutum

NT

Tetraselmis sp.

NT

Skeletonema sp.

NT

GRAS

Schizochytrium

GRAS

NT

Thalassiosira pseudonoma

NT

Porphyridium cruentrum

GRAS

Cyanobacteria Spirulina (Arthrospira) Synechococcus sp. Dinophyta

Haptophyta

Crypthecodinium cohnii GRAS

Rhodophyta

GRAS, Generally Recognized As Safe by Food Drug Administration (FDA); NT, no toxins known. Data from Enzing, C., Ploeg, M., Barbosa, M., Sijtsma, L., 2014. Microalgae-Based Products for the Food and Feed Sector: An Outlook for Europe. Joint Research Centre Scientific and Policy Reports, European Commission. Publications Office of the European Union, Luxembourg.

Algae such as Spirulina, Isochrysis galbana, H. pluvialis, C. vulgaris, D. salina, P. tricornutum and P. cruentum, among others that are commercially used in aquaculture and food supplements, do not produce toxins. Even within the same species, large differences exist between toxic and nontoxic algae. For example, dinoflagellates and diatoms are best known for their production of toxins that can affect humans, but for instance the dinoflagellate strain Crypthecodinium cohnii has a GRAS/FDA for ω-3 DHA human food consumption (Enzing et al., 2014) (Table 3.4). Consequently, in view of the application of algae for food or feed, it is very important to know their safety at their strain level. Many algae, particularly dinoflagellates, are associated with highly toxic natural products. An example of a drug being developed from a dinoflagellate toxin is Tetrodin sold by WEX Pharmaceuticals Inc. (Canada). This product is derived from tetrodotoxin (TTX), a potent neurotoxin, and mainly used in cancer-related pain management. Okadaic acid is another example of a marine biotoxin that has proven useful in medical research, because of its activity as an inhibitor of protein phosphatase 2A (PP2A), it is regularly applied in studies where inhibition of PP2A is wanted (Gallardo-Rodrı´guez et al., 2012).

3.10.3 Heavy Metals Heavy metals are often assumed to be highly toxic or damaging to the environment. Almost all microorganisms, primarily those living in an aquatic biotope, are capable of accumulating heavy metal ions by absorption and adsorption at concentrations that are several orders of magnitude higher than those present in the surroundings. According to WHO/FAO guidelines, an adult person of 60 kg body weight should not incorporate more

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than 0.3 mg of mercury, 0.5 mg of cadmium, 3 mg of lead, and 20 mg of arsenic per week through beverages and food. At present, no official standards exist for the heavy metal content of microalgal products. On a voluntary basis, some algae manufactures have established internal guidelines for metal levels in their products (Becker, 2013).

3.11 CHALLENGES SURROUNDING ALGAL USAGE 3.11.1 Food Safety Several studies show that safety hazards related to algae may include allergens, toxins, pathogens, heavy metals, and pesticides. As mentioned earlier, no toxins have been found in Spirulina and Chlorella. However, in A. flos-aqua, toxic microcystines have been detected (Kerkvliet, 2001). Extracts from products consisting of A. flos-aqua, Spirulina, and Chlorella, or mixtures thereof, were cytotoxic (Heussener et al., 2012). Under favorable conditions, pheophorbides are formed in Chlorella, which give rise to photosensibilization in some humans (Kerkvliet, 2001). Another safety aspect is the presence of pathogenic microorganisms. Spirulina and Chlorella are cultivated in open raceway ponds, which may result in microbiological contamination from birds, insects, and rodents (Spiegel et al., 2013). Algae may accumulate heavy metals. In general, Spirulina accumulates more heavy metals than Chlorella (Kerkvliet, 2001). There are therefore strict legal limits in some countries for the safe maximum exposure of heavy metals, such as mercury, arsenic, lead, and cadmium, in foods intended for human consumption. These limits are based upon the recommendations of the Joint FAO/WHO Expert Committee on Foods Additives (JECFA). In Europe, it is regulated by the Panel on Contaminants in the Food Chain of the European Food Safety Authority (CONTRAM Panel), outlined by the legislation under Commission Regulation (EC) No. 1881/2006 (Bleakley and Hayes, 2017).

3.11.2 Genetically Modified Algae Genetic modification of eukaryotic microalgae and cyanobacteria is now mainly studied in the laboratory. Several microalgae (e.g., Synechococcus, Synechocystis, Anabaena, C. reinhardtii, Nannochloropsis gaditana, Ostreococcus tari, P. tricornutum) are currently established for genetic engineering approaches. Curiously, Spirulina has proved to be extremely recalcitrant to genetic transformation (Doron et al., 2016; Ga´rcia et al., 2017). C. reinhardtii, a green alga used widely in biology laboratories as a genetic model, can produce a range of complex proteins for human therapeutic drugs, such as malaria vaccine. In malaria, a protein gene Pfs25 has been found on the surface of the malaria parasite’s reproductive cells. C. reinhardtii has been used as a host organism to synthesize Pfs25 antigens for malaria antibodies (Algae Industry Magazine, 2015). Chlorella represents an attractive alternative to currently well-established bacteria, yeast, and mammalian cell-based expression systems for production of recombinant proteins (e.g., enzymes, vaccines, monoclonal antibodies, and growth factors). In contrast to bacteria, Chlorella is a eukaryotic organism and can perform posttranscriptional and

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posttranslational modifications essential for producing functional eukaryotic proteins. Since Chlorella has long been used as a health food and is approved to be safe to human, recombinant proteins derived from Chlorella may be readily acceptable by the public (Liu and Hu, 2013). In the case of GM algae for food application, which are not still currently available in the market, two main aspects related to biosafety need to be addressed: (1) potential adverse environmental consequences; and (2) potential harm to human or animal health in case of food/feed or pharmaceutical applications. In general, all research—including microalgae research—is governed by regulations in the field of good laboratory practice. In the United States the FDA, and in Europe the EFSA are responsible for biosafety evaluation (Enzing et al., 2014). For human health risks, biosafety evaluation could refer to the methods applied in higher plants to guarantee that they are safe and that they do not produce toxic substances or allergens (Song et al., 2012). For the environmental risks, Henley et al. (2013) have published a comprehensive study on risk assessment of GM microalgae for commodity-scale biofuel cultivation which is also relevant for other applications. In particular for food applications, a history of safe use for a certain algae implies that production has proven to be safe over a longer period of time. Nevertheless, the process of bringing a GM microalgal product to market can therefore be a long and complicated issue.

3.11.3 Scalability The scalability of protein extraction from algae is a further obstacle that needs to be overcome before microalgae become a viable source. Algal protein extraction is still very much in its infancy, meaning that many of the methods that have been developed are still at a small scale. PEF and ultrasound have been suggested as being suitable for large-scale algal protein extraction. Membrane technologies may also be scalable for commercial applications, with ultrafiltration reported as being suitable for R-phycoerythrin extraction from the algae at the industrial scale (Bleakley and Hayes, 2017).

3.11.4 Price A critical factor that will determine the commercial viability of microalgae is their competitiveness compared to other protein sources on the market. For example, a major application for the production of microalgae is the extraction of lipids to be converted to biofuel. However, while they are a greener alternative for the environment, they are still not competitive compared to fossil-based petroleum fuels (Cuellar-Bermudez et al., 2015). Conventional methods of algal protein extraction are often quite wasteful as they typically dispose of the algae by-products following processing (Vanthoor-Koopmans et al., 2013). As several studies have shown, the by-products of algae can still have many applications that could be of economic value (Enzing et al., 2014). The techno-economic analysis of protein concentrate (95% water-soluble peptides and free amino acids) produced by flash hydrolysis of S. obliquus microalga (assuming a protein content of 45%wt.) has been conducted by Asiedu et al. (2017). The protein selling prices vary from US$4.31 to

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6.00 kg21 using ultrafiltration concentrated method, which is comparable (US$6.25 kg21) to 80% whey protein concentrate (Navarro da Silva et al., 2015). Asiedu et al. (2017) also emphasized that the huge amount of freshwater required to cultivate algae, which represents almost 64% of total water used, is one of the main obstacles encountered. To minimize input water demand, the use of brackish or saline water, which is more abundant, is recommended. Wastewater is another promising candidate that can be employed to grow microalgae. Using nonfreshwater will eventually reduce the costs of water treatment and nutrient additions.

3.12 CONCLUSION In the last years, there has been a growing interest from consumers in alternative source of proteins. Therefore, the use of microalgal biomass or its derived metabolites (protein, amino acids, peptides, enzymes, proteases) has become an innovative approach for the development of healthier food products. The high protein content coupled with all essential amino acids of several microalgal species have been one of the main reasons for considering these organisms as a source of food. Apart from protein composition, two important active substances (phycocyanin and phycoerythrin), both phycobiliproteins (pigment protein complexes), have fluorescent and antioxidant properties, which are marketed as high-fluorescent dyes in biological and biomedical research, and as food supplements and natural dyes in the food and cosmetics sectors. As many of the high-value products from microalgae are intended for human nutrition or related use, these compounds are subject to a range of food regulations and legislative standards, such as GRAS. To expand the sector of the microalgal food products and related materials, future paths are towards the multidisciplinary action of microbiologists for discovering new microalgae species and their properties, engineers for developing more cost-effective cultivation and biorefinery systems, food scientists for improving sensory appeal, and clinicians for evaluating algal compounds availability.

References Adrian, J., 1975. Evolution de la lysine des algues spirulines soumises a des traitments thermiques varies. Ann. Nutr. Aliment. 29, 603 613. Adrio, J.L., Demain, A., 2014. Microbial enzymes: tools for biotechnological process. Biomolecules 4, 117 139. Algae Industry Magazine, 2015. Algal malaria vaccine advances. http://www.algaeindustrymagazine.com/algalmalaria-vaccine-advances (accessed 05.09.18.). Algae Industry Magazine, 2016. $1.1B global algae market projected by 2024. www.algaeindustrymagazine.com. (accessed 12.08.18.). Algae Industry Magazine, 2017. TerraVia’s Whole Algal Protein gets Canadian approval. www.algaeindsutrymagazine.com. (accessed 14.08.18.). Andrade, L.M., Andrade, C.J., Dias, M., Nascimento, C.A.O., Mendes, M., 2018. Chlorella and Spirulina microalgae as sources of functional foods, nutraceuticals, and food supplements; an overview. MOJ Food Process. Technol. 6, 00144. Anupama, Ravindra, P., 2000. Value-added food: single cell protein. Biotechnol. Adv. 18, 459 479. Ariede, M.B., Candido, T.M., Jacome, A.L.M., Velasco, M.V.R., Carvalho, J.C.M., Baby, A.R., 2017. Cosmetic attributes of algae—a review. Algal Res. 25, 483 487.

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Plant-Based Proteins Rene´ Renato Balandra´n-Quintana1, Ana Marı´a Mendoza-Wilson1, Gabriela Ramos-Clamont Montfort2 and ´ ngel Huerta-Ocampo3 Jose´ A 1

Center for Research in Food and Development, A.C. Coordination of Technology of Foods from Vegetal Origin, Hermosillo, Sonora, Mexico 2Center for Research in Food and Development, A.C. Coordination of Food Science, Hermosillo, Sonora, Mexico 3 CONACYT-Center for Research in Food and Development, A.C. Coordination of Food Science, Hermosillo, Sonora, Mexico O U T L I N E

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4.4.1 Cereal and Pseudocereal Proteins 103 4.4.2 Proteins from By-Products of Plant Oil Refining Industry 111 4.4.3 Other Sources of Plant Proteins 118 4.5 Concluding Remarks

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4.1 GENERAL INTRODUCTION Proteins are important biomolecules because of their essential functions for life (Berg et al., 2002) and attributes they provide to many food systems (Li-Chan and Lacroix, 2018). Proteins of animal origin are of higher quality than plant proteins, due to their high digestibility, better amino acid score (AAS), and greater water solubility. An increasing population demands to consume more meat (Tilman and Clark, 2014) but meeting this demand implicates strong environmental impacts so the global food system is considered among

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the goals for sustainable development (Chaudhary et al., 2018). A solution to the problem of protein sustainability could be the shift to diets based on plant protein (Day, 2013). Digestibility and the contribution of essential amino acids, which in principle should be the most important drawbacks, can be solved by combining proteins from different plant sources (Mariotti, 2017). However, the biggest challenge is cultural (Macdiarmid et al., 2016) as the population is reluctant to replace animal proteins with plant ones. Since the act of eating is predominantly sensory, the academic and industrial sectors have two important challenges ahead: identifying alternative sources of proteins that meet the nutritional expectations and innovating to offer sensory-acceptable products (Day, 2013; Fritsch et al., 2017). In this chapter, the concept of sustainability in the food context is briefly addressed and updated scientific information on nutritional aspects, extraction, properties, and potential uses of conventional and nonconventional sources of vegetable protein is presented. The objective of gathering this information is to serve as a guide for technologists and academics interested in entering the field of protein sustainability.

4.2 IMPORTANCE OF PROTEINS IN HUMAN NUTRITION AND FOOD PROCESSING Amino acids determine both the structure and function of proteins and so their biological value (Berg et al., 2002). Eleven of the 20 amino acids found in proteins are synthesized in the cells of the human body. The remaining nine are not synthesized and so are essential or indispensable (Reeds, 2000), and must be provided through the diet (Tessari et al., 2016). Indispensable amino acids, whose content in a food is less than the optimum recommended level, are known as limiting amino acids (Friedman, 1996). Protein quality is measured in several ways. The AAS predicts the efficiency to meet the recommended levels of essential amino acids and is estimated for different age groups according to the recommended dietary protein intake (https://www.nal.usda.gov/fnic/ protein-and-amino-acids). An AAS greater than or equal to 100 (often the value is truncated to 100) indicates that the protein completely satisfies the recommended levels of essential amino acids; however, this does not take into account their bioavailability. The latter is included in the protein digestibility-corrected amino acid score (PDCAAS), which is calculated by multiplying the AAS by the digestibility of the protein (Schaafsma, 2000). Often, in literature PDCAAS values are indicated as a fraction. Here were converted to percentage in order to homogenize style, when necessary. Since a good protein digestibility does not mean bioavailability of all the indispensable amino acids (Schaafsma, 2000), the digestible indispensable amino acid score (DIAAS) was suggested as a new measure of protein quality (FAO, 2013). In DIAAS, each amino acid is considered a nutrient, so the individual digestibility of essential amino acids is taken into account. It is calculated by multiplying by a hundred the quotient obtained by dividing the content of the indispensable amino acid digestible in the dietary protein by the content of the same AA in the reference protein. A DIAAS 5 100% indicates a quality equal to that of the reference protein. In general, proteins of animal origin have a better score of essential amino acids and greater digestibility than those of plant origin, which is why they are used as reference proteins. Tables 4.1 and 4.2 show a comparison between

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TABLE 4.1 Reported Values for Protein Content, Indispensable Amino Acids (IAA) Profile, Amino Acid Score (AAS), Protein Digestibility Corrected Amino Acid Score (PDCAAS), and Digestible Indispensable Amino Acid Score (DIAAS) of Representative Proteins From Plant Origin Corn (white, raw)

Wheat (hard winter)

Rice (white, raw)

Barley (pearled, raw)

Sorghum (raw)

Quinoa (raw)

Soy (protein isolate)

Peas (cooked)

Kidney beans (cooked)

Peanuts (roasted)

9.4a

12.6a

7.1a

9.9a

9.8d

14.1a

92.7e

8.3a

8.7a

23.7a

Tryptophan

713a

1270a

1169a

1667a

510d

1184a

1402e

1120a

1195a

970a

Threonine

3766a

2897a

3592a

3404a

2959d

2986a

3614e

3566a

3667a

3422a

Isoleucine

3585a

3635a

4338a

3657a

3776d

3574a

4725e

4145a

4713a

3515a

Leucine

12287a

6778a

8296a

6798a

1265d

5957a

7961e

7205a

8460a

6477a

Lysine

2819a

2659a

3634a

3727a

2041d

5433a

6138e

7253a

6977a

3586a

a

a

a

a

d

a

e

a

a

2511a

Protein (g/100 g) IAA (mg/100 g prot)

SAA

3904

AAA

9000a

Valine

4151

4423

4131

3061

3631

7770a

8718a

8485a

7856d

6099a

8813e

7542a

8230a

8819a

5074a

4413a

6127a

4909a

4694d

4213a

4768e

4747a

5747a

4190a

Histidine

3053a

2262a

2366a

2253a

2245d

2887a

2600e

2446a

2736a

2527a

AAS

55a

52a

71a

73a

40a

106a

96c

102a%

89a

70a

PDCAAS_(%)

59.9j

42b

63g

61l

16-24i

79k

91b

60f

65f

51f

DIAAS (%)

48d

43d

64d

51d

29d

--

99.6h

58f

59f

43f

a

2415

2554

2230

From Nutrition Data. Available from: https://nutritiondata.self.com. From Hess, J., Slavin, J., 2016. Defining “protein” foods. Nutr. Today 51(3), 117 120, (Hess and Slavin, 2016). c From Schaafsma, G., 2000. The protein digestibility-corrected amino acid score. J. Nutr. 130 (7), 1865s 1867s, (Schaafsma, 2000). d From Cervantes-Pahm, S., Liu, Y., Stein, H., 2014. Digestible indispensable amino acid score and digestible amino acids in eight cereal grains. Br. J. Nutr. 111(9), 1663-1672, (Cervantes-Pahm et al., 2014). e From Mathai, J., Liu, Y., Stein, H., 2017. Values for digestible indispensable amino acid scores (DIAAS) for some dairy and plant proteins may better describe protein quality than values calculated using the concept for protein digestibility-corrected amino acid scores (PDCAAS). Br. J. Nutr. 117(4), 490 499, (Mathai et al., 2017). f From Rutherfurd, S.M., Fanning, A.C., Miller, B.J., Mougha, P.J., 2015. Protein digestibility-corrected amino acid scores and digestible indispensable amino acid scores differentially describe protein quality in growing male rats. J. Nutr.145, 372 379, (Rutherfurd et al., 2015). g From Sung-Wook H., Kyu-Man, C., Seong-Jun, C., 2015. Nutritional quality of rice bran protein in comparison to animal and vegetable protein. Food Chem. 172, 766 769, (Sung-Wook et al., 2015); (Determined in endosperm; in rice bran, PDCAAS reaches 90%). h From Ertl, P., Knaus, W., Zollitsch, W., 2016. An approach to including protein quality when assessing the net contribution of livestock to human food supply. Animal 10 (11), 1883 1889, (Ertl et al., 2016). i From da Silva, L.S., Jung, R., Zhao, Z.-Y., Glassman, K., Taylor, J., Taylor, J.R.N., 2011. Effect of suppressing the synthesis of different kafirin sub-classes on grain endosperm texture, protein body structure and protein nutritional quality in improved sorghum lines. J. Cereal Sci. 54, 160 167, (da Silva et al., 2011). j From Naves, M.M.V., Castro, M.V.L., Mendonc¸a, A.L., Santos, G.G., Silva, M.S., 2011. Corn germ with pericarp in relation to whole corn: nutrient contents, food and protein efficiency, and protein digestibility-corrected amino acid score. Food Sci. Technol. 31 (1), 264 269, (Naves et al., 2011). k From Boye, J., Wijesinha-Bettoni, R., Burlingame, B., 2012. Protein quality evaluation twenty years after the introduction of the protein digestibility corrected amino acid score method. The Br. J. Nutr. 108, S183 S211, (Boye et al., 2012). l From Nitrayova´, S., Brestensky´, M., Heger, J., Patra´ˇs, P., 2014. Protein digestibility-corrected amino acid score and digestible indispensable amino acid score in rice, rye and barley. In: Proceedings of the XVII International Symposium, “Feed Technology”, October 25 27, 2016, Novi Sad, Serbia, pp. 90 95. ,https://www.cabdirect.org/cabdirect/abstract/20173322729. (accessed 09.11.018), (Nitrayova´ et al., 2014). b

SAA, sulfur amino acids (methionine 1 cysteine), AAA, aromatic amino acids (phenylalanine 1 tyrosine).

100

4. PLANT-BASED PROTEINS

TABLE 4.2 Reported Values for protein Content, Indispensable Amino Acids (IAA) profile, Amino Acid Score (AAS), Protein Digestibility Corrected Amino Acid Score (PDCAAS), and Digestible Indispensable Amino Acid Score (DIAAS) of Representative Proteins From Animal Origin Beefa

Chickenb

Fishc

Eggd

Milke

18.7f

21.4f

18.4f

12.6f

3.2f

Tryptophan

209f

250f

206f

167f

75f

Threonine

816f

904f

808f

556f

143f

Isoleucine

840f

1130f

849f

672f

165f

Leucine

1477f

1605f

1498f

1088f

265f

Lysine

1555f

1818f

1693f

914f

140f

SAA

687f

866f

744f

652f

58f

AAA

1357f

1571f

1342f

1181f

299f

Valine

909f

1061f

950f

859f

192f

Histidine

640f

664f

543f

309f

75f

AAS

144f

136f

148f

136f

85f

PDCAAS (%)

114h

92j

124j

118i

110k

DIAAS-(%)

112h

108k

100g

113k

114k

Protein (g/100 g) IAA (mg/100 g)

a

Trimmed retail cuts, separable lean and fat, all grades, raw. Chicken, broilers or fryers, meat only, raw (DIAAS determined in breasts). c Sea bass, mixed species, raw. PDCAAS was determined in canned in oil tuna. d Whole, raw, fresh. (DIAAS determined in hard-boiled eggs). e Whole, 3.25% milkfat. f From Nutrition Data. Available from: https://nutritiondata.self.com. g From Shaheen, N., Islam, S., Munmuna, S., Mohiduzzaman, Md., Longvah, T., 2016. Amino acid profiles and digestible indispensable amino acid scores of proteins from the prioritized key foods in Bangladesh. Food Chem. 213, 83 89, (Shaheen et al., 2016) (determined in Tilapia). h From Ertl, P., Knaus, W., Zollitsch, W., 2016. An approach to including protein quality when assessing the net contribution of livestock to human food supply. Animal 10 (11), 1883 1889, (Ertl et al., 2016). i From Schaafsma, G., 2000. The protein digestibility-corrected amino acid score. J. Nutr. 130 (7), 1865s 1867s, (Schaafsma, 2000). j From Boye, J., Wijesinha-Bettoni, R., Burlingame, B., 2012. Protein quality evaluation twenty years after the introduction of the protein digestibility corrected amino acid score method. The Br. J. Nutr. 108, S183 S211, (Boye et al., 2012). k From Marinangeli, C., House, J.D., 2017. Potential impact of the digestible indispensable amino acid score as a measure of protein quality on dietary regulations and health. Nutr. Rev. 75 (8), 658 667, (Marinangeli and House, 2017). SAA, sulfur amino acids (methionine 1 cysteine). AAA, aromatic amino acids (phenylalanine 1 tyrosine). b

representative proteins of animal and plant origin, in terms of the content of indispensable amino acids, chemical score, PDCAAS, and DIASS. Another important attribute of proteins is their contribution to physical properties of food systems due to their ability to form foams, gels, doughs, emulsions, and fibrous

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structures (Weder and Belitz, 2003). This set of capabilities, known as functional properties, provide to proteins the means to modulate texture, palatability, and, in general, the sensory properties of foods (Phillips et al., 1994; Aryee et al., 2018). There is a lot of literature dealing with the functional properties of proteins (Phillips and Williams, 2011; Lam and Nickerson, 2013; Soria-Herna´ndez et al., 2015; Li-Chan and Lacroix, 2018). A growing world population demands a wide variety of protein ingredients. The food and beverage, pharmaceutical, animal feeding, cosmetics, and personal care industries, are what drive this market, for which a value of US$58.49 billion is projected for the year 2022, with an annual growth rate of 6% from 2017 (Markets and Markets, 2017). Within this market, the segment with the highest projected growth is that of sports nutrition and functional beverages. The most demanded proteins are those of animal origin (GrandViewResearch, 2018). However, there are also consumer groups interested in health or environment subjects, who are demanding plant protein.

4.3 PLANT-BASED PROTEINS IN THE SUSTAINABILITY CONTEXT The population increase and the capacity to produce food is one of the most relevant current issues. It is not the amount of foods per se that matters most, because if these were distributed equally, the average global demand for nutrients could be met (Wood et al., 2018). What is really disturbing is the sustainability of their production. Although the concept of food sustainability is subject to many interpretations, the definition used in the statement from the World Commission on Environment and Development (Aiking and de Boer, 2004) can be adopted in a very appropriate way. In this statement, it is implicit that in order to feed an entire population, means must be used that do not have negative repercussions on the environment, and that do not compromise the natural resources of future generations. In this sense the current system of food production has a strong environmental impact (IOM and NRC, 2015) that must be reversed in the short term to avoid consequences of greater scope. In the context of mass food production, proteins are the focus of attention due to the essential biological functions that they perform (Moore and Soeters, 2015) and because for a long time it has been promoted that those of animal origin are of better quality. Thus, the greater purchasing power of the population encourages a greater consumption of proteins of animal origin (Sabate and Soret, 2014), which in turn drives the intensive production of poultry, livestock, and fish. A growing demand for animal protein has a high environmental cost. On the one hand, there is the greatest emission of greenhouse gases by livestock, contributing to global warming (Herrero et al., 2013). On the other hand, the inefficient conversion of vegetable protein to animal protein (Shepon et al., 2016) results in a high demand for animal feed, with large areas of land being allocated to monoculture of grains and causing indiscriminate deforestation, and depletion of aquifers (Rojas-Downing et al., 2017). The final effect is counterproductive because it contributes to the generation of desert areas of thousands of hectares, which leads to water scarcity, a reduction in food production and, therefore, in the migration of people to more hospitable areas with the

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FIGURE 4.1 Scheme that summarizes the series of events and impacts of an animal protein-based diets and the counterpart of shifting to a plant protein-based one. (Earth image credit: NASA).

subsequent social problems and potential risks related to public health (WHO, 2018). In Fig. 4.1 these impacts are condensed and schematized. This problem has aroused a real interest to reduce the consumption of animal protein, substituting the contribution of essential amino acids with the inclusion of plant protein. This chapter deals with proteins of plant origin that are of interest to academics and industrialists in the context of food sustainability. The objective is to provide information on nutritive aspects, extraction, and functional and bioactive properties of these proteins, in order to contribute to the formation of a criterion with scientific grounds on the factors to consider when their use is intended.

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4.4 PROPERTIES AND EXTRACTION OF PLANT PROTEINS Due to their low solubility in water, traditional methods of recovering plant protein include alkaline and alcoholic extraction, as well as isoelectric precipitation. The classical extraction presents some drawbacks, for example, low yields and using reagents or conditions that can damage biomolecules, in addition to the disposal or final handling of chemicals representing environmental risks. Thus, developing sustainable techniques for extracting plant proteins from conventional and unconventional sources is a current subject (Pojic et al., 2018). In the present chapter plant by-products are preferably discussed because their proteins are usually underutilized.

4.4.1 Cereal and Pseudocereal Proteins 4.4.1.1 Rice Bran Proteins With almost 759 million tons, in 2016 rice was the third most produced cereal in the world (FAOSTAT, 2016a). Rice is harvested as paddy rice, that is, the grains are enveloped by a husk, which is removed in the first step of a process called milling to give brown rice as product. In a subsequent step, the bran is removed to obtain white rice. Paddy rice milling yields 8% 12% of bran (Dapˇcevi´c-Hadnaðev et al., 2018) into which the rice proteins are concentrated (11.3% 14.9% by weight) (Schramm et al., 2007), and these are mainly storage proteins (Juliano, 1985), including albumins, globulins, prolamins, and glutelins (Fabian and Ju, 2011). Rice bran proteins have a true digestibility equal to casein (94.8%) and higher than proteins from rice endosperm (90.8%), soybean (91.7%), and whey (92.8%) (Han et al., 2015). Although the PDCAAS of rice bran proteins (90) is lower than casein (100) and soybean proteins (95) (Han et al., 2015) the rice bran stands as a good economic and nutritive source of protein. Rice bran is perhaps the most studied cereal by-product regarding the extraction and functional properties of proteins. This subject has been reviewed by several authors (Fabian and Ju, 2011; Phongthai et al., 2017; Al-Doury et al., 2018; Balandra´n-Quintana, 2018). Because of space limitations, here the state-of-the-art will be briefly addressed and updated. Alkaline extraction of rice bran proteins at pH 7 12 yields 30% 50% proteins and these values are raised to more than 90% if extraction is done with combinations of solvents (Fabian and Ju, 2011). Optimal conditions for the alkaline extraction of defatted rice bran have been reported at pH 10, 80 rpm and 300 minutes of stirring, 52 C, with an extraction yield of 34.51% and protein content in concentrates of 48.53% (Bernardi et al., 2018). Ultrasound-assisted water extraction of defatted rice bran results in an increased yield of protein and improves the rate of initial extraction up to 3.5-fold (Ly et al., 2018). Water and oil absorption capacity, emulsifying capacity, and emulsion stability are not affected by ultrasound, whereas increased gelation capacity and minor foaming capacity and stability were reported (Ly et al., 2018). On the other hand, ultrasound power affects significantly the total phenolic content, metal chelating activity, and antioxidant (radical scavenging) activity of rice bran proteins (Iscimen and Hayta, 2018).

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Functional properties of rice bran proteins depend on the rice cultivar and this is related to different proportions of the secondary structure of proteins between cultivars, that is, β-sheets, α-helix, or β-turns (Singh and Sogi, 2018). Another important factor to take into account is the high lipase activity of rice bran, which makes a stabilization treatment mandatory to prevent rancidity. Such a treatment must not be deleterious to functional properties or result in significant low protein yields. The advice is to monitor the effect of the stabilizing treatment on the properties of interest in order to take the best decision. For example, microwave and dry heating as stabilizing treatments cause a decrease in protein yield but a higher purity, whereas the oil absorption and the emulsifying capacity are improved, and water holding and foaming properties are slightly impaired (Lv et al., 2018). There are a lot of bioactive properties reported for rice bran proteins or their hydrolysates. Because of their high selectivity to polyphenols of high antioxidant capacity, such as the tea catechins, rice bran proteins have been proposed as a matrix to deliver catechins to gastrointestinal tract (Shi et al., 2017). Also, rice bran proteins are considered as nutraceuticals to improve the cholesterol metabolism (Wang et al., 2017a). On the other hand, hydrolysates of rice bran proteins have shown a series of activities, such as antimicrobial (Taniguchi et al., 2017), inhibition of angiotensin-I converting enzyme and glucosidase (Pooja et al., 2017; Wang et al., 2017b; Uraipong and Zhao, 2018), antioxidant activity (Moritani et al., 2017; Phongthai et al., 2018), reduction of arterial stiffening caused by metabolic syndrome (Senaphan et al., 2018), and attenuation of diabetic nephropathy (Boonloh et al., 2018). All of these reports suggest a great potential for rice bran proteins as supplements or functional foods. 4.4.1.2 Wheat Bran Proteins Wheat (Triticum aestivum L.) is the second most important cereal cultivated worldwide with 740 million tons produced in 2016 (FAOSTAT, 2016a). Wheat bran consists of a series of layers that surrounds the wheat grain and which are obtained as a flaked by-product during milling, whereas flour is the main product. Wheat bran has 12% 2 19% proteins. As bran represents 15% of the grain weight, and because it is mainly intended for animal feed, there are many million tons of proteins that are wasted or underutilized each year. The latter is important because wheat bran proteins are of better quality than those of flour due to their AAS and digestibility. For many years attempts have been made to take advantage of wheat bran proteins, however, to the best of our knowledge there are no extensive uses for them, probably due to drawbacks in extraction and low yields. Nevertheless, new approaches and modifications to classical extraction methods in order to make them more sustainable and improve protein yields are frequently proposed. Updated reviews on this subject have been performed (Balandran-Quintana et al., 2015; Balandra´n-Quintana, 2018; Balandra´n-Quintana and Mendoza-Wilson, 2018), so it will not be addressed in detail here. 4.4.1.3 Sorghum Proteins Sorghum is the fifth most important cereal in the world, after corn, wheat, rice, and barley (Awika and Rooney, 2004). World production of sorghum for the period 2017 2 18 reached 59.24 million metric tons, concentrated in the United States, Nigeria, Me´xico,

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India, Ethiopia, Sudan, China, Argentina, and Australia (USDA, 2018). Around 35% of the sorghum produced is intended for human consumption (porridges, sausages, pasta, cookies, tortillas, bread, snacks, alcoholic, and nonalcoholic beverages), the rest is used for animal feed and industrial applications, mainly bioethanol production (Awika and Rooney, 2004; Dicko et al., 2006; Tuinstra, 2008). On average, the whole grain of sorghum is constituted of 65% 2 80% of carbohydrates, 3.5% 2 18% proteins, 1.5% 2 6% lipids, 1% 2 4% ash, and 8% 2 12% moisture (Lasztity, 1996; Dicko et al., 2006; Badigannavar et al., 2016). Within the components of sorghum, the presence of fiber and tannins is highlighted (2% and 7% 2 8%, respectively), due to their impact on the digestibility of proteins and amino acid availability in the grain (Deshpande et al., 1986; Kulamarva et al., 2009). Sorghum proteins are classified in kafirins (prolamins), glutelins, albumins, and globulins. Kafirins make up 50% 82% of storage proteins located in the endosperm. Based on their molecular weight (MW) kafirins are divided into four groups: α, β, γ, and δ. α-Kafirins represent approximately 80% of total kafirins and include polypeptides with molecular weights of 23 and 25 kDa; β-kafirins (16, 18, and 20 kDa MW) range 8% 2 13%; γ-kafirins (20 2 28 kDa) content fluctuates between 9% and 21%; and δ-kafirins have a MW about 13 kDa and are present in very low amounts (Belton et al., 2006; Dicko et al., 2006; De Mesa-Stonestreet et al., 2010). In general, kafirins are distinguished by their high content of glutamic acid, proline, leucine, and alanine, but have reduced amounts of essential amino acids, such as lysine, threonine, tryptophan, and total sulfur amino acids (De MesaStonestreet et al., 2010). Glutelins are storage proteins located mainly in the endosperm, where they constitute around 16% 2 35%. Unlike kafirins, glutamic acid content in glutelins is low, but their contribution of the essential amino acid lysine is high (Taylor and Schu¨ssler, 1986; Holding, 2014). Albumins and globulins are storage proteins that abound in the germ with values of 32% 2 35%, MW of 75 2 100 kDa and 30 kDa, respectively, and are rich in essential amino acids, especially lysine (Haikerwal and Mathieson, 1971; Taylor and Schu¨ssler, 1986). Protein digestibility among sorghum varieties is highly variable; through in vitro and in vivo studies, values have been reported ranging from 30% to 70% (IAEA, 1977). Sorghum grain proteins have a significantly lower protein digestibility compared to other cereals (corn and wheat). The low digestibility of sorghum is attributed to the interaction of its proteins with tannins and fiber, which act as antinutritional factors (Badigannavar et al., 2016). Likewise, reduced solubility of kafirins and an increase in resistance of these to be digested by proteases such as pepsin has been observed when sorghum is cooked, which limits the use of sorghum grain as a primary source of protein (Anglani, 1998). In order to solve the problems of digestibility of sorghum proteins, high lysine sorghum genotypes have been developed, which have an enormous potential to be used as a food source of essential amino acids. These genotypes are distinguished from the main varieties of cultivated sorghum by having low values of kafirins (0.62 and 0.35 mg/g grain DW of α- and β-kafirins, respectively) and intermediate levels of glutelins (12.47 mg/g grain DW), albumins, (3.88 mg/g grain DW), and globulins (0.73 mg/g grain DW). Additionally, large variability in MW of proteins has been detected: α-kafirins 14 2 89 kDa, β-kafirins 16 2 56 kDa, glutelins 21 2 84 kDa, albumins 19 2 87 kDa, and globulins 15 2 64 kDa (Vendemiatti et al., 2008).

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Traditionally, differential solubility extraction methods have been used for the isolation of sorghum proteins. Following a sequential procedure, albumins and globulins are extracted with water and sodium chloride solution; kafirins with 60% tert-butanol and 2mercaptoethanol, and glutelins with alkali borate and sodium dodecyl sulfate, achieving a recovery of 26% 2 40% (Youssef, 1998; De Mesa-Stonestreet et al., 2010). However, other methods have been implemented with the purpose of improving extraction time, percentages of recovery, purity, digestibility, and functionality of the proteins, as well as the recovery of industrial by-products proteins. In this sense the addition of detergents and suitable reducing agents for food analysis have been proved to modify both the digestibility and extractability of sorghum proteins. The extraction of sorghum proteins with a 12.5 mM sodium borate buffer, pH 10.0, containing 2% of sodium dodecyl sulfate and 2% β-mercaptoethanol, using a 20:1 solvent-to-sample ratio (v/w) and three extraction intervals of 5 minutes, allows to isolate 84% of proteins reducing the time of extraction up to 80% (Park and Bean, 2003). On the other hand, treating decorticated sorghum flour with thermostable α-amylase employing extruder or batch mixer, allows to separate proteins and starch by a faster process that yields concentrates with higher protein contents (up to 80%) and more digestible proteins (De Mesa-Stonestreet et al., 2009). Sonication has also been used to make more efficient the extraction of sorghum proteins, separating them rapidly from the starch. Extracting protein from whole ground sorghum flour with 70% ethanol for 1 hour at 50 C followed by 4 minutes of sonication, increases the purity of proteins from 31% 2 52% up to 78% (Bean et al., 2006). The benefits of extraction by sonication have been remarkable and are achieved by modifying the secondary structure of kafirins, increasing their solubility, and improving their antioxidant and anti-inflammatory abilities (Cullen, 2017). The extraction of kafirins from sorghum distillers dried grains with solubles (by-product in bioethanol production), using acetic acid as solvent instead of traditional alcohols, yields 44.2% of kafirins with the highest purity (98.9%) (Wang et al., 2009). Sorghum proteins have low functionality because of their poor rheological properties with regard to cohesivity, pliability, extensibility, and rollability (Kulamarva et al., 2009; Espinosa-Ramı´rez et al., 2017). To solve the problems of sorghum protein functionality, several efforts have been performed, among these the search for efficient food-grade extraction methods (chemical, enzymatic) to isolate functional proteins, as well as the genetic manipulation to develop improved sorghum varieties. One of the chemical methods that have been developed to improve the functional properties of sorghum proteins consists of their conjugation with dextran or galactomannan. The sorghum proteins are extracted in an aqueous alkaline (pH 8) medium containing 2-mercaptoethanol, then are mixed with dextran or galactomannan at a ratio 1:5 and then freeze-dried. Both sorghum protein and conjugates increase their water solubility up to 90% 2 95% and their emulsifying capacity up to twice that of sorghum protein alone (Babiker and Kato, 1998). Regarding enzymatic methods, kafirins extracted from decorticated sorghum flour using 70% aqueous ethanol and sodium meta-bisulfite at 65 C, and subsequently treated with a protease, show higher protein purity (95%), improved protein digestibility (89%), better water holding (2.8 vs 1.9 g/g), and higher fat absorption capacities (2.4 vs 1.6 g/g) compared to extracts from ground decorticated sorghum (EspinosaRamı´rez et al., 2017). A flour obtained from a variety of genetically improved sorghum,

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which is characterized by having proteins with high digestibility and high lysine content, has shown significantly improved extensibility properties compared to normal sorghum varieties (Goodall et al., 2012). Although sorghum proteins have low digestibility and functionality, their potential as bioactive molecules is important. Kafirins have been shown to be useful in the inhibition and downregulation of expression of signal molecules generating inflammatory response (Holding, 2014). Such bioactive effect is enhanced when kafirins are extracted by means of sonication, since it alters the secondary structure of these proteins, increasing their solubility and ability to halt the production of proinflammatory cytokines through inhibition of reactive oxygen species in THP-1 human macrophages. 50 2 100 μg kafirin/mL are enough to induce these effects (Cullen, 2017). One hydrolysate rich in peptides of α-kafirinin, obtained with 40 μg chymotrypsin/mg α-kafirin, was able to inhibit in vitro the angiotensin-I converting enzyme, which suggests it may be useful in treating high blood pressure (Kamath et al., 2007). Peptides generated from kafirin in vivo hydrolysis in rats fed with diets supplemented with sorghum kafirin extract, induced a decrease in the levels of total cholesterol, and increased the serum antioxidant potential as well as the low density lipoprotein cholesterol levels, suggesting a role for kafirins to reduce the risk of cardiovascular diseases (Ortı´z-Cruz et al., 2015). Enzymatic deamidation, used to improve solubility, emulsification, stability, and foaming of cereal storage proteins, is a food-compatible method that has not been tested in sorghum proteins and which could be advantageous for the exploitation of these proteins. Likewise, in vitro and in vivo studies of the bioactive properties of sorghum proteins have been limited by their digestibility drawbacks. Both issues represent an area of opportunity to generate relevant research for future applications of sorghum proteins. 4.4.1.4 Barley Proteins Barley is the fourth largest cereal in the world (FAOSTAT, 2016a). On a dry basis, carbohydrates predominate (78% 83%) its chemical composition, among which starch (60% 65%), arabinoxylans (4.4% 7.8%), β-glucans (3.6% 6.1%), and cellulose (1.4% 5%) are found in the largest proportions. Proteins are second (8% 15%), while lipids (2% 3%), minerals (2% 3%), and others, including B vitamins, are minor components (Izydorczyk et al., 2014). About 75% of the cultivated barley is used as forage. Most of the remaining 25% is directed to produce alcoholic beverages, mainly beer, while a small proportion is processed to obtain β-glucans or consumed directly by humans. Protein content of barley grain is cultivar-dependent (Yu et al., 2017). These proteins have nutritional and functional properties that could compete with those of soybean (Houde et al., 2018). Barley flour proteins are extracted by different methods. For the classical alkaline extraction and isoelectric precipitation, different results are reported. For example, Alu’datt et al. (2012) obtained concentrates with 32.9% protein and 60.15% extraction yield, while Mohamed et al. (2007) reported concentrates with 90.5% protein content and 70% yield. On the other hand, Houde et al. (2018) reported yields of 51.4% with protein content of 68.9%. Isoelectric precipitation after alkaline extraction is beneficial, because although the yield is lower (57.1% vs 51.4%) the purity increases (33% vs 68.9%) due to selective isolation of the rest of the components. In order to increase yield and/or purity, other methods have been assayed. The use of amylase and amyloglucosidase to hydrolyze

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carbohydrates resulted in a protein recovery of 25.7% and, if a step of digestion with β-glucanase is included, this increases to 71.6% but the protein content decreases from 49% to 37%. If isoelectric precipitation is added to the tri-enzymatic treatment, the protein content and recovery increase to 41.4% and 78%, respectively (Houde et al., 2018). According to results, if purity is sacrificed the tri-enzymatic treatment could be a good alternative to alkaline extraction and isoelectric precipitation, since the recovery is higher. Economic factors of the process deserve a separate consideration. When barley is processed to produce beer, large amounts of brewers’ spent grain (BSG) are produced. BSG is a solid residue that remains from the must, having relatively highprotein content because this is concentrated as most of the starch is removed during the mash and used in fermentation (Negi and Naik, 2017). The protein content of the BSG is between 14.2% and 24.7% (Lynch et al., 2016), which are mostly storage proteins, that is, hordeins and globulins (Bi et al., 2018). BSG is generally used in animal feed (Ikram et al., 2017) or sent to landfills, so the proteins are wasted or underutilized and there is a growing interest in their recovery and use. Extraction of BSG proteins has been recently reviewed (Ikram et al., 2017; Balandra´nQuintana, 2018). Alkaline extraction and further isoelectric precipitation (Connolly et al., 2014) are usual methods, through which proteins can be recovered up to 85% (Vieira et al., 2014). Sequential extraction with salt and isopropanol, and further isoelectric precipitation, are reported to obtain the nonprolamin and prolamin fractions, respectively, of defatted BSG (50% wheat, 50% barley) (Negi and Naik, 2017). Combining solvent extraction with physical methods, e.g., pressing followed by sifting in the presence of water; sifting, with different combinations of solvents; wet milling, and ultrafiltration, are also reported. Concentrates with no more than 60% proteins have been obtained in most of these methods, except the alkaline extraction. Treatments prior to alkaline extraction and acid precipitation, e.g., steam explosion and proteolysis, can improve extraction yields. Proteolysis increases the solubility of proteins from 15% (untreated BSG) to almost 100%. Steam explosion reduces the enzymatic solubility of proteins, but allows a greater recovery during a subsequent centrifugation step. The drawback of these preextraction methods is the coextraction of lignin (Rommi et al., 2018). A nonconventional method is the extraction with deep eutectic solvents, for example using sodium acetate:urea in a 1:2 molar ratio, which permits to obtain concentrates with 50% protein and extraction yields up to 79%, due to dissolution of proteins which are insoluble by nature or that are denatured during the malting process (Wahlstrom et al., 2017). Regarding functional properties, alkali-extracted BSG proteins are poorly soluble in water, which affects other properties such as the capacity of emulsification and foam formation. Water solubility and therefore functional properties of BSG proteins can be improved by partial hydrolysis with proteases (Celus et al., 2007; Treimo et al., 2008; Yalc¸ın et al., 2008) and/or carbohydrases (Niemi et al., 2013) prior to extraction. Barley flour glutelins have higher oil absorption capacity and greater emulsion stability than hordeins, which in turn have a good foam capacity (Wang et al., 2010). In the nonprolamin fraction of wheat barley BSG, emulsion stability is 80% up to 110 minutes, whereas in the prolamin fraction it decreased, reaching 40% at 90 minutes. This behavior is due to a more balanced proportion of hydrophobic and hydrophilic amino acids in the nonprolamin fraction, according to authors (Negi and Naik, 2017). In this fraction, both the emulsion

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activity index and stability were higher at pH 9. The results suggest that the nonprolamin fraction of the wheat and barley BSG may have applications as emulsifier in the food industry (Negi and Naik, 2017). BSG proteins and some of their peptides produced by enzymatic hydrolysis can act as immunomodulators (McCarthy et al., 2013; Crowley et al., 2015) and possess antithrombotic activity (Cian et al., 2018). Hydrolysates of BSG (,10 kDa) released by different proteases also exert protective effects against toxicity by free radicals in Caco-2 and HepG2 cell lines; the effect depends on the protease used (Vieira et al., 2017). The presence of a peptide with dual inhibitory activity against angiotensin-I converting enzyme and DPP-IV has been found in BSG after enzymatic hydrolysis (Connolly et al., 2017). DPP-IV is an aminopeptidase that rapidly degrades the incretin hormones, which potentiate insulin secretion stimulated by glucose, so the inhibition of DPP-IV is one of the approaches used for the management of Type 2 diabetes (Connolly et al., 2017). On the other hand the angiotensin-I converting enzyme is key in the regulation of blood pressure and the target of inhibitory drugs that are prescribed to patients with hypertension (Daskaya-Dikmen et al., 2017). 4.4.1.5 Quinoa Proteins Quinoa (Chenopodium quinoa, Willd.) is not a true grain but a pseudocereal native to the Andes (Valencia-Chamorro, 2009). Its cultivation has also been introduced into Africa, Asia, Australia, Europe, and North America because of its great ability to adapt to different agro-ecological conditions (Valencia-Chamorro, 2016). Outside of South America the volumes of cultivated quinoa still do not seem to be very important. In 2016 148,720 tons of quinoa were produced in the world, distributed among only three countries: Peru, 53% of total; Bolivia, 44%; and Ecuador, 3% (FAOSTAT, 2016a). Quinoa is considered a complete food due to the quantity and quality of its proteins, as well as its content of vitamins, minerals, phytosterols, flavonoids, omega-6, and vitamin E (Abugoch, 2009; Vega-Galvez et al., 2010). Quinoa is recognized as “one of the grains of the 21st Century” (Vilcacundo and Herna´ndez-Ledesma, 2017) and 2013 was designated by FAO as “The International Year of Quinoa” (Valencia-Chamorro, 2016). There is extensive literature on the composition and nutritive, functional, and nutraceutical properties of quinoa (Lamothe et al., 2015; Tang et al., 2015; Navruz-Varli and Sanlier, 2016; Vilcacundo and Herna´ndez-Ledesma, 2017; Pellegrini et al., 2018). The outer layer or pericarp of quinoa grains contains between 2% and 5% of saponins (Medina-Meza et al., 2016; Woldemichael and Wink, 2001). Although saponins do not affect the nutritional quality of quinoa proteins (Ruales and Nair, 1992), they provide bitter flavors and are considered antinutrients that can damage the mucous membranes of the small intestine (Gee et al., 1993). Processing reduces both concentration and membranolytic activity of saponins (Gee et al., 1993) but it is recommended to remove them from grains before consumption (Medina-Meza et al., 2016). The latter can be achieved by water washing (Repo-Carrasco-Valencia and Serna, 2011). In addition, production of saponins in quinoa can be regulated by controlling the cultivation conditions, for example, saline stress and drought decrease the content of saponins in the grain by 50% and 45%, respectively (Go´mez-Caravaca et al., 2012).

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Protein content of quinoa is between 13.7 and 16.7 g/100 g of edible material, being higher than cereals such as rice, barley, corn, and rye (reviewed by Filho et al., 2017). The largest storage proteins are albumins and globulins, while prolamins are found in very low concentrations (Prakash and Pal, 1998; Abugoch et al., 2008; Vilcacundo and Herna´ndez-Ledesma, 2017). The latter is relevant since quinoa has potential as an ingredient in the manufacture of gluten-free products (Alvarez-Jubete et al., 2010; Jan et al., 2018; Kurek et al., 2018). Quinoa storage proteins are separated by SDS PAGE into four groups (Abugoch et al., 2008). The group with the highest molecular mass (  60 kDa) corresponds to type 11S storage proteins, named collectively as Chenopodina (Brinegar and Goundan, 1993). Chenopodina, in turn, consists of a basic and an acid subunit, with masses in the ranges 22 23 and 32 39 kDa, respectively (Brinegar and Goundan, 1993; Brinegar et al., 1996). Finally, peptides with molecular mass ,20 kDa are identified as albumins (Abugoch et al., 2008; Toapanta et al., 2016), among which a fraction of 8 9 kDa has been characterized as type 2S (Brinegar et al., 1996). The latter is interesting because of its high content of arginine and histidine, amino acids that are essential for children (Brinegar et al., 1996). Proteins of quinoa seeds have an AAS higher than cereals (Ruales and Nair, 1992). The content of lysine in the quinoa grains is relatively high since this amino acid is not very abundant in plant proteins (Table 4.1). The in vitro protein digestibility of raw quinoa fluctuates between 76% and 78%, being lower than that of proteins of animal origin but very similar to that of other plant proteins (Lo´pez et al., 2018). The protein efficiency ratio (PER) of raw quinoa seeds devoid of saponins is between 78% and 93% relative to casein, and increases to 102% 105% when the seeds are cooked. Alkaline extraction and subsequent acid precipitation is the best method for obtaining quinoa protein isolates; the higher the extraction pH, the greater the amount of recovered protein (Abugoch et al., 2008; Elsohaimy et al., 2015; Navarro-Lisboa et al., 2017). Isolates with 77% and 83% (w/w) of protein have been reported at extraction pH of 9 and 11, respectively (Abugoch et al., 2008). However, if the pH is too alkaline it could affect the water solubility of proteins due to denaturation and formation of agglomerates, since average solubility of 30% has been reported when the extraction is done at pH 11, versus 75% 98% when extracting at pH 9; in both cases the solubility has been measured in a pH range of 3 11 (Abugoch et al., 2008). On the other hand, the extraction pH in the alkaline range (9 11) does not significantly affect the content of essential amino acids or other functional properties, such as the water absorption capacity, which is around 4 mL H2O/g protein, a value very similar to that of soy protein isolates (4.3 mL H2O/g protein) (Abugoch et al., 2008; Elsohaimy et al., 2015). The oil absorption capacity of quinoa proteins (extraction at pH 10 and precipitation at pH 4.5) is 1.88 mL/g, close to 2.1 which is the value reported for soybeans. The foaming capacity is lower than the average value for egg white albumin, which is used as a reference (69.28% vs 178%). However, in terms of foam stability, reported values are very similar for both proteins (around 54%) (Elsohaimy et al., 2015). The average emulsification capacity index of quinoa proteins is 2.1 m2/g calculated within a range of protein concentration from 0.1% to 3%, at pH 10. At the same conditions, the stability of the emulsion is 38.4 minutes on average (Elsohaimy et al., 2015). Aluko and Monu (2003) reported emulsification capacity indexes of almost 50 m2/g for a protein concentration of 1% (w/v), pH 8, which is a huge

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difference since in both studies the same method was used. In the latter case, the stability of the emulsion was almost 100% after 30 minutes (Aluko and Monu, 2003). Although quinoa is a promising food, it is necessary to systematize its production, as well as to make protein extraction methods more efficient in order to make it a sustainable process (Scanlin and Lewis, 2017).

4.4.2 Proteins from By-Products of Plant Oil Refining Industry 4.4.2.1 Soybean Proteins Soybean (Glycine max L. Merr.) is an annual legume plant (Leguminosae or Fabaceae family) native to East Asia, widely grown in America, China, and India, and consumed worldwide as a cooking oil, animal feed or food ingredient (FAOSTAT, 2016a). Soybean seeds are rich in oil (15% 25%), protein (35% 45%), and carbohydrate (33%, insoluble and soluble) contents. Seed composition can be affected by genetic and environmental factors (Zarkadas et al., 2007). Salt-soluble globulin proteins glycinin (300 380 kDa, 11S), and β-conglycinin (150 200 kDa, 7S) are the major dominant storage proteins (65 85%) in soybean seed. Other minor proteins (2% 5% of the total seed proteins) include β-amylases, Kunitz trypsin inhibitor (20 25 kDa), Bowman Birk inhibitor (8 kDa), chymotrypsin inhibitor, urease, lipoxygenases, and soybean lectins (120 kDa, 7S) (Fukushima, 2011). Oleosins account for 8% 20% of seed proteins (Murphy, 2008). Methionine and cysteine are the most nutritional limiting amino acids of soybean proteins. However, soybean proteins have a PDCAAS of 90 99 indicating that they provide a good balance in amino acid composition for human consumption (Young, 1991; Fukushima, 2011). Defatted soybean flakes, a by-product remaining after oil extraction, is the major source of commercial soybean proteins. A less usual extraction process uses the whole seed to obtain a soy-base (a water extract of dehulled soybeans) from which soy milk, tofu, and soy fruit beverages are produced (Preece et al., 2017b). Before being processed into soybean meal, ground defatted soybean flakes receive a desolventizing-toasting process in order to remove residual hexane and to inactivate antinutritional factors like lectins and protease inhibitors. The resulting soybean meal is mainly used as a protein source in animal feed (Fukushima, 2011). Soy protein products for human consumption are obtained from soybean meal. These include soy flour (with a finer particle size than soybean meal, 56 59% protein); soy protein concentrates (65% 72% protein); soy protein isolates ($90% protein); and texturized soy proteins. To process soybean meal in food products it is necessary to minimize protein denaturation in order to obtain high water soluble proteins. This is achieved by replacing the desolventizing-toasting process for a superheated hexane vapor process. Commercial production of this process is discussed in detail by Liu (1997). Soy flour is obtained by grinding the soybean meal to make a powder whose particles pass through a 100-mesh screen. Soy protein concentrates are prepared by removing soluble carbohydrates from soy flour. This can be achieved by protein denaturation using aqueous ethanol (50% 80%) extraction; by attaining the protein isoelectric point (pH 4.5); or by a moist heat treatment. After separation by centrifugation, the proteins are dispersed in water and spray-dried to produce the soy concentrates (Fukushima, 2011).

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For soy protein isolates production, both soluble and insoluble carbohydrates are removed. In the commercial processes, proteins and soluble carbohydrates are first extracted using water or a mild alkali extraction (pH 7 10). The precipitate (mostly insoluble carbohydrates) is separated by centrifugation. Then, the protein is precipitated by isoelectric point, resuspended in water and spray dried (Preece et al., 2017b). Other methods developed to produce SPI include the conventional extraction followed by ultrafiltration and diafiltration; alkali extraction followed by ultrafiltration reverse osmosis and diafiltration. These membrane processes yield more soluble proteins because of the exclusion of the acid precipitation step (Liu, 1997). For research purposes, the protein extraction methodology depends mainly on the research objective. For example, glycated proteins were usefully extracted using a solution which contained phosphate buffer, sodium metabisulfite, ascorbic acid, and sodium chloride (Serra et al., 2016) using enzyme-assisted microwave followed by ultrasound- and cavitation-assisted extraction (Wang et al., 2014; Lu et al., 2016; Preece et al., 2017a). These and other extraction processes were discussed in detail by Luthria et al. (2018). Off-flavors and allergenic proteins are two main factors, which limit the expansion of soy protein consumption in western countries. Off-flavors are produced by lipoxygenases (beany, grassy flavors), isoflavones, and saponins (bitter, astringent, chalky flavors). Despite many studies, a process that removes or mask these components satisfactorily has not been developed (Fukushima, 2011). There are at least 18 soybean proteins considered as potential allergens (Gly m 1 to Gly m 8; Gly m BD 28 K and 30 K; lipoxygenase; soybean hemagglutinin, among others). However, data on a single potential soybean allergen protein is difficult to obtain because clinical reactivity analysis uses crude soybean extract or soybean formulas to evaluate allergenicity (Selb et al., 2017). Soybean allergy worldwide prevalence is 10-fold less common in adults (0.27% prevalence) than in children (Katz et al., 2014). It is not possible to remove the soybean allergens with the current methodology. However, some research showed that application of high-intensity ultrasound, microwave, high-pressure homogenization, and high hydrostatic pressure reduced SPI allergenicity by approximately 19%, 25%, 30%, and 47%, respectively (Li et al., 2016). In addition to their nutritional value, soybean proteins have an important role in food functionality. Because of their gelling/textural capabilities, fat and water absorption, and emulsifying properties they are recognized worldwide as versatile ingredients with great consumer acceptability. 4.4.2.2 Rapeseed/Canola (R/C) Proteins Rapeseed (Brassica napus L. spp oleifera) is the second largest oilseed crop in the world cultivated mainly in the colder temperate regions of Canada, China, Australia, Europe, and India (FAOSTAT, 2016b). A group of rapeseed varieties with special characteristics (,2% of erucic acid in oil and ,30 μM of glucosinolates in its solid content) was registered as canola by Western Canadian Oilseed Association in 1978 (Wanasundara et al., 2017). Cruciferin (salt-soluble globulin, 230 300 kDa) and napin (water soluble albumin, 12 14.5 kDa) are the two major proteins of R/C. They account for 65% and 25% of total R/C seed proteins, respectively (Perera et al., 2016). Napin may cause allergic symptoms in hypersensitive individuals (Palomares et al., 2002). Other minor proteins are the oil body proteins oleosins (B18 kDa) and caleosins (27 kDa) (Jolivet et al., 2009). R/C protein

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fractions can be separated by electrophoresis, anion exchange chromatography, membrane filtration, and ultrafiltration (Aider and Barbana, 2011). The amino acid composition of R/C proteins is highly influenced by the method of protein extraction (Wanasundara et al., 2017). For example, alkaline extraction produces protein with lower levels of lysine which might be due to the formation of lysinoalanine (Shahidi et al., 1992). In general, R/C proteins have high contents of arginine, glutamic acid, glutamine, and isoleucine and leucine, and low amounts of methionine and cysteine. Lysine is the first limiting amino acid followed by valine (Ivanova et al., 2016). R/C meal, a by-product remained after oil extraction, is the major source of R/C proteins (B50% of meal dwt). Other R/C meal compounds are carbohydrates (B20%) and some antinutritional factors, such as phenolics (tannins and sinapine), glucosinolates (3-butenyl glucosinolate, 4-pentenyl glucosinolate, 2-hydroxy-3- butenyl glucosinolate, and 2-hydroxy-4-pentenyl glucosinolates), and phytates (Wanasundara et al., 2017). These antinutritional factors limit the use of R/C proteins in food applications. Commercial R/C meal is mainly used as animal feed in aquaculture and livestock industries. However, concentrations of some of these compounds can be diminished by developing special rapeseed cultivars using genetic selection tools or through chemical modifications, membrane filtration, or microbial treatments (Aider and Barbana, 2011). Thus the development of innovative technologies could allow the use of R/C proteins for human consumption. The most common procedure to concentrate or isolate R/C proteins (obtained 30% of initial meal protein content) is based on aqueous extraction in the presence of alkali (pH, near neutrality) followed by acidic precipitation (Aider and Barbana, 2011). However, higher pH is needed to increase protein recuperation, allowing phenols and phytates extraction and delaying salt elimination (Perera et al., 2016). Normally, alkaline extraction yields low solubility proteins with dark colors because of the dramatic change of pH during extraction and also the formation of protein polyphenol interactions and/or polyphenol oxidation (Wanasundara et al., 2017). The addition of sodium bisulfite or sodium metaphosphate may produce less discolored protein isolates (Mahajan and Dua, 1994). Ghodsvali et al. (2005) implemented a three-step procedure for R/C protein isolation. After alkali extraction and acidic precipitation, an ultrafiltration (10 kDa) step was implemented, followed by diafiltration and drying. The obtained isolate was high in protein content (90%) and low in glucosinolates (2 μM) and phytates (1%). Diosday et al. (1984) obtained a protein isolate (89% protein content) free of glucosinolates through aqueous extraction followed by ultrafiltration. A solvent-free oil extraction by cell wall enzymatic degradation and/or cold-press followed by flash chromatography produced a 40% 50% protein-enriched meal (Wanasundara et al., 2017). Other alternative technologies such as enzyme-assisted wet fractionation and dry fractionation have been studied in order to save water and energy consumption. Fractionation during recovery optimizes biological, chemical, and technological functionality of R/C proteins. For example, the formation of a protein micelle mass (PMM) by protein salting-in followed by hydrophobic aggregation promotes the obtaining of a cruciferin-rich fraction and a napin-rich fraction (Burcon Nutrascience MB Corp 2008). Expanded bed absorption-ion exchange chromatography (EBA-IEC) was used by Pudel et al. (2015) for the scale-up isolation of napin (98%) and cruciferin (95%).

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Functional properties of R/C proteins largely depend on the protein composition, type of processing, and protein-associated components (Wanasundara et al., 2017). In general, isolates prepared from alkaline extraction possess unacceptable water-holding capacity and poor emulsification and poor foaming capacities (Aider and Barbana, 2011). Protein chemical modification, including acetylation, succinylation, glycation, and enzymatic hydrolysis have been conducted in order to improve these functional properties (Gruener and Ismond, 1997; Alashi et al., 2014; Pirestani et al., 2017). Potential uses of R/C protein peptides with bioactive activity have been reported. Bioactivity includes mainly antihypertensive (He et al., 2013a) and antioxidant (Alashi et al., 2014). Akbari and Wu (2015) reported the synthesis of chitosan cruciferin nanoparticles for carotene delivery while Hong et al. (2017) studied the role of amphipathic peptides in the obtaining of cruciferin nanoparticles by cold gelation. All these methodologies need the development of scale-up and competitive technologies in order to incorporate R/C protein products in human food (Wanasundara et al., 2017). 4.4.2.3 Cotton Proteins Cotton (Gossypium hirsutum L.) is the third largest oilseed crop in the world and also a source of relatively high-quality protein (Campbell et al., 2014). Globulins (salt soluble, vicilin, and legumin families) are the major dominant storage proteins in cottonseed and account for 60% 70% of seed proteins. Albumins (water soluble) and gliadins (alkali soluble) are in low concentrations (Bellaloui et al., 2015). The most abundant amino acid is arginine (15% 34% of total protein) while methionine and cysteine are the least abundant (1% 2%) (He et al., 2015). Cottonseed meal (CM), a by-product remained after oil extraction, is the major source of commercial cottonseed proteins. CM is of two types depending on oil extraction, solvent extraction (more common) and expeller extraction. Both meals contain 33% 41 % of protein and also a harmful terpenoid derived from (1)-δ-cardinine named gossypol which is cardio- and hepatotoxic for humans and other monogastric animals (Gadelha et al., 2014). Part of gossypol present in the CM binds to the proteins forming Schiff bases with lysine (Pelitire et al., 2014). Because of this, raw cottonseed proteins (CM) are mainly used as fertilizer and as a protein supplement for ruminant animals which possess a detoxifying system for gossypol (Campbell et al., 2014). Nonfood potential protein products include bioplastics and films (Yue et al., 2012) and bio-based wood adhesives (Cheng et al., 2016). In 2014 17 the global production of cottonseed was about 39 44 million metric tons/ year, which means between 8.3 and 9.4 million metric tons of available protein. Options to produce free gossypol cottonseed include the transfer of gossypol glandless mutant into commercial cultivars and the use of RNA interference (RNAi) technology. Sunilkumar et al. (2006) used tissue-specific RNAi mediate suppression to disrupt the synthesis of gossypol to develop transgenic seeds with 99% reduction of gossypol content. Richardson et al. (2016) used meal from these transgenic seeds as feed ingredient for shrimps without toxic effect. Pelitire et al. (2014) explored the gossypol extraction from cottonseed proteins using ethanol-based solutions in the presence of phosphoric acid to promote the hydrolysis of protein-bound gossypol. However, the toxic compound reduction was only 5% 10%.

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Cottonseed protein isolates can be prepared from defatted CM by alkaline protein solubilization followed by isoelectric precipitation (Zhang et al., 2009). Optimized extraction conditions (stirring, pH, temperature, solvent:defatted meal ratio) resulted in 40% 70% protein isolation (Zhang et al., 2009; He et al., 2013b). Gerasimidis et al. (2007) prepared an edible cottonseed protein concentrate (72.2%) using a two-solvent extraction method utilizing acetone in aqueous and anhydrous form. Gossypol reduction was approximately 65%, producing a concentrate with oil and water absorption higher than wheat flours and good foaming properties. Ma et al. (2018) prepared cottonseed protein isolates (free gossypol contain ,0.012%) using different methods of solvent extraction (hot, cold, and supercritical). Isolates obtained by cold solvent extraction and supercritical fluid extraction showed good functional properties (high emulsifying capacities, high water/oil adsorption, and surface hydrophobicity) allowing their use as food ingredients. Overall, proteins isolated from gossypol-free CM could be used as food additives. However, more studies are required. 4.4.2.4 Sunflower Proteins Sunflower (Helianthus annuus L.) meal, a by-product of the sunflower oil industry, comprises 30% 50% protein (Weisz et al., 2009). Albumin (17% 30%) and globulins (mostly helianthinin protein, 300 350 kDa) are the major dominant storage proteins, although other minor proteins, oleosins included, are also described (Gonza´lez-Pe´rez, 2015). Lysine is the first limiting amino acid of sunflower proteins. Sunflower meal is mostly used as low-value animal feed. This is mainly due to the presence of phenolic compounds (1% 4%), like 5-O-caffeoylquinic acid (the most abundant) and caffeic acid (Weisz et al., 2009). Conventional vegetable protein extraction under alkaline condition causes polyphenol oxidation and simultaneous interaction of this compounds with sunflower proteins (Gonza´lez-Pe´rez, 2015). These interactions reduce protein digestibility and solubility, impart astringency and bitter taste, and affect the sensorial properties (mainly color) of the protein extract (Weisz et al., 2009). A novel pilot plant process using salt-assisted protein extraction under mild acidic conditions, combined with adsorption and ion exchange resin columns, was developed by Weisz et al. (2013) and Pickardt et al. (2015). This process prevents the protein polyphenol interaction, allows the removal and recovery of polyphenols, and leads to the production of light-colored sunflower protein isolates. Dephenolized protein isolates showed higher solubility, foaming properties, dispersibility, and emulsifying activity than protein isolates with higher phenolic content (Malik and Saini, 2017). Bioactivities associated with polyphenols and the good quality of sunflower protein could lead to the economic viability of sunflower meal utilization for human food consumption. 4.4.2.5 Palm Kernel Meal Palm kernel meal (PKM) is the by-product generated by oil extraction from the African oil palm (Elaeis guineensis Jacq.) (Alimon, 2004). Palm oil is extensively produced also in Central and South America as well as in East Asia (Sharmila et al., 2014). Palm oil and palm kernel oil are the two main marketable products obtained from African oil palm, representing 22% and 4% 6% of the fresh-fruit bunch weight, respectively (Boateng et al., 2008). Several by-products (fronds, trunks, press fiber, empty fruit bunches, kernel cake,

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kernel shells, and mill effluent) are generated from oil extraction and used for livestock feeding (Marini et al., 2005). PKM world production exceeded 9.5 million metric tons in 2017, with Indonesia and Malaysia producing together just over four-fifths of that. After a first stage of oil extraction from palm, the kernel is obtained. Crushed palm kernels are extracted in the second stage from which the PKM results (Sharmila et al., 2014). Several strategies are used in palm kernel oil extraction that generate the high-protein PKM, such as mechanical extraction, solvent extraction, prepressing followed by protein extraction, as well as hydrothermal techniques. More recently, supercritical carbon dioxide extraction has been shown to be an effective strategy to produce low-priced de-oiled PKM as a suitable source of protein and fiber for human and animal consumption (Saw et al., 2012; Boateng et al., 2013; Sharmila et al., 2014; Hossain et al., 2016). Chemical and therefore nutrient composition of PKM depend on a number of factors, such as the palm sources, efficiency of oil extraction from the kernel, the residual endocarp, and the oil extraction method (Onuh et al., 2010). Protein content of PKM ranges 14.4% 20%. It also contains a high amount of carbohydrates (50.3%) and crude fiber (16.7%). Nutritive values of PKM can be enhanced by biological processes such as solid-state fermentation with cellulolytic and hemicellulolytic bacterial cultures in order to increase the protein value and the availability of nutrients (Marini et al., 2005; Alshelmani et al., 2014). A protein concentrate can be obtained by extraction of ground PKM with alkali (1 N NaOH) followed by precipitation with HCl (pH 3.5), with a protein yield of around 55% 60% (Manaf, 2008). Although the isoelectric method and modifications to it are widely used, there is no universal or standard method for isolating proteins from PKM. Protein extraction can also be performed with alkaline (30 60 mM NaOH, 35 45 C) or saline solution (200 400 mM NaCl, pH 7 9) and drying at 50 C (Arifin et al., 2009). An alternative method used for PKM protein isolation is the extraction with sodium hexametaphosphate and further isoelectric precipitation of soluble protein at pH 3.7 (Chee and Ayob, 2013). Because of the low cost and high availability of PKM, it is used to partially substitute feed ingredients such as soybean and maize in livestock feeds, poultry broiler, swine, and freshwater fish (Zahari and Alimon, 2005). However, PKM is also a potential source of valuable components for human nutrition and raw materials for several industries. Searches for bioactive peptides that allow the generation of functional products with high added value from PKM have been performed. Peptides of variable molecular weight and antioxidant activity have been obtained after hydrolysis of PKM with different proteases (trypsin, flavourzyme, chymotrypsin, bromelain, alcalase, pepsin, and papain). It is noteworthy that, the use of papain as hydrolytic enzyme produced the hydrolysate with the highest antioxidant activity (Saw et al., 2012). Remarkably, PKM protein obtained by hexametaphosphate-assisted extraction shows better solubility at pH 7, as well as higher oil binding capacity and emulsion activity, but lower emulsion stability, foaming capacity, foaming stability, and water binding capacity, when compared to soybean protein isolate. Nevertheless, PKM protein obtained by hexametaphosphate-assisted extraction also displayed an essential amino acid profile limited in tryptophan. Consequently, it can be used as a complementary source by supplementing with a tryptophan-rich source (Chee and Ayob, 2013).

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The palm oil industry generates millions of tons of low cost PKM as by-product per year. PKM is widely used as a complement for animal feeding because of its high-protein content. However, despite being used as a source of bioactive antioxidant peptides, PKM isolated proteins have not being used for human consumption. There is a requirement for a more rigorous evaluation of the physicochemical properties of the protein extracted from PKM to successfully incorporate these proteins into food systems and products with high added value. 4.4.2.6 Peanut Meal The groundnut or peanut (Arachis hypogaea L.) is a legume native to South America (Devi et al., 2013). The peanut is the fifth most used vegetable source worldwide for the production of vegetable oil (Sibt-e-Abbas et al., 2015). Peanut by-products are rich in functional compounds like proteins, fiber, polyphenols, antioxidants, vitamins, and minerals, which can be included in many processed human foods, besides being used as a replacement for conventional components in livestock and fish diets (Arya et al., 2016; SantosDias et al., 2018). The residue generated after the extraction of peanut oil is called oil cake/meal, or peanut meal, which is obtained in the form of flakes or grits (Kain et al., 2009). This by-product is rich in crude protein (20% 45%) and contains about 6% of residual oil (Fapohunda, 2008; Kain and Chen, 2008). Peanut meal world production exceeded 7.3 million metric tons in 2017, with China and India together accounting for over 71%. It is used commonly for animal feeding; however, its incorporation at high levels results in poor growth because of the presence of antinutritional compounds (Ghosh and Mandal, 2015). It also represents an economical and underutilized by-product with essentially the same health and nutritional benefits as peanut, and possesses remarkable functional properties that are important for food processing and food product formulation (Kain and Chen, 2008). Peanut meal protein isolate is regularly obtained by alkali extraction at pH 8, followed by isoelectric precipitation at pH 4.5 (Jain et al., 2015). This protein isolate contains 86% protein. Due to their excellent functional properties, peanut meal protein isolates can be used in supplementation of several food products, also enhancing their nutritional value (Sibt-e-Abbas et al., 2015). Peanut meal is often processed to obtain defatted peanut flour, which is a cheap high-protein source with equal dietary attributes of peanut but reduced in fat. Defatted peanut meal flour contains 40% 60% protein, 20% 30% carbohydrate, 3.8% 7.5% crude fiber, and 4% 6% minerals (Devi et al., 2013). Protein from peanut defatted flour has been concentrated up to 72% by ultrafiltration after being treated with cellulase, and finally freeze dried (Jain et al., 2015). Peanut meal flours with high oil and water absorption are desirable for use in meats, sausages, breads, and cakes, whereas flours with high emulsifying and whipping capabilities are more suitable for salad dressing, soups, bologna, confectioneries, frozen desserts, and cakes (Kain and Chen, 2008). It is noteworthy that some of the functional properties of peanut meal flour can be modified by the processing and production methods. For example, peanut meal protein concentrate prepared using membrane technology showed superior functional and sensorial characteristics compared to acid-precipitated peanut protein isolate (Jain et al., 2015).

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4.4.2.7 Cashew Meal The cashew tree (Anacardium occidentale L.) is native to South and Central America. It is widely cultivated for its nuts and derived products throughout Australia, Asia, and Africa (Ogunwolu et al., 2010; Liu et al., 2018). Cashew nut production amounted to 4.28 million tons in 2015, with Nigeria, India, and Vietnam producing 53%. Cashew kernel is rich in lipids (40% 57%), carbohydrates (23% 25%), and high-quality proteins (20% 25%). Also, it is rich in monounsaturated (61%) and polyunsaturated (17%) fatty acids (Lima et al., 2012; Liu et al., 2018). During processing large amounts (around 30%) of cashew kernels are rejected because they are not suitable for sale. These full-fat and defatted rejected meals are rich in proteins and are used in animal feeding (Akande et al., 2015). Defatted flour is obtained by grinding cashew nuts and removing lipids with petroleum ether. Defatted flakes are air-dried to completely remove the solvent and finally, ground to obtain the defatted cashew flour (Liu et al., 2018). A protein isolate can be obtained by dispersing defatted flour in deionized water and adjusting pH to 9.0 before stirring for 2 hours. The suspension is centrifuged and the supernatant collected. Sediments are suspended again in water to repeat the extraction as described previously and supernatants are pooled and pH adjusted to pH 4.6. Precipitated proteins are recovered by centrifugation, suspended in deionized water, neutralized, dialyzed against cold water, and freezedried (Liu et al., 2018). Osborne fractions of cashew meal have also being obtained using a previously reported method (Deng et al., 2011). Results have shown that Osborne fractionation is practical to preliminarily purify cashew nut meal protein where the main fractions are globulins (51.4%), glutelins (23.2%), and albumins (22.9%), whereas prolamins represent a very small proportion (2.5%) (Liu et al., 2018). Functional properties of cashew protein isolate and concentrate as well as albumin, globulin, and glutelin fractions have been evaluated and the results were comparable to those of peanut, lupin, and soybean proteins currently used as functional ingredients in numerous food products (Ogunwolu et al., 2009; Liu et al., 2018). According to FAO/WHO recommendations, the content of essential amino acids lysine, isoleucine, and valine in albumin, globulin, and glutelin, as well as in cashew nut protein isolate can satisfy young children’s requirements (Liu et al., 2018). Increased global production of cashew makes necessary the exploitation of cashew meal as a cheap source of protein concentrates, whereas isolated fractions with interesting functional properties may be relevant for the food industry.

4.4.3 Other Sources of Plant Proteins Alongside the usual sources of plant oil (e.g., soybean, canola, cotton, sunflower, palm, and peanut), there are other oil seeds like sesame, pecan, coconut, and macadamia nut. Also, there are other less extensively exploited plant oil sources like neem seed (Indica azadirachta), hemp seed (Cannabis sativa), and several cucurbitaceous seeds (Cucurbita maxima, C. pepo, C. moshata). From the processing of all of them, by-products are obtained which represent alternative plant protein sources useful as food for livestock, fishes, poultry, and human, as well as being sources of bioactive peptides (Usman et al., 2005; Aguilar et al., 2011; Chambal et al., 2012; Ranganayaki et al., 2012; Van Ryssen et al., 2014; de Menezes Lovatto et al., 2015; Rezig et al., 2016;

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Ozuna and Leo´n-Galva´n, 2017; Hadnaðev et al., 2018; Marchetti et al., 2018). Studying molecular and functional properties of such protein concentrates/isolates will determine their suitability as functional food ingredients (Liu et al., 2018). Oils extracted from castor bean (Ricinus communis) seeds and Jatropha curcas are widely used for biodiesel production. Castor bean seed meal is rich in proteins but cannot be used for animal feed because of the presence of toxic components like ricin, ricinin, agglutinin, and allergen CB-1-A (Madeira et al., 2011). However, protein can be extracted and concentrated to be used in the production of biodegradable materials (Lacerda et al., 2014). Due to the presence of toxic and antinutritional compounds, J. curcas seed meal (60% 63% protein) and oil are not suitable for animal or human consumption. However, a nontoxic J. curcas genotype not containing phorbol esters, but containing insignificant amounts of curcin that are not enough to cause toxic effects has been reported, and their seeds are traditionally used as food in Me´xico (He et al., 2011; Perea-Domı´nguez et al., 2017; Leo´n-Villanueva et al., 2018). By-products of fruit industrialization are also of current interest, since as an outcome of fruit processing, the seeds, peel, rind, and unusable pulp are commonly discarded (Reis et al., 2012). As an example, the residues of jackfruit (Artocarpus heterophyllus) constitute 70% of the fruit weight, whereas seeds may represent 8% 15% and contain 18% 37% protein, depending on the fruit variety. Functional and nutritive properties of jackfruit seed protein isolate have been determined, showing a good balance of essential amino acids and good functional properties. Therefore, it can be a novel protein source suitable to be added to breads, cakes, beverages, ice cream, sausage, and meat products (Ulloa et al., 2017).

4.5 CONCLUDING REMARKS The growing interest in plant proteins as an alternative to animal proteins is due to their comparative low cost as well as the increase in consumers’ demands originating from health and environmental concerns, and vegetarianism trends. Numerous residues coming from the food industry, especially those from vegetable oil extraction are low-cost plant protein sources. Due to the low solubility in water, conventional methods of recovering plant protein include the alkaline extraction and isoelectric precipitation. The classical extraction presents some drawbacks, for example, low yields and using reagents or conditions that can damage biomolecules, in addition to the disposal or final handling of chemicals representing environmental risks. Thus, developing sustainable techniques for extracting plant proteins from conventional and unconventional sources is a current subject.

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Senaphan, K., Sangartit, W., Pakdeechote, P., Kukongviriyapan, V., Pannangpetch, P., Thawornchinsombut, S., et al., 2018. Rice bran protein hydrolysates reduce arterial stiffening, vascular remodeling and oxidative stress in rats fed a high-carbohydrate and high-fat diet. Eur. J. Nutr. 57 (1), 219 230. Serra, A., Gallart-Palau, X., See-Toh, R.S.-E., Hemu, X., Tam, J.P., Sze, S.K., 2016. Commercial processed soy-based food product contains glycated and glycoxidated lunasin proteoforms. Sci. Rep. 6, 26106. Shaheen, N., Islam, S., Munmuna, S., Mohiduzzaman, Md, Longvah, T., 2016. Amino acid profiles and digestible indispensable amino acid scores of proteins from the prioritized key foods in Bangladesh. Food Chem. 213, 83 89. Shahidi, F., Naczk, M., Hall, D., Synowiecki, J., 1992. Insensitivity of the amino acids of canola and rapeseed to methanol-ammonia extraction and commercial processing. Food Chem. 44 (4), 283 285. Sharmila, A., Alimon, A., Azhar, K., Noor, H., Samsudin, A., 2014. Improving nutritional values of palm kernel cake (PKC) as poultry feeds: a review. Malays. J. Anim. Sci. 17 (1), 1 18. Shepon, A., Eshel, G., Noor, E., Milo, R., 2016. Energy and protein feed-to-food conversion efficiencies in the US and potential food security gains from dietary changes. Environ. Res. Lett. 11 (10), 105002. Shi, M., Huang, L.Y., Nie, N., Ye, J.H., Zheng, X.Q., Lu, J.L., et al., 2017. Binding of tea catechins to rice bran protein isolate: interaction and protective effect during in vitro digestion. Food Res. Int. 93, 1 7. Sibt-e-Abbas, M., Butt, M., Sultan, M., Sharif, M., Ahmad, A., Batool, R., 2015. Nutritional and functional properties of protein isolates extracted from defatted peanut flour. Int. Food Res. J. 22 (4), 1533 1537. Singh, T.P., Sogi, D.S., 2018. Comparative study of structural and functional characterization of bran protein concentrates from superfine, fine and coarse rice cultivars. Int. J. Biol. Macromol. 111, 281 288. Soria-Herna´ndez, C., Serna-Saldı´var, S., Chuck-Herna´ndez, C., 2015. Physicochemical and functional properties of vegetable and cereal proteins as potential sources of novel food ingredients. Food Technol. Biotechnol. 53 (3), 269 277. Sung-Wook, H., Kyu-Man, C., Seong-Jun, C., 2015. Nutritional quality of rice bran protein in comparison to animal and vegetable protein. Food Chem. 172, 766 769. Sunilkumar, G., Campbell, L.M., Puckhaber, L., Stipanovic, R.D., Rathore, K.S., 2006. Engineering cottonseed for use in human nutrition by tissue-specific reduction of toxic gossypol. Proc. Natl Acad. Sci. U.S.A. 103 (48), 18054 18059. Tang, Y., Li, X., Zhang, B., Chen, P.X., Liu, R., Tsao, R., 2015. Characterisation of phenolics, betanins and antioxidant activities in seeds of three Chenopodium quinoa Willd. genotypes. Food Chem. 166, 380 388. Taniguchi, M., Kameda, M., Namae, T., Ochiai, A., Saitoh, E., Tanaka, T., 2017. Identification and characterization of multifunctional cationic peptides derived from peptic hydrolysates of rice bran protein. J. Funct. Foods 34, 287 296. Taylor, J.R.N., Schu¨ssler, L., 1986. The protein compositions of the different anatomical parts of sorghum grain. J. Cereal Sci. 4 (4), 361 369. Tessari, P., Lante, A., Mosca, G., 2016. Essential amino acids: master regulators of nutrition and environmental footprint? Sci. Rep. 6, 26074. Tilman, D., Clark, M., 2014. Global diets link environmental sustainability and human health. Nature 515, 518. Toapanta, A., Carpio, C., Vilcacundo, R., Carrillo, W., 2016. Analysis of protein isolate from quinoa (Chenopodium quinoa Willd.). Asian J.Pharm. Clin. Res. 9 (2), 332 334. Treimo, J., Aspmo, S.I., Eijsink, V.G.H., Horn, S.J., 2008. Enzymatic solubilization of proteins in brewer’s spent grain. J. Agric. Food Chem. 56 (13), 5359 5365. Tuinstra, M.R., 2008. Food-grade sorghum varieties and production considerations: a review. J. Plant Interact. 3 (1), 69 72. USDA, 2018. World Agricultural Production [Online]. United States Department of Agriculture. Foreign Agricultural Service. Circular Series WAP, 9 18 September 2018. ,https://apps.fas.usda.gov/psdonline/circulars/production.pdf. (accessed 10.10.18). Ulloa, J.A., Villalobos Barbosa, M.C., Resendiz Vazquez, J.A., Rosas Ulloa, P., Ramı´rez Ramı´rez, J.C., Silva Carrillo, Y., et al., 2017. Production, physico-chemical and functional characterization of a protein isolate from jackfruit (Artocarpus heterophyllus) seeds. CyTA J. Food 15 (4), 497 507. Uraipong, C., Zhao, J., 2018. In vitro digestion of rice bran proteins produces peptides with potent inhibitory effects on glucosidase and angiotensin I converting enzyme. J. Sci. Food Agric. 98 (2), 758 766. Usman, L., Ameen, O., Ibiyemi, S., Muhammad, N., 2005. The extraction of proteins from the neem seed (Indica azadirachta A. Juss). Afr. J. Biotechnol. 4 (10).

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C H A P T E R

5

Protein Isolates From Meat Processing By-Products Cristina Chuck-Herna´ndez1 and Ce´sar Ozuna2 1

Tecnologico de Monterrey, School of Engineering and Sciences, Monterrey, Mexico 2Food Department, Bioscience Graduate Program, Division of Life Sciences, Campus IrapuatoSalamanca, University of Guanajuato, Guanajuato, Mexico O U T L I N E

5.1 Meat Processing By-Products 5.1.1 Blood as a Source of Serum Albumin 5.1.2 Bone, Cartilage, and Skin as Sources of Collagen 5.1.3 Sources of Keratin

131 133 135 136

5.2 General Procedures in the Production of Protein-Based Ingredients From Animal By-Products 136 5.2.1 Gelatin Production 136 5.2.2 Feather Meal Production 137 5.2.3 Blood Transformation Processes 138 5.2.4 Protein Isolates, Hydrolysates, and Bioactive Peptides 140

5.3 Food and Biomedical Applications of Proteins Derived From Meat Processing By-Products 149 5.3.1 Proteins From Meat Processing By-Products 149 5.3.2 Proteins From Poultry Processing By-Products 151 5.3.3 Proteins From Fish Processing By-Products 154 5.4 Future Trends and Conclusions

155

Acknowledgments

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References

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5.1 MEAT PROCESSING BY-PRODUCTS Meat can be defined as the “edible postmortem component originating from live animals, particularly from domesticated cattle, pigs, sheep, goats, and poultry” (Kauffman, 2001; Tarte´, 2009). According to the Code of Federal Regulations (CFR), meat is “the part

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of the muscle of any cattle, sheep, pigs, or goats, be it skeletal or found in the tongue, diaphragm, heart, or esophagus, either with or without the accompanying and overlying fat, and parts of bone (in bone-in products such as T-bone or porterhouse steak), skin, sinew, nerve, and blood vessels, all of which normally accompany the muscle tissue and are not separated from it in the dressing process” (9 CFR 301.2). Therefore, all animal body parts and products derived from animals which are not primarily meant for human consumption can be considered meat processing by-products (Lasekan et al., 2013). Table 5.1 summarizes the main by-products derived from meat production, which may include blood, bone, skin, and other collagen-rich tissues. Current European legislation classifies meat processing by-products according to the level of risk they present in transmitting pathogens and toxic substances. Level 1 includes by-products aimed for incineration or burial, whereas level 2 includes meat-derived material for biogas and compost production. Finally, level 3 encompasses by-products obtained from healthy animals, used as feed or food ingredients, as well as in cosmetic products, and for other useful applications (Lasekan et al., 2013; Lynch et al., 2017). The proportion of by-products from meat-based processing can be higher than 50% of the live animal weight (Table 5.1), especially for cattle, pigs, and lambs (Jayathilakan et al., 2012). Animal by-products are rich sources of valuable components, such as proteins, peptides, enzymes, lipids, but they are a potential source of food. Their disposal can be very expensive, both from environmental and economic points of view. This is the main reason why a lot of work has been done using new processes to add value to meat processing byproducts aimed at human consumption. TABLE 5.1 By-Products of Meat and Poultry Industry by Weight By-Product

Pig (%)

Cattle (%)

Poultry (%)

Live weight

100

100

100

Whole carcass

77.5

63.0

Blood

3.0

2.5

Fatty tissue

3.0

4.0

3.2 3.7

Hide, skin, feathers

6.0

6.0

7.0 8.0

Organs

7.0

16.0

3.5 4.2a

Head

5.9

Viscera

10.0

16.0

8.5 9.0

Feet

2.0

2.0

3.5 4.0

Tail

0.1

0.1

Brain

0.1

0.1

2.5 3.0

a

In the case of poultry, organs are gizzard and proventriculus. Bah, C.S.F., Bekhit, A.E.D., Carne, A., Mcconnell, M.A., 2013. Slaughterhouse blood: an emerging source of bioactive compounds. Compr. Rev. Food Sci. Food Saf. 12 (3), 314 331. Available from: https://doi.org/10.1111/ 1541-4337.12013; Jayathilakan, K., Sultana, K., Radhakrishna, K., Bawa, A.S., 2012. Utilization of byproducts and waste materials from meat, poultry and fish processing industries: a review. J. Food Sci. Technol. 49 (3), 278 293. Available from: https://doi.org/10.1007/s13197-011-0290-7.

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Industrial uses

Consumption general

Human consumption

• • •

• •

• •

Fertilizers Energy production Chemicals

Livestock feed Pet food

•

Food ingredient Bioactive ingredients Enzymes

FIGURE 5.1 Main uses for meat and poultry processing by-products. Source: Figure self-developed based on data by Toldra´, F., Mora, L., Reig, M., 2016. New insights into meat by-product utilization. Meat Sci. 120, 54 59. https://doi. org/10.1016/j.meatsci.2016.04.021.

The use of meat by-products can be classified into industrial uses, general consumption, and human consumption (Fig. 5.1). Industrial uses include fertilizer production, energy generation, and preparation of chemicals. General consumption comprises uses in pet food and livestock feed. As for human consumption, meat processing by-products can be used directly as food or as a source of food ingredients (in this case also identified as meat processing co-products), but also as raw material for generation of enzymes, protein isolates, and functional food derivatives, among which bioactive and antimicrobial peptides stand out. A lot of research has been carried out on bioactive peptides (Hou et al., 2009; Lapen˜a et al., 2018; Martı´nez-Alvarez et al., 2015; Phanturat et al., 2010; Sarbon et al., 2018; Toldra´ et al., 2012; Villamil et al., 2017) and their production will be explored in more detail in the following sections. In order to understand and visualize the potential applications and uses of animal byproducts, it is important to review their main components (Table 5.2) and to learn about their characteristics. The most important solid components in meat by-products are proteins, namely serum albumin, collagen, and keratin, which are the main components of blood, bones, and feathers, respectively.

5.1.1 Blood as a Source of Serum Albumin Blood is an important edible animal by-product and it contains 75% 82% of moisture and around 18% of protein (Table 5.2) of which around 20% is serum albumin (3.6% 3.8% out of total blood weight), the main protein present in blood (Bah et al., 2013; Tarte´, 2009). Blood represents an important part of an animal’s body mass, taking up to 4% of the total live weight. The average mass percentage of blood that can be recovered from pigs, cattle, and poultry is about 3% (Table 5.1), which roughly translates to 3 kg in pigs and 15 kg in cattle (Bah et al., 2013; Jayathilakan et al., 2012; Tarte´, 2009). In several countries and regions, blood is used to produce sausages, such as Spanish blood sausages, or blood pudding and blood bread (Bah et al., 2013; Ghosh, 2001). In the United States, blood can be used as food ingredient when it has been removed by bleeding from an animal that has been inspected and has qualified for use in meat food products. However, it may not be used in uncooked products. Besides its application in food, blood is also used for non-food applications, such as fertilizers, feedstuffs, and binders. Blood from cow, sheep, pig, or chicken has also been reported as an ingredient in palatability enhancers composed of blood, reducing sugars, yeast, and fat, which are treated with enzymes (lipase and protease) and applied as an ingredient in animal foods, such as balanced dog food (Castro-Lugay et al., 1978, US4089978A). The main disadvantages of using blood as an ingredient are its dark color

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TABLE 5.2 Protein Percentages in the Main By-Products From Meat and Poultry Industry, Their Main Uses in Industry, and Processes Used for Isolation or Application

Product

Protein (%)

Main Type of Protein

Main Uses in Industry

Main Process or Technique for Isolation or Application

Blood cattle 17 18

Serum albumin

Blood meal, functional agents, thrombin, fibrinogen, protein hydrolysates and antimicrobial peptides

Blood meal

Blood porcine

18.5

Serum albumin

Blood meal, functional agents, thrombin, fibrinogen, protein hydrolysates, and antimicrobial peptides

Blood meal

Blood plasma

6 8

Serum albumin

Blood meal, functional agents, thrombin, fibrinogen, protein hydrolysates, and antimicrobial peptides

Blood meal

Connective tissue

85.0

Collagen

Gelatin type A and B, peptides

Collagen, gelatin, or hydrolysate

Feather and 85 99 feather meal

Keratin

Meal, hydrolysates, or peptides

Feather meal, keratin hydrolysate

Heads and feet (chicken)

16.0

Collagen

Gelatin

Protein isolates, hydrolysates

Bone (chicken)

23-24

Collagen

Gelatin

Collagen hydrolysate

Bone

-

Collagen

Gelatin type B, edible bone collagen (ossein), bone collagen hydrolysate (stocks and broths), edible bone phosphate, and edible fat

Collagen hydrolysate

Collagenrich tissues

-

Collagen

Concentrated collagen

Collagen hydrolysate

Table self-developed, based on published data from Lasekan, A., Abu Bakar, F., Hashim, D., 2013. Potential of chicken by-products as sources of useful biological resources. Waste Manag. 33 (3), 552 565. Available from: https://doi.org/10.1016/j.wasman.2012.08.001; Mullen, A.M., A´lvarez, C., Zeugolis, D.I., Henchion, M., O’Neill, E., Drummond, L., 2017. Alternative uses for co-products: harnessing the potential of valuable compounds from meat processing chains. Meat Sci. 132, 90 98. https://doi.org/10.1016/j.meatsci.2017.04.243; Tarte´, R. 2009. Meatderived protein ingredients. In: Tarte´, R. (Ed.), Ingredients in Meat Products: Properties, Functionality and Applications. Springer New York, New York, NY, pp. 145 171. https://dx.doi.org/10.1007/978-0-387-71327-4-7.

due to hemoglobin, as well as some adverse sensory properties. In order to overcome the color problem, the blood fraction devoid of blood cells (plasma) is of the greatest interest, also thanks to its good functional properties when used in food. Plasma comprises around 60% of the total blood weight (Jayathilakan et al., 2012). This represents 67.5%, 56.5%, and 72.0% of the total amount of blood in cattle, pigs, and sheep, respectively. The total protein content in plasma is around 6% 8% (albumin, globulins, and fibrinogen), with 3.6% 3.8% of total blood weight being albumin and 0.46% 0.65% of total blood weight being fibrinogen (Bah et al., 2013). When plasma is devoid of fibrinogen, the material is known as blood serum. Serum albumin is a globular protein with a molecular size of approximately 66 kDa, whereas fibrinogen, defined as a glycoprotein, is bigger (341 kDa) and it has two subunits with three polypeptide chains stabilized by 29 disulfide bonds (Bah et al., 2013). PROTEINS: SUSTAINABLE SOURCE, PROCESSING AND APPLICATIONS

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Blood proteins exhibit excellent nutritional quality because of the bioavailability of nutrients, but with deficiencies in a couple of essential amino acids, such as methionine and isoleucine, yielding a total percentage of Essential Amino Acids (EAA%) of 60% and 58% for cattle and pig blood, respectively (Lynch et al., 2017; Mullen et al., 2017; Tarte´, 2009).

5.1.2 Bone, Cartilage, and Skin as Sources of Collagen Collagen, another important component from animal by-products (Table 5.2), is the main protein in bones, cartilages, and skins. Bones represent around 11%, 15%, and up to 16% in pork, beef, and lamb carcasses, respectively. These by-products have been used for centuries in making gelatins and soups. Bone marrow represents 4% 6% of the carcass weight and can also be used as food (Jayathilakan et al., 2012; Matak et al., 2015a,b). Most of the bones obtained from meat processing have adhered meat, cartilage, and connective tissues, a fact that makes them a good source of protein. Other by-products that are sources of collagen are skins and hides, typically used for valuable non-food products, such as leather shoes and other clothing items, leather bags, rawhide, sausage skins, and glue. Hides represent 5.1% 8.5% of the total live animal weight in cattle, 11.0% 11.7% in sheep, and 3.0% 8.0% in pigs (Benjakul et al., 2009; Jayathilakan et al., 2012). Collagen is not only the main protein in skins, hides, and bones, but also the most abundant protein in animal by-products in general. It can be described as a family of fibrous proteins found in all multicellular organisms, representing around 25% 30% of total body protein, 1% 2% of bovine skeletal muscle, and 4% 6% of high connective muscle tissue (Tarte´, 2009). Collagen structure depends on the animal tissue it constitutes. The main amino acids present in collagen are glycine (33%), proline (12%), hydroxyproline (11%), and alanine (11%). Being almost devoid of EAA%, such as tryptophan, its EAA% is below 12%, depicting a lower nutritional value compared to other types of protein (Mullen et al., 2017; Tarte´, 2009). The basic unit of collagen is tropocollagen with a molecular weight of 300 kDa made up of three polypeptide chains (α-1, α-2, and α-3) coiled around into a triple helix stabilized with hydrogen bonds. There are variations in the composition of the α chains, resulting in different collagen types: fibrous (I, III, and V) and non-fibrous (IV). While all of them are abundant in meat, type I predominates in bone, sinew, and skin (Tarte´, 2009). Four to eight collagen molecules in cross-section are stabilized and reinforced by covalent cross-links to constitute the basic unit of collagen fibrils. These cross-links are reducible and they are capable of linking only two collagen molecules together (divalent), but as the animal ages, the cross-links become more stable, non-reducible, and trivalent, yielding toughness in animal tissue (Gomez-Guillen et al., 2011; Tarte´, 2009). In recent decades, collagen has been explored as a source of functional peptides (Fu et al., 2015; Gomez-Guillen et al., 2011; Herregods et al., 2011; Mora et al., 2014; Saiga et al., 2008; Toldra´ et al., 2012, 2016), with its outstanding antihypertensive capacity being reported when compared to hydrolysates or peptides from other sources, such as keratin. This difference is due to the fact that it is rich in proline, an amino acid reported as key in antihypertensive roles (Gime´nez et al., 2009; Gomez-Guillen et al., 2011; Lasekan et al., 2013). The soluble and denatured product obtained through a process of extraction from collagen is called gelatin. The first raw material used as a gelatin source, back in the 1930s, was PROTEINS: SUSTAINABLE SOURCE, PROCESSING AND APPLICATIONS

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pig skin. However, a rise in the use of fish and poultry by-products in recent years has made these alternative sources of collagen and gelatin (Gomez-Guillen et al., 2011). Nowadays, the most common sources of gelatin production are pig skin (46%), bovine hide (29.4%), and pork and cattle bones (23.1%), with fish gelatin accounting for less than 1.5% of total gelatin production (Gomez-Guillen et al., 2011).

5.1.3 Sources of Keratin Keratin is the main protein found in hairs, nails, feathers, and outer layers of skin. It is rich in glycine, serine, and proline, but deficient in some other amino acids, such as histidine, methionine, and lysine. Feathers represent about 7% 8% of live poultry weight and they are commonly used as bedding material, for decorative purpose, in sporting equipment, as manure or fertilizers, and for the production of feather meal (Jayathilakan et al., 2012). Among poultry processing by-products, feathers are the main source of keratin. They have high resistance to proteolysis and therefore they are commonly processed through thermochemical treatment to produce feather meal. Unlike collagen hydrolysates and peptides, keratin hydrolysates exhibit a higher antioxidant activity (Lasekan et al., 2013; Toldra´ et al., 2016).

5.2 GENERAL PROCEDURES IN THE PRODUCTION OF PROTEINBASED INGREDIENTS FROM ANIMAL BY-PRODUCTS Fig. 5.2 summarizes the main by-products from meat and poultry industry and the principal value-adding procedures that are used in their processing. One of the most important processes is rendering, a common way of transforming trimmings and other solid wastes into more valuable products, namely fat and protein meal. The rendering process applies high temperatures and pressures (around 133 C and 3 bar, respectively) for 20 30 minutes in order to separate fat and protein. There is also a low temperature (49 C) alternative to this process, producing better quality protein as one of the final products (Jayathilakan et al., 2012). The oily fraction can be used as a raw material for the manufacture of cooking oils, soaps, detergents, and cosmetics, whereas the protein fraction is dried and ground and used in livestock feed formulas (Shareefdeen et al., 2005).

5.2.1 Gelatin Production Following the rendering process, collagen can be transformed into gelatin, that is, a soluble and modified form of the native collagen. Because of the structural differences in collagen from different species and differences in its amino acid content, mainly because of hydroxyproline, the denaturation temperature as well as the gelatin production process can vary. This process includes a pretreatment with acid or base to produce type A or type B gelatin, respectively, with an isoelectric point (pI) of 8 9 and 4 5, respectively (Gomez-Guillen et al., 2011). The objective of this pre-treatment is to break down noncovalent bonds, disorganize the protein structure, and allow for collagen solubilization. After the chemical pre-treatment, a water-heating step is performed to cleave both PROTEINS: SUSTAINABLE SOURCE, PROCESSING AND APPLICATIONS

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5.2 GENERAL PROCEDURES IN THE PRODUCTION OF PROTEIN-BASED

Trimmings

Fat

Rendering

Protein residues

Bones (chicken or beef) Pretreatment (thermal or chemical)

Slaughterhouse

Blood

Fermentation (Bacillus pumilus A1)

Feathers

Thermochemical treatment

Protein meal

Collagen

Extraction heat treatment

Gelatin

Protein hydrolyzates

Hydrolysis (enzymes)

Enzymatic hydrolysis

Peptides

Mixing anticoagulant Meal Coagulation

Centrifugation Cell fraction

Water removal

Fluidized spray drying

Plasma Dry hemoglobin

Drying

Blood meal

Concentration (MF, evaporation)

Recovery of proteins Enzymatic hydrolysis Thrombin, fibrinogen, etc.

Spray drying Freezing

Peptides

Dry plasma Frozen plasma

FIGURE 5.2 Flow diagram with the main by-products from meat and poultry industry, as well as the principal processes to addition of value. Source: Figure self-developed with information from Bah, C.S.F., Bekhit, A.E.D., Carne, A., Mcconnell, M.A., 2013. Slaughterhouse blood: an emerging source of bioactive compounds. Compr. Rev. Food Sci. Food Saf. 12 (3), 314 331. https://doi.org/10.1111/1541-4337.12013; Gomez-Guillen, M.C., Gimenez, B., LopezCaballero, M.E., Montero, M.P., 2011. Functional and bioactive properties of collagen and gelatin from alternative sources: a review. Food Hydrocoll. 25 (8), 1813 1827. https://doi.org/10.1016/j.foodhyd.2011.02.007; Toldra´, F., Aristoy, M.C., Mora, L., Reig, M., 2012. Innovations in value-addition of edible meat by-products. Meat Sci. 92 (3), 290 296. https://doi.org/ 10.1016/j.meatsci.2012.04.004.

covalent and hydrogen bonds, destabilize the triple helix, and convert the molecules into soluble gelatin. The last step in gelatin production is the recovery of the final product which includes filtration, evaporation, drying, milling, and sifting (Gomez-Guillen et al., 2011; Tarte´, 2009; Toldra´ et al., 2012). As described in Fig. 5.2, gelatin can be also used to obtain bioactive peptides. More information about the different types of processes used in bioactive peptide production is provided in the following sections.

5.2.2 Feather Meal Production Another interesting process that adds value to an animal processing by-product is the thermochemical treatment of feathers and their conversion to feather meal. Keratin is highly resistant to proteolysis because of its extensive cross-linking with high amount of PROTEINS: SUSTAINABLE SOURCE, PROCESSING AND APPLICATIONS

138

5. PROTEIN ISOLATES FROM MEAT PROCESSING BY-PRODUCTS

disulfide and hydrogen bonds. Thus, in order to increase the availability of amino acids, a thermochemical treatment is required. This treatment involves the use of elevated temperatures and pressures in combination with an alkaline or acid solution. This treatment results in a feather meal with much higher digestibility (at least 75%) compared to the feathers (5%). Feather meal has a high protein content (around 90% of dry weight) but with an inferior protein quality compared to feathers, due to the destruction of thermo-labile amino acids (Lasekan et al., 2013; PW, 2017; Sangali and Brandelli, 2001; Wageningen, 2015; Wang and Parsons, 1997). After the thermochemical treatment, the feather product can contain up to 65% moisture. Therefore, a post-treatment usually includes filtration, drying, grinding, and sieving. The final product (feather meal) must contain about 8% of moisture to guarantee its shelf-life and a possibility to be used as an ingredient in animal feed or pet food.

5.2.3 Blood Transformation Processes Blood is another main by-product of animal processing for food. Protein concentration in blood and its components may depend on several factors, among them the particular animal species and the animal’s age, but on average, protein constitutes 17% 18% of blood, 6% 8% of plasma, 34% 38% of the cellular fraction, and 70% 95% of dried plasma (Tarte´, 2009). From the nutritional point of view, blood has an EAA of 58% 60%, being deficient in methionine and isoleucine (Bah et al., 2013; Kowalski et al., 2011; Mullen et al., 2017; Tarte´, 2009). Blood from slaughterhouses can be collected either by means of an open or a closed system, the latter of which makes use of a hollow knife, allowing for the blood to circulate from the animal towards the final storage at 0 C 3 C without exposure to air. Once the blood is stored at low temperature, it needs to be processed at least in the following three days to maintain its quality and safety. Blood meal can be produced directly from blood. The most important steps in blood meal production are coagulation, water removal, and drying. On some occasions, two additional steps are carried out, namely grinding and shifting (Fig. 5.2) (Kowalski et al., 2011). Blood can also be separated into cellular fraction and plasma. Plasma represents 60% 80% of the total blood weight, and it is built up from 90% to 92% moisture and only about 6% 8% protein. Plasma contains over 100 types of proteins, mainly albumin, α-, β-, and γ-globulin, as well as fibrinogen. In commercial terms, albumin is the most important protein in plasma (Kowalski et al., 2011; Tarte´, 2009). The cellular blood fraction yields 20% 40% of the total blood weight. It contains 60% 62% moisture, a high amount of red cells (erythrocytes), and a small amount of white cells and platelets. The separation of the two blood fractions is carried out using high-speed centrifugation, performed after mixing the blood with an anticoagulant, such as citric acid, sodium citrate, or EDTA (Fig. 5.2). Anticoagulants convert the calcium present in blood to its non-ionized form to inhibit its role in coagulation (Lynch et al., 2017). If sodium citrate is used as anticoagulant, it is added as a 20% solution at a ratio of 1:20 regarding the total volume of blood. Centrifugation is performed at temperatures below 3 C and the speed must be carefully assessed to avoid cellular lysis. The cellular fraction can have up to 38% of dry matter, so concentration or evaporation is not required, and it can be fed directly into a

PROTEINS: SUSTAINABLE SOURCE, PROCESSING AND APPLICATIONS

5.2 GENERAL PROCEDURES IN THE PRODUCTION OF PROTEIN-BASED

139

fluidized-spray dryer. The product obtained after dehydration is dried hemoglobin or dried red blood cells, a dark brown powder with a density of 0.6 0.65 kg/L, containing 90% of protein and less than 10% of moisture (Kowalski et al., 2011). Dried hemoglobin has limited applications in food products because of its dark color and off-flavors. Therefore, it is mainly used in meat products as a color enhancer (Ofori and Hsieh, 2011; Tarte´, 2009). In order to avoid the dark color in these products, some attempts have been made to remove the heme component from hemoglobin (Hsieh and Ofori, 2011; Tybor et al., 1975). The obtained product (devoid of the heme group) is known as decolorized blood, with high water-holding capacity, limited gel-forming ability, and good capacity to replace fat in meat products (Tarte´, 2009; Toldra´ et al., 2012; Viana et al., 2005). Following Fig. 5.2, the use of plasma in its different presentations is one of the processes that adds most value to blood as a meat processing by-product. Plasma is a liquid with a color that goes from yellow to light pink, and a density of 1.030 kg/L. It consists of about 91% moisture and most of the solids are proteins (Kowalski et al., 2011). The simplest process applied to plasma is freezing out the material high in moisture. If a higher shelf-life is required, a drying process can be carried out in order to produce spray-dried plasma (SDP), a product described as a powder of cream to light beige color, with a density of 0.60 0.65 kg/L, and 88% of water solubility. Protein content of 70% (or higher) stands out in the proximal composition of dried plasma (Kowalski et al., 2011). According to Kowalski et al. (2011), plasma can be dried using a BallTec Dryer (Marel Meat, Denmark). This dryer is characterized for low use temperature and its high thermal efficiency. The principle of this dryer is the use of balls in order to enlarge the contact surface during drying. These balls are wetted with the product to be dried and are circulated within a drying chamber. Most of the air runs counter flow (70% 80%), whereas the rest is in the same direction as the balls, allowing for the dry product to be removed from the surface. The equipment can dry up to 500 L of water per hour (Marel-Meat, 2018). The concentration is made in a vacuum evaporator and, depending on the process, a step of decolorization (with hydrogen peroxide, sodium hydroxide, and hydrochloric acid) can be carried out as well. To obtain 1 ton of powdered plasma, 9600 kg of liquid plasma or around 14,000 kg of fresh blood must be used (Kowalski et al., 2011). Plasma can be used as a food ingredient. Due to its functional properties, such as gelation, foaming, and emulsification, it can be used as a substitute for other food ingredients; for example, its use has been reported in cakes to replace egg whites (Lee et al., 1991; Myhara and Kruger, 1998; Ofori and Hsieh, 2011; Toldra´ et al., 2012). Plasma is a source of useful proteins with a wide array of possible applications in food, chemical, analytical, pharmaceutical, and microbial areas. Plasma proteins can be further fractioned. According to Lynch et al. (2017), the methods for fractioning can be classified as: (1) differential solubility; (2) chromatographic methods; and (3) membrane filtration. The former method relies on a precipitant that decreases the interaction between the protein and the solvent, whereas the latter two are based on the interaction of the protein with a solid medium. The type of protein fraction will depend on the process parameters, such as pH, ionic strength, protein concentration, and temperature. Some of the materials used in the differential solubility method are ethyl alcohol, polyethylene glycol (PEG), anionic polysaccharides, and ammonium sulfate. The use of solvents, such as ethyl alcohol, displaces water from the protein surface, leading to protein

PROTEINS: SUSTAINABLE SOURCE, PROCESSING AND APPLICATIONS

140

5. PROTEIN ISOLATES FROM MEAT PROCESSING BY-PRODUCTS

aggregation. Using this method, fractions that can be obtained from plasma include fibrinogen (with 8% ethanol and pH 7.2), γ-globulins (with 19% ethanol and pH 5.5), α- and β-globulins (with 40% ethanol and pH 5.5), and albumin (with 40% ethanol and pH 4.5). The precipitated proteins can be re-dissolved in phosphate buffer with pH value of 7.2 ´ lvarez et al., 2009; Lynch et al., 2017). Thus, four protein-rich fractions can be recovered (A using this method, and the supernatant rich in ethanol can be recovered by distillation in order to be reused (Lynch et al., 2017). In the case of fractionation with PEG, this synthetic polymer has the advantage over ethanol in forming less foam and being less expensive. The disadvantage is a higher effort that must be made in removing PEG from the supernatant, and the use of techniques such as ultrafiltration and gel permeation. With PEG (4 6 kDa), three fractions can be recovered from the process. Step 1 yields 91% of fibrinogen (pH 5.5, PEG 9.06% and NaCl 1.4 M), whereas step 2 produces 88% of precipitated immunoglobulins using the supernatant from step 1 (PEG 12.6% and NaCl 0.35 M, pH 8.22). Additionally, step 3 yields 92% of serum albumin from the supernatant obtained from the step 2 (Lee et al., 1987). Some proteins from beef blood plasma, such as fibrinogen and thrombin (E.C. 3.4.21.5), are used as binders in restructured meat products under the trade name Fibrimex (Bah et al., 2013; Toldra´ et al., 2012). The function of thrombin is to convert soluble fibrinogen into fibrin, promoting aggregation and yielding a final fibrin clot that increases the binding strength in meat pieces (Beltra´n-Lugo et al., 2005; Lennon et al., 2010; Mullen et al., 2017; Toldra´ et al., 2012; Yoon et al., 2006). The content of fibrinogen in blood is about 0.6% in both cattle and pig, and about 0.46% in sheep (Bah et al., 2013). Blood also can be used as a source of nutrients in growth media in microbiology. Fibrinogen, thrombin, and other components are isolated for chemical and medical uses. Bovine albumin has applications in animals recovering from fluid loss, as a stabilizer for vaccines, and in antibiotic sensitivity tests (Jayathilakan et al., 2012; Kurbanoglu and Kurbanoglu, 2004).

5.2.4 Protein Isolates, Hydrolysates, and Bioactive Peptides Besides the traditional treatment of animal by-products, there are other alternatives for its processing and use, such as production of protein hydrolysates and bioactive peptides. In order to obtain these materials, protein isolates or concentrates must be produced from the animal by-products using water, alkali, or acid, depending on the range of pH for protein solubilization. Soluble protein can be recovered from the clarified solution through precipitation, followed by a step of spray drying (Elizondo-Garza et al., 2017; Klompong et al., 2007; Neklyudov et al., 2000; Wu et al., 2003). This process is called isoelectric solubilization precipitation (ISP) and relies on the change of pH to dissolve protein and separate it from lipids, skins, bones, or other non-soluble fractions. The solubilization is followed by a separation, where pH is changed to the pI of the proteins, thus obtaining a precipitated protein known as isolate (90% of purity) or concentrate (purity of about 65% or more). This process has been successfully applied to sources such as fish, chicken, and beef by-products and krill (Matak et al., 2015a,b; Elizondo-Garza et al., 2017). Besides solubilization precipitation, a process based on chemical precipitation can be used, as well as ultrafiltration and extrusion (Galanakis, 2012). All these methods yield

PROTEINS: SUSTAINABLE SOURCE, PROCESSING AND APPLICATIONS

5.2 GENERAL PROCEDURES IN THE PRODUCTION OF PROTEIN-BASED

Raw material

Freeze dry protein hydrolyzate

Size reduction

Freeze drying

Homogenization

Protein hydrolyzate

Protein extraction

Centrifugation and filtration

Centrifugation

Sludge

Enzymatic reaction

Suspension of isolate in water/pH adjustment

Protein precipitation

Centrifugation

141

Protein isolate

Drying

Whey

FIGURE 5.3 Flow diagram of protein isolate and protein hydrolysate production from different meat and poultry processing by-products. Source: Modified from Lasekan, A., Abu Bakar, F., Hashim, D., 2013. Potential of chicken by-products as sources of useful biological resources. Waste Manag. 33 (3), 552 565. https://doi.org/10.1016/j. wasman.2012.08.001.

different final compounds because of the changes in structure and chemical properties of the final proteins. Treatments with a heating step, for example, can lead to the destruction of heat-sensitive peptides or amino acids, cross-linkage, or even protein denaturation. Changes in pH can lead to racemization, oxidation, or the destruction of amino acids. The concentrations of proteins on membranes can change their amino acid composition. All of these modifications can reduce protein functionality, cause losses in their nutritive value or bioactivity, and change the final applicability of the protein. Once the isolate or concentrate is produced (Fig. 5.3), the use of enzymes, chemicals, or simultaneous application of both, produces a mixture of peptides with different lengths and free amino acids. Partially-hydrolyzed protein has improved functional properties, such as solubility, fat absorption, foaming stability, and emulsifying capacity (Klompong et al., 2007;

PROTEINS: SUSTAINABLE SOURCE, PROCESSING AND APPLICATIONS

142

5. PROTEIN ISOLATES FROM MEAT PROCESSING BY-PRODUCTS

Ozuna and Leo´n-Galva´n, 2017). Some peptides obtained from chemical and/or enzymatic hydrolysis can also be described as bioactive, thanks to their antioxidant properties, cholesterollowering activity, enhancement of mineral absorption, opioid activity, and antimicrobial and antihypertensive properties, such as angiotensin-converting-enzyme (ACE) inhibitors (Bah et al., 2013; Gomez-Guillen et al., 2011; Herna´ndez-Ledesma et al., 2011; Lasekan et al., 2013; Ozuna and Leo´n-Galva´n, 2017; Toldra´ et al., 2016). According to Dziuba et al., (2012), function or bioactivity in peptides can be classified as related to: circulatory system (antihypertensive and antithrombotic activities); nervous system (specifically, peptides with activity similar to opioids); immune system (immunomodulating and antimicrobial activities); gastrointestinal system (regulation or inhibition of enzymatic activities and metal ionbinding peptides); and 5. functional properties (sensory peptides, surface-active peptides, and antioxidants). 1. 2. 3. 4.

Among the first discovered peptides were those with opioid activity (reduction of the perception of pain) from wheat gluten and from alpha casein (Zioudrou et al., 1979). In the early 1990s, the first protein-based antihypertensive sequences were found in milk (Nakamura et al., 1995) after fermentation with Lactobacillus helveticus and Saccharomyces cerevisiae as starters (derived from Calpis, a popular soft drink in Asia). The two peptides with ACE-inhibitory activity were Val-Pro-Pro and Ile-Pro-Pro (Nakamura et al., 1995). Peptides can be used in a variety of ways, for example, as food ingredients in patients that cannot digest protein, as flavors in foodstuffs, and even as a source of nitrogen in growth media in microbiology (Fallah et al., 2015; Lapen˜a et al., 2018; Sukkhown et al., 2018; Tarte´, 2009; Van Boekel, 2006). A functional component in food can be described as something with the ability to exert beneficial effects in the human body beyond basic nutrition (Gul et al., 2016; Kris-Etherton et al., 2004). Thus, bioactive peptides are short functional sequences of proteins, from two to twenty amino acids in length, with a physiological benefit beyond nutrition. Hydrolysates, on the other hand, are defined as sequences of more than 20 amino acids in length. The method for their production from protein isolates and concentrates is based either on commercially available digestive or microbial enzymes or on proteolytic microorganisms from fermentation processes, the most common procedure being based on digestive enzymes. However, a fermentative alternative with microorganisms such as L. helveticus is popular in the industrial environment. (Bah et al., 2013; Davies et al., 2005; Shimizu, 2012; Vercruysse et al., 2005). The production process of protein hydrolysates is described in Fig. 5.3. Moreover, Tables 5.3 5.5 include several examples of functional peptides, their activity, and the type of hydrolysis used in their preparation. Most protein hydrolysates and peptides are obtained by means of enzymatic hydrolysis, using commercial proteases, such as trypsin, pepsin, alcalase, flavourzyme, bromelin, and papain (Tables 5.3 5.5). Alcalase has been widely used when collagen and its derivatives are used as the source of antioxidant and antihypertensive biopeptides (short for bioactive peptides). These sequences are characterized by glycine-proline and hydroxyproline structures, also yielding antimicrobial activity, and mineral-binding and lipid-lowering activities (Gomez-Guillen et al., 2011). The enzymatic reaction described in Fig. 5.3 can be carried out in batch, batch-fed, or continuous reactors. Pepsin immobilized on an alumina support can be used for the enzymatic PROTEINS: SUSTAINABLE SOURCE, PROCESSING AND APPLICATIONS

TABLE 5.3 Hydrolysis Parameters of Meat Processing By-Products and Bioactive and Functional Properties of Their Hydrolysates Process Parameters

t (h)

8

T (oC) % 55

Molecular Weight of Hydrolysis Hydrolysates Degree (%) (kDa)

0.5 7

52.3

1.35 0.13

50 (U/g)

6

60

0.5 7

36.6

. 1.35

Pepsin

2/100 (w/w)

3.5

37

4

Sequential (pepsin 1 trypsin)

2/100 (w/w)

3.5

37

4

Not reported

Sequential (pepsin 1 alcalase)

2/100 (w/w)

Bovine hemoglobin

By-Product

Enzyme

E/S Ratio

pH

Bovine collagen

Alcalase

100 (U/g)

Papain Deer skin

0.5/100 (w/w) 8

References

ACE-inhibitory activity

Fu et al. (2015)

Not reported

DPP-IV inhibitory activity

Jin et al. (2015)

3 37

4

0.5/100 (w/w) 8

50

3

Pepsin

1/100 (w/v)

5.5

23

Not 1 reported

0.66 4.4

Adje et al. ACE-inhibitory and antimicrobial activity (2011a,b)

Bovine hemoglobin

Pepsin

1/100 (w/v)

4.5

23

Not 3 reported

0.37 3.5

Antimicrobial activity

Catiau et al. (2011)

Porcine hemoglobin

Pepsin

0.7 2.1/100 (w/w)

1.3 2.7

32 42

1

2.78 7.05

Not reported

Antioxidant activity

Sun et al. (2011)

Porcine hemoglobin

Pepsin

1/3 (w/w)

3

37

6

Not reported

3

ACE-inhibitory activity

Ren et al. (2011)

Porcine plasma

Pepsin

1/25 (w/w)

2

37

5

3 12

Antioxidant activity

Papain

1/20 (w/w)

8

37

16

Not reported

Xu et al. (2009)

Porcine plasma

Flavourzyme

1/50 (w/w)

7

50

8

Not reported

1.1

Calcium-binding

Lee and Song (2009)

Deer, sheep, and pig plasma

Papain

1/10 (w.b.)

6.5

55

1 24

. 20

Antioxidant activity

6.5

50

Not reported

Bah et al. (2015)

6.5

50

B3.5

6.5

50

B3.5

Bromelain a

FP400 FPII

a

3.5

Hydrolysates Properties

3 7

20 60

(Continued)

TABLE 5.3 (Continued) Process Parameters

t (h)

3.5

T (oC) % 23

Molecular Weight of Hydrolysis Hydrolysates Degree (%) (kDa)

0 24

18

0.653

Antimicrobial activity

Przybylski et al. (2016)

6.5

65

24

6

Not reported

Pepsin

2

37

Not reported

Antioxidant and anti-inflammatory activities

O’Sullivan et al. (2017)

Alcalase

9.5

60

28

6.5

65

B13

Not reported

Alcalase

9.5

60

B40

,2

DPP-IV and PEP inhibitory activity

Lafarga and Hayes (2017)

Collagenase

7.5

37

B8

Not reported

8

50

1.22 23.56

Not reported

Trypsin

8

37

1.24 26.82

Verma Antioxidant and antimicrobial activity et al. (2017)

Papain

6.5

50

1.18 19.12

By-Product

Enzyme

E/S Ratio

pH

Bovine cruor

Pepsin

1/11 (mole/ mole)

Bovine lung

Papain

1/100 (w/w)

Bovine lung

Porcine liver

Papain

Alcalase

1/100 (w/w)

1/100

Pork and beef byproducts (intestines, spleen, fat, liver, and tails)

Alcalase

Pork by-products (colon, heart, and neck) and beef byproducts (kidney, pancreas, and lung tissue)

Simultaneous 1/1000 (w/w) (Alcalase 1 Protamex)

Papain

0.5/100 (w/w) 8 and without pH control

0 60

Not 55 reported

24

0 6

Hydrolysates Properties

References

2

Not reported

6.5 0.24

Growth medium

Lapen˜a et al. (2018)

2

Not reported

20 0.1

Antioxidant activity

Damgaard et al. (2015)

Goat viscera (liver, lungs, and heart)

Beef bone

Alcalase

0.1 0.8/100 (w/w)

7

60

1 3

8.58 17.63

Brauzyn

0.1 0.8/100 (w/w)

6.5

70

1 3

4.61 14.42

Protamex

0 4/100 (w/ w)

6

40

2

B4

5

55

B4

Flavourzyme

6

50

B12

Simultaneous (P 1 F)

Not Not reported reported

Simultaneous (B 1 F)

Not Not reported reported

Sequential (P 1 F)

6

Bromelain

Sequential (B 1 F)

a

B15

40

2

50

2

5

55

2

6

50

2

FP400, A peptide peptidohydrolase derived from Aspergillus oryzae; FPII, protease preparation derived from A.oryzae. Table self-developed, based on published data.

Not reported

Solubility, oil retention capacity, emulsifying property, emulsion stability and antioxidant activity

de Queiroz et al. (2017)

10 30, 1 5 and ,1

Not reported

Chiang et al. (2018)

TABLE 5.4 Hydrolysis Parameters of Poultry Processing By-Products and Bioactive and Functional Properties of Their Hydrolysates Process Parameters

t (h)

Molecular Weight of Hydrolysis Hydrolysates Degree (%) (kDa)

8

28.31

By-Product

Enzyme

E/S Ratio

pH

Duck skin (gelatin)

Alcalase

0.2 g

8

T (oC) % 50

Collagenase

7

37

48.7

Flavourzyme

7

50

30.01

Neutrase

6

50

29.27

Papain

7

37

25.31

Pepsin

2

37

33.14

Protamex

6

40

22.79

Trypsin

7

37

29.98

α-Chymotrypsin

7

37

36.09

Hydrolysates Properties

Not reported

ACE-inhibitory and antioxidant activity

References Lee et al., (2012)

Chicken skin (gelatin)

Alcalase

1/20 (w/w)

8

50

3

Not reported

Not reported

Omar and Antioxidant Sarbon activity, (2016) emulsifying activity, waterholding capacity, oil-binding capacity, and foaming capacity

Chicken skin (gelatin)

Sequential use (alcalase, pronase E, collagenase)

1/50

8

50

6

, 10

1/33

8

50

6

Not reported

Antioxidant activity

Sarbon et al. (2018)

1/100

7.5

37

6

1 4/100 (w/v)

8

55

4

79.43

Sequential use 1 4/100 (w/v) (pepsin 1 pancreatin) 1 4/100 (w/v)

2

37

2

77.82

ACE and renin inhibitory activity

Onuh et al. (2013, 2015)

7.5

37

4

Chicken skin (elastin)

Elastase

8.5

37

2 24

8.5

60

ACE-inhibitory activity

Yusop et al. (2016)

Chicken blood meal

Alcalase

8

50

ACE-inhibitory activity

Huang and Liu (2010)

Chicken skin

Alcalase

Not reported

Alcalase 0.5 10/100 (w/w)

,5 ,2 , 1 to 5 10

47.25 65.08 # 3 47.20 66.45

2 or 5

B25 90

Prozyme-6

7

B85 90

Protease-N

8

B60 85

3 10

6

41.59 58.41 3.32 6.68

Not reported

,3

Antioxidant activity

Zheng et al. (2018)

0.5/100 (w/w)

8 and without pH control

0 60

2

Not reported

1.3 0.24

Growth medium

Lapen˜a et al. (2018)

Alcalase

1.5/100 (v/w)

Not 45 reported

2.5

26.12

Not reported

Antioxidant and antibacterial activity

Chakka et al. (2015)

Chicken foot

Protamex

0.4 (AU/g)

7

2

18.85

Not reported

ACE-inhibitory activity

MasCapdevila et al. (2018)

Chicken foot

Papain

1/100 (w/v)

Not 4, 30, and reported 56

20, 24 and 28

Not reported

25 150

Solubility, emulsification, foaming, water and oil-holding capacity

Dhakal et al. (2018)

Chicken leg collagen

Proteases (protease FP, protease A amano G, and Protease N)

0.1/100

7

50

4 or 24

Not reported

3

ACE-inhibitory and antihypertensive activity

Pepsin and trypsin/ chymotrypsin

1/100

7

37

1

Saiga et al. (2008), Shimizu et al. (2010)

Alcalase

1/50

8

50

12

B70

36 and ,20

Pepsin

1/50

3

37

B35

. 66 and ,14

ACE-inhibitory activity

Cheng et al. (2008)

Trypsin

1/50

8

37

B70

36 and ,20

Pepsin

1/100 (w/w)

2

37

1.5

13.5

, 3.4

Sequential use 1/100 (w/w) (pepsin 1 pancreatin) 1/100 (w/w)

2

37

1.5

18.6

, 1.7

Antihypertesive and antioxidant activity

Udenigwe et al. (2017)

7.5

37

1.5

Chicken blood corpuscle

Simultaneous (flavourzyme 1 papain)

1.6 3.34/100

Chicken byproducts (heart, liver, and digestive tract)

Alcalase

Chicken liver

Chicken bone

Spent hen meat

1/100

Papain

Table self-developed, based on published data.

50

TABLE 5.5 Hydrolysis Parameters of Fish Processing By-Products and Bioactive and Functional Properties of Their Hydrolysates Process Parameters

t (h)

7.5

T (oC) % 50

Molecular Weight of Hydrolysis Hydrolysates Degree (%) (kDa)

2.5

6.1

2.53

10

55

2.5

9.6

0.5/100 (w/w)

8 and without pH control

0 60

2

Alcalase

1.5/100 (v/w)

8.5

55

Trypsin

1.5/100 (w/w)

7

Silver carp by-products (fish meat leftover on bones, head, skin, and viscera)

Alcalase

3000 (U/g)

Protamex

Ark shell Fish (Epinephelus malabaricus) skin

Hydrolysates Properties

References

1.21

Antioxidant and antibacterial activity

Bougatef et al. (2018)

Not reported

1.3 0.24

Growth medium

Lapen˜a et al. (2018)

2

4.94

Not reported

Growth medium

37

4

4.6

Fallah et al. (2015)

8.5

60

Not 10, 20, and reported 30

. 150 and1.3

Liu et al. (2014)

2400 (U/g)

7

50

10, 20 and 30

. 150 and ,2.4

Solubility, emulsifying, and foaming properties

Pepsin

1/100 and 1/500

2

37

Not reported

1.5 0.5

Antioxidant activity Jin et al. (2018)

Pepsin

0.5 8/100

1.6 2.4

30 50 2 6

B10

2

Not reported

Papain

1.5 8/100

5 7

20 40

B18

Hema et al. (2017)

Protease from bovine pancreas

0.05 0.8/100

6 8

30 50

B25

7

50

Not reported

Emulsifying, foaming, and antioxidant properties

Sukkhown et al. (2018)

By-Product

Enzyme

E/S Ratio

pH

European eels (Anguilla anguilla) head and viscera

Protamex

3 (U/g)

Salmon viscera

Savinase Alcalase Papain

Silver carp by-products

Squid head

Flavourzyme 1/100 (w/w)

Table self-developed, based on published data.

1 4

0 3.5

23.02

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149

reaction. After their production, protein hydrolysates and peptides are fractioned and purified (Ticu et al., 2005; Toldra´ et al., 2012). At laboratory level, the most common techniques used for fractioning and isolation of peptides and protein hydrolysates are highperformance liquid chromatography in reverse phase (RP-HPLC) followed by ion-exchange and size-exclusion chromatography (Sato and Hashimoto, 2012). Anspach et al. (1999) reported the recovery of proteins and peptides directly from crude extracts (cultivation of microorganisms or preparation of disrupted cells) without the removal of suspended solids using expanded bed chromatography. On a pilot scale, ultrafiltration is applied to fractionate proteins and peptides. The separation principle is the different size of target molecules, but when aiming for peptides with similar molecular mass, other techniques must be used. Electrophoresis is used to fractionate peptides with different charges and isoelectric points besides different molecular size. The disadvantage is the difficulty to scale-up gelbased electrophoresis. Isoelectric focusing, on the other hand, does not require a gel matrix and can be used to separate small peptides, based on the fact that proteins are amphoteric molecules (Sato and Hashimoto, 2012). Traditionally, amphoteric polymers with different isoelectric points are used in this technique (under the commercial name of Ampholines). When an electric current is applied on the solution, molecules migrate to their isoelectric point until a stable pH gradient is formed and then used to separate the molecules. In the case of the autofocusing technique proposed by Sato and Hashimoto (2012), the amphoteric nature of peptides or protein hydrolysates was used by just applying a direct electric current without the use of either additional polymers or gel, a process with a scale-up potential and the advantage of reduced costs when compared to liquid chromatography. In recent years, several emerging technologies have been reported that could improve protein extraction and reduce the negative influence of traditional technologies on the functionality of protein concentrates and isolates. Emerging technologies that stand out are ultrasound-assisted extraction, pulsed electric fields, subcritical water hydrolysis, and high hydrostatic pressure, among others (Mullen et al., 2017; Ochoa-Rivas et al., 2015; Ochoa-Rivas et al., 2017; Ozuna et al., 2015). In meat processing by-products, high-power ultrasound has been used in the preparation of collagen from soft-shelled turtle calipash (Zou et al., 2017a), duck-liver protein isolate (Zou et al., 2017b), porcine cerebral hydrolysate (Zou et al., 2016), and cattle-tendon collagen (Ran and Wang, 2014). On the other hand, sub-critical water hydrolysis of hog ´ lvarez et al., 2016) has been hair (Esteban, Garcı´a et al., 2010) and porcine hemoglobin (A reported in the preparation of peptides and amino acids. Recently, pulsed electric fields have been applied to extract proteins from waste chicken meat (Ghosh et al., 2019). Finally, high-hydrostatic pressure processing has been used to produce hemoglobin hydrolysates from porcine blood (Toldra´ et al., 2012),

5.3 FOOD AND BIOMEDICAL APPLICATIONS OF PROTEINS DERIVED FROM MEAT PROCESSING BY-PRODUCTS 5.3.1 Proteins From Meat Processing By-Products Proteins from meat by-products represent about one-eighth of total protein content in lean meat (Toldra´ et al., 2016; Webster et al., 1982). These proteins can be used as food PROTEINS: SUSTAINABLE SOURCE, PROCESSING AND APPLICATIONS

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ingredients because of their water-holding capacity, viscosity, gelation capacity, emulsification and foaming capacity, solubility, and bioactivity (De Queiroz et al., 2017; Mullen et al., 2017). The applicability of proteins from by-products depends on several factors, with the most important being the intrinsic factors (amino acid profile, molecular structure, and surface hydrophobicity) and the extrinsic factors (pH, temperature, and ionic strength) (Mullen et al., 2017). Collagen transformed into gelatin is widely used in soups, gravies, and dairy products, but it can also be included in edible packaging or other novel casing techniques (GomezGuillen et al., 2011). As examples of biomedical applications, proteins such as collagen are ideal raw materials for tissue engineering and drug/gene delivery applications (Abbah et al., 2015; Mullen et al., 2017; Thomas et al., 2016). Regarding the bioactive properties of collagen, it has been reported that bovine collagen hydrolysates and their peptide fractions possess ACE-inhibitory activity (IC50 values ranging from 3.95 to 7.29 μg/mL), which is maintained after the exposure to simulated gastrointestinal digestion in vitro (Fu et al., 2015). The ACE-inhibitory activity implies the inactivation of angiotensin II, an enzyme that constricts blood vessels, increasing the blood pressure. The inhibition of this enzyme is part of the treatments for high blood pressure and heart and kidney diseases. The enzymes tested to obtain hydrolysates from bovine collagen were alcalase and papain, reaching a hydrolysis degree of 52.3% and 36.6%, respectively (Table 5.3). Jin et al. (2015) obtained dipeptidyl peptidase IV (DPP-IV) inhibitory peptides from deer skin hydrolysates (IC50 values ranging from 83.3 to 1638.3 μM), using pepsin as well as simultaneous and sequential hydrolysis with pepsin 1 trypsin and pepsin followed by alcalase, respectively (Table 5.3). DPP-IV has the capacity to inactivate gut-derived hormones [incretins such as glucose-dependent insulinotropic polypeptide (GIP) and glucagon-like peptide 1 (GLP-1)] that exert an important role in glycemic regulation. The inhibition of DPP-IV has been then explored as a management strategy for type 2 diabetes. Blood and blood plasma, as described above, is a versatile substrate that can be applied as a color enhancer in sausages and other food products. Blood plasma can be used as a food ingredient because of its good emulsifying, gelling, foaming, and solubility properties. Its uses as a binder in meat products and an egg substitute in baking have been widely reported (Hsieh and Ofori, 2011; Mullen et al., 2017). Proteins obtained from plasma, such as immunoglobulins, fibrinogen, and albumin, have also several applications as food/feed ingredients because of their good gelling and emulsifying properties. When the blood cellular fraction is hydrolyzed (see Table 5.3), antimicrobial peptides are generated that can be used on Gram-positive bacteria, fungi, molds, and yeasts (Fogac¸a et al., 1999; Przybylski et al., 2016). Moreover, ACE-inhibitory activity (Adje et al., 2011a,b) and antioxidant activity (Bah et al., 2013; Sun et al., 2011) have been reported in meat and blood peptides. An enzymatic globin peptide (VVYP) is obtained from bovine or pig red blood cells and it is commercialized in a soft drink (seishou sabou) from Morinaga and Co. This product is used to improve the dietary life of people with diets rich in fat (Hajfathalian et al., 2017). Bah et al. (2015) hydrolyzed plasma separated from deer, sheep, and pig blood using plant and fungal proteases (see conditions in Table 5.3). For all three animal plasmas, protein hydrolysates generated with fungal proteases exhibited higher antioxidant activity than those generated with plant proteases. This effect was attributed to the fact that fungal

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proteases were capable of generating smaller peptides (,3.5 kDa) in comparison to plant proteases (20-60 kDa). Similarly, porcine and bovine hemoglobin hydrolysates generated with pepsin have been proven to possess ACE-inhibitory, antioxidant, and antimicrobial activity (Adje et al., 2011a,b; Sun et al., 2011). Via hydrolysis with flavourzyme (a peptidase preparation from A. oryzae), porcine plasma has also been used to obtain peptides with calcium-binding capacity that are useful as a supplement to increase calcium absorption and bioavailability (Lee and Song, 2009). Cattle viscera are other by-products which represent a potential source of protein hydrolysates and peptides with functional and bioactive properties. De Queiroz et al. (2017) reported that goat viscera hydrolysates prepared by Alcalase and Brauzyn enzymes (see conditions in Table 5.3) are rich in glycine, glutamic acid, lysine, and aspartic acid. The amino acid profile of these hydrolysates was correlated with their antioxidant activity and functional properties, namely solubility, oil-retention capacity, emulsifying properties, and emulsion stability. In addition, it has been reported that hydrolysates with high glutamic acid content could be used as flavoring agents and flavor enhancers (Witono et al., 2016). Regarding the bioactive properties of cattle viscera, bovine lung hydrolysates have been shown to possess DPP-IV and prolyl endopeptidase inhibitory activity (Lafarga and Hayes, 2017), as well as antioxidant and anti-inflammatory activities (O’Sullivan et al., 2017). Verma et al. (2017) reported that porcine liver hydrolysates prepared with alcalase, trypsin, and papain exhibited DPPH (40.32%-57.49%), ABTS (86.79%-70.63%), and FRAP (12.65%-14.92%) radical scavenging activity. Finally, protein hydrolysates can also be used in the nutritional management of individuals who cannot digest whole or intact proteins (Table 5.6). Similarly, they can be used as a way of improving sources of minerals, such as calcium, iron, and magnesium; and vitamins, such as niacin and folate. Moreover, the bioavailability of iron can be enhanced by the use of heme iron from meat processing by-products.

5.3.2 Proteins From Poultry Processing By-Products Traditionally, collagen production has been associated with porcine and bovine byproducts. However, poultry processing by-products, such as skin and feet, are also an abundant source of this protein (Lee et al., 2012). Nowadays, many investigations have focused on studying functional (Dhakal et al., 2018; Wan Omar and Sarbon, 2016) and bioactive properties (Lee et al., 2012; Onuh et al., 2013, 2015; Sarbon et al., 2018; Yusop et al., 2016) of collagen/gelatin obtained from poultry processing by-products (Table 5.4). In the case of the functional properties of collagen obtained from poultry by-products, Wan Omar and Sarbon (2016) studied the effect of the drying method (freeze-drying and vacuum-drying) on chicken-skin gelatin prior to obtaining alcalase hydrolysates. The hydrolysates produced from freeze-dried gelatin showed good antioxidant activity and functional properties (emulsifying activity, and water-holding and oil-binding capacities at different pH). On the other hand, vacuum-dried hydrolysates exhibited better emulsifying stability, foaming capacity, and foaming stability. Dhakal et al. (2018) extracted collagen from chicken feet by papain hydrolysis and obtained collagen with a molecular weight distribution in the range of 25 150 kDa. The isolated collagen presented functional

PROTEINS: SUSTAINABLE SOURCE, PROCESSING AND APPLICATIONS

TABLE 5.6 Amino Acid Composition (%) of Protein Hydrolysates From Meat, Poultry, and Fish Processing By-Products Pork Beef Processing By-Products Heart Amino Acid

Lung

Kidney

Pancreas

Neck

Colon

Alcalase 1 Protamex

Pork Beef Processing ByProductsa Alcalase

Papain

Goat Viscera

Chicken Processing ByProductsb

Salmon Viscera

Alcalase

Alcalase

Alcalase

Brauzyn

Papain

Papain

ESSENTIAL AMINO ACID Histidine (His)

2.56

2.35

2.62

2.39

2.63

2.36

2.01

1.97

1.04

1.95

2.26

2.40

2.53

2.5

Threonine (Thr)

4.38

3.38

4.70

4.78

3.58

4.59

4.09

4.00

4.93

3.91

5.10

5.15

5.86

5.86

Valine (Val)

4.72

4.91

5.72

6.06

4.29

5.11

5.21

5.18

2.53

1.82

5.70

6.04

6.84

6.84

Methionine (Met)

2.04

1.63

1.84

1.72

1.54

2.20

1.89

1.84

3.13

2.00

2.23

1.24

2.87

2.84

Phenylalanine (Phe)

3.71

3.62

4.15

4.13

3.02

3.83

3.62

3.60

5.50

4.21

3.96

4.15

4.08

3.92

Isoleucine (Ile)

3.67

3.18

4.12

4.24

2.93

4.07

3.89

3.84

4.13

2.76

4.61

4.75

5.44

5.35

Leucine (Leu)

8.15

7.37

8.34

8.16

6.73

7.58

6.84

6.74

6.48

4.85

7.83

8.09

8.39

8.16

Lysine (Lys)

8.66

7.32

7.53

7.73

8.46

7.46

6.43

6.38

7.65

7.87

7.47

7.59

4.52

4.72

Tryptophan (Try)

0.86

0.77

1.1

1.43

0.63

0.98

0.75

0.73

0.81

0.98

1.12

1.17

2.69

3.04

1.52

1.62

1.28

1.41

1.27

1.31

9.75

9.73

9.00

9.02

NON-ESSENTIAL AMINO ACID Tyrosine (Tyr)

2.77

2.14

1.76

1.49

2.01

2.83

2.46

2.66

Cysteine (Cys)

0.95

0.99

1.48

1.67

0.70

1.36

1.06

1.13

Aspartic acid (Asp)

9.45

8.58

9.39

9.71

8.19

9.22

8.72

8.58

10.63

11.40

Glutamic acid (Glu)

16.69

14.63

14.56

13.32

15.62

14.77

13.70

13.50

23.88

22.55

Serine (Ser)

4.46

4.18

5.01

5.39

4.40

4.99

4.65

4.62

3.98

2.97

5.09

5.09

5.48

5.36

Glycine (Gly)

7.39

12.96

8.94

8.88

11.34

8.86

12.70

4.89

5.53

6.91

6.72

8.66

8.85

Arginine (Arg)

6.76

6.42

5.96

6.27

6.17

6.91

7.05

7.13

4.78

5.96

6.49

6.54

4.18

4.13

Proline (Pro)

5.45

7.90

6.03

5.86

10.02

6.39

7.95

8.06

Traces

Traces

5.71

5.62

6.18

6.15

Alanine (Al)

7.33

7.68

6.77

6.79

7.75

6.51

7.25

7.21

5.15

4.26

6.46

6.41

7.50

7.51

a

12.2

4.08

3.28

15.5

14.9

14.4

14.6

Intestines, spleen, fat, liver, and tails. Heart, liver, and digestive tract. Table self-developed, based on published data from Damgaard, T., Lametsch, R., Otte, J., 2015. Antioxidant capacity of hydrolyzed animal by-products and relation to amino acid composition and peptide size distribution. J. Food Sci. Technol. 52 (10), 6511 6519. https://doi.org/10.1007/s13197-015-1745-z; De Queiroz, A.L.M., Bezerra, T.K.A., de Freitas Pereira, S., da Silva, M.E.C., de Almeida Gadelha, C.A., Gadelha, T.S., et al., 2017. Functional protein hydrolysate from goat by-products: optimization and characterization studies. Food Biosci. 20, 19 27. https://doi.org/ 10.1016/j.fbio.2017.07.009; Lapen˜a, D., Vuoristo, K.S., Kosa, G., Horn, S.J., & Eijsink, V.G.H., 2018. Comparative assessment of enzymatic hydrolysis for valorization of different protein-rich industrial byproducts. J. Agric. Food Chem. 66 (37), 9738 9749. https://doi.org/10.1021/acs.jafc.8b02444. b

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153

properties such as solubility, emulsifying and foaming capacity, as well as water and oilholding capacity. In addition, these authors reported that biopolymeric fibers produced from chicken feet collagen showed such physical properties that made them possible to be used in drug delivery, enzyme immobilization, and other advanced biomedical applications. Mas-Capdevila et al. (2018) tested chicken foot hydrolysate (Hpp11) in spontaneously hypertensive rats (SHR) to obtain the best dose (mg/kg) below the traditionally used 100 mg/kg. They concluded that 55 mg/kg was the most effective dose to increase ACE-inhibitory activity. Regarding bioactive properties of gelatin, Lee et al. (2012) reported that pepsin hydrolysate obtained from duck-skin gelatin exhibited the strongest scavenging activities (DPPH, hydroxyl, and alkyl radical scavenging activity of 1.230, 0.554, and 1.193 mg/mL, respectively), in comparison to the other enzyme hydrolysates tested in their study (Table 5.4). In addition, pepsin hydrolysates protected DNA from hydroxyl radical-induced damage at 0.125, 0.25, 0.5, and 1.0 mg/mL, respectively. Sarbon et al. (2018) used three different enzymes sequentially (alcalase, pronase E, and collagenase) on chicken-skin gelatin and obtained peptides with 200 800 Da of molecular weight. These peptides, rich in hydroxyproline, glycine, alanine, proline, and lysine (11.57%, 33.49%, 7.78%, 15.07%, and 12.87%, respectively), showed antioxidant properties, such as reducing power, DPPH and superoxide anion radical scavenging, and ferrous chelating activities. Onuh et al. (2013) obtained enzymatic hydrolysates from chicken thigh and breast skin using alcalase or a mix of pepsin/pancreatin at enzyme concentrations of 1% 4%. They also obtained different molecular weight peptides using ultrafiltration (from 5 to 10 kDa to below 1 kDa). They found that alcalase hydrolysates had higher ACEinhibitory activity when compared to pepsin/pancreatin counterparts and this activity was inversely correlated to the molecular size of the peptides. Poultry viscera is another by-product which represents an interesting source of hydrolysates with bioactive and antimicrobial properties. Lapen˜a et al. (2018) compared chicken-viscera hydrolysates prepared by enzymatic hydrolysis (alcalase and papain) and by autolysis. The results revealed that enzymatic hydrolysis improved protein recovery, reaching maximum levels of 77% in comparison to 50% obtained by autolysis. However, both types of hydrolysates were rich in leucine, lysine, phenylalanine, alanine, arginine, and glutamic acid (Table 5.6). In addition, chicken-viscera hydrolysates represented an optimal medium for growth of Candida utilis yeast. In the same way, Chakka et al.(2015) compared the antioxidant and antibacterial properties of chicken-liver hydrolysates produced by fermentative hydrolysis [sugar 15% (w/w), inoculum 10% (w/v), NaCl 2% (w/w), agitation 120 rpm, 24 h, and 37 6 2 C] and enzymatic hydrolysis (see conditions in Table 5.4). As for antioxidant properties, both hydrolysates scavenged close to 90% of the radicals used for testing, except for ABTS radicals (32.16%). On the other hand, fermentative hydrolysates showed the highest antagonistic activity against Listeria monocytogenes (30 mm) and Bacillus cereus (28 mm), while enzymatic hydrolysates showed antibacterial activity only against Micrococcus luteus (12 mm). Poultry blood has been traditionally limited to its use as a nutritional ingredient in food and feedstuffs. However, several authors have reported its bioactive properties, including antihypertensive and antioxidant activities (Huang and Liu, 2010; Zheng et al., 2018). Zheng et al. (2018) found a novel antioxidative peptide derived from chicken blood with an amino-acid sequence of Ala-Glu-Asp-Lys-Lys-Leu-Ile-Gln (AEDKKLIQ, 943.5 Da).

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The obtained peptide showed similar reducing power to reduced glutathione (p..05), demonstrating its capacity to donate electrons or hydrogen. On the other hand, Huang and Liu (2010) reported that chicken-blood peptides with a molecular weight of 470 190 Da possess high ACE-inhibitory activity (inhibitory efficiency ratio of 1071.66 6 1.03% per mg/mL) in comparison to peptides with a higher molecular weight. Protein hydrolysates from animal by-products, prepared by means of chemical or enzymatic reactions, are also used as flavor ingredients because of the small peptides and free amino acids they may contain (Maehashi et al., 1999; Toldra´ et al., 2012, 2016). From chicken protein extracts, some specific dipeptides (Glu-Glu and Glu-Val) and tripeptides (Ala-Asp-Glu, Ala-Glu-Asp, Ser-Pro-Glu, and Asp-Glu-Glu) with umami taste can be produced using enzymatic hydrolysis (Maehashi et al., 1999).

5.3.3 Proteins From Fish Processing By-Products Nowadays, aquaculture and the entire fishery chain have shown important growth worldwide. This fact has been related to the generation of a large amount of waste that negatively impacts the environment (Villamil et al., 2017). In order to reduce these wastes, alternatives have been sought for their processing and implementation into balanced diets as food supplements, taking advantage of the valuable protein content these by-products may have (Klomklao and Benjakul, 2017). It has been reported that fish processing byproducts, such as skin (Hema et al., 2017), viscera (Lapen˜a et al., 2018), bones (Liu et al., 2014), and shells (Jin et al., 2018), could be an excellent source of functional and bioactive ingredients for the food and pharmaceutical industries (Rustad et al., 2011). Several investigations have reported the functional properties of protein hydrolysates obtained from fish by-products (Table 5.5). In this sense, silver carp by-products (fish meat leftover on bones, head, skin, and viscera) represent an excellent source of hydrolysates with good solubility, and emulsifying and foaming properties (Liu et al., 2014). Fallah et al. (2015) produced fish peptone from silver carp filleting by-products with enzymatic hydrolysis (alcalase and trypsin). They tested the efficiency of the hydrolysates as a nitrogen source in growth media for Staphylococcus aureus, obtaining the best results with peptone produced by alcalase when compared with TSB as the commercial medium for this bacterium, whereas trypsin peptone was not as good as the reference. Sukkhown et al. (2018) reported that hydrolysates from squid-head proteins could be used as functional ingredients due to their emulsifying, foaming, and antioxidant properties. In addition, squid-head proteins contain high amounts of glutamic acid and volatile compounds, such as trimethylamine and toluene, which are associated with the umami taste. Therefore, squid-head protein hydrolysates represent a promising flavoring agent or flavor enhancer for the food industry (Sukkhown et al., 2018). Fish viscera, one of the most important fish processing by-products, contain significant protein and lipid fractions as well as vitamins and minerals (Klomklao and Benjakul, 2017; Villamil et al., 2017). Lapen˜a et al. (2018) compared the effect of enzymatic hydrolysis (see conditions in Table 5.5) and autolysis of salmon-viscera protein. The results revealed that endogenous proteases possessed a similar capacity to generate hydrolysates without the help of externally added enzymes. The generated hydrolysates, rich in leucine, alanine,

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5.4 FUTURE TRENDS AND CONCLUSIONS

155

and glutamic acid (Table 5.6), proved to be an optimal medium for the growth of C. utilis yeast. On the other hand, Bougatef et al. (2018) reported that peptides obtained by enzymatic hydrolysis of viscera and heads of European eels (Anguilla anguilla) showed antioxidant and antibacterial activity against S. aureus, Enterococcus faecalis, Salmonella enterica, Klebsiella pneumoniae, and Escherichia coli. Thus, the incorporation of these peptides into minced meat that was then stored for 11 days in the cold, diminished lipid oxidation and inhibited microbial growth. On the market, there exists at least one bioactive product derived from fish (sardine) to control blood pressure and it is commercialized by a Japanese company (Shimizu, 2012). Without any doubt, fish processing by-products are a promising source of valuable protein isolates and hydrolysates.

5.4 FUTURE TRENDS AND CONCLUSIONS Meat processing by-products have been widely used both as a protein-rich food ingredient and a nutraceutical agent. Despite the amount of published research and the promising results that have been obtained in this field, many aspects of meat processing by-product use still need to be investigated. Technological advances have made it possible to extract different protein fractions from meat, poultry, and fish processing by-products. Moreover, enzymatic, chemical, and fermentative hydrolysis in vitro has been implemented to simulate the breakdown of these proteins in digestion. However, in order to evaluate the real effect these protein hydrolysates from meat processing by-products might have on living organisms, it is necessary to validate the findings by means of in vivo studies, both in laboratory animals as well as human subjects. Bioactive properties of protein hydrolysates from meat processing by-products that have been investigated include their antioxidant, antimicrobial, and ACE-inhibitory and antihypertensive activities. As for the functional properties of these protein hydrolysates, their solubility has been investigated thoroughly since all the other functional properties depend on it. However, it might be of great interest to explore the relationship between the distribution of peptide molecular weight in the protein hydrolysates from meat processing by-products and their bioactive and functional properties. The body of research reviewed in this chapter seems to suggest that a specific range of molecular weights might guarantee both bioactive and functional properties of protein hydrolysates from meat processing by-products. Therefore, future research should focus on purifying and characterizing the individual bioactive peptides within protein isolates and hydrolysates from meat, poultry, and fish processing by-products. Determining the bioavailability of these peptides, both in animals and humans, would have to follow in order for them to be successfully added to novel functional foods or pharmaceutical products. In order to fully benefit from meat processing by-products, finding cost-effective methods for protein isolation and hydrolysis would be necessary for the industrial implementation of such processes. Future research in this area should focus on applying hydrolysis pre-treatments to proteins obtained from meat, poultry, and fish processing by-products. Attention should be paid to the use of emerging technologies, such as high-power ultrasound, pulsed electric fields, subcritical water hydrolysis, and high hydrostatic pressure, with the aim of both reducing the costs and minimizing the impact they may have on the

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environment. Nevertheless, if emerging technologies were to be applied as pre-treatments to hydrolysis, it would be necessary to deepen our knowledge of the conformational changes they may provoke in meat protein isolates and hydrolysates, depending on the type of protein used. The specific amino acid composition and bioactive properties of protein isolates, hydrolysates, and peptides from meat, poultry, and fish processing by-products makes their use as functional food ingredients plausible. However, functional food are complex systems and studying protein isolates, hydrolysates, and peptides on their own is not enough in order to predict their behavior in such systems. Therefore, a lot of future research is needed in these areas as well.

Acknowledgments This product forms part of the activities carried out by a Mexican Research Network within CONACYT (294768) “RED 12.3, Para Reducir y Valorizar las Pe´rdidas y Desperdicio de Alimentos: Hacia Sistemas Alimentarios Sostenibles” and by the Center for Research and Protein Development (CIDPRO) of Tecnolo´gico de Monterrey. The authors would like to thank Stanislav Mulı´k, MA (Applied Linguistics), for his valuable contribution in writing the English version of this chapter.

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Villamil, O., Va´quiro, H., Solanilla, J.F., 2017. Fish viscera protein hydrolysates: production, potential applications and functional and bioactive properties. Food Chem. 224, 160 171. Available from: https://doi.org/10.1016/j. foodchem.2016.12.057. Wageningen, 2015. Processing of feathers to proteins—from fundamental insight to application. Retrieved from ,https://www.wur.nl/en/project/Processing-of-feathers-to-proteins-from-fundamental-insight-to-application.htm.. Wan Omar, W.H., Sarbon, N.M., 2016. Effect of drying method on functional properties and antioxidant activities of chicken skin gelatin hydrolysate. J. Food Sci. Technol. 53 (11), 3928 3938. Available from: https://doi.org/ 10.1007/s13197-016-2379-5. Wang, X., Parsons, C.M., 1997. Effect of processing systems on protein quality of feather meals and hog hair meals. Poult. Sci. 76 (3), 491 496. Webster, J.D., Ledward, D.A., Lawrie, R.A., 1982. Protein hydrolysates from meat industry by-products. Meat Sci. 7 (2), 147 157. Available from: https://doi.org/10.1016/0309-1740(82)90080-8. Witono, Y., Taruna, I., Windrati, W.S., Azkiyah, L., Sari, T.N., 2016. “Wader” (Rasbora jacobsoni) protein hydrolysates: production, biochemical, and functional properties. Agric. Agric. Sci. Procedia 9, 482 492. Available from: https://doi.org/10.1016/j.aaspro.2016.02.167. Wu, H.-C., Chen, H.-M., Shiau, C.-Y., 2003. Free amino acids and peptides as related to antioxidant properties in protein hydrolysates of mackerel (Scomber austriasicus). Food Res. Int. 36 (9 10), 949 957. Available from: https://doi.org/10.1016/S0963-9969(03)00104-2. Xu, X., Cao, R., He, L., Yang, N., 2009. Antioxidant activity of hydrolysates derived from porcine plasma. J. Sci. Food Agric. 89 (11), 1897 1903. Available from: https://doi.org/10.1002/jsfa.3670. Yoon, W.B., Kim, B.Y., Park, J.W., 2006. Rheological characteristics of fibrinogen-thrombin solution and its effects on surimi gels. J. Food Sci. 64 (2), 291 294. Available from: https://doi.org/10.1111/j.1365-2621.1999.tb15885.x. Yusop, S.M., Nadalian, M., Babji, A.S., Mustapha, W.A.W., Forghani, B., et al., 2016. Production of antihypertensive elastin peptides from waste poultry skin. Int. J. Food Eng. 2 (1), 21 25. Zheng, Z., Si, D., Ahmad, B., Li, Z., Zhang, R., 2018. A novel antioxidative peptide derived from chicken blood corpuscle hydrolysate. Food Res. Int. 106, 410 419. Available from: https://doi.org/10.1016/j. foodres.2017.12.078. Zioudrou, C., Streaty, R.A., Klee, W.A., 1979. Opioid peptides derived from food proteins. The exorphins. J. Biol. Chem. 254 (7), 2446 2449. Available from: http://www.jbc.org/content/254/7/2446.abstract. Zou, Y., Wang, W., Li, Q., Chen, Y., Zheng, D., et al., 2016. Physicochemical, functional properties and antioxidant activities of porcine cerebral hydrolysate peptides produced by ultrasound processing. Process Biochem. 51 (3), 431 443. Available from: https://doi.org/10.1016/j.procbio.2015.12.011. Zou, Y., Wang, L., Cai, P., Li, P., Zhang, M., Sun, Z., et al., 2017a. Effect of ultrasound assisted extraction on the physicochemical and functional properties of collagen from soft-shelled turtle calipash. Int. J. Biol. Macromol. 105, 1602 1610. Available from: https://doi.org/10.1016/j.ijbiomac.2017.03.011. Zou, Y., Wang, L., Li, P., Cai, P., Zhang, M., Sun, Z., et al., 2017b. Effects of ultrasound assisted extraction on the physiochemical, structural and functional characteristics of duck liver protein isolate. Process Biochem. 52, 174 182. Available from: https://doi.org/10.1016/j.procbio.2016.09.027.

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Proteins From Fish Processing By-Products Daniel Ananey-Obiri, Lovie G. Matthews and Reza Tahergorabi Food and Nutritional Sciences Program, North Carolina Agricultural and Technical State University, Greensboro, NC, United States O U T L I N E 6.1 Introduction 6.2 Recovery Methods of Fish Protein From Fish Processing By-Products 6.2.1 Enzymatic Hydrolysis 6.2.2 Chemical Hydrolysis 6.2.3 Fermentation Hydrolysis 6.2.4 Isoelectric Solubilization and Precipitation

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6.5.1 Nutritional Properties of Fish Protein Isolates 6.5.2 Functional and Physicochemical Properties 6.6 Comparative Differences Between Fish Protein Hydrolysates and Other Protein Hydrolysates 6.6.1 Plant Protein Hydrolysates 6.6.2 Other Food Protein Hydrolysates

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6.1 INTRODUCTION Proteins are macromolecules that can be obtained from both plant and animal sources and are generally regarded as sources of nutrients for body building and for growth. However, their functionalities and bioactivities have been unraveled over the past years and these have contributed to health promotion and the diverse food processing industries. The characteristic functionality and bioactivity of proteins have been correlated with biologically active peptides that are encrypted in the native sequence of the proteins (Gobbetti et al., 2007). These biologically active peptides, known as bioactive peptides, could be inactive if they remain in the encrypted sequence of their native proteins. However, their physiological roles can be released upon the breaking down of these sequences through enzymatic hydrolysis (Lahl and Braun, 1994), even though some of these properties are exerted directly (Korhonen et al., 1998). Hydrolysates are the resulting products of hydrolysis. Schaafsma (2009) defined protein hydrolysates as a blend of short- and long-chain peptides and amino acids that result from partial hydrolysis. Hydrolysates are considered the most important source of protein and bioactive peptides. Native proteins could have their functional, physicochemical, and sensory properties improved by hydrolysis, with no adverse effect on their nutritional value (Kristinsson and Rasco, 2000). It has become noticeable over the years that fish protein hydrolysates (FPHs) serve as the most important source of protein hydrolysates and bioactive peptides (Chalamaiah et al., 2012). Fish accounts for 15% of animal protein intake globally (Tahergorabi et al., 2012). With shrimp, krill, and fish fillets, there are low protein recoveries of 10%, 15%, and 30%
40%, respectively (Gehring et al., 2009). There is increasing demand and consumption of seafood and seafood products, resulting in an increase of fish processing byproducts or waste. The by-products include the viscera, carcass, head, skin, and bones. Fish processing byproducts in commercial settings constitute about 60%
70% of the live fish weight (Ananey-Obiri and Tahergorabi, 2018). The by-products, which are discarded, could be used for plant fertilizer, livestock feed, and value-added specialty foods such as fish oil, which could be isolated efficiently and used as a dietary supplement. Large quantities of by-products arising from fish processing pose serious disposal issues in both industrialized and developing countries. However, by-products are a significant source of protein, phospholipids, soluble vitamins, and bioactive compounds (Villamil et al., 2017). Fish peptides are known to have antioxidative, antihypertensive, and antithrombotic effects (Hamed et al., 2015). The health-related benefits that are associated with FPHs have been exploited, and they have been confirmed by many in vitro and in vivo studies. Bioactive peptides have been well recognized as having possible roles in reducing the risk of cardiovascular diseases (CVDs) (Erdmann et al., 2008). Also, they provide vitamins, minerals, and other growth factors, contributing to productivities and higher yields (Pasupuleki and Braun, 2010). Hence, there is an upsurge in demand for functional foods, dietary supplements, and pharmaceuticals containing peptides (Rizzello et al., 2016). Hydrolysates are recovered through hydrolysis and are considered a functional food. Numerous studies have focused on hydrolysates or peptides for their commercial value as

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added ingredients for food products. Fish muscle proteins are easily susceptible to denaturation. The choice of hydrolysis method employed and the processing conditions are critical factors in obtaining hydrolysates with the desired qualities. The quality and functionality of the hydrolysate is reliant on the type of fish and the handling and processing conditions of the recovery method (Kristinsson and Ingadottir, 2006). There have also been some physicochemical challenges associated generally with protein hydrolysates. The bitter taste of hydrolysates has been a major challenge in developing hydrolysate products. The degree of hydrolysis, which is defined as the number of peptide bonds that have been broken, divided by the total number of peptide bonds in the native protein and multiplied by 100% (Pasupuleki and Braun, 2010), has been associated with the bitter taste of hydrolysates. This in a way has hampered the commercialization and marketing of hydrolysates. However, according to Temussi (2012), the outweighing importance of the biological function of peptides barely makes bitterness a pertinent issue of discussion. This chapter provides an exposition on FPHs from fish processing by-products, with an emphasis on available recovery methods, and the structure, function, and bioactivity of FPHs and peptides. It also gives highlighted accounts of the challenges and future applications of FPHs.

6.2 RECOVERY METHODS OF FISH PROTEIN FROM FISH PROCESSING BY-PRODUCTS There are three known methods of protein recovery from fish processing by-products that have been used over the years. They all primarily aim either at breaking the peptide bonds to release peptide fragments and amino acids from the encrypted native proteins or use pH changes to isolate the protein. According to Li-Chan (2015), the employed approach to producing bioactive protein hydrolysates and peptides first involves correctly identifying the native protein, and then the subsequence freeing of the peptides using one of the three methods, namely, enzymatic, chemical, or fermentation processes. Desired product quality, cost, and time influence the choice of recovery method that is employed for fish protein. Hydrolysis is followed by a separation process using either centrifugation or microfiltration to remove the insolubles from the FPH that has been produced. This step might be repeated until a refined color of the hydrolysate is obtained. The filtered hydrolysate is pasteurized to kill microorganisms, dried, and packaged thereafter (Pasupuleki and Braun, 2010).

6.2.1 Enzymatic Hydrolysis Enzymatic, also known proteolytic, hydrolysis is the most common method of protein hydrolysate recovery from fish processing by-products. This process is carried out using endogenous enzymes or is an accelerated and controlled process using exogenously sourced enzymes. Enzymatic hydrolysis is usually a partial hydrolysis, and by this method the functionalities of native proteins are improved (Althouse et al., 2018). Prevalent proteolytic enzymes used include Alcalase, α-chymotrypsin, Neutrase, papain, pepsin, trypsin,

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pancreatin, Flavourzyme, bromelain, Pronase E, Protamex, Orientase, thermolysin, Validase, Protease A amano, Protease N amano, and cryotin F (Chalamaiah et al., 2012). Most of these commercial enzymes used as proteolytic agents are prepared from a bacterial origin, for example, Alcase, Neutrase, Protease N, and Protamex, with some reported successes with plant proteases like Papain Bromelain and Ficin (Aspmo et al., 2005). The selection of the appropriate enzyme in hydrolysis is a salient determinant of the production of hydrolysates and peptides with bioactive and functional properties. According to Pasupuleki and Braun (2010), the choice of enzyme and enzyme system that is employed in hydrolysis influences the degree of hydrolysis, peptide chain length, molecular weight distribution profiles, free and total amino acids. It was demonstrated by Jun et al. (2004) that protein hydrolysates prepared from the frame of yellowfin sole (Limanda aspera) using different proteases had different antioxidant activities. The processes involved in the enzymatic hydrolysis of fish processing byproducts is illustrated diagrammatically in Fig. 6.1. This process involves a high production cost due to the long reaction time. However, the resulting FPHs are more marketable and valuable (Shahidi et al., 1995). Functional properties such as water holding capacity, oil-holding capacity, emulsification, solubility, and sensory attributes among others can be improved using enzymatic hydrolysis with controlled reaction conditions (dos Santos et al., 2011). Processing conditions such as temperature and pH must be controlled for optimal activity of the enzyme and the hydrolysis time is critical in the production of protein hydrolysate or peptides with antioxidative activity (Samaranayaka and Li-Chan, 2011; Aspmo et al., 2005). Many recent research studies have established that protein hydrolysates or peptides produced from aquatic by-products using enzymes are good sources of antioxidants (Yarnpakdee et al., 2015; Yang et al., 2008; Jemil et al., 2014).

6.2.2 Chemical Hydrolysis The chemical method of recovery of hydrolysates from fish protein involves the breakdown of the proteins into peptides of different sizes, and amino acids using either acid or alkaline. Generally, this method of hydrolysate recovery from proteins is simple and relatively inexpensive. However, it is a process that is difficult to control, due to the unspecified peptide cleavage during the hydrolysis. In acid hydrolysis of fish protein, either hydrochloric acid or sulfuric acid is used to completely hydrolyze the proteins at high temperatures, and most times under high pressure (Kristinsson and Rasco, 2000). Afterwards, the product is then brought to pH 6.0
7.0, and it is further processed into the desired form (Kristinsson and Rasco, 2000). Hydrolysates recovered using this method have high solubility, but they are bitter and have reduced nutritional qualities and poor functionality (Chobert et al., 1996). Pasupuleki and Braun (2010) noted that the use of an acid in hydrolysis typically breaks down the proteins into individual amino acids and smaller peptides, but that some of the essential amino acids, such as cystine, cysteine, methionine, and tryptophan, are destroyed. Alkaline hydrolysis of fish protein involves rapid cleavage of the protein to produce water-soluble polypeptides, and subsequent breakdown at a slower rate. Kristinsson and Rasco (2000) demonstrated that hydrolysis done at a lower pH and higher pH temperature releases more

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FIGURE 6.1 A diagrammatic representation of enzymatic hydrolysis of fish processing by-products to produce fish protein hydrolysate.

protein and has a higher degree of hydrolysis. However, during alkaline hydrolysis hydrogen is removed from the alpha carbon of the amino acid causing the racemization of L-amino acids, which produces D-amino acids, and these are not absorbed by humans.

6.2.3 Fermentation Hydrolysis Fermentation biochemically uses microorganisms to break down fish proteins into peptides and amino acids. Different microorganisms have been identified and used to produce

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hydrolysates from fish protein. Due to the differences in microorganisms used in the culture for fermentation, the functionality of FPHs recovered by this method may vary (Daliri et al., 2017). Protein hydrolysates have been produced through fermentations using bacteria such as Enterococcus faecium NCIM5335 (Balakrishnan et al., 2011), and lactic acid bacteria (LAB) Pediococcus acidilactici NCIM5368 (Chakka et al., 2015). Jemil et al. (2014) prepared FPH from sardinelle, zebra blenny, goby, and ray using proteolytic bacterium, Bacillus subtilis A26. The FPH produced possessed outstanding solubility and interfacial properties, and showed antibacterial and antioxidant activities. Ruthu et al. (2014) isolated three proteolytic LAB (P. acidilactici NCIM5368, E. faecium NCIM5335, and P. acidilactici FD3) from fish processing. The bacteria were used to ferment fish head waste under the following conditions: 10% (w/w) glucose, 2% (w/w) NaCl, and 10% (v/w) LAB cultures at 37 C. It yielded a 38.4% degree of hydrolysis, and the process did not affect the fatty acid profiles of the lipid. In a similar experiment, Rai et al. (2011) used five different LAB recovered from fish processing waste that were used in fermentation under the same conditions described above. They observed that the fermentation liquor which predominantly contained FPH demonstrated antioxidant as well as antagonistic properties against several bacterial pathogens. It is also interesting to note that fermentation of fish protein can help remove hyperallergic or antinutritional components that are found in the ingredients (e.g., trypsin inhibitors, glycinin, β-conglycinin, phytate) (Hou et al., 2017). This offers an advantage that is not obtained from other FPH recovery methods.

6.2.4 Isoelectric Solubilization and Precipitation Isoelectric solubilization and precipitation (ISP), also known as the pH-shift method, is a protein recovery method that relies on a pH called the isoelectric point (pI) of proteins. This pH is protein specific, and thus differs from protein to protein. ISP is a mild, nonthermal pasteurization process, due to the extreme pH shifts involved (Lansdowne et al., 2009). It has successfully been employed in protein recoveries from fish and fish proces´ lvarez et al., 2018; Tahergorabi et al., 2012; Chen sing by-products (Choi and Kim, 2005; A et al., 2009); beef and chicken meat (Dewitt et al., 2006; Tahergorabi et al., 2012); from plant sources including soy (Foh et al., 2011; Rickert et al., 2006), and wheat protein (Liu et al., 2013). Among many applications, ISP has been successful in reducing Escherichia coli populations in fish protein isolate (FPI) (Lansdowne et al., 2009). ISP has been used widely because it permits selective and efficient recovery of protein, while separating lipids and other materials such as bones, skins, and scales that are not intended for human consumption (Ananey-Obiri and Tahergorabi, 2018). After ISP, the protein produced from fish is referred to as FPI. This resulting protein recovered using ISP technology is stable, and has high functionalities (Lee et al., 2016). According to Liu et al. (2013), many research findings have accorded its functionalities such as emulsification, gelation, and water absorption of isolates to the significant role of pH. According to Abugoch et al. (2008), the composition, yield, and the extent of unfolding of protein isolate can be manipulated with the method of extraction and precipitation pH. The adoption of ISP as a protein recovery method resulted in a significant yield of protein.

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The solubilization and precipitation processes are necessary steps; they contribute immensely to the amount of protein produced (Liu et al., 2013). The principle behind the pH shift involves adjusting the pH to solubilize the protein and a subsequent reduction to pH suitable for the precipitation of the protein. At this pH, the charges on the protein assume equilibrium, and the net ionic charge on the protein becomes statistically zero. When an acid is added to the protein solution, it dissociates to produce hydronium (H3O1). The low pH induces protonation of negatively charged side chains on glutamyl or aspartyl. On the other hand, with the addition of a base it dissociates to produce hydroxide ions (
OH). This reaction encourages side chains on tyrosyl, tryptophanyl, cysteinyl, lysyl, argininyl, or histidine residues to lose hydrogen ions, thus becoming deprotonated. ISP processing involves first, the rupturing of the fish muscle cells to release the protein. Subsequent to this, the muscles are homogenized with deionized water at 1:6 ratio (ground fish:water, w-v). The resulting homogenate is adjusted to a pH of 11.5 6 0.05 for 10 minutes to allow for protein solubilization. The mixture is centrifuged to produce three layers, namely: top layer—oil; middle layer—fish muscle protein solution; and bottom layer— insoluble (bones, proteins, membrane lipids, etc.). The middle layer is recovered and the pH is isoelectrically adjusted to 5.5 6 0.05 for 10 minutes to allow for protein precipitation. The mixture is centrifuged into two layers: bottom layer—process water; and bottom layer—FPI.

6.3 STRUCTURAL AND FUNCTIONAL PROPERTIES OF FISH PROTEIN HYDROLYSATES Different fish muscles and tissues are composed of stromal, myofibrillar, and sarcoplasmic proteins. Myofibrillar proteins consist of myosin, actin, tropomyosin, a-actinin, b-actinin, and other minor proteins. Myosin consists of about 60% myofibrillar protein, 5% tropomyosin protein, and 25% other regulatory/structural proteins. It is an asymmetrical molecule that has a long-coiled tail and two globular heads with 500 kDa. Myosin has been studied for its gelation properties and was found to be dependent upon the habitat of the fish. Myosin from cold water fish denatured and formed gels at lower temperatures (Tahergorabi et al., 2011). Sarcoplasmic proteins consist of myoglobin, hemoglobin, cytochrome proteins, and enzymes associated with the citric acid cycle and the electron transport chain. Endopeptidases, such as proteinases, are classified according to the chemical group of their active site. Proteinases can be grouped according to the optimum pH of their activity on muscle proteins. The optimal pH can affect water binding capacity, as water binding depends on the composition and conformation of the protein molecule (Tahergorabi et al., 2012). Much of the water in the muscle is entrapped in the structures of the cell, including the intra- and extramyofibrillar spaces. Higher final muscle pH improves the solubility of sarcoplasmic proteins, water holding capacity, cooked color, and emulsion stability of cooked muscle compared to initial muscle pH. Functional properties are defined as overall physiochemical properties of proteins in food systems during processing, storage, and consumption. Many functional properties of hydrolysates have been mentioned, chiefly solubility, emulsifying properties, foaming

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properties, water holding capacity, and fat binding capacity. These properties are affected by the molecular weight and structure, which play a role in their applications as gelling agents, nutritional supplements, and emulsifiers (Tahergorabi et al., 2015). Solubility is the property measuring how well the solute can dissolve in a liquid, called the solvent. The net charge of peptides that increases as the pH moves away from isoelectric points and the change in surface hydrophobicity promotes aggregation and causes a variation in solubility. More available peptides create an optimal environment for solubility, as the polar residue can form more hydrogen bonds with water (Tahergorabi et al., 2011). A decrease in hydrogen bonds due to larger molecular weight causes reduced solubility. The hydrolysis process generates more carboxylic and amine groups, which provide more exposed groups to surrounding water. The emulsifying properties of the hydrolysate is determined by the reduced tension of hydrophobic and hydrophilic components and a greater exposure to interaction at the oil
water interface. Hydroxylated amino acids contributed to the emulsion of hydrolysate from rainbow trout viscera (Taheri et al., 2013). Foaming properties are governed by penetration and rearrangement at the air
water interface (Halim et al., 2016). Hydrophobic regions of the molecules determine the adsorption at the air
water interface. Protein
protein interaction affects film formation and determines foam stability. Protein dispersion lowers tension at the interface. Water holding capacity is the ability of the protein to capture water in the food matrix. The content of water and its distribution have a profound influence on properties of meat, especially its toughness, juiciness, firmness, and appearance. Low molecular weight peptides are more hydrophilic and can affect water holding capacity more. Fat binding capacity affects taste which is an important quality in value-added products. It involves the entrapment of oil and increased protein density leading to increased protein absorption. Molecular size, chemical instability, and aggregation affect the bioavailability of hydrolysates. Higher weight peptides lose solubility, making them less likely to be able to cross the permeable membrane. Any swift changes in the processing of the hydrolysates will cause further protein denaturation than necessary. Aggregation is the misfolding of proteins due to environmental, functional, or metabolic factors (Stein, 2011). This process can cause protein denaturation and render them nonfunctional.

6.4 BIOACTIVE PROPERTIES OF FISH PROTEIN HYDROLYSATE Bioactive properties have been of interest in developing FPH. Bioactive properties are subsets of bioactivity, measuring the beneficial effects of drugs on a living organism. Bioactive properties exist via hydrolysis and exhibit antioxidation, antiproliferation, cytoimmunomodulation, antihypertension, and antidiabetic activities. These specific activities are of great importance to researchers, considering the grave afflictions they could remedy. Cancer, type 1 and 2 diabetes, and high blood pressure are some of the main causes of death in the world, attributed to the disruption of the body’s homeostasis.

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6.4.1 Antiproliferative Properties Cancer is the proliferation of abnormal cells to various parts of the body, forming malignant tumors, and eventually leading to death. One in six adults die from cancer worldwide. The development of the disease is characterized by three phases: initiation, promotion, and progression (Kim, 2013). During initiation, the normal cell regulatory processes, that is, the cell cycle, is challenged by carcinogens. Most challenges are neutralized by detoxification and DNA-repair systems, but occasionally these fail, and mutations of the DNA occur. During promotion, the mutated cells grow under the influence of several promotion factors, they evade apoptosis, and may develop into damaging lesions, which in turn can develop into invasive lesions of cancer. Progression is an irreversible process characterized by rapid cell division, increased invasiveness, and metastasis. Immunomodulation is responsible for the increased invasiveness during cancer progression and alters the immune system’s response or function. Peptides may enhance the growth and maturation of immune cells, regulate the synthesis of antibodies, and inactivate inflammatory compounds (Maestri et al., 2016). Hsu et al. (2011) produced and identified peptides that are active against human breast cancer cell line MCF-7 from tuna dark muscle. The isolated amino acid sequences for the peptides were Leu-Pro-His-Val-Leu-Thr-Pro-Glu-Ala-Gly-Ala-Thr and Pro-Thr-Ala-GluGly-Gly-Val-Tyr-Met-Val-Thr, respectively. The authors concluded that tuna dark muscle by-product would be a good source for producing antiproliferative peptides. Moreover, proteins from tuna cooking juice showed 25% antiproliferative activities. Lee et al. (2004) described a peptide isolated from anchovy sauce that demonstrated antiproliferative activity in human U937 lymphoma cells through the induction of apoptosis. Later, a study by Sae-leaw et al. (2016) demonstrated that gelatin hydrolysates from seabass skins could reduce interleukin-6 and IL-1β production in macrophage cells. Low molecular weight peptides have greater molecular mobility and diffusivity than high molecular weight peptides, thus improving interactions with cancer cell components and enhancing antiproliferative activity (Song et al., 2011). One of the immunomodulatory responses was examined to determine if the protein hydrolysates from tilapia mince would cause antiinflammatory activity. Tilapia mince hydrolysates enhanced immunity through the induction of IL-1β and cyclooxygenase-2 (COX-2) expression (Toopcham et al., 2017). Interleukin-beta cells regulate immune response by stimulating growth of disease-fighting cells, and COX-2 is an enzyme that promotes inflammation, fever, and pain through prostaglandins. In another study, collagen from the backbone of Alaskan pollock was hydrolyzed to trypsin protein hydrolysate (TPH). TPH enhanced the proliferation of spleen lymphocytes, T cells, and peritoneal macrophages, indicating high immunomodulatory activity (Hou et al., 2012). TPH also had high solubility, low viscosity, and high stability in acidic and alkaline conditions.

6.4.2 Antioxidant Properties Oxidative stress is the intracellular imbalance created between production of reactive oxygen radicals, also known as free radicals, and reactive metabolites, referred to as reactive oxygen species (ROS). ROS are normally produced through cellular metabolism and

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FIGURE 6.2 A diagrammatic representation of the effect of antioxidants against diseases caused by free radicals.

they are actively involved in the simulation of signaling pathways in both plant and animal cells, in response to changes in intra- and extracellular environmental conditions (Jabs, 1999). When oxidative stress continues over a period of time, ROS are also generated and accumulated over time, thus affecting cellular viability, metabolism, and function which can lead to chronic diseases (Kim and Yim, 2015). Research over the years has linked this continuous disturbance to chronic diseases including diabetes and cardiovascular and pulmonary diseases, which are mediated by chronic inflammations (Reuter et al., 2010). Fig. 6.2 represents the effect of antioxidants against disease caused by free radicals. Antioxidants are chemicals that can prevent, intercept, or repair free radicals. They are also able to prevent/or improve different diseases that are at different stages (Knight, 2000). Antioxidants inhibit the oxidation of other molecules. Antioxidants work through three mechanisms: free-radical scavenging, quenching of singlet oxygen, and metal chelation. Free-radical scavenging inhibits the initiation phase or propagation phase through scavenging radicals (lipid alkoxyl, lipid peroxyl, and protein carbonyl). Quenching singlet oxygen transfers energy from oxygen to the antioxidant or adds to endoperoxides. Metal-ion chelators inhibit transition metals with multiple valence states (Kim, 2013). Natural antioxidants are more prone to be investigated and discovered due to the toxicity of synthetic antioxidants and their subsequent regulation changes. They are characterized by their peptide sequences containing tyrosine, histidine, and valine (Halim et al., 2016). Antiproliferative activities prevent further oxidative damage to proteins and DNA that causes cancer. Mainly nonpolar hydrophobic amino acids (phenylalanine, alanine, and proline) and hydrophobic amino acids (such as tyrosine, histidine, and valine) existed in the sequences. They serve as active electron donors with low molecular weights (0.5
1.5 kDa) that react with free radicals. The antiproliferative amino acids were not specifically stated, but some activity has been shown in tuna and anchovy studies (Song et al., 2011; Hsu et al., 2011). These processes include the fermentation of microbes in various mediums. Picot et al. (2006) examined the antiproliferative activity of various species

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(salmon, blue whiting, Atlantic cod, and plaice) in cell culture. All the species mentioned showed cancer cell growth reduction of at least 20%. The specific molecules responsible for the activity have yet to be determined. Some hypotheses include the composition of the hydrolysates and a binding competition between the fish peptides and the fetal calf serum used to grow the cancer cell lines. A study was conducted by Godinho et al. (2016) to determine if fermentation would promote antioxidant activities. Bacillus species were used to prepare hydrolysates and compared to commercial protein hydrolysates (CPH). Reducing power and DPPH radical scavenging activity were higher compared to CPH, and angiotensin I-converting enzyme (ACE)-inhibitory activity was similar to CPH. In addition, the antioxidant potential was lower with the modified peptide than CPH. In another study done by Je et al. (2005), the hydrolysates with antioxidant activity were identified, and weighed. The peptide with the strongest activity was determined to be Leu-Pro-His-Ser-Gly-Tyr (MW 672 Da). Because it contains histidine, it was noted to have some antioxidative effect caused by the chelating and lipid radical-trapping ability of the imidazole ring. In addition, this peptide contained a tyrosine residue, which is a potent hydrogen donor in its sequence. These results suggest that the antioxidative activity of the isolated peptide was dependent on the amino acid residue and molecular weight. Considering the all-important health benefits that are attributable to antioxidants, it is extremely appropriate to have antioxidants in our diets. Raw or processed proteins can contain peptides that have antioxidant properties. Different types of fish and fish processing by-products, including Nile tilapia (Oreochromis niloticus) (Yarnpakdee et al., 2015), sardinelle (Sardinella aurita) (Bougatef et al., 2010), Alaska pollack (Theragra chalcogramma), herring (Clupea harengus) Sathivel et al. (2003), have been reported to have strong antioxidant activity. Antioxidant peptides inhibit several different oxidation pathways, including inactivation of ROS, scavenging of free radicals, and chelation of prooxidative transition metals (Fang et al., 2002; Guiotto et al., 2005; Seth and Mahoney, 2001). The most reactive amino acids are usually those containing either nucleophilic sulfur-containing side chains (taurine, cysteine, or methionine) or aromatic side chains (tryptophan, tyrosine, and phenylalanine). The amino acid residues histidine, glutamic acid, aspartic acid, and phosphorylated serine and threonine are particularly active metal chelators (Kim, 2013). Antioxidant peptides exist in fish muscle as carnosine, anserine, and glutathione. Glutathione is also involved in the enzymatic defense system. The enzymes cleave the peptide bonds that release active peptides that have the ability to block oxygen radicals, chelating prooxidant metal ions and constraining lipid peroxidation in the food system (You et al., 2010). Kim et al. (2001) established that amino acids, including histidine, proline, and alanine, can scavenge free radicals. The antioxidant activity of a protein hydrolysate is dependent on the degree, sequence, and composition of its free amino acids and peptides (Chalamaiah et al., 2015). It is worth noting that, the hydrophobic amino acids and one or more residues of His, Pro, Met, Cys, Tyr, Trp, Phe, and Met have the ability to improve the antioxidative activities of peptides (Ren et al., 2008). Generally, shorter peptides exhibit greater antioxidant activity than their native proteins or larger polypeptides (Ma et al., 2010; You et al., 2010).

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TABLE 6.1 Experimental Results Showing the Effectiveness of Fish Protein Hydrolysates from Different Kinds of Fish as Antimicrobial Kind of Fish

Scientific Name

Habitat

Peptide

Source

Flounder

Pleuronectes americanus

Marine

Pleurocidin

Cole et al. (1997, 2000)

American plaice

Hippoglossoides platessoides

Marine

Piscidins

Patrzykat et al. (2003)

Antarctic eelpout

Lycodichthys dearborni

Marine

Hepcidin

Xu et al. (2008)

Tilapia

Oreochromis mossambicus

Freshwater, brackish

Hepcidin

Huang et al. (2007)

Ayu

Plecoglossus altivelis

Marine, brackish

Cathelicidins

Lu (2011)

Atlantic cod

Gadus morhua

Marine, brackish

Beta-defensin

Ruangsri et al. (2013)

Rainbow trout

Oncorhynchus mykiss

Marine, brackish

Defensins

Falco et al. (2008)

Grayling

Thymallus thymallus

Freshwater and brackish

Cathelicidins

Scocchi et al. (2009)

Common carp

Cyprinus carpio L.

Freshwater and brackish

Defensin

Marel et al. (2012)

6.4.3 Antimicrobial Properties Bacteria are important in food processing and microbiology because of the role they play in food safety and quality loss. A wide range of approaches, including both natural and synthetic, have been used to control the growth of pathogenic bacteria to curb their disease-causing and food spoilage potential. Lactoferrin, nisin, and lysozyme among others have been evaluated to varying degrees, and they have been effective against bacteria (Bhatia and Bharti, 2015). However, unbridled use of antibiotics in the field of medicine and in food preservation has resulted in bacteria developing resistance to these antibiotics. Discovering new antibiotics that could be effective against bacteria is therefore very crucial. Many research papers have reported the effectiveness of protein hydrolysate from almost all fish types against both Gram-negative and Gram-positive bacteria. Over 1600 cationic antimicrobial peptides have been isolated from many organisms, or synthesized chemically depending on the sequence of the isolated peptide (Sila et al., 2014). Peptides produced by the hydrolysis of protein have been identified as having the potential for reducing health concerns (Sato et al., 2006). Fish peptides have very strong antimicrobial properties, which are used by the fish to adapt to the high level of pathogen exposure in their environment. The antimicrobial properties exhibited by fish peptides, as described by Rajanbabu and Chen (2011), are exhibited as antibacterial, antifungal, antiparasitic, antiviral, antitumor, and immunomodulatory activities. It has been established that β-defensin (BD)-1 peptide fights against hemorrhagic septicemia infection in rainbow trout (Wang, Kung, and Chen, 2010). Table 6.1 lists the effectiveness of FPHs from different kinds of fish as antimicrobial agents.

6.4.4 Antihypertensive Properties Cardiac muscle responds more readily to the fatty acid composition, that is dietary fats such as saturated and unsaturated fat. Thus, the organs containing them, that is, heart and

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blood vessels, will be more affected by hypertension. Salt is regulated via excretion from the kidneys. When salt intake levels are very high, the kidneys do not function well, resulting in salt in the bloodstream. Generally, salt attracts water, so high salt in the blood draws more water into the bloodstream, which increases blood volume, raising blood pressure (Ahhmed and Muguruma, 2010). High blood pressure transpires from prolonged tension of these vessels that carry blood from the heart to the rest of the body. It can often have no symptoms until it has escalated to CVDs such as stroke and heart disease. Antihypertensive/angiotensin I-converting enzyme, angiotensin-II, and bradykinin are a part of the vasoconstriction process. Antihypertensive/ACE aids in vasoconstriction by cleaving to angiotensin-II and inactivating bradykinin. ACE inhibitor activity is identified through branched-chain amino acids and C-terminal amino acids which are aided by aromatic and aliphatic side chains (Kim and Wijesekara, 2010; Ghassem et al., 2011). ACE is affected by molecular weight and amino acid composition. Peptides derived from FPH exhibit antihypertensive properties. These peptides inhibit vasoconstriction resulting in high blood pressure via the ACE. Dietary ACE-inhibitory peptides may be classified into three groups (Fujita and Yoshikawa, 1999): true inhibitors, substrates for ACE, and prodrug peptides. True inhibitors are not changed by preincubation with ACE. Substrates for ACE are converted to weaker or inactive peptides by ACE. Prodrug peptides are converted to true inhibitors by ACE or by gastrointestinal proteases (Kim, 2013). Dark tuna muscle contained ACE-inhibitory proteins that formed enzyme
substrate
inhibitor and enzyme
inhibitor complexes that lower ACE efficiency (Qian et al., 2007). In another study, rats with high blood pressure were orally administered sardine protein hydrolysate dissolved in distilled water at different concentrations (Huang et al., 2016). Sardine protein hydrolysates were also determined to alleviate oxidative stress, reduce lipid peroxidation, and decrease the ACE activity and angiotensin-II concentration in the rats’ systems.

6.5 NUTRITIONAL, FUNCTIONAL, AND PHYSICOCHEMICAL PROPERTIES OF FISH PROTEIN ISOLATES Previously, the functional properties of FPH were discussed, that is, water binding capacity, oil binding capacity, foaming and emulsion capabilities. The same properties can be associated with FPI as well. Multiple studies have concluded that these properties benefit from FPI processing through ISP (Nolsøe and Undeland, 2009; Thawornchinsombut et al., 2006; Yongswawatdigul and Park, 2004). In this section, further emphasis will be placed on the attributes of the functional, nutritional, and physicochemical properties of FPI.

6.5.1 Nutritional Properties of Fish Protein Isolates To become a valuable addition as a functional food for human consumption, the nutritional properties of FPI must be evaluated and improved upon, if necessary. These properties include high quality protein based on essential amino acid content and proximate composition. As mentioned previously, proteins are important molecules that are needed

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for growth as well as providing diverse functional foods. Nutritional value is of prime importance as the essential amino acids are found in fish. Tian et al. (2017) studied the effects of ISP on FPI from the common carp. There were not huge differences in the nutritional value between the acid and base isolates, as they had similar solubility (80%
90%), essential amino acid content (44
45 g), and protein content (70%
80%). Tian et al. (2017) suggested that FPI processed using the ISP method have much more potential in being used for functional foods, than fresh muscle. Fish muscle also contains ω-3 and ω-6 which are considered superfoods due to their protective benefits. These ω-3 and ω-6 acids include eicosapentaenoic acid, docosahexaenoic acid, linoleic acid, and arachidonic acid. In addition, Pacific Ocean perch by-products were found to contain high levels of essential amino acids, minerals like calcium and phosphorus, and high levels of ω-3 fatty acids (Bechtel et al., 2010).

6.5.2 Functional and Physicochemical Properties Functional properties influence sensory attributes positively or negatively, for example, water holding capacity, oil binding capacity, solubility, emulsifying capacity, and foaming capacity. This determines the types of food products the isolates can be used in. During ISP, the pH transforms the 3D (unfolding, misfolding, and refolding) structure of the proteins, affecting the functional properties. Physicochemical properties are essential to sensory attributes and include color and texture. For instance, red snapper’s functional properties were examined after ISP (Pramono et al., 2018). It was determined that the water holding capacity, oil binding capacity, and amino acid content improved, which concurred with other studies on krill and tilapia (Chen et al., 2009; Foh et al., 2011). In addition, water holding capacity, oil binding capacity, solubility, emulsifying capacity, and foaming capacity all improved in a study of Lanternfish isolates under alkaline conditions (Oliyaei et al., 2017). Water holding capacity is influenced by pH, as the protein conformation increases the exposed amount of water binding sites. Oil binding capacity influences the emulsifying properties of foods; as hydrophobic groups of the proteins are exposed to water. Proteins are absorbed at the water
oil interface and help stabilize the system. Foaming capacity increases as the soluble proteins are diffused at the air
water interface (Oliyaei et al., 2017). The isolates were also determined to have the desired color properties, as the hemoglobin is coprecipitated at the isoelectric point. In another study, it was also determined that the pH changes during ISP do not affect the composition of ω-3 or ω-6 (Gehring et al., 2011). This could be due to the extreme protein shift and nonthermal pasteurization stabilizing the fatty acid composition (Ananey-Obiri and Tahergorabi, 2018). ISP conducted under basic conditions yielded more positive changes to the gel formation due to increased hydrophobicity and the presence of
SH groups oxidized to form
SS-linkages. The
SS-linkages also contribute to gelation and texture development. Texture is a measure of the mechanical properties through analysis by the human senses. It can be determined using texture profile analysis which measures properties such as resilience, springiness, and hardness. Various studies have analyzed the texture of protein gels and surimi (Lee et al., 2017; Hsu and Chiang, 2002; Kasapis, 2009). In a study by

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Abdollahi et al. (2017), ISP benefitted gel formation and its texture. Blended protein gels had a higher breaking force due to enhanced dispersion of myofibrillar proteins, enhanced charge distribution, and higher amounts of
SH and hydrophobic groups. Protein gels have also had improved texture due to ω-3 oils (Tahergorabi et al., 2012). Color is another sensory attribute that is critical to customer acceptance. Whiteness has to be corrected in isolates because the dark pigment from the by-products remains. Titanium dioxide and ω-3 oils have been used to improve whiteness in surimi and protein isolate gels (AnaneyObiri and Tahergorabi, 2018; Taskaya et al., 2010).

6.6 COMPARATIVE DIFFERENCES BETWEEN FISH PROTEIN HYDROLYSATES AND OTHER PROTEIN HYDROLYSATES FPHs have been used for a wide variety of food applications. Plant protein hydrolysates have had little use in food applications, accounting for only 33% of use (Faostat, 2016). In addition, animal protein hydrolysates can be up to 10 times costlier than plant proteins hydrolysates (Zayas, 2012). Various plant and animal protein hydrolysates have been used based on their functionality, including egg whites, soy, wheat and maize starch, beans, chicken, and beef muscle. The functionality of these proteins can be the difference in their uses for application, sustainment during processing, and further development as health aids. Like FPHs, decreased solubility during the hydrolysis of plant proteins is promoted by protein
protein interactions and results in precipitation. This can impact the texture of the final product negatively. Water and fat-holding capacities contribute to favorable texture properties like juiciness and tenderness. Water holding capacity can be determined by balancing a decreased molecular mass and a higher availability of hydrophobic groups (Wouters et al., 2016). Fat-holding capacity can be decreased due to a lower entrapment of oil and liberation of ionizable polar groups. Gelation is most dependent upon the molecular forces (covalent bonds, hydrophobic interactions, and hydrogen bonds) that form the protein network. Foam formation is affected by surface tension and can be destabilized due to disproportionation and coalescence. Emulsification stabilizes with decreased surface tension and film formation involving oil
water droplets.

6.6.1 Plant Protein Hydrolysates Plant protein hydrolysates contain bioactive peptides that are pharmaceutically inactive and help defend the plant against pathogens. They are generated by microorganisms, Gastrointestinal (GI)-digestive enzymes, or proteolytic enzymes in extreme conditions (Maestri et al., 2016). Even after generation, they have to be maintained during digestion for efficacy. The bioavailability during absorption, distribution, metabolism, and excretion has been studied recently at each stage. Once determined, the hydrolysates may be transported with effective carrier compounds. During adsorption, certain peptides are more prone to degradation by proteases, while others can easily pass through the intestinal epithelium (Maestri et al., 2016). This can happen through transcellular entry, paracellular entry, and endocytosis. Microfold cells are

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capable of endocytosis, allowing small peptides to enter the bloodstream. Peptides in plasma exist in low concentrations (picograms per milliliter). Small oligopeptides can be transported from the GI tract to the blood without degradation (Foltz et al., 2007). This could be due to protease inhibitors that increase the half-life during distribution. Peptides are catabolized in the host cell and peptidases are present at the brush border, in enterocytes, in plasma, and inside the target cells. They can be digested in the lysosome. When below 25 kDa, peptides can be filtered at the glomerular site and reabsorbed or hydrolyzed during elimination. Maestri et al. (2016) mentioned a variety of plant species that are bioactive. They possess most of the same characteristics as the fish hydrolysates, with the specifics stated below. 6.6.1.1 Soy Protein Hydrolysates Soy protein is used in a variety of human food applications. At its isoelectric point (4
4.5), its functionality drastically decreases. Its solubility in an acidic environment causes decreased functionality, as solubility is the prerequisite for all other functional properties (Horax et al., 2017). Horax et al. (2017) optimized soy protein hydrolysates at acidic pH values. At a pH of 3.0 solubility increased to 75% and emulsifying properties were enhanced. No changes were observed in ACE-inhibitory properties. Lamsal et al. (2007) studied the gelation of soy protein hydrolysates. Gel formation was affected when using mixtures of hydrolyzed soy flour versus nonhydrolyzed soy flour. When treated with transglutaminase, however, the gelation properties were restored. Changing prepreparation steps impacted foaming properties as glycinin peptides increased foam capacity. de la Barca et al. (2000) studied the improvement of soy protein hydrolysates in various fractions bound with methionine. The molecular weight and content of bound Met were interacting together to modify and determine the functional properties. All fractions (FII, FII-E, FIII, FIII-E) displayed improved functional properties specific to various products. For instance, FII-E could be used in hypoallergenic formulas for babies and medical formulas for adults due to its improved amino acid content. FII and FIII could be used for carbonated fortified beverages because of their solubility and clarity at low pH. 6.6.1.2 Wheat Starch and Maize Gluten Hydrolysates Starch is the primary source of stored energy in many plants and consists of amylose and amylopectin α-1,4 and α-1,6 bonds. Variations in the amount of amylopectin and amylose are produced through cross-breeding, mutagenesis, or transgenic breeding. Plants synthesize granules which form crystalline rings (Waterschoot et al., 2015). Amylopectin is responsible for the crystalline character of starch. Modification through the hydrolysis of starch was shown to improve gelatinization, solubility, and retrogradation (Horstmann et al., 2017). This is due to a disruption of the molecular order of the granules. A lower crystalline order, characteristic of short amylopectin chains, leads to a lower gelatinization temperature. The opposite is also true as well. Increased gel firmness correlates with higher amylose content. The swelling properties of the starch are affected by these factors. Amylopectin contributes directly to swelling properties (Hamzah and Sandra, 2010). Pasting properties describe the events that occur after gelatinization. They influence the quality and esthetic (texture) of the product (Adebowale et al., 2005). Wheat starch hydrolysates have been found to require additional enzymes during hydrolysis, such as

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lysophospholipases and pentosanases, due to increased viscosity and amylose
lipid (AML) bonds hindering enzyme access. The problems of AML linkages and resistant starch have increased interest in determining and combating genetic mutations to resolve this. Through genetic engineering, swelling-resistant wheat starch could be developed to improve dietary fiber intake, enhance fiber content of white flour products, and reduce glycemic and insulin response (Sjo¨o¨ and Nilsson, 2017). Corn is the most produced cereal crop in the world, with more than 1 billion metric tons produced annually (Yuan-Qing et al., 2018). The corn kernel consists of starch, nonstarch polysaccharides, proteins, and lipids. After carbohydrates, proteins are the most abundant component. When processed, however, corn gluten meal (a source of the protein) cannot be absorbed and is subsequently discarded. Yuan-Qing et al. (2018) studied the bioavailability of corn gluten meal hydrolysates and their effects on the immune system through rats. Rats displayed an increased body mass when weighed on the corn gluten meal hydrolysate diet, and after examining the salivary gland and thymus gland, it was suggested that the hydrolyzed corn gluten meal positively affected the immune system. This is due to the production of small peptides, proteins, and amino acids that produce beneficial microbes in the GI tract. In addition, as mentioned before, a vast amount of research has been conducted supporting bioactive protein hydrolysates (Chobert et al., 1996; Erdmann et al., 2008; Rizzello et al., 2016). 6.6.1.3 Kidney Bean Hydrolysates and Rice Bran Hydrolysates Legumes or beans are considered an inexpensive source of many nutrients. Their use in an edible coating is due to the interest in developing biodegradable films and providing an alternative to the major allergens. Many of the same properties observed for films were examined in the process. Moisture permeability and thermal properties contribute to the tensile strength of the film, as moisture is needed for the gelation process, and protein denaturation by heat promotes intermolecular bonds that strengthen protein films (Shevkani and Singh, 2015). Riceberry bran hydrolysates were observed to be a suitable replacement for commercial rice bran due to its high antioxidant content. Riceberry bran hydrolysates from Oryza sativa L. did not affect the protein solubility and heat stability (Thamnarathip et al., 2016). Emulsification properties were affected by the type and size of peptides. Smaller peptides increased the interaction between biopolymers and the stability of emulsions. Specific enzymes produced during hydrolysis have been shown to exhibit food preservation properties. For instance, endohydrolase has a reduced action toward branched β-D-glucan substrates, an integral part of the fungal cell wall. This protects the plant from pathogen attack and is vital for food preservation (Mine et al., 2011).

6.6.2 Other Food Protein Hydrolysates 6.6.2.1 Egg White Hydrolysates Egg proteins are an important source of amino acids and exhibit versatile functional and biological properties. However, they only account for 2% of human consumption (Chen and Chi, 2012). The proteins contained in egg whites such as ovalbumin, ovotransferrin, and lysozyme contain hydrolysates that have antihypertensive, antioxidant, antiinflammatory, and

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antidiabetic properties (Garce´s-Rimo´n et al., 2016). The proteins in egg whites exhibit multifunctional properties. However, no studies have shown that these multifunctional properties could combat metabolic syndrome. Metabolic syndrome can be defined as a group of health problems characterized by at least three of the following risk factors: abdominal obesity, high serum triglycerides, low high-density lipoprotein (HDL) levels, elevated fasting plasma glucose, and hypertension. In a study conducted by Garce´s-Rimo´n et al. (2016), egg white hydrolysates were proteolyzed with a variety of enzymes to determine their biological capabilities and defense against metabolic syndrome. Alcalase 2.4 LFG, Flavourzyme 1000 L, Neutrase 0.8 L, Trypsin PNT 6.0 salt-free, BC Pepsin 1:3000, Pancreatin 4XNF, Peptidase 433P, and Promod 144P were used at their optimum conditions. ACE-inhibitory activity, antioxidant and antiinflammatory activity, hypocholesterolemic activity, and dipeptidyl peptidase-inhibitory activity (DPP-IV) were examined. Egg white hydrolysates with peptidase 433P and BC pepsin 1:3000 were determined to have the most active ACE-inhibitory, antioxidant, and hypocholesterolaemic activity. The peptide sequences within these hydrolysates were listed (RADHPFL, IVF, SALAM, YQIGL, and YAEERYPIL) and correspond to studies in previous literature (Miguel et al., 2005, 2007). Majumder and Wu (2010) examined the functional and bioactive properties of egg proteins based on pH. Egg white hydrolysates have been shown to contain ACE-inhibitory properties, as well as a variety of necessary amino acids. The ACE-inhibitory properties are linked to a substrate preferred structure. The preferred structure includes branched amino acid residues at the N-terminal position and hydrophobic residues at the Cterminal position. Higher pH showed better orientation at the oil
water interface and molecular repulsion resulted in a more stable interface. Foaming properties were not consistent as the interfacial surface is influenced by the polypeptide’s amino acid composition and structural conformation (Waniska and Kinsella, 1979). 6.6.2.2 Chicken and Beef Muscle Hydrolysates As mentioned previously, many marine and plant products contain various hydrolysates that can be useful in providing functional foods. These functional foods would have bioactive properties such as antimicrobial, antioxidant, antihypertensive, and antiproliferation activities. Certain animal products, recently discovered and unknown, may have these beneficial properties as well. These animal products include chicken liver, chicken skin, and beef muscle. Decreasing liver lipid accumulation and lipid absorption or increasing serum/liver antioxidant capacity are most effective at reducing the occurrence of cardiovascular or liver disorders (Yang et al., 2014). Chicken liver hydrolysates (CLHs) have been shown to decrease total cholesterol (Yang et al., 2014). The benefits of CLHs on the cardiovascular system and livers in a high-fat dietary habit were demonstrated, via the regulation of lipid homeostasis. Hamsters were fed high-fat diets consisting of 12% fat and 0.2% cholesterol or chow diets containing CLHs in powder form for 8 weeks. The organs were then extracted for further analysis. Weight comparisons were made to determine the effect of the hydrolysates on the organs. The CLH diet reduced the fatty tissues on the organs. The major gene expressions were investigated to determine lipid homeostasis effects. Acetyl coA carboxylase and fatty acid synthase were downregulated, while fatty acid β-oxidation was upregulated. The lipid profile improved, cholesterol metabolism increased, and energy metabolism increased. In a study conducted by Onuh et al. (2014), chicken skin from the thigh and breast were hydrolyzed to compare antioxidant activity.

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Overall both skin hydrolysates showed antioxidant activity. These peptide products exhibited varying abilities to scavenge or quench DPPH, hydroxyl, peroxyl, and superoxide radicals, as well as chelate metal ions (Onuh et al., 2014). Chicken thigh skin hydrolysates had better antioxidant activities and chicken breast skin hydrolysates had better freeradical scavenging activities. Beef hydrolysates were shown to have antihypertensive activity (Jang and Lee, 2005). The specific peptide sequence with the highest activity was Val-Leu-Ala-Gln-Tyr-Lys. This sequence has aliphatic valine and lysine residues at the N-terminal and C-terminal, respectively. A further study was done by examining the peptide in vitro on rats given diets containing 0.2, 0.5, and 1.0 g of the peptide per kg of body weight every day for 8 weeks. Total and LDL cholesterol were lower in the rats given the diet and systolic blood pressure was suppressed when rats were given higher concentrations of the peptide supplement. This peptide may be a potent ACE inhibitor for clinical use and have potential as a valueadded functional material for food.

6.7 PHYSICOCHEMICAL CHALLENGES ASSOCIATED WITH FISH PROTEIN ISOLATES AND HYDROLYSATES Physicochemical properties of fish and fish products are characterized by color, texture, and taste. These properties are salient determinants of the quality of fish and fish products, which translate into consumer acceptability. Evidently, hydrolysis of protein produces the improved desired functionalities such as solubility, emulsification, gelation, and water holding capacity (Foh et al., 2011). The degree of hydrolysis which measures the extent of protein degradation is a controlling parameter for the process, and critically influences the properties of the resultant protein hydrolysate (Quist et al., 2009). Together with the degree of hydrolysis, the kind of substrate and the proteases used impact on the physicochemical properties of the resulting hydrolysate. Specific enzymes cleave peptides to produce specific peptide patterns. This method of proteolytic modification of food proteins to improve the palatability and shelf-life of the available protein resource dates back into the ancient times (Taha and Ibrahim, 2007; Adler-Nissen, 1986). Comparatively, protein hydrolysis produces peptides with improved functional, bioactive, and sensory properties than their native proteins (Cheison and Wan, 2003). However, the physicochemical properties of fish FPH and isolates are tampered with as they go through the various processing steps. More and more, mass and large-scale productions of FPH have been constrained by taste defects and the economic consequences that come with hydrolysates. Even though the bitter taste associated with FPH is still not fully understood, it is speculated to be caused by the exposure of the hydrophobic tail of the amino acid during protein breakdown. Maehashi and Huang (2009) also posits that insufficient hydrolysis of the protein possibly results in the unfavorable bitter taste and unpleasant flavors. The unfortunate bitter taste associated with these peptides and hydrolysates has been masked by suitable formulation of the peptides with fructose, pectin, and other natural and artificial flavors (Li-Chan, 2015). Also, porcine kidney cortex homogenate or activated carbon could be used to reduce the bitter taste associated with protein hydrolysates (Hou et al., 2017).

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The method of hydrolysis significantly affects the nature, composition, and physicochemical qualities that characterize the hydrolysate. Enzyme hydrolysis has been the widely used method of protein hydrolysis. Unlike acid and alkaline protein hydrolysis, enzyme hydrolysis of protein is noted to be mild and does not result in any loss of amino acids (Hou et al., 2017). The specificity of the proteases used in enzymatic hydrolysis, and the ability to regulate these enzymes have promoted their use. However, the bioactivity of peptides produced through fermentation and enzymatic proteolysis could be decreased if the hydrolysis process is not controlled. This situation of the vulnerability of the peptides to be further hydrolyzed, coupled with the different peptide bonds, have made the kinetic study of the process intricate. Solubility is considered as an important physicochemical and functional property of protein hydrolysate as it impacts other functional properties, including foaming and emulsification (Kristinsson and Rasco, 2000). The solubility of FPH is defined as the amount of soluble nitrogen from the total nitrogen, calculated as: Solubility ð%Þ 5

nitrogen content of the supernatant total nitrogen content of the sample

Jemil et al. (2014) produced FPH through the fermentation of various fish types. They realized that the solubility of FPH was improved by more than 70%, when compared to nonhydrolyzed fish proteins. However, according to Li-Chan (2015), peptides possessing bioactivity are often hydrophobic in nature and exhibit poor aqueous solubility at high concentrations. Amino acid composition is important in fish hydrolysate because of its rich nutritional value and its influence on functional properties (dos Santos et al., 2011). The antihypertensive, antioxidant, and immune modulatory properties are due to the bioactive amino acids. Both acid and alkaline hydrolyses of protein to produce peptides have been less patronized because of their deleterious effect on some amino acids.

6.8 CURRENT AND FUTURE APPLICATION OF FISH PROTEIN HYDROLYSATE Protein hydrolysates have found many uses both in the medical field and industrially, even as other possible usages are currently being explored. The potential use of FPH is based on their bioactivity, functionality, and nutritional value. FPH properties have spurred the search for the possible use of fish peptides and hydrolysate in diverse areas. The bioactivity and functionalities of FPH allow opportunities for their use in different food formulations. The numerous properties of FPH, such as high water holding capacity, solubility, emulsion ability, foaming capacity, and gelling ability, could be utilized in many different food formulations (Chalamaiah et al., 2012). FPHs have been shown to have potential for nutritional or pharmaceutical applications. It has been established that FPH has the potential for applications in pharmaceuticals and nutrition. Wergedahl et al. (2004) demonstrated in Zucker rats that FPH reduces plasma

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total cholesterol, increases HDL cholesterol proportion, and lowers acyl-CoA:cholesterol acyltransferase activities. The nutritional value of FPH makes it viable for use as a food that can stimulate the growth and survival of aquatic animals. Kotzamanis et al. (2007) incorporated FPH into four different diets for start-feeding sea bass (Dicentrarchus labrax) larvae. They made two FPH formulations, namely, 10% and 19% of the total ingredient. They realized from their observation that 10% FPH gave better growth, survival, and intestinal development of the larvae, compared to the 19% FPH, and consequently concluded that a lower dose of FPH seems appropriate and optimal for larval development. In another study, Delcroix et al. (2014) supplemented diets for sea bass (D. labrax) larvae with different proportions of diand tripeptides supplied by five different companies, and by raw materials. They observed that two of the diets stimulated larval growth, while two other diets produced substandard growths, when compared to the control. However, they could not conclude that high portions of small peptides are not an adequate way to assess the dietary value of hydrolysates. Also, Xu et al. (2017) realized the potential applicability of FPH as an alternative protein source when used at a low level in high protein diets for juvenile turbot (Scophthalmus maximus). Nitrogen remains one of the most expensive constituents of bacterial growth media. Ghorbel et al. (2005) demonstrated the ability of FPH prepared from sardinella (S. aurita) as a nitrogen source for the production of extracellular lipase by filamentous fungus Rhizopus oryzae. The best results were realized with defatted meat
FPHs. They also realized that extensive hydrolysis of fish proteins produced higher amounts of lipase. In another study involving the enzymatic hydrolysis of silver carp filleting by-products, Alclase and trypsin were used to produce fish peptone. Fallah et al. (2015) prepared fish peptone. The fish peptone was used as a source of nitrogen for Streptococcus thermophilus. It was evident from their study that fish peptone can sufficiently provide nitrogen to support the proliferation of S. thermophilus. Comparatively, fish peptone produced by Alcalase significantly outperformed commercial tryptic soy broth as a growth media. Wang et al. (2017) concluded from their study, after hydrolyzing grass carp fish skin, that molecular weights below 1000 Da in the hydrolysates are the best for the proliferation of S. thermophilus. Cryoprotectants are very useful in the frozen food industry, in that they are able to protect the food against the loss of textural and other qualities during freezing. FPHs have been shown to have cryoprotective effects, allowing their potential use as cryoprotectants. Jenkelunas and Li-Chan (2018) investigated the cryoprotective effect of FPH for cod fish mince subjected to freeze
thaw abuse. Cheung et al. (2009) investigated FPH produced by Alcalase and Flavourzyme hydrolysis of Pacific hake (Merluccius productus) for a potential use as a cryoprotectant of frozen fish mince. They strongly asserted after their experiment that FPH could be a new cryoprotectant for maintenance of the quality of frozen fish and must be given due attention. The current trend of research in fat-uptake reduction in deep fat-fried foods is skewed towards using a coating to serve as a barrier to fat absorption during frying. Many edible materials have been explored and extensively studied. FPHs were produced by four different processes (enzymatic, microwave-intensified enzymatic, chemical, and microwaveintensified chemical) from Yellowtail Kingfish (He et al., 2015). They coated fish (fish cakes and battered fish), with a coating prepared from the FPH, and fried them. This resulted in

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a significant reduction of fat uptake in deep-fried battered fish from 7% to 4.5% by replacing 1% (w/w) batter with FPH, and a significant reduction in fat uptake from 11% to about 1% in deep-fat fried fish cakes coated with FPH. The numerous studies that support the antimicrobial and antioxidant properties of FPH and peptides are overwhelming. The need for another antibacterial to solve the problem of bacteria resistance to current antibiotics is imminent. Also, the health-related benefits of antioxidants in counteracting free radicals is laudable. Both antioxidant and antibacterial properties of FPH would need further studies to ascertain their various health benefit claims.

6.9 CONCLUSIONS FPH offers many useful capabilities, which makes its applicability in food processing and even outside food processing desirable. The functionality, bioactivity, and nutritional value of FPH widen its scope of use. The choice of method used in the hydrolysis process prodigiously impacts the physicochemical, functional, and bioactive characteristics of the FPH, and consequently its use as a food ingredient. The undesirable bitter taste that has been a constraint to the use and acceptability of hydrolysates needs further study in order to correctly ascertain its cause and so that appropriate measures can be taken. Even though strides have been made through considerable research, there is still much to be done. Despite the physicochemical challenges that have been associated with FPH, its outstanding nutritional and functional properties create opportunities for its use in both the food and health industries.

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7. Zayas, J.F., 2012. Functionality of Proteins in Food. Springer Science & Business Media.

Further Reading Li, X., Luo, Y., Shen, H., You, J., 2012. Antioxidant activities and functional properties of grass carp (Ctenopharyngodon idellus) protein hydrolysates. J. Sci. Food Agric. 92 (2), 292
298.

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C H A P T E R

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Whey Proteins Rajeshree A. Khaire and Parag R. Gogate Chemical Engineering Department, Institute of Chemical Technology, Mumbai, India O U T L I N E 7.1 Introduction

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7.2 Whey Proteins: Overview

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7.3 Available Approaches and Recent Advances in Recovery of Whey Proteins 7.3.1 Precipitation 7.3.2 Chromatographic Separation 7.3.3 Membrane Separation 7.3.4 Other methods for Whey Protein Separation 7.3.5 Drying 7.3.6 Proposed Improvements in the Whey Protein Manufacturing

196 197 199 200 203 206 208

7.4 Important Applications of Whey Proteins 7.4.1 Healthcare Applications 7.4.2 Applications in the Food Industry 7.4.3 Application as Edible Films and Coatings 7.4.4 Whey Protein Hydrogels and Nanoparticles

212 212 215 216 216

7.5 Global Whey Protein Market

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7.6 Conclusions and Future Aspects

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References

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Further Reading

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7.1 INTRODUCTION Whey proteins are one of the sustainable products of milk processing (often neglected currently) which can have various applications in the food and healthcare industries due to their high nutritional value and specific functional properties. The whey proteins are typically obtained from whey, a yellow/green liquid remaining after coagulation and separation of caseins from the milk using an enzyme (chymosin) or acid (mineral/organic)based approach during the cheese-making process (Smithers, 2008). Currently the whey is not utilized completely for reprocessing but the environmental issues relating to a high amount of biochemical oxygen demand (BOD) and chemical oxygen demand (COD) in the Proteins: Sustainable Source, Processing and Applications DOI: https://doi.org/10.1016/B978-0-12-816695-6.00007-6

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© 2019 Elsevier Inc. All rights reserved.

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7. WHEY PROTEINS

whey and the quantity of wastewater, and the high amount of whey production worldwide (B190 million tons/year) are driving research into the proper utilization of whey. Whey protein also has a great biological value in the human diet and provides an array of functionality in food systems. Whey proteins can be effectively utilized by humans and provide considerable amounts of essential amino acids for growth (Hambraeus and Lonnerdal, 2003). Whey protein has also long been popular in the exercise industry as a muscle-building supplement with its high protein quality score and high percentage of branched chain amino acids. It has been also demonstrated that whey proteins have far wider applications as a functional food in the management of conditions such as cancer, Hepatitis B, HIV, cardiovascular disease, and even chronic stress. Whey proteins have also found application in the food industry as high nutritional functional foods and as a substitute for various ingredients for improved sensory properties. They can be used as ingredients in foods due to their unique functional properties, that is, emulsification, gelation, thickening, foaming, and fat and flavor binding capacity (McClements, 1995). A specific constituent of whey protein (α-lactalbumin) has good emulsifying and foaming properties, (Ibanoglu and Ibanoglu, 1999). Besides, whey demonstrates a range of immune-enhancing properties as an anticarcinogenic agent and as a precursor for bioactive peptides. Overall it can be said that effective processing of whey into whey proteins is very important considering the environmental concerns associated with whey disposal and the high nutritional value/specific healthcare related applications of whey proteins. Whey processing for the recovery of valuable products in general involves membrane separation followed by spray drying to obtain proteins. The traditional methods proposed for whey proteins recovery from whey offer processing problems often making the recovery uneconomical. Hence the evolution of newer whey protein recovery approaches with possible improvisation/intensification in the processing is very important for obtaining high-quality product in the most economic manner possible. The processing of whey for the recovery of whey proteins has grown over the years and different methods have been implemented for enhanced whey protein recovery both from quality and economic perspectives. The current chapter provides an in-depth understanding into the different available approaches for the recovery of whey proteins and also highlights the recent advances. The issues involved in whey protein recovery process that need to be considered for achieving commercial applications and emerging applications in the health and food sectors have been discussed too.

7.2 WHEY PROTEINS: OVERVIEW Whey proteins are obtained from whey and constitute about 20% of the total proteins present in the milk, while casein accounts for the remaining 80%. Whey proteins are comprised of β-lactoglobulin (β-Lg), α-lactalbumin (α-La), bovine serum albumin (BSA), and immunoglobulins (Ig) representing about 50%, 20%, 10%, and 10% of whey protein fractions, respectively. Among the major fractions, β-Lg is a small globular protein and contains two disulfide bridges and a free thiol. α-La is also a small globular protein and contains four disulfide bridges and needs calcium for its functional fold. BSA is a large globular protein with an alpha-helical structure and contains 17 disulfide bridges and a free thiol (Madureira et al., 2007). Besides these major fractions, whey contains some minor fractions (about 10% content) of the low abundance proteins such as lactoferrin (LF), PROTEINS: SUSTAINABLE SOURCE, PROCESSING AND APPLICATIONS

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TABLE 7.1 Whey Protein Components, Properties, and Benefits (Madureira et al., 2007; Marshall, 2004) Whey Protein Concentration Components (g/L)

Molecular Weight (kDa)

Number of Amino Acid Residues

β-Lg

3.2

18,277

162

Source of essential and branched chain amino acids, Retinol and fatty acid binding, possible antioxidant.

α-La

1.2

14,175

123

Primary protein found in human breast-milk. Source of essential and branched chain amino acids, helps in lactose production, calcium transport, acts as immunomodulator, anticarcinogen.

Ig

0.7

Light chain: 25,000, heavy chain: 50,000 70,000

BSA

0.4

66,267

582

Antioxidant, growth inhibition effect on human breast cancer cells, opioid agonist.

LF

0.1

80,000

700

Antioxidant, antibacterial, anticarcinogen, antiviral, antifungal, promotes growth of beneficial bacteria, together with betalactoglobulin and alpha-lactalbumin it can suppress tumor development.

LP

0.03

70,000

612

Antibacterial and antioxidant.

Benefits/Functions

Primary protein found in colostrum. Pathogen binding inhibition (chelates iron), antimicrobial, activation of phagocytosis, antiinflammatory, response to allergens.

lactoperoxidase (LP), proteose peptone (PP), osteopontin (OPN), and lisozyme (LZ) among others. The properties of important whey fractions with typical concentrations and their functions are shown in Table 7.1. The different important forms of whey proteins that are of commercial interest are listed as follows: • Whey Protein Powder (WPP) is produced from the whey obtained from cheese production based on the steps of clarification, pasteurization, and drying to provide a fine white powder. This form has applications in the food industry for confectionery, dairy, beef, snack, and bakery products. It is also available in different varieties including acid whey, demineralized whey, sweet whey, and reduced forms (Kassem, 2015), depending on the source of whey. • Whey Protein Concentrate (WPC) is a processed form of whey protein which has the lowest level of fats and cholesterol, as compared to other forms of commercially available whey. This form has a high level of bioactive compounds. It also contains carbohydrates in the form of lactose and has protein content in the range of 65% 70% (Levin et al., 2016). It has a mild to slightly milky taste. These forms are very rich in lysine and sulfur-containing amino acids and hence WPC can be a good complement in cereal-based (lysine-deficient) diets (Siso, 1996).

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• Whey Protein Isolate (WPI) is typically obtained after processing of whey to remove fats and lactose. It has lower quantities of bioactive compounds but a higher protein content (.90%). Similar to WPCs, it has a mild to slightly milky taste. It has an excellent amino acid profile and is ideal for both fat loss and muscle building-related applications (Shankar and Bansal, 2013). • Whey Protein Hydrolysate (WPH) are obtained using process of hydrolysis that breaks down the protein into smaller segments called peptides. This conversion makes the protein easier to digest and reduces the potential for allergic reactions (Geiser, 2003). WPHs show various beneficial impacts such as improvement in heat stability, enhanced digestibility and absorption, improvement in the texture of nutritional bars and shelf life, improvement of foaming and emulsification properties, reduction or elimination of the allergenicity of whey proteins, and creation of bioactive peptides (Khan, 2013). However, the added processing steps make WPHs expensive as compared to the WPCs and WPIs.

7.3 AVAILABLE APPROACHES AND RECENT ADVANCES IN RECOVERY OF WHEY PROTEINS Cheese making presumably originated some 8000 years ago after it was noticed that an acid-coagulated milk gel separates into curds and whey. The health benefits of whey led to the development of processes to isolate the solids by concentration and drying. In the 1920s, the initial industrial attempts at concentrating and drying whey typically involved conventional hot roller milk driers with processing steps as heating until a concentrated liquid was obtained, cooling to solidification, and then extruding in a tunnel (Gillies, 1974). Roller drying, in which whey is dried on the surface of a hot drum and removed by a scraper, is still used by some processors as part of whey powder production. The first important development came in 1933 when the long-tube multiple-effect evaporator was applied to whey processing (Gillies, 1974) to evaporate water to produce a concentrated whey stream. Later use of a spray dryer for whey processing came in 1937, which was a huge evolutionary step in the dairy sector. Around these years, as no separation technology was available for lactose removal from whey, lactose was a major part of the final dried whey product. Thus, the hygroscopic nature of the lactose in the dry product as well as the excessive cost of the process, mainly due to inefficient operations for concentration/ removal of water, prohibited industrial applications. Precrystallization of lactose before the drying in order to minimize problems of hygroscopicity as well as the careful manipulation of the heat conditions to minimize problems caused by the heat sensitivity of whey proteins were thus thought of as important research areas. Subsequent advances in the processing allowed the recovery of the hygroscopic and amorphous lactose as nonhygroscopic α-monohydrate lactose. In the case of nonhygroscopic whey powder production, a holding period was required to allow the crystallization of lactose into a nonamorphous, nonhygroscopic form prior to drying. However, there were a lot of processing problems, such as rapid drying resulting in amorphous α-lactose, which is highly hygroscopic. The absorption of moisture from air resulted in the formation of a hydrate that occupies more space than the amorphous form (Tsakali et al., 2010).

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FIGURE 7.1 A general flowsheet for whey processing to produce whey protein concentrate (WPC) and lactose from whey, which is a byproduct from cheese manufacturing. UF, ultrafiltration; RO, reverse osmosis; De-lac WP, de-lactosed whey permeate or mother liquor from crystallization (Yang, 2007).

After the recovery of lactose, the concentrated whey was typically dispersed by a rotary wheel or nozzle atomizer into a drying chamber through a hot-air stream, producing a powder with a final moisture content of 10% 14% (Deis, 1997). Lactose removal improved the purity and physical properties of dried whey powder. However, whey powder was available only in a heat-denatured, water-insoluble, yellowish-brown powder form until the 1970s and was reported to have limited use (Wingerd, 1971). In 1965, application of Membrane filtration in food processing came into light and ultrafiltration-based separation of whey was introduced in 1971, which subsequently proved a better alternative to efficient separation of lactose and proteins (Fig. 7.1) The principal processing methods in the manufacture of whey protein are typically dependent on the physicochemical properties of the whey available as raw materials. We now discuss the important processes applied in recent years in the overall manufacturing, citing the important operating conditions and recent trends based on an overview of the reported literature.

7.3.1 Precipitation Precipitation typically involves a change in the physical properties of a solution to instigate the insolubility of a solute for easy removal. Factors such as pressure, temperature, agitation rate, and holding time are the important operating parameters that affect the product characteristics, specifically the protein conformation and yield. Mainly, two types of precipitation methods, heat and chemical precipitation, have been reported for whey proteins. 7.3.1.1 Heat Precipitation The exposure to heat induces the aggregation of whey proteins which can precipitate out. Precipitation of whey proteins by heating is generally favored at acidic or near neutral pH conditions. Proteins are typically least soluble at a pH near the isoelectric point (pI) or in low ionic strength solutions, and are most likely to aggregate under these conditions (Mollea et al., 2013). Among the specific forms of whey proteins, α-La precipitates and aggregates better at acidic pH (3.5 5.5) and moderate temperature conditions (50 C 65 C) though requiring long reaction times, and is usually accompanied by the precipitation of BSA, Igs, and Lf, while β-Lg remains soluble. The precipitate so formed at acidic pH conditions is firmer and more readily separated than that obtained in unacidified whey. β-Lg precipitates rapidly and selectively at high

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temperatures (70 C 120 C) and pH near neutral or slightly alkaline condition (Wang et al., 2006; Fernandez et al., 2011). Processes for recovery of La have been described by Marshall (1982). Whey is typically heated and held for a specific time in a vessel (in batch or continuous mode) and then the precipitated protein is removed by settling (static or accelerated). The obtained precipitate is then washed, separated, and dried to obtain the final product. High speed centrifuges such as clarifiers and decanters can be used to effect both primary and secondary (after washing) separations. Ring, fluid bed, roller, and spray driers can be used to obtain the finished product with an average yield of 4.2 5.2 kg/m3. Yadav et al. (2014b) reported the application of heat-induced precipitation to recover the residual soluble protein after yeast cultivation in cheese whey which was continuously fermented with a cell recycle system at 40 C. The yeast was separated by centrifugation, and residual soluble protein from the fermented whey supernatant was then precipitated by heat treatment (100 C, pH 4.5, and 10 minutes incubation) with a maximum soluble protein recovery of 53% at pH 4.5, also giving 54% residual COD removal in the treated solution. The particle separation by gravity sedimentation at a pH of 3.5 was reported to yield 47% protein recovery, whereas a reactor scale-up study conducted at pH of 3.5 with agitation demonstrated a higher soluble protein recovery of 68% and a residual COD removal of 62%. Though there have been reports of effective separation based on easy methods, Heat precipitation has shortcomings such as the probability of whey protein denaturation and is a highly energy intensive process which may not be economical in large-scale processing. 7.3.1.2 Chemical Precipitation The process of selective chemical-induced precipitation is based on the use of chemicals like acetone, hydrochloric acid, and ammonium sulfate. The selection of chemicals is very important as their addition to whey may lead to the alteration of chemical properties due to changes in ionic strength, and sometimes chemicals may also make the whey unusable for specific products such as biopeptides. β-Lg has been reported to be effectively isolated from bovine whey using differential precipitation with ammonium sulfate followed by cation-exchange chromatography without altering its structure, and resulting in overall yield of 14.3% with .95% purity (Lozano et. al., 2008). β-Lg and BSA were also reported to be selectively precipitated with the use of 7.5 mM FeCl3 aqueous solution at pH 4.2 and 4 C (Kaneko et al., 1985), also giving a supernatant concentrated in α-La and Igs. Precipitation of soluble protein was also reported to be effective based on the addition of carboxymethylcellulose (CMC) by Yadav et al. (2014b). It was reported that thermal treatment followed by CMC addition applied sequentially resulted in an increase in protein precipitation to as high as 81% of the total soluble protein present initially. A comparative study of different precipitation methods has been reported by Jime´nez et al. (2012). It was reported that, although ultrafiltration (UF) showed the best results in terms of protein recovery and purity, the products obtained via ammonium sulfate and acetone precipitation had recovery of 84% 86%, while samples obtained via other methods had recovery in the range of 75% 80%. In addition, the use of ammonium sulfate was reported advantageous in that it limits bacterial growth and protects proteins from denaturation, enabling the recovery of nondenatured globulins. However, the acetone and ammonium

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sulfate-based precipitation methods demonstrated higher levels of ash and sugar contents in the final recovered product as compared to other precipitation methods due to the use of solvents and salts (Foegeding and Luck, 2002). An additional disadvantage of this chemical precipitation method is the need for dialysis or UF to remove salt. The water holding capacity of whey proteins is also negatively affected by acidic pH conditions and salts, which can lead to aggregation and an increase in the viscosity, a very important fact that must be considered while designing the precipitation method and also the subsequent step of hydrolysis of whey proteins, since the protein solubility is affected by these physical changes. Finally, it can be said that, although the thermal and chemical precipitation processes have generated considerable commercial interest, they have not been widely implemented for large-scale whey protein purification because of their complexity, high cost, low overall yield, poor sensitivity, and/or unacceptable product degradation associated with the extremes of heat, pH, or salt used during the processing (Tsakali et al., 2010).

7.3.2 Chromatographic Separation In the development stage for the recovery of whey proteins after the 1990s, emphasis was given to the cost-effectiveness of the process, with a need for preserving the functionality and bioactivity of native protein wherever possible. In this regard, chromatographic techniques, including traditional fixed bed and continuous approaches, offered more promise for whey protein isolation and fractionation (De Silva et al., 2003). A chromatographic method of separation is easy to develop, but the process parameters, such as choice of column matrix, salt, buffer, organic solvent, temperature, and gradient, make the optimization of the process complex (Bonnaillie et al., 2014). With the emergence of the chromatographic technique as a novel whey protein separation method, the dairy industry became more receptive to new and novel processing technologies based on chromatography for the cost-effective manufacture of whey ingredients, especially for those intended for valuable functional food applications. Chromatographic methods such as gel permeation, hydrophobic interaction, ion-exchange, and affinity chromatography have been popular for the separation and purification of whey proteins or of individual whey proteins. Ion-exchange and affinity chromatography are well known for the separation and purification of bovine lactoperoxidase (bLp) from whey. The principle behind ion-exchange chromatography is the reversible interaction between the target protein and membrane functional groups. It provides an additional level of selectivity beyond membrane processing because additional factors other than molecular size determine protein absorption, as the separation of proteins is based on the isoelectric points. The minor whey proteins, such as Lf and Lp, that hold a positive net charge at the pH of whey can be retained using cationic resins, while the major whey proteins, β-Lg, BSA, and α-La, which are negatively charged at the pH of rennet whey (typical pH range of 6.2 6.4) are easily separated in the same process, obtaining a fraction rich in these proteins (Tsakali et al., 2010). WPI grade protein, with virtually all the lactose removed was reported to be obtained using an ion-exchange tower in conjunction with membrane filtration (Foegeding and Luck, 2003).

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The use of affinity chromatography allows the direct adsorption of a protein at low concentration but the process cost can be a big concern especially considering the high price of ligands. A recent study reported that the triazine dyes offer good promise as effective ligands as they can be immobilized on different supports, present chemical and thermal stability, and allow the purification of whey proteins with a favorable cost/selectivity ratio (Wolman et al., 2005). Urtasuna et al. (2017) also reported the use of 18 triazine dyes immobilized on Sepharose 6B with a screening study for their performance as possible ligands for the purification of b-Lf from whey without any conditioning using affinity chromatography. Whey processed using the Reactive Red 4-Sepharose matrix in batch mode was reported to demonstrate the highest bLp purification yield (86.5% 6 3.8%), higher purification factor (46.1 6 1.1), and a relative purity higher than 80% according to SDS-PAGE gel densitometry. Even though some reports of successful application of chromatography for whey protein separation are available, in general, the dairy industry has not exploited this technology to its maximum potential considering the intricacy involved in the development of the complex chromatographic protocol and limitations of efficient scale up approaches (TovarJime´nez et al., 2012).

7.3.3 Membrane Separation The application of the membrane separation technique revolutionized the dairy sector as it provided a much better alternative for the recovery of the whey proteins as compared to the energy intensive thermal denaturation or complex chromatographic separations. Whey processing can employ four types of membrane filtration, including UF, microfiltration (MF), nanofiltration (NF), and reverse osmosis (RO), either individually or in combination. Membrane processes have been successful because they can be effectively and economically implemented at the large scale required for most dairy applications. The development of robust, synthetic, cleanable membranes and the refinement of a continuous operation using multistage operations and recycle loops have been the significant factors contributing to the success of this approach. UF works on the basis of the molecular weight cut-off (MWCO) and pore sizes, resulting in better protein rejection under the recommended values of 1 100 kDa and 20 200 nm respectively. Whey protein separation by UF is normally performed at temperatures below 55 C and an inlet pressure of 300 kPa (Ganju and Gogate, 2017). The retentate stream after the UF processing typically consists of protein, fat, and insoluble salts while permeate contains lactose, soluble minerals, and much of the water. The retentate is further evaporated and spray dried to achieve around 30% 60% whey protein by weight. UF is the most widely used process for the recovery of soluble WPC. In the early development stages, the use of cellulose acetate membranes was prominent, though these were later replaced by more resistant and durable membranes made of polysulfones or polyethersulfones. Preliminary initial processing steps such as filtration and centrifugation to remove suspended cheese or casein particles and fat, as well as other pretreatment procedures, are typically required to improve the performance of the UF plant (Matthews, 1984). Subsequent diafiltration (DF) after the UF processing offers

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further removal of lactose and mineral content and this combined UF DF approach can yield a high value retentate containing about 85% protein. Microfiltration is an approach by which a small pore size, often #1 μm, is employed to retain larger particles, such as fat and aggregated proteins, while allowing whey proteins, lactose, and salts to permeate (Modler, 2000). Thus, MF serves as a useful pretreatment step to UF, as the MF helps in retaining larger molecular mass particles (Neville et al., 2001), which can minimize the fouling in the actual UF operation. Microfiltration has also been applied for the removal of residual lipids from whey, prior to UF in combination with heat treatment (Merin et al., 1983). Heat treatment and/or pH adjustment helps to aggregate lipids and calcium phosphates (Gesan et al., 1995) leading to better separations. Microfiltration can also be used to remove microorganisms from whey thus eliminating the need for high temperature pasteurization. NF can be used in whey processing for concentration of the whey up to 20% 24% w/w solids or in combination with UF for obtaining high concentration retentate. Usage of RO has been reported for the recovery of whey proteins, removing two-thirds of the water in whey and leaving a concentrate that can be dried or shipped more efficiently. In this case, the applied MWCO was only 150 Da and, thus, membrane-fouling was reported as the main problem (Muller and Harper, 1979). Among the different membrane-based separation techniques, UF has been the most commonly applied and effective and, hence, we now discuss some of the important developments in the application of UF-based separations for whey processing along with some illustrations. 7.3.3.1 Case Studies and Developments in UF Process An evaluation of commercially available, wide-pore UF membranes was performed by Metzger et al. (2011) for the production of α-La enriched WPC. The feed whey was first mechanically clarified and pasteurized at 63 C for 30 minutes and then subjected to MF as a preliminary filtration step. The pilot-scale MF unit used in the work contained a 0.2 mm spiral-wound polyvinylidene fluoride (PVDF) membrane with a filtration area of 5.2 m2. The trans membrane pressure (TMP), temperature, and pH of the MF process were 55 kPa, 31 C, and 6.3, respectively. The prepurified stream devoid of casein fines, lipid materials, and aggregated proteins obtained as a permeate from the MF process was then used as the feed for the wide-pore UF studies performed using three membrane types (PVDF50, PVDF100, and PES300) at three different TMP (110, 207, and 310 kPa). The PVDF50 membrane operated at a TMP of 207 kPa was reported to yield the most effective separation of whey protein among the three types of wide-pore membranes used. An α-La purity of 0.63 and α-La:β-Lg ratio of 1.41 was reported to be obtained with an overall flux of 49.46 L/m2 h. An economic analysis of this process for a hypothetical plant presented in the work indicated a significantly lower production cost value of $17.92/kg compared to the market value for the cost of one kilogram of α-La enriched WPC 80, demonstrating the practicability of this study for commercial membrane-based α-La enriched WPC production. Typically the high cost and need for speciality equipment for UF membrane plants for whey processing are the constraints which should be looked at as an important research area. A cheaper equipment design was reported recently with successful operation in small dairy sector processing with capacity up to 50,000 L/day milk (Kukuˇcka and Kukuˇcka, 2013). The suitability of polysulfone UF membranes designed for commercial

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water treatment was investigated for the separation of protein from sweet whey as a cheaper alternative at pilot plant scale using the configuration represented in Fig. 7.2. A polysulfone spiral-wound membrane with 33.9 m2 as the area and 50 100 kDa as the MWCO was used. The feed, retentate, and permeate volume and compositions reported in the study are given in Table 7.2. The influence of two whey temperatures (9 C and 30 C) on the efficiency of protein concentration was also examined. It was reported that using the MWCO of 50 100 kDa resulted in the very successful concentrating of whey proteins of predominantly lower molar weights than 50 100 kDa. The application of investigated UF elements gave WPC with five to six times excess protein content compared to the initial

FIGURE 7.2 The flow sheet of an ultrafiltration pilot plant. WM, thermally insulated tank with a mixer for

whey; WT, tank with demineralized water; HE, heat exchanger; MF, polypropylene microfilter of 5 μm; UF, ultrafiltration modules; PT, tank of permeate; RT, tank of retentate; CIP, vessel for the storage of the solutions for UF membrane cleaning; CP1, feed pump; CP2, booster pump of UF modules; CP3, pump for demineralized water transport; R1, continuous flow meter of permeate, R2, continuous flow meter of retentate; G1/2, pressure gauges before and after MF; G3/4, pressure gauges before and after the membranes; F1, cumulative permeate volume meter; F2, cumulative retentate volume meter (Kukuˇcka and Kukuˇcka, 2013).

TABLE 7.2 Compositions and volume of feed Whey, retentate and permeate obtained after processing (Kukuˇcka and Kukuˇcka, 2013) Sample

T ( C)

Protein (%)

Lactose (%)

Whey

9

1.03

4.51

5.99

11,657

Retentate

5.22

4.41

11.79

1199

Permeate

0.21

3.79

5.78

10,458

0.99

4.12

5.33

4460

Retentate

5.24

4.68

12.03

424

Permeate

0.21

3.65

5.67

4036

Whey

30

TS (%)

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product. Better results were reported to be obtained with cold whey filtration. The obtained WPC was reported to be used successfully daily over a period of 6 months in the dairy production of ricotta and mixed with the milk for the production of semihard cheeses. Patil et al. (2014) reported the use of a three-stage ultrafiltration cascade for the continuous purification of whey protein isolate. Three different cascade configurations were examined and it was reported that a relatively constant and optimum performance was achieved using a 2 g/L solution concentration with 7.2 as the pH of the solution containing 5 mM NaCl at a cross-flow velocity of 0.1 m/s. It was also reported that all three cascade systems gave superior results as compared to a single-stage system, and higher degrees of separation were observed with a high ratio of product stream flow rate to waste stream flow rate. It was thus demonstrated that the separation efficiency can be enhanced with a suitable integrated cascade design. In addition, it has been reported that the separation efficiency can be improved based on hybrid approaches, such as ceramic microfilters combined with polyethersulfone or using modifications in flow configurations, such as use of tangential flow filtration modules, or by using regenerated cellulose membrane (Chollangi and Hossain, 2007).

7.3.4 Other methods for Whey Protein Separation 7.3.4.1 Use of Catalytic Membranes The whey protein separation can be made more effective by increasing the protein size using catalytic membranes during the separation process. Generally, small proteins like α-La and β-Lg permeate through membrane or get stuck in the membrane pores, thus reducing the permeate flux and whey protein recovery, as well as leading to potential fouling. Increasing the size of the protein, which can be achieved using different enzymes, can result in better separation and less fouling (Norazman et al., 2013). The effects of different parameters, such as protein rejection rate, whey protein recovery rate in the permeate, volumetric processing rate, lactose rejection rate, on the relative permeate flux and the overall membrane filtration performance, were assessed for the cheese whey in the presence of transglutamase as the catalyst (Wen-Qiong et al., 2017). It was reported that an enzyme concentration of 40 U/g, pH of 5, temperature of 40 C, and a retention time of 60 minutes gave an increase in whey protein recovery of about 15% and about 30% increase in the permeate flux with a substantial decrease in the membrane resistance. Considering the benefits, the use of transglutamase or in general a catalyst can be considered as a promising approach for the effective separation of whey proteins. 7.3.4.2 Electrodialysis Traditionally, the membrane separation process has been based solely on differences in molecular mass but recently it has been also possible to achieve separation of proteins with little or no difference in the molecular mass based on the principle of electrostatic rejection. Slight residual charges on the molecules obtained from adjustment of the solution pH and ionic strength can promote electrostatic rejection. The effective diameter of a protein increases as the ionic strength decreases. The difference in effective hydrodynamic size can be maximized by operating near the pI of the smaller protein and far from the pI

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of the larger protein, thus enhancing the separation. The use of low salt concentrations (1 20 mmol/L) increases the electrostatic and steric rejection by the membrane. Based on the use of different salts at varying loading, a multistep adjustment of the pH and ionic strength of the whey can be achieved which can allow fractionation of proteins using a sequence of membrane separation processes (Etzel, 2004). Almecija et al. (2007) studied the effect of pH on the separation efficacy, employing a 300 kDa tubular ceramic membrane in a continuous DF mode, based on measurements of the flux time profiles and the retentate/permeate yields of α-La, β-Lg, BSA, Ig, and LF. It was reported that a 300 kDa membrane could be effectively employed to fractionate the original array of whey proteins into two parts: α-La and β-Lg in the permeate; and BSA, Ig, and LF in the retentate. Thus it is clearly demonstrated that the adjustment of pH or the introduction of salts allows for enhancement of the separation efficiencies. 7.3.4.3 Colloidal Gas Aphrons Colloidal gas aphrons (CGAs) were first reported by Sebba (1971) as microbubbles (size of around 10 100 μm 1) composed of a gaseous inner core surrounded by a thin surfactant film, which are created by intense stirring of a surfactant solution. Their large interfacial area per unit volume, short separation times from bulk phase, and low viscosity make them particularly attractive for protein separation. Proteins interact with the surfactant in the aphrons due to the electrostatic and/or hydrophobic forces. Once mixing ceases, the aphron phase enriched in protein is separated easily from the solution due to its buoyancy. Generally the stability of aphrons should be high enough to allow protein adsorption. A disadvantage of the method is that in some cases, protein surfactant interaction can cause protein denaturation (Attwood and Florence, 1983). Fuda et al. (2004, 2005) investigated the fractionation of whey proteins using CGAs generated with either sodium bis-2-ethylhexyl sulfosuccinate or acetyl trimethyl ammonium bromide and reported that CGAs are analogous to ion exchangers when applied to whey. It was also reported that the type of surfactant, pH, and ionic strength played important roles in determining the selectivity of the process. The recovery and separation of LF, LP, or β-Lg from whey was demonstrated to be effectively achieved using CGAs. 7.3.4.4 Molecular Imprinting Molecular imprinting is known as a technique for creation of tailor-made binding sites inducing separations based on the shape, size, and functional groups of the template molecules (Chen et al., 2011). Molecular imprinting technology is also described as a method of making a molecular lock to match a molecular key (Chen et al., 2016). The method consists of the selection of a template molecule, which is associated with some functional monomers through noncovalent binding, and polymerization forming a template monomer complex and resulting in a molecularly imprinted polymer that has a cavity to recognize the template molecule, allowing its capture and separation from the complex mixture. Few studies have been reported related to the preparation of protein imprinted polymers using different strategies, despite the difficulties involved due to the large molecular size, fragility, and complexity of the molecules. Recovery of LF as a functional monomer using ethylene glycoldimetacrilate as a crosslinker with molecular imprinting was reported by Mendez-Palacios et al. (2006).

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The obtained imprinted polymers when tested against a protein mixture containing LF, showed an efficiency of 27%, while the control polymer retained only 1.6% of the proteins, indicating that the retention of the protein can be enhanced using the cavity formed by the template. Jime´nez-Guzma´na et al. (2014) reported that molecular imprinting using vinyl pyridine resulted in an efficiency of 34.5% when tested against a mixed solution of proteins, while the control did not retain any proteins, clearly demonstrating that the retention of the protein is not due to unspecific adsorption in the polymer, but rather to a selective retention in the cavity formed by the template. It is important to note that this approach has been applied only recently and there are many limitations currently, such as the complex nature of biological macromolecules, heterogeneous binding sites, template leakage, incompatibility with aqueous media, low binding capacity, and slow mass transfer (Chen et al., 2011), that are restricting its application at an commercial level.

7.3.4.5 Membrane Chromatography Membrane separation and chromatography are the most widely used methods for LF isolation from bovine milk and whey. Membrane separation is preferable compared to chromatography for whey fractionation as it does not include adsorption and elution steps and does not provide problems such as the costs of chromatographic material, buffers, and effluent disposal. However, membrane filtration is usually not effective in separating similar size proteins, such as LF and BSA, and other whey proteins (Johanson, 1960; Hahn et al., 1998). The application of chromatography alone is also not possible as the large volumes and high protein concentrations in whey may cause fouling of columns, long cycle times, large pressure drops, and offer complicated process control (Orr et al., 2013). The combination of a membrane and chromatography can overcome the limitations of the individual processes and enhance the separation process. Membrane chromatography (MC) is becoming more established as an efficient protein purification unit operation with respect to protein binding and high volumetric throughput. Several studies have been reported for the fractionation of whey proteins using MC. Further, to minimize the fouling and to enhance the LF binding, the MC system can be operated in a cross-flow mode. Teepakorn et al. (2015) reported that the selectivity of LF and BSA were independent of flow rate when strong anion and cationexchange membranes were applied for separation of LF and BSA mixture, which allowed the application of high flow rates. LF recovery of around 80% was reported to be obtained on the cation-exchange device using 25% ethylene glycol in phosphate buffer with pH of 12.0 and at the reduced flow rate of 1.0 BV/min. Similarly, BSA recovery of about 94% was obtained using the anion exchange membrane using 25% ethylene glycol in citrate buffer at pH of 3.0 as the eluent and at the lowest flow rate of 1.0 BV/min. MC can thus be said to be a very effective technique for BSA LF mixture separation and could be applied successfully to the separation of other proteins of similar size and different isoelectric points. Though promising separations have been reported in the discussed case studies, certainly the major challenge for industrial applications especially for whey processing would be the higher price of the available MC devices.

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7.3.5 Drying Drying is an integral operation after the separation of whey proteins, and is required to obtain the final commercial grade dried whey powder with possibly enhanced physical properties. Spray drying and freeze drying are the two approaches generally considered for whey powder processing and hence discussed in this chapter. 7.3.5.1 Spray Drying Spray drying involves atomizing the liquid whey and introducing it into predried hot air, causing the evaporation of water (Anandharamakrishnan et al., 2010). WPC and WPI in dry powdered form are normally produced from the concentrated liquid whey using a spray drying process. Spray drying is continuous in operation and ideal for heat-sensitive products because of its short drying time. In this process, hot air enters the drying chamber, evaporating the moisture from sprayed liquid, reducing the air temperature as it passes through the chamber. In order to remove dry products from humid air, several cyclones are placed outside the dryer, which recover the product through vortex separation, minimizing the loss of dry powder. Fig. 7.3 shows the typical spray drying process for whey powder manufacture based on spray drying. The operating conditions of atomization, such as the type of spray/air contact, drying air temperature, and feed parameters (concentration, temperature, and the degree of feed aeration), play important roles in determining the physical and morphological properties of the final produced powder. A detailed investigation into the effect of operating conditions and the parameters of spray drying on the final recovered whey powder has also been reported (Chegini and Taheri, 2013). Despite advantages, such as rapid drying, large throughput, and continuous operation, spray drying also offer drawbacks, including the denaturation of proteins and higher

FIGURE 7.3 Sketch of the industrial spray-drying process (Donz et al., 2014).

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costs of operation. Some variations in the mean particle size and size distribution can also be expected, which can be a problem based on the specific application requirements. The use of ultrasonic atomizers can overcome this limitation of nonuniform particle size distribution and can provide an alternative and possibly efficient approach for spray drying. Two configurations of ultrasonic atomizers, simple nozzles and nebulizers, are commonly available and can be selected depending on the expected particle sizes. In the ultrasonic nozzles, the liquid feed is passed through a vibrating horn generating standing waves or capillary waves to produce an aerosol-like structure. The ultrasonic nebulizers produce a fountain-like structure in a thin liquid film and are operated at higher frequencies (Ashokkumar et al., 2009a). Ultrasonic nozzles are more precise, reliable, and controllable. They can handle very low velocities and do not clog. Ultrasonic nozzles prevent the wasteful overspray and waste generation thus reducing feed material consumption (Gajendragadkar and Gogate, 2016). Ultrasonic atomizers typically produce spray with a very low velocity, eliminating the requisite for a large-sized drying chamber thus cutting down the capital and operating costs for the process (Ashokkumar et al., 2009a). It is also important to note that currently the application of ultrasonic nozzles in the dairy industry is very limited, partly because of the lack of suitable designs operating at the required scales of operation for whey processing in an energy efficient manner. 7.3.5.2 Freeze Drying Freeze drying, also known as lyophilization, is a popular method of producing stable particulate products, and most suitable for drying heat-sensitive materials, which can be damaged by higher temperature processing methods, such as spray drying. In the freeze drying technique, the component to be dried is frozen first and then exposed to heat under vacuum which causes sublimation of the frozen liquid. The major driving mechanism for heating is conduction, by bringing the frozen materials in contact with the heated shelf, and radiation from the upper and lower shelves (Ganju and Gogate, 2017). Freeze drying is considered to be the best method for producing high-quality dried products that retain their native properties with minimized deterioration of components. However, this method involves high capital and operating costs, mostly due to the requirement of low temperatures, high vacuum, and long residence times. In addition, the throughputs of such dryers are limited and hence the applicability for the recovery of whey proteins really needs to be looked at considering the requirement of processing of much higher volumes (Anandharamakrishnan et al., 2010). Spray freeze drying (SFD), which is a two-step process consisting of spray-freezing followed by freeze-drying as shown in Fig. 7.4, can be a very useful technique as it avoids the long residence time required in only freeze-drying process. Spray freezing is similar to spray drying but the only difference is that the generated spray is freezed. In the case of freeze drying, the latent heat of sublimation is supplied by conductive or radiative heating. However, this is difficult to apply uniformly to a powder (used to take advantage of the small particle size and reduced drying times in the case of SFD) taken on a tray without the risk of particle melting and collapse (Anandharamakrishnan et al., 2010). A process modification whereby fluidization is performed at subatmospheric pressures allows rapid freeze drying and using much less gas, as demonstrated by Anandharamakrishnan et al. (2010) for WPI. The SFD rig in which the fluidized bed freeze drying section is capable of

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FIGURE 7.4 Atmospheric spray freeze-drying equipment. (Left) Photograph of equipment. (Right) Schematic of chamber with spray and drying gas inlets (Sebastia˜o et al., 2017).

operating at reduced pressures is reported in the work. The fluidized bed was fitted with thermocouples to measure the temperatures of the particle bed and the exit gas (located B3 cm above the bed). The study demonstrated the efficient working of the fluidized bed SFD technique at subatmospheric pressures (0.1 bar) using whey protein isolate solution (20% w/w solids) at gas inlet drying temperatures ranging from 210 C to 230 C. The final dried powder consisted of highly porous particles with little loss of activity for β-Lg and α-La, the principal proteins in the isolate. A wet basis moisture content of 8.1% was achieved after freeze-drying at 210 C for only 1 hour, while at 30 C a longer drying time (100 minutes) produced a wetter product (14% wet basis) in the conventional operation of freeze drying. Despite some limitations, this technique appears to be able to produce powdered pharmaceutical and food products more quickly than is normally possible by vacuum freeze drying processes. It is important to also note that the transformation of this process on a commercial scale is constrained due to the complexity involved and the need for very large areas.

7.3.6 Proposed Improvements in the Whey Protein Manufacturing 7.3.6.1 Pretreatment of Whey During the long storage involved in the transport from the dairy industry to the whey processing unit, whey has a typical tendency to form a sticky syrup-like solution and there may also be microbial contamination due to the presence of lactose. These problems need processing mainly for microbial inactivation. The conventional method for microbial inactivation is a thermal treatment approach, however, one of the main disadvantages in thermal treatment is that the use of high temperatures usually induces the formation of whey

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protein aggregates and hence problems like gelling or thickening of whey streams are observed. Denaturation of whey proteins is observed at the high temperatures associated with heat treatment, particularly when higher treatment times are used. Precautions can be taken that the temperatures required for preheat treatment for whey do not exceed 70 C, so as to maintain the integrity and stability of whey proteins (Ashokkumar et al., 2009b), but this may lead to inefficient treatment in terms of a reduction in microbial activity. As a result of heat treatment, sourness based on the acidity and formation of higher amounts of sediment is also likely to occur, reducing the nutritional and sensory qualities of the finally recovered product (Jeliˇci´c et al., 2008). Thus, it is important to look for alternative or complementary techniques to thermal treatment with an objective of retaining the quality of recovered products. One such treatment is based on the use of ultrasound or sonication either alone or in combination with thermal treatment. In recent years, the application of sonication has been explored for both microbial and enzyme inactivation. Cavitation is believed to be the main bactericidal effect of ultrasound (Piyasena et al., 2003) and is usually a local phenomenon giving hot spots and shear effects. Pretreatment using ultrasound for a short period of time (20 kHz frequency, ,5 minutes as treatment time) following a preheat treatment (80 C, 1 minute as the reduced time) for the processing of whey proteins (4% 15% (w/w) WPC) was reported to reduce viscosity and size of aggregates in addition to the expected inactivation (Ashokkumar et al., 2009b). Sonication alone can cause microbial inactivation but sonication with heat (thermosonication) provides a greater effect for the inactivation of microorganisms which can be additive or even synergistic (Lopez-Malo et al., 2005). In addition, no negative impacts on crude protein, casein content, fat content, or lactose content in milk were reported as a result of thermosonication (Cameron et al., 2009), which is quite important as the quality of processed whey proteins and lactose will be retained. A combination of ultrasound and heat during the pretreatment can also be beneficial to reducing the problem of thickening or swelling of whey protein-based dairy streams. It can also increase the heat stability of whey proteins (Gajendragadkar and Gogate, 2016). Jeliˇci´c et al. (2008) studied the effect of ultrasonication and thermosonication on microbiological quality and sensory properties of fresh cheese whey. It was reported that ultrasonication-only treatment had no impact on the reduction in the content of microorganisms, whereas thermosonication showed microbial inactivation, the extent of which increased with an increase in the ultrasonic power input and/or exposure times. It was also reported that the sensory properties after ultrasonication and thermosonication were considerably improved in comparison to the simulated pasteurization processes. Khaire and Gogate (2018) studied the effect of different pretreatment methods on whey UF and the final recovery of the value-added products. It was observed that thermosonication showed better results compared to thermal and ultrasonic pretreatments, delivering a higher permeate flux and better protein and lactose separation. It can be thus established that a properly optimized thermosonication approach helps in extending the shelf life of whey and also gives better product characteristics. 7.3.6.2 Avoiding Membrane Fouling The major problem affecting the cost and efficiency of many whey processing industries is the fouling of membranes, especially the UF membranes which are the most used

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membranes for separation. Fouling is mainly due to the accumulation of feed constituents, such as colloids, proteins, macromolecules, and inorganic materials, as a cake layer on the membrane surfaces and pores (Lee et al., 2004). Membrane fouling is generally characterized by the reduction in permeate flux as a result of pore blockage and accumulation of material near the surface of the membrane, reducing the throughput value during largescale processing. Methods like forward flushing and back flushing are well known for membrane cleaning. Further cleaning using chemicals and electric field inducement are in use but have shortcomings such as membrane damage, secondary pollution, high cost, etc. The use of ultrasound in membrane cleaning has emerged as a new and efficient technique in recent years. A number of physical effects, such as turbulence, microstreaming, microjet formation, and shear effects on collapse of cavitation bubble, can lead to the removal of a fouling layer and the disruption of the physical aggregates of whey proteins (Muthukumaran et al., 2005; Ashokkumar et al., 2009a). The collapse of a bubble/cavity due to the pressure fluctuations releases a high amount of energy which is sufficient to overcome the attraction between the membrane and the foulant particles and dislodge the particles. Cavitation produces turbulence near the membrane surface, which also assists in preventing the cake layer formation of particles over the membrane, and thus, results in enhanced permeate flux and also yields better separation of lactose and proteins during the whey processing. Many studies have reported the use of ultrasound for membrane cleaning and improved processing. Muthukumaran et al. (2005) used low-frequency ultrasound to facilitate cross-flow UF of dairy whey solutions. Flat sheet polysulfone UF membrane was placed between two acrylic manifolds, which were in turn held in place by stainless steel plates. The membrane unit was completely immersed in 5000 mL of water contained in the ultrasonic bath with the membrane unit kept 3 cm above the bottom of the ultrasonic bath throughout the entire experimental period. The schematic representation of the applied configuration is shown in Fig. 7.5A. Experimental results established that ultrasonic irradiation at low power levels significantly enhanced the permeate flux with an enhancement factor between 1.2 and 1.7. It was reported that the use of ultrasound acts to (A)

(B)

Permeate

Connect to generator Ultrasonic horn

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Ultrasonic bath

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Transducers Membrane Gear pump

Computer Whey solution

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FIGURE 7.5 US-assisted UF process: (A) Using bath configuration (Muthukumaran et al., 2005). (B) In-line direct irradiation of ultrasonic waves for MF process (Mirzaie and Mohammadi, 2012).

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lower the compressibility of both the initial protein deposit and the growing cake. It is also important to note that the application of ultrasonic baths for membrane cleaning involves a high amount of energy consumption and all the irradiated waves might not be used for cleaning purposes. An alternative to an ultrasonic bath is the use of an in-line direct irradiation approach as shown in Fig. 7.5B, which was reported for the flux enhancement of cellulose ester membrane based MF with fresh cow milk as feed by Mirzaie and Mohammadi (2012). A detailed study into the effects of the different parameters established that the highest flux enhancement factor of 490% was obtained at a pressure of 0.5 bar, ultrasonic power of 40 W, and distance of ultrasound source from membrane unit of 2.6 cm. Continuous irradiation of ultrasonic waves was reported to increase the flux enhancement factor by a degree of 33% as compared to the pulse irradiation. It was also reported that both SEM analysis and permeation flux measurement of pure water confirmed that ultrasonic irradiation had no destructive effects on the membrane surface. The application of ultrasound for improved UF processing needs to be analyzed in greater detail from an energy and economic viewpoint for industrial application. The use of low ultrasonic power levels can impact directly on the economic viability of any large-scale application and can also minimize the possibility of membrane damage. Thus the ultrasonic power needs to be properly optimized to arrive at the best treatment protocol. Large-scale trials of ultrasonic application to membrane modules need to be studied in order to analyze the energy requirements and impacts on the membrane configuration for the eventual industrial application. 7.3.6.3 Improving the Whey Protein Stability Whey proteins are stable in milk in their native form. However, the application of heat treatment above 60 C may cause denaturation and aggregation of them through the noncovalent thiol disulfide interactions (Guyomarc’h et al., 2015). Aggregated whey proteins offer processing problems during cheese and yoghurt manufacture as well as in any processing of the recovered whey proteins. For example, during cheese making, renneting is adversely affected by heat-treated and denatured whey proteins because of interactions between whey proteins and casein micelles. In addition, denaturation of whey proteins is accompanied by the release of small sulfur-containing compounds, such as hydrogen sulfide and methane thiol, which are highly flavorsome compounds and cause cooked flavors in processed products (Al-Attabi et al., 2009). The formation of whey protein aggregates or complexes can lead to the fouling of heat exchangers, which, in turn, reduces heat transfer, increases pressure drop, and also limits run times of industrial plants (Visser and Jeurnink, 1997; Bansal and Chen, 2006). Whey protein aggregation can be controlled using different methods, including blocking free SH groups by chemical modification, addition of hydrophobic compounds, addition of chaperone proteins, addition of carbohydrates, cross-linking with TGase enzymes, conjugation with carbohydrates, and chelation of minerals. Furthermore, modification of the native proteins to a more stable state can help in producing stable whey protein products (Wijayanti et al., 2014). The reported modifications include hydrolysis and partial or total denaturation/aggregation based on the enzymatic treatment, microparticulation, and microencapsulation. The controlled denaturation of proteins during pretreatment of whey using ultrasound treatment can also be useful in obtaining a final stable product without

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any processing problems. Thermosonication pretreatments have shown favorable results and have emerged as an alternative approach to high temperature processing for microbial inactivation. Moreover, ultrasound treatment either alone or in combination with thermal treatment has also been reported to increase the whey protein stability (Gajendragadkar and Gogate, 2017). Chandrapala et al. (2011) reported the sonication effect on reconstituted solution of WPC 80 powder with MilliQ water. Some minor changes in whey proteins were indeed reported to be observed with the application of ultrasound but no aggregation or denaturation of protein was reported. It was thus established that the use of only ultrasound in the recovery does not induce any significant changes, thus maintaining the nutritional value of whey proteins. Likewise, the functional properties were retained even after postheating, spray and freeze drying, and subsequent reconstitution. Zisu et al. (2010) investigated the heat stability of whey proteins using ultrasound combined with heat at an industrial pilot-scale operation using various whey protein products. It was demonstrated that the combination of heat treatment and sonication improved the heat stability of reconstituted WPC (5% w/w), whey protein retentate, and milk protein retentate. Thus it can be established that ultrasound also offers a promising approach for enhancing the stability of the final whey protein products depending on the applied sonication conditions.

7.4 IMPORTANT APPLICATIONS OF WHEY PROTEINS 7.4.1 Healthcare Applications Whey protein is considered as an excellent protein for individuals of all ages to improve and maintain their health. Traditionally, whey protein was only used by athletes and bodybuilders to promote muscle growth. But in the past few years, whey protein has found applications in clinical treatments and for overcoming many health issues. Whey protein is a rich source of bioactive peptides which may play a role in the dietary management of chronic diseases. In addition, whey protein provides immunity support, increases muscle mass, boosts metabolism, helps in weight management, and also benefits overall health. The established and emerging benefits of whey protein in healthcare applications are discussed in the following sections. 7.4.1.1 Antiinflammatory and Antioxidant Inflammatory or oxidative stress results in cystic fibrosis, pneumonia, diabetes, cancer, atherosclerosis, myocardial infarction, aging, and a host of other degenerative diseases (Essick and Sam, 2010). Plasma levels of LF have been found to be elevated due to release from neutrophils during infection, inflammation, tumor development, and iron overload. Thus it is expected that external dosage of LF can be supportive treatment. The feasibility of using pressurized whey protein for lowering the risk of pulmonary infection by the pathogen Pseudomonas aeruginosa was investigated by Kishta et al. (2013). It was reported that the whey-fed mice showed decreased levels of inflammatory response, oxidative stress, and lung damage. In addition to this, whey has been reported to offer potent antioxidant activity, likely by contributing cysteine-rich proteins that aid in the synthesis of glutathione, a potent intracellular antioxidant. The amino acids, methionine and cysteine, are precursors of glutathione and taurine, which serve as the “body’s own” antioxidants. PROTEINS: SUSTAINABLE SOURCE, PROCESSING AND APPLICATIONS

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Overall, whey proteins act as an important component to increase endogenous antioxidant enzymes and to reduce oxidative stress markers (Kassem, 2015). 7.4.1.2 Infant and Expected Mothers Nutrition Whey proteins are used as a vital ingredient in a wide variety of infant formulas especially for premature infants owing to the fact that it contains many of the components found in human breast-milk (Solak and Akin, 2012). The infant formulas containing whey proteins are thus considered as the best alternatives to breast feeding. The addition of LF (an important whey protein component) to a feeding formula can increase the levels of Bifidobacteria in bottle-fed babies, which decreases the potential of developing atopic disease for at-risk infants with family history, as well as improved gastrointestinal immunity. Certain types of whey proteins-based infant formulas have also been shown to help to reduce crying in the case of infantile colic. In addition, whey proteins are an excellent choice for the expectant mother who needs an increased amount of proteins. Pregnancy can increase the body’s protein needs by up to 33% (Gupta et al., 2012), this increased requirement can be effectively achieved based on a diet involving whey proteins. 7.4.1.3 Wound Healing Protein and its amino acids are the building blocks that initiate the growth of new skin during the healing process and inadequate amounts of protein or diets high in poor quality proteins, such as gelatin, may delay it. Whey proteins are a very high-quality protein containing the desired constituents for healing and are often the preferred choice for high protein products recommended by physician for postsurgical wound healing (Gupta et al., 2012). 7.4.1.4 Antidiabetic Whey proteins are high biological activity proteins which are of great benefit for people who have diabetes. Whey proteins have been demonstrated to reduce serum glucose level in individuals, maintain muscle mass, boost the release of satiety hormones [cholecystokinin, leptin, and glucagon like-peptide 1 (GLP-1)], and also lower the secretion of the hunger hormone ghrelin (Sousa et al., 2012). Whey protein has been reported to be one of the candidates, along with its constituents, leucine, isoleucine, valine, lysine, and threonine, for mediating the insulin increment (Salehi et al., 2012). However, the broader and continuous use of whey proteins in diabetes management is not well proven and needs to be explored in greater detail. 7.4.1.5 Hepatitis Treatment The supplementation of whey protein demonstrates beneficial effects in patients infected with Hepatitis B or C. Bovine LF has been reported to prevent Hepatitis C virus (HCV) infection in vitro in a human hepatocyte line (Ikeda et al., 1998). In another open study on patients with Hepatitis B virus (HBV), it was reported that the use of whey proteins showed decreased levels of serum lipid peroxidase and increased IL-2 and NK activity. In six of eight HBV patients, serum alanine transferase levels were reduced and plasma glutathione levels increased in five of the same eight on whey protein treatment. This trial confirmed the effective use of whey protein in the treatment of HBV (Marshall, 2004).

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7.4.1.6 Cardiovascular Disease Prevention Heart disease is considered to be one of the leading causes of death for both men and women. Whey protein helps fight against hypertension and reduces the elevated cholesterol level, a main factor associated with heart disease. Whey protein intake reduces cardiovascular disease (ischemic stroke) risk, though the precise role of its peptides in the regulation of vascular endothelial function has not been adequately investigated (Patel, 2015). Fermented milk with WPC has been reported to lower triglyceride levels in the blood (Solak and Akin, 2012), thus reducing the potential of heart disease. Hydrolyzed whey protein isolate reduces cholesterol, which is again beneficial. It was also reported that whey proteins improve blood pressure and vascular function in overweight and obese individuals (Pal and Ellis, 2010). Some amino acids of the whey proteins, for example, α-La and β-Lg, are precursors of peptide inhibitors of angiotensin-converting enzyme (Luhovyy et al., 2007), which is a key enzyme in the regulation of blood pressure. Whey derived extract (NOP-47) ingestion was reported to increase the impaired brachial artery flow-mediated dilation (improved endothelial function). The overall literature suggests that whey protein offers significant antihypertensive effects and whey protein has the potential to be used as an added component in dietary plans and in functional foods aimed at reducing cardiovascular disease risks. 7.4.1.7 Anticancer Several studies have suggested that whey protein may confer benefits on cancer patients. It has been also demonstrated that the hydrolysis of the protein might improve the anticancer efficacy (Patel, 2015). Diets supplemented with LF or with β-Lg showed enhanced protection against the development of putative tumor precursors. Also, diets containing whey have been shown to reduce intestinal, mammary, and colon cancers (Hakkak et al., 2001). The possible mechanism behind the role of whey proteins in anticancer activities may be related to their sulfur amino acid contents (cysteine, methionine) (McIntosh et al., 1998). A 48-year-old caucasian female with recurrent cervical cancer was administered with whey protein (10 g thrice daily) and a weekly intramuscular injection of testosterone enanthate before and during the standard-of-care (SOC) chemotherapy. As a result of this applied combination therapy, improvement in the lean body mass, physical activity, and overall quality of life was reported (Dillon et al., 2012). The use of whey protein also provides an excellent choice for meeting the nutritional requirements of cancer patients and help in fighting the effects of cancer drugs. 7.4.1.8 Immunomodulation The immune system is a vast and complex network of cells, organs, and molecules that work together to defend the body against foreign microorganisms, such as bacteria, parasites, and viruses, and anything that triggers an immune response to these invaders is referred to as an antigen (McIntosh et al., 1995). Whey protein concentrates have been reported to enhance innate mucosal immunity during early life and offer a protective role in some immune disorders (Pe´rez-Cano et al., 2007). In particular, three whey peptides (α-LA, β-Lg, and LF) are known to boost the immune system by increasing the production of glutathione. The effects of WPCs on blood parameters, plasma cytokine profiles,

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immune cell proliferation, and migration were investigated in mice by Badr et al. (2012). Significantly reduced levels of plasma and cholesterol were reported in the whey proteintreated group compared to the control group. In addition, in another group the intake of whey proteins was reported to improve the concentration of glutathione, confirming the mechanism for enhancing immunity. 7.4.1.9 Treatment of Human Immunodeficiency Virus Whey protein isolate has been reported to be very effective in treating HIV patients. Glutathione deficiency is a common problem for individuals infected with HIV. Whey protein helps in elevating the levels of glutathione and thus provides an extremely important antioxidant involved in the maintenance of functional and structural integrity of muscular tissue undergoing oxidative damage during exercise and aging (Kassem, 2015). With an effort to increase cysteine, and ultimately glutathione content, several studies have been reported on the use of whey proteins in HIV-positive individuals (Marshall, 2004, Micke et al. 2002). In the study by Micke et al. (2002), 30 subjects with HIV were randomized to receive a daily dose of 45 g whey proteins from one of two sources, Protectamin or Immunocal, and the Protectamin-supplemented group demonstrated significantly elevated glutathione levels. Whey protein-supplemented HIV patients demonstrated that although a whey protein supplement could not result in weight gain in these patients, it was able to strengthen their immune system.

7.4.2 Applications in the Food Industry Different food products can be enriched with different whey protein depending on their utilization in specific applications. Using whey proteins can generally give advantages such as enhanced biological values, a superior physical, textural, or other functional properties, the ability to formulate high protein and/or low-lactose products, and improved sensory attributes. The important properties of whey protein, such as gelation, thermal stability, foaming, and emulsification, has made whey protein favorable in food applications (Patel, 2015). Whey protein can be directly applied as an ingredient in many foods, such as dairy products (particularly cheeses, yogurts, dairy-based dry mixes, and dairy-based beverages), medical foods, sport foods, beverages, soups, and protein bar. WPC and WPI are used for producing different cheeses like feta, ricotta, processed, and spread cheeses. Other uses of whey protein include baked goods, meat products, low-fat spreads, desserts, and toppings. The use of about 25% 50% skim milk powder which is the key ingredient in dairy ice cream manufacturing has been now replaced by WPC or demineralized delactosed whey powder. Whey proteins have a number of properties common with egg white proteins and hence can be used as a replacement. The beating of egg is a crucial step in cake formation and it is important that the egg is not overwhipped. For this and economical reasons, many attempts have been made to substitute egg white proteins with WPC. The ability of nonheated whey protein high methoxyl pectin complexes to act as a fat-replacer and texturing agent in reduced-fat yoghurt was also reported in one recent study (Krzeminski et al., 2014). Additionally, Pearl millet supplemented with barley flour, WPC,

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carboxy methyl cellulose, and water was used successfully to make pasta (Yadav et al., 2014a). New methods and technologies need to be explored for the effective whey protein applications in the food sector at commercials scale and as an auxiliary/replacement to different ingredients in food manufacturing processes, wherever possible, for deriving the improved functional and nutritive properties and economic benefits.

7.4.3 Application as Edible Films and Coatings Edible or biodegradable films constitute a convenient means to prolong the shelf life of foods and also maintain their quality. These films are “green” alternatives to traditional plastics and thus also help in controlling environmental pollution. Based on its excellent oxygen barrier properties, whey protein films can be competitive biodegradable materials replacing nylon or polyesters, which are typically used as oxygen barriers currently (Jooyandeh, 2011). Edible films can be eaten together with the food with or without further removal. Edible films and coatings can replace and fortify natural layers and prevent the loss of moisture and important components (Soares de Castroa et al., 2017). Moreover, films and coatings can allow a controlled exchange of important gases and provide surface sterility (Huber and Embuscado, 2009). The material composition of films and coatings is mostly similar with the thickness being the only difference (Ramos et al., 2012). Edible films can be first molded as solid sheets and then applied as a wrapping on the product. They can also be applied in a liquid form to the material, typically by immersion in a polymer solution. However, with consideration to some limitations on the mechanical features, plasticizers, such as sorbitol or glycerol, are required to improve the resistance to moisture transfer and to enhance flexibility significantly (Basiak et al., 2017; Ramos et al., 2012). Therefore, when blended with suitable plasticizers, whey protein-based films and coatings offer a potential technology to develop new ecoefficient food packaging products. Whey protein was also reported to reinforce the oxygen barrier properties of commercial compostable plastic film. The coating not only upgraded the barricade but also bestowed fast biodegradability (Cinelli et al., 2014). A recent and interesting study by Boyacı et al. (2016) demonstrated the development of an antimicrobial film composed of whey proteins, beeswax, oleic acid, and lysozyme for the preservation of opened packaged food at home. The overview has clearly highlighted the potential application in food packaging though much work is required for effective translation to commercial scale operation.

7.4.4 Whey Protein Hydrogels and Nanoparticles Whey protein has found applications as hydrogels and nanoparticle carriers for encapsulation and controlled drug delivery. Hydrogels are considered polymeric three-dimensional networks that have the ability to assimilate high amounts of water or biological fluids. The presence of hydrophilic groups, such as OH, CONH , CONH2 , and SO3H, in polymers forming hydrogel structures determine their affinity to absorb water. Hydrogel nanoparticles have been the center of attention in recent studies on drug delivery systems due to their unique potential of combining the characteristics of a hydrogel system with a nanoparticle (Soares de Castroa et al., 2017). Nanostructured particles from whey protein

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have gained more attention in recent years because of their protein gelling ability, easy preparation, relative low cost, and based on the fact that the size distribution can be effectively monitored. Sadeghi et al. (2014), using a cold gelation method, reported effective microemulsification of whey protein, glucono-δ-lactone, date pit extract, and CaCl2. The use of date palm pit was due to its polyphenol richness, which is known to have biological activity as an antioxidant, antiinflammatory, and antibacterial material. The nanoparticles presented a controlled release character based on the phenolic content measurement, indicating potential for a future formulation as a carrier for date palm pit extract. Additionally, several changes can be induced in the whey protein matrix contributing to the formation of complexes with other biopolymers, chiefly polysaccharides, as a starting point for the development of different nanosystems (Ramos et al., 2014). Despite their ability to bind hydrophobic compounds, nanoparticles from β-Lg present instability at a pH range below their pI values with and without high salt concentrations, causing secondary aggregation, which indeed is the current challenge in producing an effective whey protein isolate microgel system (Dai et al., 2015).

7.5 GLOBAL WHEY PROTEIN MARKET The global market value for whey powder and whey proteins amounted to approx. US$7.7 billion in 2016, up significantly compared to 2015 given increasing volumes, but more importantly due to the global price recovery in the wake of the global dairy crisis. According to the report—Whey Book 2017—The Global Market for Whey and Lactose Ingredients 2017 21, the global whey protein (powder and concentrate) market grew at a CAGR of around 4% during 2010 17 reaching a volume of 4.1 million metric tons in 2017. At constant 2017 prices, the market value is forecasted to surpass US$9 billion by 2021, corresponding to average annual growth of 4%. The global market value for pharmaceutical and conventional lactose, which is typically obtained as co-product to whey proteins, along with permeate powder increased to almost US$2.3 billion in 2016, also benefitting from recovering prices, and is expected to reach US$2.7 billion in 2021, implying a compounded annual growth rate of 4% over the forecast period at constant prices. In developed countries, a clear product trend of adding value to the whey pool has become apparent as high-end protein products, that is, WPC80, WPI, as well as demineralized WP90, to some extent, have been growing strongly, whereas the production of whey powder and other low-end products is stagnating. Thus it is imperative that the changes in the product profile is a must to sustain the changing market scenario. The EU and US represent the major producers as well as the major markets for whey ingredients. In recent years, Asia has become the major market for permeate powder and has surpassed North America in terms of market size for permeate. Although India is the leading dairy milk producer, the whey protein market in India is not growing from a global standpoint. China continues to remain the dominating importer of whey and lactose ingredients, while the EU and the US are the key global suppliers by a long shot (IWC, 2017).

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7.6 CONCLUSIONS AND FUTURE ASPECTS The present chapter has focused on providing an in-depth understanding of the different available approaches for the recovery of whey proteins and has also highlighted the recent advances. The development of technologies, such as membrane separation, spray/ freeze drying, membrane chromatography, electrodialysis, and the other methods mentioned, has opened the door to far more proficient, cost-effective, and high-grade commercial production of whey protein products suitable for numerous applications. Whey protein has been considered as a substitute to traditional ingredients owing to its specific structural and biological functions that allow products with desirable characteristics and potential applications to be obtained. Whey protein-based edible films, hydrogels, nanoparticles, microencapsulated products have directed the attention of the scientific community towards the distinct biological and functional characteristics of whey proteins. Moreover, the clinical indications have incorporated whey proteins in the treatment of various diseases and the further potential needs to be fully recognized. Overall, it can be said that fulfilling the growing demand for high quality whey products and exploring novel technologies to meet the requisites of the consumer are the needs of the hour and the pathway to the future.

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Further Reading Castro, G.A., Maria, D.A., Bouhallab, S., Sgarbieri, V.C., 2009. In vitro impact of a whey protein isolate (WPI) and collagen hydrolysates (CHs) on B16F10 melanoma cells proliferation. J. Dermatol. Sci. 56, 51 57. Cheang, B., Zydney, A.L., 2004. A two-stage ultrafiltration process for fractionation of whey protein isolate. J. Membr. Sci. 231, 159 167. Cordova, M., Griebenow, K., Zale, S.E., Tracy, M.A., 2007. Protein spray-freeze-drying-effect of atomization condition on particle size and stability. Pharm. Res. 17, 1374 1380. De Wit, J.N., 2001. Lecturer’s Handbook on whey and whey products, first ed. European Whey Products Association, Belgium.

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Ghasemi, S., Nematollahzadeh, A., 2017. Molecularly imprinted polymer membrane for the removal of naphthalene from petrochemical wastewater streams. Adv. Polym. Technol. 37 (6), 2288 2293. Muthukumaran, S., Kentish, S.E., Stevens, G.W., Ashokkumar, M., Mawson, R., 2007. The application of ultrasound to dairy ultrafiltration: the influence of operating conditions. J. Food Eng. 81, 364 373. Neocleous, M., Barbano, D., Rudan, M., 2002. Impact of low concentration factor microfiltration on the composition and aging of cheddar cheese. J. Dairy Sci. 85 (10), 2425 2437. Pan, K., Song, Q., Wang, L., Cao, B., 2011. A study of demineralization of whey by nanofiltration membrane. Desalination. 267, 217 221. Sinha, R., Radha, C., Prakash, J., Kaul, P., 2007. Whey protein hydrolysate: functional properties, nutritional quality and utilization in beverage formulation. Food Chem. 101, 1484 1491.

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Bioanalytical Aspects in Enzymatic Protein Hydrolysis of By-Products Sileshi G. Wubshet, Diana Lindberg, Eva Veiseth-Kent, Kenneth A. Kristoffersen, Ulrike Bo¨cker, Kathryn E. Washburn and Nils K. Afseth Nofima AS - Norwegian Institute of Food, Fisheries and Aquaculture Research, Norway O U T L I N E 8.1 Introduction

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8.2 Factors Influencing EPH Processes 227 8.2.1 Protease Stability 229 8.2.2 Inhibitors 229 8.2.3 Accessibility of Substrate Proteins and Peptides 230 8.2.4 Protease Specificity 231 8.3 Biomass Characterization 8.3.1 Classical Methods 8.3.2 Spectroscopic Methods

231 232 234

8.4 EPH Process Characterization and Monitoring 236 8.4.1 Classical Methods 236 8.4.2 Electrophoresis 239

Proteins: Sustainable Source, Processing and Applications DOI: https://doi.org/10.1016/B978-0-12-816695-6.00008-8

8.4.3 Calorimetry 8.4.4 NMR Spectroscopy 8.4.5 Infrared Spectroscopy

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8.5 Product Characterization 8.5.1 Chromatographic Methods 8.5.2 Amino Acid Composition Analysis 8.5.3 Isolation and Identification of Bioactive Peptides 8.5.4 Proteomic Approaches

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8.6 Future Perspectives

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8.1 INTRODUCTION There are several industrially applied technologies for the valorization of by-products, including rendering, chemical hydrolysis, and the ensilage process. In recent years, enzymatic protein hydrolysis (EPH) has gained significant attention as a more versatile technology than these methods. EPH is regarded as a mild biotechnological process, resulting in high product yields without impacting the nutritional quality (Aspevik et al., 2017). The EPH process relies on the catalytic nature of proteases, which liberates peptides enclosed in the primary sequence of proteins by cleavage of peptide bonds. By applying selected processing conditions, the EPH industry is able to fine tune the hydrolysis products towards different markets. Several value-added products, including nutraceuticals, sports nutrition, and food supplements, have been developed from by-products using state-ofthe-art EPH technology. One example is Lacprodan, which is a stable whey protein hydrolysate with a low bitterness profile and high solubility that is sold as nutrition for hospital patients by Arla Foods Ingredients Group P/S, Denmark. ProGo is another product successfully developed from EPH of salmon by-products, sold as a fast-absorbing and digestible protein supplement by Hofseth BioCare, Norway. The general layout of an EPH processing plant comprises several steps. For pretreatment, one or several grinding units (depending on the by-product at hand) are used. For EPH, a hydrolysis reactor with either batch or continuous mode is used. The homogenized byproduct is suspended in an appropriate amount of water, and after adjusting the processing conditions (e.g., temperature), a selected protease is added to initiate the hydrolysis and protein recovery. After the appropriate amount of hydrolysis time, the reaction mixture is transferred to a dedicated protease deactivation (typically thermal) unit. Finally, downstream operations and processing such as phase separation (de- or tricanter), ultrafiltration, desalting, and drying are performed to yield protein hydrolysates of intended quality. The general scheme and major unit operations involved in EPH are illustrated in Fig. 8.1.

FIGURE 8.1 A generic scheme of the major processing steps in EPH processing of industrial by-products. Inserted on top are summaries of the important analytical aspects at different stages of processing. Source: Reproduced and modified from Wubshet, S.G., Wold, J.P., Afseth, N.K., Bo¨cker, U., Lindberg, D., Ihunegbo, F.N., et al., 2018. Feed-forward prediction of product qualities in enzymatic protein hydrolysis of poultry by-products: a spectroscopic approach. Food Bioprocess Technol. 11 (11), 2032
2043.

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The success of a given EPH process can be measured by its ability to produce the maximum possible yield of a high-quality product within the required specifications and with limited or no batch-to-batch variation. Analytical methods for characterization of raw materials and products, as well as process monitoring techniques, play vital roles in achieving production goals, including high-quality products and optimal yield. This chapter covers the analytical methodologies used to reach these goals in relation to laboratory, pilot, and industrial-scale EPH protein recovery processes. Moreover, specialized analytical approaches for the discovery and development of more specified products, such as bioactive peptides, from protein hydrolysates of by-products are discussed. In order to highlight how the different biochemical phenomena that occur during EPH of complex by-products affect product qualities, the main factors influencing protease activity during digestion of proteins are discussed first.

8.2 FACTORS INFLUENCING EPH PROCESSES Irrespective of the scale of the EPH process, there are many factors that determine the production outcome. The general key EPH-regulating factors can be classified into the following subgroups: • Process-specific parameters, such as substrate concentration, enzyme to substrate ratio, time, temperature, and pH. • Substrate-specific factors, such as origin, age, feed regimen, and complexity. • Protease-specific factors, such as specificity, stability, and sensitivity to inhibitors. It is not within the scope of this chapter to cover how all these factors determine product quality parameters during EPH processing, along with postprocessing steps such as downstream treatments and product formulation. The reader is encouraged to seek this information in other reviews or book chapters, for example, a review by Pasupuleti and Braun (2010). In the following, the focus will be set on describing how the EPH reaction kinetics during processing is influenced by protease-specific factors. The creation of proteins from by-products using EPH most often involves the use of commercial proteases. Some consist of only one protease in the commercial formulation, but many of these consist of a mixture of enzymes. The choice between a single protease and a protease cocktail influences the level of specificity during hydrolysis, but also product yield and the degree of hydrolysis (DH). The vast number of existing proteases in organisms from different habitats all around the world indicates that proteases are highly specialized for the activities and/or organisms they originate from. Some of this specialization is utilized for industrial applications, shown by the fact that proteases are one of the market-leading industrial enzymes worldwide (Li et al., 2013a,b; Tavano, 2013). As proteases degrade proteins, it is important for the organisms out of strict necessity to be able to regulate their activity on a molecular level. Many of these protease-specific regulatory options are also available for commercial proteases applied in EPH processing of by-products. However, when aiming at understanding how different factors influence the reaction rate of EPH processes and the process outcome, it is necessary to be able to monitor the reaction kinetics. Deviations from a PROTEINS: SUSTAINABLE SOURCE, PROCESSING AND APPLICATIONS

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“normal” reaction curve are indicative of problems during processing, which cause a departure from optimal processing conditions. Deviations from optimal conditions affects key product qualities, such as molecular weight distribution (MWD), amino acid composition, and yield. When monitoring the time-dependent progression of an enzymatic reaction, normally the initial linear phase is followed by a decline in reaction rate, which is seen as a consequence of the substrate being depleted (see Fig. 8.2A and B). This results in a hyperbolic reaction curve, shown in Fig. 8.2. Many studies have shown that substrate depletion alone is not sufficient to explain the premature decline in reaction rate seen in EPH reactions. A direct consequence of this is that it can be difficult to reach the theoretical maximum degradation of substrate proteins and peptides. In practice, the decline in reaction rate is caused by one or several factors working in parallel in the reaction mixture, hindering the substrate from being digested to the maximum possible degree. Many causes have been proposed for this reduction in rate seen in EPH processes. Protease denaturation and/or autolysis over time, substrate and/or product inhibition, and problems associated to substrate accessibility are the most frequently proposed reasons. Indeed, much of the detailed work on the kinetics of proteases on natural substrates has been performed with the aim of elucidating these ratelimiting causes. In the sections below, some of the major factors influencing EPH are discussed.

FIGURE 8.2 Typical reaction progression curves resulting from an enzymatic reaction. In (A) and (B), two reaction curves that might be resulting from two concentrations of a single enzyme used on the same concentration of substrate. The initial linear rate is declining as the substrate is being depleted. In both cases, all substrate is used up, converted to a total product concentration of 100, albeit at two different rates reflecting the differences in enzyme concentration. In (C), the same substrate concentration, but in this case, the enzyme used is unstable, making it impossible to reach the same product concentration as in (A) and (B), as the enzyme is inactivated before all substrate is converted. Source: Reproduced from Tipton, K.F., 2002. Principles of enzyme assay and kinetic studies. In: Eisenthal, R., Danson, M.J. (Eds.), Enzyme Assays: A Practical Approach, second ed. Oxford University Press, pp. 1
47 with permission (Tipton, 2002).

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8.2.1 Protease Stability A protease that gradually loses its activity due to autolysis, denaturation, and/or aggregation during an EPH reaction would lead to an apparent reduction in reaction rate (see Fig. 8.2C), and ultimately to a reduced yield. Although many proteases are initially produced as inactive precursors called zymogens, that are in need of auto- or proteolysis for activation (Khan and James, 1998), Li et al. (2013a,b) stated that industrial proteases are normally stable within their specified pH and temperature ranges. This is also evident by the relatively few reports on autolysis and protease denaturation during the processing of animal-based proteins. If needed, advanced techniques such as immobilization of proteases can be used for stabilization of the proteases during EPH processing. Interested readers are encouraged to read Tavano et al. (2018) for an overview of protease immobilizations on various matrixes in different biotechnological applications.

8.2.2 Inhibitors The existence of substrate and/or product inhibitor(s) in a reaction mixture during EPH processing would effectively inhibit the protease from working, in the best case leading to a reduction in reaction rate and in the worst case, a complete termination of the process. Partly due to the pronounced impact on the EPH process, inhibition has been extensively investigated as the cause of rate reductions in protease kinetics. In an early study by O’Meara and Munro (1985) on hydrolysis of lean meat by Alcalase at 60 C, substrate inhibition was ruled out by the lack of effects of varying substrate concentration on the initial reaction rate. The study concluded that the major causes for the rate reduction seen were the combined effect of peptide product inhibition and protease inactivation. Later, Gonzalez-Tello et al. (1994) proposed that the rate decrease over time was caused by irreversible inhibition of proteases taking place very early in the EPH process of whey at 50 C, using several different proteases. Margot et al. (1997) studied the kinetics of tryptic digestion of whey protein concentrate at different temperatures (55 C, 60 C, and 70 C). As a result of the study, it was proposed that the main influences on protease kinetics seem to be inhibition by the peptides formed, as well as inactivation of the enzymatic activity by autohydrolysis and thermal unfolding. In contrast, in a later study on Alcalase used in fish protein hydrolysis, the authors concluded that substrate inhibition and enzymatic inactivation was unimportant in rate reduction, instead supporting earlier studies proposing the major rate-reducing factor to be peptide product inhibition (Valencia et al., 2014). During the last two decades, more studies supported product inhibition as the main cause of the observed time-dependent decrease in reaction rate (Valencia et al., 2014, 2016; Kasper et al., 2014; Qu et al., 2015; Sousa et al., 2004; Trusek-Holownia et al., 2016). From an EPH processing perspective, it is important to be able to counteract the effects of product inhibition to be able to reach maximum yield. One option that has been extensively investigated is the use of EPH reactors that continuously remove the protease inhibiting low molecular weight peptides during processing. To support the suitability of the method, many studies report successful examples of EPH processes on various substrates in both batch and continuous reactors that use membrane technologies for peptide removal (Tavano et al., 2018; Qu et al., 2015; Trusek-Holownia et al., 2016; Bhat et al., 2015).

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8.2.3 Accessibility of Substrate Proteins and Peptides When a substrate binds to the active site of proteases, the actual point of cleavage is the individual peptide bonds of peptide chains. It might be regarded as an obvious fact, but it is important to highlight that a successful EPH process relies on the fact that all parts of the substrate molecules are made available for digestion. Fundamental work on substrate accessibility was done early in the 1970s by Archer et al. in studies on fish protein concentrate, an insoluble substrate (Archer et al., 1973). The starting hypothesis was that the initial rate is proportional to the surface area of the insoluble substrates, as had been stated earlier. The notion of the reaction rate being proportional to the surface area is in contrast to solubilized substrates, where the initial rate is proportional to the total amount of enzyme. The study instead showed that what happened was a mix of these two theories, where proteases initially interact with the substrate while also digesting protein fragments solubilized by the proteases interacting with the insoluble material. The study also suggested that for this insoluble substrate, the first step of the reaction constitutes protease adsorption to the surface of the particles to hydrolyze the easily accessible peptide bonds. The second step is then hydrolysis of less accessible substrates. In a later study, O’Meara and Munro (1985) reported results supporting the notion of a two-step process in digestion of insoluble proteins: the first where the protease acts on the more easily accessible peptide chains on the surface of the meat substrate, and secondly as the reaction progresses, the structure opens up and more substrate will be accessible for the proteases. Studies on solubilized protein substrates show that the rate of hydrolysis for these substrates is also affected by the folding state of the proteins, that is, the accessibility of individual peptide bonds. The process of unfolding of proteins is known as demasking (Adler-Nissen, 1986). Demasking therefore results in increased accessibility of the polypeptide chain within the folded proteins, leading to an increased velocity of the hydrolysis reactions (Adler-Nissen, 1976; Hubbard, 1998). Aiming to understand the effect of demasking in EPH in greater detail and to link the kinetics in demasking to the overall protease kinetics, Vorob’ev and coworkers published a number of reports on the subject (Vorob’ev et al., 2011, 2013; Vorob’ev and Kochetkov, 2016). Initially, the studies proposed two rates involved in the hydrolysis of proteins, that is, the rate of demasking and the rate of the hydrolysis, arguing that the peptide bonds in proteins cannot be hydrolyzed if they are not first made accessible through demasking of the protein substrates. The latest study by Vorob’ev et al. covers all of the above factors discussed in this section. The study shows that more accessible bonds are cleaved first in line with first-order enzyme kinetics, that some bonds are not cleaved at all, and lastly, that the rate of hydrolysis of the remaining bonds are dependent on the demasking rate of the proteins in the EPH process (Vorob’ev and Kochetkov, 2016). Deviations from ideal initial rate kinetics, that is, the linear part of the curve (B) in Fig. 8.2, in EPH reactions have also been interpreted as an effect of formation of peptide aggregates (Creusot and Gruppen, 2007a). During EPH processing, aggregate formation limits both the accessibility of the substrate protein/peptides for further proteolytic activity, but also causes problems in the downstream processing steps, as it might hinder both filtration and drying of the product hydrolysate. Creusot and Gruppen (2007b) summarized several probable reasons for aggregation of peptides and its mechanisms in

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a review. The review highlights that it is the aggregating properties of hydrophobic parts of the peptide chain, whether in the partly denatured protein or resulting peptides, and environmental causes that are the most important factors for aggregation. In a later review, Gong et al. (2015) collectively named protease-induced peptide aggregates as plastein, and focused on possible new markets of the plastein hydrolysis products. In the review, the authors included summaries of two more reported causes for plastein formation, one being peptide condensation, apparent by formation of higher molecular weight complexes, and another being increased viscosity by the occurrence of transpeptidation reactions.

8.2.4 Protease Specificity Protease specificity determines where the peptide chain in the substrate is to be cleaved, with the exact interaction between the substrate molecule and the protease substrate binding area contributing to protease specificity (see for example Bode et al., 1999; Perona and Craik, 1997; Turk, 2006). Ideally this means that knowledge regarding the peptide chain sequence and the specificity of single proteases should enable hydrolysis product peptides to be tailor-made, and hence control product properties. Knowledge of protease specificity might allow for tailor-made hydrolysis end-product peptides, but several factors determine the outcome of such an approach. One is that the specificity of single proteases are more or less relaxed. Proteases exhibiting relaxed specificity are able to hydrolyze more peptide bonds than proteases with a very strict specificity. From the perspective of EPH of complex substrates, such as animal by-products, generally a more relaxed specificity is preferred as it can be expected to result in a better product yield than proteases with strict selectivity. However, if aiming at developing EPH processes where a single peptide is of extra interest, for example, one exhibiting a particular bioactivity, the use of a protease exhibiting a stricter specificity is the natural choice. There are studies that have looked into the formation of all peptides resulting from EPH on single proteins. These studies result in values of the velocity index, that is, the rate of hydrolysis of each scissile bond within the substrate protein (Su et al., 2007; Tauzin et al., 2003). When monitoring digestion of the cleaved bonds in molecules, it is possible to extract the t1/2 values, defined as the reaction time required to reach 50% of the maximal quantity of each peptide extracted. These studies are also excellent for determining the resulting protease specificity in a specific EPH process on real substrates. Finally, a recent study has shown that substrate concentration influences the DH, that is, yield, but also that the extent of this effect depends on which protease that was used in EPH processing (Deng et al., 2018).

8.3 BIOMASS CHARACTERIZATION In EPH of by-products with an inherent complexity, such as meat or fish by-products, there are day-to-day variations in composition. These differences arise from seasonal variations in marine fish species. For poultry and aquaculture fish species, differences in fat content and cartilage hardness and content can be attributed to age, feeding regimen, and

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cage differences. In addition, the composition of by-products for EPH will inherently depend on the processing settings of the original raw material, for example, fillet production or mechanical deboning. In many EPH processing plants, the amount of protease added to the process is a fixed quantity. A consequence of a fixed enzyme addition in an EPH processing plant where the by-product protein concentration fluctuates with time is that the hydrolysis product MWD varies with time. Therefore, it is vital to characterize the raw material composition (e.g., fat, protein, and ash content) over time, if one aims at building a robust and optimal process that leads to an efficient production of defined high quality hydrolysis peptide products. While the majority of the currently used analytical methods for the characterization of by-products are based on classical wet chemistry, recent applications of spectroscopic techniques have shown potential as tools for the rapid estimation of the proximate composition of by-products.

8.3.1 Classical Methods The normal procedure in raw material characterization of many animal and marine byproducts involves a so-called proximate analysis to extract the percent-wise partitioning of the major components in these raw materials. For most animal-based by-products composition analysis usually covers protein, water, ash, and fat measurements, since the carbohydrate fraction in these materials is normally very low. The most commonly used approach for ash and water measurements is the gravimetric measurement of the raw material after a drying step. There are more types of methods available for measuring protein and lipid content. Although there have been several reviews and book chapters on this matter (Chutipongtanate et al., 2012; Moore et al., 2010; Silvestre, 1997; Srigley and Mossoba, 2016), for the sake of comprehensiveness, an introduction to the most commonly used methods is included here. 8.3.1.1 Protein Measurements The two most used analytical methods to determine total protein content by far are the combustion (Dumas) and the Kjeldahl methods (Etheridge et al., 1998), both of which rely on measurement of the total nitrogen content. A total nitrogen-to-protein conversion factor is then used to calculate the crude protein content based on the total nitrogen value reported by these methods. The conversion factor of 6.25 is frequently used, based on the assumption that proteins contain 16% nitrogen and that all nitrogen in the sample originates from proteins. Other conversion factors have also been proposed, see for example Mariotti et al. (2008). Conversion factors for both the Dumas and the Kjeldahl methods specific to different raw materials and applications are listed in standards by the International Organization for Standardization (www.iso.org). Kjeldahl is an extremely versatile analytical technique used to determine the nitrogen content in a wide range of sample types, including food matrixes. The analytical procedure in Kjeldahl constitutes four major steps: 1. digestion, which converts nitrogen to ammonium sulfate; 2. neutralization of ammonium sulfate to ammonia;

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3. distillation of ammonia into boric acid; and 4. back titration of excess boric acid. Several adaptions of these basic steps exist (Moore et al., 2010). The total Kjeldahl nitrogen determined from the above procedure is then converted to protein content using an established protein conversion factor for the material under study. In protein and peptide recovery from by-products, Kjeldahl has been used to evaluate the protein content in complex matrixes including fish and chicken. For example, Aspevik et al. (2016) have used Kjeldahl for determination of total protein content in salmon byproducts, and Wubshet et al. (2018) have used it for several different by-products from the poultry industry. Although several combustion procedures for measuring protein were developed during the 18th century, Dumas has been credited for developing the most reliable method of the time. Combustion methods were refined in the 1980s with the advent of reliable combustion nitrogen analyzers (Moore et al., 2010). During a combustion procedure, samples are combusted to form water, carbon dioxide, sulfur dioxide, nitrogen oxides, and nitrogen gas. After removal of carbon and sulfur dioxide, the different nitrogen oxides are reduced to nitrogen gas, followed by measurement of the total content of nitrogen gas. As for the Kjeldahl method, specific nitrogen-to-protein conversion factors can then be used to calculate the crude protein content of the samples (Owusu-Apenten, 2002). Studies have been performed on whey proteins to evaluate the performance of the Dumas combustion method with the Kjeldahl one, showing that Dumas indeed is comparative while also representing a faster option (Etheridge et al., 1998; Chiacchierini et al., 2003). An interlaboratory research study came to a similar conclusion that Dumas was the preferred technique after sending several fish meal samples to more than 10 laboratories to compare results from the two methods (Miller et al., 2007). A comprehensive summary comparing the precision of Kjeldahl and Dumas methods on a range of food matrixes can be found in Moore et al. (2010). These frequently used methods can be said to show low selectivity, as is evident by the inability to distinguish protein-based nitrogen from nonprotein nitrogen. No other viable alternatives currently exist, although many ways to measure proteins as well as peptides and amino acids are available and have been reviewed many times. For more information on alternative methods, see for example Silvestre et al. (1997). 8.3.1.2 Fat/Lipid Measurements With complex materials, the fat needs to be separated from the matrix to yield accurate figures on the amount of fat within the material. Methods for crude lipid analysis can be based either on gravimetric or hydrolytic approaches (Srigley and Mossoba, 2016). The Soxhlet, Folch, and Bligh and Dyer methods are examples of commonly used gravimetrybased methods in proximate analysis measurements; all are dependent on a solvent extraction step. While the Soxhlet method originally was based on the use of a semicontinuous Soxhlet extractor for lipid extraction (Soxhlet, 1879) using solvents such as hexane or petroleum ether, the Bligh and Dyer and Folch methods rely on the use of a combination of chloroform and methanol for lipid extraction (Bligh and Dyer, 1959). There are several variations of all these methods (Srigley and Mossoba, 2016). For the Soxhlet method,

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though it can be performed conventionally by refluxing of organic solvents in specific glassware, these days the analysis industry more commonly uses automated or semiautomated instruments that are now available (Srigley and Mossoba, 2016). For some materials, such as dairy products (Vaghela and Kilara, 1995), a hydrolytic procedure is recommended before performing the solvent extraction. The main benefit of hydrolysis before fat extraction is that a full digestion of the material enables all lipids in the material to be extracted, whereas only some lipids can be extracted in undigested materials. Studies have been performed to compare the Soxhlet and Bligh and Dyer methods of lipid extraction from complex matrixes such as fatty and lean fish muscles (Ewald et al., 1998), and the Soxhlet, Bligh and Dyer, and Folch methods for lipid extraction from meat and meat products (Pe´rez-Palacios et al., 2008). The latter paper also includes a comparison of the method with and without prehydrolysis steps. Fat determination has also been used in raw material characterization from by-products of different animal origin (Wubshet et al., 2018; Aspevik et al., 2016).

8.3.2 Spectroscopic Methods All the analytical measurements discussed in the previous section are performed in laboratories, typically on a small sample taken from a large batch of by-products. This approach reflects the usual practice for the industry, which is to send small sets of byproduct samples to external laboratories, and in the best case receive results within a few days. During the wait period, large quantities of by-products are processed with the original processing settings, not taking into account any potential change in raw material composition in this time. This assumption that the raw material does not change with time may lead to suboptimal processing resulting in compromised yield and quality of hydrolysis. In addition, the heterogeneity of meat and fish-based by-products poses a significant challenge for representative sampling. Recent advances in spectroscopy and data modeling have enabled the use of rapid spectroscopic techniques as on-line or real-time characterization tools in the food and biotech industries. Spectroscopic techniques are fundamentally based on interaction between matter and electromagnetic radiation. By studying these interactions, information regarding both quantitative and qualitative aspects of chemical substances may be obtained. Vibrational spectroscopy is the branch of spectroscopic techniques that mainly deals with probing of vibrational states of a molecule. Fourier-transform infrared spectroscopy (FTIR), near-infrared spectroscopy (NIR), and Raman spectroscopy constitute important members of the group of vibrational spectroscopic techniques. Vibrational spectroscopic instrumentation for food analysis has developed rapidly in recent decades. The precision, stability, flexibility, and speed of these instruments is continuously improving, making them versatile tools for food analysis at different levels and providing detailed chemical characterization of both major and minor food components. The combination of instrument development and application research is pushing the limits of spectroscopic applications in food analysis, and new application areas are constantly being developed. Common to all these techniques is the possibility to perform rapid and nondestructive analysis, potentially also in an industrial environment.

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8.3.2.1 Near Infrared Spectroscopy Over the past four decades, NIR spectroscopy and multivariate modeling have proven to be reliable and versatile tools in industrial food applications. NIR analysis enables rapid, nondestructive, and environmentally safe assessment of several parameters simultaneously, in a variety of products and processes. The method has traditionally been restricted to rather homogeneous materials and batch processes, but recent developments now facilitate adequate measurements on heterogeneous products. The development of NIR instruments relevant for the EPH industry builds these on previous successful measurements of complex food matrixes, proving the applicability of NIR for proximate composition measurements in by-products. Dedicated NIR hyperspectral imaging systems have been used to measure fat content in meat trimmings and fish fillets, meat in live crabs, and protein in chicken fillets (ElMasry and Wold, 2008; Wold et al., 2010, 2011, 2017). Deep penetrating NIR has enabled the determination of dry matter and fat in whole potatoes and live salmon (Folkestad et al., 2008; Helgerud et al., 2015). Lastly, the fat content in entire pork bellies can be measured by NIR transmission imaging in a fraction of a second (Wold et al., 2016). On-line methods allow the producer to actively control and optimize the process by collection of continuous data during processing. This approach, which is often termed process analytical technology, can lead to improved and more predictable hydrolysis product quality, optimal use of raw materials, and less loss, all of which lead to increased profit. For example, this has been proven for the situation of optimal sorting of meat trimmings (Ma˚ge et al., 2013). In an EPH-industry relevant study, a process analytical technology approach utilizing NIR measurements of raw materials for process control and optimization has recently been demonstrated in protein recovery from poultry by-products (Wubshet et al., 2018). 8.3.2.2 Raman and FTIR Spectroscopy Compared to NIR, Raman and FTIR are less frequently used as on-line analytical tools. The techniques are gaining increasing interest, not only for determining differences in crude composition as with NIR, but first and foremost for their ability to capture subtle distinctions and detailed composition of food matrices. Because of their capacity for qualitative analysis of proteins, Raman and FTIR are increasingly employed to characterize complex food matrixes. Hence, applications of these technologies have recently been shown as attractive tools for the characterization of animal-based protein-rich by-products (Wubshet et al., 2018, 2019). Information on both crude fatty acid composition and single fatty acids are potentially available from Raman and FTIR spectra (Afseth et al., 2010; Tyburczy et al., 2013). The nature of Raman spectroscopy allows measurements in more heterogeneous food matrixes, dominated by water and proteins (Næs et al., 2013). Raman spectroscopy has inherent limitations with regards to sampling of heterogeneous materials, but in recent years we have seen that deep penetration is possible by spatially resolved Raman spectroscopy and transmission measurements (Afseth et al., 2014; Schulmerich et al., 2012). Recently, approaches for scanning of larger sample surfaces have also become available. With such an approach, Wubshet et al. (2019) showed the first application

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of Raman spectroscopy as a rapid tool for estimating calcium and ash contents in bone and meat mixtures after mechanical deboning of chicken meat, one of the relevant substrates for EPH. FTIR is a versatile spectroscopic technique, and its potential for high-throughput screening of fairly homogeneous samples like fluids, cells, and microorganisms is well documented. This technique has been successfully applied in recent years for at-line, on-line, and in-situ bioprocess monitoring of extracellular metabolites and substrates (e.g., glucose, lactic acid, and ethanol). The development of industrial applications of FTIR has also been closely linked to the dairy industry, a major producer of whey by-products. As early as 1980, the second generation of MilkoScan instruments (Foss, Hillerød, Denmark) was reported to match the AOAC specifications for fat, protein, and lactose analysis. As instrumentation has developed from fixed-wavelength filter methods to covering the full infrared spectral range, the accuracy and precision of measurement of the main sample constituents has improved. Today, applications range from estimations of casein content, total solids, lactoferrin, and antibiotics to the identification of microorganisms and microbial spoilage in milk. Soyeurt et al. (2011) also showed that the MilkoScan IR instrument could be used to estimate the overall fatty acid composition of milk. Despite some applications for milk by-products, the application of FTIR for analysis of complex by-products is limited. In contrast, FTIR finds most of its application in EPH as a process monitoring tool (see section 8.4.5).

8.4 EPH PROCESS CHARACTERIZATION AND MONITORING A multitude of analytical methods have been used to follow, understand, and optimize EPH processes. These methods have been used in investigations on everything from the enzyme kinetics of simple single enzyme/single protein EPH processes, to the more intricate reaction kinetics of industrial-like EPH processes. Enzyme activity is measured by following either the rate of substrate disappearance or the rate of product formation. The rates can also be extracted from fitting rate equations to data reporting on the number of bonds broken over time. There are a multitude of commercially available methods to measure both specificity and activity of proteases, based on synthetic or derivatized natural proteins for activity measurement in many conditions. References are therefore deemed redundant, but for the interested reader there are some recent reviews focusing on highthroughput assays (Acker and Auld, 2014; Ong and Yang, 2017). However, for studies on the kinetics of EPH on by-products for different applications, the number of methods available is more limited. In the following section, both the classical and emerging approaches used for monitoring EPH reactions will be discussed.

8.4.1 Classical Methods The degree to which the hydrolysis has been executed, the DH, is a well-established parameter, both for describing the extent of hydrolysis in the resulting peptide product

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and for monitoring the EPH of by-products. DH is defined as the percentage of cleaved peptide bonds in a protein hydrolysate and is calculated according to Eq. (8.1): DH 5

h 3 100% htot

(8.1)

where h is the measured amount of cleaved bonds and htot is the total number of available peptide bonds (Nielsen et al., 2001). Several methods to determine DH have been proposed. The most commonly used of these include the pH-stat (Ficara et al., 2003; Jacobsen et al., 1957), trinitrobenzenesulfonic acid (TNBS) (Adler-Nissen, 1979), o-phthaldialdehyde (OPA) (Nielsen et al., 2001), formol titration (Levy, 1934), and trichloroacetic acid soluble nitrogen (Margot et al., 1994). These methods are based on different principles for measuring cleaved peptide bonds, and it should also be noted that multiple versions of each of the mentioned methods have been published. The pH-stat method is one of the most commonly used and simplest methods for determining DH in hydrolysates derived from simple proteins to more complex substrates. Using this method, the EPH process is monitored by keeping the pH at a set value during the course of the reaction. Most frequently, this is achieved by automated titration with a base of known concentration. DH is then calculated based on the number of protons released during hydrolysis, estimated from the amount of base consumed (Rutherfurd, 2010). This makes it possible to monitor both enzyme and reaction kinetics and to calculate the DH. This is, however, not necessarily a direct measurement of cleaved peptide bonds as many factors, such as the nature of the enzyme used, the size of the hydrolyzed peptides, and the reaction temperature will influence the measurements. In the pH-stat method, DH is calculated according to Eqs. (8.2)
(8.3): DH 5 α5

B 3 NB 3 ð100%Þ ðMP 3 α 3 htot Þ

(8.2)

10ðpH2pKÞ ð1 1 10ðpH2pKÞ Þ

(8.3)

where B is the base concentration, NB is the normality of the base, MP is the protein content, htot is the number of peptide bonds in the protein, α is the average dissociation of α-amino groups, and pK is the average pK of α-amino groups liberated during hydrolysis (Rutherfurd, 2010). In one of the early studies using the pH-stat methodology, Baumann et al. used a modified pH-stat method to evaluate the contribution of interactions around the site of cleavage of the scissile bond during hydrolysis of chymotrypsin (Baumann et al., 1973). Since then, the pH-stat method has been used either as a stand-alone methodology or together with other analytical methods to follow the reaction progression in EPH, in either single or more complex mixtures of proteins. There are many examples of using a pH-stat measurement method in EPH of by-products. Two examples are in the processing of yellowfin tuna by-products (Guerard et al., 2001) and salmon by-products (Aspevik et al., 2016). Formol titration is another method used to measure the DH during EPH. As with the pH-stat technique, this method is based on the number of liberated protons, as measured with base titration, but the basic principle differs. Formol titration is based on a reaction between an amino group and formaldehyde, as shown in Fig. 8.3. This reaction will occur

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FIGURE 8.3 Formol titration: reaction between amino acids and formaldehyde in the presence of sodium hydroxide.

FIGURE 8.4 Chemical reactions of (A) OPA with amino acids and (B) TNBS with amino acids.

under both neutral or alkali conditions, resulting in a one-to-one liberation of protons and a concurrent reduction of the pKa value of the amino acid
formaldehyde derivative. There are two approaches for carrying out formol titration, the direct and the indirect approach. In the direct method, formaldehyde is added directly to the sample solution before base titration is performed. In the indirect method, the sample solution is first pHadjusted before formaldehyde is added. In a study comparing these approaches, the direct method was found to be superior to the indirect in terms of reproducibility (Taylor, 1957). An early example of the use of formol titration originates from 1973, where this approach was used in a sensory study of peptides from fish protein hydrolysates (Fujimaki et al., 1973). Formol titration has also been used to study kinetics of tryptic digestion of chicken heart protein (Belikov et al., 1986). In a different study, formol titration has been used to calculate DH and study the effect of hot-pressure extraction of peptides from chicken bones, in combination with proteolysis by Flavourzyme (Dong et al., 2014). The TNBS and OPA methods are among the most commonly used approaches for monitoring the extent of protein hydrolysis in EPH of by-products. Both methods are based on measurement of the number of free N-terminals, and both methods are carried out via derivatization of their amino groups to enable ultraviolet-visible or fluorescence detection, as shown in Fig. 8.4 (Herna´ndez et al., 1990; Satake et al., 1960). For these techniques,

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however, the derivatization reagents are known to exhibit different reactivity towards some amino acids. This will affect the accuracy of the measurement. For example, the OPA method will not be accurate when applied on proline- and cysteine-rich hydrolysates (Rutherfurd, 2010). TNBS and OPA have been used for optimization of process settings in the production of hydrolysates for feed, food, and functional food applications. For example, TNBS has been used in a study aiming at optimizing process parameters for hydrolysis of skipjack fish by-product for feed applications (Chotikachinda et al., 2013). Opheim et al. (2015) used OPA for analysis of industrial samples when aiming to understand the influence of various processing factors on composition, nutritional value, and presence of bioactive peptides in salmon by-product hydrolysates. OPA has also been used for the physicochemical characterization of dairy protein hydrolysates and to study the potential correlation of DH with sensory attributes (Newman et al., 2014). Fu et al. (2018) used OPA to monitor DH in the production of low bitter and high umami-tasting hydrolysates from bovine muscle and porcine plasma. The trichloroacetic acid soluble nitrogen method, also known as the trichloroacetic acid-solubility index, is another method used to estimate DH (Margot et al., 1994, 1997, ). This is not a method that detects the amount of cleaved peptide bonds. Instead, the amount of trichloroacetic acid soluble nitrogen is measured and is assumed to stem exclusively from amino acids and small peptides. The trichloroacetic acid solubility index value is determined by precipitating the nonhydrolyzed protein with trichloroacetic acid, followed by centrifugation. The nitrogen content of the supernatant and the original sample material is then determined by calculating the trichloroacetic acid soluble nitrogen index value according to Eq. (8.4): Trichloroacetic acid solubility index; % 5

trichloroacetic acid solubleN 3 100% TotalNin the sample

(8.4)

Numerous studies have been conducted to compare the different methods used for determining DH of food protein hydrolysates (Rutherfurd, 2010; Spellman et al., 2003). In a study by Spellman et al. (2003), DH was determined using the pH-stat, OPA, and TNBS methods on whey protein concentrate hydrolysates. It was reported that the measured DH values differed by as much as 60% depending on the method used. The pH-stat gave the lowest DH value, while TNBS gave the highest (Fig. 8.5). The resulting differences in measured DH, diversity in methods, and principles of measurement seen from the Spellman study clearly demonstrate the challenges arising when comparing reported DH values in literature. Therefore, while the methods discussed above provide tools for monitoring relative changes during EPH of specific by-products, comparison of DH values between different materials should be performed with caution.

8.4.2 Electrophoresis Electrophoresis is a methodology based on the migration and separation of charged particles under an electrical field (Fritsch and Krause, 2003). This methodology is not frequently used to monitor EPH of by-products; methods like sodium dodecyl

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FIGURE 8.5 DH (%) values obtained by the pH-stat (Δ), TNBS (&), and OPA (e) methods for the hydrolysis of a 20% (w/v) whey protein concentrate with Debitrase HYW20. Source: Reproduced from Spellman, D., McEvoy, E., O’Cuinn, G., FitzGerald, R.J., 2003. Proteinase and exopeptidase hydrolysis of whey protein: comparison of the TNBS, OPA and pH stat methods for quantification of degree of hydrolysis. Int. Dairy J. 13 (6), 447
453.

sulfate polyacrylamide gel electrophoresis (SDS-PAGE) are more commonly used for characterizations of molecular weight and purity of, for example, protein hydrolysates. However, in an extensive study on peptide product inhibition in native-state proteolysis of two proteins, Kasper et al. (2014) used SDS-PAGE for hydrolysis product separation. Subsequently, the size of the colored product bands was used to quantify the products. Using this methodology in combination with mathematical modeling, inhibition constants were calculated for two proteases after hydrolysis of the two native-state proteins. Møller et al. used a combination of capillary electrophoresis (CE) and chromatography to study both enzyme kinetics and specificity in hydrolysis of κ-casein using two different chymosins. Kinetic parameters were extracted by monitoring degradation of κ-casein by CE, while the soluble peptides were separated and analyzed by chromatography coupled to mass spectrometry (MS) (Møller et al., 2012).

8.4.3 Calorimetry Similar to electrophoresis, calorimetry is one of the less frequently used methods to evaluate enzyme and/or reaction kinetics related to EPH. Williams and Toone introduced this technique as a viable option in cases where enzyme kinetic studies are to be executed on substrates without optical properties or ionic components (Williams and Toone, 1993). In their publication, they examined the feasibility of using underivatized peptide substrates and microcalorimetry for kinetic studies (Williams and Toone, 1993). A few years later, Todd and Gomez (2001) revisited the subject in a study where the kinetics of enzymes from all enzyme commission (EC) classifications were investigated with isothermal titration microcalorimetry, aiming at introducing this as a universal and nondestructive assay for enzyme activity measurements. Isothermal titration calorimetry has recently been used to investigate pepsin digestion under various conditions (Luo et al., 2018). In the study, isothermal titration calorimetry was used to understand the enzyme kinetics of this digestive tract protein on natural substrate proteins, successfully verifying that isothermal titration calorimetry as a method could be used to follow the time-dependent kinetics in a continuous manner.

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8.4.4 NMR Spectroscopy Nuclear magnetic resonance (NMR) is a powerful spectroscopy technique with versatile applications. The technique has the capability to monitor several aspects of EPH processing. One of the most straightforward aspects is measuring the amount of hydrolysis products present in the solute. The NMR signal is linear, that is, doubling the amount of a constituent will double its signal. Therefore, if a constituent can be identified, it can easily be quantified. There are many approaches that can be used to identify constituents. One of the most straightforward methods is the standard one-dimensional (1D) 1H NMR experiment. However, more complex experiments, such as NOESY, are more frequently used in order to suppress the contribution of water in the NMR spectrum to make detection of the hydrolysis products easier. Most methods that are used to monitor EPH require deactivation of the proteases before analysis can be performed. Therefore, there is some lag time and uncertainty between when a sample is taken and when the protease is deactivated. In contrast, NMR measurements can be performed directly on a sample without the need to deactivate the protease (Sundekilde et al., 2018). Many of the simpler NMR experiments can be performed in seconds to minutes, making them practical for on-line monitoring. Recent work used NMR spectroscopy to monitor an EPH process (Sundekilde et al., 2018). The study used proton (1H) NMR spectroscopy for monitoring the EPH of muscle protein under both at-line and real-time conditions. For the at-line measurements, samples were taken at several time points during the EPH process, and proteases were heatinactivated prior to NMR analysis. For real-time measurements, the hydrolysis was performed directly in the NMR tube. The chemical shift in the 1H NMR spectrum was used to monitor the formation of amino acids during hydrolysis. Several different products were tracked as a function of reaction time, as shown in Fig. 8.6. From this, the reaction rates of the two proteases used were estimated. These results show NMR to be a promising method for the monitoring of the EPH process. NMR has many advantages as a measurement technique, being a noninvasive, relatively rapid method that allows for measurements of opaque samples. The magnet setup also makes it easy to perform flow-through experiments for potential on-line applications. A disadvantage of the method is that more advanced measurements can often take a long time to perform. Despite its utility for characterizing the hydrolysis products, NMR has not been used industrially for EPH process monitoring. The main reason for this has been the cost and complexity of equipment. In order to obtain the necessary spectral resolution, magnetic fields of certain strengths are needed. Previously, this could only be obtained using cryogen-cooled superconducting magnets, which require regular fills of liquid nitrogen and helium. This limited NMR spectroscopy to universities or industrial research centers due to the high cost and cumbersome upkeep of the equipment. In recent years, the development of cryogen-free magnets has brought down both the price and the required maintenance of NMR equipment. These developments have opened up the possibility for the industrial use of NMR spectroscopy and it is expected that this will become a reality in the near future.

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FIGURE 8.6

Evolution of different amino acids during EPH of chicken meat. The quantifications are based on H NMR spectroscopy. Star symbols represent samples treated with Alcalase, triangles represent samples treated with papain, and open symbols represent control samples without enzyme. A.U., arbitrary unit. Source: Reproduced from Sundekilde, U.K., Jarno, L., Eggers, N., Bertram, H.C., 2018. Real-time monitoring of enzyme-assisted animal protein hydrolysis by NMR spectroscopy—an NMR reactomics concept. LWT95, 9
16.

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8.4.5 Infrared Spectroscopy The use of FTIR for bioprocess monitoring is gaining considerable attention as a way to provide solutions in industrial biotechnology (Franco et al., 2006; Landgrebe et al., 2010). To this end, fermentation has been one of the major bioprocesses where FTIR-based monitoring has proven to be successful (Kosa et al., 2018; Pollard et al., 2001; Wu et al., 2015). In addition to high-end optical advancements, the technical means for using FTIR for monitoring purposes is rapidly improving. As will be discussed in this section, the technique is also a highly relevant analytical tool in the field of EPH processing. FTIR is an established method for the characterization of protein and polypeptide structures, and FTIR is one of the major tools for molecular evaluations of secondary protein structures. The amide groups of the protein and polypeptide backbones give rise to nine characteristic IR absorption bands, that is, amides A, B, and I
VII, with amide I and II being the most prominent Barth (2007). These absorption bands are also sensitive to changes in the surrounding electronic environment. Consequently, FTIR has been widely used to study the molecular structure of proteins in biological samples ranging from simple aqueous protein solutions (Barth, 2007) to complex matrixes in food (Bo¨cker et al., 2017; Perisic et al., 2011) and medical research. Secondary effects such as hydrogen bonding, and intrinsic parameters such as pH and water holding capacity have also been extensively studied (Barth, 2007; Andersen et al., 2017). One of the intriguing research fields that builds on the extensive research related to FTIR and protein structure is the application of FTIR for probing protein modifications induced by enzymes. Hydrolysis of peptide bonds results in C-terminal carboxylate (COO2) and N-terminal amino (NH31) groups at the specific site of cleavage. In addition to the apparent changes in the primary structure, shortening of the protein chain also affects the secondary structure. Both primary and secondary structural changes have pronounced effects on the overall FTIR absorption fingerprints of the protein. Ruckebusch and coworkers, as one of the first groups to report on the monitoring of EPH using FTIR, followed the degradation of bovine hemoglobin by collecting transmission infrared spectra throughout the hydrolysis reaction (Ruckebusch et al., 1999a,b). The same group could also predict the progress of the hydrolysis and showed how the DH could be estimated from the FTIR spectra (Ruckebusch et al., 2001). Gu¨ler and coworkers combined real-time measurements of FTIR and ultraviolet-circular dichroism to follow a protease reaction. In these studies, the hydrolysis of β-lactoglobulin, β-casein, and bovine serum albumin, respectively, were investigated (Gu¨ler et al., 2011, 2016). Recently, Poulsen et al. (2016) also showed that FTIR can be used to predict DH for trypsin-catalyzed hydrolysis of whey proteins. Bevilacqua et al. (2017) used FTIR to study the solubilization of five industrial grade plant proteins (i.e., potato, soy, corn gluten, wheat gluten, and pea) by EPH. The authors also showed how kinetic parameters from the measurements could be derived and subsequently be used to describe the suitability of the protein sources as substrates for hydrolysis. Also enzymes from other EC classes than proteases have been followed in the hydrolysis of proteins. Pacheco et al. (2005) have demonstrated the use of FTIR in kinetic evaluations of hydrolysis and synthesis reactions, catalyzed by a recombinant amidase. The abovementioned examples show that FTIR can be used to follow enzyme kinetics. However, the examples mostly encompass pure or homogenous mixes of proteins.

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Moreover, a majority of the FTIR studies in this field have been performed directly in the liquid phase. Bo¨cker et al. (2017) presented recently the first application of so-called dryfilm FTIR analysis as a prospective tool for the characterization of protein and polypeptide chain reduction during EPH processing of complex raw materials. In dry-film FTIR, aliquots of liquid samples are dried on multiwell silicon plates prior to transmission FTIR analysis. To evaluate the analytical approach on complex substrates with industrial relevance, salmon- and chicken-based substrates were digested for 80 minutes using Alcalase. A total of 12 FTIR spectra were acquired during the course of hydrolysis, and the observed changes in the IR spectral features as a function of hydrolysis time were found to be in agreement with breakdown of the amide backbone and formation of amino and carboxylate terminals, respectively. Some of the most consistent markers for hydrolysis time were the bands at 1516 cm21 (
NH31) and B1400 cm21 (
COO2) (Fig. 8.7). Moreover, principal component analysis (PCA) of the FTIR spectra demonstrated the systematic relationship between hydrolysis time and key wavelengths in the protein backbone region (800
1800 cm21). Scores of the first principal component versus the hydrolysis time were also shown to provide an overview of the process dynamics related to protein structural changes. To follow up this study, Wubshet et al. (2017) looked at the quantitative aspects of this tool by developing a multivariate approach for monitoring the change in average sizes of protein hydrolysates during EPH of chicken muscle and mechanical chicken deboning residues, respectively. For a total of 129 protein hydrolysates, average molecular weights derived from size-exclusion chromatographic analysis was established as a pragmatic measure of the extent of hydrolysis. FTIR spectra acquired from dry films of the hydrolysates were used to build multivariate calibration models using partial least squares regression. For both groups of hydrolysates, very good correlations were found between the FTIR spectra and the corresponding average molecular weights of the hydrolysates. To the authors’ knowledge, there is no commercial FTIR system available or in industrial use for monitoring EPH processes. As described, several instrument configurations

FIGURE 8.7 The 2nd derivative FTIR spectra (1800
1200 cm21) from Alcalase-catalyzed hydrolysis of (A) salmon processing by-products and (B) mechanical chicken deboning residue. Each spectrum represents the sampling time given in the legend to the right. Source: Reproduced from Bo¨cker, U., Wubshet, S.G., Lindberg, D., Afseth, N. K., 2017. Fourier-transform infrared spectroscopy for characterization of protein chain reductions in enzymatic reactions. Analyst 142 (15), 2812
2818.

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are available that could be used as a commercial industrial tool for EPH process monitoring, and all have pros and cons. The liquid analysis approach is closest to commercial use as generic probe systems and in-line systems are available. The challenge herein lies in the fact that water is a strong absorber in the infrared region, leading to lower sensitivity and less available protein-specific information. This challenge is minimized in the dry-film approach as water is removed prior to analysis. However, the total measurement time will be longer as sample pretreatment requirements are more demanding. Thus, dry-film analysis might be better suited for analysis in continuous processing modes rather than in batch-type processing conditions.

8.5 PRODUCT CHARACTERIZATION Depending on the intended application, protein hydrolysates recovered from EPH of by-products have to meet a set of desired specifications. For example, the MWD of protein hydrolysates have been associated with important quality attributes including nutritional profile, therapeutic value, and functionality (Gbogouri et al., 2004; Wasswa et al., 2007; Wu et al., 1998). Therefore, protein hydrolysate characterization has been a crucial element of product development as well as quality control.

8.5.1 Chromatographic Methods Size exclusion chromatography is perhaps the most commonly used chromatographic technique for characterization of protein hydrolysates in terms of the MWD. Size exclusion chromatography is a technique where constituents of a mixture carried by a mobile phase through a column packed with porous particles are separated according to molecular size and, in some cases, molecular weight. The eluents of the column are then directed to a dedicated detector, for example, diode array detector. The resulting chromatogram will then serve as a MWD profile of a given sample. In such chromatograms, the large proteins elute earlier while the smaller peptides and amino acids elute later. This technique has been widely used in analytical scale characterization of protein hydrolysates as well as in preparative scale for isolation of peptides with specific molecular weight ranges (Chi et al., 2015; Pownall et al., 2010; Slizyte et al., 2016). MWD profiles of protein hydrolysates have been shown to correlate with important processing parameters of production such as hydrolysis time and type and concentration of proteases used for hydrolysis (Guerard et al., 2002; Li-jun et al., 2008). Slizyte et al. (2016) studied the MWD profiles of salmon backbone protein hydrolysates produced using seven different commercial proteases. The study shows that depending on the choice of protease, three types of hydrolysis products with different MWD can be obtained. The possibility of altering the MWD of protein hydrolysates recovered from specific animal by-products by adjusting the choice of protease is one of the major advantages of EPH. The apparent relationship between hydrolysis time and MWD has been studied for several animal-based by-products (Aspevik et al., 2016; Wubshet et al., 2017). Fig. 8.8,

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FIGURE 8.8 Size exclusion chromatograms (at 214 nm) of chicken fillet (A) and by-products (B) with hydrolysis time ranging from 0.5 to 60 minutes. Ranges of chromatograms displaying a pronounced decrease in signals for early eluting large proteins (i) accompanied by an increase in smaller molecular weight region (j) are highlighted in gray. Source: Reproduced with permission from Wubshet, S.G., Mage, I., Bocker, U., Lindberg, D., Knutsen, S.H., Rieder, A., et al., 2017. FTIR as a rapid tool for monitoring molecular weight distribution during enzymatic protein hydrolysis of food processing by-products. Anal. Methods 9 (29), 4247
4254.

illustrates the MWD profiles of chicken fillet (A) and chicken by-products (B) with hydrolysis time ranging from 0.5 to 60 minutes (Wubshet et al., 2017). Several studies have highlighted the correlation between the MWD of protein hydrolysates and the corresponding functional and nutritional properties. In this regard, size exclusion chromatographic analysis can serve as a diagnostic tool to study and optimize the desired properties of a product. In a study by Jeon et al. (1999), cod frame protein hydrolysates subjected to ultrafiltration membranes of different molecular weight cut-offs were shown to afford hydrolysis products with different MWD profiles and functional properties. The MWD distribution profiles were then used as an insight into the relevant molecular weight ranges for functional properties, such as emulsifying and foaming properties. Similarly, Li et al. (2013a,b) have shown that the average molecular weights of fish cartilage collagen hydrolysates, derived from MWD profiles, are linearly and inversely correlated to solubility. The same study has shown that, in contrast to solubility, improved emulsifying and foaming capacities of the hydrolysates are correlated with a higher average molecular weight. Therefore, the MWD profile of protein hydrolysates can serve as a tool to verify and control desired functional properties of a given product. Another widely used chromatographic technique for characterization of protein hydrolysates is reversed phase high-performance liquid chromatography (RP-HPLC). In RP-HPLC, constituents of a mixture are injected on to a column packed with hydrophobic stationary phase. Elution is typically performed using a gradient mixture of water and organic solvent. This generally results in a chromatogram where constituents of the mixture sample are separated based on lipophilicity. The more hydrophobic the constituents of the sample are, the later they elute from the column. In the characterization of protein hydrolysates, RP-HPLC finds most of its applications as a peptide mapping tool. In an interesting study, van der Ven et al. (2001) have investigated the relationship between the RP elution profile and the MWD of milk protein hydrolysates. In the study, a regression was performed on RP retention profiles of whey and casein hydrolysates to

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predict proportions of molecular weight fractions. It was shown that the proportion of a molecular weight fraction in the range from 0.5 to 1 kDa could be accurately predicted from the RP elution profile of the crude hydrolysates.

8.5.2 Amino Acid Composition Analysis Amino acid composition is considered a nutritional profile of protein hydrolysates and is therefore a typical parameter measured for both the substrate (i.e., by-products) and the product. For EPH products, the measurement of amino acid composition is typically performed using chromatographic methods. The first step in amino acid analysis involves a complete digestion of protein and peptides into amino acids. Typical digestion involves addition of a strong acid, such as HCl, and heating to a temperature as high as 110 C (Heu et al., 2003). The identification and quantification are then performed using chromatographic analysis. Nowadays, there is an increased use of commercial amino acid analyzers that can automatically identify and quantify amino acids directly from the digested samples (Chi et al., 2015). In addition to serving as a nutritional profile, the amino acid composition of a given protein hydrolysate can also be used as a chemical interpretation of important product qualities, such as bioactivity. Pownall et al. (2010) studied the amino acid composition of different chromatographic fractions of pea seed (Pisum sativum L.) enzymatic protein hydrolysate. The study showed that the peptide fractions with a higher content of hydrophobic and aromatic amino acids exhibited the strongest radical scavenging and metal chelating activities (Pownall et al., 2010). Similarly, Chi et al. (2015) studied the effect of the amino acid composition of peptide fractions on antioxidant capacities of protein hydrolysates from skipjack tuna (Katsuwonus pelamis) dark muscle. This study also attributed the potent antioxidant activity of selected peptide fractions to the high content of hydrophobic and aromatic amino acid residues.

8.5.3 Isolation and Identification of Bioactive Peptides Bioactive peptides are one of the most sought-after, valuable ingredients in protein hydrolysates from by-products. The analysis and discovery of bioactive compounds is a challenging analytical subject due to the complexity of protein hydrolysates. However, several analytical approaches have been developed and applied for the identification of bioactive peptides towards diseases and complications such as hypertension, diabetes, and oxidative stress. Bioassay-guided fractionation is one of the classical analytical approaches for the discovery of active constituents in a complex mixture. In this approach, a crude, bioactive matrix is subjected to multiple cycles of fractionations, while performing bioassays on fractions collected from each cycle, until a pure and bioactive molecule is isolated. This molecule will then typically be analyzed using MS. This method has been applied for the identification of bioactive peptides in protein hydrolysates in several studies. For example, Tao et al. (2018) followed a series of fractionation steps, involving membrane ultrafiltration, anion-exchange chromatography, gel filtration chromatography and RP

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chromatography, to isolate and identify antioxidant peptides from cartilage protein hydrolysate of Spotless smooth-hound (Mustelus griseus). After isolating the final bioactive fraction, MS was used for identification of three bioactive peptides (i.e., GAERP, GEREANVM, and AEVG). Similarly, Zhang et al. (2015) used a series of chromatographic fractionations in combination with liquid chromatography
mass spectrometry (LC-MS/MS), for isolation and identification of dipeptidyl peptidase IV-inhibitory peptides from trypsin/ chymotrypsin-treated goat milk casein hydrolysates. Examples of typical mass spectrometric data used for elucidation of the bioactive peptides are presented in Fig. 8.9.

8.5.4 Proteomic Approaches Proteomics is defined as the large-scale study of proteins, and is now largely reliant on the use of various LC-MS/MS techniques and bioinformatics tools. Bottom-up proteomics is an approach commonly used to determine the protein composition of a given tissue, cell, or organelle, with respect to protein composition, protein quantification, and identification of posttranslational modifications of proteins. Firstly, a controlled proteolytic digestion of proteins is performed, before the resulting peptide mixture is analyzed using LC-MS/MS methods, and finally bioinformatics analysis is performed. The main advantage of bottom-up proteomics is its ability to unravel systematic effects at a functional level, without a priori knowledge, by combining LC-MS/MS analysis with bioinformatics and statistics. Thus, bottom-up proteomics has become a popular and powerful approach to determine the protein composition of a given sample. Likewise, this approach can be used as a screening tool to unravel changes in peptide composition of EPH products. Indeed, Holton et al. (2016) performed a LC-MS/MS comparison of the peptide profiles after digestion with five different proteases of the same skim milk powder. They demonstrated that it was possible to group the peptide profiles of the different hydrolysates based on the digestion enzyme used, and that the most efficient grouping was achieved by comparing the residues at the peptide termini together with the relative peptide abundance data. Similarly, Lambers et al. (2015) compared the peptide profiles of several commercial infant formulae characterized by LC-MS/MS analysis and coupled these with multivariate clustering analysis. They showed that the peptide profiles from the various formulae provided descriptive and distinct signatures, particularly when the identified peptide sequences were coupled to their position in their corresponding parental proteins. Another application of proteomics related to protein hydrolysates or digests is to perform in silico analysis, either by itself or in combination with LC-MS/MS analysis, aiming at predicting the release of bioactive peptides (for a comprehensive review, see Rani et al., 2018). Such analyses are thus reliant on information regarding protein and/or peptide sequences in addition to protease substrate specificity. As an example, to predict the release of bioactive peptides from meat digestion, Sayd et al. (2018) combined the use of LC-MS/MS analysis to detect peptides generated during in vivo gastric digestion, with a subsequent in silico digestion of those peptides mimicking the action of intestinal proteases using the “Enzyme actions” tool of the BioPep database. Similarly, Lafarga et al. (2016) used LC-MS/MS to identify peptides from bovine blood globulin hydrolysates, and combined that with in silico analysis of allergenicity and toxicity using AlgPred

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FIGURE 8.9 Identification of DPP-IV-inhibitory peptides. (A) LC-MS spectrum and (B) LC-MS/MS spectrum of single-charged ion with m/z 1268.63. Identified peptide sequence is displayed with ion fragments observed in the spectrum. Source: Reproduced with permission from Zhang, Y., Chen, R., Ma, H., Chen, S., 2015. Isolation and identification of dipeptidyl peptidase IV-inhibitory peptides from trypsin/chymotrypsin-treated goat milk casein hydrolysates by 2D-TLC and LC-MS/MS. J. Agric. Food Chem. 63 (40), 8819
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(Saha and Raghava, 2006), AllerTOP (Dimitrov et al., 2014), and ToxinPred (Gupta et al., 2013), respectively (Lafarga et al., 2016). Proteomics tools have also been used to study substrate specificity of proteases. O’Donoghue et al. (2012) developed a strategy for Multi Substrate Profiling by MS, where they created a peptide library with 124 peptides with different chemical properties based on the nature of the protease of interest. These peptides were initially characterized by LC-MS/MS, and the relative abundance of each peptide was determined based on the chromatographic profile. The peptides were then exposed to the protease of interest, before their relative abundance was measured again using LC-MS/MS. The substrate specificity was then determined by investigating which of the peptides showed a decrease in abundance over the exposure time. Proteomics tools are well suited for characterizing and comparing peptide profiles in different protein hydrolysates. By applying bottom-up proteomics to protein hydrolysates both during processing and of the final EPH products, more knowledge regarding substrate specificities in complex systems as well as more detailed characterization of the peptide compositions will be achieved. Moreover, Multi Substrate Profiling-MS could be an important tool to study substrate specificities more in detail. These approaches can provide the essential information needed to understand the complex enzyme kinetics in protein hydrolysates. In addition, the combination of LC-MS/MS for peptide profile characterization and in silico analysis is a useful and efficient tool to screen the biological potential of protein hydrolysates produced from different raw materials and/or with different proteases, without the need to perform time-consuming and often expensive assays.

8.6 FUTURE PERSPECTIVES Process control is essential in all types of bioprocessing due to the inherent biological variations. Several of the analytical techniques described in this chapter have the potential to be used in industrial environments to fit into a process optimization and control scheme. Processes can be controlled either in a feed-forward or feed-backward approach, or a combination of the two, as shown in Fig. 8.10. The feed-backward scheme is most

FIGURE 8.10 Feed-forward and feed-backward process control and optimization schemes in EPH. Source: Reproduced and modified from Wubshet, S.G., Wold, J.P., Afseth, N.K., Bo¨cker, U., Lindberg, D., Ihunegbo, F.N., et al., 2018. Feed-forward prediction of product qualities in enzymatic protein hydrolysis of poultry by-products: a spectroscopic approach. Food Bioprocess Technol. 11 (11), 2032
2043.

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commonly used, and works by deciding a suitable control action based on end-point measurements. The disadvantage of this approach for EPH processing is that the control action is taken too late, that is, a lot of product that is off-specification is produced before action is taken. Since we know that the raw material quality varies and highly affects the hydrolysis product, a feed-forward scheme is more appropriate. In feed-forward control, a predefined model decides settings of the controllable process parameters, using measured raw material characteristics as input. We have already shown that there is a potential for feed-forward control of EPH processes by using spectroscopic measurements of raw materials (Wubshet et al., 2018). This was however a small feasibility study focusing on raw material variation, and hydrolysis time was the only controllable process parameter that was studied. In order to use the feed-forward approach in an industrial setting, we need to know how other process parameters, such as temperature and protease (type and amount), affect the yield and product quality. The hypothesis is that there are significant interactions between raw material properties and process parameters, and also within the process parameters themselves. A feed-forward approach depends on a reliable mathematical model that describes the dependencies between input factors (raw material quality and process parameters) and output factors (e.g., yield and DH). The complexity of bioprocesses and their raw materials, as described earlier in this chapter, calls for multivariate models, where many variables are summarized by so-called latent structures. Such models can be fitted using partial least squares regression and multiblock approaches (Ma˚ge et al., 2008), and also can be applied for process optimization (Ma˚ge and Næs, 2007). The models are empirical and depend on reliable data that span a relevant variation range. The optimal way to collect such data is using statistical Design of Experiments, but this is usually not feasible in full-scale EPH production. A huge challenge is therefore to obtain mathematical models that are representative at the industry scale. This can be solved by using a combined strategy where extensive experiments (based on a Design of Experiment approach) are performed in a small-scale laboratory environment. These data can then be used to identify the critical process parameters, and most importantly their interactions with raw material characteristics. In parallel, daily samples from full-scale industrial EPH production can be collected. These samples will not have the same variation range and statistical properties as the lab-scale results, but can be used to validate the lab-scale findings and adjust the model to fit an industrial setting. Several strategies for feed-forward process control can be applied. The most straightforward approach is to measure the raw materials as they enter the process and adjust process variables accordingly. Another approach is so-called prediction sorting, where raw materials are sorted into homogeneous groups before processing. These groups can then be processed together (using different production settings) or even be processed into different product categories (Berget and Næs, 2002). This approach may be especially useful in batch processes and continuous processes where changing the protease type is an option.

CONCLUSION EPH is one of the advanced technologies used in the recovery of protein, peptides, and amino acids from food processing by-products. Analytical technologies play a vital role in

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areas ranging from characterization of the by-product biomass (i.e., the by-product used as the raw material) to studies of product quality. In this chapter, both classical analytical methodologies and emerging advanced technologies were reviewed. The classical analytical methodologies, such as OPA for measuring the degree of protein hydrolysis, are perhaps the most frequently used approaches both by the industries and research laboratories. However, poor reproducibility and relatively time-consuming and labordemanding experiments are the major limitations associated with the classical method. In order to overcome these challenges, emerging analytical approaches have focused on the use of advanced and rapid techniques, such as FTIR for the characterization of protein hydrolysates. These advanced analytical approaches have, in contrast to the classical methods, a promising potential to serve as on-line process and quality control tools. In conclusion, various analytical strategies have been developed and shown to serve as core research tools in the recovery of protein from food processing by-products using EPH.

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Production and Bioengineering of Recombinant Pharmaceuticals Tatiana Q. Aguiar, Sı´lvio B. Santos, Ivone M. Martins, Lucı´lia Domingues and Carla Oliveira CEB

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9.2 Phage Display 262 9.2.1 Identification of New Protein/Peptide Ligands by Phage Display 262 9.2.2 Production of Phage Display-Derived Proteins/Peptides in Escherichia coli 264 9.2.3 Therapeutic Applications of Phage Display-Derived Proteins and Peptides 265 9.3 Antimicrobial Peptides 268 9.3.1 Heterologous Expression Systems Used in Recombinant Antimicrobial Peptide Production 269 9.3.2 Generic Strategies Used for Increasing Recombinant Antimicrobial Peptide Expression 271 9.3.3 Fusion Technology for Recombinant AMP Production and Purification 272

Proteins: Sustainable Source, Processing and Applications DOI: https://doi.org/10.1016/B978-0-12-816695-6.00009-X

9.4 Endolysins 274 9.4.1 Application of Endolysins to Control Pathogenic Bacteria 274 9.4.2 Understanding the Structure of Endolysins for Protein Engineering 275 9.4.3 Improving the Activity of Endolysins by Protein Engineering 275 9.4.4 Improving the Solubility of Endolysins by Protein Engineering 277 9.4.5 Endolysins as Tools in Heterologous Protein Production and Purification 278 9.5 Lectins 280 9.5.1 Overall Molecular Cloning Strategy for Recombinant Production of Lectins in Pichia pastoris 280 9.5.2 Recombinant Lectin Production in Pichia pastoris and Purification Strategies 282

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9.1 INTRODUCTION Proteins and peptides are the mainstay of the biopharmaceutical sector, but unlike other drugs, proteins are not synthesized chemically but recombinantly in different host cells. Since the early recombinant production of the well-characterized peptide, insulin, for human therapeutic use in 1982, recombinant techniques and manufacturing platforms have evolved quite significantly. On the other hand, the evolution of bioinformatics and high-throughput techniques have been and still are pivotal for the discovery of novel peptides and proteins, increasing the amount of the biological drugs produced recombinantly. Thus, this is a quite dynamic field with quick changes enabling the improvement of the production and bioengineering of therapeutic proteins. On a 2015 update, Sanchez-Garcia et al. (2016) referred that around 650 protein drugs were approved worldwide, and from these 400 were obtained by recombinant techniques while another 1300 recombinant pharmaceuticals were under development. In fact, product development shows no sign of stopping, 183 biotech products were in Phase III clinical trials in 2014 (Mattews et al., 2017). In this development, there is a clear tendency towards bioengineered versions with improved performance and new functionalities regarding the conventional, plain protein (Sanchez-Garcia et al., 2016). Pivotal to this development is the expansion of the genetic engineering toolbox together with the ever increasing understanding of cell physiology and stress. These are unquestionably driving the development of recombinant protein production cell platforms. Since the foundation of genetic engineering, genetic techniques and resources have significantly evolved from a few DNA modification techniques and scarce genetic materials (plasmids, genome sequences, etc.) to much more controlled, broad, and high-throughput gene manipulation techniques and wide commercial availability of genetic resources (Oliveira et al., 2017). This expanded toolbox of synthetic promoters, fusion partners, signal sequences, and strains, integrated with bioprocess development and downstream processes, enables the manufacturing of tailor-made recombinant proteins. Among the many conventional and emerging cell-based systems for protein production, bacteria, yeast, and mammalian cell lines are the most common in biopharma (Sanchez-Garcia et al., 2016). One of the main bottlenecks of the Escherichia coli expression system is endotoxin production, but recently an endotoxin-free strain of E. coli has been developed (Mamat et al., 2015). This, together with other abovementioned and downstream processing developments (Oliveira and Domingues, 2018), makes this system a cost-efficient and versatile production of therapeutic proteins by skipping endotoxin removal steps, thus gaining in biosafety and reducing production costs. For the Pichia pastoris expression system, the main drawback is the hypermannosylation of the proteins produced. In this regard, the engineering of Pichia glycosylation has enabled the production of valuable biopharmaceuticals with a more homogeneous, “humanized” N-glycosylation pattern (Jacobs et al., 2009). This, together with the

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development of new platform strains and molecular cloning tools (Yang and Zhang, 2018), have made P. pastoris an important host for biopharmaceutical production. On the other hand, production processes in mammalian CHO cells have become well-developed, surpassing the limitation of low production titers and reaching typical commercial values of 1 5 g/L today (Mattews et al., 2017). In such a vast and growing field, there are emerging many interesting examples of recombinant pharmaceuticals, either proteins or peptides. In this chapter, this is exemplified by presenting a technique that enables the discovery of novel therapeutic peptides and proteins, phage display (Section 9.2), and by addressing novel approaches and microbial cell platforms used for the recombinant production of interesting peptides, such as antimicrobial peptides (AMPs) (Section 9.3), and proteins, such as endolysins (Section 9.4) and lectins (Section 9.5), all with a wide range of applications. The discovery of novel peptides and/or proteins is one of the drivers for therapeutic protein development. Different approaches have been explored in that regard, with phage display being one of the prominent techniques. In Section 9.2, phage display is addressed with the aim of showing the potentialities of this technique, which allows the discovery of novel peptides and proteins targeted to a specific receptor/ligand. In the context of expanding protein drug markets, there is a generic consensus about the need to enable drugs for cell- or tissue-targeted delivery to reduce doses, production costs, and side effects (Sanchez-Garcia et al., 2016). In this sense, production of phage display-derived proteins/peptides and their applications in cancer research, regenerative medicine, and neurosciences are also described. Antibiotics resistance is one of the biggest health threats to global health, food security, and development today. The excessive use and misuse of conventional antibiotics in medicine, agriculture, and food industry led to one of the greatest health challenges of our time: the worldwide emergence of multidrug resistant microbes, against which there are no effective therapies. To counteract this issue, compounds such as AMPs and endolysins from phages have been extensively evaluated as novel alternatives to antibiotics. Nevertheless, for the implementation of these alternatives, one would need large-scale production facilities that can be encountered by the development of microbial production systems. Moreover, the use of recombinant systems has allowed for the bioengineering of AMPs and endolysins. This has been pivotal for the successful development of improved and/or tailor-made molecules (Sections 9.4 and 9.5). Lectins are glycan binding proteins that selectively recognize glycan epitopes of free carbohydrates or carbohydrates bound to cell membranes, such as glycoproteins, glycolipids, or polysaccharides. As the recognition of these carbohydrates is specific, lectins will differentially bind to certain molecules and cells (Martı´nez-Alarco´n et al., 2018). Lectins’ selectivity has been explored as a tool in glycobiology, for instance, as an array for assessing glycosylation of therapeutic proteins (Zhang et al., 2016), but also in the analyses of changes occurring in cellular interactions during physiological and pathological processes (Oliveira et al., 2013a), providing evidence for a wide spectrum of industrial and pharmaceutical applications (Martı´nez-Alarco´n et al., 2018). However, the need for large-scale production of these molecules, as well as the production control of specific isoforms, can only be achieved in recombinant expression systems. Recent developments and strategies used for this goal are described in Section 9.5.

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9.2 PHAGE DISPLAY The identification of new proteins and peptides with the capacity to bind to their target molecules and to manipulate their biological function was enabled with the discovery of phage display—expression of a given molecule at the surface of a bacteriophage particle. Bacteriophages (abbreviated to phages) are viruses that recognize and infect only bacterial cells, and are composed of a densely packaged genetic material (DNA or RNA) encapsulated in a protein coat. As natural parasites of bacteria, phages are considered the most diverse and abundant entity on Earth and are thought to exist in every ecosystem, being consequently present in mammalian organisms as important components of the human microbiome (Merril, 1974; Dabrowska et al., 2005). They were discovered in 1915 by Frederick Twort, an English physician, and later in 1917 by Felix d’Herelle, a Canadian microbiologist, who named these bacteria-infecting viruses bacteriophages. Given their capacity to kill bacteria, phages have been extensively studied and used in clinical practice since the 1920s (Abedon et al., 2011, 2017; Domingo-Calap et al., 2016; Nobrega et al., 2015; Sharma et al., 2017, Vlassov et al., 2017). Phage display was first reported in 1985 by George Smith (awarded with the Nobel Prize in Chemistry 2018) as a simple combinatorial molecular biology technique where, most commonly, peptides and proteins are displayed on the surface of phage particles. Using filamentous phages, Smith was able to produce a viable modified E. coli virus with a foreign protein on its surface (Smith, 1985). For phage display experiments, E. coli filamentous phages are the most used, in particular the group of the F-pilus-specific phages (Ff), which comprises the f1, fd, and M13 phages. Besides being very easy to handle and manipulate, with low-cost maintenance and production, they are very stable and versatile, as they can be modified genetically and/or chemically (Barbas et al., 2001; Smith and Petrenko, 1997). Structurally, filamentous phages are tube-shaped filaments formed by thousands of helically arranged copies of a major coat protein pVIII and, at the extremities, by two different pairs of proteins: on one side by pVII and pIX, and on the opposite side by pIII and pVI (Fig. 9.1A). The major advantage of this technology stems from the direct linkage between the genotype and phenotype of the phage displaying the protein/peptide of interest. All the phage’s coat proteins can be genetically modified by recombinant DNA technology and a mixture of various filamentous phages can be generated, each expressing different proteins (including antibodies) or peptides, displayed as fusions to the phage’s coat proteins—combinatorial phage libraries. The most common method to generate phage libraries involves the cloning of the foreign sequence (coding for a protein/peptide) in the gene 3 of the phage genome, which will result in the display of the protein/peptide of interest on the amino terminus of the minor coat protein pIII (Fig. 9.1B). Although each phage particle displays a unique foreign protein/peptide, the entire library represents a vast diversity of billions of protein/ peptide candidates that can specifically bind to a target.

9.2.1 Identification of New Protein/Peptide Ligands by Phage Display Phage display has been widely used as an efficient molecular selection technique to identify, from a phage library, specific phage particles bearing sequences that have some

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FIGURE 9.1 Schematic representation of a phage display experiment. Bacteriophages can be genetically manipulated to display at their surface a given molecule (A). The cloning of random DNA sequences on the phage genome originates combinatorial phage libraries with a diversity of billions of phage particles, each one displaying a single protein/peptide (B). Phage libraries are screened by a process called biopanning, where 4 5 rounds of incubation with the target will promote the selection of the phage containing the sequence with more affinity towards that target (C). One step of the biopanning comprises the infection of Escherichia coli with the eluted phages for replication and posterior enrichment of the phages (D). The phage particles identified can be further modified chemically with imaging compounds and/or therapeutic drugs for targeted delivery (E).

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special properties, such as binding to a given protein receptor or organic molecule. Random libraries are screened by affinity selection, through a biological evolutionary selection process called biopanning (Fig. 9.1C). Several rounds of selection are needed, usually between four to five rounds, each comprising several steps: 1. the incubation of the phage library with the target immobilized on plates. Alternatively, selection can be performed with the target in suspension (Giordano et al., 2001); 2. a washing step to remove the unbound phages; 3. the elution of the phages that specifically bind to the target by changing the binding conditions (such as changing the pH or adding a competing ligand, a denaturant, or a protease), to disrupt the interaction between the displayed ligand and the target; 4. infection of the bacterial host E. coli for replication/production of the eluted phages (Fig. 9.1D). It is noteworthy that phages eluted in each biopanning round will be used for the following round, promoting the enrichment of phages displaying a given protein/peptide of interest; and 5. the identification of the selectants by phage DNA sequencing, to determine the sequence of the protein/peptide with affinity to the target. After multiple rounds, a specific phage (or phages) can be isolated from the population and replicated in bacteria to produce an infinite number of descendants.

9.2.2 Production of Phage Display-Derived Proteins/Peptides in Escherichia coli Despite being the most numerous life forms on Earth, phages cannot replicate on their own. To replicate, they need to infect their bacterial host. However, and contrarily to other phages, filamentous phages do not cause cell lysis and are continuously produced by infected cells (Kehoe and Kay, 2005; Rakonjac et al., 2011). The eluate recovered from the biopanning rounds, containing the phage with affinity towards the target, is incubated with the phage’s bacterial host to allow phage infection. The bacterial culture should be at early-log phase and incubation should be performed under agitation for 4 5 hours at 37 C. The pellet containing the bacterial cells and debris is discarded by centrifugation and the supernatant containing the phages is respined to discard the remaining pellet. The upper phase of the supernatant (approximately 80%) is transferred to a fresh tube and stored at 4 C. Alternatively, during the production, when the culture turbidity decreases after 4 5 hours of infection, the culture can be transferred to 50-mL centrifuge tubes and chloroform can be added to a final concentration of 10% (v/v). After mixing, the suspension is filtered with a 0.22-μm filter. This is the amplified phage stock and can be stored at 4 C for several weeks with little loss of titer. For long-term storage (up to several years), dilute 1:1 with sterile glycerol and store at 20 C (Clokie and Kropinski, 2009; Sambrook and Russell, 2001). After production, purification is performed to ensure a pure phage suspension. Phages can be purified from crude bacterial lysates by several methods, but the addition of polyethylene glycol (PEG) is a common and fast procedure that does not affect the phage’s infectivity. Briefly, a PEG/NaCl solution is added to the phage solution, to allow the phages to precipitate at 4 C, preferably overnight. The supernatant is then decanted by centrifugation and the phage pellet is resuspended in Tris buffer saline (TBS). The suspension is again PROTEINS: SUSTAINABLE SOURCE, PROCESSING AND APPLICATIONS

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centrifuged to pellet residual cells and the supernatant is reprecipitated again with PEG/ NaCl. The pellet is obtained by centrifugation, resuspended in approximately 200 μL of TBS, and stored at 4 C (Clokie and Kropinski, 2009; Sambrook and Russell, 2001). Phage titration is required to determine the amount of phage particles that are present in a given sample. For that, 10 103-fold serial dilutions of phage solution should be prepared in Luria Bertani (LB) medium or SM buffer, in a final volume of 1 mL. Infection must be carried out in bacteria at mid-log phase. Ten microliters of each phage dilution must be dispensed into 200 μL of bacterial suspension, mixed, and briefly incubated at room temperature. The infected cells should be transferred to culture tubes containing prewarmed top agar, mixed briefly, and immediately poured onto a prewarmed LB plate containing isopropyl-β-D-thiogalactoside (IPTG) and 5-bromo-4-chloro-3-indolyl-β-D-galactoside (X-gal) for blue/white screening. The blue/white screening is permitted because the library cloning vector M13KE contains the lacZα-peptide cloning sequence. The plates must be incubated overnight at 37 C and on the next day blue colonies can be counted (only the plates containing 3 30 colonies). The phage titer is calculated in colony forming units (CFUs) per 10 μL, by multiplying each number by the dilution factor of each plate (Clokie and Kropinski, 2009; Sambrook and Russell, 2001). It is noteworthy that for filamentous Ff phages to infect bacteria two important receptors must be present on a F-pilus positive E. coli strain. The pili are the primary receptor—a long filamentous structure present on the surface of bacterial cells—and the tolQRA complex is the secondary receptor, present in the internal membrane of bacterial cells. The infection process initiates when the pili are recognized by the phage’s coat minor protein pIII and attach to it. When the pili retract on the cell surface they bring the phage closer to the internal membrane, allowing the communication of pIII with the tolQRA complex. Consequently, the minor protein pVI “opens” and the phage’s genetic material (ssDNA) is inserted into the cell cytoplasm, triggering a cycle of phage production and transforming the bacterial cell in a phage factory. ssDNA replicates first, followed by the synthesis of structural proteins of the phage particle, including the major and minor coat proteins, and also an extra protein, the pV, which transports and protects the genetic material to the bacterial membrane. As ssDNA crosses the membrane, the pV protein dissociates and is replaced by the major protein pVIII. New phage particles are assembled by the addition of the remaining coat proteins and when the DNA is completely enclosed by the proteinaceous capsid, the new phage is released without causing any damage to the host cell (Fig. 9.1D).

9.2.3 Therapeutic Applications of Phage Display-Derived Proteins and Peptides Although the main application of phage display is to identify proteins/peptides of interest, its application has also been broadened to use the phage particle itself as a scaffold to develop biologically active protein/peptide platforms. These platforms can be engineered not only by genetic manipulation of the phages’ surface to display a given molecule of interest, but also by chemical modification of their coat proteins. Chemical modifications are usually performed on the major protein coat pVIII (Fig. 9.1E) using standard chemical modulation techniques to label the phage’s coat protein with several compounds (e.g., a specific imaging agent or a therapeutic drug), without interfering with the targeting ability of the displayed molecule or with the phage’s ability to be internalized into mammalian cells, revised in Bernard and Francis (2014) and Chung et al. (2014). PROTEINS: SUSTAINABLE SOURCE, PROCESSING AND APPLICATIONS

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As bacterial viruses, the therapeutic potential of phages was primarily exploited against bacterial pathogens causing infections. Nevertheless, given their versatility, phage engineering and its phage display products (proteins and peptides) have broadened their application to the most diverse areas of medicine. Bacteriophages and phage display have been widely used and exploited in cancer research, neurosciences, regenerative medicine, microbiology, infections, and other medical fields, contributing to the discovery and generation of a wide range of therapeutic peptide and protein ligands with therapeutic, diagnostic, and imaging applications. Their biomedical applications range from the development of probes for sensing, imaging, diagnosis, and detection, the development of gene and therapeutic drug carriers for targeted delivery, the development of new vaccine vectors, and also the development of new biomaterials as supramolecular building blocks (Fig. 9.2) (Farr et al., 2014; Ferreira and Martins, 2016; Henry et al., 2015; Kelly et al., 2006; Li et al., 2010; Martins et al., 2016; Molek and Bratkovic, 2015; Omidfar and Daneshpour, 2015; Tan et al., 2016; Wu et al., 2016). Every year hundreds of protein and peptide sequences are identified by phage display and some of them are deposited in different databases (PepBank, Tree of Medicine, BDB), which can be accessed free of charge. Currently, the biopanning data bank (BDB, at http://immunet.cn/bdb/) contains 30,383 peptide sequences (Huang et al., 2012).

FIGURE 9.2 Applications of bacteriophages and phage display.

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Cancer research is one of the main fields exploited, taking advantage of the potential of phages and phage display. Some applications rely on the identification of biomarkers or receptors expressed on cancer cells (Brissette et al., 2006; Gray et al., 2013; Nobrega et al., 2016; Silva et al., 2016b). Using phage display, new peptides specific for the MDA-MB-231 cells (a representative of the claudin-low subtype of breast carcinomas) were identified, PRWAVSP and DTFNSFGRVRIE, exhibiting a strong binding towards these cells and a good specificity, demonstrated by the low binding to other breast cancer cells (Nobrega et al., 2016). A similar study from the same group also reported the use of phage display for the identification of other peptides, CPTASNTSC and EVQSSKFPAHVS, using the triple negative breast cancer murine mammary carcinoma cell line 4T1. Interestingly, those peptides have also shown affinity towards the human MDA-MB-231 cell line, promoting the translational application of these novel ligands between species (Silva et al., 2016b). The identification of those biomarkers enables the development of synthetic platforms, phage-based or not, for targeted drug delivery to optimize the effect of cytotoxic drugs and to reduce the toxic side effects of chemotherapy (Li and Mao, 2014; Petrenko and Jayanna, 2014). Moreover, those targeted phage-based carriers can also be coupled with imaging molecules, through chemical modification of the phage particles, for the development of imaging and biosensing platforms (Condeelis and Weissleder, 2010; Ghosh et al., 2012). The same approach can be used for regenerative medicine applications, namely the engineering of new biomaterials. Several different types of biomaterials can be functionalized with the phage-selected peptide to: 1. develop new synthetic substrates for cell culture that can control cell expansion and/or differentiation, and which recreate the in vivo environment, to promote tissue repair; 2. serve as nanocarriers for gene and drug delivery in targeted therapy; or 3. develop imaging probes for cells’ and tissues’ imaging in vivo (Brown, 2010; Farr et al., 2014; Hamzeh-Mivehroud et al., 2013; Laakkonen and Vuorinen, 2010; Martins et al., 2016; Petrenko and Jayanna, 2014). In a recent review paper (Martins et al., 2016), the authors have compiled some sequences of peptides identified by phage display that bind to regenerative medicine targets. Moreover, a follow-up of those sequences summarizes the subsequent use of those peptides in diverse experimental studies, including in vitro and in vivo testing/characterization and optimization/formulation towards their medical application (Martins et al., 2016). The neurosciences field has also benefited from phage display. Several phage displayderived peptides and proteins have been reported for application in this field, mainly due to the fact that phages are able to cross the blood brain barrier (BBB). By sheltering the central nervous system from the systemic circulation, the existence of the BBB is a major bottleneck for effective therapeutic applications for neurodegenerative diseases (Modarres et al., 2018). By in vivo phage display, through intravenous injection of a peptide phage display library in mice, an increased number of phages were observed in several organs, like the brain and kidney, and peptide sequences with affinity to those organs were identified (Kolonin et al., 2006; Pasqualini and Ruoslahti, 1996). More recently, and also by in vivo phage display, a peptide denominated Pep7 was identified with high affinity towards the brain tissue (Li et al., 2012). In vitro the M13 capsid protein motif has been reported to bind to and to remodel multiple types of misfolded protein aggregates, including amyloid-beta

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AB, the prime suspect in causing Alzheimer’s Disease (AD) (Krishnan et al., 2014; Levenson et al., 2016). Given this, once inside the brain, phages may be used to selectively destabilize or neutralize AB peptide aggregates and promote AD therapy. Phage display has also allowed the identification of AMPs from various sources, most of which can be found at the AMPs database (http://aps.unmc.edu/AP/main.php) (Wang et al., 2016). Several peptides have been identified using this approach: from peptides with antimicrobial activity against bacteria and viruses (Flachbartova et al., 2016; Pini et al., 2005; Sainath Rao et al., 2013; Tanaka et al., 2008), to peptides with activity against pathogenic parasites, such as the one responsible for malaria (Lanzillotti and Coetzer, 2008), the protozoan parasite Cryptosporidium parvum (Chen et al., 2003), and avian coccidiosis causal agents (da Silva et al., 2002). These peptides make it possible to develop a therapy that targets the antigens involved in host parasite interactions and in the pathogen’s ability to attach and invade host cells.

9.3 ANTIMICROBIAL PEPTIDES AMPs are small polypeptides produced naturally by nearly all living organisms as components of nonspecific basal defense mechanisms against invading pathogens or competitor species (Ageitos et al., 2017; Holaskova et al., 2015; Silva et al., 2016a). In mammalians, they are also involved in immunomodulatory activities and inflammatory processes (da Costa et al., 2015; Li et al., 2017a). AMPs typically contain fewer than 50 amino acids, present strong alkaline and thermal stability, and provide fast and broad-spectrum antimicrobial effects. They have been reported to exhibit biocidal activity against a wide range of pathogenic organisms (bacteria, fungi, viruses, and parasites) and even transform cancer cells (Ageitos et al., 2017; Holaskova et al., 2015; Wang et al., 2018). The specificity and biological activity of each AMP is defined by its composition, physicochemical properties, and structure (Lee et al., 2016; Wang et al., 2018). The most widely recognized mechanism of action of AMPs involves their interaction with the membrane of target cells, which ultimately results in membrane disruption and cell lysis (da Costa et al., 2015; Lee et al., 2016; Silva et al., 2016a). However, some AMPs can quickly pass through the membrane without causing membrane damage and target intracellular molecules, leading to cell death through inhibition or inactivation of important cellular functions (da Costa et al., 2015; da Cunha et al., 2017; Lee et al., 2016; Silva et al., 2016a). The cationic and amphiphatic nature of most AMPs promotes the establishment of electrostatic and hydrophobic interactions, specifically with the surface of negatively charged microbial membranes (Lee et al., 2016). Thus, cationic AMPs display low toxicity to healthy mammalian cells (da Costa et al., 2015; Lee et al., 2016; Silva et al., 2016a). Moreover, since AMPs mainly target microbial membranes and do not interact with specific receptors, their targets have a low propensity to develop resistant phenotypes (da Cunha et al., 2017). Owing to their attractive properties, AMPs are of particular interest as a new generation of antimicrobial agents for use as alternatives to conventional antibiotics and biocides in human and animal therapy (in monotherapy or combined with other drugs), in crop protection, and also in food preservation (Ageitos et al., 2017; Holaskova et al., 2015; Mishra et al., 2017; Silva et al., 2016a). Although a large number of AMPs have been discovered, only a

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few have reached the clinical trials stage and even fewer are commercially approved (da Costa et al., 2015; da Cunha et al., 2017; Mishra et al., 2017; Silva et al., 2016a). The road to market of new AMPs is usually long and costly, and the high manufacturing costs are one of the major obstacles to overcome (da Costa et al., 2015; da Cunha et al., 2017; Silva et al., 2016a). Given the small molecular weight of AMPs and the importance of their structural characteristics to the antimicrobial activity (Lee et al., 2016; Wang et al., 2018), the obtainment of high-purity AMPs with high biological activity in a cost-effective way is challenging. Traditional extractions methods from natural sources, while having the advantage of obtaining AMPs with high biological activities, are labor-intensive, time-consuming, and yield low amounts of purified AMPs (Deng et al., 2017; Gaiser et al., 2011). Therefore, these methods do not provide a cost-effective way for the large-scale production of AMPs. Chemical synthesis processes, namely solid-phase synthesis, solution-phase synthesis, and hybrid approaches, provide an interesting alternative for large-scale production of highquality AMPs, namely of a synthetic or hybrid nature (da Costa et al., 2015). These processes have the advantage of producing satisfactory yields of high-purity AMPs in a faster way and of providing more flexibility in terms of peptide design, by allowing the incorporation of nonnatural amino acids or modifications into peptides (da Costa et al., 2015; Wang et al., 2018). Indeed, most of the commercially approved peptide-based drugs, which are frequently of small to medium size, are produced by chemical synthesis (da Costa et al., 2015; da Cunha et al., 2017). Nevertheless, the chemical synthesis of large peptides or of peptides with complex posttranslational modifications (e.g., multiple disulfide bonds) constitutes a long, highly costly and laborious process (Gaiser et al., 2011; Sousa et al., 2016). Therefore, this is not an ideal platform for the large-scale production of AMPs with these characteristics. Another way to produce AMPs involves their biological synthesis in recombinant production systems. This is the most commonly used method to produce therapeutic proteins and has been described as the most cost-effective procedure for the large-scale production of high-quality AMPs (Li, 2011). Compared to chemical synthesis, it presents the following advantages (Deng et al., 2017; Li, 2011): 1. 2. 3. 4.

short production periods and low production cost, even for long and complex peptides; reasonably high production yields of short and large AMPs; easily scalable; and the existing facilities certified for the recombinant production of therapeutic proteins can be used for AMP production.

This production technology also provides flexibility in terms of peptide engineering and design (Li, 2011; Li et al., 2017a; Mishra et al., 2017; Wang et al., 2018). Many AMPs have been successfully obtained through recombinant production in various heterologous hosts (Table 9.1) (Deng et al., 2017; Holaskova et al., 2015; Li, 2011; Li et al., 2017a; Mishra et al., 2017).

9.3.1 Heterologous Expression Systems Used in Recombinant Antimicrobial Peptide Production Among the various heterologous expression systems that have been used to produce AMPs in large-scale, microbial systems (bacteria and yeast) have been the most widely PROTEINS: SUSTAINABLE SOURCE, PROCESSING AND APPLICATIONS

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TABLE 9.1 Reported Yields for Recombinant AMPs Produced in Different Heterologous Expression Systems, With Indication of the Expression Strategies Used Expression System

Promoter AMP

Partner(s)

Expressiona

Yield (mg/L)b References

Escherichia coli

T7

LL37

CBM3

Soluble

2

Ramos et al. (2010)

T7

Pa-MAP 2

ELP-intein

Soluble

96

Sousa et al. (2016)

T7

Histatin 1

FK-intein

Insoluble

6.6 [180] Zhao et al. (2017)

T7

Snakin-1

Coexpression partner HLA

Insoluble

2

Kuddus et al. (2017)

T7

Cecropin A

Intein-sup35NM

Secretion (CsgA)

294

Wang et al. (2017b)

Bacillus subtilis glv

CAM-W

His-tag-EDDIE

Secretion (SacB)

159

Ji et al. (2017)

glv

Peclastin

His-tag-SUMO

Secretion (SacB)

5.5 [41]

Zhang et al. (2015)

AOX1

Snakin-1

His-tag

Secretion (αMF)

40

Kuddus et al. (2016)

AOX1

PaDef

His-tag

Secretion (αMF)

80

Meng et al. (2017)

AOX1

Apidaecin Ia HSA-His-tag-TEV cleavage site

Secretion (αMF)

[.700]

Cao et al. (2018)

AOX1

Plectasin

His-tag

Secretion (αMF)

880

Chen et al. (2016)

GAP

Plectasin

His-tag

Secretion (αMF)

370

Chen et al. (2016)

GAP

MP1102

None

Secretion (αMF)

377 [538]

Mao et al. (2015)

Pichia pastoris

a

Between parenthesis are indicated the signal peptides used. Between square brackets are indicated the yields for the fusion protein before AMP purification.

b

employed, mainly because of easy manipulation, rapid growth to high cell densities, and relatively low-cost fermentation processes (Deng et al., 2017; Li, 2011; Li et al., 2017a). Nevertheless, AMPs have been also produced in plant-based heterologous expression platforms, namely for molecular farming (i.e., large-scale AMP production in plant cell factories) and plant protection purposes (Chahardoli et al., 2018; Holaskova et al., 2015). Bacterial expression systems, in particular E. coli, have been preferred over yeast systems for recombinant AMP production (Deng et al., 2017; Li, 2011). Nevertheless, in recent years, several studies have shown that many AMPs can be obtained in higher yields using the P. pastoris expression system (Table 9.1) (Amorim et al., 2018; Cao et al., 2018; Deng et al., 2017; Kuddus et al., 2016, 2017; Li et al., 2017a). P. pastoris has been shown to efficiently secrete large amounts of AMPs to the medium, which significantly facilitates downstream purification and allows circumventing toxicity issues associated with

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intracellular AMP accumulation (Amorim et al., 2018; Deng et al., 2017). Bacillus subtilis expression hosts also have the capacity to secrete AMPs into the extracellular space with their biological activity intact, but at yields considerably lower than those obtained with P. pastoris (Deng et al., 2017; Ji et al., 2017). Moreover, many AMPs present higher antimicrobial activities when expressed in P. pastoris, because this yeast is capable of efficient disulfide bond formation and of performing eukaryotic posttranslational modifications (e.g., glycosylation) that are likely critical for the appropriate folding of some AMP molecules and thus for the conservation of their functions (Cao et al., 2018; Deng et al., 2017; Kuddus et al., 2016; Meng et al., 2017). However, there are E. coli host strains that were engineered to provide a more favorable oxidizing intracellular environment for disulfide bond formation (e.g., Origami) and which can be used for the production of AMPs with disulfide bridges (Li, 2011; Li et al., 2017a). Cell-free expression systems have also been used for recombinant AMP production, but at a microscale (Pardee et al., 2016). The major advantage of these production systems is their flexibility, as they allow the incorporation of nonstandard amino acids and constitute an attractive platform for portable, on demand biomanufacturing of AMPs (Pardee et al., 2016).

9.3.2 Generic Strategies Used for Increasing Recombinant Antimicrobial Peptide Expression Most of the strategies developed to boost the heterologous expression of AMPs in different hosts combine the optimization of multiple stages of protein synthesis, including transcription, translation, posttranslational modification, and/or subcellular targeting (Deng et al., 2017; Li, 2011; Li et al., 2017a). Transcriptional regulation of AMP encoding-genes with strong promoters is the primary strategy used to obtain high transcription levels (Deng et al., 2017). Two types of promoters have been used in recombinant AMP production, inducible and constitutive ones. In E. coli and P. pastoris, the inducible promoters T7 and alcohol oxidase 1 (AOX1), respectively, have been favorably used, because the highest AMP expression levels have been achieved with these promoters (Deng et al., 2017; Li et al., 2017a). However, constitutive promoters are usually preferred for industrial-scale production, because inducible promoters require inducing agents to regulate them, which adds extra economic and/or environmental burden to the production process (Deng et al., 2017; Mao et al., 2015). In this regard, the constitutive promoter GAP represents a good alternative to the methanol-inducible AOX1 promoter for efficient AMP expression in P. pastoris (Chen et al., 2016; Li et al., 2017a; Mao et al., 2015). Indeed, the recombinant AMP peclastin produced with the GAP promoter was shown to exhibit superior antimicrobial activities than when produced with the AOX1 promoter, though higher yields were obtained with the latter (Chen et al., 2016). This shows how high production yields may not guarantee high-quality AMP production, because the host cells’ folding capacity is negatively affected by increasingly high activity of its protein biosynthesis machinery (Aguiar et al., 2014). The expression of AMPs as tandem multimers (instead of being expressed as monomers) has been also explored as a strategy to increase the transcription levels. High AMP

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expression could be obtained in E. coli, B. subtilis, and P. pastoris using this strategy, but since the expression levels in tandem design do not linearly correlate with the degree of multimerization, the appropriate gene copy number has to be determined for each AMP in each expression system (Deng et al., 2017; Li, 2011). The tandem multimeric strategy also allows alleviating problems related to AMP instability and toxicity to the host, which altogether contribute for the improvement of AMP production yields. Optimization of the translation efficiency is another strategy commonly used to enhance the heterologous expression of AMPs (Chahardoli et al., 2018; Deng et al., 2017; Li, 2011). Because each organism has its own codon preference, the substitution of codons that are rarely used by the host by synonymous codons that are recognized by more abundant tRNAs has resulted in remarkable improvements in heterologous AMP expression both in bacterial and yeast systems (Deng et al., 2017; Li, 2011; Li et al., 2017a). In E. coli, host strains engineered to contain higher abundance of tRNAs for rare codons (e.g., Rosetta) have been used as an alternative strategy to overcome low AMP expression problems caused by codon usage bias (Deng et al., 2017; Gaiser et al., 2011). The addition of multiple stop codons has been also used to provide stronger termination efficiency (Deng et al., 2017; Gaiser et al., 2011).

9.3.3 Fusion Technology for Recombinant AMP Production and Purification Among all the strategies developed to enhance recombinant AMP production, the heterologous expression of AMPs in fusion with carrier peptides/proteins has led to the most remarkable AMP production yields (Deng et al., 2017; Li, 2011; Li et al., 2017a). The utilization of fusion technology allowed the overcoming of major problems associated to the production of low molecular weight and highly cationic AMPs in microbial hosts (Deng et al., 2017; Li, 2011). Indeed, this technology is used in recombinant AMP production with multiple objectives: 1. to mask the intrinsic toxicity of AMPs to the producing host (Li, 2011); 2. to provide stabilization to the small, labile AMPs, shielding them from intracellular proteolytic degradation (Li, 2011); 3. to boost the expression and/or solubility of recombinant AMPs (Deng et al., 2017; Li, 2011); 4. for subcellular targeting of recombinant AMPs (e.g., to inclusion bodies or extracellular space) (Cao et al., 2018; Ji et al., 2017; Yang et al., 2018); 5. to facilitate downstream purification (Li, 2011; Yang et al., 2018; Zhao et al., 2017); 6. to produce chimerical peptides with higher expression yield, increased antimicrobial activity, and/or lower cytotoxicity against host cells (Deng et al., 2017; da Cunha et al., 2017); and 7. to functionalize biomaterials (Guerreiro et al., 2008; Oliveira et al., 2015). Therefore, fusion expression is the most common and successful strategy used for recombinant AMP production and purification. The carrier peptides/proteins used in fusion technology usually have multiple roles, they facilitate the expression, stability, and/or the purification of the fusion protein

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(Costa et al., 2014; Oliveira and Domingues, 2018). Most AMPs have been successfully expressed as soluble fusions using solubility enhancers such as thioredoxin A (TrxA), small ubiquitin-related modifier (SUMO), glutathione S-transferase (GST), maltose-binding protein (MBP), among other (Deng et al., 2017; Li, 2011). These tags help stabilizing the AMPs and boost their expression both in bacteria and yeast hosts (Deng et al., 2017; Li et al., 2017a). Among these, GST and MBP can also be used as affinity tags for purification purposes and SUMO can be cleaved by a specific protease that allows the release of AMPs with native N-termini (Li, 2011). CBMs (carbohydrate-binding modules) have been used as fusion partners in the recombinant production of AMPs in E. coli, both as soluble expression enhancers and affinity purification tags (Guerreiro et al., 2008; Ramos et al., 2010, 2013). For these and other fusions, the addition of a cleavage site between the tag and the AMP is usually necessary to allow the AMP release by chemical or proteolytic cleavage (Li, 2011; Ramos et al., 2013). Tags that promote the formation of protein aggregates in vivo, such as the N-terminal autoprotease (Npro) variant EDDIE (Ji et al., 2017), the ketosteroid isomerase (KSI) and others (Li, 2011; Yang et al., 2018; Zhao et al., 2017), have been used to induce the insoluble expression of some AMPs as inclusion bodies, as this helps preventing AMP toxicity and degradation during protein expression in bacteria, and facilitates the recovery of the fusion (Li, 2011; Yang et al., 2018). A similar strategy includes the use of cleavable selfassembling tags (cSATs), which are composed by a self-assembling tag that induces soluble expression in vivo and aggregates in vitro (e.g., elastin-like polypeptides; ELPs) fused to a intein-based self-cleaving tag (Sousa et al., 2016; Zhao et al., 2017). The main advantage of this strategy over the former is that it allows the removal of the tag within the aggregate, thus reducing the number of steps required to achieve purification (Yang et al., 2018). Alternatively, coexpression of the AMPs with aggregation-prone partner proteins has also been used to promote insoluble AMP expression (Kuddus et al., 2017). Compared with the fusion approach, this strategy has the advantage of eliminating the cleavage step for AMP retrieval (Li, 2011), which has many practical difficulties associated (Oliveira and Domingues, 2018). Other fusion strategies have been used to facilitate AMP purification, namely direction of the AMPs secretion to the culture medium using signal sequences such as the alpha mating factor (αMF) secretion signal, used in P. pastoris (Cao et al., 2018; Meng et al., 2017), the signal peptide SacB, used in B. subtilis (Ji et al., 2017), and the first 42 residues of CsgA, a recently developed signal peptide based on the E. coli curli secretion system (Wang et al., 2017b). When these tags are used, usually additional tags are fused to the AMPs to facilitate their production and purification (Ji et al., 2017; Meng et al., 2017). For example, the human serum albumin (HSA) was recently used as an additional tag to facilitate AMP secretion in P. pastoris (Cao et al., 2018). Moreover, the polyhistidine tag (Histag) was also added to this fusion to facilitate its purification (Cao et al., 2018). This is still the most used tag for AMP purification purposes (Deng et al., 2017; Li, 2011) and its permanent fusion to AMPs usually does not affect their antibacterial activities (Chahardoli et al., 2018; Meng et al., 2017). Another classic affinity tag used in recombinant AMP production is the self-cleavable intein chitin-binding domain (CBD), but the large size of the intein chitin domain and the uncontrolled autocleavage of the intein fusions are disadvantageous for final AMP yield (Li, 2011).

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9.4 ENDOLYSINS Ensolysins (or lysins) are hydrolase proteins encoded in the genomes of (bacterio)phages, the viruses of bacteria. These hydrolases are employed by phages to enzymatically degrade the peptidoglycan from the cell wall of the host bacteria at the end of the phage replication cycle in order to lyse the host and release the newly formed progeny (Drulis-Kawa et al., 2015; Melo et al., 2017; Oliveira et al., 2013b). They act naturally from within the cell, accumulating during phage replication without harm due to the protective effect of the inner membrane that prevents the contact of endolysins with the peptidoglycan. At a given time, holins, small proteins encoded in the phage genome, are expressed and produce holes in the inner membrane, giving endolysins access to degrade the peptidoglycan. However, in the case of Gram-positive bacteria that have their peptidoglycan exposed, the endolysins are highly efficient when added externally (Oliveira et al., 2013b; Schmelcher et al., 2012). The high efficiency of endolysins has attracted increasing interest for their potential as antimicrobial agents (Fischetti, 2010; Nelson et al., 2012; Schmelcher et al., 2012), and antimicrobial resistance—the second leading cause of death worldwide (WHO, 2017)—has driven research on the use of these peptidoglycan hydrolases as effective alternatives to antibiotics.

9.4.1 Application of Endolysins to Control Pathogenic Bacteria The high hydrolytic murein activity of endolysins immediately suggested their antimicrobial potential against Gram-positive pathogens. The endolysin from the Streptococcus phage C1 was able to sterilize a culture of 107 Streptococcus in just 5 seconds. Besides this in vitro demonstration, the endolysin efficacy was also validated in vivo by decreasing the Streptococcus load on heavily colonized mice to undetectable colony numbers in just 2 hours. Moreover, this was carried out without disturbing other nontargeted streptococci or commensal bacteria (Nelson et al., 2001). Research has proven the capacity of these enzymes to control localized and systemic infections of Gram-positive pathogenic bacteria both in vitro and in vivo. The combined therapy of endolysin MR-10 (targeting Staphylococcus aureus) and minocycline resulted in 100% survival of mice with a systemic methicillin-resistant S. aureus infection, with only one dose. This combined therapy was also effective in the treatment of localized burn wound infection, resulting in the early resolution of the infection followed by fast healing (Chopra et al., 2016). The endolysin PlyC was shown to cross the epithelial cell membranes and consequently to control Streptococcus pyogenes, demonstrating its potential to control refractory infections caused by the internalized pathogen (Shen et al., 2016). Recently, the first in vivo administration of an endolysin targeting Bacillus anthracis showed that endolysins could protect mice from lethal anthrax infections and consequently could be used therapeutically against this disease (Park et al., 2018). Importantly, the exposure of S. aureus to the endolysin LysH5 was unable to identify any resistant clone, even after 10 cycles of exposition, and to date, resistance development was not yet reported (Gutie´rrez et al., 2014; Rodrı´guez-Rubio et al., 2013). Endolysins have also shown promise in controlling biofilms. Biofilms are communities of bacteria formed on biotic and abiotic surfaces that provide an increased resistance to

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bacteria against host defenses, antibiotics, and other antimicrobials, thus increasing bacterial pathogenicity. The ability of endolysin LysH5 to reduce biofilms of S. aureus and S. epidermidis, including antibiotic resistant clones, raises a new hope to control this problem with a high impact on the food industry and clinical health (Gutie´rrez et al., 2014). The reduction of methicillin-resistant S. aureus by endolysin LysSA11 in milk and ham in just 15 minutes, even at refrigerated temperatures (4 C), and the complete elimination of S. aureus on utensils in 30 minutes, demonstrates the efficacy of these proteins on food and food utensils (Chang et al., 2017). Also, the combination of endolysins with high hydrostatic pressure showed a synergistic effect, leading to the inactivation of Listeria monocytogenes, an important foodborne pathogen. Such combination has important applications in food safety, especially in low processed, ready-to-eat food products (van Nassau et al., 2017). Many other studies exist demonstrating the tremendous potential of these enzymes.

9.4.2 Understanding the Structure of Endolysins for Protein Engineering The structures of phage endolysins usually differ between those enzymes targeting Gram-positive and Gram-negative bacteria. The endolysins derived from phages infecting Gram-positive hosts (and mycobacteria) present a modular structure, generally between 25 and 40 kDa in size, typically composed of a N-terminal enzymatically active domain (EAD) responsible for the catalytic activity, and a C-terminal cell wall binding domain (CBD) that recognizes the substrate, two functional domains that are separated by a linker. The orientation and the number of these two domains may be different in some cases. On the other hand, endolysins from phages infecting Gram-negative hosts usually present a globular structure, with a molecular mass of 15 20 kDa, composed of only one EAD. However, some exceptions exist presenting a modular structure (Oliveira et al., 2013b; Oliveira et al., 2018; Roach and Donovan, 2015; Schmelcher et al., 2012). The different endolysin structures reflect the cell wall architecture of these two major bacterial groups. While the peptidoglycan of Gram-positive bacteria is exposed from the outside, Gram-negative cells present an outer membrane. Consequently, phages infecting a Grampositive host have evolved to possess CBDs that target the protein by its substrate and keep it tightly bound to the cell wall debris after cell lysis, preventing diffusion and destruction of surrounding intact cells not yet infected, assuring phage survival (Oliveira et al., 2018; Schmelcher et al., 2012). The outer membrane in Gram-negative cells prevents the access of endolysins from the outside, avoiding such collateral damage (Schmelcher et al., 2012).

9.4.3 Improving the Activity of Endolysins by Protein Engineering The modular structure of endolysins targeting Gram-positive bacteria and their relatively independent functions rapidly suggested the possibility to change and combine domains between different endolysins, while maintaining the function of each isolated domain. Such a possibility enabled the engineering and design of endolysins through domain swapping, leading to the production of new chimeras with improved properties, such as higher specificity, broader range, or higher lytic activity (Santos et al., 2018) (Fig. 9.3).

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FIGURE 9.3 Protein engineering to improve endolysins. Endolysins of bacteriophages infecting Grampositive bacteria have a modular structure composed of an enzymatically active domain (EAD) and a cell wall binding domain (CBD) connected by a linker (L). The modular structure of these enzymes led to strategies of engineering based on domain swapping or combinations of CBDs with other hydrolytic enzymes. By doing so, endolysins with high catalytic activity and low specificity (EAD1-CBD1) may be combined with endolysins with low catalytic activity and high specificity (EAD2-CBD2) to obtain a chimeric protein with both high catalytic activity and specificity (EAD1-CBD2), to strongly target specific bacteria, or with unspecific and high catalytic activity (EAD1), for disinfection purposes.

The number of possible combinations to create a new endolysin is enormous. Endolysins present a high diversity with more than 89 unique native architectural organizations identified, composed of 24 different EADs and 13 different CBDs (Oliveira et al., 2013b); and this is just considering combinations of functional domains only found in endolysins. If we think that many other protein domains can be combined with endolysins or endolysins’ domains, the number of possibilities grows exponentially. An important characteristic of endolysins is their specificity, sometimes limited to a single species. This allows for targeting of a specific pathogen without disturbing the normal flora (Drulis-Kawa et al., 2015; Roach and Donovan, 2015). On the other hand, such specificity may constitute a limitation when treating an infection caused by multiple pathogenic bacteria. Considering that the specificity of endolysins is usually conferred by its CBD and that endolysins with a lytic spectrum across different genera exist, it is reasonable to envisage that protein engineering of endolysins through domain swapping can originate new chimeric endolysins with a wider spectrum (Fig. 9.3). This was proved through the fusion of an EAD from an endolysin with a limited spectrum with the CBD of an endolysin targeting different genera (Dong et al., 2015). But endolysins’ improvement is not limited to its lytic spectra. The combination of endolysins’ domains has also originated chimeras with improved lytic activity (Mao et al., 2013). Although the CBDs are usually necessary for the lytic activity of endolysins, cases exist showing that the CBD may not be required and sometimes its removal can significantly improve the endolysin activity or/and broaden its host range (Fenton et al., 2010; Horgan et al., 2009; Loessner et al., 1999; Low et al., 2005).

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More interesting is the fusion of multiple domains, as the inclusion of different CBDs targeting different bacteria in the same endolysin can result in a wider spectrum, and the duplication in tandem of an EAD may increase its activity (Schmelcher et al., 2011). The efficacy of recombinant endolysins is such that some formulations are already in clinical trials or reaching the market. The company Micreos commercializes Staphefekt SA.100, an engineered phage endolysin with bacteriolytic activity on methicillin-sensitive and methicillin-resistant S. aureus skin infections. SAL200, another formulation targeting methicillin-resistant S. aureus, is now in a first-in-human phase 1 study and was successful in treating S. aureus, reporting no adverse effects or resistance development when administered intravenously (Schmitz et al., 2011). In fact, swap or combination of endolysins’ domains has proven successful in producing proteins with improved characteristics, allowing the production of new tailor-made antibacterial products.

9.4.4 Improving the Solubility of Endolysins by Protein Engineering Understanding and exploring endolysins’ functions and potential applications requires their production in a heterologous context and, like for other proteins, E. coli has been the workhorse for both cloning and protein expression. Heterologous expression of many of these proteins frequently results in a number of problems that can impair research on this field. Problems such as low yields and production of insoluble proteins resulting from the formation of insoluble inclusion bodies are very common (Becker et al., 2009; Fernandes et al., 2012; Manoharadas et al., 2009; O’Flaherty et al., 2005; Schmitz et al., 2011; Yoong et al., 2004). Proteolytic degradation and protein misfolding are also frequent problems during the overexpression of endolysins (Haddad Kashani et al., 2017). The available technical approaches used to increase production and solubility, or to recover and refold proteins from inclusions bodies, are usually time-consuming and most of the time present a low efficiency. Protein engineering emerged as a potential solution for these problems. We have seen that the modular structure of endolysins enables the swapping of their domains to create new endolysins with improved features. Such improved features also include the overproduction of more soluble and stable proteins with higher yields, enabling their study and application in different fields. One of the most well studied endolysins is LysK, the endolysin of the staphylococcal phage K, known for its strong lytic activity against Staphylococcus. The modular structure of this endolysin is composed of two catalytic domains, a cysteine- and histidine-dependent amidohydrolase/peptidase (CHAP) domain, a central Amidase-2 domain (N-acetylmuramoyl-L-alanine amidase), and a C-terminal SH3b CBD (Horgan et al., 2009). Heterologous expression of LysK in E. coli consistently resulted in its insoluble production as inclusion bodies (O’Flaherty et al., 2005). Truncation of LysK to its N-terminal CHAP domain, besides maintaining its native lytic activity (Fenton et al., 2010; Horgan et al., 2009), increased its solubility compared to that of the parental full-length LysK (Horgan et al., 2009). Further studies on the S. aureus phage endolysin Lys16 were impaired due to protein insolubility in E. coli. The identification of the EAD was conducive to the production of a truncated version of the endolysin, but in this case it was still insoluble. By combining this

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EAD with the CBD of protein 17 it was possible to obtain a new and soluble protein, leading to possible further studies (Manoharadas et al., 2009). The same approach was used with the Staphylococcus Twort phage endolysin, also insoluble when produced in E. coli, by combining its EAD with the CBD from the Staphylococcus phage phiNM3 endolysin. The resulting chimera (ClyS) was efficiently expressed in E. coli in a soluble form. It is important to emphasize that in this study more than 15 chimeras were engineered against S. aureus with different EADs and CBDs and only this one was successful (Daniel et al., 2010). Another example includes the S. aureus endolysin Lys87, which is produced in inclusion bodies in E. coli. Swapping its EAD with those from two enterococcal endolysins (Lys170 and Lys168) resulted in an easy expression in E. coli in a soluble form. These new chimeras “inherited” the solubility of the parental enterococcal endolysins and the staphylococcal lytic ability and specificity (Fernandes et al., 2012). Apart from the current methodologies to improve protein solubility, protein engineering, in the particular case of endolysins through domain swapping/addition/deletion, has been shown to be an interesting and feasible approach in the heterologous production of these important bacteriolytic enzymes. Endolysins are now more amenable to large-scale production, and to obtaining higher yields of soluble protein, which are crucial to fully explore their potential applications and enable their commercialization. It is important to highlight here the main role of bioinformatics. The presently available bioinformatics tools are essential in the identification of functional domains in proteins and in the prediction of their primary, secondary, and tertiary structures (Haddad Kashani et al., 2017; Oliveira and Domingues, 2018; Santos et al., 2018). These allow the design of new domain combinations and the deletion of peptide sequences that contribute to instability and insolubility, resulting in chimeras optimized for heterologous expression and with improved features.

9.4.5 Endolysins as Tools in Heterologous Protein Production and Purification Recombinant heterologous production of proteins is critical for functional analysis of unknown or uncharacterized proteins, to study their structure, and to exploit their biotechnological applications and product development. Protein production must result in large amounts of the desired protein in a soluble and active form to allow their study. Overproduction of proteins in E. coli, the most used microorganism for this purpose, is largely dependent on an efficient and rapid method to lyse the cells in order to recover the expressed protein. The most common methods are sonication and high pressure homogenization with some disadvantages associated that include slowness, lytic efficiency, proper equipment requirement, inactivation, insolubility, aggregation, and degradation of the protein of interest (in part due to the heat generated during the process) (Doulah, 1977; Feliu et al., 1998). This will obviously have negative implications on the protein functionality. The lysozyme domain of mycobacteriophage D29 endolysin, shown to lyse E. coli (Pohane et al., 2014), was cloned in E. coli BL21 (DE3) using a vector with two multiplecloning sites in order to produce an efficient lysis of the cells at the end of the expression period. The protein to be overproduced, α subunit of RNA polymerase or GFP, was inserted in the second multiple-cloning site. The endolysin was produced together with

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the protein of interest, accumulating inside the cells and remaining nontoxic due to the cell inner membrane that protects against contact with the peptidoglycan. After the expression period the cells were harvested and chloroform was added, permeabilizing the cell membrane and enabling the diffusion of the endolysin into the peptidoglycan. This led to peptidoglycan degradation through the endolysin’s activity and consequently to cell lysis and protein release in an efficient and rapid way. The high yields of protein recovery, quickness of cell lysis, efficiency, and dispensability of traditional mechanical methods demonstrates its applicability and usefulness in high-throughput and large-scale protein overproduction (Joshi and Jain, 2017). Cyanobacteria have a great potential in the production of carbohydrates, fatty acids, or alcohols as renewable sources of biofuels due to their fast growth, efficient conversion of carbon dioxide gas into biomass, their genetic tractability, and dispensability for fermentable sugars and arable land for growth (reducing competition with agricultural lands and resources) (Sarsekeyeva et al., 2015). Their role has become more important with the need for renewable energies alternative to fossil fuels. Nevertheless, the requirement for huge amounts of water is one of the greatest issues in cyanobacteria-based biofuel production (Ducat et al., 2011). If any chemical needs to be added (e.g., for proteins’ induction, cell disruption) it will contaminate the water and require proper treatment with the consequent extra costs. Coupled with this disadvantage is the lack of an efficient method to lyse the cells and collect the synthesized compounds. As in E. coli, the most common methods used are the mechanical. Other methods have also been used, such as freeze thaw, organic solvents, and detergents, which require additional steps for removing the chemicals (Miyake et al., 2014). Transformation of cyanobacteria with the P22 lysis genes encoding for the endolysin and holin under a nickel ion inducible promoter reduces the impact on water resources by avoiding the use of chemicals for cell disruption. Moreover, it also allows the recovery of compounds with minimal contaminants, effort, and energy. However, the use of a nickel ion (or other chemical) induction system limits its feasible application for future biofuel production processes due to the requirement for such production of the addition of the inducer to a large amount of water (Liu and Curtiss, 2009). An interesting solution for the issues raised, including water contamination with inducers, seems to be the introduction of phage lysis genes in cyanobacteria under the control of a promoter regulated by green light. Cyanobacteria modified with the T4 bacteriophage lysis genes expressed the self-lysis system when exposed to green light, which greatly increased the fragility of the cell membrane and enhanced the intracellular compounds’ release, leading also to cell death. Such a strategy (the use of a photosynthetic modified organism) avoids the use of chemical inducers, limiting the impact on the scarce water resources. Moreover, it is important to highlight that these genetically modified organisms (GMOs) are photosensitive and thus unable to survive under sunlight (which contains green light) if accidentally released into the environment, a fact that contributes to alleviate the safety issues related to GMOs (Miyake et al., 2014). Lately, the endolysin from phage T4 has been used as a fusion partner during protein production in E. coli to facilitate subsequent crystallization of several proteins (Cherezov et al., 2007; Kellosalo et al., 2011; Miyake et al., 2014; Rosenbaum et al., 2007; Wang et al., 2014; Yang et al., 2015; Zou et al., 2012). Membrane-bound pyrophosphatase proteins were better recombinantly expressed when the T4 endolysin was fused and also yielded better

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crystals by improving crystal contacts (Kellosalo et al., 2011). Moreover, the T4 endolysin fusion was found not to interfere with the ligand binding and importantly, with the protein folding. Thus, besides improving production, the T4 endolysin enabled the solving of the protein structure. More recently, a metal ions-binding mutant of the T4 endolysin (mbT4L) was used as an intramolecular purification tag for recombinant proteins in E. coli. This approach presents some advantages over the commonly used His-tag in the immobilized metal affinity chromatography (IMAC), which includes a higher purity of the purified proteins as a consequence of a stronger affinity to metal ions and an improved solubility of the target protein. This fusion partner is also compatible with X-ray crystallography and seems to improve protein overexpression (Boura et al., 2017).

9.5 LECTINS Lectins are proteins that contain at least one noncatalytic domain that binds specifically and reversibly to different carbohydrate structures. They are widespread and present in almost every living organism, with the lectins of plant origin being the most studied. Lectins’ applications have been mainly related to the biomedical field. Of particular interest is their exploitation as cancer diagnostic and therapeutic tools (Oliveira et al., 2013a). A critical point in preparing pure lectin samples from their original hosts for biomedical application is the difficulty in separating a single lectin isoform by conventional techniques. Many lectins are encoded by a family of genes that deliver slightly different mature lectin sequences, called isoforms, which may have distinct activities. Heterologous protein production in different organisms has been employed to improve yield and homogeneity of the lectin samples. Furthermore, custom-made lectins with desired specificities may be obtained by recombinant means (Oliveira et al., 2013a). Martı´nez-Alarco´n et al. (2018) have recently pointed out important considerations for host selection, according to lectin synthesis and origin. In general, the bacterium E. coli is the first-choice expression host to produce recombinant lectins. However, the methylotrophic yeast P. pastoris has been increasingly used to overcome problems of insoluble expression of the bacterial system, and thus to produce high amounts of soluble lectins (Li et al., 2017b; Wahid et al., 2017), and particularly, glycosylated lectins (Biswas and Chattopadhyaya, 2016), since most lectins are glycoproteins. The yeast P. pastoris is a recognized production platform of heterologous proteins, including therapeutic and biopharmaceutical proteins approved for human use from the US Food and Drug Administration (FDA) (Gasser et al., 2013; Juturu and Wu, 2018), and thus it is a suitable host for producing lectins for biomedical use.

9.5.1 Overall Molecular Cloning Strategy for Recombinant Production of Lectins in Pichia pastoris As a general rule, the lectin gene is optimized according to the preferred codon usage of P. pastoris (Klafke et al., 2016; Li et al., 2017b; Oliveira et al., 2008), which is essential to achieve high expression levels in this host (Yang and Zhang, 2018). Many factors should

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be taken into account in codon optimization, such as secondary structure of messenger RNA, the balance of GC distribution and the codon adaption index (CAI) (Yang and Zhang, 2018). Codon optimization may be designed using bioinformatics tools (e.g., GENEius, http://www.geneius.de) or obtained from specialized companies. Optimized genes may also be created via assembly PCR and overlap extension PCR (Aguiar et al., 2017). The optimized gene is then cloned into specific versions of the pPIC expression vectors carrying the Saccharomyces α-factor preprosequence (αMF) to direct the recombinant lectin into the secretory pathway, which is where protein glycosylation occurs. Nevertheless, incorrect cleavage of this sequence has resulted in recombinant lectins with undesired N-terminal extensions, which may affect lectin properties (Al Atalah et al., 2011; Lannoo et al., 2007; Oliveira et al., 2008; Raemaekers et al., 1999). For example, although being a small addition, one E-A repeat left behind at the N-terminal of frutalin (the galactose-binding lectin from breadfruit) decreased its predicted isoeletric point (pI) from 8 to 5 (Oliveira et al., 2014). αMF has been extensively engineered to improve the efficiency of its cleavage and secretion (Lin-Cereghino et al., 2013). It is worth noting that αMF should not be used if potential cleavage sites (internal accessible dibasic amino acids, such as KR and RR) are present in the target protein sequence, as this may lead to its proteolysis. Modification of a putative Kex2 (endo protease responsible for αMF cleavage) at the C-terminus of an insecticidal peptide fused to snowdrop lectin (GNA) improved levels of intact fusion protein (Pyati et al., 2014). Alternative signal sequences have been described in the literature (Ciplys et al., 2015; Govindappa et al., 2014). Lectins are cloned with no fusion tags or, in some cases, in fusion with a six histidine tag (His-tag; provided in pPIC, downstream of the multiple-cloning site) for IMAC purification. This tag can also be directly attached at the N- or C-terminal of the protein during gene amplification by PCR, but before cloning it is important to evaluate its accessibility for purification by analysis of the tertiary structure of the lectin or by homology-based modeling (Oliveira and Domingues, 2018). The His-tag does not seem to affect the functionality of the fused recombinant lectins (Al Atalah et al., 2014). Lectins are preferentially expressed under the control of the P. pastoris promoter AOX1 (PAOX1), which is tightly regulated and strongly induced by methanol. The rate of methanol feeding is critical for the success of protein production, because methanol serves as both carbon source and inducer. However, accumulation of methanol is cytotoxic to cells and its use raises safety issues. To overcome these drawbacks, many efforts have been made in recent years to develop methanol-free PAOX1 systems, based on promoter engineering (2nd generation of AOX1 promoter variants) or strain engineering. In a recent work, a methanol-free PAOX1-based strain (MF1) was constructed by deleting three transcription repressors associated with catabolite repression and by overexpressing one transcription activator (Wang et al., 2017a). A fermentation strategy designated by “glucose glycerol-shift” was developed in a bioreactor with the MF1 strain, in which glucose was used for cell growth and PAOX1 suppression, and glycerol was used to remove the repressive effect of glucose, allowing induction of the recombinant gene (Wang et al., 2017a). Interestingly, the expression level of an insulin precursor reached 2.46 g/L in MF1, which was 58.6% of the wild-type strain using a methanol-feeding strategy (4.20 g/L) (Wang et al., 2017a). Methanol-free systems have not yet been employed for the production of recombinant lectins, but are worthy of use in future works.

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A relevant characteristic of the Pichia expression system is the spontaneous multiple genomic insertions by homologous recombination. Indeed, the protein yield can be enhanced by increasing the gene dosage within a certain range of copy numbers. Screening for multicopy recombinants is normally done by selection with increasing concentrations of antibiotics or by in situ measurement of enzymatic activity. For rapid selection of high-producing clones, Hu et al. (2011) developed a visual method using a mannanase reporter. To increase copy number, multiple expression cassettes were inserted in tandem into the integrative vector, resulting in 10-fold higher amounts of secreted insecticidal peptide fused to GNA lectin (Pyati et al., 2014). Nevertheless, the effect of high copy numbers on protein production should be carefully examined, as the overproduction of recombinant proteins may become a major burden to the host’s metabolism and lead to inefficient secretion (Aguiar et al., 2014).

9.5.2 Recombinant Lectin Production in Pichia pastoris and Purification Strategies Lectin production in P. pastoris is conducted either in a shake-flask (small-scale) or in a bioreactor (large-scale). Titers in the order of 1 500 mg/L are usually reported, regardless of the production scale (Table 9.2), but higher yields are generally obtained when Pichia cultures are grown in a bioreactor under controlled conditions. This large-scale production is typically conducted in fed-batch mode, which is an efficient operational strategy for high cell density fermentation, and thus for maximizing product formation. For example, an impressive yield of 1.2 g/L was recently reported for the production of Narcissus tazetta lectin (NTL) in a 5-L bioreactor (Li et al., 2017b). In some cases, scaling-up the fermentation has not resulted in improved yield. Klafke et al. (2016) reported an unsatisfactory yield (1.5 mg/L) of Bauhinia variegate lectin in a 7-L bioreactor operated in fed-batch under standard conditions. Also, the production yield of frutalin was lower in bioreactor than in shake-flasks, presumably due to a temperature effect. Namely, small-scale induction at 15oC resulted in a sixfold higher yield of frutalin % to a production conducted at 28 C in a 1.6-L (18 20 mg/L; Oliveira et al., 2008), compared bioreactor (3.3 mg/L; Wanderley et al., 2013). Nonetheless, yield could be raised to 13.4 mg/L by supplementation with Pichia trace minerals (PTM) during the fed-batch process (Wanderley et al., 2013). The positive effect of lower temperatures on protein production by Pichia has also been reported for other proteins, including lectins (Al Atalah et al., 2014). Induction of the recombinant protein at lower temperatures has many advantages in terms of reducing proteolytic degradation and alleviating oxygen stress (Zepeda et al., 2018). Other strategies to reduce proteolytic degradation include the use of proteasedeficient strains or addition of protease inhibitors (Yang and Zhang, 2018). Lopes et al. (2014) also showed that raising air pressure from 1 to 5 bar in a stirred tank bioreactor led to a reduction in secreted protease activity by recombinant P. pastoris. Under pressurized conditions, recombinant frutalin yield was improved three times and the specific activity of recombinant β-galactosidase was raised ninefold (Lopes et al., 2014). Downstream purification in Pichia is quite simple. A single chromatographic purification step is normally sufficient, since the recombinant protein comprises the vast majority

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9.5 LECTINS

TABLE 9.2 Reported Yields for Plant Lectins Recombinantly Produced in the Yeast Pichia pastoris Lectin Original Host

Lectin Abbreviation

Yield (mg/L)a

References

Allium sativum (garlic bulbs)

ASAI

10 20 (1.25 L bioreactor)

Fitches et al. (2008)

ASAII Artocarpus incisa (breadfruit seeds)

Frutalin

18 20 (shake-flasks)

Oliveira et al. (2008)

Bauhinia variegate

BVL-I

1.5 (7 L bioreactor)

Klafke et al. (2016)

Galanthus nivalis (snowdrop bulbs)

GNA

80 (200 L bioreactor)

Baumgartner et al. (2003)

Moringa oleifera

MoL

520 (shake-flasks)

Wahid et al. (2017)

Nicotiana tabacum (tobacco leaves)

Nictaba

6 (shake-flasks)

Lannoo et al. (2007)

Oryza sativa (rice)

Orysata

12 (shake-flasks)

Al Atalah et al. (2011)

OrysaEULS2

4 6 (shake-flasks)

Al Atalah et al. (2012)

OrysaEULD1A and its domains

0.5 3 (shake-flasks)

Al Atalah et al. (2014)

Phaseolus vulgaris (kidney bean)

PHA-E

100 (2 and 200 L bioreactor)

Baumgartner et al. (2002)

P. vulgaris and G. nivalis (snowdrop)

PHA

0.4 2 (shake-flasks)

Raemaekers et al. (1999)

1.8 (shake-flasks)

Oguri et al. (2008)

Solanum lycopersicum (tomato)

GNA Tomato lectin

a

Between parenthesis are indicated the production scales. Adapted from Oliveira, C., Teixeira, J.A., Domingues, L., 2013a. Recombinant lectins: an array of tailor-made glycan-interaction biosynthetic tools. Crit. Rev. Biotechnol. 33, 66 80.

of the total extracellular protein. This is because Pichia secretes low levels of native proteins, and also because the standard production medium (minimal medium) has low protein content. In fact, this is one of the major advantages of P. pastoris over other expression systems such as E. coli. Nevertheless, as the recombinant protein is much diluted in the fermentation broth, a concentration step is conducted before the purification procedure. This can be done, for instance, by ultrafiltration (Oliveira et al., 2008; Wahid et al., 2017) or ammonium sulfate precipitation (Al Atalah et al., 2014; Klafke et al., 2016). Then, recombinant lectins are purified by affinity chromatography, taking advantage of their ability to bind to specific carbohydrates. For example, mannose-specific lectins are purified in mannose agarose/sepharose columns (Li et al., 2017b) and galactose-specific lectins in α-lactose-agarose columns, which is a suitable matrix for lectins that recognize alphalinked galactose residues (Klafke et al., 2016). Additional chromatographic steps, such as ion-exchange and/or IMAC, can be placed upstream of the affinity chromatography for purification refining (Al Atalah et al., 2011, 2014). Affinity chromatography has many advantages, such as easy one-step purification, protein concentration, and the selection of functional species in case of sample heterogeneity (Costa et al., 2014). However, it does not

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provide information regarding the correct oligomeric structure of the recombinant lectin. Most lectins are multimeric proteins, consisting of noncovalently associated subunits, which range from dimers (e.g., banana lectin) or tetramers (e.g., jacalin) to octamers (e.g., heltuba) or higher oligomeric structures. It is this multimeric structure that gives to lectins the ability to agglutinate cells or to form precipitates with glycoconjugates. Size exclusion chromatography (SEC), also known as gel filtration, is a suitable technique for the separation and quantification of protein oligomers, allowing the purification of proteins to size homogeneity (Oliveira and Domingues, 2018). Thus SEC has been employed to purify and/or to determine the oligomeric structure of some recombinant lectins (Oliveira et al., 2008; Sumisa et al., 2004).

9.5.3 Impact of Pichia Processing on Lectin Properties Although P. pastoris possesses a sophisticated eukaryotic machinery to perform many posttranslational modifications, it did not succeed in executing complex proteolytic events involving the cleavage of a linker region between two polypeptide chains in frutalin (Oliveira et al., 2008) and garlic ASAI lectin (Fitches et al., 2008), and subsequent chains’ religation (protein splicing) in ConBr (Bezerra et al., 2006; Carvalho et al., 2008). Nonetheless, recombinant ASAI showed relevant insecticidal activity (Fitches et al., 2008), and recombinant frutalin showed remarkable capacity as a biomarker for human prostate cancer (Oliveira et al., 2009b) and as an apoptosis-inducer of cancer cells (Oliveira et al., 2011). Glycosylation is one of the most important posttranslational modifications, is quite common among secretory proteins, and may have a determinant role in protein folding, stability, and/or activity. N-linked glycosylation is a common feature in lectins synthesized in the endoplasmic reticulum (ER). N-glycosylation occurs at the asparagine residue (N) in a consensus sequence NXS/T/C, where X can be any amino acid except proline. Pichia N-linked glycosylation consists mostly of short chains of 8 14 mannoses (Man) attached at the double N-acetylglucosamine (GlcNAc) residue bound to the asparagine, and is quite close to the typical high-mannose glycosylation pattern occurring in higher eukaryotes. Indeed, most lectins glycosylated by Pichia were functional, even though glycans accounted for a substantial increase in their molecular masses, ranging from 2 kDa (Oliveira et al., 2008) to 4.5 kDa (Al Atalah et al., 2011). For example, recombinant orysata (a lectin originally from rice), which presented high-mannose structures (mostly Man9 11), showed hemagglutination activity and potent antihuman immunodeficiency virus and antirespiratory syncytial virus activities in cell culture (Al Atalah et al., 2011). Nevertheless, some glycosylated lectins produced in Pichia have lost their hemagglutination activity, while maintaining the capacity to bind to carbohydrates, as reported for frutalin (Oliveira et al., 2008), the tomato lectin (Oguri et al., 2008), PCL-F (from the mushroom Pleurotus cornucopiae) (Iijima et al., 2003), and BVL-I (from the plant Bauhinia variegata) (Klafke et al., 2016). The importance of glycosylation for the hemagglutination activity of the tomato lectin was shown by a significant reduction of this activity upon deglycosylation (Oguri et al., 2008). However, hemagglutination activity of the lectins frutalin (Oliveira et al., 2009a), PCL-F (Iijima et al., 2003), and BVL-I (Klafke et al., 2016) was found independent of glycosylation, since recombinant deglycosylated versions of these

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lectins produced in E. coli presented this activity. In the case of frutalin, different isoforms (sharing 92% of sequence identity) that may have different agglutinating abilities were produced in E. coli and P. pastoris (Oliveira et al., 2008; 2009a). In the case of PCL-F (Iijima et al., 2003) and BVL-I (Klafke et al., 2016), it was hypothesized that glycosylation inhibited the formation of the dimeric structure, but the tetrameric structure of frutalin was not affected (Oliveira et al., 2008). In a more recent study, the role of glycosylation on the Curcuma longa rhizome lectin was studied by structural and activity assays of a deglycosylated version produced in E. coli and a glycosylated version at both N residues, and its glycosylated mutants N66Q and N110Q, produced in P. pastoris (Biswas and Chattopadhyaya, 2016). Circular dichroism (CD), fluorescence spectroscopy, and hemagglutination assays showed no differences in secondary or tertiary structures or sugarbinding properties between the wild-type lectin and each of the produced recombinant lectins under physiological pH (Biswas and Chattopadhyaya, 2016). Nevertheless, at acidic pH, glycosylation was shown to be important for the maintenance of proper lectin folding, and thus to maintain the active conformation (Biswas and Chattopadhyaya, 2016).

9.6 CONCLUSIONS AND PERSPECTIVES The development of versatile and cost-efficient recombinant protein production cell platforms that enable the production of tailor-made protein or peptide pharmaceuticals is being driven by the evolution of the fields of genetic engineering and synthetic biology. With the ever increasing understanding of molecular physiology that will enable the engineering of novel proteins and peptides, there is no end to the development of novel pharmaceuticals. Phage display is very promising in this regard, as with the so far identified phage-produced proteins and/or peptides (ligands) different tools can be developed, either by using the phage ligand complex or the ligand itself, functionalized or not with other compounds, for a wide range of medical applications. For the recombinant protein production of pharmaceuticals, both the prokaryotic E. coli and eukaryotic P. pastoris microbial expression systems have shown significant advances in molecular biology and bioengineering. It is now feasible to quickly and efficiently engineer these host organisms and establish new platforms for the production of recombinant pharmaceuticals. These improvements were exemplified in this chapter by describing the novel approaches for AMP and endolysin production in E. coli and for lectin production in P. pastoris. Endolysins have been proven to be potent antibacterial agents in vivo and in vitro, with biotechnological applications in the health, food, and industry fields. We have given here an overview of endolysins’ characteristics, structures, and cases of successful studies, highlighting their potential not only in the control of pathogenic bacteria, but also as tools in protein production and purification. However, as it happens with many other interesting proteins, they need to be improved to ameliorate their expression, activity, and application in specific contexts. Protein engineering allows the improvement of lysins’ activity and also to solve common expression problems when they, or other proteins, are overproduced in E. coli.

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Despite the fact that some lectins produced in P. pastoris are processed differently than their native counterparts, their carbohydrate-binding activity has not been compromised, and, when reported, the recombinant lectin could be successfully used for the aimed application. Thus the yeast P. pastoris may be seen as a successful lectin expression platform, paving the way for the exploration of the recombinant lectins’ potential for biomedical applications. The exemplified case studies provided in this chapter are not representative of all recombinant biopharmaceuticals, but give a good overview of the actual trend of the recombinant pharmaceuticals’ growth by two main interconnected factors: the evolution of protein production microbial cell platforms, both at the cell and process level; and the appearance of more shophisticated protein constructs resulting from a rational design process. This also enables a deep understanding on molecular physiology, which in turn will fuel the design of next-generation tailor-made recombinant pharmaceuticals.

Acknowledgment The authors acknowledge the support from the Portuguese Foundation for Science and Technology (FCT) through the strategic funding of UID/BIO/04469/2019 unit, grant SFRH/BPD/110640/2015 to Carla Oliveira and Project EcoBioInks4SmartTextiles (PTDC/ CTM-TEX/30298/2017), co-funded by COMPETE 2020 (POCI-01-0145-FEDER-03029). The support from BioTecNorte operation (NORTE-01-0145-FEDER-000004), funded by the European Regional Development Fund under the scope of Norte2020 Programa Operacional Regional do Norte, is also acknowledged.

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Petrenko, V.A., Jayanna, P.K., 2014. Phage protein-targeted cancer nanomedicines. FEBS Lett. 588, 341 349. Pini, A., Giuliani, A., Falciani, C., Runci, Y., Ricci, C., Lelli, B., et al., 2005. Antimicrobial activity of novel dendrimeric peptides obtained by phage display selection and rational modification. Antimicrob. Agents Chemother. 49, 2665 2672. Pohane, A.A., Joshi, H., Jain, V., 2014. Molecular dissection of phage endolysin: an interdomain interaction confers host specificity in Lysin A of Mycobacterium phage D29. J. Biol. Chem. 289, 12085 12095. Pyati, P., Fitches, E., Gatehouse, J.A., 2014. Optimising expression of the recombinant fusion protein biopesticide omega-hexatoxin-Hv1a/GNA in Pichia pastoris: sequence modifications and a simple method for the generation of multi-copy strains. J. Ind. Microbiol. Biotechnol. 41, 1237 1247. Raemaekers, R.J.M., de Muro, L., Gatehouse, J.A., Fordham-Skelton, A.P., 1999. Functional phytohemagglutinin (PHA) and Galanthus nivalis agglutinin (GNA) expressed in Pichia pastoris—Correct N-terminal processing and secretion of heterologous proteins expressed using the PHA-E signal peptide. Eur. J. Biochem. 265, 394 403. Rakonjac, J., Bennett, N.J., Spagnuolo, J., Gagic, D., Russel, M., 2011. Filamentous bacteriophage: biology, phage display and nanotechnology applications. Curr. Issues Mol. Biol. 13, 51 76. Ramos, R., Domingues, L., Gama, M., 2010. Escherichia coli expression and purification of LL37 fused to a family III carbohydrate-binding module from Clostridium thermocellum. Protein Expr. Purif. 71, 1 7. Ramos, R., Moreira, S., Rodrigues, A., Gama, M., Domingues, L., 2013. Recombinant expression and purification of the antimicrobial peptide magainin-2. Biotechnol. Prog. 29, 17 22. Roach, D.R., Donovan, D.M., 2015. Antimicrobial bacteriophage-derived proteins and therapeutic applications. Bacteriophage 5, e1062590. Rodrı´guez-Rubio, L., Martı´nez, B., Rodrı´guez, A., Donovan, D.M., Go¨tz, F., Garcı´a, P., 2013. The phage lytic proteins from the Staphylococcus aureus bacteriophage vB_SauS-phiIPLA88 display multiple active catalytic domains and do not trigger staphylococcal resistance. PLoS One 8, e64671. Rosenbaum, D.M., Cherezov, V., Hanson, M.A., Rasmussen, S.G.F., Thian, F.S., Kobilka, T.S., et al., 2007. GPCR engineering yields high-resolution structural insights into beta2-adrenergic receptor function. Science 318, 1266 1273. Sainath Rao, S., Mohan, K.V., Atreya, C.D., 2013. A peptide derived from phage display library exhibits antibacterial activity against E. coli and Pseudomonas aeruginosa. PLoS One 8, e56081. Sambrook, J., Russell, D.W. (Eds.), 2001. Molecular Cloning—A Laboratory Manual. third ed. Cold Spring Harbor Laboratory, New York. Sanchez-Garcia, L., Mangues, R., Ferrer-Mirrales, N., Va´squez, E., Villaverde, A., 2016. Recombinant pharmaceuticals from microbial cells: a 2015 update. Microb. Cell Fact. 15, 33. Santos, S.B., Costa, A.R., Carvalho, C.M., No´brega, F.L., Azeredo, J., 2018. Exploiting bacteriophage proteomes: the hidden biotechnological potential. Trends Biotechnol. 36, 966 984. Sarsekeyeva, F., Zayadan, B.K., Usserbaeva, A., Bedbenov, V.S., Sinetova, M.A., Los, D.A., 2015. Cyanofuels: biofuels from cyanobacteria. Reality and perspectives. Photosyn. Res. 125, 329 340. Schmelcher, M., Tchang, V.S., Loessner, M.J., 2011. Domain shuffling and module engineering of Listeria phage endolysins for enhanced lytic activity and binding affinity. Microb. Biotechnol. 4, 651 662. Schmelcher, M., Donovan, D.M., Loessner, M.J., 2012. Bacteriophage endolysins as novel antimicrobials. Future Microbiol. 7, 1147 1171. Schmitz, J.E., Ossiprandi, M.C., Rumah, K.R., Fischetti, V.A., 2011. Lytic enzyme discovery through multigenomic sequence analysis in Clostridium perfringens. Appl. Microbiol. Biotechnol. 89, 1783 1795. Sharma, S., Chatterjee, S., Datta, S., Prasad, R., Dubey, D., Prasad, R.K., et al., 2017. Bacteriophages and its applications: an overview. Folia Microbiol. 62, 17 55. Shen, Y., Barros, M., Vennemann, T., Gallagher, D.T., Yin, Y., Linden, S.B., et al., 2016. A bacteriophage endolysin that eliminates intracellular streptococci. Elife 5, e13152. Silva, J.P., Appelberg, R., Gama, F.M., 2016a. Antimicrobial peptides as novel anti-tuberculosis therapeutics. Biotechnol. Adv. 34, 924 940. Silva, V.L., Ferreira, D., Nobrega, F.L., Martins, I.M., Kluskens, L.D., Rodrigues, L.R., 2016b. Selection of novel peptides homing the 4T1 cell line: exploring alternative targets for triple negative breast cancer. PLoS One 11, e0161290. Smith, G.P., 1985. Filamentous fusion phage: novel expression vectors that display cloned antigens on the virion surface. Science 228, 1315 1317. Smith, G.P., Petrenko, V.A., 1997. Phage display. Chem. Rev. 97, 391 410.

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Sousa, D.A., Mulder, K.C.L., Nobre, K.S., Parachin, N.S., Franco, O.L., 2016. Production of a polar fish antimicrobial peptide in Escherichia coli using an ELP-intein tag. J. Biotechnol. 234, 83 89. Sumisa, F., Iijima, N., Ando, A., Shiga, M., Kondo, K., Amano, K., et al., 2004. Properties of mycelial aggregatespecific lectin of Pleurotus cornucopiae produced in Pichia pastoris. Biosci. Biotechnol. Biochem. 68, 959 960. Tan, Y., Tian, T., Liu, W., Zhu, Z., Yang, C.J., 2016. Advance in phage display technology for bioanalysis. Biotechnol. J. 11, 732 745. Tanaka, T., Kokuryu, Y., Matsunaga, T., 2008. Novel method for selection of antimicrobial peptides from a phage display library by use of bacterial magnetic particles. Appl. Environ. Microbiol. 74, 7600 7606. van Nassau, T.J., Lenz, C.A., Scherzinger, A.S., Vogel, R.F., 2017. Combination of endolysins and high pressure to inactivate Listeria monocytogenes. Food Microbiol. 68, 81 88. Vlassov, V.V., Morozova, V.V., Babkin, I.V., Tikunova, N.V., 2017. Bacteriophages: 100 years in the service of mankind. Science First Hand 46. WHO, 2017. Global priority list of antibiotic-resistant bacteria to guide research, discovery, and development of new antibiotics. World Health Organization Publication. ,https://www.who.int/medicines/publications/ global-priority-list-antibiotic-resistant-bacteria/en/. (accessed 14.03.19). Wahid, M.A.A., Noor, M.J.M.M., Goto, M., Sugiura, N., Othman, N., Zakaria, Z., et al., 2017. Recombinant protein expression of Moringa oleifera lectin in methylotrophic yeast as active coagulant for sustainable high turbid water treatment. Biosci. Biotechnol. Biochem. 81, 1642 1649. Wanderley, M.S.O., Oliveira, C., Bruneska, D., Domingues, L., Lima, J.L., Teixeira, J.A., et al., 2013. Influence of trace elements supplementation on the production of recombinant frutalin by Pichia pastoris KM71H in fedbatch process. Chem. Pap. 67, 682 687. Wang, G., Li, X., Wang, Z., 2016. APD3: the antimicrobial peptide database as a tool for research and education. Nucleic acids Res. 44, D1087 D1093. Wang, J., Wang, X., Shi, L., Qi, F., Zhang, P., Zhang, Y., et al., 2017a. Methanol-independent protein expression by AOX1 Promoter with trans-acting elements engineering and glucose-glycerol-shift induction in Pichia pastoris. Sci. Rep. 7, 41850. Wang, M., Huang, M., Zhang, J., Ma, Y., Li, S., Wang, J., 2017b. A novel secretion and online-cleavage strategy for production of cecropin A in Escherichia coli. Sci. Rep. 7, 7368. Wang, X., Cui, Y., Wang, J., 2014. T4-lysozyme fusion for the production of human formyl peptide receptors for structural determination. Appl. Biochem. Biotechnol. 172, 2571 2581. Wang, Z., Wang, X., Wang, J., 2018. Recent advances in antibacterial and antiendotoxic peptides or proteins from marine resources. Mar. Drugs 16, E57. Wu, C.H., Liu, I.J., Lu, R.M., Wu, H.C., 2016. Advancement and applications of peptide phage display technology in biomedical science. J. Biomed. Sci. 23, 8. Yang, H., Tong, J., Lee, C.W., Ha, S., Eom, S.H., Im, Y.J., 2015. Structural mechanism of ergosterol regulation by fungal sterol transcription factor Upc2. Nat. Commun. 6, 6129. Yang, X., Pistolozzi, M., Lin, Z., 2018. New trends in aggregating tags for therapeutic protein purification. Biotechnol. Lett. 40, 745 753. Yang, Z., Zhang, Z., 2018. Engineering strategies for enhanced production of protein and bio-products in Pichia pastoris: a review. Biotechnol. Adv. 36, 182 195. Yoong, P., Schuch, R., Nelson, D., Fischetti, V.A., 2004. Identification of a broadly active phage lytic enzyme with lethal activity against antibiotic-resistant Enterococcus faecalis and Enterococcus faecium. J. Bacteriol. 186, 4808 4812. Zepeda, A.B., Pessoa Jr., A., Farias, J.G., 2018. Carbon metabolism influenced for promoters and temperature used in the heterologous protein production using Pichia pastoris yeast. Braz. J. Microbiol. In press. Zhang, L., Li, X., Wei, D., Wang, J., Shan, A., Li, Z., 2015. Expression of plectasin in Bacillus subtilis using SUMO technology by a maltose-inducible vector. J. Ind. Microbiol. Biotechnol. 42, 1369 1376. Zhang, L., Luo, S., Zhang, B., 2016. The use of lectin microarray for assessing glycosylation of therapeutic proteins. MAbs 8, 524 535. Zhao, Q., Zhou, B., Gao, X., Xing, L., Wang, X., Lin, Z., 2017. A cleavable self-assembling tag strategy for preparing proteins and peptides with an authentic N-terminus. Biotechnol. J. 12, 1600656. Zou, Y., Weis, W.I., Kobilka, B.K., 2012. N-terminal T4 lysozyme fusion facilitates crystallization of a G protein coupled receptor. PLoS One 7, e46039.

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C H A P T E R

10

Stability of Proteins During Processing and Storage Shivani Pathania1, Puneet Parmar2 and Brijesh K Tiwari1 1

Teagasc, Ashtown, Dublin, Ireland 2Teagasc, Moorepark, Fermoy, Co. Cork, Ireland O U T L I N E

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10.2 Types of Modifications in Native Food Proteins 296 10.2.1 Physical Changes in Proteins 296 10.2.2 Chemically Induced Changes in Proteins 300 10.3 Effect of Processing on Nutritional Properties of Food Proteins 303 10.3.1 Plant Proteins 303 10.3.2 Milk Proteins 307 10.3.3 Meat Proteins 311 10.3.4 Egg Proteins 314

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10.1 INTRODUCTION Proteins perform predetermined nutritional, functional, and physiological functions which are altered by preparation, processing, storage, and consumption. Proteins also contribute to the quality and sensory characteristics of foods. Food processing involves controlled and intentional modification of the structure and functionality of food components; and specifically for proteins, these changes are mostly desirable in terms of

Proteins: Sustainable Source, Processing and Applications DOI: https://doi.org/10.1016/B978-0-12-816695-6.00010-6

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their functionality (Li-Chan, 2004). Food processing operations such as thermal treatment, high pressure processing, freezing and frozen storage, dehydration, concentration, mixing, homogenization, extrusion, membrane processes, as well as numerous combinations of these processes, can manipulate food proteins. Various physical and chemical changes affect protein functionality, such as protein
protein interactions, coaggregation among proteins, and interactions with other molecules, such as polysaccharides or a receptor to activate biological function. Distinct proteins are present in food systems, which are influenced differently based on their amino acid composition, their presence in the inner or outer food matrix, and interactions with other food components, ultimately affecting their native and denatured polymeric state (Dutson and Orcutt, 1984). The objective of this chapter is to provide a comprehensive overview on the instability of food proteins as influenced by various processing conditions and the resulting effect on the nutritional, digestible, and physiological properties of food proteins.

10.2 TYPES OF MODIFICATIONS IN NATIVE FOOD PROTEINS Proteins in food systems are complex in nature in terms of their composition and spatial organization (Li-Chan, 2004). Size, shape, composition, and the sequence of amino acids that contain several functional groups, such as sulfydral and carboxyl, affect protein structure at the secondary, tertiary, and quaternary level. They also impact net charge and charge distribution, hydrophobic/hydrophilic ratio, flexibility and rigidity of the molecule, and the ability to interact/react with other components, while directly influencing the properties of food proteins (Damodaran, 2008; Han et al., 2009). Furthermore, steric strain, hydrogen and disulfide bonds, van der Waals electrostatic, and hydrophobic interactions all contribute to protein folding and stability (Damodaran, 2008). Throughout those interactions, proteins are able to interact with other molecules, such as polysaccharides, in the food system or with a receptor to activate biological function. Further details on protein chemistry are available in a detailed review by Damodaran (Damodaran, 2008). Food processing often involves high temperatures, extremes in pH, particularly alkaline, and exposure to oxidizing conditions and uncontrolled enzyme chemistry. Such conditions can result in the introduction of protein cross-links, producing substantial changes in the structure of proteins, and therefore the functional (Singh, 1991), nutritional (Friedman, 1999a,b,c), and physiological properties of the final product. Schematic representations of the physical changes and chemical changes induced in proteins during processing are provided in Figs. 10.1 and 10.2.

10.2.1 Physical Changes in Proteins 10.2.1.1 Denaturation The term “denaturation” is usually used to refer to changes from the original native structure of the protein, without alteration of the amino acid sequence. Denatured proteins

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FIGURE 10.1 Schematic representation of the physical changes induced in proteins during processing (the images do not correspond to a particular protein structure and are used for illustration only). Source: www.rcsb.org

are unfolded but do not undergo changes in their covalent structure with the possible exception of the breaking and reshuffling of disulfide bonds. As discussed in the introduction, this folded state is easily disrupted by environmental conditions and particularly during food processing (change in pH values, pressure and temperature, addition of organic solvents, etc.). When heated above the critical temperature (thermal denaturation), protein hydrogen bonding, electrostatic interactions, and van der Waal bonds are destabilized, while hydrophobic interactions are stabilized (Damodaran, 2008); the latter being mostly responsible for the protein unfolded state. Thus, the secondary and higher native-state structures of proteins are interrupted, changing the protein from its native state to an altered configuration, or denatured state. Inorganic salts, ionic strength, pH, solvents, and other factors also have an effect on the thermal stability of proteins (Finch and Ledward, 1973). Denaturation can occur in the solid state but is more likely to happen when the protein is

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FIGURE 10.2

Schematic representation of the chemical changes induced in proteins during processing(these images do not correspond to a particular protein structure and is used for illustration purposes only). Source: www.rcsb.org

dissolved in a liquid and during drying (Mensink et al., 2017). Generally, in the native conformation, hydrophobic parts of the protein are folded inward, and unfolding/ denaturation results in the exposure of these inward groups to the outside of the protein’s three-dimensional structure (Mensink et al., 2017). The increased surface area and exposed hydrophobic groups of unfolded or partially refolded proteins increase the risk of aggregation (Chi et al., 2003). Calorimetric experiments on proteins have determined that during denaturation or unfolding, free energy change exhibits a parabolic profile as a function of temperature. This behavior has indicated a second conformational transition caused by a temperature decrease (below room temperature), beyond the well-known heat denaturation of globular proteins, and this intriguing phenomenon is known as “cold denaturation.” At lower temperatures, a decrease in the stabilization effect of hydrogen bonding and hydrophobic interactions reduces the stability, whereas at higher temperatures, increased dissipative forces destabilize the native conformation. Calorimetrically, determined values of ΔCp, Tm,

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and ΔH of unfolding, and assuming ΔCp is constant, the standard Gibbs free energy difference between the native and denatured states of a protein at any chosen temperature is:      T T 0 12 ΔG0D ðT Þ 5 ΔHD 1 ΔCp ðT 2 Tm Þ 2 Tln Tm Tm where Tm is the midpoint of the unfolding transition (Privalov, 1990). Li-Chan and Lacroix (2018) explained that a simple two-state transition (native -denatured) is used to describe the transition from folded, native structure to the unfolded, denatured structure, for most of the single-domain proteins. They also recognized that the two-state transition model does not always adequately describe the process of denaturation and instead, a stable, partially folded state, referred to as the “molten globule,” has been characterized for certain proteins under particular conditions. The molten globule state possesses little or no tertiary structure but has a similar secondary structural composition and degree of compactness to the native protein structure (Farrell et al., 2002). Denaturation is known for its effect on protein functionality, in terms of changes in chemical, physical, or biological properties (Li-Chan, 1998). Controlled thermal denaturation in food proteins is known to improve the functionality of food proteins; unfolding of proteins improved the functionality of sunflower, potato, and soybean vegetable proteins (Gonza´lez-Pe´rez and Arellano, 2009). Furthermore, denaturation and partial unfolding of protein molecules upon adsorption at the interface with appropriate hydrophobic and hydrophilic orientation is critical for emulsion formation and stabilization. 10.2.1.2 Aggregation Heating or cooling can accelerate or slow aggregation by colonizing fully unfolded or partially unfolded conformational states that are key intermediates in aggregation processes. This occurs because the same forces that drive protein folding also drive proteins to form interprotein contacts that help bury hydrophobic residues and maximize hydrogen bonding and favorable van der Waals contacts, etc. (Roberts, 2014). The folding reaction is reversible; however, aggregation is irreversible and results in the loss of a specific type of protein monomer (involved in aggregation). More details on aggregation can be found in an article published by Roberts (2014). 10.2.1.3 Gelation According to Damodaran (2008), gel is defined as an intermediate phase between solid and liquid states. A gel is formed by polymers cross-linked through covalent or noncovalent bonds, establishing a network that can retain water or other low molecular weight molecules, and is one of the most important functional properties of proteins in food systems (Han et al., 2009). Gelation can also be induced by high hydrostatic pressure, cold gelation (salt-induced and acid-induced), and enzyme-induced gelation (Gonza´lez-Pe´rez and Arellano, 2009). The denaturation process exposes the functional groups in an unfolded protein, encouraging interaction with each other to form aggregates that may precipitate at low protein concentration and may gel at high protein concentration. Gelation, in general, increases the

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water holding capacity of the food system by entrapment of water within the gel matrix (Dutson and Orcutt, 1984). Two main types of gel networks, fine-stranded and coarse networks, are distinguished in globular proteins. Find stranded gels are generally transparent and attached to each other as bead strings. When the pH value, during gel formation, approaches the isoelectric point and the ionic strength is increased, coarse gels are formed, which are characterized as nontransparent, random aggregations of proteins into clusters (100 6 1000 times a single protein molecule). Network structure affects gel properties, including permeability, the ability to retain water, and rheological properties (Gonza´lez-Pe´rez and Arellano, 2009). 10.2.1.4 Precipitation Depending upon the food system utilized and the components within that food system, thermal processing of proteins can proceed beyond the aggregation stage and cause precipitation and/or gelation. Normally, when protein precipitation occurs, there is a decrease in water holding capacity and a reduction in the functional properties of proteins in most food systems. However, when gelation occurs, there may be an increase in the water holding capacity of the food system by entrapment of water within the gel matrix.

10.2.2 Chemically Induced Changes in Proteins 10.2.2.1 Covalent Aggregation The exact mechanism for aggregation of proteins during thermal processing is not clear, it was rightly estimated by the researchers in the past that some type of covalent bonding must be involved because of the stability of most protein aggregates (Dutson and Orcutt, 1984). It has been stated that chemical covalent aggregation, rather than physical noncovalent aggregation, is the predominant route of aggregation in the solid state (Liu et al., 1991). Chemical aggregation is, in most cases, linked to a thiol
disulfide interchange in the protein, and is accelerated by residual moisture or exposure to atmospheric water. In food systems containing several different proteins, thermal processing causes coaggregation among proteins and protein
protein interactions. These interactions could be very important in determining the properties of processed food and sometimes can proceed beyond the aggregation stage and cause precipitation. Protein precipitation decreases the water holding capacity and leads to a reduction in the functional properties of proteins in most food systems (Dutson and Orcutt, 1984). Degradation of proteins commonly leads to a loss of functionality (Mensink et al., 2017). Aggregation is in most cases irreversible. Furthermore, aggregates in liquid formulations can be qualified as either soluble or insoluble and when aggregate size increases, sedimentation (or floating) will eventually occur (Mensink et al., 2017). 10.2.2.2 Maillard Browning Maillard browning starts with a reaction between the aldehyde or ketone group of the reducing sugar and the amino group of the protein, forming a Schiff’s base, and is

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10.2 TYPES OF MODIFICATIONS IN NATIVE FOOD PROTEINS + Amino compound

N-Substituted glycosylamine

Aldose sugar

+ H2O

Amadori rearrangement

Amadori rearrangement product (ARP) 1-amino-1-deoxy-2-ketose –2H2O

> pH 7

> pH 7

Reductones

–3H2O

Fission products (acetol, diacetyl, pyruvaldehyde, etc.)

–2H

≤ pH 7

Schiff’s base of hydroxymethylfurfural (HMF) or furfural

+2H

+H2O

Dehydroreductones –CO2 + α amino acid

Strecker degradation

+ Amino compound

– Amino compound

Aldehydes HMF or furfural

+ Amino compound

Aldols and N-free polymers + Amino compound

Aldimines and ketimes

Melanoidins (brown nitrogenous polymers)

FIGURE 10.3 Maillard reaction pathways. Source: Adapted from Hodge, J.E., 1953. Dehydrated foods, chemistry of browning reactions in model systems. J. Agric. Food Chem. 1 (15), 928
943; Martins, S.I., Jongen, W.M., Van Boekel, M.A., 2000. A review of Maillard reaction in food and implications to kinetic modelling. Trends Food Sci. Technol. 11 (9
10), 364
373.

followed by a cascade of reactions, eventually leading to the formation of covalent aggregates (Martins et al., 2000). Extensive research has been carried out to date to understand Maillard reaction pathways, Maillard reaction products (MRPs) and their effect on the functionality of involved proteins, participating components, and end products. Fig. 10.3 shows the Maillard reaction, where the different pathways corresponding to the formation of melanoidins are depicted. Hydroxy methyl furfural (HMF) or the furfural route is commonly associated with browning development in bakery products because of the pH range. Other reaction pathways (pH .7) involve sugar dehydration and fragmentation, amino acid degradation (Strecker degradation), and finally polymerization and formation of melanoidins. Corresponding (intermediate) reaction products include reductones, fission products (acetol, pyruvaldehyde, and diacetyl), aldehydes, aldols and N-free polymers, and aldimines. These intermediate products react with amino acids to form low molecular weight’s (LMWs), leading to the production of high molecular weight MRPs by polymerization (Martins et al., 2000; Hodge, 1953; Purlis, 2010; Wang et al., 2011). The formation of low or higher molecular weight melanoidins depends on the degree of

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polymerization among the low molecular weight intermediates and/or the degree of cross-linking between low molecular weight chromophores and noncolored high molecular weight biopolymers. More details on these reactions can be found in review articles written by Hodge (1953), Martins et al. (2000), and Wang et al. (2011). The Maillard reaction plays a considerable function in the resulting flavor of foods following processing and storage by creating a multifaceted reaction between different components. Moreover, MRPs have also been used in the form of flavor enhancers to improve the taste, mouthfeel, and flavor longevity of foods. In foods with high moisture content such as beer, sweet wine, and grape syrup, a temperature lower than 50 C for a period of more than 30 days during fermentation and storage initiates a Maillard reaction. Low molecular weight melanoidins are produced by the reaction of sugars and amino acids at lower temperatures and long reaction times support extensive polymerization leading to the formation of high molecular weight melanoidins (Wang et al., 2011). In low moisture products such as bread, coffee, roasted malt, cocoa, and biscuits, a Maillard reaction will take place if the product is held at temperatures higher than 150 C for less than 2 hours. Under these conditions, the oligoand polysaccharides preferentially react as complete molecules at the reducing end and form high molecular weight melanoidins. Although the mechanism of melanoidin formation is different in high and low moisture foods, high molecular weight melanoidins are commonly the end product. 10.2.2.3 Oxidation and Deamidation When an increasing amount of thermal energy is applied to protein systems, degradation occurs, including destruction of amino acids, hydrolysis of peptide bonds, and other reactions. Chemical degradation mechanisms such as oxidation and deamidation are moisture dependent. Carbonylation is generally recognized as one of the most remarkable chemical modifications in oxidized proteins, especially those containing threonine, proline, arginine, and lysine residues (Este´vez, 2011). Reactive oxidation species-mediated protein oxidation can lead to the conversion of one amino acid into a different one, fragmentation of the peptide backbone, and the formation of intra- and intermolecular cross-links (Stadtman and Levine, 2000). Sulfur-containing amino acids, such as cysteine and methionine, are highly susceptible to oxidation in the presence of oxidizing lipids and yield sulfone, sulfoxide, and disulfide derivatives. Tryptophan residues are oxidized in the presence of transition metals and are regarded as an early protein oxidation manifestation (Este´vez et al., 2011). Deamidation progressively disrupts the structural integrity and biological activity of a protein. It occurs when asparagine (Asn) and glutamine (Gln) residues are converted into aspartic acid and glutamic acid, respectively. Rates of protein deamidation depend on the specific amino acids flanking the Asn and Gln residues in a given peptide chain. Susceptibility to deamidation is also affected by protein 3D structure, local pH, temperature, ionic strength, buffer ions, and numerous other environmental variables (Hao et al., 2017). Other factors affecting these chemical degradation reactions include storage temperature, excipients, the physical state of the excipients (e.g., liquid, amorphous, crystalline), and the chemical composition of the protein (Lai and Topp, 1999).

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10.3 EFFECT OF PROCESSING ON NUTRITIONAL PROPERTIES OF FOOD PROTEINS Generally, food systems are a mixture of proteins and other molecules such as air, water, fat, sugars, polysaccharides, and minerals, physically or chemically attached to each other, making food a complex system. Proteins present in similar/differing food matrices, react differently to processing steps performed as part of the food manufacturing process. The severity and complexity of the process will lead to intermolecular attractions (repulsions) with the neighboring molecules (environment). Cooking transforms the natural biological structure of meat into a final product with desirable textural characteristics, whereas milk processing aims to convert the liquid into semisolid and soft-solid colloidal structures; manufacturing of cheese and yogurt requires processing steps that concentrate the proteins and favor intermolecular interactions. One of the most common processing methods, heat treatment, does not only preserve food and improve palatability, it also modifies the nutritional properties of proteins, that is, the Maillard reaction depletes lysine, whereas fermentation elevates the amino acid content of food products. Endogenous enzymes found in many food systems, are generally denatured by heat which prevents their action (beneficial or deleterious) upon food components of a particular food system (Dutson and Orcutt, 1984).

10.3.1 Plant Proteins Recently, food trends such as vegetarianism and veganism are gaining popularity across the globe and consumer orientation necessitates the need to develop products in this food category. Economically and culturally, plant proteins such as legumes (also known as the poor man’s meat) and cereals are valuable foods, being relatively inexpensive and rich sources of protein. Additionally, the protein hydrolysates from plant sources such as soybean, egg, whey, casein, potato, chickpea, canola, corn, wheat gluten, rice endosperm, and bran have been used to improve the nutritional value of foods due to their high antioxidant, amino acid, peptide, and protein content and their low costs. 10.3.1.1 Cereal Proteins Almost 50% of global food protein supplies come from cereal seeds. Primary processing, such as dehusking (for rice) and dry/wet milling (wheat, corn, rice, rye, barley, and oats), is an essential step in grain processing to impart special characteristics and improve organoleptic properties such as expansion, softness, and flavor development. Milling of grains into flour involves total separation of the hulls and bran, as well as the germ from the endosperm. Usually the endosperm, containing the major proportion of the starch and protein, is the desired fraction for milling into food flour (Olu et al., 2013). Generally, milling processes can alter the nutritional quality of the final product leading to a reduction in nutrients (dietary fiber), phytochemicals (phytic acid, tannin, polyphenol), and antinutrients (trypsin inhibitor) present in the outer layer. Milling can also improve the digestibility of protein and carbohydrates and increase the availability of minerals (Oghbaei and Prakash, 2016). The polishing step in rice milling removes a significant amount of

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nutrients and phytochemicals. Germination and malting of grains is positively associated with nutritional content, whilst decreasing the antinutrients, thereby increasing the digestibility and availability. Drum drying, postsprouting in maize showed promising results in terms of amino acid content and increased the levels of phenylalanine, valine, isoleucine, cystine, praline, tyrosine, and arginine (Mitchell et al., 1949). Nearly all wheat grown in the world is subjected to milling and used for the production of many staple foods, primarily different kinds of bread (Edwards et al., 2007). During the mixing of bread dough, gluten proteins (gliadin and glutenin) in wheat flour unfold and build a transient network comprising mainly disulfide bonds, hydrogen bonds, hydrophobic interactions and entanglements (Singh and Khatkar, 2005; Singh and MacRitchie, 2001) which are important in the development of desired elasticity of the protein matrix of the doughs. Proteins, along with starch, are also hydrolyzed by enzymes naturally existing in the flour, further softening the dough and producing taste compounds. Glutamic and aspartic acids present in the flour are known for their “umami” flavor (Fuke and Konosu, 1991), and glycine, alanine, and aminobutyric acid exhibit a sweet taste that is comparatively half as sweet as sucrose (Ishibashi et al., 1988). Amino acids, present in the wheat flour, not only contribute directly to the flavor and taste of bread, but also react with reducing sugars during the baking process to produce MRPs, which affect the color, flavor, and taste of the bread (Kokawa et al., 2017). Amino acids are consumed by yeast during fermentation namely glutamine, cystathionine, lysine, citrulline, arginine, glycine, histidine, tryptophan, and threonine. The concentration of amino acids increases with the proteolytic enzymes in the wheat flour and decreases due to assimilation by yeast (Collar et al., 1992; Thiele et al., 2002). In a standard bread manufacturing process, amino acid levels such as alanine, are affected negatively with the fermentation time of the wheat dough up to 120 minutes (Kokawa et al., 2017; Jensen et al., 2011). Interestingly, Kokawa et al. (2017) found that yeast growth enters a stationary phase at the fermentation time of 300 minutes, increasing the concentration of amino acids (albeit sensory preference of the bread was significantly low). In another type of bread, sourdough, proteolysis by lactic acid bacteria has been shown to increase the amino acid concentration in the dough. Lactobacillus reuteri and Pediococci strains increased the basic amino acid, predominantly the amount of ornithine. Addition of lysine-producing Pediococci to wheat dough has also been recommended as an alternative in order to increase the amino acid concentration in wheat bread. Thermal treatment alters the conformation of wheat proteins and their ability to take part in thiol
disulfide interchange reactions, thus impacting their functionality in the dough formation process. Mann et al. (2014) in their experiments found that heat treatments (50 C
90 C for 3 hours) induced gluten aggregation resulting in decreased protein solubility and reduced network strength of the dough. Wheat flour obtained from microwave-treated caryopses exhibited reduced dough elasticity, but increased water absorption capacity during mixing and induced modifications such that gluten proteins became soluble (generally insoluble) in aqueous solution (Lamacchia et al., 2016). During baking, the condensation between reducing sugars and amino acids during the Maillard reaction certainly destroys the amino acids, particularly the lysine content and its bioavailability. Furthermore, browning encompasses oxidation and destruction of other essential amino acids (methionine and tryptophan) and cross-linking of proteins (also

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related to crust formation and setting), thus impairing the digestibility of the proteins involved and reducing the nutritional quality of bakery products (Morales et al., 2007). Additionally, acrylamide, a toxic compound in baked foods, is formed by the condensation of reducing sugars and the amino acid, asparagine, in the first stage of the Maillard reaction. Its concentration is strongly correlated with baking temperature and time, and the asparagine and reducing sugars content (Purlis, 2010). The type and degree of heating can alter wheat proteins differently. Gluten heated at 100 C for 12 hours showed a drastic reduction of 25% free sulfhydryl and 37% disulfide concentrations, and the antioxidant capacity was reduced by almost 80% as compared to ˇ c et al., 2012). The results obtained from studies conducted by native gluten protein (Zili´ ˇ c et al. (2012) suggested that sulfhydryl groups are oxidized Wagner et al. (2011) and Zili´ to disulfide groups resulting in disulfide cross-linking of protein molecules. In contrast to this, shearing and heating processes such as the extrusion of wheat proteins appeared to result in the reduction of disulfide bonds to sulfhydryls (Anderson and Ng, 2000). 10.3.1.2 Other Plant Proteins Among the plant-based protein sources, legume seeds are characterized by their relatively high protein content (17%
40%). 16% to 40% of the weight of oilseeds is protein and cereal grains consist of 7% to 13% proteins. The dry matter of vegetables contains 1% to 5% proteins (Gonza´lez-Pe´rez and Arellano, 2009). However, these values can substantially vary from the range provided as the composition of legumes is strongly impacted by the variety. Soaking, dehulling, fermentation, germination, and cooking effectively improve the nutritional value of legumes. Soaking is a common preprocessing technique for whole legumes to facilitate decortication or cooking. The dehulling process removes the outer layer of the grain leading to a significant reduction in soluble and insoluble dietary fiber, phytic acid, and tannins, improving the nutrient bioavailability. Germination of legumes such as cow pea can reduce the polyphenol content significantly (Oghbaei and Prakash, 2016). Cooking in boiling water is the most common processing step in domestic and commercial production. Cooking in boiling water, using both conventional and microwave methods, decreased the amount of amino acids in peas. Microwave cooking was found to be slightly more destructive to amino acids than conventional cooking, indicating that the type and amount of energy absorbed has a significant effect on the nutrient content of thermally processed foods. Plant-based sources are an alternative and novel source of protein. The proliferation of vegetarianism and veganism has driven processors around the world to manufacture vegetable protein concentrates and isolates. Novel approaches are continuously being scouted as current processing steps involve extreme physical and chemical treatments to separate and extract proteins from its native matrix. Extreme pH values, used in processing, can cause protein denaturation, altered functionality, and reduced nutritional properties. Precipitation is a commercial, conventional protein extraction process which is carried out by isoelectric precipitation, organic solvents, addition of salts (salting-out method), and reduction of ionic strength (salting-in method), exclusion polymers, or heating. Isoelectric precipitation is the most common of them; however, it can lead to extensive

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protein aggregation and modification of protein solubility, mostly caused by noncovalent interactions (Gonza´lez-Pe´rez and Arellano, 2009). It is interesting to note that limited protein unfolding is desirable in vegetable protein concentrates and isolates as it improves the functionality of vegetable proteins; however, it is usually counteracted by protein losses due to precipitation. Further, mild heat treatment can improve the antioxidant capacity of vegetable isolates. For example, heated pea protein isolate exhibited 1.5-fold higher antioxˇ c et al., 2012). idant capacity when compared to an unheated sample (Zili´ Extraction of proteins at extreme basic pH values may cause racemization of amino acids, reduced protein digestibility, and losses of essential amino acids such as cysteine and lysine (Gonza´lez-Pe´rez and Arellano, 2009). Industry continues to explore novel approaches and membrane filtration has been identified as a commercial process which is mild and can be performed at ambient temperatures. Therefore, protein products concentrated by ultrafiltration generally exhibit more enhanced functionality than those obtained by precipitation. The final step in the processing of extracted and concentrated proteins is the drying phase to obtain protein concentrate or isolate powder for direct human consumption as a functional ingredient. Spray drying and freeze drying are two methods employed in powder manufacturing. In spray drying, a protein solution is atomized by pumping it through a nozzle and exposing it to hot air, causing evaporation of the moisture and thus drying. In freeze drying, the solution is frozen and water is subsequently removed by sublimation under a vacuum. These two processes subject the protein to fundamentally different stresses. Spray drying exposes the protein to shear (during atomization), heat, air
liquid interfacial, and dehydration stresses; whereas lyophilization is associated with freezing, dehydration, and solid
liquid interfacial stresses (Grasmeijer, 2016). The processing parameters of drying (drying rate, time, temperatures, etc.) influence the stresses which determine the degradation phenomena during drying (Connolly et al., 2015). Therefore, an optimized combination of formulation and processing should be chosen to maximize the protein stability and enhance protein functionality. Additives and enzymatic treatments such as salt addition or microbial transglutaminase have also been employed to improve the functional properties of protein isolates from peas and beans (Ali et al., 2010). Microbial transglutaminase-treated isolates demonstrated increased protein solubility, suggesting that these substrates contain sites recognizable by the enzyme. Deamidation reduces the surface hydrophobicity of protein molecules and increases the electrostatic repulsion between protein chains, which increases their solubility. Further information on modifications of functional properties of food proteins by microbial transglutaminase can be found in a review published by Gaspar and de Go´es-Favoni (2015). Protein hydrolysates are commercially utilized in many food formulations for their enhanced solubility at different pH and temperatures, as compared to native proteins with reduced solubility at the isoelectric point with increased temperatures. Enzymatic protein hydrolysis is a commonly used method and it produces smaller fractions of peptides with superior nutritional uniqueness than the original protein (Amza et al., 2013). Sequential combination of Alcalase (endoprotease) and Flavourzyme (exoprotease) have been used to prepare flaxseed protein hydrolysates. Furthermore, heat treatment of these hydrolysates

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can be carried out to produce MRPs with different functionalities. The low molecular weight MRPs have the functionality to improve the mouthfeel and flavor intensity in umami soup and high molecular weight components could contribute meat-like flavors as well as influencing other sensory features. It is interesting to note that protein content does not correspond to protein functionality such as emulsification. It is the qualitative as well as quantitative differences in protein content which contribute to the emulsifying properties. Lupin flour contains higher protein content (400 g/kg) than green pea, wheat, and buckwheat flours, however, it is a weak emulsifier and it has been deduced that not all protein molecules are effective as emulsifiers (Raikos et al., 2014). Dairy milk alternatives like soymilk, rice milk, and oat milk are gaining popularity amongst young people as well as adult consumers. Heat treatment is a crucial step in plant-derived milk drinks, which not only improves the flavor and structurally alters the food proteins, bringing beneficial palatability changes, but also improves the stability of the plant-based dispersions. When compared with raw and mild treated (70 C and 80 C) equivalents, high heat treatment of soymilk (90 C, 100 C, and 115 C) increased the dispersion stability of soymilk protein or a soymilk emulsion consisting of proteins and lipids (Shimoyamada et al., 2008). Soy proteins, β-conglycinin and glycinin, present in soymilk denature at 70 C and 90 C, respectively (Zhang et al., 2004) and mild heat treatment denatures only β-conglycinin, but not glycinin. Coexistence of heat denatured β-conglycinin and native glycinin decreased the dispersion stability of soymilk but heat denaturation of both proteins improved the stability and prevented precipitation (Guo et al., 1997). Overheating can destabilize food proteins by different phenomena such as association, aggregation, or partial refolding. Shimoyamada et al. (2008) confirmed that reheating soymilk for a second time, post high heat treatment, resulted in increased protein denaturation and the formation of soluble aggregate particles which result from a combination of denatured protein molecules.

10.3.2 Milk Proteins Milk is an important part of the human diet due to its high nutritive value. It exists in an emulsion state with fat globules suspended in an aqueous solution comprising dissolved and suspended constituents such as casein, serum proteins, lactose, minerals, and vitamins (Brans et al., 2004). Milk proteins have been classified into two categories— casein present in milk in a colloidal state known as casein micelle, and whey proteins, that is, α-lactalbumin, β-lactoglobumin, immunoglobulins, and Proteose peptone, that are water soluble and heat-sensitive proteins and enzymes (Hui, 1993). Thermal processing of milk is a common practice adopted by the dairy industry to extend shelf life and enhance milk quality by reducing/removing microbial activity (McKinnon et al., 2009). Thermal processing of milk may also be used to enhance the organoleptic properties of milk-based products by influencing the milk protein functionality (del Angel and Dalgleish, 2006; Nicorescu et al., 2008; O’Kennedy and Mounsey, 2009). Milk homogenization is usually followed by pasteurization or ultrahigh-temperature (UHT) treatment. Subsequently, whey protein is denatured, and the extent of denaturation

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is proportional to the temperature used (neglectable for pasteurization, approximately 60% for UHT) (Michalski and Januel, 2006). Thus, micellar fragments and semiintact casein micelles cover the fat droplet interface, and the denatured whey protein can interact through disulfide bonds with milk fat globular membrane proteins and micellar caseins absorbed at the interface (Michalski, 2009). Depending upon the heat treatment and physicochemical properties of milk, the whey protein is reversibly or irreversibly altered. Due to denaturation, small aggregates of β-lactoglobulin are formed which are dependent upon the time and temperature of the thermal process applied. Further heat treatment leads to denaturation of α-lactalbumin which forms larger nodes with β-lactoglobulin, subsequently binding to the surface of casein micelles. This is due to protein
protein interactions, that is, SH/S-S interchange in proteins like β-Lactoglobumin, κ-casein, and α-lactalbumin, leading to irreversible accumulation of protein nodes of varying sizes dependent upon protein composition and processing conditions (Considine et al., 2007). It is critical to understand the mechanisms that proteins undergo to realize the effect of processing on milk proteins. Denaturation or aggregation of milk whey proteins is attributed to β-lactoglobulin due to its higher concentration in milk. Interactions of denatured β-lactoglobulin with α-lactalbumin and interactions with nonwhey proteins such as κ-casein are important reactions that occur in milk during thermal processing (Wijayanti et al., 2014). The denatured protein is irreversibly changed at temperatures beyond 70 C leading to the formation of either small (through interchange reactions) or large aggregates (without SH interactions). According to Verheul et al. (1998), denaturation and aggregate forming happens in two steps, that is, unfolding of β-lactoglobulin and aggregation. Aggregation, as a result of either chemical (interchange interactions S
S/S
H reactions) or physical reactions or both, is dependent upon the conditions available for reactions, that is, temperature, pH, ionic strength of β-lactoglobulin system. Chemical reactions are increasingly activated at higher temperatures ( . 85 C), higher pH system ( . 6), and low ionic strength, while physical reactions are more prevalent at low temperature (65 C
85 C), low pH (,6), and higher ionic strength ( . 1.0 M NaCl). More recent research compares the aggregation process with polymerization with higher molecular weight aggregate formation depending upon the concentration levels of dimers, trimers, tetramers, and oligomers (Surroca et al., 2002; Tolkach and Kulozik, 2007). α-Lactalbumin and bovine serum albumin (BSA) are also considered to be responsible for denaturation of whey proteins and aggregate formation. However, it has been observed that α-lactalbumin, on its own, is unable to form aggregates and is reliant upon the presence of β-lactoglobulin for all treatments under 95 C. Denaturation of α-lactalbumin is largely reversible for treatments ,90 C and irreversible denaturation only begins when the milk system is heated beyond 95 C for a minimum of 14 minutes. This is attributed to the higher heat stability of α-lactalbumin due to the nonavailability of a free SH molecule. Gelation properties of α-lactalbumin are dependent upon the protein content, pH, salt concentration, temperature, and time (Gezimati et al., 1996). BSA is an important gelling whey protein and the gelation process involves unfolding of the protein molecule and formation of aggregates. In its native form, BSA contains a lower percentage of its β-sheet which increases (nonnative form) as heat is applied and the amount of α-helix decreases prior to gel formation (Clark et al., 1981). Havea et al. (2000) and Havea

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et al. (2001) carried out studies on mixtures of major whey proteins, that is, β-lactoglobulin, BSA, and α-lactalbumin and found out that concentration of BSA is more effective in hastening aggregate formation in α-lactalbumin as compared to β-lactoglobulin. β-Lactoglobulin and BSA are more capable of undergoing
SH/S
S interchange reactions and initiating denaturation due to the presence of free
SH molecule in comparison to α-lactalbumin. Whereas α-lactalbumin is capable of forming interprotein aggregates with β-lactoglobulin and BSA. When milk is thermally treated between 75 C and 100 C for up to 60 minutes, the casein micelles increase in size by the same amount as denatured whey proteins (Anema and Li, 2003). Denatured whey proteins tend to attach to the casein micelles via
SH/ S
S interactions but the rate of denaturation for whey proteins is faster than their association with κ-casein (Considine et al., 2007). The rate of interaction between whey proteins and κ-casein is slow when temperatures are between 75 C and 85 C, while it increases rapidly between 90 C and 100 C (Anema and Li, 2003). The interaction of whey proteins with κ-casein is dependent upon the availability of a free
SH molecule and involves both hydrophobic and chemical interactions (Cho et al., 2003). As with the case of denaturation of whey proteins, interaction between whey and casein is dependent upon the conditions of treatment, that is, heating time and temperature, pH, ionic conditions, and protein concentrations (O’connell and Fox, 2003; Singh, 2004). Heating the system slowly for a longer time period leads to the formation of monomers and smaller aggregates, while rapid heating results in bigger aggregates. This happens due to more effective interaction between whey and κ-casein micelles with slower heating times. Dissociation of κ-casein is highly reliant on the temperature and pH of the system. The interaction between whey and κ-casein micelles happens in two forms. Firstly, when κ-casein dissociates from the micelle at lower heating and denatured whey proteins interact with κ-casein in serum state (Guyomarc’h et al., 2003). Secondly, the denatured whey and κ-casein interact before the κ-casein is detached from the casein micelle. Parker et al. (2005) observed that denatured whey prefers to interact with still-intact κ-casein on the micelle rather than its dissociated state in serum. Donato et al. (2007) observed that there is a small amount of space available on the surface of the casein micelle which allows denatured whey proteins to attach themselves to the surface of the micelle and interact with individual κ-casein molecules. It has been observed that the availability of free
SH molecules acts as a catalyst in irreversible denaturation of whey proteins and aggregate formation through S
S/
SH interactions. Blocking the
SH group could prevent association of the monomers and inhibit interprotein polymers. Loch et al. (2011) found that β-lactoglobulin has a strong affinity towards hydrophobic compounds like hydrolyzed lecithin and fatty acids with a hydrocarbon chain length of 12
20. The heat stability of protein is enhanced, that is, increased denaturation temperature (76 C
81 C), by the addition of hydrolyzed lecithin to heated whey protein isolate (Van der Meeren et al., 2005). Similar results of hydrolyzed lecithin on protein stability were observed by other researchers (Ikeda and Foegeding, 1999). It was observed that hydrolyzed lecithin binds to the surface of denatured whey protein (
SH groups) before they interact with casein micelles. Other compounds that have been tested for reducing the denaturation of proteins include phosphatidylcholine and fatty acids like butyrate,

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oleate, and palmitate (Ikeda et al., 2000), SDS and palmitate, and conjugated linoleic acid or myristic acid (Considine et al., 2005, 2007). Fatty acids like palmitate, oleate, and butyrate require salt to be effective on heat-induced aggregation of β-lactoglobulin at room temperature. The presence of salt enabled the formation of a very elastic gel, indicating hydrophobic interactions under these circumstances. Considine et al. (2005) stated that the addition of SDS and palmitate to heated β-lactoglobulin stabilized the native structure against thermally induced denaturation. Several minerals, such as calcium, are added to whey protein products like whey protein, milk protein isolate, and concentrate being produced commercially. It has been observed that the addition of mineral compounds like calcium chloride to whey protein products like whey protein isolate markedly affects the aggregate formation (Caussin et al., 2003) leading to gelation. Heat stability of proteins can be impacted by chelation of minerals using compounds like EDTA which decreases the gel hardness of whey protein concentrate (Kuhn and Foegeding, 1991). Keowmaneechai and McClements (2006) examined the effects of adding EDTA and trisodium citrate to the available whey proteinbased emulsions with added soybean oil and protein isolate. In the absence of chelating agents, whey protein emulsions were unstable at temperatures of 90 C but were stable with the addition of EDTA at a molar ratio to calcium of $ 1:1. Trisodium citrate proved to be less effective in controlling denaturation as compared to EDTA. This was due to the ability of chelating agents to bind free calcium ions, therefore reducing aggregation caused by denaturation (Keowmaneechai and McClements, 2002). Several whey protein-based products are known to contain sugars and the addition of sugar to protein solutions is known to have a stabilizing effect on proteins. Some early research in this domain showed that the addition of sucrose between 2% and 20% reduced the amount of aggregate formed from the heating of whey protein solutions at 80 C (Garrett et al., 1988). It was observed that the amount of sucrose added also significantly impacts the denaturation process. Low concentration sucrose solutions added to heated whey protein isolate emulsions showed a decreased rate of aggregation and gelation, while higher concentration sucrose solution increased interprotein interactions and gelation (Kulmyrzaev et al., 2000). Sucrose had a similar effect on BSA solutions as it increased the denaturation temperature of BSA solution from 72 C to 79 C (Baier and McClements, 2001). Other carbohydrates such as sorbitol and glycerol were also assessed for their impact on aggregation and gelation properties of whey protein. Chanasattru et al. (2007) found that sorbitol was more effective than glycerol in enhancing the heat stability of β-lactoglobulin. Sorbitol increased the denaturation temperature from 74 C to 86 C, while glycerol increased the temperature to 76 C which was attributable to protein
protein interactions. A drawback of adding carbohydrates to improve the heat stability of whey proteins was observed by Rich and Foegeding (2000). They identified the occurrence of the Maillard reaction and browning of ribosecontaining gels due to reactions between ribose and proteins, but no effect was observed for lactose-containing gels. Chymosin is responsible for the fracture of κ-casein molecules leading to the release of the C-terminal region of κ-casein, that is, caseinomacropeptide (CMP). During the initial stage of the reaction, a dispersal coefficient of micelles is augmented due to the removal of the “hairy” layer of the micelles (De Kruif, 1992). κ-Casein molecules begin to aggregate

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and gel (Dalgleish, 1979; Kethireddipalli et al., 2010) after 85% of κ-casein is hydrolyzed, leading to a drop in the viscosity of milk. At this stage, almost complete release of CMP is attained, which is also attributed to the partial collapse of κ-casein with a decrease in pH making the casein micelle less effective in providing steric stabilization (De Kruif, 1999). A lower pH environment is required for renneting which permits a lower degree of κ-casein collapse and gelation. The second stage of aggregation is dependent upon a reduced dielectric constant due to added ethanol, the complete collapse of κ-casein, and reduced steric repulsion (O’Connell et al., 2006). Micelles begin to aggregate due to reduced steric repulsion, caused by increased hydrophobic bonding and the presence of ionic solutions allow higher aggregation at lower CMP levels (Bringe and Kinsella, 1986). The firmness of the subsequent gel is dependent upon the concentration of protein, while the time of coagulation is not affected by protein concentration for up to four times the original concentration of casein micelles (Sandra et al., 2011). Heating of casein micelles in the presence of whey proteins leads to accelerated changes in the surface of casein micelles. The whey protein molecules tend to attach on to the surface of κ-casein micelles at temperatures .70 C due to denaturation, and form interprotein complexes (Anema and Li, 2003; Donato et al., 2007; Donato and Dalgleish, 2006). The physical state of complexes is also dependent upon the pH and heating of the system, with about 30% of denatured whey associated with casein and remaining present in a soluble state (Kethireddipalli et al., 2010). Ultrahigh pressure treatment at .250 MPa leads to irreversible changes in casein micelle size (Gebhardt et al., 2006), depletion of κ-casein and β-casein, and solubilization of calcium phosphate (Gaucheron et al., 1997). Pressure treatment ( . 100 MPa) does not affect the rennet-induced accumulation of caseins, has a lower gelling time (LopezFandino et al., 1996), and leads to the association of denatured whey and casein micelles (Needs et al., 2000). Transglutaminase can be used to bind glutamine and lysine and form cross-links between caseins to stabilize the micelles. This helps to prevent dissociation of micelles due to pressure treatments and also prevents rennet-induced hydrolysis of κ-casein (O’sullivan et al., 2002; Smiddy et al., 2006).

10.3.3 Meat Proteins Proteins, especially in protein gels, beef, and fish muscle, have the ability to soak up water and retain it within a protein matrix through protein
water interactions. This ability of proteins to trap water is associated with the juiciness and tenderness of ground meat products and the desirable textural properties of breads and other gel-type products (Gaspar and de Go´es-Favoni, 2015). Approximately 20% of the muscle weight is made up of meat proteins which undergo a significant amount of change when thermally processed during cooking. Thermal processing is a vital step which makes meat and meat products microbially safe and edible. Thermal processing of meat induces significant structural changes in meat protein which alters the quality of meat products. Muscle tissue is made up of 75% water, 20% protein, 3% fat, and 2% soluble nonprotein substances. Meat proteins may be classified into three categories, myofibrillar, sarcoplasmic, and connective tissue proteins (Tornberg, 2005).

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Myofibrillar proteins make up approximately half of the total protein content (50%
55%), while sarcoplasmic and connective tissue proteins follow with 30%
34% and 10%
15% of the total protein content, respectively (Bailey et al., 1979). Meat proteins behave in a similar manner to dairy proteins, such as whey and casein, when thermally processed. The protein unfolds from its original helical structure upon heating and subsequent heating allows for protein
protein interactions, leading to aggregation. These changes can be monitored using various techniques such as differential scanning calorimetry, optical rotary dispersion, circular dichroism, and by studying the loss of protein solubility, and/or by performing mechanical and microstructural measurements. Past research (Hamm, 1977; Tornberg et al., 1997; Mane et al., 2014) suggests that sarcoplasmic proteins are responsible for the consistency of cooked meat products, and they form gel-like aggregates, linking structural meat elements together. Sarcoplasmic proteins also impact on the tenderness of the meat, which is dependent upon the rate of heating and the temperatures reached. At lower temperatures ,60 C, collagenase remains active and heating for at least 6 hours, known as LTLT or low-temperature long-time cooking, led to reduction in shear force causing meat tenderness (Marino et al., 2014; Dominguez-Hernandez et al., 2018). Myofibrillar proteins display unfolding of their helical structure at the beginning of the heating process (at 30 C) and the helical content is observed at a minimum at 70 C (Choe et al., 1991; Han et al., 2014). During this temperature change, the surface hydrophobicity decreases with the increase in temperature instigating interprotein interactions and leading to aggregate formation and/or gelation. The gelation of myofibrillar proteins, especially myosin, is aided by the presence of ionic solutions even at low concentrations such as 0.5% by weight (Hermansson and Langton, 1988). The denaturation temperature is reduced (45 C) due to the presence of the ionic solution and the gel firmness also increases (Fretheim et al., 1986; Sharp and Offer, 1992; Zhang et al., 2017). Ionic concentrations and pH of the solution enable the determination of the myosin structure, that is, monomers or filaments. At lower ionic concentrations, myosin occurs as filaments similar to the natural fibers of muscle tissue. Gelation of myosin happens in two steps, the first step involves aggregation of myosin heads at a temperature range of 30 C
50 C. Heating beyond 50 C leads to the formation of larger myosin aggregates with myosin tails becoming indistinguishable. The second step involves network formation through hydrophobic interactions between myosin tails and the flattening of the helical structure. Connective tissue denaturation, that is, collagen breakdown, results from a collapse of hydrogen bonds and subsequent contraction of collagen occurring at temperatures of 53 C
63 C (Martens et al., 1982). Further heating leads to additional contraction of collagen leading to formation of gelatine (Burson et al., 1986; Lewis and Purslow, 1989). Heat-stable bonds are more prevalent as the animal ages due to conversion of thermally-labile cross-links to heat-stable bonds, thereby increasing tension in the connective tissue and reducing the tenderness of meat (Shimokomaki et al., 1972). High pressure denaturation of protein is dependent upon the type of protein, processing conditions, and amount of pressure applied. Proteins may dissipate or dissolve depending upon the level and duration of pressure applied. Changes in protein structure are reversible for a pressure range of 100
300 MPa, whereas they become more irreversible when pressure increases beyond 300 MPa. High pressure leads to the rupture of noncovalent interactions within protein and leads to the formation of secondary, tertiary, and

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quaternary structures of protein through intra- and intermolecular bonds. Protein denaturation due to pressure happens because of the destruction of hydrophobic bonds and the subsequent unfolding of the proteins. Balny and Masson (1993) observed significant changes to the protein structure at various levels of pressure, for example, the tertiary structure of protein unfolded at applications of pressure beyond 200 MPa, while the secondary structures deformed only at pressures greater than 700 MPa, leading to irreversible denaturation. There is an inverse relation between temperature and pressure, since pressure increases up to 100 MPa lead to an increase in the denaturation temperature. But an increase in pressure beyond 100 MPa leads to a reduction in the denaturation temperature. Several studies have been conducted to investigate the effect of high pressure on proteins like myosin (Ma and Ledward, 2004; Chen et al., 2014) and metmyoglobin (Defaye and Ledward, 1995; Bak et al., 2014; Chouhan et al., 2015). When no pressure is applied, the two heads of the myosin monomer molecule are clearly differentiated. The structural change is first observed at 140 MPa and beyond. On pressure application, the monomers interact head-to-head to form aggregates or oligomers. Under constant pressure, the size of the oligomers begins to increase with the proportion of one-headed aggregates increasing. It was observed that the helical structure of myosin didn’t change at pressures of 210 MPa (Yamamoto et al., 1993). High pressure denaturation of myosin also led to the formation of stabilized structures due to the formation of disulfide and hydrogen bonds (Angsupanich et al., 1999). Metmyoglobin dimerized at pressure conditions of 750 MPa at a pH range of 6
10, applied for 20 minutes. Acidic conditions are not favorable for dimerization of metmyoglobin (Defaye and Ledward, 1995). Lipid oxidation is another phenomenon that is observed at pressures beyond 300 MPa. The presence of air helps to expedite the oxidation process, as observed in minced meat, while pressure ranges between 300 and 400 MPa cause myofibrillar and sarcoplasmic denaturation of minced pork (Ananth et al., 1998). Other studies have also pointed to the modification of the secondary structure of myofibrillar proteins at high pressure and irreversible denaturation and aggregation at pressures beyond 300 MPa (Chapleau and de Lamballerie-Anton, 2003; Chapleau et al., 2004; Lee et al., 2007). Meat color is also impacted significantly by the application of high pressure. Meat processed at 200
350 MPa showed a pink color due to a significant increase in L color values, while the a-value decreased at pressures 400
500 MPa leading to a gray-brown appearance of the meat. A whitening effect was observed at pressures of 200
300 MPa due to denaturation of globin or the oxidation of ferrous myoglobin to ferric myoglobin at .400 MPa pressure (Cheftel, 1995). Cooking has been found to promote the carbonyl gain in meat products and, moreover, increase the susceptibility of meat proteins to undergo further carbonylation during the subsequent chilled storage. Naturally occurring constituents of muscle tissues, such as heme pigments, oxidative enzymes, and unsaturated lipids, provide a catalyst for the oxidation of muscle tissues (Youlin, 2000) through reactive oxygen species such as hydrogen peroxide, hydroperoxides, and superoxide (Butterfield and Stadtman, 1997). Reactive oxygen species frequently target peptide and other functional groups of the amino acid to initiate oxidation through abstraction of the hydrogen atom, leaving an exposed carbon-centered protein radical (Stadtman and Levine, 2003) which is constantly oxidized in the presence of oxygen to form alkyl peroxide which attaches itself to free

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radicals of metals such as copper and iron. Therefore, formation of reactive oxygen species is inevitable during aerobic metabolism and thus oxidation affects proteins and lipids in vivo (Holloszy and Coyle, 1984). Chilled storage at 0 C and reduction of pH from 7 to 5.5 leads to enhanced oxidation of beef as measured by the dinitrophenyl hydrazine method (Srinivasan et al., 1996). Proteins are also more susceptible to oxidation at other low pH conditions, for example, denaturation, aggregation, and decrease in protein solubility. Preslaughter stress has also been noted to be an inducing factor for reduction in pH in muscles leading to denaturation, aggregation, and oxidation in broiler and chicken breast meat (Wang et al., 2009; Zhang et al., 2010). Other postrigor factors that significantly contribute to the oxidation process include the release of free-catalytic iron and oxidizing enzymes, propagation of lipid oxidation and alteration of cellular compartments. The findings of several other studies are in agreement on the effect of storage and chilling on protein oxidation in beef (Rowe et al., 2004; Lindahl et al., 2010), pork (Ventanas et al., 2006; Herring et al., 2010), and poultry (Zhang et al., 2010; Rababah et al., 2004). Cooking under different circumstances leads to physicochemical changes like alterations in cellular compartments, the release of catalytic iron, and the formation of hydroperoxides, therefore inducing oxidative reactions and protein carbonylation (Vuorela et al., 2005; Salminen et al., 2006; Ganha˜o et al., 2010). Processes such as curing and drying also induce a greater protein oxidation as observed by Ventanas et al. (2006, 2007). Cured ham dried for a longer duration had comparatively much higher carbonyls (B9 nmol/mg protein) versus normal loins. The effect of carbonylation has also been studied in comminuted cooked meats such as sausages, ham, and liver pate (Este´vez and Cava, 2004; Este´vez et al., 2005; Sun et al., 2010). Certain processes like cutting and mincing, etc., promote the formation of carbonyls as noted in cooked meat products (B5 nmol/mg protein) as compared to raw meat (B1
3 nmol/mg protein). Cava et al. (2009) and Fuentes et al. (2010) have also noted the effect of high hydrostatic pressure on protein oxidation and the increase in carbonyls, that is, AAS and GGS at a pressure .600 MPa. The probable mechanism through which carbonylation may affect the sensory, nutritional, and technological traits of meat and meat products is summarized below. High temperature, lighting, and the mincing process can also enhance protein carbonylation in dried minced pork slices (Este´vez, 2011) (Fig. 10.4).

10.3.4 Egg Proteins Egg proteins are used in manufacturing cakes, meringues, souffles, custards, etc. The beneficial functional properties of egg proteins are due to their denaturation and coagulation at specific temperatures and the formation of a stable matrix upon coagulation (Dutson and Orcutt, 1984). Eggs are processed in different ways for microbial safety and shelf life extension, which in turn can have consequences on protein digestibility (Gharbi and Labbafi, 2018). Alkali-treated preserved eggs have many distinct characteristics, including unique flavors, dark green yolks, and brown transparent egg white. Alkali treatment is also effective in destroying aflatoxin in eggs of laying hens fed diets containing aflatoxin (Zhao et al., 2016). The most used processing method for

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10.3 EFFECT OF PROCESSING ON NUTRITIONAL PROPERTIES OF FOOD PROTEINS

+ Proteolysis

Carbonylation

Target

Target

Target

Proteolytic enzymes (calpain system?)

MP

MP

315

Strecker-type degradation of free amino acid

Loss of ε-NH3 groups

Cross-linking

Alteration electrical arrangement MP

Stabilization aggregates

Myofibril strengthening

Decreased proteolytic activity

Strecker aldehyde

Myofibril shrinkage

Meat toughening

Impaired tenderization

Impact in flavor?

Aggregation

Mechanical constriction muscle

Increased hardness

Denaturation

Decreased WHC

Loss functionality

Decreased juiciness

Unfolding

Increased hydrophobicity

Decreased solubility

FIGURE 10.4 Probable mechanism of carbonylation affecting sensory, nutritional, and technological traits of meat (Este´vez, 2011).

egg white is heat treatment, but the interest in emerging technologies (high pressure, high intensity ultrasound, ultraviolet, and pulsed electric field) is growing. An excellent review on the effect of processing on egg white proteins has been written by Gharbi and Labbafi (2018). Egg white is a colloidal system where proteins are dispersed in water medium; there are more than 150 proteins, including mainly ovalbumin, ovotransferrin, ovomucoid, lysozyme, ovomucin, and globulin (Mann and Mann, 2011). Egg white is well-known for its excellent nutritional and functional attributes (foaming, gelation, and emulsifying) (Mine and Zhang, 2002). Ovalbumin is the main protein in egg white and contains free sulfhydryl groups buried in the protein core. During processing, the buried sulfhydryl groups are exposed to form disulfide bonds and can alter the functional properties of the egg white proteins (Van der Plancken et al., 2005). Ultraviolet exposure of egg white leads to structural modifications leading to the formation of protein aggregates indicated by an increase in the particle size and turbid appearance. Ultraviolet treatment also induced the development of browning, the formation of large protein aggregates by disulfide exchange, and protein backbone cleavage (Manzocco et al., 2012). Egg yolk is a valuable food ingredient for the manufacture of many food products. Liquid yolk is frozen for prolonged storage of up to 1 year to prevent microbial growth and spoilage, retention of egg yolk flavor and color, and inhibition of chemical reactions, such as autoxidation of lipids and the browning reaction (Powrie, 1968; Au

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et al., 2015). However, the freezing process induces gelation in egg yolk leading to undesirable physiological changes, such as reduced yolk dispersibility in water and loss of functionality (Primacella et al., 2018). Although the mechanism for yolk gelation caused by freezing and thawing has not been fully elucidated, many existing proposed mechanisms suggest that ice crystal formation during freezing storage plays a fundamental role in yolk gelation. These undesirable changes induced by gelation, however, could be inhibited by the addition of cryoprotective agents such as glucose, arabinose, galactose, glycerol, sorbitol, propylene glycol, salt, carboxy methlyl cellulose, proline, polyethylene glycol, and Tween 80; proteolytic enzymes such as lecithinase A and papain; hydrolyzed egg white and hydrolyzed egg yolk; or mechanical treatments, such as colloid milling, to prevent ice crystal formation and changes in the yolk’s physicochemical conditions that favor aggregation of proteins (Primacella et al., 2018; Lopez et al., 1954; Powrie et al., 1963).

10.4 EFFECT OF PROCESSING ON DIGESTIBILITY OF FOOD PROTEINS Protein quality is measured as a combination of the protein content and digestibility to satisfy the daily protein requirement. Protein digestibility is significant for realizing the physiological benefits of the proteins. Interestingly, peptides may be generated from the parent proteins during food processing or intact proteins are acted upon by gastrointestinal fluids during the digestion process. In a series of metabolic studies with young rats, it was found that dry roasting or toasting of cut maize grains at 176.7 C
204.4 C for 3
4 minutes increased the digestibility of the proteins by about 8% and caused no change in their biological value (Mitchell et al., 1949). However, applying a flaking process (steamed and toasted for 20
45 seconds) reduced the digestibility of the protein by about 15% and the biological value by about 6%. Grain milling leads to the reduction of phytate, tannin, and phenolic elements, which improves the availability of minerals and the digestibility of protein and carbohydrates (Oghbaei and Prakash, 2016). Germination and malting of whole grains decreases the antinutrients and increases the digestibility and availability of nutrients. Soaking is a common preprocessing technique for whole legumes to facilitate decortication or cooking. Soaking and fermentation are known to increase in vitro protein digestibility of some common legumes (Oghbaei and Prakash, 2016). According to Neucere and Cherry (1982), excessive heat treatment causes proteins to undergo many complex reactions which decrease their digestibility. The beneficial effects of heating, high pressure, and high intensity ultrasound on the egg white proteins (EWP) digestibility have been reported (Van der Plancken et al., 2005; Nyemb et al., 2014; Stefanovi´c et al., 2014a,b; Van der Plancken et al., 2003). According to Van der Plancken et al. (2003), the extent of egg white protein digestion depends on the degree of protein unfolding. When compared with unheated ovalbumin, all types of ovalbumin aggregates (linear, linear-branched, spherical, and spherical-agglomerated) are more susceptible to digestion. The degree of ovalbumin unfolding before aggregation is a major factor in its digestibility, which is highest in the linear aggregate and lowest in the sphericalagglomerated aggregate (Nyemb et al., 2014).

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10.5 EFFECT OF PROCESSING ON PHYSIOLOGICAL FUNCTIONS OF FOOD PROTEINS Proteins, when (un)digested in the gastrointestinal tract, perform various physiological functions in the human body, including protein synthesis, gastrointestinal functions, enzyme activity, gut hormone release, insulin secretion and glucose metabolism, blood pressure and energy balance. Protein digestion commences in the stomach with hydrolysis by pepsin and then by duodenal enzymes, including trypsin, chymotrypsin, elastase, and carboxypeptidase, to liberate small peptides and free amino acids into the gut for their local or postabsorptive action (Turgeon and Rioux, 2011). The release of peptides may occur after gastrointestinal fluids act on the intact proteins, or the peptides may be generated from the parent proteins during food processing and then consumed. This section will provide specific highlights of the physiological effect of dietary proteins. Dietary proteins are essential compounds that build and repair body tissues. Specific health conditions such as illness, physiological stress, pregnancy and breast-feeding, and infections can benefit from dietary modifications such as increased protein consumption (Akin and Ozcan, 2017). Clinical studies on healthy and diabetic subjects have revealed that proteins from pulses, cereal grains, and their hydrolysates may be able to reduce blood glucose concentration and enhance insulin response (Lo´pez-Baro´n et al., 2017). Pea, wheat, rice, and soybean protein hydrolysates are potentially more potent than intact protein in inhibiting starch digestion. Protein hydrolysates generate faster hormonal responses of insulin and glucagon in healthy participants than with the intact protein alone which could be attributed to the leucine, isoleucine, phenylalanine, valine, and arginine present in the protein hydrolysate (Claessens et al., 2009). Protein synthesis is undoubtedly the major utilization of amino acids in the body. Protein consumption impacts the balance between muscle protein synthesis and muscle protein breakdown (Phillips et al., 2009). After milk homogenization, coagulation of caseins and lipid droplets occurs simultaneously during gastric digestion, and they were found to be much finer and more digestible. Gastric emptying is delayed with smaller fat droplets; however, increased lipolysis is found due to a larger interface area (Michalski, 2009). Maillard reactions are known to cause loss in nutritive value of foods, however, the interaction of proteins with reducing sugars can increase the antioxidant activity of the proteins (Gu et al., 2010). MRPs, depending on the severity of the heat treatment, produce insoluble and soluble melanoproteins which are known to possess antioxidant activity. Additionally, the antioxidant activity depends on the food matrix, thereby affecting the properties of insoluble melanoproteins to remain in the gastrointestinal tract for a long time which might help in quenching the soluble radicals that are continuously formed in the intestinal tract and that could be involved in the etiology of colon cancer (Babbs, 1990). A mixture of pea protein isolate and glucose, when heated at 180 C for 10 minutes (60.28 mmol Trolox/kg), as expected, showed 40% less antioxidant activity when ˇ c compared to the mixture heated at 180 C for 5 minutes (100.19 mmol Trolox/kg) (Zili´ et al., 2012). This showed that a difference of 5 minutes could impact the protein functionality significantly.

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Cereal β-glucan is known for its potential to lower blood cholesterol values, reduce glycemic and insulin responses, and reduce the risk of coronary heart disease. β-glucans exhibit health benefits such as lowering of low density lipoprotein (LDL) cholesterol, the exact mechanism of their action is still unclear however their association with the proteinaceous moieties to form viscous slurries during digestion (Wolever et al., 2010) and aggregation behaviour and are known to have huge impact on their physiological properties. Zielke et al. (2018) found that whey protein and gliadin provoke aggregation of β-glucan alleviating β-glucan’s health benefits. Microwave treatment of the wheat grains for 2 minutes at 1000 W to a temperature of 120 C interestingly indicated a significant reduction (B99%) in the levels of detectable gluten protein by the R5-ELISA technique. Gliadins in wheat proteins are tested for their ability to bind a specific monoclonal antibody, R5, using the high sensitivity of R5-ELISA (Valde´s et al., 2003) to monitor residual gluten levels as low as 3.2 ppm in all commercial gluten-free foods. Lamacchia et al. (2016) found that the amount of gluten detected significantly decreased from 10% to 12% in untreated flour to 60 and 40 ppm in microwave-treated durum and soft wheat grains, respectively. Treated flours did not induce production of the inflammatory cytokine, interferon gamma, when tested, after digestion and deamidation, on gut-derived human T-cell lines of celiac patients highly reactive to 33-mer, omega, and gamma peptides (Lamacchia et al., 2015). However, microwave treatment (1000 W for 1, 5, and 10 minutes) of the wheat flours, as studied by Kwak et al. (2012), increased the antigenicity of gliadins (measured by antigliadin IgG to bind gliadins) as the treatment time (from 1 to 10 minutes) and protein denaturation increased. It is hypothesized that reduced antigenicity of the whole wheat grains, when treated with microwave energy, is due to their minimal denaturation in their native matrix and induced chemical changes which otherwise are impossible in the already formed gluten. Anugu et al. (2010) reported that pulsed UV light treatment allowed the allergenicity of isolated egg white proteins to be reduced. However, Manzocco et al. (2012) did not find any difference in the immunoreactivity between egg white exposed to UV light and untreated egg white. Ovalbumin and ovomucoid, which are known to be the major allergens of egg white were shown to be barely sensitive to UV radiation. The linear sequence of amino acids within a given protein/peptide is a primary contributor to its potential to be either bioactive and/or allergenic. The main health concern is allergenic reactions of food proteins and most food allergenic portions of a protein, referred to as epitopes (linear or conformational), bind IgE and cause cross-linking on the surface of mast cells resulting in allergic response. The so-called big eight foods associated with allergenic reactions are eggs, fish, shellfish, milk, peanuts, soy, tree nuts, and wheat (Foegeding and Davis, 2011). Heat treatment can potentially reduce the allergenicity of some of the food products. In one of the studies, children with cow’s milk allergy tolerated extensively heated milk in the form of muffins or waffles. Immunological evidence suggests that patients who tolerated the extensively heated milk proteins primarily reacted to conformational epitopes, whereas patients who did not tolerate the extensively heated milk proteins primarily reacted to sequential or linear epitopes (Nowak-Wegrzyn et al., 2008). In peanuts, dry roasting enhances the IgE binding capacity of peanut proteins as compared to raw and boiled peanuts. The Maillard reaction, during dry roasting, results in protein/sugar adjuncts with enhanced reactivity as opposed to the boiled peanuts,

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wherein high moisture content prevents the occurrence of the Maillard reaction, and thus reduced allergenicity (Schmitt and Turgeon, 2011). Novel approaches such as the hydrolytic ability of new strains of bacteria, such as Lactobacillus plantarum, in the fermentation of sourdough to reduce gluten-allergen compounds have been identified and assessed by Gerez et al. (2006).

10.6 EFFECT OF STORAGE ON PROTEIN STABILITY The protein quality of the final product depends on the reactions that take place during storage, processing, transportation, circulation, and consumption. It has been emphasized that all chemical reactions are temperature dependent and any initiated during processing will continue to take place during storage. Melanoidins in food may also serve as preservatives and protect the quality of food during storage. In foods, the antioxidant properties of melanoidins can inhibit the oxidation of unsaturated lipids and functional food ingredients, such as vitamins, polyphenols, and flavonoids. Moreover, they can inhibit the growth of microorganisms and prevent the spoilage and deterioration of foods (Wang et al., 2011). Refrigerated storage maintains the UHT treated milk product as acceptable for a long period, however, UHT products do not require refrigeration and are shelf stable at ambient temperatures. Two types of deterioration have been reported in stored UHT milk; coagulation/separation/sedimentation owing to age gelation and sedimentation, and brown discoloration due to the Maillard reaction (Deeth and Lewis, 2017). Sometimes, native proteases and plasmin and bacterial proteases survive the high heat treatment and remain active during storage at room temperature instigating proteolysis and eventually leading to bitter flavor and gelation during storage. Oxidative reactions during storage lead to the deterioration in a number of properties of meat and meat products, such as loss of tenderness, flavor, texture, color, and nutritive value. Protein modifications are mostly categorized as denaturation, proteolysis, increase in carbonyl groups, polymerization, and protein oxidation (Fuentes et al., 2010; Kemp et al., 2010; Lund et al., 2007; Van Laack et al., 2000). In oxidation, the formation of reactive oxygen is inevitable due to aerobic metabolism. The total protein carbonyl content is approximately 1
2 nmol/mg protein in a wide variety of animal tissues (Requena et al., 2001), but after slaughter, certain biochemical changes occur that enable oxidation of meat (Morrissey et al., 1998). Past research shows that storage has a significant impact on the oxidation of meat proteins and their content increased from 1
2 to 3.1
5.1 nmol/mg protein for beef longissimus lumborum and 4.8
6.9 nmol/mg protein for beef diaphragma pedialis (Martinaud et al., 1997). Similarly, pH measured 2 days post mortem was 5.53 6 0.06 for beef cuts used in an experiment which promoted carbonylation after 5, 10, and 15 days of storage significantly (Lindahl et al., 2010). Another form of storage of meat products involves chilling and freezing to enhance shelf life and quality. Freezing meat also enables increased storage time and greater inventory and product control (Pietrasik and Janz, 2009). Freezing of meat impedes undesirable biochemical reactions but may lead to cell disruption and muscle fiber change due to ice crystals (Sebranek, 1982). Soyer et al. (2010) reported that protein carbonyls in chicken

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meat increased significantly as a result of 6-month frozen storage at
18 C (from 1.78 nmol/mg protein to 2.88 nmol/mg protein). The extent of protein carbonylation in storage is significantly dependent upon the origin of meat, the species, type of muscle, and storage conditions (Filgueras et al., 2010; Sante´-Lhoutellier et al., 2008; Gatellier et al., 2000). Carbonylation is also dependent upon the freezing temperature (Soyer et al., 2010), packaging conditions, and related operations such as premincing (Este´vez et al., 2011). Aging/chill storage of beef, pork, poultry, turkey, lamb, and ostrich meat leads to protein carbonylation. The extent of protein carbonylation is highly dependent on the origin of the meat, the type of muscle, the species, and the storage condition; beef has been found to be more susceptible to protein carbonylation than pork (Este´vez, 2011). Protein oxidation in dried minced pork slices showed significantly higher content of protein carbonyls after 20 days of storage (Xu et al., 2018). The carbonyls accumulation was attributed to the oxidative side chain degradation of histidine, arginine, proline, and lysine residues. Oxidation of meat has also been observed in a frozen state for pork (Este´vez, 2011; Xia et al., 2009), beef (Popova et al., 2009), and poultry (Soyer et al., 2010; Rababah et al., 2010). Soyer et al. (2010) reported a significant increase in protein carbonyls after 6 months of frozen storage (
18 C), while Xia (Xia et al., 2009) also reported a significant increase in porcine protein carbonyls. The carbonylation process or oxidation through reactive oxidative species is dependent upon the type of muscle (Este´vez, 2011; Soyer et al., 2010), the temperature (Soyer et al., 2010), and packaging and processing conditions (Este´vez, 2011). Protein carbonylation is also greatly affected by processing technologies like irradiation, cooking, ripening, fermentation, and pressure. Irradiation induces a large amount of reactive oxidative species, initiating oxidation while irradiation effects have been discussed in detail for different meats such as raw meat (Martinaud et al., 1997), poultry (Rababah et al., 2010), and sausages (Badr and Mahmoud, 2011). Freezing results in physical changes which could create different stresses that impact protein stability (Bhatnagar et al., 2007). Wagner and Anon (1985) studied the denaturation effect on myofibrillar proteins of bovine muscle and found that after thawing, the slow-freezing muscle tissue had the higher water loss. The denaturation observed during slow freezing was attributed to the pronounced partial unfolding of the myosin head. Commercially, shrimps are frozen by two methods: (1) glazing with water followed by freezing in an air-blast freezer; and (2) individually frozen using liquid CO2 or N2. They might be thawed and refrozen during transportation and repackaging, and multiple freeze
thaw cycles (Boonsumrej et al., 2007) pose challenges in maintaining the quality of the frozen shrimps. Denaturation of proteins is caused by the interaction of free fatty acids with salt soluble protein, reducing the levels of salt soluble proteins, and thus lowering overall protein solubility. In addition, myosin denaturation, as well as cross-linking and aggregation of myofibrillar proteins, leads to toughness of the frozen shrimp. The oxidation of myofibrillar proteins in fish over freeze
thaw cycles during storage, negatively affected the textural properties, such as tenderness, and resulted in a deterioration in quality (Lund et al., 2011). Myofibrillar proteins play a vital role in determining the quality of fish products due to their water holding capacity and bonding force (Chapleau et al., 2004). Controlling protein oxidation in fish products is particularly important in commercial industries as it leads to muscle toughness,

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10.7 CONCLUSION

321

reduced solubility, conformational changes, and the loss of functional properties. The freeze
thaw cycles decreased the freshness and textural properties whilst accelerating protein oxidation (Shao et al., 2018). White-shelled eggs, during storage at 22 C, had altered egg white proteins and lost their viscous nature to form a thin liquid, commonly referred to as egg white thinning (Omana et al., 2010). Egg white thinning can be observed visually after 20 days of storage and egg white proteins such as ovalbumin underwent clustering and degradation during storage. Thinned egg white is more prone to microbial infections and there is a change in the desirable functional properties of egg albumen, such as heat coagulation and the ability to form stable foams during whipping (Robinson, 1987; Scha¨fer et al., 1999; Silversides and Budgell, 2004), which is why egg white thinning is regarded as a sign of loss of quality that leads to staleness, eventually leading to its spoilage. In a recent study, Liu et al. (2018) characterized the formation of peptides in egg white during prolonged storage. The peptides were identified to be fragments of major egg white proteins, ovotransferrin, ovomucin, ovomucoid, and ovoinhibitor (antiproteases). Additionally, they observed rapid degradation of gallin/ovodefensin during storage. A study published by Primacella et al. (2018) showed that egg yolk remains dynamic at 220 C and gelation occurs not only during freezing and thawing, but also during extended freezing storage. Protein aggregation, reflected through the shift of particle size distribution towards larger particle size during freezing, has been attributed to multiple factors, such as the concentration of yolk components and dehydration of LDL micelles due to the formation of ice crystals, or the exposure of previously inaccessible hydrophobic regions due to a change in protein structure when pH or ionic strength were altered.

10.7 CONCLUSION Different proteins are present in food systems and they experience different influences based on their amino acid composition, their presence in inner or outer food matrices, their interaction with other food components, and ultimately affecting their native and denatured polymeric state. Physical and chemical changes such as protein
protein interactions, coaggregation among proteins, interactions with other molecules, such as polysaccharides or a receptor to activate biological function, will all affect protein functionality. This chapter outlined the fluctuations that proteins experience during food processing and storage as well as how these changes impact the nutritional, digestibility, and physiological properties of the food proteins. Novel processing technologies are mild and limit protein alterations; however, we recommend that a holistic approach is required in the food chain to prevent negative impacts on protein quality throughout the food chain from raw material acquisition through transport, processing, packaging, and storage to consumption.

Acknowledgment Authors would like to acknowledge Dr. Ciara McDonagh’s contribution in proofreading this manuscript.

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6.

PROTEINS: SUSTAINABLE SOURCE, PROCESSING AND APPLICATIONS

Index Note: Page numbers followed by “f” and “t” refer to figures and tables, respectively.

A AAS. See Amino acid score (AAS) Abiotic surfaces, 274 275 Accessibility of substrate proteins and peptides, 230 231 ACE. See Angiotensin I-converting enzyme (ACE) Acid hydrolysis of fish protein, 166 Acid precipitation, 110 Acrylamide, 304 305 Active substances from A. platensis and P. cruentum, 78 81 AD. See Alzheimer’s disease (AD) Additives, 306 Affinity chromatography, 200, 282 284 African cricket (Brachytrupes membranaceus), 43 African oil palm (Elaeis guineensis), 115 116 African palm weevil (Rhychophorus phoenicis), 43 AgE´ncia Nacional de Vigilaˆncia Sanita´ria (ANVISA), 86 Aggregate formation, 230 231 Aggregation, 170, 299, 308 Agricultural land requirements, 15 Alanine acid, 304 Alaska pollack (Theragra chalcogramma), 173 Albumins, 105 106, 138 Alcalase, 142, 229, 306 307 Alcohol oxidase 1 (AOX1), 271 Algal/algae, 90 physicochemical and technofunctional of, 81 82 protein application for human consumption, 82 84 Spirulina as superfood, 83 84 proteins, 72, 76 toxins, 87 88, 88t Alkali-extracted BSG proteins, 108 109 Alkaline extraction method, 110 hydrolysis of fish protein, 166 167 Allergenic proteins, 112 Allergenicity, 42 Allergens, 47 Alpha casein, 142 Alpha mating factor (αMF), 273 α-lactalbumin (α-La), 194 195, 307 309 Alternative animal product scenarios, 15 16, 20 22 Alternative diet scenarios, 23 Alzheimer’s disease (AD), 267 268

Amino acid score (AAS), 97 98 Amino acids, 72 74, 73t, 98, 105, 304 composition analysis, 247 in fish, 182 of R/C proteins, 113 of whey proteins, 214 Aminobutyric acid, 304 AML. See Amylose lipid (AML) Amnesic shellfish poisoning (ASP), 87 Amphipathic peptides, 114 AMPs. See Antimicrobial peptides (AMPs) Amylose lipid (AML), 178 179 Anabaena variabilis, 75 76 Anaphylaxis, 46 Androgens, 47 Angiotensin I-converting enzyme (ACE), 107, 173 ACE-inhibitory activity, 150 complex, 109 inhibitors, 141 142 Animal by-products, 132, 231 procedures in protein-based ingredient production, 136 149, 137f blood transformation processes, 138 140 feather meal production, 137 138 gelatin production, 136 137 protein isolates, hydrolysates, and bioactive peptides, 140 149 Animal feed and pasture, allocating areas for, 6 7 Animal nutrition, 6 Animal products, 4, 10 11 alternatives to current, 12 15 aquaculture, 14 15 cultured meat, 13 14 imitation meat, 14 insects, 13 yields of alternatives to, 20 Anthropogenic carbon dioxide emissions, 2 Antibiotics resistance, 261 Antibodies, 41 42 Anticancer, whey proteins role in, 214 Antidiabetics, whey proteins role in, 213 Antihypertensive/ACE, 174 175 Antimicrobial peptides (AMPs), 46, 261, 268 273 fusion technology for recombinant AMP production and purification, 272 273

331

332

INDEX

Antimicrobial peptides (AMPs) (Continued) generic strategies for increasing recombinant AMPs expression, 271 272 heterologous expression systems in recombinant AMPs production, 269 271, 270t Ants (Atta), 44 ANVISA. See AgE´ncia Nacional de Vigilaˆncia Sanita´ria (ANVISA) AOX1. See Alcohol oxidase 1 (AOX1) Aquaculture, 14 15, 18, 42 Aromatase, 47 Arthrospira platensis, 64, 78 81, 79f Arylphorin, 46 ASP. See Amnesic shellfish poisoning (ASP) Asparagine (Asn), 302 Astaxanthin, 66 Atta. See Ants (Atta) Autofocusing technique, 149

B Bacillus species, 173 B. anthracis, 274 B. cereus, 153 B. subtilis A26, 167 168 B. subtilis expression hosts, 270 271 Bacteria, 48, 174 Bacterial expression systems, 270 271 Bacteriophages, 262, 266, 266f BallTec Dryer, 139 Barley proteins, 107 109 BBB. See Blood brain barrier (BBB) BD-1. See β-defensin-1 (BD-1) BDB. See Biopanning data bank (BDB) Bead milling, 77 Beef muscle hydrolysates, 180 181 Beef production, 27 Beetle larvae (Rhynchophorus), 44 β-conglycinin, 111, 307 β-defensin-1 (BD-1), 174 β-glucan, 318 β-lactoglobulin (β-Lg), 194 195, 198 199 β-lactoglobumin, 307 310 Bioaccessibility, 72 Bioactive molecules, 74 Bioactive peptides, 140 149, 143t, 146t, 148t, 164, 212 isolation and identification of, 247 248 Bioactive properties of cattle viscera, 151 of collagen, 150 of fish protein hydrolysate, 170 175 of gelatin, 151 153 of protein hydrolysates, 155 Bioactivity, 72, 114

Bioassay-guided fractionation, 247 248 Bioavailability, 72 Bioenergy, 2 Biogenic toxins, 87 Bioinformatics, 260, 278 Biological processes, 70 Biological structures, 13 Biological value (BV), 70, 71t Biomass characterization, 231 236 classical methods, 232 234 spectroscopic methods, 234 236 Biomaterials, 267 Biopanning, 262 264 Biopanning data bank (BDB), 266 BioPep database, 248 250 Biopolymeric fibers, 151 153 Biosafety evaluation, 90 Biotic surfaces, 274 275 Black ants (Carebara vidua), 43 Black chafer beetle (Holotrichia parallela), 43 Black soldier fly (Hermetia illucens), 42 Bligh method, 233 234 Blister beetles (Cantharis vesicatoria), 42 Blood, 140, 150 plasma, 150 protein percentages in by-products from meat and poultry industry, 134t as source of serum albumin, 133 135 transformation processes, 138 140 Blood brain barrier (BBB), 267 268 bLp. See Bovine lactoperoxidase (bLp) Blue-green algae (Cyanobacteria), 14, 67 Bogong moth (Agrotis infusa), 44 Bone as sources of collagen, 135 136 Bovine albumin, 140 meat, 14 15 Bovine lactoperoxidase (bLp), 199 Bovine serum albumin (BSA), 194 195, 308 309 Brewers’ spent grain (BSG), 108 109 5-Bromo-4-chloro-3-indolyl-β-D-galactoside (X-gal), 265 BSA. See Bovine serum albumin (BSA) BSG. See Brewers’ spent grain (BSG) Burnet moths (Zygaena filipendulae), 47 BV. See Biological value (BV) BVL-I, 284 285

C C-terminal carboxylate (COO ), 243 C-terminal SH3b CBD, 277 5-O-Caffeoylquinic acid, 115 CAI. See Codon adaption index (CAI) Calcium, 310

INDEX

calcium-dependent serine protease, 75 76 Calorimetric experiments on proteins, 298 299 Calorimetry, 240 Cambodia roasted crickets, 43 Cancer, 171 research, 267 Candida utilis, 153 Capillary electrophoresis (CE), 239 240 Carbohydrate-binding modules (CBMs), 272 273 Carbohydrates, 310 Carbonylation, 302, 314, 315f, 319 320 Carboxymethylcellulose (CMC), 198 199 Cardiac muscle, 174 175 Cardiovascular diseases (CVDs), 164 prevention, 214 Carnivorous fish, 14 15 Carpenter ant (Camponotus spp.), 44 Carrier peptides/proteins, 272 273 Cartilage as sources of collagen, 135 136 Casein micelle, 307 Caseinomacropeptide (CMP), 310 311 Cashew tree (Anacardium occidentale L.), 118 meal, 118 Castor bean (Ricinus communis), 119 Catalysts, 75 Catalytic membranes, 203 Catfish production, 14 15 Cattle viscera, 151 CBD. See Cell wall binding domain (CBD); Chitinbinding domain (CBD) CBMs. See Carbohydrate-binding modules (CBMs) CD. See Circular dichroism (CD) CE. See Capillary electrophoresis (CE) Cell cell-disruption techniques, 76 cell-free expression systems, 271 culture, 14 disruption technique, 77 78 Cell wall binding domain (CBD), 275 276 Cellulose, 75 Central Amidase-2 domain, 277 Cereals, 303 proteins, 303 305 and pseudocereal proteins barley proteins, 107 109 quinoa proteins, 109 111 rice bran proteins, 103 104 sorghum proteins, 104 107 wheat bran proteins, 104 CFR. See Code of Federal Regulations (CFR) CFUs. See Colony forming units (CFUs) CGAs. See Colloidal gas aphrons (CGAs) Changing dietary patterns, 9 12, 11f, 12f

333

CHAP domain. See Cysteine-and histidine-dependent amidohydrolase/peptidase domain (CHAP domain) Cheese making, 196 Chemical aggregation, 300 cell disruption, 70 71 chemically induced changes in proteins, 298f, 300 302 covalent aggregation, 300 maillard browning, 300 302 oxidation and deamidation, 302 composition of microalgae, 69 76 amino acids, 72 74 enzymes, 75 76 peptides, 74 75 protein, 70 72 hydrolysis, 166 167 precipitation method, 198 199 reactions, 308 synthesis processes, 269 Chemical score (CS), 74 Chenopodina, 110 Chicken hydrolysates, 180 181 Chicken liver hydrolysates (CLHs), 180 181 Chinese Microalgae Industry Alliance (CMIA), 86 Chitin-binding domain (CBD), 47 48, 273 Chlorella, 65, 89 90 C. vulgaris, 64 Chocolate-coated roasted crickets or mealworms, 50 Chromatography, 76 methods, 245 247 separation, 199 200 ChtBD3. See Chitin-binding domain (CBD) Chymosin, 310 311 Ciguatera fish poisoning, 87 Circular dichroism (CD), 284 285 Circulatory system, 142 Classical methods, 232 234, 236 239 fat/lipid measurements, 233 234 protein measurements, 232 233 Cleavable self-assembling tags (cSATs), 273 CLHs. See Chicken liver hydrolysates (CLHs) Climate change, 7 CM. See Cottonseed meal (CM) CMC. See Carboxymethylcellulose (CMC) CMIA. See Chinese Microalgae Industry Alliance (CMIA) CMP. See Caseinomacropeptide (CMP) Coarse gels, 300 Cochineal insects (Dactylopius coccus), 42 Cockroaches (Blatella germanica, Periplaneta Americana), 46

334 Code of Federal Regulations (CFR), 131 132 Codex Alimentarius Commission, 86 Codon adaption index (CAI), 280 281 Codon optimization, 280 281 Cold denaturation, 298 299 gelation, 299 300 Collagen, 133 bone, cartilage, and skin as collagen sources, 135 136 production, 151 Colloidal gas aphrons (CGAs), 204 Colony forming units (CFUs), 265 Color, 176 177 Combustion methods, 233 Commercial large-scale production, 64 Constitutive promoters, 271 Conventional animal product consumption, 3 4 Conventional livestock consumption changes, 18 production, 17 Conventional microalgae production system, 65 Conventional protein production, 41 42 Conversion factors, 232 Cooking, 313 314 in boiling water, 305 Corn, 178 179 Cossid moth (Xyleutes leucomochla), 44 Cotton (Gossypium hirsutum L.), 114 proteins, 114 115 Cottonseed meal (CM), 114 Covalent aggregation, 300 Cow pea, 305 COX-2. See Cyclooxygenase-2 (COX-2) CPTASNTSC peptide, 267 Cricket powder, 50 Crustaceans, 42 Cryogen-cooled superconducting magnets, 241 Cryoprotectants, 183 CS. See Chemical score (CS) cSATs. See Cleavable self-assembling tags (cSATs) Cucurbitaceous seeds (Cucurbita maxima), 118 119 Culture system, 64 Cultured meat, 3, 13 14, 17, 25t and energy, 28 Curing, 314 CV-N. See Cyanovirin-N (CV-N) CVDs. See Cardiovascular diseases (CVDs) Cyanobacteria, 67, 279 Cyanovirin-N (CV-N), 74 75 Cyclooxygenase-2 (COX-2), 171

INDEX

Cysteine, 302 Cysteine-and histidine-dependent amidohydrolase/ peptidase domain (CHAP domain), 277 Cytochrome P450 enzyme, 47

D Dairy milk alternatives, 307 DC. See Digestibility coefficient (DC) Deamidation, 302, 306 Decolorization, 139 Decolorized blood, 138 139 Degree of hydrolysis (DH), 164 165, 227, 236 237 Dehulling process, 305 Dehusking, 303 304 Demasking, 230 “Demitarian” diet, 15 Democratic Republic of Congo (DRC), 49 Denaturation, 296 299 Dephenolized protein, 115 DF. See Diafiltration (DF) DH. See Degree of hydrolysis (DH) DIAAS. See Digestible indispensable amino acid score (DIAAS) Diafiltration (DF), 200 201 Diarrheic shellfish poisoning (DSP), 87 Diatoms (Bacillariophyceae), 67 Dietary change scenarios, 18 19 proteins, 41 42, 317 Digestibility coefficient (DC), 70, 71t Digestibility of food proteins, processing effect on, 316 Digestible indispensable amino acid score (DIAAS), 98 Dipeptidyl peptidase IV (DPP-IV), 150, 179 180 Disgust, 44 46 DM. See Dry matter (DM) DPP-IV. See Dipeptidyl peptidase IV (DPP-IV) DRC. See Democratic Republic of Congo (DRC) Dry matter (DM), 6, 16t Dry-film FTIR analysis, 243 244 Dry/wet milling, 303 304 Drying, 206 208, 314 freeze drying, 207 208 method, 151 153 spray, 206 207 DSP. See Diarrheic shellfish poisoning (DSP) DTFNSFGRVRIE peptide, 267 Dumas method, 232 Dunaliella salina, 64 Dyer method, 233 234

INDEX

E EAA. See Essential amino acids (EAA) EAAI. See Essential amino acid index (EAAI) EAD. See Enzymatically active domain (EAD) EAE. See Enzyme-assisted extraction (EAE) EBA-IEC. See Expanded bed absorption-ion exchange chromatography (EBA-IEC) EC. See Enzyme commission (EC) Economic approaches, 30 Edible films and coatings, whey protein application as, 216 Edible insects, 42 preparations, 44, 45f Edible weight (EW), 6, 16t EDTA, 138 139, 310 Efficient aquacultural product, 25t Efficient conventional animal products, 25t EFSA. See European Food Safety Authority (EFSA) Egg proteins, 314 316 Egg white, 315 hydrolysates, 179 180 thinning, 321 Egg yolk, 315 316 Elastin-like polypeptides (ELPs), 273 Electrodialysis, 203 204 Electrophoresis, 149, 239 240 ELPs. See Elastin-like polypeptides (ELPs) Elution, 246 Emperor moth (Gonimbrasia belina), 43 Emulsification, 177, 307 Endolysins, 274 280 to control pathogenic bacteria, 274 275 improving activity of endolysins by protein engineering, 275 277, 276f improving solubility of endolysins by protein engineering, 277 278 structure for protein engineering, 275 as tools in heterologous protein production and purification, 278 280 Endopeptidases, 169 Endotoxin production, 260 Energy, 20, 21f cultured meat and, 28 Enterococcus faecium NCIM5335, 167 168 Entomophagy, 42 43 Enzymatic cell lysis, 70 71 deamidation, 107 globin peptide, 150 hydrolysis, 75, 153, 165 166, 167f treatments, 306 Enzymatic protein hydrolysis (EPH), 226, 306 307 biomass characterization, 231 236

335

factors influencing, 227 231 accessibility of substrate proteins and peptides, 230 231 inhibitors, 229 protease specificity, 231 protease stability, 229 reaction progression curves, 228f future perspectives, 250 251 process characterization and monitoring, 236 245 calorimetry, 240 classical methods, 236 239 electrophoresis, 239 240 infrared spectroscopy, 243 245 NMR spectroscopy, 241 242 processing steps in, 226f product characterization, 245 250 Enzymatically active domain (EAD), 275 Enzyme commission (EC), 240 Enzyme-assisted extraction (EAE), 75 Enzymes, 41 42, 75 76 enzyme-induced gelation, 299 300 hydrolysis, 182 proteases, 75 76 EPH. See Enzymatic protein hydrolysis (EPH) Epitopes, 318 319 Escherichia coli, 168, 270 271 filamentous phages, 262 phage display-derived proteins/peptides in, 264 265 Essential amino acid index (EAAI), 74 Essential amino acids (EAA), 135 Estrogens, 47 Ethnic and modern entomophagy practices, 42 44 Eukaryotic organisms, 64 European eels (Anguilla anguilla), 154 155 European Food Safety Authority (EFSA), 86 EVQSSKFPAHVS peptide, 267 EW. See Edible weight (EW) Existing diets with high and low animal products, 29 30 Exogenous wealth-based factor, 3 Expanded bed absorption-ion exchange chromatography (EBA-IEC), 113 Extraction of proteins, 78 81 Extrusion, 17

F F-pilus-specific phages (Ff), 262 Fat, 13 determination, 233 234 measurements, 233 234 FDA. See US Food and Drug Administration (FDA) Feathers, 136

336

INDEX

Feathers (Continued) meal production, 137 138 Fed-batch mode, 282 Feed conversion factors, 6 7, 6t ratios, 15, 22 feed-forward approach, 251 Fermentation, 316 hydrolysis, 167 168 strategy, 281 Fibrimex, 140 Fibrinogen, 140 Ficin, 165 166 Filamentous phages, 262 Find stranded gels, 300 Fish peptides, 164 viscera, 154 Fish processing by-products. See also Meat processing by-products nutritional, functional, and physicochemical properties of FPI, 175 177 proteins from, 154 155 recovery methods of fish protein from, 165 169 chemical hydrolysis, 166 167 enzymatic hydrolysis, 165 166 fermentation hydrolysis, 167 168 ISP, 168 169 Fish protein hydrolysates (FPHs), 164 165. See also Plant protein hydrolysates bioactive properties, 170 175 antihypertensive properties, 174 175 antimicrobial properties, 174 antioxidant properties, 171 173 antiproliferative properties, 171 current and future application, 182 184 differences between FPHs and other protein hydrolysates, 177 181 food protein hydrolysates, 179 181 plant protein hydrolysates, 177 179 physicochemical challenges associated with FPI and, 181 182 structural and functional properties, 169 170 Fish protein isolate (FPI), 168, 175 functional properties, 176 177 nutritional properties, 175 176 physicochemical challenges associated with FPHs and, 181 182 physicochemical properties, 176 177 “Fish-to-fish” conversion ratios, 14 15 Fishery, 42 Fixed global average production, 27

Flavourzyme, 306 307 Fluorescence spectroscopy, 284 285 Fodder production, 2 Folch method, 233 234 Food food-grade enzymes, 75 human appropriation of land for, 4 7 intercountry variation in appropriation of land for, 7 9 neophobia, 44 46 nutrients, 22 processing, 295 296 proteins in, 98 101 production, 27 28 protein hydrolysate chicken and beef muscle hydrolysates, 180 181 egg white hydrolysates, 179 180 safety, 89 security, 2 3 systems, 3, 295 296, 303 Food commodities, allocating areas for, 5 Food industry, whey protein applications in, 215 216 Formol titration, 237 238, 238f Fourier-transform infrared spectroscopy (FTIR), 234 236, 243 FPHs. See Fish protein hydrolysates (FPHs) FPI. See Fish protein isolate (FPI) Fractionation, 113 Free-radical scavenging, 172 Freeze drying, 207 208, 306 Freezing, 315 316 Fried honey bees (Apis sp.), 43 FTIR. See Fourier-transform infrared spectroscopy (FTIR) Functional component in food, 142 Functional properties, 110, 142 of BSG proteins, 108 109 of cashew protein, 118 of fish protein hydrolysate, 169 170 of fish protein isolates, 176 177 of peanut meal flour, 117 of R/C proteins, 114 of rice bran proteins, 109 of sorghum proteins, 106 107 Fusion expression, 272 technology for recombinant AMP production and purification, 272 273

G GAP promoter, 271 Gastrointestinal system, 142 Gel, 299

INDEX

Gel filtration. See Size exclusion chromatography (SEC) Gelatin, 135 136, 150 production, 136 137 Gelation, 177, 299 300 Generally Recognized as Safe (GRAS), 82 83 Genetically modified algae, 89 90 Genetically modified organisms (GMOs), 279 GHG. See Greenhouse gas (GHG) GIP. See Glucose-dependent insulinotropic polypeptide (GIP) GlcNAc. See N-acetylglucosamine (GlcNAc) Gliadin, 318 Global average production system, 8 9 Global consumption per capita, 12 Global marine biotechnology market, 66 Global wheat production, 66 Global whey protein market, 217 Globulins, 105 106, 114 Glucagon-like peptide 1 (GLP-1), 150 Glucose-dependent insulinotropic polypeptide (GIP), 150 Glucose glycerol-shift, 281 Glutamic acid, 304 Glutamine (Gln), 302 Glutathione deficiency, 215 Glutathione S-transferase (GST), 272 273 Glutelins, 105 Glycerol, 310 Glycine acid, 304 Glycinin, 111 Glycosylation, 284 285 GMOs. See Genetically modified organisms (GMOs) Golden algae (Chrysophyceae), 67 Grain milling, 316 Gram-negative bacteria, 275 Gram-positive bacteria, 275 GRAS. See Generally Recognized as Safe (GRAS) Green algae (Chlorophyceae), 67 Greenhouse gas (GHG), 2 Groundnut. See Peanut (Arachis hypogaea L.) GST. See Glutathione S-transferase (GST)

H HACCP. See Hazard Analysis & Critical Control Points (HACCP) Haematococcus pluvialis, 64 HALF index. See Human Appropriation of Land for Food index (HALF index) Hazard Analysis & Critical Control Points (HACCP), 86 HCV. See Hepatitis C virus (HCV) HDL. See High-density lipoprotein (HDL) Healthcare applications of whey proteins, 212 215

337

anticancer, 214 antidiabetic, 213 antiinflammatory and antioxidant, 212 213 cardiovascular disease prevention, 214 hepatitis treatment, 213 immunomodulation, 214 215 infant and expected mothers nutrition, 213 wound healing, 213 Healthy diet, 31 32 Heat heat-stable bonds, 312 precipitation method, 197 198 stability of proteins, 310 treatment, 307 Heavy metals, 88 89 Hemagglutinationassays, 284 285 Hemicellulose, 75 Hemp seed (Cannabis sativa), 118 119 Hepatitis C virus (HCV), 213 Hepatitis treatment, 213 Herring (Clupea harengus), 173 Heterologous expression systems in recombinant AMP production, 269 271, 270t High animal product diet, 18 19, 25t existing diets, 29 30 High hydrostatic pressure processing, 149 High pressure denaturation of myosin, 313 of protein, 312 313 High-density lipoprotein (HDL), 179 180 High-performance liquid chromatography in reverse phase (RP-HPLC), 142 149, 246 High-throughput techniques, 260 His-tag. See Polyhistidine tag (His-tag) HIV. See Human immunodeficiency virus (HIV) Honeypot ant (Melophorus bagoti), 44 Hormones, 41 42 Horseshoe crab (Limulus polyphemus), 42 House dust mites (Dermatophagoides pteronyssinus), 46 HSA. See Human serum albumin (HSA) Human appropriation of land for food, 4 7 allocating areas for animal feed and pasture, 6 7 allocating areas for food commodities, 5 assessing land use impact of different diets, 7 consumption, 23, 42 algal protein application for, 82 84 health risks, 90 nutrition, 65 proteins, 98 101 requirements, 30 Human Appropriation of Land for Food index (HALF index), 3 4, 9f, 24t, 26f

338

INDEX

Human immunodeficiency virus (HIV), 215 Human serum albumin (HSA), 273 Hybrid approaches, 269 Hydrogels, whey protein and, 216 217 Hydrolysates, 164, 166 167 of rice bran proteins, 104 Hydrolysis, 164 165, 170, 241 chemical, 166 167 enzymatic, 165 166, 167f fermentation, 167 168 of proteins, 230 Hydrophilic groups, 216 217 Hydrophobic amino acids, 172 173 Hypermannosylation of proteins, 260 261

I IL. See Interleukin (IL) Imitation meat, 14, 17, 27 28 Immobilized metal affinity chromatography (IMAC), 280 Immune system, 142 Immunoglobulins (Ig), 194 195, 307 Immunomodulation, 171, 214 215 Immunotoxin, 47 In vitro meat. See Cultured meat Inducible promoters, 271 Infrared spectroscopy, 243 245 Inhibitors, 229 Insects, 13, 25t, 29 allergy, 47 48 consumption, 17 exoskeleton chitin, 47 insect-based foods, 48, 48f insects-based protein and solutions, 44 48 as source of sustainable proteins common edible insects and common names, 51t cricket powder, 50 ethnic and modern entomophagy practices, 42 44 insects-based protein and solutions, 44 48 sustainable food source, 49 Intercountry variation in appropriation of land for food, 7 9, 8f Interleukin (IL), 50 IL-beta cells, 171 Ion-exchange chromatography, 199 Ionic liquids, 78 IPTG. See Isopropyl-β-D-thiogalactoside (IPTG) Iron bioavailability, 151 Isoelectric point (pI), 168 Isoelectric precipitation, 305 306 Isoelectric solubilization and precipitation (ISP), 140, 168 169 Isoforms, 280

Isoleucine, 135 Isopropyl-β-D-thiogalactoside (IPTG), 265 Isothermal titration calorimetry, 240 Isotope analysis, 13 ISP. See Isoelectric solubilization and precipitation (ISP)

J Jackfruit (Artocarpus heterophyllus), 119 Japan External Trade Organization (JETRO), 86 Jatropha curcas, 119 Joint FAO/WHO Expert Committee on Foods Additives (JECFA), 89 Juvenile turbot (Scophthalmus maximus), 183

K Kafirins, 105, 107 κ-casein, 308 311 Keratin, 133 sources, 136 Ketosteroid isomerase (KSI), 273 Kidney bean hydrolysates, 179 Kjeldahl method, 232 233 Krill (Euphausia superba), 42 KSI. See Ketosteroid isomerase (KSI)

L LAB. See Lactic acid bacteria (LAB) Lab-based meat. See Cultured meat Lacprodan, 226 Lactic acid bacteria (LAB), 167 168 Lactobacillus helveticus, 142 Lactobacillus plantarum, 318 319 Lactobacillus reuteri strains, 304 Lactoferrin (LF), 194 195 Lactoperoxidase (LP), 194 195 Lactose removal, 197 Land, 2 human appropriation of land for food, 4 7 intercountry variation in appropriation of land for food, 7 9 land-based production, 18 requirements, 15 32 alternative animal product scenarios, 15 16, 20 22 alternative diet scenarios, 23 aquaculture, 18 comparisons to previous studies, 31 32 conventional livestock consumption changes, 18 cultured meat, 17, 28 existing diets with high and low animal products, 29 30

INDEX

imitation meat, 17, 27 28 insects, 17, 29 limitations of analysis, 24 27 obesity, malnutrition, and waste, 30 31 results, 23 24, 25t ruminants, 32 soybean production, 27 28 uncertainty quantification, 19 20 waste and dietary change scenarios, 18 19 yields of alternatives to animal product, 20 Land use impact assessment of different diets, 7 Lao People’s Democratic Republic (Lao PDR), 49 Large-scale outdoor microalgae cultivation, 68 69 LC-MS/MS. See Liquid chromatography mass spectrometry (LC-MS/MS) LCA approach. See Life cycle analysis approach (LCA approach) Least efficient conventional animal product, 25t Lectins, 261, 280 285 molecular cloning strategy for recombinant production, 280 282 Pichia processing impact on lectin properties, 284 285 recombinant lectin production in P. pastoris and purification strategies, 282 284 Legumes, 303 seeds, 305 LF. See Lactoferrin (LF) Life cycle analysis approach (LCA approach), 3 Lignin, 75 Lipid oxidation, 313 Lipocalin, 46 Liquid analysis approach, 244 245 Liquid chromatography mass spectrometry (LC-MS/ MS), 247 248 Liquid yolk, 315 316 Lisozyme (LZ), 194 195 Listeria monocytogenes, 153, 274 275 Live weight (LW), 6 Livestock, 2 production, 32 products, 4 5 Low animal product diet, 18 19, 25t exiting diet, 29 30 Low molecular weight melanoidins, 302 Low-temperature long-time cooking (LTLT), 312 LP. See Lactoperoxidase (LP) LTLT. See Low-temperature long-time cooking (LTLT) Lupin flour, 307 LW. See Live weight (LW) Lyophilization. See Freeze drying Lys87, 277 278 LysH5 (endolysin), 274

339

LysK, 277 LysSA11 (endolysin), 274 275 LZ. See Lisozyme (LZ)

M M13 capsid protein motif, 267 268 MAAs. See Mycosporine-like amino acids (MAAs) Maillard browning, 300 302 Maillard reaction, 74, 317 pathways, 300 302, 301f Maillard reaction products (MRPs), 300 302, 304 Maize gluten hydrolysates, 178 179 Malnutrition, 30 31 Maltose-binding protein (MBP), 272 273 Mannoses (Man), 284 285 Markets and products from microalgae, 66 Mass spectrometry (MS), 239 240 MBP. See Maltose-binding protein (MBP) mbT4L. See Metal ions binding mutant of T4 endolysin (mbT4L) MC. See Membrane chromatography (MC) MDA-MB-231 cells, 267 Mealworm larvae, 17 Meat processing by-products, 131 136. See also Fish processing by-products blood as source of serum albumin, 133 135 bone, cartilage, and skin as sources of collagen, 135 136 by-products of meat and poultry industry by weight, 132t food and biomedical applications of proteins derived from, 149 155 keratin sources, 136 procedures in protein-based ingredient production, 136 149 uses for meat and poultry processing by-products, 133f Meat proteins, 311 314 Melanoidins in food, 319 Membrane filtration, 200, 306 separation, 200 203 case studies and developments in UF process, 201 203 Membrane chromatography (MC), 205 2-Mercaptoethanol, 106 107 Metabolic syndrome, 179 180 Metal ions binding mutant of T4 endolysin (mbT4L), 280 Methanol-free PAOX1-based strain (MF1), 281 Methionine, 135, 302 Metmyoglobin, 313 MF. See Microfiltration (MF)

340 MF1. See Methanol-free PAOX1-based strain (MF1) Microalgae, 76 challenges surrounding algal usage food safety, 89 genetically modified algae, 89 90 price, 90 91 scalability, 90 chemical composition of, 69 76, 69f as potential source of proteins application of algal protein for human consumption, 82 84 chemical composition of microalgae, 69 76 commercial traditionally microalgae, 67f extraction of proteins and active substances from A. platensis, 78 81 markets and products from microalgae, 66 microalgal biorefinery plant concept, 65f microalgal production systems, 67 69 nutritional standard and regulations, 84 86 physicochemical and technofunctional of algal proteins, 81 82 protein extraction methods, 76 78 toxicological aspects, 86 89 production systems, 67 69 cultivation of different types of algae, 68f Microbial fermentation, 75 Microbial transglutaminase, 306 Microbubbles, 204 Microfiltration (MF), 201 Micronutrients, 13, 17 Microwave cooking, 305 microwave-assisted extraction, 78 Milk homogenization, 307 308 and products, 25t proteins, 307 311 MilkoScan IR instrument, 236 Milling, 303 304 Minerals, 151 Modern entomophagy practices, 42 44 Molecular cloning strategy, 280 282 Molecular imprinting, 204 205 Molecular weight (MW), 105 Molecular weight cut-off (MWCO), 200 201 Molecular weight distribution (MWD), 227 228 profiles of protein hydrolysates, 245 246 Molten globule, 299 Monarch butterfly (Danaus plexippus), 47 Monogastric animals, 6 7 Monte Carlo uncertainty method, 19 20 MR-10 (endolysin), 274 MRPs. See Maillard reaction products (MRPs)

INDEX

MS. See Mass spectrometry (MS) MW. See Molecular weight (MW) MWCO. See Molecular weight cut-off (MWCO) MWD. See Molecular weight distribution (MWD) Mycobacteriophage D29 endolysin, 278 279 Mycoprotein-based Quorn, 14 Mycosporine-like amino acids (MAAs), 74 75 Myofibrillar proteins, 169, 312, 320 321 Myosin, 169, 312 313

N N-acetylglucosamine (GlcNAc), 284 285 N-linked glycosylation, 284 285 N-terminal amino group (NH31 group), 243 N-terminal autoprotease variant EDDIE (Npro variant EDDIE), 273 NACIA. See North American Coalition for Insect Agriculture (NACIA) Nanofiltration (NF), 200 201 Nanoparticles, whey protein and, 216 217 Narcissus tazetta lectin (NTL), 282 National Health and Family Planning Commission (NHFPC), 86 Native food proteins, modifications in, 296 302 Natural antioxidants, 172 173 Near-infrared spectroscopy (NIR), 234 235 Neem seed (Indica azadirachta), 118 119 Nervous system, 142 Net protein utilization (NPU), 70 Neurosciences field, 267 268 Neurotoxic shellfish poisoning (NSP), 87 Neurotoxin, 47 Neurotransmitters, 41 42 New protein/peptide ligands identification, 262 264 NF. See Nanofiltration (NF) NF-κB. See Nuclear factor-κB (NF-κB) NHFPC. See National Health and Family Planning Commission (NHFPC) Nile tilapia (Oreochromis niloticus), 173 NIR. See Near-infrared spectroscopy (NIR) Nitric oxide (NO), 46 Nitrogen, 183 Nitrophorins, 46 NMR spectroscopy. See Nuclear magnetic resonance spectroscopy (NMR spectroscopy) NO. See Nitric oxide (NO) Nonconventional method, 108 Nonpolar hydrophobic amino acids, 172 173 “Normal” meat, 15 North American Coalition for Insect Agriculture (NACIA), 49 Nostoc commune, 64 65 Nostoc flagelliforme, 64 65

INDEX

Nostoc punctiforme, 64 65 Nostopeptins. See Cyanovirin-N (CV-N) Npro variant EDDIE. See N-terminal autoprotease variant EDDIE (Npro variant EDDIE) NPU. See Net protein utilization (NPU) NSP. See Neurotoxic shellfish poisoning (NSP) NTL. See Narcissus tazetta lectin (NTL) Nuclear factor-κB (NF-κB), 50 Nuclear magnetic resonance spectroscopy (NMR spectroscopy), 241 242 Nucleic acids, 87 Nutrients, 10 11 Nutritional properties of fish protein isolates, 175 176 processing effect on nutritional properties of food proteins, 303 316 Nutritional standard and regulations, 84 86, 85t

O O-phthaldialdehyde (OPA), 236 239 Oat milk, 307 Obesity, 30 31 Off-flavors, 112 Oily fraction, 136 On-line methods, 235 One World One Health (OWOH), 49 One-dimensional 1H NMR experiment (1D 1H NMR experiment), 241 OPA. See O-phthaldialdehyde (OPA) OPN. See Osteopontin (OPN) Optimal cell disruption method, 79 80 Osborne fractionation, 118 Osteopontin (OPN), 194 195 Ovalbumin, 315, 318 Ovomucoid, 318 OWOH. See One World One Health (OWOH) Oxidation, 302

P P. pastoris promoter AOX1 (PAOX1), 281 Pacific hake (Merluccius productus), 183 Pallid emperor moth (Cirina forda), 43 Palm kernel meal (PKM), 115 117 Palm kernel oil, 115 116 Palm oil, 115 116 Papain Bromelain, 165 166 Paralytic shellfish poisoning (PSP), 87 PCA. See Principal component analysis (PCA) PCL-F, 284 285 PDCAAS. See Protein digestibility corrected amino acid score (PDCAAS) Peanut (Arachis hypogaea L.), 117 meal, 117

341

Pediococci strains, 304 Pediococcus acidilactici NCIM5368, 167 168 PEF. See Pulsed electric field (PEF) PEG. See Polyethylene glycol (PEG) Pep7, 267 268 Pepsin, 142 149 Peptides, 74 75, 142, 171, 196, 260, 317 PER. See Protein efficiency ratio (PER) pH-shift method. See Isoelectric solubilization and precipitation (ISP) pH-stat method, 237 Phage display, 261 268, 263f new protein/peptide ligands identification, 262 264 phage display-derived proteins/peptides in E. coli, 264 265 therapeutic applications of phage display-derived proteins and peptides, 265 268 Phage titration, 265 Photobioreactors, 68 Photosynthetic cyanobacteria, 64 Phycobiliproteins, 78 79, 84 Phycocyanin, 78 79, 80f Phycoerythrin, 78 79, 80f Physical changes in proteins, 296 300, 297f aggregation, 299 denaturation, 296 299 gelation, 299 300 precipitation, 300 Physicochemical properties of algal proteins, 81 82 of fish protein isolates, 176 177 Physiological functions of food proteins, processing effect on, 317 319 pI. See Isoelectric point (pI) Pichia expression system, 282 Pichia glycosylation, 260 261 Pichia pastoris, 270 271, 280 molecular cloning strategy for recombinant production of lectins, 280 282 recombinant lectin production and purification strategies, 282 284, 283t Pichia processing impact on lectin properties, 284 285 Pichia trace minerals (PTM), 282 Pigments, 68 Pili, 265 PKM. See Palm kernel meal (PKM) Plant oil refining industry, proteins from by-products of, 111 118 Plant oil sources, 118 119 Plant proteases, 165 166 Plant protein hydrolysates, 177 179. See also Fish protein hydrolysates (FPHs) kidney bean hydrolysates, 179

342

INDEX

Plant protein hydrolysates (Continued) rice bran hydrolysates, 179 soy protein hydrolysates, 178 wheat starch and maize gluten hydrolysates, 178 179 Plant proteins, 303 307 cereal proteins, 303 305 Plant-based proteins properties and extraction, 103 119 cereal and pseudocereal proteins, 103 111 proteins from by-products of plant oil refining industry, 111 118 proteins in human nutrition and food processing, 98 101 sources, 118 119 in sustainability context, 101 102 Plant-based sources, 305 306 Plasma, 139 Plastein, 230 231 PMM. See Protein micelle mass (PMM) Polyethylene glycol (PEG), 139 140, 264 265 Polyhistidine tag (His-tag), 273 Polyvinylidene fluoride (PVDF), 201 Poor man’s meat. See Legumes Porcine liver hydrolysates, 151 Porphyridium cruentum, 78 81 Poultry processing by-products, proteins from, 146t, 151 154, 152t Poultry viscera, 153 PP. See Proteose peptone (PP) PP2A. See Protein phosphatase 2A (PP2A) Precipitation, 76, 197 199, 300, 305 306 chemical precipitation, 198 199 heat precipitation, 197 198, 197f Prediction sorting, 251 Preslaughter stress, 313 314 Pretreatment of whey, 208 209 Price, 90 91 Principal component analysis (PCA), 243 244 Process analytical technology, 235 Process-specific parameters, 227 Processing effect on digestibility of food proteins, 316 on nutritional properties of food proteins, 303 316 egg proteins, 314 316 meat proteins, 311 314 milk proteins, 307 311 plant proteins, 303 307 on physiological functions of food proteins, 317 319 ProGo, 226 Progression, 171 Promoters, 271 Protease(s), 75 76

protease-specific factors, 227 specificity, 231 stability, 229 Protein digestibility corrected amino acid score (PDCAAS), 14, 98 Protein efficiency ratio (PER), 70, 110 Protein hydrolysates, 140 149, 141f, 143t, 151, 154, 164, 166, 182, 306 307, 317 hydrolysis parameters of fish processing by-products, 148t of meat processing by-products, 143t of poultry processing by-products, 146t MWD profiles of, 245 246 Protein isolates, 140 149, 141f hydrolysis parameters of fish processing by-products, 148t of meat processing by-products, 143t of poultry processing by-products, 146t Protein micelle mass (PMM), 113 Protein phosphatase 2A (PP2A), 88 Protein stability modifications in native food proteins, 296 302 chemically induced changes in proteins, 300 302 physical changes in proteins, 296 300 processing effect on digestibility of food proteins, 316 on nutritional properties of food proteins, 303 316 on physiological functions of food proteins, 317 319 storage effect, 319 321 Protein(s), 2, 13, 20, 21f, 30 31, 41 42, 68, 70 72, 97 98, 133, 139, 164, 260, 295 296 from by-products of plant oil refining industry cashew meal, 118 cotton proteins, 114 115 peanut meal, 117 PKM, 115 117 R/C proteins, 112 114 soybean proteins, 111 112 content, 70 digestibility, 105 of microalgae, 72 engineering improving activity of endolysins, 275 277, 276f improving solubility of endolysins, 277 278 structure of endolysins, 275 extraction methods, 76 81, 112, 116 bead milling, 77 ionic liquids, 78 microwave-assisted extraction, 78 PEF-assisted extraction, 77 78 UAE, 77 food and biomedical applications

INDEX

proteins from fish processing by-products, 154 155 proteins from meat processing by-products, 149 151, 152t proteins from poultry processing by-products, 151 154 functionality, 295 296 in human nutrition and food processing, 98 101 values for protein content, 99t, 100t measurements, 232 233 modifications, 319 320 precipitation, 300 protein-based ingredient production, procedures in, 136 149 quality, 316 Proteolysis, 75 76 Proteolytic hydrolysis. See Enzymatic hydrolysis Proteolytic LAB, 168 Proteomic approaches, 248 250 Proteose peptone (PP), 194 195, 307 Protozoa, 48 Proximate analysis, 232 PRWAVSP peptide, 267 Pseudomonas aeruginosa, 212 213 PSP. See Paralytic shellfish poisoning (PSP) PTM. See Pichia trace minerals (PTM) Pulsed electric field (PEF), 77 78 PEF-assisted extraction, 77 78 Pulsed UV light treatment, 318 PVDF. See Polyvinylidene fluoride (PVDF)

Q Quinoa (Chenopodium quinoa), 109 proteins, 109 111

R R/C proteins. See Rapeseed/Canola proteins (R/C proteins) Raman spectroscopy, 234 236 Rapeseed (Brassica napus L.), 112 113 Rapeseed/Canola proteins (R/C proteins), 112 114 Reactive oxidation species-mediated protein oxidation, 302 Reactive oxygen species, 313 314 Recombinant pharmaceuticals, 260 AMPs, 268 273 endolysins, 274 280 lectins, 280 285 phage display, 262 268 Rendering process, 136 Repulsion toward arthropods, 42 Reverse osmosis (RO), 200

343

Rice (Oryza sativa L.), 179 bran hydrolysates, 179 bran proteins, 103 104 milk, 307 RNA interference technology (RNAi technology), 114 RNAi technology. See RNA interference technology (RNAi technology) RO. See Reverse osmosis (RO) RP-HPLC. See High-performance liquid chromatography in reverse phase (RP-HPLC) Ruminants, 32

S Saccharomyces cerevisiae, 142 Saccharomyces α-factor preprosequence, 280 281 Salt addition, 306 salt-assisted protein extraction, 115 Sarcoplasmic proteins, 169, 312 Sardine protein hydrolysates, 174 175 Sardinelle (Sardinella aurita), 173 Scalability, 90 Scenedesmus obliquus, 70 SCPs. See Single-cell proteins (SCPs) SDS-PAGE. See Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) Sea bass (Dicentrarchus labrax), 183 SEC. See Size exclusion chromatography (SEC) Serum albumin, 133 blood as source of, 133 135 SFD. See Spray freeze drying (SFD) SFE. See Supercritical fluid extraction (SFE) SHR. See Spontaneously hypertensive rats (SHR) Shrimps, 320 321 Silkworm (Bombyx mori, Antheraea assamensis), 42 Single-cell oils, 68 Single-cell proteins (SCPs), 82 Size exclusion chromatography (SEC), 245, 282 284 Skin as sources of collagen, 135 136 Small ubiquitin-related modifier (SUMO), 272 273 Small-scale algae production, 68 Soaking, 305, 316 SOC chemotherapy. See Standard-of-care chemotherapy (SOC chemotherapy) Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), 239 240 Solid-phase extraction (SPE), 77 Solid-phase synthesis, 269 Solubility, 170, 182 of endolysins by protein engineering, 277 278 Solubilization precipitation, 140 141 Solution-phase synthesis, 269 Solvents, 139 140

344 Solvents (Continued) extraction, 75 Sonication, 106 Sorbitol, 310 Sorghum proteins, 104 107 Soxhlet method, 233 234 Soy protein hydrolysates, 178 Soy-based products, 14 Soybean (Glycine max L.), 111 curd, 17, 25t production, 15, 27 28 proteins, 111 112 Soymilk, 307 SPE. See Solid-phase extraction (SPE) Specificity of endolysins, 276 Spectroscopic methods, 234 236 Spirulina, 65 S. maxima, 83 as superfood, 83 84 Spontaneously hypertensive rats (SHR), 151 153 Spray drying, 206 207, 306 Spray freeze drying (SFD), 207 208, 208f ssDNA, 265 Standard-of-care chemotherapy (SOC chemotherapy), 214 Staphefekt SA. 100, 277 Staphylococcus aureus, 154 Storage effect on protein stability, 319 321 Streptococcus phage C1, 274 Streptococcus pyogenes, 274 Streptococcus thermophilus, 183 Substantial environmental impacts, 2 Substrate-specific factors, 227 Sucrose, 310 Sulfhydryl groups, 305 Sulfur-containing amino acids, 302 SUMO. See Small ubiquitin-related modifier (SUMO) Sunflower (Helianthus annuus L.), 115 proteins, 115 Supercritical fluid extraction (SFE), 77 Superfood, Spirulina as, 83 84 Sustainability, 98 plant-based proteins in, 101 102 sustainability meat consumption, 19 Sustainable food source, 49 Sustainable intensification, 2 3 Sustainable proteins alternatives to current animal products, 12 15 comparison of land requirements, 15 32 global and country-level consumption patterns and trends changing dietary patterns, 9 12

INDEX

intercountry variation in appropriation of land for food, 7 9 human appropriation of land for food, 4 7 source, 42 Synthetic meat. See Cultured meat

T T4 bacteriophage lysis genes, 279 T4 endolysin, 279 280 T7 (inducible promoter), 271 Tags, 273 Tandem multimeric strategy, 271 272 TBS. See Tris buffer saline (TBS) Technofunctional of algal proteins, 81 82 Tecuitlatl, 64 65 Termite (Macrotermes natalensis), 43 44 Tetrodotoxin (TTX), 88 Texture, 176 177 Textured vegetable protein, 14 Thawing, 315 316 Thermal processing, 300 of meat, 311 312 of milk, 307 Thermal treatment, 304 Thermosonication pretreatments, 211 212 Thioredoxin A (TrxA), 272 273 Third World Food Survey, 64 Thrombin, 140 Tilapia mince hydrolysates, 171 production, 14 15 Time-dependent HALF indices, 10 Time-dependent progression of enzymatic reaction, 228 Tissue culture, 14 TNF. See Tumor necrosis factor (TNF) tolQRA complex, 265 Total nitrogen-to-protein conversion factor, 232 Toxicological aspects, 86 89 algal toxins, 87 88 heavy metals, 88 89 nucleic acids, 87 TPH. See Trypsin protein hydrolysate (TPH) Transcription factors, 41 42 Trichloroacetic acid soluble nitrogen method, 236 239 Trichloroacetic acid-solubility index. See Trichloroacetic acid soluble nitrogen method Tris buffer saline (TBS), 264 265 Trisodium citrate, 310 tRNAs, 272 TrxA. See Thioredoxin A (TrxA) Trypsin protein hydrolysate (TPH), 171 Tryptophan residues, 302 TTX. See Tetrodotoxin (TTX)

INDEX

Tumor necrosis factor (TNF), 50 Two-state transition, 299

U Ultrafiltration (UF), 75 76, 142 149, 198 199 case studies and developments in, 201 203, 202f concentrated method, 90 91 US-assisted UF process, 210f Ultrahigh-temperature (UHT), 307 308 Ultrasonic atomizers, 206 207 Ultrasound, 210 211 Ultrasound-assisted extraction (UAE), 77 ultrasound-assisted water extraction of defatted rice bran, 103 Uncertainty quantification, 19 20 Uric acid, 87 US Department of Agriculture (USDA), 49 US Food and Drug Administration (FDA), 280

V Vacuum-dried hydrolysates, 151 153 Vegetarianism, 30 Venoms, 47 Vibrational spectroscopy, 234 Virtual intermediate peptides, 230 231 Viruses, 48

W Waste, 30 31 and dietary change scenarios high and low animal product diets, 18 19 waste reduction, 18, 25t Wastewater, 91 Water holding capacity, 170 Wheat (Triticum aestivum L.), 104 bran proteins, 104 starch hydrolysates, 178 179

345

Whey protein concentrate (WPC), 195 Whey protein hydrolysate (WPH), 196 Whey protein isolate (WPI), 196 Whey protein powder (WPP), 195 whey protein-based products, 310 Whey proteins, 193 196, 307, 318 application as edible films and coatings, 216 in food industry, 215 216 approaches and recent advances in recovery, 196 212 chromatographic separation, 199 200 drying, 206 208 membrane separation, 200 203 methods for whey protein separation, 203 205 precipitation, 197 199 components, properties, and benefits, 195t global whey protein market, 217 healthcare applications, 212 215 improvements in whey protein manufacturing avoiding membrane fouling, 209 211 improving whey protein stability, 211 212 pretreatment of whey, 208 209 whey protein hydrogels and nanoparticles, 216 217 White-shelled eggs, 321 Whitening effect, 313 Wound healing, 213 WPC. See Whey protein concentrate (WPC) WPH. See Whey protein hydrolysate (WPH) WPI. See Whey protein isolate (WPI) WPP. See Whey protein powder (WPP)

X X-gal. See 5-Bromo-4-chloro-3-indolyl-β-D-galactoside (X-gal)