Innovative thermal and non-thermal processing, bioaccessibility and bioavailability of nutrients and bioactive compounds 9780128141748, 0128141743

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Innovative thermal and non-thermal processing, bioaccessibility and bioavailability of nutrients and bioactive compounds
 9780128141748, 0128141743

Table of contents :
Front Cover......Page 1
Innovative Thermal and Non-Thermal Processing, Bioaccessibility and Bioavailability of Nutrients and Bioactive Compounds......Page 4
Copyright......Page 5
Contents......Page 6
Contributors......Page 10
Section 1: Introduction......Page 14
1.1 Introduction......Page 16
1.2 Culinary techniques......Page 18
1.3 Thermal processing......Page 21
1.4.1 High-pressure processing......Page 22
1.4.3 Pulsed electric fields......Page 23
1.4.4 Ultrasound (US)......Page 24
1.5 European legislation for development and labeling of healthy food products......Page 25
1.6 Challenges and future perspectives......Page 26
Acknowledgments......Page 27
References......Page 28
Further reading......Page 34
2.1 Introduction......Page 36
2.2 Bioactive compounds and nutrients......Page 37
2.3.2 Bioactivity......Page 38
2.4 Bioavailability measurements......Page 39
2.5 Bioaccessibility measurements......Page 40
2.6.1 Gastrointestinal models......Page 41 In vitro digestion models......Page 42 In vitro dynamic digestion models......Page 44 Intestinal absorption and presystemic metabolism assessment......Page 50 In vitro studies incorporating gut microflora......Page 51 Caco-2 cell models......Page 52 In vivo experiments......Page 54 In vitro and in vivo correlations......Page 55
2.7 Advantages and disadvantages of in vivo and in vitro procedures used to assess bioaccessibility and bioavailabili .........Page 57
2.8 Conclusions......Page 58
References......Page 59
3.1 Introduction......Page 68
3.2.1 Supercritical carbon dioxide......Page 70 Extraction......Page 71 Inactivation of microorganisms and enzymes by pressurized CO 2......Page 77 Disruption of biomass components......Page 78 Encapsulation techniques......Page 79
3.2.2 High hydrostatic pressure......Page 80
3.2.3 Ultrasound in food processing......Page 82 US-assisted freezing processes and sonocrystallization......Page 84 US-assisted extraction of food components......Page 85 Emulsification and emulsion separation......Page 86 Foaming/defoaming and airborne US for enhanced drying......Page 87
3.2.4 Ohmic heating......Page 88 Current, voltage, and electric field......Page 89
3.2.5 Food packaging: Active packaging with natural additives and biodegradable edible films......Page 90 Active packaging......Page 91 Antimicrobial active packaging......Page 92 Antioxidant active packaging......Page 93 Biodegradable and edible films with antimicrobial/antioxidant activity......Page 94
3.3 Conclusions and future opportunities......Page 96
References......Page 97
Further reading......Page 116
Section 2: Processing, bioavailability and bioaccessibility......Page 118
4.1.1 Fatty acids......Page 120
4.1.2 Phospholipids......Page 121
4.2 Absorption process......Page 123
4.3 Lipid oxidation. The main factor that affects the bioavailability of lipids......Page 124
4.4 Effect of processing on lipid oxidation and bioaccessibility......Page 126
4.4.1 Thermal processing......Page 127 Microwave......Page 128 Spray-drying......Page 129 High pressure processing (HPP)......Page 130 Ultrasound (US)......Page 133 Ionizing radiation (IR)......Page 136 Pulsed electric fields (PEF)......Page 137 Other non-thermal processing......Page 140
References......Page 142
5.1 Introduction-protein quality......Page 152
5.2.1 Thermal-conventional treatments......Page 155
5.3.1 Ultrasound......Page 158
5.3.2 High pressure......Page 161
5.3.3 Pulsed electric field......Page 163
5.3.4 Enzymatic reaction......Page 165
5.3.5 Ohmic heating......Page 167
5.3.6 Irradiation......Page 169
References......Page 172
Further reading......Page 182
6.1 Introduction......Page 184
6.2 Types of dietary carbohydrates in foods......Page 185
6.2.1 Simple and easily digestible carbohydrates......Page 186
6.2.2 Non-digestible carbohydrates......Page 187
6.3.1 Utilization of dietary carbohydrates as main energy provider......Page 188
6.3.2 Non-digestible carbohydrates can protect and maintain gastrointestinal health......Page 190
6.4 The important role of food processing in influencing the bioavailability and bioaccessibility of dietary carbohyd .........Page 192
6.4.1 Effect of conventional and thermal food-processing technologies......Page 193
6.4.2 Effect of nonthermal food-processing technologies......Page 210
6.5 Conclusion......Page 212
References......Page 213
Section 3: Processing, bioavailability and bioaccessibility of micronutrients......Page 220
7.1.1 Macrominerals......Page 222
7.1.2 Microminerals......Page 223
7.2 Effect of household processing on mineral bioavailability......Page 225
7.3.1 Vegetable foods......Page 234
7.3.2 Animal foods......Page 241
7.4 Non-conventional thermal processing......Page 244
References......Page 248
8.1 Introduction......Page 254
8.2 Definition and principles of bioavailability and bioaccessibility......Page 257
8.3 Processing parameters affecting the water-soluble vitamins......Page 259
8.4 The different effects of processing......Page 261 Pasteurization and sterilization......Page 262 Thermal drying......Page 263 Dense-phase carbon dioxide (DPCD)......Page 264 Ozone processing......Page 265 Ultrasound processing......Page 266 High hydrostatic pressure processing (HHP)......Page 267
Pulsed light (PL)......Page 269
γ -Irradiation......Page 270
8.6 Conclusion......Page 271
References......Page 272
9.1 Introduction......Page 280
9.3 Processing parameters affecting the fat-soluble vitamins......Page 284 Cooking......Page 285 Effect of different processing techniques on carotenoids......Page 286
9.5 Effect of storage on α -tocopherol and beta-carotene......Page 288
9.6 Effect of heat processing on α -tocopherol and beta-carotene......Page 291
9.7 Effect of light on α -tocopherol and beta-carotene......Page 293
9.8 Effect of drying and dehydration on α -tocopherol and beta-carotene......Page 294
9.11 Conclusions......Page 295
References......Page 296
Further reading......Page 302
Section 4: Processing, bioavailability and bioaccessibility of bioactive compounds......Page 304
10.1 Introduction......Page 306
10.2 Bioactive organosulfur compounds of Allium species......Page 307
10.3 Bioavailability of organosulfur compounds......Page 312
10.4 Bioavailability of organosulfur compounds and processing......Page 314
10.5 Concluding remarks and future perspectives......Page 315
References......Page 316
Further reading......Page 321
11.1 Introduction......Page 322
11.2 Phenolic compounds: General characteristics and sources......Page 323
11.3 Digestion and absorption of phenolic compounds......Page 327
11.4 Bioaccessibility of phenolic compounds......Page 329
11.4.1 Effect of thermal processing on phenolic compounds’ activity and bioaccessibility......Page 330
11.4.2 Effect of non-thermal processing on phenolic compounds’ activity and bioaccessibility......Page 334
11.5 Thermal and non-thermal technologies’ effect on bioavailability and bioactivity of phenolic compounds......Page 338
References......Page 339
Further reading......Page 345
12.1 Introduction......Page 346
12.3 Effect of non-thermal treatment on the release of bioactive peptides......Page 347
12.4 Effect of thermal treatment on the activity of bioactive peptides......Page 350
12.5 Effect of gastrointestinal digestion on bioavailability of bioactive peptides......Page 352
References......Page 354
Index......Page 360
Back Cover......Page 372

Citation preview

Innovative Thermal and Non-Thermal Processing, Bioaccessibility and Bioavailability of Nutrients and Bioactive Compounds

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Woodhead Publishing Series in Food Science, Technology and Nutrition

Innovative Thermal and Non-Thermal Processing, Bioaccessibility and Bioavailability of Nutrients and Bioactive Compounds Edited by

Francisco J. Barba Jorge Manuel Alexandre Saraiva Giancarlo Cravotto José M. Lorenzo

An imprint of Elsevier

Woodhead Publishing is an imprint of Elsevier The Officers’ Mess Business Centre, Royston Road, Duxford, CB22 4QH, United Kingdom 50 Hampshire Street, 5th Floor, Cambridge, MA 02139, United States The Boulevard, Langford Lane, Kidlington, OX5 1GB, United Kingdom © 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: 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. Library of Congress Cataloging-in-Publication Data A catalog record for this book is available from the Library of Congress British Library Cataloguing-in-Publication Data A catalogue record for this book is available from the British Library ISBN: 978-0-12-814174-8 For information on all Woodhead publications visit our website at

Publisher: Charlotte Cockle Acquisition Editor: Megan R. Ball Editorial Project Manager: Redding Morse Production Project Manager: Joy Christel Neumarin Honest Thangiah Cover Designer: Greg Harris Typeset by SPi Global, India


Contributors ix

Section 1  Introduction 1 An integrated strategy between gastronomic science, food science and technology, and nutrition in the development of healthy food products Elena Roselló-Soto, Rohit Thirumdas, José M. Lorenzo, Paulo Eduardo Sichetti Munekata, Predrag Putnik, Shahin Roohinejad, Kumar Mallikarjunan, Francisco J. Barba 1.1 Introduction 1.2 Culinary techniques 1.3 Thermal processing 1.4 Nonthermal processing 1.5 European legislation for development and labeling of healthy food products 1.6 Challenges and future perspectives Acknowledgments References Further reading 2 Methods for determining bioavailability and bioaccessibility of bioactive compounds and nutrients Diana I. Santos, Jorge Manuel Alexandre Saraiva, António A. Vicente, Margarida Moldão-Martins 2.1 Introduction 2.2 Bioactive compounds and nutrients 2.3 Concepts 2.4 Bioavailability measurements 2.5 Bioaccessibility measurements 2.6 Methods to determine bioavailability and bioaccessibility of nutrients and bioactive compounds 2.7 Advantages and disadvantages of in vivo and in vitro procedures used to assess bioaccessibility and bioavailability of bioactive compounds 2.8 Conclusions Acknowledgments References

1 3

3 5 8 9 12 13 14 15 21 23 23 24 25 26 27 28 44 45 46 46


3 Green technologies for food processing: Principal considerations María López-Pedrouso, B. Díaz-Reinoso, José M. Lorenzo, Giancarlo Cravotto, Francisco J. Barba, A. Moure, H. Domínguez, Daniel Franco 3.1 Introduction 3.2 Innovative techniques 3.3 Conclusions and future opportunities References Further reading


55 57 83 84 103

Section 2  Processing, bioavailability and bioaccessibility


4 Lipids and fatty acids Mirian Pateiro, Rubén Domínguez, Paulo Eduardo Sichetti Munekata, Francisco J. Barba, José M. Lorenzo 4.1 Background 4.2 Absorption process 4.3 Lipid oxidation. The main factor that affects the bioavailability of lipids 4.4 Effect of processing on lipid oxidation and bioaccessibility 4.5 Conclusions Acknowledgments References


5 Proteins and amino acids María López-Pedrouso, José M. Lorenzo, Carlos Zapata, Daniel Franco 5.1 Introduction-protein quality 5.2 Influence of conventional-thermal food processes on protein quality 5.3 Influence of emerging non-thermal food processes on protein quality 5.4 Conclusions and future remarks References Further reading


6 Carbohydrates Sze Ying Leong, Sheba Mae Duque, Setya Budi Muhammad Abduh, Indrawati Oey 6.1 Introduction 6.2 Types of dietary carbohydrates in foods 6.3 Beneficial physiological functions of dietary carbohydrates 6.4 The important role of food processing in influencing the bioavailability and bioaccessibility of dietary carbohydrates 6.5 Conclusion Acknowledgment References


107 110 111 113 129 129 129

139 142 145 159 159 169

171 172 175 179 199 200 200


Section 3  Processing, bioavailability and bioaccessibility of micronutrients


7 Impact of processing on mineral bioaccessibility/bioavailability Antonio Cilla, Reyes Barberá, Gabriel López-García, Virginia Blanco-Morales, Amparo Alegría, Guadalupe Garcia-Llatas 7.1 Dietary importance of essential minerals 7.2 Effect of household processing on mineral bioavailability 7.3 Conventional thermal processing 7.4 Non-conventional thermal processing 7.5 Conclusions References


8 Water-soluble vitamins Amin Mousavi Khaneghah, Seyed Mohammad Bagher Hashemi, Ismail Es, Aliakbar Gholamhosseinpour, Monica Rosa Loizzo, Alessandra Giardinieri, Deborah Pacetti, Kiana Pourmohammadi, Daniela S. Ferreira 8.1 Introduction 8.2 Definition and principles of bioavailability and bioaccessibility 8.3 Processing parameters affecting the water-soluble vitamins 8.4 The different effects of processing 8.5 Determination of bioavailability and bioaccessibility 8.6 Conclusion References


9 Fat-soluble vitamins Seyed Mohammad Bagher Hashemi, Kiana Pourmohammadi, Aliakbar Gholamhosseinpour, Ismail Es, Daniela S. Ferreira, Amin Mousavi Khaneghah 9.1 Introduction 9.2 Definition and principles of bioavailability and bioaccessibility 9.3 Processing parameters affecting the fat-soluble vitamins 9.4 Effects of processing 9.5 Effect of storage on α-tocopherol and beta-carotene 9.6 Effect of heat processing on α-tocopherol and beta-carotene 9.7 Effect of light on α-tocopherol and beta-carotene 9.8 Effect of drying and dehydration on α-tocopherol and beta-carotene 9.9 Effect of high-pressure processes on α-tocopherol and beta-carotene 9.10 Effect of freezing condition on α-tocopherol and beta-carotene 9.11 Conclusions Acknowledgments References Further reading


209 212 221 231 235 235

241 244 246 248 258 258 259

267 271 271 272 275 278 280 281 282 282 282 283 283 289


Section 4  Processing, bioavailability and bioaccessibility of bioactive compounds 10 Bioavailability and food production of organosulfur compounds from edible Allium species Predrag Putnik, Domagoj Gabrić, Shahin Roohinejad, Francisco J. Barba, Daniel Granato, José M. Lorenzo, Danijela Bursać Kovačević 10.1 Introduction 10.2 Bioactive organosulfur compounds of Allium species 10.3 Bioavailability of organosulfur compounds 10.4 Bioavailability of organosulfur compounds and processing 10.5 Concluding remarks and future perspectives Acknowledgments References Further reading 11 Polyphenols: Bioaccessibility and bioavailability of bioactive components José M. Lorenzo, Mario Estévez, Francisco J. Barba, Rohit Thirumdas, Daniel Franco, Paulo Eduardo Sichetti Munekata 11.1 Introduction 11.2 Phenolic compounds: General characteristics and sources 11.3 Digestion and absorption of phenolic compounds 11.4 Bioaccessibility of phenolic compounds 11.5 Thermal and non-thermal technologies’ effect on bioavailability and bioactivity of phenolic compounds 11.6 Concluding remarks Acknowledgments References Further reading 12 Bioactive peptides Leticia Mora, Marta Gallego, M-Concepción Aristoy, Milagro Reig, Fidel Toldrá 12.1 Introduction 12.2 Main characteristics of bioactive peptides 12.3 Effect of non-thermal treatment on the release of bioactive peptides 12.4 Effect of thermal treatment on the activity of bioactive peptides 12.5 Effect of gastrointestinal digestion on bioavailability of bioactive peptides 12.6 Conclusions References

291 293

293 294 299 301 302 303 303 308 309 309 310 314 316 325 326 326 326 332 333 333 334 334 337 339 341 341

Index 347

Contributors Setya Budi Muhammad Abduh Department of Food Science, University of Otago, Dunedin; Riddet Institute, Palmerston North, New Zealand; Department of Food Science, Diponegoro University, Semarang, Indonesia Amparo Alegría Preventive Medicine and Public Health, Food Science, Toxicology and Forensic Medicine Department, Universitat de València, València, Spain M-Concepción Aristoy Instituto de Agroquímica y Tecnología de Alimentos (CSIC), Valencia, Spain Francisco J. Barba Nutrition and Food Science Area, Preventive Medicine and Public Health, Food Sciences, Toxicology and Forensic Medicine Department, Faculty of Pharmacy, Universitat de València, València, Spain Reyes Barberá  Preventive Medicine and Public Health, Food Science, Toxicology and Forensic Medicine Department, Universitat de València, València, Spain Virginia Blanco-Morales  Preventive Medicine and Public Health, Food Science, Toxicology and Forensic Medicine Department, Universitat de València, València, Spain Danijela Bursać Kovačević  Faculty of Food Technology and Biotechnology, University of Zagreb, Zagreb, Croatia Antonio Cilla Preventive Medicine and Public Health, Food Science, Toxicology and Forensic Medicine Department, Universitat de València, València, Spain Giancarlo Cravotto Dipartimento di Scienza e Tecnologia del Farmaco, Università di Torino, Torino, Italy B. Díaz-Reinoso  Chemical Engineering Department, University of Vigo, Ourense, Spain H. Domínguez  Chemical Engineering Department, University of Vigo, Ourense, Spain Rubén Domínguez  Meat Technology Center of Galicia, Parque Tecnológico de Galicia, Ourense, Spain


Sheba Mae Duque  Department of Food Science, University of Otago, Dunedin; Riddet Institute, Palmerston North, New Zealand; Institute of Food Science and Technology, College of Agriculture and Food Science, University of the Philippines Los Baños, College, Laguna, Philippines Ismail Es Department of Material and Bioprocess Engineering, School of Chemical Engineering, University of Campinas (UNICAMP), Campinas, Brazil Mario Estévez IPROCAR Research Institute, TECAL Research Group, University of Extremadura, Cáceres, Spain Daniela S. Ferreira Department of Food Technology; Department of Food Science, School of Food Engineering, University of Campinas, Campinas, Brazil Daniel Franco Meat Technology Center of Galicia, Parque Tecnológico de Galicia, Ourense, Spain Domagoj Gabrić  Faculty of Food Technology and Biotechnology, University of Zagreb, Zagreb, Croatia Marta Gallego Instituto de Agroquímica y Tecnología de Alimentos (CSIC), Valencia, Spain Guadalupe Garcia-Llatas  Preventive Medicine and Public Health, Food Science, Toxicology and Forensic Medicine Department, Universitat de València, València, Spain Aliakbar Gholamhosseinpour Department of Food Science and Technology, Faculty of Agriculture, Jahrom University, Jahrom, Iran Alessandra Giardinieri  Department of Agricultural, Food, and Environmental Sciences, Polytechnic University of Marche, Ancona, Italy Daniel Granato Department of Food Engineering, State University of Ponta Grossa, Av. Carlos Cavalcanti, Ponta Grossa, Brazil Seyed Mohammad Bagher Hashemi Department of Food Science and Technology, College of Agriculture, Fasa University, Fasa, Iran Sze Ying Leong Department of Food Science, University of Otago, Dunedin; Riddet Institute, Palmerston North, New Zealand Monica Rosa Loizzo  Department of Pharmacy, Health and Nutritional Sciences, University of Calabria, Rende, Italy


Gabriel López-García  Preventive Medicine and Public Health, Food Science, Toxicology and Forensic Medicine Department, Universitat de València, València, Spain María López-Pedrouso Department of Zoology, Genetics and Physical Anthropology, University of Santiago de Compostela, Santiago de Compostela, Spain José M. Lorenzo Meat Technology Center of Galicia, Parque Tecnológico de Galicia, Ourense, Spain Kumar Mallikarjunan  Department of Food Science and Nutrition, University of Minnesota, Saint Paul, MN, United States Margarida Moldão-Martins  Linking Landscape, Environment, Agriculture and Food (LEAF), School of Agriculture, University of Lisbon, Lisbon, Portugal Leticia Mora Instituto de Agroquímica y Tecnología de Alimentos (CSIC), Valencia, Spain A. Moure Chemical Engineering Department, University of Vigo, Ourense, Spain Amin Mousavi Khaneghah  Department of Food Science, School of Food Engineering, University of Campinas, Campinas, Brazil Paulo Eduardo Sichetti Munekata  Meat Technology Center of Galicia, Parque Tecnológico de Galicia, Ourense, Spain; Department of Food Engineering, Faculty of Animal Science and Food Engineering, University of Sao Paulo (USP), Pirassununga, Brazil Indrawati Oey Department of Food Science, University of Otago, Dunedin; Riddet Institute, Palmerston North, New Zealand Deborah Pacetti  Department of Agricultural, Food, and Environmental Sciences, Polytechnic University of Marche, Ancona, Italy Mirian Pateiro Meat Technology Center of Galicia, Parque Tecnológico de Galicia, Ourense, Spain Kiana Pourmohammadi Department of Food Science and Technology, College of Agriculture, Fasa University, Fasa, Iran Predrag Putnik  Faculty of Food Technology and Biotechnology, University of Zagreb, Zagreb, Croatia Milagro Reig  Instituto de Ingeniería de Alimentos para el Desarrollo, Universitat Politècnica de Valencia, Valencia, Spain


Shahin Roohinejad  Department of Food Science and Nutrition, University of Minnesota, Saint Paul, MN, United States; Burn and Wound Healing Research Center, Division of Food and Nutrition, Shiraz University of Medical Sciences, Shiraz, Iran Elena Roselló-Soto Nutrition and Food Science Area, Preventive Medicine and Public Health, Food Sciences, Toxicology and Forensic Medicine Department, Faculty of Pharmacy, Universitat de València, València, Spain Diana I. Santos  Linking Landscape, Environment, Agriculture and Food (LEAF), School of Agriculture, University of Lisbon, Lisbon, Portugal Jorge Manuel Alexandre Saraiva QOPNA & LAQV-REQUIMTE, Department of Chemistry, University of Aveiro, 3810-193 Aveiro, Portugal Rohit Thirumdas  Department of Food Process Technology, College of Food Science and Technology, PJTSAU, Hyderabad; Department of Food Engineering and Technology, Institute of Chemical Technology, Mumbai, India Fidel Toldrá Instituto de Agroquímica y Tecnología de Alimentos (CSIC), Valencia, Spain António A. Vicente Centre of Biological Engineering (CEB), University of Minho, Braga, Portugal Carlos Zapata  Department of Zoology, Genetics and Physical Anthropology, University of Santiago de Compostela, Santiago de Compostela, Spain

Section 1 Introduction

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An integrated strategy between gastronomic science, food science and technology, and nutrition in the development of healthy food products


Elena Roselló-Soto*, Rohit Thirumdas†, José M. Lorenzo‡, Paulo Eduardo Sichetti Munekata‡,§, Predrag Putnik¶, Shahin Roohinejad‖, Kumar Mallikarjunan‖, Francisco J. Barba* ⁎ Nutrition and Food Science Area, Preventive Medicine and Public Health, Food Sciences, Toxicology and Forensic Medicine Department, Faculty of Pharmacy, Universitat de València, València, Spain, †Department of Food Process Technology, College of Food Science and Technology, PJTSAU, Telangana, India, ‡Meat Technology Center of Galicia, Parque Tecnológico de Galicia, Ourense, Spain, §Department of Food Engineering, Faculty of Animal Science and Food Engineering, University of Sao Paulo (USP), Pirassununga, Brazil, ¶Faculty of Food Technology and Biotechnology, University of Zagreb, Zagreb, Croatia, ‖Department of Food Science and Nutrition, University of Minnesota, Saint Paul, MN, United States

1.1 Introduction A diverse and balanced diet, along with healthy choices, is considered as one of the necessary actions to maintain wellbeing and reduce the risk of diseases (Aranceta Bartrina et  al., 2016; Musina et  al., 2017). The World Health Organization (WHO) recognizes the importance of diet, by means of adequate consumption of nutrients and bioactive compounds, and sets important recommendations for consumers, health professionals, food industries, and researchers. These recommendations are substantiated in a large body of scientific evidence that highlights the importance of eating healthy as preventive action against new cases of the most prevalent noncommunicable diseases worldwide, such as obesity, diabetes, heart disease, stroke, and cancer (WHO, 2013). Due to the importance of diet and the role of food in this context, an important question can be raised here: “How to produce and cook foods in a way to take advantage of maximum nutritional value and natural bioactive compounds already present or subsequently added during production?” Value-added compounds from foods are mainly referred to the concepts of functional foods that can be designed from almost any foods through enrichments, fortifications, and alterations with various extracts obtained from plants or food by-products (Čukelj et al., 2016; Formato, Gallo, Ianniello, Innovative Thermal and Non-Thermal Processing, Bioaccessibility and Bioavailability of Nutrients and Bioactive Compounds © 2019 Elsevier Inc. All rights reserved.


Innovative Thermal and Non-Thermal Processing

Montesano, & Naviglio, 2013; Granato, Nunes, & Barba, 2017; Lorenzo et al., 2018; Montesano, Gennari, Seccia, & Albrizio, 2011; Musina et al., 2017; Naviglio, Gallo, Vitulano, Montesano, & Faralli, 2014; Putnik, Barba, Španić, Zorić, et  al., 2017; Vinceković et al., 2017). This challenging task involves three main knowledge areas: gastronomy (food preparation), food science and technology (food structure and composition as well as processing), and nutrition (digestion, absorption, and efficient use of nutrients and bioactive compounds) (Barba, Mariutti, et al., 2017; Carbonell-Capella, Buniowska, Barba, Esteve, & Frígola, 2014). Each area has a fundamental role to improve food processing and preparation because along with nutritional and health benefits, food properties and characteristics are also influenced (Coveney & Santich, 1997) (Fig. 1.1). Gastronomy can be defined as the set of knowledge and activities that are related to food ingredients, recipes, and culinary techniques, as well as their historical usage. In other words, it is the outcome of interaction among food and culture. The constant evolution of gastronomy has led us to develop a more scientific approach to food preparation and consumption, formally called molecular gastronomy. This new and exciting branch of food science provides the scientific-based explanations for transformation of physical and chemical properties of food (Caporaso & Formisano, 2016). However, the number of studies evaluating the effects of culinary techniques and nonconventional processing methods on bioaccessibility and bioavailability of nutrients and bioactive compounds is limited. At this stage of development, it is of great importance to have data about bioaccessibility and bioavailability of nutrients and their association with new and conventional processing techniques, as they represent a limiting factor for the development of new and healthy products.

Sensory analysis

Technological aspects

Scientific data


Alleged health claim

Quality control Final product

Fig. 1.1  Schematic representation of an integrated approach between gastronomic science, food science and technology, and nutrition in the development of healthy product.

An integrated strategy in the development of food products


1.2 Culinary techniques Cooking techniques are a set of methods and procedures for preparing, cooking, and presenting food, which have a key role in the physicochemical and organoleptic characteristics of food as well as food’s nutritional quality. The use of appropriate processing conditions and culinary techniques is essential for the development of nutritious meals and for planning a healthy diet. Food processing and preparation by consumers allow for various degrees of control over added ingredients and retention of most of natural nutrients (e.g., bioactive compounds) from foods. Generally, homemade and directly obtained fresh foods have higher nutritive value than that of canned and heavily processed ones. Commonly, thermal processing can be used to achieve such goals, although cooking foods above 100°C for a long period of time is usually not recommended. If excessive, each of the cooking techniques and technologies will tend to damage the native nutritive content of foods (Fig. 1.2) (Aranceta Bartrina et al., 2016). For example, overcooking will significantly change the physical parameters (e.g., texture and color) and chemical composition of food, and impacts on the loss of vitamins, denaturation of proteins, and bioavailability of bioactive compounds (Table 1.1). Selection of appropriate culinary techniques may also increase in vivo ­bioavailability of many bioactive compounds, when food is properly cooked (Miglio, Chiavaro,

Fig. 1.2  Impact of conventional culinary techniques on nutrient and physicochemical properties retention, fat and salt reduction.


Table 1.1  Effect of different cooking methods on the nutrient and bioactive compound composition of foods. Source


Treatment time (decrease)




60 min (53%)

Cauliflower Tomato

Glucosinolates Polyphenols

30 min (75%) 10 min (38%)

Tomato Carrot Spinach Potato Lentil Chickpea Onion Onion Onion Carrot Kale Broccoli Brussels sprouts Peas Broccoli Broccoli Peas

Polyphenols Polyphenols Folate Folate Thiamine Thiamine Flavonoid Flavonoid Flavonoids Lutein Polyphenol Vitamin C Vitamin C Folate Folate Glucosinolate Folate

10 min (11.5%) 10 min (32%) 3.5 min (50%) 60 min (17.8%) 35min (34.3%) 35 min (48.9%) 60 min (25%) 5 min (19%) 15 min (29%) 15 min (42.7%) 5 min (29.3%) 30 min (20%) 30 min (64.8%) 4 min (27%) 10 min (9%) 13 min (26%) 4 min (16.7%)

Sautéed Frying Microwaving


Reference Rodrigues, Pérez-Gregorio, García-Falcón, and SimalGándara (2009) Song and Thornalley (2007) Ramírez-Anaya, Samaniego-Sánchez, Castañeda-Saucedo, Villalón-Mir, and De La Serrana (2015) Dolinsky et al. (2016) Dolinsky et al. (2016) McKillop et al. (2002) McKillop et al. (2002) Prodanov, Sierra, and Vidal-Valverde (2004) Prodanov et al. (2004) Takenaka et al. (2007) Lombard, Peffley, Geoffriau, Thompson, and Herring (2005) Rodrigues et al. (2009) Miglio et al. (2008) Dolinsky et al. (2016) Pellegrini et al. (2010) Pellegrini et al. (2010) Stea, Johansson, Jägerstad, and Frølich (2007) McKillop et al. (2002) Pellegrini et al. (2010) Stea et al. (2007)

Innovative Thermal and Non-Thermal Processing


An integrated strategy in the development of food products


Visconti, Fogliano, & Pellegrini, 2008). Some examples of such effects can be observed for iron and food prepared in cookware made of this metal that will increase its bioavailability (Patted, Bharthi, & Sreeramaiah, 2012). Maillard reactions between reducing sugars and amino acids, commonly occurring during cooking, can increase the bioavailability of phosphorous that is an essential nutrient for skeletal mineralization and pH regulation (Roncero-Ramos, Delgado-Andrade, Alonso-Olalla, & Navarro, 2012). Furthermore, certain nutrients become bioavailable only after cooking as they are released in the process and made available for absorption. For instance, cooking of grains will inhibit the activity of several antinutritional factors (i.e., phytic acid), which reduces the absorption of minerals such as iron and hemagglutinin, a toxin with agglutinatory activity on red blood cells (Kumar, Sinha, Makkar, & Becker, 2010; Thompson, Rea, & Jenkins, 1983). Inappropriate choice of cooking technique and/or preparation vessels can induce the formation and accumulation of toxic compounds. In this context, the high consumption of processed and canned foods will increase the proportion of ingested aluminum, which is consider a toxic heavy metal involved in the increasing risk of neurodegenerative disease (Cabrera-Vique & Mesías, 2013). Additionally, cooking method affects the bioaccessibility of organic pollutants, such as polychlorinated biphenyls and polychlorinated dibenzo-p-dioxins/furans, which can induce the development of defects and lesions. Particularly for foods of vegetal origin, frying greatly increases the bioaccessibility of both toxic compounds (Shen et al., 2016). Some of the most common techniques used to prepare foods at the household level are described next. For instance, steaming is a moist-heat cooking technique that cooks food in a suspended support by direct contact with water vapor inside a covered pan. It is a very advantageous technique since the temperature of food does not exceed 100°C and requires only a few minutes to achieve the expected characteristics. In general, nutritional quality is preserved, but the processing time is dependent on the type of food (Fabbri & Crosby, 2016). Alternatively, boiling can be explained as a method where food is cooked in water at high temperature (Fabbri & Crosby, 2016). The impact of boiling on the nutritional properties of foods is related to preparation time and food type. For broccoli and cauliflower, contrasting results were reported in the literature; boiling for more than 10 min reduces the acceptance of broccoli, while no differences were observed for cauliflower (Poelman, Delahunty, & de Graaf, 2013). Boiling food for longer periods (more than 1 h) is a common technique to produce broths and soups, particularly for vegetables as the main ingredient in these dishes, wherein pressure is also an important factor for improving the acceptance of such meals (Mougin et al., 2015). Regarding griddling/grilling, intense heat leads to the rise of gas bubbles toward the surface (e.g., in griddle cakes such as pancakes), which causes the aerated and tender structure of prepared foods. In a similar way, grilling is one of the main methods for meat preparation that denatures proteins and creates a crunchy layer that allows the food to keep the juices inside (McGee, 2004). Oven cooking is an easy and clean but slow method for preparing large amounts of food with little or no added fats, at temperatures around 220°C (Varela, Salvador, & Fiszman, 2008). During the process, a toasted layer is created, sealing the food and


Innovative Thermal and Non-Thermal Processing

preserving most of the juices inside. In meats, this technique also leads to formation of the characteristic roasted flavor (Holtz, Skjöldebrand, Jägerstad, Reursswärd, & Isberg, 1985). Sautéing consists of cooking food, usually vegetables, with a little oil in a pan or wok and with constant stirring. Thus the ingredients are crispy and appetizing with little addition of fat (Fabbri & Crosby, 2016). Frying food involves the heating of food, whether or not coated, at higher temperatures by direct contact with oil, which induces evaporation of water. “Internal cooking” induces the absorption of oil and changes in sensory properties of food, thus resulting in the development of flavor and changes in texture (Fabbri & Crosby, 2016). Fritters (food coated in batter and deep-fried) should be consumed in moderation, made with extra-virgin olive oil, at a temperature around 160°C. In deep-frying, oil covers food that is incorporated with the oil just at the point of heat. This procedure avoids the deterioration of oil, the formation of undesirable products, and generates the formation of an external crust that minimizes the penetration of the oil inside the food. However, reuse of darkened oil with traces of the previous frying is not recommended due to formation of toxic compounds (Choe & Min, 2007; Saguy & Dana, 2003; Ziaiifar, Achir, Courtois, Trezzani, & Trystram, 2008). A microwave is an oven that emits high-frequency electromagnetic waves causing molecular friction among water molecules, which usually heats food up to 100°C in a short period of time. In this way, microwaving foods tends to preserve nutrients and organoleptic properties in comparison to other techniques involving conventional heating. However, texture may be adversely affected due to water migration from inner layers of food to the surface where it condenses due to lower temperature of air surrounding the food (Chandrasekaran, Ramanathan, & Basak, 2013; Fabbri & Crosby, 2016). In addition, the elderly favor the use of microwave ovens as it helps them avoid efforts to bend down in order to put foods in the oven (Wylie, Copeman, & Kirk, 1999).

1.3 Thermal processing Commonly, thermal processing of foods is required to achieve longer storage periods than those obtained for raw or unprocessed food. Although each food requires specific conditions, thermal processing mainly inactivates enzymes and microorganisms, destroying antinutritional factors along with changing the characteristics of food. The intensity of thermal processing is directly correlated with the reduction in the activity of the enzymes and microorganisms. However, the increase in shelf life may affect other characteristics of food associated with flavor, taste, color, and texture. Changes associated with nutritional profile vary according to the nutrient type and intensity of treatment (Barba, Esteve, & Frígola, 2012; Barba, Mariutti, et al., 2017; Omidizadeh et al., 2011; Petruzzi et al., 2017). From a technological point of view, thermal processing can be divided into four categories with regards to the combination of temperature and time: high temperature for a long period (HTLP), high temperature for a short period (HTSP), mild t­emperature

An integrated strategy in the development of food products


for a long period (MTLP), and mild temperature for a short period (MTSP). In HTLP treatment, food is subjected to temperatures higher than 80°C, eventually reaching temperatures above 100°C, and lasts for several minutes, while in HTSP the same range of temperature is observed, but only for a few minutes or seconds. On the contrary, MTLP and MTSP foods are treated at temperatures lower than 80°C, but for longer (several minutes) or shorter (few minutes or seconds) periods of time, respectively (Petruzzi et al., 2017). Among the many possible applications of thermal processing, blanching is a specific step in processing to inactivate endogenous enzymes (such as polyphenol oxidases and peroxidases). The importance of this step is due to microbial and enzymatic activity involved in characteristic browning of some fresh fruits and vegetables (Putnik & Bursać Kovačević, 2017; Putnik, Bursać Kovačević, Herceg, & Levaj, 2016, 2017a, 2017b; Putnik, Bursać Kovačević, Herceg, Pavkov, et al., 2016; Putnik, Bursać Kovačević, Herceg, Roohinejad, et al., 2017). Drying with hot and dry air is also a common thermal technology to preserve food that removes water and prevents the development of spoilage and pathogenic microorganisms (Nayak, Liu, & Tang, 2015).

1.4 Nonthermal processing The traditional thermal processing technologies can have a negative impact on the nutritional profile in various foods (Barba, Putnik, et al., 2017; Koubaa et al., 2017; Önür et al., 2018; Poojary, Putnik, et al., 2017; Putnik, Barba, Lorenzo, Gabrić, et al., 2017). In this section is a short discussion of the effects of nonthermal food-processing technologies on preserving the nutritional profile of foods with a focus on some of the novel nonthermal processing technologies such as high-pressure processing (HPP) (high hydrostatic pressure), high-pressure homogenization (HPH), pulsed electric fields (PEF), ultrasound (US), and supercritical CO2 (SC-CO2) (for detailed information see reviews of Barba et al., 2012, 2015; Barba, Mariutti, et al., 2017; Barba, Putnik, et al., 2017; Gabrić et al., 2018; Misra et al., 2017; Zinoviadou et al., 2015).

1.4.1 High-pressure processing HPP applied to food products at room temperature has shown negligible effects on nutritional profile and preservation of the original quality of foods with enhanced shelf life. In the early 1990s, a Japan-based Meidi-Ya company first commercialized HPP fruit jams (Hori et al., 1992). During the same decade, HPP ready-to-eat meats were authorized by the United States and Canada for the control of risk associated with Listeria monocytogenes. HPP has been reported as one of the emerging, economical, and nonthermal technologies that allows processing of heat-sensitive liquid foods in a semicontinuous method for both solid and liquid foods in batch-type equipment (Considine, Kelly, Fitzgerald, Hill, & Sleator, 2008; Nguyen, Rastogi, & Balasubramaniam, 2007; Rastogi, Raghavarao, Balasubramaniam, Niranjan, & Knorr, 2007). Hendrickx, Ludikhuyze, Van den Broeck, and Weemaes (1998) reported that the exposure of proteins to high pressures results in structural changes governed by


Innovative Thermal and Non-Thermal Processing

Le Chatelier's principle (decrease in volume is associated with an increase in pressures). Based on this principle (phase-transition phenomenon), chemical reactions take place leading to changes in molecular configuration, denaturation of proteins, and breakdown of some specific low-energy bonds. Pressures ranging from 200 to 700 MPa can sterilize and stabilize both low-acidic and acidic foods at near-room temperatures. Efficiency is independent of the packaging material, dimensions, and geometry of foods as high pressure is quickly and uniformly transmitted in all directions (Farkas, 2011). Cilla, Bosch, Barberá, and Alegría (2018) reported that HPP treatment does not induce the formation of undesirable reaction by-products, such as those formed during thermal treatments, but rather leads to improved microbial safety with the highest nutritional and sensorial quality of foods. HPP preserves flavor, color, and nutritional content by preserving the covalent bonds in food products (Farkas, 2007). The commercial HPP application for pressure-assisted pasteurization of lowacid juices and beverages in the range of 400–450 MPa can be done in less than 10 min (Oey, Faridnia, et al., 2016; Tonello, 2011). HPP treatment can also preserve and increase the content of bioactive compounds, such as anthocyanin in apples (George, Selvan, & Rastogi, 2016), carotenoids in red pepper (Hernández-Carrión, VázquezGutiérrez, Hernando, & Quiles, 2014) and honey (Akhmazillah, Farid, & Silva, 2013), vitamin A in orange juice (Plaza et al., 2011), and vitamin C in blueberry juice (Barba, Esteve, & Frigola, 2013). HPP also increases the extractability of flavonols from onion (Fernández-Jalao, Sánchez-Moreno, & De Ancos, 2017) and lycopene from tomato (Gupta, Kopec, Schwartz, & Balasubramaniam, 2011).

1.4.2 High-pressure homogenization HPH is a process applied to yield a homogenous size distribution of particles suspended in a liquid by applying pressure. The application of HPH is primarily useful for liquids, where foods are forced through a narrow valve achieving high pressure and high velocity to induce required physical changes (Misra et al., 2017). The combination of shear forces produces impact among particles and induces cavitation. Nowadays, HPH is employed in production of dairy, cosmetic, and beverage products (Misra et al., 2017). Pharmaceutical industries use HPH for microbial and enzymatic inactivation, functionality improvement, and particle size reduction. The recovery of intracellular by-products by disruption of the cells was the first notable application of HPH (Patrignani & Lanciotti, 2016).

1.4.3 Pulsed electric fields Among the nonthermal emerging technologies, PEF is one of the most promising used for pasteurization of liquid foods (Barba et al., 2015; Oey, Roohinejad, et al., 2016). The first application of PEF was reported in the 1960s (Doevenspeck, 1961). Food is placed between two electrodes that deliver high-intensity electricity (20–80 kV/cm) during a short period of time (microseconds) at room temperature. The electric fields can be applied in different forms: exponentially decaying waves, square waves, and bipolar or oscillating pulses (Puértolas & Barba, 2016; Puértolas, Koubaa, & Barba, 2016).

An integrated strategy in the development of food products


The primary application of PEF is to disintegrate biological tissues and microorganisms (Barba et al., 2015; Gabrić et al., 2018; Roohinejad, Everett, & Oey, 2014; Roohinejad, Oey, Everett, & Niven, 2014; Silva et al., 2016). The microbial inactivation mechanism involves the exposure of microbial cells to a high-intensity electric field, either temporary or permanently, to destabilize the lipid bilayer and proteins of microbial membrane cells (i.e., electroporation). This allows the entry of small molecules, such as water, to cause swelling (phase), after which microbial cells eventually rupture and became inactive (cell lysis phase) (Roohinejad, Koubaa, Sant’Ana, & Greiner, 2018). The effect of PEF on microbial load depends on several factors, including type of organism, field intensity, temperature, membrane surface potential, pH, shape of the pulse, pulse duration, and number of pulses (Barba et  al., 2015; Misra et al., 2018; Pal, 2017). Regarding the inactivation of enzymes, PEF has been associated with structural and conformational changes of proteins, most probably due to disruption of electrostatic interactions within enzymes (Buckow, Ng, & Toepfl, 2013; Poojary, Roohinejad, et al., 2017). In a dairy pudding dessert produced from yogurt, PEF combined with mild heat treatment reduced the total viable aerobic bacteria as well as total molds and yeasts during storage at both 4°C and 22°C. Authors also obtained similar scores for sensory analysis (Yeom, Evrendilek, Jin, & Zhang, 2004). In addition, PEF technology can be also exploited as a pretreatment to enhance mass transfer during drying of apple and potato slices as reported by Arevalo, Ngadi, Bazhal, and Raghavan (2004). They obtained higher diffusion coefficients for samples treated with PEF than untreated slices. The potential application of PEF for cooking food has been also evaluated with promising results (Blahovec, Vorobiev, & Lebovka, 2017).

1.4.4 Ultrasound (US) US consists of sound waves propagating with frequencies higher than 20 kHz, which is higher than the threshold level to human hearing. The sonic waves are divided into two categories according to intensity: low-intensity US waves are characterized by frequencies lower than 100 kHz with energies less than 1 W/cm2 and high-intensity US waves are characterized by frequencies higher than 100 kHz with energies more than 10 W/cm2. The cavitation is the main mechanism that results in physical and chemical changes of liquids, disruption of biological cells, emulsifications, and mixing materials, along with microbial and enzymatic inactivation (Misra et al., 2018). In early 1929, the first observation of microbial lethality of US was reported. In liquids, US waves propagate longitudinally by creating alternate compression and expansion cycles. The combination of rapid change in pressure induced by the US and liquid incompressibility produces localized regions of reduced pressure (lower than vapor pressure of the liquid) that results in formation of small bubbles (Zinoviadou et al., 2015). After eventual growth, these bubbles expand and become unstable ending with collapse that will create high-temperature and high-pressure regions (Paniwnyk, 2017). The increase in temperature is localized and the overall increase in surrounding medium is up to 5°C (Gould, 2001). The implosion of bubbles and formation of high-temperature spots induces sonolysis, which results in the formation of very


Innovative Thermal and Non-Thermal Processing

r­ eactive species. Among all the reactive species, hydroxyl radicals are mostly responsible for oxidative reactions, termed as advanced oxidation process (AOP) that will destroy microorganisms and organic compounds. Some of the areas in the food industries that US was successfully applied are microbial and enzymatic inactivation, crystallization, rheology, fractionation, filtration, drying, extraction, emulsification, meat tenderization, degassing, defoaming, freezing, and oxidation processes (Koubaa et al., 2016; Misra et al., 2018; Roselló-Soto et al., 2015, 2016; Zhu, Guan, et al., 2017; Zhu, Wu, et al., 2017; Zinoviadou et al., 2015).

1.4.5 Supercritical fluids The supercritical technology is one of most widely used techniques in food industries, mainly for the extraction of flavors and bioactive compounds as an alternative to organic solvents. Particularly for the use of CO2 as a solvent, it has the following advantages: low cost, nonflammability, abundant availability, adjustable solvent properties, nontoxicity, selective fractionation, and moderate critical temperature and pressure (31.1°C and 7.4 MPa). SC-CO2 has been used for decaffeination of coffee and extraction of flavors, bioactive compounds, and aromatic oils mainly from food of plant materials (Brown, Fryer, Norton, Bakalis, & Bridson, 2008). The use of supercritical (SC) fluids technology in the food industry combines the knowledge of both supercritical fluid and extrusion operations. In this process, supercritical fluid is injected in food mass, under specific pressure and temperature, before mass leaves the extruder. The extrudate expands due to rapid change in pressure and temperature and shrinks due to cooling of inner gases. The combination of both technologies is also suggested to facilitate the inclusion of fat-soluble micronutrients and food additives trough SC-CO2 injection. Puffed food such as morning cereals, pasta, and confectionary can be produced by this technique (Rizvi, Mulvaney, & Sokhey, 1995). In addition, Rawson et al. (2012) reported that SC-CO2 had a significant lethality for microorganisms and deactivates enzymes, deodorizes liquid foods and medicines while being nontoxic, and is easily removed by depressurizing and outgassing. Microbial inactivation involves formation of ruptures in cells by rapid pressure release and expansion of CO2, extraction of lipids from the cell membrane, and decrease in pH due to the formation of carbonic acid.

1.5 European legislation for development and labeling of healthy food products The European Parliament has already established regulations for development of new food products (Regulation No 258/97), which determines that novel foods and food ingredients must not present a danger to or mislead consumers, differ from original ingredients that are intended to be replaced in foods, and must not be nutritionally disadvantageous. This regulation requires mandatory information regarding the composition, nutritional value/nutritional facts, and intended use of the food, which must be declared on the food’s label (European Commission, 1997). Along with these aspects, the discussion about the importance of healthy eating and development of nutritious products can also be extended to health and nutritional claims, also present on food labels in Europe.

An integrated strategy in the development of food products


Healthy eating is important for the development of healthy products in both the general population and children, as discussed and established by the European Parliament. The displayed information on the food label is also related to the development of such foods. Food business operators can highlight the nutritional and health benefits associated with consumption of their product, but only after approval from the European Parliament. Nutritional claims should be understood in the context of a specific nutrient in comparison to standard products or foods (e.g., free, low, and high). Also, a health claim refers to the relationship between the nutrient or ingredient and the risk reduction for a particular disease or health-related condition. A statement such as the “reduction of disease risk” on a product label is also permissible when a food category, particularly a food or food component, reduces the risk for a specific disease (European Commission, 2006). Some nutrients and food components from the European Commission’s Regulation No 1924/2006 (December 20, 2006), such as fats, saturated fats, trans-fatty acids, salt/sodium, and sugars, are worth noting due to their negative impact on health after excessive and constant consumption. Other nutrients such as fibers, vitamins, and minerals are also incorporated in this regulation. However, such legislation does not consider the presence of important bioactive compounds, such as polyphenols and other bioactives for health relevance. In addition, caloric energy intake or food caloric value per mass of a product (or standardized portion) is included in this regulation (European Commission, 2006). The increased consumption of fruits, vegetables, and dairy products is considered crucial as one of the main preventive actions for reducing childhood obesity. Regulation No 2016/791 (May 11, 2016) facilitates and establishes specific rules for the distribution of fruit and vegetables, and milk and milk products, in nurseries, preschools, and primary and secondary levels of education. Lawmakers defined those rules due to the nutritional and health importance of such foods on children’s development (European Commission, 2016). In addition, health claims from food labels of processed products are informative for the child market, according to Regulation No 432/2012 (May 16, 2012). Many nutrients are targeted with this regulation, such as calcium, guar gum, iron, niacin, vitamin C, and zinc (European Commission, 2012). For commercialization in Europe, the newly designed and healthier products must be produced under the parameters established by current regulations of the European Parliament. Health and nutritional claims can bring advantages for food industries and consumers; to help the former to became more competitive and contribute to production of foods with improved nutritional profiles, and for the latter to select the most suitable products to maintain a healthy diet.

1.6 Challenges and future perspectives The development of alternative, novel nonthermal technologies is necessary for sustainable processing, decreased energy consumption, reduced environmental pollution, and a healthier society. Recent studies highlighted the presence of health-­ promoting compounds in fruits and vegetables that have stimulated the demand for alternative nonthermal technologies, capable of extracting such compounds in an


Innovative Thermal and Non-Thermal Processing

environmentally friendly manner. The response to consumers’ demands for minimal processing of foods has resulted in increased interest in nonthermal technologies suitable for inactivating microorganisms and enzymes, while promoting retention of quality attributes such as flavor, scent, color, texture, and nutritional parameters. The challenge to the introduction of any novel technology into the food industry (for established and startup enterprises) arises from the perception that it is disruptive, risky, and difficult to implement when faced with day-to-day competitive pressure. The knowledge derived from the integration of gastronomy, food science, and nutrition is the key information for the development of new products with improved nutritional and healthy claims. However, the use of nonthermal technologies still demands additional studies, particularly with poorly explored culinary techniques by both researchers and cooks. Specific regulations imposed by the regulatory authorities must be considered regarding the safety and nutritional profiles of the food processed by nonthermal technologies. Moreover, some important production aspects must be considered as well, namely, higher investments and cost of production, low availability of commercial equipment, and lack of underdeveloped specific regulations relevant for nonthermal technologies. In this sense, more attention should be brought to bioactive compounds and their positive influence on health and wellbeing, and legal claims relevant to food labeling. Exchange of knowledge between gastronomic sciences, professional cooks, food technologists/engineers, and nutritionists must be facilitated to develop dishes suitable for nonthermal technologies. In addition to the increase of information about unexplored aspects and the intersection between various food disciplines, the food industry and regulatory bodies are able to establish specific regulation guidelines about food safety and development of healthy competition. Thus, the development of healthier foods, either by changes in composition or processing technologies, should be better associated with the concepts of bioaccessibility and bioavailability for all the nutrients (including neglected bioactive compounds). This consideration is due to the importance of various bioactives on health, their changes in processing, and subsequent digestion by the human gastrointestinal tract. Accordingly, the next chapters are dedicated to explanations of the main aspects of these relationships regarding the evaluation of bioaccessibility and bioavailability, green technologies, and the impact of processing on foods commonly found around the world.

Acknowledgments The authors José M. Lorenzo and Paulo E.S. Munekata are members of the MARCARNE network, funded by CYTED (ref. 116RT0503). Paulo E. Munekata acknowledges postdoctoral fellowship support from Ministry of Economy and Competitiveness (MINECO, Spain) ‘‘Juan de la Cierva” program (FJCI-2016-29486). Moreover, F.J.B. and J.M.L. would like to thank Generalitat Valenciana for the financial support (IDIFEDER/2018/046—Procesos innovadores de extracción y conservación: pulsos eléctricos y fluidos supercríticos) through European Union ERDF funds (European Regional Development Fund). P. Putnik, wish to thank Croatian Science Foundation for support through the funding of the project: “High voltage discharges for green solvent extraction of bioactive compounds from Mediterranean herbs (IP-2016-06-1913).”

An integrated strategy in the development of food products


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Further reading Velazquez-Estrada, R. M., Hernandez-Herrero, M. M., Lopez-Pedemonte, T., Guamis-Lopez, B., & Roig-Sagués, A. X. (2008). Inactivation of Salmonella enterica serovar senftenberg 775W in liquid whole egg by ultrahigh pressure homogenization. Journal of Food Protection, 71(11), 2283–2288.

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Methods for determining bioavailability and bioaccessibility of bioactive compounds and nutrients


Diana I. Santos⁎, Jorge Manuel Alexandre Saraiva†, António A. Vicente‡, Margarida Moldão-Martins⁎ ⁎ Linking Landscape, Environment, Agriculture and Food (LEAF), School of Agriculture, University of Lisbon, Lisbon, Portugal, †QOPNA & LAQV-REQUIMTE, Department of Chemistry, University of Aveiro, 3810-193 Aveiro, Portugal, ‡Centre of Biological Engineering (CEB), University of Minho, Braga, Portugal

2.1 Introduction The composition of foods in bioactive compounds and antioxidant activity is of great significance in dietary habits due to the effects on the prevention of diseases related to free radicals (Pereira, Barros, Carvalho, & Ferreira, 2011). The most abundant antioxidants in ingested foods may not match the higher concentration of these active metabolites in the tissues, whereby the bioaccessibility and bioavailability of each compound varies greatly (Palafox-Carlos, Ayala-Zavala, & González-Aguilar, 2011). Human digestion is a complex process in which ingested foods are transformed into nutrients through mechanical and enzymatic alterations, whereby macromolecules are hydrolyzed into their building blocks, which are absorbed into the bloodstream. The fragmentation of food occurs predominantly in the mouth and stomach, while enzymatic digestion and absorption of nutrients and water occur predominantly in the small and large intestine (Guerra et al., 2012). The quantification of the amount of bioavailable bioactive compounds is more important than determining the quantity of these compounds existent in the foods (Carbonell-Capella, Buniowska, Barba, Esteve, & Frígola, 2014). Methods for measuring bioavailability and/or bioaccessibility of nutrients imply research in humans, mice, pigs, and other animals (in vivo) or simulation in laboratory assays (in vitro) (Parada & Aguilera, 2007). The bioavailability and bioaccessibility of nutrients and bioactive compounds can be determined by various procedures such as in vitro methodologies (simulated gastrointestinal digestion, Caco-2 cell, cell membranes, and others), ex vivo methodologies (gastrointestinal organs under controlled laboratory conditions), in situ

Innovative Thermal and Non-Thermal Processing, Bioaccessibility and Bioavailability of Nutrients and Bioactive Compounds © 2019 Elsevier Inc. All rights reserved.


Innovative Thermal and Non-Thermal Processing

methodologies (intestinal perfusion in animals), and in vivo methodologies (human and animal studies) (Carbonell-Capella et  al., 2014). One of the most frequently used methodologies is in vitro digestion, followed by experiments with Caco-2 cell cultures that aim at simulating bioactive compounds’ uptake in the small intestine (Barba et al., 2017). Numerous studies aimed at determining the bioavailability of the compounds after digestion, since, for example, the effects of antioxidants in vivo depend on their concentrations in vegetables and fruits as well as their bioaccessibility and bioavailability after ingestion (Manach, Williamson, Morand, Scalbert, & Rémésy, 2005; PérezJiménez et al., 2009). The bioavailability of nutrients for absorption in the intestine is rather difficult and changes for a given food depending on processing conditions and interaction with other compounds, chemical status of the nutrient, release from the food matrix, suppressors or cofactors present in the food composition, formation of stable complexes that are gradually metabolized, and so on (Parada & Aguilera, 2007). Therefore the purpose of the present chapter is to explore the different techniques used to evaluate the bioaccessibility and bioavailability of bioactive compounds and nutrients through in vitro and in vivo methods.

2.2 Bioactive compounds and nutrients Bioactive compounds are present in small quantities in foods, mainly in fruits, vegetables, and whole grains, and provide health benefits beyond the basic nutritional value (Gökmen, 2016). Bioactive compounds are molecules that can present therapeutic potential with influence on energy intake, while reducing pro-inflammatory state, oxidative stress, and metabolic disorders (Siriwardhana et al., 2013). Epidemiological studies indicate that high consumption of foods rich in bioactive compounds with antioxidant activity, including vitamins, phytochemicals, and mainly phenolic compounds, such as flavonoids and carotenoids, has a positive effect on human health and could diminish the risk of numerous diseases, such as cancer, heart disease, stroke, Alzheimer’s, diabetes, cataracts, and age-related functional decadence (Hassimotto, Genovese, & Lajolo, 2009; Siriwardhana et al., 2013). Bioactive compounds are capable of modulating metabolic processes and demonstrate positive properties such as antioxidant effect, inhibition of receptor activities, inhibition or induction of enzymes, and induction and inhibition of gene expression (Carbonell-Capella et al., 2014). The diversity of chemical structures of bioactive compounds influences bioavailability and biologic properties, while antinutritional factors can decrease the bioavailability of certain compounds or inhibit digestion enzymes (Septembre-Malaterre, Remize, & Poucheret, 2018). Potential health effects of bioactive compounds and nutrients are dependent on the digestion process, as this affects bioactive compounds and their stability and as a consequence affects the bioavailability and potential beneficial effects on health (Carbonell-Capella et al., 2014).

Methods for determining bioavailability and bioaccessibility


2.3 Concepts 2.3.1 Bioavailability An important issue in feeding is the bioavailability of bioactive compounds and ingested nutrients. Nevertheless, the bioavailability of nutrients is an important but also unclear concept, related with the efficiency of absorption and the metabolic application of an ingested nutrient (Gregory, Quinlivan, & Davis, 2005). The rate and extent to which the compound is absorbed and becomes available for host cellular metabolism may be defined as bioavailability. In other words, it is the “fraction of nutrient from ingested dose that is absorbed.” Bioavailability is influenced by gastrointestinal digestion, which releases the components from the food matrix, absorption by epithelial cells, metabolism, tissue distribution, and bioactivity (Gorusupudi & Bernstein, 2016). Bioavailability is usually measured by in  vivo methods through the portion of nutrients or phytochemicals that are digested, absorbed, metabolized, and reach the systemic circulation. In order to simplify the analyses, the term “bioavailability” is frequently defined as the fraction of compound or its metabolites reaching the systemic circulation without including bioactivity (Holst & Williamson, 2008). Therefore bioavailability is determined by in vivo plasma concentration in animals or humans following administration of a dose of a compound-containing food or of the compound itself (Rein et al., 2013). Physiological status, individual variability, presence, and dose of other meal components are factors influencing blood plasma determinations of humans (in vivo assay) (Faulks & Southon, 2005). The bioavailability of bioactive compounds may change due to combination with macronutrients, namely the fiber content of low-processed foods and beverages and the polysaccharides and proteins of processed foods (Dupas, Baglieri, Ordonaud, Tomè, & Maillard, 2006). The bioavailability of each polyphenol is distinct and there is no relationship between the amount of polyphenol in the food and its bioavailability in the human body, since bioavailability depends on digestive stability, food matrix release (bioaccessibility), and efficiency of transepithelial passage. In bioavailability tests, it must be confirmed that the compound is efficiently digested, assimilated, and after absorption, that it promotes a positive influence on human health. These methodologies present some experimental and ethical constraints for measuring bioactivity (Gawlik-Dziki, 2012).

2.3.2 Bioactivity Bioactivity is the physiological response to exposure to a given compound, following tissue uptake. Digestibility corresponds to the amount of food constituents that are converted by the digestion process into compounds available to the lumen, and assimilation refers to the entry of bioaccessible compounds through the epithelium by transepithelial absorption mechanisms (Etcheverry, Grusak, & Fleige, 2012). The definition of bioactivity is associated with the way bioactive compounds reach the systemic circulation, are carried and reach the specific tissue, the interaction


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with the metabolism of biomolecules in the tissues, and the physiological responses it causes (Cardoso, Afonso, Lourenço, Costa, & Nunes, 2015; Fernández-García, Carvajal-Lérida, & Pérez-Gálvez, 2009). Food components are subject to diverse physical, chemical, and biochemical conditions during digestion that change the bioavailability and bioactivity of the bioactive compounds (Hur, Lim, Decker, & McClements, 2011). For example, some ingested phenolics are not assimilated in the small intestine and pass into the large intestine, where the intestinal microbiota causes structural alterations and forms specific metabolites (Mosele, Macià, Romero, Motilva, & Rubió, 2015).

2.3.3 Bioaccessibility Bioaccessibility may be characterized as the amount or portion of a compound that is released from the food matrix in the gastrointestinal tract and becomes accessible for absorption. This definition includes the release of compounds from food matrices and the stability of the respective compounds under the gastrointestinal environment (Gawlik-Dziki, 2012; Tagliazucchi, Verzelloni, Bertolini, & Conte, 2010). An alternative definition of bioaccessibility, more rigorous and less utilized, defines bioaccessibility as the portion of a compound released from the food matrix in the gastrointestinal system, becoming thus accessible for intestinal absorption, including uptake/assimilation into epithelial cells' intestinal metabolism and also in the presystemic metabolism (hepatic and intestinal) (Cardoso et al., 2015). Bioaccessibility is frequently determined by simulating gastric and small intestinal digestion using in vitro digestion processes and sometimes monitored by Caco-2 cells uptake (Courraud, Berger, Cristol, & Avallone, 2013). The initial concentration of a compound in the food matrix, matrix composition, and factors associated to the host, such as the physicochemical characteristics of gastrointestinal fluids and the presence of digestive enzymes, influence the bioaccessibility of the compounds (Tagliazucchi, Verzelloni, & Conte, 2012). Nutrients are potentially bioaccessible, but in reality, few nutrients are fully converted into absorbable forms during digestion. The bioaccessibility and bioavailability of the nutrients are limited by the physical characteristics of the food matrix, which influence the efficiency of the physical, chemical, and enzymatic digestion processes (Boyer & Liu, 2004). A summary of the different models to study bioaccessibility and bioavailability of bioactive compounds including in vitro, ex vivo, in situ, and in vivo methodologies, is shown in Fig. 2.1.

2.4 Bioavailability measurements Bioavailability is assessed by in vivo analysis of the metabolites present in blood and/ or urine after food consumption (Carbonell-Capella et al., 2014). Numerous factors influence the bioavailability of compounds, namely their bioaccessibility and the capacity to pass through the intestinal mucosal barrier (Crozier, Del Rio, & Clifford, 2010). In most studies related to the bioavailability of polyphenols, bioaccessibility is not

Methods for determining bioavailability and bioaccessibility

Artificial gastrointestinal systems

In vitro


Ex vivo

Gastrointestinal organs

In vivo

Animal or human studies


Intestinal perfusion in animals

In situ

Fig. 2.1  Methodologies used to determine the bioavailability and bioaccessibility of bioactive compounds.

considered and bioavailability is evaluated essentially using pure, simple compounds (Manach et al., 2005; Saura-Calixto, Serrano, & Goñi, 2007). Some compounds are hydrolyzed by intestinal enzymes and by bacterial action in the large intestine prior to absorption. These compounds undergo modifications in intestinal and hepatic cells and reach the blood and tissues in forms other than those existent in foods, making it difficult to detect all metabolites and assess their biological activity (Pandey & Rizvi, 2009). Several in vivo studies use animal models and human volunteers to study the bioavailability (Lafay et al., 2006; Rondini et al., 2004; Zhao, Egashira, & Sanada, 2004).

2.5 Bioaccessibility measurements Bioaccessibility is determined by in vitro methodologies, which estimate the amount of compounds available for intestinal absorption (Carbonell-Capella et al., 2014). The evaluation of bioaccessibility can be determined by different in vitro methods that include simulated gastrointestinal digestion, intestinal sections, brush-border and basolateral membrane vesicles, enterocytes, and altered intestinal cell lines, predominantly the human Caco-2 cells (Cilla et al., 2012). The digestive process begins in the mouth through chewing and causes release of the bioactive compounds existent in food. Thereafter, during gastrointestinal digestion, the hydrolytic enzymes and the physicochemical properties of the secretions act on the food bolus and affect the bioaccessibility (Tagliazucchi et al., 2012). Gastric acidity makes phenolic compounds stable and improves the release of compounds from the food matrix (Chandrasekara & Shahidi, 2012; Tagliazucchi et  al., 2010). Phenolics suffer alterations under alkaline pH, characteristic of the small intestine. Proteins and polyphenols or digestive enzymes may interact before absorption, which may also affect the bioaccessibility of these compounds. Bioaccessibility may be reduced or improved during gastrointestinal digestion due to interaction with other food components such as lipids, sugars, and fibers (Ferruzzi, Bordenave, & Hamaker, 2012). In vitro bioaccessibility experiments do not substitute for in vivo assays, but are important for studying the influence of gastrointestinal digestion and food matrices


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on the systemic bioavailability of compounds (Chandrasekara & Shahidi, 2012). The bioaccessibility of phenolic compounds and other antioxidants from solid matrices is essential, since only the components released from the food matrix and/or assimilated in the small intestine are able to become bioavailable to exert their beneficial properties in the body (Tagliazucchi et al., 2010). Reducing foods to small particles through mastication increases the surface available to the action of digestive enzymes, thus enhancing the digestion effectiveness and gastrointestinal absorption of antioxidants. Only the antioxidants that are released from the food matrix as a consequence of the activity of digestive enzymes and bacterial microflora are available in the intestine and consequently bioavailable (SauraCalixto et al., 2007).

2.6 Methods to determine bioavailability and bioaccessibility of nutrients and bioactive compounds The bioavailability of the bioactive compounds present in foods is determined by the low bioavailability in the small intestine caused by the physicochemical interactions of the antioxidants with the indigestible polysaccharides of the cell walls. The compounds released during processing and digestion may associate with other food constituents in the gut, namely macromolecules such as fibers, and form chemical complexes and colloidal structures that impair or enhance bioavailability. The interactions and consequences of dietary fiber on the bioabsorption of components in the gastrointestinal tract influence the nutritional value and the true impact of phytochemicals (PalafoxCarlos et al., 2011). Table 2.1 summarizes methods for evaluating bioaccessibility and bioavailability by in vivo and in vitro.

2.6.1 Gastrointestinal models Models simulating gastrointestinal digestion under laboratory conditions are called gastrointestinal models and may be static or dynamic. An in  vitro gastrointestinal digestion simulates the physiological procedures, such as transit time, enzymatic ­conditions, and pH, that take place in the gastrointestinal region of the human ­digestive Table 2.1  Methods for evaluating bioaccessibility and bioavailability. Methodologies



In vivo

Balance studies Tissue analysis Intestinal absorption availability Intestinal absorption Presystemic metabolism

Animal and human

In vitro

Static digestion model Dynamic digestion model Cell culture studies

Methods for determining bioavailability and bioaccessibility


Table 2.2  Characteristics of the human gastrointestinal tract. Gastrointestinal system



Transit time


Chewing and mixing with saliva


10 s–2 min


Mechanical and enzymatic processing of ingested bolus Breaking down of macromolecules and absorption of nutrients Microbial fermentation of undigested food and water reabsorption


15 min–3 h


2–5 h

Pancreatic juice, bile, and NaHCO3


12–24 h


Small intestine


Conditions Salivary enzymes: amylase, lingual lipase HCl, pepsin, gastric lipase

Based on Guerra, A., Etienne-Mesmin, L., Livrelli, V., Denis, S., Blanquet-Diot, S., & Alric, M. (2012). Relevance and challenges in modeling human gastric and small intestinal digestion. Trends in Biotechnology, 30(11), 591–600.

system (Buniowska, Carbonell-Capella, Frigola, & Esteve, 2017). In static models, the digestion products are immobilized and do not mimic physical processes like shearing, mixing, hydration, and others. In the case of dynamic models, the physical and mechanical events and alterations in luminal conditions occur to better simulate in vivo conditions. These models allow monitoring the changes in the physical conditions of food and the released fluids during digestion over time considering mixing, diffusion, formation of colloidal phases, and others (Parada & Aguilera, 2007). The main characteristics of the human digestive system are summarized in Table 2.2. The gastrointestinal system consists of a simulation of the initial digestion with pepsin-HCl (gastric digestion), followed by digestion with pancreatin with bile salts (small intestine conditions), and ending with dialysis (absorption process) (BermúdezSoto, Tomás-Barberán, & García-Conesa, 2007). In vitro digestion systems were developed to study the physical and chemical processes occurring during the digestion of bread carbohydrates and proteins (Hoebler et al., 2002), antioxidants release from whole foods (Nagah & Seal, 2005), iron available in citrus fruit juices (Haro-Vicente, Martínez-Graciá, & Ros, 2006), and phenolic compounds from orange juices, strawberries, and strawberry jam (Gil-Izquierdo, Zafrilla, & Tomás-Barberán, 2002). The effects of gastric and pancreatic digestion in vitro on the composition and stability of the main polyphenols in chokeberry juice have also been evaluated (Bermúdez-Soto et al., 2007). In vitro digestion models In vitro digestion models were developed based on human physiology to be simple, economical, and reproducible to investigate structural modifications, digestibility, and release of food constituents under simulated gastrointestinal conditions (Oomen et al., 2002).


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The characteristics of the sample, ionic composition, enzymatic activity, digestion time, and mechanical stresses have an important influence on the results obtained by in vitro digestion methods. The relationship between in vitro digestion and enzymatic activity has been studied and it has been defined that the in vitro technique can use enzymes specifically chosen to obtain maximum digestibility values or to determine the initial rate of hydrolysis. Enzyme characteristics are the most significant factor in in vitro digestion and depend on various factors such as temperature, concentration, incubation time, pH, stability, inhibitors, and activators. The selection of the enzymes and of the incubation conditions depends on the objectives of the study. The use of a purified enzyme instead of a complex biological mixture is advantageous because it allows the standardization of in vitro digestion models and laboratory comparisons. However, digestion of a nutrient is influenced by the digestion of other nutrients and sometimes it is more representative to use a complex combination of enzymes rather than a purified enzyme (Coles, Moughan, & Darragh, 2005). A representative scheme of the digestion process is shown in Fig. 2.2.

Fig. 2.2  Scheme of in vitro digestion process.

Methods for determining bioavailability and bioaccessibility


There are several in vitro systems with different technological complexities and biological importance, from static mono-compartmental to dynamic ­multicompartmental models. Some examples of assays performed with in vitro methods for bioaccessibility determination, including the digestion phases (oral, gastric, and pancreatic), are presented in Table 2.3. In vitro static digestion models The static digestion model, despite its limitations, allows the estimation of bioaccessibility by in vitro models and can correlate with results obtained from human studies and animal models (Liang et al., 2012). Static models are the most common digestive systems in which the gastric phase consists of pepsin hydrolysis in homogenized foods at pH (1–2) and fixed temperature (37°C) for a defined period (1−3h). Subsequently, the intestinal phase can occur in the same bioreactor with the addition of pancreatic enzymes with or without bile (pH 6–7). Enormous volumes of media are frequently implanted and mechanical forces caused by continuous agitation are not representative of complex peristaltic movements (Kong & Singh, 2008). Static single-compartment models contain other parameters such as mechanical forces or elimination of final digestion products (Chen et al., 2011; Miller, Schricker, Rasmussen, & Van Campen, 1981). Static models do not replicate the dynamic processes that occur during human digestion, such as continuous changes in pH and secretions or gastric emptying (Guerra et al., 2012). This method is based in the reduction of the gastrointestinal system to a simple chemical reactor, producing a maximum value of bioaccessibility using a continuous leaching system where the digestive juices are added continuously (Chu & Beauchemin, 2004; Dufailly, Guérin, Noël, Frémy, & Beauchemin, 2008). The simple and inexpensive online leaching technique has been adapted to conduct bioaccessibility studies and combines a simple flow-injection manifold, where artificial reagents (saliva, gastric, and intestinal juice) are injected and pumped through a micro-column and the released components are monitored uninterruptedly. The  ­peristaltic pump simulates peristaltic movements in the gastrointestinal tract while the micro-column is maintained at human body temperature (37°C). Some examples of assays performed with in vitro static digestion methods for bioaccessibility determination, including the digestion phases (oral, gastric, and pancreatic), are presented in Table 2.4. In vitro dynamic digestion models Dynamic gastric models were developed based on in vivo data to reproduce the gradual acidification of gastric contents by addition of HCl, pepsin flow rate, and gastric emptying (Hoebler et al., 2002). The dynamic gastric model was developed considering the characteristics of the stomach region (Mercuri et al., 2011; Vardakou et al., 2011). These systems are often composed of two sections: the “body” where the gastric secretions are mixed with food and the “antrum” where the shear forces and grinding of the stomach are replicated. Gastric emptying is controlled by a flow regulator that separates the smaller particles and allows them to leave the stomach, while the larger ones are refluxed in the upper compartment to be digested again.


Table 2.3  In vitro methods for bioaccessibility determination. Product




Orange juice


Gastric: sample, pepsin, pH 2/95 rpm/37°C/1 h Pancreatic: bile extract, pancreatin, pH 6.9/95 rpm/37°C/2 h Oral: sample cut/artificial saliva, pH 6.7–6.9/95 rpm/37 °C/1 min. Gastric: sample, pepsin, pH 2/95 rpm/37°C/1 h. Pancreatic: porcine bile extract, pancreatin, pH 7.5/95 rpm/37°C/2 h Oral: sample, blender/1 min Gastric: pepsin, pH 2.0/37°C/2 h Pancreatic: pancreatin, bile salts, pH 7.5/37°C/2 h Gastric: sample, pepsin, 100 rpm/37°C/1 h. Pancreatic: dialysis, 100 rpm/37°C/45 min, pancreatin, porcine bile extract, pH 6.5/37°C/2 h Oral: samples, blender/1 min, α-amylase, pH 6.75/37°C/10 min Gastric: pepsin, pH 1.2/37°C/60 min Gastric: sample, pepsin, pH 2.0/120 strokes/ min/37°C/2 h. Pancreatic: pancreatin-bile salt, pH 6.5/2 h Gastric: sample, pepsin, pH 2/90 rpm/37°C/2 h Pancreatic: dialysis, pH 7.5, gastric digesta, pancreatin, and bile, pH 5.0/90 rpm/37°C/2 h

Stinco et al. (2012)

Carotenoids, flavonoids, and vitamin C

Phenolics, flavonoids, and anthocyanin

Apples (four varieties)

Phenolics, flavonoids, and anthocyanins

Tomato, onion, garlic, and lettuce


Milk- and soy-based fruit beverages

Tocopherols, carotenoids, and ascorbic acid

Blend of orange, pineapple, and kiwi juices

Vitamin C, phenolics, and carotenoids

Tagliazucchi et al. (2010)

Bouayed, Hoffmann, and Bohn (2011)

Gawlik-Dziki, 2012

Cilla et al., (2012)

Rodríguez-Roque, Rojas-Graü, Elez-Martínez, and MartínBelloso (2013)

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Red Globe grapes

Aschoff et al. (2015)

Phenolics and cinnamaldehyde

Blended fruit juice–soymilk beverage

Vitamin C, phenolics, isoflavones, and carotenoids

Papaya, mango, oranges, and oat beverage

Carotenoids, phenolics, anthocyanins, and steviol glycosides

Grape varieties, blueberry, black mulberry, and cherry wines


Pumpkin flours

Functional properties, phenolics

Fruit seeds

Antioxidants, phenolics

Dormant seeds, buckwheat sprouts

Nutritional quality and phenolics

Helal, Tagliazucchi, Verzelloni, and Conte (2014) Rodríguez-Roque, Rojas-Graü, Elez-Martínez, and MartínBelloso (2014) Carbonell-Capella, Buniowska, Esteve, and Frígola (2015)

Celep, Charehsaz, Akyüz, Acar, and Yesilada (2015)

Aydin and Gocmen (2015)

Chen et al. (2016)

Świeca (2016)


Gastric: sample, pepsin, pH 2.5/37°C/2 h Pancreatic: pancreatin, bile salts, pH 7.5/37°C/2 h Gastric: sample, pepsin, pH 2/90 rpm/37°C/2 h Pancreatic: dialysis, pancreatin-bile mixture, pH 7/90 rpm/37°C/2 h Oral: sample, saliva solution, 90 rpm/37°C/10 min Gastric: pepsin, pH 2/90 rpm/37°C/2 h Pancreatic: dialysis, pH 5.0/30 min, pancreatin-bile mixture, pH 7.5/90 rpm/37°C/2 h Gastric: sample, simulated stomach solution, 100 rpm/37°C/2 h Pancreatic: pancreatin, bile salts, 100 rpm/37°C/2 h Gastric: sample, pepsin, pH 2/37°C/1 h Pancreatic: Bile/pancreatin, pH 7.2/37°C/2.5 h Gastric: sample, pepsin, pH 2.0/95 rpm/37°C/1 h Pancreatic: sodium bicarbonate, pH 5.3, bile salts, glycodeoxycholate, taurodeoxycholate, taurocholate, pancreatin, pH 7.4/95 rpm/37°C/2 h Oral: sample, salivary fluid, pH 6.75/37°C/10 min Gastric: gastric fluid, pH 1.2, 37°C/2 h Pancreatic: pancreatin, bile extract, pH 7/2 h

Methods for determining bioavailability and bioaccessibility

Cinnamon beverages with or without bovine milk



Continued Product




Cashew apple bagasse

Phenolics and vitamin C

Fonteles et al. (2016)

Cocoa powder

Phenolics and procyanidins

Juices (apple, grape, and orange)


Extra virgin argan oil


Granny Smith apples

Bioactive compounds



Gastric: samples, pepsin, pH 2.0/100 rpm/37°C/2 h Pancreatic: bile and pancreatin, pH 6.0/37°C/2 h, bile and pancreatin, pH 6.7–7.5/37°C/2 h Oral: sample, α-amylase, pH 6.9/37°C/5 min Gastric: pepsin/HCl, pH 2.0/37°C/2 h Pancreatic: bile salts/pancreatin, pH 6.5/37°C/2 h Gastric: sample, pepsin, pH 2/120 strokes/ min/37°C/1 h Pancreatic: bile, pancreatin-lipase, pH 5.3. Duodenal conditions: pH 7.2/37°C/2 h Gastric: sample, pepsin, pH 2/110 oscillations/min/37°C/2 h Pancreatic: pancreatin, bile salts, pH 7/110 rpm/37°C/2 h Oral: sample, saliva, pH 7.0/30 s. Gastric: gastric juice, pH 1.8–2.0/100 rpm/37°C/3 h. Gastric: sample, pepsin, pH 2/37°C/2 h Pancreatic: dialysis, pH 7.5/2 h, 37°C/30 min, pancreatin, bile salts, 37°C/2 h

Gültekin-Özgüven, Berktaş, and Özçelik (2016)

He et al. (2016)

Dalmau, Bornhorst, Eim, Rosselló, and Simal (2017) de Lima et al. (2017)

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Rueda, Cantarero, Seiquer, Cabrera-Vique, and Olalla (2017)

Minerals and phenolics

Mango and papaya

Carotenoids, phenolics, and anthocyanins

Gastric: sample, pepsin, pH 1.2/37°C/2 h Pancreatic: bile salts, pancreatin, pH 6.8/37°C/2 h Oral: sample, saliva, pH 6.75/90 rpm/37°C/10 min Gastric: pepsin, pH 2/90 rpm/37°C/2 h Pancreatic: dialysis, pH 5.0 pancreatin-bile, pH 7.5/90 rpm/37°C/2 h

Schulz et al. (2017)

Buniowska et al. (2017)

Methods for determining bioavailability and bioaccessibility

Juçara fruit



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Table 2.4  In vitro static digestion methods for bioaccessibility determination. Product




Corn bran

Zinc and lead

Chu and Beauchemin (2004)



Fresh carrots


Maize grains


Oral: sample, saliva, 37°C/10 min Gastric: gastric juice, 37°C/1 h Pancreatic: intestinal juices, 37°C/1 h Oral: sample, saliva, 37°C/10 min Gastric: gastric juice, 37°C/2 h Pancreatic: intestinal juices, 37°C/2 h Gastric: sample, pepsin, pH 4/37°C/30 min, pH 2/37°C/30 min Pancreatic: pancreatin, bile extract, pH 6.9/37°C/2 h Oral: sample, amylase, 37°C/5 min Gastric: pepsin, pH 2.0/37°C/1 h Pancreatic: dialysis, pH 6.7/37°C/30 min pancreatin and bile extract, 37°C/2 h

Dufailly et al. (2008)

Knockaert, Lemmens, Van Buggenhout, Hendrickx, and Van Loey (2012) Greffeuille et al. (2011)

These systems are complex, but do not accurately replicate peristaltic forces in vivo. The human gastric simulator constituted by a latex chamber surrounded by a mechanical steering structure imitates the peristaltic movements of the stomach in intensity, amplitude, and frequency (Kong & Singh, 2010). The assays should preferably reproduce each digestion step, with an accurate transit time, and to respond to this need dynamic bi- and multicompartmental dynamic systems were designed and built (Guerra et al., 2012). The bi-compartmental models imitate the luminal environments of the stomach and proximal small intestine. These systems control temperature, pH variations in the gastric and duodenal sections, gastric emptying, addition of pepsin, pancreatic juice and/or bile, and dialysis of final digestion products (Mainville, Arcand, & Farnworth, 2005). The TNO gastro-intestinal Model 1 is a multicompartmental gastrointestinal system that combines multicompartmentalization and dynamism. This model simulates the stomach and the three fragments of the small intestine (duodenum, jejunum, and ileum), being each compartment composed of a glass capsule encasing a flexible inner silicone jacket (De Souza Simões et al., 2017; Ribnicky et al., 2014); it allows controlling the main parameters of human digestion: gastric and intestinal pH kinetics, temperature, gastric and ileal deliveries, transit time, peristaltic mixing and transport, successive addition of digestive secretions, and passive absorption of water and small particles through a dialysis (Minekus et  al., 1999; Minekus, Marteau, Havenaar, & Huis in ‘t Veld, 1995). Secretions of amylase, gastric juice, bile, and pancreatin/pancreatic juice were released into the gastrointestinal compartments through pumps. The pH is regulated by the discharge of hydrochloric acid in the stomach and sodium bicarbonate in the intestinal sections. The hollow fiber-filtration equipment, consisting of semipermeable membranes, is coupled to jejunal and ileal compartments, allowing the

Methods for determining bioavailability and bioaccessibility


reproduction of the absorption of water released/digested or liposoluble compounds less than 50 nm in size (Ribnicky et al., 2014). This model allows approximate simulation of the behavior within the lumen of the human gastrointestinal system in vivo and has been used in nutrient and microbiological studies (Blanquet-Diot et al., 2012; Martin & De Jong, 2012). Dynamic models can be advantageous in relation to static systems, since they contain specific characteristics such as the rate of stomach leakage or gastrointestinal transit time, which can change the bioaccessibility of the ingested components, affecting the release of the food matrix, stability, and solubility in the digestive tract lumen. Knowledge of the factors that influence bioaccessibility of nutrients is important in the development of functional foods and when establishing process conditions, in order to maximize bioactive compounds and consequently food health benefits (Parada & Aguilera, 2007). Gastrointestinal systems have some limitations such as the inability to fully imitate in vivo processes, feedback mechanisms, hormonal and nervous control, mucosal cellular activity, peristaltic movements, and involvement of the local immune system (Guerra et al., 2012). The enzyme concentrations, pH variability, and/or a more representative simulation of peristaltic movements all influence the bioaccessibility estimation (Torres-Escribano et al., 2011). Intestinal absorption and presystemic metabolism assessment The determination of the amount of bioaccessible nutrients or contaminants that are assimilated after digestion through intestinal cells can be done in vivo using animals via intestinal perfusion (Kunes et al., 2005) or using ex vivo methods combined with in vitro models or with a fully included in vitro system where the digested samples are moved to a cell culture in vitro (Karlsson & Artursson, 1991). The cultures of Caco-2 human colorectal cancer epithelial cell lines are the most used (Viadel, Perales, Barberá, Lagarda, & Farré, 2007). There are also other cell lines for the representation of intestinal mucosal absorption. Each cell line exhibits specific characteristics in relation to culture preservation and appropriateness for the expression of transporters. Modifications in the expression of transporters and metabolic enzymes in cellular models may underestimate or overestimate intestinal permeability (Langerholc, Maragkoudakis, Wollgast, Gradisnik, & Cencic, 2011). Cell culture tests assume that simple filtration (Knockaert et al., 2012) or centrifugation of the digested food in vitro is sufficient to determine the amount of the material that is transported through the intestinal epithelium (Versantvoort, Oomen, Van De Kamp, Rompelberg, & Sips, 2005). This assumption must be tested and validated and the contents of the decantation/centrifugation supernatant and the residue reproduce the quantities of the compound accessible in small and large intestine, respectively (Granado-Lorencio et al., 2010). Decantation/centrifugation may not exactly mimic the physical separation of the portions in the intestinal lumen or the passage of composites into the intestinal epithelium. Dialysis membranes are a ­relatively cheap and direct


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technique to try to mimic intestinal cell tissue, but the phenomena of i­ ntestinal absorption are reduced to a simple membrane separation process, thus ­excluding biochemical phenomena occurring at intestinal cells (Greffeuille et  al., 2011). Biochemical interactions are complex and the results are variable. The amount of calcium available for intestinal absorption in a spinach omelet was found to be affected by the high amount of oxalic acid, which leads to calcium precipitation. Phytic acid contents and fiber influence the availability of calcium for intestinal absorption, therefore centrifugation and dialysis methods indicate dissimilar results (Cámara, Amaro, Barberá, & Clemente, 2005). Bioaccessibility studies of lipophilic constituents, such as carotenoids, identify the restrictions of simple centrifugation as a method representative of intestinal absorption. A limiting factor is the solubilization of the lipophilic substances in the digestion after the release of the food matrix. The integration of these constituents into lipophilic absorbable micelles is considered to be crucial. A two-phase in vitro digestion system was used to determine the bioaccessibility of chlorophylls, isoflavones, and carotenoids (Ferruzzi, Failla, & Schwartz, 2001; Hornero-Méndez & Mínguez-Mosquera, 2007; Walsh, Zhang, Vodovotz, Schwartz, & Failla, 2003). The principal assumption is that in vitro processes, such as ultracentrifugation, become a portion of the digesta containing the absorbable micelles (Hornero-Méndez & Mínguez-Mosquera, 2007; Liu, Hou, Lei, Chang, & Gao, 2012). The content of the substance in the ultracentrifugation supernatant allows evaluating the efficiency of micellarization, that is, the move of carotenoids from the digest to the micelle fraction (Jiwan, Duane, O’Sullivan, O’Brien, & Aherne, 2010). The availability of absorption may be followed by a third step to determine metabolic transformation in hepatic tissues. Polyphenols suffer metabolic processes such as glucuronidation, sulfation, and methylation in the small intestine and in the liver. Caco-2 cells are able to perform these metabolic transformations (Etcheverry et al., 2012). Specific metabolic pathways of hepatic tissues are evaluated through specific cell cultures that mimic liver tissue. The difficulty is to provide hepatic cell cultures with adequate nutrients to mimic cellular functions performed in  vivo (LeCluyse, Witek, Andersen, & Powers, 2012). Quality control and the guarantee of these procedures require the use of certified reference materials for the confirmation of the analytical methods used and the authentication of mass balances (Khouzam, Pohl, & Lobinski, 2011). In vitro studies incorporating gut microflora This methodology consists of evaluating intestinal accessibility with simulated microbial fermentation under in  vitro conditions to study the influence of bacteria to bioaccessibility (Chandrasekara & Shahidi, 2012). The colonic fermentation model with a colony microbiota inoculum is used to estimate colonic metabolism in the indigestible portion obtained from intestinal simulation in  vitro. The Human Intestinal Microbial Ecosystem Simulator incorporates the entire gastrointestinal tract from the stomach to the colon, with three colon sections containing a mixed microbial community. This system was d­ eveloped

Methods for determining bioavailability and bioaccessibility


to ­determine the relations of food constituents with human resident microbiota (Marzorati et al., 2010). Phenolics present as glycosylated forms or as esters or polymers may need hydrolysis by microflora to be absorbed as aglycones (Carbonell-Capella et al., 2014). The bioaccessibility of food polyphenols was estimated with the isolated indigestible portion (small intestine) and a colonic fermentation of this portion (large intestine). The results showed that 48% of the food polyphenols were bioaccessible in the small intestine and 42% were bioaccessible in the large intestine (Saura-Calixto et al., 2007). The colonic fermentation assays may be suitable for the TNO gastrointestinal dynamic model, so that the results of the bioaccessibility evaluation of polyphenols are close to the real ones (Carbonell-Capella et al., 2014). The colonic microflora process most of the unabsorbed flavanols in the small intestine to a diversity of compounds capable of being absorbed, thus showing the significance of regarding the microflora for the realistic evaluation of bioaccessibility (Hackman et al., 2008). In vitro fermentation systems still have numerous challenges to overcome. Stability of the microbial ecosystem should be improved and evaluated, the relationship between technical complexity and biological importance should be explored, and methodologies should be validated. The selected model should be chosen according to its advantages and limitations associated to the topic being studied. Developments should consider evaluating the functional stability of the microbiome in vitro, the utilization of “-omics” technologies, “diseased” microbiota modeling, and an improved association within in  vitro methodologies (Payne, Zihler, Chassard, & Lacroix, 2012). Examples of methodologies using intestinal flora after gastrointestinal digestion, such as in vitro colonic fermentation, are presented in Table 2.5. Caco-2 cell models In  vitro methods mimic the processes of digestion and absorption (bioavailability) or merely the digestion process (bioaccessibility) and the measured response is the amount of a nutrient in the final extract. The digestion process is replicated under a controlled environment using digestive enzymes, while the final absorption is evaluated with cultures of Caco-2 cells (polarized human colon carcinoma cell line) (Verwei et al., 2005). The Caco-2 cell culture model has been utilized as part of in vitro digestion models as a tool to predict the absorption of bioactive compounds from food and pharmaceutical mixtures. In vitro digestion with Caco-2 cell culture is a rapid and inexpensive method to screen the bioavailability of food prior to human trials (Mahler, Shuler, & Glahn, 2009). The in vitro digestion and culture procedure of Caco-2 cells was used as an economical model to track the bioavailability of carotenoids from vegetable foods such as carrots, spinach, and tomato paste (Garrett, Failla, & Sarama, 1999, 2000). Iron bioavailability estimated by an in vitro digestion/Caco-2 cell culture model correlated qualitatively with human data. Quantitative tests concluded that there is an agreement between the results obtained with human in  vivo iron uptake and those obtained in vitro using Caco-2 cells (Mahler et al., 2009).

Table 2.5  Methodologies with in vitro gastrointestinal digestion and subsequent gut microflora processing. Compounds



Cereals, vegetables, legumes, nuts, and fruits


Saura-Calixto et al. (2007)

Five millet grain


Fruit (Arbutus unedo)

Phenolics and antioxidants

Juçara pulp




Plum and cabbage

Carotenoids and polyphenols

Enzymatic digestion: sample, pepsin, pH 1.5/40°C/1 h, pancreatin, pH 7.5/37°C/6 h, lipase, pH 7.5/37°C/6 h, bile extract, pH 7.5/37°C/6 h, αamylase, pH 6.9/37°C/16 h; supernatant, amyloglucosidase, 60°C/45 min, dialysis 25°C/48 h Colonic fermentation: rat caecal contents, enzymatic treatments, 4°C/16 h, incubation, 37°C/24 h Enzymatic digestion: sample, sodium chloride, 200 rev/min/37°C/10 min, α-amylase, pH 6.9/5 min, pH 2 MHz) employs very low power levels insufficient to cause acoustic cavitation, which therefore produces negligible physical and chemical alterations in the material through which the wave passes. Hence, it can be employed for food analysis and quality control without affecting the product. In contrast, low-frequency US (20–200 kHz) employs power levels high enough to generate cavitation and is capable of producing physical and chemical modifications in numerous applications. The food industry is always looking toward innovative technologies that can enhance processing efficiency, reduce energy consumption, and produce high-quality, safe products, which is why US technology feasibility has been widely investigated (Chemat & Khan, 2011; Kentish & Feng, 2014). In recent years, several examples have been reported in the literature employing US to process and interact with liquid foods, primarily dairy and fruit juices (Paniwnyk, 2016). Mechanical and shear forces, agitation, microjets, microstreaming, cavitation hot spots, and shockwaves are some of the physical forces that have been effectively used in several applications in food processing (Ashokkumar, 2015). The cavitation


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Fig. 3.2  (A) and (B). External and internal view of a 300 L batch US reactor. Martin-Bauer Italia Spa.

phenomenon (acoustic or hydrodynamic) is of great importance in food-processing applications in liquids. US protocols are safe and can operate at low temperatures, however, profitable applications need high-throughput, dedicated equipment either in batch (Fig. 3.2) or in flow mode (Fig. 3.3). High-intensity US systems become ever more standardized; the means by which energy is applied to the medium (flow cell design or number of transducers) is unique in every application, offering opportunities of patent protection, a driving force for new investments.

Fig. 3.3  Multifrequency weber ultrasonics flow reactor. Original set up at DSTF, University of Turin.

Green technologies for food processing: Principal considerations71

An appropriate energy balance is fundamental; if too much energy is applied within a high-intensity field such as that provided beneath an ultrasonic horn, then a stagnant cloud of bubbles can form that shields the transmission of further acoustic energy. In this instance, a decline in process efficiency can be observed (Kentish et al., 2008). US is typically applied in liquids; however, sonic waves can be propagated through solids and airborne, amplifying the potential applications in food processing. Recent advances in low- and high-frequency US include enhanced drying and defoaming with airborne US, emulsification and homogenization, green extraction methods and high-frequency US (megasonics) for separation of oil/water dispersions, and separation or fractionation of milk fat in dairy industries (Leong, Juliano, & Knoerzer, 2017). US effects in food liquids are evaluated by measuring the main physico-­chemical ­parameters such as color, viscosity, Brix index, and pH. Among its advantages are increase in shelf life via the reduction of contaminating microorganisms (fresh milk and juices) and higher nutritional and health benefits (increased levels of antioxidants and bioactive components). Ultrasonic cutting This is the apparently simplest application in which vibrational US energy is exploited as a highly efficient blade to improve cutting quality. During cutting the ultra-fast vibration minimizes deformations, transversal cracks and crumbling, broken edges, and damaged material as well as surface roughness (Schneider, Zahn, & Linke, 2002). Friction is reduced and the material does not stick to the blade, making it particularly effective for cutting viscoelastic and viscoplastic foods, fragile and frozen nourishments, heterogeneous products, and products that cannot be cut by a pressing force. The quality of the food cutting by an US cutter is affected by the geometry of the blade, the direction of vibration of the knife relative to the movement of the food, and the frequency and amplitude of the US (Arnold, Zahn, Legler, & Rohm, 2011). US cutting can be applied to cut thin slices of food, which conventional cutting cannot do (Liu, Jia, Xu, & Li, 2015). US-assisted freezing processes and sonocrystallization The applications of high-intensity US are to improve the efficiency of the freezing process, to control the size and size distribution of ice crystals, and to improve the quality of frozen foods (Cheng, Zhang, Xu, Adhikari, & Sun, 2015). Crystallization of ice dramatically affects the efficacy of the freezing process and the quality of frozen food. Fast freezing generates fine crystals evenly distributed both within and outside of the cells, while slow freezing generally produces large ice crystals. Acoustic cavitation induces ice nuclei and increases ice nucleation rate, while microstreaming enhances heat and mass transfer during the freezing process. Moreover, large ice crystals will fracture into smaller size crystals when subjected to the alternating acoustic stress. Resulting from these acoustic effects, power US has proved itself an effective tool to initiate the nucleation of ice crystals, control the size and shape of ice crystals, a­ ccelerate the rate of freezing, and improve the quality of frozen foods. The freeze-drying process


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comprises three steps: freezing, primary drying or sublimation, and secondary drying or desorption. Freezing is a critical step because it determines the size and distribution of ice crystals in frozen materials, both of which greatly affect efficiency of the drying process and the quality of freeze-dried products (Rhim, Koh, & Kim, 2011). The size and distribution of ice crystals are closely related to the freezing rate taking a dramatic advantage from sonication. US has been shown to promote sonocrystallization by enhancing the nucleation of crystals from saturated solutions (Ruecroft, Hipkiss, Ly, Maxted, & Cains, 2005). The sizes of the crystals produced through sonication are generally smaller, and the agglomeration of the newly formed crystals is also reduced. This can be related to the shear forces associated with US that act to slow growth processes and break up nascent agglomerates. Sonocrystallization has been applied to whey solutions to obtain lactose crystals with quasi-quantitative recovery in a few minutes of sonication. Another application of interest in food processing is the use of US to enhance the crystallization of fat with a much shorter induction time and provide smaller crystals (Martini, Suzuki, & Hartel, 2008). With the aim of reducing fat bloom during chocolate manufacturing, more stable polymorph species were crystallized (Baxter, Morris, & Gaim-Marsoner, 1997). US-assisted extraction of food components Current industrial technologies used in food-related industries show evident bottlenecks to overcome, often requiring up to 50% of investments in a new plant and more than 70% of total process energy used in food industries (CapEx and OpEx). In the last two decades, these shortcomings have led to the application of highly efficient enabling technologies amenable to automation such as US-assisted extraction (UAE) to enhance process yields and product quality (Chemat & Strube, 2015; Cravotto & Binello, 2016). UAE technology can enhance the extraction of any type of food component by acting as either a pretreatment step in a unit process, or as the main extraction reactor. Obtained extracts are more concentrated in soluble materials, which makes them easier to handle and reduces the need for additional process steps. UAE is clean, while its low bulk temperature and rapid protocols usually mean that no degradation is observed, especially if carried out in a modified atmosphere. Recent studies tried to design flow processes using ultrasonic multiprobe reactors with suitable cells with high power density (Alexandru, Cravotto, Giordana, Binello, & Chemat, 2013). The short residence time fully preserves the chemical, biological, and functional properties of vegetal cell content. The method consists of cell wall disruption by intense acoustic cavitation in a multihorn flow system (Fig. 3.4; Daghero & Cravotto, 2012). It is now feasible to consider the industrial-scale UAE of food components from plant and animal materials a source of worthwhile economic gain where the investment cost over benefit requires generally less than 1 year. The most common medium for cavitational treatment is water, which conveys the US waves and disperses primary and secondary metabolites via cell wall disruption creating dispersions or emulsions. Besides improving the quality and safety of processed foods, it can also offer the potential for developing new products with unique properties.

Green technologies for food processing: Principal considerations73

Fig. 3.4  High-power, multihorn flow system.

The higher yields obtained with UAE are of major interest for industry as this technology is extremely efficient and can be used as an “add-on” step in existing processes with minimal alterations (Chemat, Rombaut, Meullemiestre, et al., 2017). Emulsification and emulsion separation US has a strong use in the formation of stable emulsions, even in the absence of any emulsification enhancers, acting at 20 kHz, but also at higher frequencies up to 2 MHz. The acoustic waves at the interface between oil and aqueous phases generate a cloud of large droplets’ mixture, then reduced in size by shear forces, eventually helped by a surfactant. When the droplet size is reduced to less than 50 nm, then the emulsion become a transparent liquid inert to gravitational separation. This approach can be important when oil-soluble nutraceuticals or vitamins need to be added into an aqueous phase product, such as reconstituted beverages. Small droplet sizes can lead to improved mouthfeel and product texture. Low-frequency US is still proving to be most efficient for the production of stable emulsions, though the generation of radicals may cause lipids oxidation. This phenomenon can be controlled by reducing the sonication time and temperature and is to some extent dependent upon the nature of fat composition. In a contrary approach, US can also be used to break emulsions and enhance the separation of an oil and aqueous phase. Through the use of Bjerknes forces, oil and fat globules can be made to aggregate, which facilitates their coalescence and separation from an aqueous solution. Enhanced US separations of fats have been reported for milk and several vegetal oils, as well as palm oil in a pilot test (Juliano et al., 2013).


Innovative Thermal and Non-Thermal Processing Microorganisms and enzyme inactivation Several studies have shown the ability of US to inactivate spoilage and pathogenic microorganisms and enzymes in dairy products and fruit juices. This is attributed to thinning of cell wall membranes, formation of localized hotspots, and production of free radicals within the bulk medium as well as cell wall damage by mechanical forces. US as a sole method of inactivation is not feasible with most benefits being observed with combined treatments such as thermosonication, manosonication (high pressure), or use of UV irradiation. The most often quoted frequency used is around 20–25 kHz with short treatment times (few minutes) in a temperatures range of 50–60°C. Inactivation of bacteria or yeasts when employing thermosonication, the most common process employed, is effective in increasing shelf life with no reduction of flavor stability or texture. Nevertheless, temperatures are well lower than those normally employed for thermal pasteurization and sterilization thus leaving open the option of potential economic gain in terms of energy saving. Foaming/defoaming and airborne US for enhanced drying Foam generation in tanks during fermentation and food processing, and beverage bottling lines through aeration and agitation of liquids, can lead to significant losses of product and decreases the useful volume of processing equipment. Besides cooling protocols, common mechanical foam breakers and chemical antifoaming agents may negatively affect the products. Defoaming by airborne US application is a great technology improvement that reduces product loss, water, and energy consumption. US is applied to foams through air at a few centimeters’ distance (Riera, Gallego-Juárez, & Mason, 2006) causing the instantaneous breakage of foam bubbles, because energy is dissipated within the elastic foam layer causing the collapse. A common case is the fast defoaming of carbonated soft drinks and premixed alcoholic drinks. Some limitations have been observed in foams that are stabilized by high levels of proteins that make stable structures at the aqueous liquid/gas interfaces in which US can hardly defoam (i.e., residual barley proteins in beer). The drying process requires a high energy consumption; it is probably the largest energy-consuming unit operation in the food industry. The drying process is time-­ consuming, and even moderate heating may affect food quality. Recent studies on airborne US drying applications have shown that product drying time can be reduced by more than 50% operating at reduced temperatures with a remarkable energy savings (Sabarez, Gallego-Juarez, & Riera, 2012). US accelerates the drying rate by enhancing the mass transfer rate at which moisture is expelled from solids (De la Fuente-Blanco, De Sarabia, Acosta-Aparicio, Blanco-Blanco, & Gallego-Juárez, 2006). In the case of solid foods, the US transducer can be coupled in direct contact with the wet material or applied via airborne. US can be applied simultaneously during the drying process or as a pretreatment step. Several factors may influence the drying rate, in particular the applied power and the acoustic properties of the material while frequency is in general in the range of 20–25 kHz.

Green technologies for food processing: Principal considerations75 Pilot and industrial US applications The most common industrial applications of US and hydrodynamic cavitation are in the fields of extraction, emulsification (tomato ketchup), homogenization (milk), crystallization (sugar), microorganism inactivation, packaging welding, cutting, degassing, and defoaming. More specific procedures have been designed to promote fermentations and the vinification process (Gambacorta et al., 2017). Low-frequency US (20–50 kHz) can influence the course of fermentation by improving mass transfer and cell permeability leading to improved process efficiency and production rates. The use of US on a small industrial scale on crushed grapes indicates that this technology facilitates the extraction of phenolic compounds from grapes. It could be applied as a continuous pretreatment of crushed red grapes, before loading the maceration-­ vinification tanks, representing a possibility to optimize winery capacity by reducing the skin maceration time without losing the quality characteristics of the obtained wines (Bautista-Ortín et al., 2017). US have been successfully applied for the extraction of virgin olive oil, both at low frequency (20–80 kHz) (Bejaoui, Sánchez-Ortiz, Sánchez, Jiménez, & Beltrán, 2017) and megasonic treatment (585 kHz) (Juliano et al., 2017). US favors the mass and heat transport in the medium and also helps to destroy the cellular structures and the liberation of the oil from cell vacuole reducing malaxation time or even replacing this step. Compared to the traditional method, all investigations showed an oil yield increase and a higher content of polyphenols. Megasonics can be applied before or after malaxation, and the combination with low-frequency US to promote cell wall disruption premalaxation, followed by megasonic standing wave post-malaxation, is more advantageous for enhancing oil yield and facilitating process scalability.

3.2.4 Ohmic heating Ohmic heating is a novel food-heating technique for thermal processing in which heat is generated directly inside the food. In this method the food is placed between two electrodes and an electric current (usually alternating current) runs through the food liquids and solids, and the food acts as electrical resistance consequently generating heat inside the food. The amount of thermal energy is proportional to the electric field strength and the electrical food conductivity. In contrast, in conventional thermal food processing there is a temperature gradient within the food, therefore heating is generated by thermal conduction, convection, or radiation, and the heat energy is transferred through the food matrix (Varghese et al., 2014). Many factors have been found to influence the heating process, such as electrical conductivity, field strength, particle size, concentration, ionic concentration, and electrodes. The parameters that have a greater impact are electrical conductivity, current, voltage, and temperature (Kaur, Gul, & Singh, 2016; Sakr & Liu, 2014), but the first one is the most influential parameter in ohmic heating (Kaur & Singh, 2016). The most important advantage is that this technique allows us to achieve a high temperature in a short time in a uniform way and to stop the process instantaneously; hence the process is optimum for ­thermal-sensitive foods (Fig. 3.5).


Innovative Thermal and Non-Thermal Processing

Pump is used to transfer food throught the system Ohmic heating is generated by alternating current through the food Cooling is produced by heat exchangers Aseptic packaging and storage

Fig. 3.5  Basic elements of ohmic heating technology. Electrical conductivity Ohmic heating occurs when an electric current passes through a food by the Joule effect. As noted, electrical conductivity is the major contributing factor to the ohmic heating model and it can be defined as the measure of a material’s ability to permit the electrical current pass. A positive linear relationship between electric conductivity and temperature has been reported (Palaniappan & Sastry, 1991; Srivastav & Roy, 2014). As would be expected, electrical conductivity is affected by food composition, thus acids and salts improve conductivity, unlike fats or lipids. This fact has practical applications because electric field strength can be adjusted by the addition of electrolytes. According to Jaeger et al. (2016), the optimum range of electrical conductivity for the application of ohmic heating is in the interval of 0.1–10 S/m. This parameter has been studied in liquid foods, such as juices (Castro, Teixeira, Salengke, Sastry, & Vicente, 2004; Darvishi, Khostaghaza, & Najafi, 2013; Srivastav & Roy, 2014). In solid foods, additional considerations are present, like water evaporation, fat content, or the fibers’ orientation (Jin, Cheng, Fukuoka, & Sakai, 2015; Shirsat, Lyng, Brunton, & McKenna, 2004). For meat and fish products it is recommendable to evaluate the temperature effect, food composition (protein, water, salt), and even the electrical current direction and microstructure-like muscle fiber orientations (Engchuan, Jittanit, & Garnjanagoonchorn, 2014; Jin et al., 2015). Indeed, high temperatures lead to the formation of air bubbles, which decreases electric conductivity. Consequently, the possible distribution of the temperature profile is important to determine the effect of ohmic heating (Cappato et al., 2017). Finally, another effect that has to be considered is that during the ohmic heating process, changes associated to protein denaturation affect the microstructure, decreasing the electrical conductivity (Van der Sman, 2017). Current, voltage, and electric field Ohmic heating is generated by the application of alternating current (AC) through food, which is the resistance. To avoid undesirable secondary reactions, AC is used at high frequency. The electrons clouded around the atomic nuclei are distorted due to the electric field, creating a dipole. When the electric field direction is changed very fast, the equilibrium is not achieved. The most common voltages used in food

Green technologies for food processing: Principal considerations77

a­ pplications are between 400 and 4000 V with electrode gaps of 10–50 cm, producing field strengths from 20 to 400 V/cm. In the ohmic heating process, the electrode gap and/or the applied voltage should be controlled to generate the proper heating power (Jaeger et al., 2016). The effect of the electric field frequency on ascorbic acid degradation and color changes in acerola pulp using frequencies greater than 100 Hz was similar to that achieved with conventional heating (Mercali, Schwartz, Marczak, Tessaro, & Sastry, 2014). Some researchers have evaluated the resistance of bacterium spores at ohmic heating varying the electric field or voltage gradient in apple and orange juice as an effective method (Baysal & İçier, 2010; Kim, Ryang, Lee, Kim, & Rhee, 2017). Applications This is a new heating technology in which the food reaches a high temperature and cools particularly fast, preventing the loss of vitamins and nutrients as well as destroying microorganisms (Mesías, Wagner, George, & Morales, 2016). Therefore one of the most important industrial applications is sterilization and pasteurization of liquid and particulate foods such as vegetables and fruits, meat, poultry, or fish, maintaining their nutritional quality. Nowadays the ohmic systems include sterilization of solid-liquid food mixtures in continuous flow to enhance the commercial ohmic heater (Kamonpatana et al., 2013). The inactivation of the microorganisms is attributed to the thermal effects of ohmic heating like those of conventional heating methods. The advantages of this technique make possible to use it for cooking, blanching, thawing, baking, enhanced diffusion, or electroporation. In addition, it has showed that conventional and ohmic heating have a similar effect in terms of main industrial enzyme inactivation thus their kinetics are not related with the electric field (Castro, Macedo, Teixeira, & Vicente, 2004). Indeed, peroxidase and polyphenol oxidase were inactivated by ohmic heating in sugarcane juice at 80°C avoiding degradation of phenolic and flavonoid compounds (Brochier, Mercali, & Marczak, 2016). Achir et al. (2016) performed a study on the effect of ohmic heating on common carotenoids in citrus juices and they showed it as very good alternative when compared with conventional heating. It is also known that ohmic heating minimizes the amino acid content and the protein quality degradation in sterilization treatment of vegetable baby foods (Mesías et al., 2016). Continuous ohmic heating was an effective method to inactivate Escherichia coli O157:H7, Salmonella typhimurium, and L. monocytogenes achieving at the same time higher quality than conventional heating in orange juice, tomato juice, and milk (Kim & Kang, 2015; Lee, Sagong, Ryu, & Kang, 2012). Another industrial application of ohmic heating is blanching of vegetables in order to soft the vegetable fiber, enhancing the sensory quality. Recently, the study of ohmic heating and conventional heating on phenolics from bottle gourd (Lagenaria siceraria) has been realized by Bhat, Saini, and Sharma (2017).

3.2.5 Food packaging: Active packaging with natural additives and biodegradable edible films The technology of food packaging is one of the biggest challenges facing modern societies today. According to the FDA about 1.3 billion tons of food are thrown away


Innovative Thermal and Non-Thermal Processing

each year (FAO, 2011). Inside the European market are estimated losses of 89 million tons of foods wastes, and within households the loss of food constitutes 20%–30% of food purchased (EC, 2014). For this reason, the EU policies are focused on reducing this level of food waste. Hence strategies based on size optimization and designs of food packages allow for maintaining food quality during longer periods of shelf life. Maintaining food quality during shelf life also means not compromising food safety, which is a worldwide priority of current food legislation because the microbiological risks in foods are the main sources of diseases and intoxications transmitted by them. In this sense, listeriosis with a mortality rate of 13% is the largest disease transmitted by food. The microorganism that causes this disease (L. monocytogenes) can grow at refrigeration temperatures, between 2°C and 4°C, increasing the prevalence of the pathogen in ready-to-eat foods with relatively long shelf-life periods, such as heat-treated fishery or meat products as well as cheeses. For these reasons, one of the current trends in food preservation consists of active packaging that can be conveniently used for the quality and control of perishable foods. In addition, packaging science has to develop in the future following the green chemistry principles such as election of green solvents in the processes and biocompatible reactive and agents to develop energy-efficient and economic processes for producing packaging polymers. Active packaging Beyond its traditional use as a barrier against external factors and environments, food packaging has evolved from traditional containers to active and intelligent packaging. Specifically “active materials and articles means materials and articles that are intended to extend the shelf-life or to maintain or improve the condition of packaged food; they are designed to deliberately incorporate components that would release or absorb substances into or from the packaged food or the environment surrounding the food” (Commission Regulation, 2009). The active packaging consists of a material or combination of materials with the purpose of containing the food, acting as a passive barrier that separates it from the environment. The active packaging retards the adverse effects of the environment, maintaining the quality and safety of packaged foods. According to Ahvenainen (2003), the active packaging is a good example of innovative traditional packaging as the food, environment, and packaging interact to increase the shelf life, safety, or even in some cases, to improve food’s sensory properties. Another type of packaging is smart packaging, which acts to improve the safety and quality of the packaged food as well as to increase the shelf life and/or to inform the consumer of the situation of the food or its environment (Kerry, O'Grady, & Hogan, 2006). The technology of active packaging is based on the deliberate incorporation of certain compounds within the packaging that will release or absorb substances in/from the food or its environment to achieve three main purposes (Camo, Antonio Beltrán, & Roncalés, 2008): (1) increase food safety, (2) maintain the food and its organoleptic quality, and (3) extend shelf life. These active compounds either can be part of or be present inside the package ­together with the packaged food, although separated from it. In the first case, the ­advantages are that there is no possibility of manipulation by the consumer, ­decreasing

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the chances of rejection and accidental contamination. In addition, the packaging is done with conventional packaging equipment; there are no additional operations, decreasing the complexity of the packaging process. In the second case, there are more packaging technological difficulties and possible cases of toxicity due to contamination. The challenges to be overcome lie in the following points: (1) searching of active substances compatible with the packaging manufacturing processes, (2) the active agent must act at the exact moment of packaging, (3) releasing the active substance at minimum concentrations, and (4) avoiding the migration of toxic compounds. The main active compounds in packaging systems can be classified as follows: absorbers of water, oxygen, ethylene, carbon dioxide, and undesirable aromas; and generators of carbon dioxide, antimicrobials, and antioxidants. For the food industry, those active compounds with antimicrobial and/or antioxidant activities are the most attractive, because it should be noted that after microbial spoilage, the lipid oxidation phenomena is the most important related to the deterioration of the majority of foods. Antimicrobial active packaging In the case of antimicrobial compounds, although there are numerous possibilities in the market, only those based on material with silver have success in the United States and Japan. It is expected that the use of silver-based antimicrobial active packaging will grow in Europe after its inclusion in the provisional list of additives and biocides for use in materials in contact with food. Recent green analyses of the incorporation of silver nanoparticles into biodegradable polymers have received a great deal of attention due to their splendid biocompatibility and the development of economic and ecofriendly procedures for nanomaterial synthesis (Acharya et al., 2017; Božanić, Dimitrijević-Branković, Bibić, Luyt, & Djoković, 2011; Kalayci et  al., 2010). However, despite this effectiveness, this type of compound causes distrust in the consumer; consequently the most recent researches have focused on other compounds of natural origin such as: bacteriocins (nisin and pediocin), natural extracts from plants and essential oils, or enzymes (lysoenzyme) (Realini & Marcos, 2014). The bacteriocins produced by lactic acid bacteria have a broad antimicrobial spectrum and nisin, enterocin, sakacin, and lactocin have been tested with success against L. monocytogenes. Indeed the nisin is approved as an additive for meat products in some countries such as the United States (FSIS, 2002) and Australia (FSANZ, 2007) with acceptable uptake of 0.13 mg per kg per day per person (ESFA, 2006). Nisn has been used with success in antimicrobial films (polyethylene, LDPE, polypropylene (PP), soy protein, or starch) against Listeria for different foods such as turkey breast (Trinetta, Floros, & Cutter, 2010), sausages (Blanco Massani et al., 2014), and smoked salmon (ConchaMeyer, Schobitz, Brito, & Fuentes, 2011). Natural extracts from plants, rich in polyphenolic compounds with antimicrobial and antioxidant effect, have been applied with success in films for foods. For example, grapeseed extracts incorporated in edible starch films were able to control the growth of Brochothrix thermosphacta on the surface of pork loin for 4 days (Corrales, Han, & Tauscher, 2009).


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Essential oils from rosemary, garlic, oregano, and thyme have shown their effectiveness in delaying the spoilage caused by microbial growth in several foods such as salmon (Cerisuelo et al., 2013), trout (Jouki, Yazdi, Mortazavi, Koocheki, & Khazaei, 2014), and veal (Sung et al., 2014). In addition, clove essential oil was effective against pathogens, such as E. coli O157:H7 and L. monocytogenes in salmon (Lee, 2014). The antimicrobial effect of essential oils in active packaging can be compromised if the volatile active compounds are released previously to a food’s shelf-life period. To solve this issue their micro or nanoencapsulation could be useful to have a controlled liberation, but this research line has not received much attention. In addition, this strategy would have the advantages of protecting the active compound from the high temperatures that are reached during the film-extrusion process as well as increasing the contact area compared to conventional systems, which would reduce the amount of active compound necessary (Picouet, Fernandez, Realini, & Lloret, 2014). Akbar and Anal (2014) developed sodium alginate films containing zinc oxide nanoparticles capable of eliminating S. typhimurium and Staphylococcus aureus in poultry meat. Overall the main challenges are focused on overcoming the possible alterations of the mechanical properties (tear resistance, strength, plasticizer capacity) and barrier that these compounds produce in the films, as well as using compounds of natural origin. Antioxidant active packaging Regarding active packaging based on antioxidant compounds, this is an emerging sector that is continuously growing inside packaging science. The current trend is focused on replacing synthetic additives by natural antioxidants (tocopherol, plant extracts, and essential oils) with the aim of protecting the polymer itself from the normal “aging process.” Synthetic antioxidants such as butylated hydroxytoluene (BHT) are widely questioned for food safety reasons. Active packaging films based on starch and ascorbic acid were successfully developed using casting procedures (Ashwar et al., 2015). Successful results have been reported in beef (Park et al., 2012) and mackerel (Giménez, Gómez-Guillén, Pérez-Mateos, Montero, & Márquez-Ruiz, 2011). In general rosemary, oregano, and tea and others, are of great interest for the food industry as natural antioxidants because besides increasing food shelf life, in most cases it can offer beneficial health effects (Barbosa-Pereira, Aurrekoetxea, Angulo, Paseiro-Losada, & Cruz, 2014). An ideal solution would be obtaining active packaging that provides antioxidant and antimicrobial activity, which is possible with many essential oils and natural extracts (Sacchetti et al., 2005), but the problems appear later in the technological process of incorporation. To overcome these drawbacks, the most recent researches are focused on modifying polymers using plasticizers, stabilizers, and chain extenders. These changes allow modifications in the physical and chemical properties such as stability, degradability, or permeability of packaging. Regarding antioxidant active packaging these modifications could permit the insertion of hydrophilic binding points, where the polyphenolic structures of the antioxidants compounds will bind by hydroxyl groups in the polymer final formulation. These insertion points have been shown to be satisfactory with active compounds such as tocopherol in low-­density polyethylene and in PP (Castro-López, López de Dicastillo, López Vilariño, &

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González Rodríguez, 2013) and catechins linked by maleic anhydride in PP (López de Dicastillo, Castro-López, Lasagabaster, López-Vilariño, & González-Rodríguez, 2013), tea extracts (López de Dicastillo, del Mar Castro-López, López-Vilariño, & González-Rodríguez, 2013), or papain linked by curcumin (Manohar, Prabhawathi, Sivakumar, & Doble, 2015). The use of this type of compound has several advantages. Firstly, in many cases they are considered GRAS substances and potentially beneficial for human health. Secondly, depending on the modifier’s concentration different active compound release speeds can be obtained, which allows introducing less quantity because they act in a prolonged period of time with the final results of increasing effectiveness and reducing costs. The use of wastes from agroindustrial origin certainly presents advantages from an environmental point of view. For this reason recent researches within active packaging are based on the use of composites and the use of biodegradable active packaging with antioxidant compounds based on polylactic acid (Jamshidian et al., 2012). The global market for food preservation and shelf-life extension totaled $442.4 billion and $463.4 billion in 2014 and 2015, respectively. The market should total $582.8 billion by 2020, growing at a compound annual growth rate of 4.7% from 2015 to 2020 (BCC Research, 2016). Overall, the active packaging market ranks in importance behind traditional packaging and modified atmosphere packaging and it is continuously growing with an estimation of sales of $11.9 billion in 2017 (BCC Research, 2013). Other research lines are based on the use of edible films formulated with natural antioxidants, which are discussed in the next section. Biodegradable and edible films with antimicrobial/ antioxidant activity Due to environmental concern caused by plastic food packaging, consumer demands are currently focused on edible films and biodegradable plastics based on renewable sources (Babu, O'Connor, & Seeram, 2013; Gómez-Guillén, Ihl, Bifani, Silva, & Montero, 2007). Bio-based polymers are not necessarily biodegradable, because polyethylene or PP can be obtained from ethanol, but this fact does not make them more biodegradable than their petroleum-based counterparts (Otoni et al., 2017). In addition, we have petroleum-derived biodegradable polymers such as polycaprolactone or aliphatic-aromatic polyesters. Optimum candidates for replacing conventional plastic materials should be both renewable and biodegradable. Edible films and coatings can be obtained from carbohydrates (cellulose, chitosan, alginate, and starch), proteins (casein collagen, gelatin, and wheat gluten), lipids, or the combination of these compounds, and should be ingested with the food (Krochta, 2002). Not all biodegradable films are edible, and, in this case, they should comprise food-grade compounds, including the matrix, solvents, plasticizers, and other additives. Vegetables (pectin, starch, and cellulose), animals (chitosan), fruits, and plants have been employed as primary compound sources for the elaboration of polysaccharidebased edible films, and more than 35 plant species have already been used to this aim (Otoni et  al., 2017). The main problem that these edible films exhibit is related to poor mechanical properties (tensile strength and elongation), water vapor barrier, and


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thermal properties, especially compared to conventional polymers. A feasible strategy to overcome these obstacles is the production of edible composites or nanocomposites with the aim of improving all these mechanical characteristics. Apart from this technical hurdle, edible film may carry functional additives to improve characteristics (sensory, nutritional, and/or microbiological) of the packaged product or the packaging material itself. Specifically, we will focus on reviewing those studies that are based on the incorporation of compounds with antimicrobial and antioxidant properties. Natural additives have been added to edible films in order to confer antioxidant and antimicrobial activity. Within edible films based on protein, gelatin has good gelling and film-forming abilities as well as a melting temperature close to the human body’s temperature providing a good sensation because of its melt-in-your-mouth behavior. Indeed, gelatin from tuna has been utilized to produce transparent edible films with addition of natural extract with a high polyphenol content (Gómez-Guillén et al., 2007). Soy protein films have been developed with oregano to enhance their antioxidant properties (Pruneda et al., 2008). Edible films based on milk protein with oregano and paprika essential oils applied on beef slices showed antioxidant activity (Oussalah, Caillet, Salmiéri, Saucier, & Lacroix, 2004). Chitosan is a polysaccharide obtained from chitin with a wide range of applications. It has been used as both matrix and filler in edible films. Due to its abundance, nontoxicity, and biodegradability and composability characteristics, it makes an excellent candidate for use in biodegradable active packaging (Lago et al., 2011). The main disadvantages are related to mechanical and water barrier properties. The antimicrobial activity of chitosan against bacteria, yeast, and molds has been proven (Siripatrawan & Harte, 2010). Although it has been reported to have some antioxidant activity in grilled pork (Yingyuad et  al., 2006), the addition of natural antioxidant to chitosan films has been checked in order to obtain films with antioxidant and antimicrobial activities. For example, Blanco-Fernandez, Isabel Rial-Hermida, Alvarez-Lorenzo, and Concheiro (2013) have incorporated vitamin E into chitosan films. Similarly, Ojagh, Rezaei, Razavi, and Hosseini (2010) and Abdollahi, Rezaei, and Farzi (2012) have developed edible chitosan films with cinnamon and rosemary oil, respectively, with success to inhibit lipid oxidation and microbial growth. Other essential oils have been added into edible films based on fruits and vegetables for antimicrobial aim, such as allspice (Du et al., 2009), lemon-grass, oregano, cinnamon (Rojas-Graü et al., 2006), and thyme (Espitia, Avena-Bustillos, Du, Chiou, et al., 2014). Especially satisfactory were the bactericidal results obtained with essential oil from oregano and cinnamon against E. coli O157:H7 due to the presence of carvacrol and cinnamaldehyde, respectively (Rojas-Graü et al., 2006). These findings were subsequently confirmed in real foods as ham and bologna (Ravishankar et al., 2012) and lettuces and spinaches (Zhu et al., 2014), controlling the growth during a week of L. monocytogenes and Salmonella, respectively. As noted these ingredients are GRAS (Espitia, Avena-Bustillos, Du, Teófilo, et al., 2014), which is compulsory for edible industrial purposes. Natural antimicrobials are not limited exclusively to essential oils and it is known that polyphenols, flavonoids, or phenolic derivatives possess antioxidant activity (Moure et al., 2001). Indeed

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polyphenolic compounds from apple skin have been successfully incorporated into edible films and have acted against L. monocytogenes (Du, Olsen, Avena-Bustillos, Friedman, & McHugh, 2011). Overall, several researches have tested edible films with antioxidant and/or antimicrobial compounds in real foods extending their shelf life, including meat products such as chicken breasts (Ravishankar, Zhu, Olsen, McHugh, & Friedman, 2009), ham, and bologna (Ravishankar et al., 2012); vegetables, such as organic leafy greens (Zhu et al., 2014); oils, such as soybean (Hayashi, Veiga-Santos, Ditchfield, & Tadini, 2006) and palm (Reis et al., 2015); and fruits, such as apple (McHugh & Senesi, 2000) and strawberry (Peretto et al., 2014).

3.3 Conclusions and future opportunities A viable balance of the environment and food supply together with the need for improved quality and extension in shelf life is not an easy task. To address these concerns, we turn to novel technologies to replace traditional thermal processes. Firstly, SCF-based technologies are involved in a wide variety of food industrial applications such as the extraction of natural colorants and aromas, extraction of lipids, elimination of odorant compounds avoiding unpleasant sensorial characteristics in fish oil, the sterilization of milk and fruit juice, and so on. The development of SCFs is growing exponentially due to a lack of toxicity and low cost, and the mild conditions (temperature and pressure) in which they can be used, making them environmentally safe. Another key food technology is US, which is rapidly increasing due to its extreme efficiency with minimal alterations. Low-frequency sonication is widely applied for process intensification in terms of control and monitoring, whereas high-frequency sonication can produce alterations in foodstuffs. In addition, the employed temperatures to inactivate or kill microorganisms are not as high as they are in the thermal process allowing the saving of energy. It is worth noting that a novel ohmic heating technique is an alternative to conventional heating. It is considered a waste-free process, which is a clear environmental benefit, and does not require high energy consumption. Finally, recent developments in packaging have made it possible to enhance food and beverage product preservation, maintaining their nutritional quality and reducing the release of contaminants. Natural additives with antioxidant and antimicrobial activity could be used to ensure food safety in order to reduce synthetic additives. Another goal of active packaging is to enhance the protective wrapping, transforming it into biodegradable, or even edible, film to be more eco-friendly. Overall, novel food processing technologies are being developed and some are commercially available at the industrial scale. They offer advantages derived from higher efficiency, thus leading to shorter processing times, maintaining or improving the nutritional and functional properties of the processed foods. Further developments in the process scale-up are required. In some cases, coupling these emerging technologies with existing processes avoiding dramatic configuration changes could improve their performance.


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Further reading Buzrul, S. (2008). High hydrostatic pressure (HHP) applications in food science: A study on compression heating, microbial inactivation kinetics, pulsed pressure and high pressure carbon dioxide treatments [PhD thesis]. University of Bordeaux 1 and Middle East Technical University. Rosa, P. T. V., Parajó, J. C., Moure, A., Díaz-Reinoso, B., Smith, R. L., Toyomizu, M., et al. (2009). Supercritical and pressurized fluid extraction applied to the food industry. In M. A. A. Meireles (Ed.), Extracting bioactive compounds for food products (pp. 269–401). Boca Raton, FL: CRC Press.

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Section 2 Processing, bioavailability and bioaccessibility

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Lipids and fatty acids


Mirian Pateiro⁎, Rubén Domínguez⁎, Paulo Eduardo Sichetti Munekata*, Francisco J. Barba†, José M. Lorenzo⁎ ⁎ Meat Technology Center of Galicia, Parque Tecnológico de Galicia, Ourense, Spain, † Nutrition and Food Science Area, Preventive Medicine and Public Health, Food Sciences, Toxicology and Forensic Medicine Department, Faculty of Pharmacy, Universitat de València, València, Spain

4.1 Background Lipids are a class of compounds soluble in non-polar organic solvents observed in composition of living organisms and food. These molecules are formed mainly by carbon, hydrogen, and oxygen atoms, although many structures also contain phosphorus, nitrogen, and sulfur (Valenzuela & Valenzuela, 2013). This class of compounds is of great importance for the human body due to a large body of evidence from in vitro, animal, and clinical studies that associate its consumption with adequate growth, development, and normal tissue activity (Simopoulos, 2011; Valenzuela, 2009). Lipids are essential for many structures and functions in the human body including: (1) as components of biomembranes (cellular and mitochondrial membranes), (2) in the formation of intracellular constituents (lipoproteins) involved in lipid transport in the blood, (3) in regulation of metabolism, (4) as thermal insulators due to deposition in subcutaneous adipocyte tissue around organs, (5) as storage of energy (9 kcal/g or 37.62 kJ/g), (6) as an intermediate agent in the absorption of fat-soluble vitamins, (7) as a basic and essential skeleton for synthesis of steroid hormones (androgens and estrogens), and (8) as a constituent of and a contributor to the production of bile salts (Dawson, Lan, & Rao, 2009). Regarding food composition, lipids are important molecules associated with characteristics as palatability, flavor, aroma, and texture of food. They also facilitate the dispersion of fat-soluble antioxidants, pigments or dyes, and vitamins, and promote, or contribute as emulsifying agent, in emulsification and suspension of food ingredients/additives (Valenzuela, Delplanque, & Tavella, 2011). The nutritional requirements are dependent on age, physiological state, and other characteristics of individuals (Willett, 2012).

4.1.1 Fatty acids Fatty acids are basic units of fat and oils. This group of molecules is composed of four or more hydrocarbon structures (basically a carbon connected with hydrogen atoms) linked to a carboxyl group (acidic functional group) (Fig. 4.1). The number of carbons influences chemical and physical properties of fatty acids, such as solubility in non-polar solvents and melting point (Fahy et al., 2005). Innovative Thermal and Non-Thermal Processing, Bioaccessibility and Bioavailability of Nutrients and Bioactive Compounds © 2019 Elsevier Inc. All rights reserved.


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Fig. 4.1  Structure of the main lipids. Fatty acids (A), phospholipids (B), and sterols (C).

Fatty acids can be classified according to number of carbons: 4 (C4) to 10 (C10) carbons as short-chain fatty acids, 12 (C12) to 14 (C14) carbons as medium-chain fatty acids, 16 (C16) to 18 carbons (C18) as long-chain fatty acids, and 20 (C20) or more carbon atoms as very long-chain fatty acids. This classification excludes structures made of two (C2) and three (C3) carbons known as acetic acid and propionic acid, respectively. Although the occurrence of fatty acids with >20 carbons is lower than that observed for other fatty acids, the accumulation of some structures as long as 36 carbons have been reported in the brain of vertebrates, including mammals and humans (Kuipers et al., 2012). Another common classification for fatty acids divides them according to the number of double bonds. Saturated fatty acids do not contain double bounds, while monounsaturated and polyunsaturated fatty acids have one or at least two double bounds, respectively. Within polyunsaturated fatty acids (PUFA), there are essential for normal life functions. These fatty acids can be classified as belonging to two “families,” omega-3 and omega-6, which differ not only in their chemistry but also in their natural occurrence and biological functions (Cascio, Schiera, & Di Liegro, 2012). In a nutritional perspective, some unsaturated fatty acids also receive another classification: essential fatty acids (EFA) and non-essential fatty acids (NEFA) (Fig. 4.2). An EFA is a fatty acid that cannot be synthetized by metabolic routes (enzymatic addition of double bounds) from other dietary fatty acids but is crucial to maintain normal body status. EFA is also species dependent since each species requires different amounts and types of fatty acids (Goodhart & Shils, 1980; Valenzuela & Valenzuela, 2013). Particularly for humans, linoleic and linolenic acids (from omega-6 and omega-3 families, respectively) are the only known EFA due to the lack of desaturase enzymes (Whitney & Rolfes, 2008). Some other fatty acids are sometimes classified as “semi-essential,” meaning that they can become essential under some developmental or disease conditions; examples include the omega-3 fatty acids eicosapentaenoic (EPA) and docosahexaenoic (DHA), and the omega-6 arachidonic acid. In particular occasions (during development or progression of disease), EPA, DHA, and arachidonic acid can be considered as "essentials" (Robinson et al., 2017).

4.1.2 Phospholipids Phospholipids are amphipathic lipids that are composed of glycerol, fatty acids, phosphate, and (usually) an organic base or polyhydroxy compound. In this molecule, fatty

Polyunsaturated fatty acids

n-9 serie Biosynthesis from carbohydrates

n-6 serie

n-3 serie



Diet Elongase C16:0 C18:0


Elongase D 9 desaturase


C18:2n-6 D 6 desaturase








Elongase Elongase







C20:1n-9 C22:1n-9

D 5 desaturase



C22:4n-6 D 4 desaturase

C24:4n-6 C24:5n-6

Elongase D 6 desaturase Oxidation


Fig. 4.2  Endogenous synthesis of monounsaturated and polyunsaturated fatty acids.

C22:5n-3 C24:5n-3 C24:6n-3 C22:6n-3

D 4 desaturase

Lipids and fatty acids109

Monounsaturated fatty acids


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acids (usually PUFA) are linked to glycerol in positions sn-1 and sn-2 of glycerol moiety by ester bounds, while phosphate is likely to be bound in the sn-3 position of glycerol (O’Keefe, 2002). They are the main constituents of cell membranes with relevant metabolic functions. They form double-layered membranes with the water-soluble molecules on the outside of the cell membrane and the water-insoluble molecules in the inside (Green, Liu, & Bazinet, 2010). Moreover, they have structural and functional properties, activating the enzymes as messengers in the transmission of signals to the interior of the cell. They are also pulmonary surfactants, being indispensable for the proper functioning of the lungs.

4.1.3 Sterols Sterols are an important class of organic molecules. The basic structure of these compounds is formed by four aromatic rings (A, B, C, and D) derived from sterane (cyclopentanoperhydrophenanthrene). A common characteristic of all sterols is the presence of a polar hydroxyl group at carbon 3 of A ring, while the remaining structure is non-polar. This condition confers an amphipathic character similar to that observed in phospholipids (Valenzuela & Valenzuela, 2013). Among sterols, cholesterol is the most important and abundant steroid lipid in the body. Many important animal metabolic molecules are formed from cholesterol, such as steroid hormones, bile salts, vitamin D, and oxysterols. In addition, cholesterol is also involved in both cellular formation and repair (Björkhem, 2009).

4.2 Absorption process Foods contain many components that are of great importance in human nutrition. Due to the importance of healthier eating habits, consumers prefer the natural version of foods over their chemical counterparts. This scenario is also motivated by the choice for food with health claims rather than taking medicine separately (Betoret, Betoret, Vidal, & Fito, 2011). To this regard, consumers demand high-quality food products with low fat and healthier fat composition, in view of cardiovascular disease or obesity (Domínguez, Pateiro, Sichetti Munekata, Bastianello Campagnol, & Lorenzo, 2017). In addition, it is well known that food with a high fraction of PUFA per se is not necessarily healthy if the n-6/n-3 ratio it is not balanced (Simopoulos, 2004). Therefore the trend in the food industry is to enrich its products with bioactive compounds. In the particular case of lipids, one of the main strategies is enrichment with long-chained n-3 fatty acids, especially EPA and DHA, that have a positive impact on human health (Pourashouri et al., 2014). In fact, there are international recommendations about the amount of EPA and DHA intake (between 250 mg/day and 1 g/day) (Kris-Etherton et al., 2012). However, not only the content of certain lipids can be considered healthy since they are not always available. Before becoming bioavailable, they must be released from the food matrix and modified in the gastrointestinal tract (Carbonell-Capella, Buniowska, Barba, Esteve, & Frígola, 2014). Therefore it is important before ­concluding on any

Lipids and fatty acids111

potential health effect to analyze whether the digestion process affects these compounds and their stability. At this point, it is important to define bioavailability and bioaccessibility (Fig. 4.3). Bioaccessibility is the quantity of some compound, which is released from the food matrix, modified in the gastrointestinal tract that becomes available for absorption (Galanakis, 2017; Heaney, 2001). Therefore bioaccessibility includes all transformations (both intestinal and hepatic metabolism) that facilitate assimilation and absorption of a certain nutrient by intestinal epithelium cells (Carbonell-Capella et al., 2014). Food matrix and composition can influence the bioaccessibility of nutrients, which can vary depending on the antagonisms or synergies between the different compounds, improving or worsening the release and/or absorption of the compounds of interest (Fernández-García, Carvajal-Lérida, & Pérez-Gálvez, 2009; Galanakis, 2017). Alternatively, the concept of bioavailability encompasses all processes such as gastrointestinal digestion, absorption, metabolism, tissue distribution, and the specific effect upon exposure to a substance (physiological response), which also involves the concept of bioaccessibility (Fernández-García et  al., 2009). In this sense, bioavailability is defined as the portion of a given nutrient that is absorbed from its dietary source and properly stored or transported to exert a specific physiological function (Fairweather-Tait, 1993). With this in mind, the bioavailability of a given nutrient depends on the release from food matrix, the solubilization in gastrointestinal fluid, the migration and absorption by intestinal epithelial cells, and further enzymatic and chemical reactions to achieve tissues (McClements & Xiao, 2014). Taking into account the theme of this chapter, absorption requires that the lipids are released from the food matrix. Lipids (triglycerides, phospholipids, and cholesterol esters) are then transferred to bile salt-mixed micelles as free fatty acids, monoand di-acylglycerides, lyso-phospholipids, and free cholesterol (Harrison, 2012). The generated micelles diffuse through the mucin layer to the apical cell surface of the absorptive epithelial cells. Finally, fat-soluble compounds interact with brush border proteins for transfer to the cell interior, and bile salts in the micelles dissociate and are re-absorbed primarily in the ileum (Kopec & Failla, 2018).

4.3 Lipid oxidation. The main factor that affects the bioavailability of lipids Food processing is necessary to increase the amount of products available as well as to ensure their stability for long periods. However, food processing usually impacts important physico-chemical and sensory characteristics along with food nutrients. This effect on food nutrients requires special attention since bioaccessibility can be influenced. Therefore processing parameters (e.g., temperature, time, oxygen amount, and radiation) must be selected to achieve stability and prevent/reduce the impact on nutrients. Lipid oxidation (together with microbial contamination) is considered as one of the main causes of quality deterioration in food, which leads to off odors and off flavors as


Fatty acids and monoglycerides

Intestinal lumen

BIOAVAILABILITY Absolute Bioavailability

Emulsion Micelles

Epithelial cells

Triglyceride Golgi

Total intake of food

Assimilated amount used for storage and metabolic functions



Quantity of compound that is released from its matrix into the gastrointestinal tract, becoming available for absorption

Digestive transformation of foods Absorption process Pre-systemic, intestinal and hepatic metabolism

Fig. 4.3  Scheme of lipid absorption, bioaccessibility and bioavailability.

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Essential for nutritional efficiency. Includes gastrointestinal digestion, absorption, metabolism, tissue distribution, and bioactivity



Rate and extent to which the active ingredient is absorbed and becomes available

Lipids and fatty acids113 Termination

Initiation OH






+ OH



+ O2




Unsaturated Lipid



R Lipid Radical




R Lipid Peroxy Radical



R Lipid Peroxyde


Fig. 4.4  Process of lipid oxidation of unsaturated fatty acids.

well as discoloration or texture changes (Gong, Parker, & Richards, 2010). Additionally, oxidation reduces nutritional values and induces the formation of compounds that may pose continual risks to human health (Kanner, 1994; Min & Ahn, 2005; Vandamme et al., 2015). Lipid oxidation involves, in general, the irreversible formation of radicals from breakdown of double bonds of unsaturated fatty acids, the consumption of oxygen, rearrangement of double bonds, and the production of several products (such as alcohols, aldehydes, and ketones) from lipid peroxides breakdown (Fig. 4.4). Lipid oxidation is a phenomenon governed by thermodynamic laws. During heat treatment, inorganic and biological agents, naturally present in food (metal ions or enzymes), can induce lipid oxidation (Jambrak & Škevin, 2017). Two factors greatly influence lipid oxidation in food: fat content and fatty acid composition. Fatty acid profile of fat is more important than the amount of fat because the susceptibility of food lipids to lipid peroxidation depends upon the degree of polyunsaturation in fatty acids (Min, Nam, Cordray, & Ahn, 2008). The greater facility to extract a hydrogen atom in the PUFA than in the saturated fats makes them more susceptible to lipid oxidation (Gong et al., 2010). It is well known that fats or oils containing high proportions of linoleic or linolenic acids are more prone to oxidation than oils with high oleic acid contents. Thus lipid oxidation reduces the essential fatty acids content of fats (Lorenzo, Domínguez, & Carballo, 2017), resulting in lower nutritional value. In addition, lipid peroxides can also degrade lipid-soluble vitamins such as alpha- and beta-carotene (Jambrak & Škevin, 2017).

4.4 Effect of processing on lipid oxidation and bioaccessibility Conventional thermal technologies are largely applied in the food industry due to well-established reliability and efficacy. However, the use of such technologies is commonly associated with detrimental effects on food quality by destroying nutrients,


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forming off flavors, and causing color changes (Mújica-Paz, Valdez-Fragoso, Samson, Welti-Chanes, & Torres, 2011). As mentioned, lipids have beneficial effects on human health, in particular the omega-3 fatty acids EPA and DHA (Connor, 2000). Epidemiological and clinical studies showed the important role that these fatty acids have in the risk and management of cardiovascular diseases, and in normal growth and development (Kritchevsky, 2008; Simopoulos, 1999). Therefore dietary recommendations often contain high amounts of PUFA (as part of healthful nutrition), which are readily oxidized and should be considered in food processing (Jambrak & Škevin, 2017). From this scenario, alternative technologies to complement or replace traditional thermal processing have been studied and applied in the food industry. These novel technologies include high-pressure processing (HPP), ultrasound (US), ionizing radiation (IR), and pulsed electric fields (PEF).

4.4.1 Thermal processing Thermal processing is still one of the most useful tools to effectively eliminate or reduce to acceptable levels any pathogenic bacteria that may be potentially present in foods. These techniques are classified as conventional processing technologies, and use high temperatures to processed food. Pasteurization, sterilization, microwaving, and spray-drying are among the most commonly used thermal processing technologies (Fig. 4.5).

Fig. 4.5  Main thermal processing methods used in food industry. Pasteurization and sterilization (A), spray-drying (B), and microwave treatment (C).

Lipids and fatty acids115

However, previous studies support the idea that these processing techniques show disadvantages that restrict their use (Lund, 1988), which can affect the nutritional profile and characteristics of bioactive compounds (bioavailability and stability). Pasteurization and sterilization Pasteurization reduces the amount of vegetative microorganisms in foods by heating food to pre-established temperature and time (Lund, 1988). This thermal treatment destroys pathogenic microorganisms, most of the microorganisms that cause spoilage and reduce enzymatic activity of food. The purpose is to increase both safety and shelf life of treated products. This heat treatment usually uses temperatures between 60°C and 85°C. Due to the possible combinations of time and temperature, three methods of pasteurization can be distinguished: (1) low temperature holding pasteurization (LTH) or low temperature long time (LTLT), (2) high temperature short time (HTST) or continuous flow and (3) ultra-heat treatment or ultrahigh temperature (UHT). After pasteurization, a reduced amount of viable microorganisms remains in pasteurized products. To prevent their growth, pasteurized products must be stored under refrigerated conditions (0–5°C) (Heinz & Hautzinger, 2007). Regarding meat products, cooked ham and sausages packed in plastic pouches and casings, respectively, are commonly pasteurized. The treatment of such products requires that internal temperature must be held between 72°C and 78°C for sensory reasons. Alternatively, sterilization is a thermal process that treats food at temperatures >100°C, which creates a sterile condition in the final product (Lund, 1988). Properly sterilized meat products, particularly those packaged in hermetically sealed glass jars and tin cans, do not contain viable microorganisms and can be stored at ambient temperature. Although sterilization is an efficient technology to drastically reduce microbial load, certain microbial strains and spores are extremely heat resistant, which demands additional strategies to prevent their development and preserve organoleptic and nutritive value of the final product (Heinz & Hautzinger, 2007). Microwave Electromagnetic waves of frequency between 300 MHz to 300 GHz are known as microwaves. In the characteristic bands of 915 and 2450 MHz, microwave penetrates food in the range of 8–22 cm and 3–8 cm, respectively. This penetration rate is also influenced by moisture content in food (Ahmed & Ramaswamy, 2007). The heating effect observed in food subjected to microwaves can be explained by two main mechanisms: dipole rotation and ionic polarization. The first mechanism involves dipole molecules, mainly water. When an alternating current electric field is applied to food, dipolar molecules rotate to align themselves with the electric field. The constant and fast rotation induced to such molecules causes internal molecular friction, which results in heating. However, ionic polarization influences ions to perform unidirectional movements in an attempt to align themselves with the oscillating electric field produced by microwaves (Decareau & Peterson, 1986).


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There are two known microwave heating systems: batch and continuous-flow heating. In the first, heating occurs in the microwave cavity to achieve a target temperature after a pre-established time. Continuous systems increase productivity, are easier to clean up and automate, and are often used for liquids (Gentry & Roberts, 2005). Microwave treatment has been widely used in the food industry since 1970 for thawing and tempering. However, new applications can be associated with this technology, such as drying and cooking, which are used for food processing (Schubert & Regier, 2007); pasteurization and sterilization (Hamid, Boulanger, Tong, Gallop, & Pereira, 1969); and development of novel microwaveable foods packaged in suitable materials (Bertrand, 2005). Microwave processing has positive effects compared with conventional other thermal technologies. It leads to a significant reduction in processing time and energy, fast and uniform heating, and preservation of nutrients, vitamins, and sensory characteristics (e.g., flavor and color). It produces minimum fouling depositions, offers high heating efficiency, is suitable for liquids sensible to heat, has high viscosity and is composed of multiphase, does not cost a lot to maintain, and can be coupled to other equipment, such as infrared heating and regenerative heat exchangers to enhance process performance (Ahmed & Ramaswamy, 2007; Yarmand & Homayouni, 2011). In contrast, multiple researches observed an increase of lipid oxidation when meat samples were cooked with the microwave technique. To this regard, Weber, Bochi, Ribeiro, Victório, and Emanuelli (2008), Domínguez, Gómez, Fonseca, and Lorenzo (2014a, 2014b), and Lorenzo and Domínguez (2014) found that samples treated with microwave presented the highest values of TBARs, in comparison to raw meat and other cooking methods. The fact that samples cooked by microwave had high levels of oxidation compounds suggests microwaves induce oxidation on meat fat, which affects PUFA (Broncano, Petrón, Parra, & Timón, 2009). In a similar way, Yoshida, Hirakawa, Tomiyama, Nagamizu, and Mizushina (2005) observed a reduction of PUFA values in phospholipids fraction after microwave cooking in different foods. Authors suggested that PUFA were degraded to secondary oxidation products. Moreover, there are studies that suggest that total fatty acids remain stable in samples cooked with a microwave oven. Domínguez, Borrajo, and Lorenzo (2015) did not find any differences in fatty acids profile between raw and microwave-treated samples. In fact, not even the fatty acids most susceptible to oxidation, such as linolenic, EPA, DPA or DHA, showed differences in between treatments. Spray-drying Spray-drying is a well-established method of producing a dry powder from a liquid or slurry by rapidly drying with a gas. The process of microencapsulation using spray-drying involves six steps: (1) preparation of emulsion to be processed, (2) homogenization of the emulsion, (3) atomization of the emulsion into the drying chamber or feed atomization, (4) heat transfer, (5) dehydration of the atomized particles or mass transfer, and (6) particles separation (Shahidi & Han, 1993). This technique is usually applied for food fortification (addition of bioactive compounds) during processing of functional foods and encapsulation of volatile, sensitive, and functional ingredients (Drusch & Diekmann, 2015; Gharsallaoui, Roudaut,

Lipids and fatty acids117

Chambin, Voilley, & Saurel, 2007). From the product point of view, this technique leads to the formation of powders with low water activity, which reduces weight and volume, gives improved flow properties, facilitates packaging storage, transportation, and commercialization, and ensures microbiological stability. One limitation of this technology is the limited number of shell materials available and the cost of the equipment (Franco et al., 2017). Nowadays, there is a growing interest in developing products enriched in omega-3 fatty acids with a healthy purpose (Lee, Joaquin, & Lee, 2007). As previously indicated, spray-drying technology can be used to fortify food, and omega-3 oils are between the ingredients that have been encapsulated with this technique. The encapsulation of omega-3 oils with this technique allows to improve oxidative stability, mask off flavors, and transform liquid into powder (Botrel et al., 2017; Chen, McGillivray, Wen, Zhong, & Quek, 2013; Comunian & Favaro-Trindade, 2016). In meat products, spray-drying is considered as a novel technology; however, there are few studies where this technology was used to enrich in omega-3 fatty acids (Aquilani, Perez-Palacios, Crovetti, Antequera, & Pugliese, 2016; Jiménez-Martín, Pérez-Palacios, Carrascal, & Rojas, 2016; Lorenzo, Munekata, Pateiro, Campagnol, & Dominguez, 2016), and others where oil microcapsules of fish oil, olive, and fish-oil mixtures are used to improve the lipid profile by partially replacing pork back fat (Domínguez, Pateiro, Agregán, & Lorenzo, 2017; Josquin, Linssen, & Houben, 2012). These oils are considered healthy because they come from fish and other seafood, which are sources of EPA and DHA (long chain n-3 PUFA). These fatty acids are readily oxidized, which demands additional protection such as that provided by the microencapsulation technique (Jiménez-Martín, Gharsallaoui, Pérez-Palacios, Carrascal, & Rojas, 2015).

4.4.2 Non-thermal processing In contrast to thermal processing, there are emerging technologies that avoid using high temperatures. In this case, heat comes from internal energy generation. Because of that, emerging technologies are classified as non-thermal technologies (Jambrak & Škevin, 2017). These alternative processing technologies preserve food and in consequence bioactive compounds, since they do not remove functional properties or change important nutrients and sensorial characteristics (Galanakis, Barba, & Prasad, 2015). However, the use of some alternative processing technologies, such as cold plasma or US, can cause detrimental effects instead of protection to lipids. These technologies are considered as advanced oxidation processes (Galanakis, 2017). With this in mind, it is clear that more knowledge is needed about the effect of these technologies on some components of food. High pressure processing (HPP) High pressure is one of the non-thermal processing techniques most used. This emerging technology has recently been developed and adapted as a method to reduce the microbial load of food and thus increase its useful life. This processing technique is considered as minimal processing food-preservation technology (Medina-Meza,


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Barnaba, & Barbosa-Cánovas, 2014). Usually, HPP subjects food to no >900 MPa at room temperature to destroy most of microorganisms found in foods while sensitive components (such as PUFA) are preserved (Mathavi, Sujatha, Ramya, & Devi, 2013). The main advantages of this emerging technology are the ability to process food uniformly and independently of matrix size and composition in a short time, while killing bacteria and spores in a non-toxic way. The application of HPP reduced processing time in comparison with thermal treatments, and it could be applied at room temperature, which preserves nutrients, freshness, flavor, color, and taste (Mathavi et al., 2013; Medina-Meza et al., 2014). In addition, food structure and texture of certain foods can also be improved after high-pressure treatment (Hayashi, 2002). The typical pressure used in different food matrices is 350–400 MPa applied for 30 min (Mathavi et al., 2013). According to Bermúdez-Aguirre and Barbosa-Cánovas (2011), operating pressure has been increasing from 300 to 400 MPa to 800 MPa, above which reduced the usual holding time between 15 and 30 min to no >5 min. These increases of operation pressure have an important and unwanted secondary effect. The compression process increases the temperature between 3°C and 8–9°C (in foods with high fat content) per 100 MPa (Butz & Tauscher, 2002; Rasanayagam et al., 2003). Moreover, the stability of lipid fractions in food relies on hydrophobic interactions that are also sensitive to high pressure (Medina-Meza et al., 2014). This fact characterizes lipids as the most pressure-sensitive biological components (Rivalain, Roquain, & Demazeau, 2010). Different researchers conclude that lipid oxidation is triggered by pressure between 300 and 500 MPa, although other factors also influence this threshold, such as temperature, time, lipid composition, and non-lipid fractions of food (Medina-Meza et  al., 2014). As a general rule, the increase in lipid oxidation after the application of high pressures becomes more evident during food storage (Simonin, Duranton, & De Lamballerie, 2012). Particularly for meat, increased lipid oxidation after HPP treatment has been associated with the heme group of haemoproteins. This protein undergoes structural changes that expose the pro-oxidant heme group to catalyze the oxidation of unsaturated fatty acids (Bou et al., 2008). Many researchers studied the effect of HPP on different foods. Some of them are summarized in Table 4.1. Generally speaking, for intramuscular lipids, no significant effect has been observed for HPP up to 500 MPa on the total fat content and fatty acid composition. In fact, He et al. (2012) and Huang et al. (2012; 2015), for pork, and Kang et al. (2013), for goat meat, did not find any significant differences in fatty acid contents of total lipid fraction. In contrast, Canto et al. (2015) found that HPP treatment decreased the total fat content and the n-3 PUFA (linolenic acid, EPA, DPA and DHA) in caiman meat. Significant changes can be observed in fatty acid composition as well as in the concentration of major fatty acid groups such as free fatty acids and phospholipids. Lipolysis on phospholipids is induced during HPP and storage, which increases the free fatty acid content. Many authors observed similar results for the susceptibility of PUFA to hydrolysis. Phospholipids composed of PUFA displayed remarkable reduction, while the free fatty acid content increased (He et al., 2012; Huang et al., 2012; Huang et al., 2015).

Food matrix

Processing conditions

Processing time




400 & 600 MPa/35°C, 45°C and 55°C 200, 400 & 600 MPa/15°C 200, 350 & 500 MPa/20°C

20 min

Increase TBARs value. Reduce n-6/n-3 ratio

4 min


500 MPa/20°C

20 min


200, 400 & 600 MPa/20°C and 50°C

20 min


200, 300 & 400 MPa/20°C 300, 450 & 600 MPa/15°C 100 MPa/20°C

10 min

600 MPa Increase TBARs value. Other treatments did not affect the oxidation level Increase TBARs value. 500 MPa reduce linoleic acid in triglyceride fraction and linolenic acid and total PUFA in phospholipid fraction Increase TBARs value. Reduce linoleic acid, linolenic acid and total PUFA in phospholipid fraction. Increase free fatty acids Increase TBARs value. Reduce linoleic acid, linolenic acid and total PUFA in phospholipid fraction and increase these fatty acids in free fatty acids fraction. Decrease total phospholipids while increases total free fatty acids Decrease total fat. Reduce total PUFA, total n-3 fatty acids and specially linolenic, EPA, DPA and DHA fatty acids 600 MPa Increase TBARs value. Other treatments did not affect the oxidation level Not affected fatty acids profile. Changes in volatile compounds were observed

McArdle, Marcos, Kerry, and Mullen (2011) Utama et al. (2017)

Beef Pork

Beef Goat

20 min

5 min 24 h

He et al. (2012)

Huang, He, Li, Li, and Wu (2012) Huang et al. (2015)

Canto et al. (2015) Kim et al. (2014) Kang et al. (2013)

Lipids and fatty acids119

Table 4.1  Effect of high pressure processing (HPP) on lipids stability (oxidation, fatty acids and sensory properties).


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The additional release of fatty acids also induced an increase of TBARs values. This effect is mainly attributed to susceptibility of PUFA to oxidation in this fraction of lipids. In fact, the majority of the researches presented in Table 4.1 showed that the application of HPP (≥500 MPa) increased lipid oxidation. Alves, Bragagnolo, da Silva, Skibsted, and Orlien (2012) observed a clear relationship between the increasing pressure during HPP and the evolution of lipid oxidation during refrigerated storage in meat samples. These authors also conclude that pressure treatment >300 MPa accelerates lipid oxidation. This effect is more evident during storage, due to the free fatty acids interaction with other compounds. In fact, the oxidation level is much higher in the samples treated with the highest pressures during storage (Fig. 4.6). Two explanations for the effect of HPP in lipid oxidation have been indicated: catalytic effect of heme proteins and structural damage of lipid membranes (Bolumar, Andersen, & Orlien, 2014). Ultrasound (US) US is a technology of multiple applications. This characteristic is due to its wide acoustic frequency spectrum, which ranges from 18 kHz up to several MHz (Knoerzer, 2016). In the range of 18–100 kHz, this technology is known as power ultrasonic and the main mechanism involved is attributed to cavitation (Knoerzer, Buckow, Trujillo, & Juliano, 2015). Cavitation occurs in stages wherein microbubbles are formed, grow in size, and eventually collapse due to propagation of US in liquids (Knoerzer, 2016). The main advantages of US, in comparison to thermal or conventional processing, can be related to reduction of energy, time, and resources to operate the system in general (Jambrak & Škevin, 2017). Particularly for microbial inactivation, US is also considered as safe and does not produce toxic compounds (Kentish & Ashokkumar, 2011). 7

TBARs value


500 MPa

5 4 350 MPa

3 2 1 0

10 kGy induce significant changes on lipid oxidation of beef patties, while the application of 5 or 10 kGy did not affect TBARs values. Similarly, Ham et al. (2017), on pork sausage, and Li et al. (2017) on fresh pork, observed using gamma radiation (60Co) that only doses higher than 7.5 kGy had an adverse effect on lipid oxidation and sensory characteristics. In contrast to these researches, Feng et al. (2017) found that the TBARs value in turkey meat increased with all treatments (all of them 5000 K) and high pressure (more than thousands of bars) affecting the structural properties of proteins. Indeed, cell structures can modify through the formation of bubbles, varying quality properties such as texture and color and affecting microbial inhibition (Alarcon-Rojo, Janacua, Rodriguez, Paniwnyk, & Mason, 2015). Effectively ultrasound treatment could be used as a non-thermal sterilization technique inactivating microorganisms or microbial spores without affecting sensorial parameters. In recent years, ultrasound techniques have been used in two different ways: (1) directly applied on food (food preservation, extraction technique, or with the aim of modifying functional properties) and (2) indirectly applied on food (cleaning and disinfection of instruments and material). In general, ultrasound applications are divided into two mains categories: low- and high-intensity ultrasounds. Both types have been demonstrated to be useful for industrial processing.


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Amplitude pressure

Low frequency wave


Cell lysis Implosion

Cavitation bubble

Amplitude pressure

High frequency wave


Cavitation bubble


Fig. 5.2  Schematic graph of cavitation bubbles in high and low ultrasounds and their effect on cell lysis.

Low-power ultrasounds (high frequencies between 2 and 10 MHz; power up to 10 W) are used as non-destructive tools to monitor the industrial processes. The passage of the waves through the food matrix changes the food material properties. Generally, they are used in quality control (Chandrapala, Oliver, Kentish, & Ashokkumar, 2013; Ghaedian, Coupland, Decker, & McClements, 1998), extraction, functionality modification, or microbes deactivation (Ashokkumar, 2015). The quality control applications are based on wave speed, which depends on the moisture content or chemical composition. Several researches have studied the effect of low-power ultrasound on different foodstuffs, such as fruit and vegetables (Elvira, Durán, Urréjola, & de Espinosa, 2014; Mizrach, 2008), cakes (Gómez, Oliete, García-Álvarez, Ronda, & Salazar, 2008), cheese (Benedito, Carcel, Gonzalez, & Mulet, 2002) and meat products (Simal, Benedito, Clemente, Femenia, & Rosselló, 2003). Conversely, high-power ultrasounds (low frequencies between 20 kHz and 100 kHz; power up to hundreds of watts) are used to generate intense cavitation. In the review by Corzo-Martínez et al. (2017), structural modifications of proteins during food processing with high-intensity ultrasound are described. These structural changes of proteins were usually denaturalization and subsequent aggregation; even biological value and organoleptic properties decreased depending on the severity of processing. Structural properties of the proteins are affected by high-intensity ultrasound treatment due to formation of covalent and/or noncovalent bonds, enhancement in dissolution and solubilization, or foaming ability. Functional properties are also altered by inter and intramolecular interactions, and the changes induced by ultrasound ­depends on the type of protein (Ozuna, Paniagua-Martínez, Castaño-Tostado, Ozimek, & Amaya-Llano, 2015). Zou et al. (2017)

Proteins and amino acids147

reported that ultrasounds improved functional properties such as solubility, foamability, and gelation, with respect to a traditional treatment. Protein agglomerates can be disrupted, decreasing their size in aqueous solution. In addition, the ultrasonic energy can also break disulphide bonds destabilizing the secondary structure and maintaining the denatured structure (O’Sullivan, Park, Beevers, Greenwood, & Norton, 2017). Furthermore ultrasounds produce changes in the post-translational modifications of proteins varying their level of oxidation, glycosylation, hydroxylation, phosphorylation, methylation, and acylation. Moreover they can trigger chemical reactions in live organisms changing edibility and nutritional quality of proteins, such as seed germination stimulation. For example, Yang, Gao, Yang, and Chen (2015) reported improvements in the nutritional value of soybean sprouts. Stefanović et al. (2014) studied the effect of ultrasound treatment on enzymatic hydrolysis of egg protein with several proteases. They found that optimized conditions of ultrasound in the enzymatic process improved the degree of hydrolysis compared with a thermal treatment. Milk and whey have been extensively studied with power ultrasounds to achieve different aims focused on microbiological and functional properties (Cameron, McMaster, & Britz, 2009; Muthukumaran, Kentish, Ashokkumar, & Stevens, 2005; Yanjun et al., 2014). In addition, the effects of ultrasounds on milk homogenization have been previously studied (Villamiel & de Jong, 2000). These authors have tested that the ultrasonic process can influence fat separation in raw milk due to reduction in fat globule size. In the same way, Juliano et al. (2011) used ultrasound to move fat particles and generate coalescence; these larger particles have low density floating on the milk surface. When whey was subjected to ultrasounds treatment, some evidence of denaturation was indicated by Villamiel and de Jong (2000) at temperatures higher than 60°C. However, when lower temperatures (60 different foods. The volume of the irradiated foodstuffs is increasing annually in the world (Arvanitoyannis, 2010). The main effects of irradiation include: sterilization and control of pests, shelf-life improvement by modification of ripening delay and sprout inhibition of fruits and vegetables, and prevention of foodborne disease by the inactivation of pathogens. In addition, irradiation techniques have showed improvement to the rehydration process as well as reduction of anti-nutritional components of plants (Bhat, Ameran, Voon, Karim, & Tze, 2011). However, indirect effects of irradiation, such as lipid oxidation, vitamin destruction, and protein denaturation, limit its application in food processing (Al-Kahtani et al., 1998; Rahman, 2007). This is a controversial issue; according to the Food and Drug Administration (FDA), irradiation has effects on food nutritional value that is similar to those of conventional food-processing techniques. In this regard, lipids, carbohydrates, proteins, minerals, and most vitamins remain almost unaffected by irradiation (Stewart, 2001). On the contrary, high-intensity irradiation may cause the degradation of some micronutrients, such as vitamins A, B1, C, and E (Smith & Pillai, 2004) and protein (Bhattacharjee & Singhal, 2010). The intensity of the effect for each type of radiation is correlated with accumulated radiation dose and its penetrability, changing the biological effect (Jaczynski & Park, 2003, 2004). Experts reviewed the data and intervals and thus concluded that food irradiation has no risk. An international committee reported that a safety interval between 1 and 10 kGy for irradiated food presents no toxicological hazard (JECFI, 1981). Safety issues are being reviewed by international agencies in order to establish a safe process for irradiated food (EFSA, 2011; JECFI, 1981; WHO, 1994). Concerning proteins, it should be noted that radiation of proteins has been studied for >30 years (Houée-Levin & Sicard-Roselli, 2001). Irradiated food absorbs energy, which also breaks down the chemical bonds producing free radicals. Free radicals are unstable molecules that are highly reactive, and consequently new bonds are formed almost instantly. In a radiation process, proteins are affected by direct and indirect effects of ionizing, which result in conformational and structural changes (Kuan et al., 2013). The most commonly occurring changes of proteins are polymerization and


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f­ ragmentation because of the presence of free radicals. Another chemical reaction that occurs is the protein oxidation that occurs by the action of OH free radicals. The oxidative reactions occurring in muscle foods can result in the generation of carbonyls (aldheydes and ketones), protein polymers, and peptide scissions via deamination. Protein oxidation can occur by strong interaction between a protein and an oxidized lipid, and the oxidation can be easily transferred from lipids to proteins. Primary and secondary lipid oxidation products such as aldheydes and ketones can react with proteins, inducing protein oxidation. Different chemical reactions such as deamination, decarboxylation, reduction of disulphide linkages, oxidation of sulfhydryl groups, modification of amino acid moieties, peptide-chain cleavage, and aggregation can produce permanent changes (Kuan et al., 2013). The result of these chemical reactions can vary greatly depending on protein state (e.g., fibrous or globular, native or denatured, protein composition), the presence of other molecules (presence of oxidizable lipids, heme pigments, transition metal ions, water concentration and oxidative enzymes), and the irradiation conditions (e.g., dose, dose rate, temperature, and oxygen concentration) (Audette-Stuart, Houée-Levin, & Potier, 2005; Houée-Levin & Sicard-Roselli, 2001). The splitting and aggregation of proteins due to irradiation phenomenon are related to alterations of the secondary and tertiary protein structures. The protein damages in muscle food might lead to changes in protein functionality (e.g., gel-forming ability, meat-binding ability, emulsifying capacity, viscosity, solubility, and water-holding capacity with a significant impact on food quality). However, irradiation of low and medium energy has no significant effect on protein (Kuan et al., 2013). The study of the irradiation application on protein nutritional changes is rather scarce. Jaczynski and Park (2004) investigated the effects of electron-beam irradiation on surimi seafood, showing that degradation of myosin heavy chain was dose-­ dependent (6–8 kGy), while the actin integrity was only slight affected. Alternatively, Xiao, Zhang, Lee, Ma, & Ahn, 2011 showed that irradiation at 3 KGy significantly increased protein oxidation in chicken thighs during refrigerated storage of 7 days. These authors suggested that irradiation could produce hydroxyls radicals by splitting water molecules that react with peptides or amino acids prone to irradiation (e.g., cysteine, methionine, tyrosine, phenylalanine, histidine, tryptophan, and lysine). In addition, they postulated that irradiation can break the protein structures by splitting hydrogen and sulfur bonds, because secondary and tertiary protein structures can be unfolded due to SS bond reduction or oxidation of SH group. Food processing with ionizing irradiation can modify food antigenicity, reducing allergens by two different ways: firstly, by interaction with target protein or secondly, by formation of major products from water radiolysis (Kempner, 2001). A study conducted by Seo et al. (2007) about the effect of gamma irradiation (0–10 kGy) on ovalbumin confirmed that irradiation could be useful to inhibit and reduce the food allergy produced by hen’s egg albumin. They demonstrated that a band of SDS-PAGE associated with egg albumin disappears after a treatment either gamma and electronbeam irradiation. This result agrees with the findings of other studies realized on lectins, generally recognized as an important food anti-nutrient, in which high doses of gamma radiation suppress allergic effect induced by them (Vaz et al., 2013). These authors confirmed the absence of structural integrity in irradiated antigens at high

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doses (25 kGy). At this level, lectins do not maintain tertiary or secondary structure, indicating the presences of precipitation and insoluble aggregates, without trace of native structure. On the contrary, irradiation at low dose (1 kGy) does not mitigate the allergenic response; it can even be increased and cause severe allergenic inflammatory response, as the same research group demonstrated (Vaz et al., 2013). Recently, Sujatha, Hymavathi, Uma-Devi, Roberts, and Kumar (2017) reported that irradiation significantly improved in vitro protein digestibility of selected millet grains and the effect was more pronounced in dehulled grain (2.58%) than in whole grain (2.13%). Regarding UV radiation, it has been reported that UV radiation induces cross-linking in food proteins under certain conditions. Indeed, Kato, Uchida, and Kawakishi (1992) reported collagen degradation and fragmentation caused by UV irradiation, using its model peptides. Authors explained these results due to proline oxidation for fragmentation, whereas degradation was caused because collagen is a proline-rich protein. Even UV radiation has been used to produce rheological changes in order to improve some of the quality features of fish and meat gelatin. Indeed, Ishizaki, Hamada, Iso, and Taguchi (1993) studied the effect of UV in pork and sardine pastes, improving gel strength. The modifications were attributed to actomyosin denaturation in the samples, because UV irradiation produced a significant increase of the surface hydrophobicity and decrease in the total SH in samples. Ishizaki, Hamada, Tanaka, & Taguchi, 1993, in a posterior study, indicated that the myosin solubility decreased when irradiation time and intensity increased (Ishizaki, Ogasawara, Tanaka, & Taguchi, 1994).

5.4 Conclusions and future remarks This chapter describes how proteins are affected by food processing. Food quality is a global challenge in order to achieve food safety and a healthy and balanced life for our society. Dietary protein, probably more than any other food component, is indispensable to human nutrition. Therefore it should be necessary to emphasize the need to control food processing techniques in order to maintain nutrition and improve the texture and sensory qualities of food through protein composition and its structural organization. Traditional industrial-thermal processes have negative impacts on food proteins, decreasing their nutritional value. Novel and emerging technologies (ultrasound, high-pressure processing, pulsed electric field, ohmic heating, and irradiation) could improve food safety and sensorial qualities as well as protein quality. So far, however, there has been little research about the effects of these emerging technologies on nutritional value of proteins. Therefore still more researches are need to completely understand all effects produced by emerging technologies. It is expected that in the future more studies on bioaccessibility and bioavailability of food proteins will be conducted to assess these processes.

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Further reading Canadian Food Inspection Agency. n.d. 6763218/1389206811747?chap=7#s10c7 (Accessed 20 December 2017). JSGHDI (FAO/IAEA/WHO Joint Study Group High-Dose Irradiation). (1999). Whole- someness of food irradiated with doses above 10kGy. Technical report series no. 890 Geneva, Switzerland: World Health Organization. Kanatt, S. R., Chander, R., & Sharma, A. (2006). Effect of radiation processing of lamb meat on its lipids. Food Chemistry, 97(1), 80–86. WHO. (1985). Energy and protein requirements: Report of a joint FAO/WHO/UNU expert consultation. WHO technical report series no. 724 Geneva: WHO.

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Sze Ying Leong⁎,†, Sheba Mae Duque⁎,†,‡, Setya Budi Muhammad Abduh⁎,†,§, Indrawati Oey⁎,† ⁎ Department of Food Science, University of Otago, Dunedin, New Zealand, †Riddet Institute, Palmerston North, New Zealand, ‡Institute of Food Science and Technology, College of Agriculture and Food Science, University of the Philippines Los Baños, College, Laguna, Philippines, §Department of Food Science, Diponegoro University, Semarang, Indonesia

6.1 Introduction Dietary carbohydrates, namely sugars, starch, and non-starch polysaccharides (NSPs), are major energy sources in the human diet that support body metabolism. Food of any plant origin, such as fruits, vegetables, edible seeds, grains, legumes, and wholegrains, are reliable sources of dietary carbohydrates for humans. However, the nutritional quality of carbohydrates strongly depends on the type, nature or source of the carbohydrate, interaction with other food constituents, their structure, processing methods that affect their physical and chemical characteristics, particle size of plant foods due to food oral processing (or mastication) prior to digestion at the small intestine, and their digestibility properties in the normal digestion system of humans (Englyst & Englyst, 2005). This chapter provides an overview of the different types of dietary carbohydrates based on their digestibility in the human intestine, which can vary from simple and easily digestible carbohydrates to non-digestible carbohydrates. It also discusses how differences in carbohydrate digestion behavior can confer specific physiological functions in the human body upon metabolism. The digestibility of some carbohydrates can be limited by the fact that the macromolecules are physically protected in specific compartments of the grains, seeds, or plant tubers that are inaccessible to the digestive enzymes, and/or because the carbohydrates naturally exist in a structural form that prevents the digestive enzymes from breaking them down or being easily digested (Lunn & Buttriss, 2007). The digestibility of these carbohydrates can be substantially improved when appropriate food-processing techniques, such as milling, are applied to the plant food. They ease the liberation of carbohydrates from the food matrix during ingestion. Moreover, the transformation of certain carbohydrates, such as starch, through conventional thermal or emerging nonthermal food-processing technologies into a structural form that affects their digestion rate has been evidenced (Singh, Dartois, & Kaur, 2010). The effect of a wide range of conventional thermal and emerging nonthermal technologies on the digestibility, bioaccessibility, and bioavailability of dietary carbohydrates from different plant sources is also discussed in detail in this chapter.

Innovative Thermal and Non-Thermal Processing, Bioaccessibility and Bioavailability of Nutrients and Bioactive Compounds © 2019 Elsevier Inc. All rights reserved.


Innovative Thermal and Non-Thermal Processing

6.2 Types of dietary carbohydrates in foods Carbohydrates in human diets range from simple sugars to starch and NSPs (Fig. 6.1). In the human body, the digestion of carbohydrates begins at the oral cavity (mouth) through the action of salivary α-amylase, while the main site for carbohydrate absorption is at the small intestine. All carbohydrates absorbed in the small intestine must be hydrolyzed to monosaccharides by human digestive enzymes prior to absorption. Then, the monosaccharides eventually enter the blood stream, providing energy for human metabolism. Simple sugars, such as glucose, are easily absorbed in the small intestine while starch has to be hydrolyzed by digestive enzymes into glucose molecules first. However, not all the dietary carbohydrates are easily digested and readily absorbed in the small intestine (Lovegrove et  al., 2017). Some starches, including NSPs, namely cellulose, hemicellulose, β-glucans, and pectin, are resistant to digestion (Knudsen, 2001). A significant portion of resistant starch and NSPs passes into the large intestine (colon), where they are fermented by the naturally present intestinal bacteria to short-chain fatty acids (SCFAs) that provide additional energy to the body; some specific SCFAs are known to be particularly beneficial to maintain colonic health. Therefore dietary carbohydrates are generally classified based on their digestibility by the small intestine: “easily digestible” or “non-digestible” carbohydrates. Both types of carbohydrates are present in common plant foods, and consumption of these foods, either containing higher or lower amounts of digestible and/or non-­ digestible carbohydrates, can confer a range of physiological and metabolic responses in human body.


Simple sugars Simplest form of carbohydrate

• • • •

Glucose Fructose Galactose Xylose

Complex Carbohydrates/Polysaccharides


> 10 monosaccharide units

Contain 3–9 monosaccharide units


• • • •

Raffinose Stachyose Verbascose Inulin


Starch Polymers of glucose Also classified as storage carbohydrate


• Amylose (straight chain) • Amylopectin (branched)

Disaccharides Contain 2 monosaccharide units

• • • •

Sucrose Lactose Maltose Trehalose


Carbohydrate digestion and absorption: 1. Simple and easily digestible in human small intestine 2. Non-digestible in small intestine, but are likely to be fermented in the large bowel 2a. (complete) fermentable 2b. partial or poorly fermentable



Non-starch polysaccharides Also classified as cell wall polysaccharides

Insoluble components • Cellulose


Resistant starch (RS) Non-digestible

• RS1 - physically protected • RS2 - ungelatinised resistant granules

• RS3 - retrograded starch • RS4 - chemically modified • RS5 - amylose-lipid complex

• • • • • •

Soluble components Pectin b-glucan 2a Arabinoxylans Xyloglucans Glucomannans Galactomannans

Fig. 6.1  Dietary carbohydrates and their potential for digestion and absorption in human body.


6.2.1 Simple and easily digestible carbohydrates Digestible carbohydrates are the most important biological fuel provider in human nutrition. Monosaccharides, α-linked disaccharides and oligosaccharides, and starch are classified as digestible carbohydrates. Monosaccharides, such as glucose, fructose, and galactose, occur naturally in food and are absorbed directly into the bloodstream. Disaccharides, such as sucrose (glucose and fructose) from sugar beet and sugar cane, maltose in malt and beer beverages, and lactose (glucose and galactose) in milk and dairy products, are readily broken down into their monosaccharide constituents by the naturally occurring disaccharidase digestive enzymes (sucrase and lactase) secreted in the small intestine. Therefore mono- and disaccharides are generally quick sources of easily digestible carbohydrates in the human diet. Starch is considered as the most important dietary carbohydrate that can provide an abundant amount of glucose upon human digestion because starch is a polysaccharide that is made up from polymers of glucose units bound together via glycosidic linkages. The human body secretes a range of digestive enzymes that can help to degrade starch polymers into glucose molecules, which are then absorbed into blood circulation (Lunn & Buttriss, 2007). Being an energy storage carbohydrate in plants, starch exists in various botanical origins such as tubers, grains, and pulses. Hence they are always consumed as staple foods (potatoes, wheat, maize, rice, cassava) by many populations around the world (Singh et al., 2010). On average, starchy foods account for up to 60% of a human diet on a daily basis (Subar, Krebs-Smith, Cook, & Kahle, 1999). The rapid digestion of digestible carbohydrates and absorption of glucose in a human body, however, will affect the blood glucose response and insulin demand, causing metabolic disorders particularly for diabetic patients or even healthy individuals. Digestible starches can be classified by the rate and extent of their digestibility: rapidly digestible starch (RDS) and slowly digestible starch (SDS). Food containing RDS breaks down quickly during digestion and is then rapidly absorbed in the small intestine, which tends to cause elevated blood glucose responses. In comparison, foods with a high proportion of SDS require a longer time for digestion and absorption in the small intestine and thus evoke a lower response in blood glucose (Jenkins et al., 1981). Starch has two constituents: the linear chain amylose (20–30%) and the branched amylopectin (70%–80% and contributing to starch crystallinity); starch also occurs naturally in the form of semi-crystalline and water-insoluble granules, which varies depending on their polymorphic types and degrees of crystallinity. In general, SDS exists mostly in semi-crystalline structures and hence is less accessible to the action of digestive enzymes compared to RDS, which is mainly amorphous in structure (Lehmann & Robin, 2007). Factors such as the physiological status of plant foods, the chemical composition of foods and the interaction with other food components, cooking and food-processing methods, the structural feature of the carbohydrate (e.g., ­amylose-to-amylopectin ratio in starch, the nature of crystallinity of the starch granule), the extent of food disintegration, and the presence of added food components have been identified to affect the digestion properties of dietary carbohydrates, as well as conferring a physiological impact on the blood glucose and insulin responses after consumption (Björck, Granfeldt, Liljeberg, Tovar, & Asp, 1994).


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6.2.2 Non-digestible carbohydrates Compared to simple and easily digestible carbohydrates, non-digestible carbohydrates are those that cannot be hydrolyzed by human digestive enzymes and absorbed in the upper gastrointestinal (GI) tract. Non-digestible carbohydrates in the human diet include NSPs, β-linked disaccharides and oligosaccharides, and resistant starch. NSPs are plant components that make up the cell wall and all NSPs could be categorized as dietary fiber (Lovegrove et al., 2017). Most non-digestible carbohydrates are polymers of different monosaccharide units joined by β-glycosidic bonds. Enzymes in the human digestive system are not able to cleave this kind of linkage and hence indigestible carbohydrates pass into the large intestine where they are fermented by the colonic bacteria to provide numerous health benefits. Common non-digestible carbohydrates in the human diet are: ●

β-linked oligosaccharides such as galacto- and fructo-oligosaccharides are described as having prebiotic properties that have the ability to promote the growth of beneficial lactic acid bacteria (probiotics) in the gut; other oligosaccharides, such as raffinose and stachyose, that are present in many cereals, vegetables, and legumes are indigestible because the enzymes responsible for hydrolyzing them are not found in the human digestive system; cellulose is a linear (unbranched) polymer consisting of up to 15,000 of β-(1–4) d-glucose units that make up the structural component of the primary cell wall of all plants; hemicellulose is a non-cellulosic polysaccharide with shorter chains of sugar units compared to cellulose; it is present in conjunction with cellulose in almost all plant cell walls. Hemicelluloses include arabinoxylan, xyloglucan, glucomannan, and galactomannans; o arabinoxylan is a polymer of β-(1–4) d-xylose units with l-arabinose substitution at three or two and three positions. Arabinoxylan is present in wheat grain and its water-soluble properties are important for bread making (Courtin & Delcour, 2002) o xyloglucan is a polymer of d-glucose units linked at β-(1–4) that may be substituted with d-xylose with α-(1–6) linkages. Xyloglucan is present in tamarind seed and most other land plants that can be utilized as a natural food ingredient, acting as thickening agents, gelling agents, emulsifying agents, and stabilizers (Popper et al., 2011) o glucomannan is a polymer of glucose and mannose units linked at β-(1–4) but may also have α-(1–6) linkages of glucose substitutions, and are also valued as a natural food ingredient to be used as emulsifier and thickener o galactomannan is a polymer of mannose linked at β-(1–4) but may have substitution of galactose at α-(1–6). Galactomannans are widely used in the food industry as thickeners and stabilizers and they are derived mainly from guar, locust, and carob beans (seeds) galactan is a polymer of d-galactose and 6-anhydro-α-d-galactose units linked at β-(1–3) and (1–4) found in numerous red seaweeds, and is used widely in the food industry as gels and thickeners; β-glucans consist of glucose units linked at β-(1–4) but interspersed with β-(1–3) linkages (Li, Cui, & Kakuda, 2006), and are present in many cereals such as oat and barley; and pectin is an important cell wall polysaccharide that contains galacturonic acids linked at α-(1–4); it can be differentiated into homogalacturonan (HG), rhamnogalacturonan-I (RG I), and rhamnogalacturonan-II (RG II) (Willats, Knox, & Mikkelsen, 2006). Pectin is the major component of the middle lamella at the primary cell walls of plants and found abundantly in citrus fruits.


Some starches in the diet are also found to be resistant towards digestion. They are known as resistant starches as they escape intestinal digestion and pass into the colon, then undergo digestion by colonic microbiota (Lunn & Buttriss, 2007). Therefore resistant starch is considered to confer numerous health benefits and behaves like dietary fiber. There are generally five subtypes of resistant starch; they are classified based on the location of starch in the biological compartment of origin (RS1), native non-­ gelatinized starch (RS2), retrograded starch (RS3), chemically modified starch (RS4), and lipid-complex starch (RS5) (Svihus & Hervik, 2016). In summary, ●

Class 1 resistant starch refers to those starches present in starchy plant foods without any processing and physically protected within the plant structure; they are inaccessible to the digestive enzymes such as in whole- or partly milled grains and seeds, and legumes. The digestibility of RS1 in the colon can be reduced if these starchy plant foods are pre-milled; Class 2 resistant starch refers to starches freed from the food matrix but still existing in their intact native (ungelatinized) form that prevents the digestive enzymes from breaking them down in the small intestine, as exhibited by uncooked wheat particles and uncooked rice and potatoes. Food processing and cooking can help to improve digestibility of RS2; Class 3 resistant starch is gelatinized starch that has been recrystallized (or retrograded) due to a prolonged cold storage period, which makes it less easily digested. RS3 is found in cooked and cooled potatoes, rice, and stale bread; Class 4 resistant starch is chemically modified starch such as acetylated, hydroxypropylated, octenyl-succinylated, and cross-linked starch (Juansang, Puttanlek, Rungsardthong, Punchaarnon, & Uttapap, 2012); and Class 5 resistant starch is amylose-lipid complexed starch (Svihus & Hervik, 2016).

Overall, resistant starch is found in a wide range of foods, including intact wholegrains, legumes, pasta, unripe bananas, raw potatoes, cooked and cooled potatoes, and foods containing commercial sources of modified starches (e.g., bread, breakfast cereals, and nutrition bars). The determination of resistant starch in food can be performed via an in vitro digestibility test that employs three different digestive enzymes, that is, α-amylase, amyloglucosidase, and invertase, to convert starch into glucose as the final product of digestion, followed by assaying with a reducing sugar test. Technically, resistant starch is defined as starch that is not digestible after 120 min of in vitro digestibility test (Englyst, Kingman, & Cummings, 1992). The health benefits of consuming sufficient amounts of non-digestible carbohydrates (including dietary fibers) in the diet will be critically discussed in the next section.

6.3 Beneficial physiological functions of dietary carbohydrates 6.3.1 Utilization of dietary carbohydrates as main energy provider Dietary carbohydrates provide about 50% of the energy from the foods consumed (Blanco & Blanco, 2017). However, before they can supply the energy that the body requires, carbohydrates need to undergo digestion, absorption, and metabolism. Carbohydrate digestion by human digestive enzymes is important since


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p­ olysaccharides cannot pass through cell membranes; only monomeric sugars (e.g., glucose) can be transported for uptake in the blood stream. Carbohydrate digestion, particularly for polysaccharides, starts in the oral cavity (mouth) upon the action of salivary α-amylase or ptyalin that hydrolyses inner α1, 4-glycosidic bonds in linear glucose polymers, except for those located near the branching points, into disaccharides. Upon reaching the stomach, digestion temporarily stops due to the inactivation of salivary amylase by acidic gastric juices and resumes upon the action of pancreatic α-amylase in the small intestine, where the digestion of smaller carbohydrate fragments also begins. Brush border enzymes at the microvilli of the small intestine further hydrolyze those smaller carbohydrate fragments previously produced, including maltose, maltotriose, sucrose, lactose, and α-dextrin. Brush border enzyme complexes are composed of glucoamylase, sucrose-­ isomaltase, and β-glycosidase or lactase. Glucoamylase, also known as α-glucosidase, is an exoglucosidase that can hydrolyze α-1, 4-glycosidic bonds starting from the non-reducing end of glucose polymers leading to the production of glucose and/or isomaltose. Sucrose-isomaltase has two catalytic subunits with different substrate specificities. In brief, the sucrase-maltase subunit is responsible for hydrolyzing sucrose and maltose to their monosaccharide components (glucose, fructose) and the isomaltase-maltase subunit hydrolyzes the isomaltose and maltose into free glucose units. Lastly, the β-glycosidase enzyme complex (or lactase-phlorizin hydrolase) also has two subunits, in which the lactase subunit hydrolyses lactose, whereas the phlorizin subunit hydrolyzes glycolipids to its monosaccharide components and ceramide (Blanco & Blanco, 2017). After normal intestinal digestion processes, complex digestible carbohydrates are finally broken down into their simplest form, monosaccharides (glucose, fructose, galactose), which are rapidly absorbed by enterocytes or the small intestine cells. Monosaccharides, being highly hydrophilic, need to be the transported across cell membranes via various glucose transporters. Glucose and galactose are absorbed via a sodium- and energy-dependent active transport mechanism (SGLT1), while passive diffusion of fructose is facilitated by a glucose transporter 5 (GLUT5) (Adair, 2007). Once inside the cells, glucose transporter 2 (GLUT2) transports monosaccharides to the serosal side of the cell, which are then carried to the various tissues of the body via the blood stream. Glucose transporters (GLUT) are a wide group of membrane proteins that facilitate the transport of glucose over a plasma membrane. The type of GLUT transporter found in tissue cells determines its role in the metabolism of glucose (Sanders, 2016). GLUT1 transporter is expressed at highest levels in red blood cells and brain endothelial cells and due to its high affinity for glucose, this transporter ensures that cells are always supplied with glucose, even at low blood glucose concentration, to sustain respiration of all cells. GLUT2 transporter has a lower affinity for glucose, but its amount in cell membranes is the highest at tissues involved in the absorption and regulation of blood glucose, such as liver, kidney, pancreas, and intestines. GLUT3 transporter is expressed mostly in neurons. GLUT4 transporter is the insulin-regulated glucose transporter, which is important in regulating blood glucose levels and is predominantly present in skeletal muscle, cardiac muscle, and adipose tissues.


Carbohydrates are metabolized in the human body to yield cellular energy, and the released energy is stored temporarily in the cell in the form of high-energy molecule adenosine triphosphate (ATP) for use in various cellular processes. Once the glucose molecules are transported into the cells, they are phosphorylated to glucose-6-­ phosphate, which is the precursor to several metabolic pathways including glycolysis, pentose phosphate pathway, and conversion of glucose to glycogen (glycogenesis) (Bender, 2013; Landau, 2013; Zoidis & Papamikos, 2016). Glycolysis is the major cellular process that produces ATP, NADH (reduced nicotinamide adenine dinucleotide), and pyruvate, wherein the last two molecules can be further oxidized, in the presence of oxygen, to produce more ATP. Generally, the energy value (or metabolic yield) of carbohydrates is estimated to be 4 kcal/g (or 17 kJ/g). However, the presence of polyols, highly digestible starches, and soluble and insoluble fibers in the ingested foods can affect its caloric value. Different forms of dietary carbohydrates can provide a range of energy values: common monosaccharides yield 3.74 kcal/g or 15.7 kJ/g, disaccharides yield 3.95 kcal/g or 16.6 kJ/g, and starch yields 4.18 kcal/g or 17.6 kJ/g. Apart from energy production, glucose metabolism also generates precursors for other biosynthetic pathways such as amino acids and lipids formation (de Mendoza & Schujman, 2014; Reitzer, 2014). In the absence of oxygen, glucose oxidation can produce NADPH (nicotinamide adenine dinucleotide phosphate) for use in other biosynthetic pathways (e.g., lipid synthesis) and defense against cellular oxidative damage. Moreover, ribose-5-phosphate produced in the pentose phosphate pathway is a precursor for nucleic acid synthesis process. As a result of glycogenesis, uridine diphosphate glucose (UDP-glucose) is produced and becomes the precursor for the formation of glycoproteins and glycolipids (Sanders, 2016).

6.3.2 Non-digestible carbohydrates can protect and maintain gastrointestinal health The large intestine (colon) plays a major role in the lower part of the human GI tract as this is the last part of human digestive system that allows the interaction between microbial activity and human physiology to take place. Indigestible carbohydrates, such as NSPs and resistant starch, that escape digestion at the upper part of the GI tract and pass into the large intestine are virtually still maintained in their inherent polymer form, where they are eventually fermented by microbial flora naturally present in the colon. Since the movement of intestinal contents in the large intestine is rather slow (48–70 h) compared to the transit time in stomach and small intestine (4–6 h), the intestinal contents act as ideal substrate that allows the proliferation of large colonies of human gut microbiota here (Panesar & Bali, 2016). The composition of gut microbiota housed in the colon can change over the life span of a human, especially when the human ages and there is a vast change in diet, overall health condition, and geographical location. The impact of gut microbiota on human health has been widely reported. In fact, it is the carbohydrate fermentation that serves as the important driving force in maintaining the large intestinal microbial activities (Macfarlane & Macfarlane, 2012). Carbohydrate fermentation generally leads to the production of low levels of carbon


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dioxide, methane and hydrogen gases, as well as organic acids and SCFAs. Examples of SCFAs being produced in the human gut are butyrate, propionate, and acetate, and they are found to be beneficial to human health because they lower the luminal pH to restrict the growth of pathogenic and putrefactive microorganisms, regulate sodium and water absorption, reduce blood cholesterol, and increase the absorption of calcium and other minerals (Panesar & Bali, 2016). Among the SCFAs, special attention is given to butyrate, which has been reported to have multiplex physiological effects, especially on colonic epithelial cells and maintaining of the integrity of the gut wall. Macfarlane and Macfarlane (2012) have presented strong evidence that butyrate formed from dietary carbohydrates can protect against colorectal cancer by preventing the uncontrolled proliferation of abnormal cells, and has the capability to induce programmed cell death. The concentrations of SCFAs produced in the large intestine vary depending on the types of non-digestible carbohydrates fermented in the colon as well as the different regions of the colon where carbohydrate fermentation has taken place (Lunn & Buttriss, 2007). Non-digestible carbohydrates, such as resistant starch, non-digestible oligosaccharides, and NSPs, can also act as prebiotics to help stimulate the growth and activity of targeted bacteria, such as bifidobacteria and lactic acid bacteria, in the colon. The term prebiotic is defined by Valcheva and Dieleman (2016) as “a non-digestible compound that, through its metabolization by microorganisms in the gut, modulates composition and/or activity of the gut microbiota, thus conferring a beneficial physiological effect on the host.” Fructo-oligosaccharides, galacto-oligosaccharides, and inulin that occur naturally in plants, such as onion, chicory, garlic, asparagus, banana, legumes, and artichoke, have been widely studied as beneficial prebiotics for maintaining a healthy gut (Roberfroid et al., 2010). Plant foods containing these prebiotics, if consumed by humans at higher amounts, are major substrate sources for microbial fermentation and hence are able to influence the composition of intestinal microbiota, causing selective growth and proliferation of microorganisms (Valcheva & Dieleman, 2016). This can ultimately affect gastrointestinal health. Irritable bowel syndrome, constipation, and diarrhea are commonly reported GI tract-related problems (Hansen, 2017). Moreover, gastric ulceration, inflammatory bowel diseases, and colorectal cancer have become more prevalent across age groups, with the latter having an increased incidence rate of 22% from 2000 to 2013 among adults aged 30 kg/m2), with worldwide obesity prevalence doubling since the 1980s (World Health Organization, 2016). This is due to the amplified imbalance between energy intake and expenditure, mainly due to the increased consumption of energy-dense food and the sedentary nature of most activities. To address this, there is a growing interest in developing various strategies to prevent and address obesity by promoting fat mobilization and increasing energy use. One of the strategies involves dietary intervention, which seems possible based on promising results of animal experiments and human metabolic rate studies (van Dam & Seidell, 2007). Epidemiological and clinical studies have shown an inverse relationship between the consumption of dietary fiber-rich and wholegrain foods with obesity (Brownlee, Chater, Pearson, & Wilcox, 2017). The consumption of dietary fiber-rich foods was reported to benefit weight management due to lower energy intake and absorption, an increase in postprandial energy expenditure due to improved gastrointestinal motility, and efficient elimination of bile acid that drives body fat mobilization. This is because diets that are high in fibers often tend to have lower fat content and generate a strong satiety sensation, which leads to reduced total food (or energy) intake. Since dietary fibers are generally indigestible, they provide a lower energy yield to that of digestible carbohydrates, too. A recent study by Han et al. (2017) showed that dietary fiber originated from cereals could reduce obesity induced by high-fat diet through activation of a signaling pathway that increases browning of white adipose tissues. On the other hand, consumption of soluble dietary fibers (e.g., pectin, β-glucan and some hemicellulose) can reduce serum cholesterol levels because of their ability in increasing the luminal viscosity (Kim & White, 2013; Liu, Bailey, & White, 2010).

6.4 The important role of food processing in influencing the bioavailability and bioaccessibility of dietary carbohydrates In order to facilitate efficient food digestion, absorption, and metabolism of dietary carbohydrates, foods originating from plant sources are usually processed adequately in order to transform the plant tissue/organ into edible foods (e.g., in the form of puree, juice, beverage, concentrate, fresh-cut, canned, fermented, or frozen products) with palatable textures and pleasant sensory properties, which are safe to consume and nutritious. This is because the natural complex and heterogeneous structure of plant tissue can influence the accessibility of important dietary carbohydrates (as well as are other nutrients) that are only available for utilization upon plant tissue damage or plant structure manipulation through various food-processing strategies. The food-processing procedures that can be applied to plant tissues include postharvest management techniques (washing, sanitizing, storage), mechanical techniques (cutting, milling), chemical techniques (brining, fermentation), and cooking (baking, steaming, boiling, frying, roasting).


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Bioaccessibility refers to the fraction of the total amount of endogenous compounds liberated from the complex food matrix into the GI tract, modified during digestion, followed by binding and passage across the intestinal walls and therefore potentially becoming available for absorption (Gerschenson, Rojas, & Fissore, 2017). Bioavailability, on the other hand, expresses the fraction of the ingested nutrient that is released from the matrix, reaches systemic circulation, and is utilized effectively for physiological functions (Galanakis, 2017). Bioavailability includes the term bioaccessibility and both terms require digestive transformations of foods to take place first. In other words, carbohydrates must be released from the food matrix and broken down in the GI tract before becoming bioavailable. This is also when food processing becomes important in facilitating the liberation of carbohydrates, making them more bioavailable. Due to the different ways that dietary carbohydrates can undergo digestion in the human GI tract, resulting in a small intestinal digestible fraction that leads to absorption of monosaccharides and a fermentable fraction that leads to absorption of SCFAs in the large intestine, the bioavailability of all types of carbohydrates is not often measured in the current literature. Instead, the digestibility of different fractions of carbohydrates is measured and used as an indicator of bioavailability. For this reason, the digestibility of starch is the most common aspect discussed in the literature to assess the overall bioavailability of carbohydrates. It is important to note that any food-processing technique applied to the plant tissue during food preparation might induce in vitro changes to the composition, content, native structure, interaction with other constituents, and functional properties of carbohydrates. These changes then, indirectly, affect subsequent in  vivo factors upon their ingestion, such as how the plant tissues are physically transformed into bolus and then disintegrated into smaller particle sizes before being swallowed down the esophagus to the stomach for digestion, and consequently how the dietary carbohydrates are digested, absorbed, and metabolized through the normal digestion process in the human body. Overall, food processing is considered to play an important role leading to changes in the bioavailability of dietary carbohydrates, which may make significant contributions to the energy status of the human and the associated physiological functions. In the following sections, we will discuss the bioavailability of carbohydrates, predominantly starch, as affected by different processing techniques, either through conventional (also refer to Table 6.1) or emerging nonthermal processing (also refer to Table 6.2) technologies, respectively.

6.4.1 Effect of conventional and thermal food-processing technologies Conventional methods of food processing include (1) physical processing such as soaking, dehulling, or decortication, (2) heat processing such as cooking, pressure cooking, frying, baking, parboiling, and puffing, (3) extrusion, (4) fermentation, (5) chemical modification like acetylation, cross-linking, hydroxypropylation, and ozonation, and (6) enzyme modification. The impermeable outer protective coverings (husks, bark, seed coat, skin) for legumes and cereal grains are known to be very difficult for digestive enzymes to penetrate and access the storage carbohydrates, as well as to hinder water uptake that is necessary for starch gelatinization to occur during cooking. Milling and dehulling are

Table 6.1  Effect of conventional food processing technologies on carbohydrate bioavailability. Conventional processing techniques Physical

Carbohydrate of interest

Processing conditions

Plant matrix

Soaking in tap water or NaHCO3 solution (30°C or 100°C, 1 or 2 h)

Red and white kidney beans

Starch and sugars

Dehulling using “Grain Testing Mill”


Starch Dietary fibre Oligosaccharides

Dehulling using tangential abrasive dehulling device Dehulling (or decortication)


Total carbohydrate

Finger millet

Total carbohydrate Starch Dietary fibre Total free sugars NSPs

Changes observed

Overall effect on carbohydrate bioavailability

↓ Total soluble sugars ↓ Reducing sugars ↓ Non-reducing sugars ↓ Starch contents ↑ Starch ↑ RS ↓ Total, soluble, insoluble dietary fibre ↓ Raffinose ↑ Stachyose ↑ Verbascose ↓ Total carbohydrate

No significant change in % starch digestibility

↑ Total carbohydrate ↑ Total amylose ↓ Soluble amylose ↑ Soluble fibre ↓ Insoluble fibre ↓ Total free sugars ↑ Hot water soluble fractions ↑ (slight) Pectic polysaccharide ↑ Hemicellulose A ↑ Cellulose ↓ Hemicellulose B

↑ Carbohydrate digestibility

Reference Rehman, Salariya, and Zafar (2001)

Wang, Hatcher, Toews, and Gawalko (2009)

Oomah and Mazza (1997) Dharmaraj and Malleshi (2011)


Table 6.1  Continued Conventional processing techniques Heat processing

Carbohydrate of interest

Processing conditions

Plant matrix

Boiling (using tap water or NaHCO3 solution until soft) Autoclave (15 psi, 15 min)

Pre-soaked red and white kidney beans

Starch and sugars

Boiling in soaking water (30 min)

Pre-soaked, aged common bean seeds

Dietary fibre

Autoclave (15 psi, 10 min) Pressure cooking (10 min) Roasting (160°C, 5 min) Baking (180°C, 20 min)

Ragi (finger millet) flour


Neutral sugars (NS)/uronic acid (UA) ratio Arabinose/UA ratio Starch

Dietary fibre Shallow frying (150°C, 7 min) Deep fat frying (180°C, 4 min) Roasting (210°C, 25 min)

Sliced potato

Reducing sugars as a whole, glucose, fructose, sucrose

Changes observed ↓ Total soluble sugars ↓ Reducing sugars ↓ Non-reducing sugars ↓ Starch contents ↑ Soluble fibre ↓ Insoluble fibre ↑ Alkali-soluble polysaccharides ↓ NS/UA ratio in water soluble and insoluble polysaccharides ↑ Arabinose/UA ratio in water soluble polysaccharide ↑ RDS (all except baking) ↓ RDS (only baking) ↓ SDS ↓ RS (autoclave, baking) ↑ RS (pressure cooking, roasting) ↓ Total and insoluble fibre ↑ Soluble fibre ↑ Reducing sugars ↑ Glucose ↑ Fructose ↑ Sucrose

Overall effect on carbohydrate bioavailability


↑ % Starch digestibility

Rehman et al. (2001)

↓ Degradation and solubilisation of NSP ↓ Degradation of acidic polymers ↓ Degradation of galacturonans ↑ Starch digestibility

Shiga, Cordenunsi, and Lajolo (2009)

Roopa and Premavalli (2008)

Murniece et al. (2011)

Puffing (1.3–1.5 MPa, 75–85 s)

Heat processing

Puffing using hot sand at about 230°C

Heat moisture treatment, 25% moisture starch heated in electric blast oven at 120°C for 3 or 9 h

Annealing (50°C, 24 h)

Common wheat, emmer wheat, rye, barley, rice, buckwheat Finger millet (ragi) flour


Waxy maize and normal maize starch

Starch and its properties

Black bean starch Pinto bean starch



↑ Grain size ↑ Damaged starch ↑ Water absorption index ↑ Water solubility index ↓ Bulk density ↑ RDS ↓ SDS ↓ RS ↑ Water absorption capacity Agglomeration of starch granules, weaker maltose cross, more susceptible (1,6)-glycosidic bonds ↑ RDS ↑ SDS ↓ RS ↓ Double helix content ↓ Starch crystallinity ↑ RDS for black bean ↓ RDS for pinto bean ↓ SDS ↑ RS ↓ Hydrolysis index

Significant changes in structure

Mariotti, Alamprese, Pagani, and Lucisano (2006)

↑ Starch digestibility

Roopa and Premavalli (2008)

↑ Starch digestibility

Chen et al. (2017)

↓ Estimated glycaemic index

Simsek, OvandoMartinez, Whitney, and Bello-Perez (2012)


Table 6.1  Continued Conventional processing techniques

Processing conditions

Plant matrix

Parboiling (steeped for 10 h, steamed in autoclave at 98°C, 30 min)

Finger millet

Carbohydrate of interest Total carbohydrate Starch Dietary fibre Total free sugars NSPs


Co-rotating twin-screw extruder, varying temperature and screw speed Twin-screw extruder

Barley flour


Oat flour

Starch β-glucan

Co-rotating, intermeshing, selfcleaning twin-screw extruder

Oat bran

Dietary fibre

Changes observed = Total carbohydrate ↑ Total amylose ↑ Soluble amylose ↓ Soluble fibre = Insoluble fibre ↓ Total free sugars ↓ Cold and hot water soluble fractions ↓ Hemicellulose B ↑ Pectic polysaccharides ↑ Hemicellulose A ↑ Cellulose

↑ Total starch ↑ SDS ↓ RDS = Total β-glucan content and molecular weight ↑ Total carbohydrate ↑ Soluble fibre ↓ Insoluble fibre

Overall effect on carbohydrate bioavailability


↑ Carbohydrate digestibility

Dharmaraj and Malleshi (2011)

↑ Starch digestibility

Altan, McCarthy, and Maskan (2009) Brahma, Weier, and Rose (2016)

Gualberto, Bergman, Kazemzadeh, and Weber (1997)

Twin-screw extruder


Natural fermentation 30°C, 450 rpm, 4 days

Wheat extrudates enriched with wet okara Lentil seeds

Dietary fibre

↓ Insoluble fibre ↑ Soluble fibre

Rinaldi, Ng, and Bennink (2000)

Starch and sugars

↑ Fructose No sucrose, raffinose, stachyose ↓ Total starch ↓ In vitro available starch ↑ Available starch/total starch ↓ Neutral detergent fibre ↓ Cellulose ↓ Hemicellulose ↓ RS ↓ Total starch

Vidal-Valverde et al. (1993)

Dietary fibre

Chemical Modification

Natural fermentation 37°C, 36 h

Sorghum flour


Traditional method



Cross-linking using 0.05% and 0.10% phosphoryl chloride (POCl3)

Taro starch


Hydroxypropylation (etherification) using 5% and 10% propylene oxide (CH₃CHCH₂O) Ozonation by exposure to ozone gas

Taro starch


Black bean starch


Significant starch granule damage ↓ Solubility ↓ Swelling = Amylose content and granule morphology ↑ Solubility ↑ Swelling = Amylose content and granule morphology ↑ RDS ↓ SDS ↑ RS = Hydrolysis index

↑ % In vitro starch digestibility

Elkhalifa, Schiffler, and Bernhard (2004) Marcon et al. (2006) Hazarika and Sit (2016)

No significant change in estimated glycaemic index

Simsek et al. (2012)


Table 6.1  Continued Conventional processing techniques

Enzyme Modification

Carbohydrate of interest

Processing conditions

Plant matrix

Ozonation by exposure to ozone gas

Pinto bean starch


Acetylation using acetic anhydride (CH3CO)2O

Cassava starch Potato starch


Acetylation using acetic anhydride (CH3CO)2O

Black bean starch Pinto bean starch Plantain starch Mango starch



Plantain starch



Mango starch


Amylo-(1,4→1,6)transglycosylase, (1000 U, 120 h)

Waxy maize


β-amylase and transglucosidase


Changes observed = RDS ↓ SDS ↑ RS ↓ Hydrolysis index ↑ Solubility until 80°C (for cassava) ↑ Swelling power ↓ RDS ↓ SDS ↑ RS ↓ Hydrolysis index ↓ RDS ↑ SDS ↑ RS ↓ RDS ↓ SDS ↑ RS ↓ RDS ↑ SDS = RS ↑ α-1,6 branching ↓ α-1,4 branching ↓ RDS = SDS ↑ RS ↓ Total starch

Overall effect on carbohydrate bioavailability


↓ Estimated glycaemic index

↓ Estimated glycaemic index

Mbougueng, Tenin, Scher, and Tchiégang (2012) Simsek et al. (2012)

CasarrubiasCastillo, Hamaker, Rodriguez-Ambriz, and Bello-Pérez (2012)

Significant correlation was found between ↓ RDS, ↑ SDS, and ↑ α-1,6 linkages

Kasprzak et al. (2012)

Amylo-(1,4→1,6)transglycosylase (1000 U, 120 h) and β-amylase

Waxy maize


Amylo-(1,4→1,6)transglycosylase (1000 U, 120 h) and α-glucosidase

Waxy maize


Amylo-(1,4→1,6)transglycosylase (1000 U, 120 h) and amyloglycosidase

Waxy maize


Note: ↑ increase, ↓ decrease, = no significant changes.

↑ α-1,6 branching ↓ α-1,4 branching ↓ RDS ↑ SDS ↓ Total starch ↑ α-1,6 branching ↓ α-1,4 branching ↓ Total starch ↓ RDS = SDS ↑ α-1,6 branching ↓ α-1,4 branching ↓ Total starch ↓ RDS ↑ SDS


Table 6.2  Effect of emerging nonthermal food processing technologies on carbohydrate bioavailability. Emerging processing techniques High pressure processing (HPP)

Plant matrix

Carbohydrate of interest

Various treatment pressures (200– 600 MPa), 30 min, room temperature



Potato, Yam


Various treatment pressures (300– 600 MPa), 15 min, 25°C



Various treatment pressures (100–600 MPa) at varying temperatures (25°C, 60–80°C)

Sweet potato

Starch and reducing sugar

Changes observed ↓ Crystallinity Deformations and coalescence were notable on granular morphology Complete disruption of granule at 600 MPa = Granule morphology = Crystallinity

Granular structure was preserved = Pasting properties up to 400 MPa Loss of birefringence (>400 MPa) = Granular morphology ↑ Reducing sugar with ↑ P and T

Overall effect on carbohydrate bioavailability ↑ In vitro enzymatic hydrolysis at high treatment pressure (600 MPa) No significant change in the in vitro enzymatic hydrolysis Promotes “cold” gelatinization

↓ T requirement for thermal treatment at atmospheric pressure

Reference Wang et al. (2017)

Leite, de Jesus, Schmiele, Tribst, and Cristianini (2017) Shigematsu et al. (2017)

Innovative Thermal and Non-Thermal Processing

Processing conditions

Tartary buckwheat

600 MPa, 20°C and 45°C, 15 min

Buckwheat flour


Bench-scale, continuous PEF system (60 mL/ min flow rate)

Waxy rice starch


Varying electric field strengths (30, 40, 50 kV/cm) Exponential decay PEF generator Varying electric field strengths (1, 2, 3 kV/cm)

Sorghum flour



= Granule morphology at low and medium pressures Granules collapsed at ↑P ↑ Amylose content ↓ RDS ↑ SDS ↑ RS ↑ Thermostability Granule retained its morphology at 20°C but completely disrupted at 45°C ↑ Starch granule damage ↓ Relative crystallinity ↓ Gelatinization temp ↓ Gelatinization enthalpy ↑ RDS ↓ SDS ↓ RS ↑ Porosity of cellular membrane ↑ Disrupted granule ↑ Granule surface area Granules appeared in loosen structure

↓ In vitro hydrolysis

Liu et al. (2016)

↑ Starch digestibility

Zhou et al. (2015)

Changes in starch in vitro digestibility

Zeng, Gao, Han, Zeng, and Yu (2016)

Lohani and Muthu­ kumarappan (2016)



Pulsed electric fields (PEF)

Various treatment pressures (120– 600 MPa), 20 min, room temperature


Table 6.2  Continued Emerging processing techniques

Changes observed

Overall effect on carbohydrate bioavailability


↓ Molecular weight ↓ Gelatinization enthalpy = NMR and TGA analyses

PEF did not affect the chemical structure of maize starch

Native corn starch


↑ Granule damage ↑ Granule diameter ↓ Relative crystallinity ↓ Gelatinization enthalpy ↓ Peak, breakdown, and setback viscosity

Han, Zeng, Zhang, and Yu (2009)

Potato starch


↑ Granule damage ↑ Granule size Aggregated granules Gel like granules ↓ Relative crystallinity, ↓ Gelatinization enthalpy ↓ Peak and breakdown viscosity

Han, Zeng, Yu, Zhang, and Chen (2009)

Processing conditions

Plant matrix

Carbohydrate of interest

Bench-scale, continuous PEF system

Maize starch

Han et al. (2012)

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Varying electric field strengths (30, 40, 50 kV/cm) Bench-scale, continuous PEF system (60 mL/ min flow rate) Varying electric field strengths (30, 40, 50 kV/cm) Bench-scale, continuous PEF system (60 mL/ min flow rate) Varying electric field strengths (30, 40, 50 kV/cm), 1008 Hz, 40 μs pulse width, 20 unipolar pulses



24 kHz, various times (1, 2, 4, 8, and 16 min), 20°C

Corn starch


Granules with fissures and cracks on the surface ↑ Crystallinity ↓ Intensity of FTIR spectra band ↑ Gelatinization enthalpy

Ultrasound bath (42 kHz, 132 W, 60 min)

Agave durangensis leaves


Varying frequencies (25, 80, or 25 and 80 kHz), 10, 20, 30, 45, and 60 min Gamma (20 kGy)

Sweet potato starch


Cereals porridge (wheat, rice, waxy rice, maize) Sago starch

Solid content

= RDS, SDS ↑ RS ↑ Hollocellulose (Cellulose and Hemicellulose) ↓ Water soluble carbohydrates (fructan, fructooligosaccharides) Larger visible pore size Dents were observed on the granule ↓ Crystalline index ↑ Starch solubility ↑ Total solid content




↓ Intrinsic viscosity ↓ Molecular weight ↓ Degree of polymerization ↓ Swelling ↑ Solubility

Electron beam (10, 15, 20, 25, 30 kGy)

Flores-Silva et al. (2017)

ContrerasHernandez et al. (2017)

Zheng et al. (2013)

No significant change in the starch digestibility index

Lee et al. (2008)

Pimpa et al. (2007)





Table 6.2  Continued Emerging processing techniques


Plant matrix

Carbohydrate of interest

X-ray and electron beam (10, 50, 100 kGy)

Corn, potato and drum dried corn starch


↓ Molecular weight distribution ↓ Polydispersity ↑ Solubility

2450 MHz and 750 W, varying duration (0, 5, 10, 15, 20 s)



240 W for 5 min

Pound cake


2450 MHz

Barley (normal, highamylose, and waxy) Moth bean


Flaws/fractures on the granule was observed Loss of birefringence ↓ Molecular weight values ↑ RS = Retrograded starch ↓ Degree of hydrolysis (compared to white bread reference) ↓ Hydrolysis index ↑ RDS ↓ SDS ↓ RS

2450 MHz


Changes observed

↑ Maltose

Overall effect on carbohydrate bioavailability

Reference Kerf, Mondelaers, Lahorte, Vervaet, and Remon (2001) Xie, Yan, Yuan, Sun, and Huo (2013)

↓ Predicted glycaemic index

↑ Starch digestibility

↑ In vitro starch digestibility

Sanchez-Pardo, Ortiz-Moreno, Mora-Escobedo, and NecoecheaMondragon (2007) Emami, Perera, Meda, and Tyler (2011)

Negi, Boora, and Khetarpaul (2001)

Innovative Thermal and Non-Thermal Processing

Processing conditions

Dielectric barrier discharge plasma

Corn starch


Glow plasma (nitrogen or helium gas, 30–60 min)

Potato starch


Low-pressure glow ethylene plasma (10−2 Torr, 30 min)

Various starches (potato, sweet potato, waxy corn, cassava


Note: ↑ increase, ↓ decrease, = no significant changes.

↓ Starch crystallinity and pasting viscosity Starch exhibited Newtonian fluid behaviour ↓ Gelatinization temperature ↓ Gelatinization enthalpy ↓ Pasting temperature Corrosions on starch granules ↑ Crystalline lamellae thickness ↓ Amorphous lamellae ↓ Average molecular weight ↑ Reducing sugars Deposits were noted in the granule surface

Bie et al. (2016)

Zhang, Chen, Li, Li, and Zhang (2015)

↑ Starch digestibility

Lii, Liao, Stobinski, and Tomasik (2002)




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the most common and effective ways to physically break and remove the outer coverings, as well as reduce the particle size of plant structures, which in turn increases the surface area of substrate (e.g., starch) available for amylase attack during digestion. These types of physical techniques are potentially advantageous for increasing the bioavailability of dietary carbohydrates upon cooking and ingestion, but they are particularly more effective in reducing the levels of antinutrients (Pal et al., 2017). Since the major constituents of the outer protective coverings are NSPs, reduction in the levels of dietary fiber and overall amount of carbohydrates of the final products is expected, as observed in dehulled lentils (Wang et al., 2009) and flaxseed (Oomah & Mazza, 1997). Moreover, milling and dehulling may cause physical damage to a proportion of starch granules, leading to loss of starch crystallinity (Lovegrove et al., 2017). This in turns increases the water absorption capacity of damaged starches, which become more prone towards gelatinization, as well as more digestible compared to their ungelatinized form. The soaking of legumes is another common approach to soften the outer protective coverings, but due to the water solubility properties of carbohydrates, the reduction in the level of total available carbohydrates of the pre-soaked legumes and beans is always inevitable. Despite the loss of total available carbohydrate and total starch contents, the study of Rehman et  al. (2001) showed that the remaining starches in the pre-soaked red and white kidney beans experienced a higher rate of gelatinization and were found to have higher digestibility upon cooking. Cooking is a typical food-processing technique for most food prior to consumption. The heating of foods at 50–100°C in excess water causes starch granules to gelatinize. Three major molecular events take place during starch gelatinization to promote the conversion of native starch from a semi-crystalline, relatively indigestible form to an amorphous (readily digestible) form (Dona, Pages, Gilbert, & Kuchel, 2010). In brief, the first stage of gelatinization involves breaking down the intermolecular bonds of starch molecules in the presence of water and heat. Meanwhile, water is absorbed and trapped in the amorphous space of the starch, leading to swelling of starch granules. The free amyloses present in the amorphous space of starch granules eventually leach out and dissolve in surrounding water. Gelatinization is important for starch digestibility as gelatinized starch experiences a great loss of its crystalline organization and tends to be more susceptible to amylase hydrolysis, and hence is more digestible. The digestibility of cooked starches can be affected by several factors including starch gelatinization, retrogradation, starch interaction with other components in the food matrix, and the specific cooking method employed. A study by Lee, Lee, Han, Lee, and Rhee (2005) showed that the degree of starch gelatinization for rice samples cooked in various ways is in the following order: autoclave (starch hydrolysis at 75.2%) > stone pot>electric cooker>microwave (starch hydrolysis at 64.6%); this is in line with the results of an in vitro starch hydrolysis with amyloglucosidase and blood glucose response in rats upon ingestion. In another study, the application of 12 different types of heat processing treatment (pressure cooking, autoclaving, puffing, roasting, baking, frying, toasting, and others) on ragi (finger millet) flour was all found to consistently decrease the level of SDS and increased the amount of soluble fiber, leading to improved starch digestibility after cooking (Roopa & Premavalli, 2008). Another interesting finding from the study is that pressure cooking and roasting were


the only two cooking methods that increased the amount of RS by 1.7 and 3-fold, respectively, when compared to native ragi flour. Increased level of RS fraction after certain cooking methods would make a substantial contribution to the carbohydrate available for fermentation in the colon. With respect to the effect of cooking on the levels of NSPs, it was found that cooking does not impart significant changes on most of the NSPs in rice, although a redistribution of insoluble components to soluble fractions has been observed; this appears to favor the starch digestibility for cooked rice (Sagum & Arcot, 2000). On the other hand, prebiotics, such as fructo-oligosaccharides and inulin, can be quite prone to degradation during high temperature heating (e.g., dry heating, caramelization, Maillard browning, etc.). Excessive degradation of prebiotics to their mono- and disaccharides components reduced their prebiotic properties, and can render them unavailable for colonic bacteria metabolism and no longer provide selective stimulation of beneficial colonic bacteria. It has been reported that dry heating of inulin from chicory at temperatures between 135°C and 195°C, for up to 60 min, has resulted in a significant degradation of the fructans ranging from 20% to 100% (Böhm, Kaiser, Trebstein, & Henle, 2005). Fructo-oligosaccharides are also found to be hydrolyzed easily when heated (85°C for 30 min) at low pH (pH 4–7), causing a significant reduction in prebiotic activity (Huebner, Wehling, Parkhurst, & Hutkins, 2008). Both annealing and heat-moisture processing of starch necessitates a critical control of the starch-to-water ratio, temperature, and heating time that leads to a physical reorganization of starch granules (Tester & Debon, 2000). Such treatments are deliberately conducted on starch to impart novel processing characteristics. The heating of starch in excess (>40%–60% w/w) of water is referred to as annealing and usually occurs at a low temperature (50°C) to induce partial gelatinization; hence it is able to selectively modify starch characteristics, such as decrease the swelling power and starch solubility, increase the endothermic temperatures and enthalpies, or change the pasting properties (Jacobs, Eerlingen, Clauwaert, & Delcour, 1995). A previous study found that annealed starches originated from black bean and pinto have a lesser amount of SDS and a higher amount of RS, hence producing starches that break down into glucose slowly and are thus beneficial in lowering the glycemic response on blood glucose level after consumption (Simsek et al., 2012). Meanwhile, the exposure of starches to 100°C) is described as heat-moisture treatment in which this treatment can control the molecular mobility of starch under high temperatures and a limited amount of water, hence restricting the occurrence of complete starch gelatinization. Despite this, the application of heat-moisture treatment on waxy and normal maize starches has been investigated and it was found that the treated starches have reduced crystallinity and contain higher amounts of RDS and SDS, which consequently lead to an overall improved in vitro starch digestibility (Chen et al., 2017). On the other hand, food extrusion is a fully automated technology that involves exposing raw material to intense mechanical shear force under high temperature for a short time. This combined thermo-mechanical treatment of extrusion can promote starch gelatinization, hydrolyze glycosidic bonds in polysaccharides, and cause severe disruption to plant structure (Singh, Gamlath, & Wakeling, 2007). A careful control


Innovative Thermal and Non-Thermal Processing

on the extrusion process conditions, such as raw material composition and moisture content, the cooking temperature along the extruder, and the die and screw speed, is deemed critical to ensure desirable modification to the dietary carbohydrate and its digestibility. Previous studies have consistently demonstrated that the extrusion of cereal grains such as oat bran/flour, wheat, and barley flour can increase the levels of total carbohydrate, soluble dietary fibers, and starch (specifically SDS fraction), leading to better starch digestibility (Altan et al., 2009; Brahma et al., 2016; Gualberto et al., 1997; Rinaldi et al., 2000). Following cooking, gelatinized starches readily realign themselves as they cool down or are stored for an extended period. This process is known as retrogradation and involves recrystallization of the granular structure of starch that is previously gelatinized (Wang, Li, Copeland, Niu, & Wang, 2015). It is important to note that retrogradation is not a process that returns the gelatinized starch into its initial native state. In fact, the cooling of gelatinized starch allows the linear molecules, amylose, and linear parts of amylopectin molecules to retrograde and rearrange themselves again to a more ordered and tightly packed crystalline structure, stabilized by hydrogen bonding while expelling water from the recrystallized structure. Retrograded starch is known to be more resistant to α-amylase attack and therefore is difficult to digest at the initial stage of digestion (i.e., small intestine), but has an important effect on reducing glycemic response in blood glucose upon ingestion due to slower glucose release (Wang et al., 2015). Therefore starch retrogradation is desirable in some food applications, such as in the production of breakfast cereals, parboiled rice, and dehydrated mashed potatoes. Since the reheating of cooked-cooled food is part of domestic food preparation practices, Tian et al. (2016) conducted an in vitro study to investigate the effects of sequential preparation methods of potatoes on their starch digestibility. Results clearly showed that the in  vitro digestion rate of starch for freshly boiled potatoes was 82.21%, then dropped to 54.31% after the cooked potatoes were cooled for 24 h at 4°C due to starch retrogradation that restricts amylase attack. Finally, the rate of starch digestion raised to 66.98% when reheating the cooled potatoes via microwave at 1100 W for 3 min. It appeared that starch crystallinity was, again, substantially affected during the reheating step in which the starch granules become more accessible to digestive enzymes. Moreover, the presence of residual crystals formed during the cooling process in the microwaved potatoes may have slowed down the rapid digestion of the gelatinized starch granules, thus resulting in a lower rate of starch hydrolysis compared to freshly boiled potatoes. Apart from physical and heat-induced modifications, the molecular modification of the carbohydrate matrix through chemical and enzymatic processes has also been widely introduced to the food industry with the aim of improving digestibility properties and hence lowering the estimated glycemic index of starch-based food products. Chemical modification involves the introduction of functional groups into the starch molecule such as through acetylation, hydroxypropylation, oxidation, cross-linking, or dual modification, and is effective in altering the physicochemical, thermal, and functional characteristics of starch (Hazarika & Sit, 2016; Mbougueng et al., 2012). A lower estimated glycemic index has been found in modified black bean and pinto bean starches after acetylation, probably because the substitution of functional


groups has restricted enzyme access due to steric hindrance (Simsek et  al., 2012). In fact, amylose-amylose interaction was still found to be intact after gelatinization of these chemically modified starches. Starch can also be enzymatically modified to increase the degree of branching in the amylopectin fraction, leading to increased proportions of SDS. In this respect, the work of Kasprzak et al. (2012) has showed that waxy starches with higher proportion of α-(1–6) linkages can be produced with amylo-(1,4 → 1,6)-transglycosylase, which can also be used in combination with β-­amylase, α-glucosidase, or amyloglucosidase.

6.4.2 Effect of nonthermal food-processing technologies The application of emerging nonthermal processing technologies on plant foods is necessary to minimize or prevent the undesirable effects that conventional food processing can cause, particularly a substantial loss of important health-promoting compounds due to the use of high temperature. Nonthermal technologies such as High Pressure Processing (HPP), Pulsed Electric Fields (PEF), ultrasound (US), and irradiation have been shown to affect plant structure while maintaining a high retention of bioactive compounds (Rawson et al., 2011). Recently, there is an immense interest towards the use of these nonthermal technologies on starchy or carbohydrate-rich food in order to manipulate the structural and physicochemical properties of carbohydrates without the involvement of the undesirable heating step. Such changes in the properties are expected to have an effect on the digestibility properties of carbohydrates. During HPP, food products are generally exposed to pressure between 100 and 600 MPa at ambient temperature. The great advantage of HPP is that pressure at a given position and time is maintained at a similar level in all directions and transmitted uniformly and immediately through the pressure-transferring medium that is independent of the geometry of the treated product (Oey, Van der Plancken, Van Loey, & Hendrickx, 2008). Unfortunately, the effect of temperature under HPP on starch is not much discussed in literature. Depending on the pressure, temperature, and time combinations applied, starch under the influence of pressure can lose its crystallinity and promote gelatinization-like events through a mechanism that has been described to be quite different from the normal starch gelatinization induced by conventional heat treatment (Liu, Hu, & Shen, 2010). In this respect, HPP generally restricts the swelling power of starch granules, and this technology has the tendency of affecting the amorphous and ordered structure of starch during pressurization followed by the release of pressure. The extent of HPP-induced starch gelatinization can be influenced by the intensity of pressure applied, starch source and concentration, processing temperature and time, and media. When exposed to a similar pressure level (200–600 MPa, 30 min), it was found that starches sourced from potato and yam were very resistant to the effects of HPP and still retained their smooth surface, compared to wheat starch (Wang et al., 2017). The crystallinity of wheat starch dropped most dramatically— from 22.6% to 3.4%—after HPP treatment at 600 MPa, which in turn improved in vitro starch digestibility by at least 20%. Previous studies on pea starch (Leite et al., 2017), tartary buckwheat (Liu et al., 2016), buckwheat flour (Zhou et al., 2015), and potato starch (Colussi et al., 2018) have also consistently indicated that once starches


Innovative Thermal and Non-Thermal Processing

are processed at HPP beyond 400 MPa, they experienced major drastic change in their granule morphology and pasting properties. More importantly, findings from previous studies have revealed that starch exhibited different digestion behaviors when exposed to HPP applied at different conditions. Zhou et  al. (2015) reported that buckwheat starch exhibited a lower digestion rate after HPP treatment (600 MPa for 15 min) at 45°C compared to buckwheat starch treated at room temperature. Recent work by Colussi et al. (2018) further showed that HPP (400 MPa, 6 cycles×10 min) along with retrogradation reduces at least 10% in starch hydrolysis of potato starch during digestion in vitro due to increased levels of RS and SDS, compared to native starch and that treated with HPP alone. Similar to tartary buckwheat, HPP treatment promoted the reduction of RDS levels while increasing the amount of SDS and RS, leading to the production of final products with lower in vitro starch digestibility (Liu et al., 2016). Overall, there are strong indications that HPP might be useful for the preparation of starch-based, slowly digestible, low-glycemic foods suitable for specific target groups (e.g., diabetic patients). Through the mechanism of causing cell electroporation at the cell membrane, PEF has been proven as a valid nonthermal technology capable of influencing the textural properties and structural integrity of solid plant foods. The ability of PEF to induce effective cell electroporation depends upon the parameters applied (e.g., electric field strength, specific energy input, pulse width, pulse number, treatment time, and pulse frequency) (Barsotti, Merle, & Cheftel, 1999) and the physical characteristics of plant material (e.g., electrical conductivity and resistivity, particle size, size and type of cells, chemical composition, pH, and temperature) (Barsotti & Cheftel, 1999). A study by Zhu, Bals, Grimi, and Vorobiev (2012) reported that the cell electroporation effect of PEF (0.6 kV/cm) can facilitate the release of more inulin from chicory roots, which suggests that prebiotic inulin from PEF-treated plant tissue may be more readily available for fermentation by colonic bacteria. With respect to the effect of PEF on starch, previous studies have consistently shown that starches originated from corn, potato, tapioca, and waxy rice (prepared in water suspension) experienced a substantial loss of crystallinity and changes in granule shape with increasing electric field strength from 30 to 50 kV/cm (Han, Zeng, Zhang, & Yu, 2009; Han, Zeng, Yu, et al., 2009; Han et al., 2012; Han, Zeng, Fu, Yu, Chen, & Kennedy, 2012; Zeng et al., 2016). Moreover, the major rearrangement and destruction of starch molecular structure during PEF treatment at high field strengths has caused these PEF-treated starches to be more easily gelatinized. Structural changes in the PEF-treated waxy rice starch was found to influence the digestibility pattern of starch whereby the amount of RDS was found to increase while SDS decreased (Zeng et al., 2016). A recent application of PEF is to assist the acetylation of plant starches in order to modify the functional properties of starch. The application of PEF (2.5–3.75 kV/cm, 40 μs pulse width, 1000 Hz pulse frequency) on acetylated potato starch was found to decrease the RDS fraction and increase the SDS fraction, respectively (Hong, Zeng, Han, & Brennan, 2018). The resulting starch was found to be useful for specific food applications, for instance, PEF-assisted acetylation of potato starch (Hong, Chen, Zeng, & Han, 2016) and cassava starch (Hong, Zeng, Buckow, Han, & Wang, 2016) showed superior retrogradation, swelling power, and freeze-thaw stability. Despite all these advantages promoted


by PEF, there is limited study in the literature to investigate the impact of PEF on the in vitro digestibility of starch. The continuous agitation of plant tissue at ultrasonic frequencies (>20 kHz) generated with either piezoelectric or magnetostrictive transducers that create high-energy vibrations could be sufficient to disrupt cell membranes and release cellular contents. With respect to starch, US treatment has been shown to distort the crystalline region of starch granules as well as increase the swelling power and hence the water absorption capacity (Jambrak et al., 2010; Sujka & Jamroz, 2013). A recent study by Flores-Silva et al. (2017) reported that US treatment (24 kHz, 16 min) altered the compositions of maize starch upon gelatinization, that is, it increased RS content and RDS fractions. A reasonable change in the morphological and crystallinity of starch granules after US treatment has led to the formation of large amounts of short-chained amylose molecules during gelatinization, which makes them less resistant to amylase attack upon in vitro digestion. Irradiation treatment generally involves the exposure of food products to ionizing (high-energy electrons, X-rays, or gamma rays) or non-ionizing radiation (electromagnetic radiation such as ultraviolet rays, visible light, microwaves, and infrared). Plant starches can be quite sensitive to irradiation treatment, depending on the botanical source, irradiation dose, and on the type of radiation source. Previous studies reported that the application of irradiation generated from different radiation sources on sago, corn, and potato starches has led to reduced swelling power of starch and increased solubility (Kerf et al., 2001; Pimpa et al., 2007), but little investigation has been conducted on the effect of irradiation on starch digestibility.

6.5 Conclusion Overall, there is a strong indication in numerous research that some fractions of carbohydrates cannot withstand the food-processing conditions applied, making them undergo drastic structural disruption and/or very prone to degradation to simple sugars. Therefore they are more likely to be hydrolyzed by digestive enzymes and promote improved bioavailability compared to their unprocessed form. In many starchy foods, however, not all starches are completely gelatinized during processing, usually due to limited water content or insufficient heating, while some components of gelatinized starch proceed to retrogradation at a faster rate. These types of starch fractions undergo the same fates as NSPs, that is, to be either slowly digestible at the small intestine or readily fermentable at the colon, which can help in lowering glycemic responses and reducing the development of obesity-related diseases particularly for individuals with diabetes or with features of metabolic syndrome. More importantly, nonthermal processing techniques such as HPP, if applied at suitable pressure levels and on certain starch types, might be capable of modulating such digestion behavior and consequently imparting the aformentioned health benefits. It is noteworthy that knowledge on the effects of nonthermal processing technologies on the bioavailability of carbohydrates in a whole food system (not isolated starch), in relevance to their physiological functions in human, is very limited;


Innovative Thermal and Non-Thermal Processing

t­herefore, future studies (e.g., in  vivo digestion or dietary intervention) in this area are necessary. While most studies in the current literature have placed a great emphasis on examining the digestion patterns of starch after being subjected to nonthermal processing, it will be of future interest to study if the non-digestible carbohydrates are able to withstand certain nonthermal food processing conditions that allow them to reach the colon intact and to be fermented. This aspect will be particularly important in identifying whether those carbohydrates known to possess prebiotics properties are able to retain the activity under the influence of nonthermal processing.

Acknowledgment This research was supported by the Riddet Institute, a New Zealand Centre of Research Excellence, funded by the Tertiary Education Commission. Duque received her PhD scholarship from the Riddet Institute and Abduh gratefully acknowledges the Indonesia Endowment Fund for Education (LPDP) for awarding a PhD scholarship.

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Section 3 Processing, bioavailability and bioaccessibility of micronutrients

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Impact of processing on mineral bioaccessibility/bioavailability


Antonio Cilla, Reyes Barberá, Gabriel López-García, Virginia Blanco-Morales, Amparo Alegría, Guadalupe Garcia-Llatas Preventive Medicine and Public Health, Food Science, Toxicology and Forensic Medicine Department, Universitat de València, València, Spain

7.1 Dietary importance of essential minerals Minerals are classified into macro or microminerals according to their presence in foods and the human requirements. Macrominerals are required in amounts of >100 mg/day, and include calcium (Ca), phosphorous (P), potassium (K), and magnesium (Mg). Microminerals in turn are required in amounts of 300 enzymatic reactions, acting either on the enzyme itself as a structural or catalytic component, or on the substrate, especially in reactions involving ATP. This makes Mg essential in intermediary metabolism in the synthesis of carbohydrates, lipids, nucleic acids, and proteins, as well as for specific actions in various organs in the neuromuscular or cardiovascular system. Good sources include whole grains, legumes, leafy green vegetables, tofu, meat, fruit, and dairy products. Mg deficiency is observed only in two situations: as a secondary complication of primary disease (e.g., cardiovascular or neuromuscular disorders), and as the result of rare genetic homeostatic disorders. Deficiency is usually associated with tachycardia, which can be accompanied by weakness, muscle spasms, disorientation, nausea, vomiting, and seizures. Mg deficiency can cause hypocalcemia and hypopotassemia, leading to neurological or cardiac symptoms when associated to marked hypomagnesemia (EFSA, 2015b). Potassium: K is the main cytosolic cation, with 98% of the total content being found inside the cells. A high intracellular concentration of K is maintained by the Na+/K + -ATPase pump. It plays a major role in the distribution of water inside and outside cells, assists in regulation of the acid-base balance, and contributes to establish a membrane potential, which supports electrical activity in nerve fibers and muscle cells. K plays a role in cell metabolism, participating in energy transduction, hormone secretion, and the regulation of protein and glycogen synthesis. Fruits and vegetables, particularly leafy green vegetables, vine foods such as tomatoes, cucumbers, zucchini, eggplant, and pumpkin, and root vegetables are good sources of K. Hypopotassemia can cause cardiac arrhythmias, muscle weakness, and glucose intolerance, and usually results from increased K loss or intracellular shifts in K. Hypopotassemia is rare and may be associated with severe hypocaloric diets, or relative insufficiency caused by increased K requirements for tissue synthesis during recovery from malnutrition. An inadequate intake of K may increase the risk of cardiovascular problems (Cilla et al., 2014; EFSA, 2016).

7.1.2 Microminerals Iron: Fe acts as a catalyst in a broad range of metabolic and enzymatic processes. It is essential for cellular respiration, as a component of hemoglobin or myoglobin with participation in oxygen transport, and is found in enzymes that participate in electron transfer and redox reactions. There are two types of dietary Fe. Heme-Fe

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comes from hemoglobin and myoglobin, and is of animal origin (viscera, red meat, fish, and seafood). It is the most bioavailable type (15%–35%), contributing around 10%–15% of the total Fe consumption in omnivorous individuals. The non-heme form (inorganic Fe) in turn is the most abundant type of dietary Fe, and is present in plant foods (vegetables, whole grains, nuts, and oil seeds) and animal tissues. It is comparatively less bioavailable (5%–15%). Dietary factors that influence non-heme Fe bioavailability are the chemical form (absorption of the ferrous state being greater than in the case of the ferric form), Fe intake, the presence of enhancers such as protein fraction of muscle tissue and ascorbic acid, and inhibitors such as phytic acid, polyphenols, or Ca. Iron deficiency, associated with diminished work productivity, lowered immunity, and impaired cognitive development, remains the most common nutritional deficiency in developing countries, despite dietary improvements (Alegría et al., 2015; Cilla et al., 2014). Copper: Cu is essential as an enzymatic cofactor (metallothionein, ceruloplasmin, superoxide dismutase [SOD], etc.) or allosteric component of cuproenzymes with oxido-reductase activity (cytochrome c oxidase, amino-oxidase, SOD, etc.). Cu participates in growth, defense mechanisms, bone mineralization, the maturation of red and white blood cells, Fe transport, cholesterol metabolism, myocardial contractility, glucose metabolism, brain development, and as a component in gene expression. The main dietetic sources include viscera, seafood, legumes, nuts, seeds, and drinking water. Deficiency is rare, but children with poor Ca diets are more susceptible, especially if they also have diarrhea or malnutrition. Cu deficiency has been described in subjects with malabsorption syndromes such as celiac disease, tropical and non-tropical sprue, cystic fibrosis, or short intestine (Cilla et al., 2014; EFSA, 2015c). Zinc: Zn is fundamental for gene expression and the regulation of cellular growth and differentiation, since it participates as a cofactor of >200 enzymes involved in the metabolism of nucleic acids, carbohydrates, and proteins. It also plays a structural role in a large number of Zn finger proteins. Zn is essential in the immune system and in male reproductive function. Besides, it participates in the antioxidant defense system and has an antioxidant role. The main dietetic sources of Zn are viscera, meat, seafood, whole grains, nuts, and oil seeds. Its absorption is influenced by activators (picolinic acid secreted by the pancreas, vitamin B6, citrate, and amino acids) and inhibitors (phytic and oxalic acids, tannins, fiber, Se, Fe, and Ca). The food supply of nearly 50% of the world population is low in absorbable Zn, due to limited availability of animal products. In recent years Zn deficiency has become a global nutrition problem, particularly in developing countries (Akhta, Anjum, & Anjum, 2011; Alegría et al., 2015; Cilla et al., 2014; Salgueiro et al., 2000). Selenium: Se is a part of enzymes involved in the protection of tissues against oxidative stress, such as glutathione peroxidase (GPX). Other selenoproteins are thioredoxin reductases (which regulate the cellular redox balance), deiodinases (involved in thyroid function), and selenoprotein P (which could act as a transporter between the liver and other organs). It exerts antioxidative and anticancer effects, contributes to the prevention of cardiovascular diseases and to heavy metal detoxification, and participates in cerebral functions, reproduction, and the immune system. Good Se dietetic sources include enriched yeast and cereals. Se is frequently added as a supplement


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(Se-enriched foods) in broccoli, yeast, and potatoes, for example. It is present in different chemical forms: selenate, selenomethionine, and smaller amounts of selenocysteine. Methylated forms are found in plants that actively accumulate Se well in excess of the amounts found in the soil. Se deficiency is associated with health disorders including oxidative stress-related conditions, reduced fertility and immune functions, and an increased risk of cancer (Alegría et al., 2015; Finley, 2007; Thiry, Ruttens, De Temmerman, Schneider, & Pussemier, 2012).

7.2 Effect of household processing on mineral bioavailability Household processes such as dehulling, mechanical processing, soaking, germination, and fermentation alone or with thermal processing could influence the bioaccessibility of minerals in vegetable foods. The aforementioned strategies could increase the bioaccessibility of minerals due to a decrease in antinutrients (phytate) or an increase in enhancer content (organic acids, which form soluble ligands with minerals, thereby preventing the formation of insoluble complexes with phytate). In this regard, dehulling removes phytate localized in the outer aleurone layer of cereals or in the germ, increasing mineral bioavailability, though the total content is simultaneously reduced. Other methods such as soaking, with passive diffusion of water-soluble Na, K, or Mg phytate, fermentation by microbial phytase enzymes that hydrolyze to lower inositol phosphates, and germination increasing the activity of endogenous phytase activity in cereals, legumes, and oil seeds through de novo synthesis, as well as the activation of intrinsic phytase, would be particularly interesting because they reduce phytate content but not mineral content. Several of these strategies in combination are probably needed to obtain a positive effect in terms of mineral adequacy (Hotz & Gibson, 2007; Khanam & Platel, 2016a). This would be particularly interesting for vegetarians and in developing regions (e.g., Africa, India) that include germination and fermentation in their traditional cuisine. In this context, household strategies have been used in in vivo studies to enhance Fe and Zn bioavailability by including dephytinized cereals and/or legumes in porridges for infants and adults (Gibson, Perlas, & Hotz, 2006). Table 7.1 summarizes several studies that have evaluated the influence of household strategies with thermal processing upon mineral bioavailability. The influence of germination and fermentation (alone or combined with heat processing) of cereals and/or pulses upon Se bioaccessibility has been evaluated. Pressure cooking of the germinated grains (chick pea, green gram, and finger millet) produces a decrease in Se bioaccessibility (49%–59%) with respect to the raw and germinated product, a fact that has been attributed to the decrease in total Se content after heat treatment (33%–36%, depending on the cereal involved). Cooking of fermented foods habitually consumed in India produces an increase in Se bioaccessibility with respect to foods that have only been fermented (71% and 44%, respectively), though in both instances the bioavailability is lower than in the case of the native product (Khanam & Platel, 2016a).



Processing method



Fe, Zn

Fermented porridges prepared with finger millet with and without peanut butter

Fermentation flour water 24 h 25– 30°C + cooking 20 min 80–100°C

Gabaza et al. (2017)

Fe, Zn

Bred cowpeas

(a) Soaking (with or without) + regular pan 30 min at 100°C (b) Soaking (with or without) + pressure cooker 10 min at 100°C


Finger millet, chickpea, whole and decorticated green gram, decorticated black gram, rice Fermented foods of India: dhola, dosa and idli

Germination soaking water 16 h, germination 48 h + pressure cooking water 10 min 15 psi

Fe and Zn dialyzability (%): Cooked fermented (6.1 and 13.2); fermented (5.7 and 9.7); flour (7.7 and 12.7) Total soluble Fe and Zn (%)a: Cooked fermented (25 and 72); fermented (25 and 49); flour (25 and 49) Fe and Zn dialyzability (%): Raw (2.5–4.3; 37.4–40.8); soaked+pressure cooked: 6.4–40.6; 19–51.6; pressure cooked: 8.6–44.4; 37.5–56.3; soaked+regular pan: 2.4–8.9; 37.1–45.9; regular pan: 2.9–8.7; 38.8–52.7 Dialyzability (%): Heat processed (10–17); unprocessed (25–35)


Fe-biofortified bean flours

Fermentation (soaking 10 h water + fermentation 14 h ambient temperature) + cooking 10 min (idli and dhokla) or pan fried (dosa) (a) Soaking in distilled water 24 h 1:2 w/v ratio + germination 48 h +  roasting in oven 45 min 170°C (b) Extrusion (twin screw extruder with three heating sections 60°C, 130°C, and 130°C)

Pereira, Carvalho, Dellamora-Ortiz, Cardoso, and Carvalho (2016)

Khanam and Platel (2016a)

Dialyzability (%): Cooked fermented (30–36); fermented (29–30); native (41–44) Fe and Zn HCl-extractability (%): Raw flours 38.9–40.7; 51.5–55; malted/ roasted 70.5–74; 66.9–68.6; extrusion 79.4–83.4; 72.3–73.7

Nkundabombi, Nakimbugwe, and Muyonga (2015)


Impact of processing on mineral bioaccessibility/bioavailability213

Table 7.1  Effect of household and conventional thermal processing on mineral bioavailability.


Table 7.1  Continued Food

Processing method



Ca, Fe, Zn

Faba bean, azuki bean, mung bean

Ca, Fe and Zn bioaccessibility (mg): Raw, pressure cooking and microwaving: Faba bean: Ca (188, 185, 186), Fe (32.6, 30.4, 31.3) Zn (32.2, 31.8, 32) Azuki bean: Ca (313, 312, 313), Fe (42.5, 40.5, 40.8) Zn (25.5, 23, 23) Mung bean: Ca (425, 418,409), Fe (48,46.5, 46.5) Zn (28, 27.1, 27.2)

Luo, Xie, Jin, Wang, and Zai (2013)

Ca,Fe, Zn

Finger millet

Mung bean products (namkeen)

Mineral dialyzability (%): Ca: native millet 40; expanded 33; decorticated millet 55; popped millet 46 Fe: native millet 9; expanded 14; decorticated millet 35; popped millet 49 Zn: native millet 64; expanded 82; decorticated millet 88; popped millet 89 Ionizable Fe (%): Dehusked fried (22−23); roasted (2.5– 3.1); whole fried (2.5–2.7); raw (2–2.5)

Krishnan, Dharmaraj, and Malleshi (2012)


Soaking (10, 12, or 16 h prior to germination 60, 24, 20 h for raw faba bean, azuki bean, and mung bean respectively)+ (a) Pressure cooking (1.5 × 106 Pa 121°C) 1 min faba bean and mung bean, 5 min azuki bean (b) Microwave cooking (800 W) 7 min faba bean and mung bean, 15 min azuki bean (a) Soaking 30 min + decortication + high temperature and short time to prepare expanded millet (b) Unsoaking + high temperature and short time to prepare popped millet (a) Whole: soaking 12 h (b) Soaking 12 h + dehusking + deep frying 30 s (full flame) and 30 s (low flame) (c) Soaking 12 h + deep frying 30 s (full flame) and 30 s (low flame) (d) Soaking 12 h + roasting 120°C 1 min (full flame) and 3 min (low flame)

Raghuvanshi, Singh, Bisht, and Singh (2011)

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Fe, Zn

Fermented foods of India: dhola, dosa and idli

Ca, Mg

Faba beans

(a) Whole and dehulled mung bean: washing running tap water 15 min + pressure cooking at 15 lb. 5 min (b) Germination whole mung bean: soaking 12 h water, germination 24 h) + pressure cooking (seed:water ratio 1:2 w/v) 4 min (c) Fermentation dehulled mung bean: soaking overnight water/ fermentation 12 h 37°C beaten 7–10 min) + frying refined oil 1 min 190°C Fermentation soaking water 10 h + fermentation 14 h room temperature + cooking 10 min (idli and dhokla) or pan frying (dosa) Soaking 9 h room temperature in citric acid solution (pH 2.6 and 5.3) and sodium bicarbonate (pH 8.4) + cooking water 35 min

Fe absorption (%)b: Whole raw (7.32); whole cooked (8.86); dehulled cooked (9.95); germinated cooked (12.52); germinated raw (11.04); fermented cooked (11.04); fermented raw (9.55)

Barakoti and Bains (2007)

Fe and Zn dialyzability (%): Cooked fermented (13–30 and 24–40); fermented (13–25 and 48–54); raw grains (6–12 and 31–54) In vivo (rats) Ca and Mg apparent digestibility coefficient (%): Raw (79.7 and 30.8); soaked at different pH (76.4–80.2 and 41.1–47.4); soaked and cooked (77.6–83.3 and 43.2–50.1) Ca and Mg retention/absorption (%): raw (84.6 and 23.7); soaked at different pH (90.3–91.7 and 51.9–57.4); soaked and cooked (89.9–93.1 and 32.2–49.7)

Hemalatha, Platel, and Srinivasan (2007a)

Aranda et al. (2004)


Impact of processing on mineral bioaccessibility/bioavailability215



Table 7.1  Continued Mineral


Processing method




Rice bean

(a) Soaking 12 h + pressure cooking 103 × 103 N/m2 until done (b) Germination soaking 12 h, germination 37°C 48 h + pressure cooking 4 min 103 × 103 N/m2 (c) Roasting 2 min 250°C (d) Dehulling + soaking 12 h (e) Dehulling + soaking 12 h + pressure cooking 4 min 103 × 103 N/m2

In vitro: solubility (%): Dehulling pressure cooked (29) > germination pressure cooked (28.4) > dehulled raw (25.4) > soaked pressure cooked (20.2) > roasted (18.9) > whole raw (16) In vivo (rats): Similar apparent absorption and retention availability (total femur zinc content μ/100 mg): germinated, dehulled or soaked products pressure cooked (20.35–20.78) > dehulled, roasted or whole products (17.51–19) Mineral HCl-extractability (%): Ca: Ordinary or pressure cooking: unsoaked (55.2, 56.2 4), soaked (58.2, 58.3) soaked-dehulled (60.2, 62) P: Ordinary or pressure cooking: unsoaked (38.3, 40.4), soaked (41.3, 43.4), soaked-dehulled (46.3, 50.3) Fe: Ordinary or pressure cooking: unsoaked (44, 47), soaked (49.7, 51.3), soaked-dehulled (52.8, 55.8)

Kaur and Kawatra (2002)

Pigneon pea

Unsoaking seeds, soaking (12h) in double-distilled water at 30°C, 1:5 w/v ratio) or soaking-dehulling + (a) Ordinary cooking until soft (b) Pressure cooking

Duhan, Khetarpaul, and Bishnoi (2002)

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Ca, P, Fe


White beans, chickpeas, lentils

Soaked (16 h in tap water at room temperature) + (a) Traditional cooking on a hot plate (500w, 15 min lentils, 45 min beans and 60 min chickpeas) (b) Microwave oven 1400 W, 5 min lentils and 25 min chickpeas (c) Industrial processing (readyto-eat legumes)

Mineral dialyzability (%): Ca: raw legume (14–21); traditional cooked (11–14); microwave (11–14); industrial processed (14–25) Fe: raw legume (2–5); traditional cooked (1–3); microwave (1–3); industrial processed (40–47) Zn: raw legume (30–50); traditional cooked (26–34); microwave (29–35); industrial processed (46–67)

Soluble (%) = Soluble no dialyzable (%) + dialyzable (%). Fe available calculated with the following prediction equation Iron absorption (%) = 0.4827+0.4707+percentage ionizable iron at pH 7.5/iron solubility.


Sebastiá, Barberá, Farré, and Lagarda (2001)

Impact of processing on mineral bioaccessibility/bioavailability217

Ca, Fe, Zn


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The effect of dehulling, fermentation, and germination associated with thermal treatment upon Fe bioaccessibility in mung bean has been studied. The increases with respect to the raw material were as follows: pressure cooking of dehulled mung bean and cooking of whole mung bean (35% and 21%, respectively); cooking of germinated mung bean and germinated raw mung bean (71% and 50%); and fried fermented mung bean and fermented raw mung bean (38% and 19%). The reduction of phytates and the increase in ascorbic acid content—both maximum in germinated and fermented samples—could help to enhance Fe bioavailability in mung bean and improve Fe status in vegetarian subjects (Barakoti & Bains, 2007). In this sense, using ionizable Fe at pH 7.5 expressed as a percentage of total Fe as an index of Fe bioavailability, significantly higher values for dehusked fried namkeen (an Indian snack) formulated with different mung bean cultivars versus raw mung bean have been reported. However, this effect has not been observed in the case of roasted and whole fried namkeen, probably because the loss of tannins would not have been sufficient, while decortication suffices to reduce the tannins, resulting in a significant increase in ionizable Fe (Raghuvanshi et al., 2011). Increased Zn solubility has also been reported in soaked or germinated ­pressure-cooked rice beans when compared with the whole raw food. In dehulled products, a significant increase in Zn solubility has been found, greater than in the thermally treated food, probably due to the elimination of phytate and fiber in the dehulled product. A significant and negative correlation between Zn solubility and saponin and polyphenol contents has also been reported. However, nonsignificant differences in the percentage absorption and retention of Zn were obtained in rats fed with experimental diets containing ZnSO4, soaked, germinated, or dehulled pressurecooked rice beans, and whole, dehulled, or roasted rice beans. Total femoral Zn content (used as an indicator of Zn availability) and Zn content in the liver, kidney, and spleen of animals fed dehulled rice-bean diets were higher than in animals fed a whole rice-bean diet, as well as in those that received soaked or germinated pressure cooked rice beans (Kaur & Kawatra, 2002). A study in cereals and/or pulses similar to the aforementioned study on Se has been carried out in relation to Fe and Zn bioavailability. Zn bioaccessibility decreased after cooking of fermented foods with respect to fermented food; in some products reaching higher (27% increase), and in others lower values (18% and 31% decrease, respectively) than those recorded in raw (unprocessed) grains. However, Fe bioaccessibility improved with fermentation and heat processing, resulting in increases of 276% and 335% in some products, while Fe bioaccessibility was not modified in others. Significant reductions in both phytate and tannin during the fermentation of ­cereal-legume combinations could explain the increase in Fe bioaccessibility. The lack of significant reductions in phytate content or proteins that bind minerals contributed by the use of legumes (chickpea and green gram) in these products is consistent with the absence of increments in Zn bioaccessibility (Hemalatha et al., 2007a). In contrast, in fermented porridges prepared with finger millet respect its flour or an slurry of that grains fermented spontaneously, no improvement was observed for total soluble Fe (dialyzable and non-dialyzable), though an increase (47%) was reported for total soluble Zn. However, the increase in solubility did not result in increased ­dialyzability.

Impact of processing on mineral bioaccessibility/bioavailability219

Besides, similar bioaccessibility was found for different varieties and fermentation techniques (Gabaza et  al., 2017). In relation to the bioaccessibility of Fe and Zn, the effect of regular pan or pressure-cooking, with or without previous soaking, in bread cowpea has been evaluated by Pereira et al. (2016). Higher Fe dialyzability percentages (up to sevenfold) were found when cooking was done in a pressure cooker without soaking compared with regular pan cooking. For Zn dialyzability, only slight differences were found between the two cooking methods (with or without previous soaking). The lower leaching out of Zn compared with Fe can be attributed to the fact that these minerals are not linked to the same molecules (Zn is highly associated with enzymes and proteins), and they are not found within the same location in the seeds. Soaking in solutions of different pH values (acid pH 2.6 and 5.3; basic pH 8.4) of faba beans alone or combined with cooking improved the digestive utilization of Mg (determined by the apparent digestibility coefficient) in rats, without significant differences between treatments, though this was not observed with Ca. The lack of effect in the case of Ca could be due to its high digestive utilization (80%–83%), which is at the upper limit of the body absorption capacity of the rat. The metabolic utilization (% retention/absorption) of both minerals increased in rats fed with processed faba beans compared with those receiving the raw faba bean diet, being up to three times greater in processed versus raw legume diet in the case of Mg, despite the higher intake of cellulose, lignin, and phytate found in the processed faba bean diets. The improvement in mineral absorption and retention was not correlated to higher mineral contents in plasma, femur, and longissimus dorsi muscle for Ca, or in femur and muscle in the case of Mg (Aranda et al., 2004). Ca, Fe, and Zn dialyzability has been determined in processed legumes (Sebastiá et al., 2001). Traditional and microwave treatments following prior soaking reduced the dialyzability of Ca, Fe, and Zn versus raw legumes. No differences were observed for the three elements and all legumes between the thermal treatments. Industrially processed legumes have higher Ca and Zn dialyzability than traditionally or microwave cooked legumes. This effect is greater (between 20- and 40-fold) for Fe, a fact that could be attributed to soaking in EDTA solution, which protects Fe from reacting with phytic acid and therefore increases available Fe, while the addition of ascorbic acid can counteract the negative effect of phytate and polyphenols upon Fe bioavailability. Other processes such as soaking-germination and pressure or microwave cooking on in vitro Ca, Fe, and Zn bioaccessibility in faba bean, azuki bean, and mung bean seeds was evaluated by Luo et al. (2013). Although a significant reduction was observed in phytate in all three legumes after soaking (13.7%–14.8%) and during germination (45.2%–50.1%), no differences in Ca, Fe, or Zn content were detected in the bioaccessible fractions between raw legumes versus pressure cooking or microwaving. Using mineral HCl extractability as an index of mineral bioavailability, the influence of no soaking, soaking, or soaking and dehulling of pigeon peas, with ordinary or pressure-cooking, upon Ca, P, and Fe was evaluated by Duhan et al. (2002). Ordinary cooking as well as pressure-cooking increased the extractability of the minerals (Fe > P > Ca) from unsoaked pigeon pea seeds, though to a lesser degree than in the case of soaked and soaked-dehulled seeds. The effect was moreover greater with pressure-cooking. Soaked and dehulled pressure-cooked seeds showed higher Ca, P,


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and Fe extractability versus ordinary cooking. The greater decrease in phytic acid content of up to 36% in soaked-dehulled pressure-cooked seeds versus 32% with ordinary cooking could be responsible for the results obtained. During cooking at a temperature of 40–55°C, phytase activity may degrade IP6 to IP5 or to lower molecular weight forms. In this context, in different cultivars of white beans, higher Ca, P, Mg, K, Na, Fe, Cu, and Zn HCl extractability (up to fivefold) was observed in cooked and soaked-cooked versus untreated bean cultivars (ElMaki et al., 2007). This observation possibly being attributable to the decrease in phytic acid and polyphenol contents. The greater decrease in phytic acid and polyphenol contents, respectively, in soakedcooked (21%–27% and 19%–28%) versus cooked food (13%–25% and 17%–26%) may be the reason for the higher mineral extractability found for the latter treatment. After malting/roasting or extrusion applied to Fe biofortified bean flour, an increase was observed in Fe and Zn HCl extractability (1.3- to 3-fold) versus raw product, the highest mineral extractability percentages corresponding to extrusion-treated products. During germination, a decrease in phytate and polyphenol contents occurs due to the increase in the enzymatic activity of phytase and polyphenol oxidase. In addition, soaking, which is part of the malting process, reduces phytates through water solubilization and subsequent leaching of some phytic acid salts. Besides, thermal treatment (roasting and high extrusion temperatures) especially reduces phytates, resulting in higher mineral availability (Nkundabombi et al., 2015). Treatments involving high-temperature, short-time popping and expansion applied to native finger millet and decorticated finger millet, respectively, produced a decrease (about 10 g/100 g) compared with non-thermal treatments in Ca bioaccessibility but an increase in Fe and Zn bioaccessibility; the effect being most apparent for Fe bioaccessibility and expanded millet (increase of 14 g/100 g) and for Zn bioaccessibility and popped millet (increase 18 g/100 g). The influence of inhibitory factors upon mineral bioaccessibility depends on the treatment applied. Popping did not change the phytic acid content, and produced a slight decrease or increase in polyphenol content or total dietary fiber, respectively. However, expanding did not modify the polyphenol and total dietary fiber content, and decreased the phytic acid content (Krishnan et al., 2012). In bakery products (whole-wheat bread, white bread, and muffins), fermentation increased Fe (2- to 4-fold) and Zn solubility (1.2 to 2-fold) and dialysis (10- to 20- and 1- to 6-fold for Fe and Zn, respectively), due to the decrease in pH value to 5 as well as to the action of endogenous phytases, while the effect upon Ca was variable, depending on the bakery product involved. Baking decreased Fe and Zn solubility (two- to threefold), probably due to the formation of complexes with melanoidins in the case of Fe and to the negative effect of phytate content in the case of Zn, despite the decrease after baking in phytate content and the increase in Fe and Zn dialyzability. However, the effect of baking on Ca solubility or dialyzability is highly variable. Furthermore, Caco-2 cell uptake and transport efficiency of Fe increased (one- to threefold) during the fermentation of bakery products with respect to the baked state; no differences being observed for Zn, while a slight decrease was observed for Ca. Despite the effect of processing (fermentation and baking) upon the lowering of phytate content, no clear relationship was found between phytate content and the mineral bioavailability of bakery products (Frontela, Ros, & Martínez, 2011). In this cellular model, the effect

Impact of processing on mineral bioaccessibility/bioavailability221

of baking upon Fe bioavailability (ferritin synthesis) from Fe-fortified low-extraction wheat flour bread has been described by Yeung, Miller, Cheng, and Glahn (2005). Baking significantly reduced the ferritin formation of products fortified with FeSO4 and Ferronyl (reductions of about 100 and 90 ng ferritin/mg cellular protein, respectively), probably because the baking process promotes the oxidation of Fe from these Fe fortificants to ferric Fe, which is less bioavailable. No effect was observed in the case of FeCl3 fortificant.

7.3 Conventional thermal processing This type of processing is usually applied in order to preserve beneficial structures and nutrients, food safety, and/or to prepare the food product (cooking). In this sense, operations such as steaming, boiling, roasting, microwaving, and so on, can be applied to food products. Shelf-life extension of processed foods is possible by applying high temperatures and low moisture content, although many nutrients are sensitive to heat, oxygen, or water as well as to other physical operations. Compared with vitamins, minerals are more resistant to industrial production processes; however, despite the increase in safety afforded by these procedures, they can have a substantial effect upon the bioaccessibility/bioavailability of the mineral contained in the foods. Food processing can also improve the bioaccessibility of micronutrients by decreasing ­anti-nutrient levels, and causes minerals in the food matrix to be more diffusible and bioaccessible/bioavailable (Cilla, Bosch, Barberá, & Alegría, 2017; Gharibzahedi & Jafari, 2017).

7.3.1 Vegetable foods The effect of conventional processes (mainly boiling and pressure and microwave cooking) upon mineral bioaccessibility/bioavailability of vegetable foods has been mainly studied for Ca, Fe, and Zn from pulses and cereals (see Table 7.2), with variable effects depending on the sample and/or thermal processing involved. In this sense, in mangalo, pigeon pea, and cowpea beans, thermal processing significantly favored Ca bioaccessibility (increments of 15%–20%), with similar behavior in samples subjected to different types of thermal processing (pressure-cooking, oven, or microwave) (Santos et al., 2018). The authors indicated that this could be due to the gelatinization of starch and protein denaturation, which favor the action of digestive enzymes, making Ca more bioaccessible. The same trend was observed for Fe in the three pulses studied, with heat treatments generally favoring Fe bioaccessibility. This behavior was more noticeable in treatments in which water was used for cooking (microwave and under pressure). However, this study also reported significant decreases in K and Mg bioaccessibility after thermal treatment of the pulses, probably due to the high solubility of these salts. Different studies have been made of mineral (Ca, Fe, and Zn) uptake by Caco-2 cells from white beans, lentils, and chickpeas subjected to thermal processing (traditional boiling or industrial cooking) after soaking (Viadel, Barberá, & Farré, 2006a,


Table 7.2  Effect of conventional processing on mineral bioaccessibility/bioavailability in vegetable foods. Mineral


Processing method



Ca, Fe, K, Mg

Pulses (mangalo, pigeon pea, cowpea beans)

Cereals (wheat, rice, finger millet, maize, sorghum, pearl millet), pulses (chickpeaa, cowpea, horse, redb, greena and black grama), green leafy vegetables (fenugreek, spinach, amaranth, dill, moringa) Green leafy vegetables

Solubility (%) (data provided as ranges including non-thermal and thermal processed in mangalo, pigeon pea, cowpea beans, respectively): Ca: 27–61, 71–81, and 68–74. Increase vs. raw Fe: 13–76, 0–10, and 0–21. Increase vs. raw K: 12–19, 10–21, and 10–13. Decrease vs. raw Mg: 7–10, 9–10, and 7–10. Decrease vs. raw Dialyzability (%): Se total: In cereals: Decreased for cooked wheat (pressure cooked and MW: ~18 and ~15, respectively), cooked rice (pressure cooked: ~10) and cooked pearl millet (MW: ~10) vs. raw (~24, 22 and 20, respectively). In pulses: decreased for cooked whole green gram (pressure cooked and MW: ~13 and 10, respectively) and cooked decorticated (pressure cooked: ~16) vs. raw (~18 and 25, respectively). In GLV: decrease for cooked graveolens (~10 and ~14) and oleifera (~19 and ~16) in pressure cooked and MW, vs. raw (~29 and 31, respectively). For spinach: decrease for pressure cooked vs. raw (~9 vs. 25) SeMet: Decreased at pressure cooked and MW vs. raw (cereals: 3–15 vs. 24–71; pulses: 2–11 vs. 10–80; GLV: 0.1–1 vs. 2–11, respectively) SeCys2: ND in treated samples

Santos, Ribeiro, Santos, Korn, and Lopes (2018)

Se total, SeCys2, SeMet,

(a) Pressure-cooking (15 psi): 120°C/3 and 6 min (b) Oven: 200°C/20 and 40 min (c) c) MW: 100°C/6 and 12 min (a) Pressure-cooking (15 psi): 10 min. (b) MW (360 W): 20 min.


Dialyzability (%): No effect cooked (15–33) vs. raw (15–33)

Amalraj and Pius (2015)

Innovative Thermal and Non-Thermal Processing


Khanam and Platel (2016b)


Growing: peat fortified with Se (IV) and Se (VI) (1:9) at low, medium or high level (6, 21 and 169 mg Se kg−1) Boiling: 10 min

Ca, Mg, Zn

Carrots (4 landraces and 2 commercial cultivars)

(a) Pulping (in glass mortar, particles 3–8 mm) + steaming: 20 min (b) Homogenizing (particles homogenized particles). In the case of Ca, pulped carrots showed a significant increase in Ca solubility in all the cultivars (38%–86% in thermal processed versus 14%–28% in raw food), whereas homogenized carrots only favored Ca bioaccessibility in three of the cultivars (76%–94% in thermal processed versus 40%–60% in raw food). Results showed that the synergy between particle size and cooking was not of equal magnitude in all of the samples because the cell wall of these cultivars retained a large amount of Ca and/or a high content of natural chelating agents, such as phytates. Greater bioaccessibility in some carrot cultivars may be explained by differences in the organic acid contents (malic, citric, ascorbic), though carrot acidity is generally low (pH 6–7). The organic acids could function as Ca chelating agents. Moreover, it can be assumed that in the gastric digestion step (acid pH), Ca phytate would be free, though at the end of intestinal digestion (basic pH) Ca could be chelated again and retained. In general, high Mg bioaccessibility was reported when compared with other minerals, probably due to the fact that the majority of the mineral is located in cell vacuoles, which are 60%–90% water extractable and therefore cooking did not have an effect on the Mg bioaccessibility because only a small percentage of the mineral is bound to the cell wall and/or chloroplasts. In addition, the reduction in particle size by homogenization disrupts the cells to a larger extent than pulped carrots, which allows for greater Mg bioaccessibility with a smaller particle size. A lower bioaccessibility for Zn than Ca or Mg was detected. The authors argued that the complexing of Zn with phytic acid is dependent on pH and the presence of other metals, being greater than in the case of Ca at the pH of carrots, whereas the increase in citric acid and malic acid as chelating agents had little or no effect on Zn solubility. The assessment of Se (Se (IV), Se (VI), selenocystine and selenomethionine) solubility after cabbage cooking has been carried out by Funes-Collado et  al. (2015). Results showed that boiling cabbage increased the bioaccessibility of organic selenocompounds, mainly selenomethionine (8%–19% versus 1%–3% in the raw cabbage), whereas inorganic Se levels decreased. The authors attributed this effect to


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the extraction of the original inorganic mineral from cabbage during the boiling process, thus exerting a positive effect on selenomethionine solubility. Khanam and Platel (2016b) in turn evaluated the dialyzability of Se (selenocystine and selenomethionine) from cereals, pulses, and green leafy vegetables after pressure or microwave cooking. Heat treatment (both pressure-cooking and microwave cooking) exerted a significant decreasing effect on the bioaccessibility of total Se in most of the cereals, pulses, and vegetables. This reduction as a result of heat processing could be due to the volatile nature of Se, as commented by the authors, since it is reportedly volatilized as dimethylselenide or dimethyldiselenide from plants. Regarding the dialyzability of selenomethionine and selenocystine, heat processing resulted in a significant decrease in the bioaccessibility of selenomethionine from cereals and pulses subjected to pressurecooking and microwave cooking (2%–15% in thermal processed versus 24%–80% in raw food), the reduction being >90% in most of the cases. On the contrary, selenocystine was not detected in cooked samples.

7.3.2 Animal foods Depending on the way in which different animal foodstuffs are processed through thermal treatment, the latter can have a positive or negative impact on nutritional value and even mineral availability. The influence of different conventional thermal treatments on the mineral bioaccessibility/bioavailability in foods of animal origin is summarized in Table 7.3. Regarding meat products from beef, pork, and chicken, and bovine liver, the mineral bioaccessibility studies have yielded contrasting results depending on the food matrix, the mineral considered, and the type and conditions of thermal processing (Da Silva et al., 2017; Menezes et al., 2018; Sorensen et al., 2007). In this regard, a general positive effect on Ca, Cu, Fe, K, Mg, and Zn has been reported after cooking in water, sous vide cooking, and baking in the microwave oven, increasing mineral bioaccessibility from 7% to 175% versus untreated food. In addition, pork meat heattreated at 60–120°C for 1 h in sealed stainless steel cans has shown an increase in Fe dialyzability in the gastric stage from 88% to 164% at a temperature of 80°C onwards. The beneficial effect could be attributed to protein denaturalization during thermal treatment, which allows improved accessibility for the proteolytic enzymes, with the release of minerals bound to the food matrix (Sorensen et al., 2007). It is worth noting that sous vide processing results in considerably superior mineral bioaccessibility than water cooking, since mild temperatures and long periods of heating are used together with a lack of mineral leaching, thereby promoting enhanced mineral bioaccessibility (Da Silva et  al., 2017). In contrast, when the thermal cooking processing methods comprise baking in a conventional oven at 180°C for 60 min, a general decrease in mineral bioaccessibility is observed (20%–188%) versus untreated samples. This negative effect has been linked to the formation of Maillard reaction products that can tightly bind minerals (Gharibzahedi & Jafari, 2017) or reduce the binding active sites between protein nitrogen and metals (Da Silva et al., 2017). However, independently of the thermal treatment involved, Ca is always improved in all food matrixes, while Cu is decreased only in pork meat.

Table 7.3  Effect of conventional/thermal processing on mineral bioaccessibility/bioavailability in foods of animal origin. Mineral


Processing method



Ca, Cu, Fe, Mg, Zn

Beef, pork shank, chicken breast

(a) CW: 30 min (b) Baking in MW: 6 min at 650 W (c) Baking in conventional oven (B1): 45 min at 180°C (d) Baking in conventional oven (B2): 60 min at 180°C (e) GR: 10 min

Menezes, Oliveira, França, Souza, and Nogueira (2018)

Ca, Cu, Fe, K, Mg, Zn

Bovine liver

(a) CW: 20 min at 180°C (b) SV cooking under vacuum packaging: 120 min at 65°C

Dialyzability (%): Ca: Increase after CW (22−30), GR (15–19), MW (18–19), B1 (10−20) and B2 (10−13) vs. untreated (8–12) for all samples Cu: Increase in beef after CW (43), GR (25), MW (37), B1 (25) vs. untreated (19), unlike B2 (12). In chicken slight increase after CW (14) and MW (13), but decrease B2 (8) vs. untreated (12). In pork decrease after CW (21), GR (19), MW (19), B1 (11) and B2 (8) vs. untreated (23) Fe: Increase in beef after CW (25) and MW (28) vs. untreated (19). In chicken decrease slightly after MW (14), B1 (15) and B2 (8) vs. untreated (16). Increase in pork after CW (15), GR (16), MW (14), B1 (20) vs. untreated (12) except B2 (8) Mg: Increase after CW (17–27), GR (21–37), MW (26–29) and B1 (15–30) vs. untreated (8–25) for all sample, except B2 (7–15) Zn: Increase for beef and chicken after CW (15–16), GR (15–20), MW (13–16) and B1 (16–20) vs. untreated (11–15) unlike B2 (8–12). Increase in pork after CW (20), GR (15) and B2 (12), but decrease in MW (10) and B1 (10) vs. untreated (11) Solubility (%): Ca: Increase after CW (62) and SV (96) vs. untreated (40) Cu: Increase after CW (15) and SV (27) vs. untreated (9) Fe: Increase after CW (11) and SV (40) vs. untreated (9) K: Increase after CW (44) and SV (43) vs. untreated (30) Mg: Increase after CW (31) and SV (44) vs. untreated (26) Zn: Increase after SV (36), but decrease with CW (18) vs. untreated (25)

Da Silva et al. (2017)


Table 7.3  Continued Mineral


Processing method




Fullcream UHT milk Raw milk Pork meat

UHT (not specified conditions)

Human bioavailability (%): No effect vs. untreated

Talsma et al. (2017)

TT: 1 h at 60, 80, 100 and 120°C

Sorensen, Sorensen, Sondergaard, and Bukhave (2007)

Dried tuna

Microwave cooking: 4 min at 650 W

Dialyzability (%): – Gastric stage: No effect at 60°C but increase at 80, 100 and 120°C (~30, ~32 and ~60) vs. untreated samples (~16) – Gastrointestinal stage: No effect vs. untreated Solubility (%): Increase vs. untreated (~55 vs. 50)



CW, Cooking in water; MW, Microwave oven; GR, Grilling; SV, Sous vide cooking; TT, Thermal Treatment.

Cabañero, Madrid, and Cámara (2004)

Impact of processing on mineral bioaccessibility/bioavailability231

On considering other animal food matrixes, a 9.1% increase in Se solubility has been described for dried tuna cooked in a microwave oven versus untreated food. This positive effect can be ascribed to the fact that such hydrothermal processing may imply protein degradation that can improve protein digestibility and thus facilitate the release of bound Se, incrementing its bioaccessibility (Cabañero et al., 2004). Fullcream milk subjected to ultra-high temperature (UHT) processing showed no differences (25.5%) in fractional Zn absorption in young Dutch women compared to raw milk (27.8%), thus indicating a lack of effect of this thermal process on Zn bioavailability (Talsma et al., 2017).

7.4 Non-conventional thermal processing Common processing practices in the food industry mostly rely on conventional thermal technologies, which in spite of extending the shelf life of products (microbial safety) may have an undesirable impact on important food-quality attributes, such as the destruction of important nutrients, the development of off-flavors, and color changes. However, in recent years, more sophisticated and diverse processing techniques have been developed to meet consumer demands for fresh-like foods, with non-thermal processing techniques (e.g., high-pressure processing, high-intensity pulsed electric fields, ultrasound, and ultraviolet C radiation, among others) being useful tools for extending the duration and quality of products, preserving nutritional and functional characteristics, and even potentially increasing bioaccessibility/bioavailability (Cilla et al., 2017). The effect of processing on the bioaccessibility/bioavailability of minerals is dependent upon a number of factors, such as the type and conditions of processing, food matrix composition and structure, and the presence of compounds that affect absorption efficiency (Van Buggenhout et al., 2010). In the particular case of the effect of non-conventional processing upon mineral bioaccessibility/bioavailability, only studies dealing with high-pressure processing have been found in literature. As compared with classical and conventional thermal processing generally performed under atmospheric pressure, high-pressure processing can be considered a cold pasteurization technique in which products sealed in their final packaging are introduced in a vessel and subjected to a isostatic pressure (100–600 MPa) transmitted by water. The same applied pressure at a given position and time is exerted in all directions, transmitted uniformly and immediately through the pressure transferring medium, and independent of product size or geometry (Oey, Van der Plancken, Van Loey, & Hendrickx, 2008). Results on the effect of non-conventional processing attributed to high-pressure processing upon mineral bioaccessibility/bioavailability are shown in Table 7.4. On considering the effect of high-pressure processing (100, 300, and 500 MPa for 10 min at 18°C) on mineral (Ca, Cu, Fe, Mg, Mn, P, and Zn) bioaccessibility (solubility) in germinated brown rice, no effect on Mg, Mn, and Zn in processed versus untreated samples has been observed. In the case of P, a 14%–23% decrease in solubility versus untreated samples was generally recorded, attributed to the fact that h­ igh-pressure processing can release this mineral from phytate present in such s­ amples, but the released


Table 7.4  Effect of non-conventional processing on mineral bioaccessibility/bioavailability. Processing method


Mg, Mn, P

Germinated brown rice

HPP: 100, 300 and 500 MPa 0 min at 18°C

Ca, Cu, Fe, Zn

Germinated brown rice

HPP: 100, 300 and 500 MPa 0 min at 18°C

Ca, P

Milk-based (whole, skimmed and soya milk) fruit beverages

TT: 30s at 90°C HPP: 400 MPa 5 min at 40°C



Solubility (%): Mg: No effect vs. untreated Mn: No effect vs. untreated P: Decrease at 100 and 500 MPa vs. untreated (~47 and ~51 vs. ~58). No effect at 300 MPa Solubility (%): Ca: Increase at 500 MPa vs. untreated (~8 vs. ~7). No effect at 100 and 300 MPa vs. untreated Cu: Increase at 100 and 500 MPa vs. untreated (~17 and ~17.5 vs. ~16). No effect at 300 MPa vs. untreated Fe: Decrease at 100, 300 and 500 MPa vs. untreated (~7 all treatments vs. ~8.5) Zn: No effect vs. untreated Solubility (%): Ca: Decrease TT vs. untreated (89.8–93.5 vs. 84.4–99.0). HPP no effect vs. untreated P: Decrease TT vs. untreated (84.6–88.6 vs. 97.5–99.5). HPP no effect vs. untreated Uptake efficiency (%) Caco-2 cells: Ca: No effect of TT and HPP vs. untreated P: Decrease TT and increase HPP vs. untreated (15.7–18.9 and 38.1–72.5 vs. 26.7–31.1)

Xia et al. (2017)

Xia, Wang, Xu, Mei, and Li (2017)

Cilla et al. (2011)

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HPP: 500 MPa 2, 4, 8 and 10 min at 20°C

Ca, Fe, Zn

Algarrobo seeds

HPP: 500 MPa 2, 4, 8 and 10 min at 20°C

HPP, High Pressure Processing; TT, Thermal Treatment.

Solubility (%): Ca: Decrease vs. untreated (1.0–6.5 vs. 14.4) Fe: No effect, except 4 min increase vs. untreated (0.93 vs. 0.69) Zn: No effect, except 2 min increase vs. untreated (2.2 vs. 1.4) Dialysis (%): Ca: Decrease vs. untreated (4.5–15.6 vs. 79.0) Fe: No effect at 2 and 10 min; 4 min decrease and 8 min increase vs. untreated (0.38 and 1.1 vs. 0.67) Zn: Decrease at 2–4 min and increase at 8–10 min vs. untreated (0.37–0.43 and 6.38–4.29 vs. 1.12) Solubility (%): Ca: Increase vs. untreated (22.3–35.5 vs. 8.6) Fe: No effect, except 10 min increase vs. untreated (56.7 vs. 18.4) Zn: No effect, except 10 min increase vs. untreated (59.0 vs. 11.0) Dialysis (%): Ca: Decrease at 2–8 min vs. untreated (42.1–52.2 vs. 82.2). No effect at 10 min Fe: No effect at 4 and 8 min; increase at 2 and 10 min vs. untreated (77.4 and 71.9 vs. 49.3) Zn: Decrease at 2–8 min and increase at 10 min vs. untreated (12.5–27.6 and 71.1 vs. 66.4)

Briones-Labarca, VenegasCubillos, Ortiz-Portilla, Chacana-Ojeda, and Maureira (2011)

Briones-Labarca, Muñoz, and Maureira (2011)

Impact of processing on mineral bioaccessibility/bioavailability233

Ca, Fe, Zn


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P shows a strong capacity to bind to many divalent cations and proteins, forming insoluble complexes that reduce its bioaccessibility. Similarly, a 24% decrease in Fe solubility versus untreated samples has been described, probably due to the formation of complexes with phytates and/or P released from phytates. However, there an increase in solubility has been recorded for Cu (6%–9%) and Ca (12.5%) mainly at high processing pressures (500 MPa) versus untreated samples. This could be ascribed to a lesser propensity to form divalent cation or phytate complexes when the same food matrix contains other minerals, such as Fe, with a strong tendency to participate in linkages of this kind, thereby decreasing solubility (Xia, Tao, et al., 2017; Xia, Wang, et al., 2017). Regarding the effect of high-pressure processing (400 MPa for 5 min at 40°C) on Ca and P bioaccessibility/bioavailability in milk-based fruit beverages, it has been reported that samples treated with high-pressure processing show improved Ca and P solubility (6%–10% and 8%–15%, respectively) and P Caco-2 cell uptake efficiency (53%–74%) versus thermal treated samples (soya milk- and whole milk-based beverages being the samples with the highest in vitro bioavailability values). In this regard, high-pressure processing at >300 MPa can exert a disruptive effect on ionic and hydrophobic interactions of Ca phosphate with casein micelles, producing increments in soluble Ca and P levels. Therefore, and despite the fact that no differences between high-pressure processing and untreated samples were noted in some cases, it may be assumed for foodstuffs of this kind that high-pressure processing can positively modulate Ca and P bioaccessibility and P bioavailability in Caco-2 cells versus ­thermal-treated samples, and can be used as an alternative to traditional heat processing (pasteurization) in the manufacture of functional foods with improved nutritional value and health benefits (Cilla et al., 2011). Finally, studies on the effect of high-pressure processing (500 MPa for 2, 4, 8, and 10 min at 20°C) on Ca, Fe, and Zn bioaccessibility (solubility and dialysis) in two different vegetable matrixes such as apples and algarrobo seeds have yielded conflicting results. Regarding solubility, it was seen that Fe and Zn only presented increments versus untreated samples at low treatment times (2 min) for apples, but that high treatment times (10 min) were required for algarrobo seeds; this indicating the existence of food matrix-based differences. The rise in solubility was attributed to the changes induced by high-pressure processing upon the food matrix, such as the disruption of plant cell walls, with the release of minerals and making them more soluble. However, in the case of Ca, contrasting results have been reported, with reductions and increments for apples and algarrobo seeds, respectively. However, a general trend was observed in which Ca exhibited decreased dialyzability for both food matrixes at all treatment periods, and shorter treatment times (2–4 min) resulted in decreased Fe and Zn dialyzability. Nevertheless, when longer processing times were used, an increase in Fe and Zn dialyzability was recorded, attributed by the authors as possibly due to the presence of organic acids in the samples that favor the formation of soluble low-molecular weight complexes with these divalent cations, thereby increasing their dialyzability (Briones-Labarca, Muñoz, & Maureira, 2011; Briones-Labarca, Venegas-Cubillos, et al., 2011).

Impact of processing on mineral bioaccessibility/bioavailability235

7.5 Conclusions Household processes such as dehulling, mechanical processing, soaking, germination, and fermentation influence the bioaccessibility of minerals in vegetable foods, the effect being most pronounced with thermal processing. In general, these processes increase mineral bioaccessibility/bioavailability due to a decrease of phytate and/ or polyphenols and tannins, or an increase in organic acids, which form soluble ligands with the minerals thereby preventing the formation of insoluble complexes with phytate. Studies on the impact of conventional thermal processing upon mineral bioaccessibility/bioavailability in foods of vegetable origin have mainly focused on cereals and pulses cooked by boiling (traditional, microwave, or under pressure), and have yielded different results depending on the sample involved and the thermal treatment used. In the case of foods of animal origin, it seems that in general shorter times and lower temperatures positively influence mineral bioaccessibility, probably due to enhanced accessibility of proteolytic enzymes in denatured proteins that can release bound minerals, thereby making them more soluble/dialyzable. Nevertheless, baking at high temperatures and for long times is associated with lower mineral bioaccessibility, mainly due to the formation of Maillard reaction products that tightly bind minerals. Thus the sous vide cooking method could be selected as the preferred technique to improve mineral bioaccessibility. With regard to non-conventional processing (high pressure), conflicting results have been reported in terms of mineral bioaccessibility, depending on the mineral, food matrix, and time and pressure conditions considered. Therefore no general guidelines can be offered for improving mineral bioaccessibility for this processing technique. Nevertheless, since high-pressure processing affords better mineral bioaccessibility and cell uptake efficiency compared with pasteurization, it can be considered an alternative to thermal treatment for improving the nutritional value of foods. As a general conclusion, more studies are needed in order to select suitable processing conditions according to the food matrix composition and structure involved, with a view to securing maximum mineral bioaccessibility and therefore optimum nutritional efficacy.

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Nicklas, T. A. (2003). Review: Calcium intake trends and health consequences from childhood through adulthood. Journal of the American College of Nutrition, 22, 340–356. Nkundabombi, M. G., Nakimbugwe, D., & Muyonga, J. H. (2015). Effect of processing methods on nutritional, sensory, and physicochemical characteristics of biofortified bean flour. Food Science & Nutrition, 4, 384–397. Oey, I., Van der Plancken, I., Van Loey, A., & Hendrickx, M. (2008). Does high pressure processing influence nutritional aspects of plant based food systems? Trends in Food Science and Technology, 19, 300–308. Pereira, E. J., Carvalho, L. M. J., Dellamora-Ortiz, G. M., Cardoso, F. S. N., & Carvalho, J. L. V. (2016). Effect of different home-cooking methods on the bioaccessibility of zinc and iron in conventionally bred cowpea (Vigna unguiculata L.Walp) consumed in Brazil. Food & Nutrition Research, 60, 29082. Raghuvanshi, R. S., Singh, A., Bisht, K., & Singh, D. P. (2011). Processing of mungbean products and its nutritional and organoleptic evaluation. International Journal of Food Science and Technology, 46, 1378–1387. Ramírez-Moreno, E., Díez Marqués, C., Sánchez-Mata, M. C., & Goñi, I. (2011). In  vitro calcium bioaccessibility in raw and cooked cladodes of prickly pear cactus (Opuntia ­ficus-indica L. Miller). Food Science and Technology, 44, 1611–1615. Repo-Carrasco-Valencia, R. A. M., Encina, C. R., Binaghi, M. J., Greco, C. B., & Ronayne de Ferrer, P. A. (2010). Effects of roasting and boiling of quinoa, kiwicha and kañiwa on composition and availability of minerals in vitro. Journal of the Science of Food and Agriculture, 90, 2068–2073. Salgueiro, M. J., Zubillaga, M., Lysionek, A., Sarabia, M. I., Caro, R., De paoli, T., et al. (2000). Zinc as an essential micronutrient: A review. Nutrition Research, 20, 737–755. Santos, W. P. C., Ribeiro, N. M., Santos, D.C.M.B., Korn, M. G. A., & Lopes, M. V. (2018). Bioaccessibility assessment of toxic and essential elements in produced pulses, Bahia, Brazil. Food Chemistry, 240, 112–122. Sebastiá, V., Barberá, R., Farré, R., & Lagarda, M. J. (2001). Effects of legume processing on calcium, iron and zinc contents and dyalizabilities. Journal of the Science of Food and Agriculture, 81, 1180–1185. Sorensen, A. D., Sorensen, H., Sondergaard, I., & Bukhave, K. (2007). Non-haem iron availability from pork meat: Impact of heat treatments and meat protein dose. Meat Science, 76, 29–37. Talsma, E. F., Moretti, D., Chheng Ly, S., Dekkers, R., Van den Heuvel, E.G.H.M., Fitri, A., et al. (2017). Zinc absorption from milk is affected by dilution but not by thermal processing, and milk enhances absorption of zinc from high-phytate rice in young Dutch women. Journal of Nutrition, 147, 1086–1093. Thiry, C., Ruttens, A., De Temmerman, L., Schneider, Y. J., & Pussemier, L. (2012). Current knowledge in species-related bioavailability of selenium in food. Food Chemistry, 130, 767–784. Van Buggenhout, S., Alminger, M., Lemmens, L., Colle, I., Knockaert, G., Moetlants, K., et al. (2010). In vitro approaches to estimate the effect of food processing on carotenoid bioavailability need through understanding of process induced microstructural changes. Trends in Food Science and Technology, 21, 607–618. Viadel, B., Barberá, R., & Farré, R. (2006a). Effect of cooking and legume species upon calcium, iron and zinc uptake by Caco-2 cells. Journal of Trace Elements in Medicine and Biology, 20, 115–120.

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Viadel, B., Barberá, R., & Farré, R. (2006b). Calcium, iron and zinc uptakes by Caco-2 cells from white beans and effect of cooking. International Journal of Food Sciences and Nutrition, 57, 190–197. Viadel, B., Barberá, R., & Farré, R. (2006c). Uptake and retention of calcium, iron, and zinc from raw legumes and the effect of cooking on lentils in Caco-2 cells. Nutrition Research, 26, 591–596. Xia, Q., Tao, H., Huang, P., Wang, L., Mei, J., & Li, Y. (2017). Minerals in vitro bioaccessibility and changes in textural and structural characteristics of uncooked pre-germinated brown rice influenced by ultra-high pressure. Food Control, 71, 336–345. Xia, Q., Wang, L., Xu, C., Mei, J., & Li, Y. (2017). Effects of germination and high hydrostatic pressure processing on mineral elements, amino acids and antioxidants in vitro bioaccessibility, as well as starch digestibility in brown rice (Oryza sativa L.). Food Chemistry, 214, 533–542. Yeung, C. K., Miller, D. D., Cheng, Z., & Glahn, R. P. (2005). Bioavailability of elemental iron powders in bread assessed with an in vitro digestion/Caco-2 cell culture model. Journal of Food Science, 70, 199–203. Zaccari, F., Cabrera, M. C., Ramos, A., & Saadoun, A. (2015). In  vitro bioaccessibility of β-carotene, Ca, Mg and Zn in landrace carrots (Daucus carota, L.). Food Chemistry, 166, 365–371.

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Water-soluble vitamins☆


Amin Mousavi Khaneghah⁎, Seyed Mohammad Bagher Hashemi†, Ismail Es‡, Aliakbar Gholamhosseinpour§, Monica Rosa Loizzo¶, Alessandra Giardinieri‖, Deborah Pacetti‖, Kiana Pourmohammadi†, Daniela S. Ferreira# ⁎ Department of Food Science, School of Food Engineering, University of Campinas, Campinas, Brazil, †Department of Food Science and Technology, College of Agriculture, Fasa University, Fasa, Iran, ‡Department of Material and Bioprocess Engineering, School of Chemical Engineering, University of Campinas (UNICAMP), Campinas, Brazil, §Department of Food Science and Technology, Faculty of Agriculture, Jahrom University, Jahrom, Iran, ¶Department of Pharmacy, Health and Nutritional Sciences, University of Calabria, Rende, Italy, ‖Department of Agricultural, Food, and Environmental Sciences, Polytechnic University of Marche, Ancona, Italy, #Department of Food Technology, School of Food Engineering, University of Campinas, Campinas, Brazil

8.1 Introduction Water-soluble vitamins can be simply defined as the vitamin group that dissolves in water (Table 8.1). These vitamins show high solubility in the polar environment of the intestinal lumen due to one or more polar groups present in their structure and therefore are absorbed by the surface of the gut. Due to the physiological conditions, the human body is not capable of storing this vitamin group for a longer time and therefore it must be continuously supplied via diet. Consequently, properties such as bioavailability and bioaccessibility become more critical due to the lack of storing ability of the body as well as biosynthesis of vitamins by mammalian cells. Every consumed nutrient passes through common biochemical processes such as absorption, retention, and other metabolic pathways to be used for physiological functions. Most of the time passive diffusion is the main process to provoke absorption of water-soluble vitamins, while in some cases specific carriers play a role in the transport of this vitamin group when concentration gradient, as driving force, is not sufficient to absorb them (Said, 2011). In this context, the dosage of consumed vitamins can be considered a critical factor since it may be related to the absorption process. Therefore the bioavailability of water-soluble vitamins should be well studied. Vitamin bioavailability is associated with the portion of the vitamins utilized in the total quantity of vitamins present in taken nutrients. In most cases, determination of bioavailability is difficult since it depends on several factors. One of the principal factors is the significant losses of vitamins during storage of foods, processing, or cooking (Plaza et al., 2006). Moreover, the intestinal transit time of vitamins can ☆

This chapter describes comprehensively how processing (conventional and non-conventional) is affecting the bioavailability and bioaccessibility of water-soluble vitamins.

Innovative Thermal and Non-Thermal Processing, Bioaccessibility and Bioavailability of Nutrients and Bioactive Compounds © 2019 Elsevier Inc. All rights reserved.

Table 8.1  List of water-soluble vitamins. Diseases associated with absence or low intake

Recommended dietary allowance (RDA)


Food source



Thiamin B1

Bread, breakfast cereals, flour, milk, cheeses, eggs, cooked vegetables, dried yeast

Coenzyme TDP (Thiamin pyrophosphate and cocarboxylase) functions in carbohydrate metabolism and branched-chain amino acids

Caballero (2009)

Yeast, kidney, liver, cheese, eggs, milk, green vegetables, natural grains

1.1 mg/day for women, 1.3 mg/ day for men

Powers (2003)

Niacin B3

Milk, eggs, enriched bread and cereals, rice, fish

Biologically active forms participate in redox reactions Coenzyme in energy metabolism Protection against cancer and cardiovascular disease Favorable changes in the level of three major lipoproteins

Leigh’s disease, Alzheimer’s disease, Huntington’s disease, Wernicke-Korsakoff syndrome, pyruvate dehydrogenase deficiency sore throat, cheilosis, angular stomatitis, glossitis, seborrheic dermatitis

0.4 mg/4.2 MJ (daily intake depends on physical activity)

Riboflavin B2

headache, irritability, insomnia, rapid pulse, weakness, and pruritus for intakes >50 mg/kg or 3 g/day The oral administration does not show toxic manifestations

Digestive problems, inflamed skin, mental impairment

Liver, kidney, fish, milk, yogurt, avocados, broccoli

14 mg/ day; 18 mg/ day during pregnancy 4–7 mg/day

NIH (2017)

Pantothenic Acid B5

Increased blood sugar, liver damage, peptic ulcers –

Biosynthesis of coenzyme A

headache, fatigue, sensation of weakness, sleep disturbance


Tahiliani and Beinlich (1991)

Pyridoxine B6

Chickpeas, tuna, salmon, chicken breast, banana, beef liver

Coenzyme in protein metabolism Biosynthesis of neurotransmitters Maintaining normal levels of homocysteine Involved in immune function and hemoglobin formation

Biotin B7

Eggs, salmon, pork chop, sunflower seeds, tuna, spinach, almonds Liver, spinach, breakfast cereals, rice, asparagus, lettuce, avocado

Cofactor for five carboxylases Plays a role in histone modifications, gene regulations, and cell signaling

Folate B9

Cobalamin B12 Ascorbic Acid C

Animal foods, meat, milk, eggs, fish, shellfish Citrus fruits, tomatoes, red and green peppers, kiwifruit, broccoli, strawberries

Coenzyme in single‑carbon transfer reactions in nucleic and amino acid metabolism Folate is required for the synthesis of methionine and S-adenosylmethionine Cofactor in the synthesis of methionine; an intermediate of the citric acid cycle Biosynthesis of collagen, l-carnitine, and certain neurotransmitters

Chronic administration of 1–6 g oral pyridoxine per day for 12–40 months can cause severe sensory neuropathy characterized by ataxia No toxicity found in humans

Oral intake of folate shows neurological manifestations in patients with pernicious anemia No toxicity

Diarrhea, nausea, abdominal cramps, gastrointestinal disturbances

Microcytic anemia, electro­ encephalographic abnormalities, dermatitis with cheilosis, and weakened immune function

1.3 mg/day between 10 and 50 years, 1.7 mg for men, 1.5 mg for women older than 50 years

NIH (2017)

Thinning hair or hair loss, conjunctivitis, aciduria, seizures, depression, hallucinations Folate deficiency is related to depression and plays a role in cognitive function

30 μg/day

NIH (2017)

400 μg/day

Bottiglieri (2013)

Depression, dementia, confusion, poor memory Scurvy, a widespread connective tissue weakness, and capillary fragility

2.4 μg/day

90 mg/day for men, 75 mg/day for women; 85 mg/ day during pregnancy, 120 mg/day during lactation

NIH (2017)


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d­ ramatically change depending on the composition of the nutrients, which is part of dietary effects. Another important factor is physiological conditions of the human body. In elderly people, the processing time and efficiency of metabolic activities are mostly slower than in younger people. This age-related difference results in the reduction of the absorption of vitamins (Fabian, Bogner, Kickinger, Wagner, & Elmadfa, 2012). Besides the effect of age, the health status of people also affects the absorption of vitamins. Some illnesses can alter the metabolic pathways that use vitamins more efficiently. Since vitamins show essential functions as a cofactor in enzymes, their transport in cells depends on the performance of transport proteins. In some individuals, these proteins might present deficiency, and this situation directly influences the absorption of these vitamins. Since each vitamin shows specific biochemical characteristics and different mechanism of action in the body, each type of vitamin should be discussed separately regarding bioavailability, bioaccessibility, and consequent outcomes of their consumption regarding human health. Food processing is capable of modifying the bioavailability of vitamins in the relation of its natural form in food. However, studies about the influence of specific processing on vitamin content are very important for the food industry and consumers. Most of the water-soluble vitamins are liable to high temperature. Thiamin can be reduced by the Maillard reaction, because of its free amino groups, and its activity can be reduced by alkaline treatment. The same occurs with folacin, which has a free amino group. Conversely, niacin or nicotinic acid is less affected by heat, light, oxygen, or moisture. Biotin activity is also less affected by heat, but oxidative rancidity reduces biotin bioavailability. Vitamin B6 has its bioavailability reduced after canning, sterilizing, and freezing, as well as in the presence of minerals in the form of carbonates or oxides (Baker, 1995). Compared to other water-soluble vitamins, a high concentration of vitamin C or ascorbic acid is present in foods. However, consumption with a high content of pectin, zinc, iron, or copper can decrease its absorption. The antioxidant activity of vitamin C is used as an additive to prevent oxidation of the other vitamins (Baker, 1995).

8.2 Definition and principles of bioavailability and bioaccessibility Bioavailability is the proportion of ingested and absorbed vitamins that will be used for metabolic activities; the vitamins are converted to a functional form. In general, the term bioavailability refers to the analytical response of a reference compound with high bioavailability. The composition of food affects its absorption. Some components can retard or enhance vitamin bioavailability, for example, B-group vitamins are associated with protein apoenzymes, and these complexes present lower absorption than free vitamins, like supplements (Ball, 2005). Today consumers are interested in the health benefits provided by food-derived compounds. In order to be active, these compounds should release from the food

Water-soluble vitamins


Table 8.2  Vitamin bioavailability according to bioaccessibility, absorption, and transformation characteristics. Bioaccessibility

Absorption Transformation

Liberation from the food matrix Solubilization in intestinal fluids Interaction and insoluble complex formation Active transporters Chemical degradation Metabolism

­ atrix, be absorbed into the bloodstream, and then transported to their respective tarm get tissues. Nevertheless, not all ingested compounds can be utilized to the same extent since their bioavailability is different. Bioavailability can be defined as the amount of a nutrient that is available for gut absorption. It includes gastrointestinal digestion, absorption of a nutrient into the systematic circulation, metabolism utilization/storage in the body, tissue distribution, and excretion from the body via renal and biliary routes (Table 8.2). All these steps must take place through normal pathways (Aggett, 2010). So, from a different point of view, bioavailability is the amount of nutrient that exerts physiological functions (Gibson, 2007). The bioavailability of food-derived products is influenced by internal and external factors. External factors include the release of the compounds from food, the chemical form of nutrients, the ability of these compounds to pass between or enter into gut cells by using transporters, metabolizing enzymes, intestinal transit time, and gastric emptying and/or the co-ingestion with compounds, which can increase or decrease solubility and absorption (e.g., iron). The relief of compounds from food depends on different factors, such as whether the foods are consumed fresh or after processing (both mechanical and thermal). The release of bioactive compounds from the food matrix occurs only when the plant cell wall is broken during food preparation, processing, and/or mastication. Moreover, some bioactive molecules are necessary for the active transporter to cross the epithelium cells (Wilson, 2005). Vitamins are absorbed in high amount when their concentration in the non-thermal tract is low due to these active transporter mechanisms. The fraction absorbed may decrease if the transporter becomes saturated. Age, gender, nutrient status, and health condition, such us genotype, pregnancy, lactation, or diseases, are considered internal factors (Hambidge, 2010). Among gastrointestinal disorders, those that influence the functional status of the gastrointestinal tract (production of digestive enzymes and bile) or those associated with liver and kidney are the most important ones. For this reason, bioavailability is characterized by high inter-individual variability (Rodriguez-Mateos et al., 2014). Bioavailability is expressed as a percentage of intakes. In order to become bioavailable, vitamins must be released from the food and subjected to the digestion process. As mentioned, it is crucial to analyze the effect of the digestion process on the stability of nutrients since digestion can modify their healthy properties. Regarding bioaccessibility, researchers define it as the amount of nutrient available for gastrointestinal absorption/assimilation into the intestinal tract as well as the pre-systemic, hepatic, and intestinal metabolism. In order to improve the bioavailability of vitamins, several


Innovative Thermal and Non-Thermal Processing

technologies have been developed, including nanotechnology, structural modification, and colloidal systems. Due to the complex nature of food bioactive constituents and the different mechanisms of their absorption, distribution, and excretion, the knowledge regarding bioavailability helps to optimize diet (Aggett, 2010). Aspect regarding the food fortification with vitamins. Ohrvik and Witthoft (2011) evidenced that folate bioavailability derived from vegetables, fruits, and breakfast cereal products is limited to 20%–70%, so they recommend using synthetic folic acid. In another human study, Jesse III (1993) showed that vitamin C derived from orange products or cooked broccoli is equally bioavailable. However, a controversial interference with vitamin C bioavailability was reported when both vitamin C and flavonoids are present in the food matrix. In fact, Jones and Hughes (1984) showed an increased level of vitamin C bioavailability after consumption of flavonoids-rich juice, whereas Uchida et  al. (2011) showed a significant reduction in urinary excretion of vitamin C in the presence of flavonoids. Bioactive food compounds from a different source need to be bioavailable in order to exert their potential health effects. Molecular structures, food matrix release, transporters, and metabolizing enzymes are factors able to influence bioavailability. Enhanced bioavailability improves the bioefficacy of healthy compounds. Even though there are several types of research on the bioavailability and bioaccessibility of vitamins, the literature appears controversial (Loizzo, 2017). For this reason, further studies are necessary to improve the knowledge on this topic.

8.3 Processing parameters affecting the water-soluble vitamins The hydro-soluble vitamin group is composed of vitamin C (ascorbic acid) and vitamin B complex (thiamine, B1; riboflavin, B2; niacin or nicotinamide, B3; pantothenic acid, B5; pyridoxine, together with pyridoxal and pyridoxamine, B6; biotin, B7; folate, B9; and cobalmin, B12). Their stability in food depends on chemical properties of the vitamin, the food matrix, and external factors (temperature, light, oxygen) related to postharvest handling procedures, processing technologies, and storage conditions of foods. The type of foods matrix plays a key role in vitamin preservation. In fact, vitamins within the matrix come into contact with many other molecular species, so some of these interactions contribute to vitamin stability. For example, a good ascorbic acid (AA) retention was found in high total sulfur and glutathione vegetables. It was supposed that glutathione reduced dehydroascorbic acid to ascorbic acid in crucifers (Albrecht, Schafer, & Zottola, 1990). High levels of monosaccharide sugar in food influenced vitamin C content negatively. It was found that there is a loss of vitamin C in orange juice products due to the chemical interaction between the fructose and vitamin C (Nagy, 1980). Otherwise, high levels of lipid content in food have been shown to enhance riboflavin stability. The riboflavin depletion was higher in skim milk than in whole milk (Gaylord, Warthesen, & Smith, 1986).

Water-soluble vitamins


Hydroxyl, citric, and malic acids can also stabilize B vitamins since they chelate pro-oxidant metals and increase juice acidity (Nagy, 1980). Besides influencing other compounds, vitamin stability is greatly affected by the pH of the food matrix. Due to ascorbic acid’s high stability under acidic conditions, the ascorbic acid lost is greater in low-acid foods (e.g., melon flesh) compared to high-acid foods (e.g., citrus fruits). Melon flesh showed a rapid reduction of ascorbic acid within two days of storage (Kalt, Forney, Martin, & Prior, 1999). Thiamin, biotin, and riboflavin are also preserved in food with low pH values, such as honey (da Silva, Gauche, Gonzaga, Costa, & Fett, 2016). For example, alkaline pH led to the transformation of riboflavin into lumiflavin, which promotes the decomposition of vitamin C in milk (Henry & Chapman, 2002). Additionally, to the intrinsic characteristics of the food matrix, stability of water soluble-vitamins is strongly linked to food processing and storage conditions. Hydro-soluble vitamins are prone to degradation in solutions, particularly when exposed to light. Among them, vitamin C is the most affected by intrinsic and extrinsic factors (Cruz, Vieira, & Silva, 2008). Niacin content in food is not influenced by heat, light, oxygen, or pH. Cobalamin and biotin are not sensitive to air, to the neutral and acid environment, or to heat treatment. Moreover, cobalamin is decomposed by oxidizing or reducing agents and interacts adversely with thiamin, ascorbic acid, and niacin. Pantothenic acid is not air sensitive, but it is strongly affected by dry-heating conditions. During cheese manufacture, large losses can be revealed. Also during cheese ripening, the pantothenic acid can be synthesized by microorganisms. The rate of destruction of vitamin C is enhanced by prolonged heating, exposure to light and oxygen, and by the action of metals (especially copper and iron) and enzymes (ascorbic acid oxidase, phenolase, cytochrome oxidase, and peroxidase). Heat treatments lead to thermal degradation and oxidation of vitamin C. (Lucci, Pacetti, Loizzo, & Frega, 2016). Huge losses of vitamin C were found in canned peas (Lathrop & Leung, 1980). The addition of natural antioxidants, such as delactosed whey permeate, inhibited vitamin C’s thermal degradation during canning of Irish plum tomatoes. It was assessed that delactosed whey permeate was able to inhibit vitamin C oxidation by forming a protective layer on the vegetable surface (Ahmed, Patras, Martin-Diana, Rico, & Barry-Ryan, 2012). Besides thermal oxidation, ascorbic acid can be oxidized by the action of pro-­ oxidant agents during storage. Higher concentrations of oxygen in packaging are associated with increased vitamin C loss in fruit (Del-Valle, Hernández-Muñoz, Catalá, & Gavara, 2009). This loss in vitamin C is attributed to degradation promoted by oxidative processes mediated by high O2 and CO2 levels. Plastic films equipped with selective permeability to O2 and CO2 have been shown to be effective in cherries and raspberries (Mangaraj, Goswami, & Mahajan, 2009; Mditshwa, Magwaza, Tesfay, & Opara, 2017). Oxidation of ascorbate seems to be also due to mechanical stress. A remarkable vitamin C loss was revealed in cut or shredded vegetables (i.e., salad mixes, cabbage, carrots) (Dewhirst, Clarkson, Rothwell, & Fry, 2017). Unlike vitamin C, folate losses in industrial processing are mainly linked to leaching rather than to heat degradation and oxidation. Blanching treatment does not affect


Innovative Thermal and Non-Thermal Processing

the folate content in green beans and spinach. Otherwise, in the freezing process of spinach, the washing step decreased the amount of folates significantly. In canned green beans, 20% of the initial folate amount was transferred into the covering liquid. The industrial canning process, consisting of soaking, blanching, and retorting treatments, did not alter folate level in fava beans, but led to folate decrease in chickpeas (Delchier, Reich, & Renard, 2012; Delchier et al., 2013). Concerning riboflavin, heating processes weakly affect the riboflavin content in foods. During roasting of pork meat, riboflavin was weakly affected (Lassen, Kall, Hansen, & Ovesen, 2002). Otherwise, riboflavin is very sensitive to light, and its photo-oxidation produces riboflavin radicals and reactive oxygen species, which accelerate the oxidation of food components, especially of ascorbic acid. Milk lost one-third of its riboflavin to sunlight exposure but one-tenth after boiling for 30 min. The extent of riboflavin decline under light exposure changed according to the light intensity, wavelength, and packaging materials. The most destructive wavelength resulted at 450 nm. The rate of riboflavin loss was higher in milk in a clear bottle or white sachet than in milk packed in a brown bottle or carton (Choe, Huang, & Min, 2005). Unlike riboflavin, thiamin is not light sensitive. Heat and airflow treatments may destroy thiamin. The extent of thermal degradation depends on the commodity. Losses may range from 25% in asparagus to 66% in spinach (Rickman, Barrett, & Bruhn, 2007). Red and white rye malt show a reduced content of thiamine compared to that of grain, caused by air flow as well as high-temperature kilning (Dewettinck et al., 2008). In fermented foods, thiamin level is also affected by the fermentation conditions. Prolonged fermentation time resulted in thiamine increase. The increase was significantly higher in white bread with yeast than with sourdough. The lactic acid fermentations occurring in sourdough bread deplete thiamine levels (Batifoulier, Verny, Chanliaud, Remesy, & Demigne, 2005). Finally, no significant losses of pyridoxine (B6) have been reported after heat treatment of milk. High B6 retention was underlined during the canning process of mushrooms, cherries, and lentils. Differently, the pyridoxine losses are mainly related to the storage conditions. Pyridoxine is highly oxygen sensitive. Flour contained higher amounts of pyridoxine than the respective dough after kneading (Dewettinck et al., 2008). Similarly, whole wheat flour had higher pyridoxine levels than white flour (Batifoulier et  al., 2005). The storage B6 losses observed in milk have been attributed to conversion of pyridoxal to pyridoxamine and then to bis-4-pyridoxal disulfide.

8.4 The different effects of processing Vitamins are active organic biocompounds with high sensitivity against numerous factors such as heat, light, some specific components present in foods, storage, and most importantly food processing. Moreover, each water-soluble vitamin has a unique molecular mechanism, and this significantly affects the degree of degradation together with the processing and storage time in which the vegetable is being processed.

Water-soluble vitamins


Considering all these factors that may increase the degree of degradation, monitoring activities of vitamins during processing carries great importance for the food industry as well as consumers.

8.4.1 Thermal processing For the production of certain products, heat treatment is essential in order to inhibit the growth of a pathogenic microorganism or inactivate some enzymes. During the cooking process, vegetables or other food products are exposed to high temperatures for a certain time (Cruz, Vieira, Fonseca, & Silva, 2011). Therefore thermal-processing conditions should be optimized to avoid significant losses in the water-soluble vitamin content of food products. Blanching Blanching is an essential pre-treatment step applied before processing of vitamin-­ containing foods in order to inactivate some enzymes that might eliminate certain favorable properties of foods (Xiao, Bai, Sun, & Gao, 2014). However, conventional blanching methods cause more loss of water-soluble vitamins. Therefore novel, advanced techniques should be employed to avoid this problem. In the blanching processes conditions should be precisely determined for the raw material so that vitamins are maintained at acceptable levels while over-processing is avoided (Abboudi, AlBachir, Koudsi, & Jouhara, 2016). In most of the industrial processes there is a high demand for food products that pass through less processing steps. However, minimally processed foods generally do not show long shelf life. In this context, blanching seems to be an advantageous thermal treatment for food products that require minimal processing steps. Moreover, blanching also prevents possible discoloration of foods and makes them more attractive for consumption. Murcia, López-Ayerra, Martinez-Tomé, Vera, and García-Carmona (2000) showed that applying blanching for 60 s at 92– 96°C could allow 50% retention of vitamin C in broccoli. In a similar study, Agüero, Ansorena, Roura, and Del Valle (2008) analyzed the decrease in ascorbic acid content in butternut squash during blanching. They showed that higher temperatures during shorter time periods resulted in more vitamin retention. With this study, they maintained 50% of ascorbic acid by applying blanching for 30 s at 80°C. In vegetable systems, peroxidase (POD) is considered the most thermally resistant enzyme. Moreover, it is one of the principal enzymes that may catalyze undesirable reactions for the food industry, such as reactions that lead to browning; therefore, inactivation of POD can be used as a reference to determine important parameters of the blanching process (Pellicer & Gómez-López, 2017). Based on this information, it could be possible to optimize the blanching process for each food product. Pasteurization and sterilization According to the US Department of Agriculture (USDA), the thermal pasteurization process is defined as any process or treatment that is employed to food products in order to eliminate or reduce the microorganisms that may show significant risk to public health.


Innovative Thermal and Non-Thermal Processing

However, within this definition non-thermal pasteurization processes are also included (Maria, Gibbs, Nunez, Almonacid, & Simpson, 2014). In this chapter, only thermal pasteurization processes are discussed. However, thermal pasteurization is used to degrade vegetative cells of pathogenic microorganisms with potential health risks to extend the shelf life of foods and promote their safety. By starting to use mandatory pasteurization and sterilizations, outbreaks associated with milk-borne diseases have decreased significantly across the world. Pasteurization is usually used for dairy products, more specifically milk since raw milk harbors a great variety of microbial pathogens that cause foodborne illness. The pasteurization process in milk is very critical since milk is rich with critical water-­ soluble vitamins such as thiamin, riboflavin, and B12 as well as niacin, pantothenic acid, B6, and ascorbic acid (Lucey, 2015). Although heat pasteurization affects vitamin content in milk, the decrease is not significant to cause vitamin-dependent deficiency (Macdonald et al., 2011). A systematic meta-analysis of the effect of different pasteurization processes on vitamin change was conducted by Macdonald et al. (2011). According to numerous literature results, all water-soluble vitamins showed a significant decrease in the application of the pasteurization process with different conditions. Depending on the temperature applied, the alteration in the concentration of vitamins was insignificant compared to the control group, which was raw milk. Due to the concerns related to foodborne diseases, the pasteurization process seems to be a more efficient method for industrial milk. However, new improvements in pasteurization processes are required to minimize essential vitamin loss. Recently, Ward, Kerth, and Pillai (2017) developed a new electron beam pasteurization technique for raw milk. Even B2 vitamin decreased by 31.6% after the process, quality of raw milk maintained all the recommended criteria by USDA nutritional guidelines. The pasteurization process is also applied for fruits rich in water-soluble vitamins. Cerrillo et al. (2015) used fermentation, and subsequent pasteurization of orange juice, and found that the ascorbic acid level was decreased to half of its original value. Thermal drying Some of the fruits and vegetables that grow in tropical countries contain a high amount of water in their structures due to some seasonal problems. This characteristic makes them more attractive for microbial or chemical decompositions. In order to avoid this problem, the water content should be reduced by applying a thermal drying technique. This reduction usually occurs by vaporization or sublimation. Moreover, an additional dehydration step is employed to reduce overall weight and volume of food products to facilitate transport and decrease storage costs. However, especially during the mandatory drying process, there is a loss of vitamin content in food. During thermal drying, temperature and air velocity used to dry the samples carry great importance regarding the kinetics of vitamin loss. Most of the time lower air temperatures are recommended in order to dry food products with higher vitamin retention. Moreover, the degradation rate of vitamins during the thermal drying process is strongly associated with the difference between air-drying temperature and the glass transition temperature, at which the glass-rubber transition occurs, of the processed product. Kurozawa, Terng, Hubinger, and Park (2014) evaluated the degradation of

Water-soluble vitamins


vitamin C in papaya during the thermal drying process. When they used lower temperatures (40°C and 50°C), they could retain 50% of vitamin C, however, as the temperature was increased to 70°C, the degradation of vitamin C significantly increased. They also observed that degradation of vitamin C was greater when the difference between air-drying temperature and glass transition temperature was bigger. Most of the time employing thermal drying alone does not result in higher retention of water-soluble vitamins in food. Applying different techniques such as simple osmotic dehydration or pulsed vacuum osmotic dehydration together with thermal drying can enhance retention time and amount of vitamins. An et al. (2013) analyzed the retention amount of ascorbic acid in cherry tomatoes pre-treated with thermal drying. When they employed only thermal drying to tomatoes, the retention rate of ascorbic acid was 24.79%, however, when they applied thermal drying together with osmotic dehydration, the retention rate increased up to 56% without showing a negative significant effect on the quality of the product.

8.4.2 Non-thermal processing Dense-phase carbon dioxide (DPCD) Dense-phase carbon dioxide (DPCD), as a non-thermal method of processing, is used to pasteurize mostly liquid foods. Mild temperature ranges (30–50°C), removal of oxygen, and short process times of ≈ 5 min (in continuous systems) allow retention of beneficial components like ascorbic acid (Balaban & Duong, 2014; Balaban & Ferrentino, 2012). Vitamin C is the main vitamin found in fruit juices. Scientific studies have shown that the vitamin C is largely preserved after DPCD treatment in foods. Ferrentino, Plaza, Ramirez-Rodrigues, Ferrari, and Balaban (2009) processed red grapefruit juice by continuous DPCD equipment at pressures of 13.8, 24.1, and 34.5 MPa for 5, 7, and 9 min at 40°C and CO2 level of 5.7% and then stored it for 6 weeks at 4°C. They did not observe considerable differences between ascorbic acid contents of DPCD-treated and untreated samples and attributed these differences to the variability of the samples. The effect of high temperature short time (HTST; 90°C, 60 s) and DPCD (55°C, 60 min, and 35 MPa) pasteurization on physicochemical properties and flavor compounds of Hami melon juice were studied by Chen et al. (2009). The results showed that DPCD reduced only 13.3% of ascorbic acid compared to HTST, which caused a 51% reduction. Dissolving CO2 in the aqueous phase of foods results in a decrease in pH. The higher stability of ascorbic acid at low pH and replacing of air with CO2 have been suggested as the most important reasons for lower loss of ascorbic acid in DPCD treatment (Balaban & Duong, 2014). DPCD processing (34.5 MPa, 6.9 min, 8% CO2, 35°C) of guava (Psidium guajava L.) puree was reported to have minimal effect on the degradation of vitamin C during storage (4°C for 14 weeks) (Plaza, 2010). Valverde, Marín-Iniesta, and Calvo (2010) processed fresh-cut pears in a continuous DPCD system (6–30 MPa, 25–55°C, 10–90 min) and stated that the decrease in vitamin C was one of the reasons for the darkening of treated pears.


Innovative Thermal and Non-Thermal Processing Pulsed electric field (PEF) Many factors like pH, light, oxygen, and processing temperature and time affect the degradation of vitamin C. In PEF processing, the degradation of vitamin C is significantly influenced by the time and electric field strength (Odriozola-Serrano, SolivaFortuny, Gimeno-Añó, & Martín-Belloso, 2008). The influence of PEF treatment on the amount of vitamin C has been reported widely in the literature. Some scientific publications exhibit high vitamin C retention after PEF processing in orange juice (Sánchez-Moreno et al., 2004), grape juice (Wu, Mittal, & Griffiths, 2005), tomato juice (Nguyen & Mittal, 2007), and strawberry juice (Odriozola-Serrano, SolivaFortuny, & Martín-Belloso, 2009), while in others the content of vitamin C has been decreased after PEF processing (Bi et  al., 2013; Sánchez-Vega, Elez-Martínez, & Martín-Belloso, 2015). Under the same PEF conditions, the differences in vitamin C retention among PEF-treated juices may be related to their different pH, because low-pH conditions are known to stabilize vitamin C (Morales-de La Peña, SalviaTrujillo, Rojas-Graü, & Martín-Belloso, 2010; Oms-Oliu et al., 2009). The effect of PEF with the effect of thermal processing on vitamin C stability in different juices was compared by several scientists. Accordingly, vitamin C levels in PEF-treated juices appeared to be more highly degraded in comparison to thermal-treated ones (Bansal, Sharma, Ghanshyam, Singla, & Kim, 2015; Morales-de La Peña et al., 2010; Odriozola-Serrano, Soliva-Fortuny, & Martín-Belloso, 2008). The higher retention of vitamin C in juices treated by PEF in comparison to those thermally treated can be attributed to the lower temperatures used in PEF processing (200 mg destroys cell membranes of subcellular organelles of tissues. Blurred vision, nausea, headache, alopecia, liver abnormalities.

Protects vitamin A, carotene, and vitamin C in foods from oxidative destruction. Antioxidant properties. Prevention of cancer. May protect against cardiovascular diseases possibly by inhibiting LDL oxidation

Vegetable oils, vegetables, meat, poultry, and fish

For adults, EARe and RDAc are 12 and 15 mg of α-T per day,

Muscle weakness, creatinuria, and increased susceptibility of the membranes of the erythrocytes to hemolysis. Ataxia, weakness, reflex change, impaired vision, and retinopathy are associated with advanced chronic deficiency. Infants with low plasma αtocopherol develop hemolytic anemia and thrombocytopenia

It is the least toxic of the fatsoluble vitamins. Some individuals have experienced gastrointestinal distress with daily intakes of up to 300 mg. It interferes with vitamin K metabolism

Sardesai (2003); Ye and Eitenmiller (2010)

Sardesai (2003); Ye and Eitenmiller (2010)


Table 9.1  Continued Vitamin

Active form



Functions in the human body Antihemorrhagic effects. To maintain normal levels of blood clotting factors: Prothrombin and factors VII, IX, and X possess procoagulant activity, whereas proteins C and S act as anticoagulants cofactor for the enzyme carboxylase that converts specific glutamic acid residues of precursor proteins to g-carboxyglutamic acid (GLA). Residues in the new protein. Maintaining strong, healthy bones

Food sources





fresh, green, leafy vegetables such as spinach, cabbage, lettuce, broccoli, kale, and cauliflower

AI levels are 120 and 90 μg/day for adults

prolonged blood coagulation time and an increased incidence of hemorrhage

produces hemolytic anemia in rats and hyperbilirubinemia and kernicterus in some low-birthweight infants

Sardesai (2003); Ye and Eitenmiller (2010)

Dietary Reference Intake (DRI). DRIa recommendations were limited to Average Intake values (AIb) due to the lack of reliable data required to set Estimated Average Requirements (EARse) and recommended dietary allowances (RDAsc). RAE (Retinol Activity Equivalent)d.

Fat-soluble vitamins271

9.2 Definition and principles of bioavailability and bioaccessibility In order to fully understand the beneficial functions of fat-soluble vitamins, it is crucial to explore their distribution in the human body as well as their possible subforms, which can be delivered most efficiently. Vitamins consist of different chemical groups, with each group having a specific function. For example, the chroman group in vitamin E is responsible for antioxidant activity, while the phytyl chain mostly plays a critical role in the kinetics of transport or retention of the molecule, which is associated with bioavailability (Burton & Traber, 1990). Due to their solubility in fat, their absorption can occur in the presence of bile salts, which act as a surfactant allowing digestion of dietary fats. In this case, passive diffusion is believed to be the main intestinal uptake process (Hofmann & Borgström, 1964). Fat-soluble vitamins can form micelles in the small intestine along with uptake of dietary fat. Therefore it is known that dietary fat plays a crucial role in the absorption of fat-soluble vitamins (Roodenburg, Leenen, van het Hof, Weststrate, & Tijburg, 2000). Since the presence of bile salts is required for their absorption, fat-soluble vitamins can be administered in gelatin form, which will induce bile secretion in the delivered area. Most of the fat-soluble vitamins are absorbed via the lymphatic system and incorporated into the structure of lipoprotein complexes, then transported to other organs. Since these lipoprotein complexes are too large to pass through the pores of blood capillaries, lymphatic vessels play a role in this transport. Since these vitamins are fat soluble, the content of fat present in the human body also can affect their bioavailability. Bioavailability of vitamins varies according to the degree of obesity. In the case of vitamin D3, which is stored in subcutaneous fat, it can hide away more in obese than in the non-obese people, which, consequently, can decrease its bioavailability (Wortsman, Matsuoka, Chen, Lu, & Holick, 2000). This situation also influences the required daily dose of the vitamin since obese patients may take larger doses than usual in order to overcome vitamin D deficiency, which is associated with obesity. Besides diseases like obesity, daily lifestyle has a great influence on the bioavailability of these vitamins, especially alcohol consumption and smoke-induced oxidative stress, which can result in the inactivation of vitamin E and beta-carotene as a precursor of vitamin A. In general, consuming low-fat diets does not negatively affect the absorption of fat-soluble vitamins, but it has been shown that a cholesterol-lowering diet or foods with non-absorbable fat might negatively affect vitamin absorption. Therefore diet plays a crucial role in absorbability of fat-soluble vitamins (van den Berg, van der Gaag, & Hendriks, 2002).

9.3 Processing parameters affecting the fat-soluble vitamins Food processing is highly important to improve critical properties of food products regarding health, safety, taste, or preservation. Although the benefits of processing are numerous, unfortunately it can alter the nutritional quality of food products.


Innovative Thermal and Non-Thermal Processing

Consequently, processing techniques directly affect the bioavailability of some key molecules present in fat-soluble vitamins after intake. Fat-soluble vitamins are organic compounds, and due to their chemical structure, they are highly sensitive to temperature, light, or oxygen. Another interesting factor is the season when the crop matures. For example, in general, the crops maturing during winter mostly present less vitamin A content than those maturing during autumn. This situation is also related to plant genetics. Vitamin content may vary in different parts of the same tissue or even among vegetables or animals collected at different times and locations. Moreover, particularly for vitamins, geographic conditions, seasonality, and maturity of the food source are also some important factors known to affect nutrient content (Greenfield & Southgate, 2003).

9.4 Effects of processing 9.4.1 Thermal processing by steam or hot water Blanching Unlike fruits, most of vegetables are not usually consumed in fresh form, and a blanching process is required before consumption. Blanching is a heating process, which is carried out in hot stream at high temperatures to completely destroy enzymes present in the vegetable. Besides enzyme activation, this process also shortens the drying and dehydration time (Oboh, 2005). Typical commercial blanching process is conducted by applying heat at 90–95°C for 1–10 min followed by a rapid cooling process (Puupponen-Pimiä et al., 2003). However, the blanching process can cause an increase in the concentration of carotenoids due to increased extractability, enzymatic degradation, and moisture losses, which might concentrate the carotenoids. Moreover, the applied heat during blanching inactivates enzymes like oxidases and breaks down foods, which consequently increases bioavailability (Rodriguez-Amaya, 1997). Heat also promotes changes in the cis/trans isomerization of the chemical structure of vegetables, leading to the formation of different carotenoid-based derived products (Chen, Peng, & Chen, 1995). Most of the time, these changes in fat-soluble vitamins depend on the type of vegetable, the blanching procedure, temperature, and time of heat applied. Another important point to consider is the cooling process used after blanching. A freezing process applied after blanching can preserve the provitamin A carotenoid. Therefore the blanching process needs to be optimized depending on the vegetable to be processed to obtain fat-soluble vitamins in order to minimize nutrient losses. Cooking The determination of fat-soluble vitamins in cooked foods is of great importance for consumers. Different cooking methods such as microwaving, baking, grilling, and frying have been used to process foods. Fat-soluble vitamins are known to be less heat-labile than water-soluble vitamins. The presence of oxygen makes them more susceptible to destruction by high temperatures (Lund, 1988). It was found that the

Fat-soluble vitamins273

content of fat-soluble vitamins is significantly altered by the type of procedure used during cooking. For instance, Ersoy and Özeren (2009) studied the effect of different cooking processes (grilling, baking, microwaving, and frying) on fat-soluble vitamin content in African catfish. They found that vitamin A content was increased in grilled and fried fish, while it was significantly decreased after microwave processing. On the other hand, the frying process usually increases the fat content, which can be considered as a reason to increase the content of vitamin A and E since they are fat-soluble. Pasteurization and sterilization Pasteurization is widely used to inactivate pathogenic microorganisms by heating milk or milk products up to a defined temperature for a specific period without permitting recontamination (Barba, Esteve & Frígola, 2012b). A meta-analysis of 40 studies regarding the effects of pasteurization showed that the pasteurization process qualitatively decreased vitamin E content and increased vitamin A levels in milk (McDonald et al., 2011). Since milk is not a considerable source of vitamin E, the effect of pasteurization can be ignored. In another study, no significant changes in vitamin A content in milk as a result of pasteurization (75–90°C) was noted. In a similar study, the pasteurization process did not alter vitamin D content in milk (Davídek, Velisek, & Pokorny, 1989). However, these results may vary depending on the milk products. Especially in the case of bovine milk, cow breed, season, and origin of a cow can affect the concentration of fat-soluble vitamins in milk, consequently altering the effect of pasteurization. In the case of holder pasteurization, most of the time a significant level of nutrients is lost. Therefore, as a new alternative, high-pressure processing has been used in order to maintain important fatty acids and especially levels of delta-, gamma-, and alpha-tocopherols as the strong vitamin E in foods such as human milk (Barba et al., 2012a). Moreover, to avoid the negative effects of the pasteurization processes, novel systems such as liposomes as vehicles for fat-soluble vitamins can be used (Marsanasco, Márquez, Wagner, Alonso, & Chiaramoni, 2011). Effect of different processing techniques on carotenoids Depending on the type of process and conditions, the different processing procedures may have negative impacts on carotenoid content. Degradation of food texture as a result of chopping or homogenization can reduce carotenoid content, especially due to oxidation processes. For example, >30% of carotene is lost by soaking green leaves of vegetables. Carotenoids are usually stable during the heating process and cooking of fruits and vegetables, but these processes can increase their isomers (Khachik et al., 1992). As a result of the heat processing of mango puree, carotene content was reduced by 13%, but no changes were observed in carotene content in mango pieces stored for 10 months; however, carotene content decreased by 50% in months 10–14 of storage. Carotene content in mango puree stored in a bottle was more sensitive than that stored in cans; however, 50% reduction of carotene content was observed in both samples after 14 months. Preservation processes in different fruits and vegetables increased carotenoid isomers. Preservation resulted in an increase of six isomers of carotenoids in


Innovative Thermal and Non-Thermal Processing

sweet potato (39%), carrot (33%), tomato juice (20%), and cabbage (19%) (Chandler & Schwartz, 1988). Conservation (120°C for 30 min) caused the highest rate of carotenoid degradation followed by heat processing at high temperature and short time (120°C for 30 s and 110°C for 30s). Following this heating process, carrot juice color changed from orange to yellow. When the processed juice was stored, carotene content was decreased by an increase in storage temperature. The formation of 13 isomers was enhanced under light, while nine isomers were increased under darkness. Storage of carrot and tomato juice under light radiation (230 ft. at 4°C) saved the carotene content by 75%, while storage at darkness was accompanied by a negligible reduction in carotene content (Rodriguez-Amaya, 1999). Dehydration (50°C hot air), freezing at −30°C, and freeze-drying of spinach soaked in salt and bicarbonate resulted in a 12% loss of carotene content (Rodriguez-Amaya, 1999). Other authors observed similar losses in trans carotene content of spinach regardless of drying method; freeze-dried spinach lost 33% and spinach dried by sunlight lost 34% (Nyambaka & Ryley, 1996). Squash did not lose any carotenoid content during blanching or freeze-drying; however, carotene was decreased by 15%, 20%, and 53% in squashes dried by freeze-drying and stored at 30°C for one, two, and three months. Storage at 3°C for three months only preserved carotene content by 10%. In another study, Bengtsson, Namutebi, Alminger, and Svanberg (2008) reported a 77%–88% reduction of carotene content during boiling (20 min) and frying (10 min) in the proceeded tomato. These processes caused an important carotene isomerization. Ade-Omowaye, Adedeji, and Oluwalana (2015) observed a reduction in carotenoid content (55%–80%) during osmotic dehydration of bell pepper at 25–55°C. Pott, Jansonius, & Kooijman (2003) found that conventional drying of mango caused isomerization of all-trans carotene to 13 cis-carotene, while sunlight drying resulted in the formation of 9-cis carotene. In contrast, Minguez-Mosquera and Hornero-Mendez (1994) reported that drying of red paprika increased carotenoids by 40%. Other authors reported that carotenoid content increased by 2–22 times during drying of fruits (Wen-ping, Zhi-jing, He, & Min, 2008). In another work, OdriozolaSerrano, Soliva-Fortuny, Hern’andez-Jover, and Martín-Belloso (2009) demonstrated that heat pasteurization promoted carotenoid content and red color of fruit juices; meanwhile, freezing may reduce, increase, or adjust the carotenoid content of fruits and vegetables. PEF, osmotic dehydration, radiation, and HPP cause negligible degradation of carotenoids (Barba et al., 2012b; Gabrić et al., 2018; Zulueta, Barba, Esteve, & Frígola, 2010). It has been reported that carotene can be better protected at lower temperatures and short cooking times (Meléndez-Martínez, Vicario, & Heredia, 2004). For example, soaking a carrot in water at 50°C, 70°C, and 90°C for 15 min had no significant effect on carotene content; only the treatment at 90°C slightly reduced carotenoids (Meléndez-Martínez et al., 2004). Moreover, sun drying also significantly reduced carotenoid content (Rodriguez-Amaya & Kimura, 2004). For instance, mangos dried in the sun up to 10%–12% moisture and stored for two months had 4 g carotenoid/100 g mango; the value reduced up to 3.68 g/100 g after six months (Rankins, Sathe, & Spicer, 2008). In conclusion, after applying most of the heating processes, carotenoid content is poorly protected when high temperatures and long treatment times are used.

Fat-soluble vitamins275

It should be noted that carotenoid degradation might be reduced by reduction of temperature, process duration, reduction of oxygen availability, and application of antioxidants.

9.5 Effect of storage on α-tocopherol and beta-carotene Carotenoids are sensitive to temperature, light, and oxygen. Isomerization and oxidation of these compounds during processing and storage can cause loss of color, biological activity, and formation of volatile compounds, which may result in bad odor or taste of the foods. Oxidation processes depend on the presence of oxygen, metals, enzymes, unsaturated lipids, pro-oxidants and antioxidants, light, type and physical status of carotenoids, as well as the verity of the processes, packaging materials, and storage conditions (e.g., temperature and relative humidity). In this context, the high temperature causes trans-cis isomerization. Isomerization leads to increases in cis isomers and the conversion of 5,6 epoxide to 5,8 oxide furanoid, structural changes by enzyme release, and oxidative changes of poly-n by free radicals to form apocarotenals and ketones (Simpson, Rodriguez, & Chichester, 1976). These reactions result in the loss of texture and color of the products. Oxidative breakdown of carotenoids may result in the formation of aromatic and tasteful products such as ionone and dihydro actinodiolide. A notable variation has been reported regarding the stability of carotenoids of fruits and vegetables during storage. In general, loss or change in carotenoid contents of unripe fruits and vegetables that can be stored for a long time is negligible, whereas these changes occur faster in ripe fruits and vegetables. Carotenoid accumulation may occur after harvesting or during ripening through storage or transportation. Quick degradation of carotenoids and change in their composition can be correlated with unsuitable storage conditions. It should be noted that carotenoid esters are more stable than free carotenoids. Degradation of carotenoids during storage under high temperature and light or after harvest of fruits and vegetables has been reported by many authors (Ezell & Wilcox, 1962; Kopas-Lane & Warthesen, 1995; Simonetti, Porrini, & Testolin, 1991; Takama & Saito, 1974). Application of packages impermeable to light, temperature, and humidity in biodegradable films can enhance the stability of carotenoids in perishable fruits and vegetables. Storage temperature also affects carotenoid content. Carotenoids variation during storage depends on the maturity stage of fruits and vegetables (Gross, 1991). Several researchers observed an increase in carotenoid content during maturation and then a decrease during senescence; differing these changes according to the temperature used. For example, carotenoid content in tomato was increased during storage and maturation, being the increase intensified at temperatures higher than 25°C (Watada, 1987); however, no increase in carotenoid content was observed in sweet potato during storage. This can be attributed to a complete carotenoid synthesis at harvesting time (Watada, 1987). The maturation-temperature interaction was also observed in cucumber, observing a higher carotenoid content in unripe and ripe cucumbers stored at 18°C compared to those stored at 5°C. However, carotenoid content was


Innovative Thermal and Non-Thermal Processing

not affected by temperature in mature cucumbers. Storage of cucumbers at 7–20°C for 16 to 43 days resulted in a reduction of total carotenoid content even when the cucumbers had been matured under optimal conditions. Lycopene and carotenoid content in tomatoes stored at 20°C were significantly higher than those stored at 30°C, which shows that key enzymes catalyzing lycopene and carotene synthesis are mainly inactivated during maturation and can be affected by temperature. For example, carotenoid synthesis is limited at temperatures higher than 30°C (Goodwin & Jamikorn, 1952; Lurie, Handros, Fallik, & Shapira, 1996). This may be attributed to the prevention of ethylene synthesis or enzymes involved in carotenoid compounds biosynthesis (Lurie et al., 1996). Application of modified and controlled atmosphere with low oxygen mitigated carotenoid degradation (Yahia, 2009). It has been reported that a high concentration of CO2 may reduce carotenoid content. Under controlled atmosphere, six-day storage of broccoli at 5°C provided better protection of carotenoids compared to normal gas concentration at the same temperature, so that under normal storage conditions more than half of the carotenoids were lost (Kalt, 2005). Cucumber storage under controlled atmosphere for 21 days in the presence of high CO2 concentrations (15 KPa for 2 days, followed by 5 KPa for 19 days) protected carotenoids, whereas high CO2 concentrations (15 KPa) significantly reduced carotenoid content within 14 days of storage (Huyskens-Keil, Prono-Widayat, Lüdders, & Schreiner, 2005). Moreover, high concentrations of CO2 reduced the carotenoid content in spinach, watermelon, and cabbage (Kader, 2002). The color of golden apples remained green during storage at 15 KPa CO2. This fact can be due to the prevention of carotenoid biosynthesis in the presence of high CO2 concentrations (Hribar, Plestenjal, Vidrih, & Simcic, 1994). Low O2 concentrations improved carotene stability in carrot. A concentration of 5 KPa CO2 reduced carotene content, while 7.5 KPa or more CO2 significantly improved carotene biosynthesis (Weichmann, 1986). In another study, it was found that leeks stored at 1 KPa O2 and 10 KPa CO2 had more carotenoid content than those stored at normal atmosphere (Weichmann, 1986). Moreover, it has been reported that storage of pepper at controlled atmospheric conditions (5 KPa oxygen and 4 KPa CO2) for 15 days caused 92% retention of carotenoids, while its storage in the normal atmosphere only retained 52% carotenoid (Howard & Hernandez-Brenes, 1998). Other studies showed that peach pieces stored at 12 KPa CO2 had lower carotene content than those stored at 2 KPa oxygen or 2 KPa O2 plus 12 KPa CO2 for 8 days at 5°C (Wright & Kader, 1997). Hot-water treatment postpones carotenoid synthesis and makes broccoli yellow. Hot-air treatment gradually reduces carotenoid content, while fruit heating at 34°C for 24 h followed by storage at 20°C increased carotenoid content (Soto-Zamora, Yahia, Brecht, & Gardea, 2005). Papaya treated with humid hot air (50°C and 50% relative humidity) individually or in combination with tiabendazole did not affect carotene content (Perez-Carrillo & Yahia, 2004). Liang, Huang, Ma, Shoemaker, and Zhong (2013) investigated the effect of relative humidity on the storage stability of spray-dried beta-carotenenano-emulsions. To resolve the limitations of fluid-based emulsions, beta-carotenenano-emulsions stabilized by modified starch were powdered by spray drying after emulsification. The

Fat-soluble vitamins277

powders had a suitable solubility in water and particle size of reconstituted emulsions was similar to that of fresh nano-emulsions. Thirty-day-storage was conducted to study the effect of relative humidity on the storage stability of beta-carotene powders at 25°C. The results indicated that more carotene is protected in modified starches with lower film oxygen permeability. Glass transition temperature of the powder had a significant impact on beta-carotene degradation at various values of relative humidity (Liang et al., 2013). Bationo et  al. (2015) investigated the effect of storage on α-tocopherol and beta-carotene of solar-dried spirulina microalga. Spirulina has been introduced by the World Health Organization (WHO) as a functional food with high nutritional value. The results of high-performance liquid chromatography (HPLC) analysis indicated that after 6 months of storage, α-tocopherol and beta-carotene contents decreased by 28% and 24%. Reduced food value of spirulina during storage suggests that this product should be consumed fresh or stored for shorter times (Bationo et al., 2015). Oliveira, De Carvalho, Nutti, and De Carvalho (2010) studied the degradation of carotenoids and beta-carotene in bitter yellow cassava. Five cassava species were investigated, and their beta-carotene content was measured after heat processing during storage. Betacarotene degradation was 50% after the heating process and occurred after 30 days. Heat process, light, and oxygen affect beta-carotene degradation (Oliveira et al., 2010). Rastrelli, Passi, Ippolito, Vacca, and De Simone (2002) investigated the degradation rate of α-tocopherol, squalene, phenolics, and polyunsaturated fatty acids in olive oil during various storage conditions. For instance, they studied tocopherol concentration during one-year storage. Two extra virgin olive oils were kept under darkness and in colorless bottles, either full or semi-full. The authors observed major changes occurring in the concentrations of all studied compounds in bottles with higher oxygen (semi-full bottles). The first oxidated molecule was α-tocopherol (−20% and −92% after two and 12 months). In another study, Amaya et al. (1999) studied carotenoid changes during processing and storage of food products. They observed an enhanced trans carotenoid isomerization to cis-carotenoid in the presence of acid, temperature, and light, thus reducing color and vitamin A activity. The authors observed that carotenoid reduction during processing and storage is intensified by the destruction of the cellular food structure, increased porosity, lengthening of the process, storage duration, and temperature, light, and oxygen penetration to package. In contrast to lipid oxidation, whose mechanism is approved, the carotenoid oxidation mechanism is not understood well. The carotenoid oxidation process may include epoxidation, the formation of apocarotenoids, and hydroxylation. Constituting components may be low molecular weight compounds, then when they completely lose their color and functions, carotenoids may increase volatile compounds, thus promoting the development of suitable taste and aroma in tea and drinks (Rodriguez-Amaya, 1999). Comstock, Alberg, and Helzlsouer (1993) investigated the effect of freeze storage on the concentration of α-tocopherol and beta-carotene. At temperatures higher than −40°C, a slight reduction of α-tocopherol was observed, and a small part of beta-­ carotene was protected. At temperatures of −70°C and colder, both α-tocopherol and beta-carotene remained unchanged for 15 years.


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In another study, Bouzari, Holstege, and Barrett (2015) examined the content of ascorbic acid, riboflavin, α-tocopherol, and beta-carotene in corn, carrot, broccoli, spinach, pea, strawberry, and blueberry at three storage times and fresh and freezing conditions. Three products had higher α-tocopherol under freezing conditions, while the others did not show any significant differences between fresh and freezing conditions. Regarding β-carotene, it was significantly reduced in most of the vegetable study. For example, its content in corn, strawberry, and blueberry was negligible; it was lower in frozen samples of pea, carrot, and spinach. Moreover, there were no significant differences for beta-carotene in fresh and frozen samples of pea and spinach.

9.6 Effect of heat processing on α-tocopherol and beta-carotene Ryang et al. (2014) investigated the stability of tocopherol and tocotrienols extracted from rice bran at various temperatures and oxygen conditions. Rice bran contains a high amount of tocopherol and tocotrienol with antioxidant and anticarcinogenic properties. These compounds are sensitive to high temperature and oxygen. In this study, vitamin E isomers such as α, β, δ, γ tocopherol and tocotrienol (T3) extracted from rice bran were investigated at various temperatures and oxygen concentrations. Each isomer had a particular sensitivity to temperature. α-T3 was degraded faster than the others were. In the lack of oxygen, no degradation occurred for 4 h even at 95°C at 2% oxygen, 20% and 29% loss of γ-T3 and γ-T3 was observed. Organic solvents such as isooctane and hexane were more effective than edible oil in improving γ-T3 stability. Among edible oils, maize oil was more effective than soybean and rice bran oil (Ryang et al., 2014). D'Evoli, Lombardi-Boccia, and Lucarini (2013) investigated the effect of heating processes on the carotenoid content of cherry tomatoes. Tomato and its products are rich in carotenoids especially lycopene, beta-carotene, and lutein. Preserved and fresh tomatoes were studied; the whole fruit, peel, and pulp either fresh or processed. In processed tomato, a significant reduction of beta-carotene and lutein was observed, with the reduction of beta-carotene and lutein more significant in peel (−17%) and pulp (−25%), respectively. The analysis of the formed isomers during the heating process indicated that lycopene in the tomato matrix was more stable than other compounds (D'Evoli et al., 2013). Marx, Stuparic, Schieber, and Carle (2003) investigated the effect of the heating process on cis-trans isomerization of beta-carotene of carrot juice in a lab. The results indicated that pasteurization and sterilization at 121°C caused a partial isomerization, and sterilization at 130°C and blanching increased cis-isomers. The dissolution of crystalline carotenes by cellular lipids during blanching was recognized as the main cause of isomerization. Moreover, the addition of grape seed oil to carrot juice promoted isomerization in both heated and unheated juices. Heating treatment of β-­carotene dissolved in toluene caused temperature-dependent isomerization. Stabilizing compounds of β-carotene in carrot juice prevented the dissolution of carotene in n­ eutral lipids under mild heating conditions because the nucleus is coated with insoluble compounds such as pectin, cellulose, and protein (Marx et al., 2003).

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In another study, Henry, Catignani, and Schwartz (1998) investigated the oxidative degradation kinetics of lycopene, lutein, and β-carotene. Thermal and oxidative degradations of carotenoids in oil were studied. Trans β-carotene, 9-cis β-carotene, lycopene, and lutein from safflower seed oil were heated at 75°C, 85°C, and 95°C for 24, 12, and 5 h. The main isomers formed during the process were 13-cis and 9-cis. The degradation rate of the carotenoids was as follows: lycopene, trans-β-carotene, and lutein. These results indicated that lycopene is the most sensitive compound and there is no significant difference between the degradation of 9-cis and trans-β-carotene (Henry et al., 1998). Kuppithayanant, Hosap, and Chinnawong (2014) investigated the heating impact of vitamin E degradation in edible palm oil. Vitamin E is a determinant factor of palm oil quality. During the conversion of crude palm oil to edible oil, a part of vitamin E is lost. High cooking temperatures result in the reduction of vitamin E. It was indicated that vitamin E degradation at temperatures of 210–278°C is increased, and this rate was fixed after 30-min treatment at 278°C. It was concluded that long duration of treatment time during the heating process had a lower impact on vitamin E degradation compared to increased temperature (Kuppithayanant et al., 2014). Mader (1964) studied the thermal degradation of beta-carotene. Heating processing of crystalline beta-carotene (240°C at vacuum conditions) resulted in the formation of the volatile fraction containing chiefly aromatic hydrocarbons such as toluene, m, p xylene, 2–6 dimethyl naphthalene, and ionene. Achir, Randrianatoandro, Bohuon, Laffargue, and Avallone (2010) investigated trans beta-carotene degradation kinetics and trans lutein in olein palm and vegetaline at four temperatures ranging from 120 to 180°C. In both oils, the degradation rate of primary beta-carotene and trans lutein was enhanced by an increase in temperature. Trans lutein was more stable than beta-carotene. The degradation rate of vegetaline was lower than that of olein palm (Achir et al., 2010). Dutta, Raychaudhuri, and Chakraborty (2005) investigated beta-carotene degradation kinetics, and visual color of pumpkin puree blanched in 1% salt water for 2 min at 60–100°C from 0 to 2 h. The activation energy of beta-carotene oxidation and the visual color was 22.27 KJ/mol and 33.68 KJ/mol, respectively. By increasing activation energy, the sensitivity of visual color to temperature was also increased. The color change was positively related to the variation of beta-carotene (Dutta et al., 2005). Lemmens et  al. (2010) investigated the beta-carotene isomerization kinetics during heat processing (80–150°C) in carrot puree. Heating increased variation of beta-­carotene stability and caused the conversion of beta-carotene to cis isomers after long heating until reaching a balanced status. Regarding low activation energy of all compounds (11 KJ/mol), the isomerization rate constant showed a slight sensitivity to heating treatment. Temperature dependency of equilibrium concentrations varied in different compounds. Since isomerization occurs after heating processes, it can be concluded that during industrial heating processes the retention of trans-beta-carotene of carrot puree is high, which can be due to the presence of a protective food matrix. In another study, Zepka and Mercadante (2009) studied the degradation kinetics of the main carotenoids in apple juice and its impact on a color system using HPLC. The results indicated that color parameters are good markers for β-carotene ­degradation and there was a linear relationship between color change and carotene content variation.


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Azizah, Wee, Azizah, and Azizah (2009) investigated the effect of various cooking methods on β-carotene and lycopene content of pumpkin. The pumpkin was boiled and stir-fried for 2, 4, and 6 min and β-carotene and lycopene content were measured by HPLC. The results indicated that both β-carotene (2–4 times) and lycopene (17–40 times) contents were increased after pumpkin cooking. Somavat (2011) investigated the effect of ohmic heating on bioactive compounds. Industrial application of ohmic heating is limited and a continuous heat process is more common. Carotenoids were not changed by the ohmic process, and β-carotene and lycopene remained unchanged at 90–110°C at pH 3.9 and pH 4.4. Seybold, Fröhlich, Bitsch, Otto, and Böhm (2004) investigated β-carotene, lycopene, and tocopherol variation during the heating process at various time intervals. Due to the reduction of humidity during the heating process, lycopene and tochopherol were increased, while the β-carotene content was decreased. In contrast to lycopene, β-carotene was isomerized, and tocopherol content was increased during heat processing. This increase was negligible, probably due to the release of R-tocopherol from the pumpkin seeds (Seybold et al., 2004). Gao, Wu, Wang, Xu, and Du (2012) investigated the effect of different heating methods (sunlight, oven, microwave, freeze dryer) on α-tocopherol and β-carotene in jujube fruits. The results indicated that microwave and freeze-drying protected much more α-tocopherol and β-carotene, thus the combination of these two methods is a useful strategy to retain food nutrients by decreasing processing time. Zhao et al. (2006) investigated the effect of microwave and ultrasound treatments on carotenoid content, observing that microwave treatment stimulates carotenoid isomerization, which is enhanced by an increase in duration and microwave power. In contrast, ultrasound treatment promoted the conversion of carotenoids into unknown and colorless compounds with the degradation enhanced by an increase in the duration and ultrasound power. It can be concluded that although microwave and ultrasound treatments are methods with high efficiency to treat food products in a short time, caution should be taken in their application to minimize their effect on carotenoid content. Along the same lines, Kamel (2013) studied the impact of microwave heating for 1, 2, and 3 min on carotenoid compounds of parsley and dill. In both vegetables carotenoid content was increased after 1 min of heating and then gradually decreased after 2 (−32.3%) and 3 (−80%) min.

9.7 Effect of light on α-tocopherol and beta-carotene Nhan and Hoa (2013) investigated the effect of light on vitamin E content of pharmaceutical products. The authors observed that long storage of products resulted in the degradation of vitamin E and that the degradation was enhanced under direct light. Moreover, a higher concentration of vitamin E in tablets was accompanied by a faster degradation; the vitamin E content reduced to 50% within 5 h in tablets stored under sunlight or UV radiation and thus vitamin E tablets should not be stored under light radiation. Sabliov et al. (2009) investigated the effect of temperature and UV on α-tocopherol degradation in free and soluble form. They studied the effect of temperature and UV

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on free α-tocopherol, α-tocopherol dissolved in methanol, and α-tocopherol dissolved in hexane. The results revealed that the highest degradation rate occurred in samples stored at 180°C. Compared to α-tocopherol dissolved in solvents, free α-tocopherol was degraded at higher temperatures. In contrast, free α-tocopherol was not degraded when it was stored under UV for 6 h. α-tocopherol dissolved in hexane and methanol was degraded by 20% and 70% under the same light intensity, and degradation of α-tocopherol was higher in methanol than hexane.

9.8 Effect of drying and dehydration on α-tocopherol and beta-carotene Pinheiro-sant Ana, Stringheta, Brandão, Páez, and Queiróz (1998) investigated the stability of total carotene, α- and β-carotene, of carrot during dehydration and other preparations. They used different processing techniques, including steam cooking, water cooking (with or without pressure), dry or wet cooking, and conventional dehydration. The results indicated that water cooking without pressure improved the retention of α- and β-carotene and vitamin A, whereas water cooking under pressure promoted the retention of total carotenes. The highest reduction of carotenes was observed under dehydration. It can be concluded that cooking without pressure, under controlled time and temperature, is the best process to mitigate the loss of total carotene. The results of this study revealed that increasing sugar solution concentration from 30° Brix to 50° Brix did not change β-carotene content; however, a significant reduction in carotene content was observed when mango was retained in the sugar solution for four weeks. Mulokozi and Svanberg (2003) studied the variation of β-carotene in eight vegetables dried by light, either by open sun drying or solar drying. The results revealed that β-carotene content was significantly reduced under open sun drying, while it was well retained under solar-drying treatment. Siriamornpun, Ratseewo, Kaewseejan, and Meeso (2015) investigated the effect of drying methods and osmotic treatments on the carotenoid content of papaya and tomato. Drying methods included far-infrared radiation and air convection (FIR-HA) drying and hot air (HA) drying in the untreated and osmotically treated sample. The authors observed that β-carotene content was significantly reduced in all treatments (samples treated osmotically and dried with infrared radiation, untreated samples, air hot-dried, and osmotically treated by hot air), whereas lycopene and lutein content was increased in the samples untreated by osmotic method but dried by both methods. Orset, Leach, Morais, and Young (1999) studied the impact of spray drying on β-carotene of a microalga. Results show β-carotene reduction and isomerization were at a minimum during spray drying, and in samples containing butylated hydroxyl toluene (BHT) and tert-butyl hydroquinone (TBHQ). The drying process caused 52%–72% degradation of β-carotene in the samples containing tocopherol-based natural antioxidants. β-carotene was significantly degraded in all dried samples kept


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under light and air oxygen. Storage of the samples under darkness did not cause any change in β-carotene content, while β-carotene content was reduced in all samples containing TBHQ.

9.9 Effect of high-pressure processes on α-tocopherol and beta-carotene McInerney et  al. (2007) investigated the impact of HPP (400 and 600 MPa) on the antioxidative activity and carotenoid content of three vegetables. Antioxidants and carotenoids were not changed under high-pressure treatment, while the carotenoid content of the vegetables varied between 3% and 35%. HPP changed the availability of the carotenoids based on the vegetable type and pressure rate. In another study, Barba et al. (2012b) found an increase in vitamin E activity when pressures >100 MPa were applied to an orange-juice milk beverage and a vegetable soup. They attributed this enhanced α-tocopherol content to an increased extractability after high-pressure treatment. For a more detailed discussion about the impact of HPP on tocopherols and carotenoid contents see the reviews by Barba et al. (2012b) and Barba et al. (2017).

9.10 Effect of freezing condition on α-tocopherol and beta-carotene Park (1987) investigated the impact of freezing and defrosting, drying and cooking on retention of carotene in carrot, broccoli, and spinach. Regardless of the drying method, dehydration significantly reduced the carotene content in the studied vegetables. Carotene degradation rate was enhanced 6 h after defrosting, with cooked samples having more carotene. In another study, Thomas, Duewer, Kline, and Sharpless (1998) examined α-tocopherol and β-carotene content stored for 10 years at −25°C and −80°C. They did not observe any changes in α-tocopherol and β-carotene content during 10 years of storage, but the stability of the compounds was lower at −25°C. At −80°C, β-carotene, and α-tocopherol did not change for three and five years, respectively.

9.11 Conclusions Carotenoid content may increase or decrease in a product during storage. Moreover, high temperature may promote an increase or decrease carotenoid content, but it depends on several factors such as temperature, food matrix, pH, targeted carotenoid, and so on. The temperature effect is limited, meaning that carotenoid content is increased until a certain temperature, and under higher temperature carotenoid content is reduced, which may be attributed to the reduced activity of enzymes involved in

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carotenoid synthesis. Moreover, some studies show that storage of products at 8000 compounds in one of the following groups: flavonoids, phenolic acids, acetophenones, coumarins, xanthones, stilbenes, phloroglucinols, and other phenolic compounds, which also include their derivative compounds (with glucuronide and sulfur substituents for example) and oligomeric/polymeric structures. Due to the importance of the flavonoid group (the largest group of phenolic compounds with >4000 identified compounds), a further classification can also be used to discriminate flavonoid from non-flavonoid ­compounds.

Polyphenols: Bioaccessibility and bioavailability of bioactive components311

All phenolic compounds contain at least one aromatic ring with hydroxyl substituent (Crozier, Jaganath, & Clifford, 2009; Tsao, 2010). Flavonoids are the main phenolic group due to their wide distribution in plant tissue, which includes food of vegetable origin (Barba, Esteve, & Frigola, 2014). The biosynthesis of flavonoids initiates with phenylalanine that gradually undergoes enzymatic substitution and rearrangements to yield a variety of compounds that may be classified in six sub-classes: flavonols, flavones, flavan-3-ols, isoflavones, flavanones, and anthocyanidins. The structural skeleton of flavonoids (C6–C3–C6) is composed of two aromatic rings connected by a three‑carbon bridge, which can also contain hydroxyl, sugars, methyl, and isopentyl as substituents. The flavonols group includes kaempferol, quercetin, isorhamnetin, and myricetin; flavones include apigenin and luteolin; in flavan-3-ols are grouped catechin and its isomer epicatechin; the isoflavone class is comprised of daidzein and genistein; while hesperidin, narirutin, neohesperidin, and naringin belong to the flavanones group; and finally, the anthocyanidins class includes pelargonidin, cyanidin, delphinidin, peonidin, petunidin, and malvidin (Barba et al., 2014; Crozier et al., 2009). Phenolic acids (C6–C1 skeleton) are grouped into hydroxybenzoic acids and hydroxycinnamic acids sub-classes. These compounds are synthesized in the shikimate pathway that converts shikimic acid into L-phenylalanine or L-tyrosine (main compounds in the pathway), thus leading to the formation of cinnamic acid and p-­coumaric acid, respectively, at first. Then benzoic and p-coumaric acids are formed followed by several phenolic acids such as gallic acid, syringic acids, caffeic acid, protocatechuic acid, and ferulic acid (Heleno, Martins, Queiroz, & Ferreira, 2015). Acetophenones are a group of phenolic compounds synthesized from -phenylalanine that is characterized by a phenol skeleton with a carboxylic acid L substituent (C6–C2). Some of these phenolic compounds are volatile and implicated as intermediate compounds in the formation of other compounds associated with the jasmine and strawberry fragrances (Dong et al., 2012). Coumarins (C6–C3) are characterized by the basic structure that comprises the 1,2-­benzopyrone. This class can be divided into six categories, namely, simple coumarin (esculetin and ostruthin), furano coumarins (imperatorin), dihydrofuran coumarins (anthogenol), pyrano coumarins (grandivittin in linear type and inophyllum A in angular type), phenyl coumarins (isodispar B), and bicoumarins (dicoumarol) (Venugopala, Rashmi, & Odhav, 2013). Interestingly, the name coumarin is derived from the French word coumarou, which is attributed to the tonka bean tree (Musa, Cooperwood, & Khan, 2008). In the xanthones group (C6–C1–C6) many compounds have been characterized, such as mangiferin, isomangiferin (Schieber, Berardini, & Carle, 2003), garciyunnanin A and B (Xu et al., 2008; Xu et al., 2008), and α-mangostin (Kondo, Zhang, Ji, Kou, & Ou, 2009). The group of stilbenes (C6–C2–C6) is produced in plant tissues through the phenylpropanoid pathway. In this class, trans-resveratrol is the most relevant compound along with other relevant compounds: pinosylvin, piceatannol, trans-pterostilbene, astringin, and rhapontin (Chong, Poutaraud, & Hugueney, 2009). Phloroglucinols are a particular group of phenolic compounds largely found in terrestrial plants, algae, and bacteria that accounts for at least 700 compounds. The c­ ompounds grouped in this


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class can be separated according to the degree of polymerization (DP) and ­substituents in the main skeleton (C6) that is attributed to phloroglucinol. The phloroglucinol class can be separated as follows: monomeric (DP = 1; grandinol, euglobal G1, and picraquassioside D); dimeric (DP = 2; robustaol A, rottlerin, and grandinal); trimeric (DP = 3; myrtucommulone A and japonicin D); tetrameric (DP = 4; dryocrassin and hexaflavaspidic acid); and phlorotannins (DP ≥ 5; diphlorethol, trifucol-2-O-sulfate, eckol, and fucophlorethol A) (Singh & Bharate, 2006). The “other phenolic compounds” subclass is formed by unique structures that contain at least one aromatic ring. Ellagic acid along with ellagitannins belongs to a group of phenolic compounds found in few dietary sources such as pomegranate, berries, and nuts (Landete, 2011). Another group is formed by amide phenolic compounds with N-substituents in the phenol ring, such as avenanthramides. This class is found in oat and derived products (Meydani, 2009). Lignans is another relevant group of phenolic compounds present in the human diet in the composition of grains and nuts. This class is formed by several fiber-related compounds, for instance, secoisolariciresinol, matairesinol, and pinoresinol (Landete, 2012). Typically, phenolic compounds are widely distributed in food of vegetable origin, such as fruits, vegetables, whole grains, honey (in small amounts), and its processed forms (Barba et al., 2014) (Table 11.1). The amount and variety of phenolic compounds in each dietary source are influenced by many factors during production and processing (Barba, Esteve, Tedeschi, Brandolini, & Frígola, 2013). Regarding differences between varieties, significant differences in total phenolic content were observed for three raspberry varieties when grown in wild conditions. Glen Ample and Maurin Makea varieties produced more polyphenols than the Gleen Dee variety. Under controlled conditions such varieties displayed no significant differences, although higher yields were obtained in these conditions. In addition, these results highlight the importance of environmental effect on phenolic content in fruit cultivation (Palonen et al., 2017). The ripening state and season are major factors to consider in this context. Significant differences were observed between three sour cherry cultivars: Oblačinska cherry variety (140–159 mg/100 g) had the highest total phenolic content in comparison to Marela (72–87 mg/100 g) and Cigančica (91–122 mg/100 g) varieties in 2008, 2009, and 2010 seasons. Ripening state displayed a significant accumulation, particularly for the Oblačinska variety (Mitić et al., 2012). Total phenolic content can also be influenced by location as indicated by a study with olive fruits in Turkey. Olive fruits harvested in Umurbey, Turkey had the highest phenolic content in comparison to Mudanya, Çağrışan, and Kumla. Harvest dates can also influence the phenolic content in olive fruits wherein late harvesting can be associated with reduced phenolic concentration (Uylaşer, 2015). The influence of ripening in phenolic content of olive fruits is also observed in olive oil. The phenolic content of olive oil produced from Chemlali and Chétoui varieties displayed an increase followed by a decrease during six harvesting dates within 75 days of growing (Hbaieb et al., 2017).

Polyphenols: Bioaccessibility and bioavailability of bioactive components313

Table 11.1  Dietary sources of phenolic compounds. Source


Source b


Apple pulp Bananaa Barleyd Basilc Bayf Black crush-tearcurl teag Black currantsi Black goji berryj Black mulberryi

8 mg/g dw 2 mg/g dw 903.5 μg/g dw 20 mg/g dw 1 mg/g dw 11 mM

Murici Mustardc Mustard greense Okrae Oreganoc Peanuth

9907 mg/kg dw 7–9 mg/g 11,546 mg/kg dw

Pecan nuth Persimmona Pistachioh

Black Orthodox teag Blackberriesi Blond grapefruita Blue-berried honeysucklei Blueberryi Bog blueberrym Butter beanse Butter pease Chamomilec Cinnamonf Collard greensi Cornd Cranberrym Cuminf Eggplante European juneberryi Fennelc Gabirobab Gingerc Grapefruitk Green Grapea Green onione Green teag Green tea low-caffeineg Guapevab Kalee Kiwi fruita Laurelc

15 mM 23,000 mg/kg dw 15 mg/g dw 21,279 mg/kg dw

Pummelok Purple hull pease Purslanee Raspberry fruitl

222 mg/100 g 20 mg/g dw 26 mg/g dw 22 mg/g dw 74 mg/g dw 301–457 mg/ 100 g dw 1225 mg/100 g dw 4 mg/g dw 566–710 mg/ 100 g dw 801–863 mg/L 13 mg/g dw 28 mg/g dw 3–4 mg/g

23,714 mg/kg dw 504 mg/100 g 15 mg/g dw 7 mg/g dw 17 mg/g dw 6 mg/g dw 24 mg/g dw 1251 μg/g dw 350 mg/100 g 5 mg/g dw 21 mg/g dw 23,154 mg/kg dw 6 mg/g dw 851 mg/100 g 9 mg/g dw 1241 mg/L 6 mg/g dw 18 mg/g dw 24 mM 23 mM

Red goji berryj Red grapefruita Red pomeloa Rosemaryc,f Rutabagase Sagec Sour cherryn Strawberrya Sweet orangek Sweet potato greense Thong Dee pomeloa Thymef Triticaled Turmericc Walnuth Wheatd White pomeloa White teag Olive fruitso Virgin olive oilp

2–4 mg/g 15 mg/g dw 5 mg/g dw 5–46 mg/g dw 14 mg/g dw 26 mg/g dw 72–159 mg/100 g 15 mg/g dw 1173–1500 mg/L 53 mg/g dw 11 mg/g dw 3 mg/g dw 1136 μg/g dw 17 mg/g dw 1404 mg/100 g dw 1700 μg/g dw 3 mg/g dw 18 mM 405–4300 mg/kg 105–669 mg/kg

321 mg/100 g 27 mg/g dw 6 mg/g dw 37 mg/g dw

3322 mg/100 g 3647 mg/100 g 977 mg/100 g 680 mg/100 g


5 mg/g dw

Chocolate powderq Cocoa powderq Cupuassu powderq Dark cupuassu “chocolate”q Milk cupuassu “chocolate”q

540 mg/100 g Continued


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Table 11.1  Continued Source




Lemon balmc

43 mg/g dw

96 mg/100 g

Lowbush blueberrym Lowbush cranberrym

371 mg/100 g 624 mg/100 g

Mangosteena Marjoramc Mature Duriana Mintc Muricib

11 mg/g dw 49 mg/g dw 2 mg/g dw 11 mg/g dw 222 mg/100 g

White cupuassu “chocolate”q Wine—Bonardar Wine—Cabernet Sauvignonr Wine—Malbecr Wine—Merlotr Wine—Shirazr Wine—Tempranillor Honeys

3372 mg/L 3378 mg/L 4203 mg/L 3448 mg/L 1586 mg/L 3137 mg/L 96–115 mg/100 g

TPC, total phenolic content; dw, dry weight. a Park et al. (2015). b Malta, Tessaro, Eberlin, Pastore, and Liu (2013). c Fernandes et al. (2016). d Kandil, Li, Vasanthan, and Bressler (2012). e Huang, Wang, Eaves, Shikany, and Pace (2009). f Vallverdú-Queralt et al. (2014). g Carloni et al. (2013). h Rosales-Martínez, Arellano-Cárdenas, Dorantes-Álvarez, García-Ochoa, and López-Cortez (2014). i Zadernowski, Naczk, and Nesterowicz (2005). j Islam, Yu, Badwal, and Xu (2017). k Xu, Feng, et al., 2008; Xu, Liu, et al., 2008. l Palonen, Pinomaa, and Tommila (2017). m Grace, Esposito, Dunlap, and Lila (2013). n Mitić, Obradović, Kostić, Micić, and Pecev (2012). o Uylaşer (2015). p Hbaieb et al. (2017). q Genovese and Lannes (2009). r Fanzone et al. (2012). s Sant'Ana, Buarque Ferreira, Lorenzon, Berbara, and Castro (2014).

11.3 Digestion and absorption of phenolic compounds Digestion of phenolic compounds is a complex and not fully understood process that involves several process and agents (e.g., pH, enzymes, and microorganisms) as indicated in Fig.  11.1. Mastication and the reduced pH in the stomach initiate the release of phenolic compounds by softening and disintegrating the food matrix. Depolymerization initiates in the stomach wherein polymeric and oligomeric phenolic compounds are degraded to small structures. After that, the absorption of simple phenolic compounds involves the activity of intestinal enzymes secreted to hydrolyze glycosidic bonds: lactase phloridizin hydrolase located in the brush border of the small intestine and cytosolic β-glucosidase within epithelial cells of the small intestine. Then, absorption of small phenolic compounds, particularly aglycones, occurs by passive diffusion (lactase phloridizin hydrolase activity) or active transport by ­sodium-dependent glucose transporter (cytosolic β-glucosidase) (D’Archivio, Filesi, Varì, Scazzocchio, & Masella, 2010; Tarko, Duda-Chodak, & Zając, 2013).

Polyphenols: Bioaccessibility and bioavailability of bioactive components315 Food + Softening of food matrix + Breakdown of food matrix Food processing

– Oxidation – Polymerization – Reaction with other molecules

Food matrix

Simple phenolic compounds

Diffusion or active transport

Mastication, stomach pH and intestinal enzymes

Food matrix

Aglycones and monomeric phenolic compounds

Diffusion or active transport

Intestinal gut microbiota Diffusion or Monomeric and active transport metabolized phenolic compounds Absorbed phenolic compounds Liver and kidney enzymes Food matrix Non-absorbed phenolic compounds Urinary excretion

Conjugated phenolic compounds

Tissues and cells

Faecal excretion Faecal excretion

Fig. 11.1  Schematic events involved in digestion and bioaccessibility and bioavailability of dietary phenolic compounds.

Unaltered or non-absorbed phenolic compounds are now moved to the colon where gut microbiota play a critical role in the transformation of phenolic compounds for further absorption. Many oligomeric and polymeric compounds undergo biotransformation that reduces molecular weight and formation of derivatives that can be more easily absorbed. In this regard, gut microbiota produces β-glucosidase, β-rhamnosidase, and esterases to break the linkage between aglycones and sugar and related structures.


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From these reactions, simpler phenolic compounds are formed from the glycosidic, oligomeric, and polymeric phenolic compounds (Tarko et al., 2013). Although absorption can be facilitated by gut microbiota activity, the variability within individual gut microbiota is a determinant factor to increase the bioaccessibility of phenolic compounds. This condition can be observed in the case of ellagitannins, which are transformed into simple and readily absorbed phenolic compounds. Ellagitannins (e.g., vescalagin and punicalagin) are degraded by gut microbiota leading to the formation of ellagic acid and a unique group of compounds known as urolithins. Once urolithins are absorbed by intestinal cells, a series of reactions take place in the liver where methyl, glucuronide, and sulfate derivatives are formed (Landete, 2011). However, the formation of this new class of bioactive compounds requires the activity of gut microbiota that varies between individuals (Li et al., 2015). The importance of such events and agents is related to the structure of phenolic compounds in dietary sources that usually occur as esters, glycosides, oligomers, and polymers and require human or microbial enzymes to form absorbable compounds (Tarko et al., 2013). Following the sequence of events, once simple phenolic compounds are absorbed, the transformation of phenolic structure also occurs in the liver where many enzymes catalyze glucuronidation, methylation, or sulfation reactions leading to formation of new compounds with potentially different effects from those displayed by the precursor molecules. Catechol-O-methyltransferase, largely expressed in the liver and kidneys, produce methyl-derivatives of quercetin, cyanidins, and catechins; sulfotransferase catalyzes the formation of sulfate derivatives of phenolic compounds, and uridine-5′-diphosphate is involved in the inclusion of glucuronide acid to structure of phenolic compound. After the event in former organs, phenolic compounds and/or derived compounds are transported to the tissue where their bioactivity may be similar or not to precursor compounds (Tarko et al., 2013; Walle, 2004). The percentage of bioaccessible phenolic compounds is usually