Inulin: chemical properties, uses and health benefits 9781536123241, 1536123242

Table of contents :
INULIN:CHEMICAL PROPERTIES, USESAND HEALTH BENEFITS......Page 2
INULIN:CHEMICAL PROPERTIES, USESAND HEALTH BENEFITS......Page 4
NOTICE TO THE READER......Page 5
CONTENTS......Page 6
PREFACE......Page 8
ABSTRACT......Page 12
1. INTRODUCTION......Page 13
2. CHEMICAL STRUCTURE AND PROPERTIES......Page 14
3. NUTRITIONAL AND HEALTH BENEFITS......Page 17
4. FOOD APPLICATIONS......Page 23
CONCLUSION......Page 28
REFERENCES......Page 29
BIOGRAPHICAL SKETCH......Page 41
ABSTRACT......Page 44
2. BRIEF HISTORY OF VACCINES......Page 45
3. BASIC REVIEW OF IMMUNE RESPONSES TO VACCINES......Page 46
4. OVERVIEW OF VACCINES......Page 51
5. DESCRIPTION OF INULIN......Page 57
6. GAMMA INULIN-BASED VACCINE ADJUVANTS......Page 58
7. DELTA INULIN-BASED VACCINE ADJUVANTS......Page 65
8. INULIN-BASED ADJUVANTS IN ANIMAL VACCINES......Page 75
10. OTHER USES OF GAMMA INULIN, ALGAMMULIN,DELTA INULIN, AND ADVAX......Page 79
11. DELIVERY VEHICLES –ALTERNATIVE APPROACH TOFORMULATING INULIN ADJUVANTS......Page 80
CONCLUSION......Page 88
REFERENCES......Page 90
ABSTRACT......Page 120
INTRODUCTION......Page 121
PREBIOTICS......Page 122
FAT PROPERTIES AND USE OF FAT REPLACERS......Page 127
INULIN AS FAT REPLACER IN YOGHURT......Page 131
INULIN AS FAT REPLACER IN ICE CREAM......Page 145
INULIN AS FAT REPLACER IN CHEESES......Page 147
INULIN AS FAT REPLACER IN DAIRY DESSERTS......Page 153
CONCLUSION......Page 156
REFERENCES......Page 157
INDEX......Page 164
Blank Page......Page 0

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BIOCHEMISTRY RESEARCH TRENDS

INULIN CHEMICAL PROPERTIES, USES AND HEALTH BENEFITS

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BIOCHEMISTRY RESEARCH TRENDS

INULIN CHEMICAL PROPERTIES, USES AND HEALTH BENEFITS

CHRISTIAN R. DAVIS EDITOR

Copyright © 2017 by Nova Science Publishers, Inc. All rights reserved. No part of this book may be reproduced, stored in a retrieval system or transmitted in any form or by any means: electronic, electrostatic, magnetic, tape, mechanical photocopying, recording or otherwise without the written permission of the Publisher. We have partnered with Copyright Clearance Center to make it easy for you to obtain permissions to reuse content from this publication. Simply navigate to this publication’s page on Nova’s website and locate the “Get Permission” button below the title description. This button is linked directly to the title’s permission page on copyright.com. Alternatively, you can visit copyright.com and search by title, ISBN, or ISSN. For further questions about using the service on copyright.com, please contact: Copyright Clearance Center Phone: +1-(978) 750-8400 Fax: +1-(978) 750-4470 E-mail: [email protected].

NOTICE TO THE READER The Publisher has taken reasonable care in the preparation of this book, but makes no expressed or implied warranty of any kind and assumes no responsibility for any errors or omissions. No liability is assumed for incidental or consequential damages in connection with or arising out of information contained in this book. The Publisher shall not be liable for any special, consequential, or exemplary damages resulting, in whole or in part, from the readers’ use of, or reliance upon, this material. Any parts of this book based on government reports are so indicated and copyright is claimed for those parts to the extent applicable to compilations of such works. Independent verification should be sought for any data, advice or recommendations contained in this book. In addition, no responsibility is assumed by the publisher for any injury and/or damage to persons or property arising from any methods, products, instructions, ideas or otherwise contained in this publication. This publication is designed to provide accurate and authoritative information with regard to the subject matter covered herein. It is sold with the clear understanding that the Publisher is not engaged in rendering legal or any other professional services. If legal or any other expert assistance is required, the services of a competent person should be sought. FROM A DECLARATION OF PARTICIPANTS JOINTLY ADOPTED BY A COMMITTEE OF THE AMERICAN BAR ASSOCIATION AND A COMMITTEE OF PUBLISHERS. Additional color graphics may be available in the e-book version of this book.

Library of Congress Cataloging-in-Publication Data ISBN: H%RRN

Published by Nova Science Publishers, Inc. † New York

CONTENTS Preface Chapter 1

Chapter 2

Chapter 3

Index

vii Inulin: Technological Applications and Health Benefits Celso Fasura Balthazar, Adriano Gomes da Cruz and Marcus V da S Ferreira Immunostimulatory Inulin Adjuvants in Prophylactic Vaccines against Pathogens Matthew D. Gallovic, Eric M. Bachelder and Kristy M. Ainslie Inulin as a Fat Replacer in Dairy Products Tatiana Colombo Pimentel, Suellen Jensen Klososki, Michele Rosset, Carlos Eduardo Barão and Gislaine Silveira Simões

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PREFACE In the food industry, inulin is widely used as ingredient in food stuff, due to its technological and functional properties. Chapter One describes inulin as an ingredient that provides low caloric intake and is a source of fiber itself. The authors argue that inulin gathers beneficial characteristics to enhance health, when added to many different products. Chapter Two discusses the history, development, and application of preclinical and clinical vaccines that contain inulin formulations. Chapter Three covers the performance of inulin as a fat replacer in dairy products, giving an overview of the influence of inulin addition on the textural, rheological, prebiotic and sensory properties of the dairy products. Chapter 1 - Inulin is a carbohydrate polymer of fructose units (2 - 60 fructose units) constituted by a β (2,1) linked to fructosyl residues ending with a glucose residue. This compound is found in many vegetables such as onion, garlic, banana and others. The main content of inulin is found in chicory root (15 – 20%). In food industry, inulin is widely used as ingredient in food stuff, due to its technological and functional properties. This fiber may module the gut flora and brings many positive effects to host wellbeing, being considered a prebiotic. Some studies pointed other beneficial effects resulted in inulin consumption as food ingredient: rise in calcium absorption; reduction in blood stream lipids; weight control; and immune system stimulation. However, to provide inulin’s beneficial effects, the host must ingest a minimum amount of fiber, which is in

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accordance to legislation of each country. The capability of inulin to increase texture and softness of food stuff is the mainly importance of this fiber use for fat replacement by industry. It may also increase the sweet taste and mouthfeel. Inulin’s protective effect favors probiotics survival and viability in products and through gastrointestinal transit. Nowadays, consumers are more conscious about what they eat and the consequences for their healthy. Thus inulin is an interesting food ingredient which provides low caloric intake and is a source of fiber itself. Inulin gathers beneficial characteristic to enhance health, when added to many different products. Chapter 2 - Vaccines that protect against pathogens are considered one of the medical world’s greatest achievements. Several types of vaccines exist, including subunit or inactivated/live-attenuated pathogen formulations. A component known as an adjuvant is commonly included in subunit vaccines to boost immunogenicity, and can be added to the other vaccine types for the same purpose. Traditional adjuvants such as aluminum (alum)-based preparations have several significant shortcomings, including the inability to drive Th1-biased cellular immunity. Because protection against many pathogens (e.g., viruses) often requires this type of immune response, novel adjuvants are needed. Two particulate isoforms of the naturally-occurring inulin polysaccharide have emerged as new adjuvant candidates. Gamma inulin was the first of these isoforms used in vaccine development. A popular gamma inulin formulation known as Algammulin consisted of gamma inulin cocrystallized with the antigen-adsorbing alum. The immunostimulatory effects of the second isoform, delta inulin, were discovered more recently. It has emerged as an enticing alternative to gamma inulin formulations because of its enhanced stability, as well as reproducibility in both manufacturing and immunostimulation. Delta inulin, known as AdvaxTM in its adjuvant formulation, has been examined preclinically in many vaccines and has also reached clinical testing for safety and immunogenicity against several pathogens. In addition to Algammulin and Advax, a select number of inulin preparations have been engineered into dual-functioning systems that serve as both an antigen delivery vehicle and adjuvant. This chapter

Preface

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will discuss the history, development, and application of preclinical and clinical vaccines that contain inulin formulations. Chapter 3 - Dairy products are regarded by consumers as healthy products because they aid in the digestion process, are essential for the bones, and help the immune system. However, due to the negative relationship between consumption of saturated fats and heart disease, reducing animal fat in the diet has been recommended by nutritionists, increasing the consumption of low-fat or fat-free products. Milk fat has an important role in the development of texture, flavor and color of dairy products; and its reduction can cause defects such as loss of flavor and consistency or lack of texture, which makes indispensable the use of fat replacers in obtaining fat-free products with technological and sensory properties similar to those of full-fat products. Inulin is a prebiotic dietary fiber and can be used in a wide range of healthy products due to its ability to improve texture and its neutral and slightly sweet taste. The low caloric value makes it an ideal ingredient. Inulin has been used as a fat replacer in the dairy industry and has shown positive effects on rheology and product stability. This chapter aims to describe the performance of inulin as a fat replacer in dairy products, giving an overview of the influence of inulin addition on the textural, rheological, prebiotic and sensory properties of the dairy products.

In: Inulin Editor: Christian R. Davis

ISBN: 978-1-53612-301-2 © 2017 Nova Science Publishers, Inc.

Chapter 1

INULIN: TECHNOLOGICAL APPLICATIONS AND HEALTH BENEFITS Celso Fasura Balthazar1,*, Adriano Gomes da Cruz2 and Marcus V da S Ferreira3 1

Department of Food Science and Technology, Universidade Federal Fluminense, Rio de Janeiro, Brazil 2 Instituto Federal de Educação, Ciência e Tecnologia do Rio de Janeiro, Rio de Janeiro, Brazil 3 Universidade Federal Rural do Rio de Janeiro, Brazil, Seropédica, Brazil

ABSTRACT Inulin is a carbohydrate polymer of fructose units (2 - 60 fructose units) constituted by a β (2,1) linked to fructosyl residues ending with a glucose residue. This compound is found in many vegetables such as onion, garlic, banana and others. The main content of inulin is found in chicory root (15 – 20%). In food industry, inulin is widely used as ingredient in food stuff, due to its technological and functional properties. This fiber may module the gut flora and brings many positive effects to

*

Corresponding author: [email protected].

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C. Fasura Balthazar, A. Gomes da Cruz and M. V da S Ferreira host wellbeing, being considered a prebiotic. Some studies pointed other beneficial effects resulted in inulin consumption as food ingredient: rise in calcium absorption; reduction in blood stream lipids; weight control; and immune system stimulation. However, to provide inulin’s beneficial effects, the host must ingest a minimum amount of fiber, which is in accordance to legislation of each country. The capability of inulin to increase texture and softness of food stuff is the mainly importance of this fiber use for fat replacement by industry. It may also increase the sweet taste and mouthfeel. Inulin’s protective effect favors probiotics survival and viability in products and through gastrointestinal transit. Nowadays, consumers are more conscious about what they eat and the consequences for their healthy. Thus inulin is an interesting food ingredient which provides low caloric intake and is a source of fiber itself. Inulin gathers beneficial characteristic to enhance health, when added to many different products.

Keywords: fiber, functional ingredient, health, inulin, prebiotic

1. INTRODUCTION Inulin is a soluble dietary fibre (Roberfroid, 2005), which do not form highly viscous solutions (Tungland; Meyer 2002). It is presented in over 3000 vegetables and is extensively distributed worldwide (Wichienchot et al., 2011). It also has been contributing to human daily food intake for centuries with its nutritional properties and technological benefits (Giarnetti et al., 2015). Inulin was first discovered in early eighteenth century by a german scientist Valentine Rose from the roots of elecampane herb (Inula helenium), also known as horseheal or scabwort. Inulin spherocrystals were detected in dahlia, Jerusalem artichoke and elecampane by Julius Sachs in 1864. Likewise, the natural sources of inulin include chicory roots, Jerusalem artichoke, dahlia tubers, yacon, asparagus, leek, onion, banana, wheat, agave and garlic (Roberfroid, 2007). Chicory (Cichorium intybus) is the principal commercial source of food grade inulin and its tubers contain approximately 15–20% (w/w) inulin, which is extracted in a process similar to the extraction of sucrose from sugar beet (Karimi et al., 2015).

Inulin: Technological Applications and Health Benefits

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Inulin is widely used by food industry as fat or sugar replacer, due to its fat like technological features. It gives between 25 to 35% of energy compared to the digestible carbohydrates and also presents 10% of sweetness in sucrose equivalent (Shoaib et al., 2016). Due to inulin’s complex structure, human enzymes are not capable of digest it, being this fiber assimilated by benefic bacteria in colon. Therefore it is considered a prebiotic dietary fiber (FAO 2007; Rastall & Gibson, 2015; Wu et al., 2016), which its benefits are associated with many benefits such as constipation prevention (Marteau et al., 2011) blood sugar management (Gargari et al., 2013; Han et al., 2013), bifidogenic effect (Meyer et al., 2011), immunological system stimulation (Lomax; Calder, 2009), gastrointestinal infections resistance (Cummings et al., 2001), mineral absorption (Meyer; Stasse-Wolthuis, 2006), blood stream lipids reduction (Brighenti, 2007), weigh control (Parnell ; Reimer, 2009) and colon cancer prevention (Rafter et al., 2007).

2. CHEMICAL STRUCTURE AND PROPERTIES Inulin, classified as a oligossacharide fructan, is a polysaccharide structure: α-D-Glu[-(1-2)-β-D-Fru]n (n = 10 – 60) (Figueroa-González et al. 2011; Roberfroid et al. 2010). The β-(2-1)-d-frutosyl fructose bonds are presented between the fructose units of inulin and configuration of anomeric carbon, which makes it indigestible in the human small intestine. Although, inulin can be fermented in the large intestine by the intestinal microflora (Apolinario et al., 2014). Structure of inulin molecule could be seen in Figure 1. Specifically, inulin-type fructan consists in linear (2 → 1)-linked -dfructosyl units attached to the fructosyl moiety of sucrose. In chicory inulin, the number of fructose units varies from 2 to 60 indicating a combination of oligomers and polymers (Jeurink et al., 2013). The degree of polymerization (DP) and branches have an effect on the functionality of the inulin. Plats containing inulin usually has relatively low DP (maximally < 200), which depends on the plant species, climatic conditions and the

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plant’s physical condition. Inulin presented in bacteria has a very high DP, ranging from 10,000 to above 100,000; furthermore, a bacterial inulin is 5% more branched than the plant one. Synthetically, inulin-type fructans are made from sucrose (Cooper et al., 2015). Chicory is the most common and economic plant used commercially to produce inulin (Rastall et al., 2015). Although, dahlia and Jerusalem artichoke are also considered good sources for industrial production in tropical areas (Flamm et al., 2001). Chicory grows twice a year and belongs to the Asteraceae family. During the first year of growth, its plants persist in the vegetative phase by forming only leaves, taproots and roots. The rootstocks have considerable resemblance with small oblong sugar beets (Kelly, 2008). Inulin production presents to stages, such as extraction and initial purification of raw syrup. Furthermore it is refined to produce commercial product (above 99.5%). Advanced technologies are also being applied in the inulin extraction process in order of getting higher yields of purified final product with less energy consumption, such as supercritical carbon dioxide (Mendes et al., 2005), ultrasound assisted extraction (Lingyun et al., 2007), simultaneous ultrasonic/microwave (Lou et al., 2009) and pulsed electric field (Loginova et al., 2010).

Figure 1. Structure of inulin molecule. From Wikimedia Commons.

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Chicory roots are sliced into chips and inulin is extracted with hot water partially hydrolyzed by endo-inulinase. Furthermore by carbonatation process the proteinaceous and other contaminants are removed. Afterwards, raw inulin is chromatographically demineralized and decolored prior to spray drying (Rastall et al., 2015). In classical purification process, inulin pass through clarification, which requires multiple steps (i.e., pre-liming, liming and carbonation) at relatively high temperature (80–90°C) in order to remove impurities from the extracted juice (Franck; De Leenheer, 2005). Moreover, inulin molecules are hydrolyzed and additional calcium ions are introduced to clarify the juice, which requires further purification treatments (Kim et al., 2001). Microfiltration and ultrafiltration i.e., membrane technology, are also reported to facilitate those steps. The result comes from inulin ranging between 3 up to 60 DP. In addition a high quality long chain inulin with more than 23 DP is also obtained (Cho; Samuel, 2009). As an ingredient, inulin offers many important dietary benefits and industrial properties in food applications (Roberfroid, 2005). Chicory inulin is a white powder with fine particles, whicg have better clarity. Moreover, the neutral flavor of the inulin offers no aftertaste. Despite its long-chain inulin is not sweet i.e., 10% sweetness level, when compared to sucrose (Valluru; Van den Ende, 2008). Also, inulin behaves similar to bulk ingredients and along with high levels of artificial sweeteners such as aspartame or acesulfame K, which provide a good mouthfeel with a little aftertaste (Franck, 2002). Moreover, this compound is moderately dissolved in water i.e., nearly 10% at 25°C, which enables it to be added in aqueous system without any precipitation. Use of heated water or milk at 50–100°C is also recommended in order to make inulin solution (Balthazar et al., 2015, 2016, 2017). Chicory inulin solutions have relatively low viscosity, i.e., 5% solution, 1.65 mPa.s and 30% solution, 100 mPa.s both at 10°C (Kalyani Nair et al., 2010). Freezing and boiling points of water are affected by inulin in a small extent i.e., 15% chicory inulin reduces the freezing point by 0.5°C. Low pH, high temperature and less dry-substance environments are the critical parameters for inulin hydrolysis (Roberfroid,

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2007). Furthermore, β-(2-1) bonds between the fructose units can be partially hydrolyzed in highly acidic environment. Inulin shows gelling properties at high level (for standard chicory inulin > 25% and for long-chain inulin > 15%), which produces a gelling structure that afterwards pass thorugh a shearing process. When the inulin is totally dissolved in water or any other aqueous medium, with shearing instrument like a rotor mixer or homogenizer, it results in the formation of a white creamy structure that can easily be added in foods as a fat replacer up to 100% (Imeson, 2010). Even Though, inulin is not affected by pH between 4 and 9, the gelling property of inulin is influenced by some variables, such as the concentration, quantity of total dry substance, shearing factors and by the kind of shearing instrument used. Moreover, inulin gels are made up of a 3-D structure which are normally non-soluble submicron inulin fragments in water (Zimeri; Kokini, 2002).

3. NUTRITIONAL AND HEALTH BENEFITS Currently, dietary fiber is considered as a key ingredient to improve human health and the attention towards the dietary fiber with enriched foods has been intensified due to its health properties. The basic characteristics of dietary fiber is the fact that it is not digested by the gastrointestinal secretions or absorption in the small intestine whereas, it is fermented by the microflora in the large intestine (FAO 2007; Rastall & Gibson, 2015; Shoaid et al., 2016; Wu et al., 2016). The summarized description of health promoting inulin fiber characteristics is presented in Table 1. Inulin is a storage carbohydrate in plants, having fructose moieties joined by β-(2-1)-d-frutosyl linkages and is resistant to digestion in human small intestine due to the β-configuration of anomeric C-2 but it can be fermented in large intestine by micro flora (Apolinario et al., 2014). Almost 90% of the inulin passes to the colon where it is digested by bacterias (Cherbut, 2002). Due to inulin is not digested by humans, its caloric value is low i.e., 1.5 kcal/g or 6.3 kJ/g.

Inulin: Technological Applications and Health Benefits

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Table 1. Summarized health promoting inulin fiber characteristics Low caloric Decrease of lipidemia

Intestinal pH reduction Short chain fatty acids metabolization Bifidogenic effect Decreases risk of intestinal diseases Colon cancer prevention Immune modulation Mineral absorption

As a non digestible by human, this fiber is considered low caloric (1.5 kcal/g or 6.3 kJ/g). Studies have shown reduction of triacylglycerol in bloodstream, as well cholesterolemia and reabsortion of circulating bile acids. Also a reduction in hepatic fatty acid and triacylglycerol synthesis. Human enzymes cannot break inulin molecules and absorb its fractions, resulting in lowering the pH of intestine. Thus this fiber may increase stool frequency and consistency. Inulin alter the constitution of SCFA, promoting constipation relieve. Improves the development and metabolic action of bacteria in the colon, as Bifidobacteria and Lactobacilli genus among others. By manipulating the composition of intestinal microorganisms of the intestine. Deceases of amount and mass lesions, lowering the malignancy of cancer and modulates microorganisms, chancing SCFA composition and promotes immune system improvement by stimulating immune cells. reduction of intestinal pH by SCFA and organic acids production, promoting calcium absorption and increase bioavailability.

By the action of intestinal bacteria, inulin is metabolized into shortchain fatty acids, such as acetate, propionate, and butyrate; lactate, and gases (Nyman, 2002). Merely, short chain fatty acids and lactate can add energy to the metabolism of a host organism. Bacteria and host cell can also use some part of short chain fatty acids. There are evidences in both in vitro and in vivo, showing that the energy rate of inulin ranges from 1 and to 1.5 kcal/g (Flamm et al., 2001). Many studies on humans shows that inulin reduces triglyceridemia. The addition of inulin on a diet can decrease the risk of high triacylglycerol concentrations in blood. A study showed that supplementation of inulin reduced significantly the levels of triacylglycerol in bloodstream of

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volunteers, whereas other volunteers did not have effect on blood lipids (Williams; Jackson, 2002). Animal tests have shown that inulin influences lipid metabolism mainly by lowering triglyceridemia and decreasing cholesterolemia (Delzenne et al., 2005). Concerning the inulin mechanism the addition of its compound to diet can lower the lipid breakdown in the liver by suppressing the production of the genes responsible for lipogensis (Roberfroid, 2007). The lipid-lowering action of inulin is possibly due to some factors such as changes in the hepatic triacylglycerol synthesis, lowers in the density lipoprotein cholesterol secretion and decreases in the reabsorption of circulating bile acids (Trautwein et al., 1998). At low pH, the amount of soluble bile acids decreases. As a result, lipid absorption decreases and fecal bile acid excretion increases (Vanhoof; De Schrijver, 1995). Thus, higher bile acid excretion leads to an increased utilization of liver cholesterol to re-synthesize bile acids. Moreover, what usually happen is that, while hepatic pools of free cholesterol decreases to reach a new steady state, endogenous cholesterol synthesis increases. This leads to a high activity of the 7-α-hydroxylase3-hydroxy-3-methylglutarylcoenzyme, which is a reductase i.e., HMG-CoA that compensate the loss of bile acids and cholesterol from liver stores. Furthermore, hepatic low density lipoprotein (LDL) receptor-related protein (LRP) is regulated to restore hepatic cholesterol stores, which will lead to a decreased serum cholesterol concentration (Ellegard; Andersson, 2007; Theuwissen; Mensink, 2007). Studies in rodents demonstrate that inulin can decrease the plasma cholesterol and triacylglycerol. Additionally, it can stop the triacylglycerol’s accumulation in the liver with favorable influences on hepatic steatosis. The decrease in liver lipogenesis is perhaps the main accountable mechanism to the decrease in plasma triacylglycerol’s levels, both in humans and animals. However the mechanism of the hypocholesterolaemic action is still unclear (Shoaib et al., 2016). Inulin may cause a reduction in hepatic fatty acid and triacylglycerol synthesis through a coordinated reduction in the activity of all lipogenic enzymes. The triacylglycerol lowering the effect of short-chain inulin observed in rats was caused by its antilipogenic action in the liver, which was achieved by reducing the activity and possibly the expression of all

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lipogenic enzymes. Due to prebiotic intake significantly reduces serum insulin and glucose, which induces lipogenic enzymes (Delzenne; Kok, 1999). The inviability of human enzymes to digest inulin results in the effectiveness of the gut, lowering the pH in the intestine, also providing assistance in relieving constipation and increasing stool load, which is known as bulking effect (Anderson et al., 2009). Inulin increases about 1.5–2 g of the wet fecal weight, which improves stool frequency and consistency (Den Hond et al., 2000). Inulin may alter constitution of the short chain fatty acids and microbiotic colonies, resulting in variations in the intestinal activity. In old age, probiotic and prebiotics strains facilitate to reduce constipation (Kolida; Gibson, 2007). Due to prebiotic action of inulin, it acts as a substrate for probiotics. Thus, chicory inulin i.e., 20–40 g/day, had a sufficient cathartic result in relieving constipation (Fernández-Banares, 2006; Kolida; Gibson, 2007). The bifidogenic effect is the development and metabolic action of a limited number of bacteria in the colon, which was stimulated by inulin, in a particularly Bifidobacteria and Lactobacilli, which are known for its high quality and health promoters (Eckburg et al., 2005; Kamarul Zaman et al., 2015). The colonic fermentation depends on the chain length of the inulin, as well the fermentation time of short degree of the polymerization fractions, which is two times higher than long chain inulin. Therefore, the long-chain inulin exert activity in distal areas of the colon due to its high polymerization levels (Allsoop et al., 2013; Gallego; Salminen, 2016). Inulin can be used as a fat replacer in non-fat functional dairy products, providing approximately the same sensory characteristics (Akın et al., 2007; Cruz et al., 2010; Balthazar et al. 2015). However this substance improves firmness as a result of increased microbial activity caused by metabolic relations between lactic acid bacteria i.e., Lactobacillus acidophilus, Lactobacillus rhamnosus and Bifidobacterium lactis, and partial inulin metabolization (Oliveira et al., 2011). Microbial stability of patients suffering from intestinal irregularity are re-established by inulin incorporation on a diet, favoring microbial balance and assists in

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overcoming the difficulties of the epithelia by prebiotic action. The reason for that is it may provide the host some defense against attacks and translocation of endogenous or exogenous microbes and inhibit gastrointestinal diseases (Bosscher et al., 2006). Inulin exerts favorable properties in decreasing the risk of many diseases of the intestinal tract, such as irritable bowel disease, ulcerative colitis, Crohn’s disease, and inflammatory bowel disease. Manipulating the composition of intestinal microflora by using probiotics and/or prebiotics can be applied as therapeutic approach to prevent chronic intestinal inflammation (Mendis et al., 2016) Colon cancer is one of the most common reasons of death in developed countries. It results from colon cell mutation or in the appendix (Cancer Genome Atlas, 2012). Studies demonstrated that combination of inulin, oligofructose and probiotic bacteria i.e., Lactobacillus rhamnosus and Bifidobacterium lactis, prevent colon carcinogenesis in rats and mice (Pool-Zobel, 2005) and in humans (Rafter et al., 2007). However, the use of long-chain inulin is more beneficial than short-chain inulin and oligofructise, since the long-chain molecules take more time for fermentation in large intestine, thus extending its influences in the distal colon. Long-chain inulin take action generally in the progression of the cancer, by decreasing the amount and mass of lesions as well as lowering the chance of malignancy on those lesions (Poulsen et al., 2002). The beneficial roles of inulin in colon are specifically modulation microflora, which changes shot-chain fatty acids composition through anaerobic fermentation, mainly butyrate; and immune system of gastrointestinal improvement and promotion (Van Loo et al., 2005). Many theories have been proposed about the contribution of inulin to the enhanced mineral absorption. One way might be the reduction of intestinal pH, as colonic fermentation of inulin, which produces short chain fatty acids or additional organic acids that results in decreased pH in the large intestine (Coxam, 2005). Microflora lows the pH, which increases the bioavailability of calcium. Thus, it results in an improvement of calcium absorption through passive diffusion in the small intestine and the first part of the large intestine. Furthermore, by ion exchange system there is a

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possibility that short chain fatty acids have an influence the calcium absorption. Additionally, by changing the action of vitamin D receptor and increasing calbindin D9K, inulin can amend trans-cellular active calcium transport where vitamin D controls this action by producing cytosolic calcium binding protein and calbindin D9K. Another way of increasing mineral incorporation is amplifying butyrate yield or some polyamine, which inulin can stimulate the cell growth and increase the intestinal absorptive region (Scholz-Ahrens et al., 2001; Scholz-Ahrens; Schrezenmeir, 2002). Research trials have shown on rats that inulin-type fructans enhanced the mineral absorption, particularly calcium and magnesium (Weaver, 2005), increasing whole body bone mineral content and bone mineral density (Bosscher et al., 2006). As a result, that mineral incorporation reflects in bone density, bone mineralization, bone growth and re-absorption, such as bone turnover (Ghoddusi et al., 2007). In humans, inulin-type fructans have little influence in small intestine for mineral absorption, so their beneficial impact on calcium or magnesium absorption are probably influenced by changes in the last part of the intestine, which is facilitated by the action of the microflora (Holloway et al., 2007). The beneficial was evident in adolescents (Griffin; Abrams, 2005; Griffin et al., 2003) and postmenopausal women (Tahiri et al., 2003; Takahara et al., 2000). Current studies also determine the effects of inulin on Fe absorption under certain conditions such as in a case of its deficiency (Clark, 2009); and sulfide degree decreases in the colon (Yasuda et al., 2006). Inulin may regulate food intake and appetite via short-chain fatty acids i.e., acetate, propionate and butyrate, produced by fermentation in the colon (Tarini & Wolever, 2010). High level of short-chain fatty acids in the colonic lumen may enhance the expression of glucagon-like peptide-l (GLP-l) in the mucosa, thus it increases the blood GLP-l levels while lowers the levels of hormone ghrelin (Cho; Samuel, 2009). GLP-l is an appetitehiding peptide, which when in the bloodstream at a certain level is linked to subjective hunger assessment and the decrease in food intake, whereas ghrelin hormone stimulates hunger and consequently food

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ingestion (Drucker, 2002). However, more human investigation are required (Delzenne et al., 2005). The immune system is indirectly stimulated by T cell functions, which are natural killer cells and phagocytic responses. The alteration in concentration of lactic acid bacteria in the gastrointestinal tract may protect the body from pathogens and tumor (Kelly-Quagliana et al., 2003). Also studies in mice have shown that the mixture of inulin and oligofructose (70/30) increases the oral vaccine efficacy by stimulating immunoglobulin G, fecal immunoglobulin A and natural killer cell activity against Salmonella typhimurium (Benyacoub et al., 2008; Kelly-Quagliana et al., 2003). Inulin intake improved immune response to vaccination against Influenza virus and Pneumococcus in elderly and infants (Bunout et al., 2004; Hegar et al., 2004). There was postulated that inulin and its derived compounds has a direct interaction with the gut-associated lymphoid tissues of mice (Roberfroid, 2005). However, this interaction has not yet been found in humans. As an indigestible fiber, inulin exerts osmotic effects, enhancing the presence of water in the colon, which leads to a purgative effect. Moreover it produces gases by the fermented goods (Cho; Samuel, 2009) where the flatulence is a recognized and a frequently after effect of inulin ingestion, when it is consumed over than10 g per day (Bonnema et al., 2010; Turner; Lupton, 2011). Futhermore, its comsume of 30 g per day or more can lead to diarrhea. (Den Hond et al., 2000). However, short and long-term intakes of inulin-rich soluble chicory extract when taken in daily doses of 5 g are well tolerated by healthy subjects (Ripoll et al., 2010).

4. FOOD APPLICATIONS The wide use of inulin in the food industry is based on its technogical attributes and the great interest to develop healthy products aiming the consumers requirement such as fiber-enriched, prebiotic, low fat and low sugar (Franck, 2002). As a dietary fiber, inulin is undigested by human gastrointestinal enzymes, and as a prebiotic it is selectively fermented by

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the beneficial micro flora, which enhances their growth and strengthens its action against pathogenic microorganisms (Van Loo, 2004). As a technological characteristic, highly branched inulin polymers have more solubility, within the presence of water inulin is capable to develop a particulate gel, enhancing taste and texture. Thus altering the product texture and providing a fat-like mouthfeel (Karimi et al., 2015), while short-chain molecules enhance flavor, sweetness and are used to partially replace sucrose (De Castro et al., 2009). Technical and dietetic benefits of inulin make it a good choice to be used as an essential ingredient in diet and it is mostly used to offer dual benefits: a better sensorial character and a nutritious value (Roberfroid, 2007). In breakfast cereals and bakery items, the inulin offers important improvement when compared to other fibers (Franck, 2002). Incorporation of inulin in baked products not only keeps them moist and fresh for a long period but also improve their crispiness (Gulati, 2012). Its solubility allows the fiber addition in aqueous environment such as drinks, dairy products, thickened beverages and table spreads (Dahl et al., 2005). For an efficient prebiotic, a molecule should have some qualities, such as it should not be hydrolyzed or absorbed in the upper part of the gastrointestinal tract; gut micro-flora should be fermented and beneficial bacteria of colon should be stimulated (Gibson, et al., 2004; Van Loo, 2004). The addition inulin has demonstrated higher stability and suitable sensory characteristics in fermented milks. In addition, the use of supplements, such as whey and whey concentrates, can potentiate the probiotic viability and the physicochemical and sensory characteristics of probiotic dairy products (Castro et al., 2013a, b). As supplement in skim milk, inulin considerably increases the growth and sustainability of Lactobacillus spp and Bifidobacterium spp in non-fat fermented milk (Closa-Monasterolo et al., 2013). Various prebiotic dairy desserts with low fat content have been prepared using inulin as a prebiotic, in which inulin supplementation not only was giving a prebiotic effect but also was reducing the fat content and sugar content without affecting its acceptability within consumers (Arcia et al., 2011; Balthazar et al., 2015, 2017).

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Inulin products consisting mainly of long-chain molecules are applied as fat replacement, within the presence of water they are capable to develop a particulate gel, thus alter the product texture and provide a fatlike mouthfeel (Karimi et al., 2015; Tungland & Meyer, 2002). In non-fat functional dairy foods inulin can be used as a fat replacer and provides them nearly the same sensory characters as a full fat products (Akın et al., 2007; Baltahzar et al., 2017; Cruz et al., 2010; Solowiej et al., 2015). Scientists have analyzed the effect of long chain inulin addition on physical and sensorial features in dairy foods whether to yogurt or custard. Long-chain inulin has been used in low-fat yogurts to replace fat where it was shown a considerably improve on creaminess, mouthfeel and smoothness (Modzelewska-KapituŁ; Bukowska, 2009). Moreover, the addition of long-chain inulin to low fat custards enhanced creaminess and consistency (Salvatore et al., 2014). Fat replacer can additionally be used in meal replacers, meat products, sauces and soups, which will contribute todecrease fat in meat products, however enhancing some attributes such as the juicy, creamy mouthfeel and an enhanced firmness, due to its water control (Cho; Samuel, 2009). The addition of inulin to added-fat containing meat products i.e., sausages, could be a way to health conscious consumers regarding its significance to human nutrition following the dietary guidelines (Keenan et al., 2014; Menegas et al., 2013). Inulin addition in bakery could be used to obtain fat replacement and good sensory properties (Laguna et al., 2014). Inulin has become an important component providing new ways for the food manufacturing in the development of a novel food, which has a better sensorial and nutraceutical properties by replacing sugar in chocolate and custards (Shah et al., 2010; Palazzo et al., 2011). This fiber products contain mainly short-chain molecules, which enhances the sweetness of sucrose up to 35%, thus it is useful to partially replace sucrose molecule’s flavor (De Castro et al., 2009; Villegas et al., 2010). The addition of inulin into yoghurt did not influence attributes such as acetaldehyde, pH and titratable acidity (Guven et al., 2005). The amount of fat replacer and storage time had a significant effect on the physical, chemical, textural, and sensory properties of strained yoghurts (Yazici;

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Akgun, 2004). Yoghurts containing inulin had less syneresis than the control and had a better body and texture compared to other yoghurts (Aryana et al., 2007). The analysis showed that stickiness, airiness, and thickness all contributed to the creamy mouthfeel of the yoghurts. It also demonstrate that consistence was significantly affected by inulin, which the values were higher for those samples that was extracted with ethanol and lyophilized Al-Sheraji et al., 2013). However the rheological characterization was performed by dynamic, shear, and compression– extrusion assays and did not show any significant differences (Dello Staffolo et al., 2004). Regarding the color in food fiber, it reduces the clarity and gives a yellow-greenish color to the yogurt, which also varied depending on the method of extraction and drying. The yogurts with water-extracted fibers are usually more colorful (Al-sheraji et al., 2013). Thus the sample best ranked according to a consumers test was the yoghurt containing waterextracted and oven-dried fiber. Those performed for some parameters such as aroma, taste, texture, overall acceptance, ethanol-extracted and lyophilized for color. Fiber obtained by all methods was equally compatible with yoghurt enrichment (Sanz et al., 2008). The addition of inulin to food products as a prebiotic could improve the viability and activity of probiotic bacteria (Nazzaro et al., 2009), such as L. casei-01 (Paseephol; Sherkat, 2009). Populations of Bifidobacterium, Lactobacillus and Enterococcus genus and the Atopobium group are all significantly increased after konjac glucomannan hydrolysate and inulin fermentation (Connolly et al., 2010). Kiwifruit pectin and inulin also enhance the adhesion of L. rhamnosus and decrease the adhesion of Salmonella typhimurium to CaCO-2 cells. Inulin and citrus pectin significantly enhance the adhesion of Bifidobacterium bifidum to CaCO-2 cells (Parkar et al., 2010). The use of soymilk and whey protein isolate plus inulin replacing the milk showed to be feasible in order to obtain a food matrix, which may provide protection to the probiotic strains under gastrointestinal stress conditions when compared to the milk-based counterpart. Most importantly, the ice cream mixtures containing soy extract and/or whey

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protein isolate plus inulin may expand the range of symbiotic products for individuals varying the degrees of lactose intolerance or lactose sensitiveness (Matias et al., 2016). Other feature of inulin is its capacity to produce a frozen yogurt with acceptable flavor and texture attributes, except for the melting quality due to the occurrence of very few ice crystals that was perceived by the panelists. Thus it is possible to produce an attractive frozen yogurt product with the incorporation of inulin with no added sugar and reduced fat (Isik et al., 2011). It should be possible to produce a symbiotic functional ice cream with inulin with a minimum of 3% and still have a potential probiotic product. Strains of Lb. casei and Lb. rhamnosus showed high survival by the end of production and good survival during frozen storage, which maintained counts of 106 to 107 CFU/g, even in the presence of inulin. Furthermore, 10% inulin doses altered the sensorial and physical properties of prebiotic ice creams. Using formulations with 2.5% of inulin content, which did not adversely affect ice cream characteristics, it is possible to cover the 40% intake needed for beneficial effects (Di Criscio et al., 2010). Balthazar et al. (2015) have studied different concentrations (2 or 6 g/100 g) of inulin in smoothie yogurt made from sheep milk. The results showed that Inulin did not alter the pH values of the yogurts but resulted in products with higher lightness and lower yellowness i.e., higher L∗ value and lower b∗ value. Due to its particle nature, inulin can act as a lightscattering center and increase the opaqueness. However, Inulin did not interfere with the sensory acceptance of the products, such as appearance, aroma, texture, and overall liking, and it improved the flavor acceptance. An increase in the viscosity and creaminess of sheep milk yogurt containing inulin was observed only in laboratory tests, indicating that inulin has limited effect on the sensory viscosity and creaminess of the products. According to penalty analysis, prebiotic yogurt containing 6 g/100 g inulin had minor penalties, which indicates its advantage in the quest to improve consumer demand. The authors concluded that the addition of prebiotic inulin fiber had a positive effect on the technological aspects of yogurt.

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Balthazar et al. (2016) studied the effect of the addition of inulin (0 to 6 g/100 g) on the physicochemical and microbiological characteristics of sheep milk yogurts during refrigerated storage (4°C for 28 d). Inulin delayed the post acidification during storage, which is an advantage from sensory and technological perspective. The lower acidic environment is an ideal condition to add the probiotic bacteria into food matrix, which is sensitive to the post acidification in yogurt. Furthermore, more acidic products generally have lower acceptances by consumers. Inulin addition did not interfere in the cell counts of the starter culture (S. thermophilus and L. delbruekii bulgaricus) and no significant changes have been observed in the fatty acid levels. The authors concluded that the supplementation of inulin to sheep milk dairy products is interesting for the functional foods market because of the beneficial effects of its prebiotic fiber.

CONCLUSION The concern of people about the diet and how it affects their health is a part of our daily live. Therefore, consumers demand low caloric foods with better health enhancing properties. The role of inulin as a food ingredient has been more explored in this century. This fiber is considered a functional food ingredient due to its prebiotic value, also nutritious aspect. Thus, inulin possesses tremendous effects on growth and health status of humans. Furthermore, it can be an ideal replacement of carbohydrates and fat. It also can be a good source of fiber to enrich different food products, thus develop the desired sensory characteristics. Moreover, Inulin has a significant effect on the activity of starter probiotic cultures and in the pH value, when it is used as prebiotic in dairy industry. Therefore, more research work are needed for further specification and characterization of the prebiotic potential of inulin as a dietary fiber, so it can be use in the development of new functional foods.

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Oliveira, R. P S., Perego, P., De Oliveira, M. N. & Converti, A. (2011). Effect of inulin as a prebiotic to improve growth and counts of a probiotic cocktail in fermented skim milk. LWT—Food Science and Technology, 44, 520–523. Palazzo, A. B., Carvalho, M. A. R., Efraim, P. & Bolini, H. M. A. (2011). The determination of isosweetness concentrations of sucralose, rebaudioside and neotame as sucrose substitutes in new diet chocolate formulations using the time-intensity analysis. Journal of Sensory Studies, 26, 291–297. Parkar, S. G., Redgate, E. L., Wibisono, R., Luo, X., Koh, E. T. H. & Schröder, R. (2010). Gut health benefits of kiwifruit pectins: Comparison with commercial functional polysaccharides. Journal of Functional Foods, 2, 210–218. Parnell J. A. & Reimer, R. A. (2009). Weight loss during oligofructose supplementation is associated with decreased ghrelin and increased peptide YY in overweight and obese adults. American Journal of Clinic Nutrition, 89, 1751-1759. Paseephol, T. & Sherkat, F. (2009). Probiotic stability of yoghurts containing Jerusalem artichoke inulins during refrigerated storage. Journal of Functional Foods, 1, 311–318. Pool-Zobel, B. L. (2005). Inulin-type fructans and reduction in colon cancer risk: review of experimental and human data. British Journal of Nutrition, 93, S73–S90. Poulsen, M., Molck, A. M. & Jacobsen, B. L. (2002). Different effects of short- andlong-chained fructans on large intestinal physiology and carcinogen-inducedaberrant crypt foci in rats. Nutrition and Cancer-an International Journal, 42, 194–205. Rafter, J., Bennett, M., Caderni, G., Clune, Y., Hughes, R. & Karlsson, P. C. (2007). Dietary synbiotics reduce cancer risk factors in polypectomized and colon cancer patients. American Journal of Clinical Nutrition, 85, 488–496. Rastall, R. A. & Gibson, G. R. (2015). Recent developments in prebiotics to selectively impact beneficial microbes and promote intestinal health. Current Opinion in Biotechnology, 32, 42–46.

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Ripoll, C., Flourie, B., Megnien, S., Hermand, O. & Janssens, M. (2010). Gastrointestinal tolerance to an inulin-rich soluble roasted chicory extract after consumption in healthy subjects. Nutrition, 26, 799–803. Roberfroid, M., Gibson, G. R., Hoyles, L., McCartney, A. L., Rastall, R., et al. (2010). Prebiotic effects: metabolic and health benefits. British Journal of Nutrition, 104, S1–63. Roberfroid, M. B. (2005). Introducing inulin-type fructans. British Journal of Nutrition, 93, S13–S25. Roberfroid, M. B. (2007). Inulin-type fructans: functional food ingredients. The Journal of Nutrition, 137, 2493S–2502S. Salvatore, E., Pes, M., Mazzarello, V. & Pirisi, A. (2014). Replacement of fat with long-chain inulin in a fresh cheese made from caprine milk. International Dairy Journal, 34, 1–5. Sanz, T. A., Salvador, A., Jiménez, A. & Fiszman, S. M. (2008). Yogurt enrichment with functional asparagus fibre. Effect of fibre extraction method on rheological properties, colour, and sensory acceptance. European Food Research and Technology, 227, 1515–1521. Scholz-Ahrens, K. E. & Schrezenmeir, J. (2002). Inulin: oligofructose and mineral metabolism—experimental data and mechanism. British Journal of Nutrition, 87, S179–S186. Scholz-Ahrens, K. E., Schaafsma, G., van den Heuvel, E. G. & Schrezenmeir, J. (2001). Effects of prebiotics on mineral metabolism. The American Journal of Clinical Nutrition, 73, 459s–464s. Shah, A. B., Jones, G. P. & Vasiljevic, T. (2010). Sucrose-free chocolate sweetened with Stevia rebaudiana extract and containing different bulking agents − effects on physicochemical and sensory properties. International Journal of Food Science and Technology, 45, 1426– 1435. Shoaib, M., Shehzad, A., Omar, M., Rakha, A., Raza, H., Sharif, H. R., Shakeel, A., Ansari, A. & Niazi, S. (2016). Inulin: Properties, health benefits and food applications. Carbohydrate Polymers, 147, 444–454. Solowiej, B., Glibowski, P., Muszynski, S., Wydrych, J., Gawron, A. & Jelinski, T. (2015). The effect of fat replacement by inulin on the physicochemical properties and microstructure of acid casein

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processed cheese analogues with added whey protein polymers. Food Hydrocolloids, 44, 1–11. Tahiri, M., Tressol, J. C., Arnaud, Y., Bornet, F. R. J., BouteloupDemange, C., Feillet-Coudray, C., et al. (2003). Effect of short-chain fructooligosaccharides on intestinal calcium absorption and calcium status in postmenopausal women: a stable-isotope study. American Journal of Clinical Nutrition, 77, 449–457. Takahara, S., Morohashi, T., Sano, T., Ohta, A., Yamada, S. & Sasa, R. (2000). Fructooligosaccharide consumption enhances femoral bone volume and mineral concentrations in rats. Journal of Nutrition, 130, 1792–1795. Tarini, J. & Wolever, T. M. S. (2010). The fermentable fibre inulin increases postprandial serum short-chain fatty acids and reduces freefatty acids and ghrelin in healthy subjects. Applied Physiology Nutrition and Metabolism-Physiologie Appliquee Nutrition Et Metabolisme, 35, 9–16. Theuwissen, E. & Mensink, R. P. (2007). Simultaneous intake of β glucan and plant stanol esters affects lipid metabolism in slightly hypercholesterolemic subjects. Journal of Nutrition, 137, 583–588. Trautwein, E. A., Rieckhoff, D. & Erbersdobler, H. F. (1998). Dietary inulin lowers plasma cholesterol and triacylglycerol and alters biliary bile acid profile in hamsters. Journal of Nutrition, 128, 1937–1943. Tungland, B. C. & Meyer, D. (2002). Nondigestible oligo- and polysaccharides (dietary fiber): their physiology and role in human health and food. Comprehensive Reviews in Food Science and Food Safety, 1, 90–109. Turner, N. D. & Lupton, J. R. (2011). Dietary fiber. Advances in Nutrition: An International Review Journal, 2, 151–152. Valluru, R. & Van den Ende, W. (2008). Plant fructans in stress environments: emerging concepts and future prospects. Journal of Experimental Botany, 59, 2905–2916. Van Loo, J. A. (2004). Prebiotics promote good health: the basis, the potential: and the emerging evidence. Journal of Clinical Gastroenterology, 38, S70–S75.

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Van Loo, J., Clune, Y., Bennett, M. & Collins, J. K. (2005). The SYNCAN project: goals, set-up: first results and settings of the human intervention study. British Journal of Nutrition, 93, S91–S98. Vanhoof, K. & De Schrijver, R. (1995). Effect of unprocessed and baked inulin on lipid metabolism in normo- and hypercholesterolemic rats. Nutrition Research, 15, 1637–1646. Villegas, B., Tárrega, A., Carbonell, I. & Costell, E. (2010). Optimising acceptability of new prebiotic low-fat milk beverages. Food Quality and Preference, 21, 234–242. Weaver, C. M. (2005). Inulin: oligofructose and bone health: experimental approaches and mechanisms. British Journal of Nutrition, 93, S99– S103. Wichienchot, S., Thammarutwasik, P., Jongjareonrak, A., Chansuwan, W., Hmadhlu, P. & Hongpattarakere, T. (2011). Extraction and analysis of prebiotics from selected plants from southern Thailand. Journal of Science and Technology, 33, 517–523. Williams, C. M. & Jackson, K. G. (2002). Inulin and oligofructose: effects on lipid metabolism from human studies. British Journal of Nutrition, 87, S261–S264. Wu, W., Xie, J. & Zhang, H. (2016). Dietary fibers influence the intestinal SCFAs and plasma metabolites profiling in growing pigs. Food and Function, 7, 4644–4654. Yasuda, K., Roneker, K. R., Miller, D. D., Welch, R. M. & Lei, X. G. (2006). Supplemental dietary inulin affects the bioavailability of iron in corn and soybean meal to young pigs. Journal of Nutrition, 136, 3033–3038. Yazici, F. & Akgun, A. (2004). Effect of some protein based fat replacers on physical, chemical, textural, and sensory properties of strained yoghurt. Journal of Food Engineering, 62, 245–254. Zimeri, J. E. & Kokini, J. L. (2002). The effect of moisture content on the crystallinity and glass transition temperature of inulin. Carbohydrate Polymer, 48, 299–304.

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BIOGRAPHICAL SKETCH Celso Fasura Balthazar Affiliation: PhD student in Department of Food Science from Animal Origin at Veterinary School, Universidade Federal Fluminense, Brazil. External PhD student in Dipartimento di Scienze delle Produzionidell’Ingegneria della Meccanica e dell’Economia Applicate al Sistemi Agro-Zootecnici, Università degli Studi di Foggia, Italy. Education: Veterinary Physician - Veterinary School, Universidade Federal Fluminense, Brazil. Specialist in Food and Nutritional Quality and Security – Instituto Federal do Rio de Janeiro, Brazil. Master in Veterinary Hygiene and Technological Processing Animal Products - Veterinary School, Universidade Federal Fluminense, Brazil. Business Address: Via Napoli, 25, 71100 Foggia, Italy. Research and Professional Experience: Experience sheep production and milk technology and derivatives. He works in the area of Technology and Inspection of products of animal origin, focusing on technology of milk and derivatives, quality, analytical and nutritional analyzes. Hs research aims on elaboration of dairy products with functional appeal by the addition of prebiotics and probiotics, with emphasis on the development of functional dairy products of sheep’s milk. Publications from the Last 3 Years: [1]

Balthazar, C.F.; Pimentel, T.C.; Ferrão, L.L.; Almada, C.N.; Santillo, A.; Albenzio, M. ; Mollakhalili, N.; Mortazavian, A.M.; Nascimento, J.S.; SILVA, M.C.; Freitas, M.Q.; Ana, A.S. Sant’; Granato, D.; Cruz, A.G.. Sheep Milk: Physicochemical Characteristics and

Inulin: Technological Applications and Health Benefits

[2]

[3]

[4]

[5]

[6]

[7]

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Relevance for Functional Food Development. Comprehensive Reviews in Food Science and Food Safety, v. 00, p. 1, 2017. Balthazar, C.F.; Júnior, C.A. Conte; Moraes, J.; Costa, M.P.; Raices, R.S.L.; Franco, R.M.; Cruz, A.G.; Silva, A.C.O. Physicochemical evaluation of sheep milk yogurts containing different levels of inulin. Journal of Dairy Science, v. 99, p. 1-9, 2016. Balthazar, C. F.; Silva, H. L. A.; Moraes, J.; Vieira, A. H.; Neto, R. P. C; Granato, D.; Freitas, M. Q.; Calado, V. M. A.; Tavares, M. I. B.; Cruz, A. G.. Assessing the effects of different prebiotic dietary oligosaccharides in sheep milk ice cream. Food Research International, v. 91, p. 38-46, 2016. Balthazar, Celso Fasura; Gaze, Leonardo Varon; Azevedo Da Silva, Hugo Leandro; Pereira, Camila Serva; Franco, Robson Maia ; ConteJúnior, Carlos Adam; De Freitas, Mônica Queiroz; De Oliveira Silva, Adriana Cristina. Sensory evaluation of ovine milk yoghurt with inulin addition. International Journal of Dairy Technology, v. 67, p. 281-290, 2015. Balthazar, C.F.; Silva, H.L.A.; Celeguini, R.M.S.; Santos, R.; Pastore, G.M.; Junior, C. A. Conte; Freitas, M.Q.; Nogueira, L.C.; Silva, M.C.; Cruz, A.G.. Effect of galactooligosaccharide addition on the physical, optical, and sensory acceptance of vanilla ice cream. Journal of Dairy Science, v. 98, p. 4266-4272, 2015. Costa, Marion P. ; Balthazar, Celso F.; Rodrigues, Bruna L.; Lazaro, Cesar A.; Silva, Adriana C. O.; Cruz, Adriano G.; Conte Junior, Carlos A.. Determination of biogenic amines by high-performance liquid chromatography (HPLC-DAD) in probiotic cow’s and goat’s fermented milks and acceptance. Food Science & Nutrition, v. 3, p. n/a-n/a, 2015. Costa, M.P.; Balthazar, C.F.; Franco, R.M.; Mársico, E.T.; Cruz, A.G.; Conte, C.A.. Changes on expected taste perception of probiotic and conventional yogurts made from goat milk after rapidly repeated exposure. Journal of Dairy Science, v. 97, p. 2610-2618, 2014.

In: Inulin Editor: Christian R. Davis

ISBN: 978-1-53612-301-2 © 2017 Nova Science Publishers, Inc.

Chapter 2

IMMUNOSTIMULATORY INULIN ADJUVANTS IN PROPHYLACTIC VACCINES AGAINST PATHOGENS Matthew D. Gallovic, Eric M. Bachelder and Kristy M. Ainslie* Division of Pharmacoengineering and Molecular Pharmaceutics, Eshelman School of Pharmacy, University of North Carolina, Chapel Hill, NC

ABSTRACT Vaccines that protect against pathogens are considered one of the medical world’s greatest achievements. Several types of vaccines exist, including subunit or inactivated/live-attenuated pathogen formulations. A component known as an adjuvant is commonly included in subunit vaccines to boost immunogenicity, and can be added to the other vaccine types for the same purpose. Traditional adjuvants such as aluminum (alum)-based preparations have several significant shortcomings, including the inability to drive Th1-biased cellular immunity. Because protection against many pathogens (e.g., viruses) often requires this type

*

Corresponding author: Email: [email protected].

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Matthew D. Gallovic, Eric M. Bachelder and Kristy M. Ainslie of immune response, novel adjuvants are needed. Two particulate isoforms of the naturally-occurring inulin polysaccharide have emerged as new adjuvant candidates. Gamma inulin was the first of these isoforms used in vaccine development. A popular gamma inulin formulation known as Algammulin consisted of gamma inulin co-crystallized with the antigen-adsorbing alum. The immunostimulatory effects of the second isoform, delta inulin, were discovered more recently. It has emerged as an enticing alternative to gamma inulin formulations because of its enhanced stability, as well as reproducibility in both manufacturing and immunostimulation. Delta inulin, known as AdvaxTM in its adjuvant formulation, has been examined preclinically in many vaccines and has also reached clinical testing for safety and immunogenicity against several pathogens. In addition to Algammulin and Advax, a select number of inulin preparations have been engineered into dual-functioning systems that serve as both an antigen delivery vehicle and adjuvant. This chapter will discuss the history, development, and application of preclinical and clinical vaccines that contain inulin formulations.

Keywords: inulin, vaccine, adjuvant, Algammulin, Advax, delivery system

1. CHAPTER OVERVIEW Before inulin vaccine adjuvants are discussed in detail, it is important to introduce pertinent background. This chapter will begin with an overview of vaccines, the immune system, and the types of adjuvants currently used in research settings and the clinic. Following this introductory material, the chapter will then move to its focus – inulin vaccine adjuvant formulations. The chapter will conclude with a discussion of engineered delivery vehicles and the next-generation inulin adjuvants that have been designed using these platforms.

2. BRIEF HISTORY OF VACCINES Communicable diseases can have a profound detrimental effect on human life. Pandemics like the Black Death during the Middle Ages,

Immunostimulatory Inulin Adjuvants in Prophylactic Vaccines … 35 Spanish flu of 1918, Asian flu in the late 1950s, and the current human immunodeficiency virus (HIV) crisis have left millions sick, disabled, or dead. Although preventative epidemiological measures are one of the best ways to reduce the spread of infection, failure of these measures often necessitates medical intervention to successfully protect the human population. Vaccines may be considered one of the most powerful forms of preventative medicine and greatest medical achievements of the modern era. They have protected populations against many new infections and led to the near or complete eradication of devastating diseases (e.g., smallpox, polio). While the Chinese used variolation (also known as inoculation) techniques to immunize people against smallpox during the 10th century, Edward Jenner is typically credited with testing the first “modern” vaccine in the late 1700s [1]. He immunized a young boy with a cowpox viral load that had been isolated from another infected human. The boy demonstrated immunity upon future re-infection with the more virulent smallpox virus. One of the first laboratory vaccines, designed against fowl cholera, was prepared by Louis Pasteur in the 1870s [2]. His subsequent work involved formulating vaccines against bovine anthrax, as well as conducting the first known clinical tests of a laboratory-developed vaccine, which was against rabies. There are currently over eighty U.S. Food and Drug Administration (FDA)-approved vaccines on the market [3], with countless more approved around the world. Vaccine development continues to be a medical research priority because of ongoing risks, including communicable diseases still lacking an effective vaccine (e.g., HIV), bioterrorism concerns, and emerging pathogens that may threaten life in years to come.

3. BASIC REVIEW OF IMMUNE RESPONSES TO VACCINES Vaccines stimulate the host immune system, which involves a complex coordination of many acellular and cellular components. Proper

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coordination can eventually lead to successful immunity against pathogens. A basic overview of the immune system’s two types of responses, innate and adaptive, is needed to understand the immunological pathways vaccines engage. In brief, innate immune responses are non-specific, occur in the short-term, and have no memory of the pathogen. Adaptive immune responses, on the other hand, are pathogen-specific, develop over time, and exhibit memory. Within each of these responses, both humoral and cellular immunity can take place. Humoral is named after Hippocrates’ use of ‘humors’ to describe bodily fluids. Hence, humoral immunity is used to classify immune system components that are contained in bodily fluids outside cells. Cellular immunity, as the name implies, refers to more direct functions by cells. A generalized depiction of these pathways can be found in Figure 1. Other resources, such as Janeway’s Immunobiology [4], can be referenced for a more detailed explanation of the immune system.

Source: Figure design adapted from Ref. 34. Figure 1. Overview of major aspects of the immune system that vaccines engage.

Immunostimulatory Inulin Adjuvants in Prophylactic Vaccines … 37

3.1. Innate Immune Response When a vaccine is administered to a patient, it must first activate the innate immune response, which is composed of a few notable arms. One major arm occurs when immune cells recognize a broad family of molecular moieties not inherent to host cells. These moieties, known as pathogen-associated molecular patterns (PAMPs), consist of structures including, but not limited to, single-stranded ribonucleic acid (ssRNA), double-stranded RNA (dsRNA), and bacterial deoxyribonucleic acid (DNA). Certain specialized immune cells, such as macrophages (Mϕs) and dendritic cells (DCs), are important for initiating the innate immune response because they have pattern recognition receptors (PRRs) that detect PAMPs [5-7]. This detection of foreign moieties is a component of innate cellular immunity. Innate cellular immunity stemming from other cells such as natural killer cells and neutrophils is also involved. These cells are primarily involved with functions like pathogen phagocytosis and direct anti-microbial responses. Another important arm of the innate immune system involves humoral immunity. While there are two significant aspects of humoral immunity, antibodies and complement, the former is part of the adaptive immune response and will be discussed later (Section 3.3). The complement system involves 30+ plasma proteins that work to drive an enzymatic cascade down three pathways: classical, lectin, and alternative [8]. The classical pathway is initiated when a cascade component binds to an antibodyantigen immune complex on a pathogen or directly to the surface of a pathogen. The lectin pathway commences when lectins, or other certain molecules, bind to sugars located on a pathogen surface. The alternative pathway (ACP) can be activated by either of the other two pathways or via a spontaneous ‘tick-over’ effect [9]. Each of the three pathways eventually converge when one of two complement enzymes is formed. Downstream of this step, innate immune responses such as inflammation, pathogen lysis by a structure known as a membrane attack complex, or pathogen

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phagocytosis by Mϕs and DCs occur [9]. Direct stimulation of B cells can also occur [9]. One of B cells’ primary functions will be discussed in Section 3.3.

3.2. Bridging the Innate and Adaptive Immune Responses While B cell stimulation by the complement system could be considered one bridge between innate and adaptive immune responses, antigen-presenting cells (APCs) like Mϕs and DCs also have an important role in bridging these two phases via a process called antigen presentation. Mϕs and DCs phagocytose pathogens, digest the pathogen, and present pathogen-specific peptides on plasma membrane-bound proteins formed into structures known as major histocompatibility complexes (MHCs). Although both cell types are regarded as APCs, DCs are deemed “professional” APCs [10, 11] because unlike Mϕs, which also clear cell debris and perform other functions, DCs’ primary function appears to be presentation of antigen. For this reason they are a key player in forming a bridge between the innate and adaptive immune system [12]. In order to fulfill this role, a secondary signal, such as PRR stimulation by a PAMP, is needed to alert the immune system that the MHC-bound antigen is derived from a foreign source. This is referred to as a co-stimulatory signal. Lack of a co-stimulatory signal can lead to antigen-specific immune tolerance in the form of regulatory T cells, anergy, or T cell deletion [13]. Upon recognizing foreign antigens at the site of vaccine administration, DCs express co-stimulatory molecules on their surface and traffic to lymph nodes with the “message” that the host has been “infected.” In the lymph nodes, which are highly immuno-active organs found throughout a host’s body, they present MHC-bound pathogen-specific antigen peptide epitopes and co-stimulatory molecules to other naïve immune cells. This interaction with naïve immune cells, which have not previously been stimulated by an APC, initiates the immune response that can eventually lead to long-term memory of the antigen [5-7].

Immunostimulatory Inulin Adjuvants in Prophylactic Vaccines … 39

3.3. Adaptive Immune Response This path to long-term memory occurs via adaptive immune responses, which when mounted properly, result in both cellular and humoral immunity. This dual adaptive immunity is in addition to the previously described innate cellular and humoral immunity (Section 3.1). Adaptive cellular immunity is focused, in part, on the activation of T cells and B cells. T cell activation depends on APC presentation of peptide sequences on one of two MHCs. This omprises a large portion of cellular immunity. Typically, antigen peptides from phagocytosed pathogens are presented on MHC II, whereas antigen peptides from an intracellular pathogen are presented on MHC I. These forms of presentation are not mutually exclusive and may occur together in what is known as crosspresentation [14], albeit with unique peptide sequences. Cluster of differentiation 8 cytotoxic T cells (CD8+ T cells) and cluster of differentiation 4 helper T cells (CD4+ T cells) recognize antigenic fragments on MHC I and II, respectively. The primary function of CD8+ T cells, also known as cytotoxic T lymphocytes (CTLs), is to interpret MHC I presentation of pathogen peptide fragments as a cue that the host is infected and to subsequently initiate a process to kill the host cells which have presented that peptide on their MHC I molecules. Whereas CD8+ T cells can lyse cells directly, CD4+ T cells work indirectly to clear infected cells. CD4+ T cells have several important functions, including a few that are relevant to this chapter’s discussion of vaccines. The first pertinent function of CD4+ T cells is to activate B cells which, in turn, produce antibodies. Antibody production is one of the primary arms of adaptive humoral immunity. As a reminder, this occurs in addition to complement, a part of innate humoral immunity. Antibodies are proteins that exist extracellularly, and bind to specific epitopes on pathogens to alert the immune system to clear the pathogen. The most common type of antibodies that exist in the serum and mucosa are immunoglobulin G (IgG) and immunoglobulin A (IgA), respectively.

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Similar to other immune cells, CD4+ T cells also produce groups of molecules known as cytokines that help polarize adaptive humoral and cellular immunity down one of two biases [15]. Th1-biased CD4+ T cells lead to increased production of certain IgG antibody subtypes (e.g., IgG2, IgG3) and cytokines (e.g., IFN-γ) that result in re-stimulation of other cells, such as CD8+ T cells, necessary for clearing pathogens. Th2-biased CD4+ T cells result in skewing toward an IgG1 antibody-based response and production of another collection of cytokines (e.g., IL-4, IL-5). In general, Th1-biased IgG subtype antibodies are better for binding to, and subsequently neutralizing, pathogens such as viruses. The most successful vaccines initiate a strong innate response, and lead to appropriate adaptive responses that ultimately result in sufficient immunity against the target pathogen. This immunity is achieved when long-lasting memory T and B cells, with specificity against pathogenassociated antigen(s), are generated. These cells are able to respond very rapidly to a future secondary infection, resulting in effective protection for the host.

4. OVERVIEW OF VACCINES 4.1. Types of Vaccines 4.1.1. Live-Attenuated and Inactivated Pathogen Vaccines Traditional vaccines can be classified as either live-attenuated or inactivated. Jenner and Pasteur’s vaccines consisted of live-attenuated pathogens, where pathogenicity is weakened to a level allowing relatively safe administration to the patient/host. Live-attenuated vaccines sometimes require just a single dose to provide long-term, or even lifelong, immunity. There always remains, however, a slight risk that the live-attenuated pathogens could replicate in patients, leading to adverse events of varying severity. Alternatively, pathogens can be inactivated by heat or chemicals. These inactivated vaccines often require multiple boosters, which can lead to patient non-adherence. Additionally, inactivation processes can

Immunostimulatory Inulin Adjuvants in Prophylactic Vaccines … 41 sometimes be ineffective. One notable example of such an occurrence was the Cutter incident in the mid-20th century. Live poliovirus was incompletely inactivated, causing disease and death in patients who received the vaccine [16]. Although manufacturing methods and quality control have vastly improved since this incident, either of these traditional vaccine formulations can still be risky for patients that are immunocompromised because of inherent medical conditions or infections with other pathogens, or for patients that have weaker immune systems related to age.

4.1.2. Subunit Vaccines Subunit vaccines can avoid some of the concerns associated with traditional vaccine formulations. They are generally acellular and contain limited portion(s) of a pathogen, typically referred to as the subunit antigen(s). The original subunit vaccines were created by Dr. Gaston Ramon in the 1920s when he used formaldehyde to inactivate the diphtheria and tetanus protein toxins, which are constructs termed toxoids [17]. More advanced subunit vaccine formulations consist of antigens such as polysaccharides [18], polysaccharide-protein conjugates [19], nucleic acids [20-22], adenoviral vectors [23], or recombinant proteins either in a soluble form [24] or assembled into virus-like particles (VLPs) [25-27]. Subunit antigens alone generally do not, however, create a strong enough immune response for long-term protection. They often require many boosters and stringent adherence to an immunization schedule in order to achieve protective immunity.

4.2. Adjuvants One way to increase a vaccine’s immunogenicity is to include a component known as an adjuvant, derived from the Latin word that means ‘to help’. Adjuvants are typically added to subunit vaccines, but liveattenuated and inactivated vaccines can be supplemented with them as well. In this section of the chapter we will define four of the most common

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classes of adjuvants: (I) aluminum-based salts, (II) emulsions, (III) PAMPs, and (IV) natural products. Adjuvants from these classes can also be used in tandem, and such combinatorial formulations may be considered a fifth class. Many literature reviews serve as excellent resources for additional historical perspective and discussion of each adjuvant type [2838].

4.2.1. Adjuvant Class I: Aluminum-Based Salts Alum is a term used to identify an entire family of aluminum-based salts, including aluminum hydroxide, aluminum phosphate, and others. Alum’s adjuvant activity was first identified in the 1920s when a potassium alum salt was co-precipitated with a diphtheria toxoid [39]. It has an established clinical safety record and demonstrated efficacy in many FDA-approved vaccines (e.g., BioThrax® [anthrax], Infanrix® [diphtheria and tetanus], Gardasil® [herpes/cervical cancer], Recombivax HB® [hepatitis B]). Despite these advantages, alum actually has several notable drawbacks. First of all, its precise mechanism of action is unclear [40]. For a long time it was believed that alum’s adjuvant effect was derived from its ability to act as an antigen depot [41], but more recent studies have suggested that its adjuvant activity may be the result of other mechanisms [42-47]. Alum can also lead to adverse effects such as local lesions [48] and granulomas [49]. Finally, alum is Th2-skewing, and does not lead to CTL activity, which restricts its use to immunizing against only certain pathogens [40, 50]. 4.2.2. Adjuvant Class II: Emulsions The first type of emulsion adjuvants emerged in the 1930s. Dr. Jules Freund incorporated killed Mycobacterium tuberculosis components in water and emulsified them in a non-degradable mineral oil to create a water-in-oil emulsion [51, 52]. One such emulsion, stabilized by a mannide monooleate surfactant called Arlacel A [53], later became known as complete Freund’s adjuvant (CFA). Although CFA was deemed too reactogenic for use in prophylactic vaccines [54], CFA was tested

Immunostimulatory Inulin Adjuvants in Prophylactic Vaccines … 43 clinically in several trials from the late 1960s to 1980s as part of therapeutic vaccines against cancer [55]. A less reactogenic water in mineral oil emulsion without the bacterial components, termed incomplete Freund’s adjuvant (IFA), was first used in the 1940s [56]. IFA reached widespread clinical testing in prophylactic vaccines in the 1960/70s [57-61], however, the emulsion was ultimately deemed too reactogenic for widespread use [60, 62, 63]. Interestingly, IFA incorporated within therapeutic vaccines against HIV or cancer has been extensively tested in clinical trials of the past few decades [61, 64, 65]. Montanides are a more recently developed family of water-in-oil emulsions. There are two primary Montanide formulations. The first, a close relative of IFA, is known as Montanide ISA 51TM. It is comprised of an antigen-containing water phase emulsified in a mineral oil. Montanide ISA 720TM, on the other hand, incorporates an antigen emulsified in a vegetable oil [66]. These emulsions are stabilized using a more purified surfactant than the ones used in CFA or IFA, leading to potentially increased safety of the formulations [66]. Montanides have been tested in more than 200 clinical trials for prophylactic and therapeutic applications [67], but they are not marketed in any clinical prophylactic vaccines. A therapeutic lung cancer vaccine containing Montanide ISA 51 has been licensed in Cuba [68]. A second major type of emulsion adjuvants is composed of squalene, an oil derived from shark livers (that can also be produced synthetically) [69]. Squalene alone does not create a significant innate immune response – an emulsion of squalene oil droplets within a surrounding water phase is required (i.e., oil-in-water emulsion) [70]. Two of these formulations, known as adjuvant system number three (AS03) [71] and microfluidization 59 (MF59®) [72], have been FDA-approved for use in certain influenza vaccines (Influenza A (H5N1) Virus Monovalent Vaccine Adjuvanted and FluadTM, respectively). They are under development for other vaccines as well [29]. The emulsions seem to activate resident APCs that subsequently produce chemokines to recruit more phagocytic cells to the injection site [72]. The adjuvants do not, however, drive strong Th1-biased cellular immunity [72].

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4.2.3. Adjuvant Class III: Pathogen-Associated Molecular Patterns A third class of adjuvants, PAMPs, are typically Th1-skewing both humorally and cellularly. Additionally, they have very well-defined, specific cellular targets. Interaction of PAMPs with PRRs of host APCs leads to cell activation, as well as responses such as the production of proinflammatory cytokines and chemokines that attract additional immune cells [5, 73]. PRRs can exist either in cells’ cytosol or on cell membranes. The cytosolic PRRs include proteins such as nucleotide-binding oligomerization domain receptors (NLRs), retinoic acid-inducible gene (RIG)-1-like receptors (RLRs), and the stimulator of interferon genes (STING). NLR [74], RLR [75, 76], and STING [77] agonists have all been used as adjuvants in preclinical vaccines. The most well-known membrane-bound PRRs are toll-like receptors (TLRs). An entire TLR family and their corresponding agonists have been characterized [78]. TLRs 1, 2, 4, and 6 are found on plasma membranes, and TLRs 3, 7, 8, and 9 are located on phagolysosomal membranes. In addition to natural PAMPs that are TLR agonists, a wide range of synthetic TLR agonists also exist. These synthetic analogs were being synthesized as early as the 1960s [79]. Several examples of synthetic TLR agonists that have demonstrated promise as adjuvants in clinical trials [32] include: (i) TLR 7/8 agonists imiquimod and resiquimod, ssRNA analogs [80]; (ii) TLR 3 agonist polyinosinic:polycytidylic acid (poly I:C), a double stranded RNA analog [81]; and (iii) TLR 9 agonist CpG, a bacterial DNA analog [82]. Another notable example is TLR 4 agonist monophosphoryl lipid A (MPL). It is a biocompatible fragment of gram-negative Salmonella enterica’s lipopolysaccharide [83], and is approved for use in several combinatorial clinical vaccines (Section 4.2.5) [32, 84]. 4.2.4. Adjuvant Class IV: Natural Product-Derived Adjuvants A fourth class of adjuvants are derived from inherently immunogenic natural products. One prominent example is saponin, a glycoside derived from the bark of Quillaja saponaria [85]. The whole water-soluble fraction of Q. saponaria’s bark is known as Quil A, and although it is immunogenic, it is not safe to use in its naked form [86]. A certain fraction

Immunostimulatory Inulin Adjuvants in Prophylactic Vaccines … 45 of Quil A, known as QS-21, is more tolerable [87]. Despite still having some toxic side effects [86], QS-21 has reached the clinic most notably as part of GlaxoSmithKline (GSK)’s adjuvant system (AS) family, in the form of AS01 (Section 4.2.5) [35]. Another saponin adjuvant, a selfassembled cage-like structure termed an immunostimulating complex adjuvant (ISCOM), is composed of Quil A, cholesterol, and phospholipids [88]. It also has demonstrated a promising safety profile [89] and reached clinical testing [32]. It is worth mentioning that Q. saponaria tree destruction resulting from Quil A production has forced countries where the plant grows to restrict access to the natural product [90]. Quil A’s availability is further limited by the fact that it has yet to be synthetically produced at the potency of its natural form [91]. Inulin, the focus of this chapter, is another prominent example of a natural product with inherent immunostimulatory properties. The most common source of inulin is the tubers of chicory and dahlia from the Asteraceae plant family. Applications of inulin as vaccine adjuvants will be discussed beginning in Section 5.

4.2.5. Adjuvant Class V: Combinatorial Adjuvants A common strategy in adjuvant development has been to combine multiple adjuvants to generate a broader or stronger immune response. To create a more broad response, Th2-skewing adjuvants (e.g., alum, squalene-based emulsions) have been combined with Th1-skewing PAMPs. In the most successful formulation to date, GSK’s alum plus MPL (AS04) [92, 93] was clinically approved for use in an FDA-approved vaccine against human papillomavirus (Cervarix®) and a European Unionapproved vaccine against hepatitis B (Fendrix®). Industrial leaders in the vaccine field such as Novartis and GSK are also formulating their respective squalene emulsions in combination with TLR agonists. Many of these formulations have demonstrated substantial progress in clinical testing [31, 32, 35, 84]. To achieve a stronger immune response, combinations of Th1-polarizing adjuvants have been pursued. One of the most notable formulations is GSK’s AS01, which contains both QS-21 and MPL [94]. Recently a malaria vaccine composed of an AS01-adjuvanted

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recombinant protein, termed RTS,S or Mosquirix [95, 96], was recommended by both the European Medicines Agency and World Health Organization (WHO) for use in certain countries [97]. Inulin has similarly been used in combination with other adjuvants, and such instances will be discussed later in the chapter.

5. DESCRIPTION OF INULIN As many of the chapters in this book may describe, inulin is a fructan polysaccharide containing β-D-(2-1) poly(fructo-furanosyl) α-D-glucose chains. In other words, it is composed of fructose monomers (n = 3 to 100) with a single terminal glucose [98-100]. Inulin is non-antigenic and nonnephrotoxic [101]. Inulin is known to have several isoforms/polymorphs (e.g., alpha, beta, gamma, delta) that are identified by their solubility in water [98]. Alpha and beta are the most common ‘soluble’ isoforms, and both are highly soluble in water at >25ºC [99]. At lower temperatures, beta inulin precipitation requires addition of ethanol into water, while alpha inulin can spontaneously precipitate out in 100% water [98-100, 102, 103]. Gamma and delta inulin isoforms, on the other hand, are quite water-insoluble at temperatures cooler than 37ºC. At the physiological temperature of 37ºC, gamma inulin has slight solubility and delta inulin remains completely insoluble. Delta inulin does not solubilize until temperatures greater than 40ºC [99]. Structural analysis of these inulin isoforms can be found in [104-106]. The existence of an insoluble form of inulin was first noted by G.F. Phelps in 1965 [103]. Six years later Götze et al. identified that this insoluble inulin, which would later be termed gamma inulin [107], could activate an arm of the complement system different than the classical pathway [108]. This pathway became known as the ACP (Section 3.1). Further mechanistic studies showed that enzyme fragments involved in the ACP accumulate on APCs following incubation with gamma inulin, and the APCs’ antigen presentation capabilities are dependent on this

Immunostimulatory Inulin Adjuvants in Prophylactic Vaccines … 47 deposition [109]. Delta inulin, which can be formed from alpha, beta, or gamma inulin using certain heating and precipitation steps, activates the ACP to an even greater extent than gamma inulin [100]. The adjuvant formulation of delta inulin is known as AdvaxTM, which is also referred to as DeltinTM in some publications [110]. Currently, not much is known about Advax’s mechanism of action. It has been reported that Advax does not cause Mϕs to secrete pro-inflammatory cytokines [99, 110], as PAMPs do, nor does it induce the immune complex called an inflammasome [99] as alum has been shown to do. Despite further investigation of Advax’s mechanism of action by Hayashi et al., many questions remain [111]. It is generally reported that alpha and beta inulin do not activate complement [98, 99].

6. GAMMA INULIN-BASED VACCINE ADJUVANTS 6.1. Early Development Although this section’s focus will be gamma inulin-based vaccine adjuvants used in prophylactic vaccines against pathogens, it is important to mention that gamma inulin first drew interest as an anti-cancer therapeutic. Dr. Peter Cooper’s group published several studies in the mid1980s demonstrating that complement-activating compounds had anticancer activity. Their first study showed this with protein A from Staphylococcus aureus cells [112]. Their next study used ACP-activating zymosan and isolated ACP components [113] to further substantiate the anti-cancer therapeutic effect. In a follow-up report, they studied ACPactivating inulin and observed increased survival time of melanoma mice after treatment with gamma inulin compared to a soluble inulin fraction [114]. They were able to further increase the survival time by two-fold in a subsequent study and suggested that this anti-cancer activity was a result of ACP activation [115]. Their data also showed that dissolved soluble isoforms had no therapeutic effect. In the same year, Dr. Cooper’s group published methods for preparing stable endotoxin-free (i.e., undetectable

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levels of TLR 4 agonist lipopolysaccharide) gamma inulin ‘ready for injection’ [107]. Gamma inulin prepared in this way resulted in greater APC activity than other strong complement-activators such as the bacterium S. aureus. Based on the results of these gamma inulin cancer studies, the next step forward was to consider gamma inulin as a vaccine adjuvant (reviewed in [110, 116-124]). Cooper and Steele used gamma inulin ‘ready for injection,’ as developed previously [107], to adjuvant a model protein antigen, keyhole limpet haemocyanin (KLH) [125]. In this study they showed that gamma inulin could enhance a Th1-skewed humoral antibody production and cellular immunity compared to the unadjuvanted KLH antigen. The authors also improved the protection offered by a liveattenuated influenza vaccine against a lethal viral challenge by formulating it with a gamma inulin adjuvant. The next step in gamma inulin adjuvant development was to combine it with alum. At the time, it had already been established that alum was able to induce potent humoral antibody immunity, but could not generate strong cellular immunity. The first published paper using a complex of alum and gamma inulin, known as Algammulin, was not completed in Dr. Cooper’s group. They gifted batches of un-optimized gamma inulin and Algammulin to Leslie et al. [126]. In the study, the authors used recombinant protein hepatitis B surface antigen (HBsAg) and evaluated only humoral antibody responses induced by gamma inulin, alum, and Algammulin adjuvants. The gamma inulin performed on par with alum for total serum antibody levels as well as Th1-biased subtypes. Alum seemed to outperform Algammulin, and the Algammulin failed to induce more antibodies than the soluble antigen alone. Following this work, Dr. Cooper and co-authors wanted to optimize the formulation of Algammulin. They published two accompanying papers describing the complete characterization and model vaccine application of an optimized 1-2 µm ovoid particulate Algammulin formulation [127, 128]. In addition to possibly inducing a broader immune response, as Leslie et al. had noted [126], the authors also suggested that Algammulin’s alum component could also enable protein-adsorption. This is a feature that

Immunostimulatory Inulin Adjuvants in Prophylactic Vaccines … 49 gamma inulin particles did not exhibit [125]. The group’s first paper introduced a preparation method of Algammulin, where alum was embedded within a matrix of gamma inulin [127]. They compared the immunogenicity of Algammulin to gamma inulin, and found that Algammulin was able to activate the ACP as much as gamma inulin. Its antigen loading capacity, however, decreased about 10-fold compared to alum. In the group’s second paper, KLH was used as a model protein antigen for evaluating the activity of alum, gamma inulin, a mixture of the two, or Algammulin [128]. The major results from this work were that Algammulin induced synergistic humoral immunity that was better than alum, gamma inulin, or a mixture of alum and gamma inulin. They also observed that Algammulin shifted the IgG antibody isotype profile from alum’s Th2 bias to gamma inulin’s Th1 bias, and could generate total antiKLH antibody levels nearing that of CFA. Cellular immunity was not investigated. After completing this proof-of-concept KLH study, Dr. Cooper’s group moved on to using the clinically relevant HBsAg [129]. To begin, the authors wanted to address Leslie et al.’s poor results when HBsAg was adjuvanted with an “unverified” Algammulin batch [126]. Using electron microscopy and centrifugation studies, it was shown that Leslie et al.’s Algammulin batch was physically equivalent to just a mixture of alum and gamma inulin, as opposed to a complex of alum embedded within gamma inulin. Dr. Cooper, therefore, used an optimized Algammulin to adjuvant HBsAg [129]. Unlike Leslie et al.’s data [126], a several fold increase in IgG titers was observed when using Algammulin compared to an equivalent alum-only dose. It is also notable that little to no toxicity from the Algammulin was observed [128, 129].

6.2. Further Application of Gamma Inulin Adjuvants Following Dr. Cooper and his collaborators’ foundational work with gamma inulin and Algammulin, several other groups adopted these adjuvants for vaccines against the following diseases/complications:

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diphtheria, malaria, acquired immunodeficiency syndrome (AIDS), meningococcal diseases, and herpes/cervical cancer. A summary of these studies is located in Table 1, and study specifics are discussed in subsequent sections. Table 1. Summary of gamma inulin or Algammulin-adjuvanted vaccines reported during the mid-1990s to early 2000s. Disease/ Condition

Causative Agent

Type of Vaccine

Inulin Formulation

Reference

Diphtheria

Corynebacterium diphtheriae

Subunit

Algammulin & Gamma inulin

Gupta et al. [130] Jones et al. [131] Jones et al. [132] Saul et al. [133] Jones et al. [134] Silva et al. [118]

Malaria

Plasmodium falciparum

Subunit

Algammulin or Gamma inulin

Acquired Immunodeficiency Syndrome

Human immunodeficiency virus

Subunit

Algammulin & Gamma inulin

Harris et al. [135]

Meningococcal Diseases

Neisseria meningitidis

Subunit or Inactivated

Algammulin

Gonzalez et al. [136]

Herpes/Cervical Cancer

Human papilloma virus

Subunit

Algammulin

Fernando et al. [137] Fernando et al. [138] Frazer et al. [139]*

*Indicates clinical trial.

6.2.1. Diphtheria Corynebacterium diphtheria is the causative bacterium of diphtheria, which was at one time, among the most devastating childhood diseases [140]. In the 1930s, diphtheria was one of the leading causes of death for children in England and Wales, and the U.S. saw 15,000 deaths per year [141]. Vaccines based on diphtheria toxoid, first prepared by Dr. Ramon in the 1920s [17], received U.S. licensure in the late 1940s [142]. Current day diphtheria vaccines are still based on the toxoid, and at least 86% of infants worldwide were immunized against diphtheria using combinatorial

Immunostimulatory Inulin Adjuvants in Prophylactic Vaccines … 51 diphtheria-tetanus-pertussis vaccines in 2015 [143]. Whereas larger outbreaks have occurred in areas around the world with lower immunization rates, the U.S. experienced just 55 reported cases of diphtheria between 1980 and 2011 [141]. While existing diphtheria vaccines are highly efficacious, the diphtheria toxoid still remains relevant in vaccinology research as a model antigen to test adjuvant immunogenicity. Gupta et al. took this approach by adjuvanting the toxoid with either gamma inulin or Algammulin to demonstrate increased Th1biased IgG antibody titers compared to alum [130].

6.2.2. Malaria Malaria, which is caused by the parasite Plasmodium falciparum, has a global incidence of over 200 million and an at-risk population of over 3 billion [144]. Pertaining to inulin adjuvant development, researchers initially conducted studies using a variable peptide region, P2122, from P. falciparum’s non-conserved merozoite surface antigen 2 (MSA-2). They first demonstrated that the peptide can inhibit parasite invasion [145]. The authors then conjugated P2122 to a diphtheria protein toxoid, and showed that an Algammulin-adjuvanted formulation induced comparable antibody titers as CFA in rabbits [131]. They also showed that the Algammulin group generated higher titers in mice than groups adjuvanted with one of several Montanide formulations, CFA, a squalene-based oil-in-water emulsion stabilized by Arlacel surfactant, or alum. Based on the promising results of the P2122 study, the authors proceeded to using several more conserved MSA-2 peptide regions (E-71, G-5, and G-12) [132]. Having previously tested these peptides only in a diphtheria toxoid-conjugate vaccine adjuvanted with CFA, they now wanted to compare CFA to alum and Algammulin. Each of the adjuvanted formulations resulted in similar antibody titers. In a follow-up challenge model using a mouse-adapted P. chabaudi parasite, the three adjuvanted formulations showed similar protection in the E-71 vaccines, while alum performed more poorly compared to the other two adjuvants when using G-5 [133]. Using the conserved peptides, they showed that anti-peptide antibodies did not necessarily cross-react against polymorphic full recombinant proteins,

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independent of the choice of adjuvant [134]. Several years later, inulin expert Dr. Cooper and collaborators did conduct further research using gamma inulin-adjuvanted MSA-4 or MSA-5 and demonstrated similar IgG antibodies and cellular immunity as a CFA-adjuvanted group [118]. Although no inulin adjuvant research in malaria vaccines has been published since, malaria vaccine research recently reached a milestone. It was announced on World Malaria Day 2017 that a pilot population of ~375,000 children in three sub-Saharan countries will receive the AS01adjuvanted RTS,S vaccine [146].

6.2.3. Acquired Immunodeficiency Syndrome AIDS was first identified in the early 1980s in the U.S. state of California. HIV was identified as the causative agent of AIDS in 1984, and by the mid-1990s a half-million cases of AIDS were reported in the U.S. alone [147]. Currently, more than 1 million people die from AIDS-related complications and more than 2 million new HIV infections occur worldwide each year [148]. Unfortunately, there is still no clinically approved HIV vaccine. Although earlier HIV vaccine development focused on creating both neutralizing antibodies and CTL responses [149], inulin adjuvant researchers of the mid-1990s were most interested in evaluating the CTL against VLPs carrying a portion of the HIV surface glycoprotein gp120 [135]. Although the unadjuvanted VLPs resulted in the greatest CTL activity, the gamma inulin formulations performed better than NLR agonist glucoaminylmuramyl dipeptide, a Mϕ-activating βglucan particulate system named Chemivax, a combinatorial adjuvant Syntex Adjuvant Formulation (SAF-MF) comprised of NLR agonist Nacetylmuramyl-L-threonyl-D-isoglutamine in a squalene oil-in-water emulsion, and a highly reactogenic Detox adjuvant composed of Mycobacterium phlei’s cell wall skeleton and MPL. When either alum or Algammulin was used, however, the vaccines did not lead to any CTL activity, and it was hypothesized this was due to the VLPs’ instability following adsorption to alum.

Immunostimulatory Inulin Adjuvants in Prophylactic Vaccines … 53

6.2.4. Meningococcal Diseases Neisseria meningitidis is a gram-negative bacterium that causes meningococcal diseases such as meningitis or bacteremia. The bacterium is endemic to sub-Saharan Africa, where ~100,000 people died from N. meningitidis-related meningitis between 1991 to 2010, with ~25,000 deaths attributed to a mid-1990s epidemic [150, 151]. Globally, up to 135,000 deaths occur each year from these diseases [152]. In 1998, inulin adjuvant researchers were interested in pursuing a more “universal” protein-based vaccine that could protect against multiple bacterial serotypes [136]. It should be noted that only polysaccharide-based vaccines against certain N. meningitidis serotypes (A, C, Y, or W-135) existed at the time, and protein-based vaccines against serotype B and polysaccharide-protein conjugate vaccines against serotype C were not clinically approved until later [153]. The authors used one “universal” protein, a recombinant outer membrane protein known as P64k, and compared it to inactivated wholebacteria vaccine formulations. Of the many adjuvants tested (Algammulin, CFA, IFA, three alums, and Quil A), the Algammulin-adjuvanted group was medial for antibody titers production against the recombinant P64k, and was the best for inducing titers against the whole meningococcal bacteria. 6.2.5. Herpes and Cervical Cancer More than 250,000 women worldwide are diagnosed with cervical cancer each year [154]. Herpes papilloma virus (HPV) is one of the primary causes of cervical cancer, with more than 95% of cervical cancer cells containing genetic material from HPV [155]. Although a recombinant subunit L1 protein vaccine against HPV/cervical cancer (Cervarix) was FDA-approved in 2009, previously it had been shown that HPV’s E7 protein was a potential oncogenic antigen [156]. In their first study, Fernando et al. adjuvanted a recombinant E7 protein either entrapped in liposomes (these systems are further described in Section 11), or mixed with Quil A, CFA, or Algammulin [137]. The Quil A group resulted in the highest anti-E7 IgG antibody titers, as well as the highest Th1-biased IgG2

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titers and CTL activity. In addition, T cell-containing splenocytes from the Quil A-vaccinated mice were pulsed with E7, and their resulting cytokine response was more Th1-skewing than the Algammulin group. In contrast to the other studies discussed in the chapter, Algammulin exhibited Th2polarization. In an attempt to skew the response from Th2 to Th1, the authors used a booster immunization with Quil A. Mice immunized with Algammulin-adjuvanted E7, however, maintained a persistent Th2-bias even after this booster. In their second study, Fernando et al. moved to testing the efficacy of the Quil A-adjuvanted E7 formulation against a tumor challenge [138]. Algammulin was used as a Th2-skewing control group. Similar to the previous study, the Algammulin group induced low anti-E7 IgG2a titers and minimal CTL activity, and therefore resulted in poor protection against the tumor challenge. Interestingly, Quil Aadjuvanted E7 did not lose its immunogenicity or protective efficacy when mice were immunized simultaneously with Algammulin-adjuvanted E7. Despite these poor preclinical results, an Algammulin-adjuvanted E7 fused to glutathione-S-transferase was tested for safety and immunogenicity in a Phase I/II clinical trial [139]. The trial consisted of five subjects with untreatable cervical cancer. The administered vaccine was composed of various E7 antigen doses combined with a high 25 mg dose of Algammulin, which is several fold higher than most clinically acceptable doses of alum-based adjuvants [157]. There were only minor local adverse events at the injection site. Anti-E7 IgG antibodies were formed and two of three patients’ T cells proliferated, an indicator of cellular immunity, but CTL responses were not achieved. It does not appear that any further development of this formulation was pursued.

7. DELTA INULIN-BASED VACCINE ADJUVANTS As discussed, gamma inulin and the more advanced Algammulin formulation showed promise in both preclinical and clinical studies. These formulations, however, exhibited two substantial drawbacks: (i) batch-tobatch variability in particle morphology and size, and (ii) marginal

Immunostimulatory Inulin Adjuvants in Prophylactic Vaccines … 55 solubility at human physiological temperature (37ºC) which detrimentally affected the consistency of its immunogenicity [99]. Delta inulin, on the other hand, can be consistently manufactured and is completely insoluble at < 40ºC. This solubility state enables it to maintain an active particulate structure at physiological temperature. Table 2. Summary of Advax-adjuvanted vaccine research beginning in 2010. Disease/ Condition

Causative Agent

Type of Vaccine

Reference

Encephalitis

Japanese encephalitis virus

Inactivated

Lobigs et al. [161] Larena et al. [163] Petrovsky et al. [164]

Acquired immunodeficiency syndrome

Human immunodeficiency virus

Subunit

Cristillo et al. [165] Menon et al. [166]

Anthrax

Bacillus anthracis

Subunit

Feinen et al. [167]

Listeriosis

Listeria monocytogenes

Subunit

Calderon-Gonzalez et al. [168] Rodriguez-Del Rio et al. [169] Calderon-Gonzalez et al. [170]

Severe acute respiratory syndrome (SARS)

SARS-associated coronavirus

Subunit

Honda-Okubo et al. [171] McPherson et al. [172]

Respiratory complications

Respiratory syncytial virus

Live (nonattenuated)

Wong et al. [173]

Hepatitis B

Hepatitis B virus

Subunit

Cooper and Petrovsky [100] Saade et al. [174] Gordon et al. [175]*

Inactivated

Layton et al. [176] Honda-Okubo et al. [177] Honda-Okubo et al. [178] Honda-Okubo et al. [179] Honda-Okubo et al. [160] Gordon et al. [180]* Gordon et al. [181]* Li et al. [182]* Murugappan et al. [183]

Influenza

*

Indicates clinical trial.

Influenza virus

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Using the inulin expertise of Dr. Cooper, Australia’s then Director of the National Health Sciences Center, Dr. Nikolai Petrovsky, founded the company known as Vaxine Pty Ltd (termed Vaxine for short; www.vaxine.net) in 2002 [158, 159]. One of the company’s leading product candidates was a delta inulin-based particulate adjuvant called Advax, as reviewed in [99, 120-122]. It was designed through the U.S. National Institutes of Health’s Adjuvant Development Program [160] and first termed as Advax in 2010 [161]. Vaxine and collaborators have since pursued the formulation of numerous vaccines containing current good manufacturing practices-grade (cGMP) Advax [98, 100], which can also be terminally sterilized with gamma irradiation [162]. As summarized in Table 2, they have targeted several diseases and complications, including encephalitis caused by Japanese encephalitis virus (JEV), AIDS, anthrax, listeriosis, severe acute respiratory syndrome (SARS), respiratory complications caused by respiratory syncytial virus (RSV), hepatitis B, or influenza. While much of this research remains in preclinical evaluation, vaccines against the hepatitis B and influenza have reached clinical testing. Subsequent sections will discuss the major results from each of these preclinical and clinical studies.

7.1. Encephalitis Caused by JEV JEV is a flavivirus that causes encephalitis in humans, horses, and pigs. It is endemic to many countries in the western Pacific, and up to 70,000 clinical cases with a ~30% fatality rate are reported worldwide each year [184]. JE-VAX®, a reactogenic vaccine formulated from an inactivated JEV grown in mouse brains, was first licensed in the 1960s by the Japanese government. Another JEV vaccine known as IXIARO® (or JESPECT®) was approved by the FDA in 2009. It consists of an inactivated JEV grown in African green monkey kidney epithelial cells (Vero cells) adsorbed to alum. Lobigs et al. evaluated a vaccine comprised of a different Vero cell-grown JEV strain adjuvanted with Advax. They demonstrated that mice immunized with this formulation achieved greater

Immunostimulatory Inulin Adjuvants in Prophylactic Vaccines … 57 serum antibody titers, balanced Th1/2 IgG subtypes, viral neutralization, and survival against an intranasal JEV challenge compared to mice immunized with JE-VAX or the unadjuvanted Vero cell-grown JEV [161]. The Advax-adjuvanted group also showed higher antibody titers and viral neutralization compared to alum-adjuvanted IXIARO. The authors later used mechanistic studies to show that their vaccine induced memory B cells that could protect against a viral challenge independent of CD8+ T cells and pre-existing neutralizing titers [163]. Petrovsky et al. demonstrated that a similar mechanism drove cross-protection against the related flavivirus West Nile virus (WNV) when using the JEV Advax vaccine [164].

7.2. Acquired Immunodeficiency Syndrome While the previous gamma inulin-based HIV vaccine induced cellular immunity [135], Cristillo et al. were interested in evaluating mucosal antibody responses since HIV infects via mucosal regions like the vaginal cavity [165]. Furthermore, they introduced a different subunit antigen strategy consisting of a DNA plasmid encoding the full HIV-1 envelope protein, as well as a recombinant gp120 protein, which is the cleaved portion of the full envelope protein. Two different “Advax” formulations were used: (1) Advax-P, which was the common plant derived delta inulinbased adjuvant; or (2) Advax-M, which was a non-delta inulin, marine sponge-derived glycolipid α-galactosyl ceramide-based adjuvant. They showed that three unadjuvanted DNA immunizations followed by two Advax-adjuvanted gp120 immunizations could induce both systemic and vaginal IgA titers that were greater than just three Advax-adjuvanted gp120 protein immunizations. The serum from Advax groups was neutralizing to homologous HIV-1 isolates, and the sera titers for the Advax groups persisted out to 23 weeks post-final boost. The DNA prime + Advax-adjuvanted protein boost group also generated greater Th1skewing IgG subtype antibodies and T cell cytokine responses than the protein only group. In a follow-up study, Menon et al. immunized rabbits

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and non-human primates using a similar DNA prime followed by delta inulin Advax-adjuvanted protein boost regimen [166]. Fairly broadly neutralizing titers were observed.

7.3. Anthrax Anthrax is a disease caused by the Bacillus anthracis bacterium and came to the forefront of popular concern following the Amerithrax postal service incidents in the early 2000s [185]. BioThrax®, an alum-adjuvanted subunit anthrax vaccine containing a protective antigen (PA) protein, has been FDA-approved for pertinent military and lab personnel since the 1970s. Due to the various reported drawbacks of BioThrax, Feinen et al. examined how Advax or Advax plus murabutide, an NLR agonist, would compare to alum only [167]. While alum provided 100% protection against a lethal dose of aerosolized bacterial anthrax spores, neither Advax nor murabutide alone provided this level of protection. Combining Advax with murabutide, however, afforded anti-PA antibody titers equivalent to alum, as well as 100% protection. The antibody titers and bacterial toxin neutralization, a correlate for survival, were still present 11 months after the immunization. Lastly, the authors showed that the Advax + murabutide adjuvant caused less local in vivo inflammation than alum, signifying the formulation’s encouraging tolerability.

7.4. Listeriosis Listeriosis, a common food-borne illness [186, 187], is caused by the bacterium Listeria monocytogenes (Lm). Recent instances of contaminated food products such as vegetables, cheese, and ice cream has led to numerous deaths and recalls in the U.S. [188]. Currently there are no FDAapproved vaccines against listeriosis, but efforts to develop one are ongoing [189, 190]. Calderon-Gonzalez et al. used bioinformatics to predict MHC-presented epitopes of two Lm protein antigens, listeriolysin

Immunostimulatory Inulin Adjuvants in Prophylactic Vaccines … 59 (LLO) and glyceraldehyde-3-phosphatedehydrogenase (GAPDH), known to induce T cell responses [168]. They used Advax-adjuvanted epitopes to confirm their bioinformatic predictions in two strains of mice. They demonstrated antigenicity of an LLO91-99 epitope and two GAPDH epitopes via induction of T cell responses and protection against an Lm challenge. In a follow-up study, researchers from the same group covalently conjugated LLO91-99 to gold nanoparticles (GNPs, a formulation strategy discussed in Section 11), and then combined the GNPs with Advax to increase the peptide’s antigenicity [169, 170]. A major portion of this study was also to compare this formulation against an idealized “DC vaccine,” where DCs were loaded with the antigen ex vivo to force antigen presentation – an impractical method for widespread immunizations. In addition to being superior to unadjuvanted GNP LLO91-99, the GNP LLO9199 + Advax also performed on par with the DC vaccine with respect to T cell production of Th1 cytokines and protection against an Lm challenge.

7.5. Severe Acute Respiratory Syndrome SARS-associated coronavirus (SARS-CoV) is a positive stranded RNA virus that was first identified in 2002 in Chinese patients. There were 8,000 cases of infection during the subsequent pandemic [191], with an overall mortality rate of 10% and a ~50% mortality rate for elderly patients [192]. A further concern associated with SARS is that infected individuals do not necessarily gain immunity against future infection [193]. There is currently no clinically approved SARS vaccine, and while the alum-adjuvanted subunit or inactivated virus vaccines in development have shown encouraging results, lung pathological issues are still observed following experimental viral challenges. Honda-Okubo et al., therefore, used Advax or a combination composed of Advax and CpG, a TLR 9 agonist, to adjuvant a recombinant spike protein (rSP) [171], which is involved in receptor-mediated endocytosis of the virus into host cells. One year postimmunization, the rSP adjuvanted with the Advax + CpG formulation demonstrated greater Th1-biased IgG antibodies and antibody-secreting B

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cells than rSP adjuvanted with Advax or CpG alone. The Advax-only formulation, on the other hand, appeared to induce higher-proliferating CD8+ T cells and greater production of Th1 cytokines. Following a viral challenge four weeks post-immunization, all rSP groups (alum, Advax, and Advax + CpG) provided 100% protection, and showed similar viral lung titers six days post-challenge. When evaluating the extent of undesired lung pathology, all Advax-adjuvanted groups had minimal infiltration of eosinophils, a type of innate immune cell. Conversely, the unadjuvanted rSP, alum-adjuvanted rSP, and inactivated virus formulations showed substantial eosinophilic infiltration. Following this work, these Vaxine researchers published a book chapter describing methods on how to produce the rSP, as well as a list of experiments to measure the immunogenicity of Advax-adjuvanted rSP [172].

7.6. Respiratory Complications Caused by RSV RSV is part of the Paramyxoviridae family of viruses, and causes substantial respiratory complications in babies and young children [194] that lead to high hospitalization rates [195]. There is currently no FDAapproved vaccine to prevent RSV infections. Previous efforts to formulate a vaccine with an alum adjuvant failed in clinical testing due to enhanced respiratory disease [196]. In a study investigating substitutes for the alum adjuvant, Wong et al. evaluated how well the Advax formulations used in the SARS-CoV vaccines, alone or combined with CpG, could adjuvant a fully live, non-attenuated, RSV vaccine [173]. The Advax + CpGadjuvanted group generated enhanced anti-RSV F protein serum IgG titers that were more skewed toward Th1 subtypes than Advax or alum adjuvant groups, as well as the unadjuvanted virus. Advax + CpG resulted in equivalent viral neutralization as alum, and enhanced over Advax alone. All adjuvanted RSV vaccines resulted in sterile lungs following an intranasal challenge, but they also showed more lung pathology than mice that were immunized with adjuvant alone controls sans the live RSV. As

Immunostimulatory Inulin Adjuvants in Prophylactic Vaccines … 61 expected, these controls offered neither sterility nor detectable neutralizing titers.

7.7. Hepatitis B Currently at least 250 million people are infected with hepatitis B virus (HBV) worldwide, making it one of the most prevalent communicable diseases [197]. In 2011, Cooper and Petrovsky first reported delta inulin’s adjuvant activity [100] using the same HBsAg as the gamma inulin/Algammulin adjuvant vaccine studies of the early 1990s [126, 129]. At the time of publication, the fully formulated Advax adjuvant had yet to be reported. The authors demonstrated that a delta inulin adjuvant enhanced anti-HBsAg antibody titers compared to a gamma inulin adjuvant, and the delta inulin-adjuvanted antigen resulted in higher CD4+ and CD8+ T cell proliferation than the alum-adjuvanted group. Saade et al. expanded upon this initial study by using HBsAg with the fully formulated Advax adjuvant [174]. Here they showed that mice immunized with the Advax group resulted in greater anti-HBsAg IgG titers than the alumadjuvanted Engerix-B® HBV vaccine, which was first approved by the FDA in 1989. Similar to the previous study by Cooper and Petrovsky, the Advax vaccine achieved higher Th1-skewing antibody titers compared to the alum group, as well as increased CD4+ and CD8+ T cell proliferation. In guinea pigs, a next-generation protein antigen known as preS, which is part of the viral envelope, was adjuvanted with Advax. No adverse events were observed. The preS + Advax vaccine also had higher Th1-skewed titers and T cell responses than Engerix-B. Based on these encouraging preclinical studies, in 2014 Vaxine moved to a first-in-man Phase I clinical trial where they assessed three immunizations of HBsAg alone, or HBsAg plus 5 or 10 mg of Advax [175]. The HBsAg dose was less than half of typical alum-adjuvanted clinical vaccines. The safety and immunogenicity of the Advax groups were very encouraging. There were not any increases in molecular inflammatory markers, white blood cell counts remained within a normal range, and no increased injection site pain was reported.

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Four weeks following the third immunization, the anti-HBsAg antibody titers for both Advax groups were higher than antigen alone. There were no differences in antibody levels between the two Advax doses, and the Advax adjuvant groups also exhibited higher CD4+ T cell activity. As of April 2017, no further reports of this vaccine’s development have been published.

7.8. Influenza Much of Vaxine’s efforts have been geared towards development of Advax-adjuvanted influenza vaccines. Influenza virus causes millions of serious infections each year [198] and is a substantial economic burden [199]. There are three different types of FDA-approved influenza vaccines: live-attenuated virus (FluMist®), inactivated virus (many formulations), and subunit recombinant proteins (Flublok®). It is notable that current vaccines protect against just a limited subset of viral strains. Two squalene oil-in-water emulsions (AS03 and MF59) are approved as adjuvants for certain inactivated influenza vaccines, but they have limited immunogenicity (Section 4.2.2). In Vaxine’s first influenza vaccine study, they immunized ferrets, the most relevant animal model to human influenza infections, with an Advax-adjuvanted inactivated H5N1 “bird flu” virus [176]. Higher virus neutralizing titers were achieved with the Advax groups than the inactivated virus alone. Furthermore, all ferrets receiving the Advax-adjuvanted inactivated virus were 100% protected versus only 67% for animals immunized with the virus alone. No gross adverse events were observed and the central nervous pathology of the Advax survivors was better than survivors who received the unadjuvanted virus. In the next study, Honda-Okubo et al. tested an Advax-adjuvanted clinical seasonal trivalent inactivated influenza vaccine in mice [177]. Serum antibody titers and T cell proliferation were enhanced in the presence of Advax. Using one mouse-adapted H1N1 influenza strain, similar results were observed with antibodies, virus neutralization, and T cell responses. The Advax group offered similar protection against a lethal

Immunostimulatory Inulin Adjuvants in Prophylactic Vaccines … 63 challenge as an MF59-adjuvanted group. Furthermore, adjuvant only injections demonstrated that Advax caused less local injection site reactogenicity compared to MF59, Montanide ISA 720, Quil A, and CFA. The encouraging results from the two preclinical studies enabled Vaxine to quickly progress to a clinical trial just three months after the WHO had isolated the 2009 H1N1 pandemic influenza strain. In this trial they used Advax-adjuvanted recombinant hemagglutinin (rHA) [180]. The rHA was made by Protein Sciences Corporation, which has since received FDA approval for their rHA-based subunit influenza vaccine, Flublok. Two hundred eighty-one non-pregnant patients were enrolled into one of six groups containing three rHA doses with or without an Advax adjuvant. The adjuvanted rHA formulations showed encouraging immunogenicity without any increase in reported adverse events by the subjects. A recently published study followed up on Gordon et al.’s clinical trial [160]. The authors discussed their initial struggles with determining the exact sequence of the WHO’s isolated pandemic flu HA protein antigen. This subsequently made it difficult to determine the proper rHA protein to use for the hemagglutinin inhibition (HAI) assay, a standard test for clinical flu vaccine immunogenicity [200, 201]. Remarkably, despite the HAI assay’s broad use, it does not provide a direct functional output. The authors, therefore, re-tested the clinical samples using both the HAI assay, as well as a more functional microneutralization (MNT) assay. MNT evaluates antibodies’ ability to prevent viral infection in vitro. The data showed that although the HAI assay correlated with the MNT assay, the HAI assay was less successful in measuring cross-reactivity of antibodies against other non-vaccine HAs. These results suggest that a recombinant protein platform might one day be used to formulate “universal” flu vaccines that are protective against all strains of influenza virus. Since influenza vaccines are administered to a very heterogeneous population, following Gordon et al.’s 2012 clinical trial, Vaxine reverted back to a preclinical study, and this time immunized pregnant murine dams with rHA or Advax + rHA [178]. The adjuvanted rHA offered improved antibody titers, as well as protection against a lethal viral challenge. The dams’ litter size and weight of their pups were not detrimentally affected

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by Advax. In a follow-up study, Honda-Okubo et al. immunized neonatal pups and showed that they could be protected against a lethal viral challenge and overcome the T cell hyporesponsiveness often observed following neonatal flu vaccination [179]. In a follow-up clinical trial, Gordon et al. considered the safety and immunogenicity of an Advaxadjuvanted reduced dose seasonal flu vaccine versus a full dose unadjuvanted seasonal flu vaccine [181]. Encouraging antibody responses and tolerability were observed. In a parallel clinical trial, Li et al. examined blood samples from Gordon et al.’s trial to evaluate Advax’s mechanism of action [182]; their work can be referenced for details of the study and its results. In recent research, Vaxine has evaluated preclinical pulmonary delivery of an Advax-adjuvanted inactivated flu vaccine to achieve mucosal immunity at the site of infection. Murugappan et al. demonstrated higher overall antigen-specific serum antibodies, similar Th1/2 IgG antibody skewing, and increased mucosal IgA titers compared to the unadjuvanted inactivated flu vaccine [183]. A needle-free pulmonary vaccine, which could promote higher patient compliance, might be especially valuable if FDA-approved FluMist continues to demonstrate poor efficacy [202].

8. INULIN-BASED ADJUVANTS IN ANIMAL VACCINES In addition to human applications, inulin adjuvants (gamma inulin, Algammulin, and Advax) have also been used in several animal vaccines. These vaccines have been formulated against pathogens that infect either just animals, or animals as well as humans. The results of these studies, as discussed in subsequent sections, demonstrate that inulin can successfully be used in animal vaccines against several pathogens. Table 3 contains a summary of the studies.

Immunostimulatory Inulin Adjuvants in Prophylactic Vaccines … 65 Table 3. Summary of inulin-adjuvanted animal vaccines. Disease/ Condition

Causative Agent

Type of Vaccine

Inulin Formulation

Reference

Taeniasis

Taenia ovis

Subunit

Gamma inulin & Algammulin

Deol et al. [203]

Glanders & African horse sickness

Burkholderia mallei & African horse sickness virus

Inactivated

Advax

Eckersley et al. [204]

Encephalitis

Japanese encephalitis virus

Inactivated

Advax

Lobigs et al. [161] Bielefeldt-Ohmann et al. [205]

Peste des petits ruminants

Peste des petits ruminants virus

Inactivated

Advax

Ronchi et al. [206] Cosseddu et al. [207]

8.1. Taeniasis Pork or beef-derived Taenia spp., the causative agents of parasitic tapeworm infections, known as taeniasis, are endemic to many developing countries and a growing concern in developed countries as immigration to these regions increases [208]. The species from pork, T. solium, causes the severe human neurological disease cysticercosis, which was added to the list of Neglected Tropical Diseases in 2010 [209]. Another Taenia species, T. ovis, is asymptomatic in humans. It does, however, cause “measles” in sheep and can form cysts in sheep meat that results in rejection for human consumption. Deol et al. evaluated a recombinant GST-45W T. ovis antigen adjuvanted with alum, gamma inulin, Algammulin, or CFA in a sheep model [203]. The proliferation of cells from sheep immunized using gamma inulin, Algammulin, or CFA were not statistically different than each other, but each of the proliferation indices was greater than the alum group. CFA was superior to all other adjuvants for inducing antibody responses. Alum, gamma inulin, and Algammulin performed on par with each other. Unlike other vaccines tested in mice, gamma inulin and

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Algammulin did not induce greater Th1-biased IgG subtypes than alum. This could suggest differences in species’ responses. Finally, only the CFA group protected sheep from a T. ovis egg challenge. A correlation between antibody responses and protection was noted.

8.2. Glanders and African Horse Sickness Vaxine’s first reported veterinary application was completed in collaboration with a United Arab Emirates research group interested in camel vaccines. They evaluated the reactogenicity and humoral immunogenicity of a substantial list of adjuvants: Gerbu Vet (cationic liposomes), Gerbu Pharma (cationic liposomes encapsulating glucoaminylmuramyl dipeptide, an NLR agonist), Montanide ISA 763 A VG (emulsified plant-derived oil), Montanide IMS 3012 VG PR (another oil emulsion), Montanide Pet Gel A (non-emulsified polyacrylic polymer in water), Advax Horse and Camel Excel (Advax HCXLTM, a delta inulin formulation specially made for horses and camels), and alum [204]. Camels were injected with adjuvants only, adjuvants plus an inactivated Burkholderia mallei bacterium (causative agent of glanders), or adjuvants plus inactivated African horse sickness virus strain 4 (AHSV4). No substantial inflammation, as measured by total inflammation area, was induced with Advax HCXL or Montanide Pet Gel A adjuvant only groups, or inactivated formulations adjuvanted with Advax. Additionally, Advax did not increase injection site skin thickness or temperature. In fact, the authors used a reactogenicity index to demonstrate that Advax showed no more reactogenicity than saline only injections. Furthermore, the AHSV4 antibody titers generated by the Advax-adjuvanted group were equivalent to several of the other adjuvants, but the Advax-adjuvanted B. mallei vaccine was unexpectedly the only one to induce antibody production. Overall, Advax demonstrated promising tolerability and immunogenicity.

Immunostimulatory Inulin Adjuvants in Prophylactic Vaccines … 67

8.3. Encephalitis Caused by JEV As discussed earlier in the chapter, JEV infects not only humans, but also domesticated animals such as pigs and horses. Lobigs et al. demonstrated that adult horses immunized with the same vaccine as the mouse study discussed in section 7.1, cell-grown JEV + Advax, attained higher viral neutralizing titers than unadjuvanted cell-grown JEV or JEVAX [161]. The cell-grown JEV + Advax vaccine additionally showed cross-neutralizing titers against the related flaviviruses WNV and Murray Valley encephalitis virus (MVEV). In a follow-up study, BielefeldtOhmann et al. evaluated the same vaccine in pregnant mares and young foals [205]. Only mild infection site inflammation was noted in both populations. A high percentage of the foals and pregnant mares, 75 and 100%, respectively, achieved JEV-specific neutralizing titers more than 7 months following their booster shot. With respect to cross-reactivity titers against WNV and MVEV, the foals had little to none against either pathogen. Some of the pregnant mares, on the other hand, demonstrated cross-reactivity against both viruses. At the time of foaling, the mares also had cross-neutralizing titers in their colostrum. Furthermore, foals born to naïve mares responded much quicker to a prime immunization than foals born to immunized mares. A booster vaccination administered to the same animals 10 months later generated increased responses against both JEV and the related flaviviruses.

8.4. Peste des Petits Ruminants Sheep and goats experience up to 90% mortality when infected with peste des petits ruminants virus (PPRV), a pathogen that is endemic to many African and Asian countries [210]. Of the four viral lineages that exist, two of them, I and IV, are currently used in live-attenuated veterinary vaccines [211]. In an effort to replace the live-attenuated vaccines with an inactivated formulation, Ronchi et al. added either Montanide ISA 71 VG (water-in-oil emulsion), or Advax supplemented

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with or without the TLR 9 agonist CpG [206]. A concentrated viral dose adjuvanted with Advax + CpG (AFSA2) demonstrated the highest antibody titers in both rats and goats. Furthermore, the goats’ rectal temperatures did not rise following vaccination, and there was no evidence of adverse events. In a follow-up study, Cosseddu et al. demonstrated that 60 days following a boost, goats vaccinated with the inactivated virus plus AFSA2 reached peak neutralizing titers [207]. Following an intranasal viral challenge, the vaccinated goats were able to mount neutralizing titers faster than unvaccinated goats. The AFSA2 group also did not exhibit any viremia, whereas the unvaccinated animals developed a fever, as well as had higher viremia and viral loads in numerous lymph nodes and organs.

9. OTHER INULIN FORMULATIONS AS VACCINE ADJUVANTS AND ADDITIVES While this chapter’s primary focus has been on particulate gamma and delta inulin isoforms, it is important to mention that other inulin fractions have also been used as preclinical vaccine adjuvants. These include (i) a complete polysaccharide extract of inulin for a Chagas disease vaccine [212]; (ii) alpha inulin, which may have been misidentified and actually be a particulate inulin isoform, for a filariasis vaccine [213]; and (iii) a squalene-emulsified whole extract of inulin chemically modified with a sulfate group and lipid for pseudorabies or influenza vaccines [214]. Additionally, inert whole soluble extracts of inulin can also be used as cryoprotectants and/or storage stabilizers for vaccines [215-220], as reviewed by Mensink et al. [121].

10. OTHER USES OF GAMMA INULIN, ALGAMMULIN, DELTA INULIN, AND ADVAX In addition to gamma or inulin-based adjuvants in prophylactic vaccines against pathogens, there is other literature of potential interest that

Immunostimulatory Inulin Adjuvants in Prophylactic Vaccines … 69 is beyond the scope of this chapter, including a preclinical contraceptive vaccine using gamma inulin [221], preclinical Alzheimer’s vaccine with Advax or Advax + CpG [222], and clinical trial for honey bee venom immunotherapy using Advax [223].

11. DELIVERY VEHICLES –ALTERNATIVE APPROACH TO FORMULATING INULIN ADJUVANTS Although Advax has very promising adjuvant characteristics, it is not engineered to offer controlled delivery of antigens [99] or secondary adjuvants such as PAMPs. Thus, a potential window of opportunity exists to further improve inulin adjuvants. Technologies known as delivery systems (e.g., lipid bilayer structures called liposomes, polymeric particles, polymer conjugates) offer a promising strategy to fulfill this potential while continuing to achieve safe and efficacious vaccines with inulin-based adjuvants. Drug delivery systems have various advantages, as summarized by Drs. Robert Langer and Nicholas Peppas in their seminal review papers [224, 225]. When applied to vaccine delivery, these systems can: (i) facilitate antigen or secondary adjuvant dose-sparing resulting from increased uptake by APCs [226, 227]; (ii) induce higher APC antigen cross-presentation on MHCs I and II [228, 229]; (iii) improve secondary adjuvant safety profiles by decreasing systemic exposure [230, 231]; and/or (iv) enable a shorter immunization schedule due to sustained antigen/adjuvant release from the vehicles [232, 233], which can also improve patient adherence [234]. The 1960-80s saw use of the first antigen or adjuvant-loaded delivery vehicles. The initial antigen delivery systems included polymeric micelles [235], liposomes [236-238], polyelectrolyte polymer conjugates [239], and polymeric particles [240-244]. The potential for sustained antigen release from a vehicle was first demonstrated in 1976, when Langer and Folkman reported the use of non-degradable ethylene-vinyl acetate copolymer pellets that exhibited a sustained 100 day protein release [245]. A few

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years later, Preis and Langer reported their seminal work showing that a single dose of these antigen-loaded polymer pellets could generate longterm antibody titers on par with two doses of CFA-adjuvanted antigen [246, 247]. The first adjuvant delivery vehicles to be reported were PAMPloaded liposomes [237, 248, 249], and self-assembled cage-like ISCOMs composed of saponins were discovered soon after [88]. Over the last several decades, numerous other antigen and/or adjuvant delivery vehicles have been engineered, including polymeric micelles [250], viral vectors [251], dendrimers [252], lipid-based systems [253, 254], virosomes [255], hydrogels/scaffolds [256], and biodegradable or non-degradable nano/microparticles [226, 257]. Several of the inulin studies previously discussed in the chapter used one of these antigenloaded systems (liposomes [137] or non-degradable gold nanoparticles [169, 170]). While none of the above mentioned antigen-loaded vehicles have reached clinical testing, the potential of the strategy is highlighted by other antigen or adjuvant-loaded systems that have successfully attained this milestone [258]. A few notable adjuvant vehicles in clinical trials are the micellar Quil A-based cage-like structures known as ISCOMTM, where the antigen is entrapped within the complex, and ISCOMATRIXTM, where the antigen is mixed with a preformed complex [89]. Antigen or adjuvant systems that have earned full approval by non-U.S. regulatory agencies include an antigen mixed with liposomal AS01 for malaria (RTS,S) [95, 96, 146, 259], a polymer-antigen conjugate for influenza (Grippol®) [239], an inactivated virus adsorbed to a liposome-like virosome for hepatitis A (Epaxal®), and a protein antigen entrapped in a virosome for influenza (Inflexal®) [30]. To date, the FDA has not approved any analogous systems, and it is notable that Epaxal and Inflexal are being phased out of production [254].

Immunostimulatory Inulin Adjuvants in Prophylactic Vaccines … 71

11.1. Inulin-Based Antigen Delivery Systems While Vaxine has effectively moved forward with their Advax adjuvant platform, a few research groups have recently engineered antigenloaded inulin vehicles with dual functionality: (i) controlled antigen delivery and (ii) immunostimulatory properties stemming from the vehicles’ inulin component. This strategy can be first credited to Dr. Viktor Kabanov’s work with antigen-immunostimulatory polyelectrolyte conjugates in the 1970s [239], and Kohn et al.’s design of antigen-loaded biodegradable polymer implant with immunostimulatory hydrolytic byproducts in the mid-1980s [260]. Here we will describe the four inulinbased studies that take this approach. Table 4 provides a summary of the studies, and Figure 2 depicts the vehicle platforms. Table 4. Summary of dual-functioning inulin-based antigen delivery vehicles. Disease/ Condition

Causative Agent

Type of Vaccine

Inulin Formulation

Reference

Subunit

Soluble inulin microparticles

Kumar et al. [261]

Subunit

Inulin-chitosan conjugate

Yu and Hu [262]

N/A (model system)

Subunit

Acetalated inulin microparticles

Gallovic et al. [263]

N/A (model system)

Subunit

Silylated inulin microparticles

Gallovic et al. [264]

N/A (model system)

Tuberculosis

Mycobacterium tuberculosis

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Figure 2. Representative schematics of the two types of inulin-based antigen delivery systems: (A) antigen bulk encapsulated within modified-inulin microparticles, and (B) antigen covalently conjugated to inulin/chitosan.

In the first study, Kumar et al. used an overnight ethanol precipitation method to extract a soluble inulin fraction that was presumably some combination of alpha and/or beta inulin [261]. Soluble inulin microparticles (sIMs) encapsulating a model ovalbumin (OVA) protein antigen were formed by a water-in-oil emulsion. The OVA-loaded sIMs were then precipitated out with acetone, washed, and collected. They were tested for endotoxin contamination and were within the acceptable limits for parenteral formulations. The sIMs had an average hydrodynamic diameter of 1.5 µm, encapsulated OVA at 75% efficiency, and attained an OVA loading capacity of 7.5% w/w. All of the OVA was released in 16 hours when incubated at physiological buffer conditions, suggesting moderate controlled release. DC uptake of OVA loaded in sIMs was drastically increased compared to soluble OVA. When mice were

Immunostimulatory Inulin Adjuvants in Prophylactic Vaccines … 73 immunized with the OVA-loaded sIMs, anti-OVA antibody titers were enhanced relative to OVA adsorbed to alum or OVA mixed with empty SIMs. The sIMs showed no detrimental effects according to histological analysis of the injection site. The authors also demonstrated that sIMs functioned independently of the ACP, establishing an unconventional platform for using soluble inulin adjuvants. These are interesting results based on soluble fractions’ established lack of adjuvant activity, and emphasize the favorable utility of particulate delivery vehicles. Future studies with pathogen-associated antigens would be valuable. In another inulin-based delivery vehicle study, Yu and Hu formulated a vaccine against the causative agent of tuberculosis, M. tuberculosis (Mtb) [262]. Tuberculosis is one of the leading causes of death worldwide, where ~1.4 million people succumb to the disease per year [265]. While there is an FDA-approved live-attenuated tuberculosis vaccine that has been used clinically for almost 100 years, Bacillus Calmette-Guérin (BCG), its efficacy can be short-lived in children, sporadic across populations, and inadequate against pulmonary tuberculosis in adults [266]. Many tuberculosis vaccines are in development [267], and in this study the authors used a fusion protein (CT) consisting of two Mtb antigens, CFP10 and TB10.4. The adjuvant was prepared from a whole extract of inulin chemically attached to an immunostimulatory chitosan polysaccharide (inulin-Cs). The CT was, in turn, covalently conjugated to the inulin-Cs adjuvant to create inulin-Cs-CT. Mice were immunized three times with the following groups: unadjuvanted CT, CT adjuvanted with alum, an inulin-CT conjugate, a chitosan-CT conjugate, or inulin-Cs-CT. The inulin-Cs-CT resulted in the highest amount of total anti-CT IgG and Th1skewed IgG subtype antibody titers, as well as the greatest T cell cytokine responses. Finally, the inulin-Cs-CT levels displayed a more long-lasting plasma half-life than any of the other CT formulations, as well as complementary sustained IgG titers. This suggests that the formulation could offer dose-sparing. The use of this platform may be expanded to other protein antigens as well. The next study to take this approach was conducted by Gallovic et al. [263]. A summary of the data reported in this manuscript can be found in

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Figure 3. They implemented a one-step acetalation chemical reaction that has been widely used with a dextran polysaccharide (reviewed by Bachelder et al. [268]). In this work, they converted a whole extract of inulin, which is water-soluble at room temperature, into an insoluble form by protecting the fructose monomer’s pendant hydroxyl groups with hydrophobic acetal groups. The modified inulin, termed acetalated inulin (Ace-IN), could be processed into polymeric particles using emulsion chemistry or other particle fabrication methods. Acetal groups are much more hydrolytically labile at acidic pH 5.0 than physiological pH 7.4. This was particularly appealing because of APCs’ acidic phagolysomal environment, which can range in pH from ~4.5 to 6.0 [269]. The Ace-IN particles had more sustained stability at pH 7.4, and underwent triggered degradation and payload release upon particle phagocytosis. Furthermore, the extent of acetalation could be tuned based simply on the one-step reaction time. The authors used an oil-in-water emulsion procedure to make blank Ace-IN microparticles (ranging in size from ~0.5 to 2 µm) with different extents of acetalation. They showed that the particles’ degradation half-life could be tuned and that their degradation was acid-sensitive. In order to accurately assess the Ace-IN microparticles immunogenicity and prevent false-positive immune responses, they were fabricated via a process that prevented endotoxin contamination. A model Mϕ cell line was treated in vitro with these “clean” Ace-IN microparticles. It was shown that the clean particles could induce production of pro-inflammatory cytokines. The authors further demonstrated that cytokine production was contingent on having particulate inulin. They accomplished this by making particles containing various blends of Ace-IN and an inert acetalated dextran (Ac-DEX) polysaccharide, and showed that cytokine production was dependent on the particles’ inulin mass content. Following these proof-of-concept studies with the empty particles, the authors then used a water-in-oil-in-water emulsion to encapsulate a model OVA antigen within the Ace-IN microparticles. They showed that the OVA can be sustainably released for at least one week at physiological pH. Additionally, APCs could be passively targeted with this formulation in vivo, because APCs are the only

Immunostimulatory Inulin Adjuvants in Prophylactic Vaccines … 75 cells capable of phagocytosing particles of this size. When vaccinated with the OVA-loaded Ace-IN particles, mice produced as many anti-OVA antibodies as OVA adsorbed to alum. Unfortunately, cellular immunity was not evaluated in the study.

Source: Reproduced from Ref. 263 with permission from the Royal Society of Chemistry. Copyright 2016. Figure 3. (A) Synthesis of acetalated inulin (Ace-IN) polymer. DMSO = dimethyl sulfoxide, CHCl3 = chloroform; (B) scanning electron micrograph of Ace-IN microparticles (MPs), where the scale bar is 5 µm; (C) release kinetics of Texas Redlabeled ovalbumin (TR-OVA) from Ace-IN MPs at acidic and neutral pH; and (D) anti-OVA serum IgG antibody titers, where 26.7% represents the extent of polymer acetalation. PBS = saline control.

In a follow-up study, Gallovic et al. implemented another synthetic platform to chemically modify inulin [264]. A summary of the data reported in this manuscript can be found in Figure 4. Here they converted a whole inulin extract’s pendant hydroxyl groups into an acid-sensitive trimethylsilyl (TMS) ether moiety. The modified trimethylsilyl inulin (TMS-IN) was rendered organic-soluble to enable particle fabrication. The

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TMS-IN particles exhibited degradation half-lives of 16 and 130 hours at pH 5.0 and 7.4, respectively. Additionally, TMS-IN microparticles (with no measurable endotoxin) were cytocompatible and independently induced high levels of a pro-inflammatory cytokine. An OVA protein antigen was encapsulated in the particles using a water-in-oil-in-water emulsion process. Mice immunized with these particles generated anti-OVA antibody titers equivalent to alum-adjuvanted OVA. Unlike the previous Ace-IN study, the authors also evaluated the cellular immunity induced by the particle-encapsulated OVA. They showed that the TMS-IN particle group induced a strong Th1-polarizing cytokine response that was much greater than unadjuvanted OVA, OVA mixed with TMS-IN particles, and alum-adjuvanted OVA.

Source: Reproduced from Ref. 264 with permission from the Royal Society of Chemistry. Copyright 2016. Figure 4. (A) Synthesis of trimethylsilyl inulin (TMS-IN) polymer. Et3N = triethylamine, DMSO = dimethyl sulfoxide, THF = tetrahydrofuran; (B) scanning electron micrograph of TMS-IN microparticles (MPs), where the scale bar is 5 µm; (C) anti-ovalbumin (OVA) serum IgG antibody titers; and (D) OVA-specific cellular response as indicated by IFN-γ cytokine production. PBS = saline control.

Immunostimulatory Inulin Adjuvants in Prophylactic Vaccines … 77 These recent two studies conducted by Gallovic et al. demonstrate that “smart” stimuli-responsive inulins can be designed to enhance vaccine efficacy. While these systems have exhibited many advantages, follow-up studies are needed to realize the platforms’ full potential. First of all, evaluating how the extent of chemical modification (acetalation or silylation) affects vaccine outcomes would assist in further optimizing the platform. It is known that chemical characteristics such as hydrophobicity affect particle immunogenicity [270]. Encapsulation of secondary adjuvants (e.g., PAMPs) within Ace-IN particles could also be a promising avenue to achieve more broad immune responses. Furthermore, the particles’ safety and efficacy need to be tested in clinically relevant immunization models, similar to previously published Ac-DEX studies. Determining the particles’ mechanism of adjuvant activity would be beneficial as well.

CONCLUSION Among the many adjuvants in development, inulin formulations have demonstrated an especially encouraging balance of safety and efficacy. Inulin’s long journey to becoming a vaccine adjuvant began in 1971, with the first report of its immunostimulatory properties (Figure 5). Particulate gamma inulin-based formulations exhibited promise as adjuvants in the late 1980s, but ultimately were deemed suboptimal. The continued efforts of inulin expert Dr. Peter Cooper, in collaboration with many researchers, eventually enabled the company Vaxine to be founded in 2002 by one of his collaborators, Dr. Nikolai Petrovsky. In the 15 years since, Vaxine has effectively developed its particulate delta inulin-based Advax formulation for application across a wide variety of prophylactic vaccines. Advax reaching clinical trials as the adjuvant for influenza and hepatitis B vaccines is a significant step, but it is important to remember that regulatory agencies are often slow to approve new adjuvants unless they are involved with breakthrough or essential vaccines. Very few adjuvants are clinically licensed, and demonstrating sufficient safety to achieve this

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Figure 5. Timeline of significant milestones in the development of inulin-based vaccine adjuvants. ACP = alternative complement pathway.

Immunostimulatory Inulin Adjuvants in Prophylactic Vaccines … 79 licensure is a long and arduous process. GSK’s AS01 adjuvant, for instance, was first designed in the 1990s, and it has taken 20+ years for it to reach a clinical market. It will continue to be the job of vaccinologists to further optimize existing adjuvant systems, while also conceiving new adjuvants and vaccination strategies. Since Advax does not offer any control over antigen delivery kinetics, one next-generational strategy is to design inulin-based antigen delivery vehicles at the intersection of vaccinology and pharmaceutics. These dual-functioning engineered delivery systems, with features such as biological stimuli-responsive moieties, could bolster vaccine efficacy. The reported inulin-based antigen delivery systems have demonstrated very encouraging results, and further development, application, and optimization will be necessary for realizing their full potential.

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Immunostimulatory Inulin Adjuvants in Prophylactic Vaccines … 105 [247] Langer, R. Production of Antibodies. Polymers for the sustained release of macromolecules: Their use in a single-step method of immunization. Methods in Enzymology. 1981, 73(Pt B), 57-75. [248] Siddiqui, WA; Taylor, DW; Kan, SC; Kramer, K; Richmond-Crum, SM; Kotani, S; et al. Vaccination of experimental monkeys against Plasmodium falciparum: a possible safe adjuvant. Science. 1978, 201(4362), 1237-1239. [249] Dancey, GF; Yasuda, T; Kinsky, SC. Enhancement of liposomal model membrane immunogenicity by incorporation of lipid A. The Journal of Immunology. 1977, 119(6), 1868-1873. [250] Wilson, JT; Keller, S; Manganiello, MJ; Cheng, C; Lee, CC; Opara, C; et al. pH-Responsive nanoparticle vaccines for dual-delivery of antigens and immunostimulatory oligonucleotides. ACS Nano. 2013, 7(5), 3912-3925. [251] Bråve, A; Ljungberg, K; Wahren, B; Liu, MA. Vaccine delivery methods using viral vectors. Molecular Pharmaceutics. 2007, 4(1), 18-32. [252] Heegaard, PM; Boas, U; Sorensen, NS. Dendrimers for vaccine and immunostimulatory uses. A review. Bioconjugate Chemistry. 2009, 21(3), 405-418. [253] Moon, JJ; Suh, H; Bershteyn, A; Stephan, MT; Liu, H; Huang, B; et al. Interbilayer-crosslinked multilamellar vesicles as synthetic vaccines for potent humoral and cellular immune responses. Nature Materials. 2011, 10(3), 243-251. [254] Alving, CR; Beck, Z; Matyas, GR; Rao, M. Liposomal adjuvants for human vaccines. Expert Opinion on Drug Delivery. 2016, 13(6), 807816. [255] Bungener, L; Serre, K; Bijl, L; Leserman, L; Wilschut, J; Daemen, T; et al. Virosome-mediated delivery of protein antigens to dendritic cells. Vaccine. 2002, 20(17), 2287-2295. [256] Singh, A; Peppas, NA. Hydrogels and scaffolds for immunomodulation. Advanced Materials. 2014, 26(38), 6530-6541.

106 Matthew D. Gallovic, Eric M. Bachelder and Kristy M. Ainslie [257] McHugh, KJ; Guarecuco, R; Langer, R; Jaklenec, A. Single-injection vaccines: progress, challenges, and opportunities. Journal of Controlled Release. 2015, 219, 596-609. [258] Irvine, DJ; Swartz, MA; Szeto, GL. Engineering synthetic vaccines using cues from natural immunity. Nature Materials. 2013, 12(11), 978-990. [259] Fox, CB; Kramer, RM; Barnes, VL; Dowling, QM; Vedvick, TS. Working together: interactions between vaccine antigens and adjuvants. Therapeutic Advances in Vaccines. 2013, 1(1), 7-20. [260] Kohn, J; Niemi, SM; Albert, EC; Murphy, JC; Langer, R; Fox, JG. Single-step immunization using a controlled release, biodegradable polymer with sustained adjuvant activity. Journal of Immunological Methods. 1986, 95(1), 31-38. [261] Kumar, S; Tummala, H. Development of soluble inulin microparticles as a potent and safe vaccine adjuvant and delivery system. Molecular Pharmaceutics. 2013, 10(5), 1845-1853. [262] Yu, W; Hu, T. Conjugation with an Inulin–Chitosan Adjuvant Markedly Improves the Immunogenicity of Mycobacterium tuberculosis CFP10-TB10.4 Fusion Protein. Molecular Pharmaceutics. 2016, 13(11), 3626-3635. [263] Gallovic, MD; Montjoy, DG; Collier, MA; Do, C; Wyslouzil, BE; Bachelder, EM; et al. Chemically modified inulin microparticles serving dual function as a protein antigen delivery vehicle and immunostimulatory adjuvant. Biomaterials Science. 2016, 4(3), 483493. [264] Gallovic, MD; Bandyopadhyay, S; Borteh, H; Montjoy, DG; Collier, MA; Peine, KJ; et al. Microparticles formulated from a family of novel silylated polysaccharides demonstrate inherent immunostimulatory properties and tunable hydrolytic degradability. Journal of Materials Chemistry B. 2016, 4(24), 4302-4312. [265] World Health Organization. The Top 10 Causes of Death - Fact Sheet #310, 2017. Available from: http://www.who.int/mediacentre/ factsheets/fs310/en/.

Immunostimulatory Inulin Adjuvants in Prophylactic Vaccines … 107 [266] Ottenhoff, TH; Kaufmann, SH. Vaccines against tuberculosis: where are we and where do we need to go?. PLoS Pathogens. 2012, 8(5), e1002607. [267] Kaufmann, SH. Tuberculosis vaccines: time to think about the next generation. Seminars in Immunology. 2013, 25(2), 172-181. [268] Bachelder, EM; Pino, EN; Ainslie, KM. Acetalated Dextran: A Tunable and Acid-Labile Biopolymer with Facile Synthesis and a Range of Applications. Chemical Reviews. 2016, 117(3), 1915-1926. [269] Deshayes, S; Kasko, AM. Polymeric biomaterials with engineered degradation. Journal of Polymer Science Part A: Polymer Chemistry. 2013, 51(17), 3531-3566. [270] Petersen, LK; Ramer-Tait, AE; Broderick, SR; Kong, CS; Ulery, BD; Rajan, K; et al. Activation of innate immune responses in a pathogenmimicking manner by amphiphilic polyanhydride nanoparticle adjuvants. Biomaterials. 2011, 32(28), 6815-6822.

BIOGRAPHICAL SKETCHES Dr. Matthew D. Gallovic Dr. Matthew D. Gallovic is a postdoctoral researcher under Drs. Erc. M. Bachelder and Kristy M. Ainslie in the Division of Pharmacoengineering and Molecular Pharmaceutics at the University of North Carolina at Chapel Hill. After receiving his B.S. in chemical engineering from Northwestern University, he worked in the industrial sector designing and characterizing biopharmaceutical delivery systems. Following this experience, he returned to academia to pursue his Ph.D. in chemical engineering at the Ohio State University. He joined the labs of Drs. Barbara Wyslouzil and Ainslie, where he worked on developing improved formulation and delivery strategies for drugs and vaccines using acid-degradable biopolymeric nano/microparticles. He completed his doctoral studies in 2016, and has been in his current position since.

108 Matthew D. Gallovic, Eric M. Bachelder and Kristy M. Ainslie Dr. Eric M. Bachelder Dr. Eric M. Bachelder initially gained expertise in immune responses and cell interactions while mentored by Drs. William Velander and Polly Matzinger. Following completion of his graduate studies, he became a postdoctoral research fellow for Dr. Jean Fréchet at the University of California, Berkeley. In this position he invented various biopolymers for drug and vaccine delivery platforms. In both his appointments as Assistant Research Professor, previously at the Ohio State University and currently at the University of North Carolina at Chapel Hill, his research has involved the development of novel drug delivery systems for vaccines, as well as immunotherapies against cancer and autoimmune diseases. Dr. Bachelder holds a Ph.D. in chemical engineering from the University of Nebraska. Dr. Kristy M. Ainslie Dr. Kristy M. Ainslie has always maintained a very interdisciplinary research program, centered on developing strategies for delivering vaccines and pharmaceuticals. She currently holds an Associate Professor appointment in the Division of Pharmacoengineering and Molecular Pharmaceutics within the Eshelman School of Pharmacy at the University of North Carolina at Chapel Hill, and is affiliated with the Department of Biomedical Engineering as well. Her research focuses on the design and application of numerous formulations for prophylactic or therapeutic treatments against infections, cancer, and autoimmune conditions. She previously held a position as an Assistant Professor in the Division of Pharmaceutics and Pharmaceutical Chemistry at the Ohio State University. Prior to this, she was a postdoctoral scholar at the University of California, San Francisco under Dr. Tejal Desai, where she fabricated oral drug delivery vehicles and studied immune responses to nanomaterials. Dr. Ainslie holds both an M.S. and Ph.D. in chemical engineering from the Pennsylvania State University.

In: Inulin Editor: Christian R. Davis

ISBN: 978-1-53612-301-2 © 2017 Nova Science Publishers, Inc.

Chapter 3

INULIN AS A FAT REPLACER IN DAIRY PRODUCTS Tatiana Colombo Pimentel1,*, PhD, Suellen Jensen Klososki1, PhD, Michele Rosset2, PhD, Carlos Eduardo Barão1 PhD and Gislaine Silveira Simões3, PhD 1

Federal Institute of Paraná (IFPR), Paranavaí, Paraná, Brazil Federal Institute of Paraná (IFPR), Colombo, Paraná, Brazil 3 Federal Institute of Paraná (IFPR), Foz do Iguaçu, Paraná, Brazil 2

ABSTRACT Dairy products are regarded by consumers as healthy products because they aid in the digestion process, are essential for the bones, and help the immune system. However, due to the negative relationship between consumption of saturated fats and heart disease, reducing animal fat in the diet has been recommended by nutritionists, increasing the consumption of low-fat or fat-free products. Milk fat has an important role in the development of texture, flavor and color of dairy products; and its reduction can cause defects such as loss of flavor and consistency or *

Corresponding author: [email protected].

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T. Colombo Pimentel, S. Jensen Klososki, M. Rosset et al. lack of texture, which makes indispensable the use of fat replacers in obtaining fat-free products with technological and sensory properties similar to those of full-fat products. Inulin is a prebiotic dietary fiber and can be used in a wide range of healthy products due to its ability to improve texture and its neutral and slightly sweet taste. The low caloric value makes it an ideal ingredient. Inulin has been used as a fat replacer in the dairy industry and has shown positive effects on rheology and product stability. This chapter aims to describe the performance of inulin as a fat replacer in dairy products, giving an overview of the influence of inulin addition on the textural, rheological, prebiotic and sensory properties of the dairy products.

Keywords: inulin-type fructans, fat substitute, prebiotic, dairy products

INTRODUCTION In the last decades there has been an increase in awareness about health and quality of life, which encouraged people to exercise, acquire healthy eating habits and decrease the consumption of foods rich in sugar, salt and fat. Therefore, the consumption of foods with low caloric value and with reduced concentration of fat has increased considerably, since the consumers have associated their consumption to the reduction of the risk of coronary diseases and obesity. These products can also be added substances that have potential human health benefits, such as prebiotics. The food industry has a great challenge to associate in the same product the characteristics of a healthy and pleasing product to the consumer. Prebiotics are non-viable food components that confer health benefits to the host associated with the modulation of its microbiota (Fao/Agns, 2007). One group of prebiotics currently used is the inulin-type fructans, fructooligosaccharides formed by β-1,2 bonds, not hydrolyzed by the human organism; thus being considered soluble dietary fibers. Inulin-type fructans consumption, in addition to their prebiotic effect, has been related to inhibition of intestinal pathogen growth and other physiological effects, such as increased calcium absorption; reduction of the risk of atherosclerosis; and no change in the glycemic index and insulin levels in the blood.

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Inulin has been used as a food ingredient due to its technological properties as a fat replacer, calorie reducer and texturizing agent. In low-fat or fat-free dairy products; such as fresh, creamy or processed cheeses; the addition of small percentages of inulin results in a creamier texture and a more balanced flavor. In frozen desserts, it can promote easy processing, creamy mouthfeel of fat, excellent melting properties and stability to freezing and thawing without any residual flavor. In yoghurts, inulin addition improves the texture and offers synergistic flavor in combination with aspartame and acesulfame K or both. Furthermore, it improves the processability of aerated dairy desserts, resulting in products with typical structure for more time and a fat-like sensation (Franck, 2002). The fat replacer ability is dependent on the degree of polymerization of the inulintype fructan and to the concentration used. Inulin has been applied in several dairy products, including ice cream, yoghurt, dairy dessert, kefir and imitation cheese/fresh cheese, due to the moisture content of these products (higher than 70%), short shelf life, and storage temperatures (lower than 10°C), factors that contribute to the reduction of the hydrolysis of the functional component, maintaining their physicochemical and functional properties (Buriti et al., 2007; Pimentel et al., 2012a). This chapter aims to describe the performance of inulin as a fat replacer in dairy products, giving an overview of the influence of inulin addition on the textural, rheological, prebiotic and sensory properties of the dairy products.

PREBIOTICS The term prebiotic was defined in 1995 by Gibson and Roberfroid as a nondigestible food ingredient, which results in beneficial effects on host health through selective stimulation of the growth and/or activation of the metabolism of a limited number of bacteria in the colon (Gibson & Roberfroid, 1995). A FAO technical meeting in 2007 brought the currently accepted definition: “Prebiotics are non-viable food components that

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confer health benefits to the hosts associated with the modulation of their microbiota” (Fao/Agns, 2007). Prebiotics are a category of nutritional compounds grouped together by the ability to promote the growth of specific beneficial (probiotic) gut bacteria, resulting in individual health benefits (Table 1). Such benefits are only achieved when the prebiotic components resist to the absorption in the gastrointestinal tract, the acidity of the stomach and the action of intestinal enzymes. Because they are nondigestible substances, the action of prebiotics occurs by stimulating the growth or activity of gut bacteria (Kely, 2008; Moroti, 2009). Table 1. Claimed gastrointestinal effects of prebiotics Mechanism or target of action Through fermentation in the large bowel

Products and effects

Production of short-chain fatty acids and lactate Production of gas, mainly CO2 and H2 Increase in biomass Increased faecal energy and nitrogen Mild laxative properties On the Selective increases in Bifidobacteria and Lactobacilli in planktonic microbiota and biofilm communities Reduction in clostridia Increase in colonization resistance to pathogens Potential benefit in preventing pathogen invasion Small intestine Osmotic effect of short-chain prebiotics (DP3-4) which occasionally causes diarrhoea Improved calcium, magnesium and iron absorption Interaction with mucus to change binding sites for bacteria, lectins, etc Mouth Protection against caries Other effects Variable effects on microbial enzymes with potential to affect carcinogenesis Stimulation of apoptosis Adapted from Cummings & Macfarlane (2002).

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To be classified as a prebiotic, the component can not be hydrolyzed or absorbed in the upper gastrointestinal tract; must be a selective substrate for one or a limited number of colonic bacteria; must change the composition of the microbiota to a healthier composition; and, preferably, to induce beneficial effects on host health (Manning & Gibson, 2004). The technological properties required are: be stable during storage of the products, do not require refrigeration and be easily and efficiently incorporated in processed foods. Most of the studies related to the evaluation of prebiotic effects in humans refer to inulin-type fructans, which belong to the family of fructans, considered as functional ingredients, since they have a positive influence on the biochemical and physiological processes of the human organism, resulting in improvement health and reducing the risk of diseases. Inulin-type fructans are carbohydrates in which most of the glycosidic bonds are made of fructosyl-fructose bonds, and usually have a terminal glucose unit. They are divided based on their degree of polymerization (number of individual units of monosaccharides that make up the molecule) (DP) or the method of obtaiment. The three generic terms most often found are inulin, oligofructose and fructooligosaccharide. Regarding oligofructose and fructooligosaccharides (FOS), they are inulin-type fructose mixtures with a maximum DP of 10, being classified as short-chain inulin. Fructooligosaccharides (FOS) are composed of 1kestose (GF2), nystose (GF3) and fructofuranosylnistose (GF4) compounds, in which the fructosyl units (F) are bound in the β-2,1 position of sucrose. FOS are obtained through an enzymatic reaction of transfructosylation in sucrose residues, resulting in linear and branched chains of oligosaccharides, with degree of polymerization ranging from 1 to 5 units of fructosyl. Oligofructoses are commercially produced through the enzymatic hydrolysis of inulin, resulting in linear fructosyl units with or without a final glucose unit, having a degree of polymerization ranging from 1 to 7 fructosyl units.

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Inulin is a linear β- (2 → 1) -linked fructose polymer that occurs in garlic, asparagus root, Jerusalem artichoke, dahlia tubers or chicory root. Inulin occurs naturally as a reserve carbohydrate, is soluble in water, with temperature-dependent solubility. At 10°C, the solubility of inulin is 6%, being increased by 35% when the temperature reaches 90°C (Toneli et al., 2008). Medium-chain inulin has a medium DP of 10 and contains about 6 to 10% free sugars, represented by glucose, fructose and sucrose. These sugars are present in the plant from which the inulin has been extracted and therefore do not result from the processing. The long-chain inulin consists of mixtures of inulin-type fructan exclusively with DP ≥ 10 and therefore has a higher molecular weight. Because of the beta-configuration of the anomeric C2 in their fructose monomers, inulin-type fructans resist hydrolysis by intestinal digestive enzymes, being ‘non-digestible’ carbohydrates, and are dietary fibers. These substances provide a glycemic index approximately equal to zero to 1.5 kcal/g. Moreover, inulin is used in the production of functional foods because it favors the growth of beneficial microorganisms (bifidobacteria and lactobilli), contributing to the appropriated gastrointestinal functioning. The prebiotic dose of 5 g/day of inulin, oligofructose or FOS is enough to beneficially alter the colonic microbiota, and in specific cases this value can reach 8 g/day (Kolida, Meyer & Gibson, 2007). According to Carabim & Flam (1999), the inulin-type fructans, when administered in the diet at high levels, do not result in mortality, morbidity, target organ toxicity, reproductive or developmental toxicity, or carcinogenicity. Several in vitro studies have also shown the absence of mutagenic or genotoxic potential. The only basis for limiting use of inulintype fructans in the human diet relates to gastrointestinal tolerance. A series of clinical studies has been reported that up to 20 g/day of inulintype fructans is well tolerated. The labels of the products state both the quantity per serving size and the corresponding percentage of the daily value (% DV); therefore, the consumers can make appropriate choices and decisions about daily consumption without exceeding individual tolerance.

Inulin asofaprebiotic Fat Replacer Dairy Products 115 Figure 1 shows the mechanism action ofininulin-type fructans in the human organism.

Figure 1 - Health effects of inulin-type fructans

Figure 1. Health effects of inulin-type fructans.

The prebiotic effects of inulin such as calcium uptake, lipid metabolism and modulation of the intestinal microbiota also result in reduced risk of osteoporosis, arteriosclerosis and constipation (Kaur & Gupta, 2002; Saad, 2006). In 1995, Fiodarliso et al. observed a reduction of 8.5% in the total cholesterol concentration and 14.4% in the LDL-cholesterol concentration after ingestion of 18 g per day of inulin. Passos & Park (2003) observed a reduction of 15% in total cholesterol, a 15% decrease in phospholipids, and 25% in triglycerides in rats after ingestion of 10% of FOS. Experimental studies showed the bifidogenic action of inulin-type fructans favors the increase of the number of bifidobacteria in the colon and acts in the reduction of colon cancer risk. Among the possible mechanisms of action for the anti-carcinogenic effect are the increased immune response, decreased inflammation, inhibition of tumor cell formation, and the decreased conversion of pre-carcinogenic substances into carcinogenic ones. Figure 1 shows the mechanism of prebiotic action of inulin-type fructans in the human organism.

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Table 2. Saturated fatty acids related to the increase of LDL levels Fatty acid Myristic Palmitic Stearic

Number of carbons 14 16 18

Natural sources Milk, butter and milk derivatives Animal fat and palm oil Cocoa fat

The production of nutrients and vitamins, reduction of toxins and pathogenic bacteria, as well as the improvement of irritable bowel syndrome and tolerance to lactose are considered to be beneficial effects of inulin-type fructans prebiotics.

FAT PROPERTIES AND USE OF FAT REPLACERS Fats perform various functions in the body, such as providing energy, facilitating absorption of fat-soluble vitamins, maintaining body temperature, protecting organs against mechanical shocks, among others. Furthermore, they are important sources of calories, i.e., each gram contributes with nine calories, more than twice of the energy provided per gram of protein and carbohydrates (4 kcal/g). However, excessive consumption of fats can interfere with the general health of individuals, which is directly influenced by their eating habits. The higher consumption of saturated fatty acids is directly related to the increased risk of coronary heart disease (CHD), LDL cholesterol levels, type 2 diabetes, obesity and cancer. For these reasons, there are recommendations for reducing food intake that contains saturated fatty acids. The main saturated fatty acids related to the health effects are those with long chain (higher than 14 carbons), such as myristic, palmitic and stearic (Table 2). Considering the adverse health impacts and economic outcomes associated with the overconsumption of dietary fat, reduced-fat or fat-free foods are a reasonable and practical choice. However, the presence of fat provides specific characteristics to the products, influencing their appearance, flavor and texture, besides the caloric value and nutritional

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quality. Sensory and physiological aspects are considered of higher quality in full-fat products, such as flavor, appearance, creaminess, aroma, softness, juiciness and sensation of satiety after ingestion. The interaction of fat with the other components in food influences the texture properties, such as consistency and cohesiveness. These properties are strongly influenced by the removal of this component in dairy products. The perception of aroma is also directly related to the fat content present in the product, as well as flavor and color. The reduction of fat is associated to decreased apparent viscosity, storage modulus, yield stress, smoothness and compliance in ice creams. For yoghurts, fat-free products are associated to increased apparent viscosity and consistency index. Considering cheeses, increased hardness, firmness, and springiness; and decreased adhesiveness, cohesiveness and meltability were observed in fat-free products. Furthemore, undesirable aromas and changes in color are reported. To perform the technological function exerted by fats, compounds called fat replacers are used. However, it is very difficult to replace the function of fats in foods, as they affect the technological and sensory properties of the products, such as flavor, color, aroma and consistency. The substitution of fat by a single substitute generally does not provide the dairy product with all the attributes conferred by the original component, and it is difficult to select which substitute to use. A fat replacer should be free of toxic compounds, not produce harmful metabolites, be completely eliminated from the body, and be widely recognized as safe (Pinheiro & Penna, 2004). Furthermore, it should have low energy density so that the total caloric content of the product is reduced. Fat replacers are classified according to their chemical nature. Carbohydrates and proteins isolated from biosources are among the most suitable fat replacers because of their generally safe status in food applications and low energy density (0–4 cal/g) compared with fat. Compared with protein-based fat replacers, carbohydrate-based fat replacers are generally more cost-effective (Peng & Yao, 2016). Figure 2

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shows some fat replacers, their classification, the technological functions they perform, and the dairy products that they are applied. Many carbohydrates can be used as fat replacers because they alter the texture properties, giving thickening to foods and promoting a sensory perception similar to fat. The main carbohydrates used as fat replacers are starch, dextrin, maltodextrin, polydextrose, grain-based fibers, cellulose, carboxymethylcellulose, microcrystalline cellulose, gums (guar, xanthan and carrageenan), and inulin-type fructans, among others. Some of these carbohydrates are digestible and provide 4 kcal/g, whereas others are partially or non-digestible, presenting low caloric value. The non-digestible carbohydrates are used in low calorie products conferring characteristics related to the technological role of fat. The fat replacer potential of inulin-type fructans was discovered and patented in 1992 by Orafti. Since then, inulin-type fructans have been used as fat replacers in the dairy industry and had shown positive effects on rheology and product stability. This is due to the fact that inulin-type fructans promote a sensation similar to fat in the mouth. This characteristic is the result of their ability to form microcrystals when mixed with milk. These microcrystals are not perceived in the mouth, but interact to form a finely creamy texture that promotes the sensation of fat. The degree of polymerization of oligofructose or FOS is insufficient to form microcrystals. Only medium and long-chain inulin, that have DP higher than 10, can develop a gel structure formed by a network of crystalline particles. Inulin is available as a white, odorless and neutral taste powder with high purity and known chemical composition. The physicochemical properties of inulin are linked to the degree of polymerization (DP). Shortchain inulin, that have low DP, are slightly sweet (10-30% compared to sucrose), while medium and long-chain inulins are more viscous. In general, it is moderately soluble in water (maximum 10% at room temperature) and has low viscosity (less than 2 mPa.s for a 5% solution in water), but the solubility and viscosity depend on the DP and temperature of the media. The solubility of inulin allows its use in large proportions and

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does not confer to the products the common feel of sandiness that occur with insoluble grain-based fibers. DAIRY APPLICATION

TYPE

FUNCTIONAL PROPERTIES

CARBOHYDRATES

Yoghurt, ice cream, cheese, dairy dessert

Starch

Dairy desserts and other dairy products

Dextrin

Dairy desserts and other dairy products

Maltodextrin

Dairy desserts Dairy desserts and other dairy products Dairy desserts, yoghurts, cheese and fermented whey beverages

Polydextrose Cellulose, CMC, microcrystalline cellulose Pectin

Gelling, thickener, stabilizer and texturizing agent

Water retention, bulking agent and texturizing agent Water retention, texturizing agent, softener and stabilizer

Gelling agent, thickener and softener

Ice cream and dairy dessert

Dairy products

Guar, Carrageenan and Xanthan Gums

Water retention, texturizing agent, stabilizer, thickener, and softener

Inulin PROTEINS

Yoghurt, ice creams, fermented creams, dairy spreads

Microparticulated protein Softner

FATS AND SYNTHETIC COMPOUNDS

Modified whey protein concentrate Sucrose polyesters

Ice cream and cheese

Softner Dialkyl dihexadecyl malonate

Figure 2. Fat replacers used in dairy products.

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In creams, inulin addition can result in products with pleasant oral sensory perception due to the texture acquired, as well as spreadability for products such as margarine, low-calorie milk candy, among others. For products such as cheese, cream cheese, melted cheese and curd, inulin promotes creaminess, body and balance in flavor. Thus, it is possible to perceive advantages in the use of inulin-type fructans in dairy products to obtain low-calorie foods with prebiotic potential. When inulin-type fructans are added to food in low concentrations, the rheological properties and the sensory quality of the product will not be affected strongly due to the neutral or slightly sweet taste and their limited effect on viscosity. However, as inulin-type fructan content increases, its effect on product structure and texture becomes important, because at these higher levels and due to its physicochemical properties, inulin can modify the texture of dairy products and may significantly influence their sensory quality (Meyer et al., 2011). The quantity of inulin-type fructan to alter the characteristics depends on the dairy product and the type used.

INULIN AS FAT REPLACER IN YOGHURT Yoghurt is a fermented dairy product with specific rheological and textural characteristics. The texture of yoghurt is a result of the development of a three-dimensional network of milk proteins due to the aggregation of casein micelles with denaturated whey proteins, through hydrophobic and electrostatic bonds (Paseephol et al., 2008). Good quality set type yoghurt should maintain strong curd integrity without any sign of shrinkage and disintegration into lumps and whey-off. The defect of syneresis relates to the appearance and mouthfeel and can adversely affect acceptability or preference of consumers (Srisuvor et al., 2013). The stirred yoghurt must have a homogeneous texture, with no phase separation. Both categories should also possess pleasant odor and flavor. Milk fat has an important role in the development of texture, flavor and color of dairy products. The reduction in fat content may cause some yoghurt defects such as loss of flavor and consistency or lack of texture. In

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addition, fat influences other product characteristics such as handling, stability and appearance. Although the manufacture of low-fat and fat-free yoghurts has been possible for many years, the use of fat substitutes is relatively recent. Inulin is added to the yoghurt process in the initial phase, concomitantly to the sugar and skim milk powder (or other ingredient that increase the total solids, when necessary). This is because inulin has to be part of the structural network formed during fermentation and gel formation, therefore, the addition of inulin after fermentation results in less favorable results, mainly for creaminess of the product. Table 3 presents studies that evaluated the addition of inulin as fat replacer in yoghurts. Inulin generally has no effect on the pH, titratable acidity and color of the products. Increases in the total soluble solids and carbohydrate (fiber) contents with consequent reduction in the moisture content are observed, because inulin-type fructans are soluble oligosaccharides/polysaccharides, which cause an increase in total solids content when added to foods. The maintenance in the acidity of the yoghurt is interesting from a sensory point of view, because an increase in acidity could result in a decrease in the acceptance of the products by consumers. Furthermore, high acidity is related to decreased viability of the probiotic cultures in the products that these beneficial microorganisms are added. The main influence of inulin addition is on the texture/rheological and sensory aspects of the products. The effect of the addition of inulin on the texture and rheological properties depends on the structure and composition of the product, the fat content (low-fat or fat-free), type (set or stirred yoghurt) and the concentration and degree of polymerization of the inulin used. For some yoghurt (mainly stirred-type) supplemented with inulin, an increase in apparent viscosity, firmness and/or yield stress of the products after inulin addition (Kip et al., 2006, Guggisberg et al., 2009) was observed, resulting in low-fat products with texture and rheological parameters comparable to the whole milk products and higher than the low-fat products. In these products, inulin can complex (via H-bridge formation) with the protein aggregates, becoming part of the structural network that is formed during fermentation and structuring them. Inulin

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forms elongated gelled structures that intermingled into the protein network and secondary gelled structures, which fortified the yoghurt network by acting as a water structuring agent (Crispin-Isidro et al., 2015). In other cases (mainly set-type yoghurt), the addition of long-chain inulin can decrease the texture (firmness, cohesiveness, adhesiveness and gumminess) and rheological (apparent viscosity, maximum compression force, dynamic oscillatory) values (Paseephol et al., 2008, ModzelewskaKapitula & Klebukowska, 2009, Pimentel et al., 2012b) of the low-fat products, also resulting in products similar to the full-fat ones. Generally, in these cases, inulin was used as a fat replacer by substituting partially the skim milk powder, maintaining the total solids of the low-fat products. The substitution results in a lower net protein content, what causes a lower cross-linkage of the gel network, resulting in a less dense and rigid gel structure. Therefore, in these cases, the attenuation of the texture/ rheological parameters can be more associated to the protein content than to the effect of inulin addition. The low-fat product, thus, have higher textural and rheological parameters than the low-fat added inulin and the full-fat products, because of the higher protein content. Anyway, it is important to observe that inulin make the textural and rheological properties of low-fat products similar to their full-fat counterparts. It is note worth; however, that not only the type of yoghurt (set or stirred) and the protein content are important to the texture and rheological characteristics and the influence of inulin addition. It is suggested that these parameters are also dependent on the inulin concentration. In low concentrations (up to 4%), inulin could complex (via H-bridge formation) with the protein aggregates, increasing the texture/rheological parameters. At higher levels (higher than 6%), the protein (casein) flow units are increasingly covered with inulin and the casein aggregates are partially sterically stabilized. This will reduce the effective volume fraction, reducing viscosity, and increasing water holding capacity, because the casein network begins to lose structural integrity and expel serum phase. Therefore, in this case, there is more solvation rather than aggregation, and this would yield the yoghurt gel with remarkably lower consistency.

Table 3. Inulin as fat replacer in yoghurts Yoghurt type

Inulin type

Inulin concentration

General characteristics

Texture/ Rheological results

Sensory results

Reference

Stirred fat-free

Long-chain (DP > 23) and mediumchain (DP > 9)

1.5, 3 and 4% of long-chain or 3% of medium-chain

NE

More thick, airy and sticky Long-chain inulin more effective

Kip, Meyer & Jelema (2006)

Stirred fat-free

Medium-chain (DP > 10)

2, 4 and 6%

Reduced syneresis

Stirred low-fat (1.3%)

Medium-chain (DP > 10)

2, 4 or 6%

No effect on titratable acidity Reduced syneresis for 4 and 6% addition

Increase in viscosity with higher inulin addition Increased firmness with long-chain inulin Increase in viscosity, firmness and consistency Increase in G’ and G” values

Improvement in creaminess and smoothness Improvement in flavor, viscosity, creaminess and overall acceptance with 4 and 6% addition

Brennan & Tudorica (2008) Crispin-Isidro et al. (2015)

Table 3. (Continued) Yoghurt type

Inulin type

Inulin concentration

General characteristics

Texture/ Rheological results

Sensory results

Reference

Stirred fat-free

Oligofructose, Medium-chain inulin (DP > 10) and Long-chain inulin (DP > 23)

4%

The acidity increased with the decrease of inulintype fructans DP No influence on pH after storage (4 weeks)

No influence in aroma, color and flavor Consistency improved with longchain inulin Inability to substitute fat

Glibowski & Rybak (2016)

Set fat free or low-fat

Long-chain (DP > 23)

1, 2, 3 and 4%

No effect on pH

Long-chain inulin yoghurt with similar apparent viscosity to full-fat yoghurt Decrease in apparent viscosity with oligofructose and medium-chain inulin Higher yield stress with inulin addition

Higher creaminess for low-fat products, no effect in fat-free

Guggisberg et al. (2009)

Yoghurt type

Inulin type

Inulin concentration

General characteristics

Texture/ Rheological results

Sensory results

Reference

Set fatfree

Long-chain (DP > 23)

2%

Increase in syneresis

Decrease in firmness, cohesiveness, adhesiveness and gumminess making it similar to the whole products

Higher brightness, firmness and syneresis No effect on creaminess

Pimentel et al. (2012b, 2013)

Set fatfree

Long-chain (DP > 23)

1-3%

No influence in pH, titratable acidity and acetaldehyde content Increase in syneresis

Higher consistency

Negative impact in color, appearance, taste and aroma scores No influence in body and texture scores 1% addition is efficient to obtain products similar to the whole ones

Guven et al. (2005)

Table 3. (Continued) Yoghurt type

Inulin type

Inulin concentration

General characteristics

Texture/ Rheological results

Sensory results

Reference

Set lowfat (1.5%)

Long-chain (DP > 23)

1-3%

No influence in pH, titratable acidity and syneresis Increase in total soluble solids

Influence is dependent of the quantity added Low concentration resulted in higher apparent viscosity, consistency and firmness Higher concentration resulted in lower apparent viscosity, consistency and firmness

1% addition resulted in better appearance, color, texture and overall acceptante 2 and 3% addition improved texture and overall impression

Srisuvor et al. (2013)

Yoghurt type

Inulin type

Inulin concentration

General characteristics

Texture/ Rheological results

Sensory results

Reference

Set fatfree

Oligofructose, Medium-chain inulin (DP = 10) and Long-chain inulin (DP = 23)

4%

No effect on pH and titratable acidity

NE

Paseephol Small & Sherkat (2008)

Set fatfree

Long-chain inulin (DP = 23)

0.7 and 2.7%

No effect on pH

Decrease in firmness, apparent viscosity, yield stress, complex viscosity, storage modulus and loss modulus Long-chain inulin yoghurt presented similar rheological behavior to the full-fat product Decrease in apparent viscosity on 1st and 21th days of storage

Negative impact in color, aroma and taste Yoghurt with 2.7 added inulin only with slightly lower scores than full-fat yoghurt

ModzelewskaKapitula & Klebukowska (2009)

Table 3. (Continued) Yoghurt type

Inulin type

Inulin concentration

General characteristics

Texture/ Rheological results

Sensory results

Reference

Set fatfree

Medium-chain (DP = 10)

4, 6 and 8%

No effect on pH Decreased syneresis

8% addition resulted in better spoon viscosity, firmness and mouthfeel

Rudra et al. (2016)

Set fatfree

Oligofructose (DP = 4), Medium-chain inulin (DP = 10) and Long-chain inulin (DP = 23)

1.5%

Decrease in pH and increase in syeneresis of oligofructose added products No effect in pH and decrease in syneresis for medium and longchain inulin No effect in L*, a* and b* color parameters

Increase in consistency index Higher stability of protein networks (time sweep and frequency sweep) No effect on apparent viscosity

Higher flavor scores in oligofructose yoghurts compared to long-chain Higher body and consistence scores in long-chain inulin

Aryana et al. (2007)

NE – not evaluated.

Table 4. Inulin as fat replacer in ice cream Ice cream type

Inulin type

Inulin concentration 5, 7 or 9%

General characteristics Improvement of melting resistance

Texture/ Rheological results Increase in consistency coefficient and stickiness Decrease in firmness

Low fat (5%)

Mediumchain (DP > 10)

Low-fat (24%) and Fatfree

Long-chain inulin (DP > 23)

4, 6 or 8%

Increased overrun and decrease of melting rate (6 and 8% addition)

Long-chain inulin (DP > 23)

4%

Improvement of melting resistance Overrun reduction

No influence on hardness up to 6% addition Increase in consistency coefficient, hardness and pseudoplasticity at 8% Decrease in consistency coefficient, apparent viscosity and pseudoplasticity Increase in hardness

Low-fat (3 and 6%)

Sensory results

Reference

Improvement in smoothness Decrease in grittiness, iciness, coarseness and hardness Negative impact on flavor and taste (4 and 8%) Negative impact on body and texture scores (8%)

El-Nagar et al. (2002)

NE

Akalin et al. (2008)

Karaca et al. (2008)

Table 4. (Continued) Ice cream type

Inulin type Mediumchain (DP > 10)

Inulin concentration 5.7 or 6.2%

Low-fat (5%)

Not specified

2 or 4%

Low-fat (2%)

Longchain ( > 23)

Low-fat (3.5 vegetable fat)

Not specified

Low-fat (1.6%) and fatfree

General characteristics Decrease of overrun No effect on melting rate Maintanence of overrun and melting resistance

Texture/ Rheological results Increase in viscosity

2, 3 or 4%

Maintanence of pH, color and melting rate

Decrease in hardness and adhesiveness

1, 2, 3 and 4%

Decrease in melting rate, hadness and compression force

Increase in apparent viscosity and overrun

Increase in consistency, viscosity and hardness

Sensory results

Reference

Improvement on body and texture No effect on appearance, color and flavor Flavour, texture, colour and overall acceptability of ice cream samples, containing inulin were more appealing than control, mainly eith 2% inulin addition Improved appearance and texture Improved flavor with 3 and 4% addition Only 4% added yoghurts had melting properties similar to full-fat products 3% added low-fat ice cream is similar to the fullfat product

Aykan et al. (2008)

Mahdian & Karazhian (2013)

Akbari et al. (2016)

Pintor et al. (2013)

Ice cream type Low-fat (approximately 3%)

Inulin type Longchain (DP > 23)

Inulin concentration 5%

Low-fat (3.8 to 7.7%)

Not specified

2, 4 and 6%

Low-fat (3%)

Not specified

2.5 and 5%

NE – not evaluated.

General characteristics Maintanence of pH, total solids and melting rate Increase in overrun Maintanence of total solids, protein content, water activity and overrun Increase in melting rate Decrease in ash content and L* color parameter Increase in total solids No effect on overrun and fat instability Decrease in meltdown rate

Texture/ Rheological results Increase in viscosity

Sensory results

Reference

No effect

Hashemi et al. (2015)

Increase in hardness and adhesiveness Decrease in viscosity and consistency coefficient

Negative impact with 6% addition

Tiwari et al. (2015)

Increase in viscosity and adhesiveness at 2.5% and decrease at 5% Increase in hardness, cohesiveness, gummines and elasticity

Improved in the sensory properties of the low-fat ice cream, including the mouthfeel, texture, body, and flavour

Ismail, AlSaleh & Metwalli (2013)

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Syneresis is defined as a formation of a liquid layer at the top of the yoghurt surface and is considered one of the main defects in this type of dairy product. The addition of long-chain inulin could result in increased syneresis in yoghurts (mainly set-type) where inulin was used as a partial substitute of skim milk powder, because, as mentioned before, these products were less firm than the conventional low-fat products. A lower firmness makes yoghurts high susceptible to rearrangements within their structure and therefore more susceptible to serum separation. On the other hand, a lower syneresis was observed in inulin supplemented yoghurts (mainly stirred), probably due to entrapment of higher total solids and through hydrogen bridges with charged moieties on the surface of the protein. Furthermore, inulin, like the fat globules, may limit casein aggregation, preventing the shrinkage and rearrangement of the three-dimensional network into a more compact structure, reducing the syneresis. As discussed for the texture parameters, inulin content is important to evaluate its influence on syneresis. Crispin-Isidro et al. (2015) reported that 2% addition of inulin had no effect on syneresis, but the addition of 4 or 6% reduced the values of this parameter. These results may be explained in terms of the ability of inulin molecules to bind water, preventing its free movement; as well as its interaction with the milk constituents (mainly proteins), that provide stability to the protein network. In addition, higher concentrations of inulin originated a protein matrix less prone to whey expulsion, probably due to entrapment of higher total solids. It is important to mention that there are many forms to evaluate syneresis in yoghurts, what make difficult to compare the available results. The main methods are: espontaneos release of whey, quantity of whey released after some time of evaluation or centrifugation. When evaluating a fat replacer, the primordial characteristic in the product is creaminess, as this property is provided by fat. Generally, the increase in inulin content (1-3.5%) causes an increase in the perception of creaminess in low-fat products. However, when considering fat-free products, this improvement was not observed in some studies (Guggisberg et al., 2009; Pimentel et al., 2013), while other studies reported increased

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creaminess even in fat-free products (Brennan & Tudorica, 2008, CrispinIsidro et al., 2015). Possibly, inulin is capable of increasing the creaminess of yoghurts only when it is used as a partial replacer for fat; that is, when at least a small amount of fat is present in the product. The attribute creaminess is complex and dependent of many other texture attributes. Kip et al. (2006) observed that the attributes thick, airy and fatty contribute positively to creamy mouthfeel, whilst rough/astringent, dry and heterogeneous attributes have a negative contribution. Furthermore, stickiness is likely to be important for the formation of a coating in the mouth, which is an essential part of creamy mouthfeel and after feel, and of the sensory functionality of fat in general. Creaminess is evaluated in different manners in the studies, what makes the results not comparable. Making an overall evaluation of the studies, inulin is able to improve creaminess of yoghurt, making them similar to the full-fat products, since appropriated concentrations and the right type (long-chain) is used. Other attributes could also be improved with inulin addition, such as stickiness, airiness, smoothness and viscosity. The addition of inulin can improve the acceptability of low-fat yoghurts, making the low-fat products added inulin similar accepted to their full-fat counterparts. Considering that the dairy products are developed to meet the consumer expectations, this is of major interest for dairy industry. This is because no matter how good results we can obtain due to instrumental analysis, the analysed model food product will never be commercially produced until it is accepted by a potential customer (Globowski & Rybak, 2009). However, some authors reported negative impact on color, appearance, taste and/or aroma (Guven et al., 2005, Modzelewska-Kapitula & Klebukowska, 2009). In these cases, the full-fat yoghurt had the highest amount of acetaldehyde compared to low-fat yoghurts with inulin addition. One of the most important aroma compounds in yoghurt is acetaldehyde. In practice, generally each gram of fat is replaced by 0.25 grams of inulin. Consequently, replacement of fat in most foods would result in concentrations of 2-6 grams of inulin per serving.

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From the studies of inulin application as fat replacer in yoghurts it can be observed that inulin is able to improve the textural and rheological properties and reduce syneresis, in a manner that the low-fat added inulin yoghurts have similar characteristics to the full-fat products. In addition, no negative impact on the physicochemical characteristics is observed, with maintenance of the acidity and color. The ability to improve creaminess depends on the concentration of inulin used and the fat content of the products, and, in the majority of the studies, inulin can improve the acceptance of low-fat yoghurts, making it similar to the full-fat products, even if the creaminess was not increased.

INULIN AS FAT REPLACER IN ICE CREAM Ice cream is a frozen food made from a mixture of dairy ingredients, milk fat and bulk ingredients, such as flavorings and sweeteners. Milk fat is the ingredient of major importance in ice cream because its correct use is vital not only to balance properly the mix but also to satisfy legal standards. Milk fat affects textural attributes such as viscosity, tenderness, elasticity, emulsification, ice crystallization and other desirable attributes such as richness and smoothness. The primary difficulties to be overcome in low-fat or fat-free ice creams include improving the mouthfeel and flavor perception to resemble that of full-fat products. To meet this challenge, ice cream manufacturers have been focusing on eliminating the problems resulting from the replacement or removal of milk fat using fat replacers. The utilization of inulin as fat replacer in ice creams has been evaluated by many authors and the studies are presented in Table 4. The addition of inulin as fat replacer in ice creams does not alter the pH, titratable acidity and L*, a* and b* color parameters of the products. An increase in the density was observed. In ice creams, inulin could reduce the freezing point depression, increase the melting resistance and overrun and alter positively the texture and rheology by increasing chewiness, flow consistency, pseudoplasticity,

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consistency index, apparent viscosity and stickiness. This is because inulin may act as a stabilizer, binding water and immobilizing the molecules, which become unable to move freely among other molecules of the ice mix, retarding the product melting. Inulin can modulate ice recrystallization acting as a stabilizer agent, improving first dripping time and reducing melting. Melting rate is normally inversely proportional to overrun, therefore, higher overrun values result in slower melting, since air cells act as an insulator medium. The increase in viscosity could be explained by the interactions of inulin and liquid components of ice-cream and because inulin exhibit a viscous behavior, imbibing water, and increasing the viscosity of the system. Furthermore, inulin could interact with milk proteins. The influence of inulin in the hardness of ice cream seems to be dependent on the concentration used. Low concentrations (2-4% of longchain type) result in reduction of this parameter. Water absorption by inulin increases the unfrozen water content with consequent decrease of the ice crystals in ice cream structure. This could probably be the reason of the lower hardness of the inulin-containing low-fat ice creams compared with the inulin-free ice cream. An alternative reason for the decrease in hardness may be related to decreases in freezing point as a result of higher solute concentrations. However, when higher concentrations are used (higher than 4%) an increase in the hardness is observed (El-Nagar et al., 2002; Akalin & Erisir, 2008). An ice cream with softer texture is the preference of the consumers. The addition of inulin has also an impact in the compression force of the products, decreasing it value. A similar compression occurs in the mouth during eating, for example when the ice cream is squashed between the tongue and the roof of the mouth. In fact, long-chain inulin have the ability to form micro crystals when sheared in water or milk to form a creamy texture, binding water to form a particulate gel network, resulting in a soft texture. Inulin addition has no effect on fat instability. Fat instability refers to the clustering, flocculation, and clumping process (partial coalescence) of the fat globules, which leads to the development of a continuous internal

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fat network or matrix structure in the product. The fat instabilisation mechanism in ice cream involves the breakage of protein-lipid membranes, which surround the fat globules during freezing, as well as a part of the liquid fat release, which acts as a cementing agent between the damaged globules (Ismail, Al-Saleh & Metwalli, 2013). This characteristic is important in low-fat products. Low-fat ice creams are usually described as hard, icy, coarse and gritty, while full-fat ice creams have a softer and smoother texture. Addition of inulin to the low-fat base can improve the sensory characteristics of the samples, mainly the mouthfeel, what can be associated to the decreased meltability (El-Nagar et al., 2002). From the studies of inulin application as fat replacer in ice cream it can be observed that inulin is able to improve the textural and rheological properties, in a manner that the low-fat added inulin ice creams have similar characteristics to the full-fat products. In addition, no negative impact on the physicochemical characteristics is observed, with maintenance of the acidity and color. The ability to alter hardness and probably other attributes depends on the concentration of inulin used, and, in the majority of the studies, inulin has positive effects in the acceptance of low-fat ice creams by consumers.

INULIN AS FAT REPLACER IN CHEESES Consumption of dairy products is encouraged because of the significant presence of essential nutrients such as calcium, phosphorus and protein, but cheeses, in addition to providing essential nutrients to the human body, are a significant source of fat. Due to the incentive to reduce fat consumption, there is an increase in the production of low-fat or fat-free cheeses. In the structure of the cheeses matrix the fat globules remain physically trapped in a calcium phosphate crosslinked protein network. The matrix becomes elastic when the casein remains intact but decreases with the evolution of proteolysis during cheese ripening. The entrained fat globules

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serve to limit deformation of the cheese matrix and their distribution determines the uniformity and degree of crosslinking of the casein matrix. When part of the fat is removed, casein starts to play a larger role in the cheese texture. With fat reduction there is insufficient casein breakdown, especially αs1-casein hydrolysis. Thus, the texture of a low-fat or fat-free cheese is characterized as hard and elastic. Removal of fat from cheese causes rheological, textural, functional and sensory defects such as rubbery texture, lack of flavor, bitterness, offflavor, poor meltability and undesirable color. Furthermore, it can cause lower acceptability by consumers, in addition to low yield of production. The most common texture defects are the increase in hardness and elasticity, while the flavor problems comprise low intensity, aroma reduction, and presence of astringency and bitterness. It is not easy to make low-fat or fat-free cheeses with desirable properties (Fadaei et al., 2012). As fat content decreases, the protein matrix becomes more compact and the cheese texture is more chewy. When fat ingredients are reduced in food formulations, other ingredients are often required to fulfill their functional role in maintaining sensory qualities (Karimi et al., 2015). Several alternatives have been presented to improve the quality of lowfat or fat-free cheeses. The main strategies used are technological modifications of the manufacturing process, use of adjunt cultures, use of enzymes and use of fat replacers. Inulin-type fructas has been used as a fat replacers in several types of cheese, such as fresh cream cheese (Alves et al., 2013, Buriti, Cardarelli, Filisetti & Saad, 2007, Miri, Habibi and Najafi, 2011), wheyless cream cheese (Fadaei et al., 2012), petit Suisse cheese (Cardarelli et al., 2008a), cottage cheese (Araújo et al., 2010), imitation cheese (Hennelly et al., 2006), fresh kashar cheese (Koca & Metin, 2004), mozzarella cheese (Pagliarini & Beatrice, 1994; Wadhwani, 2011), cheddar cheese (Wadhwani, 2011) and karish cheese (Alnemr et al., 2013). The ability of inulin-type fructans to act as a fat replacer in fresh or melted cheeses is due to the fact that inulin provides adequate texture, pleasant taste, and symbiotic properties. However, the addition of inulintype fructans in cheeses is a challenge, since the emulsifying capacity of

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inulin is lower than that of casein, as casein has more hydrophobic sites with emulsion capacity. In addition, the interaction between casein and inulin has less emulsifying capacity than isolated inulin. The fat replacement property of inulin is also based on its ability to stabilize the aqueous phase structure, which creates improved creaminess. Creamy mouthfeel is obtained when inulin is used as a fat substitute in dairy products because of its interactions with whey protein and calcium caseinate (Bot et al., 2004). Long-chain inulin is the inulin-type fructan most indicated as a fat replacer in cheeses because long chains reduce the solubility of inulin and result in the formation of inulin microcrystals when mixed with water or milk. These microcrystals are discreetly perceptible and have a smooth and creamy mouth feel. Inulin can also form parts of the protein’s structural network through complexation with protein aggregates (Kip et al., 2006). The moment of the addition of inulin into the cheeses is an important point to be evaluated and depends on the type of cheese. In cream, petitsuisse, processed and fresh cheeses, it is suggested to incorporate inulin into the cheese base through homogenization, during which a smooth and homogeneous cream is formed. If inulin is added before the drainage of the whey, a high concentration of inulin can be lost with the whey (Buriti et et al., 2007; Alves et al., 2013). In the production of karish cheese, Alnemr et al. (2013) indicated heating the skim milk (74°C for 15 seconds) and then adding inulin directly to the milk. For low-fat fresh cheese spreads, inulin is added to the cream, heated to approximately 80◦C and mixed during several minutes to hydrate. This resultant is then mixed to the fresh cheese followed by pasteurization at 80◦C and homogenization at 200 bar (Karimi et al., 2015). Another point to be controlled is the temperature used in the process of cheesemaking, as high temperatures result in the hydrolysis of inulin at both neutral and acid pH. Therefore, raising the heating temperature increases the minimum concentration of inulin required for gel formation. Hydrolysis of inulin at neutral pH is not significant for processing at temperatures up to 60oC, but may be significant at higher temperature

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grades (over 80oC). In addition, prolonged heating of the inulin solution adversely affects the rheological characteristics of inulin gels. In addition to the processing temperature, it is necessary to mention that the pH value has an effect on the action of inulin. The effects of temperature on inulin hydrolysis should be observed under acidic conditions (pH of 2.0-4.2), but this condition does not occur in the processing of several types of cheese, since the normal range of pH in different stages of processing and storage of various types of cheese varies between 4.3 and 5.6. Therefore, the pH of most cheeses in which inulin is used as fat replacer is higher than 4. In addition, this acidity does no appear during the ripening or storage of the cheese, where the maximum temperature is 43-45°C. As inulin degradation does not occur in the products with pH ≥ 5 heated up to 100°C, the pH (higher than 4) and temperature of cheese ripening or storage (maximum 43–45°C) are adequate (Karimi et al., 2015). Koca and Metin (2004) studied fresh Kashar cheese with 70% fat reduction and long-chain inulin as partial fat replacer. They reported that the low-fat content of the cheeses dificulted the formation of the mass and the products had harder texture due to their high protein content. The addition of 5% inulin as a fat replacer to the cheese resulted in a decrease in the hardness of the low-fat cheese, but slightly higher hardness than the full-fat product. The effect of inulin on the texture of fresh Kashar cheese was due to the increase in the moisture and in the filler volume, which decreased the amount of protein matrix. Inulin improved cheese texture until the 30th day of storage, but reduced the shelf life of the products. Fadaeiet al. (2012) studied the chemical characteristics of wheyless cream cheese containing long-chain inulin as a fat replacer. They found that 10% of inulin was sufficient to obtain a low-fat cream cheese with chemical attributes close to the full-fat product. They also reported that inulin had an excellent water-binding ability that inhibits syneresis. In this way, it was highlighted that the long-chain inulin had water retention capacity and consequently capacity to avoid syneresis. Wadhwani (2011) studied the effect of four fibers (inulin, low methoxyl pectin, polydextrose and resistant starch) on the quality of low-

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fat mozzarella and cheddar cheeses and concluded that inulin had a better effect on final texture than the other three fibers. The incorporation of inulin provided a better texture in cheeses with reduced fat content, decreasing hardness and gumminess and maintaining cohesiveness, adhesiveness and springiness. Salvatore et al. (2014) evaluated the effect of the substitution of fat (0 to 7%) by long-chain inulin on the texture and microstructure properties of caprine milk fresh cheese. Using scanning electron microscopy and penetrometry, they found that cheese containing inulin as a fat replacer had a more open structure compared to the full-fat cheese, due to the lower fat distribution in the protein matrix. Inulin appeared as structures embedded in the gel system, the size of which tended to increase with increasing inulin concentration in the product. The authors verified that the presence of inulin, the size of which is strongly dependent on its concentration, interrupts the casein network, resulting in a softening effect, which increases with increasing replacement levels of fat with inulin. Furthermore, they concluded that samples containing inulin were characterized by lower values of compressive force, stiffness, viscosity and adhesiveness. In low-fat processed cheese spread, inulin (7-8%) provided a positive effect on the spreading property (spreading ability). Furthermore, inulin had significantly higher yield-stress values than either the full-fat cheesespread control (20% fat without inulin) or low-fat spreads with relatively low inulin contents. The results indicated that the low-fat processed cheese spreads with 7% and 8% inulin achieved yield-stress values and spreadability similar to the full-fat processed cheese spreads (Dave et al., 2012). In relation to the microstructure of inulin-added cheeses, Miocinovi’c et al. (2011) observed that the addition of 3% inulin in low-fat cream cheese provided a more compact structure, a dense protein matrix and a greater arrangement of protein chains with pores between the chains, when compared to cheeses without addition of inulin. In this way, it has been suggested that inulin can become part of the structural protein network in cheeses after coagulation.

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The characteristic flavor of the cheeses is due to different types and concentration of fatty acids presented in the milk, and the fat influence is related to the hydrolysis of the fat during the ripening process. Furthermore, due to the biochemical transformations, other flavoring compounds are formed, such as ketones, methyl ketones and lactones, which can contribute to the development of cheese flavor. Flavor defects in low-fat cheeses include the low flavor intensity and characteristic aroma in relation to the full-fat version, as well as bitter taste and astringency. These defects are related to the alteration of the balance between fat, protein and moisture in the cheese, which results in deficiencies of fatty acids. Furthemore, there is interaction of the products generated in the lipolysis and proteolysis during ripening, and the increased moisture results in a lower proportion of salt, interfering with the flavor of the product (McMahon, 2010). Many fat replacers can confer unpleasant flavors to the products, but inulin can act as a fat replacer without causing unpleasant flavor. Cardarelli et al. (2008a) verified the sensory acceptance of potentially symbiotic petit suisse cheeses prepared with the addition of 10% of different fibers (medium-chain inulin, oligofructose and oligofructose from honey) and L. acidophilus and B. lactis as probiotic cultures. They found that acceptance increased significantly during storage only for cheeses supplemented with oligofructose and/or inulin; the highest acceptance was observed for the trial containing oligofructose. The combination of inulin and oligofructose provided better functional, sensory and technological characteristics to the petit suisse symbiotic cheese. Therefore, inulin has a significant effect on improving the flavor of low-fat cheese. This effect may be a result of inulin’s ability to form microcrystals when dissolved in water or milk. These microcrystals can not be sensed in the mouth, but directly influence the formation of the smooth and creamy structure of the product, and the creaminess effect increases with increasing inulin content in the cheese. Juan et al. (2013) studied the effect of the addition of 5% of long-chain inulin on the sensory properties of low-fat fresh cheese. The sensory panel described the low-fat cheese added inulin as more acceptable than its counterpart without inulin. They also found that cheeses produced with

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inulin had the lowest lightness and the highest yellowness values, although these color differences were not detected by the panelists. Inulin particles can also act as light scattering centers and increase the opacity of cheeses. In addition, the addition of inulin has modified the texture characteristics of fresh cheeses with reduced fat content, making them more similar to cheeses with full fat content, with less hardness, springiness, cohesiveness and chewiness. The reduction of fat in cheeses is related to the reduction in yield, since the fat content may constitute 50% or more of the dry matter of the cheeses. Cheese yield is important due to economic aspects and processing control. The addition of 2 and 4% of long-chain inulin in low fat karish cheese significantly increased cheese yield and moisture content as compared to control (low-fat cheese). The increase in yield in cheeses containing texturized inulin may be due to the form a gel network thus increasing the water holding ability (Alnemr et al., 2013). Considering the diversity of cheese manufacturing procedure, most important factors affect the final quality of different types of cheeses containing inulin depends on chain length of inulin, inulin concentration, preparation temperatures and amount of shear. Generally, application of inulin in cheese is of great interest in the development of prebiotic cheese that maximizes nutritional benefits and sensory characteristics to meet consumer demands. The inclusion of inulin in cheese as a fat replacer has different influences on the rheological and textural properties depending on the structure and composition of each type of cheese matrix. Sensory properties can vary among the different inulin contents and/or blends. For modification and optimization of different applications of inulin in cheese, an adequate understanding of the physiochemical characteristics of inulin polymers is needed (Karimi et al., 2015).

INULIN AS FAT REPLACER IN DAIRY DESSERTS A large number of semi-solid dairy products are currently available to the consumers, such as dairy desserts, which are considered to be healthy

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products with pleasant sensory properties. Dairy desserts are primarily formulated with milk, thickeners (starch, sodium carboxymethylcellulose and other hydrocolloids), sucrose, flavoring and colorants. Because they are added thickeners, they exhibit viscoelastic properties typical of weak gels. The use of inulin in dairy desserts aims to produce options with lowfat content or fat-free, improving their functional properties. The interaction between starch and inulin is an important factor to be highlighted in starch-based dairy desserts. Generally, inulin does not interact synergistically with the starch forming a three-dimensional network, but inulin acts as a diluent for the starch, interfering in the formation of the three-dimensional network of the gel. Zimeri and Kokini (2003) investigated the interaction of inulin with waxy maize starch in an aqueous system, evaluating the morphological, rheological and physicochemical characteristics. They found that the samples had two individual glass transition temperatures (Tg) corresponding to the different ingredients presented in the system and concluded that there is phase separation and formation of weak gels. Schaller-Povolny and Smith (2002) observed that, in the presence of inulin, soluble fractions of the milk protein become insoluble, probably because of hydrophobic interactions. It should be noted that inulin has different effects on different concentrations of starch, due to competition for water from the system. Thus, in lower concentrations of starch there is a higher content of free water in the system for inulin action, but with the increase of the starch concentration there is a limitation of free water content, consequently there will be competition between starch and inulin for free water. Thus, with increasing inulin concentration, the viscosity of the product will be compromised and part of the inulin may form crystals or crystalline aggregates in the product during storage, influencing the sensory properties. Lobato et al. (2009) analyzed the effect of interactions between milk, starch and medium-chain inulin on texture profile parameters and sensory characteristics of pudding-type dairy dessert and observed that the addition of inulin affected the cohesiveness of the product, with increasing inulin

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concentration decreasing cohesiveness. This observation can be explained by the fact that inulin weakens the starch network because it competes with water. In addition, the increase in inulin concentration increased the syneresis of the gels by competition for water or the diluent action of inulin. Tárrega and Costell (2006) evaluated the inclusion of inulin on the rheological and sensory properties of low-fat dairy desserts containing different concentrations of starch. These authors concluded that the ideal maximum starch concentration was 3.25% combined with 6% inulin. The addition of inulin to low-fat dairy dessert increased sweetness, viscosity, and creaminess of the final product, resembling the full-fat product. It should be noted that the effects of the addition of 6% of inulin on the rheological behavior of these desserts are clearly dependent on the starch concentration as a function of competition between inulin and starch by water. The different types of inulin significantly interfere on the viscoelastic properties of low-fat dairy desserts, with long-chain inulin having better effects on product viscosity and consistency. González-Tomás et al. (2009a,b) found that the addition of 7.5% of long-chain inulin significantly increased the consistency and viscosity of low-fat dairy desserts. However, the addition of inulin adversely affected the sensory acceptance of the final product, reduced softness and increased the sensation of roughness. This fact can be explained by the fact that long-chain inulin in aqueous media can form small crystals during storage, which cause a sensation of roughness to the palate. The crystallization process of inulin depends on both the inulin-chain size and the inulin initial concentration (Bot et al., 2004). Thus, reducing the concentration of long-chain inulin decreases the formation of inulin crystals, although decreasing their fat replacement capacity in gel systems. One possibility is to use a mixture of short and long-chain inulins as a fat replacer. Microstructural observations of starch-based dairy desserts with fullfat, low-fat and fat-free milk added long-chain inulin showed that the starch granules remain immersed in a continuous network composed

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mainly of milk proteins independently of milk fat concentration. In the fatfree samples with inulin, some large particles were observed confirming the inulin crystals aggregation in the continuous phase, which explains the important rheological effects and the sensory changes observed in low-fat dairy products with addition of long-chain inulin (Meyer et al., 2011). In addition to the effect of inulin on increasing the consistency and creaminess of low fat dairy desserts, the use of inulin presents the possibility of producing symbiotic dairy desserts when combining the addition of inulin with the addition of probiotic cultures. In this sense, Cardarelli et al. (2008b) evaluated the effect of the addition of a probiotic culture (Lactobacillus paracasei) along with 5% of medium-chain inulin on the texture and sensory attributes of chocolate mousse. The authors verified that the symbiotic product presented greater value in instrumental firmness and adhesiveness and was perceived in the sensory evaluation as significantly firmer than the formulations without addition of inulin, besides obtaining a product with symbiotic characteristic.

CONCLUSION The effect of inulin on rheological behaviour and on texture of different dairy products depends on the concentration of inulin, the degree of polymerization of the inulin, the structure and composition of the dairy product, the type and concentration of other carbohydrates present in the product, the inulin interactions with other ingredients and the process characteristics. Only medium and long-chain inulins can develop a gel structure formed by a network of crystalline particles, therefore, are recommended as fat replacers. In dairy products, inulin is able to improve the textural and rheological properties, in a manner that the low-fat added inulin products have similar characteristics to the full-fat products. In addition, no negative impact on the physicochemical characteristics is observed, with maintanance of the acidity and color. The ability to improve creaminess depends on the concentration of inulin used and the fat content of the products, and, in the

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majority of the studies, inulin can improve the acceptance of low-fat products, making it similar to the full-fat products. The ability of inulin to improve the creaminess and provide a fat-like mouthfeel to the dairy products is a result of its ability to form microcrystals when mixed with milk. These microcrystals are not perceived in the mouth, but interact to form a finely creamy texture that promotes the sensation of fat.

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INDEX A acetalated inulin (Ace-IN), 74, 75 acetaldehyde, 14, 125, 133 acidity, 14, 112, 121, 123, 124, 125, 126, 127, 134, 136, 139, 145 acquired immunodeficiency syndrome (AIDS), 50, 52, 56, 93, 94 adaptive immune responses, 36, 37,38, 39, 79, 97 adaptive immunity, 39, 83, 88 adjuvant, viii, 33, 34, 41, 42, 43, 45, 47, 48, 49, 51, 52, 53, 56, 57, 58, 59, 60, 61, 63, 66, 69, 70, 71, 73, 77, 79, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 94, 95, 96, 97, 98, 100, 101, 102, 103, 104, 105, 106 Advax, viii, 34, 47, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 71, 77, 79, 88, 90, 95, 96, 97, 98 adverse event, 40, 54, 61, 62, 63, 68 African horse sickness, 65, 66 agonist, 44, 48, 52, 58, 59, 66, 68, 86, 88 Algammulin, viii, 34, 48, 49, 50, 51, 52, 53, 54, 61, 64, 65, 68, 91, 93, 100 alpha inulin, 46, 68 Alum, 42, 48, 65, 83

anthrax, 35, 42, 56, 58, 96 antibody(ies), 37, 39, 40, 48, 49, 51, 52, 53, 54, 57, 58, 59, 61, 62, 63, 64, 65, 66, 68, 70, 73, 75, 76, 84, 86, 95, 96, 100, 102, 103, 104 antigen, viii, 34, 37, 38, 39, 40, 41, 42, 43, 46, 48, 49, 51, 53, 57, 58, 59, 61, 63, 64, 65, 69, 70, 71, 72, 74, 76, 79, 80, 81, 83, 89, 91, 92, 93, 94, 96, 97, 100, 103, 104, 106 antigen-presenting cells (APCs), 38, 39,43, 44, 46, 48, 69, 74, 83, 89

B B cells, 38, 39, 40, 57, 60, 95 bacteria, 3, 4, 7, 9, 10, 12, 13, 15, 17, 18, 20, 53, 111, 112, 113, 116 bacterium, 48, 50, 53, 58, 66, 146 beneficial effect, vii, 2, 16, 17, 111, 113, 116 beneficial microbes, 26 benefits, 2, 3, 5, 13, 18, 20, 26, 27, 110, 112, 142 beta inulin, 46, 47, 72 beverages, 13, 19, 21, 29

154

Index

bifidogenic, 3, 9, 115, 150 bile, 7, 8, 21, 28 bioavailability, 7, 10, 29 bioinformatics, 58 biomaterials, 103, 107 blood, vii, 2, 3, 7, 11, 64, 110 blood stream, vii, 2, 3, 7, 11 bonds, 3, 6, 110, 113, 120 bone, 11, 20, 21, 25, 28, 29 bone growth, 11 bone mineral content, 11 bone resorption, 21 bone volume, 28

C calcium, vii, 2, 5, 7, 10, 23, 28, 110, 112, 115, 136, 138 caloric, vii, viii, ix, 2, 6, 7, 17, 110, 116, 117, 118 calorie, 111, 118, 120, 147, 149 cancer, 7, 10, 19, 26, 43, 47, 48, 50, 53, 84, 87, 90, 108, 116 carbohydrates, vii, 1, 3, 6, 17, 22, 25, 113, 114, 116, 117, 118, 121, 145 casein, 27, 120, 122, 132, 136, 138, 140 CD8+, 39, 40, 57, 60, 61, 95 cellular immunity, viii, 33, 36, 37, 39, 40, 43, 48, 52, 54, 57, 75, 76, 97, 104 cervical cancer, 42, 50, 53, 94 chain molecules, 10, 13, 14 challenges, 22, 59, 85, 88, 106 cheese, 24, 27, 28, 58, 98, 111, 120, 136, 137, 138, 139, 140, 141, 142, 146, 147, 148, 149, 150, 151 chemical, 14, 24, 29, 74, 77, 90, 107, 108, 117, 118, 139 chemical characteristics, 24, 77, 139 chemokines, 43, 44 chicory, vii, 1, 2, 3, 5, 6, 9, 12, 19, 21, 23, 24, 25, 27, 45, 114

children, 50, 52, 60, 73, 88, 99 cholera, 35, 85, 104 cholesterol, 8, 28, 45, 115, 116, 148 clinical trials, 43, 44, 70, 77 cluster of differentiation, 39 colon, 3, 6, 7, 9, 10, 11, 12, 13, 19, 26, 111, 115 color, ix, 15, 109, 117, 120, 121, 124, 125, 126, 127, 128, 130, 131, 133, 134, 136, 137, 142, 145 complement, 37, 38, 39, 46, 47, 78, 89, 90 complete Freund’s adjuvant (CFA), 42, 43, 49, 51, 53, 63, 65, 70 compliance, 64, 117 complications, 49, 52, 55, 56, 60 composition, 7, 10, 20, 113, 118, 121, 142, 145 compounds, 12, 47, 112, 113, 117, 133, 141 compression, 15, 122, 130, 135 constipation, 3, 7, 9, 21, 22, 115 consumers, viii, ix, 2, 12, 13, 14, 15, 17, 109, 110, 114, 120, 121, 135, 136, 137, 142 consumption, vii, ix, 2, 27, 28, 65, 109, 110, 114, 116, 136 crystalline, 118, 143, 145 crystallization, 134, 135, 144 crystals, 16, 79, 135, 143, 144, 145 culture, 17, 145, 146, 147 cytokines, 40, 44, 47, 59, 60, 74

D Dairy, v, ix, 18, 19, 20, 21, 23, 25, 27, 31, 109, 142, 143, 146, 148, 149, 150, 151, 152 dairy industry, ix, 17, 110, 118, 133 dairy products, vii, ix, 9, 13, 17, 25, 30, 109, 110, 111, 117, 118, 119, 120, 133, 136, 138, 142, 145, 150 deaths, 50, 53, 58, 94

Index defects, ix, 109, 120, 132, 137, 141 delivery system, 34, 69, 72, 79, 91, 102, 106, 107 delivery vehicles, 34, 69, 70, 71, 73, 79, 108 delta inulin, viii, 34, 46, 47, 56, 57, 61, 66, 68, 77, 88, 90, 95, 96, 97, 98, 100, 102 dendritic cells (DCs), 37, 38, 59, 83, 85, 86, 96, 105 deoxyribonucleic acid( DNA), 37, 44, 57, 81, 87, 96, 99 Diabetes, 22 diarrhea, 12, 102 diet, ix, 7, 9, 13, 17, 19, 23, 26, 109, 114 dietary fat, 116 dietary fiber, ix, 3, 6, 12, 17, 18, 22, 24, 28, 110, 114, 147 diphtheria, 41, 42, 50, 51, 82, 93 diseases, 7, 10, 19, 34, 35, 49, 50, 53, 56, 61, 94, 110, 113 distribution, 137, 140 drug delivery systems, 69, 103, 108 drying, 5, 15, 101, 102

E emulsions, 42, 43, 45, 62, 85 encephalitis, 55, 56, 65, 67, 95, 100 energy, 3, 4, 7, 23, 112, 116, 117 energy consumption, 4 energy density, 117 engineering, 107, 108 enzymes, 3, 7, 8, 9, 12, 37, 46, 112, 137, 150 epitopes, 38, 39, 58, 96 ethanol, 15, 46, 72 evidence, 22, 24, 28, 68, 91 extraction, 2, 4, 15, 24, 27

155 F

fat, vii, viii, ix, 2, 3, 6, 9, 12, 13, 14, 16, 17, 18, 23, 24, 25, 27, 29, 109, 110, 111, 116, 117, 118, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152 fat reduction, 137, 139 fat substitute, 24, 110, 121, 138 fatty acids, 7, 9, 10, 11, 28, 112, 116, 141 FDA, 35, 42, 43, 45, 53, 56, 58, 60, 61, 62, 63, 64, 70, 73 FDA approval, 63 fermentation, 9, 10, 11, 15, 112, 121 fiber, vii, ix, 1, 2, 3, 6, 7, 12, 13, 14, 15, 16, 17, 18, 20, 21, 22, 24, 25, 27, 28, 29, 110, 118, 119, 121, 139, 141, 147 firmness, 9, 14, 117, 121, 123, 125, 126, 127, 128, 129, 132, 145 flavor, ix, 5, 13, 14, 16, 109, 111, 116, 117, 120, 123, 124, 128, 129, 130, 134, 137, 141 Food and Drug Administration, 35, 79 food industry, vii, 1, 3, 12, 110 food intake, 2, 11, 116 food products, 15, 17 force, 59, 89, 122, 130, 135, 140 formaldehyde, 41, 80 formation, 6, 84, 89, 115, 121, 122, 132, 133, 138, 139, 141, 143, 144 fragments, 6, 39, 46, 89 freezing, 5, 111, 134, 135, 136 fructan, 3, 46, 111, 114, 120, 138 fructose, vii, 1, 3, 6, 20, 46, 74, 89, 113, 114 functional food, 17, 18, 27, 114 functional ingredient, 2, 113

156

Index G

gamma inulin, viii, 34, 46, 47, 48, 49, 50, 51, 52, 54, 57, 61, 64, 65, 69, 77, 91, 100 gastroesophageal reflux, 23 gastrointestinal tract, 12, 13, 112, 113 gel, 13, 14, 22, 24, 118, 121, 122, 135, 138, 140, 142, 143, 144, 145 glanders, 66 glass transition, 29, 143 glass transition temperature, 29, 143 glucose, vii, 1, 9, 20, 23, 46, 89, 113, 114 glutathione, 54 glycoside, 44 growth, 4, 11, 13, 17, 26, 110, 111, 112, 114

H hardness, 117, 129, 130, 131, 135, 136, 137, 139, 140, 142 health, vii, viii, 2, 6, 7, 9, 14, 17, 20, 24, 25, 26, 27, 28, 29, 83, 94, 110, 111, 112, 113, 116, 149, 151 health effects, 116 health status, 17 heart disease, ix, 109 hepatitis B, 42, 45, 48, 56, 61, 77, 81, 91, 97, 102, 103, 104 HIV-1, 57, 85 hospitalization, 60 host, vii, 2, 7, 10, 35, 37, 38, 39, 40, 44, 59, 110, 111, 113 human, 2, 3, 6, 7, 9, 12, 14, 19, 21, 22, 23, 26, 28, 29, 34, 35, 45, 55, 58, 62, 64, 65, 82, 83, 85, 91, 96, 104, 105, 110, 113, 114, 115, 136, 148, 151 human body, 136 human health, 6, 28, 110, 151 human immunodeficiency virus (HIV), 35, 43, 52, 57, 85, 93, 94, 96 humoral immunity, 36, 37, 39, 49

hybrid, 91 hydrogels, 70 hydrogen, 132 hydrolysis, 5, 111, 113, 114, 137, 138, 139, 141 hydrophobicity, 77 hydroxide, 42, 83, 103 hydroxyl groups, 74, 75

I imitation, 111, 137, 149 Immune, 7, 35, 37, 38, 39, 101 immune function, 24 immune response, viii, 12, 19, 34, 36, 38, 41, 45, 48, 74, 77, 83, 90, 91, 92, 100, 101, 102, 105, 108, 115 immune system, vii, ix, 2, 7, 10, 12, 34, 35, 36, 37, 38, 39, 41, 109 immunity, 25, 35, 36, 37, 39, 40, 41, 48, 49, 59, 64, 80, 81, 94, 99, 100, 106 immunization, 41, 51, 54, 58, 59, 62, 67, 69, 77, 84, 92, 96, 97, 98, 104, 105, 106 immunogenicity, viii, 33, 41, 49, 51, 54, 55, 60, 61, 62, 63, 64, 66, 74, 77, 85, 92, 93, 98, 100, 102, 103, 105 immunoglobulin, 12, 39 immunostimulatory, viii, 34, 45, 71, 73, 77, 83, 105, 106 immunotherapy, 69, 90, 102, 103 in vitro, 7, 63, 74, 91, 114 in vivo, 7, 58, 74, 81 Incomplete Freund’s Adjuvant (IFA), 43, 53 individuals, 16, 59, 116 induction, 59, 92, 103 infants, 12, 23, 50, 88 infection, 24, 35, 40, 59, 64, 67, 96, 99, 100, 101 inflammasome, 47, 83 inflammation, 10, 37, 58, 66, 67, 115 inflammatory bowel disease, 10

Index influenza, 43, 48, 56, 62, 63, 68, 70, 77, 81, 84, 86, 95, 97, 98, 100, 101, 102, 104 influenza vaccine, 43, 48, 62, 63, 68, 81, 84, 95, 97, 98 influenza virus, 63, 81, 84, 86, 104 ingestion, 12, 115, 117 ingredient, vii, ix, 1, 2, 5, 6, 13, 17, 20, 27, 110, 111, 113, 121, 134, 137, 143, 145 inhibition, 63, 110, 115 innate immune response, 36, 37, 43, 88, 107 intervention, 29, 35 intestine, 7, 9, 10, 112 Inulin, v, vii, viii, ix, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 31, 33, 34, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 61, 64, 65, 68, 69, 70, 71, 72, 73, 74, 75, 77, 78, 79, 88, 89, 90, 91, 95, 97, 98, 101, 102, 106, 109, 110, 111, 113, 114, 115, 116, 118, 120, 121, 122, 123, 124, 126, 127, 128, 129, 130, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152 inulin-type fructans, 4, 11, 23, 24, 27, 110, 113, 114, 115, 116, 118, 120, 121, 124, 137, 148 irritable bowel disease, 10 irritable bowel syndrome, 116 Italy, 22, 30, 148

L lactic acid, 9, 12 Lactobacillus, 9, 10, 13, 15, 18, 25, 145, 146, 147, 151 lactose, 16, 116 large intestine, 3, 6, 10 lipid metabolism, 8, 23, 28, 29, 115 lipidemia, 7 lipids, vii, 2, 3, 8, 83

157

liposomes, 53, 66, 69, 70, 102, 104 Listeria monocytogenes, 55, 58, 98, 99 listeriosis, 56, 58 long-term memory, 38, 39 lymph node, 38, 68 lymphoid tissue, 12

M macromolecules, 104, 105 macrophages (Mϕs), 37, 38, 47, 103 magnesium, 11, 112 major histocompatibility complex, 38, 103 majority, 134, 136, 146 malaria, 45, 50, 52, 70, 88, 93, 104 manufacturing, viii, 14, 34, 41, 56, 137, 142 mass, 7, 10, 23, 74, 139 matrix, 15, 17, 49, 132, 136, 137, 139, 140, 142 membranes, 44, 136 memory, 36, 40, 57, 88, 95, 99 memory B cells, 57, 95 meningococcal diseases, 50, 53 meta-analysis, 19, 24 metabolism, 7, 23, 27, 111 methodology, 18, 151 MHC, 38, 39, 58, 103 mice, 10, 12, 19, 24, 47, 51, 54, 56, 59, 60, 61, 62, 65, 72, 75, 90, 91, 92, 98, 101, 102, 103 microbiota, 20, 22, 23, 25, 110, 112, 113, 114, 115, 148 microcrystalline, 88, 118 microorganisms, 7, 13, 114, 121 microparticles, 70, 71, 72, 74, 75, 76, 103, 106, 107 microstructure, 27, 140, 152 moisture, 29, 111, 121, 139, 141, 142, 152 moisture content, 29, 111, 121, 142 molecular weight, 92, 114

158

Index

molecules, 5, 7, 37, 38, 39, 40, 102, 103, 132, 135 Montanide, 43, 51, 63, 66, 67, 85

N nanoparticles, 59, 70, 103 National Academy of Sciences, 86, 88, 94, 103 National Institutes of Health, 56 natural killer cell, 12, 37 neurological disease, 65 neutral, ix, 5, 75, 110, 118, 120, 138 nucleic acid, 41 nucleoprotein, 94 nutraceutical, 14 nutrients, 116, 136 nutrition, 14, 24, 149

O oil, 25, 42, 43, 51, 52, 62, 66, 67, 72, 74, 76, 84, 85 oligomerization, 44, 85 oligosaccharide, 3, 91 optimization, 18, 79, 142 ovalbumin, 72, 75, 76

P pathogens, viii, 12, 33, 35, 36, 38, 39, 40, 42, 47, 64, 68, 112 pathway, 37, 46, 78, 89, 90 peptide, 11, 21, 26, 38, 39, 51, 59, 92, 93, 96 peste des petits ruminants, 67, 100, 101 pH, 5, 6, 7, 8, 9, 10, 14, 16, 17, 74, 75, 76, 105, 121, 124, 125, 126, 127, 128, 130, 131, 134, 138, 139 phagocytic cells, 43

phagocytosis, 37, 38, 74, 103 pharmaceutical, 18, 81, 90, 91, 108 phosphate, 42, 136 phospholipids, 45, 115, 148 physical properties, 16, 89 physicochemical characteristics, 20, 134, 136, 143, 145 physicochemical properties, 27, 103, 118, 120, 146 plasma membrane, 38, 44 platform, 63, 71, 73, 75, 77 polymer, vii, 1, 20, 66, 69, 70, 71, 75, 76, 89, 103, 104, 106, 114 polymer chain, 20, 89 polymeric particles, 69, 74 polymerization, 3, 9, 23, 111, 113, 118, 121, 145 polymethylmethacrylate, 104 polysaccharide, viii, 3, 26, 28, 34, 41, 46, 53, 68, 73, 74, 80, 88, 90, 91, 97, 98, 106, 121 population, 35, 51, 63 prebiotic, vii, ix, 2, 3, 9, 10, 12, 13, 15, 16, 17, 18, 19, 20, 21, 22, 24, 25, 26, 27, 29, 31, 110, 111, 112, 113, 114, 115, 120, 142, 149, 150 precipitation, 5, 46, 47, 72, 82 preparation, 49, 91, 142 prevention, 3, 7, 19, 20, 81 primary function, 38, 39 probiotic, 9, 10, 13, 15, 16, 17, 18, 19, 20, 21, 26, 31, 112, 121, 141, 145, 146, 147, 152 pro-inflammatory, 44, 47, 74, 76 proliferation, 61, 62, 65 prophylactic, 42, 43, 47, 68, 77, 108 protection, viii, 15, 33, 40, 41, 48, 51, 54, 57, 58, 59, 60, 62, 63, 66, 81, 95, 96, 97, 98, 100 proteins, 38, 39, 44, 80, 87, 101, 104, 117, 120, 132, 135, 145, 152 proteolysis, 136, 141

Index Q QS-21, 45, 82, 87 quality control, 41 quality of life, 110 Quil A, 44, 53, 63, 70

R receptor, 8, 11, 37, 44, 59, 79, 86 recombinant proteins, 41, 51, 62, 92 rectal temperature, 68 regulatory agencies, 70, 77 Replacer, v, 109, 120, 134, 136, 142 researchers, 51, 52, 53, 59, 60, 77 resistance, 3, 25, 112, 129, 130, 134 respiratory syncytial virus (RSV), 56, 60, 97, 99 response, 18, 40, 45, 54, 76, 84, 93, 96, 99, 100, 104, 151 rheology, ix, 110, 118, 134, 147 ribonucleic acid, 37 risk, 7, 10, 26, 40, 51, 83, 98, 99, 110, 113, 115, 116 risk assessment, 83, 99 RNA, 37, 44, 59, 86 room temperature, 74, 118 RSV infection, 60 RTS, 46, 52, 70, 88

S safety, viii, 34, 42, 43, 45, 54, 61, 64, 69, 77, 82, 85, 88, 91, 97, 98, 147 Salmonella, 12, 15, 19, 44, 87 saponin, 44, 87 SARS, 55, 56, 59, 60, 97, 99 SARS-CoV, 59, 60 saturated fats, ix, 109 saturated fatty acids, 116

159

scanning electron microscopy, 140 sensation, 111, 117, 118, 144, 146 serum, 8, 9, 19, 28, 39, 48, 57, 60, 64, 75, 76, 90, 100, 104, 122, 132, 148 severe acute respiratory syndrome (SARS), 55, 56, 59, 60, 97, 99 sheep, 16, 17, 18, 19, 30, 31, 65, 100 small intestine, 3, 6, 10 smallpox, 35, 79 smoothness, 14, 117, 123, 129, 133, 134 solubility, 13, 46, 55, 89, 114, 118, 138 soluble, 2, 6, 8, 12, 24, 27, 41, 46, 47, 48, 68, 72, 75, 106, 110, 114, 118, 121, 126, 143 solution, 5, 118, 139 stability, viii, ix, 9, 13, 26, 34, 74, 102, 110, 111, 118, 121, 128, 132, 148, 151 stabilization, 80, 101 stabilizers, 68, 150 starch, 118, 139, 143, 144, 150, 152 starch granules, 144 stimulation, vii, 2, 3, 38, 40, 83, 111 storage, 6, 14, 16, 17, 25, 26, 68, 102, 111, 113, 117, 124, 127, 139, 141, 143, 144, 151 stress, 15, 28, 117, 121, 124, 127, 140 structural changes, 95 structural protein, 140 structure, 3, 6, 37, 45, 55, 87, 89, 111, 118, 120, 121, 122, 132, 135, 136, 138, 140, 141, 142, 145 sucrose, 2, 3, 5, 13, 14, 26, 113, 114, 118, 143 supplementation, 7, 13, 17, 20, 22, 24, 26, 149 surfactant, 42, 43, 51 survival, viii, 2, 16, 25, 47, 57, 58, 146, 151 sweeteners, 5, 134 symptoms, 23 syndrome, 55, 96 synthesis, 7, 8, 21

160

Index T

T cell, 12, 38, 39, 40, 54, 57, 59, 60, 61, 62, 64, 73, 80, 88, 95, 103 T lymphocytes, 39, 81 taeniasis, 65 technology, 5, 18, 30, 150 temperature, 5, 46, 55, 66, 114, 116, 118, 138, 139 testing, viii, 34, 35, 43, 45, 54, 56, 60, 70, 85, 97 tetanus, 41, 42, 51, 84, 103 tetrahydrofuran, 76 textural character, 25, 120 texture, viii, ix, 2, 13, 14, 15, 16, 25, 109, 111, 116, 118, 120, 121, 122, 125, 126, 129, 130, 131, 132, 133, 134, 135, 136, 137, 139, 140, 142, 143, 145, 146, 147, 150, 151 TLR, 44, 45, 48, 59, 68, 103 TLR agonists, 44, 45, 103 total cholesterol, 115 toxic side effect, 45 trial, 20, 50, 54, 55, 61, 63, 69, 85, 88, 98, 141 triglycerides, 115, 148 trimethylsilyl inulin (TMS-IN), 75, 76 tuberculosis, 42, 71, 73, 106, 107 tumor, 12, 54, 92, 94, 115 type 2 diabetes, 22, 116

U ulcerative colitis, 10 ultrasound, 4

underlying mechanisms, 80 uric acid, 83

V vaccine, viii, 12, 19, 33, 34, 35, 37, 38, 41, 43, 45, 47, 48, 51, 52, 53, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 66, 67, 68, 69, 73, 77, 78, 79, 80, 81, 82, 84, 85, 86, 87, 88, 89, 90, 91, 93, 94, 95, 97, 98, 99, 100, 101, 102, 103, 105, 106, 108 vaccine design, 87 vehicles, 34, 69, 70, 71, 73, 79, 108 very low density lipoprotein, 148 viral infection, 63 viral vectors, 70, 105 virology, 80 viruses, viii, 33, 40, 60, 67, 87, 100 viscoelastic properties, 143, 144 viscosity, 5, 16, 117, 118, 120, 121, 122, 123, 124, 126, 127, 128, 129, 130, 131, 133, 134, 135, 140, 143, 144 vitamin D, 11

W water, 5, 6, 12, 13, 14, 15, 42, 43, 44, 46, 51, 52, 62, 66, 67, 72, 74, 76, 85, 101, 114, 118, 122, 131, 132, 135, 138, 139, 141, 142, 143, 144 white blood cell count, 61 World Health Organization (WHO), 46, 63, 84, 85, 93, 94, 98, 99, 100, 101, 106 worldwide, 2, 50, 52, 53, 56, 61, 73, 94