Michelle Wyatt, Michelle Wyatt-Whey Proteins_ Functional Properties, Production and Health Benefits-Nova Science Publishers, Inc. (2014)

PROTEIN BIOCHEMISTRY, SYNTHESIS, STRUCTURE AND CELLULAR FUNCTIONS

WHEY PROTEINS FUNCTIONAL PROPERTIES, PRODUCTION AND HEALTH BENEFITS

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PROTEIN BIOCHEMISTRY, SYNTHESIS, STRUCTURE AND CELLULAR FUNCTIONS

WHEY PROTEINS FUNCTIONAL PROPERTIES, PRODUCTION AND HEALTH BENEFITS

MICHELLE WYATT EDITOR

New York

Copyright © 2015 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. For permission to use material from this book please contact us: [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:  (eBook) Library of Congress Control Number: 2014951171

Published by Nova Science Publishers, Inc. † New York

CONTENTS Preface Chapter 1

vii Fractionation of Peptides by Means of Ultrafiltration and Nanofiltration Techniques Francisco Amador Riera, Ayoa Fernández and Claudia Muro

1

Chapter 2

Italian Valorization of Dairy Waste Gigliola Camaggio and Vera Amicarelli

59

Chapter 3

Encapsulation of Whey Proteins and Active Peptides Claudia R. Muro Urista, Francisco A. Riera Rodriguez and Yolanda Alvarado Pérez

75

Chapter 4

Formation, Stability, Flow and Viscoelastic Properties of Foams Prepared with Whey Protein Isolate and Its Individual Mixtures with Xanthan, Guar and Refined Sugar Eva M. García-García, Mariana Ramírez-Gilly and Alberto Tecante

117

Use of Computational Fluid Dynamics in the Manufacture Process of Whey Protein Products Bárbara E. Meza and Juan Manuel Peralta

149

Chapter 5

vi Chapter 6

Index

Contents The Potential of Cow‟s Milk Proteins for Prophylaxis against Intestinal Infection Mizuho Inagaki, Tomio Yabe and Yoshihiro Kanamaru

163

179

PREFACE Whey proteins are now one of the most important products in food processing industries. Profit of whey proteins in food applications include its high amino acid content; low calorie, fat, and sodium content; high emulsification and capacity and compatibility with other ingredients. This book discusses the functional properties of whey proteins along with the production and health benefits of consuming these proteins. Chapter 1 - Some peptides produced after controlled or natural enzymatic hydrolysis of proteins show, not only interesting nutritional and technological properties, but also biological activities that can be of interest for human health. In recent years, the therapeutic potential of some food components have had the attention of food manufacturers, clinicians, and consumers. Only a few peptides obtained after protein hydrolysis have been deeply studied so far, and consumers are unaware of their potential properties. These peptides are inactive within the protein sequence and must be isolated in order to take advantage of their biological activities. In addition, some of the peptides present in the hydrolyzed proteins do not show any biological activity and can even present antagonistic properties. For this reason, it is important to have technologies that are able to fractionate these peptides. These techniques must be compatible with foods and they should be easily scaled up. Pressure-driven membrane (PDM) processes such as ultrafiltration (UF) and nanofiltration (NF) have been viewed as efficient techniques for the fractionation of hydrolysates from different protein sources. In the first part of this chapter, useful information about membrane techniques applied to peptide fractionation is included. The second part resumes the main peptides obtained after the hydrolysis of different protein sources, and it describes their biological

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properties as well as the advances in their fractionation by means of PDM technologies. Chapter 2 - An economic system, even more oriented to sustainable development models, should provide a new way of managing production processes optimizing resources utilization and minimizing the amount of waste produced. In this context, waste valorization becomes a crucial option to improve the economic and the environmental performances of a company and/or of an industrial sector. The dairy waste represents an interesting example of this type of approach considering that in Italy, dairy industry plays a key role in the agri-food sector in terms of production, employment and turnover. The possibility to transform this waste into resources is related to their composition, they are rich in high value-added substances such as proteins, protein derivatives, potentially prebiotic sugars, minerals and vitamins. The aim of this chapter is to evaluate the different possibilities of dairy industry waste utilization to produce new raw materials useful in the food, pharmaceutical, and cosmetic industries. The authors will focus their analysis on Italian situation related to the dairy by-products valorization. Chapter 3 - For several years, many researchers have focused on study of whey proteins and derived active peptides. Its functional and therapeutics properties have caused a particular interest in pharmaceutical and food products and in different areas of cosmetology. In the context of health, serum molecules have been a subject of growing research, because they can provide specific health benefits. However proteins and whey peptides are currently in the circle of sensitive biological molecules which require an effective oral administration, since its degradation, stability and permeability are not yet controlled. The peptides mainly suffer degradation in the gastrointestinal tract, including its physicochemical instability and inactivation, so these aspects are an important research topic to achieve the desired effects in medicine field. Under this perspective, various approach enhance have been studied as alternatives to increase stability and oral bioavailability of some biomolecules which could to impact in the use and application of proteins and whey peptides as therapeutic products. In this chapter the author‟s make mention on different strategies to enhance oral bioavailability of sensible molecules and their application status. A wide range of compounds are exposed summarizing main alternatives of enhance systems.

Preface

ix

Studies of encapsulation of whey proteins and peptides in nano systems are the origin the future trends on approach enhances in active molecules to their oral administration and delivery. Chapter 4 - Currently, food foams have become increasingly important due to the wide variety of textures that can be obtained by incorporating air into a precursor medium, and the additional advantage of including an ingredient without cost. The purpose of this chapter is to discuss the characteristics of stability, overrun, flow and viscoelastic properties of foams formed with whey protein isolate (WPI) and its individual mixtures with xanthan, guar and refined sugar, to understand the factors affecting their formation and rheological properties. A concise presentation of the most relevant information on the stability and rheological properties of foams made from mixtures of proteins and polysaccharides is presented first, followed by the author‟s results with WPI, WPI-xanthan, WPI-guar and WPI-refined sugar precursor solutions and foams. As part of the author‟s methodology, the suitable foaming conditions in a rotor-stator agitator, i.e. rotational speed, time and WPI concentration, considering stability and overrun are discussed first. Then, the stability, overrun, flow and viscoelastic properties of the WPI, WPIxanthan, WPI-guar and WPI-refined sugar for the suitable foaming conditions are analyzed and discussed to understand the role played by these components on the behavior and characteristics of these edible foams. Chapter 5 - Whey proteins are well known for combining high nutritional value with specific functional properties. In general, they are commercialized as concentrates and isolates that can be different in their functional properties due to variances in milk composition as well as in manufacturing conditions used to obtain those products. Computational fluid dynamics (CFD) is one of the most important technique used to solve the transport phenomena balances. The main advantage of this method is the possibility to obtain a high detailed description of the fluid system without using expensive experimental measurements. CFD is widely used in food science and engineering to study heat and mass transfer, phase change, solid and fluid interactions, and chemical reactions in food processing. As a consequence, the application of CFD to simulate the transport phenomena in several food processing steps (for instance heat treatments, membrane filtration, evaporation, and spray-drying) was documented in literature. For this reason, this chapter is a brief review about the use of CFD as a tool for simulating and optimizing the industrial manufacture of whey protein products, such as concentrates and isolates. Chapter 6 - Milk provides breast-fed infants a wide array of molecules, including nutrients and various proteins with defense functions, resulting in

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their healthy growth. In this review chapter, the author‟s describe experimental results that demonstrate the protective efficacy of the cow milk proteins against viral intestinal infection. The protective efficacy against viral intestinal infection of the anti-viral milk components, IgG, PAS6/7, and LP, contained in a microfiltration retentate fraction (MFRF) prepared from industrial sweet whey was studied and proven both in vitro and in vivo. Therefore, the author‟s conclude that milk whey concentrates, such as MFRF, are promising prophylactic food additives against intestinal infection. The author‟s also describe the HRV inhibitory mechanism of bovine -casein, focusing on the glycan structures of -casein.

In: Whey Proteins Editor: Michelle Wyatt

ISBN: 978-1-63463-116-7 © 2015 Nova Science Publishers, Inc.

Chapter 1

FRACTIONATION OF PEPTIDES BY MEANS OF ULTRAFILTRATION AND NANOFILTRATION TECHNIQUES Francisco Amador Riera1,*, Ayoa Fernández2 and Claudia Muro3 1

Department of Chemical Engineering and Environmental Technology. Faculty of Chemistry. University of Oviedo. C/Julián Clavería, Oviedo, Principado de Asturias. Spain 2 Department of Food Chemistry and Technology, Teagasc Food Research Centre, Moorepark, Fermoy, Co. Cork, Ireland 3 Department of Chemical Engineering and Investigation. Instituto Tecnológico de Toluca. Avda. Tecnológico s/n Ex-Rancho la Virgen. Toluca, C.P., México

ABSTRACT Some peptides produced after controlled or natural enzymatic hydrolysis of proteins show, not only interesting nutritional and technological properties, but also biological activities that can be of interest for human health. In recent years, the therapeutic potential of some food components have had the attention of food manufacturers, clinicians, and consumers. Only a few peptides obtained after protein hydrolysis have been deeply studied so far, and consumers are unaware of their potential properties. *

Correponding author: E-mail: [email protected].

2

Francisco Amador Riera, Ayoa Fernández and Claudia Muro These peptides are inactive within the protein sequence and must be isolated in order to take advantage of their biological activities. In addition, some of the peptides present in the hydrolyzed proteins do not show any biological activity and can even present antagonistic properties. For this reason, it is important to have technologies that are able to fractionate these peptides. These techniques must be compatible with foods and they should be easily scaled up. Pressure-driven membrane (PDM) processes such as ultrafiltration (UF) and nanofiltration (NF) have been viewed as efficient techniques for the fractionation of hydrolysates from different protein sources. In the first part of this chapter, useful information about membrane techniques applied to peptide fractionation is included. The second part resumes the main peptides obtained after the hydrolysis of different protein sources, and it describes their biological properties as well as the advances in their fractionation by means of PDM technologies.

1. INTRODUCTION Biofunctional peptides obtained from food and food wastes have gained great interest in the last two decades. Sources, such as milk derivatives (cheese whey, caseins), fish wastes, cereals, seeds, and so forth, can be used to produce peptides with different biological properties. All of these proteinaceous compounds can be hydrolyzed by means of enzymes in batch or continuous reactors, and the obtained broth can be fractionated, as probably not all peptides obtained have the same biological properties. The separation of peptides with molecular weights between 200 and 2000 Dalton (Da) is difficult, do to their small physicochemical differences. Some separation technologies, such as chromatography and electrophoresis, are selective enough for analytical purposes, but they are not appropriate to obtain concentrated peptides at an industrial scale, owing to difficulties in scale-up and prohibitive costs (Garem et al., 1997). Ultrafiltration (UF) and nanofiltration (NF) are membrane technologies that can be used for this fractionation and later concentration of interesting peptides. In the first part of this chapter, practical information is given about the use of UF and NF in the fractionation of peptides from a hydrolyzed proteinaceous broth. In the second part, a review of practical works on peptide fractionation is given.

Fractionation of Peptides by Means of Ultrafiltration …

3

2. MEMBRANE TECHNOLOGIES Membranes can be defined as selective barriers that allow the transmission of certain components of a liquid solution or suspension, while retaining others according to their size, shape, or other characteristics such as charge or hydrophobicity. The driving forces for the process can be pressure, concentration, or electrical fields. The main characteristics of the membranes used at industrial scale are certain mechanic and chemical resistances that withstand work pressures as well as alkaline and acidic media (especially in cleaning steps), low resistance to fluid permeation, narrow pore size, and so forth. Ultrafiltration (UF) and nanofiltration (NF) are classified as pressuredriven membrane techniques, together with microfiltration (MF) and reverse osmosis (RO). The main characteristics of these different techniques are detailed in Table 1. Membrane techniques are commonly used in the concentration, purification, and fractionation of compounds and molecules present in biological fluids. They do not need external additives (such as solvents or others); they can operate at low and moderate temperatures (with minimal impact on biological compounds), have low energy consumption, are easily scaled up, and their use do not imply high environmental impacts. Among these techniques, “tight” UF and “loose” NF are especially adequate for the fractionation of peptides from hydrolyzed broths, owing to the fact that their pore sizes are in the same size range as the peptides obtained after the hydrolysis of protein sources (generally lower than 5000 Da). To select an adequate membrane for peptide fractionation, several aspects must be taken into account: Table 1. Pressure-driven membranes characteristics Average pore Permeate flow size (nm) rate (L.h-1.m-2)* MF >200 >300 UF 2-200 20-200 NF 1-2 5-100 OI Robs). To estimate the 

Rreal value is not easy and some expressions have been proposed (Koyuncu &

 1  Robs  Robs

Topacik, 2002): Ln

  1  Rreal  J   Ln   , where J is the water   Rreal  k

flux and k is the mass-transfer coefficient that depends on Reynolds number and the module geometry, process conditions, and fluids properties. A number e

d  of correlations can be used to estimate k : Sh  a.Re .Sc . h  , where Sh ,  L kd Re ,and Sc are the dimensionless numbers of Sherwood ( Sh  h ), D d ρv  Reynolds ( Re  h ), and Schmidt ( Sc  ), respectively; a , b , c , D μ and e are parameters that depend on the flow regime (laminar or turbulent). The d h value is the hydraulic diameter that depends on the module geometry. b

c

Diffusivity ( D ) is a parameter that is more difficult to estimate. The intensity of CP can be measured through the polarization modulus (

PM ) (Schäfer et al., 2005), which is defined as PM 

Cw J  exp  .This Cb k

equation comes from the well-known film model, obtained after a mass

 C w  Cp   . The PM values  C C  b p  

balance on the membrane surface: J  k .Ln

depend on the feed nature. For proteins, PM can be substantially higher than 10 (in the case of ions, PM is lower than 2). As high PM values indicate

Fractionation of Peptides by Means of Ultrafiltration …

11

fouling, it is interesting to work in conditions to minimize it. All techniques or strategies to increase mass-transfer coefficient k will reduce the CP and delay the membrane fouling. Fluid properties, such as density, viscosity, and diffusivity, which are included in k correlations, are fixed by the feed. Turbulent regimes (high lineal velocities on the membrane surface) in the membrane modules are recommended to increase k. However, high velocities imply a higher channel pressure loss (P) and extra energy consumption. Some module configurations, such as tubular ones, are especially recommended if high lineal velocities are necessary.

2.3. Membrane Fouling Membrane fouling is the main drawback of membrane techniques. The operating conditions and the membrane and solutes characteristics (including solutes concentration) are major factors that affect fouling. In general, fouling is more important in MF and loose UF, but it must not be neglected in NF and tight UF. The detection of fouling can be quantified by calculating the flux reduction parameter ( FR ): FR 

J wb  J wa x100 , where J wb and J wa are J wb

the water permeate flux before filtration (permeate flux with the clean membrane) and after filtration and rinsing, respectively, and represent the part of flux caused by fouling compounds remaining in the membrane after feed filtration and rinsing with water. When a membrane fouls, not only the productivity is reduced (lower permeate flow rate, higher membrane surface necessary, and increasing costs), but membrane selectivity is modified and the efficiency of the separation process can be affected. Fouling can be caused by the presence of soluble inorganic salts, colloidal or microparticulated matter solutes, dissolved organics, chemical reactants, and microorganisms on the membrane surface or inside the pores (Speth et al., 2000); it is difficult to avoid it and the filtration process must be designed to delay or minimize this effect. Once the membrane has fouled, cleaning is the only way to recover the original membrane permeability. Fouling is considered irreversible but, as mentioned before, CP not (van der Bruggen et al., 2002). Fouling mechanisms are varied, including precipitation of solutes or colloids on the membrane surface, chemical reaction of compounds at the membrane boundary layer or with the membrane

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Francisco Amador Riera, Ayoa Fernández, Claudia Muro

material, solutes adsorption on the membrane surface, gel formation, bacteria growth, scaling, and so forth. Many times, the final fouling can be described by a combination of the mechanisms. Fouling mechanisms have been studied in several monographs and articles (Belfort et al., 1994; Schäfer et al., 2005). They are dependent on the solute/membrane pore ratios (colloids or solutes can be absorbed inside the membrane pores when dsolute < dpore, reducing the effective pore size, and can completely block the pore when dsolute ≈ dpore, or can form a cake on the membrane surface when dsolute > dpore), that is, the tendency of colloids and solutes to aggregate, forming loose or tight aggregates on the membrane surface or the solute, and the interaction solute/membrane due to hydrophobic/hydrophilic properties. Enzymatic hydrolyzed protein broths are composed of organic, inorganic, and biological compounds, and interactions between some of them can affect the extent and characteristics of fouling.

2.3.1. Organic fouling Organic fouling depends on the affinity of nonhydrolyzed protein and peptides towards the membrane material. Proteins, amino acids, and saccharides can be adsorbed onto the membrane material or on the colloids present in the broth, and they can also form a cake or a gel layer, depending on the CP, and then enhancing fouling. The adsorption of these compounds is dependent on the pH value, and the highest fouling occurs when charge repulsion between charged solutes and membrane is minimized (Mänttäri et al., 2000). Proteins and peptides can be neutral or positively/negatively charged, depending on their isoelectric point (IP) and the pH. When pH < IP, proteins, peptides, and amino acids are charged positively, and they are negatively charged when pH > IP. At pH ≈ IP, the solutes are uncharged. Additionally, peptide–peptide interactions also influence fouling, but these mechanisms are less studied. The presence of polysaccharides and other aromatic compounds increase fouling when peptides and proteins are present (Mackey, 1999). The organic compounds can be adsorbed on the membrane surface. 2.3.2. Inorganic fouling Protein-hydrolyzed broths always contain different types of salts and ions. They can be intrinsic (presence of calcium and phosphorous salts in case of using milk, cheese whey, and other derivatives) and extrinsic (acids or alkalis to control the pH during the enzymatic process, or added to adjust the broth

Fractionation of Peptides by Means of Ultrafiltration …

13

ionic strength that affects the membrane performance). These salts can be partially retained by UF/NF membranes (especially bivalent ions in case of NF), and they can be concentrated on the membrane surface. If the salts overpass their solubility limit, they can precipitate and foul the membrane. The risk increases when the membrane operation requires high recovery values. The term “membrane scaling” is used when this phenomenon takes place at a hard scale, and it is one of the reasons to stop the process and even to spoil the membrane, as severe scaling cannot be removed with intensive cleaning in some cases. Knowing the exact composition of the broth is essential to prevent membrane scaling. Special attention has to be paid to compounds such as CaCO3, CaSO4.2H2O, silica, Ca3(PO4)2, as well as aluminum and ferric hydroxides, owing to their low solubility. In protein solutions, some ions (

Ca 2 , PO 43 , and others) can associate to proteins, peptides and organic compounds (intermolecular bridges), changing their net charge and solubility, increasing fouling.

2.3.3. Biological fouling Biofouling is caused by the accumulation of bacteria (and fungi to a less extent) on the membrane surface. The enzymatic broths obtained after the enzymatic hydrolysis of protein sources have all the compounds and conditions that facilitate microorganism growth (sugars, organic matter, as well as moderate pH, temperature, and high water activity). If bacteria count grows, biofilms are formed on the membrane surface. These films can increase in importance for two reasons: the attachment of new bacteria present in the feed or by the growing of bacteria previously attached, which use the organic matter and other nutrients supplied in the feed to produce cell mass and other compounds. Biofouling is different to other fouling mechanisms mentioned above, because it can continuously increase (dynamic fouling) if prompt corrective actions are not taken. Aerobic microorganisms previously attached to the membrane surface beforehand, but anaerobic bacteria can grow in the internal layers. The presence of biofilms causes similar effects to inorganic and organic fouling, notably reducing the permeate flow rate of the membrane. Biofouling can be detected by taking samples of CP or by using destructive methods (membrane autopsy).

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Francisco Amador Riera, Ayoa Fernández, Claudia Muro

2.4. Fouling Prevention Some actions can be taken to reduce or delay fouling. Some of them are related to the ways of performing filtration, and others are linked with feed pretreatments or membrane modification. This last case is, however, out of the scope of this chapter. In general, hydrophobic membranes are more susceptible to fouling and adsorption than hydrophilic ones, and most of the membrane modifications have as objective to convert them into more hydrophilic surfaces. Membrane surface modification can be performed during manufacture, but some attempts have been made to modify them “on-line” by adsorbing monomers and derivatives of different natures. Thus, modified membranes tend fouled to be less extent by organic fouling agents, preventing them from entering the pores, but membranes usually lose flux (Lindau & Jönsson, 1999; Gilron et al., 2001).

2.4.1. Feed pre-treatments Feed pretreatments are always recommended when using “tight” UF and NF membranes, not only to delay fouling or to reduce CP, but to protect the membranes and to increase their lifetimes. Pretreatment can reduce solids and colloids that can damage membrane surfaces, as well as reduce biological fouling, scaling effect, and remove reactive compounds that can interact chemically with the membrane surface, modifying their permselective properties. Some indexes can be used in order to evaluate the opportunity of a pretreatment. Of them, the silt density index (SDI) is one of the most recommended (Boerlage et al., 2000). Determination of SDI is easy and only simple devices are required to measure it. Most NF membranes demand SDI values lower than 3. If the water used for protein solubilization contains a large amount of salts (high hardness), softening will be the best process to prevent fouling. The reduction of hardness (Mg+2 and Ca+2, mainly) can be achieved by precipitating these compound in their hydroxide- forms (generally by adding lime or soda ash). Ionic exchange is another alternative. Solids and colloids can be removed by MF pretreatment, using bag filters of dead-end filters depending on their concentration and size. The advantage of using MF (0.22 or 0.45 m) is the sterility of permeate, thus reducing the risk of biofouling in the following steps. With an adequate pore selection, the retention of proteins is negligible in the pretreatment. Other organic substances that can be adsorbed onto the membrane surface are difficult to be removed if proteins and enzymes are present, as substances

Fractionation of Peptides by Means of Ultrafiltration …

15

in the first group are usually smaller in size. Coagulation and the use of carbon-active columns cannot be used in these applications. Fouling due to organic compounds must be reduced by modifying the process conditions. Finally, biological compounds are also removed by means of MF, whereas treatment with chlorinated compounds, ozonation, and UV irradiation should be discarded, because these treatments can affect the protein and enzyme structures. Maintaining sterile conditions during all peptide production and fractionation processes is the best way to reduce or avoid biological fouling. MF of the feed is the best solution or, if the feed does not need any pretreatment, it should be sterilized prior to use.

2.4.2. Effects of process conditions During the fractionation process, there are a number of process parameters that must be optimized in order to increase the membrane selectivity to separate peptides, maintain the permeate flow rate as high as possible, and reduce fouling. The most important include lineal velocities on the membrane surface, transmembrane pressure, temperature, pH value, ionic strength, additives, and feed (protein) concentration. 



High lineal velocities on the membrane surface will reduce the CP and subsequent fouling, owing to the fact that, at turbulent regimes, the turbulence reduces the CP layer thickness and increases the masstransfer coefficient (k). High velocities are possible in tubular, plate and frame, and spiral-wound membrane configurations if adequate spacers are used. Common lineal velocities are in the range of 1–4 ms-1; values > 5ms-1 can be considered as high velocities. Velocities in plate and frame and spiral wound configurations are lower (0.5 – 3 ms-1. The turbulent regime implies the use of powerful pumps with increasing energy consumption. When high lineal velocities are not possible, concentration polarization and fouling can be minimized using different spacers and turbulence promoters. Increasing the transmembrane pressure in the pressure-controlled region, leads to a higher membrane permeate flux, but, at the same time, more solutes can be accumulated at the membrane surface by convection, so the risk of membrane fouling increases. In general, at higher TMP, salts are more retained when using NF membranes. The TMP values selected must be a compromise between these two aspects, and these TMP values should be lower than those corresponding to the critical flux.

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Francisco Amador Riera, Ayoa Fernández, Claudia Muro





In some equipments, constant permeate flux is the mode of operation. In those cases, the permeate flux is fixed according to the membrane and manufacturer recommendations (usually in spiral-wound membranes), and transmembrane pressure increases when the flux decays. The important point in these cases is not to exceed the limit permeate flow rate recommended. The fact that membrane technology usually works at mild temperatures is of great interest in biological applications. Some proteins can be denatured at medium/high temperatures. This aspect can be of great importance with some proteins, as is the case of bovine serum albumin (BSA), in which moderate heat treatment unfolds the protein, enhancing the enzymatic attack. In other cases, extreme heat treatment can first unfold proteins and then aggregate them, reducing the enzymatic hydrolysis degree. Temperature can slightly diminish the salt rejection, owing to the increase of the apparent activation energy associated with the solvent-transport process (Arrhenius equation) (Drioli & Giorno, 2010), but it increases the permeate flux (owing to a decrease in viscosity and increase in diffusivity of fluids), thus reducing the necessary membrane area. Another effect of temperature is related to salt precipitation on the membrane surface. Most salts increase their solubility with temperature and, thus, the risk of precipitation diminishes. If the broth contains inverse solubility salts (such as Na2SO4), this effect must be taken into account. Finally, working at mild temperatures has an influence on microorganism growth, and biofouling can occur throughout the experiments. Most of the processes of peptide fractionation take place at temperatures similar to those used in the hydrolysis steps (between 30 and 60°C). High pH values are preferred to diminish the organics, and the protein and peptide fouling. The reason for this behavior is the negative charge that most membranes have at these pH values. Then, the electrostatic repulsion between solutes and membrane surfaces reduces the fouling risk. Fortunately, most peptidases have optimum conditions at neutral or alkaline pH. Some of the enzymes used in protein hydrolysis and the optimum conditions are shown in Table 3.

Fractionation of Peptides by Means of Ultrafiltration …

17

Table 3. Most common enzymes used in peptides production* Enzyme Trypsin Alcalase® Pepsin A Chymotrypsin Papain Carboxypeptidase Proteinase K * Several sources. 





pH interval (optimum) 7,5-8,5 6-10 (7,5) 1-4 (1,5) 8-9 (7,8) 6-7 7-8 7,5-12 (7,5-9)

Temperature (ºC) 30-60 (37) 50-60 (60ºC) 37 45-55 (50) 65 20-60 (37)

Molecular Weight (kDa) 23,3 27 34,6 25 23,4 28,9

The influence of pH on membrane selectivity and permeability is related with possible interactions between solutes and membrane surfaces. Proteins and peptides have low charge density (or no charge at all) at pH values similar to their IP, decreasing their solubility and enhancing CP formation. At high pH, the rejection of negative peptides increases. Peptides with a MW higher than the membrane pore sizes are always retained by the membranes, but some with a lower MW can also be retained (even completely), due to electrostatic repulsion. The effect of pH can also be taken into account if salts are in high concentrations, as they can easily precipitate at alkaline pH (generally in hydroxide forms). The prediction of rejection in NF and tight UF for charged solutes is difficult, as different mechanisms (such as hydrodynamic convection, particle–wall interactions, Donnan effects, hydrophobicity /hydrophilicity) can play important roles. In fact, there are no models to predict transport through membranes in which pH is included as a parameter. Ionic strength can play an important role in membrane selectivity when fractionating peptides. On the one hand, the added salts behave as inorganic compounds with respect to fouling and scaling and, on the other hand, when the feed is diluted, the salts can surround charged proteins, increasing their effective radius and changing their transmission through the membranes. Low ionic strength can enhance size differences between peptides. Sometimes different antiscaling agents are added to the feed in order to avoid salt precipitation and fouling. Some classic antiscaling compounds are sodium hexametaphosphate (SHMP), phosphonates,

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Francisco Amador Riera, Ayoa Fernández, Claudia Muro



and polymers such as polyacrylic acid and polysulfonates, as well as their derivatives. Antiscalants must be selected once a deep knowledge of the feed salt composition has been obtained. On the other hand, the possibility of interactions of these compounds with the membrane surface and the solutes in the feed must be considered, apart from the effects of contamination of permeate or concentrate. Generally, hydrolysis broths do not contain too many salts that can precipitate, and antiscalant addition is not usually necessary. The first consequence of increasing the feed concentration is to increase the osmotic pressure and, then, the effective TMP is reduced. Most of the models proposed to predict solute retention use diluted real or synthetic solutions. At higher concentrations, the effects of charges and hydrophobicity/hydrophilicity are masked by the presence of the CP, as it acts as a real dynamic second membrane, affecting the permeate flow rate as well as the rejection properties (Fernández & Riera, 2012). From an industrial point of view, a concentrated feed is recommended, as the productivity increases but all problems mentioned before (concentration polarization, fouling, etc.) will be noticeably increased.

2.5. Membrane Cleaning Sooner or later, the membranes must be cleaned. The cleaning frequency and the detergent formula used depend on each application. When treating low polluted feeds, it is possible to work several days without cleaning. In the food industry and in biotechnological applications, cleaning must be performed daily, either because some processes can be developed in discontinuous ways or due to the risk of bacterial growth. Cleaning methods can be of different natures, including physical, chemical, enzymatic, addition of biocides, and so forth. Despite the number of research articles published on this topic, many times, selecting adequate cleaning methods and conditions is based on a trial-and-error approach. It is important to know the possible causes of fouling (type of foulants), and, if this is not possible, methodologies based on statistical planning (factorial designs) represent a good way to get quick information about the best cleaning method. Cleaning conditions and agents must be adapted to each foulant and to the membrane characteristics.

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Physical cleaning: Different techniques, such as back washing, back flushing, scrubbing, two-phase (liquid–gas) cleaning, vibration, and sonication, have been assayed with different membrane geometries, materials, and feeds. In general, physical cleaning is not sufficient to recover original membrane permeability, but, in some cases, the working time can be reasonably extended. On the other hand, some of these techniques, such as vibration and sonication, are not compatible with some membrane materials (ceramic membranes), as the active layer can be damaged. The physical methods are useful to remove the CP temporarily and part of the non-adsorbed fouling, but are less efficient at removing fouling inside the pores (Schäfer, et al., 2005). Among the physical methods, rinsing can be included as a previous and intermediate step in chemical or enzymatic cleaning methods. Cabero et al. (1999) demonstrated the effectiveness of rinsing performed in two steps (1. close permeate valve, 2. open permeate valve) when cleaning membrane fouled with protein solutions. Chemical cleaning: Detergent ingredients react with the soil components (fat saponification, protein and peptide hydrolysis, salt solubilization, etc.) in different ways. Most detergents contain an alkaline (NaOH) or acid component (ClH or HNO3) as well as surfactants (SDS or others) to disperse the fouling agents and sequestering agents (EDTA and others) to remove multivalent ions and to avoid foulant re-deposition. In general, alkaline agents are designed to remove organic compounds, as is the case for proteins and peptides, and acid cleaners are recommended for removing inorganic fouling. In both cases, the detergent solutions show extreme pH values (between 1 and 12) that should be compatible with membrane materials. Transmembrane pressure must be moderate during the cleaning step, as high pressures can push the foulants inside the pores; additionally, high pressure during cleaning can compact the fouling layer (Razavi et al., 1996). The efficiency of cleaners is increased with temperature, but special attention should be paid to the membrane tolerance. Some membranes can withstand high temperatures during short periods of time (30–45 min), but they can be damaged if contact at extreme pH and/or temperature is excessive. For protein and peptide fouling, the alkaline cleaning is the most effective, and acid cleaning is only used after several alkaline cycles if membrane permeability is not recovered. Enzymatic cleaning: There has been a strong development in the production of a wide variety of enzymes at competitive prices that are tolerant to large pH ranges and temperatures intervals, which has led to great interest in their use as membrane cleaners. Protease- and lipase-based enzymatic cleaners can remove fouling of proteins/peptides and fats. Enzymes show, in some

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Francisco Amador Riera, Ayoa Fernández, Claudia Muro

cases, high specificity and can be useful when the exact foulant is known. The addition of biocides (as bisulfite) is recommended to control bacteria growth, especially when there is no processing period. The efficiency of the cleaning methods can be measured by determining the value of either the water permeate flux after cleaning (Jw) or the membrane resistance (Rm). After each step in the cleaning method, values of R should decrease (Figure 2). When Jw ≥ 95% of the initial value, the cleaning process is considered finished.

Rm: Membrane resistance; Ruf: Total resistance after fouling; Rcw: total resistance after cleaning; Rres: residual resistance; Rif: Resistance due to fouling; Rrf: resistance due to reversible fouling. Figure 2. Evolution of hydraulic resistances during fouling, rinsing and cleaning steps (Argüello et al., 2003).

Finally, the recovery or regeneration of cleaning solutions is another aspect to keep in mind for either environmental or economic reasons (Dresch et al., 1999; Gésan-Guiziou et al., 2002). Detergent concentration is generally lower than 2% w/w, and the water consumption in these steps is very high (Fernández & Riera, 2010). In some cleaning in place (CIP) systems, the detergents are discharged, maintaining residual activity as some detergent compounds can be recovered by means of UF and NF processes (Suárez et al., 2013). Additional information about the methodologies for recovering water and detergent compounds can be seen in the report by Suárez & Riera (2012).

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3. BIOACTIVE PROPERTIES OF FOOD PROTEIN-DERIVED PEPTIDES Bioactive peptides have been defined as specific protein fragments that have a positive impact on body functions and may, ultimately, influence health (Kitts & Weiler, 2003). Although some food proteins are able to show physiological functions in their intact form, bioactive peptides are not usually active within the sequence of the parent protein. However, they can be released through proteolysis by means of enzymes, be it through digestive enzymes or enzymes derived from microorganisms or plants. The range of functionalities attributed to food protein-derived bioactive peptides is diverse. Thus, for example, although there is plenty of information about food-derived peptides showing antihypertensive effects, both in vitro and in vivo, antiallergenic or immunomodulatory peptides have also been described. Table 4 summarizes the main beneficial pharmacological properties attributed to bioactive peptides that are released from food proteins, and the possible effect on different body systems and diseases. Bioactive peptides with properties relevant to cardiovascular health have been broadly investigated during the last two decades. Cardiovascular disease (CVD) is one of the leading causes of death in highly developed countries, and, apart from therapeutic drug regimens focused on its treatment, special attention has been paid to diet ingredients that may contribute to the prevention of causative risk factors such as high blood pressure, obesity, and oxidative stress. Hypertension is one of the major independent risk factors for developing CVD and a key element for the control of CVD-related mortality. Bioactive peptides that can interfere in the renin-angiotensin system (RAS), that is, the main system in human body responsible for blood pressure control, are among the most extensively studied peptides in the nutraceutical field. In this sense, there are two common targets in the RAS pathway: angiotensin-I-converting enzyme\9ace0 and renin. The inhibition of any of these enzymes will potentially reduce blood pressure. A number of peptides derived from food proteins have been said to exert a beneficial effect on high pressure, at least in vitro, by inhibiting ACE, which catalyzes the conversion of angiotensin-I to angiotensin-II (a potent vasoconstrictor). Milk proteins are by far the most studied substrate for the release of ACE-inhibitory peptides or, as they are also known, lactokinins (Cadee et al., 2007). Not only casein (Yamamoto et al., 1994, Maeno et al.,

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1996, Pihlanto-Leppälä et al., 1998, Mizuno et al., 2005, Jiang et al., 2010) and whey protein (Abubakar et al., 1998, Hernández-Ledesma et al., 2002) hydrolysates, but also fermented milk products (Yamamoto et al., 1999, Leclerc et al., 2002), are a source of ACE inhibitory peptides. In addition, ACE inhibitory peptides have been identified in a broad variety of food protein hydrolysates, such as fish and shellfish (Hardeny & FitzGerald, 2012, Kim et al., 2012), eggs (Liu et al., 2010), soybeans (Back et al., 2010), legumes (Boschin et al., 2014), and meat (Saiga et al., 2003, Jang and Lee, 2005). The mechanism of action of ACE inhibitory peptides has been found to be via competitive inhibition, that is, the peptides compete with the ACE substrate for the enzyme catalytic sites (Sato et al., 2002). The amino acid sequence of a given peptide and its length are the main parameters to influence its potential ACE inhibitory effect. Short-chain sequences, with hydrophobic amino acid residues close to the C-terminal position, especially Pro, are important structural features for potency. In addition, N-terminal aliphatic amino-acid residues such as Leu, Phe, and Tyr are necessary structural features for di- and tripeptides that exhibit ACE inhibition (Wu et al., 2006). Thus, for example, two of the most potent ACE inhibitory peptides derived from milk proteins, IPP and VPP, meet those characteristics. ACE inhibition is not the only possible pathway that has been studied for hypertension therapy. Some recent studies have demonstrated the inhibition of renin activity by the action of food-protein-derived peptides. The first study on this topic was carried out by Udenigwe et al. (2009), who reported the inhibition of human recombinant renin in vitro by a flaxseed protein hydrolysate. Subsequent studies on renin inhibition have been published using tuber (Ajibola et al., 2013), chicken skin (Onuh et al., 2013), pea and hemp seeds (Girgih et al., 2011), and seaweed hydrolysates (Fitzgerald et al., 2012). Interestingly, most of the hydrolysates and peptides displaying renin inhibition have dual roles, as they also inhibit ACE. Apart from ACE and renin inhibition, bioactive peptides can potentially exert an antihypertensive effect by means of other mechanisms, including angiotensin-II receptor blocking (Yu et al., 2014), calcium channel blocking (Tanaka et al., 2008), acting over the kinin–nitric oxide system (Nurminen et al.,2000; Sipola et al.,2002), and inhibiting the endothelin-converting enzyme (Okitsu et al., 1995).

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Table 4. Physiological/Pharmacological properties of some peptides Physiological / Pharmacological properties Antihypertensive activity ACE-inhibition Renin-inhibition Other mechanisms Antioxidant activity Antithrombotic activity Hypocholesterolemic activity Lipid-lowering activity Anti-appetizing activity Opioid-like activity CKK-release induction Other mechanisms Mineral binding capacity (Calcium-binding) Antimicrobial activity Anti-inflammatory activity Antiproliferative effects Hypoglucemic activity DPP-IV inhibition

Effects on: Cardiovascular system

Prevention / Treatment: CVD

Cardiovascular system Cardiovascular system Cardiovascular system Cardiovascular system Cardiovascular system, digestive system, nervous system

CVD, Cancer CVD CVD CVD CVD

Digestive system

Osteoporosis, caries Infections

Digestive system, Immune system Immune system

Arthritis, colitis, etc.… Cancer Diabetes

Although most of the work has been done so far with in vitro tests, a number of in vivo studies have confirmed the potential hypotensive effect of some food-protein-derived peptides. In a study published in 1995, the famous casein-derived peptides IPP and VPP (active ingredients of the commercial products Calpis and Evolus) were reported to reduce blood pressure in spontaneous hypertensive rats (SHR) after oral administration (Nakamura et al., 1995). In addition, Hata et al. (1996) observed a decrease in blood pressure, both systolic and diastolic, in middle hypertensive human subjects after Calpis intake. Moreover, the consumption of peptide IPP has also been proven to reduce blood pressure in Caucasian subjects with stage one hypertension (Boelsma &Kloek, 2010). BioZate, a whey protein hydrolysate commercialized by Davisco Foods Int., has also been subjected to human intervention trials, in which it exhibited hypotensive activity in hypertensive human volunteers (Pins & Keenan, 2002). Despite the favorable results

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Francisco Amador Riera, Ayoa Fernández, Claudia Muro

dispatched by these studies, not all human intervention studies have been so successful. Thus, for example, two recent studies have not found any significant blood-pressure-lowering activity of lactotripeptide-containing products that were administrated to pre-hypertensive and hypertensive subjects following 24 h ambulatory monitoring studies (Van Mierlo et al., 2009; Usinger et al., 2010). Oxidative stress, that is, an increase in the production of reactive oxygen species (ROS), is, in combination with outstripping endogenous antioxidant defense mechanisms, a significant causative factor for the initiation or progression of several vascular diseases. ROS can cause extensive damage to biological macromolecules such as DNA, proteins, and lipids. Dietary consumption of antioxidants appears to provide extra benefits to the endogenous antioxidant defense strategies in the fight against oxidative stress (Fang et al., 2002). Owing to the complexity of oxidative reactions in biological systems, several cell-free in vitro methods have been developed to measure the antioxidant potential of food-derived peptides [see the review by Niki (2010)]. These methods are useful for preliminary screening, but they do not offer any information about the relevance of potential antioxidants in biological systems. Cell-based assays have been extensively used recently. These methods are an intermediate step between the aforementioned cell-free methods and in vivo studies and they allow the evaluation of the antioxidant effect of a given peptide/hydrolysate, the elucidation of the mechanism of action of the peptides within cells, and they are useful for the determination of the necessary dose for a beneficial antioxidant effect. Finally, only a few in vivo models with antioxidant peptides have been conducted to date (Manso et al., 2008; Xue et al., 2009). A number of food protein hydrolysates and peptides have shown antioxidative properties, such as scavenging or quenching of ROS/free radicals, inhibition of ROS-induced oxidation of biological macromolecules including lipids, proteins, and DNA, transition-metal chelating, and ferricreducing power (Udenigwe & Aluko, 2012). As in the case of antihypertensive effects, milk proteins and their derived peptides have been extensively studied for antioxidant activity (Suetsuna et al., 2000; Kansci et al., 2004; Contreras et al., 2011; Zhang et al., 2013; Di Pierro et al., 2014). The hydrolysis of food proteins from marine organisms such as blue mussels (Wang et al., 2013), salmon (Girgih et al., 2013), and other fish species (Harada et al., 2010; Ko et al., 2013; Farvin et al., 2014) have also been shown to release peptides with different antioxidant activities. The potential use of plant extracts as antioxidants is well known, owing to the abundant presence of polyphenolic

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compounds. However, there is also some evidence highlighting the significance of plant-derived peptides as novel antioxidant agents (Li et al., 2008; Zhang et al., 2010; Zhou et al., 2012). Antioxidative activity is not only related to the prevention of cardiovascular disease, the use of food-derived peptides for food preservation has also generated great interest in the food industry (Elias et al., 2008). In addition, the application of antioxidative peptides to prevent and control cancer, which may be a consequence of oxidative DNA damage, has been studied (Phelan et al., 2010). Despite the big effort made during recent years to study antioxidants, including peptides and other compounds, the European Food Safety Authority (EFSA) has not yet approved any health claim in this area, owing to a lack of evidence for the potential health benefits of antioxidants. High cholesterol is one of the main factors leading to heart disease. The enzymatic hydrolysis of food proteins can also release amino-acid sequences that possess cholesterol- and lipid-lowering activities. It is well known that dietary intake of soy protein reduces circulating triglycerides and cholesterol levels, but different soy hydrolysates have also been reported to have a stronger serum cholesterol-lowering effect than intact soy proteins (Sugano et al., 1990; Zhong et al., 2007). Those soy protein peptides exhibit hypocholesterolemic activity by altering gene expression (Mochizuki et al., 2009) by binding bile acids and neutral sterols in the intestine, leading to increased fecal removal (Cho et al., 2007). Although most previously published papers on lipid-lowering peptides have focused on soy proteins and hydrolysates, other protein sources such as milk (Nagaoka et al., 2001; Kirana et al., 2005), buckwheat (Kayashita et al., 1997), egg whites (Manso et al., 2008), chickpeas (del Mar et al., 2012), and fish (Wergedahl et al., 2004) have also demonstrated the release of hypocholesterolemic peptides. Another functionality related to the cardiovascular system is antithrombotic activity. Caseinomacropeptide, the C-terminal fragment of bovine κ-casein, contains peptides that inhibit both the aggregation of ADPactivated platelets as well as the binding of human fibrinogen γ-chain to its receptor region on the platelet surface (Jolles et al., 1986, Maubois et al., 1991). In particular, the docapeptide MAIPPKKNQDK has shown positive results in vivo and could potentially be used to treat or prevent thrombosis (Maubois et al., 1991). Peptides isolated from a pork-meat papain hydrolysate have also shown antithrombotic activity (Shimizu et al., 2009). Obesity is a serious health issue in highly developed countries, where it is usually associated with a higher incidence of CVD. Proteins are well known to

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be the most satiating macronutrient (Anderson & Moore, 2004). Apart from that, some peptides codified in the primary sequence of food proteins contribute to the initialization of satiety signals (Pupovac & Anderson, 2002). Two of the main sources of anti-appetizing peptides studied to date are soya proteins and caseins. Thus, for example, residue 51–63 of β-conglycinin acts as an appetize suppressant, interacting directly with the intestinal mucosal cells to stimulate cholecystokinin (CKK) release (Nishi et al., 2003). In the case of caseins, peptides such as casomorphins and caseinomacropeptides control food intake through different mechanisms. Casomorphins, which are opioid-like peptides, interact with gastric opioid receptors to slow gastrointestinal motility, preventing further food intake (Daniel et al., 1990). In addition, they are also able to modify the postprandial levels of metabolic hormones involved in satiety (Schusdziarra et al., 1983). Caseinomacropeptides have shown both opioid- and CKK-release activities (Pedersen et al., 2000). Apart from these mechanisms, a number of peptides have been claimed to stimulate satiety through other pathways [see the review by Erdmann et al. (2008)]. In addition to its potential use as a satiety-promoting compound, caseinomacropeptide has also been reported to interact with some of the functions connected to the gastrointestinal system. Thus, it seems to contribute to an anticariogenic effect by inhibiting the adhesion and growth of plagueforming bacteria in oral mucosa (Brody, 2000), and it has been used as an ingredient in some dental care products. This κ-casein-derived peptide may have a beneficial role in modulating the gut microflora, owing to its carbohydrate content (Manso & López-Fandiño, 2004), but its main nutritional role is derived from its use as an ingredient in diets designed for people suffering from phenylketonuria, that is, those who are unable to metabolize phenylalanine (Marshall, 1991). In addition to this particular peptide, other milk-derived peptides display a variety of activities connected to the gastrointestinal system. Thus, for example, caseinophosphopeptides (CPPs) could potentially lead to the enhancement of mineral (primarily calcium) absorption, and they have been considered to be beneficial in the prevention of osteoporosis, dental caries, hypertension, and anemia [see the review by Korhonen & Pihlanto (2006)]. However, animal and human studies investigating the effect of CPPs on calcium absorption have shown conflicting effects (Yuan & Kitts, 1991; Narva et al., 2003). Antimicrobial peptides, displaying a broad range of activities against Gram-positive and Gramnegative bacteria, have been identified in milk proteins [see reviews by Clare et al. (2003) and Pellegrini (2003)], with the most studied being the

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lactoferricins. In addition, fish proteins [see the review by Najafian & Babji (2012)] and egg proteins (Abdou et al., 2007) release antimicrobial peptides. As mentioned before, milk proteins are a source of opioid peptides. The effects of these peptides in the nervous system is not only limited to appetite satiety, but also include respiratory depression, behavior modulation, and gastrointestinal mobility, depending on the opioid receptors involved in the process (Teschemacher et al., 1997). Generally, the opioid peptides released from α- and β-casein elicit agonist responses, including inhibition of diarrhea, prolongation of gastrointestinal transit time, modulation of amino-acid transport, and prolongation of analgesia. On the other hand, opioid peptides released from κ-casein, also called casoxins, rise to antagonist responses, potentially leading to appetite regulation and modulation of insulin secretion by the pancreas (Rutherfurd-Markwick & Moughan, 2005). It is not only milk that has been explored as a raw material for the production of opioid peptides. Other protein sources such as cereals (Zioudrou et al., 1979; Fukudome & Yoshikawa, 1992) have been explored for this purpose. Bioactive peptides have also shown immune-related functions, as they can act as regulators of cytokine expression in antibody production (Meisel & FitzGerald (2003). Mercier et al. (2004) reported the presence of immunomodulating peptides in whey proteins, which are able to increase the proliferation of lymphocytes. Takahashi et al. (1994) reported the influence of rice protein digestion on the improvement of different immune functions, and Mine & Kovacs-Nolan (2006) supported the use of egg-derived peptides during cancer immunotherapy. In addition, the effect of immunomodulatory peptides derived from whey proteins on the newborn immune system has also been studied (Gill et al., 2000), highlighting the potential use of these peptides in infant formulas. Inflammation is a defensive reaction triggered by harmful stimuli of different natures (of physical, chemical, and pathogen origin). It is characterized by increased endothelial permeability, leakage of protein-rich exudates, and infiltration of leukocytes into extravascular tissues (Chakrabarti et al., 2014). Although inflammation has a key role in the prevention of microbial infections and wound healing, uncontrolled anti-inflammatory changes often lead to health problems and chronic diseases such as atherosclerosis. The anti-inflammatory activity of food-derived bioactive peptides has mainly been proven in vitro for mammalian cell lines. In this sense, milk- and soya-derived peptides have been studied the most, to date. Casein-derived peptides have shown anti-inflammatory effects on activated macrophages (Nielsen et al., 2012) and in leukocyte–endothelial interactions

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in vitro (Aihara et al., 2009). In addition, whey protein hydrolysates have shown promising results in respiratory and intestinal epithelial cells (Piccolomini et al., 2012; Iskandar et al., 2013). Lactoferricins, lactoferrinderived peptides, have also demonstrated potential anti-inflammatory effects in arthritis management (Kim et al., 2013; Yan et al., 2013). Eggs (Huang et al., 2010; Majumder et al., 2013), fish (Fitzgerald et al., 2005), and casein hydrolysates have also been reported to show anti-inflammatory effects in vitro. As for in vivo effects, models including experimentally induced colitis in mice (Hatori et al., 2008; Marchbank et al., 2009; Mochizuki et al., 2010; Chatterton et al., 2013; Pescuma et al., 2013) are the most frequently studied. Peptides with anticancer activity in cell systems have also been reported from some foods. The most famous example is lunasin, a soybean-derived peptide that has shown anticancer activity against chemical and viral oncogene-induced cancers (Wang et al., 2008; Hernández-Ledesma et al., 2009). Despite the promising results obtained in cell assays, the large molecular size of this peptide may be a drawback in its absorption and use as an oral-health-promoting agent (Dia et al., 2009). Other soy-protein-derived peptides have shown anticancer activities against leukemia cells (Wang et al., 2008) and macrophage-like murine tumor cells (Kim et al., 2000). In addition, peptides from dark-muscle byproducts of tuna (Hsu et al., 2011) and oysters (Wang et al., 2010) have been reported recently. Detailed animal and human trials are needed in order to evaluate and validate the anticancer activities of these peptides. Food protein hydrolysates and peptides may be useful functional ingredients for the management of type-2 diabetes. In this sense, several peptides derived from milk proteins (Nongonierma & FitzGerald, 2013) and βlg (Lacroix & Li-Chan, 2012) have been shown to inhibit the action of DPPIV, an enzyme that can cleave incretin hormones responsible of glucose homeostasis. In addition, particular peptide sequences have been shown to exert more than one biofunctionality. Power et al. (2014) reported the complimentary functionality of β-lg peptides (namely antioxidant, ACE, and DPP-IV inhibition) and suggested the use of these multifunctional peptides in a preventative strategy for the dietary management of increasingly prevalent diseases such as hypertension and type-2 diabetes. Despite a large worldwide effort by the research community in the field of bioactive peptides during recent decades, more in vivo and long-term human intervention studies are needed in order to clarify the real effects of these functional preparations.

Table 5. Research studies involving conventional membrane fractionation processes for the fractionation of protein hydrolysates containing bioactive peptides Protein source Bovine caseinomacropeptide Whey proteins Goat whey Bovine α-la and β-lg

Biological activity Antithrombotic

Dried bonito Soy protein

ACE-inhibition ACE-inhibition

Soy proteins: βconglycinin and glycinin Alfalfa white protein Alfalfa leaf protein

ACE-inhibition

Wheat gluten

Antioxidant

Fish protein

Antioxidant and ACE-inhibition

ACE-inhibition ACE-inhibition ACE-inhibition

ACE-inhibition Antioxidant

Fractionation process Enzymatic membrane reactor

Enzymatic membrane reactor Two steps UF process (30kDa + 1kDa membranes) after hydrolysis UF through 3kDa MWCO membrane Fractionation of the hydrolysate by means of different UF membranes (MWCO: 20, 10 and 5 kDa) followed by further fractionation on a cationic exchange resin UF through 10, 3 and 0,5 kDa Enzymatic membrane reactor Single UF step through 3kDa polysulphone membrane Fractionation of the hydrolysate through three UF membranes with different MWCO: 10, 5 and 3 kDa Successive fractionation on UF and NF membranes

Reference Bouhallab & (1995a) and Bouhallab et al., (1995b) Mullally et al. (1997) Bordenave et al. (1999) Pihlanto-Leppälä et al. (2000) Fujita et al. (2001) Wu & Ding (2002)

Kuba et al. (2005) Kapel et al. (2006) Xie et al. (2008) Kong et al. (2008)

Picot et al. (2010)

Table 5. (Continued) Protein source Whey proteins

Biological activity DPP-IV inhibition

Soy proteins

Soy protein

Antioxidant and muscle glucose uptake Antioxidant

β-lg

Immunomodulation

β-lg

Antioxidant, ACEinhibition and DPP-IV inhibition ACE-inhibition and ferrus chelating activity ACE-inhibition

Whey proteins

Whey proteins

Fractionation process Fractionation of the hydrolysate through 5 and 2 kDa membranes Fractionation of the hydrolysate using hollow fiber or spiral-wound membranes

Reference Nongonierma & FitzGerald (2013) Roblet et al. (2012)

Sequential UF and NF

Ranamukhaarachchi et al. (2013) Rodríguez-Carrio et al. (2014) Power et al. (2014)

Fractionation of the hydrolysate using 5, 2 and 1 kDa organic membranes Fractionation of the hydrolysate using 5, 2 and 1 kDa organic membranes Pilot-plant scale fractionation via membrane cascade (0,14μm and 30, 10, 5 and 1 kDa membranes) Fractionation of a 1kDa fraction by membrane cascade

O'Loughlin et al. (2014a)

O'Loughlin et al. (2014b)

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4. FRACTIONATION OF PEPTIDE MIXTURES USING PRESSURE-DRIVEN MEMBRANE TECHNIQUES Many different techniques have been used for the fractionation of peptide mixtures and the enrichment of bioactive peptides. However, pressure-driven membrane techniques such as UF and NF seem to be the most promising techniques, mainly due to their low processing costs and ease of scale up. Thus, the publication of research results in this field has been a constant hot topic during the last two decades. Table 5 summarizes the research studies, in which UF and NF have been used for the fractionation of bioactive protein hydrolysates, be it as an independent separation step or combined with enzymatic proteolysis in a single-operation enzymatic membrane reactor (EMR). Two of the first studies to deal with the application of UF membranes to the production of preparations enriched in bioactive peptides were published in 1995 (Bouhallab). In both cases, the separation process and the enzymatic hydrolysis of caseinomacropeptide were combined in a single-unit EMR. Two years after, Mullally et al. (1997) suggested the use of 1 and 3 kDa UF membranes to enrich the ACE inhibitory peptides present in different β-lg and WPC digests. Pihlanto-Leppälä et al. (2000) also confirmed the improvement in terms of ACE inhibition activity, obtained after the fractionation of different whey protein hydrolysates, using a membrane cascade consisting in 30 and 1 kDa MWCO membranes. As a result, permeate ACE inhibitory activity obtained through the 1 kDa membrane, was, in many cases, higher than that observed in the other fractions tested. UF has also been reported to increase the DPP-IV inhibitory activity of a whey protein hydrolysate (Nongonierma & FitzGerald, 2013). The permeates generated by UF through 5 and 2 kDa membranes were significantly more potent DPP-IV inhibitors than the whole whey protein hydrolysate. Power et al. (2014) also reported a selective enhancement of the biofunctional properties of a β-lg hydrolysate when filtered through a 1 kDa polyethersulfone membrane. The permeate sample showed antioxidant activity that was 1.7 times higher, as well as a threefold decrease in IC50 for DPP-IV inhibition and a twofold reduction in IC50 for ACE-inhibition. Data about the immune effect of these samples supported the use of membrane techniques for obtaining immune bio-enriched fractions (Rodríguez-Carrio et al., 2014). Most previously published studies used lab-scale conditions to perform the fractionation. However, there are also some interesting results at pilot-plant

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scale, which suggest the good performance of UF and NF separation processes under industrial-scale conditions. Thus, for example, O´Loughlin et al. (2014a) fractionated a 400 L whey protein hydrolysate using membrane cascade and reported an increase in bioactivity of the 1 kDa permeate fraction. Milk and whey proteins have been the most studied source of bioactive peptides and, by extension, the most used raw material in studies involving membrane fractionation of protein hydrolysates. However, there are also studies that deal with other protein sources. Enzymatic hydrolysis of fish and shellfish proteins has been reported to release bioactive peptides. In this sense, Fujita et al. (2001) improved the ACE inhibitory activity of a dried bonito hydrolysate when filtering the original formulation through a 3 kDa UF membrane. The new product showed antihypertensive activity in the spontaneously hypertensive rats after oral administration that was two times higher than the original hydrolysate. Results were also supported by a human intervention study. In a different study, Picot et al. (2010) used UF and NF membranes to refine fish hydrolysates. However, this process was not as successful and the biological activity of the obtained fractions was not increased in comparison with the starting hydrolysate. Membrane technology has also been used for the fractionation of soy protein hydrolysates to produce ACE-inhibitory food ingredients (Wu & Ding, 2002). Kuba et al. (2005) used UF membranes as part of a complex fractionation process directed towards the purification and identification of ACE inhibitory peptides. In another study, Kapel et al. (2006) described the production of ACE inhibitory-peptide preparations from alfalfa white protein at pilot-plant scale. In this case, two UF ZrO2 mineral membranes modules of 10 kDa MWCO were employed during the fractionation step. Alfalfa was also the protein source used by Xie et al. (2008) to produce antioxidant peptides. In this case, the alfalfa leaf hydrolysate was filtered through a 3 kDa polysulfone membrane. The peptides present in permeate were shown to possess interesting antioxidant activities. Kong et al. (2008) employed three membranes with different MWCO (10, 5 and 3 kDa) to fractionate a wheat gluten hydrolysate, permeate obtained with the 3 kDa membrane displayed the best antioxidant properties. Although membrane separation techniques have widely been used for the improvement of the bioactive properties of peptide mixtures, the optimization of the fractionation process is not usually included amongst the main goals of most of the aforementioned studies. Only a few authors have focused their objectives on the study of the mechanism behind the fractionation of complex peptide mixtures or protein hydrolysates. However, understanding how this separation takes place and the mechanisms governing the processes may result

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in an improvement of the final results, in terms of bioactivity and efficient use of membrane techniques. Table 6 summarizes fundamental research papers focused on the fractionation of amino acids and simple peptide mixtures by means of UF and NF. Although these studies do not usually involve complex peptide mixtures, they should be taken into account as a starting point to gain insights into the membrane fractionation process of complex hydrolysates. According to the studies included in Table 6, the molecular weight of the peptides, their charge, and, ultimately, their hydrophobicity influence the transmission of peptides through UF and NF membranes. Understanding how these parameters can be slightly modified, at least apparently, by small changes in filtration conditions may lead to more selective fractionation processes, in terms of peptide separation. As mentioned in the first part of this chapter, pH value and the ionic strength seem to be amongst the most important parameters influencing membrane selectivity when working with peptides. In addition, the concentration of the feed solution and the type of membrane used for each specific application (material and cutoff) have an important role in the fouling phenomenon and in the formation of the polarization layer, especially in the case of protein hydrolysates and complex peptide mixtures. Recent research studies clarified the effect of each one of the previous parameters, using a β-lg tryptic hydrolysate as an example (Fernández & Riera, 2012, 2013a). Peptide transmission has clearly been shown to depend on the pH value of the feed solution (Fernández et al., 2013a). Thus, the highest transmission values for different peptides have been found when the pH of the mixture is close to the isoelectric point of the peptide (see Figure 3). Repulsive electrostatic interactions between the peptide and the membrane are minimized, making it easier for the peptide to approach the membrane surface. The progressive decrease in peptide transmission is especially significant as the peptides acquire negative charge, that is, as the pH of the feed solution increases from the PI of a given peptide to high pH values. This is mainly attributed to the formation of repulsive electrostatic interactions between the peptides and the membrane surface, which has a negative charge at pH values higher than 5, in most cases.

Table 6. Pressure-driven membrane studies involving amino acids and peptides (adapted from Muro et al., 2013) Reference Tsuru et al. (1994)

Solution Single AA solutions mixtures of dipeptides

 pH variation experiments  Separation experiments of mixed dipeptides

Garem et al. (1997)

Mixtures of AA

 Separation of a mixture of 9 AAs on the basis

Martin-Orue et al. (1998)

-Single AA solutions -AA mixtures -Peptides (from protein hydrolysate) -Single AA solutions -AA mixtures

Timmer et al. (1998)

Experiments

of differential electrostatic interactions with the membrane  Membrane selectivity as a function of pH, AA concentration and Ionic Strength  NF of charged AA (single solutions and

mixtures) and peptides(similar MW but different pI)  Influence of concentration and ionic

composition (salt concentration and kind of salt added) on single AA retention.  Separation of AA mixtures

Membrane Flat-sheet membranes Materials: PA, TFC, SPES and SPS. MWCO: 0.2-3 kDa Charge at pH 7: negative (SPES, SPE and TFC) or amphoteric (PA) Material: Inorganic membrane, chemical modification of the ZrO2 layer of a UF membrane with cross linked PEI Charge: positive Material: ZrO2 filtering layer on a mineral support Charge: weakly negative charge at pH 8.0 Material: cellulose acetate, SPES, SPS and PS MWCO: 35-45% (NaCl retention), 1 kDa, 3, 6 kDa respectively

Reference Garem et al. (1998)

Solution Protein hydrolysate

Pouliot et al. (1999)

Protein hydrolysate

Grib et al. (2000)

-Single AA solutions -AA mixtures

Experiments  Separation of a mixture of 10 small peptides  Influence of physicochemical conditions (ionic

strength and pH) on the fractionation (permeate flux and Tr)  Effect of adjusting pH and ionic strength in the fractionation of the hydrolysate.

 Influence of experimental conditions on the

steady-state regime.  pH effect on retention coefficients of single

AA solutions and AA mixtures.  Influence of ionic strength and transmembrane

Wang et al. (2002)

AA mixtures

pressure on retention coefficients of an AA mixture  Separation performance of two different NF membranes.  Influence of pH and operation pressure on the selectivity of the separation.  Simulation NF process system for separation and concentration of L-Phe and L-Asp

Membrane M5+PEI: ZrO2 modified with PEI Kerasep Solgel: microporous active layer of ZrO2 Flat sheet thin film composite membranes. Material and MWCO: PA (2.5 kDa), cellulose acetate (0.5, 0.8, 1-5 and 8-10 kDa). Charge: anionic characteristics Cross-flow NF membrane Material: ceramic alumina γ with an average pore radius of 2.5 nm. Charge: zero point charge in the range of pH 8-9. Positively charged in the pH range tested. Composite thin-film membranes with asymmetric structure Material: aromatic PA and SPS

Table 6. (Continued) Reference Lapointe et al. (2003)

Solution Protein hydrolysate

Experiments  Concentration polarization phenomena: effect of hydrodynamic conditions on the Tr of selected peptides from the hydrolysate

Li et al. (2003)

-Single AA solutions -Fermentation broth

 Effect of pH, concentration and

Groleau et al. (2004)

Protein hydrolysate

Lapointe et al. (2005b)

Protein hydrolysate

physicochemical environment (ionic strength and kind of salt added) on single AA rejection  Effect of operating pressure and concentration of fermentation broth on NF (selectivity and AA rejection)  Effect of aggregating peptides on the fractionation of a protein hydrolysate

 Effect of feed concentration, pH,

transmembrane pressure and feed velocity in the ability of a “loose” composite NF membrane to fractionate acid, neutral and basic peptides.  Evaluation of the effect of peptides fouling on sieving and electrostatic characteristics of the

Membrane Flat sheet membrane Material: cellulose acetate MWCO: 2.500 kDa Charge: anionic charge characteristics at basic pH Material: SPES Charge: high negative charge at neutral pH

Flat sheet membrane Material: PA (propietary) MWCO: 2.5 kDa Charge: negatively charged at alkaline pH Flat sheet membrane Material: PA (propietary) MWCO: 2.5 kDa Charge: negatively charged at alkaline pH

Reference

Solution

Butylina et al. (2006)

Peptide mixture

Hong & Bruening (2006) Tessier et al. (2003)

AA mixtures

Experiments membrane: PEG and NaCl retention measurements.  Selectivity estimation in the separation peptides from lactose and effect of pH in fouling  Separation of neutral AA using multilayer

polyelectrolyte NF membranes Protein hydrolysate

 Fractionation of small peptides using a 1 kDa

NF membrane.  Influence of pH and ionic strength on Tr

Kovacs & Samhaber (2009)

Single AA solutions

Permeation of single AA solutions in the whole range of their solubility with a stepwise pH scan ranging from 0 to-1 total net charge

Shirley et al. (2011)

Single AA solutions

 Study solute rejection versus concentration of

5 different AA.  Comparison of experimental data against a

combined steric and charge rejection model.

Membrane

Material: SPES MWCO: 1 kDa Charge: negatively charged at neutral pH Material: Bi-layers of PSS/PAH on porous alumina support Cross-flow filtration Material: cellulose acetate MWCO: 1 kDa Membrane discs MWCO and material: 0.150.30 kDa (proprietary), 1 kDa (proprietary), 2.5 kDa (proprietary), 0.15-0.30 kDa (permanently hydrophilic PES) and 1kDa (permanently hydrophilic PES) Material: SPES MWCO: 1kDa

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Francisco Amador Riera, Ayoa Fernández, Claudia Muro

Figure 3. Transmission coefficient of neutral peptides in a β-lg tryptic digestion through a 5kDa PES membrane as a function of pH. Neutral peptides are classified from the smallest to the biggest in X-axis and each bar represents the Tr calculated for a given pH, the charge of the peptide at each pH value being indicated over this information. (Fernández et al., 2013a).

This study also demonstrated the role of peptide hydrophobicity in the fractionation process, as represented in Figure 4 (increasing transmission when hydrophobicity increases). In the absence of electrostatic interactions (membrane–peptide) and when the MWCO of the membrane is not a limiting factor, the higher is the peptide hydrophobicity the higher is transmission through organic membranes.

Figure 4.Relationship between peptide hydrophobicity and experimental transmission when the peptides have zero net charge (pH ≈ pI), using a 5 kDa PES membrane. (Fernández et al, 2013).

Fractionation of Peptides by Means of Ultrafiltration …

39

Ionic strength is the other main factor influencing membrane performance in terms of peptide separation. A charged protein/ electric double layer”, peptide is surrounded by a diffuse ion cloud, usually known as the “the thickness of which depends on the ionic strength, as reflected in the following 

1

equation: LD  0.304 xIS 2 , where LD is the Debye length (nm) and IS the ionic strength (molL-1). In this way, the higher the salt contents of the solution, the smaller the size of the molecule. In agreement with this equation, salt addition would increase the transmission of some peptides, and this has indeed been observed when electrostatic interactions between peptides and membrane are negligible (see Figure 5). As an example, the selectivity of membranes (PES) with a 1000 Da cutoff between basic and acid peptides changed from 174.8 to 3.5 by adding 0.5 M NaCl, and the selectivity between neutral and acid peptides changed from 69.9 to 3.0 when adding 0.5 M NaCl. Similar trends were found for a 5000 Da membrane, although the differences were not so important (Fernández & Riera, 2013). However, the change in the effective ratio of charged molecules is not the only effect of the ionic strength. A partial decrease in the charge of the membrane surface has also been reported at high salt concentration levels (Garem et al., 1998; Pouliot et al, 1999; Grib et al., 2000), and this observation has a different effect on the transmission of positively and negatively charged molecules. Figure 5, extracted from Fernández and Riera (2013), illustrates the aforementioned statements. In addition to its effect over membrane fouling, as reflected in permeate flux decline, the increase in the concentration of the hydrolysate also plays a role in the selectivity of the process. According to Fernández & Riera (2012), an increase in feed concentration results in an increase in the transmission of anionic peptides, whereas the opposite effect was observed in the case of anionic peptides. This resulted in a less selective process and suggests that the dual size/charge separation mechanism is lost, at least partially, when working with concentrated hydrolysates or peptide mixtures. Finally, the composition of the membrane and its hydrophilic/hydrophobic character also has an effect in peptide transmission. Although highly hydrophilic membranes have been recommended for working with protein solutions, owing to their good performance in terms of permeate flux, their application in peptide separation purposes is not always recommended. Thus, for example, the poorer selectivity and lower peptide recovery observed in comparison with common hydrophobic membranes played against their use in the fractionation of a tryptic β-lg hydrolysate (Fernández et al., 2013b). In

40

Francisco Amador Riera, Ayoa Fernández, Claudia Muro

addition, Roblet et al., (2012) studied the application of both spiral-wound and hollow-fiber membranes and reported differences in the composition of the recovered fractions as well as in their bioactivity.

Peptides identification: 1-ALK; 2-FDK; 3-IIAEK; 4-IDALNENK; 5-GLDIQK; 6Aggregates:WEND (G)ECAQK and WEND(G)ECAQKK; 7-TPEVDDEALEK; 8-LIVTQTMK; 9-ALPMHIR; 10-VAGTWY; 11-IPAVFK; 12-VLVLDTDYK; 13-TPEVDDEALEKFDK; 14-WEND(G)ECAQKK+LSFNPTQLEEQCHI; 15WEND(G)ECAQK+LSFNPTQLEEQCHI; 16-SLAMASSDISSLLDAQSAPLR; 17-VYVEELKPTPEGDLEILLQK. Figure 5. Theoretical (Trthe) and experimental (Trexp) transmission coefficients of different peptides (obtained from -Lg hydrolyzed with trypsin) through a 5 kDa PES membrane as a function of pH and ionic strength (NaCl). Peptides are classified from the smallest to the biggest in X-axis (Fernández et al., 2013).

Fractionation of Peptides by Means of Ultrafiltration …

41

Apart from the composition and physicochemical composition of the peptide mixture and the membrane characteristics, hydrodynamic conditions may play an important role in the separation process, probably because of peptide–peptide interactions in the weakly attached layer over the membrane surface (Lapointe et al., 2003). Some advances have been made during recent years in the use of conventional pressure-driven membrane processes for the production of bioactive peptide preparations. However, the efficiency of the separation process must be improved. The application of an external electric field, which acts as an additional driving force for the pressure gradient in the case of charged molecules, has been used to improve the efficiency of conventional UF and NF membranes. Lapointe et al., (2005a) studied the effect of the feed solution pH and hydrodynamic conditions in the selective separation of ACE inhibitory peptides from a β-lg tryptic hydrolysate using electrofiltration. In a more recent study, Leeb et al., (2014) applied cross-flow electro-membrane filtration for the fractionation of a tryptic micellar casein hydrolysate. The efficiency of the process was examined by analyzing the yield of ACE peptides in the fractions (retentate and permeate) and by measuring their ACE inhibitory activities in vitro. In vitro ACE inhibitory tests showed an important reduction in the IC50 value of the permeate stream, which was up to six times lower than the IC50 value obtained for the raw hydrolysate. However, this new methodology has not been shown to be able to avoid fouling problems that are typically associated with conventional membrane techniques. Other approaches have proposed the fractionation of peptide mixtures using a conventional electrodialysis cell, but by replacing some of the ionexchange membranes with UF membranes. Electrodialysis with UF (EDUF) membranes couples the size-exclusion capabilities of UF membranes with the charge selectivity of electrodialysis, allowing separation of molecules according to their electric charge and their molecular weight. Thus, for example, this technique has been used for the fractionation of snow crab byproducts (Doyen et al., 2012) and β-lg tryptic hydrolysates (Poulin et al., 2006), displaying a significant improvement in the separation of peptides with a different charge state. In a similar approach, Firdaous et al. (2009) applied this technique to the fractionation of an alfalfa protein hydrolysate. These authors reported that EDUF can overcome some of the fouling problems in conventional UF and they also observed an improvement in the selectivity of the process. Indeed, only eight out of the 70 peptides present in the original hydrolysate permeated through the membrane, and the first study on the comparison of pressure-driven membrane techniques and electrically-driven

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Francisco Amador Riera, Ayoa Fernández, Claudia Muro

processes for the fractionation of bioactive peptides was published for Langevin et al. (2012). According to these results, the combination of NF and electrodialysis using an UF membrane would be useful for the production of more specific peptide fractions.

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Yu, Z. P., Yin, Y. G., Zhao, W. Z., Chen, F. & Liu, J. B. (2014). Antihypertensive Effect of Angiotensin-Converting Enzyme Inhibitory Peptide RVPSL on Spontaneously Hypertensive Rats by Regulating Gene Expression of the Renin-Angiotensin System. J Agr Food Chem, 62, 912917. Yuan, Y. V. & Kitts, D. D. (1991). Confirmation of Calcium-Absorption and Femoral Utilization in Spontaneously Hypertensive Rats Fed Casein Phosphopeptide Supplemented Diets. Nutr Res, 11, 1257-1272. Zeman, L. & Wales, M. (1981). Steric rejection of olymeric sokutes by membrane with unifor pore sizes distribution. Sep Sci Technol, 16, 275290. Zhang, L., Li, J. R. & Zhou, K. Q. (2010). Chelating and radical scavenging activities of soy protein hydrolysates prepared from microbial proteases and their effect on meat lipid peroxidation. Bioresource Technol, 101, 2084-2089. Zhang, Q. X., Wu, H., Ling, Y. F. & Lu, R. R. (2013). Isolation and identification of antioxidant peptides derived from whey protein enzymatic hydrolysate by consecutive chromatography and Q-TOF MS. J Dairy Res, 80, 367-373. Zhong, F., Liu, J. M., Ma, J. G. & Shoemaker, C. F. (2007). Preparation of hypocholesterol peptides from soy protein and their hypocholesterolemic effect in mice. Food Res Int, 40, 661-667. Zhou, K. Q., Sun, S. & Canning, C. (2012). Production and functional characterisation of antioxidative hydrolysates from corn protein via enzymatic hydrolysis and ultrafiltration. Food Chem, 135, 1192-1197. Zioudrou, C., Streaty, R. A. & Klee, W. A. (1979). Opioid peptides derived from food proteins. The exorphins. J Biol Chem, 254, 2446-2449.

In: Whey Proteins Editor: Michelle Wyatt

ISBN: 978-1-63463-116-7 © 2015 Nova Science Publishers, Inc.

Chapter 2

ITALIAN VALORIZATION OF DAIRY WASTE Gigliola Camaggio* and Vera Amicarelli University of Bari Aldo Moro, Department of Business and Law Studies, Unit of Commodity Science and Ecology, Largo Abbazia Santa Scolastica, Bari, Italy

ABSTRACT An economic system, even more oriented to sustainable development models, should provide a new way of managing production processes optimizing resources utilization and minimizing the amount of waste produced. In this context, waste valorization becomes a crucial option to improve the economic and the environmental performances of a company and/or of an industrial sector. The dairy waste represents an interesting example of this type of approach considering that in Italy, dairy industry plays a key role in the agri-food sector in terms of production, employment and turnover. The possibility to transform this waste into resources is related to their composition, they are rich in high value-added substances such as proteins, protein derivatives, potentially prebiotic sugars, minerals and vitamins. The aim of this chapter is to evaluate the different possibilities of dairy industry waste utilization to produce new raw materials useful in the food, pharmaceutical, and cosmetic industries. The authors will focus their analysis on Italian situation related to the dairy by-products valorization. *

Corresponding author: E-mail: [email protected].

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Keywords: Dairy wastes, dairy by-products, whey, whey protein, lactose

INTRODUCTION The economic system, more and more oriented to sustainable development patterns, should provide a new production processes management focused on optimizing resources utilization and minimizing waste production. In this context, waste valorization becomes a crucial option to improve economic and environmental performances of a company and/or of an industrial sector. The agricultural and agro-industrial sectors are also becoming more and more oriented towards higher sustainability both for their weight in Italian economy and for their by-products valorization. Particularly in dairy industry by-products valorization is not a recent theory. In the USA, during F.D. Roosevelt administration, some experts already became aware of agricultural by-products high content of potential industrial raw materials. It is the case of peanuts and soy proteins suitable for artificial textile fibers production, or the case of some plants cultivated in areas with same climate of Murge (a vast karst plateau in Puglia region) suitable for extraction of a rubber similar to the one derived from Hevea Brasiliensis, and even more the case of several types of starch and cellulose suitable for gasoline surrogate fuels (bioethanol) (Nebbia, 2007). This scenario indicates that agro-industrial waste and byproducts valorization may lead to a new vision of economy and to a new and more environmental protective agriculture. Over the years, besides other agroindustrial sectors specific attention was paid to the dairy one which handles, each year, million tons of effluents. They includes a) milk processing byproducts mainly whey, buttermilk, so-called “scotta” (intending with this word the remaining effluent after ricotta cheese production) and stretchwater and b) wastewater like washing water for milk coagulation tanks, machineries and workplaces; exhausted brines, conditioning water utilized in cooling systems. While the by-products (a), due to their composition, are likely to further valorization and value creation the wastewater (b) are at the base of environmental damages. Moreover, effluents have a modest pollution load (COD average 5,000-10,000 mg/L) requiring conventional disposal systems and procedures, by-products show a very high content of organic substances requiring complex and expensive disposal processes (evaluated in up to 20-25 €/m3). For instance, 1 liter of whey with an average COD equal to 25,000-70,000 mg/L, has the same pollution load of a person by day (Pizzichini et al., 2001).

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Whey, even if does not contain toxic agents or bacterial activity inhibitors cannot be discharged directly into water bodies and furthermore it is very complex to dispose it by standard biological systems. Currently dairy byproducts are classified as special waste even if they are non-hazardous. Valorizing their components (protein and lactose) means decreasing the volume of non-hazardous waste to be disposed and increasing “secondary raw materials” for various industrial sectors (Figure 1). On the basis of this brief introduction, the aim of this chapter is to analyze the potential for the enhancement of dairy products with special reference to whey. After outlining sector main features, all potentially obtainable products and their related markets will be described.

ITALIAN DAIRY INDUSTRY Milk and dairy sector is one of the most important compartments of the Italian food industry. On a revenues basis, and despite the difficult general economic crisis, in 2013 such compartment, with approximately 15 million of Euro (M€), was the most profitable one, followed by the confectionery (13 M€) and oenological (more than 10 M€) industry. In 2012, Italy produced 1.2 million ton (Mt) of cheeses resulting the 4th world producer after the United States of America (5.2 Mt), Germany (about 2 Mt) and France (1.28 Mt) and the 3rd European producer (FAOStat, 2014). Food

Zootechny

Whey

Fuels

Cheeses, beverages, protein additives, lactose and derivatives, principles for functional foods.

Feed and additives for pigs, bovine and ovine feeding.

Bioethanol and Biodiesel.

Pharmaceutical and Cosmetic

Components for drugs, creams, shampoos and other cosmetics.

Other

Fertilizer, insecticide, fungicide and other products.

Source: personal elaboration by the authors. Figure 1. Potential dairy by-product applications.

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Besides, it must be remembered that less than 40% of the Italian production involves high quality products that already gained different European Union (EU) schemes for their standards. In 2012, EU recognized 1,138 schemes of which 248 in Italy and out of these products listed in the Register of Protected Denominations of Origin (PDO), Protected Geographical Indications (PGI) and Traditional Speciality Guaranteed (TSG), 45 are Italian cheeses (ISTAT, 2014a). Italy is, after France (with 47 of them), the second DOP cheese community producer. In the same period, in Italy 43 DOP cheeses, 1 IGP and 1 STG were listed. All these schemes attest the high quality standards of Italian agro-industrial sector mainly recognized by foreign countries where interest and appreciation for products "made in Italy" is more and more increasing. Confirming this, still in 2012 cheese import/export trading balance was positive (+354 M€), enhancing export sales value equal to 1,975 M€ higher than the import ones (1,621 M€). To this result mainly contributed export of Grana Padano and Parmigiano Reggiano (7% compared to 2011), mozzarella and other fresh cheeses (+ 9 compared to 2011) (Assolatte, 2014). In 2012 (tab. 1), Italian production units were approximately 2,076 and 67% of them were cheese factories and milk plants, approximately 25% were agricultural cooperatives plants and the remaining quotas were milk farms and milk collecting centers (MCC) (ISTAT 2014b). Their national geographic distribution is not homogeneous; in fact, 47% (970 unities) is in the North, mostly in Lombardia and Emilia Romagna, around 44% (924 unities) in the South, mostly in Campania and Puglia and more than 8% is in Central Italy. While in northern Italy the sector includes mainly cooperatives, dairy plants and milk plants, in southern and central Italy the majority of production units are dairy (70%) and milk plants (88%). Table 1. Production units operating in Italian dairy sector in 2012 Dairy plants and milk plants Northern Italy 443 Central Italy 129 Southern Italy 821 Italy 1,393 Source: ISTAT, 2014b.

Milk farm

Agricultural cooperative.

47 9 25 81

438 30 53 521

Milk collecting centers 42 14 25 81

Total 970 182 924 2,076

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The raw material supply chain consists of bovine, caprine, ovine and buffalo milk. Still in 2012, Italy produced over 11.2 Mt of milk equal to 2% of bovine milk worldwide production. Among all different typologies prevails bovine's milk (less than 95%) followed by caprine milk (roughly 3.5%), buffalo milk (about 1.5%) and ovine milk (0.2%). The whole production includes direct use of milk (2.6 Mt), cheeses (1.2 Mt), butter (0.10 Mt) yogurt (0.33 Mt) and other derived products such as milk-based beverage, milk crème and etc. production. More than half of Italian yearly milk production (51%) is made out of partly skimmed milk while 45% of it is whole milk and a very small quantity less than 104,000 t is skimmed milk (ISTAT, 2014c). Second to direct milk use, the most productive segment of the Italian dairy sector is cheese production. Out of 1.2 Mt produced, approximately 73% (0.87 Mt) comes from Northern Italy, about 20% (0.24 Mt) from Southern Italy and the remaining 7% (0.09 Mt) from Central Italy. Based on their hardness (hard, semi hard, soft and fresh) fresh and hard cheeses represent the main national production quotas respectively equal to 41% and 36%. Soft cheeses with 0.17 Mt account to 14% of total production while the semi hard ones with about 0.10 Mt are 8.5%. The higher production of cheese, as already mentioned, occurs in Northern Italy where there are plants of considerable production capacity, with an output of approximately 0.37 Mt of hard cheeses, 0.27 Mt of fresh cheeses, 0.16 Mt of soft cheeses and 0.074 Mt of semi hard cheeses. Southern Italy instead mainly produces fresh cheeses, approximately 0.16 Mt, over 50% of its overall production. In the same area, hard cheeses account to 0.048 Mt, semi hard ones to 0.02 Mt and soft ones to 0.006 Mt. In Central Italy production is more limited: 0.059 Mt fresh, 0.018 Mt hard, 0.02 Mt soft and 0.0049 Mt semi hard (ISTAT, 2014d). These data do not only represent the national milk-cheese compartment scenario, but they also allow the mapping of by-products production centers. This information is essential to their valorization because first they provide quantity data and secondly, remembering that by-products composition is direct linked to cheese typologies, they give quality specifications too. Table 2 shows the average composition of dairy byproducts obtained by the production of different varieties of cheeses.

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Gigliola Camaggio and Vera Amicarelli Table 2. Italian cheese whey average composition (g/100 ml) Dry mater 6.88

Parmigiano Reggiano Grana Padano 7.11 Provolone 7.55 Gorgonzola 6.83 Mozzarella 7.1 Source: Corradini, 1995.

Ash

Calcium

Lipids

Lactose

0.54

//

0.33

//

Protein (N x 6, 38) 0.83

0.53 0.54 0.57 0.5

0.06 0.05 0.04 0.05

0.35 0.7 0.2 0.65

5.35 5.32 4.45 5.1

0.86 0.88 0.9 0.82

Table 3. Dairy by-products average chemical composition (%) Sweet whey Acid whey Lactose 4.5 4.5 Protein 0.75 0.75 Lipid 0.4 0.4 Ash 0.7 0.7 c Vitamin 12 12 pH 5.8-6 4.9-5.5 CODc 50,000 50,000 a ( ) suspended solids 8,3%. (b) N x 6.38. (c) mg/L. Source: Losacco, Faccia, 2009.

Scotta 4 0.15 0.2 1 // 5.-6.2 25,000

Stretchwater 0.1 0.2 0.05 1.3 // // 5,000-10,000

Buttermilka 4.2 3.4b 0.7 0.8 // // 70,000

Type and size of dairy companies may also influence the possibilities of dairy by-products valorization. The northern Italy for instance, mainly characterized by cooperatives and medium/big size plants with good attitude to integration among producers, shows a high level in financial possibilities to afford all innovation costs to make the whole productive chain much more environmental friendly. On the contrary, southern Italy shows a different scenario with numerous small companies, high productive and territorial fragmentation, and low attitude to integration among producers. This makes much more difficult to implement the right strategies necessary to dairy byproducts collection, treatments and valorization often encouraging illegal spillages losing in environmental and economic sustainability.

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Dairy Industry By-Products Valorization As just mentioned before, fresh milk processing produces different byproducts mainly whey, buttermilk, scotta and stretchwater. They have different composition (tab. 3) and so they cannot be used and/or valorized and/or disposed in the same way. Whey is the most abundant by-products and it is the watery part of milk remaining after the precipitation and removal of milk casein during cheese manufacturing and it is equal to 85-88% of milk treated, therefore 1 kg of cheese produces 7-9 kg of whey (ANPA, 2001; Pizzichini, 2006). It is an opalescent liquid with a thinned yellow-green color and a 90-95% water content. Its chemical and microbiological composition is varying but it is very similar to the treated milk. Thus, there are several types of whey such as sweet and acid, upon which milk has been used, which cheese has been produced and which coagulation method has been adopted. Sweet whey, with a pH higher than or equal to 5.6, is usually obtained from whole milk after rennet-coagulation as happened in most cheese making process worldwide and acid whey with a pH lower than or equal to 5.1 is usually obtained by direct acidification of milk and/or skimmed milk to produce cottage cheeses. Buttermilk is also a watery by-product obtained during butter production process and in its Italian diction “latticello” is often used by mistake, in southern Italy, to indicate stretchwater obtained during mozzarella cheese production. Stretchwater is the hot water used in the stretching step for “pasta filata” cheeses and scotta is leftover from ricotta cheese production. These last organic matrices (stretchwater and scotta) are characterized by a seasonal factor and they are typical of some southern Italian regions (Puglia, Campania, Sardinia) where it is widespread the production of pasta filata and ricotta cheese. In general and related to the chemical composition of all these by-products (tab. 3), carbohydrate fraction is the most abundant (0.1-4.5%) even if lactose is a not too soluble sugar and has a low sweetening power (0.2-0.4 versus sucrose). The proteins (0.75-3.4%) are those residual after milk processing and for their quality their recovery could be a good source of nitrogenous compounds. The fat portion (0.05-0.7%) is “easily” recoverable. By skimming, it is used in whey butter production. With centrifugal separators, the whey is

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separated into a fat-rich cream and a diluted aqueous fraction containing mainly proteins, salts, and lactose. The fat-rich whey cream is pasteurized and cooled to be used to obtained whey butter. The valorization of all these by-products, after their separation, refrigeration and concentration, is strictly linked to the possibility to recovering single high value fractions, like whey proteins and lactose, useful in food, pharmaceutical and bio-fuels industries. In an energy perspective, the valorization of dairy industry by-products high in lactose, like buttermilk and scotta, may lead to biogas and bioethanol production. In the first case the anaerobic digestion of the substrate would lead to biogas production useful in energy and/or steam generation. In order to ensure a good level of efficiency of such type of plants, it may be implemented a codigestion with another biomass, like animal manures, to ensure better environmental condition to methanogenic bacteria. High acidity and COD values characterizing dairy by-products, limit the activity of these bacteria (Kavacik, Topaloglu, 2010; Migliardi et al., 2008). Bioethanol instead is obtained by lactose fermentation with yeasts like Kluyveromyces lactis, Kluyveromyces marxianus and Candida pseudotropicalis. The limits of bioethanol production are represented by two basic elements: the too low lactose concentration and the scarce availability of yeasts capable of fermenting this disaccharide. Therefore, the efforts in this field should be mainly addressed to genetic yeasts improvement (Guimarâes et al., 2010).

Dairy by-Products No-Energy Valorization The interest and the possibilities in no-energy exploitation of these byproducts are not only due to the quantity of their components but also to their quality. Whey proteins represent the non-casein protein fraction of the milk, do not precipitate at casein isoelectric point (pH 4.6) and show remarkable water solubility. They are: bovine serum albumin, α-lactalbumin, β-lactoglobulin, lactoferrin and immunoglobulin. The concentration of whey proteins depends on the type of whey (acid or sweet), the source of milk (bovine, caprine or ovine), the time of the year, the type of feed and the quality of processing. The ability to absorb water, to form gels and foams and the aptitude to be a "fat replacer" for instance in ice-cream recipes are their functional properties allowing their profitable utilization. (Yilsay et al., 2006; Sodini et al., 2006).

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The evolution of separation technologies during the last two decades have made available different whey components (proteins, minerals, lipids and sugars) with specific qualitative and quantitative profiles useful and appreciated in just mentioned food and no-food sector. Whey may be treated with membrane-based separation technologies to concentrate proteins or to remove most lactose, minerals and low molecular weight components and thus produce whey protein concentrates (WPC). Based on their concentration WPC are commercialized in different ways. There are WPC containing 35%, 50%, 65% and 80% (w/w) of protein. A higher quality and purity protein concentrate, so called Whey Protein Isolated (WPI) is obtained when a percentage of 90% (w/w) of protein is reached. Due to their high biological and purity values, both those products (WPC and WPI) are added to food to improve daily diet or special diet for athletes and bodybuilders. Even if whey proteins several properties are long known, there are still not adequate studied concerning the interactions that occur both in milk and whey among proteins, carbohydrates, lipids and minerals. A deep knowledge of these molecular mechanisms would be very helpful to improve the efficiency and the innovation in separation processes technology of whey components. Scientific research in recent years has helped to clarify the beneficial functions of whey proteins demonstrating their synergistic action with vitamins B1 and B2 in the increased body weight and lean body mass, especially in people with HIV, and in the reduction of cancer and pneumonia incidence (Cayot, 1996). Also remarkable are the bioactive peptides obtained from whey proteins whose activities are responsible for the reduction of cholesterol, blood pressure and sugar content in the blood (Fiat, Jolles, 1989; Butylina et al., 2006). In Italy there is a fermented milk whose main component is formed by these peptides, adjuvants in the regulation of blood pressure; its trade name of EVOLUS. From whey is possible to obtain lactose that, as already mentioned, is the largest component. Lactose is obtained from the permeate of ultrafiltration (UF) through concentration, crystallization, centrifugation, washing, drying and grinding processes. Depending on concentration (on a dry basis) there are several lactose grades: edible (powder not less than 99%), refined (powder not less than 99.5%) and pharmaceutical (powder, not less than 99.8%). Very interesting is also the perspective of lactose derivatives valorizing due to their prebiotic properties favoring the growth of bifidobacteria and lactic acid bacteria in the intestinal tract and including enhancing mineral

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uptake, serum lipids and reducing the risk of intestinal infection and colon cancer. Lactulose, lactitol, lactobionic acid, lactosucrose and galactoseoligosaccharides (GOS) are lactose main derivatives obtained through enzymatic, chemical and microbiological processes. They are primarily commercialized as food (mostly in functional foods), pharmaceuticals and cosmetics ingredients. In particular, lactulose, the first commercialized lactose derivative, is enzymatically obtained using β-galactosidase and compared to sucrose it has a sweetening power of 0.6. Thanks to its recognized therapeutic characteristics it is used in encephalopathy treatment and also as a laxative. The lactitol produced through lactose chemical hydrolysis, has a sweetening power equal to 0.3-0.4 and is used as a non-caloric sweetener for low-calorie food and dietary additives. It is also used in pharmaceutical field because it has the same therapeutic qualities of lactulose. The lactobionic acid is used in chelation therapy for some type of metals intoxication treatment and it is also used in pharmaceutical applications, as food additives and in cosmetics as humectant. The lactosucrose is mainly produced and used in Japan as an ingredient in functional foods. From lactose hydrolysis, through the β-galactosidase enzyme, are obtained glucose and galactose monosaccharides that after a recombination phase, generate the GOS, with prebiotic properties. These properties have a beneficial impact on the intestinal flora in particular, on bifidobacteria to whom scientific community has long recognized anticancer properties (Yaeshima, 1996). GOS biomedical effect is to increase the nutrients absorption and the protection from pathogenic bacteria like Escherichia coli, Salmonella and Staphylococcus aureus (Gibson, Wang, 1994). They are also cholesterol and blood pressure reducers, and blood glucose level regulators (Tomomatsu, 1994). The hydrolysis producing the GOS may happen in different conditions and using various enzymes of microbial origin; the bacteria involved are Kluyveromyces fragilis, Aspergillus oryzae and Escherichia coli. The GOS find a place in the healthy-food sectors in particular they are baby-food ingredients, due to their fermentation and intestines absorption properties. The currently available main techniques used in other countries of Northern Europe, New Zealand and United States of America, to recover whey components are based on the use of specific membrane technologies. They consist of semi permeable filters, also called ion-selective, which allow the separation of organic and inorganic solutes and solvents. These technologies

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use a tangential flow filtration, opposite to the classical perpendicular one, in order to smooth the progress of solutes removal from the membrane surface. The difference between the various techniques is the membrane and the operating conditions. The combination of different filtration systems grants the production of high added value products. The microfiltration retains 1 microns (μm) size solutes. The ultrafiltration retains about 1 kdalton macromolecules like proteins. The nanofiltration, instead, retains molecules with molecular weight greater than 200 daltons and therefore useful for lactose recovery, and the last one, the reverse osmosis retains electrolytes such as sodium chloride. The main advantage of these techniques is their capability to operate at low temperatures (10-20°C), avoiding the degradation of the various whey components. Since several years, ENEA has developed an integrated membrane treatment cycle for all dairy industry by-products, able to obtain from 1 m3 treated: 16 kg of protein powder, 49 kg of pure lactose powder besides 580 kg of low salt content water (Pizzichini et al., 2010). The whey along with all other industry by-products (buttermilk, scotta and stretchwater), has always been considered a waste, at least in Italy, accounting for the dairy traditional industry a problem due to high disposal costs and pollution load (COD between 50,000 -70,000 mg/L). On the contrary, in other countries, like Germany, Switzerland or New Zealand, there is a long-standing culture of such precious organic matrix recovery. According to Italian available data for the year 2012, it is estimated that more than 7 Mt of whey have been produced of which 31% is used for livestock, 11% for ricotta cheese and 25% for concentrate (14%) and powders (11%) (ISTAT, 2014c). The remaining quota, less than 2.4 Mt, is disposed complying to pertinent rules. This means that Italy, in 2012 "wasted" less than 18,000 tons of whey proteins and more than 105,000 tons of lactose, (these quantities are calculated based on average whey composition recorded in table 3) and in the same time imported more than 53,000 t of whey powder used for livestock and food industry spending more than 67 M€ considering a price equal to 1,260 €/t (CLAL.it, 2014). Nevertheless, it is not easy to implement technical and economic feasible integrated solutions for dairy by-products valorization, considering the just mentioned small size of most of the factories engaged in this sector (mostly in Southern Italy) and their fragmented distribution in the territory, It is necessary to adopt collective and appropriate waste and by-products management practices instead of individual solutions. To achieve this result all

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supply chain stakeholders have to be involved in order to creating consortiums, companies and cooperatives with the aim to organize a sort of common “whey recovery and valorization center” maximizing environmental and economic benefits and sharing costs and risks. It must be considered at the end, that dairy by-products are perishable and therefore need immediate refrigeration and subsequent concentration in order to obtain high added value products as previously mentioned. (Nicoletti, Camaggio Sancineti, 1996).

The Whey Village The idea of planning a whey village has been the subject of studies and investigations also by Italian National Agency for New Technologies, Energy and Sustainable Economic Development (ENEA) in order to obtain high added value products integrating strategies towards sustainable management for the valorization of dairy by-products and good disposing practices for resulting wastewater. The project consisted in a plant made out of three single modules. In the first module whey is concentrated and then protein fraction is isolated. The second is for biogas production from scotta and buttermilk lactose-rich fractions. The third module is for the thermovalorization of stretchwater and dairy wastewater which are the poorest fractions not useful for further exploitation. The output of the first module consists, essentially, in WPC. However, WPI which are more profitable, may also be produced, treating WPC with a chromatographic process whose by-product is the ultrafiltration permeate. The latter, along with scotta and buttermilk, is conveyed in the second module to be fermented for biogas production, partially used by the plant itself. The residual fraction of anaerobic digester, due to its features, may be placed on the fertilizers market as soil amendment. The plant is completed by the third module whose function is, as just mentioned before, the thermovalorization of the less profitable dairy waste and by-products. Preliminary operation, in this latter stage, is the input concentration by evaporation, producing a semi-solid (slurry) residual and an aqueous phase. The slurry is sent in a rotary hearth furnace where salts like sodium chloride are obtained and used for instance as antifreeze agent for winter road maintenance. The aqueous phase have to be treated to make lower COD value for instance before to be reused or sent to municipal water disposal system.

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The whey village also included in a European project still under review and herein briefly described, achieve two scopes. The first one is all dairy wastewater and by-products recovering, while the second one is their energy content (biogas) or their high added value fractions (WPC and WPI) valorization (Di Luccia, 2011; Losacco, Faccia, 2009). This type of plant may reduce environmental impacts answering to the urgent and growing need of a sustainable and environmental friendly solutions to the dairy wastewater and by-products issue.

CONCLUSION Having assessed the range of possibilities for dairy by-products valorization as basis of new valuable raw materials in different manufacturing segments, particularly food industry, the first result is that Italy, one of the main European cheeses producers is still far from sustainable managing of these by-products. Italian dairy compartment is still affected by several limits like: small and medium-sized factories, high productive and territorial fragmentation, low attitude to integration among producers and diffused operators ignorance of heterogeneity of produced dairy waste and by-products and innovative practices to dispose and/or to valorize them. Membrane technologies for instance, briefly already described, are widely used in other countries of Northern Europe and in United States of America. Whey is very perishable organic matrix and it must be refrigerated and concentrated as soon as it is produced to better valorize its fractions, particularly protein and lactose. Of course this means that collecting and transportation costs are critical issues to implement good and sustainable management practices. Furthermore, because dairy industry production is affected by seasonal factor, ensuring an annual steady whey supply flow it could be difficult. So flexible plants, able to treat different agro-industrial matrixes or useful for other industrial applications such as sterilizing treatments and/or milk concentration, could be one of the possible solution to valorize dairy byproducts and waste avoiding or reducing environmental impacts, decreasing import of products like WPC and WPI and increasing in the same time, new green business opportunities.

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produttiva - Anno 2012. Retrieved July 12, 2014, from http://agri.istat.it/ jsp/dwExcel.jsp?q=plL050000010000010000&an=2012&ig=1&ct=707&i d=8A|11A ISTAT (Istituto Nazionale di Statistica). (2014d). Produzione industriale di latte alimentare, di burro e di formaggio. Dettaglio per ripartizione geografica - Anno 2012. Retrieved July 12, 2014, from http://agri.istat.it/ jsp/dawinci.jsp?q=plL060000010000011000&an=2012&ig=1&ct=157&id =8A|11A Kavacik, B. & Topaloglu, B. (2010). Biogas production from co-digestion of a mixture of cheese whey and dairy manure. Biomass and Bioenergy, 34, 1321-1329. Losacco, A. & Faccia, M. (2009). Sierovalore: scenari e prospettive per la valorizzazione dei reflui caseari. Documento finale Progetto PIC INTERREG III A Italia-Grecia. Modugno (Ba): Grafica 080. Migliardi, D., Patuzzo, E., Vecchiet, A., Tonutto, A. & Moro, R. (2008). Energia dal siero di latte, sfruttamento del potenziale energetico mediante digestione anaerobica con liquami bovini. Ambiente Risorse Salute, 119, 41-46. Nebbia, G. (2007). Il valore dell’agricoltura. Retrieved May 15, 2011, from http://www.fondazionemicheletti.it/altronovecento/articolo.aspx?id_artico lo=12&tipo_articolo=d_eventi&id=21. Nicoletti, G. M. & Camaggio Sancineti, G. (1996). Il siero di latte in Puglia, disponibilità e possibilità di utilizzo, Bari Economica, 1, 93-104. Pizzichini, M. (2006). Tecnologie di processo per la valorizzazione delle componenti del siero di latte. Viterbo: Primaprint. Pizzichini, M., Montani, R. & Ruscio, F. (2001). Il siero di latte: da rifiuto zootecnico a materia per alimenti e farmaci. L'Informatore agrario, 16, 49-53. Pizzichini, M., Russo, C., Vitagliano, M. & Pizzichini, D. (2010). Valorizzazione commerciale del siero di latte bufalino nel rispetto dell'ambiente. Alimenta, 18, 115-120. Sodini, I., Mattas, J. & Tong P. S. (2006). Influence of pH and heat treatment of whey on the functional properties of whey protein concentrates in yoghurt. International Dairy Journal, 16, 1464-1469. Tomomatsu, H. (1994). Health effects of oligosaccharides. Food Technology, 48, 61-65. Yaeshima, T. (1996). Benefits of bifidobacteria to human health. Bullettin of the International Dairy Federation, 313, 36-42.

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Yilsay, T. O., Yilmaz, L. & Bayizit, A. A. (2006). The effect of using a whey protein fat replacer on textural and sensory characteristics of low-fat vanilla ice cream. European Food Research and Technology, 222, 171-175.

In: Whey Proteins Editor: Michelle Wyatt

ISBN: 978-1-63463-116-7 © 2015 Nova Science Publishers, Inc.

Chapter 3

ENCAPSULATION OF WHEY PROTEINS AND ACTIVE PEPTIDES Claudia R. Muro Urista*, Francisco A. Riera Rodriguez2 and Yolanda Alvarado Pérez1 1

Department of Chemical Engineering and Research, Technological Institute of Toluca, México 2 Department of Chemical Engineering and Environmental Technology, University of Oviedo, Asturias, Spain

ABSTRACT For several years, many researchers have focused on study of whey proteins and derived active peptides. Its functional and therapeutics properties have caused a particular interest in pharmaceutical and food products and in different areas of cosmetology. In the context of health, serum molecules have been a subject of growing research, because they can provide specific health benefits. However proteins and whey peptides are currently in the circle of sensitive biological molecules which require an effective oral administration, since its degradation, stability and permeability are not yet controlled. The peptides mainly suffer degradation in the gastrointestinal tract, including its physicochemical instability and inactivation, so these aspects are an important research topic to achieve the desired effects in medicine field. *

Corresponding author: E-mail: Email: [email protected].

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C. R. Muro Urista, F. A. Riera Rodriguez and Y. Alvarado Pérez Under this perspective, various approach enhance have been studied as alternatives to increase stability and oral bioavailability of some biomolecules which could to impact in the use and application of proteins and whey peptides as therapeutic products. In this chapter we make mention on different strategies to enhance oral bioavailability of sensible molecules and their application status. A wide range of compounds are exposed summarizing main alternatives of enhance systems. Studies of encapsulation of whey proteins and peptides in nano systems are the origin the future trends on approach enhances in active molecules to their oral administration and delivery.

INTRODUCTION Whey proteins are one of the most important products in food processing industries. Profit of whey proteins in food applications include its high amino acid content; low calorie, fat, and sodium content; high emulsification and capacity and compatibility with other ingredients (Cabuk et al., 2014). Whey proteins have currently also a significant role in field of medicine. They are recognized as active substances with therapeutic potential and thus they are seen as an attractive alternative for health and as possible replacement of conventional small molecule drugs. Extensive scientific evidence has proved that whey proteins have beneficial effects upon human health; their biological activity and functional properties have led to the development of some food and products designed to suit specific health concerns (Möler et al., 2008). To date, there are some examples in which whey proteins have been used successfully for therapeutic purpose and under clinical applications (Orsi 2004; Hallgren et al., 2008; Kamau et al., 2010). Hydrolyzed whey proteins have also led the development of biologically active nutritional components like bioactive peptides to promote health-giving opportunities for the use of dairy ingredients and drug substances in commercial products. Peptides with different amino acid content and bioactivity are liberated from protein hydrolysis depending to enzymatic degradation process. Several reports on the bioactivity of peptides in vitro and animal model systems, such as opiate activity, antihypertensive, immunomodulatory, bioconversion of metal ions (Ca2+), antithrombotic activity and antibacterial properties are found in the literature. Some bioactive peptides have been incorporated as ingredients in functional foods, nutraceuticals and pharmaceuticals where their biological activities may assist

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in the control and prevention of diseases. A comprehensive review on active peptides from whey milk and their implementation degree can be seen in Muro et al., (2011) and Riera et al., (2012). However more researches on effect active substances in vivo and clinical studies have been needed to firmly establish their therapeutic potency. In addition today there are various chalengues in the application field of active whey proteins and peptides that could addressed. Whey substances are prone to a variety of physical and chemical degradation pathways, which require the development of strategies that may permit structural and functional stabilization of substances to increase their applicability in formulations and competitive delivery alternatives (Korhonen 2009; Srinivas et al., 2010). While stability of whey substances in food products implies, their retaining within the shelf life of the product and to avoid their early alteration during food processing, or some interaction with complex food ingredients (ions, enzymes and fat). Adding up, to these inconveniences, more studies of viable processes for large-scale production of whey peptide therapeutics have been exposed as necessary to increment their application (Riera et al., 2012; Agyei and Danquah, 2012). In the health perspective, use of these active substances as therapeutic agents is also limited due to lack of an effective route and method of delivery. Stability of formulations certainly still remains one of the most important hurdles for their successful oral administration. The fast degradation of active substances in the gastrointestinal tract (GI) and poor permeability across the intestinal epithelium should be improved to achieve appropriate concentrations in blood and to prolong the residence time in the body since to exert their health benefits, the active substances have to be delivered in the site of absorption. These factors including stability and circulation time in the gastrointestinal tract, strongly affect the effective absorption of oral-delivered substances, which causes the inability of the peptides to reach their targets in an active form in vivo. Therefore, route of administration is a critical factor which governs both the pharmacokinetics and efficacy of whey products. Formulations must be able to release an intact active moiety and actually, the ability to demonstrate molecule stability inside pharmaceuticals and during delivery which becomes a requisite for reaching the product approval stage (Renokuntla et al., 2013). Effective oral delivery of active substances from whey is not exclusive problem of their origin. Certain classes of drugs especially biologics (peptides and proteins) present this restriction. In past few decades several studies have intensified their research towards many oral therapeutic molecules delivery

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because of their poor metabolic stability and low bioavailability (Shaji and Patole, 2008); this difficulty is due in part to the inability to readily cross intestinal membrane barriers, limiting their oral administration. An evident case is the insulin protein; where less than 0.1% of the orally dosed insulin reaches the blood stream intact (Blanchette et al., 2004); thus due to their excessive degradation it must be administer through injections to ensure their effect. Enzymes presents in the GI are the main cause of rapid degradation of therapeutic molecules and they are a significant barrier to their administration via the GI route. Peptide bonds as that of an aspartyl-proline bond contribute also to a poor oral bioavailability. The combination of exopeptidases, aminopeptidases and carboxypeptidases in the GI, break down sequences from the N- and C-termini of proteins and peptides, whereas endopeptidases break in recognized cleavage sites within amino acid sequence before it reaches the bloodstream. These characteristics lead to low oral bioavailability (< 1-2%) and short in vivo half-lives (< 30 min) (Iyer et al., 2010). The principal aim of biologics therapeutic molecules is, therefore, to develop formulations to facilitate both aqueous solubility at near-neutral pH and lipid layer penetration in order to cross the intestinal membrane and then basal membrane for entry into the bloodstream. Detailed understanding about of structural features of therapeutic molecules is also necessary for improving oral active molecules bioavailability. Proteins and peptides are molecules with large molecular weight and size. Their structure contains hydrophilic and hydrophobic appendages which restricting entry into cells and other body compartments. Consequences as a poor permeability through various mucosal surfaces and biological membranes are often caused by these factors. In addition proteins and peptides have very short biological half-lives in vivo due to their rapid clearance in liver and other body tissues by proteolytic enzymes. Tertiary structure may also easily lose under various physical and chemical environments, resulting their denaturation or degradation and thus loss of biological activity. A precise clinical dosing is also of utmost importance. The body may mount an immune response against the therapeutic substance and this immune response may neutralize the molecule and even cause a harmful reaction in the recipient. Other aspects of interest complementing this perspective can be seen in Muheem et al., (2014). There are currently three recognized strategies to improve the oral bioavailability of drug molecules (Morishita and Peppas (2006); the methods are applied singly or in combination to achieve best effects. 1) Addition of absorption enhancers for increasing the intestinal absorption of active

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molecules and enzyme addition inhibitors of proteolytic enzymes present in the GI. 2) Modification of physical and chemical molecules (lipophilicity and susceptibility to enzymes) nature. 3) The use of a carrier as a protective and delivery system. An accurate overview that provides a current perspective on technologies for oral stabilization and delivery of active molecules can be seen in Brayden and Mrsny (2011). In this case delivery technologies are considered primarily for molecules that fall into the biopharmaceutical as class III molecules (drugs with poor intestinal permeability). Commercially relevant peptides include: insulin, glucagon-like-peptide 1 and analogues as salmon calcitonin, octreotide, parathyroid hormone and LHRH, analogues such also as leuprolide. Nonpeptide macromolecules with permeability issues include unfractionated heparin, low-molecular weight heparins, antisense oligonucleotides and vancomycin. Small molecules such as bisphosphonates, acyline, amphotericin, camptothecin and gentamycin are also recognized as relevant peptides. At present there are very few attempts on application of these strategies for oral delivery systems for active substances from whey proteins; oral administration remains poorly bioavailable mainly. Nonetheless this review article provides an overview into novel pharmaceutical approaches to improve oral bioavailability of therapeutic proteins and peptides which could be explored in to develop next researches on the oral delivery of protein and peptide or to develop new delivery systems capable of delivering therapeutic products from whey in an effective manner.

1. ADDING OF INTESTINAL ABSORPTION ENHANCERS AND ENZYMES INHIBITORS Bioavailability of drugs with molecular mass above 500-700 Da, decreases sharply, but for drugs of less than 500-700 Da, their bioavailability depends of adhesion power in the gastrointestinal tract. Currently, pharmaceutical strategies aim to enhance the permeability and develop safe, efficacious and highly-potent proteinous drugs (Muheem et al., 2014). Adding of absorption enhancers (promoters) allows particularly that therapeutic compounds permeate across biological membranes and thus that they reach the site of action to exert pharmacological effect (Shaji and Patole, 2008). Promoters can directly increase paracellular or transcellular

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permeability under the following mechanisms: Temporarily disrupting the structural integrity of the intestinal barrier; decreasing the mucus viscosity; opening the tight junctions and increasing the membrane fluidity (Renokuntla et al., 2013). The effective application of a promoter depends on the physicochemical properties of the drugs, regional differences in intestinal membrane, nature of the vehicle and other excipients. Promoters should also be safe and non toxic, pharmacologically and chemically inert, non-irritant, and non-allergenic (Jitendra et al., 2011; Senel and Hincal, 2001). Some substances as surfactants, chelating agents, bile salts, cationic and anionic polymers, swell able polymers like starch, polycarbophil, and chitosan; acylcarnitines, fatty acids and their derivatives have been reported as absorption enhancers, some of which have „generally recognized as safe‟ (GRAS) status as food additives (Maher et al., 2009). Researches on use of sodium caprate (C10) as absorption agent are of the most advanced in clinical trials. It has generally recognized as safe (GRAS) status in dietary supplements and there is extensive knowledge from its widespread use as an excipient, which may suggest that it may have less safety hurdles than new chemical entity-type promoters (Maher et al., 2009). In successive preclinical studies, it was demonstrated that C10 improved oral macromolecule permeation across Caco-2 monolayers, isolated rat and human intestinal mucosa, as well as in rat intestinal instillations and perfusions (Maher et al., 2009a, 2009b; Kamm et al., 2010). Their oral bioavailability has also been successfully demonstrated in clinical studies for enteric coated soliddosage forms (Leonard et al., 2006; Hardee et al., 2008; McHug et al., 2009; Amory et al., 2009). The most important classes of proteins and peptides that have been investigated with this approach include insulin, gonadotropin-releasing hormone, calcitonin, the enkephalins, the glucagons, other hormone analogs, enzyme inhibitors and vaccines. Use of absorption enhancers is currently the most studied alternative to improve oral bioavailability of therapeutic proteins and peptides. For nearly half a century, various agents have been analyzed, but to-date none have been approved for commercial use (Wang et al., 2010).

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Special focus is also seen in dendrimers as promotors system. They comprise a category of branched materials with diverse functions that can be constructed with defined architectural and chemical structures. Dendrimer conjugates have so far been studied for their versatile capabilities to enhance stability, solubility and absorption of various types of therapeutics. These systems can be made with monodisperse molecular weight, tunable sizes and nanoscale starburst branches, making them ideal scaffolds for the creation of functional nano-biomaterials (Liu et al., 2014). Mucoadhesive polymeric are also other systems used as enhancers, they adheres to the intestinal mucus and thus increases the drug concentration gradient. The most common systems which have been studied extensively as mucoadhesives, are chitosan, poly [lactic-co-glycolic acid] (PLGA), thiolated polymer and alginate. The addition of further specific substances as protease inhibitors have different role; their aim is inhibits enzymes present in de tract GI. Inhibitors are used to retard the action of enzymes which could degrade peptides and proteins present in the GI system. Conjugate adding of absorption enhancer and enzyme inhibitor have also been tested to overcome the barriers of oral administration of therapeutic compounds (Aungst, 2012; Checkoway et al., 2012). Major results on these systems are found on insulin in presence of excipients that include pancreatic inhibitor, soybean trypsin inhibitor and aprotinin. Inhibitors of insulin degrading-enzyme include phenantrolyne and bacetracin (Agarwual and Khan 2001). Polymer inhibitor conjugates such as carboxymethyl cellulose-Elastin (CMC-Ela) have principally been studied as possible additives for administration of active molecules, where they have shown in vitro and in vivo protection against enzymes trypsin, a-chymotrypsin and Elastase (Marsüchtz and Bernkop-Schnurch, 2000a, 200b). Other enzyme inhibitor or conjugate systems can see in Park et al., (2011). To date the use of conjugate systems or individual enzyme inhibitor to enhance bioavailability of molecules, still stay in study because of possible absorption of unwanted proteins, disturbance of the digestion of nutritive proteins and stimulation of protease secretion as a result of feedback regulation (Morishita et al., 2006; Muheem et al., 2014). Table 1 shows recent researches on some additive substances used to enhance the bioavailable of protein and peptide drugs.

Table 1. Absorption enhancers used to enhance or promote the absorption of protein and peptide drugs Absorption enhancers or enzyme inhibitor N-trimethyl chitosan (TMC 60, 40)

Proteins and peptides drugs Hydrophilic [14C]mannitol

Polymer-inhibitor conjugates carboxymethylcellulose (NaCMC) Bowman–Birk inhibitor (BBI) and elastatinal, respectively Polymer-inhibitor conjugates carboxymethylcellulose (NaCMC)-Bowman-Birk inhibitor (BBI) and CMC-elastatinal compressed to 2 mg microtablets and enteric coated with a polymethacrylate.

Insulin

Fatty acid (MCFA) Sodium captrate (C10) and ethyleneglycol-bis-(b-aminoethyl ether)-N,N9-tetraacetic acid (EGTA)

[3H]mannitol and [14C]dextran

Polycarbophilcysteine conjugate, insulin, and mannitol

Enhancement action Permeability and integrity of intestinal Caco-2 cell monolayers Inhibitory activity of Polymer-BBI. Insulin remained stable towards enzymatic attack after 4 h of incubation Strong protective effect for insulin. High cohesiveness of the dosage form. Controlled release of insulin in a time period of 10 h. Decrease in basal glucose levels of 20% to 40% during a time period of 80 h in rats. Increase in permeability coefficients using C10 for both molecules

Authors Thanou et al., (2000)

Application trials In vitro

Marsüchtz and Bernkop-Schnurch (2000a)

In vitro

Marsüchtz and Bernkop-Schnurch (2000b)

In vitro and in vivo

Coyne et al. (2000)

In vitro and in vivo

Absorption enhancers or enzyme inhibitor Fatty acids (MCFA) Sodium captrate C10, C12 and taurocholate (NaTC)

Proteins and peptides drugs Cyclopeptidic alpha(nu)beta(3)antagonist (EMD 121974)

Fatty acid (MCFA) Sodium captrate (C10)

Antisense oligonucleotides ISIS 2503 (phosphorothioate) and ISIS 104838 (methoxyethyl modified phosphorothioate) Clodronate

Palmitoyl carnitine chloride (PCC), N-trimethyl chitosan chloride (TMC), sodium caprate (C10), and ethylene glycol-bis(βaminoethyl ether)-N,N,N′,N′tetraacetic acid (EGTA) EDTA and sodium captrate

Norfoflaxin

Enhancement action

Authors

Significant absorption enhance with MCFAs and increase in permeability coefficients using NaTC Increase of oral delivery of molecules in pigs, membrane-permeation effect was rapid, shortlived and dose independent.

Kamm et al. (2000)

Application trials In vitro

Raoof et al. (2002)

In vitro

Increase of permeability through Caco-2 cell by enhancers

Raiman et al.(2003)

In vitro

Absorption kinetic of noforflaxin was markedly accelerated with enhancers. In vitro enhancers increased norfoflaxin dissolution kinetic

Dos Santos et al. (2003)

In vitro

Table 1. (Continued) Absorption enhancers or enzyme inhibitor Mixture of Labrasol and D-atocopheryl polyethylene glycol 1000 succinate (TPGS)

Proteins and peptides drugs Glycopeptide antibiotic Vancomycin hydrochloride (VCM)

Labrasol

Heparin (LMWH)

Epinephrine and α y β-adrengic receptor antagonist and agonist

Dextran (Mw 4, 10 and 40 KDa)

Fatty acids (sodium caprylate and caprate), cyclodextrins (βcyclodextrin, hydroxypropyl βcyclodextrin) and bile salts (sodium cholate and deoxycholate)

Cyclosporin A and lovastatin

Enhancement action Labrasol and TPGS improve intestinal absorption of hydrophilic macromolecular drug, VCM Increase the intestinal LMWH absorption. Maintaining high drug concentration gradient across intestinal wall is important to obtain increased intestinal LMWH absorption. Intestinal absorption of dextran (4000 Da) with no toxic effect Enhance permeability of drug by decreasing lipid membrane fluidity and/or interacting with hydrophilic domains of membrane, and has the

Authors Rama Prasad et al. (2003)

Application trials In vitro

Rama Prasad et al. (2004)

In situ

Kamio et al. (2005)

In situ

Sharma et al. (2005a)

In vitro

Absorption enhancers or enzyme inhibitor

Proteins and peptides drugs

Sodium caprate (SC, Piperine and sodium deoxycholate (SD) separately

Clase III efotaxime sodium (CX)) and Class IV (cyclosporin A (CSA))

Different doses of product GIPET®, which is based on medium-chain fatty acids

Alendronate, desmopressin and low-molecularweight heparin Ophiopogon japonicus polysaccharide (OJP) Gentamicin sulfate (GM)

Sodium caprate (SC)

Caprylocaproyl macrogol-8 glyceride (Labrasol) in Microemulsifying systems

Deoxycholic acid (DOCA)

Heparin (LMWH)

Enhancement action potential to improve oral delivery. Increased intestinal permeability and substantial absorption of molecules and promotion of peroral bioavailability in rats Increase in permeability on the human intestine, action of short-lived and reversible in vivo Increase of residence times and fast dilution by intestinal fluids in rats In rat ileum, only 25% Labrasol solution enhanced GM permeability and Labrasol micelles increase membrane lipid fluidity The absorption of orally administered LMWH–

Authors

Sharma et al. (2005b)

Application trials

In situ and in vivo

Leonard et al. (2006)

In vivo and clinical

Lin et al. (2006)

In situ

Koga et al. (2006)

In vitro

Lee et al. (2006)

In situ

Table 1. (Continued) Absorption enhancers or enzyme inhibitor

Proteins and peptides drugs

Chitosan oligomers

dextran 4kDa (FD4) e insulin

Fatty acid (MCFA) Sodium captrate (C10) and melittin

[(14)C]-manitol and FITC-dextran 4kDa (FD4)

Enhancement action DOCA occurred in all parts of the small intestine, particularly in the ileum without causing any damage. The absorption of FD4 was improved by chitosan hexamer and dimer. Increase in plasma insulin levels and a significant hypoglycemic effect after jejunal administration of insulin with chitosan hexamer. Melittin and C10 improved bioavailability of polar sugars across the jejunum and colon of rats in situ, which was associated with some degree of mucosal damage.

Authors

Application trials

Gao et al. (2008)

In situ

Maher et al. (2009a)

In situ

Absorption enhancers or enzyme inhibitor Fatty acid (MCFA) Sodium captrate (C10) and melittin

Proteins and peptides drugs [(14)C]-manitol and FITC-dextran 4kDa (FD4)

Different doses of product GIPET®, which is based on medium-chain fatty acids

Acyline antagonist of Gonadotropin releasing hormone (GnRH)

Fatty acid (MCFA) Sodium captrate (C10)

FITC-dextran 4kDa (FD4)

Polyamidoamine (PAMAM) dendrimers

5(6)Carboxyfluorescein (CF), fluorescein isothiocyanatedextrans (FDs), calcitonin and insulin

Enhancement action

Authors

C10 and melittin increased the transport of paracellular flux markers across isolated human and rat colonic mucosae Suppression of serum LH, FSH and serum testosterone by all doses of GIPET®-enhanced oral acyline after 6 h, with suppression reaching a nadir 12-24 h after dosing. Enhancement with C10 is narrow, compromised intestinal epithelial barrier of rats is rapidly restored to its native state within an hour. The absorption of CF, FD4 and calcitonin from the rat small intestine was significantly enhanced in the presence of lower concentrations of PAMAM dendrimers

Maher et al. (2009b)

Application trials In vitro

Amory et al. (2009)

In vitro and in vivo

Wang et al. (2010)

In vitro and in vivo

Lin et al. (2011)

In vitro

Table 2. Modifying of physicochemical nature of some proteins and peptides Enhance modification Cristallization Acylation with butyric acid and the application of a protease inhibitor Glycosylation on Ser-NH2

Proteins and peptides drugs b12, a human IgG1k antibody Enkephalin peptide and lipophilic derivatives of insulin Synthetic opioid peptide

Cocrystalization

Octanoyl-NepsilonLysB29-human insulin (C8-HI)

Monomeric and dendrimeric tetrabranched Form by residue substitution or sequence shortening

Synthetic antibacterial peptide

Enhancement action Crystallization of molecule Absorption of proteins across the intestinal membrane Increase of analgesic potency, duration of analgesia, metabolic stability of peptide and permeability without significantly altering the initial volume of distribution. Increase of transference of insulin across the mucosal membranes of the large intestine and improved its stability against intestinal enzymatic degradation in beagle dogs. Increase of peptide stability to blood proteases, low hemolytic activity, and low cytotoxic effects on eukaryotic cells

Authors Ollmann et al., (2000) Uchiyama et al. (2000)

Application trials No application In vitro

Egleton et al., (2001)

In vitro and in vivo

Brader et al. (2002)

In vitro and in vivo

Pini et al. (2005)

In vitro and in vivo

Enhance modification Substitution of two polycationic lipophilic-core carbohydratebased dendrons 2a-b and five polycationic lipophilic-core peptide dendrons 3-6, containing four arginine or lysine terminal residues Cyclization between the two allylglycine residues

Proteins and peptides drugs Heparin (LMWH)

Synthetic opiode peptide

Attaching of 2-aminooctanoic acid moiety to the N-terminus

Thyrotropinreleasing hormone

Chemical conjugate of heparin (LHD) and deoxycholic acid (DOCA) by conjugating the carboxylic group of each substance Complexation with acid derivative cationic deoxycholylethylamine (DCEA)

Heparin (LHD)

Heparin (LMWH)

Enhancement action Penetration enhances. Dendron‟s containing terminal lysine had a significant antifactor Xa activity was obtained when low molecular weight heparin was coadministered with dendron 5. Increase of peptide activity and stability under hydrolysis conditions Permeability of peptides, increased stability and increase lipophilicity which results in enhanced passive diffusion. Increase of LHD absorption in the jejunum and ileum of the small intestine in Caco-2 cell monolayers for mimicking the intestine. Improve in lipophilicity of drug. Complex was absorbable through all regions of the small intestine of rats without causing tissue damage

Authors Ayes et al. (2006)

Application trials In vitro and in vivo

Berezowska et al. (2007)

In vitro

Wessling et al. (2007)

In vitro

Kim et al. (2007)

In vitro and in vivo

Lee et al. (2007)

In vitro and in vivo

Table 2. (Continued) Enhance modification Glycosylation at both N- and Ctermini by lipoamino acids

Proteins and peptides drugs Synthetic dipeptide

Conjugation with lipoamino acid to the N-terminus into phosphatidylcholine liposomes.

Neuroactive peptide

Acetylation, and Pegylation (PEG of N-and C-termini by Npyroglutamate and C-amidation) into micelles Coupling the succinimidyl active esters of the acidic retinoids with appropriately protected amino acids or peptides followed by deprotection

Synthetic antibacterial peptide

Hyperbranche drendimer by residue substitution

Conjugates of amino acids with all-transretinoic acid (ATRA) and shorter polyene chain analogues CLB anticancer drug

Enhancement action

Authors

Peptides stability towards enzymatic degradation from exopeptidases and the permeability across biological membranes Significant increases both aqueous solubility and metabolic stability. In vitro endothelial permeability of the liposomal formulation Protection of peptides from exopeptidases and increase antimicrobial potency

Bergeon et al. (2008)

Conjugates with α-amino acids with lipophilic side chain, such as 7, or linear amino acids, such as 9, significantly decreased prostate cancer LNCaP cell number. Appropriate delay of the release of drug and adjust of the ratio of drug released in total drug loading

Application trials In vitro

Koda et al. (2008)

In vitro

Zhang et al. (2008)

In vitro

Sadikoglou et al. (2009)

In vitro and in vivo

Yuanyuan et al. (2009)

In vitro

Enhance modification Chemical conjugate of heparin (LHD) and deoxycholic acid (DOCA) by conjugating the carboxylic group of each substance Replacement of the disulfide bonds by thioether, selenylsulfide, diselenide, and ditelluride bridges Chemical conjugate of heparin (LHD) and deoxycholic acid (DOCA) by conjugating the amine group of Ndeoxycholylethylamine (EtDOCA) with the carboxylic groups of heparin at various DOCA conjugation ratios. Peptides cyclization with thioether bridges between a D-amino acid and an L-amino acid by applying an enzymatic method.

Proteins and peptides drugs Heparin (LHD)

Enhancement action

Authors

Application trials In vivo

Increase of the patency of oral LMWH-DOCA (10 mg/kg) from 15% to 45% in microvascular anastomosis of the crushed artery in rats, after a microsurgery. Peptide stability in human plasma and increase of their potent nicotinic antagonist

Eom et al. (2010)

Muttenhaler et al. (2010)

In vitro

Antiangogenic heparin (LHD)

Inhibition of tumor growth in SCC7 mice model and A549 mice xenograft model. LHD4 was orally absorbable, showed minimal anticoagulant activity and inhibited tumor growth

Park et al. (2010)

In vitro and in vivo

Decapeptide Luteïnizing Hormone Release Hormone (LHRH)

Increase of therapeutic potential and stability thioether LHRH isomer with strongly enhanced proteolytic resistance compared to natural LHRH.

Rink et al. (2010)

In vitro

Synthetic peptide Oxytoxin

Table 2. (Continued) Enhance modification Conjugating 2-O- or 6-Odesulfated LMWH with deoxycholic acid (DOCA) or bisDOCA

Proteins and peptides drugs O-desulfated heparin (ODS-LMWH)

Replacing the second Lys in the sequence by the polar noncharged amino acid threonine

Synthetic antagonist peptide

N-terminal modification with lipoamino acid and lactose succinamic acid, and the inclusion of D-amino acids. Develop of LHT7-ApoPep-1-C, an apoptosis-homing by peptideconjugated variant of LHT7

Endogenous opioid peptide Endomorphin-1 Heparin (LMWH)taurocholate conjugate LHT7-ApoPep-1

Enhancement action

Authors

6ODS-LHbD showed the highest oral bioavailability in rats (19.3%). Inhibited neovascularization in the joint, the increase of hind-paw thickness, and the structural damage in the bone. Increase of peptide activity to inhibit of TNFα secretion by synovial cells from rheumatoid arthritis patients Metabolic stability towards enzymatic degradation in human plasma

Hwang et al. (2012)

Decreasing vessel formation and increased apoptotic area in tumor tissues. Conjugate drug exerted antitumor effects by suppressing tumor vessel growth and homing to apoptotic cells within the tumor.

Application trials In vitro and in vivo

Santos et al. (2012)

In vivo, clinical studies

Mansfeld and Toth (2012)

In vitro

Bae et al., (2013)

In vitro and in vivo

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2. Modifying of Physicochemical Nature of Active Molecules Oral bioavailability of active molecules can be enhanced by modifying of their physicochemical nature with the aim of to increase membrane permeability, penetration of product in the tract GI and proteolytic stability in this place to be absorbed transcellularly. Physicochemical modifications are performed into specific sites of molecule or into structure by phase or pH changes. Some modification alternatives include crystallization, lipidization, glycosylation, substitutions with non natural amino acids or pseudo-amino acids and peptide cyclization; use of other unnatural residues, modification of peptide bonds, termini protection/modifications and conjugation to carrier constructs, such as biocompatible polymers (Vlieghe et al., 2010). Significant advances have been found under this approach. Table 2 shows actual relevant structural modifications on proteins and peptides to enhance their physicochemical properties. Prodrugs are an example of the molecules modification; they are currently recognized as an active pharmacological moiety which has been converted into inactive form through chemical modification. These approaches enhance the solubility, permeability and targeting of small molecules but it faces challenges, such as limitation in methodology, stability of proteins and structural complexity (Hsieh et al., 2009). Crystallization of molecules has particularly shown major benefits in biopharmaceutical applications due to advantages in better handing and improves stability of drugs. An evident result is the insulin modification which is used in crystals form as therapeutic molecule. Long acting insulin formulations are the best characterized naked protein crystals with control release properties. To date, various commercial formulations of this protein are available for their use (Basu et al., 2004). Additional formulations containing various proteins and peptides have been studied and patented, however crystallized products are still very few recognized and commercialized; large quantities required for clinical efficacy have limited their application. Other recognized alternative as strategy to improve the uptake of peptide therapeutics is the lipidation of molecules, which is continually used to form a pro-drug. Lipidation process consists in the modification of a molecule by attachment of a lipid moiety on an unstable peptide without decreasing its activity or selectivity (Hsieg et al., 2009). The N-terminal conjugation with lipidic amino acids can increase the half-life of active molecules, to improve absorption by passive diffusion across epithelial barriers, notably in the gastrointestinal tract and the central nervous system (Goodwing et al., 2012).

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Glycosylation is also often used to increase the half-life of therapeutic substances by addition of carbohydrate moieties. This modification produces changes in the molecular structure of proteins (glycoprotein) and peptides (glycopeptide) with effects on their pharmacodynamic and/or pharmacokinetic properties while maintaining optimal activity. The physico-chemical properties of active molecules can also be enhanced by the substitution of natural amino acids with unnatural ones. Dconformation, N-methylation, tetra-substitution, -amino acids, side chain methylation(s), cyclization, terminal capping, truncation, and disulfide bond engineering can also improve stability to proteolytic degradation of peptides (Muttenhaler et al., 2010). Chemical modification to peptide bonds by the replacement of the carbonyl or amide groups with esters, sulfides and alkyls, particularly produces stable peptides which are classified as peptidomimetics (Liskamp et al., 2011). Particular attention in modification of synthetic peptides by dendrimer bonds has also been seen in several reports. For drug delivery purposes, dendrimers may serve at least two functions: to present bioactive ligands to enable targeted cellular interactions carry drug molecules and transport them to the diseased tissue. Therapeutic compounds may associate with the interior branches of dendrimers through hydrophobic interactions or through covalent bonding, which is convenient when the molecule contains functional groups that are readily ligated to dendrimer (Liu et al., 2012). The core and the interior branches of a dendrimer can contain a natural o synthetic peptide. The branches of drendimer grow radially with each successive generation, so the size and functionality of the peptide-dendrimer can be simultaneously tuned and used in the development of artificial proteins, antimicrobial/anti-viral reagents, and drug delivery and as release materials (Dutta et al., 2010). Scaffolds dendrimers provide extra functional groups for drug conjugation; in this case hydrophilic dendrimer conjugates may enhance the solubility of drugs or drug-loaded devices by drug inclusion into the pit of the cyclic oligosaccharides (Kojima et al., 2009). This method has been used for the solubilization of hydrophobic small molecules involved in anti-cancer, depressant, -inflammatory and -microbial applications. Therapeutic compounds can also directly forms complexes with dendrimers containing counter-charged groups. This method has been widely studied for nucleic acidbased therapeutics and other negatively charged therapeutic.

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3. USE OF CARRIER SYSTEMS AS VEHICLES OR ENTRAPMENT AND DELIVERY ACTIVE MOLECULES Currently effective alternatives by biomolecules delivery systems are associated with encapsulation or adsorption on carriers. Advances in this topic have led to develop of an ever-increasing number of them, as are polymeric systems hydrogels, emulsions, nanoparticles, microspheres pellets, implants, films and liposomes. These systems are principally designed to enhance stability of molecules, to prolong their activity and protect the drugs from the harsh environments of the stomach before releasing the drug into more favorable regions of the GI tract, specifically the lower regions of the intestine. However the encapsulation and adsorption of molecules in carriers have not been easy, since each molecule requires its own specific condition for stability, solubilization and control releases immune elimination. In addition permeation problems have been exposed in several reviews (Morishita and Peppas, 2006; Brayden and Msrny 2011; Muhem et al., 2014). Recently specific combinations of approach enhance have facilitated permeation of proteins through paracellular opening. Among of carrier systems are found those that are recognized by endogenous cellular-transport systems in the GI tract. In this case attaching an active substance to a carrier that is accepted by a peptide-influx transporter, improve its oral absorption (Morishita and Peppas, 2006). The use of this type of carriers has been the most practical and safer strategy for increasing intestinal absorption of peptides and proteins (Han and Amidon, 2000; Brayden and Msrny 2011). Table 3 shows some of the most studied methods for delivery proteins and peptides using different carrier and combination with other approach enhance. Special focus is seen in emulsion formulations, as functional method of entrapping or load drugs. Emulsions are complex microstructure where the dispersed droplets contain smaller droplets of active biomolecules inside (emulsions can be constituted by various layers). Emulsions are used to encapsulate, protect molecules and delivering them to specific sites within the human body. Multiple layers presents advantages over simple oil-in-water (O/W) emulsions for encapsulation, such as the ability to carry both polar and non-polar molecules, and a better control over their releasing.

Table 3. Carrier systems as approach enhance to active molecules Carrier systems Poly(vinyl alcohol)-graftpoly(lactic-co-glycolic acid), PVAL-g-PLGA w/o/w doublé emulsion technique. Amphipathic peptide (Pep-1)

Proteins and peptide drugs bovine serum albumin, ovalbumin, cytochrome c and FITC-dextran

Peptides and proteins

Lipospheres (LS)

Two lipophilic drugs, retinyl acetate and progesterone, and one hydrophilic drug, sodium cromoglycate (SCG).

Solid triglyceride nanostructures coated with chitosan orPoly (ethylene glycol)

Salmon calcitonin (cST)

Enhance

Authors

Status

Offers additional degrees of freedom to manipulate properties molecular weight, glass transition temperature and hydrophilicity.

Pistel, et al. (2001)

No application

Stability in physiological buffer Deliver. A variety of peptides and proteins into several cell lines in a fully biologically active form. The SCG release reached the 100% of the total amount of drug after 5 h the retinyl acetate release efficacy within the first 8 h was 27%, in the same period, the amount of progesterone released was 63%. Reduction in the transepithelial electrical resistance of the cell monolayer Caco-2, increase transport of drug and reduction in the serum calcium levels in rats

Morris, et al. (2001)

No application

Cortesi, et al. (2001)

No application

Fuentes et al. (2005)

In vitro and in vivo

Carrier systems Nanoparticles, CScoated oil nanodroplets (nanocapsules) and CScoated lipid nanoparticles Microemulsifying systems (SMEDDS) of Caprylocaproyl macrogol-8 glyceride (Labrasol) Poly(D,L-lactic acid) and poly(D,L-lacticcoglycolic acid) nanoparticles

Encapsulation into nanoparticles prepared with a blend of a polyester and a polycationic polymethacrylate by the double emulsion method

Proteins and peptide drugs Salmon calcitonin (cST)

Enhance

Authors

Status

intestinal absorption of peptide, and long-lasting decrease in the calcemia levels observed in rats.

Prego et al. (2005)

In vitro and in vivo

Manitol

Significantly increase in mannitol permeability across the monolayer Caco-2 cell

Sha et al. (2005)

In vitro

Tetanus toxoid, lysozyme, and Insulin

Tetanus toxoid, 80% entrapment efficiency and retained more than 95% of its antigenicity. Lysozyme 90% entrapment efficiency did not damage the primary structure Of insulin. Oral absorption of nanoparticles between 4 and 10 or 12 h, with a delayed onset of action ranging from 3 to 4 h in rabbits. Bioavailabilities of 51% and 59%. Anticoagulant effect prolonged up to 8 h.

Bilati et al. (2005)

No application

Hoffart et al. (2006)

In vivo

Tinzaparin

Table 3. (Continued) Carrier systems Chemically modified heparin–DOCA nanoparticles in aqueous condition VB12- dextran NPs conjugate

INS nanoparticulate system of chitosan (CS), triethylchitosan (TEC), and dimethylethylchitosan (DMEC) MicrospheresofPLGA RG 503H Mucoadhesion of casted free pectin/chitosan combination films

Proteins and peptide drugs Dexorubicin

Insulin

Insulin

Glial cell line-derived neurotrophic factor (GDNF) glycosylated Paracetamol

Enhance

Authors

Status

Nanoparticles induced tumor volume reduction

Park et al. (2006)

In vitro and in vivo

High insulin entrapment and faster insulin release with low levels of cross-linking. These VB12-NPs conjugates (150-300 nm) showed profound (70-75% blood glucose reductions) and prolonged (54 h) anti-diabetic effects with biphasic behaviour in STZ diabetic rats. In vitro studies, a sustained release characteristic for 5 hours

Chalasani, et al. (2007)

In vitro

Bayat et al. (2008)

No application

A PC-12 cell neurite outgrowth bioassay demonstrated that the in vitro GDNF released was bioactive. Increasing of mucoadhesion and drug permeability into porcine small intestine

Garbayo, et al. (2008)

In vitro

Hagesaether et al. (2009)

In vitro and in vivo

Carrier systems Composite microparticle based on chitosan, alginate and pectin with improved pH sensitivity Microspheres of low degree of esterification (DE) pectin with calcium and coated with chitosan (PCaC) Rat erythrocytes

Proteins and peptide drugs bovine serum albumin (BSA)

Enhance

Authors

Status

The releases of BSA increased significantly, in the presence of pectinase

Yu et al. (2009)

In vitro

Mangiferina

The highest release (7.8 mg of mangiferin/g of bead)

De Souza et al. (2009)

In vitro and simulation study

Antiretroviral Zidovudine (AZT)

Variables such as the initial AZT concentration, the dialysis time, and the dialysis bag/buffer volume ratio. Increased lipolysis in the presence of amylase and/or trypsin was attributed to the digestion of the encapsulant, which facilitated displacement of the interface of oil droplets by bile salts.Facilitated lipase adsorption at the oil–water interface Conservation of the bioactivity of insulin. Increasing the loading ability and optimizing the release profile. Reduction in blood glucose level and powerful therapeutic effects in rats

Briones, et al. (2010)

In vitro

Chung, et al. (2010)

In vitro

Wei et al. (2010)

In vitro and in vivo

Nanocapsules of sodium caseinate and starch

fish oil powders protein and resistant starch

Chitosan microspheres by the SPG membrane emulsification technique and glutaraldehyde cross-linking method

Insulin

Table 3. (Continued) Carrier systems Alginate-chitosan microspheres by membrane emulsification with ion (Ca2+) and polymer (chitosan) Chitosan nanoparticles (CS NPs) by ionic crosslinking with hydroxypropyl methylcellulose phthalate (HPMCP) Encapsulation of drug with BaSO4, sugar and dextrin as the excipient.

Proteins and peptide drugs Insulin

Enhance

Authors

Status

Activity maintenance (99.4%) of the drug. Stable and sustained insulin release (14 days). Chemical stability of insulin into the simulated gastric of rats for 2 h.

Zhang et al. (2011)

In vitro and in vivo

Insulin

Improvement in the intestinal mucoadhesion and penetration in rats. Stability and biological activity of the peptide. Increased the hypoglycemic effect of insulin

Makhlof et al. (2011)

In vitro and in vivo

Heparin (LMWH)

Protecting of drug from releasing in stomach and small intestine. Decreasing of serum levels of TNFα, IL-6 as well as FXa Entrapment efficiency is 94%. The pravastatin loaded cells has no change in the morphology. Pravastatin releasing from carrier cell was 83% The drug release in simulated gastric fluid (0-2 h), simulated intestinal

Luo et al. (2011)

In vitro and in vivo

Harisa, et al. (2011)

In vitro

Dutta and Sahu (2012)

In vitro

Encapsulation in human erythrocytes by endocytosis method

Pravastatin

Encapsulation in spherical magnetic

Diclofenac sodium (DS)

Carrier systems nanocarrier of 100-150 nm dimensions made of pectin and chitosan (MPCh-DS0.05) Chitosan–Pluronic nanoparticles

Proteins and peptide drugs

Enhance

Authors

Status

fluid (69% in 2-5 h), simulated colonic fluid (5-60 h)

Anticancer Gemcitabine (GC; 2′,2′difluorodeoxycytidine)

Nanoencapsulation within lipid nanovesicles in a multiple water-in-oil-inwater emulsion. Encapsulation into polylactide-co-glycolide (PLGA) based microspheres

Lactoferrin

Whey microbeads

riboflavin and peptides

Immunoglobulin G

Increase in the cytotoxicity of gemcitabine embedded. Thedrug was dispersed in its amorphous. Mucoadhesive for the nanoparticles was of 90 %. Effectiveness of the encapsulation procedure and Lactoferrin nanovesicle were successfully employed at lab-scale antimicrobial trials. Loading of up to 6% (w/w) and an encapsulation efficiency of up to 60% (w/w). Preserving the integrity of the encapsulated antibody.

Hosseinzadeh, et al. (2012)

In vitro

Balcao et al., 2013

In vitro

Marquette, et al. (2013)

No application

The migration of riboflavin, amino acidsand peptides from solution into the whey microbeads was shown to be via a process of partition influenced by hydrophobic

O´Neill, et al. (2014)

No application

Table 3. (Continued) Carrier systems

single carbon nanotubes (CNTs)

Proteins and peptide drugs

α-helical peptide

Enhance interactions between the compound and the microbead. The peptide could be modulated through the CNTs‟ geometry (armchair and zigzag).

Authors

Zhang, et al. (2014)

Status

No application

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Polymeric micelles nanomaterials to delivery active molecules are particularly seen today as one promise in the pharmaceutical area. There are two categories: nanocapsules and nanospheres, nanocapsules are vesicular systems with a polymeric shell and an inner core, their hydrophobic core acts as a micro reservoir for the encapsulation of hydrophobic drugs and they can cross the intestinal barrier after oral administration (Yadav et al., 2011) whereas nanospheres are polymeric matrices. Feasible nanoformulations are prepared by spray drying, lyophilisation or solvent evaporation to obtain spheres or capsules, in any system, their size is of 10 to 1000 nm, thus overcoming the instability of emulsion. Carriers as solid dispersions are studied as the most successful strategies to improve drug release of poorly soluble drugs. Solid dispersions can improve molecules stability and performance by increasing drug-polymer solubility, amorphous fraction, particle wettability and particle porosity. Some advantages that nanocarriers offer are their controlled release properties and the protection to the compound of interest; therefore, they might be useful for the oral delivery of macromolecules. However the physicochemical properties of active substances and conditions of absorption from the gastrointestinal, has limited their function (Dollo et al., 2003; Mahato et al., 2003). In addition protein adsorption and denaturation at the water/solvent interfaces is one of the major issues that lead to a decrease in the protein bioactivity occurring during the encapsulation process. Other result have showed that polymeric nano carriers can reduce the transepithelial resistance (Prego et al., 2005), therefore, their potential use for clinical applications is still uncertain (Muheem et al., 2014). Applications of polymerized compounds with covalent double bonds to improve the stability of emulsions and biomolecules are reported in Muheem et al., (2014). Emulsions are equipped with the envelope liposomes or coated with a mucoadhesive polymer to significantly improve of hydrophilic macromolecular drug absorption from the intestine. Successful cases of the application of these systems are the vaccines. The integration of functional viral envelope proteins into liposomes has led to an antigen carrier and delivery system termed a virosome, a clinically proven vaccine platform for subunit vaccines with an excellent immunogenicity and tolerability profile (Felnerova et al., 2004). Synthetic hydrogels present also a possibly efficient and convenient way to administer peptides and proteins. In the method, the therapeutic agent is diluted into the initial monomer mixture prior to polymerization; thus the diluted agent is trapped inside the polymer network that forms around it

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(Blanchette et al., 2004). Characteristics of hydrogels make these copolymers model also carriers for oral delivery of proteins and peptides. They are considered as ideal to protect the entrapped active molecules in gastric fluid since they can protect them from degradation by digestive enzymes in the stomach and they can also inhibit the activity of Ca2+ dependent proteolytic enzymes. After of the review about approach enhance to oral administration of proteins and peptide, we have observed significant advances in this topic and relevant data have been identified in these results. Since last decade several methods have been designed and tested to find an adequate system to active molecules delivery. Protection of proteins from degradation, prolonged or modified release and increase of potential activity are the principal objectives in developing of these methods. Adding of promoters, molecule modification and encapsulation or use of solid carriers, are the principal strategies to enhance the oral bioavailability of molecules. However few of them are today applied and commercialized and reports on clinical studies are minimal. Regarding to oral administration of active molecules of whey, isolated reports on encapsulation of peptides and proteins were found in some manuscripts. In general, the aim of the encapsulation methods has been the increase their half-life against proteases. Then we do a count soon of these relevant investigations. Pistel et al. (2001) obtained brush-like branched polyesters by grafting poly(lactic-co-glycolic acid), PLGA, onto water-soluble poly(vinyl alcohol) (PVAL) for the microencapsulation of hydrophilic macromolecule bovine serum albumin using a w/o/w double emulsion technique. Surface morphology, particle size, encapsulation efficiencies and protein release profiles were characterized in these tests. Trials in vitro showed that shorter PLGA chains increased drug release in the erosion phase; release profiles from 2 to 12 weeks could be attained by modification of composition and molecular weight of PVAL-g-PLGA. Pini et al. (2005) synthesized antibacterial peptides (similar structure of molecules from bovine whey) in dendrimeric form (multiple antigen peptides MAP). Their antibacterial activity against E. coli was much higher than that of the monomeric form. Dendrimeric peptides showed also stability to blood proteases, low hemolytic activity, and low cytotoxic effects on eukaryotic cells. These results signify that dendrimeric peptides are promising candidates for the development of new antibacterial drugs.

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Yu et al. (2009) synthesized composite microparticle drug delivery systems based on chitosan, alginate and pectin with improved pH sensitivity for oral delivery of bovine serum albumin. The releases of BSA at pH 7.4, pH 6.8 and pH 6.0 increased significantly, especially in the presence of pectinase. These results clearly suggested that the microparticles had potential for sitespecific protein drug delivery through oral administration. Marquette et al. (2013) analyzed the stability of bovine IgGs into polylactide-co-glycolide (PLGA) microspheres. In this case, stability of molecule was achieved in these systems which were also resistant against attack by the enzymes. Balcao et al. (2013) analyzed the stabilization of three-dimensional structure of Lf. Authors tested nanoencapsulation of bovine protein within lipid nanovesicles, integrating a multiple water-in-oil-in-water nanoemulsions. Encapsulate proteins produced antimicrobial effects in laboratory tests. The above data show that approach enhances is a new but promising concept to improve the oral bioavailability of bioactive peptides from whey. In this context, future perspectives will be probably to apply these innovative processes in other whey molecules to guarantee their effect per oral administration and perform clinical trials that demonstrate these results. Other alternatives of molecules administration may possibly also be considered as potential strategies for protect the stability of whey therapeutic products. Some advantages of administration as nasal, rectal, vaginal, ocular, and oral cavity route are exposed in Patel et al. (2011). Oral transmucosal delivery, especially buccal and sublingual delivery, have progressed far beyond the use of traditional dosage forms with novel approaches which could be engineered to deliver complex molecules of proteins and peptides from whey.

CONCLUSION Whey proteins and peptides are potential therapeutic molecules that could be administered to reduce different diseases, however oral administration possibility still is few considered. The challenge of these compounds are similar to many other biological molecules or drugs that continue been studied to their application in pharmaceutical area. Currently reports on oral drugs delivery, show different methods of approach enhance to increase their bioavailability which that could be a

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promising alternative to overcome their limitations. In all cases, approaches have been tested in vitro and in others, trials and in vivo have been demonstrated in rats; but the current commercially available formulations are very few still. Consequently peptides and proteins from whey milk have been the minority studied for their oral administration. Stability and faster degradability are the principal causes of this situation; however favorable opportunities and many formulations possibly will improve the efficacy of these molecules that have already been approved. Other alternatives of administration could also be evaluated to their use as therapeutic molecules.

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In: Whey Proteins Editor: Michelle Wyatt

ISBN: 978-1-63463-116-7 © 2015 Nova Science Publishers, Inc.

Chapter 4

FORMATION, STABILITY, FLOW AND VISCOELASTIC PROPERTIES OF FOAMS PREPARED WITH WHEY PROTEIN ISOLATE AND ITS INDIVIDUAL MIXTURES WITH XANTHAN, GUAR AND REFINED SUGAR Eva M. García-García, Mariana Ramírez-Gilly and Alberto Tecante Departamento de Alimentos y Biotecnología, Facultad de Química, Universidad Nacional Autónoma de México, Cd. Universitaria, D.F., México

ABSTRACT Currently, food foams have become increasingly important due to the wide variety of textures that can be obtained by incorporating air into a precursor medium, and the additional advantage of including an ingredient without cost. The purpose of this chapter is to discuss the characteristics of stability, overrun, flow and viscoelastic properties of foams formed with whey protein isolate (WPI) and its individual 

Corresponding author: e-mail: [email protected].

118 Eva M. García-García, Mariana Ramírez-Gilly and Alberto Tecante mixtures with xanthan, guar and refined sugar, to understand the factors affecting their formation and rheological properties. A concise presentation of the most relevant information on the stability and rheological properties of foams made from mixtures of proteins and polysaccharides is presented first, followed by our results with WPI, WPI-xanthan, WPI-guar and WPI-refined sugar precursor solutions and foams. As part of our methodology, the suitable foaming conditions in a rotor-stator agitator, i.e. rotational speed, time and WPI concentration, considering stability and overrun are discussed first. Then, the stability, overrun, flow and viscoelastic properties of the WPI, WPI-xanthan, WPIguar and WPI-refined sugar for the suitable foaming conditions are analyzed and discussed to understand the role played by these components on the behavior and characteristics of these edible foams.

INTRODUCTION Preparation of foods, such as "mousses", "soufflés", meringues, whipped cream, ice cream and biscuits, among others, involves obtaining a disperse system called foam. Currently, studies on food foams have gained importance due to the variety of textures that can be obtained by incorporating air into a precursor medium, and the added bonus of including an ingredient without cost. Foams are dispersions of gas bubbles, usually air, in a continuous phase which can be liquid or semi-solid, stabilized by the presence of a surface active agent (Walstra, 1996). The gas phase dispersed as small bubbles, modifies not only the microstructure but also the sensory properties of the matrix in which it is dispersed. Aeration helps to obtain specific textures, e.g. fragility in confectionery, lightness in whipped cream, or "scoopability" in "mousses" (Nicorescu et al., 2010). Today, the stability of foams is an important research subject and interest in their study is primarily driven by the sensory pleasure that these products can cause (Green et al., 2013). Foams are generally formed and stabilized by proteins which are capable of forming cohesive viscoelastic membranes at the air-water interface to prevent bubble coalescence. Traditionally, egg protein has been used as a foaming agent but it has been shown that the foam-forming capacity of whey proteins is comparable to egg protein and has potential to replace this protein (Pernell et al., 2002; Davis & Foegeding, 2007). However, whey protein foams have lower stability (Yang & Foegeding, 2011) and for stable foams to be produced it is necessary to use stabilizing agents; the most common ones are polysaccharides (Hemar et al., 2001). Texture and overall stability of foams

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depend not only on the intrinsic properties of proteins and polysaccharides, but also on the nature and strength of protein-polysaccharide interactions. The resulting synergistic effect of the mixture of these biopolymers is of great importance and can be applied to improve many prepared foods, e.g. to reduce their cost and also to create different textures.

FOAMS: DEFINITION, FORMATION AND FACTORS AFFECTING STABILITY Foams are dispersions of a gas or mixture of gases entrapped in a dispersing phase; air bubbles are surrounded by the liquid that separates them from each other, the barrier or wall is called the lamella. Gas bubbles are mostly polyhedral in shape and usually have diameters from 10 to 100 m. Foam bubbles are organized in polyhedrons along the edge of the lamella, three lamellae are joined at 120° angles, called angle of Steiner, and the boundary where they are located is called the Plateau border; in this region pressure is different than in the rest of the lamella and this promotes foam destabilization (Schramm, 2005). Foam instability is due to three main causes (Walstra, 1996): 1) Diffusion of gas from the smaller to the larger bubbles, as pressure is higher in smaller than in larger bubbles; 2) Fluid drainage from and through the foam layer, due to gravity, and 3) Bubble coalescence due to film instability between bubbles. Foams can be characterized by their bulk properties: rheology, texture, stability, air content and foamability, i.e. ease of foam formation. Air content can be calculated from the density or specific volume, but this requires the density of free gas to be known, and this can be difficult to determine, so other parameters such as density, specific volume and overrun are reported. Overrun is used to characterize liquid food foams with high air content as whipped cream, ice cream and "mousse", among others (Campbell & Mougeot, 1999). All these variables are important to predict and control the properties of the prepared products, the foaming operation aims to prepare lighter products, as well as to modify the appearance and texture of foodstuffs to confer cohesion and flexibility with a homogeneous appearance and a more even distribution of flavor. Therefore, the foaming operation has been applied to develop new products tailored to the choice and needs of consumers, using air as a free-of-cost ingredient (Narchi et al., 2009).

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ROLE OF PROTEINS IN FOAMING In addition to their nutritional importance, proteins are important ingredients in food products because of their functional properties. These properties can be considered as any physicochemical property of a given polymer, e.g. a protein, that affects and modifies some characteristics of a food and contributes to the quality of the product. Proteins can stabilize emulsions and foams as they are amphiphilic molecules that spontaneously migrate to the air-water interface. Unlike surfactants of low molecular weight, proteins form a viscoelastic film that resists mechanical stresses during storage and handling. The various proteins differ greatly in their surface properties; proteins with an appropriate activity for technological purposes have three attributes: 1) Rapid adsorption at the interface; 2) Rapid unfolding and reorientation at the interface; and 3) Ability to interact with neighboring molecules to form a viscoelastic film that supports strong mechanical and thermal movements, once positioned and oriented at the interface. The adsorption rate of a protein at the air-water interface depends on the distribution of hydrophilic and hydrophobic zones on its surface; with an increasing number of hydrophobic regions on the surface, spontaneous adsorption at the interface becomes more and more likely. The firmness with which the protein molecule remains at the interface largely depends on the flexibility of its structure (Damodaran, 1996). After adsorption, proteins unfold and set intermolecular interactions with other proteins at the interface or near of it leading to the formation of a protein film. These interactions can be electrostatic, hydrogen bonds and hydrophobic (Carp et al., 2001); a proper balance of attraction, repulsion and hydration forces is required to form stable viscoelastic films.

POLYSACCHARIDES AS FOAM STABILIZERS Polysaccharides are biopolymers of high molecular weight used as functional ingredients in the food industry to control the microstructure, texture, flavor and shelf life. Because of their great capacity to retain water, they produce highly hydrated colloidal particles, and for this reason they are also called hydrocolloids. Hydrocolloids include many of the polysaccharides extracted from plants, marine algae, microbial sources, gums derived from plant exudates, and modified natural polymers produced by chemical or

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enzymatic treatment of starch or cellulose (Dickinson, 2003). Hydrocolloids perform at least three functions in food processing: emulsify, stabilize and thicken. In addition, some of them are also gelling agents. A stabilizer is any material that reduces the rate at which changes occur within a product during storage, transport and handling; stabilizers retard or prevent any of the following processes: gravity sedimentation of suspended particles, drops or bubble aggregation in a fluid medium, flocculation, coagulation or coalescence of dispersed fractions, aggregates disaggregation and skimming, among others. The main stabilizing action of food polysaccharides is by modification of viscosity or gelation of the aqueous continuous phase. Gas diffusion, liquid drainage and coalescence in foams depend on the interactions between the stabilizing layers and the nature and strength of protein-polysaccharide interactions. Proteins and polysaccharides can be associated by covalent and non-covalent interactions, e.g. electrostatic and hydrophobic interactions, steric exclusion, hydrogen bonding; partial unfolding of proteins at the interface can make them more susceptible to complex formation with polysaccharides. A mixture of proteins with polysaccharides may have three behaviors; 1) Miscibility in dilute solutions, 2) Thermodynamic incompatibility, as biopolymers repel each other forming two different aqueous phases, one rich in protein and one rich in polysaccharide and 3) Complexation, when biopolymers attract each other (de Kruif, 2001). The main reasons for protein-polysaccharide complexes to be used as foam stabilizers are their high surface activity, ability to increase the viscosity of the dispersion medium and their ability to form charged and thick gel-like adsorbed layers. Conformation of the films formed by the proteinpolysaccharide complexes at the interface depends on the electrostatic charge of such complexes and adsorption order at the interface, i.e. simultaneous or sequential. Formed layers are usually not homogeneous in thickness; a protein monolayer can be formed, followed by a layer which is always less dense in polysaccharide, in sequential adsorption, or by a protein-polysaccharide complex, in simultaneous adsorption (Rodríguez & Pilsof, 2001).

FLOW AND VISCOELASTIC PROPERTIES OF FOAMS The stability and foamability of liquid foams are of primary importance. The former is largely correlated to the rheological characteristics of the system as shown in Table 1 (adapted from Campbell & Mougeot, 1999).

Table 1. Life time and rheological attributes of food foams Attribute Solid

Seconds

Minutes

Hours

Soft solid High viscosity Intermediate viscosity Low viscosity

Wine foam

Soufflé Beaten eggs Batter Milkshakes Beer foam Cappuccino

Waffles Pancakes Whipped cream

Days

Weeks

Months RTE cereals Chocolate

Bread

Cakes Marshmallows

Swiss cheese

Mousse

Years Meringues Snacks Crackers Confectionery Ice cream

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Foams do not strictly behave as elastic materials, even for small deformations they show slow viscoelastic relaxations. The viscoelastic properties of foams may be determined using small amplitude oscillatory shear tests (Germain & Aguilera, 2014). Dynamic or oscillatory mechanical measurements allow the viscoelastic properties of the material to be characterized. In such experiments the applied strain varies sinusoidally with time according to the expression: γ = γ°senωt

(1)

The stress varies with the same frequency ω, but is out of phase by an amount δ with respect to the strain: σ = σ°sen (ωt +δ)

(2)

Considering the equations describing the behavior of shear strain and stress, it is possible to develop the constitutive equation to arrive at the expression: σ = γ° (G'senωt + G"cosωt)

(3)

This equation defines the storage modulus G'(ω) and the loss modulus G"(ω). The first is associated with the stored energy and the second with the energy dissipated by the material (Steffe, 1992). Germain & Aguilera (2014) and Laporte et al. (2014), report a behavior in which the storage modulus is greater than the loss modulus with low frequency dependence. These features indicate gel-like behavior. Data in the literature related to the analysis of flow properties of edible foams made up with whey protein concentrate and different hydrocolloids are not abundant. Some authors (Weaire, 2008; Liszka-Skoczylas et al., 2014) report shear-thinning behavior in mixtures formed by whey protein and xanthan and acacia gum systems. They also conclude that foams do not disintegrate during high temperature flow and their apparent viscosity, especially when the shear rate increases, does not decrease when temperature decreases. They associate this phenomenon to the fact that foams behave as viscoelastic solids.

124 Eva M. García-García, Mariana Ramírez-Gilly and Alberto Tecante

PREPARATION AND CHARACTERIZATION OF FOAMS Our work was carried out with whey protein isolate (WPI: Nutrical, Lactoprot, MÉXICO), food grade xanthan (CPKelco, USA), guar (Gomas Naturales, MÉXICO) and refined sugar (Alcoholes y melazas de México, MÉXICO). Moisture was determined with a thermobalance (MB45-2A, Ohaus, USA) at 100 °C for 10 min. Sugar moisture was determined according to an official method (NMX-F-294-SCFI-2011). Table 2. Moisture and soluble protein content (%) of materials used for preparing precursor solutions Component WPI Moisture 8.50 ± 0.24 Soluble protein 87.5 ± 0.05 n.a. = not applicable.

Sugar 0.057 ± 0.006 n.a.

Guar 10.2 ± 0.02 n.a.

Xanthan 11.1% ± 0.02 n.a

The soluble protein content was determined by Lowry et al. (1951) method. Absorbance at 500 nm was read in a spectrophotometer (Thermo, Genesys 10 series) and protein concentration was determined by reference to a bovine serum albumin curve. Solutions were prepared with 100 mM NaCl, stirred for 2 hours at room temperature and pH was adjusted to 7.0 with 0.1 M NaOH. Solutions were kept at 4° C overnight before experiments. Table 2 shows the analyses of WPI, sugar, guar and xanthan.

Preparation of Precursor Solutions The amount of WPI was weighed and dissolved in 100 mM NaCl at room temperature (20° C) with stirring for 2 h. Subsequently, pH was adjusted to 7.0 with 0.25 M NaOH and the solution was kept at 4 °C overnight. The co-solutes were added to the WPI solution before storage at 4 ºC and after pH adjustment.

Foaming A volume of 500 mL of the precursor solution was placed in a 1 L beaker and strongly agitated with a rotor-stator unit (Turbotest 37/750P, Rayneri,

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125

France). The rotor-stator head was completely immersed into the liquid and centered in the beaker to let it rotate freely. Additionally, a small-capacity rotor-stator device (Ultra-Turrax, T25 D51, Germany) was used and 100 mL of the precursor solution were stirred in a 600 mL beaker under the same conditions as for the bigger rotor-stator unit.

Overrun Overrun, i.e. the foaming capacity or yield, was calculated as:  foam volume - initial liquid volume  Overrun   100 initial liquid volume  

(4)

Stability The foam was carefully transferred into a measuring graduated cylinder and the volume of drained liquid was recorded every 10 minutes.

Rheological Measurements Rheological tests were performed at 25 °C in a rheometer (ARES-RFS III, TA Instruments, USA). Solutions were examined in a double wall Couette fixture; diameter of internal cup; 27.94 mm, inner diameter of bob; 29.5 mm, outer diameter of bob; 32.0 mm and inner diameter of cup; 34 mm, and foams in a Vane tool. Tests included strain sweeps at an angular frequency of 6.28 rad/s to determine the zone of linear viscoelasticity (ZLV), frequency sweeps at a given strain within the ZLV to examine the viscoelastic behavior and steady shear tests to examine the variation of viscosity with shear rate.

Statistical Analysis Results are expressed as the mean of several repetitions and the standard deviation is included. Statistical differences were determined by an analysis of

126 Eva M. García-García, Mariana Ramírez-Gilly and Alberto Tecante variance (ANOVA) and a least significant difference test of Fisher to a 95% level (α = 0.05).

DEFINING OPERATING CONDITIONS For foaming to occur protein has to unfold and adsorb at the interface. The initial concentration was 4.0 wt% WPI and foaming was induced by stirring at speeds of 900 to 2700 rpm for 10, 20 and 30 minutes. Then, overrun and stability were measured. Overrun is defined as the volume of gas entrapped, expressed as a percentage relative to the initial volume of liquid. As shown in Table 3, Overrun (%) increased with increasing WPI concentration. For example, at 1800 rpm and 30 min, Overrun was 216.7% for 10.0 wt% WPI and 160.0% for 4 wt% WPI. This means 26.2% more Overrun for 10 wt% WPI. Likewise, at 1800 rpm and 20 minutes the Overrun for 10 wt% WPI was 12.7% superior to those for 4 or 6 wt% WPI. This occurred because the increased concentration of protein tends to increase its foaming ability. Overrun also increased with agitation speed; greater speeds allow more air to be incorporated. For example, for 4 and 6 wt% WPI the Overrun increase from 900 to 2700 rpm for 10 min, was 72.8%. Stability also increased with WPI concentration and decreased with increasing agitation speed. When speed was greater than 2100 rpm, stability decreased with increasing stirring time. As shown in Table 3 for 10.0 wt% WPI at 2400 rpm for 10 to 20 minutes of stirring, the volume of drained liquid increased 2.5 times and for 10 to 30 minutes, it did it 3.5 times. Stability improved by increasing WPI concentration, because viscosity increased and facilitated formation of a cohesive film made up by several layers of protein molecules at the interface (Damodaran, 1996). However, excessive agitation, as for speeds greater than 2100 rpm, produced high stretching in the film formed and rupture occurred almost immediately; hence, when the stirring time increased, stability decreased. From these results, the chosen conditions were 10 wt% WPI, at 2700 rpm for 20 minutes.

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WPI-XANTHAN, WPI-GUAR AND WPI-SUGAR FOAMS After obtaining the proper operating conditions for foaming, the rheological properties of precursor solutions and foams were determined. Subsequently, xanthan (XAN) was added to the formulation in three different concentrations to examine the effect on stability, overrun, flow and viscoelastic properties. Figure 1 shows the flow behavior of xanthan and guar gum solutions. 101 0.05 wt% XAN 0.10 wt% XAN 0.15 wt% XAN 0.15 wt% GG 0.27 wt% GG 0.30 wt% GG

Viscosity (Pa.s)

100

10-1

10-2

10-3 10-2

10-1

100

101

shear rate (s-1)

102

103

Figure 1. Flow behavior of xanthan and guar solutions.

The remarkable shear-thinning behavior of xanthan contrasts with the moderate shear-thinning flow of guar. Guar concentrations (GG) were fixed in such a way as to obtain approximately the same level of viscosity than XAN at some intermediate region in the flow curve. As can be observed, the viscosity of 0.15 wt% GG overlapped with 0.05 wt% XAN from 8 to 12.5 s-1, 0.27 wt% GG matched with 0.10 wt% XAN from 10 to 12.5 s-1, and 0.30 wt% GG did it with 0.15% XAN from 8 and 10 s-1.

Table 3. Overrun and stability for diferentes protein concentrations, speed and agitation time

Time (min)

10

20

30

1

Speed (rpm) 900 1200 1500 1800 2100 2400 2700 900 1200 1500 1800 2100 2400 2700 900 1200 1500 1800 2100 2400 2700

WPI concentration (wt%) 4 Overrun Stability1 57.1 77.1 100.0 66.7 114.3 71.4 160.0 60.0 170.0 50.0 210.0 50.0 210.0 54.0 57.1 77.1 100.0 66.7 114.3 71.4 160.0 60.0 210.0 60.0 250.0 70.0 210.0 70.0 57.1 77.1 100.0 66.7 114.3 83.3 160.0 60.0 210.0 70.0 250.0 70.0 210.0 74.0

6 Overrun 57.1 100.0 114.3 160.0 170.0 210.0 210.0 57.1 100.0 114.3 160.0 210.0 250.0 210.0 57.1 100.0 114.3 160.0 210.0 250.0 210.0

Percentage of drained liquid after 10 min of having stopped agitation.

Stability 87.5 65.0 63.3 50.0 70.0 50.0 34.0 87.5 60.0 63.3 50.0 70.0 70.0 70.0 87.5 60.0 66.6 50.0 70.0 70.0 80.0

8 Overrun 100.0 116.7 160.0 130.0 150.0 150.0 210.0 100.0 116.7 160.0 170.0 170.0 170.0 250.0 100.0 116.7 160.0 210.0 170.0 210.0 250.0

Stability 83.3 66.7 60.0 66.0 46.0 40.0 10.0 83.3 66.7 60.0 70.0 80.0 60.0 70.0 83.3 66.7 60.0 70.0 80.0 80.0 70.0

10 Overrun 100 116.66 140 133.33 150 170 210 100.0 133.3 160.0 183.3 170.0 210.0 250.0 100.0 150.0 180.0 216.7 170.0 210.0

Stability 83.3 60.0 57.1 50.0 30.0 20.0 36.4 83.3 60.0 57.1 50.0 30.0 50.0 63.6 83.3 60.0 57.1 50.0 50.0 70.0

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Strain sweeps of guar solutions (not shown), obtained at 6.28 rad/s, showed predominance of G" over G' for all concentrations, whereas only for 0.15 wt% XAN solutions this behavior was observed. For 0.10 and 0.15 wt% XAN, G' predominated over G" this behavior being clearer for 0.15 wt% XAN. These behaviors demonstrate the different nature of each polysaccharide; for GG the viscous character predominated over the elastic one and for XAN at concentrations greater than 0.15 wt%, the opposite occurred. The zone of linear viscoelasticity (ZLV) of xanthan and guar solutions extended up to 100% strain which is typical of fluid polymer solutions. The viscoelastic behavior of xanthan solutions is shown in Figure 2. The variation of dynamic moduli with frequency was characteristic of dilute solutions for 0.05 wt% XAN. Dynamic moduli exhibited a notorious dependence with frequency with a crossing point at 14.5 rad/s. This frequency separates the flow region, in which G” was greater than G‟, from the beginning of the elastic zone in which G‟ was greater than G”. For 0.10 wt% XAN, G' and G'' were congruent (tan = 1) over a large frequency interval and above 10 rad/s, G‟ was greater than G”. For 0.15 wt% XAN the congruency of G‟ and G” extended only up to 1 rad/s and beyond this frequency G' was greater than G". The different viscoelastic behaviors of xanthan solutions, going from viscous to elastic depending on polysaccharide concentration, make this polymer an interesting material as a foam stabilizing agent. Guar gum solutions exhibited a behavior typical of viscoelastic polymers with a predominantly viscous character, with moduli highly dependent on frequency and moduli overcrossing at high frequencies (Figure 3). Unlike xanthan, guar is not an anionic macromolecule and is not a branched polysaccharide. This makes guar chains considerably more flexible than xanthan molecules and this explains their different viscoelastic behaviors. It is therefore expected that foam stabilization with guar gum is mainly due to a viscous rather than to an elastic contribution.

Addition of Sugar Refined sugar (RS) was added at concentrations of 10, 20, 30 and 50 wt%. Figure 4 shows RS solutions to be Newtonian in nature. Viscosities were

130 Eva M. García-García, Mariana Ramírez-Gilly and Alberto Tecante around 1.4, 1.9, 2.6 and 10.8 mPa·s for 10, 20, 30 and 50 wt% RS, respectively. Strain sweeps (not shown) showed that the ZLV of these solutions extended up to 100% strain. RS has a thickening power considerably lower than xanthan and guar, but it is interesting to examine the differences among these co-solutes as foam stabilizing agents as they have different viscous and viscoelastic natures. In aqueous solution, xanthan is a rigid molecule, guar is flexible and sugar is only a low viscosity and Newtonian solute. 101 0.05% 0.10% 0.15%

G'; G'' (Pa)

100

10-1

10-2

10-3 10-1

100

101

102

 (rad/s)

Figure 2. Variation of dynamic moduli with frequency for xanthan solutions of different concentrations; G‟ (black symbols), G” (white symbols).

Overrun and Stability The flow behavior of precursor solutions is shown in Figures 5, 6 and 7, for WPI-XAN, WPI-GG and WPI-RS, respectively. Individual 10 wt% WPI solutions were slightly shear-thinning with low viscosity. Addition of xanthan, guar or sugar to WPI increased viscosity and accentuated shear-thinning. Overrun was affected by increasing the viscosity of the liquid in which air was dispersed because greater viscosities prevent gas entrapment (Carp et al.,

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131

2001). WPI-GG mixtures were more viscous than WPI-XAN mixtures yet comparable reductions in overrun were observed for 0.15 wt% xanthan and guar and 0.27 and 0.30 wt% guar reduced overrun further as seen in Figures 8 and 9. For 0.15, 0.27 and 0.30 wt% guar, overrun decreased 33, 39 and 44%, respectively, compared with the foam containing only protein. For 0.05, 0.10 and 0.15 wt% xanthan, overrun decreased 20, 28 and 36%, respectively. In sugar containing solutions, the decrease was 10, 16, 34 and 30% for 10, 20, 30 and 50 wt% RS, respectively. Linear correlation parameters between overrun and polysaccharide and sugar concentrations are shown in Table 4. Even if the correlation coefficients are not close to unity, it is possible to identify a trend. The slope decreased in the order WPI-XAN > WPI-GG > WPI-RS, that is, overrun decreased more when xanthan concentration increased than when guar and sugar concentration increased. 101 0.15% 0.27% 0.30%

G'; G'' (Pa)

100

10-1

10-2

10-3 10-1

100

101

 (rad/s)

102

103

Figure 3. Variation of dynamic moduli with frequency for guar gum solutions of different concentrations; G‟ (black symbols), G” (white symbols).

132 Eva M. García-García, Mariana Ramírez-Gilly and Alberto Tecante 10-1

viscosity Pa.s)

10% 20% 30% 50%

10-2

10-3 100

101

102

103

shear rate (s-1)

Figure 4. Viscosity of sugar solutions of different concentrations (wt%); 10 (circle), 20 (square), 30 (triangle up) and 50 (triangle down).

Viscosity (Pa.s)

10-1

10-2

10-3 100

101

102

Shear rate (s-1)

103

Figure 5. Viscosity of precursor solutions with 10 wt% WPI alone (circle) and 10 wt% WPI with 0.05 wt% (square), 0.10 wt% (triangle up) and 0.15 wt% (triangle down) xanthan.

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133

Viscosity (Pa.s)

10-1

10-2

10-3 100

101

102

Shear rate (s-1)

103

Figure 6. Viscosity of precursor solutions with 10 wt% WPI alone (circle) and 10 wt% WPI with 0.15 wt% (square), 0.27 wt% (triangle up) and 0.30 wt% (triangle down) guar.

Viscosity (Pa.s)

10-1

10-2

10-3 100

101

102

103

Shear rate (s-1)

Figure 7. Viscosity of precursor solutions with 10 wt% WPI alone (circle) and 10 wt% WPI with 10 wt% (square), 20 wt% (triangle up), 30 wt% (triangles down) and 50 wt% (diamond) refined sugar.

134 Eva M. García-García, Mariana Ramírez-Gilly and Alberto Tecante 260

240

Overrun (%)

220

200

180

160

140

120 0.00

0.05

0.10

0.15

0.20

0.25

0.30

Polysaccharide concentration (wt%)

Figure 8. Effect of xanthan (circle) and guar (square) concentration on Overrun of foams with 10 wt% WPI. Continuous lines are regressions of data points. 260

240

Overrun (%)

220

200

180

160

140

120 0

10

20

30

40

50

Sugar concentration (wt%)

Figure 9. Effect of sugar concentration on Overrun of foams with 10 wt% WPI.

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135

The overrun decrease with increasing sugar concentration was 339 times lower with respect to xanthan and 205 times lower with respect to guar. This correlation reflects the thickening power of these polysaccharides over sugar. The thickening power is related to the intrinsic viscosity which depends on molecular weight, hydrodynamic volume and conformation in solution. The intrinsic viscosity of xanthan in aqueous media is high (5000-7000 mL/g) because of its high molecular weight, and its rigid conformation in solution. The intrinsic viscosity of guar in aqueous media is considerably lower (675 mL/g), and this can explain why the decrease in overrun was greater with higher concentrations of xanthan. Table 4. Correlation between Overrun and polysaccharide and sugar concentration Solutions WPI with xanthan WPI with guar WPI with sugar

Ordinate (%) 238 236 241

slope -550 -332 -1.62

r 0.9761 0.9598 0.8867

Foam stability, expressed as fraction of drained liquid, as a function of time is shown in Figures 10, 11 and 12 for mixtures of WPI with xanthan, guar and sugar, respectively. Foams formed with WPI and RS, except with 10 wt% WPI and 50 wt% RS, and WPI-GG exhibited a behavior than can be described by equation (5):

Vliq Vf



Kt t 1/2  t

(5)

where Vliq is the volume of drained liquid (mL), Vf is the final volume of drained liquid once all foam has collapsed (mL), t1/2 is half life, i.e. the time to obtain half the final volume of drained liquid (min), t is time (min) and K is a dimensionless constant. The stability of the rest of the foams could be described by the linear behavior described by equation (6):

Vliq Vf

 mt  b

(6)

136 Eva M. García-García, Mariana Ramírez-Gilly and Alberto Tecante where Vliq, Vf and t have the same meaning as in equation (5) and m and b are the slope and ordinate of the straight line, respectively. Note that b is dimensionless and m has the units of reciprocal time.

Fraction of drained liquid

1.0

0.8

0.6

0.4

0.2

0.0 0

20

40

60

80

100

120

140

160

Time (min) Figure 10. Kinetics of foam stability for 10 w% WPI alone (circle) and 10 wt% WPI with 0.05 wt% (square), 0.10 wt% (triangle up) and 0.15 wt% (triangle down) xanthan. Continuous lines are regression values from equations (5) and (6).

Values obtained from equations (5) and (6) are shown in Table 5. This table also presents the significant difference in the stability of different formulations. Greater stability was observed in foams prepared from WPIXAN solutions. The half-life time with 0.05 wt% xanthan increased 6.8 times in relation to the foam formed with protein alone. Stability of formulations with 10, 20 and 30 wt% sugar and with 0.15 wt% guar are not significantly different with the individual WPI foam.

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137

Fraction of drained liquid

1.0

0.8

0.6

0.4

0.2

0.0 0

20

40

60

80

100

120

140

160

Time (min) Figure 11. Kinetics of foam stability for 10 w% WPI alone (circle) and 10 wt% WPI with 0.15 wt% (square), 0.27 wt% (triangle up) and 0.30 wt% (triangle down) guar. Continuous lines are regression values from equation (5).

Foam stability may be improved if the viscosity of the dispersing liquid increases. In this case, as shown in the flow curves (Figures 5, 6 and 7) 0.27 and 0.30 wt% WPI-GG and 50 wt% RS solutions were more viscous than WPI-XAN solutions, hence, it seems that the effect of viscosity was not predominant on the stability of the formed foams. The molecular structure of hydrocolloids and their interactions with proteins may be a more important factor than viscosity itself. Unlike guar, xanthan is an anionic branched polyelectrolyte that in an aqueous medium bears negative charges, and therefore can establish electrostatic interactions with proteins. Once the protein film is formed around the air bubbles, xanthan molecules can interact with proteins forming multiple layers and as a result improve the stability of the foam. Also as already mentioned, xanthan has a larger molecular weight than guar and the stability of the foam can be increased or decreased depending on the molecular weight and size of the protein-polysaccharide complexes formed (Istarova et al., 2005).

138 Eva M. García-García, Mariana Ramírez-Gilly and Alberto Tecante

Fracction of drained liquid

1.0

0.8

0.6

0.4

0.2

0.0 0

20

40

60

80

100

120

140

160

Time (min) Figure 12. Kinetics of foam stability for 10 w% WPI alone (circle) and 10 wt% WPI with 10 wt% (square), 20 wt% (triangle up), 30 wt% (triangle down) and 50 wt% (diamond) sugar. Continuous lines are regression values from equations (5) and (6).

Table 5. Parameters of foam stability Formulation (wt%) 10 WPI 10 WPI + 10 RS 10 WPI + 20 RS 10 WPI + 0.15 GG 10 WPI + 30 RS 10 WPI + 0.27 GG 10 WPI + 0.30 GG 10 WPI + 50 RS 10 WPI + 0.05 XAN 10 WPI + 0.10 XAN 10 WPI + 0.15 XAN Different letters mean significant difference with  = 0.05.

t1/2 (min) 12.92a 17.04ab 23.17ab 24.97abc 32.70abc 33.44bc 32.97bc 62.96cd 88.44d 205.95e 269.88e

r 0.9807 0.9856 0.9856 0.9857 0.9838 0.9758 0.9758 0.9683 0.9957 0.9955 0.9920

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139

Viscoelastic and Flow Properties of Foams Strain sweeps (not shown) showed significant changes in sensitivity to strain between precursor solutions and foams. The former exhibited viscous fluid behavior with a large ZLV extending up to 100% strain. Separation between dynamic moduli increased with increasing concentrations of polysaccharides or sugar. Foams, however, are elastico-viscous materials in which G' was greater than G". Predominance of G' can be the result of the interaction of proteins at the air-water interface caused by unfolding and orientation; the film formed by proteins and protein-polysaccharide or proteinsugar interactions resulted in solid-like properties. This makes foams more sensitive to strain; the limit of the ZLV changed drastically from 100% strain for precursor solutions to 1% for foams. Foaming, changed viscous fluid behavior to solid-like elastic behavior irrespective of xanthan, guar or sugar addition. However, the extent of this change depended on the nature of the solute added to WPI. 100

G'; G'' (Pa)

10-1

10-2

10-3

10-4 100

101

 (rad/s)

102

Figure 13. Variation of dynamic moduli with frequency for precursor solutions of WPI and WPI with xanthan. Individual 10 wt% WPI; G‟ (dashed line), G” (continuous line). 10 wt% WPI with 0.15 wt% xanthan; G‟ (black triangle down), G” (white triangle down).

140 Eva M. García-García, Mariana Ramírez-Gilly and Alberto Tecante 100

G'; G'' (Pa)

10-1

10-2

10-3

10-4 100

101

 (rad/s)

102

Figure 14. Variation of dynamic moduli with frequency for precursor solutions of WPI and WPI with guar gum. Individual 10 wt% WPI; G‟ (dashed line), G” (continuous line). 10 wt% WPI with 0.30 wt% guar; G‟ (black triangle down), G” (white triangle down). 101

100

G'; G'' (Pa)

10-1

10-2

10-3

10-4 100

101

102

 (rad/s)

Figure 15. Variation of dynamic moduli with frequency for precursor solutions of WPI and WPI with sugar. Individual 10 wt% WPI; G‟ (dashed line), G” (continuous line). 10 wt% WPI with 50 wt% sugar; G‟ (black diamond), G” (white diamond).

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141

Frequency sweeps confirmed changes in nature of rheological behavior. Figures 13, 14 and 15 show the variation of dynamic moduli with frequency for precursor solutions for 10% wt WPI with xanthan, guar and sugar, respectively. The viscoelastic behaviors were clearly typical of viscous liquid solutions. Addition of polysaccharides and sugar increased the dynamic moduli with respect to individual 10 wt% WPI solutions and shifted the overcrossing point to higher frequencies. Moduli for intermediate concentrations of each co-solute lied between those shown in the corresponding figures. Overcrossing frequencies were around 20, 30 and 80 rad/s for precursor solutions added with xanthan, guar and sugar, respectively. Even if dynamic moduli were of the same order of magnitude for the two polysaccharides and sugar, it is clear that the elastic nature of precursor solutions depends on the type of added co-solute. 103

G'; G'' (Pa)

102

101

100

10-1 10-1

100

 (rad/s)

101

102

Figure 16. Variation of dynamic moduli with frequency for foams of WPI and WPI with xanthan. Individual 10 wt% WPI; G‟ (black circle), G” (white circle). 10 wt% WPI with 0.05 wt% xanthan; G‟ (black square), G” (white square) and 0.10 wt% xanthan; G‟ (black triangles up), G” (white triangles up).

Figures 16, 17 and 18 show the mechanical spectra of foams with added xanthan, guar and sugar, respectively. The rheological behavior changed remarkably when precursor solutions were foamed, but the extent of this change depended on the added co-solute. The rheological behavior of foams in

142 Eva M. García-García, Mariana Ramírez-Gilly and Alberto Tecante which xanthan was present was more sensitive to polysaccharide concentration (Figure 16). The overall effect was moduli depression with respect to foams with individual WPI; addition of a small amount, 0.05 wt%, of xanthan largely reduced dynamic moduli and also the loss angle (not shown). Further increase in xanthan concentration to 0.10 wt%, increased the dynamic moduli but not enough to individual WPI and continued reducing the loss angle and when xanthan concentration was 0.3 wt%, tan  was 0.9 to 1.0. 103

G'; G'' (Pa)

102

101

100 10-1

100

 (rad/s)

101

102

Figure 17. Variation of dynamic moduli with frequency for foams of WPI and WPI with guar. Individual 10 wt% WPI; G‟ (black circle), G” (white circle). 10 wt% WPI with 0.30 wt% guar; G‟ (black triangle down), G” (white triangle down).

This indicates a more structured system in which the elastic nature increased and explains why these foams were largely more stable, although at the expense of less overrun as previously discussed. In the presence of guar and sugar, the dynamic moduli of individual WPI were not significantly modified as shown in Figures 17 and 18, respectively. The effect of guar and sugar on the viscoelasticity of individual WPI foams was practically the same. Therefore, here again the nature of the added co-solute clearly has an influence on the rheological behavior. Guar is a neutral polysaccharide and therefore it is not expected to interact with protein chains as xanthan does. The different behavior between WPI-XAN, WPI-GG and WPI-RS foams indicates greater interaction between proteins with xanthan than with guar or sugar.

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G'; G'' (Pa)

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 (rad/s)

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Figure 18. Variation of dynamic moduli with frequency for foams of WPI and WPI with sugar. Individual 10 wt% WPI; G‟ (black circle), G” (white circle). 10 wt% WPI with 50 wt% sugar; G‟ (black diamond), G” (white diamond). 103

Viscosity(Pa.s)

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10-1

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Figure 19. Variation of viscosity with shear rate for foams of individual 10 wt% WPI (circle) and 10 wt% WPI with 0.05 (square), 0.10 (triangle up) and 0.15 wt% (triangle down) xanthan.

144 Eva M. García-García, Mariana Ramírez-Gilly and Alberto Tecante 103

Viscosity (Pa.s)

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101

100

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Figure 20. Variation of viscosity with shear rate for foams of individual 10 wt% WPI (circle) and 10 wt% WPI with 0.15 (square) and 0.30 wt% (triangle down) guar. 103

ViscosityPa.s)

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101

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Figure 21. Variation of viscosity with shear rate for foams of individual 10 wt% WPI (circle) and 10 wt% WPI with 10 (square) and 50 wt% (diamond) sugar.

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Figures 19, 20 and 21 show the variation of viscosity with shear rate for foams with added xanthan, guar and sugar, respectively. Shear-thinning was the common feature of all foams and a power law relationship of the type

η  Kγ n 1 adequately described (r > 0.9900 in all cases) the variation of viscosity with shear rate. The consistency index, K, ranged from 1.3 to 2.7, 12.4 to 15 and 13 to 23 Pa·sn for xanthan, guar and sugar, respectively. The flow behavior index ranged from 0.07 to 0.48, 0.18 to 0.28 and 0.13 to 0.37 for xanthan, guar and sugar, respectively. Low concentrations of added co-solute exhibited low flow index, therefore, for these concentrations shearthinning was more accentuated. This behavior is the macroscopic expression of the existence of macromolecular entanglements. Shear causes a progressive disentanglement that results in shear-thinning. Foams with added guar and sugar were more viscous than those with xanthan. Furthermore, in foams containing 0.10 and 0.15 wt% xanthan, viscosity decreases to a lesser extent than for the rest of the foams, indicating that WPI-XAN interactions were stronger and less sensitive to the shear rate, i.e. the foams formed from 10 wt% WPI with 0.10 and 0.15 wt% xanthan have a stiffer structure than the rest. As already mentioned regarding stability, the carboxyl groups in xanthan structure and the helix conformation this polysaccharide adopts, allow greater interaction with the proteins conferring greater stability to shear.

CONCLUSION The protein-polysaccharide ratio has a strong influence on the stability and rheological properties of foams and the impact on these properties depends on the strength of their interactions. Foams exhibited characteristics similar to the rheological behavior of a solid-like material, since G' dominated G" and showed low frequency dependence. Among the studied foams, the more stable were WPI-XAN in which protein-polysaccharide interactions are expected to be stronger than for guar and sugar. WPI-XAN interactions possibly enable formation of multilayer complex around air bubbles that makes them more resistant to coalescence and therefore of superior stability. Not only the nature of the selected polysaccharide to stabilize foams, or proteins that form such foams determine the characteristics of the foam, foaming conditions also directly influence the properties of the final system.

146 Eva M. García-García, Mariana Ramírez-Gilly and Alberto Tecante

REFERENCES Campbell, G.M., & Mougeot, E. (1999). Creation and characterisation of aerated food products. Trends in Food Science & Technology, 10, 283296. Carp, D.J., Bartholomai, G.B., Relkin, P., & Pilosof, A.M.R. (2001). Effects of denaturation on soy protein–xanthan interactions: comparison of a whipping–rheological and a bubbling method. Colloids and Surfaces B: Biointerfaces 21, 163-171. Damodaran, S. (1996). Aminoacids, Peptides and Proteins. In: Fennema, O.R. (Ed.). Food Chemistry (3rd edition, pp. 321-430), New York, Marcel Dekker, Inc. Davis, J.P., & Foegeding, E.A. (2007). Comparisons of the foaming and interfacial properties of whey protein isolate and egg white proteins. Colloids and Surfaces B: Biointerfaces, 54, 200-210. Dickinson, E. (2003). Hydrocolloids at interfaces and the influence on the properties of dispersed systems. Food Hydrocolloids, 17, 25-39. Germain, J., & Aguilera, J.M. (2014). Multi-scale properties of proteinstabilized foams. Food Structure, 1, 55-70. Green, A.J., Littlejohn, K.A., Hooley, P., & Cox, P.W. (2013). Formation and stability of food foams and aerated emulsions: Hydrophobins as novel functional ingredients. Current Opinion in Colloid & Interface Science 18, 292-301. Hemar, Y., Tamehana, M., Munro, P.A., & Singh, H. (2001). Viscosity, microstructure and phase behavior of aqueous mixtures of commercial milk protein products and xanthan gum. Food Hydrocolloids, 15, 565-574. Istarova, T.A., Semenova, M.G., Sorokoumova, G.M., Selishcheva, A.A, Belyakova, L.E., Polikarpov, Y.N., & Anokhina, M.S. (2005). Effect of pH on the interactions of sodium caseinate with soy phospholipids in relation to the foaming ability of their mixtures. Food Hydrocolloids, 19, 429–440. de Kruif, C.G, & Tuinier, R. (2001). Polysaccharide-protein interactions. Food Hydrocolloids, 15, 555-563. Laporte, M., Della Valle, D., Loisel, C., Marze, S., Riaublanc, A., & Montillet, A. (2014). Rheological properties of food foams produced by SMX static mixers. Food Hydrocolloids (in press). Liszka-Skoczylas, M., Ptaszek, A., & Zmudzinski, D. (2014). The effect of hydrocolloids on producing stable foams based on the whey protein concentrate (WPC). Journal of Food Engineering, 129, 1-11.

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Lowry, O.H., Rosebrugh, N.J., Lewis Farr, A., & Randall, R.J. (1951). Protein measurement with the Folin phenol reagent. Journal of Biological Chemistry, 193, 265-275. Narchi, I., Vial, Ch., & Djelveh, G. (2009). Effect of protein–polysaccharide mixtures on the continuous manufacturing of foamed food products. Food Hydrocolloids, 23, 188-201. Nicorescu, I., Vial, C., Loisel, C., Riaublanc, A., Djeveh, G., Cuvelier, G., Legrand, J. (2010). Influence of protein heat treatment on the continuous production of food foams. Food Research International, 43, 1585-1593. NMX-F-294-SCFI-2011 (2011). Industria Azucarera y Alcoholera. Determinación de humedad en muestras de azúcares cristalizados (Sugar and Alcohol Industry. Determination of dampness in samples of cristallized sugar). Secretaría de Economía. México. pp. 8. Pernell, C.W., Foegeding, E.A., Luck, P.J., & Davis, J.P. (2002). Properties of whey and egg white protein foams. Colloids and Surfaces A: Physicochemical and Engineering Aspects, 204, 9-21. Rodríguez Patino, J.M., & Pilosof, A.M.R. (2011). Protein–polysaccharide interactions at fluid interfaces. Food Hydrocolloids, 25, 1925-1937. Schramm, L.L. (2005). Emulsions, Foams and Suspensions: Fundamentals and Applications. Wiley-VCH, Germany. Steffe, J.F. (1992). Rheological Methods in Food Process Engineering (2nd edition, pp. 294-348). East Lansing MI, Freeman Press. Yang, X., & Foegeding, E.A. (2011). The stability and physical properties of egg white and whey protein foams explained based on microstructure and interfacial properties. Food Hydrocolloids, 25, 1687-1701. Walstra, P. (1996). Dispersed Systems. Basic Considerations. In Fenema, O. (ed.) Food Chemistry 3rd ed., pp. 146, Marcel Dekker, Inc., New York. Weaire, D. (2008). The rheology of foam. Current Opinion in Colloid & Interface Science, 13, 171–176.

In: Whey Proteins Editor: Michelle Wyatt

ISBN: 978-1-63463-116-7 © 2015 Nova Science Publishers, Inc.

Chapter 5

USE OF COMPUTATIONAL FLUID DYNAMICS IN THE MANUFACTURE PROCESS OF WHEY PROTEIN PRODUCTS Bárbara E. Meza and Juan Manuel Peralta Instituto de Desarrollo Tecnol gico para la Industria Qu mica (INTEC), Universidad Nacional del Litoral – CONICET, Guemes, Santa Fe, Argentina

ABSTRACT Whey proteins are well known for combining high nutritional value with specific functional properties. In general, they are commercialized as concentrates and isolates that can be different in their functional properties due to variances in milk composition as well as in manufacturing conditions used to obtain those products. Computational fluid dynamics (CFD) is one of the most important technique used to solve the transport phenomena balances. The main advantage of this method is the possibility to obtain a high detailed description of the fluid system without using expensive experimental measurements. CFD is widely used in food science and engineering to study heat and mass transfer, phase change, solid and fluid interactions, and chemical reactions in food processing. As a consequence, the application of CFD to simulate the transport phenomena in several food processing steps (for 

Email: [email protected].

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Bárbara E. Meza and Juan Manuel Peralta instance heat treatments, membrane filtration, evaporation, and spraydrying) was documented in literature. For this reason, this chapter is a brief review about the use of CFD as a tool for simulating and optimizing the industrial manufacture of whey protein products, such as concentrates and isolates.

1. COMPUTATIONAL FLUID DYNAMICS Computational fluid dynamics (CFD) involves the analysis of the fluid flow, heat transfer, and associated phenomena (for example chemistry reactions) in the studied system by computational simulations (Versteeg and Malalasekera, 2007; Ansys, 2013). The CFD codes are developed based on numerical algorithms that solve non-linear partial differential equations of the fluid flow, the energy transfer, and the associated phenomena. CFD is used in a wide range of industrial and non-industrial applications by many researchers and engineers. Typical uses include: process industry (mixing containers, chemistry reactors, heat exchangers, etc.), constructions services (building ventilation), automobile industry (combustion modeling and vehicle aerodynamic), and electronic (energy transfer within circuit panels) (Xia and Sun, 2002; Versteeg and Malalasekera, 2007). Specifically in the food field, applications of CFD are related to unit operations and processes such as mixing, drying, cooking, sterilization, cooling, and freezing (Xia and Sun, 2002; Norton and Sun, 2007; Erdoğdu, 2008; Norton and Sun, 2009; Blei and Sommerfeld, 2007; Kaushal and Sharma, 2012; Anandharamakrishnan, 2013; Norton and Tiwari, 2014). When computational fluid dynamic analysis is used, a great amount of information related to the problem to solve is required in order to obtain a precise solution (Norton and Sun, 2007). Firstly, the human analyst must apply the scientific knowledge to express the problem to solve mathematically. Then, the CFD software package will give body to the problem expressed in scientific terms. Finally, the computer program will perform calculations and the human analyst has to interpret the obtained results. This way, three important and different steps are required to carry out a CFD simulation (Figure 1) (Xia and Sun, 2002; Norton and Sun, 2007; Versteeg and Malalasekera, 2007): Pre-processing: all tasks that are performed before the numerical resolution processes are called pre-processing. This step is related to the input

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of the fluid flow problem into the CFD program by a friendly operator interface. User activities involved in this step are: 1) problem analysis, where the human analyst should consider the flow problem and try to understand as much as possible about it; 2) definition of the geometry of the interested region to create the computational domain, where it is usually carried out using a computer design program; 3) generation of a mesh of cells (control volumes or elements) to subdivide the domain into a number of small, nonoverlapping sub-domains; 4) selection of physical and chemical phenomena that require to be modeled; 5) definition of the fluid properties; 6) specification of appropriated initial and boundary conditions for the cells that match or contact the domain edge; and 7) solution of the flow problem (speed, pressure, temperature, etc.) at nodes within each cell, where the accuracy of the CFD solution is governed by the number of cells in the grid. Spray dryer

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Figure 1. Example of computational fluid dynamics steps using a spray dryer.

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Solving: the solving step involves the use of a computer to solve fluid flow mathematical equations. Numerical resolution methods, such as finite difference, finite element or volume element are used. Solving activities involved in this step are: 1) approximation of the unknown variables by simple functions; 2) discretization of the balance equations by substitution of approaches and subsequent mathematical manipulations; and 3) resolution of the algebraic equations. Post-processing: the post-processing program is used to make an assessment of the data generated by CFD analysis. When the model has been solved, the obtained results can be analyzed both numerically and graphically. Some examples of the activities performed in the post-processing step are: visualization of the geometry and the control volume, visualization of the vector field showing the flow magnitude and direction, variation of scalar variables through the domain, and animation and plotting of domain variables in 2-D and 3-D.

2. MANUFACTURE PROCESSES OF WHEY PROTEIN PRODUCTS Whey proteins are well known as the nitrogenous compounds that remain soluble in the whey after the isoelectric precipitation of caseins at pH 4.6 (de Wit, 1981). These proteins have different structure and properties than caseins, being susceptible to heat denaturation and aggregation. Whey proteins combine high nutritional value and specific functional properties such as gelification, water retention, emulsification, and foaming or thickening capacity (de Wit et al., 1988; Ennis and Mulvihill, 2000). Whey proteins are commonly commercialized as powdered concentrates (WPC) and isolates (WPI). The composition of these products can be different due to differences in the raw whey source and also in the manufacture process (De Wit et al., 1986; Morr and Foegeding, 1990; Huffman, 1996). WPC products have protein contents between 20% and 89%, while WPI ones have protein contents greater than 90%. The WPI production process usually involves many steps and high energy costs, therefore whey protein isolates tend to be used for more specific applications than whey protein concentrates (Bryant and McClements, 1998). The principal steps of the manufacture process of whey protein products are shown in Figure 2 and described below (Walstra, 2006; Tunick, 2008):

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Liquid whey (from cheese-making process)

Filtration and centrifugation Liquid whey (without traces of curd and less residual fat)

Pasteurization (heat treatment)

Membrane filtration (ultrafiltration, diafiltration, microfiltration, electrodialysis, nanofiltration, reverse osmosis)

Permeate (with high lactose and mineral contents)

Evaporation Concentrated whey (with 45% solids content)

Spray-drying Whey protein powder (with 10-14% moisture content)

Vibrating fluid bed

Whey protein powder (with 3-5% moisture content)

Figure 2. Flow diagram of the general manufacture process of whey protein products.

Filtration and centrifugation: the liquid whey from the cheese-making process vats is subjected to filtration in order to eliminate traces of curd and centrifugation to remove some of the residual fat. This step is important because the presence of lipids reduces the efficiency of subsequent steps, such as membrane filtration, and also can decrease the ion-exchange capacity. The purified whey is cooled to 5 °C and stored in insulated steel tanks. Pasteurization and concentration by membrane filtration: the stored liquid whey should be pasteurized by heat treatment, using for example high temperature short time technology. Heat treatment is mandatory to ensure the microbial safety for human consumption and to increase the shelf life of the

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final product. After that, liquid whey has to be concentrated by membrane filtration. This technology allows the separation and fractionation of whey proteins, retaining their solubility. In general, whey industries employ different types of membrane filtration: ultrafiltration, diafiltration, microfiltration, electrodialysis, nanofiltration, and reverse osmosis. Combinations of these technologies are used to create whey protein powders with different protein contents. For example, ultrafiltration is used to obtain a WPC with 35% of protein content. However, a combination of ultrafiltration and diafiltration removes minerals and lactose from the retentate, allowing the production of WPC with protein contents higher than 50%. On the other hand, a high removal of lactose is required in WPI production. For this reason, the use of an ion-exchange tower (which separates components by ionic charge instead of molecular size) is common in conjunction with membrane filtration. Evaporation and drying: the concentration of liquid whey by membrane filtration is followed by evaporation under vacuum conditions and spraydrying in order to obtain a dry powder product with less than 5% of moisture content. Evaporation is developed using multiple-effect evaporators (for example, the first effect takes place at around 77 °C and in the second one at around 45 °C). Water is evaporated from the whey, obtaining a product with 45% of solids content. After that, the concentrated whey is dispersed into a drying chamber through a hot air stream by a rotary nozzle atomizer, producing a powder with 10-14% of moisture content. The evaporation of water keeps the temperature down, decreasing the possibility of protein denaturation. In addition, lactose (which is amorphous and hygroscopic) is cooled and crystallized into non-hygroscopic lactose monohydrate. The obtained powder is dried to 3-5% of moisture content in a vibrating fluid bed. As consequence the final powder product with specific whey protein content is ready to be packaged for storage and subsequent marketing.

3. USE OF CFD TO SIMULATE AND OPTIMIZE THE INDUSTRIAL PRODUCTION OF WHEY PROTEINS The use of CFD in the industry has several advantages (Xia and Sun, 2002): 1) provides a detailed understanding of flow distribution, weigh lost, particle separation, energy and mass transfer, etc.; 2) allows evaluate changes in the geometry with much less time and cost than in laboratory tests; 3) the operator can answer many "what if" questions in a short period of time; 4) the

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scaling problems can be reduced because mathematical models are independent of the scale; and 5) it is particularly useful in conditions where experimental measurements are not possible (for example, at high temperatures in an oven or in dangerous environments). According to the literature, computational fluid dynamics was used to simulate the transport phenomena in many food processing steps: heat treatment, membrane filtration, evaporation, and spray-drying. As was discussed before, the industrial production of powder whey protein products involves some of these unit operations that are also common in the food and dairy industry in general. Surprisingly, the use of CFD in research related to whey protein production was not extensively considered in literature. Consequently, CFD models that were obtained for the dairy industry can be taken into account as starting point for further studies in the optimization and control field applied to the manufacture process of whey protein products.

3.1. Heat Treatment The application of CFD to heat treatment of dairy products was established. Although the major contributions were done using milk, those investigations could be considered as a basis for further studies using liquid whey. Anand Paul et al. (2011) developed a 3-D CFD model for low and high temperature pasteurisation of milk in a stationary and rotational can. Authors investigated the effect of thermal processing time on the temperature distribution inside the can and the effect of different can rotation on the temperature distribution and processing time. The model was validated with experimental measurements. Grijspeerdt et al. (2003) studied the use of CFD as a tool to model the flow patterns in corrugated plate heat exchangers for optimal design of milk processing. According to the results, the 2-D and 3-D complementary simulations have shown the influence of the corrugation shape and orientation. Calculations could help to identify regions of turbulent backflows and regions of high temperature near the wall. Those zones are the most sensitive to fouling and should be avoided to obtain a better heat exchanger design. Fouling by deposition of milk components onto the heat transfer surface is undesired. It reduces the heat transfer coefficient and increases the pressure drop over the equipment, generating a high tendency of product loss and increasing the operating costs. Denaturation and aggregation of whey proteins (specially -lactoglobulin) are involved in the deposition mechanism.

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Recently, Bouvier et al. (2014) applied a 2-D CFD model as a tool for simulating the -lactoglobulin local heat induced denaturation and aggregation in a plate heat exchanger. The inclusion of a denaturation kinetic model in the CFD calculations was considered. The flow regime was turbulent and two configurations were tested using a pure -lactoglobulin solution and a skim milk suspension as testing materials. Temperature and velocity heterogeneities occurring in heat exchanger were established. According to the obtained results, the CFD model could be used as a tool for giving operational recommendations in order to prevent protein deposits at the stainless steel surface of the plate heat exchangers.

3.2. Membrane Filtration The application of CFD analysis to dairy membrane filtration was not found in literature, although fractionation of milk by membrane technology has been extensively studied (Brans et al., 2004). However, investigations related to general module design, process parameters, and selectivity could be considered as starting point for further studies using liquid whey as a feed, considering that membrane filtration has to be reliable and safe for food processing. Membrane filtration is considered as a main technology to warrant the purity and safety during water and effluents treatment. According to the large number of publications available in literature, CFD simulations were used to improve membrane performance, suggesting a high potential of this technique in membrane process design (Ghidossi et al., 2006). For example, CFD simulations were used to study the fluid flow in complex geometries in order to find the best configuration of inserts and spacers to reduce particle deposition in membrane modules by microturbulence (Karode and Kumar, 2001; Schwinge et al., 2002). Wiley and Fletcher (2003) developed a novel diffusion-convection CFD model approach for flow in membrane channels, taking into account the dynamic behavior of the dissolved components and the cake layer formation. In addition, Dufreche et al. (2002) used a CFD model for particle deposition on the membrane in order to study the modification of effective permeability during cake growth.

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3.3. Evaporation and Spray-Drying The application of CFD to drying of dairy products was amply established, being the spray-drying of milk the most studied case. CFD is considered a useful tool to predict operational conditions in spray-drying design, such as gas flow pattern and temperature, velocity, residence time, and impact position of particles inside the chamber (Verdurmen et al., 2002; Kuriakose and Anandharamakrishnan, 2010). Jin and Chen (2010) developed a mathematical model for particle deposition and they incorporated this model in CFD simulations of an industrial milk spray dryer. Particle deposition is an undesirable problem in spray dryers. For this reason, those authors developed the mathematical model considering several particle characteristics, such as diameter, temperature, moisture content, velocity, air humidity, etc. The reentrainment of particles into the dryer was considered by the wall shear stress. Simulations were compared with industrial-scale experimental data. The obtained results indicated that the reentrainment of particles at the chamber walls was significant and the intensity of the deposition was higher near the adjunctions of the ceiling, vertical, and conical wall. Although the deposition rate was high at the conical wall, most of depositing particles were predicted to reenter the dryer due to the high shear stress. Chen and Lin (2005) used CFD simulations to study the air drying of a milk droplet under constant and time-dependent conditions. Two models were investigated: one model was based on the characteristic drying rate curve approach and the other one was based on the reaction engineering approach. Experimental data were obtained measuring the drying kinetic under a wide range of constant and variable time conditions of a single droplet drying using an improved glass filament system. A good agreement between theoretical and experimental data was obtained with both mathematical models. Some studies were carried out using liquid whey to obtain powder products. Lin and Chen (2007) investigated the spray-drying of a whey protein concentrate suspension using CFD and a reaction engineering approach model. This model was able to predict the droplet drying under non-isothermal and high humidity drying conditions, reducing the computational time in CFD simulation. The change of droplet diameter during drying was measured experimentally, obtaining a good agreement with the theoretical data. An interesting investigation was done by Anandharamakrishnan et al. (2007) who studied the effects of process variables during the spray-drying of a whey protein suspension on the denaturation rate of proteins. The authors analyzed the effect of feed concentration and outlet temperature on whey

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protein denaturation and solubility at pH 4.6. According to the results, low outlet gas temperatures (60-80 C) are required to avoid excessive denaturation of whey proteins. Both slightly more denaturation and loss of solubility were observed in a feed with high concentration (40%). Authors hypothesized that crust formation could generate particle with high temperatures and a wet core, leading high levels of protein denaturation. Although computational fluid dynamics was not used in this research, the obtained results could be complemented with CFD simulations in further studies.

CONCLUSION Computational fluid dynamics is one of the most important methods used to solve the transport phenomena balances. The main advantage of this technique is the high detailed description of a fluid system without using expensive experimental measurements. In this chapter, a brief review about the use of CFD to simulate and optimize the industrial manufacture of whey protein products, such as concentrates and isolates, was proposed. The industrial production of powder whey protein products involves unit operations that are also common in the food and dairy industry in general: heat treatments, membrane filtration, evaporation, and spray‐drying. Computational fluid dynamic technique was widely used in food science and engineering literature to simulate the transport phenomena during those unit operations. Surprisingly, the use of CFD in research related to whey protein production was not extensively considered. For this reason, the CFD models that were obtained for the dairy industry could be taken into account as starting point for further studies in the optimization and control field applied to the manufacture process of whey protein products.

ACKNOWLEDGMENTS This work was supported partially by Universidad Nacional del Litoral (Santa Fe, Argentina), Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET, Argentina), and Agencia Nacional de Promoción Científica y Tecnológica (ANPCyT, Argentina).

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REFERENCES Anand Paul, D.; Anishaparvin, A.; Anandharamakrishnan, C. (2011). Computational fluid dynamics studies on pasteurisation of canned milk. International Journal of Dairy Technology, 64(2), 305-313. Anandharamakrishnan, C. (2013). Computational fluid dynamics applications in food processing. Springer, Springer, Germany. Anandharamakrishnan, C.; Rielly, C. D.; Stapley, A. G. F. (2007). Effects of process variables on the denaturation of whey proteins during spray drying. Drying Technology, 25, 799-807. Ansys. (2013). Ansys-CFX theory solver guide. Ansys Inc. Canonsburg, PA, USA. Blei, S.; Sommerfeld, M. (2007). CFD in drying technology – Spray-dryer simulation. In: Modern Drying Technology. V. Tsotsas and A. S. Mujumdar (Eds.). Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim, Germany. Bouvier, L.; Moreau, A.; Ronse, G.; Six, T.; Petit, J.; Delaplace, G. ( 2014). A CFD model as a tool to simulate -lactoglobulin heat-induced denaturation and aggregation in a plate heat exchanger. Journal of Food Engineering, 136, 56-63. Brans, G.; Schröen, C. G. P. H.; van der Sman, R. G. M.; Boom, R. M. (2004). Membrane fractionation of milk: state of the art and challenges. Journal of Membrane Science, 243, 263-272. Bryant, C. M.; McClements, D. J. (1998). Molecular basis of protein functionality with special consideration of cold-set gels derived from heatdenatured whey. Trends in Food Science and Technology, 9, 143-151. Chen, X. D.; Lin, S. X. Q. (2005). Air drying of milk droplet under constant and time-dependent conditions. AIChE Journal, 51(6), 1790-1799. de Wit, J. N. (1981). Structure and functional behaviour of whey proteins. Netherlands Milk and Dairy Journal, 35, 47-64. de Wit, J. N.; Hontelez-Backx, E.; Adamse, M. (1988). Evaluation of functional properties of whey protein concentrates and whey protein isolates. 3. Functional properties in aqueous solution. Netherlands Milk and Dairy Journal, 42, 155-172. de Wit, J. N.; Klarenbeek, G.; Adamse, M. (1986). Evaluation of functional properties of whey protein concentrates and whey protein isolates. 2. Effects of processing history and composition. Netherlands Milk and Dairy Journal, 40, 41-56.

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Dufreche, J.; Prat, M.; Schmitz, P.; Sherwood, J. D. (2002). On the apparent permeability of a porous layer backed by a perforated plate. Chemical Engineering Science, 57(15), 2933-2944. Ennis, M. P.; Mulvihill, D. M. (2000). Milk proteins. In: Handbook of Hidrocolloids. G. O. Phillips and P. A. Williams (Eds.). Woodhead Publishing Editors, CRC Press, Cambridge, UK. Erdoğdu, F. (2008). Computational fluid dynamics for optimization in food processing. In: Optimization in Food Engineering. F. Erdoğdu (Ed.). CRC Press, Boca Raton, FL, USA. Grijspeerdt, K.; Hazarika, B.; Vucinic, D. (2003). Application of computational fluid dynamics to model the hydrodynamics of plate heat exchangers for milk processing. Journal of Food Engineering, 57, 237242. Ghidossi, R.; Veyret, D.; Moulin P. (2006). Computational fluid dynamics applied to membranes: state of the art and opportunities. Chemical Engineering and Processing, 45, 437-454. Huffman, L. M. (1996). Processing whey protein for use as a food ingredient. Food Technology, 50, 49-52. Jin, Y.; Chen, X. D. (2010). A fundamental model of particle deposition incorporated in CFD simulations of an industrial milk spray dryer. Drying Technology, 28, 960-971. Karode, S. K.; Kumar, A. (2001). Flow visualization through spacer filled channels by computational fluid dynamics. I. Pressure drop and shear rate calculations for flat sheet geometry. Journal of Membrane Science, 193, 69-84. Kaushal, P.; Sharma, H. K. (2012). Concept of computational fluid dynamics (CFD) and its applications in food processing equipment design. Journal of Food Processing & Technology, 3(1), 100-138. Kuriakose, R.; Anandharamakrishnan, C. (2010). Computational fluid dynamics (CFD) applications in spray drying of food products. Trends in Food Science & Technology, 21, 383-398. Lin, S. X. Q.; Chen, X. D. (2007). The reaction engineering approach to modelling the cream and whey protein concentrate droplet drying. Chemical Engineering and Processing, 46, 437-443. Morr, C. V.; Foegeding, E. A. (1990). Composition and functionality of commercial whey and milk protein concentrates and isolates: a status report. Food Technology, 44, 100-112.

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Norton, T., Sun D.-W. (2009). Computational fluid dynamics in thermal processing. In: Engineering Aspects of Thermal Food Processing. R. Simpson (Ed.). CRC Press, Boca Raton, FL, USA. Norton, T.; Tiwari, B. (2014). Computational fluid dynamics in food processing. In: Introduction to Advanced Food Processing Engineering. J. K. Sahu (Ed.). CRC Press, Boca Raton, FL, USA. Norton, T.; Sun, D.-W. (2007). An overview of CFD applications in the food industry. In: Computational Fluid Dynamics in Food Processing. D.-W Sun. (Ed.). CRC Press, Boca Raton, FL, USA. Schwinge, J.; Wiley, D. E.; Fletcher, D. F. (2002). A CFD study of unsteady flow in narrow spacer-filled channels for spiral-wound membrane modules. Desalination, 146(1-3), 195-202. Tunick, M. H. (2008). Whey protein production and utilization: a brief history. In: Whey Processing, Functionality and Health Benefits. C. I. Onwulata and P. J. Huth (Eds.). John Wiley & Sons Inc., IA, USA. Verdurmen, R. E. M.; Straatsma, H.; Verschueren, M.; van Haren, J. J.; Smit, E.; Bargeman, G.; de Jong, P. (2002). Modelling spray drying processes for dairy products. Lait, 82, 453-463. Versteeg, H. K.; Malalasekera, W. (2007). An Introduction to Computational Fuid Dynamics - The Finite Volume Method. Second Edition. Longman Scientific & Technical, Essex, UK. Walstra, P.; Wouters, J. T. M.; Geurts, T. J. (2006). Dairy Science and Technology. Second Edition. CRC Press, Boca Raton, FL, USA. Wiley, D. E.; Fletcher, D. F. (2003). Techniques for computational fluid dynamics modelling of flow in membrane channels. Journal of Membrane Science, 211, 127-137. Xia, B.; Sun, D. -W. (2002). Applications of computational fluid dynamics (CFD) in the food industry: a review. Computers and Electronics in Agriculture, 34, 5-24.

In: Whey Proteins Editor: Michelle Wyatt

ISBN: 978-1-63463-116-7 © 2015 Nova Science Publishers, Inc.

Chapter 6

THE POTENTIAL OF COW’S MILK PROTEINS FOR PROPHYLAXIS AGAINST INTESTINAL INFECTION Mizuho Inagaki, Tomio Yabe and Yoshihiro Kanamaru United Graduate School of Agricultural Science, Faculty of Applied Biological Sciences, Gifu University, Japan

ABSTRACT Milk provides breast-fed infants a wide array of molecules, including nutrients and various proteins with defense functions, resulting in their healthy growth. In this review chapter, we describe experimental results that demonstrate the protective efficacy of the cow milk proteins against viral intestinal infection. The protective efficacy against viral intestinal infection of the anti-viral milk components, IgG, PAS6/7, and LP, contained in a microfiltration retentate fraction (MFRF) prepared from industrial sweet whey was studied and proven both in vitro and in vivo. Therefore, we conclude that milk whey concentrates, such as MFRF, are promising prophylactic food additives against intestinal infection. We also describe the HRV inhibitory mechanism of bovine -casein, focusing on the glycan structures of -casein.



To whom correspondence should be addressed: Tel & Fax: +81-58-293-2913, E-mail: [email protected]

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INTRODUCTION Human rotavirus (HRV), a double-stranded RNA virus of family Reoviridae, is the most common viral cause of viral gastroenteritis and diarrhea in infants and young children worldwide. Improvement of sanitary condition has no apparent effect on the incidence of HRV infection. Before the age of 5 years, 95% of all children are infected with HRV resulting in approximately half a million deaths annually [1]. Two vaccines, Rotarix® and RotaTeq®, have been licensed for use in more than 100 countries worldwide. However, to reduce the risk of side effects, administration of the first doses of both vaccines is strictly limited to infants between 6 and 15 weeks of age, and the entire dose schedule needs to be completed by 6-8 months of age. Elderly people occasionally suffer from this infection. Moreover, the immune system of young infants is immature and they are incapable of active production of adequate amounts of antibodies. Therefore, other prophylactic options against HRV infection should be pursued. Milk, the natural food of the newborn mammal, is endowed with protective components against pathogens. Here, we discuss the possible use of cow milk proteins to protect against HRV gastroenteritis.

APPROACHES FOR PREVENTION AGAINST HRV INFECTION VIA MILK PROTEINS 1. Passive Immunity via Antibody Young mammals depend on passive immunity through breast-feeding for their resistance against infectious diseases, because their immature immune systems cannot produce antibodies immediately after birth. The mother is able to produce components that protect against infectious agents that are passively transmitted to the offspring though breast milk. Bovine milk is known to be rich in immunoglobulins, such as immunoglobulin G (IgG), which play important roles in the immunity of newborn animals, because the IgG produced by mother cows is transmitted to the offspring through milk after parturition, but not through the placenta to fetal animals. Therefore, bovine milk contains specific and diverse bovine IgGs, which are produced by cows immune response to the wide spectrum of antigens in living environment. Bovine IgGs could include antibodies specific

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for infectious human pathogens that have protective effects that may be valuable for human passive immunization.

IgGs derived from hyper-immunized bovine colostrum and normal bovine late colostrum Ten years after the discovery of rotavirus, ingestion of hyper-immunized bovine colostrum and immunoglobulin concentrates have been proposed for passive oral prophylaxis against HRV gastroenteritis [2, 3]. Hyper-immunized bovine colostrum was produced by immunizing pregnant Holstein cows with HRV and harvesting colostrum after delivery; hence, it contains rotavirusspecific, neutralizing IgG. Although previously studies show that hyperimmunized bovine colostrum was clinically beneficial [2, 4], its large-scale production is difficult. We previously investigated whether skimmed, concentrated, and pasteurized bovine milk obtained from healthy lactating cows at 6-7 d postparturition, referred to as late colostrum, prevents HRV-induced diarrhea in suckling mice, as an alternative to hyper-immunized bovine colostrum. We found that the late colostrum exhibits significant anti-HRV activity; its effect was ten times weaker than hyper-immunized bovine colostrum but one hundred times stronger than mature milk [5]. Surprisingly, IgG isolated by affinity chromatography from the late colostrum and IgG isolated from mature milk exhibited the same level of anti-HRV activity in the in vitro neutralization assay. This similarity indicates that the effectiveness of bovine milk is not attributable to the anti-viral titer of IgG but to the quantity of IgG contained therein, regardless of the lactation period [6]. As shown in Figure 1A, using a pretreatment assay, we clearly observed that interaction of IgG with host cells did not inhibit HRV infection. In the experiment, cells were first allowed to interact with the milk samples during incubation, then washed, then inoculated with HRV, as described in the figure legend. This result indicates that IgG interacts with viral particles but not with host cells. Furthermore, we demonstrated that the administration of late colostrum as early as possible and repeatedly during the course of diarrhea are important for reduction of diarrheal symptoms in the experimental diarrhea suckling mice model [6]. It was reported that late colostrum reduced the binding of virus-like particles of Norovirus (Ueno-7k strain) and Sapovirus (NK24 strain) to human intestinal Caco-2 cells in a concentration-dependent manner [7] and that treatments with late colostrum could shorten the duration of the disease in the upper respiratory tract caused by a virus (i.e. rhinovirus, coronavirus, and influenza virus) in clinical trials [8].

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Figure 1. Effects of IgG and PAS6/7 on HRV infected MA104 cells. MA104 cells were plated into the wells of a 24-well heavy Teflon-coated slide (AR Brown, Tokyo) and grown to full confluence. A suspension containing 1 × 10 5 - 1 × 106 fluorescent cell focus-forming units (FCFU)/ml of infectious virus was treated with 20 mg/ml trypsin (Sigma-Aldrich, St. Louis, MO) for 30 min at 37°C. After appropriate dilution with Eagle‟s minimum essential medium (E-MEM) containing 2% fetal calf serum to obtain approximately 103 FCFU per 100 ml, aliquots were mixed with equal volumes (100 ml) of one-half serially diluted samples in microtubes for 1 h at 37ºC. The diluted mixtures (20 l/well) were added to the confluent monolayer of MA104 cells. The controls, which lacked the test samples of milk, produced approximately 100 infected foci per well. The cells were further cultured for 1 h at 37°C in an atmosphere of 5% CO2. After removal of the inoculums, the cells were washed once with E-MEM to remove unbound virus, followed by incubation at 37°C in an atmosphere of 5% CO2. After 17 h of incubation, the cells were fixed with cold methanol. Infected cells were detected by an indirect immunofluorescence assay. The numbers of infected foci were measured by observation of fluorescence microscopy. Neutralizing activity was expressed as the percentage reduction in the numbers of infected foci as compared to controls (100%). The minimum inhibitory concentration (MIC), the minimum concentration inducing a 50% reduction in infected cells, was calculated for each sample from a logarithmic regression of the concentration-dependent percentage focus reduction. The experiments were performed in triplicate at least 3 times, and representative results for each sample are given as the mean (SD). Pre-treatment assays previously described [29] were conducted to investigate whether interactions of the milk samples with cells achieve anti-viral activity. Serially diluted milk samples were placed onto the confluent monolayers of MA104 cells and incubation for 1 h at 37ºC in 5% CO2. Following incubation, the cells were washed once with the same media, and inoculated with trypsinized HRV, approximately 2 x 10 3 FCFU per 100 mL, diluted in MEM containing 2% FBS. The controls, which weren‟t pre-incubated with milk test samples, showed approximately 100 infected foci per well. After removal of the viral inoculation, the cells were washed and incubated for 17 h at 37ºC in 5% CO2. Subsequent fixation, detection of infected cells, and MIC determinations were performed as described above. (A) anti-HRV effects of IgG were evaluated in the neutralization assay (closed circles) and the pretreatment assay (closed triangles). (B) anti-HRV effects of PAS6/7 were evaluated in the neutralization assay (closed circles) and in the pretreatment assay (closed triangles).

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Additionally, late colostrum was reported recently to have preventative and healing effects on small intestinal injury induced by non-steroidal, antiinflammatory drugs, such as indomethacin [9], suggesting that it may be an effective therapy relevant to human intestinal diseases. The utilization of bovine IgG as functional food needs treatment, for instance, heating. Additionally, in the case of oral administration, it is essential for the activity of IgG to survive pH variation and degradation by various proteases in the gastrointestinal tract. Song and Kanamaru [10] reported that the activity of IgG derived from bovine colostrum just after delivery, exhibited extreme stability against heat treatment (70ºC, 60 min), pH variation (pH 3-9) and was pepsin-, trypsin-, and chymotrypsin-resistant. Taken together, the above information indicates that normal healthy unimmunized bovine late colostrum containing high amounts of IgG may provide an alternative for the prevention and management of intestinal infectious disease, such as severe rotavirus diarrhea, in immunocompromised children.

2. Protective Effect of Non-Immunoglobulin Components Breast-feeding has been recognized as important for protection against environmental pathogens. In fact, breast-feeding lessens the severity of diarrhea associated with HRV infection in hospitalized children [11,12,13]. However, it was reported that the degree of protection conferred by human milk only partially correlates with the level of anti-HRV antibody measured in the milk [14, 15]. These reports suggested that non-immunoglobulin factors may contribute to the protective function of milk and stimulated the search for anti-HRV effects of milk components other than immunoglobulin. In this section, we describe the anti-viral properties of these components.

Lactadherin and PAS6/7 Lactadherin, a 46 kDa mucin-associated glycoprotein, is a major milk fat globule membrane (MFGM) component of human milk and the first anti-HRV non-immunoglobulin component reported by Yolken et al [16]. They found that milk mucin complex in the delipidated human MFGM, which is free of detectable immunoglobulin, exhibits anti-HRV activity in cell culture and prevents the development of gastroenteritis in the HRV-induced diarrhea mice model. Then lactadherin was identified as the major anti-viral component in this complex. They demonstrated that lactadherin can bind to both HRV and

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rhesus rotavirus. Deglycosylation of lactadherin results in the loss of HRV binding activity. This finding suggested that glycans in lactadherin play a major role in its viral binding activity. PAS6/7, a counterpart of lactadherin in bovine milk MFGM, was also investigated for its inhibitory activity against HRV. One report indicated that bovine lactadherin did not have anti-HRV activity [17]. The study was performed using the HRV Wa strain infection of 80% confluent Caco-2 cells and a short-term (1 h) incubation of cells with PAS6/7. In contrast, we reported the inhibitory activity of PAS6/7 against HRV infection [18]. The study was performed using the HRV MO strain infection of a MA104 cell suspension and long-term (22 h) incubation of cells with PAS6/7. Recently, we observed that PAS6/7 exhibits a plateau of 50% inhibitory activity and the inhibition that never reached 100%, in the neutralization assay under the Kvistgaard‟s experimental condition, which has 80% confluence MA104 cells and a short-term (1h) incubation of cells with PAS6/7 (Figure 1B). Although the reasons for our inconsistent results are unclear, these observations suggested that the experimental cell conditions might be an important factor for the inhibitory activity of PAS6/7. PAS6/7, consists of 2 N-terminal epidermal growth factor (EGF)-like domains followed by 2 repeated C domains with homology to the C1 and C2 domains of blood clotting factors V and VIII [19]. Interestingly, PAS6/7 has an EGF-like domain containing 2 glycosylation sites (Asn41 and Ser9 or Thr16), whereas human lactadherin has defects in this domain [19]. The roles of PAS6/7 glycans in anti-HRV effect are also poorly understood and their inhibitory mechanisms of PAS6/7 remain controversial. Further studies are needed to elucidate the details of the HRV inhibitory mechanism of lactadherin and PAS6/7, focusing on their respective molecular structures.

Lactophorin (proteose peptone 3) Lactophorin (LP), referred to as proteose peptone (PP) component 3, was initially found to be a phosphorylated glycoprotein in the heat- and acid-stable PP fraction [20]. LP has an average concentration of 0.3 g/l in normal bovine milk and is present in both whey and the soluble glycoprotein fraction prepared from the MFGM fraction. Interestingly, LP is found in bovine milk but not in human milk [21]. Initially, we found that the 16 kDa LP C-terminal fragment (LP16) separated from cow milk whey strongly inhibited the replication of HRV MO strains in MA104 cells [18]. Because it is known that LP consists of 2 major glycopeptides, 28 kDa (LP28) and 18 kDa (LP18) [20], we investigated the

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anti-HRV activity of these glycopeptides. We found that LP28 and LP18 inhibited HRV infection to the same degree (Figure 2). The MIC values of LP28 and LP18 were 0.034 and 0.18 g/mL, respectively.

Figure 2. Effects of LP28 and LP18 on HRV infection. Inhibitory activities of LPs were determined using the neutralization assay. The samples tested were: LP28 (closed circles) and LP18 (closed triangles). Results are expressed as means ± SD of 3 independent experiments conducted in triplicate.

LP28 contains 5 partial phosphorylation sites (Ser29, Ser34, Ser38, Ser40, and Ser46), 3 O-glycosylation sites (Thr16, Thr60, [22], and Thr86), and 1 Nglycosylation site (Asn77) [20]. LP18 has an amino acid sequence corresponding to the 54-135 C-terminal portion of LP28, and this sequence is thought to be a proteolytic degradation product of LP28 [20]. The observation of similar anti-HRV effects of these LPs suggested that the consensus structure of LP28 and LP18 (i.e., the 54-135 C-terminal portion of LP) was involved in their inhibitory activities. Further studies are needed to elucidate the details the HRV inhibitory mechanism of LPs.

PREVENTION AND REDUCTION OF DIARRHEAL SYMPTOMS CAUSED BY HRV VIA MILK PROTEINS In this section, we will describe the potential utilization of milk proteins mentioned above. Because the production of sweet whey, a byproduct of cheese production, increases with greater cheese consumption, its routine

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disposal will become a profound problem in the dairy industry. Therefore, the greater utilization of sweet whey should be pursued. Based on the above findings that IgG, LP and PAS6/7 exhibit inhibitory activities against HRV infection, we attempted to investigate the potential utilization of sweet whey as a food additive protective against HRV gastroenteritis. We prepared a microfiltration retentate fraction (MFRF) from sweet whey to concentrate IgG, LP and PAS6/7 by microfiltration on an industrial scale, and then pasteurized it using a high-temperature short-time method sterilization (HTST) method consisting of heating at 72°C for 15 s, followed by spray drying. We confirmed that MFRF contains these anti-HRV components using chromatography and protein analysis, and that MFRF exhibits in vitro inhibitory activity against both HRV and bovine rotavirus strains derived from field breeds [23]. To evaluate whether multiple doses of MFRF prevented or reduced the diarrhea symptoms of a HRV-induced suckling mice model, we investigated the effects of three different dosages of MFRF (0.1, 0.5, and 1.0 mg). In brief, pregnant BALB/c mice were purchased. Litters of 5-day-old mice were orally inoculated with the HRV MO strain. The MFRF was first given orally 30 min prior to viral inoculation. Additional administrations were given orally by gavage four times, 12, 24, 48, and 72 hours post-inoculation (hpi). Control mice were given PBS following the same administration schedule. Stools were observed by gentle abdominal palpation. Observation of liquid-mucous yellow stool was judged as diarrhea. A three-point ranking system (diarrhea index) was used to characterize diarrhea [24]: 0, normal brown formed stool or no stool; 1, soft orange stool; 2, liquid yellow stool. Diarrhea scores were calculated by averaging the diarrheal index of stool observations. The obtained results were distinguished by whether diarrhea symptom presented at 24 hpi, as shown in Figure 3A and Figure 3B, respectively. As shown in PBS group, the most serious diarrhea symptoms occurred at 48 hpi, thereafter a majority of the mice presented with brown stool through 96 hpi. In the mice with non-symptom onset at 24 hpi (Figure 3A), symptomatic diarrhea was dramatically suppressed, depending on dosage, suggesting that MFRF is able to significantly protect against HRV-induced diarrhea by repeated administration. Furthermore, in the mice with symptom onset at 24 hpi, the groups given PBS, 1.0 mg MFRF and 0.5 mg MFRF presented the same level of diarrhea symptoms (Figure 3B); however, the dose of 1.0 mg MFRF was able to shorten the diarrhea symptomatic period, suggesting that multiple administration of MFRF as early as possible reduced HRV-induced diarrhea symptom from 24 hpi onward. These results clearly indicated that MFRF has a

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symptom relieving effect and is a promising prophylactic food additive against HRV gastroenteritis.

Figure 3. Effect of multiple administrations of varying doses of MFRF against HRVinduced diarrhea in suckling mice. Litters of 5-day-old mice were given either PBS (n = 15, opened diamonds) or MFRF orally 30 min prior to viral inoculation with 0.9 x 105 FCFU of HRV MO strain per mouse. After viral inoculation, MFRF was orally given by gavage four times (12, 24, 48, and 72 hpi). The dosages tested were: 0.1 mg (n = 15, closed squares), 0.5 mg (n = 13, closed triangles), and 1.0 mg (n = 11, closed circles). Stools were examined daily to assess diarrhea from 0 hpi to 96 hpi by diarrhea index as described in text. (A) non-symptom onset at 24 hpi, (B) symptom onset at 24 hpi. * P < 0.05 and ** P < 0.005, as determined by the Mann-Whitney U test.

ANTI-HRV MECHANISM OF MILK PROTEIN: THE CASE OF BOVINE KAPPA-CASEIN From the above findings of MFRF‟s protective efficacy in the HRVinduced diarrhea suckling mice model, we conclude that milk proteins have the potential to protect against intestinal infection. As shown in figure 1, the IgG exhibits anti-viral activity by direct binding to the virus. Although nonimmunoglobulin components have also been reported to exhibit the anti-HRV activity, their inhibitory mechanisms against HRV are not well understood. Elucidation of their mechanism of action is essential both for practical use against intestinal disease and to understand viral replication mechanisms. Finally, we describe the experimental results from an investigation of the antiHRV mechanism of bovine -casein.

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The phosphorylated glycoprotein -casein from both human and bovine mature milk was reported to have anti-HRV effects in an international patent [25]. Bovine -casein, the only glycosylated protein in casein components, contains three O-glycosylation sites (Thr131, Thr133, and Thr135) [26]; its glycan structure was reported to change during the transition from colostrum to mature milk [27]. Glycans, including sialic acids such as N-acetylneuraminic acid (NeuAc), were suggested to interact with HRV [28]. Therefore, we compared of the structure of glycans from late colostrum obtained from nonimmunized cows on days 6-7 after parturition and from mature milk and investigated the role of glycans in -casein‟s anti-HRV mechanism [29]. In brief, using normal-phase high performance liquid chromatography (HPLC) and matrix-assisted laser desorption ionization quadruple ion trap time of flight mass spectrometry (MALDI TOF-MS), we confirmed that the Olinked glycan compositions differed between late colostrum and mature milk; monosialylated and disialylated oligosaccharides having a core 2 structure (N-acetylglucosamine (GlcNAc) 1-6 [galactose (Gal)1-3] N-acetylgalactosamine (GalNAc)) were detected in late colostrum but not in mature milk.

Figure 4. Effects of deglycosilation of casein derived from late colostrum on HRV infection. The inhibitory activity of late colostrum casein with enzymatic modification was measured by the neutralization assay. The samples were: late colostrum casein, closed circles; late colostrum casein treated with o-glycosidase (deglycosylated late colostrum casein). Results are expressed as means ± SD of 3 independent experiments conducted in triplicate.

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To investigate the involvement of these bovine -casein glycans in antiHRV activity, we prepared the deglycosylated casein derived from late colostrum and mature milk using enzymatic treatments, neuraminidase and Oglycosidase. These enzymes release NeuAc and the unit of Gal 1-3 GalNAc, respectively. Remarkably, deglycosylated caseins from late colostrum and mature milk did not inhibit HRV infection, indicating that O-linked glycans in casein are essential for inhibitory activity against HRV infection (Figure 4). Furthermore, HRV particles bound directly to heated casein (at 95ºC for 30 min) in a viral titer-dependent manner using an evanescent-field fluorescence-assisted assay, an established method to detect weak interactions between glycans and various biological samples (or carbohydrate binding proteins) [30, 31]. This finding indicates that bovine -caseins directly bind to HRV and thereby inhibit viral adsorption onto cells via glycan residues.

CONCLUSION Milk contains components fundamental for child growth and health. In this chapter, we described the anti-HRV components, IgG, PAS6/7, and LP, and examined the effect of MFRF, which is obtained from a byproduct of cheese manufacturing and contains large amount of these anti-viral components, on prevention and relieving diarrhea symptom as an alternative therapy against HRV gastroenteritis. Because we found that MFRF inhibited several strains of HRV and animal rotavirus in vitro [23], MFRF will be able to defend against epidemic HRV strains and livestock animal rotavirus. Furthermore, the anti-HRV activity of MFRF is stable to heat treatment, so it is easy to use as a protective food additive and an alternative therapy against rotaviral gastroenteritis. Milk protects against pathogens through adoptive immunity and innate immunity. Through adoptive immunity against viral infection, milk protein blocks direct binding to the viral receptor of target cells. On the other hand, the inhibitory mechanisms of the non-immunoglobulin component(s) that confer innate immunity are still largely unknown. Viruses perform the following steps during their replication cycle; entry into host cells, replication of viral genome, synthesis of viral protein, and assembly and egress of nascent viral particles. Anti-HRV component(s) could interact with target cells, which may shield the viral receptor on target cells. These components may protect against

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HRV through mechanisms that affect any of the various viral replication steps. Future research, in particular for non-immunogloblin components, should explore this issue more thoroughly.

ACKNOWLEDGMENTS We thank Dr. Osamu Nakagomi, Dr. Toyoko Nakagomi, Dr. Takusaburo Ebina, Dr. Nobuyuki Minamoto, Dr. Makoto Sugiyama, Dr. Tohru Suzuki, and Dr. Keita Yamada for their useful suggestions, in-depth discussions and valuable advice as supervisors of our experiments. We express our gratitude to Meiji Co., Ltd. and Kobayashi Pharmaceutical Co., Ltd. for kindly providing MFRF and late colostrum, respectively. This research was supported by the Japan Society for the Promotion of Science (JSPS; Grant-in-Aids for Young Scientists (B) no. 24780124 and 26850078 (to MI)) and the Program for Promotion of Basic and Applied Research for Innovations in Bio-oriented Industry (to YK).

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[15] Jayashree, S., Bhan, M. K., Kumar, R., Bhandari, N., & Sazawal, S. (1988). Protection against neonatal rotavirus infection by breast milk antibodies and trypsin inhibitors. J. Med. Virol., 26, 333-338. [16] Yolken, R. H., Peterson, J. A., Vonderfecht, S. L., Fouts, E. T., Midthun, K., & Newburg, D. S. (1992). Human milk mucin inhibits rotavirus replication and prevents experimental gastroenteritis. J. Clin. Invest., 90, 1984-1991. [17] Kvistgaard, A.S., Pallesen, L. T., Arias, C. F., López, S., Petersen, T. E., Heegaard, C. W., & Rasmussen, J. T. (2004). Inhibitory effects of human and bovine milk constituents on rotavirus infections. J. Dairy Sci., 87, 4088-4096. [18] Inagaki, M., Nagai, S., Yabe, T., Nagaoka, S., Minamoto, N., Takahashi, T., Matsuda, T., Nakagomi, O., Nakagomi, T., Ebina, T., & Kanamaru, Y. (2010). The bovine lactophorin C-terminal fragment and PAS6/7 were both potent in the inhibition of human rotavirus replication in cultured epithelial cells and the prevention of experimental gastroenteritis. Biosci. Biothechnol. Biochem., 74, 1386-1390. [19] Mather, I. H. (2000). A review and proposed nomenclature for major proteins of the milk-fat globule membrane. J. Dairy Sci., 83, 203-247. [20] Girardet, J. M., & Linden, G. (1996). PP3 component of bovine milk: a phosphorylated whey glycoprotein. J.Dairy Res., 63, 333-350. [21] Sørensen, E. S., Rasmussen, L. K., Møller, L., & Petersen, T. E. (1997). The localization and multimeric nature of component PP3 in bovine milk: purification and characterization of PP3 from caprine and ovine milks. J. Dairy Sci., 80, 3176-3681. [22] Kjeldsen, F., Haselmann, K. F., Budnik, B. A., Sørensen, E. S., & Zubarev, R. A. (2003). Complete characterization of posttranslational modification sites in the bovine milk protein PP3 by tandem mass spectrometry with electron capture dissociation as the last stage. Anal. Chem., 75, 2355-2361. [23] Inagaki, M., Xijier, Nakamura, Y., Takahashi, T., Yabe, T., Nakagomi, T., Nakagomi, O., & Kanamaru, Y. (2012). Food Additive: Production and Functional Properties of Dairy Products Containing Lactophorin and Lactadherin. Rijeka, Croatia: InTech. [24] Takahashi, K., Ohashi, K., Abe, Y., Mori, S., Taniguchi, K., Ebina, T., Nakagomi, O., Terada, M., & Shigeta, S. (2002). Protective efficacy of a sulfated sialyl lipid (NMSO3) against human rotavirus-induced diarrhea in a mouse model. Antimicrob. Agents Chemother., 46, 420-424.

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[25] Mukerji, P., Preito, P. A., Seo, A. E., Baxter, J. H., & Cummings, R. D. inventors. (1996). Inhibition of human rotavirus infection. ABBOTT LABORATORIES. assignee. US Pat. No. WO9608269. [26] Eigel, W. N., Butler J.E., Ernstrom, C.A., Farrell, H. M., Harwalkar, V.R., Jenness, R., and Whitney, R.M., (1984). Nomenclature of proteins of cow‟s milk: fifth revision. J. Dairy Sci., 67, 1599-1631. [27] Saito, T., Itoh, T., Adachi, S., Suzuki, T., & Usui, T. (1981). The chemical structure of neutral and acidic sugar chains obtained from -casein. Biochim. Biophys. Acta., 678, 257-267. [28] Isa, P., Arias, C.F., & López, S. (2006). Role of sialic acids in rotavirus infection. Glycoconj. J., 23, 27-37. [29] Inagaki, M., Muranishi ,H., Yamada, K., Kakehi, K., Uchida, K., Suzuki, T., Yabe, T., Nakagomi, T., Nakagomi, O., & Kanamaru, Y. (2014). Bovine κ-casein inhibits human rotavirus (HRV) infection via direct binding of glycans to HRV. J. Dairy Sci., 97, 2653-2661. [30] Kuno, A., Uchiyama, N., Koseki-Kuno, S., Ebe, Y., Takashima, S., Yamada, M., & Hirabayashi, J. (2005). Evanescent-field fluorescenceassisted lectin microarray: a new strategy for glycan profiling. Nat. Methods., 2, 851-856. [31] Tateno, H., Mori, A., Uchiyama, N., Yabe, R., Iwaki, J., Shikanai, T., Angata, T., Narimatsu, H., & Hirabayashi, J. (2008). Glycoconjugate microarray based on an evanescent-field fluorescence-assisted detection principle for investigation of glycan-binding proteins. Glycobiology. 18, 789-798.

INDEX A accounting, 69 acetylation, 6 acetylcholine, 113 acid, 18, 19, 22, 25, 27, 36, 39, 50, 65, 66, 68, 81, 82, 83, 85, 86, 87, 88, 89, 90, 91, 92, 96, 97, 104, 110, 111, 112, 113, 114, 168, 172 acidic, 3, 90, 177 acidity, 66 activation energy, 16 additives, 3, 15, 68, 81 adhesion, 26, 79 adjustment, 124 ADP, 25 adsorption, 12, 14, 49, 55, 95, 99, 103, 120, 121, 172 age, 164 aggregation, 25, 121, 152, 155, 159 agonist, 27, 84 agriculture, 60 alfalfa, 32, 41, 45 algae, 120 alkaline media, 5 aluminium, 6 amine, 91 amine group, 91 amino, vii, 4, 12, 22, 25, 27, 33, 34, 76, 78, 90, 91, 92, 93, 94, 101, 114, 169

amino acid, vii, 4, 12, 22, 33, 34, 76, 78, 90, 91, 92, 93, 94, 101, 114, 169 amplitude, 123 amylase, 99 anaerobic bacteria, 13 anaerobic digestion, 66 analgesic, 88 anastomosis, 91, 108 anemia, 26 angiotensin converting enzyme, 42, 47, 52 ANOVA, 125 antibiotic, 84 antibody, 27, 88, 101, 167, 174, 175 anti-cancer, 94 anticancer activity, 28 anticancer drug, 90, 113 anticoagulant, 91 antigen, 103, 104, 114 antigenicity, 97 anti-inflammatory drugs, 167 antioxidant, 24, 28, 31, 32, 42, 44, 47, 48, 51, 53, 55, 57 antisense, 79, 109 antisense oligonucleotides, 79, 109 antitumor, 92, 106, 113 aorta, 55 apex, 115 apoptosis, 92, 106 appetite, 27, 51 aptitude, 66 aqueous solutions, 54

180

Index

Argentina, 149, 158 arginine, 89 aromatic compounds, 12 Arrhenius equation, 16 artery, 91 arthritis, 28, 46, 110 arthritis management, 28 assessment, 51, 152 atherosclerosis, 27 athletes, 67 atmosphere, 166 attachment, 13, 93 autopsy, 13

blood pressure, 21, 23, 42, 43, 46, 50, 51, 55, 67, 68 blood stream, 78 blood-brain barrier, 108 body weight, 42, 67 bonding, 121 bonds, 78, 91, 93, 94 bone, 92, 110 breast cancer, 47 breast milk, 164, 175, 176 breast-fed infants, ix, 163 buffalo, 63 by-products, viii, 44, 59, 60, 61, 63, 64, 65, 66, 69, 70, 71

B C bacteria, 12, 13, 20, 26, 66, 67, 68, 72 barriers, 3, 78, 81, 93 base, 60 beef, 47 behaviors, 121, 129, 141 beneficial effect, 21, 76 benefits, vii, viii, 24, 25, 70, 75, 77, 93 bile, 25, 80, 84, 99, 114 bile acids, 25 bioassay, 98 bioavailability, viii, 76, 78, 79, 80, 81, 85, 86, 92, 93, 104, 105, 107, 108, 109, 114, 115 bioconversion, 76 biogas, 66, 70, 71 biological activity, vii, 1, 2, 4, 32, 76, 78, 100 biological fluids, 3, 4 biological molecules, viii, 75, 105 biological samples, 172 biological systems, 24, 61 biomass, 66 biomaterials, 81 biomolecules, viii, 76, 95, 103 biopolymers, 119, 120, 121 biotechnological applications, 18 blood, 21, 23, 42, 43, 46, 50, 51, 55, 67, 68, 77, 78, 88, 98, 99, 104, 108, 168 blood clot, 168

Ca2+, 76, 100, 104 calcitonin, 79, 80, 87, 96, 97, 108 calcium, 12, 22, 26, 51, 96, 99 calorie, vii, 68, 76 cancer, 25, 27, 47, 52, 67, 113 candidates, 104 capsule, 111 carbohydrate, 26, 65, 67, 89, 94, 172 carbon, 4, 15, 102, 116 carbon nanotubes, 102, 116 carboxyl, 145 carboxylic groups, 91 carboxymethyl cellulose, 81 cardiovascular disease, 25, 44 cardiovascular system, 25 caries, 23 casein, x, 21, 23, 25, 26, 27, 28, 41, 43, 44, 46, 47, 49, 50, 52, 54, 65, 66, 72, 115, 163, 172, 173, 177 cell culture, 167 cell death, 109 cell line(s), 27, 47, 52, 96, 98, 110 cellulose, 34, 35, 36, 37, 60, 121 central nervous system, 93 ceramic, 4, 19, 35, 43 challenges, 93, 159 charge density, 17

Index cheese, 2, 12, 42, 50, 60, 62, 63, 64, 65, 69, 72, 73, 114, 122, 153, 170, 173 chemical, ix, 3, 5, 11, 18, 19, 27, 28, 34, 64, 65, 68, 77, 78, 79, 80, 93, 94, 111, 113, 120, 149, 151, 177 chemical degradation, 77 chemical properties, 94 chemical reactions, ix, 149 chemical stability, 5 chemical structures, 80 chemotherapy, 109 chicken, 22, 52, 53 children, 164, 167, 174 chitosan, 80, 81, 82, 83, 86, 96, 98, 99, 100, 101, 105, 108, 109, 112, 115 chlorine, 5 cholesterol, 25, 48, 56, 67, 68 chromatography, 2, 49, 57, 165, 170 chronic diseases, 27 chymotrypsin, 81, 167 circulation, 77 classes, 77, 80 cleaning, 3, 4, 11, 13, 18, 19, 20, 44, 46, 53 cleavage, 78 climate, 60 clinical application, 76, 103 clinical trials, 80, 105, 165 CMC, 81, 82 CO2, 166 colitis, 23, 28, 50 collagen, 110 colon, 68, 86, 111 colon cancer, 68 color, 65 colostrum, 165, 167, 172, 173, 174, 175 combustion, 150 commercial, 23, 45, 49, 50, 76, 80, 93, 146, 160 communication, 72 community, 28, 62, 68 compaction, 5, 6, 9 compatibility, vii, 4, 76 complexity, 24, 93 composition, viii, ix, 13, 18, 34, 39, 41, 46, 59, 60, 63, 64, 65, 69, 104, 149, 152, 159

181

compounds, viii, 2, 3, 4, 11, 12, 13, 14, 15, 17, 20, 25, 54, 65, 76, 79, 81, 94, 103, 105, 108, 115, 152 computational fluid dynamics, 151, 155, 158, 160, 161 computer, 150, 151, 152 conditioning, 60 conjugation, 91, 93, 94 consensus, 169 constituents, 176 consumers, vii, 1, 119 consumption, 20, 23, 24, 153, 170 containers, 150 contamination, 18 controversial, 168 cooking, 150 cooling, 60, 150 copolymer(s), 104, 107 coronavirus, 165 correlation coefficient, 135 correlations, 10, 11, 135 cosmetic, viii, 59, 68 cosmetology, viii, 75 cost, ix, 4, 117, 118, 119, 154 covalent bond, 94 covalent bonding, 94 CPPs, 26 Croatia, 176 crust, 158 crystallization, 67, 93 crystals, 93, 106 CSA, 85 culture, 69 CVD, 21, 23, 25 cycles, 19 cyclodextrins, 84, 114 cysteine, 82 cytochrome, 96 cytotoxicity, 101

D dairy industry, viii, 45, 59, 60, 66, 69, 71, 155, 158, 170 dairy waste, viii, 59, 70, 71

182

Index

damages, 60, 175 D-amino acids, 92 deaths, 164 decay, 8, 9 defects, 168 defense functions, ix, 163 defense mechanisms, 24 degradation, viii, 69, 75, 76, 77, 78, 88, 90, 92, 94, 104, 112, 167, 169 degradation process, 76 denaturation, 78, 103, 146, 152, 154, 156, 157, 159 dental care, 26 deposition, 9, 19, 155, 156, 157, 160 deposition rate, 157 deposits, 9, 156 depression, 27, 142 depth, 174 derivatives, viii, 2, 12, 14, 18, 59, 67, 68, 80, 88 desorption, 172 destruction, 110 detectable, 167 detection, 11, 166, 177 detergents, 19, 20, 45, 54 developed countries, 21, 25 diabetes, 28 dialysis, 99 diamonds, 171 diarrhea, 27, 164, 165, 167, 168, 170, 171, 173, 174, 175, 177 diet, 21, 67 dietary intake, 25 diffusion, 8, 89, 93, 121, 156 diffusivity, 11, 16 digestibility, 48 digestion, 27, 38, 42, 66, 73, 81, 99 digestive enzymes, 21, 104 dipeptides, 34, 51 discretization, 152 discs, 37 diseases, 21, 28, 77, 105, 164, 167 dispersion, 121 displacement, 99 dissociation, 176

distribution, 57, 62, 69, 88, 119, 120, 154, 155 DNA, 24, 25 DNA damage, 25 dogs, 88 DOP, 62, 72 dosage, 80, 82, 105, 111, 170 dosing, 78, 87 double bonds, 103 drainage, 119, 121 drug carriers, 116 drug delivery, 94, 105, 107, 108, 109, 110, 112, 114, 115 drug release, 90, 100, 103, 104, 115 drugs, 76, 77, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 107, 108, 111, 114, 115 drying, ix, 67, 103, 150, 154, 155, 157, 158, 159, 160, 161, 170

E economic crisis, 61 effluents, 60, 156 egg, 25, 27, 42, 49, 50, 118, 146, 147 elaboration, 61 electric charge, 41 electric field, 41 electrical fields, 3 electrical resistance, 96 electron, 176 electrophoresis, 2 elucidation, 24 e-mail, 117 employment, viii, 59 emulsification, vii, 76, 99, 100, 152 emulsions, 95, 103, 120, 146 encapsulation, ix, 76, 95, 101, 103, 104, 107, 113, 116 encephalopathy, 68 energy, 3, 11, 15, 66, 71, 123, 150, 152, 154 energy consumption, 3, 11, 15 energy transfer, 150 engineering, ix, 44, 94, 149, 157, 158, 160

Index enkephalins, 80 enteritis, 175 entrapment, 97, 98, 132 environmental impact, 3, 71 environment(s), 36, 78, 95, 155 enzyme(s), 2, 4, 14, 15, 16, 17, 19, 21, 22, 28, 42, 47, 49, 51, 52, 53, 54, 55, 56, 68, 77, 79, 80, 81, 82, 83, 84, 85, 86, 87, 105, 172 enzyme inhibitors, 80 epidemic, 173 epidemiologic, 107 epithelial cells, 28, 47, 52, 176 epithelium, 77, 115 equilibrium, 8 equipment, 155, 160 erosion, 104 erythrocytes, 99, 100, 107 ethanol, 72 ethylene, 83, 96, 107, 108 ethylene glycol, 83, 96, 108 ethylene oxide, 107 eukaryotic, 88, 104 eukaryotic cell, 88, 104 Europe, 68, 71 European Union (EU), 62 evaporation, ix, 70, 103, 150, 154, 155, 158 evidence, 25, 76, 107 evolution, 67 exclusion, 41, 121 experimental condition, 35, 168 exploitation, 66, 70 exposure, 107 extraction, 60 extracts, 24

F factories, 62, 69, 71 farms, 62 fat, vii, 19, 65, 66, 74, 76, 77, 153, 167, 176 fatty acids, 80, 85, 87, 114 fermentation, 36, 49, 66, 68 fertilizers, 70 fiber(s), 30, 40, 53, 60

183

fiber membranes, 40 fibrinogen, 25 filament, 157 films, 13, 95, 98, 109, 120, 121 filters, 14, 68 filtration, ix, 8, 9, 11, 14, 33, 37, 41, 49, 69, 150, 153, 154, 155, 156, 158 financial, 64 fish, 2, 22, 24, 25, 27, 28, 32, 44, 45, 46, 48, 50, 51, 52, 99, 108 fish oil, 44, 99, 108 fixation, 166 flavor, 119, 120 flexibility, 119, 120 flight, 172 flocculation, 121 flora, 175 flour, 53 flow curves, 137 fluid, ix, 3, 7, 100, 101, 104, 121, 129, 139, 147, 149, 150, 151, 152, 154, 156, 158, 159, 160, 161 fluorescence, 166, 172, 177 foams, ix, 66, 117, 118, 119, 120, 121, 122, 123, 125, 126, 134, 136, 137, 139, 141, 142, 143, 144, 145, 146, 147 folic acid, 113 follicle, 112 food additive, x, 68, 80, 163, 170, 171, 173 food applications, vii, 76 food industry, 18, 25, 61, 69, 71, 120, 161 food intake, 26, 42 food products, viii, 75, 77, 120, 146, 147, 160 force, 41 formaldehyde, 107 formation, ix, 9, 17, 33, 92, 118, 119, 120, 121, 126, 145, 156, 158 formula, 18 fouling, 4, 5, 8, 9, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 33, 36, 37, 39, 41, 43, 45, 49, 155 fragility, 118 fragments, 21 France, 61, 62, 124

184

Index

free radicals, 24 freedom, 96 freezing, 150 functional food, 46, 68, 76, 167 fungi, 13

G gastroenteritis, 164, 165, 168, 170, 171, 173, 174, 175, 176 gastrointestinal tract, viii, 75, 77, 79, 93, 167 gel, 12, 121, 123 gel formation, 12 gelation, 121 gene expression, 25 gene transfer, 108 genome, 174 geometry, 10, 102, 116, 151, 152, 154, 160 Germany, 61, 69, 125, 147, 159 glass transition, 96 glass transition temperature, 96 glucagon, 79 glucose, 6, 28, 30, 68, 82, 98, 99 glutamic acid, 116 glutamine, 49 glycans, 168, 172, 177 glycol, 84 glycopeptides, 169 glycosylation, 93, 108, 111, 168, 169, 172 grades, 6, 67 grants, 69 GRAS, 80 gravity, 119, 121 growth, x, 12, 13, 16, 18, 20, 26, 67, 72, 91, 92, 110, 114, 156, 163, 168, 173 growth factor, 110, 168

H half-life, 93, 94, 104, 137 hardness, 14, 63 harvesting, 165 hazardous waste, 61

healing, 167 health, vii, viii, 21, 25, 27, 28, 75, 76, 77, 112, 173 health effects, 112 health problems, 27 heart disease, 25 heat transfer, 150, 155 hemp, 22 heterogeneity, 71 high blood pressure, 21 history, 159, 161 HIV, 67 homeostasis, 28, 115 hormone(s), 26, 28, 80, 87, 112 host, 165, 173 human, vii, 1, 21, 22, 23, 25, 26, 28, 32, 47, 52, 73, 76, 80, 85, 87, 88, 91, 92, 95, 100, 109, 110, 111, 116, 150, 151, 153, 165, 167, 168, 172, 174, 175, 176, 177 human body, 21, 95 human health, vii, 1, 73, 76 human milk, 167, 168 human subjects, 23 humidity, 157 hydrogels, 95, 103 hydrogen, 120, 121 hydrogen bonds, 120 hydrolysates, vii, 2, 22, 24, 25, 28, 29, 31, 32, 33, 39, 41, 43, 44, 47, 49, 51, 52, 53, 55, 57 hydrolysis, vii, 1, 3, 4, 5, 13, 16, 18, 19, 24, 25, 29, 31, 32, 52, 57, 68, 76, 89 hydrolyzed proteins, vii, 2 hydrophilicity, 17, 18, 96 hydrophobicity, 3, 17, 18, 33, 38 hydroxide, 14, 17 hypertension, 22, 23, 26, 28, 42, 50, 55 hypotensive, 23

I ideal, 7, 81, 104 identification, 32, 40, 47, 49, 57 IL-8, 47, 52 ileum, 85, 86, 89

Index immune function, 27 immune response, 78, 164 immune system, 27, 164 immunity, 164, 173 immunization, 165 immunocompromised, 167 immunofluorescence, 166 immunogenicity, 103 immunoglobulin(s), 66, 112, 114, 164, 165, 167, 172, 173, 174 immunomodulatory, 21, 27, 46, 76, 106 immunotherapy, 27 implants, 95 in vitro, x, 21, 22, 23, 24, 27, 41, 53, 56, 76, 81, 98, 104, 106, 108, 109, 111, 112, 163, 165, 170, 173 in vivo, x, 21, 23, 24, 25, 28, 47, 77, 78, 81, 82, 85, 87, 88, 89, 90, 91, 92, 96, 97, 98, 99, 100, 106, 112, 163 incidence, 25, 67, 164 incompatibility, 121 individuals, 55 induction, 23 industrial sectors, 60, 61 industry, vii, viii, 59, 61, 66, 69, 76, 150, 154, 155, 158 infants, ix, 163, 164, 174, 175 infection, x, 68, 163, 164, 165, 167, 168, 169, 170, 171, 172, 173, 175, 176, 177 infectious agents, 164 inflammation, 27, 51, 56 influenza, 165 influenza virus, 165 ingestion, 165 ingredients, vii, 19, 21, 23, 28, 32, 46, 68, 76, 120, 146 inhibition, 21, 22, 23, 24, 27, 28, 29, 30, 31, 42, 48, 52, 55, 168, 176 inhibitor, 81, 82, 83, 84, 85, 86, 87, 88, 112 initiation, 24 injections, 78 injury, 167 innate immunity, 173 inoculation, 166, 170, 171 INS, 98

185

insulin, 27, 78, 79, 80, 81, 82, 86, 87, 88, 93, 97, 98, 99, 100, 106, 107, 112, 115 integration, 64, 71, 103 integrity, 80, 82, 101 interface, 10, 99, 118, 120, 121, 126, 139, 151 intermolecular interactions, 120 intervention, 23, 28, 32 intestinal flora, 68 intestinal tract, 67 intestine, 25, 43, 85, 89, 95, 103 intoxication, 68 intrinsic viscosity, 135 inventors, 177 ion-exchange, 41, 49, 153, 154 ionization, 172 ions, 7, 10, 12, 19, 77, 116 Ireland, 1 iron, 47 isolation, 45, 48, 49, 115 issues, 71, 79, 107 Italy, viii, 59, 61, 62, 63, 64, 65, 67, 69, 71

J Japan, 68, 174 jejunum, 86, 89

K kinetic model, 156

L laboratory tests, 105, 154 lactation, 165 lactic acid, 67, 97 Lactobacillus, 49, 56 lactoferrin, 28, 66, 106, 113 lactose, 37, 60, 61, 65, 66, 67, 68, 69, 70, 71, 72, 92, 154 lamella, 119 laminar, 10 large intestine, 88

186

Index

leakage, 27 lean body mass, 67 legend, 165 leucine, 115 leukemia, 28, 56, 107 leukocytes, 27 lipid peroxidation, 57 lipids, 24, 67, 68, 153 lipolysis, 99, 108 liposomes, 47, 90, 95, 103 liquid chromatography, 172 liver, 56, 78 livestock, 69, 173 living environment, 164 localization, 176 lovastatin, 84 low temperatures, 69 Luo, 55, 100, 111 luteinizing hormone, 112 lymphocytes, 27 lysine, 89 lysozyme, 42, 97

M macromolecular entanglements, 145 macromolecules, 24, 69, 79, 103 macrophages, 27 magnitude, 141, 152 major issues, 103 majority, 62, 170 MALDI, 172 mammal(s), 164 mammalian cells, 112 management, 28, 53, 60, 69, 70, 71, 167 mannitol, 82, 97 manufacturing, ix, 65, 71, 147, 149, 173 manure, 73 mapping, 63 marketing, 154 mass, ix, 10, 13, 15, 149, 154, 172, 176 mass spectrometry, 172, 176 materials, 19, 80, 94, 123, 124, 139, 156 matrix(es), 69, 71, 113, 118, 172 matter, 11, 13

measurement(s), ix, 37, 123, 147, 149, 155, 158 meat, 22, 25, 54, 57 mechanical stress, 120 media, 3, 135, 166 medicine, viii, 75, 76 membrane permeability, 11, 19, 93, 110 membranes, 3, 4, 5, 6, 7, 13, 14, 15, 16, 17, 18, 19, 29, 30, 31, 32, 33, 34, 35, 37, 38, 39, 41, 42, 43, 44, 45, 46, 48, 49, 53, 78, 79, 88, 90, 118, 160 metabolic, 92 metabolism, 51 metal ion(s), 76 metals, 6, 68 meter, 7 methanol, 166 methodology, ix, 41, 44, 93, 118 methylation, 94 methylcellulose, 100 MFRF, x, 163, 170, 171, 173, 174 mice, 28, 57, 91, 111, 165, 168, 170, 171, 174, 175 microorganism(s), 5, 11, 13, 16, 21 microparticles, 105 microscopy, 166 microspheres, 95, 99, 100, 101, 105, 109, 113, 115 microstructure, 95, 118, 120, 146, 147 migration, 101 mixing, 150 model system, 76 modelling, 160, 161 models, viii, 7, 17, 18, 24, 28, 51, 59, 107, 155, 157, 158 modifications, 14, 93 modules, 8, 11, 32, 70, 156, 161 modulus, 10, 123 moisture, 124, 154, 157 moisture content, 154, 157 molecular mass, 79 molecular structure, 94, 137, 168 molecular weight, 2, 4, 7, 33, 41, 67, 69, 78, 79, 80, 89, 96, 104, 106, 108, 109, 110, 111, 114, 120, 135, 137

Index molecules, viii, ix, 3, 39, 41, 69, 75, 76, 77, 78, 79, 81, 82, 83, 85, 93, 94, 95, 96, 103, 104, 105, 106, 120, 126, 129, 137, 163 monolayer, 96, 97, 121, 166 monomers, 14 Moon, 108 morbidity, 175 morphology, 8, 100, 104 mortality, 21 MRI, 116 mucin, 167, 176 mucosa, 26, 80 mucus, 80, 81 muscle extract, 53 mussels, 24

N Na2SO4, 16 NaCl, 34, 37, 39, 40, 124 nanomaterials, 103 nanoparticles, 95, 97, 98, 100, 101, 107, 109, 112 nanostructures, 96, 108 national product, 63 natural food, 164 natural polymers, 120 neovascularization, 92, 110 nervous system, 23, 27 Netherlands, 159 neutral, 7, 12, 16, 25, 36, 37, 38, 39, 48, 78, 142, 177 New Zealand, 68, 69 NH2, 88, 107 nitric oxide, 22 nitrides, 6 nodes, 151 non-polar, 95 nucleic acid, 94, 108 nutraceutical, 21 nutrients, ix, 13, 68, 163 nutrition, 44

187

O obesity, 21 ODS, 92 oil, 95, 97, 99, 101, 105, 108, 112 OJP, 85 oligomers, 86 operating costs, 155 operations, 9, 44, 150, 155, 158 opportunities, 71, 76, 106, 160 optimization, 32, 155, 158, 160 oral administration, viii, ix, 23, 32, 75, 76, 77, 78, 79, 81, 103, 104, 105, 106, 109, 112, 167 oral cavity, 105 organic compounds, 5, 12, 13, 15, 19, 55 organic matter, 13, 49 organize, 70 osmotic pressure, 7, 18 osteoporosis, 26 oxidation, 5, 24, 47 oxidative reaction, 24 oxidative stress, 21, 24, 52 oxygen, 46 oysters, 28 ozonation, 15

P palpation, 170 PAN, 5, 6 pancreas, 27 parathyroid, 79 parathyroid hormone, 79 partial differential equations, 150 partition, 101 pasta, 65 pathogens, 164, 165, 167, 173 pathways, 26, 77 PCR, 9 pepsin, 167 peptidase, 49, 51, 53 perfusion, 112

188

Index

permeability, viii, 4, 5, 17, 27, 75, 77, 78, 79, 80, 82, 83, 84, 85, 88, 90, 93, 97, 98, 109, 110, 115, 156, 160 permeation, 3, 80, 83, 95, 114 permit, 77 PES, 5, 6, 37, 38, 39, 40 pharmaceutical(s), viii, 59, 66, 67, 68, 75, 76, 77, 79, 103, 105, 114 pharmacokinetics, 77 phenol, 147 phenylalanine, 26 phenylketonuria, 26 phosphatidylcholine, 90 phospholipids, 146 phosphorous, 12 phosphorylation, 169 physical properties, 147 physicochemical instability, viii, 75 physicochemical properties, 80, 93, 103 Physiological, 23 pigs, 83, 114 placebo, 50 placenta, 164 plants, 21, 60, 62, 63, 64, 66, 71, 120 platelets, 25 platform, 103 pleasure, 118 pneumonia, 67 polar, 86, 92, 95 polarization, 8, 10, 15, 18, 33, 36 pollution, 60, 69 polyesters, 104, 113 polymer, 5, 81, 100, 103, 112, 120, 129 polymer solubility, 103 polymer solutions, 129 polymeric matrices, 103 polymerization, 103 polymers, 4, 5, 6, 18, 80, 93, 129 polysaccharide(s), ix, 12, 50, 85, 111, 118, 119, 120, 121, 129, 135, 138, 139, 141, 142, 145, 147 polyurethane, 6 porosity, 4, 103 prebiotic sugars, viii, 59 precipitation, 11, 16, 17, 65, 152

preparation, 45, 48, 50, 109 preservation, 25 pressure gradient, 41 prevention, 21, 25, 26, 27, 47, 77, 167, 173, 175, 176 producers, 64, 71 progesterone, 96 project, 70, 71 proliferation, 27 proline, 78 promoter, 80, 115 prophylactic, x, 163, 164, 171 prophylaxis, 165, 175 propylene, 107 prostate cancer, 90, 112, 114 protease inhibitors, 81 protection, 68, 81, 93, 103, 167, 175, 176 protein analysis, 170 protein blocks, 173 protein hydrolysates, 22, 24, 28, 29, 31, 32, 33, 43, 44, 47, 49, 52, 53, 57 protein hydrolysis, vii, 1, 16, 76 protein sequence, vii, 2 protein sources, vii, 2, 3, 13, 25, 27, 32 proteinase, 42, 44, 49, 52 proteolysis, 21, 31 proteolytic enzyme, 78, 79, 104 pumps, 15 purification, 3, 32, 176 purity, 67, 156 PVA, 96, 104

Q quality standards, 62 quotas, 62, 63

R radicals, 44 radius, 7, 17, 35 raw materials, viii, 59, 60, 61, 71 reactants, 11 reactions, 150

Index reactive oxygen, 24 reagents, 94 receptors, 26, 27, 53 recombination, 68 recommendations, 16, 156 recovery, 13, 20, 39, 44, 46, 48, 54, 65, 69, 70, 175 regeneration, 20, 45 regression, 136, 137, 138, 166 rehydration, 6 rejection, 5, 6, 7, 8, 10, 16, 17, 18, 36, 37, 57 renin, 21, 22, 52, 55 repetitions, 125 replication, 169, 172, 173, 176 repulsion, 12, 16, 17, 120 requirements, 56 researchers, viii, 75, 150 residues, 22, 89, 93, 172 resistance, 3, 4, 5, 6, 7, 20, 91, 103, 164 resolution, 150, 152 resources, viii, 59, 60 response, 44, 78, 109 restrictions, 4 reverse osmosis, 3, 43, 46, 69, 154 RFS, 125 rheological properties, ix, 118, 126, 145 rheology, 119, 147 rheumatoid arthritis, 92 riboflavin, 101, 113 risk(s), 13, 14, 15, 16, 18, 21, 44, 68, 70, 107, 164 risk factors, 21 RNA, 164 room temperature, 124 rotavirus, 164, 165, 167, 168, 170, 173, 174, 175, 176, 177 rotor-stator agitator, ix, 118 rubber, 60

S safety, 80, 153, 156 salmon, 24, 79 Salmonella, 68

189

salt concentration, 34, 39 salts, 7, 11, 12, 14, 15, 16, 17, 18, 48, 66, 70, 80, 84, 99, 114 scaling, 12, 13, 14, 17, 155 science, ix, 44, 110, 149, 158 scientific knowledge, 150 scope, 14 secretion, 27, 47, 52, 81, 92 sedimentation, 121 seed, 47 selectivity, 5, 8, 11, 15, 17, 33, 34, 35, 36, 39, 41, 93, 156 selenium, 113 sensitivity, 99, 105, 139 serum, viii, 16, 25, 66, 68, 75, 87, 96, 99, 100, 104, 105, 106, 112, 116, 124, 166 serum albumin, 16, 66, 96, 99, 104, 105, 116, 124 shape, 3, 119, 155 shear, 123, 125, 127, 132, 143, 144, 145, 157, 160 shelf life, 77, 120, 153 shellfish, 22, 32, 46 shoot, 115 showing, 21, 152 sialic acid, 172, 177 side chain, 90, 94 side effects, 164 signals, 26 silica, 13 simulation(s), 99, 150, 155, 156, 157, 158, 159, 160 skimming, 65, 121 skin, 22, 52 small intestine, 86, 87, 89, 98, 100 sodium, vii, 17, 69, 70, 76, 80, 83, 84, 85, 96, 99, 100, 108, 111, 114, 115, 146 software, 150 solubility, 13, 16, 17, 37, 66, 78, 80, 90, 93, 94, 154, 158 solution, 3, 4, 15, 33, 39, 41, 71, 85, 101, 124, 131, 135, 150, 151, 156, 159 solvents, 3, 4, 5, 68, 107 soybeans, 22 Spain, 1

190

Index

species, 7, 24, 42 specifications, 63 Sprague-Dawley rats, 55 stability, viii, ix, 5, 75, 76, 77, 78, 80, 88, 89, 90, 91, 92, 93, 94, 95, 100, 103, 104, 105, 107, 110, 115, 117, 118, 119, 121, 126, 128, 135, 136, 137, 138, 145, 146, 147, 167 stabilization, 77, 79, 105, 129 stabilizers, 121 stakeholders, 70 standard deviation, 125 starch, 60, 80, 99, 108, 121 state, 35, 41, 87, 159, 160 steel, 6, 153, 156 sterile, 15 sterols, 25, 48 stimulation, 81 stomach, 95, 100, 104 storage, 120, 121, 123, 124, 154 stress, 24, 123, 157 stretching, 65, 126 structural modifications, 93 structure, 35, 56, 78, 93, 97, 104, 105, 120, 145, 152, 169, 172, 177 substitution(s), 88, 90, 93, 94, 152 substrate, 21, 22, 66 sucrose, 65, 68 sulfate, 85, 110 Sun, 57, 150, 154, 161 supervisors, 174 supply chain, 63, 70 suppression, 87 surface layer, 5 surface modification, 14, 46 surface properties, 120 surfactants, 19, 80, 120 susceptibility, 79 suspensions, 42 sustainability, 60, 64 sustainable development, viii, 59, 60 Switzerland, 69 symptoms, 165, 170, 175 synergistic effect, 119 synthesis, 107, 174

T tanks, 60, 153 target, 173 techniques, vii, 2, 3, 4, 7, 8, 9, 11, 19, 31, 32, 41, 68, 69, 107 technological properties, vii, 1 technology, vii, 2, 16, 32, 54, 67, 69, 71, 79, 153, 154, 156, 159 temperature, 5, 6, 7, 9, 13, 15, 16, 19, 114, 123, 151, 153, 154, 155, 157, 170 territorial, 64, 71 territory, 69 testing, 156 testosterone, 87, 106 Tetanus, 97 texture, 119, 120 therapeutic agents, 77, 107 therapeutic effects, 99 therapeutics, viii, 75, 77, 80, 93, 94, 109 therapy, 22, 68, 167, 173, 175 thinning, 123, 127, 132, 145 thrombosis, 25 thyrotropin, 115 tissue, 89, 94, 175 titanium, 6 TMC, 82, 83 TNF, 47, 100 TNF-alpha, 47 TNF-α, 100 total cholesterol, 56 total product, 63 toxic effect, 84 toxicity, 111, 114 transference, 88 transmission, 3, 8, 17, 33, 38, 39, 40 transport, ix, 16, 17, 27, 87, 94, 95, 96, 113, 115, 121, 149, 155, 158 transportation, 71 treatment, 7, 15, 16, 21, 52, 68, 69, 73, 121, 147, 153, 155, 156, 166, 167, 173, 174 triglycerides, 25 trypsin, 40, 81, 99, 166, 167, 176 tumor, 28, 91, 92, 98, 109 tumor cells, 28, 109

Index tumor growth, 91 turbulence, 8, 15 turnover, viii, 59 type 2 diabetes, 53

191

W

U ulcerative colitis, 111 ultrasonography, 109 United States, 61, 68, 71 upper respiratory tract, 165, 175 USA, 43, 60, 124, 125, 159, 160, 161 UV irradiation, 15

V vaccine, 103 valorization, viii, 59, 60, 63, 64, 66, 69, 70, 71, 72 valve, 8, 19 vancomycin, 79, 113 variables, 119, 152, 157, 159 variations, 46 varieties, 56, 63 vascular diseases, 24 vasodilation, 55 vector, 152 velocity, 9, 36, 156, 157 ventilation, 150 vibration, 19 viral gastroenteritis, 164 viral infection, 173 viscoelastic properties, ix, 117, 123, 127 viscosity, 7, 11, 16, 80, 121, 122, 123, 125, 126, 127, 131, 132, 135, 137, 143, 144, 145 vision, 60 visualization, 152, 160 vitamin B1, 107 vitamin B12, 107 vitamins, viii, 59, 67

Wales, 7, 57 Washington, 110 waste, viii, 46, 59, 60, 61, 69, 70, 71 wastewater, 60, 70, 71 water, 10, 11, 13, 14, 20, 44, 60, 61, 65, 66, 69, 70, 95, 99, 101, 103, 104, 105, 108, 112, 115, 118, 120, 139, 152, 154, 156 weak interaction, 172 wells, 166 wettability, 103 whey concentrates, x, 163 whey peptides, viii, 75, 76 whey protein isolate (WPI), ix, 52, 53, 67, 70, 71, 117, 124, 126, 128, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 152, 154, 159 whey proteins, vii, viii, ix, 27, 32, 43, 50, 52, 66, 67, 69, 72, 75, 76, 79, 118, 154, 155, 158, 159 worldwide, 28, 63, 65, 164 wound healing, 27

X xanthan, ix, 118, 123, 124, 126, 127, 129, 130, 131, 132, 134, 135, 136, 137, 139, 141, 142, 143, 145, 146 xanthan gum, 146 X-axis, 38, 40

Y yield, 41, 125

Z zirconium, 6