Engineering Aspects of Milk and Dairy Products

Engineering Aspects of Milk and Dairy Products Contemporary Food Engineering Series Editor Professor Da-Wen Sun, Dir...

Author: Jane Selia dos Reis Coimbra | Jose A. Teixeira

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Engineering Aspects of Milk and Dairy Products

Contemporary Food Engineering Series Editor

Professor Da-Wen Sun, Director

Food Refrigeration & Computerized Food Technology National University of Ireland, Dublin (University College Dublin) Dublin, Ireland http://www.ucd.ie/sun/

Engineering Aspects of Milk and Dairy Products, edited by Jane Sélia dos Reis Coimbra and José A. Teixeira (2009) Processing Effects on Safety and Quality of Foods, edited by Enrique Ortega-Rivas (2009) Engineering Aspects of Thermal Food Processing, edited by Ricardo Simpson (2009) Ultraviolet Light in Food Technology: Principles and Applications, Tatiana N. Koutchma, Larry J. Forney, and Carmen I. Moraru (2009) Advances in Deep-Fat Frying of Foods, edited by Serpil Sahin and Servet Gülüm Sumnu (2009) Extracting Bioactive Compounds for Food Products: Theory and Applications, edited by M. Angela A. Meireles (2009) Advances in Food Dehydration, edited by Cristina Ratti (2009) Optimization in Food Engineering, edited by Ferruh Erdoˇgdu (2009) Optical Monitoring of Fresh and Processed Agricultural Crops, edited by Manuela Zude (2009) Food Engineering Aspects of Baking Sweet Goods, edited by Servet Gülüm Sumnu and Serpil Sahin (2008) Computational Fluid Dynamics in Food Processing, edited by Da-Wen Sun (2007)

Engineering Aspects of Milk and Dairy Products Edited by

Jane Sélia dos Reis Coimbra José A. Teixeira

Boca Raton London New York

CRC Press is an imprint of the Taylor & Francis Group, an informa business

CRC Press Taylor & Francis Group 6000 Broken Sound Parkway NW, Suite 300 Boca Raton, FL 33487-2742 © 2010 by Taylor and Francis Group, LLC CRC Press is an imprint of Taylor & Francis Group, an Informa business No claim to original U.S. Government works Printed in the United States of America on acid-free paper 10 9 8 7 6 5 4 3 2 1 International Standard Book Number: 978-1-4200-9022-2 (Hardback) This book contains information obtained from authentic and highly regarded sources. Reasonable efforts have been made to publish reliable data and information, but the author and publisher cannot assume responsibility for the validity of all materials or the consequences of their use. The authors and publishers have attempted to trace the copyright holders of all material reproduced in this publication and apologize to copyright holders if permission to publish in this form has not been obtained. If any copyright material has not been acknowledged please write and let us know so we may rectify in any future reprint. Except as permitted under U.S. Copyright Law, no part of this book may be reprinted, reproduced, transmitted, or utilized in any form by any electronic, mechanical, or other means, now known or hereafter invented, including photocopying, microfilming, and recording, or in any information storage or retrieval system, without written permission from the publishers. For permission to photocopy or use material electronically from this work, please access www.copyright. com (http://www.copyright.com/) or contact the Copyright Clearance Center, Inc. (CCC), 222 Rosewood Drive, Danvers, MA 01923, 978-750-8400. CCC is a not-for-profit organization that provides licenses and registration for a variety of users. For organizations that have been granted a photocopy license by the CCC, a separate system of payment has been arranged. Trademark Notice: Product or corporate names may be trademarks or registered trademarks, and are used only for identification and explanation without intent to infringe. Library of Congress Cataloging-in-Publication Data Engineering aspects of milk and dairy products / editors, Jane Selia dos Reis Coimbra, Jose A. Teixeira. p. cm. -- (Contemporary food engineering) Includes bibliographical references and index. ISBN 978-1-4200-9022-2 (hardcover : alk. paper) 1. Dairy processing. 2. Milk. 3. Dairy products. I. Coimbra, Jane Selia dos Reis, 1962II. Teixeira, José A. (José António), 1957SF250.5.E54 2010 637--dc22 Visit the Taylor & Francis Web site at http://www.taylorandfrancis.com and the CRC Press Web site at http://www.crcpress.com

2009032737

Dedication Our gratitude to God for the life

Contents Series Editor’s Preface...............................................................................................ix Preface.......................................................................................................................xi Series Editor............................................................................................................ xiii The Editors................................................................................................................ xv Acknowledgment....................................................................................................xvii Contributors.............................................................................................................xix Chapter 1. Physical Chemistry of Colloidal Systems Applied to Food Engineering...........................................................................................1 Ana Clarissa dos Santos Pires, Maria do Carmo Hespanhol da Silva, and Luis Henrique Mendes da Silva* Chapter 2. Bioseparation Processes...................................................................... 27 Jane Sélia dos Reis Coimbra and José Teixeira Chapter 3. Applications of Membrane Technologies in the Dairy Industry......... 33 Antonio Fernandes de Carvalho* and J.-L. Maubois Chapter 4. Aqueous Two-Phase Systems Applied to Whey Protein Separation............................................................................................ 57 Abraham Damian Giraldo Zuniga, Jane Sélia dos Reis Coimbra,* José Teixeira, and Lígia Rodrigues Chapter 5. Techniques Applied to Chromatographic Product Manufacturing..................................................................................... 81 Rafael da Costa Ilhéu Fontan,* António Augusto Vicente, Renata Cristina Ferreira Bonomo, and Jane Sélia dos Reis Coimbra Chapter 6. Crystallization of Lactose and Whey Protein................................... 121 Everson Alves Miranda,* André Bernardo, Gisele Atsuko Medeiros Hirata, and Marco Giulietti Chapter 7. Novel Technologies for Milk Processing.......................................... 155 Ricardo Nuno Pereira and António Augusto Vicente* vii

viii

Contents

Chapter 8. Active and Intelligent Packaging for Milk and Milk Products......... 175 Nilda de Fátima Ferreira Soares,* Cleuber Antônio de Sá Silva, Paula Santiago-Silva, Paula Judith Pérez Espitia, Maria Paula Junqueira Conceição Gonçalves, Maria José Galotto Lopez, Joseph Miltz, Miguel Ângelo Cerqueira, António Augusto Vicente, José Teixeira, Washington Azevedo da Silva, and Diego Alvarenga Botrel Chapter 9. Microcalorimetry: A Food Science and Engineering Approach...... 201 Ana Clarissa dos Santos Pires, Maria do Carmo Hespanhol da Silva, and Luis Henrique Mendes da Silva* Chapter 10. Potential Applications of Whey Proteins in the Medical Field......... 221 Lígia Rodrigues* and José António Couto Teixeira Index....................................................................................................................... 253

Series Editor’s Preface CONTEMPORARY FOOD ENGINEERING Food engineering is the multidisciplinary field of applied physical sciences combined with the knowledge of product properties. Food engineers provide the technological knowledge transfer essential to the cost-effective production and commercialization of food products and services. In particular, food engineers develop and design processes and equipment in order to convert raw agricultural materials and ingredients into safe, convenient, and nutritious consumer food products. However, food engineering topics are continuously undergoing changes to meet diverse consumer demands, and the subject is being rapidly developed to reflect market needs. In the development of food engineering, one of the many challenges is to employ modern tools and knowledge, such as computational materials science and nanotechnology, to develop new products and processes. Simultaneously, improving food quality, safety, and security remain critical issues in food engineering study. New packaging materials and techniques are being developed to provide more protection to foods, and novel preservation technologies are emerging to enhance food security and defense. Additionally, process control and automation regularly appear among the top priorities identified in food engineering. Advanced monitoring and control systems are developed to facilitate automation and flexible food manufacturing. Furthermore, energy saving and minimization of environmental problems continue to be an important food engineering issue and significant progress is being made in waste management, efficient utilization of energy, and reduction of effluents and emissions in food production. The Contemporary Food Engineering series, consisting of edited books, attempts to address some of the recent developments in food engineering. Advances in classical unit operations in engineering applied to food manufacturing are covered as well as such topics as progress in the transport and storage of liquid and solid foods; heating, chilling, and freezing of foods; mass transfer in foods; chemical and biochemical aspects of food engineering and the use of kinetic analysis; dehydration, thermal processing, nonthermal processing, extrusion, liquid food concentration, membrane processes, and applications of membranes in food processing; shelf-life, electronic indicators in inventory management, and sustainable technologies in food processing; and packaging, cleaning, and sanitation. The books are aimed at professional food scientists, academics researching food engineering problems, and graduate level students. The books’ editors are leading engineers and scientists from many parts of the world. All the editors were asked to present their books to address the market need and pinpoint the cutting-edge technologies in food engineering.

ix

Furthermore, all contributions are written by internationally renowned experts who have both academic and professional credentials. All authors have attempted to provide critical, comprehensive, and readily accessible information on the art and science of a relevant topic in each chapter, with reference lists for further information. Therefore, each book can serve as an essential reference source to students and researchers in universities and research institutions. Da-Wen Sun Series Editor

Preface Nowadays, it is impossible to imagine a diet not incorporating dairy products. The dairy industry has been able to meet consumer needs by offering a wide range of products that go from the traditional milk to the new and high-value-added products. In addition to the products that consumers traditionally associate with milk, such as cheese, butter, and yogurts, several products contain milk as a source of nutrients with important and unique properties. This reinforces the importance of milk as a raw material in the food industry, and consequently, the relevance of several processing technologies used for milk transformation. The complex nature of this unique material as well as its biological properties are a major challenge for process engineers. The development of new dairy products and the improvement of their safety are due to the developments of food technology which have been able to reply successfully to the challenges of consumers and the industry. Separation processes also play a major role in the processing of milk products, going from the “conventional” defatting to the purification of active proteins, passing by the crystallization of lactose. More recently, evidence of therapeutic properties of several milk proteins available in small amounts reinforced the importance of the application of advanced separation processes in the dairy industry. This book focuses on engineering aspects of food manufacture using the integration of concepts, unit operations, and physical chemistry. Aspects of packaging are also presented. The processing of milk and milk-based products is used as a case study to illustrate what happens in the production chain and to present applications of the bioseparation process. Jane Sélia dos Reis Coimbra José Teixeira

xi

Series Editor Born in Southern China, Professor Da-Wen Sun is a world authority in food engineering research and education. His main research activities include cooling, drying and refrigeration processes and systems, quality and safety of food products, bioprocess simulation and optimization, and computer vision technology. Especially, his innovative studies on vacuum cooling of cooked meats, pizza quality inspection by computer vision, and edible films for shelf-life extension of fruit and vegetables have been widely reported in national and international media. Results of his work have been published in over 200 peer-reviewed journal papers and more than 200 conference papers. He received a first class BSc Honours and MSc in mechanical engineering and a PhD in chemical engineering in China before working in various universities in Europe. He became the first Chinese national to be permanently employed at an Irish University when he was appointed college lecturer at National University of Ireland, Dublin (University College Dublin) in 1995, and was then continuously promoted in the shortest possible time to senior lecturer, associate professor, and full professor. Dr. Sun is now Professor of Food and Biosystems Engineering and director of the Food Refrigeration and Computerised Food Technology Research Group at University College Dublin. As a leading educator in food engineering, Professor Sun has significantly contributed to the field of food engineering. He has trained many PhD students, who have made their own contributions to the industry and academia. He has also given lectures on advances in food engineering on a regular basis in academic institutions internationally and delivered keynote speeches at international conferences. As a recognized authority in food engineering, he has been conferred adjunct/ visiting/consulting professorships from ten top universities in China including Zhejiang University, Shanghai Jiaotong University, Harbin Institute of Technology, China Agricultural University, South China University of Technology, and Jiangnan University. In recognition of his significant contribution to food engineering worldwide and for his outstanding leadership in the field, the International Commission of Agricultural Engineering (CIGR) awarded him the CIGR Merit Award in 2000 and again in 2006, the Institution of Mechanical Engineers (IMechE), based in the United Kingdom, named him Food Engineer of the Year 2004; in 2008 he was awarded CIGR Recognition Award in honor of his distinguished achievements as the top one percent of agricultural engineering scientists in the world. He is a Fellow of the Institution of Agricultural Engineers and a Fellow of Engineers Ireland. He has also received numerous awards for teaching and research xiii

xiv

Series Editor

excellence, including the President’s Research Fellowship, and has twice received the President’s Research Award of University College Dublin. He is a member of the CIGR executive board and honorary vice-president of CIGR, editor-in-chief of Food and Bioprocess Technology—An International Journal (Springer), series editor of the Contemporary Food Engineering book series (CRC Press/Taylor & Francis), former editor of Journal of Food Engineering (Elsevier), and editorial board member of Journal of Food Engineering (Elsevier), Journal of Food Process Engineering (Blackwell), Sensing and Instrumentation for Food Quality and Safety (Springer), and Czech Journal of Food Sciences. He is also a registered chartered engineer.

The Editors Jane Sélia dos Reis Coimbra is an associate professor who teaches unit operations at undergraduate and graduate levels at the Food Technology Department, Federal University of Viçosa, Brazil. She earned her B.S. in chemical engineering at Federal University of Minas Gerais, Brazil, and her D.Sc. degree in food engineering at State University of Campinas, São Paulo, Brazil, and at Heinrich-Heine Universität, Düsseldorf, Germany. Dr. Coimbra earned her postdoctoral degree in nanotechnology at the University of Minho, Portugal, and in protein adsorption at the State University of Campinas, Brazil. Coimbra’s research interests are focused on unit operations, bioseparation, and the design of nanostructures to food applications. José Teixeira is a professor at the Biological Engineering Department, Universidade do Minho, Portugal. He graduated in chemical engineering at Porto University, where he also earned his Ph.D. in 1988. His research interests include nonconventional food processes, advanced bioreactors for food and biotechnology applications, bioreactor hydrodynamics, and medical applications of dairy proteins. Dr. Teixeira supervised 15 Ph.D. theses and several postdoctoral researchers, was the coordinator of 21 research projects, four of them international, and is the editor of two books and author/co-author of 200 peer-reviewed papers. He also has an extensive cooperation with the Portuguese food industry.

xv

Acknowledgment To the students and our collaborators who helped us to conduct the investigative work.

xvii

Contributors André Bernardo Georgia-Pacific Resinas Internacionais Jundai, São Paulo, Brazil Renata Cristina Ferreira Bonomo Universidade Estadual do Sudoeste da Bahia Itapetinga, Bahia, Brazil Diego Alvarenga Botrel Universidade Federal de Viçosa Viçosa, Minas Gerais, Brazil

Marco Giulietti Instituto de Pesquisas Tecnológicas do Estado de São Paulo Universidade Federal de São Carlos São Paulo, Brazil Maria Paula Junqueira Conceição Gonçalves Universidad de Santiago de Chile (USACH) Estación Central, Santiago, Chile

Antonio Fernandes de Carvalho Universidade Federal de Viçosa Viçosa, Minas Gerais, Brazil

Gisele Atsuko Medeiros Hirata Universidade Estadual de Campinas Campinas, São Paulo, Brazil

Miguel Ângelo Parente Ribeiro Cerqueira IBB (Institute for Biotechnology and Bioengineering) Universidade do Minho Braga, Portugal

Maria José Galotto Lopez Universidad de Santiago de Chile (USACH) Estación Central, Santiago, Chile

Jane Sélia dos Reis Coimbra Universidade Federal de Viçosa Viçosa, Minas Gerais, Brazil Paula Judith Pérez Espitia Universidade Federal de Viçosa Viçosa, Minas Gerais, Brazil Rafael da Costa Ilhéu Fontan Universidade Estadual do Sudoeste da Bahia Itapetinga, Bahia, Brazil

Jean-Louis Maubois Dairy Research Laboratory INRA (Institut National de la Recherche Agronomique) Rennes, France Joseph Miltz The Goldstein Packaging Laboratory Haifa, Israel Everson Alves Miranda Universidade Estadual de Campinas Campinas, São Paulo, Brazil

xix

xx

Ricardo Nuno Correia Pereira IBB (Institute for Biotechnology and Bioengineering) Universidade do Minho Braga, Portugal Lígia Rodrigues IBB (Institute for Biotechnology and Bioengineering) Universidade do Minho Braga, Portugal Ana Clarissa dos Santos Pires Universidade Federal de Viçosa Viçosa, Minas Gerais, Brazil Cleuber Antônio de Sá Silva Universidade Federal de Viçosa Viçosa, Minas Gerais, Brazil

Contributors

Washington Azevedo da Silva Universidade Federal de Viçosa Viçosa, Minas Gerais, Brazil Paula Santiago-Silva Universidade Federal de Viçosa Viçosa, Minas Gerais, Brazil Nilda de Fátima Ferreira Soares Universidade Federal de Viçosa Viçosa, Minas Gerais, Brazil José Teixeira IBB (Institute for Biotechnology and Bioengineering) Universidade do Minho Braga, Portugal

Maria do Carmo Hespanhol da Silva Universidade Federal de Viçosa Viçosa, Minas Gerais, Brazil

António Augusto Martins de Oliveira Soares Vicente IBB (Institute for Biotechnology and Bioengineering) Universidade do Minho Braga, Portugal

Luis Henrique Mendes da Silva Universidade Federal de Viçosa Viçosa, Minas Gerais, Brazil

Abraham Damian Giraldo Zuniga Universidade Federal do Tocantins Palmas, Tocantins, Brazil

Chemistry of 1 Physical Colloidal Systems Applied to Food Engineering Ana Clarissa dos Santos Pires, Maria do Carmo Hespanhol da Silva, and Luis Henrique Mendes da Silva* Contents 1.1 1.2 1.3 1.4

Introduction.......................................................................................................1 General Concepts...............................................................................................2 Capillarity..........................................................................................................6 Adsorption.........................................................................................................8 1.4.1 Monolayers........................................................................................... 10 1.4.2 Factors Affecting Adsorption.............................................................. 14 1.5 Micellization.................................................................................................... 14 1.6 Stability of Colloidal Systems......................................................................... 17 1.7 Double Electrical Layer................................................................................... 19 1.8 Colloidal Systems in Food Engineering and Technology............................... 21 1.9 Concluding Remarks....................................................................................... 22 Acknowledgments..................................................................................................... 23 References................................................................................................................. 23

1.1 Introduction Formal studies of interface and colloid science began in the early nineteenth century; however, humans observed and made use of such phenomena thousands of years earlier. For example, the preparation of inks and pigments, baked bread, butter, cheeses, glues, and other substances all represent interfacial and colloidal phenomena of great practical importance to ancient cultures (Myers, 1999). The scientific approach of interfacial phenomena started in the second half of the eighteenth century. Later, in the nineteenth century, the first quantitative studies of the properties of monolayers of surface-active substances in liquid–air interfaces were realized (Norde, 2003). Colloidal dispersions were first described by Selmi in 1845 as “pseudosolutions.” In 1861 the name colloids (from the Greek, meaning “glue”) was assigned to the 1

2


particles in Selmi’s pseudosolution. By choosing this name, Graham intended to emphasize the low rate of diffusion indicating a particle size of, at least, a few nanometers in diameter (Norde, 2003). There is great interest in studying and understanding the colloidal systems. In addition, the presence of colloids in food either as ingredients or natural constituents, as well as their importance as cleaning agents increases the involvement of food engineering and technology researchers in this area. In this chapter, an introduction to colloid science is presented, including basic concepts and definitions.

1.2 General Concepts A colloidal system can be defined as a heterogeneous system, wherein one phase is finely dispersed in another continuous phase, as can be observed in Figure 1.1. Because the dimensions of the dispersed phase are too small, colloidal systems show a large interfacial area (Norde, 2003; Vicent, 2005). It is important to emphasize that the colloidal state is not a physical state but is an aggregation state. In many practical cases, the system can be more complex, presenting more than one dispersed phase, and each of the phases can be multicomponent. Table 1.1 lists some common examples of colloidal systems present in everyday life. Traditionally, colloids are classified as suspension, emulsion, foam, sol, gel, and aerosol. Table 1.2 shows examples of each type of colloidal system. Colloids are an important class of materials, intermediate between bulk and molecularly dispersed systems. The colloid particles may be present in spherical form, but sometimes, one dimension is larger than the other two, such as with a needle shape. Generally, the designation of colloid is applied to particles that are in the range 10 –9 m < r < 10 –6 m. Therefore, the colloid size cannot be determined by either the naked eye or optical microscope, with light scattering the main method used to investigate colloidal particles (Voets et al., 2008).

Phase α

Phase β

Figure 1.1 A colloidal dispersion, where a is the continuous phase and b is the dispersed phase.

Physical Chemistry of Colloidal Systems Applied to Food Engineering

3

Table 1.1 Common Examples of Colloidal Systems Detergent Shampoo Aerosol spray Cosmetic cream Mayonnaise

Ice cream Butter Fruit juice Milk Beer foam

Wastewater Dust Blood Digestive fluid Smoke

A phase of a colloidal system can be defined as a region formed by volume elements, dV, where the intensive thermodynamics properties are constants. In a system with more than one phase, there is a region where molecules of phase a go to phase b, and vice versa, interacting with each other. This boundary place is called the interface (Figure 1.2). Interfaces are the boundaries between immiscible phases, wherein the intensive thermodynamic properties are intermediate between the properties of phases a and b. They can be formed between solid/liquid, solid/gas, liquid/gas, and liquid/liquid. The common thickness of an interface is around 3 to 4 × 10 –10 m (three times more than the diameter of a molecule). Some authors call the interface of a surface when one of the immiscible phases is a gas or vacuum. To define an interface physicochemically, it is necessary to think about energy and keep in mind that nature will always act to reach a condition of minimum free energy in a system. Therefore, if the presence of the interface increases the total free energy, this region will be spontaneously reduced; consequently, the two phases tend to separate (Myers, 1999). In an interface region, there is an excess of energy in relation to the two phases of the system, because in this area, the intermolecular interactions between a-phase molecules and b-phase molecules are unfavorable. Interfaces are the region of excessive Gibbs energy, which occurs as a consequence of the unbalanced intermolecular interactions field between molecules of phases a

Table 1.2 Classification of Colloidal Dispersions Dispersed Phase Continuous Phase Solid Liquid Gas

Solid Solid suspension (e.g., bone, wood) Sol, suspension (e.g., blood, ink) Aerosol (e.g., smoke, dust)

Liquid

Gas

Solid emulsion (e.g., Solid foam (e.g., bread, pearl) loofah) Emulsion (e.g., milk, Foam (e.g., detergent shampoo) foam, beer foam) Aerosol (e.g., fog, spray)

4


Phase α Interface Phase β

Figure 1.2 The interface between phases a and b.

and b. This excess of Gibbs free energy gives rise to various interfacial phenomena, such as interfacial tension (g), wetting, adsorption (Γ), and adhesion. The resulting interfacial properties govern the interactions between colloidal particles and therewith the macroscopic behavior and characteristics of a colloidal system, such as its rheological and optical properties and its stability against aggregation. The interfacial tension, g, can be thermodynamically defined as the increment of Gibbs free energy when reversibly extending the interfacial area by one unit, at constant temperature, pressure, and composition of the system (Norde, 2003). To achieve this definition, some thermodynamics aspects must be considered. For a reversible change in a heterogeneous system, the energy change can be demonstrated as in Equation 1.1:

dG = Vdp − sdT +

∑ µ dn + γ dA i

i

(1.1)

where G is the Gibbs energy of the system, T is the temperature (in K), S is the entropy, p is the pressure, V is the volume, m is the chemical potential of the component i, ni is the number of moles of i in the system, g is the interfacial tension, and A is the interfacial area. The term TdS refers to the heat energy absorbed by the system from its surroundings, and the other terms are related to the work (mechanical and chemical) performed on the system (Norde, 2003). In practice, p and T are constants, and the number of mols of components i between the phases does not vary. Therefore, the interfacial tension can be defined as shown in Equation 1.2:

dG  γ =   dA  T ,P ,ni

(1.2)

A rigorous definition of g can be based on Figure 1.3 and Equations 1.3, 1.4, and 1.5. Consider the prism shown in Figure 1.3, which has edges perpendicular to the interface. This prism is formed by the phase a side, by the volume Va , and by the


fβ

5

Phase β + G´´ G´ –

z x

Phase α

y

fα

Figure 1.3 A binary system and its interface, where f is the energy density, and fa and fb are different, because the molecules of phase a and b are different.

region associated with phase b with Vb . A limit –d is defined, below which the volumetric density of Gibbs free energy at the interface, f int = (dG/dV)P,T, is equal to fa , being, f int ≠ fa , above –d. For regions below +d, fint ≠ fb , and above +d, fint = fb . Because of the variation of density of free energy in the interface, in comparison with the values found in the phases, the free energy of the real system is bigger than the energy of the idealized system, where there were no interfaces—that is, Greal > Ga+ Gb = faVa + fbVb . Therefore, it can be defined that Gint = Greal – (faVa + fbVb) = gS. With regard to the continuous variation, in the z axis, the density of Gibbs free energy can be written as Equation 1.3:

+∞ 0  Gα + Gβ =  fα ( z ) dz + fβ ( z ) dz    −∞ 0

∫

∫

(1.3)

The Gibbs free energy of the surface can be expressed as Equation 1.4:

+∞ 0  Greal − (Gα + Gβ ) =  ( fint ( z ) − fα ( z ))dz + ( fint ( z ) − fβ ( z )) dz  S = γ S (1.4)   0  −∞ 

∫

∫

Considering that f int ≠ fb and f int ≠ fa only in the region between –d < z < + d, Equation 1.4 can be rewritten, changing the limits of integration (Equation 1.5):

+δ 0  Greal − (Gα + Gβ ) =  ( fint ( z ) − fα ( z ))dz + ( fint ( z ) − fβ ( z )) dz  S = γ S (1.5)   0  −δ 

∫

∫

6


Dividing Equation 1.5 by the interface area gives the definition of g (Equation 1.6):

+δ  0 γ =  ( fint ( z ) − fα ( z )) dz + ( fint ( z ) − fβ ( z )) dz    0   −δ

∫

∫

(1.6)

1.3 Capillarity The term capillarity comes from the Latin “capillus” and describes the rise of liquids in fine glass tubes. Laplace showed that the rise of fluids in a narrow capillary was related to the difference in pressure across the interface and the surface tension of the fluid (Birdi, 2003b). There are a lot of phenomena where curved interfaces play an important role. Figure 1.4 illustrates a capillary rise and a capillary depression. The angle formed between the liquid and solid is called contact angle (q), which we will discuss in coming sections. The quantitative interpretation of the capillary events requires an introduction to capillary pressure—the pressure difference across a curved interface as a function of the interfacial tension (Myers, 1999). Consider the formation of an air bubble in a liquid medium. To blow this bubble, some pressure should be applied. This excess pressure is called capillary pressure (Norde, 2003). To understand the relation between capillary pressure, interfacial tension, and the size of the bubble, we will begin with a picture of a cross section of a bubble with radius (R) (Figure 1.5). Any infinitesimal change in the bubble volume is described by Equation 1.7: dV = 4π r 2dr

(1.7)

and any change in the bubble area by Equation 1.8: dA = 8π rdr

(1.8)

θ

(a)

(b)

(c)

Figure 1.4 Capillarity effects: (a) capillary rise, (b) capillary depression, and (c) contact angle (q) formed between the liquid and solid surface.


R

7

dR

Figure 1.5 Cross section of a bubble with radius R. dR corresponds to the change in the bubble radius. The bubble volume is V = 43 π r 3 .

There are two forces that control the bubble size. The first drives the bubble expansion (Equation 1.9) and another force is the contraction (Equation 1.10). At equilibrium, both forces are equal, as demonstrated in Equation 1.11, describing the relation between capillary pressure and interfacial tension (Equation 1.12) (Adamson, 1990):

dw = − P 4π r 2dr

(1.9)

dG = γ 8π rdr

(1.10)

− P 4π r 2dr = −8π rdr

(1.11)

∆P =

2γ r

(1.12)

Equation 1.12 enables formulation of the balances between interfacial forces (F1) and body forces (F2) (Norde, 2003), allowing the calculation of the height of a liquid in a capillary (Equations 1.13 through 1.16):

F1 =

2γ A r

(1.13)

F2 = mg

(1.14)

2γ A = ∆ρVg r

(1.15)

at equilibrium:

Therefore,

h=

2γ r∆ρ g

(1.16)

8


1.4 Adsorption Adsorption is particularly important in surface and colloid science, because it is one of the main ways in which high-energy interfaces can be altered to reduce the overall energy of a system (Myers, 1999). Adsorption of molecules from solution on interfaces is important in controlling a variety of interfacial processes. Adsorption is a consequence of energetically favorable interactions between the molecules at interface and the solute species and also of the interactions between the solute and the molecules solution, which reflects the chemical potential. Several interactions, such as electrostatic attraction, covalent bonding, hydrogen bonding, or nonpolar interactions between the adsorbate and the adsorbent species, as well as the lateral interaction between the adsorbed species, and their desolvation, can contribute to the adsorption process (Somasundaran et al., 2003). The basic concepts behind the factors governing the adsorption of surface-active molecules at interfaces are often mentioned in terms of surface excess concentration of the adsorbed species, Γi, to the surface or interface of the system (Myers, 1999). Mathematically, Γi can be defined as Equation 1.17: Γi

(N − N = i

α i

S

− N iβ

) =  (C (z) − C ∫

+δ

0

i

α i (z)

 −δ



) dz + ∫ (Ct (z) − Ciβ (z)) dz  0

 

(1.17)

where Ni, N iα , and N iβ are the total amount of substance “i” in the system and in the phases a and b, respectively. Consider that the component “i” does not move spontaneously to phase b. Therefore, the second integration term of Equation 1.17 is equal to zero, and this equation can be written as Equation 1.18:

0  Γ i =  (Ci ( z ) − Ciα ( z ) ) dz     −δ 

∫

(1.18)

Applying the Integral Mean Value Theorem to Equation 1.18, Equation 1.19 is obtained:

Γ i = (Ciint − Ciα )δ

(1.19)

If Ciint is much higher than Ciα , the amount of adsorbed material can be defined as shown in Equation 1.20:

Γ i = (Ciint )δ

(1.20)

Equation 1.20 indicates that the amount of adsorbed material is not equal to the concentration of the compound in the interface, but it is equal to the multiplication of the interface compound concentration and the interface thickness.


9

If the interfacial tension of a liquid is reduced by the addition of a solute, the solute must be adsorbed at the interface (Prpich et al., 2008). Equation 1.21 shows the fundamental Gibbs equation for the adsorption phenomena occurring in a binary system: GT = γ + Γ1µ1

(1.21)

where GT is the energy required for adsorption to occur, g is the energy change in the interface area, and the term Γ1m1 is related to the energy change associated with the chemical work of solute transfer from the solution to the interface. Using Equation 1.21, it is possible to obtain an equation that enables us to calculate the amount of adsorbed molecules in a system (Equations 1.22 through 1.25):

dGT dγ dΓ µ = + Γ1 + 1 1 dµ1 dµ1 dµ1

(1.22)

dGT dGT dΓ1 = dµ1 dΓ1 dµ1

(1.23)

Joining both equations and recognizing that dGT / dΓ1 = m1,

µ1dΓ1 dγ µ dΓ = + Γ1 + 1 1 dµ1 dµ1 dµ1

(1.24)

It is possible to obtain Γ1 = −

dγ dµ1

(1.25)

where dm1 can be defined as follows (Equations 1.26 and 1.27):

dµi = dµ 0 + RT ln ai

(1.26)

where ai is the activity. It is possible to express Equation 1.26 in terms of concentration:

dµi = dµ 0 + RTd ln γ 1[C1 ]

(1.27)

where g 1 is the activity coefficient, and C1 is the solute concentration. Some approximations are usually done to make the calculation easier, as can be seen in Equations 1.28 and 1.29. In very diluted solutions, dµ1 = RTd ln[C1 ]

(1.28)

with C1 as the solute concentration. Therefore,

Γ1 = −

dγ C dγ =− 1 RTd ln[C1 ] RT dC1

(1.29)

10

Engineering Aspects of Milk and Dairy Products Y

C

Figure 1.6 Interfacial tension versus concentration. The slope in any point of the curve allows for the calculation of the amount of adsorbed molecules in the interface.

This is a very important equation, as it allows us to obtain the amount of adsorbed molecules (Γi) in an interface in an easier way than with Equation 1.25. Measuring the interfacial tension as a function of the total solute concentration, it is possible to construct a graphic of interfacial tension versus concentration (Figure 1.6). From the slope of this representation, the value of Γ can be obtained. The main advantage of using Equation 1.29 in comparison with Equation 1.25 is related to the difficulties in obtaining the chemical potential of the solute in the solution (m), which is necessary to calculate the Γ. However, it is important to emphasize that Equation 1.29 is only an approximation, and Equation 1.25 is the precise definition of Γ.

1.4.1 Monolayers The term monolayer refers to a layer of amphiphilic molecules that adsorb in an interface. Monolayers are well-defined systems formed by only one layer of amphiphilic molecules (Shah and Moudgil, 2002). Amphiphilic molecules contain a polar head and an apolar tail (Figure 1.7); therefore, these types of molecules are able to interact either with hydrophilic or hydrophobic medium. Surfactants are common examples of amphiphilic molecules. Adsorbed monolayers are formed by allowing the surfactant molecules to adsorb from either one of the adjoining phases. Spread monolayers are obtained with molecules that, at least on the time scale of the experiment, do not or barely dissolve in

Hydrophobic tail

Figure 1.7 An amphiphilic molecule.

Hydrophilic head


11

Aqueous sub-phase

Figure 1.8 A surfactant monolayer.

the adjoining phases. Such monolayers are called insoluble monolayers or Langmuir monolayers (Norde, 2003). According to the fundamental Gibbs equation (Equation 1.30), a monolayer is formed if the adsorption of molecules on the interface reduces only the total free energy (Eastoe, 2005). Considering the formation of a surfactant monolayer in the air–water interface (Figure 1.8), the interaction between the hydrophobic tails with the water is stronger than with the air. However, the energy involved in this interaction is not enough to break the hydrogen binding between the water molecules. Therefore, the hydrophobic tails are outside the water, lowering the enthalpy and raising the entropy, because the water molecules are free to interact with each other; this means that this conformation is enthalpic and entropic favorable, according to the fundamental Gibbs equation (Equation 1.30): dG = dH − TdS

(1.30)

where G is the free Gibbs energy, H is enthalpy, T is temperature, and S is entropy. Langmuir monolayers are formed by depositing amphiphilic molecules in an interface. The most common procedure is spreading (Yam et al., 2008). The amphiphilic molecules are dissolved in a solvent and then this solution is applied at the interface, in which it is not soluble. The most often used equipment to form and study monolayers is the Langmuir trough (Figure 1.9). In a Langmuir trough, the amphiphilic molecules are spread on the subphase, and the solvent disappears by evaporating. Spreading molecules at one side of the barrier results in a difference between the interfacial tension at both sides. This difference

Aqueous sub-phase Mobile barrier

Surface pressure sensor

Aqueous sub-phase (a)

(b)

Figure 1.9 (a) A Langmuir trough. (b) Cross section of a Langmuir trough, showing the amphiphilic molecules in the aqueous subphase.

12


exerts a force on the pressure sensor, which measures this force called superficial pressure (p) (Equation 1.31):

π = γ 0 − γ film

(1.31)

where g 0 is the interfacial tension of the pure solvent, and g film is the interfacial tension of the film. The mobile barrier moves in a controlled way, compressing the molecules in the available area. At very low values of p, the monolayers display gaseous behavior, because the amphiphilic molecules are far from each other and the interaction between them is weak. Inasmuch as the compression gradually increases, the monolayer changes from the gaseous (G) state to the liquid-expanded (LE) state. A further increase in the compression allows a new transition to a liquidcondensed (LC) state, wherein the interaction forces between the amphiphilic molecules become higher, because these molecules are near each other. With a higher compression, the available area between the molecules reduces, and the molecules become closer to each other, this being the state called solid (S) state (Adamson, 1990). If further compression occurs, the collapse pressure is reached, and the film is not in a molecular conformation (Ferreira et al., 2005). In Figure 1.10 and Figure 1.11, it is possible to observe the different aggregation states of molecules in a monolayer and the molecule conformations in these different states, respectively. Monolayers formed in an air–liquid interface can be transferred to a solid support. The transference can be carried out moving the support vertically (Langmuir–Blodgett

50

Collapse pressure

∏/mN m–1

40 S

30

20 LC 10 LE

0 3000

4000

5000

Area/cm2

mg–1

6000

G 7000

Figure 1.10 The different aggregation states of a Langmuir monolayer: (G) gaseous, (LE) liquid expanded, (LC) liquid condensed, and (S) solid. The collapse pressure can also be seen.

13


(a)

(b)

(c)

(d)

Figure 1.11 Different conformations of molecules in the different aggregation states of a monolayer: (A) gaseous, (B) liquid expanded, (C) liquid condensed, and (D) solid.

technique) (Seto et al., 2007) or horizontally (Langmuir–Schaeffer technique) through the monolayer (Carpick et al., 2004; Miyano and Maeda, 1986). The last one may be done above or under the monolayer. Figure 1.12 shows the different techniques used for Langmuir monolayer transference. There are many new developments involving the use of solid support containing monolayers in the food industry, such as their use as biosensors to identify microorganisms, toxins, antibiotic residues, and pesticides.

Solid support

Aqueous sub-phase

(a)

(b)

(c)

(d)

Figure 1.12 The transference process of Langmuir monolayers to solid support: (a) monolayer in an air–water subphase before the transference process, (b) vertical transference (Langmuir–Blodgett technique), (c) horizontal transference (Langmuir–Schaeffer [LS] technique) above the monolayer, and (d) LS technique under the monolayer. In the last case, the subphase is removed by aspirating.

14


1.4.2 Factors Affecting Adsorption Several factors can affect the mechanisms of the adsorption phenomenon. The nature of the surface, for instance, determines the area available for adsorption, and the chemical nature drives the interaction that occurs between adsorbent and adsorbate (Das et al., 2006; Somasundaran et al., 2003). Another important point is the chemical nature of the solute and the solvent, and the interaction between both. For example, according to Equation 1.25, it is possible to promote adsorption even if the interfacial tension is increasing. To reach this condition, the chemical potential of the solute in the solution must be reduced. Temperature can also influence the adsorption process, because it may alter the properties of the solute, surface, and solvent, as well as their interactions (Karadag et al., 2007). In food engineering and technology, this is especially important, because thermal processes are broadly used.

1.5 Micellization In addition to forming oriented interfacial monolayers, amphiphilic molecules can aggregate to form micelles (Figure 1.13). According to Eastoe (2005), micelles are clusters of around 50 to 200 molecules, whose size and shape are governed by geometric and energetic considerations. Micelle formation occurs when the concentration of amphiphilic molecules in solution increases and overcomes the critical micelle concentration (CMC), which is an important parameter (Figure 1.14). Above the CMC, the amphiphilic molecules form micelles, whereas under the CMC, the molecules are in solution. When amphiphilic molecules are added to a solution, they are able to reduce the interfacial tension, because they are adsorbing on the interface, as can be seen in part 1 of Figure 1.14. Inasmuch as the concentration increases, no more reduction in the interfacial tension occurs, and micelles are formed, as shown in part 2 of Figure 1.14. According to Holmberg et al. (2002), in addition to this, osmotic pressure takes on an approximately constant value, light scattering starts to increase, and self-diffusion starts to decrease. It is also important to highlight that the higher the amphiphilic molecule concentration, the higher the number of micelles (not the size of the micelles). But, why is the interfacial tension reduction stopped for concentrations above CMC? The answer to this question is related to the energetic saturation on the interface. In this sense, if one molecule above the CMC goes to the interface, there would

Figure 1.13 A micelle.


15

Y

1 CMC 2 [C]

Figure 1.14 The change of interfacial tension as a function of the concentration of amphiphilic molecules in the solution. At the critical micelle concentration, micelles start to form.

be an increase of enthalpic content as a function of the repulsive forces occurring between the amphiphilic molecules. The entropy would be lower, and according to the fundamental Gibbs equation (Equation 1.30), an increase in energy content on the interface would occur, which is energetically unfavorable. Another important question is “Why are micelles formed?” To answer this question, it is essential to know that the intermolecular interaction between the hydrophobic tails is smaller than the one between the hydrophobic tail and the water molecules. Therefore, it must be clear that the micelle formation is not only related to “protection” of hydrophobic tails from water. Actually, micelles form to liberate water molecules to interact between them, because this binding is more enthalpic favorable. In addition, the system entropy rises even though there is a reduction in the entropic content of amphiphilic molecules. However, the water molecules are free to form different bindings between them, and consequently, the system entropy increases. These facts contribute to the reduction of Gibbs free energy of the total system (Equation 1.30). To understand the thermodynamics of micelle formation, it is necessary to consider the micelle as a phase, presenting intensive thermodynamic properties different from the solvent phase and also from its hydrophilic interface—it means its hydrophilic part. Based on this, Equations 1.32 and 1.33 can be written:

sol = µ °sol + RT ln a sol µamph amph amph

(1.32)

sol where µamph is the chemical potential of the amphiphilic molecule in the solution, sol µamph ° is the chemical potential of the amphiphilic molecule in a very diluted solusol tion, and aamph is the activity of the amphiphilic molecule in a solution:

mic = µ mic + RT ln a mic µamph ° amph amph

(1.33)

mic where µamph is the chemical potential of the amphiphilic molecule in the micelle, mic is the chemical potential of the pure amphiphilic molecule, and a mic µamph ° amph is the activity of the amphiphilic molecule in a micelle.

16


Amphiphilic molecules have different chemical potential when they are in solution or in micelles, as different kinds of interactions take place in the solution and in the micelle. For example, in the solution, there are more water molecules solvating the hydrophilic and hydrophobic regions, and in the micelle, there are almost no water molecules solvating the hydrophobic tails of the amphiphilic molecules. sol At the thermodynamic equilibrium, there is no difference between µamph and mic . Therefore, Equation 1.32 can be subtracted from Equation 1.33, resulting in µamph Equation 1.34: mic − µ sol + RT ln 0 = µ°amph °amph

mic aamph sol aamph

(1.34)

By rearranging Equation 1.34, the required energy for 1 mol of amphiphilic molecule to go from solution to micelle, the Gibbs free energy of micellization (DGmic) is obtained (Equations 1.35 and 1.36):

(

)

mic − µ sol = RT ln − µ°amph °amph

mic aamph sol aamph

(1.35)

mic = µ mic at any temperature, In a = 0 (a = 1). Because the amphiphilic conAs µamph °amph sol sol ] can be considered. Hence, centration is very low, aamph = [Camph

− ∆ micG = RT ln

1 ⇒ ∆ micG = RT ln CMC CMC

(1.36)

The CMC is a fundamental characteristic of an amphiphilic molecule (Liu et al., 2008), because by knowing this parameter, important thermodynamic properties, such as the Gibbs free energy, the entropy, and the enthalpy of micellization, can be calculated. The relevance of this phenomenon in the food industry can be demonstrated if a simple and practical application is considered. In the cleaning step, detergents are used to remove the organic residues from food-contact surfaces. In order to solubilize the fat residues, for instance, the surfactants present in a detergent must be in a concentration above the CMC, because the fat globules are solubilized inside the micelles (Figure 1.15).

Fat

Figure 1.15 A fat globule inside a surfactant micelle.


17

Figure 1.16 A reverse or inverted micelle.

Several factors can influence the CMC. For example, it varies according to the chemical composition of the molecule (Colafemmina et al., 2007). Inasmuch as the alkyl chain increases, the CMC decreases strongly. The temperature and presence of salts can also affect the CMC (LaRue et al., 2008). It is important to emphasize that in a nonaqueous solution, amphiphilic molecules can associate with their polar head, exposing their apolar tails (Figure 1.16) and forming reverse micelles. The thermodynamic of inverted or reverse micelle formation is similar to the micelle formation.

1.6 Stability of Colloidal Systems A colloidal dispersion is considered stable if the dispersion is able to resist aggregation into larger entities that would then segregate from the medium (López-León et al., 2008). A colloidal system to be considered as thermodynamically stable requires that the size and the size distribution of the system particles are not altered and cannot sediment or float. On the other hand, colloidal systems can also be classified as kinetically stable. These systems are stable for a period of time but will destabilize in the future. Colloidal stability is a main issue in applications in food technology and engineering. In most cases in the food industry, stable dispersions are desired, as is the case of milk, fruit juices, and processed foodstuffs, such as butter, mayonnaise, and salad dressings; many times, the product shelf life is related to its colloidal stability (Jang et al., 2005). On the other hand, in some applications, such as wine clarification, aggregation is needed (Norde, 2003). Therefore, it is essential to understand the stability of colloidal systems and manipulate the state of the dispersions for specific applications (Cruz-Silva et al., 2007; Eastman, 2005). Colloid systems can be classified as lyophilic and lyophobic. The first refers to systems that are thermodynamically stable, and the other is related to unstable systems. Lyophobic particles tend to aggregate, because they try to minimize contact with the continuous phase.

18


(a)

(b)

Figure 1.17 Colloid formation: (a) comminution and (b) condensation.

There are many factors contributing to the instability of a colloidal system (Meyer et al., 2006; Zhang et al., 2008) which will be discussed in this section. First, the mechanisms of colloid formation will be presented. There are two ways to form colloids. The first is related to breaking down large pieces to the size required, known as comminution, and the other refers to starting with a molecular dispersion and building the size by aggregation—that is, by condensation (Myers, 1999). Both colloid formation mechanisms are presented in Figure 1.17. There are three basic mechanisms for the destabilization of colloidal systems: isothermic distillation, coalescence, and coagulation. The basic principle of isothermic distillation is that smaller particles transfer molecules to bigger particles. Hence, smaller particles become increasingly smaller, and bigger particles become increasingly larger, destabilizing the colloidal system. This process occurs as a function of a transference process from a region with higher chemical potential to a region with smaller potential, reducing the free Gibbs energy. The difference in chemical potential is related to the Gibbs energy excess on the interface, and this energy excess is a consequence of the closeness between molecules in the smaller particles, which promotes repulsive forces and reduction in entropy. To avoid the particle increase, it is possible to add a surfactant in the solution to reduce the interfacial tension, as when a stabilizer is added in a food formulation, improving the stability. Coalescence is the collision phenomenon between two particles, producing just one particle. This mechanism promotes the diminution of the interfacial area (Figure 1.18) and, consequently, the free Gibbs energy. Food emulsions often undergo coalescence (Akartuna et al., 2008).

(a)

(b)

Figure 1.18 The coalescence process: (a) particles present smaller radius and bigger interfacial area and (b) particles have bigger radius and smaller interfacial area.


(a)

19

(b)

Figure 1.19 The coagulation process: (a) particles present bigger interfacial area and (b) particles together have smaller interfacial area.

There are some strategies to stop the increase of the colloidal particles, such as using a surfactant to reduce the interfacial tension. In food systems, proteins are often used as an adsorbed layer to stabilize fat (Jang et al., 2005). There are other ways to avoid coalescence, such as diminishing the system temperature, because this action decreases particle movement and, consequently, the frequency of collisions; an increase in the system viscosity to reduce the speed of particles also results in a diminution of collisions. Sherman (2007) studied the colloidal stability in ice cream and observed that the size of the oil globule, as well as the number of globules and variation in holding temperature, influences the coalescence process. The author found that globules of diameter greater than 0.95 m allow a sharp reduction in coalescence rate, because decreasing the interfacial area with increasing diameter of the globule leads to a more stable colloidal system. Coagulation can be defined as the aggregation of particles that start moving together (Figure 1.19). This phenomenon occurs aiming to reduce the interfacial area, but this reduction is smaller than in the coalescence process. Sometimes the coagulation phenomenon is desirable, as shown when a practical example in the dairy industry is considered. The milk stability is mainly attributed to the presence of casein. When rennin enzymes are added in the milk, the casein micelles are destroyed. Therefore, cheese formation is a coagulation process that results from the destabilization of a colloidal system, the milk. On the other hand, the acid coagulation of milk, as a result of removing calcium bound between casein micelles, causes destabilization of casein which aggregates and forms a curd, compromising milk and yogurt shelf life (Shaker et al., 2000). To avoid the coagulation process, similar procedures to those applied to avoid coalescence can be adopted.

1.7 Double Electrical Layer Interfaces in contact with water or an aqueous solution can develop small or large electrical charge (Nikolov et al., 2007). The presence or absence of charge in colloid particles is extremely important, as it implies significant features related to stability of the systems. The presence of charges in surfaces is essential to food technology. A surface can acquire charge by different mechanisms, such as ionization of surface groups, dissolution of ionic solids, and preferential ion adsorption (Riley, 2005).

20


Figure 1.20 The double electrical layer.

The electrical charge generated at the interface gives rise to an electrical field around the interface that may modify the ion and molecule spatial distribution close to the interface (Figure 1.20). The ionic distribution around the interface aims at reducing the Gibbs free energy of the system. When the thermodynamic equilibrium is achieved, the electricalchemical potential of all ionic species is kept constant, as can be observed in Equation 1.37:

µ = µi + zieϕ ( x ) N A = µio + RT ln ni + zieϕ ( x ) N A = Const

(1.37)

where µi is the electrical-chemical potential, Z i is the ion charge (positive for a cation and negative for an anion), e is the electron charge, j(x) is the difference in the electrical potential between the interface and a point P placed at a certain distance from the interface. m i is the chemical potential, ni is the amount of ions per m3, NA is the Avogadro constant, R is the gas constant, and T is the temperature in Kelvin. Equation 1.37 shows that there are three main factors that drive the ion configuration around the interface. The first, ( µio ), is the energy of intermolecular interaction between ions and molecules present in the interface. The second (RT ln ni) is associated with the configurational entropy of the ions, determined mainly by the thermal movement of charged species. The third factor (Ziej(x)NA) is the energy due to the electrostatic interactions occurring between an ion with charge Z and the ionic environment that generates the electrical potential j(x). The intermolecular ( µio ) and electrostatic (Ziej(x)NA) interactions will mainly determine the ion packaging in a dense layer formed by nonsolvated chemical species. This layer is closer to the interface, and it is called the Stern–Helmholtz layer. More distant from the interface, a diffuse layer, named the Gouy–Chapman layer, is formed where the ion distribution depends on the entropy (RT ln ni) and the electrostatic interactions (Figure 1.21). The double electric layer is responsible for all electrical properties related to colloidal systems: electrophoresis, electroosmosis, flow, and sedimentation potentials.


21

Stern-Helmholtz layer

Gouy-Chapman layer

Figure 1.21 Stern–Helmholtz and Gouy–Chapman layers.

1.8 Colloidal Systems in Food Engineering and Technology Colloidal systems are often present in food processes. In this section, studies involving colloids in different areas of food engineering and technology will be presented. Complexation between proteins and carbohydrates has been used to stabilize food emulsion and foams. In this context, Semenova et al. (2009) used static and dynamic light scattering to determine various structural and thermodynamic parameters of particles formed from sodium caseinate and dextran sulfate in aqueous solution and at interface, with different molar ratio. They observed that the structure formed in the bulk aqueous phase was able to provide a more effective stabilization of the mixed emulsions, as compared with the interfacial complexes. Many studies have been done in edible coatings applications. Due to their hydrophobicity, lipid compounds have been used as a moisture barrier to coat food products. The influence of polymer (agar and cassava starch) on the structure and the functional properties of emulsified films were evaluated, with observation directed at the formation of an aggregate of lipids in the film formed by vegetable oil and cassava starch. There was no coalescence required to the formation of a continuous lipid phase necessary for the existence an effective barrier. The authors concluded that the application of agar is better suited for most applications (Phan The et al., 2009). Many food products are made up of emulsions, and the stability of these emulsions is one of the key factors that determine the food shelf life. It is known that the interactions between emulsions and other ingredients present in the food may affect the emulsion stability. In this context, Chuah et al. (2009) evaluated the effect of chitosan (CHI) on the stability of monodisperse modified-lecithin (ML)-stabilized soybean oil-in-water emulsion. The stability of the ML-stabilized monodisperse emulsion droplets was investigated as a function of CHI addition at various concentration,

22


pH, ionic strength, thermal treatment, and freezing–thawing treatment by means of particle size and x-potential measurements as well as microscopic observation. The emulsion was stable in the presence of NaCl, and aggregation was observed in the presence of CHl. In the presence of CHl, the emulsion was more stable at higher temperatures, such as 70°C. These results demonstrate the importance of the food components for emulsion stability. Beverage emulsions are often stabilized by arabic gum, xanthan gum, or hydrophobically modified starch. The effects of different concentration levels of arabic gum, xanthan gum, and orange oil on physicochemical emulsion properties and flavor release from orange beverage emulsion were investigated (Mirhosseini et al, 2008b). In another work, Mirhosseini et al. (2008a) evaluated the effects of pectin and carboxymethylcellulose on physical stability, turbidity loss rate, cloudiness, and flavor release of orange beverage emulsion stored for 6 months. It was observed that the stability of orange beverage emulsions decreased during the stored period and that pectin was generally more effective. In relation to flavor release, it was concluded that the type and concentration of hydrocolloid as well as the storage time were important factors. The results exhibited that a decrease in the release content of some volatile compounds appeared to be in parallel with the decrease in emulsion stability. Mayonnaise is a much studied food colloidal system because of its stability issues. Iota-carrageenan (IC) and wheat protein (WP) were evaluated as emulsifier alternatives to egg yolk in a model mayonnaise system. According to the authors, the main motivation for this work was based on the need to replace egg yolk, due its cholesterol content. A 0.1% IC and 4% WP solution was prepared and used as an emulsifier in five different mayonnaise formulations. The obtained mayonnaises were analyzed for viscosity and stability at different temperatures. The authors concluded that the mayonnaise formulation containing a high proportion of IC and WP were stable at 4°C (Ghoush et al., 2008). This kind of study enables us to understand the importance of different compounds on colloidal system stability.

1.9 Concluding Remarks Colloidal systems are present in many areas, including the food sector. In this chapter, the most important concepts and issues involving colloids from the point of view of food engineering and technology were presented. We cited some examples within the chapter, aiming to clarify some aspects of colloids in a food system. We also presented some of the numerous studies, including issues and developments in the colloid world, applied to food research. Foods are complex matrices, containing a lot of different ingredients. Many of these ingredients are in the colloidal state, making it of fundamental importance to understand colloid properties in order to obtain a deep knowledge of food systems. In addition, as has been emphasized, one of the most important factors governing food shelf life is related to its colloidal stability. Another important point is the increasing interest in fat replacement in food, as food researchers and technologists are asked to develop lighter and healthier products with the same quality and stability as their counterparts. This points out how relevant will be the knowledge of colloidal science and technology for food application.


23

Acknowledgments The authors wish to acknowledge the National Council of Technological and Scientific Development (CNPq) and the Foundation to Research Support of the Minas Gerais State (FAPEMIG) for their financial support.

References Adamson, A.W. Physical Chemistry of Surfaces, 5th ed., John Wiley and Sons, Chichester, 1990, 777p. Akartuna, I., Studart, A.R., Tervoort, E., Gonzenbach, U.T., Gauckler, L.J. (2008). Stabilization of oil-in-water emulsions by colloidal particles modified with short amphiphiles. Langmuir, 24 (14), 7161–7168. Birdi, K.S. Introduction to surface and colloid chemistry. In: Birdi, K.S. (ed) Handbook of Surface and Colloid Chemistry, 2nd ed., CRC Press, Boca Raton, FL, 2003a, pp. 11–14. Birdi, K.S. Surface tension and interfacial tension of liquids. In: Birdi, K.S. (ed) Handbook of Surface and Colloid Chemistry, 2nd ed., CRC Press, Boca Raton, FL, 2003b, pp. 76–125. Chuah, A.M., Kuroiwa, T., Kobayashi, I., Nakajima, M. (2009). Effect of chitosan on the stability and properties of modified lecithin stabilized oil-in-water monodisperse emulsion prepared by microchannel emulsification. Food Hydrocolloids, 23 (3), 600–610. Colafemmina, G., Fiorentino, D., Ceglie, A., Carretti, E., Fratini, E., Dei, L., Baglioni, P., Palazzo, G. (2007). Structure of SDS micelles with propylene carbonate as cosolvent: a PGSE−NMR and SAXS study. Journal of Physical Chemistry B, 111 (25), 7184–7193. Cruz-Silva, R., Arizmendi, L., Del-Angel, M., Romero-Garcia, J. (2007). pH- and thermosensitive polyaniline colloidal particles prepared by enzymatic polymerization. Langmuir, 23 (1), 8–12. Das, S.K., Bhowal, J., Das, A.R., Guha, A.K. (2006). Adsorption behavior of rhodamine B on Rhizopus oryzae biomass. Langmuir, 22 (17), 7265–7272. Eastman, J. Colloid stability. In: Cosgrove, T. (ed) Colloid Science: Principles, Methods and Applications, Blackwell, Ames, IA, 2005, pp. 36–49. Eastoe, J. Surfactant aggregation and adsorption at interfaces. In: Cosgrove, T. (ed) Colloid Science: Principles, Methods and Applications, Blackwell, Ames, IA, 2005, pp. 50–76. Ferreira, M., Caetano, W., Iltri, R., Tabak, M., Oliveira Junior, O.N. (2005). Técnicas de caracterização para investigar interações no nível molecular em filmes de Langmuir e Langmuir-Blodgett (LB). Química Nova, 28, 502–510. Ghoush, M.A., Samhouri, M., Al-Holy, M., Herald, T. (2008). Formulation and fuzzy modeling of emulsion stability and viscosity of a gum–protein emulsifier in a model mayonnaise system. Journal of Food Engineering, 84 (2), 348–357. Holmberg, K., Jönsson, B., Kronberg, B., Lindman, B. Surfactants and Polymers in Aqueous Solution, John Wiley and Sons, Chichester, 2002, 545p. Jang, W., Nikolov, A., Wasan, D.T., Chen, K., Campbell, B. (2005). Effect of protein on the texture of food emulsions under steady flow. Industrial and Engineering Chemistry Research, 44 (14), 4855–4862. Karadag, D., Turan, M., Akgul, E., Tok, S., Faki, A. (2007). Adsorption equilibrium and kinetics of Reactive Black 5 and Reactive Red 239 in aqueous solution onto surfactant-modified zeolite. Journal of Chemical and Engineering Data, 52 (5), 1615–1620.

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LaRue, I., Adam, M., Zhulina, E.B., Rubinstein, M., Pitsikalis, M., Hadjichristidis, N., Ivanov, D.A., Gearba, R.I., Anokhin, D.V., Sheiko, S.S. (2008). Effect of the soluble block size on spherical diblock copolymer micelles. Macromolecules, 41 (17), 6555–6563. Liu, J., Liu, D., Yokoyama, Y., Yusa, S., Nakashima, K. (2009). Physicochemical properties of micelles of poly(styrene-b-[3-(methacryloylamino)propyl]trimethylammonium chloride-b-ethylene oxide) in aqueous solutions. Langmuir, 25 (2), 739–743. López-León, T., Santander-Ortega, M.J., Ortega-Vinuesa, J.L., Bastos-González, D. (2008). Hofmeister effects in colloidal systems: influence of the surface nature. Journal of Physical Chemistry C, 112 (41), 16060–16069. Meyer, M., Le Ru, E.C., Etchegoin, P.G. (2006). Self-limiting aggregation leads to long-lived metastable clusters in colloidal solutions. Journal of Physical Chemistry B, 110 (12), 6040–6047. Mirhosseini, H., Tan, C.P., Aghlara, A., Hamid, N.S.A., Yusof, S., Chern, B.H. (2008a). Influence of pectin and CMC on physical stability, turbidity loss rate, cloudiness and flavor release of orange beverage emulsion during storage. Carbohydrate Polymers, 73 (1), 83–91. Mirhosseini, H., Tan, C.P., Hamid, N.S.A., Yusof, S. (2008b). Effect of Arabic gum, xanthan gum and orange oil contents on x-potential, conductivity, stability, size index and pH of orange beverage emulsion. Colloids and Surface A: Physicochemical and Engineering Aspects, 315 (1–3), 47–56. Miyano, K., Maeda, T. (1986). Photoluminescence, absorption and Raman spectra of a polydiacetylene monolayer. Physical Review B, 33 (6), 4386−4388. Myers, D. Surfaces, Interfaces and Colloids: Principles and Applications, 2nd ed., John Wiley and Sons, Chichester, 1999, 519p. Nikolov, V., Lin, J., Merzlyakov, M., Hristova, K., Searson, P.C. (2007). Electrical measurements of bilayer membranes formed by Langmuir−Blodgett deposition on single-crystal silicon. Langmuir, 23 (26), 13040–13045. Norde, W. Colloids and Interfaces in Life Sciences. Marcel Dekker, New York, 2003, 430p. Phan The, D., Debeaufort, F., Voilley, A., Luu, D. (2009) Influence of hydrocolloid nature on the structure and functional properties of emulsified edible films. Food Hydrocolloids, 23 (3), 691–699. Prpich, A.M., Biswas, M.E., Chen, P. (2008). Adsorption kinetics of aqueous n-alcohols: a new kinetic equation for surfactant transfer. Journal of Physical Chemistry C, 112 (7), 2522–2528. Riley, J. Charge in colloidal systems. In: Cosgrove, T. (ed) Colloid Science: Principles, Methods and Applications, Blackwell, Ames, IA, 2005, pp. 14–49. Semenova, M.G., Belyakova, L.E., Polikarpov, Y.N., Antipova, A.S., Dickinson, E. (2009). Light scattering study of sodium caseinate þ dextran sulfate in aqueous solution: relationship to emulsion stability. Food Hydrocolloids, 23 (3), 629–639. Seto, K., Hosoi, Y., Furukawa, Y. (2007). Raman spectra of Langmuir–Blodgett and Langmuir– Schaefer films of polydiacetylene prepared from 10,12-pentacosadiynoic acid. Chemical Physics Letters, 444 (4-6), 328–332. Shah, D.O., Moudgil, B.M. Highlights of research on molecular interactions at interfaces from the University of Florida. In: Mittal, K.L., Shah, D.O. Adsorption and Aggregation of Surfactants in Solution. Marcel Dekker, New York, 2002, pp. 1–48. Shaker, R.R., Jumah, R.Y., Abu-Jdayil, B. (2000). Rheological properties of plain yogurt during coagulation process: impact of fat content and preheat treatment of milk. Journal of Food Engineering, 44 (3), 175–180. Sherman, P. (2007). Colloidal stability of ice cream mix. Journal of Texture Studies, 1 (1), 43–51.


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Somasundaran, P., Markovic, B., Yu, X., Krishnakumar, S. Colloid systems and interfaces— stability of dispersions through polymer and surfactant adsorption. In: Birdi, K.S. (ed) Handbook of Surface and Colloid Chemistry, 2nd ed., CRC Press, Boca Raton, FL, 2003, pp. 393–439. Vicent, B. Introduction to colloidal dispersions. In: Cosgrove, T. (ed) Colloid Science: Principles, Methods and Applications, Blackwell, Ames, IA, 2005, pp. 1–13. Voets, I.K., Moll, P.M., Agil, A., Jérôme, C., Detrembleur, C., Waard, P., Keizer, A., Stuart, M.A.C. (2008). Temperature responsive complex coacervate core micelles with a PEO and PNIPAAm corona. Journal of Physical Chemical B, 112 (35), 10833–10840. Yam, Q., Gao, L., Sharma, V., Chiang, Y.M., Wong, C.C. (2008). Particle and substrate charge effects on colloidal self-assembly in a sessile drop. Langmuir, 24 (20), 11518–11522. Zhang, H., Liu, Y., Zhang, J., Wang, C., Li, M., Yang, B. (2008). Influence of interparticle electrostatic repulsion in the initial stage of aqueous semiconductor nanocrystal growth. Journal of Physical Chemical C, 112 (6), 1885–1889.

2 Bioseparation Processes Jane Sélia dos Reis Coimbra and José Teixeira Contents 2.1 Introduction..................................................................................................... 27 2.2 Techniques for the Separation of Biocompounds............................................ 29 Bibliography............................................................................................................. 31

2.1 Introduction In a trip to the supermarket, you will find yourself in front of various products, and your mind will probably be stimulated to consider how the raw materials were transformed into the products available on the shelf. You may wonder about the history behind a liter of pasteurized milk, a chocolate bar, fresh fruit, or the various types of pasta. Food industrialization aims to establish preservation conditions for foods and guarantee food safety to consumers. Foods are considered as being ready for industrialization after being submitted to a sequence of steps that alter the biological, physical, or chemical properties of the raw materials. Therefore, the whole pasteurized milk found on the supermarket shelf is the result of a series of processes: It was initially collected, either mechanically or manually, cooled, transferred to a dairy processing plant, filtered, stored at low temperatures, and finally processed in a pasteurizer. Following these steps, it was then packed in different containers, stored at adequate conditions, and distributed on a commercial network that allows us to find it in our supermarkets. Low-fat powdered milk, for example, is obtained from a process with a slightly greater number of integrated steps. The initial stages are the same as those for pasteurized milk. After pasteurization, the milk is submitted to centrifugation to separate the fat from the milk in one or more centrifugation cycles. Milk, free of fat, is submitted to a preconcentration step in evaporators and transferred to an atomizer to obtain the solid particles. Once dehydrated, it is separated from the fine particles in a cyclone and then passed through a size standardization process. The final product is then packaged and ready for commercialization. Each step inserted in the food processing line is known as “unit operation.” Employment of the term unit operation for each processing step was proposed by Arthur D. Little in 1915 for a group of common operations in the petrochemical industry and was then extended to other industrial operations. For the production of whole pasteurized milk, the operations of filtration, refrigeration, and heating in the 27

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pasteurizer can be identified. The unit operations of centrifugation and drying are added for the processing of low-fat powdered milk. Therefore, it can be stated that the processing line for the production of a determined food is composed of a series of integrated unit operations, where contact is made between the substances to be processed, some physicochemical properties of the system components are altered, and separation of the two or more system phases is performed. The same is valid for the fabrication lines of other products, including cosmetics, pharmaceuticals, chemicals, petrochemicals, textiles, plastics, cements, among others. The vast majority of processing lines present one or more unit operations that involve the separation of a compound from the material being processed. These are physicomechanical processes involving mass transfer and/or heat transfer. For example, when processing whole milk, the unit operation for physical separation is filtration used to remove impurities found in the milk; operations including heat transfer are, for example, heating and cooling in the pasteurizer. In the case of lowfat powdered milk, separation in cyclones is another of the physical separation operations, whereas concentration in evaporators and drying in an atomizer are cases of unit operations involving heat and mass transfer. Therefore, unit operations for separation include the separation between different types of solids, such as sieving, magnetic separation, and electrostatic separation; between solids and liquids, including filtration, centrifugation, precipitation, decantation, separation in hydrocylones, lixiviation, adsorption, and drying; between liquids, such as liquid–liquid extraction and centrifugation; between liquids and gases, including distillation, adsorption, and humidification; and between solids and gases, as is the case in the use of cyclones. Agitation techniques, including the mixture and transport of solids and fluids, are not considered unit operations but include, principally, applications of the transfer of the momentum concept. However, they are fundamental for the development of plant processing projects and therefore are normally clarified in introductory courses on unit operations. It should also be observed that some authors make a distinction between the terms unit operations and unit processes, considering that a unit process is employed when a unit operation is conducted along with a chemical reaction. In this text, the two terms are used interchangeably. Separation techniques have been developed with the objective of using traditional methodologies for the separation of new substances as well as increasing the efficiency of existing processes. Some of these techniques have already been applied at the industrial scale, such as membrane technologies, which involve the use of semipermeable membranes in separation processes (reverse osmosis, ultrafiltration, microfiltration, nanofiltration); ion exchange chromatography; and drying by lyophilization or by vacuum. Other techniques being tested at the industrial scale are liquid–liquid extraction with aqueous biphasic systems and extraction with supercritical fluids; other classes of chromatography, such as molecular exclusion, hydrophobic interaction, affinity, and immunoaffinity; absorptive membranes; crystallization; and flotation, among others. In case you are responsible for modifying, designing, or developing process lines or supervising production processes, it is necessary to understand the basic principles underlying the unit operations present in the production plant under your supervision. Then you will have the required skills to solve any problems you might encounter. Some of the unit operations for separation technologies that have potential for

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the separation of biological composts of high aggregated value and have potential for being employed in the food industry will be discussed in this chapter. In general, the aggregated value of a product when purified offsets the cost of concentration, as is the case of cheese whey, a product with elevated nutritional and functional value, which is frequently used for the nutrition of athletes. The cost of the food supplement “whey protein concentrate” is roughly $25 per kilogram, but the cost of one of the proteins present in cheese whey known as beta-lactoglobulin, with a purity grade of 80%, is $60 per gram.

2.2 Techniques for the Separation of Biocompounds The development of processes for the production of compounds of biological origin with high aggregated value, such as proteins, enzymes, vitamins, essential oils, antibiotics, hormones, and several others, requires their separation from complex mixtures. The separation of these compounds is characterized by its elevated cost, often due to the need to integrate various purification steps required to achieve the desired purity grade. The complexity of the starting material, such as fermented medium, grains, vegetable oils, or fruits, as well as the small concentration of the target molecule are key factors that contribute to the increase in the final product cost. The scale-up of the separation is another issue that has to be considered. The biotechnology industry is therefore searching for new strategies to reduce costs, including the development, adaptation, and control of new separation techniques. Advances in chemistry, biology, physics, and technological areas have accounted for greater understanding and optimization of separation processes. Development of a strategy for the purification of biomolecules can be conceived with the methodology proposed by Belter (1987) which considers that a process contains four steps with various unity operations in each of them: clarification (removal of insoluble compounds), isolation of the product (capture/concentration), intermediate purification, and polishing (finishing). Generally, the first step of the purification process employs a low-cost solid– liquid separation technique, such as filtration or centrifugation. In this stage, both the purity grade and solute concentration are low. In the second step, a concentration operation is utilized, such as liquid–liquid extraction or adsorption. Purity of the product increases but remains low because the acquisition of a pure compound is not the objective of this step. Concentration of the solute in the medium increases drastically and is approximately four times greater than its initial concentration. In the third step, a chromatographic technique is normally used, significantly increasing the purity of the product, sometimes reaching values up to 99%. This is the principal objective of this phase. The concentration of the solute does not increase considerably and can even be reduced as in the case of chromatographic elution. In the final phase, a technique for removal of the solvent (generally water) and/or trace impurities with drying or crystallization is employed. Purity is not easily altered after reaching values near 100%. The concentration of the solute is drastically modified, reaching the level of roughly 100%. Some of the unit operations to be applied in the development of a purification strategy for biocompounds are listed in Table 2.1.

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Table 2.1 Techniques Used for the Development of Purification Strategies Stage Operation Filtration Microfiltration Centrifugation Precipitation Extraction with aqueous biphasic systems Evaporation Ion exchange Affinity chromatography Hydrophobic interaction chromatography Molecular exclusion chromatography Crystallization Ultrafiltration Diafiltration Lyophilization

Clarification

Concentration

Purification

X X X X X

X X X

X X

X X

X

X

X

X

X

X

X

X

X X X X

X X

Polishing

In the next three chapters, the unit operations of liquid–liquid extraction using aqueous two-phase systems, adsorption, ion exchange, and molecular exclusion chromatography will be described. The objective of these chapters is to give the reader the freedom to propose different ways to integrate these operations to obtain new routes for the concentration, separation, and purification of the compounds found in cheese whey, including the major and minor proteins and lactose. For the traditional unit operations used in milk or milk products processing lines, such as centrifugation, filtration, and precipitation, a list of references containing its description is presented. The importance of the separation and purification of compounds found in cheese whey results from the fact that there are many processed foods that incorporate whey, lactose, and their derivatives, as their application increases the foods’ functional and nutritional characteristics. Although whey powders and whey protein concentrates together with lactose are the most used whey derivatives, there is a growing interest in the separation and purification of several of the whey proteins—b-lactoglobulin, a-lactalbumin, lactoferrin, lactoperoxidase, caseinomacropeptide—and on the obtention of high-purity lactose for further transformation in high-added-value compounds. The application of these bioseparation techniques will play a crucial role in the development of high-added-value products from whey, a high-volume by-product of cheese processing. Aiming to give the reader more information about the food science area, some references are presented as follows.

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Bibliography Beckett, S.T. Physico-Chemical Aspects of Food Processing, Springer, New York, 1996. Bylund, G. Tetra Pak Dairy Processing Handbook, Tetra Pak Processing Systems AB, Lund, Sweden, 1995. Farral, A.W. Engineering for Dairy and Food Products, Krieger, New York, 1963. Fellows, P.J. Food Processing Technology—Principles and Practice, 2nd ed, CRC Press, Boca Raton, FL, 2000. Hartel, R.W., Heldman, D.R. Principles of Food Processing, Springer, Heidelberg, 1997. Ibarz, A., Barbosa-Canovas, G.V. Unit Operations in Food Engineering, CRC Press, Boca Raton, FL, 2003. Kessler, H.G. Food Engineering and Dairy Technology, Verlag A. Kessler, Freising, Germany, 1981. Nakai, S., Modler, H.W. Food Proteins Processing Applications, Wiley-VCH, New York, 2000. Onwulata, C. Whey Processing, Functionality and Health Benefits, Wiley-Blackwell, New York, 2008. Pomeranz, Y., Meloan, C.E. Food Analysis: Theory and Practice, Springer, New York, 1994. Rahman, M.S. Handbook of Food Preservation, 2nd ed, CRC Press, Boca Raton, FL, 2007. Rao, M.A., Rizvi, S.S.H., Datta, A.K. Engineering Properties of Foods, 3rd ed, CRC Press, Boca Raton, FL, 2005. Robinson, R.K. Dairy Microbiology Handbook: The Microbiology of Milk and Milk Products, 3rd ed, Wiley-Interscience, New York, 2002. Robson, B., Garnier, J. Introduction to Proteins and Protein Engineering, Elsevier Science, New York, 1988. Singh, R.P., Heldman, D.R. Introduction to Food Engineering, 4th ed, Academic Press, New York, 2008. Smit, G. Dairy Processing: Improving Quality, CRC Press, Boca Raton, FL, 2003. Spreer, E. Milk and Dairy Product Technology, CRC Press, Boca Raton, FL, 1998. Tamime, A.Y., Law, B.A. Mechanisation and Automation in Dairy Technology, Blackwell, New York, 2001. Toledo, R.T. Fundamentals of Food Process Engineering, 3rd ed, Springer, New York, 2006. Yada, R.Y. Proteins in Food Processing, CRC Press, Boca Raton, FL, 2004. Walstra, P., Wouters, J.T.M., Guerts, T.J. Dairy Science and Technology, 2nd ed, CRC Press, Boca Raton, FL, 2005. Zadow, J.G. Whey and Lactose Processing, Elsevier Applied Science, New York, 1992.

of 3 Applications Membrane Technologies in the Dairy Industry Antonio Fernandes de Carvalho* and J.-L. Maubois Contents 3.1 3.2 3.3 3.4

Introduction..................................................................................................... 33 Definitions.......................................................................................................34 Membrane Design and Configuration.............................................................34 Applications of Membrane Technologies for the Production of Liquid Milks................................................................................................ 36 3.5 Applications of Membrane Technologies for the Separation of Milk Proteins............................................................................................... 39 3.6 Applications of Membrane Technologies for the Production of Cheese.........40 3.6.1 Buffering Capacity.............................................................................. 42 3.6.2 Rheological Changes........................................................................... 43 3.6.3 Rennet Coagulation............................................................................. 43 3.6.4 Adjustment of Aqueous Phase of Cheese Milk...................................44 3.6.5 Cheese Made by Membrane Technologies..........................................44 3.7 Applications of Membrane Technologies for the Treatment of Whey............46 3.8 Applications of Membrane Technologies for the Treatment of Colostrum.................................................................................................... 49 3.9 Applications of Membrane Technologies for the Treatment of Brine and Dairy Wastewaters.................................................................................... 50 3.10 Conclusions and Perspectives.......................................................................... 51 References................................................................................................................. 52

3.1 Introduction Laboratory curiosities until the late 1960s, membrane technologies started to enter in an industrial reality with the pioneering work of Loeb and Sourirajan (1963), who developed the first anisotropic membranes, made from cellulose acetate, able to deliver reasonable fluxes and permeabilities for sea water desalination by reverse osmosis. Then, remarkable progress was accomplished as well in the development of more robust membranes and better designed equipment, as in the applications of this ubiquitous family of technologies which includes separation of molecules or particles 33

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based on size differences: reverse osmosis (RO), nanofiltration (NF), ultrafiltration (UF) and microfiltration (MF), separation based on ionic charge—electrodialysis (ED), and separation based on chemical potential difference (pervaporation). Among the food industries, dairy has undoubtedly known the largest introduction of most of the membrane technologies, MF, UF, NF, and RO (the total installed area is more than 500,000 m² according to Gezan-Guiziou, 2007), except for pervaporation, which has not known any application in milk treatment to our knowledge. Numerous reasons have contributed to the success of membrane technology: deep knowledge of biochemical characteristics of milk and of the coproducts (mostly whey) that helped in the optimization of the wished differential separation, dynamism of several research teams, temperature of operation which did not cause irreversible damages to the biological properties of milk components, high unacceptable environmental pollution induced by the discharge of cheese whey, and so forth. In many countries, the presence of membrane equipment in a dairy plant is as common as the presence of a cream separator. Before describing the many major innovations that originated in dairy processes and new product development by membrane technologies, we will define each separation process.

3.2 Definitions Microfiltration (MF): a pressure-driven membrane separation process using porous membranes with average pore size diameter above 0.1 mm, allowing the retention of all milk particles (somatic cells, fat globules, bacteria, and casein micelles). Ultrafiltration (UF): a pressure-driven membrane separation process using porous membranes with average pore size diameter in the range of 0.001 to 0.1 mm in which the cut-off is preferably expressed by the molecular weight of the retained macromolecule in kD. UF allows permeation of lactose, soluble minerals, and other small milk molecules and water. Nanofiltration (NF): a pressure-driven membrane separation process using porouscharged membranes with average pore size diameter under 0.001 mm (1 nm). NF retains lactose and all other larger milk components but allows permeation of monovalent soluble mineral ions and water. Reverse osmosis (RO): a pressure-driven membrane separation–diffusion process using nonporous membranes. RO is a concentration process that allows permeation of water, only. Electrodialysis (ED): a membrane process in which separation of electrically charged ions results from an electric field.

3.3 Membrane Design and Configuration Four basic membrane configurations are currently available for MF, UF, NF, and RO applications in the dairy industry: (1) tubular, (2) hollow fiber, (3) plate and frame, and (4) spiral wound. Interests and disadvantages of each configuration are detailed in the review of Mistry and Maubois (2004). The most widely used configurations in the world dairy industry are the spiral wound for UF, NF, and RO, and tubular for MF.

Applications of Membrane Technologies in the Dairy Industry

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Spiral-wound membranes essentially made with polysulfone material are considered relatively inexpensive, but they have limitations in terms of acceptable pH range (1 to 12), chlorine resistance (200 ppm for cleaning and up to 50 ppm for short-term storage) (Cheryan, 1998), and temperature (maximum 80°C); moreover, the spiral wound design is not fully satisfactory for cleaning and disinfection efficiently, especially when high concentration factors in milk proteins and fat are required. On the contrary, tubular membranes, essentially made with ceramic material (totally alumina or membrane layer either in zirconium oxide or in titanium oxide or mixture of both oxides supported by alumina) have no pH limitation, can be operated at temperatures to 350°C, and are not affected by high doses of chlorine (up to 2000 ppm) (Cheryan, 1998), but they are relatively expensive. Membrane lifetime is between 6 and 18 months for polysulfone and likely higher than 14 years for ceramic. The choice of a membrane (for MF, UF, and NF) must take into account the pore size distribution of the constituting material, which will determine its physicochemical and mechanical resistances. Unfortunately, to our knowledge, this major characteristic is not well defined because of the difficulties in measuring it. Except for the MF Nuclepore® membrane (Porter, 1990), which is close to being an ideal filter, all other industrial membranes show more or less wide pore size Gaussian distribution, which will determine their selectivity. MF membrane pore size distribution, generally given as the average pore size expressed in mm, is improved by the deposition on the support of at least two membrane layers. Selectivity of UF and NF membranes is generally given as molecular weight cut-off (MWCO), which refers to the molecular weight of a test solution that is rejected at 90% by the membrane under standard processing conditions (Cheryan, 1998). Cleaning and disinfection of membrane equipment used in the dairy industry require the use of good quality water. Soft drinkable water filtrated on 0.2 mm pore size filters in order to get a total bacterial count of 4.5) because of their large apparent cellular volume when they are in milk (Trouvé et al., 1991). Synthesis of the studies done by Madec et al. (1992), the Pasteur Institute, and the Institut


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Figure 3.1 Raw MF Marguerite® liquid milk.

National de la Recherche Agronomique (INRA) has shown for Listeria monocytogenes, Brucella abortus, Salmonella typhimerium, and Mycobacterium tuberculosis DR of 3.4, 4.0, 3.5, and 3.7, respectively. Considering the usually described contaminations of milk at the farm level, such results will assure that MF 1.4 mm skim milk will contain less than 1 CFU.L –1 of these pathogenic bacteria (Saboya and Maubois, 2000) which means 1.4 mm MF milk can be considered as safe as pasteurized milk. France is the only country that has officially allowed the commercialization of extended shelf-life (ESL) MF raw milk. The MF skim milk is mixed with the amount of heated cream (95°C–20 s) requested for fat standardization; the mixture is homogenized and aseptically filled. The authorized shelf life at 4°C to 6°C is 3 weeks. The yearly volume of this MF milk proposed, to our knowledge, by only one dairy company under the trademark Marguerite®, (see Figure 3.1) reached in 2008, 10 million liters. Other plants in many countries apply to the homogenized mixture before conditioning a high-temperature short-time (HTST) (72°C–20 s) pasteurization leading to a claimed shelf life of 5 weeks (Eino, 1997). In many countries, the commercial success encountered by these MF milks is high because of their improved flavor (no cooked taste) and storage ability (Eino, 1997). In some plants, use of 1.4 mm MF has been extended as a pretreatment in the production of UHT milk in order to decrease the intensity of heat treatment (decreased to 140°C–4 s or less) with, consequently, a less cooked taste and an improved storage capability coming from the removal by MF of thermoduric enzymes present in dead bacterial cells and in somatic cells. Use of MF membrane with a smaller pore diameter (0.8 mm instead of 1.4 mm) proposed by Lindquist (1998) was studied in Sweden, France (AFSSA, 2002), and Canada. At 50°C, the obtained flux was in the range of 400 L.h–1.m–2, and the observed DR with this MF 0.8 mm membrane was higher than 13 on Clostridium botulinum, a value that means sterility of the product. After mixing with UHT cream

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(142°C–4 s) for fat standardization, homogenization at 80°C, a heat treatment limited to 95°C–6 s is applied with the only purpose to inactivate endogenous milk enzymes, followed by aseptic conditioning and packaging at 20°C. The obtained milk, called Ultima® milk by the Tetra-Laval Co., was recognized as commercially sterile (AFSSA, 2002). It is stable at 40°C for 62 days and for more than 8 months at room temperature. Its organoleptic quality was judged as similar to that of an HTST pasteurized milk. Its lactulose content was reduced by 71% compared to UHT milk. But until now, to our knowledge, this Ultima process was not commercially developed by the Tetra Pak Co., for unknown reasons. Nevertheless, today, in some dairy plants, the MF 1.4 mm membrane is substituted by the 0.8 mm membrane for the production of ESL MF pasteurized milk in order to extend storage ability. Ultrafiltration offers the possibility of adjusting the protein content of consumer milks either by their specific concentration or by addition of UF milk permeate to the collected milk in order to overcome natural variations in milk composition depending on the cow’s breed, its feed, the season, and its stage of lactation. Surprisingly, although fat standardization is commonly accepted and has been legally authorized for many years, the proposal to deliver consumer milks with defined protein content has encountered incomprehensible and illogical (protein content is one of the payment criteria to the milk producers) opposition, and until now, to our knowledge, no country in the world has modified its legislation for allowing protein standardization of consumer milks despite the fact that adjustment is allowed for milk and whey powders. Questions that arose by protein standardization of consumer milk were summarized by Maubois (1989): ethical acceptance and logic face to fat standardization, one unique level (for example, 32 g.L –1) or several ranging from 29 g.L –1 (minimum defined in EU [J.O.U.E., 2007] and required on a nutritional point of view) to 34 g.L –1 (content found in many developed countries), technologies to be used, and economical consequences. Somatic cells (SCs) that range in size from 15 to 6 mm contain numerous thermoresistant enzymes (protease, lipase, catalase). They are very sensitive to mechanical treatments and consequently are able to release their enzymes into the milk with potential impacts on the quality of the dairy products derived from that milk (pasteurized and UHT milks). They have been shown to protect Listeria monocytogenes during heat treatment, and it has been suggested that milk leukocytes could also contain bovine spongiform encephalopathy (BSE) prions, but no demonstration of this hypothesis has been made either in milk or in colostrum (Maubois and Schuck, 2005). Specific removal of SC from raw whole milk by MF membranes having an average pore size ranging from 12 mm (Le Squeren and Canteri, 1995) to 5 mm (Maubois and Fauquant, 2004) was studied by the group of one of the authors of this chapter. Permeation fluxes between 2000 L.h–1.m–2 and 1460 L.h–1.m–2 were respectively obtained over a running time of 8 h. In the MF retentate, 93% to 100% of the SC were retained which represented 4% to 5% of the volume of treated milk. Permeation rates of the globular fat were, respectively, 89% and 83%. In addition to being the solution for treating milk if the presence of prions was eventually demonstrated, these results open new avenues for researching, for example, the specific effects of varied numbers of SC in normal milks (most of the published studies have been done


39

with mastitis milks of which the composition is highly modified) on the stability of UHT milk in comparison with the residual activity of the endogenous milk plasmin or the proteases of Pseudomonas, the potential creation of microheterogeneity in the microstructure of cheese and, on the other hand, its use as tracers for identifying cows or herds that have produced the used milk raw material, as all their genetic patrimony is contained in the SC. For fermented milks such as yogurts, enrichment of milk either by RO or by NF has led to products considered as better in terms of texture and flavor than those made from milk added with milk powder (Tamime and Robinson, 1985). Such results probably originated by a drastic reduction of the Maillard reaction always initiated in milk powders and the absence of insoluble particles that are more or less present in even high-quality powders. The specific increased flavor improvement found in yogurts made from milk concentrated by NF likely originates from the specific decrease in monovalent ions (Na and Cl) to which consumers are particularly sensible.

3.5 Applications of Membrane Technologies for the Separation of Milk Proteins Proteins are undoubtedly the milk component of most concern by membrane separation technologies. These technologies have opened new avenues profit from their diversity and their unique properties in numerous fields such as technofunctionality (solubility, emulsifying, whipping and foaming abilities, water entrapment, viscosity adjustment) and nutritional quality (amino acid requirements and regulation by their derived biopeptides of major physiological functions of human beings) (Maubois and Ollivier, 1997; Maubois, 2002a). If the first applications concerned only separation and purification of all proteins, nowadays the dairy technologist disposes of numerous means for extracting from milk all the major and numerous minor proteins either by membrane technologies alone or by combination with other techniques such as chromatography. The development of industrial processes was relatively easy and rapid because of the knowledge accumulated by dairy biochemists on the physical properties of milk proteins. By using ultrafiltration and diafiltration, milk protein concentrates (MPCs) containing 50% to 90% proteins in TS can be prepared in order to be used as food ingredients in the meat industry, fermented milks, production of cheese from recombined dry dairy products, and coffee creamers (Novak, 1992). Running UF temperature must be around 50°C to 55°C because apparent viscosity of UF retentates shows a minimum in this range and also because bacterial growth is minimized in this temperature range. In order to avoid some problems in the use of MPCs, such as an unacceptable proteolytic activity, caused by a high microbial count originated by growth during UF in spiral-wound equipment, it is preferable to pretreat the skim milk by MF 1.4 mm before UF to improve the bacteriological quality of the UF retentates and consequently to reduce the intensity of heat treatment before spray-drying. This MF pretreatment will also reduce the UF concentration of the residual fat present in skim milk and consequently will extend the storage capability of MPC powder. Rehydration of highly purified MPC requires the same adjustments as the rehydration

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of purified native micellar casein (Schuck et al., 1994)—that is, an increase to 50°C of the temperature and a higher rate of stirring of the solution. By using MF membrane with an average pore size of 0.1 mm and diafiltration with RO water, pure native casein (PPCN) is prepared (Pierre et al., 1992) and then spraydried (Schuck et al., 1994). The MF separation is carried out first until a concentration of 3:1 either at 37°C to avoid detrimental effects on whey proteins (denaturation of Ig or lactosylation of b-lactoglobulin) or at 50°C. These temperatures will respectively lead to permeate fluxes around 42 L.h–1.m–2 and 75 L.h–1.m–2. Then, diafiltration is done with four volumes of RO water and MF concentration is continued until a concentration of 6:1. By itself, PPCN in solution or in powder has the potential to replace actual commercial caseinates in most of their uses in the food industry. It also has a specific property such as the preservation of stallion sperm (Batellier et al., 2000; Leboeuf et al., 2003) which was used to develop a patented stallion sperm diluter named INRA 96®. It is also an excellent starting substrate for preparing either individual caseins, by exploiting, for example, the temperature-dependent property of b-casein to leave the micelle at low temperature (Terré et al., 1987) and its main biopeptides (Léonil et al., 1991) or the C-terminal part of k-casein, the glycomacropeptide (GMP) of which numerous bioactivities have been shown (Brody, 2000), particularly that inducing the secretion of cholecystokinin (CCK) on human beings (Corring et al., 1997) with its positive regulation consequence on food intake (Portmann, 2002).

3.6 Applications of Membrane Technologies for the Production of Cheese Complete control of the bacteriological quality of the cheese milks can be obtained by a pretreatment by MF 1.4 mm with the same technology as described for fluid milk production. If the MF is done at a temperature of 35 to 37°C, the raw milk labeling that means the production of many “appellation d’origine protégée (AOP)” cheese varieties is respected. It can be claimed from the obtained aforementioned results (Saboya and Maubois, 2000) that cheeses made from 1.4 mm MF skim milk added with pasteurized cream are at least as safe from a hygienic point of view as cheeses made from pasteurized milk. Moreover, because the MF pretreatment removes, at a very high level, spore-forming bacteria such as Clostridium tyrobutyricum, the addition of nitrate at a level of 15 g per 100 kg of milk, as is done in a few countries such as the Netherlands, to prevent late blowing of semihard or hard cheeses, could be suppressed with positive consequences for the environment, quality of resulting whey, and consumer health (Meershon, 1989). On the other hand, use of 1.4 or 0.8 mm MF milk opens new avenues for the cheese scientist to determine and precisely characterize the exact role, such as proteolysis, lipolysis, biogenesis of flavor components, and metabolic commensalism, played in cheese by each component of the acidifying (added starter) and ripening (NSLAB, yeasts, molds, propionibacteria, etc.) ecosystem present in cheese milk, notably the natural flora (De Freitas, 2006; Demarigny, 1997; Maubois et al., 2000; Maubois, 2002b). For example, studies already done have shown the major role played by Hafnia alvei


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Figure 3.2 Cantal cheese obtained from milk added with specific yeast strains.

in the genesis of sulfur aroma compounds (methanethiol and its derivatives DMS, DDS, DTS, and 2,4-DTP) which characterize camembert flavor (Cousin, 1994), as well as that resulting from the metabolism of some particular yeast strains in Cantal flavor and presentation (De Freitas, 2006) (Figure 3.2). Studies done on cheddar and emmental cheeses conduce to the required addition of NSLAB (nonstarter lactic acid bacteria) such as Lb paracasei, Lb casei, Lb rhamnosus, Lb plantarum, Lb curvatus, Lb brevis, or Lb fermentum to cheese milk at a level not yet determined for a positive contribution to cheese flavor during ripening (Lawrence et al., 2004). Whole protein enrichment of cheese milk by UF is widely used for making cheese in many countries. The presence of UF equipment in a cheese plant is now becoming as typical as that of a cream separator. Specific increase of milk proteins ranges from a simple standardization in order to cancel variations due to the cow’s lactation stage to feeding until obtaining a protein and fat level similar to that existing in the drained curd, a product called by Maubois et al. (1969) a liquid precheese. This patented process, named MMV after its inventors (Maubois, Mocquot, Vassal), has completely modified the traditional way of transforming milk in cheese by doing the differential concentration of proteins and fat on the milk itself instead of drainage of the heterogeneous mixture of curd and whey. Despite its numerous significant advantages, including an increase in cheese yielding capacity reaching 20% at the maximum, improvements in plant efficiency including the possibility of developing a continuous process and new cheese varieties, considerable reduction of standard deviation of individual cheese weights, and 80% saving in rennet, representing a net economical benefice between 8% and 12% of cheese milk value, it took almost 10 years for the MMV process to be used at a large scale, probably because it requires a totally new approach to cheesemaking. Since the end of the 1980s, MF using an average 0.1 mm pore size has emerged as a new tool in the cheesemaking industry for a specific enrichment in micellar

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casein of the cheese milk in order to produce cheese through an integrated and high-value use of all milk components (Maubois et al., 2001). The use of caseinenriched milk has encountered fast-growing success in many plants for making numerous cheese varieties. Concentration of all solid contents of cheese milk was proposed either by RO or by NF (Jeantet, 1995), but it is doubtful whether it will find widespread acceptance in industrial cheesemaking because of the organoleptic defects that originate by the excess of lactose (postacidification during ripening) and minerals (sandy texture) in the curd as well as increased fat losses in drained whey (Mistry and Maubois, 2004). To successfully make cheese by UF or 0.1 mm MF, specific properties of the protein-enriched cheese milk must be well understood, because they strongly determine the quality of the final cheese as well as the benefits of the use of membrane technology.

3.6.1 Buffering Capacity If milk is enriched in micellar casein either by UF or by 0.1 mm MF done at normal pH (6.6–6.8), mineral phosphocalcic salts bound to the casein micelles are concentrated in the same proportion as casein. This results in an increase in the buffering capacity of UF and MF retentates, which will consequently modify the basic parameters of the cheesemaking process: acidification kinetics by the lactic starters, ultimate pH value, rennet coagulation kinetics and rheological characteristics of the curd, autolysis properties of mesophilic lactic bacteria during ripening (Hannon et al., 2006; Saboya et al., 2001), activity of ripening enzymes, growth and rate of eventual survival of spoilage flora (Rash and Kosikowski, 1982), and water holding capacity of the cheese mass during ripening because of the resulting increase in ionic strength (Mistry and Maubois, 2004). According to the VCF (ratio of the volumes of milk and retentate), higher production of lactic acid by lactic starter bacteria is required to the optimum pH in the cheese variety, usually 5.2 in semihard and hard cheeses and 4.6 in soft and fresh cheeses. For the latter category, the increase in required lactic acid production was quantified by Brulé et al. (1974) and was expressed as QL = 4.4 VCF + 1.5, where Q L is in grams of lactic acid per kg of pH 6.7 UF retentate. Consequently, for most cheese varieties, use of pH 6.7 retentates without adjustments of the parameters of cheese technology controlling both Ca salt total content and its partition between curd matrix and aqueous phase will create texture and taste defects described in the review of Mistry and Maubois (2004). Thanks to the pioneering study of Brulé et al. (1974) and to the numerous experimentations done after, solutions to adjust Ca salt distribution between casein matrix and aqueous phase of cheese and to correct all these defects have been found and industrially applied with success (Mistry and Maubois, 2004). For example, fresh unripened cheeses such as Quarg, cream cheese, or French fromages frais with almost no micellar Ca must be made by UF through UF of pH 4.6 acidified and renneted milk or buttermilk by using membranes and equipment causing minimum shear stress (Mahaut, 1990). Soft cheese varieties made by the MMV process require initial addition of NaCl until an amount of 0.8% to the UF liquid precheese (LPC) before renneting at pH around 5.4 to 5.2 in order to reduce ionization of phosphoseryl groups of casein molecules with


43

the consequences: an increase of the solubilization of colloidal calcium in the aqueous phase of the curd and a lowering of the isoelectric point (pI) of casein micelles, which increases the security margin to the cheesemaker for handling acidified and renneted LPC products. Satisfactory stretching and melting properties of mozzarella cheeses made from cheese milk enriched 2.3 times in native casein by 0.1 mm MF require an adjustment of Ca salts partition as shown by Dong et al. (2009).

3.6.2 Rheological Changes Milk has a Newtonian rheological behavior, which means its viscosity is not influenced by shear stress. On the contrary, products enriched either in native casein by 0.1 mm MF or in whole protein by UF show not only a sharp exponential increase in their viscosities according to the increase of protein content but also an accentuation of the pseudoplastic characteristics (Culioli et al., 1974), which must be taken into consideration in the design (length and hydraulic diameter of the membranes, centrifuge or positive-displacement recirculation pumps, valve openings, radius of curvature of the pipe elbows) and in the operating parameters (restarting procedure) of membrane equipments. Another consequence of the high viscosity of highly concentrated UF retentates is a strong entrapment of milk gases, which requires the use of a special vacuum device before renneting to avoid getting a spongy curd, and then use of a special mixing tool (static and dynamic) to enable thorough blending of lactic starters and rennet (Maubois, 1987). Acidification of milk to pH 4.6 before UF in order to produce lactic unripened cheeses such as Quarg, French fromages frais, or cream cheese exacerbates the need for a deep knowledge of the effects of UF equipment design and of operating parameters because of the extreme sensibility of the final texture of these products to mechanical shear stresses (Mahaut, 1990). No detrimental effect was seen on the texture of UF retentate by the use of recirculation centrifuge pumps until a protein content of 7.3%, but for obtaining higher concentrated curds with texture identical to traditionally made cheeses, it was necessary to use a recirculation positive-displacement pump, to implement half-length membrane in the last stage (Figure 3.4) and static cooling by cold ventilated air of the packed product.

3.6.3 Rennet Coagulation As shown by Maubois and Mocquot (1971) and Garnot et al. (1982), the use of cheese milks in which casein is concentrated by UF or by MF 0.1 mm leads to a saving in rennet proportional to the volumetric concentration factor. When protein content of milk is increased, there is an increase of the enzymatic reaction velocity, and the required degree of proteolysis at gelation also decreases. With the usual casein content of milk at pH 6.6, coagulation occurs when 80% to 90% of the k-CMP is released. In a 4:1 UF retentate, hydrolysis of only 50% of k-casein is necessary for curd formation (Dalgleish, 1980) due to the sharp increase of the rate of aggregation (Garnot, 1988). On the other hand, it is well known that heat treatment of milk has a detrimental effect on rennet coagulation, weak curds being obtained, and even no curd if rennet is added to UHT milk despite the fact that the primary phase of k-casein hydrolysis by chymosin is almost unaffected. The covalent binding of

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b-lactoglobulin with k-casein which increases the electronegativity of the casein micelles originates this phenomenon (Dalgleish, 1990). Surprisingly, an increase of the protein content by UF before or after UHT restores the curd-forming ability of milk (Maubois et al., 1972). It was hypothesized by Ferron-Baumy et al. (1991) that because UF induces reduction of the distances between casein micelles with bound b-lactoglobulin, there was some screening of the negative charges, apprehended by a lower zeta potential of casein micelles and thus reestablishment of rennet coagulation of UF milk, which allows for the making of cheese with UHT milk.

3.6.4 Adjustment of Aqueous Phase of Cheese Milk Both 0.1 mm MF and UF technologies offer the cheesemaker the opportunity to precisely adjust the composition of the aqueous phase of the cheese milk by using the technology of diafiltration. Moreover, the simple dilution of the components (lactose, soluble mineral salts, and nonproteic compounds by UF plus whey proteins by MF 0.1 mm) by adding water, traditionally used in the making of semihard cheeses, can be used as an alternative to create new tastes and new textures of cheese by substituting the milk original aqueous phase with different solutions. The addition of various salts (NaCl, MgCl, MnCl, citrate, lactate) or sugars (glucose, for example) could be a way through further research to influence the in situ metabolism of either lactic starters or of the other ripening microorganisms present in the cheese ecosystem. The addition of acids also offers multiple ways of research either by inducing a simple solubilization of micellar Ca salts or by influencing the redox potential of the medium (through the addition of ascorbic acid, for example). As proposed by Yvon et al. (1998), addition of a-ketoglutarate between 0.9 and 3.6 mg.g–1 of cheese could enhance conversion of amino acids into aroma compounds. Because citrate is present at a low level in milk, specific addition of this compound should be a way to increase biosynthesis of diacetyl and acetate in some cheese varieties for which they are important flavor components. On the other hand, it is known that the increase of salting decreases the release of hydrophobic peptides from casein curd during ripening and consequently reduces bitterness (Alais, 1984). LAB exopolysaccharides (EPS) have the property to bind water and make it nonsoluble. Consequently, their addition in the aqueous phase of cheese curd will increase concentrations of all soluble components, including NaCl, in the remaining water of hydration and thus allows for reduction of the high salt content which avoids bitterness, in the case, for example, of blue-veined cheeses, the most salted cheese variety.

3.6.5 Cheese Made by Membrane Technologies As described in detail by Mistry and Maubois (2004), a number of cheese varieties are now industrially made all over the world by using UF and MF according to specific recipes that have been continuously improved thanks to the implementation of dairy science results. The most recent developments concern the production of fresh unripened cheese and the adjustment of flavor and texture of soft cheese to answer the wishes of consumers and to improve the quality of cheeses made from


45

milk powders. Thanks to the mechanical resistance of the ceramic membrane and to the knowledge acquired on the effect of shear stress on their rheological properties (Mahaut, 1990), use of UF for making lactic curd cheese varieties such as Quarg, cream cheese, mascarpone, and fromages frais has replaced in many plants the use of filtration clothes or of centrifugal separators either from milk or from buttermilk (Mistry and Maubois, 2004). As reviewed by Hannon et al. (2006), many reports have shown that the ripening of UF cheese, made from fully concentrated milk with the use of mesophilic starters, is retarded or even absent in comparison to traditional cheese ripening. That was because bacterial lysis is significantly delayed. Such a phenomenon that does not occur in cheese made with thermophilic lactic starters was erroneously attributed by Creamer et al. (1987) and Lawrence (1989) to the presence in the UF cheese of a large amount of whey proteins inhibiting the proteolytic activity of residual rennet. The same retarded Lactococcus lysis is observed in cheeses made from milk concentrated only in native casein by 0.1 mm MF (i.e., with the same whey protein content as traditional cheeses) but with the same high Ca salts content as UF cheeses (i.e., the same high buffering capacity which is the cause of this inhibition of lysis). Milk production varies more or less during the year according to the countries (habits of animal husbandry, the climate, the available feed, etc.) and to the producing animals (goats and ewes are almost nonlactating during 44 weeks per year). For satisfying the needs of cheese consumers during the year, transfers of milk cheese components collected when milk production is high are required. Freezing of UF retentates for this purpose was developed in the 1980s for avoiding flavor defects (oxidized and tallow tastes) often observed in cheeses made from frozen fresh curds (Le Jaouen, 2000). In line with UF, the LPC is wrapped in bags of 25, 50, or 100 kg, which are quickly frozen at –20°C and then stored several months at this temperature. Contrary to what is recommended for frozen curds, thawing of LPC must be as quick as possible through the use, for example, of defrosting cabinets in which frozen LPC plates are placed on bundles of stainless steel tubes containing 30°C circulating water. Cheeses made from this thawed LPC did not show any flavor defect if the milk submitted to UF was of good quality. In many countries milk production during the year is insufficient for a regular supply of dairy plants that must therefore import milk powder. It is well known that the higher the heat treatment applied during the manufacture of milk powder, the lower is its cheesemaking ability and the lower the quality of obtained cheeses. As it was for heated milk, use of UF in the framework of the MMV process greatly improves the cheesemaking ability of the reconstituted milk powder and the organoleptic qualities of the obtained cheeses. On the other hand, because the poor renneting coagulation of heated milk powder is originated by the covalent binding of b-lactoglobulin on the micellar k-casein, partial removal of whey proteins by MF 0.1 mm before spray-drying leads to a new milk powder having after reconstitution cheesemaking abilities similar to those of a raw milk and thus offering, after reconstitution or recombination, possibilities to make all cheese varieties (Garem et al., 2000; Quiblier et al., 1991).

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3.7 Applications of Membrane Technologies for the Treatment of Whey Whey is the coproduct of the cheese- and casein-producing industries. Its composition varies according to the process from which it comes, but it can be characterized roughly as milk from which 90% to 95% of the casein and the fat have been removed. Consequently, whey still contains a lot of components of high nutritional value but some (proteins and lactose) in an unbalanced ratio for human nutrition, and others (minor proteins, growth factors, etc.) in very low concentration. Classically, two types of whey (sweet and acid) are distinguished by the dairy industry according to their pH, >6.4 and from 6.4 to 4.6, respectively. Until the end of the 1960s, whey was mainly used for animal (pig) feeding or even was spread over the fields or directed into the sewage system, with adverse environmental consequences: With a BOD5 of 30 to 50 g.L –1, 1000 L of whey has the same polluting power as 400 people (Marshall et al., 1968). Thanks to the membrane technologies, a new whey industry has emerged, and it represents one of the best demonstrations of what can be done with well-thought-out uses of separation techniques and biotechnologies. In Figure 3.3, the current state of the art is summarized. The use of 0.1 mm MF for treating cheese milk has originated a new category of whey named ideal whey by Fauquant et al. (1988). This ideal whey or MMF (milk microfiltrate) is sterile, its eventual virus count is reduced by at least

Classical Whey

Pre-Treatment NF

Phospholipid Enriched Retentate

MF 0.1 µm

Lipid Agregation

UF

Defatted Whey

Casein Enriched Retentate

Milk MF 0.1 µm

UF UF

Partially Demineralized Whey Concentrate

Whey Protein Concentrate

Permeate

Defatted Whey Protein Concentrate

Diafiltration

NF Partially Demineralized Concentrate

Figure 3.3 Membrane technologies and whey.

Lactose Recovery

Whey Protein Isolate


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Figure 3.4 Ultrafiltration (UF) equipment for treating pH 4.6 milk.

2.8 log (Gautier et al., 1994), it has no fat and no k-GMP, and if milk has not been heated, in industrial conditions, its protein and salt contents are those of the aqueous phase of raw milk. When treated by UF, MMF leads with the introduction of a diafiltration step to either a WPC (whey protein concentrate) or a WPI (whey protein isolate), according to the used VCF, having, respectively, protein/total solids ratios of 0.77 and 0.975 (Maubois et al., 2001) and showing high nutritional and technicofunctional qualities (foaming, gelling, solubility) that are better than those obtained for WPC and WPI made from classical whey (Bacher and Konigsfeldt, 2000). As previously mentioned, whey processing was one of the first applications of membrane technologies in the dairy industry. Use of RO instead of vacuum evaporation for preconcentration of whey has allowed for a large saving in energy. Energy consumption is 9 kWh per ton of removed water for RO versus consumption between 90 and 150 kWh for vacuum evaporation (Daufin et al., 1998a). A large part of the RO membrane area carrying out this concentration on sweet whey is replaced by NF membrane in order to simultaneously perform concentration (until a total solids content of 22% to 25%) and partial demineralization (removal of 25% to 50% of the mineral salts, mainly the monovalent species). Moreover, this double effect obtained by the use of NF leads to a saving of energy compared to RO (the used transmembrane pressure is reduced to 30 bar or less), a reduction of effluents, and a significant improvement of the spray-drying of whey because of a better crystallization of lactose. On the other hand, use of NF has provided to the dairy industry new possibilities to commercialize, with a reasonable added value, components of acid whey which were previously difficult to adapt for animal feeding because of its high mineral content and were also the source of adverse effects on the environment. Use of UF was extensively applied to whey to allow the development of a broad array of WPCs with a protein/total solids ratio ranging from 35% to 80%

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(Pearce, 1992). Numerous studies related to the manufacture of WPCs (Hobman, 1992) and to their properties (Mangino, 1992) have been published. In summary, the manufacture of high-quality WPCs required particular care of the technological treatments applied to the milk used for making cheese: Heat treatments have a cumulating effect on the thermosensibility of whey proteins. The presence of proteolytic enzymes issued from psychotrophic or thermoduric bacteria causes casein degradation and increases the NPN (nonprotein nitrogen) content. Regarding the whey, careful control of bacteria growth is essential because during UF, initial bacteria count can be concentrated by up to 30 to 50 times. Lastly, removal of casein fines and of globular residual fat has to be done. To our knowledge, no study has been concerned with the potential detrimental effect of the somatic cell content of whey, in spite of the fact that about 15% of the cells of the cheese milk are going in the whey and then are concentrated as bacteria 30 to 50 times, which means a count in the WPC ranging from 1.5 × 106 to 3 × 106 cells per mL or even more according to the initial count. The preferred temperature for the ultrafiltration of whey is generally 50°C. At this temperature, acceptable fluxes are achieved, and thermal denaturation of protein is minimized. However, most of the manufacturers of WPC preferred to operate at a lower temperature (10°C to 12°C) in spite of a lower flux: half of the flux was achieved at 50°C (Nielsen, 1988) because of a much lower growth of thermoduric bacteria in the spiral-wound membrane equipment and the increase of solubility of calcium salts at this temperature which slows fouling. Residual fat of whey affects the functionality (emulsifying, foaming, and gelling characteristics) of whey proteins, impairs the UF membrane flux during the manufacture of WPC, and can promote the development of off-flavors (Rosenberg, 1995). To remove residual lipids from whey, a thermocalcic aggregation process of these components was simultaneously proposed by Maubois and al. (1987) and Pearce (1987). The optimized method is summarized in Figure 3.3. Whey is first concentrated by UF until a concentration of 4 to 5, then the pH of the retentate is adjusted to 7.5 by the addition of sodium hydroxide, the temperature is maintained at 55°C for 8 minutes, and finally, the lipoprotein-Ca aggregates as well as the small fat globules and the bacteria are separated by 0.1 mm membrane MF. The absence of fat in the resulting microfiltrate strongly reduces the fouling in subsequent UF; consequently, the UF running time is increased, and the UF flux is at least doubled (Maubois et al., 1987) despite the fact that the used UF membrane must have a low MWCO (no more than 5000 Da) for avoiding losses in small-sized whey proteins such as a-lactalbumin. The introduction of a diafiltration step in the UF process allows us to easily obtain WPI with a protein/TS ratio higher than 80% and show high foaming and gelling properties, although slightly lower than those observed for WPI issued from “ideal whey” as aforementioned. Nevertheless, for example, in a meringue-like formulation, egg white can be totally substituted by a 10% WPI protein solution in both overrun and stability. As shown by Pearce (1987) and Maubois et al. (1987), defatted WPI are excellent starting materials for industrial production of purified b-lactoglobulin and a-lactalbumin through a process based on the property of a-lactalbumin to reversibly aggregate (Pearce, 1983) at low pH (3.8 by addition of HCl or preferably citric acid) with a moderate heat treatment (55°C for 30 min). If highly purified b-lactoglobulin is obtained through this process (Léonil et al., 1997), there are still problems, to our


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knowledge, related to the purity of the industrially recovered a-lactalbumin (70% to 75% due to presence of some denatured immunoglobulins, b-lactoglobulin, and bovine serum albumin) despite the numerous studies carried out (Bramaud, 1995). Further work is required for better knowledge of the structural conformation of this protein and of its interactions with the other proteinaceous components present in whey, because a-lactalbumin has a great potential market due to its already shown biological properties (Maubois and Ollivier, 1997), both in nutraceutics (brain hormone precursors due to its high content in tryptophane, four residues per mole) and in therapeutics (apoptosis of lung carcinogen cells as shown by Hakansson et al., 1995). On the other hand, owing to its high content of phospholipids, whey MF retentate that represents a volume of no more than 2% of the initial volume of whey (Baumy et al., 1990) has potential as an effective emulsification agent for food applications (low-fat dairy products or sausages) or cosmetics. As shown by these authors, it constitutes an excellent starting material for producing purified phospholipids (phosphatidylethanolamine, phosphatidylinositol, phosphatidylserine, phosphatidylcholine, sphyngomyelin, and ceramids) with a yield of 150 g per 1000 L of whey. As it was for milk UF permeate, use of UF whey permeate will be either for animal feeding or manufacture of lactose after partial demineralization by NF. However, in some countries, production of edible ethanol from UF whey permeate through yeast fermentation, either for drinking (Carbery process) or for fuel is an industrial reality (Barry, 1982). NF performs simultaneously separation of salts (mainly monovalent species Na, K, H+, and Cl) and concentration. Treatment of milk and UF permeates by NF leads to a demineralization rate around 35% (42% if a diafiltration step is added) and it results in the following claimed benefits (Kelly et al., 1992): reducing costs in condensing the permeate to 62% TS before crystallization (75% of the water is removed by NF), reducing deposit in the finishing evaporator, and improving the lactose crystallization process (higher yields and less washings of the crystals to reach the wished purity). In addition to this improvement in lactose production, NF is the best solution to convert acid and salty whey to normal whey and consequently solve a disposable environmental problem (Kelly et al., 1992). Spray-drying of acid whey treated by NF showed a significant improvement in running parameters and a three times reduction in the hygroscopicity of the powder (Jeantet et al., 1996).

3.8 Applications of Membrane Technologies for the Treatment of Colostrum Use of MF with membrane having an average pore size of 0.1 mm offers an elegant way to solve the poor bacteriological quality of colostrum, the first secretion of mammals after parturition. The obtained microfiltrate, named serocolostrum (Piot et al., 2004), is crystal clear and sterile. It represents the whey part of colostrum and contains, at a level highly superior to milk or cheese whey, numerous interesting components (IgG, growth factors, lactoferrin, etc.) that could be subsequently concentrated by UF for either preserving health through the stimulation of the immature immune system of offspring (piglets, foals, calves, lambs, kids) or preparing the aforementioned components in a purified form required for use in veterinary

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or human medicine. Moreover, recent studies have shown that colostrum contains bioactive components, notably a protein complex with a proline-rich polypeptide named colostrinin shown through a clinical study on about 100 human beings to stabilize Alzheimer’s disease (Leszek et al., 1999). Colostrum also has a high content of MBPs (milk basic proteins), shown by Toba et al. (2000) to regulate growth of osteoblasts and osteoclasts and so prevent osteoporosis. If all these bioactivities are confirmed, production of serocolostrum could grow rapidly.

3.9 Applications of Membrane Technologies for the Treatment of Brine and Dairy Wastewaters Despite the numerous actions implemented for reducing them, the dairy industry was still a large producer of wastewaters (between 1 and 5 L of water used per liter of treated milk). Membrane technologies have allowed not only for reducing the volume and the pollution generated by this used water but also for recycling a significant part of the milk water. The first industrial applications concerned the milk components contained in “white waters” resulting from the condensation of milk water during vacuum evaporation of milk, which are now treated either by RO or by a cascade of NF + RO (named polishing step) in order to reduce their volume at least 20 times (Daufin et al., 1998b). The resulting permeate can be used as a source of heat, as a washing water, or as a water source for steam production (IDF, 1988). The wide range of pH utilization has led to envisage the treatment of all the waste water generated by milk transformation by membrane technologies and even to recycle the CIP (cleaning in place) solutions. According to Daufin et al. (1998), the treatment by NF + RO of 1000 L of an industrial dairy wastewater (pH varying between 6.5 and 9.0) containing 0.8 g.L –1 of total solids and having a BOD5 of 2.0 g.kg–1 leads to 950 L of permeate with no TS and a BOD5 100 times lower. The remaining 50 L of retentate with a TS content of 30 g ⋅ kg–1 and a BOD5 of 20 g ⋅ kg–1 could be ultimately depurated by membrane fermentors (Kulozik, 1991; Maubois, 1974). Use of ceramic MF, UF, and NF membranes that can be carried out on the pH scale as well as some particular organic membranes resistant to high pH has been proposed for recycling acid and caustic soda solutions employed in the dairy industry in CIP systems. The obtained savings on both solutions in terms of maintaining cleaning efficiency and decreasing final pollution could lead to a payback of the membrane equipment ranging from 1.5 to 5.3 years (Daufin et al., 1998). Efficient sanitation of cheese brine is requested to prevent postcontamination of cheeses by microorganisms able to grow in 20% salt solution (Staphylococcus, Listeria, yeasts, and molds). MF 0.1 or 0.8 mm might be an interesting technique in the substitution of the actually used pasteurization and Kieselguhr treatment, which, in addition to the inactivation of contaminating microorganisms, changes the protein and mineral balance of the brine and thus modifies Ca and Na mineral salts transfers between brine and cheese (Pedersen, 1992). MF is carried out with a concentration factor between 1:30 and 1:100 and a permeation flux reaching 600 l.h–1.m–2, at 20°C for avoiding precipitation of Ca salts, either periodically on the whole brine or continuously on a fraction of the daily used brine.


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3.10 Conclusions and Perspectives Membrane processes have already allowed for huge improvements in the quality of existing dairy products, many developments of new products, and enhanced process efficiency and profitability (Rosenberg, 1995). The world dairy industry has new powerful and flexible tools for dramatic improvements in the hygienic safety of all dairy products while avoiding cumulative intense heat treatments always detrimental to the intrinsic biological properties of most of the milk components. Moreover, removal by MF of somatic cells (SCs) and contaminating bacteria of raw milk opens for dairy scientists new avenues to determine without any bias the precise role of endogenous thermoduric enzymes of SC and the complex metabolism of microbial ecosystems involved in the production and in the ripening of fermented dairy products (particularly cheese varieties). Among the specific separations of milk particles, if preparation of casein-enriched milks or purified native milk solutions or powders are now an industrial reality thanks to the use of 0.1 mm MF, that is not yet the case for milk fat globules in spite of the process developed by Goudédranche et al. (2000) which allows separation of small and large fat globules. Removal of contaminating bacteria present in cream remains for membrane technologists a challenge that could be faced positively in the near future by an adapted pretreatment of cream before MF. With the different membrane technologies (MF, UF, NF, and RO) eventually combined with other separation methods such as chromatography, new horizons for fractionation of numerous milk components, especially proteins, have been opened. Through collaborative studies with nutritionists and medical research teams, dairy scientists now have the capability to determine the nutritional and eventually physiological impacts of purified bioactive milk components on human beings. If demonstration of functionality requires highly purified milk derivatives, it must be noticed that commercial functional products, issued from these studies, will not often need a high purity, contrary to organosynthetic drugs produced by the pharmaceutical industry, the worst contaminants being nutriments with naturally no side effects. On the other hand, in our opinion, the enormous potentialities offered by the membrane reactors either with enzymes or with microorganisms have not yet been exploited thoroughly. The membrane enzymatic reactor technology allows not only the possibility to prepare very well-defined peptidic mixtures, for example, but also to deepen the knowledge of complex enzymatic reactions through the characterization of all intermediary products. With the membrane fermentor technology, it should be possible to continuously produce highly concentrated biomass and also excreted metabolic products and equally to do in-depth studies on bacterial commensalism in ecosystems (e.g., the use by one or several species of metabolites or the intracellular content released by lysis of one or several others). Finally, if the dream by one of the authors in 1970 during an informal exchange with A. Michaels, a membrane pioneer and creator of one of the first membrane producing companies, of a dairy plant in which all milk treatments will be done by membrane technologies, is not yet a reality, the applications described in this chapter show how dairy scientists and the dairy industry have worked together to expand the capabilities of membrane processes with the goal of improving the quality and creating new milk derivatives answering to the constant needs of world dairy consumers.

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Maubois, J.-L., Fauquant, J., Famelart, M.-H., Caussin, F. (2001). Milk microfiltrate, a convenient starting material for fractionation of whey proteins and derivatives. The importance of whey and whey components in food and in nutrition. B. Behr’s Verlag, Hamburg, Germany, Third International Whey Conference, Munich, Germany, pp. 59–72. Maubois, J.-L., Caudron, B., Daviau, C., Madec, M.-N., Pierre, A. (2000). Membrane technologies: tools for a total control of the cheesemaking process. IDF Symposium on Cheese: Ripening and Technology, Banff, Canada. Maubois, J.-L. (2002a) Protéines du lait, protéines de toujours, protéines d’avenir. In: Lactoprotéines et lactopeptides: propriétés biologiques. Ed. Pierre Jouan, INRA Editions, Paris, pp 11−16. Maubois, J.-L. (2002b). Membrane microfiltration: a tool for a new approach in dairy technology. Australian Journal of Dairy Technologies, 57, 92–96. Maubois, J.-L., Schuck, P. (2005). Membrane technologies for the fractionation of dairy components. IDF Bulletin 400, pp. 2–7. Meershon, M. (1989). Nitrate free cheesemaking with Bactocatch. North European Food Dairy Journal, 55, 108–113. Mistry, V.V., Maubois, J.-L. (2004). Application of membrane separation technology to cheese production. In: Cheese: Chemistry, Physics and Microbiology, vol. 1, Ed P.F. Fox, P.L.H. McSweeney, T.M. Cogan, T.M. Guinee, Elsevier, London, pp. 261–285. Nielsen, P.S. (1988). Membrane filtration for whey protein concentrate. Marketing bulletin. APV Pasilac AS, Aarhus, Denmark. Novak, A. (1992). Milk protein concentrate in new applications of membrane processes. IDF special issue 9201, Brussels, Belgium, pp. 51–66. Pearce, R.J. (1983). Thermal separation of beta-lactoglobulin and alpha-lactalbumin in bovine Cheddar cheese whey. Australian Journal of Dairy Technologies, 38, 144–148. Pearce, R.J. (1987). Fractionation of whey proteins. Australian Journal of Dairy Technologies, 42, 75–78. Pearce, R.J. (1992). Whey protein recovery and whey protein fractionation. In: Whey and Lactose Processing, Ed J.G. Zadow, Elsevier, New York, pp. 271–316. Pedersen, P.J. (1992). Microfiltration for the reduction of bacteria in milk and brine. IDF special issue 9201, New applications of membrane processes, pp. 33–50. Pierre, A., Fauquant, J., Le Graët, Y., Piot, M., Maubois, J.-L. (1992). Préparation de phosphocaséinate natif par microfiltration sur membrane. Lait, 72, 461. Piot, M., Fauquant, J., Madec, M.-N., Maubois J.-L. (2004). Preparation of “serocolostrum” by membrane microfiltration. Lait, 84, 333–342. Porter, M.C. (1990). Handbook of Industrial Membrane Technology, William Andrew Publishing, Noyes, Park Ridge. Portmann, R. (2002). Composition for reducing caloric intake. US Patent 2004/0077530 AI. Rash, K.E., Kosikowski, F.V. (1982) Behaviour of enteropathogenic Escherichia coli in Camembert cheese made from ultrafiltred milk. Journal of Food Science, 47, 728−732. Quiblier, J.P., Ferron-Baumy, C., Garric, G., Maubois, J.-L. (1991). Procédé de traitement des laits permettant au moins de conserver leur aptitude fromagère. Fr Patent 2 681 218 A 1. Rosenberg, M. (1995). Current and future applications for membrane processes in the dairy industry. Trends in Food Science and Technology, 6, 12–19. Saboya, L.V., Maubois, J.-L. (2000). Current developments of microfiltration technology in the dairy industry. Lait, 80, 541–553. Saboya, L.V., Goudédranche, H., Maubois, J.-L., Lerayer, A.L.S., Lortal, S. (2001). Impact of broken cells of lactococci and propionibacteria on the ripening of Saint-Paulin UF cheeses: extent of proteolysis and GC-MS profiles. Lait, 81, 689–713.

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Schuck, P., Piot, M., Mejean, S., Fauquant, J., Brulé, G., Maubois, J.-L. (1994). Déshydratation des laits enrichis en caséine micellaire par microfiltration; comparaison des propriétés des poudres obtenues avec celles d’une poudre de lait ultra-propre. Lait, 74, 47–63. Tamime, A., Robinson, R. (1985). Yoghurt: Science and Technology. Pergamon Press, New York. Terré, E., Maubois, J.L., Brulé, G., Pierre, A. (1987) Procédé d’obtention d’une matière enrichie en caséine beta, appareillage pour la mise en oeuvre de ce procédé et application des produits obtenus par ce procédé comme aliments, complémentaires ou additifs en industrie alimentaire et pharmaceutique ou dans la préparation de peptides à activité physiologique. French Patent no. 2 595 769 A1. Toba, Y., Takada, Y., Yamamura, J., Tanaka, M., Matsuoka, Y., Kawasaki, H., Itabashi, A., Aoe, S., Kumegawa, M. (2000). Milk basic protein: a novel protective function against osteoporosis. Bone, 27 (3), 403–408. Trouvé, E., Maubois, J.-L., Piot, M., Madec, M.-N., Fauquant, J., Rouault, A., Tabard, J., Brinkman, G. (1991). Rétention de différentes espèces microbiennes lors de l’épuration du lait par microfiltration en flux tangentiel. Lait, 71, 1–13. Yvon, M., Berthelot, S., Gripon, J.C. (1998). Cheese flavour formation by amino acid catabolism. International Dairy Journal, 8, 889–898.

Two-Phase 4 Aqueous Systems Applied to Whey Protein Separation Abraham Damian Giraldo Zuniga, Jane Sélia dos Reis Coimbra,* José Antonio Couto Teixeira, and Lígia Rodrigues Contents 4.1 4.2 4.3 4.4 4.5 4.6

Introduction..................................................................................................... 57 Types of Aqueous Two-Phase Systems........................................................... 58 Phase Equilibrium Diagrams...........................................................................60 Physicochemical Characteristics of ATPS...................................................... 62 Applications of ATPS...................................................................................... 63 Biomolecule Distribution in ATPS..................................................................64 4.6.1 Molar Mass (MM) of the Polymer......................................................64 4.6.2 Polymer Concentrations....................................................................... 65 4.6.3 pH........................................................................................................ 65 4.6.4 Salts...................................................................................................... 65 4.6.5 Polymer Charge................................................................................... 65 4.6.6 Hydrophobic Groups............................................................................66 4.6.7 Temperature.........................................................................................66 4.7 Affinity Protein Partitioning............................................................................66 4.8 Extractive Bioconversion in ATPS.................................................................. 67 4.9 Recycling of Constituent Reagents of the ATPS............................................. 68 4.10 Conventional Liquid–Liquid Extraction Equipment Operated with ATPS......69 4.11 Case Study: Separation of Serum Proteins in a Graesser Extractor................ 72 4.11.1 Protein Partitions in ATPS Using a Graesser Extractor...................... 73 4.12 Conclusions...................................................................................................... 74 Acknowledgments..................................................................................................... 74 References................................................................................................................. 74

4.1 Introduction The separation of components in a liquid mixture by means of direct contact of the solution with a solvent, in which one of the compounds is preferentially soluble, is known as liquid–liquid extraction. This unit operation is used in the processing of fuels and in the separation of hydrocarbons in the petroleum industry. It is also 57

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applied in the chemical, pharmaceutical, metallurgical, and food industries, as well as in sewage treatment. In recent years, an increase in the variety of biotechnological products and the parallel need for separation of compounds that have low relative volatility, are thermal sensitive, and have proximate boiling points has resulted in the rapid industrial diffusion of liquid–liquid extraction. Conventional liquid–liquid extraction, using an aqueous solution and organic solvents, is not adequate to separate biomolecules such as proteins, because their stability is low in organic solvents. An appropriate alternative to traditional bioseparation processes is partitioning in aqueous two-phase systems, which has been successfully used for the isolation of proteins and other biological organic material. Extraction with aqueous two-phase systems (ATPSs) allows for the isolation of biomolecules in complex mixtures and offers advantages such as short processing time and simple scale-up, as well as the use of a medium suitable to work with compounds of biological origin. Phases from the majority of ATPSs are composed of 70% to 90% water, which favors the stability of biomolecules during separation, different from traditional systems composed of organic solvents. Recent improvements in the technique, including the employment of new ATPSs composed of polymer + salt, polymer + polymer, copolymer + salt, or copolymer + polymer permit their use at the industrial level. Extraction with ATPS was successful in the separation and purification of different enzymes and proteins.

4.2 Types of Aqueous Two-Phase Systems The ATPSs are formed when two polymers or one polymer and one salt are mixed above their critical thermodynamic conditions. They are composed of two immiscible phases that promote the separation of components in a proper environment that preserves the principal characteristics of the products being separated. These types of systems result in the incompatibility between two polymers in a solution, for example, polyethylene glycol (PEG) and dextran (Dex), or between a polymer and a salt, such as PEG and potassium phosphate (PPP). However, the formation mechanism of two phases is not yet well known. According to Albertsson (1986), the formation of ATPSs was first observed by Beijerinck in 1896 when mixing agar, gelatin, and water at the correct concentrations. The upper phase became rich in gelatin and the lower phase in agar. Beijerinck also noted the formation of phases in systems composed of agar + starch + water. Soon after, Dobry and Boyer-Kawenoky studied the systematic miscibility of pairs of polymers in the presence of water or inorganic solvents, observing the occurrence of phase separation. It was only in 1956 that the works of Albertsson (1986) were employed in ATPSs for the separation of biomolecules. The author found that mixtures of two structurally different polymer solutions could also be used for the formation of ATPS. This technique was then applied for the partitioning of molecules with biological activity, such as proteins, enzymes, and cells. There are a large variety of hydrophilic polymers, natural or synthetic, capable of forming phases when mixed with a second polymer or a salt, as can be seen in Table 4.1. The ATPSs composed of PEG + Dex or PEG + salts are widely utilized due to their availability in large quantities on the market and the fact that they are not toxic.

Aqueous Two-Phase Systems Applied to Whey Protein Separation

59

Table 4.1 Typical Aqueous Two-Phase Systems Applied to Dairy Products Polymer Polyethylene glycol (PEG)

Polymer Dex

MD HPS Repall PES Polypropylene glycol Dex

Polymer PEG

MD Guar gum Ucona EOPOb Salt Potassium phosphate

Polymer EO50PO50

Sodium sulfate Magnesium sulfate Water Water

a b

Reference Han and Lee, 1997a; Lu and Tjerneld, 1997; Truust and Johansson, 1996; Zaslavsky et al., 2000 Silva and Meirelles, 2000 Venâncio et al., 1996 Berggren et al., 1995; Venâncio and Teixeira, 1995 Silva and Meirelles, 2000 Simonet et al., 2000 Carlsson et al., 1996 Planas et al., 1998 Chen, 1992; Coimbra et al., 1994; Han and Lee, 1997b; Harris et al., 1997; Papamichael et al., 1991 Rito-Palomares et al., 2000; Save et al., 1993 Harris et al., 1997; Li et al., 2000 Johansson et al., 1999

Ucon: copolymer with an equal content of EO and PO. EOPO: copolymer composed of ethylene oxide (EO) and propylene oxide (PO).

For use at the industrial scale, Dex is very expensive. Therefore, the PEG + salt systems have been employed for the extraction of enzymes on large scale due to low cost, low viscosity, and elevated selectivity (Husted et al., 1985; Kim and Rha, 2000b). Saline ATPSs (PEG + salt) are formed at room temperature, where the upper phase is rich in PEG and the lower phase rich in salt, as shown in Figure 4.1. However, these systems still present some limitations such as the denaturation of biomolecules when salt concentrations are high. To overcome these limitations, new compounds are being used as substitutes for Dex or salt in the ATPS mainly for large-scale processing. For example, a system composed of PEG + maltodextrin (MD) was used for the separation of Lactobacillus acidophilus H2B20 UFV cells from a fermented medium, for the partition of bovine serum albumin (BSA) and for the separation of a-lactalbumin (a-la) and b-lactoglobulin (b-lg) (Alves et al., 2000; Silva and Meirelles, 2000). BSA, a-la, and b-lg were also partitioned in ATPS composed of polypropylene glycol (PPG) l400 + MD. Sarubbo et al. (2000) used a system formed of cashew-nut tree gum (CTG) + PEG for the separation of BSA. Lysozyme and lactic acid BSA partitions were also evaluated by Johansson et al. (1999), who tested ATPSs composed of aqueous solutions of only one compound formed by a linear copolymer of ethylene oxide (EO) and propylene oxide (PO) hydrophobically modified with miristic groups

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System mixture PEG 18,75% Salt 13,42% Water 67,83%

Upper phase rich in PEG PEG 35,49% Salt 3,45% Water 61,06%

Lower phase rich in salt PEG 1,67% Salt 23,24% Water 75,09%

Figure 4.1 Phase composition (% w/w) system mixture of PEG1500 + potassium phosphate + water, at 25°C and pH 7.

(C14H29) (HM-EOPO). This HM-EOPO copolymer, which produces an ATPS when mixed with water, presents potential for bioseparation because a single polymer and water are capable of phase formation and can be used at moderate temperatures and salt concentrations. Additionally, the copolymer can be recovered with only moderate heating. A greater number of systems and their respective phase diagrams are detailed in Albertsson (1986), Zaslavsky (1995), and Walter et al. (1985). Literature related to the formation of two phases, for nearly all types of polymer + polymer + solvent systems, generally occurs because of the low molar concentration of the polymers in the solution (less than 0.05 mol/L), causing a small increase in entropy while mixing. On the other hand, because polymeric chains have a greater surface area per molecule than compounds with low molar mass, the interaction energy between the two polymers overlaps the Gibbs free energy of the system.

4.3 Phase Equilibrium Diagrams Phase equilibrium data for ATPSs can be represented in equilibrium diagrams, at a given temperature and pressure. Graphical representation of ATPS thermodynamic data is of great importance for the study of biomolecule separation, because they are used as a basic tool for the development of an extraction process. For the construction of the diagrams, values referring to the concentration of the components in the equilibrium phases can be obtained from different mixture points in the system constituents (Albertsson, 1986). Typically, the type of representation for the equilibrium diagram utilized in the ATPS reference literature is the rectangular diagram. Figure 4.2 shows a typical phase diagram for a salt + polymer ATPS. Concentration of one of the components is shown on the horizontal axis and the other on the vertical axis. The amount of water (or of the third component) is calculated as the difference. The CEB curve, which divides the biphasic region from the monophasic region, is known as the binodal curve or equilibrium curve. In the region above the binodal curve, two phases are


Top phase (rich in Q)

61

Bottom phase (rich in P)

C

Salt Q (%)

C1

A1

A

E B1

B

Polymer P (%)

Figure 4.2 Phase diagram for a salt–polymer system.

formed (biphasic region), and below the curve the mixture is completely miscible (monophasic region). Supposing that point A represents the composition of an aqueous solution containing the polymer P and salt Q, after reaching thermodynamic equilibrium, the compositions of the resultant phases are represented by points B and C. At the critical point (E), the two phases have identical compositions and volumes, therefore being indistinguishable. The curve segments EC and EB represent the phases rich in the salt Q and polymer P, respectively. All systems with a mixture point located on the CAB line segment, denominated as the tie-line, will possess final compositions identical to the upper (rich in Q) and lower (rich in P) phases. Tie-lines unite the points that represent the equilibrium of the phases; however, the volumes of these phases are different because the composition variation of the mixture point along the tie-line produces changes in the phase volume. If composition were expressed as a mass fraction, the ratio between the masses of the phases rich in P and Q would be equal to the ratio between the lengths of lines AC and AB (Zaslavsky, 1995). For phase separation studies in ATPSs, a standard numeric measurement for the composition of the phases is necessary. It was empirically determined that the tie-line length, usually referred to as TLL, is adequate for this measurement. The TTL can be calculated from the concentrations of the components in the phases using Equation 4.1: TLL = {[C(P)1 – C(P)2 ]2 + [C(Q)1 – C(Q)2 ]2 }

0,5

(4.1) in which C(P)n and C(Q)n are, respectively, the concentrations of the polymers P and salt Q in the phase n, being that n = {1,2}. Another important characteristic of the phase diagrams is the slope of the tie-line (STL), calculated from Equation 4.2. The STL is used to deduce the proportion of compounds to be used for the formation of two phases:

STL = ΔC(P)/ΔC(Q)

in which ΔC(P) = [C(P)1 – C(P)2] and ΔC(Q) = [C(Q)1 – C(Q)2].

(4.2)

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For the construction of the equilibrium diagrams, a significant amount of experimental data is needed, which signifies an increase in cost and time required in order to perform the experiments. Computational thermodynamic models were developed in this specific field with the goal of minimizing time and expenses to predict the equilibrium of phases in multicomponent systems, requiring a minimal number of experimental data. Models proposed in the literature to predict the behavior of phases in ATPSs are based on the osmotic viral expansion theory and on the “Lattice” theory (Li et al., 1998). The osmotic virial expansion is derived from the understanding of the osmotic pressure of the solvent in the solution and has been used to predict the behavior of phases in polymer + polymer ATPSs and the partitioning coefficient of biomolecules. Another model based on the Lattice theory is the UNIQUAC model that incorporates the effect of polymer polydispersion on system behavior. Other thermodynamic models found in literature to predict ATPS equilibrium are the UNIFAC and NRTL. A review of these and other models was performed by Cabezas Jr. (1996).

4.4 Physicochemical Characteristics of ATPS Physical-chemical properties of ATPSs such as density, viscosity, and interfacial tension are affected by the concentration of constituents of the system. For polymeric ATPSs, phase densities are not very different from the density of water, commonly between 1.0 and 1.1 g/mL. This is due to one of the peculiar characteristics of ATPS as is the case of the high water content in the phase. The PEG + salt ATPSs present a difference in densities in the range of 8% to 14%. For example, for a PEG 1500 + potassium phosphate ATPS maintained at 25°C, the lower phase, rich in potassium phosphate, is denser than the upper phase rich in PEG. An increase in the phase densities was noted with the elevation of the constituent concentration in the system (PEG and salt), being the system with the greatest difference in density composed of 18% PEG 1500 and 18% potassium phosphate. A greater difference in density is found in systems containing soluble proteins, which can be attributed to the unequal partition of biomolecules. The low interfacial tension (g) of polymeric ATPSs, from 1.0 × 10 –4 to 0.35 mN/m, provides friendly conditions for the extraction of biomolecules, such as enzymes and fragile cells. These values are relatively low compared with conventional liquid–liquid extraction systems composed of organic solvents, such as hexane + water, glycerin + hexane, and toluene + water that have interfacial tensions of approximately 48.5, 34.9, and 35.7 mN/m, respectively (Contreras and Olteanu, 2000; Forciniti et al., 1990; Rydén and Albertsson, 1971). Interfacial tension values between 1 × 10 –3 and 2.0 mN/m were obtained for PEG + salt ATPSs (Albertsson, 1986; Kim and Rha, 2000a). Mishima et al. (1998) evaluated the influence of temperature and MM of PEG on the interfacial tension of PEG + dibasic potassium phosphate, verifying that the interfacial tension increased with both the increase in PEG MM and with the increase in the TLL. The effect of temperature on interfacial tension was insignificant. In respect to viscosity of the ATPS phases, found values were 17 cP and 2.6 cP for the polymeric and saline phases in an ATPS composed of 14% PEG 1550 + 18%


63

potassium phosphate (pH 7), respectively; 25 cP and 2.5 cP for the polymeric and saline phases, respectively, for the ATPS composed of 18% PEG 1500 + 18% potassium phosphate (pH 7). Viscosity of the polymeric phase was always significantly greater than the saline phase. Venâncio et al. (1996) reported elevated viscosity values in the lower phase rich in HPS, for the PEG + HPS ATPS, in relation to values in the upper phase. Phase viscosity also increases when incorporated with biomass systems containing compounds to be separated.

4.5 Applications of ATPS Aqueous two-phase systems have been successfully employed for the separation of diverse biomolecules. A list of some published works in the dairy industry can be consulted in Table 4.2. Bovine serum albumin (BSA) is, usually, the reference protein for ATPSs. Purification of proteins using ATPS on the large scale, for example, involves an alternative technique that is economically feasible compared to traditional biomolecule purification processes.

Table 4.2 Aqueous Two-Phase System Applications in the Dairy Industry Compound

ATPS

Reference

b-lg, antitrypsin, casein

PEG + PPP PEG + Dex PEG + Dex PEG + MgSO4

Da Silva et al., 2007 Großman et al., 1998 Nerli et al., 2001 Harris et al., 1997

a-la, b-lg

PEG + PPP

Chen, 1992; Coimbra et al., 1994; Giraldo-Zuniga et al., 2005 Rodrigues et al., 2001 Kaul et al., 1995 Venâncio and Teixeira, 1995 Venâncio et al., 1996 Johansson et al., 1999 Persson et al., 1999 Planas et al., 1998, 1999 Sarubbo et al., 2000 Simonet et al., 2000 Chen et al., 1999 Silva and Meirelles, 2000 Zaslavsky et al., 2000 Rodrigues et al., 2003

CMP* BSA BSA e ovalbumin

Escherichia coli BSA BSA BSA BSA, lysozyme Lactic acid BSA b-lg, BSA, casein BSA, lysozyme a-la, b-lg, BSA Lysine, glycine Proteose peptone *

CMP: caseinomacropetide

PEG + (NH4)2SO4 PEG + PPP PEG + HPS PEG + goma guar HM + EOPO EO50PO50 + HM-EOPO EOPO + Dex PEG + GAC Dex + guar gum PEG + PPP PEG + MD PEG + Dex PEG + Reppal PES 100

64


4.6 Biomolecule Distribution in ATPS Biological materials added to ATPS distribute themselves between the two phases, without losing their biological activity. The relationship between the correct biomolecule concentrations in phases 1 and 2 defines the partition coefficient (K) in aqueous systems (Albertsson, 1986):

K = C1/C2

(4.3)

where C1 is the biomolecule concentration in phase 1 (mg/mL) and C2 is the biomolecule concentration in phase 2 (mg/mL). Albertsson (1986) proposed a simpler model to calculate K, breaking it down into

Ln K = ln Kel. + ln K hidrof. + ln K hifil. + ln Kconf. + ln Klig

(4.4)

where the subscripts el., hidrof., hifil., conf., and lig. refer to the electrostatic, hydrophobic, hydrophilic, conformation, and ligand interaction contributions. Empirical studies with ATPS have shown that protein distribution is a function of various factors, such as those discussed below.

4.6.1 Molar Mass (MM) of the Polymer The MM of the polymer acts on the partition, altering the equilibrium and interactions between the polymer and the protein. In general, increase in the polymer MM, which enriches one of the phases, causes the migration of the biocompound to the other phase. However, this effect is diminished with the increase of the polymeric chain (Baskir et al., 1989). The effect of polymer MM alteration is dependent on the MM of the protein to be partitioned. Proteins of high MM are more influenced by changes in the MM of the polymer which form the phase than proteins with low MM. As an example, according to Albertsson et al. (1987), the partition coefficient of the cytochrome c was little affected (from 0.18 to 0.17) when the MM of dextran was increased by using the systems PEG 6000 + Dex 40 and PEG 6000 + Dex 500. For b-galactosidase, which has a greater MM than cytochrome c, the partition coefficient increased from 0.24 to 1.59 under the same conditions. This behavioral tendency can also be observed for different MM of PEG + Dex 500. Polymers with different MM can be used to optimize the separation of proteins of various sizes. Studies with a PEG + MD system for microbial cell partitioning reported that with the increase in MM of the PEG, the microbial cells in the lower phase, rich in MD, experienced a decrease in K. Increase in the MM of the PEG from 4000 to 8000 Da provokes a 70-fold decrease in K. The use of a PEG + potassium phosphate ATPS for the separation of proteins in cheese whey showed that the partition coefficient of the a-la diminished with the increase of the MM of the PEG. For b-lg, the opposite was verified, being observed an increase in K with the increase of PEG MM, except for PEG 8000.


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4.6.2 Polymer Concentrations Particles such as organelles and fragmented cells are absorbed more forcefully at the ATPS interface with the increase of polymer concentration. Elevation in the concentration of polymers normally causes the displacement of the binodal curve and the critical point, as well as an alteration in the composition of the phases. As a result, soluble substances, such as proteins, preferentially distributed to one of the phases, presented changes in their partition coefficient. In the evaluation of microbial cell partitioning in the ATPS composed of PEG 4000 + MD, it was observed that the increase in PEG concentration caused the decrease in K being that, as the PEG concentration increased, more Lactobacillus acidophilus cells migrated to the maltodextrin-rich lower phase.

4.6.3 pH System pH alters the surface charge of the proteins and, consequently, their partition coefficient. A classic example is the denaturation of proteins due to the reduction in pH. The distribution of denatured proteins in liquid solutions is different from that obtained from proteins in their natural state due to their significantly greater surface area.

4.6.4 Salts The presence of salt in polymer + polymer ATPS is important for the successful partition in practically all molecular species and cellular particles (Asenjo, 1990). The addition of salts (0.1 to 0.2 mol/L) in polymeric ATPSs generates a charge difference between the phases, resulting in the salt’s preference for one of the phases (Baskir et al., 1989). The presence of monovalent cations and anions diminishes the K of the negatively charged proteins according to the order Li+ < NH4 < Na+ < Cs+ < K+ and F– < Cl– < Br < I–, respectively. For positively charged proteins, this order is inverted. The presence of phosphate, sulfate, and citrate bivalent anions causes a greater increase in K in relation to the monovalent anions. Han and Lee (1997) observed for a PEG + Dex ATPS that the incorporation of phosphate salts brought on a reduction of K in BSA which has a negatively charged surface protein. However, the K for lysozyme, which is positively charged, increased with the addition of phosphate. In saline ATPS, Harris et al. (1997) reported that for PEG + magnesium sulfate systems the K for b-lg, casein, and a-antitrypsin increased with the elevation of NaCl concentration. The same behavior was also observed by Lu and Tjerneld (1997) for the partition of b-galactosidase.

4.6.5 Polymer Charge Ionically charged polyethylene glycols have been used to drive protein partition. Positively charged, in the form of PEG-trimethylamine (TMA-PEG), concentrate compounds with a negative charge in the PEG rich upper phase. Compounds with a

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positive charge are therefore excluded from the PEG-rich phase. Negatively charged PEG, consequently, has an inverse reaction (Asenjo, 1990).

4.6.6 Hydrophobic Groups When low concentrations of PEG modified with hydrophobic groups (roughly 1 mM), palmitate for example, are used, the affinity of hydrophobic proteins for the upper phase is increased (Asenjo, 1990). Berggren et al. (1995) observed that the K for some proteins with low hydrophobicity was not significantly affected by the presence of hydrophobic polymers and salts in the makeup of the phases. However, the K of a protein with high hydrophobicity was greatly influenced by the increase in the hydrophobicity of the constituent polymer of the ATPS.

4.6.7 Temperature The influence of temperature on biomolecule partitioning is seen indirectly. Temperature can cause changes in the viscosity of the phases or in the structure of the polymers, altering the form of the binodal curve in the phase diagram (Baskir et al., 1989). Systems with phase compositions near the critical point are more affected by changes in temperature because an inherent instability might situate the system in the region of the critical point. A displacement of the binodal curve may also place the system in the monophasic region (Walter et al., 1985).

4.7 Affinity Protein Partitioning Many proteins present interactions with small molecules known as ligands. These interactions facilitate alterations in the partition of the proteins, increasing the selectivity of the system. This type of selective extraction, called affinity partitioning, which uses specific ligands added to one of the ATPS phases or immobilized on one of the polymers formed in the ATPS, has been quite effective for the separation of proteins and enzymes. Usually a single fraction of the polymer which forms the phase is used as the ligand-transporter. The bonding forces that occur between the ligand and protein are basically Van der Waals, hydrophobic, and electrostatic forces. Interaction between a protein and a ligand is usually more complex the enzyme– substrate interaction, for example (Brocklebank, 1987; Johansson, 1998). The function of the ligand on protein partitioning can be observed by the relative change in the K of the protein. The partition coefficient, in the presence of ligands (K*), can be defined by Equation 4.5:

K* = K/Kaff

(4.5)


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Table 4.3 Use of Ligands for Affinity Protein Partitioning Ligand Nucleotides Palmitic acid Triazine

Protein Dehydrogenases, kinases BSA Lactic dehydrogenase

Reference Johansson, 1998 Asenjo, 1990; Johansson, 1998 Johansson and Joelsson, 1985

of which Kaff incorporates the effect of affinity partitioning with a quantitative factor. Table 4.3 presents a list of some ligands used for affinity protein partitioning. Silva et al. (1997) used the specific p-aminophenyl 1-thio-b-D-galactopyranoside (APGP) ligand bonded to PEG for the purification of b-galactosidase from Kluyveromyces lactis and observed an enzyme recovery of 83% in the PEG rich upper phase of the PEG-APGP + potassium phosphate system. The use of the APGP ligand increased the enzyme purification factor by 1.6 times.

4.8 Extractive Bioconversion in ATPS Aqueous two-phase systems have also been used in the extractive bioconversion of enzyme, cells, and organelles. The catalyst used for bioconversion is retained in one of the phases and formed product migrates to the other phase. In the majority of studies with different ATPS, the biological catalyst is partitioned to the lower phase and the product to the upper phase. However, in other cases, the product was equally distributed between the phases. Extractive bioconversion in ATPS can be conducted either continuously or semicontinuously and integrated with other techniques for purification such as ultrafiltration. One case of extractive bioconversion was reported by Stred’ansky et al. (1994) who studied the hydrolysis of lactose by b-galactosidase in PEG + Dex ATPS. The majority of both yeast cells and free enzymes remained in the lower phase, rich in Dex. The produced carbohydrates (glucose and galactose) and lactose were equally distributed among the phases. The partition of the products grew with the increase in MM and concentration of involved polymers. Extractive bioconversion in ATPS allows for the reuse of the catalysts. Table 4.4 shows some of the applications involving bioconversion in ATPS. Table 4.4 Bioconversion in Aqueous Two-Phase Systems Bioconversion Lactose into glucose Monosaccharides into oligosaccharides Glucose into ethanol

Reference Nguyen et al., 1988 Bartlett et al., 1992 Kuhn, 1980

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4.9 Recycling of Constituent Reagents of the ATPS The quantity of chemical reagents consumed, both polymers and salts, can determine the competitiveness of extractions with ATPS in relation with other bioseparation techniques. For this reason, the possibility for reuse of the phase constituents must be considered when sizing the project, because the cost of the phase components increases linearly with the production scale. The recycling of reagents has been basically studied for ATPS composed of PEG + salts. Recycling of PEG can be easily integrated in the process, reaching recovery levels of 90% to 95%. The PEG from an intermediate step can be directly reused for the first step of a new extraction process, making the process more economically feasible (Husted et al., 1988; Papamichael et al., 1992; Rito-Palomares et al., 2000). The recirculation of the constituent reagents from the ATPS can reduce processing costs by reducing its consumption and effluent treatment. According to Papamichael et al. (1992), in a two-step process for the purification of the fumarase enzyme, direct recirculation of the upper phase of the second extraction to the first extraction causes a reduction in reagent costs of 43% in the discontinuous process and 24% in the continuous process. Figure 4.3 shows the schematic representation of an extraction process with PEG recirculation, as suggested by the authors. Recently, the use of thermoseparating polymers in ATPSs has been introduced (Persson et al., 2000). When such polymers are heated above a lower critical solution temperature (LCST), the solubility of the polymer will decrease and a system composed of a water and a polymer phase is formed. This makes it possible to perform

Receiver of PEG

Receiver of salt Water

Protein solution Recycle of PEG Mixer

Product Centrifuge

Centrifuge Receiver of salt

Figure 4.3 An extraction process with polyethylene glycol (PEG) recirculation. (Modified from Papamichael, N., Boerner, B., Husted, H. 1992. Journal of Chemical Technology and Biotechnology. 54 (1): 47–55. With permission.)


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temperature-induced phase separation whereby a target protein can be separated from the polymer and recovered in the water phase. In addition, polymer can be recovered and recycled in the other phase. Many thermoseparating polymers contain ethylene oxide groups. PEG is also a thermoseparating polymer, but its LCST is too high (above 100°C) for separation of labile molecules. Several random copolymers of ethylene oxide and propylene oxide (EOPO) have LCST low enough to be applied for separation of biological molecules. This property makes these copolymers suitable as substitutes for PEG in the conventional polymer–salt systems, as shown in Figure 4.4 representing a separation scheme for endo-PG recovery using UCON (Ucon 50 HB-5100, an EOPO random copolymer of 50% ethylene oxide and 50% propylene oxide [mass] with an average molar mass of 3900) as a phase-forming polymer. The discard of salts is generally more problematic. In systems containing cells, nucleic acid, soluble proteins, or insoluble proteins, the separation of salts from the primary phase by mechanical separation techniques, such as centrifugation or ultrafiltration, is difficult to perform efficiently. Electrodialysis is considered a general method for the recycling of salts and desalination of the PEG-rich phase (Husted et al., 1980). Salts were also recovered using an aliphatic alcohol + salt + water mixture. Specifically for the separation of potassium phosphate, cooling to temperatures lower than 6°C promoted the precipitation of the salt which could therefore be reutilized (Papamichael et al., 1992).

4.10 Conventional Liquid–Liquid Extraction Equipment Operated with ATPS The various types of extractors can be subdivided into two distinct categories: stage columns and differential columns. Stage columns are composed of a series of stages, in which the phases are in contact with the compound until equilibrium is established. The phases are then separated and passed to a new stage. Differential columns are constructed so that the composition of the phases changes along the length of the extractor. These columns can be further classified as a function of the phase dispersion method of the flow regime. Phase dispersion can be obtained by gravity, pulses, mechanical agitation, or centrifugal force (Coulson et al., 1996). Application of ATPS for biomolecule separation on the large scale requires the use of continuous operation. Equipment available on the market employed in conventional liquid–liquid extraction can be used for extraction with ATPS. Table 4.5 shows a few liquid–liquid extractors that were operated with ATPS. For example, the spray column is one of the most studied due to its extreme simplicity of construction and operation. It is basically composed of a cylindrical vessel with a distributer at the base where the dispersed phase is fed into the extractor. The distributor is normally formed by a plate with orifices. Drops formed at the distributor ascend along the length of the column, coalescing at the top of the column (Treybal, 1980). Among the liquid–liquid extraction equipment available on the market, the Graesser extractor (Raining Bucket Contactor) showed to be well suited for ATPS

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Clarified broth Salt 528 ml

Ucon 11 g

Stage 2#

Stage 1#

1 Separator 1# Top 1# (2.4% Ucon + 6.4% salt) 109 ml, enzyme-rich

Polymer recycling

Water 99.5 g Ucon 2.1 g

(27% Ucon + 2.2% salt) 800 g, 30°C Bottom 1# (30% Ucon + 2.0% salt) 651 ml

Heating to 80°C

Separator 3#

Separator 2#

(27% Ucon + 2.4% salt) 310 g, 30°C Bottom 1# (30% Ucon + 2.0% salt) 252 ml

Heating to 80°C

Separator 4# Polymer recycling Top 3# Waste

Bottom 3# (84% Ucon) 244 g

Top 4# Waste Bottom 4# (84% Ucon) 94 g

Top 2# (2.4% Ucon + 6.4% salt) Centrifugator Top 5# (0.5% Ucon + 6.8% salt) 42 ml, enzyme-rich 38 ml, enzyme-rich 1 Dialysis Heating to 40°C Ucon 2g

Bottom 5# for polymer recycling

Product

Stage 3#

Figure 4.4 The proposed separation scheme for endo-PG recovery. The data in brackets are phase compositions, and others are the amounts of fluid (see Pereira et al., 2003).


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Table 4.5 Liquid–Liquid Extractors Operated with Aqueous Two-Phase Systems Extractor Type

System

Reference

Spray Spray Spray Spray Kunhi Graesser

PEG + MD PEG + Dex PEG + Na2SO4 PEG + HPS PEG + PPP, PEG + Dex PEG + PPP

Rotating disc York Sheibel Podbielniac Westfalia Perforated plate

PEG + PPP PEG + Na2SO4 PEG + Dex, PEG + PPP PEG + PPP PEG + Na2SO4

Mixer-separator

PEG + PPP

Raghav-Rao et al., 1991 Sawant et al., 1990; Patil et al., 1991 Rostami-Jafarabad et al., 1992a Venâncio and Teixeira, 1995 Husted et al., 1989 Husted et al., 1989; Coimbra et al., 1994; Giraldo-Zuniga et al., 2005 Coimbra et al., 1998; Porto et al., 2000 Rostami-Jafarabad et al., 1992b Husted et al., 1980 Papamichael et al., 1991 Bhawsar et al., 1994; Hamimi et al., 1999 Husted et al., 1980

(Coimbra et al., 1995; Husted et al., 1980). Figure 4.4 shows the experimental setup of a Graesser extractor. The Graesser extractor was patented by Coleby (1962) and since then has been applied industrially for the purification of herbicides and deodorization of naphtha. It can be used for the processing of liquid mixtures containing solids, as in the polishing of bearings with kerosene, recovery of metal from effluent streams in the metallurgy industry, removal of dyes from fragmented material, extraction of pharmaceutical products, treatment of municipal residues, and separation of proteins. In the apparatus shown in Figure 4.5, the two phases are introduced at the two opposing ends of the device and flow in countercurrent directions; contrary to the majority of conventional extractors, it is operated horizontally. The mixture of the

Figure 4.5 Graesser extractor.

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phases is due to the movement of a series of partially open cylindrical baskets that are secured in circular supports. These supports are fixed to a horizontal axis connected to a variable speed rotor.

4.11 Case Study: Separation of Serum Proteins in a Graesser Extractor In recent years, ATPSs have been used for the separation of whey proteins (Boaglio et al., 2006; Capezio et al., 2005; Chen, 1992; Coimbra et al., 1994, 1995; Da Silva et al., 2007) and for the concentration and purification of other biomolecules (Albertsson, 1986). Da Silva et al. (2007) reported the viability of the use of ATPS to recover caseinomacropetide (CMP) from whey using PEG 1500, potassium phosphate systems at pH 7.0 and room temperature. Coimbra et al. (1995) and Giraldo-Zuñiga et al. (2005) analyzed the hydrodynamic behavior of a Graesser extractor measuring 100 cm in length and 10 cm in internal diameter, operating with an ATPS composed of polyethylene glycol and potassium phosphate (PEG + potassium phosphate ATPS). The device was utilized for the continuous separation of a-la and b-lg from cheese whey, with satisfactory results at low rotation speeds. Tests were therefore conducted for the hydrodynamic characterization of the device and the mass transfer data between the phases were obtained. These tests were performed at different operating conditions with the objective of selecting those with the best mass transfer values and also those that provide the best economical return with the process. The variable operating conditions of the Graesser extractor are, for example, rotation velocity of the rotor and relationship between the polymeric and saline phases flow. The following hydrodynamic characteristics were evaluated for the Graesser extractor: • Retention Time Distribution: The retention time distribution (RTD) is applied in the dynamic study of processes and in the calculation of hydrodynamic parameters of the device (Steiner et al., 1988). In the food industry, the RTD is extensively used in what is referred to as aseptic food processing, commonly employed for liquid products such as milks, juices, fruit concentrates, yogurts, eggs, and in liquid suspensions containing small particles such as baby foods and tomato concentrates (Torres and Oliveira, 1998). Residence time is defined as that during which a fluid element remains inside the equipment. The distribution of this time is expressed as the function of retention time distribution (Torres and Oliveira, 1998). According to Levenspiel (1992), the general procedure for RTD determination comes from the response of stimuli provided by the system. For this author, the simplest measurement method consists of the introduction of a tracer in the form of a pulse and concentration measurement at the exit of the device if performed in standardized time intervals. The residence time distribution is characterized by the average residence time.


•

•

•

•

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For RTD determination in a Graesser extractor operating with an ATPS, the injection of a concentrated dye in the form of a Dirac pulse was used, and the open system dispersion model was successfully applied in the characterization of the RTD in the Graesser extractor. The Holdup Fraction: In different industrial applications with liquid–liquid extraction columns, the phase holdup is important for the mass transfer calculation, one of the fundamental parameters for the selection of an adequate extractor (Coulson et al., 1996). The holdup is a parameter that allows for the prediction of the drop size, flooding point, and, consequently, the mass transfer interfacial area and the operating limits of the equipment. Holdup varies frequently with the height of the extractor, as well as the distribution of drop size and not only its average diameter. Steiner et al. (1988) reviewed some techniques employed for holdup determination: gamma radiation, ultrasonic and plug flow, among others. With the plug-flow technique, a column or section of the column is abruptly blocked, and the volume of the phase of interest and the total volume are measured, providing average holdup values. Axial Dispersion: The phenomenon of axial or longitudinal dispersion in liquid–liquid extractors is the result of the combination of various factors that diminish the concentration gradient and therefore negatively influence the mass transfer rate. Axial, radial, or retroactive mixing phenomenons are based on the vertical or horizontal liquid flow in the opposite direction of the natural flow (Godfrey and Slater, 1994). Physical-Chemical Characteristics of the Aqueous Two-Phase Systems: Physical-chemical characteristics such as density, viscosity, and interfacial tension must be known and vary with the concentration of the constituents in the system. The Phase Diagrams: For the study of protein partitioning in ATPS, the phase diagram for the corresponding system must be determined. For example, Albertsson (1986) can be consulted for the use of the systems composed by polyethylene glycol and potassium phosphate at different temperatures and molar masses of the PEG.

4.11.1 Protein Partitions in ATPS Using a Graesser Extractor For the separation of a-lactalbumin and b-lactoglobulin proteins from cheese whey, ATPSs containing polyethylene glycol (PEG) with different average molar masses of 1500, 4000, 6000, and 8000 daltons and both monobasic and dibasic potassium phosphate were used. One of the criteria used to determine the best ATPS for the separation of a-lactalbumin and b-lactoglobulin is the largest partition coefficient. A suitable technique is needed for the quantification of the component of interest in different systems. In the case of a-lactalbumin and b-lactoglobulin proteins from cheese whey, high-performance liquid chromatography (HPLC) with a reversedphase column can be used.

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Before using the equipment in continuous operation, it is also necessary to perform laboratory tests for the initial determination of the operational variables that allow for the scale-up of the equipment. Laboratory tests performed for the separation of targeted proteins were done using an ATPS composed of stock solutions of PEG (50 mass %) and potassium phosphate (30 mass %) with pH 7. The components from the stock solutions were weighted to obtain the desired concentrations, and the systems were constructed in test tubes. The tubes were agitated and then left standing to allow the phases to separate and later quantify the proteins in both phases. With these data, the partition coefficient of the proteins could be determined for each of the evaluated systems. For the experiments in the Graesser extractor operating in a continuous cycle on the prepilot scale, systems were prepared with larger quantities. After agitation, the system was allowed to stand for 12 hours to reach the equilibrium state and separate the phases. The separated phases were introduced into the extractor. The conducted experiments provided the following results: the proteins a-lactalbumin and b-lactoglobulin were satisfactorily separated using the ATPSs. Study of protein separation in the ATPS was quantified by calculating the partition coefficient, being verified that almost all b-lactoglobulin remained in the saline phase and the majority of a-lactalbumin was transferred to the polymeric phase.

4.12 Conclusions The liquid–liquid extraction of biomolecules, using ATPSs, presents advantages such as lower material cost, good reproducibility, and easy scale up. The distribution of a biocompound in an ATPS can be analyzed using the partition coefficient (K), which is the ratio between the concentrations of biomaterial in the phases. The degree of separation can be altered by the variation of factors such as electric charge, hydrophobicity, the addition of biospecific ligands, and others. The ATPS shows great potential for the separation of biomolecules, being economically competitive for the separation of proteins and cellular components.


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5 Chromatographic Techniques Applied to Dairy Product Manufacturing Rafael da Costa Ilhéu Fontan,* António Augusto Vicente, Renata Cristina Ferreira Bonomo, and Jane Sélia dos Reis Coimbra Contents 5.1 Introduction..................................................................................................... 82 5.2 Adsorption....................................................................................................... 83 5.2.1 Chemical and Physical Adsorption......................................................84 5.3 Nature and Types of Adsorbents..................................................................... 86 5.4 Sorption Equilibrium....................................................................................... 87 5.4.1 Solute Adsorption in Dilute Solutions................................................. 87 5.4.1.1 Linear Isotherm.................................................................... 88 5.4.1.2 Freundlich Isotherm.............................................................. 88 5.4.1.3 Langmuir Isotherm............................................................... 89 5.4.1.4 Bi-Langmuir Isotherm.......................................................... 89 5.4.1.5 Toth Isotherm........................................................................90 5.4.1.6 Jovanovic Isotherm...............................................................90 5.4.1.7 Exponentially Modified Langmuir Isotherm........................90 5.4.2 Determination of Adsorption Isotherms.............................................. 91 5.4.2.1 Batch or Stirred Tank Method.............................................. 91 5.4.2.2 Frontal Analysis Method......................................................92 5.4.3 Solute Desorption................................................................................ 93 5.4.4 Sorption Hysteresis.............................................................................. 93 5.5 Conservation Equations Involved in Adsorption.............................................94 5.5.1 Mass Balances.....................................................................................94 5.5.2 Energy Balance.................................................................................... 95 5.6 Kinetics of the Adsorptive Process..................................................................97 5.6.1 Intraparticle Transport Mechanisms...................................................97 5.6.2 Extraparticle Transport Mechanism.................................................... 98 5.6.3 Axial Dispersion Coefficient............................................................... 98 81

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5.7 Adsorption Operation Modes..........................................................................99 5.7.1 Batch Adsorption.................................................................................99 5.7.2 Fixed-Bed Adsorption....................................................................... 100 5.7.2.1 Bed Capacity and Scale-Up................................................ 103 5.7.3 Adsorption in Expanded Beds........................................................... 104 5.7.3.1 Stable Fluidization.............................................................. 105 5.7.3.2 Critical Parameters............................................................. 106 5.8 Ionic Exchange.............................................................................................. 107 5.8.1 Ionic Exchangers................................................................................ 107 5.8.2 The Ionic Exchange Mechanism....................................................... 109 5.8.3 Ion Exchange Equilibrium................................................................. 109 5.9 Molecular Exclusion Chromatography.......................................................... 111 5.9.1 General Aspects of MEC................................................................... 112 5.9.2 Basic Principles of MEC................................................................... 113 5.10 Final Remarks................................................................................................ 115 Acknowledgments................................................................................................... 115 References............................................................................................................... 116

5.1 Introduction Adsorption is defined as a spontaneous process during which one or more components of system concentrates on the interfacial region. Adsorption is, in most cases, a transient process, involving a solid and a fluid. The use of a solid is the main distinguishing feature from other processes such as absorption, distillation, or extraction. Solids are able to adsorb only traces of solute, therefore making this method useful for dilute solutions. The adsorbed solute, the adsorbate, does not dissolve into the solid; it stays on its surface or inside the pores. The process of adsorption is mostly reversible, and changes in pressure or temperature can easily provoke the removal of that solute from the solid. Adsorption has been used by the chemical and food industries for quite some time, and is taking its place in the purification and isolation of biocompounds in the pharmaceutical and fine-chemistry areas. The applications of adsorption in the food industry removal of color, odor, and other compounds that are a negative influence on the sensory characteristics of foods, such as liquid and crystal sugars, alcoholic drinks, fruit juices, fats and edible oils, among others; industrial purification of air and other gases, wastewater recycling, and organic solvents recovery; separation and purification of high-added-value products, such as various acids, vitamins, enzymes, and proteins (e.g., cheese whey proteins). The adsorption of proteins at solid–liquid interfaces has been reported, and various inorganic carriers were shown to be able to adsorb proteins from horse serum. Since then, adsorption has been used in the analysis and purification of biomolecules by the pharmaceutical and biotechnological industries. Table 5.1 shows some applications of adsorption in the dairy industry. It should be noted that the main objective of a purification process, such as cheese whey proteins, is not only to remove undesired contaminants but also to concentrate

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Table 5.1 Applications of Adsorptive Techniques in the Dairy Industry Application

Technique

Reference

General protein fractioning

Anion exchange Cation exchange

Separation of immunoglobulin G and glycomacropeptide Separation of caseinomacropeptide fractions Isolation of bovine lactoferrin and lactoperoxidase Purification of lactoferrin Isolation of lysozyme

Anion exchange

Gerberding and Byers (1998) Hahn et al. (1998), Doultani et al. (2004) Xu et al. (2000)

Anion exchange

Kreuß et al. (2008)

Cation exchange

Plate et al. (2006)

Immunoaffinity Ion exchange and hydrophobic interaction Ion exchange Size exclusion and ion exchange Expanded bed Liquid–liquid extraction and conventional adsorption Supercritical CO2 and adsorption on alumina Conventional adsorption and size exclusion Immunoaffinity Reversed phase Immunoaffinity Reversed phase Gas chromatography

Noppe et al. (2006) Noppe et al. (1996)

Recovery of antithrombin III Separation of lactose Purification of orotic acid Separation of cholesterol from butter oil

Extraction of pyrethroid pesticide residues Analysis of bovine growth hormone Detection of polychlorinated biphenyls Analysis of ochratoxin A Determination of methionine sulfoxide Determination of galactose

Özyurt et al. (2002). Geisser et al. (2005) Baumeister et al. (2003) Sundfeld et al. (1993) Mohamed et al. (1998) Di Muccio et al. (1997) Cho et al. (1996) Picó et al. (1995) Bascarán et al. (2007) Baxter et al. (2007) Chiesa et al. (1999)

the desired product and to transfer it to a medium where it is stable and able to keep its properties unaltered. The same objective holds true for the application of adsorption to the purification of other biological compounds.

5.2 Adsorption The knowledge that a porous solid can accumulate significant volumes of a condensable gas dates from 1777, when Fontana observed that recently calcinated coal was able to retain considerable amounts of different gases. It was mentioned that the retained volume was dependent on the type of coal used and on the type of gas, and that the effectiveness of the process was a function of the exposed area and of the porosity of the material. The word adsorption was introduced by Kayser, in 1881, when referring to the condensation of gases on surfaces, in order to emphasize the differences between that phenomenon and absorption, during which the molecules of

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the gas penetrate inside the solid. Nowadays, adsorption is defined as the concentration of one or more components at an interfacial region. In general, adsorption is used when referring to both adsorption and capillary condensation in the pores of a solid; however, in such cases it would be more correct to use the term sorption. As opposed to absorption, in which molecules of the solute diffuse from a gaseous phase to a liquid phase, in adsorption the molecules of the solute diffuse from a fluid to the surface of a solid adsorbent, thus forming a different adsorbed phase. In such a process, the accumulation of solute per unit area is small; therefore, highly porous solids (with very high values of surface area per unit volume) are used to overcome this problem. Adsorption surfaces are, in general, physically and/or chemically heterogeneous, and therefore the energy required to establish a bond changes considerably from place to place. Residual surface forces, well known as van der Waals interactions, are common to all surfaces, and the only reason why some solids are adsorbent is their highly porous nature, resulting from their particular manufacturing process, which generates a great internal surface area. Once the adsorbed components concentrate on the external surface of the solid, the bigger the external surface per solid unit mass, the most favorable its adsorption will be. This is why adsorbents are usually porous particles. Adsorption features peculiar characteristics, such as the high degree of recovery from dilute solutions, as well as the high selectivity in the separation of molecules by mass transfer. The scale and complexity of an adsorption unit may range from that of a chromatography column, with few millimeters of diameter, or from simple stirred tanks, to that of fluidized bed reactors used in vapor recovery, with diameters of several meters, or to highly automated moving beds. In all units, the common occurrence is the saturation of the adsorbent throughout the course of the process, which implies that it must be periodically regenerated or replaced.

5.2.1 Chemical and Physical Adsorption Adsorption applies to the physical or chemical transfer of a solute in a fluid, gas, or liquid, to a solid surface where it is retained as a consequence of microscopical interactions with the solid. It may occur as a result of imbalanced forces at the surface of the solid which attract the molecules of the contacting fluid, during a finite time, once this fluid that contains the solute to be retained is flowing through the empty volumes in the exterior of the adsorbent particles. The solute is transported by diffusion, from the volume of the fluid, through an external, eventually stagnant, film, to the solid particle, being adsorbed on its outer surface or in its pores. This adsorption takes place on an unoccupied adsorption site due to physical or chemical interactions. A given molecule may be adsorbed and deadsorbed several times while it is inside a single solid particle. The adsorbed solute (the adsorbate) does not dissolve in the solid; rather, it stays on the surface or in the pores. The adsorption process is often reversible and changes in pressure or temperature may result in the removal of the adsorbate. In fact, when in equilibrium, the adsorbate has a partial pressure that equals that of the fluid phase in contact,


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and through a simple change of the operation temperature or pressure, that equilibrium is changed and the solute may be removed from the solid. The process rate is governed by the diffusion outside the particle and in the pores and by the ability that a given adsorbent has to adsorb a certain solute. Knowing the mechanics of this process is a fundamental step to design and develop adsorption equipment at the industrial scale. The existence of residual free forces at the surface of the adsorbents is a known fact; such forces create active points. When molecules present in the fluid phase are placed into contact with those active points of the solid, the attractive forces between the adsorbent and those molecules may cause these to concentrate on the surface of the solid. The intensity of such attractive forces depends on the nature of the adsorbent (i.e., on the characteristics of its surface), and on the type of adsorbate; it also varies with factors such as temperature and pressure under which adsorption occurs. In general, the phenomenon of adsorption can be divided in two categories: physical adsorption and chemical adsorption. • Physical adsorption, or van der Waals adsorption: A reversible phenomenon that results from weak intermolecular attraction forces between the adsorbent and the adsorbate. Physical adsorption of a gas or a vapor is similar to condensation and occurs with energy release. However, it differs from condensation in that it also occurs when, at a given temperature, the solute’s partial pressure in the vapor phase is lower than the corresponding vapor pressure at that temperature. Physical adsorption can also occur in a liquid phase, in which equilibrium between the adsorbate and the fluid phase is reached very quickly due to the low amount of energy required. Another important feature of van der Waals adsorption is the possible superposition of several layers of adsorbed molecules. • Chemical adsorption, or chemisorption: Is the result of chemical interaction between the adsorbant and the adsorbate through attractive forces that are much stronger than those involved in physical adsorption. The energy released in this case is high, of the same order of magnitude of the enthalpy change in chemical reactions. It is a frequently irreversible process, and the desadsorption of the original substance often leads to its chemical modification. In this type of adsorption, there is only one layer of chemically adsorbed molecules on the surface of the adsorbant, which can be complemented with further layers of physically adsorbed molecules. When a molecule moves from the fluid phase to the adsorbed phase, it loses degrees of freedom, and its free energy is reduced. In this case, adsorption is accompanied by energy liberation, in higher or lower amounts. If this energy is not dissipated by any reason, the adsorbant capacity will be reduced due to the consequent temperature increase. Adsorption can be thought of as a three-stage process which occurs with the increasing concentration of the adsorbate. For a gas–solid system, the stages are as follows: first stage—a single layer of molecules binds to the solid’s surface. If that monolayer results from chemical adsorption, a free energy change will be associated to the process which will influence the attraction forces present; second stage—the molecules present in the fluid phase form several layers over the first one, through

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physical adsorption. The number of layers allowed is primarily determined by the size of the pores of the adsorbant; third stage—when the fluid phase is a gas, capillary condensation occurs in the pores, filling them with condensed adsorbate as soon as the partial pressure reaches a critical value with relation to the pore size. In a real system, these stages of the adsorption phenomenon occur simultaneously in different portions of the adsorbant, due to its heterogeneity. It is noteworthy to mention that the same substance may be adsorbed either physically or chemically on the same adsorbant, depending on the operational conditions. In general, processes at lower temperatures favor physical adsorption, and processes at higher temperatures favor chemical adsorption. This allows the control of both the intensity of the bonds and the reversibility of the process. Also worth mentioning is the fact that the activation energy is, in general, null or very low in physical adsorption and higher in chemical adsorption, in which case, however, the values are lower than those typical of a chemical reaction.

5.3 Nature and Types of Adsorbents In general, adsorbents are found as irregular granules, extruded pellets, and spherelike particles, with diameters ranging from 50 mm to 12 mm. They can be natural or synthetic, presenting either an amorphous or a microcrystalline structure. The size of the solid is a compromise between the need to obtain a higher surface area per unit volume when preparing a packed bed and the minimization of the flow pressure drop through the bed. In order to be commercially attractive, an adsorbent should feature high selectivity when adsorbing specific molecules; high adsorbing capacity; high internal surface area, with pores of sufficient size to allow the access of the adsorbate molecules but small enough to exclude large molecules, therefore increasing the selectivity of the adsorbent; easy regeneration and stability of the adsorptive capacity even after several regeneration cycles; high mechanical resistance, chemical inertia, and favorable cost–benefit relationship. An adsorbent’s specific surface area (surface area per unit mass) is undoubtedly the characteristic that most influences its adsorptive capacity. Mechanical resistance is also important, once a low resistance adsorbent may fragment easily, increasing the pressure drop through a reactor bed or leading to effectiveness losses in a batch process. In general, industrial applications use adsorbents of bigger sizes in continuous packed-bed reactors and smaller size adsorbents are used in batch processes, followed by a filtration step. The main adsorbents with industrial importance are as follows: • Alumina: Has a high affinity for water and hydroxyl groups, it is used to dry gases and liquids. • Activated clay: Some clays, such as bentonite, show low adsorptive capacity. However, when treated with sulfuric or hydrochloric acid, washed, dried, and milled, they present better adsorptive properties than Fuller’s earth. They are specifically used in discoloration operations. • Bauxite: Used for clarification and drying of gases.


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• Activated carbon: One of the adsorbents which is most frequently used, especially in the food industry. Due to its low affinity for water, it is preferentially used to adsorb components from aqueous solutions or wet gases. It is used as a powder that, after being used, can be removed by filtration. • Molecular sieves: Also known as zeolites, they are synthetic, porous crystals of metallic aluminosilicates, with a very defined structure, allowing the separation of molecules based on their molar weight, and promoting further separation by adsorption according to the polarity and insaturation of the molecule. They are used in gas and liquid drying operations and in a variety of other processes. • Polymers and resins: Substances used in water purification, recovery and separation of biological compounds, and in chromatographic processes in general. They are composed of a crystalline core covered with a polymeric matrix that confers porosity to the structure, and to which specific and selective chemical groups are bonded which are responsible for adsorption. • Silica gel: Together with activated carbon, is one of the most popular adsorb ents. It is used in gas drying and for the removal of saturated hydrocarbons. • Fuller earth: A natural clay that must be dryed and subjected to a fine milling before being used as an adsorbent. It is used in blanching, clarification, and neutralization of mineral, vegetal, and animal oils.

5.4 Sorption Equilibrium Phase equilibrium between the fluid and the adsorbed phase for one or more components is the most important factor for the performance of the adsorptive process. In most cases, this factor is even more important than mass and heat transfer rates: doubling the stoichiometric capacity of the adsorbent or significantly changing the shape of the isotherm has often a more significant impact in the unit operation than doubling the mass and heat transfer rates. The graphical description of sorption equilibrium for the adsorption of a single component is usually presented in terms of the sorption isotherm, where the relationship between the solute concentrations in the fluid and adsorbed phases, at a given constant temperature, is established. Sorption equilibrium is a dynamic concept that describes the situation in which the rate of adsorption of a given type of molecules on the solid surface equals the rate of desorption of those molecules from that surface. The physical and chemical concepts involved in these phenomena may become very complex, and there is no single theory on adsorption describing satisfactorily all the possible systems. Fortunately, for engineering purposes, the only information needed is an accurate representation of sorption equilibrium, and some of the first theories on adsorption are still widely applied.

5.4.1 Solute Adsorption in Dilute Solutions When an adsorbent is added to a binary solution, it is possible that either the solute or the solvent will adsorb to it. Since measurement of total adsorption is impossible, an apparent or relative adsorption is determined, instead. Total adsorption is not used because the adsorption of the solvent causes a slight, not measurable change in the enthalpy, in the volume, or in the mass of the solution. This makes it impossible

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to differentiate the amount of solvent adsorbed from the amount of solvent simply retained in the pores of the adsorbent. Therefore, the procedure to determine the parameters involved in the apparent adsorption of the solute consists of treating a predefined volume of solution with a known mass of the adsorbent. The ratio between these values is noted by v. As a result of the preferential adsorption of the solute, its concentration in the solution diminishes from an initial value c0 to a final value in equilibrium, c*. If changes in the volume of the solution are disregarded, the apparent adsorption of the solute can be given by v(c0 – c*). This relationship is satisfactory for dilute solutions when the fraction of the solvent that can be adsorbed is very small. An increase of the initial solute concentration in the solution leads to a corresponding increase of the amount of solute adsorbed. However, if the solvent is also adsorbed and if the extension of this adsorption is close to that of the solute, a preferential adsorption of the solvent may occur from a given value of concentration onwards. This situation corresponds to an inversion of the apparent adsorptivity and, for a well-defined concentration value, apparent adsorptivity reaches the unity, in a situation similar to that of the formation of azeotropes in distillation. The phenomenon of adsorption in liquids is less understood than in gases. In principle, the equations applicable to gases are also applicable to liquids, except in those cases in which capillary condensation occurs. 5.4.1.1 Linear Isotherm In general, for physical adsorption on a homogeneous surface and at low concentrations, the isotherm assumes a linear shape, with a constant slope (K), and this relationship may be expressed using Henry’s Law, represented by the following equation:

q = k ⋅ C

(5.1)

q = k′ ⋅ p

(5.2)

or

where q is the concentration of the adsorbed phase, C is the concentration of the fluid phase, and p is the partial pressure of the fluid phase (in the case of gases). Henry’s Law is very useful at low concentrations of the adsorbate, but for higher concentration values, the interactions between the adsorbate molecules increase, and the saturation on the adsorbed phase occurs. This means that for higher concentrations of the adsorbate, isotherms may have more complex shapes. 5.4.1.2 Freundlich Isotherm One of the most widespread equations representing the adsorption isotherms for liquids is the one proposed by Freundlich in 1926, which was developed from studies on the adsorption of organic compounds in vegetable coal, when in aqueous solution. This equation takes the following form:

CS = a ⋅ (C*)1/n

(5.3)


89

where CS is the mass of adsorbate per unit mass of adsorbent; C* is the concentration of solute in solution in equilibrium with the solid; a and n are constants, with n usually much higher than 1. This isotherm nearly corresponds to an exponential distribution of the adsorption enthalpy values, but in order to represent all data, it also needs to make use of the linear region of Henry’s Law. It can be used to correlate data obtained from heterogeneous adsorbents in a wide concentration range. 5.4.1.3 Langmuir Isotherm The Langmuir model (1916) is another classical model for isotherms, which is by far the most used and frequently the first choice for the fitting of experimental data; it can be represented by the following equation: qi =

qis ⋅ K i ⋅ ci 1 + K i ⋅ ci

(5.4) where qis is the number of adsorption sites of the monolayer (saturation capacity), ci is the concentration of species i, and Ki is an equilibrium constant. This model assumes that there is a constant number of adsorption sites available, only a single layer (monolayer) of adsorbed molecules is formed, adsorption is reversible, equilibrium is achieved, and interaction between the adsorbed molecules is null. Figure 5.1 shows a graphical scheme of these models.

Solute Concentration in the Solid Phase

5.4.1.4 Bi-Langmuir Isotherm The Bi-Langmuir isotherm model was proposed by Graham, in 1953, in order to evaluate adsorption behavior in nonhomogeneous surfaces. This model considers the surface

Langmuir Freundlich

Linear

Solute Concentration in Solution, in Equilibrium

Figure 5.1 Adsorption isotherms: linear, Freundlich, and Langmuir.

90


as having two different types of chemical domain, which behave independently. This means that the proposed model is the result of the sum of two Langmuir isotherms:

q = qs ,1

b1C bC + qs ,2 2 1 + b1C 1 + b2C

(5.5)

This model has two saturation capacities, qs,1 and qs,2, corresponding to each of the two types of chemical domain. The total saturation capacity is the sum of these two capacities, and b1 and b2 are the equilibrium constants. 5.4.1.5 Toth Isotherm The Toth isotherm model has three parameters and was originally derived from gas– solid equilibrium studies. Similar to Langmuir’s model, it can be applied in the case of solid–liquid equilibrium. This isotherm is used to fit experimental equilibrium data obtained in nonhomogeneous adsorbents:

q = qs

bC [1 + (bC )n ]1/ n

(5.6)

In this equation, qs and b have the same meaning as q is and Ki in Langmuir’s isotherm, and n is the heterogeneity parameter (0 93.5°C and be converted to anhydrous a-lactose. • Crystallization in a supersaturated solution at T < 93.5°C leads to a -lactose monohydrate. • Crystallization in a supersaturated solution at T > 93.5°C leads to anhydrous b-lactose. • a -Lactose monohydrate at T > 93.5°C in the presence of water vapor is converted to anhydrous b-lactose. • a-Lactose monohydrate at T > 93.5°C in an alcohol solution is converted to anhydrous b-lactose. • Anhydrous b-lactose is dissolved at T < 93.5°C to form a-lactose monohydrate. • a-Lactose monohydrate at T > 93.5°C in vacuum is converted to unstable anhydrous a-lactose. • Unstable anhydrous a-lactose can uptake water at T < 93.5°C and be converted to a-lactose monohydrate. • Unstable anhydrous a-lactose can uptake water at T > 93.5°C and be converted to anhydrous b-lactose. • a-Lactose monohydrate at T > 150°C in the presence of water vapor is converted to stable anhydrous a-lactose. • Stable anhydrous a–lactose is dissolved at T < 93.5°C and converted to a-lactose monohydrate. • Stable anhydrous a–lactose is converted to b-lactose when mediated by methanol and NaOH.

143

Crystallization of Lactose and Whey Protein

• When b-lactose is added to stable anhydrous a–lactose, this is it converted to b-lactose. • Crystallization in a supersaturated solution with ethanol leads to anhydrous a-lactose. • a-Lactose monohydrate in a methanol solution in the presence of HCl forms the crystal compound a5b3, anhydrous. The two forms of lactose, a and b, differ considerably in solubility and temperature dependence. If a-lactose is added to water, a smaller amount is dissolved at the beginning than later. This is a consequence of mutarotation. In the beginning, a-lactose is converted to b-lactose, diminishing the concentration of a-lactose in solution and allowing more a-lactose to dissolve. If b-lactose is added, solubilization is higher at the beginning than later. On mutarotation, more a-lactose is formed than the amount that can stay in solution and crystallization of a-lactose can occur (Olano and Rios, 1978; Parrish et al., 1980; Walstra et al., 1984). Equilibrium constant (K) depends on temperature, presence of salts, and pH of the solution. Higher K values are obtained at high and low pH values. Ammonium acetate also increases the K values and NaCl and KCl have no effect (Herrington, 1934c). Figure 6.13 shows the solubility curve (one of most important tools to analyze and study crystallization) of a-lactose, b-lactose, and the equilibrium relation between b/a as a function of temperature. The following relations can be used to evaluate the solubility of lactose in water q (in g anhydrous lactose/100 g water): a-lactose: log q = 0.613 + 0.0128T

(6.29)

b-lactose: log q = 1.64 + 0.003T

(6.30)

Concentration (g/100 g H2O)

1.5 =2 tion ion = tura rsaturat a s r Supe Supe

100 β-lactose

10

1

mo

briu

ili Equ

0

10

20

nd fαa

30

40

tose

β-lac

cto α-la

50

se

60

70

80

90

100

Temperature (°C)

Figure 6.13 Solubility of a-lactose, b-lactose, and the equilibrium relation between b/a as a function of temperature.

144


Concentration (g/100 g solvent)

60

H 2O EtOH EtOH:H2O Acetone:H2O 2‒Propanol:H2O Ethylene Glycol:H2O Propylene Glycol:H2O

50 40 30 20 10 0

10

20

30

40

50

60

70

Temperature (°C)

Figure 6.14 Solubility of lactose for different solvents. Mixtures are 50 wt%.

In equilibrium,

q = 12,48 + 0.2807T + 5.067 × 10 –3.T2 + 4.168 × 10 –6.T3 + 1.147.10 –6.T4 (6.31)

for T < 93.5°C. The solubility of lactose in other solvents is low as shown in Figure 6.14. This fact favors the crystallization of lactose in solutions having these solubility depressors, in a process called drowning out crystallization. Some organic solvents or cosolvents also modify the mutarotation equilibrium constant, like alcohols and glycerol (Herrington, 1934d; Nickerson and Lim, 1974). The colloids of milk do not have any material influence on the solubility of lactose. The presence of sucrose diminishes the solubility of lactose (Hunziker and Nissen, 1926). Solubility of lactose can be predicted by the group contribution method UNIFAC using special groups developed for sugars (Ferreira et al., 2003; Machado et al., 2000). For nonaqueous sugar solutions, the modified UNIFAC model can also be used (Spiliotis and Tassios, 2000). The modified extended Hansen method that utilizes the partial solubility parameters was also successfully applied to predict the solubility of lactose (Peña et al., 2000).

6.4.4 Metastable Zone Width Lactose solutions can be supersaturated easily and to a considerable extent as roughly indicated in Figure 6.13. At concentrations over two times the saturation concentration, a rapid spontaneous crystallization occurs, due to primary nucleation, the formation of nuclei in the solution. Below this value, the supersaturation can persist without the formation of nuclei, in a metastable solution. At less than 1.5 times the saturation concentration, seeding with crystals usually is needed to induce crystallization. Mechanical stimulus or ultrasound can also be used for this purpose. A saturated lactose clear solution can be cooled rapidly for more than 30°C without the appearance of crystal, meaning that the metastable zone width can reach values from


145

20 to 40°C without nucleation of new crystals. This means that in order to obtain high-quality lactose crystals—high purity, homogeneous, and uniform size—it is necessary to seed the supersaturated solution with a good-quality small crystal.

6.4.5 Nucleation Only a few studies in lactose nucleation have been published. Due to large metastable zone width (>30°C) in pure solutions, long induction times and high primary nucleation rates are expected. At saturation temperatures of about 60°C, induction times of about 20 to 60 h were observed by Herrington (1934a). With these high values, seeding and sonication can be a good strategy in order to obtain high productivity and high-quality crystals. Sonocrystallization is a recent technique used to recover lactose by crystallization. Bund and Pandit (2007) used this technique, and by controlling the sonication time, standing time, lactose concentration, and pH, lactose crystals of desired crystal shape, size, and size distribution (CSD) were obtained. Sonocrystallization not only enables rapid crystallization but also guarantees a relative uniformity of CSD and prevention of agglomeration in comparison to nonsonicated samples. Because of the relative stability of supersaturated lactose solution, the preparation of a solid supersaturated solution of lactose, generally referred to as lactose glass, is possible. This is done by quickly removing most of the water from a highly concentrated solution, or drying it, so that neither the alpha nor the beta form has the possibility to crystallize (Choi, 1958) as a consequence of a rapid increase in the solution viscosity (Gänzle et al., 2008).

6.4.6 Growth Rate It is well known that all forms of lactose exhibit a complex crystal growth mechanism in water. In spite of this, the design of suitable crystallization conditions and the use of structurally related additives allows for the control of the mean crystal size and crystal habit. Garnier et al. (2002) pointed out that the mean crystal size of a-lactose monohydrate grown in water at room temperature is significantly increased by the use of high supersaturation, mainly due to the presence of b-lactose that acts as a strong growth inhibitor at low supersaturations. b-Lactose influences the type and habit of crystals normally formed. It can inhibit the growth of needle or prism forms, allowing for the more commonly observed a-lactose crystals types to develop (Nickerson and Moore, 1974a). As an example of the influence of additives in growth rate, small quantities of a-glucosamine. HCl decrease the growth rate and lead to more elongated crystals, the formation of larger crystals being observed when large quantities of this compound are present. a-Galactose and maltitol induce the formation of flattened morphology crystals (Garnier et al., 2002). Addition of LiCl led to an increase in growth rate and a decrease in lactose solubility, and K2HPO4 had opposite effects (Bhargava and Jelen, 1996). Another factor that can also influence the crystal growth rate is the pH of the solution where crystallization occurs. High acidity (pH < 1) obtained with sulfuric acid greatly accelerated crystallization, but this does not happen when acetic and lactic acids are used (Nickerson and Moore, 1974b).

146


The crystal growth kinetics order is of about 2 as determined by Twieg and Nickerson (1968), Jelen and Colter (1973) and Brito (2007). The following relation was proposed by Jelen and Colter (1973) for the growth rate of lactose crystals:

R = –68.480 + 12.627T – 43.845ΔC – 0.173T2 + 1.635ΔC2 + 0.582TΔC

(6.32)

with supersaturation as ΔC = C – Cs (g lactose/100 g water); concentration of the solution as C (g lactose/100 g water); saturation concentration at T as Cs (g lactose/100 g water); solution temperature as T (°C); mass growth rate as R (mg/(m2.min); and growth rate as G = R/rc (m/min); crystal density of lactose as rc = 1590 (kg/m3). An increase in temperature from 30°C to 50°C doubles the lactose crystal growth rate, but for temperatures above 50°C, no significant increase is observed. Berglund and De Jong (1990) suggested that growth rate dispersion or dependence with size can be observed for sugars. This may also occur for lactose, but, so far, it has not been confirmed.

6.4.7 Kinetics of Crystallization Crystallization is one of the most important unit operations in the process of production of lactose, being the most well-studied crystallization technique used. Lactose can be supersaturated either by increasing the content of lactose in relation to the water content by evaporation or by cooling the solution as lactose becomes less soluble in water at lower temperatures. The addition of a water-miscible nonsolvent could accelerate the nucleation by reducing the metastable zone width. These techniques of supersaturation generation can also be combined. When using whey as raw material, it is necessary to concentrate the solution by vacuum evaporation to a solid content as high as possible prior to cooling the concentrate. As seen before, the removal of a-lactose from the solution as monohydrate crystal as a consequence of the crystallization process results in a change in the relative amounts of a- and b-lactose, so that the solution contains more b-lactose than that corresponding to equilibrium. Due to mutarotation, the a-lactose solution again becomes supersaturated, so that crystallization continues. This process will continue as long as the solution is supersaturated and will not stop until the saturation point is reached. The kinetics of crystallization is an important tool for the design of the overall lactose crystallization process. This is also important for other dairy systems like milk fat (Herrera et al., 1999). Thurlby (1976) presents values of three to four for the overall order of the crystallization kinetics in the temperature range of 15 to 50°C and also indicates that surface integration is the rate-controlling step instead of the diffusion of a-lactose to the crystal surface when crystals are suspended in solution. Griffiths et al. (1982) and Shi et al. (1990) used the population balance methodology developed by Randolph and Larson (1988) to determine the kinetics of lactose crystallization in 2 L and 0.2 L laboratory-scale MSMPR (mixed suspension, mixed product removal) continuous cooling crystallizers. Although these values were obtained at a laboratory scale, they can be used in the design of industrial units if appropriate care is taken. The determined relations were as follows:

147


Griffiths et al. (1982):

G = 4.67 × 10−5 ΔC3.55

(6.33)

B = 1.65 × 10 ΔC

(6.34)

−7

0

2.33

for T = 30°C; 0.064 < ΔC < 0.123; 0.4 < t < 3.4, with corresponding mean values of G = 1.5 × 10 –8 m/s; B 0 = 0.1 (#/L.s); and L D = 110 (mm). Shi et al. (1990):

G = 1,02 × 108 exp(–22.1/RT)(S-1)2.5

(6.35)

B = 3,32 × 10 exp(–17.0/RT)(S-1)

(6.36)

0

13

1.9

for 30°C < T < 60°C; 0.25 < S < 1.77; 0.1 < t < 1, with corresponding mean values of G = 3.5 × 10 –8 m/s; B0 = 0.7 (#/L.s); L D = 90 (mm). For the above, supersaturation is ΔC (g lactose/g solution); supersaturation ratio is S = {C/ [Cs-FK(C – Cs)]}; mutarotation equilibrium constant is K; mutarotation temperature dependence factor is F; growth rate is G (m/s); nucleation rate is B0 [#/(L.s)]; crystal mean size is LD (mm); and reactor residence time is t (h). Raghavan et al. (2001) discussed the difficulties in crystallizing lactose. The crystallization was performed in a 20 mL crystallizer with saturation temperatures of 50 and 60°C. Induction times longer than 10 hours as well as long periods for crystal growth (larger than 10 h) were measured. The main results of the developed work are as follows: • • • • •

Metastable zone width from 10 to 35°C Total crystallization time from 22 to 72 h Supersaturation calculated from 22% to 130% Yields from 18% to 72% Mean particle size from 24 to 62 mm

Based on the obtained results, these authors suggest seeding as the best technique for lactose crystallization. Mimouni et al. (2005) determined the kinetics of lactose crystallization in a batch cooling seeded crystallizer with a 400 mL volume. The solution with an initial concentration of 70 g/100 g H2O was cooled from 80 to 30°C without a predefined cooling rate. The kinetics of crystal growth resulted in a first-order relation, an average rate constant of 8.6 × 10 –3 min–1 and a mean crystal size of 100 mm. Westhoff and Bermingham (2008) reported industrial tests of lactose batch cooling crystallization. The tests were conducted in a 15 m3 jacketed crystallizer with an initial temperature of 80°C and a 60% dry matter solution cooled down to 25°C. The values obtained for the crystal growth rate were in the range of 0 to 5 × 10 –8 m/s, comparable with those from Griffiths et al. (1982) and Shi et al. (1990). The nucleation rate was in the range of 4.1 × 106 to 1.7 × 108 (#/L.s) with a specific power input of 2 kWh/m3. These values differ from those of Griffiths et al. (1982) and Shi et al. (1990), probably due to the difference in the test scale. Although differences are observed in the values of the kinetic parameters for lactose crystallization, its range of variation can be considered small for engineering

148


purposes, meaning that the parameters can be used with caution in the design of industrial crystallization units.

6.4.8 Industrial Aspects Lactose is produced in large-scale industrial units. Plants with more than 10,000 tpy of capacity are used worldwide, most of them using crystallization as the main process. In addition to the single-batch cooling crystallizer and the large continuous process, other arrangements have been proposed. Thurlby and Sitnai (1976) proposed the combination of batch and continuous mode of operation, with a better nucleation control, increasing yield, and purity. Crystallization is the key step in the manufacture of lactose from whey. In the same way, lactose crystallization is a key step in the manufacture of whey powders. Although the crystallization process is not fully understood, it is a well-known fact that crystallization takes place on the surface of already existing crystal. As seen before, seeding of a supersaturated solution must be done in order to promote good crystallization, with the small crystals required to create the needed surface area to grow the lactose crystals. Quantities from 0.1 to 1 wt% are adequate. Crystallization by cooling must be conducted in a gentle way, with cooling rates of about 1 to 3°C/h. In order to get a high yield, the final temperature of 15°C is recommended. This allows the mutarotation to proceed at a reasonable speed resulting in the crystallization of a high amount (80%) of lactose. The suspension must be vigorously and continuously mixed in order to transport the supersaturated solution to the surface of the crystals with a simultaneous replacement of the saturated solution. In these conditions, crystal size can reach values from 30 to 50 mm (GEA, 2008). As pointed by Garnier et al. (2002), the rational approach to crystal engineering constitutes an important step toward a more predictive approach and a better control of physical, thermal, and mechanical properties of solid samples used in the food and pharmaceutical industry. The Industrial Crystallization (Nývlt et al., 2001) approach as presented here is the basic tool to a successful design of a dairy crystallization system.

Acknowledgments The authors wish to acknowledge the National Council of Technological and Scientific Development (CNPq) and the Foundation to Research Support of São Paulo State (FAPESP) for their financial support.

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Littlechild, J. A. Protein crystallization: magical or logical: can we establish some general rules? Journal of Physics D: Applied Physics, v. 24, p. 111–118, 1991. Lu, J.; Wang, X. J.; Ching, C. B. Batch crystallization of soluble proteins: effect of precipitant, temperature and additive. Progress in Crystal Growth and Characterization of Materials, v. 45, p. 201–217, 2002. Ma, D. L. Simulation and optimization of batch crystallization processes, PhD thesis, University of Illinois at Urbana-Champaign, 2002. Machado, J. J. B.; Coutinho, J. A.; Macedo, E. A. Solid-liquid equilibrium of a-lactose in ethanol/water. Fluid Phase Equilibria, v. 173, p. 121–134, 2000. McCommins, D. B.; Bernhard, R. A.; Nickerson, T. A. Recovery of lactose from aqueous solutions: precipitation with calcium hydroxide and sodium hydroxide. Journal of Food Science, v. 45, p. 362–366, 1980. McPherson, A. Handbook of Crystal Growth, v. 2. Amsterdam: Elsevier Science, 1994, Chap. 8: Crystallization of biological macromolecules, p. 418–463. McPherson, A. Crystallization of Biological Macromolecules. Cold Spring Harbor Laboratory Press, New York, 586 p., 1999. McPherson, A. Introduction to protein crystallization. Methods, v. 34, p. 254–265, 2004. Mersmann, A. Particle size distribution and population balance. In: Crystallization Technology Handbook, edited by Mersmann A., Marcel Dekker, New York, 2001. Miller, S. M. Modelling and quality control strategies for batch cooling crystallizers, PhD thesis, University of Texas at Austin, 1993. Mimouni, A.; Schuck, P.; Bouhallab, S. Kinetics of lactose crystallization and crystal size as monitored by refractometry and laser light scattering: effect of proteins. Lait, v. 85, p. 253–260, 2005. Monaco, H. L.; Zanotti, G.; Spadon, P.; Bolognesi, M.; Sawyer, L.; Eliopoulos, E. E. Crystal structure of the trigonal form of bovine beta-lactoglobulin and of its complex with retinol at 2.5 Å resolution. Journal of Molecular Biology, v. 197, n. 4, p. 695–706, 1987. Mullin, J. W. Crystallization, 4th ed. Butterworth-Heinemann, London, 2001. Myerson, A. S.; Ginde, R. Crystals, crystal growth and nucleation, in: Handbook of Industrial Crystallization, edited by Allan S. Myerson, 2nd ed., Butterworth, Woburn, MA, 2002. Nickerson, T. A.; Lim, S. G. Effect of various alcohols on lactose. Journal of Dairy Science, v. 57, p. 1320–1324, 1974. Nickerson, T. A.; Moore, E. E. Alpha lactose and crystallization rate. Journal of Dairy Science, v. 57, p. 160–164, 1974a. Nickerson, T. A.; Moore, E. E. Factors influencing lactose crystallization. Journal of Dairy Science, v. 57, n. 11, p. 1315–1319, 1974b. Nývlt, J.; Hostomský, J.; Giulietti, M. Crystallization (in Portuguese). São Paulo, Brazil: EDUFSCar – IPT, 2001. O’Brien, C. S. A mathematical model for colloidal aggregation, MSc thesis, University of South Florida, 2003. Olano, A.; Rios, J. J. Treatment of lactose with alkaline methanolic solutions: production of betalactose from alpha-lactose hydrate. Journal of Dairy Science, v. 61, p. 300–302, 1978. Oliveira, K. M. G.; Valente-Mesquita, V. L.; Botelho, M. M.; Sawyer, L.; Ferreira, S. T.; Polikarpov, I. Crystal structure of bovine beta-lactoglobulin in the orthorhombic space group C222(1)—structural differences between genetic variants A and B features on the Tandfod transition. European Journal of Biochemistry, v. 268, n. 2, p. 477–483, 2001. Ortin, A.; Cebrian, J. A.; Johansson, G. Large-scale extraction of alpha-lactalbumin and betalactoglobulin from bovine whey by precipitation with polyethylene-glycol and partitioning in aqueous 2-phase systems. Preparative Biochemistry, v. 22, n. 1, p. 53–66, 1992.

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Technologies 7 Novel for Milk Processing Ricardo Nuno Pereira and António Augusto Vicente* Contents 7.1 Introduction................................................................................................... 155 7.2 Thermal Processing....................................................................................... 156 7.3 Novel Thermal Processing Technologies...................................................... 157 7.3.1 Ohmic Heating (OH) Technology..................................................... 157 7.3.1.1 Microbial Inactivation......................................................... 160 7.3.1.2 Enzyme Inactivation........................................................... 161 7.3.1.3 Effects on Physical-Chemical Properties........................... 161 7.3.2 Microwave (MW) Heating................................................................ 162 7.3.2.1 Potential Effects.................................................................. 163 7.3.2.2 MW and Microorganisms................................................... 163 7.3.3 Infrared Heating (IH)........................................................................ 164 7.4 Novel Nonthermal Processing Technologies................................................. 165 7.4.1 Pulsed Electric Field (PEF)............................................................... 165 7.4.1.1 Inactivation Studies............................................................. 166 7.4.1.2 Effects of PEF on Milk Quality.......................................... 167 7.4.1.3 Current Limitations............................................................ 167 7.4.2 High-Pressure Processing.................................................................. 168 7.5 Final Remarks—The Hurdle Concept........................................................... 169 References............................................................................................................... 169

7.1 Introduction During the last 25 years, consumer demands for more convenient and varied milk food products, together with the need for faster production rates, improved quality, and extension in shelf life have brought significant improvements to the processing of fluid milk and milk products. Many technological developments have been directed toward unit operations such as separation, standardization, pasteurization, and packaging, leading to considerable advances in mechanization, automation, energy efficiency, hygiene, and quality within the processing plant (Goff and Griffiths 2006). In particular, extending the shelf life of milk and milk products without compromising their quality and safety has been a prime goal of milk processors. In general, the use of heat is still 155

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a common practice of the dairy industries in order to guarantee the microbiological safety of milk and its subproducts. However, the processing of milk through heating has had a noticeable evolution during the twentieth century, which has continued until the present time. The technological improvements together with the efforts and diligence of processors, technologists, and dairy researchers in bringing superior quality products to consumers, has been triggering the investigation and development of new technological approaches for milk processing capable of substituting the traditional well-established preservation processes. Thermal technologies such as ohmic heating, dielectric heating, and inductive heating have been developed and can replace, at least partially, the traditional heating methods that rely essentially on conductive, convective, and radiative heat transfer. Nonthermal approaches to milk processing, such as pulsed electric fields, high pressure, among others, may also be valuable alternatives to thermal processing, because they have the ability to inactivate microorganisms at near-ambient temperatures, avoiding the undesirable effects of heat on the organoleptic properties of foods. The purpose of this chapter is to provide a general perspective of the main thermal processing technologies currently available and, in particular, to give the reader an overview of the novel thermal and nonthermal processing technologies of fluid milk, while providing examples of recently conducted research.

7.2 Thermal Processing Biological and physical-chemical changes can occur in milk during and after thermal processing, which normally affect its nutritional, organoleptic, or technological properties and can also lead to interactions between its principal constituents (Fox and McSweeney 1998). Several heat treatments exist and are applied to milk processing according to its different applications. Thermal approaches such as ultrahigh-temperature (UHT) sterilization, high-temperature short-time (HTST), and higher-heat short-time (HHST) pasteurization are widely used in the dairy industry today. UHT is a sterilization process that heats milk within a range of 138°C to 150°C for 4 to 15 seconds followed by aseptic packaging. UHT processing typically extends the shelf life of milk from up to 6 months without refrigeration (Raynal-Ljutovac et al. 2007), although gelation and flavor changes are very likely to occur during storage (Fox and McSweeney 1998). UHT commercial sterilization is achieved most successfully by direct heating systems, such as steam injection, or steam infusion, in which the temperature of milk is rapidly raised to 140°C by direct mixing with steam, followed immediately by rapid cooling though flash vacuum evaporation of water that condensed in the product from the steam (Goff and Griffiths 2006). Pasteurization is the name given to heat processes typically applied for up to a few minutes below the boiling point of milk, within in the range of 60 to 80°C. The HTST pasteurization has been effectively used for decades as a method to extend the shelf life of fluid milk (Raynal-Ljutovac et al. 2007; Steele 2000) and is truly associated with the following purposes: reduction of the number of any harmful microorganisms to a level at which they do not constitute a significant health hazard; reduction of the level of activity of undesirable enzymes and spoilage bacteria; and increase of the keeping quality while achieving the preceding two goals without destroying the original characteristics of the product (Hudson, Wong, and Lake 2003). Under

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optimal processing and refrigerated storage conditions, HTST thermal pasteurization is able to extend the shelf life of milk to around 3 weeks, depending on the initial microbiological quality of the raw milk and the degree of refrigeration (Sepulveda et al. 2005). For an effective HTST pasteurization, every particle of milk will have been heated in properly operated equipment to 71.7°C (161°F) for 15 seconds (Food and Drug Administration 2003). Likewise, the Europe EC Directive 92-46-EEC of June 1992 indicates that pasteurized milk must have been obtained by means of a treatment involving a high temperature, for a short time, or a pasteurization process using different time and temperature combinations to obtain an equivalent effect. HTST pasteurization of milk is normally carried out by indirect heating systems, such as plate and tubular heat exchangers, where the milk and the heating medium (superheated steam or hot water) are separated by heat-conducting material, and heat is transferred to the product by conduction and convection. The plate heat exchangers (PHEs) are broadly used for heating and cooling applications in the dairy industry, because they offer high degrees of compactness and effectiveness (Bansal and Chen 2006; Ghosh, Sarangi, and Das 2006). These features lead to high rates of heat transfer, small terminal temperature difference with a small overall size of the exchanger, flexibility in stream arrangement, high turbulence, and ease of cleaningin-place (Morison 2005). However, a problem during UHT and HTST processing is fouling and deposit of proteins and minerals on the surface of the heat exchangers (Johansson 2008). Actually, the fouling of heat exchanger surfaces by milk and its products is a major problem experienced by the dairy industry (Simmons, Jayaraman, and Fryer 2007), because it reduces heat transfer efficiency and increases pressure drop and hence affects the economy of a processing plant (Toyoda et al. 1994). As a result of fouling, there is a possibility of deterioration of the product quality because the process fluid cannot be heated to the required temperature for pasteurization (Bansal and Chen 2006). Despite the fact that the thermal technologies referred to above are still prevalent and well established in the industry today, development of new technologies for continuous thermal dairy food treatment, such as ohmic heating and microwave and radiofrequency heating are still of great industrial and scientific interest (Ayadi et al. 2004; Pereira et al. 2008). They all have a common feature: heat is generated directly inside the food, and this has direct implications in terms of both energetic and heating efficiency.

7.3 Novel Thermal Processing Technologies 7.3.1 Ohmic Heating (OH) Technology Ohmic heating (OH), also called Joule heating, electrical resistance heating, direct electrical resistance heating, electroheating, and electroconductive heating, is one of the earliest applications of electricity in food pasteurization and is defined as a process where electric currents are passed through foods to heat them. Heat is internally generated due to electrical resistance (De Alwis and Fryer 1990a). The OH technology is distinguished from other electrical heating methods by the presence of electrodes contacting the foods (in microwave and inductive heating, electrodes are absent); the

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frequency applied (unrestricted, except for the specially assigned radio or microwave frequency range); and waveform (also unrestricted, although typically sinusoidal) (Vicente 2007). A successful application of electricity in food processing was developed in the nineteenth century to pasteurize milk (Getchel 1935). This pasteurization method was called the Electropure Process, and by 1938 it was used in approximately 50 milk pasteurizers in five U.S. states and served about 50,000 consumers (Moses 1938). This application was abandoned apparently due to high processing costs (De Alwis and Fryer 1990a). Also, other applications were abandoned because of the short supply of inert materials needed for the electrodes, although electroconductive thawing was an exception (Mizrahi, Kopelman, and Perlaman 1975). However, research on ohmic applications in food products, such as fruits, vegetables, meat products, and surimi has been undertaken by several authors, more recently Palaniappan and Sastry (1991a), Palaniappan and Sastry (1991b), Wang and Sastry (1997), and Castro, Teixeira, and Vicente (2003). In fact, OH technology has gained interest recently because the products are of a superior quality to those processed by conventional technologies (Castro, Teixeira, and Vicente 2003; Kim et al. 1996; Parrott 1992). The potential applications are very wide and include, for example, blanching, evaporation, dehydration, and fermentation (Cho, Yousef, and Sastry 1996). Presently the focus of OH is being addressed to thermal processing operations, such as sterilization and pasteurization. This technology can be accomplished in a continuous in-line heater for cooking and sterilization of viscous and liquid food (Icier and Ilicali 2005). OH can be used for HTST pasteurization of liquid proteinaceous food products which tend to denature and coagulate when thermally processed conventional technologies are used. Due to its extremely rapid heating rates, OH technology enables higher pasteurization temperatures to be applied, with consequent increase in refrigerated shelf life, without inducing coagulation or excessive denaturation of the constituent proteins (Parrott 1992). The major benefits claimed for ohmic heating technology are as follows:

1. Temperature required for HTST processes can be achieved very quickly 2. Suitable for continuous processing without heat transfer surfaces 3. Uniform heating of liquids with faster heating rates 4. Reduced problems of surface fouling or overheating of the product compared to conventional heating 5. Fresher-tasting, higher-quality products than with alternate heat preservation techniques 6. No residual heat transfer after the current is shut off, and very low heat losses 7. Useful in preheating products before canning 8. Low maintenance costs (no moving parts) and high energy conversion efficiencies 9. Environmentally friendly system

For all these reasons, OH is now receiving increased attention by the dairy industry, once it is considered to be an alternative for the indirect heating methods of milk pasteurization, such as shell and PHE exchangers where heating of milk is achieved through direct contact with a hot surface. In OH, heat is generated directly within milk (volumetric heating) and, hence, the problems associated with heat transfer surfaces are eliminated

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Novel Technologies for Milk Processing 1.00E–02 9.00E–03

Conductivity (S · cm–1)

8.00E–03 7.00E–03 6.00E–03 5.00E–03 4.00E–03

20.6 V/cm

3.00E–03

37.5 V/cm

28.9 V/cm 53.8 V/cm

2.00E–03 1.00E–03 0.00E+00

15

25

35

45 55 65 Temperature (°C)

75

85

95

Figure 7.1 Relationship between electrical conductivity and temperature in milk, at different field strength values.

(Bansal and Chen 2006). The electrical conductivity of foods together with the electrical field strength applied play a major role during OH processing. Furthermore, other properties related to the type of food, such as kind of phase (solid or liquid), size and shape of the particles, moisture content of the solids (if present), solids/liquids ratio, viscosity of the liquid component, possible occurrence of electrolysis, pH, and specific heat are also very important for the effectiveness of this technology (Fellows 2000). Milk contains sufficient free water with dissolved ionic salts and therefore conducts sufficiently well for the ohmic effect to be applied (Palaniappan and Sastry 1991b), and because electrical conductivity increases with temperature, OH becomes more effective at higher temperatures. Furthermore, for materials of uniform electrical conductivity, such as milk, the energy generation is far more uniform than microwave heating (Sastry et al. 2002), where the limited penetration of the microwave radiation often promotes significant temperature gradients. Figure 7.1 shows a linear relation between electrical conductivity and temperature for the different field strengths applied during the heating of milk. Overall, this technology provides a rapid and uniform heating and can be considered a HTST process (Castro et al. 2004b; De Alwis and Fryer 1990b; Reznick 1996; Zareifard et al. 2003). Despite OH features, some disadvantages, namely those related to the high initial operational costs and the lack of generalized information or validation procedures, the absence of a hot wall should provide a considerable advantage for milk processing applications, by avoiding the degradation of thermosensitive compounds due to overheating and by reducing the fouling of the surfaces during processing (Ayadi et al. 2004a; Leizerson and Shimoni 2005).

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7.3.1.1 Microbial Inactivation The principal mechanisms of microbial inactivation in OH are thermal in nature. The destruction of microorganisms by nonthermal effects such as electricity is still not well understood and generates some controversy (Vicente 2007). Moreover, most of the published results do not refer to the sample temperature or cannot eliminate temperature as a variable parameter (Food Safety and Nutrition 2000; Palaniappan et al. 1990). However, studies such as that of Cho et al. (1996) provide evidence that OH may be useful in the dairy industry to shorten the time for processing yogurt and cheese production. Recently, the influence of OH on the heat resistance of Escherichia coli, which frequently contaminates dairy products when their manufacture conditions are unsanitary, was studied in goat milk and compared to that of conventional heating. The results have shown that the microorganism’s inactivation was faster when the OH was applied, indicating that in addition to the thermal effect, the presence of an electric field provided a nonthermal killing effect over vegetative cells of E. coli (Pereira et al. 2007b). Sun and coworkers (2008) studied the effects of OH (internal heating by electric current) and conventional heating (external heating by hot water) on viable aerobes and Streptococcus thermophilus 2646 in milk under identical temperature history conditions. It was found that both the microbial counts and the calculated decimal reduction time (D value) resulting from OH were significantly lower than those resulting from conventional heating. The main reason for the additional killing effect of ohmic treatment observed in different microorganisms seems to be linked with the electrical current and frequency applied during OH inactivation (Sastry et al. 2002; Sun et al. 2008). Several authors suggest that a mild electroporation mechanism may contribute to cell death, bringing a nonthermal effect to inactivation (Imai et al. 1995; Kulshrestha and Sastry 1999; Wang 1995). However, further research is needed to understand the inactivation mechanisms of various microorganisms in different types of foodstuffs. Data on nonthermal effects are scarce (see Table 7.1), and more studies are needed to Table 7.1 D-Values for Various Microorganisms in Milk under Conventional and Ohmic Heating Microorganism Viable aerobes

a

Staphylococcus thermophilusa Escherichia colib

Conventional D(57°C) /min 11.25 ± 1.45 D(70°C) /min 7.54 ± 0.37 D(55°C) /min 10.9 ± 1.08

D(60°C) /min 9.39 ± 0.85 D(75°C) /min 3.30 ± 0.42 D(63°C) /min 3.9 ± 0.50

D(72°C) /min 0.44 ± 0.00 D(80°C) /min 0.20 ± 0.03 D(65°C) /min 3.5 ± 0.2

Ohmic D(57°C) /min 8.64 ± 1.08 D(70°C) /min 6.59 ± 0.35 D(55°C) /min 14.2 ± 0.2

D(60°C) /min 6.18 ± 0.44 D(75°C) /min 3.09 ± 0.55 D(63°C) /min 1.9 ± NA

D(72°C) /min 0.38 ± 0.00 D(80°C) /min 0.16 ± 0.03 D(65°C) /min 0.86 ± NA

Adapted from Sun, H.-X., Kawamura, S., Himoto, J.-I., Itoh, K., Wada, T. & Kimura, T. (2008). Food Science and Technology Research, 14(2):117–123. With permission. b Adapted from Pereira, R., Martins, J., Mateus, C., Teixeira, J. & Vicente, A. (2007b). Chemical Papers, 61(2):121–126. With permision. Note: NA, not available. a


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determine, for example, the effect of electricity on the physiological characteristics of microorganisms, changes in glycosylation degree of proteins and lipids, and other elements that can affect the heat resistance of microorganisms (Pereira et al. 2007b). However, it is clear that by reducing the time required for inactivation of microorganisms, the use of OH could diminish negative thermal effects of pasteurization on fluid milk, opening a new perspective for shorter, less aggressive aseptic processing. 7.3.1.2 Enzyme Inactivation There is still limited information about the effects of OH technology on the activity of enzymes, particularly those used as time–temperature integrators in the dairy industry. In recent years, inactivation assays were performed (Castro et al. 2004a) under conventional and OH conditions, where the thermal history of the samples (conventionally and ohmically processed) was made equal to determine if there was an additional inactivation caused by the presence of an electric field. Among others, two important enzymes for the dairy industry were tested: alkaline phosphatase (ALP) and b-galactosidase (b-GAL). Results have shown that all the enzymes followed first-order inactivation kinetics for both conventional and OH treatments, and that the presence of an electric field did not cause enhanced inactivation of b-GAL (in the range from 55 to 80°C), and a reduction of the D value was observed for ALP (in the range from 52 to 78°C). In case of the first enzyme, this result seems to be quite interesting, once b-GAL allows for the production of dairy products that can be consumed by lactoseintolerant individuals. In the case of ALP, enhanced inactivation is obtained when an electric field is present (Wilinska et al. 2006), thus reducing inactivation time. 7.3.1.3 Effect on Physical-Chemical Properties Despite the reduced amount of information available, the technology of OH appears to be promising and highly effective on the inactivation of some microorganisms and enzymes. However, the information concerning the effects of this technique on specific food components compared to conventional pasteurization is even scarcer. Conventional thermal processing always implies the loss of nutritional and organoleptic qualities of the end product, be it milk or, for example, the cheese made from it. For example, whey proteins, typical globular proteins with high levels of secondary and tertiary structures, are very susceptible to denaturation by heat (Fox and McSweeney 1998). Another negative aspect due to the technological treatment of milk is the increase of free fatty acids (FFAs) concentration (Antonelli et al. 2002; Morgan and Gaborit 2001). During thermal processing, significant changes in physical properties of milk lipids can occur, especially at the level of the milk fat globule membrane which is a delicate structure and can easily be ruptured by either physical or thermal shock (Muir 1988), leading to excessive accumulation of FFA in milk, which is frequently associated with the appearance of undesirable flavors. Recently, Pereira et al. (2007a) studied the effects of HTST ohmic pasteurization on quality of goat milk by assessing physical and chemical properties such as pH, total solids, and total fatty acids, and concluded that the technology based on OH provides products with physical-chemical properties similar to those of the products obtained by conventional treatment. Likewise, when degrees of protein denaturation in OH and conventional heating at different temperatures (ranging from 40 to 80°C)

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were studied, no significant differences were noticed between the two types of treatments, which led the authors to conclude that electrical current had no additional effect on protein denaturation (Sun et al. 2008). To assess the value of electroheating in dairy processing, a OH system developed by Raztek Corporation in Sunnyvale, California, was used to superheat pasteurized milk for up to 4 seconds The ohmic heated milk was then compared to a commercially available UHT milk sample. Results indicate that the cooked, sour, and stale flavors in the electroheated samples were much lower than in the commercial variety. Their analysis also showed that protein denaturation, a measure of the chemical and flavor changes caused by heat exposure, was of ca. 67% in the commercial UHT sample and dramatically lower in the ohmic treated milk, only 30% (Dairy Management 2001). The short-chain and medium-chain free fatty acids profiles were characterized in raw milk and processed milk by conventional and ohmic HTST pasteurization, at 72°C for 15 seconds to determine the influence of each treatment on the final quality of the milk. In particular, it was possible to conclude that ohmic HTST pasteurization did not promote an extended modification of free fatty acid contents in goat milk when compared to that of conventional pasteurization, indicating that the OH technology can be introduced in goat milk pasteurization without affecting negatively the quality of goat milk flavor (Pereira et al. 2008).

7.3.2 Microwave (MW) Heating In conventional thermal processes, slow heat conduction from the heating medium to the cold spot often results in treatment of the material at the periphery of the container that is far more severe than that required to achieve commercial sterility (Meredith 1998). Since the early 1960s, microwave energy has been used for cooking. Hamid et al. (1969) were the first group to use the technology for milk pasteurization. Heating with microwave involves primarily two mechanisms: dielectric and ionic. Water in the food is often the primary component responsible for dielectric heating. Due to their dipolar nature, water molecules try to follow the electric field associated with electromagnetic radiation as it oscillates at the very high frequencies and such oscillations produce heat. The second major mechanism of heating with microwaves and radio frequency is through the oscillatory migration of ions in the food that generates heat under the influence of the oscillating electric field (Food and Drug Administration 2000). MW has the potential to replace conventional processes once it can eliminate excessive heating with rapid and more uniform heating from a direct interaction between microwave energy and the food. Other advantages of MW heating systems, some depending on the application, are as follows: • • • •

It can be turned on or off instantly. The product can be pasteurized after being packaged. It allows space saving or reduced noise levels. Heating can be selective (microwaves couple selectively into materials that are more absorptive of the energy; although greater efficiency can be achieved, temperature profiles can develop in multicomponent food systems).


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These advantages often yield an increased productivity or an improved product quality (Food and Drug Administration 2000; Vicente 2007; Wang et al. 2003). Studies of the application of MW for commercial pasteurization and sterilization in milk have been reported for years (Decareau 1985; Hamid et al. 1969; Knutson et al. 1988; Kudra et al. 1991; Villamiel et al. 1997) and have been described as offering great potential benefits to the dairy industry (Sierra and Vidal-Valverde 2000). 7.3.2.1 Potential Effects Lopez-Fandiño et al. (1996) studied the effect of continuous-flow microwave on quality of milk by using indicators of the heat treatment intensity (β-lactoglobulin denaturation, inactivation of alkaline phosphatase and lactoperoxidase). Results were compared with those obtained using a conventional process having the same heating, holding, and cooling phases. Continuous microwave treatment proved to be an effective system for pasteurizing milk, with the inclusion of a holding phase to maintain the time and temperature conditions required; at high pasteurization temperatures, the extent of thermal denaturation observed with the microwave treatment was lower than that obtained with the conventional system. These results have been attributed to a better heat distribution and the lack of hot surfaces contacting the milk in the case of the microwave unit. There are also some published studies considering the effects of MW on vitamins of milk (Medrano et al. 1994; Sieber et al. 1993, 1996; Vidal-Valverde and Redondo 1993). However, literature on the effect of microwave heat treatment of milk on vitamins is not always consistent due to different conditions of heat treatment (combination of time and temperature). Despite that, Sierra and Vidal-Verde (2000) observed that when milk was heated in a continuous MW system, at 90°C without a holding phase, no vitamin B1 and vitamin B2 losses were observed. However, holding times of 30 to 60 seconds lowered the content of vitamin B1 (3% and 5%, respectively), and the content of vitamin B2 was not modified. Analogous results were obtained when the milk was submitted to a similar heating process using a conventional system. These authors have concluded that continuousflow MW of milk at high temperature does not offer any advantage with respect to vitamin B1 and B2 retention compared with a conventional heating process having the same heating, holding, and cooling times. 7.3.2.2 MW and Microorganisms The inactivation curves for microorganisms using MW heating are similar to those obtained using conventional heating methods. Microwave treatment may be adequate for inactivating L. monocytogenes at a temperature similar to the conventional pasteurization process (Galuska et al. 1989) and inactivation of all cells of L. monocytogenes by MW at 71.7°C/10 minutes were reported (Choi et al. 1992). More recently (Clare et al. 2005), microbiological and biochemical parameters of microwave processed fluid milk were compared with conventionally prepared, indirectly heated UHT milks. They concluded that microwave processing may afford new opportunities to develop fluid milk products that exhibit a long shelf life, with sensory characteristics that are equivalent to, if not better than, those achieved with indirect UHT-treated milk. There are conflicting works in the literature with respect to the lethal effects of microwaves on microorganisms; in particular, there is no consensus

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on if they are exclusively of a thermal nature or not (Valsechi et al. 2004). Four major theories were proposed to explain nonthermal inactivation by microwaves: selective heating, electroporation, cell membrane rupture, and magnetic field coupling (Kozempel et al. 1998). It is currently accepted that MW energy may complement or magnify thermal effects by causing nonlethal injuries to the cells (Vicente 2007). However, it is still very difficult to precisely compare the effectiveness of MW to conventional heating based on the literature results. The effects of microwaves on inactivation of microorganisms present in foods are influenced by several factors, such as intrinsic characteristics of the microorganism (stage of development and their initial amount) and the products being processed (pH, chemical composition); and extrinsic factors related to temperature, frequency, and intensity of the radiation, time of exposure, position of the foods in relation to the effective radiation field, among others (Valsechi et al. 2004). Overall, continuous-flow microwave treatments of milk could still be advantageous (Sierra and Vidal-Valverde 2000). However, it is important to notice that one of the major disadvantages claimed for MW is the eventual nonuniform heating and unpredictability of cold spots which may put at risk safety of the food. In addition, the difficulties in controlling the process and the high energy costs associated with this technology are main obstacles to industrial setting up of MW heating processes. The changes of dielectric properties of food products during the heating processes are not yet fully understood or modeled and, consequently, the validation of the processes has to be done almost individually for each food product, slowing the dissemination of MW industrial lines (Vicente 2007).

7.3.3 Infrared Heating (IH) Infrared heating (IH) has been widely applied to various thermal processing operations in the food industry such as dehydration, frying, and pasteurization (Sakai and Hanzawa 1994). By exposing an object to infrared (IR) radiation, the heat energy generated can be absorbed by food materials. Basically, any product has its own inherent reaction to infrared which is called “heat absorption factor.” IR radiation transfers thermal energy in the form of electromagnetic waves and can be classified into three regions depending on the wavelength, namely, near-infrared (NIR), corresponding to the spectral range of 0.7 to 2.0 μm and with temperatures above 1000°C; mid-infrared (MIR) corresponding to the spectral ranges of 0.7 to 2.0 μm, when temperatures range from 400 to 1000°C; and far-infrared (FIR), when temperatures are below 400°C and spectral ranges vary from 4.0 μm to 1 mm. In general, FIR radiation is advantageous for food processing because most food components absorb radiative energy in the FIR region (Sandu 1986). However, new applications using short waves have been arising (Vicente 2007) because of several advantages, similar to the other types of electromagnetic heating, namely, faster heating and higher energy efficiency (there is no need for heat buildup because electric IR systems produce heat instantly); minimal deterioration in food quality; high degree of process control parameters, and space saving along with clean working environment; high heat transfer coefficients; reduced operating costs (depending on the insulation, type of construction, and other factors, the energy savings can reach 50%); and equipment can be compact and automated


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with a high degree of control of process parameters (the IR energy does not propagate, it is absorbed only at the area it is directed into) (Krishnamurthy et al. 2008; Nowak and Lewicki 2004; Sakai and Hanzawa 1994; Vicente 2007). IR heating can be used to inactivate bacteria, spores, yeast, and molds in both liquid and solid foods. Efficacy of microbial inactivation by IH depends on the following parameters: infrared power level, radiator efficiency, infrared reflection/ absorption properties and IR penetration properties, temperature of food sample, peak wavelength, and bandwidth of IH source, sample depth, types of microorganisms, moisture content, physiological phase of microorganism (exponential or stationary phase), and types of food materials. Recent research (Krishnamurthy et al. 2008) demonstrated that IR heating can effectively inactivate pathogenic microorganisms in dairy products, preserving their quality; IR demonstrated potential for effective inactivation of Staphylococcus aureus in milk, which was reduced from 0.10 to 8.41 log10 cfu/mL, depending on the treatment conditions. In spite of these advantages of this technology, application of infrared energy in milk processing is rather scarce, but further investigation on sensory and quality changes during IR heating can shed light on the efficacy of this process and may provide a potential novel pasteurization method for the dairy industry (Krishnamurthy et al. 2008).

7.4 Novel Nonthermal Processing Technologies The availability of less processed foods that are safe to consume and have a similar or better shelf life than traditional foods prompted the research into alternative nonthermal processes for the destruction of microorganisms. Novel nonthermal techniques for the processing of raw milk include ultraviolet irradiation, gamma irradiation, ultrasounds, high-pressure (HP) processing, and pulsed electric field (PEF) treatment. These treatments do not involve heat or a subsequent heat treatment to kill the microorganisms, avoiding the deleterious effects that heat has on the flavor, color, and nutrient value of foods.

7.4.1 Pulsed Electric Field (PEF) PEF has the greatest potential for successful shelf-life extension of milk, as it is highly applicable to liquids; has minimal effects on the nutritional, flavor, and functional characteristics of milk; has a demonstrated ability to inactivate microorganisms; and is under development to commercial scale (Bendicho et al. 2002a; Leadley 2003). The origin of PEF can be found in electroporation, a biotechnology process used to promote bacterial DNA interchange by perforating microbial membranes with induced electric fields. The main idea behind the use of electric fields as a food preservation method is to take advantage of the lethal effect observed in electroporation to inactivate undesirable bacteria in food products (Góngora-Nieto et al. 2002). PEF technology is based on the application of pulses of high voltage (typically 20 to 80 kV/cm) delivered to the product placed between a set of electrodes that confine the treatment gap of the PEF chamber. PEF treatment can be conducted at ambient temperature for less than 1 second, and energy loss due to the heating of foods is minimized.

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In the last decade, several studies have been performed in order to develop nonthermal electrical pasteurization processes. Different types of equipment for the application of PEF have been patented, and several studies have demonstrated the effectiveness of this nonthermal technique in food processing (Bendicho et al. 2002b). Preservation of milk and fluid dairy products seems to be one of the main market niches for PEF technology, because it is mainly intended for preservation of pumpable fluid or semifluid foods (Qin et al. 1996). 7.4.1.1 Inactivation Studies Overall, PEF technology is considered a novel processing technology valued for its ability to eliminate bacteria from milk, without increasing their temperature, and thus avoiding the detrimental changes of the milk’s sensory and physical properties. Therefore, most of the studies carried out on milk have been performed to evaluate the PEF effect on microbial and enzyme inactivation. Inactivation of important spoilage microorganisms, such as Escherichia coli (Evrendilek and Zhang 2005; Martín et al. 1997), Salmonella dublin (Sensoy et al. 1997), or Staphylococcus aureus (Sobrino-López and Martín-Belloso 2006a, 2006b), Pseudomonas isolates (Craven et al. 2008), and Listeria innocua and Pseudomonas fluorescens (Fernandez-Molina et al. 2006) by applying a PEF treatment on skim, whole, and simulated ultrafiltered milk (SMUF), has been demonstrated by several authors (see Table 7.2).

Table 7.2 D-Values for Various Microorganisms in Milk After Pulsed Electric Field Treatment Microorganism

Type of Milk

Pseudomonas fluorescens

Skim milk

Pseudomonas species

Whole milk

Staphylococcus aureus Listeria monocytogenes Listeria monocytogenes

Whole milk

Salmonella dublin

Skim milk

Whole milk Whole milk

Treatment Conditions

Log Reduction

Reference

28 kV/cm, 1 pulse, 2 µs, repetition rate of 200 pulses per second, 40°C 31 kV/cm, 1 pulse, 2 µs, repetition rate of 200 pulses per second, 55°C 35 kV/cm, 150 bipolar pulses, 8 µs, 25°C 30 kV/cm, 400 pulses, 25°C 30 kV/cm, 400 pulses, 25°C

2

Craven et al. (2008)

>3

Craven et al. (2008)

4.5 2.5

Sobrino-López et al. (2006b) Reina et al. (1998)

4

Reina et al. (1998)

1

Sensoy et al. (1997)

25 kV/cm, 100 pulses, 30°C


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PEF treatments that are more severe than those applied on microorganisms are needed to obtain a significant reduction on enzyme activity (Ho et al. 1997); therefore, milk could be treated to destroy microorganisms while maintaining the activity of enzymes. Regarding the studies about the effects of PEF on enzymes, contradictory results have been obtained; in some cases, high levels of inactivation have been achieved, whereas in other cases no effect or an increase in the initial activity has been detected (Bendicho et al. 2001, 2002b; Castro et al. 2001; Van Loey et al. 2002; Vega-Mercado et al. 1995, 2001). Differences in electrical pulsation parameters applied (treatment time, pulse wave shape, treatment temperature), product factors (pH, ionic strength, and conductivity), microbial factors (stage of development and concentration and type of microorganisms), and the use of a different PEF system, can explain the different conclusions obtained. 7.4.1.2 Effects of PEF on Milk Quality Bendicho et al. (2002b) evaluated the effect of PEF on water-soluble vitamins in milk (riboflavin, thiamin, and ascorbic acid) and fat-soluble vitamins (cholecalciferol and tocopherol), applying treatments of up to 400 ms at field strengths from 18.3 to 27.1 kV/cm. No changes were reported in the vitamin content except for ascorbic acid; it was observed that milk retained more ascorbic acid after a 400 ms treatment at 22.6 kV/cm (93.4%) than after either a LTLT (low-temperature long-time, 30 minutes at 63°C, 49.7% retained) or a HTST (15 seconds, –75°C, 86.7% retained) heat pasteurization treatments. Xiang et al. (2007) studied the extent of protein denaturation of whole milk, through their thermal behavior using differential scanning calorimetry (DSC) and fluorescence spectroscopy (FS). The results have shown that both apparent enthalpy and transition temperatures of PEF-treated whole milk were modified by PEF; protein was denatured at a level of about 25% with a PEF treatment performed at electric field intensity of 22 kV⋅cm–1 and with a number of pulses of 80. On the other hand, fluorescence intensity decreased for higher numbers of pulses. These results indicated that the effects of PEF on milk proteins in whole milk may have significant implications for properties of products made from PEF-treated milk. 7.4.1.3 Current Limitations The most challenging aspects in PEF technology are related with the generation of high electric field intensities, the design of chambers that impart uniform treatment to foods with minimum increase in temperature, and the design of electrodes that minimize the effect of electrolysis Additionally, the lack of methods to accurately measure treatment delivery, number, and diversity in equipment, limits the validity of conclusions that can be drawn about the effectiveness of particular process conditions (Dairy Management 2001). Commercial application of PEF technology in milk processing has not yet been implemented, mainly due to the reasons enunciated before and also due to lack of regulatory approval, high initial investment, and high maintenance costs (Góngora-Nieto et al. 2002).

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7.4.2 High-Pressure Processing High-pressure (HP) treatment of food products is a novel processing technique during which the product is treated in a vessel of suitable strength at a high pressure, generally in the range of 100 to 1000 MPa (Huppertz et al. 2006). The first studies on the application of high pressure (HP) in food technology and, particularly, in milk, were carried out at the end of the nineteenth century (Hite 1899). Under pressure, biomolecules obey the Le Chatelier-Braun principle (i.e., whenever a stress is applied to a system in equilibrium, the system will react so as to counteract the applied stress); thus, reactions that result in reduced volume will be promoted under HP (Huppertz et al. 2002). Therefore, HP is considered an interesting alternative for milk heat pasteurization and possibly sterilization, because under HP conditions, microorganisms (vegetative cells) and certain enzymes are inactivated and fresh flavor, color, taste, and vitamins are only minimally affected (Anema et al. 2005; Balny and Masson 1993; Cheftel 1992; Mussa and Ramaswamy 1997). Many review papers about the effect of high pressure on various properties of milk and dairy-based products can be found in the literature (see, for example, Balci and Wilbey 1999; Datta and Deeth 1999; Huppertz et al. 2002; Knorr 1993). The technique offers several advantages: • Preserved products with characteristics similar to those present before processing • Homogeneity of treatment due to the fact that pressure is uniformly applied around and throughout the food product • Shelf lives similar to thermal pasteurization, while maintaining the natural food quality parameters (nutrients, flavor, and sensorial preservation) However, full commercialization of HP for low acid food processing such as milk has not been realized yet mainly because of the inability of this process to destroy spores without added heat and absence of large-scale industrial equipment (Anema et al. 2005; Ramaswamy et al. 2007). Despite the fact that pressure treatment can be used for the preservation of food products, research in HP processing has been centered on the effects that this technology may have on physical and chemical properties of milk. Huppertz et al. (2002) have extensively reviewed the current state of knowledge of the effects of HP on constituents and properties of milk and possible applications of HP treatment of milk prior to the production of yogurt and cheese. This review clearly states some of the most important pressure-induced changes in milk, such as • Disruption of casein micelles and denaturation of whey proteins at pressures of a-lactalbumin and b-lactoglobulin (ranging from 400 to 800 MPa), with the former being more resistant to pressure than the latter • Milk enzymes seem to be quite resistant to pressure • Shifts in the mineral balance in milk • Crystallization of milk fat at moderately high pressures (100 to 400 MPa) • Increased pH and reduced turbidity of milk following HP treatment • Reduced times for rennet coagulation and increased cheese yield


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Further research is required to evaluate the full commercial potential of HP treatment of milk through a complete understanding of the effects of pressure on preservation and the nutritional and technological values of milk. Several aspects have received only little attention to date, such as the reversibility of HP-induced changes in milk, the stability of HP-treated milk during subsequent storage, the heat and alcohol stabilities, and age-gelation behavior of HP-treated milk, for example (Huppertz et al. 2002). Likewise, the amount of kinetic data regarding microbiological destruction as well as denaturation, inactivation, or formation of compounds under HP-temperature conditions is insufficient, and the criteria based on the application of heat markers for HP processing of milk will be different from criteria established for thermal treatment of milk (Claeys et al. 2003).

7.5 Final Remarks—The Hurdle Concept The hurdle concept exploits synergistic interactions between traditional preservation treatments. In general, many nonthermal processes require very high treatment intensities to achieve adequate microbial destruction with consequences on sensory proprieties of low acid foods such as milk. According to the hurdle concept, thermal and nonthermal preservation techniques combined at lower individual intensities have additive or even synergistic antimicrobial effects, and their impact on organoleptic properties of the food is minimized (Leistner 1992). Further gains in shelf life and quality of milk can be achieved through intelligent application of the hurdle concept to the combination of “old” and “new” technologies, always thinking about the consumers’ demands for more convenient and varied food products, with an improved quality and extended shelf life.

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and Intelligent 8 Active Packaging for Milk and Milk Products Nilda de Fátima Ferreira Soares,* Cleuber Antônio de Sá Silva, Paula Santiago-Silva, Paula Judith Pérez Espitia, Maria Paula Junqueira Conceição Gonçalves, Maria José Galotto Lopez, Joseph Miltz, Miguel Ângelo Cerqueira, António Augusto Vicente, José Teixeira, Washington Azevedo da Silva, and Diego Alvarenga Botrel Contents 8.1 Importance and Definition of Active and Intelligent Packaging................... 176 8.1.1 A Brief Historical Introduction of Package Evolution....................... 176 8.1.2 More Consumer Demand, More Packaging Functions..................... 177 8.1.3 Concepts and Application of Active Packaging................................ 179 8.1.3.1 Antimicrobial Packaging.................................................... 179 8.1.3.2 Edible Packages.................................................................. 180 8.1.3.3 Oxygen Absorber................................................................ 182 8.1.3.4 Ethylene Absorber............................................................... 183 8.1.3.5 Humidity Absorber............................................................. 184 8.1.4 Concepts and Application of Intelligent Packaging.......................... 184 8.2 Development of Antimicrobial Packaging.................................................... 185 8.2.1 Commonly Used Antimicrobial Substances...................................... 186 8.2.2 Antimicrobial Incorporation into Plastic Polymers........................... 187 8.2.3 Antimicrobial Immobilization in Polymers...................................... 188 8.2.4 Surface Modification......................................................................... 189 8.2.5 Factors to Consider in the Production of Antimicrobial Films......... 190 8.3 Nanotechnology—Applications in Food Packaging..................................... 190 8.4 Potential Use of Active and Intelligent Packaging in Milk and Milk Products......................................................................................... 192 Acknowledgments................................................................................................... 196 References............................................................................................................... 196

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8.1 Importance and Definition of Active and Intelligent Packaging 8.1.1 A Brief Historical Introduction of Package Evolution In the distant past, food was consumed at the place it was found. One day, most likely when someone noticed that drinking water was much easier when hands were arranged as a shell to carry the water, instead of drinking it directly in the river or lake, man invented the package in its elementary form. The function to contain was defined. Later, man must have realized that the use of naturally available resources such as gourds, leaves, or shells, made it possible to take goods home. With this step forward, the function to transport was incorporated. This moment is the starting point for package evolution. As time went by, this elementary concept was developed and containers made from wood, skin, bones, and organs of animals were built. The discovery of ores and other compounds led to the development of packages made of metals and ceramics. These packages had different shapes and were used to store foods, always with the aim of conserving them. The function to protect then became associated with the food package. Economic growth led to an increase in the exchange of foods that had to be transported from one place to another. This raised the concern of transporting the largest possible amount of products with minimum risk. With this, the full concept of packaging was developed. As societies developed, there was a need to incorporate new functions in the packages, as in the case of antiquity merchants who started to identify the content of the packages to facilitate their businesses. Later, with the appearance of food companies and the development of new products, information such as manufacturers’ identification, preparation process, storing conditions, shelf life, and nutritional value had to be incorporated in the package. The function to inform became a major issue in packaging. Simultaneously, responding to the evolution of the market and the increasing competition between producers/products, the package started to be used as a vehicle to influence the consumer’s purchase decision and to sell the product. The industrial revolution played a major role in package development, as new technologies, enabled better packaging, consequently augmenting the stability of the produced foods and consumers’ ease of access. An increase in package production was also observed in postwar periods. In the 1970s, the arrival of supermarkets and food self-service required packages to be more attractive to the consumer, as packaging became fundamental to attract consumers’ attention and guarantee the sale. Although packages can have other functions, the main ones (passive) can be summarized as follows: To contain—the package offers the facility of handling and storing. To transport—the package enables the product to easily go through the logistical chain. Packaging has a crucial impact on the efficiency of transport and

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handling and storage of goods. Easy handling and space-saving storage and stowage are main properties for packages. To protect—the package ensures that the packed product reaches the consumer as it left the factory. The packaging must protect the food from biological agents, from mechanical damage (product abrasion, compressive forces, and vibration), and from chemical degradation (oxidation, moisture transfer, and ultraviolet light). To inform—the package serves as a vehicle through which the manufacturer can communicate with the consumer. Nutritional information, ingredients, producer, package material, discard and recycling of packaging, and often, the way to prepare recipes are found on a food label. To sell—the package acts as a “silent seller” and through its design, color, appearance, convenience, and cost tries to persuade the consumer to buy the product. Usually, less than 10% of the packaged products in a supermarket are advertised. So, the remaining argument depends exclusively on the package to convince the consumer. Overall, the food packaging concept can be considered as the association of two main areas—art with science and technology—that together have as the main objective to deliver packaged products to the market and to sell them. The packaged product must be delivered to the end user, in good condition and at a low cost. As art, packaging involves aspects related to printing, design, practicality, hygiene, convenience, and consumer identification with the product. It has a direct connection with the function of selling. Through art, packaging may be an important marketing tool. As science and technology, packaging involves aspects related to barrier functions (gases, light, and humidity), convenience, and practicality. As both areas are very dynamic, the concept and the function of food packaging have changed in recent years. Instead of being made of an inert material and having a minimal interaction with the food, the package became active and intelligent, interacting with the food material.

8.1.2 More Consumer Demand, More Packaging Functions Packaging has played a major role in the food supply chain as an integral part of both the food processes and the whole food supply chain (Ahvenainen, 2003). Although packaging has contributed greatly to the early development of food distribution systems, novel functions are required to answer the demands of today’s society. Modern living patterns have been reflected in eating habits and consumption patterns. The demand for natural and minimally processed products has considerably increased. In addition, changes in retail sales and distribution practices, as the centralization of the sale activities, the use of the Internet, and internationalization of the market, result in the need for an increase in the storage time of the food products. This requires the development of new packages to ensure adequate shelf life of the product. Traditional concepts are not enough to meet consumers’ demands. The traditional concept in which a minimal interaction between the package and the product has

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been changed, and a new concept based on a passive interaction between the product (food) and the package has been developed, giving rise to the appearance of new active and intelligent packaging. Innovative packaging with enhanced functions is constantly sought in response to consumer demands for minimally processed foods with fewer preservatives, increased regulatory requirements, market globalization, concern for food safety, and the recent threat of food bioterrorism (Yam et al., 2005). To attend these demands, rethinking and shifting the original concept of food packaging is required as well as the introduction of new functions. Recently, a host of new packaging materials have been developed to provide “active” protection for the product, and a new function was incorporated in this system—to interact. The food package became able to feel and to communicate some of the changes occurring in food products during its shelf life. A new function was attributed to the package—to feel. These new packaging systems have been the object of intense research activity. Active packaging and intelligent packaging will become key elements in food processing, allowing for improvement in the longevity and nutrient value of food products. The main aspects associated with the application of these packaging systems will now be considered in detail. Active packaging has been defined as a system in which the product, the package, and the environment interact in a positive way to extend shelf life or to achieve some characteristics that cannot be obtained otherwise (Miltz et al., 1995). Packaging may be termed active when it performs some desired role, other than providing an inert barrier to external conditions. The word desired is important in this definition because it clearly differentiates between unwanted interactions and desired effects (Rooney, 1995). Vermeiren et al. (1999) defined active packaging as a packaging system that actively changes the condition of the package to extend shelf life or improve food safety or sensory properties, while maintaining the quality of the food. All active packaging technologies involve some physical, chemical, or biological action for altering the interactions between the package and the product and the package headspace to achieve the desired outcome (Brody et al., 2001). The goal of active packaging in conjunction with other food processing and packaging techniques is to enhance preservation of contained food and beverage products. Intelligent packaging has been defined as a system that monitors the condition of packaged foods to give information about the quality of the packaged food during transportation and storage (Ahvenainen, 2003). Intelligent packaging (also more loosely described as smart packaging) is packaging that in some way senses some of the properties of the food it encloses or the environment in which it is kept and is able to inform the manufacturer, retailer, and consumer of the state of these properties. Although distinctly different from the concept of active packaging, intelligent packaging can be used to check the effectiveness and integrity of the active packaging systems (Hutton, 2003). Yam et al. (2005) proposed a more precise and useful definition of intelligent packaging. This definition is consistent with the historical development of packaging. According to them, a package is “intelligent” if it has the ability to track the


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product, sense the environment inside or outside the package, and communicate with the consumer. For example, an intelligent package is one that can monitor the quality/ safety condition of a food product and provide early warning to the consumer or food manufacturer. Expressions such as responsive packaging, diagnostic packaging, and clever packaging were not included here, as that would further complicate the already confusing terminology. Intelligent packaging is a system that involves not only the package, but also the food product, the external environment, and other aspects. This model points out that the uniqueness of intelligent packaging lies in its ability to communicate: As the package and the food move constantly together throughout the supply chain cycle, the package is the food’s companion and is in the best position to communicate the conditions of the food, active packaging being a provider of enhanced protection. Thus in the total packaging system, intelligent packaging is the component responsible for sensing the environment and processing information, and active packaging is the component responsible for taking an action (for example, release of an antimicrobial) to protect the food product. The terms intelligent packaging and active packaging are not mutually exclusive, and some packaging systems may be classified either as intelligent or active or both, but this does not diminish the usefulness of these expressions. Intelligent packaging, active packaging, and traditional packaging functions work together to provide a total packaging solution.

8.1.3 Concepts and Application of Active Packaging The most important concepts associated with the use of active packaging are antimicrobial films, edible coatings, absorbers of oxygen, ethylene, flavors and odors, humidity regulators, releasers of carbon dioxide, antimicrobial agents, antioxidants, and flavors. Their applications are numerous, and their use is in evident growth. 8.1.3.1 Antimicrobial Packaging Within the concept of active packaging, antimicrobial packaging is defined as the package that incorporates active antimicrobial agents, replacing its direct addition to the food. The concept of antimicrobial packaging involves the gradual release of the antimicrobial agent from the packaging into the food, inhibiting or slowing the growth of microorganisms in the food surface (Appendini and Hotchkiss, 2002). Because microbial contamination of most foods occurs primarily at the surface, due to postprocessing handling, attempts have been made to improve safety and to delay spoilage by use of antimicrobial sprays or by dipping the substrate in the antimicrobial coating. However, direct surface application of antibacterial substances has limited benefits, as they are neutralized on contact or diffuse rapidly from the surface into the food matrix. On the other hand, incorporation of bactericidal or bacteriostatic agents into food formulations may result in a partial inactivation of existing active substances and is therefore expected to have a limited effect on the surface microflora (Quintavalla and Vicini, 2002). Therefore the use of packaging films containing antimicrobial agents can be more efficient as a slow migration of the agents from the packaging material into the surface of the product will occur, helping to maintain controlled concentrations where and when they are needed. If an antimicrobial can be released from the

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package during an extended period of time, its activity can also be extended into the transport and storage phase of the food chain. Active compound diffusion between the packaging material and the food and partitioning at the interface are the main migration phenomena involved in this system. Initially incorporated antimicrobial agents migrate into the food through diffusion and partitioning (Han, 2000). There are several ways to prepare antimicrobial packaging: addition of sachets containing volatile antimicrobials within packages, incorporation of volatile and nonvolatile antimicrobial agents directly in the polymer, adsorption of antimicrobial agents in the surface of the polymer, immobilization of antimicrobials in the polymer by ionic or covalent links, and use of polymers with antimicrobial activity (Appendini and Hotchkiss, 2002). Antimicrobial substances incorporated into packaging materials can control contamination by reducing the microbial growth rate and the maximum microbial concentration, extending the lag-phase of the target microorganism or direct contact microbial inactivation (Quintavalla and Vicini, 2002). Several compounds have been proposed and tested for antimicrobial activity in food packaging, including organic acids such as sorbate, propionate, and benzoate or their respective acid anhydrides; bacteriocins (e.g., nisin, natamicin, and pediocin); enzymes such as lysozyme; and natural compounds as chitosan and essential oils. Table 8.1 pre sents some examples of antimicrobials used in food packages and their application, while Table 8.2 lists examples of antimicrobial agents allowed for food use. 8.1.3.2 Edible Packages Edible packages are presented in two ways: as a film and as a coating. Frequently these two terms have been used without any distinction between them. However, a film is a fine skin formed separately from the food and later applied, while a coating is a suspension or emulsion applied directly on the surface of the food, where film formation occurs (Gennadios and Weller, 1990). The fine coating acts as a barrier Table 8.1 Examples of Antimicrobials Used in Food Packages Antimicrobial Compound Pediocin Acetic acid and chitosan Basil (linalool and methylchavicol) Sorbic acid Sorbic acid Sorbic acid anhydride

Substrate

Packaging Materiala

Reference

Ham Garlic

Cellulose Agar-agar

Santiago-Silva et al. (2009) Geraldine et al. (2008)

Cheddar cheese

LDPE

Suppakul et al. (2008)

Pastry dough Culture media Culture media

Cellulose polymer WPI PE

Silveira et al. (2007) Cagri et al. (2001) Weng and Chen (1997)

LDPE, low-density polyethylene; WPI, whey protein isolate; PE, polyethylene. Source: Modified from Yamada, Boletim CTC Tecno Carnes, 2004 and Han, Innovations in Food Packaging, 2005. a


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Table 8.2 Examples of Antimicrobial Agents Class Organic acid Acid salts Acid anhydrides Benzoic acids Alcohol Bacteriocins Fatty acids Fatty acid esters Chelating agent Enzyme Metal Antioxidant Antibiotic Fungicide Sanitizing Polysaccharide Phenolics Volatile oils of plants Plant/spice extracts Probiotics

Examples Acetic, benzoic, lactic, citric, malic, propionic, sorbic, succinic, tartaric acids Potassium sorbate, sodium benzoate Sorbic anhydride, benzoic anhydride Propyl paraben, methyl paraben, ethyl paraben Ethanol Nisin, pediocin, subtilin, lacticin Lauric and palmitoleic acid Glycerol mono laureate EDTA, citrate, lactoferrin Lyzozyme, glucose oxidase, lactoperoxidase Silver, copper, zirconium Butyl hidroxyanysole, butyl hidroxytoluene, terc-butyl hidroquinone, iron salts Natamcyin Benomyl, imazalyl, sulfur dioxide Ozone, chlorine dioxide, carbon monoxide, carbon dioxide Chitosan Catechin, cresol, hydroquinone Allyl isotiocyanate, cinnamaldehyde, eugenol, linalaol, terpineol, thymol, carvacrol, pinene Grape seed extracts, grapefruit seed extract, brassica erucic acid oil, rosemary oil, oregano oil Lactic acid bacteria

Source: Modified from Yamada, E. Desenvolvimento de sistema de embalagem antimicrobiana. In Boletim CTC TecnoCarnes, Vol XIV. Ital, Campinas–SP, 2004; and Han, J.H. Antimicrobial packaging systems. In Han, J. (Ed). Innovations in Food Packaging. Amsterdam: Elsevier Science. 2005.

to external elements such as humidity, oil, or organic compounds, protecting the product and extending its shelf life (Guilbert et al., 1996; Krochta and De MulderJohnston, 1997). The expression edible implies that the materials used in the elaboration of the packaging are safe for human consumption. In other words, they are considered GRAS (Generally Recognized as Safe) and processed according to the Good Manufacturing Practices (GMPs) for foods. Also, the polymer, typically a biopolymer, used in the preparation of the packaging must have a long chain needed to give a certain insolubility and stability to the polymeric matrix in an aqueous environment. The increasing demand for fresh foods that present high quality, long shelf life, and are ready for consumption has motivated the development of minimally processed fruits and vegetables covered with films and edible coatings. These films and

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coatings guarantee the fresh appearance, firmness, and shine, thus adding value to the product (Lin and Zhao, 2007). The main functions of the edible coatings are to inhibit the humidity, oxygen, carbon dioxide, aromas, lipids, and other solutes’ migration; to carry food additives and antimicrobial agents; and to improve the mechanical integrity and the handling characteristics of foods (Krochta and De Mulder-Johnston, 1997). The effectiveness of edible coatings depends on coating composition, with proteins, polysaccharides, and lipids being extensively used in its preparation (Perez-Gago et al., 2006). As these compounds can be recycled and completely biodegraded in a short time, the use of edible coating also contributes to the decrease of environmental pollution (Guilbert et al., 1996). Generally, films formed by proteins and polysaccharides have good mechanical properties but form a poor barrier to humidity due to their hydrophilic nature. On the contrary, lipids give rise to coatings that are a good moisture barrier but are less effective in their mechanical properties and present undesirable sensory attributes (Guilbert, 1986). Due to their different properties, advantages can be obtained in the preparation of coatings by the simultaneous utilization of proteins, polysaccharides, and lipids (Cerqueira et al., 2009; Fabra et al., 2008). 8.1.3.3 Oxygen Absorber The presence of high levels of oxygen in food packages may facilitate microbial growth, the generation of off-flavors and off-odors, color changes, and nutritional losses, thereby causing significant reduction in the shelf life of foods. Therefore it is important to control the oxygen level in food packages to limit the rate of such deteriorative and spoilage reactions in foods. Oxygen-absorbing systems provide an alternative to vacuum and gas flushing technologies as a means of improving product quality and shelf life. Residual oxygen inside the package can react biochemically with the contained food and cause long-term adverse oxidative effects that increase as the temperature rises. Expressions such as antioxidants, interceptors, absorbers, and scavengers have been used to describe the materials employed in the process of removing oxygen or preventing it from entering the in-package environment of food products subject to undesirable oxidative reactions. Antioxidants are compounds that react with lipid or peroxide radicals or, in the presence of light, with singlet oxygen and that are themselves oxidized to generate what are generally innocuous nontoxic compounds. For many years, antioxidants were incorporated into fatty foods to preferentially react with intermediate oxidation products in the surrounding air or dissolved in the food product. Nowadays, BHA (butylated hydroxyanisole) and BHT (butylated hydroxytoluene) are incorporated into polyolefin films not only to retard oxidation of plastic materials, but also to act as agents that diffuse to the surface, sublimate into the package environment, and are incorporated into dry food products (Brody et al., 2001). The expression oxygen scavenger has been applied to materials incorporated into packaging structures that chemically combine with and remove oxygen from the inner package environment. Oxygen scavengers are able to remove oxygen from the food product by permeating through the polymer structure by diffusion as a result


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of the existing pressure gradients. Most of the commercially available oxygen scavengers contain iron as the oxygen absorber and are marketed in the form of a sachet. Water is required for their action, according to the following equation applicable at ambient or chilled temperatures—1 g of iron is capable of absorbing 300 cc of oxygen, requiring 0.5 g water for its action (Miltz and Perry, 2005):

4 Fe + 3O2 + 6 H2O → 4 Fe(OH)3 → 2 Fe203.3H20

4 Fe (OH)2 + O2 + 2 H2O → 4 Fe(OH)3 → 2 Fe203.3H20

Some scavengers contain water in their carrier materials (self-activated types) that start scavenging upon exposure to oxygen. Others use moisture from the product and are therefore suitable for moist products only when other scavenger components such as organic compounds need to be activated by ultraviolet light. One of the first applications was the removal of oxygen from canned milk powder involving the use of oxidizable metal powders; later, the use of a palladium catalyst attached to the inside of the can was introduced. New developments in the polyester industry have resulted in a range of approaches to achieve oxygen scavenging by polymeric and low-molecular-weight compounds within polyester bottle walls. One of the approaches was the oxidation of MXD-6 nylon by the permeating oxygen in the presence of a transition metal catalyst (Cochran et al., 1991). These developments include multilayer systems as well as blends with polyethylene terephthalate (PET) in monolayer bottles. 8.1.3.4 Ethylene Absorber Ethylene is a hormone that produces different physiological effects on fresh fruit and vegetables such as acceleration of the respiration process leading to ripeness, senescence, and softening of several fruits. Ethylene accumulation causes yellowing of green vegetables and is responsible for a great number of postharvest detrimental changes in fresh fruit and vegetables, reducing their quality and shelf life. Taking these aspects into consideration, it is clear that ethylene should be avoided inside the package. The double bond of ethylene makes it a very reactive compound. Ethylene can be removed inside packaging through chemical (absorption) or physical reactions (adsorption). Clay materials such as cristobalite and zeolite have been reported as having ethylene absorbing capacity. Also, activated charcoal impregnated with KBrO3 and H2SO4 has been used to eliminate ethylene. Another ethylene scavenger is based on activated carbon and the subsequent breakdown by a metal catalyst (Ahvenainen, 2000). The most effective ethylene absorber is KMnO4 which is also absorbed in an inert carrier, such as silica gel, celite, perlite, or glass at 4% to 6% of KMnO4. The process is described as follows:

3CH2CH2 + 2 KMnO4 + H2O → 2MnO2 + 3CH3CHO + 2KOH

3 CH3CHO + 2 K MnO4 + H2O → 3 CH3COOH + 2 MnO2 + 2KOH

3 CH3COOH + 8 KMnO4 → 6 CO2 + 8 Mn02 + 8 KOH + 2 H2O

3 CH2CH2 + 12 KMnO4 → 12 MnO2 + 12 KOH + 6 CO2

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in which ethylene is oxidized to acetaldehyde (CH3CHO) which is also oxidized to acetic acid that is further oxidized to carbon dioxide and water. KMnO4 absorbers change color, becoming brown when MnO4 is reduced to MnO2. KMnO4 are used in a sachet due to its toxicity. The incorporation of ethylene absorbers in the polymer matrix has also been done with the inclusion of finely dispersed minerals such as zeolites or sepiolites. However, its inclusion produces opaque films and also modifies films O2 permeability as oxygen will diffuse more rapidly than through pure PE. 8.1.3.5 Humidity Absorber In food packaging, moisture control is usually directed to dry foods or to fresh fruits and vegetables. The susceptibility of foods to moisture damage requires their packaging with a high humidity barrier material. However, a certain amount of moisture is trapped during the packaging or developed along the distribution chain. This excessive water content causes softening of dry crispy products such as biscuits and crackers, and caking of milk powder and instant coffee. However, excessive water evaporation through the packaging material may result in desiccation of the packed foodstuff, or it may favor lipid oxidation. Desiccants are successfully being used for a wide range of foods such as cheeses, meats, chips, nuts, popcorn, candies, gums, and spices (Anon, 1995). In those cases, silica gel has been the most effective moisture absorbent included in a sachet inside the package, with a capacity to absorb water up to 35% of its own weight. Molecular sieves such as zeolites are able to absorb up to 25% of their weight and present high affinity to water. Technology is oriented to include a moisture absorber inside the polymer matrix, such as the desiccant blended in a polymer melt like a filler or additive. Better results have been obtained by using desiccant in a sachet.

8.1.4 Concepts and Application of Intelligent Packaging Intelligent packaging is defined as a packaging system that monitors the condition of packaged food and informs on food quality during transport and storage. Immobilized enzymes and antibodies are frequently used components of intelligent packaging systems that act as time–temperature integrators, spoilage indicators, and indicators of chemicals or other types of contamination. The following are the main applications of intelligent packaging:

1. Enhance food safety and biosecurity. Intelligent packaging is a useful tool for tracking products and monitoring their conditions, providing real-time data. and enabling rapid response and timely decision making. Intelligent packaging can be integrated into existing traceability systems to create more effective communication links. Bar codes and radio-frequency identification (RFID) tags can enable electronic record keeping and information sharing, especially when interfaced with external instruments capable of rapidly measuring quality attributes and monitoring food safety. 2. Enhance food quality and convenience. TTI (time–temperature indicator), small self-adhesive labels attached to shipping containers or individual


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consumer packages that provide visual indicators of temperature history during distribution and storage, are particularly useful for warning about temperature abuse. Of great interest is the use of biosensors, compact analytical devices that detect, record, and transmit information about pollutants and detection and identification of pathogens present in packaged food.

8.2 Development of Antimicrobial Packaging Antimicrobial films can be obtained through the addition of antimicrobial agents by incorporation or immobilization. In the case of incorporation, there is a release of the antimicrobial agent into the food, whereas in immobilization, the compound acts only at the surface. Incorporated antimicrobials increase the lag phase and reduce the rate of microbial growth. They prolong the food shelf life and confer greater security to the products, being beneficial to the food industry and consumers (Han, 2003). Since microbial growth in foods occurs mainly at the surface of solid or semisolid packaged foods, research has been carried out with the objective of incorporating antimicrobial substances into the packaging to maintain quality and prolong the shelf life of these foods (Appendini and Hotchkiss, 2002; Weng and Hotchkiss, 1993). The packaging system can inhibit the microbial growth in nonsterilized or pasteurized foods, preventing postprocessing contamination. According to Han (2003), the development of active packaging incorporated with antimicrobials must take into consideration the following points: 1. The type of antimicrobial agent to incorporate 2. The chemical nature of the antimicrobial agent 3. The physicochemical characteristics of the food 4. The physiology of the target microorganism and the microbiota of food 5. The kinetics of migration of the antimicrobial agent to the food 6. The environment and storage temperature 7. The film or container manufacture process 8. Toxicity and regulatory aspects 9. The sensorial properties of the antimicrobial agent 10. The adequacy to the process of the antimicrobial packaging It is important to control the migration rate of antimicrobials from the package to the product. The released antimicrobials must be able to control microbial growth and to maintain the antimicrobial level above the minimum inhibitory concentration (Brody et al., 2001). In some cases, it is necessary that the package have an activation mechanism to initiate its activity only when in contact with the food. This avoids loss of packages efficiency during transport and storage before its use. An example of the activation mechanism is when the humid food comes in contact with the active packaging and the antimicrobial agent is solubilized, allowing its migration to the food-package contact surface.

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8.2.1 Commonly Used Antimicrobial Substances Most of the antimicrobials incorporated into packaging materials are food grade or GRAS additives. They can be substances naturally found in foods, antimicrobial polymers, biotechnology products, or other products authorized for use. Table 8.1 presents some substances that can be used in antimicrobial packages. Antimicrobial agents for use as additives in packages that come in contact with foods have to follow the recommendations and approval of regulatory agents. Various substances received authorization for use in active packages by the United States Food and Drug Administration (FDA) (Suppakul et al., 2003). In the European community, up to 2004, Directive 89/109/EEC, which deals with food packaging legislation, established the limit of overall migration of substances from packages (60 mg.kg–1) and was impeditive for active packaging. In the European Union between 1999 and 2001, the “Actipak” study on the use of active packaging and needs of modification of the legislation was carried out, resulting in Regulation CE 1935/2004 of the European Parliament (2004). This regulation allows the migration of substances from packages and regulates the registration of these substances and specific labeling. All the new active and intelligent packaging systems need to be evaluated by the European Food Safety Authority (EFSA) (Jong et al., 2005). Taking into consideration the fact that foods have different chemical characteristics, different foods offer distinct environmental conditions for microorganisms and antimicrobial agents (Brody et al., 2001). For example, the pH of the food alters the ionization of the majority of active chemical substances, affecting the antimicrobial activity and the microbial growth rate. The water activity affects the antimicrobial activity and the chemical stability of incorporated substances, among other characteristics. Benzoic anhydride in low-density polyethylene (LDPE) (Weng and Hotchkiss, 1993), chitosan in polyvinylacetate (Cho et al., 2000), grapefruit seed extract in LDPE (Lee et al., 1998), and propolis extract in LDPE (Hong et al., 1998) have been evaluated as antimicrobial packages. Silver zeolites (Ag-zeolites) have been used as antimicrobial agents in plastic materials. They act against a variety of bacteria, yeasts, and molds by altering their metabolisms but show no effectiveness against thermoresistant bacterial spores. Silver ion is adsorbed by the microbial cell surface and, by active transport, incorporated inside the cell. As these ions react with proteins, after their incorporation by the cell they can react with proteases, inhibiting the cell metabolic process, resulting in the inhibition (Brody et al., 2001). Antimicrobial substances of natural occurrence can also be applied to packaging materials to inhibit microbial growth. The use of natural antimicrobials, like plant spice extracts, is a promising alternative because of the appeal of a natural product, consumer preference, and less conflict with legislation. Cinnamon, clove, thyme, rosemary, oregano, garlic, and mustard are among the antimicrobial substances originated from spice extracts as bacteriocins, mainly nisin, pediocins, natamycin, enterocins are obtained from microorganisms. The bacteriocins are produced by different lactic acid bacterial strains, with action normally targeted to pathogen control. Nisin is a hydrosoluble protein produced by lactic bacteria, with a specific action against gram-positive bacteria, including sporulated ones, but inefficient against gram-negative bacteria and molds. Nisin is considered as being of reduced efficiency in the conservation of meats


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in the presence of psychotrophic gram-negative species, like Pseudomonas spp. Studies demonstrated the efficiency of nisin in combination with a chelating agent on Listeria monocytogenes control in some types of processed meats. Nisin has also been proven to be active against lactic bacteria and total aerobic microorganisms in a cooked ham and sliced cheese conditioned in a modified atmosphere (Brody et al., 2001). Conte et al. (2007) evaluated the effectiveness of different antimicrobial packaging systems on the microbial quality of mozzarella cheese. Lemon extract was used as the active agent in combination with brine and with a gel made of sodium alginate. Results showed an increase in the shelf life of all active packaged mozzarella cheeses, confirming that the investigated substance may exert an inhibitory effect on the microorganisms responsible for spoilage phenomena without affecting the functional microbiota of the product. Han and Floros (1998) determined the diffusivity of potassium sorbate in mozzarella cheeses, showing that this kind of cheese would maintain the surface concentration of potassium sorbate above the critical fungistatic level two times longer than American processed cheese. Also Limjaroen et al. (2005) used polyvinylidene chloride films containing sorbic acid in surface-inoculated beef bologna and cheddar cheese with L. monocytogenes. The results have shown that films containing sorbic acid inhibit the common spoilage organisms in both products, Buonocore (2005) showed the efficiency of monolayer and multilayer films of polyvinyl alcohol (PVOH), a cross-linking agent (glyoxal), and lysozyme against Micrococcus lysodeikticus. Chlorine dioxide (ClO2), known in food industries as a wide-spectrum antimicrobial, not forming trihalomethanes or dioxines, is used in antimicrobial films and is commercially available as MicroatmosphèreTM. This film is capable of a controlled and constant release of chlorine dioxide. The generation of the antimicrobial in the interior of the packaging is activated by the conditions of the product, normally humidity (Brody et al., 2001) and the amount of generated ClO2, and its duration can be modulated to eliminate diverse types of microorganisms, including yeast spores.

8.2.2 Antimicrobial Incorporation into Plastic Polymers Active packaging systems with antimicrobial action are based on the incorporation of antimicrobial substances in the polymer. They act on the food under one of the two forms: The antimicrobial is immobilized and acts only at the contact surface, or the active material is placed in contact with the humidity of the food, leading to the release of the antimicrobial. In both cases, the objective of the system is to prolong the shelf life of the food, inhibiting the microbial growth and preserving its sensorial properties (Buonocore, 2005). The incorporation of an antimicrobial in packaging material needs an adequate evaluation of the rate of migration for the surface of the food. Also, it is necessary to evaluate the growth kinetics of the target microorganism. When the migration rate of the antimicrobial is bigger than the growth rate of the microorganism, the antimicrobial concentration will be inferior to the minimum inhibitory concentration (MIC) before the expected end of the storage time, and microbial growth will occur after the reduction of the antimicrobial agent (Han, 2003). On the

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other hand, when the migration rate is very slow, the microorganism will grow before the action of the antimicrobial compound. The antimicrobial concentration in the surface of the food depends on the migration rate and is highly dependent on the solubility of the active agent in the food. The profile of antimicrobial agent migration must be known so that its concentration is always above the MIC, and a constant antimicrobial action is guaranteed during the shelf life of the food (Han, 2003). Reduction on the growth of filamentous molds and yeasts in butter was obtained by employing an active film incorporating 7% of sorbic acid. In the experiment, the initial microbial count in the product was 3 × 106 UFC.g–1, and it was reduced to 9 × 105 UFC.g–1 and 8 × 104 UFC.g–1, after 10 and 20 days of storage, respectively. The majority of the works developed in antimicrobial films consider a doublelayer structure in which the internal layer, in contact with the product, displays antimicrobial action, and the external layer has a structural and barrier action. Buonocore (2005) comments on the importance of extending studies with multilayer films. In these multilayer films, each layer has a specific function: • The first layer, external, prevents the loss of the active substance for the environment and acts as a barrier to the oxygen, humidity, and gases. • The second layer, intermediate, contains the antimicrobial substance and allows for its fast migration. • The third layer, internal and in contact with the food, has the function of controlling the migration of the antimicrobial agent to the surface of the food. Relevant factors for its preparation are the diffusivity of the material and the layer thickness. Wan et al. (1997) successfully incorporated nisin into a matrix of calcium alginate and ground into microparticles smaller than 150 mm. The incorporation efficiency was 87% to 93%, and the nisin in the alginate-incorporated form was 100% active against an indicator culture of Lactobacillus curvatus both in De Man, Rogosa, and Sharpe (MRS) broth and reconstituted skim milk. Oliveira et al. (2007) developed and evaluated the antimicrobial efficiency of natamycin-incorporated film in the production process of Gorgonzola cheese. Films with different concentrations of natamycin were produced and tested in Gorgonzola cheeses to evaluate their efficiency against Penicillium roqueforti on the cheese surface. The films with natamycin incorporation presented satisfactory results as fungal inhibitors, with the amount of natamycin being released to the cheese below the value allowed by legislation.

8.2.3 Antimicrobial Immobilization in Polymers In some cases, to be active, polymers do not need to allow the migration of the bioactive compounds to the food. Polymers may have, on their own, an antimicrobial activity, as is the case of chitosan, or the bioactive molecule may be linked to the polymer making impossible its release. The immobilization of the antimicrobial substance in the polymers can be done by covalent linkage, adequate for the cases


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where the legislation does not allow migration of antimicrobials for the food, and the inhibition must occur in the contact surface of the packaging/product. Chitosan is a polymer with antimicrobial properties, derived from chitin (polyN-acetyl-glucosamine) existing in crustaceans. The mechanism of its antimicrobial action, still being studied, occurs by the rupture of the external membrane of bacteria (Steven and Hotchkiss, 2003). There are indications that the antimicrobial activity is due to the adsorption of the bacteria (Appendini and Hotchkiss, 2002), because once the polymer molecule is big, it does not have to penetrate the cell for the antimicrobial action to occur. For several polymers, an initial step for the creation of reactive groups that will act as linking sites for the immobilization of antimicrobials may be required (Steven and Hotchkiss, 2003). In these situations, the active agent is not available to migrate from the polymer to the surface of the food, and its applications are limited, as its main use is in the surface of equipment for food contact. Glutaraldehyde cross-linking between proteins or other biological molecules and the polymers is one of the most used methods to link bioactive agents to polymers. Other methods include the use of carboimides and active esters of succinimidil succinate (Steven and Hotchkiss, 2003). Nariniginase immobilization is one of the cases that make use of glutaraldehyde cross-linking (Soares, 1998). The immobilization of the bacteriocins nisin and lacticin 3147 in packaging materials was studied by Scannella et al. (2000). The stability of both cellulose-based bioactive inserts and antimicrobial polyethylene/polyamide pouches was examined over time, against the indicator strain Lactococcus lactis subsp. lactis HP, and in addition to Listeria innocua DPC 1770 and Staphylococcus aureus MMPR3. When applied to food systems, the antimicrobial packaging reduced the population of lactic acid bacteria in sliced cheese and ham stored in modified atmosphere packaging at refrigeration temperatures, thus extending the shelf life of those products. Nisinadsorbed bioactive inserts reduced the levels of Listeria innocua by ≥2 log units in both products, and Staphylococcus aureus by ≈1.5 log units in cheese. Millette et al. (2007) successfully immobilized nisin into palmitoylated alginate-based films and activated alginate beads. They were tested against Staphylococcus aureus inoculated in ground and muscle slices of beef, showing a decrease in the content of S. aureus over 1.8 log CFU/g after 14 days.

8.2.4 Surface Modification In antimicrobial packaging, the development of materials through physical action on the surface of films is of great interest. Paik et al. (1998) studied the use of ultraviolet (UV) irradiation of polyamide (nylon 6,6) films. This provokes the transformation of the amide group of the film into amines, these groups acting as surface sites with antimicrobial action. Additionally, they observed that UV treatment provoked an alteration in the surface topography, leading to an increase in the contact area between packaging and product. However, it is not proved that this increase leads to greater antimicrobial activity. The film was tested against Staphylococcus aureus, getting three decimal reductions after 6 hours, for an initial concentration of 8 × 103 UFC.mL –1. The film was less effective against Enterococcus faecalis

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and Pseudomonas fluorescens. The film was radiated with photons of UV light at 193 nm, at 200 kW.cm–2 for 16 nanoseconds, with a total dosage of 2.7 J.cm–2. Cho et al. (2000) synthesized a polymer containing a lateral chain of chitosan oligosaccharide introduced in polyvinylacetate (PVA) by cross-linking with the bifunctional compound N-metylacrylamide (NMA). S. aureus growth was almost completely suppressed in this packaging.

8.2.5 Factors to Consider in the Production of Antimicrobial Films The maintenance of the antimicrobial action, the homogeneity in the distribution of the active agent in the film, the chemical compatibility, the integrity, and the physicomechanical and optical properties of the films are important properties to be considered. Morphologic studies of polymers are important to predict the loss of integrity caused by the addition of the antimicrobial agent. According to Han (2003), antimicrobials in the form of small particles can be mixed to the polymeric material and located in amorphous regions of the structure. The use of great amounts of antimicrobials leads to a saturation of the amorphous spaces, and the added substance starts to interfere with the polymer–polymer interactions in the crystalline region, thus reducing the integrity of the packing material. Beyond the conditions of storage and distribution of the packing material, the antimicrobial activity of an active film can be modified as a result of the conditions of its manufacture as casting, conversion, or extrusion, The incorporation of antimicrobial additives in plastic films during the extrusion process depends on the thermal stability of the additives and on the pressure and the shear force that they are submitted to during extrusion (Brody et al., 2001). Moreover, the polarity and the molecular weight of the additive are parameters that must be considered in the interaction with polymeric matrix and in the release of the additive to the food product. Other factors to be considered are the effect of operations such as conversion, lamination, impression, adhesives, solvents, drying, and storage. For the production of films, the antimicrobial agents and packaging materials must be chemically compatible. The use of water and ethanol as solvents for homogenization is recommended. The use of other solvents must consider the possibility of migration of residues for the food. The physical properties (tensile strength, elongation, hot sealing) as well as other mechanical and optical properties of the materials used in the production of the antimicrobial packaging must remain unchanged. Generally, when small antimicrobial chemical compounds are mixed to macromolecular packaging materials, the mechanical properties are not significantly modified. However, optical properties like color, transparency, and brightness may be more affected.

8.3 Nanotechnology—Applications in Food Packaging Nanotechnology is recognized as a major area of research and development. Nanotechnology seeks to create new materials and develop new products and processes by handling atoms and molecules in the 1 to 100 nm scale. At this scale,


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the material properties can be very different from the conventional, because the nanomaterials may represent a considerable increase of its superficial area. These factors can modify or enhance properties like mechanical strength, reactivity, and electric characteristics (The Royal Society and The Royal Academy of Engineering, 2004). Nanotechnology can be used to produce packaging with a larger mechanical and thermal resistance or sensors that can be placed into the package can alert the consumer about the food safety of the product (Sorrentino et al., 2007). In vitro studies show that there are nanocompounds with different adsorption capacity. Zeolite, bentonite, kaolin, and sodium and calcium hydrated aluminosilicates are some of the materials that are used at the nanoscale (Huff et al., 1992; Maryamma et al., 1991). Montmorillonite (MMT), known as bentonite as this is the name of the rock it is extracted from, is a type of silicate that belongs to the 2:1 phospho-ossilicates structural group. This composite has the capacity to adsorb organic substances in its external surface as well as in the interlaminar spaces (Rodríguez et al., 1989). MMT is constituted by structural layers having two tetrahedral silica leaves, with an octahedral central leaf of aluminum, linked by oxygen atoms (Qiu et al., 2006) MMT is widely used in the production of nanocomposites, hybrid materials in which at least one of the components has nanometrics dimensions. They are constituted by organic and inorganic materials, being the inorganic phase dispersed in the polymeric matrix. Materials with better mechanical resistance, thermal stability, and optic, magnetic, or electrical properties can be obtained by the incorporation of an inorganic load into the polymers (Ray and Okamoto, 2003). These modifications in the polymer properties are related to the fact that nanoparticles have small dimensions and consequently are highly superficial. This improves the dispersion in the polymeric matrix as a result of the specific chemical interactions between the nanoparticles and the polymer and the subsequent change in the molecular dynamic of the polymer. Three types of structures are obtained when MMT is spread in the polymeric matrix: separate phase structure—when the polymeric chains are not inserted between the clay layers, producing a structure with properties similar to a conventional composite; inserted structure—when the polymeric chains are placed between the clay layers, forming a better organized multilayer structure that has improved properties as compared to the conventional composite; and exfoliated structure—when the clay is completely and evenly dispersed in the polymeric matrix, maximizing the polymeric–clay interactions and improving the mechanical properties. Composites are prepared by a simple mixing procedure, with MMT being the most used inorganic in the preparation of polymeric nanocomposites. In addition to being very well known, MMT is from a natural origin, has good lamination capacity, has high resistance to solvents, and has high thermal stability which is required for the polymerization and extrusion processes. Polymeric matrices like polymethyl methacrylate, polyamide, polyethylene, polypropylene, polystyrene, polyethylene terephthalate, polyvinyl chloride, copoly(acrylonitrile-butadiene-styrene) are used with clay in the preparation of nanocomposites. These nanocomposites can be applied in the automobile, packaging,

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medical and textile materials areas as well as drug-release controlled systems (Ray and Okamoto, 2003). The polymer/MMT nanocomposite is extraordinarily interesting because low levels of inorganic clay can introduce significant improvements in some polymer properties, such as inflammability reduction and enhancement of the mechanical and barrier properties as well as improved thermal stability (Qiu et al., 2006; Zhang and Wilkie, 2006). Kampeerapappun et al. (2007) produced cassava starch films added with chitosan, glycerol and MMT, by the casting method. These researchers observed a reduction in the water vapor permeability and an increase in the mechanical parameters like tensile strain and Young’s module when materials were incorporated with 10% of MMT. The use of nanotechnological solutions can reduce the amounts of antimicrobials that are usually used. The incorporation of nanocomposites with antimicrobial, antifungal, and bacteriostatic properties was studied by Rudra et al. (2006) and Cioffi et al. (2005). Calcium carbonate nanoparticles were used in isotactic polypropylene films, showing good dispersion in the polymeric matrix as well as an improvement of the mechanical parameters, together with a reduction of gas permeability. The shelflife analysis showed that these materials are able to preserve apple slices, limiting oxidation and microbial growth (Avella et al., 2006).

8.4 Potential Use of Active and Intelligent Packaging in Milk and Milk Products Active and intelligent packaging are emerging areas of food technology that can provide better food preservation and extra convenience for the benefit of consumers. The use of these two packaging technologies, together or separately, will improve product quality, enhance the safety and security of foods, and consequently decrease the number of retailer and consumer complaints. Edible coatings can be used in cheese to prevent moisture loss and control the exchange of gases, such as carbon dioxide and oxygen, with the outside environment. Kampf and Nussinovitch (2000) used hydrocolloid coatings based on k-carrageenan, alginate, and gellan to coat cheeses. All coatings reduced weight loss during 46 days of storage in the tested semihard cheese, contributing to a better color and gloss. Cerqueira et al. (2009) showed a decrease of the respiration rates (O2 and CO2) of a semihard cheese when coated with a galactomannan coating, presenting the uncoated cheese with extensive mold growth at the surface when compared with the coated cheese. The use of antimicrobial compounds in cheese products was also explored by several authors. Duan et al. (2007) showed that chitosan-lysozime films and coatings can be applied in mozzarella cheese packaging to control the postprocessing microbial contamination, improving the microbial safety of cheese products. Conte et al. (2007) applied lemon extract in combination with brine and with a gel solution made of sodium alginate also in mozzarella cheese. Results show an increase in the shelf life of all active packaged mozzarella cheeses. Limjaroen et al. (2005) used polyvinylidene chloride films containing sorbic acid in surface-inoculated cheddar


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cheese with L. monocytogenes. The results have shown that films containing sorbic acid inhibit the common spoilage organisms in both products. Films incorporated with antimicrobial substances nisin (NI), natamicine (NA), and the mixture of both were evaluated by Pires et al. (2008) in sliced mozzarella cheese. The films incorporated with NA and NI + NA were capable of delaying the growth of filamentous fungi up to the ninth day of storage at 12°C ± 2°C, but Staphylococcus sp. growth was not affected during the storage time. The lag phase for psychotrophic microorganisms was extended to 6 days. Cellulosic films incorporated with 50% of a commercial product containing 2.5% of nisin (NI), 8% of a commercial blend having 8% of natamicine (NA), and the mixture of both blends were developed by Pires et al. (2008). The antimicrobial efficiency of the films was evaluated against Staphylococcus aureus, Listeria monocytogenes, Penicilium sp., and Geotrichum sp., by the agar diffusion method, in the proper culture medium. The films incorporated with NI and the mixture of NI and NA were effective against S. aureus and L. monocytogenes, although no diffusion of the antimicrobial from the film to the culture medium was observed. The films containing NA and the mixture of both NI and NA presented antifungal effects against Penicilium sp., with Geotrichum sp. being more sensitive to the NA. These films have potential application as food active packaging materials. On the other hand, there was no synergistic effect by the simultaneous addition of both antimicrobials. This might be due to the characteristics of the culture medium, because they influence the migration of the additive incorporated into the films. Antimicrobial migration from the film to the food was evaluated during the storage time. Nisin was not detected in the cheeses in contact with the films incorporated with NI and NI + NA, the opposite being observed for natamicine. The antimicrobial levels in the sliced cheeses packed with the film incorporated with NA were not significantly different (p ≥ 0.05) during the storage time. The films incorporated with the mixture of NI and NA showed a larger natamicine migration to the mozzarella slices, and this migration was time dependent (p < 0.05). The natamicine levels in the cheeses packed with NA and NI + NA were above the maximum limit allowed by legislation. Obtained results show that the simultaneous addition of nisin and natamicine resulted in the reduction of the mechanical properties of the films and in a larger natamicine migration (Pires et al., 2008). Cellulosic films incorporated with lactase aiming at the decrease of lactose levels in milk were produced by the casting method, were immersed in flasks containing 100 mL of pasteurized milk, and were stored at 25°C for 25 hours or at 7°C for 48 hours. The films remained stable when stored at room and refrigeration temperatures. Migration tests showed that 21.94% of lactase incorporated in the film migrated to the medium after 14 hours of contact. Moreover, after 24 hours at 70°C, a 78% and 85% reduction in lactose concentration was observed for films added with 1 and 1.5 mL of lactase, respectively. This reduction was of 92% and 100% after 25 hours at 25°C. The developed films showed potential to be used as internal layers of a multilayer milk carton packaging. Laminated active film incorporated with 0%, 4%, and 8% of natamicine were used for cheese application. These films showed good adherence to Gorgonzola, and its very purpose was to inhibit slime formation on the product surface. After 30 days

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of storage, the control group (submitted to a natamicine bath—conventional treatment) presented superior natamicine levels in their surface to the cheeses packaged with the laminated active film. In these samples, the presence of the antimicrobial substance was not detected at a 5 mm depth from the surface of the cheese. The antimicrobial activity of the laminated active film against Penicillium roqueforti, Aspergillus niger, and Penicillium sp. was studied by the agar diffusion method. An antimicrobial effect was observed for the film with natamicine, and this effect increases with the increase of the antimicrobial concentration. These results were satisfactory because the microbial growth on the cheese surface could be inhibited by the use of the laminated active film. Furthermore, the cheese had a better quality and was healthier because the amount of ingested additive was reduced. Yildirim et al. (2006) studied the effects of casein coating on some properties of Kashar cheese and its effectiveness in carrying natamycin to prevent mold growth. The results showed that the samples coated with casein containing natamycin presented lower mold growth when compared with those samples having no coating, coated only with casein or dipped only in natamycin. The casein coating with natamycin showed that it can suppress mold growth for about 1 month without any adverse effects for cheese quality. Aromatized active film incorporated with fine herbs and bacon and ham flavor was applied to butter packaging. Microbiological and sensorial analysis as well as rancidity tests were made on the butter packaged with the aromatized active films, at 0, 5, 10, 20, and 40 days of storage, at refrigeration temperatures. The microbiological analysis results complied with legislation, and no rancidity was detected in the butter. The sensorial analysis demonstrated that this product had a large acceptance. Overall, these results show that aromatized active films present great potential for use as a primary packing, resulting in a differentiated and widely accepted product. Suppakul et al. (2008) studied the feasibility of low-density polyethylene (LDPE)based films containing the main basil (Ocimum basilicum L.) constituents as antimicrobial agents. Linalool and methylchavicol were successfully incorporated into LDPE films, and their inhibitory effects against microbial growth were successfully tested in model (i.e., solid medium) and real (Cheddar cheese) systems. The LPDE with linalool and methylchavicol presented suppression effects against Escherichia coli and Listeria innnocua, more pronounced at 12°C than at 4°C. Linalool or methylchavicol did not influence the results of sensory evaluation tests. Therefore, these additives were shown to be useful in the antimicrobial packaging of some foods by enhancing microbial stability and food safety. The importance of the studies of multilayer films and their application was already commented on by Buonocore (2005). Multilayer coextruded films made of high-density polyethylene (added with titanium dioxide), ethylene vinyl alcohol, and a layer of low-density polyethylene containing the antioxidants butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), and a-tocopherol (4%) were applied for the release of the antioxidants from the films to whole milk powder. Whole milk powder stability was measured by monitoring vitamin A, hexanal, pentanal, and heptanal content for 30 days at 30°C. BHT and BHA migrated quickly from the films to the milk powder, and a-tocopherol migrated gradually. Multilayer coextruded films provided an adequate light-barrier for whole milk powder, and the film added


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with a-tocopherol contributed to a better protection of vitamin A degradation when compared with the other films. The electrostatic power coating, widely used in the manufacturing industries, was used by Elayedath and Barringer (2002) in shredded Cheddar and mozzarella cheeses. They were electrostatically and nonelectrostatically powder coated with a mixture of natamycin and powdered cellulose. The electrostatic powder coating for shredded cheese improves its shelf life significantly, when compared with nonelectrostatic powder coating. The use of electrostatic coating is also a more environmentally friendly process, which reduces the exposure of workers to inhalation of dust and the risk of dust explosions. One of its advantages would be an increase in particle transfer efficiency and decreased dustiness as compared to a nonelectrostatic coating. Amefia et al. (2006) applied electrostatic coating in mozzarella cheese slices with sodium erythorbate or cellulose with natamyacin at 0 kV and −25 kV. When the same amount of powder on each sample was compared, electrostatically coated samples showed a greater color development and less mold growth than nonelectrostatically coated samples. Packaging may also be a shelf-life relevant factor depending on its capability to protect the product from the influence of oxygen and light. One of the most studied packaging solutions was the one developed for the packaging of milk. Saffert et al. (2006) studied the influence of different light transmittance properties, under fluorescent light at 8°C, in the vitamin content of pasteurized whole milk. Milk packed in pigmented PET bottles with low values of light transmittance, stored in the dark under the same experimental conditions, served as the control sample. In clear polyethylene terephthalate (PET) bottles, a reduction of 22% was observed for vitamin A and 33% for vitamin B2, and vitamin B12 content remained almost stable. In all pigmented PET bottles, the vitamin retention was significantly higher; the losses were 0% to 6% for vitamin A and 11% to 20% for vitamin B2, depending on the pigmentation level, as compared to 6% for vitamin A and no significant loss for vitamin B2 in the control sample. Perkins et al. (2007) studied processed UHT milk packaged in Intasept™ aseptic pouches with (treatment) and without (control) an oxygen-scavenging film, with the samples being analyzed for dissolved oxygen, stale flavor volatiles (methyl ketones and aldehydes), and free fatty acids. The oxygen-scavenging film was shown to significantly reduce dissolved oxygen content by 23% to 28% during storage. Significant reductions of 23% to 41% were also observed for some stale flavor volatiles, including three methyl ketones and two aldehydes. Free fatty acid levels remained far below threshold values, indicating that lipolytic rancidity would not interfere with the subjective analysis. However, the consumer panel failed to detect a significant difference in odor between the treatment and control samples. Artificial intelligence (AI) tools such as knowledge-based expert systems, fuzzy logic, inductive learning, and neural networks can also be used to monitor milk and milk products. AI tools are designed to deal with complex real-life data and transfer expert knowledge to quantitative functions that can be processed by computers. The use of those tools has been demonstrated in the control of cheese ripening (Perrot et al., 2004) and other food applications (Linko, 1998).

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9 Microcalorimetry A Food Science and Engineering Approach Ana Clarissa dos Santos Pires, Maria do Carmo Hespanhol da Silva, and Luis Henrique Mendes da Silva* Contents 9.1 Introduction................................................................................................... 201 9.2 Techniques..................................................................................................... 203 9.2.1 Isothermal Titration Microcalorimetry............................................. 203 9.2.1.1 Fundamentals......................................................................203 9.2.1.2 The ITC Equipment............................................................204 9.2.1.3 Experimental Part...............................................................206 9.2.2 Differential Scanning Microcalorimetry........................................... 212 9.2.2.1 Fundamentals...................................................................... 212 9.2.2.2 The mDSC Equipment......................................................... 213 9.2.2.3 Experimental Part............................................................... 215 9.2.2.4 Practical Applications......................................................... 217 9.3 Final Remarks................................................................................................ 218 Acknowledgments................................................................................................... 219 References............................................................................................................... 219

9.1 Introduction Food is a complex matrix that contains a great variety of molecules. These molecules interact to create assemblies of molecules with specific supramolecular structures that raise a particular food structure. The components are autoaggregated during processing, and one or more effects govern the structure created, such as physical (e.g., interparticle interactions, phase separations), chemical (e.g., formation of specific covalent bonds between molecules), or biological (e.g., fermentation, enzyme action) (Dalgleish 2004). Many events occurring in food process involve the interaction between molecules, such as proteins, lipids, carbohydrates, among others. Remarkable progress has been made in detecting and imaging structural properties of biological systems. Nevertheless, structure data are only the first step in the direction of understanding a biological process (Heerklotz 2004). The examination 201

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of structural information alone does not tell us about the motriz power (thermodynamic potential) that drives complex formation and is responsible for maintaining the conformation of biological macromolecules in a system (O’Brien and Haq 2004), as a food, for example. The knowledge about the functions of biological molecules requires additional information on dynamics and on the molecular interactions governing its behavior. Such issues can be obtained by thermodynamics and calorimetry approaches (Heerklotz 2004). In recent years, there have been an increasing number of detailed thermodynamics studies on biological systems, including food systems. All biological processes involve the generation or loss of energy as heat. It is possible to measure a very small amount of heat output by using microcalorimetry. The total amount of heat evolved is a measure of the extent of the process and can be related to thermodynamic properties (Tortoe et al. 2007). Microcalorimeters are instruments that directly and quantitatively measure the heat of a thermodynamic process (reaction, phase transition, molecular interaction). Even though this equipment has been used since the eighteenth century, only in the last 40 years has it been used to study biological molecules and their interactions, as a result of the development in electronics, design, and temperature sensing that allows this kind of study (O’Brien and Haq 2004; Zielenkiewicz and Margas 2002). An increasing number of researchers recognize the great potential of these methods, as getting insight into the forces governing a system is essential for understanding its behavior and function. Calorimetric methods provide a depth of information that is not, or is barely, available through the use of other techniques (Heerklotz 2004). Excellent microcalorimeters and a variety of calorimetric techniques have been developed over the last decade and are now available to a broad spectrum of users (Heerklotz 2004). Because of its high sensitivity, high accuracy, nondestructivity, and automaticity, microcalorimetry has been recently used in food engineering and technology, microbiology, pharmacological analysis, biotechnology, ecology, genetics, and environmental sciences (Chen et al. 2006). Microcalorimetry has proven to be an invaluable tool for understanding the forces that stabilize the conformations of biologic molecules (Silva and Loh 2000), being able to give qualitative and quantitative information about the energy involved in reactions between biological molecules. Moreover, the application of this technique adds a new perspective, in addition to the more widespread studied parameters involving food engineering and technology, improving the understanding of complex phenomena. Microcalorimetry is a simple and direct method based on the measurement of the energy (heat) quantity involved in a state of thermodynamic change. It allows the determination of precise values for the change in enthalpy, ∆H (P, T constants) or internal energy, ∆U (T, V constants) (O’Brien et al. 2001). No other secondary reactions are needed, and no particular pretreatment of the samples must be done (Antonelli et al. 2002, 2008). The advantages of the microcalorimetric method also include its nondestructive and noninvasive way to conduct a large variety of analyses. In this chapter, our main goal is to emphasize the diversity of potential applications of microcalorimetry in food engineering and technology. We aim to demonstrate the power and the variety of possible uses of this method to clarify thermodynamic

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information on food systems. To demonstrate the importance and the usefulness of this method in thermodynamics studies, examples from the recent literature will be used.

9.2 Techniques There are various microcalorimeters, and all of them are thermodynamic instruments. Depending on the type of experiment the instruments are intended for, different measurement principles are employed, and practical designs vary (Wadsö 1997).

9.2.1 Isothermal Titration Microcalorimetry The term isothermal microcalorimetry is employed for calorimeters developed to work in the microwatt (some cases in nanowatt) range under isothermal conditions (Wadsö 1997). As is well known, the enthalpy changes can be obtained, indirectly, from the temperature dependence of the equilibrium binding or dissociation constant (van’t Hoff analysis). However, there are severe limitations associated with indirect approaches of this nature (O’Brien et al. 2001), especially because of its low accuracy (Wadsö 1997). Isothermal titration (micro)calorimetry (ITC) measures, directly and without the need of a predetermined model, the enthalpy change for a series of interesting thermodynamic processes, as, for example, bimolecular binding interaction at a constant temperature (O’Brien and Haq 2004). ITC methods can lead to a simultaneous determination of equilibrium constants (Kb) and enthalpy changes (ΔH), from which the changes in standard Gibbs energies and entropies can be derived (Wasdö 2001). In addition, the process (reaction or molecular interaction) stoichiometry (n) can be determined. A modern ITC is able to measure the energy of interaction (molar enthalpy ΔH) with excellent precision. Typically the minimum heat pulse is of the order of 1.0 to 0.02 mcal (Thomson and Ladbury 2004). 9.2.1.1 Fundamentals In the ITC experiment, one compound A is titrated into the other compound B, and the energy change measured (in the form of heat) is used as a proportion parameter of the extent of A–B molecular interaction. As a consequence, the concentration of the complex formed is known at any point in the titration, and thus the equilibrium binding constant (K B) can be obtained (Thomson and Ladbury 2004). Once the KB and the ΔH have been determined, the full thermodynamic characterization of the reaction can be done, such as change in free energy (ΔG) and entropy (ΔS), using well-known expressions shown in Equations 9.1 and 9.2: ∆G = − RT .ln K b where R is the gas constant, and T is the absolute temperature (Kelvin).

∆G = ∆H − T∆S

(9.1)

(9.2)

For an interaction to occur spontaneously, ΔG must be negative. From the above equation, it is possible to perceive that a negative ΔH and a positive ΔS term contribute for binding (O’Brien et al. 2001).

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(a)

(b)

Figure 9.1 Configurational entropy change resulting from the interaction between two molecules: (a) molecular distribution of each molecule (pure compounds) and (b) molecular distribution after the interaction (mixture) between the both molecules.

The ΔG and ΔH values correspond to the measure of heat energy associated with going from the free to the bound state at a given temperature. It is important to emphasize that, in addition to the bounds associated with ligand binding in the other component site, this value includes the bounds related to the solvent rearrangement and conformational changes occurred by the interaction. The ΔS parameter can be defined as the thermodynamic property that describes the way molecules are distributed into the different quantum states or in different spatial distribution (configurational or orientational entropy) of a system. Clearly, the entropy changes when there is a binding (interaction) between two molecules in comparison with each separated molecule, mainly because of the new possibilities of rearrangement between the molecules (Figure 9.1). An additional thermodynamic parameter that can be gained from ITC experiments is the change in constant pressure heat capacity (ΔCp), because this term is related to the temperature dependence of ΔH, as can be observed in Equation 9.3 (O’Brien et al. 2001; Thomson and Ladbury 2004):

∆C p =

∆H ∆T

(9.3)

9.2.1.2 The ITC Equipment An ITC instrument consists of two identical cells made with a highly efficient thermal conducting material. One of the cells is called a reference cell, and it is usually filled with water or buffer and does not participate in the titration. The other cell is the

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Syringe

Thermoelectric sensors Thermal conducting jacket Stirrer Reference cell

Sample cell

Water bath

Figure 9.2 An isothermal titration (micro)calorimetry (ITC) instrument.

sample cell, where the thermodynamic process occurs (reaction, phase separation, molecular aggregation). Both cells are located in a thermal conducting jacket that is commonly thermal equilibrated by a circulating water bath (Figure 9.2). The temperature of both cells is kept constant and identical with a precision of about 10 –4 K. The ligand molecule is injected in the sample cell by a syringe. A continuous power is applied on the reference cell. Thermoelectric sensors measure temperature differences between reference cell, sample cell, and the water bath. On interaction between ligand and the receptor molecules, heat is released or required. Depending on the nature of binding, a circuit is activated and increases or decreases power to the sample cell, aiming to keep equal the temperature in both cells and water bath. The heat per unit of time supplied to the sample cell or released from it is the signal obtained in an ITC experiment, and it is equal to the product between the temperature change and the heat capacity of the calorimetric vessel. Hence, a direct measure of the heat involved in the association between two molecules is obtained. The heat absorbed or released during an ITC experiment is proportional to the fraction of bound ligand. Therefore, it is of fundamental importance to have a precise determination of the initial concentration of both ligand and receptor molecules (Pierce et al. 1999). The data are presented as a plot of power (mjoule/s or mcal/s) versus time (s), resulting in energy peaks. As can be observed in Figure 9.3, as the system returns

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2

µcal/sec

0 –2 –4 –6 0

20

40

60

80

100

120

Time (min)

Figure 9.3 Data obtained from an isothermal titration (micro)calorimetry experiment. Peaks are plotted as power versus time.

to equilibrium, the power comes back to its initial value. In most of the experiments, for the initial injections, all or almost all the added ligand is interacting with the receptor molecules placed in the sample cell, resulting in a large energy signal. On the following injections, as the ligand (A) concentration increases, the receptors (B) become saturated, and less of B is available for interacting. Subsequently, less heat is absorbed or released when A is added. Because binding sites on B have already been occupied, the heat observed is provided from the dilution of compound A into the solution in the cell. This heat is called heat of dilution and must be subtracted from the heat of binding. The peaks obtained of each injection are then integrated with respect to time and plotted against the molar ratio of components (Figure 9.4). This curve is now appropriated to calculate the enthalpy, the equilibrium binding constant, and the stoichiometry of the reaction. If the enthalpy and Kb of molecule interaction are known, then the free energy and entropy are easily determined. The thermodynamic parameters obtained from an ITC experiment for an equilibrium binding event are the sum of all the individual changes in noncovalent interactions occurring on the formation of a new state in the system. The measured ΔH has a direct relation to the number and power of formed or broken bounds when the molecules go from the free to the bound state, including those associated with the solvent (O’Brien et al. 2001). 9.2.1.3 Experimental Part Before beginning an ITC experiment, some details should be carefully considered, from the sample preparation to the data analysis. This section will be divided into steps to facilitate the understanding of an ITC experiment.

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Microcalorimetry 1000 0

Cal/Mole of Injectant

–1000 –2000 –3000 –4000 –5000 –6000 –7000 –8000 –9000

0

0.5

1

1.5

2

2.5

Molar Ratio

Figure 9.4 Isothermal of the integrated experimental data, plotted as molar change in enthalpy versus molar ratio, obtained in an isothermal titration (micro)calorimetry experiment.

9.2.1.3.1 Concentration Requirements and Sample Preparation The concentrations of each compound that will interact are a critical factor in an ITC, especially due to the large amount required. This is one of the main disadvantages of the ITC method used for equilibrium constant (Kb) determination (O’Brien et al. 2001; Pierce et al. 1999). The affinity of the interaction should be regarded when choosing the concentration to be used. The capacity of the technique to obtain a satisfactory estimate of Kb depends, to a certain extent, on the dimensionless c value, which is a product of Kb, the concentration of receptor molecule [r], and the stoichiometry of the reaction (n), as shown in Equation 9.4:

c = K b [r ]n

(9.4)

ITC data are reliable if the c value is between 1 and 1000. Large c values are undesirable because the transition is sharp and few points are collected near equivalence, meaning that saturation can be reached in a unique injection of the ligand. On the other hand, low c values avoid the Kb determination, because the characteristic sigmoidal shape is lost, and the equivalent point cannot be identified. The effect of c values in the binding isothermal can be observed in Figure 9.5. Based on the ΔH values commonly associated with equilibrium interaction between biological molecules and the limitations of the c values, the sensitivity of the technique limits the binding constants to between 103 and 108 M–1 (O’Brien et al. 2001).

208

Engineering Aspects of Milk and Dairy Products 1000 0 “c” value lower than 1

Cal/Mole of Injectant

–1000 –2000 –3000

“c” value between 1 and 1000

–4000 –5000 –6000 –7000

“c” value higher than 1000

–8000 –9000

0

0.5

1 1.5 Molar Ratio

2

2.5

Figure 9.5 Plot of the effect of different c values on the binding isotherm.

The choice of the buffer solution is another critical point as the heat of dilution of compound A into the solution should be maintained at minimum, and this is not the case if the chosen buffer presents a large enthalpy of ionization (Pierce et al. 1999). If a large heat of dilution needs to be subtracted from the heat of binding, then the precision of the data will be negatively affected (Thomson and Ladbury 2004). In the absence of detailed information about the behavior of the compounds in a given buffer, it is recommended that solutions be chosen where these are stable and easily soluble (O’Brien et al. 2001). In some cases, it is necessary to use organic solvents or surfactants to help with the solubilization of one or both compounds. Nevertheless, particular attention should be given to organic solvents, as the cosolvent can compete for the binding sites. Both titrant and receptor molecules should be completely dissolved in the buffer, and immediately before loading the sample cell and the injection syringe, the ligand and receptor solutions must be degassed to remove air bubbles. The presence of bubbles can cause interferences in the feedback circuit, and instable baselines can be generated. 9.2.1.3.2 Loading the Sample, the Reference Cells, and the Syringe Usually, the reference cell is supplied with water or buffer solution. The sample cell should be filled with care to avoid air bubbles. Approximately 2.0 ml of B component is necessary to completely fill the sample chamber and the tube, although the active volume of microcalorimeter cell is 1.3 ml. Care is also needed when filling the syringe with the A component solution. The concentration of ligand solution has to be such that the molar ratio of ligand to receptor, following the last injection, is around 2. Typically, a complete titration involves 15 to 20 injections of ligand (Pierce et al. 1999). The handling of the injection syringe must be done carefully to avoid bending of the needle, which can result in poor quality baselines.

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9.2.1.3.3 Experimental Parameters The number, volume, and time of injections are critical points and should be carefully considered. To achieve a precise value of enthalpy of binding, it is fundamental that the first injections define a baseline at which all ligand molecules are bound to receptor molecules. It is necessary that chosen concentrations allow for amounts of free and bound ligand to be in equilibrium within a titration zone defined by the injections (Pierce et al. 1999). Another important consideration in an ITC experiment is the need to carry a control experiment aiming to determine the heat of dilution of compound A into the solvent used in the system. In a typical ITC experiment, in addition to the heat of the reaction occurring between components A and B, there are other sources of heat that should be accounted for. These additional heat values must be verified, by carrying out a control experiment, and then subtracted from the raw data for the calculation of binding energy. The control experiment is done by the titration of the solution containing component A into the buffer used in the binding step, in the absence of compound B. As can be seen in Figure 9.6, the obtained peaks of heat are considerably lower in comparison with those observed in Figure 9.3. Another interesting contribution of the control experiment is that it can bring to view some complications that could not have been predicted. At the high concentrations required for an ITC experiment, it might occur that the compound A is in an associated form. If the injections of this solution into the sample cell lead to a dissociation of this component, a progressive decrease in the heat of dilution will be observed. Because the dissociation effect is concentration dependent, experiments conducted at different concentrations (Thomson and Ladbury 2004) can be done to verify its occurrence.

2

µcal/sec

0 –2 –4 –6 0

20

40

60

80

100

120

Time (min)

Figure 9.6 Control isothermal titration (micro)calorimetry experiment showing the intensity of heat of dilution.

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The high sensitivity of the ITC equipment requires a final control experiment. This experiment is called machine blank; it is done by the addition of buffer into buffer and considers the heat associated with the equipment operation. 9.2.1.3.4 Data Analysis The method used for data analysis depends on the system of interest. The first steps in analyzing the obtained data include the normalization of the heats of binding as a function of ligand concentration and the correction of the volume due to dilution of receptor molecules during the injections. The following actions are to integrate the peaks using the chosen model. Usually, the data analysis is based on one independent binding site where the stoichiometry term can be neglected. Therefore, the heat derived from an injection Q is related to the charge in enthalpy by Equation 9.5: Q = [ B]total V 0 ( F2 − F1 ) ∆H

(9.5)

where [B]tot is the total concentration of B molecules, V0 is the cell volume, F is the fraction of bound B, and the numerical subscripts refer to the terms when moving from injection 1 to injection 2. On the other hand, if the multiple independent binding sites model is applied, the determination of n, Kb, and ΔH is obtained by Equation 9.6: Q=

(n[ B]tot V0 ∆H ) 2

(9.6)

2  (1 + [ A] )  1 4[ A]toot  1 + [ A]tot 1 tot − − + −  +   (n[ B]tot ) (nK b [ B]tot )  (n[ B]tot ) (nK b [ B]tot )  (n[ B]tot )   

9.2.1.3.5 Troubleshooting Several problems can occur during the ITC experiment. Sometimes, the enthalpy of binding measured from the first injection is lower than that of the following injections. This can happen as a result of a slow leakage of the ligand solution from the syringe or due to an incorrect placement of the syringe in the calorimeter device. A simple reduction in the length of time that the syringe is in contact with the receptor solution should help to keep the ligand solution from slowly leaking from the syringe (Pierce et al. 1999). Another common problem in an ITC experiment occurs when the cell signal does not return to the equilibrium value before the next injection. This can be solved by making sure of the required time between injections (O’Brien et al. 2001). Baseline instability is also a frequent issue, and, as previously pointed out, may be solved by the removal of the air bubbles from both sample cell and syringe solution and by carefully handling the syringe to avoid its bending,

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When the cell feedback signal often changes, this means that the heat capacity has undergone an alteration, indicating aggregation or precipitation. In this case, the sample conditions must be changed (O’Brien et al. 2001). 9.2.1.3.6 Practical Applications A wide variety of methods have been used to provide information about food and its components. The ITC technique has been highlighted because it is a direct and nondestructive method. In this section, some examples of application of ITC in food engineering and technology will be described. Chitosan is a cationic biopolymer that has potential for application in food and other areas due to its nutritional and physicochemical properties. Thongngam and McClements (2004) used the ITC instrument to evaluate the interaction between chitosan and a model anionic surfactant, sodium dodecyl sulfate (SDS). Aliquots of 10 ml of SDS solution were injected into a 1.48 ml cell containing 0.1 wt% chitosan in acetate buffer. The experimental temperature was 30°C, there were intervals of 300 s between each injection, and the solution was stirred at 315 rpm during the experiment. The authors integrated the curves resulting from the injections and obtained the enthalpy change per mole of SDS injected into the sample cell. It was observed that in the presence of chitosan, the enthalpy changes from exothermic to endothermic inasmuch as the concentration of surfactant titrated into the chitosan solution increased. Relative large endothermic peaks were observed at concentrations higher than the saturation concentration due to the micelle dissociation, which was also observed in an experiment carried out without chitosan. This kind of study is a powerful tool to promote the rational design of chitosan-based food ingredients, because it provides a wealth of information about the origin and characteristics of molecular interactions between chitosan and anionic surface-active lipids. Casein is the main protein of milk, and it has important functional properties. Portnaya et al. (2006) carried out an ITC experiment to study the effect of temperature on the thermodynamics of micellization of b-casein. For this purpose, micellar b-casein solutions (1.67 mM) were titrated into degassed phosphate buffer. The duration of each injection was 10 s, and the equilibrium time between consecutive titration was 3 min. The experiment was conducted at different temperatures. Large exothermic enthalpy changes in the initial injections, probably related to the micelle dilution, demicellization, and dilution of individual b-casein molecules were observed. The small enthalpy changes observed at the final injections are associated with the micelle dilution. At 18°C, there was a gradual change in the dilution enthalpy, suggesting that the association of b-casein into micelles is gradual, taking place over a certain concentration range. It was also observed that the higher the temperature, the lower the protein concentration at which the micellization process began and ended, characteristic of increased hydrophobic interactions. In this work, the authors also evaluated the effect of ionic strength on the b-casein micellization, having observed that at a low ionic strength, the critical micellization concentration (CMC) was higher as the increased electrostatic repulsion forces under low ionic strength require higher protein concentration to initiate micellization. This is the type of work that allows for the obtention of important physicochemical parameters for food engineering and technology applications.

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The presence of polyphenols such as tannins is common in foods and beverages. A research line that is gaining interest in food technology is related to the understanding of the interaction occurring between these compounds and some proteins present in the human saliva. Pascal et al. (2007) studied the interactions between tannin molecules and praline-rich proteins using the ITC technique. Tannin solution was titrated into the stirred sample cell containing the proteins as a sequence of 30 injections of 10 ml each. A dilution experiment was also carried out, and it was subtracted from interaction raw data. During the titration experiments, four phases were observed. At the very beginning of the experiment, the ΔH increased, and the binding isotherm presented a sigmoid shape as if site saturation had been reached, indicating an interaction between the two molecules. In the second phase, ΔH was stable and slightly negative, meaning that there was an exothermic reaction. In the third phase, exothermic peaks of higher amplitude were present, and the threshold to achieve the third phase depended on the protein concentration. At the end of the titration, the fourth phase, an endothermic phenomenon related to the dilution of the tannin was observed. This work contributed to a better understanding of molecular and colloidal causes of these interactions, and therefore it made astringency control, an important issue in the beverage industry, easier.

9.2.2 Differential Scanning Microcalorimetry Differential scanning microcalorimetry (mDSC) is an experimental technique that measures the heat energy changes in a sample during controlled increase or decrease in the temperature. At the simplest level, it may be used to determine the melting temperatures of samples in a solution, solid or mixed phases. A more sensitive instrument allows the determination of several thermodynamics parameters (Cooper et al. 2001; Gabbott 2008), making it possible to characterize a material as a function of heat changes. The calorimeters are extremely sensitive, and they are able to measure small changes in the thermal properties of materials at levels of a few milligrams per milliliter (MacNaughtan and Farhat 2008). mDSC is often the preferred thermal technique, because it is able to provide us with a wealth of information related to the physical and energetic properties of a system, which cannot be obtained precisely, quickly, and simply using other available techniques (Clas et al. 1999). It has been widely used, for instance, for studying conformational transitions in biological molecules, such as proteins, lipids, carbohydrates, and polymers. Its application can be extended to organizational changes in molecular assemblies, such as vesicles. The use of mDSC has become increasingly popular as it is based on a direct measure of thermal properties of samples, with the advantage of being a noninvasive technique and not requiring specific pretreatment of the sample. 9.2.2.1 Fundamentals Although having the same fundamentals of traditional DSC, mDSC can measure heat flows resulting from low energy reactions (Saunders 2008), as larger amounts of sample can be analyzed.

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In a mDSC experiment, a sample and a buffer are heated in a controlled way to eliminate any temperature difference between them. As a result, the equipment senses a signal that represents the difference of heat capacity at constant pressure (Cp) between the two components (Heerklotz 2004). The Cp of a substance can be defined as its capacity of absorbing or releasing energy without altering the temperature. This is a fundamental property from which all thermodynamics quantities can be obtained. For example, the enthalpy (H) and the entropy (S) can be derived from the total heat energy uptake involved in the heating process of a material, as can be seen in Equations 9.7 through 9.9:

H=

∫

T 0

C p ⋅ dT + H 0

 Cp  dH  T  dS = = T dT S=

∫

T

0

 C p  ⋅ dT  T 

(9.7)

(9.8) (9.9)

The Cp value depends on the numbers of possibilities of distributing added heat energy to the system. For example, in a system that has a small number of ways to dispense the received energy, a little amount of energy will be needed to increase the temperature; hence, the system presents a low Cp. On the other hand, if there are many ways to distribute the uptaken energy, such as using this energy to break bounds or to vibrate molecules, more energy will be necessary to promote the temperature rise. Once Cp, ΔH, and ΔS have been determined, the free energy involved in the process can be calculated (see Equation 9.2). 9.2.2.2 The mDSC Equipment A typical mDSC instrument consists of two identical cells. One cell is named the reference cell and contains a buffer. Another cell (sample cell) is filled with the diluted sample, and both are heated at a constant rate of temperature increase. In Figure 9.7, a schematic representation of mDSC equipment is presented. The two cells (reference (R) and sample (S)) are contained within a thermal shield. A power is applied so that the heaters increase the temperature of the cells at a constant rate, while controlling the temperature or energy differences between both cells (ΔT1) and between the cells and the adiabatic jacket (ΔT2). A heater/cooler on the jacket permits the thermal shield to have a similar temperature as the cells, and feedback heaters on the cells counterbalance any temperature change between the cells during the experiment. In a heat-flux mDSC, a sensor measures the temperature difference between the sample and the reference cell, with this being proportional to the difference in the heat capacity of the sample and the buffer reference (Senin et al. 2000). On the other hand, in a power-compensation mDSC, the energy applied to or removed from the calorimeter to maintain the same temperature in the cells is measured. The amount of power required to keep the thermal equilibrium in the system is

214


P Thermal shield

Feedback heater (∆T1)

Jacket (∆T2) heater/cooler

∆T2 ∆T1

R

S Feedback heater (∆T1)

Principal heaters

Figure 9.7 A mDSC instrument.

proportional to the energy changes occurring in the sample. The power-compensation mDSC is more precise than the heat-flux mDSC, because the direct measurement of energy involves fewer errors than the temperature evaluation. In addition, in this equipment design, no heat-flux equations are necessary, because the energy is directly measured. At the end of the microcalorimetric scan, a plot of ΔCp against temperature is constructed, as can be observed in Figure 9.8. The integrated area below the peak represents the total heat energy uptake by the sample undergoing the transition—that is, the variation in the enthalpy. This heat depends on the amount of the sample in the cell (Cooper et al. 2001).

Cp (kcal/mol/deg)

15

10

5

0 20

30

40

50

60

70

80

Temperature (°C)

Figure 9.8 Data obtained from a mDSC experiment. The peak represents the difference in the Cp values of the tested sample and the reference buffer.

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Cp (kcal/mol/deg)

15

0% A

5% A

10

5

10% A

0 20

30

40

50

60

70

80

Temperature (°C)

Figure 9.9 Data obtained from a mDSC experiment to determine enthalpy of interaction (ΔHint.) between compounds A and B. It can be observed that as the concentration of A increases, the ΔCp decreases, indicating that the ΔHint. is lower.

A mDSC experiment can also give information about the liquid–solid transition temperature, the enthalpy of fusion, the melting point, and the enthalpy of interaction between two molecules. To evaluate the enthalpy of interaction between two molecules using a mDSC, solutions with different concentrations of ligand compound (A) should be made. These solutions will be used to dissolve the other substance involved (B) in the interaction and also as a reference. The following step is to proceed with the calorimetric scans. From the measured variations in Cp (Figure 9.9), information on the enthalpy of interaction can be obtained using Equation 9.7. Also, the crystalline melting point is often measured by mDSC, as melting is an endothermic process in which the sample absorbs energy in order to melt. The integration of the peak area gives the heat of fusion (ΔHf) (Gabbott 2008). 9.2.2.3 Experimental Part The success in a mDSC experiment requires that several items, from the sample preparation to the data analysis, are taken into account. This section will be divided into steps to facilitate the understanding of a mDSC experiment. 9.2.2.3.1 Concentration Requirements and Sample Preparation The precision of a calorimetric ΔH measurement is extremely dependent on the purity of the sample and on the knowledge of its concentration (Cooper et al. 2001). Therefore, the use of a reliable method for concentration measurement is recommended.

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The cells of mDSC equipment are able to accommodate dilute solutions with volumes ranging from 0.5 to 2.0 ml. Before starting the experiment, it is important to remove air bubbles of the sample mixture and of the buffer. This can be achieved under vacuum with stirring. Also, froth formation in the sample should be avoided, aiming at the obtention of a stable baseline. 9.2.2.3.2 Loading the Sample and Reference Cells Because the mDSC is used for studying different chemical compounds, the cells should be inert to the action of diverse chemicals. Glass cells, for instance, have a high resistance to most chemicals, besides being strong, durable, and smooth (Senin et al. 2000). The sample and reference cells have to be filled with the buffer without air bubbles, and a baseline should be obtained using an appropriate temperature range and scan rate. After cooling of the cells, the sample cell is refilled with the solution of interest. The same conditions should be used in the following run in order to assure reversibility and reproducibility of the data. 9.2.2.3.3 Calibration The reliability of mDSC results depends on the care taken in calibrating the equipment as close as possible to the transition temperatures of interest. The calibration parameters are important especially when a comparison with results obtained in different instruments or at different times (Clas et al. 1999) is needed. Generally, the calibration stage uses metals such as indium, tin, bismuth, and lead. 9.2.2.3.4 Experimental Parameters The initial and final temperature of the heating and cooling stages as well as the scan rate are important mDSC experimental parameters and must be carefully selected. In order to make sure that the precise control temperature is obtained, it is recommended to program the equipment to start at least 30 K below and to end at least 10 K above the temperature of interest (Clas et al. 1999). 9.2.2.3.5 Data Analysis The output obtained from the microcalorimetric scan is a thermogram showing the excess ΔCp —the value obtained by subtracting the reference Cp from the sample Cp —as a function of temperature (Figure 9.8). The analysis of the data is done using the instrument-associated software, which commonly involves subtraction of buffer baseline and concentration normalization, followed by deconvolution of the resultant thermogram using a suitable model (Cooper et al. 2001). 9.2.2.3.6 Troubleshooting Some problems can occur during the mDSC experiment. For instance, the cells can be contaminated and distort the results. This requires that the presence of contaminant agents be checked and the cell be carefully cleaned. The establishment of the sample baseline is a difficult stage. If the Cp baseline does not return to the same value after the transition, the analyst has to be able to

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estimate what the sample baseline might have been in the region under the endotherm peak in the absence of the transition. Some strategies to correct the baseline are recommended, for example, the use of linear, cubic, progress, and step (Cooper et al. 2001) models. It is important to emphasize that the differences between the calorimetric and the van’t Hoff enthalpies is due, besides other reasons, to a poor baseline correction. 9.2.2.4 Practical Applications Differential scanning microcalorimetry can provide a complete thermodynamic characterization of the thermo-induced process, by allowing the determination of, for example, the heat capacities of native and denaturated protein, the enthalpy and entropy of denaturation, as well as the melting temperatures (Blanco et al. 2007). Heat-induced gelation is one of the most important properties of muscle myofibrillar proteins, being mainly responsible for the texture characteristics of meat products. Vega-Warner and Smith (2001) evaluated the effect of pH in the thermally induced unfolding and aggregation properties of two types of myosin. To achieve this goal, a mDSC experiment was carried out. Buffer solutions were run before each protein run to obtain the baseline for following calculations. The myosin types 1 and 2 were tested at pH 5.50 and 6.05, from 25°C to 80°C at a heating rate of 1°C.min –1. The following parameters were determined: heat capacity (Cp), initial transition temperature (T0), endothermic peak temperatures (Tm), calorimetric enthalpy (ΔHcal), and van’t Hoff enthalpy (ΔHvH). It was observed that the ΔHcal of both myosins at pH 5.50 did not differ from the ΔHcal at pH 6.05, meaning that the two tested myosins presented a high conformational stability at lower pH. The endotherms of the two types of myosins were different and showed multiple transitions at pH 5.50 and 6.05, indicating that the proteins have multiple unfolding domains. This kind of study is a powerful tool to understand the distinct changes that occur with meat and meat products during the heating process, and it can be advantageously used for food formulations that are submitted to heat processing. Isoflavones are commonly found in soybeans. The isoflavone content and composition in food vary as a result of the food manufacturing process. Some thermal processes, for instance, alter the isoflavone profile in the food. Ungar et al. (2003) used the mDSC instrument to evaluate the isoflavone degradation and modification induced by a thermal process. Diluted isoflavone solution (1.0 mM) and borate buffer were scanned in the temperature range of 50 to 120°C at a rate of 3.8°C.h–1, followed by a rescan. The analysis of data provides the ΔCp and ΔH of degradation. Isoflavones exhibit large exothermic peaks, indicating that degradation occurs under the scanning conditions. In works such as this one, the stability of food components could be measured as well as the bioactivity and bioavailability of food constituents after thermal processing. The study of protein unfolding is a classical use of mDSC technique. This method allows the determination of the ΔCp of protein denaturation and the ΔH of the process. In this kind of experiment, a baseline of the buffer is collected, and the protein solution undergoes a scan. The obtained data provide a plot as illustrated in Figure 9.10.

218


Cp (kcal/mol/deg)

15

10

5

2 3

1 0 20

30

40

50

60

70

80

Temperature (°C)

Figure 9.10 Data obtained from a mDSC experiment for the thermal unfolding of a protein. The peak represents the difference in the Cp values of the tested sample and the reference buffer.

Three distinct regions can be observed. In the first region (1), the protein and the solvent are undergoing a similar process, and the heat energy is equally distributed in the kinetic and potential forms. As the temperature increases, the Cp also increases, and when the protein starts unfolding, a sharp increase is observed and a peak is achieved (region 2). In this stage, the protein transfers the received heat energy to the potential form, meaning that the energy is used for breaking bonds and changing protein conformation. In region 3, the protein is already unfolded; hence, the heat energy is transferred again to the kinetic and potential forms either in the protein or in the solvent. This experiment provides important information about proteins, and it is extremely important for food research.

9.3 Final Remarks Calorimetry has a special significance in studies involving thermal-induced changes and binding between two or more molecules. It is a straightforward and nondestructive method that can be used to measure several physicochemical parameters. Examples of application of microcalorimetry to food research have been presented, and the importance of the obtained parameters on the understanding of the biological and thermal processes that occur in food processing and storage was demonstrated. In most cases, food is a multiphase system complex matrix, and this makes it difficult to understand the changes caused by heat processing or instability of its components. Microcalorimetry is a useful technique that can help with the understanding of these changes and will allow for a detailed study of different components of food.

Microcalorimetry

219

Acknowledgments The authors wish to acknowledge Mafalda Aguiar de Carvalho Quintas Baylina for the revision and the National Council of Technological and Scientific Development (CNPq) and the Foundation to Research Support of the Minas Gerais State (FAPEMIG) for their financial support.

References Antonelli, M.L., D’Ascenzo, G., Lagana, A., Pusceddu, P. (2002). Food analyses: a new calorimetric method for ascorbic acid (vitamin C) determination. Talanta, 58(5), 961–967. Antonelli, M.L., Spadaro, C., Tornelli, R.F. (2008). A microcalorimetric sensor for food and cosmetic analyses: l-Malic acid determination. Talanta, 74(5), 1450–1454. Blanco, E., Ruso, J.M., Sabín, J., Prieto, G., Sarmiento, F. (2007). Thermodynamic study of the thermal denaturation of a globular protein in the presence of different ligands. Journal of Thermal Analysis and Calorimetry, 87 (1), 143–147. Chen, X.J., Miao, W., Liu, Y., Shen, Y.F., Feng, W.S., Yu, T., Yu, Y.H. (2006). Microcalorimetry as a possible tool for phylogenetic studies of Tetrahymena. Journal of Thermal Analysis and Calorimetry, 84(2), 429–433. Clas, S.D., Dalton, C.R., Hancock, B.C. (1999). Differential scanning calorimetry: applications in drug development. Pharmaceutical Science and Technology Today, 2(8), 311–318. Cooper, A., Nutley, M.A., Wadood, A. Differential scanning microcalorimetry. In: Harding, S.E., Chowdhry, B.Z. Protein-Ligand Interactions: Hydrodynamics and Calorimetry. Oxford University Press, New York, 2001, pp. 287–318. Dalgleish, D.G. Food emulsions: their structures and properties. In: Friberg, S.E., Larsson, K., Sjöblom, J. Food Emulsion, 4th ed., Marcel Dekker, New York, 2004, pp. 1–44. Gabbott, P. A practical introduction to differential scanning calorimetry. In: Gabbot, P. Principles and Applications of Thermal Analysis, Blackwell, Ames, IA, 2008, pp. 1–50. Heerklotz, H. (2004). The microcalorimetry of lipids membranes. Journal of Physics Condensed Matter, 16(15), 441–467. MacNaughtan, B., Farhat, I.A. Thermal methods in the study of foods and food ingredients. In: Gabbot, P. Principles and Applications of Thermal Analysis. Blackwell, Ames, IA, 2008, pp. 331–402. O’Brien, R., Haq, I. Applications of biocalorimetry: binding, stability and enzyme kinetics. In: Ladbury, J.E., Doyle, M.L. Biocalorimetry 2: Applications of Calorimetry in the Biological Sciences. John Wiley and Sons, Chichester, 2004, pp. 1–34. O’Brien, R., Ladbury, J.E., Chowdhry, B.Z. Isothermal titration calorimetry of biomolecules. In: Harding, S.E., Chowdhry, B.Z. Protein-Ligand Interactions: Hydrodynamics and Calorimetry. Oxford University Press, New York, 2001, pp. 263–286. Pascal, C., Poncet-Legrand, C., Imberty, A., Gautier, C., Sarni-Manchado, P., Cheynier, V., Vernhet, A. (2007). Interactions between a nonglycosylated human proline-rich protein and flavan-3-ols are affected by protein concentration and polyphenol/protein ratio. Journal of Agricultural and Food Chemistry, 55(12), 4895–4901. Pierce, M.M., Raman, C.S., Nall, B.T. (1999). Isothermal titration calorimetry of proteinprotein interactions. Methods, 19(2), 213–221.

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Portnaya, I., Cogan, U., Livney, Y.D., Ramon, O., Shimini, K., Rosenberg, M., Danino, D. (2006). Micellization of bovine b-casein studied by isothermal titration microcalorimetry and cryogenic transmission electron microscopy. Journal of Agricultural and Food Chemistry, 54(15), 5555–5561. Saunders, M. Thermal analysis of pharmaceuticals. In: Gabbot, P. Principles and Applications of Thermal Analysis. Blackwell, Ames, IA, 2008, pp. 287–327. Senin, A.A., Potekhin, S.A., Tiktopulo, E.I., Filomonov, V.V. (2000). Differential scanning microcalorimeter SCAL-1. Journal of Thermal Analysis and Calorimetry, 62(1), 153–160. Silva, L.H.M., Loh, W. (2000) Calorimetric investigation of the formation of aqueous twophase systems in ternary mixtures of water, poly(ethylene oxide) and electrolytes (or dextran). Journal of Physical Chemistry B, 104(43), 10069–10073. Thomson, J.A., Ladbury, J.E. Isothermal titration calorimetry: a tuturial. In: Ladbury, J.E., Doyle, M.L. Biocalorimetry 2: Applications of Calorimetry in the Biological Sciences. John Wiley and Sons, Chichester, 2004, pp. 37–58. Thongngam, M., McClements, D.J. (2004). Characterization of interactions between chitosan and an anionic surfactant. Journal of Agricultural and Food Chemistry, 52(4), 987–991. Tortoe, C., Orchard, J., Beezer, A., O’Neil, M. (2007). Potential of calorimetry to study osmotic dehydration of food materials. Journal of Food Engineering, 78(3), 933–940. Ungar, Y., Osundahunsi, O.F., Shimoni, E. (2003). Thermal stability of genistein and daidzein and its effects on their antioxidant activity. Journal of Agricultural and Food Chemistry, 51(15), 4394–4399. Vega-Warner, V., Smith, D.M. (2001). Denaturation and aggregation of myosin from two bovine muscle types. Journal of Agricultural and Food Chemistry, 49(2), 906–912. Wadsö, I. (1997) Trends in isothermal microcalorimetry. Chemical Society Reviews, 26(2), 79–86. Wasdö, I. (2001). Isothermal microcalorimetry: current problems and prospects. Journal of Thermal Analysis and Calorimetry, 64(1), 75–84. Zielenkiewicz, W., Margas, E. Theory of Calorimetry. Kluwer, New York, 2002, 188p.

Applications 10 Potential of Whey Proteins in the Medical Field Lígia Rodrigues* and José António Couto Teixeira Contents 10.1 Introduction................................................................................................... 222 10.2 Whey Protein Concentrates and Whey Protein Isolates................................ 225 10.2.1 Antimicrobial and Antiviral Activity................................................ 226 10.2.2 Immunomodulation........................................................................... 227 10.2.3 Anticancer Activity............................................................................ 228 10.2.4 Nutrition Effects and Other Metabolic Features............................... 228 10.3 b-Lactoglobulin............................................................................................. 229 10.3.1 Immunomodulation........................................................................... 229 10.3.2 Nutrition Effects and Other Metabolic Features............................... 229 10.4 a-Lactalbumin............................................................................................... 230 10.4.1 Immunomodulation........................................................................... 230 10.4.2 Anticancer Activity............................................................................ 230 10.4.3 Nutrition Effects and Other Metabolic Features............................... 230 10.5 Bovine Serum Albumin (BSA)...................................................................... 231 10.5.1 Anticancer Activity............................................................................ 231 10.5.2 Nutrition Effects and Other Metabolic Features............................... 231 10.6 Lactoferrin..................................................................................................... 231 10.6.1 Antimicrobial and Antiviral Activity................................................ 232 10.6.2 Immunomodulation........................................................................... 232 10.6.3 Anticancer Activity............................................................................ 234 10.6.4 Nutrition Effects and Other Metabolic Features............................... 235 10.7 Lactoperoxidase............................................................................................. 236 10.7.1 Antimicrobial and Antiviral Activity................................................ 236 10.8 Immunoglobulins........................................................................................... 237 10.8.1 Antimicrobial and Antiviral Activity................................................ 237 10.8.2 Immunomodulation........................................................................... 238 10.8.3 Nutrition Effects and Other Metabolic Features............................... 238 10.9 Others............................................................................................................ 238 10.9.1 Proteose Peptones.............................................................................. 238

221

222


10.9.2 Glycomacropeptide.......................................................................... 239 10.9.3 Osteopontin...................................................................................... 239 10.10 Future Trends...............................................................................................240 10.11 Conclusion...................................................................................................240 References............................................................................................................... 241

10.1 Introduction Over the past few decades, clinical and mechanistic studies have indicated many relations between nutrition and health; thus, evidence that diet is a key environmental factor affecting the incidence of many chronic diseases is overwhelming (Rodrigues et al. 2009). In recent years, milk constituents have become recognized as functional foods, suggesting their use has a direct and measurable effect on health outcomes (Gill et al. 2000). Whey, a liquid by-product, once considered a waste product, is now widely accepted to contain many valuable constituents (Madureira et al. 2007; Marshall 2004). These include proteins that possess important nutritional and biological properties regarding health promotion and disease prevention (Korhonen 2006; Madureira et al. 2007; Rodrigues et al. 2009). As a result, there is a growing interest by the dairy industry and other food and even pharmaceutical industries to design and formulate products that incorporate specific bioactive components derived from whey. In fact, the dairy industry has achieved a leading role in the development of functional foods and has already commercialized products (whey protein concentrates [WPC], reduced lactose whey, whey protein isolated [WPI], demineralized whey, and hydrolyzed whey) that boost the immune system, kill pathogenic microorganisms, or reduce blood pressure (Horton 1995; Korhonen 2006; Smithers 2008). Today, whey is a popular dietary protein supplement alleged to provide antimicrobial activity, immune modulation, improved muscle strength and body composition, and to prevent cardiovascular disease and osteoporosis (Smithers 2008). Advances in processing technology, including ultrafiltration, microfiltration, reverse osmosis, and ion exchange, have resulted in the development of several different finished whey proteins (Marshall 2004; Smithers et al. 1996; Zall 1984). Current challenges in the exploitation of bioactive components are their maximal recovery from whey, their stability in different food matrices, and their optimal bioavailability in the body in order to deliver the expected health effects (Korhonen 2006). Milk contains two primary sources of protein: the caseins (insoluble) and whey (soluble) (Madureira et al. 2007; Marshall 2004). After processing occurs, the caseins are the proteins responsible for making curds (caseins account for 80% (w/w) of the whole protein inventory) and can easily be recovered from skim milk via isoelectric precipitation or rennet-driven coagulation, while whey remains in an aqueous environment as a by-product. The components of whey include b-lactoglobulin, a-lactalbumin, bovine serum albumin, lactoferrin, immunoglobulins, lactoperoxidase enzymes, glycomacropeptides, peptones, lactose, and minerals (Smithers 2008; Walzem et al. 2002). Whey proteins are globular molecules with a substantial content of a-helix motifs, in which the acidic/basic and hydrophobic/hydrophilic amino acids are distributed in a fairly balanced way along their polypeptide chains (Madureira et al. 2007). Table 10.1 depicts the whey protein profile, including general chemical and physicochemical properties,

400

Bovine serum albumin

8–20

1200

5–60

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