Engineering Aspects of Milk and Dairy Products

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

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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.

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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

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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

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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

Engineering Aspects of Milk and Dairy Products

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)

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Engineering Aspects of Milk and Dairy Products

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

Physical Chemistry of Colloidal Systems Applied to Food Engineering

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 

∫

∫

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Engineering Aspects of Milk and Dairy Products

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.

Physical Chemistry of Colloidal Systems Applied to Food Engineering

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)

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Engineering Aspects of Milk and Dairy Products

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.

Physical Chemistry of Colloidal Systems Applied to Food Engineering

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)

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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

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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.

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Engineering Aspects of Milk and Dairy Products

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.

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Physical Chemistry of Colloidal Systems Applied to Food Engineering

(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.

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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.

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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.

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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.

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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.

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Engineering Aspects of Milk and Dairy Products

(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.

Physical Chemistry of Colloidal Systems Applied to Food Engineering

(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).

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Engineering Aspects of Milk and Dairy Products

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.

Physical Chemistry of Colloidal Systems Applied to Food Engineering

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,

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Engineering Aspects of Milk and Dairy Products

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.

Physical Chemistry of Colloidal Systems Applied to Food Engineering

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.

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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 <1/100 mL is the best (Saboya and Maubois, 2000). Cleaning and disinfection are on average done after 10 hours of running according to the following sequence:



1. Rinse with water at the same temperature as the treated dairy product. 2. Alkaline step at temperature higher than 60°C for at least 20 min with a ternary detergent of which caustic soda causes “peptization” of the protein part of the fouling and solubilization of fat enhanced by the surfactant while mineral sequestering agent solubilizes the mineral deposit. 3. Rinse with hot water (50 to 60°C). 4. Acid step at a temperature between 20°C and 50°C for 15 min with preferably nitric acid; use of phosphoric acid leads to insoluble calcium salts deposition on membrane. 5. Rinse with hot water (50 to 60°C). 6. Sanitize at 20°C for 20 min generally twice a week or more, with hypochlorite (NaOCl solution giving 100 to 200 ppm of active chlorine) or equivalent peroxyacetic acid solution. 7. Final rinse with water.

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Engineering Aspects of Milk and Dairy Products

According to the membrane material, the pH of the last cleaning solution has a major influence on the water flux and consequently on the equipment performance on a dairy product. It must be alkaline if a polymeric membrane is used and acid if an inorganic membrane is used (Saboya and Maubois, 2000). An initial low water flux will induce an enhanced concentration polarization layer when the dairy product is put in contact with the membrane.

3.4 Applications of Membrane Technologies for the Production of Liquid Milks Decontamination of collected milk is generally achieved through heat treatments that use various combinations of time–temperature parameters to obtain the desired bactericidal effect: thermization, pasteurization, or sterilization by autoclaving or ultra-high-temperature (UHT) treatment. Even though these heat treatments ensure the safety of milk and dairy products, they almost always induce irreversible modifications of milk components, alter physicochemical calcium salts and protein equilibrium, and also adversely affect the organoleptic quality of fluid milk and dairy products as well as the cheesemaking ability. Moreover, the dead cells of killed bacteria remain in heated milk with their potentially active enzymes, which with the metabolic activity developed by the growth of the remaining thermoduric bacteria will cause alterations of liquid milks during storage, thus reducing commercial shelf life. Membrane MF offers an interesting alternative to heat treatments. Initially proposed by Holm et al. (1984), it has led to the technology and equipment called Bactocatch® (Tetra Laval Co.). Numerous studies conducted in Sweden and in France, summarized by Saboya and Maubois (2000), have optimized the original parameters described in the patent of Holm et al. (1984). Skim milk heated to 50°C is circulated at a velocity of 7.2 m.s–1 along a membrane having an average pore size of 1.4 mm (Sterilox® or equivalent) according to the hydraulic concept of an uniform transmembrane pressure (UTP), in the range of 0.5 bar, obtained either by recirculation of the permeate (Meershon, 1989) or by a specially designed MF membrane having continuous variation of the porosity of its support (Membralox GP®) or continuous variation of the thickness of the membrane layer (Isoflux®). All the somatic cells and most of the residual fat and the contaminating microorganisms are concentrated 20 times in the MF retentate. In MF industrial equipment, this retentate is then concentrated 10 times more in a second MF apparatus, thus leading to a volumetric concentration factor (VCF) of 200. Fluxes obtained industrially are in the order of 500 L.h–1.m–2 during 10 h. According to VCF of 20 or 200, the observed permeation rates are, for proteins 99% and 99.4% respectively, and for total solids 99.5% and 99.9%. Average observed decimal reduction (DR) of bacteria is above 3.5 for the milk collected in the developed dairy countries (initial Total Count [TC] < 200 000 CFU.mL –1); it can be higher than 6 in milks with a poor bacteriological quality collected in some emergent countries. Spore-forming bacteria that represent the main surviving species to pasteurization are highly retained by MF membrane (DR > 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

Applications of Membrane Technologies in the Dairy Industry

37

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

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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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Engineering Aspects of Milk and Dairy Products

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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Engineering Aspects of Milk and Dairy Products

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

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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

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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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Lawrence, R.C., Gilles, J., Creamer, L.K., Crow, V.L., Heap, H.A., Honoré, C.G., Johnston, K.A., Samai, P.K. (2004). Cheddar Cheese and Related Dry-Salted Varieties in Cheese: Chemistry, Physics and Microbiology, vol. 2, 3rd ed., Major Cheese Groups Ed: Fox, P. et al., Elsevier, Amsterdam, the Netherlands, pp. 71–102. Leboeuf, B., Guillouet, P., Batellier, F., Bemelas, D., Bonne, J.L., Forgerit, Y., Renaud, G., Magistrini, M. (2003). Effect of native phosphocaseinate on the in vitro preservation of fresh semen. Theriogenology, 60, 867–877. Le Jaouen, J.C. (2000). Curd Carry Over in Cheesemaking Ed. A. Eck and J.-C. Gillis, Lavoisier, Paris, pp. 341–349. Léonil, J., Nau, F., Mollé, D., Maubois, J.-L. (1991). Procédé d’obtention à partir de caséine b de fractions enrichies en peptides à activité biologique et les fractions peptidiques obtenues, Brevet FR 2,650,955. Léonil, J., Mollé, D., Fauquant, J., Maubois, J.-L., Pearce, R.J., Bouhallab, S. (1997). Characterization by ionization mass spectrometry of lactosyl b-lactoglobulin conjugates formed during heat treatment of milk and whey and identification of one lactose binding site. Journal of Dairy Science, 80, 2270–2281. Le Squeren, J.C., Canteri, G. (1995). Procédé pour éliminer les cellules somatiques des milieux alimentaires ou biologiques et produits correspondants. Brevet FR 2,731,587. Leszek, J., Inglot, A.D., Jjanuz, M., Lisowski, J., Krukowska, K., Georgiades, J.A. (1999). Colostrinin®: a Proline-rich polypeptide (PRP) complex isolated from ovine colostrum for treatment of Alzheimer’s disease. A double blind, placebo controlled study. Archivum Immunologiae et Therapiae Experimentalis, 47, 377–385. Lindquist, A. (1998). A method for the production of sterile skimmed milk. PCT Patent WO no. 57549. Loeb, S., Sourirajan, S. (1963). Advances in Chemistry Series, 38, 117. Madec, M.N., Mejean, S. and Maubois, J.L. (1992) Retention of Listeria and Salmonella cells contaminating skim milk by tangential membrane microfiltration (“Bactocatch process”), Le Lait, 72, 327–332. Mahaut, M. (1990). Approfondissement des connaissances sur la nature des mécanismes physiques et biochimiques intervenant sur les propriétés rhéologiques des fromages frais. PhD thesis, ENSAR–Rennes University, France. Mangino, M.E. (1992). Properties of Whey Protein Concentrates in Whey and Lactose Processing. Ed J.G. Zadow, Elsevier, New York, pp. 231–270. Marshall, P.G., Dunkley, W.L., Lowe, E. (1968). Fractionation and concentration of whey by reverse osmosis. Journal of Food Technology, 22 (8), 969–970, 974, 976, 978. Maubois, J.-L., Mocquot, G., Vassal, L. (1969). Procédé de traitement du lait et de sous produits laitiers French Patent no. 2,052,121. Maubois, J.-L., Mocquot, G. (1971). Préparation de fromage à partir de préfromage liquide obtenu par ultrafiltration du lait. Lait, 51, 495–533. Maubois, J.-L., Mocquot, G., Vassal, L. (1972). Procédé de traitement du lait et de sous produits laitiers. French Patent no. 2166315. Maubois, J.-L. (1974). Utilisation des techniques à membrane: osmose inverse et ultrafiltration dans les industries agricoles et alimentaires. Bulletin Technique d’Information 291. Maubois, J.-L., Pierre, A., Fauquant, J., Piot, M. (1987). Industrial fractionation of main whey proteins. IDF Bulletin, 212, 154–159. Maubois, J.-L. (1987). Proceedings of the Cornell Symposium Honoring Frank Kosikowski Cheese, Biotechnology and International Food Development, Cornell University, Ithaca, p 24. Maubois, J.-L. (1989). Applications of membrane techniques in the dairy industry—proposals for a new IDF group of experts. IDF Bulletin, 244, 26–29. Maubois, J.-L., Ollivier, G. (1997). Extraction of Milk Proteins in Food Proteins and Their Applications. Ed Damodaran S. and Paraf A., Marcel Dekker, New York, pp. 579–595.

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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

Aqueous Two-Phase Systems Applied to Whey Protein Separation

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%

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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

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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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65

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)

Aqueous Two-Phase Systems Applied to Whey Protein Separation

67

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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Engineering Aspects of Milk and Dairy Products

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.)

Aqueous Two-Phase Systems Applied to Whey Protein Separation

69

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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Engineering Aspects of Milk and Dairy Products

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).

Aqueous Two-Phase Systems Applied to Whey Protein Separation

71

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.

Aqueous Two-Phase Systems Applied to Whey Protein Separation

•

•

•

•

73

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.

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.

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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)

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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.

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Engineering Aspects of Milk and Dairy Products

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 < n < 1). When n = 1, Toth’s isotherm equals Langmuir’s isotherm. The parameters b and n provide an independent fitting of the initial slope and of the curvature of the isotherm. 5.4.1.6 Jovanovic Isotherm This model was derived for adsorption in a solid, homogeneous surface, considering the phenomenon as nonlocalized, without lateral interactions, and allowing for a monolayer of solute to be formed. The corresponding equation is as follows:

q = qs [1 − exp(− b ⋅ C )]

(5.7)

where qs is the surface concentration for solute saturation, and b is the appropriate binding constant. 5.4.1.7 Exponentially Modified Langmuir Isotherm In the case of molecules of biological origin, such as proteins, Langmuir’s isotherm provides a less than satisfactory description of hydrophobic adsorbents due to two main reasons: • The binding of many proteins in hydrophobic adsorbents is based in multivalent interactions. • The adsorption of proteins in a hydrophobic medium is highly influenced by the concentration of other components, such as salts, but Langmuir’s model alone is unable to express this behavior despite the fact that the parameters of the model are implicit functions of the concentration of salts.

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In order to circumvent this problem, an exponentially modified Langmuir isotherm model was proposed which contains a parameter related to the contribution of salt concentration to protein adsorption isotherms: q=

λ b exp(− kCs )C 1 + b exp(− kCs )C

(5.8)

where l, b, and k are parameters of the equation; Cs is the salt concentration in the liquid phase; and C is the protein concentration in the liquid phase.

5.4.2 Determination of Adsorption Isotherms There are several methodologies that can be used to experimentally determine adsorption isotherms. The most widespread and best understood are the batch method and the frontal analysis method, which is used in packed-bed systems. 5.4.2.1 Batch or Stirred Tank Method The procedure for the determination of adsorption isotherms in batch, or in a stirred tank system, is used when the adsorbent’s capacity and time for equilibrium are sufficiently high to ensure that the saturation of the adsorption sites is quickly achieved in a single step, as sought in most laboratory experiments. Due to the simplicity of the mass balances involved, the stirred tank methodology is also used for mass transfer studies between the fluid phase and the adsorbent. These data are calculated based on the values of substrate concentration in the fluid phase. Batch adsorption experiments generate information on the amount of solute in equilibrium which cannot be obtained in continuous flow experiments. That information allows the establishment of an exact relationship between the flux in diffusive processes and the accumulation of the adsorbate inside the adsorbing materials. A simple way to determine an adsorption isotherm is to use a series of stirred tank reactors, as shown in Figure 5.2. The reactors are filled with predefined amounts of adsorbent which are put into contact with predefined volumes of solutions (liquid or gaseous) containing the adsorbate in increasing concentrations. The reactors are kept under constant agitation, at controlled temperature and pressure, until the equilibrium is attained. The quantification of the amount of adsorbate in a solution is performed before and after the adsorptive process takes place. This allows determination of the corresponding equilibrium condition—that is, the equilibrium concentration of the adsorbate in the adsorbent. Therefore, for each stirred tank, the concentration of adsorbate in the equilibrium is given by the following:



qi =

Vi (Ci − Ceq ,i ) mi

(5.9)

where qi is the concentration of the adsorbate in the adorbent; Vi is the volume of solution in the stirred tank; Ci is the initial adsorbate concentration in the solution in the tank; Ceq,i is the equilibrium concentration of the adsorbate in that solution; and

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V1

=

V2

=

V3

=

V4

=

V5

=

...

=

Vn

... 1

2 < =

C1 m1

3

C2 m2

< =

4

C3 m3

< =

C4 m4

5 < =

C5 < m5 =

n ... ...

< Cn = mn

Keep under agitation until equilibrium is reached Ceq1

≠

Ceq2

≠

Ceq3

≠

Ceq4

≠

Ceq5 ≠ ...

≠ Ceqn

Figure 5.2  Experimental setup for the determination of adsorption isotherms using the batch methodology.

mi is the adsorbent weight. Index i represents the tanks used (i varies from 1 to n, n being the last tank of the series). If in each tank a solution of the adsorbate with a different initial concentration is placed, a different equilibrium concentration will be achieved, thus providing a data point for the experimental adsorption isotherm (Figure 5.3).

Concentration of the Adsorbent, in Equilibrium

5.4.2.2 Frontal Analysis Method The frontal analysis (FA) method was developed and used for the first time by James and Philips and by Schay and Szekely in the determination of adsorption isotherms.

n 5

...

4 3 2 1 Concentration in Solution, in Equilibrium

Figure 5.3  Experimental adsorption isotherm obtained by the batch method.

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This method consists of provoking successive changes in the adsorbate concentration at the inlet of a packed column and determining the rupture curves. Among the available methods to determine the adsorption isotherms of simple components, FA is the most precise. Among other applications, it has been used to determine isotherms of peptides and proteins in different types of chromatographic techniques. This method is adequate for adsorption works in small-diameter columns due to advantages such as a reduction in the amounts of adsorbent, adsorbate, and solvent needed in the experiments. For intensive work, it is recommended that a series of solutions of the adsorbate with known concentrations be prepared. In FA, the amount of adsorbate, qi+1, can be determined by



qi +1 = qi +

(C 1+i −Ci )(VF ,i +1 − V0 ) Va

(5.10)

where qi and qi+1 are the amounts of compounds adsorbed in a volume of adsorbent after the ith and the (i + 1)th step, when at equilibrium with the concentrations Ci and Ci+1, respectively; VF,i+1 is the retention volume of the (i + 1)th inflexion point of the rupture curve; V0 is the void volume of the column; and Va is the volume of adsorbent in the column.

5.4.3 Solute Desorption The desorption phenomenon is the opposite of the adsorption phenomenon—that is, it is when molecules of a given solute initially bound to a solid surface are transferred to a fluid phase. Once, in general, the conditions at the fluid–solid interface are of thermodynamic equilibrium, it will be necessary to step away from that equilibrium when performing research on adsorption/desorption dynamics. This can be achieved by several means: • Through an isothermal desorption process (IDP), consisting in the reduction of the gas phase pressure at the surface of the solid. • Through the rapid heating of the adsorbate, a technique known as flash desorption process (FDP), or, according to some simple relationship of temperature as a function of time, also known as programmed temperature desorption (PTD). • Through the use of some external influence (electrons, electromagnetic radiation, etc.). In the beginning of the process, the adsorbate is taken to an excited state, and when starting to relax to a new configuration, energy is released which will increase the degrees of freedom of the adsorbate, thus setting it free to the fluid phase. A method using this concept is infrared photodesorption.

5.4.4 Sorption Hysteresis While adorbing or desorbing, some compounds have the same equilibrium relationship with the adsorbent; therefore, such equilibrium can be represented in a single

94 Adsorbed Weight/Unit Weight of the Adsorbent

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Adsorption

Desorption

Solute’s Concentration at Equilibrium

Figure 5.4  Sorption isotherm with hysteresis.

equilibrium curve, totally reversible. This is the case, for example, of the adsorption and desorption of acetone in activated carbon. Occasionally, a difference may exist between the adsorption and desorption equilibrium, leading to adsorption and desorption curves that do not coincide. In this case, adsorption is represented in one direction, and desorption is represented in the other direction. Figure 5.4 shows a sorption isotherm with hysteresis.

5.5 Conservation Equations Involved in Adsorption Mass and energy balances, and eventually momentum balances, are used when representing an adsorptive process. Balance equations can be presented in several ways: as a function of the specific application and of the accuracy needed. Examples of the use of such equations are given below, where mass balances for two types of adsorptive processes and an energy balance for a fixed packed bed are presented.

5.5.1 Mass Balances At the microscale, a given adsorbate can exist in three locations: at the adsorbed phase, in the fluid inside the pores, or in the fluid phase out of the adsorbent particles. As a consequence of this, a mass balance must consider terms involving ni (adsorbed weight of the adsorbate per unit weight of the adsorbent), cpi (concentration of the adsorbate inside the pores), and ci (concentration of the adsorbate in the fluid outside the adsorbent particles). Once it is impossible to determine the local concentration inside the particles of the adsorbent, the terms qi and cpi will be used as average concentration values. Figure 5.5 shows the regions involved in this mass balance where the adsorbate may be present.

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Pores

Stagnant fluid around the adsorbent

Fluid

Figure 5.5  Scheme of the structure of an adsorbent particle.

For batch processes, or in stirred tanks, the mass balance for a given component i is given by m⋅



dqi d (V ⋅ ci )  + = Vin ⋅ ci ,in − Vout ⋅ ci ,out dt dt

(5.11)

with ε   ε  qi = qi +  p  ⋅ c p,i = qi + (1 − ε ) ⋅ p  ⋅ c p,i ρb    ρp 



(5.12)

where .m is the adsorbent mass; V is the fluid volume external to the adsorbent particle; V is the volumetric flow rate entering (in) and exiting (out) the particle; r p and r b are the densities of the particle and of the fluid, respectively; and e and e p are the volume fraction outside the particle and the particle’s porosity, respectively. For a process in a fixed bed, the mass balance for component i is given by

ρb ⋅

∂qi ∂c ∂( v ⋅ ci ) ∂  ∂y  +ε ⋅ i +ε ⋅ = ε ⋅ DL ⋅  c i  ∂t ∂t ∂z ∂z  ∂z 

(5.13) where v is the interstitial velocity of the fluid; DL is Fick’s axial dispersion coefficient; and yi is the molar fraction of component i in the fluid phase.

5.5.2 Energy Balance The energy balance for a fixed bed, ignoring dispersion, is given by



ρb ⋅

∂U s ∂(ε b ⋅ c ⋅ U f ) ∂(ε ⋅ v ⋅ c ⋅ H f ) 2 ⋅ hw ⋅ (T − Tw ) + + =− ∂t R ∂t ∂z

(5.14)

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where Us and Uf are the internal energies of the stagnant phase and of the fluid phase, respectively; Hf and Hw are the enthalpy of the fluid phase and the heat transfer coefficient at the wall of the column, respectively; and Rc is the radius of the column. The second term in Equation 5.14 expresses the contributions in terms of energy, both for the fluid in the pores and for the fluid outside the particle. It is necessary to use thermodynamics to evaluate the enthalpy (or the internal energy) of the fluid phase and the internal energy of the stagnant phase. For a process in which the fluid is a gas at low or moderate pressures, the contribution of this second term for enthalpy calculation may be ignored. Therefore, it is necessary to define the reference state for each pure component as being that of an ideal gas at a given temperature T°, and the reference state for the stagnant phase as being that of the adsorbent free from the adsorbate, also at T°. Therefore, it holds for the gaseous phase that Hf =

∑

( yi ⋅ H f ,i ) =

i

∑ i

T   yi  H 0f ,i + C p0 dT    T0  

∫

(5.15)

and P c

Uf = Hf −



(5.16)

where Cp0 is the heat capacity of the ideal gas, and P is the pressure in the gas phase. And for stagnant phase, U s = Ul + q ⋅ U a ≈ Ul + q ⋅ H a



(5.17)

and T

Ul = Ul0 +

∫C

p ,s

dT

(5.18)

T0

where Cp,s is the heat capacity of the adsorbent, and q is the weight of the adsorbate per unit weight of the adsorbent. The enthalpy of the adsorbed phase, Ha, is evaluated considering that each component of the gas phase goes through a temperature shift from T° to T, followed by an isothermal adsorption, resulting in

Ha =

∑ i

1 xi H f ,i −    n

qi

∑ ∫ f (q , T ) dq i

i

0

where xi is the molar fraction of the adsorbed phase.

i

(5.19)

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Chromatographic Techniques Applied to Dairy Product Manufacturing

For the condition of isosteric energy of adsorption (the isosteric condition is given by the change of pressure with temperature, for a constant adsorptive capacity, ni), qist depends on the system’s composition. The sum of integrals in Equation 5.19 becomes difficult to evaluate for multicomponent adsorption if the isosteric energy of adsorption of each component is, in fact, dependent on the weights (contributions) of each of them. Once the isosteric energy of each component depends on the contribution of all the other components, the sum must be evaluated starting from a clean adsorbent condition and finishing with the contributions of all components. If the isosteric energy is constant, as it is usually considered to be, then the energy balance in Equation 5.14 becomes   ρ  c +  b s  

=−

∑ i

  ∂T qiC p0,i  + ε bcC p0  − ρb   ∂t  

∑ f (q , T ) ∂∂qt − ∂(ε∂tP) + ε vcC i

i

b

2hw (T − Tw ) R

0 p

∂T ∂z

(5.20)

where Equation 5.13 has been used assuming DL equals zero. Equation 5.20 is a common expression for the energy balance in a fixed bed. Often, the first sum of the lefthand side of Equation 5.20, involving gas phase heat capacities, is neglected, or the gas phase heat capacities are replaced by the heat capacities of the adsorbent phase. Nonisothermic processes with a liquid phase involved may be conducted by changing the temperature at the feed stream, or by heating or cooling the column through its wall. This means that adsorption energies and pressure effects are minor influences in this case, and the energy balance becomes



ρ bcs + ε bcC p0  ∂T + ε vcC p0 ∂T = − 2hw (T − Tw ) R ∂t ∂z

(5.21)

5.6 Kinetics of the Adsorptive Process The adsorption kinetics of a given compound on an adsorbent involve a sequence of steps, which occur both inside and outside of that adsorbent. In short, the adsorbate will approach the adsorbent solid, migrate to its surface, and from there migrate to its pores, where it will finally be adsorbed at the active sites. This sequence involves mechanisms such as those described below.

5.6.1 Intraparticle Transport Mechanisms Intraparticle transport may be limited by diffusion in the pore, diffusion in the solid, kinetic reactions at the pore surroundings, or two or more of these mechanisms together: • Diffusion in the pores: Pores are sufficiently large to allow the adsorbate molecules to escape from the force fields at the adsorbent surface. The forces governing this diffusive process may be estimated through the molar fraction gradient or, if the molar concentration is constant, through the concentration gradient of the diffusing species inside the pores.

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• Diffusion in the solid: This occurs when the diffusion in the pores is sufficiently small so that the adsorbate molecule does not escape from the force fields at the adsorbent surface. Transport may occur through an activated process involving “jumps” between the adsorption sites. This phenomenon is usually called surface diffusion. The forces governing this process may be estimated through the concentration gradient of the adsorbate. • Kinetic reactions at the pore surroundings: Adsorption and desorption rates in the porous adsorbents are usually controlled by mass transfer inside the pores and not by surface sorption kinetics. Exceptions are the cases of chemisorption and affinity adsorption, used for biological separations, where the kinetics for the establishment of bonds may be excessively slow. Intraparticle convection may also occur in packed beds when the adsorbent particles have big, well-interconnected pores. However, in general, the flow through the pores of the adsorbent particles represents only a small fraction of the total flow. Once intraparticle convection may affect the transport of molecules that diffuse very slowly, the force governing convection, in this case, is the pressure drop of each particle, generated by the frictional resistance of the flow through the packed bed.

5.6.2 Extraparticle Transport Mechanism Extraparticle transport is affected by the configuration of the equipment designed for adsorption and by the hydrodynamic conditions outside the adsorbent particles. External mass transfer between the surface of the adsorbent particles and the neighborhood of the fluid phase is governed by forces related to the concentration difference between phases and the degree of mixing, agitation, or turbulence, of the fluid phase. In some cases, the mixing conditions of the fluid phase may lead to the occurrence of dead zones in packed beds or to inefficient mixing in stirrers, thus changing the hydrodynamics of the flow and, consequently, changing the mass transfer rate from the fluid to the adsorbent. In packed beds, mixing is usually described in terms of an axial dispersion coefficient, where all the effects related with axial mixing are included.

5.6.3 Axial Dispersion Coefficient As mentioned before, the axial dispersion coefficient considers all the effects caused by the axial mixing of the fluid phase when flowing through a packed bed: it includes the diffusive and convective effects and also inequalities in the local velocities throughout the column. Assuming a uniform velocity throughout the column, the axial dispersion coefficient, DL , is given by



d v DL (Re)( Sc) = γ1 +γ 2 p = γ1 +γ 2 Di Di ε

(5.22)

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99

Or, in terms of the Peclet number (Pe = dpv/DL ),



1 γ 1ε = +γ2 Pe (Re)( Sc)

(5.23)

The first term on the right-hand side of Equations 5.22 and 5.23 considers the diffusive effects, and the second term considers the mixing effects. In short, a solute molecule in the fluid phase, influenced by mixing effects, approaches a steady fluid film around a particle of adsorbent. Due to the solute’s concentration difference, this solute diffuses through the film, eventually reaching the surface of the solid adsorbent. Still diffusing, the molecules of the solute migrate inside the pores where they are finally adsorbed at the adsorption sites. The molecules may further diffuse inside of the solid structure, but the rate at which that occurs is very low, thus rendering this effect negligible.

5.7 Adsorption Operation Modes Adsorption is unique in its various possibilities of application. It is used in a wide variety of processes such as vapor recovery from a mixture of gases, solute recovery, and removal of contaminants from liquid solutions and fractioning of liquid and gaseous mixtures. In operational terms, adsorption may be conducted in batch, semibatch, or continuous mode. In each of these categories, it is possible to find characteristics that are similar to those of other mass transfer processes. In fact, when only one component of a fluid mixture (gaseous or liquid) is strongly adsorbed, it is possible to establish an analogy with the approach used for gas absorption, being that in the adsorption process, the adsorbent plays the role of the insoluble phase in absorption; when more than one component is strongly adsorbed, separation requires a fractioning procedure that is analogous to liquid extraction, and the adsorbent corresponds to the solvent in extraction. The following text addresses batch adsorption and packed-bed adsorption; for this latter process, reactor dimensioning will also be discussed.

5.7.1 Batch Adsorption Batch adsorption (Figure 5.6) is frequently used to adsorb solutes from liquid solutions when the amount of solution to be treated is small, such as in some cases in the pharmaceutical industry. Keeping in mind that in many batch extraction or purification processes, equilibrium relationships, such as Freundlich or Langmuir isotherms, and a mass balance are needed for engineering calculations, and the mass balance for the adsorbate is given by

q0 M + c0 S = qM + cS

(5.24)

where q 0 and q are the initial and final solute concentrations in the adsorbent, respectively; c 0 and c are the initial and final solute concentrations in the solution, respectively; M is the adsorbent weight; and S is the volume of the solution to be treated.

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Figure 5.6  Batch adsorption system.

Once the adsorption process has occurred, the solute’s equilibrium concentration values, both in the adsorbent and in the solution, can be obtained from the same graph as the adsorption isotherm and the mass balance. The interception of both curves provides the values of the equilibrium parameters, as shown in Figure 5.7.

5.7.2 Fixed-Bed Adsorption Many techniques used to adsorb solutes from liquids or gases use packed-bed reactors containing adsorbent particles. Such operation, also called percolation, is used in some steps of liquid discoloration processes, in oil clarification (both vetegable

Isotherm qeq Adsorptive Capacity

Mass balance

Ceq

Equilibrium Concentration

Figure 5.7  Calculation of the adsorption equilibrium parameters.

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101

oils or oil derivatives), and in sugar production from which both ashes and color compounds are removed. The fluid to be treated is passed through the adsorbent bed, commonly called a filter. This filter is usually installed in a vertical cylindrical vessel, with dimensions of up to 4.5 m diameter and 10.0 m height. It may contain up to 50,000 kg of adsorbent, supported by a sieve on a perforated plate. Fluid flow is constant, but this process is a more complex situation than the batch process, once the equilibrium state is not reached. The global dynamics of the system defines the process efficiency better than equilibrium considerations alone. The liquid flow is usually descendant, either natural or forced. A very useful practical tip is to leave the column permanently flooded, thus avoiding the presence of air in the bed and prolonging the life of the adsorbent. In discoloration processes, the concentration of impurities in the initial effluent is usually much lower than the product’s specifications, and the rupture curve has a small slope. It is therefore common practice to accumulate the effluent generated during the operation in a buffer tank placed after the reactor and to allow the process to continue until the product’s specification is met, thus achieving the maximum tolerable level of impurity. This also allows that the maximum concentration of adsorbate is accumulated on the adsorbent. When this happens, the adsorbent needs to be regenerated. This process is preceded by the interruption of the flow of the liquid being treated and by draining what is left of it inside the column. The regeneration can then start, using the same column, by washing the adsorbent with the appropriate solvents or by removing the adsorbent from the column and performing a reactivation step (e.g., by submitting it to high temperatures). The desorption of the adsorbate, using solvents, is called elution. The solvent provoking desorption is called the eluent, and the solution resulting from the process is called the eluate. In order to perform a successful elution, the amount of solute removed must be equal to the amount that was adsorbed inside the column; if this does not happen, the column will gradually lose its efficiency. The solute’s concentration in the liquid phase and in the adsorbent changes with its time and position along the bed during the adsorptive process. At the inlet of the bed, when the process starts, it is considered that the solid is free from any solute. Once the fluid flow starts, mass transfer starts as well, more precisely at the inlet of the bed. With the flow of the solution throughout the bed, the solute’s concentration in the fluid phase drops sharply with the distance until its value eventually reaches zero, before the outlet, at a time t0. After a short period, the solid closer to the inlet is practically saturated, and most of the mass transfer now happens somewhat deeper in the bed. This is a gradual process that will eventually lead to the complete saturation of all the solid adsorbent in the bed, after a certain time tf . The saturation of the packed adsorbent by the adsorbate as a function of time is shown in Figure 5.8. If we now turn our attention to the outlet of the column, as a fixed observer, the observation will be that the concentration of solute in the effluent is approximately zero for a large period of time, until practically all the packed adsorbent is saturated. From this moment on, the solute’s concentration rises slowly, until a value cb is attained. The time at which this happens is called the break point. After the break point, the solute’s concentration rises sharply until cd, which is usually equal (or very close) to the value

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Solute Concentration

t1 < t2 < . . . tf

t1

...

t2

tf

Bed Length

Figure 5.8  Packed-bed saturation.

of the initial concentration of solute in the solution, c0. This point signals the end of the rupture curve, indicating that the bed is saturated and has lost its effectiveness. The value of cb represents the maximum concentration of solute which is allowed to be discarded, which usually varies between 1% and 5% of c0. When cb is attained at the outlet of a packed bed undergoing an adsorptive pro­cess, such a process is interrupted to regenerate the column and to provoke the de­sorption of the solute. The flow of solution may then be deviated to a second column while the first one is regenerated. A typical rupture curve is shown in Figure 5.9. For a narrow mass transfer region, the rupture curve is very steep, where the most significant part of the bed’s capacity is used until the break point is attained. This renders the use of the adsorbent more efficient, besides reducing its regeneration costs.

c/c0

1

cd

Mass transfer region

cb Time

Figure 5.9  Characteristic rupture curve.

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5.7.2.1 Bed Capacity and Scale-Up The shape and width of the mass transfer zone depend on the adsorption isotherm, flow rate, mass transfer rate for the particles, and diffusion in the pores. The prediction of the mass transfer zone as well as of the concentration profile in the bed may be performed using several theoretical methodologies; however, practical experiments are needed to perform a scale-up and to validate predicted data. The full capacity of the packed bed, if it reaches the equilibrium state with the feed flow, is proportional to the area between the curve and the line c/c0 = 1, as shown in Figure 5.10. The total shadowed area represents the total capacity of the bed and may be described mathematically as ∞

tt =



c

∫  1 − c  dt

(5.25)

0

0

where tt is the time equivalent to total capacity. The usable capacity of the bed is that measured until the break point tb, which is represented by area 1 in Figure 5.10. Mathematically, it can be described by tb

tu =



c

∫  1 − c  dt

(5.26)

0

0 where tu is the time equivalent to the usable capacity (i.e., the time elapsed until the solute’s concentration in the effluent reaches the maximum value allowed). The value of tu is generally very close to the value of tb. The ratio tu/tt represents the fraction of the total capacity (or length) of the bed which is used before reaching the break point. If the total length of the bed is represented by HT, and the length used to reach the break point is HB, then

HB =



tu HT tt

(5.27)

1

Cd

c/c0

2 1

Time

tb

Cb

Figure 5.10  Total and usable capacity of a packed bed.

td

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Engineering Aspects of Milk and Dairy Products

In turn, the length of the unused bed, HUNB, is given by the unused time fraction of the total length:



 t  HUNB =  1 − u  HT tt  

(5.28)

The unused length, HUNB, represents the mass transfer zone. It depends on the fluid velocity, and it is essentially independent of the column’s total length. HUNB may be experimentally obtained in laboratory columns, of small diameter, packed with the adsorbent being studied. This means that the usable part of the bed may be calculated for large-scale equipment by means of simply calculating the length of the column, HB, used until the break point is reached. HB is directly proportional to tb. Finally, the total length of the column, HT, can be obtained simply by adding HB and HUNB. This procedure is widely used to scale packed beds, and its validation depends on the working conditions of the laboratory column being similar to those of a largescale unit. The small diameter of the first column is necessary to ensure that similarity with the large adsorption towers occurs, once in these cases the process is essentially adiabatic. The mass flow rate in both cases must be equal, and the bed should be sufficiently long to contain a mass transfer region at steady state. Axial dispersion or axial mixing are not the same in both conditions, but if care is taken, this is a very useful method.

5.7.3 Adsorption in Expanded Beds Adsorption in expanded beds is a one-step operation where unwanted compounds, such as protein, are adsorbed from a particularly dirty feed with no need for its clarification, centrifugation, and initial purification, which are, in general, demanded operations for fixed-bed processes (Figure 5.11). The expansion of the bed creates a distance between the adsorbent particles—that is, it increases the bed porosity, which in turn opens the way to cells, cell fragments, and other particles during the application of the solution. The principle of adsorption in expanded bed considers that the adsorbent is expanded and equilibrated by the application of an ascending flow in the column. A stable fluidized bed is formed when the adsorbent particles are suspended in equilibrium due to the balance between the settling velocity of the particles and the velocity of the liquid flow. The unprocessed feed is subsequently fed to the expanded bed, maintaining the same liquid flow rate. Proteins adsorb (by ionic exchange, hydrophobic interactions, affinity) to the adsorbent and fragments of cells, whole cells, particles, and contaminants pass through the bed. Once the adsorption step is finished, the materials that are loosely bound are washed with the initial buffer solution; when this operation has ended, the flow is stopped, therefore promoting the sedimentation of the adsorbent

Chromatographic Techniques Applied to Dairy Product Manufacturing

Fixed-Bed Process

Expanded-Bed Process

Fermentation

Fermentation

105

Clarification Concentration

economy in process steps

Expanded Bed

Initial Purification Intermediate Purification

Intermediate Purification

Final Purification

Final Purification

PRODUCT

PRODUCT

Figure 5.11  Comparison between the purification processes using adsorption in fixed bed and in expanded bed.

particles. This procedure is followed by the elution of the adsorbed proteins; their adsorption is promoted by the reverse flow of a buffer solution containing the appropriate salt concentration. Following the elution step, the sedimented bed is regenerated by a descending flow of buffer solutions, specifically chosen for the chromatographic principle applied in the separation. This regeneration removes those proteins that are more strongly bound and therefore were not removed during the elution phase. Finally, a cleaning-in-place procedure is applied to remove nonspecifically bound, precipitated, and denaturated substances, so that the original performance of the adsorbent is restored.

5.7.3.1 Stable Fluidization Adsorption in expanded beds is based on the control of a stable fluidization, which combines the hydrodynamic properties of a fluidized bed with the chromatographic properties of a fixed bed. The achievement of stable fluidization with minimal backmixing, vortexing, and turbulence in the bed leads to the formation of several mass transfer units, or theoretical plates, in the expanded bed, thus increasing the performance of a traditional packed column.

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5.7.3.2 Critical Parameters In addition to the adsorbent and the column, there are other critical parameters in adsorption which may be divided in physical and chemical parameters. Chemical parameters are those related with selectivity and volumetric capacity of the separation processes and include pH, ionic strength, and types of ions and buffers used. The influence of these parameters in the separation performance of expanded-bed adsorption and traditional fixed-bed chromatography is virtually the same. Physical parameters are those related with hydrodynamics and stability of a homogeneous fluidization in the expanded bed. Some physical parameters are related with the concentration of the bulk, such as viscosity or density; others are related with operation conditions, such as temperature, flow velocity, and bed height. In general, chemical parameters are optimized in fixed-bed operation, and physical parameters are optimized in expanded-bed operation, once these are related with the hydrodynamic properties of the bed. Example 5.1 b-Lactoglobulin, the main allergenic component of cheese whey, will be partially removed from 1000 L of whey by a batch adsorption process. Whey has 3.5  g/L of that protein, and the process will use 15 kg of an adsorbent that behaves according to the Langmuir isotherm model. Knowing that the whey and the adsorbent will be kept in contact until equilibrium is reached, determine the remaining concentration of b-lactoglobulin in the whey and the efficiency of the process. Given: Absorption isotherm equation ⇒ q =



179,3 + C 0,2 + C

where q is in g/kg and C in g/L. Solution: assuming that the amount of adsorbed protein will not significantly change the total volume of whey and the total weight of adsorbent, the mass balance for this process will be

( )

0 × 15( kg) + 3, 5 g L × 1000(L) = qeq × 15( kg) + Ceq × 1000(L)



⇒ qeq =

3500 1000 − Ceq ⇒ qeq = 233, 33 − 66, 67Ceq 15 15



(5.29)

The diagram of q versus c, shown in Figure 5.12, is a graphical representation of the mass balance and the adsorption isotherm for the case evaluated in Example 5.1. From this graphical representation, it is possible to conclude that for the given operational conditions, equilibrium is achieved for a concentration of b-lactoglobulin in the whey (Ceq) equal to 1.2 g/L and a concentration in the adsorbent (qeq) of 155 g/kg. This means that the efficiency of the process equals



E (%) =

C0 ⋅ S − Ceq ⋅ S 3, 5 − 1, 2 × 100 = × 100 ⇒ E (%) = 65, 7% C0 ⋅ S 3, 5

(5.30)

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107

225 200

Isotherm

q (g/kg)

175

qeq = 155 g/kg

150 125 100

Mass balance

75 50 25 0

Ceq = 1.2 g/L 0

0.5

1.0

1.5 2.0 C (g/L)

2.5

3.0

3.5

Figure 5.12  Equilibrium condition for the process described in Example 5.1.

5.8 Ionic Exchange Ionic exchange may be defined as a physical-chemical phenomenon in which a solution changes ions with the surface of a porous solid, as depicted in Figure 5.13. The techniques used in ionic exchange are so similar to those used in adsorption that, for most engineering applications, it can be considered as a special case of adsorption. In ionic exchange, the fluid phase is an aqueous electrolytic phase, and the solid is an electrolyte that is insoluble in the liquid, usually called ion exchange resin. The result of the operation is an exchange reaction between the electrolyte in solution and the resin. Ionic exchange has several applications, such as water softening for steam generation, water deionization, purification of pharmaceutical products (such as vitamin B and antibiotics), metallurgic processes, and the fractioning of mixtures through chromatography. The development and scale-up of equipment which make use of ionic exchange are made in the same way as for adsorptive processes. The ionic exchange rate is dependent on the processes of mass transfer of ions from the solution to the surface of the solid particle, diffusion through the pores to the interior of the solid, ion exchange, and diffusion of the exchanged ions outside the pores, in a process similar to that described for adsorption.

5.8.1 Ionic Exchangers Ionic exchangers are porous, insoluble solids, with ions and water in their structure. There is a network of fixed ions and also moving ions (anions or cations) that are susceptible to exchange by ions of the same electrical charge present in electrolytic solutions. The solid acts in a selective way, by removing from those solutions the ions for which it has more affinity.

108

Engineering Aspects of Milk and Dairy Products Ionic Exchange –

–

–

Solvent flow

+

–

–

+

– –

–

+ –

–

+ –

–

–

+

–

+

– –

+ –

–

Adsorbent

– –

+

–

Counter-ions

+

–

+ –

–

+

–

+ –

–

+

–

–

– – – +

+

–

– + –

–

– –

– +

–

–

+

Ions in solution

+

–

–

–

–

– –

+

–

–

+

– –

+

–

+

– – + –

Figure 5.13  An anionic exchange process.

According to their structure, ion exchangers are divided into mineral (either natural or synthetic) and organic. According to their function, they can be cationic, anionic, or amphoteric, depending on the ions that can be exchaged with an external solution. They can be further divided into weak or strong exchangers, depending on the degree of exchange that can be achieved. Cationic exchangers are those that have acid functional groups in their structure, thus rendering them negatively charged; they are neutralized by cations that can be exchanged with cations present in solution. The main acid functional groups used to produce cationic exchangers are sulfonic acid and its derivatives (for strong exchangers) and carboxilic acids and their derivatives (for weak exchangers), and sodium (Na+) and hydrogen (H+) ions are the most frequently used exchange agents. Anionic exchangers, in turn, have basic functional groups in their structure, rendering it positively charged; it is neutralized by anions that can be exchanged with anions present in solution. The main functional groups used are quaternary ammonium compounds and their derivatives (for the strong exchangers) and derived tertiary amines (for the weak exchangers), and chloride (Cl–) and hydroxide (OH–) ions are commonly used exchange agents.

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Finally, amphoteric exchangers have both types of functional groups in their structure, thus acting as anionic and cationic exchange agents simultaneously.

5.8.2 The Ionic Exchange Mechanism The mechanism describing ionic exchange is not yet fully understood, but there are some theories that explain satisfactorily the phenomena taking place. The electric double-layer theory is based on the principle that the contact between two phases with different chemical compositions containing charged molecules generates a difference of potential where the separation of charges can be observed. There are several possible structures for the electric double layer. Adopting an anionic exchanger as an example, where the solid phase is positively charged and the ions to be exchanged are negative (anions), three structures may be described: • Helmholtz’s double layer: it considers that all the anions needed to neutralize the electric potential of the solid form a single layer around the solid, situated at a minimal distance d from its surface (Figure 5.14a). • Gouy’s double layer: its structure is completely diffuse, diminishing the charge intensity as a function of the distance to the solid’s surface (Figure 5.14b). • Stern’s double layer: this is an intermediate structure between the previous two: part of the anion layer is electrically dense and close to the solid’s surface, and the other part, farther from that surface, is diffuse (Figure 5.14c). The same mechanisms are valid when the solid is negatively charged and there are cations in solution. It is worthwhile to note that electric layers are not as static or homogeneous as the above definitions may suggest; in fact, at different locations on the exchanger’s surface, different structures may be observed.

5.8.3 Ion Exchange Equilibrium An ion exchanger consists basically of a solid matrix with charged ionic groups (positive or negative) bound to it. Ions with opposed charges, called counterions, neutralize those charges, thus keeping the system in equilibrium. When placed in contact with an electrolytic solution, the ion exchanger allows the replacement of the ions in solution by the ions (of the same charge) in its structure; this induces a perturbation in the system until a new equilibrium is attained. Ionic exchange occurs in a similar way as a stoichiometric reaction: with the decrease of the concentration of the counterions on the adsorbent’s surface, their concentration in the solution increases; simultaneously, the concentration of the ions in the original solution is decreased as their concentration increases at the surface of the adsorbent.

110

Surface of the Adsorbent

Engineering Aspects of Milk and Dairy Products + + + + + + + + + + + + + + + + + + + + + +

– – – –

δ

– – – – – – –

– – – – – – – – – – –

– – – – – – – – – – –

– – – – – – – – – – –

– – – – – – – – – – –

– – – – – – – – – – –

Surface of the Adsorbent

(a) + + + + + + + + + + + + + + + + + + + + + +

– – δ

– – – – – – – – –

– –

–

– –

– –

–

– –

– –

– –

–

–

– – –

–

– – –

–

–

(b)

Figure 5.14  Three different types of electric double layer in ionic-exchange processes: (a) Helmholtz, (b) Gouy, and (c) [following page] Stern.

Surface of the Adsorbent

Chromatographic Techniques Applied to Dairy Product Manufacturing + + + + + + + + + + + + + + + + + + + + + +

– – – – δ

– – – – – – –

– – – – – – – – – – –

– – – –

–

– – – – –

– –

– –

–

– –

–

111

– –

– – –

– – –

–

–

–

(c)

Figure 5.14  (Continued)

5.9 Molecular Exclusion Chromatography Chromatography is a physicochemical separation method for components in a mixture. It is performed by the distribution of compounds between two phases that are in close contact. One of the phases remains stationary, and the other (the mobile phase) passes through it. During the passage of the mobile phase through the stationary phase, the components of the mixture are distributed between the two phases; selective retention of each component by the stationary phase results in the differential migration of these components. In the purification process of a biological compound, different chromatographic techniques can be employed that differ fundamentally from the various forms in which each of the components may interact with the stationary phase. Chromatography is used to obtain countless compounds of high added value. The success of chromatographic protein separation is due to its ability to achieve high purity grades from complex mixtures in which component concentrations are minimal. This is a powerful separation method, because it can promote and facilitate the individual removal of various components from a mixture under experimental conditions in which the two phases of a system are always near their equilibrium point, therefore causing the rapid mass transfer between the two phases. The separation performance of a column is a direct function of the mass transfer rate and of the axial dispersion coefficient. The mass transfer phenomenon in a chromatography column includes the effects of diffusion, mass transfer resistance, viscosity, and adsorption and desorption kinetics.

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Chromatography can be applied at the analytical or preparative level. The main objective of analytical chromatography is the achievement of an elevated resolution for the separation of solutions during the retention time. Diluted samples are applied in small volumes (1% to 5% of the total column volume), to minimize interactions between each component of the chromatographic system. On the other hand, the focus of preparative (commercial) scale separation is not only resolution but also the overall cost of the process; in this case, the basic method to increase productivity is to maximize the use of the column by increasing the injection volume for separation. Preparative liquid chromatography is a viable technique for the isolation and purification of biomolecules such as proteins, peptides, amino acids, and enzymes. The use of molecular exclusion chromatography (MEC) as the final step of a purification process aims at changing the mobile phase to one that can be vaporized during a subsequent lyophilization or concentration step, and removing the contaminant molecules of low molecular weight, such as salts, polypropylene, nonionic detergents, among others. The most well-known MEC application in the purification of protein solutions is desalination, which occurs under conditions in which there is no protein denaturation. MEC is also applied for the determination of native or denatured protein molecular mass at different pH, temperature, and ionic force conditions. It can also be used to determine the molecular mass of natural or synthetic polymers, which is essential for the quality control of these polymers.

5.9.1 General Aspects of MEC This technique was introduced in 1959 with the name of gel filtration. It is a liquid– liquid chromatography method, capable of separating components from a mixture based on their molecular dimensions using a stationary phase composed of electrically uncharged particles. Partition occurs due to the differential access of molecules with varying sizes as they pass through the pores of the particles which make up the filtration gel. Substances with low molecular masses, which are small compared with the size of the pores in the filtration gel (e.g., salts), penetrate the pores and have greater retention volumes, virtually equal to the internal volumes of the pores occupied by the solvent. If the molecules present intermediate sizes, they will only penetrate a portion of the pore volume. When they are very large, their retention volumes will be equal to the interstitial volume of the column; this is the case of proteins. In MEC, the largest molecules are the fastest to migrate through the solid phase, and the smaller molecules are retained for the longest time. For certain substance groups, an extremely intimate correlation exists between molecular mass (molecule size) and their retention time, meaning that, in practice, retention time is directly related to molecular dimensions. Therefore, the largest molecules leave the column first, the medium-sized particles are slowed down, and the smallest particles are retained in the particle pores and their retention time is increased even more. The egression of molecules at different times is expressed as the degree of separation that, in chromatography, is named resolution.

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Ideally, a small volume of a solution of a multicomponent mixture injected in a chromatography column should be totally separated if each solute egresses with different retention volumes. Resolution can be increased by various methods, for example, using a longer column, using a gel with more selective particles, using smaller sample volumes, and using lower flow rates. The selectivity factor quantifies the separation capacity for two components, which is very important for resolution determination. This selectivity factor should be different from 1, because in this case there would be no resolution. Selectivity can be altered by changes in temperature or in the composition of either the mobile or the stationary phase. The best method is to change the nature of the mobile phase, which implies better selectivity with little influence on the capacity of the process. Changing the stationary phase is not practical, and modifying temperature has a stronger effect on ion-exchange chromatography than on other chromatographic methods. The optimal selectivity value is typically achieved by a trial-and-error procedure. It should be emphasized that variations in selectivity cause changes in molecular diffusion. In situations where many different compounds are removed in tight succession, the retention factor varies little and can have a negligible influence on resolution. The number of theoretical plates expresses the efficiency factor of the column and can be increased by using a longer column, diminishing the gel particle size, or optimizing the flow rate of the process feed.

5.9.2 Basic Principles of MEC The main mechanism governing separation by MEC is the steric effect during exclusion, or the retention of molecules of different sizes which pass through the chromatographic bed. The behavior of the system is related to entropy changes produced by molecules that penetrate the stationary phase. Theoretically, two types of separations can be identified in MEC for applications both at the preparative scale as well as at the analytical scale. One of these is the separation of molecules with high molecular masses from those with low molecular masses. It is a separation by compound groups, also known as desalination, even though no salts are involved in the process. In the other separation method, substances that have proximate molecular masses are separated by fractioning in the gel, in which no one class is defined for compounds that penetrate the pores of the gel. Superpositioning can occur during the migration of the constituents during the elution through the column. The use of desalination precedes a concentration step in the purification process and is a fast and efficient technique compared to other methods, such as dialysis. Desalination of bovine serum albumin (BSA) on the laboratory scale can be conducted with good results using, for example, a Shepadex® G-25 (superfine) resin. For applications at the industrial level, chromatography columns must be evaluated according to their utilization conditions. For example, a BPSS (Bio Processes Stainless Steel, Pharmacia Biotech) was used for industrial desalination of general biological compounds, connecting three BPSS columns in series. The best results were obtained when the columns were used in series.

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Fractioning of substances for separation of molecules with similar molecular masses can be used for the analysis and purification of peptides, proteins, enzymes, and hormones. A particular characteristic of MEC when compared to other types of partitioning chromatography is that the material that makes up the stationary phase in the chromatography bed is composed of an uncharged gel. This gel presents swelling in the presence of the solvent as well as of the mobile phase when they are passing through the bed. Typical gels used in MEC are composed of macromolecules with high affinity for the solvent used. The gel is a tridimensional network, being its mechanical stability due to its crosslinked structure. Vacancies unoccupied with structural material are filled with the liquid used as solvent. Many substances, such as polysaccharides from fruits or roots, proteins, inorganic silicates, and phosphates can make up gels. An adequate combination of these compounds with a reticulation agent can create a gel in the presence of compatible solvents. The gels react differentially to the removal or addition of liquids. In some cases, when the liquid is retained (during drying) or reincorporated, the gel may not return to its initial state. The microstructure of the gel can be homogeneous or heterogeneous. In the first case, gels present properties that indicate a homogeneous distribution of the matrix; these gels are usually softer and allow for the entrance of molecules with low molecular mass. Gels with heterogeneous structures have regions with high concentration of the matrix and others with nearly no matrix content; such structure, with large spaces, allows for the entrance of larger molecules. It is desirable that the gels present chemical constituents possessing different fractioning regions, thus enabling the adaptation of the chromatographic technique to diverse situations. For gels with microlattices (homogeneous structure), the fractioning interval is determined by properties during swelling which, in turn, are dependent on the matrix density. Varying the matrix density from 50% to 5% (w/v) in water leads to changes in the exclusion limit, in terms of the molecular mass, between 1000 and 500,000 Da. Gels with macrolattices (heterogeneous structure) can be produced with the same matrix density but exhibit variable properties due to their structural composition. The different types of gels employed in MEC vary with the material used, with the way it is produced, and with the molecular exclusion limit (MEL). Among the main gel types, the most common are those composed of dextran (Shepadex®), polyacrylamide (Bio-Gel®) (the first to be studied), agar and agarose (Sheparose®, Gelarose®, Bio-gele®, Sagarav®, Shepacril®, Sheparoce®), and rigid divinylbenzene (Shodex®). Gels that are adequate for MEC must present specific characteristics, such as being chemically inert and stable, having low ionic group levels, having uniform size and particle distribution, and having a high mechanical rigidity in order to avoid deformation caused by the liquid flow through the column. Small particles have the best resolution and separation efficiency, but a high flow resistance; on the other hand, an increase in particle size results in the diffusion of the peak. This means that the best flow conditions must be associated with the lowest resistance at which the

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optimal resolution is achieved. In general, gels are fabricated with spherical particles to facilitate flow. Viscosity is an important physical parameter that assists in the characterization of flow properties of a solution. Various factors, including temperature, pressure, time, and molecular structure, affect viscosity. High viscosity values cause instability and irregular flow and alter the separation of peaks of the solutes which make up a mixture, reducing column performance. Samples with low viscosity tend to provide good resolutions. Because viscosity depends on interactive forces between molecules, and these forces are related to the distances between molecules, an increase in pressure can result in an increase in viscosity. The viscosity of the sample influences the chromatographic separation efficiency of the solutions because greater viscosity causes high flow resistance in the column. In many cases, in analytical chromatography, high sample viscosity can significantly increase analysis time. The same happens in preparative chromatography: greater solvent concentrations and high injection volumes in the system produce fractioning among the bands and migration of the solution at different velocities. Finally, column packing is another important factor and affects the separation quality of the solvent. The homogeneity of column packing can be verified by the flow through the stationary phase of a solution of blue dextran (2 mg/mL). The movement of the colored region is observed to be uniform in a homogeneously packed column. A light transmittance test along the column is used to detect flaws, such as air bubbles.

5.10 Final Remarks Adsorptive techniques are being widely used in many industrial sectors, from oil refining and metallurgy to the pharmaceutical and food industries, thus showing their great potential for application. The study of the phenomena involved in adsorptive processes improves the understanding of the types of interactions on which they are based, thus allowing the development of more selective and more efficient adsorbents, leading to the optimization of, for example, purification, extraction, and clarification operations, among others. This chapter has presented the basics on adsorptive processes, such as type of adsorbents, equilibrium relationships, mass and energy balances, operation modes, and applications. It is hoped that the text will raise interest in adsorption and its potential uses.

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.

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Di Muccio, A.; Pelosi, P.; Barbini, D. A.; Generali, T.; Ausili, A.; Vergori, F. Selective extraction of pyrethroid pesticide residues from milk by solid-matrix dispersion. Journal of Chromatography A, v. 765, p. 51–60, 1997. Doultani, S.; Turhan, K. N.; Etzel, M. R. Fractionation of proteins from whey using cation exchange chromatography. Process Biochemistry, v. 39, p. 1737–1743, 2004. Encyclopedia of Emulsion Technology, Becher Ed, Vols. 1,2,3,4, Marcel Dekker, New York. Fang, F.; Aguilar, M. I.; Hearn, M. T. W. Influence of temperature on the behaviour of proteins in cation-exchange chromatography. Journal of Chromatography A, v. 729, p. 49–66, 1996. Gallant, S. R. Modeling ion-exchange adsorption of proteins in a spherical particle. Journal of Chromatography A, v. 1028, p. 189–195, 2004. Geankoplis, C. J. Transport processes and unit operations, 3rd edition, Prentice Hall, Englewood Cliffs, NJ, 921p., 1993. Geisser, A.; Hendrich, T.; Boehm, G.; Stahl, B. Separation of lactose from human milk oligosaccharides with simulated moving bed chromatography. Journal of Chromatography A, v. 1092, p. 17–23, 2005. Gerberding, S. J.; Byers, C. H. Preparative ion-exchange chromatography of proteins from dairy whey. Journal of Chromatography A, v. 808, p. 141–151, 1998. Graham, D. The characterization of physical adsorption systems. I. The equilibrium function and standard free energy of adsorption. Journal of Physical Chemistry, v. 57, p. 665–669, 1953. Grandison, A. S. Ion-exchange and electrodialysis—chapter 6. In: Grandison, A. S.; Lewis, M. J. (editors) Separation process in the food and biotechnology industries. Woodhead, Cambridge, p. 153–176, 1996. Gritti, F.; Gotmar, G.; Stanley, B. J.; Guiochon, G. Determination of single component isotherms and affinity energy distribution by chromatography. Journal of Chromatography A, v. 988, p. 185–203, 2003. Gritti, F.; Guiochon, G. Effect of the mobile phase composition on the isotherm parameters and the high concentration band profiles in reversed-phase liquid chromatography. Journal of Chromatography A, v. 995, p. 37–54, 2003. Gritti, F.; Guiochon, G. Heterogeneity of the surface energy on unused C18-Chromolith adsorbents in reversed-phase liquid chromatography. Journal of Chromatography A, v. 1028, p. 105–119, 2004. Gritti, F.; Guiochon, G. Influence of a buffered solution on the adsorption isotherm and overloaded band profiles of an ionizable compound. Journal of Chromatography A, v. 1028, p. 197–210, 2004. Guiochon, G. Preparative liquid chromatography. Journal of Chromatography A, v. 965, p. 129–161, 2002. Guiochon, G.; Shirazi, S. G.; Katti, A. M. Fundamentals of preparative and nonlinear chromatography. Academic Press, New York, 700p., 1994. Hahn, R.; Schulz, P. M.; Schaupp, C.; Jungbauer, A. Bovine whey fractionation based on cationexchange chromatography. Journal of Chromatography A, v. 795, p. 277–287, 1998. Hearn, M. T. W.; Hodder, A. N.; Fang, F. W.; Aguilar, M. I. High-performance liquid chromatography of amino acids, peptides and proteins. Journal of Chromatography, v. 548, p. 117–126, 1991. Holmberg, K. Handbook of applied surface and colloid chemistry, Vols. 1, 2, Ed., John Wiley & Sons, Chichester, 2002. Huang, J.; Horváth, C. Adsorption isotherms on high-performance liquid chromatographic sorbents—I. Peptides and nucleic acid constituents on octadecyl-silica. Journal of Chromatography, v. 406, p. 275–284, 1987. Huang, J.; Schudel, J.; Guiochon, G. Adsorption behaviour of albumin and conalbumin on TSKDEAE 5PW anion exchanger. Journal of Chromatography, v. 504, p. 335–349, 1990. Jacobson, J.; Frenz, J.; Horváth, C. Measurement of competitive adsorption isotherms by frontal chromatography. Industrial and Engineering Chemistry Research, v. 26, p. 43–50, 1987.

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of Lactose 6 Crystallization and Whey Protein Everson Alves Miranda*, André Bernardo, Gisele Atsuko Medeiros Hirata, and Marco Giulietti Contents 6.1 Engineering Aspects of Crystallization......................................................... 122 6.1.1 Population Balance............................................................................ 122 6.1.2 Mass and Energy Balances................................................................ 123 6.1.3 Crystal Growth.................................................................................. 125 6.1.4 Nucleation.......................................................................................... 126 6.1.5 Thermodynamic Aspects: Solubility, Supersaturation, and Heat of Crystallization................................................................ 127 6.1.6 Method of Moments........................................................................... 127 6.1.7 Agglomeration and Its Implications.................................................. 128 6.2 Protein Crystallization................................................................................... 129 6.2.1 Special Characteristics of Protein Crystals and Crystallization....... 130 6.2.2 Methods for Protein Crystallization.................................................. 131 6.2.3 Large-Scale Protein Crystallization.................................................. 131 6.2.4 Milk Protein Crystallization.............................................................. 132 6.3 Crystallization of Sugars............................................................................... 132 6.3.1 Introduction....................................................................................... 132 6.3.2 Solubility and Supersaturation Generation........................................ 133 6.3.3 Metastable Zone Width..................................................................... 135 6.3.4 Growth of Crystals............................................................................ 136 6.3.5 Crystal Product Characteristics......................................................... 136 6.4 Crystallization of Lactose.............................................................................. 138 6.4.1 Introduction....................................................................................... 138 6.4.2 Crystalline State and Polymorphs..................................................... 139 6.4.3 Solubility............................................................................................ 140 6.4.4 Metastable Zone Width..................................................................... 144 6.4.5 Nucleation.......................................................................................... 145 6.4.6 Growth Rate....................................................................................... 145 6.4.7 Kinetics of Crystallization................................................................. 146 6.4.8 Industrial Aspects.............................................................................. 148 Acknowledgments................................................................................................... 148 References............................................................................................................... 148

121

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6.1 Engineering Aspects of Crystallization The Latin word salarium, from which the word salary is derived, means “of salt” and was the periodic quantity given to Roman soldiers to purchase salt. Romans produced salt in a manner similar to the current process, evaporating seawater in shallow basins by sunlight incidence to crystallize salt. This is a simple history that shows us that crystallization is important to mankind since antiquity. Crystallization is a general name for several different operations—crystallography, melt crystallization, solution crystallization, industrial crystallization, and precipitation—that have in common two sequential phases: the birth of a solid phase (called nucleation) and its growth. In this text, we will be focused in the solution crystallization, or the crystallization from a solution. The driving force of nucleation and crystal growth is supersaturation which is the difference between concentration in the solution and equilibrium concentration at those conditions (of temperature and pressure). The magnitude of supersaturation affects differently nucleation and crystal growth, but if crystallization occurs, the solution is necessarily supersaturated. The existence of particles in suspension requires the description of the behavior of this population dispersed in a continuous phase. The developed mathematical support to describe, in general, dispersed phase systems is known as population balance.

6.1.1 Population Balance Population balance was introduced by Hulburt and Katz (1964) and then detailed to crystallization processes by Randolph and Larson (1988). The formulation of the population balance is based on population density n(L) that is derived from the number of particles per unit volume N:



n = lim

∆L→0

∆N dN = ∆L dL

(6.1)

In a practical view, population density is calculated as n=

∆m kV ρC L3 (∆L )

(6.2)

where ∆m is the mass retained in one sieve, kV is the volumetric shape factor, rC is the crystal density, L is the average size of sieve opening in which crystals were retained, and ∆L is the size difference between the sieve where crystals were retained and the sieve that is above in the sequence of sieves. The general equation to the population density balance of a crystallizer of volume V is



∂n ∂(Gn) ∂V + +n + D ( L ) − B( L ) + ∂t ∂L V∂t

i

∑ VVn = 0 k

i

(6.3)

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where n is population density, G is crystal growth rate, L is the characteristic size of the crystals, V is the crystallizer volume, D(L) and B(L) are, respectively, the rates of disappearance and appearance of crystals due to the agglomeration of crystals or their attrition and breakage as function of the size, and Vi is the volumetric flow of stream i with population density ni. In Equation 6.3, the term ∂n/∂t gives the change in number density with respect to time in the batch crystallizer and disappears in the case of a steady-state system. The expression ∂(Gn)/∂L describes the difference between crystals growing into and out of a crystal size interval dL due to the crystal growth rate G. The term n(∂V/V∂t) takes into account changes in crystal volume with respect to time (e.g., the decrease in volume in batch-operated evaporative crystallizers due to the evaporation of the solvent). The term Σk(Vi ni /V ) gives the sum of all particle flows entering and leaving the crystallizer (Mersmann, 2001). The characteristic size L deserves an explanation: it is inherent to an amount of crystals or population, which means that they would have a distribution of sizes (the crystal size distribution, CSD) with a mean and a standard deviation. Nevertheless, as crystals are three-dimensional structures (with three size distributions), usually calculations refer to one dimension—the characteristic size—associated with a size measurement methodology. For instance, sieving measures the second largest dimension of the crystals, and light scattering measures the equivalent diameter of a projected area. Admitting that there is no crystal breakage or agglomeration, that volume stays constant during the process, and that crystals nucleate with a negligible small size L 0, Equation 6.3 may be simplified to



∂n ∂(Gn) + − B 0δ ( L − L0 ) + ∂t ∂L

i

∑ VVn = 0 i

(6.4)

k

where B 0 is the nucleation rate and d(L – L 0) is the Dirac delta function acting to L = L 0 . The boundary condition to Equation 6.4 is n ( L0 , t ) =

B0 G L = L0



(6.5)

It must be observed that the population balance equation describes the evolution of particulate material in the system, only. Characterization of the system is only complete with mass and energy balances description.

6.1.2 Mass and Energy Balances Mass balance of solute in a crystallization process is



d (εVC ) =− dt

∑ε Q C k

k

k

k

−R

(6.6)

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where V is suspension volume, C is solute concentration, e is the volumetric fraction free of solids, Q is volumetric flow, k refers to k-esim stream and R is the global rate of mass transport from solution solute to the crystals. The term R may be obtained from the population balance of the crystallizer. Multiplying Equation 6.4 by Lj (j = 1, 2, …) and integrating in L results in



d (Vµ j ) +V dt

∞

∫ 0

∞

∑ ∫

(6.7)



(6.8)

∂(Gn) j L dL − VB 0 L0j + ∂L

Qk nk L j dL = 0

k

0

where m j is the j-esim moment, defined by ∞

µj =

∫ nL dL j

0

Integrating by parts and substituting in Equation 6.7, ∞



d (Vµ j ) = jV GnL j −1 dL + VB 0 L0j − dt

∫ 0

∞

∑ Q ∫ n L dL k

k

k

j

(6.9)

0

Notice that rCkVm3 is the mass of crystals by volume of suspension. That is why, assuming rC and kV constants and using M to denote the mass of crystals in the crystallizer, Equation 6.9 becomes ∞



dM = ρC kV 3V GnL2 dL + ρC kV VB 0 L30 − ρC kV dt

∫ 0

∞

∑ ∫

Qk nL2 dL

k

(6.10)

0

The first two terms of Equation 6.10 are related to mass transport from solution to the crystals; the latter term is the net flow of crystals in the crystallizer. Therefore, ∞

∫

R = ρC kV 3V GnL2 dL + ρC kV VB 0 L30

(6.11)

0

In a similar way, the energy balance of the crystallizer can be described as

ρVc p

dT d ( PV ) = − dt dt

∑ ρ Q c (T k

k k p

out k

− Tkin ) − ∆HC R − Hext

(6.12)

where r is suspension density, P is pressure, cp is specific heat, ∆HC is the heat of crystallization, Tkin is the temperature of the entering stream k, Tkout is the temperature

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125

of the outing stream k, and Hext is the heat withdrawn from the system (normally expressed as UA∆T in systems with jackets and coils, where U is the global heat exchange coefficient, A is the heat exchange surface, and ∆T is the mean logarithmic difference between suspension temperature and the heating or cooling temperature) (Miller, 1993). In order to fully characterize the system, mass, energy, and population balances have to be completed with the determination of the values of crystal growth and nucleation rates.

6.1.3 Crystal Growth A unified theory for crystal growth does not exist, but a group of complementary theories was developed, each one dealing with a single aspect of the process of crystal growth. According to Sgualdino et al. (1996), morphologic theories completely reject the parameters of kinetic growth. Beyond morphologic theories, which try to explain crystal shape by energetic interactions of each crystal face, there are the theories of adsorption layer and diffusion layer. The first ones consider that the limiting step to crystal growth is the integration of the growth-unit to the adsorption layer, and the last ones consider that the diffusion to crystal surface of the growth-unit is the limiting step. The term growth-unit must be understood as the basic “brick” of the crystal walls. It may be a single molecule (as in sucrose crystallization) or a group of molecules (as the insulin hexamer bound by a zinc atom in insulin crystallization). Although these models give good insight into the physics of crystal growth, they imply many parameters, such as surface-diffusion coefficients and kink densities, that are difficult or even impossible to determine or predict. Even though this might be possible one day, it would lead to a single-face growth rate of an ideal crystal rather than to an overall growth rate of a crystal collective in an industrial crystallizer (Mersmann, 2001). During crystallization, crystals are subjected to mechanical and thermal stresses. Therefore, dislocation generation in crystal structure is very likely, which implies density variation from one crystal to another, as among equivalent faces of a crystal. As a consequence, the phenomena as the dispersion of crystal population size and size-dependent crystal growth rate appear and cannot be explained by previous cited theories (Sgualdino et al., 1996). Theories supply knowledge to correlate experimental data and to determine kinetic parameters from the data, in order to be used in industrial crystallization process models. The overall crystal growth rate G is the the time variation of crystal characteristic size L:

G=

dL dt

(6.13)

Generally, the basic expression to describe the relationship between supersaturation (the driving-force of process) and the growth rate is

G = k g ∆C g

(6.14)

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where ∆C is the supersaturation, defined in this equation as the difference between solute concentration and equilibrium concentration. Usually, the exponent g, also named crystal growth order, does not depend on the type of the used equation and typically is a number between 1 and 2. The growth constant kg is dependent on temperature and is typically adjusted by an Arrheniuskind of equation:



E k g = k g0 exp  − G   RT 

(6.15)

where kg0 is a constant, and EG is the activation energy, which may be used to determine if crystal growth is diffusion limited or surface integration limited. Low values of activation energy point to the first hypothesis (Myerson and Ginde, 2002).

6.1.4 Nucleation Nucleation is the process of nuclei formation. Nuclei are the interface between fluid and solid phase from which crystals grow. Nucleation can be called primary or secondary nucleation depending on if it is governed by physical-chemical or purely physical phenomena, respectively. Primary nucleation can be homogeneous, if the nuclei appearance occurs in a solution free of other surfaces, or heterogeneous, if it occurs supported in an alien surface (vessel wall, impeller blades, dust particles, etc.). Primary nucleation depends exclusively on the supersaturation of the system and may be modeled by quantum mechanics principles based on the mechanism of continuous additions until a critical size:



 16π ( fγ )3 ν 2  J = A exp − 3 3 2   3k T ln S 

(6.16)

where J is the primary nucleation rate, A is a constant, n is monomer volume, k is the Boltzman constant, T is absolute temperature, S is relative supersaturation (defined as the ratio between current concentration and equilibrium concentration), g is interface tension, and f is a factor indicating system heterogeneousness (equal to 1 in homogeneous nucleation and lower than 1 in heterogeneous) (Bernardo et al., 2004). Secondary nucleation, a complex phenomenon not totally understood, results from the presence of crystals in a supersaturated solution, and it is the result of the interaction between these crystals and the medium—friction with the fluid and collision with the impeller, with the crystallizer wall, or with other crystals. There is no theory to predict secondary nucleation. In industrial crystallizers, the secondary nucleation rate is a function of agitation, suspension density, and supersaturation, modeled according to a power law:

B 0 = k N W i M Tj ∆C n

(6.17)

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127

where B 0 is the secondary nucleation rate, kN is a nucleation constant, W is agitation speed, MT is suspension concentration, and ∆C is the supersaturation (Myerson and Ginde, 2002).

6.1.5 Thermodynamic Aspects: Solubility, Supersaturation, and Heat of Crystallization Thermodynamics (from Greek, heat change) is the study of the laws that conduct relations among heat, work, and other kinds of energy; more specifically, the transformation of one kind of energy into another, and the availability of energy to do work and heat exchanges. Dealing with processes in which systems change mass or energy, thermodynamics is not concerned with the rate of the processes, but with their occurrence and in what conditions. That is why the term thermodynamics normally refers to equilibrium thermodynamics (Atkins, 1995). The main thermodynamic properties related to crystallization are solubility (along with the supersaturation it determines) and the heat of crystallization. The term solubility normally refers to a part of the solid–liquid equilibrium, at moderate temperatures, between solvent freezing and boiling. Solubility is, therefore, the saturation concentration of a given substance in a given solvent. Solubility normally increases with temperature, but there are systems in which solubility remains practically unaltered and others in which solubility decreases with temperature (Atkins, 1995). In a general way, supersaturation is the distance from equilibrium, normally measured as the difference between the system concentration and equilibrium concentration at that temperature. Supersaturation is the driving force of crystallization processes, which means that crystallization only occurs if the system is supersaturated. Supersaturation may be imposed to a system by cooling, solvent evaporation, antisolvent addition, or chemical reaction (Jones, 2002). Crystallization deals with the ordering of the solute molecules (i.e., in the decrease of system entropy). As the spontaneity of a process results in the decrease of free energy, that entropy decrease must be compensated for by a decrease in the enthalpy, characterized by a heat release during the process—the heat of crystallization. This process is easily comprehended by the analysis of the expression that describes Gibbs free energy (Atkins, 1995):

G = H − TS

(6.18)

Industrial cooling crystallization processes must have, therefore, a heat transfer system and are limited by the maximum quantity of heat that can be removed from the system.

6.1.6 Method of Moments The method of moments substitutes the partial differential equations of population balance—Equations 6.3 and 6.4—by a group of ordinary differential equations, which

128

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simplify simulation and optimization of a batch crystallizer. Considering that the crystallizer is perfectly mixed, that agglomeration and breakage are negligible, and that growth rate is independent of crystal size, Equation 6.4 may be written in a batch process as



∂n ∂n +G = B0 ∂L ∂t

(6.19)

The moment equations are derived multiplying Equation 6.19 by Lj, integrating in L, and utilizing solvent mass as calculus base (constant during process),



dµ j = jGµ j−1 + Br0j , dt

j = 0 , 1 , 2

(6.20)

where r0 is the nucleus size (assumed constant during process), and m j is the j-esim moment already defined in Equation 6.8 (Ma, 2002): ∞

µj =

∫ L ndL j

(6.8)

0

Equations 6.1 and 6.8 allow one to infer the physical meaning of moments. m 0, m1, m2, m3 are, respectively, number, length, area, and total volume of the crystals. In the same way, ratios m1/m 0, m2/m1, m3/m2, m 4/m3 are, respectively, mean sizes pondered in number, length, area, and volume. This method simplifies energy and mass balance equations of a crystallization system, as previously described by Equations 6.9 through 6.12. In batch crystallization systems, with crystal growth rate independent of size, the method of moments simplifies the mass balance equation (Ma, 2002). With the additional hypothesis that total volume does not vary during the process (constant suspension density),



dC ρ k = − C v (3Gµ 2 + B0 L3N ) dt msolvente

(6.21)

where C is solute concentration in a solvent mass basis. In a similar way, the energy balance to the batch crystallizer may be written as



dT ρ k UA = − C v (3Gµ2 + B0 L3N ) ∆Hc − (T − TC ) dt mc p mc p

(6.22)

6.1.7 Agglomeration and Its Implications Agglomeration is the process of particle size increase in which little particles, more than ions or molecules, are bound in a group within a precipitation or crystallization

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129

process, as well as the cohesion of dry powder by a liquid binder in rotary disks, drums, or pans, where the process is generally known as granulation. Crystal agglomeration is a process of particle formation that carries out a fast increase in particle size, particularly during precipitation processes where the supersaturation degree is very high. Particle movement affects this growth process, but the same movement also induces particle breakage. So, crystal agglomeration is the net result of two opposite processes—aggregation and dispersion (Jones, 2002). Agglomeration involves particle association to form groups (clusters) and depends on two distinct phenomena: particles move in order to collide, and particles repel each other and are said to be stable, because they do not form aggregates. Almost all particulate systems involve in higher or lower degree particles that are some kind of aggregates of smaller system particles. Insofar as particles suffer agglomeration, particle size distribution may dramatically change, depending on the fractal structure of the aggregate (O’Brien, 2003). According to Tavare (1995), the equation of moments may be written as follows:



∂µ j + ∇  ( veµ j ) = r0j B0 + jGµ j−1 + B − D, j = 0,1, 2 ∂t

(6.23)

where B and D represent the rates of appearance and disappearance of crystals as functions of size due to agglomeration. Seckler (1994), utilizing the model of population balance proposed by Hulburt and Katz (1964) and the work of Hounslow et al. (1988), proposed an agglomeration rate based on the method of moments:



1 j Bag , j (t ) − Dag , j (t ) = − 1 −  βµ0 (t )µ j (t ), j = 0, 1, 2 2  3

(6.24)

where b is the agglomeration kernel. In turn, the agglomeration kernel may be dependent or independent of particle size (Jones, 2002; O’Brien, 2003; Seckler, 1994). Agglomeration can make the comprehension of the evolution in time and the handling of a particulate system very complex. Using a model (Derjaguin, Landau, Vervey, and Overbeek, DLVO) in which interparticle forces were considered, Braun et al. (2004) identified a product whose apparent growth was mainly the result of nucleation and agglomeration. They concluded that when less energy is dissipated by using a jet crystallizer (crystallizer with a concentric tube in which agitation is made by air injection), larger particles are obtained.

6.2 Protein Crystallization The crystallization of proteins, a group of very diverse and complex biomolecules, as any other crystallization process, is a function of many parameters that involve the already discussed steps of nucleation and growth in which the system containing the molecule to be crystallized is led to a supersaturated state, themodynamicaly

130

Engineering Aspects of Milk and Dairy Products

unstable. Protein crystallization is commonly referred to as an art more than a science, as its sensibility for system conditions make, most of the times nonapplicable, the conventional crystallization methods, based on evaporation, high pressure, large change in temperature, and the use of organic solvents (Littlechild, 1991; McPherson, 2004). The manipulation of the system for achieving supersaturation must be done through the manipulation of variables such as concentration of the protein, temperature, pH, and concentration of crystallization agents. In the last 30 years, many techniques for the crystallization of proteins have been developed, but no method is considered better than the other. At a small scale, four are more frequently used: batch crystallization, vapor diffusion, liquid–liquid diffusion, and dialysis. The choice of a specific method is a function of the specific protein, the amount of protein available, and the preference of the person who does the crystallization, especially when at small scale (Chayen, 1999). Although difficult, protein crystallization is common at small scale due to the crucial need to have a crystal to be used in X-ray diffraction for protein structure determination. Regarding large-scale processes, a final purification and polishing step unit operation is frequently used, and most of the time, optimal conditions are determined by trial and error and are not based on strong theoretical basis (Jacobsen et al., 1998; Lu et al., 2002; Velev et al., 1998). The use of crystallization as an initial recovery step is still a major challenge.

6.2.1 Special Characteristics of Protein Crystals and Crystallization Although protein crystals are visually similar to crystals of simple and small organic and inorganic compounds, there are many differences in their physical properties as well as in their growth kinetics, nucleation, and growth conditions, and in the type of internal interaction in the structure that forms the crystal lattice (McPherson, 1999). The crystallization of proteins is complex due to many factors. Proteins have a molecular mass, in the order of a thousand of kilo-Daltons, which is much higher than most of the crystallized organic molecules. Generally, due to their size and composition (20 amino acids with different side chains), a protein molecule is not symmetrical and does not have many contacts with other protein molecules in the crystalline lattice. The bond energy between the molecules is small, the crystals are sensitive to changes in system conditions, and the amount of solvent in the crystal structure is high (about 50%) which usually makes them very fragile (Littlechild, 1991). Conventional crystals can grow to sizes of many centimeters virtually without limits for final size in a few minutes, hours, or days, while protein crystals rarely exceed 1 millimeter during the growth period of a few days or weeks. Protein crystals also differ regarding growth conditions and the factors that affect the degree of perfection: macromolecular crystals nucleate at high supersaturations—supersaturations in the range of 3 to 30 which are much higher than the case of conventional inorganic crystals (Kierzec and Zielenkiewicz, 2001; McPherson, 1999). The number of interactions and the strength of the bonds (saline and hydrogen bridges and hydrophobic interactions) proteins have in the crystalline structure are fewer and much weaker when compared with the crystalline structure of conventional crystals. These contacts are of paramount importance because they are responsible for the stability of the crystal (McPherson, 2004).

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131

Sensitivity to harsh conditions such as low water activity and high temperature is typical of almost all of the proteins that may undergo denaturation under such conditions. This is a limitation in almost any protein separation process when compared with small organic molecules. Therefore, common methods used for crystallization of conventional molecules such as solvent evaporation, large temperature changes, or organic solvent addition are usually not applicable (Feigelson, 1988; McPherson, 1994). Water is a structural component or a form of solvent impurity inclusion in crystals of small molecules. In protein crystals, the irregular shape of these molecules and their high molecular masses favor the inclusion of water. There is also evidence that the contacts and interactions that hold the protein structures depend on the large network of water molecules and on the hydrogen bonds between them and the protein (Feigelson, 1988; McPherson, 1999).

6.2.2 Methods for Protein Crystallization There are many methods to generate a supersaturated state for macromolecules, including proteins, some more frequently used than others (Table 6.1). The yield and purity of protein crystals will depend on factors related to their biological and physical-chemical properties. The biological factors include the presence of impurities that vary enormously depending on the system by which the protein was produced, posttranslational modification (not correctly done and depending on the expression system for the case of recombinant proteins), and aggregation.

6.2.3 Large-Scale Protein Crystallization Although intensively used for structure determination by x-ray diffraction (Shukla et al., 2007), protein crystallization has its potential as a unit operation in downstream processing at large-scale still significantly untapped. The industrial crystallization of proteins is a technology with many not-well-known aspects. However, if

Table 6.1 Methods to Generate Supersaturation in Protein Crystallization 1. Mix the protein with excess of precipitant to instantaneously create a condition of supersaturation (batch method) 2. Change temperature 3. Change salt concentration to induce salting out or salting in 4. Increase or lower the pH 5. Add a ligand that changes the solubility of the protein 6. Remove water (evaporation) or concentrate the protein (e.g., ultrafiltration) 7. Add a polymer that excludes the protein out of the solution (volume exclusion) 8. Remove a solubilizing agent Source: Based on McPherson, A., Crystallization of biological macromolecules. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 586 p., 1999.

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well designed and operated, a protein crystallization process can generate products of high purity and yield and economically compete with chromatography, the “workhorse” of large-scale proteins (Hekmat et al., 2007; Schimdt et al., 2005; Ulrich and Jones, 2004). A well-known example of large-scale protein crystallization is the crystallization of the hormone protein insulin (swine, bovine, and recombinant human insulin). In addition to the need to consider the factors that control the crystallization process of conventional molecules, the high instability of proteins and their diversity in composition, structure, and size make it a challenge to translate experimental results in theoretical models simulating the protein crystallization process. There is a lack of background information when compared with conventional molecules which makes the scale-up of protein crystallization to be done based more on empirical results than on theoretical simulations. In fact, these experimental results are analyzed in conjunction with theoretical knowledge on small molecules to generate theories applicable to protein crystallization at a large scale (Carbone et al., 2005).

6.2.4 Milk Protein Crystallization As protein crystallization is only possible or has a high efficiency, in terms of yield and purity, if the feed already has a significant purity and the process costs are high, it is commonly considered for high-value products. Despite the potential applications some milk proteins have as nutraceuticals and as pharmaproteins (e.g., a-lactalbumin, ß-lactoglobulin, lactoferrin, growth factors, and lactoperoxidase) (Bargeman, 2003), there is no report of their crystallization at large scale. In fact, the literature recognizes insulin as the only crystalline protein pharmaceutical product in the market until 2004 (Basu et al., 2004). The development of a crystallization process for whey proteins at large scale depends only on the market demand, because precipitation and crystallization processes at small scale for these proteins have been reported. Precipitation processes developed for the separation of a-lactalbumin and ß-lactoglobulin using polyethylene glycol (PEG) and isoelectric precipitation, the latter more commonly used for a-lactalbumin (Bramaud et al., 1997a, 1997b; GésanGuizou et al., 1999; Ortin et al., 1992), and crystallization of lactoperoxidase (Polis and Shmukler, 1952), b-lactoglobulin (Monaco et al., 1987; Oliveira et al., 2001), and a-lactalbumin (Acharya et al., 1991; Fenna, 1982) have been described.

6.3 Crystallization of Sugars 6.3.1 Introduction Sugar is a generic name for a family of chemical compounds found in nature in different organic sources, including plants, leaves, and fruits. There are several kinds of industrially important sugars that are used in several processes and for several applications. In general, sugars are used in the solid form, mainly as a crystalline product, with crystallization being the most used process in its production. Industrially and commercially, the most used sugars are sucrose, glucose, fructose, lactose, xylose, and galactose.

133

Crystallization of Lactose and Whey Protein

In general, in the crystallization process, the following information is required: • Solubility curves, or equilibrium diagram, that will define the method of supersaturation generation and the type of crystallization • Metastable zone width that will define how difficult is the crystallization and the importance of nucleation • Growth kinetics that will define the extent of crystallization and the size of crystallizers • Secondary phenomena that can play an important role in the final product characteristics: agglomeration, impurity inclusion, morphology

6.3.2 Solubility and Supersaturation Generation In general, crystallization of sugars is made in aqueous solutions. Figure 6.1 shows the solubility data of some sugars in water as a function of temperature (Mullin, 2001). The equilibrium phase diagram can be much more complex than the solubility curve as shown in Figure 6.2 (Schuck et al., 2004) and Figure 6.3 (Young, 1957). The generation of supersaturation (the driving force for crystallization) in solution will depend on the dependence of equilibrium concentration on temperature and on its magnitude. Thus, supersaturation can be generated by the following methods: • Precipitation if the solubility is low, less than 1%. Precipitation can be chemical if there is a chemical reaction involved or physical if a nonsolvent is added. • Evaporation if the solubility is high, more than 20%, but the slope of the solubility curve is low, dC/dT < 5%/°C.

500 Glucose.H2O Lactose.H2O Maltose.H2O Raffinose.5H2O Sucrose Frutose

450

Solubility (g/100gH2O)

400 350 300 250 200 150 100 50 0

0

10

20

30

Figure 6.1  Solubility of some sugars.

40 50 60 Temperature (°C)

70

80

90

100

134

Engineering Aspects of Milk and Dairy Products 100 80

Temperature (°C)

60

Solution (Emulsion)

40

Solution and lactose crystals

20 0 –20

Solution and ice crystals

–40 –60

Glassy state 0

10

20

30

40

50

60

70

80

90

100

Composition (% total solids)

Figure 6.2  Milk phase diagram.

• Cooling for high solubility, higher than 20%, and high temperature dependence, dC/dT > 5%/°C. • Combination of the previous methods. For example, for the sugars presented in Figure 6.1, one can choose evaporation or precipitation for fructose and cooling for glucose and sucrose.

60

0 –10

se

Ice

–20 –30

on

uco

10

m

-Gl

uc Gl Dα

20

e os

β-D

30

te ra yd h o

α-D -G luc

Temperature (°C)

40

ose

50

0

10

20

30 40 50 60 Composition (% Glucose wt.)

Figure 6.3  Phase diagram of glucose–water system.

70

80

90

135

Crystallization of Lactose and Whey Protein

Several methods can be used to predict the solubility of sugars. UNIQUAC and UNIFAC group contribution methods fail if used directly (Peres and Macedo, 1997), and special group’s contribution is needed for them to work correctly (Ferreira et al., 2003; Gabas and Laguérie, 1993; Spiliotis and Tassios, 2000). Methods that directly correlate experimental data are more precise but fail to predict the solubility sugars, in most of the situations (Brito and Giulietti, 2007).

6.3.3 Metastable Zone Width

Concentration (g/100 g H2O)

Metastable zone is the region where the supersaturation exists. The width of this region can be defined for each equilibrium temperature as the difference between the temperature at which the first crystals appear by primary nucleation and the equilibrium temperature. The greater the metastable zone width, the larger is the need of free energy to nucleate the crystals. Seeding the supersaturated solution lowers the required free energy for nucleation. In general, the nucleation of sugars is difficult. Metastable zone width values can be higher than 50°C in the absence of seeds, and the metastable zone width can depend on the system temperature, cooling, or evaporation rate. Walstra et al. (1984) report a metastable zone width of 15°C for a-lactose at 80°C and 25°C at 30°C (Figure 6.4). Seeding the supersaturated solution is always a good strategy to decrease the metastable zone width for sugars. Other methods can be used, such as the addition of external energy to the supersaturated solution, as it stimulates the nucleation and the formation of crystals. Mechanical energy in the form of stirring and ultrasound were also successfully applied (Devarakonda et al., 2004).

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

Temperature (°C)

Figure 6.4  Metastable zone widths for lactose.

70

80

90

100

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Engineering Aspects of Milk and Dairy Products

6.3.4 Growth of Crystals The mechanism of crystal growth is one of the most studied phenomena in crystallization. Theories explaining the mechanisms of crystal growth can be grouped as surface energy, adsorption layer, kinematic, diffusion-reaction, birth and spread models, and combination of effects. The crystal surface can grow only at supersaturated solutions. The structure of the crystal surface, roughness, or entropy factor at its interface with the supersaturated solution has an important role on the particular mode of crystal growth mechanism. Different growth rates are observed for each crystal face. In general, the crystal face growth rate is proportional to the relative supersaturation as described by Equation 6.14. The crystal growth of sugars is generally diffusion controlled (Mullin, 2001). A global value for the growth rate of all faces can also be expressed by an equation like Equation 6.13. Depending on the sugar, the crystal growth rate can vary significantly, as is the case for fructose and sucrose that have values of 10 and 100 mm/h, respectively (Green and Perry, 2008). A general expression for the sucrose crystallization kinetics is

B 0 = 5.106N0.7.MT 0.3G 0.4

(6.25)

where Bo is the nucleation rate [#/ (L.s)]; N is the agitator rotational speed (1/s); and MT is the slurry density (g/L). As reported by Mullin (2001), other secondary phenomena, such as the following, can have an important role in sugar crystallization: • Agglomeration. • Aging and ripening: is the tendency for the smaller particles to dissolve and the solute to be deposited on the larger particles, thus the small particles disappear and the large grow. • Phase transformation: following the Ostwald’s rule of stages, first deposited crystals are the less stable form than that which normally crystallizes. This formation can be followed by a phase transformation to the final product. The metastable phase may be an amorphous, a polymorph, or a hydrated species. All these phenomena will define the final characteristics of the crystalline product.

6.3.5 Crystal Product Characteristics The characteristics of the final crystalline product determine its behavior during storage, handling, and use, the most relevant. The properties to be considered are • Particle or crystal size distribution: taking into account the requirements for use, a dominant size as well as homogeneity or monodispersity in size distribution are usually required. The particle size distribution can be monitored during the crystallization process by controlling the supersaturation aiming at the obtention of a predefined dominant size.

Crystallization of Lactose and Whey Protein

137

Figure 6.5  Different morphology of a-lactose obtained from different solvents: acetone and 2-propanol.

• Morphology: although the crystal lattice is unique for each substance, the difference in the face growth rate during the crystallization process can result in a different final morphology for the bulk crystalline product (Raghavan, 2001). Figure 6.5 presents the different morphologies occurring for a-lactose using different solvents (Brito, 2007). Different morphologies can also result in different handling and storage properties. Morphology can be modified by changing the solvent media, additives, and impurities. • Inclusions: during the crystallization process, differences occur in the growth rate of crystal faces, partially rounded surfaces growing faster and entrapping mother liquor in the bulk crystal. Figure 6.6 shows liquid inclusions in sucrose crystals. This inclusion can cause problems such as caking during storage. • Cocrystallization: during crystallization, in high-density slurries, different molecules can be incorporated in the solute crystals, giving different properties and degree of purity to the end crystalline product. Fructose and glucose can cocrystallize with sucrose (Bhandari and Hartel, 2002).

Figure 6.6  Liquid inclusions in sucrose crystals.

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New developments are continuously occuring, and several patents on the industrial aspects of sugar crystallization are deposited every year, mainly due to the market requirements on productivity and quality.

6.4 Crystallization of Lactose 6.4.1 Introduction Lactose (C12H22O11) is a disaccharide consisting of one galactose and one glucose moiety. It is the only commercially available sugar that is derived from animal rather than from plant sources. Lactose is found in mammalian milk with concentrations ranging from 3 to 6 wt%. In cow milk products, the lactose concentration ranges from 4 wt% in whole milk to 70 wt% in sweet dried whey. Commercially, lactose is mainly produced as the crystalline a-monohydrate and is obtained from whey, a by-product of cheese and caseinate production. Other forms are also available, such as the b-anhydride, resulting from a high temperature crystallization (Hull, 1958; Kirk-Othmer, 1995; Westhoff, 2008). Figure 6.7 presents the general process for the production of pharmaceutical-grade lactose from cheese whey. Other methods used to produce lactose at the commercial scale are as follows: • Melt crystallization: after evaporation of a solution of lactose, the melted sugar is solidified and removed from the solution, even in the presence of other compounds such as fats and other sugars (Heikkila et al., 2003). • Precipitation with calcium and sodium hydroxide: the complexation reaction of lactose with calcium forms a stable complex that can be removed from the whey solution. The complex can react in an acid solution to recover the purified lactose (McCommins et al., 1980).

Figure 6.7  Production of pharmaceutical-grade lactose.

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Crystallization of Lactose and Whey Protein

• Drying of whey: lactose, not purified, can be directly obtained from the whey solution by drying, using spray, fluidized-bed, or freeze-drying techniques (Gänzle et al., 2008; Schuck, 2002; Schuck and Dolivet, 2002; Schuck et al., 2004). • Chromatographic separation: using the permeate of whey ultrafiltration, chromatographic columns are used to purify lactose (Theobald, 2008). Although all these techniques are used, evaporation followed by crystallization is the most-used process to produce lactose (GEA, 2008; Terlet, 2008). Lactose has many different final applications such as raw material in the chemical and food industry, as intermediate in the pharmaceutical industry, and as commercial final product (Elliott et al., 2001; Gänzle et al., 2008). Lactose can be hydrolyzed to give the interesting products galactose and glucose. The hydrolysis can be made by acid catalysis, enzymatic conversion, or resins. Shah and Nickerson (1978) detail the procedure and yield of the lactose hydrolysis process. Catalytic hydrogenation of lactose to the sugar alcohol lactitol can be made using, for example, ruthenium in silica and alumina supports. Hydrogenolysis is also an important reaction where lactose used as reactant can lead to a number of interesting polyol ether products (Elliott et al., 2001). Lactose may also isomerize into lactulose.

6.4.2 Crystalline State and Polymorphs Lactose can exist in three different crystalline states or polymorphs: a-lactose, b-lactose, and a-lactose monohydrate. Table 6.2 presents the crystal cell parameters for these forms (CSD, 2007). All the forms are monoclinic sphenoidal (space group P2) and belong to class C2. Figure 6.8 presents a typical habit of a lactose crystal, also called the tomahawk form (Garnier et al., 2002). This is the expected habit of lactose when obtained from aqueous solution crystallization. Lactose can be formed in different habits depending on several factors. A prism is formed at very high growth rates of the alpha lactose monohydrate. Diamond shapes can appear at a slower growth rate. Pyramids and the tomahawk habit, a tall pyramid with bevel faces at the base, can also be formed (Herrington, 1934b). The lactose habit also changes according to the solvent used or if impurities or additives are present in solution. In water, lactose crystals tend to grow as shown in

Table 6.2 Crystal Cell Parameters of Lactose Parameters b-lactose a-lactose a-lactose monohydrate

a

b

c

a

B

10.81 7.716 7.895

13.32 19.78 21.58

4.909 4.947 4.820

90.00 91.01 90.00

91.37 105.0 108.2

g 90.00 93.58 90.00

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Engineering Aspects of Milk and Dairy Products

–b (1–50) (1–10)

(0–10)

(110) (110)

a (010)

c

Figure 6.8  Tomahawk crystal habit of lactose.

Figure 6.9 as proposed by Hunziker and Nissen (1927). When sucrose is present in the aqueous solution, the habit can change, and the crystal growth occurs as shown in Figure 6.10. When the crystallization is made in different solvents, it is possible to obtain different crystal habits as shown in Figure 6.11 (Brito, 2007). This is due to the differences in the attachment energy in crystal faces. Clydesdale et al. (1997) made the molecular modeling of the a-lactose monohydrate using the HABIT95 program. Their aim was to predict the effect of additives in the change of crystal morphology, making it possible to obtain more convenient habits to be used in the formulation of pharmaceutical products.

6.4.3 Solubility As others sugars, lactose solubility is influenced by several factors: temperature, pH, impurities, and the presence of salts and other sugars. In addition, lactose pre­ sents the property called mutarotation. In aqueous solution, the lactose molecule is present in a and b forms that differ only in one spatial position of a OH terminal group as shown in Figure  6.12. These two forms are in a reversible equilibrium called mutarotation. The equilibrium constant of these forms is determined by the temperature.

Figure 6.9  Development of crystal of lactose growing in aqueous solution.

141

Crystallization of Lactose and Whey Protein

Figure 6.10  Development of crystal of lactose growing in aqueous solution in the presence of sucrose.

All three forms can be obtained by an adequate manipulation of the operational process conditions. In a simplified way, these conditions are as follows: • In solution, an equilibrium between a and b forms occurs

a <=> b

(6.26)



K = [b]/[a] = 1.64 + 0.0027T

(6.27)

• Rapid drying or freezing of a saturated solution leads to amorphous lactose with K = [b]/[a] = 1.25



(6.28)

(a) Ethanol

(b) Acetone

(c) Isopropanol

(d) Agglomerate in isopropanol

Figure 6.11  Crystals obtained from different solvents.

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Engineering Aspects of Milk and Dairy Products

H HO

H

OH

OH

H

H

CH2OH H

H O

O

CH2OH

H

O

OH

H

H

OH

H OH

α-Lactose

H HO

H

OH

OH

H

H

O

CH2OH H

H O

CH2OH

H

O

OH

H

H

OH

OH H

β-Lactose

Figure 6.12  Lactose molecule in a and b configurations.

• Amorphous lactose can uptake water from the environment at T < 93.5°C and be converted to a-lactose monohydrate. • Amorphous lactose can uptake water from environment at T > 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.

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Engineering Aspects of Milk and Dairy Products

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

Crystallization of Lactose and Whey Protein

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

Engineering Aspects of Milk and Dairy Products

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

Crystallization of Lactose and Whey Protein

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

Engineering Aspects of Milk and Dairy Products

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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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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Sobrino-López, A., Raybaudi-Massilia, R. & Martín-Belloso, O. (2006b). High-intensity pulsed electric field variables affecting Staphylococcus aureus inoculated in milk. Journal of Dairy Science, 89:3739–3748. Steele, J. (2000). History, trends, and extent of pasteurization. Journal of the American Veterinary Medical Association, 217(2):175–178. Sun, H.-X., Kawamura, S., Himoto, J.-I., Itoh, K., Wada, T. & Kimura, T. (2008). Effects of ohmic heating on microbial counts and denaturation of proteins in milk. Food Science and Technology Research, 14(2):117–123. Toyoda, I., Schreier, P. & Fryer, P. (1994). A computational model for reaction fouling from whey protein solutions. In Fouling and Cleaning in Food Processing, Jesus College, Cambridge, England; March 23–25, pp. 222–229. Valsechi, O.A., Horii, J. & De Angelis, F. (2004). The effect of microwaves on microorganisms: the effect of microwaves on microorganisms. Revista Arquivos do Instituto Biológico, 71(3):399–404. Van Loey, A., Verachtert, B. & Hendrickx, M. (2002). Effects of high electric field pulses on enzymes. Trends in Food Science and Technology, 12:94–102. Vega-Mercado, H., Powers, J., Barbosa-Cánovas, G.V. & Swanson, B.G. (1995). Plasmin inactivation with pulsed electric fields. Journal of Food Science, 60:1143–1146. Vega-Mercado, H., Powers, J.R., Martín-Belloso, O., Luedecke, L., Barbosa-Cánovas, G.V. & Swanson, B.G. (2001). Change in susceptibility of proteins to proteolysis and the inactivation of an extracellular protease from Pseudomonas fluorescens M3/6 when exposed to pulsed electric fields. In G.V. Barbosa-Cánovas & Q.H. Zhang (Eds.), Pulsed Electric Fields in Food Processing: Fundamental Aspects and Applications (pp. 105–120). Lancaster, PA: Technomic. Vicente, A.A. (2007). Novel technologies for the thermal processing of foods. Proceedings of “8° Encontro de Química dos Alimentos: alimentos tradicionais, alimentos saudáveis e rastreabilidade” [CD-ROM]. Beja: Instituto Politécnico de Beja, 2007, pp. 499–506. Vidal-Valverde, C. & Redondo, P. (1993). Effect of microwave heating on the thiamin content of cows’ milk. Journal of Dairy Research, 60:259–262. Villamiel, M., Lopez-Fandino, R. & Olano, R. (1997). Microwave pasteurization of milk in a continuous flow unit: effects on the cheese-making properties of goat’s milk. Milchwissenschaft, 52(1):29–32. Wang, W.-C. (1995). Ohmic heating of foods: physical properties and applications. Columbus, OH. The Ohio State University. Wang, W. & Sastry, S. (1997). Changes in electrical conductivity of selected vegetables during multiple thermal treatments. Journal of Food Process and Engineering, 20:499–516. Wang, Y., Wig, T.D., Tang, J. & Hallberg, L.M. (2003). Dielectric properties of foods relevant to RF and microwave pasteurization and sterilization. Journal of Food Engineering, 57:257–268. Wilinska, A., Annus, J., Bryjak, J., Illeova, V. & Polakovic, M. (2006). Investigation of stability of food enzymes during ohmic heating. In Session: Workshop: Ohmic Heating for Food Processing. Proceedings 33rd International Conference of Slovak Society of Chemical Engineering. Hotel Hutnik. Tatrasnké Matliare, Slovakia. May 22–26, p. 33. Xiang, B.Y., O Ngadi, M., Gachovska, T. & Simpson, B.K. (2007). Protein denaturation in whole milk treated by pulsed electric field. ASAE Annual Meeting 076008. Published by the American Society of Agricultural and Biological Engineers, St. Joseph, Michigan, www.asabe.org. Zareifard, M., Ramaswamy, H., Trigui, M. & Marcotte, M. (2003). Ohmic heating behaviour and electrical conductivity of two-phase food systems. Innovative Food Science and Emerging Technologies, 4:45–55.

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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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.

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Geraldine, R.M.; Soares, N.F.F.; Botrel, D.A.; Gonçalves, L.A. Characterization and effect of edible coatings on minimally processed garlic quality. Carbohydrate Polymers, 72, 403–409, 2008. Guilbert, S. Technology and application of edible films. In M. Mathlouthi (Ed.), Food Packaging and Preservation (pp. 371–394). New York: Elsevier Applied Science. 1986. Guilbert, S.; Gontard, N.; Gorris G.M. Prolongation of the shelf-life of perishable food products using biodegradable films and coatings. Lebensmittel- Wissenschaft und- Technologie, 29(1–2), 10–17, 1996. Han, J.H. Antimicrobial food packaging. Food Technology, 54(3), 56–65, 2000. Han, J.H. Antimicrobial food packaging. In Ahvenainen, R. (Ed). Novel Food Packaging Techniques. Cambridge: CRC Press–Woodhead, Inglaterra. 2003. Han, J.H.; Floros, J.D. Potassium sorbate diffiisivity in American processed and Mozzarella cheeses. Journal of Food Science, 63(3), 435–437, 1998. Han, J.H. Antimicrobial packaging systems. In Han, J. (Ed). Innovations in Food Packaging. Amsterdam: Elsevier Science. 2005. Hong, S.I.; Park, J.D.; Kim, D.M. Antimicrobial and physical properties of food packaging films incorporated with some natural compounds. Food Science Biotechnology, 9(1), 38–42, 1998. Huff, W.E.; Kubena, L.F.; Harvey, R.B. Efficacy of hydrated sodium calcium aluminosilicate to reduce the individual and combined toxicity of aflatoxin and ochratoxin A. Poultry Science, 71, 64–69, 1992. Hutton, T. Food packaging: an introduction. Number 7. Chipping Campden, Gloucestershire, UK: Campden and Chorleywood Food Research Association Group (p. 108). 2003. Jong, A.R.; Boumans, H.; Slaghek, T.; Veen, J.; Rijk, R.; Zandvoort, M.N. Active and intelligent packaging for food: is it the future? Food Additives and Contaminants, 22(10), 975–979, 2005. Kampeerapappun, P.; Aht-ong, D.; Pentrakoon, D.; Srikulkit, K. Preparation of cassava starch/ montmorillonite composite film. Carbohydrate Polymers, 67(2), 155–163, 2007. Kampf, N.; Nussinovitch, A. Hydrocolloid coating of cheeses. Food Hydrocolloids, 14, 531– 537, 2000. Krochta, J.M.; De Mulder-Johnston, C. Edible and biodegradable polymer films: challengers and opportunities. Food Technology, 51(2), 61–74, 1997. Lee, D.S.; Iiwang, Y.I.; Cho, S. Developing antimicrobial packaging film for curled lettuce and soy-bean sprouts. Food Science Biotechnology, 7(2), 117–121, 1998. Limjaroen, P.; Ryser, E.; Lockhart, H.; Harte, B. Inactivation of Listeria monocytogenes on beef bologna and Cheddar cheese using polyvinylidene chloride films containing sorbic acid. Journal of Food Science, 70(5), M267, 2005. Lin, D.; Zhao, Y. Innovations in the development and application of edible coatings for fresh and minimally processed fruits and vegetables. Comprehensive Reviews in Food Science and Food Safety, 6, 60–75, 2007. Linko, S. Expert systems—what can they do for the food industry? Trends in Food Science and Technology, 9(1), 3–12, 1998. Maryamma, K.I.; Rajan, A.; Gangadharan, B. In vitro and in vivo studies on aflatoxin B1 neutralization. Indian Journal of Animal Science, 61, 58–60, 1991. Millette, M.; Le Tien, C.; Smoragiewicz, W.; Lacroix, M. Inhibition of Staphylococcus aureus on beef by nisin-containing modified alginate films and beads. Food Control, 18, 878– 884, 2007. Miltz, J.; Passy, N.; Mannheim, C.H. Trends and applications of active packaging systems. In Ackerman, P.; Jagerstad, M.; Ohlsson, M. (Ed). Food and Packaging Materials— Chemical Interaction. London: The Royal Society of Chemistry. 1995. Miltz, J.; Perry, H. Evaluation of the performance of iron-based oxygen scavengers with comments on their optimal applications. Packaging Technology and Science, 18(1), 21–27, 2005.

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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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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

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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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Microcalorimetry

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.

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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.

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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

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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

<400

Lactoperoxidase

Glycomacropeptide

Osteopontin

Proteose peptones

18,000–30,000 (PP3 component)

69,000

6700

70,000

80,000

25,000 (light chain) + 50,000–70,000 (heavy chain)

66,267

14,175

18,277

Molar Mass (kDa)

135 (PP3 component)

262

64

612

700

—

582

123

162

Number of Amino Acid Residues

3.3–3.7

3.5

—

9.5

8.5–9.0

5.5–8.3

4.7–4.9

4.2–4.8

5.2

Isoelectric Point

Immune modulating benefits

Bone mineralization

Source of branched-chain amino acids; lacks the aromatic amino acids phenyalanine, tryptophan, and tyrosine

Inhibits growth of bacteria

Antioxidant, antibacterial, antiviral, antifungal, promotes growth of beneficial bacteria

Immune modulating benefits

Source of essential amino acids

Source of essential and branched amino acids

Source of essential and branched-chain amino acids

Benefits

Sources: Data adapted from Marshall K (2004) Alt Med Rev 9(2): 136–156; Madureira AR, Pereira CI, Gomes AMP, Pintado ME, Malcata FX (2007) Food Res Int 40: 1197–1211; Smithers GW (2008) Int Dairy J 18: 695–704; De Wit JN (1998) J Dairy Sci 81: 597–602; Farrell HM, Jimenez-Flores R, Bleck T, Brown EM, Butler JE, Creamer LK, Hicks CL, Hollar CM, Ng-Kwai-Hang KF, Swaisgood HE (2004) J Dairy Sci 87: 1641–1674; Rodrigues LR, Teixeira JA, Schmitt F, Paulsson M, Lindmark-Mansson H (2007) Cancer Epidemiol Biomarkers Prev 16: 1087–1097; Rodrigues LR, Teixeira JA, Schmitt F, Paulsson M, Lindmark Måsson H (2009) Crit Rev Food Sci Nut 49: 1–15; Zydney AL (1998) Int Dairy J 8: 243–250; Sugahara T, Onda H, Shinohara Y, Horii M, Akiyama K, Nakamoto K, Hara K (2005) Biochim Biophys Acta 1725: 233–240; Girardet J, Linden G (1996) J Dairy Res 63: 333–350; Sodek J, Ganss B, McKee MD (2000) Crit Rev Oral Biol Med 11: 279–303; and Chatterton DEW, Rasmussen JT, Heegaard CW, Sorensen ES, Petersen TE (2004) Trends Food Sci Tech 15: 373–383.

50–70

Lactoferrin

300–600

1200

a-Lactalbumin

Immunoglobulins

1300

b-Lactoglobulin

Protein

Concentration (mg/L)

Table 10.1 Protein Profile of Cheese Whey: Content, Primary Structure, Basic Properties, and Benefits

Potential Applications of Whey Proteins in the Medical Field 223

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as well as their benefits. Together, whey proteins have all the essential amino acids and in higher concentrations compared to various vegetable protein sources such as soy, corn, and wheat gluten (Walzem et al. 2002). In addition to having a full spectrum of amino acids, the amino acids found in whey are efficiently absorbed and utilized, relative to free amino acid solutions (Daenzer et al. 2001). As compared to other protein sources, whey has a high concentration of branched-chain amino acids (leucine, iso­ leucine, and valine), as mentioned above (Marshall 2004). In addition, whey proteins are rich in the sulfur-containing amino acids cysteine and methionine. With a high concentration of these amino acids, immune function is enhanced through intracellular conversion to glutathione (GSH). All the whey proteins have at least been implicated in a variety of nutritional and physiological effects, and apart from the major whey proteins—b-lactoglobulin, a-lactalbumin, and glycomacropeptide—whey contains a number of other proteins with potent bioactivity (Table 10.2). This review aims to provide an overview on the whey proteins’ properties, functions, and mechanisms of interaction which could be further exploited in developing its potential therapeutic applications. Table 10.2 Biological Functions of Whey Proteins Protein

Biological Function

References

Whole whey proteins

Prevention of cancer (breast, colon, and prostate) Increment of glutathione levels (increase of tumor cell vulnerability and treatment of patients with HIV) Antimicrobial and antiviral activities Increment of satiety response Immunomodulating effects Prebiotic activity

Bounous 2000; Gill and Cross 2000; Madureira et al. 2007; Rodrigues et al. 2009; Rowlands et al. 2001; Smithers 2008 Marshall 2004; Micke et al. 2001, 2002; Middleton et al. 2003; Parodi 1998 Bojsen et al. 2007; Clare et al. 2003; Rodrigues et al. 2009; Sitohy et al. 2000; Smithers 2008 Hall et al. 2003; Marshall 2004 Hoerr and Bostwick 2000; Mercier et al. 2004 Mercier et al. 2004

b-Lactoglobulin

Transporter (retinol, palmitate, fatty acids, vitamin D, and cholesterol) Enhancement of pregastric esterase activity Transfer of passive immunity Regulation of mammary gland phosphorus metabolism

Chatterton et al. 2006; Puyol et al. 1991; Wang et al. 1997; Wu et al. 1999 Perez et al. 1992 Warme et al. 1974 Farrell et al. 1987

a-Lactalbumin

Prevention of cancer Lactose synthesis Treatment of chronic stress-induced disease

Chatterton et al. 2006; Marshall 2004; Smithers 2008 Markus et al. 2002 Ganjam et al. 1997 (continued)

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Table 10.2 (Continued) Biological Functions of Whey Proteins Protein

Biological Function

References

Bovine serum albumin

Fatty acid binding Antimutagenic function Prevention of cancer Immunomodulation

Walzem et al. 2002 Bosselaers et al. 1994 Marshall 2004; Rodrigues et al. 2009 Madureira et al. 2007

Immunoglobulins

Prevention and treatment of various microbial infections (upper respiratory tract infections, gastritis, dental caries, diarrhea, among others)

Mehra et al. 2006; Pan et al. 2006

Lactoferrin

Antibacterial, antiviral, and antifungal activities Prevents several microbial infections and several types of cancer Prebiotic activity

El-Fakharany et al. 2008; Madureira et al. 2007; Pan et al. 2006; Rodrigues et al. 2009; Smithers 2008; Wakabayashi et al. 2006

Lactoperoxidase

Biocidal and biostatic activities Prevention of colon and skin cancer

Boots and Floris 2006 Smithers 2008

Glycomacropeptide

Interaction with toxins, viruses, and bacteria (mediated by the carbohydrate fraction) Control of acid formation in the dental plaque Immunomodulating activity

Thoma-Worringer et al. 2006

Osteopontin

Bone mineralization, participates in bone remodeling, inflammation, cancer, and immunity to infection disease

Rodrigues et al. 2007

Proteose peptones (PP3)

Immunostimulation effects Prevention of caries

Sugahara et al. 2005 Aimutis 2004; Grenby et al. 2001

Aimutis 2004 Matin and Otani 2000

10.2 Whey Protein Concentrates and Whey Protein Isolates The evolution of the membrane separation technologies (ultrafiltration, microfiltration, and diafiltration) in the last years led to the widespread use of a number of protein whey components as food additives (Smithers et al. 1996; Zall 1984). Several whey treatments have been studied conducting to a great diversity of products with specific qualitative and quantitative profiles of proteins and other whey components (Marshall 2004). Therefore, it is possible to remove most lactose, minerals, and low molar mass components using ultrafiltration or diafiltration, producing the whey protein concentrates (WPCs) with different concentrations (35%, 50%, 65%, and 80% w/w). Moreover, in some cases when the threshold of 90% (w/w) protein is reached, a whey

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Table 10.3 Summary of Clinical Trials Using Whey Protein Condition Cancer

Dose of Whey Protein

Study Duration

Results

30 g daily

6 months

Two of five patients showed tumor regression Results suggested an increase in glutathione levels in healthy cells and a decrease in cancer cells

40 g daily Stage IV malignancies Used other natural therapies

6 months

16/20 survivors at the end of the treatment Increased natural killer (NK) cell function and glutathione levels Increased hemoglobin and hematocrit Improved quality of life

Hepatitis B

12 g daily

12 weeks

Decreased serum lipid peroxidise levels Increased IL-2 and NK activity Decreased serum alanine transferase activity Increased plasma glutathione levels

HIV

45 g daily

2 weeks and 6 months

Increased glutathione levels in both trials

Exercise

1.2 g/kg body weight daily

12 weeks

10 g twice daily

3 months

Improved lean tissue mass Improvement in one of four muscle strength measurements Significant improvements in peak power Significant increase of 30-second work capacity Increased lymphocyte glutathione levels

Source: Adapted from Rodrigues LR, Teixeira JA, Schmitt F, Paulsson M, Lindmark Måsson H (2009) Crit Rev Food Sci Nut 49: 1–15.

protein isolate (WPI) is accordingly obtained (Madureira et al. 2007). It is important to notice that both WPC and WPI are used as vectors for the promotion of many biological properties upon addition to foods (Table 10.2). Furthermore, some clinical trials have been conducted using whey protein, as can be seen in Table 10.3. Whey proteins as a whole have been reported to have several biological features in addition to their recognized nutritional value (Smithers 2008).

10.2.1 Antimicrobial and Antiviral Activity The antimicrobial activity of whey protein products (WPC and WPI) has been extensively reviewed in the last years (Clare et al. 2003; Marshall 2004; Sitohy et al. 2008;

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227

Smithers 2008). Enrichment of WPC with Helicobacter pylori-specific antibodies produced by lactating cows has proven to prevent infections (Early et al. 2001). Moreover, Oevermann and coworkers (2003) chemically modified, by 3-hydroxyphthalic anhydride (3-HP), the proteins BSA, a-lactalbumin, and b-lactoglobulin, and tested their antiviral activity against several enveloped viruses (human herpes simplex virus type 1 [HSV-1], bovine parainfluenza virus type 3, and porcine respiratory corona virus). Of those three viruses, only HSV-1 was sensitive to 3-HP-proteins. The study conducted by Bojsen and collaborators (2007) also demonstrates that WPC inhibits rotavirus infections both in vitro and in vivo. Furthermore, esterified whey proteins were found to inhibit poliovirus and Coxsackie virus (Sitohy et al. 2008).

10.2.2 Immunomodulation Whey has potent antioxidant activity, likely by contributing cysteine-rich proteins that aid in the synthesis of glutathione (GSH), which is a potent intracellular antioxidant naturally found in all cells of mammals (Walzem et al. 2002). GSH primary structure is composed of glycine, glutamate, and cysteine. Cysteine contains a thiol (sulfhydryl) group that serves as an active reducing agent in preventing oxidation and tissue damage; however, cysteine incorporation in GSH is the rate-limiting step for its synthesis. Both cysteine and glutamine are major players in the coordinated T-cell response of macrophages and lymphocytes. Note that whey proteins are rich in cysteine and glutamate residues, suggesting that their ingestion may contribute to increase the free cysteine levels and, consequently, the production of GSH (Bounous et al. 1989; Wong and Watson 1995). GSH has been reported as an important peptide in immune regulation and cancer prevention in animals, in improvement of immune and liver functions, and in helping overcome GSH deficiency in patients who are seropositive and have Alzheimer’s disease (Grey et al. 2003; Madureira et al. 2007; Micke et al. 2001, 2002). Furthermore, during immune deficiency states, WPC was shown to be an effective cysteine donor for GSH replenishment (Gomez et al. 2002; Wong and Watson 1995). Cell culture studies and in vivo experiments (Gomez et al. 2002) have demonstrated that whey proteins may enhance nonspecific and specific immune responses. Dietary supplementation with a whey-based product increased lymphocyte GSH levels in patients suffering from lung inflammation associated with cystic fibrosis (Grey et al. 2003). Moreover, whey proteins were found to suppress in vitro lymphocyte mitogenesis and alloantigen-induced proliferation, when included in mature murine lympocytes solution (Barta et al. 1991). Other studies with modified WPC demonstrated that it can also suppress the mitogen-stimulated secretion of g-interferon, as well as the surface expression of interleukin-2 receptor, when added to T- and B-lymphocyte cultures (Cross and Gill 1999). Moreover, Mercier and coworkers (2004) claimed that addition of WPI to cell culture media stimulates in vitro proliferation of murine spleen lymphocytes. Furthermore, the (long-term) ImmunocalTM supplement is a WPC that has proven effective toward improvement of liver disfunction in patients exhibiting chronic hepatitis B (Madureira et al. 2007; Marshall 2004).

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10.2.3 Anticancer Activity WPCs have been researched extensively in the prevention and treatment of several types of cancer (Gill and Cross 2000). Tsuda and coworkers (2000) used animal models (female rats) to study the potential of whey proteins being part of a diet, as cancer preventive agents, namely for breast and intestinal cancers. GSH stimulation is thought to be the primary immune-modulating mechanism (Bounous 2000). The amino acid precursors to GSH available in whey might increase GSH concentration in relevant tissues, stimulate immunity via the GSH pathway, and detoxify potential carcinogens. Immunocal actually caused depletion of GSH and inhibition of proliferation of human breast cancer cells in in vitro assays, at concentrations that induce GSH synthesis in healthy human cells (Madureira et al. 2007; Smithers 2008). Other authors conclude that the iron-binding capacity of whey may also contribute to anticancer potential, as iron acts as a mutagenic agent causing oxidative damage to tissues (Weinberg 1996). A recent in vitro study by Kent and coworkers (2003) demonstrated that a hydrolyzed WPI increased GSH synthesis and protected human prostate cells against oxidant-induced cell death. Furthermore, WPI may also protect from cancer by acting as a co-adjuvant of baicalein—an anticancer drug; the cytotoxicity of this molecule is enhanced by inducing more apoptosis in the human hepatoma cell line Hep G2, which is in turn associated with depletion of GSH (Tsai et al. 2000). To date, few clinical trials on whey and cancer have been conducted (Table 10.3). It has been proposed that GSH concentrations are high in tumor cells, giving them resistance to chemotherapeutic agents (Kennedy et al. 1995).

10.2.4 Nutrition Effects and Other Metabolic Features It is widely recognized that whey proteins apparently facilitate the attainment of favorable weight and composition (Marshall 2004). Whey proteins were found to be more effective in satiation than caseins, when both these proteins were studied in terms of food intake—based on ratings of hunger and fullness by selected subjects, and on postprandial metabolite and gastrointestinal hormone responses (Hall et al. 2003; Roberts et al. 2002). Furthermore, whey proteins were found to lower plasma and liver cholesterol, as well as plasma triacylglycerol levels in model animals fed with cholesterol-containing diets (Beena and Prasad 1997; Zhang and Beynen 1993). Whey protein supplements have been readily utilized in the consumer market because of their high protein quality score and high percentage of branched-chain amino acids (Tawa and Goldberg 1992). Although human studies documenting the beneficial effect of whey protein supplementation on muscle size and strength are limited, some reports claim this effect for WPCs, as well as a reduction of the tendency for bone breaking, based on their amino acid profiles (Bos et al. 2000; Burke et al. 2001; Lands et al. 1999; Walzem et al. 2002). The advantages of whey proteins in terms of muscle anabolism are in their fast adsorption, coupled with the leucine abundance required to initiate synthesis, as well as in their amino acid composition that provides substrates for protein synthesis (Ha and Zemel 2003). The loss of bone mass during menopause is a well-documented realization; whey proteins may

Potential Applications of Whey Proteins in the Medical Field

229

provide help toward bone formation and activation of the bone cells (osteoblasts) (Takada et al. 1996).

10.3 -Lactoglobulin b-Lactoglobulin represents approximately half of the total protein in bovine whey (Table 10.1), and human milk contains no b-lactoglobulin (Chatterton et al. 2006). When in isolated form, it exhibits a low solubility (despite its globular nature) and a low ionic strength (Table 10.1). Synthesized in the mammary gland of ruminants (and other species) and designed to be included in milk, this protein has several genetic variants, of which b-lactoglobulin A is the most common (Farrell et al. 2004). It is composed mainly of b-sheets motifs and consists of 162 amino acid residues, which lead to a molar mass of ca. 18,227 kDa. Its quaternary structure depends on the medium pH. It occurs mainly as a stable dimer, with a molar mass of 36,700 kDa, at pH values between 7 and 5.2; as an octamer, with a molar mass of ca. 140,000 kDa, at pH values between 5.2 and 3.5; and as a monomer, with two-cysteine residues per monomer, at pH 3.0 and above 8.0 (de Wit 1989). In addition to being a source of essential and branched-chain amino acids, a retinol-binding protein has been identified within the b-lactoglobulin structure (Guimont et al. 1997). This protein, a carrier of small hydrophobic molecules including retinoic acid, has the potential to modulate lymphatic responses.

10.3.1 Immunomodulation The amino acid content of this protein is rather important, because, in addition to fueling muscle growth, it is a source rich in the essential amino acid cysteine, which is important for the synthesis of GSH (de Wit 1998). b-Lactoglobulin has been reported to play a role in the transfer of passive immunity to the newborn (Warme et al. 1974), as well as in regulation of phosphorus metabolism at the mammary gland (Farrell et al. 1987, 2004) (Table 10.2).

10.3.2 Nutrition Effects and Other Metabolic Features Although several studies have been conducted in the last decades, the actual biological function of b-lactoglobulin is still unclear (Smithers 2008). However, it often binds to small hydrophobic ligands, such as retinol, fatty acids, protoporphyrin IX, triacylglycerols, alkanes, aliphatic ketones, aromatic compounds, vitamin D, cholesterol, palmitic acid, and calcium (at pH 5.0) (Brown 1984; Chatterton et al. 2004, 2006; Cho et al. 1994; Farrell et al., 2004; Futterman and Heller 1972; O’Neil and Kinsella 1987; Patocka and Jelen 1991; Puyol et al. 1991; Said et al. 1989; Smith et al. 1983; Wang et al. 1997; Wu et al. 1999). Specifically, this protein binds to free fatty acids as they are released by pregastric lipases, in order to facilitate digestion of milk fat (Perez and Calvo 1995). Because of its high stability at low pH, b-lactoglobulin protects hydrophobic molecules during passage through the stomach, in order to deliver such ligands to a specific receptor located in the intestine of the sucking neonate (Cho et al. 1994).

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10.4 -Lactalbumin a-Lactalbumin is one of the main proteins found in human and bovine milk (Chatterton et al. 2004, 2006) and is fully synthesized in the mammary gland. Here a-lactalbumin acts as coenzyme for biosynthesis of lactose—an important source of energy for the newborn (de Wit 1998). It comprises approximately 20% to 25% of whey proteins (Table 10.1) and contains a wide variety of amino acids (123 amino acids), including a readily available supply of essential and branched chain amino acids (its sequence is quite homologous to that of lysozyme). Three genetic variants have already been identified—A, B, and C (Fox 1989). Its globular structure is stabilized by four disulfide bonds, at pH values in the range of 5.4 to 9.0 (Evans 1982).

10.4.1 Immunomodulation In a murine study, a-lactalbumin, in both the native and hydrolyzed states, was found to enhance antibody response to systematic antigen stimulation (Bounous and Kongshavn 1982). The same group proved a-lactalbumin has a direct effect on B-lymphocyte function, as well as suppressing T-cell-dependent and -independent responses (Bounous and Kongshavn 1985).

10.4.2 Anticancer Activity Based on its effect on cell division, a-lactalbumin was reported to contribute to reduce the risk of incidence of some cancers (Ganjam et al. 1997). Additionally, this protein can be a potent Ca2+-elevating and apoptosis-inducting agent (Hakansson et al. 1995). Sternhagen and Allen (2001) demonstrated the antiproliferative effects of a-lactalbumin in colon adenocarcinoma cell lines (Caco-2 or HT-29 monolayers).

10.4.3 Nutrition Effects and Other Metabolic Features Purified a-lactalbumin is most readily used in infant formula manufacturing, as it has the most structurally similar protein profile compared to breast milk. However, due to cost-effectiveness measures, most dairy-based infant formulas contain ingredients such as demineralized whey with higher levels of b-lactoglobulin, making them less similar to human milk (Marshall 2004). One of the most interesting features of a-lactalbumin is related to its association with the treatment of chronic stress-induced cognitive decline (Markus et al. 2002). In fact, an imbalance in brain serotonin function was claimed to be a factor mediating the negative effect of chronic stress on cognitive performance; serotonin release decreases under exposure to chronic stress, thus decreasing the available concentrations of brain serotonin and tryptophan (a precursor of serotonin), both of which cause serotonin activity to fall below functional needs. Due to its high tryptophan content (ca. 6% (w/w)) (Chatterton et al. 2006), a diet based on a-lactalbumin increases the plasma tryptophan-to-large neutral amino acids (Trpp-LNAA) ratio.

Potential Applications of Whey Proteins in the Medical Field

231

10.5 Bovine Serum Albumin (BSA) Bovine serum albumin (BSA) is a large protein that makes up approximately 10% to 15% of total whey protein (Table 10.1) and is not synthesized in the mammary gland, but appears instead in milk following passive leakage from the bloodstream. It contains 582 amino acid residues, which lead to a molar mass of 66,267 kDa; it also possesses 17 intermolecular disulfide bridges and one thiol group at residue 34 (Fox 1989). Because of its size and higher levels of structure, BSA can bind to free fatty acids and other lipids, as well as to flavor compounds (Kinsella et al. 1989). Its heat-induced gelation at pH 6.5 is initiated by an intermolecular thiol-disulfide interchange, similar to what happens with b-lactoglobulin (de Wit 1989). BSA is a source of essential amino acids, but there is very little available information regarding its potential therapeutic activity (Table 10.2).

10.5.1 Anticancer Activity BSA has been reported to inhibit tumor growth (Rodrigues et al. 2009; Smithers 2008). In vitro incubation with human breast cancer cell line MCF7 has provided adequate evidence thereof, which lays on modulation of activities of the autocrine growth regulatory factors (Laursen et al. 1990).

10.5.2 Nutrition Effects and Other Metabolic Features The aforementioned binding properties of BSA depend on the fatty acid (or other small molecules) at stake (Brown and Shockley 1982). Binding to fatty acids that are stored in the human body as fat allow it to participate in the synthesis of lipids, which are a part of all outer and inner cell membranes, and which provide energy (Choi et al. 2002). Furthermore, BSA has been reported to possess important antioxidant activities (Tong et al. 2000).

10.6 Lactoferrin Lactoferrin (LF), an iron-binding glycoprotein, is a nonenzymatic antioxidant found in the whey fraction of milk as well as in colostrums. The whey lactoferrin consists of approximately 700 amino acid residues (Table 10.1) and is composed of a single polypeptide chain with two binding sites for ferric ions (Rodrigues et al. 2009). The concentration of lactoferrin in bovine milk and colostrums is approximately 0.2 mg/mL and 1.5 mg/mL, respectively. The concentration of lactoferrin in most of the commercial whey protein powders is only 0.35–2.0% of the total protein content (Marshall, 2004). Structurally, LF is folded into two lobes, representing its N- and C-terminal halves (Baker 1994) that show sequence homology with each other and can each reversibly bind one ferric ion along with a synergistic anion, usually bicarbonate (Baker 1994; Steijns and Hooijdonk 2000). In these respects, it closely resembles transferrin, although its affinity for iron is somewhat higher, allowing iron to be retained at lower pH values (Peterson et al. 2000). This gives LF a more potent iron withholding ability (Baker et al. 2002). The lobes are connected by a peptide of 10 to 15 residues, which in LF forms a three-turn a-helix, but in transferrins is irregular and flexible.

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Ready availability of large quantities of purified lactoferrin has allowed intensive study of the biological effects of this protein in vitro and in vivo (Brock 2002; Rodrigues et al. 2009; Vorland 1999; Wakabayashi et al. 2006) (Table 10.2). The mechanisms of the well-known antimicrobial effects of lactoferrin have been established (Farnaud and Evans 2003). In an elegant work reported by Cornish and collaborators (2004), lactoferrin has been shown to have potent bone growth enhancement properties manifested through stimulation of the growth of osteoblasts and inhibition of osteoclasts. This research is helping form a strong science foundation for the use of lactoferrin as a complement to various strategies in the prevention and treatment of osteoporosis. Because of its close resemblance to transferrin, initial research on LF function was directed toward establishing functions related to its iron-binding properties— namely, iron absorption, antimicrobial activity, and modulation of iron metabolism during inflammation. However, despite their structural similarities, LF differs from its serum counterpart in several important aspects, including location and functional activity. LF has been proposed to play a role in intestinal iron absorption, regulationw of cellular proliferation and differentiation, protection against microbial infection, anti-inflammatory responses, regulation of myelopoiesis, immunomodulation, and cancer prevention (Baveye et al. 1999; Iyer and Lönnerdal 1993; Levay and Viljoen 1995; Min and Krochta 2005; Naidu 2002; Nuijens et al. 1996; Pan et al. 2007; Sanchez et al. 1992; Steijns and van Hooijdonk 2000; Ward et al. 2005). Table 10.4 summarizes some of the established physiological roles for LF and its mechanisms.

10.6.1 Antimicrobial and Antiviral Activity The reported antimicrobial activities (Levay and Viljoen 1995; Santagati et al. 2005; Valenti et al. 1998) of LF highlight the many possible modes by which it can contribute to host protection against microbial infections at the mucosal surfaces—namely, by growth inhibition as a result of iron scavenging (Brock 1980; Nemet and Simonovits 1985), disruption of the bacterial cell membranes (Al-Nabulsi and Holley 2006; Ellison and Giehl 1991; Yamauchi et al. 1993), or blocking of cell–virus interactions (Andersen et al. 2001; Giansanti et al. 2002; Ikeda et al. 2000; Siciliano et al. 1999).

10.6.2 Immunomodulation Regarding LF anti-inflammatory activity, several mechanisms of action by blocking or inhibiting key mediators of the inflammatory response have also been proposed, such as the binding to lipopolysaccharide (LPS) (Miyazawa et al. 1991); the inhibition of several cytokines (TNF-a and IL-1b) (Baveye et al. 1999; Crouch et al. 1992; Machnicki et al. 1993; Slater and Fletcher 1987), or the binding to bacterial CpG motifs (Britigan et al. 2001). Moreover, LF was found to elevate the number and increase the activity of T and B lymphocytes and NK cells (Dhennin-Duthille et al. 2000; Goretzki and Mueller 1998), stimulate the release of a number of cytokines (Hangoc et al. 1991), increase phagocytic activity and cytotoxicity of monocytes/macrophages (Birgens et al. 1984; Van Snick and Masson 1976), accelerate the maturation of T and B cells, and elevate the expression of several types of cellular receptors (Adamik et al. 1998; Bennett and Davis 1981; Frydecka et al. 2002; Zimecki et al. 1991, 1995). Many immunological

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Table 10.4 Reported in vitro and in vivo Phsiological Roles of Lactoferrin Physiological Role

Mechanism

References

Iron absorption

Increasing solubility and receptor-mediated uptake

Levay and Viljoen 1995; Ward et al. 2005

Antioxidant

Iron scavenger Lactoferrin (LF) has the ability to bind to cell membranes enhancing its ability to prevent iron-mediated lipid peroxidation

Konishi et al. 2006; Larkins 2005

Antimicrobial

Growth inhibition by iron scavenging or membrane disintegration LF has a powerful iron-binding capacity and shows a strong interaction with other molecules and cell surface

Lee et al. 2005; Nemet and Simonovits 1985; Qiu et al. 1998; Santagati et al. 2005; Weinberg 2007; Yamauchi et al. 1993

Antiviral

Prevention of virus attachment, inhibition of virus replication, blocking of cell–virus interactions

Ammendolia et al. 2007; Andersen et al. 2001; Giansanti et al. 2002; Ikeda et al. 2000; Longhi et al. 2006; Mistry et al. 2007; Pan et al. 2006

Anti-inflammatory, immune modulating

LPS binding, stimulation of natural killer (NK) cells, reduction of pro-inflammatory cytokines, T-cell maturation LF modulates the migration, maturation, and function of the immune cells at the cellular level and at the molecular level

Adamik et al. 1998; Baveye et al. 1999; Berlutti et al. 2006; Britigan et al. 2001; Crouch et al. 1992; Dhennin-Duthille et al. 2000; Fischer et al. 2006; Hangoc et al. 1991; Kruzel et al. 2006; Legrand et al. 2005; Senkovich et al. 2007; Yamauchi et al. 1993; Zimecki et al. 1991, 1995

Anticancer

Regulation of NK cell activity, modulation of expression of G1 proteins, inhibition of VEGF(165)-mediated angiogenesis, enhancement of apoptosis

Baumrucker et al. 2006; Bezault et al. 1994; Damiens et al. 1998, 1999; Giuffre et al. 2006; Kawakami et al. 2006; Kim et al. 2006; Kuhara et al. 2006; McKeown et al. 2006; Mohan et al. 2006; Norrby et al. 2001; Sekine et al. 1997; Yoo et al. 1997

Source: Adapted from Rodrigues LR, Teixeira JA, Schmitt F, Paulsson M, Lindmark Måsson H (2009) Crit Rev Food Sci Nut 49: 1–15.

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mechanisms are critically dependent upon cell–cell interactions; the number and affinity of interactions between two cells can often affect the nature of downstream events. The ability of LF to bind to cell surfaces is likely to affect these parameters and could thus give rise to altered immune responses (Legrand et al. 2005).

10.6.3 Anticancer Activity Several studies have been done to evaluate the effect of orally administrated LF in healthy or diseased human beings and animals (Teraguchi et al. 2004; Tomita et al. 2002). To date, it has become evident that the oral administration of LF exerts various beneficial effects against diseases (Table 10.5)—namely, as a chemopreventive agent.

Table 10.5 Effectiveness of Orally Administered bLF in Humans Disease Bacterial Flora Fecal flora in low birth weight infants

Fecal flora in infants

Efficacy

Increase of Bifidobacterium, decrease of Clostridium

Administrated Agent and Dose

bLF; 1 mg/ml in infant formula

Increase of Bifidobacterium

Infection (Digestive Tract) Gastric infection with Increase of eradication Helicobacter pylori by triple therapy Infection (Other than Digestive Tract) Neutropenic patients Decrease of incidence of bacteremia and severity of infection

References

Wakabayashi et al. 2006

Roberts et al. 1992

bLF; 0.2 g/body

Di Mario et al. 2003

bLF; 0.8 g/body

Trumpler et al. 1989

Chronic hepatitis C

Decrease of ALT and HCV RNA in serum

bLF; 1.8 and 3.6 g/ body

Iwasa et al. 2002; Konishi et al. 2006; Tanaka et al. 1999; Yamauchi et al. 1993

Influenza

Attenuation of pneumonia through the suppression of infiltration of inflammatory cells in the lung

62.5 mg per mouse

Katsuaki 2006

Source: Adapted from Rodrigues LR, Teixeira JA, Schmitt F, Paulsson M, Lindmark Måsson H (2009) Crit Rev Food Sci Nut 49: 1–15.

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Table 10.6 Effectiveness of Orally Administered Lactoferrin (LF)-Related Compounds on Cancers

Cancer Model

Efficacy

Animal

Administrated Agent and Dose

References

Carcinogeninduced tumor in colon, lung, esophagus, bladder, liver, tongue

Inhibition of tumor development

Rat

bLF 0.2 and 2% in diet

Fujita et al. 2002; Masuda et al. 2000; Mohan et al. 2006; Sekine et al. 1997; Tanaka et al. 2000; Ushida et al. 1999

Spontaneously developed intestinal polyposis

Inhibition of polyp development

ApcMin mouse

bLF 2% in diet

Ushida et al. 1998

Tumor cell injection

Inhibition of lung metastasis

Mouse

bLF 0.3 g/kg

Iigo et al. 1999

Tumor cell injection

Inhibition of tumor development

Mouse

rhLF 1 g/kg

Varadhachary et al. 2004

Source: Adapted from Rodrigues LR, Teixeira JA, Schmitt F, Paulsson M, Lindmark Måsson H (2009) Crit Rev Food Sci Nut 49: 1–15.

The protective effect of LF against chemically induced carcinogenesis, tumor growth, and metastasis have been demonstrated in an increasing number of animal model experiments, namely directed to specific organs, such as esophagus, tongue, lung, liver, colon, and bladder (Bezault et al. 1994; Fujita et al. 2002; Iigo et al. 1999; Kuhara et al. 2000; Masuda et al. 2000; Sekine et al. 1997; Shimamura et al. 2004; Tanaka et al. 2000; Tsuda et al. 2002; Ushida et al. 1999; Varadhachary et al. 2004; Wang et al. 2000; Yoo et al. 1997). Table 10.6 summarizes the effectiveness of orally administrated LF-related compounds on cancer. Despite the evidence that LF possesses chemopreventive activity, little is known about its anticancer activity against established tumors; its ability to potentiate chemotherapy, as described with other immunotherapeutics; or the immune mechanisms by which its antitumor activity is mediated. Moreover, no human clinical studies on the potential chemopreventive effects have been done so far.

10.6.4 Nutrition Effects and Other Metabolic Features Supplementing the diet of dairy calves with lactoferrin has been tried, along with monitoring its effect on their performance (Joslin et al. 2002); lactoferrin was able

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to improve the average weight gain and decrease weaning time in dairy calves. An in vivo research effort conducted by Kajikawa and collaborators (1994) led them to hypothesize that bovine lactoferrin may act as an antiatherogenic agent, by inhibiting accumulation of cholesterol esters in macrophages. Furthermore, this protein can survive gastric digestion in adults (Troost et al. 2001) and bears no toxicity when orally administered to rats (Yamauchi et al. 2000); these two reasons account for its regular use as a bioactive ingredient for therapeutic administration.

10.7 Lactoperoxidase Whey contains many types of enzymes, including hydrolases, transferases, lyases, proteases, and lipases. Lactoperoxidase, an important enzyme in the whey fraction of milk, is the most abundant enzyme, and the majority of it ends up in whey following the curding process. Lactoperoxidase accounts for 0.25% to 0.5% of total protein found in whey (Table 10.1). It has the ability to catalyze certain molecules, including the reduction of hydrogen peroxide (Bjorck 1978). This enzyme system catalyzes peroxidation of thiocyanate and some halides (such as iodine and bromium), which ultimately generates products that inhibit or kill a range of bacterial species (Kussendrager and van Hooijdonk 2000). During the pasteurization process, lactoperoxidase is not inactivated, suggesting its stability as a preservative. Lactoperoxidase’s biological function has been mainly associated with its antimicrobial activity, and there is no report on other direct functions such as immunomodulation or cancer prevention.

10.7.1 Antimicrobial and Antiviral Activity Lactoperoxidase is an important part of the natural host defense system in mammals, which provides protection against invading microorganisms (de Wit and van Hooydonk 1996). The mechanism of action of lactoperoxidase was explained in detail elsewhere (Boots and Floris 2006; de Wit and van Hooydonk 1996); such an enzyme is more active at acidic pH (Wever et al. 1982). The antimicrobial activity of peroxidases depends on the ion acting as electron donor. The LP system is completed when lactoperoxidase, thiocyanate ion (SCN–), and hydrogen peroxide (H2O2) are present together (Reiter and Harnulv 1984). The thiocyanate anion, which is necessary for the antibacterial activity of lactoperoxidase be expressed, appears to significant extents in saliva, milk, and airway secretions (Reiter and Perraudin 1991). The amount of SCN– in cow’s milk ranges from 0.1 to 15 mg/kg (Perraudin 1991). Studies pertaining to the antimicrobial effects of lactoperoxidase have focused on its role upon thermal resistance of Salmonella spp. (Doyle and Mazzotta 2000), in both raw (Heuvelink et al. 1998) and mature milk (Shin et al. 2000). The natural antimicrobial action of lactoperoxidase is being exploited in a range of oral health care products (Boots and Floris 2006) and is finding application in products directed toward the prevention and treatment of xerostomia (dry mouth) (Tenovuo 2002). The lactoperoxidase-containing products have been clinically proven to inhibit harmful microorganisms associated with gingivitis and oral irritation, to promote the healing of

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bleeding gums and reduce inflammation, and to combat both the causes and effects of halitosis (bad breath) (Tenovuo 2002).

10.8 Immunoglobulins Immunoglobulins constitute a complex group, the elements of which are produced by B lymphocytes; they make a significant contribution to the whey protein content, in addition to exerting an important immunological function (especially in colostrums). These proteins are present in the serum and physiological fluids of all mammals; some of them attach to surfaces, where they behave as receptors, whereas others function as antibodies, which are released in the blood and lymph. There are five classes of antibodies: IgA, IgD, IgE, IgG, and IgM. IgG constitutes approximately 75% of the antibodies in an adult. IgG is transferred from mother to child in utero via cord blood and by breast-feeding and serves as a child’s first line of immune defense, referred to as “passive immunity.” IgA is secreted in breast milk and ultimately transferred to the digestive tract in the newborn infant, providing better immunity than a bottlefed child (Bonang et al. 2000; Mehra et al. 2006). Colostrums contain significantly greater concentrations of immunoglobulins than mature milk. Immunoglobulins reach maximum concentration in the first 24 to 48 hours postparturition and decline in a time-dependent manner following peak concentration (Kelly 2003). Similarly, the whey fraction of milk appears to contain a significant amount of immunoglobulins, approximately 10% to 15% of total whey proteins (Mercier et al. 2004) (Table 10.1).

10.8.1 Antimicrobial and Antiviral Activity Immunoglobulins have been reported to prevent and treat various microbial infections (upper respiratory tract infections, gastritis, dental caries, and diarrhea, among others) (Mehra et al. 2006; Pan et al. 2006) (Table  10.2). An in vitro study demonstrated that bovine-milk-derived IgG suppresses human lymphocyte proliferative response to T cells at levels as low as 0.3 mg/mL of IgG (Kulczycki and MacDermott 1985). The authors further concluded that bovine milk IgG typically ranges between 0.6 and 0.9 mg/mL and is therefore likely to confer immunity that could be carried to humans. Studies show that raw milk from nonimmunized cows contains specific antibodies to human rotavirus, as well as antibodies to bacteria such as Escherichia coli, Salmonella enteriditis, S. typhimurium, and Shigella flexneri. Furthermore, antibody concentrates derived from immune colostrum and milk collected from cows immunized with inactivated human rotavirus (HRV) serotypes 1 (Wa) and 2 (S2), as well as simian rotavirus serotype 3 (SA11), were shown to possess preventive (or treatment) features in enteric diseases (Sarker et al. 1998). Additionally, immune bovine colostrum was demonstrated to be effective against Cryptosporidium parvum infections common in patients with HIV (Okhuysen et al. 1998). There is also evidence of protection via bovine antibodies against dental caries caused by cariogenic streptococci (Loimaranta et al. 1999).

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10.8.2 Immunomodulation As described above, immunoglobulins provide protection against diseases in the newborn through passive immunity (Bonang et al. 2000; Mehra et al. 2006). Most studies have focused on newborn ruminants, but more recent research has started exploring the effects on nonruminants and adults.

10.8.3 Nutrition Effects and Other Metabolic Features Immunoglobulins represent the most abundant of the established bioactive proteins found in whey. Although colostrum represents the preferred choice of raw materials for the manufacture of immunoglobulin-enriched milk products (Huffman and Harper 1999), cheese whey has been used for the preparation of similar ingredients (Ayers et al. 2003). These immunoglobulin-rich isolates all impart passive immunity to the consumer, and evidence is building that they combat infections, improve athletic performance and recovery times, assist those who may be immunocompromised, and enhance gut health (Mehra et al. 2006). Furthermore, immune milk was also suggested to lower blood pressure (Sharpe et al. 1994). In a double-blind, clinical-trial study, the effects on reduction of plasma cholesterol and blood pressure of immune milk produced by dairy cows previously hyperimmunized with a multivalent bacterial vaccine were assessed, involving human hypercholesterolemic subjects who consumed 90 g of immune milk dairy, versus regular milk; the former was a useful adjunct in the dietary management of hypercholesterolemia (Sharpe et al. 1994).

10.9 Others 10.9.1 Proteose Peptones The potential exploitation of selected milk proteins as ingredients in functional food products has been the reason for an increasing interest in their fractionation (Andrews et al. 2000). Heating of skimmed milk (95°C, 30 min) followed by acidification promotes the denaturation of whey proteins and their coprecipitation with caseins, which are insoluble at pH 4.6 (Girardet and Linden 1996). In spite of these drastic conditions, a heterogeneous fraction called proteose peptone remains soluble. The total proteose peptone fraction of bovine milk represents about 10% of total whey protein (Table 10.1). Its principal components have been designated as components 3, 5, and 8 (PP3, PP5, PP8) (Innocente et al. 1999). The primary structure of PP3 includes a polypeptide backbone of 135 amino acid residues containing five phosphorylated serines, two threonine-linked O-glycosylations, and one N-glycosylation, with an apparent molecular mass of 28 kDa (Sorensen et al. 1997). Also, a glycoprotein with apparent molecular mass of 18 kDa is associated with component PP3, corresponding to the 54-135 fragment released by plasmin hydrolysis in milk (Sousa et al. 2007).The PP3 is extremely hydrophobic and particularly interesting because of its functional properties, such as its emulsifying power, strong affinity for oil–water interface, strong foaming properties, and biochemical role (Innocente et al.  1999;

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Rodrigues et al. 2003). Although not many studies have been conducted on the biological functions of PP3, some researchers demonstrated its immunostimulation (Sugahara et al. 2005) and prebiotic effects (Etienne et al. 1994) as well as its role in caries prevention (Aimutis 2004; Grenby et al. 2001) (Table 10.2).

10.9.2 Glycomacropeptide Glycomacropeptide (GMP), also referred to as casein macropeptide, is biologically active and of particular interest in food science and nutrition for the manufacture of novel functional foods (Thoma-Worringer et al. 2006). GMP is a protein present in whey at 10% to 15%, due to the action of chymosin on casein during the cheesemaking process (Table 10.1) (Marshall 2004). GMP is only present when chymosin is used during processing; therefore, cheeses such as cottage cheese not made with chymosin do not produce GMP in the curding process (Brody 2000). GMP is high in branched-chain amino acids including phenylalanine, tryptophan, and tyrosine. It is one of the few naturally occurring proteins that lack phenylalanine, making it safe for individuals with phenylketonuria (PKU) (Thoma-Worringer et al. 2006).

10.9.3 Osteopontin Osteopontin was identified independently, together with bone sialoprotein (BSP), as a major sialoprotein in the mineral extracellular matrix of bone (Prince et al. 1987; Zhang et al. 1990). The name osteopontin was introduced to reflect the potential of the bone protein to serve as a bridge between cells and hydroxyapatite (HA) through RGD (arginine-glycine-aspartate motif) and polyaspartic acid motifs discovered in the primary sequence of the protein (Oldberg et al. 1986). Generally, osteopontin is extremely hydrophilic with a low isoelectric point (pI: 3.5), and it displays an unusual amino acid composition (Christensen et al. 2005). The conservation among mammalian species of certain residues is presumably indicative of the functions osteopontin performs (Sodek et al. 2000). For instance, osteopontin has been found to play a role in bone mineralization due to its characteristic amino acid composition and its interaction with integrin receptors on cells lining these surfaces (Hunter et al. 1994; Oldberg et al. 1986). The RGD motif that is particularly exposed in the osteopontin molecule represents a major, although not unique, binding ligand for the family of integrin receptors and was found to be involved in cell attachment, cell migration, and intracellular signalling (Denhardt and Noda 1998). Osteopontin has been suggested as having a protective role in interactions between epithelial surfaces and the external environment, as well as other biological functions (Table 10.2). In milk, osteopontin is likely to have a physiological role (Senger and Perruzzi 1989), as it was noticed that milk is a rich source of the protein. In bone, osteopontin is produced by the matrix-synthesizing osteoblasts at the mineralizaton front and by bone-resorbing osteoclasts (Butler 1989; Oldberg et al. 1986). Osteopontin preferentially accumulates at cell–matrix and matrix–matrix interfacial structures in bone. Hence, it has multiple presumed functions, including the attachment of osteogenic cells to bone matrix, control of mineralization (Goldberg and Hunter 1995; Hunter

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et al. 1996; Nagata et al. 1991), coupling of bone formation, and resorption (Giachelli and Steitz 2000; Ross et al. 1993). Osteopontin is not only produced by epithelial and bone cells but also by activated macrophages and lymphocytes (Ashkar et al. 2000; Giachelli et al. 1998; Patarca et al. 1989, 1993; Santana and Rosenstein 2003), as well as kidney tubule cells, arterial endothelial and smooth muscle cells, and fibroblastic cells in embryonic stroma and in wound healing sites (Liaw et al. 1998). Finally, several studies have described a link between osteopontin and tumor progression and metastasis (Chambers et al. 1992; Rodrigues et al. 2007; Senger et al. 1989a,b).

10.10 Future Trends As referred by Smithers (2008), the transformation of whey from “gutter to gold” over the past approximately 50 years, founded on advances in science and technology, has resulted in increasingly sophisticated products. Concomitant increases in the value of these products in an increasingly sophisticated marketplace have resulted in enhanced wealth to dairy manufacturers and the communities that rely on them. Whey components, particularly the proteins and peptides, will increasingly be preferred as ingredients for functional foods and nutraceuticals, and as active medicinal agents, built upon the strong consumer trend for health and well-being, and continuing discovery and substantiation of the biological functionality of whey constituents. Current challenges in the exploitation of these bioactive whey constituents include 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). Therefore, the future encompasses the development of emerging technologies for nonthermal processing and preservation of whey (e.g., high-pressure processing, high-power ultrasound, pulsed electric field, microfluidization (Devlieghere et al. 2004), new avenues for modulation of protein functionality, and continuing expansion of the applications base. Furthermore, novel separation technologies will be developed, refined, and introduced to whey processing for the cost-effective manufacture of whey-based products. Possibilities include vibratory shear enhanced processing, further enhancements to continuous separation in combination with high-power ultrasound and other technologies, ion exclusion, and molecular recognition-based isolation techniques (Smithers 2008).

10.11 Conclusion A host of interesting whey protein products are currently available and allow for the tailoring of their use to specific clinical indications. Hydrolyzed whey provides readily available di- and tripeptide fractions attractive to athletes and other individuals desiring a quickly absorbed, low allergenicity protein source. Undenatured whey provides the highest concentrations of intact native proteins, such as lactoferrin and immunoglobulins, which can be provided as functional food for immunomodulation or antimicrobial purposes. Glycomacropeptide isolates do not contain the amino acids phenylalanine, tryptophan, or tyrosine, providing a valuable protein source for individuals with PKU.

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In summary, the transformation of whey from a waste into a valuable dairy stream containing a multitude of components available for exploration in the agri-food, biotechnology, medical, and related markets, is currently both a reality and a growing tendency.

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Index a Acid coagulation, 19 Acid whey, spray-drying of, 49 Actipak study, 186 Active packaging, see Packaging, active and intelligent Adsorption, 83–86 activation energy, 86 active points, 85 apparent, 87 batch, 91 categories, 85 chemical and physical adsorption, 84–86 colloidal systems, 8–14 factors affecting adsorption, 14 Gibbs equation, 9 insoluble monolayers, 11 Integral Mean Value Theorem, 8 interactions contribution to, 8 interfacial tension, 9, 10 Langmuir–Blodgett technique, 13 Langmuir monolayers, 11, 12 Langmuir–Schaeffer technique, 13 monolayers, 10–13 surfactant monolayer, 11 conservation equations involved in, 94–97 energy balance, 95–97 mass balances, 94–95 features, 84 gas–solid system, 85 isothermal, 96 operation modes, 99–106 adsorption in expanded beds, 104–106 batch adsorption, 99–100 fixed-bed adsorption, 100–104 process, 85 relative, 87 solute transport, 84 stages, 85 surfaces, 84 Affinity partitioning, 66, 67 AI tools, see Artificial intelligence tools Alkaline phosphatase (ALP), 161 ALP, see Alkaline phosphatase Amphiphilic molecules adsorption, 10 chemical potential, 15, 16 fundamental characteristic, 16 Langmuir monolayers, 11

micellization, 14 mobile barrier, 12 nonaqueous solution, 17 repulsive forces, 15 Anionic exchangers, 108 Antimicrobial packaging, development of, 185–190 antimicrobial immobilization in polymers, 188–189 antimicrobial incorporation into plastic polymers, 187–188 commonly used antimicrobial substances, 186–187 production of antimicrobial films, 190 surface modification, 189–190 AOP cheese, see Appellation d’origine protégée cheese Appellation d’origine protégée (AOP) cheese, 40 Aqueous two-phase systems (ATPSs), 57–79 affinity protein partitioning, 66–67 applications, 59, 63 aseptic food processing, 72 binodal curve, 60 bioconversion, 67 biomolecule distribution, 64–66 hydrophobic groups, 66 molar mass of polymer, 64 pH, 65 polymer charge, 65–66 polymer concentrations, 65 salts, 65 temperature, 66 case study, 72–74 conventional liquid–liquid extraction equipment, 69–72 dairy industry applications, 63 discard of salts, 69 electrodialysis, 69 endo-PG recovery, 70 equilibrium curve, 60 extractive bioconversion, 67 formation of ATPSs, 58 Graesser extractor, protein partitions using, 73–74 continuous cycle on prepilot scale, 74 determination of operational variables, 74 high-performance liquid chromatography, 73 Graesser extractor, separation of serum proteins in, 72–74

253

254 axial dispersion, 73 holdup fraction, 73 phase diagrams, 73 physical-chemical characteristics, 73 retention time distribution, 72 HM-EOPO copolymer, 60 hydrophilic polymers, 58 isolation of biomolecules, 58 Lattice theory, 62 ligands, 66 liquid–liquid extraction, 57, 71 open system dispersion model, 73 partition coefficient, 64 phase diagram for salt–polymer system, 61 phase equilibrium diagrams, 60–62 phase separation studies, 61 physicochemical characteristics, 62–63 Raining Bucket Contactor, 69 recycling of constituent reagents, 68–69 salt + polymer, 60, 61 thermodynamic models, 62 types, 58–60 UNIQUAC model, 62 viscosity of phases, 62 Arabic gum, 22 Artificial intelligence (AI) tools, 195 Aseptic food processing, 72 Aspergillus niger, 194 ATPSs, see Aqueous two-phase systems Avogadro constant, 20 Axial dispersion coefficient, 98

b Bacteria IR heating used to inactivate, 165 lactic starter, 42 nonstarter lactic acid bacteria removal of, 51 spore-forming, 36 Bacteriocins, 186, 189 Bactocatch®, 36 Batch crystallization, 128 Beverage emulsions, stabilized, 22 BHA, see Butylated hydroxyanisole BHT, see Butylated hydroxytoluene Bi-Langmuir isotherm, 89–90 Bio-Gel®, 114 Bio-gele®, 114 Biomolecule(s) partitioning, influence of temperature on, 66 purification of, 29 separation, ATPS application for, 69 Bioseparation processes, 27–31 centrifugation, 28 liquid–liquid extraction, 28 milk preconcentration step, 27

Index physicomechanical processes, 28 purification of biomolecules, 29 purification techniques, 30 solute concentration, 29 techniques for the separation of biocompounds, 29–30 unit operations, 27, 28 whey protein concentrate, 29 Bone sialoprotein (BSP), 239 Bovine serum albumin (BSA), 59, 63, 113, 231 Bovine spongiform encephalopathy (BSE), 38 Break point, 101, 103 Brine treatment, 50 cleaning in place solutions, 50 Kieselguhr treatment, 50 water source for steam production, 50 white waters, 50 Brucella abortus, 37 BSA, see Bovine serum albumin BSE, see Bovine spongiform encephalopathy BSP, see Bone sialoprotein Butylated hydroxyanisole (BHA), 194 Butylated hydroxytoluene (BHT), 194

c Capillarity effects, 6 Capillary pressure, 6 Carbery process, 49 Casein, 19, 42, 168, 211 κ-Casein, 40 Caseinomacropetide (CMP), 30, 72 Cationic exchangers, 108 CCK, see Cholecystokinin Centrifugation, 28 Cheese Appellation d’origine protégée, 40 brine, sanitation of, 50, see also Brine treatment Cheddar, 195 compounds, antimicrobial compounds in, 192 cream cheese, 42, 43 French fromages frais, 42, 43 Gorgonzola, 188 mozzarella, 195 production (membrane technologies), 40–45 acidification kinetics, 42 adjustment of aqueous phase of cheese milk, 44 appellation d’origine protégée, 40 buffering capacity, 42–43 cheese made by membrane technologies, 44–45 LAB exopolysaccharides, 44 liquid precheese, 42 milk production, 45

Index MMV process, 41 rennet coagulation, 43–44 rheological changes, 43 ripening enzymes, 42 ripening of UF cheese, 45 unripened cheeses, 42 Quarg, 42, 43 Chemical adsorption, 85 Chitosan, 21, 189, 211 Cholecystokinin (CCK), 40 Chromatographic techniques, 81–119 activated carbon, 87 activated clay, 86 adsorbate, 82 adsorption, 83–86 activation energy, 86 active points, 85 categories, 85 chemical and physical adsorption, 84–86 features, 84 gas–solid system, 85 introduction of word, 83 process, 85 solute transport, 84 surfaces, 84 adsorption operation modes, 99–106 adsorption in expanded beds, 104–106 batch adsorption, 99–100 fixed-bed adsorption, 100–104 alumina, 86 anionic exchangers, 108, 109 applications of adsorptive techniques in dairy industry, 83 bauxite, 86 break point, 101, 103 cationic exchangers, 108 characteristic rupture curve, 102 cleaning-in-place procedure, 105 column packing, 115 conservation equations involved in adsorption, 94–97 energy balance, 95–97 mass balances, 94–95 counterions, 109 desalination, 113 electric double-layer theory, 109 eluate, 101 eluent, 101 elution, 101 Fick’s axial dispersion coefficient, 95 filter, 101 Fuller’s earth, 86, 87 gel filtration, 112 gel microstructure, 114 Gouy’s double layer, 109 Helmholtz’s double layer, 109 Henry’s Law, 88, 89

255 ionic exchange, 107–108 electric double layer, 110 ion exchange equilibrium, 109 ionic exchange mechanism, 109 ionic exchangers, 107–109 kinetics of adsorptive process, 97–99 axial dispersion coefficient, 98–99 extraparticle transport mechanism, 98 intraparticle transport mechanisms, 97–98 mechanical resistance, 86 molecular exclusion chromatography, 111–115 basic principles, 113–115 general aspects, 112–113 molecular sieves, 87 nature and types of adsorbents, 86–87 packed-bed saturation, 102 percolation, 100 polymers and resins, 87 pore diffusion, 97 resolution, 113 selectivity factor, 113 silica gel, 87 sorption equilibrium, 87–94 batch or stirred tank method, 91–92 Bi-Langmuir isotherm, 89–90 determination of adsorption isotherms, 91–93 exponentially modified Langmuir isotherm, 90–91 Freundlich isotherm, 88–89 frontal analysis method, 92–93 Jovanovic isotherm, 90 Langmuir isotherm, 89 linear isotherm, 88 solute adsorption in dilute solutions, 87–91 solute desorption, 93 sorption hysteresis, 93–94 Toth isotherm, 90 Stern’s double layer, 109 surface diffusion, 98 total and usable capacity of packed bed, 103 van der Waals interactions, 84 zeolites, 87 CIP solutions, see Cleaning in place solutions Cleaning-in-place procedure, 105 Cleaning in place (CIP) solutions, 50 Clostridium botulinum, 37 tyrobutyricum, 40 CMC, see Critical micelle concentration CMP, see Caseinomacropetide Coagulation, definition of, 19 Coalescence, 18 Colloidal stability, ice cream, 19

256 Colloidal systems (food engineering), physical chemistry of, 1–25 acid coagulation of milk, 19 adsorption, 8–14 factors affecting, 14 Gibbs equation, 9 insoluble monolayers, 11 Integral Mean Value Theorem, 8 interactions contribution to, 8 interfacial tension, 9, 10 Langmuir–Blodgett technique, 13 Langmuir monolayers, 11, 12 Langmuir–Schaeffer technique, 13 monolayers, 10–13 surfactant monolayer, 11 beverage emulsions, stabilized, 22 binary system, 5 capillarity, 6–7 capillary pressure, 6 casein, 19 charge in colloid particles, 19 chitosan, 21 classification of colloidal dispersions, 3 coagulation, 19 coalescence, 18 colloidal stability, 17 colloid formation, 18 colloid particles, forms of, 2 comminution, 18 contact angle, 6 critical micelle concentration, 14 cross section of bubble, 7 double electrical layer, 19–20 edible coatings applications, 21 examples, 3 fat globule, 16 fat replacement, 22 first description of colloidal dispersions, 1 food engineering and technology, 21–22 general concepts, 2–6 Gibbs energy excess, 3, 18 Gouy–Chapman layer, 20 history, 1 interface, 3 interfacial tension, 4, 7 ion configuration around interface, 20 isothermic distillation, 18 lyophilic colloid systems, 17 lyophobic colloid systems, 17 micellization, 14–17 critical micelle concentration, 14, 16 detergents, 16 Gibbs equation,15 interfacial tension reduction, 14 surfactants, 16 thermodynamics, 15 milk stability, 19

Index model mayonnaise system, 22 pseudosolutions, 1 rennin enzymes, 19 solid state, 12 stability of colloidal systems, 17–19 Stern–Helmholtz layer, 20 superficial pressure, 12 Colostrinin, 51 Colostrums, 237 Colostrum treatment, 49–50 bioactive components, 50 colostrinin, 51 milk basic proteins, 50 serocolostrum, 49 ultrafiltration, 49 Comminution, 18 Contact angle, 6 Counterions, 109 Cream cheese, 42, 43 Critical micelle concentration (CMC), 14, 211 amphiphilic molecule, 16 factors influencing, 17 Cryptosporidium parvum, 237 Crystal agglomeration, 129 Crystallization, see Lactose and whey protein, crystallization of Crystal size distribution (CSD), 123, 145 CSD, see Crystal size distribution

d Dairy wastewater treatment, 50 cleaning in place solutions, 50 Kieselguhr treatment, 50 water source for steam production, 50 white waters, 50 Decimal reduction (DR), 36 Desalination, 113 Diafiltration, milk protein separation, 39 Differential scanning calorimetry (DSC), 167 Differential scanning microcalorimetry (µDSC), 212–218 equipment, 213–215 experiment, 215–217 calibration, 216 concentration requirements, 215–216 data analysis, 216 loading of sample and reference cells, 216 parameters, 216 sample preparation, 215–216 troubleshooting, 216–217 fundamentals, 212–213 instrument, 214 practical applications, 217–218 protein unfolding, 217 Dirac delta function, 123

257

Index Direct electrical resistance heating, 157, see also Ohmic heating technology DLVO model, 129 DR, see Decimal reduction Drowning out crystallization, 144 DSC, see Differential scanning calorimetry µDSC, see Differential scanning microcalorimetry

e ED, see Electrodialysis Edible coatings applications (colloidal systems), 21 Edible packages, 180–182 EFSA, see European Food Safety Authority Electrical resistance heating, 157, see also Ohmic heating technology Electric double-layer theory, 109 Electroconductive heating, 157, see also Ohmic heating technology Electrodialysis (ED), 34, 69 Electroheating, 157, see also Ohmic heating technology Electropure Process, 158 Elution, 101 Enterococcus faecalis, 189 EO, see Ethylene oxide EPS, see Exopolysaccharides Equilibrium thermodynamics, 127 Escherichia coli, 160, 166, 194, 237 ESL MF raw milk, see Extended shelf-life MF raw milk Ethylene absorber, 183–184 Ethylene oxide (EO), 59 European Food Safety Authority (EFSA), 186 Exopolysaccharides (EPS), 44 Extended shelf-life (ESL) MF raw milk, 37

f FA, see Frontal analysis Far-infrared (FIR), 164 FDA, see United States Food and Drug Administration FDP, see Flash desorption process Fermented milks, 39 FFAs, see Free fatty acids Fick’s axial dispersion coefficient, 95 FIR, see Far-infrared Flash desorption process (FDP), 93 Fluorescence spectroscopy (FS), 167 Food industrialization, aim of, 27 Free fatty acids (FFAs), 161 French fromages frais, 42, 43 Freundlich isotherm, 88–89 Frontal analysis (FA), 92

FS, see Fluorescence spectroscopy Fuller’s earth, 86, 87 Fuzzy logic, 195

g β-GAL, see β-Galactosidase β-Galactosidase (β-GAL), 161 Gelarose•, 114 Gel filtration, 112 Generally Recognized as Safe (GRAS), 181 Geotrichum sp., 193 Gibbs free energy aqueous two-phase systems, 60 density of, 5 excessive, 3 interfacial tension, 4 ion configuration, 20 reduction, 15 Glucose–water system, phase diagram of, 134 Glutathione (GSH), 224, 227 Glycomacropeptide (GMP), 40, 239 GMP, see Glycomacropeptide GMPs, see Good Manufacturing Practices Good Manufacturing Practices (GMPs), 181 Gouy–Chapman layer, 20 Graesser extractor operating conditions, 72 photograph, 71 protein partitions using, 73–74 continuous cycle on prepilot scale, 74 determination of operational variables, 74 high-performance liquid chromatography, 73 separation of serum proteins in, 72–74 axial dispersion, 73 holdup fraction, 73 phase diagrams, 73 physical-chemical characteristics, 73 retention time distribution, 72 Granulation, 129 GRAS, see Generally Recognized as Safe Growth-unit, 125 GSH, see Glutathione

h HA, see Hydroxyapatite Hafnia alvei, 40 Heat absorption factor, 164 Heat of dilution, 206 Henry’s Law, 88, 89 High-performance liquid chromatography (HPLC), 73 High-pressure (HP) processing, 168–169 advantages, 168 kinetic data, 169

258 low acid food processing, 168 microorganism inactivation, 168 milk treatment, 168 High-temperature short-time (HTST) pasteurization, 156, 161 HPLC, see High-performance liquid chromatography HP processing, see High-pressure processing HRV, see Human rotavirus HTST pasteurization, see High-temperature short-time pasteurization Human rotavirus (HRV), 237 Humidity absorber, 184 Hurdle concept, 169 Hydrogenolysis, 139 Hydrophobically modified starch, 22 Hydroxyapatite (HA), 239

i IC, see Iota-carrageenan Ice cream, colloidal stability in, 19 Ideal whey, 46, 48 IDP, see Isothermal desorption process IH, see Infrared heating Immunoglobulins, 49, 237–238 Inductive learning, 195 Infrared heating (IH), 164–165 application, 164 bacteria inactivation, 165 pathogenic microorganisms, 165 Insoluble monolayers, 11 Intasept™ aseptic pouches, 195 Intelligent packaging, see Packaging, active and intelligent Interfacial tension, 4, 7 Ion exchange resin, 107 Ionic exchange, 107–108 electric double layer, 110 ion exchange equilibrium, 109 ionic exchange mechanism, 109 ionic exchangers, 107–109 Iota-carrageenan (IC), 22 Isoflux•, 36 Isothermal desorption process (IDP), 93 Isothermal titration microcalorimetry (ITC), 203–212 baseline instability, 210 casein, 211 chitosan, 211 control experiment, 209 data reliability, 207 dilution experiment, 212 experiment, 206–212 fundamentals, 203–204 ITC equipment, 204–206 polyphenols, 212

Index thermodynamic parameters, 206 thermoelectric sensors, 205 Isothermic distillation, 18 ITC, see Isothermal titration microcalorimetry

j Joule heating, 157, see also Ohmic heating technology Jovanovic isotherm, 90

k Kieselguhr treatment, 50 Kluyveromyces lactis, 67 Knowledge-based expert systems, 195

l α-Lactalbumin aqueous two-phase systems, 59 bioseparation, 30 medical field, 230 milk protein crystallization, 132 whey treatment, 48 Lacticin, 189 Lactic starter bacteria, 42 Lactobacillus acidophilus, 59, 65 curvatus, 188 Lactoferrin (LF), 30, 231–236 β-Lactoglobulin aqueous two-phase systems, 59 bioseparation, 30 chromatographic techniques, 106 medical field, 229 milk protein crystallization, 132 whey treatment, 48 Lactoperoxidase, 30, 236–237 Lactose catalytic hydrogenation of lactose, 139 crystal cell parameters, 139 difficulties in crystallizing, 147 formation, habits, 139 forms of, 143 glass, 145 hydrolysis, 139 metastable zone widths for, 135 molecular configurations, 142 mutarotation, 140 pharmaceutical-grade, 138 solubility, 143, 144 tomahawk form, 139, 140 Lactose and whey protein, crystallization of, 121–153 agglomeration kernel, 129 batch crystallization, 128

259

Index chromatographic separation, 139 cocrystallization, 137 crystal agglomeration, 129 crystal cell parameters of lactose, 139 crystal growth rate, 123 crystallization of lactose, 138–148 crystalline state and polymorphs, 139–140 growth rate, 145–146 industrial aspects, 148 kinetics of crystallization, 146–148 metastable zone width, 144–145 nucleation, 145 solubility, 140–144 crystallization of sugars, 132–138 crystal product characteristics, 136–138 growth of crystals, 136 metastable zone width, 135 solubility data, 133 supersaturation generation, 133–135 crystal size distribution, 123 crystals obtained from different solvents, 141 difficulties in crystallizing lactose, 147 Dirac delta function, 123 DLVO model, 129 drowning out crystallization, 144 energy balance of crystallizer, 124 engineering aspects, 122–129 agglomeration and its implications, 128–129 crystal growth, 125–126 mass and energy balances, 123–125 method of moments, 127–128 nucleation, 126–127 population balance, 122–123 solubility, supersaturation, and heat of crystallization, 127 thermodynamic aspects, 127 equilibrium thermodynamics, 127 glucose–water system, phase diagram of, 134 granulation, 129 growth kinetics, 133 growth-unit, 125 Industrial Crystallization approach, 148 lactose formation, habits, 139 lactose forms, 143 lactose glass, 145 lactose solubility, 143, 144 liquid inclusion in sucrose crystals, 137 melt crystallization, 138 methods to generate supersaturation in protein crystallization, 131 milk phase diagram, 134 mutarotation, 140 nucleation, 122, 126–127 power law, 126 primary, 126

rate, 123 secondary, 126 particle association, 128 particle movement, 129 population density, calculation of, 122 precipitation processes, 132 production of pharmaceutical-grade lactose, 138 protein crystallization, 129–132 large-scale protein crystallization, 131–132 methods, 131 milk protein crystallization, 132 special characteristics, 130–131 secondary phenomena, 133, 136 sonocrystallization, 145 tomahawk crystal habit of lactose, 140 Langmuir–Blodgett technique, 13 Langmuir isotherm, 89 Langmuir monolayers, 11, 12 Langmuir–Schaeffer technique, 13 Lattice theory, 62 Lb brevis, 41 Lb casei, 41 Lb curvatus, 41 Lb fermentum, 41 Lb paracasei, 41 Lb plantarum, 41 Lb rhamnosus, 41 LCST, see Lower critical solution temperature LDPE, see Low-density polyethylene LF, see Lactoferrin Ligands, 66, 67 Liquid–liquid extraction, 28, 57, 69–72 Liquid milk production, 36–39 decontamination of collected milk, 36 extended shelf-life raw milk, 37 fermented milks, 39 microfiltration, 36 somatic cells, 38 spore-forming bacteria, 36 ultrafiltration, 38 Liquid precheese (LPC), 41, 42 Listeria innocua, 166, 189, 194 monocytogenes, 37, 163, 166, 187, 193 Low-density polyethylene (LDPE), 186, 194 Lower critical solution temperature (LCST), 68, 69 LPC, see Liquid precheese Lyophilic colloid systems, 17 Lyophobic colloid systems, 17

m Machine blank, 210 Maltodextrin (MD), 59

260 Marguerite® milk, 37 MBPs, see Milk basic proteins MD, see Maltodextrin MEC, see Molecular exclusion chromatography Medical field, potential applications of whey proteins in, 221–252 biological functions of whey proteins, 224–225 bovine serum albumin, 231 anticancer activity, 231 nutrition effects and other metabolic features, 231 branched-chain amino acids, 224 colostrums, 237 future trends, 240 glycomacropeptide, 239 immunoglobulins, 237–238 antimicrobial and antiviral activity, 237 immunomodulation, 238 nutrition effects and other metabolic features, 238 α-lactalbumin, 230 anticancer activity, 230 immunomodulation, 230 nutrition effects and other metabolic features, 230 lactoferrin, 231–236 anticancer activity, 234–235 antimicrobial and antiviral activity, 232 immunomodulation, 232–234 nutrition effects and other metabolic features, 235–236 β-lactoglobulin, 229 immunomodulation, 229 nutrition effects and other metabolic features, 229 lactoperoxidase, 236–237 antimicrobial and antiviral activity, 236 biological function, 236 osteopontin, 239–240 passive immunity, 237 phenylketonuria, 239, 240 proteose peptones, 238–239 whey protein concentrates and isolates, 225–229 anticancer activity, 228 antimicrobial and antiviral activity, 226–227 immunomodulation, 227 nutrition effects and other metabolic features, 228–229 MEL, see Molecular exclusion limit Melt crystallization, 138 Membralox GP•, 36 Membrane technologies, applications of, 33–56 Bactocatch•, 36 brine treatment, 50

Index cleaning in place solutions, 50 Kieselguhr treatment, 50 water source for steam production, 50 white waters, 50 cheese production, 40–45 acidification kinetics, 42 adjustment of aqueous phase of cheese milk, 44 appellation d’origine protégée, 40 buffering capacity, 42–43 cheese made by membrane technologies, 44–45 LAB exopolysaccharides, 44 liquid precheese, 42 milk production, 45 MMV process, 41 rennet coagulation, 43–44 rheological changes, 43 ripening enzymes, 42 ripening of UF cheese, 45 unripened cheeses, 42 cleaning of membrane equipment, 35 colostrum treatment, 49–50 bioactive components, 50 colostrinin, 51 milk basic proteins, 50 serocolostrum, 49 ultrafiltration, 49 dairy wastewater treatment, 50 cleaning in place solutions, 50 Kieselguhr treatment, 50 water source for steam production, 50 white waters, 50 definitions, 34 first anisotropic membranes, 33 Isoflux®, 36 liquid milk production, 36–39 decontamination of collected milk, 36 extended shelf-life raw milk, 37 fermented milks, 39 microfiltration, 36 somatic cells, 38 spore-forming bacteria, 36 ultrafiltration, 38 liquid precheese, 41 Membralox GP•, 36 membrane design and configuration, 34–36 milk protein separation, 39–40 cholecystokinin, 40 diafiltration, 39 glycomacropeptide, 40 milk protein concentrates, 39 pure native casein, 40 ultrafiltration, 39 MMV process, 41 perspectives, 51 pervaporation, 34

Index removal of bacteria, 51 somatic cells, 38 Sterilox®, 36 ultra-high-temperature treatment, 36 uniform transmembrane pressure, 36 whey treatment, 46–49 Carbery process, 49 demineralization rate, 49 denatured immunoglobulins, 49 development of off-flavors, 48 ideal whey, 46, 48 membrane technologies, 46 milk microfiltrate, 46 nonprotein nitrogen, 48 processing, energy consumption, 47 purified phospholipids, 49 residual fat, 48 spray-drying of acid whey, 49 MF, see Microfiltration MIC, see Minimum inhibitory concentration Micellization (colloidal systems), 14–17 critical micelle concentration, 14, 16 detergents, 16 Gibbs equation,15 interfacial tension reduction, 14 surfactants, 16 thermodynamics, 15 Microcalorimetry, 201–220 basis of, 202 differential scanning microcalorimetry, 212–218 equipment, 213–215 experiment, 215–217 fundamentals, 212–213 instrument, 214 practical applications, 217–218 protein unfolding, 217 heat of dilution, 206 isothermal titration microcalorimetry, 203–212 baseline instability, 210 casein, 211 chitosan, 211 control experiment, 209 data reliability, 207 dilution experiment, 212 experiment, 206–212 fundamentals, 203–204 ITC equipment, 204–206 polyphenols, 212 thermodynamic parameters, 206 thermoelectric sensors, 205 machine blank, 210 microcalorimeters, 202 motriz power, 202 reference cell, 204, 216 Micrococcus lysodeikticus, 187

261 Microfiltration (MF), 34 Bactocatch•, 36 definition of, 34 extended shelf-life raw milk, 37 Marguerite• milk, 37 membrane configurations, 34 skim milk, 37 Microwave (MW) heating, 162–164 microorganisms, 163–164 potential effects, 163 Mid-infrared (MIR), 164 Milk(s), see also Liquid milk production acid coagulation of, 19 basic proteins (MBPs), 50 fermented, 39 HTST pasteurization of, 157 Marguerite• milk, 37 microfiltrate (MMF), 46 Newtonian rheological behavior, 43 pasteurization, 27 preconcentration step, 27 processors, prime goal of, 155 protein concentrates (MPCs), 39 protein separation, 39–40 cholecystokinin, 40 diafiltration, 39 glycomacropeptide, 40 milk protein concentrates, 39 pure native casein, 40 ultrafiltration, 39 shelf life, 155 skim milk, 36, 37 stability, casein and, 19 Ultima® milk, 38 Minimum inhibitory concentration (MIC), 187 MIR, see Mid-infrared ML-stabilized monodisperse emulsion, see Modified-lecithin-stabilized monodisperse emulsion MM, see Molar mass MMF, see Milk microfiltrate MMT, see Montmorillonite MMV process, 41 Model(s) Bi-Langmuir isotherm, 89 DLVO, 129 Langmuir, 89 mayonnaise system, 22 multiple independent binding sites model, 210 open system dispersion model, 73 phase equilibrium, 62 population balance, 129 thermodynamic models, aqueous two-phase systems, 66 Modified-lecithin (ML)-stabilized monodisperse emulsion, 21

262 Molar mass (MM), 64 Molecular exclusion chromatography (MEC), 111–115 basic principles, 113–115 general aspects, 112–113 most well-known application, 112 Molecular exclusion limit (MEL), 114 Molecular weight cut-off (MWCO), 35 Montmorillonite (MMT), 191 MPCs, see Milk protein concentrates Mutarotation (lactose), 140 MWCO, see Molecular weight cut-off MW heating, see Microwave heating Mycobacterium tuberculosis, 37

n Nanofiltration (NF), 34 definition of, 34 membrane configurations, 34 whey treatment, 47, 49 Nanotechnology (food packaging), 190–192 antimicrobials, 192 cassava starch films, 192 exfoliated structure, 191 inserted structure, 191 materials, 191 polymeric matrices, 191 purpose of, 190 separate phase structure, 191 structures, 191 Nariniginase immobilization, 189 Natamicine, 193 Neural networks, 195 NF, see Nanofiltration Nisin, 189, 193 Nonprotein nitrogen (NPN), 48 Nonstarter lactic acid bacteria (NSLAB), 41 Novel technologies (milk processing), 155–174 Electropure Process, 158 heat absorption factor, 164 hurdle concept, 169 nonthermal processing technologies, 165–169 high-pressure processing, 168–169 pulsed electric field, 165–167 pasteurization, 156 plate heat exchangers, 157 relationship between electrical conductivity and temperature in milk, 159 thermal processing technologies, 157–165 infrared heating, 164–165 microwave heating, 162–164 ohmic heating technology, 157–162 TST ohmic pasteurization, 161 value of electroheating, 162 NPN, see Nonprotein nitrogen

Index NSLAB, see Nonstarter lactic acid bacteria Nucleation, 122, 126–127 power law, 126 primary, 126 rate, 123 secondary, 126

o Ocimum basilicum L., 194 Ohmic heating (OH) technology, 157–162 disadvantages, 159 effects on physical-chemical properties, 161–162 enzyme inactivation, 161 microbial inactivation, 160–161 OH technology, see Ohmic heating technology Open system dispersion model, 73 Osteopontin, 239–240 Oxygen absorber, 182–183 Oxygen scavenger, 182, 195

p Packaging, active and intelligent, 175–199 Actipak study, 186 active packaging, 179–184 antimicrobial packaging, 179–180 definition of, 178 desired role, 178 edible packages, 180–182 ethylene absorber, 183–184 goal of, 178 humidity absorber, 184 oxygen absorber, 182–183 antimicrobial packaging, development of, 185–190 antimicrobial immobilization in polymers, 188–189 antimicrobial incorporation into plastic polymers, 187–188 commonly used antimicrobial substances, 186–187 production of antimicrobial films, 190 surface modification, 189–190 bacteriocins, 186, 189 consumption patterns, 177 demand for fresh foods, 181 functions of packages, 176 importance and definition, 176–185 active packaging, 179–184 antimicrobial packaging, 179–180 consumer demand, 177–179 edible packages, 180–182 ethylene absorber, 183–184 history, 176–177 humidity absorber, 184

263

Index intelligent packaging, 184–185 oxygen absorber, 182–183 industrial revolution, 176 intelligent packaging, 184–185 definition of, 178 main applications, 184 product tracking, 178–179 uniqueness of, 179 nanotechnology, 190–192 antimicrobials, 192 cassava starch films, 192 exfoliated structure, 191 inserted structure, 191 materials, 191 polymeric matrices, 191 purpose of, 190 separate phase structure, 191 structures, 191 nariniginase immobilization, 189 natural antimicrobials, 186 packaging as art, 177 packaging as science and technology, 177 potential use, 192–195 antimicrobial migration, 193 aromatized active film, 194 artificial intelligence tools, 195 cellulosic films, 193 cheese compounds, 192 edible coatings, 192 electrostatic power coating, 195 Intasept™ aseptic pouches, 195 laminated active film, 193 multilayer films, 194 shelf-life relevant factor, 195 Pasteurization, 27, 156, 161 PEF, see Pulsed electric field PEG, see Polyethylene glycol Penicillium roqueforti, 188, 194 Penicilium sp., 193 Percolation, 100 Pervaporation, 34 PET, see Polyethylene terephthalate Phenylketonuria (PKU), 239, 240 PHEs, see Plate heat exchangers Physical adsorption, 85 Physicomechanical processes, bioseparation, 28 PKU, see Phenylketonuria Plate heat exchangers (PHEs), 157 PO, see Propylene oxide Polyethylene glycol (PEG), 60, 66, 132 Polyethylene terephthalate (PET), 183, 195 Polymers, antimicrobial immobilization in, 188–189 Polypropylene glycol (PPG), 59 Potassium phosphate (PPP), 58 PPCN, see Pure native casein PPG, see Polypropylene glycol

PPP, see Potassium phosphate Primary nucleation, 126 Programmed temperature desorption (PTD), 93 Propylene oxide (PO), 59 Protein, see also Milk protein separation affinity partitioning, 66, 67 complexation between carbohydrates and, 21 crystallization, 129–132 large-scale protein crystallization, 131–132 methods to generate supersaturation in, 131 milk protein crystallization, 132 special characteristics, 130–131 distribution, aqueous two-phase systems, 64 enzymatic reaction velocity, 43 milk basic proteins, 50 partitioning, ATPS, 73 partitions using Graesser extractor, 73–74 unfolding, 217 whey, see also Lactose and whey protein, crystallization of; Medical field, potential applications of whey proteins in denaturation of, 168 purification of, 30 Pseudomonas fluorescens, 166 Pseudosolutions, 1 PTD, see Programmed temperature desorption Pulsed electric field (PEF), 165–167 current limitations, 167 effects on milk quality, 167 electrical pulsation parameters, 167 inactivation studies, 166–167 Pure native casein (PPCN), 40

q Quarg, 42, 43

r Radio-frequency identification (RFID) tags, 184 Reference cell, 204, 216 Rennet coagulation, 43 Rennin enzymes, 19 Retention time distribution (RTD), 72 Reverse osmosis (RO), 34 definition of, 34 membrane configurations, 34 whey treatment, 47 RFID tags, see Radio-frequency identification tags RO, see Reverse osmosis RTD, see Retention time distribution

264

Index

s

t

Sagarav®, 114 Salmonella dublin, 166 enteriditis, 237 typhimurium, 37, 237 Salt–polymer system, phase diagram for, 61 Salts, presence of in ATPS, 65 SCs, see Somatic cells SDS, see Sodium dodecyl sulfate Secondary nucleation, 126 Selectivity factor, 113 Serocolostrum, 49 Shepacril•, 114 Shepadex•, 114 Sheparoce•, 114 Sheparose•, 114 Shigella flexneri, 237 Shodex•, 114 Skim milk, 36, 37 Slope of the tie-line (STL), 61 Sodium dodecyl sulfate (SDS), 211 Somatic cells (SCs), 38, 51 Sonocrystallization, 145 Sorption equilibrium, 87–94 batch or stirred tank method, 91–92 Bi-Langmuir isotherm, 89–90 determination of adsorption isotherms, 91–93 exponentially modified Langmuir isotherm, 90–91 Freundlich isotherm, 88–89 frontal analysis method, 92–93 Jovanovic isotherm, 90 Langmuir isotherm, 89 linear isotherm, 88 solute adsorption in dilute solutions, 87–91 solute desorption, 93 sorption hysteresis, 93–94 Toth isotherm, 90 Spore-forming bacteria, 36 Staphylococcus aureus, 166, 189, 193 Sterilox®, 36 Stern–Helmholtz layer, 20 STL, see Slope of the tie-line Streptococcus thermophilus, 160 Sucrose crystallization kinetics, 136 Sugars, crystallization of, 132–138 crystal product characteristics, 136–138 growth of crystals, 136 metastable zone width, 135 solubility data, 133 supersaturation generation, 133–135 Superficial pressure, 12 Supersaturation, 127, 131 Surface diffusion, 98

Thermodynamic models, aqueous two-phase systems, 66 Tie-line length (TLL), 61 Time–temperature indicator (TTI), 184 TLL, see Tie-line length α-Tocopherol, 194 Toth isotherm, 90 TTI, see Time–temperature indicator

u UF, see Ultrafiltration UHT treatment, see Ultra-high-temperature treatment Ultima• milk, 38 Ultrafiltration (UF), 34 cheese, ripening of, 45 colostrum treatment, 49 definition of, 34 liquid precheese, 42 membrane configurations, 34 milk protein separation, 39 whey treatment, 47 whole protein enrichment of cheese milk by, 41 Ultra-high-temperature (UHT) treatment, 36, 156 Uniform transmembrane pressure (UTP), 36 UNIQUAC model, 62 United States Food and Drug Administration (FDA), 186 Unit operations bioseparation, 27 unit processes vs., 28 Unripened cheese, 42 UTP, see Uniform transmembrane pressure

v van der Waals interactions, 84 VCF, see Volumetric concentration factor Volumetric concentration factor (VCF), 36, 47

w Whey drying of, 139 protein (WP), 22, see also Lactose and whey protein, crystallization of; Medical field, potential applications of whey proteins in concentrate (WPC), 29, 47, 222, 225

265

Index denaturation of, 168 isolated (WPI), 222, 226 separation, see Aqueous two-phase systems treatment, 46–49 Carbery process, 49 demineralization rate, 49 denatured immunoglobulins, 49 development of off-flavors, 48 ideal whey, 46, 48 membrane technologies, 46 nonprotein nitrogen, 48 processing, energy consumption, 47 purified phospholipids, 49 residual fat, 48 spray-drying of acid whey, 49

Whole pasteurized milk, unit operations, 27 WP, see Wheat protein WPC, see Whey protein concentrate WPI, see Whey protein isolated

x Xanthan gum, 22

y Yogurts, 39

z Zeolites, 87