Characterization of Food

CHARACTERIZATION OF FOOD Emerging Methods

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CHARACTERIZATION OF FOOD Emerging Methods

edited by ANILKUMAR

G. GAONKAR

Technology Center Kraft Foods, Inc. Glenview, IL 60025 USA

1995 ELSEVIER Amsterdam - Lausanne - New York-

Oxford - Shannon - Tokyo

ELSEVIER SCIENCE B.V. Sara Burgerhartstraat 25 EO. Box 211, 1000AEAmsterdam The Netherlands

Library

oF Congress C a t a l o g i n g - I n - P u b l i c a t i o n

Data

Characterization oF Food : e m e r g i n g methods / e d i t e d by A n i l k u m a r O. Gaonkar. p. cm. Includes bibliographical references and i n d e x . ISBN 0 - 4 4 4 - 8 1 4 9 9 - X 1. F o o d - - A n a l y s i s . I . G a o n k a r , A n l l k u m a r G . , 1954. TX541.C42 1995 95-35144 664'.07--dc20 CIP

ISBN 0 444 81499 X 9 1995 Elsevier Science B.V. All rights reserved. No part of this publication may be reproduced, stored in a retrieval system or transmitted in any form or by any means, electronic, mechanical, photocopying, recording or otherwise, without the prior written permission of the publisher, Elsevier Science B.V., Copyright & Permissions Department, P.O. Box 521, 1000 AM Amsterdam, The Netherlands. Special regulations for readers in the U.S.A. - This publication has been registered with the Copyright Clearance Center Inc. (CCC), 222 Rosewood Drive, Danvers, MA 01923. Information can be obtained from the CCC about conditions under which photocopies of parts of this publication may be made in the U.S.A. All other copyright questions, including photocopying outside of the U.S.A,. should be referred to the copyright owner, Elsevier Science B.V., unless otherwise specified. No responsibility is assumed by the publisher for any injury and/or damage to persons or property as a matter of products liability, negligence or otherwise, or from any use or operation of any methods, products, instructions or ideas contained in the material herein. This book is printed on acid-free paper. Printed in The Netherlands

To my Family, Relatives, Teachers, Friends and Colleagues,

and to all Children and Senior Citizens of the World

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vii

Preface

Rapid and continued developments in electronics, optics, computing, instrumentation, spectroscopy, and other branches of science and technology led to considerable improvements in various methodologies. Due to this revolution in methodology, we are now able to solve problems which were thought to be extremely difficult to solve a few years ago. The new methods enabled us to better characterize foods and enriched our understanding of foods. The aim of this book is to assemble, for a handy reference, various emerging, state-of-the-art methodologies used for characterizing foods. Although the emphasis is placed on real foods, model food systems are also considered. The book contains invited chapters contributed by scientists actively involved in research, most of whom have made notable contributions to the advancement of knowledge in their field of expertise. It is not possible to discuss all the methods available for characterizing foods critically and systematically in a single volume. Methods pertaining to interfaces (food emulsions, foams, and dispersions), fluorescence, ultrasonics, nuclear magnetic resonance, electron spin resonance, Fourier-transform infrared and near infrared spectroscopy, small-angle neutron scattering, dielectrics, microscopy, rheology, sensors, antibodies, flavor and aroma analysis are included. This book is an indispensable reference source for scientists/engineers/technologists in industries, universities, and government laboratories who are involved in food research and/or development, and also for faculty, advanced undergraduate, graduate and postgraduate students from Food Science, Food Engineering, and Biochemistry departments. In addition, it will serve as a valuable reference to analytical chemists, and surface and colloid scientists. I wish to thank all the contributing authors for their dedication, hard work and cooperation and the reviewers for valuable suggestions. Last, but not least, I would like to thank my family, friends, relatives, colleagues, and the management of Kraft Foods Research for their encouragement.

April 1995

Anilkumar G. Gaonkar Kraft Foods, Inc. Glenview, IL 60025

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ix

CONTRIBUTORS

Niels M. Barfod Grindsted Products A/S, Edwin Rahrs Vej 38, DK-8220 Brabrand, Denmark.

Wendy E. Brown BBSRC Institute of Food Research, Whiteknights Road, Reading RG6 2EF, United Kingdom.

Charles R. Buffler Microwave Research Center, 126 Water Street, Marlborough, NH 03455, USA.

S. Chakrabarti Kraft Foods, Inc., 801 Waukegan Road, Glenview, IL 60025, USA.

S.G. Greg Cheng Kraft Foods, Inc., 801 Waukegan Road, Glenview, IL 60025, USA.

D.C. Clark Institute of Food Research, Norwich Laboratory, Norwich Research Park, Colney, Norwich NR4 7UA, United Kingdom.

Mika Fukuoka Food Science and Technology Department, Tokyo University of Fisheries, Konan 4, Minato, Tokyo 108, Japan.

R.G. Fulcher Department of Food Science and Nutrition, University of Minnesota, St. Paul MN 55108, USA.

R. Gray Food Science Division, Department of Agriculture for Northern Ireland, Newforge Lane, Belfast BT9 5PX, Northern Ireland.

M.C.M. Gribnau Unilever Research Laboratorium, Olivier van Noortlaan 120, 3133 AT Vlaardingen, The Netherlands.

Sumio Kawano National Food Research Institute, 2-1-2 Kannondai, Tsukuba 305, Japan.

K. Koczo Department of Chemical Engineering, Illinois Institute of Technology, 10 West 33rd Street, Chicago, IL 60616-3793, USA.

D.J. McClements Department of Food Science, University of Massachusetts, Amherst, MA 01003, USA.

Zohar M. Merchant Kraft Foods, Inc., 801 Waukegan Road, Glenview, IL 60025, USA.

M.M.W. Mooren Unilever Research Laboratorium, Olivier van Noortlaan 120, 3133 AT Vlaardingen, The Netherlands.

A.D. Nikolov Department of Chemical Engineering, Illinois Institute of Technology, 10 West 33rd Street, Chicago, IL 60616-3793, USA. D.G. Pechak Kraft Foods, Inc., 801 Waukegan Road, Glenview, IL 60025, USA. Peter Schieberle Bergische Universitat/GH, Food Chemistry/FB 9, Gauf~straB e 20, D-42097 Wuppertal, Germany.

M.G. Smart Kraft Foods, Inc., 801 Waukegan Road, Glenview, IL 60025, USA.

Philip H. Stothart 33, Betchworth Avenue, Earley, Reading, Berkshire RG6 2RH, United Kingdom.

K. Toko Department of Electronics, Faculty of Engineering, Kyushu University 36, 6-10-1 Hakozaki, Higashi-Ku, Fukuoka 812, Japan.

xi M.A. Voorbach Unilever Research Laboratorium, Olivier van Noortlaan 120, 3133 AT Vlaardin gen, The Netherlands.

D.T. Wasan Department of Chemical Engineering, Illinois Institute of Technology, 10 West 33rd Street, Chicago, IL 60616-3793, USA. Hisahiko Watanabe Food Science and Technology Department, Tokyo University of Fisheries, Konan 4, Minato, Tokyo 108, Japan. Tokuko Watanabe Food Science and Technology Department, Tokyo University of Fisheries, Konan 4, Minato, Tokyo 108, Japan.

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xiii

Contents

Page No. Preface

Vll

Contributors

ix

o ~

Interfacial Characterization of Food Systems D. T. Wasan, K. Koczo and A. D. Nikolov .

Application of State-of-the-Art Fluorescence and Interferometric Techniques to Study Coalescence in Food Dispersions

23

D. C. Clark

3. Methods for Characterization of Structure in Whippable Dairy-based Emulsions

59

Niels M. Barfod

4. Ultrasonic Characterization of Foods

93

D.J. McClements

5. Recent Advances in Characterization of foods using Nuclear Magnetic Resonance (NMR)

117

Hisahiko Watanabe, Mika Fukuoka and Tokuko Watanabe

6. Determination of Droplet Size Distributions in Emulsions by Pulsed Field Gradient NMR

151

M.M. W. Mooren, M. C.M. Gribnau and M.A. Voorbach ,

The Application of EPR Spectroscopy to the Detection of Irradiated Food

163

R. Gray

Progress in Application of NIR and FT-IR in Food Characterization Sumio Kawano

185

xiv Developments in the Application of Small-Angle Neutron Scattering to Food Systems

201

Philip H. Stothart

10. Advances in Dielectric Measurement of Foods

213

Charles R. Buffier

11. Recent Developments in the Microstructural Characterization of Foods

233

M.G. Smart, R.G. Fulcher and D.G. Pechak

12. Some Recent Advances in Food Rheology

277

S. Chakrabarti

13. The Use of Mastication Analysis to Examine the Dynamics of Oral Breakdown of Food Contributing to Perceived Texture

309

Wendy E. Brown

14. Biosensors in Food Analysis

329

S.G. Greg Cheng and Zohar M. Merchant

15. Developments in Characterization of Foods Using Antibodies

347

Zohar M. Merchant and S.G. Greg Cheng

16. Taste Sensor

377

K. Toko

17. New Developments in Methods for Analysis of Volatile Flavor Compounds and their Precursors

403

Peter Schieberle

Index

433

Characterization of Food: Emerging Methods A.G. Gaonkar (Editor) 9 1995 Elsevier Science B.V. All rights reserved.

Chapter 1 Interfacial characterization of food systems D. T. W a s a n , K. K o c z o and A. D. Nikolov D e p a r t m e n t of C h e m i c a l Engineering, Illinois Institute of T e c h n o l o g y , Chicago, I L 60616, U S A

1. INTRODUCTION Many food products (salad dressings, whipped toppings, ice cream etc.) are dispersed colloid systems, such as emulsions, suspensions or foams. Texture, structure and stability of these dispersions have fundamental importance for the food manufacturer. Our chapter presents new methods, most of them developed in our laboratory, and mechanisms which can be very helpful for the food researcher or developer. The interfacial area of fine dispersions is very high and thus these interfaces strongly influence the behavior of the dispersions. The rheological characteristics of interfaces can be investigated by a new, versatile, but very simple technique, the controlled drop tensiometer, as will be described. The stability of foams and emulsions strongly depends on the structure and stability of the liquid films which form between approaching bubbles, emulsion drops or a bubble and an oil drop, respectively. Another application of the controlled drop tensiometer as well as optical interferometric techniques, will be discussed which allow the study of liquid films. A new mechanism of film stability involving thin film microlayering by small particles (sub-micron particles, surfactant micelles, macromolecules or protein aggregates) is also presented. The texture and s~cture of foods is very delicate, therefore experimental methods which cause no or very little structural damage has to be applied in their investigations. Such techniques, the surface force balance and back-light scattering methods and dielectrometry will be also discussed in the chapter. 2. INTERFACIAL RHEOLOGY Interfacial rheology deals with the flow behavior in the interfacial region between two immiscible fluid phases (gas-liquid as in foams, and liquid-liquid as in emulsions). The flow is considerably modified by surface active agents present in the system. Surface active agents (surfactants) are molecules with an affinity for the interface and accumulate there forming a packed structure. This results in a variation in physical and chemical properties in a thin interfacial region with a thickness of the order of a few molecular diameters. These

surfactants alter the hydrodynamic resistance to interfacial flow. Therefore, study of this variation, especially of the rheological properties, is important since many properties of dispersions, such as foam and emulsion stability, emulsification (making emulsions) and demulsification (breaking up emulsions) processes, are controlled by the interfacial flow behavior. To study the flow behavior in the interfacial region we use interfacial rheometers. In these instruments, a stress is imposed on an interface containing surfactants and the response is studied by measuring the velocity profile or the capillary pressure change. We can distinguish between two types of stresses on an interface: a shear stress and a dilatational stress. In a shear stress experiment, the interfacial area is kept constant and a shear is imposed on the interface. The resistance is characterized by a shear viscosity, similar to the Newtonian viscosity of fluids. In a dilatational stress experiment, an interface is expanded (dilated) without shear. This resistance is characterized by a dilatational viscosity. In an actual dynamic situation, the total stress is a sum of these stresses, and both these viscosities represent the total flow resistance afforded by the interface to an applied stress. There are a number of instruments to study interfacial rheology and most of them are described in Ref. [1]. The most recent instrumentation is the controlled drop tensiometer. The controlled drop tensiometer is a simple and very flexible method for measuring interfacial tension (IFT) in equilibrium as well as in various dynamic conditions. In this technique (Fig. 1), the capillary pressure, Pc, of a drop, which is formed at the tip of a capillary and immersed into another immiscible phase (liquid or gas), is measured by a sensitive pressure transducer. The capillary pressure is related to the IFT and drop radius, R, through the Young-Laplace equation [2,3]: 2o Pc = Pa-Pb = R

(1)

where o is the instantaneous IFT, and Pa and Pb are the pressures inside and outside the drop, respectively. Deformation of the drop by gravity can be avoided by using capillaries with sufficiently small radius (Re). The size of the drop is varied by using a computer controlled microsyringe attached to the capillary and the output of the transducer is also fed into a computer. The volume and radius of the drop at any instant are determined by the position and speed of the microsyringe plunger. For the measurement of equilibrium IFT, a drop is formed at the capillary tip and maintained at that size. After sufficient time, chemical equilibrium is achieved and the equilibrium thermodynamic IFT can be calculated from the measured, steady state capillary pressure and drop radius by Eq. 1. The adsorption and desorption kinetics of surfactants, such as food emulsifiers, can be measured by the stress relaxation method [4]. In this, a "clean" interface, devoid of surfactants, is first formed by rapidly expanding a new drop to the desired size and, then, this size is maintained and the capillary pressure is monitored. Figure 2 shows experimental relaxation data for a dodecane/aq. Brij 58 surfactant solution interface, at a concentration below the CMC. An initial rapid relaxation process is followed by a slower relaxation prior to achieving the equilibrium IFT. Initially, the IFT is h i g h , - close to the IFT between the pure solvents. Then, the tension decreases because surfactants diffuse to the interface and adsorb, eventually reaching the equilibrium value. The data provide key information about the diffusion and adsorption kinetics of the surfactants, such as emulsifiers or proteins.

Figure 1. Schematics of controlled drop tensiometer Desorption kinetics can also be studied by contracting a drop from a known state to a new state. The sudden reduction in interfacial area causes desorption of surfactants, which is deduced from the IFT change over time. Dynamic interfacial tension of dilating or contracting liquid-liquid interfaces can be measured by monitoring the capillary pressure for an expanding (or contracting) liquid drop. The IFT, as a function of time, is computed from the capillary pressure change and radius change with time. To study the effect of dilation of the interface under controlled conditions, first, a drop is formed and an initial equilibrium state is established by maintaining this drop size for sufficient time. Then, in expansion experiments, the drop volume is increased with constant flow rate and the capillary pressure is monitored over time. In pure systems, or in systems where surfactant adsorption is fast (high surfactant concentration) the IFF does not change during drop expansion and the capillary pressure decreases, as shown by Eq. 1. In surfactant systems, if the surfactant adsorption is not fast, the dynamic IFT can be significantly higher than the equilibrium values and the capillary pressure can increase during interface expansion. Figure 3 shows the dynamic IFT of soybean oil/water interfaces under expansion with constant flow rate as a function of the relative change of the interfacial area, with various surfactants in the oil and aqueous phases, respectively. The IFT is lowest if both phases contain surface active additives, and it hardly changes due to the presence of the fast adsorbing, low molecular emulsifier SPAN 80 in the oil phase. The increase of the dynamic IFT with the interface expansion is most pronounced with 0.01% BSA in the aqueous phase due to the slow adsorption of the protein.

Figure 2. Stress relaxation of the dodecane/aqueous Brij 58 interface at c=10 -6 mol/dm 3, Rc---O.141 mm, 25 ~ A similar technique can be used to study the rheological properties of liquid films. Figure 4 shows the formation of a W/O/W emulsion film with two, identical aqueous phases (such as in water-in-oil emulsions) at the tip of the capillary. A pre-requisite of the experiment is that the surface of the capillary must be well wetted by the film phase, i.e., it should be hydrophobic in this case. First, an aqueous drop is formed inside the oil (film liquid) and the aqueous phase is in the bottom of the cuvette. Then, the level of the aqueous phase is slowly increased. As the oil/water interface passes the drop, a cap shaped oil film, bordered by a circular meniscus, covers the drop. This film can be studied in equilibrium and in dynamic conditions, similar to the single interfaces (See above). The technique can be used to study films from oil or aqueous phase which can be sandwiched between identical or different liquid or gas phases. For relatively thick films (higher than about 30 nm), the pressure drop at the film is the sum of the capillary pressures at the two film interfaces. In this case, the Young-Laplace equation for the film can be written as

_2f I where the film tension (f) is given by:

t2)

Figure 3. Dynamic interfacial tension of soybean oil/water systems. expanding in water, pH=7, flow rate: 3.10 .4 mm3/s, Rc=0.141 mm.

Soybean oil drop

Figure 4. Method to form and study an oil film between aqueous phases at the tip of capillary

f-

4 0 to o Ol+O o

(3)

where Rf is the film radius, t~i and % are the interfacial tensions at inner and outer film interfaces, respectively. For emulsion (or foam) films: ~i=% and f=2t~. Figure 5 shows dynamic film tension of soybean oil films stabilized by 0.5 wt % SPAN 80 emulsifier between aqueous phases under expansion by various flow rates. The increase in film tension from equilibrium is higher at higher rates of interface expansion because the flux of surfactant that can adsorb during expansion is lower at higher rates.

Figure 5. Dynamic film tension of soybean oil film containing 0.5 wt% SPAN 80 between water phases, expanding with various flow rates as a function of the relative film area. Rc=0.32 mm, h=0.03 mm, pH=7, at 25 ~

3. INTERFEROMETRY 3.1. Common interferometry - mechanisms of liquid film stability

Liquid films which form between approaching drops or bubbles are important structural elements of dispersed systems. The stability of these films controls the dispersion stability because the drops or bubbles cannot coalesce until the intervening film ruptures. The drainage and stability of thin liquid films attracted the attention of scientists already centuries ago [5,6]. The thinning process of plan-parallel liquid films have been generally observed using reflected light interferometry [7-9]. The experimental setup to form and study such film contains a small, vertically oriented cylindrical tube of hydrophilic inner walls with a horizontal capillary side arm, as shown in Figure 9 of Chapter 2. The film liquid is filled into the vertical tube and, then, a horizontal liquid film encircled by a biconcave meniscus is formed by slowly sucking out the liquid through the side arm. The driving force for film thinning is the capillary pressure which, for small film contact angles and complete wetting of the capillary wall, is given for this film configuration by:

PC

(4)

Rc2

2

where R c is the inner radius of the capillary, Rf is the film radius and c is the interfacial tension. Thus, an increase in Pc leads to an increase in Rf and the film area. The film is observed by a microscope using reflected light. The film holder and the objective are immersed in air in the case of foam (i.e., air/liquid/air) film and in the oil phase, in the case of an O/W/O emulsion film, respectively. The film thickness can be determined by measuring the intensity of the light reflected from the film surfaces [9]. Further details of the technique will be discussed in Chapter 2. We have used film interferometry to reveal a new mechanism for the stabilization of foams and emulsions due to layering inside the thinning films, as will be discussed below. When two emulsion drops or foam bubbles approach each other, they hydrodynamically interact which generally results in the formation of a dimple [10,11]. After the dimple moves out, a thick lamella with parallel interfaces forms. If the continuous phase (i.e., the film phase) contains only surface active components at relatively low concentrations (not more than a few times their critical micellar concentration), the thick lamella thins on continually (see Fig. 6, left side). During continuous thinning, the film generally reaches a critical thickness where it either ruptures or black spots appear in it and then, by the expansion of these black spots, it transforms into a very thin film, which is either a common black (10-30 nm) or a Newton black film (5-10 nm). The thickness of the common black film depends on the capillary pressure and salt concentration [8]. This film drainage mechanism has been studied by several researchers [8,10-12] and it has been found that the classical DLVO theory of dispersion stability [13,14] can be qualitatively applied to it by taking into account the electrostatic, van der Waals and steric interactions between the film interfaces [8]. The hydrodynamic stability of such films is controlled by the capillary pressure, film

Figure 6. Mechanisms of liquid film stability

area, the interfacial rheological properties (such as surface shear viscosity etc.) and the surface tension gradients (Gibbs-Marangoni effect) of the surfactant adsorption layer at the film interfaces [8,15-21]. The properties of these films, in relation to food systems, are discussed in Chapter 2. Film studies in the past decade have revealed the existence of another film stability mechanism: If the continuous phase contains not only a small amount of surface active substances but also a "sufficient amount" of "small particles", these particles can form layers inside the draining film (see Fig. 6, right)[9,22-32]. As a result, such films thin step-wise, by several step-transitions (also called stratification) when at a step transition a layer of small particles leaves the film. Sodium caseinate is commonly used in foods as emulsion and foam stabilizer. The photomicrographs of Figure 7 show the phenomenon of film step-transitions for a foam film which was formed from 2 wt% sodium caseinate solution at 40 ~ Shortly after lamella formation, a dimple forms (See Fig. 7, picture a). After the dimple leaves the lamella the film drains continually. After about 100 nm film thickness, however, the film thinning becomes step-wise. First, most of the film turns uniformly bright. Then, uniform, light grey (i.e., thinner) areas with sharp borders appear and start to cover the bright areas (See Fig. 7, pict. b), i.e., the first step-transition occurs. Shortly later dark grey spots form near the border of the film, inside the light grey region (See Fig. 7, pict. c, upper left section). The dark grey spots expand, unify and occupy the film area (second transition). During this process, a black, thinner spot forms inside the dark grey film (See Fig. 7, pict. d), followed by the formation and expansion of several other black spots (See Fig. 7, pict. e). Finally, the black spots unify (See Fig. 7, pict. 3') and the film turns uniformly black (third steptransition). No more step-transitions could be observed and the color, i.e., the thickness, of the foam film did not change any more. These observations show that microlayering takes place in the foam film containing 2 wt% caseinate. During a step-transition a layer leaves the film, until no layer is left (black film). Thus, the bright film contained three layers, the light grey two, the dark grey one and the black film contained zero layers. The average thickness differences between films containing zero, one, or two layers, respectively (i.e., the heights of the step-transitions), were measured by interferometry and it was found that they are approximately equal and about 20 nm. It was found by several researchers [33-36] that casein molecules form aggregates in aqueous solutions the so-called casein sub-micelles with approximately the same size as these step-transitions. (The casein miceUes are much larger particles and they form from the sub-micelles by calcium [33,34]. In the sodium caseinate there is practically no calcium and thus, the caseinate solution contains a significant amount of sub-micelles.) It can be concluded that the foam film containing caseinate solution thins by step-transitions because the caseinate sub-micelles form layers in the film. The step-transition phenomenon resembles the common black film/Newton black film transition (Fig. 6, left), however, there are basic differences between the two processes. The step transitions, due to microlayering, can occur at very high thicknesses (depending on the size and concentration of the small particles, as high as several hundred nanometers [27]) and the number of the step-transitions can be much higher than one [27]. The investigations in our laboratory showed that the film microlayering mechanism is a universal phenomenon which fundamentally differs from the classical film thinning mechanism by common black film/Newton black film transition as summarized in Fig. 6. It has been found that the "small particles" can be virtually any kind of isotropic structures with about 10-100 nm size,

10

Figure 7. Photomicrographs on the various drainage stages of a foam film containing 2 wt% sodium caseinate, at 40 ~ Film diameter: 0.35 mm.

11 including micelles of ionic or non-ionic surfactants [9,22-24] - fine solid particles, such as silica or latex particles [9,27] - macromolecules, such as globular protein molecules or random coil shaped polysaccharide molecules protein aggregates, such as caseinate sub-micelles, as was shown above [29] for the occurrence of film microlayering. Note that all of these substances are commonly used in foods. The reason for film microlayering is that the restrictive geometrical conditions, i.e., the presence of the "walls", the film surfaces, force the (sub)-micelles or Brownian particles inside the film to be layered and organized [30]. A pre-requisite of the film microlayering phenomenon is that the effective volume fraction of the small particles should be sufficiently high, at least about 5-1.0 vol% [27]. It is important to emphasize that the effective volume fraction of such sub-micron sized particles, i.e., the volume that the particles really occupy in the solution, is much higher than their geometrical volume fraction. Thus, the above volume fraction range can be reached with about 0.1-1 wt% of small molecule surfactants or with less than 0.1 wt% macromolecules [29]. The number of layers increases with the effective volume fraction of the small particles [127]. It is of great importance to the film microlayering phenomenon that these concentration ranges are typical in practical applications such as in food emulsions and foams. (It can be mentioned that at very high concentrations, from about 10 wt%, ordering of the small particles, such as surfactant micelles, takes place not only inside the films but also in the bulk phase [37]. These concentrations are, however, impractical and therefore, this phenomenon will not be discussed here.) Lower polydispersity enhances the film microlayering process, thus the film stability [27]. Liquid films containing layers cannot be described by the DLVO theory because their disjoining pressure is controlled by the repulsive particle/particle and particle/interface interactions and not by the interface/interface interactions because the interfaces are too far apart when layers are inside the film [25]. Due to these interactions, the disjoining pressure isotherm of a film containing layers is oscillatory (Fig 6.), which explains that the film thinning has several steps [30]. Because of this, the occurrence of layering and the height of the step-transitions do not depend on the nature of the interface: the same steps (by number and height) can be observed in a foam and in an O/W/O emulsion film, respectively, if the two types of films contain the same amount of small particles, such as sodium caseinate [29,32]. A very important feature of the film microlayering phenomenon is that the occurrence of a step-transition also depends on the area (diameter) of the film. If the film area is smaller than a critical value, the step-transition is inhibited and a layer or layers of fine particles stay inside the film for an unlimited time [27,29,31,32]. It is interesting to note that the capillary pressure of drops or bubbles, which is the driving force of film drainage, increases with decreasing drop or bubble size, i.e., film area (See Eq. 1). However, the presence of strong structural forces in the film overrides the effect of capillary pressure in this case. The phenomenon can be explained by the vacancy mechanism of the step-transitions [28]. The existence of critical film size has great practical importance: when layer or layers of small particles stay trapped in the liquid films between small drops (or bubbles) the stability of these films is extremely high. -

-

12

3.2. Differential interferometry- characterization of the pseudoemulsion film When an oil drop in an aqueous phase rises to the surface of the solution or an oil drop approaches a bubble inside a foam an asymmetrical, oil/water/oil film, the so-called pseudoemulsion film forms between the oil and air phases (Figure 8.) The importance of

Figure 8. Formation of a pseudoemulsion film drop between an oil drop and air this film is that the effect of oil drops in foam stability is controlled by the stability of the pseudoemulsion film [31,32]. If this film is unstable, that is, it ruptures, the oil drop enters the air/water surface and spreads on it, generally resulting in antifoam action [38]. If, however, the pseudoemulsion film is stable, the oil drops cannot spread and instead of breaking it, the oil drops stabilize the foam [39]. Due to its asymmetrical nature, the pseudoemulsion film is always curved. Similarly, foams or emulsions which form between bubbles (drops) of differing size are not plane parallel but curved (cap) shaped [4]. When two fluid interfaces have a high radius of curvature, such as in the pseudoemulsion film, the distance between the interference patterns is too small to be measured by common reflected light interferometry. In this case, differential interferometry can be used for imaging the interface profile 140-45]. (Another technique for studying curved films is the controlled drop tensiometer, as was shown in section 2.) The basic principle of differential interferometry consists of splitting the original image into two images. An Aus Jena Epival Interphako microscope was used in our laboratory for film studies with common and differential interferometry. This microscope is capable of viewing objects in transmitted light as well as in reflected light and also equipped with a Max Zhender interferometer. The interferometer splits the original beam of the image into two beams of different optical paths which, when recombined, give a sheafing type differential interference pattern - this can be used to measure curvature of surfaces [43-45] (See Figure 9). The two images are shifted at a distance d at which the beams reflected by the interfaces Z(x,y) and Z'(x,y), respectively, interfere. As a result, a characteristic interference pattern forms, which contains streaks, tings and mustaches [41,42] (Fig. 9). The optical path length between the two beams is

A = 2(Z-Z')n/

(5)

where n r is the refractive index in the phase between the surface and the objective. When A =iL/2, (where i=0,1,2.., is the order of interference and ~, is the wavelength of

13 the monochromatic light used) dark fringes form for odd and bright ones for even i. By measuring the distance between the parallel bright and dark fringes the curvature of the film, Rf, can be calculated [44]. Lobo and Wasan [41] determined the exact profile of pseudoemulsion films, their meniscus and the film contact angles by using common interferometry (Newton tings) and differential interferometry in conjunction with the Laplace equation for the film menisci [42]. The differential interference image of a pseudoemulsion film between an octane drop and air inside a 4 wt% micellar solution of the non-ionic surfactant C1215AE30 (ethoxylated alcohol with C~z-C~5alkyl chain and 30 ethoxy groups) is shown in Figure 10. The drainage characteristics of the pseudoemulsion film were also observed, in reflected light by common interferometry, by submerging the oil (octane) drop and allowing it to rise in the solution. As the rising oil drop reached the gas-aqueous interface, a thick, nonuniform film (with a dimple) was initially formed as seen in Fig. 1 la. Fig. 1 lb and c show the film which was undergoing similar thickness transitions as the foam film in Figure 7. The step-wise thinning phenomenon observed here for the pseudoemulsion film is the result of microlayering of non-ionic surfactant micelles, as described in the previous section for foam or emulsion films. In Figure 11 it is also seen that the thin pseudoemulsion film appears bright, as opposed to the thin foam and emulsion films, which are black (see Fig. 7). The reason for this is the optical path difference between the reflected rays from the two film surfaces. Light rays which are incident on the film reflect from the two surfaces

Figure 9. Principle of differential interferometry

14

Figure 10. Photomicrograph of the differential interference pattern of a pseudoemulsion film (octane drop in 4 wt% C1215AE30 solution).

Figure 11. Thinning pseudoemulsion film. a) Thick film with dimple, b) Film undergoing stratification - two thickness transitions - and, c) Enhanced image of film undergoing stratification. The film has three discrete thicknesses resulting from the first two transitions.

15 of the film and these reflected light rays interfere. When light rays of wavelength k, are incident on a thin (zero thickness)foam or emulsion film, they encounter, alternately, optically dense and optically rare medium (the order depends on the type of emulsion). As a result, the two reflected rays from each of the film surfaces differ by a path length of ~./2. This is the condition for the destructive interference of light, which is why for film with thicknesses less than 100 nm the foam and emulsion films appear black in reflected light. On the other hand, light rays incident from the air side of an aqueous pseudoemulsion film encounter an optically denser medium at both the film surfaces (air to water and water to oil). Thus, the reflected rays from film surfaces are shifted by ~./2 and the path difference is ~,. This is the condition for constructive interference, which is why the pseudoemulsion film appears bright [42].

3.3. Capillary force balance The texture and stability of food foams or emulsions strongly depend on the interaction between the fat or other dispersed particles in the system. Aggregation of paricles, drops by polysaccharide macromolecules has been observed in food systems [46-48]. Aggregation phenomena and interparticle interactions can be directly observed by using transmitted light interference microscopy in conjunction with the capillary force balance technique recently developed in our laboratory. First, the emulsion or dispersion is filled in the film holder, then, the formed conical interfaces are pushed together by sucking out the liquid through the capillary side arm (Fig. 12). A thick film (lamella) several micrometers thick forms as a result of this increase in the capillary pressure. The film structure, its response to external stress, which can be manipulated by the capillary pressure and the stability of the lamella can be directly observed using transmitted light microscopy. Draining of the lamella can proceed until a thin liquid film is formed. A great advantage of the method is that the observed emulsion layer is "free", without having any connection to other surfaces (such as a glass slide, etc.). Figure 13 demonstrates the particle aggregation phenomenon induced by gums as observed using the surface force balance method. The figure shows photomicrographs of thick foam lamellae from O/W food emulsions containing 20 vol% fat, in the presence and in the absence of gums, respectively. The elementary particle size of these emulsions, as determined by light scattering after strong dilution, was mainly in the sub-micron range. It is seen, however, that in the undiluted emulsion containing gums (Fig. 13 a) the fat particles appear as 5,101am aggregates. It could be also observed that the particle aggregates move together as the lamella thickness is changed. In the emulsion without gums (Fig. 13 b) only a few larger particles can be seen, the rest of the particles are very small and cannot be seen under the low magnification used.

4. BACK-LIGHT SCATTERING - KOSSEL DIFFRACTION Another optical technique, called the back-light scattering (Kossel-diffraction) method can also be used to investigate structure in food emulsions and foams. In this method, the emulsion (or foam) in a transparent vessel is illuminated by a collimated laser beam (See

16

Figure 12. Principle of capillary force balance

Figure 13. Photomicrographs of lamellae formed from O/W food emulsions containing 20 wt% emulsified fat, illuminated by transmitted light. a) Particles aggregate in the presence of gums. b) Negligible aggregation without gums.

17 Fig. 14). A portion of the light rays are scattered from the emulsion particles through the wall of the vessel and form a concentric interference pattern. The back-scattering phenomenon is analogous to the operation of diffraction gratings [49]. The measurement can be used to characterize the structure of the emulsion, because the shape of the light intensity profile depends on the particle size (2a), the average distance between the particles (d), the wavelength of the laser light used (~.), the angle of observation (O) and the regularity of the spacial arrangement of the particles (S) (See Fig. 14). The interference image can be recorded and the intensity profile along a vertical line, going through the center of the image, can be measured by an image analyzer. The optical conditions (laser beam diameter, magnification etc.) and the wall of the sample holder influence the intensity profile, thus, these parameters must be kept constant in the measurements. Figure 15 shows a typical light intensity profile of a food emulsion as a function of distance in arbitrary units. The curves are symmetrical with a large, primary maximum in the center, which is surrounded by minima and secondary maxima at both sides. When the parameters of the emulsion (d, a and S) change, it is generally reflected on i.) the width of the shoulder of the primary maximum; ii.) the depth and position of the minima and iii.) the height and position of the secondary maxima of the intensity profile. The shape of the intensity profile reflects the shape of the radial distribution function of the particles. The radial distribution of a highly ordered structure, such as a crystal, is periodic, i.e., the concentration of particles changes periodically as a function of the radial distance from a given point. If the order, the regularity of the structure, is lower, such as in a liquid, the radial distribution function is less periodic: the difference between the maxima and minima

Figure 14. Principle of back-light scattering measurement.

18

Figure 15. Intensity profile of light back-scattered from a food emulsion containing 20 wt% emulsified fat, at 5 ~ using green light (543 nm). are much smaller than in the ordered structure. If there is no order, such as in a gas, the periodicity of the radial distribution function vanishes and the intensity decreases monotonously as a function of distance. The effect of particle aggregation on the intensity profile is illustrated in Fig. 16 showing the profiles of a food emulsion without gums, in which no aggregation takes place, and the same emulsion in the presence of gums, where the particles aggregate, respectively. Aggregation changes several emulsion properties at the same time: it increases the particle size, polydispersity and the distance between the particles. As a result~ the shoulder of the primary maximum becomes wider (See Fig. 16). Moreover, aggregation generally decreases the order of the particles too, which results in a decrease of the secondary maximum. It is seen that the emulsion with the aggregates has, indeed, a very small secondary maximum.

5. D I E L E C T R O M E T R Y The dielectric properties of water have been extensively used to determine moisture content in food systems. However, only very recently have we used the complex dielectric properties of emulsions in the microwave frequency region to characterize both emulsion type and water content [50-52]. We have developed both a cavity resonance dielectrometer capable of operating at 8-11 GHz and an interference dielectrometer operating at 23.45 GHz.

19

Figure 16. Effect of particle aggregation on the back-scattered light intensity profile of food emulsion, containing 20 wt% emulsified fat, at 5 ~ (green light). We have employed these dielectric techniques to study the hydration characteristics of hydrocolloids widely used in food systems 1531. The rotational relaxation of water molecules is influenced by its immediate environment. The microwave dielectric characteristics of associated or bound water molecules are markedly different from those of free water molecules. During the hydration of hydrocolloids, water molecules go from a bound state to an unbound state and the change is detected dielectrically. In food systems, hydrocotloids are added to impart increased product viscosity as well as to stabilize the emulsions. The stabilizing action arises from the formation of complex structures between the water molecules and gums. The water molecules are held in a bound state through avariety of bonding mechanisms. The existence of continuous phase structures prevents the emulsion drops from approaching each other and therefore prevents coalescence. In addition to enhancing emulsion stability, the bound water is not available for microbial growth and this is clearly an important feature in food emulsion systems. The extent of hydration of hydrocolloid is important in determining the efficacy of inhibiting the coalescence of emulsion drops. The microwave dielectric measurements exclusively measure the rotational relaxation of the water molecule.

20 Figure 17 shows the change in permittivity as a function of time of hydration for 0.5 wt% ~c-carrageenan dissolved in double deionized water. The measurements were made by monitoring the changes in dielectric response of the hydrocolloid sample solution held in the cavity of resonance dielectrometer operating at 9.505 GHz. The measurements indicate ,hat the hydration process is complete after a period of 6 hours. During the early stages of hydration, a high dielectric permittivity value was measured corresponding to the large amount of free water present in the system. As the water molecules attach themselves to the numerous hydratable groups present in the hydrocolloid molecule, the permittivity values decline. When all the water molecules are held in a bound state, the hydration process is complete and no change in permittivity was observed.

Figure 17. Experimentally measured variation in permittivity with extent of hydration of 0.5 wt% K:-carrageenan hydrocolloid at 23.45 GHz.

6.

CONCLUDING REMARKS

New experimental techniques and several of their applications were presented which help in the understanding of structure, texture and stability of food systems. For future research, the mechanism of film stability by the microlayering of colloid particles seems to be the most promising - especially in food emulsions and foams. Work is in progress in our laboratory to calculate the oscillatory disjoining pressure inside liquid films containing microlayers [30]. The structure and stability of foamed emulsions, such as whipped cream, ice cream or whipped toppings, strongly depend on the interparticle interactions and on the orientation of drops/particles at the foam films. Further development of the surface force balance and

21 back-light scattering techniques will aid in the understanding of the stability mechanisms in food dispersions.

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22 27. 28. 29. 30. 31. 32. 33. 34. 35. 36. 37. 38. 39. 40. 41. 42. 43. 44. 45. 46. 47. 48. 49. 50. 51. 52. 53.

A.D. Nikolov and D.T. Wasan, Langmuir, 8 (1992) 2985. P.A. Kralchevsky, A.D. Nikolov, D.T. Wasan, and I.B. Ivanov, Langmuir, 6 (1990) 1180. K. Koczo, A.D. Nikolov, D.T. Wasan, R.P. Borwankar and A. Gonsalves, paper submitted to J. Colloid Interface Sci. (1994). X. L. Chu, A.D. Nikolov and D.T. Wasan, Langmuir, 10 (1994) 4403. D.T. Wasan, A.D. Nikolov, L.A. Lobo, K. Koczo and D.A. Edwards, Progr. Surf. Sci., 39 (1992) 119. D.T. Wasan, K. Koczo and A.D. Nikolov, in Foams: Fundamentals and Applications, L.L. Schramm (ed.), ACS, Chapter 2, 1994. D.G. Schmidt and T.A.J. Payens, Surface and Colloid Science, E. Matijevic (ed.), pp. 165-229, Wiley-Interscience, New York, 1976. P. Walstra and R. Jenness, Diary Chemistry and Physics. Wiley, New York, 1976. D.G. Schmidt and J.A.J. Payens, J. Colloid Interface Sci., 39 (1972) 655. T.F. Kumosinski, H. Pessen, H.M. Farrell and H. Bumberger, Arch. Biochem. Biophys., 266 (1988) 548. S. Friberg, S.E. Linden and H. Saito, Nature, 251 (1974) 495. K. Koczo, J.K. Koczone and D.T.Wasan, J. Colloid Interface Sci., 166 (1994) 225. K. Koczo, L. Lobo and D.T.Wasan, J. Colloid and Interface Sci., 1992, 150, 492. Nikolov, A.D., Dimitrov, A.S. and Kralchevsky, P.A., Optica Acta, 33 (1986), 33. L.A. Lobo, A.D. Nikolov, A.S. Dimitrov, P.A. Kralchevsky and D.T. Wasan, Langmuir, 6 (1990) 995. L.A. Lobo and D.T. Wasan, Langmuir, 9 (1993) 1668. H. Beyer, Jenaer Rdsch., 16 (1971) 82. A.D. Nikolov, A.S. Dimitrov and P.A. Kralchevsky, Optica Acta, 33 (1986) 33. A.S. Dimitrov, P.A. Kralchevsky, A.D. Nikolov and D.T.Wasan, Colloids Surfaces, 47 (1990) 299. Y. Cao, E. Dickinson and D.J. Wedlock, Food Hydrocolloids, 4 (1990) 185. Y. Cao, E. Dickinson and D.J. Wedlock, Food Hydrocolloids, 5 (1991) 443. E. Dickinson and V.B. Galazka, in Food Polymers, Gels and Colloids, E. Dickinson (ed.), Royal Soc. of Chem. Spec. Publ. No. 82, pp. 494-497, 1991. R.D. Guenther, Modem Optics, John Wiley & Sons, pp. 361-431, 1990. J.P. Perl, C. Thomas and D.T. Wasan, J. Colloid Interface Sci., 137 (1990) 425. C. Thomas, J.P. Perl and D.T. Wasan, J. Colloid Interface Sci., 139 (1990) 1. J. Rudin and D.T. Wasan, J. Colloid Interface Sci., 162 (1994) 252. C. Thomas, PhD Thesis, Illinois Institute of Technology, Chicago, 1990.

Characterization of Food: Emerging Methods A.G. Gaonkar (Editor) 9 1995 Elsevier Science B.V. All rights reserved.

23

Chapter 2 A p p l i c a t i o n of state-of-the-art f l u o r e s c e n c e and i n t e r f e r o m e t r i c techniques to study c o a l e s c e n c e in food d i s p e r s i o n s D.C. Clark Institute of Food Research, Norwich Laboratory, Norwich Research Park, Colney, Norwich NR4 7UA, United Kingdom.

1. INTRODUCTION Coalescence is an important mechanism of destabilization of food foams and emulsions [1]. The coalescence process involves fusion of two adjoining gas bubbles in a foam or oil droplets in an oil-in-water emulsion by rupture of the thin aqueous film or foam lamella which keeps the dispersed phase separated. Foams generally contain a high phase volume of gas and the thin planar films form very rapidly as entrained liquid drains from the foam. In contrast, formation of thin films in emulsions is a much slower process. This is because the phase volume of the dispersed phase in emulsions is usually not as high as in a foam, the average droplet size is smaller than the average bubble size and there is generally a comparatively small difference in the density of the continuous and dispersed phases. A combination of these factors, coupled with the inclusion of stabilizing agents such as polysaccharide thickeners, means that the rate of creaming in emulsions is relatively slow. However, it is worth considering the thin films that may form between oil droplets in an emulsion when they pack together closely in the cream layer of an emulsion or during collision processes. Thin films are stabilized by two distinct mechanisms. The mechanism that prevails is dependent upon the molecular composition of the interface. Low molecular weight surfactants such as food emulsifiers or polar lipids congregate at the interface and form a fluid adsorbed layer at temperatures above their transition temperature (Figure l(a)). When a surfactantstabilized thin film is stretched, local thinning or dimple formation occurs in the thin film. This is accompanied by the generation of a surface tension gradient across the locally thin region. Surface tension is highest at the thinnest point of the stretched film, due to decreases in the surface concentration of emulsifier in the region of the stretch. Equilibrium surface tension is restored by lateral diffusion of surfactant in the adsorbed layer towards the region of highest surface tension. This surfactant drags interlamellar liquid into the thin region and contributes to the restoration of equilibrium film thickness. This process is often referred to as the Marangoni effect [2]. In contrast, the adsorbed layer in protein-stabilized thin films is much stiffer and often has viscoelastic properties [3]. These derive from the protein-protein interactions that form in the adsorbed layer (Figure 1(b)). These interactions result in the formation of a gel-like adsorbed layer in which lateral diffusion of molecules in the adsorbed layer is inhibited. Multilayer formation can also occur. This serves to further mechanically strengthen the adsorbed layer.

24 When pure protein films are stretched, the change in interfacial area is dissipated across the film, due to the cohesive nature of the adsorbed protein layer and the deformability of the adsorbed protein molecules.

Figure 1. Schematic diagram showing the possible mechanisms of thin film stabilization. (a) The Marangoni mechanism in surfactant films; (b) The viscoelastic mechanism in proteinstabilized films; (c) Instability in mixed component films. The thin films are shown in cross section and the aqueous interlamellar phase is shaded.

25 Thin film instability can result in systems that contain both proteins and low molecular weight surfactants, as is the case in many foods. The origin of this instability may rest in the incompatibility of the two stabilization mechanisms; the Marangoni mechanism relyifig on lateral diffusion, the viscoelastic mechanism on immobilisation of the protein molecules that constitute the adsorbed layer. One can speculate that in a mixed system, competitive adsorption of low molecular weight surfactant could weaken or interfere with the formation of protein-protein interactions in the adsorbed layer thus destroying the integrity and viscoelastic properties of the adsorbed layer (Figure l(c)). This could be a Progressive process, with the presence of small quantities ~of adsorbed surfactant initially introducing faults in the protein film. Adsorption of more surfactant could induce the formation of protein 'islands' in the adsorbed layer, which were capable of slow lateral diffusion; too large to participate in a Marangoni type of stabilization. Adsorption of progressively more surfactant would reduce the size of the protein aggregates still further until the adsorbed protein was in its monomeric form. Ultimately, all the protein would be displaced from the interface by the surfactant. The properties of the adsorbed layers in thin films have been inferred from the results of many detailed studies of macroscopic air-water (a/w) or oil-water (o/w) interfaces. Whether such models accurately reflect the interfaces found in thin films is a matter of some contention. Certainly, the volume of bulk solution that is present beneath the adsorbed layer of a macroscopic interface is of infinitely larger volume than that found in the interlamellar region of a thin film. In the former case, surface tension has been shown to fall slowly over many tens of hours [4], consistent with conformational rearrangements of the adsorbed protein but also formation of multilayers of adsorbed protein. The timescale of such changes is irrelevant when compared, for example, to the lifetime of the foam that forms the head on a glass of beer. In addition, macroscopic interfaces are relatively insensitive to processes that can lead to the rupture of a foam lamella. For example, the adsorption of a lipid micelle and the subsequent spreading of lipid causes film rupture by a Marangoni effect (Figure 2). Interlamellar liquid associated with the polar head groups of the lipid is dragged away by the spreading lipid causing local thinning of the thin film and increasing the probability of film rupture.

Figure 2. A Schematic representation of the stages whereby a spreading particle causes local film thinning leading to film rupture [1].

26 Thus, there is a strong incentive to develop methods that allow controlled formation and characterisation of the adsorbed layer properties of thin liquid films.

2. PREPARATION OF AIR-WATER AND OIZ,-WATER THIN FILMS Although methods were available to prepare and investigate isolated air-suspended thin liquid films many years ago [5], they have only been developed further comparatively recently. The most extensive studies have been performed on surfactant-stabilized films using molecules such as sodium dodecyl sulfate [6]. Our apparatus has been developed from the film holders used by this Bulgarian group. Microscopic thin films [7,8] have generally been formed by introduction of a droplet of solution into a ground glass supporting ring or annulus (Figure 3). This device is crudely analogous to a miniaturized version of the loop children use to blow bubbles from soap solutions. However, rather than relying on gravity to drain off surplus bulk liquid as in the childs toy, film formation is initiated by withdrawal of liquid by applying controlled suction. This is achieved via a capillary sidearm that is connected to the film supporting ring. Liquid withdrawal is stopped once a thick horizontal planar film of appropriate diameter (e.g., 0.3mm) has been formed. Drainage of the thick film proceeds from this point mainly as a result of suction from the adjoining wedge shaped region that surrounds the film. This region is referred to as the Plateau border.

Figure 3. A schematic diagram of an air-water suspended thin liquid film held in a ground glass annulus.

27 Microscopic thin films are relatively fragile structures and are highly sensitive to changes in temperature, mechanical disturbance and evaporation. We have designed a dedicated chamber that allows the necessary control of the environment surrounding the film whilst not impeding observation of film drainage and measurement of equilibrium thickness or surface diffusion in the adsorbed layer. A photograph of our film chamber and a film support ring is shown in Figure 4. The film chamber is surrounded by a temperature-controllable brass housing. Crown glass optical windows allow observation of the thin film from either above or below the housing. Evaporation from the thin film, once formed in the chamber is controlled by the presence of a horseshoe-shaped trough which can be filled with the solution under investigation, prior to formation of the film in the chamber. The brass housing is suspended beneath an aluminium holder via a kinematic mount which allows levelling of the film using the micrometer adjusters. The aluminium holder fits directly onto the stage of an inverted microscope (Nikon Diaphot TMD) equipped with an epi-illumination attachment.

Figure 4. A photograph of the,thin~ film holder and temperature controlled chamber. More recently, we have developed a device that allows formation of thin films in a liquid bath [9,10]. The apparatus opens up many more opportunities for film formation under different conditions but is rather more difficult to operate than the film ring. The former apparatus, which can be used for formation of a/w or o/w thin films is shown in Figures 5 and 6. The liquid b a t h requires chemical treatment prior to introduction of the continuous phase solution. This involves thorough cleaning of the cell using chromic acid followed by drying,i Controlled silanation is used to create a highly localized hydrophobic patch on the optical window that forms the base of the cell. A 10 ~1 droplet of octadecyl trichlorosilane was found to bean effective silanation agent for this purpose [ 11]. Unreacted silanation reagent can be removed by washing with anhydrous heptane. The cell can then be filled with the continuous phase of interest which could be an aqueous protein solution, an oil-in-water emulsion or the separated continuous phase of an emulsion. An oil droplet or air bubble is then immediately introduced onto the hydrophobic patch by careful delivery using

28

Figure 5. A schematic diagram showing the apparatus used to form a thin aqueous film between oil droplets. Reproduced from reference [ 10] with the permission of Academic Press.

Figure 6. A photograph of an aqueous thin film formed between two droplets of ntetradecane. The laser beam illuminating the thin film (misaligned for clarity) can be used to measure lateral diffusion in the adsorbed layer or film thickness.

29 a hypodermic syringe and needle. The droplet will remain captive on the hydrophobic patch, provided it is of relatively small volume. The second droplet suspended from a concavetipped nozzle attached to a micrometer-controlled l ml glass syringe is then lowered into position above the captive droplet creating a thin aqueous film in the region of contact. Thin film drainage behavior can be viewed through the lower droplet by means of the inverted microscope in reflected light. It is relatively simple to convert the chamber to allow modelling of thin oil films between water droplets by application of the hydrophobic coating to all the inner surfaces of the chamber. A small hydrophilic spot can then be generated in the centre of the baseplate by judicious application of a small droplet of acid using a micropipette. In this case, captive and suspended water droplets are brought into close contact, thus providing a model for thin film formation in water-in-oil emulsions.

3. THIN F I L M DRAINAGE AND THICKNESS MEASUREMENTS Observation of the drainage process from thick film to equilibrium thin film can be very informative. Both a/w and o/w thin films have very poor contrast and are impossible to observe under the microscope under bright field (background) illumination conditions. However, it is possible to observe the films in reflected light mode using epi-illumination. This is possible because although the films are virtually transparent, a small quantity of illuminating light is reflected from both upper and lower interfaces (see inset Figure 9). This phenomenon is also exploited in the measurement of film thickness which is described later in this section.

Figure 7. A photographic sequence showing the drainage behavior of a thin film formed from 2 mM SDS in 2 mM sodium phosphate buffer, pH 7.0 containing 0.1 M NaC1. See text for description.

30 The drainage properties of surfactant-stabilized films [8], can easily be distinguished from protein-stabilized film drainage [12]. Surfactant-like drainage behavior is illustrated in the sequence of photographs shown in Figure 7. The sample shown in the figure is 2 mM sodium dodecyl sulfate in 2 mM phosphate buffer, pH 7.0 containing 0.1 M NaC1. The initial 20 seconds of drainage are characterized by rapid movement of regions of different thickness, distinguishable by their different bright colors, sweeping towards the periphery of the film (Figure 7(a)). This accurately conveys the fluid nature of the interfacial layer in these films. This phase of drainage terminates with the disappearance of colors leaving a white film which is 100-200 nm in thickness. Drainage proceeds for 2-3 minutes with darkening of the periferal region of the film to form thinner gray patches. The interlamellar liquid trapped in the central region of the film appears to be squeezed out into the Plateau border by the advancing thinner gray regions and forms white arcs around the edges of the gray regions (Figure 7(b)). This process continues in several discrete waves and the film darkens with each stage. This is followed approximately 3 minutes after film formation by spontaneous nucleation of black spots at random points in the film (Figure 7(c)). The black spots grow in size and coalesce (Figure 7(d) and (e)), and eventually approximately 5 minutes after formation the whole film becomes black (Figure 7(f)). This is termed the primary or common black film and has an equilibrium thickness of the order of 12nm. Most other low molecular weight surfactants, including polysorbate emulsifiers [ 13], sucrose esters [14], mono and diglycerides, lysolecithins [15] and lecithins, follow this type of drainage behavior with minor differences. Firstly, the surfactant must be above its transition temperature and in the liquid crystalline phase. Indeed, it is generally impossible to form stable films if the surfactant is in the gel state. Secondly, the chosen solution conditions may not favour drainage to the common black film stage. Quite often equilibrium thickness is achieved at some intermediate gray film stage or thicker. Alternatively, drainage can proceed to thickness regimes that are considerably less than that of the common black film. The observed equilibrium thickness represents the film dimensions where the attractive and repulsive forces within the film are balanced. This parameter is very dependent upon the ionic composition of the solution as a major stabilizing force arizes from the ionic double layer interactions between any charged adsorbed layers confining the film. Increasing the ionic strength can reduce the repulsion between layers and at a critical concentration can induce a transition from the primary or common black film to a secondary or Newton black film. These latter films are very thin and contain little or no free interlamellar liquid. Such a transition is observed with SDS films in 0.5 M NaC1 and results in a film that is only 5 nm thick. The drainage properties of these films follows that described above but the first black spot spreads instantly and almost explosively to occupy the whole film. This latter process occurs in the millisecond timescale. In contrast, protein-stabilized thin films display very different drainage characteristics [7]. Until recently, the work on protein-stabilized thin films was limited to preliminary measurements of equilibrium film thickness and determination of contact angle [16-19]. A sequence of photographs depicting stages in the drainage of a typical protein film are shown in Figure 8. The initial appearance of protein films immediately after formation is distinct from that of surfactant-stabilized films. The protein-stabilized thin film is characterized by a series of concentric white, black and brightly colored fringes or Newton's rings. These correspond to constructive and destructive interference patterns of light reflected from: the upper and lower interfaces of the film and interconnect regions of similar thickness. Initially, the fringes are closely spaced indicating that the film is thick. In addition, there is a steep

31 thickness gradient across the film (Figure 8(a)), which is thinnest in the central region. As drainage proceeds the fringes become more widely separated and the region bordering the contact line at the perimeter of the film lightens in color (Figure 8(b)). This is followed by darkening of the periphery of the film (Figure 8(c)) which eventually becomes black (Figure 8(d)) after approximately 10 minutes drainage (Figure 8(e)). Formation of a continuous black ring around the perifery of the film (Figure 8(0) traps liquid in the thicker central region and slows down the rate of drainage. Nevertheless, drainage proceeds, albeit at a slow rate due to the constriction at the perifery of the film and results in a shrinkage in the dimensions of the lighter central region of the film (Figure 8(g)). Eventually, the whole film becomes black (Figure 8(h)) but this may take in excess of 20 minutes. The drainage rate of the film is very sensitive to the time history of the interface. Aged interfaces generally result in films that are very slow to drain, due to their increased interfacial shear viscosity [3].

Figure 8. A photographic sequence showing the drainage behavior of an a/w thin film formed from BSA in distilled water, adjusted to pH 8 containing 25 mM NaC1. See text for description. Reproduced from reference [12] with the permission of Academic Press. Equilibrium film thickness can be measured by interferometry [7,8] using an apparatus of the type shown in Figure 9. When studying a/w films, we use an interferometer mounted above the stage of the inverted microscope. The interferometer comprises an interrogating beam from a 3 mW helium-neon laser (632.8 nm) which is passed through an optical chopper and is directed down onto the surface of the film by means of a beam splitter. The beam is focused onto the thin film using an extra long working distance objective lens (Nikon M-plan, magnification x20 or x40). The diameter of the illuminated spot on the film surface is 25 50/~m. The majority of the incident light is transmitted through the film and care must be taken with the inverted microscope to ensure that appropriate barrier filters are fitted to the

32 eyepieces to avoid injury to the operator. A small fraction of the incident light is reflected from both the upper and lower interfaces of the film and passes back up the optical axis of the interferometer (inset Figure 9). These reflected beams are transmitted by a 633 nm narrow pass filter, positioned above the beam splitter and the combined signal is detected at a photodiode. The detected intensity fluctuates between the two extremes of totally constructive and totally destructive interference, thus producing in the photodetector output a varying signal that is a measure of film thickness. The signal from the photodiode is amplified (AMP) and fed into a phase sensitive detector (PSD) referenced to the chopper frequency. The PSD, which reduces signal noise and improves signal stability, is set up with a constant negative offset to compensate for background reflected light from the chamber windows etc. The signal is output via a chart recorder.

Figure 9. A schematic diagram of the interferometer used to measure thin film thickness. The inset shows that light is both transmitted and reflected by the thin film. Reproduced from reference [7] with the permission of the Royal Society of Chemistry. The equilibrium film thickness (h) is calculated using the expression:

~,

[

h . . . . . sin "l ~ 27rn [

I/Ira 1 + [4R/(1-R)2].[1-I/Im]

] 0.5 } J

(1)

33 where (n-l) 2 R

-

(2)

(n+l) 2 and k is the wavelength of the laser, n is the refractive index of the film, I is the intensity of light at the photodiode at equilibrium, and Im is the intensity at the last maximum. In the ideal situation the chart output resembles the interferograph shown in Figure 10, and this can be achieved relatively easily with protein films by careful positioning of the spot near the central region of the film. Often it is more difficult to achieve such output with surfactant films due to their fluid nature and the fact that regions of differing thickness sweep across the interrogating beam of the interferometer. Accurate determination of I and Im can be difficult due to uncertainty in the position of the minimum. We find that the most reproducible results are obtained if the minimum value is taken as the signal observed after removal of the film annulus at the end of the experiment.

Figure 10. Interferograph from a draining a/w suspended thin film showing the calculated change in film thickness with time. Reproduced from reference [20] with the permission of the Institute of Brewing.

34 If a complete interferograph is obtained it is possible to construct a drainage curve for the film (Figure 10) since the film thickness at each maximum and minimum can be calculated from equation (1). In the case of aqueous thin films between oil droplets (Figure 5), the interferometer beam is brought into the microscope through the epi-illumination attachment whereby the objective lens is used to both observe the film and focus the interferometer beam. The contrast of the observed image is much improved in stray light is minimized by positioning a pinhole at the image plan of the epi-illumination device. The thickness calculations remained the same as for the a/w films as the refractive index of the aqueous thin film was the same in both cases.

4. SURFACE DIFFUSION MEASUREMENTS BY FRAP Observations of film drainage behavior provides an indication of the structural properties of the adsorbed layers. It is simple to distinguish between protein or surfactant-stabilized films. However, most food systems contain mixtures of both proteins and low molecular weight surfactants. Detailed study of the thin film properties of protein solutions containing increasing levels of surfactant reveals a corresponding decrease in film thickness with increasing surfactant concentration [ 10,13,15,21 ]. In addition, at certain critical molar ratios of the two components, we have observed a transition in drainage behavior to a intermediate type of drainage [15,22]. The latter possess features of both surfactant-like and protein-like drainage. Typically, distorted Newton's rings are observed as the once rigid protein stabilized interface becomes more fluid. Clearly, important changes in the adsorbed layer structure and surface rheological properties are occurring but it is difficult to identify a method that would allow their direct investigation in the thin film. Certainly such delicate structures would not be amenable to study by conventional surface shear or surface dilational methods. Indeed, these methods are still not widely available for the investigation of macroscopic interfaces. A radical alternative was sought and found. A technique referred to by several different names including fluorescence recovery (or redistribution) after photobleaching (FRAP), fluorescence microphotolysis and fluorescence photobleaching recovery (FPR) was first developed in the mid 70's and has proved a useful technique for the study of lateral diffusion processes in biological cell membranes and the cytoplasm [23,24] and has been reviewed recently [25]. The previous use of the technique in interfacial studies was limited to investigation of the lateral diffusion of lipid at the a/w interface of a Langmuir trough [26]. Several variations of the FRAP technique exist but the simplest are based on the principals outlined in the schematic diagram shown in Figure 11. The method requires that the molecular species of interest is fluorescent labelled or alternatively that an independent fluorescent probe molecule is partitioned into the environment of interest. In the case of thin films, the surface diffusion properties of a given protein in the adsorbed layer can be measured by forming a thin film (diameter 100-200 #m) as described above which includes fluorescent-labelled protein. An attenuated laser beam is used to illuminate a small spot (approximate diameter 5 #m) on the surface of the thin film, eliciting fluorescence from labelled molecules contained within the spot, which is recorded (Figure l l(a)). These fluorescent molecules are then irreversibly photodestroyed (bleached) by increasing the intensity of the laser beam approximately 1000x for a few milliseconds (Figure 1 l(b)), before returning the laser intensity to its attenuated level. Fluorescence returns to the bleached spot only if the bleached molecules are free to diffuse laterally away from the spot to be replaced

35 by non-bleached molecules in the surrounding film diffusing into the spot (Figure 1 l(c) and(d)). Measurement of the time dependence of this process and knowledge of the dimensions of the bleached spot, allows calculation of the surface diffusion coefficient.

Figure 11. A schematic diagram showing the various stages in typical spot FRAP experiment. See text for explanation The design and construction of a FRAP apparatus has been recently reviewed [27]. The purpose of the majority of the optical components is to deliver a well-defined, microscopic Gaussian or uniform circular cross section beam, that can be rapidly modulated, to the sample. A schematic of our FRAP apparatus is shown in Figure 12. The light source used is an Argon ion laser (Coherent Innova 100 - 10). We have examined three different beam modulation arrangements during the course of our studies. The first device we tried was an acousto-optic modulator (Coherent 304A) which contains a crystal, that diffracts the input laser beam into a number of secondary beams. The intensity of the secondary beams can be modulated by application of an RF signal to the crystal [28]. The disadvantage of this device is that the output beam was of ellipsoidal rather than circular cross section and therefore did not have a true Guassian intensity profile. Since the uniform circular beam is obtained by projecting the Guassian beam from the laser through a microscopic pinhole, it was not possible to deliver a uniform circular or Gaussian cross section bleach pulse to the sample as required. The second modulation method involved the positioning of an LCD light valve (Displaytech) between two crossed Glan Thompson polarizers. Application of a DC voltage to the light valve caused rotation of the plane of polarisation of the laser beam from that of the first polarizer such that it was no longer extinguished by the second polarizer. Although this method produced acceptable beam profiles, the LCD had a rather limited lifetime before laser-induced damage significantly reduced its performance resulting in a reduction in the intensity difference between the monitor and bleach beams. Our preferred modulation method is one of the first described [26], and involves generation of an attenuated beam by reflection off glass flats as shown in Figure 12. When the fast electronic shutter (c) is closed, only the monitoring laser beam (a), which has been attenuated by multiple reflection illuminates the

36 sample. When the shutter is open the intense bleaching beam (b) which is transmitted through two of the glass fiats passes through to the sample. The crucial factor with this modulator arrangement is beam alignment to ensure that both attenuated and bleach beams are coincident at the sample.

Figure 12. A schematic diagram of a FRAP apparatus. See text for a key to the abbreviations. The beam provided by the modulator passes through a beam monitor (beam splitter and photodiode), the signal from which is used to electronically compensate for minor fluctuations in the laser beam intensity. The beam is then launched through a pinhole aperture (A~) located at the image plane, at the entrance port of the epi-illumination attachment of the fluorescence microscope. Our apparatus can be used with both upright (Nikon Optiphot) or inverted (Nikon Diaphot TMD) microscopes but the latter is most convenient for thin film measurements. The filter block in the epiillumination attachment is selected to match the laser line used for excitation andthe emission peak of the fluorescent probe. The 488nm line is the most popular for FRAP measurements with the Argon ion laser, as it can be used to excite a number of different fluorophores including fluorescein, 4-chloro-7-nitrobenz-2-oxa-l,3-diazole (NBD chloride) and members of the carbocyanine family. The use of the well-defined laser-line for

37 excitation renders the short band pass interference filter usually supplied with the filter block for wavelength in the filter block redundant. A 510 nm dichroic mirror (DM), mounted at an angle of 45 ~ is suitable for reflection of the 488 nm excitation beam used for the fluorophores above, through the extra long working distance objective (Nikon) lens (magnification x40 or x20) of the microscope and onto the sample. This will also allow acceptable transmission of the emitted light from the above fluorescent labels. A 520 nm long pass filter (LBF) removes stray excitation light and prevents it reaching the photon counting photomultiplier tube (PMT; Thorn-EMI 9816B) positioned at the cine camera port of the inverted microscope. The PMT is protected during the bleaching pulse by an electronic gating circuit and a mechanical shutter (MS). Prior to entering the detector, the emitted light beam passes through a second aperture (A2) again positioned at the image plane. The two apertures have equivalent diameters (e.g., 200/~m) and serve to make the apparatus confocal. This feature is not so important in the case of thin films, since these can be considered 2dimensional systems once they have drained to equilibrium thicknesses. However, the confocal arrangement is most useful if diffusion measurements are planned using 3-dimensional systems (e.g., probe diffusion in a gel etc). System timing and control, data acquisition and data analysis are performed using a VME microcomputer system (Motorola 68020). The diameter of the focused laser spot on the sample was measured using a beam profile measuring device (BeamScan, Model 2180; Photon Inc.). FRAP data were analysed by a non-linear least squares fit to an expression [8,23,29], defining the time dependence of the fluorescence recovery (F(t)). The apparatus as described above delivered a laser spot of uniform circular cross sectional intensity to the sample and the recovery curves obtained could be analysed with the expression:

F(0 = Fo~

1 -2(1 - Fo/F ~)[O.5(rD/t)e2~D It(Io(2 ro/t) 100 (m + 1)!(2m + 2)! + I2(2rD/t)) +

/.

(-rD/t)m+2] }

(3)

m!2(m + 2)! 2

m=l

where F0 is the fluorescence intensity after the bleach, F o~ is the fluorescence intensity to which the signal recovers and I0 and 12 are modified Bessel functions. The lateral diffusion coefficient, D, is given by

D = w2/4ro

(4)

where w is the radius of the circular spot and ro is the characteristic diffusion time. It is simple to modify the apparatus to include a spacial filter that provides a Gaussian beam profile at the sample. T h e recovery curves obtained with such an experimental arrangement could be analysed by a much simpler expression [29] of the form:

38 F0 + Foo(t//3ro) F(t) = --

(5)

1 + t/~r D

where/3 is related to the proportion of bleach, P (i.e., the prebleach fluorescence intensity F0 divided by the prebleach fluorescence intensity). In practice, the value of/3 is obtained from a lookup table in the analysis programme.

4.1. Fluorescence-labelling of samples Measurements of surface diffusion in thin liquid films by the FRAP method requires the presence of fluorescent molecules in the adsorbed surface layer. The low molecular weight of surfactant molecules and absence of a reactive side groups makes fluorescent-labelling difficult. In addition, conjugation with a fluorophore is likely to significantly alter the surface active properties of the molecule. Therefore, it is preferable to adulterate the surfactant solution of interest with trace quantities of fluorescent lipid or surfactant analog. A range of molecules are commercially available [31] and we have had considerable success using samples such as negatively charged, 5-N-(octadecanoyl)-aminofluorescein (ODAF), positively charged, 3,3'-dioctadecyl oxacarbocyanine perchlorate (DiO), neutral NBD-dihexyldecylamine and phospholipid analogs such as NBD-phophatidylethanolamine. FRAP measurements of protein diffusion at interfaces can be achieved in one of several ways (Figure 13). One option involves controlled covalent labelling of the protein molecule of interest with a reactive fluorescent molecule as in Figure 13(a), [12,32]. An alternative and in some cases simpler approach which we have used recently involves direct addition of trace quantities of a fluorescent lipid analog (Figure 13(b)). For example, we have shown that an amphipathic fluorescent molecule such as ODAF, which has low solubility in water, when added in very low concentrations preferentially partitions into the adsorbed layer, where it can be used to probe the global surface viscosity [8,33]. One major advantage of this approach is that it eliminates the requirement to isolate a protein of interest from a complex mixture (e.g., /3-1actoglobulin from whey protein isolate), the covalent labelling and reconstitution of the system by adding back the labelled protein. This could alter the properties of the total system. The third possible approach, which has been widely used in FRAP studies in cell biology, involves indirect, selective fluorescent-labelling of the protein of interest by interaction with a fluorescent-labelled antigen binding fragment of an antibody raised against the protein (Figure 13(c)). This opens up the opportunity of selective labelling of a protein in a complex mixture, without the need to isolate, label and then reconstitute the system. Considerable care needs to be exercised during fluorescence-labelling of proteins to avoid alteration of the surface properties of the protein. Many reactive fluorescent derivatives are now available from most major chemical companies and specialist suppliers such as Molecular Probes Inc [31]. The isothiocyanate derivative of fluorescein (FITC) has been widely used in our work to label the e-amino group of lysine residues in proteins. Efficient labelling is achieved if the pH of the protein solution is raised to approximately 9.2 to ensure significant deprotonation of the amine groups on the protein surface. Under such conditions, effective labelling of BSA can be achieved in the presence of a 2-fold excess of FITC. The predominant product obtained under these conditions is singly labelled FITC-BSA [12]. Covalent reaction of this fluorophore will occur at lower Ph [32], but the reduction in rate of reaction means it is necessary to add higher molar ratios of fluorophore to the stock

39 protein solution. Labelling under less alkaline conditions is necessary in the case of ~lactoglobulin, since this protein undergoes an irreversible denaturation under our normal labelling conditions [34].

Figure 13. A schematic diagram showing three different approaches to introducing a fluorescent label into thin films to measure surface diffusion in the adsorbed layer. Fluorescein is highly fluorescent at neutral pH. The quantum yield of this fluorophore is very significantly reduced as the pH is reduced below neutrality. This is caused by the protonation of a negatively charged carboxylic acid group on the fluorescein molecule. Thus, labelling of a protein by a single FITC molecule results in the loss of a positively charged

40 amino group and the introduction of a negative charge at neutral pH, a change in net charge of 2. Therefore, it is important to ensure that extent of labelling is minimized and that the properties of the mildly labelled protein do not differ significantly from those of the unlabelled protein. We have examined the foaming properties, thin film drainage and thickness properties of labelled BSA, ~-lactoglobulin, ot-lactalbumin and ~-casein and have not identified significant alteration in their properties provided the prepared conjugate contains on average less than 1 mole of fluorophore per mole of protein. 4.2. Surface concentration measurements by fluorescence The FRAP apparatus can also be used in a semi-quantitative manner to measure the surface concentration and subsequent competitive displacement of adsorbed labelled species, such as the fluorescent-labelled protein in the adsorbed layer of a/w or o/w thin films [ 10]. This can be achieved by focusing the low power 488 nm beam on the film and detection of the emitted fluorescence using the FRAP photon counting photomultiplier. The detected fluorescence signal is proportional to the amount of adsorbed protein at the interfaces of the thin film provided that the incident laser intensity is kept constant. Calculations have proved that the contributions from non-adsorbed protein molecules in the interlamellar region of the film are negligible [12]. 4.3. FRAP measurements of surface diffusion in surfactant or lipid-stabilized thin films Thin films stabilized by SDS were selected as the test system during the construction and commissioning of our FRAP apparatus [8]. Most measurements were performed on samples containing 1 mole of ODAF per 150 moles of SDS. Results obtained using lower concentrations of ODAF ( > two-fold) confirmed that the data were not influenced by the presence of ODAF. Surface excess measurements were performed using a modification of the method of Weil [35]. The two-fold increase in concentration of ODAF between solution and collected foam showed that it was preferentially associated with the a/w interface although not as effectively as SDS which showed a five-fold increase in concentration. The solution conditions chosen were appropriate for formation of common black films and measurements were undertaken once the films had reached equilibrium thickness. Fluorescence recovery was rapid necessitating use of a very short bleach pulse [8]. The signal-to-noise ratio of individual curves was quite poor and acceptable data curves were only obtained after summation of 10 or more experimental curves. A typical curve is shown in Figure 14(a). An average surface diffusion coefficient of 6.8x10 7 cm2/s was obtained for ODAF in SDS-stabilized films. Two different spot sizes were used to determine whether ODAF mobility was due to lateral diffusion or linear flow in the thin film. These phenomena can be distinguished since the characteristic recovery time is proportional to the increase in the spot diameter under conditions of flow and to the increase in spot diameter squared when diffusion is the dominant process [29]. The results obtained supported the conclusion that the surface molecular mobility observed in these films resulted from diffusion rather than flow. The observed lateral diffusion coefficient was dependent upon the positioning of the laser spot in the film. A 25 % reduction in the diffusion coefficient was observed in the region within 25-50 #m of the periphery of the film. This may result from the presence of thinner regions at the film periphery or other competing processes such as marginal regeneration. Increasing interlamellar viscosity by addition of glycerol reduced the rate of thin film drainage and decreased the lateral diffusion coefficient.

41

Figure 14. Typical FRAP data curves obtained with (a) 2 mM SDS in 2 mM sodium phosphate buffer, pH 7.0 containing 0.1 M NaC1 and 14/zM ODAF; (b) FITC-BSA (0.5 mg/ml) in distilled water, pH 8.0, at an equilibrium film thickness of 83 nm; (c) FITC-BSA (0.2 mg/ml) in 50 mM Na acetate buffer, pH 5.4 at an equilibrium common black film thickness of 14 nm. This study comprised the first reported direct experimental measurement of surface diffusion in air-suspended thin liquid films.

4.4. Surface diffusion measurements in protein-stabilized films The solution diffusion properties ofFITC-labelled BSA were measured by FRAP [12]. The results showed that the protein diffused freely in solution with a diffusion coefficient of approximately 3x10 7 cm2/s. This was in reasonable agreement with previously published values [36]. FRAP measurements were also made on thin films stabilized by FITC-BSA. The films were allowed to drain to equilibrium thickness before measurements were initiated. Thin films covering a range of different thicknesses were studied by careful adjustment of solution conditions. BSA stabilized films that had thicknesses up to 40 nm showed no evidence of surface diffusion as there was no return of fluorescence after the bleach pulse in the recovery part of the FRAP curve (Figure 14(c)). In contrast, experiments performed with thin films that were > 80 nm thick showed partial recovery (55 %) of the prebleach level of fluorescence (Figure 14(b)). This suggested the presence of two classes of protein in the film; one fraction in an environment where it was Unable to diffuse laterally, as seen with the films of thicknesses < 45 nm, and a second fraction that was able to diffuse with a calculated diffusion coefficient of l x l 0 -7 cm2/s. This latter diffusion coefficient was 3 times slower than that

42 observed for FITC-BSA in solution. Care needs to be exercised in the interpretation of these data. Firstly, the slow drainage of the protein films especially once the perimeter of the film reaches black thicknesses suggests that these films contain very little interlamellar liquid. Therefore, it is reasonable to assume that the vast majority of the fluorescence signal from the < 45 nm thick films originates from protein in the adsorbed layer. The complete immobility of the fluorescent-labelled protein in these structures over the timescale of our experiments suggests that diffusion in the interlamellar liquid region is very restricted or highly compartmentalized. Indeed, it is possible that protein molecules bridge between the two interfaces [37]. The partial recovery observed in films > 80 nm thick (Figure 14(b)), is consistent with abolition or a significant reduction in the impediments to diffusion in such films. However, the diffusion coefficient is significantly lower than that observed in aqueous solution. Calculations predict a significant enhancement (several orders of magnitude of concentration ) of protein in the adsorbed layer compared to the interlamellar solution. Therefore, it is necessary to define a mechanism that can account for an increase in protein concentration in the interlamellar space to explain the observed 55 % recovery, whilst impeding protein diffusion compared to bulk solution. One hypothesis involves a low affinity interaction and exchange of protein adsorbed in the secondary layers with that in the interlamellar space. This would be consistent with a previous FRAP result of mobile and immobile fractions of BSA bound at a macroscopic quartz-water interface [38]. In this study, partial recovery was attributed to adsorption/desorption processes in the adsorbed multilayers.

5. CHANGES IN THIN FILM PROPERTIES AS A FUNCTION OF INCORPORATION OF L O W M O L E C U L A R W E I G H T SURFACTANT IN THE ADSORBED PROTEIN LAYER 5.1. Air-water thin films We have predicted that transitions in surface diffusion behavior will be observed under certain conditions in mixed protein/low molecular weight surfactant systems (Figure 1). The behavior of these systems depends on the protein and surfactant type. The effect of protein type may be studied separately by examining the effect of a given surfactant, for example the polysorbate emulsifier, Tween 20 (polyoxyethylene (20) sorbitan monolaurate) on different proteins. This is a non-ionic emulsifier which is water soluble, has a critical micelle concentration of approximately 35 /zM and has a bulky polar headgroup [39]. In our experiments, we have formed films from a range of samples composed of a fixed concentration of the protein of interest but containing increasing levels of surfactant. To facilitate comparison, the data obtained are uniformally presented in terms of the molar ratio of Tween 20 to protein (R). We have been able to categorise the effect of this emulsifier on a range of proteins into three classes of behavior.

5.1.1. Type I: Globular protein with surfactant binding activity. ~-lactoglobulin (~-lg) and Tween 20 is a classic example of a mixed component system that displays Type I behavior [13,22]. A summary of film thickness, surface concentration of FITC-/~-lg and FITC-~3-1g surface diffusion is given in Figure 15. All these data were obtained at a protein concentration of 0.2 mg/ml.

43

Figure 15. A summary of the film thickness (o), surface concentration of FITC-B-lg (x) and FITC-/~-lg surface diffusion (A) as a function of the molar ratio of Tween 20 to protein (R) at the interfaces of a/w thin films. Reproduced from Faraday Discussion 98 with the permission of the Royal Society of Chemistry. Fluorescence measurements reveal that the displacement of FITC-~-lg from the a/w interfaces occurs in several distinct steps, The initial phase of FITC-~-lg displacement begins at R = 0.1 [10]. Paradoxically, this coincideswith an increase in film thickness. Observation of the films reveals the appearance of coexisting regions of two distinct thicknesses in the thin film in the R value range of 0.4 - 0.8 [13,22]. We interpret the pseudo plateau in the displacement data as an indication that no further displacement of protein occurs in certain regions of the film (e.g., in parts of the thicker regions) in this R value range. However, further displacement of FITC-/3-1g is observed in the R value range of 0.8 to 1.0, which shows good correlation with the onset of surface diffusion in this a/w thin film system. Only minor displacement is observed between R = 1.0 and 2.0, which corresponds to the concentration ratio where the surface diffusion coefficient is increasing sharply. Further gradual displacement is observed at higher R values. Major changes in all three measured parameters in Figure 15 occur at R = 0.9 - 1.0. It is significant that this is also the point where instability is first observed in the bulk foam [13,22]. Thus, there is strong evidence that suggests a link between changes in the adsorbed

44 layer structure in the thin films and bulk foam stability in this system. Complete understanding of the behavior of this system is only possible with knowledge of the surfactant binding properties of the protein. We have measured the binding of Tween 20 by ~-lg, and found it to be characterized by a dissociation constant (Kd) for the complex of 4.6 ~M [10]. This has allowed calculation of the relative concentrations of free/3-1g, Tween 20 and ~-lgTween 20 complex present in a given solution of these two components. From the binding data, it is evident that at R = 1, the solution contains effectively equivalent amounts of all three species, free Tween 20,/3-1g and complex [40]. Using these data and further evidence [22,40], we have been able to construct an elaborate mechanism that explains different stages in the breakdown of the adsorbed layer structure in this system, which is shown in Figure 16.

Figure 16. A schematic representation of the change in interactions and composition of the adsorbed layer at the a/w interface in solutions of/~-lg containing increasing levels of Tween 20. Reproduced from reference [40] with the permission of the Royal Society of Chemistry. In this schematic, the B-lg molecules are depicted as jigsaw puzzle pieces, since the proteinprotein interactions in the interface generated by this particular protein are very strong [3]. The experimental evidence is consistent with the complex formed between/3-1g and Tween 20 being unable to interact with B-lg or other molecules of the complex. It is convenient to depict the complex in our schematic model by shielding the protein-protein interaction site on the icon with the hydrophilic polyoxyethylene chain of the Tween 20 molecule. This is

45 reasonable since the complex has been shown to have a much larger hydrodynamic radius than /3-1g alone or the/3-1g/Span 20 complex [22]. Span 20 is sorbitan monolaurate and therefore lacks the polyoxyethylene side chains that are present on the Tween 20 molecule. This inability to interact may explain the film thickening (Figure 15) observed at low R values (0.1 - 0.6), since the complex may become trapped in the adsorbed layers or interlamellar space of the draining film. More importantly, at R > 0.4, the presence of the complex appears to induce loss of multilayers from the film and the appearance of local thin regions (Figure 15). As R reaches 0.9, complex begins to appear in the primary adsorbed layer, breaks the cohesive nature of the adsorbed/3-1g layer and causes the onset of surface diffusion. The sharp increase in surface diffusion coefficient of the FITC-/3-1g/Tween 20 complex is superseded by a more gradual rate of increase at R > 1.3, as the appearance of free Tween 20 in the primary adsorbed layer decreases surface viscosity by diluting the adsorbed complex. Finally, at R > 5, the complex is almost completely displaced from the interface. Several other proteins that bind emulsifiers follow the general trends of this model. For example, the properties of the lipid binding protein from wheat called puroindoline has broadly similar properties [15]. 5.1.2. Type II: Globular protein which does not bind surfactant. Solutions containing mixtures of c~-lactalbumin (o~-la) and Tween 20 are a classic example of a two component system that displays Type II behavior [21]. A summary of foam stability, film thickness and FITC-c~-la surface diffusion is given in Figure 17. Alone, a-la produced less stable foams than/3-1g, and it was necessary to increase the stock protein concentration to 0.5 mg/ml (35.4 #M).

Figure 17. A summary of the bulk foam stability (Fq), equilibrium thin film thickness (o), and FITC-a-la surface diffusion (zX)as a function of molar ratio of Tween 20 to protein (R). The concentration of a-la was 0.5 mg/ml (35.4/zM). Reproduced from reference [41] with the permission of VCH Verlagsgesellschaft.

46 Tween 20 was considerably more effective at reducing the stability of foams of o~-la than was the case with/3-1g. There was a significant decrease in o~-la foam stability in the presence of Tween, at R values as low as 0.05. Minimal foam stability was observed at R = 0.15. There was no observed change in film drainage behavior or onset of surface diffusion in the adsorbed protein layer up to this R value. The only observed change was a progressive decrease in film thickness. Therefore, it is likely that disruption of adsorbed multilayers is responsible for a reduction in the structural integrity of the adsorbed protein layer and that this increases the probability of film rupture. An improvement in foam stability was observed as R was increased to > 0.15 (Figure 17). This was accompanied by the onset of surface diffusion of c~-la in the adsorbed protein layer. This is significantly different compared to our observations with/3-1g, where the onset and increase in surface diffusion was accompanied with a decrease in foam stability. Fluorescence and surface tension measurements confirmed that a-la was still present in the adsorbed layer of the film up to R = 2.5. Thus, the enhancement of foam stability to levels in excess of that observed with o~-la alone supports the presence of a synergistic effect between the protein and surfactant in this mixed system (i.e., the combined effect of the two components exceeds the sum of their individual effects). It is important to note that Tween 20 alone does not form a stable foam at concentrations < 40 ~M [22]. It is possible that o~-la, which is a small protein (Mr = 14,800), is capable of stabilizing thin films by a Marangoni type mechanism [2] once ot-la/o~-la interactions have been broken down by competitive adsorption of Tween 20. A schematic model showing the Tween 20-induced change in the structure of the adsorbed layer of c~-la is shown in Figure 18. In this schematic diagram, the o~-la molecules are depicted as shapes that interact together (Figure 18(a)), but in a much weaker fashion than the /3-1g molecules in Figure 16. This is consistent with the lower interfacial viscosity observed with this protein [3]. In this simpler two component system, competitive adsorption of low levels of Tween 20 (0< R < 0.2), may cause faults to occur in the primary adsorbed layer of protein (Figure 18(b)). One can envisage the presence of large regions or plates of interacting ot-la molecules at the interfaces of the thin film, which are not capable of independent surface diffusion on the timescales of the FRAP experiment. However, the ability of such thin films to withstand thermal or mechanical stretching would be significantly reduced by faults or weaknesses in the adsorbed layer due to the presence of low levels of Tween 20. Incorporation of higher levels of Tween 20 into the adsorbed layer would progressively increase the breakdown of ot-la interactions such that at R = 0.2, surface diffusion of FITC-o~-la is observed (Figure 18(c)). Ultimately, as the concentration of Tween 20 is increased further (R = 2.5), the protein is completely displaced from the interface. 5.1.3. Type III: Random protein that does not bind surfactant. /3-casein (/3-cas) and Tween 20 is an example of a mixed component system that displays Type III behavior [42]. A summary of foam stability, film thickness and FITC-/3-cas surface diffusion is given in Figure 19. All these data were obtained at a/3-cas concentration of 0.5 mg/ml. The foam stability of /3-cas foams progressively decreased with added Tween 20. In contrast, there was a very sharp transition in equilibrium film thickness at R = 0.5. Surprisingly, surface diffusion of/3-cas was not detected at any R value in these films. This was unexpected since it has been reported that adsorbed layers of 13-cas are characterized by a very low surface viscosity [3], signifying that protein-protein interactions in/3-cas films are very weak. We had expected to observe surface diffusion either in the films stabilized by

47

Figure 18. A schematic representation of the change in interactions and composition of the adsorbed layer at the a/w interface in solutions of c~-la containing increasing levels of Tween 20. protein alone or in the presence of low quantities of Tween 20. The data suggest an alternative mechanism of destabilization operates which involves phase separation of the/3-cas and Tween 20 in the adsorbed layer. A very speculative schematic representation of this mechanism is shown in Figure 20. Evidence in support of this model comes from observations of the coexistence of two regions of differing thickness in the thin films at R = 0.5 which coincided with the observed transition in film thickness. Photographs of thin films depicting this condition are shown in Figure 21.

48

Figure 19. The foam stability, film thickness and surface diffusion of adsorbed ~-cas as a function of the concentration of added Tween 20 in a/w thin films. The ~-cas concentration was held constant at 0.2 mg/ml (8.33 /~M). Reproduced from reference [41] with the permission of VCH Verlagsgesellschaft.

Figure 20. A schematic representation of the change in interactions and composition of the adsorbed layer at the a/w interface of solutions of/3-cas containing increasing levels of Tween 20.

49 Fluorescence measurements revealed that the concentration of adsorbed protein was much reduced in the thinner regions, but high in the thicker regions. There was only sufficient protein adsorbed to allow FRAP measurements to be made in the thicker regions of the film. The results showed that the protein present in the thicker region was immobile. The absence of significant fluorescence from the thinner regions of the film suggested that these regions contained very little protein. However, /3-cas must be able to influence the interface in these regions since the stability of the foams was still minimal even at high concentrations of Tween 20. If the protein had been totally displaced the concentration of Tween 20 alone should have been sufficient to stabilize the foam.

Figure 21. Photographs of the drainage behavior and coexistence phenomena in thin films formed from solutions of/3-cas and Tween 20 of composition R = 0.5. (a) Early stages of drainage of the thin film showing protein-like (concentric rings) and surfactant-like (distortions) features; (b) A sample showing a few dark (thin) regions in a predominantly gray film; (c) A sample showing light (thick) regions in a predominantly dark film. In summary, three different types of emulsifier-induced transitions in thin film behavior have been observed. The mechanisms of destabilization depend on the strength of proteinprotein interactions in the adsorbed layer. The stronger the interactions, the more emulsifier is needed to destabilize the thin film. Knowledge of the mechanism of destabilization allows the formulation of scientific strategies for control of stability. Preliminary results have shown that enhancing the interactions in the adsorbed layer through the addition of natural crosslinking agents is a promising approach [43]. Alternatively, introduction of a component capable of selective binding of the low molecular weight destabilizing agent (e.g., lipid) is another possibility [15,44]. 5.2 Oil-water thin films We have used our thin film techniques to compare the behavior of the protein adsorbed layers of a/w and o/w thin films [10,45]. The results revealed significant differences between these two related systems. Data from film thickness, FRAP and surface concentration

50 measurements from o/w thin films stabilized by mixtures of ~-lg and Tween 20 are presented in Figure 22.

Figure 22. A summary of the film thickness (o), surface concentration of FITC-/~-lg (x) and FITC-~-lg surface diffusion (zX) as a function of the molar ratio (R) of Tween 20 to protein at the interfaces of o/w thin films. Reproduced from Faraday Discussion 98 with the permission of the Royal Society of Chemistry. These data can be compared with those for a/w films shown in Figure 15. Such comparison suggests that there is substantially less protein at the interface in o/w thin films, indeed almost five times less. However, care needs to be exercised when equating surface concentration to fluorescence intensity. It is possible that the fluorophore is located in different environments in the two types of thin film and that the difference in fluorescence intensity is a fluorescence quantum yield effect. However, this is unlikely since the surface concentration, as judged by the surface fluorescence signal at which surface diffusion is first observed, in both a/w and o/w films is very similar at approximately 600 counts per channel. It is reasonable to assume that the structure of the adsorbed layer is similar at the point where surface diffusion is first observed. The presence of similar surface counts indicates that the quantum yield of fluorescence is similar at both o/w and a/w interfaces. Thus, this strongly supports the

51 presence of multilayer structures in the adsorbed layers of/3-1g in a/w thin films. These multilayers need to be removed by competitive adsorption of Tween 20 before surface diffusion is observed. However, in the case of the o/w thin films, the surface concentration data confirms that these films are initially comprised of an adsorbed monolayer. Displacement of the protein from the adsorbed layer in o/w thin films shows very different behavior from its a/w counterpart. Although displacement of protein from the o/w interfaces initiates at approximately the same solution composition (i.e., R = 0.1), there i s little evidence for the stepwise displacement observed in the a/w thin films. This observation is further confirmation of the monolayer versus multilayer structure at the o/w and a/w thin films. The displacement of/3-1g has also been investigated in oil-in-water emulsions of ntetradecane [46,47]. In these reports it was shown that the protein was not completely displaced until R = 10, which was considerably higher than R = 1 - 2 in Figure 22. This will be discussed further below. The onset of surface diffusion of adsorbed FITC-/3-1g in both a/w and o/w film coincides with the initiation of displacement of protein from the monolayer (or primary adsorbed layer). However, the R value at which this occurs is different for the a/w and o/w systems. The origin of this difference is not clear, particularly if the onset of surface diffusion is explained by the adsorption of complex as is the case with the a/w films (Figure 16). The experimental results shown in Figure 22 were obtained from thin films prepared by adsorption from aqueous solutions containing 0.2 mg/ml/3-1g and appropriate concentrations of Tween 20, to captive oil droplets (as in Figure 5). Under these solution conditions, only 6 % of the/3-1g was in the complexed form rather than the 50% present at the point where surface diffusion is first observed in the a/w thin films. It is possible that this small amount of complex is sufficient to disrupt the monolayer in the o/w thin film allowing surface diffusion of the remaining adsorbed protein. An alternative explanation involves the emulsifier (/3-1g and Tween 20) concentration to interfacial area ratio. Our o/w thin film experiments involved a protein concentration of 0.2 mg/ml and an interfacial area of approximately 6x10 -5 m 2. This amounts to a protein load per unit area 200x greater than used in previous studies of emulsions of these two components [46,47]. In the latter, complete displacement of/3-1g required the presence of a 10-fold higher Tween 20 concentration than reported in our thin film experiments. Thus a considerably larger fraction of the total protein present was adsorbed in the emulsion experiments. Therefore, at an equivalent R value there was more Tween 20 present in the thin film system relative to the amount of adsorbed protein, than in the emulsion. This could explain the displacement of/3-1g at lower R values in the thin film experiments. We have tested this hypothesis in some recent o/w thin film experiments [45]. It was not practical to reduce the protein load per unit area of interface to that found in the emulsion experiments, since the very low concentrations required would have been very slow to reach equilibrium adsorption. We circumvented this problem in a unique way. Rather than adsorb emulsifier mixtures from aqueous solution, we formed the oil droplets and the thin film in a preformed emulsion. Therefore, the adsorbed layers on the captive droplets formed by adsorption of surfactant from the continuous phase of the emulsion. The results are shown in Figure 23, where surface diffusion data of FITC-/3-1g in o/w and a/w thin films as a function of added Tween 20 are summarised.

52

Figure 23. The lateral diffusion coefficient of adsorbed FITC-/3-1g in thin films as a function of added Tween 20. ( 9 o/w thin films formed from aqueous non-homogenized solutions of /3-1g at 3 mg/ml; (s), o/w thin films formed from 10% v/v n-tetradecane emulsion or emulsion subnatant samples of FITC-/3-1g, initial protein concentration 3 mg/ml; (.), a/w thin films formed from aqueous non-homogenized solutions of 13-1g at 0.2 mg/ml. The diffusion behavior of the protein in the o/w thin films formed from emulsified samples was completely different from that observed from non-homogenized samples used to form o/w or a/w thin films, since the onset of diffusion occurred at R = 4. This was in closer agreement with previous reports [46,47] since some displacement of/3-1g by Tween 20 has been reported to occur at R = 4 but not R = 0.1. However, again the transition point at R = 4 does not comply with our destabilizati0n model shown in Figure 16. It is now evident that at least part of the shift to R = 4 is explained by a shear-induced conformational change in/3-1g. This complicates matters further by introducing a second class of free protein into the solution with altered Tween 20 binding capacity. Nevertheless, the results are beginning to converge towards equivalent solution compositions being required to induce surface diffusion in both o/w and a/w systems.

6. THE RELATIONSHIP OF FRAP MEASUREMENTS OF SURFACE DIFFUSION AND OTHER SURFACE R H E O L O G I C A L MEASUREMENTS The FRAP data described above report molecular self diffusion in the adsorbed layers at the interfaces of thin films. The measurements are sensitive to the strength of interactions

53 between the molecules at the interface. Once the strong protein-protein interactions have been weakened or destroyed by competitive adsorption of low molecular weight surfactant, surface diffusion ensues. The magnitude of the measured diffusion coefficient of adsorbed protein will be dependent upon the density of molecular packing at the interface, since this will influence the surface viscosity.

Figure 24. A comparison of the data obtained from a range ot surface rheological measurements of samples of/3-1g as a function of Tween 20 concentration. (R), The surface diffusion coefficient of FITC-/3-1g (0.2 mg/ml) at the interfaces of a/w thin films; (X), the surface shear viscosity of/3-1g (0.01 mg/ml) at the o/w interface after 5 hours adsorption; (o), the surface dilational elasticity and (o) the dilational loss modulus of/3-1g (0.2 mg/ml). It was of interest to compare the results obtained with the FRAP technique with those obtained with classical surface rheological techniques. Our detailed knowledge of properties of solutions of/3-1g containing Tween 20 made this an ideal system on which to compare the methods. Firstly, surface shear viscosity measurements were performed on the Tween 20//3-1g system [47] using a Couette-type torsion-wire surface rheometer as described previously [3,48]. All the experiments were carried out at a macroscopic n-tetradecane-water interface at a fixed protein concentration of 0.01mg/ml. In the absence of Tween 20, the surface shear

54 viscosity of the adsorbed interfacial film of/3-1g rapidly increased to over 500 mN.m 1 during the first hour of adsorption, followed by a more gradual increase to 720 mN.m -1 after 20 hours. Samples containing Tween 20 in the concentration range between R = 0 to 1, showed a time dependent increase in surface shear viscosity, but values obtained at a given time were always significantly lower than that observed with/3-1g alone. The surface shear viscosity data obtained with samples of/3-1g containing Tween 20, 5 hours after formation of the o/w interface are shown in Figure 24. The data show that increasing the concentration of Tween 20 caused a progressive drop in the observed surface shear viscosity. At R = 1, the surface shear viscosity was too low to measure accurately, without using a much finer torsion wire. There is a clear complementarity between the surface shear viscosity and FRAP measurements; the former is sensitive when the surface viscosity is high and molecular diffusion is zero due to protein-protein interactions, the latter is sensitive when the surface viscosity is very low due to the abolition of interactions. The surface rheological properties of the/3-1g/Tween 20 system at the macroscopic a/w interface were examined by a third method, namely surface dilation [40]. Sample data obtained are presented in Figure 24. The surface dilational modulus, (E) of a liquid is the ratio between the small change in surface tension (AT) and the small change in surface area (AlnA). The surface dilational modulus is a complex quantity. The real part of the modulus is the storage modulus, e' (often referred to as the surface dilational elasticity ,Ea). The imaginary part is the loss modulus, E", which is related to the product of the surface dilational viscosity and the radial frequency (~Tao~). Experiments with the/3-1g/Tween 20 system were performed at a macroscopic a/w interface at a/3-1g concentration of 0.2 mg/ml [40]. The data obtained relate to the properties of the interface 20 minutes after formation. Up to R = 1, the storage modulus (dilational elasticity) was large and relatively constant, whereas the loss modulus (dilational viscosity) increased with increasing R. As R was increased to higher values there was a marked decrease in the storage modulus (dilational elasticity) and a gradual increase in the loss modulus (dilational viscosity). In summary, the data show the presence of a transition in surface dilational behavior in this system at a solution composition of approximately R = 1. At this point, there is a transformation in the adsorbed layer properties from elastic to viscous. The results of these studies show that all three surface rheological methods give results that correlate with each other. In addition, the results add further evidence in support of our model for Tween 20-induced changes in adsorbed layers of/3-1g (Figure 16). The three surface rheological techniques described here are very complementary, each providing different but related data and providing sensitivity across different ranges of interfacial layer stiffness. However, it is important to note that only the FRAP method can be applied to both macroscopic and thin film samples.

7.

FUTURE PROSPECTS

The progress made using the methods described in this report has opened up a number of opportunities. There are consumer and health pressures to reduce the consumption of synthetic emulsifiers used in processed foods. Therefore, a need exists to identify alternative 'natural' replacement emulsifiers. One approach is to develop 'natural', biodegradable emulsifiers through biosynthetic routes using enzymes. Alternatively, more widespread use of proteins as emulsifiers would be an option if their functional properties were more

55 predictable. We have identified a number of mechanisms of destabilization of proteinstabilized foams involving competitive adsorption of surfactant and the breakdown of proteinprotein interactions in the adsorbed layer. The knowledge that this approach allows development of scientific strategies for controlling destabilization in protein-stabilized systems. Currently, we are examining two different approaches, The first involves inclusion or exploitation of existing natural ingredients in the food system of interest, which are capable of enhancing interactions in the adsorbed layer by crosslinking. It has proved possible to test candidate molecules for this role using one of our characterized systems (e.g., /3-1g/Tween 20). Preliminary studies with this system have identified that the beer foam stabilizing activity of the iso-a-acid fraction in hop extract operates through a protein crosslinking mechanism [43]. Our knowledge of the mechanisms of destabilization in foams allows strategic targeting of a number of other natural compounds (e.g., divalent ions, bifunctional acids e.g., tartaric acid, phenolics and mixtures of polysaccharides or proteins). The second approach is most effective when the destabilizing agent is only present at low concentrations (e.g., egg yolk lipid in meringue). Here, removal of the destabilizing component by selective binding has potential. Preliminary work has demonstrated the effectiveness of exploiting the lipid binding properties of the protein, puroindoline from wheat flour [15], for selective removal of destabilizing components (e.g., lipid) from model and real beverage systems [44]. However, the effectiveness of this protein is not explained by binding alone and needs further study [15]. Our understanding of the influence of competitive adsorption on emulsion stability is less secure. Recent work has identified several marked differences between the adsorbed layer properties at air/water and oil/water interfaces (e.g., multilayer versus monolayer formation). Advancing our knowledge of the stabilization of emulsions by protein merits further investigation, since emulsions comprise a major sector of processed foods. If competitive adsorption of surfactants influences the stability of protein emulsions in a similar manner to foams, use of the strategies outlined above may be appropriate for controlling destabilization. If we are successful, food processors specialising in the preparation of food dispersions (e.g., foaming and sparkling beverages, salad dressings, sauces, ice cream etc) will benefit from the results of this research. The work provides the underpinning knowledge that will allow food ingredient manufacturers to supply 'natural' emulsifier proteins and functionality enhancing components to meet the legislative demands for food ingredients in the future, whilst satisfying consumer demands for the elimination or reduction of use of synthetic additives in foods.

ACKNOWLEDGEMENTS The author would like to acknowledge the involvement of Alan Mackie, Peter Purdy and Dr. Andrew Pinder in the design, construction and continued development of the FRAP apparatus. The experimental results described in this paper were obtained in collaboration with Mark Coke, Peter Wilde and David Wilson. The author wishes to thank AM and PW for discussions relating to the text and PW for his assistance in preparing the figures. This work was funded by the AFRC.

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57 29. D. Axelrod, in 'Spectroscopy and Dynamics of Molecular Biological Systems', P.M. Bayley and R.E. Dale (eds.), Academic Press, London, 1985, p. 163. 30. J. Yguerabide, J. Aschmidt and E.E. Yguerabide, Biophys. J., 39 (1982) 69. 31. R.P. Haugland, in 'Handbook of Fluorescence Probes and Research Chemicals', Molecular Probes Inc. 1992. 32. G.E. Means and R.E. Feeney in 'Chemical modification of Proteins' Holden Day, San Francisco, 1971. 33. I.S.K. Craig, P.J. Wilde and D.C. Clark, Coll. Surf. B:Biointerfaces, in press. 34. H.F. Swaisgood, in Developments in 'Dairy Chemistry - 1', P.F.Fox (ed.), Applied Science Publishers, London, 1982, p. 1. 35. I. Weil, J. Phys. Chem., 70 (1966) 133. 36. G. Barisas and M.L. Leuther, Biophys. Chem., 10 (1979) 221. 37. O.D. Velev, A.D. Nikolov, N.D. Denkov, G. Doxastakis, V. Kiosseoglu and G. Stanlidis, Food Hydrocolloids, 7 (1993) 55. 38. T.P. Burghardt and D. Axelrod, Biophys. J., 33 (1981)455. 39. D.C. Clark, Encyclopaedia of Food Science, Food Technology and Nutrition, Academic Press, 1993, p. 1577. 40. D.C. Clark, P.J. Wilde, D. Bergink-Martens, A. Kokelaar and A. Prins in 'Food Colloids and Polymers: Structure and Dynamics', E. Dickinson and P. Walstra (eds.), Royal Society of Chemistry Special Publication No. 113, Cambridge, 1993, p. 354. 41. D.C. Clark, A.R. Mackie, P.J. Wilde and D.R. Wilson, in 'Food Proteins Structure and Functionality', K.D. Schwenke and R. Mothes (eds.), 1993, p. 263. 42. D.R. Wilson, P.J. Wilde and D.C. Clark in 'Food Colloids and Polymers: Structure and Dynamics', E. Dickinson and P. Walstra (eds.), Royal Society of Chemistry Special Publication No. 113, Cambridge, 1993, p. 415. 43. D.C. Clark, P.J. Wilde and D.R. Wilson, J. Inst. Brew., 97 (1991) 169. 44. D.C. Clark, P.J. Wilde and D. Marion, J. Inst. Brew., in press 1993. 45. A.R. Mackie, P.J. Wilde, D.R. Wilson and D.C. Clark, Royal Chem. Soc. Faraday Trans. 89 (1993) 2755. 46. J-L. Courthaudon, E. Dickinson, Y. Matsumura and A. Williams, Food Struct., 10 (1991) 109. 47. J-L. Courthaudon, E. Dickinson, Y. Matsumura and D.C. Clark, Coll. Surf., 56 (1991) 293. 48. E. Dickinson, B.S. Murray and G. Stainsby, J. Colloid Interf. Sci., 106 (1985) 259.

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Characterization of Food: Emerging Methods A.G. Gaonkar (Editor) 9 1995 Elsevier Science B.V. All rights reserved.

59

Chapter 3 M e t h o d s for characterization of structure in whippable d a i r y - b a s e d emulsions Niels M. Barfod Grindsted Products 38, E d w i n Rahrsvej DK-8220 Brabrand Denmark

1.

INTRODUCTION

This chapter describes some methods to study physical characteristics and ingredient interactions in whippable dairy-based emulsions. The story of whippable emulsions begins with natural dairy cream. From this starting point a range of dairy-type whippable emulsions has been developed over the years. In unhomogenized dairy cream the natural phospholipids contribute to the whipping properties of the cream. However, after homogenization the particle size of the fat globules decreases, and the total fat surface area increases. This means that the interracial concentration of polar lipids decreases because milk serum proteins adsorb at the newly formed interfaces, and the whipping properties are lost. Consequently, additional polar lipids or emulsifiers are needed to obtain good whipping properties in most industrially manufactured products. This chapter will deal with the following types of whippable emulsion: 9 9 9 9

Whipped topping Natural and imitation whipping cream Ice cream Aerated desserts

The formulations of these products vary greatly, and therefore only principal and important aspects of product stability and functional properties will be discussed. Most studies have dealt with ice cream, because commercially this product is the most important whippable emulsion. Thus, methods for characterization of ice cream will be highlighted more than other types of whippable emulsions. There is a fundamental problem which must be solved when dealing with whippable emulsions. Before use the emulsion must be sufficiently stable. On the other hand, it must b e p o s s i b l e to destabilize the emulsion by mechanical treatment combined with air incorporation (whipping, air pressure, cooling, freezing). The partly destabilized fat globules in the whipped emulsion are important for the stability of the foam structure. There is a

60 delicate balance between emulsion stability and instability. If the emulsion is too stable, it will not whip, if it is not stable enough, the foam formed will collapse after a short whipping time. The ingredient composition and manufacturing process are important for the different types of whippable emulsions. In many industrially produced whippable emulsions, functional ingredients, such as food emulsifiers and hydrocolloids are used to improve functionality and product stability.

Product description of whippable emulsions Toppings are spray-dried emulsions made from sodium caseinate, vegetable fat such as palm kernel or coconut fat, and emulsifiers with low polarity, such as acylated (acetylated or lactylated) monoglycerides or propylene glycerol monostearate. Whipping cream (30-40% butterfat) can be made from natural cream, but its whipping properties may be improved by changing the manufacturing process or by using additional milk protein fractions and/or food emulsifiers. Whipping cream with reduced fat content (25 % or less tat) can be made by incorporating food emulsifiers and hydrocolloids ~. Imitation whipping creams are made from skimmed milk powder or sodium caseinate, vegetable fats, and emulsifiers as described above for toppings. More polar emulsifiers in low dosage may be incorporated to ensure storage stability. Sodium alginate may be used to prevent syneresis of the foam after whipping 2. In general, the emulsifier dosage in imitation whipping cream is 10 tilnes less than that in toppings. Ice cream is made from skimmed milk, condensed skimmed milk or skimmed milk powder in combination, and dairy cream, butter or butter oil. In some countries vegetable fat is used to replace dairy fat. Usually, monoglycerides or mono-diglycerides are used, but other more polar emulsifiers can also be used. The emulsifier dosage is similar to that used in imitation cream. Ice cream also contains sugar and hydrocolloids, which mainly influence the freezing behaviour of the ice cream mix. Aerated desserts are products with a stabilized foam structure based on dairy ingredients or dairy analogues. They may be based on neutral, acidified (yogurt-type) or concentrated milk, and are typically low in tat ( < 10%) and high in sugar (8 to 15 %). The foam structure may be stabilized by selected emulsifiers/hydrocolloids for different products and different manufacturing processes 2.3. The main difference between aerated dessert products and other whippable emulsions is the gelation of the continuous water phase. The most common hydrocolloids used for this purpose are gelatine, alginate and carrageenan. Aerated desserts may be whipped in continuous aerators (cold-stored products) or in ice cream freezers (frozen products). 1.1.

General mechanisms for stabilizaiion of whippable emulsions The subject of this chapter is whippable emulsions, and some background theory on foams may be appropriate. To produce a foam, stable or metastable, it is necessary for surface-active molecules 1.2.

61 such as emulsifiers or proteins to be adsorbed at the gas-liquid interface of the air bubbles to build a stabilizing film. The hydrophobic residues of the molecules will be attracted towards the air phase and the hydrophilic residues will be attracted towards the water phase. The main factor determining the stability of such foams is the rate and extent of drainage from the thin liquid film. In general, this type of foam is relatively unstable. The stability may be enhanced by increasing the viscosity of the liquid by increasing the dry matter content or adding certain hydrocolloids. The foam stability may also be enhanced with hydrocolloids, in particular microcrystalline cellulose. In addition to surface-active molecules, the foam of whippable emulsions contains particles in the form of tat globules trapped in the continuous phase. During whipping, fat globules penetrate and partially replace the protein fihn at the air-water interface 4. The foam stability is affected by the degree of aggregation of fat globules in the vicinity of the air-water interface. The tat composition, and in particular its crystallization behaviour, exerts a dominant influence on the quality of whippable emulsions. Adsorption of fat globules to the air bubbles depends on the hydrophobicity of the fat globules. The hydrophobicity depends on the amount of protein bound on the surface of the fat globules. In general, proteins act as emulsion stabilizers whereas certain food emulsifiers induce controlled emulsion destabilization during whipping. Later in this chapter it will be shown how emulsifiers, such as polar lipids, control protein binding to the surface of fat globules and thus the aggregation and whipping properties of these products. Hydrocolloids may be used to increase viscosity and inhibit syneresis of the foam and gel the water phase in whippable emulsions. In frozen systems such as ice cream, hydrocolloids have the additional effect of inhibiting the growth of ice crystals thus enhancing foam stability and improving texture. 1.3.

Methods for characterization of whippable emulsions In the food industry a range of practical or descriptive tests are used to evaluate product quality and the stability of whippable emulsions. Using such methods a number of reliable and commercially valuable whippable emulsions have been developed over the years. To develop new whippable emulsion systems which are more difficult to stabilize, i.e. primarily low-fat products, lnore advanced physical methods have been used to elucidate the fundamental mechanisms behind the behaviour of whippable emulsions. In this chapter the physical methods for analyzing whippable emulsions are divided into analyses of 1) the emulsified fat phase 2) the fat-water interface and air-water interface, and 3) the continuous water phase. The descriptive tests are mentioned at the end of the chapter as it is easier to explain the meaning of these tests after the fundamental mechanisms have been described.

2.

EMULSIFIED FAT PHASE

As already mentioned above, the flmctional properties of whippable emulsions depend largely on the properties of the tat globules they contain. The fat globules form the skeleton of the foam. The crystallization behaviour inside the fat globules of whippable emulsions is decisive for the stabilization of the foam structure after aeration. It is a well-known fact in the food industry that whippable emulsions made with liquid fats are totally devoid of functionality.

62 The quality of the fat crystallization in whippable emulsions is important, e.g. crystallization rate, and shape, form and size of the fat crystals formed. In some creams, needle-shaped crystals at the oil-water interface have been shown to be associated with partial coalescence resulting in defects in whipping properties s,6. The fat phase in many oil-water emulsions is in a supercooled state, since nucleation followed by crystal growth is greatly reduced if fat is present in a large number of isolated droplets with small particle size7. Nucleation may be enhanced with emulsifiers present in the fat phase by increasing the number of nucleation centres, resulting in the formation of many small fat crystals. This will result in improved functional properties. The various aspects of the importance of fat crystallization with regard to the functional properties are beyond the scope of this chapter, but methods to analyze this important phenomenon in whippable emulsions are described below. 2.1.

Thermal analyses Differential Thermal Analysis (DTA) and Differential Scanning Calorimetry (DSC) are techniques used to measure the heat changes which occur in a small sample (1 to 30 mg) subjected to heating or cooling at a known linear rate (typically 1 to 30~ per minute) 8. One example of this type of analysis is described. Ice cream emulsions (mixes) are normally subjected to a cooling period of several hours at 0 to 5~ before freezing and whipping. During this treatment several physical changes take place 9. These changes are described later in this chapter. One change is the crystallization of tat globules in the mix, which can be followed by DSC analysis as shown in Figure 1.

Figure 1 Melting enthalpy of bulk fat and emt, lsified fat of ice cream mix with (+E) and without (-E) emulsifier alter cooling at 5~ measured by DSC.

The melting of crystalline tat took place between 15~ and 40~ and was analyzed at a heating rate of 10~ ~~ In non-emulsified state fat crystallizes very quickly. However, in emulsified state a reduced crystallization rate is observed. In the presence of emulsifier (saturated mono-diglyceride) the crystallization rate is enhanced, and the supercooling is reduced. The melting enthalpy in Figure 1 is expressed per gram of fat in the sample analyzed.

63 Solid Fat Content (SFC) by Low Resolution NMR (wide-line or pulse) Analysis of solid tat content by NMR has replaced older techniques such as dilatometry ll. The material may be studied in an equilibrium without melting. The analytical time is less than l0 seconds using NMR in contrast to more than 15 minutes using DSC, and the amount of sample material is about 100 times higher when using NMR than when using DSC. This is important if the sample material is not completely homogenous. One drawback with NMR is that the liquid signal from water in o/w emulsions has to be subtracted to obtain the true SFC. This can be done by analyzing emulsions and fat blends with no tendency to supercooling and making calibration curves. Another possibility is to measure reference samples without fat and calculate the true SFC by subtracting the signal from the water and water-soluble components 9. 2.2.

Figure 2 shows the same experiment as in Figure 1, but analyzed by NMR. The same information is obtained, but the NMR method is easier and quicker to use.

Figure 2 Fat crystallization of bulk fat and emulsified fat of ice cream mix with (+E) and without (-E) elnulsifiers after cooling to 5~ measured by pNMR.

The NMR method may also be used to study supercooling phenomena in spray-dried topping emulsions 12. Figure 3 shows that the %solids content is lower in topping powders than in the corresponding simple dry mixtures. With effective emulsifiers (propylene glycol monostearate (PGMS)) supercooling is slightly reduced, and with ineffective emulsifiers (glycerol monostearate (GMS)) hardly any supercooling takes place. Supercooling increases with increased protein (sodium caseinate) content due to both reduction in fat particle size and

64 to increased lipid-protein interaction. Sodium caseinate with 3 % peptide bonds hydrolysed results in hardly any supercooling, but a similar fat particle size as intact sodium caseinate. Other globular food proteins tested have been found to be less effective than sodium caseinate. The lipid-protein interaction is specific to high lauric fats such as hardened coconut oil or palm kernel oil, and is not evident with other fats such as partially hydrogenated soybean oil, fish oil or normal butterfat ~3.

Figure 3 Per cent solids of topping powders and corresponding dry mixtures measured by pNMR at temperatures from 5~ to 25~ Reprinted from reference 12, courtesy of the American Oil Chemists' Society.

Crystallization of supercooled fat in topping powders may be studied by NMR afterreconstitution in heavy water. Below room temperature spontaneous fat crystallization takes place under isothermal conditions in the presence of effective emulsifier (PGMS) but not with ineffective emulsifiers or without emulsifiers (Figure 4).

65

Figure 4 Crystallization of supercooled fat at 15~ measured by pNMR in the absence or presence of emulsifiers (PGMS or GMS).

The time scale of tat crystallization is much shorter for topping powders than for ice cream mix as presented in Figure 2. This is due to the much higher emulsifier content in topping powder. The induction of tat crystallization in whippable emulsion systems is due to interfacial protein desorption from the tat globules of the emulsion mediated by the emulsifiers. This phenomenon is described in section 3.1. Other methods to study fat crystallization in whippable emulsions may be used, e.g., a recently developed technique using ultrasonic velocity ~4. 2.3.

X-ray diffraction This technique is particularly suited for studying polymorphism of fats and ordered structures of emulsifier in water, e.g., liquid crystals or 'gel' phases. The concentration of emulsifiers in food emulsions is often too low to allow the formation of multi-layered liquid crystals at oil-water interfaces ~s. In systems in which the emulsifier concentration is sufficiently high, such as toppings, the formation of 'gel' phases appears to play a role. Studies of topping tat phases by X-ray diffraction analysis show that the triglycerides from hydrogenated coconut oil do not co-crystallize with the PGMS emulsifier added 16. The coconut fat crystallizes in a beta-prime form with long spacings of 36 A, whereas the emulsifier crystallizes in alpha-form with long spacings of 49 A. After contact with water at 5~ the long spacings of emulsifier in the tat phase increases from 49 A in the bulk phase to 56 A in the interfacial phase. This increase of 7 A can only be due to the penetration of water into the polar regions of the emulsifier caused by the so-called hydration force ~7. Water absorption into the tat phase of the topping results in interfacial protein

66 desorption, and with regard to crystallization results in a more stable foam structure. For further details see reference 16. A model of the emulsifer-water gel structure formed near the oil-water interface of the emulsion is shown in Figure 5. Similar results on toppings have been presented by Westerbeek and Prins l~.

Figure 5 A schematic model of the formation of lipid gel phase by hydration of the polar groups in crystalline regions of emulsifiers, d = interplanar Bragg spacing; d~ = thickness of lipid bilayer; dw = thickness of water layer. Redrawn from reference 15, courtesy of Marcel Dekker Inc.

In a standard topping formulation 10 to 20% of the emulsifier in the fat phase is used to produce the desired foam stability and overrun after whipping. This is due to protein desorption and tat crystallization during whipping in cold water. Practical tests have shown that low-fat topping powders (down to 80% fat reduction) may be produced by a concomitant increase in emulsifier concentration (up to 50%) in the fat phase 19. A higher concentration of emulsifier tacilitates enhanced formation of alpha crystalline gel structure, which is obviously important for the whipping properties and foam texture of low-tat topping products. 2.4.

Electron microscopy (EM) Various electron microscopy techniques have been used to study the structures of whippable emulsions such as normal and cryo-scanning electron microscopy or transmission electron microscopy using various preparation methods such as freeze fracturing, freeze etching, etc. The literature is quite extensive, and only a few important papers will be discussed in this chapter. EM studies of whipped cream show that the air bubbles are completely surrounded by a layer of tat globules which protrude partially into the air bubbles. These parts of the fat globules no longer have their original membrane layer, but exhibit surface layers of crystallized tats. The fat globules adsorbed around the air bubbles are bonded together with

67 coalescent tat. The cross-linking of fat globules adsorbed to the adjacent air bubbles by chains of coalescent globules establishes a stabilizing infrastructure in the foam 4'2~ Although the lipid phase in powdered toppings is finely dispersed in the form of small globules (< 1 /zm), strong destabilization takes place after reconstitution in cold water, resulting in platelet-like crystal agglomerates (Figures 6 and 7).

Figure 6 a. Structure of topping powder in the dry state with close-packed globular fat particles (f) (diameter less than 0.5 txm). b. Crystallization and transformation of the globular structure of fat particles into thin layers of crystal platelets (c). Reprinted from reference 21, courtesy of Verlag Th. Mann.

During whipping these crystalline lipid platelets accumulate at the air-water interface of the air bubbles and also form bridges between them 22'23. The structure of whipped topping is thus completely different from that of whipped dairy or liquid imitation creams. In the latter systems the air bubbles appear to be covered in a monolayer of fat globules, which are rarely deformed and which protrude with a substantial part of their volume into the air phase of the bubbles. If large fat crystals are present, they are considered detrimental to foam stability, in contrast to whipped toppings 6 (Figure 7).

68

Figure 7 Left: Surface of air bubbles (a) in whipped topping emulsion is completely covered with a thick layer of plate-shaped tat crystals (c). w = water phase. Right: Surface of air bubbles (a) of whipped imitation cream is covered with a monolayer of only slightly destabilized globules (f). Reprinted from reference 22, courtesy of Scanning Microscopy International.

The flocculated fat globules of whipped cream contain fewer contact points, and the foam is therefore not as stiff as in toppings in which the aggregated crystal platelets have a large surface area, many contact points and thus increased stiffness. This means that an acceptable topping foam may be tbrmed at a much lower tat content than is the case with liquid whipping creams. Ice cream is a partially frozen fat-stabilized foam ~. The interaction of the fat globules (0.5 to 1.0/~m) of the ice cream mix and the air bubbles in the finished product depends to a great extent on the presence of emulsifiers such as mono-diglycerides. In the absence of emulsifiers, micellar casein adsorbs strongly to the dispersed fat phase, thus preventing adsorption of tat globules to the air bubbles. In the presence of emulsifiers, interfacial protein layers are desorbed during cold storage (ageing), and during whipping and freezing in the ice cream machine, thereby facilitating the binding of partially crystalline fat globules to air 23. This air bubble stabilization is important for the physical properties of this product (Figure 8).

69

Figure 8 Air bubbles in ice cream (a). Interface (arrows) between a large air bubble (A) and water phase (W) in an ice cream sample without emulsifier. There is very little adsorption of fat globules to the air-water interface, which is stabilized by a thin protein film only. (b) Corresponding structures in an ice cream with emulsifier (saturated lnono-diglycerides). Fat globules interact strongly with the air-water interface. Reprinted from reference 23, p 242, courtesy of Marcel Dekker Inc.

2.5.

Particle size distribution The characterization of emulsions by particle size distribution analysis has been facilitated in recent years by a range of new instruments. Most of these instruments employ laser light diffraction principles, and have replaced older spectrophotometric methods. To obtain optimal functional properties, the range of average fat particle size of most dairy type whippable emulsions is from about 0.5/xm to 1.0/xm. If the average fat particle size increases, the emulsion will not be stable in the long term and the whipping properties will not be as good. This is most critical for systems which are to be stored for a long time before whipping, e.g., UHT-treated imitation cream. Particle size analysis may also be employed to detect the degree of fat globule agglomeration. In liquid imitation whipping crealn weak agglomeration of fat globules before whipping is beneficial for the whipping properties. The degree of tat globule agglomeration,

70 as well as the size and mechanical stability of the aggregates formed after whipping the emulsions, may be estimated by particle size distribution analysis as shown in Figure 9. The Figure shows analysis of frozen ice cream melted at 0~ to 5~ using a Malvern 2600 Particle Sizer.

Figure 9 Particle size distribution analysis of air bubbles stabilized with aggregated fat globules in ice cream atier thawing and dilution 100 x with water at 0~ to 5~ Effect of emulsifier concentration (MDG = saturated mono-diglyceride) on air bubble size and stability. Upper row: Samples analyzed without degassing. Lower row: Samples analyzed after degassing for 5 minutes.

No stable aggregates form in the absence of emulsifiers. The agglomeration of fat globules can be observed in tile presence of emulsifiers (0.3 and 1.5 % mono-diglycerides). The stability of air bubbles with aggregated tat globules may be tested by evacuating the samples, which will expand all unstable air bubbles and induce a breakdown. If the amount of emulsifier is overdosed (1.5%), large aggregates forln and the air bubble stability is reduced, resulting in a reduction of aggregate size after degassing due to the collapse of the

71 air bubble structure. When the dosage of emulsifiers is optimal (0.3 %), smaller, more stable air bubbles form. These bubbles do not collapse after degassing. This type of analysis requires careful sample preparation and experience to get reproducible results. It is only possible to analyze relatively stable air bubbles with this technique. Large unstable air bubbles may break down during dilution before analysis. Particle size and particle aggregate size distribution is now being used for monitoring product stability and functional properties in a range of food emulsion systems 24. A new foam analyzer has recently been developed in Holland 25. The foam is illuminated by continuous light from an opto-electronic unit, and the light reflection is measured by an optical glass fibre probe, which is moved down through the foam at a known speed. More light is reflected when the probe tip is in a gas than when it is in a liquid. The reflected light is converted into an electronic analogue signal from which the bubble size distribution in the foam is calculated by a computer 25. The advantage of this method is that samples can be studied without dilution, and it is quicker than electron microscopy methods. It is believed that the method will provide valuable information on foams in the fixture. Using this method also makes it possible to measure the rate of foam drainage and collapse, as well as the gas fraction in the foam. 2.6.

Light microscopy This technique does not provide information as detailed as the above-mentioned methods, but may be used as a rough quality check of whippable emulsions. The method is suitable for detection of the presence of large tat globules in the emulsion. Fat globule agglomeration may be distinguished from tat coalescence by using a combination of phase contrast and polarized light illumination. The detection of small fat globules with quick Brownian motions may be made easier by diluting the sample with a polar solvent with a higher viscosity than water, e.g., glycerol 26. 2.7.

Free Fat Esthnate (FFE) FFE is an extraction method using heptane which measures the churning out of fat during emulsion stabilization 9. High protein and low fat content reduce destabilization, whereas the presence of emulsifiers, cold treatment at 5~ and mechanical treatment (whipping, possibly combined with freezing) increases destabilization. The FFE method is of great practical use to verify the level of mechanical treatment applied to ice cream mix during aeration and freezing. It also provides an indication of the storage stability and creaminess of the product tested 27. The total fat content in whippable emulsions may be estimated by the Gerber method 28 or by the gravimetric method 29.

3.

INTERFACIAL EFFECTS

The interfacially bound protein layer on fat globules is influenced greatly by the emulsifier and hydrocolloid content as well as by processing conditions. During homogenization of whippable emulsions at high temperatures, emulsification is facilitated by emulsifiers, whereas protein binding to the fat globules acts as an emulsion

72 stabilizing mechanism 15. However, the effect of emulsifiers on the long-term properties of emulsions is far more important than their influence on particle size distribution during homogenization. In general, protein-fat binding is weakened in emulsions containing emulsifiers 3"'31. The effect is temperature-dependent and increases at low temperatures (5~ to 10~ 3-~. In whippable emulsions, such as ice cream mix, toppings and homogenized creams, weakening the protein-tat binding by emulsifiers results in an improvement of the whipping properties 9,12,33,34.

3.1.

Protein-fat binding The amount of protein bound to fat globules is usually estimated by high-speed centriti~gation followed by quantitative protein analysis (e.g. Kjeldahl method) of the isolated cream layer or fat-free water phase 9"35,36. This phenomenon may be studied in greater detail by fast protein liquid chromatography 37, or by confocal scanning laser microscopy 38. It is recommended that the temperature in these types of studies be strictly controlled, as protein-tat binding is highly dependent on temperature. The effect of temperature and whipping on three whippable emulsion systems is shown in Table 1. For further details and results, see references 9'12'13'16. Table 1 Protein-fat binding in three whippable emulsion systems % Fat-adsorbed protein

System

Control 25~ 5~

Foam

With Emulsifiers 25~ 5~ Foam

Imitation cream 1~ Ice cream Topping

83 38 42

69 162~ 22

80 30 34

1) 2)

80 21 24

38 12 1

4 42) 0

The amount of adsorbed protein is initially high due to the high fat content (approx. 30%) in this system Analyzed after defrosting the frozen foam at 0~

Low temperatures, whipping, and the presence of emulsifiers all increase protein desorption. Protein desorption in toppings takes place very quickly after reconstitution in cold water, due to the high emulsifier content, but in liquid systems such as ice cream mix the process is much slower, and takes many hours. This is the primary reason why a long ageing period at 5~ in ice cream production is required Q. Protein desorption in ice cream mix during ageing at 5~ with and without saturated mono-diglycerides is shown in Figure 10. In the presence of emulsifiers protein desorption is accelerated.

73

Figure l0 Protein desorption from the fat globules into the water phase during ageing of ice cream lnix with (+E) and without (-E) emulsifier (saturated mono-diglyceride).

Figure 11 Protein binding to fat globules in ice cream mix at various temperatures and after ice cream production (I.C.). The latter analyzed at 5~ after thawing ice cream at 0~ Effect of hydrocolloid blend and emulsifier.

Milk protein desorption at low telnperature is due to stronger hydrophilic and weaker hydrophobic forces, and is caused mainly by dissociation of beta-casein 39. Hydrocolloids are used in ice cream to increase viscosity and inhibit ice crystal growth. In general, hydrocolloids also increase the protein load on the fat globules during the manufacture of emulsions 4~ This may be due to direct protein-polysaccharide binding at the o/w interface and/or protein-polysaccharide incompatibility in the water phase41. This

74 phenomenon has not been fully recognized in ice cream and should be studied in greater detail since it may give rise to important functional effects. Figure 11 shows the results of an ice cream mix containing a commercial hydrocolloid blend in combination with monodiglycerides. The protein load increases in the presence of hydrocolloids. In the presence of additional emulsifiers a very effective desorption of protein takes place during whipping and freezing in the ice cream machine. Effective protein desorption is facilitated by the increased viscosity of the mix due to increased surface shear forces, which makes the ice cream continuous freezer work better. The desorption of thick protein layers from fat globules of ice cream mix containing emulsifiers and hydrocolloids during ageing and mechanical treatment may also be observed by transmission electron microscopy (Figure 12). The protein bound to the surface of fat globules is desorbed as a thick coherent skin 23.

Figure 12 Transmission electron microscopy study of protein desorption in ice cream mix containing emulsifiers and hydrocolloids. (a) Immediately after homogenization the fat globules (t) are stabilized by adsorbed partially dissociated casein micelles (arrows). (b) During ageing the mix at 5~ the previously adsorbed protein film is released in the form of coherent protein layers (arrows) into the water phase (w). (c) After mechanical treatment in the ice cream freezer, desorbed protein layers are seen more often in the water phase without association to tat globules (arrows). From reference 48, courtesy of Dr. W.Buchheim, Kiel, Germany.

75

3.2. Interracial protein hydration The ageing at 5~ of whippable emulsions such as ice cream mix will enhance the hydration of milk proteins in the system. This is due to a property of casein micelles in milk. At low temperatures, the hydration or voluminosity of casein increases. The voluminosity is the volume of hydrated protein per gram of protein. This can be studied by analyzing the protein and water content in the sedimented casein pellet after centrifugation of skimmed milk. The increased hydration at low temperature is due to lower protein content in the pellet owing to dissociation of protein from the micelle (mainly beta-casein), and corresponds to data from the literature 42. During ageing of the mix, interfacial milk protein hydration also increases simultaneously with protein desorption from the fat globules. The water content of the isolated cream layers after centrifugation of ice cream mix can be analyzed by Karl Fischer titration. From such analyses, interfacial protein hydration can be calculated (Figure 13).

Figure 13 Desorption and hydration of protein bound to fat globules of ice cream mix during ageing at 5 ~ The voluminosity or hydration of interfacially bound protein may be calculated from the amount of water bound per gram of fat divided by the amount of protein bound per gram of fat. This corresponds to the volume of water per gram interfacial protein. Calculations show that emulsifiers facilitate interfacial protein hydration. This property is probably connected with their ability to desorb protein from the interface (Figure 14).

76

Figure 14 Effect of low temperature on hydration of bovine casein micelles and of interfacially bound protein in ice cream mix with (+ E) and without (-E) emulsifier (saturated mono-diglyceride).

Figure 15 Effect of temperature on average particle size of ice cream mix with and without emulsifier.

The increased interfacial hydration in the presence of emulsifiers gives rise to a slight increase in the particle size of the fat globules in the ice cream mix (Figure 15). The volume of cream layers after centrifugation also increases up to 100% when lowering the temperature

77 from 30 oC to 5 oC. The increased interfacial hydration at 5~ described below.

gives rise to an increased mix viscosity as

3.3.

Interfacial tension Interracial tension analysis may be used to study the interaction of emulsifiers and milk protein at the oil-water interface of whippable emulsions. The interfacial activity of proteins is affected only slightly by temperature changes. In general, emulsifiers can reduce interfacial tension much more than protein, and this effect is especially pronounced at low temperatures. The relationship between surface tension and temperature in emulsifiers was observed two decades ago by Lutton et al. 43. They explained that this relationship is due to a transition from a liquid-expanded type of monolayer existing at high temperatures (above 40~ to a solid condensed monolayer existing at a lower temperature (below 20~ In solid condensed monolayers the molecular packing of the emulsifier molecules is much denser than in the liquid expanded monolayers, and these differences result in lower or higher surface tension, respectively. Models of such surface films are shown in Figure 16. Emulsifier molecules are packed more closely in the solid condensed film than in the liquid condensed film. B

Water

Solid condensed film Surface areaJmol" 20-25/k 2

Water

Liquid condensed film Surface area/mol: 35-60/k 2

Figure 16 A schematic model of a solid condensed surface film (A) at temperatures below the melting point of the emulsifier, and of a liquid condensed fihn (B) at high temperatures (adapted froln reference 43). Such types of study may be performed using the Wilhelmy plate as a measuring device for interfacial tension analysis. This makes it possible to measure interracial tension continuously during temperature changes in the sample vessel controlled by external heating and cooling equipment 9. It is important to use a very pure triglyceride oil which is liquid down to 0~ to avoid disturbance of the analysis due to triglyceride crystallization. The connection between interfacial activity and emulsifier crystallization is easily

78 demonstrated in a system with a high emulsifier concentration in the oil phase, such as in toppings. Figure 17 shows measurements of interfacial tension between sunflower oil containing 5% emulsifier (propylene glycol monostearate) and distilled water. Separate samples of the oil phase containing emulsifier were analyzed for solid fat content.

Figure 17 Crystallization of the oil phase (sunflower oil) during cooling from 50~ to 0~ and interfacial tension ('7) between 5 % propylene glycol monostearate in sunflower oil and distilled water.

At temperatures above 25~ the presence of emulsifier results in only a slight reduction in interracial tension compared to a pure oil-water interface ('7 - 2 5 mN/m). When the temperature is decreased further, a significant drop in interfacial tension (,7) is registered due to interfacial crystallization followed by crystallization of emulsifier in the bulk oil phase below 15~ The increase in 7 observed at temperatures below 10~ is artificial being caused by a viscosity increase due to the crystal network which has formed. Interracial tension studies in relation to ice cream were also carried out using model two-phase systems similar to those mentioned above in connection with whipped toppings 9. These studies were carried out to analyze the interplay of emulsifiers and milk proteins at the oil-water interface. Emulsifiers were dissolved in sunflower oil, and protein in the water phase. With increasing amounts of saturated mono-diglycerides in the oil phase, increased interfacial activity was observed at low temperatures. At a concentration of 0.1%, which is usual in ice cream mix, the drop in interracial tension starts just below room temperature (15~ At this concentration no visible crystallization of emulsifier takes place in the oil phase. When both skimmed milk proteins and emulsifiers are present, a mixed film of both types of surtace active species forms at 40~ (Figure 18). When cooled, the emulsifier

79 crystallizes and dominates the interfacial tension. This will accelerate protein desorption from the oil-water interface. After reheating, the emulsifier melts and gives rise to readsorption of protein previously repelled from the interlace.

Figure 18 Interfacial tension of sunflower oil/water with and without protein (0.25% skimmed milk) in the water phase, and with and without 0.1% emulsifier (saturated monodiglyceride) in the oil phase. The two-phase systems were heated to 40~ for 1 hour, cooled to 5~ and reheated again to 40~ Symbols O = Oil; W = Water; P = Protein; E = Emulsifier. Reproduced from reference 44, courtesy of The American Institute of Chemical Engineers 9 1990 AIChE. All rights reserved.

Reversible interfacial effects are also observed in ice cream emulsion systems as regards protein desorption and readsorption. The interfacial interaction of milk proteins and emulsifiers during temperature changes is believed to be the keystone in explaining the physical changes which take place in ice cream mix during ageing. Protein desorption, fat crystallization, and flocculation of fat globules appear to correlate with the interfacial activity of emulsifiers during cooling. In the absence of emulsifiers, the physical changes at low temperature appear to be reduced considerably 9. 3.4.

Surface tension In whippable emulsions with a high fat content, the air-water interface of the foam after whipping is dominated by adsorbed deproteinated fat globules. In whippable emulsions with a low fat content other foam stabilizing lnechanisms come into play, such as proteinhydrocolloid and protein-emulsifier interactions. The former subject may be studied by

80 spectrophotometric analysis, the latter by various surface monolayer techniques 45,46 Increased emulsifier and hydrocolloid content is necessary to obtain stable foams when the fat content is reduced. Figure 19 shows results from practical tests of ice cream systems47.

Figure 19 Recommended dosages of commercial integrated emulsifier/hydrocolloid blend (CREMODAN'"SE 47) in ice cream mix. There are several reasons for this relationship. First, smaller fat globules with increased surface area tbrm in a low-tat recipe due to the use of higher homogenization pressure in such systems. Second, the protein-fat ratio is higher in low-fat recipes, resulting in stronger and thicker protein coverage on the tat globules which is more difficult to desorb. Third, the emulsifier takes over the function of tat in low-fat recipes, and will concentrate at the interface between air and serum, i.e., the emulsifiers will stabilize the air cells in a similar way to that of agglomerated tat. The adsorption of emulsifier to the air-water interface can be detected clearly by surface tension measurenaents because emulsifiers result in far greater depression of surface tension than proteins. Such analyses may also give intbrmation regarding the binding mechanisms of emulsifier in low-fat ice cream mix described below. Very surface-active emulsifiers (high HLB value) are capable of forming micelles in water. The latter is in equilibrium with emulsifiers at the air-water interface. At a certain concentration (= critical micelle concentration, CMC) the surface will be saturated with emulsifier and no further reduction in surface tension will be observed. The CMC can be found by surface tension measurenaents according to Figure 20.

81

Figure 20 A schematic figure showing how to find critical micelle concentration (CMC) from surface tension analysis at varying emulsifier concentrations. Monoglycerides and mono-diglycerides have low HLB values and cannot form micelles. They build up a multi-layer at the surface, resulting in a constantly decreasing surface tension as their concentration increases. However, in systems with proteins such as fat-free ice cream mixes, these emulsifiers behave as if they have a CMC. A possible explanation for this observation is that the unbound emulsifiier in the fat-free mix is in equilibrium with the protein-bound emulsifier. Above a certain concentration of emulsifier in the mix, any surplus of emulsifier will adhere to the protein in the water phase after the surface has been saturated. The unadsorbed emulsifier is seen as very small crystals less than 200 nm by electron microscopy analysis 4s. Without proteins the emulsifier will normally adsorb quickly to the surface, but in the presence of proteins adsorption takes up to 1 hour at 25 ~ (Figure 21).

Figure 21 Effect of protein, fat (oil) and emulsifier on surface tension of low-fat ice cream mix at 25 ~

82 Increased fat and increased protein content in the rnix delay adsorption of emulsifiers to air. Low temperature also has an inhibiting effect on this phenomenon. Surface tension analysis may be used to measure dosage effect in low-fat ice cream mixes. Such studies show that on a weight basis emulsifier is bound 10 times more strongly to tat than to milk protein in the nlix 49. As little as 1% fat in the mix has a very strong effect on the stability of the final ice cream (mentioned later under Descriptive Tests). Due to this strong effect the fat phase is believed not to be in a globular but in a more expanded crystalline state in such systems. This would give better possibilities for covering the air bubbles in the foam. This theory is highly speculative, and requires ti~rther studies for clarification.

4.

W A T E R PHASE

The properties of the water phase in whippable emulsions are important for product stability. The water phase is influenced by the soluble components of the systems, i.e., sugars, proteins and hydrocolloids. Interfacial hydration may also influence the properties of the water phase, particularly in high-fat systems. 4.1.

NMR Pulse NMR techniques, both low-field and high-field, were applied to study the properties of water in food systems. All three possible nuclei, ~H, 2H and 170, were probed, and various models for data interpretation were developed. An extensive review of the subject may be found in Schmidt and Lai 5~ Most of the data were collected probing the ~H nucleus owing to high sensitivity, although problems of data interpretation due to chemical exchange and cross-relaxation are under debate -s~. These types of analysis are most useful in monitoring changes or trends in hydration. T2-relaxation analysis may be used to study the effect of ingredient composition on the properties of water in whippable emulsions ~6. In food systems non-exponential relaxation curves are often found. This can be accounted for by the presence of 2, 3 or more recognizable components representing species of hydrogen atoms with different mobility 51. Figure 22 is an example of such an analysis of ice cream mix. A data program from Bruker was used to resolve relaxation curves into two components. From such analyses the relative abundance (%) of each hydrogen species and their corresponding T2-values may be calculated. The figure shows the effect of emulsifier (E) and hydrocolloids (H) on the properties of H atoms with short T2 (usually called bound water).

83

Figure 22 Effect of emulsifiers (E) and hydrocolloids (H) on properties of bound water in ice cream mix (T2 time and percentage of hydrogen atoms with low T2). Both hydrocolloids and emulsifiers increase the water-binding capacity in the mix (increased % of hydrogen atoms with low T2 and decreased T2 values). A synergistic effect is observed when both ingredients are present. From studies described earlier in this chapter, the effect of hydrocolloids is assumed to be due to simple water binding and increased thickness of protein layers around the fat globules, whereas the effect of emulsifiers may be due to the increased hydration of interfacially bound protein as well as increased hydration of polar groups of emulsifier at the oil-water interface. Water crystallization in frozen whippable emulsions such as ice cream or aerated desserts, may be analysed by the NMR technique similar to that described for solid fat content analysis. Again, this technique is best used for only relative studies on the effects of ingredient composition on freezing/melting behaviour. 4.2.

Thermal analysis Differential scanning calorimetry is a very suitable method to study the behaviour of melting and freezing of water in frozen food systems. Using this technique it is also possible to measure the glass transition temperature. However, this may be of minor interest because the glass transition temperature in traditional ice cream is much lower than the storage temperature in ordinary freezing cabinets 52. Freezing point determination A successful calculation of the freezing points of ice cream mixes was made using the freezing points observed for sucrose solutions after correction for effects of lactose and milk proteins. Good agreement was obtained between the calculated and observed freezing point values in a series of experimental mixes 53. This is due to the fact that fat, protein and hydrocolloids in general have a negligible effect on the freezing point of the water solutions in which they are dispersed. Freezing point analysis then makes it possible to calculate the amount of water that will be frozen at any particular temperature during freezing, hardening,

4.3.

84 and storage of ice cream. For details see Doan and Keeney 53. The characteristic freezing curve for ice cream can be used to explain why relatively low freezer drawing temperatures help facilitate a smooth-textured ice cream.

Figure 23 A typical freezing curve for ice cream showing the percentage of water frozen at various temperatures. Redrawn from reference 53.

More than 50% water is converted into ice crystals in ice cream at -5~ to -6~ which is the common drawing temperature for correctly operated continuous freezers. This portion of the water freezes very rapidly, often in less than one minute. Fast freezing induces the formation of small ice crystals, a critical prerequisite for smooth ice cream. At slightly higher temperatures (such as -4~ which is the common drawing temperature for batch freezers), less than 40% water is frozen and the freezing time will be longer. This is one of the reasons why ice cream frozen continuously is smoother in texture than batch-frozen products. A coarse texture may also develop as a result of heat shock, which involves alternate thawing and freezing of the water in the ice cream owing to temperature fluctuations in the hardening and storage cabinet. This results in a reduction of the textural quality of the ice cream. 4.4.

Size distribution of ice crystals Microscopic analysis is the only method available for estimating ice crystal size in ice cream. Light microscopy, equipped with cold stage and image analysis, may be used for this purpose 54. Low temperature scanning electron microscopy may also be used 55. Apart from the processing conditions discussed in section 4.3, hydrocolloids are important ingredients for controlling ice crystal growth in ice cream 56. Despite considerable scientific research in this area, the mechanism of this action remains obscure 57'58. Hydrocolloids do not influence the amount of water frozen or the glass transition point in ice cream which was believed to be involved in the stabilizing effect of hydrocolloids52. When ice cream starts to freeze, ice nucleation begins and water will freeze out of the solution in the form of pure crystals. As water is removed from the mix in the form of ice, the concentration of dissolved solids in solution increases. The unfrozen portion of the mix becomes increasingly concentrated as freezing continues, and contains dissolved sugars, milk

85 proteins, salts, and the hydrocolloids. During freeze concentration, the viscosity of the unfrozen phase becomes very high, primarily due to the increased hydrocolloid concentration, and this is believed to restrict the diffusion of water to existing ice crystals during fluctuations in temperature, or simply slowing down the latter process 52. Numerous hydrocolloids have been used in ice cream to inhibit ice crystal growth during distribution and storage. Useful hydrocolloid combinations and concentrations have been found for various ice cream products 59. The air cell stabilizing effect of agglomerated fat globules, promoted by emulsifiers and the ice-crystal-growth-controlling eft'ect of hydrocolloid stabilize the foam structure of ice cream to a great extent. This is evident by melt down analysis (see section 5.2) of ice cream exposed to heat shock.

4.5.

Wheying-off test In addition to their role in primary stabilization related to viscosity increase, some hydrocolloids (particularly carrageenan) are traditionally used as secondary stabilizers. Many of the primary stabilizing hydrocolloids, including locust bean gum and carboxy methyl cellulose induce precipitation of the milk proteins in the mix. This phenomenon in ice cream mix is known as wheying-off, and may be due to direct protein-polysaccharide binding and/or protein-polysaccharide incompatibility in the water phase 4~ The latter phenomenon may be due to decreased 'solvent quality' due to the competition between protein and polysaccharide for solubilisation. Carrageenan can prevent this wheying-off from occurring. Carrageenan binds directly to milk proteins forming a gel network which will protect the proteins from precipitation by the other hydrocolloids. Carrageenan is usually used at a much lower concentration than other hydrocolloids. This combined use of carrageenan and other hydrocolloids is very important in the stabilization of pasteurized chill-stable and UHT-treated ice cream premixes in softserve ice cream production. The effect of carrageenan is magnified in the freeze-concentrated aqueous phase of deep frozen ice cream, resulting in firm, cohesive gelation 6~ The wheying-off preventing activity may be estimated by making ice cream mix with locust bean gum as the main stabilizing hydrocolloid. The test carrageenan is added in different concentrations and the mixes are heated to 70~ for 30 minutes, cooled to 25~ with occasional stirring, and kept for 16 to 20 hours at 5~ The concentration at which wheying-off starts is estimated by visual inspection of graduated cylinder, and compared to a standardized carrageenan. From such studies the relative strength of the carrageenan being tested can be calculated 61.

5.

DESCRIPTIVE TESTS

A range of methods are used to test the textural quality of whippable emulsions. These methods are used to quantity the mechanical properties of the various products.

5.1.

Viscosity The viscosity range varies, depending on the whippable emulsion system in question. In whipped toppings viscosity increases as soon as the topping powder is reconstituted in cold water. This is due to the tbrmation and aggregation of hydrated fat crystals which will

86 stabilize the foam during whipping ~2. In UHT imitation whipping cream, a low viscosity of the emulsion before whipping is essential. The undesirable increase in viscosity during storage of cream is due to aggregation of fat globules, and this will reduce the pourability. If the agglomeration is too strong, the whipping properties will also be reduced. The viscosity of cream may be kept low by incorporating a sufficient amount of milk proteins and ionic emulsifiers, which will improve the emulsion storage stability before whipping. Fat globule aggregation is also minimized by quick cooling of the hot emulsion immediately after homogenization. In frozen whipping cream products, hydrocolloids are often used for ice crystal control 59. This will, of course, give higher emulsion viscosity. The viscosity of ice cream mix is important for processing in the ice cream freezer and must be within certain limits. Factors which may increase viscosity are increased %solids content, particularly hydrocolloids and protein, and low drawing temperatures in the freezer. Viscosity is usually measured on a simple comparison basis using a 50 to 100 ml capacity pipette, marked at an arbitrary place below the bulb. The flow time required to discharge the sample to the lower mark may be determined for water and then for the sample being tested for comparative purposes, and recorded in seconds62. More sophisticated rotation viscometers may also be used. The viscosity effect of hydrocolloids on ice cream mix is due to several factors. Hydrocolloids have a direct viscosity effect in binding large amounts of free water in the mix. Some hydrocolloids, such as kappa-carrageenan, form a gel network in the mix by binding to the milk proteins 6~ In general, hydrocolloids increase the thickness of the interfacial protein layer around the tat globules, and increased interfacial hydration is also obtained (see sections 3.1 and 3.2). Increased interfacial hydration is correlated to increased viscosity of mixes made with different hydrocolloids (Figure 24).

Figure 24 Viscosity of ice cream mix with different hydrocolloid types determined by the pipette method (flow time in seconds). Relation to interfacial hydration of fat globules

(%H20).

87 The viscosity effect increases exponentially when the ice cream is frozen 33. The effect of hydrocolloids becomes particularly dominant as free water crystallizes out during freezing 6~ The viscosity of aerated dessert mixes should be sufficiently low to withstand pasteurization, homogenization and ageing. On the other hand, the viscosity should be sufficiently high at low temperatures to stabilize the foam structure of the products. The foam should not gel or set before it is tapped, and should remain stable for several weeks without collapsing or showing signs of syneresis 2. Comlnon types of hydrocolloids for aerated desserts are gelatine, alginate and carrageenan. These hydrocolloids are all lnore or less shear-reversible gelling agents and are therefore suitable for use in aerated desserts 2. Only gelatine, which is acid-stable, can be used in low-pH desserts (yogurt-type desserts). When gelatine is used, the ageing temperature must be above 20~ and the mix must be agitated continuously to prevent the mix from gelling before it enters the aerator~. If hydrocolloids are used in sufficient quantities to enable them to gel the mix, then they will also be able to tbrm a stable foam when whipped. Starch and emulsifiers can also be used to provide aerated desserts with more body and a creamier consistency 2. 5.2.

Rheology of whipped emulsions After whipping whippable elnulsions obtain more solid-like properties. This means that ordinary viscometry measurements are not useful. The solid-like properties may be measured by non-destructive dynamic rheology analysis or by destructive methods using a Penetrometer, Jelly Tester, Instron instruments, or other types of texture analyzers. The latter methods are the most useful due to their simplicity and speed. Texture analysis of whippable emulsion must always be compared with the amount of air incorporated into the foam, which is known as percentage overrun and is calculated as follows: %

Where

Overrun

=

W1

-

W2

x

i00

1 -Weight of a given volume of whippable emulsion before whipping W2 = Weight of the same volume of whippable emulsion after whipping

W

Other useful parameters are whipping time and estimation of syneresis (serum separation from the foam). In ice cream the percentage of overrun is controlled in the ice cream machine, where the mix is whipped and frozen to a certain predetermined overrun. Only very few studies regarding the rheology of frozen ice cream are reported 63'64. This area should be studied in further detail to relate organoleptic and visual evaluations to instrulnental analysis. 5.3.

Melt-down analysis To test the melt-down properties, a rectangular block of ice cream of defined size is taken from the storage cabinet (e.g., at -20~ and is placed on a wire gauze (mesh size. e.g., 4 ram) at a controlled temperature between 15 and 25~ The melting may be followed

88 by weighing the melted ice cream collected in a beaker below the wire gauze. The time until the first drop falls, the amount of ice cream melted after 60 minutes, and the shape (stand-up quality) of the ice cream remaining at the top of the gauze are often used for evaluation 60,65. An example of melt-down analysis is shown in Figure 25. As little as 1% fat gives an enormous quality improvement in the texture of fat-reduced ice cream which has been properly stabilized by emulsifiers and hydrocolloids 66.

Figure 25 Melting resistance of non-fat and low-fat ice cream (redrawn from reference 65) In most countries consumers regard good ice cream melting properties as being synonymous with minimuln drip loss and good shape retention on melting. By contrast, in North America the retention of shape in melted ice cream is regarded as a defect 67.

5.4.

Organoleptic evaluation Organoleptic evaluation and product stability are usually assessed by a small expert panel trained to evaluate product appearance and ice cream consistency including smoothness, firmness, creaminess, sandiness, body, icy texture, and other properties. For a review of common body and texture defects, scoring and grading see Arbuckle 62. Although organoleptic evaluation is basically the most important analysis in practical ice cream product development, it is difficult to use that as the basis for exact conclusions. Despite these difficulties, it is always organoleptic analysis which has the highest priority due to its direct relationship with consumer acceptance. This argument is also valid for other types of whippable emulsions.

REFERENCES .

2. .

4.

Mann, E.J., Dairy Industries International 52 (1987) 15. Groven, S." Application of Emulsifiers and Stabilisers in Selected Dairy Products. Grindsted Technical Paper 215 (1989). Nielsen, H: Aerated Desserts. Grindsted Technical Paper 220 (1993). Brooker, B.E., M. Anderson & A.T. Andrews, Food Microstructure 5 (1986) 277.

89

,

6.

7. 8. ,

10.

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14. 15.

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19. 20. 21. 22. 23.

24. 25. 26. 27. 28. 29. 30.

Van Boekel, M.A.J.S and Walstra. P., Colloids Surf. 3 (1981) 109. Brooker, B.E., Food Structure 9 (1990) 223. Skoda, W. and Van den Tempel, M., J. Colloid Science 1___88(1963) 5687. Berger, K., in "Food Emulsions", second edition, K. Larsson and S.E. Friberg (Eds). Marcel Dekker, New York (1990) 367. Barfod N.M., Krog N., Larsen G. and Buchheim W., Fat Sci. Technol. 9.3 (1991) 24. Barfod N.M., Effect of emulsifiers on fat crystallisation in ice cream emulsions, in Proceedings from 15th Scandinavian Symposium on Lipids, Rebild Bakker, Denmark, V.K.S. Shukla and G. H6hner (Eds.), Lipidforum, G6teborg (1989) 133. van Putte, K. and van den Enden. J., J. Am. Oil Chem. Soc. 5 (1974) 316. Barfod, N.M., and Krog, N., J. Am. Oil Chem. Soc. 64.4(1987) 112. Krog, N., Barfod N.M. and Buchheim W., Protein-fat-surfactant interactions in whippable emulsions, in "Food Emulsions and Foams", E. Dickinson (Ed.), Royal Society of Chemistry, London (1987) 144. McClements, D.J. and Povey, M.J.W., Int. J. Food Sci. Technol. 2__33(1988) 159. Krog, N., Food emulsifiers and their chemical and physical properties, in 'Food Emulsions', second edition, K. Larsson and S.E. Friberg (Eds.), Marcel Dekker. New York (1990) 127. Barfod N.M., Krog, N. and Buchheim, W., Lipid-protein emulsifier-water interactions in whippable emulsions, in "Food Proteins", J.E. Kinsella and W.G. Soucie (Eds.), Am. Oil Chem. Soc., Champaign, Illinois (1989) 144. Le Neveu, D.M., Rand, R.P., Parsegian, V.A. and Gingell, D., Biophys. J. 1__88(1977) 209. Westerbeek, J.M.M. and Prins, A., Function of alpha-tending emulsifiers and proteins in whippable emulsions, in "Food polymers, gels and colloids", E. Dickinson (Ed.), Royal Society of Chemistry, Cambridge (1991) 147. Bern, M.B., Topping powder, internal Grindsted report (1992). Buchheim, W., Gordian 7___88(1982) 184. Buchheiln, W., Kieler Milchwirtschafte Forschungs Berichte 4__33(1991) 247. Buchheim, W., Barfod, N.M. and Krog, N., Food Microstructure 4 (1985) 221. Buchheim, W. and Dejmek, P., Milk and dairy-type emulsions, in "Food Emulsions", second edition, K. Larsson and S.E. Friberg (Eds.). Marcel Dekker, New York (1990) 203. Anonymous: Unilever uses Mastersizer to monitor particle size. Ice Cream and Frozen Confectionery. March 1993, 189. Bisperink, C.G.J, Ronteltap, A.D. and Prins, A., Adv. Colloid Interface Sci. 3___88(1992) 13. Anonymous: Phase contrast microscopy of ice cream mix, Technical Memorandum 217, Grindsted Products (1993). Andreasen, T., Grindsted system for stick novelties, paper presented at the INTER-EIS Seminar 1987, Solingen, Technical Paper 214. Grindsted Products. Anonymous: Determination of fat in ice cream (ice cream mix) according to the Gerber method, Technical Memorandum 214, Grindsted Products (1993). Anonymous: Determination of fat in ice cream - gravimetric. Technical Memorandum 215. Grindsted Products (1993). de Feijter, J.A., Benjamins, J., Tamboer, M., Colloids Surf. 27 (1987) 243.

90 31. 32. 33. 34. 35. 36. 37. 38. 39.

40. 41.

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49 50.

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54. 55. 56. 57. 58.

Darling, D.F., Birkett, R.J., Food Colloids in Practice, in "Food Emulsions and Foams", E. Dickinson (Ed.), Royal Society of Chemistry, London (1987), 1. Dickinson, E. and Tanai, S., J. Agric. Food Chem. 4__9_0(1992) 179. Keeney, P.G., Food Technol. 3__6_6(1982) 65. Towler, C. and Stevenson, A., New Zealand J. of Dairy Sci. and Technol. 2__33(1988) 345. Goff, H.D., Loboff, M., Jordan, W.K. and Kinsella, J.E., Food Microstructure 6 (1987) 193. Oortwin, H. and Walstra, P., Neth. Milk Dairy J. 3_33(1979) 134. Dickinson, E., Rolfe, S.E. and Dalgleish, D.G., Food Hydrocolloids 3 (1989) 193. Heertje, E., Nederlof, J1, Hendrickx, H.A.C.M. and Lucassen-Reynders, E.H., Food Structure 9 (1990) 305. Reimerdes, E.H., Changes in the proteins of raw milk during storage, in "Developments in dairy chemistry", vol. 1, P.F. Fox (Ed.), Applied Science Publishers, London (1982) 271. Tolstoguzof, V.B, Food Hydrocolloids 4 (1991) 429. Dickinson, E. and Euston, S.R., Stability of food emulsions containing both protein and polysaccharide, in "Food polymers, gels, and colloids", E. Dickinson (Ed.), Royal Society of Chemistry, Cambridge (1991) 132. Bloomfield, V.A. and Morr, C.V, Neth. Milk Dairy J. 2__7_7(1973) 103. Lutton, E.S, Stauffer, E., Martin, J.B. and Fehl, A.S, J. Colloid Interface Sci. 3___00 (1969) 283. Krog, N. and Barfod N.M., AIChE Symposium, Series 8___66,No. 277 (1990), 1. Rahman, A. and Sherman, P., Colloid Polym. Sci., 260 (1982) 1035. La Libert6, M.-F., Britten, M. and Paquin, P., Can. Inst. Food Sci. Technol. J. 2__.!_1 (1988) 151. Anonymous, CREMODAN*"SE 47, Product Description 214, Grindsted Products (1988). Buchheim, W., Structures and interactions in ice cream mixes. In Proceedings of the Penn State Ice Cream Centennial Conference, M. Kroger (Ed.), Pennsylvania State University, College Park, PA, (1992) 281. Barfod, N.M., Unpublished results. Schmidt, S.J. and Lai, H.-M., Use of NMR and MRI to study water relaxations in foods. In "Water relationships in foods", H. Levine and L. Slade (Eds.), Plenum Press, New York (1991) 405. Brosio, E., Altobelli, G. and DiNola, A., J. Food Technol. 1___99(1984) 103. Goff, H.D. and Caldwell, K.B., Modern Dairy 7__Q0(1991) 14. Doan, F.J. and Keeney, P.G., Frozen dairy products. In "Fundamentals of Dairy Chemistry", B.H. Webb and A.H. Johnson (Eds.), AVI Publishing Co., Westport, Conn. 1965, 771. Donhowe, D.P., Hartel, R.W., and Bradley, R.L., J. Dairy Sci. 7__44(1991) 3334. Caldwell, K.B., Goff, H.D. and Stanley, D.W., Food Structure 1__!1(1992) 1. Caldwell, K.B. Goff, H.D. and Stanley D.W., Food Structure 1_1_1(1992) 11. Muhr, A.H. and Blanshard, J.M.V., J. Food Technol. 2__!_1(1986) 683. Buyong, N. and Fennema, O., J. Dairy Sci. 7__!_1(1988) 2630.

91 59. 60. 61. 62. 63. 64. 65. 66. 67.

Knightly, W.H., J. Food Technol. 22 (1968) 73. Dea, I.C.M., Int. Food Ingred., No. 1 (1991) 9. Anonymous. GENU Control Method C306-1, The Copenhagen Pectin Factory Ltd. (Hercules Inc.) (1978). Arbuckle, W.S., Ice Cream. Third Edition. AVI Publishing Company Inc., Westport, Conn., 1977. Shernlan, P., J. Food Sci., 30 (1965) 202. Windhab, E., ZFL 5 (1989) 242. Larsen, G., "The principle of homogenisation of an ice cream mix", paper presented at the INTER-EIS Seminar 1988, Solingen, Technical Paper 216, Grindsted Products. Christensen, E.S., "hnprovement of creaminess in non-fat and low-fat frozen desserts", paper presented at INTER-EIS Seminar, Solingen 1991. Mahdi, S.R. and Bradley, R.L, J. Dairy Sci. 5_]_1(1968) 931.

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Characterization of Food: Emerging Methods A.G. Gaonkar (Editor) 9 1995 Elsevier Science B.V. All rights reserved.

93

Chapter 4 U l t r a s o n i c c h a r a c t e r i z a t i o n of foods D.J. McClements Department of Food Science, University of Massachusetts, Amherst, MA 01003, USA

1. I N T R O D U C T I O N Ultrasound is the study and application of sound waves whose frequency is too high to be detected by the human ear, i.e., above about 16 kHz [1]. This is a purely arbitrary cut-off point, determined by the limitations of the human ear. The physics describing the propagation of ultrasonic waves is the same as that describing the propagation of sound waves. Ultrasound is already an established technique for characterizing the physical properties of many biological and non-biological materials [ 1]. It is routinely used in medicine to detect tumors and to determine the health and sex of fetus' in the womb [ 1]. In materials testing it is used to characterize the position and size of cracks in metals and plastics [2]. Oceanographers use acoustics to map the contours of the sea-bed, and to determine the location, number and size of fish swimming in shoals [1,3]. The chemical processing industry uses ultrasound to determine the concentration of solutes in aqueous solutions and to determine the flow rate of liquids and particulates in pipes [2]. It is not suprising therefore that ultrasound can also be used to characterize food materials. The possibility of using ultrasound to characterize foods has been realized for over half a century [5-8]. The wide variety of different applications investigated during this period (see Section 5), reflects the diversity and complexity of food materials, as well as the versatility of the ultrasonic technique. Even so, there are still few areas in the food industry where ultrasound is recognized as an established technique for characterizing foods, with perhaps the exception of the inspection of meat quality. This situation will almost certainly change in the near future, and ultrasound will become as important a tool as NMR for characterizing foods. Advances in microelectronics have made available sophisticated electronic instrumentation capable of making accurate ultrasonic measurements at relatively low-cost. The interaction between ultrasound and microheterogeneous materials is fairly well understood, and there are mathematical formulae available for interpreting ultrasonic measurements in a number of systems relevant to the food industry. Finally, ultrasound offers a number of advantages over alternative techniques used to characterize food: it is capable of rapid and precise measurements, it is non-intrusive and non-invasive, it can be applied to systems which are concentrated and optically opaque, it is relatively inexpensive and it can easily be adapted for on-line measurements. There are two distinct types of applications of ultrasound in the food industry: high and low intensity [7]. High intensity ultrasound is used to physically alter the properties of a material through which it propagates. It utilizes relatively high power levels (> 1 W cm -2) and low frequencies (< 0.1 MHz). Typical applications of high intensity ultrasound are cleaning, homogenization, cell disruption, promotion of chemical reactions and extraction [7]. Low intensity ultrasound is used to provide information about the physical properties of materials. The power levels used are lower than those used in high intensity applications (< 0.1 W cm -2) and the frequencies higher (0.1 - 100 MHz). Low intensity ultrasound does not alter the

94 properties of a material and is therefore non-destructive. Only low intensity applications are reviewed in this chapter. The objectives of this chapter are to introduce the basic concepts of ultrasonic propagation in materials, to describe some of the most important methods for measuring and interpreting ultrasonic measurements, and to outline existing and possible applications of the technique in the food industry. 2. U L T R A S O N I C P R O P A G A T I O N IN M A T E R I A L S

2.1. General considerations Ultrasound is used to obtain information about the properties of a material by measuring the interaction between a high frequency sound wave and the material through which it propagates. This interaction depends on the frequency and nature of the ultrasonic wave, as well as the composition and microstructure of the material. The parameters most commonly measured in an ultrasonic experiment are the velocity at which the wave travels and the extent by which it is attenuated. To understand how these parameters are related to the properties of foods it is useful to consider the propagation of ultrasonic waves in materials in general.

Figure 1. Ultrasonic compression and shear waves generated by the application of a sinusoidal force F(t) to the material. An ultrasonic wave can propagate through a material in a number of different ways. Consider a material to consist of a series of imaginary layers of particles (Figure 1). If a force is applied to one end of the material it will act throughout the material due to restoring forces between the layers. When an oscillating mechanical wave is applied perpendicular to the surface of the material a compression wave is generated, which m o v e s t h r o u g h the material as a series of expansions and compressions. The oscillation of the layers is in the same direction as the propagation of the ultrasonic wave. If the ultrasonic wave is applied parallel to the surface of the material a shear wave is generated. In this case the layers move perpendicular to the direction of propagation of the ultrasonic wave. Other types of wave are also possible, e.g., surface or lamb waves [2], although these are seldom used in the food industry at present. There is no net movement of the particles in a material: each layer

95

Figure 2. Dependence of the displacement of a particle from its equilibrium position on the time and distance the wave has traveled. simply oscillates around its equilibrium position and returns to this position when the energy stored as ultrasound is dissipated. An ultrasonic wave is represented graphically by considering the displacement (~ of the layers of particles from their equilibrium positions (Figure 2). The displacement varies with the distance (x) traveled by the wave and the time (t). The amplitude of the particle displacement decreases with distance because of attenuation of the ultrasonic wave (see later). The important characteristics of an ultrasonic wave are the amplitude and frequency (f), which are chosen by the investigator, and the wavelength (;~) and attenuation coefficient (a), which are characteristic of the material. The ultrasonic velocity (c) is simply related to the wavelength and frequency: c = Z.f, so that it is also a characteristic of the material. Measurement of the ultrasonic velocity (or wavelength) and attenuation coefficient is the basis of the ultrasonic testing of materials. A mathematical description of an ultrasonic wave must describe the dependence of the particle displacement on distance and time, and the reduction of its amplitude with distance traveled through the material. For plane sinusoidal waves the following equation is appropriate: 927rx 2nt - ~ oe'(T-T)

e-aX

(1)

The first term describes the sinusoidal variation of the particle displacement with distance, the second term the variation with time, and the final term describes the attenuation of the wave. In most text books equation 1 is written in the following form:

- ~o ei(~-~

(2)

Here co is the angular frequency ( = 2rcf) and k is the wave number ( = o~/c + M), which contains information about the ultrasonic properties of the material, i.e., the velocity and

96 attenuation coefficient. The oscillatory variations in the particle displacement are accompanied by variations in the velocity of the particle, and the local pressure, temperature and density of the material [ 1]. Variations in these quantities can be described by equations with a similar form to that for particle displacement and are the starting point for the derivation of most mathematical formulations used to describe ultrasonic propagation in materials. In practice ultrasound is usually propagated through materials in the form of pulses rather than continuous sinusoidal waves. Pulses contain a spectrum of frequencies, and so if they are used to test materials that have frequency dependent properties the measured velocity and attenuation coefficient will be average values. This problem can be overcome by using Fourier Transform analysis of pulses to determine the frequency dependence of the ultrasonic properties.

2.2 Relationship between the ultrasonic and physical properties of a material A simple relationship can be derived between the ultrasonic properties of a material and its physical properties by a mathematical analysis of the propagation of plane waves in a material. A general wave equation can be derived by differentiating equation 2 twice with respect to distance and twice with respect to time: d2

2

(3) This equation is applicable to the propagation of electromagnetic waves, as well as to ultrasonic waves, although the terms in the wave number have different meanings. It is fairly straight forward to derive an equation which describes the propagation of high frequency sound waves in a material by considering the restoring forces acting on an element of the material as the wave passes through [ 1]:

d2~

p d2~

dx 2

E dt 2

(4)

Here E is the appropriate elastic modulus (which depends on the physical state of the material and the type of wave propagating) and p is the density. By combining equations 3 and 4 the physical properties of a material (E and p) can be related to its ultrasonic properties (c and a).

(5) Thus a measurement of the ultrasonic properties can provide valuable information about the bulk physical properties of a material. The elastic modulus and density of a material measured in an ultrasonic experiment are generally complex and frequency dependent and may have values which are significantly different from the same quantities measured in a static experiment. For materials where the attenuation is not large (i.e., a , of the spins during a time interval A (A being the time between the two field gradient pulses). This mean displacement is given by < x2> = 2DA, where D is the diffusion coefficient. Typical values for D are around 10-l~ m2s-~ and for A around 100 ms. This means that displacements on a linear scale of microns for molecules in low molecular weight liquids can be monitored. A relatively short spin-spin relaxation time, T2, limits the maximum diffusion time if the spin-echo method is used. Therefore, instead of the above mentioned two pulse Hahn spinecho sequence it is sometimes better to use a three-pulse stimulated echo sequence [4]. By using the latter pulse sequence the effects of a possible residual background gradient are eliminated between the second and the third 90 ~ pulse. Furthermore, because in most emulsion systems the spin-lattice relaxation time, T1, is (much) larger than the T2-relaxation time, a significant gain in signal-to-noise ratio is obtained by using this three-pulse stimulated echo sequence instead of the conventional two-pulse Hahn echo.

154 2.2. Low resolution versus high resolution The low resolution NMR method can be adapted for routine determinations of water droplet size distributions of spreads such as margarines and halvarines [5]. Because of the modest price and the relative simplicity of the low-resolution NMR equipment, this method can be used in a factory environment. When using this method all protons, originating from oil or water, resonate at the same position of the spectrum. Because we are only interested in the contribution of the protons of the discontinuous phase (water, in case of an W/O emulsion), the contribution of the ~H nuclei in the continuous phase (the oil phase in a W/O emulsion) has to be selectively eliminated. This can be done by applying an additional 180 ~ pulse. The resulting pulse program for a W/O emulsion, using the three-pulse stimulated echo sequence, is given in Fig. 2. Typical time delays are given in the captions to the figures. The protons in the oil have a T1 different from the T1 of the pure oil of the emulsion. The value of the delay time ~- therefore depends on the T1 of the pure oil of the emulsion. A high resolution NMR spectrometer is built around a superconducting magnet with a very strong and homogeneous magnetic field and, consequently, is very expensive. Homogeneity of the field is important and the method is therefore very sensitive to disturbances. Measurement times may be much longer than on a low-resolution NMR spectrometer. These aspects make a high-field spectrometer not readily applicable in a factory environment.

Figure 2. Pulse sequence diagram of a Hahn spin-echo experiment with field gradient pulses. Rf- and field gradient pulses are denoted by 90 ~ 180 ~ and FGP, respectively. The FGP pulses have a length t5 and are separated by an interval A as in the spin-echo sequence given in Fig. 1. VD is a time delay which may be variable in which case also A is variable. A PFG NMR experiment may also be performed with variable t5 or gradient strength (G) and fixed/x. Normally, t3 is chosen between 0 and 10 ms and A between 0 and 400 ms. The time delay r depends on the T1 relaxation time of the pure oil of the emulsion but is normally between 130 and 180 ms.

155 Nevertheless, high resolution NMR spectroscopy has some important advantages over low resolution NMR. By changing magnetic field strengths (e.g. from 0.47 T when using a spectrometer operating at 20 MHz when protons are to be detected to 7.05 T for a 300 MHz spectrometer) the sensitivity can be increased to a great extent, since the quanta absorbed are larger and the resonance is correspondingly stronger [6]. Secondly, the high resolution method is much more selective. The acquired free induction decay can be Fourier transformed and the peaks in the spectrum, originating from the oil and water are very well separated. In this way one can obtain information about the continuous as well as the discontinuous phase at the same time. An example is given in Fig. 3, which shows experimental proton spectra of cheese, measured with and without field gradient pulses.

Figure 3. High resolution proton NMR spectra of cheese, obtained by application of a Hahn spin echo pulse sequence with and without field gradient pulses. Measurements were performed on a Bruker MSL-300 spectrometer, operating at 300 MHz. The field gradient unit used with this spectrometer was home-built and the strength was calibrated to 0.25 T/m, using a 1-octanol sample for which the diffusion coefficient is known at several temperatures.

156 2.3. Analysis of the NMR R-values The most difficult step in the performance of PFG-measurements is the analysis of the experimentally measured R-values. For unrestricted diffusion (i.e. the quantity v/2DA is much smaller than the distance between the barriers) the PFG-NMR echo attenuation is given by:

lnR=lnE*2A E T2 y2G262D(A- 38)

(1)

where T2 is the spin spin relaxation time, 3' is the gyromagnetic ratio of the protons, G is the strength of the field gradient, ~i is the duration of the gradient pulses, A is the time between the gradient pulses and D is the self diffusion coefficient. In the situation where ~/2DA is of the same order or larger than the distance between any diffusional barriers in the system, so-called restricted diffusion is observed. In a W/O emulsion, for example, the water molecules are restricted in the extent of their diffusion by the presence of the boundaries of the water droplets. The extent of the restriction of the diffusion of the water molecules is reflected in the ratio R = E'/E. An expression for the echo attenuation R-factor as a function of droplet diameter has been derived by Murday and Cotts for uniform spherical droplet sizes [7]:

lnR=ln( •

=__~._2,r __• 2A

)

1

a2,,,(a2,.a2-2)

x

26

2 +exp(- a2~D(A - 6)) -2exp(- a2 D6)

a2mD

(a2mD)2

(2)

2exp(- a 2,,,DA) - exp(- a 2,,,D(A +8)) ) (a2.D) :2

Here T2 is assumed to be independent of R and C~mis the m m positive root of the Bessel function equation: 1

--J3/2( aa) =Js/2(aa ) ixa

(3)

Eq. 2 reduces to Eq. 1 if v/2DA < < a. In this connection it should be noted that Eq. 2 is derived for cavities which are not mobile during the experiment. Indeed, a sphere of radius 1 /~m will have a diffusion coefficient in water of about 2.10 -13 m2/s [8]. Thus, the contribution to the decay of the echo from the diffusion of the sphere will be negligible. Several research groups have used NMR restricted diffusion measurements to determine size distributions of emulsion droplets (see below). The measurements can be made by variation of either the field gradient, G, the time interval, A, or the duration of the gradient

157 pulses, ~5. In their analysis o f the data the different authors extended the work of Murday and Cotts by incorporating effects due to emulsion polydispersity. Packer and Rees [3] extended the expression derived by Murday and Cotts [7] to include the effects of a droplet size distribution, assuming a log-normal distribution. By curve fitting they were able to determine the principal parameters of such a distribution from the experimental R-values. In the presence of a distribution of sizes, the observed echo attenuation ratio Robsis expressed in terms of the calculated attenuation of individual droplets, R: f a 3P(a)R( A , f , G,a)da

(4)

Ro~- o m

f a3p(a)da 0 where R(A,5,G,a) is given by Eq. 2, P(a) is the droplet size distribution and the factor a3 allows for the fact that the signal from a sphere of radius a is proportional to a 3. The algebraic form of P(a) is not unique. It was found in the literature that a log-normal distribution function was representative of a broad class of emulsions: P(a)=

1 exp[ (ln(2a)-lnD~176] 2ao(2n) ~ 202

(5)

In (5) D0,0 is the median diameter and ~ is the standard deviation of the distribution. By fitting the experimental R-values, the parameters D0,0 and a can be determined and hence the size distribution of the droplets in the emulsion can be obtained. For microbiological safety aspects D3,3 is more important. D3, 3 is the volume weighted mean droplet diameter and cr is the standard deviation of the logarithm of the droplet diameter. The parameter 133,3is related to the parameter D0,0 according to: Do,o = D3,3exp( _ 3 o 2)

(6)

The value of D3,3 indicates that 50% of the volume of the water occurs in droplets with a diameter smaller than D3, 3 and 50% of the volume is present in droplets with a larger diameter. A plot of the volume-weighted and number-weighted log-normal distribution for D3,3 = 20 #m and a = 0.7 is shown in Fig. 4. This figure clearly shows the elongated tail of the log-normal distribution for larger droplet diameters. It can be observed that the highest probability density for the volume weighted distribution occurs at a greater droplet diameter than that of the number-weighted diameter. This can easily be understood as the larger droplets contribute more to the volume-weighted distribution than the number-weighted distribution.

158

Q2

L~ o~ oml

o

Droplet radius a (~m) Figure 4. The number-weighted (Poo) and volume-weighted (P33) log-normal distribution for ) P33; ( ....... ) PooD3,3=20#m and tr=0.7. (

Eq. 1 showed that in the case of unrestricted diffusion the echo attenuation value R depends upon the durations t5 and A. This is also true in the case of restricted diffusion, although in a different manner. The dependence of the R-value upon these two parameters is shown in Fig. 5. This figure clearly shows that the echo attenuation factor R steadily decreases with increasing A in the case of unrestricted diffusion, but becomes independent of this parameter in the case of restricted diffusion. It may be deduced from this figure that it is necessary to determine the parameters of the log-normal droplet size distribution R as a function of A or by measuring R as a function of t5 for a fixed large value of A. Measurement of only o n e R-value, at a chosen ~ or A, is not sufficient for a careful determination of the droplet size distribution: in Fig. 5 a given In R-value can be found on more than one In R versus A-t3/3 curve. This means that the In R-values have to be determined for different values of A and/or tS. Depending on hardware configurations, measurements can also be performed by variation of the field gradient strength, but we had to adopt the approach of measuring the NMR attenuation as a function of A or tS. Unfortunately, the first part of an R versus A curve cannot always be measured owing to technical limitations. In this situation one is left with the alternative of measuring the R versus t5 pattern for a fixed large value of A. The measured list of R(tS) values forms a unique fingerprint of the emulsions [9] which can be used for the determination of the droplet size distribution in emulsions. Several years ago it was verified in our laboratory that different droplet size distributions occurring in food emulsions always result in different fingerprints [9]. The actual calculation of the parameters of the log-normal distribution from the measured values can be performed in two ways. The

159

Figure 5. Echo attenuation R versus the time interval A between field gradient pulses for different widths ~ of the field gradient pulses in the case of unrestricted (A) and restricted (B) diffusion.

first approach is based upon the use of a large matrix of R(~) data sets [9]. First,a large number of such theoretical datasets for a series of 6 values and fixed A value were calculated as a function of D3,3 and a. This resulted in an array of R(5) datasets. The calculated range of datasets represented more than 90% of the droplet size distributions found in the emulsions of interest to the food industry. After storing the complete data matrix in the computer, a simple computer program was used to obtain the best match between a set of experimental R-values and a set of theoretical R-values. This approach resulted in a very fast calculation of the droplet size distribution parameters for the assumed log-normal distribution from a measured set of R-values as a function of 6. In the second approach, the actual calculation of the parameters D3,3 and ~ of the log-normal distribution from the measured values was done by iterative curve fitting. This is the approach that is more being used in our and some other laboratories. A comparison between the results of the fingerprint approach and the iterative curve fitting program has shown that the agreement between the two methods is very satisfactory [9].

3. EARLIER NMR CHARACTERIZATION OF EMULSIONS As shown above, the pulsed field gradient NMR technique was first described by Tanner and Stejskal [1,2]. In addition to their work on unrestricted diffusion they also performed theoretical analyses of restricted diffusion and tested their results on octanol-in-water emulsions stabilized by surfactants. Packer and Rees [3] extended the work of Tanner and Stejskal by the development of a theoretical model using a log-normal size distribution function. Measurements made on two water-in-oil emulsions are used to obtain the self-diffusion coefficient, D, of the water in the droplets as well as the parameters a and D0,0. Since then, NMR has been widely used for studying the conformation and dynamics of molecules in a variety of systems, but NMR studies on emulsions are sparse. In first instance pulsed field gradient NMR was used to measure self-diffusion coefficients of water in plant cells (e.g. ref. [10]). In 1983 Callaghan

160 et al. [11] presented a paper about the diffusion of fat and water in cheese as studied by PFG-NMR. The water diffusion coefficients found were one-sixth of that of bulk water at the same temperature. The authors suggested that water diffusion is confined to surfaces within the protein matrix. The fat is present in the form of small droplets within the cheese. The data were fit to a Gaussian distribution of sphere volumes. Fleisher et al. [12] studied the self-diffusion of oil and water in rape seeds. The selfdiffusion of oil was found to be completely restricted. The experiments could be explained in terms of the model of diffusion within spherical droplets and a Gaussian mass distribution of the droplet radii. At the same time Van den Enden et al. [9] introduced the technique described above. It is a rapid method for the determination of water droplet size distributions in spreads by using low resolution pulsed field gradient NMR. Their method was based on the recognition that a set of echo attenuation values (R) as a function of the field gradient pulsed width, obtained under conditions where R is independent of the time allowed for diffusion, contains all the necessary information on the water droplet size distribution (see above). A log-normal distribution of water droplet sizes was assumed. Cory and Garroway [13] introduced the NMR pulsed gradient stimulated echo method to study compartments which are too small to be observed by conventional NMR imaging. They showed so-called proton displacement profiles of bulk water and dimethyl sulfoxide. The displacements are due to free diffusion and are Gaussian shaped. The profile of water in yeast cells showed restricted diffusion with a characteristic cell width of approximately 5 #m. L6nnqvist et al. [14] performed NMR experiments on emulsions stabilized by surfactants to obtain information about the droplet size and size distribution and whether a particular emulsion is of the O/W or the W/O type. Murday and Cotts' equation [7] was used with different droplet radii, each radius being weighted by its normalized volume fraction. S&lerman et al. [8] gave an overview of NMR self diffusion studies of emulsion systems. They stated that a log-normal distribution function gives a better fit than a normal distribution. Several examples are given including margarine and hydrocarbon gel emulsions. Hills and Snaar [15] used the PFG-NMR technique to study cellular tissue and related multicompartment systems. By fitting their data they showed how to obtain dynamic information about membrane permeability, and they intended to use the given strategy to study plant tissue and food preparations. Recently, Li et al. [16] performed PFG-NMR experiments on oil-in D20 emulsions. D20, with similar chemical properties as H20, was chosen because the NMR resonance frequency of deuterium is quite different from that of hydrogen. Therefore they could select the experimental parameters so that only NMR signals from oil molecules are observed. In their calculations they assumed a log-normal distribution. Because of the very different diffusion coefficients of the two oils used, they were only able to obtain stable converged distribution parameters for the n-octane sample during the non-linear fitting procedure.

4. EXPERIMENTAL RESULTS Spreads such as margarines and halvarines are systems where control of the droplet size distribution is very important. These systems are W/O emulsions. A very few methods are available for determining the droplet size distribution in W/O emulsions. As an illustration of the theory presented in the previous paragraphs, it will be shown that NMR can be readily

161

Figure 6. Theoretical curves, showing the echo attenuation R versus the time interval 6. Parameters, used in these calculations are: A=210 ms, D=l.31.10-9m2s ~, G=2.0T/m.

applied to this type of systems. In Fig. 6 some calculated In R versus ~ curves are shown for different values of D3,3 and a. For two spreads, encoded sample A and B pulsed field gradient NMR experiments were performed at 5 ~ on a Bruker PC120 Minispec operating at 20 MHz in combination with a home-built field gradient unit and a specially programmed application PROM. The gradient strength used was 2.00 + 0.01 T/m which was calibrated using a water sample [5]. The time ~i was varied between 0.1 and 3.5 ms and A was fixed at 210 ms. The diffusion coefficient of the water at this temperature was determined to be 1.31.10 -9 m2s "1. From the In R versus 8 curves (or alternatively from a In R versus (A-8/3) curve as shown in Fig. 5), the droplet size distributions have been calculated. The two spreads give different results as is shown in Table 1. The 97.5 % and 2.5 % intervals given in the table indicate that 97.5 % and 2.5 %, respectively, of the volume in droplets is larger than the given value. The intervals can be calculated as follows: 97.5% interval = D3,Jexp(2a) 2.5% interval = D3,3*exp(2tr) Although it did not happen in the examples given above it may be that the experimental Rvalues remain below 0.12. This points towards (very) large water droplets of the order of 200/xm and above, called free water. R-values above 0.98 indicate small droplets of the order of 1/xm or below. We have experienced that it may sometimes be necessary to allow for a small percentage of free water when fitting experimental data of W/O emulsions. In case of high percentages of additives the NMR signal may be absent, because the T2 relaxation time of the water molecules in the presence of high concentrations of these additives may become too fast.

162 Table 1 Droplet size distribution data, obtained for two spread samples ,

Sample

D3.3 (~m)

exp(a)

97.5 % interval (#m)

2.5 % interval (/~m)

A.

2.6

2.1

0.6

11.7

B.

8.0

3.6

0.6

106.4

5. CONCLUSION The pulsed field gradient NMR technique can be readily used for the determination of the water droplet size distribution in W/O emulsions or the oil droplet size distributions in O/W emulsions. Important advantages are the non-invasive nature, the ease of sample preparation, and the fact that pulsed field gradient NMR measures the droplet size distribution of the bulk in contrast with microscopic methods which estimate the size distribution of the surface. Both the proposed matrix method and the iterative curve fitting procedure can be successfully applied in a factory environment. The method can be implemented on a high as well as on a low resolution NMR soectrometer.

REFERENCES

1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16.

E.O. Stejskal, J. Chem. Phys., 43 (1965) 3597. J.E. Tanner and E.O. Stejskal, J. Chem. Phys., 49 (1968) 1768. K.J. Packer and C. Rees, J. Colloid Interface Sci., 40 (1972) 206. J.E. Tanner, J. Chem. Phys., 52 (1969) 2523. K.R. Harris and L.A. Woolf, J. Chem. Faraday Trans. I, 76 (1980) 377. C.P. Slichter, Principles of Magnetic Resonance, Springer-Verlag, New York, 1989. J.S. Murday and R.M. Cotts, J. Chem. Phys., 48 (1968) 4938. O. S6derman, I. L6nnqvist, B. Balinov, Nato Asi Ser. Ser. C (1992) 363 (Emulsions: Fundam. Pract. A00r.) 239. J.C. van den Enden, D. Waddington, H. van Aalst, C.G. van Kralingen and K.J. Packer, J. Colloid Interface Sci., 140 (1990) 105. P.T. Callaghan, K.W. Jolley and J. Leli~vre, Biophys. J., 28 (1979) 133. P.T. Callaghan, K.W. Jolley and R.S. Humphrey, J. Colloid Interface Sci., 93 (1983) 521. G. Fleisher, V.D, Skirda and A. Werner, Eur. Biophys. J., 19 (1990) 25. D.G. Cory and A.N. Garroway, Magn. Reso Med., 14 (1990)435. I. L6nnqvist, A. Khan and O. S6derman, J. Colloid Interface Sd., 144 (1991) 401. B.P. Hills and J.E.M. Snaar, Mol. Phys., 76 (1992) 979. X. Li, J.C. Cox and R.W. Flumerfelt, A1ChE J., 38 (1992) 1671.

Characterization of Food: Emerging Methods A.G. Gaonkar (Editor) 9 1995 Elsevier Science B.V. All rights reserved.

163

Chapter 7 The Application of E P R S p e c t r o s c o p y to the Detection o f Irradiated F o o d R.Gray Food Science Division, Department of Agriculture for Northern Ireland, Newforge Lane, Belfast BT9 5PX, Northern Ireland 1. I N T R O D U C T I O N As more and more use is made of the process of food irradiation in the national and international food chains it becomes obvious that some form or forms of internationally accepted methods of detection are required. In 1988 a conference on "The Acceptance, Control of and Trade in Irradiated Food" recommended that :"governments should encourage research into methods of detection of irradiated foods so that administrative control of irradiated food once it leaves the facility can be supplemented by an additional means of enforcement". (Anon., 1989). Considerable international effort has since been directed towards these goals and substantial progress has been made in a number of directions. The development of accepted detection procedures not only gives enforcement authorities the ability to check that products are correctly labelled but also gives the consumer confidence that adequate independent controls are available. Perhaps the detection method that is, to date, the most internationally accepted is Electron Paramagnetic Resonance (EPR) spectroscopy which can also be referred to as Electron Spin Resonance (ESR) spectroscopy. 2. P R I N C I P L E S

OF ELECTRON

PARAMAGNETIC

RESONANCE

Spectroscopy is the measurement and interpretation of the energy differences between atomic or molecular states. This energy difference can be measured because, according to Planck's Law, energy will be absorbed if the difference in energy AE = hv where h is Planck's Constant and v is the frequency of the radiation. The energy differences studied by EPR spectroscopy are predominantly due to the interaction of unpaired electrons with the combined effects of a strong magnetic field and a source of microwave energy. The sample is placed in a metal box or resonant cavity within the spectrometer and the microwave radiation, generated in the microwave bridge, is conducted down a waveguide into the cavity. This cavity is designed to ensure that, at one particular frequency, the microwaves resonate within the cavity in a similar fashion to sound waves resonating in an organ pipe and this increases the sensitivity of the instrument. The resonant cavity is placed between the poles of a strong electromagnet which provides the intense magnetic field required.

164 EPR spectroscopy detects species with unpaired electrons and as electrons are normally paired, any species, except a transition metal or rare earth ion, with an odd number of electrons is referred to as a free radical. These species are highly reactive and normally very short lived surviving for only milliseconds in the liquid phase. Electrons may be visualised as spinning negative charges and since a moving charge generates a magnetic field, each electron in effect acts as a minute bar magnet. In species with even numbers of electrons the effect of the individuals in each pair are cancelled out. However, in the case of free radicals the magnetic effects do not cancel and the species is said to be paramagnetic. If an external magnetic field is applied to such paramagnetic species the unpaired electron can only occupy one of two states :either (a) parallel to the external field (lower energy state) or

(b) anti-parallel to the external field (higher energy state) Electrons can be made to resonate between these two states by the application of microwave energy. In EPR spectroscopy samples are subjected to microwave radiation of constant frequency and the magnetic field strength is increased until energy absorption is detected - this occurs when the energy difference between the two spin states matches the energy of the microwave radiation. When foodstuffs are subjected to ionising radiation the absorption of some of the incident energy can lead to the ejection of electrons from chemical bonds. These free electrons may immediately recombine or may pass along a chain of highly reactive entities each of which can interact with the foodstuff to produce a stable end product. In the majority of foods the moisture content is sufficiently high to ensure that these radicals are rapidly neutralised. However, in food components and packaging materials of high dry matter e.g. bone, seeds, shells and cellulose the radicals are trapped in an environment which ensures a relatively long lifespan. These are the radicals which can be detected by EPR.

3. U S E S O F I R R A D I A T I O N

IN THE FOOD INDUSTRY

The use of ionising radiation for the preservation of food is not a new technology. In fact, about 90 years ago, a patent was issued in the United Kingdom (UK) which detailed the use of the process for the preservation of foods, especially cereals. Despite this initial interest in the technology, progress was hindered because of the limited availability of suitable sources of ionising radiation. About 1950, 6~ which generated gamma photons, and machines producing high energy electrons began to become available and as a consequence extensive research programmes commenced in the United States. Since then, many countries throughout the world have been involved in evaluation of the technology for the preservation of a wide range of foods and the process is used commercially in a number of countries (Anon., 1991). In combination with good hygienic practices, the process is effective in enhancing food safety by reducing the numbers of pathogenic micro-organisms including Listeria, Salmonella

165 and Campylobacter which are otten implicated in food poisoning outbreaks involving poultry, meat, fish and shellfish. The process also kills spoilage micro-organisms and so the shelf-life of these foods can be extended. Ripening of fruits can be delayed by irradiation and the technology provides an alternative to the chemicals which have been or are still being used to decontaminate spices and herbs, disinfest cereals and tropical fruit and inhibit sprouting in tubers such as potatoes. As well as the effects of the technology on the chemical, microbiological, sensory and nutritional quality of foods, the safety of irradiated foods has been repeatedly evaluated by a number of independent expert groups. In 1981, the Food and Agriculture Organisation (FAO)/Intemational Atomic Energy Agency (IAEA)/World Health Organisation (WHO) Joint Expert Committee concluded that:"the irradiation of any food commodity up to an overall average dose of 10 kGy, presents no toxicological hazard and introduces no special nutritional or microbiological problems" (Anon., 1981). Similar conclusions were reached by the UK Advisory Committee on Irradiated and Novel Foods in their report on the safety and wholesomeness of irradiated food (Anon., 1986). Following the favourable response of this committee, steps were taken to amend the existing UK legislation which permitted only the radiation sterilisation of foods for people whose immune response was compromised. New regulations came into force in early 1991 which permitted the irradiation of seven groups of foods under strictly controlled conditions (Anon., 1990a) (Table 1).

Table 1 Foods which may be treated with ionising radiation in the UK

Food group

Bulbs and tubers Cereals Vegetables Fruit and mushrooms Fish and shellfish Poultry meat Spices and condiments

Maximum dose (kOy) 0.2 1.0 1.0 2.0 3.0 7.0 10.0

(From Anon., 1990a). Scientific evidence has shown that on health grounds irradiated foods need not be labelled, but it is generally felt that consumers should be able to either choose or avoid irradiated foods. In the UK, surveys carried out using questionnaires indicated that consumers harboured considerable reservations about the consumption of food treated with ionising radiation (Anon., 1990b). On the other hand, in countries where test marketing of irradiated foods was undertaken, and where consumers had been given unbiased information about the process,

166 the response was very favourable (Anon., 1988; Bruhn and Noell, 1987; Marcotte, 1992). Nevertheless, it was accepted that in order to enforce labelling regulations, a method or methods to detect irradiated food was required. In addition, the availability of detection methods would help to promote international trade in irradiated foods. 4. D E V E L O P M E N T

OF DETECTION

METHODS

When food is treated with ionising radiation, the changes which occur are minimal and similar to those induced by other processes, such as cooking. As a consequence, considerable scientific effort has been necessary to develop methods which fulfil the technical and practical criteria considered necessary for an effective identification method (Delinc6e, 1993) (Table 2). It is accepted that several techniques will be needed to cover the range of foods which are likely to be treated with irradiation. A number of different approaches based on the physical, chemical, biological and microbiological changes occurring in irradiated food have been investigated in order to establish their potential as detection methods and these have been reviewed by a number of workers (Delinc6e, 1991; Leonardi et al., 1992; Raffi and BeUiardo, 1991 a; Stevenson, 1992; Raffi et al., 1993, Schreiber et al., 1993a).

Table 2 Some requirements for an identification procedure of irradiated food

Discrimination Specificity Robustness

radiation-induced response should be distinct and separable comparable response not induced by other processing, different breeds or varieties, different growth or storage conditions insensitive or predictable response for: variation of radiation parameters (dose-rate, temperature, gaseous environment etc.) - presence of other food components - further processing reproducible, accurate, validated throughout storage life no falsification possible rapid, simple, low cost, no complicated instruments, small sample size, applicable to a wide range of foods estimation of absorbed dose -

Reliability Stability Confidence Practicability Dose-dependence Proof in court (From Delinc6e, 1993).

Although only qualitative identification of irradiation will be required to enforce labelling regulations, it has been recommended that any method should be capable of providing an estimate of the absorbed dose. One such technique is electron paramagnetic resonance spectroscopy.

167 5. A P P L I C A T I O N

OF EPR TO IRRADIATED

FOODS

The suitability of the technique for the identification of a number of products including food containing bone or shell, fresh and dried fruits and vegetables, spices and nuts has been investigated. Although the vast majority of the work has dealt with primary food products, there is evidence which indicates that the procedure can be used to detect the presence of irradiated components such as mechanically recovered meat (MRM) in secondary food products. In addition, some materials used to package food can give a radiation-induced signal which would indicate that the packaging, and so perhaps the food contained within it, had been irradiated.

5.1. Foods containing bone When bone is treated with ionising radiation, free radicals are trapped in the crystal lattice of the bone (Gordy et al., 1955) and consequently can be detected by EPR spectroscopy. Prior to its application for the identification of irradiated food, the technique was used to date archaeological specimens (Ikeya and Mild, 1980) and as an in-vivo dosimeter to determine the level of human exposure to radiation (Pass and Aldrich, 1985). Detection methods which use bone samples examine only the mineralised tissue thereby avoiding the additional free radical species in the marrow. Two predominant paramagnetic species are generated in mineralised tissues following irradiation at room temperature (Ostrowski et al., 1980). The species which is derived from the organic material, probably from collagen, is characterised by a symmetric doublet, but it is not stable and so cannot be used to identify irradiated bone containing food. On the other hand, a much more stable paramagnetic centre localised in the crystalline hydroxyapatite gives an asymmetric singlet. Evidence has been presented to show that the trapped species corresponded to the CO2"radical (Geoffroy and Tochon-Danguy, 1982) but recently it has been suggested that more than one trapped radical may contribute to the EPR signal (Rossi et al., 1992). The EPR signal induced in irradiated fragments of bone or ground bone is different in shape to the signal present in unirradiated bone (Figure 1) and independent of the origin of the bone. This suggests that EPR spectroscopy has potential for the identification of a range of foods including chicken (Stevenson and Gray, 1989a), duck, turkey, goose (Dodd et al., 1988), whiting (Stewart et al., 1991), salmon, pork, (Goodman et al., 1989), carp (Stachowicz et al., 1992) and frog legs (Raffi et al., 1989a). Moreover, the signal has been found to be a specific indicator of irradiation. Using mainly chicken bone, it has been shown that the radicals trapped in the hydroxyapatite are not generated by other conventional processes such as grinding (Stevenson and Gray, 1989a) and cooking (Gray and Stevenson, 1989a) and they are sufficiently stable during storage (Stevenson and Gray, 1989b) and following cooking (Dodd et al., 1992; Gray and Stevenson, 1989a) to be useful for the identification of both raw and cooked chicken throughout its expected shelf-life. The technique is also very sensitive and the EPR signal can be derived using samples weighing as little as 20 mg and be detected at doses as low as 50 Gy (Dodd et al., 1988). One of the characteristics of an ideal detection method is that it should be capable of providing an estimate of the absorbed dose. Thus considerable effort has been directed towards studying the parameters which might influence the intensity of the radiation-induced signal and the conditions under which it might no longer be detectable.

168

[G] Figure i. EPR spectra from Irradiated and Unirradiated bone.

5.1.1. Variables affecting signal intensity Variables associated with several aspects of the processing chain from primary production through irradiation processing and storage conditions to consumption have been examined using mainly chicken bone. The intensity of the EPR signal, can be quantified by integration of the area under the absorption spectra (double integration of the first derivitive spectrum) (Stevenson and Gray, 1989a, b) or can be estimated by the measurement of peak height (the distance between the maximum and minimum of a particular spectral peak) (Dodd et al., 1988). Research has shown that the method used to prepare the bone for analysis can greatly influence signal intensity. In comparison to fragmented bone, which had been fir-dried, combinations of freeze-drying, oven drying, microwave drying and grinding produced considerable variation in signal intensity (Table 3). ~ l s t the most intense signals were obtained from samples which were fragmented and freeze-dried, most research has been performed using freeze-dried and ground samples because of the greater homogeneity of these samples. The possibility of using the technique to provide an estimate of the absorbed dose was demonstrated when it was shown that the intensity of the signal induced in chicken bone increased linearly with irradiation dose over the range 2.5 kGy to 10 kGy (Stevenson and Gray, 1989b) (Figure 2).

169 "Fable 3 Effect of sample preparation on the relative EPR signal strengths from irradiated chicken bones

Sample preparation method

Relative signal intensity

Basic

Fragmented & Fresh Fragmented & Freeze-dried Fragmented & Oven-dried Microwaved & ground Freeze-dried & ground Oven-dried & ground SEM Significance

Control

DM conc.

lg DM

2.317 2.846 2.945

0.955 1.467 0.830 1 160 1 560 0.983

0.949 1.466 0.794 1.174 1.591 0.903

6 889 9 965 5 188 6 643 9 390 5.005

0.0757 ***

0.0811 ***

0.0593 ***

0.3883 ***

1.138 1.813 1.888

*** P Q2 is a Guinier plot (Sec. 6.1).

4. SAMPLE P R E P A R A T I O N

4.1. Overview Neutron scattering experiments are usually at central facilities such as ILL, Grenoble, France or ISIS, Rutherford-Appleton Laboratory,UK. Beam time is scarce and costly, so experimental proposals are submitted months in advance of the scattering experiment, and refereed. Proposals must show why neutron scattering is an appropriate technique, and give a clear statement of the reasoning and objectives of the experiment. If a proposal is accepted, then beam time allocated may typically be half a day to two days. Food or biological samples usually have to be prepared well in advance, and thought has to be given to sample preservation before and during the experiment. For a structural technique such as SANS, a small degree of proteolysis or bacterial growth may be less significant than a change in the state of aggregation of the sample. Such aggregation/dissociation may occur in a time-dependent manner even in the absence of enzymatic or bacterial action. As the delay between sample preparation and experiment may be longer than for most experiments, the experimenter has to ask how reproducible the experiment will be. For very labile samples, the sample preparation may have to be done at the SANS facility. However, few "real" food samples will require this treatment. In addition to travel delays, a day may be spent in dialyzing the sample against D20 buffers for contrast variation experiments.

4.2. Preparation Details Sodium Azide is often used (at 0.01% concentration) as an anti-bacterial preservative. The same concentration should be present in an all the buffers against which the sample is dialyzed (see below). Soya Bean trypsin inhibitor is often added to protein samples at 10 ~tg/ml, but not usually to dialysis buffers. For contrast variation the H20 content of a solution has to be replaced by D20. Usually a series of samples is prepared with varying H20/D20 ratios. For each solution in the series, there must be a buffer with exactly the same composition of aqueous phase, including small solute molecules and ions. The exchange of H20 for D20 is usually performed by dialysis, and so the last dialysis must be taken to equilibrium.

4.3. Dialysis Technique Dialysis against D20 buffers can be speeded, and the quantity of expensive D20 reduced, if a multistep approach is taken. For most dilute solutions and colloids, the rate of dialysis exchange is unlikely to depend on the macromolecular sample

205 itself. However, exchange rates may be more variable in the case of emulsions, microemulsions, gels and membrane-bound vesicles. To measure quantitatively the rate of approach to dialysis equilibrium, an oscillation density meter (Anton Paar KG, Graz, Austria) can be used. A complete and cost effective dialysis can be performed by dialyzing 5 or 6 times, each time using 3 volumes of D20 solvent for each volume of sample. Dialysis tubing (8/32 size) should be boiled, soaked overnight in distilled water and the excess water squeezed out. A length of tubing is double knotted at one end, 4 ml sample inserted, and the tubing double knotted at the top to leave 1 to 2 ml air above the liquid. The dialysis tube is placed in a 20 ml boiling tube and 13 ml D20 dialysis solution added. The tube is sealed with a rubber bung wrapped in "Parafilm" or similar. The dialysis tube should be completely covered by the dialysis solution, and should be free to slide up and down inside the boiling tube. The boiling tube is placed on a test tube rotator consisting of a circular sheet of plywood with clips to hold a number of tubes on the sheet, pointing radially outwards. An axle perpendicular to the sheet and through its centre is turned by an electric motor. The circular sheet, lying in a vertical plane, is then slowly rotated about a horizontal axis. The speed of rotation (around 3 rpm) is chosen so that the two air bubbles (one inside, one outside the dialysis sac) could both travel the full length available to them during each rotation. The movement of the air bubbles is important to promote continual mixing and fast dialysis. The tube is inverted each half-revolution, and the dialysis tube must at all times be immersed in the dialysis liquid. With this arrangement, it was discovered that dialysis generally went to over 90% of equilibrium in one hour, i.e., the final density difference between solutions (excluding macromolecular components) was less than 10% of the initial difference. The rate of approach to equilibrium was rather less on the later cycles, when the initial density difference was small. Some food samples may contain groups with protons which exchange rather slowly. Hence the last cycle of dialysis is given much more time than any other. For six cycles of dialysis, the first five might typically run for one hour each while the last cycle runs overnight.

5. E X P E R I M E N T A L S C A T T E R I N G T E C H N I Q U E Intensity of scattered neutrons is measured as a function of scattering angle 20. The measured response of the neutron detector is the sum of the coherent scattered intensity of the sample particles (Eq.2), the scattering from the solvent, the scattering from the sample cell, and the electronic noise in the detector. To obtain the scattering from the sample particles, background scattering due to solvent and sample cell, and noise counts in the detector, must be subtracted from the experimental scattered intensity. The result is normalized to an

206 absolute scale using the incoherent scattering (assumed isotropic) from a vanadium sample, or from a H20 sample. Samples are usually placed in l m m thick quartz spectrophotometer cells sealed with 'Parafilm' or similar. Samples in which the aqueous phase has a very high D to H ratio are sometimes thicker, as the level of incoherent scatter due to H will be low. Samples may be in the scattering apparatus for several hours, and so H20/D20 exchange due to faulty sealing can cause errors. For gel-like samples, it is very important that there are no air bubbles trapped in the sample. Gel or viscous samples can be centrifuged to the bottom of cells, and air bubbles removed, using a Helma 'Roto-Vette' or similar. Samples with high voluminosity (e.g.,casein micelles or sub-micelles) have intrinsically weak scattering. To give valid Guinier plots (Sec.6.1) at smallest angles, inter-particle interference effects must be minimized and concentrations must be low. Interference effects between solute particles are greater for increased particle concentration and reduced concentration of screening electrolyte. Sometimes a series of scattering curves is taken at reducing concentrations, and the results extrapolated to zero concentration. Concentrations of 10 mg/ml or less are typical. For casein sub-micelles screened by 0.07 M NaC1 solvent, a concentration of 16 mg/ml protein gave minimal interparticle interference for Q > 0.012/~ (Stothart and Cebula, 1982). Concentrations can be increased for measurements at higher angles, as the length scale of interparticle interference effects is reduced. Signal to noise ratio is improved by using highly deuterated solvent to obtain higher contrast. Where samples in a range of D20/H~O ratios have been used, it is important that the measurement of solvent background should have the same D20/H20 ratio as the sample. Samples with a given D20/H20 ratio can be produced by mixing measured volumes of two stock samples, one in 100% H20 solvent, the other in 100% D~O solvent. Volumes are measured into the quartz cell with a micropipette. The solvent background is produced by mixing similar volumes of the stock dialysis liquids against which the stock samples were dialyzed. The D20/H20 ratio of the solvent background may differ slightly from that of the sample due to pipetting errors, absorption of atmospheric H~O, etc. This can be corrected for by a background subtraction technique based on the assumption that sample and background solvent should have the same neutron transmission (Stothart, 1987). Correction is generally straightforward except in the case of samples with a high volume fraction of solute in a solvent with a high D20 content at higher angles (weak scattering), in which case particular care is required.

207 6. DATA ANALYSIS Data analysis methods depend upon the level of order in the sample. The degree of order, in turn, depends upon the scale of distance on which the sample is viewed. For example, casein micelles show great variation in size (20 to 300 nm diameter) and so must be treated as a polydisperse system. However, the density variations ('submicelles') within the whole micelle are much more uniform in size. They can be treated as a quasi-monodisperse system (Stothart and Cebula, 1982) and analyzed in terms of inter-particle interference (Stothart, 1989). One scattering curve may show features of polydisperse and monodisperse systems within different angular ranges. The scattering from casein micelles at lowest angles is dominated by the external shape of the polydisperse whole micelle, while that at higher angles is dominated by the interference between quasi-monodisperse sub-micelles within the whole micelle. This is illustrated by the similarity in scattering of dilute whole micelles and a pellet of centrifuged whole micelles (Stothart, 1989). Sometimes a monodisperse system can be derived from a polydisperse one. For example, free sub-micelles (produced on disintegration of whole micelles) are quasi-monodisperse, and can be analyzed by Guinier plots and other techniques applicable to monodisperse systems. Some concentrated food systems showing little ordering may be approximated as random two-phase systems. The methods developed for SAXS of solid polymers may then be appropriate. A number of parameters can be extracted including specific interfacial area, and the correlation length Lo. Correlation length is obtained from the Fourier transform of the intensity, and is a measure of the length scale of inhomogeneities in the sample. Full details are given by Kratky (1966). With SANS, a 3-phase system (e.g., protein, fat, water) can be effectively reduced to a 2-phase system by matching the scattering density of the aqueous phase (H20/D20) to that of one of the non-aqueous phases. For t h e s t u d y of colloidal interactions, SANS gives higher signal to noise ratio and can be used to lower Q than SAXS. It has been widely used for the characterization of synthetic microemulsions. Application to food systems has been more limited, but one example is described below. A study of voids in food solids is noted. For food systems in solution and showing no preferred orientation, the most useful data analysis methods are radius of gyration R~, variation of R~ with contrast, and particle mass. Experimental data are frequently obtained using contrast variation with a series of samples with varying H20/D20 ratios in the aqueous phase of the sample.

208

6.1. Radius of Gyration For particles that are approximately isometric, Eq.(3) holds for QR~ Q2 (Guinier plot) is linear with slope (-R~/3). Solutions must be dilute to avoid inter-particle interference effects. This implies that Qd>> 1 where d is the average interparticle distance. If inter-particle interference effects are present, then the Guinier plot flattens at low Q (Guinier & Fournet,1955). In difficult cases such as weakly scattering anisometric particles, it may be necessary to run a series of Guinier plots for decreasing concentrations, and extrapolate the resultant Rg values to zero concentration. The radius of gyration is a useful parameter to quantitatively describe a monodisperse colloidal solution, since no assumptions are made as to particle shape. Polydisperse samples often show a flattening of the Guinier plot at higher Q. For such samples, the absolute value of Rg has less significance, but the relative values (before and after treatment or processing) can be significant.

Figure 1. Guinier plot of SANS for casein sub-micelles in 0.07 M NaC1 solution in D20. Protein concentration is 16.1 mg/ml. I is coherent neutron scattered intensity. Stothart and Cebula (1982) and Stothart (1989) obtained linear Guinier plots of sub-micelles of whole casein, giving a radius of gyration of 64 ,s (Figure 1). On a uniform sphere model, this would indicate sphere radius of 84/k. This sphere size was input to a model fitting calculation (below). Thurn et al (1987) found similar sizes for sub-micelles of kappa-casein.

209 A Guinier plot of deuterated calmodulin-peptide complex (Heidorn et a1,1989) showed a sharp change of behaviour at the match point for the peptide. The Guinier plot was effectively due to the calmodulin part of the complex, with little contribution from the peptide.

6.2. Particle Mass and V o l u m i n o s i t y From Eq.(4), a plot of (I(0)) ~ v e r s u s X should be linear, provided that particles are monodisperse. A sample consisting of particles of identical size but varying internal distributions of scattering density would give a curved plot. If sample concentration (mg/ml) is known, and I(0) is measured on an absolute scale (see below), then ~ b i and V, the dry volume for one particle, can be found. If the ratios of elements in the sample is known, then the mass and volume of the dry particle can be calculated from I(0) (Jacrot,1976; Jacrot and Zaccai,1981). If an accurate value of partial specific volume is known then the volume of the hydrated particle (excluding non-solvating water trapped in the cavities) can be determined. If a hydrated volume can be inferred from the radius of gyration then the voluminosity, which includes non-solvating trapped water, can be obtained (Stothart and Cebula, 1982). 6.3. V a r i a t i o n of Rg with Contrast If the dissolved particles (e.g., microemulsion) consist of two or more phases then information on the distribution may be obtained from a plot of Rg2 v e r s u s 1/p where p is the contrast (P~m " Pso~)(Jacrot,1976).

Figure 2. Plot of Rg 2 v e r s u s

1/p for casein sub-micelles.

For typical compact proteins this plot has a positive slope, as the hydrophilic residues on the outside of the dissolved protein have a higher scattering density than the hydrophobic residues on the inside. For casein sub-micelles, the slope is negative (Stothart and Cebula, 1982) (Figure 2). This seems surprising at first sight, but the sub-micelles are so highly hydrated that all the constituent protein

210 molecules are likely to be fully exposed to water. Hence, there is no reason why electrostatic shielding should place the beta-casein (the most hydrophobic of the caseins) on the inside rather than the outside. Study of a deuterated calmodulin-peptide complex (Heidorn et al, 1989) by this method showed that calmodulin lay towards the outside of the complex.

6.4. Model Fitting For highly monodisperse biological structures consisting of subunit complexes (e.g.,ribosomes) it is possible to estimate the size of subunits in situ by deuterating hydrogen atoms in covalent positions (Moore,1981).

Figure 3. SANS intensity for wet pellets of whole casein micelles made with (a) 96% D20, 4% H20; (b) 74% D20, 26% H20; (c) 41% D20, 59% H20. Casein concn. approx. 250 mg/ml. Calculated intensities for models with subunits in close packing are :(---) for 74% D20 and Nl=2, N2=l, N3=2, D=168/~.

211 Covalent deuteration is rarely justified for food systems, but it is possible to perform simple model calculations assuming different forms of subunit packing, and compare with contrast variation data. For casein micelles, the experimentally observed scattering of free sub-micelles was used to derive a scattering amplitude for a sub-micelle within a whole micelle. Calculated intensities for various packings of subunits were compared to experimental scattering curves from whole micelles (Stothart,1989). A model with Nl=2,N2=l,N3=2,D=168 .~ gave best fit, where N1,N2,N3 are lattice repeats in the a,b,c directions of a hexagonal unit cell and D is the shere diameter (Figure 3). Casein micelles are highly hydrated which results in reduced scattered intensity. To increase intensity, centrifuged pellets of casein, with protein concentrations of approx. 250 mg/ml were used. 6.5. V o i d s i n S o l i d s Cebula et a1(1990) compared SANS curves from trilaurin at different temperatures. They interpreted the results in terms of molecular-sized voids within the crystalline material. 6.6. C o l l o i d a l I n t e r a c t i o n s If inter-particle colloidal interactions are assumed to be negligible at high dilution, then a structure factor S(Q) can be defined by

S(Q) = (Ih(Q)/I~(Q))(o/o h)

(5)

where h and 1 refer respectively to high and low values of volume fraction 9 (de Kruif and May, 1991). de Kruif and May studied kappa-casein micelles before and during clotting by chymosin, and interpreted the results in terms of an initial clustering of micelles, followed by coalescence and phase separation. It is difficult to obtain meaningful results on colloidal interactions unless the samples have low polydispersity. Studies of colloidal interactions between whole casein micelles can be affected by the polydispersity of native casein micelles. (Stothart,1987b). To circumvent the problem of polydispersity, the food system can be deposited on monodisperse silica spheres (Rouw and de Kruif,1989).

7. C O N C L U D I N G REMARKS Because neutron scattering experiments use central facilities, they require a strong justification. For food systems, the justification could be that the system is of great economic or nutritional significance in itself, or the results will be of significance for a wide range of food systems. SANS is most effective on systems that are monodisperse or have a monodisperse feature, the size of which can be measured by SANS. Subunits of uniform size

212 and surface layers of uniform thickness are examples of such features. If samples do not contain such monodisperse features, then useful information can be derived by comparing samples before and after processing. The "contrast variation" method gives the ability to "highlight" or "blank out" different phases of a multi-phase system and adds greatly to the power of SANS.

REFERENCES

Cebula, D.J., McClements, D.J. and Povey, M.J.W., J. Am. Oil. Chem. Soc., 67 (1990) 76-78. Guinier, A. and Fournet, G., Small-Angle Scattering of X-rays (Chapman and Hall, London), 1955. Heidorn, D.B., Seeger, P.A., Rokop, S.E., Blumenthal, D.K., Means, A.R., Crespi, H. and Trewhella, J., Biochemistry, 28 (1989) 6757-6764. Jacrot, B., Rep. Prog. Phys., 39 (1976) 911-953. Jacrot, B. and Zaccai, G., Biopolymers, 20 (1981) 2413-2426. Kneale, G.G., Baldwin, J.P. and Bradbury, E.M., Quart. Rev. Biophys., 10 (1977) 485-527. Kratky, O., Pure Appl. Chem., 12 (1966) 483-523. de Kruif, C.G. and May,R.P., Eur. J. Biochem., 200 (1991) 431-436. Moore, P.B., J. Appl. Cryst., 14 (1981) 237-240. Rouw, P.W. and de Kruif, C.G., Phys. Rev. A, 39 (1989) 5399. Stuhrman, H.B., J. Mol. Biol., 77 (1973) 363-369. Stuhrman, H.B., J. Appl. Cryst., 7 (1974) 173-178. Stothart, P.H. and Cebula, D.J., J. Mol. Biol., 160 (1982) 391-395. Stothart, P.H., J. Appl. Cryst., 20 (1987) 362-365. Stothart, P.H., Unpublished report to Institut Laue-Langevin (1987b). Stothart, P.H., J. Mol. Biol., 208 (1989) 635-638. Thurn, A., Burchard, W. and Niki, R., Colloid Polym. Sci., 265 (1987) 653-666.

Characterization of Food: Emerging Methods A.G. Gaonkar (Editor) 9 1995 Elsevier Science B.V. All rights reserved.

213

Chapter 10 A d v a n c e s in dielectric m e a s u r e m e n t of f o o d s Charles R. Buffier Microwave Research Center Marlborough, NH 03455 USA 1.

INTRODUCTION

The ability to measure the dielectric properties of foods and materials has provided a technique for diagnosing and monitoring these parameters for over 40 years. Knowledge of dielectric properties has been used both for research purposes, to better understand the structures and composition of foods, as well as in commercial applications for controlling manufacturing process parameters. In the last five years, technological advances in the speed and convenience of microwave instrumentation has provided the impetus for the accelerated interest in microwave techniques in the food industry. This interest has spurred the development of numerous successful instruments for the monitoring and control of food parameters in manufacturing processes. Instrumentation advances in wide spectrum m e a s u r e m e n t equipment has promoted widespread research and development interest in a new diagnostic field, electromagnetic dielectric spectroscopy. The use of these techniques promise to uncover new fundamental knowledge of food and material structures. This chapter introduces the reader to dielectric properties of foods and materials and then describes the technology, instrumentation and equipment advances which have applicability to the food industry. 2.

I N T R O D U C ~ O N TO DIELECTRIC MATERIALS

2.1. Definition of Dielectric Properties Similar to the principles of optics, materials interact with radio frequency and microwave radiation in three ways; they reflect radiation impinging upon them, they transmit radiation, and finally absorb some of the energy which is being transmitted through them. Mathematical equations, presented by Maxwell in 1864, are able to predict the behavior of microwave radiation's interaction with any type of food in any geometry. In order to do this, a single pair of parameters describing the electrical (or dielectric) properties of the food are required. This pair of parameters is known as the complex permittivity, or as is more commonly called in the United States, the complex dielectric constant. This parameter pair is defined as:

214 a=r

~"

(1)

Here ~ represents the relative values of permittivity with respect to that of free space. The term permittivity is not in common usage in the United States in the field of food science. Instead, the term dielectric constant is used. Thus, in Equation 1, is called the complex dielectric constant, ~', the dielectric constant and ~", the dielectric loss factor. The values of ~; and E" of a food material play a critical role in determining the interaction of the microwave electric field with the material. A discussion of these interactions follows. A "map" of foods plotted against their dielectric parameters was introduced by Bengtsson and Risman (1971). Table I gives values for the dielectric constant, loss factor and penetration depth, and Figure I shows a "map" of these values for common foods. Table 1. Dielectric and Thermal Properties of Common Foods

Food or Material Distilled water Water + 5% NaC1 Ice Vegetables Potatoes (raw) Peas (cooked) Carrots (cooked) Vegetable soup Fish (cooked) Fruit (raw) Banana Peach Meat Beef (lean, raw) Beef (cooked) Beef (cooked) Turkey (cooked) Pork (lean, raw) Ham Ham Cooking oil Butter (salted) Gravy Catsup Mustard Bread

2.2.

Dielectric Constant E'

Loss Factor E"

77.4 67.5 3.2

9.2 71.1 0.003

62 63.2 71.5 70 46.5

Penetration Depth dp(cm)

Density (kg/m 3)

Heat Thermal Capacity Conductivity Temperature (2. (J/kg K) k (W/m K)

t"

1.7 0.25 1162

1000 1034 920

16.7 15.8 17.9 17.5 12

0.93 1.0 0.93 0.94 1.13

950

0.55

720

0.5

61.8 71.3

16.7 12.7

0.93 12.7

930 930

3350 3770

50.8 35.4 32.1 39 53.2 57.4 85 2.5 4.4 73.4 54 56 4

16 11.6 10.6 16 15.7 33.2 67 0.1 0.5 26.4 40 28 2

0.87 1.0 1.1 0.8 0.9 0.46 0.3

1080

3600

1050

3810 3800 2350

23.7

8.2 0.64 0.36 0.52 2

4180 3725 2090

0.6 2.25

24~ 24~ 0~

0.5 60~ 0.5 60~

910

2010 2010

1000

3345

0.17

Reflection and Absorption of Microwaves in Foods

2.2.1. Reflection In order to gain an understanding of how microwaves interact with materials, it is instructive to examine a simple case, i.e., that of a plane wave impinging upon

215

Figure 1. Food Map of Dielectric Properties of Common Foods (Buffier and Stanford 1991) an infinite slab of material. A plane wave is defined as microwave radiation whose electric field directions in space are all parallel. For small loads in a microwave oven or processing system, the assumption of plane waves impinging upon the load may have somewhat more validity than for large loads, particularly--i'fthe load is thin and flat. For microwave radiation incident upon a slab from a direction perpendicular to its surface, a fraction of the energy will be reflected from the surface, Pr, depending upon its complex dielectric constant a. The main contribution to the magnitude of reflection however, is from the dielectric constant ~'. Errors due to neglecting r are less than 5% for virtually all foods as is indicated by the 5% line in Figure 1. Neglecting the loss factor, an approximate equation for the fraction of microwave power reflected from an infinite slab food surface is given by: =

Lqg+

It should be noted that ~

(2)

is equivalent to the well known optical index of refrac-

tion n. This value can thus be thought of as the microwave index of refraction in future discussion.

216

2.2.2. Absorption If the fraction of power reflected is Pr, the fraction transmitted into the medium, Po, is given by Po = 1 - Pr" Once the microwave energy, Po, enters the food, it propagates internally, perpendicular to the surface, toward the opposite face of the slab. If the material is microwave absorptive or lossy, the propagating energy will decrease as it traverses the slab as more and more of the energy is absorbed (Figure 2). The parameter which measures the microwave absorptivity of a material is the loss factor E'. The loss factor is zero for a non-absorbing medium and increases to 20 to 30 for highly absorbing foods such as ham and salted products. The fundamental equation for microwave power absorption is given by: Pv = 5.56 x 10 -4 x f x r x E2

(3)

Where Pv is the power absorbed per unit volume (watt/cm3), f, the frequency (GHz), r the dielectric loss factor and E the rms electric field (volt/cm). T h u s , as m i c r o w a v e energy propagates through a food, both the power at any point, P, as well as the power dissipated per unit volume, Pv decreases. For m a t e r i a l s w i t h h i g h loss factors, p o w e r decreases rapidly and the microwave energy does not penetrate d e e p l y . For l o w e r loss m a t e r i a l s the m i c r o w a v e energy may penetrate extensively. A parameter, designated penetration depth, will be defined later and is extremely important Figure 2. Penetration Of Electric Field and Power in determining how into a Sample (Buffler 1993) microwaves interact with foods.

2.3. The Dielectric Spectrum There are two major mechanisms by which the microwave electric field is converted to heat within a food. The first, the ionic interaction, comes from the linear acceleration of ions by the field. These ions are primarily from various salts within the product. The second interaction is molecular rotation of polar molecules, primarily water, as well as weaker interactions with carbohydrates and fats. In order to u n d e r s t a n d why different materials have different dielectric properties as well as understand the temperature and frequency behavior of the

217 microwave interaction, it is necessary to have some knowledge of the fundamental physics of each of these two absorption m e c h a n i s m s . 2.3.1. Ionic Interaction

Ions in a food oscillate transversely under the influence of the microwave electric field, colliding with their neighboring atoms or molecules. These collisions impart molecular motion which is defined as heat. Materials with mobile ions are conductive. The more available ions in a food, the higher the electrical conductivity. Microwave absorption in a food thus increases with its ionic content. The portion of microwave absorption due to ionic conduction can be described as a portion of the dielectric loss factor, t o. Geyer (1990) recently discussed this concept in his publication. In commonly used units with or, the conductivity, in m m h o / c m and f in GHz, the portion of dielectric loss due to conductivity becomes: e" o = 1.80 o(mmho/cm)/f(GHz)

(4)

Thus at 2.45 GHz, a salted food with a conductivity of 11 m m h o / c m would produce a contribution to the loss factor of r = 8.08. Equation 4 shows that the loss factor in conductive foods decreases monotonically with increasing frequency. The temperature dependence of this conductivity contribution depends upon the temperature dependence of the dc conductivity. The frequency and temperature behavior are illustrated in Figure 3. 2.3.2. Polar Interaction

If a material of polar molecules, such as water, is exposed to a fixed or static electric field, the molecules will all rotate in an attempt to orient themselves in the direction of the field. The magnitude of separated charges of a polar molecule is defined as the dipole moment, and determines the strength of interaction with the field. The dipole moment is also a measure of the dielectric constant ~'. A symmetrical molecule, with no dipole moment, is said to be non-polar and does not react with an electric field. If an electric field impinging upon a polar molecule is alternating, the molecules will rotate, following reversals of field. Because the polar molecules interact with other molecules in the material, they transfer their motion, which has been imparted to them from the electric field, to the entire sample as heat. As the frequency of the electric field is increased, the molecules will continue their attempt to rotate with the field, but will be more and more impeded by the damping caused by their interaction with neighboring molecules. The molecules will no longer be able to rotate fully, and the measured dielectric constant will decrease. The dielectric loss or absorption behaves differently. At very low frequencies the dipole follows the field freely, but little energy is transferred to the surrounding molecules and thus little absorption occurs. As the frequency increases, molecular motion increases and more energy is transferred to the surrounding molecules. As the frequency increases further, molecular inertia begins to impede motion and a maximum absorption is reached. As the frequency is raised still further, the dipoles

218 can no longer move in response to the rapidly oscillating field and can no longer transfer energy to its surroundings. In this region the a b s o r p t i o n decreases towards zero. The maximum absorption point is defined as the relaxation frequency or critical frequency, fc" A plot of the loss factor as function of freq u e n c y for the polar contribution, r is s h o w n for w a t e r in Figure 3. Note that the relaxation frequency is Figure 3. Dielectric Loss Factor vs. Frequency approximately 18 GHz. (Adapted from Roebuck et al. 1972) The relaxation or critical frequency of a material is related to its structure. Many organic liquids, including cooking oils (Pace, Westphal and Goldblith 1968), are polar and have relaxation frequencies in the low MHz range. These molecules can be thought of as inertia bound and cumbersome to move. Thus, very little change in loss factor is seen with increasing frequency above fc" Liquid water is more free to move and thus has a higher relaxation frequency. Solids not containing ions, such as ice and plastics, cannot strictly be thought of as polar. These molecules are locked into place by their structure and are unable to move easily. They thus are unable to participate readily in dielectric absorption and consequently have low values of dielectric constant and loss factor (see Table 1). The temperature and frequency dependence of the dielectric properties of polar molecules such as water was first modeled by Debye (1929). Early work on dielectric properties has been described by von Hippel (1954a, 1954b). Excellent recent reviews have been published by Ohlsson and Bengtsson (1975), Mudgett (1985; 1990) and Geyer (1990).

2.4. Penetration Depth The more absorptive a material, i.e., the higher the loss factor ~', the less deep microwave energy will penetrate into that material. A parameter, penetration depth, dp, has been defined which measurers this penetration, dp is a function of both ~' and ~" and serves as a guideline to the heating effectivity of a material. The term, penetration depth, has three common, slightly different definitions. Their definitions and differences have been discussed (Buffler 1993). The internationally accepted definition is that distance in which the microwave power, once

219 entered into the material, declines to 1/e (37%) of its original value. Here e is the Napierian base = 2.718. The equation describing the power at a point within an infinite slab of material for an incident plane wave is given by: P(z) _ e - z / dp

(5)

Po

Here, P(z)/P o represents the fraction of power remaining as a function of distance into the material. The units of distance, z, and penetration depth, d are P the same and are arbitrary as they occur as a ratio; centimeters are most commonly used in the literature. The penetration depth of a material depends on both the dielectric constant, a' and loss factor a" of the material. An approximate formula which holds to better than 5% for foods is: (6)

dp2~

E:"

The 5% error boundary line is shown in Figure 1. Table 1 presents penetration depths for various foods and materials. An excellent detailed review of penetration depth has been presented by Metaxas (1985). 2.5.

Temperature Dependence of Dielectric Properties of Films The temperature dependence of the dielectric properties of foods has been extensively measured and reviewed by Bengtsson and Risman (1971) and Buffler (1993). Mudgett et al. (1977) has pioneered the prediction of dielectric properties of foods as a function of constituency and temperature. Prediction of the temperature behavior of dielectric properties is crucial for accurate mathematical modeling of foods. Many workers today still use constant room temperature values or a look-up table at best. In the author' s opinion, dielectric prediction of food properties is still a very fertile and useful research field. Other important works containing copious references on dielectric theory, measurement techniques and data tabulation have been published. Pioneering work was done by von Hippel (1954 a; b and c). Buckley and Maryott (1958) have tabulated data on liquids. Nelson (1991) and with Tinga (Tinga and Nelson 1973) and ElRays and Ulaby (1987) have tabulated dielectric information on agricultural as well as other materials. Ohlsson and Bengtsson (1975) and Kent (1987) have published data on foods . . . . . 3. MEASUREMENT OF DIELECTRIC PROPERTIES 3.1. ~ Transmission Line Techniques Early efforts characterizing dielectric properties of materials was carried out at the Massachusetts Institute of Technology (Roberts and yon Hippel 1946; yon

220 Hippel 1954b). The values of r and r were derived from microwave theory by placing a sample of material against the end of a short-circuited transmission line, such as a waveguide or a coaxial line. This technique has applicability to high and low loss materials and in the present day has found applicability for the measurement of powders, grains and pulses (Nelson 1972, 1991).

3.2. Cavity Perturbation Techniques A very sensitive and accurate technique for the determination of low loss sample properties is called the perturbation technique. This measurement utilizes both the change in frequency and absorption characteristics of a tuned resonant cavity. Full theory and design details are available as a standardized procedure published by the American Society for Testing and Materials (ASTM 1986). A detailed review of these former techniques, with substantial references, has been published by Buffier (1993). 4.

ADVANCEMENTS IN DIELECTRIC MEASUREMENT TECHNIQUES

4.1. Open Ended Probe Technique A method which circumvents many of the disadvantages of the transmission line and cavity perturbation technique was pioneered by Stuchley and Stuchley (1980). This technique calculates the dielectric parameters from the microwave characteristics of the reflected signal at the end of an open-ended coaxial line inserted into a sample to be measured. This technique has been commercialized by Hewlett Packard with their development of a user-friendly software package (Hewlett Packard 1991) to be used with their network analyzer (Hewlett Packard 1985). This technique is outstanding because of its simplicity of automated execution as well as the fact that it allows measurements to be made over the entire frequency spectrum from 0.3 MHz to 20 GHz. Some care must be exercised with this technique, as errors are introduced at very low frequencies and at very high frequencies, as well as for low values of dielectric constant and loss factor. The technique is valid for the frequencies of 915 and 2,450 MHz, for materials with loss factors greater than 1. The temperature range of the probe is limited to approximately 60~ However, new probe development is nearing completion. Interpretation for lower loss materials such as fats and oils must be treated with caution. Typical open-ended probes utilize 3.5 mm (0.138 in) diameter coaxial line. For the measurement of solid samples, probes with flat flanges may be utilized (Hewlett Packard 1991). A photograph of an open-ended probe system is shown in Figure 4. The practical aspects of this technique have been described in detail by Engelder and Buffler (1991). 4.2. Underheating Mode Technique Solid food materials have dielectric properties dependent upon their composition. In many instances, particularly when developing microwavable food products, it is necessary to know the effective bulk microwave properties of the product, crushed, as is, or when agglomerated together. Typical examples are peas, beans, corn, pasta, flour

221

Figure 4. Dielectric Properties of Measurement Systems (Courtesy Hewlett Packard Company) and meal, etc. which may be utilized for plated meals, frozen entrees to be microwave heated, or foods which might be considered for undergoing microwave processing. Techniques for the measurement of the dielectric properties of small granular agricultural products such as grains as well as larger inhomogeneous products such as peas, beans and other pulses have been studied extensively by Nelson (1991). At present, the transmission line technique is used for this measurement (Nelson 1972), but it is not particularly "user friendly". Vertically oriented waveguide sample holders must be used; and the sample must be filled to an optimum height for best sensitivity. For simplicity of calculation, a computer program (Nelson et al. 1974) must be used to obtain results and an ambiguity in calculated results requires an approximate knowledge of the dielectric constant before the measurement is commenced. These difficulties may be overcome in the future by a new technique using underheating, longitudinal section magnetic mode (LSM) technology. This technology was first described in 1907 and provided the understanding of the means of propagation of radio waves around the surface of the earth. The theory of these waves has been recently explored and applied to microwaves by Risman (1994). Understanding these waves (as well as in their contained mode form) has led to the understanding of how food loads heat from the bottom while resting on the ceramic shelf of a microwave oven. This understanding led to the development of a very successful microwave oven which has recently been introduced in Europe. The technique for using LSM technology to determine the dielectric properties

222

Figure 5. Underheating Mode Sample Holder (Risman 1994) of large volume, inhomogeneous materials is based on microwave measurements as a microwave signal is propagated between the sample and a metal trough. A sketch of the sample holder for use at 2.45 GHz is shown in Figure 5 (dimensions in mm). A microwave signal is inserted via a coaxial to waveguide transition into the waveguide. A portion of the top of the waveguide is removed and replaced w i t h a thin (3.4 mm) sheet of plastic. The waveguide is provided with a moving short circuit plunger whose depth into the waveguide can be accurately determined. A small loop is affixed to the center of the face of the plunger to measure the amplitude of the signal reaching it. A removable pick-up probe is also inserted into the bottom of the waveguide. A microwave detector attached to either the loop or the probe is used to measure microwave signal amplitude. The effective bulk dielectric constant is d e t e r m i n e d by measuring the distance between a maximum and minimum value of amplitude. The bulk loss factor is determined by measuring the amplitude of the signal under the sample with the loop, as a function of plunger distance from the beginning of the sample. The fundamental understanding of LSM modes has only recently been published. The adaptation of the fundamental equations for the extraction of the dielectric properties from the measurements can be accomplished by reference to Risman (1994). Simple algorithms with charts and a computer program are presently under development and should be available in the near future (MRC 1994).

223

.

ADVANCES IN MICROWAVE DIAGNOSTIC TECHNIQUES Commercial Applications to Process Control

5.1. Introduction For several decades (since 1955) there has been considerable interest in using microwave energy for the determination of food and material parameters. This interest has been driven by the commercial need for process monitoring and control in manufacturing facilities. Microwaves can be used by making primary measurements of dielectric parameters, r and r and then relating them to the proportion of the various constituents in the process flow. (See Electromagnetic Dielectric Spectroscopy, following). Extensive use of this technique has only recently become practical due to the introduction of the computerized network analyzer (Figure 4) and dielectric software (Hewlett Packard 1985, 1991). Early work using microwaves as a diagnostic tool relied upon measuring a secondary effect of the dielectric properties of the material under interrogation, i.e., reflection, absorption and transmission. The two fundamental microwave parameters, ~' and ~" are related to the food or material composition. These two fundamental parameters also determine the reflection, absorption and transmission of the materials exposed to a microwave signal. Thus by measuring the amplitude and phase of the reflected or transmitted wave, or the characteristics of absorption of a wave through the material, one is able to empirically establish a relationship to the constituency of the product. One of the most successful early measurements was the use of microwave interrogation for the determination of moisture content of foods and materials. An issue of the Journal of Microwave Power was dedicated to the subject of microwave aquametry and contains an extensive bibliography on the subject from 1955 through 1979 (JMP 1980). A more recent compilation has been edited by Kraszewski (1994). Refinements of these techniques in recent years has been very successful in producing commercially available instrumentation with very accurate process monitoring capabilities. A number of examples of various techniques will be described in the following sections. 5.2.

Phase and Amplitude Techniques

5.2.1. Phase Dynamics, Inc. Phase Dynamics utilizes a unique, patented microwave concept to diagnose and measure molecular transformation process parameters with high sensitivity and accuracy (Phase Dynamics 1992). While originally developed for fluid measurements, the instrumentation is adaptable to most pumpable process lines and to some batch applications. The technique has been utilized for compositional analyses of true solutions as well as complex solid-liquid systems such as colloids and emulsions. Monitoring of molecular transitions which occur in cooking processes, hydrogenation, gelatinization and hydrolysis can also be monitored. The measurement technology involves a sensor section inserted into the

224 process line, which measures the frequency shift of an oscillator connected to a coaxial microwave transmission line inside the stainless steel insertion section (Figure 6). It is well known that the frequency of an oscillator shifts or "pulls" when the reflection and absorption of its load changes. In commercial and military a p p l i c a t i o n s , this trait is d e t r i m e n t a l a n d is p r e v e n t e d . By measuring this frequency shift however, the characteristics of the load F i g u r e 6. F o o d P r o c e s s A n a l y t i c a l can be extremely accurately deterInstrumentation mined. Specifications indicate that (Courtesy Phase Dynamics, Inc.) this technique is 100 times more sensitive to process variable changes than other types of measurement systems, with accuracies to 1% and for some applications to .01%. Process streams from 1/2 to 4 inches in diameter and temperatures from -40 ~ F to 300 ~ F can be accommodated. Temperature sensors are incorporated into the insertion measurement section.

5.2.2. Berthold Systems, Inc. EG&G Berthold has d e v e l o p e d a microwave s y s t e m p r i m a r i l y for the m e a s u r e m e n t of percentage moisture in foods and materials (Berthold 1992). Techniques for adapting the hardware for the measurement of food process parameters have been successful and are being explored for custom applications. The measurement technique depends upon the determination of the attenuation and phase shift of a microwave signal transmitted through the sample at 22 discrete frequencies. These values are processed via an algorithm to provide an accurate measure of the moisture content. Other types of interrogation schemes are available such as antennas which allow measurements to be made on products on a conveyor system. The instrumentation can be used on line or off line for laboratory applications. Since attenuation and phase shift are determined for whatever sample fills the interrogation volume, density must be determined if per weight data is required. Standard g a m m a ray absorption techniques can be used as an adjunct to compensate for mass. Since the microwave measurement signal is attenuated during transmission through the product, the size of the sample which can be interrogated will depend u p o n the penetration depth. Lossy products will require a smaller interrogation region. The system has been used successfully for foods such as cream cheese, butter and margarine, caramel, potato products, and other vegetables.

225

5.2.3. Distell Industries, Ltd. Distell Industries (1993) has developed hand-held instruments for the measurement of fat content in fish and meat. The technology evolved from the knowledge that the dielectric loss factor of fish has a reasonably linear dependence on water content (Ohlsson et al. 1974). In fish, fat accumulates at the expense of water and protein, making estimation of fat content based on water content practical (Kent 1990). In meat products, fat accumulation is independent of water and protein, i.e., it is additive, and thus makes the calculation of fat based on water amount more difficult. A hand-held meat instrument has been developed, but requires calibration depending on the type of meat measured. The unit works on the principal that the microwave attenuation of a strip transmission line depends upon the loss factor of the material with which it is in contact. Data indicates accuracies of 5% can be attained by the hand-held instruments, depending upon the range of fat measured. The instruments sell for approximately s Copies of references are available from Distell. 5.3.

Cut-Off Frequency Techniques

5.3.1. Epsilon Industrial, Inc. Epsilon Industrial has pioneered a concept for the m e a s u r e m e n t of constituents in pumpable process described as guided microwave spectrometry (GMS) (Epsilon 1994). GMS was originally developed as a means for determining moisture content (0 to 100%), salinity and other molecular concentrations but has been recently been expanded to foods as well as many other applications. The GMS technique depends upon a unique microwave property of a signal which is propagated or "guided" through a rectangular or circular tube. As an interrogating microwave signal is reduced in frequency, its wavelength increases. At a critical wavelength, d e p e n d e n t u p o n the dimension of the tube, the microwave signal can no longer propagate and is said to be cutoff (Marcuvitz, 1986). The attenuation of the signal increases very rapidly and the amplitude of the propagated signal decreases as the frequency is decreased through this cutoff region. This phenomenon is demonstrated in Figure 7. If the tube is filled with a dielectric material, the cut-off frequency will change d e p e n d i n g u p o n the values of r and r Thus, changes in constituency can be determined by measuring the cut-off characteristics of the propagating signal. In the impleF i g u r e 7. A m p l i t u d e Versus m e n t a t i o n of the GMS s y s t e m , the Frequency of Guided Wave microwave attenuation of the measurement (Courtesy Epsilon Industrial) cell, through which is passed the process

226 stream, is measured at 1700 discrete frequencies. The slope and position of the attenuation versus frequency curve are a measurement of the process flow parameters. Circular flow configurations are possible from 1 to 3 inches in diameter. Rectangular configurations are possible from 0.625 to 4 inches in maximum diameter. Temperatures up to 350 ~ F and pressures to 250 psi can be accommodated. Sensor construction is of stainless steel with polyetherimide windows for introducing and receiving the microwave signals. A d v a n t a g e s of the GMS system compared to other systems are threefold. There are reportable fewer potential sources of error in the system due to the very high number of data points utilized. Downstream process stream perturbation is reduced since no discontinuities are introduced into the process flow stream by the m e a s u r e m e n t sensor. Finally, restrictions due to high loss materials are reduced since the system depends upon the measurement of the transition from high signal strength to low signal strength. Thus, there is thus always enough signal strength above the cut-off frequency to make an accurate determination of cut-off frequency and initial attenuation slope.

5.4.

Resonant Frequency Techniques

5.4.1. KDC Technology, Inc. KDC Technology has developed a cost-effective microwave sensor technique for monitoring constituents and moisture in a wide variety of products including foods (KDC 1993). The KDC sensor is adaptable to measurement of process parameters of products contained in tubes, chutes, bins, vessels as well as moving on conveyer lines. The sensor consists of a patented resonator which is designed to have one side make physical contact with the sample under test. The shift in resonant frequency of the cavity, as well as the change in amplitude of reflection are a measure of the dielectric properties, r and E" of the sample. By measuring these two parameters and correlating them to process parameters, one is able to develop an algorithm which can be used for process monitoring. The sensors are available as a stand-alone entity for R & D and development purposes and can be utilized with any network analyzer operating over the frequency range from 0.5 GHz to 10 GHz. KDC can also provides a cost-effective network analyzer with internal software to calibrate microwave parameters against process parameters. Custom design programs to meet specific applications can also be undertaken. Sensor costs are on the order of $3,000; KDC network analyzers cost approximately $24,000. As has been indicated, the sensor can be adapted to almost any process application. The sensor is designed to withstand pressures to 300 psi and can be designed to withstand process temperatures as high as 450 ~ C using sapphire window materials. Accuracies between 0.01 an 0.5 % are typical. Sensitivities to 0.01% have been obtained and are primarily limited by the sophistication of the network analyzer utilized.

227 6.

ELECTROMAGNETIC DIELECTRIC SPECTROSCOPY

6.1. Introduction An exciting new area which has only recently come to maturity for analyzing and monitoring properties of foods and other materials has been termed electromagnetic dielectric spectroscopy (EDS). The fundamental concept, that of analyzing intrinsic properties of materials as a function of frequency, has been used for many years at various frequencies in the electromagnetic spectrum. EDS has also been used with good success, but on a limited basis, in commercial applications (see previous section). EDS, however, has only recently been made convenient as a diagnostic tool with the commercial availability of computer controlled network analyzers (Figure 4) coupled with dielectric measurement software (Hewlett Packard 1985, 1991). With such instrumentation, the complete spectrum of dielectric properties of a food or material from 0.3 MHz to 20 GHz can be measured and displayed within a few seconds. If the fundamental mechanisms of dielectric properties of mixtures and combinations are understood, a tremendous amount of information about the constituency of these mixtures can be ascertained. 6.2.

Combined Mechanisms (Dielectric Mixtures) A detailed review of the various mixture formulas has been presented by Kraszewski (1977), and Nelson, Kraszewski and You (1991). Nelson (1994) also has an extensive bibliography on the subject. Following is a brief description of the major mixture models. 6.2.1. Distributive Model The most simple model for the dielectric properties of foods is called the distributive model. Here, the dielectric properties of each constituent of the food are added together according to their fractional make-up of the total product. The model assumes that the various constituents of the food are distributed uniformly throughout the product. For example, Figure 3 shows the total dielectric loss factor for a 0.5 molar aqueous solution of water at two temperatures. Note that the total loss factor, a"t is the sum of the ionic and polar contributions, r and a'd. An example of loss factor properties of mustard, ketchup, m a y o n n a i s e and w a t e r is shown in Figure 8. A comparison of food constituents important in determining dielectric properties is shown in Table 2, (USDA 1963).

Table 2. Percentage Constituents of Sauces (Wet Basis) Food Water Mayonnaise Ketchup Mustard (yellow)

Water (%) 100 15 69 80

Fat (%) 0 80.0 0.4 4.4

Salt (%) trace 1.5 2.5 3.2

228 The loss factor for mayonnaise is very low both at high and low frequencies due to the low amount of salt in w a t e r a n d h i g h v a l u e of fat content. Tap water shows its characteristic a b s o r p t i o n peak at 18 GHz and a small increasing tail at low frequencies due to the small amount of diss o l v e d ions ( d e i o n i z e d water would show no tail). Figure 8. Loss Factor of Sauces M u s t a r d shows a slightly (Engelder and Buffier 1991) h i g h e r v a l u e at 20 G H z than ketchup because of its .higher water content and a considerably higher low frequency tail because of its higher salt content. From this example it is readily seen how electromagnetic dielectric spectroscopy can be a powerful and sophisticated diagnostic tool.

6.2.2. Complex Models It is interesting to note that the synergistic effect noted among some mixtures of solutions (Roebuck, Goldblith and Westphal 1971; Engelder and Buffier 1991) cannot be easily explained by the distributive model. For example, mixtures of glycerol and water and ethanol and water show a maximum in loss factor at 3 GHz at concentrations of 50% and 22% respectively with values that are considerably higher t h a n the loss factor of either constituent. These o b s e r v a t i o n s are u n d e r stood w h e n it is realized that water has a critical freq u e n c y a r o u n d 18 G H z and the heavier organic liqu i d s h a v e a critical freq u e n c y q u i t e low in the MHz range. If the dielectric properties were simply additive, the total loss factor w o u l d be a b i m o d a l d i s t r i b u t i o n w i t h the a m p l i t u d e s of the p e a k s v a r y i n g as a f u n c t i o n of concentration. Instead, an Figure 9. Loss Factor of Water-Methanol Mixture i n t e r a c t i o n takes place (Engelder and Buffler 1991) between the two molecules

229 forming a solution with a loss factor peak that depends upon both molecules. As the polar liquid is added to water, the critical frequency of the mixture will decrease in frequency until it reaches the critical frequency of the organic liquid in the MHz range, a", measured at any frequency will at first increase as a function of concentration, reach a maximum when the critical frequency equals the measurement frequency and then again decrease. The lower the measurement frequency, the higher the concentration required to reach the maximum ~". An example of this behavior is shown in Figure 9 for a mixture of water and ethanol. Note that the loss factor for the mixture at 2.45 GHz is higher than either of the constituents alone. Note also the lack of low frequency ionic contribution tail for the deionized water. It is thus again demonstrated that an understanding of the mechanisms of dielectric behavior of mixtures must be understood in detail in order to extract diagnostic information from the microwave spectrum. 6.2.3. Fricke Model

Finally, an area which is in need of much further research is that of the dielectric properties of two-phase systems such as frozen foods, emulsions, whips and foams. It is well known that the dielectric behavior of particles of one dielectric property imbedded in a substrate of another, behave very differently from a distributive mixture of both. Fricke (1955)developed a model for randomly oriented oblate spheroids suspended in a continuous medium. It is expected that this model may be used successfully to model two-phase food systems, but to date there is very little literature reporting such studies.

7.

CONCLUSIONS

Advances in electromagnetic dielectric measurement technology over the past five years have opened up many research opportunities for the understanding of multiphase systems such as frozen foods and emulsions. In addition, many examples of technology commercialization have resulted in excellent instrumentation for measurement and monitoring of process lines. It behooves the prospective customer to evaluate the available equipment, examine the advantages of each system in light of process requirements and make an educated judgement. With increasing interest in the application of microwave technology to food science, continuing advances should be experienced over the next five years, in both electromagnetic dielectric spectroscopy as well as process instrumentation.

8.

REFERENCES

ASTM 1986. S t a n d a r d M e t h o d s of Test for C o m p l e x P e r m i t t i v i t y (Dielectric Constant) of Solid Electrical Insulating Materials at Microwave Frequencies and Temperatures to 1650~ Document D 2520-86 (Reapproved 1990). Philadelphia, PA. American Society for Testing and Materials (ASTM).

230 Bengtsson, N. and Risman, P. 1971. Dielectric properties of foods at 3 GHz as determined by a cavity perturbation technique. Measurement on food materials. Journal of Microwave Power. 6(2):107-123. Berthold 1992. Microwave Moisture Analyzer LB 354 MICROMOIST. Berthold, 101 Corporation Drive, Aliquippa, PA 15001-4863.

EGG

Buckley, F. and Maryott, A. 1958. Tables of Dielectric Dispersion Data for Pure L i q u i d s and Dilute Solutions. National Bureau of S t a n d a r d s Circular 589. Washington, DC. (Available through National Technical Information Service, Springfield, VA. Buffier, C. 1993. Microwave Cooking and Processing: Engineering Fundamentals for the Food Scientist. Van Nostrand Reinhold. New York, NY. Buffier, C. and Stanford, M. 1991. The effects of dielectric and thermal properties on the microwave heating of foods. Microwave World 12(4):15. Debye, P. 1929. Polar Molecules. New York: Reinhold Publishing Corp. (Reprinted 1945 by Dover Publications, New York, NY). Distell Industries 1993. Distell Industries, Ltd. Unit 6, Old Levenseat, Fauldhouse, West Lothian EH47 9AD Scotland. El-Rays, M. and Ulaby, F. 1987. Microwave Dielectric Behavior of Vegetation Material. Ann Arbor, Michigan Radiation Laboratory, University of Michigan. (Available through National Technical Information Service, Springfield, VA). Engelder, D. and Buffier, C. 1991. Measuring dielectric properties of food products at microwave frequencies. Microwave World. 12(2):6-15. Epsilon 1994. Guided Microwav~ Spectroscopy. Epsilon Industrial. 2215 Grand Avenue Parkway. Austin, TX 78728. Fricke, H. 1955. The complex conductivity of a suspension of stratified particles of spherical or cylindrical form. Journal of Physical Chemistry 59:168-170. Geyer, R. 1990. Dielectric Characterization and Reference Material~. NIST Technical Note 1338. National Institute of Standards and Technology, Boulder, CO. Hewlett Packard 1985. Measurin~ Dielectric Constant with the h / o 8510 Network Analyzer. Product Note 8510-3. Hewlett Packard Corp., Palo Alto, CA. v

Hewlett Packard 1991. Dielectric Probe Kit 85070A. Hewlett Packard Corp. Palo Alto, CA. JMP 1980. Journal of Microwave Power 15(4).

231 KDC, 1993. The MDA-1000 Microwave Dielectric Analyzer for Process Monitoring and Control. KDC Technology Corp., 2011 Research Dr., Livermore, CA. Kent, M. 1987. Electrical and Dielectric Properties of Food Materials. Science and Technology Publishers. Hornchurch, UK. Kent, M. 1990. Hand-held instrument for fat/water determination in whole fish. Food Control, Jan. 1990, pp. 47-53. Kraszewski, A. 1977. Prediction of dielectric properties of two-phase mixtures. Journal of Microwave Power 12(3):215-222. Kraszewski, A. 1994. Proceedings of the Workshop o__n_nElectromagnetic Wave Interaction with Wa.ter and Moist Substances. IEEE - MTT Conference, June 1993. Atlanta, GA. Marcuvitz, N. 1986. Waveguide Handbook. (reissued on behalf of Institute of Electrical Engineers; available through IEEE Service Center, Piscataway, New Jersey). Peter Peregrinus, Ltd. London, UK. Metaxas, A. 1985. A unified approach to the teaching of electromagnetic heating of industrial materials. IJEEE. 22:108-118. MRC 1994. Microwave Research Center, 126 Water Street, Marlborough, NH. Mudgett, R. 1985. Dielectric properties of foods. In Microwaves in the Food Processing Industry, R. Decareau (ed.), pp. 15-37. Academic Press. New York, NY. Mudgett, R. 1990. Developments in Microwave Food Processing. In Biotechnology and Food Process Engineering, H. Schwartzberg and M. Rao (eds.) pp. 359-404. Marcel Dekker. New York, NY. Mudgett, R., Goldblith, S., Wang, D., and Westphal, W. 1977. Prediction of dielectric properties in solid fOod of high moisture content at ultrahigh and microwave frequencies. Journal of Food Processing and Preservation 1:119-151. Nelson, S. 1972. A method for determining dielectric properties at frequencies from 8.2 to 12.4 GHz. Trans ASAE. 15(6): 1094-1098. Nelson, S. 1991. Dielectric properties of agricultural products-measurements and applications. IEEE Transactions on Electrical Insulation. 25(5):845-869. Nelson, S. 1994. List of Available Publications. c/o S. Nelson, U.S. Department of Agriculture, Russell Agricultural Research Center, Box 5677, Athens, GA 30613. Nelson, S., Kraszewski, A. and You, T. 1991. Solid and particulate material permittivity relationships. Journal of Microwave Power. 26 (1):45.

232 Nelson, S., Stetson, L. and Schlaphoff, C. 1974. A general computer program for the precise calculation of dielectric properties from short circuited waveguide measurements. IEEE Transactions on Instrumentation and Measurement. 23(4): 455-460. Ohlsson, T., Enriques, M. and Bengtsson, N. 1974. Dielectric properties of model meat emulsions at 900 and 28 MHz in relation to their composition. Journal of Food Science. 39:1153. Ohlsson, T. and Bengtsson, N. 1975. Dielectric food data for microwave sterilization processing. Journal of Microwave Power. 10(1)" 93-108. Pace, W., Westphal, W. and Goldblith, S. 1968. Dielectric properties of common cooking oils. Journal of Food Science. 33:30-36. Phase Dynamics 1992. Process Monitoring Using a Microwave Based Measurement Analyzer. S. Shortes and B. Scott. Advances in Instrumentation & Control. 7(1):633640. (Available from Phase Dynamics, Inc., 1343 Columbia Dr., Richardson, TX). Risman, E 1994. Confined modes between a lossy slab load and a metal plane as determined by a waveguide trough model. Journal of Microwave Power. 29 (3):161-170. Roberts, S. and von Hippel, A. 1946. A new method for measuring dielectric constant and loss in the range of centimeter waves. Journal of Applied Physics. 17:610. Roebuck, B., Goldblith, S. and Westphal, W. 1972. Dielectric properties of carbohydrate-water mixtures at microwave frequencies. Journal of Food Science. 37:199-204. Stuchley M. and Stuchley S. 1980. Coaxial line reflection methods for measuring dielectric properties of biological substances at radio and microwave frequencies a review. IEEE Transactions on Instrumentation and Measurement. 29(3):176-183. Tinga, W. and Nelson, S. 1973. Dielectric properties of materials for microwave processing- tabulated. Journal of Microwave Power. 8(1):23-65. USDA 1963. ComposifiQn of Foods. Agriculture Handbook No. 8. U.S. Department of Agriculture. Washington, D.C. von Hippel, A. 1954a. Tables of Dielectric Materials, Dielectric Materials and Applications. MIT Technology Press. Cambridge, MA (out of print). von Hippel, A. 1954b. editor. Dielectric Materials and Applications. MIT Technology Press. Cambridge, MA (out of print). von Hippel, A. 1954c. Dielectrics and Waves. John Wiley and Sons, New York, NY (out of print).

Characterization of Food: Emerging Methods A.G. Gaonkar (Editor) 9 1995 Elsevier Science B.V. All rights reserved.

233

Chapter 11 Recent D e v e l o p m e n t s in the Microstructural Characterization o f Foods. M.G. Smart l, R.G. Fulcher 2 and D.G. Pechak 1 i Kraft Foods, Technology Center, 801 Waukegan Road, Glenview, IL 60025, USA 2 Department of Food Science and Nutrition, University of Minnesota, St. Paul, MN 55108, USA

1. I N T R O D U C T I O N The Art and Science of microscopy are quite old [e.g., see 1] and, from early times included studies of food structure [2]. As one might expect with such long usage in fields of endeavor as diverse as Geology, Botany, Medicine, Zoology, Materials and Food Science, a vast array of techniques has been developed. Many of the methods complement one another and are applicable across disciplines. For nearly two hundred year's microscopy as a science depended on the use of visible and, rarely, near UV electromagnetic radiation. In the early part of this century developments in theoretical Physics opened other avenues of "seeing" objects. The following is not an exhaustive list but does illustrate the expansion in the science of microscopy which began earlier this century and which continues today. First came the use of electrons in the forms of transmission and scanning electron microscopies [TEM and SEM; 3,4]. Then, relatively recently, came the use of sound as an imaging medium in the development of acoustic microscopy [5,6]. Most recently, near-field optical microscopy [7] and the family of scanning probe microscopies have been developed [8]. This explosive process of fundamental technique development is ongoing within microscopy broadly and is even gathering pace as a consideration of the Scanning Probe Microscopies will show. Apart from recent developments in microspectrofluorimetry and microchemistry, however, microscopy of food has had a different driving force from that of other microscopies, vi__.zz.,the development of sample preparation techniques which will allow the use of methods which are considered "standard" to other sciences. The reasons for this are clear. The range of materials considered as food is very broad, ranging from relatively unprocessed agricultural products such as tubers, seeds and fresh meats to natural cheeses, chocolate, and such highly processed products as bread, ice cream, process cheese and lunchmeats. As a brief example of the need to perfect preparative methods of difficult specimens, consider the ongoing search for improved or convenient methods of liquid sample preparation for SEM or TEM. Early methodologies employed agar tubes or capsules [9-14]. Veliky and Kalab [15] described the use of calcium alginate gel tubes for the encapsulation of liquid dairy products. Their method avoided elevated temperatures common to previous methods using agar and had benefits with samples containing relatively high levels of fat.

234 The principle took advantage of the gelation of alginate at room temperatures in the presence of calcium ions. Recently, a new method was described by Alleyene et al. [16] based on a "microcube" concept, and compared with older methods. The significance for microscopy of having to deal with widely divergent physical conditions found in foods cannot be overstated. The effect of product pH and buffering capacity on the efficacy of fixatives, the choice of fixative buffers, the restraints imposed by low viscosity products, etc., are all factors that must be considered on an individual product basis. By no means do we wish to diminish the difficulties faced by microscopists in the other sciences, particularly the biological sciences, but contrast these widely variable physical parameters with the narrower ranges experienced in Biology. Extracellular considerations aside, cytoplasts are rather similar physiochemically and structurally whether one considers an alga, a leaf cell or a human liver cell. Food Science, it seems, must rely on microscopy in other areas of science for advances in fundamental methods while it concentrates on finding ways in which to adapt them for its own use after finding suitable means of specimen preparation. In this Chapter we will concentrate on the methods of food microscopy rather than the details of results of individual investigations. The reader is referred to Holcomb and Kalab [17], Holcomb [18], and Kalab [19], for an introduction to the details of the microscopical literature of Food Science. We begin with a discussion of some of the more common microscopical techniques in use in Food Science, especially their more uncommon applications. These are rarely published and they are usually confined to industrial applications. Included is a section on quantitative light microscopy. Finally we deal with some of the newer technologies which have yet to be applied widely to food. For the sake of brevity, we must assume a basic familiarity with traditional microscopical methods on the part of the reader. If not, the reader is referred to texts by Hayat [20], Glauert [21 ], O'Brien and McCully [22], Pearse [23], Munck [24] and Vaughan [25] for details.

2. L I G H T M I C R O S C O P Y

2.1. Specimen Preparation The published literature of microscopy in Food Science falls into two broad categories: The first is focused on expanding our fundamental understanding of product structure/function relationships while the second is focused on practical issues of interest to manufacturers. both cases the images published tend to be selected partly for their aesthetics, naturally enough. Hence, the overwhelming majority of published micrographs are of specimens that have been fixed, usually in an aldehyde, dehydrated in alcohol, embedded in plastic, sectioned and stained. This is a classical response, rewarding in its wealth of data but time consuming in its execution. In Industry one is more often concerned with the rapid resolution of an issue using the microscope as a problem-solving tool. This can lead to the development of methods rarely referred to in the Literature, especially those which do not involve some or all of the steps necessary to produce sections for observation. There are three basic preparation techniques in light microscopical methods. Which is used depends on such factors as the necessity to retain structural relationships for high resolution work, the nature of the issue (basic chemico-structural information, resolution of organoleptic issues), etc. The preparative methods used are: smears (or comminution); handsections or cryosections and sections of fixed, embedded product.

235

2.1.1. Smears and Comminution Smear or comminution techniques are enormously rewarding because they provide a wealth of informational detail without a large time investment. The methods are more suited to industrial applications and have found little application in the literature. The techniques necessarily disrupt the long range structure of a product but are well suited to the identification and enumeration of particulates such as crystals (Figure 1), or the structure and microchemical composition of proteinaceous particulates, either of which can be the source of organoleptic issues such as graininess.

Figure 1. Crystals of calcium phosphate (arrows) in an unstained smear of a cheese sauce product. A small sample of the product was placed on a slide and observed by Kohler Illumination. If quantification is necessary, sample preparation is easily standardized to allow for particulate enumeration. (x 80)

Comminution also may be used to examine the stability of dispersed phases such as oil droplets. Depending on the viscosity of the product one simply mixes it or breaks it up in a solvent (usually water but, for example, use fresh soybean oil for chocolate), a buffer or the appropriate dyes (below). For instance, we mix easily dispersible foods (cream cheese, ice cream mix or tablespreads) with dyes on slides in a ratio of about 1:1 before examination. Where the dye is a diachrome (that is, highly colored) or is fluorescent in the absence of the substrate (for example, Acridine Orange) some attempt must be made to remove excess, uncomplexed dye molecules which might confound the interpretation. This can be done by reduction of the dye concentration or by making the preparation thinner. The advantage of these simple techniques is that a battery of microchemical tests to identify protein, lipid and carbohydrate can be completed on multiple samples in a very short time period.

2.1.2. Handsections and Cryosections. Where the product firmness is high enough, for example in seeds, most cheeses, processed meats, chocolate etc, we either handsection or cryosection. The advantage of handsectioning

236 or cryosectioning techniques is that, while they too allow the comparison of many samples within a short time, these methods retain much of the structural relationships. On the other hand, because of the rapidity of these techniques one can perform replicates of a particular experiment or vary more parameters than would be feasible by the processes involved in obtaining sections of plastic-embedded material. In industry, this speed is an extremely attractive attribute. In academia, it can serve as a rapid first screen of results. The methods are simple and largely have been borrowed from the botanical sciences [22]. All that is required is a steady hand, some practice and, depending on the application and the sample, sections as thin as 20 to 40 lam can be obtained. Handsectioning is accomplished by one of two methods. The first employs the traditional botanical technique using a single razor blade [22]. The product is held in one hand between the forefinger and thumb while the other hand gently passes a single-edged razor blade through the product. This method requires a good degree of firmness in the product. For softer products one uses the twin razor blade method. For the latter, two, single-edged razor blades are held together and passed through the specimen which is resting on a solid surface. This produces a section whose thickness can be controlled somewhat by varying the pressure on the sides of the blades. If the products are unstable in water (for example, some processed cheeses) they may be fixed briefly in 2% unbuffered formaldehyde for 10 min. This treatment will prevent product dissolution in aqueous dyes without changing reactions to most of them. See Figure 2 for an example of the result of handsectioning and staining a processed cheese loaf.

Figure 2. Handsection of a processed cheese loaf stained for fat with Nile Blue. The fat fluoresces yellow (white in the image). The figure shows good resolution of the droplets with minimal sample preparation. Epifluorescence optics (x 650).

All the imaging modes of light microscopy are amenable to handsectioning methods but none more so than epifluorescence. This is shown clearly by the work of Fulcher and later by that of Yiu [26]. A range of products is examined in these papers, from cereal seeds to cheese and yet the resolution obtained approaches that of sections from embedded product.

237 Freezing foods for cryosectioning in the thickness range of 10 to 20 lam is most often accomplished without any special treatment. Foods such as cream cheese, chocolate and ice cream mix all give satisfactory results. For other products such as process cheese, we have found that the glacially slow freezing rate of the cryostat leads to massive ice crystal damage. This issue can be alleviated partially by rapidly cooling very small blocks of material in liquid Nitrogen - as if one were preparing for Freeze Fracture TEM [20]. If necessary, the Nitrogen can be frozen by applying a low vacuum in a plastic dessicator. When the vacuum is released, the Nitrogen begins to melt, forming a "Nitrogen slush" and small blocks (5 mm cubes) of material plunged into such a mixture will usually freeze better. This is because there is less Nitrogen vapor at the product surface, leading to faster cooling rates and so to smaller ice crystals. Whether simply plunged into liquid Nitrogen or frozen in slush, the block is allowed to equilibrate to the cryostat temperature (usually -20~ and sectioned as quickly as possible since ice crystals will grow at that temperature.

2.1.3. Sections of Embedded Materials Micrographs from sections of fixed, embedded products are the most time consuming to obtain but have the best resolution and the largest range of staining schedules to choose from. A stained section of product embedded in glycolmethacrylate or LR White can take upwards of 10 working days to prepare. Sections of embedded material is the process of choice in the literature but in industry is reserved for those times when smears or handsectioning techniques fail to provide the requisite structural preservation, resolution or, in some cases, when the use of immunomicrochemical or lectin-based methods are called for. The figure (Figure 3) shows an example of an embedded cereal caryopsis sectioned and stained to reveal cell wall chemistry.

Figure 3. Section of cooked rolled oat sample fixed in 3% glutaraldehyde, dehydrated and embedded in glycolmethacrylate. The stain is Calcofluor which is specific for 13-Glucans in cell walls of cereals. Epifluorescence optics. (x 200).[From 26] Curiously there have been few examples in the literature (outside of work with cereals) which use light microscopy of embedded sections as anything other than an adjunct to TEM or SEM [27,28]. Thus, fixation and embedding schedules relate to the electron microscopies:

238 Most are variations of the standard biological non-coagulant protocols which use buffered aldehyde fixatives followed by osmication. See Hayat [20], O'Brien and McCully [22], and Pearse [23] for details. Exceptions in the food literature to this use of schedules common to Biology is the practise of Kalab and coworkers to match the pH of the fixative to that of the product, particularly in natural and processed cheeses [see references in 17,19]. Generally they have done this by eliminating the use of buffers. As far as we know, the rationale for this has not been spelled out by the authors but probably is an attempt to avoid protein conformational changes - even protein solubility changes - caused by the process of fixation itself. The pH of some products is near the isoelectric points of the major milk proteins and moving the pH away during buffered fixation theoretically may have drastic effects on structure, particularly ultrastructure. For example, if the product has a pH of 5.5, fixation at the traditional (biological) point of pH 6.8 may alter the structure of the protein matrix, particularly since it is well known that the buffers precede the arrival of the fixative in the center of a specimen block [20]. Of course, matching the pH of the fixative to that of the product must be done with the realization that the efficacy of fixatives, particularly glutaraldehyde, decreases markedly at lower pH values [20]. This is important because the image obtained from fixation at a pH where glutaraldehyde is less effective may introduce artifacts from subsequent coagulant fixation of the sample by the dehydration fluids. However, a vast amount of work has proceeded in food science with fixation at the higher pH and, when judged comparatively with like-fixed material, is a reasonable means of comparing samples. Another exception to traditional fixation methods is in immunomicrochemical work, where fixation schedules in the biological literature tend to be brief to allow for structural preservation while retaining antigenicity. In Food Science, immunomicrochemistry has not found much application as yet [29,30, see Chapter 15 in this book] and so these issues have not been addressed, particularly with respect to the very large concentrations of proteins of interest in some foods.

2.2. Staining Procedures. Specific staining to mark sites of chemical identity is an old part of microscopy [23]. In the biological literature it is properly called histochemistry or cytochemistry for observations of tissues and cells, respectively. In most of Food Science the more general term, microchemistry [23] is appropriate and will be used here. Microchemistry of foods has a long history in the cereals but only recently has been applied to other foods [31,32]. There are literally hundreds of staining schedules available in the literature, many of them of dubious specificity and use. The selection which follows shows details of methods for the most commonly used specific dyes. It is a general guide to the staining of major food constituents, particularly in dairy foods but may be generalized for other food types. Dyes more specific to cereal science have been discussed previously [29]. The first and fastest tests simply categorize product components as protein, lipid, carbohydrate, ions or nucleic acids. Man~ of these techniques have been documented elsewhere for biological materials [18,20,22,23,29,30] but are repeated here to emphasize their utility in all staining, especially in the more difficult foods such as process cheese, lunchmeats etc., which are heavily buffered. Where such is the case, the pH of the unembedded product must be matched to that of the dye so as not to confound interpretation.

239 This is because most dyes rely on electrostatic interaction for specificity. Hence, such factors as buffering capacity, pH and sometimes ionic strength can influence staining results. Once embedded, of course, these issues are not relevant.

2.2.1. Protein Markers The dye 1-Anilino 8-Naphthalene Sulfonic acid (ANS) has high specificity for protein. It fluoresces only when bound to protein [30]. In smears and handsections (i.e. unembedded materials) we have never observed it to effect emulsion stability in the manner more traditional protein dyes such as Coomassie Brilliant Blue or Fast Green often do. This relative pH independence probably is due to the mode of action of this dye. It becomes fluorescent in hydrophobic pockets on protein molecules [30] in contrast to the ionic bonding necessary for Fast Green FCF and Coommassie Blue [22]. We have not observed a strong cross-reaction with lipids, either, although a fluorescence of different spectral characteristics sometimes is seen. Depending on the circumstance, either a drop of a 0.01% W/V aqueous solution is mixed with the product in a test tube before making a slide or the dye is placed with the sample on a slide. A coverslip is added and the slide observed (see Figue 4) after UV excitation (360 nm). The dye-substrate complex often fades quickly in the strongly ionic environment encountered in foods so that photomicrography must be accomplished soon after moving to a spot on the slide.

Figure 4. Fluorescence micrograph of a cryosection of chocolate stained for protein with ANS. The protein particles in chocolate are discrete and there is little cross-reactivity of the dye with the lipids. Epifluorescence optics (x 230).

For sections of embedded material, we generally do not use ANS to localize protein. Instead we use Coomassie Brilliant Blue or Fast green FCF which are used as diachromes. Either dye is used at 0.1%W/V in 7% acetic acid [22]. Slides having a puddle of stain over the sections are gently warmed for up to 5 minutes, rinsed with distilled water and allowed to dry before mounting in immersion oil for observation. Figure 5 shows an example of cream cheese treated in this manner to reveal protein.

240

Figure 5 Cream cheese fixed in glutaraldehyde and embedded in glycolmethacrylate. The section was stained for protein with Fast Green. Protein has variable staining intensity, perhaps reflective of compositional differences.The fat (white) does not stain. (x 1300).

2.2.2. Lipid Colorants Nile Blue is used as a 0.01 to 0.1%W/V aqueous solution and is simply added to or mixed with the substrate. The active component of the dye is actually a minor "contaminant" of the solution, not the blue-colored material [31]. The preparations are viewed with 450-490 nm excitation (an "FITC" filter set, Figure 6). Emulsion stability is sometimes an issue in the presence of the cationic blue component of Nile Blue. In this case we use Nile Red, the pure form of this colorant. Nile Red solution is made fresh from a stock solution (0.1%W/V in acetone). This stock is added dropwise to water until a moderate blue color is seen and the solution is used immediately (it deteriorates quickly). For either colorant, the active molecule is fluorescent only when it is in a suitably hydrophobic environment. This usually means neutral lipid droplets [31] but other sites (aggregates of surfactants, the center of casein micelles, cutin plates in some seeds) are possibilities. To probe sections of plastic embedded material one can use either of these colorants or use a diachrome such as Sudan Black, Oil Red O or Sudan IV [22]. The first of these diachromes is the best because of its color gives good contrast.for photomicrography. The colorant is made up as a filtered, saturated solution in 70% ethanol or in 70% glycerol. Sections are covered for 5 or more minutes and rinsed with the appropriate solvent. Note that 70% ethanol tends to lift sections from slides. Also note that, since generally the lipid has not been fixed into the plastic in the process of obtaining light microscope sections, those dispersed phase lipid areas having a diameter greater than the thickness of the section will simply lose the fat on sectioning or staining, leaving a space.

241

Figure 6. Cryo-section of chocolate stained with Nile Blue. The fat (white areas) forms a continuous phase around protein particles and sugar crystals. (darker areas). Epifluorescence optics.(x 490).

2.2.3. Methods for Carbohydrates The only widely used method for carbohydrates is Lugol's iodine method for starch. The common formulation is to mix 0.3 g of Iodine, 1.0 g of potassium iodide and 100 ml water until the iodine dissolves [32]. The stain is added to samples and viewed by Bright Field optics, ff mobility of the substrate is an issue, allow iodine vapor to act on the sample (usually a handsection) in a small Petri dish for half an hour and mount the specimen dry on a slide for observation. Some moisture in the sample is critical to a successful result. For example, dry pasta will not react with iodine vapor to locate starch granules. The staining of starch is problematic in embedded sections as most solubilized starch fails to be retained throughout the process of obtaining sections. Miller et al [33] have successfully used lectin localization methods on sections of cereal grains to localize starch by relying on the specific recognition of terminal alpha-linked glucose by the lectin. A more general method for carbohydrates that can be used equally well on handsections and on sections of plastic embedded materials uses the Periodic acid Schiff's (PAS) reaction or one of its variants [29] for vicinal hydroxyl groups [22,23]. Sections are treated with 1%W/V Periodic acid at room temperature for 10 minutes and rinsed. The aldehydes created by this treatment are detected with Schiff's reagent (decolorized para-rosaniline) by immersion for 30 minutes. A red color (or fluorescence with excitation at 540 nm) indicates vicinal hydroxyl groups in starch and many other carbohydrates. If the specimen has been fixed with aldehydes then an aldehyde-blocking step must precede this reaction [22]. The best treatment is immersion for 24 hr in a saturated solution of dimedone.

2.2.4. Markers for Negatively Charged Groups Acridine Orange is a fluorescent marker that will reveal negatively charged groups. A 0.01 to 0.0001%W/V aqueous solution is added to the specimen - the concentration used depends on the difficulties posed by the background fluorescence of the dye. That is, the effective

242 concentration is product specific and is found by trial and error. The "FITC" filter set used for Nile Blue (excitation at 490 nm) is best here. Toluidine Blue O, a diachrome, can also be used for negatively charged groups, particularly in sections. A 0.05% solution in benzoate buffer, pH 5.5, is used on smears or sections. The dye is metachromatic (see 22 for discussion), meaning that the color observed in a dye-substrate interaction depends on the concentration of the negative charges on the macromolecule of interest. Therefore, such things as the nature of the charges, the distances between charges, pH and the composition of the molecule on which the charges reside all can effect the result. These characteristics are useful in Botany where morphological cues are available - for example, in the identification of certain cell wall components. In Food Science, negatively charged groups (such as carboxyl and phosphate) are so pervasive and on so many different molecules of diverse structure that this dye is not generally useful microchemically.

2.2.5. Markers for Positively Charged Ions A freshly made 0.001M aqueous solution of chlortetracycline (CTC) is used for the localization of calcium. Excitation wavelengths around 360 nm ("UV" filter set) give a yellow/green fluorescence, usually against a blue autofluorescence background. The dye is actually specific for divalent cations but in dairy foods this usually translates as calcium. At higher concentrations (0.01M approx.) CTC acts as a calcium chelator. This often gives valuable additional information about composite structures that are being studied: For example, dissolution of the particles in the more concentrated CTC constitutes evidence for a role for calcium in the integrity of the structures. Methods for other ions such as sodium, potassium etc., exist in the literature [see 23,30]. Based on the results using chlortetracycline, such methods should be relatively easily transferred to foods but have not yet been tried as far as we know.

2.2.6. Methods for Nucleic Acids The dye 4, 6-Diamidino-2-Phenylindole (DAPI) in 0.001%W/V aqueous solution can be used directly on smears, cryosections and embedded specimens to locate and count culture bacteria, without regard to their viability, in cheese and other cultured products. The dye reacts with nucleic acids by intercalation. Excitation at 360nm is best for this dye. It is worth noting two other facts about its use. DAPI cross reacts with dairy proteins, but the color of the protein-dye complex is different from that of the nucleic acid-dye complex (the latter is a steely blue/white) and so the two reactions may be discriminated. The dye also may take up to 15 minutes to enter bacterial cells, particularly spores, before fluorescence is observed. An alternative nucleic acid dye, Ethidium Bromide, has less contrast between the fluorescence induced in cells and the fluorescence of cross-reacting dairy proteins. It should be tried in other products such as meats if DAPI is not successful.

2.2.7. A Note on Autofluorescence of Proteins Autofluorescence can be very useful for localizing proteins without staining or other perturbations of the product but it can be a major issue in the interpretation of stained dairy foods. It remains as background fluorescence in the presence of fluorochromes giving similar colors, particularly the protein dye, ANS and FITC-conjugated molecules such as lectins.

243 Presumably because of the relatively high content of phenolic amino acids, products based on milk are strongly autofluorescent when viewed after either 360 nm or 490 nm wavelength excitation. Methods for fluorescence reduction such as prior staining with Toluidine Blue O or Evan's Blue can be employed, especially on sectioned material where artifactual collapse of the emulsion which is caused by the charged nature of the dye, is not an issue [22].

2.2.8. Differentiating among Proteins Commercially available antibodies to some proteins exist (e.g., bovine whey proteins) and caseins should be amenable to specific localization with the anti-phosposerine monoclonal antibody available from Sigma. Antibodies can be purchased with appropriate fluorescent (or electron dense) tags. The range of possibilities for selection of tags, fixation schedules, staining schedules and other observational parameters is enormous and has been explored only by a few groups in Food Science [34,35, see Chapter 15 in this book]. Of all microchemical methods, those involving antibodies are the most difficult to apply since their development relies on trial and error changes in parameters. More often than not these conditions are product specific. For the most part antibodies must be used on embedded, sectioned products but the requirement is not absolute. An important caveat is that because products often experience high heat and/or shear during manufacture, antibodies are not always the best choice for localization as they have relatively stringent requirements for specific interaction. In particular, protein denaturation at the active site which involves conformational changes can lead to false negatives. As a starting point, the reader is referred to the detailed protocols that are available for antibody staining methods [23,36]. An often overlooked fact is that many dairy and egg proteins are glycoproteins whose terminal sugars are known or can be deduced and, using standard techniques, should be amenable to sugar localization using lectins, thus localizing the protein. Lectin methods are highly specific and have the advantage over antibody methods in that they can be expected to be less susceptible to false negative results due to protein denaturation by processing conditions. That is, denaturation of the substrate molecule would only rarely entail loss of the sugar moieties. Horisberger et a1.[37], localized ~:-casein on thin sections of casein micelles using a lectin and Miller et al. [33] have reported a preliminary study of the uses of lectins in the elucidation of food structure. We have used FITC-labeled Concanavalin A to localize whey proteins in natural cheese. It cannot be stressed enough here that all the ingredients of the product must be known in great detail so as to avoid contaminating cross-reactions (false positives).

2.2.9. Differentiating among Carbohydrates (Gums) Our methods for gums rely on the diachrome, Alcian Blue, for carrageenan and agar, and fluorescently tagged lectins for xanthan, guar and locust bean gum (LBG). Of the most important gums, only CMC and pectin currently lack specific methods.

2.2.9.1. Carrageenan and Agar Carrageenan and agar can be localized in products using Alcian Blue, a positively charged diachrome, at pH 1.0. At this pH, only sulfate groups in products remain negatively charged and so will react, provided that the pH of the substrate has been dropped to match that of the

244 stain [23]. This is especially important for processed cheeses (see Figure 7) and lunchmeats with their heavily buffered formulations which, if not treated with acid, will keep the actual pH in the range where carboxyl and phosphate groups will react (pH > approx. 2.5) thus giving false positives.

Figure 7. The left hand side shows an image of comminuted Process Cheese Spread stained for sulfate groups by application of Alcian blue at pH 1.0. Carrageenan is localized as the darker staining material in the micrograph. The right hand side of the same Figure shows a similar cheese particle not stained with Alcian Blue. Kohler illumination. (x 750)

We obtained similar results in sections of embedded product. As far as we can ascertain, agar and carrageenan are the only common macromolecular food ingredients containing sulfate groups. Since this product did not contain agar, the stain was specific for carrageenan. Control slides of unstained, comminuted product did not show the characteristic carrageenan particles within the cheese matrix. There are several published methods for the detection of sulfated polysaccharides in the medical literature [23]; the one we have chosen is the fastest and simplest that retains specificity.

2.2.9.2. Locust Bean Gum and Guar Gum Sections of products that contain guar with or without LBG are probed with lectins by using minor modifications to standard procedures [38]. Briefly, sections of material embedded in glycolmethacrylate or Lowicryl are hydrated for 10 min. in 0.01 M phosphate buffered saline (PBS), pH 7.2. The slides are shaken gently to remove excess buffer and sufficient lectin applied to cover the sections (approximately 20 1~1 of solution at 250 mg protein/ml). The lectin used for guar and LBG is Type 1, Isolectin 4, from Banderia simplicifolia, tagged with fluorescein isothiocyanate (BS 1-FITC, Sigma Chemical Co., St. Louis, MO) This lectin has specificity for terminal ~-galactose [39]. Slides are incubated at room temperature in a humid atmosphere, protected from light, for 1.5 to 2 hr. While minimizing exposure to light, the slides are then thoroughly rinsed in PBS and distilled water, allowed to dry and the sections

245 mounted in fluorescence-free immersion oil for observation. As a control for non-specific binding, the lectin is incubated with its hapten, D+galactose (Sigma Chemical Co.), at a concentration of at least 0.1M, for 20 min. prior to following the procedure above. The product, cream cheese, contained both of the galactomannans Guar and LBG, and since the lectin recognizes both gums, they are indistinguishable by this test. When the hapten, D+galactose, was preincubated with the lectin, only residual autofluorescence of low intensity was visible on the sections and, when photographed and printed using the same exposure conditions, results in a dark micrograph (see Figure 8).

Figure 8 shows the result of probing a cream cheese product with BS 1-FITC, a lectin having specificity for cx-linked galactose (left hand side). The fluorescence is rather evenly distributed over the aqueous phase of the section. The right hand side of the micrograph, a control, shows the result of incubation of the lectin with its hapten before probing a section. No fluorescence is seen. Epifluorescence optics. (x 300).

2.2.9.3. Xanthan Gum To detect xanthan one could use the procedure of fixing, embedding and sectioning as used for LBG and guar, with the addition of the LBG incubation step as described below. However, we will describe an alternate technique in which structural integrity is sacrificed in order to obtain rapid information about the presence or absence of a gum in a product: No detailed structural information about the state of the molecules in the product can be inferred except for gross aggregation (non-dipersion) of the gum. The advantage of this modification is that it gives a reliable result in about three hours rather than the one to two weeks necessary for the embedding procedure. A quantity of the product is infiltrated into a glass fiber filter with the aid of a Buchner funnel and vacuum. If necessary, viscous and solid products can be diluted or comminuted in Sorenson's buffer, pH 7.0, to allow penetration into the glass fibers. Small squares of the filter (about 2 mm on a side) are cut and placed in PBS for 10 min. The squares are moved to a multiple well spot dish and covered with a 0.1%W/V aqueous solution of LBG, pH 7, for

246 lhr at room temperature. They are rinsed thoroughly in three changes of PBS and blotted gently to remove excess buffer. In a new well, the squares are just covered with BS 1-FITC (about 50 lal at 250 mg/ml) and left in the dark, at room temperature for 1 hr. Again, they are rinsed thoroughly before observation by epifluorescence (450-490 nm excitation, Figure 9). Two controls are run concomitantly. Either the LBG step is omitted or the lectin is reacted with its hapten (galactose) prior to staining. Previous studies in this laboratory have shown that non specific binding of LBG does not occur in the absence of xanthan.

Figure 9 shows the result of testing a Pourable Dressing for the presence of xanthan. The sample was infiltrated into a glass fiber filter and the filter probed sequentially with LBG solution and BS1-FITC. White areas on the micrograph are sites of xanthan localization. Epiflurescence optics. (x 300).

Although no microstructural inference can be made because of the mechanical disruption caused by vacuum infiltration into the filter, and in this case there is apparently no aggregation issue, Figure 9 clearly shows the presence of the gum. We must point out, however, that the use of LBG as a probe is not without caveats. LBG will cross react with carrageenan if it is present in the product also and will cross react to a lesser extent with carboxymethylcellulose. Any use of LBG as a probe for xanthan in products containing both carrageenan and xanthan (or, for that matter xanthan with guar/LBG) must be interpreted cautiously. However, the autofluoresence intensity of the controls was quite low and, when photographed under the same conditions, resulted in featureless, black micrographs (not shown). In the absence of fluorescence intensity quantification (Section 2.3, below), it is valuable to compare micrographs taken under identical conditions because the human eye is notorious for its ability to accommodate to low light intensities, making visual comparisons at the microscope unreliable. At Kraft Foods, we have had occasion to use this rapid sandwich technique on unembedded cream cheese, viscous dressing and ice cream mix, looking for xanthan or guar/LBG. We have also probed sections of embedded materials for xanthan. In other words, the rapid and embedded techniques are fully complementary.

247

2.3. Quantitative Light Microscopy In Food Science quantitative analyses derived from light microscopy remain relatively infrequent, despite the increasing use of computerized detectors, automatic scanning stages and stabilized light sources. In the past few years, however, a number of quantitative instruments have found their way into routine use, ranging from relatively inexpensive and simple systems which primarily use visible light, to modular systems capable of quantitative assessment of ultraviolet, visible, and near infrared signals. The latter is typical of a range of instruments developed in the past few years by Carl Zeiss Ltd., and many of the examples cited in the remainder of this segment were developed using the Zeiss UMSP80 instrument. It is equally capable of characterizing absorption and/or fluorescence properties of cellular components or applied microchemical reagents, in either the reflectance or transmittance mode. The UMSP80 shares several properties with other commercial systems, and instruments of this type provide extraordinary insights into composition, structure, and consequent functionality of important food materials and constituents. Where practicable, fluorescence probes are preferred for maximum sensitivity and specificity. Current sensors are quite adequate to detect relatively faint fluorescence signals, and other advantages, relating primarily to improved specificity are readily apparent. Diachromic probes or reagents which are detected in traditional bright field applications are also extremely useful, however, and the well equipped microscope photometer adapts readily to either mode. In addition, optical systems capable of focusing and detecting either transmitted or reflected light signals are also well developed. Quantitative light microscopy centers on the direct, in situ, characterization of naturally occurring substances, of applied fluorochromes, diachromes or of other highly fluorescent or absorbing substances found in the cells and tissues of raw or processed foods. Measurement may involve spectral characterization, mapping of component distribution, or kinetic analysis of changes in absorption or fluorescence intensities. Provided that the investigator adheres to the basic principles of spectral analysis defined so thoroughly by Piller [40] and Dhillon et al. [41], exquisite definitions of constituent properties are possible, with concomitantly high levels of sensitivity rarely matched by other analytical techniques. Equally important, food scientists now may enjoy the opportunities provided by a rapidly expanding list of highly specific cellular probes which are often readily adapted to food applications [30]. Other sources of specific reagents are also available, and a useful list of fluorescent probes for food analysis has been compiled by de Francisco [42]. These lists are expanding daily, and the range of applications is limited only by the investigator's imagination. In the following sections, a few selected methods are described in an attempt to provide insights into the many different and diverse applications of microscope photometry. More extensive details have been published elsewhere [29,43]. New applications are under constant development and many more will occur as the challenges of food chemistry accelerate. Because fluorescence offers such dramatic advantages in sensitivity and selectivity the following remarks will focus primarily on fluorescence detection only.

2.3.1 Advantages of Fluorescence Analysis Because fluorescent objects are essentially self-luminous and viewed against a dark background, both the resolution and sensitivity are maximized. Resolution is maximized because the objects essentially emit light, often at relatively short wavelengths. Sensitivity is

248 maximized because the high contrast between the luminous object and dark background offers maximum contrast, and hence detectability. In addition, an object can be smaller than the limit of resolution of the light microscope and still be detected as a luminous source. The high contrast offers increased ability to differentiate the diverse mixtures of biological compounds normally encountered in food materials. Reflectance optics (epi-illumination see [29]) also improve sensitivity, precision, and selectivity rather dramatically in comparison to older transmission techniques, in part by minimizing the effects of specimen thickness. An essential component of microscopic photometry is the adaptation of microcomputers for operation of the wide range of filters, monochromators and detectors which are necessary for routine use in food and biological research programs. The accelerating improvements in microcomputer architecture and software, and the associated developments in optical systems ensures that these technologies will combine to provide unprecedented ease, speed and precision of microchemical characterization of foods. Many software programs are now available for routine use on IBM compatible 486 MHz systems (or better), meaning that significant opportunities exist for rapid accumulation of large amounts of spectral data. Furthermore, the ease with which data are transferred to other analytical programs for statistical evaluation (e.g., principle component analysis and a range of regression analyses) improves the utility of the measurements rather dramatically. Multivariate calibration and analysis of fluorescence data in particular is now a well developed tool and provides interpretative opportunities previously unavailable to occasional users of fluorescence photometry instruments [44]. Commercial software programs such as Unscrambler (CAMO A/S, UUC, Trondheim, Norway) are now available for detailed analysis of multiple spectra, and these have been used widely in the food industry. These powerful interpretive tools provide opportunities for the microscopic spectroscopist to unravel the very many different signals which may emanate from the complex biological structures typical of foods and raw materials. It provides systems for differentiating background light, stray scattered light, and other sources of non-specific energy which contribute to the detected signal. Use of such interpretative programs is highly recommended if comparative and continuing calibration is desired.

2.3.2. Instrumentation Fluorescence microspectrophotometers consist of several essential components and a number of additional items which are optional but may be useful in selected applications. The first component, the light microscope, is the obvious core of the system and for many uses, needs only to be a high quality research microscope, including fluorite or similar objectives which optimize fluorescence. These are available from most major microscope manufacturers. If ultraviolet analyses are needed, however, the instrument must include UV transparent quartz optics throughout the system. This adds considerable expense to the system, restricting the use of UV methods. It is, however, of some use in special applications relating to natural autofluorescence of proteins, and to measurement of structures which contain significant concentrations of phenolic polymers such as lignin. The latter are found in most fibers of plant origin, and at the University of Minnesota we have used this approach in both absorbance and fluorescence mode to differentiate food fibers derived from a range of grain products [45]. An appropriate instrument which combines scanning stage,

249 UV/Visible/NIR optics and both epi and transmitted illumination is described in some detail elsewhere [29]. Briefly however, the instrument with most flexibility and utility includes a scanning stage with 0.25 I.tm matrix step-scanning capability, a photomultiplier and PbS detector for UV/VIS and NIR detection respectively, and appropriate monochromators to allow illumination in all spectral regions. For fluorescence analysis, a minimum of two monochromators is necessary, to allow characterization of both excitation and emission wavelengths. Illumination is provided by mercury (HBO) illuminators for routine fluorescence work, xenon (XBO) illuminators for short wavelength (UV) analysis, and/or halogen illuminators for bright field and long wavelength fluorescence analysis. In addition, for highest sensitivity and efficiency in the fluorescence mode, an epi-illuminating system and infinity corrected optics are also common components of modem fluorescence microscopes and both components are compatible with quantitative analysis. Fluorescence microspectrophotometry typically provides chemical information in three modes: spectral characterization, constituent mapping in specimens, and kinetic measurements of enzyme systems or photobleaching. All three approaches assist in defining chemical composition and properties in situ and one or all may be incorporated into modem instruments. Software control of monochrometers allows precise analysis of absorption and/or fluorescence emission characteristics in foods, and routine detailed spectral analysis of large numbers of food elements (e.g., cells, fibers, fat droplets, protein bodies, crystals, etc.) is accomplished easily. The limit to the number of applications is really only that which is imposed by the imagination - there are quite incredible numbers of reagents which are capable of selective fluorescence tagging of food components, and their application is as diverse as the variety of problems in the research laboratory. Spectral scanning of food materials is a useful method for determining their composition and relative concentrations. Briefly, the object is illuminated with the range of wavelengths appropriate for the specimen in question, and both the excitation (or absorption) wavelengths and emission wavelengths obtained by sequential scanning and intensity detection through the appropriate wavelengths. For example, most cereal grains (and many of their products) contain high concentrations of low molecular weight phenolic compounds such as ferulic acid, as well as a range of higher molecular weight phenolic polymers (e.g., lignin). These compounds are major determinants of grain and product quality and because they are fluorescent and may be examined directly using fluorescence optics without other chemical enhancements, they are prime candidates for quantitative microscopy. Figure 10 shows the relative fluorescence intensities of cell components in different portions of the outer regions of the wheat kernel, and includes both excitation and emission properties of these naturally occurring phenolic compounds. Current software allows acquisition of large numbers of individual scans in a relatively short time, which in turn ensures that measurement systems such as this climb from the realm of the research analysis to routine applications in the food industry.

250

Figure 10. Excitation (left) and emission (fight) spectra optimized for aleurone tissue showing intensity differences between aleurone, endosperm, and pericarp tissues. The emission monochromator was set at 445 nm for excitation spectral scans and the excitation monochromator was set at 350 nm for emission spectral scans. RFI = relative fluorescence intensity. (From [29])

This observation has become an important contributor to the development of rapid, automatic scanning of outer tissues of grains (primarily bran tissues) which contribute both strong color and taste characteristics to grain products such as wheat flour. The ability to measure both the concentration and distribution of such components is paramount to quality control in bakeries, and to definition of raw materials. An example of the systems necessary for routine analysis is included in a following section. Similar approaches are also available for both UV and NIR absorption measurements. The former provides a useful potential method for characterizing and identifying food fiber sources based on lignin absorption spectra. For example, fibers from diverse seed sources, and perhaps non-seed sources can be characterized and differentiated simply on the basis of UV absorption properties. Microspectrophotometry was used to differentiate and characterize several common insoluble fibers used in bakery and dietary products. Thirteen fiber samples were analyzed: oat (7), cottonseed (1), soy (2),pea (1), alpha-cellulose (1), and raw ground oat hulls. A computer-controlled microspectrophotometer with high pressure xenon lamp and a grating monochromator was used to measure absorbance spectra at 230-350 nm. Each fiber type had a distinctive absorption spectrum, and showed characteristic spectra that could be used to distinguish one fiber from another. Alpha-cellulose, soy, cottonseed, and pea fibers spectra were distinctly different from oat fibers and hulls (Figure 11, Table 1).

251

Figure 11 :--Mean absorption spectra: a composite plot comparing three oat fibers identified in Table 1" raw ground oat hulls (- --), oat fiber #2 (---), and oat fiber #4 ( ). Table 1 Absorption characteristics of fiber samples a Sample Raw Ground Oat Hulls Oat Fiber #1 #2 #3 #4 #5 #6 c #7 Cottonseed Fiber Alpha-Cellulose Soy Fiber #I #2 Pea Fiber

a b c

Maximum absorption wavelengths (nm) b 240 + 5 240 + 5

280 + 4 278 _+ 4

-318 (sh) - 3 2 6 (sh)

241 + 4 242 + 5 242 + 5 233_+3 246 252 _+ 2 265 _+ 2 272 _+ 5 253 _+ 3

282 _+ 4 285 _+ 1 281 _+ 4 275 + 1 277 - 295 (sh) - 295 (sh) -295 -295

-320 -325 -325 ND ND ND -320 ND ND

< 230 < 230

- 295 (sh) -280

ND ND

(sh) (sh) (sh)

(sh)

Mean of 50 determinations for each fiber sample; standard deviations based on six randomly chosen spectra and given for definitive peaks. sh = shoulder, ND = none detected. Sample contained 2 sub-populations, each with separate standard deviation determinations

252 Similarly, near infrared (NIR) analysis is a useful method for characterizing strong IR absorbing substances, such as fats and water in foods and raw materials. Applications of NIR in the characterization of foods are described in Chapter 18 of this book. At the University of Minnesota we have used the scanning microspectrophotometer in NIR mode to assess a wide range of components in grains and foods, including water content in grains and grain products, and solubilized sugar droplets on the surfaces of stored bakery products. Once again, the primary advantage of this approach is that very small amounts of material can be characterized chemically, and in a short time. A typical NIR spectrum of a section of both air-dried and soaked wheat kernel sections shows clearly the potential for measuring water content in situ using these techniques (Figure 12). Note that although the spectrum is not different from those associated with conventional NIR measurements of grain products, it differs in the fact that the signal was obtained on regions of the sample approximately 20-30 I.tm in diameter!

Figure 12: NIR reflectance scans of microscopic sections of wheat endosperm (measuring spot approximately 50 x 50 I.tm diameter) before (solid line) and after drying.

Once a particular food constituent has been characterized and/or identified by spectral analysis, it becomes a relatively simple matter to quantify the material further by mapping its distribution or by quantifying the material by associated imaging procedures.

253 Mapping involves the measurement of absorption or fluorescence intensities at fixed intervals across a specimen. This approach requires that the instrument be equipped with a scanning stage, and a number of these is available commercially, ranging in capability from very fine and relatively slow matrix step scans (0.25 ktm intervals) to scans at 10.2000 lxm intervals. This permits either overlapping scans such that continuous images of fluorescence or absorbance values are created, or it permits scans which are essentially sampling procedures which provide statistical evaluations of the total fluorescence or absorbance across a specimen. Figure 13 provides an excellent example of the former, in which high resolution scans have allowed detailed analysis of the distribution of the proteins in individual starch granules. In this example, an isolated starch granule was placed in glycerol on a quartz microscope slide and scanned in absorbance mode at 1.0 x 1.0 ~m matrix intervalS at 280 nm to show the distribution of protein(s) in the granule. In combination with detailed extractive and SDS eletrophoretic procedures, this approach has allowed development of both compositional and distribution data regarding starch granule proteins

[57].

Figure 13: "Map" of protein distribution in single wheat starch granules before (left) and after (fight) sodium dodecyl sulfate extraction. Granules were scanned after staining with acid fuchsin (from [46]).

Specimen scanning procedures are also useful on a larger, semi-micro scale. We routinely use the scanning microspectrophotometric approach in fluorescence mode to evaluate distribution of functionally important constituents in raw materials such as grains. For

254 example, Figure 14 shows the result of scanning a complete cross section of a wheat kernel with fluorescence filters set at 365 nm excitation and 450 nm emission. Using relatively crude scan step intervals (--100 x 100 ~tm). This represents a simple procedure to exploit natural fluorescence to map phenolic compounds (primarily ferulic and coumaric acids) in grains. Similar scans have shown the distribution of 13-glucan polymers in oats and barley [47,48]; and Figure 15). On an even larger scale, the same instrumental approach has been used to map protein foulants on ultrafiltration membranes used in the dairy industry [49].

Figure 14: Distribution of natural fluorescence in an unstained oat kernel (a) Profile: plot of distance (x-axis) Vs relative fluorescence intensity (y-axis) in scan through the mid-point of a kernel cross section. (b) Intensity profile showing fluorescence of phenolic constituents in an oat cross section.

255

Figure 15" Fluorescence intensity profiles of cross sections of three oat kernels with different levels of mixed-linkage beta-glucan after staining with the beta-glucan specific fluorochrome Calcofluor (highest peaks indicate highest concentrations of beta-glucan). Note that the polymer is not uniformly distributed throughout the kernel. Instruments of this type may also be used quite effectively to evaluate kinetics of timedependent changes in foods, be they enzymatic or reactive changes of other types. The computerized data-acquisition capabilities of these instruments allow precise measurement of absorbance or fluorescence changes, often over very brief time periods (~ milliseconds). This is particularly useful for analysis of fluorescence decay rates, and in measurement of enzymatic activity in situ. A number of enzyme substrates is available commercially which, although non-fluorescent initially, release fluorescent reaction products after hydrolysis by appropriate enzymes. This kinetic approach is a relatively underused capability of computerized microspectrophotometers, but one which has considerable capability for comparing activities in individual cells or cellular components. Fluorescein diacetate, for example, is a non-fluorescent compound which releases intensely fluorescent fluorescein on hydrolysis. This product is readily quantified in individual cells which have high levels of esterase [50]. Changes in surface or internal color of foods may also be evaluated over time by these methods.

2.3.3. Hybrid Systems The scanning microspectrophotometer obviously provides a range of opportunities for detailed evaluation of the intimate association between composition and structure in raw and

256 processed foods. However, these instruments tend to be rather expensive, and are rarely useful for routine, on-line or at-line analysis in food processing environments. Recently, hybrid systems which combine relatively simple spectral measurements with digital image analysis of microscopic images have evolved to the extent that they are capable of providing rapid analysis of microscopic structures with minimal specimen preparation. The rapid evolution of microprocessors with which large data fields can be processed easily, the recent improvements in fluorescence standardization, inexpensive scanning stages, and high speed image processing boards when combined as a package now allow numerous measurements which previously had been possible only with microspectrophotometers. One of these instruments, the Dipix I440F (Dipix Technologies, Inc., Ottawa, Canada) exploits the principle that any food component clearly identified under a microscope can be quantified accurately and rapidly using automated, filter-mediated digital image analysis (DIA). The I440F uses several different common illumination systems, including (a) incident light for fluorescence analysis of natural constituents or added fluorochromes, (b) transmitted light illumination for diachromes and detection of food components with measurable absorbances, and (c) combinations of the above. The instrument is modular, relatively easy to operate, and provides measurements of several hundred fields of view in a few minutes. Although the I440F continues to evolve as applications are clearly identified, it is becoming apparent that the approach embodied in the instrument, n a m e l y t h e coincident rapid measurement of structure and chemistry, is both a novel and rewarding one. For each programmed measurement module provided by the Dipix instrument, the instrument records images at fixed wavelengths from a large number of microscopic fields of view. The number of fields is established according to the standard deviation and reproducibility of the data from each module, and this value is determined by the user. The instrument employs a standard commercial light/fluorescence microscope fitted with several modifications for routine analysis. Two types of samples are in common use for analysis by the I440F and each includes customized software and sample holders calibrated for individual instruments. The sampling methods include use of (a) dry, powdered samples (approximating those in use in common near infrared reflectance analyzers), and (b) wet monolayers which are essentially samples placed on a large scale microscope slide and a cover glass. A holder for dry powdered samples (e.g., flour, starch, bran, fiber) consists of a glass window bonded to a shallow rigid frame to form a rectangular container. The sample is placed in the container, leveled with a scraper, and covered with a metal backing designed to provide uniform compression. Samples are mounted on a fast scanning stage, illuminated with a mercury lamp through appropriate filters systems to selectively highlight specific objects, and several hundred microscopic fields of view are obtained through the glass window and analyzed in a few minutes. Each field is obtained and measured in well under one second. Although there are a number of important food components which are naturally fluorescent (e.g., cereal brans, lignified materials such as pea, soy and cotton fiber, and even proteins and pigments), detection of many food components requires application of specific fluorochromes or diachromes. Therefore, quantitative analysis using microscopic imaging also requires judicious use of sensitive dyes or stains suitable for visualization and rapid measurement. The dyes must be stable, non-toxic to liing cells, easily and inexpensively

257 obtained, and of consistent quality. A number of such dyes is available, and even food grade colors can be employed for rapid measurements, provided they exhibit specificity for particular components. To measure dyed microscopic components rapidly, instruments such as the I440F include a "wet" sample holder designed to allow examination of a relatively large field of view (--2.5 x 4.5 inches) of uniformly distributed particles with little, if any, evaporation. Consequently, this holder also consists of a glass window bonded to a shallow metal frame. A trough is etched around the edge of the glass window to provide a seal for the liquid once a cover glass has been added. The samples (usually specimens suspended in liquid fluorescent or absorbing dye) are spread over the surface of the window and a cover glass added to provide a "monolayer" of sample. The advantages of this system are that a wide range of dyes can be used for different chemical constituents, more than one dye can be used at a time (and measured sequentially), and the specimen can be examined with either epi-illumination or transmitted illumination. Common assays include measurement of the percent of damaged starch granules in a flour or starch preparation, and frequency distributions of particulates in liquids. Table 2 and Table 3 illustrate typical measurements obtained on common food materials using the Dipix instrument. Table 2 Comparison of ash and bran components in selected flours using DIA(a) Whole Wheat Samples

Ash (%)

Aleurone Pericarp Frangments (%) Fragments (%)

Total Bran (%) (Alerone and Pericarp)

HRS1 HRS2 HWW1 HWW2

1.59 1.54 1.46 1.49

6.94 7.90 7.45 11.47

13.87 11.80 18.77 19.20

6.93 3.90 11.32 7.73

(a) HRS and HWW refer to hard red spring and hard white winter wheat types respectively. The grains were ground and analyzed for aleurone fragments and pericarp fragments using the Dipix I440F image analyzer in fluorescence mode. Although there were few differences in ash content (a traditional measure of variation) among the four flours, the bran components were very different. In addition, the ash test represents a 16-24 hr analysis while the imaging approach is complete within 3-5 minutes.

258 Table 3 Damaged Starch in Flours: Analysis by Enzymatic Vs Imaging Methods Flour Type

% Damaged (Enzyme)

Undamaged Damaged Granules / Field Granules / Field

% Damaged (DIA)

HRS1 HRS2 HWW1 HWW2 SRW

5.70 6.94 ~l.S1 5.53 nd

8.65 6.23 6.39 6.77 11.88

14.34 11.19 13.81 13.06 6.38

1.44 0.79 1.02 1.02 0.78

(a) Flours from selected hard red spring (HRS), hard white winter (HWW) and soft red winter (SRW) wheats were analyzed for damaged starch content using conventional enzymatic methods, and by digital image analysis in fluorescence mode with the Dipix I440F. The latter provides values for damaged starch in a few minutes while the traditional methods take several hours.

A number of additional automatic measuring protocols is under development, but the primary advantage of the instrument is that it is essentially an automatic measuring microscope, with automatic control of light intensity and filter systems, extremely rapid autofocusing of images, and no condensers or diaphragms to adjust. It provides data and views of biological materials which are often difficult or impossible to obtain with conventional microscopic or chemical techniques, and further developments of similar systems will allow unprecedented analysis of interactions among diverse food ingredients.

3. C O N F O C A L

MICROSCOPY

The two major forms of Confocal Microscopy (CF) have excited a great deal of interest in the biological sciences. Despite operational differences, both forms of the technique have one basic principle: A thick specimen can be optically sectioned to a minimum thickness of about 0.5 l.tm, depending on structure and the numerical aperture of the objective lens [51]. Sections may be recorded in the xy or zx or zy planes and, if necessary, subsequently rearranged as three dimensional reconstructions. As an example of the power of this method of imaging objects, B. R. Masters [52] has a video of a reconstructed rabbit cornea. Using confocal microscopy, he was able to optically section the living tissue at a thickness of approximately one lam, through about 1200 lam, from the base of the cornea to its surface. With the aid of a computer software package, he created a movie of the tissue as it "revolves" in space. Admittedly, the cornea is the near perfect tissue to use for this technique because it has evolved to transmit light without distortion. Nevertheless, the demonstration of the power of the technique is impressive. The history of the development of confocal microscopes is beyond the scope of this Chapter. Parts of the developmental history of CF

259 can be found in the book edited by Pawley [53]. This book also gives an excellent account of the Physics of the technique. A bibliography of CF to 1993 is available [54]. Confocal microscopy of foods is not as straightforward as that of biological specimens. The reasons are not difficult to discern. Biological specimens often contain up to 90% water. In some plant cells the large central vacuole may increase this percentage even further. The occurrence of dispersed phases, such as crystals or oil, is uncommon, at least for structures other than seeds, and so the passage of light is not influenced strongly by refractive or opaque objects and the resolution is usually limited by microscope and preparation specific aberrations [51 ]. For foods, on the other hand, the spherical dispersed phase of emulsions and foams and the concentration, reflectivity and density of protein in food gels both efficiently scatter light, limiting the depth one can "see" into a thick preparation. To take an extreme example, we estimate that we can optically section mayonnaise to about 14 lam by confocal laser scanning microscopy - 10% as far as in a cornea! There has been little published in the literature of Food Science which has used CF. Heertje and co-workers have explored the instrument for a number of foods, including tablespreads (Figure 16), cheese, mayonnaise and rising dough [55]. In a separate paper they have imaged the dynamics of eri~lsifier replacement at a phase boundary [56]. At Kraft Foods, CF has been used to image~a range of products with issues ranging from air cell fusion in dessert toppings to graininess in process cheese. An issue in any form of CF is that one obtains an image that differs from "conventional" images from SEM, TEM or light microscopy. The problem is most acute in CF systems which do not give a "real time" color image. One has to sort out which parts of the image are due to reflectance, autofluorescence or selective staining. This can be done with suitable controls to eliminate elements systematically. For example, an unstained preparation will reveal the reflectivity and autofluorescence patterns. Subsequent elimination of autofluorescence with non-specific staining [e.g., Evan's Blue, osmium tetroxide, see 29] will reveal the pattern due solely to reflectance. Subsequent staining with a fluorochrome will then be interpretable as specific.

Figure 16. Confocal Laser Scanning micrograph of a 40% fat spread stained with FITC for protein. The aqueous phase is clearly visible (the white areas) and fat droplet sizes in the micrograph are comparable to SEM imagery. (x 3750).

260 4. E L E C T R O N

MICROSCOPY

Electron microscopy in both its transmission and scanning modes has been the technique of choice for the vast majority of literature studies, particularly structural studies [see compilations in 17,18,19]. Other reviews and texts which cover the techniques of microscopy used in Food Science include Aguilera and Stanley [2] and the earlier work of Vaughan [25]. In addition to covering many of the current uses of microscopy in Food Science the former book [2] covers the history of food microscopy and an introduction to the major microscopy instruments. It also presents the information in such a manner as to demonstrate how microscopy can be a useful tool in food science for both the product developer and the basic food scientist. Some of the more noteworthy EM techniques which were first described prior to 1990, deserve mention because of their contribution to the advancement of food structure knowledge. The utility of cryo-SEM in food systems was clearly illustrated by Sargent [57] including over 40 micrographs of diverse food systems as visualized by cryo-SEM techniques. Since that time numerous papers can be found in the literature utilizing cryoSEM methods. An ever expanding list of unique and novel applications to new foods has come from the papers of Heertje and his co-workers and is summarized by Heertje [58]. Here we must focus on the newer techniques which have been developed in recent years; the first of these is environmental SEM.

4.1. Environmental Scanning Electron Microscopy The recent introduction of environmental SEM (ESEM) [see 59] was met with excitement, at least from microscopists who understood the limitations of the usual SEM techniques. This new method held promise of convenient circumvention of the tedious (and artifact inducing!) preparative methods. The structural preservation steps commonly used before introducing the sample to the harsh environment in conventional SEMs are specimen fixation and dehydration followed by coating to increase conductivity. So the promise of observation of unfixed, non-dehydrated, uncoated samples seemed too good to be true. Even with some sacrifice of resolution, the potential to watch materials hydrate and even video-record events in real time were enough to generate great excitement. Table 4 compares the conventional SEM and the ESEM relative to an number of performance characteristics. The differential vacuum chambers of the ESEM are indicated in the cross-sectional diagram (Figure 17). Not only is the specimen chamber at significantly higher pressure than the remainder of the column, the gaseous environment of the chamber itself can be controlled. However, the technique has by no means displaced conventional scanning electron microscopy and indeed has not generated an extensive body of literature demonstrating the unique capabilities of this technology. Currently we are aware of only one manufacturer of the instrument and the number of published papers found is quite small. The reasons for this are not clear.

261 Table 4 Operating Conditions and Performance -- Colnparison of SEM and E-SEM Operating conditions

Conventional SEM

Enviromnental SEM

Imaging Modes:

Secondary Electrons (ET) Backscattered Electrons

Secondary Electrons (ESD) Backscattered Electrons

Working Distance:

6-40 mm

6-15 mm, resolution limited by beam scattering in gas

Accelerating voltage:

1-30 kV, normally 20 kV

1-30 kV, normally 20 kV

Vacutma Conditions:

10-5 to 10-3 Pa High vacuum

10-4 to 0.9 kPa Normally 10-250 Pa. Atmospheric pressure is 100 kPa. The imaging gas is usually water vapor, but air, helium, oxygen and nitrogen can also be used.

Magnification:

10 to 100,000 times

70 to 100,000 times

1.8 to 6.0 nm, usually 4.5

7 or 5 nm

Resolution:

nln

Sample Requirement:

Compatible with high vacuum. Dry and conductive samples only

Any sample type (including liquids, solids, powders, and insulators) plus dynamic reactions.

Sample exchange time:

3-5 minutes

30-60 seconds

Approximate Cost:

$65,000-250,000*

$179,000-250,000"

*Depending on resoland configuration

Table 4 compares the operating and performance characteristics between conventional SEM and ESEM [From E. Doehne and D.C. Stulik, Scanning Microscopy 2 (1990), 275-286.]

262 The first direct observation of the hydration of a protein powder (subtilisin) was reported by Roziewski et al [60] utilizing an ESEM. This was accomplished by varying the temperature and pressure in such a manner as to increase the humidity within the sample chamber. The resulting water vapor was used as the imaging gas. The ESEM showed the powder to be flake-like, in contrast to the spherical powders observed by standard SEM where preparation requirements appear to have affected the sample. As the authors point out, correctly determining the shape and size of powdered compounds "is critical to determining the role of diffusion in nonaqueous enzymology". The advantages of the ESEM technology was thus critical to helping advance the understanding of enzymology data acquired by other technologies, such as electron spin resonance.

Figure 17. Cross-sectional diagram of ESEM illustrating differential pressures at various sites along the column, culminating in a specimen chamber at relatively high pressures. [From E. Doehne and D.C. Stulik, Scanning Microscopy 2, (1990), 275-286.]

While trying to determine the source of a particular structural feature present on some but not all types of starch granules, Fannon et al [61] used conventional SEM and ESEM to determine if the pores in some starch varieties were a result of drying in the kernel, produced by in situ amylases, an artifact of preparation or a natural feature of the granule. While earlier work had been done in this area [62], the ESEM could provide a distinct advantage. By the use of conventional SEM and the ESEM, the list of possible causes of the pores was

263 shortened to their production during granule formation. While techniques were employed with conventional SEM which addressed some of the issues of sample preparations, the use of ESEM contributed to the higher level of confidence that the pores were not artifactual in nature. One of the more interesting applications of ESEM is found in the work of McDonough et al [63]. The objective of their work was to describe the structural changes occurring in corn tortilla chips during frying and "to determine where, when and how oil enters baked tortilla chips during frying". Since conventional SEM preparations typically require the removal of the oil phase prior to observation, the use of ESEM has great advantage in this study. Samples of chips were simply mounted on SEM stubs and viewed directly in the ESEM (Figure 18). The authors' enthusiasm for the ESEM as an adjunct to conventional SEM was quite apparent. ESEM afforded them the opportunity to view samples with relative ease, in their "natural state", document the location of oil at various stages of processing and achieve quick turnaround time for sample exchange permitting numerous samples to be analyzed. The addition of the ESEM technology allowed the authors to describe in detail the structural configuration of baked cereal products as well as follow the cooking process which resulted in under, optimum and over cooked product. Similar advantages could be expected by the application of ESEM technology in a wide range of food products.

4.2 Cryo-SEM of Frozen and Refrigerated Products Frozen food products such as ice cream would seem a natural subject for a technique such as cryo-SEM, but, as pointed out by Caldwell et al. [64], earlier microscopy work for frozen products involved TEM replicas [65,66] or microencapsulation of ice cream mix [67]. These earlier works all contributed to the understanding of ice cream but did not give a good representation of intact product characteristics such as large air cells. These can only be appreciated by observation at lower magnifications. Using cryo-SEM, the work of Caldwell et al [64] gives a detailed description of the overall structure of ice cream and its four phases: ice crystals, air bubbles, fat globules and serum. They discuss the pros and cons of three types of specimen holders, the impact of various sublimation times and the preferred sublimation environment in the SEM chamber. These considerations should precede any major study utilizing cryo-SEM as they set the limits of the technique, define the tradeoffs of various choices in preparation methods and so deliver a fuller understanding of the sample. As an example, the use of "excessive" sublimation gave insight into the bridging of ice crystals which had been seen previously only in fractured profiles (Figure 19).

264

Figure 18. Tortilla chip fried for one minute and observed directly in an ESEM (A and C) showing the presence of oil in air tunnels. Defatted samples were prepared for conventional SEM observations (B and D). (A x300; B x 25; C x 200; D x 25). [From 63].

265

Figure 19. Cryo-SEM images of ice cream which, in addition to showing the major structural features of the product, illustrate the impact of variable sublimation times and subsequent learnings, viz., bridging among ice crystals (arrow in D). (x 480). [From 64].

A second paper by the same authors [68] used the working knowledge established in the first paper in order to determine the influence of selected ingredients and processing. The use of cryo-SEM is critical for this type of analysis on a frozen product where the object of interest is water in the form of ice crystals. The study helped demonstrate that ice creams

266 with the smallest initial ice crystals were not the most stable. Rather, an optimum freezing rate and ice crystal size should be determined. Cryo-SEM methodology also facilitates the observation of highly hydrated systems. Harker and Sutherland [69] used the ability of cryo-SEM to preserve the structural integrity of the aqueous phase to characterize differences between mealy and non-mealy nectarines. The presence of juice on the surface of cells in non-mealy nectarines was observed after tensile tests produced a fractured surface. Such observations would not have been possible with conventional methods where dehydration and critical point drying are essential steps. A strong point to this study was the extensive use of other physical and chemical methodologies to help correlate textural difference based on storage parameters for nectarines. Although studies on potato structure had been carried out previously using conventional SEM, van Marie et al [70] used cryo-SEM to advantage in this high moisture material. The fracture planes of cooked and uncooked samples were used to help characterize cell wall adhesion in the four potato cultivars. In particular, differences in cell wall contact area and surface detail were used to explain the mealy v e r s u s firm textural attributes in the cultivars. By determining the parameters which contributed to the texture of potatoes, processing conditions and selection of suitable raw materials could be facilitated. Such information would be difficult to obtain with conventional, chemically fixed material due to the high moisture content and the inability of standard chemical fixation to retain carbohydrate-based structures. Freeman et al [71] present a body of work initiated by comparing 14 methods of sample preparation which resulted in the determination that cryo-SEM was the superior preparation method for determining the ultrastructure of glutens and doughs. The use of preparative cryotechniques was used also to prepare large samples for low magnification observation with a dissecting light microscope. The combination of the two techniques again demonstrates the advantage of multiple approaches to microscopy. While the lower power of observation allowed large samples to be observed, such preparations would be inadequate for even modest SEM observations because of the preponderance of ice crystal artifacts. For SEM observations at approximate magnifications of 100 to 2,000 times, ice crystal formation was minimized by faster freezing times associated with the smaller sample volume. This type of study demonstrates the impact of processing parameters on major ingredients of dough and helps support the contention that an interconnected network of holes is natural and not an artifact of sample preparation. The use of cryo-SEM imaging combined with rheological characterization of food material can be shown in the work of Lorimer et al [72]. Specifically, the impact of using non-wheat flour blends in dough preparations was investigated by combining rheology and microscopy. The use of cryo-SEM allowed the rapid stabilization of samples so that structural characteristics could be compared reliably with the rheological properties as measured by farinograph analysis. Additionally, the structural characteristics of doughs were correlated with thiol and disulfide bond interactions to obtain a clearer understanding of protein-protein interactions. The use of cryo-SEM helped support and explain the rheological findings. Likewise, cryo-SEM was used in conjunction with other physical methods by Taneya et al [73] to understand more fully how processing conditions impact the stringiness characteristic of a particular type of Mozzarella cheese. The study used four modes of microscopy (LM conventional SEM, cryo-SEM and freeze fracture TEM. In addition, several other

267 physicochemical techniques (compression, stress relaxation and flow characteristics) and a stringiness measurement were made. In total, the combination of techniques represented a powerful methodology to help describe and understand the effects of pH, temperature and physical processing on string cheese quality. Generally speaking, the appropriate use of several methodologies often results in a much deeper understanding of the subject matter and sounder approach to problem solving. Fannon et al [61] demonstrated the usefulness of cryo-SEM to characterize various starch granules both as individual granules and after hydration leading to gel and paste formation. Although the authors admit that the freezing process may cause some artifactual images from ice crystal formation, the information is significant when compared to the poor preservation resulting from traditional SEM (solvent exchange and critical point drying). Starches with different functional properties clearly responded to freezing methods in ways that lead to insight into their physical chemistry. A major advantage of cryo-SEM is its ability to accommodate the need to preserve lipid or fatty components within food systems when they are the main subject of study. Kawanari et al [74] used cryo-SEM in combination with polarized light microscopy to characterize structural differences in butter due to manufacturing differences and their impact on volumetric changes in pastry applications. Such a study would have been very difficult to accomplish via conventional SEM techniques.

5. U N D E R U S E D

OR NEW METHODS

5.1. Negative Staining Traditionally, this technique has found application in the study of casein micelle structure [75 and references therein]. As a technique, it suffers from the fact that long range structure is necessarily lost. But, of all the TEM methods, it is the fastest and has some exciting possibilities. Negative staining should be the method of choice for determining the presence of "string proteins" associated with the creaming reaction in processed cheese [76]. Even more pragmatically, it is a rapid means of visualizing bacteriophage which periodically are issues in manufacturing plants producing cultured products. When combined with immunomicrochemical staining, the possibilities for advances at fundamental and applied levels are fascinating. Aside from the need for a transmission electron microscope the technique requires no other sophisticated equipment and is relatively easy to apply. Negative staining in combination with quality light microscopy can give insight at the macromolecular level. In combination with other analytical techniques, negative staining was used to help visualize the structural components which make up apple haze [77]. Based on the substructure observed, the authors suggest that negative staining could be used as a diagnostic test for the presence of protein-phenol complexes in fruit juice haze. Harada et al [78] used negative staining in combination with rotary shadowing to characterize the macromolecular interaction of several polysaccharide gels including agar, carrageenan, xanthan, locust bean gum, starch and their gel of interest, curdlan (Figure 20). The effect of preparation methods, impact of cations and interaction between combinations of gel types were studied. The use of rotary shadowing, while minimal in this study, was used

268 to verify observations without the risk of artifact from the use of uranyl acetate solutions. The resolution achieved in this study was facilitated by the use of a field emission TEM, [78].

Figure 20. Although not a new technique, the use of negative staining and TEM to characterize macromolecular structure is probably underused in Food Science. Here the technique is used to compare the effects of zynolase and sulfuric acid on the structural alignment of curdlan. (x 120,000). [From 78].

5.2. Freeze Fracture The details of the methodology for this demanding technique can be found readily [20,79]. Briefly, one rapidly freezes the sample in a nitrogen slush or uses some other suitable means of rapidly freezing the sample. Then the material is fractured at low temperature and replicas of the newly exposed surfaces made with carbon and platinum. Once the original sample is dissolved or digested away, the replica can be viewed at high resolution in the TEM. The method has had its widest application in the study of casein micelle structure and function and as a means of studying theories on the stability of foams [79]. In the latter instance, Bucheim and co-workers have shown the submicellar nature of the micelle and its attachment to interfaces, particularly air/water interfaces in foams. The drawback of the technique is that it requires some patience to learn and is somewhat fickle in its operation. Structures such as large air cells are difficult to replicate with carbon. However, the advantage of the technique is that it gives high resolution views of samples which have had no chemical fixation [20]. As such, it is an appropriate and powerful method for fundamental studies into the nature of interfaces, particularly since fixation methods for the stabilization of emulsifiers must rely on

269 osmium, which necessarily creates a dependence on unsaturated carbon-carbon bonding Protein emulsifiers, of course, may be stabilized by glutaraldehyde. A potential application of Freeze Fracture techniques is in the microchemical localization of components - for example in the distribution of gums in foods. All current techniques for the localization of gums rely on plastic sections with the unavoidable artifact that the gums will precipitate in situ at about 70%V/V ethanol, requiring some reconstructive thinking as to the "true" location of the gums. Freeze fracture could be combined with en bloc staining [20,36] for immunomicrochemical staining including the use of lectins for gums and high resolution localization of non-glutaraldehyde sensitive macromolecules such as carbohydrates and lipids. As far as we are aware, no attempts to do this have been reported.

5.3. Scanning Probe Microscopy Since the invention of the first scanning probe microscope (SPM), actually a scanning tunneling microscope (STM) in 1982 by Binnig and Rohrer [80], there has been rapid expansion both in SPM applications in Biology and in the forms it may take. Most people think of SPM in terms of molecular and even atomic resolution. Now, although there are occasions where such high resolution is useful in Food Science [81], there should be more occasions when lower resolution images would be particularly useful [82]. But before we detail potential applications of low or high resolution SPM to Food Science, what is this new form of microscopy? SPM is actually the generic term for a family of imaging modes which can be applied to materials in many different circumstances. There are essentially two approaches which may be described [83]. The first is scanning tunneling microscopy. In this mode, a conducting probe having a point of atomic dimensions, is scanned in a raster pattern across the sample. The microscope depends on the quantum mechanical "tunneling" of electrons between the tip and the sample. This tunneling is analogous to covalent bond formation [83]. Piezoelectric scanners keep the distance between tip and sample constant. The variation in tip position can be measured (at better than 0.1 nm resolution in the z-axis) and a 3-dimensional image built up pixel by pixel. The largest disadvantage of STM is the requirement for electrical conductivity. The second approach is Atomic Force microscopy (AFM) which uses a probe attached to a cantilever arm having a small spring constant [83]. Again, the distance between sample and tip is kept constant and the measurement of the deflection required to accomplish this generates the image, again, pixel by pixel. Usually, measurement of the deflection of the cantilever is made by the displacement of the reflection of a laser light shone on the arm but other methods are available. Sample preparation for most forms of AFM is minimal: many samples can be examined in air at room temperature. Non-Food Science applications of the technique have allowed measurement of topographical, magnetic and even theological (lateral force) properties of materials simultaneously [84]. More detailed explanations of the theory of these microscopies are beyond the scope of this Chapter. As with any technique in science, SPM has its own set of artifacts which must be recognized. Most of these are due to contamination of or damage to the tip [85]. The reader is referred to [85] for an introduction into this important aspect. Passing acquaintance with the development of SPM might lead one to think it has application only in atomic resolution of molecules. While some exciting work has begun in

270 this area of Food Science such as the study of wheat seed storage proteins [81], it is the possibilities at lower resolution which intrigue us. The paper by Kordylewski et al. [82], shows a comparison of AFM and Freeze fracture TEM imagery of rat atrial tissue. In Food Science an application example might be the simultaneous acquisition of surface rheological and microstructural data which could relate structure and texture to sensory values.

5.4. Other Microscopies Enhanced imaging of several dairy products has been demonstrated through the application of a relatively elaborate preparative technique in combination with a cold-field emission scanning electron microscope (FESEM) [86]. The preparative methods include a metalimpregnation technique, termed tannin-ferrocyanide-osmium (TA-F-O, Figure 21), which was adapted from Hirano et al. [87]. The potential resolution is maximized by reducing the thickness of the metal coating (2-5 nm of iridium as opposed to 20-100 nm of silver or gold in conventional methods) and operating the FESEM at low kV and nA settings.

Figure 21. Yogurt prepared by the TA-F-O method and observed using a field emission SEM. In addition to clearly imaging casein micelles (CM) and submicelles (SM); the micrograph documents a resolution of 3 nm. (x 100,000). [From 86].

Indeed the images of casein micelles and submicelles in yogurt are impressive. As pointed out in the "discussions with reviewers" section of the paper the technique is exhaustive and utilizes a highly sophisticated SEM and therefore is not likely to find wide use. The capability to resolve particulates at the 3 nm size range in the SEM mode is truly noteworthy and this reference represents a step forward in defining new capabilities to address specific questions requiring these higher resolutions. The use of freeze drying as a preparative method for SEM has not enjoyed extensive popularity, but the method can be used to advantage under special situations when other methods would be excessively difficult or fraught with potential artifacts [88]. Gels of rennet-coagulated milk are very fragile and susceptible to collapse or distortion during handling and critical point drying with conventional methods of sample preparation. The

271 quick freezing of small samples and selection of undistorted regions (Figure 22) proved to be an acceptable technique [88].

Figure 22.Two examples of rennet-coagulate milk gel examined after freeze drying of the sample. (x17,000). [From 88].

The presence of ice crystal damage could be detected and therefore avoided when making comparisons between samples. This work shows how adaptations of older, less used methodologies continues to be used to advantage in Food Science. In this study, it was the fabrication of a sample collecting device which could retrieve the coagulated milk with minimal disturbance and protect the sample during initial freezing in liquid Freon 22 [88] which was critical to the result. We have not exhausted the possibilities for the application of newer techniques to Food Science. However, space limitations preclude discussion of them. Readers are encouraged to consider the advantages of Image Analysis [89], Scanning Acoustic microscopy [5,6] and Near Field microscopy [7], for their particular applications. The first is a set of techniques for the quantitative interpretation of phenomena and should assist in the resolution of dynamic issues. The second technique can give rheological data directly (elasticity etc., see 5) and so would be a useful adjunct to lateral force measurements in SPM, particularly as the latter generally are measurements of surface phenomena. Finally, Near field light microscopy offers the hope of very high resolution without tedious specimen preparation.

6. FUTURE TRENDS Microscopy in Food Science is in an exciting state of flux. Traditional techniques of specimen preparation and observation will continue to give essential data on the structure of foods. However, the emphasis in the future will probably lie in the development of faster methods and in the quantification of individual components, both aiming at definition of structre/function relationships. This will be true of particulates as they relate to sensory scores and to the characterization of dispersed phases in emulsions and foams. At the same time, the use of microchemical methods should become more common as a means of

272 problem resolution in manufacturing plants and in theoretical studies. Where the untried microscopies - SPM, near field, acoustic etc. - will take us cannot be predicted with any certainty but will no doubt allow for ever finer control of processes and quality control.

ACKNOWLEDGEMENTS One of us, RGF, would like to express appreciation to E.L. Armstrong and S.S. Miller for their help in this Chapter. MGS and DGP would like to thank Angela Eng and Saideh Safavi for their help, also. Finally, MGS acknowledges his debt to the kidney donor. REFERENCES 1. T.P. O'Brien, Cereal Structure: An Historical Perspective. In: New Frontiers in Food Structure, D.B. Bechtel (ed.), American Association of Cereal Chemists, St. Paul MN, (1983), pp3-26. 2. J.M Aguilera and D.W. Stanley, Microstructural Principles of Food Processing and Engineering, Elsevier Applied Science, New York, (1990). 3. M.A. Hayat, Principles and Techniques of Electron Microscopy, van Nostrand Reinhold, New York, (1973) Vol. 3. 4. V.K. Zworykin, J. Hillier and R.L. Snyder, American Soc. for Testing and Materials Bulletin, 117 (1940) 15-23. 5. H.W. Israel, R.G. Wilson, J.R. Aist and H. Kunoh, PNAS (USA) 77 (1980), 20462049. 6. H. Hafsteinsson and S.S.H. Rizvi, Scanning Electron Microscopy, III (1984), 12371247. 7. C.J.R. Sheppard (ed), Scanning 16 (6) (1994). 8. V.J. Morris and T.J. McMaster, Trends in Food Science & Technology, (April, 1991), pp80-84. 9. S. Henstra and D.G. Schmidt, Naturwissenschaften 57 (1970), 247. 10. S. Henstra and D.G. Schmidt, LKB Application Note # 150 (1974). 11. G.G. Jewell, Scanning Electron Microscopy, III (1981), 593-598. 12. P. Allan-Wojtas and M. Kalab, Food Microstruct. 3 (1984), 197-198. 13. M. Kalab, Electron Micros. Soc. Am. Bull. 17 (1987), 88-89. 14. M. Kalab, Food Microstruct. 7 (1988), 213-214. 15. I.A. Velicky and M. Kalab, Food Struct. 9 (1990), 151-154. 16. M.C. Alleyne, D.J. McMahon, N.N. Youssef and S. Hekmat, Food Struct. 12 (1993), 21-30. 17. D.N. Holcomb and M. Kalab (eds.), Studies of Food Microstructure, Scanning Electron Microsc. ( 1981), 342pp. 18. D.N. Holcomb, Food Struct. 9 (1990), 155-173. 19. M. Kalab, Food Struct. 12 (1993), 93-114. 20. M.A. Hayat, Principles and Techniques of Electron Microscopy. Biological Applications, CRC Press,Boca Raton, FL, (1989) Third Edition.

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Chapter 12 Some recent advances in food rheology S. Chakrabarti Kraft Foods Technology Center 801 Waukegan Road, Glenview IL 60025, USA

1. I N T R O D U C T I O N The American Society of Rheology was founded in 1929 and the term 'Rheology' was adopted to mean the science of flow and deformation of matter. During the early formative years of the Society of Rheology, much of the growth in the area of rheology came from work on foods (1). A basic knowledge of rheology is now considered essential for scientists employed in many diverse industries, as it relates laboratory measurements to product performance. Amongst these varied industries are the polymer processing industries, detergents, lubricants, cosmetics, oil exploration, biotechnology and foods. Most industries that manufacture and/or use material substances need rheological information for characterizing, processing and modifying their products. For foods, product performance is just what the proverb says: "The proof of the pudding is in the eating", i.e., the rheology measurements have to relate to sensory evaluation of foods. This is difficult, as rheologists are not sensory experts and food industries have not shown a wholehearted commitment to funding and developing food rheology. So although foodstuffs were considered a part of materials research, with the rise of the plastics industry, the interest in food rheology temporarily faded. One immediate consequence of this loss of interest in food rheology has been that in the food industry many industrial processes are based on operations similar to those carded out by housewives or craftsmen several centuries ago or borrowed without modifications from the plastics industry. Subjective assessment of quality is widely used, both for raw materials as well as for the final product. Attempts to measure consistency under conditions that are relevant to those employed by the consumer have resulted in a large variety of instruments and test methods for which extensive reviews are available (2-4). Such imitative tests are useful for control purposes and in providing correlations with sensory perception of food texture, but they do not provide physically meaningful parameters nor can they be used to understand processing conditions. A comparative discussion is given below to distinguish between empirical and fundamental rheological tests.

278

1.1. Empirical vs. fundamental rheological tests for foodstuffs In most empirical tests on foodstuffs, a force is applied which results in measurable deformation of the test material. Clearly, the degree of deformation will be determined by both extrinsic (force type, strength, temperature etc.) and intrinsic factors (material composition, morphology, physicochemical and mechanical properties). Such force and deformation data have been widely used to compare the texture of food products. Generally, empirical measurements correlate well with the sensory perceptions of texture, especially for firmness of food products. The Cone Penetrometer test, widely used in the food industry to compare the firmness of soft foods, is an example of such empirical measurements. In this test, a cone attached to a load cell is inserted into a sample at a constant rate and the force needed to penetrate a certain distance within the sample is noted. The force data are used to compare the firmness and hence, the sprcadability of soft, solid foods, e.g., butter, cream cheese etc. In the penetration test, the force data will vary with any changes in the cone angle. In another empirical test, the Marsh Funnel viscosity test, the time for a fluid to flow through a given funnel is taken as a measure of its viscosity. Any changes in the funnel size or dimensions will produce a different "viscosity" value for the same fluid. The point is that the data measured in empirical tests depend on sample size as well as on test geometry. Hence, empirical data do not represent intrinsic properties of the test sample. In contrast, in fundamental rheological tests, material properties of substances are measured using precise test protocols which ensure that the data obtained are independent of sample size, dimension and test geometry. By definition, the material properties define the relationships between stress and strain and/or strain rates and has to be independent of the sample size, shape and test geometry. However, it must be emphasized that as with the empirical measurements, fundamental material properties also vary with deformation rate, type of deformation and temperature. To understand the significance of material properties, one only has to think of density, which is also a material property. The density of a substance defines how much volume, let's say, of water, that the substance will displace on immersion. Instead of measuring the volume of water each time one drops this substance in any container of water, one measures its density and calculates how much water it will displace on immersion. Clearly, the material property concept leads to predictive abilities. It is now known that the relative ordering of the molecules in a simple bodymatter defines its density and that by altering the ordering of the molecules, one can change its density ..... the reason why the density changes with the change in crystallinity of a given solid as in fatty acids. Broadly speaking, the mechanical properties can be divided into two classes: 'bulk' and 'interfacial'. Within the 'bulk' properties are included the shear and extensional viscosities, moduli and yield stresses (material constants that relate stress to strain or strain rate), and within interfacial rheology are included the 'wall-slip' and 'friction' effects. The interfacial properties are independent of bulk mechanical properties and governed by the frictional or surface forces which are thought to operate at relatively

279 short distances (at a length scale of less than 10 times the size of the unit particles). Generally, interfacial properties are not considered to be material properties, as they can depend on the surface characteristics. Wall-slip effects in viscometric flows are a consequence of interfacial effects between a fluid and the solid boundary.

1.2. Areas of growing interest in food rheology research The measurement of viscosity is well established in the food industry. However, wall-slip effects can only be detected from fundamental measurements, not from empirical tests. In the above example of the cone penetration test, if the material does not 'stick' to the cone due to wall-slip effects, the force data will be erroneously interpreted as the product's overall bulk property, the yield stress. Similarly, in the funnel test, if the fluid slips off the wall during flow, measurement of viscosity will be unreliable. The occurrence of wall-slip during flow is, in general, a desirable parameter from an engineering point of view, as the energy required to pump the fluid is lessened. Food products are known to be prone to slip during flow. However, empirical measurements cannot reveal the wall-slip characteristics of foods and so such data cannot be used to model process flows. Although wall-slip effects have been known and studied for polymeric substances, it has received little attention to date by the food industry. It is expected to be a growing area of interest to food rheologists. A second area of increasing interest lies in measuring the extensional viscosities of foods. The importance of extensional viscosity in the characterization and processing of polymers is better understood than for foodstuffs. Intuitively, it is easy to understand how the stretchability of pizza cheeses, or the extensibility of doughs may be influenced by the extensional viscosity. Often high pressure homogenizers are used to process food emulsions. The flow is typically extensional in nature in the homogenizers. Often, difficulties are encountered in producing the emulsion having similar characteristics following an operational scale up from pilot plant to production plant. Although the effect of extensional viscosity on drop break-up is well-recognized, food emulsion drop break-up has been little studied. The measurement of extensional viscosity is not yet a routine practice in the food industry; however, its importance in food processing cannot be overlooked. The third area of growing interest is in solid mechanics. It is important to recognize that food theology goes beyond the realms of classical theology - foods do not just flow and deform, they also break. Different foods break differently. Some break sharply, without much warning, e.g., cream cracker biscuits, potato chips, fresh carrots; others deform extensively before breaking, such as bread dough, caramels, hot pizza cheeses, chewing gums, stale carrots. Clearly, fracture properties, which influence the break behavior are also relevant for describing the food quality. To study food rheology is to study both solid and fluid mechanics. This is a very special aspect of industrial food rheology, as rheological journals do not generally include fracture mechanical topics, just as a training course on rheology does not include a discussion on fracture. However, the industrial food rheologist needs to be aware of the fracture properties of foods.

280 At the present time, there is a resurgence of interest in food rheology as the food industries focus on utilizing non-food technology to obtain a competitive edge in the highly competitive marketplace. Hence, the interest in rheological properties and in their accurate measurements. The study of mechanical properties encompassing rheology and fracture mechanics is a vast, dynamic and an exciting area and can scarcely be reviewed in one chapter. In this review, the focus is to define the material functions of foods and to discuss the current measurement techniques for the material properties that are of growing interest in the industry. The discussion centers on measurement techniques for the following three topics: (1) wall-slip effects during flow, (2) extensional rheometry of food products, and (3) fracture properties of foods.

2.0. W A L L - S L I P E F F E C T S In deriving his law of fluid viscosity, Newton wrote in the "Principia", published in 1687, "The resistance which arises from the lack of slipperiness of the parts of the liquid, other things being equal, is proportional to the velocity with which the parts of the liquid are separated from one another" (5). This lack of slipperiness is known as 'viscosity' and led to Newton's law of viscosity, i.e., x =rl.7

(1)

where, x = Shear stress 7 = shear rate 11 = coefficient of viscosity or the viscosity. Figure 1 schematically depicts the buildup of a steady velocity profile for a fluid contained between two plates, where one plate is held stationery and the other set in motion. The lack of slip condition requires that the fluid velocity be zero at the stationery wall and the same as the solid at the moving wall. The lack of slip at the walls is the basic premise of rheometry and the interpretation of rheometrical data is usually based on the assumption of no slip at the boundary. As rheological research into complex substances of industrial importance advanced, anomalous flow behavior near solid walls was noted and the concept of 'wall-slip' or 'wall effect' was born. Highly heterogeneous materials, such as foods, detergents,

281

Fluid at rest

Lower plate set in motion

Velocity build-up in unsteady flow

Final velocity distribution in steady flow

Figure 1.

No slip boundary condition steady laminar velocity profile for fluid sheared between two plates.

cement slurry, cosmetics as well as less heterogeneous materials such as polymer solutions and polymer melts have all been reported as exhibiting slip effects. Most of the reported experiments infer slip indirectly from macroscopic behavior such as slope discontinuities in the curves of global flow rate versus applied pressure. This indirect determination of slip is probably the reason why wall-slip effects can still be missed, despite its important practical and fundamental implications. Present day literature suggests at least two different slip mechanisms; true slip and apparent slip.

2.1. True Slip True slip is generally associated with flow instabilities and has been reported for unfilled polymer melts (6-8). Brochard et al (9 and references therein) has summarized various experimental flow conditions in which true slip (non-zero velocity at the wall) has been reported - (a) screw extruders, (b) rheological studies on molten Polystyrene in a plate - plate geometry with small gaps, (c) studies on thin films i n wetting or dewetting processes and (d) measurements on multilayer extrusions. Migler et al (10), who applied novel optical techniques with a resolution of better than 100 nm near the wall, confmned slip effects to originate from polymer surface interactions; they used well-characterized surfaces and branched and linear polymer polydimethylsiloxane melts. Generally, slip is assumed to occur beyond a critical stress. However, Migler et al reported slip as a function of shear rate (10).

282

Figure 2.

Photomicrographs of pigment flow through a capillary showing wall-slip (ref. 11).

2.2. Apparent Slip For polymer solutions and/or concentrated suspensions, slip is thought to occur through an 'apparent slip' mechanism. This involves the formation of a thin stratum of liquid of a lower viscosity adjacent to the wall and can be considered as a particle-free suspending medium. Evidence for this phenomenon can be seen from photomicrographs, taken as early as 1949 (11), of pigment flow through tubes (Fig. 2). It should be noted that the photographs were not intended at the time to be proof of slip flow, but for the presence of yield stress in highly concentrated filled suspensions. Additional evidence for polymer molecules migrating from near the wall towards regions of low stress was provided by the flow visualization work of

283 Muller-Mohnsson et al (12, 13). Using a laser anemometer they measured the velocity profiles of aqueous polyacrylamide gel solutions flowing through rectangular slits and found that slip velocity depended on the wall material type and was particularly rapid for glass cleaned with chromosulphuric acid. The data was interpreted as indicating two separate regions, a bulk flow region and a thin slip layer of a certain thickness. The fluid velocity in the slip layer is known as the slip velocity. Various mechanistic theories of the slip phenomenon have been summarized by Cohen (8), some of which are schematically presented in Fig. 3. The practical implications of slip effects in engineering operations have been reviewed by Agarwal et al (14). With heterogeneous substances such as foodstuffs, many of which are filled suspensions of proteins, carbohydrates and triglycerides, it can be theorized that complex ionic interactions generating repulsive forces could occur and cause wall-slip.

Figure 3. Possible mechanisms for slip flow (Reproduced with permission, ref. 8).

284

2.2.1. Slip Measurement- detection and quantification In principle, slip can only be confirmed by comparing measurements of the velocity profile with the predicated velocity profile calculated from shear viscosity measurements of the fluid in a slip-free viscometer. This explains, in part, why attempts to define slip have followed three main approaches: i) Directly determining slip presence using flow visualization techniques, ii) Quantifying slip effects by modeling flow within a well-defined boundary, and iii) Devising methods to eliminate slip from the chosen experimental system.

2.2.1.1. Visualization of Wall-slip Possibly the simplest optical technique for detecting slip involves the use of a straight marker line across the edges of the parallel-disk or cone and plate fixtures in a torsional rheometer. The presence or absence of slip is schematically shown in Fig. 4. Depending on the severity of slip effects, slip can be detected with the naked eye, as in the case of mayonnaises (15) and greases (16) or with video cameras with magnifying capabilities (17). The marker line technique has also been used by Mani et al (18) to detect slip of wheat flour doughs in cone and plate fixture. They used the cyanoacrylate adhesive to eliminate the slip phenomenon and reported the dough viscosity to be the viscosity of such bonded systems.

Figure 4. Schematic drawing of patterns to detect slip effects in parallel plate geometry. The effectiveness of the marker line technique to detect wall-slip is a function of the thickness of the slip layer. For polymer solutions for which the slip layer could be of the order of microns or less, more sophisticated techniques have been applied.

285 Examples are Laser Differential Microanemometry (LMA) and Total Reflection Microscopy (TMA) (8). Both LMA and TMA measure the velocity profile of the fluid in tube flow. However, such optical techniques are generally not suitable for opaque and/or heterogeneous substances such as foods. Acoustic velocimetry seems to be more promising for determining the velocity profiles of opaque substances. Such an acoustic technique has been applied by Brunn et al (19) as an on-line viscometer for flow of mayonnaises in pipes. Wall-slip is not an easy phenomenon to detect. Although in principle, the velocity profile should reveal whether or not the fluid velocity is zero at the stationary wall, in reality determining the velocity profile with sufficient resolution near the wall is very difficult. So alternate means, e.g., checking for viscosity variation with appropriate changes in the test geometry, are also widely used in practice.

2.2.1.2. Quantification of Wall-Slip With the supposition that the slip layer is thin and the slip velocity is constant, various analyses have been developed in the search for the ideal experimental method to define slip. The Mooney analysis (20) for both tube flow and concentric cylinder flow has been applied to a wide range of materials including polymer solutions (21), filled suspensions (22), semisolid foods (23), fruit purees (24), and ketchups (25). Alternate estimates of slip velocity have been determined experimentally from, parallel plate torsion flow (26), from flow data in channels and inclined planes, and from porous medium geometries (8). As Mooney analysis is widely used for estimating wall-slip, a short summary of the analysis is given below. The major assumptions in Mooney analysis were: (1) the shear stress is linearly distributed over the pipe diameter, as in a fully developed laminar flow, (2) the slip layer thickness, a, is much smaller than the pipe diameter, a/R 1024) in roasted (180~ sesame seeds [52]

30 min) black and white FD-factor in

Odorant black (E,E)-2,4-Decadienal 2-Methoxyphenol 2-Pentylpyridine 2-Furfurylthiol 2-Ethyl- 3,5-dim ethylp yrazine 4-Hydroxy-2,5-dimethyl-3(2H)-furanone 2-Phenylethylthiol

4096 2048 2048 1024 1024 1024 128

white seeds 128 2048 256 4096 256 512 1024

2.5. Combined Headspace/AEDA Odorants present in the headspace above a food are of particular interest since they elicit the first sensory impression attracting a consumer. A first approach to analyze such volatiles is the application of the AEDA on extracts prepared by dynamic headspace extraction. An apparatus used for the extraction especially of solid foods is shown in Figure 5 [55]. The powdered material is placed into a rotating cylinder and the volatiles are continuously flushed onto a polymer material (Tenax(R)) by using a stream of helium (1 L/min). After 3 hr the volatiles are desorbed from the polymer by elution with a small amount of diethyl ether and evaluated by AEDA after concentration. Since different yields may change the composition of the volatiles during headspace extraction [7], it is essential to sensorially evaluate the flavor of the extracts in comparison with the food flavor itself. The following examples show applications of this method on fresh and stored wheat bread crust [55] and on fresh rye bread crust [P. Schieberle and W. Grosch, unpublished results].

Figure 5. Apparatus used for the dynamic headspace extraction of solid foods. (1) Carrier gas (He) inlet, (2) belt transmission, (3) glass cylinder (99 x 8.5 cm), (4) Tenax trap (16 x 1.8 cm), (5) thermostat [adapted from reference 55].

410 On the basis of high FD-factors (Table 3) the sensory significance of 3-methylbutanal and 2-acetyl-l-pyrroline with malty, roasty odors previously identified as the key odorants in fresh wheat bread crust [21 ] was established. During storage for 4 days the FD-factors of both odorants decreased significantly, while especially butanoic acid (rancid) and (E)-2-nonenal remained unchanged. The fatty, green note of the latter odorant especially contributes to the stale note detectable in the overall crest flavor of the stored wheat breadl Table 3 Changes in the FD-factors of key crust odorants during storage of wheat bread [55] FD-factor after a storage time of Odorant 3-Methylbutanal 2-Acetyl- 1-pyrroline 2,3-Butandione 1-Octen-3-one

2-Ethyl-3,5-dimethylpyrazine (E)-2-Nonenal 2,3-Pentanedione Butanoic acid

0h

96 h a

128 64 64 32 32 32 16 16

16 8 16 16 16 32 2 16

a The breads were stored in linen bags. In an headspace extract of flesh rye bread crust, 3-methylbutanal, (E)-2-nonenal and methional showed the highest FD-factors (Table 4), while 2-acetyl-l-pyrroline, the key odorant of wheat bread crust (cf. Table 3), did not significantly contribute to the rye crust flavor. Quantitative measurements established [45, 55] that especially the higher odor activity (cf. 3, this chapter) of the boiled potato-like smelling methional in the rye bread crust in combination with the much lower odor activity of the roasty-smelling 2-acetyl-l-pyrroline mainly contribute to the overall flavor differences in rye and wheat bread crusts. Table 4 Odorants showing high FD-factors in a headspace extract of flesh rye bread crust [P. Schieberle and W. Grosch, unpublished results] Odorant FD-factor 3-Methylbutanal (E)-2-Nonenal Methional 2, 3-Diethyl-5-methylpyrazine 2,3-Pentandione 2-Phenylacetaldehyde 2-Acetyl- 1-pyrroline

128 64 64 32 32 16 2

411 It should be stressed that for many compounds the yields were too low for identification experiments. It is, therefore, a prerequisite in the application of the AEDA on headspace extracts that the key odorants have already been identified in preliminary experiments and can be used as reference compounds to correlate the odor-active regions with the chemical structures on the basis of retention indices. Significant progress in the evaluation of odorants present in the headspace above a food has been recently made by combining the AEDA principle with static headspace [49, 56-57]. The method, called s_tatic headspace a_roma extract dilution analysis (SHA) is illustrated in Figure 6: a definite volume of the headspace above a food is taken off by means of a gastight syringe and injected onto a precooled GC-column (-100~ to focus the volatiles present in the gas volume. The trap and the oven temperature are then raised and odor-active compounds eluting from the GC column are detected by GCO. By decreasing the headspace volumes from e.g., 40 mL to 0.5 mL in subsequent runs, the relative odor potencies of the odorants become evident.

Figure 6. Illustration of the static headspace/aroma extract dilution analysis (SHA) [adapted from Guth and Grosch, Annual report of the Deutsche Forschungsanstalt ftir Lebensmittelchemie 1993, p. 27]. The results obtained by application of this method to the flavors of two different olive oils are summarized in Table 5. In the oil sample A, fruity, green apple-like odor notes predominated, while the overall odor of oil B was characterized as fatty, stale. The SHA reflected these flavor differences. Only 0.1 mL or 1 mL, respectively, of the headspace of oil A were necessary to detect the odors of the fruity smelling esters ethyl 2-methylbutanoate and ethyl 2-methyl propanoate as well as the green smelling (Z)-3-hexenal indicating high odor activities of these odorants in oil A (Table 5). On the contrary, the fatty, soapy smelling octanal which was detectable in only 0.2 mL of the headspace of oil B, followed by

412 acetaldehde, hexanal and ethyl 2-methylpropanoate were the most odor-active compounds in this oil. Table 5 Static headspace analysis/AEDA (SHA) of two cold-pressed olive oils [56] Olive oil a Odorant A Acetic acid (Z)-2-Nonenal Acetaldehyde 1-Penten-3-one Hexanal Octanal 1-Octen-3-one

(E,E)-2,4-D ecadi enal (E)-2-Hexenal Ethyl 2-methylpropanoate (Z)-3-Hexenal Ethyl 2-methylbutanoate

B (volume: mL)

>20 >20 >20 10 5 5 5 5 2.5 1 1 0.1

10 10 1 20 1 0.2 10 2.5 10 10 5 1

a Oil A exhibited a fruity, green odor, while oil B was described as fatty, stale. 3. ODOR ACTIVITY VALUES (OAV) 3.1. Odor thresholds

Gas chromatography/olfactometry (GCO) methods have been developed as screening procedures to detect potent odorants in food extracts. The FD-factors or CHARM values determined in food extracts are not consequently an exact measure for the contribution of a single odorant to the overall food flavor for the following reasons. During GCO the complete amount of every odorant present in the extract is volatilized. However, the amount of an odorant present in the headspace above the food depends on its volatility from the food matrix. Furthermore, by AEDA or CHARM analysis the odorants are ranked according to their odor thresholds in air, whereas in a food the relative contribution of an odorant is strongly affected by its odor threshold in the food matrix. The importance of odor thresholds in aroma research has been recently emphazised by Teranishi et al. [58]. As shown in Table 6, odor thresholds in air are generally much lower than those in an oil. But, the most important point is that the ratios oil/air differ significantly between odorants. For example, compared with 4-hydroxy-2,5-dimethyl-3(2H)-furanone (HDF) the odor threshold of 2,3-butandione is fifteen-fold higher in air, but is eleven-fold lower in oil (Table 6). This implies that compared with HDF the flavor contribution of 2,3-butandione to a fat-rich product might be under-estimated on the basis of FD-factors. Furthermore, FD-factors and CHARM values are not corrected for losses during the isolation steps, caused by the volatility of the odorants during isolation and concentration procedures or by their chemical stability. As shown in Table 7, significant differences in the yields of odorants have been observed [59]. For these reasons and to assure that the important odorants are included, the identification experiments should be focused on the odor-active compounds detected by AEDA in a wide FD-factor range of at least 1 to 100.

413 Table 6 Odor thresholds of selected food odorants in oil and air Odor threshold Odorant

air a (ng~)

4-Hydroxy-2, 5-dimethyl-3 (2H)-furanone 2-Acetyl- 1-pyrroline 3-Methylbutanal Methional 2,3-Butandione

1 0.02 3 0.15 15

oil b ( g g ~ ) 50 0.1 5.4 0.2 4.5

Ratio oil/air x 103 50 5 1.8 1.3 0.3

a Odor threshold were determined by GCO [20] using (E)-2-decenal (2.7 ng/L air) as the reference. b Odor thresholds were determined in sunflower oil by the triangle test. Values detected by at least eight of ten panellists are presented. Table 7 Yields of odorants obtained in a model study a [59] Odorant

Yield (%)

1-Octen-3-one (Z)- 1,5-Octadien-3-one (E,Z)-2, 6-Nonadienal Hexanal (E)-2-Nonenal 1-Octen-3-hydroperoxide (E,E)-2,4-Decadienal 4,5-Epoxy-(E)-2-decenal

78 70 30 24 23 16 2 1

a Definite amounts of the odorants (20 gg to 160 gg) were dissolved in diethylether, then added to sunflower oil (400 g) and the volatiles were isolated by sublimation in vacuo. 3.2. Quantification of odorants A first step to approach the situation in the food is a calculation of odor activity values (OAV) concentrationx OAV x = odor threshold x which are defined as ratio of concentration to odor threshold. The OAV is equivalent to the previously used terms aroma value [60], odor unit [61 ] or odor value [62]. A prerequisite for the calculation of OAVs are exact quantitative data. Aroma compounds, which are relatively stable and are present in food extracts in higher concentrations (>100 gg/kg food) are often quantified by using an internal standard containing a similar pattern of functional groups as the analyte. In a quantitative study on cherry odorants [63] it has been shown, that the results are significantly influenced by the isolation technique used and by the structure of the odorant. However, under appropriate conditions the values differed only between 7 % (benzaldehyde) and 26 % ((E,Z)-2,6-

414 nonadienal). This method has also been used to quantify key odorants in lemon oil [64, 65] and dill herb [66]. However, in the quantification of trace volatiles which may additionally be labile such as 2-acetyl-l-pyrroline [67] or very polar like 4-hydroxy-2,5-dimethyl-3(2H)furanone [68] and are, therefore, isolated in low yields from aqueous media, a stable isotope dilution analysis (SIDA) is the method of choice [ 11, 59, 67, 68]. Since the labelled internal standard has identical chemical and physical properties as the analyte, losses during work-up or chromatography are ideally compensated. The major effort in the development of the SIDA is the synthesis of the internal standards, since most of them are commercially not available. But, even a less effective synthetic route is acceptable since e.g., ten milligrams of the labelled standard will enable the quantification of an odorant occurring in a concentration of e.g., 20 gg/kg in about 500 samples. Table 8 Important food odorants, for which a quantification by a stable isotope dilution assay has been developed Reference Reference Odorant Odorant 2-Acetyltetrahydropyridine [70] 2-Acetyl-2-thiazoline [72] 2-Acetyl- 1-pyrroline [67] Acetylpyrazine [67] Bis(2-methyl-3-furyl)disulfide [69] 2,3-Butandione [28] Butanoic acid [28] (E)-13-Damascenone [ 11] (E,E)-2,4-Decadienal [59] 8-Decalactone [28] 2,3-Diethyl-5-methylpyrazine [72] (Z)- 6-D o d eceno- ~-1 actone [28 ] 4, 5-Epoxy- (E)-2- decenal [59] trans-2, 3-Epoxy-o ctanal [39 ] Ethyl cyclohexanoate [71 ] 2-Ethyl-3,5- dim ethylpyrazine [72 ] 5-Ethyl-3-hydroxy-4-methyl-2(5H)[73 ] furanone (Abhexone) 5-Ethyl-4-hydroxy-2-methyl-3 (2H)- [30] furanone (Ethylfuraneol) Ethyl 2-methylbutanoate [71 ] Ethyl 3-methylbutanoate [30] 2-Ethyl-3-methylpyrazine [67] Ethyl 2-methylpyropanoate [71 ] 2-Furfurylthiol [69] 2-Heptanone [30] (Z)-4-Heptenal [74] Hexanoic acid [28, 39] Hexanal [39, 59] (E)-2-Hexenal [71 ] (Z)-3-Hexenal [59]

[59] (Z)-3-Hexenol [71] (Z)-3-Hexenyl acetate 4-Hydroxy-2, 5-dimethyl-3 (2H)-furanone [68] 3 -Hy droxy-4, 5-dim ethyl-2(5H)[73] furanone (Sotolon) 4-Hydroxy-non-2-enoic acid lactone [39] 3-Mercapto-2-pentanone [69] Methional [69] 4-Methoxy-2, 5-dimethyl-3 (2H)-furanone [68] 4-Methoxy-2-methyl-2-butanethiol [71 ] 2-Methoxyphenol [39, 72] 3-Methylbutanal [55] 3-Methylbutanol [ 18] 5-Methyl-5H-cyclopenta(b)pyrazine [67] 2-Methyl-2-furanthiol [69] 3 -Methyl-2,4-non andione [59] 12-Methyltridecanal [27] (E,E)-2,4-Nonadienal [39] (E,Z)-2, 6-Nonadienal [59 ] (E)-2-Nonenal [59] (Z)-2-Nonenal [59] (Z)- 1,5 -Octadi en- 3 -o n e [59] 1- O cten- 3 -hy drop eroxi de [59 ] 1-Octen-3-one [59] 2-Pentylpyridine [46] 2-Phenylethanol [ 18] 2-Phenylethylthiol [52] Skatol [30] 2,4, 5-Trimethylthiazol [69] Vanillin [39]

In the meantime, stable isotope dilution assays have been developed for nearly 60 important food odorants (Table 8). The OAV concept has been applied to characterize the

415 flavors of potato chips [75], flesh tomatoes [76], tomato paste [77], flesh strawberry juice [48], beer [ 18,78], Swiss cheese (Emmentaler) [30], roasted sesame [46], wheat and rye bread crusts [45, 55], stewed beef [79], virgin olive oils [71] or roasted beef [72]. Following is a discussion of the OAV concept using SIDA results illustrated by studies on the flavor of roasted sesame seeds [46] and Emmentaler cheese [30]. 3.2.1. R o a s t e d s e s a m e seeds

In Figure 7, the labelled internal standards used for the quantification of eight selected key sesame odorants identified on the basis of AEDA results [46, 52] are shown. Most of them were labelled with deuterium since their preparation was relatively inexpensive. In the case of 4-hydroxy-2,5-dimethyl-3(2H)-furanone, the labelling had to be performed via the more expensive carbon-13 labelled intermediates [68], since the deuterated standard underwent significant protium-deuterium exchanges (unpublished data).

Figure 7. Labelled internal standards used for the quantification of key sesame odorants, e: deuterium label, *: carbon- 13-1abel. Once an internal standard is available, the analysis can be easily performed: the food sample is spiked with an appropriate amount of the labelled standard and then the volatile fraction is isolated by solvent extraction and sublimation under a high vacuum (cf. Figure 1). If required, the odorants and the internal standards are enriched by liquid chromatography

416 (SC, HPLC) and finally analyzed by gas chromatography in combination with mass spectrometry, usually in the chemical ionization mode (MS/CI). The labelled standard and the odorant are separately quantified by using traces of their protonated molecular ions or main fragments as exemplified for 2-phenylethylthiol in Figure 8. From the amount of the added standard and by using calibration factors obtained with definite mixtures of standard and analyte [ 11 ] the concentration of the odorants in the food can be exactly determined.

Figure 8. Differentiation between 2-phenylethylthiol (m/z 105) and its labelled standard (m/z 107) by mass chromatography. Fragments resulting from the elimination of H2S from the protonated molecular ion are used [P. Schieberle, unpublished results]. Then, on the basis of odor thresholds determined in the matrix predominating in the respective food (e.g., vegetable oil, water or cellulose can be used), the OAVs are calculated. The results obtained by an application of the OAV concept on the key odorants of white and black sesame seeds are shown in Table 9. In both roasted seeds 2-furfurylthiol with a roasty, coffee-like odor followed by 2-phenylethylthiol with a burnt, rubbery note contributed with the highest OAVs to the overall flavors thereby confirming the results of the AEDA (cf. Table 2). On the other hand, the sensory contribution of (E,E)-2,4-decadienal (cf. Table 2) has been over-evaluated by AEDA due to its comparatively high odor threshold in oil (cf. Table 9). In summary, the data implied that the higher OAV of the tallowy smelling 2pentylpyridine in the black seeds in combination with the lower OAVs of 2-furfurylthiol and 2-phenylethylthiol mainly contribute to the flavor differences between both roasted seeds. 3.2.2. Swiss cheese (Emmentaler) A further advantage of the OAV concept is that the sensory contribution of an odorant to the overall flavor can additionally be evaluated on the basis of retronasal aroma thresholds. In Table 10 the OAVs of the six most important odorants of Emmentaler cheese are compared on the basis of their nasal and retronasal odor thresholds in oil [30]. The data revealed that the potato-like, sweet smelling methional and *he caramel-like, sweet smelling 4-hydroxy-2,5dimethyl-3(2H)- and 5-ethyl-4-hydroxy-2-methyl-3(2H)-furanone are the key odor compounds in Emmentaler cheese. The significantly increased OAVs of these three odorants calculated on the basis of the retronasal aroma thresholds implied that, besides taste compounds, these aroma compounds mainly contribute to the overall sweet flavor impression perceived during eating of Emmentaler cheese.

417 Table 9 Concentrations (gg/kg) and odor activity values (OAV) of selected key odorants in roasted (_180~ 30 min) black and white sesame seeds [46, 52] Odorant

Odor threshold a (gg/kgoil)

(E,E)-2,4-Decadienal 180 2-Methoxyphenol 19 2-Pentylpyridine 5 2-Furfurylthio 1 0.4 2-Ethyl-3,5-dimethylpyrazine 3 4-Hydroxy-2, 5-dimethyl-3 (2H)-furanone 50 2-Acetyl- 1-pyrroline 0.1 2-Phenylethylthiol 0.05

Black

White seeds

Conc. OAV 1103 2652 904 673 394 11685 n.a. 12

6 139 181 1682 131 234 n.d. 240

Conc. 212 4974 255 2461 238 9155 12 44

OAV 1 262 51 6152 79 183 120 880

a Odor thresholds were determined in sunflower oil. Table 10 Odor activity values of the six key odorants of Emmentaler cheese [30] OAV a Odorant nasal b Methional 4-Hydroxy-2, 5-dimethyl-3 (2H)-furanone 5-Ethyl-4-hydroxy-2-methyl-3 (2H)-furanone 3-Methylbutanal Diacetyl Ethyl hexanoate

77 33 37 16 10 4

retronasal c 306 206 148 8 10 7

a Calculated on the basis of thresholds in sunflower oil (triangle test). b Evaluation was performed by sniffing of the oil samples. c Evaluation was performed by tasting the oil samples. 3.2.3. Off-flavors

The OAV concept is also a very useful tool to detect compounds causing undesired flavors in foods. Compounds established by the OAV concept as main contributors to offflavors in foods are summarized in Table 11. As an example, data [44] on a flavor defect occurring in puff-pastry (40 % fat) will be discussed. Puff pastries prepared with butter have a high consumer acceptance. On the other hand, substitution of butter with baking margarines (shortenings) is preferable in dough processing but may lead to puff-pastries showing an offflavor, which is described as fatty, tallowy or lard-like [44]. To analyze the compounds causing this off-flavor, puff-pastries were prepared with butter and a margarine and compared by AEDA on the basis of the same amounts of pastry. Compounds showing significant differences in their FD-factors were quantified and their OAVs were calculated on the basis of odour thresholds in sunflower oil. The data summarized in Table 12 revealed the metallic smelling 4,5-epoxy-(E)-2-decenal and (E,Z)2,4-decadienal (fatty, green) as the compounds mainly causing the flavor differences between both products. The epoxydecenal has also recently been reported as a cause for the warmed-

418 over flavor in boiled beef, the light-induced flavor defect in butter and the storage defect of soybean oils stored in the dark (cf. Table 11). Model studies as well as the isolation of its precursors from a baking margarine have revealed that the flavor compound is formed during baking from triacylglycerides containing 9- and 13-hydroperoxy-octadecadienoic acids via 2,4-decadienal or 12,13-epoxy-9-hydroperoxy-octadecenoic acid, respectively, as the key intermediates. [83]. Such compounds, established as the main cause for food off-flavors, can then be used as indicator odorants to assess the development of the respective off-flavor during storage or processing. Table 11 Odorants established by AEDA or the OAV concept to cause an off-flavor in foods Odorant

Food

trans-4, 5-Epoxy-(E)-2-decenal

Puff-pastry, Soybean oil (dark storage) Butter Boiled beef (warmed over flavor) Sesame oil Soybean oil (light induced) Butter (light induced) Boiled beef Extruded oat meal Beer Beer Extruded oat meal Butter oil Boiled beef Butter oil Lemon oil (autoxidation)

2-Pentylpyridine 3-Methyl-2,4-nonanedione Hexanal 3-Mercapto-3-methylbutylformate Phenylacetaldehyde Hexanoic acid 1-Octen-3-one (E)- and (Z)-2-Nonenal Carvone

Reference [44] [53, 59] [80] [81 ] [46] [53, 59] [80] [81 ] [39] [ 18] [ 18] [39] [29] [81 ] [29] [82]

Table 12 Odour thresholds and odour activity values (OAV) of important pastry odorants (PB: butter pastry; PM: margarine pastry) [44] Odorant

(Z)-2-Nonenal (E,Z)-2,4-Decadienal (E,E)-2,4-Decadienal 4, 5-Epoxy-(E)-2-decenal d-Decalactone

Odour threshold a (gg/kg oil) 4.5 10 180 1.3 120

a Odour thresholds were determined in sunflower oil by the triangle test.

OAV PB