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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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.
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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.
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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 << to/c) the difference is negligible and can usually be ignored. This is true for most homogeneous materials encountered in the food industry, e.g., water, oils, solutions.
2.3. Measurable ultrasonic parameters Ultrasonic velocity. The velocity at which an ultrasonic wave travels through a material
97 depends on the physical properties of the material. For many materials the attenuation of ultrasound is small (i.e. c~ << m/c) and equation 5 can be simplified:
E
c2
(6)
The velocity is therefore determined by two fundamental physical properties of a material: its elastic modulus and density. The less dense a material or the more resistant it is to deformation the faster an ultrasonic wave propagates. Usually, differences in the moduli of materials are greater than those in density and so the ultrasonic velocity is determined more by the elastic moduli than by the density. This explains why the ultrasonic velocity of solids is greater than that of fluids, even though fluids are less dense [ 1]. The modulus used in the above equation depends on the physical state and dimensions of the material being tested. For bulk solids the appropriate modulus is K+4G/3, where K is the bulk modulus and G is the shear modulus, for solid rods it is Young's modulus, Y. (A rod is a material which has a diameter much smaller than the wavelength of ultrasound, i.e., d << )~/20). For liquids and gasses the appropriate modulus is the bulk modulus, which is the reciprocal of the adiabatic compressibility ~:. Shear waves will propagate through solids (E=G) but are highly attenuated in liquids and gasses and do not usually travel far enough to be detected. The ultrasonic velocity is determined in one of two ways: either the wavelength of ultrasound is measured at a known frequency (c = )if), or the time taken for a wave to travel a known distance is measured (c = d/t). Some of the techniques available for measuring the ultrasonic velocity of materials are discussed in section 3. Attenuation coefficient. All materials attenuate ultrasound to some degree. Attenuation is observed as a decrease in the amplitude of the ultrasonic wave as it travels through a material. The major sources of attenuation by a material are adsorption and scattering. Adsorption is due to mechanisms which convert some of the energy stored as ultrasound into other forms and ultimately into heat [9]. In gasses and liquids the most important forms of adsorption are shear and bulk viscosity, thermal conduction and molecular relaxation [9]. The situation is more complex in solids, and various other mechanisms also contribute to the adsorption [9]. Scattering is important in heterogeneous materials, and occurs when an ultrasonic wave is incident on a discontinuity (e.g. a particle, crack or void) and is scattered in directions which are different from that of the incident wave. Unlike adsorption the energy is still stored as ultrasound, but it may not be detected by a receiver in the forward direction because its propagation direction and phase have been altered. The characteristics of particles can be determined by measuring the angular dependence of the scattered ultrasound. The attenuation coefficient of a material has units of Nepers per meter (Np m-1)when defined by the following equation:
A - Ao e-ax
(7)
Here A is the amplitude of the wave, and x is the distance traveled. The attenuation coefficient is determined by measuring the dependence of the amplitude of an ultrasonic wave on distance and fitting the measurements to the above equation. The attenuation is often given in units of decibels per meter (dB m -1) where 1 Np = 8.686 dB. Impedance and reflection. Another important characteristic of a material is its specific acoustic impedance (Z). The specific acoustic impedance is defined as the ratio of the acoustic excess pressure (P) and the particle velocity (U): P 09t9 Z = -- = (8) U k
98 In general Z is complex and can be divided into a real and imaginary part: Z = R+iX, where R is the resistive component and X is the reactive component. For materials where the attenuation of ultrasound is small the imaginary part can be ignored, so Z = R = pc, which is called the characteristic impedance. The impedance is practically important because it determines the proportion of an ultrasonic wave which is reflected from a boundary between materials. When a plane ultrasonic wave is incident on a plane interface between two materials of different acoustic impedance it is partly reflected and partly transmitted (Figure 3). The ratios of the amplitudes of the transmitted (At) and reflected (Ar) waves to that of the incident wave (Ai) are called the transmission (T) and reflection coefficients (R), respectively. At
2Z1
Ai
Z 1 q- Z 2
Ar Zl - Z 2 R - ~ = Ai Z1 + Z 2
(9)
(10)
The greater the difference in acoustic impedance between the two materials the greater the fraction of ultrasound reflected. This has important consequences for the design and interpretation of ultrasonic experiments. For example, to optimize the transmission of ultrasound from one material to another it is necessary to chose two materials with similar acoustic impedance. To optimize the reflection coefficient materials with very different acoustic impedance should be used. The acoustic impedance of a material is often determined by measuring the fraction of ultrasound reflected from its surface. Solids usually have larger ultrasonic velocities and acoustic impedance, than liquids, which have larger values than gasses. Air has a very low acoustic impedance compared to liquids or solids which means that it is difficult to transmit ultrasound from air into a condensed material. This can be a problem when ultrasound is used to test dry materials, e.g., biscuits or egg shells. A small gap of air between an ultrasonic transducer and the sample to be tested can prevent ultrasound from being transmitted into the material. For this reason coupling materials (often aqueous or oil based) can be placed between the transducer and sample to eliminate the effects of the air gap, or alternatively soft-tip ultrasonic transducers can be used. 2.4. Ultrasonic characterization of materials An ultrasonic experiment consists of two stages: measurement of the ultrasonic properties of the material, e.g., velocity, attenuation or impedance; interpretation of these measurements to provide information about the relevant properties of the material. These may either be fundamental physico-chemical properties (such as composition, microstructure or molecular interactions) or functional properties (such as stability, rheology or appearance). Relationships can either be established in an empirical fashion, or by using theories which describe ultrasonic propagation in materials (see section 4). To utilize the full potential of ultrasound for characterizing food materials it is important to choose the most suitable method of carrying out the measurements and to carefully analyze the data. Many applications have failed in the past because workers have used poorly designed experiments or have interpreted measurements in an inappropriate manner. 3. E X P E R I M E N T A L T E C H N I Q U E S At present there are few commercial ultrasonic instruments which are specifically designed for characterizing food materials. This is one of the major reasons why ultrasound is not
99
Figure 3. Reflection and transmission of an ultrasonic wave from a boundary between two materials. used more frequently in the food industry. This situation is already starting to change, and a number of manufacturers have recently developed ultrasonic instrumentation for application to food materials (e.g., Cygnus Instruments, Dorchester, Dorset, UK; Nusonics Inc., Tulsa, OK, USA). These instruments are simple devices which measure the velocity of ultrasound of a liquid at a single frequency. Once the full potential of ultrasound is recognized by the food industry more sophisticated instruments are likely to be developed, e.g., instruments which automatically measure ultrasonic properties over a wide range of frequencies. At present, however, it is usually necessary for investigators to design and set-up their own experimental technique. The careful design and implementation of an experimental technique can make the difference between a successful and unsuccessful application of ultrasound, and requires a certain level of understanding of the factors which affect ultrasonic propagation in materials. There are a number of different experimental configurations which can be used to carry out ultrasonic experiments [ 10,11 ]. The choice of a particular configuration depends on the material to be tested and the requirements of the operator, e.g., finances available, accuracy, type of information required, and whether measurements are on-line or off-line. Most techniques can be divided into two categories: those which utilize pulsed ultrasound and those which utilize continuous wave (c.w.) ultrasound.
3.1 Pulse techniques Techniques which utilized pulse ultrasound are the most widely used for testing materials because the experimental configuration is simple to design and operate, measurements are rapid, non-invasive and non-intrusive, there are no moving parts and the technique can easily be automated. Even though pulse techniques are less accurate than c.w. techniques, their accuracy is usually sufficient for most applications in the food industry. The simplest and most widely used technique for making ultrasonic measurements is called the pulse-echo technique. More sophisticated pulsed methods have been developed to improve the accuracy of measurements (e.g., pulse echo overlap, sing-around, pulse interferometer [10,11]), however, the operating principles are basically the same as those of the pulse-echo technique. For this reason, only the pulse-echo technique is described and some of the modifications are mentioned in passing. A typical experimental configuration consists of a measurement cell which contains the sample, a pulse generator, an ultrasonic transducer and an oscilloscope (Figure 4). The pulse generator produces an electrical pulse of an appropriate frequency and amplitude. This pulse is converted into an ultrasonic pulse by the transducer. It then propagates through the sample until it reaches the far wall of the cell where it is reflected back to the transducer. The
100
Figure 4. Schematic diagram of the experimental configuration for an ultrasonic p u l s e - e c h o experiment. transducer now acts as a receiver and converts the ultrasonic pulse back into an electrical pulse which is displayed on the oscilloscope. Because each pulse is partially transmitted and partially reflected at the cell walls a series of echoes are observed on the oscilloscope (Figure 5). The velocity and attenuation coefficient are determined from these echoes. Each echo has traveled a distance twice the cell length d further than the previous echo and so the velocity can be calculated by measuring the time delay t between successive echoes: c = 2d/t. The cell length is determined accurately by calibration with a material of known ultrasonic velocity, e.g. distilled water: 2d = Cw.tw (where the subscripts refer to water). The attenuation coefficient is determined by measuring the amplitudes of successive echoes: A = Aoe -2c~d, and comparing them to the values determined for a calibration material. A number of sources of errors have to be taken into account if accurate measurements are to be made, e.g., diffraction and reflection (see below). 3.2. Continuous wave techniques Continuous wave methods are the most accurate means of making ultrasonic measurements. Even so, they are used less frequently than pulse methods because measurements are more time consuming and laborious to carry out, are more difficult to automate, and the measurement cell requires a high degree of precision engineering. These techniques therefore tend to be used in specialized research laboratories where accurate measurements are important. Continuous wave ultrasound is utilized in a variety of different techniques, but the most commonly used is the i n t e r f e r o m e t e r [ 10,11 ]. A simple interferometer is illustrated in figure 6. The same basic components can be used for a c.w. experiment as for a pulsed experiment, i.e., a signal generator, a transducer, a measurement cell and an oscilloscope. Nevertheless, there function and arrangement are slightly different. The sample is contained in the measurement cell, between an ultrasonic transducer and a reflector plate which moves vertically through the sample. The signal generator applies a continuous sine wave of suitable frequency and amplitude to the transducer. The transducer generates an ultrasonic sine wave which propagates into the sample and is reflected back and forth between the reflector plate and transducer. Standing waves are set up in the sample, and the amplitude of the signal received by the transducer
101
Figure 5. An ultrasonic pulse travels back and forth across the measurement cell so that a series of echoes is observed on the oscilloscope. goes through a series of maxima and minima as the reflector plate is moved vertically through the sample and destructive and constructive interference occurs. The amplitude of the maxima decreases as the distance between the reflector plate and transducer is increased because of attenuation by the sample, reflection at the boundaries and diffraction (see later). The distance between successive maxima is equal to half the ultrasonic wavelength of the material and so the velocity can be calculated: c = )ft. The accuracy of the measurements can be improved by measuring the distance between a large number of maxima.
3.3. Components of an ultrasonic experiment In this section some of the important factors influencing the selection and design of the various components in an ultrasonic experiment are highlighted. Measurement cell. The measurement cell should be made of a material which does not react with the sample. The cell walls should be of an appropriate thickness and acoustic impedance so that any reverberations in the cell walls do not interfere with the signal from the sample. The internal walls of the cell should be smooth and parallel so that scattering or oblique reflection of the ultrasonic wave do not cause errors in the velocity and attenuation measurements. Ultrasonic measurements are particularly sensitive to temperature and so it is important to either use a thermostated measurement cell, or to measure the temperature and make a suitable correction. Transducer. The most commonly used ultrasonic transducers for testing materials are based on the piezoelectric effect [12]. Piezoelectric materials generate an electrical potential when they are deformed along a certain axis, and deform when an electrical potential is applied across them [ 12]. They can therefore be used as both receivers and generators of ultrasound. An ultrasonic transducer consists of a piezoelectric crystal, bonded to a backing material, which dampens its oscillations, and a front-plate, which protects it from damage (figure 7). The acoustic impedance and thickness of the front-plate have to be chosen so as to optimize the energy output of the transducer: the front plate is usually manufactured to be quarter of a
102
Figure 6. Ultrasonic interferometer. C.w. ultrasound is generated by the transducer, and the amplitude of the received signal is measured as the distance between the reflector plate and transducer is varied. wavelength thick [ 12]. There are a number of factors which have to be considered when deciding which transducer to use for a particular application. The most important of these are the frequency, crystal diameter and acoustic matching. An ultrasonic transducer generates ultrasound over a range of frequencies which depends on its resonant frequency and the degree of damping of the crystal. The resonant frequency fr of a transducer is determined by its thickness and the
Figure 7. Schematic representation of a piezoelectric ultrasonic transducer.
103 ultrasonic velocity of the material from which it is manufactured (fr = c/2d). If ultrasound is applied to a crystal which is undamped it oscillates for a relatively long time and produces most of its energy over a narrow range of frequencies centered at the resonant frequency. If the crystal is damped it oscillates for a shorter time and produces energy over a wider range of frequencies. Short duration pulses are used in pulse-echo experiments to resolve successive echoes from the material. A highly damped transducer can either be forced to a particular frequency by applying a tone-burst electrical input signal (i.e. a pulse which contains a number of cycles at a certain frequency) or it can be used to generate a pulse which contains a wide range of frequencies. Highly damped transducers are used to carry out frequency scanning measurements by using Fourier Transform analysis of the pulses [ 13].
Figure 8. Diffraction of an ultrasonic wave emitted by a transducer. It is often assumed that an ultrasonic wave propagates through a material with a crosssectional area equal to that of the transducer which generated it. In fact the ultrasonic wave spreads out after it has traveled a distance L (= D2fr/4C) from the transducer (Figure 8), where D is the diameter of the crystal, fr is its resonant frequency, and c is the ultrasonic velocity of the material the wave propagates through. The region from the transducer face to L is called the nearfield, and the region after this is called the farfield. Diffraction often has a significant affect on both the velocity and attenuation of an ultrasonic wave. Even if there was no attenuation of the ultrasonic wave by the material being tested the intensity detected by a transducer placed in the far-field would be reduced because some of the wave is diffracted. In addition, the phase of the wave is altered which affects the measured velocity. Velocity and attenuation measurements should therefore be corrected for diffraction effects if accuracy is important. Analytical equations have been derived and tabulated values are also available [ 14, 15]. The ultrasonic properties of many food materials are frequency dependent, i.e., the values of the velocity and attenuation depend on the frequency at which the measurement is made. This means that only average values of velocity and attenuation are determined if a pulse containing a wide range of frequencies is used to test a material. For this reason measurements are usually carried out at approximately a single frequency (using a tone-burst pulse) or Fourier Analysis is used to determine the frequency dependence of the velocity and attenuation [ 13]. More information about the properties of a material can usually be obtained by making measurements over a range of frequencies, rather than at a single frequency, e.g., microstructure.
104
Signal generator. A signal generator produces an electrical output of a given amplitude, duration and frequency. The type of output used depends on the nature of the experiment. In a continuous wave experiment a sine-wave with a frequency equal to the resonant frequency (or odd-harmonics) of the transducer are used. In a pulse-echo experiment pulses with a short time duration (and therefore wide range of frequencies) are used. The frequency of the electrical pulse should match the range of frequencies which the transducer can generate (which depends on the resonant frequency of the transducer and the degree of damping). For some types of experiments (e.g. tone-burst or pulse interferometry) gated sine-waves are used, i.e., pulses which contain a number of cycles. Oscilloscope. The oscilloscope is used to display the signal received from the sample. Digital storage oscilloscopes (D.S.O.) are often used because the signal can be displayed, stored and various mathematical functions carried out, e.g., averaging, smoothing, Fourier Transform. A D.S.O. must have a sufficiently high sampling rate so that a representative image of the ultrasonic signal is shown. D.S.O. are relatively expensive and so it is often convenient to use some other form of digitizer once an application has been developed and it is not necessary to observe the signal. 3.4. Other sources of error Side wall reflections. If the angle of diffraction of an ultrasonic wave leaving a transducer is large enough, reflections may occur from the side walls of the cell. This reflected ultrasound will interact with the ultrasound which has traveled directly through the sample and affects both velocity and attenuation measurements. It is therefore important to calculate the diffraction angle of the transducer and ensure that the side walls are far enough apart so that side-wall reflections do not interfere with the measurements [ 1]. Transmission and reflection at multilayer boundaries. The fraction of energy reflected or transmitted from a boundary which consists of a number of layers depends on the acoustic impedance of the layers and the relationship between their thickness and the wavelength of ultrasound. Both the amplitude and the phase of the ultrasonic wave can be significantly altered on reflection or transmission, which can cause errors in attenuation and velocity measurements if it is not taken into account. Equations have been derived to account for transmission and reflection of ultrasound at multilayer boundaries [16,17]. Transducers consist of a number of layers (backing material, crystal, wear-plate) and therefore may introduce errors into measurements. For this reason it is sometimes useful to use a buffer rod, i.e., a piece of material of suitable length and diameter which is placed between the transducer and the sample to be measured [ 13]. The walls of a measurement cell can often be used as a suitable buffer rod. Buffer rods can also be used to isolate the transducer from harsh conditions which might damage it, e.g., high temperatures or chemically reactive materials [2].
4. I N T E R P R E T A T I O N OF ULTRASONIC M E A S U R E M E N T S Once the ultrasonic properties of a material have been measured it is necessary to relate them to the physical properties which are of interest to the food scientist. There are two approaches commonly used to do this, these are the so-called empirical and theoretical approaches. 4.1 The empirical approach In the empirical approach the ultrasonic parameters of a range of samples with known properties are measured. Empirical relationships are then established between the property of interest and the measurable ultrasonic parameters. A typical example of this approach is the determination of the sugar content of fruit drinks [18]. A series of sugar solutions of different sugar concentration are prepared and their ultrasonic velocities are measured. This data is then used to make up a calibration curve which relates the sugar content to the
105
Figure 9. Dependence of ultrasonic velocity of the sugar content of a series of aqueous glucose solutions at 20oc [ 18]. ultrasonic velocity. The sugar content of an unknown sample, such as a fruit juice, can then be determined by measuring its ultrasonic velocity (Figure 9). This approach is suitable for many food materials, however, it should be used with caution. It must always be remembered that a calibration curve is only strictly applicable to the set of conditions over which was established. If some parameter changes which was not included in the original calibration, e.g., the temperature or the type of solute, then the calibration curve may no longer be valid. For example, the dependence of the ultrasonic velocity on sugar concentration actually depends on the type of sugar present (fructose, glucose, sucrose, etc.). Thus errors may occur if sugars are present in a fruit drink which were not included in the calibration experiments. The empirical approach also has the disadvantages that it does not give any information about the fundamental physical processes occurring in the system and it does not have predictive power. Nevertheless, there are many materials whose properties are too complex to be described by the existing theoretical approaches and so the empirical approach is the only option at present.
4.2. Theoretical approach There is a good understanding of the interaction between ultrasound and matter for many materials, and mathematical theories are available to relate the measurable ultrasonic parameters to the composition and microstruture of these systems. The most important examples of these types of system in the food industry are homogeneous liquids, emulsions and suspensions. Homogeneous liquids do not scatter ultrasound because they contain no discontinuities, and so the attenuation is due solely to absorption processes. If the attenuation coefficient is relatively low (ct << odc) the velocity is given by the following equation: c
2
= ~
1
top
(11)
106 Thus a measurement of the ultrasonic velocity and density can be used to determine the adiabatic compressibility (or bulk modulus) of the material. For homogeneous solids measurements of the compression and shear velocities can be used to determine the bulk and shear moduli (see section 2.4). The Young's modulus of rod-like materials (e.g. spaghetti) can be determined by measuring the velocity of ultrasound. Most food materials contain a number of different components, and are heterogeneous rather than homogeneous. The above equation must therefore be extended to multicomponent systems. In a two phase system the values of the density and adiabatic compressibility given by equation 11 are modified. For an ideal mixture the density and adiabatic compressibility are given by their volume average values: K"-- ~K"2 + (1-- ~)X' 1 P = r
(12)
+ (1 - r
Here the subscripts 1 and 2 refer to the two components in the system, and 0 is the volume fraction of component 2 (01 + 02 = 1). The attenuation of an ideal mixture is given by the volume average of the attenuation coefficients of the component phases: a = q~a 2 + (1 - q ~ ) a 1
(13)
For ideal mixtures there is a simple relationship between the measurable ultrasonic parameters and the concentration of the component phases. Thus ultrasound can be used to determine their composition once the properties of the component phases are known. Mixtures of triglyceride oils behave approximately as ideal mixtures and their ultrasonic properties can be modeled by the above equations [ 19]. Emulsions and suspensions where scattering is not appreciable can also be described using this approach [20]. In these systems the adiabatic compressibility of particles suspended in a liquid can be determined by measuring the ultrasonic velocity and the density. This is particularly useful for materials where it is difficult to determine the adiabatic compressibility directly, e.g., powders, biopolymer or granular materials. Deviations from equations 11 - 13 in non-ideal mixtures can be used to provide information about the non-ideality of a system. In non-ideal mixtures, or systems where scattering of ultrasound is significant, the above equations are no longer applicable. In these systems the ultrasonic properties depend on the microstructure of the system, and the interactions between the various components, as well as the concentration. Mathematical descriptions of ultrasonic propagation in emulsions and suspensions have been derived which take into account the scattering of ultrasound by particles [20-21]. These theories relate the velocity and attenuation to the size (r), shape (x) and concentration (0) of the particles, as well as the ultrasonic frequency (co) and thermophysical properties of the component phases (TP). c = f(co, ~), x, r, TP) = f(o3, r
(14)
x, r, T P )
Measurements of the velocity and attenuation, usually as a function of frequency, can be used to provide valuable information about a system, e.g. microstructure. Theories are available which describe ultrasonic propagation in emulsions, suspensions, bubbly liquids, laminated solids, porous solids, fibrous materials and a number of other materials [20-29].
107
5. A P P L I C A T I O N S OF ULTRASOUND TO FOODS Over the past half a century or so a wide variety of different applications of ultrasound to food materials have been developed, which reflects the complexity and diversity of food materials, as well as the versatility of the ultrasonic technique. In this section, previous applications of ultrasound to foods are discussed, as well as possible future applications. 5.1. Measurement of distance Ultrasound can be used to make precise measurements of the thickness of materials [2]. An ultrasonic transducer is pressed against the side of a material and the time taken for a pulse to travel across the material and back is measured (Figure 10). If the velocity of ultrasound in Ultrasound is the material is known then the distance can simply be calculated: 2d = ct. particularly useful for measurements on materials which are difficult to access by conventional methods e.g. the determination of the thickness of a pipe when access is only available to the exterior of the pipe. It can also be used to measure the thickness of individual layers in multilayer systems (Figure 10).
Figure 10. Ultrasonic pulse-echo technique for determining the thickness of layers in multilayer materials. The determination of the thickness of the layers of fat and lean tissue in animal flesh is the most popular use of ultrasound in the food industry at present [5, 6]. In fact there are over a hundred references pertaining to this application of ultrasound in the F o o d Science and Technology Abstracts (1969-1993). In contrast to most other applications of ultrasound in the food industry, which have rarely developed further than use in the laboratory, there are a number of commercial instruments available for grading meat quality [6, 30-32]. This application is based on measurement of time intervals between ultrasonic pulses reflected from boundaries between layers of fat, lean tissue and bone. Ultrasonic techniques have the advantage that they are fairly cheap, easy to operate and give predictions of meat quality of live animals. Other examples of thickness determinations include: liquid levels in cans or tanks, thickness of coatings on confectioneries, egg shell thickness.
108
5.2. Determination of composition The composition of foods plays an important role in determining their overall quality and cost. In addition, many foods must meet strict legal requirements concerning their composition if they are to be labeled in a certain way, e.g., mayonnaise, low-fat products. It is therefore of considerable importance to have reliable methods of determining composition. Many of the traditional techniques used for this purpose are based on chemical, gravimetric or extraction methods which are time consuming and laborious to carry-out, and so there has been great emphasis on the development of rapid analytical techniques. A number of workers have realized the potential of ultrasound for determining the composition of foods, e.g., fat:lean ratio of meats [30-32], oil content of fatty foods [33-36], milk composition [3746], sugar concentration [18,47-53], alcohol content of drinks [49-51], triglycerides in oils [54-58], air in aerated foods [59-61], salt concentration of brine [62], biopolymer concentrations in gels and aqueous solutions [63-67].
Figure 11. Dependence of ultrasonic velocity on the tristearin concentration of tristearin/paraffin oil mixtures at 18oc. This application of ultrasound relies on their being a significant change in the ultrasonic properties of a material as its composition changes. Figure 11 shows the variation of ultrasonic velocity with tristearin content for mixtures of tristearin and paraffin oil at 18 ~ (Figure 11). At this temperature the tristearin is completely solid and so its concentration is equal to the solid fat content (SFC). Once a calibration curve such as figure 11 has been established the SFC of an unknown sample can be determined by measuring its ultrasonic velocity. The accuracy of the concentration determination depends on how accurately the velocity can be measured and the magnitude of the change in velocity with composition: the greater the change the more accurately the concentration can be determined. The velocity increases by about 3 m s-I per 1% increase in SFC. There are commercial instruments which can measure the ultrasonic velocity to better than 0.2 ms-1 and so the SFC can be measured to better than 0.1% Similar figures can be obtained for aqueous solutions of sugars, salts, proteins and carbohydrates. The ultrasonic properties of most materials are strongly temperature dependent and so it is important to take this into account in the analysis. One has to be careful when using ultrasound to determine the composition of microheterogeneous materials, because the measurements depend on the structure of the
109 sample as well as the composition. For these systems it is often necessary to make measurements as a function of frequency and to use theoretical equations which describe ultrasonic propagation in microheterogeneous materials to relate the ultrasonic measurements to their physical properties. Ultrasound has a number of advantages over other techniques used for composition determinations: it is capable of rapid and precise measurements, it can be used in opaque systems, it is non-destructive and it can be used on-line.
5.3. Particle size analysis Ultrasonics can be used to determine the size of particles in microheterogeneous materials in a manner analogous to light scattering. An ultrasonic wave incident upon an ensemble of particles is scattered by an amount which depends on the size of the particles and the ultrasonic wavelength. The scattered waves, interact with the incident wave, which modifies its phase and amplitude. Thus velocity and attenuation measurements can be used to determine particle size.
Figure 12. Dependence of velocity and attenuation on particle size for an oil-in-water emulsion The dependence of the velocity and attenuation on the particle size and frequency, for a typical oil-in-water emulsion in the long wavelength limit (i.e. r << ~,) is shown in figure 12. The ultrasonic parameters are plotted against rVf because there is a unique relationship between this combination of parameters, i.e., measurements of c and c~)~made on emulsions at different frequencies and particle sizes fall on the same curve. The velocity increases as the droplet size increases, whereas the attenuation multiplied by the wavelength has a maximum value at some characteristic droplet size. The shape of the curves depends on the particle size distribution and the concentration of particles. By comparing experimental measurements with predictions made using multiple scattering theory it is possible to determine both the concentration and size of droplets in an emulsion [20]. For many emulsions and suspensions there is a good agreement between theory and experiment up to concentrations of 30 or 40% [68]. The possibility of using ultrasound to measure droplet sizes in real foods has so far been demonstrated for casein micelles [69], salad creams [35] and milk globules [70]. However,
110 there are many other food emulsions where ultrasound would also be useful, e.g., mayonnaise, cream liqueurs, margarine. Ultrasound has advantages over many of the existing methods of particle sizing because it can be applied to systems which are optically opaque or concentrated without the need for dilution or any other form of sample preparation. It can therefore be used to determine particle sizes on-line which would be useful for monitoring processes in the food industry, e.g. homogenization. The main drawbacks of the technique are that a considerable amount of data about the thermophysical properties of the component phases are needed in the theories used to interpret the measurements (e.g., specific heat capacity, thermal conductivity, coefficient of cubical expansion, density and viscosity), and there are no commercial instruments available at present which measure the frequency dependence of the velocity and attenuation coefficient.
5.4. Determination of creaming profiles An application which is related to the previous two is the determination of creaming profiles in emulsions and suspensions [71-73]. The particles in these systems usually have different densities to that of the continuous phase and so will move under gravitational forces. This movement affects the appearance and stability of the system. By measuring the ultrasonic velocity or attenuation as a function of sample height and time it is possible to quantify the rate and extent of creaming (Figure 13). This technique can be fully automated and has the advantage that creaming can be detected before it is visible to the eye, and a detailed creaming profile can be determined rather than just a single boundary. By measuring the ultrasonic velocity as a function of frequency it is possible to determine both the concentration and size of the droplets as a function of sample height in an emulsion [20].
Figure 13. Ultrasonic determination of creaming profiles. ~ is the disperse phase volume fraction, t is the time and x is the height of the emulsion.
5.5. Phase transitions Many foods contain fat or aqueous phases which can undergo some form of phase transition during manufacture, storage or consumption, e.g. melting or crystallization. The ultrasonic
111 properties of a material change significantly when it melts or crystallizes and so ultrasound can be used to monitor phase transitions. The variation of ultrasonic velocity with temperature for a typical fatty material is shown in figure 14. The curve can be separated into three regions. At low temperatures the fat is all solid and the decrease in velocity with increasing temperature is simply due to the negative temperature coefficient of ultrasonic velocity of solid fat (region I). As the temperature is increased further the fat starts to melt and the velocity decreases more dramatically because liquid oil has a lower velocity than solid fat (region II). The range of temperatures over which this region extends depends on the type of triglycerides the fat contains. When the temperature is increased to a point where all the fat has melted the slight decrease in velocity with temperature is due to the negative temperature coefficient of the velocity of liquid oil (region III). Ultrasound has been used to monitor phase transitions in margarine, butter, shortening, meat and various triglyceride/oil mixtures [ 19, 34, 74].
Figure 14. Dependence of the ultrasonic velocity on temperature for a fatty material. The solid fat content of a material can be determined by measuring its ultrasonic velocity: 1
SFC-
1
c2 c2 1-----~
(15)
4 This equation is derived from the equation describing ultrasonic propagation in ideal mixtures assuming that the densities of the solid and liquid phases are approximately equal (equations 11-13). Here CL and cs are the velocities in the system if all the fat were completely liquid or completely solid, respectively. These values are determined by extrapolating measurements from the higher and lower temperatures into the region where the fat is partially crystalline (Figure 14). A similar equation has been derived for three phase systems consisting of solid fat, liquid oil and water [34]. This equation can be used to determine the SFC of partially crystalline emulsions, such as cream, margarine and spreads. The values of SFC determined using this approach are in good agreement with those determined using traditional techniques
112 such as dilatometry [75] and NMR [76], In addition, the ultrasonic technique is more sensitive to low concentrations of solid fat and is therefore more suitable for application to low fat products.
5.6. Miscellaneous applications. There are a variety of applications of ultrasound in the food industry which fall under this heading. Ultrasonic velocity and attenuation measurements correlate well with conventional rheological measurements of the texture of biscuits and wafers [77,78], and have the advantage that they were non-destructive and quicker and simpler to carry-out. Ultrasonic absorption measurements have been used to monitor enzymatically induced changes in milk [79] and in starch solutions [80]. Velocity measurements have been used to determine the properties of foods during extrusion [81]. Ultrasound has also been used to detect flaws and air cells in cheeses [82, 83] and to determine the ripeness of fruits and vegetables [84-89]. Measurements of the ultrasonic velocity and density of aqueous solutions of sugars, salts, amino-acids and biopolymers have been used to obtain information about their structure and degree of hydration [29, 63-67]. Ultrasonic spectroscopy has been used to study chemical and molecular equilibria in food biopolymer solutions and gels [90]. There are an increasing number of applications of Acoustic Emission in the food industry. Acoustic emission techniques involve the use of passive transducers, i.e., they measure (ultra)sound generated by materials or processes, rather than measuring the response of a sample to an applied pulse. Acoustic emission techniques have been used to assess the crispiness, crunchiness and hardness of potato chips and other foods [91-98]. They have also been used to determine the moisture content of grain flowing along conveyors belt [99-101] and to detect the presence of parasites in stored grain [102]. Acoustic emission is in its infancy in the food industry. Its success depends on workers being able to establish relationships between the amplitude-frequency response of acoustic emissions and the various physical mechanisms producing them. Potentially, acoustic emission could provide a powerful low-cost tool for characterizing foods. Electroacoustic techniques have been developed to measure the electrokinetic properties of colloidal systems [103]. A charged particle subjected to an oscillating electrical field will move with the field. This movement generates a pressure wave which can be detected by ultrasonic transducers (if the appropriate frequency electromagnetic wave is used). Alternatively, an ultrasonic pressure wave applied to a suspension of particles with densities different from that of the surrounding medium will cause the particles to oscillate. Charged particles generate an electromagnetic wave which can be detected by suitable probes. The magnitude of the electroacoustic effect depends on the size and charge of the particles, and it is possible to obtain information about these parameters using appropriate theoretical relationships [103]. Commercial instruments are available which utilize this phenomenon (e.g. Electrokinetic Sonic Analyzer, Matec Applied Sciences, Hopkinton, MA, USA). 5.7. On-line measurements One of the most promising applications of ultrasound in the food industry is as an on-line sensor for measuring the properties of food materials during processing. There are a number of important attributes which any on-line sensor must have. It must be capable of rapid and reliable measurements, be non-invasive and non-destructive, be robust, low cost, easily automated and hygienic [ 104]. Sensors based on ultrasound have all of these attributes. 5.8. Limitations and advantages It is useful to give a brief overview of some of the major advantages and limitations of ultrasound as a tool for characterizing the properties of food materials. Ultrasound is fairly inexpensive to purchase and operate, it is robust and can therefore be used in factories, it is capable of rapid and reliable measurements, in a non-destructive and non-invasive manner. In addition, measurements can easily be automated and so the technique is suitable for on-line measurements as well as an analytical instrument in the laboratory. The major disadvantages
113 are: there are few commercial instruments specifically materials at present, although this situation is changing; specific, i.e., the approach used for one application may be ultrasound is highly attenuated by materials which contain its application to certain foods.
designed for application to food the technique is fairly application different from that for another; and small air bubbles, which may limit
6. CONCLUSIONS Ultrasound has considerable potential for characterizing the physical properties of food materials. Research over the past 50 years or so, in food science and in other areas, has led to a fairly good understanding of the interaction between ultrasound and biological materials, such as foods. Even so, the complexity and diversity of foods means that more fundamental research is still needed in many areas. The benefits that the food industry can gain from the development of ultrasonic techniques are substantial. On-line sensors give manufacturers greater control over the properties of the product during manufacturing which will lead to improvements in product quality and reduction in costs. Fundamental studies can give valuable information about the relationship between the molecular properties of foods and their functional properties. The continued development of ultrasound in the food industry depends on the availability of appropriate ultrasonic instrumentation, and workers using a systematic approach to the measurement and interpretation of ultrasonic data. There are already a number of areas where the application of ultrasound would prove extremely fruitful, e.g., the characterization of fats, aqueous solutions and colloidal systems, and as an on-line sensor for measuring the properties of foods during processing. Future research may lead to the development of many more useful applications. Ultrasound should therefore be regarded as a useful addition to the array of techniques already used to characterize foods. Ultrasound may have advantages over alternative techniques for certain applications, or it may be useful to use it in combination with other complementary techniques. Future instruments may combine a variety of different technologies in a single instrument, e.g., ultrasound, NMR and dielectric measurements. REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12.
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Characterization of Food: Emerging Methods A.G. Gaonkar (Editor) 9 1995 Elsevier Science B.V. All rights reserved.
117
Chapter 5 R e c e n t a d v a n c e s in c h a r a c t e r i z a t i o n o f f o o d s u s i n g n u c l e a r m a g n e t i c r e s o n a n c e (NMR) Hisahiko Watanabe, Mika Fukuoka and Tokuko Watanabe Food Science and Technology Department, Tokyo University of Fisheries Konan 4, Minato, Tokyo 108 Japan
1.
INTRODUCTION
NMR has two very important advantages over other physicochemical methods when applied to food science and engineering. These are: that it is non-invasive and that information may be obtained over a wide variety of length and time scales. Because of these two properties NMR is well suited to probing the complexities of heterogeneous systems such as food. The range of time and length scales that are available derives from the different sorts of information that NMR can give. In general terms three types of information are available which relate to chemical environment, molecular dynamics and position in space. Often environmental results contain mixtures of these types of information and detailed analysis is required before separation is possible [ 1]. The explosive advancements in NMR technology in recent years have dramatically expanded the avenues and techniques available.: NMR techniques for the study of food range from inline/on-line oil and water analysis, to characterization and authentification of foods and beverages, water relations in foods and biological tissues, mobility of water in food and to probing microstructure of food. The applications of NMR techniques in food research were recently reviewed [2, 3]. In this chapter, we describe NMR spectroscopy with an emphasis on one of the most important recent developments in NMR: the methods which concern the application of a linearly varying magnetic field, known as a field-gradient, across the sample. This method provides information on the position of the target molecules. Application of a field gradient in the form of a series of pulses associated with radio frequency pulses, enables collection of NMR signals non-destructively in which information on the density profile of water and fat in food are encoded. By a process of reconstruction through calculating Fourier transformation using a computer, a two-dimensional image of water and/or fat in food at a cross section of interest can be formed. Since the NMR signal is sensitive to the chemical environment and molecular dynamics, the reconstructed images may be contrasted reflecting such factors, demonstrating the physical and chemical changes in food. Moreover, the application of a pulsed field gradient enables measurement of translational motion (diffusion and flow) in the food and since this motion reflects the microstructure of food as well as dynamic parameters such as membrane permeability, measuring translational motion using pulsed field gradient NMR provides a good tool for exploring the structural and dynamic characteristics of food.
118 2.
A B R I E F O U T L I N E O F T H E P R I N C I P L E OF N M R
2.1. P r i n c i p l e of N M R There are excellent textbooks available on the principle of NMR for a variety of readers, ranging from the readers who may have relatively little background in physical sciences [4, 5, 6] to NMR specialists [7, 8]. A brief outline on selected aspects of the principle of NMR is given in this section. Elementary particles such as a proton or a neutron behave like a spinning top or a gyroscope and possess angular momentum, or spin. Atomic nuclei which are composed of these elements possess angular momentum. They also possess an electronic charge, which produces a magnetic field along the axis of rotation of the nucleus except several nuclei with even number of protons and neutrons. The magnetic behavior is characterized by the magnetic moment, 1~, which is determined by the gyromagnetic ratio, u Planck's constant, h, and the spin quantum number, I, as follows,
~t= y h(2z-t)-1 [I(I+l)] g
(1)
When nuclei are placed in a static magnetic field Bo, nuclear magnetic moments actually precess about the direction of the applied magnetic field at a small angle, at a Larmor frequency, Vo (= yBo(2~)-l), due to their gyroscopic properties as shown Figure 1. The number of the energy levels allowed for the magnetic moment is limited to 2I+ 1 in which magnetic moments direct with such angles that projections of the magnetic moment to the magnetic field direction (z-axis) become the magnetic quantum number, mi (mi = I, I-1, ..- , -(I-1), -I). Each allowed situation possesses different energy determined by E (= -u h(2n) -1 Bomi). The energy difference, AE, between two levels is AE = y h(2~t) -1 B o. Nuclear magnetic moments can exist in all allowed energy levels at room temperature. The population distribution of nuclear magnetic moments obeys a Boltzman equation. For spin 1/2 nuclei, the ratio of nuclear magnetic moment populations between two levels is given by the Boltzman factor, exp(-AE/kT), where k is Boltzman constant and T is the absolute temperature. A change in the energy state of a nuclear magnet can be induced by an oscillating magnetic field, if the energy of the oscillating field is equal to the energy difference between the neighboring two levels, that is, Ami = + 1 and hv = AE = u h(2~t)-a Bo, where v is the frequency
Figure 1. Nuclear magnets (I=1/2) precessing about the d i r e c t i o n of the a p p l i e d magnetic field Bo. Only two energy levels are allowed.
119 of the oscillating magnetic field. Notice that the frequency v is equal to the Larmor frequency, Vo. NMR is a spectroscopy to detect the Larmor frequency of nuclear magnets. In terms of angular frequency, m (= 2nv), a resonant condition is generally described as m = ~Bo.
(2)
This equation depicts a basic principle of NMR and the experimentally observed quantity. For many nuclei such as proton (1H), 13C, 19F, 31p, I =1/2, and mi = _+1/2, where two energy states are allowed. For simplicity, we shall here be concerned only with I=1/2 nuclear magnets. The precessing axis of nuclear magnet is either aligned parallel (spin up) or antiparallel (spin down) with the magnetic field, which corresponds to the lower or the higher energy state, respectively. Slightly more of nuclear magnets occupy the lower energy state in thermal equilibrium. Thus, net magnetic moment of the ensemble of nuclear magnets aligns parallel to the magnetic field. We call the net magnetic moment a macroscopic magnetization, M. In NMR, the behavior of the macroscopic magnetization is detected. NMR resonance frequencies are in the radio frequency (r.f.) range (~MHz) which depends on the strength of the magnetic field used and the gyromagnetic ratio of the nucleus investigated. When an r.f. pulse (a pulse of oscillating magnetic field with a radio frequency) is introduced, magnetic resonance (net absorption) is induced on the basis of the resonant condition. 2.2.
Fourier transform NMR
To visualize the spin behavior in FT-NMR, it is easy to use the classical description, where the nuclei are regarded as an ensemble of small magnets spinning at their Larmor frequency along the direction (z-axis) of the applied magnetic field, Bo. The macroscopic magnetization points to the z-axis. Instead of the laboratory frame (x,y,z-axis), let us consider a rotating frame of reference which rotates with an angular frequency tar relative to the laboratory. In this frame of reference, the nuclear magnets appear to precess with the angular frequency mo- mr. If o3r is
Figure 2. The revolution of macroscopic magnetization in a rotating frame of reference: (a), application of additional magnetic field (BI) along x'-axis as a 90 ~ pulse which tips the macroscopic magnetization into the x'y'-plane; (b), as they precess in the x'y'-plane, the macroscopic magnetization diminishes because nuclear magnets actually precess at slightly different frequencies which causes dephasing.
120
Figure 3. The acquired NMR signal: (a), Free Induction Decay (FID) in the time domain response.Two transient signals each 90 ~ out of phase comprise the real and imaginary part of the FID; (b), its frequency domain response using Fourier transformation. chosen to be equal to tOo, then tOo - Oaris equal to zero, i.e. the nuclear magnets do not appear to precess at all. This means that the apparent static magnetic field must also be zero. We label the coordinates of the rotating frame x', y', and z' (z' is equivalent to z). Let us apply along the x'-axis of the rotating frame of reference a field B 1 which is static in this frame. Then just as the application of Bo causes the nuclei to precess about its direction, so the application of B 1 in the rotating frame causes the nuclear magnetization to precess about 131 with angular frequency v B1 (Figure 2a). If the field B1 is applied for time tp, the nuclei will rotate through an angle O - yB 1tp. A pulse of B 1 field that has a duration which will rotate the nuclei through an angle of 90 ~ is known as a 90 ~ Application of a 90 ~ pulse tips the magnetization, M, into the x'y'-plane [4]. When the magnetization is tipped into the x'y'-plane, the 90 ~ pulse of B 1 field is turned off and thereafter the nuclear magnets experience only the static Bo field. If the nuclear magnets precess exactly at the reference frequency, the magnetization tipped in the x'y'-plane remains constant in its magnitude and direction. However, nuclear magnets actually experience a slightly different magnetic field due to the inhomogeneity of Bo and other local magnetic field due to interactions between nuclei. Each nuclear magnet has a different precessing frequency determined by equation (2), slower or faster than the reference, resulting in phase separation (this phenomena is called dephasing). Macroscopic magnetization in the x'y'-plane decreases to zero (Figure 2b). When we revert to the laboratory frame, the field B1, which is static in the rotating frame, must correspond to a field that rotates about the z-axis with an angular frequency tOo. A method of generating this rotating 131 field is to apply an oscillating field along a given direction (e.g. the x-direction) in the xy-plane [4]. The rotating magnetic dipole moment precessing at their resonance frequency, Vo, in the xyplane induces an electromotive force (e.m.f.) in the receiver coil nearby the sample, and NMR signal is detected. Since the phase of nuclear magnet changes continually in the x'y'-plane, the NMR signal decreases. If we detect two e.m.f.s in the receiver coil each 90 ~ out of phase, the transient signal changes as shown in Figure 3, which describes the evolution of the macroscopic magnetization as it rotates in xy-plane and is known as Free Induction Decay
121 (FID). FID is a time domain response of all nuclei irradiated by r.f. excitation pulse and can be analyzed by Fourier transformation into the frequency domain. The applied Bo induces electronic currents in atoms and molecules, and these produce a further small field Boo at the nucleus which is proportional to Bo. The total effective field, BelT, at the nucleus can therefore be written Beff - Bo(1-o), where a expresses the contribution of the small secondary field generated by the electrons. Using equation (2) we find that Vo = y(2~)Bo(1-o). The magnitude of o is dependent upon the electronic environment of the nucleus, and therefore nuclei in different chemical environments give rise to signals at different frequencies. The separation of response frequencies from a reference frequency is termed the chemical shift, and expressed in terms of the dimensionless units of parts per million (ppm)
[4].
2.3.
Relaxation
As the result of irradiation of the r.f. pulse, the state of nucleus deviates out of thermal equilibrium state in two ways ( Figure 4): i) some nuclei convert the spin state from the spin up to the spin down, resulting in a decrease of the macroscopic magnetization, and ii) all nuclei become phase coherent, resulting in a net transverse magnetization. In general, relaxation processes work to return the nuclear spin system to the equilibrium condition. In NMR, two relaxation processes are induced; one is a release of the excess energy that has just acquired from the r.f. excitation into surroundings, resulting in return of nuclei from the spin down to the spin up states (so called, spin-lattice relaxation, longitudinal relaxation or T 1 relaxation) and the other is a spread apart of the magnetization to random phase of nuclei due to spin-spin interactions which alter the local field and I_armor frequencies of nuclei (so called, spin-spin relaxation, transverse relaxation or T2 relaxation). The net magnetization is exponentially
Figure 4. Schematic explanation of T1 and T2 relaxation phenomena. The equilibrium macroscopic magnetization vector, Mo, is tipped away from the direction of the magnetic field (z-axis) by application of a radio frequency field. After the rf field is removed, it continues to rotate in x'y'-plane about the z'-axis. Two relaxation processes occur: recovery of the magnetization Mz (component along the z-axis) to equilibrium value Mo and decay of Mx,y, (magnetization in the x'y'-plane) to zero due to the loss of phase coherence.
122 recovered with the rate of the spin-lattice relaxation time, labeled T1, and the transverse magnetization exponentially decays at the rate of the transverse relaxation time, labeled T2. T 1 and T2 strongly relate to the line shape and line width of the NMR signal (see 5 . 1 .).
3. NMR IMAGING (K-SPACE IMAGING) 3.1. Theory of k-space imaging Various imaging methods are discussed in a number of reviews[9 - 11] including an excellent textbook by P. T. Callaghan [12]. We shall be concerned only with a compact description of selected fundamentals of N M R imaging.(NMR imaging is sometimes called Magnetic Resonance Imaging(MRI).) Let us consider a simple experiment where a 90 ~ r.f. pulse is applied to the sample in equilibrium in a static magnetic field Bo along the z-axis, and the signal induced in the xyplane is collected by r.f. receiver. The laboratory frame magnetization at time t following the pulse is [ 12]: M(t) - Mx + My - [i Mo cos coot + j Mo sin COot]exp(-t/T 2)
(3)
where COo=u o, T 2 is transverse relaxation time, and i and j represent the unit vector in the xaxis and the y-axis, respectively. In complex number notation, this becomes M = Mo exp(i COot)exp(-t/T 2)
(4)
The modern r.f. receiver works through a process known as heterodyning in which the signal is mixed with the output from a reference r.f. oscillator with a frequency cor. When separately mixing the receiver coil e.m.f, with two heterodyne references each 90 ~ degree out of phase, separate in-phase and quadrature phase output signals are obtained which are each respectively proportional to orthogonal phases of the magnetization, in effect detecting M• and My [ 12]. At any reference frequency COt,the signal will oscillate at the offset frequency ACO = COo-~ . The heterodyne signal at offset ACOis therefore, S(t) ~
exp(iACOt)exp(-t/T2*).
(5)
where T2* is apparent transverse relaxation time which includes the effect of inhomogeneity of the static magnet (T2 > T2*).
3.1.1. Pulsed field gradient which enables encoding position information The conventional basic NMR technique does not provide any information about the position of the nuclei within the sample, because magnetic field, Bo and r.f. field are designed and adjusted to vary as little as possible across the sample by careful removal of field inhomogeneities. In NMR imaging we are concerned with magnetic field profiles which have been purposely designed to vary linearly across the sample space, by applying a pulse of additional field much smaller than the polarizing field magnitude, Bo, giving a measurable, spatially dependent frequency shift onto the local Larmor frequency as co(r) = u Bo + y G . r
(6)
where G is defined as the grad of the pulsed gradient field component parallel to B o. Magnetic field gradients can be generated by additional sets of coils inside the main magnet of the NMR spectrometer. Manipulation of this pulsed-field-gradient in three dimensions with associated r.f. pulses makes it possible to measure the distribution of target nuclei in the sample.
123
3.1.2. Principles of one-dimensional imaging using Fourier transform Consider the nuclei at position r in the sample, occupying a small element of volume dV. If the local density of nuclei is p(r) then there will be p(r) dV nuclei in this element. Following equations (5) and (6), the NMR signal from this element may be written as dS(G, t ) ~ p(r) dV exp[i(u Bo + y G . r - tar)t].
(7)
For simplicity we shall neglect the constant of proportionality. The term representing the decay of the signal due to transverse relaxation may be neglected, because the dephasing of the transverse magnetization due to the spread in y G . r is much more rapid than that due to T2, and the spread due to inhomogeneities of the static magnetic field Bo. By choosing the reference frequency to be yBo, the on resonance condition, the signal finally obtained oscillates at y G . r as S(t) -
fffp(r)
exp[iyG, rt] dr
(8)
where dr is used to represent volume integration. This sum of oscillating terms has the form of a Fourier transformation. To make this more obvious, the concept of a reciprocal space vector, k, given by k = ?Gt/(2~) is introduced [12]. In the formalism of k-space imaging, S(k) p(r) -
fffp(r) exp[i2~k, r] dr, fffs(k)exp[-i2~ck'r] dk.
(9a) (Pb)
A simplified pulse sequence shown in Figure 5 illustrates a one-dimensional visualization of the relationship expressed in equation (9). Setting G in the x-direction as Gx, the one-dimensional
Figure 5.An illustration depicting one-dimensional NMR imaging of water in two cylinders. A 90 ~ rf pulse, which excites all hydrogen nuclei of water molecule in the cylinder, is followed by imposing a field gradient along:x-axis and the start of signal acquisition as well. Fourier transform of the collected FID data enables to reconstruct the one-dimensional image. In an actual imaging, a modified sequence which generates an echo is used. The use of echo enables to enhance the signal intensity compensating the signal decay due to imposition of read gradient as well as to the inhomogeneity of the static field Bo.
124 image, which is the nuclei density averaged over the yz-plane perpendicular to the x-direction, may be written as 9'(x): p'(x) - yyp( x, y, z) dydz. The NMR signal collected in the presence of a magnetic field gradient Gx may be therefore, S(kx)-
f [yyp(x,y,z)dydz]exp[i2~ckxx]dx-, fp'(x)exp[i2~kx x] dx.
(10)
In practice, the sampling of k-space takes place as we sample the NMR signal at successive time intervals along the time axis of data acquisition. Substituting k• = u Gx t/(2g) and COx =u Gx x/(2~), kx x= t rex. Hence equation (10) is rewritten as S(kx) -
fp' (O~x)exp[i2nmxt] dmx
- S(t).
(11)
Because of this, S(k) is measured in the time domain. We obtain a one-dimensional imaging in the frequency domain using the Fourier transform. This process is carried out by a computer. p'(x) =f_ S(k x) exp[-i2~tkxx]dkx - f_ S(t) exp[-i2~tmxt]dt- p' (cox)
(12)
The pulse sequence shown in Figure 5 is an oversimplified one for an easy understanding of the imaging principle. In an actual imaging, a pulse sequence which generates an echo (spin echo or gradient echo) is used which enables data acquisition avoiding the disturbance due to the inhomogeneity of the static magnetic field and rapid switching of r.f. and gradient pulses.
3.1.3. Two-dimensional imaging Cross-sectional NMR imaging experiments consist of three steps: excitation of nuclei in the plane of detection, spatial encoding of the signal from nuclei in the excited plane, and detection of the NMR signal. The excitation of nuclei in a selected cross section of the sample consists of the application of a narrow frequency range r.f. pulse (the "selective pulse") in the presence of amagnetic field gradient (the "slice" selection gradient ), e.g., in the z-direction, that is
Figure 6. A pulse sequence for a two-dimensional Fourier imaging using a spin echo. A Gy gradient for a fixed period is imposed for a "phase" modulation to the signal, encoding position dependent information along the y-axis. The magnitude of Gy gradient is varied with a fixed increment for each scan of the sequence.
125 perpendicular to the plane of detection. At the end of r.f. selective excitation and after switching off the slice selection gradient, the NMR signal from the entire cross section can be detected. For two-dimensional imaging, this signal has to be spatially encoded by application of a second and third magnetic field gradient [ 13]. When the NMR signal is sampled in the presence of a Gx gradient as is described in the previous section, signal points are obtained along a line in kx space (or t axis in equation(11)), and the associated gradient is known as the 'read' gradient (Figure 6). The intercept of this line along the orthogonal axis can be changed by imposing the Gy gradient (the magnitude of which varies stepwise) for a fixed period before sampling begins. The Gy gradient is then named the 'phase' gradient since it imparts a phase modulation to the signal, dependent on the position of volume elements along the y-axis [ 12]. Following equation (9), the signal is therefore /2[
S(kx, ky)
00
00
- ; a / 2 Y-~Y-~op(x'y'z)exp[i2~(kxx+kyy)]dxdy]dz)
(13)
where a is the slice thickness. For convenience we will ignore the outer integral which merely represents the process of averaging across the slice, and write o0
0o
S(kx, ky) = f_oo ~oo p(x'y) exp[i2n(kxx+kyy)]) dx dy.
(14)
It is clear that S(kx, ky) is the two-dimensional Fourier transform of the nuclei density function p (x,y) (i.e. the volume density function p(x,y,z) averaged normal to the slice). Reconstruction of p(x,y) from S(kx, ky) simply requires that we calculate the inverse Fourier transform p(x,y) - f-~0 f-~S(kx'ky ) exp[-i2~(kxx+kyy)]) dkxdky.
(15)
This process is carried out using a computer, subsequent to obtaining the two-dimensional signal, S(kx, ky). A pulse sequence for two-dimensional Fourier imaging is shown in Figure 6. An example of the two-dimensional Fourier transform process is shown for two circular objects in Figure 7.
Figure 7. Corresponding time (kx,ky) and frequency (x,y) domain data in twodimensional image using Fourier transform for two uniform circular objects. The time domain S(kx,ky) is successively one- and two-dimensionally Fourier transformed to arise the spectra S'(kx,y) and S"(x,y).
126
3.2 Application of NMR imaging (k-space imaging) 3.2.1. Internal quality evaluation of foods Product quality and quality evaluation are important in the production and marketing of fruits and vegetables. At present, most quality detection and sorting methods evaluate only external appearance. Interest in non-destructive methods for internal quality evaluation of fruits and vegetables is increasing, and researchers have tried various techniques (such as optical, sonic, and x-ray transmission) with some success. Since NMR measures physical properties that are not observable by other techniques, NMR can detect quality factors that cannot be detected by other methods. Although NMR imaging has been used commercially in the medical field to detect tumors and other abnormalities in humans, its potential for detecting defects and other quality factors in foods has not been fully explored [ 14]. Bruises are clearly visible in NMR images of apples, peach, Asian pear, and onion [ 14]. A relatively new bruise is brighter than the surrounding tissue due to a decrease in the magnetic susceptibility variations in the tissue. This decrease is a result of cell damage and subsequent diffusion of moisture into air pockets in the tissue of fruit [ 15]. However, the very old bruise does not show up as a brighter region, but instead appears darker because of dehydration[ 14]. A pit of a stone fruit consists of a hard shell which contains very little free water, and an inside seed with high water and oil content. The pit and the seed in peach, olive and prune are visible in an NMR image [ 14]. This means that the unpitted olive which has the image of a dark ring of pit shell and a bright image of the seed inside the shell can be discriminated from the pitted olive which has the image of a dark spot at the center of the fruit where the cavity is. The maturation process of fruit and vegetable often results in an increase of free water (juice) and oil, which can be detected by NMR imaging. The translucent (over-ripe) region of pineapple is clearly visible as a brighter region. The image of the ripe avocados, which has higher oil content, is brighter than that of the green fruit [ 14]. Whole-body water and fat content in bothlive animals and carcasses have been determined using NMR imaging for internal quality detection [ 16]. The in vivo NMR imaging of a variety of aquatic organisms (carp, gold fish, trout, Arabian carpet shark, tirapia and eel) by wholebody and experimental machines was examined for understanding of physiology, biochemistry and biology [ 17, 18]. These results are promising to evaluate internal quality of fish. 3.2.2. Chemical shift contrast image and relaxation contrast image When the sample has more than one resonance absorption in the NMR spectrum, such as water and oil as is the usual case in biological samples, the application of a modified pulse sequence to selectively excite the nuclei with only target Larmor frequency (e.g.,water or oil) enables reconstruction of a chemical shift contrast image representing the density of the target nuclei. Figures 8a, 8b, and 8c show three images of the same cross section of a sardine, slightly salted and dried, taken with a standard spin echo image (left), chemical shift water-only image (center), and chemical shift oil-only image (fight) [ 19]. In spin echo imaging, the signal strength of an imaging element is given by S = So(1-exp(-TR/T 1))exp(-TEF['2 ) where TR is the delay between successive experiments, and TE is the echo delay (the delay between excitation pulse and the echo). Adjustment of echo delay (TE) allows T2 relaxationweighted image contrast, resulting in a profound effect on image enhancement of specific features of the specimen. Figures 8d, 8e show two chemical shift water-only images of the same sample as in Figures8a, 8b, and 8c, taken with increased echo delays. The contrast between the dark and light meat increases as the echo delay is increased [19].
127
Figure 8. NMR imaging of a sardine (slightly salted and dried) :(a), standard spin echo image consisting of both water and oil signals; (b), chemical shift water-only-image ;(c), chemical shift oil-only-image. TE= 21.6 ms, Tr=ls. T2 relaxation weighted water-onlyimage obtained by increasing echo delay:(d), TE = 45.2 ms; (e),TE = 61.2 ms. T2 of water in dark meat (D) is obviously shorter than that in light meat (L).
Figure 9. A contour plot of oil volume fraction as a function of time and position showing the dynamic of creaming in a 40% (v/v) oil/water emulsion. This plot was calculated analyzing the data obtained by a fast, spatially localized technique for T1 determination [Reproduced with permission from Ref.23]
3.2.3. D y n a m i c s in foams and emulsions Foams and emulsions are other multiphase systems to which research on the application of NMR imaging is on-going. There are a great many practical uses of aqueous foams and emulsions in food, pharmaceuticals, and engineering. Understanding and particularly assigning specific functional roles to individual components in the foam has been severely
128 limited by the inability to analyze such unstable colloidal systems. Application of NMR imaging elegantly removes this limitation [20]. Foam from cream, egg white and beer were imaged using a Fourier imaging spin echo pulse sequence, and the signal intensities contributed by hydrogen nuclei were recorded sequentially over the life time of the foams. Using these signals to reconstruct the one-dimensional image, densities, drainage rates, and collapse throughout the foam structure were estimated [20]. To satisfy the desire for accurate values of density, however, one must take care of the need to calibrate coils, the influence of experimental parameters used such as TR and TE and the influence of relaxation time constants to which drainage and collapse contribute. [21]. Foam characteristics in several commercial beers were evaluated using one-dimensional NMR image. Foam texture differences and regions of cling, head, and liquid beer can be clearly distinguished. Significant differences in rates of foam collapse were measured [22]. When a relaxation time T1 for oil/water emulsion is known, the volume fraction of each for the emulsion can be calculated using an equation component phase ~(oil) and r 1/(T l(obs)) = ~(oil)/(T l(oil))+ ~(water)/(T l(water)). A fast (5 sec), spatially localized technique for T1 determination was developed to simultaneously estimate volume fractions along an entire emulsion profile [23]. Using this technique, the dynamics of creaming in a 40 % (v/v) oil/water emulsion was observed successfully. Figure 9 shows the contour plot of oil volume fraction as a function of time and position. The rise of the lower concentration contours towards the final interface can be used to estimate the mean creaming velocity of the emulsion [23]. Crystallization of fat in fat-water emulsion, indicated by a decrease in intensity of the oil magnetization has been observed using NMR imaging and localized NMR spectroscopy [24]. The state of cocoa butter in chocolate was followed using NMR imaging. Two pure chocolate bars were subjected to different cooling rates, one very rapid and one very slow. The variation in signal intensity of spin echo NMR obtained was interpreted in terms of variations in the solid/liquid ratio of the cocoa butter, and hence the existence of different polymorphic forms in the two samples was suggested. This interpretation was confirmed by DSC [25]. The crystallization kinetics of bulk triglycerides and oil-in-water emulsions has been characterized by both NMR imaging and localized spectroscopy. The rate of lipid crystallization in an oil-in-water emulsion was affected by the addition of a second homopolymer (addition of trilaurin to trimyristin in this case). The addition of the second homopolymer of higher chain length was observed to slow the rate of crystallization [26].
3.2.4. Drying, freezing, and cooking NMR imaging has been applied to measure transient moisture profiles during drying of an ear of sweet corn. Three-dimensional hydrogen nuclei density images were obtained, and transient moisture profiles were determined. Moisture transfer and the shrinkage of the sample were analyzed from the profile. Mass transfer occurred in individual kernels, but the mass transfer between kernels was negligible. [27]. The change of moisture profile measured by NMR imaging has been used to calculate the effective diffusion coefficient of moisture in apples [28] and potatoes[29] during drying. The change of moisture profile in barley and soybean seeds during maturation was measured using NMR imaging [30]. The process of seed maturation was discussed in relation to the biological and morphological characteristics of the crop. The freezing of food products can be monitored by following the freezing interface using NMR imaging. Combining the NMR imaging data with calorimetric studies will provide a good test of assumption of local equilibria in calculations of freezing rate. [31 ]
129 The appearance of the NMR imaging of courgette dramatically changes when it is frozen and thawed [32]. The image of the fresh and thawed samples differ from each other in two important respect. First, the overall intensity of the image of the thawed vegetable is greater than that of the fresh courgette; second, the relative image contrast between different type of tissue is less distinct in the thawed sample compared to the fresh courgette. The change in the image contrast may arise because freezing and thawing the courgette alters the morphology of the tissue, which in turn increases the transverse relaxation time (T2) of the hydrogen nuclei of water in the tissue. The ice crystals formed on freezing rupture the cell walls and destroy the cells turgor pressure, and on thawing the courgette's texture becomes very flaccid. The intercellular fluid is then free to drain into the air cavities in the tissue [32]. A satisfactory explanation rather than speculation needs a precise theory of the mechanism which causes water proton relaxation in tissues. NMR imaging has been used to study the distribution of water in wheat grains during cooking in steam and boiling water [33]. Cooking involves diffusion of water into the grain, followed by gelatinization and melting. Starch gelatinization was marked in the images as well defined "reaction front", which was not observed for the steamed samples [33].
4.
MEASURING TRANSLATIONAL MOTION USING SPIN ECHOES
4 . 1 . Diffusion measurement using pulsed-field-gradient spin echoes (PGSE) 4.1.1. Theory The effect of diffusion on the result of experiments relevant to spin echo was described by Carr and Purcell [34]. The first experiments and the detailed analysis on pulsed-field-gradient spin echo (PGSE) to study diffusion were presented by Stejskal and Tanner [35]. Several modifications to the PGSE technique were later suggested and tested, and were mostly designed for the purpose of extending the measurement range to slower diffusion rate and to heterogeneous systems. NMR provides a molecular label via the characteristic Larmor frequencies of the component nuclei. This label may be given a spatial dependence by the imposition of a well defined magnetic field gradient over the sample space. Very sensitive precessional phase displacements may be detected through the degree of phase refocusing in an NMR spin-echo, and these echo signals may be used to measure molecular translational motion. The field gradient may be applied in the form of pulses inserted between the transmit and receive periods of the NMR spin-echo experiment [36].
Figure 10. The basic pulse field gradient NMR spin echo experiment.
130 PGSE method in its simplest form consists of a two-r.f.-pulse spin-echo experiment with identical magnetic field gradient pulse of magnitude g, duration 8 and separation A applied respectively during the dephasing and rephasing segments of the echo cycle. (Figure 10). When a magnetic field gradient, g, is imposed over the sample at time tl, a rapid precessional phase shift (dephasing) takes place because the nuclei precess with a different frequency depending on the position of each nucleus in the sample. In between the first and second gradient pulses, A, the molecules containing the nuclei change position because of diffusion. The 180 degree r.f. pulse inverts all prior phase shift so that the second gradient pulse has the effect of refocussing, which is incomplete to the extent that the diffusional motion has occurred. The degree of failure in refocusing results in the attenuation of the spin-echo, Y, which gives a measure of the average translation of nuclei. The attenuation, Y, in the special case of narrow gradient pulses, ~5<
Y-f_0o
00
p(r)f_0o Ps(rlr',A)cos[u
dr
(16)
where Ps(rtr',A) is the conditional probability for the arrival at r', at time A of nuclei which originated at r. p(r) is the initial density distribution. For molecules undergoing unhindered isotropic Brownian motion, Ps(rlr',A) must be Gaussian and the attenuation, Y, is given by Y = exp[-u (17) For finite 8, the effective diffusion observation time A in equation (17) takes the reduced value Ar = A-~5/3 [35].
4.1.2. Application Equation (17) has been validated via experiments using simple liquids [35, 37], and has been applied to studies on solutions, and gels, as well as solid foods. PGSE has been used in studies on the obstruction and solvation effects related to the macromolecular shape factor for ovalbumin [38], wheat starch amylopectin [39], and for agarose gel [40]. Moisture diffusion coefficients in soybean seed during moisture soaking have been obtained via PGSE method [41] as well as via gravimetric methods [42, 43]. The moisture diffusion coefficients in soaked soybean measured by PGSE were larger by a factor of ten than those measured through gravimetric method. This discrepancy may be explained by the difference in the scale of diffusion detected through the methods used. It is interesting to note that the moisture diffusion coefficient in dry soybean seed (0.14 g H20/g solid) as measured by PGSE was unexpectedly large; it was larger than that measured by sorption/desorption method [44] by a factor of 100. Since moisture monolayer values [45] in foods range from 0.05 g H 2 0 / g solid (spray dried skim milk) to 0.11 H20/g solid (starch), a considerable percentage of moisture in dry soybean seed might be expected to be absorbed on biopolymers and immobilized. Hence, this large diffusion coefficient measured by NMR suggests an existence of less immobilized water molecules in the dry soybean seed in addition to the absorbed water. A chemical shift selective imaging of a dry soybean seed with more than a hundred times data acquisition successfully revealed the image of movable water which is located at the inter-cotyledon face. This inhomogeneously distributed moisture is surrounded by a fat layer which might prevent the outward movement of moisture [41]. PGSE may be incorporated in an NMR imaging experiment to provide an image contrast dependent on local molecular diffusion. The consequent image attenuation may exhibit a dependence on applied magnetic field gradient and localized diffusion coefficient can be available using equation (17). Moisture diffusion in muscular tissue of fatty fish (sardine, partially dried) was measured [46] using a chemical shift imaging sequence added with a couple of pulsed field gradients for imaging contrast dependent on diffusion. The water-only images shown in
131
Figure 11. Chemical shift selective water-only-image of partially dried sardine. Images are contrasted by the effects of water diffusion. (a), g=0 mTm-1; (b), g=167 mTm-1; (c), g=300 mTm -1. The light meat appears as the brighter central region. The signal intensity profile along the cross section a-a' is shown at the top. Light meat (L), dark meat (D) [46].
Figure 12. Plot of water image attenuation against a product of factors that reflects the water diffusion rate in a region of interest in (V1) light meat and (m) dark meat of partially dried sardine. Ag and Ao are the image intensity with and without the gradient pulse for detecting diffusion, respectively. The slope gives the diffusion coefficient, D (cm2/s): D=I 1.8 x 10-6 for light meat; 8.33 x 10-6 for dark m e a t after eqn.(17) [46].
132 Figure 11 are constructed using spin echo signals attenuated by the effect of diffusion over a diffusion time A= 28 ms with pulse field gradient of varied magnitude. A plot of signal attenuation against y2 c52g2(A_6/3) in a region of interest in light meat as well as in dark meat (Figure 12) gives straight lines where the slope of each line denotes the localized moisture diffusion coefficient; water molecules are more hindered in dark muscle than in light muscle. Measurement of the moisture diffusion coefficient as a function of position in wheat grain was performed [47]; the moisture diffusion rate normal to the transverse 1.3 mm section of wheat grain was measured in structural features at a 150 ~m resolution. The motion of water was most severely hindered in the endosperm, and water appeared less hindered in the vascular bundles.
4.2. Restricted diffusion 4.2.1 Theory One particular model of a biological system is a set of barriers in an otherwise homogeneous medium. If diffusion is observed over a short enough time, very little of the substance experiences the effect of the barriers, and the observed motion is characteristic of the medium alone. As the time of observation is extended, more of the substance is reflected at barriers; and thus, its total displacement is less than would have been the case without the barrier bringing about less attenuation of the PG spin-echo [48, 49]. From an analysis of such data one can, in principle, obtain information on the geometry of the domains in which restricted diffusion occurs; information on cell size, droplet size distribution [50] etc. In restricted diffusion, the conditional probability Ps(rlr',A) in equation(16) is no longer Gaussian and the solutions to the problem of echo attenuation, Y, as a function of system geometry and dynamics are in most cases quite complicated. The attenuation has been derived for homogeneous media bounded by spherical, cylindrical or planar barriers which are impermeable. The dependence of Y on observation time A has been demonstrated for water in an artificial system on thin liquid layers [48]. Restricted diffusion measurement has been applied to the study of plant [48] and animal [51, 52] tissues with some success. However, interpretation of restricted diffusion experiments in biological systems have proven very difficult. This is seemingly because the attenuation Y is affected not only by diffusion but also by relaxation/exchange and/or permeable feature of bio-membranes. Such a generalized analysis is tried in dynamic q-space imaging [53].
4.2.2. Application Tanner [49] measured diffusion coefficients of water in three different types of frog muscle cells. He used a variety of magnetic field gradient techniques so as to cover a wide range of diffusion times: A= 1 ms to 1 s. The time dependence of the diffusion coefficient was analyzed to obtain the intracellular diffusion coefficients and estimates of the permeability of the cell membranes. In restricted diffusion studies three 90 degree r.f. pulse sequences (stimulated echo) are often used which provides PG-NMR experiments with long diffusion times to explore the dependence of diffusion time on the echo attenuation [49]. Diffusion coefficients of moisture and fat in cheese were measured and restricted feature of fat signal facilitated estimation of fat droplet size distribution [50], while the water diffusion was suggested to be confined to the surface within the protein matrix [50]. Diffusion of water in polysaccharide gels (dextran and sephadex G hydrogels) were measured [54] over a range of diffusion time (A=30-300 ms), and the restricted diffusional behavior was analyzed using a planar semipermeable barrier model [55].
133
4.3.
q-space imaging and dynamic q-space imaging
4.3.1. Theory The attenuation of spin echo in a standard PGSE diffusion experiment, described by equation (16), may be written in complex number notation as 00
P(r)f_oo Ps(r I r+R,A)exp[i),tSgR]dR dr (30
YA(g) - f-oo
(18)
where R is the net displacement defined by R = r'- r. A function called average propagator Ps (R,t), defined by .
(30
Ps (R,t)
=f_oo
p(r)Ps(rl r+R,t) dr
(19)
gives the average probability for any particle to have a displacement R over a time t [ 12]. The use of average propagator in equation (18) makes the phase shifts appearing in the integrand of equation(18) to depend only on the displacement R. Hence equation(18) may be rewritten as o0
YA(q) - f-0o
Ps (R,A)exp[i2:tqR]dR
(20)
where a reciprocal space q defined by q = y~Sg/(2:t) is used in order to express an analogy to k-space imaging. Equation (20) expresses a simple Fourier relationship between YA(q) and Ps (R,A)" acquisition of signal in q-space permits us to image Ps (R,A) just as acquisition in k-space permitted us to image p(r)[ 12]. Because PGSE is sensitive to the averaged propagator, Ps (R,t) will be equivalent to Ps(rlr+R,A) only when Ps(rlr+R,A) is independent of starting position r and depends only on the net displacement. This situation occurs in unrestricted self-diffusion as well as in restricted diffusion with a long time scale limit, A---~0o.The long time limit means that the molecule loses all memory of its starting position and can be anywhere in the structure. In the long time limit, the attenuation of the spin echo may be written as 0o
Y~q)
o0
- f_oo p(r)exp[-i2~qr]dr f_oo
p(r')exp[i2nqr']dr' - S*(q)S(q) -IS(q)12.
(21)
This relationship states that the PGSE signal is precisely the power spectrum of the reciprocal lattice. This means that the PGSE experiment in the long time limit is an imaging experiment, returning not the reciprocal lattice as in k-space imaging, but the modulus squared of the reciprocal lattice [12]. The q-space imaging method, which deals with signals only after long diffusion times, discards all information relevant to dynamic aspects of water diffusion and transport, especially the restriction of water transport by membrane and cell wall permeability barriers in cellular tissues. This information is contained in the functional dependence of the pulsed gradient spin echo amplitude S(q,A,x) on the three independent variables q, A, and 1: (x is the 90-180 degree pulse spacing) [53]. As the tool to explore the q and A dependence of S(q,A,x), generalized diffusion times and their associated fractional populations are introduced and a multiple exponential time series expansion is used to analyze the dependence [53].
4.3.2. Application Diffraction-like effects in PGSE experiment, which had been discussed for diffusion in both impermeable and connected structure, were experimentally confined for a sample of watersaturated, loosely packed array of monodisperse polystyrene spheres. This success suggests
134 that q space imaging experiment may be used to provide an indirect, averaged image of the internal structure of porous solids at a resolution higher than that achievable with conventional NMR imaging[56]. An application of dynamic q-space imaging for pardnchyma tissue of apple was reported where relaxation/exchange and permeable feature of bio-membrane was considered [53]. 4.4. Flow measurement 4.4.1. Theory The phase shift experienced at time t by a nucleus following the path r(t') in a gradient g(t') is given by [57] t
qb(t)-~'f0 g(t') r(t')dt'.
(22)
If the nucleus moves at a constant velocity, Vz, in the direction of a field gradient, r(t') is Zo + Vzt'. Hence the phase shift may be t
t
q~(t)- yzof0 gz(t')dt'+ yvzf0 t'gz(t')dt'- u
vzml)
(23)
where mo and m l are the zeroth and first moments of the gradient waveform in the time domain. If the gradient is designed such that mo=0 but ml ~ 0, this phase shift will be proportional to the velocity of the nucleus. A properly designed gradient allows the phase to measure the distribution of velocities in a sample. The interpretation and analysis of velocity spectrum was discussed for the rheological measurements of heterogeneous materials like food [57]. Conventional spin echo imaging sequence may be modified to provide a velocity contrast imaging sequence, when a bipolar gradient is inserted between the slice selection and imaging segments so as to impose a phase shift (mo=0 but m l ;e 0) in each pixel dependent on the net nuclei displacement occurring over the time scale of velocity determining gradient pulses [ 12, 57a]. Another method to measure flow is time-of-flight method, the use of which is widespread in medical application of magnetic resonance imaging. One of the simplest time-of-flight techniques involves the destruction of magnetization in a selected plane (for example by a 90 degree selective pulse followed by a homospoil gradient pulse) and the subsequent imaging of nuclei residing in that plane at some later time. Only fresh inflowing nuclei contribute to the image so that static nuclei signals are suppressed while moving nuclei have a signal amplitude proportional to the overlap between the tagged and target planes [ 12].
4.4.2. Application NMR imaging techniques were applied to the measurements of velocity field in opaque systems such as tomato juice and paper pulp suspensions [58-60]. In both cases, the particle concentrations are sufficiently high that widely applied techniques such as hot film and laser Doppler anemometry could not be used. The velocity profile for a 6 % tomato juice slurry clearly showed a power-law behavior [58, 59]. Flow NMR images for a 0.5 % wood pulp suspension provided direct visual of three basic types of shear flow: plug flow, mixed flow and turbulent flow as mean flow rate was increased. Detailed analysis of flow NMR image is able to reveal the complex interaction between the microstructure of suspensions and the flow [60]. Extrusion processing is one of the most highlighted food processing to which potential application of a flow NMR image (a modified time-of-flight) have been demonstrated [61]. A vast array of food and feed products are produced by extrusion processing, an efficient method
135 for simultaneously mixing, pressuring and heating food products. A single-screw extruder, constructed of non-magnetic materials, operated within the magnet of an NMR spectrometer. During signal acquisition, one per revolution, the signal intensity from a horizontal section of fluid was nulled by a spatially selective r.f. pulse in the presence of a magnetic field gradient. The nulled section appeared as a dark horizontal band through each side of the bright ring of fluid in the plane of the image (Figure 13). The dark, magnetically "tagged" liquid deformed during the time interval, revealing the flow behavior of the fluid. The velocity of any fluid volume element could be determined by measuring the distance it traveled between the creation of the dark band and signal acquisition [61 ]. Using phase encoded flow method, velocity image for water in the plant stem, and in a wheat grain were measured. A flow rate of 50 ~m s-1 in the region of the vascular tissue of a wheat grain was obtained [ 12].
Figure 13.. Comparison of theoretical analysis and empirical NMR imaging of fluid flow during extrusion. Limiting cases for theoretical analysis:(a), the velocity profile as a function of position with no pressure gradient in the z-direction;(b), the velocity profile as a function of position with no net flow through the extruder. Limiting cases for empirical analysis by NMR flow imaging: (c), no pressure gradient in the z-direction (die open);(d), no net flow through the extruder (die closed). [Reproduced with permission from Ref.61].
136 5. R E L A X A T I O N
PHENOMENA
IN F O O D M A T E R I A L S
5.1. R e l a x a t i o n p h e n o m e n a of water in food materials Water in food materials is the most important component, which influences not only the processes where water itself mainly relates, such as freezing, dehydration and freeze-drying, but also the processes where water indirectly relates to chemical changes of the other components, such as protein denaturation, Millard browning, enzyme activity, and so on. Water also strongly relates to freshness, stability and aging of food. A precise measurement of the "nature", "state" or "availability" of water in food is needed to understand the stability or the deteriorative processes of food. Water activity, which is defined as the ratio of the partial pressure of water in the air above the food in a closed chamber to the vapor pressure of pure water at the same temperature, is mostly used as a measure of the "availability" of water. In food systems, however, it is quite difficult to keep the food at constant temperature and pressure and in thermodynamic equilibrium, which is a condition theoretically assumed in the measurement of water activity. Consequently, true water activity does not exist in many food systems. NMR method is proved to be one of the successful techniques for measuring of availability of water. From such viewpoint S. J. Schmidt and H. M. Lai reviewed on use of NMR and MRI to study of water relation in such food systems as plant protein, wheat flour, milk protein, egg white and cereal grains [2]. In this section, we would discuss chemical and physical properties of water in food materials from the view point of theoretical background on the NMR relaxation behavior of the nuclei (1H, 2D and 170) and review some typical applications. 5.1.1. R e l a x a t i o n m e c h a n i s m of water p r o t o n s Both T1 and T2 relaxations of water protons are mainly due to fluctuating dipole-dipole interactions between intra- and inter-molecular protons [62]. The fluctuating magnetic noise from all the magnetic moments in the sample (these moments are collectively termed the lattice) includes a specific range of frequency components which depends on the rate of molecular motion. The molecular motion is usually represented by the correlation time, ~:c, i.e., the average lifetime staying in a certain state. A reciprocal of the correlation time corresponds to the relative frequency (or rate) of the molecular motion. The distribution of the motional frequencies is known as the spectral density function. In T1 relaxation process, energy corresponding to the Larmor frequency of the observed proton is going in and out and the efficiency of the energy transfer from the spin system to the lattice controls T1 relaxation rate. The more closely the motional frequency approaches the Larmor frequency, the higher is the efficiency of energy transfer for T1 relaxation. Therefore, T1 relaxation time strongly depends on the magnetic field used, temperature and motional frequency (or correlation time). In pure water the motional frequency is about 106 MHz and the Larmor frequency is in the order of 10 to 100 MHz. This means the motion of water is too rapid to cause T1 relaxation of water proton, hence T1 becomes long, about 2 to 3 s. With decreasing the rate of Brownian motion, the spectral density function may contain significant components at the resonant frequency, so that the T1 relaxation becomes faster. T1 reaches the minimum values at the point 2:tv = 1/1u After this point T1 becomes longer again to result in quite long T1 in the solid state, because the molecular motions are even slower than the resonance frequency. On the other hand, T: relaxation time, the rate at which spin exchanges occur, is controlled by a randomness of the magnetic field. Rapid Brownian motion in aqueous state can cancel the
137
Figure 14. Relation between correlation time and relaxation times of protons of water in isotropic motion at 200 MHz spectrometer. randomness, resulting in the slow T2 relaxation. Slow thermal motion will produce random small changes of magnetic field. Each spin experiences different magnetic field, causing a spread of Larmor frequencies. The phase coherence of the proton spins in xy-plane is lost, resulting in the faster T2 relaxation (or conversely, the shorter relaxation time). The slower of molecular motion accelerates the faster T2 relaxation. Therefore, T2 relaxation time of liquid pure water is long and T 2 relaxation time of protons in solid samples is very short. The relation between molecular correlation times and relaxation times in pure solution is shown in Figure 14. NMR spectrum is obtained by Fourier transformation as the distribution of frequency components that are present in an FID or an echo signal sampled over a period in time. Therefore, a rapidly decaying signal (short T2) has a broad function over frequency because of a wide spread of Larmor frequencies and vice-versa. The linewidth in I--Izat half height, AVl/2, is related to T2 relaxation time by the equation, l/T2 = 71;AVl/2.
(24)
Actually, inhomogeneities of the static magnetic field, B0, and the diamagnetic susceptibility of the sample, ;~m, additionally causes the spread of Larmor frequency, which results in a combined relaxation called T2*. T2 in the equation (24) should be replaced by T2*. The magnetic interactions which govern the relaxation rate or linewidth are listed in Table 1. Relation between the relaxation rate and various physical parameters are given in the condition of extreme narrowing limit, i.e., ~o0xc<< 1. Various physical behaviors, some of which are intrinsic in the heterogeneous samples such as food materials, affect the linewidth and the line shape. For example, magnetic susceptibility inhomogeneities (mentioned above) and magnetic interactions (listed in Table 1) that are only partially averaged by molecular motion, chemical exchange and diffusive exchange cause the line broadening.
138 Table 1 The magnetic interactions governing the relaxation rate, or linewidth. Interaction
Relaxation rate (extreme narrowing condition )
Remark
Dipolar interaction
(1/T 1)i~(intra) - (1/T2)m(intra) = (4/3)y12 y s 2 [h(27t)-a] 2 S(S+ 1)'tRriS -6
*1
(1/T1)DD(inter)=(16/Z7)JtCs yi 2 ~tS2 [h(2Jt)-l] 2 S(S+l)/aD
*2
Quadrupolar interaction
(1/T1)Q = ( 1/T2)Q
*3
Spin-rotation interaction
(I/T1)SR - {2IikT/2[h(2~t)-1]2}Ceff2T J
*4
= {ii2/9[h(2~)- 1] 2 }Ceff2/.tc
*5 *6
Chemical shift anisotropy
=(3/10)~ 2 {(21+3)/12(21-1)} CQ2(1+n2/3) Xc
(1/T1)cSA - (2/15) y2B02Ao2 Xc (1/T1)cSA / (1/T2)csa - 7/6
* 1: Relaxation rate of nuclear spin I. Spin I and spin S belong in the same molecule. *2: Spins I and S belong in different molecules A and B. *3: Quadrupole-electric field gradient interaction. *4: Magnetic fields are generated by the rotation of a molecular magnetic m o m e n t and modulated by molecular collisions. Nuclear spin interacts with the fluctuating magnetic field. *5: Xc'tj = Ii/6kT *6: Anisotropic nuclear screening tensor is modulated by Brownian motion. Remarks: YI and Vs = gyromagnetic ratio of spin I and spin S nuclear spin, ris = internuclear distance, "tR= rotational correlation time, "tc = reorientation correlation time, "tj = angular momentum correlation time, Cs = concentration of spin S, CQ = e2qzzQ/h = quadrupole coupling constant, qzz = the electric field gradient, Q = nuclear electric quadrupole moment in 10 -24 cm 2, Ceff = effective spin-rotational coupling constant, a = closest distance of appropriate of spin I and spin S, D = (DA+DB)/2 = mutual translational self diffusion coefficient of the molecules containing I and S, I i = moment of inertia of the molecule, Ao = o//- OL. In aqueous food materials T1 and T2 relaxation behavior of water are related to different aspects of the interaction and motion of the water molecules. The relationship is not so simple, especially in heterogeneous food materials [63-65]. There are at least four types of protons to be considered, namely free (or bulk) water, bound (or hydrated) water, exchangeable macromolecule protons such as those found in hydroxyl and amino groups, and unexchangeable macromolecule protons. Under such circumstances measurement of T1 is more reliable than T2 measurement, but can be complicated by the spin diffusion, while T2 relaxation can be complicated by slow translational diffusion and proton exchanges. Generally, in almost all cases, water molecules quickly exchange between bulk state and hydration sphere of macromolecules by molecular diffusion (at the rate of sub-nanoseconds in the water-rich regime) [66]. In such cases, a weight-averaged T1 or T2 is observed in the single-exponential FID or the C P M G echo decay envelope on the basis of the following equation, 1/Ti - Xb (1/Tib) + xf(1/Tif),
(i=l, 2)
(25)
139 where the subscripts b and f indicate hydrated water and bulk water, respectively and x is proton fraction in each state. When the proton exchange between hydration water and exchangeable macromolecule proton is much faster than T2, (i.e., lifetime, km, of an exchangeable proton on the polymer is shorter than about 1 ms), only one T 2 relaxation time is obtained from the single exponential curves. With decreasing proton exchange rates, however, the proton exchange affects T 2 relaxation time and the observed T2 value becomes shorter[67, 68]. T2 is given by the equation, l/T2 = Xb(1/Tzb) + Xm{1/(T2m+ 1/km)}
(26)
where the subscripts b and m indicate hydration water and macromolecule proton, respectively and x is proton fraction in each state. It is important to note that the measured T2 is not, in general, given by the above equation because of the dephasing effect of the frequency offset, 6m(=mm-mb) [64]. The unexchangeable macromolecule protons have much shorter T2 relaxation times and appear as much faster decaying components in FID or CPMG envelope profile. From the multiexponential analysis of the decay curve, in principle, non-exchanging protons can be distinguished. But, actually a precise measurement of T2 relaxation time of such protons is not so easy. The dipolar cross relaxation between water and macromolecule protons results in magnetization transfer (MT), and the T1 relaxation times of all protons coupled by dipoledipole interaction tend to become equal. Therefore, the observed T1 of water apparently appear shorter than actual T1. This leads us to the wrong conclusion that water becomes less mobile, or tightly bound to the macromolecules, although water is actually mobile. The steady state magnetization for a liquid component (A) interacting via intermolecular dipole-dipole interaction with a solid component (B) was discussed in detail by Grad and Bryant [69]. When crossrelaxation between the spin populations is rapid, the magnetization for the liquid component, MZA, is written as follows: ~ZA'-( 1/2)ml 2T1BT2B/[(l+4~;ZT2BZA2)( I+T 1B/f TIA) +ma 2T1BTzB]
(27)
where f refers to the ratio of the number of B spins (solid) to the number of A spins (liquid), A denotes the frequency offset of the preparation radio frequency field from the A resonance frequency, and o)1 = ~tB 1. The linewidth of the cross-relaxation spectrum increases with decreasing T2B, i.e., increasing rigidity. The area of the cross-relaxation spectrum increases with an increase i n f . 5.1.2. A p p l i c a t i o n to food materials Theoretical and experimental approaches to study of molecular dynamics of water in foods and related model systems are reviewed by Baianu et al. [70], where the mul ti nuclear spin relaxations are compared with theoretical calculations. In the system of lactate/water and sucrose/water, T2 relaxation of proton varied with pH, but that of D nuclei varied only slightly as a function of pH. This result indicates the difference in the contribution of chemical exchange to the T2 relaxation experienced by both nuclei [71]. T2 relaxation of a70 reflects directly the water mobility in sugar-water systems. In the case of potato starch in aqueous suspensions, relaxation measurements of 1H, 2D, and 170 nuclei allowed analysis of contributions to water relaxation contributed by water binding, chemical exchange and crossrelaxation [72]. Weakly bound water in potato starch suspensions has an average correlation time of about 20 ps (determined by 17 0 NMR), compared with 5 ps (determined by 2D NMR) for bulk water at 20~ The three hydration regions were observed and from 1H and 2D relaxation data the three kinds of water, i.e., bulk water, weakly bound water and trapped water, were distinguished. 170_T2 relaxation rate showed water mobility in starch-sucrosewater systems, that is, both sucrose and starch resulted in an increase in T2 rate or decrease in water mobility [73]. Water states in work-free flour dough [74], hard wheat dough [75] and
140 staling bread [76] were estimated by 1H, 2D, 13C and 170 relaxation behaviors. Recently, T2 relaxation rate of 23Na was measured to analyze the binding of the Na + cation in Gum solutions. The transverse relaxation rate indicated less mobility of Na + ion in ionic than nonionic system. Ionic gums correspondingly suppressed saltiness perception compared to nonionic gums. Food components that bind Na + may suppress saltiness perception. These findings may be useful to evaluate sensory quality [77]. 5.2.
Low resolution NMR
Accurate determination of the amount of solid fat in edible fats and oils is an essential requirement for process control in food industry during hydrogenation, interesterification and blending. Moreover, important physical properties, such as hardness, heat resistance, mouthfeel and flavor release, can be predicted via measurements of solid fat content at different temperatures using low resolution (low-field) NMR. Low resolution transient NMR spectrometers, which are dedicated, low cost, bench-top NMR instruments developed for specific food applications, have been widely applied to the study of food systems in industry. Low resolution NMR provides a rapid method of determining either the solid or liquid fat content in plant and animal products, whereas chemical shifts are used to discriminate between different molecular species in high resolution NMR, this is achieved in low resolution NMR by means of their different relaxation properties. The proton resonance free induction decay signal following a strong r.f. pulse contains two components having distinctly different decay time constants, which represent the solid and liquid contents within the sample. By the "direct method", the solid fat content is determined by sampling of the decay signal at two different points. The first data point is taken at the end of the dead time (-10 ~zs time delay for stabilizing the receiver coil after the r.f. pulse); the second data point is taken at --70 Fts after the r.f. pulse. By this time, the NMR signal of the protons in the solid state has disappeared completely, whereas the NMR signal from liquid state protons has hardly decayed [78]. Combining these two points of data with an extrapolation factor which might have been determined in advance by the use of calibration samples, provides a tool for rapid measurement of solid fat content for process control and quality assurance. Low resolution NMR spectrometer has been applied to determination of oil and moisture in oilseeds [79], water content in food [80], and "unfrozen" water content during freezing[81,82]. Quality control and analysis in the agro-food industry by the low resolution NMR was reviewed by D. N. Rutledge [83], in which NMR method is described as faster, more precise, simpler and cheaper technique than those traditionally used. For on-line monitoring of wine fermentation, EtOH and sugar alcohol beverage and fermenting musts were determined to a precision of 0.005,--0.1% for EtOH and 4~8g/L for sugar by low resolution NMR [84]. 6.
HIGH RESOLUTION
N M R IN F O O D M A T E R I A L S
Recent papers on food materials are reviewed in this section. Subjects are limited to only intact food materials such as fruit tissue, juice, wine, meats, starch and so on, and therefore particular pure molecules isolated from food materials and/or model compounds obtained by chemical reaction are not included. Ni and Eads reviewed papers on the quantification of liquidphase components of agricultural materials, including whole fruits and vegetables or samples of their intact tissues by 1H-NMR in introduction of their papers[85, 86]. Several review papers are available on assessment of meat (components, quality and energy metabolism of muscle tissue [16], foods and ingredients as solid materials investigated by solid state 13C NMR [87], 13C NMR data (structure and microenvironmental characteristics) of native, gelled heat- and chemically-denatured soy glycinin and b-conglycinin at natural pH[88].
141
6.1. Quality control and analysis Assessment of oxidative degradation during storage, heating and processing is a big topic in food science. NMR method proved useful for this purpose. From the IH-NMR of thermally degraded vegetable oils (peanut, rapeseed, sunflower, corn, soybean, olive and olive husk oils), two indexes were proposed to characterize oil degradation [89]: One is NV value related to vinyl protons, which is defined as NV=(A-B/4), where A is the area corresponding to the multiplet at 5.34 ppm and B is the area corresponding to the methylene protons of glycerol at 4.22 ppm) . The second parameter is NM value related to methylene protons which was calculated by the equation, NM=C/B, where C is the area of the multiplet at 2.77 ppm. NV is an index of overall unsaturation, whereas NM is an index of polyunsaturation. During thermal oxidation, NV and NM decrease, while NM/NV ratio remains constant. Oxidative deterioration of oil in salted dried fish was evaluated from the ratios of olefinic protons(Ro) and divinylmethylene protons(Rm) to aliphatic protons. Both ratios decreased steadily during storage. Ro was shown to serve as an index of the oxidative deterioration of the oil [90]. In the case of spray-dried egg powder, a new approach for quantitative analysis of cholesterol oxides was reported [91]. Quantification of 7-keto-cholesterol, 7ct-hydroxycholesterol, 7-[3hydroxycholesterol, cholesterol t~-epoxide and cholesterol [3-epoxide was performed in the range from 4.9 to 9.1 ppm with detection limit of 0.3 ppm (51~g/16g matrix). This method should be useful for investigating intermediates and products due to chemical transformation of cholesterol during storage and heating of food. 13C-NMR can distinguish the differences between oleate (cis-C18: 1) and elaidate (trans-C18:1). The chemical shifts are at 27.2 ppm for the ~-C in the double bond and 129.7~ 129.9 ppm for ethyleic C in oleate. The corresponding shifts in elaidate are at 32. 5 ppm and 130.18~130.4 ppm, respectively. The proportion of elaidate to total C18:1 was estimated to be 0~44% in commercial margarines on the basis of the ~-C signals [92]. Post mortem changes in pig muscle were investigated by 31p_NM R [93]. The rate and the extent of post mortem pH changes largely determines pork quality. Fast pH fall to low ultimate pH leads to pale soft exudative (PSE) meat and high ultimate pH leads to dark firm dry (DFD) meat. Values of pH and the contents of ATP, inorganic phosphate and phosphomonoester were estimated simultaneously. The time dependence of peak heights of creatine and lactic acid in HNMR of an extracted pork muscle observed for 14 days after slaughter suggested that aging for days was favorable [94]. A method for the rapid and quantitative determination of 1,2- and 1,3-diglycerides in olive oil, which is based on the reaction of diglyceride hydroxy groups with trichloroacetyl isocyanate, is developed by using H-NMR and 13C-NMR [95]. Discrimination of virgin from neutralized olive oils becomes possible on the basis of the structure and the amount diglycerides. Relation between the degree of unsaturation of dietary fatty acids and adipose tissue fatty acids were assessed by 13C-NMR in man [96]. The ratio of unsaturated fatty acids to total fatty acids in adipose tissue correlated significantly with the ratio of fat in the diet compounds estimated by a dietitian according to food records. The results indicate that in vivo 13C-NMR is capable of assessing the degree of unsaturation of dietary fatty acids consumed during the preceding months. Sugar content in fruit tissue was evaluated by high resolution 1H-NMR (200 MHz) [97] and low speed magic angle spinning(MAS)/13C-NMR [98]. The sugar peak from muskmelon tissues with low (<8.0 %) sugar contents was not detectable with single pulse measurement, because of a broad linewidth and low resolution from the water peak. Eads et al. successfully applied inversion recovery (IR) pulse sequences for water suppression, which resulted in separation of sugar peak due to the difference of T 1 relaxation values between water proton and sugar proton[85, 97]. This allowed the detection of sugar in lower (1.8 %) sugar samples. As is shown in Figure 15, total sugars of ripe banana (15~20% of the fresh weight) are easily
142 observed in a simple MAS experiment (B,C), but further improvement in S/N is achieved upon water suppression by presaturation (D). High resolution MAS/13C-NMR spectra of intact fruit tissues (grape, peach, persimmon, banana and apple) were obtained[85, 98]. Only low speeds (a few hundred Hz) are enough to improve resolution and to reduce susceptibility broadening. Fructose, glucose and sucrose resonances were assigned and quantified. NOE and T 1 w e r e discussed. Potential applications of MAS/NMR to fruit include quality assessment, analysis of chemical changes accompanying ripening and senescence, and measuring the in situ physical state of components. Aging of gelatinized starch was studied by cross-relaxation NMR method [99]. Relatively immobile starch components increased and components with liquid-like mobility decreased during aging. Two spectral components were observed in cross-relaxation spectra. According to the theory mentioned above(5.5.1.), the broader one correlates with the degree of crystallinity and the narrower one with starch chains having mobility intermediate between
Figure 15. 1H-NMR spectra(200 MHz) of intact banana fruit tissue: (A) non-MAS spectrum obtained in a conventional high resolution probe with sample axis parallel to magnetic field; (B)MAS spectrum obtained without water peak suppression; (C) vertical expansion of (B); (D)MAS spectrum obtained with water peak suppression, the signalto-noise ratios (S/N) in spectra C and D are 55 and 1137, respectively. The magic angle spinning (MAS) frequency was 1.05 kHz.[Reproduced with permission from Ref.81].
143 those in crystal and liquid-like states. The kinetics of crystallization-induced immobilization were analyzed by fitting the intensity of the broader component to Avrami equation. The increase in solid-like component during starch retrogradation (starches from corn, potato, rice waxy rice, mungbean and sago during storage at various temperatures) was monitored by a low resolution pulsed NMR method[ 100]. Analysis of the restricted diffusion observed during retrogradation of potato starch showed a decrease in permeability of structural barrier and an increase in self-diffusion coefficient of intrabarrier water [101]. This result on diffusional motion of water suggests the same behavior during aging with that observed by crossrelaxation NMR and a pulsed NMR mentioned above. 6.2. D e t e r m i n a t i o n of c o m p o n e n t s Several new constituents or products recently detected by NMR methods are listed in Table 2.
Table 2 List of constituents and products quantitatively determined by NMR method. Matrix cider vinegar black tea
Riesling wine fish oil
Component 2,3-butanediol 1,3-propanediol theogallinin theaflavonin desgalloyl theaflavonin n~nin two phenylpropanoids m3-fatty acids methy esters glycerol ester
gum Arabic food products
trifluralin
apple, banana, grape
water, sugars, lipids, acids(malic, citric tartaric), sucrose, glucose, fructose rigid component
pulp of banana green to ripe
sucrose _glucose fructose
Remarks
Ref.
13C, new constituent 0.02-0.15%, new cons. new fermentation product
[lo2] [lO21 [lo3]
new shikimic acid metabolite new category of wine phenol list of chemical shift
[104]
13C_ N M R
[lo63
revised JECFA specification from pesticides, 19F_NMR min. detectable amounts for 25 mg samples 0.008 mg/kg for trifluoromethyl 0.023 mg/kg for monofluoromethyl high-resolution with MAS detection limit = 0.01% of fresh weight
20% to 2.0% of fresh weight by wide line 2.2 to 8.5 % 0.5 to 5.3 % 0.4 to 4.3 %
[1051
[107]
[811
[82]
144 6.3. Characterization and authentication of foods and beverages In the adulteration of expensive commodities, such as alcoholic beverage and flavoring materials, the process of adulteration has become so sophisticated that conventional means of detecting adulteration are no longer of value. In recent years, the traditional methods such as chromatography or p roximate analysis have been supplemented with techniques based on the analysis of C and H isotopes in low-molecular weight molecules such as water, ethanol, or flavor molecules. The most specific of these techniques uses NMR and mass spectroscopies (MS) to detect site-specific isotope ratios [108, 109]. The natural site-specific 2D content and the overall 13C content of acetic acid extracted from vinegars and synthesized chemically were determined by NMR and MS. A careful analysis of the repeatability of the entire analysis (isotope ratios 2D/1H, 2D/13C, 13C/12C for each group, 813C) and a study of known mixtures of natural and synthetic acids showed that as low as 5% synthetic acid in a natural vinegar can be detected in a comparative analysis. The site-specific natural abundance 2D distribution of synthetic (R)-6-decanolide was determined by 2D-NMR. Comparison of the site-specific 2Dcontent provided a method to distinguish between 'natural' 6-decanolide biogenerated from 6-2dccanolide (massoi lactone) isolated from Cryptoca la massola with bakers' yeast and other 6decanolides obtained in different 'non-natural' ways [110]. 31p-NMR spectra of milk from cow, goat, rabbit, baboon and human were examined and they may be of value in authentication of samples [111]. Identification of 4-methylsterols is useful to assure the origin (plant or animal) of a fat and to detect the exact biochemical pathway leading to the sterols specific to a given plant. Mass spectra and 1H-NMR spectra of different methylsterols were reviewed [112]. 6.4. in situ localized spectra Localized NMR spectroscopy, which is often called as MRS in comparison with MRI, is not so familiar technique in food science, because a specific pulse sequence such as ISIS and a facility which can precisely follow the pulse sequence without any contamination from other position is needed for localization of position. The localized NMR is usually used together with NMR imaging. The study of solid/liquid ratios, fat structure and polymorphism and the kinetics of fat crystallization was reviewed [24]. The potential of applications in food process development and control was offered. The localized spectra of sausages in areas of 0.3 mm x 0.05 mm (thickness of sample = 1.5 mm) were obtained by the spin echo 2DFT method [113], in which the difference in the tissue structure was discussed with relation to the process and original materials. McCarthy et al. determined mobility of water in foams by using a localized spectroscopy [114]. T2 relaxation time varies in the foam as function of diameter and its variation was analyzed by the classic 2-state fast exchange model. Cerebral metabolism and blood flow in a functioning region of cerebral cortex are enhanced during the activation responding to the specific task performed. (This is known as a functional image in MRI). A sensitivity to salt was checked by a differential NMR method localized at the taste-sensory area in the fight hemisphere [ 115]. An evoked signal enhancement was observed when a 501~1 of the saline solution was dropped on the left side of the tongue of normal, righthanded male volunteers. The evoked signal intensity depended on the concentration of saline solution. The salty taste threshold was decreased from 85 mM to 17 mM after taking salt-free diets, although this value was different among individuals.
145
7.
TRENDS AND P R O S P E C T S FOR F U T U R E D E V E L O P M E N T
Pulsed-field-gradient NMR and NMR imaging are still in their early stages of development. Rapid development in methods and apparatus is expanding the field for potential applications. It is not by food industry but medicine and pharmaceuticals and biotechnology industries that many of the new developments in NMR technology have been financially supported. This situation will continue. NMR has a merit of particular importance to medical applications: it allows one to obtain internal structure of biological systems non-destructively and noninvasively. Its greatest potential lies in its ability to provide NMR spectroscopic information from spatially resolved regions within the image. This should open up completely new avenues of research, which will allow many of the types of NMR experiments developed over the years for the study of small subsamples of larger biological materials to be performed on an intact sample. These future developments in NMR are expected to provide invaluable tools for the study in food science and engineering. It should allow dynamic events to be probed during processing and storage, as well as time studies of the migration of small molecules in complex composite structures[ 116]. The new avenues in NMR imaging may be focused on (a) improving spatial resolution, (b) imaging protons with short relaxation time T2, and (c) imaging nuclei other than proton. The attainable resolution of the NMR micro-imaging is limited by physical and technical factors. Physical factors are: the line width and the chemical shift of the NMR signal, diffusion processes, and susceptibility gradients, both within the object and its boundaries. Technical factors include magnetic field inhomogeneity or instability, non-linearity of the magnetic field gradients, and the achievable signal-to noise ratio [ 117]. The maximum obtainable spatial resolution, Ax, for a given spin-spin resolution time T2 may be described as Ax=2/(yGx T2), where Gx is the gradient strength. This means that application of larger gradient strength may help in the improvement of spatial resolution. However, very strong gradients which are rapidly switched would induce enormous eddy currents in the electrically conducting parts of the magnet, which would have a strong effect on the magnetization. Effect of eddy currents on magnetization also causes serious deterioration in the measurement of diffusion and flow. Technical improvement in actively shielding eddy current is required. Freezing and drying are among major processes which foods are subjected to. In the course of freezing and drying, free water goes out or become frozen, leaving water molecules in hydration shell, whose relaxation time T2 is too short to be covered by ordinary NMR imaging methods. Hence, further development is required in NMR imaging techniques suited for imaging solid materials, such as by the combination of rotating gradients with the magic-angle spinning frequency; it may be hopefully applicable to the study of dried or frozen foods. Molecular diffusion is among the most serious limitation to the spatial resolution in NMR micro-imaging if the nuclei diffuse into a neighboring voxel during the period between excitation and detection. In a spin echo experiment the voxel resolution Ax is only defined if, during the echo time TE, the spins do not leave the voxel in which they were excited: (Ax) 2 ___2D TE. In biological materials the diffusion coefficient, D, of water is often close to 5.10-1~ m 2 s-1. This means that spatial resolution of 1 ~m is realized only when the spin echo time is shorter than 1 ms, resulting in the need for rapid imaging methods. Since NMR signal is sensitive to chemical environment, molecular dynamics, and position in space, obtained signal data contain mixture of these types of information. Separation of these types of information often requires detailed analysis using appropriate mathematical model. Improvement in methods and apparatus of NMR may allow us to use more precise mathematical models which are helpful for the separation.
146 Relaxation phenomena include the information not only about dynamic processes which are experienced by the molecule (or nuclei) detected, but also about heterogeneous characteristics in food materials. Dynamic processes, such as dipole-dipole interaction modulated by the thermal motion, spin-spin interactions, quadrupolar interaction, chemical and diffusive exchanges, spin rotational interaction and so on, are strongly related to the properties and qualifies of food materials. The water activity, though grasped as a macroscopic concept for the present, will possibly be explained at the molecular level, if relaxation mechanisms of water in a complex system like foods are elucidated. Detection of the magnetization transfer due to the cross relaxation will be promising to investigate the structure of food materials including both liquid phase and solid phase components[86]. High resolution NMR spectroscopy has acquired an already established position in food science and technology. Non-invasive and non-destructive property in NMR measurement is one of the most important factors for living subjects and for materials which show intrinsic properties as they are. Multi-quantum NMR and multi-dimensional NMR methods will be found useful to analyze complex spectra observed in heterogeneous food materials. Recently, the field gradient system has been equipped within the high resolution NMR spectrometer and used for a quick dephasing and rephasing of the spin packets in multi-dimensional NMR instead of 180-degree of radio frequency pulse. It is quite interesting that the technique specifically developed for NMR imaging is fed back to the original NMR spectroscopy . Furthermore, some localization techniques such as the ISIS pulse sequence and the detection by the surface coil in NMR imaging have been available in high resolution NMR spectroscopy. This technique will be inevitable as well in characterizing foods.
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Characterizationof Food: Emerging Methods A.G. Gaonkar (Editor) 9 1995 Elsevier Science B.V. All rights reserved.
151
Chapter 6 Determination of droplet size distributions in emulsions by pulsed field gradient NMR M.M.W. Mooren, M.C.M. Gribnau and M.A. Voorbach Unilever Research Laboratorium, Olivier van Noortlaan 120, 3133 AT Vlaardingen, The Netherlands
1. INTRODUCTION Emulsions are colloidal dispersions of liquid droplets in another liquid phase, sometimes stabilized by surface active agents. Emulsions thus consist of a discontinuous phase, dispersed in a continuous phase. The most common types of emulsions are water-in-oil (W/O) in which oil is the continuous phase, and oil-in-water (O/W) in which water forms the continuous phase. However, this traditional definition of an emulsion is too narrow to include most food emulsions. For example, in foods the dispersed phase may be partially solidified, as in dairy products or the continuous phase may contain crystalline material, as in ice cream. It may also be a gel, as in several desserts. In addition to this, air bubbles may have been incorporated to produce the desired texture. There are several reasons why it is important for a food company to examine emulsions carefully. In systems containing both oil and water, knowledge of the emulsion structure may be important with respect to palatability, oral melting behaviour, texture and general appearance. For example, olive oil as such has a greasy taste, but it is highly acceptable in an emulsified oil-and-vinegar dressing. Furthermore, some flavour ingredients are insoluble in the salad oil, whereas others are insoluble in the vinegar. Variation in the structure of the emulsion, e.g. by changing the type of ingredients, their quantity or the way of processing, will lead to products with different characteristics. ~ In an emulsion the dispersed phase is disrupted into droplets. These droplets must be protected from immediate coalescence because emulsions are inherently unstable. Even in a system that appears to be perfectly stable, with a shelf-life of several years, the total number of droplets, their size distribution, and their arrangement in space, are all changing with time. The size of the droplets in food emulsions varies considerably and may range from 0.1 ~m to more than 20 ~m. Consequently proper characterization of the droplet size distribution is important, not only because of the above mentioned texture related reasons, but also in view of microbiological aspects. Because of this importance, different techniques have been developed to characterize the droplet size distribution in emulsions, each with its own pros and cons. Light microscopy, for example, is qualitative and only suited for particles larger than about 1/~m. When using electron microscopy, correct sample preparation is crucial to the examination and interpretation of the dispersions. The Coulter method is an indirect method which detects a
152 change in impedance if a droplet passing through a fine orifice. The emulsion needs careful and extensive dilution prior to the measurement. Finally, size characterization by light scattering is fast and has practically no lower particle size limits. However, here again, the emulsion has to be extensively diluted, which in a food emulsion such as e.g. cheese, may disrupt its structure and thus influence the original droplet size distribution. In fact, in all these techniques the integrity of the sample is disrupted. We have chosen a completely different approach for the determination of droplet size distributions, namely pulsed field gradient nuclear magnetic resonance spectroscopy (PFGNMR). This technique was first described by Tanner and Stejskal in 1965 [1,2]. Originally the pulsed field gradient spin-echo technique was used to determine diffusion coefficients in well-defined single phase systems, like pure liquids or microemulsions, where no boundary restrictions of the molecular or micellar movements exist. In the course of time the method has succesfully been extended and applied to study a variety of systems exhibiting restricted diffusion. The principle of this extension is that in emulsions the molecules inside the droplets will have a mean displacement during the measuring time that is of the same order of magnitude or larger than the droplet size and will therefore undergo restricted diffusion. Consequently, these molecules will show a different diffusion behaviour in the PFG-NMR experiment, compared with a pure liquid. This idea was used by Packer and Rees who in 1972 showed that PFG-NMR may be used for the determination of the droplet size distribution in emulsions [3]. Their results led us to investigate whether this method can be used for the routine determination of droplet size distributions in a factory environment, preferably with the aid of relatively cheap and simple equipment.
2. THE PFG-NMR TECHNIQUE
2.1. Principles The NMR technique observes the behaviour of the magnetic moments of specific nuclei in a sample. In most PFG-NMR experiments hydrogen nuclei (protons) will be observed. In typical food emulsions hydrogen atoms are abundantly present and the ~H isotope with spin % has a natural abundance of 99.98 %. These nuclei also have a relatively high gyromagnetic ratio, ~,, resulting in a high sensitivity. By comparision, the NMR sensitive carbon-isotope ~3C with spin 1/2 has a natural abundance of only about 1% and also its .y-value is rather low, making it difficult to detect these nuclei. In a static magnetic field the magnetic moments orient themselves parallel with the magnetic field. By the application of rf-pulses of a specific frequency ('on resonance') the orientation of the magnetic moments is changed: each rf-pulse results in a redistribution of orientations of the magnetic moments. The detected NMR signal reflects the redistribution after the if-pulse sequence. More precisely, the detected NMR signal is proportional to the net magnetization in the plane perpendicular to the main magnetic field. In the PFG-NMR experiment the spin-echo sequence can be used, which has to be applied with and without two gradient pulses (see Fig. 1). The height of the NMR signal, the echo, is measured in the presence (echoheight E') and absence (echoheight E) of the two magnetic field gradient pulses. The presence of the field gradients induces an additional change in the orientations of the magnetic moments. In the absence of molecular diffusion, the effects of the two gradient pulses counterbalance each other. However, in the presence of diffusion no compensation occurs and a widening
153
Figure 1. Pulse sequence diagram of a spin-echo experiment with field gradient pulses. The rf-pulses are denotedby 90 ~ and 180 ~ and the field gradient pulses by FGP. The FGP pulses have a length ~ and are separated by an interval A.
of the distribution of orientations occurs. This is reflected in a lower value for the average orientation and a decrease in the echoheight E*. The larger the effect of diffusion the stronger the decrease in E*. In fact, one measures the mean displacement, < x2>, 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<0.001; DM 9dry matter; SEM: Standard Error of Mean,'Basic" uncorrected integral, 'Control': unirradiated spectrum subtracted, q)M conc.': corrected for differences in sample DM, '1 g DM': corrected to a standard 1g DM in cavity. (From Stevenson and Gray, 1989a).
Figure 2. Response of EPR signal to dose in irradiated chicken bone. (From Stevenson and Gray, 1989b).
170 In the case of chicken, even at doses down to 1 kGy (Desrosiers and Simic, 1988) and as high as 25 kGy (Dodd et al., 1988), signal response was proportional to dose. The intensity of the signal induced by a given irradiation dose appears to be species dependent, being much lower for fish (Stewart et al., 1991) than chicken (Dodd et al., 1988). Since the trapped radicals are associated with the hydroxyapatite fraction of bone, the concentrations of calcium and phosphorus might be expected to affect signal intensity. However, using birds of different ages (4 to 8 weeks), it was shown that the increase in EPR signal strength with increasing age was not due to a change in mineral composition since the latter was consistent over the age range investigated (Gray et al., 1990a). Work published by Ostrowski et al. in 1981 has shown that the degree of crystallinity in bone structure can influence the EPR signal intensity. The crystallinity of a bone can be defined as the ratio of the crystalline fraction of tissue mineral to the total amount of mineral and can be estimated by calculating the ratio of the concentration of stable free radicals induced by a saturating dose of ionising radiation to the ash content of the tissue. In work with chicken bone it has been demonstrated (Table 4) that as the birds aged there was a linear increase in crystallinity coefficient which is a measure of bone crystallinity (Gray et al., 1990a). Table 4 Effect of age on the EPR signal strength in irradiated chicken bone
EPR signal intensity Age (weeks)
4 5 6 7 8 SEM CV% Sig.
Dose Corr'd
Ash Corr'd
Ca Corr'd
P Corr'd
Crystallinity Coefficient
1.913 2.021 2.099 2.323 2.489
1.946 2.036 2.107 2.304 2.464
1.787 1.997 1.912 2.227 2.367
1.761 1.840 1.854 2.008 2.215
2.32 2.42 2.48 2.55 2.60
0.0638 11.8 ***
0.0630 11.6 ***
0.0752 14.6 ***
0.0604 12.5 ***
0.080 13.0 ***
*** P<0.001; SEM: Standard Error of Mean; CV%: percentage Coefficient of Variation; Sig.: Significance (From Gray et al., 1990a).
The differences in crystallinity accounted for some, but not all, of the variation in EPR signal strength associated with bones from birds of different ages. Similar results have been reported for pork and salmon bones with pork bone giving a more intense signal than salmon bone and X-ray diffraction of pork bone showed it to be more crystalline (Goodman et al., 1989). When bones from different sites within the carcass of an irradiated chicken are examined the intensity of the EPR signal varies depending on the part of the skeleton from
171 which the bones are removed (Figure 3). Correction of the various signal intensities for any variation in calcium or phosphorous content of the bones did not remove the effect but there was a strong linear relationship between the EPR signal intensity and the crystallinity coefficient. More crystalline bones, such as the tibia and femur of the leg and the humerus gave a greater response to a given irradiation dose than the more cartilaginous rib and sternum (Dodd et al., 1992; Gray and Stevenson, 1990b).
Figure 3. EPR spectra from different skeletal bones Chicken can be retailed either frozen or chilled and irradiation can also be applied under both of these temperature regimes. In samples irradiated in the frozen state, the movement of free radicals is hindered and as expected fewer radicals are trapped in the bone. Consequently, the intensity of the signal induced in bone from irradiated frozen chicken was lower than in samples from chilled poultry, carcasses given the same irradiation dose (Dodd et al., 1992; Stevenson and Gray, 1990) (Figure 4). On the other hand, the rate at which an irradiation dose was applied did not significantly affect the intensity of the induced signal (Dodd et al., 1992; Gray and Stevenson, 1991). Yields of radicals induced using a machine source producing 10 MeV electrons (dose rate = 600 kGy h-a) were similar to those produced by either 137Cs or 6~ gamma rays (dose rate range 0.2 - 20 kGy h'a). Consequently, the EPR technique can be used to identify products treated using either of the major irradiation sources which are permitted for the treatment of food (Anon., 1984).
172
Figure 4. Effect of processing treatment on EPR signal intensity in chicken bones. (From Stevenson and Gray, 1990). In commercial practice, chicken might be cooked either before or after irradiation and it was important to establish that the cooking procedure neither completely destroyed the radiation-induced signal nor generated radicals which could interfere with the signal induced by irradiation. It has been shown that the cooking process does not generate signals similar in shape to those induced by irradiation but it may affect signal intensity. In one case, cooking chicken after it had been irradiated did not significantly affect EPR signal strength (Dodd et al., 1992) while another report recorded approximately a 23% reduction in signal intensity (Gray and Stevenson, 1989a). On the other hand, if chicken was cooked before irradiation, the concentration of free radicals was significantly increased (Figure 4) (Dodd et al., 1992; Stevenson and Gray, 1990). It has been suggested that because the water content of cooked bones w'as reduced, there was less opportunity for free electrons to react with water. As a consequence, more free radicals were trapped in bone thus ~ving an enhanced EPR signal (Davidson, 1988). Alternatively, the observable change in the structure of the bone and, in particular, the loss of collagen and consequent relative increase in calcium content may contribute to the greater signal intensity (Dodd et al., 1992). The stability of the trapped radicals appeared to be similar in all types of chicken bone (Dodd et al., 1992; Gray and Stevenson 1990b). Storage at chill temperatures (+5~ had either no significant effect (Dodd et al., 1992) or caused a small decay in signal intensity (Stevenson and Gray 1989b) while storage at -20~ for 10 months caused no apparent change in free radical concentration (Dodd et al., 1992).
173
5.1.2. Measurement of absorbed dose The demonstration that the EPR signal strength is affected to some extent by a number of processing variables indicates that there may be difficulties in accurately estimating the dose received by samples whose processing history is unknown. Re-irradiation has been suggested as a way of estimating dose which does not require a detailed knowledge of the sample history. However, there are still likely to be difficulties encountered with cooked samples or those irradiated in the frozen state (Dodd et al., 1992). The procedure of re-irradiation was first used by Dodd and his co-workers to estimate the dose received by chicken, pork and fish bones (Dodd et al., 1988) and has subsequently been used in a number of blind trials (Desrosiers et al., 1990; Raffi et al., 1992) with varying degrees of success. The procedure is essentially a standard addition technique whereby the sample is irradiated several times with the signal strength being recorded after each re-irradiation. An estimate of the original absorbed dose is then extrapolated from a graph of signal height versus applied dose. However the mathematical function used to fit the data is critical to the accuracy of the dose estimate (Desrosiers et al., 1991). A practical drawback to the re-irradiation technique is the need to have access to an irradiator and it has been proposed that the derivation of a dose response calibration curve could provide an alternative procedure for the quantitative evaluation of samples of unknown background (Bordi et al., 1993).
5.1.3. Detection in secondary products As well as using EPR spectroscopy to identify primary food products containing bone, the technique has also been successfully used to identify irradiated MRM. This product is manufactured by hydraulically removing meat from carcass components and normally contains less than 1% bone by weight. As a consequence of the carcass components used in the process the end product frequently has an unacceptably high bacterial count and is therefore a prime candidate for treatment with ionising radiation. The small quantity of bone in the MRM may be extracted by digestion with alcoholic potassium hydroxide at a temperature of 85~ for 90 minutes and subsequent vacuum filtration (Gray and Stevenson, 1989b). The fact that a positive EPR signal can be detected in the bone fragments after such a severe digestion process is a further indication of the stability of the free radicals trapped in the bone. Indeed, so stable are the radicals that not only can unmarked samples be identified as irradiated or non-irradiated but accurate dose estimates may be obtained from comparative measurements of peak intensity (Gray and Stevenson, 1989b). Recently this work has been extended to detect the presence of irradiated MRM in tertiary food products i.e. burgers, where as little as 3% of the MRM had been included in the burger. (Figure 5). However this work will almost certainly only allow qualitative detection of irradiation as the small sample size and consequent high signal noise would make any form of quantification virtually impossible.
5.2. Foods with shells A radiation-induced signal can be detected in the exoskeleton of Norway lobster (Nephrops norvegicus) (Stewart et al., 1992) and other species of prawn and shrimp (Morehouse and Desrosiers, 1993). In the case of Norway lobster, the signal in both irradiated and unirradiated cuticle is complex because of the presence of the six resonance peaks due to Mn 2+. In the irradiated samples there is an additional free radical peak in the centre of the Mn 2+ signal at 349.5 mT (Figure 6). This signal is more easily seen when the
174
[G] Figure 5. EPR signal from irradiated MRM bone fragments. There is no detectable signal from non-irradiated bone fragments.
Figure 6. EPR signals from irradiated and unirradiated Norway lobster cuticle.
[G]
175 effect of the Mn z+ signal is removed using the software associated with the spectrometer. The resulting peak appears to be specific for irradiation as it was neither generated nor completely destroyed by cooking (Figure 7).
[G] Figure 7. Effect of processing on the radiation-induced EPR signal in Norway lobster cuticle. (From Stewart et al., 1993b).
Figure 8. Effect of irradiation dose and storage temperature on the radiation-induced EPR signal in Norway lobster cuticle.
176 As for bone, there was a linear relationship between the intensity of the radiation-induced signal and irradiation dose over the range 1 to 5 kGy (Figure 8), so the EPR method had potential for the quantification of dose (Stewart el al., 1992). However, the fact that the signal decayed during storage (Stewart et al., 1992) (Figure 8) and was influenced by cooking (Figure 7) could affect the accuracy with which the absorbed dose can be estimated. The shape of the radiation-induced signal was similar in different componems of the exoskeleton of Norway lobster but the intensity of the peak varied (Stewart et al., 1993a). Consequently, the part of the cuticle used for EPR analysis will not affect identification of irradiation treatment but could influence the estimation of dose in samples of unknown processing history. Work with other species of prawn and shrimp has indicated that the situation with Crustacea is more complex than for foods containing bone. There is evidence that the shape of the radiation-induced EPR signal is species dependent (Figure 9) and even the same species when examined by several workers gave radiation-induced signals which were quite different in shape (Desrosiers, 1989; Stewart, 1993). The reason for these conflicting results is unknown but it is possible that some species may not have been identified correctly. At a recent meeting in Budapest, it was recommended that in all future work each species should be given its scientific name (Anon., 1992).
Figure 9. EPR spectra from the irradiated (I) and non-irradiated (N) cuticle of various species of Crustacea. (From Stewart et al., 1994.).
177 The origin of the free radicals responsible for the radiation-induced signals in the exoskeleton of Crustacea has not been conclusively identified. It has been suggested that the complex signal observed in the cuticle of pink shrimp may be derived from chitin, which is a major component of the exoskeleton (Desrosiers, 1989). However, other components of the shell matrix, such as calcium, may also contribute to the radiation-induced signal (Dodd et al., 1985). A knowledge of the origin of the radical or radicals responsible for the EPR signal may help to explain the varying responses which have been observed.
5.3. Fruits and vegetables The water content of flesh fruits and vegetables varies from approximately 80 to 95%, so radicals induced in the pulp by irradiation are not stable. However, the seeds, shells or skins can trap free radicals and so could be used to monitor radiation exposure. Among fruit, the strawberry is a likely candidate for irradiation since the process can be used to extend shelf-life and inhibit mould growth. A multicomponent signal has been detected in strawberry achenes (Dodd et al., 1985; Raffi et al., 1988). In both irradiated and non-irradiated samples, the spectrum consists of a six line signal due to Mn 2+ and a single central line, the intensity of which varies with water content and is thought to be related to a quinone radical. In irradiated achenes there is a novel radiation-induced signal and it has been suggested that it originates from cellulose (Merlin and Fouassier, 1980; Sultanov, 1991). Unlike the singlet, this signal is not affected by water content (Raffi et al., 1988). The fact that this radiation-induced peak originates from cellulose suggests that it should form the basis of a test for all parts of fruits and vegetables which contain considerable amounts of cellulose in high dry matter environments. A similar signal has also been observed in the seeds of grapes, raspberries, redcurrants and bilberries (Goodman et al., 1989; Raffi and Agnel, 1989b). The seeds, pips, shells and skins of other fruits and vegetables including onions, garlic, apples, oranges, pears, cherries and mangoes have also been examined (Stachowicz et al., 1992; Desrosiers and McLaughlin, 1989), but the cellulose triplet was not observed. It is possible that inappropriate EPR operating conditions were used since it has been shown recently that, in certain circumstances, the use of microwave power greater than 0.5 mW results in a loss of sensitivity and so an inability to detect the cellulose radical (Deighton et al., 1993). Further research is needed to define the operating conditions which are most appropriate for the identification of the cellulosic radical in a range of foods. In addition, the stability of the signals induced in irradiated fruits and vegetables needs to be studied in greater depth since there are indications that stability varies with the food being examined (Stachowicz et al., 1992). The EPR spectra of irradiated dried fruits have also been examined. They are complex and quite different to the non-irradiated samples which generally show no EPR signal thereby making identification of irradiated samples relatively easy. The spectra are similar in shape to those derived from irradiated component sugars which make up 60 to 75% of the composition of dried fruits. Irradiated dried pineapples, figs, papayas and apricots showed spectra similar to irradiated saccharose which is present in high concentrations in these fruits while irradiated raisins, dates, currants and sultanas contain less saccharose and they displayed spectra of a different shape (Helle and Linke, 1992). For some of these fruits, for example, raisins and dates, the spectral shape changed during storage (Helle and Linke, 1992; Raffi et al., 1991b) but detection of irradiation even after several months of storage was still considered to be possible. More research is needed to validate and extend the results already obtained.
178 The multicomponent signal present in dried mushrooms irradiated at a dose of 3 kGy was quite different to the EPR signal in non-irradiated samples, thus facilitating the identification of samples treated with ionising radiation (Stachowicz el al., 1992). Conversely, irradiated dried vegetables (unspecified) and garlic gave simple broad singlets which were similar in shape but different in intensity to those present in non-irradiated samples (Stachowicz el al., 1992~ Desrosiers and McLaughlin, 1989). The similarity in signal shape would therefore make it difficult to obtain conclusive evidence of irradiation treatment in these dried foods.
5.4. Spices and herbs Early research with irradiated paprika (Beczner et al., 1973), white pepper, nutmeg and ginger (Tjaberg et al., 1972) concluded that the technique was not suitable for the detection of these spices since the free radicals formed were short lived and could not be distinguished from intrinsic free radicals already present in the non-irradiated samples. More recently, a radiation-induced stable EPR signal has been observed for up to three months in paprika, white mustard and chilli (Stachowicz et al., 1992). The EPR signal derived from irradiated cellulose has also been observed in paprika. However, it has been pointed out that the detection of the cellulose lines can be difficult or impossible in the presence of the manganese signal especially if the spices have a low cellulose content (Helle and Linke, 1992).
Figure 10. EPR spectra from irradiated cellulose packaging during storage for up to 58 days
179
5.5. Nuts The EPR spectra of irradiated shells from walnuts, peanuts, hazelnuts and pistachio nuts are similar to those from irradiated strawberries and the radiation-induced signal, which is thought to be due to cellulose, has potential for the identification of these products (Raffi et al., 1992, Helle and Linke, 1992). 6. P A C K A G I N G Irradiated paper-based packaging shows the characteristic cellulose signal which exhibits a rapid decay in signal intensity during the first 24 hours after irradiation but thereafter is relatively stable showing only a gradual decline over many days (Figure 10). EPR examination of samples of packaging has the advantage of being relatively quick and simple but it should be remembered that the presence of the characteristic signal due to irradiation treatment only indicates that the packaging has been treated. It is not evidence that the contents have been irradiated but would suggest that the contents merit further investigation. 7. R E S U L T S O F B L I N D T R I A L S The ultimate proof that the EPR method is useful for the identification of irradiated food is its performance in blind trials. A number of studies have already been completed (Table 5), and the results have been very encouraging. In the majority of cases it has been possible to identify foods treated with irradiation at doses well below those likely to be used commercially. In trials reported by Desrosiers et al., 1990, Scotter et al., 1990, Desrosiers, 1992 and Schreiber et al., 1993 all irradiated and non-irradiated samples were correctly identified. In the more extensive trial reported by Raffi et al., 1992, the numbers of correct identifications depended on the foods examined and the doses applied. In this trial the results for meat bones, dried papaya and dried grape were good but those obtained with fish bones and pistachio nuts were not as conclusive as the results from the trial reported by Schreiber et al., 1993. However further development of the protocols involved with these products has been undertaken and the results from future trials on these products should be more acceptable. Following the success of these collaborative trials, standard methodologies for the application of the EPR method for the identification of irradiated meat bones, fish bones and some fruits have been prepared and are about to be submitted to the European Committee of Normalization. At the time when these trials were carried out there was not sufficient information available to permit inclusion of Crustacea among the products being examined and thus validation of the method for the identification of these irradiated foods has to be undertaken. As well as qualitative identification of irradiation, doses received by bones have also been estimated in three of the collaborative trials using the re-irradiation technique (Desrosiers et al., 1990; Raffi et al., 1992; Scotter et al., 1990). Although the differences between the applied and estimated doses were sometimes quite large and there were differences between laboratories, it was possible to distinguish between samples given low, medium and high doses of irradiation.
180 Table 5 International collaborative trials using EPR
Organiser
Number of Laboratories
Food Product
Total number of samples
Desrosiers et al., 1990
4
Chicken bone Pork bone Frog leg bone
12 12 16
Scotter et al., 1990
6
Chicken bone
36
Desrosiers, 1992
11
Chicken bone Pork bone Frog leg bone
99 33 22
Raffi et al., 1992
21
Beef bone Trout bone Sardine scales Pistachio nut shell Dried grapes Dried papaya
126 126 126 126 126 126
Schreiber et al., 1993
18
Chicken bone Trout bone Pistachio nut shell
108 108 72
8. SCOPE FOR FUTURE R E S E A R C H The technique of EPR spectroscopy has proved to be a non-destructive technique with the potential for the quick and easy identification of a number of irradiated products. However considerable research is still required into products such as Crustacea, exotic fruits and various spices. There would also appear to be considerable potential in the identification of irradiation in a number of packaging materials providing additional identification which would reinforce the results of other techniques. This should help to reassure the consumer and ensure that the technique of food irradiation is not abused.
181 ACKNOWLEDGMENTS The author wishes to thank the Ministry of Agriculture Fisheries and Food, United Kingdom for providing partial funding for some of the work carried out in Belfast and the Commission of the European Communities, Community Bureau of Reference for assistance in a number of aspects of the work that has been referred to in the text.
REFERENCES Anon. (1981). Joint FAO/IAEA/WHO Expert Committee on Wholesomeness of Irradiated Food (JECFI). Report of the Working Party on Irradiation of food; WHO Technical Report Series 659, WHO, Geneva. Anon. (1984). Codex 'General Standards for Irradiated Foods' and 'Recommended International Code of Practice for the Operation of Radiation Facilities used for the Treatment of Foods.' Codex Alimentarius Commission, XV, 1st edn. Rome. Anon. (1986). Advisory Committee on Irradiated and Novel Foods Report on the Safety and Wholesomeness of irradiated food, HMSO, London. Anon. (1988). Test market for irradiated strawberries in France. Food Irrad. Newsl. 11, 45. Anon. (1989). International document on food irradiation. In: Acceptance, Control of and Trade in Irradiated Food, IAEA, Vienna, 135. Anon. (1990a). The Food (Control of Irradiation) Regulations 1990a No. 2490, HMSO, London. Anon. (1990b). Consumer report: Food irradiation - the consumer's view. Survey Research Group, The Association for Consumer Research, London. Anon. (1990c). The Food Labelling (Amendment) (Irradiated Food) Regulations 1990, No. 2489, HMSO, London. Anon. (1991). Supplement to Food Irrad. News. 15. Anon. (1992). Coordinated Research Programme on Analytical Detection Methods for Irradiation Treatment of Foods (ADMIT). Second Research Co-ordination Meeting, Hungary, 15-19 June, IAEA, Vienna, 15. Beczner, J., Farkas, J., Watterich, A., Buda, B. and Kiss, I. (1973). Study into the identification of irradiated ground paprika. Pages 255-267 in: The identification of irradiated foodstuffs, CEC, Luxembourg. Bordi, F., Fattibene, P., Onori, S. and Pantaloni, M. (1993). An alternative procedure for ESR identification of irradiated chicken drumsticks. Appl. Radiat. Isot. 44, 443. Bruhn, C.M. and Noell, J.W. (1987). Consumer In-store response to irradiated papayas. Food Technol. 41, 83. Davidson, I.G (1988). ESR studies on gamma-irradiated foods. Ph.D. Thesis, University of Aberdeen, Scotland. Deighton, N., Glidewell, S.M., Goodman, B.A. and Morrison, I.M. (1993). Electron paramagnetic resonance of gamma-irradiated cellulose and lignocellulosic material. Int. J. Food Sci. Technol. 28, 45. Delincee, H. (1991). Analytical methods for irradiated foods. A review of the current literature IAEA-TECDOC-587, International Atomic Energy Agency, Vienna. Delincee, H. (1993). Control of irradiated food: Recent developments in analytical detection methods. Radiat. Phys. Chem. 42, 351.
182 Desrosiers, M.F. (1989). Gamma - irradiated seafoods: Identification and dosimetry by ESR spectroscopy. J. Agric. Food Chem. 37, 96. Desrosiers, M.F. (1992). EPR methods for food irradiation: 1. Results of ADMIT co-trials on irradiated meat detection. Second Research Co-ordination Meeting, Hungary, 15-19 June, IAEA, Vienna, 62. Desrosiers, M.F. and Simic, M.G. (1988). Post-irradiation dosimetry of meat by electron spin resonance spectroscopy of bones. J. Agric. Food Chem. 36, 601. Desrosiers, M.F. and McLaughlin, W.L. (1989). Examination of gamma-irradiated fruits and vegetables by electron spin resonance spectroscopy. Radiat. Phys. Chem. 34, 895. Desrosiers, M.F., McLaughlin, W.L., Sheahen, L.A., Dodd, N.J.F., Lea, J.S., Evans, J.C., Rowlands, C.C., Raffi, J.J. and Agnel, J-P. L. (1990). Co-trial on ESR identification and estimates of gamma-ray and electron absorbed doses given to meat and bones. Int. J. Food Sci. Technol. 25, 682. Desrosiers, M.F., Wilson, G.L., Hunter, C.R. and Hutton, D.R. (1991). Estimation of the absorbed dose in radiation-processed food. - 1: Test of the EPR function by a linear regression analysis. Appl. Radiat. Isot. Part A 42, 613. Dodd, N.J.F., Swallow, A.J. and Ley, F.J. (1985). Use of ESR to identify irradiated food. Radiat. Phys. Chem. 26, 451. Dodd, N.J.F., Lea, J.S. and Swallow, A.J. (1988). ESR detection of irradiated food. Nature 334, 387. Dodd, N.J.F., Haishun, J., Lea, J.S. and Swallow, A.J. (1992). Factors influencing the yield of free radicals in irradiated chicken. Int. J. Food Sci. Technol. 27, 371. Geoffroy, M. and Tochon-Danguy, H.J. (1982). ESR identification of radiation damage in synthetic apatites: a study of the C-Hyperfine synthetic coupling. Calc. Tiss. Int. 46, 99. Goodman, B.A., McPhail, D.B. and Duthie, D.M.L. (1989). Electron spin resonance spectroscopy of some irradiated foodstuffs. J. Sci. Food Agile. 47, 101. Gordy, W., Ard, W.B. and Shields, H. (1955). Microwave spectroscopy of biological substances. Paramagnetic resonance in X-irradiated amino acids and proteins. Proc. Natl. Acad. Sci. Wash. 41,983. Gray, R. and Stevenson, M.H. (1989a). The effect of post-irradiation cooking on the ESR signal in irradiated chicken drumsticks. Int. J. Food Sci. Technol. 24, 447. Gray, R. and Stevenson, M.H. (1989b). Detection of irradiated deboned turkey meat using ESR spectroscopy. Radiat. Phys. Chem. 34, 899. Gray, R., Stevenson, M.H. and Kilpatrick, D.J. (1990a). The effect of irradiation dose and age of bird on the ESR signal in irradiated chicken drumsticks. Radiat. Phys. Chem. 35, 284. Gray, R. and Stevenson, M.H. (1990b). Effect of length of storage on the ESR signal from various bones in irradiated chicken carcasses. Int. J. Food Sci. Technol. 25, 506. Gray, R. and Stevenson, M.H. (1991). Effect of dose rate and length of storage on the ESR signal strength in irradiated chicken bone. Int. J. Food Sci. Technol. 26, 669. Helle, N. and Linke, B. (1992). ESR for detecting gamma-irradiated foodstuffs. Broker Report 91/92, 8. Ikeya, M. and Miki, T. (1980). Electron spin resonance dating of animal and human bones. Science. 207, 977. Leonardi, M., Raffi, J.J. and Belliardo, J.-J. (1992). Recent Advances on Detection of Irradiated Foods, EUR- 14315, CEC, Luxembourg. Marcotte, M. (1992). Irradiated strawberries enter the U.S. market. Food Technol. May, 80.
183 Merlin, A. and Fouassier, J-P. (1980). Photochemical investigations in cellulose materials. I. Free radical generation in cellulose by photochemical excitation. Angew. Makromol. Chem. 86, 109. Morehouse, K.M. and Desrosiers, M.F. (1993). Electron spin resonance investigations of gamma-irradiated shrimp shell. Appl. Radiat. !sot. 44, 429. Ostrowski, K., Dziedzic-Goclawska, A. and Stachowicz, W. (1980). Stable radiation-induced paramagnetic entities in tissue mineral and their use in calcified tissue research. Pages 321-344 in: Free radicals in biology, Academic Press, London. Ostrowski, K., Dziedzic-Goclawska, A., Stachowicz, W. and Michalik, J. (1981). Crystallinity of tissue mineral as evaluated by electron sin resonance spectrometry. Basic Appl. Histochem. 25, 79. Pass, B. and Aldrich, J.E. (1985). Dental enamel as an in-vivo radiation dosimeter. Med. Phys. 12, 305. Raffi, J.J., Agnel, J.-P.L., Buscarlet, L.A. and Martin, C.C. (1988). Electron spin resonance identification of irradiated strawberries. J. Chem. Soc. Faraday_Trans. 1 84, 3359. Raffi, J.J., Evans, J.C., Agnel, J-P., Rowlands, C.C. and Lesgards, G. (1989a). ESR analysis of irradiated frog legs and fishes. Appl. Radiat. Isot. 40, 1215. Raffi, J.J. and Agnel, J.-P.L. (1989b). Electron spin resonance identification of irradiated fruits. Radiat. Phys. Chem. 34, 891. Raffi, J.J. and Belliardo, J.J. (1991a). Potential New Methods of Detection of Irradiated Foodstuffs, EUR-13331EN, CEC, Luxembourg. Raffi, J., Agnel, J.-P. and Ahmed, S.H. (1991b). Electron spin resonance identification of irradiated dates. Food Technol. 3/4, 26. Raffi, J., Stevenson, M.H., Kent, M., Thiery, J.M. and Belliardo, J.-J. (1992). European intercomparison on electron spin resonance identification of irradiated foodstuffs. Int. J. Food Sci. Technol. 27, 111. Raffi J., Delinc6e, H., Marchioni, E., Hasselmann, H., Sj6berg A-M., Leonardi, M., Kent, M., B6gl, K.W., Schreiber, G., Stevenson, M.H. and Meier, W. (1993). Final Report of CEC Contract No. 5415/1/5/340/90/11/ BCR-F(10) on "New methods for the detection of irradiated food", EUR ..... EN, CEC, BCR, Luxembourg. Rossi, A., Poupeau, G., Chaix, O., Raffi, J., Agnel, J-P. and Jeunet, A. (1992). Paramagnetic species induced in bioapatites by foodstuff ionisation. Pages 151 - 166 in: Electron Spin Resonance (ESR) Applications in Organic and Bioorganic Materials. Proceedings of the First European Meeting, January 1990, Lyon, France. Springer Verlag, Berlin. Schreiber, G.A., Helle, N. and Bogl, K.W. (1993a). Detection of irradiated food - Methods and routine applications. Int. J. Radiat. Biol. 63, 105. Schreiber, G.A., Helle, N., Schulzki, G., Spiegelberg, A., Linke, B., Wagner, U. and B6gl, K.W. (1993b). Intercomparisons to evaluate the suitability of gaschromatographic, electron spin resonance spectrometric and thermoluminescence methods to detect irradiated foods in routine control. Radiat. Phys. Chem. 42, 391. Scotter, S.L., Holley, P. and Wood, R. (1990). Co-operative trial of methods of analysis to detect irradiation treatment of chicken samples. Int. J. Food Sci. Technol. 25, 512. Stachowicz, W., Strzelczak-Burlinska, G., Michalik, J., Wojtowicz, A., Dziedzic-Goclawska, A. and Ostrowski, K. (1992). Application of electron paramagnetic resonance (EPR) for control of irradiated food. J. Sci. Food Agric. 58, 407. Stevenson, M.H. (1992). Progress in the identification of irradiated foods. Trends Food Sci. Technol. 3, 257.
184 Stevenson, M.H. and Gray, R. (1989a). An investigation into the effect of sample preparation methods on the resulting ESR signal from irradiated chicken bone. J. Sci. Food Agric. 48, 261. Stevenson, M.H. and Gray, R. (1989b). The effect of irradiation dose, storage time and temperature on the ESR signal in irradiated chicken drum-sticks. J. Sci. Food Agric. 48, 269. Stevenson, M.H. and Gray, R. (1990). Can ESR spectroscopy be used to detect irradiated food? Pages 80 - 96 in: Food irradiation and the Chemist, Royal Society of Chemistry, Cambridge. Stewart, E.M. (1993). The use of ESR spectroscopy for the detection of irradiated Crustacea with particular reference to Nephrops norvegicus (Norway lobster). Ph.D. Thesis, The Queen's University of Belfast, N.Ireland. Stewart, E.M., Stevenson, M.H. and Gray, R. (1991). Use of ESR spectroscopy for the detection of irradiated whiting (Merlangiusmerlangus). J. Sci. Food Agric. 55, 653. Stewart, E.M., Stevenson, M.H. and Gray, R. (1992). Detection of irradiation in scampi tails effects of sample preparation, irradiation dose and storage on ESR response in the cuticle. Int. J. Food Sci. Technol. 27, 125. Stewart, E.M., Stevenson, M.H. and Gray, R. (1993a). The effect of irradiation dose and storage time on the ESR signal in the cuticle of different components of the exoskeleton of Norway lobster (Nephrops norvegicus). Appl. Radiat. Isot. 4_, 433. Stewart, E.M., Stevenson, M.H., Gray, R. and McMurray, C.H. (1993b). The effect of processing treatments on the radiation-induced ESR signal in the cuticle of irradiated Norway lobster (Nephropsnorvegicus). Radiat. Phys. Chem. 42, 367. Sultanov, K. (1991). Spectrum of native cellulose gamma-irradiated at 300K. Vysokomol. Soedin. Sev. B. 33, 392. Tjaberg, T.B., Underdal, B. and Lunde, G. (1972). The effect of ionising radiation on the microbiological content and volatile constituents of spices. J. Appl. Bact. 35, 473. -
Characterization of Food: Emerging Methods A.G. Gaonkar (Editor) 9 1995 Elsevier Science B.V. All rights reserved.
185
Chapter 8 Progress in A p p l i c a t i o n of N I R and F T - I R in Food C h a r a c t e r i z a t i o n Sumio Kawano National Food Research Institute, 2 - 1 - 2 Kannondai, Tsukuba 305, Japan.
1. INTRODUCTION More than 20 years ago K.H. Norris first introduced near infrared spectroscopy as a powerful technology in the field of composition analysis of cereals [1]. However, classical spectroscopists did not want to recognize its potential. This tendency still persists but there is no doubt that NIR is now an established technique for characterization of food and provides a convenient analytical tool for quality and process control. In the beginning, NIR was used for compositional analysis of only grains, beans and seeds which are relatively low moisture products. NIR is now being used for compositional analysis of variety of products including fruits and vegetables which are high moisture products. As the software and hardware of NIR improved, the scope of NIR application has been expanding to diversified fields such as textile, oil, pharmacy, and medical science. In this chapter, the principle of NIR will be explained, and new trends of R & D on NIR will be described. In addition, use of FT-IR in food characterization will be described briefly.
2. MID INFRARED (IR) AND NEAR INFRARED (NIR) SPECTROSCOPY 2.1. Principle of IR and NIR Spectroscopy The near infrared and mid infrared regions of the spectrum encompasses radiation with wavelength raging from about 800 to 2500 nm and from 2500 nm (4,000 cm -1) to 25,000 nm (400 cm-1), respectively. 1 Infrared light when absorbed increases the vibrational and rotational energy of the molecule. Fundamental vibration modes mainly consists of stretching vibration and deformation vibration. Infrared light is consumed as vibrating or rotating energy by excited molecules. In the case of molecules made up of two atoms, fundamental vibration is made only with the stretching vibration between two atoms. But in the case of a molecule having more than 2 atoms, the vibration becomes much more complicated because deformation vibration also occurs. Generally, in a non-linear molecule made up of N atoms, there are (3N-6) possible
1Spectroscopists conventionally describe the position of a mid-IR absorption band in terms of its wavenumber, but in the NIR literature the wavelength is used. The conversion from wavelength (nm) to wavenumber (cm -1) can be done by dividing 107 (nm/cm) by wavelength (nm).
186
Figure 1. Fundamental vibration modes of a water molecule. modes in the fundamental vibration. For example, a water molecule, consisting of three atoms, has three modes of fundamental vibration as shown in Figure.1. But, in the case of a molecule having a symmetric structure around itself, such as carbon tetrachloride, the dipole moment does not change and absorption does not occur. When absorption band caused by fundamental vibration occurs in the IR region, weak absorption bands also occur at frequencies (wavenumber) of almost the same as a integral multiple of fundamental vibration frequency (wavenumber). This is the so-called "absorption due to overtone", and its wavenumber V. can be obtained from the fundamental vibration wavenumber V 0 by using the following equation: V. = n Vo {1-(n+l)X}
(1)
where n is an integer, X is anharmonicity constants which is much less than 1. The absorption at the overtone is observed at a wavenumber of almost the same as a integral multiple of the fundamental vibration wavenumber. When absorption is caused by more than one fundamental vibration, the sum or difference of multiples of their fundamental vibration wavenumbers are observed. This is the so-called "absorption due to combination vibration", and the wavenumber Vr is obtained by using the following equation" V c - nlVl --- nzV2 -+
(2)
where nl, n 2, ... are integers, and V1, V 2 ... are wavenumbers of fundamental vibrations. As was stated previously, absorption in the NIR region always occurs due to the vibration caused by overtones or by combinations of fundamental vibrations in the infrared (IR) region. In particular, the absorption mainly occurs through functional groups that have a hydrogen atom such as O-H, N - H and C-H. Figure 2 shows the NIR spectra of soybeans and rice, including their main components such as water, protein, starch and oil. The more light absorbed, the higher the absorbance becomes. The absorption bands of each component depend on their specific functional groups. Absorption bands observed are due to their components in both rice and soybeans.
187
Figure 2. NIR absorption spectra of rice, soybeans and their main components. The absorption band at 1935 nm observed both in rice and in soybeans are mainly due to water. The band at 2100 nm, observed in rice, are mainly due to starch. This is not clearly observed in soybeans, which contain less starch. The absorption band of protein at 2180 nm and the band of oil at 2305 and 2345 nm are clearly observed in soybeans, which contain much proteins and oil. As described above, the NIR spectrum of food contains so much information due to more than one component that interesting information can be drawn and analyzed from these spectra by using statistical methods.
2.2. Multiple linear regression analysis To perform quantitative analyses of food using NIR, it is necessary to initially establish a calibration equation which relates spectral data to objective chemical data. Multiple linear regression (MLR) analysis is typically used to make a calibration equation using a calibration sample set analyzed accurately by a conventional chemical method. The general calibration equation can be written as: C (%) = K o + K I A 1 + K 2 a 2 + K 3 A 3 + ....
(3)
where A1, A2, A3 .. are optical data (say, absorbance) at voluntary wavelength, and Ko, K1, K2, K 3 .. are regression coefficients. All K values and wavelengths are calculated by a statistical method. In the case of protein determination of flour for example, the following calibration equation can be obtained using log(1/R2as0)(absorbance at 2180 nm), which is the key absorption band for the protein. Cp(%) = 12.68 + 493.7 log(1/R218o ) -323.1 log(1/i21oo ) - 243.4 log(1/i168o )
(4)
188 where, log(1/R21oo) is absorbance at the characteristic absorption wavelength of starch and works as an adjusting term that eliminates any influence caused by starch. Log(i/R1680) is the reference which eliminates any effects caused by the particle size of the sample. In the same manner, calibration equations for quantitative analyses of moisture, fat, carbohydrates, sugar, etc. can be established.
2.3. Principal component analysis (PCA) PCA is a statistical method that compress many correlated variables to one or a small number of non-correlated variables [2]. This method compresses the information observed in the NIR spectrum. A limitation of PCA is that it is often difficult to establish the correspondence between principal components obtained and concrete characteristics of samples. Figure 3 shows the application of the PCA to spectra of flours with different processing qualities. On a plane consisting of the first and the second principal components, the difference between each flour can be distinguished. The first principal component shows information related to sample particle size in this study.
Figure 3. Principal component analysis on NIR spectra of flours with different processing qualities for bread (v), chinese noodle(o), confectionery(,,) and Japanese noodle(m).
2.4. Discriminant analysis Discriminant analysis is used to classify each sample into groups according to the information represented by n independent variables. To perform this analysis, Mahalanobis' generalized distance [3] or multiple discriminant analysis [4] is usually used. The former method employs the principle that each sample belongs to a group in which the Mahalanobis' generalized distance is minimized, and the later method employs the principle that the each sample is classified according to the value of the linear combination that is made in such a way that between-groups sum of squares is maximized with respect to the overall sum of squares. Figure 4 shows an example of the classification of flour into three groups based on their quality for bread making [4]. In this example, the multiple discriminant analysis was used on the basis of the score obtained by PCA.
189
2.5. Principal component regression (PCR) and partial least squares regression (PLSR) Wavelength selection is necessary to make a calibration equation using MLR. This is a time consuming and tedious work. Less time intensive methods such as principal component regression (PCR) and partial least squares regression (PLSR), which use full spectrum, have been developed. PCA is performed on the original spectral data, and then the reference data are related to the first few principal components using MLR. PLSR is similar in many respect to PCR. A small number of factors are constructed as linear combinations of the original spectral data, and then calibration equation based on the factor scores is made. Use of PCR and PLSR is expanding at present. The detailed description of both methods can be found elsewhere [2].
Figure 4. Multiple discriminant analysis for bread making quality of wheat flour.
3. A P P L I C A T I O N S OF NIR IN F O O D Many results have been reported since NIR has been applied as nondestructive method for food quality evaluation. Table 1 shows major applications of NIR to food. In the beginning, NIR was used for the analysis of grain such as wheat, soybean and rice. More recently, however, the method has been applied to wide areas including processed food, beverages, fruits and vegetables. The following examples are selected to demonstrate that NIR is a useful tool in characterization of a wide variety of foods.
190 Table 1 Major applications of NIR to food Products
Characterization of foods
Barley protein, moisture, lysine, amino acid, 13-glucan Buckwheat protein, moisture, ash Cotton seed gossypol, moisture, glucose, fructose Green tea protein, moisture, total nitrogen, caffeine, theanine, total free amino acid Hop moisture, o~-acid, essential oil Rapeseed fat, chlorophyll Pea protein, starch Red pepper capsaicin Rice starch(amylose), protein, moisture, ash, amino acid, taste value Soy bean protein, moisture, fat, 7S, llS Sunflower seed fat, moisture, fiber Wheat starch, protein, moisture, ash, hardness, damaged starch, o~-amylase activity, amino acid, color value, ratio of contaminated bran, bread making quality, discrimination of cultivar Cheese fat, protein, solid content, moisture Milk moisture, fat, protein, lactose, TMS, casein Whey moisture, fat, protein, lactose <Meat> Fish meat water state Meat (product) protein, moisture, fat, salt, calorie Beer alcohol Corn syrup fructose, solid content Fruit juice glucose, fructose, sucrose Sake alcohol, acidity, amino acid, total sugar Soya milk protein, moisture Wine alcohol, extract content, sugar, titration acidity Bread protein, moisture, fat Biscuit(dough) fat, sucrose, flour, moisture Cereal product fiber, gelatinization degree Cocoa protein, fat, starch Edible oil iodine number Soy sauce salt, nitrogen, alcohol, lactic acid, glutamic acid, glucose Apples Brix, titration acidity Cantaloupe soluble solids Onion Brix, moisture, dry matter Oranges Brix Peaches Brix Sugarcane crude fiber, moisture, Brix
191
3.1. State of water in foods It is well known that NIR is an excellent tool for analyzing total amount of water in foods [5]. NIR also has the potential to determine the state of water in foods as well [6]. The state of water has been the subject of numerous investigations and of much controversy for many years. Several hypotheses of structural models have been proposed in order to explain the behavior of water. A mixture model where water is postulated to consist of an equilibrium mixture of molecular species with different numbers of hydrogen bonds per water molecule is most popular [7]. In this model, water is assumed to be composed of different molecular species such as free water molecules (So), molecules with one OH group engaged in hydrogen bond ($1) and molecules with two OH groups engaged in hydrogen bond ($2). In the second derivative spectrum of water shown in Figure 5, three absorption bands which are associated with So, $1 and S 2 species, respectively were observed [6]. As temperature decreases, absorbance of S o decreases, while absorbance of S 1 and S 2 increases.
Figure 5. Second derivative spectra of water at different temperatures in the range from -15 to +30 ~
3.2. Effect of secondary structure of protein Absorbance of peptide bonds at 2170 nm is usually used as a key band for the calibration of protein content measurement. However, the intensity of the absorption depends on the structure and conformation of the protein. Yamashita et al [8] investigated the change in absorption at 2170 nm caused by the conformational change of protein using bovine serum albumin (BSA) as the model protein. A mixture of dithiothreitol (a reducing agent) and BSA (5%) was taken in a quartz cell attached to the NIR instrument, and then spectra were recorded at 10-minute intervals. Dithiothreitol reduced the disulfide bond in BSA. The
192 absorption at 2170 nm became weaker with the elapse of reaction time, suggesting that the absorption is affected by the reduction of the disulfide bond in the protein. The extent of the contribution of the secondary structure to the absorption around 2170 nm was also investigated using nine different proteins, namely, lysozyme, myoglobin, cytochrome C, ribonuclease, chymotrypsin, subtilisin, trypsin, pepsin and ovalbumin. The secondary structure of these molecules consists of c~-helix, [A-sheet and random coil structures. It was found that the relative extent of the a-helix, [A-sheet and random coil structures to the absorption was approximately 2:1:1.
3.3. On-line monitoring system for glucose in starch hydrolysis In order to control the enzymatic starch hydrolysis, it is necessary to make a real-time measurement of the concentration of substrate and products in the reactor. An on-line glucose monitoring system has been developed for this purpose [9]. The starch suspension was maintained with glucoamylase at 40~ in a reactor, and then the solution was circulated from the reactor to a NIR flow-through cell. NIR measurement was made at the wavelengths varying from 1000 to 2500 nm. The glucose concentration was analyzed by high performance liquid chromatography (HPLC). As a result of MLR based on the spectral data and the chemical data, accurate calibration equation could be obtained which consisted of log(l/R) values at two wavelengths of 2008 and 2148 nm. The absorption at 2008 nm was assigned to 2 x OH def. + CO def., and the absorption at 2148 nm was postulated to be CH str. + CO str. The standard error of prediction (SEP) was 0.077 % when glucose concentration ranged from approximately 1.5 to 5.5 %. 3.4. Development of the sensor for determining iodine number of fats and oils Iodine number as well as melting point is one of the most important factors for quality control of processed fats and oils. However, a time-consuming chemical analysis is generally used for determining iodine number which sometimes stops production. In order to overcome this problem, an on-line NIR sensor has been developed [10]. Transmittance spectra of many kinds of oils were measured in the wavelength region from 1100 nm to 2500 nm to make a calibration equation. Significant changes in absorption were observed at 1,720 nm (-CH2-) and 2,140 nm (-CH= CH-) as iodine number changed. These are absorption bands of oil. In the case of calibration for rapeseed, correlation coefficient (R) and standard error of calibration (SEC) were 0.9993 and 0.476, respectively. However, the selected wavelengths did not appear in the report. As a result, it was found that NIR had the same accuracy as the conventional method when using individual calibration for each variety. To design an on-line NIR sensor, the effects of sample temperature, moisture, air bubbles, and shape of flow cell were simulated. Based on the these results, an on-line sensing system has been developed. 3.5. Development of an automatic sorter for removing mould infected nuts An automatic sorter for removing mould infected nuts has been developed [8]. Transmittance through an individual nut was measured in the wavelength region from 500 to 1500 nm to detect peanuts internally infected by mould (Figure 6). It was found that the ratio of transmittance at 700 and 1100 nm (T7~/Tl100) is related to the degree of mould infection. That is, the ratio decreases with the degree of mould infection. A commercial nut sorter is now available which has a sorting rate of 100 k~hr.
193
Figure 6. NIR spectra of good nuts and moldy nuts; T7oo/TlloO is related to degree of mould infection.
3.6. Determination of soluble solids in some fruits NIR has been applied for determining dry matter of onion [11], soluble solids of cantaloupe [12], and sugar contents of peach [13] and Satsuma mandarin [14]. Kawano et al [13] applied the intcractance method using fiber optics, which is a type of reflectance method, for the determination of sugar content in intact peaches. A commercially available "Interactance Probe (NIR systems Inc.)", having a concentric outer ring illuminator and an inner ring receptor, was used as the fiber optics. A cushion made of urethane foam was pasted onto the end of the probe to hold a sample. The NIR measurement is made by placing a sample at the end of a probe. Using MLR based on NIR spectral data and chemical data of Brix value, good correlations between NIR estimated Brix value and actual Brix value were obtained. The highest multiple correlation coefficient (R) was 0.97 with a standard error of calibration (SEC) of 0.48 ~ The standard error of prediction (SEP) and bias were 0.50 ~ and 0.01 ~ respectively. The authors concluded that this method has an acceptable accuracy for measuring sugar content of peaches. It is very difficult, however, to determine the composition in fruits such as Satsuma mandarins which have a thick peel using this method. Therefore, an NIR transmittance method was used [14]. A schematic diagram for sample placement is shown in Figure 7. The top of the sample was illuminated by monochromatic light using fiber optics, and the amount of light transmitted through the sample was measured by a silicon detector located just below the sample. As a result of MLR based on normalized NIR spectral data and chemically determined data of Brix value, good correlations between NIR values and actual values were obtained. NIR spectra were normalized not to be affected by fruit size. The best multiple correlation coefficient was 0.99 with a SEC of 0.28 ~ The NIR Brix value calculated with the best calibration equation using a prediction sample set agrees well with the actual Brix value. It was concluded that the NIR transmittance method yields an accurate estimate of Brix value in intact Satsuma oranges.
194
Figure 7. Schematic diagram of sample placement.
3.7. Rice taste analyzer Rice has big differences in taste between varieties. Therefore, different varieties of rice are blended in the milling plant to provide rice with a desired taste. Since the blending ratio is decided on the basis of sensory evaluation by a few experts, blending can not be conducted automatically. It is well known that rice taste is a function of chemical constituents such as protein, moisture, amylose, fatty acid, and minerals. It is impractical to use the results of timeconsuming chemical analyses to control the blending process. In order to overcome this problem, rice taste analyzer based on NIR principles was developed several years ago [15]. At present, there are five different types of analyzers, as shown in Table 2, which are commercially available. A total of more than 300 analyzers are being used in the milling plants as well as experimental stations at present. Table 2 Commercially available rice taste analyzers using NIR technique. Company
Factors
Treatment Sample
Measurement
Satake
Moisture, Protein, Amylose, Fatty acid Mg, K, N, Amylose
Ground
Milled rice
R*
Ground or Whole Ground
Milled rice or Brown rice Milled rice
R or T R
Ground
Milled rice
R
Whole
Milled rice
T
Nireco
Shizuoka Seiki Yamamoto Kubota
R :reflectance,
Protein, Iodine blue value Moisture, Protein, Stickiness Moisture, Protein, Amylose T :transmittance
195 The rice taste analyzer, developed first by Satake Engineering Co., Ltd., consists of NIR instrument provided by Bran & Luebbe Company. This analyzer is based on the experimental result that rice taste is fixed by the balance of moisture, protein, amylose, and fatty acid. From a practical stand point, milled rice is ground, and the ground sample is kept at a constant temperature oven for more than one hour, after which the NIR measurement is performed to determine the amount of different constituents. From these constituents, taste scores can be calculated using taste-related equation which relates the constituents to taste score. A taste score can be generated in only a few minutes by the NIR instrument. The rice taste analyzer includes the software that calculates the blending ratio to perform lowest price at the same taste, or to perform best taste at the same price.
3.8. Fruit sweetness sorting machine In 1989, Mitsui Mining and Smelting Co., Ltd. developed and introduced the first operational nondestructive automatic peach sweetness grading machine. Peaches graded by this system were differentiated as "sweetness guaranteed". The company has now developed the Multi-Purpose Sensor (MPS) for grading apples and Japanese pears, in addition to peaches, with a single unit. Fruits on a line-up conveyor are illuminated by two focused tungsten halogen lamps, and the scattered reflected radiation is measured by the MPS unit. The reflected radiation is converged by a lens and projected on a spectroscope to extract required wavelength and intensity data. The intensity of the radiation at respective wavelengths is measured by the line sensor (Figure 8). The sugar content of a peach is calculated from the measured reflection intensity of NIR radiation by using a statistically developed calibration equation. The calculation requires only 0.13 sec. The sorting rate is 3 fruits/sec/lane. If the MPS system is introduced to a packing house, taste-oriented grading will become possible. If the quality data from the MPS system are correlated with data corresponding to cultivation conditions such as soil and weather, technical guidance for the production of highquality products will also be realized.
Figure 8. Multi-purpose sensor for determining sweetness of peaches, apples and Japanese pears.
196 3.9. Automatic composition analyzer of soy sauce Kikkoman Corp., a soy sauce making company, developed an automatic chemical composition analyzer of soy sauce (Figure 9) [16]. The analyzer consists of an InfraAlyzer 400 or InfraAlyzer 500, a temperature controller, an automatic sampler and pumps. A certain amount of soy sauce collected by the automatic sampler is sent to the NIR analyzer at a constant flow rate by a pump through a temperature controller at 20~ NIR measurement is made automatically. After the NIR measurement, the sample cell and tube are washed with cleansing liquid. It takes about 3 minutes to analyze one sample including washing process. Certain amounts of soy sauce from different fermentation vessels are blended in the bottling process to maintain the favorite quality of soy sauce. Raw soy sauce in each fermentation vessel has different chemical compositions. Up to now, the blending ratio was decided on the basis of results of time-consuming chemical analyses of each lot of the soy sauce. At present, however, chemical compositions are analyzed automatically using the NIR.
Figure 9. Automatic chemical composition analyzer of soy sauce.
4. F F - I R S P E C T R O S C O P Y In FT-IR spectroscopy, a pattern known as an interferogram is obtained in place of the normal spectrum. The interferogram is the Fourier transform of the normal spectrum. Therefore, the normal spectrum can be obtained by transforming the interferogram. The advantages of the FT-IR are simultaneous spectral acquisition and high signal to noise ratio. A detailed description of FT-IR can be found elsewhere [17]. IR spectra have many sharp absorption bands corresponding to fundamental vibrational transitions of different functional groups, the positions of which are influenced by the chemical environment of the group. Therefore, IR spectroscopy is well suited for the structural elucidation and identification of organic compounds. However, the use of IR spectra is limited for the analysis of food because sample preparation is more complicated in comparison with NIR. Dehydration, homogenization, dissolution or dispersion of samples is necessary. In addition, to make an IR transmission spectra of liquid samples, it is necessary to use a cuvette with very narrow path length of 0.001 - 0.1 mm because the absorptivities are very high, which causes a sample loading
197 problem. In order to overcome theses disadvantages, Attenuated Total Reflectance (ATR) and dry extract techniques are now used for spectra acquisition of food. Recent applications of FT-IR in food are shown in Table 3.
Table 3 Recent applications of FT-IR in food Products
Characterization of foods
Method
Ref.
Soybean Carbonated beverage Liquid egg Sugar cane juice Fruit juices Milk
fat,protein, carbohydrates sugar, carbon dioxide protein, total lipid, total solid sucrose sucrose, glucose, fructose, citric acid, malic acid water
Syrup
glucose
IR, ATR [18] IR, ATR [19] IR [20] FT-IR, ATR [21] FT-IR, dry extract [22] FT-IR, capillary cell [23] FT-IR, ATR [24]
4.1. Quantitative determination of sugar cane sucrose FT-IR has been applied for determining the sucrose content of sugar cane .juice [21]. In place of the more familiar transmission cell, an attenuated total reflectance (ATR) cell and clarified sugar cane juice were used to record FT-IR spectra from 800 to 1250 cm -1. In the spectra, significant wavenumbers (927.59, 997.02, 1054.87, 1116.51, and 1137.80 cm -1) have been identified for sucrose. The application of PCR has been proposed for the development of a calibration equation for sucrose content. PCR is basically a MLR applied to scores assessed by PCA. On the basis of FT-IR spectra and sucrose content, an accurate calibration equation could be obtained by the application of PCR. The root mean square difference between predicted FT-IR values and the actual values were 0.12 % (w/v) with a bias of-0.03 % (w/v). The accuracy of FT-IR for determining sugar cane sucrose is almost equal to that of NIR [25]. 4.2. Determination of sugars and organic acids in fruit juices The high intensity and thc widcncss of the water absorption bands in thc IR spcctrum are important obstacles to ovcrcomc to achieve accuratc and scnsitivc quantitative analysis by FT-IR. To avoid the problem of water, a dry extract systcm, similar to that used for NIR analyscs by FT-IR has bccn applicd for the dctcrmination of sugars and organic acids in fruit juices [22]. This measuring method has advantages such as speed of FT-IR, elimination of solvents and better peak resolution. The dry extract system is based on the use of silicon windows covered with a thin layer of CaF2 powder. The liquid sample is spotted on the CaF 2 layer and dried in a microwave oven. MLR, PCR and PLSR were used to develop calibration equations for determining sugars and organic acids in fruit juices. The best results for the dry extract system were obtained with the PLS program. The standard errors of prediction
198 obtained by the PLS regression are : 1.63 g/L for the sucrose; 1.19 g/L for the glucose; 1.27 g/L for the fructose; 0.213 g/L for the citric acid; 0.025 g ~ for the malic acid. The results predicted by this method are comparable to the equivalent results achieved by HPLC.
5. CONCLUSION As described above, NIR is a promising method for food analyses and provides a convenient analytical tool for quality and process control. It is still important to expand applications of NIR and FT-IR to new sorts of food as well as constituents. However, research and development should produce not only simple application as substitute methods for time-consuming conventional chemical analyses, but also more sophisticated applications in order to utilize the ability of NIR and FT-IR to the fullest extent.
REFERENCES
1. B.G. Osbome and T. Fearn, Near Infrared Spectroscopy in Food Analysis, Logman Scientific & Technical, Essex, England, UK, (1986). 2. H. Martens and T. Naes, Multivariate Calibration, John Wiley & Sons, Chichester, 1989. 3. H.L. Mark and D. Tunnell, Analytical Chemistry, 57 (1985) 1449. 4. M.F. Devaux, D. Bertrand and G. Martin, Cereal Chem. 63 (1986) 151. 5. P. Williams and K. Norris (eds.), Near-Infrared Technology in the Agricultural and Food Industries, AACC, USA, 1987. 6. M. Iwamoto, K. Nishinari, N. lshida and H. Watanabe, The first Intemational NIR Spectroscopy Conference, Norwich, UK, (1989). 7. G. Nemethy and H.A. Sheraga, J. Chem. Phys. 36 (1962) 3382. 8. H.K. Yamashita, M. Tatara, H. Takamura and T. Matoba, Nippon Shokuhin Kogyo Gakkaishi 41 (1994) 65. 9. K. Nishinari, R.K. Cho and M. Iwamoto, Starch 41 (1989) 110. 10. On-line Sensor R & D Association (ed.), Food Industries and Sensor (in Japanese), Korin Publishers, Tokyo, 1991. 11. G.S. Birth, G.G. Dull, W.T. Renfroe and S.J. Kays, J. Amer. Soc. Hort. Sci. 110 (1985) 297. 12. G.G. Dull, G.S. Birth, D.A. Smittle and R.G. Leffler, J. Food Sci. 54 (1989) 393. 13. S. Kawano, H. Watanabe and M. Iwamoto, J. Japan. Soc. Hort. Sci. 61 (1992) 445. 14. S. Kawano, T. Fujiwara and M. Iwamoto, J. Japan. Soc. Hort. Sci. 62 (1993) 465. 15. Y. Hosaka, The proceedings of International Symposium on Agricultural Mechanization and International Cooperation in High Technology Era, Tokyo, 1987. 16. K. Kobayashi, K. Iizuka, T. Okada and H. Hashimoto, Proc. of the 2nd International NIRS Conference, Korin Publishing, Tokyo, Japan, (1990) 178. 17. P.R. Griffiths, J.A. de Haseth, Fourier Transform Infrared Spectroscopy, John Wiley & Sons, 1986. 18. J.M. Wilson, A. Kramer and I. Ben-Gera, J. Food Sci. 38 (1973) 14. 19. M.S. Frant, G. LaButti, Anal. Chem. 52 (1980) 1331A.
199 20. B.G. Osborne, G.M. Barrett, J. Food Technol. 19 (1984) 349. 21. F. Cadet, D. Bertrand, P. Robert, J. Maillot, J. Dieudonne and C. Rouch, Applied Spectroscopy 45 (1991) 166 . . . . . . 22. N. Dupuy, M. Meurens, B. Sombert, P. Legrand and J.P.Huvenne, Applied Spectroscopy 46 (1992) 860. 23. E. Hop, H.-J. Luinge and H.Van Hemert, Applied Spectroscopy 47 (1993) 1180. 24. F.De Lene Mirouze, J.C. Boulou, N. Dupuy, M. Meurens, J.P. Hevenne and P. Legrand, Applied Spectroscopy 47 (1993) 1187. 25. S. Kawano, K. Takehara, T. Sato and M.Iwamoto, The proceeding of the third international conference on Near Infrared Spectroscopy, Agricultural Research Centre Publishing, Gembloux (Belgium), (1991) 510.
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Characterization of Food: Emerging Methods A.G. Gaonkar (Editor) 9 1995 Elsevier Science B.V. All rights reserved.
201
Chapter 9 D e v e l o p m e n t s i n t h e A p p l i c a t i o n of S m a l l - a n g l e N e u t r o n S c a t t e r i n g to Food Systems P h i l i p H. S t o t h a r t 33, Betchworth Avenue, Earley, Reading, Berkshire RG6 2RH, United Kingdom
1. I N T R O D U C T I O N Small-angle neutron scattering (SANS) can be applied to food systems to obtain information on intra- and inter-particle structure, on a length scale of typically 10-1000 A. The systems studied are usually disordered, and so only a limited number of parameters can be determined. Some model systems (e.g., certain microemulsions) are characterized by only a limited number of parameters, and so SANS can describe them fully without complementary techniques. Food systems, however, are often disordered, polydisperse and complex. For these systems, SANS is rarely used alone. Instead, it is used to study systems that have already been well characterized by other methods, viz., light scattering, electron microscopy, NMR, fluorescence, etc. SANS data can then be used to test alternative models, or to derive quantitative parameters for an existing qualitative model. SANS and SAXS (Small-angle X-ray scattering) are similar techniques in principle, and complementary in practice. Both are able to study systems in their native, solution state. However, SANS has some advantages for food and colloidal systems. In SANS, the scattering of deuterium is large and opposite in phase to that of hydrogen. Hence food systems can be studied against aqueous backgrounds of different contrast by replacing H20 in the sample by D20. This method of "contrast variation" (see below) is much more effective for SANS than for SAXS. The largest wavelengths usable for SANS and SAXS are >10 A and about 1.5 respectively. The longer wavelength of SANS makes it more effective than SAXS for the study of the large scale colloidal structures found in food systems. SANS on disordered food systems is quite different from X-ray crystallography of protein crystals. The former studies systems in their native solution state, with information limited by various averaging effects. The latter can obtain very detailed information on single proteins in a highly ordered crystal, which is a non-native state.
202 2. N E U T R O N S O U R C E S Traditional neutron sources for SANS have been nuclear reactors, such as the High Flux Reactor at Institut Laue-Langevin (ILL), Grenoble. As a neutron source, it is necessary t h a t a reactor should have a high neutron flux, not necessarily a high power. More recently, pulsed neutron sources have been developed, such as ISIS, at Rutherford-Appleton Laboratory, UK. These sources use pulses of high energy protons to smash into a u r a n i u m target, releasing pulses of neutrons with a wide energy range. Neutron wavelength ~. is related to velocity by the de Broglie relationship ~.=h/p where p = m o m e n t u m = m v Neutrons with a wavelength of 5 to 10/~ are often used. A narrow range of wavelengths is usually selected to give a quasi-monochromatic beam. This can be achieved by a crystal monochromator or, more usually, by a velocity-chopper system of rotating discs which select neutrons of a given velocity. Pulsed sources may use a wide range of neutron wavelengths in the beam incident on the sample. The velocity of individual scattered neutrons is determined by the detector electronics, from the time delay between the burst of neutrons leaving the u r a n i u m target, and the detection of the scattered neutron by the detector.
3. T H E O R Y
There are a number of reviews of SANS as applied to biological systems (Jacrot,1976; Kneale et a1,1977). The SANS method depends on the interference effects shown by the scattered neutrons, arising from their wave-like properties. Neutrons are scattered by their interaction with nuclei. Only part of scattering, the coherent scattering, contributes to the interference process. Another part, the incoherent scattering, contains no structure information, but adds to the background intensity. For atoms of biological or food interest, only hydrogen has a significant level of incoherent scatter. A third component of scattering, the inelastic scattering, is negligible for scattering from macromolecules. The coherent scattered amplitude F(r) of neutrons, wavelength ~., at a displacement r from a nucleus j of coherent scattering amplitude (or scattering length) b~ is F(k,r) = -b~exp(ik.r)
(1)
203 where the wavevector k has magnitude I k l =21-I/~ The total intensity I(Q) comes from summing for interference over all pairs of atoms m,n I(Q) = z Znbmb"exp[iQ.(rm-r )]
(2)
where I QI = 41-IsinO/k and 20 is the angle between incident and scattered beams. If the scattering in a particular direction is regarded as having been "reflected" from an imaginary plane in the sample, then the direction of Q is perpendicular to this plane. In a disordered sample, there are in fact no actual reflecting planes. However, the scattering is produced by fluctuations in scattering density perpendicular to these planes and so the idea of reflecting planes is useful in visualizing the sample. In X-ray scattering, the scattering amplitudes of atoms increase steeply with increasing atomic number, so the scattering from a sample may be dominated by a small percentage of heavy atoms. For many samples, hydrogen makes negligible contribution to SAXS even when present in large molar fractions. In SANS, by contrast, the magnitudes of the scattering lengths of C,O,N,P,H,D are similar (within a factor of 2). Hence SANS can give information on the distribution of hydrogen and water in food samples, which is significant advance over SAXS for food systems. The phase of the scattering from H is opposite to that from the other atoms, so the range of scattering densities of H20/D20 mixtures spans the range of scattering densities of nearly all food materials. This is utilized in the "contrast variation" technique (Stuhrmann,1973,1974; Jacrot,1976). The solvent scattering density Pso~ is varied by varying the H20/D20 ratio in the solvent. Hence the "contrast" (P,m-Pso~)can be varied over a range of positive or negative values, where P~m is the scattering density of a sample particle. If the scattering density varies over the volume of the particle, then different parts of the particle can be "blanked out" by matching P~o~to that part of the particle. At the match point, P~o~ equals the value of P,m averaged over the sample particle. At small Q, the scattering from a dilute solution of monodisperse particles becomes I(Q) --- I(0)exp[-(RgQ)2)/3]
(3)
where I(0) = (Zibi - Oso~V)2
(4)
204 where R~ is the radius of gyration (Guinier and Fournet,1955) and the sum Zi is over all atoms in the particle of volume V. A plot of ln(I) -> 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~ <1.0. For highly anisometric particles, QR~ must be smaller. A plot of lnI(Q) -> 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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Characterization of Food: Emerging Methods A.G. Gaonkar (Editor) 9 1995 Elsevier Science B.V. All rights reserved.
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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 <<1, and (3) the slip velocity V s is independent of tube length. Mooney obtained the following relationship: 32(3_ ~; D 3
8Vs + D
4 ~w 3
to
/
J
where, Q = Volume flow rate D = pipe diameter x w = wall shear stress y = Apparent shear rate
y~2d~
(2)
286 For a given value of the wall shear stress, a plot of 32Q/rID 3 against 1/D should give a straight line of slope 8V s. The value of V s can be used to obtain the contribution of slip flow, Qs, to the total flow rate Q; Qs
-
rtD2Vs
(3/
and the slip-free apparent shear rate, ~,ns q'ns
= 32 (Q-Qs) rt D 3
(4)
Tube flow data for aqueous solutions of polyacrylamide (Separan AP-30) and carboxymethylcellulose (8,21) provide confirmation for all of Mooney's assumptions. The slip velocity was found to be invariant with tube length and was a function of the wall shear stress only, increasing with increase in the wall shear stress. Estimating wall-slip is by no means an easy task, as recent data suggests that contrary to Mooney's assumptions, slip velocity can vary with tube length as well as with time. Using xanthan gum solutions, Perez-Gonzalez and coworkers (27) showed that the xanthan slip velocity increased (Fig. 5) with an increase in the ratio of capillary length to diameter at a given stress indicating a slip velocity that varied over
Figure 5.
Variation of Xanthan slip velocity with tube length in capillary flows (Reproduced with permission, ref. 27).
287 the tube length. For a highly filled polymeric suspension Aral and coworkers (17) found the slip velocity to change with time before attaining a steady value (Fig. 6). Time-dependent slip has been found also with mayonnaises (15,28). For the latter food product slip effects were found to dominate its low shear rate behavior, but disappear beyond some high stress value.
Figure6.
Time dependence of slip velocity of a concentrated (Reproduced with permission, ref. 17).
suspension
Mooney analysis is not the only technique for detecting and estimating wall-slip in tube flow. The Bagley pressure drop method can also be applied to detect the presence of slip as well as the variation of slip velocity with tube length (8,29). The Bagley correction is applied to detect the linearity of capillary flow pressures plotted as a function of capillary length to diameter, L/D. A non-linear plot generally indicates the presence of slip, shown schematically in Fig. 7. However, a non-linear Bagley plot is not necessarily a confirmation for wall-slip effects, as other factors, for example, temperature rise, material degradation etc. could also give rise to a non-linear plot. Further confirmatory tests using different test geometry, parallel plates, concentric cylinders, should also be carried out. The Bagley pressure drop method was used by Smith et al (30) to determine flow properties of cheeses. Based
288
on the type of Bagley plot, linear or non-linear, obtained for a variety of cheeses, it was concluded that slip effects were dominant for melted Cheddar and process
Figure 7. Detection of slip from Bagley plots, 1: No slip, 2: Slip effects present. cheeses, but absent for Mozzarella cheeses. The author's own unpublished work on the capillary viscometry of cheeses has also shown that steady state flow conditions were not obtained with some process cheeses and that the transient viscosity varied with changes in the capillary diameter. The results for polymeric suspensions, mayonnaises, cheeses and gums indicate that apparent slip could be an evolutionary process. A general conclusion that can be made after analyzing the flow behavior of these diverse materials is that wall-slip is affected by pressure. The onset of slip occurs at a certain applied stress (slip velocity must be zero at zero flow) and slip effects are lost above a certain higher stress. It is possible that the onset and the disappearance stress levels for wall-slip could be material specific for a given flow surface conditions. However, little work has been reported using wall-slip as a material characterization parameter.
2.2.1.3. Elimination of slip occurrence The approach of eliminating wall-slip effects has received a lot of attention. The assumption is made that slip occurs at smooth walls but not at rough walls. Experimentally, wall roughening has been achieved by sandblasting, but there is no certainty that the slip effect is entirely eliminated by this process. Thus, Jiang (31) has shown that for polymer gels, slip effects are reduced at rough surfaces but are not totally eliminated. Similar results were obtained by Plucinski et al (28) with mayonnaises who found that with rough surfaces onset of slip effects were delayed but not eliminated. The use of an adhesive to 'glue' the test sample to a solid boundary has been
289 another commonly employed technique in an attempt to eliminate slip. Navickis and Bagley (32) used a cyanoacrylate ester adhesive to glue a dispersion of wheat starch granules in a compressed flow system. Magnin and Piau (16) have used the 'Primaire MB' adhesive to attach the fluid component of a silicone grease prior to testing the lubricant in a cone and plate rheometer. The effectiveness of the adhesive in preventing slip was confirmed by visual monitoring using the marker line technique. The use of a rotating vane has become very popular as a simple to use technique that allows slip to be overcome (33,34). Alderman et al (35) used the vane method to determine the yield stress, yield strain and shear modulus of bentonite gels. In the latter work it is interesting to note that a typical torque/time plot exhibits a maximum torque (related to yield stress of the sample) after which the torque is observed to decrease with time. The fall in torque beyond the maximum point was described loosely as being a transition from a gel-like to a fluid-like behavior. However, it may also be caused by the development of a slip surface within the bulk material. Indeed, by the use of the marker line technique, Plucinski et al (15) found that in parallel plate fixtures and in slow steady shear motion, the onset of slip in mayonnaises coincided with the onset of decrease in torque (Fig. 8). These authors found slip to be present for
Figure 8. Single and double slip in mayonnaises (ref. 15). mayonnaises in all the commonly used rheometry fixtures including the cone and plate and concentric cylinders. The torque/time plot for mayonnaises were characteristic for concentrated suspensions exhibiting yield stresses. The above
290 described results (17, 27, 15) strengthen the hypothesis that the observed material yielding could be a consequence of the onset of slip and that slip velocity may be a time-dependent property. As yield stress is a very important rheological property of many foods, the slip phenomenon has many important textural and practical processing implications for food products.
2.3. Summary There are at least two aspects of slip that are of fundamental importance to food rheology as they provide important processing and textural information for foodstuffs. These are: (1) characterization of slip effects, i.e., the stresses that determine the onset and disappearance of wall-slip and the variation of slip velocity with time, and (2) control of slip effects in foods through manipulation of formulation and/or processing conditions. To date, most wall-slip studies have concentrated on either eliminating or quantifying slip effects in laboratory rheometry. Slip effects have been traditionally treated as flow anomalies, which make the measurement of material properties a challenging task. There is less evidence on how to utilize this flow anomaly to industry's advantage. A comprehensive understanding of the slip phenomenon as it relates to food texture and food processing is of crucial importance to the food industry, where the goal is to produce quality products at reduced cost.
3.0. E X T E N S I O N A L F L O W Just as the resistance to flow in a shear field is measured through shear viscosity, the resistance to being pulled into threads is measured through extensional viscosity. When working with liquids that are made up of small molecules, measures of extensional viscosity can be obtained from the shear viscosity data. However, for macromolecular solutions or polymer melts, stretching properties cannot be reliably obtained from the shear viscosity data. The extensional viscosity can be several orders of magnitude larger than the shear viscosity and the strain rate dependence could be drastically different from that of shear viscosity. Indeed, examples are given in the literature where a shear thinning fluid was found to be tension thickening in extensional flow fields (5, 36). The importance of polymer melt elongation has long been recognized in the polymer processing industries where measures of both uni- and biaxial extensional viscosities are obtained on a routine basis for defining melt spinning, film and bottle blowing and in the biaxial stretching of extruded sheets. In this overview, discussion is restricted to only uni- and biaxial extensional properties.
291 3.1. Basic definitions 3.1.1. Simple extension Also called uniaxial extension, in this type of deformation a rod-shaped sample is pulled in one direction keeping the other end fixed (Fig. 9a). The infinitesimal extensional strain, dE, can be expressed as de = dL/L o, L o = Initial length of sample. By integration, the strain for finite deformation then becomes, e = In (L/L o) This is also known as the Hencky Strain. The strain rate, E, is given by, = de/dt = 1.
d__L
L
(5)
dt
As dL/dt is the velocity, V, =V/L Since the flow is axially symmetric, the stress is also symmetric and analogous to shear viscosity, the extensional viscosity can be defined as
T~e
"Cll - "c22
=
E
=
"Cll - ~33 E
(6)
For a Newtonian fluid, 11e = 311
3.1.2. Biaxial extension For a sample in the shape of a circular disk (Fig. 9b) having a radius R, the biaxial strain rate, E b is the following: a = T
dR " T
(7)
292 The biaxial extensional viscosity, rl b is qb
=
I;11
-
I;22
s
For Newtonian fluid, rl b = 611
Figure 9. a) Simple uniaxial extension, b) Biaxial extension.
3.2. Measurement techniques There is a growing awareness in the field of rheology that the extensional viscosity of polymeric systems are important not only for polymer melts, but also for polymer solutions. The data are required as much for determining the flow characteristics as for polymer characterization purposes. Many papers presented at the 2nd International Meeting on Extensional Rheology, St. Andrews, Scotland (1994), emphasized experimental data that verified the greater sensitivity of extensional rheology to molecular structure for a variety of macromolecular substances. Clearly, it is important to ensure that the data generated are accurate. Ensuring accuracy in experimental data has been a true challenge for measuring extensional viscosity, as it is not easy to create the extensional flow field such that equilibrium conditions are attained. To reach steady state, the residence time of the fluid in a constant stretch rate needs to be sufficiently long. For some polymer melts, this has been attained; however, for polymer solutions this has proved to be a real challenge. It was not until the results of a world wide round robin test using the same polymer solution, code named M1, became available that the difficulties in attaining steady state in most extensional rheometers became clearer. The fluid M1 consisted of a 0.244% polyisobutylene in a mixed solvent consisting of 7% kerosene in polybutene. The viscosity varied over a couple of decades on a logarithmic scale depending on the instrument used. The data analysis showed the cause to be different residence times in the extensional flow field
(8)
293 as well as the lack of pure extension in the flow field studied. The evolution of our understanding of the challenges in measuring the extensional viscosity of polymeric substances is now very well documented (37, 38 and references therein). A comparative assessment of the extensional rheometers currently available has been compiled by James and Waiters (38). The lessons learnt from the M1 program has a bearing on food products testing. Hence, a brief discussion of the M1 program is given before discussing the applications of extensional rheometry to food products.
3.2.1. The M1 program The techniques used in the M1 project were those of fibre spinning, open siphon, filament stretching, contraction flows, converging channel, opposing jets, climbing constants and falling drop methods. The results led to two major findings: 1) good agreement on shear measurements between different laboratories, 2) lack of agreement for elongational flow measurements.
and
The imprecision noted for extensional viscosity measurements has been attributed to the failure to obtain steady-state extensional flow in the various flow devices used in the M1 project. The elongational viscosity-strain rate data fell into three broad zones depending on whether spin-line, falling drop or opposing-jet method was used. When a third dimension, time, is added to the viscosity-strain rate plot, the results for a Newtonian liquid fall on a horizontal plane. However the surface for a non-Newtonian liquid is convoluted and the elongational viscosity-strain rate results depend on where the experiment lies on the surface. At steady state, i.e., when the experimental times are prolonged, the surface value corresponds to the steady state test data (39). The over-riding conclusion from the analysis of the M1 data is that one can obtain a__anextensional viscosity but not th.__eeextensional viscosity. However, despite the challenges in measuring extensional viscosity, these techniques are continually being applied to polymer characterization and practical problem solving. One major consequence of the M1 project was the development of a modified filament stretching instrument by Sridhar. In this device, the test sample is held horizontally between two Teflon discs and pulled equally at both ends at a programmable exponential rate such that a constant strain rate is achieved and the stress growth at a constant stretch rate is obtained (40). It appears though that the test sample has to adhere to the plates as the technique does not use aids to clamp samples. Consequently, it is not clear if the technique can be applied to products that are non-sticky or exhibit slip, which could be limiting factors for testing food products. A second device that is also able to generate truly extensional flow field has been developed by Meissner (41) utilizing the concept of rotary clamps. At a constant strain rate this instrument can measure stress growth and thus allow the steady state flow measurements.
294
3.2.2. Commercial extensional rheometers At the time of writing this article, there are only two instruments in the market in the U.S.A. - the Rheometrics Extensional Rheometer, the RFX, and the Rheometrics Elongation Melt Rheometer, based on Meissner's rotary clamp method. The RFX is based on the opposing jet method. The RFX has been successfully used to characterize gum solutions (42); it has, however, been found to be unsatisfactory for testing yield stress fluids (43).
3.3. Applications to foodstuff There are numerous examples where food extensional properties are relevant from the product quality perspective - some obvious examples include the extensibility of doughs, stretching of Mozzarella or Pizza cheese melts, extrusion-cooking of cereals, flow of honey and treacle. Overall, dough products have received the most attention. The importance of extensional viscosity in food processing has been reviewed by Escher (44 and references therein). 3.3.1. Doughs Dough quality has been one of the most elusive properties in the food industry. Dough quality influences the finished product quality as much as the processibility of the dough. A number of empirical tests, such as the Mixograph, the Farinograph or the Extensiograph etc. are routinely used in the industry. In these tests, forces corresponding to certain actions, e.g., mixing with water, or pulling a strand of dough with a hook etc. are measured and the doughs are characterized directly by the force values. No analysis of data are made in terms of deformation type or stress and strain and/or strain rate. However, recent publications are indicating a switch to describing dough quality by dough rheology, including extensional rheometry. Some of these rheology-based methods are summarized below.
3.3.1.1. Rotary Clamp method The influence of ascorbic acid on bread dough rheology was investigated using Meissner's elongational rheometer (41). The results showed an increase of almost 50% in the tensile stresses in the dough following addition of ascorbic acid. The differences between the doughs were evident from their different stress-growth profiles. Steady state flow x~as not attained even after 30 seconds of residence time at the stretch rate of 0.06 sec- . Continuous exposure to the stretching field only led to necking and eventually fracture in the product. These results highlight the genuine need for carrying out steady state experiments for accurate measurement of dough extensional viscosity. At the present time, there is no other instrument available that can provide stress-growth profiles for doughs.
295
3.3.1.2. Converging flows The comparative ease of operation of contraction flow devices render them suitable for use in food analysis. Many composite food products are semisolid in nature which limit the use of the rotary clamp, the opposed-jet or the spinning-fiber techniques. As a fluid flows from a larger cross-sectional circular or planar channel to a smaller cross-sectional channel, the flow streamlines converge imparting an extensional component to the flow. Frequently, when the fluid is highly elastic, vortex enhancement occurs on the upstream side of the contraction or orifice, and the flow in the central core is extensional. To limit the effect of this reduction in the cross-sectional area and still achieve flow, excess energy dissipates from the fluid. This net loss of energy, expressed as the 'entrance pressure drop' is measured by a transducer which can detect pressure changes across the orifice plate of a contracting fluid. The transducer should be located before the vortex, that is, before the flow starts to converge. The 'Bagley correction' was developed to correct for the excess entry pressure drop and allow calculation of shear viscosity from capillary flow data. This entry pressure effect was analyzed in terms of extensional flow first by Cogswell (45) and later by Binding (46). At the present time, there is no obvious preferred treatment and both methods are currently being applied to the complexities of dough rheology. In Binding's analysis both shear and extensional properties are modeled by the Power law whereas in Cogswell's analysis only shear viscosity is modeled by the Power law and the extensional viscosity is assumed to be Newtonian. Both methods are contributing towards further understanding of rheology of wheat flour doughs. Recently Menjivar (47) has demonstrated a relationship between dough extrudate swelling and Trouton's ratio, which is defined as the ratio between the shear and the extensional viscosity. The extensional viscosity parameter is being utilized to develop automated slit rheometers mounted on screw extruders which can generate both shear and extensional viscosity data for dough systems. Padmanabhan et al (48, 49) have used such a system to study corn meal extrusion. A commercial slit rheometer has recently been developed and marketed by Rheometrics Inc. as an on-line dough rheometer.
3.3.1.3. Biaxial extension A further application of extensional rheometry to food products has been the characterization of the Chopin Alveograph by Dobraszczyk (50). Simulating conditions close to those of dough expansion during the baking process he was able to demonstrate reproducible stress-strain data for wheat flour doughs. Dough expansion is considered to be a process governed by high strain but by low biaxial strain rates. In the operation of the Chopin Alveograph, a gas bubble is grown under controlled conditions. From measurements of the bubble size and internal pressure over time, the stress and strain rates are calculated. Results indicated that bubble failure strain correlated with strain hardening which, in turn, correlated with baking expansion.
296 These important fundamental observations when combined with the operational ease of the instrument make the Chopin Alveograph an attractive candidate for dough research. Biaxial extension properties were measured and applied by Chakrabarti et al (51) to detect flour quality differences in bagel doughs. Using an Instron Material Tester they obtained the biaxial extensional viscosity from lubricated squeeze flow tests and obtained a positive correlation of dough viscosity with dough spread during proofing, but not with baked bagel quality. Similarly, Wikstrom et al (52) have reported that the biaxial extensional viscosity can be used to differentiate between wheats of different origin. 'Perfect slip' during squeezing has also been claimed by Wikstrom; this is an important observation as loss of lubrication can take place during compression introducing significant inaccuracy due to friction effects, as noted by Bagley (53). Extensional rheometry represents an increasingly important research tool for our understanding of dough quality. Results are of interest to both the food and agricultural industries.
3.3.2. Food emulsions The semisolid consistency and the occurrence of slip effects during flow could limit the use of the rotary clamp method, the convergent flow method or even Sridhar's extensional rheometer for a large number of food products such as mayonnaises and salad dressings. A novel rheometer (15) based on the concept of filament stretching has been developed by Plucinski et al (Fig. 10) to measure the extensional properties
Figure 10. Extensional rheology of mayonnaise (ref. 15).
297 of such products. As measurements correspond to a free-surface filament, wall-slip effects are avoided in obtaining the viscosity data. Mayonnaises were found to be less elastic than polymer solutions and the memory effect, which had plagued the M1 project, was negligible. Plucinski et al have also derived the viscous flow curve by modeling mayonnaises as a Bingham fluid. A ballpark agreement was obtained between the predicted and the experimentally determined flow-curve of mayonnaise in the slip-free region. The filament stretching device could play a new role in rheometry by providing a new direction for studying substances that exhibit severe slip effects during conventional rotational rheometry.
3.4. Summary The following could be added to provide perspective to the content described earlier: (1) These are early days in the development of extensional rheometers for foods and in our understanding of the extensional rheology of foodstuffs. The lack of commercial availability of extensional rheometers could be stalling the application of extensional rheology in characterizing products and processing operations in the food industry. For industrially important food products, product specific rheometers may need to be developed. (2) The comparatively less elastic nature of many food products makes the analyses of extensional rheology data easier than has been possible with polymeric fluids. This relative ease of data interpretation provides an opportunity both for learning the extensional behavior of materials and for effective application of extensional rheometry in the food industry.
4.0. S O L I D M E C H A N I C S The need to eat is not only for nutritional requirement, it is also for pleasure. Biting and chewing are essential elements of the experience of eating (4). Many tests exist, including the GF Texturometer (4), which were developed to imitate the action of teeth biting and thereby giving instrumental insight into failure modes and associated deformations. Research in the development of dentures (54, 55) as well as directly determining the forces during biting have shown that food samples experience a complex state of stress, i.e., a combination of tension, compression and shear. Such tests may mimic the action of teeth, but cannot give information about the fracture properties of foods. Without determining fracture properties, it is not possible to identify what other properties (including size, thickness) of the test piece contribute to
298 the assessment of texture or how to relate texture to food structure. Consequently, the effectiveness of imitative tests is limited to correlations between instrumental and sensory perceptions. In fundamental solid mechanics including fracture mechanics studies, the requirement is to determine the stress-strain curves and to study the propagation of a crack in a substance. Understanding the behavior of crack propagation was essential for the modern day development of plastic products. The application of fracture mechanics to foods is novel and has a different purpose than the one traditionally used in the plastics industry. In the latter industry, the goal was to apply fracture mechanics to develop stronger, tougher plastics, so as to avoid fracture. For foods, the requirements are different - food products are required to break and should do so in a desired fashion. This requirement holds as much for mastication purposes as for cutting foods with knives in the kitchen.
4.1. Basic Definitions The basic tenets of fracture mechanics are the following (56): 1) All materials contain flaws or cracks. 2) The strength of solid materials is governed by the presence of these flaws. 3) Fracture takes place when the flaw grows creating new surfaces. The energy necessary to create a new surface is given by Griffith's law, which states that for an infinitely wide sheet containing a crack of length 2a and loaded with a constant stress a the true surface energy e s is given by 2
rla = E 2e s
(9)
where E = Young's modulus. Griffith's law was derived for the surface energy for a perfectly brittle, elastic material undergoing no plastic work. However, for many materials, plastic work is not negligible and when included, 2e s = G, where G can include both plastic and surface work. Therefore, G = ~ 217a / E
(10)
At fracture G = G c. 2 Griffith's law states that at fracture, ~ a = constant. Hence, 2e s or G c is a constant and can be thought of as a material constant for a given material. The energy criterion arising from Griffith's work implies that fracture occurs when sufficient energy is released by the growth of the crack to supply the requirements of new fracture surface. The energy released comes from the stored elastic or potential
299 energy of the loading system. This approach provides a measure of the energy required to extend a crack over unit area and this is termed the fracture energy, G c. G c can be measured directly from fracture experiments by detecting crack initiation and measuring the work in initiating the crack.
4.2. Terminology Confusion in food rheology, especially in its fracture mechanics aspects, frequently arises because sensory words are commonly used to express precise technical properties. For an understanding of what is to follow, the fracture mechanical definitions of these words, as given by Williams (56), are summarized in Table 1. It is essential to understand that brittleness is a behavior, not a material property. Hence, a brittle substance under different thermodynamic conditions, for example, at a different temperature, could be converted into a ductile material. Similarly, rubbers show brittle fracture behavior; its rubberiness is given by its stress-strain curve. Basically, when fracture is brittle, the broken parts may be refitted to its original dimensions. Refitting is difficult with ductile behavior - the lid of an opened can of Sardine can be put back into its original location only after uncurling and that also to give a poor fit. Bread doughs show ductile behavior, as the strand of dough is irreversibly stretched before breakage occurs.
Table 1 Technical definitions of sensory terms
Terms
Common Language
Fracture mechanics
Brittle
Liable to break
Sharp crack with small amounts of deformation around the crack tip. Brittleness is a behavior, not a material property.
Ductile
The ability to be drawn into threads
Large-scale deformation around the crack tip; not a material property.
Toughness
Not easily broken
Used with the prefix Fracture energy per square area necessary to give a new crack surface; a material property.
Strength
Power of resistance
Stress carrying capacity of a material given by the maximum in the stress-strain plot; a material property.
300
4.3. Measurement techniques 4.3.1. Compression tests The shape of the stress- strain curve plays an important role in determining the resistance to yielding of materials. In the food rheology literature, the yield stress is often expressed as the fracture stress. Figure 11 summarizes the typical stress-strain
Figure 11. Stress-strain curves for Hookean solids, rubber-type materials and extensible biological substances (adapted from ref. 57). plots for a range of substances (57). Examples of the Hookean type are Wood or highly cross-linked or filled plastics; skin or similar biological tissues follow the J-shape curve, which is clearly different from that of rubber. The Young's modulus is obtained from the slope of linear part of the stress-strain plots and the Yield stress, i.e., the maximum load carrying capability of the substance, is given by the maximum in the stress-strain plot. The stress-strain data can be generated from either tension or from compression tests. For soft solids, compression tests are preferred since it avoids the need to clamp the sample ends. Consequently, compression tests are widely used in testing foods. However, the friction between the loading plates can be significant, as noted by several investigators (58-61). Typically, in compression tests a cylindrical piece of the test sample is compressed between smooth plates using a Material Tester. Assuming constant volume, the stress and strain (Hencky strain) are calculated from the force, displacement data. However,
301 the top and the bottom of the test sample can be frictionally constrained by the plates, leaving the middle portion free to deform. This friction constraint causes 'barreling' of the samples (58), as shown in Fig. 12.
Figure 12. Barelling due to friction (adapted from ref. 58) Just as researchers found an increased stress level by bonding samples to fixture walls to prevent slip (see section 1) during rheological measurements, Christianson et al (59) found an increased stress level if compression test was carried out with bonded samples. They also found the stress level to decrease if lubricated plates were used. Bagley et al (53) reported lubrication to be insufficient in eliminating friction for doughs. On the other hand, Ak et al (62) reported no significant change in the mechanical properties of Cheddar cheese when lubrication was used. They explained this observation as the release of the fat during compression which acted as a lubricant. Similar conclusions were derived by Luyten (63) for Gouda cheese. To conclude, the visual inspection of samples during compression testing is important; if barreling is observed, ways should be sought to eliminate the friction effects prior to collecting data.
4.3.2. Fracture Tests The fracture properties of foods have been extensively studied and comprehensive reviews (64 and references therein) exist that address testing and application of fracture data in the food industry. As mentioned earlier, in the food industry compression tests are viewed also as fracture tests and the yield stress is often regarded as the fracture stress. However, in this review, the discussion is confined to only those types of fracture tests that lead to estimates of the fracture toughness and not the yield stress. The fracture toughness tests are relatively recent in the food industry. A number of different fracture toughness tests have been applied to foods including, the microtome test (65), the wedge fracture test (66-68) and tension tests (69). The 3-point bend test was applied by Charalmabides et al (70) in testing Cheddar cheeses. Apart from the Microtome or the Wedge fracture test, a notch is introduced
302 intentionally to control the crack initiation. The work to fracture is obtained from the area under the load-displacement curve (71)up to the point of crack initiation, i.e., up to the point of maximum load. Friction effects are avoided only in the tension and the 3-point bend tests. A schematic representation of the load-deformation behavior during a fracture test is shown in Fig. 13.
Figure 13. Schematic drawing of force - deformation curve for brittle and ductile fracture (adapted from ref. 71). As the science of fracture mechanics grew from the need to develop stronger plastics, a number of test protocols have been defined for measuring fracture toughness : of plastics using the Linear Elastic Fracture Model (LEFM) (56, 57). In these test protocols, the mode by which the sample fractures, i.e., in shear or in tension, is carefully controlled. In the plastics industry, these tests are used for setting product specifications as well as for developing new products. The LEFM theory has also been applied to the analysis of food fracture tests, although strictly speaking, linear behavior was not found (67-70). No specific test protocols exist for foods at the present time.
5. C O N C L U S I O N In this article we have noted that the scientific topics covered within Food Rheology are broader than the rheological topics of fluid flow and deformation. It is the author's view that to accurately represent the scientific disciplines currently
303 studied under the name of Food Rheology, the latter should really be known as Food Mechanics. Although not emphasized in this chapter, most rheometers used to test food products were designed for polymer melts or polymer solutions and not for semisolid products. Most of the applied fracture mechanical tests were originally developed for much tougher substances such as plastics, and not for foods. In terms of consistency, many foods of commercial interest lie at the boundary between 'solids' and 'fluids'. These food substances have been little studied until now. This review has attempted to address the importance of making scientific measurements for complex foodstuffs. It is through the process of ensuring accuracy in the scientific measurements of mechanical properties that much of the uniqueness and the complexity of food products are revealed. Accurate measurements that are also easy to perform are the requirement in today's busy industrial laboratories and demands sensitive, state-of-the-art rheometers designed to study food material properties. Often, the torque transducer needs to be changed when going from low shear to high shear rate viscometric tests making measurements cumbersome. Many rheometers are not designed to cover low shear rate viscosity measurements, which provide valuable information for non-Newtonian fluids. A new generation of food specific rheometers and established test protocols for food material properties will ease the application of fundamental mechanical properties in the industry and should help to bring major advances in the area of food materials science. To envision the future direction of food materials science, one has to take a look at the marketplace. It is now a global marketplace in a changing world. Food companies will need to cater to a diverse set of consumers with diverse food habits. Some of the changes in eating patterns are already evident in the USA with the greater emphasis put on fat reduced, sugar reduced food products. However, the trend in the market as desired by the consumer is to still retain the same taste quality, i.e., fatty-food taste even in the absence of fat. In a global marketplace, one can anticipate that even for the same food product, consumers in different countries may have different expectations. The food industries will need to fulfill these expectations in order to be competitive and successful. The challenges to the food companies will be to supply the consumer desired quality in the food products quickly and efficiently. Technically, the task will be to master the technology and the engineering know-how to alter food texture and flavor qualities to meet the changing demands of the consumer with minimal cost. A sensory-based assessment scheme for developing food products is no longer sufficient to provide the necessary guidelines for texture/flavor modifications or processing alterations. A material science approach is gaining momentum and the companies utilizing non-traditional food technology efficiently will gain the desired technological advantages.
304
Acknowledgement Permission by Kraft Foods Research management to publish this article and the valuable discussions with Dr. David Binding, Dr. Iman Kamyab, Dr. Janusz Plucinski, Dr. Ed Bagley and Dr. Rakesh Gupta are gratefully acknowledged.
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Characterization of Food: Emerging Methods A.G. Gaonkar (Editor) 9 1995 Elsevier Science B.V. All rights reserved.
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Chapter 13 The use of mastication analysis to examine the dynamics of oral breakdown of food contributing to perceived texture Wendy E. Brown BBSRC Institute of Food Research, Whiteknights Road, Reading, RG6 2EF, UK
1. INTRODUCTION Foods are complex chemical entities with measurable physical characteristics, but their purpose is to be eaten and, preferably, enjoyed. The enjoyment of eating is derived from the sensory experience gained from breaking down the food' s structure and liberating flavours and tastants from the food matrix. For many foods the degree of breakdown achieved during mastication has little influence on the subsequent nutrient release and absorption, and hence onthe nutritional value of the foods 1. Exceptions to this are foods, such as whole seeds and grains, which if not comminuted during mastication may escape later digestion, or food combinations which may enhance or inhibit subsequent absorption, or rate of absorption of, specific nutrients (see2). However, the way the food breaks down in the mouth determines the sensory experience for the consumer. Characterisation of foods in terms of perceived qualities and consequent human enjoyment requires that we understand the source and evaluation of this sensory information. Whilst texture is only one factor in the acceptability of foods 3, it is an important, and possibly increasing, consideration for the consumer 4. However~ texture cannot be measured directly using instrumental methods since its assessment is a psychophysiological process relying on an interaction of a human subject with the food. Logical systems of texture definitions, which specify the type of interaction used to assess particular textural qualities in foods, have been developed by Szczesniak 5'6, Kokini et al 7 (for liquid and semi-solid foods), and Jowitt 8. Using these systems it is possible to obtain a sensory profile of a food's textural characteristics which is both qualitative and quantitative. Such texture profiles have formed the basis of much subsequent work in the development of both sensory and instrumental measurements of texture, and have been widely utilised throughout the food industry and food research centres. Quality assurance, product manipulation and new product formulation traditionally rely heavily on instrumental measurements of food properties, for reasons of expense, reproducibility etc. In most cases instrumental characterisation of a product runs parallel to assessments by sensory panels, and instrumental methods which correlate highly with consumer assessments of textural characteristics are keenly sought. Szczesniak 9 discussed the need for such tests as well as the factors affecting correlations between instrumental and sensory texture measures. The success of instrumental measurement of texture is reflected in the literature which abounds with examples of "good" (i.e.,high) correlations between instrumental measures and sensory ratings. Such good fits are usually obtained within defined
310 product specifications and, most frequently, with trained sensory assessors. These methods have a significant part to play in monitoring food quality. However, Kapsalis and Moskovitz 1~ state that they "believe that correlations between sensory and instrumental texture measurements are usually of an associative, indirect (at times even coincidental) nature". Thus, perhaps it is not surprising that it is difficult to achieve meaningful correlations over wider ranges of food specifications, and with naive consumers who have not been trained to assess textural attributes in ways defined by the researcher. The problem lies in the fact that we do not know specifically how the various sources of sensory information contribute to assessment of particular textural qualities, nor how the information is interpreted by cognitive processes.
2. P R O B L E M S ASSOCIATED WITH I N S T R U M E N T A L M E A S U R E M E N T OF FOOD TEXTURE
Traditionally, evaluation of the textural properties of food has centred on examination of the food. Difficulties with this approach may arise at any of the following stages. 2.1. Selection of appropriate tests It is difficult to know what type of test to use to measure perceived texture without knowledge of the sensory cues used by human subjects. The types of instrumental tests currently available for texture measurement fall broadly into three groups; fundamental, empirical and imitative. These methods have been reviewed recently l~~3, and detailed discussion of available tests is not within the scope of this chapter, although their relevance in measuring perceived texture will be considered. In contrast to the mechanical and rheological properties of materials, which have defined physical meanings, no such definitions exist for the psychophysical assessment of equivalent textural properties of foods. To identify material properties, or combinations of these, which are able to model sensory assessments requires a mixture of theory and experimentation. Scientific studies of food texture began during the twentieth century by the analysis of the rheological properties of liquid or semi-solid foods. In particular Kokini ~4 combined theoretical and experimental approaches in order to identify appropriate rheological parameters from which to derive mathematical models for textural attributes of liquid and semi-solid foods, namely, thickness, smoothness and creaminess. Rheological concepts cannot be used in the examination of solid foods, which have a far greater elastic component. For these foods larger scale deformations of the sample are necessary, but similar problems arise regarding exactly what to measure. For example, material scientists define hardness as the resistance of the surface of a body to penetration ~5. However, Boyd and Sherman ~6 concluded, from examination of a wide range of foods, that different mechanisms were used in sensory estimations of hardness for hard, as compared to soft, foods. Moreover~ Peyron and Mioche ~7 have shown that human subjects assessed the hardness of elastic products (elastomers) according to the deformation resulting from the application of a constant force, while they assessed the hardness of plastic products (waxes) according to the force needed to deform the product. Since foods are rarely purely elastic or purely plastic, but rather exhibit both properties to varying degrees, it is difficult to select instrumental methods which are wholly appropriate for each food or range of foods. For some textural
311 characteristics (crispness, crunchiness) the sound emission on sample fracture has been shown to be a more appropriate means of assessment 18-2~ The wide adoption of a fundamental approach to the measurement of the rheological and mechanical properties of foods has resulted in a greater understanding of the physical properties of food products, but has not been so successful in promoting our understanding of sensory appreciation of texture. In some cases an empirical approach has been used, for example the use of the cone penetrometer to represent penetration by the teeth, the bite tenderometer 21 to represent squeezing the sample between the teeth, or the use of 3-point bending tests to approximate snapping food samples between the teeth. However, while being appropriate for evaluation of certain foods these methods have achieved limited general application. 2.2. Selection of appropriate test conditions Most foods are viscoelastic in nature, and hence values obtained for many mechanical and rheological properties are dependent on the conditions of the test. Kokini ~4 demonstrated that different shear rates were needed to measure thickness and smoothness of semi-solid foods of different viscosity. For solid food%Sharma and Sherman ~2 showed that either one of two cheese samples was measured as being harder (requiring a greater force to attain a given deformation) according to the compression rate in an instrumental compression test. Boyd and Sherman 16 demonstrated that measures of hardness of a wider range of foods also showed variation with compression rate. They found that good correlations with sensory measurements could be obtained under predetermined conditions, but these conditions differed for soft and hard foods. For solid foods, the compression rates used during mastication are typically an order of magnitude greater than those used in conventional instrumental tests (800-1,200 mm/min compared to 10-100 mm/min). Voisey and Larmond 23, Peleg and Normand 24 and Marshall 25 all reported improvements in correlations between sensory and instrumental measures of textural properties by modifying compression rates. A further complication in identifying appropriate test conditions was highlighted by Bourne 26, and Langley and Marshall 27, who demonstrated that the compression rates used during mastication depended on the individuals as well as the foods. There is no doubt that it is possible to identify specific instrumental tests under defined test conditions, which correlate highly with sensory scores, for a range of foods. However, it cannot be assumed that this indicates a causal relationship, that these tests can be used to predict sensory characteristics of foods having a slightly different composition, or that the prediction is valid for all consumers. 2.3. Accommodation for temporal aspects of texture assessment Instrumental measurements of food properties are usually obtained as single events. Human subjects assess the textural characteristics of foods usually during or after eating a sample. Although an instrumental test may approximate the first bite or chew onto a sample, consumers may not judge the sample's texture on its response to the first bite, relying more on how it breaks down over successive chews. This may be considerably more important for textural characteristics such as chewiness or tenderness, than for hardness or brittleness. The General Foods Texturometer 28 was developed to introduce a time element in the measurement of texture, and has been used extensively, but unfortunately interpretation of the results "in
312 terms of well defined physical properties of the samples appears difficult ''29. This apparatus is not able to reproduce the conditions of temperature change and lubrication which occur during mastication, nor indeed the action of forming a bolus from the comminuted sample. All of these factors may play a part in a subject's perceptions of a food's texture, yet it is extremely difficult to determine, instrumentally, how a food will react under such conditions. A further complication arises from the different food breakdown paths sustained by different individuals, both in the extent and the rate of breakdown 3~ both of which may be important in sensory assessment of various textural characteristics. 2.4.
Accommodation for variations in chewing behaviour
Chewing behaviour is probably the result of many physiological, anatomical and psychological factors, for example the coordination of jaw movement, the positions, relative sizes and strengths of the masticatory muscles and teeth, and the learned and habitual patterns of chewing. Differences in the way individuals chew influence their perceptions of texture 31. For example, an initial impression of texture may be attenuated during the chewing process. Individuals may agree on the relative hardness of a food if asked to bite it once, but they may differ in their assessment of hardness after eating the sample, possibly depending on the length of their chewing sequences. Individuals who chew longer may be able to discern differences in chewiness of samples which are not apparent to short chewers. People with low saliva flow rates may perceive some foods to be drier or stickier than high salivators. Clearly current instrumental measures of texture, based on analysis of the food, take no account of such differences between human subjects.
3. P R O B L E M S TEXTURE
ASSOCIATED
WITH
SENSORY
MEASUREMENT
OF
FOOD
Texture is a sensory property of food. It arises from the food's physical structure, which is derived from the interactions of its constituent parts, and is perceived by monitoring how the structure responds to externally applied conditions. During development human beings learn to associate particular sensory responses from handling and eating foods, with specific textural characteristics. From verbal interactions with other humans they develop their textural vocabulary. The natural approach to characterising a food's texture is to ask human subjects to detail their assessments of texture. To measure texture would involve measuring all the sensory inputs from which texture judgements are derived and combining this information, with appropriate weighting factors, in the same way as occurs during signal processing in the brain. In the absence of technology and knowledge to achieve this, access to the sensory information is only possible by asking subjects to record their assessments. A variety of sensory methods have been developed to quantify such assessments based on difference testing, ranking, and linear, magnitude and category scaling. However, requesting a subject to interpret, quantify, and translate his or her sensory information onto some type of scale inserts an obligatory cognitive step in the process, during which the original sensory information is interpreted in the light of the current status of the subject in terms of physiology, psychology, expectations, and experience. During the early 1960s the need for a systematic sensory method to characterise food textures was addressed, and the General Foods Texture Profile Method (TPM) was developed 32. The aim
313 was to diminish variability in subjects' responses arising from differences in interpreting texture descriptors, due to cultural or experiential differences, and from the cognitive process involved in recording sensory assessments. This was achieved firstly, by rationalisation of the texture terminology 5, including a classification of texture characteristics, and agreement on precise definitions for each texture descriptor, and secondly by the use of reference standards to describe varying degrees of attributes 33. With appropriate training and evaluation, subjects using this method can produce consistent and reliable sensory scores without interference from outside factors, such as the time of day, their level of satiety or awareness. The General Foods Texture Profile Method has enjoyed widespread use over many years, it has been modified, expanded and improved 34'35, and has provided an excellent means for characterisation of a wide range of food, and non-food, products. However, whilst this method can yield a description of the product in terms of the sensory response obtained when submitting it to a series of manual or oral manipulations, this may not adequately describe the texture of the food as perceived by the consumer under normal preparation or eating conditions. Clearly, knowledge of the texture perceptions of the normal consumer is important to the food industry, and to sensory scientists. To this end we need to know how foods break down under the whole range of normal chewing patterns, and the sensory evaluations which result from different breakdown patterns. Sensory data about food products evaluated using the Texture Profile Method may mask the variability in human experience by defining, not only how a particular attribute is assessed, but also in some cases the chewing behaviour to be used during assessment. In developing the Texture Profile Method, definitions for assessment of particular textural attributes were derived from the consensus of panel members 36. In fact all subjects may not use the same strategies for assessing a particular textural attribute or may not use the same strategy for all types of foods. In a study of baked products Bramesco and Setser 37 reported definitions for a variety of textural attributes developed by both trained and untrained sensory assessors. For several attributes the definitions differed between the panel types, indicating that strategies adopted by some naive panellists in assessing textural characteristic may differ from those defined by the TPM. Untrained consumers and trained panellists may agree on the categorisation of most textural characteristics, and the foods which exemplify these characteristics 38. Discrepancies are more likely to become apparent when considering small scale differences between products. Although variability among untrained individuals in their sensory assessments of food products are commonly found, few studies have addressed these (see 39,40), or compared the results of trained and untrained subjects 41'42.
4. A N E W A P P R O A C H TO THE A S S E S S M E N T OF FOOD TEXTURE.
Clearlysit may be possible to define and accurately measure many aspects of the mechanical and rheological properties of foods, but to try and relate these measures to consumer perceptions of the texture of the foods, is fraught with difficulties. Conversely, it is possible to train human subjects to assess textural characteristics of foods in defined and consistent ways (training them to mimic an instrumental response), however this may be missing the diversity of perceptions of food texture experienced by normal consumers. In recent years there has been a trend away from the more traditional approach in
314 mechanical testing of foods, involving measurements of fail stress, strain and moduli in compression, tension, shear and bending tests. The current aim has been to adopt more a physiological approach, particularly for characterisation of solid foods which have not been as amenable to instrumental modelling as liquids. This is reflected in the use of more physiological conditions in many of the traditional tests 23. It is also evident in the recent application of methods used for examining the fracture mechanics of materials to foods 43"48. Since many foods undergo fracture during mastication, fracture tests may be more appropriate than compression tests for characterisation of texture in these foods. Developments in the sensory characterisation of food texture are also occurring from a different direction. They are based, not primarily on characterisation of the food, but on examination of the interaction between the food and the human consumer. In 1970 Pierson and Le Magnen 49 reported chewing and swallowing patterns for a variety of foods, and demonstrated magnitude and temporal differences in the chewing pattern between foods, and also between different subjects. This confirmed the results of several previous studies of mastication that chewing patterns exhibited inter-individual and inter-food variations, but suggested the use of this method for obtaining objective assessments of the sensory stimuli involved in texture assessment. Attention to this approach has been renewed recently with the evolution of more sophisticated data handling possibilities. This research seeks to identify the sensory cues determining the perception of texture of foods, by concentrating on what the consumer is doing, but to reduce or eliminate the cognitive and subjective aspects of perception involved in sensory measurements, which could be considered second order effects. The aim is to examine the mastication process during which our texture assessments are made, and which is tailored to the changing physical properties of the food. The techniques used are non-invasive and non-verbal, and are intended neither to interfere with the mastication process, nor to rely on cognitive appraisal by the subject. Boyar and Kilcast 5~ reviewed the available techniques for examining mastication, and reported the potential of using electromyography (EMG), to monitor the activity of the masticatory muscles during chewing. The technique was subsequently developed 5255 and has been reviewed together with conventional methods of texture measurement 11.56. The remainder of this chapter will address the possibilities of EMG for identifying sensory cues in the perceptions of textural characteristics originating predominantly from chewing and biting, i.e.~ those referred to as mechanical characteristics by Szczesniak et al. 5
5. THE MASTICATION PROCESS The mastication process serves to break down and lubricate the food sample prior to swallowing (see recent review by Thexton57). What is achieved by chewing will depend on the original properties of the food and how it reacts under the specific action of the teeth and tongue of a particular individual. For example, mastication may result in the breakdown of the structural integrity of the sample which is then reformed into a bolus and swallowed whole. It may cause the food sample to melt, or dissolve, and to be swallowed together with saliva, or it may result in fragmentation of the food sample, portions of which are swallowed separately. Individuals differ considerably in their ability to break down different foods to a state they are comfortable swallowing. For example, in a sample of 52 dentate consumers the time taken to eat a small cube of raw carrot varied from 12 to 125 sec. and for salami from
315
Fig 1. Scanning electron micrographs of Gouda cheese eaten by 2 different subjects and expectoratedafter 5 chews. Spitout material was collected in a sieve and rinsed in cold water. Particles were spread on individual aluminium trays and frozen in liquid nitrogen prior to examination.
316 9 to 85 sec 31. These differences may arise from differences in the consistency or particle sizes which individuals are able to swallow, but also from the efficiencies with which they achieve this consistency, or size. Factors determining the state of a food sample an individual swallows include habit and situational circumstances. Chewing efficiency is influenced by many factors: the occlussal contact area of the teeth 586~, the chewing forces used in breaking down the sample 62, the ability to manipulate the sample within the mouth and the saliva production during mastication (which may vary between individuals63). Fig. 1 shows, at low magnification, particles of Gouda cheese expectorated after 5 chews. Clearly in this short time span the first subject (top half) has saturated the sample more completely with saliva and caused more extensive melting of the fat, than the second subject (lower hal0. Such factors may greatly influence the subjects' perceptions of the textural character of the cheese. From examination of mastication patterns it may be possible to determine which factors are influential in assessment of food texture.
Fig 2. Subject prepared for EMG recording. Under normal conditions the keyboard and monitor would be relocated away from the subject, and the screen would not be within the subject's view.
317 6. ADVANTAGES OF ELECTROMYOGRAPHY FOR MONITORING MASTICATION Since the mastication process is tailored to the food being eaten, and mastication patterns for the same food may differ between people, examination of mastication patterns should assist in describing breakdown paths for different foods, and highlight differences in the way individuals' break down foods which may underlie their different perceptions of texture. To monitor mastication by means of electromyography (EMG) involves placing small electrodes on the skin overlying the superficial masticatory muscles (masseter and anterior temporalis muscles) which contribute to the jaw closing phase of chewing and which generate a large proportion of the force involved in crushing or grinding food (Fig 2). Monitoring activity from all four muscles can help to identify different strategies by which individuals' distribute the effort involved in chewing between the masticatory muscles; both between the left and right sides, and between the temporalis and masseter muscles. Placement of the electrodes is a relatively simple operation and once in place they do not encroach on normal mastication. Collection of EMG data may be computer operated, and can be accommodated in a clean environment. Moreover, in the author's experience, untrained subjects recruited from the general public have reacted with mild curiosity to this means of recording, but easily accepted the preparation for recording, and reported that it did not interfere with eating. Mastication patterns revealed by EMG records from the masticatory muscles have been shown to be reproducible for individuals, but exhibit differences both between individuals and between food types 52'54'55. Fig 3. shows electromyographs from the masseter muscles; each burst of activity constitutes the closing phase of the chewing cycle and the periods between activity bursts, the opening phase. The major advantages of using electromyographs to examine mastication patterns lie in their ability to register the changes in the chewing activity which occur within the course of a chewing sequence (Fig 3.) (see also64), to reveal differences in the mastication pattern for different foods (Fig 3, compare carrot and toffee) (see also 52'64-66), and to demonstrate pattern differences between individual human subjects (Fig 3, compare subject A and B for toffee),(see also54'55). Mastication may also be examined in a continuous and non-invasive way by monitoring the movement of the jaw. Observation of the spatial dynamics of the jaw during biting and chewing has been used largely in dental practice, where the effect of dental interventions aimed at achieving a functional and unobstructed movement may be monitored. Obviously jaw movement is synchronised with the muscle activity, but this is a complex interaction since the movement changes in speed and direction throughout each chew cycle 67"69. It is possible with this technique, as with EMG, to observe the changes in mastication throughout the chewing sequence 7~ to discern differences between mastication patterns for different foods 72" 74 and differences in patterns between individuals 7~ Although there is considerable potential in proceeding with the use of kinematic measures of jaw movement in assessing food texture, to date this approach has received little attention from food scientists. The remainder of this chapter will concentrate on the electromyographic technique, but there are clear advantages to be gained from using both techniques in combination 11.
7. USE OF MASTICATORY ELECTROMYOGRAPHS TO ASSESS FOOD TEXTURE Mastication is a highly iterative process during which the force development and spatial
318
Fig 3. Electromyographs showing differences in pattems for foods and between individuals. Patterns only from left and right masseter muscles are shown. Horizontal axis - time (see). Vertical axis - EMG units; I EMG unit = 0.5mvolts.
319 dynamics of one chew are determined by sensory feedback from the preceding chew - or, in the case of the first chew, by visual and/or tactile feedback and/or previous experience. Consequently~ changes in the EMG pattern from chew to chew reflect the changes in shape, size and consistency of the food sample. An indication of the usefulness of EMG in monitoring the consistency of the food within the mouth comes from comparing the muscle activity profile for a single chew with the instrumental force / deformation pattern for the same food. Fig 4 shows the EMG pattern arising from the jaw closing phase for the first chew for each of 3 different food samples. The activity profile has been aligned with the corresponding force diagrams obtained from compression tests using an Instron Universal Measuring Instrument. Clearly~there is a striking resemblance between the two patterns for each food. For both the carrot and biscuit samples, muscle activity ceases for short periods during the jaw closing phase. These pauses arise from reflex inhibition of contraction following sudden unloading of the muscle on fracture of the sample 77. Sequential inhibitions may occur as smaller subparticles, held in place between the teeth by the tongue and cheeks, are also fractured. In some cases a further activity pause occurs when the teeth are close to occlusion, before the activity is reinitiated, accompanying a lateral (grinding) movement of the jaw. It can be seen that the amplitude of the EMG signal for carrot is considerably greater than that for biscuit, reflecting the greater muscle activity force required to fracture the former. The pattern for the salami sample presents a generally smoother outline, the pause in activity being altogether gentler in appearance and probably related to separation of the particles comprising this comminuted product; such pauses are rarely seen in unprocessed meat (results not shown). Fig 4. EMG profiles for the first chew in chewing sequences aligned with corresponding force deformation diagrams. Top traces: Lincoln biscuit (1/4 circle, Lincoln, McVities), middle traces: salami (1 cm x 0.5 dia Pepperami, Mattessons Ltd), bottom traces: raw carrot. EMG axes as in Fig 2. Note differences in deformation for different foods.
First chew
Biscuit
Salami
Carrot
320 Fig 5. EMG profiles for chews at the middle and end of the chewing sequences for Lincoln biscuit (top traces), salami (middle traces) and raw carrot (bottom traces). For axes see legend for Fig 4. Note chew numbers indicating length of chewing sequences for the foods. Mid-sequence
End of chewing sequence
321 8. TEXTURAL SIGNATURES WITHIN E L E C T R O M Y O G R A P H S The food types shown here correspond to crisp, crumbly (biscuit), hard, brittle (raw carrot), and firm, chewy (salami) products. These represent distinct textural types, separation of which causes little confusion to the measuring instrument, or the consumer. However, they offer the opportunity for us to develop pattern recognition techniques which may be useful in the analysis of products differing in more subtle ways. If we can identify the essential features of the EMG which correspond to, for example, crumbliness, it would be possible to compare these features between different types of biscuit, not only for the first chew, but for each successive chew in the chewing sequence. Fig. 5 shows the EMG profiles for single chews occurring later in the same chewing sequence (profiles for the chews at the middle and the end of the sequence are shown). There are clear changes in the duration, amplitude and shape of these profiles during the chewing sequence. Monitoring how, for example, the "crumbliness features" of the profile change during the eating of different biscuits should enable us to predict which biscuits the consumer perceives as being more, or less, crumbly. Eves 53 monitored several features of the EMG profile for subjects eating a variety of commercial chocolate samples and demonstrated clear differences in the way the peak height of the integrated EMG signal changed during the chewing sequence for the different samples. However she was unable to predict the perceived texture of the samples from any of the EMG parameters analysed. With more computer power, sophisticated software and advanced data handling capabilities it is now possible to analyse these signals in more detail in order to discern and measure the qualitative differences shown in Fig 4. Such techniques are currently being developed in this laboratory. When the "fingerprints" for different textural characteristics, such as elasticity or brittleness, have been identified, being able to predict consumer perceptions of texture may be a possibility for the future. In recent years, techniques for examining the jaggedness of signals, using Fast Fourier Transforms and fractal analysis, have been applied to instrumentally measured stress-strain relationships of food 78,79. This has resulted in methods to describe the jaggedness of such relationships which may reflect a crunchiness/brittleness dimension 8~ Although operating within a different time scale, and different compression rates, the EMG pattern obtained for mastication shows some similarity with the instrumental stress-strain response (Fig 4). It would be interesting to apply such an approach to analysis of EMG signals recorded for different foods.
9. DIFFERENCES
BETWEEN
INDIVIDUALS
IN
PERCEPTIONS
OF
FOOD
TEXTURE. A considerable problem for both the food industry and sensory scientists is the degree of individual variation in texture perceptions. The differences in breakdown pathways in the mouth for standard samples may underlie some of the variability. Indeed Brown et al 31 have demonstrated an influence of chewing behaviour on texture perceptions in a model food system. Even if all individuals shared a common system for assessing a particular textural characteristic, the differences in the way they masticate a sample may cause them to come to different conclusions regarding its texture. However, there is also the real possibility that subjects may use different measuring systems for assessment of a textural characteristic they
322 call by the same name. Careful intra-individual correlations will be needed to examine these possibilities.
10. MODELLING FOOD TEXTURE. In 1988 Hutchins and Lillford presented a three dimensional model of food breakdown in the mouth s2. They indicated that perceived food texture could be determined from such a model, but presented no clear way of measuring the "degree of structure" and "lubrication" parameters, which formed two of the axes of the model, relative to the third axis - time. Identification of appropriate parameters, of particle size and consistency, from mastication patterns may allow us to solve this model in due time. The point at which the bolus is considered ready to swallow forms an important aspect of this model. People have different swallow thresholds 11,61,83,presumably controlled by the bolus consistency or particle size they consider ready to swallow, and also in the amount and rate of saliva production. Examination of the EMG profiles for the three foods presented in Fig 5 demonstrates a qualitative similarity of the profiles for all foods just prior to swallowing. Perhaps for this individual such a profile acts as the signal to swallow, having achieved some favoured relationship between particle size and consistency. For any modelling approach there is considerable importance in knowing what determines the end point of a process. We can determine what features of the chewing sequence influence assessment of particular textural characteristics of food by using this approach to examine the interaction between food and consumer during the mastication process. We should then be able to develop mathematical models for perception of textural qualities which take into account different texture combinations (for examplepassessment of hardness in both elastic and brittle foods), and different breakdown patterns. Although currently at an early stage, mastication analysis shows promise for enhancing our measurement of perceived texture in foods.
11. CONCLUSIONS There would be considerable advantage for both sensory scientists and the food industry in knowing what consumers are measuring in order to assess particular textural properties. Despite many real advances in the instrumental measurement of food texture, we are not significantly closer to understanding the sensory cues used in consumer assessment of texture. The mastication process is adjusted to the consistency of the food bolus in real time. From studies of this process is emerging a novel approach to characterisation of food texture.
Acknowledgements The author is grateful to Keith Langley for information and assistance on instrumental methods for measuring food properties, and for preparation of force deformation diagrams in Fig 4., and also to Alan Martin for preparation of samples and examination by SEM.
323
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Characterization of Food: Emerging Methods A.G. Gaonkar (Editor) 9 1995 Elsevier Science B.V. All rights reserved.
329
Chapter 14 B i o s e n s o r s in F o o d A n a l y s i s S. G. G r e g C h e n g a n d Z o h a r M. M e r c h a n t K r a f t F o o d s , Inc. 801 W a u k e g a n Road, Glenview, IL 60025 U S A
1. INTRODUCTION Controlling process conditions and testing finished products are traditional means that food processors used for quality control. On-time computer control system has been widely used today to control process conditions such as temperature, time, and pressure. However, food processors still rely on laboratory testing of the finished products to ensure the quality and safety. These testing methods including traditional analytical chemistry techniques such as spectroscopy and chromatography are labor intense, costly, and time consuming. C u r r e n t advanced instrumentation and automation enable food processors to reduce the testing time and cost in the QC laboratory. Consumer trends towards high quality, and low cost foods force food processors to look for product quality control through the process stream. This approach provides not only assurance of the product quality, but also improved quality, reduced cost, less waste, and ultimately a competitive edge. Traditional quality control systems are no longer sufficient. More measurements targeting the chemistry in the processing stream are required, for example, protein denaturation, hydration of dry ingredients, enzyme reactions, microbiological reactions, etc. These m e a s u r e m e n t s either require sophisticated instruments, trained chemists, complicated sample preparation, or a slow response time. This imposes certain constraints on the applicability outside the R&D laboratory. Therefore, developing rapid, simple, and accurate analytical methods, to at least transfer the tests from the R&D to QC laboratories, becomes analytical chemists' goal in the food industry. Fortunately, other industries such as chemical, pharmaceutical, a n d biotechnology have devoted these efforts longer t h a n the food industry. Increased u n d e r s t a n d i n g of biomolecules and biomolecular interactions from biotechnology research resulted in a lot of test kits being commercially available which use enzymes, immunoassay, DNA probes, and polymerase chain reaction (PCR)techniques. Applications of these techniques in food analysis are being emphasized widely in the food industry and academia (1, 2, 3). Food chemists have successfully adapted some techniques to analyze the specific food components such as organic acids, cholesterol, protein species, and pathogens. With advances in electronics, research in the area of m e a s u r e m e n t by incorporating biological components to electronic sensing device has been
330 growing rapidly (4, 5). It is known as the biosensor technology. Merging the advent of new technologies from areas of fiber optics, m e m b r a n e immobilization, medical diagnostics, biotechnology, and electronics allow biosensors to provide direct m e a s u r e m e n t s of biological and chemical components in a short time frame. This certainly fits the need for a real time process control that the food industry is looking for and will take food processing into a new era.
2. BIOSENSOR DEVICE Biosensor is one unit of analytical device that uses a biological molecule to measure a biological or chemical species directly. It consists of a biological component and a transducing or signal generating component (5). A biological reaction of a specific compound occurs in the biological component. The t ra n s d u c in g component, a transducer, detects and/or converts the reaction products such as heat, electrons, light, colors, mass, into an electronic signal. A typical biosensor can be illustrated by a glucose enzyme sensor. Figure 1 shows the schematic of a glucose enzyme sensor that uses glucose oxidase (GOD) and a hydrogen peroxide electrode. The reactions involved in the glucose determination has also been included in the figure. The enzyme is immobilized onto a membrane material such as cellulose diacetate located at the inside end of the electrode. Glucose molecules penetrate the electrode surface and react with glucose oxidase. Hydrogen peroxide, the reaction product, is electrochemically converted into electrons. Two electrons are transported between anode and cathode per molecule of glucose oxidized. I n this enzyme sensor, the immobilized glucose oxidase is the biological component providing the biological reaction (an enzymic glucose oxidation), while the electrode is the transducer which converts the reaction product into electrons and provides a signal for read-out.
2.1. Biological Component The specificity is the main reason for using these biological materials in biosensors. Any biological materials possessing a molecule recognition function can be used in this component. They may be enzymes, antibodies, receptors, nucleic acids, organelles, microorganisms, whole cells and tissues from animal and plants. They all fall into two groups: biocatalysts and biobinding agents. Enzymes are the widely used biocatalysts. Multiple enzymes have also been used in coupled reactions to either magnify the resul t ant signals or produce detectable signals. Glucose sensor using glucose oxidase probably is the first and the most well developed biosensor (6, 7, 8). Antibodies and receptors are known to have the characteristics of binding the specific biological and chemical components. The specific binding reaction between antibody and antigen has been known for a long time. Scientists have developed n u m e r o u s immunotechniques based on this reaction. The next chapter in this book is devoted to the use of antibodies in characterization of food. In the past two decades, developments in monoclonal antibodies, antibody fragmentation and conjugations with enzymes and chromophores, have made the immunotechniques powerful analytical tools. Antibodies labeled with electron dense elements are also widely used today for
Figure 1" Glucose oxidase (GOD)
- H202
Electrode for Glucose Determination
332 m i c r o s t r u c t u r e analysis (immunocytochemistry). Utilization of receptors in biosensor development is also based on the specific binding characteristics between receptor and donor. The techniques used in i m m u n o s e n s o r s can also be applied to the receptor-biosensor. Use of m i c r o o r g a n i s m s and plant and animal tissues as a biological component of biosensor are also described in the literature (9, 10). The principle is based on the use of the n a t u r a l bio-reactive systems. They have several advantages over the isolated enzymes and receptors. Isolation of enzymes and receptors are often required to increase the response time. Enzymes and receptors retained in the cells are more stable and have longer lifetimes. Cell-based biosensors are also economical as no purification step is required. 2.2~ T r a n s d u c i n g Component The t r a n s d u c e r p a r t of a biosensor transduces and amplifies the signals generated by biological reactions into a read-out device. Four basic methods used for signal t r a n s d u c i n g are electrochemical, optical, thermal, and m a s s (Table 1) (11). Electrochemical transducers are commonly available. Potentiometric type such as electrode was first used by researchers. It has a wide response range but slow response time. The response to a change is logarithmic. Electrical interference and protein fouling are the disadvantages of amperometric transducers, although the response to dose change is linear. Electrode is a common device for construction of potentiometric a n d a m p e r o m e t r i c t r a n s d u c i n g components. A disadvantage is its low sensitivity. Conductometer is the most inexpensive and rugged. However, g e n e r a t i n g conductance by other biochemical molecules existing in the sample leads to a poor signal to noise ratio. Field effect transistors are small, low cost, able to mass produce, and suitable for automation. However, the disadvantage is its sensitivity to the t e m p e r a t u r e . With faster, smaller, and stable semiconductor chips, the sensitivity and response time are tremendously increased. For example, it is capable of detecting a minute change in potential during the antibody-antigen complex formation. Optical devices have also been used as transducers. Laser fiber-optics allows high intensity light to travel a long distance using fibrous size carrier. The stable and intense light beam not only provides calibration stability but also makes all the detecting techniques faster and more sensitive. In addition to the UV-VIS absorbance and fluorescence intensity, m e a s u r e m e n t s of multiple reflections, surface plasmon resonance, and total internal reflection fluorescence had recently been used (12, 13, 14). Heat is the most common product of biological reaction. Heat m e a s u r e m e n t can avoid the color and turbidity interferences that are the concerns in photometry. M e a s u r e m e n t s by a calorimeter are cumbersome, but t h e r m i s t o r s are simple to use. However, selectivity and drift need to be overcome in biosensor development. Changes in the density and surface properties of the molecules during biological reactions can be detected by the surface acoustic wave propagation or piezoelectric crystal distortion. Both techniques operate over a wide t e m p e r a t u r e range. Piezoelectric technique provides fast response and stable output. However, mass loading in liquid is a limitation of this method.
333 Table 1 Transducers Used in Biosensors Method
Detection
Transducer
Electrochemical
Potential
Ion-selective electrodes Gas selective electrodes Field effect transistors (ISFET, semiconductors) Amperometers Oxygen electrode Conductometer Impedimetric m e a s u r e m e n t s
Current Conductance Impedance Capacitance Optical Laser Fiber-optic
Fluorescence Optical density Light scattering Light polarization Reflection Surface plasmon resonance
Fluorometers Photometers Spectrophotometers Diode arrays
Thermal
Heat
Thermistors Calorimeters
Mass
Mass change
Measurements of surface acoustic waves, piezoelectric crystal resonance
The characteristics of transducer including the physical properties, electronics, device fabrication, and the specification for the application have recently been reviewed by Sethi (15) and is not covered in this chapter.
2.3. Unification of Biological a n d T r a n s d u c i n g Components One of the characteristics of a biosensor is that the biological materials a n d transducing devices are constructed in one unit to give the re-usability of biological materials. Therefore, attaching biological material to the transducer is required for the design. They can either be immobilized directly onto the surface of the transducer or indirectly on a thin film coated onto the transducer. Several techniques can be selected depending on the n a t u r e of the materials, types of transducers, analytes and conditions to be measured (16). 9A d s o r p t i o n - A solid material having high affinity to the biological components is used. Physical adsorption is the only force binding the biological components to the matrix. The system is very susceptible to the changes in pH and temperature due to the weak binding. Although matrix is easily regenerated, biological components are easily lost. Other approaches should be selected for high cost biological components. 9E n t r a p m e n t a n d E n c a p s u l a t i o n - Biological components are physically included in a compartment. No direct chemical modifications of molecules are
334 Table 2 Biosensors Based on Biological Components Used (4). Biological Type
Recognition Element
Biosensors
Proteins
Enzymes Antibodies Receptors
Enzyme sensors Immunosensors Receptor sensors
Organelles
Organelles
Organelle sensors
Cells and Tissues
Microorganisms Animal and plant cells Animal and plant tissues
Microbial sensors Cell sensors Tissue sensors
performed. The activity and specificity are retained. Diffusion of analytes into the compartment is required prior to the occurrence of specific reaction that becomes a limiting step for the measurement. Reducing the diffusion barrier oi~en causes the loss of the biological components. High viscosity of the samples may also hamper the reaction. 9C r o s s - l i n k i n g -Biological components are cross-linked with themselves or with other protein molecules. This approach is often combined with entrapment to reduce the loss of biological components. 9C o v a l e n t B i n d i n g - B i o l o g i c a l components are chemically modified and covalently bound to a solid matrix. The biological components are directly exposed to the sample environment. No physical barriers exist for the analytes. The losses of the biological components are expected to be minimum due to the strong binding force between the components and matrices. System has high tolerance in adverse conditions of pH and ionic strength. Although h a r s h conditions and toxic chemicals are often used to activate the matrices and to bind the materials covalently, this approach is by far the most preferred. This technique can also be applied to immobilize biological components directly onto the surface of the transducers. 2.4. Terminology of Biosensors Types of biosensors can be named either by the biological components, physical transducing devices, or the measured analytes. Researchers were originally using biological components to define types of biosensors (Table 2). Types of transducers had also been included in the name to identify the physical transducing device, i.e., enzyme electrodes, acousticimmunosensors, optical biosensors, piezoelectric-immunosensors, and biochips. Analytes are also used to specify tl:e application. Glucose enzyme sensor is an enzyme biosensor measuring the glucose. Characteristics and commercial varieties of enzyme electrodes, especially using glucose oxidase, have been extensively reviewed by Kuan and Guilbault (17).
335
3. B I O S E N S O R S IN F O O D ANALYSIS
Knowledge of the composition and properties of raw materials, their changes during processing and the quality and safety of finished products are essential to food manufacturers. The substances to be analyzed are very broad and require test methods from areas of analytical chemistry, biochemistry, microbiology, and biotechnology (Table 3). While current methods are adequate for the majority of food tests, they are either costly or time c o n s u m i n g . Analysis of certain specific compounds often requires multiple sample preparations and sophisticated instrumentation. Biosensor is an ideal tool for food analysis that requires complicated procedures. Numerous designs have been reported for detecting food analytes. However, only a few systems have been practically used in food analysis. None has been used in on-line analysis. 3.1. E n z y m e Sensors The most successfully used biosensors in food applications are the enzyme reaction-based electrochemical types. The microbial enzymes are commonly used in the biosensor design (18). Clark and Lyons (6) first developed a glucose electrode by combination of glucose oxidase reaction and electrochemical determination of oxygen and hydrogen peroxide. Since then, the principle of oxidase-reaction has been extended to develop biosensors for other compounds. More than 50 oxidases were identified to act on various food compounds (19). Oxidase-electrodes m e a s u r i n g sugars (20, 21, 22, 23, 24, 25), cholesterol (26), acids (27, 28, 29), amino acids (30, 31), alcohols (32, 33), and phenols (34) h a d been prepared and tested in various foods such as fruit juices, soft drinks, beer, wines, soy sauce, milk, and yogurt. Dehydrogenases are other type of enzymes t h a t have been evaluated. They utilize the electron transfer capability of enzyme cofactors, NAD and NADP, to generate detectable signals. Numerous dehydrogenase-based sensors were reported that analyze glucose, fructose, lactose, gluconate, lactates, ethanol, and amino acids in foods a n d fermentation products (24, 35, 36, 37). Enzyme coupling technique had also been applied in the biosensor design (9). The principle is to link multiple enzyme reactions that convert the analytes into a measurable compound so as to increase the sensitivity. This technique has been widely used in food analysis (38) and numerous enzyme kits are commercially available. With wide ranges of selection of enzymes a n d reaction linkages, more compounds can be detected. Ability to quantify a specific analyte in a complex sample matrix with m i n i m u m interference is also an advantage of this technique. Coupling of ~-galactosidase with glucose oxidase and catalase in enzyme electrode was used to determine lactose in milk (25, 39, 40). Matsumoto et al (41) prepared a multi-enzyme electrode using glucose oxidase, invertase, mutarotase, fructose-5-dehydrogenase, and catalase to simultaneously detect glucose, fructose, and sucrose in fruit juices and soft drinks. Detection of multi-components by enzyme sensors was also reported in analysis of sucrose and glucose in honey (42) and drinks (43), and L-malate and L-lactate in wines (44). Although electrode is used by most researchers, other t r a n s d u c e r s including non-glass electrode also have been investigated. Thermistor is
336 Table 3 Typical Food Analytes Analysis
Analytes
Proximate
Moisture, protein, fat, ash
Carbohydrates
Glucose, fructose, sucrose, lactose, maltose, galactose, maltose, oligosaccharides, starches, gums
Acids/Alcohols
Acetic, citric, lactic, phosphate, ascorbic, fatty acids, ethanol, methanol
Elemental Analysis
Ca, ionic Ca, P, C1, Na, K, Fe, heavy metals
Nutritional labeling
Minerals, cholesterol, vitamins, fibers, fatty acids, amino acids
Additives/Preservatives sorbic acid, benzoic acid, BHT, BHA, tocopherol Proteins
Individual protein species and denatured forms
Inhibitors/Toxins
Trypsin inhibitors, aflatoxins, exotoxins (botulinum), endotoxins, PCBs, vomitoxin
Pesti cid e s/H erbi ci des
Carbamates, chlorinated aromatics, atrazine, 2.4-D DDT, diazinon
Drugs/Hormones
Chloramphenicol, penicillin, sulfamethazine, growth hormones
Microorganisms
Bacteria, yeast, molds, pathogens, phages
another transducer used in early enzyme-based biosensor development. It is more rigid and stable than the electrode (45, 46, 47). Alcohol oxidasethermistor had been used to monitor ethanol fermentation (48). Sevilla III et al (49) immobilized ~-galactosidase and glucose dehydrogenase on the surface of field effect transistor's gates for lactose analysis. Fiber optic enzyme sensors have also been used in food analysis (11, 50). Electrodes made from platinum, graphite, and carbonic composite material had been used in the construction of the enzyme sensor (51, 52, 53). The rigidity of these electrodes provides great opportunity in food applications and are suitable for on-line analysis. Appelqvist and Hansen (37)used a glucose oxidase-graphite electrode in the determination of glucose in wine fermentation. In addition to analyzing compounds, enzyme sensor has been used to determine the freshness of meats. Xanthine oxidase has been used to determine the levels of xanthine and hypoxanthine that are accumulated from purine degradation during muscle aging so as to monitor fish freshness for a long time. Traditional methods including the automated colorimetric method (54) were time consuming. J a h n et al (55) developed a dipstick test by
337 immobilized xanthine oxidase and dye on dry paper. Suzuki et al. (56) designed a disposable xanthine oxidase electrode. Xanthine oxidase enzyme sensor for fish freshness was one of the earlier commercial biosensors. Recently, biotinylated bilayer lipid membrane technology (57)was used to design an amperometric xanthine oxidase mini-biosensor with less than one minute response time and 5 days stability (58). Stability, duration, sensitivity, interference, and availability of substrates to contact enzymes are the criteria for the success of an enzyme sensor. These criteria depend on sources of enzymes, immobilization techniques, and transducers used. Food matrices are much more complicated than the clinical samples, hence, these criteria become extremely important for the application of the enzyme sensor in food analysis. An extensive list of the response time, detection limits, and stability of biosensors was summarized by Wagner (59). 3.2. I m m u n o s e n s o r s Development of immunosensors is at a racing pace. It uses antibodyantigen reaction in biological component of the biosensor. The specificity and sensitivity of the reaction make antibody an ideal molecular recognition element in biosensor design. The optical, acoustic, and electrical changes during the reaction can be monitored by any of the transducers described previously. Enzyme-linked antibody techniques such as ELISA have also been used by researchers to either magnify signals or to generate a measurable signal. Although amperometric and potentiometric transducers were used the most, piezoelectric crystal became attractive to the researchers lately (60, 61). Immunotechniques have recently been developed to detect food contaminants, e.g., toxins, growth hormone, antibiotics, pesticides, and herbicides. Penicillin (62)in milk, aflatoxins and mycotoxins (63, 64, 6 5 ) i n milk, cheeses, yogurt, corn have been detected by i m m u n o s e n s o r s . Characteristics of protein and receptors in or on the cell surface were used in detecting pathogens such as Listeria and Salmonella by immunosensors (11, 66). The principle of immunosensors has also been applied in pesticide determinations (67, 68). 3.3. Cell a n d Tissue Sensors Utilization of whole cells and tissues in biosensor has increasingly been used. Enzyme stability, availability of different enzymes and reaction systems, and characteristics of cell surface are the advantages of using cells and tissues in biosensor designs. Multi-step enzyme reactions in cells also provide mechanisms to amplify the reactions that result in an increase in the detectability of the analytes. The presence of cofactors such as NAD, NADP, and metals in the cells allows the cofactor-dependent reactions to occur in the absence of reagents. (34, 50, 69). However, the diffusion of analytes through cell wall or membrane imposes constraint to this type of biosensors and results in a longer response time compared to the enzyme biosensors. Oxygen electrode is commonly used in such biosensors. N u m e r o u s microorganisms and animal and plant tissues were used to detect various compounds such as sugars, amino acids, organic acids, and vitamins. Cell biosensors were also successfully used to monitor biological oxygen d e m a n d (BOD) in drinking water and waste stream (Table 4). Whole cell and tissue biosensors have great potential to replace the traditional and tedious analysis
338 Table 4 Examples of Cell and Tissue Based Biosensors (10, 70). Analyte
Microorganism and Tissue Cells
Reference
Glucose, Sucrose, Fructose
Pseudomonas fluorescens, Bacillus subtilus, Brevibacterium lactofermentum
71, 72, 73
Glutamic acid, Lysine, Tyrosine
Escherichia coli, Bacillus subtilus, Aeromonas phenologenes, Porcine kidney mitochondria, Sugar beet
74, 75, 76, 77, 78, 79
Acetic acid
Trichosporon brassicae
80
Nicotinic acid, NAD § Nicotine amide, Ascorbic acid
Lactobacillus arabinosus, Escherichia coli, Enterobacter agglomerans, C u c u m b e r
81, 82, 83, 84, 85
Biological oxygen demand (BOD)
Pseudomonas sp., Bacillus subtilus, 86, 87, 88, 89 Trichosporon cutaneum, 90 Thermophilic organisms
of amino acids and vitamins. The detailed information on types of microorganism used, analytes detected, response time, detection range, and the stability has been reported in literature (9, 70). 3.4. C o m m e r c i a l Biosensors One of the first commercial biosensors in food applications is the glucose electrode (91), and, by far, the most successful one. Yellow Spring I n s t r u m e n t Co. in Ohio marketed the first one used for the dairy and beverage industries in 1979. Three types of glucose analyzer based on glucose oxidase electrode are commercially available today (Table 5). Since 1979, a vast number of companies have begun to supply various enzyme sensors to food industries (Table 6). Most commercial enzyme electrodes monitor the oxygen consumption or hydrogen peroxide production amperometrically and potentiometrically. Bioanalyzer can be considered another version of commercial biosensors for off-line analysis. It was developed to have capabilities of complete analysis, short response time, specificity, and sensitivity that allows a quick clinical test. Abbott Vision, Boehringer-Mannheim Reflectron, and Kodak Ektachem DT60 (IBI Biolyzer is the new name) are used for cholesterol m e a s u r e m e n t in doctors' offices. Bioanalyzer consists of biological and transducing component that are not physically connected. The uniqueness of this separation provides the versatility of analysis, i.e., use of disposable and different biological component for multi-components' measurements. In authors' laboratory, Kodak Ektachem DT60 was used successfully to determine cholesterol in some food matrices as well as in off-line process control. The analysis time was only 10 minutes compared to 1-2 days for the GC and HPLC methods. Complicated
339 Table 5 Selected Commercial Glucose Analyzer Based on Glucose Electrode (59, 92) Analyzer Manual: Yellow Spring Inst. (USA) Model 23A Model 2000 Fuji Electric (Japan) Gluco 20A Solea-Tacussel (France) Glucoprocesseur
Detection Range
Samples/hr
Stability
1.0 - 45.0 mM 1.0 - 8.0 mM
40 40
300 samples 21 days
0 - 27.0 mM
80
500 samples
0.0001- 1.0 mM
40
1000 samples
Automatic 9 Eppendorf-NethelerHinz G m b H (German) ESAT 6660
0.6- 45 mM
120
2000 samples
OnLine 9 Life Science Inst., Miles Lab. (USA) Biostator GCIIS
up to 27.5 mM
50 hours
sample p r e p a r a t i o n and use of expensive i n s t r u m e n t and t r a i n e d c h e m i s t s can be eliminated. The sensitivity is lower t h a n the GC and H P L C m e t h o d s . However, this problem can be overcome by a simple sample c o n c e n t r a t i o n step. Very few i m m u n o s e n s o r s are commercially available. The c o m m e r c i a l i m m u n o s e n s o r s are either the detector or bioanalyzer types. The PZ 106 i m m u n o s e n s o r from Universal Sensors Inc. (New Orleans, LA) has been u s e d as a detector to m e a s u r e antibody-antigen reaction. Ohmicron (Newtown, PA) developed a series of pesticide i m m u n o - b i o a n a l y z e r s t h a t have been used in field tests. P h a r m a c i a Biosensor USA (Piscataway, N J ) r e c e n t l y i n t r o d u c e d BIAcore i m m u n o d e t e c t i o n system. A combination of a unique flow injection device and surface plasmon resonance ( S P R ) d e t e c t i o n technique provides a real time analysis. A c a r b o x y l m e t h y l d e x t r a n layer added to p l a s m o n g e n e r a t i n g gold film is a hydrophobic, activatable, and flexible polymer t h a t provides high antibody and low non-specific bindings. System d e m o n s t r a t i o n at the Institute of Food Technologists (IFT) 1994 meeting in A t l a n t a d r e w attention of food scientists. It should easily be adapted for food protein characterization. 4. F U T U R E AND TODAY The a d v a n t a g e s of biosensor are specificity and rapid response time. It offers food scientists and processors tests which are inexpensive, fast, a n d easy-to-use. Also, it allows one to detect extremely small a m o u n t s of analytes
340 Table 6 Some Commercial Biosensors Used in Foods and Fermentation (4, 16) Biosensor
Company
Immobilized glucose oxidase with an oxygen electrode
Oriental Electric Co. Ltd., J a p a n Toyo Jozo Co. Ltd., J a p a n
Immobilized glucose oxidase with a hydrogen peroxide electrode
Yellow Springs I n s t r u m e n t Co., USA TOA Electronics Ltd., J a p a n Fuji Electric Co. Ltd., J a p a n
Immobilized oxidase systems with oxygen electrode (lactic acid, alcohol)
Toyo Jozo Co. Ltd., J a p a n
Immobilized oxidase systems with an electrode
Analytical I n s t r u m e n t Co., J a p a n
Immobilized xanthine oxidase with a polarographic electrode (fish freshness)
Pegasus Industrial Specialties Ltd., Canada
Soluble oxidase with oxygen electrode (fish freshness)
Oriental Electric Co. Ltd., J a p a n
Enzyme (customer specified) trapped at the end of an oxygen electrode
Provesta Corp., USA
Immobilized yeast cells with an oxygen electrode (BOD monitoring)
Nissin Electric Co. Ltd., Japan
Immobilized enzyme (wide range) m e m b r a n e s with oxygen, ammonia, or carbon dioxide electrodes
Universal Sensors, USA
in foods. Although biosensor development, by far, was focused in non-food areas, the analytes are also found in foods. Research and development in food application are drastically growing. Controlled immobilization, increase in sensitivity and stability, reproducible signal to noise ratio, multi-component detection in foods, and robustness should be continually emphasized in future biosensor development. I m m u n o s e n s o r s for food protein characterization are the future biosensor application in the food analysis. Antibody-antigen reaction is a highly selective protein-protein interaction, which can differentiate protein species in a complex food matrix and measure the conformational changes in specific protein such as denaturation, disulfide crosslinking, and interactions with non-protein substances. The protein denaturation and interactions with other compounds have been known to be critical in food processing and product texture. Future development of immunosensors in this area will allow food scientists to u n d e r s t a n d the changes in properties of proteins and their interactions with other food ingredients during processing. Furthermore, food
341 scientists are able to concisely select ingredients and processing to produce foods with desirable and designed texture. Presence or contamination of proteases and lipases in some foods is detrimental to the shelf life stability. Peptides generated from proteolysis and fatty acids generated from lipolysis give food undesirable characteristics, e.g., soft texture, deformed structure, bitterness, and rancidity. Although the enzyme test kits provide the convenience of detection, the sensitivity and assay time are not adaquate for the detection of minute amounts of these enzymes in foods. Specificity of antibody against the enzyme molecule enables the immunosensor to detect the presence of those enzymes, which allows food processors to take a corrective action at an early stage of processing. Antibody reaction between inactive and active enzyme molecule needs to be differentiated, or an antibody reacting only with the active enzyme needs to be developed for this application. Immunosensor will become key analytical tools in future food safety monitoring. Capability of detecting varieties of pathogens will be needed. D NA biosensor is also a powerful tool to detect microorganisms. It is a newly developed area. DNA techniques have been developed and used in food microbiological analysis recently. Integrated Genetics in Massachusetts is developing a DNA probe for the detection of Salmonella in food. Use ofbilayer lipid membranes as a generic electrochemical transducer is an exciting future for food biosensors. A taste sensor with m u l t i c h a n n e l e d lipid membrane electrode was recently developed (93). The electric p a t t e r n s generated from the sensor are similar to h u m a n response. The sensor can distinguish different brands of beer. More details on the taste sensor can be found in Chapter 16 of this book. Biosensors provide great potential for on-line food quality and safety controls. Researchers and manufacturers will have to overcome the h u r d l e s such as instability of biological compounds and consistency of immobilization in mass production. Food scientists and processors shGuld also consider the complication arising from food matrices, processing conditions, and safety regulations in biosensor applications, especially for on-line applications. Massive food components may hinder analytes available for biological reaction. Processing conditions such as heat and pH are often detrimental to biological components of the biosensor. The severe conditions in sanitation m a y completely destroy the functions of biological recognition elements and possibly damage the transducing device. Possible contaminations due to compounds from the recognition elements and the biological reactions are also of concern. An ideal on-line biosensor for food processing should have the characteristics listed in Table 7. Because of the great opportunity provided by the biosensor technology, food scientists and processors should aggressively and actively participate in the research and provide the critical information needed to impact and accelerate the development of biosensors for the food industry. Several r e s e a r c h programs, specifically for food applications, have been established in the United State and other countries, i.e., Center for Process Analytical Chemistry (CPAC) at the University of Washington, WA, Electronic Design Center at the Case Western Reserve University, OH, Research Center for Advanced Science and Technology (RCAST) at the University of Tokyo, Bio-Resources Division at Tokyo Institute of Technology, Japanese Food Sensor Research Association,
342 Table 7 Characteristics of A On-line Biosensor P~rf0rmance
Physical Operation
Long-term stability Fast responding time Highest accuracy and precision Lowest drift Low protein clogging Self-cleaning capability Electrolyte flexibility Electrode lifetime Low flow dependence
Sanitizable, autoclavable, no drift Sterilizable in place without drift Simple to use, minimum training Low cost Low maintenance Small size Retractable and reusable
Multi-divisional program in biosensors at the Food Research Laboratory of Commonwealth Scientific and Industrial Research Organization (CSIRO)in Australia, Food Hygiene Department of the Campden Food and Drink Research Association (CFDRA) in the United Kingdom, and the Center of Chemical Sensors/Biosensors at the Swiss Federal Institute of Technology in Switzerland. REFACES
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Characterization of Food" Emerging Methods A.G. Gaonkar (Editor) 9 1995 Elsevier Science B.V. All rights reserved.
347
Chapter 15 D e v e l o p m e n t s i n c h a r a c t e r i z a t i o n of foods u s i n g a n t i b o d i e s Zohar M. Merchant and S. G. Greg Cheng Kraft Foods Inc., 801 Waukegan Road, Glenview, IL 60025, U.S.A. 1. INTRODUCTION A typical food material is composed of carbohydrates, proteins, lipids, nutrients, flavorants and other additives. In a processed food, these ingredients are subjected to processing conditions, long storage and distribution cycles prior to consumption. Maintaining the quality of food without spoilage over its long shelf-life is the challenge facing food producers today. This, coupled with the introduction of the new labeling guidelines, makes it critical to develop newer and better methodologies for characterization of food components. Antibodies have been known for over a hundred years. However, their application has seen a significant growth in the last 20 years. In a pioneering work, Kohler and Milstein (1)demonstrated that combination of the nuclei of normal antibody-forming cells with a malignant counterpart resulted in monoclonal antibodies. Developments in hybridoma technology, recombinant technology, protein engineering and other areas of biotechnology havc resulted in numerous antibody-based products in the market place. The most predominant application of antibodies has been in the area of diagnostic assays, known as immunoassays, which exploits the specific interaction between the antibody and antigen. The world i m m u n o a s s a y market for clinical and food diagnostics, environmental analysis and other applications exceeded $ 1.2 billion in 1 9 9 0 . ELISA (Enzyme-Linked Immunosorbant Assay) represents 60% of that market (2). Annual growth rates have been projected at 10-15%. The food diagnostics is projected to grow to $ 500 million by the year 2000 (3). This chapter will review recent developments in the use of antibodies in food characterization covering immunoassays, their development , application and value in ingredient identification (section 2), immunoadsorption or immunoaffinity purification(enrichment of ingredients present at low levels, section 3), immunocytochemistry (site-specific localization of ingredients within the food matrix, section 4), quality control/functionality determination (section 5), process control (section 6)jand future directions (section 7). 2. IMMUNOASSAYS A food analyst performs numerous physical and chemical analysis r a n g i n g fromspectroscopic, chromatographic, electrophoretic on one side, to elemental and mass analysis on the other, They may be easy to perform or complicated, requiring sophisticated equipment providing qualitative, semi-
348 quantitative and quantitative information. Some of these conventional methods are often laborious, time consuming and inconvenient. This results in cosily holding of perishable foods. Clearly, a need exists for rapid, sensitive and convenient methods of food analysis. One such rapid method is an immunoassay. Immunoassays are analytical techniques involving binding between an antibody and an antigen. They are highly specific and sensitive, requiring minimal sample preparation. The last 10 years has seen a significant growth in the use of immunoassays in the food industry (4-14). The early efforts were focused on the generation of appropriate antibodies against different types of antigens. These ranged from large molecular weight materials having several immunodeterminant regions to very small molecular weight (< 5000 Daltons) haptens conjugated to carrier proteins. They were used for detection of microorganisms (15-19), toxins (20-27), adulterants (28-33), allergens (34-37), and other contaminants/additives (38-40) present in the food systems. Recently, the emphasis has been on increasing the sensitivity, shortening the assay time, increasing laboratory-to-laboratory reproducibility, reduction of false positives or negatives and greater ease of use. This has lead to the development of different types of amplification systems, production of more specific monoclonal antibodies, more accurate dipsticks for in-field use and development of sophisticated automatic or semi-automatic computer-controlled detection systems (11-13). A typical immunoassay entails 6 steps: 1) Antigen: preparation/purification of antigen, and coupling of hapten to carrier~ i f needed. 2) Antibody: generation/purification of polyclonal or monoclonal antibodies in animals or by tissue culture. 3) Antibody-antigen reaction: via direct observation using precipitation and agglutination methods. 4) Conjugation: to different types of labels, i.e., radio, enzyme, fluorescent, precipitation, luminescent, etc. 5) Amplification: via enhanced binding, faster cell growth and coupling to coagulation cascade etc. 6) Detection: colorimetric/spectrophotometric, potentiometric, scintillation counter, turbidity, precipitation, etc.
2.1. Antigens A variety of substances can simulate antibody production, including, proteins, polysaccharides, nucleic acids, pollens, viruses, toxins, etc. These molecules which solicit an immune response and react with a specific, corresponding antibody are called antigens. These can range in size from large molecular weight polysaccharides or proteins which may have several immunodeterminant sites to small molecules (below 5000 Dalton molecular weight) which are not immunogenic and need to be conjugated to a macromolecule carrier such as a protein to induce antibody formation (5, 10, 41). These small molecules are known as haptens and include drugs, hormones, vitamins, toxins and pesticides. The carrier proteins are usually bovine serum albumin (BSA), Keyhole Limpet Hemocyanin (KLH), polylysine
349 or other large peptides. Another way to increase the immune response of an antigen is by use of adjuvants like Freund's complete and incomplete adjuvants consisting of dead M. tuberculosis cells in an oil-in-water emulsion. 2.2. Antibodies Antibodies are complex molecules that recognize and bind to foreign substances targeting them for destruction. The typical antibody molecule (Fig. 1) consists of two identical glycosylated heavy chains (H) of 450 to 575 amino acid residues (relative molecular weight 50,000 to 75,000 daltons) and two identical light chains (L) of 220 amino acids (molecular weight 50,000 daltons). The heavy and light chains can be divided into variable (V) and constant (C) regions. The variable domain formed from a light and heavy chain is responsible for the recognition and binding of antigens and the constant domain formed from two heavy chains mediates binding to the host tissues and cell receptors. The H and L chains associate via the disulfide bonds. Antibodies are susceptible to proteolysis, particularly in the hinge region (Fig. 1). Treatment with papain produces two antigen-binding fragments, Fab and one crystallizable fragment Fc while reaction with pepsin yields a bivalent antigen-binding fragment, F(ab')2 (41, 42). The specificity of antibody-antigen interactions is a result of a structural fit between the antibody binding site and the antigenic determinant. An understanding of the mechanism of the antigen-antibody interactions has revealed the existence of hypervariable and conserved regions. The
Figure 1. Structure of antibody.
350 hypervariable regions are called the complementarity-determining regions (CDR's). There are 3 CDR's in each of the Light and Heavy chains resulting in a total of 6 CDR's forming the antibody binding site. The conserved regions are called the framework regions (FR's) and are responsible for supporting the structural conformations of variable domains (42). For a more detailed description of the antibody structure one should refer to Davies and Chacko's (43) article. The forces responsible for the antigen-antibody interactions are electrostatic, hydrogen bonding, van der Waals and hydrophobic effects. Typical equilibrium binding constants range from 10-5 to 10-12 M. Antigen-antibody interactions are usually stable over a pH range from 4 to 9 and a wide salt concentrations (0 to 1.0 M NaC1). Dissociation of the antigen-antibody bonds requires use of strong denaturing conditions, such as pH of less than 3.0 or greater than 10.0 or high concentration of chaotropic agents (i.e., urea (9M), guanidinium-HC1 (6M)) (41). Animals produce several clonotypes (polyclonal, Fig. 2 ) o f antibodies upon i m m u n i z a t i o n with a given antigen such that the recovered serum contains antibodies against different determinants having variable specificities whose affinity and yield varies from bleed to bleed; thus, m a k i n g it very difficult to
Figure 2. Preparation of polyclonal antibodies.
351 obtain a consistent and reproducible supply of antibody containing s e r u m . Additionally, significant amounts of contaminating immunoglobulins may be present, entailing purification. Also, pure antigen may be required. For most routine applications they are still the antibodies of choice. Monoclonal antibodies, on the other hand, are homogeneous, have uniform specificity a n d constant affinity for a single determinant site. They are produced by fusing the spleen cells from a mouse with myeloma cells (Fig. 3). A single clone that can be maintained in vitro is used to generate large quantities of a single antibody cell line. The limitations with monoclonal antibodies is that they may be too specific for certain requirements and unexpected cross reactions may occur.
Figure 3. Preparation of monoclonal antibodies in mice.
352
2J]. Antibody-antigen reactions A wide variety of methods (45) have been developed for detecting the antibodyantigen reactions based on direct observation of antibody-antigen reaction by precipitation and agglutination. 2.3.1. P r e c i p i t a t i o n Precipitation is a secondary interaction dependent upon the divalent n a t u r e of the antibody and presence of a multivalent antigen resulting in direct observation of antibody-antigen reaction. Here, soluble antigens are mixed directly with corresponding antibodies to form a complex which m a y precipitate under appropriate conditions. In a typical i m m u n o d i f f u s i o n system, the antibody or antigen are in a solution in a tube or in wells formed in a gel matrix. The point where the two meet after diffusion results in the formation of a precipitate. The precipitation patterns give information on the presence of antigens (Fig. 4). The detection limits range from 0.1 to 10 ~g/ml (46, 47).
2.3.2. A g g l u t i n a t i o n The agglutination reaction is another type of precipitation reaction in which one of the components is present in a particulate form. The use of solid support like latex beads on which to coat a soluble antigen for an agglutination reaction with the corresponding antibody has greatly facilitated its ease of use. These assays are easy to perform; however, they can give misleading results at low analyte concentration or interference from the sample matrix (48).
2.4. Conjugation Here, the antibody-antigen reactions are observed indirectly by use of labels which are attached to either the antibody or antigen. The labels can be conjugated covalently and include radioisotopes, enzymes, labeled second antibodies, fluorescent tags, luminescent molecules and phages (12, 41, 45, 48). The use of labels helps in increasing the sensitivity of the assays considerably compared to the precipitation or agglutination assays. The labeled i m m u n o a s s a y s can be liquid-phase or solid-phase. The solid supports employed in solid-phase assays include polystyrene 96-well plates or dipsticks, glass beads, nylon or nitrocellulose membranes, agarose or magnetite coated agarose to allow efficient separation of bound from unbound reactants. Immobilization of antibodies or antigens can be achieved via adsorption or covalent coupling methods (49). The most common form of solidphase assay employs 96-well plates which are coated with either the antibody or antigen of interest (Figs. 5, 6). The coating step is followed by a blocking step employing nonspecific proteins (such as gelatin, bovine serum albumin or milk proteins) or surfactants (such as Tween 80 or Triton X-100) to minimize nonspecific binding. These same proteins and/or surfactants can also be used in the sample and rinse buffers to prevent non-specific binding. The plates are read in a detector.
2.4.1. Radio Immunoassay (RIA) In a RIA, the antibodies or antigen are labeled with a radioisotope such as iodine 125 or 131, tritium, or carbon-14. There are two types of RIA: a liquid-
353
Figure 4. Immunoprecipitation assay. (Reprinted by permission of Chapman & Hall, NY)
354
Figure 5. Solid-phase binding assay for specific antibody.
Figure 6. Solid-phase binding assay for specific antigen. phase RIA and a solid-phase RIA (12, 50, 51, 52, 53). They are more sensitive than the precipitating immunoassays. However, the cost of maintaining a lab with radioisotopes including both cost of equipment and supplies and disposal of the spent radioactive material has lead to a search for other types of labels.
2.4.2. Enzyme-l,inked Immunosorbant assay OELISA) ELISA's (54, 55)are one of the most commonly used immunoassays in the food industry for detection of a wide variety of substances including contaminants, toxins, adulterants, herbicides and carcinogens. They use an enzyme as a label and visualization is achieved via conversion of the substrate to a colored product. There are different types of ELISA's i.e., competitive (Fig. 7), non-competitive (Fig. 8), sandwich (Fig. 9) and homogeneous enzyme immunoassay (48). The enzymes commonly used are pure and have a high turnover n u m b e r (48). They include horseradish peroxidase (with o-phenylene diamine as substrate which yields a yellow colored product, 2,2'azino-(3ethyl)-benzosulphonic acid gives green colored product, or tetramethyl benzidine gives a blue p r o d u c t ) a n d alkaline phosphatase (with p-nitro phenyl phosphate as substrate to give a yellow/orange colored product). The ELISA techniques offer advantages of longer shelf life of the labelled reagents and elimination of the use of radioisotopes which require expensive scintillation/gamma counters and special disposal needs (38, 56). They are also more sensitive than the RIA's. ranging from ~g/ml to pg/ml depending on the size of the molecules, affinity of the antibody and the assay format used (48).
355
Figure 7. Competitive ELISA. (Reprinted by permission of Chapman & Hall, NY)
356
Figure 8. Non-competitive ELISA.
2.4.3. Immunofluoresence Assay (IFA) The IFA is an i m m u n o a s s a y in which the antibody or antigen are labeled with a fluorescent probe. These can be direct or indirect (8, 41, 57). The I F A technique is usually used to locate cellular constituents. The commonly used fluorescent probes are rhodamine B isothiocyanate and fluorescein isothiocyanate. The sensitivity of the assay ranges in the ng/ml range.
357 Adsorb the antibody to a solid support such as the surface of a plastic tube, multiwell plate, latex particle, magnetic particle, nylon, nitrocellulose, or glass fiber filter.
Add the sample to the solid phase antibody. The solid phase antibody will specifically capture any target antigen from a complex sample. Rinse the unbound sample material from the solid phase antibody.
Add enzyme-labelled antibody, specific for the target antigen, to the solid phase. The "tagged" antibody will attach to any antigen captured by the solid phase antibody. Rinse the unbound enzyme-labelled antibody from the solid phase complex.
Add enzyme substrate. The amount of colored reaction product (O) that developed is proportional to the amount of antigen in the sample.
KEY:
Figure 9. Sandwich ELISA. 2.4.4. L u m i n o I m m u n o a s s a y (LIA) The LIA is an i m m u n o a s s a y in which the antigen or antibody are labeled with either a c h e m i l u m i n e s c e n t or bioluminescent tags (41, 58). L u m i n e s c e n t molecules are produced by oxidation reactions. Bis-phenyl oxalates in presence of hydrogen peroxides are used for c h e m i l u m i n e s c e n t assays and luciferin in presence of luciferase enzyme is used for bioluminescent assays. The sensitivity of the LIA's are in the pg/ml or lower range.
358
2.5. Amplification Systems for Immunoassays The need for shortening the time and increasing the sensitivity for detection of antigens has lead to development of different amplification systems. Some of the initial efforts focused on use of more pure antibodies (59, 60), more specific monoclonal over less specific polyclonal antibodies (61) and use of a combination of monoclonal antibodies (62). The next phase saw the incorporation of labels as discussed in the previous section. The use of labels does increase the sensitivity; however, there is a need to go down in detection levels to enable faster turnaround time for immunoassays. This can m e a n significant savings in the food industry. In the case of Salmonella, the assay time is being reduced from several days to less than a day (63, 64).
2.5.1. Biotln-Avidin amplification system Avidin (present in eggwhite) binds very strongly with biotin (K=10-15/mol). Advantage has been taken of this tight binding by conjugating either antibody or antigen with avidin (or biotin) and have the enzyme complex with biotin (or avidin). This may result in one to two orders of magnitude amplification in the signal. This approach was applied for detection of whey proteins in meat products and led to an improvement of detection levels from 4~g/g to l~tg/g (65). Avidin may exhibit some non-specific binding to tissues due to its highly basic nature with an isoelectric point at pH 10. This may be overcome by use of nonfat dry milk (66). The other limitation with the use of avidin is in products which may contain eggs. One may then have to use streptavidin. 2.5.2. Coupled enzyme systems Coupled enzyme systems have been used in enzymology to significantly increase the sensitivity of detection of enzyme-substrate reactions. The addition of coupled enzyme systems to an enzyme immunoassay should lead to several orders of magnitude increase in the sensitivity (48, 67). 2.5.3. E n z y m e linked coagulation assay for amplification of ELISP. This assay was developed by G.J. Doellgast (68, 69). The principle of the technique is a combination of ELISA with the enzyme linked coagulation assay (ELCA) amplification system (Fig.10). The ELISA phase consists of a captag or sandwich immunoassay employing Russell's viper venom factor X activator (RW-XA) as the labeling enzyme which produces antibody-antigen-RVV-XAantibody complex. The ELCA phase consists of (i) generation of thrombin by use of a substrate comprising of factors X, II and V and (ii) formation of a alkaline phosphatase-fibrin-solid fibrin complex by interaction of thrombin with alkaline phosphatase-fibrinogen plus solid-phase fibrinogen. The ELCA complex is detected by monitoring a change in color upon addition of substrate for alkaline phosphatase. By use of this amplification system, an increase in sensitivity of 100 to 1000-fold was achieved compared to a traditional ELISA for detection of Clostridium botulinum Neurotoxins A, B and E, at sensitivity levels of 5-10 pg/ml (70, 71, 72). This is comparable to mouse bioassay, but unlike the mouse bioassay which takes 3-5 days this can be done in 1-2 days. The limitation with this assay is that numerous washing steps are required and there is a need to optimize and establish concentration ranges. The authors (71) have eliminated the need for a single multiwell microtiter plate per toxin by
359
Figure 10. ELISA-ELCA combination assay. (Reprinted by permission of CAE. IAMPES, Inc.)
360 use of avidin-biotin system or chicken antibody, measurement of several toxins with a single plate.
thereby,
allowing
2.5.4. Continuous turbidimetric system In a typical immunoassay, the sample of the food product undergoes a certain level of preparation, depending on the antigen to be measured. This is followed by time consuming dilution/washing and incubation steps. If one wants to follow a process on-line for monitoring specific analytes produced during a process, it is difficult. Freitag et al (73) made use of an immunoprecipitation reaction between bivalent antibodies and multivalent antigens which causes light scattering, leading to turbidimetric detection. Assay automation was achieved by utilizing the flow injection analysis principles. They were able to reduce the time for an assay cycle down to 2.5 minutes and a detection limit of I ~tg/ml. One needs to take into consideration, however, the intrinsic turbidity of the sample and make correction for it in the calibration curves. This on-line system should be useful for continuous processes where the antigen concentration is above the detection limit of 1 ~g/ml. 2.5.5. Other systems for shortening immunoassay time Some other forms of IA shortening protocols involve the use of different formats; i.e., dipsticks instead of microtiter plates. The dipstick protocol typically requires very small amounts of sample (<1 ml), is user friendly with minimal to no washing steps and no expensive is equipment needed. L u m a c has come out with a Salmonella dipstick assay which takes 20 minutes to perform. It has 2 colored lines developing when Salmonella is present and only I line in its absence (74, 75). The dipstick protocols are good for determining the presence or absence of antigens. However, it can give only qualitative to semiquantitative results which may be useful for field trials or rapid screening of large batches of samples. 2.6. The detection systems for immunoassays The detection system employed for monitoring antibody interactions with antigens will depend on the type of IA performed. The majority are batch systems and are performed off-line. Quantification is achieved by either measuring the absorbance with spectrophotometer, excitation or emission with flourometer, radioactivity decay with a scintillation counter or visualization of direct precipitation. Typical formats used include microtiter plates or membranes or dipsticks or gels. The assay time can range from less than a minute for precipitation/agglutination assays to minutes for the dipsticks onfield assays and hours for microtiter plate-based laboratory assays (48). 2.6.1. Abbott TDx analyzer An interesting detection system which needs no separation step and is amenable to automation is the Abbott TDx analyzer. It is based on m e a s u r e m e n t of increase in polarization of the fluorescence of a small fluorescent labeled antigen when bound by a larger molecular weight antibody. It has been used for detection of contaminants, especially smaller antigens like drugs and steroids in food (76). Its detection range is ~g/ml which can be
361 lowered to ng/ml by use of larger sample size. It is specifically useful in cases where sensitivity is not vital or where it is possible to pre-enrich analytes from samples prior to assaying. It takes 30 minutes to perform the polarization fluorescence immunoassay. 2.6.2. Biomolectflar interaction analysis system A more expensive and sophisticated detection system which can, unlike any of the other systems, perform measurements in real time without the use of labels is the Pharmacia Biomolecular Interaction Analysis (BIA) system (77). The BIA analysis uses the optical phenomenon of Surface Plasmon Resonance to monitor the physical interaction of biological molecules, providing information on association, equilibrium/steady state and dissociation. The system has a gold sensor chip to which the antibody can be covalently bound, followed by a blocking buffer. This is followed by injection of the analyte and the rate of change of reflectance corresponding to the absolute concentration of the analyte. The sensitivity is 10-3M to 10-1OM. This system, unlike RIA or EIA, requires no labels for detection and hence does not alter either the antigen or antibody due to labeling. Furthermore, it eliminates the rinsing and w a s h i n g steps, as there is no physical separation between the interaction and detection steps. Analysis after the covalent binding step takes only minutes. A standard curve plotting the rate of change in reflectance units versus analyte concentration needs to be established. Also, the dilution analysis to be performed h a s to be within the standard curve range. The bound antibody on the gold plated sensor can be re-used 50 times. The system is fully automated. 2.6.3. CREAM video image processing system The trend in the detection system employed is more towards automation. A CREAM video image processing system developed by Brogan and co-workers (78) enables one to evaluate and store ELISA data for further statistical manipulation. The CREAM EIA software gives an accurate estimation of sample concentration. It can also be used for quantitation of DOT-blots. 2.7. The value of i m m u n o a s s a y s Prior to application of immunoassays for detection of antigens, it is important to do a cost/benefit analysis to establish whether it adds value as compared to other analytical techniques. The value of i m m u n o a s s a y depends on its (1) performance versus other analytical techniques, ( 2 ) e a s e of use, (3) type of environment i.e., on site or in the field, (4) cost, and (5) return on investment (ROI) (48, 79, 80) 2.7.1. P e r f o r m a n c e The type of factors one needs to consider include the sensitivity, specificity, reproducibility, robustness and reliability of the IA. IA, because of their selectivity and sensitivity, lend themselves to a wide range of applications in the food area. They can detect antigens from ~tg/ml to pg/ml and this can be increased by 2-3 orders of magnitude by use of amplification systems. The coefficient of variance can be from 10-15%. The antigen of interest may be present in a number of different types of food systems and the IA needs to be robust. The reliability of IA has to be monitored carefully; there can be
362 variability from laboratory-to laboratory due to the type of reagents used, especially the source of antiserum/cross-reactivity/non-specific binding leading to false positives or false negatives, the level of training of analysts and the type of IA format used i.e., RIA, EIA, IFA, LIA. 2.7.2. Ease of use Here, one needs to consider the logistics; i.e., sample loading, sample preparation time (some samples may require complicated treatment like preenrichment steps for Salmonella detection), assay procedures; i.e., time consuming protocols entailing numerous pipetting and washing steps could be automated to free up personnel time. Other considerations include assay design for multiple assay to be performed at the same time.
2.7.3. Type of Environment Here, the type of assay format selected will depend on whether it is performed on the field or in the lab. For field testing, dipstick formats may be more appropriate versus the multiwell microtiter plates read on spectrophotometers. It is simple and easy to use and can be performed in minutes (74, 75). 2.7.4. Cost This will depend on the level of skill and experience needed, equipment cost for spectrophotometers or ELISA readers, cost of consumables and finally the throughput. (79) Food immunoassays can cost typically anywhere from $1 to $15 per test. 2.7.5. ROI This can improve considerably depending on the type of product analyzed. I n industries where stock movements are fast or huge volume/tonnage are involved, or when ingredients are expensive, rapid testing saves time which allows faster release of uncontaminated foods and results in significant cost savings (80). 2.8. Application o f i m m u n o a s s a y s in food systems Food products are prone to accidental or deliberate abuse anywhere in the food processing/storage/distribution/consumption cycle, starting from r a w materials to finished products. Faced with this problem a food analyst needs rapid, sensitive, reliable and cost effective techniques. Immunoassays are ideally suited for this type of tasks and their role in food diagnostics is expanding due to the numerous analytes like adulterants/additives, allergens and contaminants/toxins that it can detect. For more detailed information on applications of immunoassays one can refer to some of the recent review articles and books (11-13, 38, 44). 2.8.1. A d u l t e r a n t s / A d d i t i v e s Adulterants are any material deliberately added to the food material usually to reduce cost. Typical examples are incorporation of cheaper meats into more expensive ones or substituting non-meat proteins for meat. Additives, on the other hand, are added to impart improved flavor and/or texture characteristics to foods. However, either case creates problems for some of the consumers on health, economic and/or religious grounds. Therefore, it became imperative to
363 develop specific, rapid assays able to monitor large sample loads for detecting adulterants and additives in food. Immunoassays are capable of fulfilling these requirements. In this section meat specification will be covered and nonmeat proteins will be dealt with under allergens. According to P. Goodwin (81)for species content identification in foods, the preferred technique should be capable of testing processed meat, raw, cooked and heat-treated samples, dairy foods and other commodities in meat samples. Different immunoassay techniques have been used for meat specification (see Table 1). For more details one can refer to articles by Kangethe (82) and others (12, 13).
2.8.2. Allergens Food allergy occurs as a result of an adverse immunological reaction to an ingested food. It has been recognized for a long time that some of the foods identified as exhibiting allergic symptoms based on clinical observations include, cows milk, cereal, cod fish, legumes, eggwhite, etc. Identifying the exact component causing the allergic reaction would be useful (83). Towards this end, immunoassays have been developed against some of the potential proteins and glycoproteins (see Table 1)(84-86). In the future, one should see greater effort placed against characterizing the common allergenic and antigenic epitopes in the protein and then raising monoclonal antibodies against these epitopes to realize increased sensitivity of detection (87). 2.8.3. C o n t a m i n a n t s f r o x l n s According to Smith (88), food products are prone to accidental and deliberate abuse. Contamination can occur at any point from the raw material to finished product upon consumption. Public concern related to food borne illness is growing and rapid detection of these pathogens and contaminants is important for food safety. Salmonella and Listeria are a source of numerous food borne illness. As a result, a lot of attention has been focused on these microorganisms. The effort in the Salmonella area focused on obtaining antibodies which can detect several serotypes (14-16, 61, 64, 90) and shortening the assay time from 3-4 days to 1-2 days by use of more sensitive formats and enrichment protocols (74, 75, 91, 92). Numerous immunoassay kits for Salmonella were developed, such as the Salmonella-Tek (74, 75), Tecra Salmonella (93) and Bio-Enza Bead (94), to name a few. More information on kits is given in section 2.9. Additional information is available for Salmonella, Listeria and other microbiological contaminants and toxins (see Table 1)in review articles and books (5, 7,11-13, 88, 95-99 ). In the case of toxins, a lot of work has been done in the area of mycotoxins arising from molds (100). Molds are used in foods such as cheese for fermentation. On the other hand, molds are also responsible for food spoilage, especially in reduced water activity foods. According to De Pruiler and coworkers (101), considerable progress has been made in the development of I A based on specific recognition of immunoreactive extracellular polysaccharides excreted by molds. These immunoreactive polysaccharides can be detected at 1 ng/ml level and, hence, no pre-enrichment is necessary. Commercial kits for detection of Penicillum Aspergillus molds using the latex agglutination format developed by Kamphuis and co-workers (102) at ng/ml concentration have been
364 Table 1 F o o d A p p l i c a t i o n s of I_m_munoassay i n F o o d s
Analyte
Antibody
Adulterants Meat Species(Identification)
Assay
Concentration Detected
Referenences
P,M
I, R, E
0.5-5%
81, 82
P,M P,M P,M P,M
I,E I,E I,R,E,F I,E
10-25~tg/ml 10ng- 10~tg/ml 0.1-10% 0.5ng-l.0~g!ml
84, 85 12, 85 12, 84, 85 84,86
Bacterial: Salmonella Listeria Staph. Enterotoxin Clostridium perfringens Clostridium botulinum
P,M P,M M P P,M
E E E E E
104- 106 cells/ml 2 7 . 2 8 eells/ml < lng/ml lpg/ml 5-10 pg/ml
74,92 95, 96 97, 99 98 70-72
Fungal: Molds Aflatoxin Ochratoxin Trichothecenes Ze arale n on e Potato glycoalkaloids
P P,M P P,M M P
E E E E,F E E
ug-ng/ml >15 ng/ml ng-pg/ml ng-pg/ml n g/m I ng-pg/ml
100 104,105 106 107 108 109
Pesticides" Herbicides Insecticides Fungicides
P.M P,M P
E,R E,R E,R
3-250 ng/ml 4-50 ng/ml 0.5-2.5 ng/ml
110,111 111 111
Antibiotics (residues in milk)
P,M
E
1 ng/ml
112
Hormones (anabolic hormones in meat)
P.M
E,R
10-70 pg/ml
113-115
Flavanone Neohesperidosides (bitterness in citrus)
P
E
0.1-20 ng/ml
116
Allergens (non-meat vroteins): _
Milk Protein Egg Protein Soy Protein Wheat Protein Con tamin an t/T oxins
*P = polyclonal antibodies; M = monoclonal antibodies; E = ELISA; R = RIA; I = immunoprecipitation; F = IFA
365 introduced by Holland Biotechnology b.v. (Leiden, Netherlands) and Sanofi Diagnostics P a s t e u r (Genk, Belgium). IA are of limited use for mold identification at the species or strain level. Aflatoxins are potent carcinogenic, mutagenic and teratogenic metabolites produced by molds. The major food affected with aflatoxins are corn, p e a n u t s , rice, cottonseeds, dried fruit and milk from ingestion (103). The US action standards established by FDA are 20 ~ g ~ for foods consumed by h u m a n s a n d 0.5 pg/kg for milk. In the case of animal feed, the levels are from 100 to 300 ~g~g. Therefore, assays capable of detecting at these levels have to be developed. (see Table 1 (104, 105)). Detection of aflatoxins entails conjugation of these small molecules with carrier proteins like bovine serum albumin to produce antibodies (20). A number of commercial kits for aflatoxins are available (see sections on kits and immunoaffinity purification). Some of the other contaminants/toxins being monitored by i m m u n o a s s a y s include pesticides, insecticides, herbicides, hormones, antibiotics, etc. (see Table 1, (106-116)). More information in these areas can be had from n u m e r o u s books and review articles (5, 10-13). 2.9. Im m unoassay
kits
A number of immunoassay kits are commercially available today (Table 2 (3, 18, 74, 101, 104, 117-132)). The majority of them are concentrated in the area of Salmonella, Listeria, aflatoxins, enterotoxins, meat proteins, antibiotics, etc. Before a kit gets released in the m a r k e t it has to be validated by independent users. According to Skerritt et al (133) diagnostic kits for food require performance trialing but formal approval by a government body is not mandatory prior to marketing. Getting official methods recognition from the AOAC would aid in wider acceptability of the kit. However, the associated h i g h cost makes it not a very viable proposition for informal field tests. A partial list of i m m u n o a s s a y s with and without the AOAC official methods status or first action status having commercial kits available is given in Table 2. 3. I M M U N O A D S O R I ~ I O N OR IMMUNOAFFINITY P U R I F I C A T I O N Enzyme i m m u n o a s s a y s although faster t h a n traditional c h r o m a t o g r a p h y , methods are often difficult to assess visually and may not have the sensitivity to detect antigens at 0.1 to 10 ppb. One technique which overcomes this hurdle is immunoadsorption or immunoaffinity purification (IAP). It is a powerful method for enrichment of antigens that exploits the specificity between a n antibody-antigen complex. By performing IAP one could detect two of the major mutagenic heterocyclic amines formed in heated beef products at ppb levels. The enrichment was performed using monoclonal antibodies a g a i n s t bovine g a m m a globulin haptenized 2-amino-3-methylimidazo [4,5-t] quinoline or 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline. By this approach, 15 g of fried beef could be assayed at ppb levels (134). F u r t h e r m o r e , the purification by IAP was adequate for subsequent quantitative analysis by HPLC with UV detection. IAP may not work with every immobilized antibody due to reduced selectivity and binding efficiency for the antigen in a complex m i x t u r e .
Table 2 Official methods and commercial kits used for immunoassays Analyte
Method/Kit
Salmonella in Foods
Fluorescent Antibody Screening Method (FA Salmonella Poly) Colorimetric Monoclonal Enzyme IA Screening Method (Bio-Enza Bead Screen) Colorimetric Polyclonal Enzyme IA Screening Method (TECRA) Fluorogenic Monoclonal Enzyme IA Screening Method (Q-TROL) Colorimetric Monoclonal Enzyme IA Screening Method (Salmonella-TeK) Colorimetric Monoclonal Enzyme IA Screening Method (Bio-Enza Bead Screen ) Immunodiffusion Screening Method Polyclonal Enzyme IA Method Colorimetric Polyclonal ELISA (Salmonella-Path-Stik) Colorimetric Monoclonal ELISA (Listria-Tek) Colorimetric ELISA (TECRA)
Salmonella in Low Moisture Foods
Motile Salmonella in Foods Motile and Non-Motile Salmonella in Foods Salmonella in Foods Listeria monocytogenes in foods
Manufacturer
AOAC Methods
References
Difco Laboratories Clinical Sciences Inc.
975.54
Organon Teknika Corporation
986.35
Bioenterprises Pry Ltd.
989.14
118
Dynatech Laboratories, Inc.
989.15
119
Organon Teknika Corp.
993.09
120
Organon Teknika Corp.
987.11
121
BioControl Systems,Inc.
989.14
122
BioControl Systems,Inc.
992.11
123
Lumac b.v.
117
74
Organon Teknika Corp.
121, 124
Bioenterprises Pry., Ltd.
125
Table 2 (Contd.) Official methods and commercial kits used for immunoassays
Analyte
Aflatoxin B1 in Cottonseed Products and mixed Feed Aflatoxin B1 in Corn and Roasted Peanuts Aflatoxin B1, B2, and G1 in Corn, Cottonseed, Peanuts and Peanut Butter Aflatoxins in Corn, Raw Peanuts and Peanut Butter Total Aflatoxin Levels in Peanut Butter Aflatoxin in Corn
Staphylococcal Enterotoxins in Foods Gluten in Foods Penicillium/
Aspergillus Molds Antibiotics Pesticides
Method/Kit
Manufacturer
AOAC Methods
Referen(~s
ELISA Screening Method (Agri-screen)
Neogen Corp.
989.06
126
ELISA Screening Assay (Agri-screen)
Neogen Corp.
990.32
127
ELISA Screening Assay (ImmunoDot Screen Cup)
International Diagnostic Systems Corporation
990.34
104
Immunoaffinity Column Method (Aflatest P column)
Vicam
ELISA Method (Biokits)
Cortecs Diagnostics Ltd.
ELISA (Agri-screen) ELISA (EZ-screen) ELISA (TECRA SET)
Neogen Corp. Environmental Diagnostics, Inc. Bioenterprises Pty.,Ltd.
Colorimetric Monoclonal Antibody ELISA Method Latex Aglutination Immunoassay ELISA ELISA
Medical Innovations Ltd. Cortecs Diagnostics Transia SA Holland Biotechnology Sanofi Diagnostics Angenics Igen
128
991.45
129
993.06
130 130 131
991.19
132 101
_
_
.
368 Therefore, immobilized antibodies need to be characterized for their selectivity and binding efficiency prior to quantitative analysis. Another area where IAP is found to be beneficial is in the testing of aflatoxins. The development of Aflatest using IAP enrichment has allowed sample testing in minutes with limited laboratory facilities and without need for hazardous aflatoxin standards (135). An AOAC/IUPAC collaborative study was conducted to evaluate the effectiveness of the immunoaffinity column (Aflatest) for determination of aflatoxin. The method has been given official first action by the AOAC (128). Additional application of IAP has been in the production of species-specific antibodies. Martin et al (136) first purified crude animal protein extracts by IAP. These purified proteins were then used to raise horse-specific monoclonal antibodies. This approach helped in significantly shortening the isolation time of species-specific monoclonal antibodies. A similar approach was used for pig-specific soluble muscle protein polyclonal antibodies (137). The limitations of IAP are the harsh conditions needed for the recovery of antigens and the cost associated with use of antibodies on a large scale. To overcome these hurdles approaches such as more efficient immobilization protocols, increased life of immobilized antibodies (including electro-elution), milder recovery protocols, proper screening to select the appropriate antibodies are under investigation (138-140). In summary, the IAP technique provides the analyst with another way to increase sensitivity and reduce assay time. 4. IMMUNOCYTOCHEMISTRY An added dimension was introduced in characterization of antigens in foods through the development of immunocytochemistry. Immunocytochemistry deals with the use of antibodies in the detection of antigens in specific locations within a food matrix via the use of microscopy. Either light or electron microscopy can be used to detect the location and quantity of the antigen. Using fluorescent labeled antibodies, contamination of wild type yeast in brewing yeast was detected under ultraviolet light (141). Another way to detect antigens is to use antibodies coupled to electron-opaque materials for transmission electron microscopy or latex beads or gold particles for scanning electron microscopy (142). Immunohistochemical staining using immuno-gold with silver staining technique was applied to confirm the presence of soy protein in meat (143). This usually entails embedding of materials prior to detection. Alcock and co-workers (144)were able to detect microorganisms in thin sections of tissues in-situ. They came up with an alternate approach to embedding that involves rapid freezing of a specimen prior to sectioning between-20~ to-40~ This resulted in a minimal alteration in structure and allowed detection within 3 hours. They used polyclonal antibodies produced against Brochothrix thermosphacta which causes spoilage of red meat. Enzymatic detection allowed visualization of brown stained bacterial cells. By this approach growth was observed on the surface and subsurface but not in the center. This information could be exploited to alter the microenvironment around the growth regions, thereby leading to a longer shelf-life.
369 In addition to detection of contaminants and spoilage organisms, this approach could be used to localize specific proteins or other important components in food products. 5. QUALITY CONTROL/FUNCTIONALITY DETERMINATION Quality control is an important requisite in processed foods. It entails monitoring the quality of raw materials prior to their use, process control to produce foods possessing desired texture/flavor characteristics which can withstand spoilage over shelf-life and are safe for the consumer. Immunoassays can serve as a rapid tool not only to monitor contaminants in raw materials but also to test ingredient functionality. The role of immunoassays has been expanded for functionality testing in several areas including, wheat quality for dough strength (145-147), emulsification properties of gum acacia (148), quality of malt and hazing/foaming of beer (146), t h e r m a l properties of proteins during processing and for process control either off-line or on-line. Proteins are important constituents of food. They perform different functions in different ingredients. Identification of the proteins which are responsible for providing functional properties in these ingredients allows them to be used for monitoring ingredient functionality. This was what was done in the case of the examples listed below, by raising antibodies against these proteins and developing immunoassays to rapidly detect them. In the case of wheat, I A ' s were developed against proteins associated with dough strength to test baking quality (145-147). Another area of interest is in the emulsification properties of gum acacia. The protein-rich fractions which are amphiphillic in nature are responsible for stabilization of oil-in-water emulsions by gum arabic. I m m u n o a s s a y s developed against these protein-rich fractions can be used to m e a s u r e emulsification capability. This was tested out (148) using heated and unheated gum samples. The heated samples exhibit poor emulsification properties versus the unheated and ELISA was able to distinguish these two g u m s , indicating the viability of using IA for testing the emulsification properties of gum acacia. Chillproofers are added to freshly brewed beers to prevent the formation of haze upon cold storage. Papain-based enzymes have been used as chillproofers and their addition have to be monitored carefully, as overdosage results in flat beer. According to Skerritt et al (146), enzymatic methods currently used lack sensitivity or are slow and tedious. They have developed a polyclonal antibodybased kit for determination of these chillproofers. Thermal treatment of proteins present in processed foods may have implications beyond food safety to the functionality of these proteins. For this purpose, specific antibodies raised against native and denatured proteins which could distinguish the two forms would enable one by immunoassay to determine the extent of protein denaturation in the product and its associated implication in safety and functionality of the finished food. P a r a f (87) discusses this aspect in his review on monoclonal antibodies in the analysis of food proteins. The ability of IA's to distinguish native versus heat-denatured ovalbumin dependent on the assay protocol used. One can foresee this
370 approach extending to other cases besides native and denatured proteins to the detection of active versus inactive enzymes. For example, the presence of active amylases may be detrimental to starch-based foods or certain active proteases may cause breakdown of protein network in dairy products and cause deleterious effect on texture. Another area may be characterization of starter cultures based on intra-cellular enzymes present by immunoassays developed against these enzymes. 6. P
~
CONTROL
Normally, probes are available which can monitor temperature, pressure, pH, moisture and certain ingredients like acids etc., for process control. If one wants to monitor specific ingredients in a process, it is done off-line by sampling at different times and performing analysis afterwards. Tremendous savings would be achieved if an on-line system could be developed. On-line techniques for process control provide rapid detection and normal ELISA's are not easily amenable for this purpose due to the number of washing and incubation steps required. Dipstick-type immunoassays which are faster are not suited for automation. Immunosensors are a possibility, but they have not been developed to the point of being generally used on a routine basis for on-line systems. Freitag and co-workers (73) have developed a unique turbidimetric immunoassay for on-line monitoring of proteins in cultivation processes. The authors made use of an immunoprecipitation reaction between bivalent antibodies and multivalent antigens which causes light scattering, leading to turbidimetric detection. Assay automation was achieved by utilizing flow injection analysis principles. They were able to reduce the time for an assay cycle down to 2.5 minutes and decrease the detection limit to 1 ~g/ml. One needs to take into consideration, however, the intrinsic turbidity of the sample and make a correction for it in the calibration curves. This on-line system should be useful for continuous processes where the antigen concentration is above the detection limit of 1 ~tg/ml. 7. FUTURE DIRECTION The advent of genetic engineering has resulted in the production of antibodies including single chain peptides in E.coli and yeast (42, 44). This, along with chimeric antibodies (42) and antibody libraries (149) will result in a significant rise in the production of tailor made antibodies performing specific and unique roles to fit the needs of food technologists. In the future, one can anticipate increases in sensitivity, specificity and laboratory-to-laboratory reproducibility of cost effective and user friendly IA's. Growth will be witnessed in the areas of (i) immunoaffinity chromatography to selectively isolate, enrich or remove components from foods, (ii) immunocytochemistry to localize and understand role of ingredients in foods, (iii) process control with advent of sensors and other on-line detection systems, and (iv) alternate uses.
371 One of the alternate uses is currently being pursued by Biocode (Cambridge, MA) to covertly fingerprint food products to prevent counterfeiting, patent infringements or subsidy frauds (150). Biocode intentionally adds an inert chemical or marker to a product or its package that can be detected and tracked rapidly via IA technology. The key to its success lies in marking without deleteriously affecting the food. One can see this expanding to issues of quality assurance, process control, product liability and contaminant prevention. In conclusion, the area of antibodies for food characterization has m a d e significant inroads since its pioneering days. This chapter has made an attempt to highlight some of the more recent advances which have impacted the food industry.
.
2. 3. @
@
.
7.
.
9.
10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21.
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Characterization of Food" Emerging Methods A.G. Gaonkar (Editor) 9 1995 Elsevier Science B.V. All rights reserved.
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Chapter 16 Taste Sensor K. Toko Department of Electronics, Faculty of Engineering, Kyushu University 36, Fukuoka 812, Japan
1. I N T R O D U C T I O N Taste is constructed from five basic taste qualities. The first is sourness. Hydrogen ions produce sourness. The second is saltiness produced, mainly, by NaC1 and KC1. The third is bitterness. Quinine and caffeine produce bitterness. The fourth is sweetness, produced by sucrose, glucose, aspartame and so on. The last is umami. Monosodium glutamate (MSG), disodium inosinate (IMP) and disodium guanylate (GMP) show umami [1-5]. In biological taste reception, taste substances are received by the biological membrane of gustatory cells in taste buds on tongue (Figure 1). Then the information on taste substances is transduced into an electric signal, which is
Figure 1. Taste cell and biological membrane.
378 transmitted along the nerve fiber to the brain, where the taste is perceived. Recently, we have developed a multichannel taste sensor whose transducer is composed of lipid membranes. This sensor can detect tastes in a manner similar to human gustatory sensation. The output of the sensor is not the a m o u n t o f taste substances but the taste quality (and also magnitude), because different output patterns were obtained for different taste groups such as sourness and saltiness. On the other hand, similar patterns were obtained for taste substances in the same group such as MSG, IMP and GMP, which have an umami taste, and NaCI, KCI and KBr for saltiness. Development of this sensor was based on a concept very different from that of conventional chemical sensors, which selectively detect specific chemical substances such as glucose or urea. However, taste cannot be measured in those terms even if all the chemical substances contained in foodstuffs are measured. Humans do not distinguish each chemical substance, but express the taste in itself; the relationship between chemical substances and taste is not clear. It is also not practical to arrange so many chemical sensors with r e s p e c t to the number of chemical substances, which amounts to over 1000 in one kind of foodstuff. Moreover, there exist interactions between taste substances, such as the synergistic effect or the suppression effect. A taste sensor should measure these effects; the intention is not to measure the amount of each chemical substance but to measure the taste itself, and to express it quantitatively. The recently developed sensor satisfies this request. In fact, this sensor could detect the interactions between saltiness and sourness. We here mention the principle of the taste sensor and applications to aqueous solution constructed of five basic taste substances and several foodstuffs such as beer, coffee and tomatoes. Quantification o f the taste is possible using such a taste sensor, and hence we can discuss the taste objectively.
2. R E S P O N S E S O F L I P I D M E M B R A N E S T O T A S T E One method to realize the taste sensor may be the utilization of similar materials to biological systems as the transducer. The biological membrane is composed of proteins and lipids. Proteins are main receptors of taste substances. Especially for sour, salty, or bitter substances, the lipid-membrane part is also suggested to be the receptor site [6]. In biological taste reception, taste stimulus changes the receptor potentials of taste cells, which have various characteristics in reception [7,8]. Then the pattern constructed of receptor potentials is translated into the excitation pattern in taste neurons (across-fiber-pattern theory).
379 In the present study, therefore, lipid membranes were used as transducers of taste information. Artificial lipid materials, such as dioleyl phosphate (DOPH) or dioctadecyl-dimethyl-ammonium, were used to construct a lipid membrane and responses of electrical potential and resistance of the membranes were measured [9-15]. It was confirmed that the lipid membranes could discriminate five primary taste substances. Moreover, they could detect the interactions between taste substances observed in biological systems. The response properties w e r e different in different types of lipids. If a hydrophobic part of a lipid was different, taste substances which can be detected were different. These facts indicate that the taste sensor can be realized by the use of various kinds of lipid membranes as transducers. Figure 2 shows the changes in the electrical potential difference across the membrane filter adsorbed with DOPH molecules by application of five taste substances [11,14,15]. The potential is taken as relative. Unity (one unit) in Figure 2 implies a b o u t - 1 1 8 mV of membrane potential. The electrical potential changes in the following order: quinine (bitter), HCI (sour), NaC1 (salty), MSG (umami) and then sucrose (sweet). This order and also the threshold magnitudes (e.g., about 10pM for quinine) agree with those known in biological systems. The responses are different for five basic taste substances. It was the first finding that the lipid membrane can respond to five taste substances. However, the sensing reproducibility was not good, because the standard deviations were about 10%. Therefore, it is not easy to distinguish between quinine and HCI.
Figure 2. Responses of the DOPH adsorbed membrane to five basic taste substances. The membrane potential is taken as relative:
380 Effects of bitter substances were also studied [10]. Strychnine, quinine and nicotine which are known to be strong bitter substances affected the potential of DOPH membrane largely. Caffeine and theobromine belonging to weak, bitter substances scarcely changed the potential. Thus, strong bitter substances are discriminated from weak bitter substances. However, picric acid showing the strong bitterness did not affect the potential. This is very unfavorable for developing a taste sensor. However, this problem has already been solved, as will be mentioned later. Let us now proceed to umami substances. Monosodium glutamate, disodium inosinate and disodium guanylate exhibit umami. Umami is a Japanese term implying "deliciousness" of meat, mushrooms, some vegetables and cheese, but is now acknowledged widely as the fifth taste [5]. Umami substances exhibit a very interesting phenomenon, i.e., a synergistic effect. In the coexistence of MSG and IMP, we experience drastic increase in umami. The membrane resistance of the DOPH membrane was largely changed with MSG coexistent with IMP. The response was amplified by a small amount of coexistent IMP [15]. It is nothing but a synergistic taste effect. In this condition, the quantity of MSG adsorbed to the membrane was found to increase; this fact agrees with an experiment in vivo [5]. Figures 3(a) and (b) show surface structures of DOPH membrane treated with 100 mM MSG and 1 mM IMP, respectively. Note that the peculiar structures which are circularly projecting are formed. Figure 3(c) shows the surface structure treated with mixed solution of 1 mM IMP and 100 mM MSG. The structure is different from that of single umami solution. That is, the different structure of lipid membranes is formed when the synergistic taste effect appears.
Figure 3. Surface structures of the DOPH adsorbed membrane treated with 100 mM MSG (a), 1 mM IMP (b) and 100 mM MSG+I mM IMP (c).
381 The DOPH membrane did not respond to picric acid, which is a strong bitter substance. We, therefore, studied a response by a different lipid membrane [12]. The synthesized lipid is dialkyl-dimethyl-ammonium, which has two hydrocarbon chains and an ammonium group positively charged. This lipid was cast on a silicon film with one hole, 100 p m in diameter. It was shown that picric acid changes the membrane potential of this membrane largely. The D O P H m e m b r a n e and the a m m o n i u m salt m e m b r a n e responded to taste substances in different ways. The above results suggest that taste substances can be perceived satisfactorily using various kinds of lipid materials. Furthermore, we must improve the sensing reproducibility. As a second step, we have developed a multichannel lipid m e m b r a n e taste sensor. Taste substances can be discriminated by the output pattern from several lipid membranes.
3. M U L T I C H A N N E L
TASTE SENSOR
Although the above lipid membranes had the ability to sense the taste by responding to many taste substances, information was insufficient to recognize quality of the taste. This weakness was overcome by means of a multichannel sensor, where transducers were composed of lipid m e m b r a n e s i m m o b i l i z e d with a p o l y m e r [16-23]. We investigated responses of the sensors to various taste solutions. The electrode showed five different response patterns to five primary tastes with small experimental deviations. The patterns looked alike when the applied substance elicited the same taste in humans.
Table 1
Lipids used in the preparation of membranes
Channel
Lipid Dioctyl phosphate (2CsPOOH) Cholesterol Oleic acid Decyl alcohol Trioctyl methyl ammonium chloride (TOMA) Oleyl amine Distearyl dimethyl ammonium bromide Trimethyl stearyl ammonium chloride
382 3.1. M u l t i c h a n n e l e l e c t r o d e Eight kinds of lipid analogues were used for preparing the membranes [16]. Lipids used are summarized in Table 1. The major parts of functional groups in the biological membrane are lined up with these lipids. Depending upon the object to be measured, we prepared different lipid materials [ 19, 21, 23]. F o r example, the mixed membranes composed of 2CsPOOH and T O M A were used for measurements of amino acids. Each lipid was mixed in a test tube with poly vinyl chloride (PVC), and a plasticizer (dioctyl phenylphosphonate), which were dissolved in tetrahydrofuran. Successively, the mixture was dried in a glass plate, which was placed on a hot plate controlled at ca. 30"C. The lipid membrane thus prepared was a transparent, colorless and soft film with ca. 200 p m thickness. The surface structure of a lipid-PVC membrane is illustrated i n F i g u r e 4. Lipid membranes were fitted on a multichannel electrode. Figure 5 shows a front view and a cross section of the electrode. The electrode was made from Ag wires, which were 1.5 mm in diameter, embedded in a basal acrylic board. The lipid membranes cut into rectangle pieces were put on the Ag wires, and then the electrode was dried in air for 1 hour and dipped in 1 mM KCI solution. The multichannel electrode was connected to an 8-channel scanner through high-input impedance amplifiers (Figure 6). The selected electric signal from the sensor was converted to a digital code by a digital voltmeter and was fed to a computer. Then, a voltage difference between the lipid membrane electrode and an Ag/AgCI reference electrode was measured.
Figure 4. Illustration of the surface state of the lipidPVC membrane used in the multichannel electrode.
Figure 5. The multichannel electrode.
383
Figure 6. The experimental setup. The potential of each channel may be composed of two potentials. One is an oxidation-reduction potential generating at the boundary surface between the Ag electrode and the lipid membrane. The other is a Donnan potential at the boundary between the lipid membrane and the aqueous medium or more generally a Gouy-Chapman electrical double-layer potential formed in the aqueous medium [24]. Figure 7 shows a potential profile near the lipid membrane. The oxidation-reduction potential would not be affected by the outer solution in short time, because the lipid membrane had low permeability for water. Then the measured potential change by application of the taste solution is mainly due to the change in the surface electrical potential.
Figure 7. Electrical potential profile near the membrane surface. The surface potential is mainly changed by taste substances.
384
3.2. Responses to five p r i m a r y tastes Typical five primary taste substances, HC1 (sour), NaC1 (salty), quinine-HCl (bitter), sucrose (sweet)and MSG (umami) were studied [16]. In general, the response of each lipid membrane was nonspecific to various taste substances. The membrane of channel 1 responded to HCI above 10 /zM at the slope near the Donnan type. NaCI also increased the potential of channel 1. Quinine increased the potential significantly at the maximum slope of ca. 135 mV against logarithm of the concentration. This large increase is due to the adsorption of quinine molecules into the lipid membrane; the adsorption of positively charged quinine molecules decreases the surface charge density of the membrane [10, 24], which is negatively charged due to ionization of phosphoric acid of dioctyl phosphate. MSG decreased the potential of channel 1 at low concentrations but increased it at high concentrations. The decrease in the membrane potential is explained by the adsorption of negatively charged glutamate ions [15, 25], and the increase at high concentrations is caused by the coexisting sodium ions. This phenomenon would account for the side (secondary) taste of MSG; i.e., MSG tastes salty at the same time. The electrical charge densities of the membrane surface of channels 2 and 4 were lower than that of channel 1. Then the response behaviors to electrolytic substances were caused by lipid materials, the plasticizer and PVC. The channel 3 behaved as a negatively charged membrane due to ionization of carboxyl groups. The potentials of channels 5 to 8 changed in almost the same way. These membranes are considered to be positively charged by an amino group or an ammonium group. The potentials of these membranes are, therefore, considered to have decreased, mainly, by anions. The potential did not change significantly through channels 1 to 8 by the application of sucrose. The potential slightly decreased above 30 mM of sucrose in channels 2 to 4, and increased in channels 5 to 8. These tendencies were opposite to HCI, NaCI and quinine, which give unpleasant feeling at high concentrations, and it might be a reason for the hedonic feeling of sucrose. The change in potential of the lipid membranes may occur through a change of ionic distribution near the surface of the electrically charged membrane by taste substances such as HCI, NaCI, quinine and MSG, which are electrolytes. On the other hand, a large change in the ionic distribution may not occur in the case of sucrose, since sucrose is a nonelectrolyte. Sucrose will be received with a conformational change in the lipid membrane or a slight neutralization of head groups of lipid molecules by a weak dipole of a sucrose molecule, which leads to the change in the surface electrical charge density to cause the change in membrane potential.
385 Figure 8 shows the electrical potential pattern from 8 channels for five kinds of taste qualities of sour, salty, bitter, sweet and umami. The pattern o f each taste substance is different, and hence each taste substance can be easily discriminated. The reproducibility is very high, because the standard deviations are smaller than 1% or ~o. The taste sensor shows similar response patterns to the same group o f taste. As examples of sour substances, HC1, citric acid and acetic acid show similar response patterns. Bitter substances such as quinine, MgSO4 and phenylthiourea show similar patterns.
Figure 8. Responses of the multichannel sensor to five taste qualities. The origin of the electrical potential was taken to 1 mM KC1.
386 Therefore, we can conclude that this taste sensor can respond to the taste in itself. This fact is very important. We must measure the taste (not chemical substances), because we want to develop the taste sensor. 3.3. T a s t e o f a m i n o acids Taste of amino acids was studied using the taste sensor [23]. Taste of amino acids has had the large attention so far because each of them elicits complicated m i x e d taste itself; e.g., L-valine produces sweet and bitter tastes at the same time. Thus, there exist detailed data on taste intensity and taste quality of various amino acids by sensory panel tests [26]. The response of the sensor to amino acids was compared with the results of the panel tests, and response potentials from the eight membranes were transformed into five basic tastes by multiple linear regression. This expression of five basic tastes reproduced human taste sensation very well. Amino acids are generally classified into several groups which correspond to each characteristic taste. Since amino acids show mixed tastes as above, the taste interactions such as synergistic effect and repression effect are automatically included in their taste. The taste sensor should express this situation. The present study is the first trial to study the taste of amino acids using artificial sensing devices. Figure 9(a) shows the response patterns to typical amino acids, each of which elicits different taste quality in humans [23]. Each channel responded to them in different ways depending on their tastes. L-Tryptophan, which elicits almost pure bitter taste, increased the potentials of channels 1, 2 and 3 greatly. This tendency was also observed for other amino acids which mainly exhibit bitter taste: L-phenylalanine and L-isoleucine. L-Valine and L-methionine, which taste mainly bitter and slightly sweet, decreased the potential of channel 5; the responses of channels 1 and 2 were small. On the other hand, L-alanine, glycine and L-threonine taste mainly sweet [26]. Only for these amino acids, the potentials of channels 1 and 2 decreased. L-Glutamic acid and L-histidine monohydrochloride, which taste mainly sour, increased each of the potentials of channels 1-5 to almost the same degree. Only monosodium L-aspartate elicits mainly umami taste in humans among amino acids used here; the response pattern was different from those of the other amino acids. Figure 9(b) shows the data points plotted in the scattering diagram on PC 1 and PC 2 by the principal component analysis. The first principal axis reflects bitterness and sweetness. The second principal axis reflects sourness and umami. Amino acids are classified clearly into five groups by the taste sensor.
387
Figure 9. (a) Response patterns for several amino acids. Symbols O, O, I , ~ and A denote L-glutamic acid, L-tryptophan, monosodium L-aspartate monohydrate, L-valine and L-alanine, respectively. (b) Scattering diagram of amino acids on PC 1 and PC2.
In addition, we tried to discriminate optical isomerism of MSG using a multichannel taste sensor. It is generally known that the L - f o r m of amino acid exhibits a taste different from its D-form. For example, L-MSG exhibits strong umami taste in humans, but D-MSG does not. Most D-amino acids taste sweet while L-amino acids exhibit various taste qualifies. However, the present sensor's outputs to both optical isomerisms resembled each other very well, as was already shown for the membrane electrical potential [15]; the previous study showed that the electrical resistance of a membrane filter adsorbed with DOPH changed in different ways for L-MSG and D-MSG although the electrical potential showed the same change. However, the present membrane, where lipids are immobilized with polyvinyl chloride, showed no change in resistance. The development of membrane materials with flexible structure such as the DOPH-adsorbed membrane may be necessary together with the improvement in the sensor system to measure the membrane electrical resistance. It is a very natural approach, because the membrane conductance is changed in biological systems at taste reception [7, 8]. Transducer materials of L and D forms of chiral lipids are also necessary for further development of the taste sensor. Development of the taste sensor may contribute to the study of reception mechanisms of taste sensing.
388 4. E X P R E S S I O N O F T A S T E BY F O U R B A S I C T A S T E S We attempted to make an artificial taste solution, which shows a similar taste to some commercial aqueous drinks, by combination o f basic taste substances by comparing electrical potential patterns of the taste sensor [22]. As the basic taste substances, HCI, NaCI, sucrose and quinine were chosen for sourness, saltiness, sweetness and bitterness, respectively. Four different concentrations we,re prepared for each of these substances: 1, 3, 10, 30 mM for HC1; 30, 100, 300, 1000 mM for NaC1 and sucrose; 0.03, 0.1, 0.3, 1 mM for quinine. The lowest concentrations correspond nearly to the thresholds to be detected by humans. We prepared 44(=256) mixed solutions with different compositions by combination of these four types of basic solutions. The above-mentioned 256 mixed solutions were measured with the multichannel taste sensor. Therefore, data on the output electrical potential pattern were taken for the 256 solutions. While the data on each channel output were dispersed discretely in the four-dimensional space constructed from four different concentrations, we approximated them by a quadratic function of the concentrations. As a result, eight quadratic functions were obtained. The data can be regarded as expressed by a set of eight different functions (corresponding to 8 channels) of concentrations of four taste substances. As test aqueous drinks, two commercial drinks of different brands were chosen. Figure 10 shows the electrical potential patterns of these two drinks. While they are not so different, the discrimination is easy because of standard deviations of • We attempted to fit the above patterns constructed from eight functions to the output electrical potential pattern of one of commercial drinks (let us call "drink A" for convenience) by minimizing the following value.
L=
8 )2 ~ (Vmi-VAi , i=l
(1)
where V~ and V~ are the output electrical potentials for the above mixed solutions (expressed by the quadratic function) and drink A, respectively, with i denoting the output from channel i. As a consequence, we obtained one mixed solution whose output pattern was nearest to that of drink A. The best combination of the concentrations for basic taste substances was obtained: 2 mM HCI, 50 mM NaCI, 0.2 mM quinine and 100 mM sucrose. As can be seen from Figure 10, the pattern for the mixed solution is surely closer to that of the drink A compared to another drink. The sensory evaluation by
389
Figure 10. Output patterns for two brands of commercial aqueous drinks and the mixed solution. Drink A is shown by a solid line with another drink by a dot-dashed line. The mixed solution (2 mM HC1, 50 mM NaC1, 0.2 mM quinine, 100 mM sucrose) is shown by a dashed line, which is closer to the solid line than the dot-dashed line.
humans was also made. The above-mentioned two commercial d r i n k s and the mixed solution were tasted, and their tastes were compared to one another. The sensory tests by humans showed that this mixed solution p r o d u c e d almost the s a m e taste as drink A. As is well known, the r scale is effective to express the taste strength [1, 2]. The concentration of each taste substance can be transformed into the taste strength. While tartaric acid is used in the r scale, we can safely consider that HC1 has the strength two times as large as tartaric acid. The above mixed solution is thus composed of 4.04 sourness, 2.03 saltiness, 5.01 bitterness and 2.24 sweemess in terms of the r scale. Therefore, drink A has the above taste strength. The taste of every drink can be quantified using the multichannel taste sensor. In other words, q u a n t i t a t i v e m e a s u r e m e n t s o f taste are possible by the sensor m o r e accurately than the sense of humans because the present taste sensor has higher reproducibility, durability and sensitivity. Now let us discuss the sensitivity of the taste sensor. The sensor had detection e r r o r s ( in the unit of logarithmic concentration) 0.73% for saltiness, 0.65% for sourness and 3.4% for bitterness in the mixed aqueous solution [27]. Humans usually cannot distinguish two tastes with a concentration difference below 20% [4]. Here, 20% means the error of 7.9% ( - l o g 1.2). T h e r e f o r e , ability of detection of the sensor is superior to that of
390 humans. Of course, too much sensitivity may not be necessary always when attempting to profile a consumer product; however, a high sensitivity is required for assurance of the quality of foods reliably.
5. A P P L I C A T I O N
TO FOODS
The present sensor could easily discriminate between some kinds of commercial drinks such as coffee, beer and aqueous ionic drinks (Figure 11) [22]. Since the standard deviations were + 2 mV at maximum in this experimental condition, these three output patterns are definitely different. If the data are accumulated in the computer, any food can be easily discriminated. Furthermore, the taste quality can also be described quantitatively by the method mentioned below. In biological systems, patterns of frequency of nerve excitation may be fed into the brain, and then foods are distinguished and their tastes are recognized [4-8]. Thus, the quality control of foods becomes possible using the taste sensor, which has a mechanism of information processing similar to biological systems.
Figure 11. Output patterns for coffee (----), beer ~----) and commercial aqueous drink (---). 5.1. Taste of beer Figure 12 shows the response patterns to 8 different brands of beer among 36 brands measured [20]. The patterns were measured relative to a certain beer as a standard. Although the difference in electrical potential between different brands was a few mV or more, each beer was easily distinguished from the other by these patterns because of the high reproducibility and
391 sensitivity of the sensor represented by • 0.2 m V o f standard deviations (smaller than the above usual condition by one order) as already reported [18]. This superior ability is due to the following methods: the first is the use of a certain beer as an initial reference of the electrical potential pattern although the KCI solution was used as the initial reference in Figures 7 and 11. This initial preconditioning implies the long-term preservation of the electrode in the reference beer; it prevented the electrode from a discrete large change in ionic condition from the KCI solution to the test beer. The second is the use of regular repetitive measurements, the period of which was 20 seconds or so. This eliminated the hysteresis effect associated with long-term measurements.
Figure 12. Output patterns for eight brands of beer. The origin of the electric potential was taken to some beer K1. Figure 13 shows the effect of temperature on the electrical potential pattern of a certain brand of beer [22]. The pattern was drastically affected by temperature. It implies that the taste was largely changed with temperature, as experienced usually. It is to be noted that the chemical component is almost the same even at different temperatures. A taste map is proposed for expressing the taste quality of beer [28]. This map is based on sensory tests made by humans, and is composed of the abscissa expressing "rich taste" or "soft taste" and the ordinate expressing "sharp touch" or "smooth touch". These expressions cannot be replaced by the terms of the five basic taste qualities. The rich or soft taste may be mainly related to the concentration of wheat, whereas the sharp or smooth touch may arise from the concentrations of alcohol, hops and so on. We tried to express
392
these taste qualities quantitatively by transforming the output pattern of the taste sensor using a simple relationship such as a linear equation [20]. The axes were divided into 8 levels. Three kinds of beer (K1, K2 and K3) were chosen as standards for determining two independent or opposite directions, which depend on the linear transformation equation. A total of 15 brands of beer were arranged on the taste map using the following equation: 8
degree of sharp-smooth touch = ]~ ai Vi + b , i=l 8 degree of rich-soft taste = ]~ ci Vi + d , i=l
(2)
where V~ is the output value from channel i of the multichannel electrode. The numerical constants a~, b, c~ and d were determined as I 0
for i=1-3,
ai--
-0.29 (1/mV)
for i=4-8,
-1.33 (1/mV) for i=l, Ci -"
0 b =
0.7,
for i=2-8, d =
0.5.
(3)
Figure 13. Effect of temperature of the output pattern of a certain beer. 4"C(----),6"C(------), 22"C(----9 and 40"C(-----).
393 In Figure 14, the same letters indicate beer p r o d u c e d by the same company. The results were similar to those of human taste sensation of beer [28]. It is interesting that five kinds of beer from company K are aligned on a line at a 45 ~ angle with the abscissa. The direct transformation from the output pattern to the taste quality was performed here as one trial of expressing the actual human sensation using the output electrical pattern. A similar trial was done for evaluation of the strengths of sourness and saltiness, which will be mentioned later. These two trials depend on the utilization of simple transformation equations by extracting typical properties of output patterns. This method is effective if some data on sensory tests, using humans as a standard, can be obtained to compare with the sensor outputs. However, the expressions for the tastes of beer are obscure because they are not described by the five basic taste qualifies. The purpose of the application of the taste sensor is also to express these kinds of obscure terms of human sense in scientific terms. The output patterns in Figure 12 may contain much more information than the properties extracted by eq. (2); for example, a positive peak or negative peak appears at channel 5 for different brands of beer. Although beer A2 shows a pattern much different from beer B, they are located at nearby positions in Figure 14. Unfortunately, direct information concerning the taste quality and quantity cannot be obtained from channel 5, because the original property studied for basic taste substances (HCI for sourness, quinine for bitterness and so on in Figure 8) was modified by preserving the electrode in
Figure 14. Taste map of beer obtained from the output patterns using transformation equation (2).
394 the standard beer. Of course, useful information can be obtained if a preliminary experiment is performed using a modified beer to which taste substances have been added. The quality and magnitude of the tastes of beer should be analyzed in more detail together with the taste map. The level is limited to 3 for "rich taste" and "sharp touch" in Figure 14. In fact, there were types of beer showing output patterns exceeding this level, although they are not shown in Figure 14. For example, the category of beer called "DRY" showed such large patterns as to exceed level 3. However, simple transformation using eq. (2) did not give a property of taste correlating well with our sensory tests. The resultant transformation to express human sensation was nonlinear (or sometimes multivalued). Therefore, the transformation to express the full range of taste properties of beer may be very complex. The study of the characteristics of such a transformation is very important and is left as a future task. A principal component analysis was also performed [20]. It was found that relative positions among different brands of beer (for example, with respect to K2 as an origin) are similar to those in Figure 14. This assures the conventional taste expressions such as "sharp touch" and "rich taste" in the taste map. Simultaneous consideration of output patterns with various methods will make it possible to describe these obscure human taste expressions using the five basic taste qualities. 5.2. Taste of tomatoes While the sensor was applied to beverages as shown in Figures 11 and 12, it can also be used for analysis of the taste ofgelatiniform or solid foods [21]. For quantification of the taste of tomatoes, the taste sensor was applied to commercial canned tomato juice, to which four basic taste substances had been added. Data were analyzed by means of principal component analysis. The taste of several brands of tomatoes was expressed in terms of four basic taste qualities by projecting the data obtained from these tomatoes onto the principal axes. This expression agreed with the human taste sensation. The used multichannel electrode was basically the same as that reported before, but was improved in a few respects. The detecting electrode of each channel was made up of Ag wires whose surface was plated with Ag/AgC1, which were embedded in a basal acrylic board of 2 mm thickness. Another acrylic board of 1 cm thickness which had eight cone-shaped holes was affixed to this board. The holes were filled with 100 mM KCI solution, and the eight membranes were fitted on the board to cover the holes. Basic taste substances had been added to standard tomato juice. We used NaCI for saltiness, citric acid for sourness, MSG for umami and glucose for sweetness. No taste substance for bitterness was added because tomatoes taste
395 only slightly bitter. The concentrations (weight percentage) of the four chemicals were 20% for NaCI, 50% for citric acid, 30% for MSG and 25% for glucose. Changes in electrical potential upon addition of the basic taste substances were measured as a preliminary experiment. The tastes of the five kinds of tomatoes were predicted by comparing the output electrical potential patterns for tomatoes with the results of the preliminary experiment. The test foods studied here were five kinds of tomatoes. When eating food, humans first masticate the food with their teeth and then taste it. Therefore, we used a mixer in place of teeth and crushed tomatoes before measuring them. The preconditions were established by keeping the electrode immersed in standard juice, i.e., commercial canned tomato juice without NaC1 added, for a long period of time. The origin of the output pattern was taken under these preconditions. Standard juice was used for the reference electrical potential pattern. The standard deviations between different lots of membrane were about 3mV. The same set of the eight membranes was used throughout the measurements for all tomatoes. The five kinds of tomatoes were fresh-market tomatoes: Ryokken, Kiss, Fukken, TVR-2 and a kind of tomato for processing (hereafter named PT). Ryokken and Kiss were deliberately cultivated to be sweeter. Figure 15 shows the response patterns for several samples of one brand, TVR-2. Each sample was measured more than five times; the typical example of standard deviations were 0.17 mV, 0.49 mV, 0.37 mV, 0.34 mV, 0.40 mV, 0.27 m V , 0.68 mV and 0.14 mV for channels 1 - 8 , respectively. The
Figure 15. Output patterns for several samples of TVR-2. Different symbols denote different samples.
Figure 16. Output patterns for five brands of tomatoes. O, C), m, [3 and A denote samples of TVR-2, Fukken, Ryokken, Kiss and PT, respectively.
396 magnitude of response was very different for each sample. However, the response patterns resembled each other in their shapes. The same situation was also observed for the other brands. Figure 16 shows examples of the response pattern for one sample each of five brands of tomatoes. Different brands of tomatoes were distinguished by the shapes of the output electrical potential patterns. Therefore, tomatoes of the same brand can be considered to have a taste with similar proportions; the difference in taste among tomatoes of the same brand may be due, mainly, to the difference in magnitude of taste, because the output electrical potential changed linearly with the concentrations of taste substances in a narrow range [18,21]. Figure 17 shows the results of projection using the transformation matrix obtained in the preliminary experiment. We can predict the taste of each tomato as follows. The first principal component scores for TVR-2 tend to be so highly positive that TVR-2 can be considered to taste sour. This result agrees well with the human sensation, because TVR-2 is a relatively old brand of tomato and is generally acknowledged as a sour tomato. F o r sweet tomatoes such as Ryokken or Kiss, the third principal component scores tend to be negative; hence these tomatoes are presumed to be sweet, as expected. The first principal component scores for most tomatoes are highly positive compared to PT; hence PT is presumed to have a relatively strong umami taste. Tomatoes tasting strongly of umami are generally used for processing; this prediction also agrees with human sensation.
Figure 17. Projection of the response patterns for tomatoes to the two principal axes. O, O, II, I-] and A denote samples of TVR-2, Fukken, Ryokken, Kiss and PT, respectively. Different points with the same symbol indicate different samples of the same brand of tomato.
397
5.3. D e g r e e o f s o u r n e s s Let us here show another example of quantification of taste made with the taste sensor [19]. Intensities of sourness and saltiness were quantified, as seen in Figure 18(a), where "degree o f sourness" is introduced as a function of tartaric acid concentration. The degree was expressed as an algebraic function of outputs of two channels among eight channels. Once the degree of sourness is defined by means of the response electrical potential of the sensor, it can be applied to other sour substances. Figure 18(b) shows correspondence between this sensor and the sense of human taste. The sensor and human sense matched each other; hence this sensor reproduces the human taste sensation. In the coexistent situation of tartaric acid and NaCI, they enhance each other [4]. This phenomenon was reproduced using the degree in Figure 18. Thus, the taste sensor expresses the taste interactions experienced by humans. The sensor could detect minute differences of taste between NaC1, KC1, KBr, NaBr, NH4CI, LiC1 and KI [19]. In fact, the response patterns are different, as can be seen from Figure 8. It implies that such taste substances as KBr, NaBr and KI do not show pure saltiness elicited by NaCI. Therefore, the sensor can detect large differences between five basic taste qualifies, and furthermore can distinguish between these small differences of similar taste quality.
Figure 18. Degree of sourness defined by tartaric acid concentration using the sensor output (a) and comparison between the sensor and humans (b).
398
6. S U M M A R Y Taste was measured with a fashion similar to the human gustatory sensation because the sensor showed a similar pattern in the same group of taste, while quite different patterns were obtained for different taste groups. In fact, the similar outputs were obtained for, e.g., NaCI, KCI and KBr eliciting saltiness, whereas their patterns differed clearly from those for other basic taste qualifies. The taste of amino acids was also classified into several groups in accordance with human taste sense. These results suggest a possibility of molecular recognition; adequate selection of membranes will enable us to detect minute difference in molecules and classify them accordingly. Some types of commercial drinks such as coffee, beer and aqueous ionic drinks were discriminated easily using the taste sensor. The taste sensor has the sensitivity, reproducibility and durability higher than those of humans. The taste quality was quantified and the taste interactions were reproduced. We tried three methods to quantify the taste of the foodstuffs. The first method is to compare output patterns between test solution and the mixed solutions by performing many measurements of various mixed solutions (Figure 10) [22]. The taste of commercial aqueous drink was reproduced by blending four basic taste substances (HCI, NaC1, quinine, sucrose) so that the response pattern could get closest to that of an aqueous drink. With this attempt, the best combination of the concentrations of basic taste substances was obtained: 2 mM HCI, 50 mM NaCI, 0.2 mM quinine and 100 mM sucrose. This mixed solution produced almost the same taste as the aqueous drink. It is very important to note that this method automatically contains the interactions between taste substances. If many measurements of various mixed solutions are made, comparison of output patterns between test solution and the mixed solutions can be easily made by adequate algorithms such as in neural networks. The second method to quantify the taste by the sensor may be to extract the characteristics of output patterns by adopting some algebraic functions [19, 20]. We can know the taste quality and estimate the taste strength of test solution by using the functions (Figures 14 and 18). However, it may not be easy to get such reliable, simple functions for expressing the taste strength for each taste quality. As the third method, recent experiment has been successful in the quantification of taste of various brands of tomatoes [21]; a preliminary measurement was performed to study the change in electrical potential pattern with addition of basic taste substances to the standard taste solution, e.g., commercial tomato juice without NaC1, together with the measurements on
399 tomatoes. Quantification was made by taking into account these two kinds of measurements and by using a principal component analysis (Figure 17). Improvements in the membranes have also been made according to the purpose of measurements, e.g., by adopting the Langmuir-Blodgett method [29] or the monolayer of thiol-containing lipids [30]. The taste sensor will be applicable for quality control in food industry and help automation of the production. The sense of taste is vague and largely depends on subjective factors of human feelings. If we compare the standard index measured by means of the taste sensor with the sensory evaluation, we will be able to assess taste o b j e c t i v e l y . Moreover, the mechanism of information processing of taste in the brain as well as the reception at taste cells will also be clarified by developing a taste sensor which has output similar to that of the biological gustatory system.
Deliciousness Figure 19. Objective measure of deliciousness.
REFERENCES 1. R. M. Pangborn, Food Res., 25 (1960) 245. 2. L. M. Bartoshuk, Physiol. Behav., 14 (1975) 643. 3. S. Yamaguchi, J. Food Sci., 32 (1967) 473.
400 4. C. Pfaffmann, The sense of taste, J. Field (ed.), Handbook of Physiology, See. 1 Neurophysiology, Vol. 1, American Physiological Society, Washing -ton D.C., 1959, p.507. 5. Y. Kawamura and M. R. Kare (eds.), Umami: A Basic Taste, Marcel Dekker, Inc., New York, 1987. 6. K. Kurihara, K. Yoshii and M. Kashiwayanagi, Comp. B iochem. Physiol., 85A (1986) 1. 7. S. D. Roper, Ann. Rev. Neurosci., 12 (1989) 329. 8. S.C. Kinnamon, TINS, 11 (1988) 491. 9. K. Toko, M. Tsukiji, S. Iiyama and K. Yamafuji, Biophys. Chem., 23 (1986) 201. 10. S. Iiyama, K. Toko and K. Yamafuji, Agric. Biol. Chem., 50 (1986) 2709. 11. S. Iiyama, K. Toko and K. Yamafuji, Maku (Membrane) 12 (1987) 231 [in Japanese]. 12. K. Hayashi, K. Yamafuji, K. Toko, N. Ozaki, T. Yoshida, S. Iiyama and N. Nakashima, Sens. Actuators, 16 (1989) 25. 13. S. Iiyama, K. Toko, K. Hayashi and K. Yamafuji, Agric. Biol. Chem., 53 (1989) 675. 14. K. Toko, K. Hayashi, S. Iiyama and K. Yamafuji, 4th Int. Conf. on Solid-State Sensors and Actuators, (Transducers '87), Tokyo, 1987, Digest of Technical Papers, p.793. 15. K. Hayashi, Y. Matsuki, K. Toko, T. Murata, Ke. Yamafuji and Ka. Yamafuji, Sens. Materials, 1 (1989) 321. 16. K. Hayashi, M. Yamanaka, K. Toko and K. Yamafuji, Sens. Actuators, B2 (1990) 205. 17. K. Toko, K. Hayashi, M. Yamanaka and K. Yamafuji, Tech. Digest 9th Sens. Symp. (1990) p. 193. 18. H. Ikezaki, K. Hayashi, M. Yamanaka, R. Tatsukawa, K. Toko and K. Yamafuji, Trans. IEICE Japan, J74-C-II (1991) 434 [in Japanese]. 19. T. Murata, K. Hayashi, K. Toko, H. Ikezaki, K. Sato, R. Toukubo and K. Yamafuji, Sens. Materials, 4 (1992) 81. 20. K. Toko, T. Murata, T. Matsuno, Y. Kikkawa and K. Yamafuji, Sens. Materials, 4 (1992) 145. 21. Y. Kikkawa, K. Toko and K. Yamafuji, Sens. Materials, 5 (1993) 83. 22. K. Toko, T. Matsuno, K. Yamafuji, K. Hayashi, H. Ikezaki, K. Sato, R. Toukubo and S. Kawarai, Biosens. Bioele. 9 (1994) in press. 23. Y. Kikkawa, K. Toko, T. Matsuno and K. Yamafuji, J. J. Appl. Phys., 33 (1994) 5731. 24. K. Nomura and K.Toko, Sens. Materials, 4 (1992) 89. 25. K. Torii and R. H. Cagan, Biochim. Biophys. Acta, 627 (1980) 313.
401 26. T. Ninomiya, S. Ikeda, S. Yamaguchi and T. Yoshikawa, Proc. 7th Congress Sensory Tests (1966) p. 109 [in Japanese]. 27. H. lkezaki, K. Toko, K. Hayashi, R. Toukubo, M. Yamanaka, K. Sato and K. Yamafuji, Tech. Digest 10th Sens. Symp. (1991) p. 173. 28. Kirin Brewery Co., Ltd., (ed.), Biru no Umasa wo Saguru [Study of Deliciousness of Beer], Shokabo, Tokyo, 1992, p.81 [in Japanese]. 29. H. Akiyama, K. Toko, K. Yamafuji and K. Hayashi, Transaction of the Society o f Instrument and Control Engineers, 29 (1993) 1012 [in Japanese]. 30. S. liyama, Y. Miyazaki, K. Hayashi, K. Toko, K. Yamafuji, H. Ikezaki and K. Sato, Sens. Materials, 4 (1992) 21.
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Characterization of Food: Emerging Methods A.G. Gaonkar (Editor) 9 1995 Elsevier Science B.V. All rights reserved.
403
Chapter 17 N e w D e v e l o p m e n t s in M e t h o d s for A n a l y s i s of Volatile F l a v o r C o m p o u n d s and their Precursors Peter Schieberle Bergische Universit~it/GH, Food Chemistry/FB 9, Gaul3stral3e 20, D-42097 Wuppertal, Germany
1. INTRODUCTION Besides texture and color, flavor (taste and smell) is an important property of foodstuffs. Smell is caused by volatile compounds coming into contact with a distinct area in the nose, the so-called "regio olfactoria" [1]. Volatile flavor compounds are denoted odorants or odor compounds, if they have been perceived nasally (before eating) and aroma compounds if they have been perceived retronasally via the throat (during eating). Therefore, in the literature the terms flavor, odor or aroma compounds are often synonymously used. The overall sensory impression induced by the flavor compounds strongly effects the acceptance of a product by the consumer. Therefore, analysis of flavor compounds provides the knowledge needed to objectify and to improve food flavor quality. Aroma compounds are present in the volatile fractions of foods. Since flavor chemists previously did not discriminate between an odorless volatile compound and a flavor compound, until now, more than 6000 volatile compounds have been identified in foods [2, 3]. With several foods it has now become evident that only a few compounds are actually involved in the aroma [4-6]. Therefore, the main goals of modern flavor chemistry are (i) to identify those compounds predominantly contributing to a food flavor (ii) to characterize their precursors in the raw materials and (iii) to clarify the reaction pathways governing their formation during food processing and storage. Such data are prerequisites for the improvement of food flavors as well as the inhibition of off-flavors by technological steps. In the following chapter, methods enabling the selection of those odorants contributing mainly to food flavors will be discussed with special emphasis on sensory experiments aimed to verify the flavor potency of the proposed key odorants. Furthermore, strategies to characterize flavor precursors in the raw materials will be described. 2. METHODS F O R IDENTIFICATION OF KEY ODORANTS 2.1. Isolation of the volatile fraction
The analysis of aroma compounds starts with the isolation of the volatile fraction from the food. Techniques used in the preparation of flavor extracts from foods have recently been reviewed [7-9]. The most important task in the choice of the isolation procedure is to test whether the flavor of the extract is identical or at least similar to the flavor of the food itself. This has to be checked by a sensory evaluation of the food extract (e.g., after dilution with an appropriate medium like water or oil) before a time consuming chemical analysis is performed.
404
Food
Extraction (Diethyl ether)
Flavor extract
Flavor distillate Separation into neutral/basic and acidic volatiles Gas chromatography olfactometry (GCO) Determination of retention indices Evaluation of odor quality Aroma extract dilution analysis Determination of FD factors Identification of odorants Comparison of mass spectra, retention indices, odor quality and odor threshold with reference compounds Quantification of key odorants by stable isotope dilution assays
Calculation of odor activity values
Sensory experiments
Figure l. Experimental design for the isolation and characterization of key odorants in foods
405 In Figure 1, a general scheme used in our group for the careful isolation of food volatiles is shown. Usually, the food is extracted with diethyl ether at room temperature and, after concentration of the extract, the volatiles are separated from the non-volatile material by high vacuum sublimation [10, 11]. In case of fat-rich products (e.g., butter, sesame seeds or soybean oil) a modified version of the high vacuum sublimation apparatus is used [ 12]. To avoid interferences during gas chromatography, the aroma distillate has to be separated into neutral/basic and acidic fractions by treatment with sodium bicarbonate, if higher amounts of volatile acids, such as acetic acid or butanoic acid, are present. Both fractions are then concentrated to the same volume and separately analyzed by gas chromatography olfactometry (GCO). To separate the acidic volatiles a free fatty acid stationary gc phase (FFAP) is very appropriate.
2.2. Gas chromatography olfactometry (GCO) A first approach to distinguish between the odor-active compounds and the many odorless volatiles present in such aroma extracts is the application of gas chromatography/olfactometry (GCO, formerly called "sniffing-technique" [ 13-17]).
Figure 2. Gas chromatogram of the neutral/basic volatile fraction isolated from a pale lager beer. Numbers characterize an odor-active position. The extract of the volatiles is separated by high resolution gas chromatography (HRGC.) and the odor of the compounds is assessed by sniffing the effluent of the GC column m parallel with the FID-detection. This technique allows the detection of odor-active volatiles, the determination of their odor qualities and, most important, the combination of these sensory data with an analytical parameter, the retention index (RI). In Figure 2, the results of
406 an application of this method to an aroma extract of the neutral/basic volatiles from fresh pale lager beer is shown [18]. Twenty-three odor-active positions were detected. Examples are peak 4 (malty, alcoholic); peak 17 (sweet, flowery) and peak 20 (spicy). The evaluation of only one GC run has an important drawback. Since it is not possible to exactly evaluate the intensity of the odor during sniffing, the results cannot be used to decide whether a compound is a key odorant in beer flavor or contributes little to the overall odor. Furthermore, the number of the compounds detected depends on accidental factors, e.g., the amount of the food used for the isolation of the volatile fraction or the degree of the concentration. 2.3. Odor dilution techniques To overcome these problems and to gain some information on the odor activities of the odorants detected during sniffing, the GCO method has been further developed by Acree and coworkers [ 19] and by Grosch and his group [20, 21 ]. The advantage of both techniques, the CHARM-analysis created by Acree and the aroma extract dilution analysis (AEDA) developed by Grosch is that the relative odor activity of single odorants in a complex mixture can be determined without knowing their chemical structures. The analyses are performed in the following way: the volatile fraction is diluted stepwise with the solvent and each dilution is then evaluated by GCO. This procedure is performed until no odorant is perceivable in the GC effluent. The highest dilution at which a compound can be smelled is defined as its flavour dilution (FD) factor in the AEDA method. Although in CHARM-analysis the duration of the smell is also taken into account, the peak maximum of a CHARM value is identical to the FD-factor. Since these techniques rank the odorants present in a food extract according to their relative flavor potencies, the often time-consuming identification experiments are now focused on the key odorants showing high FD-factors or CHARM values, respectively. Several factors have been reported to influence the results of the AEDA such as stability of odorants on a given stationary gc phase [22] or the appearance of "gaps" during sniffing of the dilution series [23]. To minimize these influences, the AEDA should be performed as detailed in the following: The original extract and two dilutions, e.g., 1:20 and 1:200 are analyzed by GC/O on at least two stationary phases of different polarity. This procedure should be performed by at least three panellists. The column on which the most odor-active regions are detectable at the highest dilution is then selected for AEDA. To avoid "gaps", the sensory analysis of a given extract should be performed within two days. This can be achieved by sniffing the original extract and e.g., the dilutions 1:4, 1:16, 1:64, 1:256 and 1:1024 at the first day and by sniffing the dilutions 1:2, 1:8, 1:32, 1:128 and 1:512 at the second day. Furthermore, each odor-active region detected in the original extract should be sniffed throughout the entire dilution series. To our experience, the differences in the relative FD-factors determined by a group of six panellists amount to not more than two dilution steps or FD-factors, respectively, (e.g., 64 and 256) implying that the key odorants in a given extract will undoubtedly be detected, even if the AEDA is performed by only one or two panellist. The results of the AEDA are usually displayed as diagram of the FD-factors vs. the retention indices (RI), the so-called FD-chromatogram. The FD-chromatogram obtained by application of the AEDA on the neutral/basic volatiles of the pale lager beer is shown in Figure 3 [18]. The highest FD-factors were found for compounds 4 (malty, alcoholic), 6 (fermented wort), 14 (fruity), 17 (flowery), 20 (spicy) and 22 (cooked apple-like). The identification experiments were focused on the twenty-three odorants detected in the FD-factor range 16 to 1024. The chemical structures of five of the most odor-active compounds in the neutral/basic fraction (nos. 4, 14, 17, 20, 22) are displayed in Figure 4. Additionally, the six most important odorants identified on the basis of AEDA results in the acidic volatile fraction of pale lager beer are shown (I-V; Figure 4). In the meantime, several food flavors have been evaluated by odor dilution techniques. The foods analyzed and the corresponding references are listed in Table 1.
407
Figure 3. Flavor dilution (FD) chromatogram of the neutral/basic odorants in a pale lager beer. The numbering follows Figure 2. RI; retention index on a silicone SE-54 GC stationary phase [adapted from ref. 18]. Table 1 Applications of odor dilution techniques on foods Food Reference Food Apple Beef, boiled roasted stewed Butter Butter oil Cheese (Emmentaler) Coffee roasted Cucumber Dill herb Grapes (labruscana) Honey (different varieties) Lemon oil Lovage Melon
[24] [25] [26] [27] [28] [29] [30] [22, 31 ] [32] [33 ] [34] [35] [36] [37] [32]
Mushroom Oat meal Olive oils Orange juice Parsley Popcorn Puff pastry Rye bread crust crumb Sesame, roasted Strawberry Tea (green and black) Trout (Sweet water fish) Wheat bread crust crumb
Reference [38] [39]
[40]
[41] [42] [43] [44] [45] [451 [46] [47, 48] [49]
[50] [21]
[51]
408
Figure 4. Key odorants in pale lager beer. The numbering follows Figs. 2 and 3. Odorants with roman numbers were identified in the acidic fraction (FD-factor). 2.4. Detection of flavor changes The overall flavors of food products may be significantly influenced by storage, processing conditions or raw materials. A comparative AEDA (cAEDA) is a useful tool to study such influences on food flavors. The cAEDA is performed in the following way: the volatiles from the same amounts of the two food batches to be compared (e.g., 600 g of flesh and stored product) are isolated by using the same isolation procedure (cf. Figure 1). Furthermore, the distillates obtained have to be concentrated to the same volume (e.g., 100 gL) and the same volumes have to be used for GCO (e.g., 0.5 gL). Assuming identical losses of the odorants during the work-up procedure, a comparison of single odorants in both samples on the basis of their FD-factors reflects changes in their concentrations between the two samples and indicates which odorants are mainly responsible for the flavor differences. Following is a discussion of the application of the comparative AEDA on the flavor differences between roasted white and black sesame varieties. Roasting of white sesame seeds resulted in a material exhibiting an intense roasty, sweet, burnt odor. On the contrary, in the overall odor of black seeds roasted under the same conditions a tallowy, fatty note was predominant, besides the roasty odor. The results of a
409 cAEDA [46, 52] reflected these flavor differences by higher FD-factors for the fatty, tallowy smelling (E,E)-2,4-decadienal and 2-pentylpyridine and by lower FD-factors of the roasty, burnt smelling 2-furfurylthiol and 2-phenylethylthiol in the black seeds (Table 2). The cAEDA has further been used to detect flavor defects during storage of beer [ 18], butter oil [29], extruded oat meal [39], trout [50] and soybean oil [53]. Furthermore, the influence of the processing on the flavor of white sesame seeds [46], strawberries [48] and apples [54] has been studied. Table 2 Comparison of important odorants (FD _> 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 <1 10 1.5 29 16
PM 3.6 110 16 206 <1
419 4. FLAVOR SIMULATION During AEDA, interactions between the odorants are not taken into consideration, since every odorant is evaluated individually. Therefore, it may be possible that odorants are recognized which are possibly masked in the food flavor by more potent odorants. Furthermore, the odor activity values only partially reflect the situation in the food, since OAVs are mostly calculated on the basis of odor thresholds of single odorants in pure solvents. However, in the food system, the threshold values may be influenced by nonvolatile components such as lipids, sugars or proteins. The following examples will indicate that systematic sensory model studies are important further steps in evaluating the contribution of single odorants to the overall food aroma. 4.1. Beer
Applications of AEDA on the flavors of a pale and a dark Bavarian beer exhibiting a caramel-like flavor revealed that in both beers the same key odorants were present but in slightly different concentrations [18]. The main difference was found for the caramel-like smelling 4-hydroxy-2,5-dimethyl-3(2H)-furanone (HDF). Its OAV was increased by a factor of nearly four in the dark beer (Table 13). However, even in this beer the OAV of the HDF was much smaller than the OAVs of further important odorants like (E)-13-damascenone or 3methylbutanol. To reveal the flavor significance of HDF for the dark beer, the following sensory experiment was performed: a pale beer was spiked with increasing amounts of HDF and the appearance of the caramel-note was judged by a sensory panel. As shown in Table 14 the caramel-note was detected by each panellist when the concentrations of HDF were equal to those present in the dark beer. In contrast to the OAVs (cf. Table 13), these results indicated a significant contribution of HDF to the dark beer flavor. In an alcohol-free beer, the concentrations of the beer odorants were 5 to 10-fold lower than in the pale lager beer [ 18] suggesting that the former beer is a very appropriate matrix for the determination of odor thresholds. A determination of the odor thresholds in the alcohol-free beer revealed (Table 15) that, compared to water, the odor threshold of all odorants increased, but to a different extent. For instance, the threshold of (E)-13-damascenone increased by a factor of 2500, while that of HDF was enhanced only by a factor of eight. Odor activity values calculated on the basis of the odor thresholds in the alcohol-free beer (Table 15) now confirmed the significant contribution of HDF to the dark beer flavor. Table 13 Comparison of odor activity values of important odorants in pale and dark beer [ 18] OAV Odorant pale 3-Methylbutanol 2-Phenylethanol 3-Methylbutanoic acid 4-Hydroxy-2, 5-dimethyl-3 (2H)-furanone a 4-Vinyl-2-methoxyphenol Ethyl hexanoate Ethyl butanoate (E)-13-Damascenone
49.6 17.5 5.2 3.5 2.4 14.9 30.5 300
dark beer 42.9 9.1 7.4 13.2 2.5 7.5 21.5 264
a Equals a concentration of 350 gg/L in the pale and of 1320 g g ~ in the dark beer.
420 Table 14 Influence of additions of 4-hydroxy-2,5-dimethyl-3(2H)-furanone on the odor of a pale lager beer [ 18] Addition (gg/L)
Concentration in the beer sample (gg/L)
Number of judges a detecti~;g a caramel-like odor note
0 200 500
354 554 854
0 1 3
1000
1354
8
a The sensory evaluation was performed by a panel of 8 trained female and male persons using the triangle test. Table 15 Comparison of odor thresholds of important beer odorants in water and alcohol-free beer [P Schieberle, unpublished results) Odour threshold (mg/L)
OAV a
Odorant H20
alcohol-free beer
pale
dark beer
3-Methylbutanol Phenylethanol 3-Methylbutanoic acid 4-Vinyl-2-methoxyphenol 4-Hydroxy-2, 5-dimethyl-3 (2H)-furanone Ethyl hexanoate Ethyl butanoate (E)-13-Damascenone
1 1 0.1 0.1 0.1 0.01 2 x 10-3 4 x 10-6
60 200 0.75 0.3 0.8 0.1 3 x 10-2 1 x 10-2
0.82 0.09 0.70 0.81 0.44 1.5 2.0 0.1
0.72 0.05 0.99 0.83 1.70 0.75 1.4 0.08
a Calculated from odor thresholds in alcohol-free beer. 4.2. Dill herb
By AEDA the four odorants shown in Figure 9 were detected with the highest FD-factors in dill herb [33 ]. A mixture of these compounds, dissolved in water at the same concentration ratios occurring in the herb (Table 16) very much resembled the typical odor of the dill herb. If (S)-a-phellandrene or the dill ether (B and A; Fig. 9), respectively, were omitted, the mixture lost its typical odor note. On the contrary, omission of myristicin and methyl 3methylbutanoate (D and C; Fig. 9) did not significantly influence the overall dill herb aroma of the model mixture [33, 66]. The data indicated that (S)-ct-phellandrene and the dill ether are the character impact compounds of the dill herb. Since, on the basis of AEDA or calculation of OAVs further odorants have been shown to contribute to the dill herb flavor [33, 66], the results of the simulation experiments revealed that obviously the two monoterpenes are able to mask the flavor contributions of these compounds. 4.3. Butter
In a similar study on butter flavor [28] we recently showed that a mixture of diacetyl, 5decalactone and butanoic acid dissolved in sunflower oil in the same concentrations occurring in a cultured butter, closely matched the flavor of the butter itself.
421
Figure 9. Important odorants of dill herb A (3R,4S,8S)-3,9-epoxy-l-p-menthen, B (S)-aphellandren, C methyl 2-methylbutanoate, D myristicin [33]. Table 16 Model solution simulating the flavor of dill herb [33] Odorant (S)-a-Phellandrene Dillether Myristicin Methyl 2-methylbutanoate
Conc. a (mg/kg water) 11.3 2.3 0.12 0.007
OAV b 56 77 4 18
a Amounts present in 10 g of dill herb were dissolved in water (1 L). b Calculated from odor thresholds in water. 4.4. Tomato
In studies on the flavors of fresh and processed tomatoes, Buttery and coworkers [76, 77] also found that the number of odorants needed to simulate either the fresh tomato flavor or the flavor of a tomato paste was much lower than the number of aroma compounds detected by the OAV concept [4]. In summary, these results corroborate the statement of Acree [5] that "response to mixtures of stimuli are characterized by inhibition and suppression and not by synergy". 5. F L A V O R P R E C U R S O R S
During food processing or storage j odorless food ingredients may be converted by biochemical or chemical reactions into odor-active volatiles. Knowledge about such precursors and reactions leading to the formation of odorants during food manufacturing
422 would offer the possibility either to enhance the flavors of products or to prevent off-flavors by selecting appropriate raw materials or optimized processing conditions. It should be stressed that it is a prerequisite of successful flavor precursor studies that the contribution of the odorant under investigation to a food flavor or off-flavor has been established. Sometimes the structure of a precursor can be assumed on the basis of structural elements in the odorant. In such cases, additions of the respective isotope-labelled precursor to the food system is commonly used to elucidate the precursor and to clarify reaction pathways governing the formation of the odorant. This method has been frequently applied, especially, in studies on the enzymatic generation of odor-active aldehydes (e.g., (Z)-3hexenal in tea leaves) or alcohols (e.g., 1-octen-3-ol in mushrooms) [cf. reviews in 84, 85] as well as lactones [86] from unsaturated fatty acids. A further approach to gain an insight into flavor precursors is the degradation of single food components in model systems and numerous studies have been performed to investigate the formation of heterocyclic volatiles from sugar/amino acid reactions [cf. review in 87]. However, mostly qualitative data on the volatiles formed are reported, whereas only a few investigations are available correlating, on the basis of precise quantitative data, the amount of the precursor(s) with the yield of an odorant. Furthermore, the reaction conditions applied in the model systems (e.g., concentration of the reactants, temperature) often significantly differ from the situation in the food under investigation. In the following, strategies to localize precursors of key odorants on the basis of systematic quantitative studies on food fractions and model systems will be illustrated for the important odorants 4-hydroxy-2,5-dimethyl-3(2H)-furanone and (E)-13-damascenone.
5.1.4-Hydroxy-2,5-dimethyl-3(2H)-furanone (HDF) The caramel-like smelling HDF has been established as a main contributor to the flavors of several processed foods (Table 17). In addition, it should be noted that in all these foods, on the basis of a high FD-factor, HDF was also by far the most important caramel-like smelling odorant. In the following, the strategy in the HDF precursor analysis will be shown using wheat bread crust, popcorn [88] and malt as the examples. Quantitative measurements were performed by using a stable isotope dilution assay (cf. Section 3.2.). Table 17 Concentrations and odor activity values of 4-hydroxy-2,5-dimethyl-3(2H)-furanone (HDF) in processed foods HDF Food
Reference (lag&g)
Wheat bread crust Rye bread crust Popcorn Dark beer Beef (stewed) Pork (stewed) Cocoa (commercial powder) b Sesame (roasted, white) Coffee (arabica)
1920 4310 1370 1320 11660 6500 6470 9155 6600
OAV a 26 57 18 18 155 87 86 122 88
[55, 88] [45] [88] [18] [79] [79] [46] [89]
a The OAVs were calculated on the basis of a mean odor threshold of 75 lag/kg medium (water: 100 lag/L, oil: 50 lag/L, cellulose: 75 lag/L). b Schieberle P, unpublished results.
423 In the crust of a model wheat bread prepared from a dough in which yeast had been replaced by a commercial leavening agent, the concentration of HDF was ten-fold lower than in the crust of the bread prepared from the yeasted dough indicating that bakers yeast supplied the precursors of HDF [88]. To localize these precursors, disrupted yeast cells were fractionated by centrifugation and ultrafiltration (Figure 10). As detailed in Table 18 (expt. 1), significant amounts of HDF were liberated simply by boiling a yeast fraction containing the water-soluble low molecular weight compounds (LMW; cf. Figure 10). Since HDF was already formed in significant amounts at 100~ it was concluded that the precursors are relatively labile.
Figure 10.Scheme for the isolation of precursors of 4-hydroxy-2,5-dimethyl-3(2H)-furanone (HDF) from bakers yeast. HMW- (MW > 1000), LMW-compounds (MW < 1000). HDF has been identified as reaction product of a thermal treatment of sugars, especially the 6-desoxy sugar rhamnose [90]. Analysis of the free sugars in the LMW-ffaction revealed fructose-l,6-biphosphate (FBP) as the predominating carbohydrate in the LMW fraction (5.75 g/kg yeast), but no rhamnose was present [88]. To elucidate the contribution of FBP as precursors of HDF in the yeast fraction, the sugar phosphate was thermally degraded under the same conditions as used for the LMW fraction of yeast. The results revealed FBP, as effective precursor of HDF in aqueous model systems at lower reaction temperatures (100~ Table 19). It should be stressed that additions of the amino acids proline or alanine did not increase the concentrations of HDF from the carbohydrates listed in Table 19 (unpublished results). The data implied that FBP which was the predominant carbohydrate in yeast, is the
424 main precursor of HDF in wheat bread crust. In similar experiments it was recently shown [79] that sugar phosphates (i.e. glucose-6- and fructose-6-phosphate) are important precursors of HDF in roasted beef. Table 18 Generation of 4-hydroxy-2,5-dimethyl-3(2H)-furanone (HDF) from fractions of watersoluble, low molecular weight compounds (LMW; cf. Fig. 10) isolated from yeast, maize and barley [88 and P. Schieberle, unpublished results] Heating
HDF ([ag/kg)
HDF (gg/kg)
Expt. T (~
Time (min)
yeast
maize
barley not germinated
1b 2b 3c
100 150 160
60 45 15
7825 13071 8596
<10 93 1103
<10 130 2923
germinated a <10 223 11959
a Barley was germinated (5 days) by a common process. b The extract (from 5 g of yeast or 20 g of cereal) was reacted in phosphate buffer (20 mL, 0.1 mol/L, pH 7.0). A laboratory autoclave was used. c The freeze-dried extract (from 5 g of yeast or 20 g of cereal) was mixed with silica gel (3 g), placed in glass tubes and reacted in a metal bloc. Table 19 Amounts of 4-hydroxy-2,5-dimethyl-3(2H)-furanone (HDF) liberated by thermal degradation of carbohydrates under different conditions HDF (gg/mmol) Carbohydrate a 100~ (60min) a Fructose- 1,6-biphosphate Sucrose Glucose F ructos e-6-phosphate Glucose-6-phosphate Fructose Maltose Rhamnose
88 <0.1 <0.1 82 3.0 3.0 <0.1 102
150~ (45min)a 232 <0.1 4.5 257 149 15 7.8 1410
160~ (15min) b 44 <0.1 60 32 166 94 56 1685
a The carbohydrate (0.2 mmol) dissolved in phosphate buffer (20 mL, 0.1 mol/L, pH 7.0) was reacted in a laboratory autoclave. b The carbohydrate (0.2 mmol) was mixed with silica gel (3 g) and reacted in small glass vials in a metal bloc. The fractionation scheme used in the yeast experiments (cf. Figure 10) was also applied to isolate the precursors of HDF in popcorn and dark beer from the corresponding raw materials maize and barley (malt --* beer). Contrary to the yeast experiments, the respective
425 LMW-fractions from maize and non-germinated barley, which contained the HDF precursors, did not produce significant amounts of the odorant when reacted in aqueous solution (expts. 1 and 2, Table 18). However, dry-heating of the freeze-dried extract significantly increased the amounts of HDF by factors of eleven or twenty, respectively (cf. expts. 2 and 3, Table 18). In an LMW extract prepared from germinated barley, about four-fold more HDF was formed than in the extract from the non-germinated barley (expt. 3, Table 18). Analysis of the carbohydrates in the respective LMW fractions revealed maltose, glucose, fructose and sucrose as the predominant free sugars in both cereals (Table 20). Neither sugar phosphates nor rhamnose were present in significant amounts. Model studies revealed (cf. Table 19) that under dry-heating conditions (160~ significant amounts of HDF were liberated from maltose, glucose and fructose with the latter sugar being the most effective precursor among the sugars present in the cereal extracts (Table 20). Assuming the same sugar degradation pathway in popcorn and the model systems, one can estimate that the free sugars present in maize should liberate 1227 gg HDF/kg popcorn, which is very close to the value measured in popcorn (cf. Table 17). The validity of this assumption is further corroborated by the fact that a four fold increase in the free sugars during germination of barley (Table 20) also led to a four fold increase in HDF after thermal treatment (Table 18). Table 20 Concentrations of free sugars (mg/kg) in the LMW-fractions from maize and barley [Schieberle P, unpublished results] . barley Carbohydrate a
maize non-germinated
germinated
Maltose Glucose Fructose
2452 1190 880
2965 1998 1806
8161 11258 8835
total
4522
6759
28254
a The concentrations were determined enzymatically. On the basis of these results a general pathway for the formation of HDF from sugars was assumed as exemplified for fructose-l,6-biphosphate in Figure 11 [88]. Elimination of the phosphate group at C-6 of the 1-deoxyosone-6-phosphate, results in acetylformoine which was established as the key intermediate in HDF formation [88]. Reduction of acetylformoine either by a disproportionation reaction with a second molecule of acetylformoine or by further reducfive agents present in foods, like vitamin C, then, after elimination of water, generates HDF. The key reaction governing the amounts of HDF during food processing is the formation of acetylformoine. If non-phosphorylated sugars like fructose or glucose are present, water instead of phosphate has to be eliminated from carbon atoms 1 and 6 of the sugar moiety (cf. Figure 11). This seems to be possible only under dry-heating conditions, like popping of corn or drying of malt. Similar reactions may lead to HDF formation during roasting of coffee, cocoa beans or sesame seeds. However, this has to be proven by further experiments. The data indicate that if the respective precursor is present and if the processing conditions are similar, the same odorant will contribute to the flavors of different foods. However, HDF has been detected also as a potent odorant in unprocessed foods like strawberries [48], pineapple [91] and, very recently, in Emmentaler cheese [30] indicating alternative biochemical pathways in HDF formation.
426
Figure 11. Formation of 4-hydroxy-2,5-dimethyl-3(2H)-furanone (HDF) from fructose-l,6biphosphate via acetylformoine as the intermediate.
5.2. (E)-fl-Damascenone
(E)-g-Damascenone (1; Fig. 12) has been identified as volatile constituent of many foods [92], e.g., apples [93], grapes [94] or tea leaves [95]. By application of CHARM analysis, AEDA or the OAV-concept, the odorant has been established as a key aroma compound in different honeys [35], tomato paste [4, 77], heated apple juice [54] or heated strawberry juice [48]. In a systematic study on damascenone precursors in grapes, Braell and coworkers [96] used the CHARM analysis to localize components generating damascenone upon heat treatment. A methanolic extract from the grape skins was fractionated on C 18 reversed phase adsorbent, followed by a polyamide separation and finally I-IPLC on C18 adsorbent again. Fractions showing 13-damascenone generating activity were detected by sniffing the volatiles generated by heating the grape fractions in the presence of acid. In a subfraction, which had been enriched in precursor material at least 22.000 fold on the basis of CHARM, several precursors were detectable. The authors assumed that these precursors were glycosidically bound terpenes. The formation of damascenone by acid catalyzed degradation of a precursor has also been reported in several further studies on fruit flavors [54, 77, 97, 98]. A general scheme proposed for the isolation and characterization of glycosidic flavor precursors in fruit or plant extracts is shown in Figure 13 [99]. The glycosides are enriched by adsorption on Amberlite XAD-2. Precursor fractions liberating e.g., (E)-13-damascenone after heating are further separated and, especially countercurrent chromatographic techniques have been demonstrated as powerful tools to pre-separate the glycosides before HPLC purification [98]. Using droplet counter current chromatography at least three different precursors of the odorant were separated in an extract of Riesling wine [98]. In a study on the characterization of 13-damascenone precursors in apple cultivars, Zhou et al [54] also found evidence for at least seven glycolytic precursors.
427
R=H or [t-D-gtucose Figure 12 Norisoprenoids characterized as precursors of (E)-13-damascenone (1) [ 10 I, 102]. I PLANT EXTRACT ]
I I
Adsorptionon RP-C18 or XAD-2 FaOA cC~eOtl-e lutio n
(GLYCOSIDIC MIXTURE,I
Hydrolysis
AGLYCONS
Preseparation
(LC, HPLC, SEC, CCC)
Derivatized r'
9 ~ Non-derivatized lSt~-ntAcrtoNsJ
.......
: r .........
I
HRGC-
'
HRGC-
MS
v-t-~t
'
!
Purification
I I I I NMR MS t.-t-~t Chiroptieal Methods
1 Hydrolyses (H § I ~ e )
Figure 13. Scheme for the isolation of glykosidic precursors (adapted from reference [99]). The precursor structures can be elucidated by HRGC~S and HRGC/FT~ of the acetylated derivatives or aglycons, respectively. Very recently, direct LC/MS measurement of a non-derivatized precursor glycoside of (E),13-damascenone isolated from apples has been reported [100]. Application of such techniques on precursor fractions from wine [97, 101]
428 and the leaves of Lycium halimifofium Mil [102] recently led to the identification of two precursors of (E)-f3-damascenone whose structures are displayed in Figure 12. Model studies on the degradation of the corresponding glycoconjugates as well as the non-glycosidic allenic triol and enynediol (cf. Figure 12) have recently revealed that the acid catalyzed degradation of especially the aglycon precursors "can account for damascenone formation during fruit processing" [103]. However, up to now a correlation between the amounts of precursors present in the flesh fruit or leaves and the amount of (E)-f3-damascenone formed after processing is lacking. Further investigations aimed to identify the precursors of key food odorants by correlating the amounts of precursors present in the food or raw materials with the yield of an odorant in the food product are summarized in Table 21. Table 21 Key odorants in foods and their precursors in the raw materials Odorant
Food
Precursor(s)
2-Acetyl- 1-pyrroline
Wheat bread crust Popcorn Roasted beef Boiled beef Stewed beef Soy beans Tea Butter
Ornithine/2-Oxopropanal Proline/2-Oxopropanal Alanine/2-Oxopropanal Thiamine/eysteine Plasmalogens Furanoid fatty acids
2-Ethyl-3,5-dimethylpyrazine 2-Methyl-3-furanthiol
12-Methyltridecanal 3 -Methy 1-2,4-nonanedione
Reference [ 104] [ 105] [ 106] [ 107] [ 108] [80]
6. CONCLUSIONS The progress in the development of methods directly combining sensory evaluation and analytical data such as AEDA or CHARM analysis has led to the identification of the most aroma-active compounds in several foods. Since FD-factors or CHARM values are not a direct measure for the odor potency of an aroma compound in the food itself, a calculation of OAVs on the basis of exact quantitative data are a necessary further step to evaluate the contribution of the flavor compounds identified. Sensory experiments to match the food flavor or off-flavor, respectively, are consequently the last step in flavor analysis to transfer the data obtained by using sensory responses to single odorants on the food flavor itself. Odorants showing high OAVs can then be used as indicators to objectively assess flavor differences in foods. Furthermore, knowledge about the key odorants as well as exact quantification methods at hand will enable successful precursor studies to clarify the formation of food flavors during processing or storage. Much progress has also been made in the development of methods for authenticity control of flavor compounds, such as chiral separations or isotope ratio analysis. However, a comprehensive discussion of this subject would have gone beyond the limits of this chapter. The interested reader is, therefore, referred to some recent articles published in this field [109-111].
Acknowledgements. I am grateful to Mrs. R. Berger for carefully typing and laying-out the manuscript.
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433
Index Absorption of Microwaves (see Microwave, absorption of, in food) 2-Acetyl-l-pyrroline, wheat bread flavor, 410, 428 Acid fuchsin, 253 Acoustic emission, 112 Acoustic impedance (see Ultrasonic impedance) Acridine orange, 235, 241 Adiabatic compressibility, 97, 106 Adsorbed layer structure, 42, 46-48, 51 monolayers in, 51 multilayers in, 45, 51 Adsorption kinetics, 2 Adsorption, competitive, 25, 42, 46, 51, 53, 55 Adulterants, 348, 354, 362, 364 AEDA (see Aroma extract dilution analysis) Aerated desserts, 59, 60, 83 Aflatoxins, 365, 367, 368 Agar, 243, 244, 267 Aggregation, 15, 16, 18, 19, 60, 86, 204 Air bubble stabilization (see Foam stability) Alcian Blue, 243, 244 Alkaline phosphatase, 354, 358, 359 Allergens, 348, 362, 364 Alpha-lactalbumin, 40, 45, 46 Amino acids response patterns of, 387 taste of, 386, 398 Amplification system for immunoassay, 358, 359 Anemometer, 283 1-Anilino 8-napthalene sulfonic acid (ANS), 239, 242
ANS (see 1-Anilino 8-napthalene sulfonic acid) Antibiotics, 354, 365 Antibody, 38, 39, 243, 330,341, 347-352, 354, 357-361, 363, 366 agglutination, 352 characterization of food, in, 347-371 fluorescent-labelled, 368 immobilization of, 352, 368 monoclonal, 330, 347, 348, 351, 358, 365, 366, 368, 369 preparation of, 351 polyclonal, 350, 358, 366, 368, 369 preparation of, 350 precipitation, 352 solid-phase binding assay for, 354 structure of, 349 Antibody-antigen reaction (complex), 332, 337, 340, 341, 348, 349, 352, 365 Antigen, 38, 330 immobilization of, 352 solid-phase binding assay for, 354 Apples, 126, 128, 143, 407 NIR application to, 190 Aroma compounds, 403 Aroma extract dilution analysis (AEDA), 406, 409, 411, 415-420, 426 comparative, 408, 409 illustration of, 411 static headspace, of olive oils, 411 Aroma value (see Odor activity value) Atomic force microscopy (AFM), 269, 270 Attenuated total reflectance (ATR), 197 Autofluorescence, 242, 243, 259 Avidin, 358 Avocados, 126
434 Back-light scattering, 1, 15-18 Bagley's correction, 287, 295 Bagley's plot, 288 Banana, 141, 143 Beef, 180, 407, 415, 418, 422, 428 Beer, 25, 378, 390-394, 406, 409, 415, 418-420, 422, 424 alcohol-free, 419, 420 chillproofers in, 369 flavor dilution (FD) chromatogram of, 407 flavor simulation, 419 flavor, 406-408, 420 foam collapse, 128 foaming of, 369 key odorants in, 408 NIR application to, 190 odor activity values of, 419 sensor for, 335 taste map of, 393, 394 taste of, 390-394 Beta-casein, 46-49, 210 Beta-galactosidase, 335, 336 Beta-glucans, 237, 255 Beta-lactoglobulin, 38-40, 42-45, 49-54 adsorbed layer structure, 44 FITC-labeled, 39, 42, 50, 51 Tween 20, binding by, 44 Beverages, FTIR application to, 197 NIR application to, 190 taste sensor for, 390-394 Binding constant, 350 covalent, 334 protein-fat, 72, 73 protein-polysaccharide, 73, 85 Bingham fluid, 297 Biological membrane, 377, 382 Bioluminescent assays, 357 Biosensor based on biological components used, 334 cell and tissue type, 337, 338 commercial, 338-340 device, 330-334 biological component, 330, 332-334, 338
[Biosensor, device] transducing component, 330, 332, 333, 338, 341 unification of biological and transducing components, 333, 334 electrochemical, 332, 333 enzyme, 335-337 immuno, 337 in food analysis, 335-342 on-line, characteristics of, 342 optical, 332, 333 terminology of, 334, 335 thermal, 332, 333 Biotin, 358 Bis-phenyl oxalate, 357 Biscuits, 321 EMG profiles, 319, 320 NIR application to, 190 Bitterness, 377, 379, 384-386, 388, 389, 393, 394 Black film common, 9, 30, 40 Newton, 7, 9 Boltzman constant, 118 Bone, 164, 167, 170-173, 179, 180 Bovine serum albumin (BSA), 3, 31, 38, 40-42, 191, 348, 352, 365 Bread, 299 crust, 407, 409, 410, 422, 428 dielectric and thermal properties of, 214 flavor, 409, 410 making qualities of wheat by multiple discriminant analysis, 188, 189 by principle component analysis, 188 NIR application to, 190 Brij 58, 2,4 Brittleness, 311, 321 Brix value, 193 Brochothrix thermosphacta, 368 BSA (see Bovine serum albumin)
Butter, 224, 405, 407, 417, 418, 420, 428 dielectric and thermal properties of, 214 fat, 60, 64 flavor, 420 oil, 60, 407, 409 spreadability of, 278
435 Caffeine, 377, 380 Campylobacter, 165 Capillary force balance, 15, 16 Capillary pressure, 2-4, 7, 11, 15 Caramels, 279 Carbohydrates markers for differentiation of, 243, 244 staining procedures for, 241 Carboxymethylcellulose (CMC), 243, 246, 286 Carrageenan, 85, 86, 267 markers for staining, 243, 244 Carrot, 279, 317 EMG profiles, 319, 320 Casein micelles, 74-76, 109, 243 in yogurt, 270 SANS study of, 206-208, 210, 211 structure by microscopy, 267, 268 Casein sub-micelles in yogurt, 270 SANS study of, 206-209, 211 Cellulose diacetate, 330 Cellulose, 164, 177-179 Cereals disinfection of, 165 NIR application to, 190 CHARM analysis, 406, 412, 426, 428 Cheese, 112, 152, 160, 235, 236, 238, 243, 259, 266, 267, 287, 407 Cheddar, 288 mechanical (fracture) properties of, 301 contaminants in, 337 diffusion coefficient of moisture in, 132 Gouda, 315, 316 high resolution NMR spectra of, 155 microscopy of, 236, 238, 259, 267 Mozzarella, 288, 294 NIR application to, 190 particle size distribution by NMR, 132 processed, 288 sauce, identification of calcium phosphate by microscopy, 235 spread, microscopy of, 238, 244 strechability of, 279 Swiss (Emmentaler), 415, 416 odorants of, 416, 417, 425 Chemical shift, 121, 145
Chemiluminescent assays, 357 Cherries, 177 Chew cycle, 317 Chewiness, 311, 312 Chewing behavior, 312 Chewing efficiency, 316 Chewing gum, 279 Chewing sequence, 312, 317, 320-322 Chicken, 167, 169, 170-173, 180 Chitin, 177 Chlortetracycline (CTC), 242 Chocolate, 235 cryo-section of, 241 fluorescence microscopy of, 239 Cholesterol, sensor for, 338 Citric acid, 394 Clostridium botulinum, 358, 364 CMC (see Critical micelle concentration) Coagulation factors, 359 Coalescence, 23 Cobalt-60, 164 Cocoa butter, 128 Cocoa, 422 NIR application to, 190 Coconut fat, crystallization of, 65 Coefficient of viscosity (see Viscosity) Cofactors, 337 Coffee, 378, 390, 422 Collagen, 167 Combined headspace aroma extract dilution analysis, 409-412 Complexes Span 20 and beta-lactoglobulin, 45 Tween 20 and beta-lactoglobulin, 44 Compression rate, 311 Compression tests, 300, 301 Concanavalin A, 243 Cone penetrometer, 278, 311 Confocal microscopy, 258, 259 Consumer perception, 313 Contact angle, 7, 30 Contaminants in food, 364, 354, 362, 365 Controlled drop tensiometer (see Tensiometer, controlled drop) Cooking, 108 Coomassie Brilliant Blue, 239 Correlation time, 136, 138 Coumaric acid, 254
436 Covalent deuteration, 210, 211 Cream cheese, 224, 235, 239, 240, 245, 246 spreadability of, 278 Creaming, 110, 111, 128 Crispness, 311 Critical micelle concentration (CMC or cmc), 2, 7, 42, 80, 81 Cross-linking, 334 Crumbliness, 321 Crunchiness, 311, 321 Crustecea, 176, 177, 179, 180 Cryo-scanning electron microscopy, 263-268 of ice cream, 265 of refrigerated and frozen products, 263-266 Cryo-sectioning, 234, 236 Cryo-SEM (see Cryo-scanning electron microscopy) Crystal network, 78 Crystallinity coefficient, 170, 171 Crystallization, 128, 144 of emulsifiers, 78 of oils, 78 Cytochemistry, 238 Degradation, oxidative, 141 Degree of sourness, 397 Deliciousness, objective measure of, 399 Demascenone, (E)-beta-, isolation of precursors, 419, 426-428 Demulsification, 1 Density, effect on ultrasonic velocity, 96, 97, 106 Dephasing, 120 Desorption kinetics, 2 Desorption, of surfactants, 3 Diachrome, 235, 242 4, 6-Diamidino-2-phenylindole (DAPI), 242 Dielectric constant, 20, 214, 217-221 critical frequency, 218, 229 loss factor, 214, 216-220, 226-228 measurements of foods, 213-229 permittivity (see Dielectric constant)
[Dielectric] properties of foods, 213-222, 226-229 complex models, 228, 229 definition of, 213, 214 distributive model, 227, 228 Fricke model, 229 temperature dependence of, 219 measurement of, 219, 220 cavity perturbation techniques, 220 open ended probe technique, 220 transmission line techniques, 219, 22O underheating mode technique, 220-222 relaxation frequency, 218 spectrum, 216-218 ionic interactions, 217 polar interactions, 217, 218 spectroscopy, 227-229 Dielectrometry, 1, 18-20 Differential scanning calorimetry (DSC), 62, 63, 128 Differential thermal analysis (DTA), 62, 83 Diffraction grating, 17 Diffusion coefficient, 41, 42, 48, 53, 128, 130-132, 138, 145, 152, 153, 156 lateral, 37, 40 self, 156, 159, 160 Diffusion, 2, 129 lateral surface, 23, 25, 28, 34, 43, 45, 50-53 in protein-stabilized films, 34, 35, 41-46 in SDS or lipid-stabilized films, 40 restricted, 132, 156, 159 unrestricted, 159 Dilatational stress, 2 Dilation of interface, 2, 3 Dilational elasticity (see Storage modulus) Dilational viscosity (see Loss modulus) Dilatometry, 63 Dill herb, 407, 420 odorants of, 421 Dip stick test, 352, 360, 370 for salmonella, 360
437 Dipole moment, 186, 217 Dipole-dipole interaction, 136, 139, 146 Disodium guanylate (GMP), 377, 378, 380 Disodium inosinate (IMP), 377, 378, 380 DLVO theory, 7, 8, 11 DNA probes, 329, 341 Donnan potential, 383 Doppler anemometry, 134 Dough, 139, 259, 266, 299, 423 extensibility of, 279 processing, 417 quality, 294, 296 rheology, 294, 295 effect of ascorbic acid, 294 on-line rheometer for, 295 Drainage behavior, of thin films (see Thin films)Dressing pourable, 246 detection of xanthan by microscopy, 246 viscous, 246 Drinks, aqueous ionic, 390 Droplet size distribution (see Particle size distribution) Drying, 128, 145 Echo attenuation ratio, 156-161 Egg white, 128 Egg, liquid, FTIR application to, 197 Elastic modulus, effect on ultrasonic modulus, 96, 97, 106 ELCA (see Enzyme-linked coagulation assay) Electrical potential pattern, 385, 388, 396, 398 Electrical potential, 379, 383, 387, 390, 391, 395, 397 Electromagnetic dielectric spectroscopy (EDS), 227, 229 Electromotive force, 120, 122 Electromyography (EMG), 314, 316-322 for assessing food texture, 317-320 for monitoring mastication, 317, 318 textural signature, 321 Electron microscopy, 66-69, 81, 151, 260-267
[Electron microscopy] cold-field emission scanning (FESEM), 270 comparison of SEM and ESEM, 261 cryo-scanning, 260, 263-267 environmental scanning (ESEM), 260-263 freeze etching, 66 freeze fracture, 66, 268-270 negative scanning, 267, 268 scanning (SEM), 233, 260-267, 270, 368 of Gouda cheese, 315 scanning probe (SPM), 269, 270 scanning tunneling (STM), 267, 270, 271 transmission (TEM), 66, 74, 233, 267, 268, 270, 368 Electron paramagnetic resonance (EPR) spectroscopy, 163-180 applications in foods containing bone, 167-173 in foods containing shell, 173-177 in fruits and vegetables, 177, 178 in nuts, 179 in spices and herbs, 178, 180 for detection of irradiation in foods, 163-180 principles of, 163, 164 ELISA (see Enzyme-linked immunosorbant assay) EMG (see Electromyography) Emmentaler cheese (see Cheese, Swiss) Emulsifiers, 23, 30, 60-65, 68, 70-72, 74-83, 85 Emulsion, 23, 51, 52, 106, 109, 127, 151, 152, 159-162 dielectric properties of, 229 oil-in-water (O/W), 4, 15, 23, 27, 51, 62, 109, 128, 151, 160, 162, 369 creaming in, 127, 128 oil-in-water-in-oil (O/W/O), 7 stability, 2, 7, 19, 45, 55 effect of markers on, 240 stabilization mechanism for whippable emulsions, 60, 61 water-in-oil (W/O), 4, 29, 151, 156, 160-162 water-in-oil-in-water (W/O/W), 4
438 [Emulsion] whippable, 59-88 characterization of structure of, 59-88 fat crystallization in, 62-65 particle size distribution of, 69 rheology of, 87 Encapsulation, 333 Enterotoxin, 365, 367 Entrapment, 333 Environmental scanning electron microscopy (ESEM), 260-263 Enzyme complex, 358 Enzyme electrode, 331, 335 Enzyme sensor, 336, 337 Enzyme-linked coagulation assay (ELCA) 358, 359 Enzyme-linked immunosorbant assay, 337, 347, 354-369 competitive, 354, 355 ELCA combination, 358, 359 non-competitive, 354, 356 sandwich, 354, 357 Epi-illumination, 27, 29, 34, 36 Epifluorescence, 236, 237, 239, 241, 246, 248, 257 EPR spectroscopy (see Electron paramagnetic resonance) Ethidium bromide, 242 Evan's Blue, 243, 259 Extensiograph, 294 Extensional rheometry, 280, 290-297 Extensional viscosity (see Viscosity, extensional) Extruder, single-screw, 135, 281 Extrusion, 134, 135, 295 cooking, 294 Fat crystallization, 144 in whippable emulsions, 62-66, 79 globule agglomeration, 69-71, 86 globule crosslinking, 67 polymorphism in, 65, 144 supercooling of, 62-65 FD chromatogram (see Flavor dilution chromatogram) FD factor (see Flavor dilution factor)
Ferinograph, 266, 294 Ferulic acid, 254 Fiber optics, 193, 336 Fiber, absorption spectra of, 250, 251 FID (see Free induction decay) Field gradient, 117, 118, 135 Film drainage, mechanism of, 7, 10 tension, 4 dynamic, 6 thickness, 7, 30, 32-34, 37, 40-43, 45-50 Fish dielectric and thermal properties of, 214 fat content from dielectric loss factor, 225 freshness, sensor for, 336, 337 irradiated, 165 oil, 143 Flavor changes, detection of, 408, 409 compounds (see Flavor volatiles) dilution (FD)chromatogram, 406 dilution (FD)factor, 406, 408-410, 412, 417, 420, 428 precursors, 403, 421-428 isolation of glycosidic precursors, 427 quality, 403 simulation of beer, 419, 420 of butter, 420, 421 of dill herb, 420 of tomato, 421 volatiles isolation of, 404, 409, 411,412 methods of analysis, 404-428 Fluid flow, imaging of, 134, 135 Fluorescein, 39 Fluorescein diacetate, 255 Fluorescein isothiocyanate, 244 Fluorescence intensity, 249, 250 Fluorescence microspectrophotometers, 248, 249 Fluorescence, 23, 43 labeling, 34, 38 measurement of surface concentration, for, 40 microphotolysis (see FRAP)
439 [Fluorescence] microscope, 36 photobleaching recovery (see FRAP) recovery after photobleaching (FRAP), 34-38, 40-54 apparatus for, 36 definition of, 34 in emulsion subnatants, 52 in protein-stabilized thin films, 41, 42 of alpha-lactalbumin, 45-47 of beta-casein, 46-49 of beta-lactoglobulin, 42-45, 50-52 in surfactant-stabilized thin films, 40, 41 Fluorescent probes, 247 Fluorophores, 36, 38 Flurochrome, 255 Foam, 127 analyzer, 71 collapse, 128 dielectric properties of, 229 stability, 2, 7, 12, 45, 46, 48, 61, 67 lamella, 23 (see also Thin films) stability of, 45, 48, 49 control of, 55 Food analytes, 336 Food contaminants, 337 Food dispersion, 21, 23, 55 Food emulsion, 9, 11, 15-17, 19, 23, 65, 71, 151, 152, 158, 279 structure by Kossel diffraction, 15-18 wall-slip effects in, 296, 297 Food foam, 23 Food products chewing patterns of, 314 contaminants in, 348 covert finger printing of, 371 fat-reduced, 303 sugar-reduced, 303 swallowing patterns of, 313 Food rheology, 277-303 areas of growing interest, 279, 280 empirical versus fundamental tests, 278, 279 recent advances in, 277-303 terminology, 299 Food texture (see also Food rheology) instrumental measurement of, 310-312
[Food texture] modeling of, 322 new approach for assessment of, 313-314 perception of, 321, 322 selection of test conditions, 311 sensory measurement of, 312-314 temporal aspect of assessment, 311, 312 variation in chewing behavior, accommodation for, 312 Force-deformation curve, 302 Fourier transform infrared (FTIR) spectroscopy, 185, 196-198 in determination of sugar cane sucrose, 197 in food characterization, 185, 196-198 recent applications of, 197 Fourier Transform, 103, 104, 117, 119-121, 123-125, 155, 207, 321 two-dimensional, 125 Fracture mechanics, 298 Fracture properties, of food, 279, 280, 297 Fracture tests, 301,302 FRAP (see Fluorescence recovery after photobleaching) Free fat estimation, 71 Free induction decay, 120, 121, 140, 155 Free radical, 164, 170, 171 Freeze fracture electron microscopy, 268-270 Freezing point, determination of, 83 Freezing, 128, 145 Friction, 278, 301 Frozen food, dielectric properties of, 229 Fruit juice (see Juice) Fruit purees, 285 Fruits dielectric and thermal properties of, 214 dried, 167, 177 irradiated, 177, 178 NIR application to, 190 ripening of, 165 soluble solids by NIR, 193 sweetness sorting machine based on NIR, 195 FTIR spectroscopy (see Fourier transform infrared spectroscopy)
440 Fundamental vibration, 185 frequency, 186 water molecules, of, 186 wave number, 186 Garlic, 177, 178 Gas chromatography (GC), 405, 416 high resolution (HRGC), 405 Gas chromatography olfactometry (GCO), 405, 406, 412, 413 Ginger, 178 Glass transition temperature, 83, 84 Glutaraldehyde, 237, 238, 240, 269 Gluten, 266 Gradient phase, 125 pulsed field (see Pulsed field gradient) slice selection, 124 Graininess, 235 of process cheese, 259 Grapes, 143, 177, 407 Griffith's law, 298 Guar gum, 243- 246 markers for staining, 244, 245 Guided microwave spectrometry (GMS), 225 Guinier plot, 204, 206, 208, 209 Gum Acacia (see Gum Arabic) Gum, 16 Arabic, 143, 369 Gustatory sensation, 378, 398 Gyromagnetic ratio, 118, 119, 138, 152, 156 Hahn spin echo sequence, 153-155 Hand-sectioning, 234-236 Hapten, 245, 246, 348 Hardness, 311 Harmonicity constant, 186 Headspace isolation, combination with odor dilution techniques, 409-412 Headspace, 409-411 Heterodyning, 122 High performance liquid chromatography (HPLC), 192 Histochemistry, 238
Honey, sensor for sugar determination, 335 Hookean solids, 300 Horseradish peroxidase, 354 Hydration, 138 Hydrocolloids, 19, 60, 61, 73, 74, 79, 83-87 Hydrodynamic radius, 45 Hydrogen peroxide electrode, 330, 331, 335 Hydrogen peroxide, 357 Hydrophobicity, of fat globules, 61 4-Hydroxy-2,5-dimethyl-3(2H)-furanone (HDF), 422-426 formation from precursors in barley, 423,424 bread crust, 423, 424 maize, 423, 424 yeast, 423, 424 key odorant in, beer flavor, 419, 420 sesame flavor, 417 Swiss (Emmentaler) cheese flavor, 417 Ice cream, 20, 55, 59-61, 68, 69, 74, 79, 83-85, 87, 88, 151, 263, 265 freezing curves of, 84 melt-down, 87, 88 mix, 60, 62, 65, 68, 71, 72, 74-76, 78, 82, 86, 235, 246, 263 fat crystallization in, 62-66, 79 low-fat, 80-82, 88 no-fat, 88 viscosity of, 85-87 wheying off of, 85 water phase of, 82-85 freezing point determination of, 83, 84 ice crystals in, 84, 85 NMR of, 82, 83 Ice crystals, 84, 85 size reduction of, 84 Ice, dielectric and thermal properties of, 214 Image analysis, 271 Imaging chemical shift contrast, 126
441 [Imaging] k-space, 122-129 q-space, 133-135 relaxation contrast, 126 two dimensional, 124, 125 velocity contrast, 134 Immunoadsorption purification (see
Immunoaffinity purification) Immunoaffinity chromatography, 370 Immunoaffinity purification (IAP), 347, 365, 367, 368 Immunoassay, 329 amplification system for, 358-360 biotin-avidin system, 358 coupled enzyme system, 358 application in food systems, 362-365 allergens, 363 contaminants and toxins, 363-365 detection of adulterants, 362, 363 commercial kits, 365-367 detection systems for, 360, 361 functionality determination, for, 369 on-line monitoring, for, 370 performance of, 361, 362 quality control, for, 369 value of, 361 Immunocytochemistry, 347, 368-370 Immunomicrochemical staining, 267 Immunoprecipitation assay, 353 Immunosensors, 337, 339-341, 370 Ingredient composition of aerated desserts, 60 of ice cream, 60 of toppings, 60 of whipping cream, 60 Interfacial activity, 77 crystallization, 78 effects, in whippable emulsions, 71-82 hydration, 76, 77, 82, 86 properties, 279 rheology, 1-6, 278 shear viscosity, 31 tension (IFT), 2, 3, 5-7, 77-79 dynamic, 3, 5, 6 equilibrium, 2 Interferogram, 196 Interferograph, 33, 34
Interferometry, 7-15, 23, 31-34 common, 7, 13 differential, 12-14 reflected light, 7 Iodine number NIR sensor for, 192 of fats and oils, 192 Ionic interactions,:217 Irradiated foods, safety of, 165 (see also
Irradiation) Irradiation adsorbed dose, 166 measurement of, 173 detection methods, 166 requirements for, 166 dose rate, 171 dose response, 169 dose, 176 labeling regulations, 166 legislation, 165 quantitative identification of, 166 uses in food industry, 164-166 Isoelectric point, 238, 358 Isolation of volatiles, 404, 409-412
Juice, 104, 105, 140, 267 sensor for sugar determination, 335
Kappa-Carrageenan, 20 Kappa-casein, 243 Ketchup, 285 Kohler illumination, 244 Kossel diffraction (see Back-light
scattering) Lactic acid, 397 Laminar flow, 285 Laminar velocity, steady, 281 Lamor frequency, 118, 119, 121, 122, 126, 129, 136, 137 Langmuir trough, 34 Langmuir-Blodgett method, 380 : Laser differential microanemometry (LMA), 285 Lactose in milk, 335
442 Lecithin, 30 Lectin, 241-246, 269 Lemon oil, 414, 418 Light microscopy, 71, 151, 234-258 near field, 271 polarized, 267 quantitative, 247-258 fluorescence analysis, advantages of, 247, 248 hybrid systems, 255-258 instrumentation for, 248-255 specimen preparation for, 234-238 hand-sections and cryo-sections, 234-237 sections of embedded materials, 237, 238 smears and comminution, 234, 235 staining procedures, 238-246 for carbohydrates, 241 for negatively charged groups, 241, 242 for nucleic acids, 242 for positively charged ions, 241 for protein, 239, 240 Lignin, 248-250 Line broadening, 137 Line width, 137, 138, 145 Lipid colorants, 240, 241 Lipid membranes, 378, 379, 381-384 lipids used in, 381 response to taste, 378-381 surface structure of, 380, 382 transducers, as, 379 Lipid, gel phase, 66 Lipid-protein interaction, 64 Lipolysis, 341 Liquid chromatography, 415 Liquid crystals, 65, 66 Liquid films, 1, 4, 7 emulsion film, 4, 5, 7, 15, 49-52 foam film, 10, 15, 42-49 microlayering in, 9-11, 13, 20 Newton black film, 7, 9 pseudoemulsion film, 12-15 stability of, 7-11 step transitions, 8-11, 13, 14 Listeria, 164, 337, 363-366 Lobster, EPR signals from, 173-175
Locust bean gum, 85, 243-246, 267 markers for staining, 244, 245 Longitudinal relaxation (see Relaxation, spin-lattice) Longitudinal relaxation, 121 Low-fat products application of ultrasound in, 108, 112 Luciferase, 357 Lumino immunoassay (LIA), 357 Lysolecithin, 30 Macroscopic magnetization, 119 Magnetic field gradient, 117, 135, 145, 152 Magnetic moment, 118 Magnetic resonance imaging (see Nuclear magnetic resonance, imaging) Magnetic susceptibility inhomogeneities, 137 Maillard browning, 136 Marangoni effect, 23, 25, 46 Margarine, 110, 111, 141, 154, 224, 417, 418 particle size distribution by NMR, 154, 160 Mass spectrometry, 416 Mastication, 298, 309,, 312, 316, 322 patterns, 316, 317, 322 process, 314, 316, 317, 322 Mayonnaise, 108, 110, 227, 228, 285, 287-289 confocal microscopy of, 259 extensional rheology of, 296 wall-slip effects in, 289, 296, 297 Meat, 93, 107, 111, 140, 225, 368 adulteration, 362, 363 dielectric and thermal properties of, 214 freshness sensor for, 336 identification of species of, 363, 364 irradiated, 165, 167-177 mechanically recovered (MRM), 167, 173 NIR application to, 190 processed, 235 proteins, 365 quality by ultrasound, 93 Melt-down, of ice cream (see Ice cream)
443 Melting enthalpy, 62 Membrane potential, 379, 384 3-Methylbutanal, rye crust flavor, 410 Micelles, 11, 13 Microemulsion, 152 SANS study of ,201, 205, 207, 209 Microlayering, 9-11, 13, 20 Microorganisms, 330, 338, 348 Microscopy, for food, 233-272 Microtiter plates, 360, 362 Microwave, 213-216 absorption of, in food, 214-216 diagnostic tool, as, 223, 226 cut-off frequency techniques, 225 phase and amplitude techniques, 223-225 resonant frequency technique, 226 energy, 216 penetration depth, 218, 219 reflection of, in food, 214, 215 sensor, 226 Milk composition by ultrasound, 108, 109 FTIR application to, 197 NIR application to, 190 31-P NMR spectra of, 144 rennet-coagulated gel, 271 sensor for, 335 Mixograph, 294 Modulus, 278 bulk, 97, 106 loss, 54 dilational, 2, 54 shear, 97, 106, 289 storage, 54 Young's, 97, 106, 298, 300 (see also Young's modulus) Moisture content, from microwave measurements, 224-226 Monoglycerides, 30, 60, 63, 68, 70, 72, 74, 76, 78, 81 Monolayer liquid-condensed, 77 liquid-expanded, 77 solid-condensed, 77 Monosodium glutamate (MSG), 377-380, 384, 387, 394
MRI (see Nuclear magnetic resonance, imaging) Multichannel electrode, 382, 392 Multichannel taste sensor (see Taste sensor, multichannel) Multilayer, 23 Multiple discriminant analysis, 188, 189 Multiple linear regression analysis (MLR), 187, 189, 192, 193, 197 Mushrooms, 165, 178, 407 Near field microscopy, 271 Near infrared spectra of nuts, 193 of rice, 186, 187, 190 of soybean, 186, 187, 190 of water, 191 Near infrared spectroscopy, 252 applications in food, 189-196 automatic composition analysis of soy sauce by, 196 food characterization, in, 185, 189-196, 198 fruit sweetness sorting machine, based, 195 iodine number of fats and oils, for, 192 on-line sensor, in, 192 principle of, 185-187 rice taste analyzer based on, 194, 195 secondary structure of protein, for, 191, 192 soluble solids in fruits, for, 193 state of water in food, for, 191 Negative staining, 267, 268 Neural networks, 398 Neutron scattering (see Small-angle neutron scattering) Newton black film, 7, 9, 30 Newton rings, 13, 30, 34 Nicotine, 380 Nile Blue, 236, 240-242 Nile Red, 240 NIR spectroscopy (see Near infrared spectroscopy) Nitrogen, liquid, 237 NMR (see Nuclear magnetic resonance) Non-Newtonian fluids, 303
444
Nuclear magnetic resonance, 63-65, 82, 83, 93, 112, 113, 117-146 authentication of foods and beverages, for, 144 carbon-13, 140, 141, 143 characterization of food, for, 117-146 CPMG echo, 138, 139 cross relaxation, 142, 143 determination of food components, for, 143 deuterium, 144 Fourier transform ,119 high resolution, 140-144, 146, 154, 155 imaging, 122-135, 145, 160 (see also Imaging) cocoa butter in chocolate, for studying, 128 cooking studies, in, 128, 129 creaming in O/W emulsion, for studying, 127 crystallization of fat, in emulsion, 128 drying studies, in, 128, 129 freezing studies, in, 128, 129 internal quality evaluation, for, of fish, 126, 127 of fruits, 126 of vegetables, 126, 129 in situ localized spectra, 144 low-resolution, 140, 143, 154 on-line monitoring of wine, for, 140 solid fat content, for, 63, 140 magic angle spinning (MAS), 141, 142, 145 phosphorous-31, 144 principle of, 118, 119 proton, 63-65, 140, 141, 143, 144 pulsed field gradient, 122, 124, 130, 145, 151-162 applications, 130-132, 159-162 drop size distribution by, 151-162 principles of, 152 spin echo, 129 theory of, 129, 130 solid fat content by, 63-65, 140 spin echo, 128-130, 145 supercooling study, for, 63 Nucleation, 62 Nucleic acids, 242, 330
Nuts, 167 irradiated, 179 sorter based on NIR, 192, 193 Oat kernel, 254, 255 Oat, rolled, microscopy-of, 237 Octadecyltrichlorosilane, 27 Odor activity value (OAV), 412-422, 428 Odor dilution techniques, 406-408 applications in food, 407 Odor threshold, 412, 413, 418-420 of beer odorants, 420 of selected food odorants, 413 Odor unit (see Odor activity value) Odor value (see Odor activity value) Odorants in beer, 408 in bread crust, 410 isolation of volatile fraction, 403-405 methods of identification, 403-412 quantification of, 413-415 Off-flavor, 403, 417, 418, 422 Oil Red O, 240 On-line monitoring, 140 biosensor for, 341 Onions, 177 NIR application to, 190 Optical isomerism, 387 Oral breakdown, dynamics of, 309 Oranges, 177 NIR application to, 190 Osmium tetroxide, 259 Overrun, 67 Oxidation-reduction potential, 383 Oxygen electrode, 337 Partial least square regression (PLSR) (see Principal component regression) Particle mass, from SANS, 207, 209 Particle size distribution, 69-72, 76, 151, 152, 157-162 .~ ice cream, in, 70 pulsed field gradient NMR, by,, 151-162 ultrasonic measurements, by, 109, 110 whippable emulsions, in, 69
445 Pastry odorants, 418 Pattern recognition, 321 Peach, 126 Pears, 177 Pectin, 243 Penetration depth, of microwave, 218, 219 Penicillum Aspergillus, 363, 367 Perception of food texture, 321 Periodic acid, 241 Permittivity (see Dielectric constant) Phenylthiourea, 385 Piezoelectric crystal, 337 Piezoelectric effect, 101, 102 Pineapple, 126, 425 Planck's constant, 118, 163 Plasticizer, 382, 384 Plateau border, 26, 30 Polar interactions, 217, 218 Poly vinyl chloride, 382, 384, 387 Polymerase chain reaction (PCR), 329 Polyoxyethylene [20] sorbitan monolaurate (see Tween 20) Popcorn, 407, 422, 424, 425, 428 Pork, 173, 180 Potato, 128 chips, 279, 415 sprout inhibition of, 165 starch, 139 Pourable dressing (see Dressing, pourable) Power law, 295 Prawn, other species of, 173, 176 Principal component analysis (PCA), 188, 197, 386, 394, 399 Principal component regression (PCR), 189, 197 Process control, microwave diagnostic techniques for, 223-226 Propylene glycol monostearate, 60, 63, 65, 78 Protein autofluorescence of, 242, 243 binding with fat, 72, 73 with lipid, 45, 49, 55 with polysaccharide, 73, 85 denaturation, 340 desorption, 65, 66, 72-74, 75, 79
[Protein, desorption] effect of emulsifiers on, 72 displacement, 51 -fat binding, 72, 73, 75 globular, 45 hydration, interfacial, 75-79 interaction with emulsifiers, 79 with hydrocolloid, 79 load, 74 markers, 239, 240, 243 milk, from, 85, 86 secondary structure by NIR, 191 Protein-protein interaction, 23, 25, 44, 46, 49, 54, 55, 266, 340 Proteolysis, 341 Prune, 126 Pulse 90 degree, 120 radio frequency, 117, 119, 121-124, 135, 140, 152-154, 156, 159-162 sequence, 124, 128, 144, 145 Pulsed field gradient (see Nuclear magnetic resonance) Quantum number magnetic, 119 spin, 118 Quantum yield, of fluorescence, 50 Quinine, 377-380, 384, 385, 388, 389, 393 Radial distribution function, 17, 18 Radicals radiation induced, 167, 176, 177, 179 stability of, 172 Radio frequency pulse, 117, 119, 121-124, 135, 140, 152-154 Radio immunoassay (RIA), 352-354 Radioisotope, 352, 354 Radius of gyration, from SANS, 204, 207-209 Rancidity, 341 Raspberries, 177 Re-irradiation, 173, 179 Receptors, 330
446 Reflection of microwaves (see Microwave, reflection of, in food) Relaxation cross, 139, 142, 143 mechanism of water protons, 136-139 phenomena, in food, 136-146 spin-lattice, 121, 128, 136, 138, 139, 141, 153, 154 spin-spin, 121-123, 129, 136-140, 153, 156, 161 time, 122, 137, 139, 144, 153, 154, 156, 161 Rheology, 87, 277-303 Rice NIR application to, 190 taste analyzer, 194, 195 Rubberiness, 299 Salad dressings, 55, 296 Salami, EMG profiles, 319, 320, 321 Salmonella, 164, 337, 341,358, 362-366 Saltiness perception, 140 Saltiness, 377-389, 384, 385, 388, 389, 393, ~394, 397, 398 SANS (see Small-angle neutron scattering) Sardine, 180 NMR imaging of, 127, 130, 131 Sauces, 227 dielectric and thermal properties of, 214 loss factor of, 228 Sausages, 144 SAXS (see Small angle X-ray scattering) Scanning acoustic microscopy, 271 Scanning electron microscopy (SEM) (see Electron microscopy, scanning) Scanning probe microscopy (SPM) (see Electron microscopy, scanning probe) Scanning tunneling microscopy (STM) (see Electron microscopy, scanning tunneling) Schiffs reagent, 241 Sensors, 329-342 (see also Biosensors and Taste sensors) for glucose, 330, 331, 335 for soy sauce, 335
Sensory characteristics, 311 Sensory perception, 298 Sensory terms, technical definitions of, 299 Sesame flavor (see Sesame seeds) Sesame seeds, 405, 407-409, 415-417, 422 key odorants in, 409 roasted, 415 odor activity values of, 417 Shear modulus (see Modulus, shear) Shear rate, 280, 285, 311 Shear stress, 280, 285 Shortenings, 417 Slip (see also Wall-slip) apparent, 282 true, 281 velocity, 283, 285, 286 time dependence of, 287 Slip effect (see Wall-slip effect) Slipperiness, 280 Small-angle neutron scattering (SANS), 201-211 advantages of, 201 coherent, 202 colloidal interactions from, 211 colloidal systems, to study, 201, 206-208 contrast variation, 201-203, 207, 209, 212 correlation length from, 207 data analysis of, 207-210 experimental technique, 205, 206 Guinier plot, 204, 206-209 inelastic, 202 incoherent, 202, 206 interface effects in, 202, 203, 211 model fitting, 210 neutron sources, 202 of casein micelles, 206-208, 210, 211 of casein sub-micelles, 206-209, 211 of kappa-casein, 208, 211 of microemulsions, 201, 205, 207, 209 of monodisperse samples, 203, 207-209, 211, 212 of polydisperse samples, 207, 208, 211, 212 of trilaurin, 211 of two-phase systems, 207 of voids in solids, 211
447 [Small-angle neutron scattering (SANS)] particle mass from, 207, 209 pulsed neutron source, 202 radius of gyration from, 204, 207-209 sample preparation for, 204, 205 dialysis technique, 204, 205 theory of, 202-204 voluminocity from, 209 Small-angle X-ray scattering (SAXS), 201-203, 207 Sniffing technique, 405 Sodium alginate, 60 Sodium caseinate, 9-1160, 63, 64 sub-micelles, 9-11 Sodium dodecyl sulfate (SDS), 26, 30, 40, 41, 253 Solid fat content, 63-65, 78, 83, 108, 111, 140 Solid mechanics, 297-302 measurement techniques, 300-302 basic definitions, 298, 299 compression tests, 300, 301 fracture tests, 301, 302 Solvent quality, 85 Sorbitan monolaurate (see Span 20) Sourness, 377-379, 384-386, 388, 389, 393, 394, 397 degree of, 397 Soy sauce, composition by NIR, 196 Soybean FTIR application to, 197 NIR application to, 190 Soybean oil, 3, 5, 6 Soybean seeds, 128, 130 Span 20, 45 Span 80, 3,6 Spices, 165, 167 decontamination of, 165 irradiated, 178, 180 Spreadability, 278 Spreads, 154, 160, 162 confocal scanning micrograph of, 259 Stable isotope dilution analysis (SIDA), 413-417 application to food odorants, 412 Staining, immunohistochemical, 368 Starch, 139, 140, 142 gelatinized, 142
[Starch] granules, microscopy of, 253, 267 hydrolysis on-line monitoring of by NIR, 192 retrogradation of, 143 staining of, 241 Static headspace aroma extract dilution analysis (SHA), 411,412 Step transitions, 9, 11, 13 Stone fruit, 126 Stratification, 9, 14 (see also Step transitions) Strawberry, 177, 407, 409, 415, 425 Stress relaxation, 2-4 Stress-strain curves, 300, 301 Sucrose esters, 30 Sudan Black, 240 Sudan IV, 240 Sugar cane juice, FTIR application to, 197 Surface concentration measurement of, 40 of beta-lactoglobulin in thin films, 43, 50 diffusion, 27, 34, 40-42, 45, 46, 51-53 coefficient, 35 measurement by FRAP, 40-42 dilational elasticity, 53 (see also Storage modulus) dilational modulus, 54 (see also Loss modulus) energy, 298 fluorescence, 50 force balance, 20 force, 278 potential, 383 rheology, 52 dilation, 53, 54 shear, 53, 54 shear viscosity, 53, 54 tension, 23, 25, 46, 77, 79-82 equilibrium, 23 gradients in, 23 viscosity, 46, 53 Susceptibility broadening, 142 Sweetness, 377, 379, 384, 385, 388, 394 Swiss cheese (Emmentaler) flavor, 416, 417
448 Syrup, FTIR application to, 197 Tx relaxation (see Relaxation, spin-
lattice) Te relaxation (see Relaxation, spin-spin) Tablespread, 235, 259 (see also Spreads) Tartaric acid, 397 degree of sourness of, 397 Tartaric acid, 397 Taste cells and biological membrane, 377 expression of, 388-390 map of beer, 391, 393, 394 of amino acids, 386, 398 of aqueous drinks, 390 of beer, 390-394 of tomatoes, 394-396, 398 qualities, 377, 378, 381, 388, 394, 397 quantitative measurement of, 389 substances (see Taste, qualities) Taste sensor, 341, 377-399 application to foods, 390-397 multichannel, 378, 381-387, 389 response to taste qualities, 385 output patterns effect of temperature, 392 of aqueous drinks, 390 of beer, 390-392 of coffee, 390 Tea, 143, 190, 407, 428 Tenderness, 311 Tenderometer, 311 Tensiometer controlled drop, 1, 2, 3, 12 Textural attributes, 310 characterization (see Textural,
properties) fingerprints, 321 properties, 311, 312,321 Texture assessment, temporal aspect of, 311, 312 profile method (TPM), 313 profile, 309 consumer perception of, 321 perceived, 309, 310, 317, 321 Texturometer, 297, 311
Thermal analysis, 62, 83 of ice cream fat phase, 62 of ice cream water phase, 83 Thermal properties of common foods, 214 Thermistor, 332, 335 Thin films, 23, 25, 26, 281 adsorbed layers of, 25, 47-51 air/water, 26, 29-34, 40, 42-49, 51 apparatus, 26, 27 bovine serum albumin-stabilized, 41, 42 common black, 40 difference in oil/water and air/water, 50-52 drainage behavior of, 29-31 emulsifier-stabilized, 30 multilayers in, 51 oil]water, 26, 27, 40, 49-52 protein-stabilized, 23, 30, 40 stabilizing mechanisms in, 23-25 surfactant-stabilized, 23, 30 thickness measurement of, 29-34, 41, 43 Time-of-flight technique, 134 Toffee, 317 Toluidine Blue O, 242, 243 Tomato juice, 134 Tomatoes, 378, 415, 421 juice, 395 response patterns for, 396 sensor output patterns of, 395 taste of, 394-396, 398 Topping, 63-68 powders, 64, 65, 85 low-fat, 66 structure of, 67 Tortilla chips, 263 microscopy of, 264 Total reflection microscopy, 285 Toxins, 348, 354, 363-365 Transducer, 2, 330, 332, 333, 337, 378, 379, 381, 387 in biosensors, 333 Transition temperature, 23 Transmission electron microscopy (TEM)
(see Electron microscopy, transmission) Transverse relaxation (see Relaxation, spin-spin) Trilaurin, SANS study of, 211
449 Tristearin, 108 Triton X-100, 352 Transverse magnetization, 123 Tween 20, 42-55 Tween 80, 352
Ultrasonic (see also Ultrasound) adsorption of, 97 attenuation of, 95-98 interpretation of, 104-106 measurement of, 98-104 characterization of air bubble, 108 of alcohol content, 108 of biopolymers, 106, 108 of biscuits, 98, 112 of casein micelles, 109 of cheese, 112 of composition, 106, 108, 109 of cream, 109 of creaming profiles, 110, 111 of distance, 107 of egg shell thickness, 98, 107 of emulsions, 106, 109 of fats, 107, 108, 111 of fruit drinks (juice), 104, 105 of fruits, 112 of gels, 108 of liquid levels, 107 of margarine, 110, 111 of meat, 93, 107, 111 of milk, 108, 109 of salt concentration, 108 of particles, 109, 112 of phase transitions, 110, 111 of solid fat content, 108, 111 of spaghetti, 106 of sugar concentration, 104, 105, 108 of triglycerides, 106, 108 of volume fraction (concentration), 109, 111 diffraction, 101, 103 impedance, 97, 98, 101, 104 interferometer, 100, 102 measurement techniques, 98-104 continuous wave, 99-101 pulse-echo (see pulsed)
[Ultrasonic, measurement techniques] pulsed, 99, 100, 103, 104, 107 sources of error in, 104 on-line sensors, 93, 112, 113 parameters, 96-98 power levels, 93 propagation, in materials, 94-98 properties, in relation to physical properties, 96 reflection coefficient, 98 scattering, 97, 109 transducer, 98-103, 107, 112 transmission coefficient, 98 velocity, 65, 96-98, 100, 103-105, 108-111 interpretation of, 96-98, 104-105 measurement of, 98-104 temperature dependence of, 111 wave equation, 95 wave length, 95, wave number, 95, 96 waves, 94-99, 103, 104, 109 compression of, 94 frequency of, 94-96, 99, 100, 103, 109, 110 reflection of, 98, 104 shear, 94 transmission of, 98, 104 Ultrasound, 93-113 (see also Ultrasonic) advantages of, 93, 109, 112, 113 limitations of, 112, 113 Umami, 377-380, 384-386, 394, 396
Vegetables, 165, 167, 177, 178, 224 dielectric and thermal properties of, 214 Viscosity, 85-87, 279, 280 extensional, 278, 279, 290 applications to foodstuff, 294-297 biaxial, 291, 292, 296 commercial rheometers for, 294 measurement technique, 292-294 of dough, 294 rotary clamp method, 294, 296 low-shear rate, 303 shear, 278, 291,295 Voluminocity, 75, 209
430 Wall-slip effect, 278-290, 296 elimination of, 288, 289 flow mechanics of, 283 measurement of, 284-290 quantification of, 285-288 visualization of, 284, 285 Water activity, 136, 146 bound, 19, 82, 83 bulk, 139 dielectric and thermal properties of, 214 free, 19, 20, 86 hydrated, 139 rotational relaxation of, 19 state of, 191 Wheat kernel, 252, 254 Whey proteins, 243 Wheying off test, 85 Whippable emulsions (see Emulsion,
whippable) Whipped cream, 20, 59, 60, 66-69 Whipped toppings, 20, 59, 67, 72, 78 emulsion structure of, 68 Wilhelmy plate, 77
Wine, 140, 143, 426, 427 sensor for, 335 X-ray diffraction, 65, 66, 170 X-ray scattering (see Small-angle X-ray
scattering) Xanthan, 243 marker for staining, 245, 246 slip velocity of, 286 Xanthine oxidase, 336, 337 Yeast, 422 Yield strain, 289 Yield stress, 278, 279, 289, 290, 294, 300, 301 Yogurt casein micelles and sub-micelles in, 270 scanning electron microscopy of, 270 sensor for, 335 Young's modulus, 97, 106 (see also
Modulus, Young's) Young-Laplace equation, 2, 4
