FOOD EMULSIONS AND FOAMS
Food Emulsions and Foams
Based on the proceedings of an International Symposium organised by the Food Chemistry Group of The Royal Society of Chemistry at Leeds from 24th to 26th March 1986
Edited by
Eric Dickinson Procter Department of Food Science University of Leeds, England
W O O D H E A D PUBLISHING LIMITED
Published by Woodhead Publishing Limited, Abington Hall. Granta Park, Great Abington, Cambridge CB21 6AH, England
www.woodheadpublishing.com The Proceedings of a Symposium organised by the Food Chemistry Group of the Royal Society of Chemistry at Leeds from 24th to 26th March 1986 First published by The Royal Society ofchemistry. 1987; reprinted 1988 Reprinted by Woodhead Publishing Limited, 2008 0 2005, Woodhead Publishing Limited The authors have asserted their moral rights. This book contains information obtained from authentic and highly regarded sources. Reprinted material is quoted with permission, and sources are indicated. Reasonable efforts have been made to publish reliable data and information, but the authors and the publisher cannot assume responsibility for the validity of all materials. Neither the authors nor the publisher, nor anyone else associated with this publication, shall be liable for any loss, damage or liability directly or indirectly caused or alleged to be caused by this book. Neither this book nor any part may be reproduced or transmitted in any form or by any means, electronic or mechanical, including photocopying, microfilming and recording, or by any information storage or retrieval system, without permission in writing from Woodhead Publishing Limited. The consent of Woodhead Publishing Limited does not extend to copying for general distribution, for promotion, for creating new works, or for resale. Specific permission must be obtained in writing from Woodhead Publishing Limited for such copying. Trademark notice: Product or corporate names may be trademarks or registered trademarks, and are used only for identification and explanation, without intent to infringe. British Library Cataloguing in Publication Data A catalogue record for this book is available from the British Library.
t
lSbN 978-1 -85573-785-3 Printed in the United Kingdom by Lightning Source UK Ltd
Preface Amongst those working in the field of food processing and preservation, there appears to be an increasing awareness of the relevance of the principles of colloid and surface science to some of the problems involved. Nowhere is this more evident than in the area of food emulsions and foams. A manufactured food product is typically a complex, multi-component, multi-phase colloidal system, and its structural and textural properties are determined by the number and strength of interactions between the various types of particle and macromolecule making up the system. For the food technologist to be able to control properly the stability and rheology of food products, it is necessary to have some understanding of processes such as adsorption and aggregation occurring at the colloidal level. In connection with food emulsions and foams, this means understanding how the properties of the system are affected by such factors as the crystallization of the fat, the surface behaviour of the proteins, and the presence of various small molecules and ions in the aqueous phase. This book records the proceedings of a n lnternational Symposium on ‘Food Emulsions and Foams’ organized by the Food Chemistry Group of the Royal Society of Chemistry at the University of Leeds, England, on 24th-26th March 1986. The meeting was attended by over 120 people, about a quarter of whom were from overseas. The meeting began with a rousing introductory lecture entitled ‘Food Colloids in Practice’ given by Dr Don Darling of Unilever Research, Colworth House, and the programme ended with a scholarly and assertive overview of the field by Professor Pieter Walstra of the University of Wageningen. Sandwiched in between were some 17 research papers covering all aspects of the subject as reviewed by the plenary lecturers. All but one of the contributed papers are reproduced here in full, after appropriate editing for style and clarity. To the pleasure of the organizers, the papers attracted lively discussion, much of which has been collected together in this volume. Also recorded here are the abstracts of six posters which were on display throughout the meeting. While the ground covered by the meeting was rather wide-ranging, two topics which perhaps engaged the interest of participants to the greatest extent in formal and informal discussion were (i) the usefulness or otherwise of simple models to describe complex food colloids and (ii) how to interpret time-dependent changes in adsorbed layers of pure and mixed proteins. I am most grateful to Don Darling, Peter Richmond and George Stainsby for helping to arrange the scientific programme, to Bronek Wedzicha for looking after the conference accounts, and to the ‘MAFF Group’ f m assistance with the local domestic arrangements. I wish to thank authors for submitting their manuscripts on time, and discussants for taking the trouble to put their remarks down on paper. Finally, I should like to express my thanks to Dr P.G. Gardam and Mr P.W. Shallis of the Royal Society of Chemistry for their assistance and co-operation in getting this volume published. E. Dickinson May 1986 V
Contents Preface
V
INTRODUCTORY LECTURE Food Colloids in Practice
I
By D.F. DARLING* and R.J. BIRKETT
Theory and Practice of Formation and Stability of Food Foams Bv A. PRINS Colloidal Properties of Model Oil-in-Water Food Emulsions Sta ilized Separately by a,,-Casein, P-Casein and K-Casein Bv E. DICKINSON*. R.H. WHYMANand D.G. DALGLEISH Coalescence Stability of Protein-Stabilized Emulsions Bv E. TORNBERG*and N. EDIRIWEERA Aggregation Rates and Electrophoretic Mobilities of Homogenized Milk Fractions Treated with Rennet Bv E. W. ROBS0NandD.G. DALGLEISH' Adsorption Kinetics of Proteins at the Air-Water Interface B v J.A. D E FEIJTER. andJ. BENJAMINS Properties of Adsorbed Layers in Emulsions Containing a Mixture of Caseinate and Gelatin Bv E. DICKINSON. A. MURRAY, B.S. MURRAYandG. S T A I N S B P The Role of Proteins in the Stabilization/Destabilization of Dairy Foams By M. ANDERSON.. B.E. BROOKERand E.C. NEEDS
30
40 52
64 12
86 100
The Formation and Breakdown of Protein-Stabilized Foams I10 By D.C. CLARK*, J. MINGINS.. F.E. SLOAN, L.J. SM1THandD.R. WILSON Behaviour of an Aerated Food Model B v R. D. B E C . A. CLEMENTand A. PRINS
I28
Protein-Fat-Surfactant Interactions in Whippable Emulsions Bv N. KROG*. N.M. BARFODand W. BUCHHEIM.
144
Polar Lipids in Emulsions and Microemulsions Bv. L. HERNQVIST
I58
Interfacial Behaviour of Protein Mixtures at Air-Water Interfaces Bv E. K. MURRA Y
I70
Desorption of Bovine Serum Albumin from the Air-Water Interface By T.M. HERRINGT0NandS.S. S A H P
I88
A Microscopic Approach to the Structure of Food Emulsions in Applied External Fields By G.C. BARKER*andM.J. GRIMSON
207
vii
Measurement of Creaming Profiles in Oil-in-Water Emulsions By D.J. HIBBERD. A . M . H O W P , A.R. MACKIE. P. W. PURDY and M.M . ROBINS
219
Isolated and Interacting Triglyceride- Water Interfaces By L.R. FISHER+. E.E. M1TCHELLandN.S. PARKER
230
SUMMARIZING LECTURE Overview of Emulsion and Foam Stability By P. WALSTRA
242
Discussion Remarks
259
ABSTRACTS OF POSTERS A Model System for Studying Aspects of Protein Functionality in Emulsions By C.J. BROCK
211
The Effect of pH on Emulsions and Foams Stabilized by Bovine Blood Plasma Proteins By S.E. HlLLand G . M . HALL
219
Multiple Emulsions Stabilized by Protein: Nonionic Surfactant Interfacial Complexation By T.K. LAW, T.L. WHATELEYandA.T.FLORENCE
282
Comparison of Large Scale Whipping and Conductimetric Methods for the Determination of Expansion and Stability of Protein Foams By D.J. WRIGHTondJ. W. HEMMANT
284
Direct Automated Observation of Emulsion Droplet Coalescence By E. DICKINSON, B.S. MURRA Yand G. STAINSBY
286
Modelling of Colloids and Emulsions By G.C. ANSELL. E. D I C K l N S 0 N a n d S . R . EUSTON
289
viii
Food Colloids in Practice By D.F. DARLING and R.J. B I R K E l l
(Unilever Research Laboratory, Colworth House. Sharnbrook. Bedford MK44 1LQ)
Introduction Emulsions and foams, according to most definitions, represent dispersions of oil, water or air in a second immiscible fluid. In the food area, a broader definition is usually adopted, encompassing systems in which either the dispersed phase or the continuous phase is semi-solid or even solid. This may be through crystallization as in the case of ice-cream or butter, or through gelation as with meat pastes or dairy desserts. Table I illustrates some typical food emulsions that make up our daily diet, together with a brief description of their structures and methods of formation. It is evident that a wide variety of structures are utilized to make and stabilize food colloids. The complexity of mechanisms involved in formation, stabilization and destabilization can be illustrated by the manufacture of butter from milk. Milk is a natural dispersion of cu. 4% fat in a protein-containing aqueous phase. The emulsion droplets are stabilized by a lipid-protein membrane. In butter manufacture the fat phase must first be concentrated by a creaming process, the conditions of which are critical for subsequent conversion to butter. Important variables for the preparation of cream suitable for butter manufacture are its source, temperature history, aging conditions, and the feeding conditions for the cows from which the milk was obtained. The separated cream is churned by agitation in the presence of air, leading to agglomeration of fat droplets at the surface of incorporated air bubbles. This de-emulsified fat is then worked or kneaded with some of the aqueous phase from the churned cream. Nearly all the incorporated air is lost during kneading, and a water-in-oil emulsion results in which the proteinaceous aqueous phase is stabilized by a partially-crystallized fat matrix. In engineering terms, the processes in butter manufacture can be considered as independent unit operations. In contrast, the physico-chemical changes induced duringeach process stage are affected by the changes that have occurred during the preceding step. The complexity of such an operation is further increased by the fact that most of the raw materials used in the manufacture of food emulsions are based upon natural sources and so significant variation occurs between batches of the I
2
D.F. Darling and R.J . Birkerr
Table I
Typicalfood colloids (0 = oil, A = air, W = aqueousphase)
Food
Method of Prepararion
Mechanism of Srabilizarion
milk
natural product
protein membrane
cream
centrifugation
as ( I ) particle stabilization of air
ice-cream
homogenization
as (2)
butter & margarine
churning & in votator
fat crystal network
high-speed mixing
by protein & polysaccharide
sauces
+
+ ice network
& homogenization
fabricated meat products
low-speed mixing &chopping
gelled protein matrix
bakery products
mixing
starch & protein network
same ingredient. It is not surprising, therefore, that our basic understanding of the chemical aspects of the formation, stabilization and destabilization of disperse systems in food is limited. The aims of this paper are to illustrate where some of that basic understanding lies, to question its relevance to practical food colloids, and to demonstrate some of the problems with which the food scientist is faced. We first briefly summarize the salient thermodynamic mechanisms involved in colloid stability, and then proceed to illustrate aspects of the formation and destabilization of foams and emulsions. Finally, we deal with some practical approaches to studying and predicting food colloid behaviour. Colloid Stability The stability of colloids or dispersions has been the subject of scientific debate for over a century. This has led to the establishment of three main approaches to colloid stability involving electrostatic, steric and particle stabilization mechanisms. It is not the intention of this paper to discuss the theoretical basis of these approaches. Most modern contain the essential features of each of these mechanisms as well as references to specialist works. Rather, the relevance of each of the mechanisms to food colloids will be outlined. Electrostatic Stabilization and DLVO Theory.-The basis of the Derjaguin, Landau, Verwey and Overbeek (DLVO) theory of colloid stability lies in combining the van der Waals forces of attraction with the electrostatic repulsion forces arising from the diffuse electric double-layer present around the surface of charged
3
particles. The van der Waals interaction between particles arises from three main types of attractive force between molecules: dipole-dipole, dipole-induced dipole, and induced dipole-induced dipole. The third term arises from charge fluctuations within a molecule and is finite even for non-polar species. To a first approximation, the attraction energy Ea is proportional to particle size and inversely proportional to the distance between two approaching particles:
Ea= -Ad/24h
(dsh)
(1)
In equation (I), A represents the Hamaker constant as determined by the chemical nature of particle and medium, d is the particle diameter, and h is the distance between spherical .particle surfaces. The repulsive term is derived from the local accumulation of counterions at a charged surface, the concentration of these ions being dependent on the ionic strength of the medium. The presence of a layer of counterions around a particle is associated with an electrostatic potential which results in two like particles experiencing a repulsive force when they approach. The electrostatic repulsion energy Er is given approximately by Er = rdere,@n[ I +exp(-~h)]
where e, is the dielectric constant of the medium, e,, is the permittivity of free space, is the electrostatic surface potential, and K is the reciprocal of the effective double-layer thickness. The net interaction energy, Ea+Er, obtained from the combined effects of van der Waals attraction and electrostatic repulsion, determines the overall stability with respect to aggregation for a pair of interacting charged particles. While the success of DLVO theory in predicting the behaviour of charged particles in an ionic environment is readily acknowledged, and has led to many successful descriptions of colloid stability, there are few if any food colloids that rely solely on electrostatic repulsion forces for their stability. Adsorbed proteins undoubtedly carry charges, but they are also macromolecules having the capacity for enthalpic and entropic stabilizing effects (see below) which are probably more important. Simple ionic surfactants, which are both functional and permissible in foods, are rare. One such emulsifier which makes stable oil-in-water emulsions is sodium stearoyl lactylate (SSL):
C-0-CH
II
I
0
COO- Na*
Table 2 shows that it obeys the so-called Schulze-Hardy rule, which predicts that the concentration of salt counterions required to screen effectively the surface charge and cause coagulation is inversely proportional to the sixth power of the
4
Table 2
D.F. Darling and R.J. Birkeii Salt stability of SSL-stabilized emulsions as a function of counter ion valency. The critical coagulation concentration C* was determined by diluting a 20 vol% vegetable oil emulsion (droplet size ca. I pm, SSL concentration 5 mM)fifty-fold with salt solutions. and observing the minimum concentration for coagulation within 10 min at room temperature
(c*/c*,ac,)-”~
Sob
C*/mol dm
L 2xperimeni
theor;
NaCl
0.43
1
I
counterion valency. SSL is used in coffee creamers,’where it is purported to confer stability at both high temperature and high ionic strength. According to DLVO theory, an emulsion stabilized by SSL in a high ionic strength medium such as coffee should rapidly coagulate. The fact that it does not implies that some other stabilization mechanism must also be operative. Electrostatic repulsive forces must undoubtedly be involved in food colloid stability, particularly where proteins are involved, but in practice other forces appear to predominate. Thus, dairy-cream emulsion droplets, which are stabilized by milk proteins, d o not flocculate at the isoelectric point (pH 4.6) provided the temperature is kept low (
5
stabilized by milk proteins. Stabilization of emulsion droplets by milk proteins has been dealt with in aseries of publications by Walstra and c o - ~ o r k e r s . ~It- ' is usually the casein, often in an aggregated form, which acts as the main stabilizer. In protein-based emulsions, the stability of droplets to coalescence can be almost infinite. Perversely, however, the functional requirements of practical food emulsions are not for complete stability, but rather for controlled instability. Thus, a cream must be stable during production and distribution, but must destabilize during whipping. This may be achieved by displacing protein from the interface by addition of a second (low-molecular-weight) surfactant, or by controlling fat crystallization such that crystals bridge the interfacial protein membrane.* Particle Stabilization.-Stabilization of foams and emulsions by particulate material was originally described by P i ~ k e r i n gThe . ~ mechanism involves particles adsorbing at the interface, which in turn requires an appropriate balance of interfacial free energies such that particles are wetted preferentially by the continuous phase. The contact angle between the three interfaces defines the ability of the particle to stabilize or destabilize the colloid. The angle 6 in Figure I should lie between 0' and 90'. Pragmatically, the most effective stabilization occurs when 8 is in the range 60-70'. If the angle is too close to !No, physical perturbations at the interface can lead to destabilization. A classical food product in which the air phase is stabilized by particles is whipped cream (Figure 2). Fat droplets adhere to air bubbles during the whipping process forming a protective layer and preventing bubble coalescence. Stability of whipped cream is then described in terms of the interfacial energies between air, fat
Adsorbed part iclc
Figure 1
Schematic representation of the balance of forces in particle stabilization of an interjace. The contact angle 8 is given by the equation cos8 = (yap- ywp)/y,,, where yap,y,, and y,, are the tensions at the air-particle, water-particle and water-air interjaces respectively
6
Figure 2
D. F. Darling ond R.J. Birkerr
Scanning electron micrographs of air bubbles in whipped cream. The bars correspond lo lengths of I0 pm
and aqueous phases. Increasing the oil-water or air-water interfacial tension increases adhesion of droplets to air bubbles, and so enhances stability. This can be achieved in practice through the use of oil- or water-soluble emulsifiers. Mayonnaise is another example of particle stabilization, although here the size of the adsorbing particle is much smaller: egg-yolk particles (ca. 30 nm diameter) are considered the main stabilizing component.'" Margarine, and possibly butter, is another example where the particle stabilization mechanism is thought to operate, but in both products the viscosity of the continuous matrix is so high that droplets are immobile and cannot destabilize without application of an external shear field. Formation of Emulsions In processing terms, emulsion formation is accomplished with a variety of unit operations involving devices ranging from high-energy short-time (high-pressure homogenizer) to low-energy long-time (paddle stirrer). The common types of emulsification equipment used in the food industry are the high-pressure homogenizer, the continuous scraped-surface mixer (e.g., votators), and the highspeed stirrer (e.g., ultraturrax). Although a variety of theories of emulsification have been described, it is most likely that the mechanisms of droplet break-up in foods are based predominantly on turbulence.' Role of Emulsifier.-Concomitant with emulsion formation in foods is the need for stabilization-both transient (at the time of formatioa) and long-term (for product shelf-life). Various emulsifiers are used in the food industry to contribute to both short- and long-term stability. Commerciall) ,emulsifiers are frequently promoted on the basis of their ability to lower the interfacial tension between two phases and hence to facilitate emulsion formation. Whilst food emulsifiers d o indeed lower interfacial tension, it is questionable as to whether this is their key functional attribute. Of far greater importance is their influence on long-term stability as discussed below. While most theories of emulsification contain an interfacial energycomponent in the mechanism of droplet rupture, the relative importance of this term is small. For example, consider Kolmogorov's theory of isotropic turbulence as described by Walstra." The minimum size dmi,of a droplet that is just disrupted depends on the energy density E, the interfacial tension y. and density p according to:
The relative significance to the emulsification process of the parameters in equation (3) can be readily deduced from the range of values attainable in practice. The energy-density term may be varied considerably (from lo4 W m-' for a paddle stirrer to 10l2W tr3for a high-pressure homogenizer), but the range of typical interfacial tension values is very much smaller(4-40 m N m ') and the range of mass density very small indeed. The tension term therefore affords only a relatively small degree of control over droplet size. In addition, under the dynamic conditions applying during emulsification, the interfacial energy between the two phases will
8
D. F. Darling and R.J, Birkerr
Table 3
Effect ofemulsgier on mean droplet size as determined by Nanositer at 20 a C . Emulsions were made at 20 a C with 10 vol% soya bean oil by premixing in a Silverson mixer and a single-pass homogenization at 14 M Pa on a Rannie 3-piston homogenizer. Interfacial tensions were measured against decane at 20 a C by a pendant-drop technique, and viscosities at 20 a C and 0. I ss' using a Deer Rheometer
Aqueous Phase Composition
Mean Droplet Size/ p n
Equilibrium Inrerfacial Tension/ mN m-'
Aqueous Phase Viscosity 1 mPa s
I wt% Tween 60"
0.46
1.4
I .3
2 wt% sodium caseinateb
0.63
11.0
2.0
I wt% sodium stearoyl lactylateC
0.43
11.9
5.8
2 wt% methyl cellulosed M20
I .33
9.3
25.7
2wt% M20+ 1 wt% Tween 60
0.57
8.3
26.5
0.36
7.4
27.0
2.23
14.7
240
I wt% Tween 60 47 wt% sucrose
+
2 wt% hydroxypropylmethyl cellulose HPM 450
Polyoxyethylene (20) sorbitan monostearate (Atlas Chemical Industries)
' DMV spray-dried powder ' SSL 2004 (2004016) (PPF International)
' Cellulose derivatives (Courtaulds Acetate) tend to approach that for the two pure liquids, and any variability induced by the choice of surfactant will be further reduced. Table 3 shows the influence of surfactant chemistry on droplet size for emulsions processed under constant conditions. Given the range of surfactant types, the effect on droplet size is relatively small. Small-molecule surfactants (e.g.,Tween 60 and SSL) generally produce the smallest droplets, but this cannot be due solely to low interfacial tension. Probably of more significance is their ability to impart greater transient droplet stability during emulsification, and so reduce re-coalescence. This transient stability may arise through Marangoni o r surface transport effects. When two droplets approach, the intervening film may thin to a critical thickness, and coalescence may take place. However, local gradients in interfacial tension induce migration of surfactant along the films to counteract the gradient, and this movement of surfactant, together with flow of liquid associated with the surfactant layer, reduces the tendency of the droplets to coalesce. Table 3 also shows that the
9
Table 4
Irnportant$ood surfactants and their typical applications
General Classification
Surfact ant
Application
protein
caseinate
ice-cream
whey protein
cake batter
egg protein
mayonnaise
methyl cellulose
artificial cream
propylene glycol alginate
salad dressing
polysaccharide
small-molecule surfactants
monoglycerides
WIO
margarine
acid esters of monoglycerides
W/O&O/W
bakery products
sorbitan fatty acid esters
confectionery
polyoxyethylene derivativesof sorbitan fatty acid esters
toppings
lecithin
O / W & WlO
milk powder
viscosity of the continuous phase has a small effect on droplet size under the emulsification conditions employed. The more viscous aqueous phases containing the cellulose derivatives lead to larger droplet sizes. These observations are consistent with the theories of turbulence-controlled emulsification as reviewed by Walstra," and illustrate how surfactant chemistry plays a relatively minor role in the droplet formation step. Three general types of surfactant are used in foods: proteins, substituted polysaccharides, and polyol derivatives of fatty acids (see Table 4). Proteins are present in nearly all food colloids, and they represent the most important class of food surfactant. providing the principal means of stabilization in many instances. The manner in which proteins adsorb, and their subsequent interfacial rearrangements, plays a crucial role in the behaviour of food colloids. One effect that small-molecule surfactants have on the behaviour of emulsions is to modify the behaviour of the stabilizing polymeric surfactant (be it protein o r polysaccharide); this point will be dealt with in more detail later. Kinetic View of Emulsion Formation.-To illustrate some of the chemical aspects of emulsion formation in foods, it is convenient to consider emulsification as a set of kinetic processes as set out in Figure 3. The various rate constants then describe the complete emulsification process. The primary formation step of the dispersion is
10
D. F. Darling and R.J. Birkeri
Formation Coalescence
J
c
0 .c
a v)
a .-
a
c v)
D e- f I occul at ion
FI occulation
Figure 3
Kinetic description of emulsification
described by disruption and coalescence equilibria which, as described above, are fairly insensitive to surfactant type. By way of contrast, the nature of the surfactant, particularly its adsorption behaviour, is absolutely crucial in achieving stabilization of droplets once formed. Unstable droplets may undergo flocculation, or coalescence to reform primary emulsion droplets. It is these stabilization and destabilization processes that control the functional properties of foods. What then are the key aspects of food surfactant chemistry influencing the behaviour? Stabilization of Oil-Water Interfaces by Proteins Cumper and Alexander have described" up to three stages in the stabilization of interfaces by proteins: adsorption, denaturation and coagulation. Each stage is postulated to require an activation energy which, once overcome, results in a successive lowering of interfacial energy. The degree to which each process occurs depends on the chemistry of the adsorbing protein. To describe the behaviour of proteins at interfaces, it is essential to have a detailed knowledge of protein chemistry in solution. On this basis, Phillips hasgivent4a general description of the interfacial behaviour of proteins, explaining how different conformational states can exist. Figure 4 shows two extreme structures: a flexible random coil and a highly ordered globular protein. Under quiescent conditions of adsorption and at low concentrations, the adsorbed layer may be treated as a two-dimensional gas with molecules unfolded as much as the tertiary structure will permit. As protein concentration increases, the
surface layer becomes compressed and it behaves more like a condensed ‘liquid’ film. At still higher concentrations the adsorbed layer may exhibit viscoelastic o r even solid-like properties. The molecules can exist in a variety of two-dimensional states analogous to those in solution. Depending on the tertiary and quaternary structures of the protein, varying degrees of molecular unfolding may occur leading to a range of different interfacial structures. The kinetics of protein adsorption has been studied by various techniques, including radio-labelling,15 interfacial rheologyI6 and surface tension measurement.I7 The first method is the most direct but requires specialist equipment; the third is the simplest, the most frequently used, but the most indirect. In discussing the underlying .mechanisms of adsorption, TornbergI7 has considered two processes: unhindered diffusion-controlled adsorption with a low o r zero activation energy, and a penetration-controlled process in which adsorption is governed by
1
Figure 4
2
Schematic representation ofprotein structure at afluid interface (after P h i l l i p ~ ‘ ~()I:) flexible. random-coil proteins: (2) globular, highly structured proteins. The arrow denotes increasing protein concentration
12
D. F. Darling and R.J. Birkert
the creation of available surface area. A constant activation energy for the latter process is unlikely, however, since the ease of penetration should depend on interfacial protein concentration. Most commonly, adsorption has been related to the unhindered diffusion model with Fick's Law used to describe the change in surface concentration with time:
In equation (4). r is surface concentration, C is bulk concentration, t is time, and D is the diffusion coefficient. At low surface coverage, I' is proportional to surface pressure, and hence interfacial tension measurements can be used to monitor the diffusion process. Figure 5 shows the time dependent decay of interfacial tension for two commercial sources of protein, gelatin and casein, as measured by Mussellwhite's using a Wilhelmy plate technique. It is evident that casein is significantly more
15
c
I
/
E 2
'0
/
/-
1
/
E
E
/ 5
I
I
I
1
I
2
L
6
8
10
t /to3 s
Figure 5
Adsorption at the palm oil-water interfacefrom solutions of sodium caseinate or gelatin. The surface pressure n is plotted against the time 1: ,alumina-treatedpalm oil, 0.0375 wt% caseinate; - -, alumina-treated palm oil, 0.03 wt% gelatin; - - - - -, commercialpalm oil, 0.0375 wt% caseinate; * - . - * - ., commercial palm oil, 0.03 wt% gelatin
surface-active than gelatin, as might be expected from their relative hydrophobicities. For both proteins, a plot of surface pressure against t'/' yields a linear region which is consistent with a diffusion-controlled process. However, the linear portions d o not extrapolate back through the origin, indicating that the initial stages of adsorption are significantly slower than predicted. This anomaly is resolved using a more sophisticated diffusion model which assumes that not every collision between protein molecule and interface is successful in achieving adsorption. Introducing a probability index P, the diffusion equation becomes
r = (2/a)CP(Dt)"'
(5)
As a minimum requirement, P will be proportional to the hydrophobicity H and inversely related to the amount of protein already adsorbed. So, equation (5) becomes
I' = [2/d(I')]CH(Dt)"'
(6)
Unfortunately, the function f ( r ) is unknown, and so an explicit solution in terms of diffusion is unavailable. At best, a plot of r versus t'12 will be linear only for low interfacial concentrations or short times. In practice, one might consider that if a monomolecular film represents cu. 1 mg m-' we could calculate the relative time and concentration conditions for which the diffusion equation holds (making the arbitrary assumption that P = I for, say, r
I00 s) at concentrations of at least lo-) wt%. Under these circumstances, it is inappropriate to use timedependent interfacial activity data as evidence for a diffusion-controlled mechanism. There is another point, additional to the arguments given above. Naturally occurring small-molecule surfactants, which adsorb more rapidly than proteins, may provide an energy barrier against protein adsorption. This hypothesis is consistent with the observation that purification of palm oil, for example by passage through an alumina column, leads to a greater change in surface pressure (see Figure 5). Thus, small-molecule surfactants may influence the kinetics of adsorption, not by influencing the transport process, but by modifying the energy barrier to adsorption. From the foregoing discussion, it would therefore seem unwise to conclude that linearity of either F o r y with respect to t1I2is indicative of a diffusion-controlled process unless confirmatory evidence is available. Comparatively few studies of adsorption processes have been undertaken under dynamic conditions, although, as mentioned earlier, the formation of colloidal dispersions in foods virtually always occurs in turbulent flow. In these circumstances, bulk transport of surfactant to the interface is convective rather than diffusive. For conditions of isotropic turbulence, it has been shown' that transport of molecules to the interface becomes faster as the size of the surfactant molecule increases, in complete contrast to a diffusion-controlled process. Walstra and Oortwijn have described' the adsorption of milk proteins to emulsion droplets
D. F. Darling and R.J. Birkerr
14
during high-pressure homogenization. The flux J of protein to the interface is approximated by
where N is the number concentration of protein particles, dg and d, are globule and protein particle size, respectively, E is the energy density, and 7 is the viscosity of the milk plasma. Based on equation (7), it is shown that the change in interfacial protein concentration r with time t in the initial stages of adsorption is given by
-
r[r]
KCdJ I -t(dP/dK)l3t
where K is a constant and C is the protein concentration. Using equation (8), Walstra and Oortwijn explain’ how large casein micelles (cu. 300 nm) adsorb in preference to small casein micelles (cu. SO nm) or serum protein (cu. 4 nm) in milk or cream. Qualitatively, the theory further explains why the interfacial protein concentration increases with decreasing emulsion droplet size: it is because the smaller droplets preferentially adsorb the larger protein particles. While the application of isotropic turbulence theory involves many simplifications and assumptions, it has been surprisingly successful in describing adsorption behaviour under practical conditions of dairy emulsification. The approach has great merit, and its application in understanding the chemistry of emulsion formation in foods should be more widely considered. Destabilization of Emulsions The stability of all colloidal dispersions is governed by thermodynamic principles. In absolute terms, all dispersions are unstable with respect to their bulk phases. In practice, however, there are many kinetic barriers which tend to prevent or retard this destabilization. A destabilization process is a kinetic event which requires the system overcoming an energy barrier, but proper quantification of this energy barrier is possible only with simple well-defined models, well outside the realms of real food colloids. The kinetic events relating to instability must therefore be discussed in qualitative terms only; they are outlined in Figure 6. Destabilization can take the form of aggregation, separation or adsorption, or various combinations of these. A detailed desription is given e 1 s e ~ h e r e . The I ~ term flocculation is used to describe any aggregation process in which droplets retain their identity. Consideration of the kinetics of these instability processes offers a unique approach to the description of the functional properties of food colloids. The whipping of cream involves adsorption and aggregation of fat droplets; the feathering of creamers in coffee involves flocculation and creaming; and so on. Separation Processes.-Gravity-creaming phenomena will be dealt with in some detail in a subsequent paper. Here, some general observations will be made about centrifugal processes, and the need for caution in interpreting data. Centrifugation is often used as a means of accelerated emulsion stability testing.20 The approach should, however, be treated with caution. The observations of Vold
I5
Adsorption
A g gre gat ion
Separation
Stable
Emulsion Figure 6
Schematic representation of ihe various kineiic processes associated with emulsion instability
and Groot21.22 are illustrative of the problems that arise with centrifugal separation experiments. Using paraffin emulsions stabilized with sodium dodecyl sulphate, they observed that at low surfactant concentrations the oil-separation rate decreased with increasing oil phase volume, consistent with theories of hindered settling. At high surfactant concentrations, however, the separation rate increased with increasing phase volume. The apparent inconsistency arises from the fact that oil separation involves two consecutive kinetic events, creaming and coalescence. At high surfactant concentrations, the emulsions are more stable, and oil separation is controlled by the coalescence rate. An increase in phase volume increases the hydrostatic pressure on the droplets at the surface of the creamed layer and so enhances separation. The rate-controlling step with the more unstable emulsions is the creaming process, in which case hindered-settling mechanisms dominate. The work of Smith et also illustrates the need for caution in usingcreaming data as a measure of emulsion stability. From gravity-creaming experiments. they conclude that added small-molecule surfactants increase the stability of emulsions stabilized by milk protein. Under the conditions described, however, the emulsions were almost certainly flocculated by protein bridging which would enhance the creaming rate. Small-molecule surfactants displace protein from the oil-water interface (see later), which has the effect of reducing flocculation and hence
16
D. F. Darling and R.J. Birkerr
creaming. But, such displacement of protein by small-molecule surfacants often leads to a decrease in stability towards coalescence, as observed, for example, by Biswas and Haydon.2s Adsorption Phenomena.-The practical significance and technological importance
of adsorption processes in food emulsions is often overlooked. Aeration of cream, either for whipping o r churning into butter, relies on the adsorption of fat droplets onto air bubbles.26 Similarly, the properties of ice-cream are influenced by the state of the fat, which is governed in part by the interaction between emulsion droplets and the air-water i n t e r f a ~ e . ~ Aeration ’.~~ of cake batters prior to baking can also depend on similar adsorption processes,29 and in certain cases fat encapsulation of air is essential for the baking process to proceed correctly. Adsorption processes are not, however, always beneficial. During food emulsion processing, fat deposits can accumulate on pipework surfaces, stirrer blades, or the mechanical parts of a pump. During the frying of moist foods such as meat, potatoes o r pastry, the adsorption of hydrophilic particles onto the metal frying surface results in the product adhering and subsequently burning. The driving force for adsorption is described classically in terms of a balance of interfacial energies as discussed in the context of particle stabilization. In contrast to the thermodynamic arguments, the kinetic events are poorly understood. The process involves a sequence of events like those depicted in Figure 7. The droplet approaches an interface, liquid begins to drain from the intervening film, and the film thins. If the droplet is to be adsorbed irreversibly, the film must rupture spontaneously, and, having ruptured, the droplet must spread and eventually reach some equilibrium conformation as described by the balance of interfacial forces (the Young-Duprk equation), The initial rate at which droplets come into contact
Figure I
Schematic representation of the various processes involved in the adsorption of a droplet at a solid surface: (I) droplet approaches interface; (2) film thins between droplet and interface; (3) film ruptures; (4) droplet begins to spreadrapidiy; (5) the rate ofspreading diminishes; (6) droplet approaches equilibrium position
17
with the surface will be controlled by diffusion or convection, although in agitated food systems this is unlikely to be rate limiting. Once at the interface, the intervening film has to drain; this will be a function of viscosity, droplet size, and Marangoni Perhaps the most critical stage is film rupture. Local fluctuations in film thickness probably lead to spontaneous film rupture when some critical film thickness is reached. Any irregularities in the surface will promote film rupture, and the fact that fat crystals have a destabilizing effect on oil-in-water emulsions26.22supports this hypothesis. If rupture occurs before the film has thinned to its equilibrium value, the rupture process will not be rate determining. If, however, the equilibrium film thickness is reached before the rupture occurs, then the stochastic process of film rupture becomes the determining factor. Either situation can occur with food emulsions. In pre-mix tanks, where relatively coarse unstable emulsions are frequently prepared prior to subsequent processing, large droplets come into contact with stirrer blades, container walls, erc., and adsorption is controlled by the rate of film drainage. Alternatively, in a stable food emulsion like cream, the oil droplets are small (ca. I pm) with a thick protective protein layer, and the kinetics of adsorption to surfaces is likely to be governed by film rupture.26 With the film ruptured, the droplet, if sufficiently fluid, will spread out until it reaches its equilibrium position. The spreading rate is determined by the droplet viscosity and the respective interfacial energies. According to Mar and Mason,-” typical spreading rates are of the order of 0.1-1 m s-’ for I ml droplets. This corresponds to a spreading time of about I second, and, as spreading rate is inversely proportional to droplet size, the time for droplets to reach their equilibrium positions should, to a first approximation, be independent of droplet size. The argument can be applied to practical situations like cream-whipping where droplets adhere LO the surface of air bubbles. Because the collision frequency between bubbles and droplets is high, it is unlikely that an individual droplet has sufficient time to spread before others adsorb. Aggregation Processes.-The term aggregation is used to describe a variety of particle-particle interaction processes. It is not intended in this paper to review the mechanisms of flocculation and coalescence. Such information can be found in a variety of texts.I.2 Suffice it to say that flocculation is a biparticle process which frequently follows second-order kinetics. Coalescence is a uni-particle mechanism, independent of particle concentration, and is hence a first-order kinetic process. The use of kinetic plots to deduce the mechanism of aggregation is a practice to be treated with caution. In a polydisperse system, the overall aggregation rate is unlikely to follow a simple kinetic plot of I / n or logn versus time, where n is the particle number concentration. This is primarily because the rate constant is dependent on particle size. Role of Emulsifiers.-Whereas proteins generally stabilize oil-in-water emulsions and foams, emulsifiers tend to destabilize them. This at first sight appears contrary to expectation, but it is well demonstrated in a variety of food systems. Biswas and Haydon25clearly demonstrated the effect for oil-in-water emulsions stabilized by bovine serum albumin. It was found that addition of anionic surfactant markedly
18
D. F. Darling and R.J. Birkerr
increases the coalescence rate. The effect of small-molecule surfactants in proteinstabilized dispersions is to decrease the equilibrium surface concentration of protein. Walstra and Oortwijn’-’ have observed that adding monoglyceride or Tween 20 prior to emulsification results in up to 80% reduction of protein load at the interface compared to a control without the additives. They found that the protein load is affected by the type and amount ofemulsifier, and that, when Tween 20 is added to the aqueous phase after emulsification, it displaces protein from the surface of emulsion droplets. Destabilization by emulsifiers is exploited in many foodstuffs, often inadvertently. For example, Zadow and K i e ~ e k e r have ’ ~ described the preparation of recombined dairy whipping creams with the addition of monoglycerides and other emulsifiers. These impart a controlled degree of instability, and hence whippability, to the emulsions. The ability of small-molecule surfactants to displace macromolecules from interfaces is related to their higher adsorption energy compared to individual segments of the macromolecule. Provided adsorption/desorption is an equilibrium process, the surfactant that exerts the highest surface pressure will displace all those with lower surface pressures. The adsorption of macromolecules at interfaces is generally considered to be an irreversible process not amenable to the use of
-
-aelatin and casein mixture
c I
Y
I
0’
/casein
I
alone
gelatin alone
10
I
0
Figure 8
I
LO
I
I
80 120 1 1 1 0 ~ ~
I
160
Injection of casein below a gelatin film at the Nujol-water interface. The surface pressure Il is plolted against the time 1. The arrow indicates the instant of casein injection
19
classical equilibrium thermodynamics. There are always a large number of polymer segments adsorbed per molecule, and the probabilty that a complete macromolecule will spontaneously desorb is therefore low. However, desorption of individual segments is reversible, and so, if small-molecule surfactants are present, and they exert a higher surface pressure than the individual components of the macromolecule, there will be a gradual displacement. That being said, it is perhaps an over-simplification to state that adsorption from a mixture of surfactants is governed solely by surface pressure. In practice, interactions between surfactants, relative concentration, and solvent effects will all tend to modify adsorption behaviour. One must use the term irreversibility with caution, since it can imply wrongly that the adsorbed state is fixed and unchangeable. One protein, for instance, can displace another, as Mussellwhite has shown'* with casein and gelatin. From a mixed solution of casein -I-gelatin, casein is preferentially adsorbed as shown in Figure 8. The same surface pressure versus time curve is obtained for casein -Igelatin as is obtained for casein alone. Furthermore, if casein is added below a preformed gelatin interface, the gelatin is displaced until the equilibrium surface pressure of the mixture is reached. Fat Crystallization.-Fat crystallization in oil-in-water emulsions invariably leads to a reduction in emulsion stability. In dressings, for example, if the oil phase starts to crystallize, the emulsion breaks immediately leading to visible oil separation. In creams and ice-cream, some crystallization of fat is essential for whippability. Van
Fat cr y s t a I s
Membranc
Em ulri on droplet
Figure 9
Schematic representation of fat crystals penetrating the interfacial membrane at the surface of an emulsion droplet
20
Figure 10
D.F. Dorling and R.J. Birkerr
Electron micrograph o$ emulsion droplets with surface crystal irregularities. The bar corresponds to a length of 1 p m
Boekel has studied8 the mechanism by which fat crystallization leads to emulsion breakdown. Basically, it involves crystals penetrating the intervening surfactant film between two droplets, thereby producing a lipid bridge. Because of the small radius of curvature at the point of contact, interfacial forces rapidly cause the bridge to grow, and a strong sintered bond is formed between the two emulsion droplets. Figure 9 illustrates schematically the mechanism whereby crystals penetrate the layer around droplets. In practice, of course, droplets rarely contain single discrete crystals, but mixed crystals of varying composition and polymorphic form. In emulsion droplets of diameter below cu. 3-5 pm, the fat frequently supercools and remains liquid well below its normal melting point. When nucleation does finally take place, subsequent crystal growth is so rapid that formation of discrete crystals is unlikely. Figure 10 shows typical droplets in a vegetable oil emulsion stabilized by sodium caseinate. Crystals appear to form concentric layers around the surfaces of the droplets. Under these circumstances, it is not a single crystal that penetrates the interface, but imperfections in the surface crystal layer. These imperfections may come from either dislocations in the crystal structure o r recrystallization processes.
21
A major factor governing crystal penetration of the interface, and hence destabilization, concerns the relative surface free energies of the various surfaces: crystal-oil, crystal-aqueous phase and oil-aqueous phase. These determine whether crystals are wetted preferentially by the oil phase (no penetration) or aqueous phase (penetration) as discussed previously in connection with particle stabilization. Additional factors which need to be taken into account include crystal size, shape and orientation. Interfacial penetration is less likely if crystals can conform to the shape of the droplet. Mechanical deformation of fat crystals depends on polymorphism; a-crystals, having more 'liquid' character and hence being more maleable than P-crystals, are undoubtedly more likely to follow droplet contours. Formation of mixed crystals will also tend to favour softer mechanical structures. Thus, fats with a broad triglyceride composition will tend to produce more stable emulsions, for a given solids content, than fats containing a narrow range of co-crystallizing triglycerides. The thickness of the interfacial layer through which the crystals must penetrate has a major influence on emulsion stability. Whether the stabilizing mechanism is electrostatic, steric, or of a particulate nature, is of secondary importance in comparison with the actual hydrodynamic thickness of the film. With proteinstabilized emulsions, the effect of small-molecule surfactants could be to reduce the effective hydrodynamic thickness by displacing protein and hence lead to a reduction in emulsion stability. Alternatively, any environmental parameters that affect protein hydration (e.g.,temperature or pH) will also profoundly influence the hydrodynamic thickness of the surface layer and hence emulsion stability.
4,
1
6.0
6.2
64
6.6
6.8
7.0
PH
Figure 11
Eflect of pH on the whipping time t of homogenized dairy cream. Points (0)and ( 0 )correspond to two dfferent cream sources
22
D. F. Darling and R.J. Birkett
An interesting example of the effect of film thickness in a practical food emulsion is found with the whipping of homogenized dairy cream.26The whipping process involves adsorption of fat droplets onto air cells, and the gradual building of an encapsulated structure. The end-point of the whipping process coincides with the maximum amount of fat adsorbed onto air cells. The interface of emulsion droplets in homogenized creams is composed3s primarily of casein micelles and their subunits. The protein particles are about 0. I p m thick and highly hydrated, and the voluminosity is strongly dependent on pH.36 As pH is reduced, so the internal molecular repulsive forces are reduced, the casein micelle contracts, and voluminosity declines. It is found that the whipping time declines as the pH of homogenized dairy cream is reduced (see Figure 1 I). One suggested explanation is that pH reduction results in a thinner interfacial film between globule and air bubble, and therefore a higher probability of film rupture and shorter whip times. This is not the complete story: other parameters also affected by pH could lead to reduced emulsion stability (e.g., lower protein solubility). Nevertheless, there are strong indications that the above is an important contributory mechanism. Bridging Flocculation.-The presence of macromolecular surfactants during emulsion formation can often lead to polymer bridging between droplets. The flocculation of emulsion droplets in homogenized cream and related dairy products is an example of this process. A good review of the subject is given by Mulder and Walstra! The typical floc in homogenized cream exhibits protein bridging between
Table5
Effect of addition of a second surfactant on the viscosity of a ) emulsified inro a flocculated emulsion. Soya bean oil (40 ~ 0 1 % was 0.4 wt% solution of gelatin (Lainers 2SO Bloom acid gelatin) at 14 M Pa and 20 C. Second surfactant was added to emulsion at 60 OC, mixed, and equilibrated for I hour at 60 O C . Emulsion was stored for 24 hours at 10°C prior 10 measurement on a Haake Rorovisco viscomerer at 38 1 s
'
Viscosity/ mPa s Second surfactant (ar 0.4 wt%)
A
I
initially
after 10 s
aJter 100 s
41.9
38.2
31.9
Tween 60
17.3
16.2
14. I
diglycerol monoesteP
26. I
25. I
23.0
sodium caseinate
11.5
11.5
11.5
lecithin
10.5
10.5
10.5
-
1
Diglycerol monoester of tallow (stearate/palmitate 70/30) was a laboratory sample, used in preference to a commercial monoglyceride because of its greater solubility in aqueous solution.
23
globules; this leads to extensive network formation and a high viscosity, but the flocs are disrupted by shear resulting in a reduced viscosity. The thixotropic nature of homogenized creams can be used to estimate both the degree of clustering and the strength of bridging between clusters, as described by Darling.26 From a measure of the change in viscosity with time at constant shear-rate, the degree of clustering in the emulsion can be evaluated. The extent of bridging flocculation depends, amongst other things, on the size, conformation and chemistry of the adsorbing species (see, for example, Vincent’’). For a given surfactant, there is an optimum concentration for maximum clustering. At concentrations well below the optimum, there is insufficient surfactant to reduce droplet size significantly. But, as surfactant concentration is raised, so the particle size is reduced and the surface area is increased. Surfactant availability per unit area is reduced and bridging between droplets becomes more pronounced. At still higher surfactant levels, the droplet size and surface area remain unchanged, and so surfactant surface density increases and the extent of clustering declines. At constant molar concentration of surfactant, the degree of clustering increases with increasing molecular weight.” We have already observed that the addition of small-molecule surfactants can reduce interfacial protein concentration. These surfactants should therefore be able to reduce flocculation in homogenized emulsions by displacing protein from the interface. This effect is demonstrated by the data in Table 5. Flocculated emulsion droplets were prepared by homogenizing40 vol%soya bean oil into0.4 wt%gelatin solution at a pressure of 14 MPa, and the degree of flocculation was quantified by measuring the time-dependent change in viscosity of the emulsion at constant shear-rate. The initial viscosity represents a measure of the overall degree of Table 6
Surface concenrrarion of gelatin in emulsions in the presence of a second surfactant. Gelatin concentration was determined by hydroxyproline assay on hydrolysed extract. with aqueous phase separated by mild centrifugation. Droplets were sized by specrrorurbidimetry, and rensions measured against decane at 20 “C by the Du Nuoy nondetachment method. Ingredient derails are as for Table 5
Surfactant (at 0.04 wt%)
Aqueous Phase Gelatin Conc.J
wt%
Droplet Size dd-
Surface Conc.1
Interfacial Tension1
mg m-z
m N m-‘
(initial emulsion)
0.10
2.5
I .9
control (gelatin only as surfactant)
0. I4
4.0
2.6
19
Tween 60
0.36
3.6
0.4
7
diglycerol monoester
0.30
3.6
0.4
3
sodium caseinate
0.28
3.4
I .o
12
lecithin
0.16
3.3
2.0
<2
24
D. F. Darling and R.J. Birkerr
flocculation, whilst the reduction in viscosity as a result of shear represents a measure of the susceptibility of flocs to disruption. Table 5 indicates that the control emulsion is in a flocculated state that is not fully disrupted by shear. The effectiveness of the second surfactant in disrupting flocculated emulsion droplets is in the order: lecithin = sodium caseinate > Tween 60 > diglycerol monoester. The displacement of gelatin from the oil-water interface is confirmed by the data on interfacial concentrations given in Table 6. Although the ranking order for protein displacement is not in complete agreement with the viscosity data, the results clearly demonstrate that gelatin, the least surface-active component, is displaced to varying degrees by the second surfactant. These results confirm Mussellwhite's findings,I8 mentioned earlier, that sodium caseinate displaces gelatin from an oil-water interface. Bridging between droplets during emulsion formation depends on how the time-scale of formation compares with the time-scale of protein adsorption. Walstra calculates1'the average time-scale of adsorption in turbulent emulsification to be of the order of I ms. In a piston homogenizer, droplets are formed much faster than this,I2 adsorption is the rate-determining process, and bridgingcan result. In a colloid mill, on the other hand, the characteristic formation time is longer (cu. 4 ms),I2 and so clustering is less likely. And in a high-speed mixer where emulsion formation is even slower, flocculation is almost impossible. Predicting Emulsion Behaviour Most standard texts describe a variety of methods for the characterization of interfaces and the prediction of emulsion phenomena. The methods include interfacial rheology, adsorption processes, interfacial tension and pressure, hydrophilic-lipophilic balance (HLB), phase-inversion temperature (PIT), erc. In what follows, we consider the practical value of some of these methods as predictors of food colloid behaviour. Interfacial Tension and Interfacial Rheology.-In single-interface studies, it is well recognized that a gradual decay in interfacial tension occurs long after an equilibrium interfacial concentration has been reached. The continued reduction in interfacial free energy is attributed to molecular conformational changes within the adsorbed protein layer. In a random-coil protein such as casein, molecular unfolding is related to loss of internal hydrophobic interactions as surface-active residues gradually orientate themselves in the interface. In contrast, the unfolding of globular proteins may involve considerable loss of tertiary structure, rupture of hydrogen bonds, and even surface denaturation. We might expect a globular protein like P-lactoglobulin to be less susceptible to unfolding at interfaces than disordered proteins like casein and gelatin. Using interfacial rheology methods, Ismailova has demonstratedI6 that changes in a-casein at interfaces continue for up to 6 hours, whereas lysozyme, aglobular protein similar to p-lactoglobulin, reaches equilibrium within 2 hours. Time-dependent changes in interfacial rheology are a useful measure of molecular conformational change. Whilst interfacial tension measurements monitor processes occurring specifically at the interface, rheological
25
lechniques monitor changes also in the associated regions close to the interface. The :quilibrium tension is invariably reached before any limiting rheological property. The kinetics of molecular rearrangement are extremely complex and difficult to define. Whereas denaturation may frequently be considered a first- or even secondorder process, many unfolding mechanisms involve co-operative effects. The kinetics of interfacial rearrangement in food emulsions therefore remains a poorly understood subject. While there have been some excellent model studies of the rheological properties of interfaces and the kinetics of conformational change (see, for example, van den Tempel and Lucas~en-Reynders)~), there has been no application to practical systems such as foods. In a recent study,39 Dickinson er of. investigated the time-dependence of the surface rheology of casein and gelatin films at the oil-water interface. Simple 0-casein solutions show little time dependency, reaching an equilibrium surface viscosity within a few hours; but sodium caseinate and gelatin films continue to change over 3-4 days. These results indicate that, in practice. the interfacial films found in typical foods are unlikely to have reached equilibrium until many days and probably weeks after their formation. Dickinson el of. also point that the surface viscosity data on mixed protein films of casein gelatin can be qualitatively described in terms of a mixed interfacial composition. The results are again consistent with the earlier observations of Mussellwhitet8that casein can displace gelatin from the interface. The rheological properties of interfacial films are frequently invoked to account for the coalescence stability of foams and emulsions (see, for example, ref. I , p. 384). Some correlations between stability and interfacial rheology d o exist in the literature.40 but the causality of these relationships has not been defined. Indeed, from their work on proteins at interfaces, Graham and Phillips concluded4’ that surface rheological properties were not predominant in determining coalescence. They considered that film thickness and disjoining pressure might be the critical factors. In those cases where correlations exist between interfacial rheology and stability, one can invariably argue that other film properties are also changing. Srivastava has reported4zthat coalescence rate is related logarithmically to surface elasticity. However, the observation is based on the use of surfactants to reduce the surface elasticity in protein-stabilized emulsions. Similar trends for the effect of small-molecule surfactants have been observed by others. For example, Pearson demonstrated4’ that adding 0.05 M decyltrimethylammonium bromide to an emulsion stabilized with bovine serum albumin or P-lactoglobulin increases the rate of coalescence by about a factor of two. And Cumper and Alexander observed’’ that oleyl alcohol substantially reduces the coalescence stability of P-lactoglobulin-stabilized emulsions, which correlated with the change in interfacial viscosity. An alternative approach to understanding the above effects of small-molecule surfactants lies in their ability to displace protein from the interface. As mentioned earlier on, if a small-molecule surfactant exerts a higher surface pressure, it will displace protein from the interface, diminish the effective film thickness, and may thereby reduce the stability of the dispersion. Film thickness may therefore be the controlling factor, and not interfacial rheology per se, even though this may well vary also. To the authors’ knowledge, the parameters governing film rupture have
+
26
D. F. Darling and R.J. Birkerr
never been varied independently, and so definitive conclusions cannot be drawn. There are several phenomenological descriptions of the coalescence process, but there is no generally accepted theory relating the coalescence process to the rheology of the interface. Liquid Crystalline Phase Behaviour.-It is well known that lipid emulsifiers form a variety of mesophase structures or liquid crystalline phases.44 The structures and properties of these complex systems have been studied by many w0rkers,4~and it has been shown that liquid crystalline structures can effectively stabilize both oil-in-water and water-in-oil emulsions. In addition, the relationship between oil/ waterlemulsifier phase diagrams and the ability of the various mesophases to stabilizeemulsions has also been extensively studied. However, despite considerable evidence supporting emulsion stabilization by liquid crystals, there is still some doubt about its relevance to food emulsions and foams. Most food colloids d o contain lipid emulsifiers, but at levels which rarely exceed monolayer coverage. Table 7 summarizes some typical emulsifier concentrations found in common food emulsions. Only for the case of whipped toppings is the emulsifier present at a level far in excess of monolayer coverage, and in each of the other cases the emulsifier concentration is too low for liquid crystalline phases to be present at the interface. Table 7
Typical emulsifier concentrations found in food colloids. (The figures quoted are only approximate; they should not be taken 10represent all products within a given category)
Emulsion Category
Typical EmulsiJier
Disperse Phase Volume Fracrion
Droplet Size1 pm
margarine
monoglycerides
0.2
I .o
I
2
ice-cream
monoglycerides
0. I
0.5
3
3
powdered toppings
acetylated monoglycerides
0.2
0.4
10
4
salad dressing
Tweens
0.4
2.0
3
4
mayonnaise
phospholipids
0.8
5.0
10
10
Typical Conc./g I-'
Number of Monolayers
Hydrophile-Lipophile Balance (HLB). - Numerous methods have been developed to relate the lipophilic or hydrophilic character of emulsifiers to emulsion properties. In simple model systems, they serve as useful descriptions of emulsifier behaviour, and frequently they correlate successfully with emuslion stability. In foods, however, they merely serve to confuse rather than to clarify. With proteinstabilized emulsions, for example, all small-molecule surfactants tend to reduce emulsion stability. Lecithins are more effective at destabilizing emulsions than are monoglycerides despite their higher HLB numbers. Tweens make very stable
21
oil-in-water emulsions provided the oil remains liquid, as in the case of flavour oils, but the presence of crystalline lipid rapidly leads to instability, unless monoglycerides are added. Similar arguments may be advanced against the use of other simple predictors of emulsion stability, such as phase-inversion temperature (PIT) or Bancroft’s rule (‘the phase in which the emulsifier is preferentially soluble will become the continuous phase of the emulsion’). The use of such predictors is inappropriate because they start with the premise that emulsifiers stabilize emulsions.‘6 Since in foods the reverse is true, their value must be strictly limited. Predicting Food Colloid Behaviour - A Pragmatic Approach To a food technologist, classical colloid and interface science is somewhat limited in application. Whilst as a discipline it represents an excellent scientific base from which to operate, it fails to find practical application in real systems because of their immense complexity. If research is to be of value, it must be able to predict, both generally and specifically. Colloid science is excellent for generalizing, but it fails when trying to predict specific phenomena in foods. At our present level of understanding, the prediction of food colloid behaviour requires a more pragmatic approach. We need to be able to measure properties of ingredients and emulsions that will predict their performance in practice. It is the authors’view that there are only a few physical parameters currently at our disposal which may satisfy this criterion. Firstly, as discussed earlier, emulsifiers destabilize sterically-stabilized emulsions. Initial results suggest that the ability of emulsifiers to displace macromolecules is highly correlated with their surface pressure. So, can a league table of specific surface pressures at equivalent concentrations be established which will define the equilibrium interfacial composition in foams and emulsions? De Feijtel-4’ has already developed a theoretical approach which suggests that this may be possible. Secondly, the hydrodynamic thickness of the stabilizing layer around a droplet is a practical indicator of the strength of the repulsive force between two colloidal particles. While characterization techniques exist, it is rarely measured in practice. Intrinsic viscosity, precise particle-size analysis, o r packing density - all could yield useful information. Thirdly, precise measurements of changes in particle size, either under quiescent or shear conditions, could be used to predict long-term colloid stability. Modern particle-size analysis methods, particularly automated particle counters and lightscattering techniques, make this a practical possibility. Finally, to finish on a controversial note, we believe that there are undoubtedly other such pragmatic descriptors which the food technologist would find invaluable in the quest for prediction. Too much time is spent burying our heads in the proverbial ‘model system’ rather than facing up to the complex world of real food systems. There are two separate schools of approach: one directed towards the model, the other towards the product. They are seldom brought together in such a way that they can be equated. Perhaps it is time for basic scientists to step into the dirty world of food colloids in practice, and help to reduce the time wasted by product developers on unnecessary and sometimes misguided experimentation.
28
D. F. Darling and R.J. Birkerr
References
I E. Dickinson and G. Stainsby, 'Colloids in Food,' Applied Science, London, 1982. 2 Th.F. Tadros and B. Vincent, in 'Encyclopedia of Emulsion Technology,'ed. P. Becher, Marcel Dekker, New York, 1983, Vol. I , Chap. 3. 3 A. Leo and J.J. Betscher, Food Product Development, 1970,4,70. 4 H. Mulder and P. Walstra, 'The Milk Fat Globule,' Pudoc, Wageningen. 1974. 5 H. Oortwijn, P. Walstra, and H. Mulder, Nerh. Milk Dairy J . , 1977.31, 134. 6 H. Oortwijn and P. Walstra, Nerh. Milk Dairy J., 1979.33, 134. 7 P. Walstra and H. Oortwijn, Nerh. Milk Dairy J . , 1982,36, 103. 8 M.A.J.S. van Boekel, 'Influence of fat crystals in the oil phase on the stability of oil-in-water emulsions,' Ph.D. Thesis, University of Wageningen, 1980. 9 S.U. Pickering, J. Chem. Soc., 1934, I I 12. 10 C.M. Chang, W.D. Powrie, and 0. Fennema, Can. Insr. FoodSci. Technol., 1972,5,134. I 1 P. Walstra, in 'Encyclopedia of E,gulsion Technology,' ed. P. Becher, Marcel Dekker, New York, 1983, Vol. I, Chap. 2. 12 J.T. Davies. Chem. Eng. Sci., 1985.40.839. 13 C.W.N. Cumper and A.E. Alexander, Trans. Faraday Soc., 1950,46,235. 14 M.C. Phillips, Chem. Ind., 1977, 170. I 5 D.E. Graham, 'Structure of adsorbed protein films and stability of foams and emulsions,' Ph.D. Thesis, Council for National Academic Awards, London, 1976. 16 V.N. Izmailova, Prog. Surface MembraneSci., 1979.13, 141. 17 E. Tornberg, J. Sci. FoodAgric., 1978,29,762. 18 P.R. Mussellwhite, in 'Proceedings of the 4th International Congress on Surface Active Substances,'ed. J.Th.G. Overbeek, Brussels, 1964, p. 947. 19 D.F. Darling, in'Food Structure and Behaviour.'ed. J. Blanshard and P.J. Lillford, in the press. 20 P. Sherman, Soc. Pharmaceur. Chem., 1971, Nov., 93. 21 R.D. Vold and R.C. Groot, J. Phys. Chem., 1962.66, 1969. 22 R.D. Vold and R.C. Groot, in 'Proceedings of the 4th International Congress on Surface Active Substances,'ed. J.Th.G. Overbeek. Brussels, 1964, p. 1233. 23 L.M. Smith and T. Dairiki, J. Dairy Sci., 1975.58, 1253. 24 L.M. Smith, M.B. Carter, T. Dairiki, A. Acuma-Banilla, and W.A. Williams, J. Agric. Food Chem., 1977,25,647. 25 B. Biswasand D.A. Haydon, KolloidZ., 1962. 185.31; 186,57. 26 D.F. Darling, J. Dairy Res., 1982.49.695. 27 J.J. Kloser and P.G. Keeney, Ice Cream Rev., 1959,42,36. 28 P. Sherman, J. FoodSci., 1966,31,707. 29 I.S. Shepherd and R.W. Yoell, in'Food Emulsions,'ed. S. Friberg, Marcel Dekker, New York, 1976, Chap. 5. 30 S.B. LangandC.R. Wilke, Ind. Eng. Chem.. Fundam., 1971,10,329. 3 I A. Mar and S.G. Mason, Kolloid 2.. 1967,224, I6 I . 32 M.A.J.S. van Boekel and P. Walstra, Colloids Sut$, 1981.3. 109. 33 P. Walstra and H. Oortwijn, Nerh. Milk Dairy J., 1975.29,263. 34 J.G. Zadow and F.G. Kieseker, Ausr. J. Dairy Technol., 1975.30, 114. 35 D.F. Darling and D.W. Butcher, J. Dairy Res., 1978.45, 197. 36 D.F. Darling, in 'The Effect of Polymers on Dispersion Properties,' ed. Th.F. Tadros, Academic Press, London, 1982, p. 285. 37 B. Vincent, Adv. Colloid Interface Sci., 1974,4, 193. 38 M. van den Tempel and E.H. Lucassen-Reynders, Adv. Colloid Inrerface Sci., 1983.18, 281. 39 E. Dickinson, B.S. Murray, and G. Stainsby, J. Colloid Inrerface Sci., 1985,106,259. 40 J. Boyd, C. Parkinson, and P. Sherman, J. Colloid Inrerface Sci., 1972,41,359. 41 D.E. Graham and M.C. Phillips, in 'Theory and Practice of Emulsion Technology,'ed. A.L. Smith, Academic Press, London, 1976, p. 75. 42 S.N. Srivastava, J. Indian Chem. Soc., 1964,41,279. 43 J.T. Pearson, J. Colloid Interface Sci., 1968,21,64. 44 S. Friberg, in'Food Emulsions,'ed. S. Friberg, Marcel Dekker, New York, 1976,Chap. I.
29
45 N.J. Krog and T.H. Riisom, in ‘Encyclopedia of Emulsion Technology,’ed. P. Becher, Marcel Dekker, New York, 1985. Vol. 2, Chap. 5. 46 G.E. Petrowski, Adv. Food Res., 1976,22,310. 47 J.A. de Feijter, unpublished work.
Theory and Practice of Formation and Stability of Food Foams
By A. PRlNS
(Department of Food Science. Agricultural University, de Dreyen 7 2. 6703 BC Wageningen. The Netherlands)
Film Stability Effect of Spreading Particles-It is common knowledge that the presence of fatty particles, from which surface-active material may spread at the air-water interface, has a destabilizing effect on aqueous foams. Some examples of this effect are the poor foaming behaviour of whole milk with respect to skim milk, the destabilizing effect of egg-yolk on the foaming of egg-white, and the detrimental effect of milk fat on beer foam. The mechanism that is supposedly responsible is as follows. When spreading of surface-active material takes place from a particle in a thin foam film, it is not only the surface of the film that moves away from the particle; the adjacent film liquid will also tend to move in the same direction. This radial liquid movement causes a
a.
b.
d. Figure 1
C
e.
Schematic representation of film breaking as a consequence of marerial from a particle spreading over the film surface: (a) particle makes contact with the suqace; (b-d) the spreading over the sugace squeezes liquidawayfrom theparticle; (e) thefilm breaks. The vertical scale is exaggeratedfor the sake of clarity
30
31
local thinning of the film which may ultimately result in its collapse when enough liquid has been squeezed away. A schematic representation of the process is shown in Figure I . In principle, the amount of liquid squeezed away by the radially spreading layer can be calculated by boundary-layer theory’ for the penetration of liquid motion into the film liquid. Figure 2 shows the liquid velocity distribution and its penetration into the film liquid at a particular moment after the onset of spreading. In practice, such a calculation has not been carried out because the boundary conditions are such that the mathematics is too complicated.
axis of radial front
Figure 2
Schematic representation of the radialflow and the velocity profile as an oil drop spreads over a liquid surface
Another way to tackle the problem is to consider the spreading process as a longitudinal disturbance which travels over the liquid surface. Application of longitudinal-wave theory shows that the time t for the wave to travel a distance is given by2
where q and p are the viscosity and density of the liquid, and Ay represents the change in surface tension associated with the propagation of the wave. It seems reasonable to assume that A y represents the spreading pressure A y s of the spreading particle, i.e. AY = A Y , = Y ~ - YP - Y’P
(2)
where yo is the surface tension of the surrounding liquid, y, is the surface tension of the spreading layer, and y’,, is the interfacial tension between spreading layer and liquid. Strictly speaking, equation ( I ) applies to a longitudinal wave propagating in one direction only. But, for an order-of-magnitude solution to the problem, it seems justified here to use equation ( I ) also for the radially propagating wave.
32
A . Prim
The penetration P of the movement of the wave in the adjacent liquid also follows from longitudinal-wave theory, and is given by
P = (qt/p)”2
(3)
Supposing that a particle of radius R spreads completely into a layer of thickness d, the ultimate distance f over which spreading takes place follows from the conservation of mass:
4aR3/3 = af2d
(4)
Substituting f from equation (4) into equation ( I ) , and combining the result with equation (3), we get
P = R(v2/Ay,pd)’l3
(5)
From equation ( 5 ) we draw the important conclusion that the penetration of liquid motion into the film liquid is proportional to the radius of the spreading particle. In an attempt to relate the penetration of liquid motion to the occurrence of film collapse, let us assume that the film will collapse when the penetration depth becomes equal to the film thickness. This means that the chance of film collapse increases with increasing size of the spreading particle. Figure 3 shows that this effect is confirmed experimentally for an aqueous sodium caseinate foam to which
Figure 3
Foam haljllife time T of 0.05 wt% sodium caseinate solutions as a function of the volumelsurface mean diameter d,, of droplets in added soya bean oil emulsion. Oil volumefractions are 0.1% (0)and 0.15% (a)
33
has been added soya bean oil emulsion droplets of different sizes. Foam half-life measurements were carried out at a constant total amount of soya bean oil, and so the number of emulsion droplets is proportional to R-'. The chance of film collapse is proportional to the number of spreading particles in the system, which therefore implies that the half-life time is proportional to R3. We see from the above discussion that there are two ways in which particle size affects foam stability: a penetration effect which leads to stability being dependent on R-', and a particle-number effect which leads to a dependence on R3. Combining these two independent contributions, the foam stability S may be expressed in the form
where A, Band C are constants whose values are system dependent. In Figure 4 the calculated foam stability expressed as the half-life time is plotted as a function of the particle size. Values of A, B and C which give the best fit with the experimental results are given in the figure legend. The good fit between measured and calculated curves is considered to be an argument in favour of the present theory. The effect of particle size of an oil-in-water emulsion on the foaming behaviour of an aqueous solution seems to be widely accepted industrially (see, for instance, the patent ofdu Pont de Nemourss), but no explanation for the effect has previously been published. Further work is needed to explain the values of the constants in equation (6).
dvs Clm
Figure 4
Colculoredfoam srability expressed by T as o funcrion of dVsusing equotion (6). Best firs with the experiment01 doto o$ Figure 3 ore obtuined with: A = I .62 X min m, B = 5.82 X IOI7 min m-', C = - 1 1.57 min (for0.1 vol%oil) o n d A = 1.12 X min m, B = 4.60 X lOI7 min m-3, C = -8.67 min Vor 0.15 vol% oil)
34
A . Prim
Effect of a Yield Stress in the Film Liquid-Consider an aqueous film which is stabilized with a surface-active agent. It will drain until a so-called equilibrium film is formed, where the interaction between the two film surfaces prevents further film thinning. If the film liquid has a yield stress, film drainage may stop before the equilibrium thickness has been reached. A quantitative treatment of this new situation requires a more detailed description of the film-drainage process. (In general, film drainage can take place by means of viscous flow, and also by means of a rather complicated process called marginal regeneration: but the latter process is usually absent in protein-stabilized films of aerated foodstuffs, and so it is here left out of consideration.) A thin film behaves elastically due to the presence of surface-active agents. When the film is stretched, the surface tension increases and remains increased as long as stretching is maintained. The elastic behaviour is caused by the limited amount of surfactant in the film, which is not enough to re-establish both the original adsorption at the two film surfaces and the original concentration in the film liquid after the film has been stret~hed.’.~When a film originally held horizontally is placed vertically in the gravity field, the upper part of the film becomes stretched and the lower part compressed as a result of the gravitational force which tries to pull the film downwards. In turn, the surface tension at the top of the film is increased relative to the original situation, and that at the bottom decreased. The resulting gradient in surface tension along the height of the film exactly balances the weight of the film according to the equation d y / d z = pgd/2
(7)
where z is the vertical co-ordinate, and d is the film thickness. The factor of I / 2 appears in equation (7) because the film has two surfaces. The liquid flows out of the film in such a way that the two film surfaces remain motionless. In this situation (see Figure 5 ) , the parabolic velocity profile of the liquid creates a shear stress along the two film surfaces which equals the surface tension gradient according to
where v7 is the velocity of the liquid in the z-direction, and x is the horizontal co-ordinate which is perpendicular to the film surface. Liquid flow between motionless surfaces can be stopped if the liquid has a yield stress ay which is large enough to prevent establishment of the velocity gradient. This has its maximum value at the two film surfaces, and so the condition for halting the flow is: aY 2 d y / d z = pgd/2
(9)
For a film thickness of 10 pm, a yield stress as small as 5 X I0 Pa is enough to keep the liquid motionless. (We recall that I Pa corresponds to a water column of 0. I mm.) A special experimental technique is available’ for the measurement of such extremely low yield values. It has to be emphasized that this type of film stabilization cannot be realized by
35
Figure 5
Parabolic velocity distribution of liquid in verticaljilmfor which the two surfaces are motionless as a resuli of a surface iension gradient
dY/dZ
the yield stress alone. The stabilizing mechanism operates only when two mechanical conditions are fulfilled: a surface tension gradient along the film surface, and a proper yield stress in the film liquid. Consequently, the system must contain not only a chemical component responsible for the yield stress, but also a surface-active component to ensure establishment of the required surface-tension gradient. Disproportionation of Gas Bubbles The growth of bigger gas bubbles at the expense of smaller ones is called disproportionation. The driving force is the Laplace pressure difference Ap over a curved bubble surface, given by Ap=2y/r
(10)
where y is the surface tension, and r is the bubble radius. Being bigger for a smaller bubble than for a bigger one, this pressure difference leads to a concentration gradient of gas in the liquid between two bubbles of different size. Assuming that gas transport through the liquid takes place by means of diffusion and taking the bigger bubble to be infinitely large, de VriesIoderived an equation for the radius r of the smaller bubble as a function of time t:
In equation ( I I), ro is the bubble radius at t = 0, R is the gas constant, T is the absolute temperature, po is the pressure (atmospheric), D is the diffusion coefficient of the gas in the liquid, S is the solubility of the gas in the liquid, and 0 is the thickness of the liquid layer over which diffusion takes place. Figure 6 shows calculated results for bubbles filled with CO, and N, using parameter values given
36
A . Prim
0
sill
ldoo
lh
2doo
z h
3iX
3iooN2 tTc 5
Figure 6
Bubble radius as afunction of time calculated from equation (11)for bubbles filled with N , or CO,. Thefo/lowing parameter values have been used: y = 40 mN m-I, 0 = 10 pm, ro = 125 pm, T = 293 K,po = 0.1 MPa, D(N,) = 1.99 X m2 s I , D(C0,) = 1.77 X 10 ' m2 s I, S(N2)=0.688X mol m4 Pa-',S(C02)=39.2X mol m-3 Pa-'
in the figure legend. The graph shows the importance of the solubility of the gas in the liquid, which is 55 times larger for CO, than for N,. It also demonstrates that, for the case of constant surface tension, the process of disproportionation is self-accelerating. This is due to the fact that the Laplace pressure gets larger and larger as the bubble gets smaller and smaller; ultimately the bubble implodes. Most bubble surfaces, especially those in foods, are viscoelastic. This means that the surface tension of the shrinking bubble is less than the equilibrium surface tension. The surface property involved here is the surface dilation viscosity qs defined by '7, = (Y - y0)/(d In A/dO = (Y - rO)/2(dIn r/dt)
(12)
where A is the surface area, and yo is the equilibrium surface tension. With most practical systems, it appears that '7, is strongly dependent on the rate of compression of the surface, i.e., the surface behaviour is non-Newtonian. It is found, for a system such as milk or beer, that this surface behaviour can be described quite well by a power-law equation: logloqS= m log,,(d In A/dt)
+n
(13)
Typical values of the constants m and n are: m = -0.995 and n = -2.4 for skim milk,
37
and m = -0.895 and n = -1.865 for beer. The negative values of m indicate the shear-thinning character of these surfaces, i.e., the surface dilational viscosity decreases as the rate of compression increases. Equations ( I 2) and ( 1 3) are now combined with equation (1 I), which is written in the form rdr = -(2RT/ p,)(DSy/O)dt to give the differential equation 2aC(dr/dr)"'+' ir"'+*(dr/dr) - cryor"'+' = O
(15)
where Q = 2RTDS/f3po and C = 2"'IOn. To get round the problem that for a compressed surfaced In A / d t is negative, a new variable r is introduced such that T = -t. The differential equation (15) has been solved numerically with the help of a Runge Kutta routine. Figure 7 shows the calculated bubble radius as a function of time. Three points are particularly noteworthy. (i) In the early stages of the disproportionation process, the bubble behaviour is close to that prescribed by de Vrieslequation ( I I)] implying that r ti'*. This corresponds to the part of the curve in Figure 7 which is concave downwards. (ii)The quantity sdoes not itself appear in equation (IS), which means that curves of r(t) are congruent to one another, i.e., they can be superimposed by shifting parallel to the t-axis. (iii) If r approaches zero, it follows from equation (15) that d r / d s also approaches zero, and so somewhere the curve has to change from being concave downwards to concave upwards. This last point is especially of interest, because it indicates a fundamental difference from the behaviour of the de Vries equation, where dr/dt--oo as r-0 indicating that the bubble implodes. The inflection point in Figure 7 indicates the onset of the slowing down of the
-
bubble radius
0 Figure 7
time
Calculated bubble radius shown schematically as ofunction ojrimejor cases when surface tension remains constant (curve I ) and when surface has a dilational viscosity for two initial bubble radii (curves 2 and 3). Radius ri corresponds to the inflection point
38
A . Prim
disproportionation process, and it is therefore worthwhile to investigate which parameters determine its position. Differentiating equation (15) with respect to z leads to the following expression ford In r / d t at the inflection point:
The bubble radius and surface tension at the inflection point are given by:
For beer, typical values of the parameters are such as to give (d In r/dt), = -6.4 X lo3 s I, r, = 45 nm (CO,) or 6.3 nm (N,), y o = 40 mN m I and y , = 3.9 mN m-I. From these results it appears that the disproportionation process can only be slowed down when the bubbles are extremely small. The slowing down is to be understood only in a relative sense, since the absolute value of d In r / d t at the inflection point is still large, indicating that small bubbles cannot exist for long. We note that the surface tension of the bubble is extremely low at the inflection point, and according to equation (18) its value depends on the parameter m. With respect to the above calculations for beer, it should be noted that the so-called 'stiffness'of the surface, as expressd by the value of qs,is only moderate: q, = 13.6 mN m I s from equation (13) with d In A/dt = I s I . Let us now consider a bubble with a surface dilational viscosity one order of magnitude larger. This is simplyrealized bytakingn=-0.865,whichgivesqs= 136 mN m-I s f o r d I n A / d t = I. Using the values of yo and m mentioned previously, the following values at the s I, rl = 2.6 mm (CO,) or inflection point are obtained: (d In r/dt), = -1.9 X 0.36 mm (NJ, and y , = 3.9 mN m I. For this more rigid surface, the inflection point is found at a much bigger bubble size, and the rate by which the bubble size is decreasing is extremely low at the inflection point. At first sight, it seems unexpected that a larger value of the surface dilational viscosity is required t o stabilize a bigger bubble, because the driving force for disproportionation (the Laplace pressure) is smaller for a bigger bubble than for a smaller one. However, we can see that, due to non-Newtonian surface behaviour, the much lower compression rate of the bigger bubble requires a higher value of q, in order to reach the required low surface tension of cu. 4 mN m I. In the same way, it can be understood that r, is smaller for N, than for CO,. This is because, in order to realize the same low surface tension at the same rate of surface compression, the driving force for transport of poorly soluble N, must be considerably higher than for CO,. This implies that the Laplace pressure of the N, bubble must be higher, which, under conditions of constant surface tension, can only be realized with a smaller bubble radius. The ultra-low surface tension required to slow down disproportionation arises here from a theoretical consideration based on an empirical 'surface equation of state' [equation ( I 3)]. Existence of these low surface tensions has yet to be confirmed by experiments that are now in progress. For the time being, however, it may be speculated that these low surface tensions are an example of what are
39
referred to by van den Tempel and Lucassen-Reynders" as new areas of application when they say: "A possible application of the work t o (sic) dynamic surface phenomena lies in the area of ultralow tensions".
Acknowledgemenr. The author gratefully acknowledges the help provided by Dr. B.R. DamstC of the Department of Mathematics of the Agricultural University at Wageningen in carrying out the computer calculations of the disproportionating bubble.
References I See, for instance: R.B. Bird, W.E. Stewart. and E.N. Lightfoot, 'Transport Phenomena.' Wiley, New York. 1960. p. 140. 2 J. Lucassen, Trans. Faraday Soc., 1968.64.2221. 3 J. Lucassen and M.van den Tempel, Chem. Eng. Sci., 1972.27, 1283. 4 P. Joos and J. Pintens, J. Colloid Inrefface Sci., 1977,60,507. 5 H.W. Walker and R.W. Morrow, US Pat. 2,482,307 (Sept. 20, 1949). E.I. du Pont de Nernours. 6 K.J. Mysels, K. Shinoda. and S. Frankel, 'Soap Films. Studies of their Thinning and a Bibliography,' Pergamon, New York, 1959. 7 J.W. Gibbs.'Collected Works,'Dover, London, 1961, p. 301. 8 A. Prins, C. Arcuri, and M. van den Ternpel, J. Colloid Inreflace Sci., 1967,24,84. 9 T. van Vliet, A.E.A.deGroot-Mostert, and A. Prins.J. Phys. E, 1981,14,745. 10 A.J. de Vries, Rec. Trav. Chim.. 1958,77,209. II M. van den Tempel and E.H. Lucassen-Reynders,Adv. Colloid lnrerface Sci.. 1983, 18, 281.
Colloidal Properties of Model Oil-in-Water Food Emulsions Sta bilited Separately by a,,-Casein, 0-Casein and K-Casein By ERIC DICKINSON, RICHARD H. WHYMAN (Procter Department of Food Science. University of Leeds. Leeds LS2
9JV and DOUGLAS G. DALGLEISH (Hannah Research Institute. Ayr, Scotland KA6 5HL)
Introduction Dispersed particles in a stable colloid are prevented from aggregating by the repulsive interactions between their surface layers. The origin of these colloidal interactions can be explained’ using theories of electrostatic stabilization, steric stabilization, or acombination of the two. One of the ways of assessing the relative importance of the electrostatic factors in food colloid stabilization is to study the effect of added electrolytes on aggregation behaviour and electrokinetic properties. In food emulsions, the heterogeneous milk protein ‘casein’ is commonly the major macromolecular component of the adsorbed layer surrounding dispersed fat particles or oil droplets. The exceptional emulsifying properties of casein are attributed* to a molecular structure which is highly disordered and substantially hydrophobic. Casein in milk exists as ‘casein micelles’: polydisperse proteinaceous coloidal particles containing the four major monomeric caseins aSlr p, aS2 and K (in the approximate proportions 4:4: I: I ) linked by calcium ions and colloidal calcium phosphate. In this paper we shall be concerned with the caseins as,, p and K , which have fairly similar molecular masses and isoionic.points (pH E 5.2+0.2), but rather different molecular charges (-20e, -12e and -4e) at neutral P H . The ~ commercial emulsifier sodium (or potassium) caseinate lacks calcium phosphate and is consequently less aggregated than the casein in milk, but it has roughly the same protein composition. Our intention is to compare the properties of emulsions stabilized by the individual caseins aII, /3 and K with those stabilized by sodium caseinate in order to gain insight into the competitive and co-operative aspects of protein adsorption and colloid stabilization in casein-containing systems. that p-casein is more Adsorption experiments at fluid interfaces have surface-active than a,,-casein, but direct evidence for preferential adsorption of &casein in emulsions is rather limited.s-6 Replacement of complex real emulsion 40
41
systems by a model system of monodisperse polystyrene latex particles enables adsorbed proteins to be compared in a systematic manner. In this paper we build on earlier with different monomeric caseins adsorbed separately on negativelycharged latex particles. Consistent with its having a higher net charge in solution than p- or K-casein, a,,-casein adsorbed on latex gives coated particles of higher electrophoretic mobility than either of the other two caseins. However, in the presence of calcium ions at concentrations above cu. I mM, latex particles coated with K-casein carry a higher effective negative charge than those coated with a,,-o r p-casein, which is interpreted8 as being due to the strong binding ofcalcium ions to the latter, thereby reducing the net negative charge on the adsorbed protein layer. The adsorption characteristics of sodium caseinate on latices are intermediate between those for a s l and /3-caseins, which suggests that, at solid surfaces at least, /3-casein does not displace a,,-casein over a short experimental time-scale.' Experimental Materials.-Individual proteins asI-,/3- and K-casein were separated from whole casein and purified by standard procedure^.^.^^ 'Scottish Pride'sodium caseinate of low calcium content (0. I g k g ' ) was obtained from the Scottish Milk Marketing Board. Polystyrene latices (diam. 33823 nm) were prepared by emulsifier-free polymerization as described previously.x Vegetable oil was obtained from the local supermarket. AnalaR grade n-hexadecane (>99 wt.%) was obtained from Sigma Chemicals. Rennet was Hansen's KBselabpulver, made up in a 150 w/v ratio with distilled water, and filtered through Whatman No. I paper before use. Sodium chloride, calcium chloride and imidazole were BDH AnalaR grade. Emulsion Preparation.-Oil-in-water emulsions of weight fraction 0.5 (nhexadecane volume fraction 0.56) and protein content in the range 0.7-1.0 wt.% were made by high-pressure homogenization at 25 "C and 300t20 bar. Before homogenization, the oil phase (n-hexadecane o r vegetable oil) was blended with an aqueous solution of the protein in 20 mM imidazole buffer (pH 7.5). [The buffer was 20 mM T R l S (pH 6.5) in K-casein emulsions made for the renneting experiments.] For the stability and electrophoresis experiments, 10 ml samples of emulsion were prepared in a single-stage mini-homogenizer designed and built in the Procter Department." For each set of exchange experiments, a 100 ml sample of emulsion was prepared using an APV Manton-Gaulin two-stage homogenizer (second-stage pressure 40 bar). Droplet-size distributions were determined with a Coulter counter using a 30 pm orifice tube and 0.18 M NaCl solution as suspending electrolyte. In n-hexadecane emulsions made with as,-casein, p-casein o r sodium caseinate, the most-probable droplet size was in the range 1-1.5 pm, depending on protein content and the exact value of the homogenization pressure. Mostprobable droplet sizes in K-casein emulsions were more variable (2t I pm). Mobility Measurements.-Electrophoretic mobilities of latex particles and emulsion droplets were measured at 25 "C either by direct microelectrophoresis on a Rank mk 11 apparatus or by laser Doppler electrophoresis on a Malvern-Rank 'particle charge' apparatus. (We have shown previously' that, within the combined
42
E. Dickinson. R. H. Whymun. and D.G. Dalgleish
experimental uncertainty, the two techniques give identical results for caseincoated latices.) To eliminate problems of creaming, the larger emulsion droplets (-50%) were removed by gentle centrifugation (2.5 x lo3 g) before dilution and injection into the cylindrical microelectrophoresis cell. Centrifugation followed by resuspension in TRlS buffer was the procedure used to wash some of the K-caseinstabilised latices and emulsions before addition of rennet and mobility measurement. Stability Measurements.-Emulsion stability with respect to droplet aggregation was determined at pH 7.5 by monitoring the change in droplet-size distribution after adjusting the electrolyte concentration in the oqueous phase of the concentrated emulsion to 2 M NaCl or 5 , 10 or 15 mM CaCI,. Typically, I ml of emulsion sample and 0.5 ml ofsalt solution were mixed rapidly, left for 2-3 hours at 25 "C, and then quickly analysed on the Coulter counter. (With the flocculated samples, it was necessary to dilute emulsions in buffered salt solutions of the appropriate electrolyte composition in order to get reproducible droplet-size distributions.) Where salt instability was evident, the emulsion was resuspended in excess buffer solution (no NaCl or CaCI,), left without stirring for a period of 15-30 min, and then reanalysed on the Coulter counter. Exchange Experiments.-A &casein (a,,-casein) emulsion was washed free of unadsorbed protein by centrifuging at 3.7 x 10' g, resuspending the cream in imidazole buffer (pH 7.5). and then repeating the procedure. To the washed flcasein (a,,-casein) emulsion was added an equal volume of buffered a,,-casein (p-casein) solution containing an equivalent amount of protein to that contained in the emulsion. Samples were taken from the mixed system at various intervals of time. For each sample, the aqueous phase was separated from the cream by centrifugation, after which it was dialysed against distilled water, freeze-dried, and analysed later for protein composition by fast protein liquid chromatography (FPLC). Freeze-dried caseins ( 5 mg) were dissolved in 2.5 ml of buffer containing 6 M urea and 10 mM TRlS (pH 6.5). Aliquots of 0.2 ml were run on a Pharmacia FPLC apparatus using an ion-exchange column with a linear gradient of 0-0.4 M NaCI. The eluted peaks corresponding to as,-and &caseins were detected and estimated by absorbance at 280 nm. After integration, concentrations were calculated using relative absorbances of 1.00 and 0.47 for as,-and /-?-caseins, respectively. Results and Discussion Stable n-hexadecane-in-water emulsions have been prepared on a small scale using as,-casein, p-casein or K-casein as the sole emulsifying agent. Droplet electrophoretic mobilities have been measured as a function of ionic strength for comparison with data for casein-coated latices. Salt stabilities of the emulsions in NaCl and CaCI, have been determined as an extension of earlier work on caseinatestabilized oil-in-water emulsions.I2 In presenting the results we concentrate on general trends of behaviour obtained after eliminating the effects on emulsion properties (especially droplet-size distribution), of changing such things as
43
emulsifier concentration and valve settings on the homogenizers. Numerical results given here form only a fraction of those obtained, since experience shows that experiments on emulsions must be repeated several times to ensure reliability. We have found that monomeric caseins from different batches can have different emulsifying capacities. This is particularly true for u-casein, which we have found to be a very unreliable stabilizer, both in this study and previously with casein-coated lati~es.’.~ T o facilitate comparison between the different caseins, we have tried to maintain controlled conditions of emulsification and procedures of sample manipulation (time and temperature of storage. order of mixing, etc.). Electrophoretic mobilites are reported for n-hexadecane droplets at 25 O C and pH 7.5 separately stabilized with a,,-. 8- and u-caseins. Figure I shows a plot of mobility against the square root of the ionic strength of added electrolyte (either sodium chloride 0.01-0.1 M or calcium chloride 0.0003-0.045 M). The data are consistent with our earlier findings for that in I:I electrolyte a,,-casein is less effective in reducing the particle surface charge density than is p- or u-casein.
0.1
0.2
0.3
11/2/M1/2 Figure I
Electrophoreticmobilities of casein-stabilized n-hexadecane emulsion droplets at 25 OC and pH 7.5 in presence of sodium chloride or calcium chloride. The mobiliry u is plorred against the square root of the ionic strength I: 0, as,-casein:v, p-casein; a, u-casein
44
E. Diekinson. R. H . Whyman.and D. G. Dalgleish
The results in Figure I show that divalent counter ions are much more effective than monovalent ones in reducing the electrokinetic potential of casein-coated oil droplets. Since bare latex particles also show similar qualitative behaviour,8 the major effect of Ca2*ions cannot be a specific binding phenomenon associated with the protein, but is rather a general divalent counter-ion phenomenon at the colloidal particle surface. We note, however, that the absolute magnitudes of the mobilities in CaCI, d o lie in the order K > a,,> 0,which is consistent with a much weaker binding of Ca2' to K-casein than to either of the other two proteins. The same small but definite contribution of specific Ca2' binding is also found with the caseins on latices.8 Some emulsions were prepared with a food-grade vegetable oil replacing nhexadecane to investigate the effect of the nature of the oil phase on the electrokinetic behaviour. The effect is small, as one would probably have expected on the basis of the similarity of the latex and n-hexadecane mobilities. Figure 2
4
L(
I
v) L(
I
'3
I
E
m I
0
2
1
OO
0.1
0.2 Ill2/ M112
Figure 2
Electrophoreticmobilities of P-casein-stabilizedparticlesat 25 C and pH 7.5 in the presence of calcium chloride. The mobility u is plotted against thesquare root of the ionic strength I: .,polystyrene latices; A, n-hexadecane emulsion droplets; 0 , vegetable oil emulsion droplets. The solid curve f i t s the combined latex and vegetable oil data within experimental error
45
shows the results for the three types of particles in CaCI, solutions with P-casein as the stabilizing protein. At ionic strengths above ca. 1 mM, the mobility values are identical within experimental error (*5%). At low ionic strengths of CaCI,(or in NaCl solutions), n-hexadecane droplets have mobilities about 10% bigger than those for vegetable oil droplets. Reducing the p-casein content in the original emulsion from 0.8 to 0.4 wt.% leads to a further 10% increase in mobility of n-hexadecane droplets Table 1
Behaviour of emulsions in 2 M sodium chloride and 15 mM calcium chloride at 25 OC and pH 7.5 (S= stable, F = flocculated)
EmuIsiJier
2 M NaCI
aSI -casein
F*
F*
fl-casein
S
F*
K-casein
S
S
sodium caseinate
S
F
I5 mM CQC/~
*Deflocculated within a few minutes
1
I
I I
/-
" I
I
1
I
I
2
lk I
I
I
4
8
d/rm Figure 3
Salt stability results for crs,-casein emulsion (0.8 wt.% protein, nhexadecane weight fraction 0.5) in sodium chloride or calcium chloride at 25 "C and pH 7.5. The volume-weighted size distribution function P,,(arbitrary units) is plotted against the droplet diameter d: A, original emulsion and redispersed samples (see text); B. 2 M NaCl for 3 h; C, 5 mM CaCI, for 3 h; D. 10 mM CaCI, for 3 h
46
E. Dickinson. R. H . Whyman. and D. G. Dalgleish
at low NaCl concentrations, but no measurable difference at NaCl concentrations above about0.02 M or with added CaCI,. We found that mobilities of n-hexadecane droplets in emulsions stabilized by sodium caseinate were generally intermediate between those for as,-and p-caseins, with a tendency for values to lie closer to p-casein than to aSl-casein, possibly due to some preferential adsorption of the former over the latter. The salt stability behaviour for n-hexadecane-in-water emulsions made with a,,-casein, p-casein, K-casein or sodium caseinate is summarized in Table I . In emulsions stabilized by the pure monomeric caseins, flocculation when it occurred was reversible: that is, on dilution with a standard buffer, there was deflocculation within a few minutes. We find that aSl-casein emulsions are more susceptible to flocculation by calcium ions than are P-casein emulsions (5 mM CaCI, partially flocculates the former but not the latter). Figure 3 shows droplet-size distributions for a 0.8 wt.$a,,-casein emulsion, as prepared (curve A), in 2 M NaCl (curve B), in 5 mM CaCI, (curve C), and in 10 mM CaCI, (curve D). Curve A in Figure 3 also refers to the distributions measured after the samples corresponding to B, C and D had been redispersed in standard buffer for 15 minutes. The rapid reversion to the original droplet-size distribution shows conclusively that what we are dealing with here is reversible flocculation, and not irreversible coagulation or coalescence. Figure 4 shows droplet-size distributions for a 0.8 wt.% @-casein emulsion, as prepared (curve A), in 2 M NaCl (curve A), in 5 mM CaCI, (curve A), and in 10 mM CaCI2(curve B). Curve A in Figure 4 also refers to the distribution measured after the sample corresponding to curve B had been redispersed in standard buffer.
A
d/P Figure 4
Salt stability results for /3-casein emulsion (0.8 wt.% protein, nhexadecane weight fraction 0.5) in sodium chloride or calcium chloride at 25 OC and pH 7.5. The volume-weightedsize distribution function Pd (arbitrary units) is plotted against the droplet diameter d: A. original emulsion, 2 M NaCl for 3 h, 5 mM CaCI, for 3 h, and redispersed sample (see text); B. 10 mM CaCI, for 3 h
47
In this study we find that only a,,-casein emulsions are flocculated in 2 M NaCI. This is perhaps somewhat surprising in view of the fact that a,,-casein emulsion droplets have a higher (negative) surface potential than have /3-casein emulsion droplets, as measured by microelectrophoresis at low salt concentrations (see Figure 1). It just goes to show that salt-stability of casein stabilized emulsions cannot simply be explained in terms of the conventional theory of lyophobic colloidal electrostatic stabilization. I The poor emulsifying capacity of one particular batch of K-casein was markedly improved by addition of mercaptoethanol. This substantiates our earlier view' that the stabilizing capacity of K-casein may be substantially impaired by protein polymerization involving -S-S- bonding. Nevertheless, once formed, K-casein emulsions were found to be stable towards 2 M NaCl or 15 mM CaCI,. The latter behaviour is not really unexpected in view of the known calcium insensitivity of K-casein in solution. Emulsions made with sodium caseinate were readily flocculated in I5 mM CaCI,, but their deflocculation was very slow compared with what was found with a,,casein or p-casein. Figure 5 shows droplet-size distributions for a 0.7 wt.% caseinate emulsion, as prepared (curve A), after dispersion in I5 mM CaCI, for 2 hours (curve B), after subsequent redispersion in buffer for a further I hour (curve C), and after redispersion in buffer for 18 hours (curve D). The results suggest that there is a stronger calcium-induced association between the adsorbed protein layers D
2 p*
1
0
1
2
4 d/rm
Figure 5
Salt stability resultsfor sodium caseinate emulsion (0.7 wt.%protein, n-hexadecane weight fraction 0.5) in sodium chloride or calcium chloride at 25 " C and pH 7.5. The volume-weighted size distribution function Pd (arbitrary units) is plotted against the droplet diameter d: A , originalemulsion; B, 15 mM CaCI, for 2 h; C, sample Bredispersed in buffer for I h; D. sample B redispersed in buffer for 18 h
E. Dickinson. R. H. Whyman. and D. G. Dalgleish
48
on different droplets when all the casein components are present than when either a s l -or @-casein is present alone. The caseinate emulsions studied here were stable
.'~ towards 2 M NaCI, in contrast to what was found in previous w ~ r k ' ~with phosphate buffered caseinate emulsions of low protein load. This difference, together with our present finding that @-casein emulsions are stable towards 2 M NaCl whereas a,,casein emulsions are not (though both are unstable in 15 mM CaCI,), leads us to speculate that the salt stability of caseinate-stabilized food emulsions may be very sensitive to the composition of the caseinate emulsifier (ionic and protein content) and its state of aggregation. This probably explains why, in an earlier study of butter oil emulsions,'* those made with caseinate from DMV
i
N
E
?J 0 F
\
a
El
0.5 -
I
OO -OO
20
I
I
40
60
t/rnin
Figure 6
Electrophoretic mobilities of K-casein-coated latices measured by laser Doppler electrophoresis in presence of 0.2 wt.% rennet ar 25 O C and pH 6.5, The mobility u isplortedagainst the time 1: a. washedparticles (added electrolyte 50 mM NaCI); h. unwashed particles (added electrolyte 50 mM NaCI); c, unwashed particles (added eleclrolyte
50 mM NaCl+ 10 mM CaCI,)
49
Holland were more susceptible to flocculation in NaCl than those made with S M M B caseinate having lower calcium content. We have investigated the effect of rennet on the electrophoretic mobilities of u-casein-coated latices and u-casein-stabilized n-hexadecane emulsion droplets. The enzymes in rennet specifically break the peptide bond 105- 106 in K-casein, and, when casein micelles are so treated, the electrophoretic mobility decreases by approximately 40%, because of the loss of the negatively-charged macropeptide (residues 106-169). Results for K-casein-coated latices are shown in Figure 6. For this experiment, latices were suspended in buffer at pH 6.5 to enhance the enzymic action. We see that for unwashed particles the mobility drops by some 40% or so during a I hour observation period in the presence or absence of calcium ions. Based on the adsorption isotherms reported previously,' we estimate that only 25% of the K-casein in the sample is adsorbed, with the other 75% remaining free in solution. Figure 6 shows that the drop in mobility for washed latices is much slower than for unwashed ones. In the latter case, presumably what happens is that K-casein in solution is preferentially hydrolysed, and the resulting para-K-casein, being strongly hydrophobic, is rapidly adsorbed on the colloidal particles, with perhaps some displacement of the originally adsorbed K-casein. The same thing seems to happen when K-casein emulsion droplets are renneted. Table 2 compares latex mobilities after I hour with those for emulsion droplets, both washed and unwashed. Renneting kinetics for washed emulsion droplets is much slower than for casein micelles in skim milk,I4 which suggests that u-casein on emulsion droplets is less accessible to the enzymes than is K-casein in casein micelles. Further washing of u-casein emulsion droplets did not produce any measurable further reduction in the effect of rennet on the mobility; this gives us some confidence in the efficacy of the washing procedure. The renneting bchaviour of u-casein-coated latices and K-casein-stablized emulsions differs from that of casein micelles or homogenized milks.ls Steric " stabilization by K-casein, as it is believed to occur in casein m i c e l l e ~ , ~ ~is.therefore a more complicated mechanism than that involved with synthetic particles o r droplets at whose surfaces u-casein has been adsorbed. One significant difference in Table 2
Change in electrophoretic mobility, u, of u-casein-stabilized latices and emulsion droplets after 1 hour in 0.02 wt.% rennet solution at 25 "C and pH 6.5 (u, = initial mobility, LDE = laser Doppler
electrophoresis, PM E = particle electrophoresis)
Sample
UlU"
Technique
latex (unwashed)
0.59
LDE
latex (washed)
0.89
LDE
emulsion (unwashed)
0.55
LDE
emulsion (washed once)
0.86
PME
emulsion (washed twice)
0.85
PME
50
E. Dickinson. R. H. Whymon, and D. G. Dolgleish
behaviour between the natural and synthetic systems is that casein micelles flocculate after rennetingI8 whereas K-casein-coated particles d o not, even in the presence of Ca2+. In terms of the mechanism of renneting, this confirms that renneted casein micelles coagulate not simply because para-K-casein is formed on the surface, but also because of interactions involving other caseins and calcium ions.I9 Finally, we present here some preliminary results on the competitive adsorption kinetics of a,,- and p-caseins at the surface of n-hexadecane emulsion droplets. In Figure 7 the percentage of protein exchanged is plotted logarithmically against time. The data clearly show that a,,-casein is displaced by p-casein much more rapidly than is &casein by aSl-casein, which is consistent with the much lower surface activity of a,,-casein at the n-hexadecane/ water i n t e r f a ~ e .This ~ result
40 -
e,/z 30 -
20 -
0
OL -2
I
I
I
0
2
4 In (t/hr)
Figure 7
Kinetics ofexchange between adsorbed layer and aqueous bulk phase in emulsions containing (Y,,- and /&caseins. The percentage P,, by weight ofprotein emulsijier(a,,-casein or &casein)exchanged into the aqueous phase is plotted against the logarithm of the time t; 0 , 0, p-casein in bulk displacing a,,-caseinfrom droplet surface (twosets of experiments); v, aSl-caseinin bulk displacing p-caseinfrom droplet sursace. (The value P,, = 50% correspondsto exactly halfthe originally adsorbed protein being exchanged, assuming that all the protein used to make the emulsion remains associated with the droplets)
51
implies that, in caseinate-stabilized emulsions containing an excess of emulsifier, one might expect to find an increasing amount of fl-casein in the adsorbed layer on storage. The dynamic nature of the exchange between casein in solution and adsorbed casein is illustrated by the fact that a measurable, if slow, partial desorption of p-casein by cr,,-casein could be observed. Conclusions
Electrophoretic mobilities of casein-stabilized emulsion droplets are very sensitive to electrolyte concentration and counter-ion valency, moderately sensitive to the protein composition of the adsorbed layer, and rather insensitive to the nature of the non-aqueous phase. The proteins a,,-casein, &casein and K-casein differ considerably from one another, and from sodium caseinate, with respect to the stability of their emulsions in the presence of sodium chloride and calcium chloride. Rennet reduces the mobility of K-casein emulsion droplets, quickly in the presence of unadsorbed K-casein but slowly in its absence. Over a period of hours, &casein displaces a,,-casein from the adsorbed layer of emulsion droplets; the reverse exchange process also occurs, but much more slowly. Acknowledgements. We thank Dr. G. Stainsby for many helpful comments during the course of this work. R.H.W. acknowledges receipt of a SERC CASE Studentship in conjunction with the Hannah Research Institute.
References I E. Dickinson and G. Stainsby,'Colloids in Food,' Applied Science, London, 1982. 2 H.E. Swaisgood, in 'Developments in Dairy Chemistry,'ed. P.F. Fox, Applied Science, London, 1982, Vol. I, p. I . 3 J. Mitchell, L. Irons, and G.J. Palmer, Biochim. Biophys. Acfu, 1970,200, 138. 4 E. Dickinson, D.J. Pogson, E. W. Robson, and G. Stainsby, Colloids Surf, 1985.14.135. 5 H.Mulder and P. Walstra,'The Milk Fat Globule,'Pudoc, Wageningen, 1974. 6 D.F. Darling and D.W. Butcher, J. Dairy Res.. 1978.45, 197. 7 E. Dickinson, E.W. Robson, and G. Stainsby,J. Chem. Soc.. Furuduy Trans. 1,1983.79, 2937. 8 D.G. Dalgleish, E. Dickinson, and R.H. Whyman, J. Colloid h e r f a c e Sci., 1985, 108, 174. 9 W.D. Annan and W. Manson, J. Dairy Res., 1969,36,259. 10 C.A. Zittle and J.H.Custer, J. Duiry Sci., 1963.46, 1183. I I E. Dickinson, A. Murray, B.S. Murray, and G. Stainsby, this volume p. 86. 12 E. Dickinson, T. Roberts, E.W. Robson, and G. Stainsby, Lebensm.- Wiss. u. -Techno/.. 1984, 17, 107. 13 S.M. Chesworth, E. Dickinson, A. Searle and G. Stainsby, Lebensm.- Wiss. u. -Technol., 1985.18,230. 14 D.G. Dalgleish, J. Dairy Res., 1984,51,425. 15 E.W. Robson and D.G. Dalgleish, this volume p. 64. 16 C. Holt, in 'Proceedings of International Conferenceon Surface Science,'ed. E. Wolfram, Akademiai Kiado, Budapest, 1975, p. 641. 17 P. Walstra, J. Duiry Res., 1979,46,317. 18 D.G. Dalgleish. J. Dairy Res., 1979,46,653. 19 D.G. Da1gleish.J. Duiry Res., 1983,50, 331.
Coalescence Stability of Protein-stabilized Emulsions
By E. TORNBERG (Swedish Meat Research Institute. P.0. Box 504. S-244 00 Kavlinge. Sweden)
and N. EDlRlWEERA (CISIR, 363 Bauddhaloka Hawatha. P.O. Box 787. Colombo 7. Sri Lanka)
Introduction
Emulsion instabilitycan appear visually as creaming or fat separation or a mixture of both. Creaming is the movement of emulsion droplets under the action of gravity due to the difference in density between oil and aqueous phases. Creaming is enhanced by flocculation (aggregation of droplets with their identities intact) or coalescence (the joining of small droplets into larger ones). Proteins used as surfactants usually give rise to very stable emulsions. Their inherently high stability makes the measurement of coalescence in proteinstabilized emulsions a relatively difficult task. I t can be tedious and laborious to follow the change in emulsion droplet-size distribution as a function of time or processing conditions. This is because it may take a long time before any measurable increase in droplet size occurs. In addition, the measurement ofdroplet size is not a straightforward task of routine character; there are inherent problems associated with fully dispersing the emulsion and finding a method capable of measuring the whole droplet-size distribution. There is a need, therefore, for some sort of accelerated technique for rapidly assessing the coalescence stability of protein-stabilized emulsions. In this investigation we study the possibility of using a solvent extraction method. The solvent chosen is n-hexane since Tinbergen and Olsman have shown' that hexane selectively extracts fat from ruptured fat cells in comminuted sausage batters. One essential requirement of an accelerated test for the prediction of stability is that it should merely speed up the process whereby instability occurs, and not change it in any other way. So, to test its predictive value, it is necessary to check instability measured using the hexane extraction method against the increase in droplet size on storage and upon freezing. In addition, the hexane extraction method has been compared with the more commonly encountered method of 'oiling off' by centrifugation. 52
53
Coalescence stability by the hexane extraction method has been measured in emulsions stabilized by sodium caseinate, whey protein concentrate, soy protein isolate, and a blood plasma preparation. The influence of droplet size and protein load has been investigated. Also presented are results for the coalescence stability of the same emulsions on being frozen, freeze-dried and heated. Experimental Materials.-Mildly produced soy protein isolate (SP) was kindly provided by Central Soya, sodium caseinate was obtained from DMV Holland, and whey protein concentrate (WPC) was obtained by ultrafiltration and spray-drying of cheese whey. The blood plasma preparation (BP) was derived from slaughterhouse blood by centrifuging off the red blood cells and freezing into flakes (Ellco Protein, Kavlinge, Sweden). The protein contents of the four studied proteins on a wet weight basis were: 84.6% ( N X 6.43) for sodium caseinate, 60.8% ( N X 6.55) for WPC, 91.2% (N X 6.25) for SP, and 7.4% ( N X 6.25) for BP. Each protein was suspended in (i) distilled water at pH 6[denoted as (0-6)], (ii) distilled water at pH 7 [denoted as (0-7)], and (iii)O.2 M NaCl at pH 7 [denoted as (0.2-7)]. The solubilities in (0-6), (0-7) and (0.2-7). determined after centrifuging the dispersions for 30 minutes at 4 X IO'g, were respectively: 92.5%,93.9% and 93.0% for sodium caseinate, 92.3%, 91.0% and 93.6%for WPC, 69.9%, 65.3% and 64.0% for SP. and 86.3%, 100.0% and 100% for BP. Emulsion Formation.-The protein-stabilized emulsions were made from 40 wt% soya bean oil and 60 wt% protein dispersion containing 2.5 wt% protein. Emulsification was carried out using a valve homogenizer in a recirculating system,* with 10 passes and power inputs of 20 W, 40 W and 70 W. Emulsion Treatment.-Emulsions were stored overnight in a freezer at -80 "C and thawed at room temperature. In the case of the freeze-drying experiments, frozen emulsions were put overnight in a freeze-drier (Hetosicc CD3). For the heating experiments, emulsions were put in a water bath at 80 OC for 30 minutes. Emulsion Characterization.-A spectroturbimetric method'.' was used for dropletsize determinations. Regarding the application of this method to soya bean oil/ water emulsions, the reader is referred to a previous paper.5 The nthmoment of the frequency distribution, Sn,can be calculated from the data obtained. As the largest droplets are expected to be the most susceptible to coalescence, a mean diameter dS4= SJS,, which biases the droplet-size distribution towards the largest droplets, was calculated. The degree of flocculation of the emulsions was ranked on an arbitrary scale from 0 to 7 as judged by microscopic observation. Figure I gives an indication of the ranking scale used. The amount of protein adsorbed per unit area of fat surface(the protein load) was obtained by subtracting the amount of residual protein in the bulk phase after emulsification from the original protein content and dividing that value by the amount of fat surface area created. 'Oiling-off' stability was estimated by centrifuging a 10 ml sample of emulsion at 2.5 X lo4 g for 30 minutes, sucking off
54
E. Tornherg and N. Ediriweera
55
the separated oil, and calculating its amount as a percentage. Measurement of the percentage of oil extracted by hexane was carried out according to the procedure outlined by Tinbergen and Olsman.' Results and Discussion In order to check the hexane extraction method against an 'absolute'measurement of coalescence stability, the change in amount of oil extracted by hexane after storage and freezing was compared with the increase in droplet size expressed as Ad54. Thirteen different protein-stabilized emulsions were investigated. A linear regression analysis was carried out between AO, the change in percentage of oil extracted by hexane, and Adw; and a correlation coefficient of 0.90 was obtained to the equation A 0 = 3.9(Ad5,/prn)
+ 4.5
(1)
According to equation ( I ) , an increase in d,, of I pm corresponds on average to another 4% oil extracted by hexane. The other accelerated technique, namely 'oiling-off' by centrifugation, was also compared to the hexane extraction method. In general, to achieve any measurable 'oiling off', emulsions have to be frozen o r freeze-dried. Altogether, some 93 different protein-stabilized emulsions were studied, and a linear regression analysis was carried out between Oh,the percentage of oil extracted by hexane, and Oc,the percentage of oil separated by centrifugation: a correlation coefficient of 0.87was obtained to the equation
Equation (2) further suggests that .the amount of oil extracted by hexane is a reflection of coalescence instability. The results presented here indicate that oil extraction by hexane is a promising accelerated method for registering the coalescence instability of protein-stabilized emulsions. It is clearly not an 'absolute' method as it induces some coalescence during measurement, but it is linearly well correlated to the 'absolute' method of increasing droplet size, and to the accelerated technique of oil separation by centrifugation. Moreover, the hexane extraction method is capable of measuring coalescence stability over a wide range of instability, whereas centrifugation provides information only about the final stages of emulsion instability. The food proteins chosen for study here are protein preparations used relatively commonly in the food industry. Moreover, they are meant to represent proteins of increasing complexity with regard to composition. molecular-weight distribution and structure (secondary, tertiary and quaternary). Ionic strength and pH have been varied, since we know that these variables change the balance between electrical attraction and repulsion amongst protein entities, thereby affecting the state of aggregation of the proteins and their molecular flexibility. This investigation was carried out in order to see if there are any general tendencies with regard to coalescence stability in the emulsions stabilized by food proteins most
56
E. Tornberg and N. Ediriweera
commonly in use. The number of measurements performed under each set of conditions, however, is not sufficient to allow detailed prediction of how a specific protein acts as a stabilizer at a certain pH and ionic strength. The individual caseinate molecules, which are assumed to be aggregated in solution to a relatively high molecular weight of 2.5 X 105,6are random-coil-like proteins with hardly any secondary or tertiary structure. The WPC contains mainly low-molecular-weight proteins (P-lactoglobulin and a-lactalbumin) which in solution d o not form molecular complexes with high aggregation numbers.6 The protein P-lactoglobulin is relatively hydrophobic and flexible. The major protein in the frozen blood plasma preparation is bovine serum albumin (BSA, molecular weight 6.7 X lo4) comprising approximately 60% of blood plasma. BSA has a relatively ordered globular structure with 17 -S-S- bridges, a helix content of 46%, and a hydrophobicity less than the caseins and ~ - l a c t ~ g l o b u l i The n . ~ rest of the proteins in BP, as represented by fibrinogen and the immunoglobulins, are highmolecular-weight proteins in solution. The soy proteins have a complex quaternary structure which is especially pronounced in 0.2 M NaCl solution. The 7 s and I IS proteins, which constitute the major part of the soy protein isolate, possess considerable secondary structure and a compact tertiary structure stabilized by hydrophobic bonds. Figure 2 shows coalescence instability measured as a percentage of oil extracted by hexane as a function of oil droplet size expressed as d54 for fresh and frozen BP-stabilized emulsions under various conditions of pH and ionic strength. First, we consider only the fresh emulsions. As might be expected, there is, according to Figure 2, a linear (albeit relatively weak) increase in the amount of oil extracted with increasing oil droplet size. By comparing droplets of the same size, it can be further deduced from the figure that BP(0-6) are the most stable emulsions, followed by BP(0-7) and then BP(0.2-7). Table 1 lists the average degree of flocculation for all the protein-stabilized emulsions under various conditions of pH and ionic strength. It can be seen that the BP preparation gives rise to the least flocculated emulsion in (0-6) and the most flocculated in (0.2-7). These observations suggest that the more flocculated the BP-stabilized emulsion is, the less stable it is to coalescence. Figures 3 and 4 compare the capacities of the different proteins to give stable emulsions when fresh, frozen and heated in (0-6) and (0.2-7) respectively. At this point, we discuss only the fresh emulsions. The more-or-less linear dependence of the amount of oil extracted by hexane on the droplet size is observed for all the proteins except WPC. (The reason for the peculiar behaviour of WPC is discussed later.) It is observed from the figures that, in both (0-6) and (0.2-7), caseinate produces the most stable emulsions followed by BP. WPC-stabilized emulsions are mostly less stable than BP-stabilized emulsions, but under certain circumstances the roles of the two proteins can be reversed. SP in (0-6) and (0.2-7) produces emulsions that are most flocculated (Table 1 ) and most unstable towards coalescence (Figures 3 and 4). In general, the whole set of data shows a correlation between flocculation and coalescence. Though many factors are held to be responsible for the very good stabilizing power of proteins, it has long been recognized that the rigidity o r viscoelastic properties of the protein film are strongly implicated.8 In a study of the coalescence
57
1
I
I
I
FRESH
I
I
I
1
5
ie
15
28
d54
Figure 2
/
Ilrn
Effect of pH and ionic strength on coalescence stability of fresh and frozen emulsions stabilized by blood plasma protein. Thepercentage of oil extracted by hexane is plottedagainst mean droplet diameter dS4;
+, (0-6); 0,(0-7); *, (0.2-7)
Table 1
Flocculation stability of the dvferent protein-stabilized emulsions Average degree offlocculation
(0-7)
(0.2-7)
Protein
(0-6)
caseinate
0.9
0
0.3
WPC
I .3
I .2
I .2
BP
0.5
I .o
I .8
SP
2.7
1.1
5.0
E. Tornberg ond N. Ediriweero
58
stability of protein-stabilized emulsions, Graham' concludes that optimum prevention of coalescence is observed when the interfacial layers are as thick as possible and also heavily charged. I n Figure 5 , therefore, we plot coalescence stability as a function of protein load, assuming that the latter is positively related to film thickness. Contrary to what is often found in the literature, Figure 5 suggests that the thinner the protein membrane the more stable theemulsion, although there
I
1
I
I
FRES
10
Figure 3
I
I
15
aa
Comparison of coalescence stabilities of fresh, frozen and heated emulsions in distilled water at pH 6 . Thepercentage of oilextracted by hexane is plotted against mean droplet diameter d54: caseinate; 0 , WPC; BP; X, SP
*,
+,
59
are deviations observed from this behaviour in some of the WPC- and SP-stabilized emulsions (see below). The apparent observation that emulsions having thinner protein films are more stable to coalescence could be due to the fact that one of the more decisive factors ruling coalescence stability is the desorption barrier of protein from the oil droplet. A more unfolded adsorbed protein (in a thinner protein membrane) will tend to have a larger proportion of attachments to the interface. Consequently, the more attachments per molecule that there are, the higher will be the energy barrier to desorption, since the probability of all attachments desorbing simultaneously will be lower. Protein unfolding at the interface should not result in too high a flexibility of protein at the interface, since this can bring about a decreased efficiency of the Gibbs-Marangoni effect in stabilizing the emulsions, according to the following mechanism. When the surfaces of droplets are deformed on collision, the increase in FROZEN
I
I
I
I
FRESfl
I
I
5
18
d54
Figure 4
I 15
I
28
/ Prn
Comparison of coalescence stabilities offresh and frozen emulsions in 0.2 M NaCl at pH 7. The percentage of oil extracted by hexane is plotted against mean droplet diameter dS4: caseinate; 0, WPC; BP; X, S P
+,
*,
60
E. Tornberg ond N. Ediriweero 30, BO
D* D*
20,-
10.-
I
I
1
2
4
6
Coalescence stability as afunction ofprotein load in emulsions cfresh) stabilizedby(A)caseinate, (B) WPC,(C)BP,and(D)SP: +,(O-6); (0-7); 0,(0.2-7)
*,
surface area, and associated increase in surface tension, can more quickly be damped out by an already unfolded flexible protein than by a protein that first has to unfold and then extend at the interface. This could explain the high value (ca. 30%) for the amount of oil extracted by hexane in WPC(O.217) as seen in Figure 5 . As the whey proteins are the least aggregated of those studied, they have a relatively high probability of unfolding at the interface. This is beneficial with regard to coalescence stability if the degree of unfolding is as substantial as that for WPC(06) at d5, = 5 p m (see Figure 3). But, at a protein load in the range 1-2 mg m 2 , the amount of WPC unfolding is not enough to restrict its mobility at the interface, and in such cases the efficiency of the Gibbs-Marangoni effect can be reduced. The main difference in coalescence stability between WPC- and BP-stabilized emulsions, in favour of the latter, could be due to the presence at the interface of more rigid BSA molecules as opposed to the more flexible /3-lactoglobulin molecules. Therefore, the Gibbs-Marangoni effect might be greater with BP than with WPC. While individual caseins are considered to be very flexible at the interface, they d o have a tendency to form aggregates at the surface, as shown, for example, by Mulder and Walstra.'O This aggregation tendency could restrict casein mobility at the interface in comparison with the less aggregated whey proteins, thereby giving rise to a greater Gibbs-Marangoni effect. Interfacial protein mobility should not be too restricted, however, which is probably what happens with the soy proteins, which are assumed to be the most aggregated of the studied proteins at the interface, as indicated by the high protein
61
loads and large degrees of flocculation. A high energy barrier to unfolding exists for such aggregated proteins, and the adsorption behaviour resembles more that of particle adsorption, with few direct attachments per unit adsorbed. This implies a low barrier to desorption, and, in the case of highly aggregated systems, there might not even be full coverage of the interface. Both of these factors will induce greater coalescence. This may explain why SP(0-6) deviates so much from the linear dependence in Figure 5. Moreover, the greater instability of BP(0.2-7) as compared to BP(0-6), although having the same protein load, could be caused by a lesser extent of interface coverage in the former emulsion. Coalescence stability of frozen BP-stabilized emulsions is shown in Figure 2 for comparison with the fresh emulsions. Freezing reduces emulsion stability in each case, and the instability droplet-size dependence is steeper for the frozen emulsions. Figure 2 shows that the susceptibility of BP-stabilized emulsions to freezing depends on pH and ionic strength. While BP(0-6) leads to the most stable emulsions when fresh (extracted oil
Figure 6
Coalescence stability as afunction ofprotein load infrozen emulsions stabilizedby (A)caseinate. (B) WPC, (C)BP, and(D)SP: (0-6); (0-7); 0, (0.2-7)
+,
*,
62
E. Tornberg and N . Ediriweera
INITIAL dS4
/ pm
Comparison of coalescence siabilifies of frozen and freeze-dried WPC-stabilizedemulsions. The percentage of oil extracted by hexane from frozen ( ) and freeze-dried (- - - - -) emulsions is plotredagainst initial mean droplet diameter d,: (0-6);0, (0-7); (0.2-7)
+,
*,
stabilitydue to freezing for any of the systems studied. [At most there was IOo/,extra oil extracted from the caseinate-stabilized emulsions in (0.2-7).] Every protein dispersed in (0-6) shows a lower stability on being frozen amounting to at most 50% extra oil extracted; the WPC(0-6) shows a drop in stability of ca. 25% at most. In frozen emulsions WPC is superior to BP with respect to coalescence stability, whereas the reverse was found for fresh emulsions. The observed coalescence behaviour of frozen emulsions suggests that there might exist a different relationship between stability and the thickness of the protein membrane from that seen with fresh emulsions. So, in Figure 6, the percentage of oil extracted by hexane in frozen emulsions is plotted against protein load. Two distinct groups of points are discernible: one for the more stable emulsions (<40% extracted) and one for the less stable (>40% extracted). The linear dependence that can be associated with these two sets of points has opposite slope from that derived for the fresh emulsions. Evidently, with regard to coalescence stability, the mechanical properties of the protein membrane come more into play when the emulsion is frozen. This is understandable, because coalescence due to freezing is supposed to be induced by ice or fat crystals distorting the protein membrane surrounding the droplets. In Figure 7 the coalescence stability of frozen and freeze-dried WPC-stabilized emulsions are compared under various conditions of pH and ionic strength. Firstly, it can be seen that dehydration per se causes instability of WPC-stabilized
63
emulsions. Secondly, the effect is greater for WPC(0-6) and WPC(0-7) than for WPC(0.2-7). As the former emulsions are the least stable towards freezing, it could be that the more destabilization that occurs on being frozen, the more susceptible is the emulsion to damage during drying. Finally, we mention the effect of heating on coalescence stability. Figure 3 shows the percentage of oil extracted by hexane as a function of d, for the different protein-stabilized emulsions in (0-6) after heating at 80 OC for 30 minutes. It is established that there is only a minor destabilization compared with the fresh emulsions.
References I B.J. Tinbergen and W.J. Olsman, J. FoodSci., 1979,44,693. E. Tornberg and G . Lundh, J. FoodSci., 1978,43,1553. P. Walstra. Nerh. Milk Dairy J., 1965, 19,93. P. Walstra, J. Colloid Inrerface Sci., 1968.21,493. E. Tornberg, J. Sci. Food A@., 1978,29,867. R.L.J. Lyster, J. Dairy Rex. 1972.39.279. H.E. Swaisgood, Crir. Rev. Food Techno/., 1973.3.375. H.J. Rivas and P. Sherman. Colloids SurJ, 1984.11, 155. D.E. Graham,'Structure of adsorbed protein films and stability of foams and emulsions,' Ph.D. Thesis, Council for National Academic Awards, London, 1976. 10 H. Mulder and P. Walstra, 'The Milk Fat Globule,' Pudoc. Wageningen, 1974. 2 3 4 5 6 7 8 9
Aggregation Rates and Electrophoretic Mobilities of Homogenized Milk Fractions Treated with Rennet
By ELIZABETH W. ROBSON and DOUGLAS G. DALGLEISH
(Hannah Research institute. Ayr. Scotland KA6 5HL)
Introduction The particulate material in homogenized milk consists of fat globules (diameter 2 I pm) stabilized by casein micelles or fragments thereof (so-called sub-micelles).' The casein micelles are themselves composed of the four casein proteins ( a s las2, , /3 and K) together with micellar calcium phosphate. The main stabilizing factor of native micelles is the K-casein which is believed to reside mostly near the surface of the micelles.2Thisis in part confirmed by the observation' that theelectrophoretic mobility of the micelles decreases considerably when they are treated with chymosin or rennet, the enzyme acting specifically to cleave K-casein alone. Qualitatively, the same behaviour is foundSwith particles from homogenized milk, showing that the protein in the region of the surface of shear is also K-casein, and therefore that the particles in homogenized milk are also stabilized by K-casein. The consequence of adding rennet to either skim milk or homogenized milk is that the particles become destabilized and aggregate to a coagulum. However, though the rennet-induced aggregation of particles in homogenized and skim milks appears to be controlled by similar factors, there are important differences of detail in the behaviour patterns of the two milks. In particular, particle aggregation in renneted homogenized milk appears to occur at a lower extent of K-casein breakdown than in skim milk, and aggregation of the homogenized milk particles is slower than that of renneted casein micelles.6 These differences, especially the first, must be related to the structure of the fat-protein interface formed during homogenization. The fat-globule surface is apparently composed of intact casein micelles as well as smaller casein-containing structures, although the protein loads and the sizes of the fatlcasein particles vary Were casein micelles to be adsorbed at the fat surface without disruption, then the fat particles would be expected to behave essentially as large casein micelles. If, however, micellar structure were to be altered by binding to the interface, differences in aggregation behaviour might well be expected. There is evidence9-" that casein micelles are sterically stabilized; certainly, the variation in coagulation 64
65
rate of renneted micelles with ionic strength and temperature indicates that particle surface charge is not completely responsible for stability,I2 and the same is true for particles from homogenized milks! Also, there appears to be little difference between electrophoretic mobilities of particles in skim and homogenized milks,5 so that charge effects are insufficient to explain the observed differences in aggregation behaviour of these two casein-stabilized systems. Experiments on whole homogenized milk d o not allow particles that have different compositions and structural features on their surfaces to be distinguished. It is not possible to separate completely the fat and protein particles in homogenized milk, which contains intact casein micelles as well as fat/ micellar complexes. Nevertheless, fat-rich and protein-rich fractions can be obtained by Centrifugation, and some separation of particles according to size and protein load can be achieved.'This paper describes some of the properties of particles that can be separated by a simple centrifugation process. Experimental
Homogenized and skim milks were prepared from whole milk taken from the bulk tank of the Institute's farm after morning milking. Skim milk was prepared by centrifugation at 2 X lo3 g for 30 minutes at 4 "C, followed by removal of the fat layer. Homogenized milk was prepared at 45 "C usinga two-stage Manton-Gaulin homogenizer with a total pressure drop of 24. I MPa (3.4 MPa across the second valve). The homogenized milk was cooled to 20 "C before fractionation in a Sorvall RC2 centrifuge (8 X 50 ml angle rotor) at 3.77 X lo4 g for 15 minutes. Three fractions were produced: a sinking pellet (SP), a floating fraction (HF),and a middle layer (SN).The first two of these fractions were suspended in ultrafiltrate from the same milk, prepared using an Amicon TCFIOA cell with a PM30 membrane. Properties of the different fractions are given in Table I . A fraction containing casein micelles was also prepared by centrifuging skim milk at 1.07 X lo4g for 15 minutes, collecting the supernatant, and centrifuging the supernatant at 1.67 X lo4 g for 15 minutes. The resulting pellet was also resuspended in ultrafiltrate. Samples were subjected to controlled renneting with aggregation prevented by keeping the temperature low. After cooling in ice, 20 ml aliquots of the fractions were treated with 200 pI of a 5 g I - ' solution of Hansen's Klselabpulver rennet powder in distilled water. Aggregation rates and electrophoretic mobilities of the rennet treated fractions were measured at approximately 5-minute intervals. Aggregation rates were measured as described previously6J3 by diluting 100 pl aliquots of renneted samples into 900 pI of ice-cold buffer (20 mM imidazole, pH 7.0, containing50 mM NaCl and 5 mM CaCI,), and then adding 100 pI aliquots of this mixture to 1 ml portions of the same buffer in a spectrophotometer cell maintained at 35 "C. Absorbance at 450 nm was measured over a period of 2 minutes and the initial rate of change of absorbance was calculated. Particle electrophoretic mobilities were measured using a Malvern-Rank 'Particle Charge' a p p a r a t ~ s A . ~20 pl aliquot of the rennet-treated fraction was diluted into a 5 ml portion of the imidazole buffer which had previously been filtered through a 0.22 p m filter. Sample dilution effectively stops the enzymic
66
E. W. Robson and D.G. Dalgleish
reaction, enabling a steady measurement of mobility to be obtained. The diluted sample was introduced into the capillary of the apparatus, which was maintained at 35 OC. Results were obtained by averaging 32 scans of the frequency spectrum, which took about 3 minutes, after which time the next sample was run. Thus, no sample storage was required during the experiments. The diameters of particles in the fractions were measured by photon correlation spectroscopy using a Malvern 'Log-Lin' autocorrelator. Measurements were made on light scattered at an angle of 90' from suspensions of the fractions made from a 20 pI aliquot in 3 ml of imidazole/NaCI/CaCI, buffer. With the fractions from homogenized milk, a separate measurement was taken to find the sizes of the fat cores of the particles. This was done by treating the particles with S mM EDTA solution in order to dissociate casein micelles on their surfaces before measuring the diameters. Concentrations of protein and fat in various fractions were determined by standard Kjeldahl and Gerber methods, respectively.
Results and Discussion Table I shows compositional properties of the particles isolated from homogenized milk. Of the three fradtions separated by centrifugation, the HF fraction is by far the most rich in fat, having a protein/ fat ratio of ca. 5 as compared with the value of 0.3 for the protein-rich SP fraction. The HF fraction has the largest particles, with an average radius more than twice that for the SP particles. With respect to protein/ fat ratio and average particle size, the S N fraction is intermediate between H F and SP. The approximate protein loads in Table 1 suggest that the HF fraction is incompletely covered (if one takes the maximum protein load for fat globules completely covered by casein micelles to be ca. 40 mg m 2 , as defined by Oortwijn and Walstran). On the other hand, the SP fraction contains more protein than can be accommodated on the surface of the fat, and, if we take the value above for the saturation coverage, it appears that not only is the fat surface totally covered with casein micelles, but the fraction contains ca. 60% of its protein in the form of free casein micelles. This estimate is at least partly confirmed by the change in the radii of the particles when they are treated with EDTA. The HF particles change radius by over 100 nm after EDTA treatment (about the size of a casein micelle), whereas
Table I
Properties offractions of homogenized milk
Properry
Fraction H F
Fracrion SN
Fraction SP
casein content/ g I-'
24.6
25.0
28.0
fat content/ wt%
12.0
2.5
0.8
mean particle radius/ nm
331
I87
141
mean core radius/ nm
227
I I8
I02
protein loading/ mg n f 2
14.1
36.0
109. I
67
1.
1.11
0
I
60
I
1
80
1
1
120
1
I
160
I
Time after Rennet Additionlmin
Figure 1
Electrophoretic mobilities ofdij$erentfraelions of homogenized milk duringrennetingato "C (measuredat 35 OC in imidazole/NaCI/CaC12 hufler). The points refer to$raction HF. and the curves t o S P ( . - * . - .), SN (- - - - - ) andcasein micelles (----)
the apparent radius change for the SP fraction is only ca. 40 nm. Since the radius measurement represents an average over all particles in the suspension, the smaller value for the change in radius of the SP fraction must imply either that the fat in this fraction is covered with smaller micelles (for which there is no evidence), o r that the fraction consists of a mixture of caseinlfat particles and casein micelles. Changes in particle electrophoretic mobility during renneting are given in Figure I . In the course of repeated experiments, it was noted that casein micelles always gave somewhat higher initial mobilities than any of the fractions from homogenized milk, all of which were similar within experimental error. This is true even for the SP fraction, for which the protein load is largest, and where there may be small amounts of the original fat-globule membrane on the fat-particle surface. During renneting, there is a drop in electrophoretic mobility of homogenized fractions from approximately -I .6 X lo-* m2 V S - I to -I .2 X 10." m2 V-' s I over a period of about two hours. A slightly greater decrease occurs with casein micelles, although the final mobilities of homogenized fractions and casein micelles are very similar. The overall kinetics of rennetingseems to be similar for all the fractions, although it can be seen from Figure I that the mobility of the H F fraction decreases more rapidly than the others. This may be due to the concentration of K-casein in the fraction, rather than a reflection of any real difference in the behaviour of the fractions. The relationship between mobility and extent of renneting is not linear
E. W. Robson and D. G. Dalgleish
68
for casein micelles,6 and so it is not possible to relate these measured mobilities to the degree of renneting. It is reasonable, however, to assume that the rate of change of mobility with the extent of renneting is sufficiently similar for the different particles to allow observed mobility to be used as an approximate indicator of the extent of reaction. But, since the surface of shear is not well defined for particles like those in homogenized milk, an interpretation of the absolute magnitudes of the mobilities cannot be made. With respect to their aggregation behaviour during renneting, the fractions differ considerably as shown in Figure 2. As the samples are rather polydisperse, it is not possible to estimate meaningful molecular weights, and so absolute rate constants cannot be obtained. However, for each fraction, the measured rates of aggregation were normalized with respect to the maximum rate after renneting was complete. Casein micelles show a period after the start of the enzymic reaction during which no detectable aggregation occurs, after which the rate of aggregation increases rapidly with renneting time. Fraction SP shows very similar behaviour to this, although the onset of aggregation is slightly earlier than for casein micelles. Fraction S N is similar, but the onset of aggregation is earlier than for SP, and in addition some aggregation occurs at a slow but constant rate even at the early stages of renneting. In complete contrast to SP and S N fractions, HF has no lag stage at
I
1
I
I
I
I
I
I
I
I
I
1
I
I
Time after Rennet Addition I min
Figure 2
Relative aggregation rates of diJferent fractions of homogenized milk during renneting at 0 O C (measured at 35 O C in imidazolel NaCIl CaCI, bufler). A relative rate of unity corresponds to the maximum rate for each sample after renneting was complete. The points refer tofraction H F . and the curves to SP (. - . - ' - .), SN (- - - - -) and casein micelles ( 1
69
all, with rennet-induced aggregation observed from the earliest stages of the renneting reaction. The HF aggregation rate increases almost linearly with time until it reaches its maximum value. Comparison of Figures 1 and 2 shows that only during the last stages of renneting does significant aggregation of the material occur; that is, the change in mobility is complete, as far as can be measured, within I20 minutes, while the rate of aggregation continues to increase for almost one hour thereafter. Figure 3 shows that the aggregation rate is not controlled directly by the electrophoretic mobility. Results for the H F fraction differ from those for the other two fractions, insofar as aggregation starts when the particles have a higher mobility (apparently larger surface charge) than with either the SP o r S N fractions. If a change in mobility is related to the extent of splitting of Kcasein, then it appears that the HF fraction requires less K-casein to be split than d o any of the other fractions. Casein micelles do not aggregate significantly until ca. 85% of their K-casein has been d e ~ t r o y e d , ' ~at. 'which ~ point ca. 60% of the mobility change has occurred (cJFigure 3). The fractions SN and SP behave much like casein micelles in this respect, although both aggregate somewhat more readily. The presence of free casein micelles, at least in the latter fraction, will account partly for this behaviour, although it is possible that the apparently saturated coverage of the fat particles by casein micelles may indeed make them behave rather like large casein micelles. This once again demonstrates that the maximum increase in aggregation
8
I
[
60
0
20
40
60
80
100
Relative Mobility Decrease
Figure 3
Plot of relative aggregation rate against percentage change in electrophoretic mobility of the fractions. The points refer to fraction HF, and the curves to SP (- - . - . - .), SN (- - - - -) and casein micelles (
1
70
E. W. Rohson and D. G. Dalgleish
rate takes place towards the end of the the renneting reaction, even for fraction H F which shows the early onset of aggregation. The low value for the protein load in fraction HF implies that the fat surface is covered either sparsely by intact casein micelles or more evenly by fragments of casein micelles. Both types of coverage have been d e ~ c r i b e d , ' , ~but . ' ~it is not certain which applies to the samples investigated here. The results in Table I strongly support the view that some intact casein micelles are present at the surface, since the radii of H F particles decrease by>100 nm when the material is treated with EDTA to dissociate the casein micelles. This result also makes it clear that the hydrodynamic surface of shear is near to the surface of the stabilizing micelles rather than close to the surface of the fat. The electrophoretic behaviour of the fat/ protein particles is, therefore, as in casein micelles, dominated by the K-casein of the intact o r semi-intact casein micellar surface. The aggregation behaviour of H F particles can be compared with the mechanism proposed by Payens el to describe the behaviour of casein micelles during renneting. Originally, this mechanism postulated that the breakdown of one Kcasein molecule would be sufficient to allow coagulation, and that more material becomes coagulable as the enzymic reaction proceeds. While the mechanism must be modified to take account of the properties of casein micelles,'* it appears that there may be potential for its use in connection with large, fat-rich particles in homogenized milk. The difference in behaviour between this fraction and casein micelles is not obviously explained. If the stabilizing protein were to be spread thinly over the fat surface, we would expect that a similar fraction of K-casein would have to be destroyed as for casein micelles before the particles could coagulate. While casein micelles appear to be sterically stabilized:-" the loss of stability by rennet requires that large bare patches be formed on the surface: this is clearly not the case for H F particles. The observations suggest then that the fat surface is unevenly coated with intact casein micelles rather than by. fragments of them. So, although K-casein is obviously important in stabilizing HF, it seems to be relatively ineffective, insofar as only a small amount of it must be destroyed to produce instability. This being the case, it is possible that aggregation of renneted particles of the H F type occurs by a different mechanism from that applying to particles of types S N o r S P , or to casein micelles. For the latter types of particle, aggregation occurs in such a way that the points of contact between particles are via renneted micelles bound to fat. Particles of the H F type may not require proteolysis of f w o surfaces to occur, if the gaps between casein micelles on the fat surfaces are sufficiently wide for interactions to take place between the fat surface of one particle and the renneted micelle of another. This work was undertaken to try to understand, infer aha, how the curdformation reactions differ in whole and homogenized milks. Two reasons for a difference in curd structure now present themselves: compositions of the particles forming the primary curd in the two systems are different, and the sizes of the particles that aggregate to form the initial gel are very different. Since casein micelles of various sizes d o not differ markedly in their renneting or aggregation b e h a v i ~ u r , 'the ~ coagulation process will tend to be initiated by the smallest micelles which are the most numerous; so the primary curd-forming units in skim milk are of the order of 50 nm in radius. Conversely, in homogenized milk, it is the
71
largest particles that form the primary aggregates, and so the structures of the two gels will be potentially different from the earliest stages of the reaction. Acknowledgements. We thank Miss E. Noble for analyses of fat and protein, and Miss S. Caddell
for technical assistance.
References I H. Oortwijn, P. Walstra, and H Mulder, Nerh. Milk Dairy J., 1977.31, 134. 2 W.J. Donnelly, G.P. McNeill. W. Buchheim, and T.C.A. McGann. Biochim. Biophys. Acra. 1984.789, 136. 3 M.L. Green and G . Crutchfield. J. Dairy Res., 1971.38, 151. 4 K.N. Pearce, J. Dairy Res., 1976,43,27. 5 D.G. Dalgleish. J. Doiry Res., 1984, 51,425. 6 E.W. Robson and D.G. Dalg1eish.J. Dairy Res., 1984,51,417. 7 D.G. Dalgleish and E.W. Robson, J. Dairy Rex, 1985,52,539. 8 H. Oortwijn and P. Walstra. Nerh. Milk Dairy J., 1979.33. 134. 9 P. Walstra. V.A. Bloomfield, J.T. Wei, and R. Jenness. Biochim. Biophys. Acra, 1981, 669,258. 10 C. Holt and D.G. Dalgleish, J. CulkuidInrerfoce Sci.. 1986, in the press. I I D.S. Horne, Biopolymers, 1984,23,989. 12 D.G. Dalgleish, J. Dairy Res., 1983,50,31 I. 13 D.G. Dalgleish, J. Dairy Res.. 1979.46.653. 14 M.L. Green, D.G. Hobbs, S.V. Morant,and V.A. Hil1.J. Duiry Res., 1978.45.413. 15 M.L. Green, R.J. MarshalLand F.A. G1over.J. Dairy Res., 1983.50.341. 16 T.A.J. Payens. A.K. Wiersma, and J. Brinkhuis, Biophys. Chem., 1977,6,253. 17 T.A.J. Payens, Biophys. Chem., 1977.6,263. 18 D.G. Dalgleish. Biophys. Chem.. 1980. 11, 147. 19 D.G. Dalgleish, J. Brinkhuis, andT.A.J. Payens, Eur. J. Biochem., 1981, 119.257.
Adsorption Kinetics of Proteins at the Air-Water Interface
By J.A. DE FEIJTER and J. BENJAMINS
(Unilever Research Laboratorium Vlaardingen. P.0.Box 7 7 4.3 7 30 AC Vlaardingen. The Netherlands)
Introduction Adsorption at air-water or oil-water interfaces reduces the interfacial tension, but with macromolecules such as proteins it may take a long time before a constant (equilibrium) tension is reached. It has been customary’-’sto interpret the kinetics in terms of the rates of various sub-processes taking place in or near the surface layer: (a) diffusion from the bulk solution to the sub-surface region in direct contact with the surface layer; (b) penetration of macromolecules into the surface layer (the actual adsorption step); and (c) reconformation o r rearrangement of adsorbed molecules within the surface layer. Various attempts have been made to assess the relative importance of the different physical processes taking place when proteins adsorb at fluid interfaces. In many cases the interpretation is based on interfacial tension measurements alone. This requires assumptions to be made about the relationship between the increase in surface pressure (decrease in surface tension) and the surface concentration (amount adsorbed per unit area). The validity of these assumptions is not always self-evident, thus casting some doubt on the often detailed conclusions drawn from such studies. Direct information on the kinetics of protein adsorption is scarce. Graham and Phillips studied4 adsorption of &casein, bovine serum albumin and lysozyme at the air-water interface using a radio-tracer method, and we have applied ellipsometry to K-casein adsorbing at the same interfa~e,’~.’’ and have extended the work to other proteins. In this paper we present results obtained with two globular proteins, ovalbumin and lysozyme, using two independent techniques: (i) ellipsometry, which allows direct measurement of the timedependent surface concentration, and (ii) the static Wilhelmy plate method, which gives the surface pressure as a function of time. The two methods are applied simultaneously to the same surfaces. The results are compared with those of others, and conclusions are drawn about the relative rates of processes (a), (b) and (c). It would seem that certain conclusions of others, based on tension measurements alone, are not correct. 12
73
Experimental Materials.-The proteins were obtained from Sigma Chemicals: ovalbumin (grade V,4.5 X lo4daltons, P I = 4.6) and lysozyme (grade 1,1.45 X lo4daltons, P I = 10.7). They were used as received. Phosphate-citrate buffer (ionic strength co. 0.02 M) controlled the pH at 6.4 for the ovalbumin solution and 6.7 for the lysozyme solution. All solutions were made with twicedistilled water and used directly after preparation. Techniques.-Surface tensions were measured with an accuracy of 0.2 mN m I using the static Wilhelmy plate method with a roughened glass plate attached to a force transducer. Contact was made between plate and surface immediately after filling the PTFE trough (10 X 7 X 2 cm), and the plate remained in the same position until the end of the experiment (in most cases for one day). The shape of the contact line between plate and solution meniscus indicated that the plate was well wetted during the entire experiment. Surface tension was monitored continuously as a function of time. (For buffer solutions without protein, the tension was found to be y = 73.0f0.2 mN m-' at 22 "C independent of time). Solutions were poured into the trough with the help of a separating funnel, the tip of the funnel remaining above the surface of the solution in order to prevent aged surface of the original solution from entering the trough. Protein surface concentrations were determined with the ellipsometric technique described previously.'6 The trough was covered with a lid to reduce evaporation. There was a hole in the lid for the suspension wire of the Wilhelmy plate, and small slits for the incident and the reflected (elliptically polarized) light beam. The refractive index and average optical thickness of the adsorbed protein layer were obtained from the change in state of polarization of the light beam upon reflection at the surface.I6 To calculate the average protein concentration in the adsorbed layer from the refractive index, we assume a refractive-index increment of 0.18 ml g-'.I6.l8 This value, combined with the thickness of the layer, gives the surface concentration r of protein to an accuracy of 0.05-0.10 mg m 2. At high protein concentrations wt%), a correction is made for the fact that the refractive index of the buffer solution is increased by the protein. Each ellipsometric measurement takes 5-10 minutes. All of the experiments were set up in a temperature-controlled room at 2 2 f I O C . Results Figures I A and 2A show plots of surface concentration r against the square root of adsorption time t for ovalbumin and lysozyme, respectively. The initial bulk concentration ranges from lo' wt% to 10.' wt%. Figures IB and 2B give the associated surface pressure curves, the surface pressure n being defined by
where yo is the constant surface tension of the buffer solution without protein, and y(t) is the time-dependent tension of the buffered protein solution. The
J . A . clr Feijter and J . Benjomins
14
n / m N m-'
Figure 1
Adsorption of ovalbumin (pH 6.4, ionic strength 0.02 M) at the airwater interjhce. Surface concentration ( A ) and surJnce pressure I1 (B) are plotted against t'12, the square root of the adsorption time.for various initial bulk concentrations: A , 10 wt%; 0,10 wt%; 0 , 10 wt%; 0 10-1 wt%
r
'
experimental points in Figures 1B and 2B denote values of I1 determined at times when r was also measured for the same surface. Values of r obtained after an adsorption time of 24 hours are plotted in Figure 3 against the initial bulk concentration co. The curves represent apparent adsorption isotherms - not true ones - since r was still tending to increase with time even after such a long time of adsorption. The surface pressure as a function of surface concentration is shown in Figure 4
75
2.5-
y o
2.0.
A
1.5-
/'
y o
/ O
,do
I0
0.5j
p /' P
:
''
1.0.
I
a /A
/
0A
/A
-a
Figure 2
Adsorption of Iysozyme (pH 6.7, ionic sirengih 0.02 M) at the airwaier interface. Surface concentration (A) and surface pressure (B) areploiiedagainsi t'I2, the square rooi of the adsorpiion time,for various iniiial hulk concenlraiions: A , wt%; 0,10 wt%; 0 ,
r
n
wt%; 0 , 10.' wt%
for ovalbumin and Figure 5 for lysozyme. Curves were assembled by combining the concomitant 11 and r values given in Figures I and 2, with different symbols referring to different initial bulk concentrations. We see that the experimental points for each protein form a single curve independent of cg. As the adsorption time t at which a given value of 11 or r is reached depends strongly on co, it is clear that TI is a unique time-independent function of I' for each of the proteins. Most of the data were obtained during the adsorption process, before n and r had reached
16
J. A. de Fe‘eijlerand J. Benjamins r l m g m-2
I
10-
Figure 3
Nl-
*
c/,
lo-’ w t *I.
Apparent adsorption isotherms of ovalbumin ( ) and lysozyme (- - - - -) after 1 day. The surface concentration r is plotted against the initial bulk concentration co
steady-state values; hence they refer to a situation in which the surface layer was not in complete equilibrium with the bulk solution. The n-r curves of the two proteins are not coincident. Discussion
Kinetics of Adsorption.-Adsorption requires transport of material from bulk solution to surface, and this may take place by diffusion, convection, or a combination of the two. Since in our experiments the solution is believed to be at rest directly after filling the trough, transport takes place here by diffusion only. Expressions for the rate of adsorption can be derived from solutions of Fick’s diffusion equations with the proper initial and boundary conditions. In our experimental arrangement, the surface area is large in relation to the depth ofsolution, implying that side effects near the trough walls can be neglected. As well as protein, the solutions studied contain a mixture of electrolytes (the buffer salts), which are not surface-active as such, but may interact with the proteins, which carry net electrical charges under the solution conditions employed (negative for ovalbumin at pH 6.4, positive for lysozyme at pH 6.7). We assume that the buffer salts affect the adsorption of a protein only indirectly through the value of the diffusion coefficient. This assumption is commonly made when dealing with protein adsorption since it simplifies the problem enormously. The adsorption process can be viewed as a one-dimensional diffusion problem involving a single mobile solute and an infinitely large flat surface. An expression relating the rate of adsorption to the adsorption properties of the
1
,r
,~
5
O Figure 4
Figure 5
0.5
Surface equation of stale of ovalbumin at p H 6.4. The surfacepressure is plotred against surface concentration A , co = ~ 1 % ;0,c,, = 10-3 wt%; 0,c0 = 10 ~ 1 % ;0 , wt%
n
*
to-’
r:
Surface equation of state oflysozyme a1 p H 6.1. The surface pressure II is plotted against surface concentration A , co = wt%; 0,cn = 10-5 wt%; 0 , cn = 10-2 wt%; 0 , lo-’ wt%
r:
78
J . A . de Feijrer and J . Benjamins
solute was obtained by Ward and Tordai.” Assuming the diffusion coefficient D is independent of concentration, the rate is given by
and the timedependent surface concentration by
where cs is the concentration in the sub-surface region (region in direct contact with the surface). In equations (2) and (3), the first term in the curly brackets accounts for solute diffusion from bulk solution to interface, and the second term (the integral) accounts for back-diffusion from the sub-surface region to the solution. The second term can be neglected ifcs
We note that equations (2)-(5) contain onlysolution parameters (c,, cs and D). This does not mean that the surface properties d o not affect the adsorption kinetics; they certainly do, especially after long adsorption times, but only indirectly through their influence on the sub-surface concentration (see below). Equation (3) gives r = 0 at t =O, and so it predicts that curves of r versus t l l Z will pass through the origin. For the experimental curves in Figures IA and 2A, this condition is not completely satisfied. We think that this is due to the fact that t = O is not well-defined experimentally: filling the trough takes about. I minute, which According to equation ( S ) , the initial part implies an uncertainty ofca. 10 s 1 I 2in of the r-t’/2 plot should be straight. This is indeed found to be the case at the lowest concentration (co = 10 wt%) up to P = 0.5-1.0 mg m 2, and so under these conditions D can be calculated from the slope of the linear region using equation ( 5 ) . We find D = 5 X 10 I t m2 s I for ovalbumin and D = 2 X 10 I ’ m2 s I for lysozyme, corresponding to the correct order of magnitude for these proteinsZo(see Table I). The values are, however, somewhat lower than the literature values,20 especially for lysozyme where the difference is certainly beyond the experimental
Table 1
Protein di/fusion coefficients D derivedfrom the initial linear parts of l’-t1/2plots (co = lo4 wt%) as compared with literature valuesm
Prorein
D / 10 lo mZ s I experiment literature
ovalbumin (pH 6.4)
0.5
0.7
lysozyme (pH 6.7)
0.2
I .o
19
error of our technique. This could be caused by the fact that lysozyme in solution at pH >4.5 tends to aggregate,2i which will have the effect of reducing the diffusion coefficient. We also note that the quoted values2n*22.22 were determined in bulk solution at much higher protein concentration (>0.1 wt%) and at higher ionic strength. The fact that the order of magnitude of our D values iscorrect indicates that, for very low bulk concentrations, the initial stage of adsorption is described adequately by the simple diffusion equation [equation ( S ) ] . Graham and Phillips4 and Benjamins er a/. also came to the same conclusion, although their diffusion coefficients were higher than the literature values. The differences were ascribed to convection not being completely suppressed in their experiments. At high bulk concentrations (I: 10 wt%). adsorption proceeds too quickly for our ellipsometric technique to follow the initial stages of the process, and so it cannot be seen from our results whether equation ( 5 ) also holds under these conditions. But there is little reason to believe that its validity is restricted to low protein concentrations only. It can be seen from Figures IA and 2A that the adsorption rate slows down at long times and eventually reaches zero. According to various a ~ t h o r s , ' . ~ . ~this -~~).~~ would indicate the development of an adsorption barrier at the surface, but, as pointed out by Ward and Tordai,I9 this need not necessarily be the case. If the sub-surface region remains in equilibrium with the surface layer, and there is no adsorption or desorption barrier, it is clear that the slope of the I'-t1'2 plot must inevitably decrease and become zero when r has reached its equilibrium value. Under these circumstances, the surface concentration r(t) and sub-surface concentration c,(t) are interrelated at any moment in time by the equilibrium adsorption isotherm of the solute, so that, when r rises, so also will c,. When the condition cs < c, is no longer fulfilled, back-diffusion cannot be neglected, and equation ( 5 ) can no longer be used. Then, according to equation (3). the net rate of adsorption will decrease until it approaches zero as cs-c,,. Hence, when after long times the slope of the P-t'i2 curve starts to fall, it does not necessarily mean that there is a barrier to adsorption at the surface. Such a conclusion may only be drawn if the adsorption isotherm is known. Then, the entire r-t curve can be calculated from equation (3) using a numerical integration p r ~ c e d u r e . * ~ - ~ ~ Comparison between theory and experiment will then reveal whether o r not the assumption of complete equilibrium between sub-surface region and surface layer has actually been fulfilled. This procedure is not feasible here because reliable theoretical expressions for the adsorption isotherms for our proteins are not available. Neither can we use the experimental r-c,, plots given in Figure 3 since they are not true equilibrium isotherms (vide supru). Thus, from the adsorption data alone (Figures IA and 2A), it cannot be inferred whether or not there is a barrier to adsorption or desorption at the surface. We see from Figure 3 that the apparent adsorption isotherms of the two proteins are of the high-affinity type characteristic of macromolecules, i.e., strong adsorption even at very low concentrations. From the molecular dimensions in bulk solution,2nit follows that for a saturated monolayer the surfaceconcentration is 2.2 mg m-* for ovalbumin and I .8 mg m-* for lysozyme. It seems as if ovalbumin does not reach full monolayer coverage at pH 6.4, whereas lysozyme gives multilayers especially at high bulk concentrations. Multi-layer adsorption of lysozyme
J . A. de Feijrer and J . Eenjomins
80
was also observed by Graham and Phillips.35 I t is attributed to the tendency of the molecules to associate in solution. Time-dependence of Surface Pressure.-We now consider the experimental n - t t / 2 curves shown in Figures 1 Band 2B. At low bulk concentrations (510 wt%), there was observed a considerable period of time during which the tension is hardly affected and the surface pressure remains low (
’,
11= k T r = k T / A
(6)
In equation ( 6 ) ,T is the absolute temperature, k is Boltzmann’s constant, and A is the area available per adsorbed molecule. Combining equations ( 5 ) and (6) gives
Il = 2kTcO(Dt/n)’i2
(7)
81
Since equation (5) applies to diffusion-controlled adsorption, it is implied from equation (7) that a linear relation between ll and t1I2 indicates that the ratedetermining step is diffusion from solution to surface. Our results clearly show, however, that equation (6) [and therefore equation (7)] does not apply to (our) proteins. This can be seen by comparing the n-t”2curves of Figures I B and 2B with the corresponding r - t ’ / 2curves of Figures 1 A and 2A: the more-or-less linear parts of the former appear not to coincide with the linear parts of the latter, as required by equations (6) and (7). The reason becomes apparent from a consideration of the rl-r curves. Figures 4 and 5 show that n is not proportional to r, certainly for I 1 > I mN m I, and hence the proteins d o not exhibit ideal surface behaviour for n 21 mN m I . This same conclusion was arrived at by who carefully studied n-A curves of proteins spread at the air-water interface, and found that, even at mN m-I, proteins d o not behave ideally. surface pressures as low as The fact that equations (6) and (7) d o not hold for protein solutions can be illustrated through a simple numerical example. For surface concentrations as high as 2 mg m (roughly a fully packed monolayer), equation (6) predicts that n is only 0. I mN m for ovalbumin and 0.3 mN m-I for lysozyme, far below the measured values in Figures 4 and 5 , respectively. So. according to equation (6),values of n above cu. I mN m I would require surface concentrations far above monolayer coverage. As this is completely unrealistic, equations (6) and (7) should not be used for protein solutions, and any conclusions based on their use should therefore be looked at with reservation.’ It follows from the above that interpreting the time-dependence of protein surface pressures in terms of the underlying physical processes always requires additional information about the relationship between I1 and r. This was realized by MacRitchie and Alexander,’ who made use of the n-A curves ofspread proteins for this purpose, implicitly assuming that the surface equations of state of spread and adsorbed proteins are identical. The correctness of their assumption was supported by the finding that the times required for their bovine albumin solutions to reach I1 = 0. I mN m I were in good agreement with equation (5). But measured times were always lower than theoretical ones for higher surface pressures. The reason for this is clear from Figures I and 2. Equation ( 5 ) only applies in the range of r where I1 is low (say,
‘
82
J.A. de Feijtcr ond J. Benjoniins
n/rnNrn-1
25r
A/m2rng-’
Figure 6
Plots of surface pressure I I against surfoce areu A f o r ovalhumin (. . . . . . . . . .) ond(vsozyme (-
-
-
-
-)
low-molecular-weight surface-active agents. The supposition is that, at high surface coverages, adsorption requires clearance of part of the surface with an area equal to the mean molecular area AA, through compression of the molecules already adsorbed. The activation energy of the process is then equal to IIAA. If, in addition, protein adsorption is assumed to be irreversible, the rate of adsorption is given by d r / d t = k,a,exp(-nAA/ kT)
(8)
or In(dr/dt) = In(k,a,)
-
IIAA/ kT
(9)
where k , is a rate constant, and ab is the protein activity in the bulk solution. When AA is taken to be constant, equation (9) predicts a linear relationship between In(dr/dt) and n, the slope being proportional to AA. MacRitchie and Alexander* have applied equation (9) to various protein ‘ 10 the required linear solutions and have found that for 4 < II/mN m ~ < relationship holds. Linearity was also found when In(dn/dt) was plotted against 11, which supported their finding that, in the same range of surface pressure, d r / d n obtained from the 11-A curves of the spread proteins is constant, from which it follows that In(dH/dt) = K - (HAA/ kT)
83
where K is a constant. The area AA obtained from the slope of the experimental plots was, however, much smaller than expected from the dimensions of the protein in solution, and this led the authors to suggest’ that only a small part of a protein molecule need penetrate the surface layer for it to remain attached and to unfold. The same procedure has been adopted by other worker^^,^,'^.'^ who often use equation (10) directly without checking whether the condition of constant d r / d n is actually fulfilled in the surface pressure range considered. Like MacRitchie and Alexander, they usually find that AA is much smaller than expected from the molecular size in solution, from which they draw the same conclusions. We think that such conclusions are premature for two reasons. In the first place, it is uncertain whether the assumption of irreversible adsorption is actually fulfilled for proteins, as recently shown by MacRitchie.-” Secondly, it seems physically unrealistic to assume that globular proteins would be able to contact the surface through a relatively small hole in the adsorbed layer (of area <20% of the protein cross-section2). This would require major changes in macromolecular shape, which seems unlikely for such rigid proteins3* (see below). As has already been stated, we believe that definite conclusions about the existence of a barrier to adsorption o r desorption can be drawn only when the adsorption isotherm of the protein is known. The Surface Equation of State.-We have shown that protein adsorption is diffusion-controlled in the initial stages, with the rate adequately described by the simplified diffusion equations (4) and (5). From the time-dependent surface pressure alone, it is not possible to arrive at reliable conclusions about relative rates of different sub-processes. Even when combined with direct adsorption measurements, definite conclusions about the existence of an adsorption/desorption barrier cannot be made. Nevertheless, our combined n and r values allow assessment of the time-scale of the third sub-process mentioned in the introduction, reconformation or rearrangement of adsorbed molecules within the surface layer. The II-r relationship is called the surface equation of state. The surface pressure is determined by the (excess) surface concentration r and the forces acting tangentially amongst the molecules within the surface layer. These forces are affected by the spatial orientation o r conformation of the adsorbed molecules. With low-molecular-weight surfactants, molecular reorientation after adsorption is fast; the time-scale is of the order of the molecular rotation time (
84
J. A. de Feijrer and J. Benjamins
reconformation time after adsorption is much shorter than the time-scale of our experiments (minutes to many hours), or reconformation does not occur at all. The second explanation is quite unlikely, as it would mean that adsorbed protein molecules have the same conformation as those in bulk solution, independent of time, which is at variance with the observation that many proteins denature on a d ~ o r p t i o nSo, . ~ ~our finding that II is a unique function of rstrongly indicates that the time-scale of the reconformation process is shorter than the experimental time-scale, i.e., less than cu. 10 minutes. In separate dynamic experiment^?^ where we periodically compressed and expanded the surface, we have found that the relaxation time of the reconformation process of globular proteins varies between a few seconds and a few minutes. (Note that this finding applies to conformational changes insofar as they affect the surface pressure, whose magnitude is mainly determined by the area occupied per adsorbed molecule.32) We have previously shown32that the n-r relationship of adsorbed proteins can be explained by assuming that the molecules behave like deformable impenetrable particles which adjust their shapes to the local surface force field. At low coverage, the molecules tend to be expanded thereby increasing the area occupied per molecule. This tendency is strongest for a flexible molecule like &casein. For a globular protein such as ovalbumin, however, the change in effective radius is relatively small - about 30% increase as r decreases from 2 mg m2(saturated monolayer) to I mg m-2.32 Ovalbumin molecules d o not expand further for r < I mg m 2 ; rather the surface coverage decreases with further decrease in r, leading to low values for the surface pressure. The same behaviour has been observed with lysozyme and bovine serum albumin.36 The fact that II for globular proteins remains very low up to relatively high values of r can be explained by the rigidity of the molecules, which prevents them from expanding considerably at low coverage. Conclusions
Our r-t'12 curves determined from ellipsometry show that the initial stage of adsorption of ovalbumin and lysozyme is well described by the simplified diffusion equation (3,at least for very low bulk concentrations. Adsorption proceeeds too fast at higher concentrations for us to follow the beginning of the process. In the later stages, equation ( 5 ) no longer holds, but this does not necessarily mean that the process is no longer diffusion-controlled. It has been demonstrated that, if the experimental y-t'/2 (or II-t''*) curve contains a linear region, the adsorption process need not necessarily be diffusioncontrolled. This is because such an interpretation would be based on the assumption that the surface equation of state was ideal [equation ( 6 ) ] , and our experiments indicate that ideality does not hold in any measurable range of surface pressure. We believe that any linearity in an experimental plot of In(dII/dt) versus II gives no indication as to whether or not there is an adsorption barrier at the surface. In fact, we think that any conclusion about relative rates of physical processes (diffusion, attachment, reconformation, erc.) taking place at or near the surface of a protein solution, which is based on an analysis of surface pressure measurements alone, should be treated with caution.
85
From our finding that the surface pressures of ovalbumin and lysozyme are unique time-independent functions of r, it follows that the time-scale of reconformation of these proteins after adsorption is less than about 10 minutes. But this statement is true only for those conformational changes that affect the surface pressure. References 1 F. MacRitchie and A.E. Alexander, J. Colloid Sci., 1963.18.453. 2 F. MacRitchie and A.E. Alexander, J. ColloidSci., 1963.18.458. 3 F. MacRitchie and A.E. Alexander, J. ColloidSci.. 1963,18,464. 4 D.E.Graham and M.C. Phillips, J. Colloid InrerJuce Sci., 1979,70,403. 5 F. MacRitchie, Adv. Prorein Chem., 1978.32,283. 6 M.C. Phillips, Chem. Ind., 1977, 170. 7 A.J.I. Ward and L.H. Regan. J. Colloid Inlerjiuce Sci., 1980,78,389. 8 M. Blank, B.B. Lee, and J.S. Britten,J. Colloid InferJkeSci., 1975,50,215. 9 E. Tornberg, J. Sci. Food Agric., 1978,29,762. 10 E. Tornberg, J. ColloidInter/aceSci., 1978,64,391. I I A.A. Trapeznikov, V.G. Vins, and T.Y. Shirikova, ColloidJ. USSR,1981.43.262. 12 T.Y. Shirikova. N.G. Volkova, and A.A. Trapeznikov, Colloid J. USSR. 1980,42,781. 13 V.A. Igoshin, S.M. Kuz'min, G.P. Bening. and G.P. Yampol'skaya. Colloid J. USSR, 1983,45,45. 14 H.B. Bull, J. Colloid Interfuce Sci.. 1972,41,305. I5 H.J. Rivas and P. Sherman, J. Dispersion Sci. Technol., 1984.5, 143. 16 J.A. de Feijter, J . Benjamins, and F.A. Veer, Biopolymers, 1978, 17, 1759. 17 J . Benjamins, J.A. d e Feijter, M.T.A. Evans, D.E. Graham, and M.C. Phillips, Furuduy Discuss. Chem. Soc., 1975.59.2 18. 18 H.A. Sober, ed., 'Handbook of Biochemistry', Chemical Rubber Co., Cleveland, 1960. 19 A.F.H. Ward and L. Tordai, J. Chem. Phys., 1946.14.453. 20 C. Tanford, 'Physical Chemistry of Macromolecules', Wiley, New York, 1961, Chap. 6. 21 M.R. Bruzessi, E. Chiacone, and E. Antonini, Biochemisrry. 1965.4, 1796. 22 L.W. Nichol, D.J. Winzor, and J.M. Creeth,J. Phys. Chem., 1960.64,1080. 23 J.R. Colvin, Con. J. Chem., 1952,30,831. 24 R. Miller, Colloid Polym. Sci., 1981,259,375. 25 B.J. McCoy, Colloid Polym. Sci., 1983,261,535. 26 K.J. Mysels, ColloidsSur/:, 1985,16,21. 27 J.M.G. Lankveld and J. Lyklema, J. Colloid Interfuce Sci., 1972,41,454. 28 H.B. Bull, Adv. Protein Chem., 1947.3.95. 29 T. Yamashita and H.B. Bull, J. Colloid lnrerfuce Sci.. 1968.27, 19. 30 A.F.H.Ward and L. Tordai, Recueil, 1952,71,572. 3 I F. MacRitchie. J. Colloid InrerJuce Sci., 1985.105, 119. 32 J.A. de Feijter and J. Benjamins, J. Colloid Inrerfoce Sci., 1982,90,289. 33 M. van den Tempel and E.H. Lucassen-Reynders, Adv. Colloid Inrerfuce Sci., 1983.18, 281. 34 A.F. Henson, J.R. Mitchell, and P.R. Mussellwhite, J. Colloid Inrerfuce Sci., 1970, 32, 162. 35 J.A. de Feijter and J. Benjamins, unpublished work. 36 D.E.Graham and M.C. Phillips, J. Colloid Interface Sci., 1979,70,415.
Properties of Adsorbed Layers in Emulsions Containing a Mixture of Caseinate and Gelatin
By ERIC DICKINSON, ANN MURRAY, BRENT S. MURRAY and GEORGE STAl NS BY (Procter Department of Food Science, University of Leeds. Leeds LS2 9JT)
Introduction The functional properties of emulsification and foaming are associated more with food proteins than with carbohydrates; and, ofthe proteins, it is casein which often occurs as the major macromolecular constituent of the interface.' Typically, the creation of an emulsion involves competition for sites at the oil-water interface amongst the various casein components (as,, /3, as*and K ) , and also between them and any other surface-active molecules arising from the presence of other food proteins, derivatives of component triglycerides (e.g., free fatty acids), and deliberately added emulsifiers. While much useful information has accrued over the years from mainly technological observations, a complete understanding of the formation and stabilization of food emulsions is still lacking. It is our view that only through experimentation on well-characterized systems will a full understanding be gained. Food proteins vary widely in their hydrophobicity and their structural organization in solution, and so no single study can aspire to answer all the questions. Nevertheless, a relevant and representative start towards reconciling structure and functionality can be made using a model emulsifying system of sodium caseinate commercial gelatin.* Gelatin is a polydisperse hydrocolloid. Its component macromolecules are highly flexible, like those of caseinate, but they are less hydrophobic. Whereas the casein components have significantly different chemical compositions and hydrophobicities, the gelatin macromolecules are all similar in these two respects, although they d o differ considerably in chain length and in the number of covalently-joined chains per molecule. The close compositional similarity of the gelatin polymers simplifies chemical analysis, and it allows a ready means of comparing the gelatin content at the interface with that in the initial bulk solution of caseinate gelatin. that there is a close correlation between Several papers have
+
+
86
87
emulsion stability and the surface rheology of the protein film at the oil-water interface, although the contrary view is occasionally aired.6 Whatever is the actual strength of the link between emulsion stability and surface rhcology, there is no disputing the fact that the latter isextremelysensitive to thedensity and structure of the macromolecular layer. This makes surface rheology a useful probe of timedependent structural and compositional changes in interfacial films adsorbed from mixed protein solutions. T o highlight any co-operative or competitive effects associated with the proteins, we use n-hexadecane as an inert oil phase in these experiments, since it is known'that polar lipids in food oils can have an influence on protein surface rheology. We have already published some results2on caseinate gelatin in emulsions and at a planar oil-water interface under neutral pH conditions whereeach component protein carries a net negative charge. Here, under similar solvent conditions, we examine changes that can become important during the processing or storage of an emulsion, when, for instance, new protein may become available for adsorption. In particular, we consider the case in which gelatin is first adsorbed and the more surface-active caseinate is made available later, since it is already known that caseinate will displace gelatin from a planar oil-water interface*." and will inhibit gelatin adsorption when both are present as during emulsification.y.'OThere is also the possibility of competition amongst the individual casein components themselves, the study of which requires the use of highly-purified individual proteins(especial1y aII. p and K ) . Sufficient quantities of the latter are available for surface rheological experiments, but are far too limited for making fine emulsions with conventional laboratory-scale high-pressure homogenizers. To overcome this problem, we have developed a mini-homogenizer which is described below.
+
Experimental Materials.-Gelatin was food grade (pl = 5.7) with a weight-average molecular weight of (2.39f0.15) X lo5 daltons." The spray-dried sodium caseinate ('Scottish Pride') was of low calcium content (0. I g kg-I). Individual caseins were the same as those used by Dickinson er 0 1 . ' ~The aqueous phase (0.005 M phosphate buffer, pH 7.0) was made from AnalaR grade reagents and doubly distilled water. The supermarket vegetable oil contained (0.040f0.004) wt% free fatty acid. and the glyceride composition was as follows: 62.4 wt% oleate, 2 I.7 wt% linoleate, 9.3 wt% linolenate, 3.9 wt% palmitate, with the balance from similar small levels of palmitoleate, eicosanoate, docosanoate, erucate and stearate. AnalaR grade nhexadecane (>99 wt%) was obtained from Sigma Chemicals. Emulsion Preparation.-Oil-in-water emulsions ( 10 wt% oil, 0.5 wt% total protein) were made by blending the components first in a Silverson homogenizer and then passing the blend through a modified two-stage APV Manton-Gaulin valve homogenizer (model 1 SM-8TA) or the mini-homogenizer (see below). To conserve materials, the volumes of the feed hopper and the dead spaces on either side of the valve in the APV homogenizer had been reduced, and the gearing between motor and piston had been changed to give a flow rate of 200 ml min-I, thereby enabling the smaller blend volume to be handled more conveniently. Nevertheless, even after
88
E. Dickinson. A . Murray, B.S. Murray and G. Siainsby
U
I
U
ball valve
Figure 1
Sketch of section through the mini-homogenizer (not drawn to scale)
modification, the capacity of the APV laboratory homogenizer is still too large in relation to the limited availabilty of some highly-purified individual food proteins, and so a mini-homogenizer was constructed (see Figure I ) to provide 10 ml of emulsion from 12 ml of pre-mix. In the mini-homogenizer, the sample of pre-mix fills a Nylon-lined cylinder (length 75 mm, diameter 16 mm) whose outlet tube is closed by a spring-loaded stainless-steel ball bearing (nominal diameter 5 mm). Driving the stainless-steel piston at constant speed forces the whole of the pre-mix sample past the valve in a single stroke under conditions of constant pressure difference (typically 300 bar). It was found that the spring-loading on the valve could be pre-set reproducibly. This means that, after cleaning, drying and re-assembling, the equipment can be filled and used without the need for trials with water to establish the desired pressure. This facility, in association with a very small dead space, conserves emulsifier and avoids contamination with priming water, a troublesome feature of the A P V homogenizer. Constancy of pressure throughout the full piston stroke, which is essential for reproducible homogenization, can be achieved only if the pre-mix contains a low level of entrapped air. It is important, therefore, to ensure that solutions of freeze-dried proteins have been degassed prior to blending.
89
The flow rate through the mini-homogenizer is about 40 times smaller than that through the modified APV equipment. (In the APV design, a single reciprocating piston drives each sample of pre-mix past the valve during one half of each cycle.) The soft cylinder (diameter I .9 rnm) into which the ball bearing protrudes in the mini-homogenizer is only ca. 8% of the area of the equivalent tube in the APV homogenizer. The gap across which the shearing action of the pressure differential occurs is narrower in the mini-homogenizer, and the flow pattern of the fluid as it passes through the annulus between ball and tube is likely to be very different from that in the gap between flat disc and tube in the APV homogenizer. Since theoretical predictions of homogenizer performance are notoriously unreliable, a direct experimental comparison of the two instruments is required. Analysis of Emulsions.-Droplet-size distributions were determined using a Coulter counter model TAll with a 30 pm orifice tube and 0.18 M sodium chloride as suspending electrolyte. The chemical composition of the interfacial layer was determined after collecting the droplets as acream by centrifugation at 2 X lo4 g for I hour, separating the cream from the aqueous phase, breaking it with acetone, extracting the oil with ether, and dissolving the residual protein in phosphate buffer. The gelatin content was determined spectrophotometrically using chloramine-T,13 and the total protein content was determined via a semi-micro Kjeldahl p r 0 ~ e d u r e . lProtein ~ solutions of known composition were included as standards in each analysis experiment. Allowance is made for the protein entrapped with aqueous phase amongst the droplets of the cream. In the exchange experiments, 10 g emulsion samples were mixed with either 20 g of buffer solution or 20 g of 0.5 wt% caseinate solution (pH 7.0) at known times following homogenization, and one hour later the mixtures were centrifuged and the cream analysed. Emulsions were kept at 25 "C until the cream had been separated. Interfacial Viscosity Measurements.-Surface viscosities at the interface between n-hexadecane and a solution of protein in 0.005 M aqueous phosphate buffer (pH 7.0) were measured using the Couette-type surface viscometer described p r e v i o u ~ l y .The ~ ~ protein concentration (10 wt%) was such that the ratio of available protein to surface area was at least 10 times that used commercially to make a typical fine food emulsion. Since only a few percent of total protein is adsorbed, diffusion from bulk to interface takes place at essentially constant bulk protein concentration. Except at the very earliest stages of film formation, the contribution of the bulk phases to the observed viscous drag is negligible. Measurements made over a range of rotation rates (4 X 10-4-7 X rad s I) and with various gap widths (6-57.5 mm) between inner disc (diameter 30 mm) and outer boundary have shown that caseinate films are Newtonian except at the highest shear rates, whereas gelatin films show extensive shear thinning. We therefore report apparent surface shear viscosities at a gap width of 57.5 mm and a rotation rate of only 1.2 X 10 rad s I to minimise disruption of the film. Some modification of the existing was required to follow the rheological changes accompanying protein exchange. With a glass tube (external diameter 8 mm) held permanently in the Couette gap near the outer dish boundary, an adsorbed film of gelatin was formed at the n-hexadecanel buffer interface. At the
90
E. Dirkinson. A . Murra,v. B.S. Murruy and G. Sminshy
appropriate age of the gelatin film, 2.5 ml of concentrated caseinate solution was added to the aqueous phase (375 ml) through the guide tube. Experiments showed that there was no measurable change in the viscous drag of the disc due to the small change i n position of the oil-water interface arising from the addition of the caseinate solution. Mixing was effected by sweeping the lower region of the aqueous phase with an L-shaped glass rod present throughout the experiment. Using a blue dye in the absence of protein, complete mixing was achieved with 20-30 sweeps over a period of 2- 3 minutes. Interfacial Tension Measurements.-The Wilhelmy-plate torsion balance has been described elsewhere.* Phase volumes and interfacial area are in proportion to those existing in the surface rheometer; in the exchange experiments, the same guide tube and the same concentration of caseinate solution were used. Although there was no
d/P Figure 2
Droplet-size distributions f o r individual emulsion batches made at 400 bar and 25 O C with 100 g vegetable oil and 5 g caseinate per kg emulsion in (a) the mini-homogenizer and (b) the A PV homogenizer. The volume-weighted size distributionfunction P, (arbitrary units) is plotted against the droplet diameter d. Each set of symbols n, A or A represents an independent run starting f r o m a separately prepared pre-mix
91
stirring, the rate of change of tension was rapid (see below). Interfacial pressures were calculated from the differences in the tension with and without protein. Results and Discussion First we describe the performance of the high-pressure homogenizers in making stable emulsions with either caseinate o r gelatin. Then we consider the changes in surface properties when caseinate is added to a system containing gelatin already adsorbed at the oil-water interface. Finally we compare the surface viscosities of the individual caseins at the same bulk concentration. For the modified A P V homogenizer and the home-made mini-homogenizer, Figure 2 shows the reproducibility in droplet-size distribution when aliquots from a single batch of pre-mix are homogenized at a nominal pressure of 400 bar. The
4
a 1
0
I
I
1
2
4
8
5
b 1
0
Figure 3
I
I
I
I
1
2
4
8
Effect of homogenization pressure on droplet-size distributions of emulsions made at 25 O C with I00 g vegetable oil and 5 g caseinateper kg emulsion in (a) the mini-homogenizer and (h) the A P V homogenizer. The volume- weighted size distribution function P, (arbitrary units) is plotted against the droplet diameter d: A. 300 bar; B. 400 bar. A verages over several runs ( 1
E. Dickinson. A . Murray, B.S. Murray and G. Stainsby
92
mini-homogenizer gives satisfactory reproducibility, but its casein-stabilized droplets are slightly smaller, on average, than those produced by the APV machine. Similar reproducibility is seen with gelatin-stabilized emulsions (not shown) and at other operating pressures. For all the experiments reported here, the APV homogenizer is operated with a second-stage pressure of 40 bar; with one-stage homogenization, the most-probable droplet diameter is increased by ca. I pm. Figure 3 shows that the droplet-size distribution of casein-stabilized emulsions becomes slightly broader when the pressure is reduced from 400 bar to 300 bar in the mini-homogenizer, whereas it is unchanged in this range for the APV homogenizer. A significant dependence of droplet size on pressure is found when the mini-homogenizer is used to prepare gelatin-stabilized emulsions (Figure 4). but here again there is little pressure dependence with the APV equipment. Overall, we observe that either homogenizer serves equally well for caseinate emulsions; with gelatin as emulsifier, however, the mini-homogenizer can provide finer emulsions.
5
a 1
a 1
2
8
4
5
b 1
a
I
I
I
I
1
2
4
8
d/P Figure 4
Effect of homogenization pressure on droplet-size distributions of emulsionsmadeat 25 O C with 100 g vegetableoiland5 ggelatinper kg emulsion in (a) the mini-homogenizer and (b) the A PV homogenizer. The volume-weighted size distribution function P, (arbitrary units) is plotted against the dropIet diameter d: A, 300 bar; B, 400 bar. Averages over several runs ( 1
93
I t is found that the composition of the interfacial layer is insensitive to the type of homogenizer used to make the emulsion. An emulsion made with 0.5 wt%gelatin in the APV homogenizer (400 bar) had 12.0 mg protein per g oil at the interface, while the value was 11.2mgg-' oil for the same premix put through the minihomogenizer (300 bar); in each case the experimental precision was ca. 0.7 mg g-l. Small differences in the amount of adsorbed protein are in keeping with small differences in surface area (see Figure 4). The data in Table 1 show that the total protein content at the interface is essentially constant, and that the use ofgelatin to monitor surface composition in earlier s t ~ d i e s was ~ . ~ justified. The value of I 1 mg g-l oil is similar to that given previouslyZ for a pure gelatin emulsion made with the mini-homogenizer, but significantly different from that found in preliminary experiments9 with the APV homogenizer. The difference is attributable, we believe, to a combination of factors: changes in homogenization temperature and the thermal history of the premix, and changes in details of procedure for separating and breaking thecream, and correcting for the amount of unadsorbed protein entrapped amongst the droplets. Table I shows that the amount of protein associated with the interface barely increases over a period of three days, even though over three-quarters of the initial protein remains in the bulk aqueous phase. These figures d o not support the view that strongly-bound multilayers form in aged emulsions. Exploratory experiments had shownZthat interfacial gelatin in a freshly made emulsion is readily replaced by caseinate added to the continuous phase. Figure 5 extends this observation by demonstrating that the ability toexchangediminishes if the interfacial gelatin film is allowed to age before caseinate is made available. In this set of experiments, exchange was permitted for I hour at 25 O C , and then the emulsion was creamed by centrifugation and subsequently analysed. During the exchange the total protein content was kept at 5 g kg-I emulsion with caseinate and gelatin in the ratio 2:1 by weight. When both proteins are available in this proportion at the time of homogenization, the interfacial protein layer in the emulsion I hour old is devoid of gelatin.2 Figure 5 shows that, when an emulsion is made solely with gelatin and then added to a caseinate solution immediately afterwards, the mixed film still contains ca. 8% gelatin. N o change in surface
Table 1
Totalamount ofprotein at interface (Pi) in emulsions made at 300 bar in a mini-homogenizer with protein emulsifier of caseinate concentration C, andgelatin concentration C, as measured (a) I hour and (6) 72 hours ajier emulsijication pi/ mg
g-' oil
C,/ wt%
CJ wt%
(a)
(b)
0
0.5
11.2
11.4
0.25
0.25
10.6
-
0.5
0
10.2
11.4
E. Dickinson, A. Murray. B.S. Murray and G. Siainsby
94
1
0.8 fG
0.6
0.4
0.2
0
0
10
20
30 fag&
Figure 5
Change in interfacial protein cornposition I hour after adding caseinate to an emulsion stabilized by gelatin. Thefraction of gelatin displacedfrom the interface (fJ is plotted against the time of aging of the emulsion (tage)before addition of caseinate at 25 O C
composition has been found when emulsions are diluted with buffer alone. This is consistent with the view that macromolecular adsorption is irreversible to dilution on a practical time-scale, provided the solvent remains unchanged (change in pH or ionic strength may lead to desorption). When exchange is permitted for a period longer than I hour, more of the interfacial gelatin is replaced. A 24-hourexposure, for example, reduced the interfacial gelatin content from 90% to 50% in an emulsion that had been previously aged for 24 hours. Displacement of adsorbed gelatin by caseinate should be readily revealed by an increase in surface pressure. Previously, we showed* that films adsorbed from solutions of caseinate 4- gelatin develop the high surface pressure of pure caseinate in a few hours, even when as little as 5% of the total protein is caseinate. Figure 6 confirms that exchange readily occurs when caseinate solution is injected under an aged gelatin film, as suggested by Mussellwhite* many years ago. While the surface pressure data indicate that caseinate dominates the interfacial region, the surface viscosity results in Figure 7 show that the actual situation may be more complicated than a simple total displacement. When caseinate is injected under an aged gelatin film to give a total protein content of 2 X wt% and a I: 1
95
protein mixture, the apparent surface viscosity is at first much reduced, but then subsequently recovers. The longer the film has been aged before adding caseinate, the more rapid is the recovery. This result is not too unexpected, given the findings already reported* for films formed from solutions initially containing both caseinate and gelatin. Substitution of some of the interfacial gelatin by caseinate presumably weakens those co-operative interactions in the film that are responsible for the viscoelastic behaviour. What is perhaps more surprising is the subsequent strengthening of a mixed film to give one which, although apparently becoming rich in caseinate, closely resembles a pure gelatin film rheologically. The film viscoelesticity appears insensitive to the immediate phase-boundary region, which may be devoid of gelatin, and to be determined by interactions in the aqueous phase between macromolecular loops and tails, which are probably mainly gelatin. This interpretation is illustrated schematically in Figure 8. An alternative explanation
30t
c
c
E
25
z
E
\
E
20
15
10
5
0 0
4
8
12
t/hr Figure6
Change in sugace pressure n with time t following addition of caseinate to a 24-hour old gelatin film at the planar n-hexadecanel buffer interface (pH 7.0, 25 "C). Dashed lines C and G refer respectively to steady state pressures for pure caseinate and pure gelatin at bulk concentration tO-'wt% and the same solvent conditions
96
E. Dickinson. A. Murray, B. S. Murray and G. Stainsby 0.3
I'
.u)
E
>
0.2
0.1
C 20
40
60
80
t/hr
Figure 7
Change in apparent surface shear viscosity 7 with time t following addition of caseinate to a gelatinfilm aged for 24 hours (0)and 72 hours ( 0 )at theplanar n-hexadecanelhuffer interface (pH 7.0,25 "C). Five-point stars indicate the exact points of caseinate addition. The dashed line denotes the time-dependent surface viscosity of pure caseinate at hulk concentration 10 wt% and the same solvent conditions
for the increasing surface viscosity with time would involve gradual multilayer formation through incorporation of gelatin, which is well known to aggregate and gel in aqueous solution. However, the inability to remove interfacial protein by dilution and the time-independent composition of films around droplets in emulsions suggests that strongly-bound multilayers are not formed. Despite the extensive rearrangements that must occur at the oil-water interface following addition of caseinate, there is no accompanying change in emulsion droplet-size distribution. Samples taken at various times up to 24 hours after adding caseinate were all indistinguishable from the original emulsion as indicated by the Coulter counter. The large changes in surface composition and viscoelasticity evidently have no effect on emulsion stability. Caseinate no doubt competes equally successfully with other proteins at the oil-water interface. In aqueous solution most food proteins are highly structured (i.e.,globular), and some give adsorbed layers that are more viscous than gelatin films.2 I t remains to be seen whether they can also dominate the rheological properties of mixed films involving casein, or whether gelatin, with its unique gelling behaviour, is a special case. We turn finally to the surface rheology of the individual caseins. Figure 9 shows
91
time-dependent surface viscosities of as,-, p- and K-caseins adsorbed from bulk solutions of wt% protein in each case (sufficiently dilute to be free from aggregatesI6). The most hydrophobic component, p-casein, gives by far the weakest film. Since a,,-casein gives a film that is also less viscous than caseinate, it would seem that the minor components, including K-casein and some entrapped whey proteins, make a major contribution to caseinate surface rheology. At this stage, however, we are not in a position to resolve completely the individual contributions to caseinate surface rheology; this will require studies of mixed films of known composition with careful attention paid to all the factors that affect intermolecular association of these proteins. Meanwhile, current results lead us to believe that the surface properties of commercial caseinate are likely to be sensitively dependent on the whey protein content and on the extent to which K-casein has been changed during milk storage and processing.
1
0 S
2
Figure 8
Schematic representation of protein configurations in a mixed f i l m containing low-molecular-weight casein molecules and high-molecular-weight gelatin molecules. Pictures I and 2 show situations immediately before and after penetration by the more hydrophobic casein components (C) into an aged gelatin layer (G) ad.vorbed at the surface (S) between bulk oil phase (0)and bulk aqueous phase (W). Region I of the composite adsorbed layer mainly contributes to the surfacepressure; region I1 mainly contributes to the surface rheology
E. Dickinson. A . Murray, B.S. Murray and G. Stainsby
98
.
20
0
30
40
t/hr Figure 9
Surface viscosity of caseinate. aS,-casein,p-casein and K-casein at the n-hexadecanelbuffer interface (pH 7.0,25 "C) at a totalprotein bulk concentration of lo-' wt%. The logarithm of rhe apparent surfoce shear viscosity is plotted against time I : e, sodium caseinate; 0, as,casein; V, p-casein; A , K-casein
Acknowledgements. We acknowledge technical assistance from Mr. P. Nelson in designing and constructing the mini-homogenizer, and financial support from the Chief Scientist's Group at the Ministry of Agriculture, Fisheries and Food. The results are the property of the Ministry and are Crown Copyright.
References I P.J. Halling, CRCCrit. Rev. FoodSci. Nutr., 1981, 15, 155. 2 J . Castle, E. Dickinson, A. Murray, B.S. Murray, and G. Stainsby, in 'Gums and 3 4
5 6 7 8 9
Stabilisers for the Food Industry,' ed. G.O. Phillips, D.J. Wedlock, and P.A. Williams, Elsevier Applied Science, London, 1986. Vol. 3, p. 409. B. Biswas and D.A. Haydon, Proc. R. Soc. London. Ser. A , 1963,211,296. J.V. Boyd, C. Parkinson, and P. Sherman, J. Colloid Interface Sci.. 1972,41,359. H.J. Rivas and P. Sherman, ColloidsSurj, 1984.11, 155. M.C. Phillips, Food Technol., 1981,35,50. G. Doxastakis, 'The interactions of glycerides with proteins and their influence on the rheological properties of o / w emulsions,' Ph.D. Thesis, University of London, 1983. P.R. Mussellwhite, J. Colloid Interface Sci., 1966,21,99. S.M.Chesworth, E. Dickinson, A. Searle, and G. Stainsby, Le6ensrn.- Wiss. u. - Technol., 1985, 18,230.
99 10 E. Dickinson, D.J. Pogson, E.W. Robson, and G.Stainsby, ColloidsSurf., 1985.14, 135. I I E. Dickinson, W.L.-K. Lam, and G . Stainsby, Colloid Pulym. Sci., 1984,262,Sl. 12 E. Dickinson, R.H. Whyman, and D.G. Dalgleish, this volume, p. 40.
13 H.Stegemann and K . Stadler, Clin. Chim. Aclo, 1967.18.267. 14 R.J.A. Grand, "-Terminal imino-acids of gelatin - occurrence and estimation,' Ph.D. Thesis, University of Leeds. 1972. 15 E. Dickinson, B.S. Murray, and G. Stainsby, J . Colloid lnterjhce Sci., 1985,106,259. 16 D.G. Schmidt, in 'Developments in Dairy Chemistry,' ed. P.F. Fox, Applied Science, London, 1982,Vol. I. p. 61.
The Role of Proteins in the Sta bilization/ Destabilization of Dairy Foams
By M. ANDERSON, B.E. BROOKER and E.C. NEEDS (AFRC institute of Food Research. Shinfield, Reading. Berks. RG2 9AV
Introduction An overall concept of the structure of whipped cream and how it develops has been given by a number of authors.'-6 In essence, this concept considers that air bubbles are held in a three-dimensional matrix of partially coalesced fat globules. Some have interpreted this to imply that bubbles are completely surrounded by a continuous layer of fat. In normal pasteurised dairy cream, the oil-water interface before whipping consists of the natural milk-fat globule membrane (MFGM) whose morphological appearance has been described by Wooding7 and whose composition has been the subject of numerous publications, summarized most recently by McPherson and Kitchen." Previous morphological studies on the structure of whipped cream have not attempted to follow the interfacial changes that accompany the whipping process. It has been generally accepted that fat composition exerts a dominant influence on the quality of whipping cream. Since the morphology of the MFGM is known to vary and to undergo changes after ~ e c r e t i o nit, ~is possible that the nature of the interfacial layers, before, during, and at the end of whipping, has an influence on foam characteristics. Darling has suggestedSthat the structure of the oil-water interface may be important in determining the whipping performance of homogenized dairy creams. Materials and Methods Creams.-The creams used in the present work were prepared by separating milk from either the Institute Farm's bulk tank or from a commercial silo. Samples were standardized to 38 wt% fat, pasteurised, conditioned for 24 hours at 4 "C, and then whipped as described by Scurlock9using an apparatus based on the design of Mohr and Koenen.lo Stiffness was measured using the Instron Universal Food Tester. Electron Microscopy.-Samples of partially and fully whipped creams were vapourfixed with formaldehyde according to the method of Graf and Muller.' They were I00
101
subsequently prepared for examination by transmission electron microscopy (TEM) in a Hitachi 600 electron microscope using the procedure described by Brooker and Anderson." Samples of whipped creams were cryo-fixed in nitrogen slush and examined in a Philips 505 scanning electron microscope (SEM) fitted with a Hexland freezing stage and cryo-transfer device. Unwhipped creams were fixed in glutaraldehyde and prepared for TEM by the method described by Hobbs.'* Analysis of Air-Serum Interface.-Air bubble ghosts were prepared from skim milk and milk plasma according to the procedure of Brooker," and their protein composition determined by polyacrylamide gel e l e c t r o p h ~ r e s i s . Skim ~~ milk subjected to aeration was fixed with glutaraldehyde (3% w/v) and examined by TEM as described previously.') Results and Discussion The air-serum interface in skim milk consists of a layer of electron-dense material 5 nm thick, with casein micelles attached to the serum side of the interface (Figure I a). Examination of the morphological appearance of ghosts fixed 4 hours after foaming (Figure Ib), rather than immediately after (Figure la), showed the apparent association of casein micelles with the interface to be reversible. It is concluded that attachment of casein micelles to the air-serum interface is a
Figure 1
(a) Air-serum interface (I) in a foam from pasteurised skim milk showing attached casein micelles ( C ) .(b) Bubble ghosts inpasteurised skim milk I hour after$oamingshowingdissociation of casein micelles
102
M. Anderson. B. E. Brooker and E. C. Needs
secondary occurrence; they should not be considered as a primary component of the interface. When ghosts were prepared from milk plasma, it was found that the air-serum interface had characteristics identical to those found with skim milk but without the attachment of casein micelles. Analysis of the composition of ghosts isolated from milk plasma showed that the major protein constituents of the interface are /.?lactoglobulin, a-lactalbumin and /.?-casein. Quantitatively, the /.?-casein was present at a higher relative concentration than that found in skim milk, which suggests that it is preferentially adsorbed at the interface. The oil-water interface of the cream before whipping showed the typical primary and secondary MFGM components described by Wooding.’ These features were absent, however, in vapour-fixed specimens, where the fat globule was bounded by a single electron-dense layer, whose surface details were obscured by deposits of osmium-derived material arising from the prolonged fixation procedure (Figure 2). Examination of cream made from Institute Farm milk at a stage representing 25% of the total whipping time showed individual fat globules adsorbed at the air-serum interface, so that part of the fat was in direct contact with the air and partly protruding into the air cell (Figures 3 and 4). These observations are consistent with previous ~ o r k .Electron-dense ~.~ particles could be observed at the fat-airjunction, as shown in Figure 4, and these may represent vestiges of the MFGM, which is partly lost during fat-globule adsorption according to Mulder and Walstra.2 Remnants of primary MFGM could be seen in the aqueous phase of the cream. These observations support the work of Buchheim’ who showed by freeze-fracture TEM that part ofthe MFGM in contact with air is removed duringfat adsorption.
Figure 2
Fat globules in pasteurised cream. Sample vapour-fixed before whipping showing how. osmium-derived deposits (D) obscure interfacial details
I03
Figure 3
Adsorption of fat globules (G)to the air-serum interface (1) showing direct contact between fat and air. The air-serum interface is continuous with the residual M F G M ( M ) on the aqueous boundary of the globules
Figure 4
The air-far-aqueous boundary in partly whipped cream showing electron-dense particles (P) at the fat-air junction which could represent M F G M material disrupted at the time of globule adsorption
I04
M.Anderson, B. E. Brooker and E. C. Needs
In Figures 3 and 4 an air-serum interface, identical in appearance to that in skim-milk and milk-plasma foams, is clearly evident between individually adsorbed globules; it is continuous with the oil-water interface of the globules. There was little evidence to suggest that the nature of the residual MFGM is different from that in the unwhipped cream. It was noted that casein micelles were closely associated with the MFGM in globules from the serum phase of the cream (see Figure 5), suggesting that in the shear field some adsorption of serum proteins may take place. However, when fat globules in partially whipped cream were conventionally fixed in glutaraldehyde, it was evident that some primary membrane had been retained at the oil-water interface (Figure 6). It is intended to confirm serum protein adsorption using surface labelling techniques. In the completely whipped cream, the periphery of each air bubble consists not only of single and coalesced fat globules but also of a variable amount of the original air-serum interface, as shown in Figure 7. There was no evidence of liquid fat filling the spaces between adjacent fat globules as suggested by The structure was confirmed by examining cryo-fixed and fractured samples by S E M (Figure 8), where the continuous area between the protruding fat globules inside the air cells represents the air-serum interface. A similar feature is evident in the micrographs presented by Buchheim.’ Since the foam matrix consists principally of coalesced fat with fat bridges between adjacent ’air bubbles (Figure 8), it is reasonable to assume that the residual air-serum interface at the boundary of the bubble does not have a significant effect on foam stability. It is possible, however, that the properties of whipped cream may be influenced by the composition of the
Figure 5
Appearance by TEM of fat globules in vapour-fixedpartly whipped cream showing possible attachment of casein micelles (C) to the MFGM (M)
I05
Figure 6
Appearance by TEM offat globules in glutaraldehyde-jixed partly whipped cream showing the retention ofprimary M FGM (M)
Figure I
Structure offully whipped cream examined by TEM. An air bubble is held in a matrix ofpartly coalescedfat globules ( G ) with remnants of air-serum interface (I) present between adsorbedglobules
I06
M. Anderson, B. E. Brooker and E. C. Needs
Figure8
Structure o$$ully whipped cream examined by SEM. The inner sur-ace ofthe air bubble consists of a continuous air-serum interface ( I ) through which individualfat globules ( G )protrude
Table 1
Whipping characteristics of stable and unstable creams madefrom the same bulk sample of milk Overrun1 %
Stable cream Unstable cream
Whipping time1 s
Stij/ness
84
I18
I I9
103
98
78
serum phase in the unwhipped cream and the consequent nature of the air-serum interface in the early stages of aeration. Structural studies were carried out to determine what interfacial changes accompany the tendency of dairy cream to collapse after whipping. Milk from a commercial dairy was separated at the dairy and at the Institute, and both samples of cream were pasteurised at the Institute. Cream separated at the commercial dairy collapsed within an hour of whipping, whereas cream prepared at the Institute under the same conditions was stable. Table 1 shows acomparison of the whipping properties of the two creams; quoted values are averages from three different whippers. We see that there is comparatively little difference between the creams: the unstable cream was found to have a slightly shorter whipping time, a lower stiffness, and a higher overrun than the stable cream. Examination of these samples by T E M showed that the morphological features of the M FGM of the stable cream
107
Figure 9
Structure of unstable cream before whipping examined by TEM. (a) Globules show considerable coalescence and an atypical MFGM structure with almost no primary membrane. (b) Some globules are surrounded by folded membrane (F)
before whipping were indistinguishable from those in other control creams corresponding to the Wooding model? There was no manifestation of emulsion instability, and after whipping the stable cream had the same appearance as that shown in Figures 7 and 8. I n the unstable cream, on the other hand, there was observed clustering, clumping, and some coalescence (Figure 9a). Appearance of material at the globule surface was consistent with the secondary M F G M of Wooding;’ unit-membranebound primary membrane was only rarely observed. I n addition, some globules were bounded by a folded layer (Figure 9b). This i s interpreted to represent a stage i n the process involving the shedding of excess interfacial material following globule coalescence. Clearly, the emulsion in the sample was unstable, a condition which could have resulted only from a difference in the performance of the commercial and Institute separators. The nature of this difference was not identified. Differences in fat aggregation between stable and unstable creams was again highlighted’in SEM studies of the overall structure of the whipped creams. The stable sample displayed features typical of those previously described for pasteurised cream (Figures 7 and 8). Fat globules were seen to retain a spherical outline, and gross coalescence of fat was not observed. There were concave depressions on the surface, indicating the positions of globules removed at the time of fracture. I n Figure 10 the structure of the unstable cream conforms to the same general pattern, but the fat globules appear as aggregates rather than distinct spheres, and their size i s larger than seen i n the stable sample. Furthermore, depressions were absent from the fracture surface of the unstablecream, and it was
I08
M. Anderson. B. E. Brooker and E. C. Needs
Figure 10
Structure of unstable whipped cream by SEM. Fat globules ( G ) are nor spherical, and have undergone considerable aggregation. Concave depressions are absent from thefractured surfoce. The aqueous phase is nor differentiated
Figure11
Appearance by SEM of unstable cream cryo-fixed 1 hour after whipping showing gross aggregation offar
I09
difficult in the micrographs to distinguish between fat and aqueous phases. When the unstable cream was cryo-fixed I hour after whipping and then examined by SEM, it was found that a transformation of the fat matrix had occurred as shown in Figure I I . We see that definition of individual globules is obscured, and it appears as if the remaining air bubbles are separated by an almost continuous network of fat. The instability of this foam made it difficult to identify by TEM any of the interfacial changes that might have been relevant to the process of collapse. The process of whipping cream can be considered as a controlled form of churning. But, at the temperature normally used for cream conditioning and whipping (4-6 "C), the solid-fat index is sufficiently high to prevent the high rate of air-bubble collapse which occurs in churning as a result of interfacial spreading of liquid fat. Before whipping, the fat globules of the unstable cream had an atypical M FGM and were partially clumped, factors that would suggest an increased rate of clumping during churning. Mulder and Walstra2 have indicated that altered membrane composition may be an important factor in affecting the clumping rate during churning. But it seems unlikely that changes in clumping rate alone could account for the observed foam instability, since an apparently normal structure (in terms of fat-air relationships) and a transiently stable foam were observed. In the absence of any conclusive evidence, the mode of foam collapse is a matter of speculation. Our results d o indicate, however, that the nature of the MFGM before whipping is one of the factors influencing the susceptibility to collapse of whipped, pasteurised, unhomogenized, dairy creams. References I E. Graf and H.R. Muller. Milchwissenschafl, 1965,20,302. 2 H.Mulder and P. Walstta,'The Milk Fat Globule,' Pudoc. Wageningen, 1974. 3 W. Buchheim, Gordian, 1978,78,184. 4 D.G.Schmidt and A.C.M. van Hooydonk, Scanning Elecrron Microsc., 1980,111,653. 5 D.F. Darling, J. Dairy Res., 1982,49,695.
6 7 8 9 10
II 12 13 14
R.J. Birkett, Proc. Inr. Cong. FoodSci. Tech., 1983.2, 149. F.B.P. Wooding, J. Ulrrastr. Res.. 1971.37,388. A.V. McPherson and B.J. Kitchen, J . Dairy Res., 1983.50, 107. P.G.Scurlock, 'Whipping cream: effect of varying the fat and protein contents on functional properties,' M.Phil. Thesis, University of Reading, 1983. W. Mohr and K . Koenen, Deursche Molk. Zeir., 1953,74,468. B.E. Brooker and M. Anderson, Food Microsrrucrure, in the press. D.G. Hobbs, Milchwissenschaff, 1979,34,201. B.E. Brooker, FoodMicrosrrucrure. 1985,4,289. A.T. Andrews, J. Dairy Res., 1983,50,45.
The Formation and Breakdown of Protein-stabilized Foams
By DAVID C. CLARK, JIM MINGINS, FRANCES E. SLOAN, LINDA J. SMITH and DAVID R. WILSON
(AFRC Institute for Food Research, Colney Lane, Norwich NR4 7UA)
Introduction
The foaming properties of proteins are clearly related to their ability to form adsorbed layers at the air-water ( A / W ) interface. But, just as with simple surfactants, there is no ready correlation between surface activity and foamimg properties. Foamability is governed by adsorption behaviour over very short time-scales. The foamability of proteins is usually less than that for conventional surfactants because their macromolecular nature ensures a much slower response to the creation of new surface. The surface stresses attendant on interfacial movement are not readily relieved by proteins, and this results in an inherent instability in the thin films formed at bubble contacts in the initial stages of foaming. Nevertheless, depending on the nature of the protein, its concentration, and the solution conditions, the rigours of these initial stages can be combatted and persistent foams obtained with stabilities often far in excess ofsurfactant foams. To understand this long-term stability, it would seem essential to know the surface density of adsorbed protein, the conformational changes arising from its adsorption, and the nature of any interfacial association. As far as we are aware, adsorption measurements in protein foams or in model microscopic thin films have not been reported. Instead, it has been common practice to use a macroscopic A / W interface, and to call on measurements such as surface radio-counting or ellipsometry, whose congruence has not yet been established. Under certain conditions, shaking a protein solution gives surfaceinduced coagulation; the mechanism for this is not clear. The gradual onset of surface association has been suggested to explain the long timedependence of the surface pressure of adsorbed protein monolayers. Speculations about the role of protein conformational changes in adsorption and in film stability have frequently been made, but singularly few studies have addressed the actual problem of measuring interfacial structure. In this paper, we report on an indirect analysis of surface-contacted protein. Pending development of insitu methods of examining interfacial conformations at 110
the A/ W interface, we use the device of collecting a sufficient quantity of foam to obtain enough protein sample to study protein conformation in bulk solution by spectroscopic methods. By this means, any irreversible changes brought about by the presence of the foam interface will come under scrutiny. At this stage, we are interested primarily in whether or not such changes d o occur. In parallel foaming experiments, attempts are made to assess surface concentration and to check the concentration-dependence of foam stability using a conductimetric method. Standard surface pressure measurements at a macroscopic A/ W interface are also followed in order to relate to previously published work. One of our main interests is the foaming behaviour of mixtures of proteins, particularly those from the egg-white system. The basic protein lysozyme is involved in this system, and, in contrast to other egg-white proteins, it is a poor foamer, though it can confer long-term stability. However, before embarking on a programme to investigate mixing effects, we felt that the foaming and interfacial behaviour of single proteins had first to be established, and this paper describes our initial exploratory measurements in this area. We focus here o n lysozyme, and, in the absence of pure samples of ovalbumin, the standard model protein bovine serum albumin (BSA). Because of time constraints prior to this conference, we have only been able to study the foaming and conformational behaviour of BSA, and some limited surface pressure behaviour of lysozyme. More extensive studies are in hand. Experimental Materials.-Fatty acid and globulin free BSA (A-7030) was purchased from Sigma Chemicals. High-molecular-weight contaminants detectable by polyacrylamide gel electrophoresis (presumably dimerized BSA) were removed by gel filtration over Sepharose 6B-CL. The column was equilibrated and run in de-ionized and distilled water. Three-times crystallized lysozyme (L-6876) from chicken egg-white was obtained from Sigma Chemicals. Polyacrylamide gel electrophoresis showed it to be free of protein contaminants, and it was used without further purification. All other chemicals were BDH AnalaR grade. Experiments were carried out at room temperature unless specified otherwise. Surface-chemically pure water was prepared by passage of borehole water through consecutive beds of anionic and basic ion-exchange resins, and then through a mixed-bed resin, prior to batch distillation from alkaline permanganate in a 50-litre steam-heated still. Surface tensions of 72.9f0.I mN m-I at 20.0f0.1 "C were recorded, and no significant aging was discerned. No cleaning routines were instituted for the buffer salts used throughout the work, and as aconsequence there was a slight lowering of the surface tension (ca. 0.1 mN m-I in 0.1 M solution). Adventitious contamination of the borehole supply by phthalate esters in the latter stages of the work coincided with a reduction in foam stability and necessitated the short-term expedient of using ion-exchange water in the foaming runs. (There is a clear need to repeat these runs at the earliest opportunity.) Surface Tension and Surface Pressure.-Surface tensions and surface pressures were measured by the Wilhelmy plate technique using the null-buoyancy and
I12
D. C. Clark, J . Mingins, F. E. Sloan. L. J . Smith and D. R. Wilson
dipping-plate methods, respectively, as described in a previous paper.’ Forces were monitored by a Beckman electronic microbalance, any time-dependence being followed by means of a potentiometric recorder. To obviate contact-angle problems from protein adsorption, the glass cover slips used for the frequent checking of the surface tension of water were replaced by paper plates in the surface pressure measurements. The paper plates were accurately cut from Whatman No. I paper, suspended from an aluminium support, cleaned by soaking in ethanol and rinsing copiously with distilled water. Plates were stored under water when not in use. Dimensions of wet plates were used in the calculation of surface pressure. The procedure for monitoring changes in surface pressure due to protein adsorption was as follows. A known volume of water (or aqueous electrolyte of interest) was added to a double-walled glass dish thermostatted to within fO.I O C by water circulating through the outer chamber. The dish stood on a magnetic stirrer and contained a cleaned PTFE-coated stirrer bar. Once thermal equilibrium had been established, the paper plate was suspended from the microbalance and lowered until its bottom edge was 1-2 mm below the A/ W interface. Provided the surface tension forces on the plate were constant with time, as judged by the recorder trace, an accurately known aliquot of stock protein solution was added to the aqueous phase after the plate had been raised the appropriate amount t o compensate for any buoyancy change. The solution was then stirred for 5 seconds at a speed moderate enough not to dislodge the plate. (It had already been established, using a conductivity probe, that stirring under identical conditions ensured a homogeneous distribution of an equivalent volume of electrolyte within this period.) Surface pressure changes were then followed directly with the recorder. With this method, adsorption times o f < l 5 s were not accessible, and this precluded assessment of the initial slopes of the surface pressure Il versus time t curves for moderate-to-high protein concentrations. In an alternative method, the protein solution of interest was added to a clean PTFE Langmuir trough until the A/ W surface was just proud of the walls. The paper plate was again partially immersed, but this time trapped between two adjacent PTFE barriers laid across the top of the trough. Moving the barriers apart created new clean surface, and the surface pressure was then monitored in the usual way as the protein re-adsorbed. Apart from giving access to adsorption times of 5 s, this method also enabled the Il-t plot to be repeated on the same solution as often as desired merely by sweeping away any adsorbed protein layer. In addition, adsorbed monolayers at any point on the time plot could be compressed or expanded to check for any adsorption/ desorption exchange. Foaming.-The method used was that of Wallinget al.* as modified and described by Weil.’ White-spot nitrogen was bubbled through a known volume of protein solution via a sintered-glass frit (porosity 17-40 pm) at a constant rate (typically 22-23 cm’ min-I). No attempt was made to pre-saturate the gas with water vapour. Having passed up a I m glass column, the foam was collected by collapsing it under a known weight of20 vol%ethanol/water mixture. A run was timed from thestart of collection until typically 5-10 g of foam had been collected. The initial and final protein concentration in the frit chamber, and the protein concentration in the ethanolic solution, were measured spectrophotometrically. Using the freeze-frame
I I3
facility on a video camera (magnification X8.5),and a film sequence of the foam near the top of the column, bubble sizes were estimated from the diameters of over 100 polyhedra near the walls of the column. No corrections for wall effects were applied to the size distribution. The whole apparatus was scrupulously cleaned after each run t o remove adsorbed protein. This entailed an initial rinse with water, followed by soaking with 5 M hydrochloric acid, washing with warm detergent solution, and then copious rinsing with water. Foam Stability.-Drainage and stability of the foams was assessed by a conductimetric method. The cell was a modified version of that described by Kato et uL4 It w a s constructed from a Perspex cylinder containing three pairs of silverplated electrodes at different heights up the column. At the base of the cylinder is a flange which bolts to a base-plate, and sandwiched between these two components is a sintered glass frit. All joints were made gas-tight with a series of rubber O-rings and Neoprene gaskets. Typically, 10-15 ml of protein solution at the appropriate concentration in 0. I M phosphate buffer was pipetted into the conductivity cell. Oxygen-free nitrogen was sparged through the cell at cu. 50 ml min-' until the body of foam had reached the top of the cell. The gas supply was then shut off, and data were collected for 10 minutes. This was done by on-line analogue-todigital conversion using a BBC microcomputer which sequentially interrogated each of the three pairs of electrodes. Semi-logarithmic plots of the data did not produce straight lines, but it was found that the curves could be qualitatively analysed by fitting the time-dependent signal A(t) to adouble-exponential equation of the form
A(t) = A, - A,exp(-k,t) - A,exp(-k,t)
(1)
where A,, A,, A,, k, and k, are constants obtained by non-linear least-squares analysis.s Protein Conformation.-Conformational properties of protein recovered from the foam were investigated by the techniques of intrinsic fluorescence and circular dichroism. Protein samples were obtained from the apparatus used for the surface concentration measurements, but with protein recovered by bubbling the drained protein-stabilized foam through water o r phosphate buffer rather than 20 vol% ethanol/ water solutions. Tryptophan fluorescence of BSA was measured by excitation at 295 nm using a Perkin-Elmer LS-5luminescence spectrometer with 2.5 nm slits and a semi-micro fluorescence cell. Care was taken to adjust the optical density to minimize inner filter effects. The data were digitized on-line using a BBC microcomputer. Circular dichroism spectra were recorded on a Jasco 5-4 1 C spectropolarimeter equipped with a J-DPY data processor.6 Fused silica cells of pathlength 1 mm were used to record spectra in the far-UV region (190-260 nm), and pathlengths of 10 mm or 40 mm were appropriate for spectra in the near-UV region (255-340 nm). Spectra were recorded at a sensitivity of 1-2 millidegrees per cm, and were averages of four scans with an instrument time constant of 4 s. The data are presented in the form of molar circular dichroism AE based on a mean amino-acid molecular weight of I 10. The mean residue ellipticity is equal to 3.3 X lo3At. The secondary structure
I I4
D. C. Clark. J . Mingins. F. E. Sloan. L.J. Smith and D. R. Wilson
content of BSA was estimated by off-line analysis of far-UV spectra on a DEC-20 computer using the method of Provencher and Glockner.' Protein concentrations were measured spectrophotometrically with a PerkinElmer 550-S UV/visible spectrophotometer. Absorbance coefficients of 0.66 and 2.58 ml mg-' cm I were assumed for BSA and lysozyme, respectively. Results and Discussion As anticipated, our attempts to pull macroscopic soap films from solutions of lysozyme or BSA were unsuccessful. This was despite waiting for long periods for substantial adsorbed layers to build up at the A / W interface. When we examine the adsorption behaviour of lysozyme and BSA at the macroscopic A / W interface, as depicted in the coherent set of papers'.'' by Graham and Phillips, we see that lysozyme is more surface-active than BSA. In addition, lysozyme has a much higher rate of adsorption than the more surface-
0
Figure 1
1 Time I h
2
Plot of surface pressure rl versus time for lysozyme in 0.1 M sodium phosphate (pH 7.0, 19.2 "C) using the stirring method. Protein concentration is 8.4 X g I. I
I I5
Timels
Plot of surface pressure n versus time for lysozyme in 0. I M sodium phosphate (pH 7.0, 21 "C) using the sweeping method. Protein concentrutions are: ( I ) 0.2 g I-', ( 2 ) and ( 3 ) 0.16 g I-', (4) 0.02 g I-'
Figure 2
16
1.2
L
E
z
< 0.8
I=
0.4
0
200
400
600
800
Tirnels
Figure 3
Plot of sudace pressure n versus time for lysozyme in 0. I M sodium phosphate (pH7.0, 20.0 "C)using the stirring method. Protein g I-' concentrations are: 0,9X lo-' g I-'; b,33 X
D. C. Clark, J. Mingins, F. E. Sloan. L.J. Smith and D. R. Wilson
116
active &casein, in keeping with its much lower mdecular weight. (Although no rate curves for BSA are shown, the implication is that the lysozyme rate would again be higher due to molecular weight considerations.) The poorer foaming behaviour of lysozyme compared to BSA cannot, therefore, be attributed to lower levels of lysozyme in the surface at any one time. The surface pressure results of Graham and Phillipss show that, at all but high (>2 X wt%) and the lowest (10” wt%) concentrations, lysozyme has a smaller surface pressure than BSA. An inflection in the lysozyme equation of state around 7.5 mN m-I would seem to be indicative of surface association. As a result of remarkss by Graham and Phillips about contact-angle problems, and our own experience with finite angles generated by lysozyme on glass, we felt it incumbent on us to check the overall surface pressure behaviour of this protein using a paper Wilhelmy plate. Using either of the adsorption procedures described above, we report the same general picture as seen by Graham and Phillips (see Figure I). For dilute solutions, three regions can be identified: an initial region I where n is essentially zero, a region 11 where Il rises significantly, and a region 111 where Il exhibits a very slight but steady rise over several hours. Behaviour in region I11 is generally attributed to rearrangement o r association of adsorbed protein molecules, or a combination of both. Graham and Phillips have clearly shown that lysozyme adsorption proceeds monotonically throughout region I, and
0
50
Time / min
Figure 4
Plot of surface pressure Il versus time for phospholipase A-alpha in water at 20.0 OC using the stirring method. Protein concentration is I .43 x 10-3 1-1
I I7
Figure5
Surface concentration of BSA as a function of mean protein concentration in foaming solution. Results were calculated from foaming measurements in 0. I M sodium phosphate (pH 7.0; 23 "C)
that the amount adsorbed is constant throughout region 111. Intrigued by the behaviour in region I, Dickinson and Stainsby" proffered one explanation in which, although steady diffusion of protein to the interface was taking place, a finite time was needed for the protein to enter the interfacial region where the density was changing maximally and could thereby affect the surface tension. However, the disappearance of the induction period found at high concentrations is not easy to accommodate on this basis. We suggest another explanation for the induction period in Figure I . This requires adsorbed protein to form a separate surface phase leaving too few monomers to significantly affect II. Only when the surface is completely covered with the condensed phase can n start to rise. Such behaviour is frequently seen with spread condensed lipid monolayers consisting of fatty acids or alcohols, cholesterol, diacyl phosphatidyl cholines or ethanolamines, etc. Here, at high surface areas, islands of condensed monolayer are supposed to be in equilibrium with a surface vapour phase often having a minute surface pressure, and the surface pressure only starts to rise when the vapour phase has disappeared. If this model is valid, the induction period reflects the time taken to cover the surface with condensed phase, and its length should therefore decrease with increasing bulk concentration of protein. The adsorption density should then always be the same where II starts to increase. Unfortunately, this point cannot be checked with the
I I8
D.C.CJark. J. Mingins. F.E. Sloan. L.J. Smiihand D. R. Wilson
L
0 Figure6
I I I 1 2 3 Mean concentration1 mg ml-'
I
4
Surface concentration of BSA as a function of mean protein concentraiion in foaming solution. Results were calculated Jrom foaming measurements in water ( 2 3 " C )
published material currently available,* and we ourselves d o not as yet have the means to measure surface concentrations at macroscopic interfaces. Our checks of the concentration-dependence of the induction time d o confirm the generally expected trend, as shown in Figure 2, but there is a disturbing lack of reproducibility in the n-t behaviour for lysozyme. For example, a large difference in induction period can be seen for the same solution, as shown by results at 0.16 g I in Figure 2, where a shift in the time axis of ca. 300 s would produce superimposition of the curves. In contrast, it is difficult to match the shape for the curve corresponding to 0.2 g I-'. On occasion, we have also seen in dilute solution what look like phase transitions at low Il instead of the usual induction period, as illustrated in Figure 3. In view of the fact that the overall gross differences are often well outside the slight errors expected with either method, we are forced to conclude that there is variability in the solution state of the lysozyme, i.e.,different degrees of time-dependent association o r aggregation. This needs further investigation. One other experiment was to compress the surface in the induction zone. Large changes in n were seen, and, after a slight initial decrease, there was a steady rise in n as more protein adsorbed.
'
*See, however, the paper by de Feijter and Benjamins in this volume, p. 72.
I I9
The presence of an induction region is not confined to lysozyme. I t has been seen by one of usr2for another globular protein phospholipase A-alpha as shown in Figure4. and it is probably a ubiquitous featureof protein systems. However, in the context of the present work, it is tempting to attribute the poor foamability of lysozyme to the lack of response of I1 in the initial stages of adsorption. For the foaming studies, we were limited to BSA solutions because of the poor foamability of lysozyme. The concentration range covered was 0.5-5 g 1 I . and experiments weredone both in water(pH 5.5)and 0. I M phosphate buffer(pH 7.0). As foaming proceeds, the bulk concentration of the foaming solution gradually
a
h
2.0
.-l .0
. I 0-0
600.0 Timels
\
0.0
6C 1.0
0.0
Time/ s
Figure 7
Time-dependence of decay in conductivity C of a foamed sample of BSA. Plots (a) and (b) each shows the experimental curve, the computedpit, and the residual obtained by suhtraciion ofthepifrom the experimental curve. Protein concentration is 2 g I I in 0. I M sodium phosphate (pH 7.0). (a) Fit to single-exponential expression with k , = 0.0094 s and A , = 255 pS. (b) Fit to double-exponential expressionwithk,=0.0275 s ',k,=0.0058 s - ' , A , = 3 0 6 pSand A,= 77 pS (residualslightly offset from zero)
I20
D. C . Clark, J . Mingins. F. E. Sloan. L.J. Smirh and D. R. Wilson
decreases; quoted results refer to the mean of initial and final concentrations. The surface concentration (amount per unit area) was calculated from Weil's equation3:
In equation (2). Sc is the surface concentration, d, is the bubble diameter, w, is the weight of collapsed foam, Vsis the volume of the initial solution, V, is the volume of foam collected as judged by the volume of N, passed, and C, and C, are the BSA concentrations in the received foam and in the final bulk solution, respectively. The results are presented in Figures 5 and 6. Although there is a large scatter, the surprising trend of a decreasing surface concentration with increasing bulk concentration is readily distinguished for protein in both water and phosphate buffer. This runs counter to expectations of reversible adsorption and Gibbsian behaviour seen in Weil's work with simple surfactants. The results of Graham and Phillipss for BSA show that saturation coverage is reached at bulk concentrations
*
4 .O
3.6
3.2
111
2.0
P
-
\
r
2.4
2 .o
0.o
1.o
2 .o C p / m g mi-1
3 .O
4.0
5.0
3.0
L
.O
50
'
0.0
7.0
6.0
.. cn
c
5.0
\ N
Y
4-0
3 .O
0.0
Figure 8
1 .o
2.0
Cp/mg m1-l
Concentration dependence of rate of decay of conductivity offoamed BSA. The constants k , and k, areplotteda~ainsiprotein concentraiion cI, in 0.I M sodium phosphate
D. C. Clark. J . Minxins. F. E. Sloun. L.J . Smirh and D. R. Wilson
I22
rate constants and amplitudes are given in the figure legend. There is a five-fold difference in rate constant between the fast and slow components of the decay, and the amplitude of the fast decay is four times greater than that of the slow decay. I t is clear that protein solution drains from the column of foam during the course of conductivity data collection. This may be studied independently by monitoring the increase in volume of the protein solution left at the bottom of the column after the gas flow is shut off. Electrophoretic and spectrophotometric analysis of the concentration of protein solution draining out of the column of foam showed that there was no change in concentration even in drainings collected 10 minutes after sparging was stopped. Our interpretation is that reduction in foam conductivity arises principally from the drainage of protein solution. Also, thinning of the lamellae due to drainage may lead to their rupture, if they are intrinsically unstable, so providing another explanation for the reduction in conductivity. Thus, we believe that the conductivity decay curves contain information about drainage and stability of the foams, and that the analysis in terms of double-exponential expressions provides a means of ranking behaviour in a qualitative manner, thereby permittingevaluation of the gross effects of solution conditions on foam properties. Conductivity decay curves for BSA foams have been studied as a function of
0.0
1
1
I
I
1
I
I 420.0
300.0 A /nm
Figure 9
Tryptophan fluorescence emission spectra of native BSA ( 0 ) and foamed BSA (0).The intensity I is plotted against wavelength A (excitation wavelength 295 nm, slit width 2.5 nm). Protein concentration is 0.I g 1 I
I23
initial protein concentration, and the rate constants of fast and slow processes are plotted against protein concentration in Figure 8. Each point corresponds to the average of 5- 10 determinations at each concentration. Although large standard deviations are found, as depicted by the error bars, there is a significant decrease in decay rate with increasing protein concentration. Further work is needed to understand the significance of this observation. but it does seem that there is a decrease in drainage rate or an increase in film stability o r both, as the protein concentration increases. We have shown separately that varying the viscosity of the bulk solution by adding sucrose or glycerol does not significantly change the rate of conductivity decay. It is, therefore, difficult to explain the effect of protein concentration in terms of changes in the rate of drainage. The conformational properties of foamed BSA have been compared with native BSA using the technique of intrinsic fluorescence. Figure 9 compares the tryptophan fluorescence of foamed and native protein. We see that the fluorescence intensity of the foamed sample is significantly reduced (by ca. 30%) compared to that of the native sample, but that this is not accompanied by a red-shift in the Amax of emission, which usually occurs13 when a buried tryptophan is exposed to polar solvent. The shoulder at 330 nm, present in all the fluorescence spectra, is due to
0.0
!
I
I
I
300.0
I
I
120.0 A/nm
Figure 10
Tryptophan fluorescence spectra of BSA as a Junction of urea concentration. The intensity I is plotted against wavelength A: D, controlsample in distilled water; 0. in 2 M urea; A , in 4 M urea; -I-, in 6 M urea; X, in 7.2 M urea. Protein concentration is 0.075 g I I
D.C. Clark, J. Mingins. F. E. Sloan. L.J. Smith and D. R. Wilson
I24
10.0
6.0
2.0
w
-2.0
-4
-6.0
--1o.o 195-0
Figure 11
I/nm
250.0
Far- LI V C D spectra of BSA. The intensity Ac is plotted against the wavelength A: m, control sample in distilled water; 0,foamed in distilled water; A,foamed in 0 . I M sodium phosphate (pH 7.0); in 4 M urea
+,
Raman scatter. An attempt has been made to model the reduction in fluorescence intensity by treating the native BSA with denaturant. The BSA fluorescence as a function of urea concentration is shown in Figure 10. It is evident that the 2-6 M urea causes a progressive reduction in the intensity of tryptophan fluorescence, which is consistent with a change in the micro-environment of the fluorophore. A red-shift in the emission spectrum is seen, however, only at high urea concentrations (7.2 M). The change in fluorescence in 7.2 M urea is consistent with major unfolding of the BSA molecule with subsequent ejection of buried tryptophans into the surrounding polar solvent. By measuring the fluorescence intensity of BSA samples in urea at the emission maximum (345 nm), normalizing to a protein concentration of 0.1 g 1 I , and plotting the results against urea concentration, it has been shown that exposing BSA to 4 M urea produces a reduction in fluorescence intensity similar to that found for the foamed samples. Circular dichroism (CD) measurements were made on the samples referred to above in order to investigate further the conformational change induced by foaming, and to test whether BSA in 4 M urea was a good model for such conformational change. Far-UV C D spectra are shown in Figure 1 1. The spectrum for BSA in 4 M urea is considerably less intense than the others. The shape and
I25
intensity at 222 nm are indicative of a protein that contains a large amount of a-helical structure. Table I gives the results of an analysis of the secondary structure content using the Contin program.’There is a major loss of a-helix content in going from the native protein to the sample i n 4 M urea, but this difference is not seen with the foamed samples. However, the small differences in a-helix content between foamed and native samples may be significant since it is seen consistently. The estimation of /%structure in a protein containing large amounts of a-helix is subject to quite large errors, and this probably explains the variation in the &structure values reported in Table I . Near-UV CDspectra of the samples are shown in Figure 12. This region of the C D spectrum can be used qualitatively to examine tertiary structure of proteins.I4 Again, the largest difference observed is between the sample in 4 M urea and the others. The near-UV spectra are most intense in the tyrosine region (275-282 nm), which is not particularly surprising considering that BSA contains 19 tyrosines and only 2 tryptophans.I5 The tryptophan region (285293 nm) does not reveal any significant differences. I t is difficult to draw firm conclusions from this preliminary investigation of protein conformation. While the fluorescence behaviour of BSA in 4 M ureaclosely resembles that of the foamed protein, the C D data suggest that the urea treatment causes considerably greater changes in secondary and tertiary structure than does the foaming treatment. Paradoxically, we have found that the effect of urea, as judged by fluorescence, is completely reversible upon removal of urea by dialysis. Thus, treatment of BSA with urea results in a large reversible change in protein conformation, whereas foaming the protein causes a small but irreversible change. We must conclude that the two treatments affect the protein in different ways, and that general protein unfolding cannot account for the observed irreversibility of changes induced by foaming. It is worth noting that the spectroscopic measurements d o not distinguish between a small conformational change present in the majority of molecules and a large conformational change present in only a small fraction of the molecules. For urea-treated samples, it is reasonable to assume that all molecules have been exposed to the same urea concentration, and have therefore undergone an approximately equivalent conformation change. On the other hand, it is likely that there is a wide range of conformations in foamed samples. This will include BSA molecules that have adsorbed at the air-water interface and have stabilized it by surface denaturationlaggregation, and also molecules that have not adsorbed, or possibly have adsorbed but have not denatured or unfolded due to the local high surface density of protein. So, although the average conformation observed for the foamed samples appears to be only slightly different from the native state, it may actually correspond to a wide range of conformational states ranging from native to completely unfolded. After collection of the foam, the slightly denatured forms may revert back to the native structure, but the highly denatured forms may be irreversibly altered. In addition, it is highly probable that the foamed sample contains protein aggregates. The formation of stable aggregates by association of surface-denatured protein could account for the observed irreversibility of the conformational state of the foamed protein. It is unlikely that this occurs to any extent in the urea-denatured samples. The absence of a red-shift in the tryptophan emission spectrum probably means
D.C. Clark. J. Mingins. F. E. Sloan. L.J. Smith and D. R. Wilson
I26
Table 1
Calculatedsecondary structure content of BSA
Percentage of Secondary Structure P-structure p-turn and aperiodic
Sample
a-helix
control (water)
53
17
30
foamed (water)
50
16
34
foamed (phosphate)
50
8
42
4 M urea
41
II
48
255.0
330.0 h /nm
Figure I2
Near- U V C D spectra of BSA. The intensity Ac is plotted against the wavelength h: 0,control sample in distilled water; A . foamed in distilled water; +,foamed in 0.1 M sodium phosphate (pH 7.0); 0 , in 4 M urea
I27
that solvent quenching is not the main mechanism operating in this system. An alternative explanation is that quenching is brought about by the close proximity of a charged group (e.g., a deprotonated carboxylic acid). This could itself be the product of conformational change or aggregation. Further experiments with classical soluble quenchers may identify the true mechanism. Acknowledgements. We thank Drs. P.M. Bayley and S.R. Martin for allowing us
to perform CD measurements o n their equipment a t the National Institute for Medical Research, London. We acknowledge the work of David Pennington, John Stormes a n d Ben Piggott in the construction of the conductivity cell and associated electronics. We thank Dr. A. Howe for useful discussions. References I 2 3 4
5 6 7 8 9 10
II 12 13 14 15
J.A.G. Taylorand J. Mingins,J. Chem. Soc.. Faraday Trans. 1. 1975.71. 1161. C. Walling. E.E. Ruff, and J.L. Thornton. J. P~.I:F. Chem.. 1952.56,989. I . Weil, J. Phys. Chem., 1966.70, 133. A. Kato. A. Takahashi. N. Matsudomi, and K. Kobayashi, J. FoodSci., 1983,48,62. P.R. Bevington,'Data Reduction and Error Analysis for the Physical Sciences,'McGrawHill. New York, 1969. P.M. Bayley, D.C. Clark, and S.R. Martin, Biopo/vmers, 1983.22, X7. S.W. Provencher and J. Glockner, Biochemistry, 1981,20,33. D.E. Graham and M.C. Phillips, J. Colloid Inrerface Sci.. 1979.70.403. D.E. Grahamand M.C. Phillips, J. ColloidInrerfaceSci., 1979,70,415. D.E. Graham and M.C. Phillips, J. Co//oid Inrerface Sci., 1979,70,427. E. Dickinson and G. Stainsby, 'Colloids in Food,' Applied Science. London, 1982. D.J. Hanahan, J. Mingins, and M.A. Wells, unpublished work. J.R. Lakowicz,'Principles of Fluorescence Spectroscopy,' Plenum, New York. 1983. E.H. Strickland. C R C Crir. Rev. Biochem., 1974. 113. .I.R. Brown. Fed. /'roc.. 1975.34.581.
Behaviour of an Aerated Food Model By R.D. BEE,
(Unilever Research Laboratory. Colworth House, Sharnbrook. Bedford MK44 7LQ)
A. CLEMENT and A. PRlNS (Department of Food Science, Agricultural University, de Oreven 7 2. 6 703 BC Wageningen. The Netherlands)
Introduction Many foods are aerated. The gas phase may be introduced in order to provide a specific textural character, such as brittleness in confectionery, lightness in whipped cream, or scoopability in ice-cream. Alternatively, the gas phase may function as a structuring element in part of a cooking process, as with 'creamed' margarines in cake baking, o r the egg-based aerated structure in a meringue o r soufflk. Though widely different in chemical composition, the aeration processes of these foods have much in common. For example, each can be prepared with a simple mechanical whisk, the initial stage being the incorporation of large gas cells, which are then broken down during whipping by the shear field of the beater blades. In each case, the process is known to be influenced to varying extents by several physical factors, of which the most important are normally taken to be the rheological properties and the interfacial behaviour. Because of the chemical and physical complexity of all the above foods, work on aspects of aeration behaviour has tended to be relevant only to a single product. Or, studies have been made on just single components, such as the surface-active proteins at the air-water interface. With both approaches, it has proved difficult to translate the information into a general picture of aeration, or to understand how the process is influenced by interactions between the various components. In practical circumstances, gas is often introduced under several atmospheres pressure, which means that the gas-phase volume present during aeration is substantially less than that present when the product is allowed to expand to atmospheric pressure. It is important, therefore, to study systems in which the incorporated gas-phase volume is relevant to these low volume fractions, and for which the aerated system can be both sampled and analysed. Temperature specification is a significant experimental complexity.' A physical property like viscosity can change drastically for a small drop in temperature - as, I28
I29 for example, the fat crystallizes in margarine or cream, the gelling agent sets in a mousse, or ice forms during the making of ice-cream. Difficulties are also met when temperature rises: an increased partial pressure of water vapour can destabilize films between gas cells and consequently modify the aeration behaviour. The aeration process normally requires a level of mechanical energy which is enough to induce temperature changes capable of affecting the properties of the system in the ways indicated above. So, for aeration experiments to be meaningful, temperature control is vital. Equally important is the choice of a model system which is not susceptible to physical changes that are very temperature-sensitive, like gelation or crystallization. Clearly important is the amount of gas incorporated during aeration. This is easily and accurately measured by monitoring the change in bulk density as compared to the unaerated material. The nature of the distribution of gas cells, however, is much more difficult to determine, and is rarely dealt with in the published literature. This is because large gas cells are vulnerable to coalescence through film rupture, and small cells are unstable with respect to large cells through disproportionation. As well as these physical problems, there is the major practical problem that no commercial particle-sizing technique lends itself to gas-cell size determination. The investigator has mostly to resort to microscopy, where the necessity to prepare samples of suitably short path-length and optical clarity results in uncertainty as to the extent to which the observed sample represents the original structure. With the preceding factors in mind, we choose a simple maltodextrin solution as our base material. The system has the advantages of chemical stability, Newtonian viscosity and optical clarity; in addition, it is readily available and inexpensive. A set of solutions of various concentrations provides an ideal model with which the main process of gas incorporation can be studied. The effect of solid particles on the properties of aerated maltodextrin solutions will be investigated by adding small glass spheres (ballotini) to the system. In this paper, we discuss how the model can be handled, the effects of varying the viscosity and the initial surfactant concentration, and methods for satisfactorily measuring air-cell size distribution. Gas-cell surface areas are recorded throughout whipping experiments, in order that conclusions can be drawn about behaviour when surfactant concentration is the limiting parameter. The rheological consequences of introducing the gas phase are described, and the data are analysed using established relationships to account for the contributions of the dispersed phases in the system. Experimental Solutions.-The criteria of high viscosity, rheological simplicity and optical clarity suggested to us that oligosaccharide solutions would make suitable bases for the liquid phase. A commercial maltodextrin syrup (Sweetose; Ragus Sugars, Slough) containing 80-85% solids, and with a dextrose equivalence of 63, was found to be ideal for the purpose. With this material, the crystallization behaviour found with solutions of simple sugars at high concentration is avoided. In addition, there is no tendency to skin formation on standing. Nor does it show the stringiness found with
R. D. Bee. A. Clemenr and A. Prins
I30
aerated solutions of higher molecular weight maltodextrin (e.g., 17DE). Experimental solutions were prepared by diluting the maltodextrin glass, firstly with a solution containing a hydrolysed protein whipping agent (DIOO; Gunther Products, Galesgurg, Illinois), and then with distilled water to the required concentration (72 wt% maltodextrin solids). To ensure proper solubilization and equilibration, solutions were warmed to 333 K for I5 minutes before cooling to the experimental temperature of 298.3 K . Maltodextrin concentrations were determined by evaporating thin films of solution, prepared by known dilutions, to dryness in an air oven at 383 K . Refractive index measurement of the original solutions with an Abbk refractometer (sodium D line, 293.3 K ) gave calibration curves from which concentrations could easily be obtained throughout the experiments. Aeration.-Aeration experiments were carried out at 298.3 K in a Kenwood mixer with a partially covered bowl and a jacket for temperature control. In normal practical situations, aeration takes at the most a few minutes, but to allow time to analyse the process as it occurs, it is necessary to extend the process considerably. This can be achieved by increasing the viscosity of the liquid continuous phase,* which, in this case, simply means working at higher solids concentration. Concentrations of ca. 72 wt% produced experimental whip times of up to 2 hours, and were ideal for this model study. The time-scale allowed small samples to be removed at regular intervals for measurement of phase volume, viscosity, air-cell size and refractive index. Below ca. 70 wt%solids, the creaming of gas cells prevents representative sampling early on in the experiment. A disadvantage of theextended experiment, however, is that the sample suffers evaporation. Typically, this results in a 2-4 wt% increase in solids content during whipping. A solids content above 76 wt% results in excessive whip times, low phase volumes, and unacceptably large increases in viscosity due to water evaporation. Figure I gives the relationship between viscosity and solids content for the experimental material.
Viscosity/ Pa S
% Solids
Figure I
Plot ojviscosity ugainst so1id.s contentfor diluted Sweetose at 298.3 K
Viscometry.-The viscosity q of the aerated material was measured at 298.3 K in a Haake viscometer at approximately 15-minute intervals throughout each run. Simultaneously, samples were deaerated by centrifugation at I500 r.p.m. for 30 minutes, and the viscosity qoof the liquid was measured. The relative viscosity qr = q / qoreflects only the effect of incorporated gas phase. In experiments where 63 p m ballotini (Jencons Scientific) were used as a monodisperse solid phase, the relative viscosity is defined as q,b = qb/qO.A Rheometrics Mechanical Spectrometer was used to determine the relative viscous and elastic contributions to the overall rheology of the aerated models. Air-cell Sizing by Microscopy.-Bubble-size distributions were measured from photomicrographs taken with an Ortholux microscope and a Polaroid attachment, with counting done using a Zeiss particle-size analyser. Aerated samples were mounted on a haemocytometer slide with a I00 p m gap, and 500-1500 bubbles were counted in each photograph. However, early experiments identified a serious deficiency with this procedure. In order to check the validity of the average cell diameters d,,o, acomparison was made between the gas-phase volume fraction 4, calculated from d,,o and the number of cells per unit volume, on the one hand, and the gas-phase volume fraction c $ measured ~ gravimetrically, on the other. A typical calculation based on
% Volume
Diameter/( p m)
Figure 2
Bubble-size distribution in Sweetose solutions containing 0. I wt% DlOO as a/unction of whipping time. Percentage volume is plotted against bubble diameter (curves drawn through mid-points of 2 pm class width)
I32
R. D. Bee, A . Clement and A. Prins
0.041
Phase volume fraction
0.02
t
\
\.
\
0.011
0 0.2 Figure 3
1 .o Gap size/mm
0.6
1.4
1.8
Phase-volume fraction occupied by bubbles larger than gap size hetweenplates (75 wt% Sweetose. 0. I wt% 0100. total+w = 0.32)
data for the distribution measured after 50 minutes whipping (see Figure 2) is set out below. Mean volume diameter = 8 pm Average bubble volume = ( 4 / 3 ) ~ ( 4= ) ~268 p m 3 Average number of bubbles per photograph = 500 Bubble volume per photograph = 500 X 268 = I .34 X lo5 pm3 Photograph area= I 1.3 X 8.9 cm2= 1.006 X I O ' O pm2 Slide gap = 100 pm Magnification = 250 Volume represented by one photograph = ( I O ' O X 100)/(250)2 = 1.6 X lo7 pm3 r$m = ( I .3 X lo5)/(I .6X lo7) = 0.0 I Gravimetrically measured +w from density reduction = 0.27 The above analysis suggests that, if the size distribution obtained is accurate, obviously not all the cells are being counted. It may be that large cells are being obscured by a raft of small cells, or sampling to the haemocytometer slide may not be representative (i.e., some large cells may be being missed). To check on the possibility that large amounts of air were present as large bubbles (>I mtn diameter), it was necessary to examine larger volumes of material in order
I33
Table 1
Ratio of gas-phase volume fractions determined microscopically (4,,,) and gravirnetrically (4,) as a function of whipping time and DIOO surfoctant concentration
Time/ min
'0.01wt%
0.015 wt%
0.025 wt%'
55
I .04
I .05
I .24
85
-
I .04
0.70
I20
0.99
I .05
I .07
to get a statistically meaningful sample. To d o this, samples of whipped fluid were sandwiched between a pair of thin glass plates (20cm X 20cm), the sample thickness being determined by spacers separating the plates. Using this technique, up to 2 ml of sample could be observed at large plate separations. All bubbles larger than the gap between the plates are easily observed because they form cylindrical channels perpendicular to the plates. Knowing the gap size, the bubble diameters, and the volume of solution on the slide, one can calculate the phase volume accounted for by bubbles bigger than the gap size. Figure 3 summarizes the results of such a set ofcalculations for gap sizes from 100 pm to I .2 mm. A volume fraction of ca. 0.04 is accounted for by bubbles of size >100 pm, implying that in this case a volume fraction of 0.28 is occupied by bubbles of size
Results and Discussion Figure 4 charts the progress of four aeration experiments involving the
R. D. Bee, A. Clement and A . Prins
I34
maltodextrin base model (72 wt%) at 25 O C with DlOO surfactant concentrations between 0.01 wt% and 0.025 wt%. In each experiment the amount of gas phase increases smoothly to a maximum and then goes down. This type of behaviour is frequently noticed during whipping, and its cause can be attributed to a physical' or chemical4 change in the components. Such changes are brought about by the shear conditions, or by irreversible processes associated with fat-droplet aggregation and fat adsorption at the newly introduced gas-liquid interface.s*6 Behaviour of a similar type is expected during the aeration of a protein solution, where protein is irreversibly denatured through adsorption at the gas-water i n t e r f a ~ e .In ~ the
I
0-0
I
I
0.01% D 100 D 100 0.02% D 100 0.0259/0D 100
s-xO.O15% A-b
Phase volume fraction
0-0
O.!
I
I
30
60 Ti m e/m in
I
I
90
120
Figure 4
Plot of phase-volume fraction c $ ~ of air againsr whipping time at 298.3 K for various DlOO concentrations: a, 0.0 I wt%; X, 0.0 I 5 wt%; A, 0.02 wt%; 0,0.025 wt%. Sweerose concentralion = 12 wt%
Table 2
Ratio of gas-liquid surface areas at various whipping rimes as a function of DlOO surfacrunt concentration (quantity A, is proportional to surface area in measured volume after t minutes) Conren/rution/ wt%
A5SlA120
0.0 I
1.12
0.0 I5
1.13
1.13
I .oo
0.025
I .22
0.93
I .29
A851A12U
A55/AX5
~
I35
present model, however, no such chemical or physical changes can be used to interpret the whipping process. The maximum in 4, in Figure 4 must be attributed either to changes in rheological conditions caused by the introduced gas cells, or, more likely, to the more limited availability of surfactant as the gas-liquid surface area increases. Figure 4 clearly shows that the incorporated gas-phase volume increases as the surfactant concentration increases. As a higher gas-phase volume is likely to be associated with a larger gas-liquid surface area, it appears that the I
I
---
0
d
/'
4 '
-
-------- ------.
'
0-
0
A verage volume diameter d,," (
) and number ofair bubbles -) us a function of whipping time at 298.3 K Jbr various D1Oo concentrations: e. 0.01 wt%; x, 0.015 wt%; A, 0.02 wt%; 0 , 0.025 wt%. Sweetose concentration = 12 wt% (~
Table 3
e x
*c-/--
*
x-
Figure 5
- 8000
0.01% D 100 0.015% D 100
200 -
~
-
Potential interfacial concentration C, as a function of whipping time and DlOO surfactunt concentrotion CJ10 X!4Clm
Time/ min
55 x5
I20
r
0.01 Wl%
0.015 wt%
0.025 wt%
5.15
7.74
12.53
6.96
16.23
6.75
11.67
~~~
5.3 I
'
I36
R. D. Bee, A. Clement and A. Prins
surface active agent is limitinggas incorporation from the phase-volume maximum onwards. It has been possible to follow the growth in gas-liquid surface area during these experiments by sizing and counting the gas cells accurately using the techniques described above. Calculations based on measured d,,o values given in Figure 5 show that the bubble surface area is increasing up to the maximum gas-phase volume fraction d,,,, but from that point on it remains constant. Hence, during the period of diminishing gas-phase volume, the bubble surface area remains constant. This can be seen from the ratios of surface areas at different whipping times listed in Table 2. To keep the surface area constant in the system, there must be a balance between two factors: as bubbles are comminuted in the shear field, thereby increasing their surface area, so to balance the change some cells are lost, presumably by film rupture near the bulk surface. It must be that bubbles at the top end of the distribution are lost by this mechanism, since the gas bubble number increases as C#J decreases (Figure 5). This is consistent with expectations: large cells are most vulnerable to distortion, and so the liquid film between them and the external atmosphere is the most likely to rupture when extended. Additonally, it may be that, because the whipping equipment operates at constant speed, the amount of mechanical energy imparted to the system increases during the experiment, and larger cells may be more vulnerable under these conditions. We can use the experimental results to see if there is indeed a critical amount of DlOO per unit area in a whipped system. The potential interface concentration is obtained by dividing the available amount of DlOO by the total area ( N X d2,0).The values are listed in Table 3. For each individual concentration, we see that, whilst after there is observed a constant total surface area (and, within experimental error, a constant ‘surface concentration’), the actual available surfactant per unit area appears to increase with increasing surfactant concentration. This is thought to be due to different effective shear conditions resulting from increased gas-phase volumes incorporated at the various surfactant concentrations. In principle, the additional contribution to the diminution in gas-phase volume could come from the reduction in gas-cell size. This is because, according to the Laplace equation, the internal pressure in a gas cell is
+,,
where r is the radius of curvature, and y is the surface tension. When gas molecules are incorporated in an aerated system, they are at higher pressure, and hence contribute less volume, the smaller the gas cells they occupy. In addition, because of the higher pressure, more gas dissolves. In some aerated systems significant expansion occurs’ from the reverse process, coarsening, resulting from disproportionation of gas cells and the concomitant release of dissolved gas from solution. Such considerations can be discounted here, however, since the cells are relatively large (see Figure 5). Without exception, gases are incorporated into foods in order to impart particular textural characteristics. It is therfore important to know how the gas phase affects the rheological behaviour of our simple model system as it is aerated. Figure 6 shows how the viscosity changes through the course of four typical
I37
5-
Viscosity/ Pa s
P
1
P/"
/*-
I
I
30
0
60
90
120
Time/ mi n
Figure 6
Viscosity as a function of whipping time at 298.3 K for various DlOO concentrations: I wt%; x, 0.0 15 ~ 1 % A; , 0.02 wt%; 0.0.025 wt%. Sweetose concentration = 72 wt%. Shear-rate = 20 s - I
0,o.o
Relative viscosity
Time/ min
Figure 7
Relative viscosity as afunction of whipping time at 298.3 K for various DlOO concentrations: 0 , 0.01 wt%; X, 0.015 ~ 1 % ;A , 0.02 wt%; 0, 0.025 wt%. Sweetose concentration = 12 wt%. Shear-rate= 20 s I
I38
R. I>. Bee, A . Clement und A . Prim
whippingexperiments. An initial rapid rise in viscosity is followed by a plateau after 30-60 minutes, after which the increase resumes. Any proper interpretation of this behaviour must take into account the loss of water vapour during the experiment. It is therefore more appropriate to consider the relative viscosity qr. where the viscosity, modified by the presence of the dispersed gas phase, is related directly to the viscosity of the unaerated liquid at that point in the experiment. Such an analysis is given in Figure 7. where relative viscosity at shear-rate y = 20 s I is plotted against the time elapsed during the whipping experiment. Gas-bubble incorporation clearly affects the viscosity, but the relationship is not straightforward. There is a significant increase in q at extended aeration times due to evaporation. This was confirmed to be the case by determining the solids content throughout the experiments by measuring the refractive index of samples. The rheological consequences of gas-phase incorporation are clearly displayed by plotting relative viscosity against air-phase volume fraction 4 as shown in Figure 8. Most striking is the fact that the gas phase, when dispersed as cells, increases 'I, systematically as a function of 4. Indeed, all the data points, from both initial and final portions of the aeration curve, lie between the predictions of Roscoe' for a rnonodisperse system of solid spheres and those of Brinkmany for a polydisperse system. The behaviour of adispersion of particles(ballotini,d,,,=63 pm) is shown in Figure9. Wesee that, though thedatado not fit theequation of Roscoe, they d o show clearly that the contribution of gas cells is very similar to that of solid particles. Presumably, this effect is most apparent at low shear-rates and with small gas cells that are difficult to distort. Eilers"' developed an empirical equation for dispersions containing a high volume fraction of particles:
The quantity c$* in equation (2) denotes the maximum volume fraction that can be obtained when particles are close packed. The measured viscosities of ballotini in Sweetose are described precisely by equation (2) with +* = 0.60. With mixed dispersed phases, the rheological situation is more complex. Figure IOshows that the relative viscosities ofsystemscontainingaircellsand ballotini are much lower for a given total volume fraction than those measured with either dispersed phase alone. The Eilers equation is no longer satisfactory, since unrealistically high values of 4* are required. Even at the low shear-rate of these determinations (20 s I), the influence of one dispersed phase on another is quite different rheologically from the effect which it has on itself. We suggest that the explanation lies with flow alignment of the solid and gas dispersed phases, rather than with gas-cell deformation, though this has yet to be properly established. We must infer that, for more complex dispersions, theoretical prediction of the contribution of dispersed phases to bulk rheological behaviour is likely to prove an extremely difficult problem. Since the effect of solid dispersed phase is a factor of major importance, it must be studied in detail before significant progress can be made in understanding the process of aeration and gas-cell dispersion, and the contribution of gas cells to food texture. Having recognized that the dispersed gas phase acts in a manner similar to rigid inclusions, it is interesting to examine the relative contributions of elastic and
I39
Relative viscosity
0 0.1
0.3
0.5
0.7
0.9
Phase volume fraction
Figure 8
Plot of relative viscosity against volume fraction of air for 12 wt% Sweetose at 298.3 K at a shear-rate of 20 s-'from/ive experiments
Relative viscosity
0.1 0.3 0.5 Phase volume fraction Plot of relative viscosity against volume fraction of hallotini for 12 wt% Sweetose at 298.3 K
0
Figure 9
R. D. Bee, A. Clement and A. Prins
I40
viscous components to the overall rheology. This depends, of course, on the applied rate of strain, since the viscous component allows the stress to relax over a certain time scale, and a rapidly applied strain produces a more elastic response. Table 4 lists values of the storage modulus G‘ and loss modulus G“ for measurements at a frequency of lo2 radians per second on a sample containing 75 wt% Sweetose and 0.1 wt% D100.It is clear that both the elastic and the viscous components increase during gas inclusion, but that the fractional increase in G’ is much the larger. Over a whipping time of 60 minutes, the elastic component of the viscosity, q’, has increased by a factor of 6.2/0.46 = 13.5, whereas the viscous component q” has just increased by a factor of 31.59/ 14.09 = 2.3. Since the overall dynamic viscosity q* is related to elastic and viscous components by ‘I* = [(‘I’)2
+(q32]”2
(3)
it is the high (initial) value of the viscous component which also makes the predominant contribution to the total viscosity. So, even though the elastic component increases its value by more than an order of magnitude, it is the viscous effects of gas bubbles and not the elastic elements such as film stretching or bubble distortion which make up the main contribution to the overall rheology.
0
Figure 10
0.1
0.3 0.5 Phase volume fraction
0.7
Plot of relative viscosity against volume fraction of air or ballot ini or both for 72 wt% Sweetose at 298.3 K
141 Gas volumefraction whipping time
Table 4
Time/ min
$I
G'/Nm-Z
4 and rheological parameters as a function of G"/Nm
q*/Pas
q'/Pas
q"/Pas
22
406
4.07
0.22
4.06
0.44
325
1076
11.24
3.25
10.76
20
0.47
488
I586
16.60
4.88
15.86
40
0.38
619
2242
23.26
6.19
22.42
60
0.30
620
3159
32.19
6.20
31.59
60'
0
46
I409
14.10
0.46
14.09
0
0
5
* Centrifuged after 60 minutes whipping
Figure I I
Rheology of aerated and deaerated (centrifuged) samples as afunction of whipping time t in minutes. The logarithm of the stress 0 is plotted against the logarithm of the shear-rate $ . DlOO concentration =0.01 wt%
I42
R. D.Bee, A. Clemenr and A. Prim
,
1
I
6
5
4 In[*]
3
2
-.-
1
-0-
.....,.....
C 2
Figure 12
3
4
5
6
7
Rheology of aerated and deaerated (centrifuged) samples as afunction of whipping time t in minutes. The logarithm of the stress 0 is plotted against the logarithm of the shear-rate y . DlOO concentration
=0.02 wt%
Finally, we consider whether the Newtonian character of the maltodextrin/ surfactant solutions is modified by aeration. We fit the measurements of stress u as a function of strain-rate y to a power-law expression 0=I)-)J"
(4)
and so plots of Ino versus Iny reveal deviations from Newtonian behaviour as n deviates from unity. Figures I I and 12 show data plotted in this way for DlOO concentrations of 0.01 and 0.02 wt% at various times throughout the whipping experiment. Results are also shown for the liquid phases from which gas cells have been removed by centrifugation. In all cases the base solutions retain their Newtonian character, but the viscosity increases with whipping time. After 10 o r 20 minutes, the aerated systems also still show reasonably Newtonian behaviour over the range of shear-rates considered, but at longer times a deviation to shearthinning is apparent. The explanation could be simply distortion of gas cells,
I43
although, because the cell-size distribution is wide, a more likely cause is the orientation ofsmall cells behind larger ones. Comparison of the data at whip times 60 minutes and I20 minutes in Figure I I , and at 45 and 90 minutes in Figure 12, suggests that at long whip times the overall rheology is tending to become more Newtonian again. This may be a reflection of the small cell sizes at long whip times. Conclusions The model aeration system described here gives useful insight into the mechanisms of gas incorporation under conditions where minimal chemical change is taking place. The process is characterized by an increase in gas-phase volume with time, a maximum in the amount incorporated, and finally gas loss. A limiting interfacial area is reached, which then remains constant. Throughout the process, cell size diminishes and the number of cells per unit volume increases. Gascell dispersions made in this way are polydisperse, and gas-cell site analysis has pointed to the unreliability of optical techniques unless precautions are taken to cover a wide range of magnifications with samples of large size. Rheologically, gas cells behave like solid inclusions, especially when the average size is small (<200 pm). The major contribution to the viscosity comes from hydrodynamic interactions between cells, and little is contributed by elastic cell deformation at the volume fractions (<0.5) and shear-rates considered. The base maltodextrin solution is Newtonian over a range of shear-rates, and it remains so at low phase volumes of incorporated gas, but shear-thinning is seen at high shearrates. This behaviour is more strongly evident at higher gas-phase volumes, and at longer whip times where the gas cells are smaller and more numerous. References I J.J. Bikerman, 'Foams,'Springer Verlag, Berlin, 1973. 2 3 4 5 6 7 8 9 10
E. Dickinson and G. Stainsby, 'Colloids in Food,' Applied Science. London, 1982. A. Prins, in'Foams.'ed. R.J. Akers. Academic Press, London. 1976. P.J. Halling, CRCCrii. Rev. FoodSri. Nurr., 1981.15, 155. H. Mulder and P. Walstra, 'The Milk Fat Globule.' Pudoc, Wageningen, 1974. P. Walstra and R.Jenness,'Dairy Chemistry and Physics.' Wiley-lnterscience, New York, 1984. S. Ross, in'chemistry and Physics of Interfaces Il,'ed. D.E. Gushee, American Chemical Society, Washington, DC, 1971. R. Roscoe, Br. J. Appl. Phys., 1952.3.267. H.C. Brinkman, J. Chem. Phys., 1952,20,571. H. Eilers, Kolloid 2.. 1941.97.313.
Protein-Fat-Surfactant Interactions in Whippable Emulsions By N. KROG, N.M. BARFOD (GrindstedProducts A / S , 38 Edwin Rahrs Vej, DK-8220 Brabrand, Denmark)
and W BUCHHEIM (Bundesanstalt fur Milchforschung, 0-2300 Kiel. West Germany)
Introduction
The microstructure of whipped dairy cream is known to consist of partly destabilized fat globules adsorbed at the air-water interface. This type of stabilization of the air-cell structure contributes to foam texture and firmness.'V2 Spray-dried. whippable emulsions (so-called toppings) are used as substitutes for dairy whipping cream, and the destabilization of toppings after reconstitution in water has previously been investigated.' Studies of reconstituted topping powders by transmission electron microscopy (TEM) show that there is a structural change in the fat phase from a globular state in the powder to a matrix of crystal platelets in the reconstituted emulsion. This structural change is related to the structure of the whipped topping emulsion. In contrast to whipped dairy cream, or a liquid imitation cream, it was found that the air cells are stabilized by adsorbed lipid crystal platelets rather than globular fat particles. And, by using a low-resolution pulsed NMR technique to investigate topping powders and the corresponding dry mixtures of the ingredients, we have found4 that part of the fat phase in topping powders is in a supercooled state. The main factor responsible for this supercooling effect is believed to be the small particle size of the fat globules, combined with the lipid-protein interaction described by Trumbetas er aL5 This interpretation is explored further in the present work. Materials and Methods
Topping emulsions, containing 50-60% hydrogenated coconut oil (m.p. 3 I " C ) , O-lO% lipophilic surfactant, 30-38% maltodextrin and 2- 10% sodium caseinate, were prepared, homogenized at 10 M Pa and 80 "C, and spray-dried under identical conditions using a rotary atomizer at I.6 X lo4 r.p.m. as described previously.3 The surfactants used were distilled propylene glycol monostearate (PGMS) and I44
I45
distilled monoglycerides from partially hydrogenated soya bean oil (GMO), both with a minimum of 90% rnonoacyl ester content. For comparison purposes, we have also studied a whipped liquid imitation cream and a whipped dairy cream with 38 wt% fat. Topping powders, reconstituted emulsions (1:4, I hour at 5 "C) and whipped emulsions were prepared for T E M analysis by the freeze-fracture technique using a Bakers BA 360 Unit.' Topping powders were first suspended in anhydrous glycerol to form a highly concentrated suspension, and then small volumes of this suspension (I -2 mm3) were cryofixed by immersion in liquid Freon 22 at - 160 "C. Reconstituted topping emulsions werecryoprotected by mixing with glycerol to a final concentration of 30 vol%, and then cryofixed as above. Whipped topping emulsions were not cryoprotected; they were directly cryofixed. The freezefracture specimen-holders were covered with a small drop of glycerol to achieve better adhesion of the foam. The procedures for freeze-fracturing, replication, and subsequent handling of the specimens for TEM analysis. have been described previously.3 Size-distribution parameters were determined for the fat globules within the powder particles by the following procedure. Freeze-fracture electron micrographs of internally cleaved powder particles, showing clearly the fine structure of the powder matrix, were prepared at final magnifications of 1-3 X 104 depending on the average droplet size. Such micrographs show predominantly a planar fracture through the matrix and the individual fat globules, thus revealing a cross-sectional profile of globules cleaved at random. Size distributions were calculated using a graphic digitizer and an Apple microcomputer. Several hundred cross-sectional profiles of fat globules were outlined in each sample by the electronic pen of the digitizer, and the circumference C and area A of each globule cross-section were calculated and stored. The surface area per unit volume, Sv, of the dispersed fat phase is given byn
where the sum is over all particles i of circumference Ci and area Ai.Equation ( I ) is valid irrespective of the shape of the particles. If the globules are spheres, Sv is related to the volume-surface average diameter dVS(or d,2) by
where di is the diameter of particle i. The fat globules in topping powders often deviate from spherical shape, and so the calculation of size-distribution parameters other than S, requires the simplifying assumption that all visible cross-sectional profiles are true circles. The effective circumference C* is then related to the area by
C* = 2(7rA)'I2
(3)
which enables an effective surface area per unit volume, S*,, to be calculated from S*, = (8/ n")C (Ai)'h
/ ? Ai
(4)
I46
N . Krog. N.M. Barfodand W . Buchheim
The approximate volume-surface average diameter d*Vhis then given by d*,s = 6/S*v The pulsed NMR measurements were done on a Bruker Minispec PC/2OB using the built-in computer program for calculating the solid-fat ~ o n t e n t . ~ Results and Discussion
Application of the freeze-fracture technique to the TEM examination of powders results in detailed information about the internal fine-structure of the powder particles.'.' The fat phase of the various topping powders studied consists predominantly of closely-packed, spherical (or slightly non-spherical) droplets, which are embedded in the continuous solid phase (maltodextrin and protein). Figure I gives a representative view of the powder matrix in a topping powder containing 10 wt% PGMS and 10 wt% sodium caseinate. Freeze-fracturing of the specimen causes droplets to be cleaved either internally or peripherally. Depending on the composition and preparation of the powders, the sizes of fat globules may range from
+
147
Fat-globule sizes in topping powders [Sv = surface area per unit volumefrom equation (I). S*, = surface ore0 per unit volumefrom equation (4), d*,, = average diameter from equation (S)]
Table 1
Surfacranr ronrenrl
Caseinare Conrenil
wt%
wt%
PGMS
10
2
1.1
I .O
6.0
PGMS
10
3
4.6
4.3
I .4
PGMS
10
4
7.9
7.4
0.8 I
PGMS
10
7
14.0
12.7
0.47
PGMS
10
10
18.2
16.7
0.36
PGMS
7
10
17.7
15.6
0.38
PGMS
4
10
16.4
13.8
0.43
0
10
13.7
12.6
0.48
GMO
8
8
20.0
17.2
0.35
PGMS
8
8h
13.8
12.3
0.49
a
S*J m2 ml
'
dfvs/m
Average standard deviation of Sv values is 0.3 m2 ml-' Sodium caseinate with 3% of peptide bonds hydrolysed
system containing G M O is transformed more than the PGMS system; it contains predominantly very thin, but large, crystal platelets. The major structual transformation of the fat phase after reconstitution determines the whipping characteristics of the topping system and the resulting foam structure.' Freeze-fracture electron micrographs of whipped toppings containing PGMS(Figure 5)or GMO(Figure6) indicate that the large, irregularlyshaped, crystalline aggregates interact with the air cells in such a way that their inner surfaces are almost completely covered by crystals. Irregularly-shaped, crystalline fat aggregates mostly prevail within the foam lamellae, i.e., in the serum phase between different air cells. This type of foam stabilization contrasts strongly with that in fat-stabilized foams like whipped dairy cream (Figure 7) or whipped liquid imitation cream based on a vegetable fat (Figure 8). In these last two systems, the foam appears to be stabilized largely by the adsorption of partly agglomerated, but only slightly destabilized, fat globules at the surface of the air cell^.^.^ Figure 9 shows the solids content as determined by pulsed NMR for topping powders and corresponding dry mixtures containing identical amounts of tempered fat phase, maltodextrin and caseinate, intimately mixed. The solids
I48
N. Krog. N.M.Barfod and W. Buchheim
Figure 1
Electron micrograph of topping powder (10 wt% PGMS, 10 wt% caseinate) showing the internal fine-structure of a cleaved powder particle. Fat droplets are cleaved peripherally (A) or internally (B). 7he bar on this and other micrographs corresponds to a length of I Nrn
Figure 2
Reconstituted topping emulsion (no surfactant, I0 wt% caseinate). Fat droplets are individually dispersed and mostly of spherical shape. Most globules show peripheral cleavage within the f a t phase and exhibit layers of crystallized fat as indicated by arrows
I49
Figure 3
Reconstituted topping emulsion (10 wt% PGMS, 10 wt% caseinate). The fat phase has been transformed from the spherical droplets present in the powder (see Figure I ) into larger aggregates of irregularly-shaped crystalline platelets (C)
Figure 4
Reconstituted topping emulsion (8 wt% CMO, 10 wt% caseinate). Structural transformation of the fat phase results in very thin. but large, crystallineplatelets. which appear either as cross-jractures(cC) or surfoce-jractures (sC).Very rarely globularfat (G) is visible
I50
N . Krog. N . M . Barfodand W.Buchheim
Figure 5
Whipped topping emulsion with PGMS. This micrograph (like Figures 6-8) represents a very limited part of thefreeze-fracturedfoam sample. Arrows show the boundary between an air cell (A) and the serum phase (S). The inner surface of the air cell is almost completely covered with fat crystals. There are also crystalline aggregates (C) in the serum phase
Fig1
.cell (A) is covered with flat crystalline layers. Aggregates of thin crystal platelets (C)protrude very ofien into the serum phase (S)
Figure 7
Whipped dairy cream. Arrows show air-water interface between air cell ( A ) and serum phase (S). Fat globules are adsorbed at the airwater intersace andprotrude often with apart ostheir volume into the air cell (*)
Figure 8
Whipped liquid imitation cream. Air cells appear to be covered with a monolayer of largely undesormedfat droplets which protrude partly into the air phase (*)
N. Krog. N.M.Barjod and W.Buchheim
I52
100-
Dry mixtures
80-
60u1
-0
s
:: 40-
20-
0, 1 ()
5
I
10
15
20
25
TemperaturekC
Figure 9
Solids content of topping powders und corresponding dry mixtures
(60 wt% fat phase, 10 wt% caseinate. 30 wt% maltodextrin) as
.,
measured by pulsed N M R . PercentaRe solids is plotted against temperaturefor various concentrations of PGMS: 0 wt%; A, A, 4 wt%; 0,7 wt%; m. 0 , 10 wt%. All samples were temperedfor a minimum of I hour at various temperatures before measurement
+, *,
content of the dry mixtures represents contributions from maltodextrin and protein protons, which are constant over the temperature interval studied, together with the contribution from the equilibrium solid/liquid state of the fat phase at the particular temperature. The fact that the solids content of the toppings is lower than that of the dry mixtures indicates that the equilibrium state of the fat phase is not reached at temperatures below 25 O C . Based on the TEM results, which show a recrystallization phenomenon taking place after the powder is reconstituted in water at low temperature, it is assumed that the lower solidscontent in the topping powders is due to a supercooling of the lipid phase. The difference AS in percentage solids between topping and dry mixture is hence a measure of the degree of supercooling. The data in Figure 9 show that the greatest degree of supercooling is found with 10 wt% caseinate and no PGMS. Addition of PGMS leads to a moderate reduction in the degree of supercooling; but it is largely independent of PGMS concentration within practical levels (4- 10 wt%). The supercooling effect is, however, strongly influenced by the protein concentration of the topping powder, as seen in Figure 10 which shows the percentage solids in powders containing 2,4,7
I53
Dry mixtwe
It
5
10
15
20
25
Temperature/%
Figure 10
Solids content of topping powders (50 wt% hardened coconut oil. 10 wt% PGMS, 2-10 wt% caseinate. 30-38 wt% maltodextrin) as measured by pulsed NMR. Percentage solids is plorted against temperaturefor various concentrations ofcaseinate (on dry basis): A , 2 wt%; 0 . 4 wt%; e,7 wt%; A , 10 wt%
or 10 wt%sodium caseinate, 50 wt% hardened coconut oil, 10 wt% PGMS,and the balance maltodextrin on a dry basis. The solids content decreases with increasing protein content, thereby indicating that the degree of supercooling increases with increasing protein content. The T E M studies have shown that the average fat-globule size in the topping powder varies considerably (see Table I). Since it is known* that the nucleation rate of fat globules is greatly reduced with decreasing particle size, we have compared the degree of supercooling with the size-distribution parameters listed in Table I . The correlation between the degree of supercooling, AS, and the surface area per unit volume, Sv, is shown in Figure I I . For systems containing 10 wt% PGMS and varying amounts of caseinate, a linear relationship is found with a correlation coefficient of 0.99. Crystallization of fat phase in the coconut oil/ PGMS/caseinate system is therefore strongly influenced by the magnitude of the S, parameter. It is also apparent from Figure I I that, by changing either the lipid phase (GMO instead of PGMS, or no surfactant at all) or the protein (hydrolysed caseinate instead of normal caseinate), the degree of supercooling for agiven S, value can become quite different. Whether a similar linear relationship between AS and Svexists for these other systems, however, remains to be established. It is tempting to speculate that a specific interaction between the sodium
N. Krog. N. M. Barfodand W. Buchheim
I54
DNO Surf.
OM0
PGMS OX Caa.
Y
~ 7 Caa. %
v)
a
15
4% Caa.
X Caa.
10
Figure 11
OHydrOl. C.S.
Relationship between supercooling. AS, and surface area per unit volume, Sv.. of the far phase in topping powders: A , 2-10 wt% caseinate. 10 wt% PGMS. 50 wt%farand30-38 wt% maltodextrin;o, nosurfaclanr, 10 wt% caseinare. 60 wt%far and30 wt% malrodexrrin: 0,8wt% GMO, 8 wt% caseinate; 52 wt%fat and 32 wt% maltodexrrin; 0 . 8 wt% hydrolysedcaseinate. 8 wt% PCMS, 52 wt%far and32 wt% malrodexrrin
I 0
Eh 'lo
No Surf
. 20
30
min.
Time n D,O (after reconstitution)
Figure 12
Crystallization of supercooled fat i n toppings measured by pulsed N M R under isorhermal conditions at 15 "C. Percentage solids is plotted against rime in D,O: m, no surfactant: A, 10 wt% PGMS; e, 8 wt% GMO. Topping powders were rempered at 15 "C and reconstituted ( I :4) i n D,O at I5 O C
I55
caseinate and the coconut oil/surfactant lipid phase plays an important role i n determining the supercooled state ofthe lipid phase, and that this interaction is o f a different nature from the so-called 'loop and train' adsorption of proteins at the surface of lipid particles. A deeper penetration of lipophilic protein segments into the outer lipid molecular layers may prevent crystallization of fat globules. The protein-lipid interaction seems to be specific to high-lauric fats like hardened coconut oil or palm kernel oil, and is not evident with other fats like partially hydrogenated soya bean oil (HSBO), fish oil, or normal butter fat. Experiments with HSBO, having an identical melting point and solid-fat index at 20 OC, show greatly reduced supercooling of the fat phase in toppings with poor functionality, and a similar result i s obtained with butter fat or hardened fish oil. A recrystallization phenomenoni s observed i n reconstitutedemulsions based on pulsed N M R measurements of solids content under isothermal conditions. Toppings were reconstitutedin deuterium oxide at a concentration of 25 wt%. Both the toppings and the D,O had been pre-tempered to I5 OC before reconstitution, and the pulsed N M R measurements were made at 15 "C.The results are shown in Figure 12, from which it can be seen that a large increase in solids content takes place in the toppings containing G M O or PGMS immediately after reconstitution under isothermal conditions, while toppings without surfactant show no such increase. This i s consistent with the TEM investigations and the rheological behaviour of the reconstituted emulsions.3 Toppings with CMO or P G M S exhibit strong destabilization with formation of a crystalline matrix giving rise to a viscosity increase and good whippability. Toppings without surfactant destabilize very little, remaining as low-viscosity emulsions after reconstitution, with no whippability. The kinetics of destabilization and recrystallization of the
u)
40 v)
E 20
01 0
10
20
30
40
50
80 &
*
Time in 40 (after reconstitution)
Figure 13
Crystallization of supercooled far in toppings measured by pulsed N M R after reconstitution in D,O at 15 O C. Percentagesolids isplotred against rimein D,O 0 , nosurfacrant: A, 1.4 wt% GMO(rareconsranr = 1.1 X 10 min-I); 3.5 wt% G M O (rate constant = 2.4 X 10 * min I); m, 7.0 wt% CMO(rate constant = 8.7 X min I )
*,
N. Krog. N. M.Borfod and W. Buchheim
I56
reconstituted emulsions is controlled by the type and concentration of surfactant. Figure 13 shows the recrystallization rate for toppings with 0, 1.4, 3.5 and 7 wt% GMO as measured by pulsed N M R in D,O. The rate increases with the GMO concentration and follows first-order kinetics as is normally found in coalescence studies of oil-in-water emulsion^.^ The destabilization process involves a transfer of protein from the oil-water interface to the bulk aqueous phase which is promoted by surfactants like PGMS o r G M 0 . 4 This is demonstrated in Table 2, which lists the relative protein concentrations in the cream layer and aqueous phase of reconstituted and centrifuged topping emulsions. The extent of protein desorption from the oil-water
Table 2
Distribution ofprotein betweenfat creamphase andaqueousphase o/ centrifuged topping emulsions reconstituted at 5 "C and 30 OC
Surfacront Conrenil wr%
Proreinl wt%'Ol Reconsrimred at 30 " C Reconstilured or 5 "C FOI Aqueous Far Aqueous
10 (PGMS)
I .3
98.7
33.7
66.3
10 (CMO)
0.0
100.0
29.2
70.8
-
24.0
16.0
41.7
58.3
Percentage of total protein in fallcream and in aqueous phase (taken from ref. 4)
Table 3
Comparison between destabilizarion parameters of toppings and their whippability uoam stiffness measured on Stevens Texture Analyser, 2.5 cm probe, penetration I cm, speed 0.2 cm s-I) Surfactant Conrentl wt%
Relaiive Rare of Crystalkorion"
Overrun1 %
Foam Srgfness index1 g wt
PGMS
4
20
I68
25
PCMS
7
41
26 1
38
PGMS
10
54
368
67
GMO
I .4
I1
63
4
GMO
3.5
24
101
63
GMO
7
87
I35
I37
I05
97
dairy cream See legend to Figure I3
I57
interface is temperature dependent, being greater at 5 OC than at 3 0 ° C . Destabilization and whippability of the emulsions follow the same pattern. The destabilization process of reconstituted topping powders is an important factor in relation to obtaining good whippability and a satisfactory foam texture and stability. The mechanism is believed to involve the following stages: ( I ) desorption of protein from lipid particles, followed by ( 2 ) coalescence and ( 3 ) recrystallization of supercooled lipid phase leading to an increase in viscosity of the reconstituted emulsion. Table 3 shows the correlation between the physical properties of various toppings and their whippability. The powders were reconstituted 1:4 incold water (I0 " C )and whipped for 3 minutes with a household mixer, after which the overrun and foam stiffness were measured. A standard dairy cream with 38 wt% fat was also tested for comparison. There is an increase in crystallization rate, percentage overrun and foam stiffness with increasing surfactant concentration in the topping powders. The contribution of the destabilized fat phase to the foam stiffness is about four times larger (per weight unit of fat) in whipped toppings than it is in whipped dairy cream o r liquid vegetable-oil based imitation cream. References I 2 3 4
5 6 7 8
9
W. Buchheim, Gordiun, 1978.6, 184. D.G. Schmidt and A.C.M. van Hooydonk, Scunning Efecrron Microsc., 1980.111.653. W. Buchheim, N . M . Barfod, and N . Krog. FoodMicrosrrucrure, 1985.1.221. N.M. Barfod and N . Krog, J. Am. Oil Chem. Soc.. 1986, in the press. 1. Trurnbetas, J.A. Fioriti, and R.J. Sims, J. Am. Oil Chem. Soc., 1979.56.890. E.R. Weibel, in'Principlesand Techniques of Electron Microscopy.'ed. M.A. Hayat. Van Nostrand-Reinhold, New York. 1973. Vol. 3, p. 239. W. Buchheim. Scunning Electron Microsc.. 1981,111,493. W. Skoda and M.J.van den Tempel, J. Colloid Sci., 1963,18,568. M.A.J.S. van Boekel, 'Influence of fat crystals in the oil phase on the stability of oil-in-water emulsions,' Ph. D. Thesis, University of Wageningen, 1980.
Polar Lipids in Emulsions and Microemulsions
By L. HERNQVIST
(Departmentof Food Technology,University of Lund. Box 724. 5-221 00 L und, Sweden)
Introduction Polar lipids often play an important role in the structure of food. Most frequently, however, it is not the polar lipid molecules themselves which influence the structure, but aggregates of many lipid molecules in association with water. Friberg and co-workers' introduced the ternary system oil -tsurfactant 4-water as an aid to understanding the stabilization of emulsions by polar lipids. Figure I
OIL
F Figure I
C
D
Phase diagram for the system soya bean oil -I- sunflower oil monoglycerides (MG)+ water at 40 " C .Lnhels L2, F, Cond D refer fo various liquid crystallinephases. AN compositions are expressed on a weight basis I58
I59
+
shows the phase diagram of the ternary system soya bean oil sunflower oil monoglycerides -twater.* The diagram shows that several types of liquid crystalline phases can be formed:
L,: F:
C:
D:
an isotropic oily solution, consisting of reversed micelles with water aggregates in a hydrocarbon-chain medium;' a reversed-hexagonal liquid crystalline phase, consisting of cylindrical aggregates of water in a continuous medium of surfactant molecules, with polar groups orientated towards the water phase and hydrocarbon chains filling out the gaps between the water cylinders; a cubic liquid crystalline phase, which is isotropic and very viscous, and whose structure is ~uggested'.~ to be bicontinuous, consisting of lamellar bilayers separating two water-channel systems; a lamellar liquid crystalline phase, periodic in one dimension and consisting of bimolecular lipid bilayers alternating with layers of water.
Emulsions It was proposed by Friberget 01.' that the lamellar liquid crystalline phase stabilizes an oil-in-water emulsion by forming a film at the oil-water interface. Figure 2 shows that the lamellar liquid crystalline phase can direct a hydrophobic surface towards the oil and a hydrophilic surface towards the water, and so it is obvious that this phase possesses ideal structural properties for reducing the surface energy of oil-water interfaces. Extreme situations are the multilayet structure at high polar lipid concentrations (Figure 2) and the monolayer structure at low emulsifier concentrations. The reversed type (water-in-oil) has not yet been identified in any
Water
Oil droplet Figure 2
Model of a multi-layered membrane structure formed by phospholipids at an oil- waier interface
L. Hernqvisr
I60
food system containing polar lipids and water, but, if it is to be prepared, the kind of ternary phase diagram shown in Figure I will be most relevant. For such a case to occur, only a small amount of monoglyceride is needed, but the emulsion must end up in the three-phase region with oil, water and liquid crystals coexisting in thermodynamic equilibrium. The implication is that, if liquid crystalline phase is present, it will contribute in one way o r another to the stabilization of water droplets in a continuous oil phase. Microemuisions The term 'microemulsion' was introduced before the structure of such systems was known. In fact, the term is quite misleading, since microemulsions are not true emulsions, but are thermodynamically stable phases. The microemulsion discussed in this paper is the reversed micellar solution called L,. The relevance of the L, phase to food systems has already been indicated above, but tostress its importance further the ternary system of Figure I should be compared with that given in Figure 3.2.6The only difference between them is the value of the temperature. We see that, when the temperature is raised, liquid crystalline phases F, C and D disappear and are replaced by an extended L, phase. The phase diagram is not dependent on which triglycerides are present; but if the surfactant is changed the positions of the phases are substantially different. Variation in the proportions of oil, water and monoglycerides within the L, phase region indicates different structures of the L, phase. The definition given above - a
OIL
H2° Figure3
MG
Phase diagram for the system soya bean oil -I- sunflower oil monoglycerides (MC) i- waier aI 90 ' C . AN compositions are expressed on a weight basis
161
OIL
/
/ H2°
MG
Figure 4
Illustration of different reverse-micelle structures in the L2region of thephase diagram shown in Figure I
Figure 5
Electron micrograph ofthe L,phase in the monoglyceride-rich part of the L2region. The bar corresponds to a length of 0.5 p m
I62
L. Hernqvisl
reversed-micellar isotropic solution - should therefore be extended. The reversed micelles are thought to be more-or-less spherical with water inside the micelles and triglycerides surrounding them (see Figure 4). The closer the composition is to the triglyceride corner of the phase diagram, the longer is the separation between the reversed micelles. The more monoglyceride that is present, the smaller is the distance between them. With roughly equal amounts of oil and monoglycerides, it is probable that spherical reversed micelles are obtained. At high monoglyceride concentrations, the structure comprises a stack of flexible discs (see Figure 4). Figure 5 is a photograph from a freeze-etching electron microscopy study.’ White, grey and black fields can be seen in the micrograph. Differences in the grey tones are due to the orientation of the fracture. Some of the regions are oriented in such a way that parallel lines can be discerned; the distance between these lines is estimated to be ca. 5 nm, which corresponds to a bilayer. The L, phase structure has been analysed in our group by X-ray diffraction?’ and the results are fully consistent with the electron microscopy. Segregation of Oils
When the ternary system shown in Figure I is examined closely, a two-phase region is seen to the left of the pureL, phase running from the oil corner to the extended lower part of the L, phase region. In this region, the oil and L, phases coexist together in a thermodynamically stable state. Depending on the proportions, we obtain an oil-in-L, emulsion or an L,-in-oil emulsion. As oil is one of the major components of the L, phase, it could be said that in this two-phase region one oil is being dispersed in another. When two different oils are mixed without any L, phase present, a homogeneous oil mixture is obtained almost immediately, but this is not the case if one of the oils is in the L, phase. In this latter case, the L,-oil interfaces of the L, phase droplets are probably covered with a bilayer of monoglycerides as shown in Figure 6. Even if the oil and L, phases are thermodynamically stable, the segregation of the triglycerides will not last for ever. Exchange of triglyceride molecules probably occurs across the interface between the two phases, but the rate seems to be rather low. An experiment has been carried out9 to investigate the oil segregation effect. Figure 7 shows the general polymorphic transitions of triglycerides, although different triglycerides d o show slightly different polymorphic behaviour. In the experiment, tristearin (StStSt) with ‘normal’ bilayers in the crystalsi0 and 2-oleodistearin(St0St) with triple layers in thecrystalsiOwere used. When an StStSt melt is cooled, it normally crystallizes in the a-form,’2.’3and thereafter can develop the p2-form,’2-’4the P’r-form,i2and finally the most stable &form.” StOSt can follow the series ~uba~a-P;-/.3’,-P.~~ The pure triglycerides StStSt and StOSt develop totally different crystal structures when treated according to the following temperature programme: 90 “ C for 3 minutes, rapid cooling to 15 O C , 15 OC for 3 minutes, 33 O C f o r 3 minutes, isothermal storage at 23 “C. The StStSt develops the a-form with double-chain layers, and the StOSt develops the &form with triplechain layers. Now, when StStSt and StOSt are mixed in the proportions 2: I, and given the same temperature treatment, the mixture co-crystallizes and develops the d o r m with double-chain layers. On the other hand, if StOSt is used as
I63
Palissade layer
Oil (2)
48 h j h%V Figure 6
Schematic representation of the interfacial region of an L2 droplet (containing oil I) dispersed in oil 2
the triglyceride oil in preparing an L, phase, and if this L, phase is of such a composition that it gives adispersion when mixed with another oil (i.e., StStSt), a totally different crystallization behaviour is obtained. As the mixture of the L, phase (StOSt) and the oil phases (StStSt) are in the two-phase region of Figure I , two segregated thermodynamically-stable phases are obtained. After giving the mixture the same temperature treatment as previously described, the segregation can be identified from the information given in Table I and Figure 8. The difference from the ordinary mixture not containing the L, phase is obvious: both (I-and p-forms are found, indicating that double- and triple-chain layers are present at the same time. When this sample is heated to 90 "C for 30 minutes, and the same temperature treatment as above is repeated, a similar segregation of StStSt and StOSt triglycerides is obtained. This shows that the segregation is quite stable.
L. Hernqvist
164
liq. - -
I
\
\
I I
,\
I I
\
I
\
I \
I
I \
/ I
solvents Figure 7
The complete set of triglyceride polymorphic transitions. All phase changes are irreversible except the liquidla and alsuba transitions. The dotted lines recognize the possibility of a direct crystallizationinto forms other than the cr-form. Also indicated is the possibility of growing crystalsfrom solvents
Table 1
Long and short spacings in triglyceride structures as given by X-ray diffraction(s=strong, m = medium, w=weak. vw=very weak). All distances are given in angstrom units SI SI SI
SI OSI
SISISI Sl OSI
+
+
SISlSI (SIOSI in Lz)
3.63 w
3.64 m 3.84 w
3.91 s
3.95 s 4.05 w 4.15s
4.16 s
4.16s
4.29 w 4.52 m
4.48 w
4.83 s
4.73 m
5.28 m
5.25 w
14.3 m
14.6 rn 17.2 rn
17.0 m
16.9 rn
18.1 w 24.8 m
23.8 m 26.0 vw
25.8 vw 37.0 s
36.8 s
50 s
50 s
49 s
I65
37.0
i
Figure 8
Phorodensitometer (racing of X-ray di/fraction pattern ofthe mixture StStSt (StOSt in LJ. The numbers refer 10 structural spacings in angstrom units (See Table I )
+
Influence of Liquid Crystals on Fat Crystallization The influence of a segregated L, phase on the polymorphic behaviour of hydrogenated fats is illustrated in Figure 9. The binary system consists of low erucic rape-seed oil (LOBRA) hydrogenated to a melting point of 40 O C and soya bean oil (Soya) hydrogenated to a melting point of41 O C . I 6 When Soya41 is crystallized, it develops a fairly stable p’-form, whereas LOBRA 40 very quickly undergoes the p’-P transition.” Figure 9 shows the rate of the p’-P transformation for different mixtures of Soya41 LOBRA 40 stored at 23 “C. The 5 0 5 0 mixture develops the @-formafter 4 days, but this time is halved if the L, phase is introduced. Neither the amount of L, phase nor the type of oil used is important, which means that it is probably the surface introduced with the L, phase which dictates the change in polymorphic behaviour. Since the L, droplets are well distributed in the entire fat blend, the extra surface increases the probability of cry~tallization,’~ and therefore the probability of the p’-P transition. To understand more fully the influence of the L, phase on crystallization, aseries of different patterns was collected as a function of time.I8 The experimental conditions were the same in all cases: 90 O C for 3 minutes, followed by rapid cooling to 40 O C , whereupon the samples were kept isothermally and X-ray diffraction data were collected as a function of time. The results are summarized in Figure 10. Trimyristin (MMM) was chosen because it was thought to crystallize in the p’,form. When a melt of MMM iscooled slowly (0.4 OC min-I), it crystallizesdirectly
+
166
L. Hernqvisr
Ti me / d ay A
6
4
2
--
L
==
--
A
Soya 41
LOBRA 40 Figure 9
Kinetics of the /3’-/3 transition for the binary system LOBRA 40 t Soya 41 at 23 O C . The transformation time is plotted against the weight percentage composition: A, binary mixtures; m, 5050 mixtures with theaddition of either (i) 3 , 6 or 9 wt% L,phase basedon LOBRA 40 monoglycerides t water, or (ii) 9 wt% L2phase based on coconut oil monoglycerides water
+
in the p’,-form at about 32 O C . ’ j This temperature happens to be the melting point of the a-form of MMM, and, as the melting point of the MMM @’,-formis 46 O C , the temperature for the diffraction experiments was chosen to be 40 OC, which is still in the undercooled region but 8 OC above the d o r m melting point. The time before the pure M M M crystallizes was found to be 25 minutes, with the /?’-/? transition occurring after 52 minutes. When triolein (000)was introduced into the system, the crystallization was found to be slower, but the p‘-/3 transition occurred much earlier, and in fact M M M / 0 0 0 (66:30) crystallized directly in the /?-form. The mixture of MMM and 000 in L, (66:33) also crystallized directly in the p-form, but much quicker than M M M / 0 0 0 . The OOO/ L, solution obviously influences the polymorphic behaviour of MMM. The OOO/ L, solution consisted of 000-monoglycerides (MG) and water in the proportions 15: 15:3 selected to give two phases when mixed with MMM (cJthe two-phase region in Figure I). Since M M M and 000 are present in the proportions 66:33 in the L, phase, L, droplets consisting of 000 in a continuous phase of M M M are obtained. Probably the surface of L, droplets increases nucleation rate. Triglycerides can be stabilized
I67
0
MMM
20
I
I -1iq.
Time at 4 0 t (minutes )
40
:
B-
B’ j
I
I
; I
j
I
I
I
I-liq.
-
I
II
I
II
+liq.
;
I
I
I
I
M M M -000 (66
15) I
-I
MMM-000 (66
30)
liq.
B+
I
MMM-000-MG (66
15)
MMM-MG (66
MG
IS)
15
j
I
P+
II
p-’~‘
Iiq.+-+B*e---B,:
I
Figure 10
I
I
+
B+
4 I
,
b
I
Polymorphic transitions at 40 O C after rapid cooling. The type of phase, as indicated by the dffraction pattern. is plotted agoinst the time for systems containing trimyristin (MMM), triolein (000)and triolein monoglycerides (MG). Numbers in brackets refer to proportions by weighi
and packed more densely if hydrocarbon chains in the monoglycerides are arranged as a relatively stable surface on the L, droplet (Figure 6). The mixture MMM/OOO/MG (60:15:15) shows the same polymorphic behaviour as M M M / 0 0 0 (66:30),i.e., liquid-8 after 30 minutes. As this mixture is not in the two-phase region, neither segregation nor any surface from L, droplets is present. The mixture of M M M MG (66:IS) showed early crystallization (liquid--P’ after 22 minutes. followed by slow transformation into the 8-form), but after 60 minutes some of the p’-form was still present. It seems as if the monoglycerides act in two different ways in this mixture. On the one hand, crystallization occurs earlier compared with pure M M M because of MG aggregation; on the other hand, the p’-B transition is delayed in some way by the monoglyceride. T o complete the picture, polymorphic behaviour of the pure monoglyceride was followed, but no crystallization was observed. The ability to mix two different oils and still keep them separate may have technical applications. But there is much fundamental research to be done before the phenomenon could be used in practice.
+
Anti-oxidants in the L2Phase
Fat oxidation in an L, phase was studied recently in our laboratory.’’ Different L, solutions from the ternary system represented in Figure I were used, and it was observed that the presence of ascorbic acid in the water aggregates gives a
L. Hernqvisr
I68
COP
6
-=
4
-=
2
-I
Figure 1I
4OoC
rn
%
0
" A
A
A
A
A
. I
I
I
I
20
40
60
Effect of ascorbic acid onfat oxidation rate as measured by the COP value (units of absorbance in a I cm cell with a I % w/ v lipidsolution). The COP value is plotted against the storage time t f o r a microemulsion made f r o m I wt% water. 19.8 wt% sunflower oil monoglycerides and 79.2 wt% soya bean oil: A, microemulsion of 5 wt% ascorbic acid solution: m. microemulsion of water only; e, mixture of soya bean oil and monoglycerides in the proportions 19.2:19.8
pronounced reduction in oxidation rate (see Figure I I). Following storage at 40 O C for 100 days, only minor oxidation of the oil was observed, whereas the control without ascorbic acid was highly oxidized. The concentration of ascorbic acid can be very low to achieve the required effect. If the water in the L, phase contains 0.5 wt% ascorbic acid, the overall concentration of ascorbic acid may be as low as 50 ppm, but still good protection against oxidation is obtained. Because L, phases are thermodynamically stable, both water-soluble and oil-soluble anti-oxidants can be used together (e.g., ascorbic acid and the oil-soluble tocopherol). This must be of great technical importance. Conclusion
It can be said that L, phases give new technical possibilities in food technology. Up to now, we have only worked on two of them: crystallization and oxidation. Another example might involve incorporation of water-soluble flavours in oils. References I S. Friberg, L. Mantell, and M. Larsson, J . Co//oidInrerfaceSci.,1969.29, 155. 2 E. Pilrnan, K . Larsson, and E. Tornberg. J. Dispersion Sci. Techno/., 1982.3.335,
I69
3 P.Ekwall, in'Advances in Liquid Crystals.'ed. G.H. Brown, Academic Press, New York, 1975,Vol. I, p. I . 4 G. Lindblom, K. Larsson, L. Johansson, K. Fontell. and S.ForsCn, J. Am. Chem. Sue., 1979,101,5465. 5 K. Larsson, K. Fontell, and N. Krog, Chem. fhys. Lipids, 1980,27,321. 6 K. Fontell, L. Hernqvist, K. Larsson, and J. Sjdblom, J. Colloid/nter/ace Sci., 1983,93, 453. 7 T.Gulik-Krzywicki and K. Larsson, Chem. fhys. Lipids, 1984.35, 127. 8 K. Larsson, J. Colloidlnterface Sci., 1979.72, 152. 9 L. Hernqvist and 0. Leissner, unpublished work. 10 K. Larsson, Proc. Chem. Soc., 1963.87. I I K. Larsson, Fette-Sefen-Anstrichmittel,1972,74, 136. I2 L. Hernqvist and K. Larsson, Fetfe-Sefen-Anstrichmittel, 1982.84.349. I3 L. Hernqvist, Fette-Sei/en-Anstrichmittel, 1984,86,297. 14 T.D. Simpsonand J.W. Hagemann, J. Am. OilChem. Soc., 1982,59,169. 15 L. Hernqvist, 'Polymorphism of fats,' Ph.D. Thesis, University of Lund, 1984. 16 L. Hernqvist, 0.Leissner, and B. Pettersson, Feite-Sefen-Anstrichmittel, in the press. 17 L. Hernqvist, B. Hersldf, K. Larsson, and 0. Podlaha,J. Sci. FoodAgric., 1981,32,1197. 18 E. Stenhagen, Arlo Chem. Scand., 1951.5.805. 19 L. Moberger, K. Larsson, H. Buchheim, and H. Timmen, J. Dispersion Sci. Technol., in the press.
Interfacial Behaviour of Protein Mixtures at Air-Water Interfaces By ELAINE K . MURRAY
(AFRC Institute of Food Research, Shinfield, Reading. Berkshire RG2 9AT)
Introduction The interfacial properties of single protein systems have been well characterized over the years. The work of Graham and Phillips' on the kinetics, adsorption isotherms, shear and dilational properties of adsorbed films of p-casein, bovine serum albumin and lysozyme has provided evidence for a detailed theory of the processes involved in forming protein films at interfaces, and has highlighted differences between films of random and globular proteins. These results have been linked6 to the rolesof these proteins in stabilizingfoams and emulsions by reducing interfacial tension and the rates of collapse and coalescence. Proteins at interfaces are known to be unfolded to an extent dependent on their native structures and local environmental conditions; in globular proteins the unfolding process may resemble heat denaturation.' Food foams and emulsions contain many surface-active materials, and so their interfaces may contain several species. Once adsorbed, these different types of molecules may interact with one another, possibly further affecting the properties of the interfacial layer. The study of mixed interfaces in comparison with systems containing only the constituent species is therefore of interest, but it has not been extensively carried out. Work so far has included lipid-protein interactions,8-I0 interactions between proteins and emulsifiers,11.12 and the rheological properties of mixed protein interfaces.I3 The work reported here involves four common milk proteins: P-lactoglobulin, a-lactalbumin, p-casein and K-casein. A Langmuir film balance has been used to compare the effects of age, temperature and surface pressure on the interfacial behaviour, as measured by surface pressure/surface concentration (n-r)isotherms and rates of film collapse under pressure. Results are presented for each protein alone, and for 5050 wt% mixtures of P-lactoglobulin with the other three proteins. Heat denaturation of P-lactoglobulin with a-lactalbumin and with K-casein is k n ~ w n ' ~to- 'result ~ in the formation of complexes by intermolecular disulphide linkages. One aim of this study was to attempt to detect any similar interactions I70
171
under conditions of surface denaturation, and to compare the results with those for p-lactoglobulin/P-casein mixtures for which no such interactions would be possible. Heat denaturation of whey proteins may be reversible below 60 O C (see reviews by de Wit and KlarenbeckIh and Kester and Richardson”), involving partial loss of three-dimensional structure with unfolding of the protein and changes in hydration, and irreversible above the denaturation temperature, resulting in reduced solubility. Measurements of the rate of collapse of protein films on the Langmuir balance have been used to try to detect effects due to changes in structure or solubility brought about by differences in the extent of surface denaturation under different conditions. The influence in protein mixtures of possible complexing on these rates has also been investigated. Materials and Methods
The a-lactalbumin was purchased from Sigma Chemicals, and the other proteins were prepared at the Institute. The K-casein was stored in its reduced form in a buffer containing I wt% mercaptoethanol and 7 M urea. Experiments were performed on a Joyce Loebl Langmuir-Blodgett film balance. Temperature control was achieved using a glass heating and cooling coil immersed in the subphase of the trough (designed by Dr. T. Hardman of the University of Reading). For temperatures below ambient, the water circulating through the coil was thermostatically controlled by a Techne Circulator plus Flow Cooler. Water used for the subphase was distilled and passed through a Zerolit filter. The airwater interface was cleaned by successively removing the interfacial layer using a Pasteur pipette attached to a water pump, as the surface area was reduced. This treatment was performed until the change in surface pressure in compressing the interface from maximum area (966cm2) to minimum area (145 cm2) was not more than I mN m-I. Surface pressures were measured using a filter-paper Wilhelmy plate (width I cm) suspended from a microbalance. Protein isotherms were measured at a compression speed of I .06cmz s I. Areas were calculated by suitably calibrating the digitally displayed voltage output from the barrier drive. Spread monolayers were used rather than the adsorbed protein films for a variety of reasons. In the first place, the volume of the trough (approx. IS 1) made adsorption from the subphase impractical due to the large amount of protein that would have had to have been used. In addition, with spread films the criterion for a clean air-water interface could be more easily met, and bulk concentration could be eliminated as a factor in film collapse at the higher surface pressures. Proteins were spread on the cleaned subphase surface from 66% isopropanol/water mixtures using a Hamilton microsyringe. Mixture concentrations were adjusted to allow calculation of the amount of protein spread. After about 10 minutes, initial equilibrium surface pressures of 5 mN m-I were produced from 0.08 mg plactoglobulin, 0.18 mg K-casein and 0.18 mg a-lactalbumin; 0.08 mg /%casein spread over the maximum area produced a slightly higher pressure (cu. 7 mN m-I). For the protein mixtures. quantities of the component solutions were pre-mixed to give 5050 wt% mixtures, which were again spread in amounts giving initial equilibrium surface pressures of 5 mN m-I or less. Apparent surface concentrations were estimated from the amount spread and the surface area calculated from the
E. K. Murray
I72
barrier-drive voltage output. The values are likely to be overestimates of true surface concentrations at surface pressures greater than 20 mN m-I where collapse of the protein film may involve displacement of whole molecules from the interface. Isotherms of surface pressure n versus apparent surface concentration r were obtained at 510 OC, 25 OC and 40 O C . The last two temperatures were controlled to f l "C;temperatures below 10 O C were more difficult to maintain over afew hours, but they were in the region 6-10 OC. (Low temperatures were obtained by precooling the subphase water overnight at 5 O C , and circulating the cooling coil with water at 2 "C.) The pH of the subphase was 5.6f0.1. MacRitchie has published a series of on the collapse of protein films, and in this work methods based on his are used to probe the rates of collapse of spread films under different conditions. In one method, the surface area at 5 mN m-I is measured, the film is compressed at a higher surface pressure (>20 mN m-I) for a measured interval of time (usually 10 minutes), it is rapidly re-expanded to the maximum area, and then the surface area corresponding to II = 5 mN m-I is remeasured. The pressure may be held constant using the feedback system of the Langmuir-Blodgett trough. Rate constants are calculated according to MacRitchie's equation for diffusion-controlled collapse kinetics, i.e., In(A/A,) = kt1'2 where A,, is the surface area at n = 5 mN m-' before compression for time t, A is the area at this surface pressure after compression, and k is a rate constant. The rate of change of area at higher Compression pressures (>20 mN m-l) was also measured. One method of analysis was directly analogous to that of MacRitchie:
In[A(t=t)/A(t=O)]n,mmNm,
0: kt''2
In another method, the decay curve has been analysed in terms of a double exponential plus a constant term, i.e., A(t=t) = a,exp(-k,t) 4- a,exp(-k,t) -ta,,
(3)
where a,, a l , a2, k, and k, are constants. Areas were measured at suitable time intervals, and the data were subjected to either a linear least-squares fit in t1/2 [equation (2)] or a non-linear regression analysis [equation (3)]. The purpose of the above measurements was to find out whether changes in the protein film brought about by changes in conformation or complexing with other proteins might produce changes in the rate of film collapse. Possible mechanisms involved in the decrease in surface area at pressures above 20 mN m-l include expulsion of protein segments, displacement of whole molecules, and coagulation. These are likely to be affected by protein conformation, intermolecular interactions and solubility. Surface pressures of 22.5,25.0,27.5 and 30.0 mN m- I were used to find the most appropriate pressure for comparative purposes. Protein films have been aged at different surface pressures: low ( 5 5 mN m-I) and high (20 mN m-I). At low surface pressures, a large proportion of the protein molecules may be fully denatured; at higher pressures, the film is more concentrated, and denaturation will tend to be more difficult and less complete.
I73
Results and Discussion Figure I shows II-rcurves measured at 9 OC, 25 OCand 40 OCforp-lactoglobulin five minutes after spreading the film. Two factors could be responsible for the change in shape of the isotherm with temperature, and an increase in the solubility of the protein, and in its rate of diffusion from the interface, which would tend to be most noticeable at high surface pressures. In the curves at 9 O C and 25 "C, an inflection is observed at a surface concentration of 2-3 mg m2(corresponding to maximum monolayer coverage in adsorption). A similar inflection was reported by Graham and Phillips for the n-c adsorption isotherm of lysozyme, but at a lower surface pressure (8 mN m-I); it was attributed to the pressure at which molecules are prevented from unfolding by the presence of molecules already in the film. Graham and Phillips also observed that the inflections were absent from the n-c curves of reduced P-lactoglobulin and heat-denatured lysozyme.
50
40 c
I
E
$
30
\
?!
2
R
2 Q
20
0
m ) I
f
v)
10
I 1 I I I I 0 1 2 3 4 5 6 Apparent surface concentration/mgm-
n-
Figure 1
*
Eflect of temperature on the surface equation of state of placioglobulinfour minutes after spreading at the air-water interface. Surface pressure is plotted againsr apparent surface concentration at T = 9.25 and40 "C
I74
E. K . Murroy
50
r
40
-
0
1
2
3
4
5
6
7
Apparent surface concentration/mgm-
Figure 2
8
9 2
Effect of aging at low and high surfacepressures on the surface equation of state of a-lactalbumin 01 25 O C. Surface pressure is plotted against apparent sur-ace concentration afler I = 5 and 90 min
It is postulated here that the inflections at 9 "C and 25 "C in Figure I are due to refolding of proteins in the surface. At 40 OC the proteins are assumed to be sufficiently denatured for refolding not to occur. Mitchell et af.24have compared various types of results: (i) II-A isotherms of spread films, (ii) II-t data of adsorbed protein films, and (iii) n-c curves for films where the surface area was kept constant and successive aliquots of protein spread on the surface. They found that II-c and n-t curves were in better agreement with each other than with the Il-A isotherms, except for random coil proteins and denatured globular proteins. They also found that II-A curves were generally independent of protein structure. The explanation given24is that in spread I'I-A films all molecules are extensively unfolded, having entered the surface at very low surface pressure, whereas in n-c experiments the successive molecules entering the film d o so against the pressure of molecules already present, so that unfolding ofglobular proteins is inhibited, and the situation is like that during adsorption. The results reported here appear at first sight to
I75
disagree with those of Mitchell el al.24This is probably due to the use here of a higher initial concentration (0.83-1.86 mg m-2 as opposed to 0.475 mg m-2), leading to more concentrated films which are less extensively denatured. Isotherms for a-lactalbumin were found to show similar effects with increasing temperature; the inflection point was below 30 mN m-I at an apparent surface concentration of 4-5 mg m-2. The isotherm for a-lactalbumin at 40 O C was flatter than that for P-lactoglobulin. Figure 2 shows the effect of age on the n-r isotherms of a-lactalbumin at 25 O C , and the difference between aging at low pressure (2 mN m-I) and high pressure (20 mN m-I). Aging for 90 minutes at 2 mN m-I produces a flattening of the measured isotherm similar to that found in a film spread at 40 * C. It is probably due to the further denaturation of the protein during the time interval from 5 to 90 minutes. When the film is aged at the higher pressure, the isotherm is more like that observed initially, although surface pressures are a little lower for given surface concentrations (possibly due to slight loss of material from the interface); the inflection at 27-30 mN m-I is still observed, suggesting that refolding of proteincan still occur to some extent. Similar results are found with P-lactoglobulin. Figure 3 shows the effects of age and temperature on &casein n-r isotherms. The shapes change little, and are similar to those of heat- and age-denatured
e=T=7.6OC, t=2hrr
Q)
0
m
.L c
;1 0 1
0
l l l l l l r l
0 1 2 3 4 5 6 Apparent surface concentration/mgmFigure 3
7
2
Eflect of temperature and aging on the surface equation of state of p-casein. Surface pressure is plotted against apparent surface concentration
E. K. Murray
I76
40
r
Apparent surface concentration/mgm-
Figure 4
Effect ofaging on the surface equation ofstate ofrc-casein at 25 O C after spreading f r o m 66% isopropanol solution containing mercaptoerhanol and urea. Surface pressure is plotted against apparent surface concentration after t = 10 and 60 min
30 I
E
z
-E ?!
20
v)
?!
P
0
r m
;10
-
0
0 1 2 3 4 5 6 7 Apparent surface concentration/mgm
-
Figure 5
Comparison of n-r isotherm of P-lactoglobulin 3- &casein (40 " C , aged for 2 hours at low surface pressure) with those for individual proteins. Surface pressure is plotted against apparent surface concentration
I77
8-lactoglobulin and a-lactalbumin. The lower surface pressure at 7.6 OC and 40 "C is probably due to increased solubility at 7.6 OC and an increased rate of diffusion of expelled molecules from the interfacial region at 40 O C . The slight change in shape on aging at 25 O C is less easily accounted for, and it may be an artefact. Although K-casein is known to have a more ordered structure than 8-casein, its isotherms (Figure 4) are similar in shape. The K-casein was spread in its reduced form. The change in isotherm shape between 10 minutes and 60 minutes may be due to removal of mercaptoethanol from the interface, and subsequent formation of disulphide bonds. Mixtures (5050 wt%) of 8-lactoglobulin with 8-casein, K-casein and alactalbumin werespread at the air-water interface and the resulting films were aged for different lengths of time at high and low surface pressures a t 5 1 0 O C , 25 "C and 40 " C .The n-r curves of P-lactoglobulin -I-&casein show an unexpected feature (see Figure 5). When the mixed protein film had been aged at 20 m N m-I, the
50
/
/c n=20
40 r
I
2E 30 \ ?! 3
v) w
g?j20 m
r L
3 v)
10
0
0
1
2
3
4
5
6
7
8
Apparent surface concentration/mgm-
Figure 6
Comparison of ll-r isotherm of 8-lactoglobulin i- a-lactalbumin (40 O C, aged for I hour) with those for individual proteins. Sugace pressure is plotted against apparent sugace concentration
I78
E.K. Murray
resultant isotherm was approximately the average of those for the pure constituent proteins. When aged at low pressure, however, the mixture curve was steeper than for either protein alone under the same conditions, but similar in shape to that obtained at n = 20 mN m I. It is possible that the more surface-active p-casein prevents P-lactoglobulin molecules from unfolding extensively at the interface. The protein K-casein is less surface-active than &casein o r P-lactoglobulin. The isotherm of its mixture with a-lactalbumin (not shown), when aged at low pressure at 40 "C, was similar to that of P-lactoglobulin up to r = 3 mg m *,but deviated at higher concentrations. The shape of the curve was little affected by conditions of aging, and pressures were always below those for P-lactoglobulin aged at 20 mN m I. Complexing via disulphide bonds may occur between the two proteins, as happens on heating in solution; if so, the extent seems not very dependent on surface pressure. This implies that P-lactoglobulin is sufficiently denatured at the experimental temperature (40 "C) for the complex to be formed even at high surface pressures. Studies of solutions of P-lactoglobulin -I-K-casein have shown that complexing can occur when P-lactoglobulin is denatured prior to mixing. Figure 6 shows the effect of aging at high and low surface pressures on the isotherm of P-lactoglobulin a-lactalbumin at 40°C. as compared with the behaviour of the component proteins alone. We note that a-lactalbumin denatures significantly on aging. When the film is aged at 20 mN m I, the surface pressure is even further reduced at higher surface concentrations, possibly due to intermolecular interactions. Also shown in Figure 6 is the P-lactoglobulin isotherm at 40 " C after aging for 2 hours at 20 mN m-I. Results of the analysis of the rates of film collapse (i.e.,decrease in surface area with time) are listed in Tables 1-7 for the four individual proteins and the three mixtures. An applied compression pressure of 25 mN m-I was found suitable for comparing all the systems. At lower pressures, the more condensed films (Plactoglobulin and a-lactalbumin at 9 O C and 25 "C) collapsed very slowly; at higher pressures, the /.?-casein films collapsed too fast to measure with any accuracy by the methods described here. Area decays were generally monitored for 10 minutes. The rate constant k in the analysis of type 2 is accurate to ca. 5%. Errors in k, and k, in the double-exponential analysis of type 3 are more variable, but are not more than 10% in k, and 20% in k,. The error in k in the analysis according to equation ( I ) cannot be estimated since it is based on a single pair of readings per experiment. The rate constants from equation ( I ) increase with temperature, as one would expect for adiffusion-controlled process. When aged at 20 m N m a-lactalbumin and j3-lactoglobulin show a reduction in k, possibly as a result of coagulation. Analysis using equation (2) shows higher k values for p-casein than for the other proteins. For &casein, rates at 10 " C and 40 O C are faster than at 25 "C, and as explained above for the isotherms this could be due to increased solubility (10 "C) or increased rate of diffusion from the interface (40 "C). With P-lactoglobulin, k shows some temperature dependence. With K-casein, k decreases with aging; this is in agreement with the isotherm data, and might be attributable to removal of mercaptoethanol from the interface and consequent intermolecular disulphide linking. Results for P-lactoglobulin 4- fl-casein are similar to those for Plactoglobulin alone at 10 " C and 25 "C, but at 40 "C the value of k is lower and tends to decrease with age. Area decays for &lactoglobulin 4-K-casein are slightly
+
',
I79
-
13
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+ .
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l
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E. K. Murray
180
rc) r-
-3 m
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I86
faster, but also tend to get slower with age. Analysis according to equation (3) shows little temperaturedependence in either k , or k,. Rates for 8-casein areseen to be slightly higher than for the other proteins. For 8-lactoglobulin 8-casein, k, at 40 "C is slightly lower than for P-lactoglobulin alone. For 8-lactoglobulin K-casein, aging brings about no significant change, and k, is lower than for the individual proteins, particularly at 40 O C . The few experiments that have been performed on P-lactoglobulin a-lactalbumin suggest that aging under compression may reduce the observed rate constant.
+
+
+
Conclusions
Although there are many more experiments that can be done on these systems, the following conclusions may be drawn from the work described here.
[I-r isotherms of spread globular protein film changes with time and temperature in a manner that may be attributed to surface denaturation processes; this is particularly obvious when films are spread at high temperature or aged at low surface pressure. (2) The 11-r isotherms of the spread protein films investigated here are not simply related to the isotherms of the individual component proteins under the same conditions, particularly when those conditions allow extensive denaturation of B-lactoglobulin. (3) Measurements of the rate of collapse of film areas with time under an applied surface pressure of 25 mN m-I suggest that a-lactalbumin and 8-lactoglobulin may associate when aged at 20 mN m I, that complexing between plactoglobulin and K-casein may be reflected in a reduction in rate constant with age, and that the presence of 8-casein may inhibit the surface denaturation of /3-lactoglobulin.
( I ) The shape of
Acknowledgements. I should like to thank Drs. M. Anderson, M.C.A. Griffin and
E.W. Evans for helpful discussions, and Mrs. L. Chaplin and Messrs. M. Taylor, J. Price and E. Needs for providing purified milk proteins. The heating and cooling coil was a gift from Cadbury Schweppes. References
D.E. Graham and M.C. Phillips, J. Colloid Interface Sci., 1979,70,403. D.E. Graham and M.C. Phillips, J. Colloid Interface Sci., 1979,70,415. D.E. Graham and M.C. Phillips, J. Colloidlnterface Sci., 1979,70,427. D.E. Graham and M.C. Phillips, J. Colloid Interface Sci.,1980,76,227. 5 D.E. Graham and M.C. Phillips, J. Colloid Interface Sci.,1980.76.240. 6 M.C. Phillips, Chem. Ind., 1977.5, 170. 7 J.V. Boyd, J.R. Mitchell, L.lrons, P.R. Mussellwhite, and P. Sherman. J. Colloid
I 2 3 4
Interface Sci., 1973,45,478. D.G. Cornell, J. Colloid Interface Sci.,1982,88,536. M.C.A. Griffin, R.B. Infante, and R.A. Klein, Chem. fhys. Lipids. 1984,36,91. E.M. Brown, R.J. Carroll, P.E. Pfeffer, and J. Sampugna, Lipids, 1983.18, I I I. G. Doxastakis and P. Sherman, Colloid Polym. Sci., 1984,262,902. A. Rahman and P. Sherman, Colloid folym. Sci., 1982,260, 1035. 13 E. Dickinson, B.S. Murray, and G. Stainsby,J. Colloid Interface Sci., 1985, 106,259. 8 9 10 II I2
I87 14 C.A. Zittle, M.P. Thompson, J . H . Custer, and J. Cerbulis, J. Dairy Sci., 1962,45,807. 15 W.H. Sawyer, J. Dairy Sci.. 1969,52,1347. 16 J.N.de Wit and G. Klarenbeck, J. Dairy Sci., 1984,67,2701. 17 J.J. Kester and T. Richardson. J. Dairy Sci., 1984.67.2757, 18 F. MacRitchie, J. Colloid lnterjace Sci., 1963,18,555. 19 F. MacRitchie and N.F. Owens, 1. Colloid lnrerface Sci., 1969,29,66. 20 F. MacRitchie, J. Colloid Interface Sci.. 1977.61.223. 21 F. MacRitchie, J. ColloidInterjaceSci., 1981,79,461. 22 F. MacRitchie, J. Colloidlnterface Sci., 1985,105, 119. 23 H.B. Bull, J. Colloidlnterface Sci.,1972,41,305. 24 J.K. Mitchell, L. Irons, and G.J.Palmer, Biochim. Biophys. Acta, 1970,200,138.
Desorption of Bovine Serum Albumin from the Air-Water Interface By T.M. HERRINGTON and S.S. SAHl
(Chemistry Department. University of Reading. Whiteknights.Reading. Berkshire RG6 2AD)
Introduction Proteins are polymers composed of well-defined amino-acid sequences with structure defined at three different levels. The primary structure is the number and sequence of amino acids in the polypeptide. The secondary structure refers to the amount of conformational regularity in the form of either a-helices or &pleatedsheet structure. The tertiary structure represents the amount of coiling and folding of the polypeptide required to attain the globular molecular shape. Proteins can be loosely classified as fibrous o r globular. The polypeptides of fibrous proteins are laterally cross-linked to yield tough, water-insoluble structures; examples include a-keratin in hair and skin, and collagen in tendons. The water-soluble globular proteins are the biologically active macromolecules of living systems (enzymes and antibodies). A water-soluble protein presents its hydrophilic groups outermost; but, because of its amphiphilic composition, it is surface-active. Protein films adsorbed at fluid interfaces are important in the food industry since they are involved in stabilizing foams and emulsions, e.g., meringue, dairy products, mayonnaise, ice-cream, and the head on beer. When a protein adsorbs at the air-water interface, its structure changes because the molecule can minimize its hydrophobic interactions by causing non-polar groups to protrude into the air phase. Thus, in a typical adsorbed film, there are probably trains of amino-acid groups lying along the interface, and loops and tails protruding into the bulk phase. A sigmoidal isotherm of surface pressure versus surface area is typical of a protein at the air-water interface. In almost all cases, a slow collapse or hysteresis is observed at surface pressures above a few millinewtons per metre. Considerable controversy has existed as to whether proteins adsorb reversibly o r not. While proteins are highly soluble in aqueous solution, proteins in surface films are ‘insoluble’ insofar as compression to surface pressures of ca. 15 mN m-I produces little detectable loss from the monolayer. It has usually been considered that, during compression of a protein monolayer, configurational changes occur with expulsion of segments from the interface. In a I88
I89
dilute spread film the molecules are extensively unfolded, but a compressed film contains molecules with different degrees of unfolding. Many studies of spread protein films have been reported and reviewed,'*2 although it is apparent from recent developments in protein chemistry that much of the early work was done with poorly-characterized materials. In contrast, relatively little work has been done with adsorbed protein films because of the difficulty of determining the true surface concentration. Gonzalez and MacRitchie showed3that the adsorption of bovine serum albumin (BSA) at the air-water interface was reversible, and they determined equilibrium adsorption isotherms, i.e., surface pressure as a function of subphase concentration. Using a radiochemical technique to study the adsorption of ['4C]acetyl lysozyme at the air-phosphate buffer interface at pH 7, Adams er uL4 compared surface pressure II versus area A isotherms of spread and adsorbed films. They found that II-A isotherms of adsorbed films became more expanded as the concentration of protein in the subphase was decreased. A film spread on the same phosphate buffer solution (pH 7) gave a more compressed 11-A isotherm, whereas a film spread on 3.5 M KCI solution was the most expanded of all. Determination of the surface concentration from the radioactivity data showed that the isotherm on phosphate buffer was less expanded due to loss of protein to the subphase. This emphasizes the fact that II-A isotherms are functions of the experimental conditions, and that concomitant desorption usually occurs. Graham and PhillipsS obtained truly equilibrium 11-A isotherms from the surface pressure and surface concentration isotherms of adsorbed films of radio-labelled pcasein, BSA and lysozyme equilibrated with protein in the subphase. Differences in properties of surface films of proteins formed under different conditions has been demonstrated by the work of Mitchell el u1.6Theycompared the II-A isotherms of spread films with isotherms of surface pressure against concentration C obtained by adding an increasing amount of protein to aconstant surface area. The 11-A and ll-Cdata on phosphate buffer at pH 7 were superimposable only up to II = 2 mN m-I. Above this surface pressure, the II-A isotherm was more expanded. It was concluded that protein molecules added to an existing film d o not unfold to the same extent as molecules added to an unpopulated surface. However, for various proteins, the difference between the two types of isotherm was not found to correlate with the helical content of the secondary structure. (The fractional helical content for BSA is around 0.5.') The II-A isotherm of a spread protein film is characterized by a sigmoid-shaped curve, and it was suggested by Bull6 that the position of minimum compressibility, corresponding t o the inflection point in the TI-A isotherm, corresponds to the smallest area to which a film can be compressed without partial collapse of the film occurring. The appearance of collapse patterns under a dark-field ultramicroscope has been shown to coincide rather closely with the position of minimum compressibility. With equine serum albumin on acetate buffer (pH 4.8). Neurath9 found collapse patterns at II > 14 mN m-I and a minimum in the compressibility at 12 mN m-I. By extrapolating the two portions of the sigmoid curve around the point of inflection, a critical pressure n, and critical area Accan be obtained. These parameters characterize the transition from a flat extended protein conformation to one in which the chains are buckled." The isotherm is possibly reversible up to
I90
T.M . Herrington ondS.5'. Sahi
the pressure n, corresponding to the minimum compressibility; for nm < n 5 n, only desorption occurs on film compression, and for pressures above fl, both collapse and desorption may occur. The loss in area exhibited on recompressing to a I1 value below IL, may be caused by desorption alone, or it may be that buckled protein molecules d o not unfold during the experimental time-scale. Factors affecting the stability of food emulsions and foams d o not occur on a time-scale comparable with that required to obtain equilibrium adsorption isotherms and n-A data. In addition, the rheological properties of the adsorbed protein film are undoubtedly paramount in determining the stability of these dispersions. In this work, it was decided to study spread films as these offer the possibility of following the various processes occurring using Langmuir-Blodgett techniques and radio-labelling.tO By carefully standardizing conditions of temperature, subphase concentration, spreading technique, speed of compression, and so on, one is able to compare n-A isotherms for different proteins, lipids, and their mixtures. The surface concentrations from radio-labelling should indicate whether conformational changes or desorption, or both, are occurring, although there is evidence that protein modification necessary for radio-labelling may significantly alter the surface properties. MacRitchie and Saraga'O used IZ5Ilabelled BSA in their investigation. The isotope lZsl has a relatively short half-life of 60 days, but its y-radiation can be measured readily with a scintillation counter without the need for a scintillant fluid. Only one in 90 BSA molecules were iodinated, but they foundioconsiderable differences in the adsorption behaviour between native BSA and i251-labelledBSA in films held at n > 18 mN m-I; the labelled protein was desorbed more rapidly. In addition, the surface appeared to catalyse a reaction in which iodine was lost from the protein. Graham and Phillips" used [ I-14C]acetylated BSA, &casein and lysozyme with conditions chosen such that approximately 2 lysine residues per protein molecule were acetylated. The isotope I4C is attractive for radio-labelling since it has a half-life of 5.7 X lo3years and is a soft p-emitter. Graham and Phillips commenttt that the adsorption characteristics of BSA and lysozyme are changed slightly upon acetylation. Certainly, the equilibrium surface pressure versus substrate concentration isotherms reported for BSA and [ I-'4C]acetyl BSA are very different, although in the former case3 the substrate was pure water whereas in the lattert2it was phosphate buffer (the isoelectric point of BSA occurs at pH 5). Muramatsu and SobotkaI3 compared BSA with N-acetyl BSA for which only some of the lysine residues had been acetylated, and they found that the n-A curves on pure water were very different: the surface areas were 0.72 and 0.54 m2 mg-I at n = 10 mN m-I for BSA and N-acetyl BSA, respectively, and 0.80 and 0.64 m2 rng-' at IL = 5 mN m-I. Adarns el d4also compared IL-A isotherms for native and acetylated lysozyme on the same substrate. The isotherm on a 3.5 M KCI subphase was independent of the number of lysine residues acetylated, but with a 0.1 M ionic strength phosphate buffer (pH 7) the area at a given surface pressure increased with the extent of modification. The surface area at Il = 5 mN m-I is 0.16 m2 mg-I for native lysozyme and 0.41 m2 mg-I for lysozyme with one lysine residue acetylated (see Table I).This difference may be due to the change in protein structure brought about by the acetylation procedure (addition of acetic anhydride to the protein dissolved in dimethyl sulphoxide) rather than the effect of adding just one acetyl
191
group. In this connection we note that alkyl groups can be introduced into amino groups by reductive alkylation, which is a relatively mild procedure producing minimal changes in the physico-chemical properties of most proteins. The pK of each amino group is altered by about I unit.I4 Lysozyme and /3-casein, with their rigid globular and flexible-chain structures, respectively, lie at the limits of the tertiary structure scale for soluble protein. The labile globular BSA lies intermediate between these extremes: and, as BSA can be regarded as a typical example of a water-soluble protein, it was chosen here for initial study. The amino-acid sequence of BSA is known, and the tertiary structure is maintained by disulphide bridges with 9 double loops between the bridgesu5 Using [14C]methylated BSA, we report comparisons of the 17-A isotherms and desorption kinetics of cold BSA, radio-labelled BSA, and a sample of cold methyl esterified BSA. Experimental [t4C]methylated BSA was purchased from Amersham International. I t had been prepared by the method of Dottavio-Martin and Ravel,16 which involves treating BSA with ['4C]formaldehyde in the presence of sodium cyanoborohydride in phosphate buffer at pH 7 and 25 OC. followed by dialysis. A sample of 20 pg in 0.01 M sodium phosphate buffer at pH 7.2 had an activity of I pCi. This is equivalent to the labelling of 55 carbon atoms per molecule, i.e., 55 lysine residues are ['4C]methylated (BSA contains 59 lysine residues and 23 arginine residues). The unlabelled BSA was purchased from Sigma Chemicals. According to the manufacturer's assay by agarose and cellulose acetate electrophoresis, the material was essentially single-banded, although there was a I-2% contamination with unidentified protein. The purity was checked by SDS-polyacrylamide gel electrophoresis. A vertical flat-bed electrophoresis apparatus was used, the analysis being performed with 7.5 wt% acrylamide as the main separating gel and solutions like those of Laemmli." A single band was obtained (see Figure I), confirming that BSA was a single species with no sign of dimers or larger aggregates, although a slight smudging of the band indicates that a small amount of lower-molar-mass impurity was present. The unlabelled methyl ester of BSA was also purchased from Sigma Chemicals; the manufacturer's assay is the same as that for native BSA. The preparation had involved dissolving BSA in methanol and esterifying it by addition of concentrated hydrochloric acid at room temperature, followed by washing with methanol and ether, and then vacuum drying.I8 All proteins were stored at -20 OC, and solutions prepared in phosphate buffer on adaily basis were stored at 5 "C. The lecithin was crystalline synthetic distearoyl-L-a-phosphatidyl choline (purity 99 wt%) from Sigma Chemicals. The subphase was phosphate buffer (0.01 M NaH,P0,.2H20) containing 0.15 M NaCl adjusted to pH 7.3 using 0.2 M NaOH. Demineralized, distilled water was further purified by a Milli-Q water system, and high-purity analytical-grade chemicals were used throughout. The subphase was checked for surface-active impurities by compressing to the minimum surface area after standing for I hour; a seven-fold decrease in area gave negligible change in surface pressure. Protein was spread from the same buffer solution using an all-glass Agla micro-syringe with its
I92
Figure 1
T.M. Herrington and S.S. Sahi
Analysis of BSA by polyacrylamide gel electrophoresis
tip held at the surface [the methyl ester of BSA was spread from acidic chloroform/ methanol (2: 1 by volume)]. Monolayer spreading was by direct application to the surface of 40-200 MI of solution in the form of numerous small drops. Measurements were made on a Joyce Loebl Langmuir-Blodgett surface balance. The constant perimeter trough avoids the problems of monolayer leakage at the barriers. The apparatus was situated within a clear-air and dust-shielded cubicle with relative humidity maintained at 95%. The trough, with its elliptical dipping well, is machined out of a single piece of Teflon. The temperature of the subphase was maintained at 20.0f0.5 OC by passing thermostatted water through aglasscoil. A standard procedure was adopted for determining II-A isotherms. Initially, protein was spread on the maximum available area (950 cm2), and the speed of the barrier was chosen to give a constant Compression rate of 0.66 cm2 s-I. In the desorption experiments, the film was compressed and held at the chosen surface pressure for a given period of time, and then it was rapidly but reproducibly expanded to the maximum area before recompression. A surface film could be transferred to a small quartz o r glass plate at constant surface pressure using the automatic ‘take-off facility. Constancy of surface pressure while the plate was being lowered into the surface indicated that there was no film transfer on entry of
I93
the plate into the subphase. The area of slide covered by the film was measured by Vernier calipers. Slides were placed in vials, Insta-gel scintillant was added, and radioactivity was measured on a Packard model 3225 scintillation counter. A quantitative determination of the amount of protein on a slide was made by measuring the radioactivity of small quantities of stock solution. Care was taken to add the same volume of scintillant (to within 200 pl) and standard buffer solution to all vials. The amount of desorbed BSA in the substrate was determined by measuring the radioactivity of several 5 cm3 samples, with the standard amount of buffer and scintillant added to the protein following removal of the aqueous phase by freeze-drying. Table 1
Data from surface pressure versus area isothermsfor modified BSA and lysozyme. Substrates used were (i) phosphate bufler containing 0.15 M NaCl (pH 7.3, ionic strength = 0.17 mol dm-') (this work and ref. 10); (ii) phosphate bufler NaCl (pH 7.0, ionic strength = 0.1 mol dm-') (ref. 4 andref. 6): (iii)pure water (ref: 13). Il, and A, are obtained by extrapolating the two portions o$ the Il-A curve o$ di//erent slopes. F5 and rlo are surface concentrations at Il = 5 and 10 mN m-', respectively. [Im and Amare the sut$acepressure andarea at the minimum compressibility
+
Source
BSA; this work
20f0.5
17.3fO.1
0.64f 0.03
1.21f 0.04
lOfl
0.74f 0.0 I
0.81f 0.01
BSA+['4]methyl BSA (2.06: I); this work
20f0.5
17.83~1
0.74f 0.I
1.16f 0.I
lOfl
0.97f 0.I
0. I
Methyl ester of RSA; this work
20f0.5
1.71f0.3
0.72f 0.04
1.22f 0.07
lOfl
0.83f 0.04
0.91f 0.05
BSA; ref.( 10)
20f2
16fl
0.74f 0.I
1.22
lOfl
0.88f 0.I
0.9 I f 0. I
['*'I] BSA; ref.( 10)
20f2
16fl
0.74f 0.1
-
lOfl
-
-
BSA; ref.(6)
25f0.5
16.4
0.68
1.23f 0.03
lOfl
0.81
0.9I
BSA; ref.( 13)
20
16.5
0.62
1.4f
lOfl
0.72
0.80f 0. I
0. I
Lysozy me; ref.(4) Lysozyme (20% acetylated); ref.(4)
25f0.5
-
__
-
-
0.16f 0.0 I
0.4 I f
0.02
I94
T. M. Herrington and S. S.Sahi
Films of proteins were transferred to a freshly-cleaved mica substrate for electron microscopy. The Langmuir-Blodgett film was taken off with the mica slide perpendicular to the moving barriers to ensure uniform slide coverage. The film was air-dried, placed in a vacuum evaporator, and shadowed with palladium/ platinum at an angle of 10" from a distance of 10 cm, and then a thin film of carbon directly from above. The replicas were lightly scored with a sharp knife, floated onto aclean high-purity water surface, picked up on a 200-mesh copper grid, and examined in a Philips 301 transmission electron microscope. Another method of preparing the films was also tried. This involved preparing Formvar slides by dipping clean glass slides into 0.5wt% polyvinyl formal in chloroform. Electron microscope grids were prepared by floating a Formvar film on high-purity water, scooping up the floating film onto a 200-mesh copper grid, and then forming a sandwich of the grid between the Formvar film and the mica support. These sandwiches were used for Langmuir-Blodgett take-off, followed by shadowing and subsequent treatment as above. Electron micrographs prepared with both types of substrate showed exactly the same detail. The photographs featured here were taken using freshly-cleaved mica as the substrate.
Results and Discussion Compression/ expansion cycles were carried out on unlabelled BSA to determine the surface pressure above which irreversible changes in surface area occur. The film was compressed and held at a given surface pressure for 20 minutes before expanding and recompressing. Over this time period, no irreversible change in surface area was detected at I l = 5 , 10, 15or 18 mN m I, but anirreversibedropinareawasfoundat Il = 21 mN m-I. Figures 2 and 3 reproduce the compression cycles for unlabelled BSA held at surface pressures of 21 and 30 mN m I , respectively. Values of the critical pressure Ilc and critical area A, are given in Table I . They are comparable with those of MacRitchie and Saraga'O obtained with a compression rate of 0.8 cm2 s I . Isotherms for the unlabelled methyl ester of BSA held for 20-minute periods at Il = 2 I and 30 mN m-' are illustrated in Figures 4 and 5 , respectively. The values of 11, and AE given in Table I agree with those for ordinary BSA within the experimental error. Rates of desorption have been calculated from the irreversible losses in area at n = I0 mN m I. Except for the initial compression cycle, the plot of logl0Aagainst time t is linear, implying a constant rate of loss of area (see Figure 6). Rate constants k, = (d InA/dt) calculated from the data are presented in Figure 7 as a function of the surface pressure. The rate of desorption for the methyl ester of BSA is similar to that for native BSA at n = 21 mN m-I. Figure 7 also shows the results of Gonzalez and MacRitchie) and MacRitchie and Saraga'O for rates of desorption of BSA with pure water as subphase. We see that the rate of loss of area is less with the phosphate buffer subphase (ionic strength = 0.17 mol dm-)) than with the pure water subphase. Subphases of very high ionic strength (2 mol dm ') have been usedI9 to reduce the solubility of proteins in the bulk aqueous phase. Gonzalez and MacRitchie investigated' the effect of stirring the subphase. The rate of loss of surface area was determined at I1 = 25 mN m I with and without stirring of the subphase, and no difference was detected over a time period of 30 minutes. At longer times, however, the rate in the presence of stirring remained constant, whereas it fell continuously in
I95
Figure 2
Compression cycles for spread BSA films held for periods of 20 minutes at 2 I m N rn I . Surface pressure Il is plotted against area A. For clarity. cycles are displaced by twice the actual change in area. and expansion cycles are not shown
A
Figure 3
/ m 7 mg-'
Compression cycles for spread BSA films held .for periods of 20 minutes at 30 rnN rn I. Surface pressure I1 is plotted against area A
I96
T. M. Herrington and S.S. Sahi
A / m 2 mg-'
Figure 4
Compression cycles for spreadfilms of the methyl ester of BSA held forperiods of 20 minutesat 21 mN m-I. Surfacepressure II isplotted against area A. For clarity, cycles are displaced by twice the actual change in area
30
;
20
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0.2
0.4
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Figure 5
Compression cycles for spreadfilms of the methyl ester oJ BSA held for periods of 20 minutes at 30 mN m-I. Surface pressure I1 is plotted against area A
I97
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Time-dependent changes in surface areafor the dgferenr rypes of BSA
films heldfor periods of 20 minutes at constant surface pressure. The logarithm of rhe area A at II = 10 mN m-' isplorredagainsr rime t: 0 , BSA at 21 mN m-l;m, 2.06: 1 mixture of BSA 4-[14C]merhylared BSA; 0 , merhylesrer
of BSA at 30 mN m-I
the absence of stirring. Values of ca. I mm have been calcultedm for the thickness of the diffusion layer near the surface in the absence of stirring. Figure 8 shows n-A isotherms for BSA and its modifications. The similarity of the plots shows that the two different methods of methylating BSA d o not alter the surface equation of state significantly. Values of II, agree within I mN m-l, and values of A, agree within 0.1 m2 mg-I. The near-parallel nature of the isotherms indicates that the various types of BSA lead to films with similar compressibilities. The effect of removing a monolayer onto a slide in a typical experiment is shown as a plateau region on the Il-A curve (see Figure 9). The protein film is transferred to slides at a constant surface pressure of 10 mN m-I. Films were also transferred to slides after compression to 24 mN m-I. Typical results for a desorption pressure of 24 mN m-' are given in Table 2 for two different batches of radio-labelled protein. From the initial isotherm, the surface concentration of protein estimated from the radioactivity count at II = 10 mN m-I is I .22f0.05 mg m-* (including errors in film area on slide and activity counts). Maintaining the film at 24 mN m-' for multiples of
I98
T.M. Herrington and S.S.Sahi
o ! 20
25
30
n / m ~m-’ Figure I
Kinetics of desorptionfrom spreadJlms containing dgferent types of BSA held for periods of 20 minutes at constant surface pressure. The rate constant k, is plotted against the surface pressure lI. Lower scale refers to this work with phosphate buffer subphase (pH 7.3): 0 , BSA; m, methyl ester of BSA; 0,2.06: I mixture of BSA +[‘4C]methylated BSA; O! 19.3: I mixture of BSA +[‘4C]methylatedBSA. Upper scale refers to reJ 10 with pure water subphase: A , BSA; A, ‘251-labelled BSA
I99
0.11
1.u
(J.8
/ET--Ah
A /m2 m y - '
Figure 8
BSA.- - - - Plots of surfacepressure TI versus area A: 2.06:1 mixture of BSA +['4C]methylated BSA; ' - * - . - *, methyl esrer of BSA. For clarity. plots are displaced along the abscissa by 0.1 m 2mg-'
Figure 9
Compression cycles /or spread Jilms of 2.06: I mixtures of BSA +[14C]methylatedBSA held for periods of 20 minutes at 21 m N m I. Surface pressure I I is plotred against area A. Flat portions of the isotherms rejer t o Jilm transfer onto quartz substrate
A / m 2 mg-'
200
T.M. Herrington 0ndS.S. Sohi
20-minute intervals reduced the film area, but had insignificant effect on the protein surface concentration. From the slides, however, the surface concentration was found to be only0.97 mg m-', i e . , 77%of that for the initial isotherm. Checks of the amount of protein desorbed into the subphase were carried out. For a 2: I mixture of BSA 4[i4C]rnethylated BSA, 2.8f0.2 pg of protein was accounted for assuming a surface concentration of 1.22 mg m-2, and 2.1f0.3 p g of protein assuming 0.97 mg m-*, based on the loss in area at ll = 10 mN m-I after successive periods of holding at 21 rnN m-I for 20 minutes. If it is assumed that the slides give a lower surface concentration because protein is lost into the subphase on initial spreading, this means that 1 2 f 2 pg of protein has gone into the subphase out of the initial 53 pg of spread protein. Thus, the subphase should contain 1 4 f 2 p g of protein. From the radioactivity of the subphase, the total amount of protein desorbed into the substrate is 1 6 f 2 pg. These results imply that loss of protein has occurred on initial spreading. For a 19:l mixture of BSA 4- [I4C]methylated BSA, the figures are: 6 pg loss by irreversible cycling, 1 6 f 7 pg loss on initial spreading, and a subphase count of 42f6 pg protein. If these results are correct, then in the reported isotherms of BSA and radio-labelled BSA the abscissa should be multiplied by a factor of I .3 to get the area in terms of protein actually present in the film. On the other hand, it could be that quantitative Langmuir-Blodgett transfer has not occurred, or that our subphase counts are subject to an unknown experimental error. Experiments are now being designed to determine the desorption into the subphase as accurately as possible. We note that desorption of protein into the subphase on initial spreading was suggested Table 2
Surface concentration ofprotein, calculated from the radioactivity, transferred to slides at I1 = 10 mN m I, and loss in film area from holding at n = 24 mN m-I fo r variousperiods of20 minutes. Two sets of data are presented f o r spread mixtures of native BSA [i4C]methylatedBSA in the ratios (a) 19.3:l and (b) 1 4 5 1
+
Protein Concentrotion/
Area of Slidel crn'
Activity/
Specijic Activity/
counts rnin-'
counts min-' cm
(a)
3.68
l 0 2 8 f 15
236
0.97f0.10
0
3.38
9 7 6 f 19
245
I . o o f O . 10
6.5
3.16
837f16
225
0.92f0.09
9. I
4.89
1277514
22 1
0.90f0.09
14.7
5.08
I546
304
0.95f0.10
0
3.49
I196
342
I .06f0.10
4.8
4.07
1071
263
0.82f0.08
7.8
4.30
I180
274
0.85f0.09
3.0
3.42
I088
318
0.99f0.10
5.3
(b)
I
mg x 1 0 ~ / c m *
Total loss
in Film Areal %
20 I
by Langmuir and Schaefer2' to account for the large change in the surface concentration of pepsin with pH. At the isoelectnc point of pepsin (pH 2.6), only 6% was desorbed into the subphase, as compared with 18%at pH 4.2. Figures 10-17 show electron micrographs of replicas of protein and lipid films transferred to the surface of freshly-cleaved mica. The direction of shadowing shown in Figure 10 is the same as in all the other photographs. Figure 10 shows a typical replica for the methylester of BSA transferred at 20 mN m-I. The irregular oval in the centre of the picture is devoid of any features, and probably corresponds to a hole in the film. The surface irregularities over the rest of the film are probably indicative of folding of the protein monolayer. The surface micro-asperities are 1-4 nm high, and the edge of the hole is cu. 1 nm thick, which is consistent with a protein monolayer." Figure 1 I is a replica of distearoyl-L-a-phosphatidylcholine transferred at a surface pressure of 30 mN m-I [phosphate buffer subphase (pH 6.9), 70 pg of the lecithin was spread from 50 pl of chloroform, temperature = 20.2f0. I "C].For surface pressures in the range 20-30 mN m-I, the monolayer is condensed, and the area per molecule changes by cu. 0.03 nm2. Striations corresponding to film collapse, oriented normal to the direction of film compression, are clearly visible, although the film is more than 10 mN m-l below the true collapse pressure. The striations in Figure I I appear to be puckered rather than layered, and a discotic structure can be seen in the bottom right-hand corner. Figure 12 shows another area of the same slide at lower magnification. Many discotic structures are present, and the white cones cast by the shadowing technique are very distinct. The edge of the replica is well-defined to the upper right of the picture, and an approximately circular hole is seen in the replica to the left of centre, whose edge is cu. 3 nm thick, consistent with a value of 2.7 nm per monolayer found for dimyristoyl phosphatidyl choline by X-ray analysis.23 There appear to be small patches of lipid in the hole cu. 3 nm high, and small asperities of similar height over the rest of the film. The large discotic structures require some comment. Some are clearly platelets, but others are of 'molehill' shape of height . ~ ~ found flattened, oblate spheroids and structures with a 40-80 nm. Ries et ~ 7 1have multilayered disc geometry, of thickness 4-6 nm but of similar breadth to those in Figure 12, for films of egg lecithin transferred at 44 mN m-I (just above the collapse pressure) directly onto Parlodian-coated electron microscope grids. The thicker platelets of Figure 12 may be collapsed liposomes, though it should be borne in mind that lipid-film dehydration in the vacuum evaporator prior to shadowing may produce artefacts. Films of lecithin transferred at a lower surface pressure, 20 mN m-l, are shown in Figures I3 and 14. Small surface irregularities and circular holes of diameter 0.2-1 pm are shown in Figure 13, and two of the holes are enlarged in Figure 14. The larger circular hole has an edge thickness of cu. 30 nm, and within the hole the layered stack has edge faces from bottom to top of 60,30 and 30 nm, consistent with a bilayer and monolayers of lecithin. Preliminary results for films of BSA transferred at 10.20 and 30 mN m-I are shown in Figures 15-17. The film transferred at 10 mN m-I is homogeneous with irregularly-shaped platelets of width cu. 80 nm. At 20 mN m-I the film is still homogeneous but the platelets have grown to 160 nm. It can be seen in Figure 17 that the features at 30 mN m-' are much larger and more numerous; the elongated lobed structures are cu. 160 nm in width and up to 500 nm in length but only I nm high. They probably arise from collapse of the film with increasing surface pressure.
202
T. M. Herringion and S.S.Sahi
'
Figure 10
Film of the methyl ester of BSA transferred at 20 m N m onto mica substrate. The arrow indicates the shadowing direction (the same direction in Figures I I - I7 also)
Figure 11
Film of disrearoyl-L-a-lecithin transferred at 30 m N m-'onto mica substrate
203
Figure 12
As Figure 11. except lower magnijkaiion
Figure 13
Film of distearoyl-L-a-lecithin transferred at 20 mN m-I onto mica subst rare
204
T.M. Herrington and S.S. Snhi
Figure 14
As Figure 13, except higher magnification
Figure 15
Film of BSA transferred at 10 mN m-I onto mica substrate
205
Figure 16
Film of BSA trangerred at 20 m N m-'onto mica substrate
Figure 17
Film of BSA trangerred at 30 m N m-' onto mica substrate
206
T. M . Herrington and S.S. Sahi
The BSA isotherms were found to be reproducible when held at I I 5 18 mN m i for 20 minutes before expanding and recompressing. Thus, below IIc, the accepted transition from a flat conformation to chain buckling, irreversible area loss, or desorption, is not evident on this experimental time-scale. While the collapse of the film can be seen in the electron micrographs for II 2 20 mN m-I (>n,),the film is not completely homogeneous even at II = 10 mN m-I. Within experimental error, the radio-labelling indicates that the protein surface concentration is constant over several cycles of irreversible area loss at Il= 10 mN mi. For the radio-labelling technique to be valid, labelled protein must desorb at the same rate as unlabelled. Agreement amongst the adsorption rates for BSA, the methyl ester of BSA, and the mixture of BSA 4-radio-labelled methylated BSA, shown in Figure 7, confirms that this condition is satisfied. In this laboratory, investigations of mixtures of proteins and lipids are currently underway using the multiprobed approach described here.
Acknowledgements. We thank M. Blissett and A. Vaughan of the Physics Department of the University of Reading for help with the electron microscopy, and AFRC for the financial assistance which enabled this research t o be undertaken. References I H. Neurath and H.B. Bull, Chem. Rev., 1938.23.391. 2 F. MacRitchie, Adv. Protein Chem., 1978.32.283. 3 G . Gonzalez and F. MacRitchie, J. Colloid Interface Sci., 1970,32,55. 4 D.J. Adams, M.T.A. Evans, J.R. Mitchell, M.C. Phillips, and P.M. Rees,J. folym. Sci.. Part C, 1971.34, 167. 5 D.E. Graham and M.C. Phillips, J. Colloid Interface Sci., 1979.70.427. 6 J . Mitchell, L. Irons, and G.J. Palmer, Biochim. Biophys. Acta, 1970,200, 138. 7 E.R. Blout, I. Schmier, and N.S. Simmons,J. Am. Chem. SOC.,1962,84,3193. 8 H.B. Bull, Adv. Protein Chem., 1947.3.95. 9 H. Neura1h.J. fhys. Chem.. 1936.40.361. 10 F. MacRitchie and L. Ter-Minassian-Saraga. frog. Colloid folyrn. Sci., 1983.68, 14. I I D.E. Graham and M.C. Phillips, J. Colloid Interface Sci., 1979,70,403. 12 D.E. Graham and M.C. Phillips, J. Colloid Interface Sci., 1979,70,415. 13 M. Muramatsu and H. Sobotka, J. fhys. Chem., 1962,66,1918. 14 G.E.Means and R.E. Fenney, Biochemistry, 1968,7,2192. IS J.R.Brown, Fed. Proc., 1976,35,2141. 16 D. Dottavio-Martin and J.M. Ravel, Anal. Biochem., 1978,84,562. 17 U . K . Laemmli, Nature, 1970,227,680. 18 J.D. Mandell and A.D. Hershey, Anal. Biochem., 1960, I, 66. 19 T. Yamashita and H.B. Bull, J. Colloid Inrerface Sci., 1967,24,310. 20 L. Saraga,J. Chim. fhys., 1955,52,181. 21 I. Langmuir and V.J. Schaefer, Chem. Rev., 1939.24, 181. 22 D.G. Cornell and R.J. Carroll, Colloids SurJ, 1983.6.385. 23 R.H. Pearson and D. Pascher, Nature, 1979,281,499. 24 H.E. Ries, G. Albrecht, and L. Ter-Minassian-Saraga, Lnngrnuir, 1985, I , 135.
A Microscopic Approach to the Structure of Food Emulsions in Applied External Fields
By G.C. BARKER and M.J. GRIMSON (AFRC Institute of Food Research. Colney Lane, Norwich NR4 7UA)
Introduction In many colloidal dispersions, particularly food emulsions, the constituent particles are deformable and show a wide variety of non-spherical, non-symmetric shapes. Often, dispersed drops exhibit continuous shape changes on a wide range of time scales. The deformations may be in response to static or dynamic external fields, such as external walls, surrounding drops, fluid flows, erc., or simply as a result of thermal fluctuations. Shape polydispersity is clearly evident in oil droplets suspended in aqueous phases, particularly so if there is some surface-active material present,' and it can also be seen in photographs and videofilms of single droplets in extensional flow (e.g., in tip streaming*), in phase-contrast micrographs of suspensions of bilayer lipid vesicles,'and in observations of red blood cells,'as well as being frequently inferred as a significant property of other oil/ waterlsurfactant system^.^ Many experimental approaches (dispersion rheology, NM R spectroscopy, static and dynamic light scattering) are able to provide information concerning the shapes of colloidally sized objects, but invariably flexibility complicates the interpretation of data. Shape fluctuations modify both the direct and the indirect particle-particle interactions, leading to shapedependent physical properties of bulk colloidal materials. Also, ageometric dependence of interfacial properties such as permeability and transport behaviour may lead to shapedependent physicochemical properties. The theoretical treatment of highly deformable particles is very complicated. Many degrees of freedom are associated with individual shapes. Methods dealing solely with the 'most probable shape' are seriously deficient through the neglect of fluctuations, since the function space of possible drop shapes is very large. In addition, the shapedependent energy contribution is often not simple, possibly containing nonHookean elastic terms, energies of curvature, tilt and stretch, curvature-dependent surface energy terms, etc. Previous theoretical studies of drop deformation have dealt primarily with the two limiting cases in which the drop geometry is either nearly spherical6.' or very elongated The processes of forming and stabilizing oil/water emulsions in the presence of a third surface-active ingredient illustrate many of the important effects. Continuous 207
208
G.C.Barker and M.J . Crimson
application of stress fields to low-surface-tension droplets produces large deformations which ultimately cause particles to break up. For agiven field strength, there exists a limiting size of droplet which will not rupture; particles below this size will exist in a range of deformed states, whereas larger particles will experience assorted eccentric shapes (necks, lobes, eic.) prior to fission. By this means a ‘stable’ size distribution will be set up. But the equilibrium is kinetic. After removal of the external field, the particles exhibit thermallydriven fluctuations, collision-induced deformations, and shape changes due to particle aggregation and fusion. Ultimate stability will depend on a balance of terms, including shape response as a central factor. We shall consider the full range of shapes of twodimensional highly flexible particles with a small or vanishing surface tension, where the major contribution to the deformation energy comes from a term quadratic in curvature, which therefore amounts to a bendingenergy? We associate this term, and theexistence of an ultralow surface tension, with the presence of surface-active material, but we shall neglect possible tilting and stretching energy contributions, tangential motions, and finite thickness effects of the surface layer. We shall impose a geometrical constraint of either constant perimeter length or constant enclosed area, the analogous threedimensional structures being bilayer lipid vesicles and biological cells in the first case, and micelles, microemulsion droplets, and large coated emulsion droplets in the second. (We note that imposition of constraints implies assumptions concerning the functional properties of the membrane, particularly its permeability with respect to the water phase.) Our restriction to two-dimensional shapes, which is motivated by computational convenience, is a serious simplification insofar as the size invariance of the curvature energy is lost. However, though limited in direct applicability, the two-dimensional model allows us to assess the significance of large shape fluctuations under differing constraints (e.g., with respect to perturbed spherical models), and it is clearly an invaluable platform for a extension which we now have in progress to a full account of solids of revolution. In what follows, we indicate a representation for the statistical geometrical properties of deformable particles in terms of integrals over the set of two-dimensional shape functions and a scheme for globally generating individual members of this set. We also give explicit expressions for the deformational energy in terms of a small number of parameters. Then we present results of a Monte Carlo evaluation of the integrals, and address two important questions: is it possible to link physical and chemical properties of colloids with statistical information on particle shapes; and is it possible to use an ‘effective’geometry to represent a set of deformable shapes?
Geometry of Two-dimensional Closed Shapes
In order to correlate particle-shape information with the physical behaviour of colloidal dispersions, we require a detailed knowledge of the geometry of irregular flexible shapes. For each member JI of the set of particle shapes we can associate a value p($) with each property p. The statistics are then conveniently expressed in a functional integral representation. The probability distribution of p is given by
209
where
and the mean value is
In these integrals, 6[G(JI)] represents topological constraints satisfied by JI (curve closure, curve crossing, erc.), E(JI) is the energy associated with a particular shape, k is Boltzmann's constant, and T is the temperature. The probability of a particular shape is proportional to the statistical factor exp[ -E(JI)/ k q . The details of a Monte Carlo method for evaluating the integrals in equations (1)-(3) are given by Ostrowsky and Peyraud.'O We adopt their scheme, which can be summarized as follows. (i) Restrict the set fl to shapes given by a limited Fourier expansion of the tangent direction
+ +
M
4(s) = cos '(I.?) = s A, 2 [A,cos(ms)
+ B,sin(ms)]
(4)
rn' I
In equation (4),2 is a unit tangent vector, 3 is the unit vector in the x-direction, and s is . set of coefficients A,, A,, B, (m = 1.M) the normalized arc length (se[O, 2 ~ 1 )The defines a closed curve of perimeter length 2a. (ii) Further restrict the shapes by choosing the N = 2(M - 1) coefficients A,, 9, (m = 2,M)inside a N-dimensional hypersphere of radius R,. (We have consistently chosen R, to limit the systematic error in the evaluation ofequation (3) to
(5)
Alternatively, maintaining a constant enclosed area S = rrR; gives a scale factor
The smallest wavelength of excitation, M/2nR, is a property of membrane structure which is taken to be invariant. Thus, incontrast to Ostrowsky and Peyraud,Iowe link particle size to the number of modes through the relation M(R) a: R, and we arbitrarily fix M(R = I ) = 5 . However, so as to retain the efficient curve-generation
210
G.C. Barker and M.J. Crimson
procedure, we slightly change this choice of M when used in conjunction with equation (6). In this case we write M R, with M(R, = I ) = 5 , and, when the perimeter length is such that R/ R, > [M(R,) I]/ M(R,), we make an additional contribution to the Boltzmann factor in accordance with equi-partition (see below). This modified scheme maintains the important contribution of small wavelength modes to the statistical weighting factor, whilst neglecting their small effect on particle geometry. We consider two competing shape-dependent contributions to the Boltzmann factor E($)/kT a bendingenergy term9 favouring smooth extended shapes, and an external field contribution leading to squashed and elongated shapes (fields of other symmetry are easily included). To compute the bending term for shape $ we integrate (d4/ds)2 along the curve. The result may be written in the form Q:
+
U
Bc = (27rK/R)
+ (rK/R ) C [(mA,)’ + (mBJ2]
(7)
m= I
where K is a constant expressing the magnitude of the bending elastic modulus. By comparing equation (4) with an expression in terms of independent normal displacements (a two-dimensional generalization of Helfrich”), it is possible to make an approximate identification of each value of m with two independent modes. Thus, when we invoke the constant S (variable R) constraint with M R,, we compensate equation (7),according to equi-partition of energy, by adding unity for each mode. We write
Bc-Bc+
3 ((I/2)+(1/2)tanh[(R-
R,)/d])
(8)
,=I
Ri = ([M(R,)
+ ill M(R,)lR,
(9)
where d (-I /5M)ischosen to make equation (8) exhibit sharp steps at the transition points Ri. In practice, only a few steps need be included in the sum. Each perimeter element experiences a force proportional to its distance from the y-axis and directed towards the y-axis. The corresponding contribution to the Boltzmann factor is
where a expresses the field strength. The precise form of the applied field is qualitatively unimportant. Results and Discussion
We present in a systematic way results for the geometrical properties of twodimensional deformable particles whose shapes are controlled by a curvature elastic energy. The results are sizedependent directly through R in equations (5). (6). erc., and indirectly through the associated increase in the number of modes. Particles up to M = 15 have been considered. The two other independent parameters are K and a for which an appropriate scaling corresponds to a variation in temperature. Although a mean radius could consistently be used to quantify deviations from circular geometry, its definition is ambiguous for non-star-shaped particles. We
21 I
therefore use S, the normalized mean enclosed surface, and P, the normalized mean perimeter length, as appropriate deformation measures for particles with constant R and constant S, respectively. Results for a = 0 are shown in Figures 1-3. In general, large particles with smaller K values exhibit wider ranges of increasingly deformed shapes. Figure I shows a steadily falling rate of decrease of S with R which we associate with an increased influence of 'hard-core' cross-over restrictions for highly flexible particles. This behaviour is not observed in an analogous plot of Pagainst R for R <3, which remains approximately linear with P = I .4 for R = 2 and K = 0.3. Two curves in Figure 2 (K = 0.3, R = 3; K = 0. I , R = I ) illustrate the importance of increasing the number of modes with particle size. For M independent of R, these two curves would be identical (cf. Ostrowsky and Peyraud'O). Deformation is aided by increasing M. The range in Figure 2 is strictly finite, but this is not so for Figure 3: examples of curves with P >3 were produced by the generation scheme with very small weights. The most significant effect of a non-zero external field is to remove the average circular geometry. (Theeffect is imperceptible from viewing individualshapeseven at
s 1.0
0.8
0.6
0.4
0.2 0 Figure 1
1
2
3
R
Plot of mean enclosed surface S against particle size R for particles of constant perimeter ( a = 0). Dashed lines are Monte Carlo resultsfor various values of K. Solid lines correspond to the perturbed circular model
G.C. Barker and M.J. Grimson
212
P tSl1
1.5
1.0
0.5
0 0
15
0.5
Figure 2
Probability distribution P(S) for enclosed surface area S of constant perimeter particles ( a = 0 )
Figure 3
Probability distribution P(P)for perimeter length P of constant area particles with K = 0.3 and a = 0
10
1.5
2.0
2.5 P
213
........,.........*
--
-2
&
Figure 4
-2 1 Figure 5
Averageshapes of constant perimeterparticles with K = 0.3 and R. = 2 in appliedjields of a = 0.0.5, 1 .O and 3.0
214
G. C. Barker and M.J. Grimson
the highest fields considered.) Larger shapes clearly show an increased average response to squashing, and the constant-volume constraint leads to reduced eccentricity for high external fields. We note that the Monte Carlo technique becomes less efficient as the average shape becomes increasingly elongated, and it may be preferableI2 to use reduced displacements between sets of coefficients A*,...AM, B2,...BM. Details of particle deformations in external fields are shown in Figures 7-10. Statistical errors increase with field strength, but are always <5% for a <2. Small constant-perimeter particles ( R = 0.6) remain substantially unaltered by fields of strength a <3 with a narrow distribution of almost circular shapes. Larger particles show a significant response to applied fields. For R = I and K = 0.3, the distribution of S values initially broadens and shifts to smaller values as the field strength increases, in contrast to the distribution of R = 2 particles, which becomes sharper for a X. Again, we attribute this difference to the cross-over criterion, which causes the distribution functions to develop sharp small-S cut-offs as the modal value approaches a critical value around S = 0.3. We note that, as a is increased, the distributions develop significant portions with S
21
-2 Figure 6
t1
Average shapes of constant area panicles with K = 0.3 and R = I in appliedfields oJa = 0,O.S. I .O and 3.0
215
the modal value of P moves steadily to higher P for a > I .5 with R = 2. Corresponding to the behaviourdiscussed above, there is a transition region around a =0.5 where the behaviour of the P distribution is complex. Because of restrictions inherent in the two-dimensional character of the model, we shall concentrate on describing the qualitative geometrical aspects of the stability of particle-size distribution to particle deformation. Under constraints of constant R and constant S, the geometrical requirements for binary fission are S < I 12 and R 3 2 ) ' ! 2 , respectively. From Figures 2 and 3, it can be seen that in both cases, for particles with K = 0.3 and R = 3, substantial portions of the distributions for a = 0 correspond to geometrically unstable configurations. This is also true, but to a lesser degree (see above), when the flexibility is increased by lowering K (e.g., R = I and K = 0.1). Similarly, Figure 8 illustrates the disruptive effect of applying a squashing field to smaller, more rigid particles of constant R. From a full set of curves for different values of R but fixed a,we could clearly estimate the final, stable size distribution for particles with constant perimeter at a given field strength (see Kim and Martini3). From Figures 9 and 10, we see that distribution functions for constant S are less responsive to squashing, and that the initial application of the field has a stabilizing effect. A corresponding result in three dimensions would have important implications for the stability of suspensions of deformable droplets. Figures 4-6 show that, before reaching any 'geometrical instability', particles in external fields may experience seriously deformed average shapes. This means that stable distributions of'on-average'noncircular shapes may occur. We note also that,
0.8
0.6
0.4
0 Figure 1
1
2
3 u
Plot ofmean enclosed surface S against appliedjield a forparticles of constant perimeter
C.C. Barker and M.J.Crimson
216
0 Figure 8
1.0
w
0 Figure 9
1 s
0.5
Probability distribution P(S) f o r enclosed surface area S of constant perimeter particles with K = 0.3, From left to right the parameter R takes the values 2,2, 1, I , and 0.5
1
2
3 a
Plot of mean perimeter length P against applied field a f o r constant area particles wilh K = 0.3
217
even in the absence of an applied field, a perturbed circular model is not in full agreement with the Monte Carlo calculation which includes highly deformed shapes. In Figure I is plotted S = 1 - (3R/ l 6 ~ K )I( - ( I / I5R)[(20R
- I ) / ( IOR - I)])
for K = 0. I and K = 0.3. Equation (1 I ) applies to circular particles with independent surface displacements, whose amplitudes are determined in accordance with the equi-partition of energy, and whose number is in agreement with our model (strictly analogous to the three-dimensional treatment of Helfrich"). This model underestimates fluctuations of small particles, and it becomes pathological as the particle size increases because of the neglect of correlations between the large numbers of modes. We conclude that, for dilute dispersions of highly flexible particles, or for concentrated dispersions in which particles are subject to straining forces (random or from flow fields), a circular geometry is a poor starting point for theoretical discussion. It is pointed out by van de Sande and PersoonsI4that a knowledge of the geometry of flexible, non-circular particles is an essential ingredient for the extraction of shape information from scattering data and in determining shapedependent dynamics of three-dimensional macromolecular structures. Distribution functions for radius of gyration and hydrodynamic radius are ideally suited to Monte Carlo calculation. For collections of particles in aggregates or concentrated sediments, we expect individual particles to have three-dimensional shapes analogous to those shown in Figures 4-6. Details of the packing effects and excluded-volume effects of these shapes, some of which are more rod-like than circular, will be required for theoretical studies of
2 3 P Probability distribution P(P)forperimeter length P of constant area particles with K = 0.3 1
Figure 10
218
G.C. Barker and M .J . Grimson
material rheology. T o our knowledge, no study of the random packing of noncircular shapes has been reported. In three dimensions, the geometrical criterion used here could be replaced by a consideration of deformation energy contributions, which are readily obtained from the Monte Carlo treatment. Many other properties could also be realistically examined, e.g., permeability or particle transport. The method is easily extended to include small non-zero surface tensions and positive osmotic pressure differences, both of which would reduce the fluctuation amplitudes, as well as non-linear elastic terms, wave-vectordependent curvature contributions,-’ and non-zero spontaneous curvatures. It has recently been suggestedi5that shape fluctuations can be driven by density fluctuations within a membrane. The Monte Carlo procedure could easily be modified to test forcorrelations between these two fields. Also within the rangeof the Monte Carlo scheme is an investigation of shape-dependent contributions to repulsive interactions between colloidal particles. l6 Using a Monte Carlo method to sample a large range of two-dimensional shapes, we have been able to infer that a circular geometry is deficient for treating shape fluctuations of flexible particles. The method described alleviates many of the problems associated with the theoretical treatment of highly deformable particles, and it can provide reliable data on the distribution of non-spherical shapes and associated geometries under differing constraints and conditions.
References Greenspan, J . Theor. Biol., 1977,65,79. J.D. Sherwood, J. Fluid Mech., 1984,144,281. H. Englehardt, H.P.Duwe, and E. Sackmann, J . Physique Leii., 1985,46,L395. F. Brochard and J.F. Lennon, J. Physique, 1975.36, 1035. S.Ljunggren and J.C. Eriksson. J. Chem. Soc.. Faraday Trans. 2, 1984,80,489. (3.1. Taylor, Proc. R. SOC.London, Ser. A , 1932,138,41. M. Bitbol and P. Mills, J. Physique Lpii., 1984,45,L775. A. Acrivos and T.A. Lo, J. Fluid Mech., 1978,86,641. W . Helfrich, 2. Narurforsch., Teil C,1973,28,693. N. Ostrowsky and J Peyraud, J. Chem. Phys.. 1982,77,208I. W. Helfrich, J . Physique, 1986.47.321. N. Metropolis, A.W. Rosenbluth, M.N. Rosenbluth, A.H. Teller, and E. Tel1er.J. Chem. Phys., 1953.21, 1087. S. Kim and G.M.Martin, Biochim. Biophys. Aria, 1981,646,I. W.van de Sande and A. Persoons, J. Phys. Chem., 1985,89,404. S.Leibler, J. Physique, 1986,47,507. D.Sornette and N. Ostrowsky, J. Physique. 1984,45,265.
1 H.P.
2 3 4 5 6 7 8 9 10
II I2
13 14
I5 16
Measurement of Creaming Profiles in Oil-in-Water Emulsions
By D.J. HIBBERD,A.M. HOWE, A.R. MACKIE, P.W. PURDY, and M.M. ROBINS
(AFRC Institute of Food Research, Colney Lane, Norwich NR4 7UA)
Introduction In colloids, the densities of dispersed and continuous phases are rarely the same, which means that the dispersions are unstable to the processes of sedimentation or creaming. Food emulsions may cream during processing or storage, as the vegetable oils used are less dense than the aqueous continuous phase.’ Since food emulsionsare usually concentrated and opaque, the study of creaming in such systems presents difficulties.* The rate of rise of a meniscus between the creaming emulsion and the separated continuous phase may be observed,’ but this does not yield information on the change in concentration profile before the meniscus is visually apparent or during subsequent creaming. In the past, changes in concentration profile during creaming have been studied by removing samples of emulsion at different heights,’ or by freezing, sectioning, and then examining the droplet concentration.5These intrusive techniques are very timeconsuming, and they destroy the sample. Recently, we have reported6 a non-intrusive, non-invasive, instantaneous technique which uses the propagation of ultrasound through a dispersion to determine the concentration profile of alkane-in-water emulsions during creaming. If the velocity of ultrasound through the continuous and dispersed phases is different, and if the droplet size is much smaller than the wavelength of ultrasound, the velocity of ultrasound through the emulsion depends only on the volume fraction of dispersed phase. Polysaccharide thickeners are often added to watercontinuous food emulsions in order to reduce the rate of creaming.’ Aqueous solutions of polysaccharides exhibit non-Newtonian behaviour due to the effects of high molecular weight and interpolymer interactions. It has been reported’ that oil droplets in emulsions containing low concentrations of polysaccharide cream much faster than one would predict on the basis of the low-shear-rate viscosity; the behaviour can be interpreted in terms of polymer-induced (depletion) flocculation. In this paper, we report acreaming study of soya bean oil-in-water emulsions stabilized by non-ionic surfactant Tween 60 with continuous phases containing various concentrations of the anionic polysaccharide Xanthan gum. 219
220
D.J. Hibberd. A.M. Howe. A . R . Mackie. P.W. Purdy and M.M. Robins
Experimental The non-ionic surfactant Tween 60 and preservative sodium azide were obtained from Sigma Chemicals. Soya bean oil was purchased from J. Sainsbury, and food-grade Xanthan gum from Kelco/ AIL. Water was distilled from alkaline permanganate. An emulsion of soya bean oil (volume fraction 0.2) in an aqueous continuous phase containing I .O wt% Tween 60, 0.12 wt% Xanthan gum and 0.02 wt% sodium a i d e was made using a set shear-cycle programme in a Waring Commercial Blender. A 75 ml sample of this emulsion was stirred thoroughly into 225 ml of aqueous solutions of Xanthan gum and preservative to give emulsions of volume fraction 0.05 containing a range of Xanthan gum concentrations in the aqueous phase as indicated in Table I. One third of each emulsion was stored in a 100 ml measuring cylinder at 20 O C , and samples were taken regularly for droplet-size determination. The remaining 200 ml of emulsion was placed in a rectangular cell in a water bath thermostatted at 20.0f0.I O C , and the concentration profile was determined over a period of one month. Droplet-size distributions were determined using a Malvern 2600HSD Laser Particle Sizer. The instrument uses the principle of Fraunhofer diffraction to determine the size distribution in the range 1.2-1 18 pm. Samples from the middle of the emulsion region were diluted with enough distilled water to obtain 60-8W transmission at 633 nm in a cell of I5 mm path-length. Densities of oil and continuous phases were measured using a Paar DMA60density meter. Rheological properties of the various continuous phases were investigated by a range of techniques. Viscosities at 20 OC were measured in a constant-rotation mode over the shear-rate range 0.1-1300 s-I using a Contraves Rheomat I15 with a double-gap measuring cell. A creep study was undertaken using an Instron 3250 Rotary Rheometer with a coneand-plate attachment (cone radius 2 cm, cone angle 2.4O, cone/ plate gap 41 pm). The lnstron was also used as a mechanical spectrometer in a strain dependence study to detect the presence of structure in the continuous phase. Values of storage and loss moduli, G' and G", were measured at 2 Hz over a strain range of cu. 0.01-10. The volume fraction of the emulsion was determined by measuring the time for a pulse of ultrasound to cross the sample.6 A single-waveform pulse of frequency 6.2 MHz was generated by a Tektronix FG405 function generator gated by a Tektronix PG501 pulsegenerator. The time for the pulse to pass between two Mateval 601/ 5 mm crystal probes across the cell containing the emulsion was measured using a Racal Dana 9904 Universal Counter-Timer, the time between successive input pulses being adjusted manually using a lwatsu SS47I 1 four-beam oscilloscope SO that the received signal coincided with the next transmitted pulse. The ultrasonic probes were moved vertically up the cell to the top of the emulsion, and the transit time of the pulse was measured at a series of different heights. A precise value for the width of the cell at each height was determined from measurements of the time for ultrasound to pass between the probes when the cell was filled with water or nheptane, two liquids which have a wellcstablished value for the ultrasound velocity. These 'cell profiles' were stored on a Vax 1 I /750. Data for each emulsion during creaming were also transferred to the Vax, enabling the ultrasound velocity through the sample and hence the volume fraction of oil to be calculated at each height.
22 I
Results Variations of Ultrasound Velocity with Emulsion Composition.-For purposes of calibration, a series of emulsions with a range of oil volume fractions from 0.02 to 0.70 was prepared with constant aqueous-phase composition (0.017 wt% sodium azide, I wt% Tween 60,0.225 wt% Xanthan gum). Mean droplet diameters for all the emulsions were in the range 2-4 pm. Figure I shows a plot of ultrasound velocity against volume fraction of soya bean oil. The theoretical curve is calculated from the velocities through the dispersed and continuous phases, vd and vc, using the formula proposed by Urick:"
In equation (I), v is the ultrasound velocity through the sample, 4 is the volume fraction of the dispersed phase, and pd and pc are the densities of dispersed and continuous phases, respectively. The absolute values measured for the emulsions were ca. 2 m s-I lower than the predictions from equation (I), but the change in v with 4 was close to that predicted.
U
E 1480L
0 C
I
T Y /
m / S
\
f, 1470-
\
I
I
I
I
I
I
I
I
1
I
I
D.J. Hibberd. A . M . Howe, A. R. Mackie, P. W. Purdy and M . M . Robins
222
For most dispersions, there is a much greater difference between vd and vcthan the figure of 13 m s-' for the system studied here. For instance, in a typical commercial salad dressing, we find (vd - vc) 140 m s-l, and Figure 2 shows that for such an emulsion there is excellent agreement between experiment and predictions from equation (I). Good agreement has also been obtained6 for n-hexadecane-in-water emulsions. In principle, the equipment can measure velocity to M. I m s-l;in practice, within a given profile, the accuracy is f0.2 m s-l. Errors due to temperature fluctuations were not found to be significant, but errors of up to f 2 m s - I were found to be present due to the possible failure of either probe to become properly relocated o r the cell not to lie squarely between the probes. A displacement in probe position of 20 p m corresponds to a change in velocity of ca. I m s - I . Uncertainties arising as a result of probe relocation problems could be substantially reduced by placing in the cell a liquid with a known velocity (e.g.,by placing soya bean oil on top of the emulsion), and, in fact, all of the calibration emulsions and the continuous-phase samples were investigated in this way. The soya bean oil, when determined simultaneously with water (1482.3 m s-l). gave an ultrasound velocity of 1469.7 m s-l, in good agreement with published value^.^ Composition profiles in the creaming emulsions were determined by fitting the predicted curve using equation ( I ) and the velocities and densities in Table I . TO compensate for errors due t o changes in the distance between the probes, aconstant time increment was added to or subtracted from the measured propagation time until
-
1
\+
1563
" 4 E
k
15434
1463
1 0
I
I
20
I
I
40
I
I
60
I
1
80
I
I
100
% OIL
Figure 2
Ultrasound velocity at 20 O C as a function of percentage of oil in commercial solad-dressing-type emulsions: experiment; predicted from equation ( I )
+,
223
the calculated total volume fraction of oil in the sample was 0.05Of0.001.Although the creaming profiles reported below are subject to high uncertainties, they are more precise than could be measured optically, since the continuous phase containing Xanthan gum is very turbid.
Table 1
Composition andproperties of dispersedphase and continuousphases ( C , = Xanrhan gum concentration, p = density, v = ultrasound velocity, G' = low-strain storage modulus) Emulsion
C,/ wt%
p/
kg m-3
v/ m s-I
G'/ Pa
oil
-
919.96
1469.7
-
A
0. I25
999. I3
1483.0
0.7
B
0. I75
999.33
1483.4
1.2
C
0.225
999.67
1484.4
2.3
D
0.325
1000.14
1485. I
4.5
E
0.425
1000.60
1485.2
7.4
100-
80-
60%
0 I
40-
20-
-
20
Figure 3
40
60
80 100 HE I G H l / m m
120
140
160
Concentration profiles of emulsion A. Volume percentage oil is plotted against height: 0 , 0 . 3 days; 0,5 . I days; A, 10.8 days; 19.8 days
+,
D.J. Hibberd. A . M . Howe, A. R. Mackie. P. W. Purdy and M . M . Robins
224
'"1
0
I 40
20
20
Figure 4
40
60
80 100 HE I GHT/mm
120
140
160
Concentration profiles ofemulsion B. Volumepercentage oil is plotted against height: 0,2.0 days; 0,7.7 days; A , 20.9 days; 25.8 days
+,
80
%
0
I 40
20
20
40
60
80
100
120
140
160
HE I GHT/mm
Figure 5
Concentration profiles of emulsion C. Volume percentage oil is plotted against height: 0,O.O days; 0 , 13. I days; A , 19.8 days; 25.8 days
+,
225
Composition Profiles of Creaming Emulsions.-Figures 3-5 show the timedependent concentration profiles of emulsions A-C. We see that, as the Xanthan gum concentration increases, the onset of creaming is delayed, and, once started, the creaming occurs more slowly. No measurable creaming was observed in emulsions D or E over a period of 28 days. In the creaming of emulsions A, B and C, there was observed an increase in oil concentration at the top of the sample, which was soon followed by the appearance of a meniscus between emulsion and continuous phase at the bottom of the sample. Above this meniscus, the volume fraction remained constant until near the top of the sample, where it increased rapidly to 0.4-0.6. Rheology of the Continuous Phases.-All continuous phases were shear-thinning, and the viscosities at low shear-rate increased strongly with increasing Xanthan gum concentration. This indicates that the polymeric system is entangled even at the lowest concentration used.I0 Figure 6 shows continuous-phase viscosities at shear-rates in the range 0.1-1300 s-I. In creep compliance studies, no yield stress could be measured in any of the samples, and so the yield value in these Xanthan gum solutions must be less
10’
0 0 0
0
0
m a
0
loo
\
*
.. b-
s2
(0
r10-l
w
1oo SHEAR RATE
Figure 6
S-’ Rheological behaviour of continuous phases in emulsions as measured at 20 C using a Contraves Rheomat. The viscosity is plotted against emulsion A; X, emulsion B; 0,emulsion C: 0. the shear-rate: emulsion D; 0, emulsion E
+,
D.J. Hibberd. A.M. Howe. A . R. Mackie. P. W. Purdy and M.M. Robins
226
(a)
E D C
B
-1
' -3
3 I
I
-2 -1 Log S T R A I N
I
I
0
1
c
Y
4
"i 9
0
-3
-2
-1
0
1
Log S T R A I N Figure 7
Dynamic mechanical properties of continuous phases in emulsions as measured using an Instron 3250 Rotary Rheometer. (a) The logarithm of the storage modulus isplottedagainst the logarithm of thestrainfor continuous phases A-E. (b) The logarithm of the modulus is plotted against the logarithm of the strain for storage (G') and loss (G") in continuous phases A and E
227
than 30 mPa (the limit of instrumental resolution). Flow was seen to occur at shear-rates as low as 0.003 s-I. Some deformation-sweep experiments were carried out on the Instron with strains in the rangeO.O1-10. Valuesofthestorage modulusG' for the five continuous phases are plotted in Figure 7a, and values of the loss modulus G" for phases A and E are compared with G'in Figure 7b. At low strains (-0. I ) we have G'> G" for all the samples, but the reverse is the case at high strains. The storage modulus is morestraindependent than the loss modulus, but both show evidenceof a plateau region at low strains, indicating the presence of structure which is stable over time-scales of ca. 0.1 s. The samples exhibit a constant value of G' at low deformations; this value increases as the Xanthan gum concentration is increased, indicating increased 'structure' in the continuous phase. At high deformations, G' eventually becomes less than G", which is consistent with the presence of more liquid-like behaviour. Discussion
The ultrasonic technique described in this paper allows the determination of composition profiles in creaming emulsions. The concentration profiles can be used to determine the positions of moving upper and lower menisci, and their velocities can be followed during the course of creaming. It was found that, after an initial delay, the two menisci move with approximately constant speed for most of the creaming time, only slowing down when almost merging with one another. Creaming speeds of lower and upper menisci, vI and v2, are given in Table 2. where they are compared with the velocity vs from Stokes' Law:l' vs =
(2)
In equation (2), g is the acceleration due to gravity, Ap is the density difference between dispersed and continuous phases, 7 is the viscosity of the continuous phase at low shear-rates, and < d 3 is the weight-mean value of the square of the droplet diameter. The shapes of the concentration profiles during creaming are in qualitative agreement with the predictions of a solitary wave model.I2 For a dilute system of non-aggregating hard particles, the model predicts that the rate of movement of the lower meniscus is comparable with the Stokes velocity. The measured velocity vI recorded in Table 2 is, however, much larger than the Stokes velocity. Also, the modelN2does not account for the delay observed before creaming starts.
Table 2
Creaming behaviour of emulsions A, Band C(vs= Stokes velociiy, vI = lower meniscus velociiy, v2 = upper meniscus velociiy)
'
vI/vs
v2/
I10
43
-13
0.70
67
96
-7.0
-10
3
0.17
18
104
-3.6
-2 I
I5
Emulsion
vs/ nrn s
A
2.6
B
C
v I / nrn s-I
nm s-I
v2/vs -4.9
delay1 days 0.7
228
D.J. Hibberd, A . M . Howe. A . R . Mackie, P. W. Purdyand M . M . Robins
There are two possible explanations for the rapid creaming found in these emulsions: the presence of low 'microviscosity' in the polysaccharide continuous phase,13 and the effect of flocculation. The delay before creaming starts, and the observation that the creaming speed does not tend to the Stokes prediction at high polymer concentrations, indicates that 'microviscosity' is not the likely explanation. On the other hand, both these observations can be accounted for if the droplets flocculate. The gravitational stresses of individual droplets in these emulsions are ca. 2 mPa, and so the presence of only a weak structure in the continuous phase would be sufficient to prevent individual droplets from creaming. However, if the droplets were flocculated, gravitational stresses would be much higher, and creaming might occur when flocs became sufficiently large. The high creaming rate observed with all the emulsions after an initial delay is consistent with flocculation because the ratio of gravitational to viscous forces increases with aggregate size." If the explanation is correct, both the delay time and the eventual creaming rate should increase with the degree of flocculation, which in turn should be related to the degree of structure in the polymer system that has to be overcome before creaming can occur. Table 2 gives values of the delay time and the relative creaming speeds, vI/vs and v2/v,. for emulsions A, B and C. Since yield stresses could not be measured, the value of the modulus G' is used instead to indicate the degree of structure in each polymeric continuous phase. As expected, the more highly stuctured systems prevent creaming for longer times (>28 days for emulsions D and E), but the eventual creamingrates are increasingly higher than the Stokes predictions. To a first approximation, the value of v,/vJ for emulsions B and C is consistent with flocs containing of the order of lo3 droplets. Gravitational stresses on such flocs would be cu. 20 mPa, which is consistent with yield stresses in the polysaccharide solutions being<30 mPa. We believe that the cause of droplet flocculation is probably a depletion effect," with highly hydrophilic Xanthan gum molecules attracting solvent from between the droplets, and so forcing them together. The dynamics of the flocculation and creaming will depend on interactions between the droplets and on structural rearrangements in the polymercontaining aqueous phase. Further work is required to identify the relevant polymeric relaxation mechanisms. Conclusions The ultrasonic technique is clearly shown to be a powerful tool for measuring creaming in emulsions. It provides a method for detecting creaming well before visual inhomogeneities can be observed. Measurement of concentration profiles enables detailed studies of creaming mechanisms to be undertaken. For dilute emulsions of soya bean oil in continuous phases containing various amounts of Xanthan gum, the measured creaming profiles agree qualitatively with predictions from a solitary wave model. At low concentrations, Xanthan gum gives only temporary stability towards creaming, and it appears to induce flocculation with the result that eventual creaming rates are higher than expected on the basis of the viscosity of the continuous phase. At high concentrations, Xanthan gum forms an elastic structure which was found to stabilize the emulsions for at least one month. Acknowledgements. We are grateful to Professor Peter Richmond for advice and
229
guidance during the course of the work, and to Dr. Vic Morris for discussions prior to its commencement. For the lnstron data, discussions on rheology, and comments on the paper, we are grateful to Dr. Geoff Brownsey. We thank Paul Gunning for technical support, and the Ministry of Agriculture, Fisheries and Food for funding the work. References I E. Dickinson and G. Stainsby, ‘Colloids in Food,’ Applied Science, London, 1982. 2 R.S. Faranito and R.L. Rowell, in‘Encycopedia of Emulsion Technology,’ed. P. k c h e r . Marcel Dekker. New York, 1983,Vol. I, p. 439. 3 P.A. Gunning, M.S.R. Hennock, A.M. Howe, A.R. Mackie. P. Richmond, and M.M. Robins, Colloids SurJ, 1986,20,65. 4 P. Walstra and H.H.Oortwijn. Nerh. Milk Dairy J., 1975.29.263. 5 S.R.Reddy and H.S. Fogler, J. Colloid Inferface Sci., 1981,79,105. 6 A.M. Howe, A.R. Mackie, and M.M. Robins, J. Dispersion Sci. Technol., 1986.7.23 I. 7 M. Glicksman. ‘Food Hydrocolloids,’ CRC Press, Florida, 1982. 8 R.J. Urick, J. Appl. fhys., 1947,18,983. 9 C.Javanaud and R.R. Rahalkar, Ferre-Sei/en-Ansfrichmiifel, in the press. 10 G. Robinson, S.B. Ross-Murphy, and E.R. Morris. CarbohydrureRes., 1982,107,17. I I G.G. Stokes, fhilos. Mug., 1851, 1,337. I2 G.C. Barker and M.J.Grimson, J. fhys. A, in the press. 13 T.-H. Lin and G.D.J. Phillies, J. fhys. Chem., 1982,86,4073. 14 D.H. Napper, ‘Polymeric Stabilization of Colloidal Dispersions,’ Academic Press, London, 1983.
Isolated and Interacting Triglyceride-Water Interfaces By L.R. FISHER, E.E. MITCHELL, and N.S. PARKER
(CSIRO Division of Food Research, P.0.Box 52. North Ryde, N.S.W. 2 1 73,Australia)
Introduction
Most studies of the stabilization of oil-in-water emulsions by surfactants have been on systems for which the oil is a liquid hydrocarbon. Comparatively little work has been reported on cases where the oil is a triglyceride or a mixture of triglycerides,’ although such cases are clearly of more direct interest in food science. In particular, accurate measurements for the triglyceride-water interface of such a fundamental property as the interfacial tension are almost completely lacking.* To understand stabilization of food emulsions by surfactants, we need to know (a) the adsorption behaviour of food stabilizingsurfactants at the triglyceride-water interface and (b) the way in which these surfactants affect the interaction between two triglyceride-water interfaces. We have begun such a study using a model triglyceride called MCT8 10 (where MCT stands for ‘medium chain triglycerides’) derived from glycerol esterified with a mixture of octanoic and decanoic acids. This synthetic triglyceride was chosen because it is readily available in a pure state and is free of the contaminants often found in natural triglyceride mixtures such as vegetable oils. We report here results on adsorption at the MCT8IO-water interface of glycerol monooleate, some related hydroxylic surfactants (oleyl alcohol and oleic acid), and several proteins. For comparison purposes, results are also presented for adsorption at the n-decane-water interface. We also describe an optical reflectance method which we are now using to study the effect of adsorbed surfactant on the interaction between two such interfaces. Materials and Methods
The MCT8 10 (Nihon Oils and Fats, Osaka, Japan) (mean relative molecular weight = 480) was a gift from Mr. A.C. Fogerty. BDH ndecane was passed through an activated aluminacolumn to remove polar impurities. The following materials were used as received: glycerol monooleate (Nu-Chek Prep., Elysian, MN, U.S.A.), oleic acid and oleyl alcohol (Sigma), bovine serum albumin, lysozyme and p-casein (gifts 230
23 I
from Dr. R.W.Burley). The water was laboratory distilled water, which had been further treated by passage through an activated charcoal bed and then distilled in a quiescent quartz still; it was in equilibrium with atmospheric CO, (pH 6.05). All apparatus components made of glass, stainless steel and fluorocarbon (the only materials to come in contact with the experimental liquids) were cleaned with chromic acid, washed thoroughly with double-distilled water, and then dried in an oven. Glycerol monooleate, oleyl alcohol and oleic acid were dissolved in the appropriate oil phase, while the proteins were dissolved in the aqueous phase. lnterfacial tensions were measured by the pendant drop method. The apparatus was similar to that described by Ambwani and Fort’ with a 2 mW He/Ne laser, beam-expanded to give a parallel beam of diameter ca. 5 mm, as the light source. Measurements of drop profile were taken directly from the image of the drop on a screen magnified approximately 100 times. Equiiibrium tensions were reproducible to fO.1 mN m-I for a temperature of 25.0f0.2O C . The density of pure MCT810 was determined using an Anton-Paar precision density meter fitted with an ultrathermostat. Other densities were calculated assuming ideal mixing. The thickness of the film of liquid draining from between two approaching interfaces was determined from the intensity of a reflected laser beam*-6(see Figure I). The incident laser beam was normal to the common tangent plane of the closely
W
PTF Septum Figure 1
I
Kel-f cell
Apparatus for measuring the thickness of a draining f i l m from its optical reflectance. A n incident vertically-polarized laser beam is focused onto the draining f i l m through a mica quarter-wave plate, tilted to displace the unwanted reflection from its own sugace. The beam reflected from the draining f i l m passes through a horizontal polarizer placed in front of a photomultiplier. Only light reflected from the draining f i l m can enter the photomultiplier; light reflected from windows, lenses, etc., is cut out. The beam-splitter andfocusing lens are mountedon a common carriage. so that thefocused beam can be translated laterally, allowing measurement of the profile of the drainingf i l m
232
L. R. Fisher, E. E. Mitchell and N.S. Parker
apposed interfaces, and it could be translated laterally. The beam was focused to a spot of diameter cu. 10 pm. The profile of the draining film between the interfaces could be measured by scanning the incident beam across the film. Results and Discussion Figures 2-4 show the effect of changing the concentration c of glycerol monooleate, oleyl alcohol or oleic acid on the tension y at the MCT810-water or n-decanewater interfaces. We interpret the relationship between y and c in terms of the Gibbs adsorption isotherm:
I'= -(kT)~l(dy/dIn a)
= -(kT)-'(dy/d In c)
(1)
(2)
If the Gibbs model of the interface is adopted,' r is the relative adsorption (molecules per unit area), a is the activity of adsorbate, k is Boltzmann's constant, and T is the absolute temperature. For glycerol monooleate in n-decane, the replacement of activity a by concentration c is not a bad approximation.8 For want of the appropriate activity coefficient data, it is also adopted for the other systems. In all the cases considered, the slope of the curve of y versus In c increases with increasing c, becoming constant at high values of c. As the amount adsorbed is from equation (2) proportional to the slope, the experimental curves indicate that adsorption is increasing with increasing surfactant concentration, as expected, but that the amount adsorbed becomes constant at high surfactant concentration. This behaviour in other systems led van Voorst Vader to p o ~ t u l a t e ~the * ' ~existence of 'saturation adsorption.' This postulate is, however, at the very least, thermodynamically dubious, since it may be, for instance, that plots of y versus In a would not be linear. We examine the possibility that the data can be accounted for by a simple adsorption isotherm. We choose the Langmuir adsorption isotherm,' written in the form:
r = r r n a x K ~ I (+t KC)
(3)
The advantage of fitting this particular isotherm is that there is only one disposable parameter, K,to adjust in order to fit a theoretical curve t o data that may span two or three decades of concentration. To compare experiment and theory directly, a little rearrangement of the preceding equations is necessary. If y = yo when c = 0, then from equation (2) we have
py
= f=II'kT d In c
Substituting r from equation (3) and integrating, we obtain y = yo - rmax kT In ( 1
+ Kc)
(4)
233
50
I
I
v-
a
€
z E
\
40
-
-
*
4 30 v)
-
‘9
b.,
z
W
20
Y,
-
4 0 2
‘0..
-I
‘q.,
-
10-
e W
‘b
b
Z
-
0
Figure 2
I
I
Y
Interfacial tension as a function of the logarithm of the concentration of glycerol monooleate: (a) n-decane-water interface (line from equation (5) with K = 9.63 X lo4 1 rnol-I):(h) MCT810-water interface (linefrom equation (5) with K = I .35X 10’ I rnol-I)
L. R. Fisher. E. E. Mitchelland N.S.Parker
234
52
I
I
v-I
log CONCENTRATION
26
I
F
E
-. b
L-Q-4
Z
E
/M
24
-
-
\
.0
Z
4!
-
-
2 20 -
-
v)
z
W
22
c -J
9
LL
oz W c Z
-
18
Figure 3
1
-1
Interfacial tension as a function ofthe logarithm of the concentration of oleyl alcohol: (a) n-decane-water interface (line from equation (5) with K = I .5 X lo3 I rnolk'); (b) MCT810-water interface (line from equation (5) with K = 40 1 rnol-I)
235
The theoretical curves in Figures 2-4 were calculated from equation ( 5 ) with values of K chosen to give the best fits by eye. The fitted curves for oleyl alcohol and glycerol monooleate agree well with the experimental data, supporting our choice of the simple Langmuir isotherm. There is a discrepancy, however, when it comes to oleic acid (see Figure 4). We first discuss oleic acid at the n-decane-water interface. Oleic acid is soluble only in the oil phase. It is well known" that carboxylic acids dimerize in hydrocarbon solution, with a carbonyl group of one molecule hydrogen-bonding to an hydroxyl group of another. The dimerization constant of oleic acid appears not to have been reported, but an estimate can be obtained by extrapolating the values quoted for octanoic, dodecanoic and tetradecanoic acids, and making the reasonable assumption that the double bond does not affect the dimerization behaviour. Using this value (1.16 X lo4 I mol-I) the monomer concentrations of oleic acid in ndecane may be calculated, I t seems reasonable to assume that the monomer is the major surface-active species, and so to replace total concentrations (Figure 4a) by monomer concentrations (Figure 4b). When this is done, a good fit between the experimental data and the curve calculated from the Langmuir isotherm is found. A similar data treatment for oleic acid at the triglyceride-water interface does not produce such a good fit (Figure 4c), and in this case we are forced to conclude that the Langmuir isotherm does not give a good representation of the adsorption behaviour. A plausible explanation in this case is that the energy of adsorption, which can be derived directly from K (see below), is not constant but depends on the degree of adsorption. For those cases in which the Langmuir adsorption isotherm appears to be applicable, further progress can be made since K is nothing more or less than the equilibrium constant for partition of molecules between bulk phase and interface. Free energies of adsorption, AGads,calculated from
AGads= kT In K are listed in Table I . (For comparison purposes, we note that the free energy of Table 1
Adsorptionfree energies AGds of surfactants at n-decane-water and MCT810-water interfaces. (The error in AG,,,, as estimatedfrom trial Lungmuir isotherms that give an obviously badfit 10the experimental data. is approximately f0.2 kT.)
kT Surfartant
n-derane-water
MC7-8IO-wclr~r
glycerol monooleate
13.1
5.6
oleic acid
10.8
*
oleyl alcohol
8.9
* Figure 4c shows that there is no point in calculating this value.
4.4
L. R. Fisher, E. E. Mitchell and N.S.Porker
236
formation of a single hydrogen bond is of the order of 10 kT.") For the same surfactant species, the difference between the values of AGadsat n-decane-water and triglyceride-water interfaces is the free energy of transfer from the one oil to the other. The free energy of transfer of glycerol monooleate from the triglyceride to the hydrocarbon is 7.4 kT, which is nearly twice the value for transfer of oleyl alcohol (4.4 kT). This can be explained by the fact that glycerol monooleate has two
log CONCENTRATION / M
2 K w I-
..
10-
I
........
I
........
I
. --
....
*.Y
237
E
z E \ z 0 v)
Z
w
c
Figure 4
Interfacial tension as afunction of the logarithm of the concentration of oleic acid: (a)n-decane-wafer inlegace (linefromequation (5) with K = 9.51 X lo3 I mol-'); (b) as (a) but now concentration refers to actual oleic acid monomer concentration in n-decane (line from equation (5) with K = 9.26 X lo3 I mol-');(c) MCT810-water interfoce with oleic acid monomer concentration in MCT810 calculatedfrom dimerization constant of 3 X lo2 I mol-' as estimatedfrom heights of hydrogen-bonded and non-hydrogen-bonded carbonyl peaks in infraredspectrum ofoleicacidin MCTBIO(upperand1owerlinesfrom equation (5) with K = 42 and K = 65 1 mol-I, respectively)
hydroxyl groups while oleyl alcohol has just one. The data are in this respect self-consistent, thereby providing some support for the adoption of the Langmuir model. In one of the very few systematic studies of protein adsorption, Graham and Phillips reportedI2.l3 the behaviour of three proteins (lysozyme, bovine serum albumin and /3-casein) at air-water and hydrocarbon-water interfaces, measuring both the actual amounts adsorbed and the effect on interfacial tension. The proteins were chosen to represent a range of structures from globular to random coil, and we have chosen to study the behaviour of the same three proteins at the triglyceridewater interface. In this initial report, we present tensions as a function of time for the n-decane-water and MCTIIO-water interfaces, and results are shown in Figures 5 and 6. We see that &casein has a greater final effect on the interfacial tension at the n-decane-water interface than does bovine serum albumin, while the reverse is the case at the triglyceride-water interface. Furthermore, the tension at the triglyceride-water interface appears to decrease in an irregular fashion for all
L. R. Fisher, E. E. Mitchell and N.S. Parker
238
50 c I
E z
45
E
\ 40
z
-
0
z
35
W
I-
-I
5
30
0 Q:
L
K 25 W I-
Z
-
20
Figure 5
Adsorption of lysozyme. bovine serum albumin (BSA)and /3-caseinat the n-decane- water inlegace. The interfacial tension isplotted against timefor a constant bulk aqueousphase concentration of wt%
28 c
E z
26
€
\
0
24
v,
z w I-J
22
9 0
2rn 20 W I-
Z
-
18
0
20
40
60
TIME Figure 6
80
100
120
min
Adsorption of lysozyme. bovine serum albumin (BSA)and &casein at the MCT810-water interface. The integacial tension isplotted against timefor a constant bulk aqueous phase concentration of wt%
239
three proteins studied, as compared with the smooth curves of tension versus time for the same proteins at the n-decane-water interface. This suggests that there may be differences in adsorption mechanism at the two interfaces. It should be said, though, that the height and position of the irregularities in the graphs of y against time for the triglyceride-water interface are not reproducible. One could speculate about the unfolding behaviour of the proteins at the interface, since a model is availablek4which allows calculation of the mean area of the unfolding unit. Use of the model, however, requires a knowledge of the adsorption isotherm, since a complicated plot covering the region of constant adsorption is needed. Plots of our data in terms of the model (equation 7 of the paper by Graham and Phillips12) d o produce the appropriate linear regions, but we are wary of interpreting these regions in terms of the unfolding model until we have obtained firm adsorption data. In any case, the stepwise adsorption behaviour of the three proteins at the MCT810-water interface does not allow for linear plots, and it remains as yet unexplained. We move now to interactions between interfaces. As described in Figure I, we have devised an optical method6 to measure the thickness of the draining film between two hemispherical interfaces as they are brought towards each other until they begin to deform - producing a flattened region in which the liquid between the interfaces forms a thin draining film. The method is equally applicable to the study of the interaction either between two oil drops coated with adsorbed surfactant or between two thin hydrocarbon films stabilized by surfactant. The former interaction is clearly more pertinent to food emulsion problems, but work in this
200
E \ C 0
.e ;100 c
c v)
L
c
2.
-0 .m
0
-.2 Figure I
-. 1 0 .1 Lateral distance from centre of draining film
/
.2 Inrn
Profile of draining film between glycerol monooleate-n-hexadecane bilayers in pure water as afunction of time: 0,after 68 s; 0,after 92 s; A . after I28 s; 0, after I76 s
240
L. R. Fisher, E. E. Mirchelland N.S. Parker
direction is still at an early stage, and so we report here an example of the application of the method to an interaction of two thin films. Figure 7 shows data for the interaction of two bilayer lipid membranes consisting of n-hexadecane films stabilized by glycerol monooleateis. The films are cu. 3.2 nm thicki6and the aqueous phase between the bilayers consists of pure water. When the bilayers are brought together rapidly (360 p m s-I), the aqueous draining film between them adopts a dimpled configuration,” the distance between the bilayers at the centre being greater than that at the boundary ring. This configuration is simply a result of the requirement that, if the aqueous film is to drain, there must be a pressure gradient driving liquid flow from the centre to the edge of the draining fi1m.l’ In the example given, the aqueous film eventually drains to produce an equilibrium flat film. The large final thickness of the aqueous film is a consequence of a small amount of charged impurity in the bilayers, which produces a long-range electrostatic repulsion between them. When the Debye length is reduced by adding salt to the aqueous phase (ionic strength 0. I M), the bilayers are able to approach closely, and will in fact fuse to produce a structure in which the apposed parts of the original bilayers have become a single bilayer.’* Conclusions For hydroxylated surfactants and proteins, there are major qualitative differences in adsorption behaviour at n-decane-water and M C T I 10-water interfaces. The adsorption of glycerol monooleate and oleyl alcohol is consistent with a Langmuir isotherm at both n-decane-water and M C T I 10-water interfaces. Adsorption of oleic acid at the n-decane-water interface can also be described by a Langmuir isotherm once allowance is made for dimerization in the oil phase. Oleic acid at the M C T I 10-water interface is not consistent with a Langmuir isotherm; the reason for this is not obvious at present. The proteins, lysozyme, 8-casein and bovine serum albumin, yield stepwise plots of interfacial tension as a function of time at the MCT8 10-water interface, while the corresponding plots at the n-decane-water interface are smooth. We report on the development of an optical method for measuring the distance between approaching interfaces. So far, the method has mainly been used for studying interactions between approaching bilayer lipid membranes stabilized by glycerol monooleate. It is to be used to study the interaction between oil droplets separated by an aqueous phase containing surfactant. Acknowledgements. The authors are grateful to Professor Lee White (University of
Melbourne), D r Joe Wolfe (University of New South Wales) and Professor Denis Haydon FRS (Cambridge University) for helpful discussions, and D r J. D. Bignall (Biorad) for help indetermining the dimerization constant of oleic acid in MCT810. References I E. Dickinson and G. Stainsby, ‘Colloids in Food,’ Applied Science, London, 1982. 2 L.R. Fisher, E.E. Mitchel1,and N.S. Parker,J. FoodSci., 1985.50, 1201. 3 D.S. Ambwani and T. Fort, jun., in’Surface and Colloid Science,’ed. R.J. Good and R.R. Stromberg, Plenum, New York, 1979, Vol. I I, p.93.
241 4 A.P. Yelkin and G.N. Berstovskii, Biojziku, 1974,5,846. 5 L.R. Fisher, N.S. Parker, and F. Sharples. Opr.Eng., 1980.19.798. 6 L.R. Fisher, N.S. Parker, and D.A. Haydon, Faruduy Discuss. Chem. Soc.. 1986,in the press. 7 R. Aveyard and D.A. Haydon, 'An Introduction t o the Principles of Surface Chemistry,' Cambridge University Press, 1973. 8 E.M. Andrews, E.D. Manev, and D.A. Haydon, Furuduy Symp. Chem. Soc., 1970,1,46. 9 F.van Voorst Vader, Truns. Furuduy Soc., 1960,56,1067. 10 F. van Voorst Vader, Truns. Furuduy Soc., 1960,56,1078. I I G.C. Pimentel and A.L. McLellan,'The Hydrogen Bond,'Freeman, London, 1963. I2 D.E. Graham and M.C. Phillips, J. ColfoidInterfuce Sci., 1979.70.403. 13 D.E. Graham and M.C. Phillips, J. Colfoidlnterfuce Sci., 1979.70.415. 14 A.F.H. Ward and L. Tordai, J. Chem. Phys.. 1946,14,453. I5 R. Fettiplacc, L.G.M. Gordon, S.B. Hladky, J. Requena, H.P. Zingsheim, and D.A. Haydon, in 'Methods in Membrane Biology,'ed. E.D. Korn, Plenum, New York, 1974, Vol. 4,p. I. 16 J.P. Dilger, Biochim. Biophys. Actu, 1981,645,357. I7 J.D. Chen, J. Colloid Inrerfuce Sci., 1984.98.329. 18 L.R. Fisher and N.S. Parker, Biophys. J., 1984.46.253.
Overview of Emulsion and Foam Stability
By P. WALSTRA
(Department of Food Science. Agricultural University, de Dreijen 12, 6703 BC Wagenmgen. The Netherlands)
Introduction The title of this article almost suggests an overview of the greater part of colloid and surface science, which is clearly beyond the scope of this volume and the qualifications of the author. Instead, I shall briefly review some aspects of importance for food emulsions and foams. Let us begin by mentioning some differences between, on the one hand, colloidal suspensions and, on the other hand, foams and emulsions. Under most conditions, the Laplace pressure causes droplets and bubbles to assume an almost perfectly spherical shape, which greatly facilitates prediction from the theories of colloid stability. Nevertheless, the deformability of particles does make their break-up into smaller ones easier, as well as allowing coalescence to occur. In emulsions and foams, the interface between particle and continuous phase is fluid, and so thereare no specific adsorption sites for surfactant molecules, which are free to move in the plane of the interface. Because of this, gradients in interfacial tension can occur, and these in turn may lead to lateral motion of the interface, thereby exerting a tangential stress on the bordering fluids. Emulsions and foams are similar in theory, but their behaviour can be quite different in practice since several important parameters differ greatly in magnitude (see Table 1). Foam bubbles can often be easily deformed (leading, for example, to a polyhedral shape instead of a sphere), but the much smaller emulsion droplets cannot. Under mild conditions, the interface of an emulsion droplet is almost fully immobilized in the tangential direction, because of the readily developing intefacial tension gradients. Consequently, emulsion droplets are usually found to behave as rigid spheres. Not included in this review, as such, is the formation of emulsions and foams. This is governed predominantly by hydrodynamic and surface phenomena, with energy input and dynamic surface properties being of great importance.' The resulting distribution of droplet or bubble sizes is of considerable significance with respect to stability, however, as is the composition of the adsorbed layers formed. Mainly because of some recent developments, this latter aspect will be considered in the following section. 242
243
Table 1
Comparison of the magnitudes of various quantities in emulsions and foams
Property
Value in Emulsions
particle diameter
2 x w 7 t o I O - ~m
particle volume fraction
0.01 to 0.8
0.5 to 0.97
density difference
10 to 100 kg m3
lo3kg m-3
compressibility of dispersed phase
5x
interfacial tension
10-10 N-I
to
m*
N m-‘
Value in Foams
to 3 x
m
N-I m2
0.03 to 0.05 N m-’
Laplace pressure
e.g., lo4 N rn-’
e.g.. lo2 N m-2
solubility of dispersed phase in continuous phase
0 (O/ W), 0.15 vol%(W/O)
2.2 vol%
Surface Layers A system quickly reaches equilibrium if only small-molecule surfactants are present. The composition of the adsorbed layer, or, more precisely, the surface excess for the various surfactant components, can normally be calculated from their concentrations and properties by use of Gibbs’ equation.2 At the high concentrations of surfactants that can form lamellar phases, it is more difficult to predict what will happen since multilayers may be formed around droplets or bubbles3 This situation is not, however, very common in food emulsions and foams, where macromolecules (predominantly proteins) are usually present, either alone or in combination with small-molecule surfactants; in this case, predictions are far more difficult. It is commonly assumed that substances like proteins adsorb irreversibly at air-water o r oil-water interfaces. Evidence for irreversibility comes from the very steep adsorption isotherm commonly found (see Figure 1). and the fact that dilution of an emulsion or foam with the solvent of the continuous phase does not apparently cause desorption from the interface. Irreversible adsorption would have some important consequences.
(a) The surface excess is not governed by the bulk concentration of surfactant, as in Gibbs’ equilibrium adsorption, but by the total concentration before emulsification or foaming divided by the surface area eventually created.’” (b) If protein molecules/particles and emulsion droplets d o not differ too much in size, the larger particles are adsorbed preferentially over the smaller ones during emulsification.l*6 This also implies that smaller emulsion droplets acquire a thicker protein layer if protein particles of different sizes are present? (As foam bubbles are relatively large, this phenomenon does not apply to them.)
P. Walsrra
244
(c) Coalescence can occur during and after emulsification or foaming, leading to a decrease in surface area and an associated increase in surface excess. (This has been demonstrated for both emulsions' and foams.") These conclusions should, however, be viewed with at least some suspicion. If a mixture of homologous macromolecules of varying molecular weight is present, even fully reversible adsorption leads to a very steep isotherm9 which depends on concentration as described [under (a)] above. This is due to the larger molecules adsorbing preferentially over the smaller ones, the preference being stronger at a lower bulk concentration. MacRitchie has shownI0 experimentally that protein molecules can desorb from an air-water interface, albeit slowly; the desorption is quicker at a higher surface pressure (produced by compressing the surface), at a lower bulk concentration, and with a protein of lower molecular weight. The same phenomenon has also been observed by others." The implication therefore is that coalescence of droplets o r bubbles can lead to desorption of protein. What is the significance of a high surface pressure? For proteins at an air-water interface, de Feijter has clearly s h ~ w n that ~ * ~measurable ~ ~ surface pressures (21mN m-I) are obtained only at a surface excess of ca. I mg m-* o r above. This suggests that, at pressures necessary to cause measurable desorption, the surface is fully packed with protein molecules, even without considerable unfolding at the interface. It also implies that very small changes in protein conformation o r surface-layer composition may cause large changes in surface pressure. This means that great caution is needed in interpreting changes in surface pressure: inferring the kinetics of protein adsorption from changes in interfacial tension appears to be impossible.
c/ kg .m-3 Figure 1
c/
Y O
Typicalprotein adsorption isotherm. The surface excess concentration
r isplotted against the bulk concentration cfor I4Cacetylated &casein at the air-water interface (pH 7,O.Ol
M NaCI,
22 0C)4
245
After a protein has adsorbed, it may undergo considerable changes of molecular conformation. As followed by changes in surface rheological properties, this can go on for a long time.I4 Multilayer formation may also O C C U ~ . ~ - I ~In addition, some proteins (e.g., ovalbumin) can denature to such an extent that they become insoluble; repeated adsorption and desorption, as occurs during prolonged emulsification, may even lead to the loss of the ability to stabilize emulsions (at least for ovalbuminIs). When a mixture of proteins is present, the component giving the lowest surface tension may (partly) drive the others out of the i n t e r f a ~ e , ’ ~ .and ’~-” this again may take a long time. Many small-molecule surfactants readily oust proteins from an oil-water interface.’ As such molecules are always present in natural triglyceride oils, this is probably part of the explanation for the difference in behaviour between oil-in-water emulsions made with such oils and those made with a pure hydrocarbon. The interfacial tension between pure triglyceride and water seems, however, to be lower than that between pure paraffin and water.I8 We may infer that it is extremely difficult to predict the composition of surface layers in most food emulsions and foams. Knowledge about the surface layers is a prerequisite for predicting the energy of interaction between closely approaching bubbles or droplets, and this in turn is needed for predicting stability against flocculation and coalescence. Even if surface compositions were known, the problem would be far from solved, since the prediction of steric repulsion between particles is still very much in its infancy, despite important theoretical advances.I9 Types of Instability
The physical changes that may occur are: (i) Ostwaldripening, involving the growth of larger particles at the expense of smaller ones (which eventually disappear) by isothermal distillation, i.e., diffusion of molecules from one particle to another through the continuous phase; (ii) creaming (or sedimentation), which causes part of the system to become partially depleted of particles. leading to the formation of a layer in which the particles are packed close to one another; (iii)/locculafion, in which groups of particles get very close to one another for a far longer time than would occur simply on the basis of their Brownian motion, due to the presence of a net attractive interparticle free energy; (iv) coalescence, the flowing together of two particles into one; and (v) partial coalescence, in which two particles flow together only partially to form one non-spherical particle. In all these cases except (v), the particles may be either emulsion droplets or foam bubbles. However, foam bubbles cream so rapidly that a foam can be regarded as a ‘cream layer’; the process of drainage of continuous phase from this layer effectively takes the place of the creaming phase in emulsions, Flocculation is of little interest in a foam since the bubbles are normally close together in any case. The various changes affect one another.20Creaming may be enhanced by any of the others. Coalescence rarely occurs unless the particles are creamed or flocculated. Creaming may enhance the rate of flocculation. Stirring (or, more generally, the presence of velocity gradients) disturbs creaming, and enhances the rate of flocculation, but it may also cause floc disruption. Intensive stirring may even induce disruption of emulsion droplets or foam bubbles. Stirring mostly enhances, or is even a necessary requirement for, partial coalescence.
P. Wolstra
246
Ostwald Ripening.-The driving force for Ostwald ripening is the greater solubility of a substance on the concave side of a curved interface in the material on the convex side. Assuming ideal thermodynamic behaviour, which is often nearly the case so long as solubility is slight, the effect is described by the Kelvin equation,
R T In(S‘r/S’,)= 2 y M / p r
(1)
where S’, is the solubility in a particle of radius r, S’, is the solubility when the interface is planar, y is the interfacial tension, R is the gas constant, T is the temperature, and M and p are the molar mass and the mass density of the substance in the particle. Typical numerical values of the various parameters are listed in Table 2. For Ostwald ripening to occur, mass transport in the continuous phase must be possible, which implies that S’- should not be too small. Since triglyceride oil is virtually insoluble in water, we can deduce that there is no Ostwald ripening in food oil-in-water emulsions. In water-in-oil emulsions, however, some Ostwald ripening may occur, but it will tend to stop quickly if the water droplets contain some salt; since ions cannot dissolve in the oil, any shrinkage of a droplet increases its osmotic pressure, which represents an increased driving force for water uptake as opposed to loss. With foams, rapid Ostwald ripening is possible:’ since air dissolves well in water and the diffusion coefficient is high. In many foams, bubbles of diameter < I mm disappear rapidly. Table 2
a
Numerical values of different quantities involved in the Kelvin equation [equation ( I ) ] for three types of dispersed system Variable
Water-in-Oil
Oil-in-Woter
Air-in-Water
y / N m-’
0.005
0.005
0.05
P I kg m-’
990
920
I .2
M /kg mol-’
0.018
0.7
0.029
SJS‘,
I .oooO73
I .003I
2.64
a
Colculatedfrom equation ( I ) for aparticle of rodius I
pm ot
300 K
Ostwald ripening proceeds slower when the interfacial tension is lower, and thus it is slowed down if contraction of the particle surface causes y to decrease. It even stops altogether if the following condition is satisfied:22 E = - d y / d In A > y / 2
(2)
In equation (2), A is the area, and E is the surface dilational modulus. When protein is at the interface, the value of E may be fairly high. It can be determined experimentally, but there is the problem that the value tends to be lower at longer times as surfactant slowly desorbs into the continuous phase, and so a timedependent modulus has to be used. (An alternative approach uses the surface
247
dilational viscosity.2') The well-known stabilization of foams by egg white is presumably due to the protein forming a denatured, insoluble layer around the bubbles, thereby conferring a large and permanent surface modulus. Thus, one can stop Ostwald ripening by immobilizing the bubble surface. This can also be done with a packed layer of solid particles at the air-water interface, e.g., partly-solid fat globules in whipped cream20 o r fat crystals in some whipped topping^.^' Another method is to confer on the continuous phase a sufficiently high yield stress (see below), e.g., by means of a gelling agent. Creaming.-In fairly dilute dispersion of spherical particles, creaming is usually described by Stokes' equation,
(3)
v = 2gApr2/9v
where v is the linear velocity, g is the acceleration due to gravity, Ap is the density difference between particle and continuous phase, and v is the viscosity of the latter. The usefulness and limitations of equation (3) have been adequately d e s ~ r i b e d . ~ . ~ ~ . ~ ~ One problem is that thecontinuous phase is often highly non-Newtonian; in fact, in
I1/Pa s B
0.3
0.2
0.1
0 1
lo-'
shear-rate/s' Figure 2
I
0.3
1
1
1 3 10 shear stress /Pa
Examples of non- Newtonian behaviour of dispersion media (courtesy Dr. T.van Vliet). ( A ) The apparent viscosity 7' is plotted as afunction of shear-rate for evaporated milk. (9) The apparent viscosity q' is plotted as afunction of shear stressfor chocolate milk (estimatedyield stress ca. 0.28 Pa)
248
P. Wolstra
many foods, it is not a single phase, but is physically inhomogeneous. Knowledge of the structure and rheology of the dispersion medium is often of greater importance than knowledge relating to the dispersed particles. We need to know the apparent viscosity 9‘ at a shear-rate of roughly v/r, i.e., in the range 1044-.10-’s-1. Measurement of 7’ under such extreme conditions is now possible.26 The example in Figure 2A shows that 9’ can be very different from the value measured in a conventional apparatus at a shear-rate of 210 s-I. Creaming can be stopped completely if the viscoelastic dispersion medium has a sufficiently high yield stress. A yield stress of value >2rgAp is required. This corresponds to cu. Pa for emulsions, and Figure 2B shows a system for which this criterion is easily satisfied. To prevent creaming of foam bubbles, a much higher yield stress is required (say, 10 Pa); but, to prevent drainage of continuous phase from the lamellae of a polyhedral foam, a somewhat lower value may suffice.2’ If Ostwald ripening of foam bubbles is also to be prevented by the yield stress, a high value is again necessary. Creaming phenomena in concentrated dispersions are much more difficult to predict. An elegant method has now been developed2’ to study creaming of emulsions in some detail, but it will take considerable effort to explain the results obtained. An intricate interplay of colloidal, dynamic surface, and hydrodynamic forces may have to be unravelled. Flocculation.-The question whether, or how quickly, small particles will flocculate is the classical one of colloid science. Flocculation may occur if the interaction free energy between two particles is negative at some distance of separation. The flocculation rate can be estimated from the product of a frequency factor (how often d o particles encounter one another?) and a probability factor (how long d o they stay together?). The frequency factor can be well predicted, even taking into account hydrodynamic hindrance, both for the case of Brownian motion2*and also for simple types of f l o ~The . ~ probability depends on the interaction energy (the free energy needed to bring the particles from infinity to some specified distance apart), and it is unity if the energy is negative for all interparticle distances. Often there is a kind of energy barrier to close proximity as shown in Figure 3. In other cases, the interaction energy is positive for all separations, implying a zero probability of flocculation. The difficulty, then, is to calculate the interaction energy as a function of the interparticle distance. Typically, one considers three terms:24van der Waals attraction, electrostatic repulsion, and steric repulsion. The van der Waals attraction depends on the particle diameter and the Hamaker constant; the latter is known in principle if the compositions of both phases are known. The electrostatic repulsion depends on the surface potential of the particle, the particle diameter, and the ionic strength of the continuous phase; the dielectric constant and the temperature also come into play. All these variables can, in principle, be determined. Protruding, flexible, macromolecular chains are the origin of the steric repulsion (attraction is also possible). The effect depends primarily on the surface density and length of these chains, and on the solvent quality as expressed by the chi-parameter in Flory-Huggins theory.2s Calculation of the interaction energy between particles is often impossible, especially if the composition of the surface layer is unknown. There are many
249
+ d2Gint
I
I
I
I
dh2 h
0
Figure 3
unstable
'I
stable
I unstable I I I
stable
Schematic representation of the colloidal interaction between two spherical particles. The free energy Gin, and its second derivative d2Gin,/dh2are plotted against the distance h between the particle surfaces. Indicated on the diagram are the separations at which a symmetric wave on thefilm between the particles (cf. Figure54 willbe damped (stable). or. alternatively. will spontaneously grow in amplitude (unstable). according to the theory of Vrij*
complicating factors. The Hamaker constant may be uncertain, primarily because it is affected by the surface layer if this is not very thin. Even if the overall composition of the surface layer is known, the steric repulsion term cannot yet, in fact, be calculated.m One problem here is that most adsorbed macromolecules are heteropolymers. For the case of freely adsorbing homopolymers, it appears that there is always a net attraction if there is enough time for macromolecular desorption as two particles approach each other. When no such equilibrium occurs, as is far more likely, steric stabilization is favoured by a high concentration of macromolecules, a high molecular weight, and a good solvent quality. The surface potential of particles may be uncertain, since electrokinetic measurements give the
P. Walstra
250
Table 3
Variables affecting the three main types of colloidal interaction bet ween sphericalparticles in an aqueous medium. A tick denotes that the variable is important. All variables except particle size may, in turn, a/fect the composition of the surface layer.
Variable
Vander Waals A ttraciion
Electrostatic Repulsion
Steric Repulsion
particle size
J
J
(J)
particle material
J
surface layer
(J)
J
PH
ionic strength solvent quality
J
potential at a slipping plane, which is often at an unknown distance from the particle surface. At small interparticle separation (52 nm), solvation forces come into play. For an aqueous continuous phase, this leads to a repulsive energy, whose magnitude is roughly known.)' When the surfactant is a small-molecule amphiphile, multilayers may be formed; the resulting strong repulsion3 is also due to solvation forces. When low concentrations of non-adsorbing macromolecules are present, a phenomenon known as depletion flocculation may O C C U ~ ~ (and ~ * ~ *at high concentrations possibly depletion stabilization). There is also the problem that particles may separate again after flocculating. This phenomenon of deflocculation has hardly been studied, and, to the author's knowledge, not even its frequency factor can be predicted. We must conclude that prediction of stability with respect to flocculation is usually impossible, certainly if droplets are covered with protein. The protein layer is involved in all the different types of interaction energy, and, moreover, its thickness and composition are probably not known. Nevertheless, we can often infer some trends, and hence predict what kinds of changes in conditions might be required to enhance o r diminish flocculation stability. Table 3 summarizes the primary factors involved. Some fairly general conclusions are as follows.
( I ) A high surface excess normally enhances stability. This implies a high molecular weight and often a relatively high bulk concentration of the macromolecular adsorbate. ( 2 ) Because of the possibility of flocculation in the 'secondary minimum' (see Figure 3), large droplets are usually less stable than small ones. (3) Electrostatic repulsion is diminished by increasing the ionic strength or by bringing the pH near to the isoelectric point of the adsorbate. (4) Solvent quality strongly affects the steric repulsion. Adding much salt to an aqueous continuous phase usually diminishes the repulsion.
25 I
The above rules do, however, have exceptions. For instance, when a limited amount of surfactant material(e.g., protein) is present, a greater intensity of emulsification may cause the surface layer to become thinner as smaller droplets are formed, thereby increasing the tendency to flocculate. Figure 4 illustrates how flocculation may be induced by bridging. During emulsification in a high-pressure homogenizer, bridging by solid particles may particularly occur if the surfactant consists of proteinaceous particles, as in milk, and if the concentration of these particles is insufficient to cover fully the oil-water In this case, there may be a gradual transition to bridging by interface adsorbed macromolecules. The extent of bridging by macromolecules depends in practice on the way in which the emulsion is made, and on the concentration of macromolecules. In theory, adsorbing homopolymers will always give bridging flocculation between a pair of particles that are close together if equilibrium between adsorbed and bulk polymer is attained.I9 However, this does imply polymer desorption, and, as mentioned already, it is extremely unlikely that this could have time to occur during a Brownian encounter; whether it could happen slowly while particles remain close together in a cream layer is not known. Indeed, it is sometimes observed that a cream layer becomes coherent, but this may be due to a variety of other phenomena, such as the formation of covalent bonds (e.g.. -S-Sbridges) between emulsion droplets (see Figure 4), o r by partial coalescence (see below). In connection with flocculation, a final and important question is whether it leads to the formation of a coagulum or gel. If a gel is formed from emulsion droplets, and it does not show syneresis, the resulting system will probably be very stable. This is because sedimentation (creaming) cannot occur if the gel yield stress is sufficiently high (for instance,X. I Pa). And, if the yield stress is also sufficiently low (SlO Pa) for the liquid to be easily poured, so that the gel is formed reversibly, we may have an almost ideal colloidal system as far as its physical properties are concerned. It is,
Figure 4
Schemaiic representation of bridging ( A ) by solid particles, (B) by adsorbed macromolecules, and (C) through cross-linking of protruding chains
P. Walsrra
252
however, quite uncertain what conditions are needed to attain such a situation. Colloid science has almost exclusively considered either pair interactions o r very concentrated systems; it is particularly the intermediate domain of concentration that now needs to be studied. Numerical simulation of the flocculation process” may offer some help here. Coalescence.-This process is initiated by the formation of a small hole in the thin film between a pair of bubbles or droplets in close proximity (see Figure 5A). The Laplace pressure then causes the pair to flow quickly together. The probability of forming such a hole must clearly increase with the time for which the thin film has existed. Mostly, the probability is low, implying a slow process, and so the coalescence instability is characterized in terms of a rate, e.g., the rate of increase in average droplet size with time. A low probability implies that coalescence is unlikely to occur during a fleeting encounter of two droplets o r bubbles, as induced by Brownian motion or a velocity gradient. Thus, for coalescence to be possible, the particles should remain close together for a long time, in a flocculated state, or in a cream layer, or in a polyhedral foam.
! Figure 5
B
A
L
>(
Y
I
?A-
Illustration ofpossible mechanismsforfilm rupture: (A)formation of of thickness h and interfacial tension y; (B) development of symmetric waves of wavelength h on thefilm surfaces a small hole in a f i l m
While, during the formation of emulsions or foams, and during the stretching of films, Gibbs elasticity and the Gibbs-Marangoni effect are paramount, and this is at least semi-quantitatively understood,’+3Sthere is no generally accepted theory for film stability under quiescent conditions. For two particles a distance h apart, the formation of a small hole in the film as shown in Figure 5A temporarily increases the interfacial free energy by an amount roughly equal to yh2; relating this to kT would give the probability for the event occurring, and this is clearly higher for a thinner film. Repulsion between droplets or bubbles will nearly always be sufficient to prevent the very close proximity needed for film rupture. But a thin film may be subject to wave-like deformations as depicted in Figure 5B. Such a wave will normally be damped, but, depending on the relationship between the colloidal interaction energy and the film thickness, its amplitude may become enhanced, more so the longer the wavelength. In this way, the film may become locally thin enough for rupture to occur. The theory for this has been worked out by Vrij36and is illustrated in Figure 3. It turns out that the film is unstable if (d2G,,/dh2)
+ 27?y/a2 <0
(4)
253
where Gin, is the colloidal interaction energy between two spherical particles at surface-to-surface separation h, and a is the diameter of the film. A necessary condition for the theory to hold is that no lateral motion of the film must occur; it has been show# that a very small dilational surface modulus is sufficient to ensure that this condition is fulfilled. The theory indicates that film rupture and coalescence are promoted by a weak repulsion between droplets or bubbles, a low interfacial tension, and a large film diameter (i.e., large particles). The predicted dependence on y is often considered to be unlikely because a lower y means less decrease in free energy on coalescence (a lower driving force), but we should note that it is not the ultimate free energy gain but the activation free energy that governs the rate. Overall, most observations on emulsion and foam stability d o not disagree with the theory. Foams are generally far less stable than emulsions, and this fits since, although y is higher, the much larger value of a for the case of foams is overriding. Several workers have tried to correlate emulsion coalescence stability, either with dynamic surface properties, or with the maximum surface pressure before the collapse of the surface layer occurs, as determined in separate experiments at macroscopic interface^.^^ Although fairly positive correlations mainly occur, there are always exceptions. It is the author’s opinion that most of the tests somehow measure the thickness of the surface layer, and this is usually correlated with the magnitude of the interdroplet repulsion. Adding small-molecule surfactants to protein-stabilized emulsions tends to lower the aforementioned surface parameters as well as the coalescence stability; obviously, protein is displaced from the droplet surface, thereby greatly diminishing the steric repulsion. It would be worthwhile to test the Vrij theory further, possibly by taking into account also the hydrodynamic aspects of the local film thinning. The theory will clearly break down for very thick surface layers, or for those consisting of small particles (Pickering stabilization). It may be concluded from the above that it is not difficult to make stable oil-in-water emulsions with the aid of sufficient protein, provided the average droplet size is low (55 pm). Proteins differ somewhat in the stability that they provide - caseinates are often the best - but they all adsorb at air-water and oil-water interfaces, give not too low an interfacial tension, and induce steric and electrostatic repulsion. Problems only arise if the protein layer is too thin (surface excess 52 mg m-*), if small-molecule surfactants are present, if the pH is near the isoelectric point, if the emulsion is heated to such an extent that globular proteins denature and become insoluble, or if droplets contain crystals (see below). It is difficult to ensure stability of emulsions during drying or freezing, although little systematic research has been done in this area. Stabilizing edible water-in-oil emulsions is also difficult; it is usually done by a combination of Pickering stabilization and the immobilization of droplets in a network of flocculated crystaka Even excepting Ostwald ripening, it is much more difficult to make a stable foam. This is presumably because most foam bubbles are large, implying infer aliu that far compared with an emulsion) fewer films have to rupture (a factor of, say, before an appreciable change occurs. In addition, other mechanisms for film rupture come into play with foams. When the continuous phase contains solid hydrophobic particles that are bigger than the film thickness between bubbles, the
254
P. Walsrra
particles may penetrate the air-water interface causing the film to break. When emulsion droplets are present, they may, depending on the spreading pressure, spread some oil over the air-water interface, thereby causing local streaming (the Marangoni effect), film thinning, and possibly rupture.20*2' For this to happen, however, the film between the emulsion droplet and the air-water interface has first to rupture, and now we are back to an emulsion stability problem. The latter rupture presumably occurs more readily if the emulsion droplet is large and, especially, if it contains crystals (see below). Partial Coalescence.-This phenomenon can occur in emulsions if the droplets contain solid particle in such quantity or arrangement that the droplets, rather than flowing together into a spherical particle after rupturing the film between them, instead form a non-spherical lump or granule. Partial coalescence can therefore occur in oil-in-water emulsions if part of the fat is crystallized, and this will depend on the oil composition, the temperature, and, because nucleation is needed for crystallization, the droplet size and the temperature history.39 Aggregation involving partial coalsecence is different from normal coalescence in several ways.
( I ) Owing to the non-spherical shape of the aggregates, the viscosity of the emulsion increases. (2) Aggregation may proceed until a continuous network has been formed throughout the volume, thereby imparting solid-like bulk properties and immobilizing any other particles present (such as air cells in whipped cream). (3) The aggregation rate increases greatly with the degree of agitation" (strainrate), and often no aggregation occurs at all under quiescent conditions (see Figure 6). With fully liquid droplets that exhibit coalescence, the rate is usually independent of a g i t a t i ~ n . ~ ' (4) The mere existence of crystals in droplets may enhance the rate of coalescence very significantly (e.g., by a factor of lo6),particularly during agitation or in a cream layer." As indicated in Figure 6, if droplets behave as if they are fully solid, there can be no (partial) coalescence. This may be compared with the improvement in stability obtained with water-in-oil emulsions when the water droplets are allowed to gel through the addition of a few percent of gelatin$2 We have observed two mechanisms of partial coalescence. In one case," the phenomenon occurs only when at least some of the crystals are oriented at the droplet surface, as judged by polarized light microscopy. Presumably, initial rupture of the film between the globules is due to a crystal partly protruding from the globule surface and so piercing the film. This effect will be enhanced if the droplets rotate around each other, as in a velocity gradient. When the contact angle between crystal, oil and water is such that the crystal can protrude further into the aqueous phase, the rate of partial coalescence is higher.43The rate can be measured from the steady and (for some time) linear increase in average droplet size with time.a The other case of partial c o a l e ~ c e n c concerns e~~ some emulsions stabilized with protein. Here, the crystals are not oriented at the boundary of the original droplets, but they are so at the boundary of the clumps formed by partial coalescence. In this case, the average droplet size, and even the relative frequency distribution of the droplet sizes, remain unchanged for some time, while the oil
255
content of the emulsion decreases linearly with time. What happens is that any partial coalescence occurs almost instantaneously, leading to the formation of such large clumps that they cream directly out of the emulsion. While the presence of a velocity gradient and crystals in the droplets is clearly essential, the mechanism leading to the initial film rupture is still somewhat uncertain. The phenomenon of partial coalescence is important and requires further study, if only because it is often the only way in which emulsions are really unstable -mere creaming can be easily undone. Partial coalescence depends strongly on droplet size40*"(see Figure 6),and it is enhanced if small-molecule surfactants are added to a protein-stabilized emulsion. Conclusions It is evident that we can hardly ever predict ab initio the instability of emulsions or foams, except creaming in certain cases. Coalescence especially poses problems, insofar as it may even be difficult to predict what will happen in the long run for an emulsion or foam that has already been made and characterized (for example, with respect to particle size). Quick tests for coalescence stability are mostly unreliable. In many cases, we are able to anticipate general trends of instability before they happen. (We can, of course, explain trends afterwards, or at least we think we can.) Perhaps more important, however, is that we realize what is happening as an emulsion or foam alters with time - and the use of a microscope can be very informative in this connection; we are then in a position to sort out what variables may be significant, and to do some order-of-magnitude calculations. This approach
256
f . Wolsrro
can save considerable time in finding ways to improve stability. In other words, the use of the 'trial and error' method is still inevitable in developing these food products, but theory has certainly progressed far enough to allow for a significant reduction in the number of trials that may lead to error.
References 1 P. Walstra. in 'Encyclopedia of Emulsion Technology,' ed. P. Becher, Marcel Dekker,
New York, 1983,Vol. I. Chap. 2. 2 E.H. Lucassen-Reynders, in 'Physical Chemistry of Surfactant Action,' ed. E.H. Lucassen-Reynders, Marcel Dekker, New York, 1979,p. 1. 3 N. Krog and J.B. Laurisden, in 'Food Emulsions,' ed. S. Friberg, Marcel Dekker, New York, 1976,p. 67. 4 D.E. Graham, S. Levy, and M.C. Phillips, in 'Theory and Practice of Emulsion Technology,', ed. A.L. Smith, Academic Press, London, 1976,p. 57. 5 P. Walstra, in 'Proceedings of Sixth International Congress of Food Science and Technology,' Boole, Dublin, 1984,Vol. 5, p. 323. 6 P. Walstra and H. Oortwijn, Nerh. Milk Doiry J., 1982.36. 103. 7 H. Oortwijn and P. Walstra, Nerh. Milk Doiry J., 1979.33. 134. 8 D.C. Clark. J. Mingins, F.E. Sloan. L.J. Smith, and D.R. Wilson, thisvolume, p. 110. 9 M.A. Cohen Stuart, J.M.H.M. Scheutjens, and G.J. Fleer, J. Polym. Sci.. Polym. Phys. Ed., 1980,18,559. 10 F. MacRitchie, J. Colloid Inrevace Sci.. 1985.105,119. 1 I T.M. Herrington and S.S. Sahi, this volume, p. 188. 12 J.A. de Feijter and J. Benjamins, J. Colloid Inrefloce Sci., 1982,90,289. 13 J.A. dc Feijter and J. Benjamins, this volume, p. 72. 14 E. Dickinson. A. Murray, B.S. Murray, and G. Stainsby, this volume, p. 86. IS K.N. Pearce and J.E. Kinsella, J. Agric. Food Chem., 1978.26.716. 16 P.R. Mussellwhite. J. Colloid Infet$oceSci., 1966.21.99. 17 E.K. Murray, this volume, p. 170. 18 L.R. Fisher, E.E. Mitchell, and N.S. Parker, this volume, p. 230. 19 G.J. Fleer and J.M.H.M. Scheutjens, 1. Colloid Inrevoce Sci., 1986,111,504. 20 H. Mulder and P. Walstra.'The Milk Fat Globule,'Pudoc, Wageningen, 1974. 21 A. Prins, this volume, p. 30. 22 M. Blank and P.R. Mussellwhite, J. Colloid Interface Sci., 1968,27,188. 23 N. Krog, N.M. Barfod, and W. Buchheim, this volume, p. 144. 24 P. Walstra and H. Oortwijn, Nerh. Milk Doiry J., 1975,29,263. 25 E. Dickinson and G. Stainsby, 'Colloids in Food,' Applied Science, London, 1982. 26 T. van Vliet. A.E.A. de Groot-Mostert, and A. Prins, J. fhys. E, 1981.14.745. 27 D.J. Hibberd,A.M. H0we.A.R. Mackie,P.W. Purdy,and M.M. Robins.thisvo1ume.p. 219. 28 L.A. Spielman, in 'The Scientific Basis of Flocculation,' ed. K.J. Ives, Sijthoff and Noordhof. Alphen aan den Rijn, 1978,p. 63. 29 T.G.M. van de Ven and S.G. Mason, J. Colloid Inlerfoce Sci., 1976,57,505,517. 30 D.H. Napper, in'Colloidal Dispersions.'ed. J.W. Goodwin, Royal Society of Chemistry, London, 1982,p. 99. 31 J.N. lsraelachvili, Chem. Scripto, 1985.25.7. 32 G.J. Fleer, J.H.M.H. Scheutjens, and B.Vincent, in'Polymer Adsorption and Dispersion Stability,'ed. E.D. Goddard and B. Vincent, ACS Symp. Series, 1984,Vol. 240,p. 245. 33 L.V. Ogden, P. Walstra, and H.A. Morris, J. Dairy Sci., 1976.59. 1727. 34 P. Meakin, J. Colloid Interface Sci., 1984,102,491,505. 35 J. Lucassen, in 'Physical Chemistry of Surfactant Action,' ed. E.H. Lucassen-Reynders, Marcel Dekker, New York, 1979,p. 217. 36 A. Vrij, Foraday Discuss. Chem. SOC.,1966,42,23. 37 A. Vrij, F.T. Hesselink, J. Lucassen, and M. van den Tempel, froc. Kon. Ned. Akod. Werensch.. 1970,B73.124.
251 38 P.J. Halling, CRCCrif.Rev. FoodSci. Nurr.. 1981,15, 155. 39 P. Walstrr and E.C.H.van Beresteijn. Nefh. Milk Doiry J.. 1975,29,35. 40 M.A.J.S. van Bockcl and P. Walstra, Colloids SurJ, 1981,3, 109. 41 M.A.J.S. van Bockcl and P. Walstra, Co/loidsSurJ, 1981,3,99. 42 J. Madsen. Res. Discl., 1982,215,69. 43 D.F. Darling, J. Doiry Res.. 1982.49.695. 44 J.P. Melscn and P. Walstra, unpublished work.
Paper by Darling and Birkett Dr. C.J. Brock (Bristol): I refer to the comments on flocculation behaviour during emulsification under conditions of very high local energy input. A protein biochemist might interpret the observation of flocculation as arising from complete denaturation of the protein molecules to form an amorphous precipitate. Addition of surfactants would solubilize the denatured and precipitated protein, hence reversing the flocculation. Do you accept this interpretation as a possible explanation of the phenomenon? Dr. D. F. Darling (Bedford): 1 accept the principle, but I d o not think it applies to food systems. In the first place, many food proteins are in such adisordered state to start with that denaturation in the conventional sense cannot occur. Where structured proteins are used (e.g., egg protein o r whey protein), there is no experimental evidence to suggest that a high shear field causes any significant irreversible denaturation. In any case, flocculation is not a property of proteins alone. Bridging flocculation may be initiated by large, random-coil, surface-active polysaccharides such as methyl cellulose. Professor A . Prim( Wageningen): Could Dr. Darling indicate how the composition of the interface, which plays such an important role in the behaviour of foams and emulsions, can be measured? Dr. D. F. Darling: The gross composition of the interface in emulsions is most easily determined by difference. That is, we calculate the interfacial concentrations from an analysis of concentrations of components in the bulk phases and a knowledge of the total amount of each component present in the whole system. More direct methods are possible, involving radio-labelling, fluorescence probes, o r even X-ray microprobe analysis, but they are usually applicable only to simple model systems. Dr. L. R. Fisher (North Ryde): If it is correct to assume that thin-film rupture is initiated at irregularities such as triglyceridecrystals protruding from the interface, there should be some dramatic effects on emulsion stability over temperature intervals corresponding to the melting ranges of triglyceride crystals. Can Dr. Darling cite any evidence to support this? Dr. D. F. Darling: The whipping and churning properties of cream are known to be very sensitive to temperature changes, and when cream is above the melting point of 259
260
Discussions
the fat it will not churn. The presence of even a small amount of crystalline fat causes a catastrophic drop in emulsion stability as shown by van Boekel [Ph.D. Thesis, University of Wageningen, 19801. A practical example of this effect occurs with salad cream o r mayonnaise, which are rapidly destabilized if cooled to a temperature at which the oil begins to crystallize.
Professor E.R. Morris (Silsoe): Could Dr. Darling explain in more detail how intrinsic viscosity measurements are used to determine the hydrodynamic thickness of the interfacial layer? Dr. D. F. Darling: The term'intrinsic viscosity'may be inappropriate here, but what was meant was the determination of the hydrodynamic volume of the particles from the viscosity of dilute suspensions. Knowing the volume of the particles present from gravimetric/density measurements, the hydrodynamic volume yields, by difference, the volume occupied by the interfacial layer. The thickness of the interfacial layer is then readily calculated from particle-size data and the hydrodynamic volume. Paper by Prim
Professor P. Walstra (Wageningen): Your theory on the thinning of a film by spreading from an oil droplet predicts that the penetration depth becomes infinite for zero difference in surface tension. This looks impossible, since a finite difference in surface tension is necessary to provide the energy for the spreading process. Professor A. Prins(Wageningen): Under conditions where the spreadingdistance( remains the same, a lower surface tension difference implies a longer time for the spreading process, which consequently results in a larger penetration depth as follows from the penetration theory [equation (3)]. In the limitingcase for which the surface tension difference goes to zero, the value o f t also goes to zero, making the time from equation ( I ) undetermined. 1 hope to obtain a more quantitative assessment of the amount of liquid squeezed away from the spreading particle by applying boundary-layer theory to the process.
Dr. D. F. Darling (Bedford): The effect of particle size on foam stability could be explained by an alternative mechanism. It is possible that, as the droplet size increases, the extent of adsorption might also increase. Adsorption will also depend on droplet concentration, and so an expression similar to equation (6)could again be obtained demonstrating a minimum in foam stability with particle size, where the term AR-' now represents the film-rupturing dependence of adsorption. and the term BR' represents the concentration dependence of adsorption. Professor A. Prins: The advantage of the theory presented here is that it predicts in a quantitative way that, as far as the effect of droplet size alone is concerned, the penetration of liquid movement in the film is proportional to the droplet radius. I agree that the particle-number effect is an obvious one which could easily by formulated opriori.
26 I
Dr. E. Dickinson (Leeds): In your treatment of film drainage, it is assumed that the film is Newtonian, although in food systems there are usually macromolecular components present which may lead to highly nowNewtonian behaviour. Can this be allowed for the analysis? Professor A. Prins: Non-Newtonian behaviour means that the liquid has viscoelastic properties. A high viscosity can only slow down the drainage of a vertical film, whereas elastic behaviour, expressed in the form of a yield stress, is able to stop drainage completely if the yield stress is high enough. In the present paper, the purpose was to demonstrate that a very low value of the yield stress is sufficient to stop drainage. In principle, it is possible for other types of deviation from Newtonian behaviour of the film liquid to be taken into account, but predicting their effects on film drainage would be a much more complicated matter than predicting the effect of a simple yield value. Dr. F. W. Cuin (Bedford): Do you have any direct experimental evidence to support your film-thinning model? What happens if the two film interfaces touch the droplet? Professor A. Prins: In a so-called falling-film apparatus, hole formation can be seen in films containing emulsion droplets. In stroboscopic light, these holes appear to grow and move downwards with the falling film; but in the absence of emulsion droplets no holes are observed. According to the film-breaking mechanism given here, the film will rupture when its two surfaces are in contact with the spreading droplet. I have been asked why, in the calculation of disproportionation, the surface dilational viscosity is used rather than, say, the surface shear viscosity. The reason is simply that the decrease in surface area of the bubble as it becomes smaller is of a purely dilational nature. Paper by Lucassen
(Presented at the meeting, but not reproduced in this volume) Dr. D. F. Durling(Bedford): Application of the results from single interface studies to real food emulsions is not straightforward because of the different
concentrations and surface areas present. One practical method of working with a realistic system is to use an emulsion as one of the bulk phases in the single-interface experiment. If the single interface is in equilibrium with the surface of thedroplets, then the properties of the interface will be a proper reflection of what is happening at the surface of the droplets. Dr. J. Lucussen (Port Sunlight): Yes, but one must make sure that the interfacial tension measurement is not affected by the close proximity of emulsion droplets. Professor P. Wulstru (Wageningen): In experimental studies of dynamic surface properties, there is usually a much lower surface-to-volume ratio than is found in
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Discussions
real foams, and particularly so for the case of real emulsions. Surfactants in foods are generally mixtures of varying surface-activity, and so the composition of surface layers in foods may differ greatly from the situation studied in the laboratory. What are the consequences of this, and are there ways to eliminate the differences? Dr. J. Lucassen: There appears to be no simple general answer to this important question. The best approach seems to be to measure how the surface properties of bulk liquids are affected by controlled depletion through partial foaming o r emulsification.
Paper by Dickinson, Whyman and Dalgleish Dr. W.P. Edwards(York): What evidence is there for dividing the effects of calcium ions neatly into two classes? Dr. E. Dickinson (Leeds): On the one hand, we have the well-known strong calcium-binding behaviour of as,-casein and &casein but not K-casein, and the general sensitivity of dairy emulsions to destabilization in the presence of calcium salts. On the other hand, we know from electrical double-layer theory that divalent counter ions (e.g., Ca2+) have a much greater effect on the electrokinetic and stability behaviour of charge-stabilized dispersions than d o monovalent counter ions (e.g., Na'). In systems of negatively-charged, casein-coated, colloidal particles, the question arises as to what extent the specific calcium-binding properties of the different caseins can over-ride the general colloidal multivalent counter-ion effect. The results in our paper show that the change in electrophoretic mobility with increasing CaC12concentration for emulsion droplets coated with a,,-o r p-casein is qualirarively the same as that for droplets coated with the calcium-insensitive K-casein, or for latex particles coated with casein, or even for latices with no protein adsorbed at all. Superimposed on the general trend, however, is the specific calcium-binding effect to the protein, which becomes predominant at high CaCI, concentrations, eventually leading to flocculation of a s l -and P-casein-coated droplets, but not of K-casein-coated ones which remain (sterically) stabilized even at high ionic strengths. Of course, any sub-division of a physical phenomenon into discrete conceptual compartments is a simplification that normally can be dispensed with when a more complete theoretical interpretation becomes available. Dr. J . R. MifcheN(Nottingham): Displacement of some /3-casein from the interface by less surface-active asl-casein implies that single casein components adsorb reversibly, i.e., that there is a continuous dynamic exchange between proteins at the interface and protein in the subphase. For random-coil proteins, which are believed, like synthetic polymers, to be adsorbed at a large number of sites (the loop/ tail/ train model), d o you not find this result surprising? Have you any other evidence of reversible adsorption? Dr. E. Dickinson: 1 believe that the ability of one disordered protein to displace
263
another is indicative of a continuous dynamic exchange between molecules at the surface and those in the bulk. It is important to distinguish clearly here between the thermodynamics and the kinetics. In any multicomponent proteincontaining colloidal system, the direction of compositional change at the surface is determined simply by how the actual bulk phase composition at that time compares with the equilibrium value. I t is possible to approach a state of chemical or material equilibrium from two (or more) directions, and so it does not surprise me in principle that a less surface-active protein is able to displace from an interface a quantity, albeit very small, of a more surface-active protein, although it should be said here that the preliminary result reported in the paper is only just outside the estimated experimental error, and therefore needs to be confirmed by more detailed experimentation. Statistical thermodynamics tells us that entropic considerations always win out over energetic factors if the concentration ratio is large enough. Possibly, however, it is not correct to think in terms of a single type of adsorbed entity, as though surface and bulk simply represented a two-state system. There is no such thing as a typical adsorbed molecule, and on statistical grounds there must be at any instant some molecules in loosely-bound all-tail (or nearly all-tail or tail/loop) configurations. So it could be that, in a mixed protein film, train segments are provided preferentially by the more surface-active component, with loops and tails drawn predominantly from the less surface-active protein. Dr. D. F. Durfing(Bedford): If /3-casein displaces a,,-casein from the interface, one might expect that the interfacial composition in an emulsion prepared with sodium caseinate would change with time. Does this happen? Dr. D.G. Dufgfeish (Ayr): We have looked at the composition of free protein in emulsions made from milk fat and sodium caseinate, but see no selective adsorption of &casein over a period of 24 hours. The technique cannot be used to separate caseins!
Dr. D. F. Darling: In response to the comment by Dr. Dalgleish that exchange does not happen with sodium caseinate, one might therefore infer that the structures of the two interfaces are different, with sodium caseinate being aggregated while the pure caseins are in a monomeric state. The protein load of the two systems should therefore be different. Has it been measured? Dr. E. Dickinson: A direct comparison of protein loads is not available for the systems reported in the paper. However, at the same (low) overall protein content, caseinate gives an emulsion with a droplet-size distribution roughly intermediate between those for the pure a,,-and &caseins, which suggests that the protein load is similar in each case, but much less than in homogenized milk or commercial dairy emulsions for which the amount of protein available per unit area is very much higher. In a previous study of casein-stabilized polystyrene latices [J. Chem. Soc.. Furoday Trans. I , 1983, 79, 29371, we found that the saturation coverage for caseinate was close to that for &casein.
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Discussions
Paper by Tornberg and Ediriweera
Dr. C.J. Brock (Bristol): I feel that it is a little bold to say that increasing aggregation of protein leads to decreasing conformational freedom of the polypeptide chain. Bovine myosin, which is highly aggregated, starts to undergo chain separation in sodium chloride solution at temperatures as low as 25 O C . Bovine superoxide dismutase, on the other hand, is a small dimeric protein, but it is very much more thermostable in terms of conformation and enzymic activity. Dr. E. Dickinson (Leeds): Could Dr. Tornberg say more about the experimental conditions involved in freezing the emulsions and subsequently extracting them with n-hexane. In particular, was the freezing and thawing rapid? Dr. E. Tornberg (Klvlinge): Yes, the freezing can most probably be considered as rapid, since 10 ml of emulsion was placed directly into a freezer at -80 O C for storage overnight. It was found to be important to control the experimental conditions of the hexane extraction in order to get reproducible results (*I%). Instead of just shaking the emulsion with solvent, a recirculating system was used, and this helped to minimize the stabilization of hexane droplets by adsorption of protein from the bulk phase. It was essential during the extraction procedure to keep constant the size distribution of the hexane droplets, which was affected by such factors as the nature of the glass filter and the stirrer in the extraction chamber, and the height of hexane in the funnel connected to the bottom of the extraction chamber. Dr. J.R. Mitchell (Nottingham): It is interesting that caseinate, which gives a surface film of extremely low viscosity and viscoelasticity [see paper by Dickinson et a/., p. 86 of this volume], also gives emulsions that have the greatest stability against coalescence. This supports the view that the surface rheology of the adsorbed film is not important in determining emulsion stability with respect to coalescence. Dr. E. Tornberg: Certainly, one of the most unexpected results coming out of our investigation is that the emulsions with the highest protein load, and for which one therefore expects a protein membrane of high viscoelasticity, are not the most stable. Paper by Robson and Dalgleish
Dr. L.K. Fisher (North Ryde): What is the ionic strength of homogenized milk?Is it such that you would expect the Debye length to be long enough for electrostatic forces to come into play? You have commented that the calculated protein loads in some of your experiments have impossibly high values. This might be due to the adoption of a rigid sphere model for the particles. Could there not be, for example, a thicker, but porous, adsorbed layer of casein micelles, which would lead to a 'hairy' particle of
265
hydrodynamic radiusequal to that of asmaller rigid sphere, even though the former has less adsorbed protein? Dr. D.G. Dalgleish (Ayr): The ionic strength of milk is cu. 0.08 mol dm 3. This means that the Debye length is considerably shorter than the length of the macropeptide ‘hairs’ which sterically stabilize the casein micelles and, putatively, the homogenized fat globules. Protein loads were determined from measurements of protein concentrations in the samples and average diameters of the fat cores. The surface area is calculated from the latter, and the protein load is defined as if all the protein were bound. The protein load in the sinking pellet is a hypothetical quantity, since the fraction contains casein micelles as free entities. Oortwijn and Walstra[Nerh. Milk Dairy J., 1979, 33, 1341 have demonstrated that, when micellar hydration is allowed for, a layer of closely packed casein micelles on a fat surface can lead to a maximum protein load of cu. 40 mg m-*, and I have repeated the calculation coming to substantially the same result. Casein micelles d o not drain significantly apart from their outer macropeptide layer, and I doubt whether the casein micelles themselves could be regarded as ‘hairs’ on homogenized fat globules. Paper by de Feijter and Benjamins Dr. J. Lucussen (Port Sunlight): The non-ideality of protein and polypeptide monolayers leads to characteristic surface dilational modulus versus surface pressure behaviour. For small values of the surface pressure n, we have found the relationship c
= d n / d I n r = kn
where c is the surface dilational modulus, r is the surface concentration, and k is a constant (=8). On integration, this purely empirical equation gives
where a is a constant, and r, is the surface concentration as n-m: This analysis leads qualitatively to the same conclusion as reached in the paper of de Feijter and Benjamins. That is, changes in surface pressure are quite small up to high degrees of surface coverage, and induction periods in plots of surface tension versus time d o not represent the changes occurring in the surface concentration. Dr. E. Dickinson (Leeds): Your paper is a valuable contribution to the subject of protein adsorption kinetics, since it clears up a number of points in the literature which are both confusing and contradictory. 1 am still not clear, however, why the surface pressure remains so very low except at high coverage. Are you saying that there is only an experimentally measurable surface pressure when there are sufficient protein molecules adsorbed for there to be significant protein-protein interactions in the surface film?
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Dr. J.A. de Feijfer(V1aardingen): Exactly that. When interactions are neglected, it follows from the ideal equation of state (IIA=kT) that the surface pressure will remain low up to high surface concentrations (e.g., II = 0. I mN m-I for ovalbumin at r = 2.0 mg m-z, where the surface is fully occupied). The low value of II arises because of the high molecular weight of proteins. As shown previously by us [J. Colloid Inferfuce Sci., I982,90,289], the experimental II-r curves of our adsorbed proteins can be satisfactorily explained by taking into account the finite size of the protein molecules in the surface layer. From the non-ideal surface equation of state used, it follows that surface pressures above 1 mN m-I are to be expected only at high surface coverages - say, above 80% of monolayer coverage for a globular protein like ovalbumin. Dr. J. Mingins (Norwich): At the risk of pre-empting my own talk, may 1 suggest that a possible alternative explanation of the induction period would be to postulate the presence of a surface phase transition. Do you know of any examination of the low surface pressure regime at the level of p N m-I sensitivity? Dr. J.A. de Feijfer: I am not aware of any such studies, at least for proteins adsorbed from solution. Various studies have been made, however, with spread proteins in the low surface pressure regime (10-2-1 mN m I), and these give no evidence for a phase transition in the surface layer. For instance, with various globular proteins, Bull [J. Biol. Chem., 1950, 185, 271 found that the correct molecular weight is obtained by extrapolating to II = 0 using the simple non-ideal surface equation of state, II(A - A,,) = kT. This indicates that, for II < 1 mN m-I, spread protein films behave like a non-ideal gas rather than like a condensed phase. Dr. J.R. Mitchell (Nottingham): Your interesting paper shows clearly that the diffusion mechanism is far more important in determining adsorption kinetics than some workers have believed previously. I find it surprising that data from measurements on globular proteins at different concentrations superimpose so nicely on the same 11-A (II-r)curve, and that this relationship gives limiting areas not too different from those obtained with spread protein films. The Graham and Phillips work appears to show that isotherms for spread and adsorbed films are quite different. A study of lysozyme adsorption at the air-water interface by Adams ef 01. [J. Polym. Sci., Purr C, 1971,34, 1671suggests that the II-A isotherm for the adsorbed film depends on the protein concentration. This work used radio-labelled proteins to determine the surface concentration. Since the reconformation of the adsorbed molecules is fast and similarities are found between spread and adsorbed films, it must be supposed that the protein quickly approaches its lowest free energy conformation at the interface. How does Dr. de Feijter reconcile this with the well-established observation [Mitchell ef ul., Biochim. Biophys. Acfu, 1970, 200, I381 that heat-denatured o r chemicallydenatured globular proteins lower the surface tension more rapidly than native proteins, and therefore must have different adsorbed-film II-A isotherms from the native proteins? If there is one equilibrium state which is rapidly approached, then the denatured protein should reach it as well.
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Dr. J.A. de Feijter: Graham and Phillips [J. Colloid Interface Sci.,1979,10,427] found that the Il-r data of adsorbed &casein films obtained at different bulk concentrations were also superimposable. On the other hand, the n-A curves of spread and adsorbed films did not always coincide [Phillips el a!., ‘Proceeding of the 4th International Conference on Surface Active Agents, Zurich, 1972, p. 38 I]. This might have been due to partial loss of protein from the surface during spreading. Also, chemical modification is known to affect the 11-A curves of spread proteins. Adams et ul. [J. Polym. Sci., Purr C,1971,34, 1671found that the rate of change of the surface pressure of lysozyme increases strongly with increasing degree of acetylation. This is in agreement with their finding that acetylation causes expansion of the 11-A isotherm, but it does not necessarily mean that the adsorption rate is also influenced. We d o not think that protein molecules will always attain the same conformation after adsorption, independent of history (including denaturation by heat or chemical modification). Different treatments may have different effects on the protein conformation in the starting solution, and conformational changes may persist after adsorption. The term ‘denaturation’should be used with care, as it does not always refer necessarily to the same irreversible change in protein conformation. Most likely, there are several different conformations, for which the free energy is at a minimum with respect to small changes. The various minima are separated by activation barriers, and which minimum is attained in any one experiment will depend on history. Mr. B.S. Murruy (Leeds): I t is not clear to me whether the bulk concentrations quoted were maintained at their constant values throughout the experiments, or whether they are in fact initial bulk concentrations. If the latter is the case, then, depending on the surface-to-volume ratio in your apparatus, could there have been any appreciable depletion of protein from the bulk phase, especially at the lower bulk concentrations? If so, this would surely affect the plots of n and r against time. Dr. J.A. de Feijier: The depth of the solution in our experiments was 2 cm. At the wt%). approximately 10% of the protein is lowest protein concentration used ( adsorbed after complete equilibrium. In presenting the experimental results, we have always given the inifiufbulk concentrations and not the final ones. Deviations from the theoretical curves due to depletion are to be expected only after extremely long times, at which the penetration depth of the concentration gradient exceeds the actual depth of the solution, i.e., (7~Dt)I’~>0.02m, where t is the time and D is the m2 s I for t 2 lo6 s diffusion coefficient. This condition is satisfied with D = (2300 hours), whereas most experiments were stopped after cu. 20 hours. Paper by Diekinson, Murray, Murray and Stainsby
Dr. E. L. Neustadrer (Sunbury-on-Thames): Regarding the relevance of interfacial shear viscosity to emulsion stability, I should like to make the followingcornments. With crude oil systems, we generally find a good correlation between interfacial shear viscosity and the stability of water-in-oil emulsions. We believe that a high interfacial shear viscosity greatly reduces the rate of oil film drainage between water
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droplets. In addition, we have carried out some experiments in which the oil-water interface has been compressed while the shear viscosity was measured. We find that a low level of Compression (-10%) can increase the interfacial shear viscosity by several orders of magnitude. Area changes of this order are readily realized in droplet-droplet coalescence. Could Dr. Stainsby explain how reproducible alignment of the disc at the interface was ensured? We d o not find this a problem with our B.P. rheometer, as the interface locates at the knife-edge even if the alignment is not perfect. Dr. G. Stoinsby (Leeds): Our bob is also a knife-edged biconical disc. Aqueous solution is introduced into the dish until the surface level is aligned with theedge of the bob, and then the oil layer is added without delay. In our experience, the raising o r lowering of the bob by up to 1 mm produces no change in viscous drag, and so its exact position is not critical. Dr. L. R. Fisher (North Ryde): Can your model of casein adsorption account for the recovery of viscoelastic behaviour in the mixed protein system? Dr. G. Sfoinsby:Picture 2 of Figure 8 in our paper refers to the situation some time after the addition of the caseinate, when the viscoelastic recovery is largely complete. What we think happens is that the small casein molecules penetrate the existing gelatin layer, and, being more hydrophobic, displace it from the interface. The displaced gelatin remains in the neighbourhood of the interfacial region in the form of aggregates rather than individual molecules. In time, through interaction with adsorbed casein, and through the reformation of some gelatin-gelatin interactions, the viscoelasticity of the gelatin-containing composite interfacial film develops. Professor E.R. Morris (Silsoe): Is there any experimental evidence for the entangled, disordered-chain conformation shown in the picture of the adsorbed gelatin film (Figure 8)? Perhaps a gradual adoption of the triple-helix structure found in bulk gelatin gels is more likely, and might explain better the timedependent changes that you have described. Dr. G. Stoinsby: We are not sure whether the time-dependent changes in apparent surface shear viscosity are due entirely to reorganization of gelatin intermolecular interactions and associations, or whether there is some contribution from additional gelatin adsorption onto the interfacial layer. Since the gelatin in the composite layer lies entirely within the aqueous phase, it seems highly likely that there is some adoption of the organized intermolecular association which in a gel would be called a collagen-like junction-zone.
Paper by Anderson, Brooker and Needs Dr. D.G. Dalgleish (Ayr): Could Dr. Anderson explain how air-water ghosts survive if they are composed of soluble, undenatured Bcasein?
269 Dr. M. Anderson (Reading): The &casein is just one of the components at the air-serum interface. It is possible that interactions amongst components at the interface prevents their dispersion when an air bubble collapses to form a ghost. Evidence for interactions involving 8-casein can be found in Dr. Murray's paper[p. I70 of this volume]. Dr. L.R. Fisher (North Ryde): Dr. Anderson and several other speakers have emphasized the need to determine which proteins are present at interfaces and in what quantities. I t is clearly possible to tackle these problems using modern monoclonal or polyclonal antibody techniques, with antibodies labelled with gold, ferritin or a fluorophore. Is anyone planning to use such an approach? Dr. M.Anderson: We d o intend to use such techniques. Initially, we shall make use of polyclonal antibodies to individual milk proteins. Dr. D.F. Darling (Bedford): Previous studies using thin-sectioning and staining techniques to observe the fat-air interface in whipped cream are subject to artefacts. If the temperature during fixation is not kept below about 10 "C, the fat droplets adsorbed at the interface can continue to spread. Careful control of temperature is essential, and its absence is probably the reason why previous studies have shown significant spreading of fat around the air cells. My second comment relates to the over-whipping of cream. The extent of coalescence and spreading of fat globules is highly dependent on the whipping time; slight differences can cause dramatic changes in the structure of the cream.
Paper by Clark, Mingins, Sloan, Smith and Wilson Dr. L.R. Fisher (North Ryde): Dr. Clark has stated that some of the protein molecules in the foam lamellae may never see the air-water interface. For a lamellar m2 s-I, the mean lifetime of, say, 100 seconds and a diffusion coefficient of cu. distance travelled by a diffusing protein molecule is of the order of lo-' m. Most lamellae are thinner than this, making it likely that most protein molecules in the lamellae would have diffused to the air-water interface. The Plateau borders, though, may well be thick enough for some of the protein molecules in those regions not to reach the interface over the same time-scale. My question is this: in your foams, what is the ratio of the total volume of liquid in the Plateau borders to that in the lamellae? The question is also relevant to the interpretation of the conductance measurements, since most of the currents could pass along the Plateau borders rather than through the lamellae. Drs. D.C. Clark and J. Mingins (Norwich): Two seemingly unequivocal points emerge from our studies: (i) there is some protein in the collected foam which is different in some way from the original protein in the bulk of the foaming solution, and (ii) the air-water interface of the foam is responsible for the difference. What we cannot say at present is whether all (or most) of the protein molecules have suffered a small change, or whether a small fraction has undergone a large change. We wish to point out that it is feasible that some of the protein in the foam had not seen the
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interface during the residence time in the column. This could arise because of insufficient time for diffusion to take all the protein molecules to the interface. Alternatively, there may be no ready exchange at the interface, so that molecules already in residence are reluctant to leave and thereby inhibit the approach and unfolding of others. Your comment about diffusion in the lamellae is perfectly valid, but it does necessarily imply that all the molecules can indeed reach the surface. A tentative calculation, based on the mean bubble size, the weight of the foam in the column, a mean film thickness throughout the column of 250 nm (from the interface colours), and a close-packed system of polyhedra with cell coordination number 6,indicates that >99% of the solution resides in the Plateau borders. As you say, these are the main conducting channels. They are also the main channels for drainage of the protein solution, and so drainage from Plateau borders will be the main contribution to the decay in conductivity. Nevertheless, the instabilities of thinning lamellae lead to bubble collapse, and this is linked to the conductivity measurements through the attendant rearrangement of neighbouring bubbles with loss of Plateau borders. We would expect this to make a more significant contribution to one of the relaxation rates than the process of gravitational and capillary-suction drainage from the minor conducting channel of the lamellae. Dr. C.J. Brock (Bristol): Would it not be a better idea to compare foam-denatured BSA with detergent-denatured BSA rather than with urea-denatured BSA, since the former disturbs hydrophobic interactions whereas the latter breaks down polar interactions? Dr. D. C. Clark: Yes, we d o plan to extend our measurements to include the effects of other denaturants (including surfactants), as well as investigating the effects of classical quenchers and temperature on the intrinsic fluorescence of BSA. These additional experiments may help to clarify the nature of the conformational change detected in foamed BSA. Dr. W.P. Edwards (York): Could Dr. Clark explain the mode of action of the conductivity electrodes in his apparatus? Are they wired in series, o r are they sampled sequentially? Dr. D. C. Clark: During data collection, three pairs of electrodes were interrogated sequentially with switching controlled by a series of relays. A 5-volt AC signal supplied by a signal generator was used for the conductivity measurements. Polarization effects were found to be a problem when D C current was used.
Paper by Bee, Clement and Prim Professor P. Richmond (Norwich): In your excellent talk you stated that the viscosity of adispersion containing both air bubbles and solid particlescould not be predicted on the basis of simply adding together the volume fractions of air and solid particles, despite the evidence that a dispersion of air bubbles alone behaves like a hard-sphere system. Intuitively, I would also have expected this to be the case,
27 I
but I would have thought that there might be a regime in which additivity holds in an asymptotic sense (low shear-rate, high surface tension, low volume fraction, etc.). Could Dr. Bee say whether this has been considered. Dr. R. D. Bee (Bedford): Our investigation of the viscosity of a dispersion of air bubbles 4-solid particles was limited to the single set of data reported in the paper. I agree that intuitively one might expect the two phases to be additive with respect t o rheological behaviour. Presumably, the deviation from additivity is the result of significant distortions of the gas cells by the ballotini when the two are sheared together. For a bubble of radius 100 p m and surface tension 30 mN m-I, the shear stress required to deform it is given by the Laplace pressure: 2 y / r = 600 Pa. In fact, the bubbles in our experiments are smaller than this (see Figure 5). which means that the applied shear stresses are not great enough to deform the bubbles on this basis. It seems Likely that when solids are present the localshear forces can become sufficient to distort the gas cells. Professor P. Wulstru (Wageningen): Have you used other shear-rates in the
whipping experiments? Dr. R.D. Bee: No, but we d o anticipate that varying the shear-rate will have a significant effect on the aeration profile, as has been demonstrated previously by Prins [in'Foams', ed. R.J. Akers, Academic Press, London, 19761. Dr. W.P. Edwards (York): Has the method described been used to investigate any practical food system? Dr. R. D. Bee: Yes. The model represents the simplest practical aerated foodstuff, and it can be developed in any desired direction. For example, a second solid disperse phase can be added. The ballotini could easily be replaced by ice crystals to represent ice-cream, o r by starch particles to model a batter. Using this approach, a description of a practical aerated foodstuffcan gradually be built up, systematically changing just one variable at a time. Dr. J. R. Mitchell (Nottingham): Do you consider the method you have described for evaluating foaming properties to be useful for predicting the peformance of surfactants in aerated food products? If so, would it rank surfactants for 'foamability'in thesame order as a method based on bubbling, like that described in the paper of Clark el al. [p. 1 10 of this volume]? Dr. R. D. Bee: The model was developed for understanding the aeration properties of surfactants in a context relevant to foods, i.e., low phase volume, appropriate cell size, etc. As the method involves a dynamic aeration approach, it is likely to reflect the surfactant properties that are relevant to behaviour in large-scale dynamic aeration equipment. We have overcome problems associated with sampling during the process and sensitivity to temperature changes, and have removed the influence of pressure. Working in the appropriate shear regime still remains a problem, however.
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In response to your supplementary question, I doubt very much whether our ranking of foamability would be identical to that measured in a bubbling (or shaking) technique. The dynamics of foam structure formation during whipping is quite different from that associated with bubbling (or shaking).
Paper by Krog, Barfod and Buchheim
Professor P. Walstra (Wageningen): According to Skoda and van den Tempel [J. Colloid Sci., 1963, 18,5681 and Walstra and van Beresteyn [Nerh. Milk Dairy J., 1975, 29, 351, supercooling in emusified fats is determined by the number of catalytic impurities per unit volume of oil. This implies that the average droplet volume determines the degree of supercooling. Surfactants, as impurities in the oil, may form inverted micelles capable of acting as catalytic impurities for crystallization, thereby diminishing supercooling. These considerations seem to fit in well with your results. Paper by Hernqvist
Dr. L. R. Fisher (North Ryde): The microemulsion region in the phase diagram appears to be very narrow. This means that the tolerances on concentration, temperature, and so on, must be correspondingly tight. How easy will it be then to produce and maintain microemulsions in commercial practice? Dr. L.Hernqvisr (Lund): We have done some pilot-plant experiments (150 g scale) with positive results. In commercial practice (e.g., margarine production), the L, phase could possibly be mixed with the oil blend before the formation of the margarine emulsion, to be followed by cooling. The effect occurs even if the two-phase region, where L, and oil coexist, is rather narrow. The technical advice therefore is: keep to the left! Paper by Murray
Dr. J . R. Mitchell (Nottingham): The limiting areas you report for a-lactalbumin and K-casein appear to be considerably lower than the values of cu. 1 m2mg-' found for most proteins. Could this be due to loss of protein to the subphase on spreading? Dr. E. K. Murray (Reading): It is possible that protein loss from the interface may be occurring, although the spreading solvent and the method of spreading were the same for these proteins as for 8-casein and 8-lactoglobulin. which had limiting areas of cu. I mz mg in my experiments. In fact, the reason for using a 66% isopropanol solution as spreading solvent was to try to minimize loss to the subphase.
'
Dr. E. Dickinson (Leeds): Do your experiments on binary protein films provide any evidence for a preferential desorption of the less surface-active component?
213
Dr. E.K. Murray: I had hoped at the outset that the rates of film collapse might indicate which protein component was preferentially desorbed. However, it appears that no such conclusions can be drawn from these experiments. Dr. D. G. Dalgleish (Ayr): In the experiments with K-casein, mercaptoethanol was added to ensure breakage of disulphide bonds at the interface. Apparently, however, there were no analogous experiments on P-lactoglobulin films in the presence of mercaptoethanol. So, when measurements are made on films of Plactoglobulin K-casein, the effect of mercaptoethanol on the P-lactoglobulin component of the mixture is an extracomplicating factor. This means that changes in interfacial properties of the mixture cannot properly be compared with data for pure P-lactoglobulin, and so the measured isotherms cannot be used as evidence for, for example, complex formation.
+
Dr. E.K. Murray: No experiments were done on P-lactoglobulin with mercaptoethanol. 1 agree that this system should be studied in order to find out whether the change in isotherm shape in the mixture is due to the presence of mercaptoethanol. Dr. D.C. Clark (Norwich): The proteins in this study were spread from mixtures of isopropanol and water. I should like to make the comment that circular dichroism studies of protein solutions have shown that alcohols can induce changes in secondary structure, in particular causing dramatic increases in a-helix content. Paper by Herrington and Sahi Dr. H. R. Kerr(Chor1eywood): The surface behaviour of BSA is dependent on pH, ionic strength, and so on. Could Dr. Sahi say whether he has made measurements under different solvent conditions? Dr. S.S. Sahi (Reading): The experiments were performed in phosphate buffer (pH 7.3,O. 15 M NaCI) at 20 OC. No other pH conditions were investigated. Paper by Barker and Crimson Dr. E. Dickinson (Leeds): In calculating the average droplet shapes generated by the Monte Carlo procedure, you first reject any generated configurations with 'overlap' on the basis that they are unphysical. Strictly speaking, these configurations should not appear in the ensemble average because their Monte Carlo probabilities are negligibly small (being very high energy states). Are you sure your ad hoc approach gives results that are rigorously correct? Dr. G.C. Barker (Norwich): Clearly, from equation (3), our absolute rejection of overlapping shapes amounts to the introduction of a discontinuous potential into the energy function to represent the effect of curve-crossing. This gives the particles a hard-core resistance to topological change which can be taken as an integral part of the model. (This is comparable to the use of Monte Carlo methods in hard-disc
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simulations by Metropolis et a/. [J. Chem. Phys., 1953, 21, 10871.) It would be difficult, and somewhat arbitrary, to assign any other weights since the corresponding physical behaviour has not been observed. It is relevant to point out that, whilst the fraction of curves which have intersections increases with the radius of the hypersphere from which the sets of coefficients are sampled, the estimated error in mean values decreases much more rapidly. In most cases, when the systematic error in P is reduced to an acceptable level, the proportion of sampled curves having cross-overs is still quite small (
Dr. G.C. Barker: A finite interfacial tension will certainly reduce the extent of droplet deformations. However, this conference has provided evidence (in the form of electron micrographs, erc.) to support the premise that in real food emulsions and foams the dispersed particles can experience a wide variety of irregular shapes. We may estimate orders of magnitude as follows. Let us assume that the mechanical behaviour of avery small spherical drop of constant volume can be modelled simply in terms of a surface tension y , and that deformations are restricted to small, independent, normal displacements each of energy k T / 2. The relative change in surface area is then given by
K = kT/ 2 y A , where A m is the minimum wavelength of surface excitation [Ljunggren and Eriksson, J. Chem. Soc., Furuduy Truns. 2, 1984,80,489]. With typical numerical values of the parameters (A,,, = 3 nm, T = 300 K), it is apparent that surface area fluctuations of cu. 10% will be experienced for y < 3 mN m-I. Values of the interfacial tensions in many food emulsions lie in this range. Moreover, these simple shapes form only a subset of all shapes with K = 0.1 [Brochard and Lennon, J. Physique, 1975,36,1035]. Theoretical examination of the full set of droplet shapes is important. Particularly when small interfacial tensions are considered in conjunction with energy contributions from interface bending and external stresses, the problem cannot be treated by simple perturbation methods. Without making broad statements about direct applicability (our model is twodimensional!), we have simply attempted to demonstrate the impact of shape fluctuations. Finally, we should like to make two further points. (i) Our calculations use a space of particle shapes spanned by surface functions. The number of independent functions increases with surface size (linearly in two dimensions). This representation includes many asymmetric shapes that are not sampled by other methods, but which describe deformations of all sizes of particles. (ii) The term 'Monte Carlo'refers to a quick and efficient way of evaluating the multiple integral in equation (I).The same integral could be formed (at length) by any standard grid technique with individual Boltzmann weights.
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Dr. E. Dickinson (Leeds): On the question of the relevance of Monte Carlo simulations to dispersions of deformable particles, Ishould like to make two points. Firstly, the continuum hydrodynamic theories make no allowance for Brownian fluctuations, which are important in relation to the translational motion of particles smaller than cu. 1 p m and in connection with statistical variations at fluid interfaces. Since thermal fluctuations provide the primary stochastic driving force for droplet or bubble coalescence, it is essential that they be included in any statistical mechanical treatment of emulsion or foam stability. I t should be remembered, however, that the Monte Carlo method only givesequilibrium results; to get kinetic information, we need to use molecular or Brownian dynamics. Secondly, I think we need to put the preliminary and exploratory calculations of Dr. Barker into proper context. They are of little relevance as they stand, but when the methods are extended to the case of concentrated systems of interacting deformable particles they should give valuable insight.
Paper by Hibberd, Howe, Mackie, Purdy and Robins Dr. M. Povey (Leeds): Your statement that ultrasonic velocity is ulwuys independent of emulsion droplet size as long as the ultrasonic wavelength greatly exceeds the droplet size is not necessarily true. While it does apply to your soya bean oil-in-water emulsions, the assumption can break down when the two phases are significantly different in density and an inertial term appears in the equation of motion. The inertial term puts the particle motion out of phase with the motion of the continuum; under these conditions, the viscosity, particle size and the wavelength all affect the ultrasonic velocity. To say that air is a strong attenuator of ultrasound is imprecise. Certainly, air in an emulsion will scatter strongly when the bubble size satisfies the Rayleigh criterion for scattering, but air is not intrinsically a strong absorber of ultrasound. By lowering the frequency, the Rayleigh scattering (and other forms of scattering) can be reduced thereby lowering the attenuation considerably. The presence of air bubbles in an emulsion will also tend to lower the measured velocity of ultrasound. Since the use of a blender tends to incorporate agreat deal of air into an emulsion, I wonder whether the presence of air explains the departure of your experimental results from the theoretical line. Dr. A.M. Howe (Norwich): We are aware of the circumstances under which we cannot assume the ultrasonic velocity to be independent of particle size. We have measured the variation of velocity with droplet size in our emulsions, and have observed it to be negligible. In the results presented, the droplet-size distribution did not change during the experiment. We agree that the presence of a small amount of air could account for the observed effects. Professor E.R. Morris (Silsoe): We have found an almost exactly linear relationship between ultrasonic velocity and fat content in aqueous emulsions. Is it possible that the discrepancy which you have observed between n-hexadecane-inwater emulsions and oil-in-water systems thickened with Xanthan gum is related to
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the large concentration-dependent changes in ultrasonic velocity found with simple Xanthan gum solutions [Pereira el al., Carbohydrate Polym., 1982,2, 103]?
Dr. A.M. Howe: There is a dependence of ultrasonic velocity on the aqueous concentration of Xanthan gum, as shown in Table I of our paper. The value of the velocity in each continuous phase is determined, and the appropriate value is used in calculating the oil volume fraction as described in the text. In Figure I , the displacement of the measured velocity values from those predicted is probably due to the inclusion of a small amount of air in the emulsions.
Paper by Walstra
M r . B.S. Murray (Leeds): What evidence is there that fat crystals at the surface of emulsion oil droplets actually pierce the interfacial protein layer around the droplets, when we know from our own measurements, and those of others, that such films can have strong mechanical properties? The presence of a protein film covering the protruding surface of such fat crystals must strongly influence the ability of the crystals to clump together. Professor P. Walsfra (Wageningen): The evidence for crystals piercing the boundary layer of emulsion droplets is predominantly circumstantial, but elaborate, and, in my opinion, rather strong [van Boekel and Walstra, Colloids S u r - , 1981, 3, 1091. In unpublished work, others have shown by electron microscopy, in slightly different emulsions, that protruding crystals d o occur. For a thin film, the mechanical forces keeping protein molecules together will be mostly small compared to surface forces. With casein gels, for instance, we find a yield stress of 10 Pa at most. Since such age1 is quite inhomogeneous, the value for a condensed protein layer gel will be much higher - let us say 10’ Pa. Assuming a thickness of 10 nm (probably an overestimate), this would imply a two-dimensional N ,-I, while the differences in interfacial tension will yield stress of the order of be at least lo-’ N m-I.
Dr. L. R. Fisher (North Ryde): Can we justify all this work on food emulsions and foams? It is, after all, mainly applicable to processed foods, which represents only a small subset of all foodstuffs. Professor P. Walsrra: While this is really a question to the organizers of the conference, rather than to one of the speakers, I would answer it as follows. 1 agree that no ‘natural’ foods are foams, and few are emulsions - though milk is a not unimportant exception. Nevertheless, many manufactured foods are, or have been during processing, either emulsions (creams, dressings, milk products, butter, margarine) o r foams (bread, omelettes, mousse), or both (whipped cream, icecream, milk-shake, puddings, cake). In addition, foamingcan be a nuisanceduring some processing operations. Also, the organizers may have had in mind, for instance, that nicely spherical emulsion droplets can be regarded as a simple model for other dispersed particles. Indeed, there are few manufactured foods that are not colloidal systems (salad oil, treacle, some drinks). Even beer exhibits foaming, and this is generally considered as desirable - except, possibly, in Britain!
A Model System for Studying Aspects of Protein Functionality in Emulsions By CHRISTOPHER J. BROCK (AFRC Institute of Food Research. Langford, Bristol BS 18 7DY)
Physico-chemical studies of protein functionality in food emulsions are complicated by the fact that the fat particle-size distribution (and hence the interfacial area) is difficult to measure, and varies with many factors, including the nature of the proteins present during emulsification. To avoid this problem, we have developed a model system for studying protein binding at interfaces. Controlled-pore glass granules have been chemically modifed so that they are coated with a film of long-chain fatty acyl groups, analogous chemically to the surface of a fat particle. I t is planned to use this model system to elucidate the structure-function relationships of proteins at interfaces, using a wide range of physico-chemical techniques, including electron microscopy, calorimetry, and, if the refractive index of the continuous phase is suitably adjusted, spectroscopy. Acid-washed, controlled-pore glass granules (BDH,200-400 mesh particle size, 300 nm mean pore diameter, surface area = 10.7 m2 g-I) were silanized with I M octadecyltrichlorosilane in n-heptane in an evacuated desiccator over potassium hydroxide. The silane reacts mono-, di- and tri-functionally with the silanol groups of the glass. After removing excess silane by washing with n-heptane, unreacted chloride groups were blocked with octadecan- 1-01( I g per g of glass) at 60 OC. The glass granules were washed thoroughly with n-heptane at 60 O C , then washed with methanol and dried. In scanning electron micrographs, the appearances of the surfaces of the acid-washed glass granules before and after coating were indistinguishable, even though the final product was extremely hydrophobic. It was therefore concluded that the chemical modification had not changed the surface area of the glass significantly. From the increase in the weight of the glass upon modification, it was calculated that a hydrocarbon layer of 0.9 nm thickness had been added to the surface, equivalent to a monolayer of octadecyl chains about 60% extended from the surface. One application of the model system is in the quantification of protein binding under different conditions. Hydrophobic controlled-pore glass granules held in a 211
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Ahsiracts of Posters
small column were wetted with methanol, which was subsequently displaced by a degassed buffer solution containing0.5 M sodium chloride, 0.01 wt%sodium azide and 0.02 M sodium phosphate (pH 7.0). A known amount of protein [myosin, actin, bovine serum albumin (BSA) or light white casein] dissolved in the same buffer was added to the glass granules, and allowed to bind for 2 hours at room temperature. Unbound protein was removed by washing with buffer until no more protein release could be detected. The released protein was quantified from its absorbance at 280 nm. The glass granules were then washed with more of the same buffer solution, but now also containing I wt% sodium dodecyl sulphate (SDS). Any further release of protein was measured. Actin failed to bind at all to the hydrophobic glass surface: all the added protein was recovered in the buffer wash, and the granules remained clumped in the aqueous buffer as they always were in the absence of any surface-active agent. By way of contrast, BSA bound quantitatively to the glass until the protein load reached 0.7 mg m-*. Above this level, additional protein led to no further binding, and all the bound BSA was released by washing with buffer containing SDS. Myosin bound almost quantitatively to a coverage of cu. I mg m-2. Additional myosin did bind, but less completely. Washing with S D S released half of the bound myosin; this SDS-stable binding had an upper limit of 0.6 mg m-2. Casein, like myosin, bound quantitatively to I mg m-2and less completely above this level. The residual binding behaviour after SDS washing was more complicated, however, showing some concentration dependence, which was probably a reflection of the heterogeneous nature of light white casein as compared with the other three pure proteins. With casein, only 20-40% of bound material remained after S D S washing. We conclude that hydrophobic controlled-pore glass, with its covalently bound film of hydrocarbon, provides a useful substitute for fat particles in physicochemical studies of protein functionality. We have found that the binding of myosin and some components of casein is more stable to detergent than that of BSA. Actin failed to bind under the non-denaturing conditions employed.
279
The Effect of pH on Emulsions and Foams Stabilized by Bovine Blood Plasma Proteins
By S.E. HILL and G.M. HALL (Departmentof Chemical Engineering, University of Technology. Loughborough. Leicestershire LE 1 1 3TU)
Only 0.9% of blood from British abattoirs is used as a food substance for human consumption. To increase its usage, the Meat and Livestock Commission has sponsored an investigation into the processing and functional properties of bovine plasma. Here we report results on emulsion and foam stability. Sodium citrate was used to stop the clotting of fresh blood as it was being collected from local abattoirs. Plasma was separated from erythrocytes by centrifugation, and concentrated by ultrafiltration followed by spraydrying. Inlet and outlet temperatures for spray-drying were 185 O C and 75 OC,respectively. Of the ten powdered samples produced, most were a light buff colour, but afew were of brighter coloration which faded with time; this was found to bedue to the presence of &carotene, whose variable content was a reflection on the cattle's feed. The average powder composition was: 79.5 wt% protein, 5.5 wt% water, 3.3 wt% salt, 6.0 wt% lipid and 4.3 wt% ash. Over 90% of the bovine plasma powder was soluble in distilled water (pH 8.4); solubility was reduced to 76% at pH 5.0. Model tests were generally employed to assess the functionality of protein powders for use in the food industry. There are no standard procedures, and comparison between values obtained by different workers is often very difficult. Even if the same basic method is adopted, the numerical values of functional parameters may depend on the method and duration of mixing, and the concentration of powder and salt. Our standard method of making an emulsion involved mixing 25 g of a 2 wt% solution of bovine plasma powder with 75 g of corn oil. Emulsion viscosity was measured using a viscometer with a helical path. Emulsion stability was determined by heating the emulsion to 80 O C for I hour, and then estimating the amount of oil released after 24 hours o r 14 days. Particle sizes of the fat globules were determined using a Coulter Counter and a Malvern Particle Sizer after diluting the emulsion in 0. I wt% sodium dodecyl sulphate. Turbidity and Emulsion Activity Index were carried out in the manner described by Pearce and Kinsella.' Normally, emulsions formed in the first part of the procedure were very stable, and further oil could be added, with increasing viscosity, up to the break point. Adding oil too quickly or at too high a shear-rate causes the thick viscous emulsion to break, thus affecting the
Abstracts of Posters
280
Table 1
Particle sizes in emulsions at dif-ferentp H values Average Droplet Diameter/ p m Sizing Equipment
pH5
pH8.l
pH9
Coulter counter
29.0
33.6
29.1
Malvern particle sizer
21.2
26.2
21.8
measured capacity. This was at its lowest at pH 5 , corresponding to the lowest protein solubility, although the decrease in emulsifying capacity was found to be greater than that expected on the basis of the loss of soluble protein. The p H of the emulsion was always lower than that of the bovine plasma powder solution. It was noted that the manner in which the pH of the solution was altered also affected the emulsion capacity values. Good correlations wereestablished between particle sizes determined from the Malvern Particle Sizer, the Coulter Counter and the Emulsion Activity Index. Coulter values were always higher than Malvern values, and Emulsion Activity Index values were higher at pH 5 and 9 than at pH 8.1. At pH 8. I , where stability was found to be greatest, the largest globules were found (see Table 1).
Foams were made using 50 ml of 2 wt% bovine plasma powder. The solution was mixed with a Silverson homogenizer in a beaker of known internal diameter for 2 minutes. Foam solutions were transferred to a cylinder, and foam volumes were determined 5 , 25 and 60 minutes after the start of mixing. Foam stability was assessed from the foam volume at 5 minutes divided by that at 60 minutes. Foam expansion was estimated by dividing the foam volume by the original volume of solution. Foam volumes and foam stability were greatest at pH 6.0 and 8.5. The
Table 2
Foam and emulsion data f o r various protein powders (V, = f o a m volume, S , =foam stability, C, = emulsion capacity, S, = emulsion stability a/er 14 days) PH of
Vrl ml
Powder
s o h ion
5 min
25 min
60 min
Sr/ % C,/ g g '
bovine plasma
8.4
61
45
32
50
281
4.5
bovine serum
9.0
63
40
30
48
296
13.4
red blood cell
8.I
48
33
25
52
271
12.8
egg
6.1
63
52
41
16
226
3.3
casein
6.9
59
35
9
I5
336
41.0
whey
5.9
8
1
5
63
190
100.0
soya isolate
1.2
20
18
16
83
221
88.0
S,I %
28 1
decrease in total soluble protein does not relate directly to the foam volume. It seems that, at certain pH values, specific proteins may be depleted, thus affecting the foam volume or stability. Other protein powders were studied using the methods developed for bovine plasma powder. The results in Table 2 show that the plasma powder has good foam stability and foam volume as compared with the other proteins, although the egg foam is clearly more stable. The limitations of the model tests could be appreciated when the plasma was incorporated into angel cakes. Compared with cakes made with egg, high-foam cakes made with bovine plasma powder were darker in colour, and were of lower volume and coarser crumb structure. Table 2 also shows that bovine plasma powder has a high emulsion capacity and a high emulsion stability. Casein has a greater emulsifying capacity, but the stability is much better with the bovine plasma. We have found that alteration of pH affects the state of a protein and thereby affects the values obtained for the functionality parameters. No functional test is complete without specification of the ionic environment in which it is conducted. Functional protein may behave very differently when incorporated into food products of differing acidity. Reference I K.N.Pearce and J.E. Kinsel1a.J. Agric. FoodChem., 1978,26,716.
282
Abstracts of Posters
Multiple Emulsions Stabilized by Protein: Nonionic Surfactant Interfacial Complexation ByT.K. LAW. T.L. WHATELEY and A.T. FLORENCE (Department of Pharmacy. University of Strathclyde. 204 George Street, Glasgow GI lXW)
Multiple emulsions have potential uses in a range of applications such as controlled drug-release and extraction processes, but their lack of physical stability is a serious drawback. Free migration of surfactant between internal and external interfaces contributes to the inherent instability following emulsification of the primary water-in-oil system with a surfactant mixture to give the final water-in-oil-in-water (W/ O / W) emulsion. We have found'.2 that the formation of an interfacial complex between albumin and nonionic surfactant gives multiple emulsions with enhanced stability. An interfacial membrane complex between albumin and a lipophilic poloxamer surfactant (e.g., Poloxamer 31 I ) develops over a period of days. This leads to a more stable emulsion, and provides a more effective barrier to solute o r drug molecules by reducing the rate of loss through physical breakdown of the system. A water-soluble model solute, sulphane blue, has been encapsulated within the internal emulsion droplets and its rate of release has been measured. We have found that the mass transfer rate and the stability of the oil membrane is influenced by the combination of external (secondary) hydrophilic surfactants used. The secondary surfactants affect the release rate of the model solute primarily by controlling its mass transfer rate through the oil membrane. The highly stable interfacial film can withstand extensive thinning brought about, for example, by swelling of the internal aqueous droplets by osmotic transfer of water into the internal multiple globules. Recently, we have described) the non-aqueous bi-liquid microfoam structure which is found in these swollen W / O / W systems. At its thinnest, the oil-continuous stabilizing film could only be observed under phase-contrast or dark-field illumination. Single droplets carrying such thin oil membranes were stable for several weeks. The structure of the interfacial protein-surfactant membrane is not well-established, but it forms irreversibly, and once formed is highly stable. However, certain surfactants (e.g., Triton X-165)can disrupt the interfacial film in the stretched state in swollen systems. The swollen W /O / W emulsion forms a semi-solid gel within a few seconds of preparation, and it can be used to provide an emulsion system of very low oil content (as low as 2.5 vol% oil).
283
References I T.K. Law, A.T. Florence, and T.L. Whateley, J. fhar. fhormorol., 1984,36, SOP. 2 T.K. Law, T.L. Whateley. and A.T. Florence, J. Controlled Releuse, 1986. in the press. 3 A.T. Florence, T.K. Law, and T.L. Whateley, J. Colloid Interfuce Sri., 1985,107,584.
Abstracts of Posters
284
Comparison of Large Scale Whipping and Conductimetric Methods for the Determination of Expansion and Stability of Protein Foams By D.J.WRIGHT and J.W. HEMMANT
(AFRC Institute of Food Research, Colney lane, Norwich NR4 7UA)
The foaming properties of protein solutions have been determined by a conductimetric method,' and results are compared with those obtained with a standard whipping method.2 In addition, the conductimetric method has been further modified to use smaller amounts of protein (10 mg rather than 125 mg).
rme/second
Figure 1
Protein foam decay curves obtained using the single nitrogen jet conduct imetric method. Electrical conductivity is plotted against time: T, trypsin; 0, ovalbumin; H, haemoglobin; L, /3-lactoglobulin; C , a-casein; S , pea whey protein
285
In the first (semi-micro) procedure, nitrogen at 2.5 bar was sparged through a ground-glass sinter (porosity No. 4, diameter 3 cm) at a flow-rate of 60 cm' min I . Sparging was continued until the foam reached 2 cm above the conductivity electrodes, corresponding to a total height of 5.5 cm above the sinter. Conductivity values were logged by computer at pre-set voltage intervals, with 500 points collected from the commencement of sparging. All proteins used for assay were dialysed exhaustively to remove residual salts, and were made up to the required concentration in 0.1 M KH2P0, (pH 7, conductivity = 12.5 mS at 24 "C). To reduce the quantity of protein required for assay, and to overcome problems associated with the procedures (e.g., blocking of the sinter), a micro-cell was developed incorporating a single nitrogen jet emanating from a 15 p m aperture. Electrodes in the micro-cell were0.49 cm2 in area, set into the cell wall I cm apart at a distance of 3.2 cm above the aperture, and with this apparatus the nitrogen sparge was stopped when the foam level was 0.6 cm above the electrodes. Decay curves obtained for a range of proteins are shown in Figure I . The foam volume stability from the semi-micro-cell was expressed as the conductivity at 3 minutes as a percentage of the maximum conductivity; the stability from the micro-cell was expressed as the conductivity at 30 seconds as a percentage of the maximum conductivity. Foam volume stability from micro and semi-micro methods was found to be directly proportional to the percentage of foam remaining at 30 minutes using the large-scale whipping procedure. Foam expansion determined from the whipping procedure, as given by the initial total volume, was directly proportional to that found with the semi-micro method, as measured by the initial increase in conductivity (range 0-0.9 mS), and inversely proportional to the foam expansion from the micro-cell, as measured by the time taken for the foam to reach 0.6 cm up the cell. Fitting the conductivity C to an equation of the form C = A,exp(-t/T,)
+ A,exp(-t/T,) + B
where t is time and A,, A,, T I ,T, and B are constants, leads to a good correlation between observed and predicted results using the micro-cell. Initially a protein concentration of 0.5 wt% was used with the microcell, but it was found later that improved resolution of foaming behaviour was achieved when the protein concentration was reduced to 0. I wt% (corresponding to 1-3 mg). This work was supported by the Ministry of Agriculture, Fisheries and Food. References
I 2
Kato, A. Takahashi, N. Matsudorni, and K. Kobayashi,J. FoodSci., 1983,48,62. Research Report No. 358, Leatherhead Food R.A., 1981.
A.
286
Abstracts of Posters
Direct Automated Observation of Emulsion Droplet Coalescence By E. DICKINSON, B.S. MURRAY and G. STAINSBY
(Procter Department of Food Science. University of Leeds Leeds LS2 9JT)
The breakdown of emulsions due to coalescence is poorly understood, yet clearly of great importance industrially. To date, most information about the behaviour of individual droplets has had to be inferred from studies of bulk emulsion stability, or from coalescence measurements on the largest droplets in a typical emulsion. Recent developments in image analysis and video technology have prompted us to construct an apparatus which allows direct and automatic observation of the coalescence of oil droplets (>I pm) with a planar oil-water interface over a time scale of several days. The apparatus described below is also suitable for measuring creaming rates and for studying the effect of particle interactions on flocculation kinetics. A planar oil-water interface is formed in a sealed glass thermostatted cell (fO.I " C )shown schematically in section in Figure I.The interface is viewed with a microscope (Nikon, M Plan 40 objective lens, 40X,long working distance) through the cover-slip window of the cell lid. The microscope is connected to a video camera (Hitachi, HV-720K, monochrome), which in turn is connected to an image analyser (Seescan, Rd 5 12p) and a high-resolution monitor. Emulsion droplets are injected just below the interface as shown in Figure 2. When a droplet reaches the interface, it may coalesce immediately o r exist there for some time before doing so. An experimental problem that had to be overcome was the elimination of interfacial drift which may cause droplets to move completely out of view prior to coalescence. This was achieved by floating a fine network of glass fibres at the oil-water interface, and trapping the network in a fixed position using four vertical stainlesssteel needles. In a gap between the fibres, the interface is static, and droplets reaching such an interfacial region exhibit negligible convective drift over a period of 24 hours. The net magnification from a droplet to its image on the monitor screen is 1. I X lo'. A stationary droplet at the interface appears on the monitor as a d a r k ring with a sharp outer edge. Providing the illumination is even, there is a sharp change in intensity of the grey level of the picture points (pixels) on moving across the outer edge of the droplet image. The image analyser is used to search the picture for such sharp transitions, and thereby detect the droplets. In the early stages following injection, not only are droplets disappearing due to coalescence, but also many
287 6 M W T U FLOW OF
GLA6S CELL
Figure 1
Schematic representation of glass cell containing the oil- water interface. together with surrounding illuminating. thermostatring and sample introduction components
..-.- -__.. ... _..__ _.._ .__.__ _...._
....................
.. .-....-_ ........ -- ............
-_...............................
.. .. ..-. . .'-- 7
-
.
-
. : . y
INJECTION OF
Figure 2
-
-
-
-
INTERFACE
.
OIL DROPLETS
Sketch ofhow droplets are introduced near the interface at which a network of glass fibres isjloated
288
Abstracts of Posters
droplets are continually arriving at the interface from below, and so, at any one time, there may be 50 o r more droplets in view. To measure the life-times of all these droplets simultaneously, an image analyser is essential. A machine-language program has been written to enable detection of droplet appearance and disappearance events, to measure the size of existing droplets, and to record the age and position of a droplet at coalescence. We are starting t o use the apparatus described here to measure the stability of emulsion-sized oil droplets under controlled conditions. By eliminating the tedium and error of manual droplet coalescence studies, we expect to be able to collect detailed and reliable data with a view to testing possible relationships between coalescence kinetics and other properties such as droplet size or surface rheology. We thank Mr. Philip Nelson for technical support, and the Ministry of Agriculture, Fisheries and Food for financial assistance.
289
Modelling of Colloids and Emulsions By G.C. ANSELL, E. DICKINSON and
S.R.EUSTON
(Procter Department of Food Science, University of Leeds. Leeds LS2 9JT)
I n food emulsions of low or intermediate volume fraction, the mechanical properties of the stabilizing macromolecular layer ensures that the droplet shape is negligibly deformed by Brownian motion, interparticle forces, creaming or hydrodynamic flow fields. Such emulsionscan be modelled, therefore, as systems of rigid spherical particles whose degree of flocculation is controlled by the balance between the random thermal motion and the forces associated with interparticle coloidal interactions and externally applied fields (gravity, shear flow, erc.). Using the computer simulation technique of Brownian dynamics,' we are modelling the aggregation behaviour of spherical particles interacting with DLVOtype colloidal potentials under a range of conditions. In a simulation of a floc of 57 particles initially arranged in a face-centred-cubic formation, we find2 that the mechanism of fragmentation in shear flow is strongly influenced by Brownian motion and the strength of the flow field. I n the absence of Brownian motion, particles of diameter I pm and secondary-minimum well-depth of I kT remain flocculated at low shear rates (55 s-I) but melt along crystal planes at high shear rates (27s-I). The presence of Brownian motion makes the process of floc fragmentation more disorganised. In a separate study,' we are simulating coagulated sediments formed by the creaming of spherical colloidal particles. We are finding that the structure of the gel-like sediment is dependent on a large number of inter-related factors. Cream volume fractions may range from above 0.6 to below 0. I depending on the droplet size, the forces between the droplets, and the applied field strength. In food emulsions of high volume fraction, the droplets are deformed by interactions with their neighbours, and the simple rigid sphere model is no longer appropriate. To simulate the statistical properties of suspensions of deformable particles, we are exploring two models initially in two dimensions. Model A consists of an assembly of interacting lattice chains, with each necklace of segments representing a membrane separating dispersed phase from continuous phase. Monte Carlo calculations show' that particle deformability and emulsion structure are controlled by the size of the repulsive interaction between non-bonded segments on the particle membrane. Model A is appealing because it has features common to both a macromolecular solution and a conventional colloidal dispersion, but it has
290
Abstracts of Posters
the disadvantage of not maintaining constant particle volume and not being easily adapted to model droplet coalescence. To overcome these problems, we introduce’ a new model B in which droplets consist of a large number of identical sub-units each occupying a single lattice site. The distribution of droplet configurations depends on the nature of inter-sub-unit interactions, which are attractive for sub-units on the same droplet, but strongly repulsive otherwise. With both models A and B, the use of periodic boundary conditions means that the behaviour of bulk systems can be reliably simulated with assemblies of between 50 and 100 model particles.
References I 2
3 4
5
E. Dickinson, Chem. SOC.Rev., 1985,14,421; Chem. Ind., 1986, 158. G.C. Ansell and E. Dickinson, J. Colloid Interface Sci. 1986,110,73. G.C. Ansell and E. Dickinson, J. Chem. Phys., 1986,85,4079. E. Dickinson, Phys. Rev. Lett., 1984.53.728. E. Dickinson and S.R.Euston, to be published.
