Food Colloids: Proteins, Lipids and Polysaccharides (Woodhead Publishing Series in Food Science, Technology and Nutrition)

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Food Colloids Proteins, Lipids and Polysaccharides

Food Colloids Proteins, Lipids and Polysaccharides Edited by

E. Dickinson Procter Department of Food Science, University of Leeds, UK and

B. Bergensdhl Institute for Surface Chemistry, Stockholm, Sweden

WOOD HEA D PuBLI sH IN G Cambridge England

LIMITE D

Published by Woodhead Publishing Limited, Abington Hall, Abington, Cambridge CB 1 6AH, England www.woodhead-publishing.com The keynote and oral papers presented at the ‘Food Colloids -Proteins, Lipids and Polysaccharides’ meeting held in Ystad, Sweden on 24-26 April 1996. First published by The Royal Society of Chemistry 1997 Reprinted by Woodhead Publishing Limited 2004 0 Woodhead Publishing Ltd, 2004 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 the publisher. 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 ISBN 1 85573 783 3 Printed in the United Kingdom by Lightning Source UK Ltd

Preface The field of food colloids has now become established as a mature subdiscipline within the wide subject of food science. The field is concerned with the structural and dynamic aspects of multi-phase food systems-dispersions, emulsions, foams, gels-viewed from a physical chemistry perspective as assemblies of molecules and particles in various states of organization. The main molecular components of food colloids are proteins, lipids and polysaccharides. The primary objective is to relate the structural, stability and rheological properties of such systems to the interactions between the constituent components and to their distribution between the bulk phases and various kinds of interfaces. This book records most of the lecture programme at the international conference on ‘Food Colloids-Proteins, Lipids and Polysaccharides’ held at the small coastal town of Ystad in southern Sweden on 2426th April 1996. This conference, organized in collaboration with Ideon Agro Food (Sweden), was the sixth in a series of biennial Spring Symposia on the subject of food colloids to be organized under the auspices of the Food Chemistry Group of The Royal Society of Chemistry (UK). The meeting was attended by over 210 delegates from 22 different countries-statistics coincidentally the same as for the previous (1994) meeting in Dijon. The programme included invited overview lectures, contributed oral presentations, and an exhibition of over 60 posters. Most of the oral presentations are recorded in this volume. A substantial proportion of the poster presentations is scheduled to be published separately in a special issue of the journal Food Hydrocolloiu3. A highlight of the conference was the presentation to Professor Dr Pieter Walstra of the Senior Medal of the RSC Food Chemistry Group in recognition of his outstanding contributions to research and teaching in the area of food colloids. The occasion of the award was particularly appropriate in view of Professor Walstra’s strong association with this series of conferences and the close proximity of the timing of the meeting to his formal retirement as Professor in Dairying at Wageningen Agricultural University in May 1996. We are delighted that we are able to include in this volume the full text of Professor Walstra’s Senior Medal Lecture, which is entitled ‘Making emulsions and foams: an overview’. The continuing success of this series of conferences relies on the support and enthusiasm of the International Organizing Committee, and we acknowledge here the important contributions of Dr Rod Bee (Unilever Research, Colworth Laboratory), Professor Denis Lorient (ENSBANA, Dijon) and Professor

vi

Preface

Pieter Walstra. We are also extremely grateful to the members of the Local Organizing Committee: Mrs Anne-Marie Camper (Ideon Agro Food), Professor Petr Dejmek (Lund University), Professor Anne-Marie Hermansson (Swedish Institute for Food and Biotechnology, G6teborg), Professor KBre Larsson (Lund University) and Professor Eva Tornberg (Swedish Meat Research Institute, Kavlinge). Finally we thank the authors of the papers presented here for their cooperation and involvement.

E. Dickinson (Leeds) B. Bergenstihl (Stockholm) June 1996

Contents Colloidal Properties and Sensory Perception INVITED LECTURE Influences of Processing on Structure-Rheology-Texture Relationships for Colloidal Food Systems E. J. Windhab, B. Wolf, and E. Byekwaso

3

Effect of Microstructure on Sensory Perception of Particulate Gels M . Langton, A . Astrom, M . Stading, and A . - M . Hermansson

18

Microstructure as Related to the Water-holding, Textural and Sensory Properties of Emulsion Sausages K . Andersson, A. Andersson, and E. Tornberg

29

Association of Emulsifiers INVITED LECTURE Association of Emulsifiers in Aqueous Systems N. Krog

45

On the Stability of Aerated Milk Protein Emulsions in the Presence of Small-Molecule Surfactants B. M . C. Pelan, K . M . Watts, I . J . Campbell, and A . Lips

55

Interactions between Sodium Caseinate and Dioleoylphosphatidylcholine on Oil-Water Interfaces and in Solution Y. Fang and D . G . Dalgleish

67

Effect of Lipophilic Molecules on Food Protein Surface Activity at the Air-Water Interface L. A . Wasserman and M . G . Semenova

77

Monoglyceride Mixed Films: Structure and Stability C. Carrera Shchez, J. de la Fuente Feria, and J. M . Rodriguez Patino

92

...

Contents

Vlll

Aggregation Phenomena INVITED LECTURE Aggregation Processes, Particle Interactions, and Colloidal Structure E. Dickinson

107

High Pressure Processing of B-Lactoglobulin and Bovine Serum Albumin V . B. Galazka, D . A . Ledward, andJ. Varley

127

Ultrasonic Characterization of Flocculation in Oil-in-Water Emulsions D . Hibberd, A. Holmes, M . Garrood, A . Fillery-Travis, M . Robins, and R. Challis

137

Measuring Aggregation in Colloids using Ultrasound Velocity and Attenuation M. J . W. Povey

150

Structure of Fat Crystal Aggregates W. Kloek, T, van Vliet, and P. Walstra

168

Interactions between and within Interfaces INVITED LECTURE Lipid-Protein Interactions-Consequences for Surface Activity in Food Emulsions M . Le Meste, P. Tainturier, and J . -L. Gelin INVITED LECTURE Forces between Lipid-Coated Interfaces P. M . Claesson

185

20 1

/3-Casein Adsorbed Layer Structures Predicted by Self-ConsistentField Modelling: Comparison with Experiment P. J. Atkinson, E. Dickinson, D. S. Horne, J . Leaver, F. A . M. Leermakers, and R. M . Richardson

217

Adsorption Behaviour of Caseinate in Oil-in-Water Emulsions: A Nuclear Magnetic Resonance Approach Y. Mine

229

ix

Contents

Stabilization of Protein-based Emulsions by Means of Interacting Polysaccharides D. G. Dalgleish and A.-L. Hollocou

236

Effect of Neutral Carbohydrate Structure on Protein Surface Activity at Air-Water and Oil-Water Interfaces A. S. Antipova, M . G. Semenova, and A. P. Gauthier-Jacques

245

Influence of Protein-Polysaccharide Interactions on the Rheology of Emulsions K. Pawlowsky and E. Dickinson

259

Deposition and Release of Bare and Protein-covered Polystyrene Latex Particles S. Joscelyne and C. TragSrdh

266

Control of Gelation INVITED LECTURE Alginate Gelation Technologies 0. Smidsred and K. I. Draget

279

Understanding Synergistic Polysaccharide Networks using Electron Microscopy and Rheology L. Lundin and A. -M. Hermansson

294

Protein-Polysaccharide Mixtures: Structure and Effect of Shear Studied by Small-Angle Neutron Scattering D. Renard, F. Bout, and J. Lefehvre

303

Interactions in Mixtures of Gelatin and i-Carrageenan C. Michon, G. Cuvelier, B. Launay, and A. Parker

316

Rheology of Protein Gels and Protein-Stabilized Emulsion Gels Cross-Linked with Transglutaminase Y. Yamamoto and E. Dickinson

326

Rearrangements in Acid-Induced Casein Gels during and after Gel Formation T. van Vliet, J . A. Lucey, K. Grolle, and P. Walstra

335

Emulsion Behaviour of Non-Gelled Biopolymer Mixtures T. J. Foster, J. Underdown, C. R. T. Brown, D. P. Ferdinando, and I. T. Norton

346

Contents

X

Structural Properties of Heat-Set Whey Protein Gels M . Verheul and S . P. F. M . Roefs

356

Making Emulsions and Foams RSC FOOD CHEMISTRY GROUP SENIOR MEDAL LECTURE Making Emulsions and Foams: An Overview P. Walstra and I . Smulders

367

Influence of Emulsifier Adsorption Kinetics and Emulsification Machine Construction on Dispersity of Oil-in-Water Emulsions M . Stang, H . Karbstein, and H. Schubert

382

Protein-Stabilized Emulsions Prepared by the Microporous Glass Method G. Muschiolik, S. Drager, I . Scherre, H . M . Rawel, and M . Stang

393

Subject Index

401

Influences of Processing on Structure-Rheology-Texture Relationships for Colloidal Food Systems By E. J. Windhab, B. Wolf, and E. Byekwaso DEPARTMENT OF FOOD SCIENCE, SWISS FEDERAL INSTITUTE OF TECHNOLOGY, ZURICH (ETH), CH-8092 ZURICH, SWITZERLAND

1 General Relationships of Flow Processes and Food Properties During flow processes, both viscous (shear and normal) and inertial stresses act on the fluid matrix. In complex fluid systems, like multiphase foods containing dispersed components (solid particles, fluid droplets, gas bubbles) and colloidally dispersed macromolecules (polysaccharides, proteins, lipids) in the continuous fluidphase or at the dispersed interfaces, the flow stresses will tend to impede or influence the interactions of these structural components. If specificcritical stresses are exceeded, flow-induced structuring will occur. Such structural states can be of a reversible or an irreversible nature. In each case the structural changes influence the rheological behaviour of the fluid system, and consequently the flow process itself is affected. On the other hand, structuring also influences the technofunctional properties of the final food product. Filling/dosing and sedimentation (phase separation) behaviour are directly related to the rheological properties. Furthermore, the sensory perception of food texture is significantly dependent on structure and rheology. For food property/quality optimization the process-rheology-strcture/ texture relationships have to be quantified. The engineering aim is to design apparatus and process foods according to such optimization criteria and additionally to control them on-line (see Figure 1). This paper refers to dispersing processes in a continuous rotor/stator apparatus. The complex fluid food systems to be considered, for instance, are oil-inwater emulsions (mayonnaise, sauces) and foams containing polysaccharides (starch) or proteins as stabilizer/thickener colloidally dispersed in the continuous aqueous fluid phase.

4

Structure-Rheology-Texture Relationships for Colloidal Food Systems

sensorial

1 1 I1 I

- consistency

- fllling I dosing - separation stability

- - - -1

Figure 1

Overview of relationships between process and structurelrheologyltexture

2 The Dispersing Process The dispersing process device used for emulsion processing was an industrial continuous rotodstator unit (Megatron St-37 Kinematica LTD, Luzern, Switzerland). The two main dispersing gap geometries are shown in Figure 2

lo

1

Ne=Ct/Re

Ne = C2

I

10 geometry4

0.1 103

Figure 2

A geometryG

1

I

104

105

Re

106

107

Rotorlstator dispersing geometries ( K and G) and their power characteristics N e = f(Re)

5

E. .IWindhab, . B. Wolf, and E . Byekwaso

:onventional premixing (step 1)

intensive dispersing (step 11)

rotor I stator device MEGATRONB, KINEWATICA AG, CH-Luzern

c 5 premix vessel

Figure 3

--

Scheme of processing apparatus for dispersing of emulsions and foams by premixing (step I ) and intensive dispersion (step I l )

together with their ‘power characteristics’ Ne = f(Re). The power number Ne and the Reynolds number R e are calculated according to equations (1) and (2). Figure 3 shows a scheme of the dispersing process constructed for this study. For continuous foam processing a rotodstator aeration system (Mondomix, Mini-Mondo) was used. The process scheme is in principle similar to the emulsion dispersing process. The main difference relates to the rotorlstator geometry which consists of a larger number of gaps. In the case of laminar gap flow (Ne = C1/Re) the stresses acting in the gap are dominated by the viscous shear stress t [see equation (3)]. For turbulent flow (Ne = C,) the so-called Reynolds stress tR [equation ( 5 ) ] refers to the dominating inertia forces. The equations for Ne and Re are listed below:

where P is the power consumption, n is the rotational spced/r.p.m., d is the average rotor diameter, p is the fluid density, s is the average dispersing gap width, q(fJ is the viscosity function, and .ji is the shear-rate at the inner gap radius (i.e. outer rotor radius). The other quantities are defined by:


6

where v(r) is the velocity profile in the dispersing (perpendicular) gap, and v is the turbulent velocity fluctuation. The stress t or sR acts on the multiphase fluid system components (macroscopic dispersed particles and colloidally disperse macromolecules). If, for instance, in the case of dispersed emulsion droplets or foam bubbles a critical shear stress zCritis exceeded, then droplet/bubble break up occurs: *

where We, is the critical Weber number [= f(r,7disp/r,7cont)], u is the interfacial tension and x is the droplet diameter. It is expected that there will be additional critical mechanical stresses associated with structural changes of the macromolecular (colloidally dispersed) components (single molecules and networks). These are related to their chemical bonding, steric conformations, hydrodynamic volume, degree of branching, extent of entanglements, as well as the pH and the ionic content of the solvent. The interactions of these colloidal structures are of a very complex nature and a theoretical pre-calculation of the critical stresses involved is almost impossible. Based on a variety of analytical structures, an integral ‘structure stability’ can, however, be described quantitatively.2

3 Materials and Methods Materials The oil-in-water ( O N ) emulsion (mayonnaise) investigated contained sunflower oil at concentrations of 14,65 or 85 wt% (emulsion samples 1 , 2 and 3). In the aqueous continuous phase of the low-fat recipe (14 wt% oil) (emulsion l), 4 wt% of a modified waxy maize starch (National Starch, E-1422; acetyl. distarchadipate) was suspended. Emulsions of type 3 were protein stabilized (see Table 1).The foams investigated here were stabilized using a whey protein isolate (Bipro) acting as both emulsifier and thickener.

Analytical Methods and Measuring Apparatus The rheological experiments (steady and oscillatory shear) were carried out in this work using a controlled stress rheometer (Rheometric Scientific, DSR)

Table 1

Three model emulsion recipes (weight basis)

~

~

Recipe I

Ingredient

-~ - ____

~

~

~

water

32%

oil emulsifier

65 Yo

stabilizer

Recipe 2 ~

_

80.3% 13.8%

3yo -

Recipe 3

_

starch 3.9%

5Y O

85Yo egg y.lO% + protein

E. J . Windhab, B. Wolf, and E. Byekwaso

7

and a controlled strain device (Rheometric Scientific, RFS). The laser light diffraction measurements of droplet-size distribution were performed with a Mastersizer X (Malvern Instruments). Additional light microscopy was carried out with an inverse microscope (Nikon). To measure phase separation stability of the emulsion systems during short-time storage (up to 28 days), a specific conductometric cell was developed to determine the conductivity difference between the upper and lower level in a small cylindrical storage ~ e s s e l . ~

Sensorial Texture Evaluation The texture of the mayonnaise emulsion system was sensorially tested according to ‘spoonability’ and mouthfeel. On a 6-point scale, the thickness was determined from the test panel. The ‘pure’ continuous starch-containing aqueous watery phase was tested after the dispersing treatment with a spoon test. For the mayonnaise emulsions an additional qualitative hedonic test was carried out.

Process Experiments In a preliminary investigation, an experimental design was configurated taking the process variables as rotational speed n, residence time t,, gap geometry, and the number of gaps in series. Similar experimental schemes were configurated and carried out for the different emulsion and foam recipes. (Emulsions are described in detail; foams are only briefly discussed here .) In a further experimental step, the ‘pure’ continuous phases, for instance, the 4% aqueous starch solution used as the continuous phase in O/W model emulsions of type 2 (discussed here in detail), were investigated in dispersing experiments under similar conditions as for the whole emulsiodfoam systems. Specifically, for constant residence time, the mechanical power input was varied by changing the rotational speed only whilst maintaining the two main gap geometries 1G and 1Kb (see Figure 2).

4 Results Emulsions From the measured droplet-size distributions, the mean value of the volume distribution Q3(x) and the standard deviation u (indicator of the distribution width) were taken as characteristic values for further comparisons. Figures 4-6 present the mean droplet size x50,3 as a function of the volume specific energy input E, for different rotor/stator gap geometries (3HG, lHG, 3HF, IHF, lKb, lKe, 1G). Obviously the mean droplet diameter decreases asymptotically with increasing energy consumption. In the case of emulsion type 3, this general tendency changes to an increase in the mean droplet size as soon as an energy input of about lo7 J/m3 is exceeded. This is due to protein coagulation (induced by dissipated heat) in this proteidegg yolk stabilized

Emulsion 1 50 1-

40

3geometdesHG 9 1 geometry HF

--

1.OE&

Figure 4

1.OE+07

1.OE&

Mean droplet size x50,3 as a function of specific energy input E, for emulsion type 1 with various gap geometries

emulsion of type 3. Furthermore, the data in Figures 4-6 demonstrate the different ‘energy efficiency’ of the gap geometries used. The 1Kb and 1Ke gap, as well as the 1G gap (for emulsion type 3), are most efficient, leading to an average droplet size of x50,35 5 pm for E, 2 lo6J/m3.

20

i

\

3

lo;

5

-,

1. O E a

,

,

,

,L, ,

1.OE+07

1.OEM8

EV / ~ m 3 Figure 5

Mean droplet size x,, as a function of specific energy input E, f o r emulsion type 2 with various gap geometries

E. . I Windhab, . B. Wolf, and E. Byekwaso

Figure 6

9

Mean droplet size x, as afunction ofspecific energy input E,,for emulsion type 3 with various gap geometries

The comparison of rheological results from steady and oscillatory shear tests has shown that the highest sensitivity to structural changes of the emulsion systems was reached by the storage modulus G ‘ ( o ) measured with the oscillatory shear test in the linear viscoelasticdeformation range at a frequency of 1Hz. The quantity G’(w)is an elastic parameter and therefore it is expected to be most sensitive to disperse particles (droplets) or macromolecular interactions leading to changes of structural networks. The small-deformation measurement in the linear viscoelastic shear domain ensures that the measuring procedure (oscillatory shear experiment) does not influence or destroy the structural state to any significant extent. Figures 7-9 demonstrate the G’ (w = 1 Hz) dependency on the volume specific energy input E,. Plots of G ‘ ( o= 1Hz)against E, for the three emulsion types are completely different. If the data in Figures 4-6 as well as the recipe information are also taken into account, an interpretation can be given as indicated below.

Emulsion rype 1 There is no macromolecular stabilizer in this recipe. The emulsion is mainly stabilized by the high oil content leading to a concentrated oil droplet network. The emulsifier used stabilizes the droplet interface. The hydrophilic groups of the adsorbed emulsifier molecules can interact with each other in the aqueous phase due to the close ‘droplet packing’. The increase in E, reduces the average droplet size significantly. In the case of experiments using more ‘gentle dispersing’ gap geometries (1HG ,lHF), an increase in G‘ is observed at lower E,. This is the effect expected for emulsion systems where only a change in size of the droplets occurs. This leads to increased droplet

10

Structure-Rheology- Texture Relationships for Colloidal Food Systems 180 : 160

2 > Q

-4a 3

1-

0

140 :120

0

I-

100 1-

80

;-

Emulsion 1

+

1 geometfy

0 1.OE+O6

HF

1.OEM8

1.OE+07

Ev I JIm3 Figure 7

Storage modulus G’ at I H z as a function of the volume specific energy input E, for emulsion type 1 with various gap geometries

interactions, strengthening the droplet network and consequently increasing the rheological parameters (here for instance G’). A further increase of E,, as well as using more intensive dispersing gap geometries, seems to influence the sterically interacting hydrophilic chains of the emulsifier molecules. Though there is no further significant decrease in the average droplet size from E, = lo7 to 8 X lo7 J/m3 (see Figure 4),the G’ values do decrease (see Figure 7).

1.OE+O6

1.OE+07

1

EV I ~ m 3 Figure 8

Storage modulus G’ at I H z as a function of the volume specific energy input E, for emulsion type 2 with various gap geometries

11

E. J . Windhab, B. Wolf, and E. Byekwaso 2500

Emukkn 3 0

I

1500 0

a

I I

.o

+x

+.

500

X

0

3i

O T 1.oE*

X 1.oE+o8

1. E d 7

1.OE+OB

1.oE+09

Ev I Jhn3 Figure 9

Storage modulus G' at 1 Hz as a function of the volume spec@ energy input E, for emulsion type 3 with various gap geometries

Emulsion type 2 Although the droplets get finely dispersed with increasing E, (Figure 9,which is expected to lead to an increase in droplet interactions and consequently should lead to an increase in G', there is a strong permanent decrease of G' (Figure 8). Consequently the stabilizer (starch, 3.9 wt%) used in this low-oil-content emulsion (13.8 wt%) must be changed in its networking properties. The weakening of the starch network causes the decrease in G' with E V

.

Emulsion type 3 Over the whole E, domain investigated, the emulsion type 3 shows an increase in G' with increasing E, (Figure 9) though the average droplet size decreases up to E, = lo7and increases again (partly) for higher E, (Figure 6). The high percentage of oil in the type 3 recipe (85%) is dominated in the resulting rheological behaviour by the egg-yolk emulsifier and the egg protein added. These components lead to an increased network stability with increasing E, based on protein coagulation which is triggered by the mechanical forces acting in the dispersing flow, and at higher E, by the dissipated heat. Due to the high (and increasing) viscosity of this emulsion, a temperature increase of up to 40"Ccould be measured in the dispersing gap at the highest E, values investigated (E, = lo8J/m3). If the coagulated proteidegg-yolk network is further treated in the dispersing gap (at high E, values), the stabilizing network structure gets irreversibly destroyed leading to droplet recoalescence (E, 1 lo7J/m3, see Figure 6) and in extreme cases to phase inversion (complete O/W emulsion destruction). Three tests of emulsion stability were carried out. For samples 1 and 2, the experimental results for mean droplet diameter and C' (o = 1 Hz) were

12

Structure-Rheology-Texture Relationships for Colloidal Food Systems 20

18

'underprocessed '

v//

16 14

5

12 10

%"

\

2

6 4

2

0 1 .OE+05

1.OE+06

1 .OE+07

1.OE+O8

1.OE+09

EV I J/m3 Figure 10

Stability domains of emulsions (types 1-3) processed under various energy input conditions (EJ using different dispersing gap geometries

monitored over a storage time of up to 28 days. For sample 3, an additional conductivity difference (AC) measurement for phase separation detection was performed over the same time period. Figure 10 summarizes the results of these stability tests. For all three emulsion types dispersed with different rotor/ stator gap geometries, there are common stability domains which can be located in the x50,3 versus f ( E V )diagram as indicated. Underprocessed emulsions are unstable due to sedimentation of the 'large' droplets. Overprocessed emulsions separate due to a destruction of the stabilizing macromolecular network. Emulsions which showed phase inversion already during processing are not included in Figure 10. Figure 11 gives a typical scan of the sensorial texture parameter 'thickness' (from the spoon test) as a function of the rheological behaviour measured under oscillatory shear. The parameter ratio G'/G" (= l/tan 6 = dynamic Weissenberg number Wi') represents a combination of viscous and elastic properties. The emulsion (type 3) samples which are compared in Figure 11are all taken from the 'stable optimum domain' (see Figure 10) but processed at different values of E , (three groups with E, = lo6, lo7, 8 X lo7 J/m3).4 The sensorial impression of 'thickness' for the emulsions investigated clearly shows a linear dependency on the ratio G'/G. Figure 11gives an additional criterion for selecting a domain from Figure 10 where the stability and sensorial properties are 'optimum'.

Starch-containing Continuous Phase As most significantly shown in the case of emulsion type 2, the stresses acting in dispersing flow fields do not only act to disperse and homogenize the dispersed

13

E. J . Windhab, B. Wolf, and E. Byekwaso

Y

v)

.-cC

8

5.5

6.0

6.5

7.0

8.0

7.5

G'/G" Figure 11

Sensorial 'thickness'impression from spoon test in relation to the rheological emulsion property G ' I G at 1 HZfor emulsion ty e 3 sam les with optimum stabilig processed at various values of E, (1 -8 X 10 Jlrn3)

8

7

phases, but they also influence the networking properties of colloidally dispersed macromolecules in the continuous phase. To get more detailed information about the influence of rotodstator dispersing flows on macromolecular starch networks, an aqueous starch solution (3.9% starch E-1422) was treated in selected gap geometries G and K similar to those used for the emulsions in the experiments described above. The average residence time in the dispersing gap was held constant for the different rotational speeds investigated (0-27 mls perpendicular velocity). During premixing of the starch suspension in a stirred vessel at 60 "C,it could be shown that, after cu. 6 hours of mixing, a steady premixing state ( k , viscosity function constant with further premixing time) was reached. The reproducibility of this state for different batches, however, was not satisfactory. When the starch suspension was treated after premixing in the rotor/ stator gap, a reproducible dependency of the rheological parameters on power input (P,) or energy input (E,) was found. Figure 12 shows the steady shear viscosity functions a(?) in a shear-rate domain from 2 X to 500 s-l for starch suspensions dispersed in the rotor/stator gap geometry 1G after premixing for 6 hours. The dynamic storage moduli G ' ( w ) for the same five different power/energy input levels are plotted in Figure 13. Both diagrams (Figures 12 and 13) demonstrate clearly that there is a maximum in the rheological parameters if the starch suspension has been treated at a 17 m / s rotational velocity ( P , = 170 X lo6 W/m3; E, = 3.4 x lo6J/m3). As demonstrated in Figure 14, the power input P, to reach the maximum level of the rheological parameters (here G' (w = 1 Hz)), as well as the height


14

-

100

10 mls; 112.1 06

*

10

w/d

17

d s ; 170.106 W / d

10

100

tQ

d l \

E 0.1

0.01 0.001

0.1

0.01

1

;. I Figure 12

1000

s”

Steady shear viscosity function ~ ( 7 or ) premixed (60“C, 6 hours) starch suspensions dispersed at various rotational speeds in the rotorlstator gap geometry I G (4 wt% aqueous starch suspension; residence time t, = 20 ms)

1 .oooo Y

0.1000

B

4 0.0100 b 0.0010

0.0001

w I rads Figure 13

Storage modulus G’ (w) of starch suspensions (4 wt%; E-1422) dispersed under various rotational speeds in gap geometry G (residence time t = 20 ms)

15

E. J. Windhab, B. Wolf' and E. Byekwaso 10.000

8 1 .ooo \

n

0

~ r0 . 1 0 0 Y

b

0

100

50

150

200

250

300

350

Storage modulus G' (w = I HI)as a function of P, for I G and I K b rotorlstator gap geometries (4wt% aqueous starch suspension; E-1422)

Figure 14

of the maximum reached, depends strongly on the gap geometry used (compare 1G and 1Kb geometries). The 1Kb geometry was shown to reach the highest G' level and to consume the least power. Figure 15 shows the same behaviour for the low modulus G (o = 1 Hz), but as expected it is less

lo

\

n

!i F

0.1

0.01

- f . . . .

0

: 50

1

1

.

: . . * , ;, . , , ;, , . . ; , , . , ; , . . 100 150 200 250 300 350

.

Pv 1 106 W/m3 Figure 15

Loss modulus G (w = I Hz) as a function of P, for IG and I K b rotorlstator gap geometries (4 wt% aqueous starch suspension; E-1422)

16


pronounced than for the storage modulus G’. An interpretation of the existence of an optimum power or energy input in dispersing operations in order to reach maximum rheological values for G’ ( w ) , G ( w ) and ~ ( jis )given as follows. After premixing, the starch suspension takes up the state of a temporary network. With dispersing treatment in the rotodstator gap, the micro-homogeneity of the starch suspension is increased, leading to more intensive networking (more entanglements). At the ‘rheological maximum’ (G’ ( w ) , G” ( w ) and ~ ( jmaxima) ) the network structure reaches as well its maximum networking efficiency, forming a permanent macromolecular gel network (see the G’-plateau, nearly independent of w in Figure 13). If the optimum power input or energy input is exceeded, the starch network is degraded by the mechanical forces acting in the dispersing gap flow. From preliminary experimental results (determination of macromolecular structural information by light scattering and gel chromatography), it is assumed that the observed mechanical degrading reduces the number of network entanglements and/or reduces the hydrodynamic volume of the starch molecules, but it does not degrade the molecules themselves.

Protein-containing Continuous Phase For emulsions of type 3, it was shown that a significant thickening of the egg protein-containing continuous aqueous phase leads to a strong thickening of the whole emulsion structure. For protein foam systems, it has also been found that mechanical energy input during foaming in rotorhtator gaps can cause protein denaturation (combined with micro-flocculation), thereby changing the rheological and stability properties of the foam system.

5 Discussion For emulsion production in rotodstator devices dependent on gap geometry and mass (or volume) related mechanical power input o r energy input, domains of optimum separation stability and preferred rheologicaVtextural behaviour can be found. The degree of mechanical powedenergy input affects the disperse droplet structure as well as the macromolecular colloid structure (polysaccharides, proteins) in the continuous emulsion phase. For underprocessed emulsions there are found to be two reasons responsible for structure instabilities. Firstly the emulsion droplets may not be finely enough dispersed, and secondly the added macromolecular stabilizer (e.g., polysaccharide) may not reach its optimum networking capacity. For overprocessed emulsions it has to be expected that the maximum networking ability of a stabilizing (networking) macromolecular colloid (polysaccharide or protein) has been reduced or destroyed by an excessive mechanical and/or combined thermal treatment. This leads to phase instability; although the droplets are finely dispersed, they are not sufficiently strongly fixed in a colloidal network. Rheology, especially oscillatory shear rheology , was confirmed to be a very

E. J . Windhab, B . Wolf, and E. Byekwaso

17

powerful tool for the detection of flow process-structure-texture relationships. Sensorial texture analysis is sensitive for detecting consistency differences between various ‘processing degrees’ on the powedenergy input scale investigated. The design of dispersing gap geometries has been shown to be of great importance if structure, rheological/textural behaviour, and phase separation stability of multiphase systems are to be optimized all together to a maximum extent. It is clearly shown that the (newly developed) K-gap geometry is more efficient (i.e., reaching optimum structure, rheology, and stability criteria) in dispersing at remarkably lower power input than the conventional geometries with which it has been compared.

References 1. G. I. Taylor, Proc. Roy. SOC.(London), 1932, A138,41. 2. E. Windhab and B. Wolf, ‘Zum rheologischen verhalten disperser Systeme mit kleinen deformierbaren Korpern’, Proc. 3rd German Food Rheology Meeting, Granum-Verlag, Detmold, 1990. 3. B. Wolf, PhD Thesis, Swiss Federal Institute of Technology, Zurich, 1995. 4. R. G. Cox,J. Fluid Mech., 1969, 37,601. 5 . F. D. Rumscheidt and S. G. Mason, J. Colloid Sci., 1961,16,238. 6. E. Windhab and Th. zu HGne, ‘Einfluss mechanischer Beanspruchung auf die Molekulargewichtsverteilung und das rheologische Verhalten von Polysacchariden’, Proc.Hamburger MakromolekularesSymposium,TU Hamburg, 1990.

Effect of Microstructure on Sensory Perception of Particulate Gels By Maud Langton, Annika Astrom, Mats Stading, and AnneMarie Hermansson SIK-THE SWEDISH INSTITUTE FOR FOOD AND BIOTECHNOLOGY, BOX 5401, S-402 29 GOTEBORG, SWEDEN

1 Introduction The perceived texture influences the acceptance of a food product. Vital information about the perception of texture can be provided by translating the sensation of texture into structural properties that can be measured. Many products such as yoghurts, minced meat, fish, egg products, etc., can be compared with the microstructure of particulate gels.I4 This means that the structure is formed from a continuous colloidal network, which holds the product together and gives rise to its characteristic properties. A colloidal network can be formed from particles linked together forming strands, enveloping pores and/or droplets, inclusions, etc. The dimensions (size and shape) of the particles, strands and pores vary, thus creating different product properties. During mastication the material breaks down and the structure is perceived as texture. Knowledge about the relationship between microstructure and texture can consequently be used to optimize food production processes as well as to develop new products with desired sensory properties. In this study two protein model systems, whey protein and P-lactoglobulin, have been studied. These proteins were chosen as the model systems because fi-lactoglobulin in particular can form a large range of structures depending on pH, temperature, salt, e r ~ . * > ~Both - ’ ~ proteins form gels on heating and particulate gels are formed around the isoelectrical point (-pH 5.2). The microstructure of whey protein gels shows similarities with the microstructure of yoghurt. It is important to understand the relationship between microstructure and sensory quality in order to produce products with desired properties. There are still only few published results on correlation between microstructure and texture. Different commercial cheese products have been analysed, and the morphology of the fat was shown to influence the texture, as evaluated ~ a 3-level sensorily or measured instrumentally.””2 Crippen et ~ 1 . ’used 18

M . Langton, A . Astrom, M . Stading, and A.-M. Hermansson

19

factorial design to find out the effect of grind size, sucrose concentration and salt concentration on peanut butter texture. They found that an increase in grind size decreased sensory smoothness, spreadability, adhesiveness and preference rating, but increased instrumental hardness. The texture was quantified both by a consumer panel and instrumentally. microstructure to the In our laboratory we have previously rheological pro erties of /?-lactoglobulin, and the microstructure has been ~ o r r e l a t e d ' ~ ~with ' ~ ~the ' ~ sensory quality of whey protein gels. This paper shows that the microstructure of a particulate protein gel can be correlated with texture as perceived sensorily as well as measured instrumentally. The microstructure quantified by image analysis has been correlated with the sensory quality. The changes in microstructure were achieved by varying the pH, heating rate and salt addition in a 2-level, full factorial design.

2 Materials and Methods We used 13.5 wt% whey protein gels for the sensory analysis and microstruc10 wt% p-lactoglobulin (Sigma) gels for the microstructural tural analy~is,'~"~ analysis and small deformation tests, and 12 wt% /I-lactoglobulin (INRA) gels for the tensile tests. l6

Experimental Design and Statistics A full, two-level, factorial experimental design was u~ed.'*''~ Three variables were evaluated: heating rate (1 and 5 "C/min), pH (4.6 and 5.4) and salt addition (0 and 0.1 mol/dm3NaCI). The results were evaluated by MANOVA, analysis of variance. Non-linear regression between a single microstructural parameter and a single sensory property at a time was performed. Correlations between latent variables were performed by using PCA, principal component analysis, as well as multivariate regression PLS, projections to latent structures. I53l9

Sensory Analysis The sensory evaluation was done using an external panel, consisting of eight specially selected and trained panellists with previously documented ability to perform valid and reliable sensory data. A descriptive test made it possible both to describe and to quantify the relevant sensory properties of the gels simultaneously.20The sensory characterization of the whey protein gels was focused on texture properties.

Microscopy Three different microscopy techniques were used to study particulate network structures: light microscopy (LM) for pore-size distribution, transmission electron microscopy (TEM) for particle-size distributions, and scanning elec-

20

Effect of Microstructure on Sensory Perception of Particulate Gels

tron microscopy (SEM) for strand characteristic^.^^^' Small pieces of the gels were double-fixed, in 2% v/v glutaraldehyde with 0.1% w/v ruthenium red, rinsed, and post-fixed in 2% w/v Os04. The samples were then rinsed and dehydrated in a graded ethanol series. The LM samples were embedded in Technovit 7100 after dehydration. Semithin sections, -1 pm, were cut on glass knives, stained with toluidine blue, and examined under a light microscope. Dehydrated T E M samples were transferred to propylene oxide and then embedded in Polybed 812. Thin sections, -80nm, were cut on a diamond knife and double-stained with uranyl acetate and lead citrate. The sections were examined in a TEM at an acceleration voltage of 80 kV. The SEM samples were critical-point dried with C 0 2 . Dried samples were fractured before they were sputter-coated with Au/Pd. The samples were analysed in an SEM using a low acceleration voltage, 1-5 kV.

Image Analysis The average pore size and the average particle size were estimated using a stereological approach. LM images were used to estimate the pore size and TEM images were used to estimate the particle size. The so-called star volume v* was estimated unbiasedly f r ~ m ' ~ , ~ *

where lois the line intercept length and the spatial average is $. The star volume is an estimate of the volume-weighted mean volume, and is in this study used as an estimate of the mean size. The strand characteristics were analysed using an expert panel, consisting of eight microscopists, assessing microstructural descriptors on a scale. This is an analogous method to the descriptive test used for sensory evaluation. l4,I5

R heology Tensile tests were performed by fracturing the samples in an Instron 1122. A constant cross-head speed of 10 mm min-' was used which corresponds to an initial strain rate of c = 8.3 x lop3s-' for a 20 mm long sample.16 Viscoelastic measurements were performed using a Bohlin VOR Rheometer with a Couette-type cup-and-bob measuring system (DIN 53 019). The frequency was varied between 0.001 and 5 Hz, and the strain was kept in the range 4-10 x

3 Results Sensory analysis was used to analyse texture, and the descriptors used for the whey protein gels are listed in Table 1 with their definitions and grouped by

M . Langton, A . Astrom, M . Stading, and A.-M. Hermansson

Table 1

21

Descriptors used for the sensory profiling of whey protein gels

Attribute

Descriptor

Explanation

Manual Texture Soft (touching) measured by a light Springy pressure with forefinger

how much the sample resists light pressure with forefinger how rapidly the sample regains its initial shape after light pressure with the forefinger

Visual Texture (visualization) observations of a newly cut surface

Surface moisture

how much water is released from the newly cut surface

Grainy

how grainy the newly cut surface appears

Oral Texture Gritty (mouthfeel) perception during Creamy the manipulation of the sample in the Sticky mouth Falling apart

how gritty the sample is perceived during chewing how creamy and smooth the sample is perceived during chewing how much the sample adheres to the teeth after chewing how easily the sample falls apart or disintegrates during chewing

attributes. The perception of texture is sensitive to the test principle used, and thus different attributes and descriptors are influenced by different microstructural parameters. When the texture of particulate whey protein gels was evaluated, two main groups of attributes were found. Principal component analysis (PCA) of the textural sensory data identifies the two groups as: (a) grainy appearance, gritty, creamy, and 'falling apart', and (b) soft, springy, and sticky. The attributes ingroup (a) were all perceived using a larger force during the analysis-namely cutting and chewing. In group (b) the attributes soft and springy were negatively correlated and were evaluated by the application of a light pressure with the forefinger. The perception of stickiness (how much the sample adheres to the teeth after gentle chewing) effectively forms a group of its own.

Large Deformations The first group was formed of descriptors dependent on application of a large force during analysis, namely cutting and chewing. This group consisted of four descriptors: grainy appearance, falling apart, gritty and creamy texture. The first three were positively correlated, and the last one (creamy) was negatively correlated to the others. This group was sensitive to the variation in the overall particulate network dimensions, such as pore size and particle size. Figure 1 shows micrographs of samples with (a) the smallest pores and (b) the largest pores. The statistical evaluation using the response surface model and the analysis of variance showed that a faster heating rate, 5 "Umin, and lower pH, 4.6, produced gels with smaller pores and smaller particles. The size of the

22

Figure 1


L M micrographs of 13.5 wt% whey protein gels: (a) with the smallestpores formed at p H 4.6, with a heating rate of 5 "Clmin and no salt addition, and (6) with the largest pores formed at p H 5.4, heating rate 1 "Clmin and 0.I molldm3 NaCl

pores was affected by interaction between the pH and the heating rate. At the lower pH of 4.6, the pore size was independent of heating rate, whereas, at the higher pH of 5.4, the slower heating rate produced a seven times larger value of the star volume of pores. The microstructural parameters are described in detail in a previous paper. l4 Figure 2 shows the logarithmic dependence of the sensory descriptor, falling apart, on the pore size. The smallest pores are around 10pm and the largest

1000 Figure 2

10000 1O( I00 volume of pores in cubic microns

The perception of 'falling apart' as a logarithmic function of the star volume of pores (in ,.urn3)

23

M . Langron, A . Astrom, M . Stading, and A.-M. Hermansson

0.00

0.05

0.10

0.15

0.20

a Figure 3

Stress-strain curve of 12 wt% /3-lactoglobulingelsformed atpH5.3 and with a heating rate of:CI, 12 "Clmin;a, I "Clmin. The parameter a is defined by n = nla, where n is the notch depth and a is the width of the sample

around 40prn, if a spherical pore shape is assumed. The non-linear dependence of the microstructural parameters on the perceived texture is summarized in Table 2. The sample which had a grainy appearance also had a gritty texture; it had a high tendency to fall apart, and it was less creamy. The perception of texture was explained in terms of the microstructure, i.e., these samples had large pores and large particles. Large deformation tests were carried out on particulate 8-lactoglobulin gels formed at pH 5.3 and at different heating rates. Figure 3 shows that the gels formed at the faster heating rate, 12 "C/min, were more difficult to fracture,

Table 2

The non-linear correlation between microstructure and sensory quality as described by Y = A + B*log(X), n = 16

Y

X

grainy grainy gritty gritty falling apart falling apart creamy creamy

star volume of particles star volume of pores star volume of particles star volume of pores star volume of particles star volume of pores star volume of particles star volume of pores

A

B

?

39

18

0.94

-50

9 29 13 19 9 -28 - 12

0.93 0.95 0.91 0.96 0.95 0.91 0.86

43 -85 40 -48 23 139

24

Figure4


L M micrographs of 10 wt% P-lactoglobulingels formedatpH5.3 by heating at (a) 12 "Clmin and (b) I "Clmin

whereas gels formed at the slower heating rate were easier to fracture. Figure 4 shows that the gel formed at the faster heating rate had small pores, whereas larger pores were produced by the slower heating rate. This is in agreement with the results above on whey protein gels, where the perception of texture using a large force during analysis, e.g. chewing, indicated that gels with large pores fell apart more easily.

Small Deformations The second group of descriptors was associated with the application of less force during analysis, i.e., light pressure with the forefinger. This group consisted of two sensory descriptors, soft and springy, which were negatively correlated. The perception of soft and springy was related to the strand characteristics of particulate gels. Figure 5 shows examples of different strand characteristics. Figure 5a shows how particles can align into strings of beads forming a regular network. Particles can also associate together in clusters like bunches of grapes, and the formation of clusters is dependent on processing conditions, pH, heating rate, and salt content. The addition of salt led to a decreased tendency for the particles to form strings of beads. Figure 5b shows an example of cluster formation. Another strand characteristic is the formation of conglomerates, where particles are tightly glued together, as shown in Figure 5c. The interaction effects showed that the heating rate mostly influenced the strand characteristics at the higher pH of 5.4, whereas the addition of salt mostly affected the strand characteristics at the lower pH of 4.6. To fully explain the soft and springy texture in terms of microstructural parameters, a more complex relationship seemed to be needed. A multivariate model, the PLS-model, was established in order to find which variables were correlated to the soft and springy texture. Figure 6 shows a loading plot of the first and second PLS components plotted against each other.

25

M . Langton, A . Rstrom, M . Stading, and A . - M . Hermanssoti

Figure 5

SEM micrographs of 13.5 wt% whey protein gels: ( a ) particles associate iogether forming strands like a string of beads; (b) parricles attach to each other in clusters; (c) particles join togelher in conglomerates

r-

I ! 1

1

1

1I 1 I

O3 4

3*8 2 0.1

0

5*93*4

I

4*8 7*9

9'9

1

Soft

161

2*9 6*6 2*3 _______ 1*31*9 SpriW7 7t7 3*5

__

'6

2*6

4'9

73*6 4 -0'3

1 LY

Figure 6

3'3 6*8

3*1

I

-

J-0

1*8

5*33+(5

-0.2

2*8

1*2 3

1- J

-0.1

I

I

I'*.

0.2

2*2

I

2*5

i

*6

294*4

4*5

Loading plot of thejirst and second components of the PLS-model used for correlating soft and springy texture to the microstructure of whey protein gels. The microstructure parameters are assigned numbers as follows: ( 1 ) mean voiume of large pores OX), (2) mean volume of parricles, (3) proportion of ihreads within the pores, (4) mean volume of small pores ( 4 0 X ) , (5) porosity, (6)clusters, (7)conglomerates, (8)strings of beads, and (9) hairiness

26

Effect of Microstructure on Sensory Perception of Particulate Gel5 lo

T

0.001

Figure 7

0.01

0.1

1

10

Frequency dependence of the storage modulus of 10 wt% B-lactoglobulin gels formed at different heating rates at p H 5.3

The largest variation is portrayed in the first component. It is seen that soft and springy properties are negatively correlated. They are plotted close to the first component axes and opposite each other with respect to origin. The loadings of the variables which are plotted close to soft and springy, respectively, are correlated with these variables. Many microstructural parameters, as well as interactions between microstructural parameters, had an impact on the perceived springiness and softness. The strand characteristics had an impact on the perception of soft and springy texture. The results are described elsewhere. I 4 * l 5 Small-deformation, viscoelastic measurements have been performed'6 on /?-lactoglobulin gels at pH 5.3. Figure 7 shows the storage modulus at varying

Figure 8

SEA4 micrographs of I0 wt% /3-lactoglobulin gels formed at p H 5.3 by heating at (a) I2 "Clmin and ( b ) I 'Clmin

M. Langton, A. Astrom, M . Stading, and A.-M. Hermansson

21

frequency for the gels produced at heating rates of 5 , l and 0.1 "C/min. The gel formed at slower heating rate had a higher storage modulus G'. Figure 8 shows micrographs of 0-lactoglobulin gels formed at (a) 12 W m i n and (b) 1 "Urnin. The gel formed at 1 "C/min had stiff strands, formed of many particles joined together, resulting in a high value of G'. Gels formed of flexible strands (see Figure 8a) had lower values of G'. Thus, the strand characteristics can explain the gel texture as measured instrumentally with small-deformation tests.

4 Discussion We have shown that the test selected for judging the perception of texture influences the results obtained. An important consideration is to define appropriate texture descriptors for the actual samples. It is desirable that the final choice of descriptors reflects the various characteristics of the microstructure. The test principles for analysing texture of particulate protein gels can be divided into two groups: destructive and non-destructive tests. Texture as measured by destructive methods was sensitive to the overall network dimensions, whereas texture measured with non-destructive methods was sensitive to strand characteristics. When making correlations between microstructure and texture, it is vital to analyse the appropriate properties of both the microstructure and the texture. For example, to explain the changes in rheological properties of particulate protein gels, as measured by viscoelastic measurements, it seems to be more appropriate to investigate the strand characteristics and clustering phenomena than to measure the pore size or particle size. The analysis in this work has been performed on particulate gel systems consisting of compact particles and liquid-rich pores. The situation is different in mixed gel systems, where, for example, a second polymer may be located within the pores of the first polymer, forming a bicontinuous system. In mixtures, the density of the particles as well as the density of the pores can vary more gradually, which will have an impact on the textural characteristics. The perception of a gritty texture is likely to depend on the differences in the density as well as in the particle size. This work has presented information on the relationship between the microstructural and textural characteristics of particulate protein gels. The combination of microscopy, sensory analysis, rheological properties, factorial experimental design and statistical evaluation methods can be used in many applications. Thus, the effects of different process variables and ingredients can be evaluated. This can be achieved by combining quantified microstructural data and textural data into models which enable prediction of textural behaviour. The knowledge about the relationship between microstructure and texture should lead to optimized food production processes as well as to the development of new products with desirable properties.

28


References 1. M. Kalab, P. Allan-Wojtas, and B. E. Phills-Todd, Food Microstruct., 1983,2,51. 2. A. M. Hermansson, in ‘Functional Properties of Food Macromolecules’, ed. J . R. Mitchell and D. A. Ledward, Elsevier Applied Science, London, 1986, p. 273. 3. J . M. Aguilera and S. W. Stanley, ‘Microstructural Principles of Food Processing and Engineering’, Elsevier Applied Science, London, 1990. 4. M. Langton, ‘The microstructure of yoghurt-a literature review’, SIK Report No. 580, Swedish Institute of Food Research, Gvteborg, Sweden, 1991. 5. V. R. Hanvalker and M. Kalab, Milchwissenschaft, 1985,40, 65. 6. V. R. Harwalker and M. KalAb, Milchwissenschuft, 1985,40,665. 7. D. M. Mulvihill and J . E. Kinsella, J . Food Sci., 1988, 53, 231. 8. D. M. Mulvihill, D. Rector, and J . E. Kinsella, Food Hydrocolloids, 1990,4,267. 9. M . Langton and A.-M. Hermansson, Food Hydrocolloids, 1992,5,523. 10. D. Renard and J. Lefebvre, Int. J . Biol. Macromol., 1992,14,287. 11. M. Kalhb, A. G. Sargent, and D . A. Froechlich, Scanning Elecrroti Microscopy 111, 1981, p. 473,514. 12. R. J . Marshall, Food Qual. Pref., 1990, 2, 117. 13. K . L. Crippen, D. D. Hamann, and C. T. Young, 1. Text. Stud., 1989, 20. 29. 14. M. Langton and A.-M. Hermansson, Food Hydrocolloids, 1996, 10, 179. 15. M. Langton, A . Wstrom, and A.-M. Hermansson, Food Hydrocolloids, 1996, in press. 16.. M. Stading, M. Langton, and A.-M. Hermansson, Food Hydrocolloids, 1993, 7, 195. 17. M. Langton, ‘Correlating microstructure with texture of particulate biopolymcr gels’, PhD Thesis, Chalmcrs University of Technology, Goteborg, Sweden, 1995. 18. G . E. P. Box, W . G. Hunter, and J . S. Hunter, ‘Statistics for Experimenters: An Introduction to Design, Data Analysis, and Model Building’, Wiley, New York, 1978. 19, R. Carlson, ‘Design and Optimization in Organic Synthesis’, Elsevier, Amsterdam, 1992. 20. H. Stone and J . Sidel, ‘Sensory Evaluation Practice’, Academic Press, San Diego, CA, 1985. 21. A.-M. Hermansson and M. Langton, in ‘Physical Techniques for the Study of Food Biopolymers’, ed. S. B. Ross-Murphy, Blackie, Glasgow, p. 277. 22. H. J . G. Gundersen, T. F. Bendtsen, L. Korbo, N. Marcussen, A. Moiler, K . Niclsen, J . R. Nyengaard, B. Pakkenberg, F. B. S~rensen,A . Vesterby, and M. J . West, A P M I S , 1988,96, 379.

Microstructure as Related to the Waterholding, Textural and Sensory Properties of Emulsion Sausages By Kerstin Andersson, Annika Andersson, and Eva Tornberg SWEDISH MEAT RESEARCH INSTITUTE, POB 504, S-244 24 KAVLINGE. SWEDEN

1 Introduction The properties of emulsion sausages are dependent upon the formation of a meat protein matrix, a gel, which gives the product its characteristic sensory properties. Moreover, the texture and water- and fat-holding of the product (the functional properties) are mainly governed by the properties of this protein network. As illustrated in Figure 1 , the quality of the meat protein gel is influenced by a number of factors interacting in a complicated way and giving rise to a complex system that is not so amenable to study using a fundamental approach. The amount of myofibrillar proteins extracted into the aqueous phase during comminution is generally considered to contribute most to the functionality.'-' The amount of myofibrillar proteins extracted from the muscle, i.e. myosin and actin, depends upon the degree of comminution, the type of muscle, the salt content, the temperature and the pH."" Moreover, the type of matrix formed is related to the dispersed or aggregated state of the proteins prior to denaturation. The solubility, which is a measure of the dispersed-aggregated state of the extracted proteins and of those already available in the water-phase (the sarcoplasmic proteins), is also markedly affected by environmental conditions such as pH,2,12,13 temperature and ionic ~trength.'~'''Minimum solubility is usually observed in the isoelectric regions, i.e. pH -5 for aetomyosin,'6 and as the pH approaches the isoelectric point, the microstructure of the gels becomes more particulate in character." It has been ~ b s e r v e d that ' ~ myosin can form two completely different gel structures depending on the ionic strength. Fine stranded gels are formed at low ionic strength (0.25 M) and coarsely aggregated structures at high ionic strength (0.6 M). The complex meat system consists not only of the solubilized meat proteins but also of insoluble compounds, such as meat fibres, connective tissue and fat (Figure 7).

'*

29

30

Water-holding, Textural and Sensory Properties of Emulsion Sausages ionic strcngth

L' Solubility

n

c-l

temperature

4

Heating conditions

%

c

Amount and form of insoluble compounds

Protein conformation and degree of aggregation

. I

I

Amount and type of protein extracted into the aqueous phase

wpe

meat fat cells of fiber ionic strength, fiben connective and PH, typc of salt tissue amoukkt fat droplets tfmperature type of disin tegration

Figure 1

Factors affecting protein gelation in a sausuge halier

The fat in emulsion sausages is mostly squeezed out of the cells and exists in the form of small droplets and/or larger fat p00ls.'~The components of the connective tissue have also become disintegrated to a certain degree, but are not soluble, either in water or at high salt content. The amount and form of these compounds has an impact on the gelation properties. The properties of the gel are also influenced by the heating conditions. Many studies have focused on the effects of different factors such as salt concentration, temperature or pH on the gelling behaviour of purified systems, such as myosin solutions from different species. 14~19,20The texture of real meat but few researchers have elucidated products has also been in~estigated,'~-'~ the more fundamental and microstructural causes of the variation in the functional properties observed. Therefore, in this study, an attempt has been made to relate the microstructure, as revealed by light micrographs and viscoelastic measurements, of a real emulsion sausage system, to its waterholding, textural (tensile measurements) and sensory properties. The emulsion sausages were produced in such a way that a large range of different types of meat protein networks was generated. Because of the complexity of the system, as illustrated in Figure 1, a large number of measurements should be performed to characterize the microstructure of the gel matrix. However, in this study, we have confined ourselves to characterizing the variation in fat

K . Andersson, A . Andersson, and E. Tornberg

31

particle size, reflecting the degree of disintegration, and observing whether substantial protein aggregation has occurred or not (visual evaluation of light micrographs).

2 Materials and Methods Materials The sausage batters (n = 27) were prepared from pork or beef meat, rindless pork fat, nitrite salt and water. In order to generate different textures in the heated sausage batters, these were made using different recipes composed of different contents of water, fat and protein (61-79 wt%, 10-25 wt% and 8-13 wt% , respectively). The connective tissue and the salt content of the sausage batters was also varied in the ranges 0.7-3.0 wt% and 1.5-2.5'/0, respectively, as well as the p H (5.5-5.8).The sausage batters were made in a 20 I Muller bowl chopper with 6 knives at a speed of 1400/2800 r.p.m. The batch size was 7 kg. The ingredients were added and disintegrated at low speed in the chopper for fixed times in the following order: meat (20 s), nitrite salt (10s), water/ice (60s) and fat (20 s). The speed of the chopper was increased and comminution was continued for 130 s to a final temperature of 8-12 "C.The batter was stuffed into 47 mm casings using a sausage filling machine (Vemag Robot 500, type 128). The sausages were heat-treated, in accordance with the following procedure: 40 minutes at 60 "C (60% RH), 10 minutes drying at 70 "C (

n.s.

p

5

j; *I

0.001

i) i)

p 5 0.1

p 5 0.01

n.s. n.s.

37

K . Andersson, A . Andersson, and E. Tornberg

4 Discussion Comparison of Effect of Disintegration and State of Protein Aggregation on Properties of the Meat Protein Matrix In order to estimate both the effect of the size of the fat particles (degree of disintegration) and the state of aggregation of the proteins in the protein network, statistical analysis was performed on the different responses in accordance with the model:

where dALwas a continuous variable and agg$ a categorial variable (1 = non-aggregated, 2 = aggregated). Figure 6 illustrates the non-linear relationship between water-losses and dAL for the two types of sausages. The squared multiple regression coefficient for the model ( r 2 ) is 83% and there is also a significant difference between the aggregated and the non-aggregated sausages. Optimum water-holding was obtained at a fat particle size of about 35 p n , while larger and smaller particle sizes give rise to higher water-losses. This suggests that there is an optimum degree of comminution, since the fat particle size reflects the degree of disintegration of the batter as a whole. The larger water-losses at higher values of dAL(i.e. a lower degree of disintegration) can be duc to less protein bcing extracted in thc aqueous phase, which mainly affects the gel-forming ability (Figure 1) and, hence, the water-holding capacity. The increased water-losses, when the fat particle size is below 35 ,urn, are more difficult to comprehend. This might be due to interactions between the 50

,

I

45 40

t

- 0

n 35

s 30 v

5 i 0

L---i,

lv-r----T--i

20

30

40

dA,. Figure 6

50

60

70

(am)

Water-losses (Yo), as a function of average fat particle size c 1 for ~ ~ ~ non-aggregated and aggregated sausage samples

38

Water-holding, Textural and Sensory Properties of Emulsion Sausages I

8

0

.

Don-rggrqated

0

0

I

small fat droplets and the proteins during the gel-forming process, hindering the gelling process and thereby giving rise to a less coherent structure. In Figure 7, the sensorially evaluated hardness is linearly related to dA,>and there is a significant difference between the aggregated and the non-aggregated sausages. The hardness of the non-aggregated sausages decreases with the size of the fat particles, in contrast to the aggregated ones, which seem to be independent of dAL.This negative linear relationship ( r = -0.68**) (i.e. p 5 0.01) for the non-aggregated gels is most probably due to an increased amount of protein being extracted, the higher the level of disintegration (lower value of dAL).However, higher amounts of protein being extracted only improves the gel-forming ability of the non-aggregated sausages, whereas for the aggregated samples, further protein extraction does not contribute to a better gel and increased hardness. Further studies are needed to confirm these results. Similar relationships are observed for fracture strain, as determined by the tensile strength measurements, and dAL (Figure 8). The fracture strain decreases with increasing fat particle size for the non-aggregated gels ( r = -0.83***) ( i . e . p 5 0.001) and a significantly lower fracture strain was obtained for the aggregated gels. The degree of disintegration, as measured by the fat particle size, did not, however, have any impact on the viscoelastic properties, which was the case for the degree of aggregation in relation to thc phase angle (Table 1).

Sensory Properties in Relation to the Properties of the Meat Protein Matrix In order to determine how the different sensory attributes were related, a principal component analysis (PCA) was performed. The first two principal

39

K . Andersson, A . Andersson, and E. Tornberg 60 I

0

\I

I

0

OlD

0

0

I

O

0

/

O

I 0 1

I ,r A

20 25 30 35 40 45 50 55 60 65

d,, Figure 8

(rm)

Fracture strain (YO)obtained using tensile measurements, as a function of average fat particle size dAL for non-aggregated and aggregated sausage samples

components obtained describe 91% of the variation in the sensory attributes between the sausage samples. The loading plot, in Figure 9, for the first two principal components, shows how they are interrelated. The first PC describes 60% of the variation and is represented by sausages with a high overall acceptability and high scores in juiciness, flavour and texture acceptability. The second PC describes 30% of the variation and is

1.o

3 0.5

4

v

c4 0.0

Flavour

-

%I 3

4

-0.5

-1.0

-0.5

0.0

0.5

1 1.o

Loadings,PCl (60%) Figure 9

Principal component analysis ( P C A ) of the sensory data of sausages. The plot sho ws PCA loadings for principal components I and 2 ( P e l and PC2)

40

Water-holding, Textural and Sensory Properties of Emulsion Sausages

related to sausages with great rubberiness and hardness. Since overall acceptability and rubberiness have high loadings in PC1 and PC2, respectively, and they describe over 90% of the variation between the sausages, these two attributes have been analysed in more detail. The results of the statistical analysis reveals that overall acceptability is best described by the results from the tensile strength measurements. A linear relationship ( r = 0.74***) between overall acceptability and fracture strain was obtained. As the fracture strain was increased (Figure lo), i.e. as the sausage became more elastic, the sausages became more acceptable to the tasting panel. The aggregated sausages gave rise to a low fracture strain (Figure 10) and, hence, to a low overall acceptability. As seen in Figure 7, the degree of comminution is also important, since the fracture strain ( i . e . overall acceptability) increased with degree of disintegration (in any case for the non-aggregated samples), as indicated by the smaller fat particle size. This implies that optimal sensory quality can be expected from sausages with a high fracture strain (high elasticity), which is in accordance with the results found in the literature.2R73" Rubberiness. Rubberiness was best described by the phase angle, as determined by the viscoelastic measurements. The linear relationship ( r = -0.78* * *), with the aggregated and the non-aggregated sausages marked by different symbols, is shown in Figure 11.Hardness, found in the same principal component as rubberiness, also showed a good correlation with the phase angle ( r = -0.85***). As the figure indicates, the phase angle was higher for the aggregated sausages than for the non-aggregated ones, but the phase angle was not sensitive to differences in the degree of comminution.

10

20

30

40

50

60

Fracture strain (YO) Figure 10

OveruN acceptability as a function of fracture strain (tensile measurements) for aggregated and non-aggregated sausages

41

K. Andersson, A . Andersson, and E. Tornherg

7 0

6 -

0

non-aggregated

0

7-

7

8

9

10

11

12

13

Phase angle Figure 11

Ruhheriness as a function of the phase angle d (degrees), obtained from viscariastic measurements of aggregated and non-aggregated sausages

5 Conclusions The influence of two factors governing the gelling properties of an emulsion sausage, i.e. the amount of disintegration and the type of protein network (aggregated or non-aggregated), was investigated. The rheological propertics of thc different sausages, measured at small deformations (viscoelastic measurements) and large deformations (tensile strength measurements). were monitored, in addition to the water-holding and sensory properties. The sensory variation was dcscribed by two main attributes, overall acceptability and rubberiness (hardness). The results of the two different rheological methods used reflected varying sensory attributes. Overall acceptability was best correlated to fracture strain, as obtained by the tensile strength measurements, where a large fracturc strain corresponded to a high ovcrall acceptability. This sensory attribute was influenced by both the degree of comminution and the protcin aggregation. Rubberiness was best correlated to thc phase angle, as obtained by the viscoelastic measurements. Water-holding on cooking was influenced by both the aggregation of the proteins and the degree of comminution. For the latter, a non-linear relationship was obtaincd with regard to water-holding, indicating an optimum in this property with thc degree of comminution.

References 1. J . C . Acton. G. R. Ziegler, and D. L. Burge, Jr.. CRC Crii. Rev. Food Sci. Nurr.. 1983, 18,99. 2. S. F. Wang and D. M . Smith, Food Sfruct., 1973. 11.273. 3. A . Gordon and S . Barbut, FoodStrurr.. 1990, 9 , 77.

42

Water-holding, Textural and Sensory Properties of Emulsion Sausages

4. D. L. Daum-Thunbcrg, E. A. Foegeding, and J. H. R. Ball, J . Food Sci., 1992,57, 333. 5 . K. Samejima, Nam H. Lee, and M. Ishioroshi, J . Sci. Food Agric., 1992,58,385. 6. G. H. Robe and Y. L. Xiong, Food Hydrocolloids, 1993,7, 137. 7. P. B. Kenney and M. C. Hunt, Meat Sci., 1990,27,173. 8. R. I. Richardson and J. M. Jones, Int. J . Food Sci. Technol., 1987,22,683. 9. C. Meyer and B. Egelandsdal, J . Texture Stud., 1992,23, 25. 10. N . Parsons and P. Knight, J . Sci. Food Agric., 1990,51,71. 11. D. Stanley, A. P. Stone, and H. Hultin, J . Agric. Food Chem., 1994,42, 863. 12. E. A. Foegeding, E. L. Bowland, and C. C. Hardin, Food Hydrocolloids, 1995,9, 237. 13. Y. H. Lan, J. E. Novalovski, R . H. McCusker, M. S. Brewer, T. R. Carr, and F. K. McKeith, J . Muscle Foods, 1995,6,403. 14. A. M. Hermansson, 0. Harbitz, and M. Langton, J . Sci. FoodAgric., 1986,37,69. 15. Y. L. Xiong, CRC Crit. Rev. Food Sci. Nutr., 1994,34,293. 16. P. A. Morrissey, D. M. Mulvihill, and E. M. O’Neill, in ‘Developments in Food Proteins-5’, ed. B. J. F. Hudson, Elsevier Applied Science, London, 1987, p. 195. 17. E. Tornberg, A. Olsson, and K. Persson, Proceedings from the 35th International Congress of Meat Science and Technology, Copenhagen, Denmark, 1989, p. 752. 18. Y. L. Xiong and S. P. Blanchard, J . Food Sci., 1994,59,734. 19. M. Ishioroshi, K. Samejima, and T. Yasui, J . Food Sci., 1979,44,1280. 20. B. Egelandsdal, K. Fretheim and K. Samejima, J . Sci. Food Agric., 1986,37, 915. 21. C. L. Lavelle and E. A. Foegeding, J . Food Sci., 1993,58,727,760. 22. S . Barbut and G. S. Mittal, Meat Sci., 1989,26, 177. 23. S. Barbut and G. S. Mittal, J . Food Sci., 1988, 53,1296, 1311. 24. A. M. Hermansson and Luciano, J . Food Sci., 1981,47, 1955. 25. K. R. Langley, D. Millard, and E. Evans, J . Diary Res., 1986,53,285. 26. R. McClung Jones, ‘Basic Microscopic Techniques’, University of Chicago Press, Chicago, 1966. 27. A. Olsson and E. Tornberg, Food Struct., 1991, 10,333. 28. T. C. Lanier, ‘Rheology of Food Biopolymer Gels: A Practical Approach’, Course at North Carolina State University, NC, 1995. 29. D. D. Hamann, Food Technol., 1988,42(6), 66. 30. J. G. Montejano, D. D. Hamann, andT. C. Lanier, J . Texturestud., 1985,16,403.

Association of Emulsifiers in Aqueous Systems By Niels Krog DANISCO INGREDIENTS, EDWIN RAHRS VEJ 38, DK-8220 BRABRAND, DENMARK

1 Introduction Liquid crystalline lipid-water mesophases such as soap preparations have been known for more than 2000 years. However, the structure of soapwater phases were first analysed in depth in 1960 by X-ray diffraction methods. Amongst the first publications on lipid-water systems relating to food systems, the two most important contributions are the discovery of the swelling properties of lecithin in water by Bangham et a1.* and the descriptions of the mesomorphic behaviour of monoglycerides in water by Lutton3 and L a r ~ s o nComprehen.~ sive reviews on the association of polar lipids in water have been published by Tiddy,’ Small,6 and Larsson.’ Therefore, in the following, only a brief description of lyotropic mesophases of polar lipids in water will be given in relation to their applications in foods.

2 Swelling Properties of Lipids in Water The interaction between lipid and water depends on the balance between the hydrophilic and hydrophobic parts of the lipid molecule. According to Small: lipids can be classified according to their interaction with water as shown in Table 1. Aliphatic hydrocarbons (for example paraffin oil) are called non-polar lipids. Non-polar lipids do not interact with water in any way. Polar lipids are divided into three classes according to their ability to interact with water. Diand triglycerides, together with fat-soluble vitamins, belong to class I (insoluble, non-swelling lipids). These lipids spread to form monolayers on water, but they do not swell or form mesophases in water. Monoglycerides, phospholipids and glycolipids are characterized as class I1 (insoluble, swelling lipids). They swell in water to form liquid crystalline mesophases. Very hydrophilic lipids, such as lysolecithins,soaps and detergents, belong to class IIIA (soluble, swelling lipids with lyotropic mesomorphism). These lipids form micelles in water above the critical micelle concentration. Another category of biological 45

46

Association of Emulsifiers in Aqueous Systems

Table 1

Lipid classiJicationin terms of monolayer formation, solubility and liquid crystal type ~

~

Solubility in water

Classification

Type of lipid

Monolayer formation

1. Hydrocarbons,

None

Insoluble

Non-polar

Stable Monolayers

Insoluble

sterol esters, waxes 2. Triglycerides, diglycerides, acetoglycerides

3. Monoglycerides, Stable phospholipids, Monolayers glycolipids, galactolipids 4. Lysolecithins,

Unstable polysorbates, Monolayers detergents, soaps

5. Bile salts,

saponins, etc.

Unstable Monolayers

Polar; Class I: Insoluble, non-swelling amphiphilic lipids Insoluble, swells to Class 11: Insoluble, swelling amphiphilic form lyotropic, lipids liquid crystals Soluble, forms micelles above CMC, and liquid crystals at low water conc. Soluble, forms micelles but no liquid crystals

Class 111, A: Soluble lipids with lyotropic mesomorphism

Class 111, B: Soluble lipids with no lyotropic mesomorphism

Based on ref. 6 .

lipids (for example bile salts) is class IIIB: (soluble lipids which form micelles but without lyotropic mesomorphism). Lipids belonging to classes I and I1 are mainly used in food systems, whereas lipids of class IIIA are less common. Most food emulsifier types belong to the polar lipid class 11, and only a few emulsifiers (for example, polysorbate) belong to class IIIA. A characteristic feature of polar lipids is that they are ordered in the crystalline state with the polar head groups in layers separated by the layers of non-polar hydrocarbon chains. Depending on the size of the polar head group, the fatty acid chains may be ordered in bilayers (e.g., monoglycerides) as shown in Figure l(a), or in a single chain length structure with penetrating fatty acids ( e . g . , organic acid esters of monoglyceride, stearoyl-lactylates, etc.). When melting a polar lipid of class I1 or 111, the hydrocarbon chain region turns liquid, but the ordered structure of the polar head groups remains. Consequently, polar lipids have a bilayer structure in the melt, and this property contributes to the increased viscosity in a melt of an emulsifier compared to that of a triglyceride. Class IIIA polar lipids may show different structures at elevated temperatures. This is referred to as thermotropic mesomorphism.

47

N . Krog

Surfactant: Water interactions

Crystal

Figure 1

T>Tc

Lamellar Mesophase

T < Tc

* Gel Phase

Schematic models of (a) crystalline structure of emulsifiers, (b)formation of a lamellar mesophase in presence of water above the Krafft point (TJ, and ( c ) the formation of an a-gel phase on cooling below T,. The structure parameters d, d,, d, and S can be measured by low-angle X-ray diffraction. (From ref. 12, courtesy of Marcel Dekker, New York)

Formation of Lyotropic Mesophases When emulsifiers are mixed with water, the high affinity of the polar head groups for water, referred to as a hydration force,' is the driving force causing the emulsifier bilayers to swell. This results in the formation of a mesophase with layers of water separated by layers of hydrocarbon chains. Such structures have a long-range order, but there is no short-range order present as in nonaqueous crystals of the same emulsifiers. The relative hydration force for different amphiphilic lipids varies, and it is considerably weaker for monoglycerides than for phospholipids.' The transition of the hydrocarbon chains from the crystalline state to a liquid-like state takes place at a temperature called the Krafft point. This temperature is usually identical to the melting point of the a-crystal form of the emulsifier in bulk. With polymorphic emulsifiers ( e . g . , monoglycerides), the Krafft point can be 15-20 "C below the bulk melting point, whereas in a-stable emulsifiers (e.g., organic acid esters of monoglycerides), the Krafft point is identical to the bulk melting point.

Structure of Liquid-Crystalline Mesophases There are three types of liquid crystalline structures: lamellar, cubic and hexagonal. The lamellar phase consists of lipid bilayers alternating with water layers, as shown in Figure l(b). A minimum of swelling is observed with non-

48

Table 2


Structural parameters of liquid crystalline phases of emulsifier + water systems. (See text for definitions of symbols.) Structural parameters

Composition

Structure

Temperature (“C)

dlA

d,/A d w / i SlA2

65 60 60 25 25 60 23 25

50.5 54.1 154.1 64.1 230.1 58.5 60.6 73.4

35.3 15.2 32.7 38.1 16.0 32.0 38.1 116.0 32.0 54.0 10.1 22.0 54.9 175.2 22.0 20.8 37.7 38.6 31.0 29.6 68.5 59.7 13.7 145

Pure monopalmitin/water (70:30)

lamellar

DGMSa/water(25:75) pH 5.0 DGMSa/water(25:75) pH 7.0 DGMSa/water(25:75) pH 5.0 DGMS”/water (25:75) pH 7.0 SSL/water (62:38) pH 5.0 Egg lecithin (62:38) Polysorbate 80 (60:40)

lamellar lamellar a-gel a-gel hex. I1

a

lamellar

hex. I

DGMS = distilled, saturated monoglycerides (C,dC,,).

ionic, polar lipids (e.g., pure glycerol monopalmitate). In such systems the thickness of the water layers is 16-20 At this distance between the lipid bilayers, the hydration force separating them is in balance with the van der Waals attraction forces between the bilayers. When the monoglycerides contain small amounts of ionic active emulsifiers (e.g.,0.5% sodium stearate), increased swelling can be obtained due to the electric repulsion forces between the bilayers. Water layers of thickness up to 230 8, as shown in Table 2 are formed.” However, such lamellar phases can only be made in distilled water with a very low ionic concentration. If the electrolyte concentration is moderately high, the repulsion forces are shielded, and the swelling is reduced. The swelling capacity of lamellar phases is often limited to water layer thicknesses in the range 20-50 A. The hexagonal phase may exist in two modifications. The hexagonal I phase consists of cylindrical emulsifier aggregates with the polar head groups in contact with the water and with the hydrocarbon chains forming the core of the aggregates. This mesophase can be diluted with water, forming micelles. It is a typical mesophase of high-polar emulsifiers (e.g.,polysorbates). The second hexagonal phase has a reversed structure with a hexagonal array of water cylinders in a continuous lipid phase, where the polar head groups are in contact with the water phase. This type of phase is very common with class I and class I1 mixed polar lipids (e.g.,mono- and diglycerides). The cubic phase is a bi-continuous system of curved bilayers separating two channel systems of water. It is usually formed by unsaturated, polar lipids (e.g., monoolein) at ambient temperature, or by other lipids at elevated temperature. The complicated structure of the cubic phase is described in detail by ~arsson.~ Mesomorphic phases can be characterized by their optical texture under a polarizing microscope, and structural data can be obtained by low-angle X-ray diffraction analysis. From such data, information about the thickncss of the

N . Krog

49

water layer, d,, dimension of the lipid bilayer, d,, and the specific surface per molecule in contact with water, S, can be calculated. As an example, structural data of mesophases of various emulsifiers and a-gel phases of monoglycerides in water are shown in Table 2, where d = d, d,. The swelling properties of DGMS:water at p H 5.0 is very similar to that of pure monopa1mitin:water. Separation of the excess water therefore takes place. The bilayer thickness (d,) of the hexagonal TI phase is lower than that of the lamellar phase due to penetrating hydrocarbon chains. The high value of d, found for the hexagonal I phase is probably due to impurities of the commercial product solubilized in the cylindrical lipid aggregates. The differences in values of the surface area S of hexagonal IT and hexagonal I reflect the differing size of the polar head groups of the emulsifiers. If the value of S for egg lecithin is calculated per fatty acid chain, it turns out to be close to that of the value for monoglycerides. Structural data on lamellar phases with varying water content can give information relating to interfacial monolayer conformations in emulsions. I ’

+

Phase Diagrams of Emulsifier-Water Systems The phase behaviour of food-grade emulsifiers in water has been described by Krog” and Larsson . I 3 The binary phase diagrams of emulsifier-water systems are valuable for predicting the concentration and temperature conditions for making aqueous emulsifier dispersions for use in food manufacturing. In this connection a knowledge of mesomorphic behaviour is very important. Phase diagrams of typical emulsifiers + water systems are shown in Figure 2. The phase behaviour of an emulsifier in water depends on its chemical composition. High purity is important for some types of behaviour. For example, distilled monoglycerides with a 1-monoester content of ca. 95% form well-defined mesophases in water, whereas mixtures of mono-, di- and triglycerides do not form such mesophases when the triglyceride content is above 20%. Emulsifiers with a complex composition regarding ester distribution may not form any well-defined mesophascs, although they may function optimally as emulsifiers in emulsions due to their partial adsorption at interfaces. The relative size of the polar head group, together with the chain length and the degree of unsaturation of the fatty acid hydrocarbon chains, are other important factors determining the lyotropic mesomorphism of emulsifiers. The effect of the sizc and nature of thc polar head group is illustrated in the phase diagrams in Figures 2(a) and 2(c), which refer to distillcd monoglycerides of palmitic and stearic fatty acids (DGMS) and phosphatidylcholine (i.e., lecithin), respectively. Both lipids are class I1 swelling amphiphilic lipids. The monoglycerides have a relatively small polar group (the glycerol moiety with two free -OH groups). Consequently, the lamellar region in the phase diagram of Figurc 2(a) is small, and it only exists when the purity of the monoglycerides is high. Addition of less than 1% triglyceride to the lamellar phase causcs a phase change to a two-phase system of a cubic phase + water. Below the Krafft point, the lamellar phase of DGMS + water transforms to a metastable a-gel (see Figure l(c)), and with time a suspension of P-crystals in water is formed.

50


Water, % 0

10

20

30

40

50

90

Water, %

(d ) 100,

1

\

100 90

Crystals

Lamellar Water, %

Figure 2

Water, %

Binary phase diagrams of (a) distilled saturated monoglycerides ( D I M O D A e P, Danisco In redients), ( b ) dktilled unsaturated monoglycerides (DIMODA LS, Dankco Ingredients), c egg lecithin, and (d) sodium stearoyl lactylate (GRINDSTEDTL )sSL P 55). Abbreviations: T, = Krafft temperature; T, = transition temperature f o r B-crystals in water to a-gel; G = gyroidal cubic structure. (Reproduced from ref. 12, courtesy of Marcel Dekker, New York, and from ref. 6 , courtesy of J . Lipid Res.)

&

Phosphatidylcholine has a large, ion-active polar head-group, and the phase diagram of phosphatidylcholine water in Figure 2(c) shows a remarkably large lamellar region with high thermal stability. This phase can solubilize up to 3% triglycerides (triolein) without any phase change taking place. At high water contents, phosphatidylcholine forms liposomes or vesicles. These two lipids, monoglycerides. and lecithin, are commonly used in foods. However, although they both form lamellar phases, their functional properties are quite different. As an example of an anion-active food emulsifier, Figure 2(d) shows a phase diagram of sodium stearoyl lactylate (SSL) in water at pH 7. Under such

+

1M)

N . Krog

51

conditions SSL forms lamellar phases at temperatures between 45 "C and 90 "C and with a water content from 20 to 60%. A t higher water contents SSL forms a dispersion of vesicles. When SSL is dispersed in water without adjusting the pH, a reversed hexagonal phase is usually formed. This is because commercial SSL is not completely neutralized, but contains a certain amount of free fatty acids as specified by food additive regulation bodies. When SSL and DGMS are combined (l:l,w/w) vesicles form. Such vesicles are relatively stable in food systems, where they can perform various functions, such as aerating or texturizing low-fat foods. Phase diagrams of a few other food emulsifiers, such as polyglycerol esters of stearic acid and diacetyl tartaric acid esters of monoglycerides, are also available in the literature.

3 Application of Lyotropic Mesophases in Foods Nearly three decades have passed since the first phase diagrams of monoglyceride + water systems were published.374A review of the phase behaviour of several food emulsifiers in water was published in 1976.14Our knowledge about the mesomorphic behaviour of emulsifiers in water has mainly been used to optimize the functional properties of emulsifiers in various food systems, e.g., using aqueous dispersions or 'hydrates' in starch-based foods for optimal interactions with starch components, or making stable a-gels for use in aeration of low-fat cakes, etc. The concept of liquid crystals as a stabilizing factor in food emulsions has been described" for systems containing lecithins or polysorbate mixtures. However, this concept has no relevance to food emulsions containing proteins. When adding an emulsifier-water mesophase to a food formulation, it is important to realize that phase changes may take place due to the effect of the food ingredients (fats, oils, proteins, salts, etc.) on the mesophase, as well as the effect of temperature during pasteurization, sterilization or freezing of foods. Regarding saturated monoglycerides, the lamellar phase can practically only exist in pure water systems, while hydration of monoglycerides in a complex food system containing fats or oils results in the formation of cubic or reversed hexagonal phases. Phospholipids will hydrate in the presence of water, forming lamellar phases spontaneously in the temperature range from ca. 0 "C to over 200 "C(which is well above what is used in food processing). Phospholipids from, for example, egg yolk form lamellar phases with high stability, even in presence of fats and oils. These phases can stabilize emulsions by liquid crystalline structures adsorbed at the oil-water interface, as shown in the light micrograph of Figure 3. The liquid crystalline phases appear as strongly birefringent layers at the surface of the oil droplets. Electron microscopy using the freeze-fracture technique is also a powerful tool to identify liquid crystals or a-gel phases in foods. When trying to produce liquid crystalline phases in foods, it is important to realize that a mesophase of an emulsifier + water system made according to the binary phase diagram will most likely change when mixed with other .food

51

Figure 3

Association of Emulsijiers in Aqueous Systems

Liquid crystalline structures around oil droplets in an emulsion of 10 wi% soybean oil in water with 3 wt% egg lecithin as emulsifier. Polarized light microscopy; bar = 50 pm. (Couriesy of Marcel Dekker, New York)

ingredients. This is particularly the case with monoglyceride + water mesophases, whereas mesophases made by combining monoglycerides with phospholipids or anion-active emulsifiers (such as SSL) may coexist with other food ingredients in a separate phase. The patent literature gives examples of such applications. The concentration of added emulsifiers in foods is usually too low to result in any formation of liquid crystalline phases during food processing, and in most cases this is not needed either. Some liquid crystalline phases may be formed, however, during processing by the polar lipids present in the food ingredients. This is the case with bakery products, such as bread, rolls and buns, based on wheat flour which contains polar lipids such as glycolipids and phospholipids. These lipids associate with water into liquid crystalline phases during dough mixing together with any added emulsifier used as a dough-strengthener.16 Another type of example is mayonnaise and salad dressings, where the phospholipids from egg yolk can form liquid crystalline structures that may cxist in the end product."

''

Emulsifier-Water Gel Phases in Aerated Foods There are two types of u-gel phases used in foods. Commercially manufactured a-gcl phases are available for use as aerating agents in low-fat cakes, etc.

N . Krog

53

Another form of a-gel is formed in situ by low-polarity emulsifiers in whippable emulsions (e.g., creams, toppings, etc.). The types of emulsifiers used here are acetic or lactic acid esters of monoglycerides or propylene glycol monostearate. They are usually called a-tending emulsifiers. When such emulsifiers come into contact with water below their melting point, they will tend to absorb water in order to hydrate the polar groups. The crystals then swell until there is a balance between the hydration force and the van der Waals attraction forces between the lipid bilayers. This usually occurs when the water layer thickness is ca. 10 A. These emulsifiers do not form mesophases. Consequently, when the gel-phase is heated to above the melting point of the emulsifier, an emulsion of the melted emulsifier in water is formed. This gel formation can take place in emulsions at the fat globule surface, promoting destabilization and aggregation of the fat particles, and thus enhancing aeration properties.” Unsaturatcd, distilled monoglycerides (mono-oleates) show properties similar to those of a-tending emulsifiers in topping fats. Unlike the a-tending emulsifiers, mono-oleates are also very effective in ice cream emulsions.

’’

4 Conclusions Knowledge about the mesomorphic behaviour of emulsifiers in water can give valuable information about their properties and conformation at emulsion interfaces. The formation of lyotropic mesophases in food systems is not a common feature, partly due to the low concentration of added emulsifiers, and partly due to lack of stability of the emulsifier mesophases during food processing. The mesomorphic properties of emulsifiers are mainly used to optimize functionality in food systems, where aeration or interactions with water-soluble ingredients (e.g., starch components) are important. In special formulations for aerated desserts, emulsifiers function through a-gel formation directly in water or within a fat phase. In recent years, low-fat food products containing such emulsifier mesophases as structuring agents have been introduced to consumers.

References V. Luzzati, H. Mustacchi, A. Skoulios, and F. Husson, Actu Cryst., 1960,13,660. A. D. Bangham, M. M. Standish, and J. C. Watkins, J Mol. B i d . , 1965, 13,238. E. S. Lutton, J. Amer. Chem. Soc., 1965,42, 1068. K. Larsson, Zeit. Phys. Chem. Neue Folge, 1967,56, 173. G. J . Tiddy, Phys. Rep. 1980,58,1. D. M. Small, ‘Thc Physical Chemistry of Lipids’, Plenum, New York, 1986, p. 475. K. Larsson, ‘Lipids-Molccular Organization, Physical Functions and Tcchnical Applications’, The Oily Press, Dundee, Scotland, 1994, p. 47. 8. D. M. Le Neveu, R. P. Rand, V. A. Parsegian, and D. Gingell, Biophys. J., 1977. 18,209. 9. €3. BergenstPhl, ‘Topics in food emulsions’, PhD Thesis, University of Lund, Sweden, 1994. 10. N. Krog and A. P. Borup, J. Sci. Food Agric., 1973,24,691. 1. 2. 3. 4. 5. 6. 7.

54


11. N. Krog, T. H. Riisom, and K. Larsson, in ‘Encyclopedia of Emulsion Technology’, ed. P. Becher, Marcel Dekker, New York, 1986, vol. 2, p. 67. 12. N. Krog, in ‘Food Emulsions’, 2nd edn, ed. K. Larsson and S. Friberg, Marcel Dekker, New York, 1990, p. 127. 13. K. Larsson, in ‘The Lipid Handbook’, ed. F. D. Gunstone, J. L. Harwood, and F. B. Padley, Chapman and Hall, London, 1994, p. 407. 14. N. Krog and J. B. Lauridsen, in ‘Food Emulsions’, 1st edn, ed. S. Friberg, Marcel Dekker, New York, 1976, p. 67. 15. I Heertje, H. Hendrickx, A. Knoops, E. C. Royers, and H. Turksma, ‘Use of mesomorphic phases in food products’, European Patent Specification no. 0 558 523 B1, 1994. 16. A.-C. Eliasson and K. Larsson, in ‘Cereals in Breadmaking. A Molecular Approach’, ed. A.-C. Eliasson and K. Larsson, Marcel Dekker, New York, 1993, pp. 161,293. 17. N. Krog, N. M. Barfod, and W. Buchheim, in ‘Food Emulsions and Foams’, ed. E. Dickinson, The Royal Society of Chemistry, London, 1987, p. 144. 18. N. M. Barfod, N. Krog, G. Larsen, and W. Buchheim, FatSci. Technol., 1991,93, 24.

On the Stability of Aerated Milk Protein Emulsions in the Presence of SmallMolecule Surfactants By B. M. C. Pelan, K. M. Watts, I. J. Campbell, and A. Lips UNILEVER RESEARCH, COLWORTH LABORATORY, SHARNBROOK, BEDFORD MK44 ILQ, UK

1 Introduction Many food products contain not only milk proteins but also small-molecule surfactants, which are often referred to as emulsifiers. The competition between proteins and emulsifiers at the oil-water interface is of importance to the final product structure. It is therefore not surprising that emulsifiers have been extensively studied in the literature, both in model systems- and in real product^.^-^ For example, in ice-cream, which is an aerated frozen emulsion, it has been shown that emulsifiers are not required to aid emulsification, as there is sufficient protein available to create a very stable emulsion.'03" It is believed rather that emulsifiers provide controlled destabilization, by displacing some of the protein from the fat droplet interface, and therefore promoting the ' ~ fat aggregation is formation of fat aggregates in the freezing p r o ~ e s s . ' ~ -This not only important to the air cell stability, but also contributes to structure formation in the ice-cream, as can, for example, be noted from the improved melting resistance at increased levels of destabilized fat. Although the role of emulsifiers is widely discussed, it is still not well understood how different emulsifiers influence the microstructure and product properties in a complicated system such as ice-cream. In this paper some emulsifiers are evaluated in terms of their influence on protein displacement and emulsion stability. Further, an attempt is made to relate the findings to the microstructure and melting resistance of ice-creams.

2 Materials and Methods Emulsions were made with 12 wt% butteroil (Dairy Crest), 13 wt% skimmed milk powder (SMP, from Eden Vale), 15 wt% sucrose and deionized water. 55

56

Stability of Aerated Milk Protein Emulsions

The water-soluble surfactant Tween 60 (polyoxyethylene sorbitan monostearate) was obtained from Sigma Chemicals. The other emulsifiers studied included MGP (mono glycerol palmitate, containing 50 wt% saturated monoglyceride and 40 wt% diglyceride), Hymono 8903 (99 wt% pure saturated monoglyceride) and Hymono 7804 (70 wt% unsaturated monoglyceride, 30 wt% saturated monoglyceride), which were all obtained from Quest International; soyabean lecithin (BOLEC ZT) was provided by Unimills. The SMP and sucrose (and Tween 60 where applicable) were dissolved in the water at 60 "C, and mixed with the melted oil phase, in which the oil-soluble emulsifiers had been dissolved. The emulsions were prepared using a Crepaco homogenizer at an operating pressure of 140 bar and a temperature of 6S"C, and pasteurized at 85 "C for 15 seconds before cooling to 5 "C. These premixes were aged for 2 hours at 5°C before being frozen in a Crepaco C freezer at an overrun of 100% and an extrusion temperature of -5.5 "C. Ice-creams were hardened at -35 "C and stored at -20 "C. No stabilizers were added to the emulsions, in order to enable emulsifier effects to be studied more fully. Protein loading measurements involved centrifugation of the premix emulsions at 48 500g for 2 hours at 5 "C (Beckman centrifuge) to separate the oil from the aqueous phase. Surface coverage was then calculated from the specific surface area, as determined by small-angle light scattering (Malvern Mastersizer), and from the difference between the total protein content in the emulsion and the protein concentration in the aqueous phase, as measured by nitrogen analysis (Foss-Heraeus micro-nitrogen analyzer).I5 The orthokinetic stability of the premixes was determined by shearing the emulsions (200 ml) at a constant speed (900 r.p.m.) and at a fixed temperature (15 "C) in the presence of air, using an apparatus described previously. l6 The relative destabilization after 50 minutes of shearing was determined by a solvent extraction technique using petroleum spirit .I6 A higher level of extractable (sometimes referred to as destabilized) fat indicates a less stable emulsion. The solid fat content in the emulsions was measured by 13CNMR (Bruker AMX400).Phase transitions of saturated monoglyceride in water, butteroil, and premix emulsions were evaluated by differential scanning calorimetry (DSC), using a Micro DSC I1 (Setaram) operated at a (cooling) scanning rate of 1 "C min-' over the temperature range from 70 "C to 5 "C. The microstructure of the ice-creams was determined by low temperature scanning electron microscopy (SEM). This technique was used to obtain a qualitative image of the air phase state in the ice-creams, as it is still difficult to measure air cell sizes quantitatively. The stability of ice-creams upon melting was determined by placing the samples from the -20 "C freezer on a meshed grid in a temperature controlled cabinet (20 "C), whilst weighing the material that leaked through the grid over a time period of 4 hours. Pictures were taken of the structures left on the grid after 4 hours to visualize the shape resistance of the melted products.

57

B. M . C . Pelan, K . M . Watts, I. J. Campbell, and A . Lips

3 Results and Discussion Premix Emulsions All emulsions were found to have monomodal droplet-size distributions with the mean droplet diameter d32 = 0.5 p m slightly decreasing with increasing emulsifier levels. The solid fat content in the premixes was found to reach an equilibrium value of 50 f 5% after 2 hours at 5 "C,independent of the presence of emulsifiers, and equivalent to the expected solids levels in butteroil at that temperature. It was therefore assumed that differences in emulsion stability as presented below were not related to fat crystallization but mainly to changes in interfacial properties. Figure 1shows the effect of the presence of Tween 60 on the surface coverage and the amount of extractable fat after shearing the emulsions. Without emulsifier present the surface coverage was If f 2 mg m-2, much higher than previously found for SMP emulsions (6 2 1 mg m-2).16 This effect is explained by experiments (not presented here) showing that increasing the sucrose concentration in SMP emulsions from 0 to 15 wt% induces this increase in surface coverage, presumably by changing the solvent conditions and therefore the casein micelle size distribution in the emulsions. The water soluble surfactant Tween 60 was found strongly to displace the protein from the interface, resulting in a surface coverage of 0.5 2 0.5 mg m-2 at a concentration of 0.4 wt%, which is in agreement with other literature The decrease in surface coverage was accompanied by a strong decrease in emulsion stability, as indicated by the high levels of extractable fat after shear. The effect of the oil-soluble surfactants on surface coverage is presented in Figure 2. While the emulsifiers clearly displace protein from the interface, the

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scatter in the data did not enable confirmation of previous reported finding^,'^.'^ which suggested that unsaturated monoglycerides are more effective at displacing milk proteins than saturated monoglycerides. It should be noted that, even at the highest emulsifier concentrations, not all the protein could be displaced from the interface, as has been shown previously for oilsoluble emulsifier^.^,^>'^ The stability of the emulsions in the model shearing experiment is indicated in Figure 3 by the levels of extractable fat in the premixes after shearing. Increased levels of unsaturated monoglyceride and soya bean lecithin led to increasingly less stable emulsions (i.e.,higher amounts of extractable fat). The lower stability of these emulsions is probably caused by the decrease in the amount of adsorbed protein. The effect of the pure saturated monoglyceride (and mono-/diglyceride MGP) is surprising. At low levels (up to cu. 0.3 wt%) these emulsifiers gave the expected decrease in stability. However, at higher concentrations no further decrease in stability could be observed, whilst from Figure 2 there is clearly progressive protein displacement. It appears, therefore, that in the presence of saturated monoglyceride, protein coverage is not the only important factor controlling the orthokinetic stability. A factor to be considered here is the formation of multilayers/gel phases of saturated monoglyceride at the oil/water interface. This is relevant, for example, for spray-dried whippable emulsions (so-called toppings), where high emulsion stability is attributed to the presence of a-gel phases of lactylated monoglyceride (glycerol lactopalmitate) at the oil droplet In such systems very high levels of monoglyceride are typically required (greater than 10% wt/wt in the oil phase). However, the stability behaviour in Figure 3 is already observed at much lower monoglyceride concentrations (0.3 wt% in the emulsion, which corresponds to 2.5% wtlwt in the oil phase). Furthermore,

59

B. M . C. Pelan, K . M . Watts, I . J. Campbell, and A . Lips

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70 1

"0 Figure 3

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Extractable fa1 ajier shear as a function of the concenlration of the oil-soluble surfactants M G P (+),saturated monoglyceride (m), unsaturated monoglyceride (0)and lecithin (+)

because of the large interfacial area in these emulsions, it is estimated that at 0.3 wt% saturated monoglyceride there would only just be sufficient emulsifier present to form a close-packed monolayer at the interface (on the basis of the reported saturation coverage of saturated monoglyceride of 2.8 mg m-2 at a solution concentration of 0.6Y0'~). Another factor could be the possible partitioning of some of the saturated monoglyceride in the aqueous phase, as has been suggested to be the case in cream liqueur^.^ DSC measurements of monoglyceride/water solutions in Figure 4 show the phase transition from lamellar or cubic phase to a-gel phase at temperatures between 50 "Cand 55 "C,independent of monoglyceride level over the concentration range studied here. Since there was no phase transition observed at these temperatures in DSC measurements of the fully formulated emulsions in Figure 5, it was concluded that the monoglyceride was strongly partitioned (> 99%) in the oil phase. Krog' has studied the behaviour of saturated monoglyceride in model systems and demonstrated that in the melted state monoglycerides only have a minor influence on the interfacial tension, with protein dominating at the interface. When the temperature was lowered below the solubility of the nonoglyceride in the oil, the emulsifier displaced protein from the interface. Figure 6 shows the crystallization temperature of saturated monoglyceridc in butter oil, as measured by DSC. At the concentrations of saturated monoglyceride equivalent to those used in the ice-creams, the monoglyceride was found to crystallize at temperaturcs between 30 "C and 55 "C. The bulk butter oil crystallized at temperatures below 25 "C, which is not shown in the traces in Figure 6, with the crystallization temperature slightly increasing with monoglyceride concentration due to a seeding effect of ?he monoglyceride crystals.


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B. M . C. Pelan, K . M . Watts, I. J . Campbell, und A. Lips

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Figure 5 shows that the monoglyceride in the fat droplets in the emulsion crystallized out at very similar temperatures as it would in the bulk butter oil, which seemed to agree with Krog's findings of the monoglyceride being mostly located in the bulk oil phase at high temperatures. Contact angle measurements of saturated monoglyceride have shown that the crystals can be considered as fairly polar in nature." The presence of monoglyceride crystals at the interface, giving rise to Pickering stabilization, could be considered as a possible explanation for the lack of instability of the emulsions at high levels of saturated monoglyceride.

Ice-Cream Microstructure To illustrate the functionality of emulsifiers in ice-cream, the microstructure and melting behaviour of ice-creams containing saturated and unsaturated monoglycerides are discussed below. The effect of lecithin was found to be comparable to that of unsaturated monoglyceride in terms of ice-cream microstructure and melting resistance, whilst MGP was similar to saturated monoglyceride. Ice-creams without emulsifiers were found to be characterized by the presence of large air cells, some of which had coalesced, as shown in Figure 7. The coalescence indicates poor air cell stability, which is probably related to the lack of fat droplets at the air cell interface. The level of extractable fat in this product was found to. be very low at 2 wt% . Increased levels of unsaturated monoglyceride in the ice-creams resulted in an increase in extractable fat to 93 wt% at an emulsifier level of 0.5 wt%. Some small air cells could be observed in the microstructure of this ice-cream, as shown in Figure 8, but there was also extended air cell coalescence. The smaller air cells are

62


Figure 7

SEM picture of an ice-cream made without emulsifiers

Figure 8

SEM picture of an ice-cream containing 0.5 wt% unsaturated monoglyceride

B. M . C . Pelan, K . M . Watts, I . 1. Campbell, and A . Lips

63

probably an indication of improved air cell stability through the adsorption of fat droplets at the air cell interface, as the fat droplets have become more hydrophobic due to protein displacement by the unsaturated monoglyceride. The air cell coalescence is presumably similar to the process of ‘overwhipping’ of cream, when after initial increase in overrun, the air is lost just beforc the emulsion finally churns. Ice-creams containing saturated monoglyceride contained very little cxtractable fat as was found when the premixes were tested in the model shearing experiments in Figure 3. Increasing the level of saturated monoglyceride led to increasingly smaller air cells in the ice-cream, as shown in Figure 9 for 0.5 wt% saturated monoglyceride. It is not clear why saturated monoglyceride should reduce the air cell size more than unsaturated monoglyceride. A possible explanation is that unsaturated monoglyceride causes too much fat destabilization, and therefore ultimately air cell coalesccnce, whilst saturated monoglyceride promotes adsorption of fat droplets at the air cell interface but docs not lead to extensive fat aggregation.

Melting Resistance of Ice-creams It has been reported that increased fat destabilization in products is usually accompanied by a ‘dry’ appearance of the ice-cream upon extrusion (at -5 “C from the freezer), and an improvement in the melting resistance of the

Figure 9

SEM picture of an ice-creurn coniuining 0.5 wt% saturuted monoglyceride

64

Stability of Aerated

Milk Protein Emulsions

products, both of which are desirable characteristics."." Studying the melting behaviour as a function of emulsifier is useful, since in the absence of stabilizers only fat and air can contribute to the shape resistance of the product once the ice has melted. First of all, the ice-cream without emulsifier, having large air cells and a low level of extractable fat (2 wto/o), melted very quickly as shown in Figure 10. The complete sample had collapsed within 2 hours, with the original premix remaining. Increased amounts of unsaturated monoglyceride, and thus extractable fat, improved the melting resistance as can be seen in Figure 10. The fat aggregation in this system is believed to hold the structure once the ice has melted; the material that remained on the grid was analysed as containing a high fat concentration, whereas the material leaking through the grid hardly contained any fat. The melting resistance of these ice-creams did not appear to be correlated to the air phase stability, since at higher levels of unsaturated monoglyceride the melting resistance was better, despite large coalesced air cells in the microstructure in Figure 8. The melting behaviour of ice-creams containing saturated monoglyceride cannot be explained by fat aggregation; the level of extractable fat was found to be very low and independent of the emulsifier concentration. The structure remaining on the grid seemed to be related to the breakdown of the foam structure in these ice-creams. At an emulsifier level of 0.2wt% in Figure 10, the melting resistance was comparable to that observed for the ice-cream containing 0.2 wt% unsaturated monoglyceride. It is believed that, when the ice melts

Figure 10

Melting resistance of ice-creams, represented by the mass loss from the product through a grid as a function of time at 20 "C (with the numbers indicating the percentage of extractable fat in the ice-creams): (a) no emulsifier; (b) 0.2 wt% unsaturated monoglyceride; (c) 0.5 wt% unsaturated monoglyceride; (d) 0.2 wt% saturated monoglyceride; (e) 0.5 wt% saturated monoglyceride

B. M. C . Pelan, K . M . Watts, I . J . Campbell, and A . Lips

65

in this ice-cream, at 0.2 wt% saturated monoglyceride and only 16Y0 destabilized fat, some air cell coalescence takes place and some of the matrix drains from the foam, but the material remaining on the grid is effectively higher in air phase volume and viscous enough not to flow through the grid. The material collected had indeed the same composition as the initial premix. Although at monoglyceride levels about 0.5 wt% (8 wt% extractable fat) the melting resistance seemed very poor, the material flowing through the grid had retained most of the air, indicating a very stable air phase, as was already demonstrated by the small air cells observed in Figure 9. But, since there was no fat aggregation in the structure, this foam (at 50% air phase volume) was very fluid with poor shape retention. It is clear that fat aggregation is the major contributor to the melting resistance of ice-creams, although the air phase stability can influence the breakdown of the structure as well.

4 Conclusions Water-soluble surfactants (like Tween 60) are much more effective in displacing protein from the fat droplet interface than oil-soluble surfactants (such as monoglycerides and lecithin). For most systems studied the orthokinetic stability was found to decrease for lower protein loadings, as was previously demonstrated for emulsions made with milk proteins alone. I6The functionality of saturated monoglyceride (and mono/diglyceride) was found to be more complicated, as there was no strong decrease in emulsion stability whilst protein was being displaced from the interface. It was shown that all the monoglyceride is situated in the oil droplets, but at concentrations not sufficient for multilayer formation at the interface. Although the mechanism by which monoglyceride stabilizes the emulsion at high concentrations is still not fully understood, it is believed to be related to the crystallization of the monoglyceride in the fat droplets. As to the effect of emulsifiers in ice-cream, it was shown that generally protein displacement and fat destabilization result in the appearance of smaller air cells and an improved melting resistance, although at too high levels of cxtractable fat extensive air cell coalescence was observed. For saturated monoglyceride (and mono-diglyceride), however, protein displacement appeared to be related to smaller air cells in the icecreams in the absence of significant fat destabilization, with the melting resistance being related to the breakdown of the foam structure in the product. A better understanding of the functionality of small-molecule surfactants in stabilizing fat droplets and air cells is required to explain the functionality of saturated monoglyceride in a complex food system such as ice-cream.

5 Acknowledgement We thank Mark Kirkland for help with the electron microscopy.

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References 1. D. F. Darling and R. J. Birkett, in ‘Food Emulsions and Foams’, ed. E. Dickinson, Royal Society of Chemistry, London, 1986, p. 1. 2. E. Dickinson, ‘An Introduction to Food Colloids’, Oxford University Press, Oxford, 1992. 3. J. de Feijter, J. Benjamins, and M. Tamboer, Colloids Surf., 1987, 27, 243. 4. I. Heertje, J. Nederhof, H. A . C. M. Hendrickx, and E. H. Lucassen-Reijnders, Food Struct. , 1990,9,305. 5. N. Krog and K. Larsson, Fat Sci. Technol., 1992,94,55. 6. S. E. Euston, H. Singh, P. Munro, and D. G. Dalgleish, J. Food Sci., 1995, 60, 1124. 7. E. Dickinson, S. K. Narhan, and G. Stainsby, J. Food Sci., 1989,54, 77. 8. N. Krog, N. M. Barford, and W. Buchheim, in ‘Food Emulsions and Foams’, ed. E. Dickinson, Royal Society of Chemistry, London, 1987, p. 144. 9. J. M. M. Westerbeek and A. Prins, in ‘Food Polymers, Gels and Colloids’, ed. E. Dickinson, Royal Society of Chemistry, Cambridge, UK, 1991, p. 147. 10. R. Govin and J. G. Leeder, J . Food Sci., 1971,36,718. 11. B. J. Nielscn, Gordian, 1976,76,220. 12. H. D. Goff, M. Liboff, W. K. Jordan, and J. E. Kinsella, Food Microstruct., 1987, 6 , 193. 13. H. D. Goff and W. K. Jordan, J . Dairy Sci., 1989,72,18. 14. N. M. Barford, N. Krog, G. Larsen, and W. Buchheim, Fat Sci. ?echnol., 1991,93, 25. 15. H. Oortwijn and P. Walstra, Neth. Milk Dairy J . , 1979, 33, 134. 16. B. M. C. van Dam, K. M. Watts, I . J. Campbell and A. Lips, in ‘Food Macromolecules and Colloids’, ed. E. Dickinson and D. Lorient, Royal Society of Chemistry, Cambridge, 1995, p. 215. 17. E. H. Lucassen-Reijnders, Food Struct., 1993, 12, 1. 18. I. J. Campbell, in ’Food Colloids’, ed. R . D. Bee, P. Richmond and J . Mingins, Royal Society of Chemistry, Cambridge, UK, 1989, p. 272.

Interactions between Sodium Caseinate and Dioleoylphosphatidylcholineon OilWater Interfaces and in Solution By Yuan Fang and Douglas G. Dalgleish UNIVERSITY OF GUELPH, DEPARTMENT OF FOOD SCIENCE, GUELPH, ONTARIO, CANADA N1G 2W1

1 Introduction Food emulsions often contain more than one emulsifying agent. Proteins are among the widely used emulsifiers, but other small-molecule surfactants are commonly included in the ingredients. The interactions between protein and surfactant may have an important influence on the properties of these ernulsions. For example, surfactants such as egg-yolk lecithin'.' and soy lecithin3y4 have been found to enhance the stability of the products. In the case of icecream mix, where a destabilizing effect is desired, small-molecule surfactants are deliberately introduced.' Studies on the interactions between milk proteins and a variety of surfactant molecules in emulsion systems have shown that competitive adsorption occurs, with many types of surfactant molecules being capable of displacing protein from oil-water interfaces.&" The degree of displacement depends on the type of surfactant; for example, phosphatidylcholine from egg-yolk (egg-PC) can displace /3-casein7from the oil-water interface when there is a very high phospholipid to protein ratio, and protcin and egg-PC can coexist on the interface. On the other hand, watcr-soluble surfactants such as Tween-20 have a greater capacity for displacing protein from the Partial or complete displacement also depends on whether the surfactant is added before or after emulsification; in the former case, partial displacement is found, and in the later case complete displacement can occur. As well as competitive adsorption between proteins and surfactants, competitive adsorption between different proteins can also occur. 13,14 The most hydrophobic protein among the different components of whole casein is 8casein, and it adsorbs more strongly on an oil-water interface than does a,,-casein, even being able to displace a,,-casein from the i n t e r f a ~ e . Small'~ molecule surfactants can also form complexes with proteins. For example, SDS promotes polymer formation of 8-casein in solution, and there seems to be a

'"

67

68

Interactions between Sodium Caseinate and Dioleoylphosphatidylcholine

limited number of binding sites for SDS on the protein.” Complexes have also been found between casein and mono- and di-glycerides16 as well as between casein and phosphatidylcholine.” Caseins are generally considered to have little or no regular secondary structure. No crystallographic measurements can be made since no crystals of caseins or their complexes are available. Thus the nature and amount of secondary structure has been estimated from CD and Raman spectroscopy. l 8 We have used Fourier transform infrared (FTIR) spectroscopy to study the structure of /3- and aSl-caseinsand their interactions with dioleoylphosphatidyl choline (DOPC) in solution. The C-H stretching region around 2800-3010 cm-’ in the spectrum was used to study the conformation of the hydrocarbon chains of the phospholipid.’9720The C = O ester stretching region (1700-1750 cm-’) could be employed to detect conformational changes at the interfacial region of the phospholipid head group ( i e . , near the glyceryl The amide I region (1600-1700 cm-’) was used to analyse the protein secondary s t r ~ c t u r e . ~ Although ~-*~ no well-defined regular secondary structures were observed, it was found that the caseins do have their own distinctive structures in solution. Changes in the spectra of the proteins were found to be induced by the presence of phospholipid, and these could be used to infer that conformational changes were caused by phospholipid-protein interaction.

2 Materials and Methods Soybean oil, DOPC and D 2 0 (99.9%) were purchased from Sigma Chemicals (St. Louis, MO). Imidazole and 2-mercaptoethanol were purchased from Fisher Scientific (Mississauga, ON). Sodium caseinates were prepared in our laboratory from fresh milk. Casein solutions were made in imidazole buffer (20 mM imidazole, pH 7) and were filtered through a 0.22 pm filter before use. Individual p-casein and a,,-casein were prepared from whole caseinate by chromatography on Sepharose Fast Flow S gel (Pharmacia Biotech, Baie d’Urf6, Pa),using an eluent solution of 6 M urea in 20 mM acetate (pH 5.0). A gradient of NaCl between 0 and 0.4 M was applied. The individual fractions were then dialysed exhaustively against Milli-Q water and freeze dried. The quality of the individual caseins was checked using electrophoresis (PhastSystem, Pharmacia Biotech). Emulsions stabilized with caseinate were made using a Microfluidizer Model 110s (Microfluidics Corp., Newton, MA). The emulsions were analysed shortly after they were prepared and were then stored at 4 “C for future studies. The average diameter (d32,volume to surface average), the size distribution, and surface area of the emulsion droplets were measured by small-angle light scattering using a Mastersizer X (Malvern Instruments Inc., Southboro, MA). The stability of the stored emulsions was followed by measuring the average droplet size and the size distribution every day for the period of a week. The amount of casein adsorbed to the oil droplets was determined by analysing the emulsion droplets (cream phase) dire~tly.’~ The interaction between DOPC and casein (p- and aSl-casein)was studied

Y . Fang and D. G . Dalgleish

69

by Fourier transform infrared (FTIR) spectroscopy (FTS-40, Bio-Rad Laboratories Ltd., Mississauga, ON). The absorbance spectrum was recorded at 4504000 cm-’ at a resolution of 2 cm-’. Data were further analysed using a Digilab software package. The spectra were smoothed using a 9-point Savitsky-Golay procedure and then a %point second derivative was calculated. For measurement, the samples were placed in a Harrick ATR cell, and 800 scans were accumulated for each spectrum. Solutions of casein and DOPC for FTIR studies were prepared in D 2 0 and were allowed to equilibrate for 24 h before measurement. DOPC dispersions were sonicated for 10 min, and cascin was added either before or after thc sonication.

3 Results and Discussion Interaction between DOPC and Casein on the Oil-Water Interface Emulsions containing 20% (wlw) oil stabilized by casein alone are known to be stable during storage at a concentration of 0.3 wt% casein or above.28 The presence of egg-PC even enhances the stability of emulsions stabilized with lower casein concentration. However, the incorporation of DOPC had an opposite effect from egg-PC on thc emulsion ~tability.~’ For emulsions containing0.2 wt% or 0.5 wt% DOPC, at least 0.7% or 1.5% casein, respectively, was necessary to form stable emulsions. Combining the emulsion stability and the DOPCkasein molarratio, it was found that if the DOPCkasein molar ratio was above 10, the emulsions formed were not stable with time, since an increase in droplet size was observed from the Mastersizer measurement. If the molar ratio was below 10, emulsions formed were stable. This stability correlates very well with the surface concentration of casein measured from SDS-PAGE. When DOPC was present, less casein was adsorbcd initially compared to emulsions stabilized by casein only. At DOPCkasein ratios above 10, the surface concentration of casein was below 1mg m-*,which has been suggested to be a critical value for the formation of a stable emulsion.28 During the storage of the emulsions, more casein was removed from thc oil-water interfa~e.~’ This destabilization of emulsion droplets as a result of the competitive adsorption was rather peculiar, since competitive adsorption does not usually induce destabilization of the emulsion unless the emulsion is ~ h e a r e d . SDS-PAGE ~.~~ analysis on emulsions of different age revealed that the displacement of casein by DOPC was highly selective, and /3-casein was the most affected of the constituents of whole casein. During emulsion formation in the presence of DOPC, less #%caseinwas found on the interface than was expected from its proportion in solution, and even more /3-casein was removed from the interface by DOPC during storage of the emulsions. At low casein concentrations, /3-casein was completely removed from the interface, while under all conditions the adsorption of aSl-cascinwas hardly affectcd by DOPC. This indicates that the removal of /3-casein did not simply result from a general competitive displacement by DOPC for all caseins adsorbed to the interface,

70


but apparently from a specific interaction between p-casein and DOPC. Such complexes have been reported to form between casein and both mono- and di-glycerides.16 Also, it is known that SDS promotes p-casein polymer formation in solution and that there is a limited number of binding sites on the protein for SDS. Is It has been demonstrated") that p-casein and P-lactoglobulin display different binding affinities for sucrose esters with different chains; p-casein has a greater affinity for unsaturated chains, whereas saturated esters bind better to P-lactoglobulin. DOPC has two unsaturated chains, and this might explain its high affinity for p-casein. If p-casein on an oil-water interfacc interacts with DOPC via the hydrophobic domains of both molecules, this would produce a more hydrophilic complex which would dissociate from the interface into the aqueous phase. Presumably there are specific structures in p-casein which facilitate its interaction with DOPC, since the analysis showed that little interaction appeared to take place between a,,-casein and DOPC, despite the fact that the proteins share many properties.

Interaction between DOPC and Casein in Solution Measured by FTIR * The C-H stretching vibrations of hydrocarbon chains of lipids give a number of well-defined absorption bands in the region of 28OO-310O cm-' in the infrared spectrum. The frequencies of these bands are very sensitive to the environment of the acyl chains. 19,2",23 The symmetric and anti-symmetric stretching bands of the methylene group occur at ca. 2850 and 2920 cm-', respectively. The olefinic (=CH) stretching band appears at ca. 3010 cm-1.20 In the presence of protein, the symmetric stretching band at ca. 2850 cm-' has been found23to be more suitable for structure studies on lipids. We measured spectra of DOPC, DOPC + p-casein and DOPC + aSl-casein, and the positions of the peak frequencies in each spectrum are summarized in Table 1. The CH3 stretching frequencies and the CH2asymmetric stretching are not purely caused by lipid, since there are contributions from the proteins also at these frequencies. There was little or no contribution from the casein at the CH, symmetric stretching and the olefinic stretching frequency. DOPC displayed a strong CH2 band at 2853.5 cm-', which is typical for an acyl chain in the liquid crystalline In thc presence of p-casein, this band was shifted slightly to 2854.2 cm-' indicating some increasing disorder in the packing of the chains. The Table 1

C-H stretching frequencies of DOPC, DOPC DOPC + usl-casein Frequency lcm -

Sample DOPC DOPC + 8-casein DOPC + a,,-casein Vibrational mode

+

&casein and

'

2853.5 2854.2 2853.5

2872.6 2872 2871.8

2924.2 2924 2924

2958.3 2961.7 2961.8

3005.9 3009.3 3005.7

vs,CH2

us,R-CH3

u,,, CH2

uHS, K-CH3

u , =CH

71

Y . Fang and D. G. Dalgleish

presence of aSl-caseindid not change the position of this band. A major change was found to occur at the olefinic stretching frequency; DOPC on its own and in the presence of aSl-caseinshowed similar peaks near 3006 cm-'. However, the band for the mixture of DOPC and p-casein was shifted to 3009 cni-I, indicating more disorder in the olefinic region. This result strengthens our belief that B-casein complexes with DOPC by means of hydrophobic interactions and that this interaction is in some way related to the presence of unsaturated bonds in the DOPC acyl chains. The conformation of the phospholipid in the rcgion of its head group can be studied by analyzing the C=O ester stretching bands around 1700-1750 cm-'. Caseins have little contribution in this region. The deconvolved or sccondderivative spectrum of phospholipids usually gives two bands, one about 1740 cm-' which is interpreted as the contribution from the sn-1 chain in a trans conformation and the other near 1725 cm-I, which is assigned to the gauche conformation of the sn-2 chain.20q21 Figure 1 shows the second-derivative

I

1800

Figure 1

1780

1760

1740

Wavenumber (cm- 1)

1720

Second-derivative spectra of DOPC and its mixtures with a,yIorb-casein in the C = O carhonyl stretching region; the molar ratio DOPClcasein is 20:I. Upper curve, DOI'C alone; middle curve, mixture of DOPC -+ aSl-casein; lower curve, mixture of DOt'C + ,&casein

72


spectra in this region for DOPC in isolation and in the presence of aS1-or /?-caseins. In isolation, DOPC gave bands at 1744 cm-' and at 1729 cm-'. On the other hand, the mixture of DOPC /?-casein showed mainly one band at 1744cm-', the band from the trans conformer at the interfacial region, with the band from the other conformer being absent. The mixture of DOPC + asl-casein showed both trans (1744 cm-') and gauche (1729 cm-') bands similar to the isolated DOPC. This indicates that the interaction between DOPC and /&casein not only changed the packing of the hydrocarbon chains (Table l), but it also significantly modified the region of the head groups. Thus, when complexed with /?-casein, DOPC adopts a conformation very different from that of a double-chain lipid. The two carbonyl groups of DOPC have become nearly identical at the interfacial region, and the spectrum appears as if only one chain were present, like a lysolecithin. This again indicates that the interaction between DOPC and p-casein occurs via the hydrocarbon chains. In contrast to the behaviour with p-casein, the mixture of DOPC + aSl-casein gave two bands as in pure DOPC, one at 1744 cm-' and another at 1729 cm-', although the ratio between the two bands seems to change slightly with increase in the contribution of the gauche conformer. A similar kind of behaviour has been found for the interaction between myelin basic protein and dimyristoylphophatidylglycerol (DMPG) b i l a y e r ~ although ,~~ the effect was much less pronounced, with the ratio between the two bands being only slightly changed. The secondary structures of /3- and asl-caseins, as well as their complexes with DOPC, were studied by analyzing the amide I region of the spectrum between 1600 and 1700 cm-' where DOPC shows little absorption. Figure 2 shows the second-derivative spectrum in this region of a,,-casein and its mixture with DOPC. Sonication made no difference to the spectrum of the -casein mixture of a,,-casein + DOPC. The second-derivative spectrum of aS1 consists mainly of one broad peak centered around 1642cm-' which is assigned to non-ordered structure,24and a small peak at ca. 1613 cm-' . This spectrum is very similar to what has been described previouslyz4 and it confirms the non-ordered nature of the protein. The second-derivative spectrum of the mixture of aSl-casein + DOPC is similar to that of the casein alone, with a broad peak centered at 1642cm-', a small peak at 1613cm-l, and a shoulder at ca. 1670 cm-'. The spectra for /?-casein and its mixture with DOPC are shown in Figure 3. The second-derivative spectrum of p-casein has a broad band centered about 1638 cm-', rather than 1642 cm-' as was found for a,,-casein. The broadness of the band indicates unresolved structure; however, the frequency at the centre of the band is considerably lower than what is normally attributed to the non-ordered band and is in the region of the spectrum usually assigned to P-sheets. Some highly helical proteins (haemoglobin , myoglobin, cytochrome C, efc.) have also been to display bands at frequencies normally assigned to /?-sheets, even though no such structure was found in these proteins by X-ray crystallography. The spectra were therefore interpreted as arising from short irregular pieces of extended structure. The /3-casein DOPC mixture has a new but not well-defined banc It 1629

+

+

73

Y . Fang and D . G . Dalgleish

1700

Figure 2

1680

1660

16

Wavenumber ( c m - 3

1620

11 w

Second-derivative spectra of a,vl-caseinand its mixture with DOPC in the amide I region; the molar ratio DOPClcasein is 20:l. Upper curve, a,vlcasein; lower curve, mixture of a,,-casein + DOPC

cm-' which is also in a region corresponding to p-structure; this may indicate that more interaction between the protein chains occurs under the influence of DOPC. It should be noted that, in both spectra of p-casein, there is a large shoulder between 1650and 1680cm-', which is usually assigned top-turns; this part of the spectrum was less affected by DOPC. Comparisons with previous studies, where a,- and /?-casein were found to form helical structures in halogenated alcoh01,~~ show that the interactions with DOPC have different impacts on their secondary structure. DOPC appears to induce more P-sheetlike structure in /?-casein, and does not change the structure of a,,-casein significantly. Because the interactions between DOPC and p-casein have been shown to change significantly the structure of both the lipid and the protein, we can conclude that a complex has formed between the two compounds. However, since the components of the mixture exist in particulate form (casein can associate with itself and with the bilayer of the DOPC liposome), we cannot at the present time distinguish the specific sources of the changes in the

74


D

1680

1660

16

Wavenumber ( c m - 3

Figure 3

1620

1

Second-derivative spectra of B-casein and its mixture with DOPC in the amide I region; the molar ratio DOPClcasein is 20:I. Upper curve, pcasein; lower curve, mixture of p-carein + DOPC

spectrum. The results have also confirmed that caseins are not completely random coil or non-ordered proteins, even though their secondary structures are not as well defined as those of globular proteins. There is a distinctive structure difference between p-casein and a,,-casein in solution, as shown by the difference in their spectra.

4

Conclusions

In oil-in-water emulsions stabilized by casein and DOPC, competitive adsorption occurred between casein and DOPC. The presence of DOPC decreased the surfacc concentration of casein during the formation and the storage of the emulsion. This effect of DOPC in turn caused instability in emulsions which would be stable in its absence. The interaction between DOPC and casein was highly selective, since B-casein was the most affected of all the casein constituents. During the storage of emulsions made using relatively low casein

Y . Fang and D. G . Dalgleish

75

concentration, p-casein could be completely removed from the interface by DOPC. Results from FTIR studies of the interaction between DOPC and @and aSl-casein demonstrated that the structure of the acyl chain region of DOPC was significantly changed by p-casein but was little affected by a,,-casein. The secondary structure of @-caseinappeared to contain more of thep-sheet type than aSl-casein,and more/?-likestructure was also found in the DOPC + p-casein mixture. This could arise from the binding of DOPC making the hydrophobic domain of the protein turn in on itself and rendering the protein more hydrophilic as a whole. Little effect of DOPC on the secondary structure of aSl-caseinwas found. The results from FTIR studies in solution are in very good agreement with the direct observation of selective interaction between DOPC and casein in oil-in-water emulsion system. A complex is formed between DOPC and @-caseininvolving the hydrophobicdomains of the two components, whereas no such complex between DOPC and a,,-casein is evident. It is this complex formation that makes the removal of casein from oilwater interface by DOPC so highly specific.

References 1. R. Nakamura, R. Muzutani, M.Yano, and S. Hayakawa, J . Agric. Food Chem., 1988,36,729. 2. Y. Fang and D. G. Dalgleish, Colloids Surf., 1993, 1,357. 3 . E. E. Hardy-Lloyd, A. W. M. Sweetsur, I. G. West, and D. D. Muir, Milchwissenschaft, 1986,41,470. 4. D. D. Muir and A. W. M. Sweetsur, Milchwissenschaft, 1992,47,80. 5. N. Krog and N. M. Barfod, ‘Interfacial properties of emulsifier/protein films related to food emulsions’, AIChE, San Francisco, 1990, vol. 86, p. 1. 6. J. A. de Feijter, J. Benjamins, and M. Tamboer, Colloids Surf., 1987,27,243. 7. J.-L. Courthaudon, E. Dickinson, and W. W. Christie,J. Agric. Food Chem., 1991, 39, 1365. 8. E. Dickinson and S . Tanai, J. Agric. Food Chem., 1992,40, 179. 9. 1. Heertje, J. Nederlof, H. A. C. M. Hendrickx, and E. H. Lucassen-Reynders. FoodStruct., 1990,9,305. 10. J.-L. Courthaudon, E. Dickinson, Y. Matsumura, and D. C. Clark, Colloids Surf., 1991,56,293. 11. P.J. Wilde and D. C. Clark, J . Colloid fnrerface Sci., 1993, 155, 48. 12. J.-L. Courthaudon, E. Dickinson, Y. Matsurnura, and D. C. Clark, Colloids Surf., 1991,56,293. 13. E. Dickinson, S. E. Rolfe, and D. G. Dalgleish, Food Hydrocolloids, 1988,2,397. 14. T. Nylander and N. M. Wahlgren, J. Colloid Interface Sci., 1994, 162. 151. 15. L. K. Creamer, Arch. Biochem. Biophys., 1980, 199, 172. 16. G. Doxastakis and P. Sherman, Colloid Polym. Sci., 1984,262,902. 17. Y. Fang and D. G. Dalgleish, in ‘Food Macromolecules and Colloids‘, ed. E. Dickinson and D. Lorient, Royal Society of Chemistry, Cambridge, 1995, p. 146. 18. H. E. Swaisgood, ‘Chemistry of the Caseins’, Elsevier, Cambridge, 1992, vol. I , p. 63. 19. J. G. Weers and D. R. Scheuing, ‘Fouricr Transform Infrared Spectroscopy in

76

20. 21. 22. 23. 24. 25. 26. 27. 28. 29. 30. 31. 32.

Interactions between Sodium Caseinate and Dioleoylphosphatidylcholine Colloid and Interface Science’, American Chemical Society, Washington, D . C . , 1990, p. 86. H. L. Casal and H. H. Mantsch, Biochim. Biophys. A m , 1984,779,381. E. Mushayakarara and I. W. Levin, J. Phys. Chem., 1982,86, 2324. S. F. Bush, H . Levin, and I. W. Levin, Chem. Phys. Lipids, 1980,27, 101. W. K. Surewicz, M. A. Moscarello, and H. H. Mantschs, Biochemistry, 1987, 26, 3881. M. Byler and H. Susi, Biopolymers, 1986,25,469. W. K . Surewicz and H. H. Mantsch, Biochim. Biophys. Acta, 1988,952, 115. H. Susi and D . M. Byler, Biochem. Biophys. Res. Comm., 1983,115,391. Y. Fang and D. G. Dalgleish, J . Agric. Food Chem., 1996, 44,59. Y. Fang and D . G. Dalgleish, J . Colloid Interface Sci., 1993, 156, 329. J . Chen, E. Dickinson and G. Iveson, Food Strut., 1993, 12, 135. D. C. Clark, P. J. Wilde, D . R. Wilson, and R. Wiistneck, Food Hydrocolloids, 1992, 6, 173. A. C. Dong, P. Huang, and W. Caughey, Biochemistry, 1990,29,3303. M. Jackson and H. H. Mantsch, Biochim. Biophys. Actu, 1992, 1118, 139.

Effect of Lipophilic Molecules on Food Protein Surface Activity at the Air-Water Interface By L. A. Wasserman and M. G. Semenova* INSTITUTE OF FOODSTUFFS, RUSSIAN ACADEMY OF SCIENCE, VAVILOV STREET 28, 117813 MOSCOW, RUSSIA

1 Introduction Proteins and lipophilic molecules (lipids, surface-active low-molecular-weight additives, aroma compounds) are the main components of food systems contributing to structure, taste and nutritional value. It is well known that proteins are able to interact with lipophilic molecules very effectively due to their high hydrophobicity.'4 However, up to now, the general principles underlying the mutual influence of proteins and lipophilic molecules on their functional properties in food systems remains uncertain. In this paper we attempt to compare the effects of lipophilic molecules with different structures, added at rather low concentration, on the basic protein functional propcrty of surface activity at the air-water interface. We have here chosen sets of lipophilic compounds differing in the nature and length of the hydrocarbon chain. In particular, we have studied the following sets of lipophilic compounds: sodium salt of fatty acid, fatty acid, monoglyceride and triglyceride. Hydrocarbon chains of the lipophilic molecules studied consisted of either 10 or 16 carbon atoms; that is, we have studied various sets of capric and palmitic acids. Ovalbumin and 11s globulin are globular proteins differing substantially in molecular structure. The interest in studying this pair of proteins arises from a combination of scientific and practical reasons. Proteins of leguminous plants are one of the most promising protein types for formulating new forms of food products.77811sglobulin of the broad bean is hom.ologous in physical-chemical properties and biological function to other 11s globulins, which are the main Ovalbumin is the main storage proteins of leguminous plants (soy, pea, et~.).*.~ protein of hen egg white. " . 1 2 This is very widely used in food processing due to "To whom correspondence should he addrcsscd.

77

I8

Effect of Lipophilic Molecules on Food Protein Surface Activity

its excellent whipping,I3 gelling14 and foaming" properties. Moreover, native 11s globulin has a more hydrophobic surface in comparison with nativc ~ v a l b u m i n , ' ~as + 'well ~ as being more conformationally stable due to the large number of disulphide bonds in the interior of the protein globule.9

2 Method of Lipophilic Compound Addition to the Protein Solution A predetermined amount of the lipophilic compound (LC), dissolved in a small volume of ether, was added to the buffer solution. This solution was stirred uncovered for 30 minutes at room temperature to remove the ether. In order to ensure LC dispersion and solubility in the aqueous buffer solution, it was ultrasonicated for 2.5 minutes at a frequency of 22 Hz. Then the LC dispersed in the buffer was added to the protein solution at known concentration, and the protein solution containing the lipophilic compound was stirred for 1 hour at 42 "C. The ratio of lipophilic compound to protein was kept constant at 2 mM of LC per 1% w/v of protein in the solution. It was observed that extent of protein saturation by LC depends on the protein concentration in aqueous medium. Therefore, in order to get a greater extent of saturation of the protein by LC, the addition of the LC was carried out also into 10% w/v protein solution. In the last stage we diluted the prepared solutions with buffer up to the required concentration, namely 0.001% w/v protein concentration for the tensiometry measurements, 0.25% w/v for the calorimetry measurements, and 0.2-1 % w/v for the light-scattering measurements.

3 The Influence of the Lipophilic Compound Structure on 11s Globulin Surface Activity The effect of the lipophilic molecules on the protein surface activity was investigated by tensiometry measurements of the change of the interfacial pressure JC of the protein adsorbed at the planar air-water interface. Let us consider the set of lipophiliccompounds: sodium salt of capric acid, capric acid, I-monodecanoyl-glycerol of capric acid, and tricaprin. Figure 1 shows that the protein surface activity is dependent o n the structure of the lipophilic molecules in the set studied. The salt of capric acid and the capric acid itself caused a similar increase in the protein surface activity. On the contrary the monoglyceride of capric acid induces a significant reduction in protein surface activity. On the other hand, the triglyceride of capric acid induces the most dramatic increase in protein surface activity. To attempt to account for the result obtained, we have investigated the effect of these lipophilic molecules on the protein conformational stability because the conformation of the protein molecule is the most responsive parameter to the interaction of protein molecules with low- and high-molecular-weight substances in aqueous mcdia. We have followed protein interaction with the

79

L. A . Wasserman and M . G . Semenova

-

21.3

227

17.5

Figure 1

Effectof lipophilic molecules on the interfacial pressure of the 1I S globulin adsorbed layer ( p H 7.0, I = 0.1 molldm')

lipophilic compounds by measurement of the thcrmodynarnic parameters of the protein heat denaturation process using differential scanning calorimetry. The calorimetric data in Figure 2(a) indicate, first of all, an increase in protcin conformational stability for all the lipophilic molecules studied. This manifests itself as an increase in the protein specific denaturation enthalpy (AHd). This result indicates a direct interaction of the lipophilic compounds with the protcin, due to additional new bond formation in the interior of the globular protcin. As this takes place, an increase in the difference between the specific heat capacities (AdCp)of the native and denaturated proteins is observed (see Figure 2(b)). This can be attributed to an increase in the hydrophobicity of the denatured protein surface contacting with water as a result of the lipophilic molecules attachcd to the protein. The largest increase in AdCpwas observed with tricaprin. Thus, it is possible to assume, on calorimetric grounds, that the increase of the protein surface activity observed in the presence of the set of lipophilic compounds studied is mainly dictated by the growth of the net protein hydrophobicity. In general, the increasc in protein conformational stability is the factor acting against the rise in protein surface activity.'x Accordingly, the greatest effect occurs with tricaprin, which is the most hydrophobic of the set o f

80


4

100.0

36.5

F

33.1

3.0

1

I

-

1M 2 5 1

\

LO 281

11SV.F.

Figure 2

CAPRK: PlQD SaUcM SALT

Effect of lipophilic molecules on the thermodynamic parameters of the 11.7 globulin heat denaturation process: (a) AHd, (h) AdC,, (pH 7.0, I = 0.I moll dm3)

81


the lipophilic compounds studied. The greatest lowering of the 11s globulin surface activity was observed in the presence of the monoglyceride. This result, obtained in spite of similar effect on the protein conformational stability (in comparison with effect of the salt of capric acid or capric acid), apparently shows a different mechanism of protein globule modification by these different lipophilic compounds. Probably, the structure of the hydrophilic head of the monoglyceride is a crucial factor controlling the globular protein modification. We have used light scattering data to elucidate the change of protein molecular parameters in bulk aqueous solution under influence of the lipophilic molecules studied. Table 1shows that there is protein association in the aqueous medium under the influence of the lipophilic compounds, probably as a result of the protein hydrophobicity increase. The protein weight-average molecular weight increases. The greatest extent of protein association is observed for the case of the monoglyceride. Moreover, in this case we observe the most negative value of the second virial coefficient characterizing the thermodynamics of the protein-protein pair interactions in the bulk aqueous medium.'" So, evidently, the most strong attractive forces act between 11s molecules modified by monoglyceride in bulk solution. We could not see the same level of protein surface activity as for 11s globulin modified by capric acid or the capric acid sodium salt, in spite of the similarity in the modified protein conformational stability. Possibly, in the case of monoglyceride, there is some compensating process which leads to new hydrophobic bond formation together with partial protein unfolding. As a result of this we can envisage a similar level of conformational stability with absolutely different surface activity. In the case of tricaprin the light scattering data indicate ideal thermodynamic affinity relative to the aqueous medium and a similar extent of protein association in comparison with the other lipophilic compounds in the set studied. Comparison of the light scattering data with calorimetric results allows us to suppose that added tricaprin causes significant increase in the protcin hydrophobicity in the interior of the globular protein. As a result of this, the aqueous medium becomes ideal thermodynamically for the modified protein, and the greatest increase in net protein hydrophobicity appears at the interface with the non-polar phase, where possibly some partial protein unfolding occurs. Table 1

Effect of short-chain lipophilic molecules on the protein molecular parameters in bulk aqueous solution (pH 7.0, ionic strength 0.1 M ) Molecular weight1 kDa

System

A",-{, x 1041 m' moI-'

~~

11s globulin 11s globulin 11s globulin 11s globulin

+ capric acid + 1 mono-decanoyl-glycerol

+ tricaprin

350 625 714 625

-39.2 142.2 -242.9 0 -

82


Thus, on the basis of the data observed, it seems necessary to obtain more information about the change of the modified protein behaviour in the adsorbed protein layer in order to gain a complete understanding of the effect of lipophilic molecular structure on the protein surface activity.

4 Influence of the Globular Protein Structure on Globular Protein Surface-Activity Let us compare the effects of the lipophilic compounds on the surface activities of the two globular proteins, namely, 11sglobulin and ovalbumin, which differ in their structure and conformational stability. 1”716317 For example, 11s globulin has a larger surface hydrophobicity and more disulphide bonds in the interior of the molecule, whereas ovalbumin increases its hydrophobicity more dramatically as a result of protein denaturation. Figure 3 shows effect of the lipophilic compounds on the ovalbumin surface activity. The behaviour is qualitatively similar to that of 11s globulin (see Figure 1). However, a quantitative difference was observed in the case of the effect of the salt of capric acid on the protein surface activity. That is, the salt of capric acid causes a greater increase in the ovalbumin surface activity than does

I

-

21.1

OVA

Figure 3

Effect of lipophilic molecules on the interfacial pressure of the ovalbumin adsorbed layer (pH 7.0, I = 0.1 molldm?)

83

L. A. Wasserman and M . G . Semenova

86.1

7

I .62

! Figure 4

Effect of lipophilic molecules on the thermodynamic parameters of the ovalbumin heat denaturation process: (a) A H d , (b) A& (pH 7.0, I = 0.1 molldm3)

84


FATNACID FAT7yACID

FATTYACID SXXUh4 S4I.T FATIYACID

Figure 5

Comparison of the effect of the chain length of the lipophilic molecules on the interfacial pressure of the I I S globulin adsorbed layer in the set: salt of fatty acid, fatty acid, monoglyceride

capric acid. Moreover the effect is like that of tricaprin on the ovalbumin surface activity. 'To account for this difference in the behaviour of the two proteins, let us consider the effect of the lipophilic molecules on the ovalbumin conformational stability (see Figure 4). We can see a decrease in ovalbumin conformational stability under influence of the salt of the capric acid as opposed to the effect of the other more non-polar compounds in the set studied. So, it is likely that there is partial unfolding of the globular protein as a result of sodium caprate addition to the ovalbumin solution. Sodium caprate is capable of inducing ovalbumin partial unfolding, apparently due to the lower stability of the ovalbumin molecule than the 1IS globulin molecule. So, the hydrophobic chains of the sodium caprate penetrate into the inner part of the globular protein and the surfactant charged head group evidently enhances protein denaturation due to the strengthening of electrostatic repulsion between like charges. The similarity of the effects of the sodium caprate and the tricaprin on the ovalbumin surface activity apparently confirms the similarity of the protein surface hydrophobicity in the adsorbed protein layer. But for sodium caprate the mechanism mainly involves protein partial unfolding, whereas for tricaprin the mechanism involves hydrophobic chains of the

85


FATTY PCID FATTY SCCRMSALT

*

35

it

?30 c,

365

37.5

FATTY 33.1

10

50

-

-

11s V.F.

b) 1.2

h4

. M

T

*

$

54

0.8

1 I FATTY 0.4

0.c

IISVF.

TkE SET

CF PALMTlC

A131D

Figure 6

Comparison of the effect of the chain length of the lipophilic molecules on the thermodynamic parameters of the I1S globulin heat denaturation process: (a) AHd, (b) A& in the set: salt of fatty acid, fatty acid, monoglyceride (pH 7.0, I = 0.1 mol/dm3)

86

Effect o f Lipophilic Molecules on Food Protein Surface Activity

tricaprin binding to the ovalbumin molecules. In the latter case we see a significant increase in the protein conformational stability.

5 Effect of the Hydrocarbon Chain Length of the Lipophilic Compounds on the Protein Surface Activity We have investigated the influence on protein surface activity of increasing the length of the hydrocarbon chain of the lipophilic compounds from 10 to 16. That is, we go from the set of capric systems to the set of palmitic systems (sodium salt of palmitic acid, palmitic acid, monoglyceride of palmitic acid). Figure 5 compares the effects of the two sets of lipophilic molecules on the 11s globulin surface activity. The similarity of the values of the interfacial pressure of 1IS globulin in the presence of the salt of the fatty acid and the fatty acid itself is independent of chain length. The major difference in the effect of lipophilic molecules of different chain length was observed for the case of the monoglycerides. To account for these data, let us compare the protein conformational stability data for the two sets studied. Figure 6 shows a significant difference in the effect of the lipophilic compounds on the 11s globulin protein conformational stability depending on the length of the hydrocarbon radicals. A long hydrocarbon chain leads to a reduction in the protein conformational stability as opposed to an increase in

~

21.1

14.2

14.2

OVA

Figure 7

CAI’RIC AC1D

OVA

PALMTIC ACID

Comparison of the effecls of the fatty acid (capric acid and palmitic acid) on the interfacial pressure of ovalhumin (pH 7.0, I = 0.1 molldm3)

87

L . A . Wasserman and M . G . Semenova

25-

.aoM

-

i Q

.

26.3

”-

I

19.0

19.0

129

lo: 5-

0-

WA

CAPRlC AaD

WA

PALMTK: AaD

1.0 ,

Comparison of the effect of the fatty acid (capric acid and palmitic acid) on the thermodynamic parameters of the ovalbumin heat denaturation process: (a) AHd, (b) A&,, (pH 7.0, I = 0.1 molIdm3)

88


the protein conformational stability under influence of the lipophilic compounds with the short hydrocarbon chain. That is, it appears that the longer hydrocarbon chain destroys some important hydrophobic contacts in the interior of the globular protein providing the stabilization of the native conformation. From the tensiometry and differential scanning calorimetry, it is possible to infer the explanation of the effect of the lipophilic molecule chain length on the 11s globulin surface activity. There is an increase of the protein surface hydrophobicity due to added lipophilic molecules in the case of the rather short

Table 2

Effect of long-chain lipophilic molecules on the protein molecular parameters in hulk aqueous solution (pH 7.0, ionic strength 0.I M ) Molecular weight1 kDa

System

11s globulin 11s globulin 11s globulin

350 690 357

+ palmitic acid + 1 mono-palmitoyl-glycerol

-39.2 - 167

0

m.3

___

14.2

x IOJl m- mol-'

14.2

OVA I

: Figure 9

Comparison of the effect of the fatty acid sodium salt (capric acid and palmiticacid) on the interfacialpressure of ovalbumin (pH 7.0,I = 0.1 moll dm3)

89

L. A . Wasserman and M . G. Semenova

a> 25

a,

. M

c,

2^ d

I5

I

5

0

L

19.0

16.2 10.6

WA

CAPRlC PClD XlXM SALT

WA

PALMTlCXD SUXM SALT

a4

. 5

Y

M

5

0.44

0.44 0.2

4

0.24

0.c

WA

CAPRlc SCCRM

Figure 10

m

WA

7

SALT

Comparison of the effect of the fatty acid sodium salt (capric acid and palmitic acid) on the thermodynamic parameters of the ovalbumin heat ( p H 7.0, I = 0.1 molldm3) denaturation process: (a) AHd, (b)

90


hydrocarbon chain, and partial protein unfolding in the case of the rather long hydrocarbon chain. In order to elucidate the observed effect of the monoglyceride with different lengths of hydrocarbon chain, it is interesting to compare the molecular parameters of the modified proteins in the aqueous medium. Table 2 presents light scattering data for lipophilic compounds of the palmitic acid set. Comparison of the values in Tables 1 and 2 shows a large dependence on the length of the hydrocarbon chain. There is no association of the globular protein modified by the monoglyceride of the palmitic acid, and the solution is thermodynamically ideal for the modified protein. So, it seems that the molecular structure of the modified protein is dramatically dissimilar for these two cases. Thus, on the basis of the data observed, it seems necessary to obtain more information about the change of the modified protein structure in order to gain a complete understanding of the role of the length of the hydrocarbon chain of the lipophilic molecules on the protein surface activity. Let us consider the effect of the fatty acid on the ovalbumin surface activity. The data for ovalbumin in Figure 7 are identical to those for 11sglobulin for the effect of the fatty acid chain length on the globular protein surface activity. The data from the differential scanning calorimetry (see Figure 8) show also in this case that the increase in the length of the hydrocarbon chain in the fatty acid molecule causes the partial protein denaturation, in contrast to an increase in the protein conformational stability in the case of the rather short chain length. In the case of the salt of the fatty acid, there is a smaller increase in the interfacial pressure for the palmitic acid salt as compared with the salt of the capric acid (see Figure 9). We can see greater denaturation of the globular protein in the presence of the lipophilic molecules with the longer hydrocarbon chain (see Figure lo). But possibly the more extensive denaturation of the protein molecules due to added lipophilic molecules as well as the more hydrophobic nature of the salt of the palmitic acid could lead either to the formation of protein associates in bulk solution through a combination of the hydrophobic part of the protein molecules or to an intensification of proteinprotein interactions in bulk solution. As a result of this, the protein surface activity may decrease. The comparison of the light-scattering data in Table 1 and Table 2 confirms the increase in both the protein association and the intensity of the proteinprotein pair interactions in bulk solution under the influence of the palmitic acid as compared with the capric acid. But it is evident that further detailed investigations are required.

References 1 . V. B. Tolstoguzov, in ‘Gums and Stabilisers for the Food Industry’, ed. G . 0. Phillips, D. J. Wedlock, and P. A. Williams, IRL Press, Oxford, 1992, vol. 6, p. 241. 2. E. M. Brown, J . Dairy Sci., 1984, 67, 713. 3. B. Closs, M. Le Meste, J . L. Courthaudon, B. Colas, and D. Lorient, in ‘Food

L. A . Wasserman and M . G. Semenova

91

Polymers, Gels and Colloids’, ed. E. Dickinson, Royal Society of Chemistry, Cambridge, 1991, p. 571. 4. M. Miura and F. Yamauchi, J. Agric. Biol. Chem., 1984,48,2449. 5 . M.N.Jones and A. Brass, in ‘Food Polymers, Gels and Colloids’, ed. E. Dickinson, Royal Society of Chemistry, Cambridge, 1991, p. 65. 6. S. Damodaran and J. E. Kinsella, J . Agric. Food Chem., 1981,29,1249. 7 . G. Fauconneau, in ‘Plant Proteins for Human Food’, ed. C. E. Bodwell and L. Petit, Martinus Mijhoff, The Hague, 1983, p. 1. 8. H. D. Belitz and W. Grosch, ‘Food Chemistry’, Springer-Verlag, Berlin, 1987. 9. E. Derbyshire, D. J. Wright and D. Boulter, Phyrochemistry, 1976,15,3. 10. C. Tanford, ‘Physical Chemistry of Macromolecules’, Wiley, New York, 1961, p. 772. 11. J. W. Donovan, C. J. Maples, J. G. Davis, and J. A. Garibaldi, J . Sci. Food Agric., 1975,26,73. 12. P. E.Stein, A. G. W. Leslie, J. T. Finch, and R. W. Carrcll, J. Mol. Biol., 1991, 221,941. 13. R. Y. Yada, R. L. Jackman, and J. L. Smith, in ‘Encyclopediaof Food Science and Technology’, ed. Y. H. Hui, Wiley-Interscience, New York, 1992, vol. 3, p. 2191. 14. S. A.Woodward, in ‘Food Gels’, ed. P. Harris, Elsevier Applied Science, 1990, p. 175. 15. K.L.Fligner and M. E. Mangino, in ‘Interactions and Food Proteins’, ed. N. Parris and N. Barford, ACS Symposium Series, American Chemical Society, Washington, D.C., 1991, ~01454,p. 1. 16. A. Kato, Yu Osako, N. Matsdomi, and K. Kobayashi, Agric. Biol. Chem., 1983, 47,33. 17. A. Kato and S. Nakai, Biochim. Biophys. Acta, 1980,624,13. 18. E.Dickinson, J. Chem. SOC. Faraday Trans., 1992,88,2973.

Monoglyceride Mixed Films: Structure and Stability By C. Carrera Sanchez, J. de la Fuente Feria, and J. M. Rodriguez Patino DEPARTAMENTO D E INGENIERIA QUIMICA, UNIVERSIDAD

DE SEVILLA, FACULTAD DE QUIMICA, C/PROF. G A R C ~ A GONZALEZ, SIN., 41012 SEVILLE, SPAIN

1 Introduction Surfactant compounds used in commercial applications typically consist of a mixture of surfactants because they can be produced at lower cost than isomerically pure surfactants, and also because mixtures of different surfactants often exhibit better properties than each one alone.'*2The study of mixed films is important because it leads to an understanding of the preferential structuring of the interface which is of significant practical importance. Interfacial interactions and film characteristics (structure, elasticity, stability, miscibility, etc.) can be studied from measurements on mixed monolayers at the airwater interface. This work is an extension of earlier studies of mixed emulsifier monolayers spread on aqueous solutions.ss We are concerned with the study of structural characteristics of mixed films of monostearin + monoolein as a function of the nature of the sub-phase (water or aqueous ethanol) and the composition of the monoglyceride mixture. This information i s determined directly from the 11-A isotherms obtained using the Langmuir trough technique with spread monolayers of these mixed lipid components. The stability of emulsions and foams can be directly related to the molecular loss of surfactants from the interface. This molecular loss is confirmed by relaxation experiments keeping the surface pressure or the molecular area constant. The mixed monolayer stability is a function of the molecular interactions at the interface as well as the studied variables-temperature, film and sub-phase compositions, and the rheological characteristics of the monolayer (surface viscosity and elasticity).

92

C. Carrera Sdncher, J. de la Fuente Feria, and J . M . Rodriguez Patino

93

2 Experimental Monoolein (l-mono(cis-9-octadecnoyl)-rac-glycerol) and monostearin (1 monooctadecanoyl-rac-glycerol) were acquired from Sigma and used without further purification. Seven mixtures with mole fractions of monoolein, xo, ranging between 0 and 1 (0, 0.2, 0.4, 0.5, 0.6, 0.8 and 1) were made up in hexane-ethanol(9:1, v.v) solvent. The sub-phase was deionized water purified by means a Millipore device (Milli-a). Ethanol as solute in the sub-phase (acquired from Merck) was used at 0.5 and 1 mol/L concentrations. The surface pressure (II) versus molecular area ( A ) measurements were performed at the liquid-air interface by Langmuir's method using a Lauda balance. The spreading of the mixtures and the experimental conditions for measuring the isotherms has been described in detail p r e v i o u ~ l y . ~The ,~*~ experiments were carried out at temperatures ranging from 10 to 40 "C. The equilibrium spreading pressures (TI,) of the mixtures and pure components were obtained by the Wilhelmy plate method with similar experimental conditions to those described e l ~ e w h e r e Spreading .~ the mixed components from the solution repeatedly, it was possible to obtain a constant value of the surface tension that returned to the original value after a new deposition. This constant value of surface tension (ye) is the basis for determining IIe(i.e.,11, = YO - Y e ) . The instability of the mixed monolayer due to collapse or desorption in the sub-phase was determined by relaxation experiments keeping constant either the surface pressure or the molecular area (in the collapse point), using a similar method to that described elsewhere.* The analysis of the data, maintaining the molecular area constant, using the Prout-Tompkins equation' implies an instability of the molecules by collapse due to nuclei formation and further growth.

3 Miscibility of Mixed Monolayers If the monolayer-forming components are immiscible, all properties of the mixed film should be appropriate averages of those of the corresponding pure monolaycrs at the same temperature and surface pressure. Any deviation from the additivity relationship then indicates some sort of molecular interaction, and hence miscibility. If the two components of the mixed monolayer have equilibrium spreading pressures II, and II, (II, > 112),we know that the mixed film is stable to a surface pressure greater than I12.'" This can be used as a criterion for assessing the miscibility or immiscibility of the film-forming components. Knowledge of 11, for the mixtures is also significant because it represents the point at which spread monolaycrs become thermodynamically unstable with respect to the bulk lipid phase. It is important to study the mechanism of monolayer instability. Instability due to monolayer collapse must be rejected at ll < ll,. Other causes of instability, such as film dissolution, film evaporation and/or surface chemical reaction, can appear for either II < II, or ll > II,.

Monoglyceride Mixed Films: Structure and Stability

94

Figure 1 shows the equilibrium surface pressure-temperature dependence for the pure components and the mixtures for sub-phases of water or an aqueous solution of ethanol of concentration 1 M. Data for monoolein and monostearin pure monolayers on buffer solution (pH = 7 and ionic strength = 0.05 M) are practically coincident with those previously reported7 for pure water as the sub-phase. The ions present in the sub-phase apparently do not affect the nevalues for these pure components. The equilibrium surface pressure for monostearin monolayers increases with temperature. However, the equilibrium surface pressure is practically independent of temperature for monoolein and monoolein + monostearin mixed monolayers. The value of the equilibrium surface pressure in monostearin monolayers is lower than in monoolein or mixed films. The values of the surface pressures for mixed films are practically independent of the film

45 E 40

t

E 35

30

25 2 3

290

300

310

T, K ......

Q.'. ...............

30

;

0

1

280

0 ................... 0- ................ Q.,.....

290

300

310

T, K Figure 1

Equilibrium surface pressure versus temperature relationship as a function of monolayer and sub-phase composition: ( A ) water (phosphate buffer, I = 0.05 M ,p H = 7); ( B ) ethanol (phosphate buffer, I = 0.05 M , p H = 7). Monolayer composition (xJ: A,0.2; 0 , 0.5; 0, 0.8; pure monostearin (continuous line);pure monoolein (broken line)

C. Carrera Sanchez, J . de la Fuente Feria, and J . M . Rodriguez Patino

95

composition, and similar to that for a pure monoolein monolayer. Isotherms of pure and mixed films on water show that it is possible to compress the monostearin monolayers above the equilibrium surface pressure. The monostearin isotherm shows a metastable range of existence at surface pressures between the equilibrium and the collapse pressures. This behaviour is not observed with monoolein or the mixed films which collapse at a surface pressure close to the equilibrium spreading surface pressure. The independence of n, on film composition implies an immiscibility of the two components in the mixed film under the equilibrium conditions. The miscibility of the components at surface pressures different from the equilibrium can be studied from the relationship between the mean area and the composition for monostearin monoolein mixed films spread on water and on 0.5 and 1 M aqueous ethanol solutions. Figure 2 shows this dependence at 20 "C for surface pressures of 10,20 and 30 mN m-I. With water as sub-phase, a linear relationship exists between molecular area and mixture composition, and so the film-forming components must be either immiscible or ideally miscible; so, the existence of specific molecular interactions at the interface must be rejected." In fact, the resultsshow a small expansion in the structure of the mixed monolayers with water and aqueous ethanol solution. The interactions existing between the two components at the interface might be repulsive. This phenomenon is more evident with ethanol in the sub-phase and is the opposite to the result obtained for the monostearin + distearin mixed film."' The positive and negative deviations obtained in A versus mole fraction curves for the mixed monolayers can be explained in terms of the model suggested by Cadenhead and Miiller. According to this model, the positive deviations obtained in A-x mole fraction curves may be attributed to film miscibility when one of the components cannot be readily accommodated and so disturbs the packing of the second component such that it increases the intermolecular spacing. This, in turn, causes a decrease in dispersive interactions, whereas negative deviations obtained in these curves may bc attributcd to geometrical rearrangement of the two components which results in a decrease in area per molecule, causing enhanced dispersive interactions. It is important to observe that positive deviations exist in those experimental conditions in which loss of molecules by desorption in the sub-phase exists, as will be described below. This implies a disturbance in the packing of the first component by the presence of the second. Conclusive indication of film immiscibility on any sub-phase was, according to thc surface phase rule,'3 the invariance in the collapse surface values as a function of the molar ratio (see Table 1). Mixtures present a collapse zone close to the monoolcin collapse surface pressurc. The substances studied in this work had different two-dimensional phases when they were spread alone, and practically opposite interfacial orientations at the same surface pressure. The results are in agreement with those previously ~ b t a i n e d . 'It~ was possible to observe small deviation from the additivity rule for the areas as a function of the mixture molar ratios, the subphase composition, the temperature, and the surface pressure. At low values of

+

''


96

0,O 0,2

0,4 0,6

0,8 1,0

XO

1 Ethanol 0.5M

I

0,O 0,2 0,4 0,6

0,6 0,8 1,0

0,O 0,2 0,4 XO

Figure 2

0,8 1,0

.,

Area per molecule versus monoolein-monostearin mixed monolayer com10; 0,20; position at 20 "C, as a function of surface pressure (mNlm): 25; A,30

+,

the surface pressure this positive deviation is greater than at higher values. The effect of temperature on the miscibility of the monolayers is a function of the sub-phase composition. With water as sub-phase, the temperature influence on A,,, = A - A l z ,where A12is the molecular area obtained by applying the

97

C. Carrera Sanchez, J . de la Fuente Feria, and J . M . Rodriguez Patino

Collapse pressure of pure components and mixed monolayers of monoolein monostearin as a function of the monoolein content (mole fraction x,)

Table 1

+

~~

I M Ethanol

0.5 M Ethanol

Water ~~

20 "C

40 "C

20 "C

40 "C

20 "C

40 "C

52.2 44.7 43.8 44.0 43.7 44.5 44.2

50.8 42.7 42.7 42.8 42.3 42.8 42.2

42.7 34.3 33.7 34.3 34.0 34.0 33.7

41.7 32.8 33.0 33.2 32.8 32.7 32.5

36.9 29.2 29.1 29.0 29.0 28.7 28.7

35.7 27.5 27.8 27.8 27.8 27.1 27.1

0 0.2 0.4 0.5 0.6 0.8 1

additivity rule and A is the experimental area of the mixed monolayer, is practically insignificant (Table 2). However, with aqueous ethanol solution as the sub-phase, an increasing temperature is associated with an increase in the positive value of A,,,. This means that the interfacial penetration of the ethanol molecules (which possess amphiphilic character) induces a large expansion of the monolayer even in presence of monolayer instability by desorption. This phenomenon is more evident at low surface pressure values, where the monolayer structure allows the adsorption of ethanol molecules between monoglyceride ones, increasing the area per molecule of the mixed monolayer. The penetration of ethanol molecules into the mixed monolayers can induce in other cases an opposite effect (for example, for mixed monolayers of monostearin distearin where a model of association can explain the contraction of the monolayer4) for cases where negative values of A,,, were found. The behaviour depends on the surface-active character of the components and on the geometrical rearrangements of the molecules at the interface.

+

Table 2

The quantity A,, as a function of sub-phase composition, surface pressure and temperature 0.5 M Ethanol

Water 20 "C x, 0.2 0.4 0.5 0.6 0.8

10"

40

40 "C 10

1.48 0.26 4.36 4.9 1 6.32 3.06 0.4 3.55 4.04 0.08 2.38 2.22 0.24 -0.96

"Surface pressure in m N m-'.

20°C 40

10

1.06 3.22 2.7 2.75 0.84

4.46 6.82 3.95 2.78

30

I M Ethanol

40°C 10

30

20 "C 10

2.34 12.2 3.76 -1.12 3.18 17.2 7.02 4.46 1.8 8.9 5.1 2.2 1.02 6.3 4.28 1.54 3.54 1.66 3.9 3.04 4.42

40 "C 25

-0.7 2 0.9 0.4 2.1

10

25

5.56 12.9 8.1 5.58 5.84

0.76 5.52 4.95 3.98 3.74

98


4 Monolayer Elasticity Film elasticity can be a useful parameter for quantifying intermolecular and film-sub-phase interactions. The dilational elasticity is a measurement of the resistance to a change in the film area.15 From another point of view, dynamic surface pressure and film elasticity play an important role in many processes such as emulsification, foaming, extraction, distillation, and chemical or electrochemical surface reactions. l 6 The elastic modulus (-dIIldA) can be calculated directly from the &-A isotherms. The elasticity modulus depends on the surface pressure, the temperature, and the sub-phase composition. As the temperature increases, the monolayer is more expanded, and so the value of the modulus for the monolayer decreases. The monoolein monolayers that maintain a liquidexpanded structure in the temperature range between 10 and 40 "C present similar values of the elastic modulus no matter what the temperature. As the surface pressure increases, the elastic modulus also increases. That is, the greater interaction between molecules at the interface induces a more rigid monolayer. Ethanol in the sub-phase is associated with a contraction of the monolayer." In the pure and mixed monostearin + monoolein monolayers spread on aqueous ethanol solution (1 M), the values of the elastic modulus are higher than those obtained using water as the sub-phase (data not shown). Figure 3 shows the variation of the elasticity with the surface pressure for

0 Figure 3

10

20

30 40 n, mN/m

50

60

Elasticity for pure und mixed monolayers of monostearin + monoolein spread on water (phosphate buffer, I = 0.05 M; pH = 7 ) ai 20 "C, as a function of monolayer composition (xo): 0, 0; A,0.2; 0, 0.4; *, 0.5; V, 0.6; +, 0.8; 0, 1.0

C. Carrera Sdnchez, J . de la Fuente Feria, and J . M . Rodriguez Patino

99

pure and mixed monolayers spread on water as a function of monoolein content (x,) at 20 "C. It can be seen that the value of the elasticity is higher for monostearin than for monoolein monolayers, and that it increases with the surface pressure. The mixed monolayers have similar values of elasticity to those of pure monoolein monolayers, and except at low content of monoolein (x, = 0.2) the value appears independent of sub-phasc composition. The values of the elasticity calculated directly from the isotherms are practically coincidcnt with the elasticity values obtained for these systems using a surface oscillatory viscometer (designed by Prof. Prins in Wageningen University).'8 This behaviour is characteristic of a monolayer that is predominantly elastic.

5 Monolayer Stability Different relaxation mechanisms-such as desorption, collapse, surface rheology, surface chemical reaction, change in the conformation, Marangoni effect, polar group hydration, erc.-can occur depending on the nature of the interactions between molecules at the interface. The data obtained from relaxation experiments can be fitted to any one of these. However, due to the slight solubility of the monoolein molecules in these systems,* and to the fact that other mechanisms are difficult to quantify, in this work we just tested the experimental data by means of equations derived from two relaxation mechanisms: desorption and collapse. The molecular loss was studied kinetically from relaxation experiments at constant temperature. The loss was determined either from a decrease in film area as a function of time, at constant surface pressure, or by a decrease in surface pressure as a function of time, at constant area. In the first case-keeping the surface pressure constant-it is possible to determine the degree of molecular dcsorption from the monolayer. Two processes are involved in the desorption: immediately upon spreading, a portion of lipid dissolves into a thin region of the sub-solution directly beneath the surface. The maximum amount of lipid that dissolves is determined by the Gibbs adsorption isotherm, and for most systems thc concentration is the equilibrium value." Dissolution is followed by the diffusion of lipid into the bulk of the sub-solution, and the rate of diffusion depends on the concentration of lipid in the thin region beneath the surface. For lipid films that appear to be insoluble, the amount of lipid that desorbs is small relative to the total amount spread on the surface, and the rate of diffusion is extremely slow; the surface concentration of lipid appears to be unchanged for the duration of typical film balance cxpcriments.20 During the initial non-steady-state period the rate of film molecular loss is a linear function of the square root of time:

During the diffusion process the rate of film molecular loss is a linear function

loo


of time:

In equations (1) and (2), where N , and N o are the numbers of molecules remaining in the surface at time 8 , and at the initial moment, respectively, and a and b are the Coefficients of regression. The analysis of the data using equations (1) and (2) gives the values of the a and b coefficients and the mechanism change times (6.) in Tables 3 , 4 and 5 . As already observed by other investigators, the behaviour can be split into two or three regimes.'* From these data we draw the following conclusions. (a) With watcr as sub-phase at 20 "C, the mixed and pure component monolayers are stable. The surface pressure is lower than the equilibrium spreading pressure for these monolayers, and the initial decrease in the ratio N J N , is due to molecular rearrangement at the interface.8 (b) The pure monostearin monolayers are stable in all cases, i . e . , ethanol solution or water as sub-phase, at 20 "C or 40 "C, and at II>IT,. However, the mixed monolayers of monostearin monoolein are unstable with water as subphase at high temperature (40 "C) or when spread on aqueous ethanol solution at 20 "C or 40 "C. (c) With water as sub-phase at 40 "C, the data are consistent with a mechanism of desorption followed by diffusion for monoolein or mixed monolayers. The change in mechanism occurs at approximately the same value of time (6.). (d) Two regimes of desorption and diffusion appear with 0.5 M ethanol as subphase at 20 "C for mixed and pure monoolein monolayers. The mechanism is independent of the monolayer composition for mixed monolayers, and the values of coefficients obtained on fitting the data are very similar. On increasing the temperature (up to 40 "C)or the ethanol concentration (Tables 4 and 5), we find a third regime of instability which follows equation (2) with lower values of the (b) coefficient (b2). Relaxation experiments with monostearin + monoolein mixed monolayers were also carried out at the collapse area using water or aqueous ethanol solution (0.5 or 1 M) as sub-phase. Relaxation phenomena were followed at 40 "C by recording the changes in the surface pressure as a function of time. Processing the experimental data according to the Prout-Tompkins law,9it was possible to fit the data with

+

log

rI -IT IT

L -J .-

=m

log 8 + n,

(3)

where II and II, are the surface pressures at time 6 and at the start, respectively, and rn and n are coefficients which depend on the experimental conditions. The fitted values of the coefficients are shown in Table 6.

101

C. Crirrera Sanchez, J. de la Fuente Feria, and J. M . Rodriguez Patino

Table 3

+

Destabilization of mixed monolayers of monostearin monoolein (Sub-phase: water (buffer 0.05 M); surface pressure = 30 mN m-')

n,

a.ld

xo

TI "C

mNm-'

Structure

(LR)"

0 0.2 0.8 1 0 0.2

20 20 20 20 40 40

27.9 45.8 46 45.5 34.5 43.3

LCh

-

0.5

40

43.7

LE'

0.8

40

43.9

LE'

1

40

43.3

LEE

LCh

LCh LE' LCh LEC

-

b,.ld b,.ld 91 ey/ 0; (LR)" (LR)" min NIN, min min -

- 130 0.91 - 129.5 0.95 -

-

-

-

12 (0.996) 7.1 (0.999) 14.4 (0.993) 15.67 (0.994)

86 (0.999) 39 (0.998) 137 (0.999) 94 (0.999)

-

222 123.6 176.7 140

-

-

0.91 0.96 0.96 0.70 12.5

-

-

-

- 200

0.80

8.8

-

-

80

0.73 14.4

-

-

115

0.70 15.5

-

Corrclation cocfficicnt of linear regression.

' Liquid condenscd monolayer structurc. Liquid cxpanded monolaycr structure.

Table 4

Destabilization of mixed monolayers of monostearin + monoolein (Sub-phase: eihano10.5 M (buffer 0.05 M); surface pressure = 30 mN m-')

xo

TI "C

14 mNm-'

Structure

0 0.2

20 20

19.8 41.4

LC LC

0.5

20

41.4

LC

0.8

20

41.4

LC

1

20

41.4

LE

0 0.2

40 40

29.2 39.9

LC LC

0.5

40

39.9

LC

0.8

40

39.9

LC

1

40

39.9

LE-LC

a.Id b,.ld b2.1d 01 e;i e; ( L R ) a ( L R ) a (LR)' min NIN,, min min ~~

Correlation coefficient.

- 181.4 5.11 23 - 163.2 (0.995) (0.999) 15 195.6 6.55 (0.998) (0.996) 5.9 90 - 128.5 (0.995) (0.998) 219 243 151.9 (0.999) (0.999) 256.8 12.8 163 44 299.9 (0.997) (0.998) (0.986) 15.54 158 69 251.1 (0.999) (0.996) (0.999) 26.7 527 656 121.4 (0.986) (0.999) (0.999) 826 253 121 (0.987) (0.997)

0.96 0.88 26.9

-

0.88 20.6

-

0.77

47

-

0.45

-

93.66

0.93 0.66 30.8 115.4 0.49

20 107.4

0.69 10.6 0.40

-

95 15.8


102

Destabilization of mixed monolayers of monosfearin + monoolein (Sub-phase: ethanol I M (huffer0.05 M ) ; surface pressure = 20 m N

Table 5

m-')

ri,

a.I@

X"

TI "C

rnNm-'

0 0.2

20 20

13.8 30.1

LC LC

0.8

20

30

LE

1

20

30.8

LE

Structure (LR)"

~,.IoPb2.10S

61 071 0:; (LR)a (LR)" min NIN, min min ~~

-

-

153 155

-

0.97 0.92 14.4 111.9

4 20 4.63 (0.997) (0.967) (0.944) 18 9 181.9 0.90 12.1 110.9 6.05 (0.998) (0.999) (0.999) 39 22 250 0.83 25.9 90.9 4.47 (0.996) (0.998) (0.986)

'' Correlation coetficient.

Table 6

Prout-Tompkins equation. Destabilization of mixed monolayers of monostearin + monoolein at constant collapse area Ll,IrnNrn-'

x,

elmin

ml

LR"

Blmin

m2

(Sub-phase: waler (buffer 0.05 M);T = 40 "C) 0 0.2 0.4 0.5 0.6 1

50.6 42.8 42.5 42.3 42.8 41.9

40.3 31.0 31.4 18.5 62.4 89.4

0.201 0.929 0.774 0.992 0.982 1.013

0.999 0.987 0.998 0.999 0.998 0.995

L R"

-

-

-

2.5 2.5 2.7 2.7 2.3

0.552 0.568 0.446 0.424 0.470

0.999 0.999 0.996 0.995 0.999

(Sub-phase: ethanol 0.5 M (buffer 0.05 M ) ; T = 40 "C) 0 0.2 0.4 0.5 0.6 0.8 1

42.3 33.2 33.5 33.5 33.2 33.0 32.7

41.4 64.8 42.6 51.9 37.5 36.8 35.5

0.225 1.033 0.999 1.014 1.012 1.188 1.030

0.996 0.994 0.998 0.996 0.997 0.999 0.985

-

-

-

3.3 2.2 3.8 5.5 2.5 3.2

0.559 0.291 0.499 0.497 0.413 0.462

0.998 0.994 0.999 0.999 0.982 0.995

(Sub-phase: ethanol I M (buffer 0.05 M ) ; T = 40 "C) 0 0.2 0.4 0.5 0.6 0.8 1 a

35.8 27.8 28.4 28.4 28.4 27.6 27.4

Correlation coefficient.

97.8 32.0 32.5 31.0 90.1 25.0 23.6

0.262 1.016 1.295 0.878 0.926 0.947 0.990

0.995 0.994 0.999 0.981 0.979 0.997 0.996

-

-

-

3.7 5.0 5.4 4.3 3.3 3.9

0.564 0.207 0.363 0.389 0.389 0.475

0.994 0.973 0.997 0.997 0.998 0.985

C. Carrera Shchez, J . de la Fuente Feria, and J . M . Rodriguez Patino

103

In some cases, with pure monoolein and mixed monolayers, the same equation gcnerates two different rate constants, usually ascribed to the acceleration and decay periods, respectively. The collapse process takes place in several steps which is typical of nucleation and growth phenomena.” The absence of specific interactions between monostearin and monoolein molecules can explain the fact that the values of the coefficients are practically independent of the composition of thc mixed monolayers, with values close to 1 in the first step and close to 0.5 in the second onc. However, with monostearin monolayers a single period is present with either water or aqueous ethanol solution as the sub-phase. The rclaxation of II is due to the collapse by nucleation and subsequent growth, of the monoolein molecules, and it is not affected by the presence of monostearin molecules. This behaviour is the opposite to what is observed with other acylglycerol mixed m o n o l a y c r ~ where , ~ ~ the existence of interactions between the components, competing with interactions between monolayer and sub-phase molecules, affects the film stability.

6 Conclusions In this work we have studied the monostearin-monoolein monolayer structure and stability from II-A isotherms and kinetic experiments of molecular loss from the monolayers spread on water or aqucous ethanol solutions as a function of monostearinlmonoolein ratio, temperature, and surface pressure. The monolayer structure (restricted to the liquid-condensed and liquidexpanded states in these systems), the clasticity modulus, and the mechanism of the instability of the monolayer is shown to be a function of the sub-phase composition and the temperature, but not the surface composition of the mixed monolayers. The mixed systems tend to follow the same structural and stability characteristics as the pure monoolcin monolayers. From thcse experiments it can be deduced that, in the monostearin monoolein mixed films, the molecules are not miscible at the air-water interface-as is demonstrated by comparison of changes in area calculated through the additivity rule and those obtained experimentally. The plot of molecular area against molc fraction is linear overall with water as the subphasc, especially at highcr surfaces pressures and lower temperatures. The existence of clusters of monoolein in a continuous film formed principally by monostearin induces a higher value of the molecular area than that obtained by the additivity rule, although in presence of monolayer instability. This is an indication that the components are not miscible. This is demonstrated by considering both kinetic studics and changes in the equilibrium surface pressure and collapse point as a function of the monolayer composition. However, a change in the conditions could possibly induce compatibility of the components at the interface. At surface pressures lower than the equilibrium surface pressure, the monostearin + monoolein monolayers are unstable due to desorption of the monoolein molecules. For values higher than He (kinetic relaxation cxperi-

+

104


ments at constant collapse area) collapse by nucleation and growth is evidcnt for both mixed monolayers and pure components. Depending on the temperature and sub-phase composition, one or two steps can appear in the destabilization. When there are two steps in the relaxation, a deceleration of the process occurs.

Acknowledgements Professor Eric Dickinson is thanked for his assistance. The financial support obtained from the DGICYT (PB94-1459) is acknowledged.

References 1. M. J. Rosen, ‘Surfactants and Interfacial Phenomena’, 2nd edn, Wiley, New York, 1989. 2. J. F. Scamehorn, ‘Phenomena in Mixed Surfactant Systems’, ed. J.F. Scamehorn, ACS Symposium Series No. 311, American Chemical Society, Washington, DC, 1986. 3. J. de la Fuente Feria and J. M. Rodriguez Patino, AIChE J . , 1995,41, 1955. 4. J . de la Fuente Feria and J . M. Rodriguez Patino, Langmuir, 1995, 11, 2163. 5. J. de la Fuente Feria and J. M. Rodriguez Patino, A I C h E J . , 1996, 42, 1416. 6. J. M. Rodriguez Patino, J . de la Fuente Feria, and C. G6r:iez Herrera, J . Coffoid Interface Sci., 1992, 148, 223. 7. J. M. Rodriguez Patino and R. M. Martin Martinez. J . Colloid interface Sci., 1994, 167, 150. 8. J. de la Fuente Feria and J . M. Rodriguez Patino, Langmuir, 1994, 10,2317. 9. E. G. Prout and F. C. Tompkins, Trans. Faraday SOC.,1944,40, 488. 10. G. L. Gaines, Jr., J . Colloid Interface Sci., 1966, 21, 315. 11. G . L. Gaines, ‘Insoluble Monolayers at Liquid-Gas Interface’, Interscience, New York, 1966. 12. D. A. Cadenhead and F. Miiller-Landau, J . Colloid Interface Sci., 1980, 78, 269. 13. D. J. Crisp, Surface Chemistry Suppl Research, London, 1949, p. 17. 14. A. Niccolai, P. Baglioni, L. Dei, and G. Gabrieli, Colloid Polym. Sci., 1989, 267, 262. 15. S. H. Kim and J. E. Kinsella, J . Food Sci., 1985, 50, 1526. 16. J . T. Davies, ‘Turbulence Phenomena’, Academic Press, New York, 1972. 17. J. M. Rodriguez Patino, M. Ruiz Dominguez, and J. de la Fuente Feria, J. Colloid Interface Sci., 1992, 154, 146. 18. M. R. Rodriguez Nirio, P. Wilde, D. C. Clark, and J. M . Rodriguez Patino, f n d . Eng. Chem. Res., m press. 19. N. L. Gershfeld and C. S . Patlak, J . Phys. Chem., 1966, 70,286. 20. N. L. Gershfeld, Ann. Rev. Phys. Chem., 1976,27, 349. 21. P. de Keyser and P. Joos, J . Colloid Interface Sci., 1983,91, 131. 22. P. Baglioni, G . Gabrielli, and G . T. Guarini,J. Colloid InterfaceSci., 1980,78,347. 23. J . de la Fuente Feria and .I. M. Rodriguez Patino, Colloids Surf. A , 1995, 104,29.

Aggregation Processes, Particle Interactions, and Colloidal Structure By Eric Dickinson PROCTER DEPARTMENT OF FOOD SCIENCE, UNIVERSITY OF LEEDS, LEEDS LS2 9JT, UK

1 Introduction The stability and rheological properties of food colloids are dependent on the ways in which the constituent particles and macromolecules are assembled together to form colloidal structures. Different kinds of interparticle interactions lead to differcnt aggregation mechanisms, and different aggregation processes lead to different kinds of colloidal structures. In order to increasc our understanding of the link between the particle interactions and the colloidal properties, it is necessary to consider what are the main factors controlling the mechanisms of colloidal aggregation. What is meant here by the word ‘particle’ is rather wide. At the smallest cxtreme it could refer to single macromolecules (e.g. B-lactoglobulin); at the largest to droplets of emulsified oil or water; it could also be applicable to protein oligomers, protein particles ( e . g . casein micelles) and very small crystals ( e . g . of triglyceride). The only condition is that Brownian motion should be important in determining thc movement of the particlcs and hence the resulting aggregate structure. This restricts the particle size to less than a few micrometres. In categorizing types of colloidal structure, a useful distinction can be made between reversible and irreversible aggregation processes. The former are primarily determined by thermodynamic considerations; the latter by kinetic considerations. Both are influenced by the nature of the interparticle interactions. Net repulsive interactions imply colloidal stability and hence inhibition of aggregation. When the interactions are strongly attractive, aggregated particlcs become permanently bonded together. When the interactions are more weakly attractive, there is opportunity for continuous aggregatc reorganization and the setting up of an equilibrium between coexisting aggregated and unaggregated phases. More complex situations may also occur with particles that are permanently, but flexibly, bonded to other particles; in such systems other repulsive or attractive non-bonded interactions can influence further the 107

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Aggregation Processes, Particle Interactions, and Colloidal Structure

detailed colloidal structure. Despite the obvious compositional complexity of real food colloids, it appears that the main structural characteristics of these various classes of multi-particle systems can be understood in general terms using established statistical mechanical theories and computer simulation techniques applied to the behaviour of rather simple models. A crucial aspect of exploring concentrated systems of this sort is that we must learn to think in terms of the structure, not of separate individual aggregates, but of the colloidal system as a whole.

2 Particle Interactions In describing the form of the interparticle interaction, what we are usually concerned with is the change in free energy AG(h) associated with bringing together a pair of colloidal particles with surface-to-surface separation h. It is convenient in many cases to regard this free energy function AG(h) as being equivalent to a pairwise-additive potential energy function U(h)-although strictly speaking the function should be called a ‘potential of mean force’ in recognition of the major entropic contribution to the free energy of interaction.‘ The form of U(h)depends, of course, in a complicated way on the particle surface properties and on the chemical composition of both the particles and the medium in which they are dispersed. For certain idealized ~,~ cases the general shape of U(h)is known ab initio from analytic t h e ~ r y(and occasionally directly from macroscopic surface force experiments). For instance, curve A in Figure 1 shows a plot of U(h) for a pair of electrostatically stabilized spherical particles in a colloidal dispersion as described by classical DLVO theory.2For particles of a micrometre diameter or above in an aqueous

Figure 1

Realistic forms of thepair interaction potential U(h) as a function of surfaceto-surface separation h for (A} electrostatically stabilized particles (DLVO potential) and ( B } sterically stabilized particles

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109

medium of low ionic strength, the theoretical potential U(h)is characterized by a large energy barrier. This so-called ‘primary maximum’ prevents pairs from irreversibly jumping together at very close separations (in the ‘primary minimum’) over the normal experimental timescale. At larger separations (beyond the energy barrier in the DLVO potential), there is a shallow attractive region (the ‘secondary minimum’) which induces pairs to associate reversibly into what, in principle at least, should be freely rotating doublets (though, confusingly, some recent experiments4 are apparently consistent with such flocs behaving as rigid doublets!). Under different conditions, the energy barrier could be of reduced or zero height (e.g. at medium or high ionic strength), or the secondary minimum could be missing altogether ( e . g . with small colloidal particles). Curve B in Figure 1 represents the equivalent theoretical potential for particles sterically stabilized by an adsorbed layer of p o l y m ~ rWhile .~ the functional form of U(h) resembles curve A at largc h, the short-range steric repulsive region of curve B is distinctly steeper at small pair separations, and the DLVO primary minimum is absent altogether because the presence of the (irrcversibly) adsorbed polymer prevents the particles from getting so close. The ‘pseudo-secondary minimum’ of the steric potential may also be missing in many systems (e.g. with small particles, long polymer chains, a n d o r good solvent conditions). In food colloids the precise form of U(h)is never known. What is sometimes known is the likely mechanism of stabilization and the approximate magnitude of some energy barrier or attractive well depth. The latter are typically inferred from experimental observations of coagulation kinetics, phase transitions or rheological behaviour. This is done using some established theory which relates the measured quantity (turbidity, viscosity, erc.) to the interparticle interactions. As the validity of the theory normally relies on a number of assumptions that are of questionable validity when applied to food systems, the potential parameters inferred in this way can be regarded only as a very rough indication of relative interaction strengths, and not as numbers of absolute significance. The limited aim of this paper is to highlight the key aspects of interparticle interactions that are responsible for determining the main features of structure, stability and rheology in aggregated colloidal systems. So, rather than discussing realistic potentials whose forms vary considerably from system to system, it is here more useful to consider sets of idealized potentials such as the ones illustrated in Figure 2. In case (a) the potential is strongly attractive at very close separations and zero at larger separations. Particles of this hypothetical type are susceptible to fast irreversible coagulation leading to diffusioncontrolled fractal-type aggregates with little relative particle movement or aggregate rearrangement. In case (b) the potential is repulsive at close separations and attractive at intermediate separations. Particles of this type are susceptible to rapid reversible flocculation, with the resultant aggregate structure and mobility dependent on the potential well depth Umin(relative to the thermal energy kT, where k is Boltzmann’s constant and Tis the temperature). For very deep potentials of type (b), the aggregation becomes effectively

110


(4

(b)

I

Figure 2

h

I

U

h

Four idealized forms of the pair interaction potential U(h) as a function of surface-to-surface separation h. See text for full discussion of the cases (a) through (d)

irreversible [like case (a)], but extensive aggregate rearrangements may still take place [unlike case (a)]. Potential (c) combines the key features of potentials (a) and (b). Particles of this type are susceptible both to irreversible coagulation at very close separations and reversible flocculation at intermediate separations. Unlike case (a), however, the coagulation is reactioncontrolled, with the value of the rate constant decreasing exponentially with the potential barrier height U,,,, and the resulting aggregate structure being sensitively dependent on floc rearrangements prior to coagulation. Type (c) systems with large U,,, ( 2 15k7') are stable to coagulation and therefore behave like type (b) systems. A further degree of complexity occurs in case (d) with the presence of a second potential maximum at large separations. Particles of this type experience a kinetic barrier to reversible flocculation, with the flocculation rate constant dependent on the secondary barrier height Ukar So far it has been assumed that the interparticle potential U ( h ) is a simple time-invariant function characteristic of the particular colloidal system. However, this is oftcn not the case in food systems where the aggregation process is triggered gradually by heating, pH change or enzyme action. In such cases, for instance, there may be occurring a continuous change in either the well depth for a type (b) system or the barrier height for a type (c) system. These effects

111

E. Dickinson

(ii)

-

tire

Figure 3

h 3 Illustration of ways in which the pair interaction U(h) may change with time: (i) the depth of the secondary minimum of type (b) potential (see Figure 2 ) increases with time; (ii) the height of the primary maximum of type (c) potential (see Figure 2) decreases with time

are illustrated schematically in Figure 3. Time-dependent changes in U ( h )of this sort are manifest experimentally as an apparent 'lag phase' in the kinetics prior to the initial detection of flocculation or coagulation. Alternatively they may be indicated by structural changes associated with complex reorganizational events during or following the onset of aggregation.

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3 Irreversible Cluster-Cluster Aggregation While the experimental observation of the formation of dendritic and chainlike aggregate structures by irreversible coagulation had been recorded on many occasions in the colloid science l i t e r a t ~ r e it, ~was ~ only through the application of computer simulation that the origin of this type of aggregate structure was definitively as being simply the result of random irreversible collisions. The major step forward over the past decade or so has been the re'~ognition"-'~that colloidal aggregates are examples of statistical fractal objectsI4 which are on average self-similar over certain length scales. More precisely, they can be characterized as muss fractals since the mass M scales with the average size ra according to

M

- r$,

where df is the fractal dimensionality. In practice, the main factor determining df is the form of the interparticle potential U(h). Experimental studies of irreversible coagulation of particles in dilute dispersions typically give values of df = 1.8 under fast aggregation conditions and df = 2.1 under very slow aggregation conditions. These two limiting types of behaviour are well described in terms of two distinct cluster-cluster aggregation models-so-called 'diffusion-limited cluster aggregation' (DLCA) and 'reaction-limited cluster aggregation' (RLCA). In a computer simulation of cluster-cluster aggregation, the particles and clusters move by Brownian diffusion in a continuum fluid (or by random walks on a lattice), and the cluster diffusion coefficient is typically scaled in inverse proportion to the cluster radius. The DLCA model is applicable to interacting particles with U(h)like that in Figure 2(a): as soon as any two particles on different clusters get within a certain minimum distance (or, equivalently, they occupy neighbouring lattice sites), the original clusters become permanently joined together and thereafter diffuse as a new single cluster. In the RLCA model, however, a repulsive barrier must be crossed [see U ( h ) in Figure 2(c)] before the colliding clusters can contact each other to become irreversibly joined. The barrier height U,,, cannot be too large, of course, because otherwise the dispersion will be stable over the experimental (or simulation) timescale. On the other hand, it cannot be too small either, because the process then becomes diffusion-limited for large (slowly moving) clusters, implying a gradual 'crossover' from RLCA kinetics to DLCA kinetics (and hence a reduction in df) as the aggregation proceeds. A widely recognized process of irreversible aggregation in food science is the coagulation of casein particles induced by lowering of pH or enzymic hydrolysis with chymosin. It has been demonstrated experimentally's-17 that the growing aggregates which appear during the formation of an acid casein gel do indeed have a fractal character, and that the structure and rheological properties of the resulting casein gel can be understood in terms of scaling laws based on the effective characteristic fractal dimensionality. However, the df values

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fitted15-" for casein gels (d, -- 2.3 k 0.2) are significantly higher than the values quoted above for simulations based on the simple DLCA or RLCA model. The question arises, then, as to what are the important features missing from these simple aggregation models which need to be added before the computer simulations can be regarded as giving reliable representations of network structures formed from aggregating casein micelles (or other particles such as protein-stabilized emulsion droplets). There are two main factors that need to be addressed when considering the fractal properties of particle gels-(i) the effect of the finite particle concentration on aggregate growth and interpenetration, and (ii) the effect of interparticle interactions on aggregate restructuring before, during and after gelation. While the two issues are somewhat interrelated, let us first consider them separately, beginning with the issue of particle volume fraction $p. In the simplest cluster-cluster gelation m ~ d e l , ' ~ the - ' ~individual aggregates are assumed to grow exactly as they would at infinite dilution ($,, + 0) with a single fractal dimension df in the range 1.8-2.1 depending on whethcr the mechanism is DLCA, RLCA, or something in between. The number of particles of radius R in a fractal aggregate of radius ra and dimensionality d f is Nd

- (ra/R)df,

as compared with the number of particles in the equivalent close-pack:d aggregate:

N,

- (r,/R)3.

(3)

The volume fraction of particles in a fractal aggregate of radius ra is

The gel point is assumed to be reached at time t = t , when Gf becomes equal to $p, as illustrated in Figure 4. The critical aggregate radius r,* at the onset of gelation is then given by

There are two main assumptions in the above. (1) All aggregates have the same size at the gel point. This is obviously not correct because it is known" that fractal colloidal aggregates of finite extent produced by DLCA or RCLA have a wide time-dependent cluster-mass distribution N ( M , t ) . In reality, in a fairly concentrated dispersion, when the biggest clusters are just beginning to join together to form a system-spanning network, a significant proportion of the particles will still exist as monomers (or very small clusters) at t = t,; these 'free' particles will only become fully

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(ii)

Figure 4

Illustration of gelation according to the simple cluster-cluster aggregation model: (i) single fractal aggregate of particle volume fraction $f;(ii) gel point is reached when Gfequals the overall particle volume fraction @,

incorporated into the developing network structure at times well beyond the gel point (t >> t g ) . ( 2 ) Cluster-cluster interactions are neglected. This assumption must be strictly invalid for any system in the immediate pre-gelation regime ( t + tg) for which the average cluster size is approaching the critical size r z , and it is effectively invalid at all stages of the aggregation process (t > 0)for moderately concentrated systems. In the latter case, even in the very initial stages of coagulation, the local cluster growth is determined by the local particle

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concentration gradient, which in turn depends on the growth of neighbouring clusters. The evolving aggregate fractal structure in a concentrated system is therefore never at any stage the same as that for cluster-cluster aggregation at high dilution. Irrespective of whether the system is concentrated or dilute, however, once the mean cluster radius has approached r t , the increasing crowding and interpenetration of the largest fractal clusters must inevitably change the structure of aggregates in the gelling system from that in the nongelling DLCA or RLCA system at infinite dilution. Intuitively, this effect of cluster4uster 'excluded-volume' interactions would be expected to increase the compactness of the largest clusters, i.e. to increase the effective value of df. Hence, for these two important reasons, the simple cluster-cluster gelation model dcfined by equations (2) to ( 5 ) does not constitute a proper description of a particle gel system formed by diffusion-limited or reaction-limited mechanisms at finite particle volume fraction. The problem lies in part with the nonflcxibility of the aggregates formed by DLCA or RLCA. Both models assume that, once two particles on different clusters actually become joined togetherhowever little or much time this takes-the bond between them thereafter remains permanent and rigid. For a computer simulation of irreversible coagulation in a concentrated dispersion of N particles with periodic boundary conditions, this leads to the curious result'' that the final configuration is a single fractal-type cluster which does not form a percolating network. Another anomaly arising with aggregating fractal clusters which stick together rigidly and irreversibly is that, in principle, there is no lower theoretical particle concentration below which gelation cannot take place, since the volume fraction of a single fractal aggregate, &, can always become equal to the particle volumc fraction of the whole system, @p, if it is allowed to grow indefinitely (@f-+ 0 as r t - + CQ and tg-+ C Q ) .This contrasts with the behaviour in real experimental situations, where it is usually found that there is a welldcfined reproducible critical gelation concentration qbg.

4 More Realistic Particle Gel Models What is crucially missing from the DLCA model is the opportunity for clusters to deform or rearrange during aggregation. In principle, there are various ways in which this severe constraint of the DLCA model can be removed in both lattice and non-lattice simulations: (a) by allowing for bonds to break as well as to form;2"'21 (b) by introducing some flexibility into all bonds (continuum models ~ n l y ) ; ~or l - (c) ~ ~by incorporating internal cluster flexibility through more complex algorithms (bond f l u c t ~ a t i o nor~ ~aggregate shakin8'). The allowance for aggregate flexibility and/or restructuring by one of these methods does lead to behaviour that lies much closer to what is observed experimentally. True gelation occurs only above a certain critical particle volume fraction qbg, and computer simulation in a periodic cell results in a percolating network structure (@p > @.J instead of the single final cluster of the DLCA or RLCA models.

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Cluster reorganization prior to gelation ( r < tg) and restructuring of the aggregated network after gelation ( t B tg) are both dependent on the nature of the non-bonded particle-particle interaction potential U(h).Recent Brownian dynamics simulations in two and three dimensions of a model involving flexible irreversible bond formation and non-bonded particle interactions have shown21.22that the particle gel structure at constant q5p is sensitive to two key parameters: the rate of bonding as measured by the probability of bonding during a single simulation time-step, and an interaction parameter measuring the strength of the medium-range interparticle force (attractive or repulsive). For a periodic system of 1000 particles at volume fraction q5p = 0.1, Figure 5 illustrates the effect on the three-dimensional network structure of changing from (1) net repulsive non-bonded interparticle force to (2) net attractive nonbonded interparticle force. In both cases, particle pairs are allowed to stick together moderately slowly via irreversible bond formation, so that the final gel structure becomes held together by a percolating network of permancnt flexible bonds.26However, due to differences in the nature of the non-bonded pair interactions during gelation, the particle gel shown in picture 1 is characterized by a smaller average pore size and a spatially more homogeneous distribution of particle positions than that in picture 2. A general requirement for the formation of a homogeneous gel of uniform pore size is the presence of a repulsive interparticle interaction and a relatively low bonding probability. With particles having attractive non-bonded forces, the simulated gels are heterogeneous, and the network can have fine or coarse pore-size distributions depending on whether the bonding is fast or slow. In systems with non-bonded attractive interactions which are strong enough to induce phase separation [as in Figure 5 ( 2 ) ] , the emerging domains grow to some characteristic average size before they get ‘pinned’ by the irreversible cross-linking. The larger the bonding probability, the smaller the domains can grow before they become incorporated into the connected network structure. Some qualitative analogies can easily be identified between these simulation results and experimental data for particle gels made from food proteins. For instance, the known dependence of the degree of heterogeneity of globular protein gels on pH and heating conditions27328 can be readily explained in terms of the balance between two types of aggregation processes-(i) the generation of intermolecular cross-links (covalent and non-covalent) bctween denatured protein molecules which leads to permanent aggregation and network formation, and (ii) the thermodynamically reversible flocculation which leads to protein precipitation under poor solvent conditions. Let us return now to the factors affecting the fractal dimensionality of particle gels formed from aggregating spherical particles. In terms of increasing distance r between particle centres, three spatial scales of structure can be. expected:w (i) short-range liquid-like order from packing and cxcluded volume effects, (ii) medium-range disorder associated with the fractal character of pregclation clusters, and (iii) long-range homogeneity associated with a uniform bulk material. The short-range structure is reflected in the characteristic

E. Dickinson

Figure 5

117

Snapshots of three-dimensional simulated structure of bonded networks formed from Id spherical particles aggregating with ( I ) repulsive nonbonded interactions and (2) attractive non-bonded interactions26


118

oscillatory behaviour of the radial distribution function g ( r ) of a concentrated monodisperse and beyond some characteristic correlation length 5 the long-range structure is uniform [g(r) = 11. In between may lie a fractal scaling regime of necessarily limited spatial range. Irrespective of the form of U(h), with increasing overall particle concentration, the upper and lower boundaries of the medium-range fractal scaling regime will tend gradually to converge, eventually to disappear altogether for, say, &, =: 0.3. The effective value of dfcan be inferred” from the set of particle coordinates in a simulated gel by calculating the slope of the linear region of a plot of log n(r) against log r , where n(r) is the average number of particle centres lying within a distance r from a given particle. For large separations, we must have

where R is the particle radius. In the intermediate fractal scaling regime, we have

where the pre-factor no is the average number of particles in the primary clusters from which the fractal scaling regime is built. A low value of the prefactor (no 1) implies a fine-grained microstructure, whereas a high value (no >> 1) implies a coarse microstructure. The correlation length of a gel is given by”

-

This compares with 5 = r*, [equation (5)j for the DLCA model (no= 1). For an aggregating system with an extremely low bonding probability and a moderately strong attractive pair interaction [ e . g . , in Figure 2, the type (b) potential with Umin>> k T ] , a gel-like network formed in the early stages will tend gradually to change its fractal character due to restructuring and coarsening during spinodal d e c o m p o ~ i t i o nIf. ~cross-linking ~ is absent altogether, the fractal character associated with the interconnected structure will eventually disappear altogether since the system is ultimately thermodynamically unstable with respect to phase separation. This type of behaviour has recently been simulated for systems of aggregating Lennard-Jones particles using both molecular dynamics in two dimensions34 and Brownian dynamics in three dimensions.2’ The evolving microstructure is a percolating network structure composed of finely dispersed clusters arranged as filaments which gradually thicken with increasing simulating time. This is reflected in a reduction in df and a simultaneous increase in no. During the relentless drive towards phase separation, when some existing fine filament branches are becoming elongated and broken, the process of short-range densification gradually produces a coarser overall blob-like structure (i.e. increasing no) with larger voids. The

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decrease in df is explicable2’ in terms of a net movement of particles (to ‘feed’ the thickening of the filaments) from the intermediate fractal scaling regime whose structure therefore becomes more stringy. It is important to note that thc fractal dimensionality deduced from the scaling behaviour of n(r) for a particle gel simulated at moderate volume 0.1) is to be regarded as a property of the whole fractions (say, &, interconnected network and not a property of individual clusters. When the particle density is moderately high, and there is a reasonably strong attraction between pairs of closely spaced neighbouring particles, small clusters join together simultaneously at a very early stage in the aggregation process. So, when a convincing fractal scaling regime first becomes evident in the statistical analysis of the simulated n(r) data, the system already exists as a percolating gel-like network. Subsequent changes in microstructure of the ageing gel depend in a complicated way on the nature of the interparticle interactions, including both the weak reversible interactions as well as any strong irrevcrsible bonding interactions. While it is expected that many gels formed by particle aggregation do have a convincing fractal scaling regime, it seems clear also that many do not. Most gels made at particle concentrations above q!~ = 0.3, both simulated and real (e.g. adhesive oil-in-water emulsion gels j), appear to have insignificant fractal character. Homogeneous polymer-like gel networks formed at moderately low concentrations (say, @p 0.1) from bonding particles with repulsive non-bonded interactions are also non-fractal. 21,22

-

s

i=

5 Reversible Aggregation: Depletion Flocculation Reversible aggregation processes are controlled primarily by thermodynamic factors, and the transition between dispersed and flocculated colloidal states can usefully be regarded as analogous to the gas-liquid transition of simple fluid systems. 31336 This thermodynamic emphasis can bc justified by the fact that strong corrclations have been identified37,38between the solvent conditions required to reversibly flocculate dispersions and those rcquired to induce liquid-liquid phase separation in the absence of colloidal particles. One of the most important mechanisms of reversible aggregation is that duc to the attractivc ‘depletion’ interaction which exists between pairs of large spherical particles disperscd in a dilute solution of non-adsorbing polymer molecules or small solid particles. That depletion flocculation is reversible to dilution was first demonstrated by B ~ n d for y ~the ~ creaming of natural rubber in the presence of water-soluble polymers. In more recent times, qualitativcly similar behaviour was observed when non-adsorbing polymers (hydroxyethyl cellulose or sodium carboxymethyl ccllulose) were added to electrostatically stabilized latices,40 silica particles4’ or emulsion droplets,42 or when microbial polysaccharides (xanthan or dextran) were added to oil-in-water emulsions stabilized with small-molecule emulsifier^^^' or milk proteins.46 Recently, it

120


has been recognized474‘ that depletion flocculation of oil-in-water emulsions can also be induced by small surfactant micelles. The simplest working theory of depletion flocculation is that due to Asakura and Oosawas” for the case of rigid spherical molecules in the gap between a pair of particle surfaces. They showed that, because solute molecules are excluded from the region between the surfaces when the gap width h is less than the molecular diameter d , there is a net attraction between the particles arising from the lower osmotic pressure in the gap than in the bulk medium of solute volume fraction &. The Asakura-Oosawa depletion potential has the form

(-3kTR$,/d3)(d - h)’,

(h < d ) (h 2 d)

(9)

where R is the particle radius. For two particles of radius R = 0.5 p m in a solution containing solute molecules of diameter d = 10 nm at concentration qjs = 0.02 (2 vol%), equation (9) predicts a maximum depletion attraction energy at contact ( h = 0) of u d = -3 kT. This sort of value is probably large enough to be detectable as enhanced flocculation in creaming or rheology experiments. The depletion interaction for flexible polymer solute molecules is much greater than for spherical solute molecules because the configurational entropy of the chains is substantially reduced near the particle s ~ r f a c c . ~A’ -theoreti~~ cal expression by Vincent et for the depletion potential in this case has thc form Ud(h) = - hRTI(A - th)[l + (2A/3R)

+ (h/6R)],

(10)

where I7 is the osmotic pressure of the polymer solution, and A is the so-called ‘depletion layer thickness’, which is approximately equal to the radius of gyration of the unadsorbed polymer in the dilute polymer concentration regime. Further refinements of t h e depletion theory have allowed for the particle surface to be coated with a sterically stabilizing layer of adsorbing polymer,s4 and for the inclusion of electrostatic polymer-polymer and particle-polymer interaction^.^"^^ In all these variants, however, the essential physical origin of the depletion attraction remains the same as that originally proposed by Asakura and 00sawa.’~ Whilst emphasizing the predominantly destabilizing effect of the depletion mechanism, Napper also suggested56 the intriguing possibility that, for somc systems of high polymer concentration, a kinetic stabilization effcct could possibly arise due to the presence of a maximum in the depletion potential Ud(h) at larger values of h. For the case of rigid spheres in the gap between a pair of surfaces, the origin of this putative depletion repulsion is the wellknown damped oscillatory potential of mean force associated with layered particle packing at liquid-like den~ities.”,’~Recent calculations of the hardspherc depletion interaction to second order in the solute volume fraction & have shownss*s9that the depletion potential U d ( h )has a positive maximum value of

E. Dickinson

121

ud(h,ax)

=

12kTR@?/d

at

h,,,

=

d ( l - 3@,/2)

(11)

and a minimum value at contact (h = 0) of

Ud(hmin)= - 3kTR@$d - 3kTR@,2/5d.

(12)

The first term of equation (12) (to order &) is the Asakura-Oosawa result [equation (9) at h = 01. For the set of parameter values referred to above ( R = 0.5 pm, d = 10 nm, & = 0.02), equation (11) predicts Ud(hmaX) = 0.05 k T , which is negligible. However, if the solute volume fraction were to be increased to GS = 0.2 (i.e. 20 vol%), then the putative maximum in the depletion potential would become ca. 5 kT. This value would certainly be significant in retarding the aggregation, although it would still represent a barrier height insufficient to confer full kinetic stability. The depletion flocculation of large spheres by small spheres can be used to explain the flocculation of casein-coated oil-in-water emulsion droplets induced by unadsorbed sodium caseinate.m Figure 6 shows light micrographs of four undiluted groundnut oil-in-water emulsions (10 vol% oil, average droplet diameter d32= 0.56 f 0.02pm, p H 7) containing different amounts of sodium caseinate as the sole emulsifier. A t just 2 wt% protein content [picture (a)] the emulsion shows only a slight indication of aggregation. But, when the protein content is increased to 2.4 wt% [picture (b)], we can begin to see numerous individual aggregates in the size range 10-20 pm. At 2.8 wt% protcin, many of the aggregates are joined together into a percolating network structure, and at 3.2 wt% a particle gel structure is clearly evident with considerable close-range densification and possibly some medium-range fractal character. The presence of emulsion flocculation is reflected experimentallym in a greatly enhanced rate of creaming and a substantial change in the low-stress rheological behaviour. As expected for a depletion flocculation mechanism, the aggregation process is completely reversible to dilution.m Assuming that the sodium caseinate is composed of particles of diameter 8-10 nm and that the average emulsion droplet size is 0.6pm, we can estimate from equation (9) that the maximum average depletion interaction strength (h = 0) for a caseinate particle volume fraction of @s = 0.03 (3 ~ 0 1 %is) Ud(h) = 3 k T . This theoretical value is certainly sufficiently large to explain the substantially changed creaming behaviour observed experimentally in conccntrated emulsions containing excess caseinate.m There is also separate experimental evidence that ordered layers of casein particles in thin liquid films between droplets may provide a significant stabilizing mechanism against coalescence in caseinate-containing emulsions.61

6 Concluding Remarks This article has considcred the relationship between particle interactions and the structure and stability in concentrated aggregating colloidal systems. For

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E. Dickinson

123

the case of irreversible aggregation, the emphasis here has been on the effect of particle volume fraction and the form of the interparticle potential on the fractal structure of simulated particle gels formed by computer simulation. A critical analysis of the predictions of the infinite-dilution cluster-cluster aggregation models (DLCA and RLCA) has been presented. For the case of reversible aggregation, emphasis has been on the conditions inducing depletion flocculation (and possibly depletion stabilization) in systems of large particles due to the presence of small particles (or polymers). In addition to the mechanisms referred to above, of course, there are various other kinds of particle interactions that can lead to aggregated particle gel structures in food colloids. For instance, small spheres (or polymers) which adsorb onto the surface of large spheres may form network structures due to a bridging flocculation mechanism if the particle concentration is sufficiently high. Such flocculation can be modelled statistically as a simple binary mixture of spheres with a sticky-sphere unlike interaction The implication of the theoretical predictions@ is that the rheology of concentrated colloids should be extremely sensitive to the addition of weakly adsorbing ~~~~ particles (or polymers). This has recently been verified e ~ p e r i r n e n t a l l yfor the case of some concentrated protein-stabilized emulsions containing small amounts of weakly adsorbing polysaccharide. Most of the visible manifestations of colloidal aggregation in the laboratory arise as a result of the influence of gravity in inducing phase separation or density gradients in aggregating emulsions or dispersions. There is dill much to be done to understand properly the effect of interparticle interactions on the structure of aggregating systems undergoing creaming or sedimentation. While some progress can be made in simulating the settling of aggregating systems using simple lattice models,67@ this research needs to be extended to nonlattice models and to a wide range of interparticle interactions. A final point to note is that, whereas the current theories typically assume monodispersity, most real colloids are substantially polydisperse. This is particularly true of food emulsions. Polydispersity complicates the interpretation of the origin of colloidal structures because the strength of dropletdroplet interactions and the effect of gravity on droplet motion are both very dependent on the droplet size. Of particular interest, therefore, are recent experimental development^^^^'^ in the formulation of nearly monodisperse emulsions. Measurements on these surfactant-stabilized systems caii be expected to allow more systematic comparisons to be made with analytical theories and computer simulations, e.g. in studies of the fractal structure of concentrated emulsion gels.71 The widespread availability of equivalent monodisperse emulsions stabilized by milk proteins would provide exciting new experimental opportunities for food colloid scientists.

References 1. E. Dickinson, ‘An Introduction to Food Colloids’, Oxford University Press, Oxford, 1992, p. 14.

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2. E. J. W. Verwey and J.Th.G. Overbeek, ‘Theory of the Stability of Lyophobic Colloids’, Elsevier, Amsterdam, 1948. 3. G. J. Fleer, M. A . Cohen Stuart, J. M. H. M. Scheutjens, T. Cosgrove, and B. Vincent, ‘Polymers at Interfaces’, Chapman and Hall, London, 1993. 4. D. Velegol, J. L. Anderson, and S. Garoff, Langmuir, 1996,12,675. 5. F. L. Usher, Proc. R . Soc. (London),Ser. A , 1924, 125, 143. 6. A. L. G . Rees, J. Phys. Chem., 1951,55, 1340. 7. G. Dezelic, M. Wrischer, Z. Devidk, and J. P. Kratohvil, Kolfoid Z . , 1960,171,42. 8. F. G. Karioris and B. R. Fish, J. Colloid Sci., 1962, 17, 155. 9. I . L. Thomas and K. H. McCorkle, J. Colloid Interface Sci., 1971,36, 110. 10. D. N. Sutherland, Nature (London), 1970,226,1241. 1 1 . R. Jullien and R. Botet, ‘Aggregation and Fractal Aggregates’, World Scientific, Singapore, 1987. 12. P. Meakin, in ‘Phase Transitions and Critical Phenomena’, eds. C. Domb and J. L. Lebowitz, Academic Press, New York, 1988, vol. 12, p. 335. 13. D.A. Weitz, M. Y. Lin, and J. S. Huang, in ‘Physics of Complex and Supermolecular Fluids’, eds. S. A . Safran and N. A. Clark, Wiley, New York, 1987, p. 509. 14. B. B. Mandelbrot, ‘The Fractal Geometry of Nature’, Freeman, New York, 1982. 15. L. G . B. Bremer, B. H . Bijsterbosch, R. Schrijvers, T. van Vliet, and P. Walstra, Colloids Surf., 1990,51, 159. 16. P. Walstra, T. van Vliet, and L. G. B . Bremer, in ‘Food Polymers, Gels and Colloids’, ed. E. Dickinson, Royal Society of Chemistry, Cambridge, 1991, p. 369. 17. L. G. Bremer, B. H. Bijsterbosch, P. Walstra, and T. van Vliet, A d v . Colloid Interface.Sci., 1993,46, 117. 18. M. Y. Lin, R. Klein, H. M. Lindsay, D. A. Weitz, R. C. Ball, and P. Meakin, J. Colloid Interface Sci., 1990, 137, 263. 19. G . C. Ansell and E. Dickinson, Phys. Rev. A , 1987,35, 2349. 20. M. D . Haw, M. Sievwright, W. C. K. Poon, andP. N. Pusey,Adv. Colloidlnterfuce Sci., 1995,62, 1. 21. B . H. Bijsterbosch, M. T. A. Bos, E. Dickinson, J. H. J . van Opheusden, and P. Walstra, Faraday Discuss., 1995, 101, 51. 22. E. Dickinson, J . Chem. Soc., Faraday Trans., 1994,90, 173. 23. E. Dickinson, J . Chem. Soc., Faraday Trans., 1995,91,51. 24. R. Jullien and A . Hasmy, Phys. Rev. Lett., 1995,74,4003. 25. H.F. van Garderen, W. H. Dokter, T. P. M. Beelen, R. A. van Santen, E. Pantos, M. A. J. Michels, and P. A. J. Hilbers, J. Chem. Phys., 1995, 102,480. 26. E . Dickinson and M. Whittle, unpublished results. 27. A . H. Clark and S. B. Ross-Murphy, Adv. Polym. Sci., 1987, 83,57. 28. M. Stading, M. Langton, and A.-M. Hermansson, Food Hydrocolloids, 1992, 6 , 455. 29. M. Stading, M. Langton, and A.-M. Hermansson, Food Hydrocolloids, 1992, 7, 195. 30. E. Dickinson, J . Colloid Inferface Sci., 1987, 118,286. 31. E. Dickinson, in ‘Colloid Science’, ed. D. H . Everett, Specialist Periodical Report, Royal Society of Chemistry, London, 1983, vol. 4, p. 150. 32. W. van Megen and I. Snook, A d v . Colloid Interface Sci., 1984, 21, 119. 33. S.W. Koch, R.C. Desai and F.F. Abraham, Phys. Rev. A , 1983, 27, 2152. 34. B. D. Butler, H. J. M. Hanley, D. Hansen, a n d D . J . Evans, Phys. Rev. Lett., 1995, 74,4468.

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35. J. Bibette, T. G. Mason, H. Gang, D. A. Weitz. and P. Poulin, Langmuir, 1993.9. 3352. 36. E. Dickinson, in ‘Annual Reports in the Progrcss of Chemistry, Section C’, ed. M. C. R. Symons, Royal Society of Chemistry, London, 1983, p. 3. 37. D. H. Everett and J. F. Stageman, Faraday Discuss. Chem. Soc., 1978.65,230 & 314. 38. A. Kumar and D. Bcyscns, Physica, 1996,224,68. 39. C. Bondy, Trans. Faraday Soc., 1939,35, 1093. 40. P. R. Spcrry, H. B. Hoplenhcrg, and N. L. Thomas. J. Colloid Interface Sci., 1981, 82,62. 41. M. J . Snowdcn, S. M. Clegg, P. A. Williams, and I. D. Robb, J. Chem. Soc. Faraday Trans., 1091, 87, 2201. 42. A. J. Fillery-Travis, P. A. Gunning, D. J . Hibberd. and M . M . Robins, .I. Colloid Interface Sci.. 1993, 159, 189. 43. E. Dickinson, J . Ma, and M . J . W. Povey, Food Hydrocolloiris, 1994.88. 481. 44. E. Dickinson, M. I . Goller, and D. J . Wedlock, J. Colloid InkrfaceSci.. 1995, 172. 192. 45. A. Parker, P. A . Gunning, K. Ng, and M. M . Robins, Food Hydrocolloids, 1995,9. 333. 46. Y . Cao, E. Dickinson, and D. J . Wedlock. Food Hydrocolloids, 1990. 4, 185. 47. M . P. Aronson, in ‘Emulsions-A Fundamental and Practical Approach’, ed. J. Sjoblom, Kluwer, Dordrecht, 1992, p. 7.5. 48. J . Bibctte, D. Roux, and B. Pouligny, J. Plzys. /I, J992. 2.401. 49. E. Dickinson and D. J. McClements. ‘Advances in Food Colloids’, Blackie. Glasgow, 1995, p. 272. 50. S. Asakura and F. Oosawa, J. Chem. Phys., 1954.22, 1255. 51. S. Asakura and F. Oosawa, J. Polym. Sci., 1958,33, 183. 52. J. F. Joanny, J . F. Leibler, and P.-G. de Gennes, J. Polym. S c i . , Polyrn. Phys. Ed., 1979,17,1073. 53. R. 1. Feigin and D. €3. Napper, J. Colloid Interface Sci., 1980, 75, 525. 54. B. Vincent, J . Edwards, S. Emmett, and A. Jones, Colloids Surf., 1986, 18, 261. 55. J . Y. Walz and A. Sharma, J. Colloid Interface Sci., 1994, 168,485. 56. D. H. Napper, ‘Polymeric Stabilization of Colloidal Dispersions’, Academic Press, London, 1983. 57. I. K. Snook and D. Henderson, J . Chem. Phys., 1978,68,2134. 58. J. R. Hcnderson and F. van Swol, Mol. Phys., 1984, 51,991. 59. Y . Mao, M. E. Cates, and H. N. W. Lekkerkerker, Physica A , 1095,222, 10. 60. E. Dickinson, M. Golding, and M. J. W. Povey, J . Cofloid Interface Sci., in press. 61. K. Koczo, A. D. Nikolov, D. T. Wasan, R. P. Borwankar, and A. Gonsalves: J. Colloid Interface Sci., 1996, 178,694. 62. E. Dickinson, .I. Chem. Soc. Faraday Trans., 1990,86,439. 63. C. Regnaut, S. Amokranc, and Y. Heno, J. Chern. f’hys., 1995,102,6230. 64. E. Dickinson, J. Chem. Soc. Faraday Trans., 1995, 91, 4413. 65. E. Dickinson and K. Pawlowsky, in ‘Gums and Stabilisers for the Food Industry’, eds. G. 0 . Phillips, P. A. Williams, and D. J. Wedlock, Oxford University Press, Oxford, 1996, vol. 8, p. 181. 66. E. Dickinson and K. Pawlowsky, J. Agric. Food Chem., 1996,44,2992. 67. E. van der Knaap, R. Vreeker, and L.L. Hoekstra, ColloidsSiirf. A , 1994,85,265.

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68. V. J . Pinfield, E. Dickinson, and M . J. W. Povey, J. Colloid lnterface Sci., submitted for publication. 69. J. Bibette, D. Roux, and F. Nallet, Phys. Rev. Lett., 1990, 65, 2470. 70. J. Bibette, J. Colloid Interface Sci., 1991, 147,474. 71. J . Bibette, T. G . Mason, H . Gang, D. A. Weitz, and P. Paulin, Langmuir, 1993,9, 3352.

High Pressure Processing of P-Lactoglobulin and Bovine Serum Albumin By Vanda B. Galazka, Dave A. Ledward, and Julie Varley DEPARTMENT OF FOOD SCIENCE AND TECHNOLOGY, UNIVERSITY OF READING, WHITEKNIGHTS, READING RG6 6AP, UK

1 Introduction The use of high pressure as a means of preserving and processing food has been known for a long time. Over 100 years ago Bert Hite' undertook such work because of constraints and dissatisfaction with the methods of sterilization, cooling and pasteurization of milk then available. He observed that pressures of 450 MPa or greater could be used to improve the keeping quality of milk, fruit juices and meat. Further studies during the next 15 years showed that micro-organisms, such as lactic acid bacteria and yeasts, which are associated with sweet, ripe fruit were more susceptible to pressure than spore formers, which are associated with vegetables.2 Bridgmar~,~ a contemporary of Hite, reported that egg-white proteins could be denatured by high pressure treatment, demonstrating that this processing technique, in addition to killing certain micro-organisms, can alter the structure and reactivity of proteins. Because of the technical difficulties and costs associated with high pressure units and the routine handling of materials at high pressures, few other reports were published on the relationship between high pressure and food properties until about ten years ago. In 1989, a special unit was set up at Kyoto University under Professor Hayashi to develop high pressure for use in the food industry. As a result of this, a number of products, mainly fruit and vegetable based, have become commercially available in Japan. Major research efforts are now underway in the United Kingdom, the United States and in mainland Europe: some are supported by national governments, and the European Commission, and others by i n d ~ s t r y . ~ In general, pressures in the range 100 MPa to 1 GPd are used to provide a variety of effects on food systems. Key effects include microbial reduction, alteration of enzyme activity, control of phase changes, and modification of the conformation of biopolymers leading to changes in functional properties.' 127

128

High Pressure Processing of p- Lactoglobulin und Bovine Serum Albumin

It is well established&'" that, in protein molecules, the application of high pressure enhances the formation of hydrogen bonds, the scparation of' ion pairs, and the rupture of hydrophobic interactions, whilst keeping covalent bonds intact. However, hydrogen bonding is less affected than either ionic or hydrophobic interaction^.^-^ In the presence of oxygen and at pressures higher than 300 MPa, sulfhydryl groups may become oxidised to form -S-S- bridges. The result of these modifications in most water-solublc globular proteins means that the molecule will partially or completely unfold leading to denaturation, followed in many cases by aggregation.&' The extent of denaturation depends on the protein structure, solvent composition, ionic strength, temperature and pH, as well as the pressure range.8 At relatively low pressures (100 to 200 MPa) reversible effects are generally observed - these pressures favour the dissociation of oligomeric proteins into subunits. Abovc 200 MPa many proteins unfold, and reassociation of subunits from dissociated oligomers can occur. Several authors have r e p ~ r t e d ~ , ~ . " that . " pressure-induced denaturation and/or dissociation can be reversible under certain conditions. However, after pressure release, refolding of the protein can be relatively slow and both hysteresis behaviour and conformational drift can be observed. The structure of the pressure-treated protein may well differ from that found in the native form. Recent studies'* have shown that pressure induces the dimerization of metmyoglobin, and the pressure-treated forms of soy proteinI3 and o ~ a l b u m i ndiffer '~ from the native structures. At higher concentrations and under certain conditions, many globular proteins irreversibly denature (e.g., egg-white) to form gels or precipitates.' There is some e ~ i d e n c c ' ~ -that ' ~ pressure-induced changcs may alter the enzymic and functional properties of the reformed protein. Recent experiments at Reading" have demonstrated that the activity of tyrosinase is reduced with increasing magnitude of applied pressure and duration of treatment. We have also ~ h o w n ' ~ ~that " pressure processing of P-lactoglobulin or whey protein concentrate before homogenization has a detrimental effect on emulsion efficiency and stability. On the other hand, other researchers18 have observed that pressure treatment of ovalbumin and soy protein leads to improved emulsifying properties. In contrast, the foaming characteristics of egg-white are reduced because of selective precipitation under certain pressurehemperature conditions. l9 The prescnt study will discuss the effects of high pressure (800 MPa for 20 min) on the structure of bovine serum albumin (BSA) and P-lactoglobulin in aqueous solution, and will compare the effect of pressure treatment (200,350, 600 or 800 MPa for 20 min) on the foaming behaviour of these two proteins. We shall also attempt to relate changes in foaming behaviour to the known pressure effects on the aggregation and conformational properties of both proteins.

2 Materials and .Methods Bovine serum albumin (99% purity) and /3-lactoglobulin (3 x crystallized and lyophilized) were purchased from Sigma Chemical Co. (St. Louis, MO,

V . B. Galazka, D . A. Ledward, and J. Varley

129

U.S. A.), as were DL-dithiothreitol (DTT), 5,5'-dithio-bis(2-nitrobenzoic acid) (DTNB), and the buffer salts. Isoelectric focusing (IEF) gels and buffers were obtained from NOVEX" (San Diego, CA, U.S.A.). Buffer solutions for the foaming experiments (20 mM imidazole, p H 7) were prepared using deionized, double-distilled water. Solutions of protein (0.1, 0.2 or 2.5 wt%) for the structural analysis were prepared with H.P.L.C. water (Rathburn Chemicals, Ltd., Walkerburn, Scotland), and adjusted to p H 7 by addition of 0.05 M HCI or NaOH. These solutions, as well as those prepared for the foaming studies (0.01 wt%), were sealed in cryovac sachets, and subjected to pressures in the range 200-800 MPa for 20 min at ambient temperature in a prototype high pressure rig as described previously.I6 Buffering at this pH is mainly due to the histidine and a-amino groups on the protein, and because ionization of these groups does not lead to a volume change2' it is likely that the pH will not change significantly during pressure treatment. Changes in the surface hydrophobicity of both proteins were estimated by reaction with 1-anilinonaphthalene-8-sulphonate (ANS),I6 the a-helical content by circular dichroism,2' determination of free thiol content using DTNB,22 thermal stability using differential scanning calorimetry,22 and aggregate formation by IEF and gel permeation chromatography in the presence and absence of DTT.22 Foaming experiments were carried out using a sparging technique. The foam column was similar to that used by other and consisted of a graduated glass cylinder (3cm in diameter and 50 cm long) with a sintered glass disc (porosity 40-100 pm) fixed to the base. A 20 ml (0.01 wt%) aliquot of protein solution was poured into the column, and nitrogen gas passed through the sample. The nitrogen gas flow was controlled using a Platon GTV gas flow assembly with a 20-250 cm3/min air flow cell, and it was humidified prior to entering the foam column by bubbling through de-ionized water. Sparging was at a constant flow rate of 45 cm3/min for 24 min. The nitrogen supply was then turned off, and the foam height visually recorded as a function of time at ambient temperature (ca. 22 "C). All recorded values are the means of 6 determinations for /?-lactoglobulin and 3 determinations for BSA.

3 Protein Denaturation and Aggregation Recent s t ~ d i e s ' " ~ on ~ , the ~ ~solution . ~ ~ properties of BSA and B-lactoglobulin have shown a significant influence of high pressure on their aggregation and conformational properties. In Table 1we compare the effect of high pressure (800 MPa for 20 min) on some structural parameters for these two globular proteins at a pre-pressure pH of 7. Differential scanning calorimetry of native BSA (2.5 wt%) and Blactoglobulin (2.5wt%) show the characteristic endothermal peaks at a T,,, value of 59.2 "C for BSA and 73.3 "C for B-lactoglobulin. Following pressure treatment at 800 MPa, the endothermal peaks for both proteins shift to lower temperatures (51.2"C and 38.3 "C for BSA and B-lactoglobulin, respectively). In both cases the enthalpy change falls to virtually zero (not shown) which

130

High Pressure Processing of P-Lactoglobulin and Bovine Serum Albumin

Table 1

Effects of high pressure treatment (800 MPa for 20 min) on the properties of bovine serum albumin and p-lactoglobulin (0.I , 0.2 or 2.5 wt% solutions) at p H 7.0

Protein

Bovine serum albumin

P-Lactoglobulin

Endothermic peak ( T , "C) (2.5 wt% solutions)

Native 59.2; Treated 51.5

Native 73.3; Treated 38.3

ANS Fluorescence Intensity 41% Decrcase (0.1 wt% solutions)

40% Increase

a-Helix Content (0.2 wt% solutions)

Native 69"/0;Treated 44%

Native 28%; Treated 25"/0

%SulfhydrylGroups

55% Decreasc

42% Decreasc

(0.2 wt% solutions)

suggests that there is a major loss of native structure on pressure treatment in 2.5 wt% solutions, which is not recovered after pressure release. Spectrofluorometry data for the binding of ANS to native and refolded BSA (0.1 wt%) shows a decrease (41%) in the fluorescence intensity following high pressure treatment indicating a significant reduction in protein surface hydrophobicity. This change after pressure processing could well be due to intermolecular interactions which prevent hydrophobic groups binding to the ANSI4 or simply due to the hydrophobic groups becoming unavailable by being buried within the reformed protein. In contrast, fluorometry studies for plactoglobulin at this concentration indicate an increase in surface hydrophobicity (40%). Circular dichroism data for BSA andP-lactoglobulin (0.2 wt%) are shown in Table 1. We find that there is a significant reduction in the a-helical content (native = 69%; pressurized = 44%) for BSA after treatment at 800 MPa for 20 min. This is in agreement with Hayakawa el. a1.,28who have shown that pressures around 1,OOO MPa for 10 min have a substantial effect on the secondary structure of the protein. On the other hand, high pressure treatment of B-lactoglobulin at this concentration leads to only a modest loss of a-helix (native 28%; pressure treated 25%), which could be due to some refolding of the protein. At higher concentrations (2.5 wt"/,) the calorimetry results (see above) suggest that the extent of unfolding is enhanced, probably due to reduced reversibility caused by aggregation ." Both these proteins have a single free -SH group which becomes available for reaction as the temperature is increased over 60 "C, leading to the formation of covalent disulphide linkages with eventual aggregation.2s3o We see in Table 1 that there is a loss of sulfhydryl groups (BSA = 55% and 8lactoglobulin = 42%) following high pressure treatment which indicates a decrease in free cysteine. It seems probable that the free cysteine forms -S-Slinks or is irreversibly oxidised by some other mechanism.

V. B. Galazka, D. A . Ledward, and J. Varley

Figure 1

131

Isoelectric focusing of native and pressure-treated (WOO MYa jor ZU min) bovine serum albumin (BSA) and p-lactoglobulin solutions. Samples of filtered (0.2 p m Millipore) and unfiltered protein were compared to indicate the average size of aggregates formed afierpressure treatment: ( I ) and (lo) standards; (2)filtered native BSA; (3)filtered treated BSA; (4) unfiltered native BSA; (5) unfiltered treated BSA; (6)filtered native 8-lactoglobulin; (7)filtered treated 8-lactoglobulin; (8) unfiltered native 8-lactoglobulin; (9) unfiltered treated 8-lactoglobulin. (Reproduced with permission from ref. 22)

Native IEF patterns (Figure 1)for both proteins provides some evidence for aggregation in the pressurized samples. Filtered (0.2 pm Millipore) samples were compared with unfiltered samples to give an indication of the average size of aggregates produced. Gel permeation chromatography data for BSA and /3lactoglobulin are presented in Figure 2; we find that BSA behaves similarly to /3-lactoglobulin. The major peak for the naturally occurring /3-lactoglobulin (Figure 2a) elutes at approximately 20,000 Da which is in good agreement with the published relative molecular weight of 18,200. Pressurization at 800 MPa for 20 min (Figure 2b) induces the formation of a new species with a molecular weight of 40,000 Da. This corresponds to the aggregation reported by Dumay et aZ.*’ Addition of 5 mM DTT (Figure 2c) to the pressure-treated sample reduces the units to monomer status, which suggests that during high pressure

132

High Pressure Processing of P-Lactoglobulin and Bovine Serum Albumin

7d

701

i 351

b

010

20

30

40

20

10

701-

35-

i

1 d i i

35-

/":'

0-

0;

~~~~

30

40

I'\1

V . B. Galazka, D. A. Ledward, andJ. Varley

133

treatment these units are stabilized by -S-S- linkages. These findings are consistent with those of Johnston and Murphy3' who have reported that pressure treatment reduces the number of -SH groups found in milk. We notc that the data for B-lactoglobulin corresponds well with that of Nakamura elui.3z who have shown that therc is extensive aggregation of P-lactoglobulin when whey protein concentrate is processed at 200-600 MPa, and with those of Dumay e t ~ lwho . ~have ~ shown that high pressure causes extensive aggregation and unfolding of this protein. Figure 2d shows that the native BSA has a molecular weight of approxirnatcly 70,000 Da with some indication of larger units. After pressurization at 800 MPa for 20 min (Figure 2e) we see that there is extensive dimer, trimer and higher aggregate formation, which correspond to the additional bands seen in the IEF patterns (Figure 1). The presence of DTT (Figure 2f) reduces the lcvel of dimerization which suggests that polymerization is due to 4 - S - bridging occurring during or after high pressure processing.

4 Foaming Behaviour The data for BSA (0.01 wt% protein, 20 mM imidazole-HC1 buffer, pH 7) are presented in Figure 3, where the time for the initial volume to collapse to 50% of its original value (foam half-life) is plotted as a function of applied pressure. In this set of expcriments, the timc for half volume collapse for the untreated BSA foam is cu. 100 min (mean of triplicates). Replacement of the native protein by samples which had been subjected to pressurc processing (200,350, 600 or 800 MPa for 20 min) leads in all cascs to foams with lower stability. Furthermore, there is a consistent trend of decreased stability with increasing applicd pressure. A treatment of 200 MPa for 20 min gives a foam with a halflifc of 82 & 10 min (mean & S.D. of 3 determinations), whereas treatment at 800 MPa for 20 min gives a foam half-life of 52 k 5 min (mean k S.D. of 3 determinations). The initial foam volume (not shown) for all samples is ca. 60.5 cm3.The decrease in the foam stability for BSA at pH 7can best be explained in terms of protein unfolding followed by aggregation due to covalent -S-S- bond formation which inhibits solubility and hence the ability to stabilize the foam. It is also probable that exposed hydrophobic groups on the protein have reacted to form oligomers. Figure 4 shows the influence of high pressure treatment on the foam stability of native and pressure treated P-lactoglobulin. We note that the stability of the native P-lactoglobulin sample is much poorer than that of BSA. The time for half volume collapse is only ca. 5 min (6 determinations were in the range 3.5-7 min). However, with the pressurized samples there is an increase in foam stability. Thc foam stability curve reaches a maximum at around 350 MPa (foam half-lifc = 53 & 10 min) (the mean k S.D. of 6 determinations), and gradually dccreases at higher pressures. At the maximum pressure (800 MPa for 20 min) thc foam half-life is 38 k 5 rnin (mean k S.D. of 6 determinations). The initial foam volume (not shown) for nativep-lactoglobulin is 72 cm3 (mcan

134

High Pressure Processing of /3-Lactoglobulin and Bovine Serum Albumin

0

200

400

600

800

Pressure lMPa Figure 3

Effect of high pressure treatment on the foaming behaviour of bovine serum albumin at pH 7.0. Foams ( I X wt% protein) were sparged with nitrogen for 2+ min at a flow rate of 45 cm”lmin. The time for half volume collapse (mean k S. D . of 3 determinations) is plotted as a function of applied pressure for 20 rnin

of 6 determinations), and this increases to 78 cm3 for the sample treated at 350 MPa for 20 min. Comparison of the initial foam volumes for BSA and P-lactoglobulin suggests that P-lactoglobulin reaches the interface faster than BSA, possibly due to its smaller molecular weight. The increase in foam stability for pressure treated P-lactoglobulin may be due to its increased surface hydrophobicity which, in the absence of aggregation is usually associated with an increase in foam stability,33 as previously hidden hydrophobic groups become exposcd and available for adsorption at the air-water interface. As with BSA it might be anticipated that aggregation will inhibit its foam stabilizing ability, but thc increased surface hydrophobicity must override this effect for P-lactoglobulin. The decrease in stability seen on increasing the pressure from 400 to 800 MPa (Figure 4) may reflect, as with BSA, increasing pressure-induced aggregation following denaturation, since P-lactoglobulin is normally dcnatured at about 300-400 MPa.32 In a previous study16 the emulsifying propertics of Plactoglobulin decreased with increasing pressure and treatment time (from 200 MPa for 10 min to 800 MPa for 40 min). This apparent difference between emulsifying and foaming may relate to the quite different relative concentrations, since in the emulsion studies an effective concentration of 0.4 wt%

135

V. B. Galazka, D.A. Ledward, andJ. Varley

60

. %

.g 50

E

--% 40 6 0)

-$

8L: I”

30

20

L

e

?i

i= 10 0

0

200

400

600

800

Pressure I MPa

Figure 4

Influence of high pressure treatment on the foaming properties of blactoglobulin at pH 7.0. Foams (1 x lo-’ wt% protein) were sparged with nitrogen for 2t min at apow rate of 45 cm31min. The time for half volume collapse (mean k S.D.of 6 determinations) is plotted as a function of applied pressure for 20 min

was used, which may lead t o more aggregation and loss of functionality than the much lower concentration (0.01 wt%) used for the foaming studies.

References 1. B. H. Hite, Bull. W .Va. Univ. Agric. Exp. Stn., 1899.58, 15. 2. B. H. Hitc, N. J. Giddings, and C. E. Weakly, Bull. W. Va. Univ. Agric. Exp. Stn., 1914,146,3. 3. P. W. Bridgman, J . Biol. Chem., 1914,19,511. 4. V . B. Galazka and D. A. Ledward, Food Technol. Int. Eur., 1995,123. 5. D. Farr, Trends Food Sci. Technol., 1990,1, 14. 6. K. Heremans, Ann. Rev. Biophys. Bioeng., 1982,11, 1. 7. C. Balny, P. Masson, and F. Travers, High Press. Res., 1989, 2, 1. 8. P. Masson, in ‘High Pressure and Biotechnology’, ed. C. Balny, R. Hayaa..i, K. Heremans, and P. Masson, Colloquc INSERM/John Libbey Eurotcxt, Montrouge, 1992, p. 89. 9. J.-C. Cheftel, in ‘High Pressure and Biotechnology’, ed. C. Balny, R. Hayashi, K. Heremans, and P. Masson, Colloque INSERM/John Libbcy Eurotcxt, Montrouge, 1992, p. 195. 10. A. Zipp and W. Kauzmann, Biochemistry, 1973,12,4217.

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1 1 . A. €3. Defaye, D. A. Ledward, D. B. MacDougall, and R. F. Tester, Food Chem., 1995,52, 19. 12. A. B. Defaye and D.A. Ledward, J . Food Sci., 1995,6U, 262. 13. N. Kajiyama, S. Isobe, K. Vemura, and A. Nogucki, Int. J . Food Sci. Technol., 1995,30, 147. 14. I . Hayakawa, J . Kajihara, K. Morikawa, M. Oda, and Y. Fujio, J . Food Sci., 1992, 57,288. 15. M. R. A. Gomes and D. A . Ledward, Food Chem, in press. 16. V. B. Galazka, E. Dickinson, and D. A. Ledward, Food Hydrocolloids, 1996, 10, 213. 17. V. B. Galazka, D. A . Ledward, E. Dickinson, and K . R. Langley, J . Food Sci., 1995,60, 1341. 18. A. Denda and R. Hayashi, in ‘High Pressure and Biotechnology’, ed. C. Balny, R. Hayashi, K. Heremans, and P. Masson, Colloque INSERM/John Libbey Eurotext, Montrouge, 1992, p. 333. 19. D. Knorr, A . Bottcher, H. Dornenburg, M. Eshtiaghi, P. Oxen, A . Richwin, and 1. Seyderhelm, in ‘High Pressure and Biotechnology’, ed. C. Balny, R. Hayashi, K. Heremans, and P. Masson, Colloque INSERMlJohn Libbey Eurotext, Montrouge, 1992, p. 21 1. 20. S. Funtenberger, E. Dumay and J.-C. Cheftel, Lebensm.-Wiss. u.-Techno/., 1995, 28,410. 21. V. B. Galazka and D. A. Ledward, in ‘Science, Engineering and Technology of Intensive Processing’, ed. G. Akay and B. J. Azzopardi, University of Nottingham, Nottingham, 1995, p. 137. 22. V. B . Galazka, I. G. Sumner, and D. A. Ledward, Food Chem., 1996, in press. 23. J. H. Buckingham, J . Sci. Food Agric., 1970,21,441. 24. S. Ross and G. Nishioka, Colloid Polym. Sci., 1977,255,560. 25. C. W . Bamforth, J . Inst. Brew., 1985,91,370. 26. M. Ahmed and E. Dickinson, Food Hydrocolloids, 1991,4, 395. 27. E. M. Dumay, M. T. Kalichevsky and J.-C. Cheftel, .I. Agric. Food Chem., 1994, 42, 1861. 28. I. Hayakawa, T. Kanno, M. Tomita, and Y. Fujio, J . Food Sci., 1994,59, 159. 29. K . Katsuta and J. E. Kinsella, J . Food Sci., 1990, 55, 1296. 30. E. Katchalski, G.S. Benjamin, and V. Gross, J . A m . Chem. Soc., 1957, 79, 4096. 31. D. E. Johnston and R. J. Murphy, in ‘Food Macromolecules and Colloids’, ed. E. Dickinson and D. Lorient, Royal Society of Chemistry, Cambridge, 1995, p. 134. 32. T. Nakamura, H. Sado, and Y. Syukunobe, Milchwissenschaft, 1993,48, 141. 33. E. Li-Chan and S. Nakai, in ‘Food Proteins: Structure and Functional Relationship’, ed. J . E. Kinsella and W . Soucie, American Oil Chemists Society, Champaign, IL, 1989, p. 232.

Ultrasonic Characterization of Flocculation in Oil-in-Water Emulsions By David Hibberd, Andrew Holmes', Martin Garrood, Annette Fillery-Travis, Margaret Robins, and Richard Challis' INSTITUTE O F FOOD RESEARCH, NORWICH RESEARCH PARK, COLNEY, NORWICH NR4 7UA, UK 'DEPARTMENT O F PHYSICS, KEELE UNIVERSITY, KEELE ST5 5BG, UK

1 Introduction In recent years there has been a growing interest in the use of low-power ultrasound to probe colloidal systems. This interest has been driven by the noninvasive nature of the technique and hence the potential for studying concentrated colloids which are generally opaque to optical techniques. At the Institute of Food Research (IFR) we routinely use a simple ultrasonic technique to measure the creaming and sedimentation behaviour of suspensions and emulsions.' By analysing the creaming data we have been able to infer the extent of flocculation and to obtain information on the subsequent structuring of emulsion droplets undergoing depletion flocculation. A drawback to this approach is that it can take a long time to characterize the creaming behaviour of an emulsion. This makes the technique time consuming and so it is difficult to investigate dynamic processes. Additionally, structural information on the emulsions can only be inferred by fitting a hydrodynamic model to the creaming data. The advent of broad-band ultrasonic spectroscopy has enabled more detailed ultrasonic data to be collected in a few milliseconds. Workers at Keele University have concentrated on the development of rapid fast-Fourier techniques to characterize the ultrasonic properties of colloidal dispersions.* Good agreement is found between these data and the predictions of a multiplescattering theoretical model. Since the ultrasonic properties of dispersions are sensitive to particle size, these techniques have potential for the detection and characterization of instability mechanisms such as flocculation and coalescence. McClements has reported3 changes in the ultrasonic properties of an oilin-water emulsion in which flocculation was inferred, but no attempt was made to compare the results with a theoretical model. Here we report the results of a 137

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Ultrasonic Characterizationof Flocculation in Oil-in-Water Emulsions

study into the ultrasonic properties of an emulsion system which had been flocculated by the addition of non-adsorbing polymer. By manipulation of the polymer concentration and the choice of oil phase we have controlled both the extent of flocculation and the rate of creaming during the ultrasonic measurements. The behaviour of the emulsions has been confirmed by use of the ultrasonic creaming monitor and optical microscopy. We have observed changes in the ultrasonic properties of emulsions undergoing flocculation and have shown that in one case these changes are consistent with theoretical predictions where the flocculation process is modelled as an increasing effective particle size. The results clearly demonstrate the feasibility of using ultrasonic spectroscopy as a direct indicator of structural information in a flocculating emulsion.

2 Materials and Methods Emulsion Preparation and Characterization The model emulsion system was designed to have just a small density difference between the oil and aqueous phases, thereby reducing the rate of droplet creaming. Thus, changes in the ultrasonic properties of the emulsion due to flocculation were isolated from those due to the creaming of the droplets. The emulsions contained 5% v/v 1-bromo-hexadecane dispersed in an aqueous phase of 0.14% w/w Brij35 (non-ionic surfactant), 0.3% w/w sodium azide (preservative), and O%, 0.026% or 0.1% hydroxyethylcellulose (HEC, Natrosol 250HR) as flocculavt. Our previous work using alkane-in-water emulsions prepared with the same surfactant had shown' HEC to be a non-adsorbing, depletion flocculant capable of inducing flocculation to varying extents depending upon its concentration. The emulsions were all prepared from a single concentrated pre-mix (50 vol% droplets), homogenized using a Waring blender. The final emulsions were prepared by dilution of the pre-mix with either water or an aqueous polymer solution. In this way the droplet-size distribution did not vary between emulsions. When analyzed using a Malvern Mastersizer, either before or after the experiments, the same distribution shown in Figure 1was always obtained, indicating that no significant coalescence of the droplets had occurred over the time-scale of the experiments.

Characterization of Extent of Flocculation from Creaming Behaviour The IFR ultrasonic creaming monitor4 was used to detect and quantify the extent of flocculation in each emulsion. The instrument measures the group velocity of a pulse of ultrasound (frequency approximately 6 MHz) through the emulsion at a range of heights in the sample cell. The velocity of the pulse is sensitive to the proportion of oil at the position monitored, and thus is a rapid detector of vertical non-uniformity arising from creaming or sedimentation

139

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Weight in band (%)

0.05 023 0.53

123 2.83

6.52

15.0 34.7

80.0

She Cm) Figure 1

Emulsion droplet-size distribution measured using a Malvern Mastersizer light diffraction instrument

processes. The evolution of the vertical oil concentration profiles with time is characteristic of the size of the droplets or aggregates which are creaming, and detailed analysis can reveal the distribution of Stokes' velocities (and thus of droplet diameter?). Measurements were taken over a period of up to 96 days at a constant temperature of 25.0 "C.

Characterization of Flocculation by Optical Microscopy The emulsions were introduced into a number of fine capillary tubes of rectangular cross-section (0.1 mm X 1 mm x 50 mm). The capillaries were examined under a differential interference phase contrast microscope using a x 100 oil-immersion objective. The microscope was fitted with a video camera in order to allow the flocculation process to be followed with time, and the information was recorded onto SVHS video tape. For studying the short timescale behaviour of the emulsions, any flocs that had assembled during the slide preparation were disrupted by sonicating the slides for two minutes at the start of the experiment. Whilst sonication certainly would have disrupted any large flocs in the sample, it is likely that some small aggregates (i.e. doublets and triplets) would have remained.

Measurement of Ultrasonic Properties The broadband fast Fourier transform ultrasonic technique used in these studies has been extensively described elsewhere.6 The sample was held in a 25 cm3 perspex cell contained within a temperature controlled cabinet set to 25 5 0.1 "C. A small stirrer was used to shear the samples prior to measurement to ensure homogeneity and to disperse flocs. Short pulses (10 ns) were sent between two acoustically thick (5 mm) piezoelectric ceramic transducers

140


mounted in the sample cell walls, the received signal being amplified and digitized at 400 MHz. Digital signal processing techniques were used to correct for transducer effects and for ultrasonic radiation coupling between the transducers and sample. The corrected time domain signal was fast-Fouriertransformed into phase and amplitude spectra in the frequency domain. The phase velocity was calculated from the pulse transmission time and the corrected unwrapped phase spectrum, the attenuation being calculated from the amplitude spectrum and initial pulse height. The range of frequency obtained (the bandwidth) depended on the signal-to-noise ratio, which deteriorates at the high frequency end as the sample’s absorption coefficient increases, and at the low frequency end where low frequency energy is lost due to coupling effects. To improve the bandwidth, the signal-to-noise ratio was improved by coherently averaging the received pulse. This allowed simultaneous measurement of velocity and attenuation of the samples with a bandwidth of 2-45 MHz, the error in the velocity measurement being approximately 0.2 m s-’.

Theoretical Predictions of Ultrasonic Properties The scattering model used to interpret the ultrasonic data is derived from the model of Allegra and Hawley,’ modified for use with emulsions using the multiple scattering formalism of Lloyd and Berry.’ The model requires a detailed knowledge of the thermal and physical properties of the oil and aqueous phases. These were either measured directly or estimated for each emulsion system. The thermophysical constants used for each of the phases are shown in Table 1.

3 Results and Discussion Creaming Measurements The purpose of the creaming study was to characterize the amount and nature of flocculation in a bulk emulsion sample. In addition, the measurements were performed in a container with similar geometry to the sample cell of the Table 1

Thermophysical constants of phases used to make the emulsions

Parameter

Unit

Compression wave speed Density Shear viscosity Thermal conductivity Specific heat Sound attenuation Thermal expansion coefficient

m s-’ kg m-3 Pa s W m-’ K-’ J kg-’ K-’ N sC2 W’

K-‘

Continuousphase (water)

Dispersed phase (oil)

1499.3 996.0 0.000891 0.595 4179.3 2.3 x 1 0 - l ~ 0.000257

1299.4 1000.0 0.00663 0.141 2090.0 1.45 x 10-13 0.000774

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spectrometer, and under the same physical conditions. Thus we were able to establish that no significant creaming would be expected during the time-scale of the spectroscopy measurements. Monitoring of the emulsion prepared in the absence of HEC, with time, using the IFR ultrasonic instrument gave a series of gently curving creaming profilcs (see Figure 2). These are typical of the creaming behaviour of an unflocculated polydisperse emulsion. Further analysis of these profiles' confirmed that they are consistent with a completely unflocculated emulsion, of similar droplet size distribution to that measured with the Mastersizer. The early profiles show that the amount of creaming which had occurred in 1 day was barely measurable. The last profile shows that the creaming process was clearly unfinished even after three months (i.e. 93 days). Figure 3 shows the creaming profilcs for the cmulsion prepared with 0.1 YO HEC. U p to 1.2 hours, the measurements can bc superimposed, but after 2.9 hours a sharp boundary is evident at the base of the sample. This boundary moves rapidly to the top of the sample within a day. This behaviour is typical of a completely flocculated emulsion creaming as a single interconnected network of droplets. The apparcnt delay phase bcfore any creaming commences has been observed bcfore in similar systems,' and in this sample it spans a period of 2 hours. Creaming was also monitored for an emulsion preparcd at a polymer concentration (0.026%)within the range for which two populations of droplets are expected to co-exist," i.e. an unflocculated fraction and a flocculated fraction. No significant creaming was detectable for the first hour (Figure 4), but then 70% of the droplets creamed to the top of the emulsion within the next

"'i

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Cream behaviour of 5% I-bromo-hexadecane emulsion in the absence of polymer flocculant

Ultrasonic Characterization of Flocculation in Oil-in-Water Emulsions

142

Height (mm)

Base

TOP

Creaming behaviour of 5% I -bromo-hexadecane emulsion containing 0.1% HEC asjlocculant

Figure 3

six hours. Since the profiles in this region are not sharp, there is no evidence for a single interconnected network, and so we infer that this fraction of the droplets is creaming as a set of polydisperse flocs. The 30% of droplets remaining behind creamed at a very much slower rate. Our initial analysis of Volume Fraction (%v/v)

Time

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Creaming behaviour of 5% I-bromo-hexadecane emulsion containing 0.026% HEC as flocculant

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the creaming rates suggests that these latter droplets have a smaller average particle size than the parent distribution, which implies that involvement in the flocculation process is biased towards the larger droplets.

Microscopy Measurements Microscopic analysis of the emulsions confirmed the flocculation behaviour inferred from the creaming results. Figures 5 to 7 show selected images from the recorded video tape sequences. Figure 5 shows the unflocculated emulsion after 60 minutes. Because the system of droplets is unstructured and the sample is relatively thick, the image suffers from a lack of contrast. The microscopy analysis is very sensitive to the effects of creaming, and even though the droplets are near density matched to the continuous phase, some creaming does occur. This is evident in the greater number of large droplets (above that expected from the Mastersizer distribution) which are visible after 60 minutes. For 0.1% HEC small aggregates were visible after only 1 minute. By 10 minutes the aggregates had become larger, and on changing the focal plane were clearly seen to be assembled into a network. This network became coarser with increasing time, and after 60 minutes (see Figure 6) regions were formed for which there was a relatively unobstructed view through the emulsion. For the intermediate HEC concentration, where the creaming measurements indicated a partial flocculation, the video sequence showed that after 1 minute the sample was unflocculated. After 10 minutes small flocs were clcarly

Figure 5

Appearance of the unflocculated emulsion after 60 minutes

144

Figure 6

Ultrasonic Characterization of Flocculation in Oil-in-Water Emulsions

Appearance of the emulsion containing 0.1% HEC after 60 minutes, showing the development of a flocculated network

partial flocculation

D . Hibberd et al.

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visible, and by 1hour these flocs had grown larger but were still clearly discrete (i.e. no network). Figure 7 shows the emulsion after 4 days; large flocs are visible in a 'sea' of unflocculated, but creamed, droplets. The flocs appear to be separate entities and seem to be made mainly from the larger droplets within the distribution. The creaming measurements and the microscopy data thus present a consistent picture of the structures arising from the flocculation process. They confirm that creaming was insignificant during the first hour after preparation. Thus any variation within this time-scale in the ultrasonic properties between the samples, or with time, could be attributed to the flocculation alone.

Ultrasonic Spectroscopy Measurements Figure 8 shows the ultrasonic attenuation and velocity as a function of frequency for the unflocculated sample. This is effectively the control experiment. The attenuation graph shows that no significant attenuation changes are observed over 60 minutes. On the velocity dispersion graph, a small change is observed. Figure 9 shows the ultrasonic data for the fully flocculated sample over a similar period. The initial attenuation data are very similar to the unflocculated sample. They then form a steady progression with the high frequency attenuation increasing as sample age increases. The lines cross over at cu. 2.5 MHz showing that at low frequencies the trend in attenuation with time is in the opposite direction. For the velocity dispersion data, the starting point is once Attenuation (NplmlsA2) 2E-12

Velocity dispersion (mksoc) 0.8

1E-12

0.6 5E-13 0.4

2E-13 0.2

1E-13 5E-14

n

2 3

Figure 8

5 10 20 30 50 Frequency (MHr)

"

10 20 30 40 Frequency (MHz)

Ultrasonic properties as a function of frequency and time for the unflocculuted emulsion: attenuation (shown as alp) and velocity dispersion (shown as change in velocity with frequency normalized to 2 MHz)

146

Ultrasonic Characterization of Flocculation in Oil-in-Water Emulsions Velocity dispersion (mlsec) O-*

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Ultrasonic properties as a function of frequency and time for the full flocculated emulsion containing 0.1% HEC: attenuation (shown as a/ ) and velocity dispersion (shownas change in velocity with frequency normalized to 2 MHz)

fy

Attenu8tion (Nplmls"2) 2E-12

Velocity dispersion (mlsec) 0.0

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1E-13 5E-14 5 10 2030 ! Frequency (MHz)

! 3

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a

10 20 30 40 Frequency (MHz)

,

Ultrasonic properties as a function of frequency and time for the partially flocculated emulsion containing 0.026% HEC: attenuation (shown as alp) and velocity dispersion (shown as change in velocity with frequency normalized to 2 M H z )

147

D. Hibberd et al. Velocity dispersion (mlsec)

Attenuation (Np/dsAZ)

thwory 0.8 -

Frequency (MHz)

Figure 11

....--.

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Frequency (MHz)

Attenuation and velocity dispersion predicted from multiple-scattering theory, showing monodisperse emulsion with droplet diameter I pm and a bidisperse emulsion with diameters I p m and 16 p m

more similar to the unflocculated sample, but then the high frequency velocity dispersion decreases steadily up to 5 minutes. At this point the behaviour changes and the high frequency dispersion starts to increase for the remainder of the time. The steadily rising attenuation increasingly limits the range over which measurements can be made. Figure 10 shows the ultrasonic properties of the partially flocculated sample over 60 minutes. We see similar features to those obtained for the fully flocculated sample, but there is less increase in high frequency attenuation, and at low frequencies the point at which the lines cross over, reversing the trend, is shifted to a higher frequency, occurring here at ca. 4 MHz (instead of ca. 2.5 MHz in the fully flocculated emulsion). The velocity dispersion results are similar to those obtained for the fully flocculated sample, but the changeover in the trend of the high frequency data is evident at 20 minutes, where there is a point of inflection in the dispersion data. It is clear that both the attenuation and velocity ultrasonic data show progressive differences between the samples that can only be attributed to flocculation.

Ultrasonic Scattering Theory Predictions Having demonstrated differences in the ultrasonic spectra of flocculating samples, the next step is to see if ultrasonic propagation theory can be used to extract quantifiable structural information from the data. There are numerous examples in the literature, where workers using the scattering theory of Allegra and Hawley7 coupled to a multiple scattering formalism' have achieved excellent correlations between theory and experiment for unflocculated colloidal systems."-'3 However, extending this theory

148


to include a particle spatial distribution function and, possibly, particleparticle interactions is a huge task. To date we have taken a rather simpler approach. Using the existing Allegra and Hawley theory, extended to include mixtures of particles sizes, we have assumed we can describe discrete flocs as single particles of larger effective size. Figure 11 shows preliminary results from these studies. We have modelled the partially flocculated sample as initially monodisperse with a similar particle size to the mean value determined using the Mastersizer, The onset of flocculation is modelled assuming the flocs behave as a second population of larger effective droplets with particle size sixteen times larger. Thus the two lines on each of the graphs are for a completely monodisperse sample and for a mixture, respectively. These graphs have many of the features of the experimental data. In Figure 11 the attenuation predicted for the monodisperse emulsion has a similar form to the early time data for the partially flocculated emulsion. The predictions for the bidisperse mixture show a higher attenuation at high frequency, and lower attenuation at low frequency. Similarly the velocity dispersion data for the monodisperse theory are similar to the early time data in the partially flocculated emulsion, and the mixture prediction bears a strong resemblance to the measured velocity after 20 minutes, including the approximate position of the point of inflection. From this we conclude that this simple approach may have some use in modelling the ultrasonic properties of samples containing discrete flocs, but clearly the theoretical work is at a very early stage.

4 Conclusions It is clear that ultrasonic measurements are sensitive to the state of flocculation in the oil-in-water emulsions studied. The changes observed over time are consistent with the development of flocs in the two samples containing HEC, and no significant changes were observed in the unflocculated system. Preliminary theoretical analysis indicates that the measurements are consistent with increasing effective particle size, and certain features of a bidisperse model are mirrored in the experimental data. The broad-band fast-Fourier ultrasonic spectroscopic technique is rapid to use and therefore offers the potential to monitor flocculation in real time. Thus new information will be available on the kinetics of a wide range of flocculation processes.

Acknowledgements The authors are grateful to the BBSRC for funding under the ROPA scheme, and to Mary Parker and Paul Gunning for advice on the microscopic techniques used. We thank John Tebbutt for helpful discussions on the application of ultrasonic theory to colloidal dispersions.

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References 1. A. J. Fillery-Travis, P. A. Gunning, D.J. Hibberd, and M.M. Robins, J . Colloid Interface Sci., 1993, 159, 189. 2. A. K. Holmes, R. E. Challis, and D. J. Wedlock, J . Colloid Interface Sci., 1993, 156,261. 3. D. J. McClements, Colloids Surf. A , 1994,90, 25. 4. A. M. Howe, A. K.Mackie, and M. M. Robins, J . Disp. Sci. Technol., 1986,7,231. 5. C. Carter, D. J. Hibberd, A. M. Howe, and M. M. Robins, Progr. Cofloid Polym. Sci., 1988,76,37. 6. R. E. Challis, J. A. Harrison, A . K. Holmes, and R. P. Cocker, J . Acoust. SOC. Am., 1991,90,730. 7. J. R. Allegra and S . A. Hawley, J . Acoust. SOC.A m . , 1972,51, 1545. 8. P. Lloyd and M. V. Berry, Proc. I’hys. Soc., 1967,91,678. 9. A. Parker, P. A . Gunning, K. Ng,and M. M. Robins, Food Hydrocolloids, 1995,9, 333. 10. W. B. Russel, D. A. Saville, and W. R. Schowalter. ‘Colloidal Dispersions’, Cambridge University Press, London, 1989. 11. D. J. McClements, J . Acoust. SOC.A m . , 1992,92,849. 12. C. Javanaud, N. R. Gladwell, S . J. Gouldby, D. J. Hibbcrd, A. Thomas, and M. M . Robins, Ullrasonics, 1991,29,331. 13. A. K . Holmes, R. E. Challis, and D. J. Wedlock, J . Disp. Sci. Technol., 1994,168, 339.

Measuring Aggregation in Colloids using Ultrasound Velocity and Attenuation By Malcolm J. W. Povey PROCTER DEPARTMENT OF FOOD SCIENCE, UNIVERSITY OF LEEDS, LEEDS LS2 9JT, UK

1 Introduction Aggregation in colloids is an important factor determining the stability and bulk properties of colloids.' Colloidal systems may be destabilized by the formation of flocs which then cream or sediment, or they may be stabilized by floc formation and subsequent gelation. So the measurement of the initial stages of aggregation is important for understanding the structure of the weakly aggregated materials which comprise so many of the foods we consume. Low-power ultrasound is well established as a non-destructive technique for characterizing dispersed systems.2The velocity and attenuation are measured, either at a single frequency, or over a range of frequencies. The technique has been successfully applied to the determination of dispersed phase concentration or solid fat content, and to the study of emulsion creaming behaviour and phase transitions. Methods for making such ultrasound measurements alrcady exist. There is experimental evidence that ultrasound is sensitive to the formation of weakly aggregated structure^,^ characterized by the reversible nature of the aggregation, Ultrasound measurements in materials containing such aggregates may therefore allow the determination of the structural features of the system, and the development of a sensor for following the process of aggregation. Ultrasound velocity and attenuation is increasingly being used to characterize colloidal s y ~ t e m sUltrasound .~ can be transmitted through systems which are optically opaque and concentrated. This makes the technique suitable for the in-situ study of real complex colloids such as food colloids. Systems containing bubbles are normally opaque to ultrasound, although ultrasound is very effective for the characterization of single bubbles, and hence of very dilute bubbly dispersions. In this article, reversibly aggregated (or flocculated) dispersions of micelles and oil droplets in water are considered.

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M. J . W.Povey

151

2 Ultrasound Profiling In ultrasound profiling (Figure l ) , a pair of ultrasound transducers is scanned along the length of a column of emulsion which is gravitationally destabilized, exhibiting creaming or sedimentation. The velocity of ultrasound is measured as a function of height in the tube, and the volume fraction of the dispersed phase may then be calculated. A series of scans permits the time evolution of the volume fraction to be plotted. The technique has been described in detail el~ewhere.~ The ultrasound profiling technique was first developed at the Institute of Food Research in Norwich.’ We have automated the instrument, refined the acoustics, and developed techniques which account for scattering. In essence, a pulse of sound travels from the transmitter to the receiver probe. The distance through which it travels is calibrated accurately using the known velocity of ultrasound at the measuring temperature. The time of flight in the sample is then measured using timing circuitry, and the data are fed into a spreadsheet which calculates the oil volume fraction from a calibration table. The transmitted pulse may be Fourier analyzed and velocity and attenuation plotted against frequency. It is also desirable to know the temperature dependence of the velocity of ultrasound and the Cygnus velocity cell (Figure 2) is convenient for this purpose. The ultrasound probes in the profiler are scanned up and down the tube containing the experimental sample (250 mm), and the sound speed is determined at small intervals (say 1mm each). The velocity is converted to a volume fraction and the result plotted against height and time as the emulsion creams or sediments under gravity. The calibration involves the use of a modified Urick equation, where two scattering coefficients may be determined6from the first scan. The known volume fraction in the initially uniformly dispersed sample is used to determine the coefficients which are subsequently used on later scans to determine the dispersed phase volume fraction from the ultrasound velocity.

35 m m

Figure 1

Schematic diagram of the ultrasound profiler

1.52

Measuring Aggregation in Colloids using Ultrasound Velocity und Attenuation

Lid

,111111.

Jlr

b - 9 9 9 9 1

Magne

PRT remperature sensor

I Figure 2

ltraso nic transducer transrr it / receive

Water bath

I

Schematic diagram of the Cygnus ulirasotiic velociiy meter

This instrument has been used to study the time evolution of the dispersed phase volume fraction. Evidence will be presented here that this apparatus can detect the state of aggregation of oil droplets and of micelles through its ability to determine the ultrasound phase velocity and attenuation as a function of frequency .

3 Flocculation and Creaming in Oil-in-Water Emulsions Oil-in-water emulsions prepared with the small amphiphilic surfactant Tween 20 or the protein emulsifier sodium caseinate have now been studied quite extensively using the ultrasound technique. In particular, effects of nonadsorbed species on emulsion stability have been investigated. In the following eight Figures, a representative selection of types of behaviour observed in oilin-water emulsions are presented, all of which have been determined using the ultrasonic profiling method. The most striking feature is the great variety of behaviour to be observed in apparently very similar systems. Figure 3 represents a ‘stable’ emulsion in which the oil content is lowered by eight percent at the bottom of the sample, although to the naked eye this change is undetectable. This emulsion formed the stock emulsion for experiments summarized in a number of the following profiles, where the emulsion was destabilized through the addition of xanthan gum, a microbial polysaccharide. In Figure 4, the stable stock emulsion has been destabilized by the addition of a very small amount of xanthan (0.017 wt%). This causes flocculation through the depletion mechanism A distinct oil-depleted serum layer is rapidly seen at the bottom, but the boundary between the serum and the emulsion does not then move. Computer modelling indicates’ that this boundary will either move up the tube, or not be apparent, in a non-flocculated emulsion. In this experimental case, it is probable that flocculation has led to the formation of a weak gel in the emulsion, which has prevented further creaming and the development of a cream.

,’

M . J . W . Povey

0.3 U

0.1

153

! \ 0

Iz F

0 (Y P

Height (mm)

Figure 3

Plot of volume fraction against height for a creaming oil-in-water emulsion (I8vol% mineral oil, 2 wt% Tween 20,30 "C.dLY2 = 0.65 pm): a, after 3 h; 0, after I88 h. (Taken from ref. 18) 0.4

0

0 (Y P

Height (mm)

Figure 4

As in Figure 3 except with 0.017 wt% xanthan added to the continuous phase: 0 after I h; 0, after 9 h; U, after 19 h; 0, after 138 h. (Takenfrom ref. 18)

Measuring Aggregation in Colloids using Ultrasound Velocity and Attenuation

154

0.6

0.5 C 0

P

-g

0.4

B

0.3

0.2

0.1

0 0

m N

Helght (mm)

Figure 5

Crearningprofiles for 15 wt% n-tetradecane oil-in-water (0.75 wt% sodium after 43 ;B, caseinate, 0.05 wt% xanthan d,, = 1.25pm: 0, after 18 h; 0, after 127 h; 0, afier 154 h; A , after 223 h. (Taken from ref. 19)

In Figure 5, we observe the type of behaviour to be expected from an emulsion which has a narrow size distribution of particles creaming in an unhindered way. This is characterized by a sharp serum boundary moving up the tube and a cream layer which expands to meet the rising serum, leaving a sharp boundary between cream and serum. In Figure 6 are observed effects consistent with a large particle diffusion component which blurs out the serum/ emulsion boundary. We can also see, in this set of profiles, that the cream layer is undergoing compression. Figure 7 shares with Figure 4 the characteristic of a static serum/emulsion boundary. We might therefore assume that the emulsion has gelled. However, the development of a cream layer indicates that gel development was delayed until at least part of the oil had formed a cream, and this is consistent with the reduced volume fraction of oil in the emulsion phase at the end of the

155

M. J . W.Povey

0.6

0.5

s

.; E

E

0.4

B 0.3

0.2

0.1

0 0

R

F

3

Height (mm)

Figure 6

As Figure 3 except with 0.I73 wt% xanthan added: 0,after I I h; 0, after 20 h; B, after30h; 0 ,after45h; A , after99h; A , after188h. (Takenfromref. 18)

experiment. So here it seems that the processes of flocculation, creaming and gel formation are taking place together in the same system. Figure 7 contrasts with Figure 8, where a lower xanthan concentration has been sufficient to destabilize the emulsion, but is apparently not sufficient to gel the emulsion at any stage. Figure 9 stands out by displaying a serum phase which grows in volume. Rheological studies indicate that the emulsion phase has gelled in these experiments, so we must infer that polymer is being displaced from the gel into the serum which grows as a consequence. This is a concentrated system (35 wt% oil) and so it is not unreasonable to suggest that the gel is collapsing under its own weight. Finally, in Figure 10, we have a situation in which there is no significant serum, certainly not one visible to the naked eye, and the beginnings of a cream.


156

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0.5 E

.-0

c)

L IL

04

0

> 0.3

0.2

01

__t__

0

0

2

+

8

Height (mm)

Figure 7

As Figure 3 except with 0.035 wt% xanthan added: 0,after 1 h; 0, after 6 h;

M, after I 0 h; 0, after 19 h; 18)

A,after 29 h; A,after 189 h. (Taken from ref.

Overall, creaming profiles such as these can give detailed indirect information about the state of flocculation of an emulsion. However, such data would be more useful if they could be augmented with other sourc.es of direct information about the emulsion, particularly its state of aggregation.

Frequency Dependent Ultrasound Velocity and Attenuation A number of improvements to the original design of the ultrasound profiler has made it possible to measure the frequency dependent ultrasound velocity and attenuation. At present this can be done over the range 0.1 MHz to 10 MHz. In Figure 11 are plotted the group velocity (nominal frequency 2.25 MHz) and attenuation for a casein containing emulsion similar to that of Figure 9, but at

157

M . J . W . Povey

0.7

0.6

0.5

0.3

0.2

0.1

0 0

0

2

00 N

Height (mm)

Figure 8

As Figure 3 except with 0.069 wt% xanthan added: 0,after 1.5 h; 0, after 4 h; D, after 6 h; 0, after 10 h; A, after 19 h. (Taken from ref. 18)

the much lower oil concentration of 10~ 0 1 %These . emulsions creamed quickly initially, but then formed a 'stable' emulsion phase consistent with a gel. Their overall behaviour was most like that of Figure 7. One feature of interest here is the observation of a secondary creaming effect. What appears to be happening is that very fine, unflocculated oil particles which have been left behind in the serum by the larger oil drops and flocs, finally do begin to cream, but find their progress hindered by the gelled emulsion. They gather at the serum/emulsion

158


0.7

0.6

0.5 C

0

zc

0.4

03

0.2

0.1

-0 0

0

2

P 0 (Y

Height (mm)

Figure 9

Creaming profiles for 35 wt% n-tetradecane oil-in-water emulsions containing 4 wt% sodium caseinate, 30 "C, p H 6.8, dJ2 = 0 . 5 p m : . after52 , h; 0, after 64 h; U, after3 days; 0 ,after 6 days; A , after 12 days; A,after 1.5 days; after 19 days; 0, after 33 days. (Taken from ref. 20)

+,

interface forming a very thin oil-rich layer, visible to the naked eye and also apparent in both the velocity and attenuation plots. This effect is made more marked by the back-flow of liquid around the more rapidly moving large particles, which actually causes the smaller particles to move downwards.8 Secondly, the attenuation data suggest that there is a considerable variation in properties between the bottom and the top of the serum. Since the velocity data suggest that this is not due to variation in the oil volume fraction (velocity data are plotted at 5 mm intervals in this region; attenuation at 1 mm intervals), it

M. J . W.Povey

159

0.7

0.6

0.5

.-s

i0 4

-5 LL

g

0.3

0.2

0.1

f

04

0

Figure 10

.,

0 0 04

Height (mm)

As Figure 3 except with 0.05% xanthan and 0.05 M NaCl added: 0,after 1 h; 0, after 3 h; after 5 h; 0, after 8 h; A, after 43 h; A, after 138 h. (Taken from ref. 18)

may be surmised that this is a polymer concentration effect, arising from the displacement of polymer from the flocculated emulsion. The attenuation data in Figure 11 were computed from the Fourier transformed ultrasonic signal whose frequency space representation appears in Figure 12. The serum region has been plotted on an expanded scale in order to highlight the fascinating detail evident in the serum signal. There is clearly a lot of frequency dependent scattering taking place within the serum layer, which is indicative of considerable heterogeneity in this part of the sample. The difference in behaviour between the serum and the emulsion is quite dramatic. An analogy that might

160

Measuring Aggregation in Colloids using Ultrasound Velocity and Attenuation 1500

15

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z

0

Group velocity and attenuation of a 2.25 M H z centre frequency pulse through 10 wt% n-tetradecane oil-in-water emulsions containing 4 wt% sodium caseinate, 30 "C, p H 6.8, d,, = 0.5 p m . Profile was taken after three hours. The attenuation plot was obtained f r o m the data in Figure 12 and is the integrated attenuation, divided by the number of points in the Fourier transform

be useful in understanding this plot is that of light scattering. If 0.1 MHz ultrasound corresponds to the infra-red and 4 MHz ultrasound to the ultraviolet, then we can construct a hypothetical colour version of the picture in Figure 12. We could imagine the acoustic signal from the serum corresponding to a sample which appears opalescent in white light, showing a variety of

161

M . J . W . Povey

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-*O 1.OE+05 1.4E+06

2.7E+06

Frequency IHz

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4.OEt06

Ultrasonic signal amplitude plotted against frequency, relative to the water signal at the same frequency. These data are for the sample of Figure I I

colours in different parts of the sample. In this analogy the bulk emulsion would appear more or less grey, whilst the incipient cream layer would appear blue as the lower frequencies were blocked out and the higher ones were transmitted.

Detecting the Flocculation of Oil Droplets The frequency scanning apparatus at Leeds has been used directly to detect the flocculation of oil droplet^.^ Both the frequency dependent velocity and the attenuation have indicated that the scattering of ultrasound decreases appreciably when depletion flocculation takes place. This result may be explained in terms of the overlap of the thermal and viscous scattered radiation from each oil drop with its near neighbours in a floc. At 3 MHz and 20 "C the thermal and viscous skin depths are 120 nm and 500 nm, respectively. These compare with the separation of flocculated droplets which must be less than 10 nm which is the approximate diameter of the Tween 20 micelles present in these experiments. However, the changes in ultrasound velocity occurring as a result of flocculation are very small, i.e. just 2 m s-' in the experiments carried out at Leeds by M ~ C l e m e n t sIf. ~this were not the case, of course, the volume fraction determined from the ultrasound profiling (Figure 3 to Figure 10) would be in error in all cases of flocculation. In fact, the profiling technique is very accurate-its accuracy can be checked simply by integrating the oil volume from top to bottom throughout the creaming process. The total oil volume should equal the original oil volume. If it does not (within say l%),then we know that the modified Urick Equation6 is inapplicable. Attenuation changes are likely to be more valuable for studying changes in the state of aggregation.

162


4 Detecting Micelle Aggregation There is a large body of work' employing ultrasonic relaxation techniques between 0.5 MHz and 80 MHz to study the exchange of surfactant monomer between micelles and solvent. This is not particularly relevant to the present discussion, and it is mentioned here only to clarify that the technique is different to the one discussed here. The same processes that lead to changes in ultrasound velocity during oil droplet flocculation will also take place in micelle aggregation. However, at least in the case of the aggregation of casein submicelles'" into micelles, the forces involved are stronger than the reversible flocculation discussed above. It may therefore be assumed that a casein micelle behaves as a single particle, rather than a floc. In this case it is possible to calculate the effects of casein micelles, casein submicelles and Tween 20 micelles using ultrasonic scattering theory" in the long wavelength limit. The results of these calculations appear in Figures 13 and 14. In Figure 13, it should first be noted that the predicted attenuation at 1 MHz is in all cases too small to be measured with our equipment. However, at 10 MHz the effects are large, and it should in principle be possible to effectively monitor the state of aggregation of casein micelles at this frequency. The same situation applies to predicted changes in the ultrasound velocity shown in Figure 14. The frequency range available to our equipment is 0.1 MHz to 10 MHz. Apparatus is also available that can operate at 100 MHz and above. Over this frequency range the calculations lead us to expect no frequency dependent effects from Tween 20, which is what we have observed. There has also been a considerable amount of work using ultrasound measurement techniques on ordinary skimmed milk" and milk coagulation.'"I6 It will be clear from the discussion so far that ultrasound propagation depends on the state of aggregation of both oil droplets and casein micelles, and neither phenomenon has been taken into account properly in work on milk published hitherto. Once a gel forms, there may well be further effects on propagation; these are the subject of the next section.

5 Ultrasound Measurements: Rheology ,Aggregation and Gelling In general, measurable changes in ultrasound velocity are not expected at the liquid-gel transition for weak g 1s formed from reversible flocculation. This is because the rigidity modulus ( /Pa) is of the order of lo9times smaller than the bulk modulus (KPa). The v ocity of sound in a solid is given by a simple relationship between the velocity of sound (vlm s-'), the elastic modulus (M/Pa) and the density (p/kg mP3), i.e.

,.'

163

M. J . W. Povey

loo00

lo00

v

a

c

E

n

z

?= s 8

loo

10 0

2

4

6

8

10

12

Log(f IHz)

Figure 13

Frequency dependent attenuation from (a) casein micelles (volume fraction 80% corresponding to 12 wt% casein in water), (b) casein

submicelles9, (c) Tween 20 micelles (2 wt% Tween 20 in water) and ( d ) in absence of scattering. The theoretical calculation on which this plot is based is given by McClements and Povey." (Some data for the calculation have been taken from ref. 12)


164

1570

1560

1550

1540

1530

-

@ 1520

\

>

1510

1500

1490

1400

1470 Log(f IHz)

Figure 14

Frequency dependent velocity v(f) for the systernsplotted in Figure 13

It should not be surprising, then, that efforts to detect changes in the storage modulus of weak gels using the ultrasound technique have been unsuccessful. ‘7 Changes observed by Audebrand et a1.” in attenuation in weak gels during the gelling process are almost certainly associated with changes in the state of aggregation of the monomer, rather than with the appearance of a shear modulus. Benguigui et al. have, nonetheless, recently reported14 changes in velocity on rennet and acid gelation in skimmed milk. This could be explained partly by

M . J . W . Povey

165

changes in the state of aggregation of the casein micelles, which would certainly have a significant effect at the 60 MHz employed by these workers. In addition, the much larger effect observed from acid gelation (Table 1j may be explained by changes in the compressibility of casein arising from the disruption of the core of the micelle caused during acid gelation. Rennet, on the other hand, has little effect on the micelle c01-e'~ and its effects on ultrasound propagation are correspondingly much smaller. A simple relationship exists between the real and imaginary parts of the elastic modulus and the ultrasound propagation parameters, velocity and attenuation ( a / Nepers m-I):

where primed quantities are real and double primed quantities are imaginary. We then have

Equations (5) and (6) assume that the attenuation is small. These equations have been used to calculate the storage and loss moduli shown in Table 1. It should be noted when reading Table 1 that the relative contributions of K and G cannot be discerned from the available data at ultrasonic frequencies.

Table 1

Experimental results at frequencies between 0.1 H z and 60 M H z for aggregation of casein micelles

Conditions

Variable

0.1 Hz, 25 "C" 0.1 Hz, 25 "C" 1 Hz, 25 "Cb 2 MHz, 30 "CC 2 MHz, 30 "C' 60 MHz"

G'IPa GIPa G' Pa

Ungelled

0.4 x 10'

Avlm s-'

60 MHz"

M'IPa

60 MHza

AWIPa

With rennet 4.20 2.16 25-250

Avlm s-'

MIPa

With acid

2.481 x lo9

Ref. 14. Ref. 12. 'Cosgrove, O'Donncll and Povey, unpublished results

-0.5 variable 3.5 44 "C 2.492 x 10' 44 "C 20.3 x 10' 44 "C

0

0.4 x 10' 0.2 25 "C 2.482 x 10' 25 "C 0.2 x 10' 25 "C

166


6 Conclusions Ultrasound measurement techniques are a valuable addition to the arsenal of techniques available to food scientists for the study of aggregation in colloids. However, great care needs to be taken in interpreting results and a detailed understanding both of ultrasound propagation and of the system being studied is often essential to success. Ultrasound propagation is sensitive to the flocculation of oil droplets and to the aggregation of some types of micelles. The interaction mechanisms involved include the scattering and overlap of thermal and viscous waves. Ultrasound can detect changes in high frequency storage and loss moduli, but these changes are not directly associated with the formation of a weak gel, but rather with the aggregation processes which often precede gelation. Ultrasound spectrometry has a lot to offer the study of aggregation processes, but a great deal more needs to be done before the interaction of ultrasound with aggregating systems is fully understood. All these techniques have now been integrated within the ultrasound profiling technique. Whilst any one ultrasound measurement, made in isolation, can often reveal very little about the system in question, when the array of techniques are gathered together in the ultrasound profiling apparatus, a new and powerful tool for the study of aggregation in colloids emerges.

Acknowledgements I would like to acknowledge valuable discussions with Valerie Pinfield and Matt Golding of the Procter Department of Food Science, University of Leeds, and Niall Cosgrove of University College Dublin. Part of this work has been funded by a grant from the BBSRC and by a grant from Leeds University.

References 1. P . C. Hiemenez, ‘Principles of Colloid and Surface Chemistry’, 2nd edn, Marcel Dekker, New York, 1986. 2. E. Dickinson and D . J. McClements, ‘Advances in Food Colloids’, Blackie, London, 1995, chap. 6. 3. D. J. McClements, Colloids Surf. A , 1994, 90, 25. 4. M. J . W. Povey, in ‘New Physico-Chemical Techniques for the Characterization of Complex Food Systems’, ed. E. Dickinson, Blackie, London, 1995, chap. 9 . 5 . A . M. Howe, A . R. Mackie, and M. M. Robins, J . Dispersion Sci. Technol., 1986, 7,231. 6. V . J . Pinfield, M. J. W. Povey and E. Dickinson, Ultrasonics, 1996,34, 695. 7. S. Asakura and F. Oosawa, J . Polym. Sci., 1958,33, 183. 8. V . J . Pinfield, E. Dickinson and M. J . W. Povey, J . Colloid Interface Sci., 1994,166, 363. 9. A. Borthakur and R . Zana, J . Phys. Chem., 1987,91,5957. 10. B. Chu, Z . Zhou, G. Wu, and H. M. Farrell, Jr., J . Colloid Interface Sci., 1995,170, 102. 11. D. J . McClements and M. J . W. Povey, J . Phys. D : Appl. Phys., 1989, 22, 38.

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W. G . Griffin and M. C. A. Griffin, J . Acoust. SOC.A m . , 1990,87,2541. C . A. Miles, D. Shore, and K . R. Langley, Ultrasonics, 1990,28, 394. L. Benguigui, J . Emery, D. Durand, and J. P. Busnel, Lait, 1994,74, 197. S. Gunasekaran and C. Ay, Food Technol., 1994,48(12), 74. S. Bachrnan, B. Klimaczak,andZ. Gasnya,ActaAlimentariaPolonica, 1978,4,55. M. Audebrand, J.-L. Doublier, D. Durand, and J. R. Emery, Food Hydrocolloids, 1995,9, 195. 18. E. Dickinson, J . Ma, and M. J . W. Povey, Food Hydrocolloids, 1994,8, 481. 19. Y . Cao, E. Dickinson, and D. J . Wedlock, Food Hydrocolloids, 1988, 5,443. 20. E. Dickinson, M. Golding and M. J . W. Povey, J . Colloid Interface Sci.,in press.

12. 13. 14. 15. 16. 17.

Structure of Fat Crystal Aggregates By William Kloek, Ton van Vliet and Pieter Walstra DEPARTMENT OF F O O D SCIENCE, WAGENlNGEN AGRICULTURAL UNIVERSITY, P.O. BOX 8129, 6700 EV WAGENINGEN, T H E NETHERLANDS

1 Introduction One of the main functions of fats in food products is their contribution to structural and sensory properties. The liquid fat phase is interspersed with solid fat, yielding a continuous fat crystal network. The fraction of solid fat largely controls the mechanical properties. Too high fractions of solid fat yield hard products with undesirable brittleness. Lower fractions of solid fat yield products with desirable properties, like spreadability. Too low amounts of solid fat can result in oiling-off. This is due to the low elastic moduli of the product and the large pores surrounding the fat crystal network; the product cannot withstand its own weight and oil can permeate easily through the pores. The latter process can become important over long time scales (relevant to shelf life) or when high stresses are applied, for instance when the product is packed in tall containers. The fat crystal network is formed due to aggregation of crystals, which are attracted to each other by van der Waals attraction.* Description of the aggregation process is hampered by the fact that crystallization and aggregation processes proceed simultaneously. Fat crystals already start to aggregate, while nucleation and crystal growth still occur. For reasonable time-scales of aggregation and crystallization, it follows that at a volume fraction of solid smaller than -0.02 a continuous crystal network can already be formed. Ongoing crystallization will produce crystals that can either initially aggregate within the pores or can aggregate with the strands of the primary network. In addition, the crystals forming the primary network will tend to grow. The structure of the primary network is therefore of great importance for the final structure and for the mechanical properties of the fat. The goal of this study is to elucidate the structure of fat crystal aggregates at low volume fractions of solid fat and to see whether the structure observed can be used to explain the mechanical properties of fully crystallized dispersions at small deformations. The structure of crystal aggregates at low volume fractions can be analyzed by viscometry and light scattering. Mechanical properties at

'

'

168

169

W. Kloek, T. van Vliet, and P. Walstra

high volume fraction can be determined by dynamic rheological measurements.

2 Materials and Methods Materials The model system used consists of fully hydrogenated palm oil (HP) in sunflower oil (SF), these being the solid phase and the liquid phase, respectively. This model system is known to be stable in the p' polymorph when crystallized in the p' polymorph. The HP consists of numerous different triglycerides, and the solid phase therefore shows compound crystals and has a significant range. Thermodynamic properties of the model system were determined using DSC by measuring the crystallization temperature of the a polymorph (rapid cooling) and the melting temperature of the p' polymorph (after isothermal crystallization above the a melting temperature). The DSC peak temperatures of dispersions with different H P volume fractions were fitted to the Hildebrandt equation assuming ideal mixing behaviour to give the molar enthalpy of fusion and the melting temperature3

Here, x is the mole fraction of soluble HP in polymorph i at temperature T, R is the gas constant, AHf,;is the molar enthalpy of fusion of H P in polymorph i, and T,,,,i is the melting temperature of H P in polymorph i. The Hildebrandt equation can be used for systems in which the liquid and solid phases have similar chemical structure and for which there is a great difference in melting temperatures. Obtained melting enthalpies of the a and p' polymorphs were 98 and 161 kJ mol-', respectively, and the melting temperatures were 41.8 and 57.3 "C, respectively. The driving force for crystallization is the difference in chemical potential 4 4 between the supersaturated dispersion and the saturated dispersion, i.e.

where c is the mole fraction of HP, x is given by equation (I), supersaturation ratio, and In/? the supersaturation.

p

is the

Methods Low volume fraction HP/SF solutions (& 5 0.01) were crystallized at a shear-rate of 460 s-l in a concentric cylinder device for 30 minutes at various initial supersaturations in the p' polymorph. After crystallization, the dispersions were allowed to aggregate at various lower shear-rates during which the

170

Structure of Fat Crystal Aggregates

viscosity was determined as a function of time. After aggregation, a shear-rate sweep was applied. Mechanical properties of dispersions containing higher volume fractions of H P (0.06 < Go < 0.15) were determined using dynamic measurements during isothermal crystallization of the dispersions at various supersaturations. Oscillations were applied at a frequency of 0.1 Hz and a maximum strain amplitude of 0.0005. This strain amplitude is within the linear region for a fully crystallized dispersion. Two-dimensional light scattering of a few dispersions was monitored during crystallization and aggregation, using a special glass apparatus of concentric cylinder geometry. Photographs of the two-dimensional scattering patterns were taken using a digital camera and converted to intensity-wave vector curves by home-made software.

3 Results and Discussion Viscosities of Low Volume Fraction Dispersions Figure 1 shows the yielding behaviour, given as viscosity as a function of time, of an HP/SF dispersion crystallized in rest. The viscosity profile as a function of time looks very irregular. At the lowest shear rate (part (a)), a yielding is clearly visible from the stress overshoot. The shear stress, which is proportional to the viscosity, becomes constant after some time (--lo3 s). At a higher shear rate (part (b)), yielding is less pronounced, and the shear stress also becomes roughly constant although its magnitude is lower than at the highest shear rate. The stress overshoot indicates that the dispersions are gelled. When the gel is sheared, rather strong van der Waals bonds and probably much stronger sintered crystal bridges have to be broken. Inhomogeneous structures lead to an inhomogeneous stress distribution and this therefore causes the formation of shear planes with solid aggregates in between. Because fats are strongly shear thinning at the regions that are yielding first, the stress needed for further deformation will decrease strongly, which causes the flow to become even more inhomogeneous. Variation in the formation of shear planes can also explain the scatter in the results for viscosity as a function of shear rate. To study the aggregation process, it was necessary to separate the crystallization step from the aggregation step. To this end, mixtures were crystallized while being sheared at a rate of 460 s-' and subsequently allowed to aggregate at a lower shear-rate. Figure 2 shows the viscosity during aggregation at various shear-rates. The viscosity increases from roughly that of the continuous phase (about 120 mPa s) to values several times higher. The viscosity increase can be explained by the increase of the volume fraction of aggregates. Directly after crystallization this volume fraction roughly equals the initial volume fraction H P (Go = 0.005), assuming that no aggregates or very dense aggregates were formed. As aggregation occurs, fat crystals form tenuous flocs with a total volume fraction much higher than Go. Figure 2 shows that for all aggregation shear-rates the steepest increase in viscosity takes place during the first, say,

171

W . Kloek, T. van Vliet, and P . Walstra

viscosity

(a)

0

1000

2000

3000

4000

time (s)

4 .A A

0

1I

0

1000

2000

3000

4000

time (s) Figure 1

Viscosity of a 0.5% HPISF dispersion, crystallized at 5°C at rest, as a function of shearing time for various shear-rates: (a) 0,0.0146s-'; (b) 0 , 0.073s-'; A, 0.146s-'

172


I

n

0

0

I l ~ ~ ~ ~ ~ ~ ~ ~ A A ~ ~ A A A A A A A A A A A A A A A A A A A A A A A A ~ A A A A A A A A A A A A A A ~ ~ A A A A ~

IrrrrnPPPlPIPPPPIPiPPPllPPPPP~P~~~~P~~~PPP~~~PPPP~~

200 seconds. The initial rate of increase is independent of the aggregation shear-rate, indicating that aggregation is a rapid (diffusion-limited) process. The viscosities measured after 1 hour of aggregation as a function of the shear-rate at which aggregation took place are plotted in Figure 3 for dispersions with various volume fractions &. A11 dispersions were crystallized at the same initial degree of supersaturation. The viscosity decreases with increasing shear-rate, which can be explained by the increasing shear stress applied to the aggregates. This would imply that the aggregates remain smaller and more dense, and thereby the volume fraction of the aggregates decreases. The volume of fractions can be modelled by assuming the aggregates to have a fractal structure. A fractal is a geometrical structure showing, within certain length scales, the same structure at different magnifications. A fractal aggregate is characterized by a fractal dimensionality D that relates the number of particles N , in the aggregate to the dimensionless aggregate radius D

(3) where R is the aggregate radius, a the particle radius, and q5 the volume fraction of particles in the three-dimensional aggregate. Potanin has developed a

W . Kloek, T . van Vliet, and P. Walstra

173

model"' that gives the viscosity of a dispersion of non-interacting fractal aggregates as function of shear rate. We will briefly outline his approach. It is assumed that the aggregate radius as function of shear rate f scales with an exponent -m. (This relation has been validated by computer simulations.) Rewriting this proportionality in a dimensionless form yields:

where C = ( 5 D P mis a constant related to the aggregate break-up criterion and G is the dimensionless shear-rate.'The later is given by

with vo the viscosity of the continuous phase and omthe breaking stress o f an interparticle bond. The magnitude of omcan be calculated from the interaction potential between two particles divided by the cross section on which it acts. For a dispersion of aggregates it is assumed that the viscosity of the whole dispersion surrounding the aggregates equals the viscosity of the dispersion q (mean field approach), so that equation (4) can be rewritten as:

174


R

G"

a

The volume fraction of aggregates

is then given by:

(7) If it is assumed that there is only hydrodynamic interaction between the aggregates, the viscosity of the dispersion is given by the semi-empirical Krieger-Dougherty relation'

with @m the volume fraction of random close packing. Experimental results can be fitted to equations (6), (7) and (8) with m , D and urnas fit parameters. For very small dimensionless shear-rates, m and D are related by l l ( m ( 3 - D ) )= constant: consequently m and 0, were used as fit parameters for some realistic values of D . 6 The final viscosities after aggregation were fitted to this model and the results are shown in Figure 3 and Table 1. With decreasing volume fraction of HP the shear rate exponent m increases or D increases slightly. The somewhat deviating results of the fit parameters for Q0 = 0.0075 are explained by the influence of u,on m or D . The order of magnitude of u, which gives the best fit can be explained by van der Waals attraction. The van der Waals interaction force F, between spherical particles is approximated by

Table 1

Fitted m values for a number of fractal dimensionalities D as determined from the $nu1 viscosities as function of shear-rate at various volume fractions of H P . Also the optimum bond breaking stress urnis given. Dispersions were crystallized in the /3' polymorph at In /3 = 5 m

$0

0.01 0.0075 0.0050 0.0020

D = 1.7

D = 1.9

D = 2.1

D = 2.3

0.34 0.38 0.36 0.38

0.40 0.45 0.43 0.45

0.48 0.55 0.53 0.55

0.63 0.71 0.68 0.70

cr,/Nm-' 104 5 x 103 1o4 lo4

175

W . Kloek, T. van W e t , and P . Walstra

Table 2

P

Fitted m values for a number of fractal dimensionalities D as determined from the viscosities measured in a shear-rate sweep after aggregation at a shear rate of 0.046s-' for various HPISF dispersions. Also the optimum a,,, is given. The 0.5% HPISF dispersions were crystallized in the /3' polymorph at various supersaturations

D

4.5 5.0 5.5

6.0

= 1.7

0.28 0.27 0.26 0.28

D = 1.9

D = 2.1

D = 2.3

u,,,/Nm-2

0.33 0.32 0.31 0.32

0.40 0.39 0.38 0.40

0.52 0.50 0.49 0.51

16 3 x 105 7 x 16 4 x 105

where A H is the Hamaker constant and H is the separation distance between the particles. The van der Waals force acts over a cross section of nu2so that the interparticle bond strength a,,, is given by F,.,/(na2). Assuming a,,, to be lo4 Nm-2, and estimating a = m and H = 4 X lo-'' m, yields a Hamaker constant of 6.0 X lo-'' J (1.6kT),which is a realistic value. The bond strength between the crystals is influenced by particle size and should therefore be sensitive to the initial supersaturation at crystallization. This is shown (Table 2) by fitting viscosities obtained from a shear-rate sweep applied to dispersions that were crystallized at various supersaturations and aggregated at a shear-rate of 0.046s-'. The bond strength increased with increasing supersaturations up to Ins = 5.5. The deviating value for the dispersions crystallized at In/3 = 6 may possibly be explained by some crystallization occurring in the a polymorph, as the a polymorph is almost supersaturated at these conditions. Since only shear-rate sweeps were performed on dispersions aggregated at a shear rate of 0.046 s - l , somewhat higher bond strengths were obtained. This will be discussed in more detail elsewhere.'

Light Scattering Since it was not possible to distinguish between m and D,light scattering was used to estimate D.The scattered intensity is proportional to the particle form factor P and the structure factor S. The particle form factor can, for a given geometry of a single particle, be calculated for various scattering angles and particle sizes. For very small particles and at very low scattering angles, P is about unity. The structure factor S depends on the radial distribution function g ( r ) of an aggregate. For a fractal aggregate the latter is proportional to rD-3.It can be shown that if P equals one, the scattered intensity I as function of the wave vector q scales with an exponent -D.This relation is valid for 1/R < q < l / u . The wave vector q is given by 4nn0 q=Tsin(:)

176

Figure 4


Two-dimensional light-scatteringpatterns of a 0.25% HPISF dispersion: ( a ) just after crystallization at a shear rate of 460s-I; (b) after 40 minutes of aggregation at 0.04 s-I

where 1 is the wavelength, no is the refractive index of the continuous phase, and 8 is the scattering angle. The scattering profiles in Figure 4 show that, directly after crystallization, light is scattered up to high 8. As soon as the shear-rate is lowered to the aggregation shear-rate, scattering is more concentrated at lower 8 . This indicates that the scattering structures increase in size. The I-q curves in Figure 5 show that directly after crystallization there is no power-law relation. After 1 minute of aggregation the relationship is still not linear but after longer times the slopes become more linear. After 5 minutes a correlation coefficient of

L 5

10 Figure 5

6

10 Scattered relative intensity (I/Imox) as a function of wave vector q of an aggregating 0.25% HPISF dispersion after various times of shearing at 0.04s-I: 0, start; 0, I minute; A,5minute.y; X , 20minutes; *, 60 minutes

W . Kloek, T. van Vliet, and P . Walstra

177

0.995 with a slope of -1.6 is obtained. After longer times, correlation coefficients become higher with a final slope of about - 1.7 to - 1.8. The timescales over which the scattering patterns change compare well with the timescales over which the viscosity increases, and this should be caused by growth of aggregates. Fitted slopes would correspond to fractal dimensionalities of 1.7 to 1.8. These values are expected for rapid aggregation processes where no barrier exists for aggregation. Rearrangements during shear would be expected to lead to more compact aggregates, i.e. to a higher dimensionality. However, rotational diffusion of the crystals would be expected to lead to lower dimensionalities. The biggest change in scattering and also in viscosity takes place during, say, the first 60 seconds. Assuming spherical crystals and a flocculation time of 60s yields a particle radius of about O.lpm which corresponds to a rotational diffusion coefficient of about 1s-'. As this number is larger than the shear-rate during aggregation, rotation of particles will cause aggregates with smaller dimensionalities. This small particle size also allows us to neglect the influence of q on P a t the low &values used. Vreeker etal." have determined similar dimensionalities for tristearate in olive oil dispersions. Although the derived dimensionalities compare well with some theoretical values, one has to be careful applying these relations to systems containing anisometric particles, since along every axis of a crystal different length scales and refractive indices have to be used. Furthermore, there is a variation in crystal sizes and aggregate sizes. Dimensionalities of 1.7-1.8 would be expected to correspond to values of the shear-rate thinning exponent rn of ca. 0.3-0.4 (see Tables 1 and 2).

Mechanical Properties of More Concentrated Systems An important consequence of a growing fractal aggregate is that, for dimensionalities smaller than 3, the volume fraction of particles in the aggregate decreases with increasing aggregate radius. When the average volume fraction of particles in the aggregates, qj, equals the volume fraction of initial particles, &,, a gel is formed built of aggregates with an average radius a Storage moduli of gels built of fractal aggregates as function of@"scale with an exponentp where the value ofp depends on the fractal dimensionality of the aggregates and on how the stress carrying chains are arranged." The exponent p is given by xl(3 - D)where x can range from 2 for straight stress-carrying chains to 4.3 for completely flexible stress carrying strands with a kinky backbone. According to Potanin,6 for elastic aggregates x is given by Urn, where rn is the shear-rate thinning exponent in equation 4. Based on this argument, the scaling exponent p = (l/rn(3 - D))is expected to be 2.0-2.8, if aggregation starts only after all the HP is crystallized. However, in our experiments on concentrated systems the crystallization and aggregation occur simultaneously, and so other values f o r p are to be expected. Figure 6 shows the storage moduli of isothermally' crystallized dispersions after the maximum amount of solid fat is obtained. Although it is expected that the network cannot be described by a single dimensionality, there is a roughly

178


r x

A

1o3 0.01

0.1

1

40

Figure 6

Storage moduli (GI) at f = 0.1 Hz for fully crystallized HPISF dispersion as a function of the volume fraction of H P . Dispersions are crystallized at various initialsupersaturations in the B' polymorph: A ,1nB = 3.25; x, In B = 3.50; *, In fl = 3.75; 0, In B = 4.00

linear power-law relation between the storage modulus and the volume fraction of solids. The exponents are cu. 6 for dispersions crystallized at low supersaturations and 4.08 when crystallized at a high initial supersaturation of 4.0. These exponents differ considerably from the values that would be obtained when aggregation starts only after all the HP is crystallized. Exponents of ca. 4 were also obtained when the dispersions were crystallized in the a-polymorph irrespective of the applied supersaturation. Papenhuijzen found'* an exponent of 4.1 for tristearate in olive oil crystallized in the a polymorph. If we assume x to equal l l m , where m is obtained from viscometric data, x would range from 2.9 to 3.6 (Tables 1 and 2). If the fractal approach is valid for high volume fraction, an exponent of 4 would correspond to dimensionalities of 2.10 to 2.28, dependent on the value of x . These dimensionalities are not real values, but should be considered apparent dimensionalities (D*). At low volume fractions of solids a gel is formed with a fractal dimensionality of 1.7 to 1.8 according to our light scattering results. Crystals produced after the formation of the primary network will aggregate with the strands or in the pores of the primary network. As a result, the fractal aggregates will become more compact and would thereby exhibit a higher dimensionality than the primary aggregates. In reality there will be a distribution of dimensionalities depending on the length scale. l 3 If it is assumed that the primary fractal network is formed at a volume fraction with dimensionality D',the dimensionless aggregate radius ( R / u ) ~at~ the , gel point is given by @ $ D ' - 3 ) . Once a gel is formed, aggregates can n o longer grow and R l a remains constant. If the fractal nature

179

W . Kloek, T. van Vliet, and P. Walstra

of the aggregates is preserved on further aggregation, the volume fraction particles in an aggregate is given by

Substituting the relation for the dimensionless aggregate radius yields an expression for the apparent dimensionality D*:

Substituting D' = 1.7,@"= 0.10 and @gFI = 0.02 yields D* = 2.24 which is close to the observed apparent dimensionality. This relation would imply that the exponent p would increase with increasing & assuming x to be constant. However, it is expected that x decreases on further crystallization because the stress carrying strands become less flexible due to thickening. Figure 7 shows the storage moduli of various dispersions on melting. The two step curves suggest that two structures are present to give the network its

X X

300,000

-XX

X

--

200,000

i:

100,000

0

I

20

30

40

50

60

7- ("C) Figure 7

Storage moduli (GI)at f = 0.I Hz for 10% HPISFdispersions crystallized at various initial supersaturations as a function of the temperature imposed after crystallization was complete: 0, In B = 3.25;A,In B = 3.50;X I In B = 3.75;*, In B = 4.00


180

Table 3

Exponent p in the relation G' & derived from the elastic properties of partially melted HPISF dispersion at a temperature of 42 "C. The dispersions were initially crystallized at various supersaturations in the /3' polymorph; r = correlation coefficient In P

P

12

3.25 3.50 3.75 4.00

3.87 3.74 3.30 3.16

0.9760 0.9964 0.9934 0.9989

mechanical properties, while the DSC melting profile gives a smooth curve. The first part to melt will be the fat that is deposited on the outside of the crystal chain or is aggregated in the pores of the primary network. This is the fat that contains compound crystals that crystallize with most difficulty (low melting temperature and low enthalpy of fusion), and so it melts first. At a small solid fraction then a structure would remain of a geometry comparable to that of the primary network. Storage moduli were found to be correlated with the volume fraction of solids at a temperature of 42 "C. The amount of soluble H P at this temperature can be calculated using the Hildebrandt equation; it is ca. 5.8%. While the amount of solids still exceeds $gel, the structure will be more comparable to that of the primary network. Table 3 shows the correlation results at various initial degrees of supersaturation. The values of the scaling exponent p are much smaller than those obtained from the fully crystallized dispersions. Calculated dimensionalities usingx = 4 yield values that agree well with dimensionalities found from light scattering and viscometry data for low volume fraction dispersions.

4 Conclusions The aggregate structure of low volume fraction HP/SF dispersions can be described well using a fractal approach. Light scattering has yielded a fractal dimensionality of 1.7-1.8, which is indicative of rapid aggregation. Viscometric data for these low volume fraction dispersions has shown that the dimensionless aggregate radius scales with the shear-rate with an exponent of -0.3 or -0.4. Assuming the aggregates to be rigid, these shear-rate thinning exponents are indicative of hinged or flexible stress-carrying strands. Description of the elastic properties of high volume fraction dispersions using the fractal approach is hampered by the fact that the processes of crystallization and aggregation proceed simultaneously. Ongoing crystallization produces crystals that aggregate with the strands or in the pores of the primary network, whereby compaction of the primary structure occurs. This tends to yield apparent dimensionalities clearly higher than 1.7-1.8.

W . Kloek, T. van Vliet, and P. Walstra

181

Acknowledgement We thank Dr. J. Dhont and Dr. H. Verduin (Laboratory of Physical Chemistry, University of Utrecht, the Netherlands) for the use of their twodimensional light scattering shear cell and the valuable discussions.

References 1. P. Walstra, T. van Vliet, and W. Kloek, ‘Advanced Dairy Chemistry, Volume 2; Lipids’, Chapman and Hall, London, 1995, p. 179. 2. M. van den Tempel, J. Colloid. Sci., 1961, 16,284. 3 . L. H. Wesdorp, ‘Liquid-Multiple Solid Phase Equilibria in Fats-Theory and Experiments’, Ph.D. Thesis, Technical University Delft, the Netherlands, 1990. 4. P. Meakin, Phys. Lett. A , 1984, 103, 337. 5. A. A. Potanin, J. Colloid Interface Sci., 1991, 145, 140. 6. A. A. Potanin, J. Dispersion Sci Technol., 1992, 13,527. 7. A. A. Potanin, J. Colloid Interface Sci., 1993, 157, 399. 8. I. M. Krieger, A d v . Colloid Interface Sci., 1972, 3, 111. 9. W. Kloek, Ph.D. Thesis, Wageningen Agricultural University, to be submitted. 10. R. Vreeker, L. L. Hoekstra, D. C. den Boer, and W. G. M. Agterof, Colloids Surf., 1992,65, 185. 11. L. G. B. Bremer, ‘Fractal Aggregation in Relation to Formation and Properties of Particle Gels’, Ph.D. Thesis, Wageningen Agricultural University, 1992. 12. J. M. P. Papenhuijzen, Rheol. Acta, 1971,10,493. 13. M. T. A . Bos, Physica A , accepted for publication.

Lipid-Protein Interactions-Consequences for Surface Activity in Food Emulsions By Martine le Meste, Patricia Tainturier, and Jean-Luc Gelin' ENSBANA, 1 ESPLANADE ERASME, 21000 DIJON, FRANCE 'SBI, BAUPTE, 50500 CARENTAN, FRANCE

1 Introduction In food technology, the objective is often to stabilize a metastable state, such as the dispersed state in an emulsion, through the control of the dynamic properties of the molecules, i.e. by lowering the rates of the physico-chemical transformations driven by thermodynamics. Indeed, for simple as well as for more complex systems, intermolecular interactions, or interactions between a molecule and a surface, are determined by the thermodynamics; however, a system will move to a more stable state only if a suitable perturbation or sufficient energy allows it. The energy can come from the thermal motion of molecules or particles. Thus, both thermodynamic and kinetic aspects are relevant to the study of food colloids. The stability of most food emulsions is controlled by the interfacial behaviour of proteins. In order to be able to design food proteins with optimal properties and to determine the best conditions and formulation to improve protein functionality, it is thus necessary to have a good understanding of the mechanisms responsible for protein adsorption at the surface of oil droplets and for the formation of the adsorbed protein film. One general feature of lipid-protein interactions in food emulsions is complexity. Indeed, a mixture of proteins may interact with a large variety of lipids within the different phases of the food. Most of the fundamental studies dealing with the interactions between proteins and lipids within organized systems have been performed either on biological membranes or on much simpler systems such as hydrocarbon-water interfaces. Many fewer fundamental studies have been performed on food oil-water interfaces, probably because of the complexity of such systems, and may be, also, because of their lower economic or biological interest. Thus, the mechanisms of stabilization of food emulsions by proteins are still incompletely understood. One of the objectives of this paper is to discuss how intermolecular interactions involving proteins within the aqueous phase, mainly protein-lipid 185

186

Lipid-Protein Interactions

interactions, may affect the affinity of proteins for the oil-water interface. Lipid-protein interactions occurring both at the oil-water interface and in the bulk water phase strongly depend on the solubility of both types of interacting species in water. Moreover, the thermodynamic contribution resulting from the changes in the properties or structure of water, as new interactions between dissolved or dispersed species arise, is one important factor to consider. After a brief presentation of some characteristics of the oil-water interface, and of the mechanisms leading to protein adsorption, some experimental results obtained with electron spin resonance (ESR) on lipid-protein interactions will be presented.

2 The Oil-Water InterfaceCome Theoretical Aspects Upon emulsification, extended interfaces with excess free energy are created. The interfacial tension is a measure of this excess in free energy. The driving force that controls intermolecular interactions in food emulsions is the tendency of the system to get closer to thermodynamic equilibrium, i.e., to decrease the free energy of the whole system and to get closer to a state where the chemical potential of each species is the same over the whole system. Adsorption of proteins and low-molecular-weight surfactants at the surface of oil droplets contributes to this tendency. In food emulsions, stabilized by proteins and low-malecular-weight surfactants, lipid-protein interactions may occur in different phases: at the surface of the oil droplets, in the bulk aqueous phase, and, in some cases, at the surface of surfactant micelles. Dispersed systems may be in an apparently stable state, or they may evolve towards a more stable state; in both cases, the molecules-even interacting molecules-have the possibility to exchange between different locations or phases. Thus, the interfacial region in an emulsion should be described in terms of structure and composition, but also in terms of dynamic properties.

Ordered Structure The interfacial region between two separated phases is characterized by a local excess free energy mainly due to the structuring effect resulting from the proximity of phases with low mutual solubility. Ordering of molecules occurs on both sides of the interface. Because of their tendency to orient their polar groups towards water and to avoid the contact of the hydocarbon chains with water, glycerides and fatty acids form relatively ordered layers at the oil-water interface. The precise configuration at the interface depends on structural features (such as chain length) of the interfacial lipids. The polar part and some short chains protrude into the water phase.' Thus, when a protein molecule arrives at the surface of an oil droplet it generally sees the plane formed by the polar groups of the more polar lipids.

M. Le Meste, P. Tainturier, and J.-L. Gelin

187

The interfacial region is a transition region where water presents more broken hydrogen bonds ( i . e . , unsatisfied bond positions) relative to the bulk phase.* When apolar residues are in contact with water-this would be the case with hydrocarbon-water emulsions-interactions between interfacial water molecules would tend to be improved. The free energy of transfer of a hydrocarbon molecule to water is roughly proportional to the surface area of the molecule. This is an indication that the number of reoriented water molecules is more-or-less determined by the non-hydrogen-bonded areas exposed to them. The high surface tension of water may be taken as a manifestation of this mainly entropic effect, since air behaves as an inert nonbonding medium.3 Conversely, more polar residues, charged or not, would prefer to interact with water rather than with other similar residues, thus disrupting the natural ordering of pure water. Ions dissolved in water have been shown to increase the surface tension of water in accordance with the Hofmeister ion ~ e r i e s : ~

This effect is caused by two factors: (i) the change in the water structure, i.e. in the H-bond system, which has a large effect on the surface energy (entropic factor); and (ii) the change in the number of water molecules per unit area of surface caused by the ions.4

Concentration of Amphiphiles The magnitude of the interfacial tension depends on the mutual affinity between the molecules of the apolar phase and the water molecules. The lower the affinity, the higher the excess free energy. Interfacial tension y at temperature T is related to this excess free energy G by

where A is the surface area. When a solute i, dissolved in either the apolar or polar phase, exhibits some affinity for the other phase, it will adsorb at the interface. Its concentration will become higher than in its original bulk phase, and the surface energy will decrease according to

where dpi is the change in chemical potential of component i , and Ti is the surface excess concentration. Thus, amphiphilic molecules will concentrate on both sides of the interface, whereas solutes with a strong affinity for water or for the oil phase will avoid the interfacial region. Two main classes of amphiphilic molecules contribute to interfacial stabilization in food emulsions: (i) low-molecular-weight surfactants (such as fatty

188


acids or monoglycerides in a non-purified oil, or added emulsifiers) and (ii) proteins. Fatty acids and monoglycerides exhibit a much higher affinity for water than triglycerides or hydrocarbons, and thus a higher amphiphilicity . Affinity for water is higher for short and unsaturated hydrocarbon chains of glycerides.’ In a droplet of non-purified food oil, lipids with the highest affinity for water will thus concentrate at the interface. The propensity of a protein to adsorb at the lipid-water interface also depends on the affinity of this protein for water. The more soluble a protein is, the lower its tendency to adsorb at the interface (a more thorough presentation of the behaviour of proteins is given below). Thus, interactions of water with both lipids and proteins will control the tendency of a protein to adsorb at the interface. Indeed, the structure and properties of the water phase affect the solubility of surface-active components, and thus their tendency to interact and aggregate. This is illustrated by the influence of salts on the hydrophilic-lipophilic balance (HLB), and on the critical micelle concentration (CMC) of monomers that form micelles, and on protein ~ o l u b i l i t y . ~

Dynamic Equilibrium About 40 years ago it was already stressed5that “the interface between fat and aqueous phase in milk is in a state of dynamic equilibrium”. The forces that hold amphiphilic molecules together in micelles, monolayers or bilayers are weak van der Waals, hydrophobic, hydrogen bonding, or screened electrostatic interactions. The stability of these structures depends on the solubility of the interfacial lipids in water.’ When this solubility is relatively high, molecules from the interface exchange rapidly with molecules in the aqueous phase. Thus, these self-assembled structures are usually highly dynamic, with molecules in constant thermal motion within the aggregates as well as exchanging with monomers in the bulk ~ o l u t i o nThe . ~ probability for exchange is e-AE’k7, with A E being the activation energy of the process. The mean residence time of a lipid molecule within a micelle or a bilayer is of the order of lop4 and lo4 s, r e ~ p e c t i v e l yThis . ~ residence time depends on the strength of the intermolecular interactions and on the structure of the individual molecule, but not on the ordered structure formed by these molecules; this means that the residence time will be shorter for a smaller and more water-soluble fatty acid than for a triglyceride. Studies performed on biological membranes have shown that even intrinsic proteins, strongly embedded within the lipid bilayer, exhibit a relatively high mobility. Moreover, the extensive work performed by DickinsonG1(’on the competition between proteins, and between proteins and low-molecular-weight surfactants, has clearly demonstrated the reversible nature of the adsorption of most milk proteins at oil-water interfaces, as long as there is no covalent binding between proteins within the adsorbed protein layer. Obviously, reversibility has to be defined relative to an observation time.

M . Le Meste, P. Tainturier, and J.-L. Gelin

189

3 Lipid-Protein Interactions in Emulsions Adsorption is thermodynamically favoured because upon adsorption the free energy of the whole system decreases. Indeed, upon protein adsorption, some water molecules are expected to be displaced from the oil-water interface and some apolar protein residues are expected to be removed from the aqueous environment. Thus, the entropy of the whole system is expected to increase.

Influence of the Interfacial Tension As the interfacial tension y is higher for hydrocarbons than for triglycerides, less protein is expected to adsorb at the latter interface. This may be experimentally observed; however, in some cases, the results do not fulfil this expectation. Comparing protein adsorption at the surface of droplets of ntetradecane and soya oil, no significant difference was noted in the surface covered of /I-lactoglobulin, despite the expected difference in interfacial tension (approximately 50 and 25 mN/m, respectively) (see Table l)." This table also shows that adding lecithin, an oil-soluble surfactant, to the oil phases induced a significant reduction in the protein load of the n-tetradecane droplets only above a molar ratio of lecithidprotein of approximately 50. Thus, in that case the strength of the driving force y does not appear to be a critical parameter for /3-lactoglobulin adsorption. A similar study on the adsorption of B-casein at the surface of n-tetradecane droplets, without or with added Tween 20 up to a molar ratio of surfactant over protein of 10, has been published recently. l2 The surface tension decreased and the specific surface area of the emulsion increased in presence of Tween 20; however, the partition of proteins between the adsorbed and non-adsorbed states was not significantly affected by the presence of the surfactant (see Figure 1).

Table 1

Influence of the nature of the oil phase on the oil droplet diameter (dJ2) determined by laser light scattering and on the protein load I' at the surface of oil droplets in n-tetradecane and soya oil emulsions (20 wt% oil; p H 7) stabilized by 0.4% B-lactoglobulin, with and =,molar ratio of lecithin to protein) without added lecithin &I (from ref. 11) n-Tetradecane (p

- 50 mN m - I )

Soya oil ( y

Mr

d3,?fpm

Timg m-2

d3.?lpm

0 50

0.68 0.57

1.01 0.95

0.75 0.73

- 25 m N m - I ) rimg m-'

1.00 0.81

190


.1

10

1

100

Rapplied Figure 1

lnfluence of increasing Tweenlprotein molar ratio ( R applied) on the concentration of p-casein in emulsions: 0,at the interface; 0, in the subnatant. The protein concentration used to form the emulsion was fixed at 65pM (from ref. 12)

Surface Hydrophobicity Surface hydrophobicity reflects the accessibility of the hydrophobic residues of a protein to an apolar probe. A good correlation has been shownI3 between hydrophobicity and emulsifying properties (Figure 2) for heat-denatured and linoleate-bound ovalbumin molecules. Moreover, a good agreement can be noticed between the surface hydrophobicity of the main milk protein^'^ and their ability to reduce the interfacial tension of an oil-water interfaceLs (Figure 3). The same order (/?-casein > BSA > as,K-casein > /?-lactoglobulin) was obtained for both properties. Protein-surface interactions are more favourable if the non-polar domains of both the protein and surface are

r

0

G4 --

E

120

3 0

E

ao

40

0 0

400

aoo

1200

SURFACE HYDROPHOBICITY

Figure 2

Relationship between ovalbumin surface hydrophobicity (arbitrary units) and emulsifying activity: 0, heat denatured; 0, linoleate-bound; 1-5, heat denatured at 85 "Cfor I , 2, 3, 4 and 5min, respectively; 6-10, bound with 0.3, 0.9, 3.1, 4.8 and 8.2 moles of linoleate per one mole of ovalbumin, respectively (from ref. 13)

191

M . Le Meste, P. Tainturier, and J.-L. Gelin 2ly

,

7 0

;

,

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; 04

,

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0

103

;

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:

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R Concenuation

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7

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% Concentration (~10')

Figure 3

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Surface activity of milk proteins at 40°C at (a,b) the butter oil-water interface and (c,d ) at the butter oil-protein-free plasma interface: A, a,y-casein;B, x-casein; C, monodispersed casein; D, B-casein; E, euglobulin; F, pseudoglobulin; G, K-lactoglobulin; H , bovine serum albumin; I , alactalbumin; J , interface protein (from ref. 15)

removed from contact with water. However, on a relatively hydrophilic surface, such as the plane of the polar heads of phospholipids, fatty acids or monoglycerides, it may be more favourable to bury the hydrophobic regions of the protein inside the adsorbed layer. l6

Protein Amphiphilicity Amphiphilicity is related to the ratio of surface polar to apolar residues, and to the distribution of these residues within distinct regions having strong polar or apolar character. This distribution is controlled by the conformation of the protein which is very sensitive to the solution conditions. Proteins can interact with other molecules or with a surface in a great number of ways. Even for proteins with a relatively strong amphiphilic character, charged, polar and apolar residues may coexist in the same region of the surface. It has been reported that a compact and negatively charged protein like insulin can adsorb on a negative surface. l 6 Indeed the protein still contains

192


positive charges, and if the protein has the proper orientation towards the interface, electrostatic repulsion may be minimized.

Protein Solubility Medium conditions, such as pH and temperature, which may affect the amphiphilic character and solubility of proteins, influences also their surface activity. For instance, it has been shown, for protein adsorbing at solid interfaces, that the decreased solubility close to the isoelectric point of the protein can often lead to the formation of multilayers at the surface.16 Temperature affects the surface behaviour of amphiphiles in several ways: (i) by controlling the strength and extent of the hydrogen bond network within the water phase, temperature influences the solubility and CMC of the amphiphiles; (ii) the reduction in molecular mobility as temperature is reduced contributes to the decrease in the entropy at the air-water interface as expressed by the increase in surface tension; however, at the triolein-water interface, we observed the opposite effect probably because a change in the strength of the interfacial lipid-water interactions was superimposed on the entropic effect; we also observed that the influence of temperature (from 5 to 40 "C) on the interfacial tension was reduced in presence of protein^;'^ (iii) by inducing changes in the physical state of the oil phase and in the conformation of the proteins. This explains why the influence of temperature may be quite variable from one system to another. Protein loads on different types of oil droplets have been measured as a function of t e m p e r a t ~ r e . ' ~On , ' ~ n-hexadecane or ntetradecane droplets, the protein load was observed to be independent of temperature, in the temperature range from 0 to 20 "C; on soya oil droplets, the load was the same at 0 and 20 "C, but was slightly lower at 5 "C.

Interactions Occurring within the Aqueous Phase Protein amphiphilicity is expected to depend on intermolecular interactions involving the protein molecules within the bulk aqueous phase. For instance, caseins, which are relatively hydrophobic, may be considered as being less amphiphilic, and thus less available for adsorption, when they are involved in micelles where hydrophobic regions of different units may associate. Interactions occurring between proteins and other kinds of solutes dissolved in the aqueous phase, in particular other surface-active molecules, may also influence the surface activity of proteins. The influence of milk proteins on the interfacial tension at an oil-water interface and at an oil-protein-free milk plasma interface has been studied.l5 The protein-free plasma was prepared by dialysing skim milk for 48 hours against 2 litres of distilled water. Figure 3 shows that the dialysate itself exhibits some surface activity and that this appears to affect the surface activity of the milk proteins, either by an effect at the interface or

193

M . Le Meste, P . Tainturier, and J.-L. Gelin

because of interactions occurring in the aqueous phase. For instance, bovine serum albumin seems to lose its surface activity in the presence of the dialysate, maybe as a consequence of the saturation of the hydrophobic sites of the protein by the surface-active components of the protein-free plasma. Indeed, previous studies performed with both aqueous solutions and emulsions have demonstrated the higher affinity of bovine serum albumin for fatty acids dissolved in water than for fatty acids dispersed in a triglyceride interfacial layer.*O The influence of lipophilic molecules on the surface activity of proteins is discussed elsewhere in this book.*'

Dynamical Properties As mentioned previously, in an emulsion lipid molecules can exchange between different environments, the two bulk phases and the interface, in order to equilibrate their chemical potential throughout the whole system. Exchange of oil is thermodynamically favourable. Indeed, a system in which the oil is evenly distributed among all the droplets is more thermodynamically favourable than one in which some of the droplets contain a pure oil. The rate of this (a)

(b)

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0

1

Tcmpntrre 1 'C

2

3

1

5

[mlI w l *

Figure 4

DSC measurements of crystallization temperature in 20% hydrocarbon oilin-water emulsions: (a) 10% n-octadecane, 10% n-hexadecane, at various times (in days); ( b ) same emulsion with 1.1% whey protein isolate (WPI); (c) dependence of the rate constant of oil exchange on the concentration of whey proiein isolate (from ref. 22)

194


exchange is higher for the smaller (more polar) lipids. Recently, very interesting experiments, performed on hydrocarbon-water emulsions, have demonstrated that even oil with a relatively low solubility in water can exchange at a significant rate between droplet^.^^*^^ These experiments were performed with emulsions containing a mixture of two types of oil droplets (20% oil): nhexadecane and n-octadecane. Exchange of oil was quantified by measuring changes in the crystallization behaviour of the two types of droplets with time (Figure 4).The rate of exchange was observed to be proportional to the specific surface area of the emulsion and to be increased by the presence of hydrophilic surfactants. The curvature of the droplets was expected to increase the Laplace pressure and, thus, the solubility of the hydrocarbons in water. Ethanol, which increases the solubility of oil in water, dramatically increased the rate of oil exchange between droplets. The rate of exchange was also found to increase with the concentration of whey protein used to stabilize the emulsion. Proteins can be expected to bind the oil molecules and hence transport them across the aqueous phase. This indicates that proteins also exchange between droplets. Lipid-protein interactions at interfaces occur through low energy interactions; consequently these interactions should be reversible. Indeed, as already mentioned, numerous examples of protein displacement by surfactants or more surface-active proteins have been described&"' demonstrating the reversible nature of protein adsorption.

4 ESR Study of Lipid-Protein Interactions in Emu1s ions Methodological Aspects Electron spin resonance has been used to study lipid-protein interactions in solution^,^^)'^^ biological membranes,25926 and e m ~ l s i o n s . Only ~ " ~ ~paramag~ netic species are detected with this technique; stable paramagnetic species have thus to be added to diamagnetic systems. Nitroxide free radicals (also called 'spin probes') are the most widely used molecules for this purpose. Paramagnetic homologues of stearic acid were selected as a lipid model for our study on food emulsions. The nitroxide moiety could be located on different places of the hydrocarbon chain: close to the polar head (SSA), in the middle of the chain (12SA), or close to the apolar end (16SA) (see Figure Sa). The results obtained with species SSA are more particularly sensitive to the behaviour of the polar head of the fatty acid, whereas species 12SA and 16SA reflect interactions involving the hydrocarbon chain. The unpaired electron of the nitroxide group is delocalized on the 2pnorbital of the nitrogen atom as shown in Figure 5b. Because of the interaction of the free electron with the nitrogen atom, the ESR spectrum of nitroxide radicals is composed of three lines (hyperfine structure), sensitive to the orientation of the nitroxide relative to the external magnetic field (and thus to the reorientation rate of the nitroxide moiety) and to the polarity of the medium. This is

M. Le Meste, P. Tainturier, and J. - L. Celin

195 OH 5SA \

16SA

Figure 5

(a) Chemical structures of 5SA and 16SA nitroxide radicals; (b) chemical structure of the nitroxide moiety

shown in Figure 6. Radicals in an aqueous medium (spectra 1 and 2) can be distinguished from radicals in an apolar environment (spectrum 3) (due to differences in the coupling constant, i.e. in the distance between hyperfine lines); moreover, non-interacting radicals dispersed in a liquid medium are much more mobile (narrow lines) than radicals interacting with a protein (broad and distant lines) (spectra 4 , 5 and 9). Spectra of radicals dispersed in a viscous medium, as in oil, are intermediate (spectrum 3). The amplitude of the motions exhibited by the nitroxide moiety depends on its location on the fatty acid chain: in a liquid or in a liquid crystal, it increases from the polar head to the apolar end. Several spectra, originating from different populations of radicals coexisting in the same sample, may be superimposed (spectra 4, 5 , 7 and 8). In the case where one population of radicals with rapid motion coexists with radicals with slow motions, a ratio of line amplitudes can be calculated: R = Z/M where Z and M are the amplitudes of the first line corresponding to the populations with slow and rapid motions, respectively. Changes in this ratio reflect changes in the proportion of non-interacting versus interacting probes. As fatty acids exhibit a higher affinity for water than for triglycerides, in an emulsion they may concentrate preferentially at the surface of oil droplets and thus they are expected to be sensitive to protein adsorption. Moreover, their relatively high solubility in water allows us to study interactions with proteins occurring in the water phase.

Figure 6

ESR spectra obtained with nitroxide homologues of stearic acid dispersed: in a citrate-borate buflersolution (pH 7):I , 16SA; 2, 5SA; in a trioleinlwater emulsion: 3; in a bovine serum albumin solution (0.45% wlw protein, probelprotein molar ratio = 6): 4, 16SA; 5, 5SA; in an emulsion prepared with same protein solution: 6, 5SA; in a solution of %-casein (2% wlw protein, probelprotein molar ratio = 0.15): 7, I6SA; 8,5SA; in an emulsion prepared from the casein solution: 9, 5SA. I represents the first line of a spectrum of radicals with slow motion (interacting spin probes); M represents thefirst line of a spectrum with rapid motion (non-interacting spin probes)

1

2.

2

f.

P P


197

Lipid-Protein Interactions in Emulsions Stabilized by Milk Proteins Figure 6 represents some examples of ESR spectra obtained with two spin probes 5SA (numbers 2 , 5 and 8) and 16SA (numbers 1 , 4 and 7) dispersed in buffer or protein solution (pH 7). In the absence of proteins, the spectra of 5SA (spectrum 2) and 16SA (spectrum 1) are characteristic of spin probes with rapid motion (rotational correlation time z, of the order of 5 x 10-”s). In the presence of bovine serum albumin (BSA) (spectra 4 and 5), at a molar ratio of spin probe to protein between 1 and 6, almost all the probes, 5SA as well as 16SA, were found to be immobilized (T, h lOP7s) by interaction with the protein. These results suggest that both the polar head and the apolar chain of the fatty acid are involved in the interaction with BSA and confirm the high affinity of BSA for free fatty acids. Much less fatty acid appears to interact with *,-casein (less than 1 mole of fatty acid per mole of protein), and 5SA preferentially to 16SA (spectra 7 and 8). It could then he concluded that the polar head group of the fatty acid is more affected than the apolar chain by the interaction with casein. Then, the same proteins, with the same probe/protein molar ratio as previously, were observed in oil-in-water emulsions. In this case, probes were less available for interacting with the protein as they were dissolved in the oil phase and expected to partition amongst the different regions of the emulsions. In the conditions of the experiment, BSA did not exhibit any visible affinity for 5SA (spectrum 6); on the other hand, a,-casein strongly reduced the mobility of 5SA (spectrum 9). The results obtained with BSA show that, as expected, lipid-protein interactions depend on the availability of the fatty acid, i.e. on its distribution between the different phases of the emulsion. The influence of three milk proteins (a,-casein, p-casein, P-lactoglobulin) on the mobility of 5SA, 12SA and 16SA has already been published.27 For the three proteins the mobility of the polar end of the fatty acid was found to be much more greatly reduced than that of the apolar end by adsorbed proteins. Indeed, except for a,-casein, when the nitroxide moiety was located close to the apolar end of the fatty acid, it was not sensitive to the presence of the adsorbed proteins. The reduction in the mobility of the fatty acid polar head should probably be attributed to proteins sitting on the surface of the oil droplets and interacting through low energy forces or steric constraints. ’l’he a,-casein appeared to be able to interact more extensively, and in a more hydrophobic way, with the polar lipids of the emulsion. The P-lactoglobulin, which has a relatively low surface hydrophobicity and surface activity, exhibited a relatively high affinity for the nitroxide homologues of the lipids (Table 2). It appears therefore that there is no direct relationship between the surface activity of a protein and its affinity for lipids, at least for polar lipids. The polarity of the polar head of the paramagnetic fatty acid can be modified by changing the p H (at p H 7 the polar head of the fatty acid is charged, whereas at p H 2.5 it is neutral) or through methylation. Using the same previous three proteins, we have observed27that, when the polarity of the polar head group is decreased, the fatty acid becomes less sensitive to the presence of the adsorbed


198

Table 2

Influence of the polarity of the polar head of nitroxide homologues of stearic acid on ESR results: the R ratio for 5-doxy1stearic acid and methyl-5-doxy1stearic acid in emulsions stabilized by different milk proteins (R = IIM is the ratio of the I and M line amplitudes) (probe1 protein molar ratio = 0.6) ~

~~

5SA PH 7

no protein a,-casein p-casein P-lactoglobulin

~

~

5SA

~~

~~

5CHjSA PH 7

coo-

pH2.5 COOH

COOCH,

0 3.82 k 0.27 0.19 k 0.01 1.15 k 0.06

0 2.44 k 0.02 0.21 k 0.01 0.54 ? 0.004

0 0 0 0

proteins (Table 2). The differences between charged and uncharged forms were found not to be very pronounced, maybe because other factors influencing the interaction (such as protein charge, conformation and association state) were also modified by the change in pH. These results suggest that electrostatic interactions between proteins and the most polar lipids predominates at the oil-water interface. However, the less polar the lipids, the lower their propensity to concentrate at the interface, and thus to be affected by the presence of adsorbed proteins; and also the lower their solubility in the aqueous phase. The latter ESR measurements having been performed on the whole emulsion, the information obtained represents the overall behaviour of the nitroxide molecules, which can become partitioned in the different regions of the emulsion, i.e. the oil phase, the interface, and the aqueous phase. An ESR experiment was designed with a relatively complex system used as a model for ice-cream. The emulsion was composed of 8% (w/w) purified triolein oil (Sigma, France) containing the spin probe 5SA, an aqueous phase with 8 % (wlw) milk powder or whey protein concentrate (i.e. 2.7% protein) (Nestle, France), 0.18% (w/w) of a mixture of stabilizing polysaccharides (SystdmeBio-Industrie, France), and finally polysorbate 80 (Sugin F150, SystCme-BioIndustrie, France) at different concentrations. The values of the average diameter of the oil droplets, measured with laser light scattering (Mastersizer, Malvern, UK), indicate that the whey protein concentrate (d32= 0.56ym) was a more effective emulsifier than the other protein preparations (d32= 0.82ym and 0.71 y m for the milk protein preparation, with (0.1% w/w) and without surfactant, respectively), in the experimental conditions used in this study (homogenization with a Polytron, PT Kinematica, 16500 r.p.m., 4 min). As shown in Figure 7, ESR spectra were obtained at 40 "C with a Bruker Electron Spin Resonance spectrometer ESP 300E, for the whole emulsion and for the cream and aqueous phases obtained from the same emulsion by centrifugation (lOOOOg, 40min, 4°C). The ESR spectrum corresponding to the emulsion stabilized by the whey protein concentrate is composed of two populations of paramagnetic fatty acids with different mobilities (spectrum 1). The population with the lower mobility was attributed to fatty acids interacting with proteins.


34C4

3420

3440

3480

199

34.0

G Figure I

ESR spectra at 40 "C with 5SA dispersed in emulsions stabilized with milk proteins. Whole emulsion: 1, with whey protein concentrate (2.72%protein wlw); 3, with milk protein powder (2.72% protein); 5, with milk protein powder (2.72% protein) + polysorbate 80 (0.1 wlw); aqueous phase separated from the emulsion; 2, with whey protein concentrate (2.72% protein wlw); 4, with milk protein powder (2.72% protein); 6, with milk protein powder (2.72% protein) + polysorbate 80 (0.1% wlw)

This population was relatively reduced in emulsions stabilized by the milk protein powder (spectrum 3) and the mixture of milk proteins + water-soluble surfactant (polysorbate 80, O.lY0) (spectrum 5 ) . ESR spectra were also obtained from the aqueous and cream phases. For the three emulsions, the population of fatty acids with reduced motions was found to be higher in the aqueous phase (spectra 2 , 4 and 6) than in the whole emulsion or cream phase. This may be explained by the relatively high proportion of protein remaining in the aqueous phase (only 6% of the proteins were found in the cream phase of emulsions), with or without polysorbate 80 (O.lYo). This is in agreement with the results from McClements et a1.22,23suggesting that proteins and surfactants in the aqueous phase have the effect of displacing the partition of fatty acids towards this aqueous phase.

200

Lipid-Protein lnteructions

References 1. D. M. Small, ‘The Physical Chemistry of Lipids’, Plenum, New York, 1986. 2. F. G. Stillinger, in ‘Water in Polymers’, ACS Symposium Series No. 127, American Chemical Society, Washington, DC, 1980, p. 11. 3. J. Israelachvili, ‘Intermolecular and Surface Forces’, Academic Press, New York, 1991. 4. W. A. Luck, in ‘Water in Polymers’, ACS Symposium Series No. 127, American Chemical Society, Washington, DC, 1980, p. 43. 5. R. Jenness and S. Patton, ‘Principles of Dairy Chemistry’, Wiley, New York, 1959. 6. E. Dickinson, S. E. Rolfe, and D. G. Dalgleish, Food Hydrocolloids, 1988,2,397. 7. D. G. Dalgleish, S. E. Euston, J. A. Hunt, and E. Dickinson, in ‘Food Polymers, Gels and Colloids’, ed. E. Dickinson, Royal Society of Chemistry, Cambridge, 1991, p. 487. 8. E. Dickinson, in ‘Microemulsion and Emulsion in Foods’, ACS Symposium Series No. 448, American Chemical Society, Washington, DC, 1991, p. 114. 9. E. Dickinson, ‘An Introduction to Food Colloids’, Oxford University Press, Oxford, 1992. 10. J.-L. Courthaudon, E. Dickinson, and D. G. Dalgleish, J . Colloid Interface S c i . , 1991, 145,390. 11. E. Dickinson and G . Iveson, Food Hydrocolloids, 1993,6, 533. 12. A. R . Mackie, S. Nativel, D. R. Wilson, S. Ladha, and D. C. Clark, J . Sci. Food Agric., 1996,70,413. 13. A. Kato, in ‘Ingredients Interactions: Effects on Food Quality’, ed. A . Gaonkar, Marcel Dekker, New York, 1995, p. 357. 14. S. A. Townsend and S. J. Nakai, Food Sci., 1983,48, 588. 15. R. H. Jackson and M. J. Pallansch, J . Agric. Food Chem., 1961,9,424. 16. P. M. Claesson, E. Blomberg, J. C. Froberg, T. Nylander, and T. Arnebrant, A d v . Colloid Interface Sci., 1995, 57, 161. 17. J.-L. Gelin, P. Tainturier, L. Poyen, J.-L. Courthaudon, M. Le Meste, and D. Lorient, in ‘Food Macromolecules and Colloids’, ed. E. Dickinson and D . Lorient, Royal Society of Chemistry, Cambridge, 1995, p. 81. 18. E. Dickinson and S . Tanai, J . Agric Food Chem., 1992,40, 179. 19. J. Chen and E. Dickinson, J . Sci. Food Agric., 1993,62,283. 20. M. Le Meste and S. Davidou, in ‘Ingredients Interactions: Effects on Food Quality’, ed. A. Gaonkar, Marcel Dekker, New York, 1995, p. 235. 21. L. A. Wasserman and M. G. Semenova, this volume, p. 77. 22. D. J. McClernents, S. Duncan, J. B. German, and J. Kinsella, Colloids Surf. A , 1993,81,203. 23. D. J . McClements and S. R. Dungan, J . Phys. Chem., 1993,97, 7304. 24. M. Le Meste, B. Closs, J.-L. Courthaudon, and B. Colas, in ‘Interactions of Food Proteins’, ACS Symp. Series No. 454, American Chemical Society, Washington, DC, 1991, p. 137. 25. 0. H. Griffith and P. C. Jost, ‘Spin Labelling, Theory and Application’, Academic Press, New York, 1976, p. 454. 26. M. A. Hemminga, J . C. Sanders, and R . B. Spruijt, Progr. Lipid Res., 1992, 31, 301. 27. S. AyniC, M. Le Meste, B. Colas, and D. Lorient, J . Food Sci., 1992,57, 883.

Forces between Lipid-Coated Interfaces By Per M. Claesson LABORATORY FOR CHEMICAL SURFACE SCIENCE, DEPARTMENT OF CHEMISTRY, PHYSICAL CHEMISTRY, ROYAL INSTITUTE OF TECHNOLOGY, S-100 44 STOCKHOLM AND INSTITUTE FOR SURFACE CHEMISTRY, P.O. BOX 5607, S-114 86, STOCKHOLM, SWEDEN

1 Introduction The surfaces of food emulsion droplets are often coated with a complex mixture of lipids, proteins and carbohydrates.' The presence of these compounds generates repulsive forces that are essential for stabilizing the emulsion. There are several types of forces that may give rise to increased emulsion or dispersion stability.* Repulsive electrostatic double-layer forces, which essentially are entropic in origin, are present when the adsorbed species are net charged.* This is the case for some lipids, for most proteins, and for some polysaccharides. Large and flexible polymers anchored to the interface also give rise to long-range repulsive forces; these are known as steric forces. The molecular mechanism behind this repulsion is primarily an unfavourable reduction in entropy of the system due to an enhanced segment density and a reduced number of available polymer conformation^.^ A stabilizing repulsive or force like this is observed when the surfaces are coated with polysa~charides~ flexible proteins.5 The importance of interparticle forces for the properties of colloidal dispersions, biomembranes and biological cells has been realized for a long time. This has inspired the development of a range of techniques for studying how these interactions depend on surface separation and on the molecular nature of the interface. A recent review6 focuses on some of the force-measuring techniques available and their advantages and drawbacks. One of these techniques, the interferometric surface force technique,' has been used for studying surfaces coated with a range of different proteins. Much of the work done in this area was recently r e ~ i e w e dAt . ~ the present stage it appears that it is comparatively easy to understand (i) the long-range forces, and also many aspects of the short range forces, induced by the presence of small, structurally stable globular proteins (like insulin and lysozyme), and (ii) the forces induced by very flexible proteins (like B-casein and proteoheparan sulphate). The most difficult pro201

202

Forces between Lipid-Coated Interfaces

teins to understand seem to be the ones that have a globular structure but are not very stable, e.g. albumin.8 Forces between surfaces coated with polysaccharides have been studied much less. However, from the studies presented so far, it is clear that the forces generated by this class of compounds are similar to those observed in other polymer and polyelectrolyte systems. For instance, the importance of the solvency for adsorption of, and the interaction between surfaces coated by, a chemically modified polysaccharide has been investigated.’-“ Further, the forces acting between negatively charged surfaces coated with chitosan, a weak cationic polysaccharide, have been measured at various pH valuesI2 and these have been found to correlate with the stability of chitosan-coated emulsion droplets.13 Interactions between lipid layers have been very intensely investigated during the last 20 years using different techniques, mainly osmotic stress and surface force measurement^.'^^'^ This topic is also the focus of this review article. First, the principles of some of the techniques used for studying interactions between lipid-coated surfaces are presented. Then, we present some data that illustrate the interactions between uncharged lipids and how the forces generated by the lipids depend on the hydrocarbon chain fluidity and the nature of the polar group. Finally, the molecular origin of the short-range repulsion between lipid layers is discussed, a topic which is currently hotly debated in the scientific literature. 19-24

2 Methods Osmotic Stress Measurements The osmotic stress method has been employed to determine interactions between lipid and surfactant bilayers in lamellar and gel phases. This information is obtained by measuring the water activity, a , from which the osmotic pressure II in the system is calculated fromI4

where V , is the partial molar volume of water and a. is the activity of pure water. The osmotic pressure thus equals the swelling pressure for the system relative to contact with pure water. The water activity in the sample was determined by measuring the relative water vapour pressure using a headspace chr~matograph.~’ This method works well at low water activities, but the scatter in the data at high water activities, i.e. at low pressures, is too large to allow accurate measurements. In order to determine the swelling pressure curve one also has to have the means to measure the separation between the bilayers. This was done by using an X-ray diffraction technique allowing the

P. M. Claesson

203

determination of the repeat distance d. Hence, the swelling pressure versus separation curve can be measured by preparing a set of samples with different water activities and determining the repeat distance in the samples. For lipids in the lamellar or gel phase, the lipid bilayer thickness dl and the water layer thickness d, can to a first approximation be calculated from the measured repeat distance26 dl =

WlVl

((1 - W J V ,

+

d = d@, WlVl

where wIis the weight fraction of lipid, v, and v, are the partial specific volumes of lipid and water, and @ is the volume fraction of lipid. The area per lipid molecule A is26

where

v

Mv =I “

N

is the molecular volume of the lipid, M is the molecular weight, and N is Avogadro’s number. In the calculations it is assumed that the specific molar volume of lipid is independent of the pressure, that there is no water in the lipid layer, and that no lipid is dissolved in the interlamellar water (indicating a sharp boundary between the lipid phase and the aqueous phase). In reality the lipidwater interface is less sharp, and other methods to determine the location of the interface have been proposed. For instance, the electron density profile can be determined from low-resolution X-ray diffraction analysis and a maximum in electron density can be obtained corresponding to the mean location of the For a more detailed discussion of the osmotic stress centre of the polar technique, the reader is referred to the review by Rand and Parsegian.”

Interferometric Surface Force Apparatus A schematic illustration of the interferometric surface force apparatus’ (SFA) is provided in Figure 1. It consists of a stainless steel measuring chamber that encloses the two interacting surfaces. The substrate surfaces are silvered on their backside and glued onto cylindrical silica discs. These surfaces are mounted in a crossed cylinder configuration. Collimated white light is directed perpendicularly to the surfaces. The light passes through the lower surface and becomes multiply reflected between the silver layers. Due to constructive

204


560 nm

(a) Tospectrometer

t

550 nm

j m l F.E.C.O.

t White light

Mica sheets crossed cylinders configuration

Figure 1

A schematic illustration of the main components of the interferometric

surface force apparatus (part a). The stainless steel measuring chamber contains the two interacting surfaces. One mica surface is glued to a silica disc that is attached to a piezo-electric crystal. The other surface, also glued to a silica disc, is mounted on a double cantilever force measuring spring. The surfaces are oriented in a crossed cylinder configuration (part b). White light enters through the window in the bottom of the chamber. It is multiply reflected between the silver layers, and a standing wave pattern, called fringes of equal chromatic order (F.E.C.O.), is generated (part c). The standing waves exit through the top window, and the wavelength and fringe shape are analysed in a spectrometer

P. M. Claesson

205

interference for certain wavelengths, multiple-beam interference fringes (or fringes of equal chromatic order, F.E.C.O.) are transmitted through the upper surface. The F.E.C.O. wavelengths depend on the thickness T of the mica surfaces and the separation between them, D. The surface separation can be determined by comparing the wavelengths when the surfaces are in contact and apart. Under good conditions, i.e. with bright fringes produced by multiple reflections between highly reflecting layers on thin mica sheets, we have found that the distance can be measured with an accuracy of ca. 0.1 nm. We note that this distance is an absolute value measured relative to the pre-determined zero separation. When the experimental geometry is that of crossed cylinders, the fringe pattern has a parabolic shape, and the local radius of the interacting surfaces can be determined from the fringe shape. The distance between the two interacting surfaces is normally changed stepwise by either a synchronous motor or, more accurately, by a stepwise change in the voltage applied to a piezo-electric crystal. The movement of the piezo-electric crystal is calibrated at large surface separations using the F.E.C.O. The expansion of the piezo-electric crystal, AD, is linear in the applied voltage, AV, as long as no force acts between the surfaces; ix.,we have

AD = cAV.

(6)

The force F, between the two surfaces in the crossed cylinder configuration is measured by expanding or contracting the piezo-electric crystal by a known amount AD and then measuring interferometrically the actual distance that the two surfaces have moved relative to one another ADO.Any difference in the two distances when multiplied by the spring constant k gives, using Hooke’s law, the difference between the forces at the initial and the final separations

AF, = k(AD

-

ADO) = ~ ( c A V- ADO).

(7)

Spring deflections of the order of 1nm can be determined in this way. Hence, using a spring with a spring constant of about 100N/m and surfaces with a radius of about 2 cm gives a detection limit in force of N, corresponding to a force normalized by radius of ca. 5pNlm. The mechanical spring system is unstable in the regions of the force-distance curve where the gradient of the force (dFJdD) exceeds the spring constant; under such circumstances the surface separation changes suddenly, and a jump occurs.28 The force measured in the crossed cylinder geometry is related to the force between two spheres, F,, and the free energy of interaction per unit area between flat surfaces, Gf, according to the Derjaguin approximation: 2,29

206


It follows that the pressure in the flat geometry, Pf, is related to the force gradient in the curved geometry by

where R is the geometric mean radius.

Thin Film Balance A thin film balance (TFB) was to measure the forces acting between the two air-liquid interfaces in a single foam lamella. The main parts of the thin film balance are shown in Figure 2. Single thin-liquid foam films are formed in the hole drilled through a fritted glass disc, onto which a glass capillary is fused. The solution-permeable film holder is placed in a gas-tight measuring cell with the free end of the capillary tube exposed to atmospheric pressure. The gas pressure in the cell is regulated by a syringe pump. The force per unit area, known as the disjoining pressure, II, in a plane-parallel film is given by3'

I

cell

interference

..- . . -

Figure 2

. ~.~. .. I L .....~

-

A schematic illustration of the main components of the thin film balance. A macroscopic f o a m film is formed in a hole drilled in a porous glass frit. The surfactant solution is contained in the frit, in the glass capillary, and at the bottom of the closed cell. The film thickness is determined using interferometry. The reflected light is viewed by a video camera and the intensity of a selected wavelength is measured with a photomultiplier tube ( P M T ) . The pressure in the measuring cell is varied by means of a syringe p u m p and measured by a pressure transducer

P. M. Claesson

207

where PG and PRare the gas and reference (atmospheric) pressures respectively, y is the surface tension of the solution, r is the radius of the capillary tube, Ap is the density difference between the aqueous surfactant solution and the gas, h, is the height of solution in the capillary tube above the film, and g is the gravitational constant. Each term on the right hand side of equation (10) is measured independently, providing a direct measurement of n. Film thicknesses are measured interferometrically. White light from a 100W halogen lamp is passed through a heat filter and focused at normal incidence onto the individual foam film formed in the porous-plate holder. Reflected light from the film is then split and sent to a video camera and a fibre optic probe placed in the microscope ocular. The light from the fibre optic is filtered ( A = 546 nm) and analysed with a sensitive photomultiplier tube. The so-called equivalent film thickness is then calculated from the standard Scheludko interferometric equation which assumes a constant refractive index across the film.32 This equivalent thickness is slightly thicker than the true film thickness h, because the surfactant adsorption layers at each film interface have a higher refractive index than the aqueous core. To correct for this difference the multilayer correction factor system derived by D ~ y v iiss adopted. ~~ Finally, the thickness of the film's aqueous core is determined by substracting the thickness of the adsorbed layers from the total film thickness. The force curve, the disjoining pressure isotherm, is generated by measuring the equilibrium film thickness as a function of the applied capillary pressure to the film. The capillary pressure is changed by altering the gas cell pressure. This makes it possible to determine the repulsive branch of the disjoining pressure isotherm. Additional experimental details can be found elsewhere.34

3 Results and Discussion Monoglycerides and phospholipids are common stabilizers for emulsions in food and pharmaceutical applications. These compounds (see Figure 3) are nonionic and zwitterionic, respectively. This means that these emulsifiers do not generate any long-range repulsive electrostatic double-layer forces. Hence, according to DLVO theory, emulsion droplets coated with monoglycerides or phospholipids ought to flocculate due to the action of atttractive van der Waals forces. However, in practice this does not occur due to the presence of rather short-range repulsive forces. These forces have been thoroughly investigated using osmotic stress and surface force techniques. I6I8 The short-range forces acting between some monoglycerides in the lamellar and gel phase are illustrated in Figure 4.This set of data was obtained in our laboratory employing the osmotic stress technique.25 The molecules are packed into bilayer structures which in the lamellar state have fluid hydrocarbon chains. The area per molecule of monopalmitin in the lamellar phase (at 60 "C)is ca. 32 A*. In this case a repulsive force acts between the bilayers out to a separation of ca. 12 A. The force decays exponentially with increasing water layer thickness and the decay constant is ca. 2.4 A. The monopalmitin sample changes into a metastable gel phase when the temperature is decreased. The

208

Forces between Lipid-Coated Interfaces (a)

(b) H

H

/

/

0

0

1

1

0

CH2 - CH - CH2

I

/I

H3C - P - CH3

0

c=o

Figure 3

Chemical structures of three of the studied surfactants: (a) monopalmitin, (h) dimethylphosphine oxide, and (c) octyl-&$ucoside

gel phase is also a bilayer structure, but in this phase the hydrocarbon chains are frozen. The area per molecule in the gel phase is 23 A2, about 72% of that found in the lamellar phase. The repulsive short-range force in the gel state is at a given water layer thickness about a factor of 4-5 weaker than that in the lamellar state. It has a measurable range of about 8 A. Monoolein, which has the same polar group as monopalmitin, contains one cis double bond and therefore it forms a lamellar phase (area per molecule ca. 36-32 A2) at a lower temperature than monopalmitin. The forces measured between monoolein lamellar bilayers at 23°C are very similar to those found between monopalmitin lamellar layers at 60"C, and thus much stronger than those in the monopalmitin gel state at 23 "C. From these observations it is clear that the short-range force in the lamellar state is much stronger than in the gel state. Further, it is the state of the hydrocarbon chains (fluid or frozen) which is of prime importance for the strength of the force, whereas the temperature and structure of the hydrocarbon chain ( i . e . , saturated or unsaturated) is of importance primarily because these parameters determine the state of the hydrocarbon chains. It is

209

P . M . Claesson

0

5

10

15

Water Layer Thickness Figure 4

Swelling pressure as a function of water layer thickness in the lamellarphase of monopalmitin at 60 "C (O), in the gel state of monopalmitin at 23 "C (O), and in the lamellar state of monoolein at 23 "C (m). The upper solid line is an exponential force law (C ' ex ( DIA)), where the pre-expon$ntial factor has a value of 1 . 1 x l@Nlm , and the decay constant is 2 . 4 A . The lower solid line represents a force law with the same decay constant and a preexponential factor of 0.26 x 108Nlm2

4 -

worth noting that the area per molecule, and thus the area of the unfavourable hydrocarbon-water contact, is larger in the lamellar phase than in the gel phase. Nevertheless, the repulsive force is larger in the lamellar phase. What is the reason for this? At present there are two main ideas about the molecular origin of shortrange forces between uncharged lipids: hydration repulsion and steric repulsion. Let us consider how these contributions are thought to orginate and how they are expected to change when going from the gel to the lamellar state. The O H groups in the polar part of the monoglyceride (Figure 3) are hydrophilic and they interact favourably with water. Water molecules close to the interface respond to the presence of these hydrophilic groups and so they adopt a nonrandom orientation. The influence of the interface extends a few molecular layers out into the solution. As the two bilayers of monoglycerides are approaching each other, their respective solvation layers start to overlap and as a consequence the free energy of the system changes. This generates a repulsive force that is known as a 'hydration force'. When the gel phase melts, the hydrocarbon-water contact area increases and this reduces the hydrophilicity of the interface. This is expected to result in a less long-range hydration force, the opposite to what is observed. However, another effect may come into play, namely, as the gel phase melts, intra-layer hydrogen bonds between

210

Forces between Lipid-Coated Interfuces

monoglyceride groups are broken, making it possible to form more hydrogen bonds with ~ a t e r .This ~ ~effect , ~ ~is likely to make the interface more hydrophilic and increase the range of the hydration force. Clearly, since .both the hydrocarbon-water interfacial area and the number of hydrogen bonds with water increase as the monoglyceride gel phase melts into the lamellar phase, it is not easy to predict if the overall hydration force contribution should increase or decrease as a result of this phase transition. The bilayer arrangement in the lamellar or gel state is not static due to the molecular and collective motions of the lipids.23This means that the interface between the lipid bilayer and the aqueous layer is not well defined. The motions of the bilayer membranes give rise to a steric force with contributions from collective bending and squeezing motions as well as movement of individual molecules perpendicular to the interface.22 All these motions are restricted when two bilayers come close to each other and this lowers the entropy of the system, thus generating a repulsive force. It has been argued22 that it is this movement of the individual molecules, causing them to protrude from the average location of the interface, that often generates the most important force contribution, known as the protrusion force.22 Since the motion of the molecules increases as the gel phase melts into the lamellar phase, it is clear that this force contribution is also expected to increase, in line with the experimental findings for mono glyceride^.^^^^^ A comparison of the difference in swelling pressure in the gel and in the lamellar phase of a typical monoglyceride and a typical phospholipid is provided in Figure 5 . First, it is clear that phospholipids generate a stronger short-range force than do monoglycerides. Secondly, for phospholipids, just as for monoglycerides, a stronger repulsive force is observed in the lamellar phase than in the gel phase.15 It is hard to rationalize this in terms of a more hydrophilic interface in the lamellar phase, where the area per molecule is larger than in the gel phase. Instead, it appears that the steric protrusion force contribution is most important in this case. This conclusion explains why changes that increase the fluidity or the size of the polar group increase the range of the short-range repulsion. For instance, consider the following three observations. (i) The range of the short-range force increases when phosphatidylethanolamine is methylated. (ii) The range of the hydration force is often larger for lipids with unsaturated chains than for lipids with saturated chains. (iii) An increase in chain heterogeneity increases the range of the short-range force. From these findings one may draw the conclusion that an efficient phospholipid stabilizer for food emulsions should be used above its chain melting temperature. Furthermore, it ought to contain a mixture of hydrocarbon chains and a high fraction of unsaturated chains. Finally, phosphatidylcholine, due to its

211

P. M . Claesson

0

5

10

15

20

25

30

35

Water Layer Thickness (A> Figure 5

Typical swelling pressure against water layer thickness curves for the exponentially decaying part of the short-range interaction for monoglycerides and phospholipids. Upper thick line represents DPPC with melted chains (50°C);lower thick line represents DPPC with frozen chains (25 "C). Upper thin line represents monopalmitin with melted chains (60"C);lower thin line represents monopalmitin with frozen chains (23 "C)

larger headgroup size, ought to be a better stabilizer than phosphatidylethanolamine. The short-range interaction is expected to increase with increasing temperature as long as molecular protrusion is the predominant force contribution. This is what is observed for phospholipids and monoglycerides. However, there are also some nonionic surfactants which generate forces that have an opposite temperature d e p e n d e n ~ e . ~ ' -Most ~ ~ well known of these are the ethylene oxide based surfactants, but the same phenomenon is also observed for dimethylphosphine oxide (Figure 3), as illustrated in Figure 6. The reason for this inverse temperature dependence of the interaction is that the polar groups become more hydrophobic as the temperature increases. For ethylene oxide based surfactants the molecular mechanism behind this has been suggesteda to be a temperature induced change in conformation from gauche to trans of the 0442-0 segments in the ethylene oxide chain. The molecular mechanism behind the increased hydrophobicity of the dimethylphosphine oxide group at higher temperature^^^ cannot be explained in the same way. We may rationalize the behaviour note that the hydration model of Kjellander41742 of both the dimethylphosphine oxide and the ethylene oxide surfactants. In general, it is clear that both hydration and protrusion forces contribute to the short-range interactions between nonionic and zwitterionic lipids. Experi-

212

Forces between Lipid-Coated interfaces

h

Ed

!3

E 2m

2

PI

0

5

10

15

Water Layer Thickness (A> Figure 6

Swelling pressure in the lamellar phase of dimethyldodecylphosphine oxide as a function of water layer thickness. Measurements were carried out at 18°C (U) and 48 "C (@)

mentally, only the total force is measured. Conclusions about which force contribution is more important are based on data interpretation. As indicated above, the protrusion force concept is able to rationalize several aspects of the short-range forces observed in phospholipid and monoglyceride systems in a consistent way. However, other researchers have preferred to interpret the short-range force observed in these systems as a hydration force, and this point is still being debated in the scientific literature. 19-24 Lipid layers adsorbed to solid surfaces also generate short-range repulsive forces. In this case we d o not have any forces due to collective motions of the lipid molecules, but both protrusion forces and hydration forces are expected to be present. The forces acting between hydrophobized mica surfaces coated with lipid and surfactant monolayers have been investigated in several studies'7v18employing the interferometric surface force apparatus. Some data obtained in our laboratory43are shown in Figure 7, including the forces acting between deposited layers of monopalmitin. The zero distance point in this graph is defined as the contact between the hydrophobic surfaces prior to deposition of monopalmitin. The long-range repulsive force observed in this case is due to residual charges on the hydrophobic surface. At a separation of cu. 70 A, a van der Waals attraction dominates the interaction and the surfaces jump inwards to a separation of cu. 50A. The short-range force, observed in the distance range 50-40 A, is due to protrusion and hydration forces. Observe that the effective range of this force, ca. 10 A,is the same as the range of the short-range interaction observed in the monopalmitin lamellar and gel phases using the osmotic stress technique. It is not trivial to compare quantitatively the interactions between solid surfaces coated with a particular lipid, obtained from surface forcc mcasure-

213

P . M . Claesson 8 6

I

4

eE4 - 2 -4

0

50

100

150

200

Distance 0

0.4

0

0 -4

1

0,

0

-4

0.2

-5

n

-b

0

4

8

Distance lnml

Figure 3

12

0

4

8

12

16

20

24

Distance Inml

Calculated total segment density profile versus distance normal to an ideal solid-liquid interface for B-casein ‘look-alike’ representations of native (+ P) and fully dephosphorylated ( - P ) protein. Calculations from scf theory assume a total protein bulk volume fraction of 2 x I K 6 , ionic strength 0.01 M and pH 7.0. Plots (a) and (b) show the inner and outer regions of the adsorbed layer, respectively (note difference in scales)

224 /3-Casein Adsorbed Layer Structures Predicted by Self-Consistent-FieldModelling

Table 1

Neutron reflectivity results for surface coverage r, rms thickness 6 and best-fit parameters derived f o r two-layer model, obtained f o r native and dephosphorylated p-casein layers adsorbed at the airwater interface

Native

2.0.5 1.69

Dephos.

1. 65f 0. 07 0.96+0.12 1.43 k 0.06 0.86 k 0.10

l.OfO.l 0.9 k 0.1

0.15+0.01 4.40k0.18 0.15 f 0.01 3.37 k 0.20

3(b)), with the dephosphorylated ‘look-alike’ profile lying well within that of the native model. If we define the ends of the outer plateau regions in the semilogarithmic plot (Figure 3(b)) as the hydrodynamic thickness, then these theoretical predictions agree quantitatively with the experimental observations depicted in Figure 1. Considering the inner portions of the theoretical profiles (Figure 3(a)), we see that the dephosphorylated ‘look-alike’ shows a considerable shoulder after the initial fall-off from the high density innermost region ($I > 0.9). Whilst this agrees with the spirit of the neutron reflectivity analyses, with the dephosphorylated protein transferring material closer to the surface, quantitatively the calculations suggest a high concentration of material in this region, a result of the prediction of a higher surface coverage by the dephosphorylated protein. This is not observed experimentally with neutron reflectivity (Table 1) though the less accurate protein depletion method suggest a marginally higher coverage for the dephosphorylated sample. Further refinements in the theoretical calculations involving varying the interaction parameters may lead to a resolution of this discrepancy. Figure 4 shows the density profiles theoretically calculated for three different p H values. Figure 4(b) shows that, irrespective of pH, the dense inner layer starting again from the high volume fraction approaching unity retains a constant thickness of ca. 1 nm. Reducing the p H from 7.0 leads to an increase in polyelectrolyte density around the mid-part of the profiles (z 2-3 nm). At p H 5.5 a distinct shoulder is observed in the volume fraction profile, indicating an increase in the amount of polymer adsorbed. Inspection of the tail region calculated for the adsorbed layer in the plot of log[ $(z) ] versus z (Figure 4(b)) shows that the hydrodynamic layer thickness is not a strong function of pH. The calculated plots for p H values 5.5 and 6.0 superimpose, and that for p H 7.0 begins to drop from the plateau value only slightly beyond the other two curves. The behaviour of the adsorbed native p-casein observed in neutron reflectivity studies parallels these calculated trends with pH. The adsorbed amounts and rms thicknesses calculated from the model-independent Guinier treatment of the reflectivity profiles are listed in Table 2, both increasing as the p H is decreased. Table 2 also gives the best-fit values of the thicknesses and volume fractions of the inner and outer layers based on the minimum X2values returned

-

P. J. Atkinson et al.

225 0

-1

0.8 I

c

0 .-c

E

0 .-w

0

2

0.6

2

0

-E, -3

Y

a

-f

-2

LL

0

w

0.4

I

0

>

2

-4

m 0

A

0.2

-5

0

-

--k

0

12

4

0

4

Distance (nml

Figure 4

8

12

16

20

24

Distance (nml

Calculated total segment density profile versus distance normal to an ideal, planar, solid-liquid interface for p-casein ‘look-alike’protein as a function of indicated p H . Ionic strength is 0.01 M , total bulk protein volume fraction at p H 7.0. Plot (a) is on a b 4.3 x at pH 5.5 and 6.0 and 6.5 x linear scale and plot (b) on a semi-/ogarithmic scale

by the least squares fitting routine. Figure 2 shows the two-layer fits obtained at p H 7.0. Lowering the solution pH from 7.0, the best-fit behaviour with this model suggests that the increasing amounts of adsorbed protein are initially distributed in the diffuse layer at pH 6.0 with both volume fraction and layer depth increasing. By p H 5.5, however, the ever-increasing surface coverage can only be accommodated by a doubling of the thickness of the inner layer. Since little change was observed in surface coverage with bulk protein concentration at pH 7.0,6indicating monolayer formation, we have argued’ that the additional adsorption as pH is lowered points to the formation of a secondary layer piling on top of the initial primary monolayer.

Table 2

Surface coverage r, rms thickness 6 together with mean parameter values and their estimated errors derived from fitting two-layer model to neutron repectivity data for adsorbed/?-casein(0.005 wt%) at three different pH values

pH

Tlmg m-2

7.0 6.0 5.5

2.05k0.1 2.85k0.1 3.90+0.15

6/nm

@inner

dinnerlnm

@outer

1.65k0.07 0.96k0.12 1.OOk0.l 0.15k0.01 2.3950.08 0.95+0.10 1.03k0.1 0.20k0.02 2.5750.08 0.94k0.16 1.85k0.15 0.19k0.02

dinnerlnm

4.4k0.2 6.1k0.5 6.9f0.7

226 p-Casein Adsorbed Layer Structures Predicted by Self-Consistent-Field Modelling 80,

. ,

0

4

.

, . , . ,

8

12

16

.

,

. ,

20

24

Applied Protein Conc. (mg m-9

Figure 5

Apparent hydrodynamic layer thicknessplotted as a function of totalprotein (native p-casein) concentration, expressed relative to available particle surface area. Layer thickness is calculated as the difference between measured hydrodynamic radii of ‘bare’ and ‘coated’ polystyrene latex particles, measured at 20 “C in 20 mmol dm-” imidazolelHC1 buffers adjusted to the indicated p H

The formation of p-casein films on polystyrene latex particles suspended in buffers of varying pH is shown in Figure 5 where hydrodynamic thickness is plotted as a function of applied protein level. Each measurement was obtained by adding a single aliquot of protein solution to the latex suspension. The high apparent values obtained at low protein levels are undoubtedly the result of bridging flocculation, a consequence of the sharing of protein molecules between partially covered latex particles. This only becomes possible and more prevalent at the lower pH values as a result of charge neutralization. When sufficient protein is present to ensure complete protein saturation coverage then the plateau behaviour depicted in Figure 5 is observed. Somewhat unexpectedly, these experimental results show no major collapse of the hydrodynamic layer as the pH is reduced and the charges are neutralized. The average values over the plateau regions of each plot show no significant dependence on pH. This is exactly as predicted by the theoretical calculations (Figure 4(b)). A first plausible explanation of this behaviour is that electrostatic screening dictates how many charges lie along the tail and hence its extension. But if this number is exceeded at pH 5.5, then we may ask why should the hydrodynamic layer thickness be so sensitive at pH 7.0 to the removal of the charged phosphate centre (Figure l(a)) or to its neutralization

P. J. Atkinson et al.

221

by specific calcium ion binding.I2 It seems likely that the increasing density of the protein segments within the layer also plays a part in maintaining the hydrodynamic layer thickness as pH is lowered. The experimental observations and theoretical predictions of the adsorbed /?-casein conformation share some major features and common trends. Both suggest a dense inner layer close to the interface with a very high volume fraction close to unity. Such a high volume fraction leaves little room for water of hydration but the indications from other experiments, particularly enzyme accessibility and the spatial distribution of individual amino acids generated in the scf calculations,’8 suggest that this inner layer is composed of those highly hydrophobic parts of the /?-casein chain which have a high affinity for the interface and their own kind to the exclusion of water. It is also a noteworthy feature of the theory that, whilst producing the dense inner layer which parallels that implied by the neutron reflectivity observations, it also predicts an extended hydrodynamically active layer. The behaviour of this calculated layer also reproduces the collapse observed on dephosphorylating the protein and the lack of change when pH is reduced. The main difference between the density profiles obtained by theoretical calculation and by analysis of the reflectivity data lies in their overall general shape. It remains to be seen whether an input function predicted by theory can reproduce the experimental reflectivity as a function of wave vector for the adsorbed protein film. We would not wish at this stage to move to consideration of the Flory-Huggins parameters as fitting variables, for there are obvious limitations in the simplicity of the B-casein ‘look-alike’ model on which the calculations are based. We know that not all of the real protein segments are the same size, but the use of the lattice coordinate system constrains them to be so. The side-chains and their displacement of charge and polarity from the protein backbone are not fully accounted for in the assumption of a linear chain. The geometrical constraints of peptide bonds and in particular proline turns along the real /?-casein molecule are wholly neglected, but surprisingly the latter omission seems not too important for the ‘look-alike’conformation. The fitted neutron reflectivity curves show a clear two-step profile, but the distinctive step may be simply an artefact, a consequence of the fact that a twolayer model was chosen as the input function. That being said, the volume fractions and thicknesses of these layers, and the sharpness of their mutual interface, were allowed to float from their input values and a least squares minimum sought for the best-fit. Unfortunately, using this type of fitting approach, one never knows whether the solution reached is the right one, or whether some other ‘guess’ or another parameterization with a different function might yield a different and possibly superior density profile. Much effort is currently being devoted among the neutron and X-ray scattering fraternity to develop alternative, model independent procedures that hopefully will be more d e f i n i t i ~ eRecognizing .~~ the limitations of the reflectivity technique, we do feel, however, that the profiles obtained using it, considered in the light of information derived from all currently available experimental approaches, represent physically meaningful and semi-quantitative infor-

228 p-Casein Adsorbed Luyer Structures Predicted by Self-Consisterit-Field Modelling

mation on the interfacial structure of the adsorbed protein film, information that is certainly not available by any other technique.

Acknowledgements We wish to thank the EPSRC Rutherford-Appleton Laboratory (UK) for the provision of neutron beam time. E D , DSH and RMR gratefully acknowledge financial support for this research from the Biotechnology and Biological Sciences Research Council (UK). Core funding for the Hannah Research Institute is provided by The Scottish Office Agriculture, Environment and Fisheries Department.

References 1. E. Dickinson and G. Stainsby, ‘Colloids in Food’, Applied Science, London, 1982. 2. Th. F. Tadros and B. Vincent, in ‘Encyclopedia of Emulsion Technology’, cd. P. Bccher, Marcel Dckker, New York, 1983, vol. 1, p. 129. 3. H. E. Swaisgood, in ‘Developments in Dairy Chemistry-1. Proteins’, ed. P. F. Fox,Applied Science, London, 1982, p. 1. 4. E. Dickinson, D. S. Hornc, J. Phipps, and R. M. Richardson, Langmuir, 1993,9, 242. 5. E. Dickinson, D. S. Horne, and R. M. Richardson, Food Hydrocolloids, 1993, 7, 497. 6. P. J . Atkinson, E. Dickinson, D. S. Hornc, and R. M. Richardson, J . Chem. SOC. Faraday Trans., 1995,91, 2847. 7. P. J . Atkinson, E. Dickinson, D. S. Horne, and R. M. Richardson, in ‘Proteins at Interfaces II’, cd. T. A . Horbctt and J . L. Brash, American Chemical Society, Symposium Series, Washington, D.C., 1995, vol. 602, p. 311. 8. G. Fragncto, R. K. Thomas, A. R. Rcnnic, and J . Pcnfold, Science, 1995,267,657. 9. T. Cosgrove, J. S. Phipps, and R. M. Richardson, Colloids Surf., 1992,62, 199. 10. R. W. Richards and J . Pcnfold, Trends Polym. Sci., 1994,2,5. 11. D. G. Dalgleish, Colloids Surf., 1990,46, 141. 12. D. V. Brooksbank, C. M. Davidson, D . S. Horne, and J . Leaver, J . Chem. SOC. Faraday Trans., 1993, 89, 3419. 13. G. J . Fleer, M. A. Cohen-Stuart, J . M. H. M. Schcutjcns, T . Cosgrove, and B. Vincent, ‘Polymers at Interfaces’, Chapman and Hall, London, 1993. 14. J . M. H. M. Schcutjcns and G. J. Flccr,J. Phys. Chem., 1979,83, 1619. 15. J . M. H . M. Scheutjcns and G. J. Fleer, J . Phys. Chem., 1980,84,178. 16. J . M. H. M. Scheutjcns and G. J . Fleer, Macromolecules, 1985, 18, 1882. 17. M. A. Cohcn-Stuart, G. J . Fleer, J. Lyklema, W. Nordc, and J. M. H. M. Scheutjcns, A d v . Colloid Interface Sci., 1991,34,477. 18. F. A. M. Leermakcrs, P. J . Atkinson, E. Dickinson, and D. S. Hornc, J . Colloid Interface Sci., 1996, 178,681. 19. E. W. Bingham, H. M. Farrell, Jr., and R. J . Carroll, Biochemistry, 1972,11,2450. 20. R. Israels, F. A. M. Lecrmakcrs, G. J . Fleer, and E. B. Zhulina, Macromolecules, 1994, 27,3249. 21. J. Lcaver and D. G. Dalglcish, Biochim. Biophys. Acta, 1990, 1041,217. 22. D. G. Dalglcish and J . Leaver, J . Colloid Interface Sci., 1991, 141, 288. 23. P. S. Pcrshan, Phys. Rev. E , 1994, 50, 2369.

Adsorption Behaviour of Caseinate in Oil-in-WaterEmulsions: A Nuclear Magnetic Resonance Approach By Yoshinori Mine DEPARTMENT OF FOOD SCIENCE, UNIVERSITY OF GUELPH, GUELPH, ONTARIO, CANADA N l G 2W1

1 Introduction Emulsification ability is one of the most important functional properties of food proteins, and so studies of this have been made by many researchers. Hydrophobicity' and flexibility2 are generally accepted to be the major factors which decide the emulsifying properties of proteins. However, the adsorption behaviour and the conformation of the adsorbed proteins at the interface are still little understood. Elucidation of the structure of proteins at interfaces is essential to understand the mechanism of emulsification and the stabilization of emulsions by proteins. Casein (and caseinate) is well known to be a good protein emulsifier and many papers have been written on the emulsifying properties of casein. The structure of protein adsorbed to oil-water and solid-water interfaces has been studied by means of proteinase,M light scattering techniques,' CD ,* DSC9 and IT-IR" spectroscopic techniques. Among the enzymic techniques, the proteases trypsin and a-chymotrypsin are widely used. The 31PNMR nucleus has been used as a specific NMR probe to provide useful in situ information about the interfacial adsorptivity and dynamic and phospholipid-protein structures of phospholipids,".'* phosph~proteins'~ interaction^.'^-'^ Casein is a well-known phosphoprotein, and it was in fact the first phosphoprotein to be studied by 31PNMR." Thus, the 31P NMR technique can be expected to be applicable for evaluating the emulsifying properties of caseinate in situ. In this study, high-resolution 31P NMR was applied in elucidating the adsorption behaviour and dynamic structure of caseinate in oil-in-water emulsions.

2 Experimental Caseinate and trioleylglycerol (tri-18:l) were purchased from Sigma Chemical Co., St Louis, MO, USA. The soluble salts, such as phosphorus compounds, 229

230

Adsorption Behaviour of Caseinate in Oil-in-Water Emulsions

were completely removed by precipitating the protein at pH 4.5 and redissolving it at pH 7.0, then repeatedly dialysing against water, centrifuging and freeze-drying. Trioleolylglycerol was purified by silica-gel column chromatography (hexane-diethylether, 97:3) until more than 95% pure by gas-liquid chromatography analysis. The emulsions were prepared as follows. The caseinate solution (0.5 wt Yo in 20 mM imidazole buffer, pH 6.0, containing 10 mM EDTA) was mixed with various amounts of triacylglycerols. Oil-in-water emulsions were prepared using a high-speed homogenizer (Cole Parmer Instrument Co., CH, USA) with a generator shaft (10 mm diameter) at 12,000 r.p.m. for 1minute, and then further homogenized using an ultrasonic homogenizer (Model 470, Cole Parmer Instrument Co., CH, USA) operating on 40 W output power for 2 minutes. The droplet-size distribution and the surface area of the oil in each emulsion was measured by a laser diffraction particle analyser (Model SALD-2000, Shimadzu Corp., Kyoto, Japan). The result was expressed as a mean volumesurface diameter (d3,2). The determination of the amount of adsorbed caseinate was measured according to the methods described previou~ly.'~ The emulsions were centrifuged at 15 000 g for 1 hour to separate as many as possible of the oil droplets. 31PNMR measurements were performed with a Varian VXR 4000s spectrometer at 161.O MHz fitted with a probe (10 min, 45-165 MHz frequency), using a 45 " pulse (25,us) with a 32K data point, a 40 000 Hz spectral window, 20 rotations per second spinning rate, and 2.0 s pulse delay. Each proton was fully decoupled by a 9900 Hz decoupling modulation frequency, and the NMR samples were of volume 3.1 ml in 10 mm precision tubes. The line widths were measured from the resonance at half-height. The spin-lattice relaxation time ( T , )was measured by an inversion recovery (180 "-t-90 ") pulse sequence with a 90" pulse width of 50 ,us. Some 2000 acquisition times were accumulated for each 6 s delay time t.The spin-spin relaxation time ( T2)was measured by the spin-echo method with a 90 "-t-180" pulse sequence.'* Some 2000 acquisition times were calculated by a computer performing a non-linear regression of the exponential to the corresponding curves. The samples contained 20% 'H,O for internal locking.

3 Results and Discussion Figure 1 shows the mean droplet size and surface area for caseinate-stabilized emulsions as a function of oil content. The average particle size was found to increase with increasing oil content from 0.1 g to 1.0 g in 4 mL of 0.5 wt YO protein solution as shown in Figure l(a). Figure l(b) shows the specific surface area derived from the particle size distribution plotted against the oil content of emulsion. The specific surface area was found to decrease linearly with increasing oil content as a result of the formation of larger emulsion droplets. These data indicate that the droplet size and surface areas of caseinate-

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Mean diameter of oil droplets and mean surface area as a function of trioleolylglycerol oil concentration. Median diameter and surface area were derived from measuring the droplet size dktribution using a laser diffraction particle analyser: (a) oil droplet size; (b) surface area. Data points are the averages of triplicate measurements

stabilized emulsions are closely related to the ratio of oil/protein in the emulsions. Figure 2 shows typical "P NMR spectra of caseinate in solution and in emulsions of various oil contents. A single spectrum with a 19.6 Hz line width was observed for caseinate in solution (Figure 2(a)). This signal must originate from the phosphoserines in /?-casein and a,,-casein. However, most of the previous rep~rts''-*~of casein phosphoserine signals have found a number of peaks spread over a range of several ppm. Interestingly, most of these signals observed in the early NMR study of milk have since been assigned to phosphorus compounds already known to be present in milk or casein sample^.^^,*^ Therefore, it is very important to remove these phosphorus compounds from samples before use in order to avoid the detection of such interfering impurities by 31PNMR. Sleigh el al." observed respectively two and four spread multiple resonances of /?-casein and aSl-caseinat pH 6.8 using high resolution 31PNMR. The same authors also reported a pH-dependent change in the chemical shift of the 31Presonance of the phosphoserines in aSl-casein. This was also observed for the case of /3-casein phosphoserines by Humphrey and Jolley.20 However, these signals have still not been assigned in detail. In this light, the resonance of the 31PNMR spectrum of caseinate must be affected by pH. In the present work, the minor peaks which should be observed at alkaline pH values, can be hidden by the major one because the pK2 value of the downfield phosphate resonance of casein is 5.9 and that of the upfield phosphate resonance is 6.65. Thus, it is suggested that there are no differences between the environments of individual phosphoserine residues of the caseinates at p H 6.0. In the emulsion of caseinate, the phosphorus line

232


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Figure 2

3’P N M R spectra of caseinate in aqueous dispersion and in emulsions: (a) solution of 0.5 wt% caseinate; (b), (c), ( d ) emulsions. The oil contents are 0.2 g (b), 0.5 g (c) and 1.0 g (d) in 4.0 m L of 0.5 wt% protein solution, respectively

widths became broader with increase of oil content (Figure 2). The phosphorus signals of 31PNMR spectra are influenced by the dynamic properties of the phosphate moiety in the molecules, and peak broadening of phosphorus signals in 31PNMR spectra is observed on increasing the magnitude of the negative phosphorus chemical a n i ~ o t r o p yThe . ~ ~peak broadening of 31PNMR spectra with increase of oil content of the emulsion is considered to be due to reduced mobility of the phosphoserine moiety in caseinate as a result of adsorption of caseinate at the interface. These data indicate that the extensive peak broadening in the 31PNMR spectrum of caseinate in the emulsion is correlated with the adsorption behaviour of the protein adsorbed at the oil-water interface. Figure 3 shows the correlation between the line width of the 31P NMR spectrum and the total amount of protein adsorbed at the interface as a function of oil content. The total amount of adsorbed caseinate was found to

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Changes in (a) total amount of adsorbed protein and (b) phosphorus line width for caseinate-stabilised emulsions as a function of oil concentration. Data points are the averages of triplicate measurements

increase with increasing oil content. The protein was in excess when the oil content was 0.1 g, and so 80% of the caseinate remained in serum. The excess protein existing in the serum was adsorbed at the interface with increasing oil content, and the total amount of adsorbed protein reached 64% at an oil content of 1.O g. The line width of 3'P NMR spectrum was increased from 19 Hz to 101 Hz with increase of oil content, and the signal peak broadening was clearly well correlated to the amount of adsorbed protein. These data can be interpreted as indicating that the average mobility of a phosphoserine moiety in caseinate was reduced in parallel to the increase of adsorbed protein at the oilwater interface. The phosphorylated groups of aSl-casein are located between residues 41 and 81 (this sequence contains the eight phosphoseryl residues). The molecule has three strongly hydrophobic regions: residues 1-40,90-113 and 132-199.25 It has been suggested3 that some hydrophobic regions of aSl-casein,i.e., the neighbourhood of residues 21-25,94,135 and 142 from the N-terminal, might interact strongly with oil. The N-terminal section (residues 1-21) of p-casein is highly charged and contains four of five phosphoseryl residues.25 Leaver and Dalgleish reported that the N-terminal peptides 1-25 and 1-28 of p-casein, a hydrophilic region, might be protruded from the Therefore, the peak broadening of the 31PNMR spectrum of caseinate in an emulsion can be a reflection of the mobility of phosphoserines in the protein as a result of the fact that the hydrophobic regions of aSl-casein and p-casein, in the neighbourhood of the phosphorylated regions, might be adsorbed at the interface. The relaxation parameters described below do correlate with structurhl features of molecules, and particularly with their motion, and so the relaxation measurements can be possibly used to good effect in structure elucidation. In order to ascertain the mobility properties of phosphoserines in caseinate for an

234


Table 1

Values of the TI and T2 relaxation times for caseinate in solution and in an emulsion

Solution Emulsion

1.42 f 0.04 0.17 f 0.02

26 If: 4 8 f 2

emulsion, the T I and T2relaxation times for caseinate in an aqueous dispersion and in an emulsion were measured (Table 1). The value of the T , relaxation time of caseinate phosphate residues in an emulsion was markedly reduced from 1.42 s to 0.17 s. The T2relaxation time was also reduced from 26 ms t o 8 ms in the emulsion. In general, the relaxation time value strongly depends on the environment and the mobility of the observed nuclei.26The T , relaxation time is influenced by the intramolecular interaction of the molecules, and the T2relaxation time is strongly correlated with intermolecular interaction. Thus, the data obtained from the relaxation time measurements indicate not only the restriction of phosphoserine mobility derived from adsorption of the protein at the oil-water interface, but also the possibility of its conformational change at the interface. In other words, the peak broadening of the 31PNMR spectrum and the relaxation time measurement in caseinate can be used as sensitive probes in evaluating the macromolecular adsorption behaviour of caseinate at the oil-water interface. Figure 4 shows the changes of phosphorus line widths of caseinate in solution and in the emulsion containing 20 wt % of oil as a function of temperature. In

0

1 20

40

60

80

Temperature ("C)

Figure 4

Phosphorus line widths for caseinate as a function of temperature: (0), solution; (a),emulsion. The emulsion is composed of 0.5 wt% protein soliition (4 m L ) and trioleolylglycerol ( I . 0 g) at 20 "C and the temperature is raised in stages from 20 "C to 80 "C at 5 "C min-'

Y. Mine

235

the protein solution, the line width scarcely changes despite the increasing temperature. On the other hand, the line width was found gradually to increase in the emulsion of caseinate with tri-18:l when the temperature was raised at 5 “C min-’ in the sample tube. The mean droplet size of the caseinate emulsion was not changed with increasing temperature. It is suggested that the environment of individual phosphoserine residues at the interface varies with the temperature. This phenomenon also indicates that the affinity of caseinate for the oil phase changes with temperature or that aggregation of caseinate molecules occurs at the interface. Increasing the affinity or the state of aggregation might cause the broadening of the lines.

References 1 . S. Nakai, J. Agric. Food Chem., 1983, 31, 676. 2. A. Kato and K. Yutani, Protein Engineering, 1988, 2, 153. 3. M. Shimizu, A. Ametani, S. Kaminogawa, and K. Yamauchi, Biochim. Biophys. Acta, 1986,869,259. 4. J. Leaver and D. G. Dalgleish, Biochim. Biophys. Acta, 1992,1041,217. 5. D. G. Dalgleish and J. Leaver, J. Colloid Interface Sci., 1991, 141,288. 6. J. Leaver and D. G. Dalgleish, J . Colloid Interface Sci., 1992, 149,49. 7. B. W. Morrissey and C. C. Han, J. Colloid Interface Sci., 1978,65,423. 8. L. J. Smith and D. C. Clark, Biochim. Biophys. Acta, 1992,1121,111. 9. C. A. Haynes and W. Norde, J. Colloid Interface Sci., 1995, 169,313. 10. R. L. Jakobsen and F. M. Wasacz, in ‘Protein at Interfaces. Physicochemical and Biochemical Studies’, American Chemical Society, Washington, D.C., 1987, p. 339. 11. K. Chiba and M. Tada, Agric. Biol. Chem., 1990,53,995. 12. K. Chiba and M. Tada, Agric. Biol. Chem., 1990,54,2913. 13. Y . Mine, K. Chiba, and M. Tada, J. Agric. Food Chem., 1992,40,22. 14. Y. Mine, H . Kobayashi, K. Chiba, and M. Tada, J. Agric. Food Chem., 1992,40, 1111. 15. Y. Mine, K . Chiba, and M. Tada, Biosci. Biotechnol. Biochem., 1992,56,1814. 16. Y. Mine, K. Chiba, and M. Tada, J. Agric. Food Chem., 1993,41, 157. 17. C. Ho and R. J. Kurland, J. Biol. Chem., 1966,241,3002. 18. A. E. Derome, ‘Modern NMR Techniques for Chemical Research’, Pergamon Press, Oxford, 1987, p. 85. 19. R. W. Sleigh, A. G. Mackinlay, and J. M. Pope, Biochim. Biophys. Acta, 1983, 742, 175. 20. R. S. Humphrey and K. W. Jolley, Biochim. Biophys. Acta, 1982,708,294. 21. P. S. Belton, R. L. J. Lyster, and C. P. Richards, J. Dairy Res., 1985,52, 47. 22. M. Wahlgren, T. Drakenberg, H. J. Vogel, and P. Dejmek, J. Dairy Res., 1986,53, 539. 23. P. S. Belton and R. L. J. Lyster, J. Dairy Res., 1991,58,443. 24. W. Wu, F. A. Stephenson, J. T. Mason, and C. Huang, Lipids, 1984, 19,68. 25. P. F. Fox, in ‘Development in Dairy Chemistry-4‘. ed. P. F. Fox, Elsevier Applied Science, London, 1989, p. 1. 26. P. A. Hart, ‘Phosphorus-31 NMR. Principles and Applications’, Academic Press, New York, 1984, p. 329.

Stabilization of Protein-based Emulsions by Means of Interacting Polysaccharides By Douglas G. Dalgleish and Anne-Lise Hollocou DEPARTMENT OF FOOD SCIENCE, UNIVERSITY OF GUELPH, GUELPH, ONTARIO, CANADA N l G 2W1

1 Introduction Many foods contain emulsion droplets, and many of these also contain protein as an emulsifier and stabilizer for the droplets. In addition, polysaccharides may be incorporated into the formulation, mainly because they increase the viscosity of the aqueous phase of the product, or form gels. These actions help to increase the shelf-life of the products, to prevent flocculation, creaming or coalescence. In terms of molecular interactions in such complex mixtures, it is generally the case that the polysaccharides and the proteins do not interact with one another, but perform their functions separately. That is, the proteins adsorb to the surface of the dispersed lipid by means of their hydrophobic regions, and so provide stability against coalescence by creating an extended, charged, layer around the droplet surface. Because of their hydrophilic nature, polysaccharides do not generally adsorb to oil surfaces, but remain in solution and modify the viscosity because of their extended, hydrated structures. Interactions between protein and polysaccharide are only found in a few cases. However, interactions between proteins and polysaccharides have been shown to occur in some specific instances. Two particular cases of relevance to this study are (i) the interaction between the milk protein casein (specifically the x-casein part of the complex) and x-carrageenanIT2and (ii) the interactions between casein and high-methoxyl pectin at low P H . Of ~ these, ~ the first is known to be partly dependent on the presence of calcium ions; although x-casein and x-carrageenan do not require calcium for interaction,”* the presence of Ca2+ may allow the other caseins to interact also.6 Moreover, x-carrageenan interacts with casein micelles as well as with c a ~ e i n a t ei.e., , ~ the casein need not be in a dissolved state for interaction to occur. The interaction can also occur at pH values around neutral. In contrast to this behaviour, the interaction between pectin and casein occurs only at pH values around or below the isoelectric point of casein (pH 5 and be lo^).^ It has been suggested that this results from the interaction between the net positively charged casein 236

D. G . Dalgleish and A.-L. Hollocou

237

and the negatively charged pectin; above the isoelectric point the casein is overall negatively charged and the interaction is minimal. Adsorption of pectin on to casein has been shown to occur;' also we have shown that a thick layer of pectin is formed around casein particles and model colloids containing caseins (Picaut and Dalgleish, unpublished results). So it is clear that both these polysaccharides can in principle stabilize particles containing or coated with casein. The experiments to study the interaction between pectin or carrageenan with caseins have been performed only using isolated caseins or casein micelles. There have been no studies on the interactions between adsorbed caseins and the polysaccharides. As part of a survey of emulsion stabilization, we have studied a range of polysaccharides to determine their interactions with emulsions prepared with milk proteins. In this paper, emphasis is placed on the stability to acidification of emulsions containing milk proteins in the presence of x-carrageenan or high-methoxyl pectin.

2 Experimental Materials The pectin used was a commercial high methoxyl pectin from Systems Bio Industries (Carentan, France); the carrageenan used was a commercial x-carrageenan (SeaKem CM611) from Marine Colloids Division of FMC Corporation (Philadelphia, PA). Whey protein isolate (WPI), a-lactalbumin and B-lactoglobulin were provided by Protose Separations, Inc., Teeswater, Ontario. Sodium caseinate was prepared in the laboratory by acid precipitation of fresh milk to pH 4.6. The precipitate was filtered, washed with distilled water, and redissolved in NaOH (1 N) at pH 7 prior to freeze drying. All other chemicals were from Sigma Chemical Co. (St. Louis, MO) and Fisher Scientific (Mississauga, Ontario) unless otherwise specified.

Measurement of Hydrodynamic Diameters of Emulsion Droplets The hydrodynamic diameters of the particles in the presence of polysaccharide were measured by dynamic light scattering (DLS) using a 4700 optical system and a 7032 Multi-8 correlator (Malvern Instruments Inc., Southboro, MA). The correlation functions were analyzed using the method of cumulants to give the average Stokes radii of the particles. The viscosities of the citrate solutions containing different polysaccharide concentrations, at different pH values, were measured using a Cannon viscometer at 25 "C, and these values were used in the calculation of the particle diameters.

Preparation of Emulsion Droplets The proteins were rehydrated to a concentration of 1% or 2% (w/w) in citrate (20 mM sodium citrate), adjusted to pH 7.0 with 1 N HCI. Sodium azide

238

Stabilization of Protein-based Emulsions

(0.02%) was added to the buffer to inhibit microbial growth. The solutions were successively filtered through filters of pore sizes 3,0.8,0.45 and 0.22ym (Millipore Corporation, Bedford, MA). Emulsions were prepared by adding 20% (w/w) of soybean oil to the solutions of proteins and homogenizing using a Microfluidizer M11OS (Microfluidics Corporation, Newton, MA) at a pressure of 42 MPa. The emulsions were stored at 4 "C until used.

Interactions between Protein and Polysaccharide Polysaccharide solutions were prepared using either pectin or carrageenan with concentrations ranging from 0% to 0.05% (w/v). The carrageenan was dissolved in citrate while stirring at 80 "C and was then cooled to room ) diluted into 60 ml of temperature before use. Aliquots of emulsion ( 3 0 ~ 1were the polysaccharide solutions to give final concentrations of O.O005% and 0.001% of the protein in the stabilizer solution. These suspensions were titrated to pH values between 6.5 and 3.5 with 1N HCI. Measurements of the particle diameters were made by DLS at all pH values. Effects of calcium ions on the interactions were studied by adding calcium chloride solutions of defined strength to the suspension of emulsion in polysaccharide, with fast stirring. The pH was then adjusted from 6.5 to 3.5 in 0.5 unit decrements and samples were retained for DLS measurements. Effects of sodium chloride were measured in the same way.

3 Results Interactions of High-methoxyl Pectin with Emulsions Containing Casein or WPI It was evident that pectin, at nearly all concentrations used, stabilized emulsions containing caseinate against precipitation during acidification (Figure I), as found previously for acid-precipitated casein^.^ In the absence of pectin, the caseinate emulsions became unstable at pH values around 5 , and precipitated because of the reduction of net charge on the caseins as they approached the isoelectric point. Even though caseins are positively charged at pH 3.5, we found the emulsions were still unstable. However, the instability of the emulsions was prevented by the presence of even the smallest concentration of pectin (0.000625%) used in the experiments. This concentration in weight terms is in the same range as the concentration of casein in the diluted emulsions, although in molar terms the casein (molecular mass 23 kDa) will be in excess of the polysaccharide (molecular mass ca. 100 kDa). In an emulsion containing 1% casein, we know that most of the casein is adsorbed to the surfaces of the oil droplets,'and approximate calculations based on the sizes of the droplets suggest that each droplet could adsorb several thousand casein molecules and about 1 thousand molecules of pectin. These would already be sufficient to form a stabilizing layer of pectin around the emulsion droplets.

239

D. G. Dalgleish and A . - L . Hollocou

250 3.5

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PH Figure 1

Diameters of emulsion particles (20 wt% soya oil,I wt% caseinate) diluted into solutions of pectin of different concentrations and then adjusted to different pH values. Buffer solution contained 20 m M citrate. Symbols show results at concentrations (wt%,) of pectin of B, 0; 0, 6.25 x i f f 4 ; A , 0.00125; 0.0025; 0, 0.005; 0, 0.01; A,0.02; 0, 0.05 %. In all figures, particle sizes of 600 nm denote that precipitation was occurring

+,

Even at pH values above 5, there was evidence that pectin was binding to the emulsion droplets. The effective sizes of the particles increased with increasing concentration of pectin, even when a correction was made to account for the increasing viscosity of the solutions. This suggests that even when the casein and pectin were both negatively charged, the repulsion was insufficient to prevent at least some of the pectin binding to the adsorbed casein. The binding may be relatively weak, since there was no significant difference between the sizes of the emulsion droplets in the absence of pectin and the same droplets in the presence of 6.25 x % pectin. However, the presence of double this amount of pectin significantly increased the diameters of the emulsion droplets, suggesting that an interaction was occurring. This interaction, binding or not, seemed to be minimized as the pH was decreased below 6.5. In all cases, the smallest diameters were found in the pH range 5-5.5. Below pH 5 , the apparent sizes of the emulsion droplets were found to increase significantly. This may either arise from limited aggregation or from the binding of pectin to the casein-coated emulsion droplets as a result of the increasing positive charge of the latter. Indeed we may be observing a mixture of both reactions; the method cannot distinguish between them. Because of the magnitude of the increase in apparent diameter of about 150 nm, it seems likely that there is a small amount of aggregation, since the formation of a layer of pectin about 75 nm thick seems unlikely. The diameters of these acidified particles increased significantly at the highest concentrations of pectin, which may suggest that even higher order complexes may be formed in the presence of large amounts of excess pectin.

240

Figure 2


Diameters of emulsion particles (20 wt% soya oil, 1 wt% WPI) diluted into solutions of pectin of different concentrations and then adjusted to different p H values. Buffer solution contained 20 mM citrate. Symbols for pectin concentrations are the same as in Figure 1

The pectin was less effective in stabilizing emulsions prepared using WPI (Figure 2). Although the higher concentrations prevented precipitation at all pH values, the pectin at 6.25 x % did not stabilize at all and 1.25 x pH values, especially near pH 5, and in fact the lowest concentration of pectin appeared to have a destabilizing effect at all pH values. As was seen for the caseinate emulsions, increasing amounts of pectin caused increases in the diameters of the emulsion droplets, even at pH 6.5, and these increases were greatest with the highest concentration of pectin. This again may suggest that an interaction between the protein and polysaccharide was occurring. There was less of a general indication, however, that this behaviour was pHdependent between pH 5 and 6.5. Increases in the apparent diameters of the emulsion droplets were found to occur below p H 5.5, at a higher p H than with casein, in agreement with the higher isoelectric points of the whey proteins and the stability of the emulsion^.^ In the emulsions with WPI the increase in apparent diameter during the acidification was somewhat larger than for the caseinate emulsions, being about 200 nm. This almost certainly implies that some aggregation occurs; it seems most unlikely that the pectin can produce a layer 100 nm thick around individual emulsion droplets. However, it is clear that the aggregation, as in caseinate emulsions, is extremely limited.

Interactions of K-Carrageenan with Emulsions Containing Casein or WPI The x-carrageenan preparation proved to be as effective as pectin, if not more so, at stabilizing emulsions prepared using caseinate or WPI. Figure 3 shows the behaviour of the particles in the caseinate-stabilized emulsion. Once again,

241

D . G . Dalgleish and A.-L. Hollocou

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PH Figure 3

Diameters of emulsion particles (20 wt% soya oil, I wt% caseinate) diluted into solutions of carrageenan of different concentrations and then adjusted to different p H values. Buffer solution contained 20 mM citrate. Symbols for carrageenan concentrations are the same as in Figure I

only a very low concentration of the polysaccharide appeared to be necessary to achieve stabilization of the diluted emulsion at low pH. The emulsion on its own became unstable at pH 5, but, in the presence of the carrageenan, the particle size increased hardly at all as the isoelectric point of the casein was passed. No sharp transition was found in this region. Rather than decreasing and then increasing as the pH is dropped, as was found for pectin, the diameters of the particles increased steadily and monotonically in the presence of carrageenan, with a total change from pH 6.5 to 3.5 in the region of 50 nm. Whether this increase should be attributed to the formation of a layer of carrageenan around the casein/oil emulsion droplet, or whether there is a slight increase in the aggregation of the emulsion droplets, is not known. There was an increase in particle diameter with concentration of carrageenan at pH 6.5, but it was smaller than the corresponding change found with pectin, apart from at the highest concentration of carrageenan. Emulsions based on WPI behaved the same with carrageenan as they did with pectin (Figure 4). That is, the emulsions showed instability at pH 5.5, and were stabilized in the presence of the higher concentrations of carrageenan. There was only a slight improvement in stability in the presence of 6.25 X % of carrageenan, and there was also a slight tendency to instability at pH 5.0 in the presence of 1.25 x % carrageenan. Otherwise, the emulsions behaved similarly to the caseinate emulsions in being well stabilized, except that there was no significant increase in the diameters of the particles when carrageenan was added even at the highest level. The differences in apparent diameter between 6.5 and 3.5 were somewhat larger than for casein, but they were much smaller than the corresponding changes which were found to occur when pectin was used as a stabilizer or when stabilizer was absent.

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250 3.5

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PH Figure 4

Diameters of emulsion particles (20 wt% soya oil, I wt % WPI) diluted into solutions of carrageenan of difjerent concentrations and then adjusted to different p H values. Buffer solution contained 20 mM citrate. Symbols for carrageenan concentrations are the same as in Figure I

Calcium ions are known to bind to carrageenan, and to alter its structure and binding to casein. The effect of incorporating calcium ions (5 mM) into the diluted emulsions containing carrageenan was to even improve the stability to pH further than was found for the emulsions in the absence of Ca. Two effects were noticed (Figure 5). First, there was a clear, albeit small, difference in the average diameter of the emulsion droplets with and without carrageenan; second, the change in diameter with pH was almost completely eliminated. It

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Diameters of emulsion particles (20 wt% soya oil,1 wt% caseinate) diluted into solutions of carrageenan of different concentrations and then adjusted to different p H values. Buffer contained 4 mM citrate and 5 mM Ca. Symbols for carrageenan concentrations are the same as in Figure I

D . G. Dalgleish and A.-L. Hollocou

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may be surmized that the increase in diameter with carrageenan is caused by binding of the polysaccharide to the emulsion droplets in the presence of Ca2+; clearly this enhances the stability of the system to a considerable extent.

4 Discussion Both of the polysaccharides used had the property, to different extents, of stabilizing emulsions containing caseins or whey proteins at pH values where precipitation of the emulsions generally took place. The mechanism of these interactions remains to be established. The binding of pectin to casein particles is the result of the interactions between the negative charges of the pectin and the positive charges of casein below its isoelectric point. Moreover, it seems as if the casein must be partly aggregated for this interaction to occur efficiently since we found no stabilization on the molecular level (Dalgleish & Picaut, unpublished observations); a surface of casein appears to be required for adsorption of pectin. In an emulsion, the casein is spread over the surface of the fat globules, and may therefore present an appearance similar to that in the aggregate. As a consequence, we might expect pectin to stabilize the emulsion against aggregation. Limited aggregation seems, however, to occur unavoidably at pH values below 5 . Thus, the caseidpectin interaction appears to be understood, although there is still an unexplained problem of how some interactions appear to occur at high pH, where they might not be expected on the basis of net molecular charges. The efficiency with which pectin stabilized emulsions containing WPI was less than with casein, but nevertheless pectin did prevent precipitation at relatively high pectidprotein ratios. It is possible that the interactions between pectin and the emulsion droplets were of the same nature as those between pectin and casein, i.e., with the negative charges of the pectin binding to the positive charges of the whey proteins below their isoelectric points. The mechanism by which the caseinate emulsions are stabilized by carrageenan results from the interaction between x-casein and x-carrageenan, which presumably relies on specific charges, and may not be pH-dependent, on the evidence from this study. Because the casein and carrageenan are strongly linked together, the polysaccharide completely protects the emulsion droplets from aggregation and so there is not even a limited aggregation as the isoelectric point is passed. Possibly with pectin there is competition between emulsion droplets and polysaccharide for interaction. For the interaction between carrageenan and whey protein, it is perhaps surprising that it occurs at all. It has not been recorded before that this interaction occurs, either in solution or under any conditions. It may be that it is possible because the whey proteins are denatured on adsorption, and this may permit new interactions to occur between protein and polysaccharide. The similarity in behaviour between caseinate emulsions and WPI emulsions is striking, and presumably occurs from a similar cause; simply the differences between the proteins arise from the strength of their interaction with carrageenan.

244


Since carrageenan is negatively charged at low pH, it is possible that its function at low pH is similar to that of pectin, namely to interact with positive sites on the proteins; this does not, however, explain the interactions which may occur at higher pH. It remains to be seen whether these types of interactions may be useful in the formulation of, for example, beverages containing proteins in the pH range 3-5. Although the interactions have been shown to occur efficiently at low concentrations of polysaccharide, it is important to find whether they can be scaled up to realistic concentrations of protein and/or emulsion without using so much polysaccharide as to cause problems of high viscosity or to render its use uneconomic. However, if fully exploited, this may prove to be a possible way of easily introducing stability into acidified beverages containing emulsified fat stabilized by milk proteins.

Acknowledgements This work was funded by the Ontario Dairy Council and the Natural Science and Engineering Research Council of Canada. Our thanks are due to Marine Colloids for the carrageenan and Systems Bio-Industries for the pectin used in these experiments.

References 1. T. H. M. Snoeren, P. Both, and D. G. Schmidt, Neth. Milk Dairy J, 1976,30, 132. 2. T. H. M. Snoeren, T. A. J. Payens, J. Jeunink, and P. Both, Milchwissenschaft, 1975,30, 393. 3. P.-E. Glahn, Prog. Food. Nutr. Sci., 1982, 6 , 171. 4. H. C. A. Pedersen and B. B. J~rgensen,Food Hydrocolloids, 1991,5, 323. 5 . A. Parker, P. Boulenger, and T. P. Kravtchenko, in ‘Food Hydrocolloids: Structure, Properties, Functions’, ed. K. Nishinari and E. Doi, Plenum Press, New York, 1994, p. 307. 6. B. J . Skura and S. Nakai, J. Food Sci., 1980,45,582. 7. D. G. Dalgleish and E. R. Morris, Food Hydrocolloids, 1988, 2, 311. 8. Y. Fang and D. G. Dalgleish, J. Colloid Interface Sci., 1993, 156, 329. 9. J. A. Hunt and D. G. Dalgleish, J. Agric. Food Chem., 1994,42, 2131.

Effect of Neutral Carbohydrate Structure on Protein Surface Activity at Air-Water and Oil-Water Interfaces By A. S. Antipova, M. G. Semenova, and A. P. Gauthier-Jacques INSTITUTE O F FOODSTUFFS, RUSSIAN ACADEMY OF SCIENCE, VAVILOV STREET 28, 117813 MOSCOW, RUSSIA

1 Introduction It is well known that structural functions of biopolymers in food systems can dramatically change with the composition of the aqueous medium. Recently, we have observed'.* a significant effect of sucrose on the protein solubility, the character of the biopolymer-biopolymer pair interactions in aqueous solution, and the biopolymer cosolubility for a set of mixtures of proteins and polysaccharides. It was also well established3,' that the mutual influence of the proteins and polysaccharides on their functionality in food systems is one of the most important factors controlling the structure, texture and rheology of multicomponent foodstuffs. This paper will try to elucidate the effects of low- and high-molecular-weight neutral carbohydrates on globular protein surface activity at the aidwater or oil/water interface in relation to neutral carbohydrate structure.

2 Effect of Low-Molecular-Weight Carbohydrates on Globular Protein Surface Activity at the Planar Interface Table 1 presents values of the interfacial pressure n of protein adsorbing at the planar aidwater interface in the presence of the low-molecular-weight carbohydrates. The data show the similarity of the effects of sucrose and glucose on the surface activity of 11s globulin. The surface pressure was found to show a dramatic dependence on the concentration of the low-molecular weight sugars. So, at relatively low sugar concentration in the solution (0.05% w/v) (molar ratio R,, defined as moles of the sugar/moles of the protein, equals 5 x 10' and 9 x lo4 respectively for the sucrose and glucose), the interfacial pressure and 245

246

Effect of Neutral Carbohydrate Structure on Protein Surface Activity Change of interfacial pressure IT for 0.001YOwlv 11s globulin solution under influence of low-molecular-weight carbohydrates after 10 hours at the plane air-water interface (25 "C, p H 7.0, I = 0.01 mol dm-3)

Table 1

n l m N m-'

Sample 11s globulin 11s globulin 11s globulin 11s globulin 11s globulin

9.3 5.4 7.8 14.3 15

+ 0.05% w/v sucrose

+ 0.05% w/v glucose + 2% w/v sucrose + 2% w/v glucose

consequently the protein surface activity decrease significantly. A large increase of the low-molecular-weight sugar concentration in the protein solutions (2% w/v) (R, = 200 X lo4 and 360 x lo4 respectively for the sucrose and glucose) causes a significant increase in the interfacial pressure JC and consequently in the protein surface activity. A similar effect of sucrose at low concentration on 11s globulin surface activity was observed at the planar oil/ water interface (see Table 2). To account for the results obtained, let us first consider the effect of the lowmolecular-weight sugars on the thermodynamic affinity of the 11s globulin for the aqueous medium. As general, the thermodynamic affinity of a biopolymer for the solvent may be characterized by the value of the second virial coefficient A2 in the concentration expansion of the chemical potentials p of the biopolymer, which is expressed by the following e q ~ a t i o n : ~ p = p'

+ RT[ln(mlm') + Azm],

(1)

Herep" is the standard chemical potential, m0 is the standard-state molality, m is the concentration of a biopolymer (molal scale), A 2 is the second virial coefficient of the biopolymer, R is the gas constant, and T is the absolute

Table 2

Changes of interfacial pressure IT for 0.001% wIv 11s globulin solution under influence of sucrose after 10 hours at the plane airwater and oil-water interfaces (25 "C, p H 7.0, 1 = 0.01 mol d m - 3 ) n l m N m-' Sample

11s globulin 11s globulin

+ 0.05% w/v sucrose

air-water

oil-water

9.3 5.4

18.1 12.5

A . S . Antipova, M. G . Semenova, and A . P. Gauthier-Jacques

0

S

10

IS

20

247

25

Sugar concentration x 1 02, g/mI Figure 1

The I I S globulin second virial coefficient A2 versus concentration of sucrose or glucose (25”C,p H 7.0, I = 0.01 mol dm--’)

temperature. Figure 1 shows the increase of the protein second virial coefficient with rise in concentration of the low-molecular weight sugars in the system for small values of the molar ratio R, (up to 8 x 104forsucrose and 17 X lo4for glucose). This result indicates clearly the increase of the thermodynamic affinity of the protein for the aqueous medium in the presence of both sucrose and glucose. One may suppose that the effect of sucrose on the 11s globulin thermodynamic properties in aqueous solution could be explained either in terms of direct interaction or by indirect action through the effect of sucrose on the water structure, or by a combination of these two mechanisms. The calorimetric data presented in Figure 2 show an exothermic effect for both sucrose and glucose interacting with 11s globulin in the aqueous medium at a concentration of low-molecular-weight sugar up to 7.5% w/v (R, = 0.7 X lo4 for sucrose and 1.7 x lo4 for glucose). A more pronounced effect was observed in the case of the sucrose. This result apparently suggests the formation of multiple hydrogen bonds between the hydroxyl groups of the lowmolecular sugars and the functional groups of the protein molecules in aqueous medium. This supposition is supported by literature data confirming the possibility of the formation of multiple hydrogen bonds between carboxyl groups on the protein and hydroxyl groups of the sucrose.6 Some literature data also suggest’** direct interaction of sucrose with proteins in aqueous solution. As a result of direct interaction of the protein with sucrose, the protein surface hydrophobic-hydrophilic balance could shift towards an increase in the

248

Effect of Neutral Carbohydrate Structure on Protein Surface Activity

0

2

4

8

6

10

',

Sugar concentration x 10 giml Figure 2

Molar enthalpy of the interactions between I ISglobulin and low-molecularweight sugars (25 "C,p H 7.0, I = 0.01 mol dm-.')

total protein surface hydrophilicity, due to sucrose layer formation around the protein molecule, and hence towards an increase in the protein thermodynamic affinity for the aqueous medium as has been observed (see Figure 1). Apparently, this change in the thermodynamic properties of the protein in the bulk aqueous solution leads to the decrease in the protein surface activity observed under the influence of the low-molecular-weight sugars at a rather low concentration in the system (R, = lo4). On the contrary, at high sucrose or glucose concentration (R, lo6), possibly the low-molecular-weight sugars are real competitors for the water molecules, and as a consequence the protein surface hydrophobic-hydrophilic balance could be shifted towards an increase in the total protein surface hydrophobicity and hence towards an increase of the protein surface activity as was observed (see Table 1). -L

3 Effect of High-Molecular-Weight Neutral Carbohydrate on Protein Surface Activity at the Planar Interface The influence of maltodextrin of high dextrose equivalent (DE=lO) and dextran I70 (7 x lo4 daltons) on the 11s globulin surface activity at the planar interface has been investigated. Table 3 shows the interfacial pressure n at the planar aidwater interface in the presence of the high-molecular-weight carbohydrates. The results show a similarity in the effects of the maltodextrin (DE 10) and dextran T70 on the protein surface activity. Unlike with the low-

249

A . S . Antipova, M . G . Semenova, and A . P. Gauthier-Jacques

Table 3

Changes of interfacialpressure IT for0.001% wlv 1lSglobulin solution under influence of high-molecular-weight carbohydrates after 10 hours at the plane air-water interface (25 “C, p H 7.0, I = 0.01 mol dm-3) Sample

11s globulin 11s globulin 11s globulin 11s globulin

+ 0.05% w/v dextran T70

d mN m -

+ 2% w/v dextran T70 + 0.05% w/v maltodextrin (DE= 10)

’

9.3 15.4 15 17.4

molecular-weight sugars, there was no concentration dependence of n with the high-molecular-weight carbohydrates over the concentration range studied (compare Tables 1 and 3). The likely reason for the observed effect of the highmolecular-weight neutral carbohydrate on the protein surface activity lies in the increase of the thermodynamic activity of the protein in the bulk aqueous The light scattering medium in the presence of the neutral polysa~charide.~’~ data obtained confirm this explanation (see Table 4). In fact, the cross second virial coefficient (Apr-pol), which characterizes the nature and strength of the protein-polysaccharide pair interactions in bulk aqueous solution, is positive for both dextran and maltodextrin. This means that forces of mutual exclusion act between the carbohydrate and the protein to cause an increase of the protein chemical potential in the bulk aqueous medium in accordance with“’

where pL(d, is the protein standard chemical potential, mL(dris the standard-state molality, mpr is the concentration of the protein (molal scale), Apr-pris the second virial coefficient characterizing protein-protein pair interactions in the is the cross second virial coefficient characbulk aqueous medium, and Apr-pol terizing protein-polysaccharide pair interactions. There is a direct correlation between the intensity of the unfavourable interactions of the protein with the ) the protein surface activity. neutral polysaccharide ( A p r - p land

Table 4

Thermodynamic parameters characterizing the pair interactions of the mixed biopolymers in aqueous solution (pH 7.0, ionic strength = 0.01 M ) System

11s globulin-dextran T-70 11s globulin-maltodextrin (DE-10)

Apr-polx 104/m”mol kg-2

6.4 11.2

250


The Role of Protein Net Charge on Protein Surface Activity It is well known that, under conditions of low ionic strength, the net biopolymer charge may play a significant role in relation to biopolymer functional properties. Let us here consider the effect of the 11s globulin net charge on the protein surface activity in the presence of dextran at low ionic strength. Figure 3 compares the effect of dextran on the 11sglobulin surface activity at different pH values of the aqueous mixed solutions, namely at pH 7.0 and pH 7.8 where the 11s globulin has net charge equal to -30 and -68 respectively (based on data from acidlbase titration). Figure 3(a) exhibits the increase of the interfacial pressure x of protein in the presence of dextran at pH 7.0. On the other hand, Figure 3(b) shows the reduction in xfor protein adsorbed with dextran at pH 7.8. To account for these results, let us consider the thermodynamic change in the pair interactions of protein + dextran in bulk aqueous solution under conditions of pH variation. Table 5 presents values of the cross second virial coefficients characterizing the nature and intensity of the 11sglobulin-dextran pair interactions. From the data obtained, it is seen that the protein surface activity is directly correlated with the character of the pair interactions with dextran in the bulk aqueous medium. So the thermodynamic forces of mutual exclusion between the protein and the polysaccharide leads to the increase in protein surface activity, whereas the presence of thermodynamically favourable interactions causes the decrease in protein surface activity. Obviously, the growth of 'the protein net charge promotes thermodynamically favourable interactions of the protein with the dextran due to the contribution of the translational entropy of counter-ions to the free energy of the system; this implies a significant increase in the mixing entropy of the system.""* Literature data suggest a significant enhancement of the miscibility of polymers with increase of charge density on the polymer molecule for the case of mixtures of an ionic polymer with a non-ionic

Effect of Neutral Carbohydrate on Protein Surface Activity in the Presence of a Lipophilic Compound The generality of the effect of a neutral high-molecular-weight carbohydrate on globular protein surface activity may be demonstrated, additionally, by the effect of amylose o n ovalbumin surface activity. Moreover, for this biopolymer pair it is interesting to follow the effect of the neutral polysaccharide on the Table 5

Effect of p H on the thermodynamic parameter of the pair interaction of the type I I S globulin-dextran at ionic strength 0.01 M

PH

I I S globulin net charge per mole

Apr-polx l @ / m 3 mol kg-2

7.0 7.83

-30

6.4 -1.0

-68

25 I

A . S . Antipova, M . G . Semenova, and A . P . Gauthier-Jacques

a)

E . z E

+

0 001% w/v I IS globulin 0 001% w/v 1 IS globulln + 2% H / V I)e\tran I 7 0

Time, hours

I

I

I

I

I

+

0.001% w/v I IS globulin 0.001% w/v 1 IS globulin + 2% w/v Dextran T250

-

-

0

1

I

2

4

I

I

1

6

8

I0

-1 I'

Time, hours Change with time of interfacial pressure n of I I S globulin adsorbed layer: (u) air-water interface under influence of dextran T70, pH 7.0, I = 0.01 mol dm-", 25 "C.(b) oil-water interface under influence of dextran T250,pH 7.8, I = 0.01 mol dm--', 25 "C

252


I0

A

0.0

0.5

1 ,0

Ovalbumin + 0.0016%w/v Amylose

1 ,s

2,o

2,s

3,O

3,s

Time. hours Figure 4

Change with time of interfacial pressure n of ovalbumin adsorbed layer at planar air-water interface under influence of amylose (25”C,p H 7.0, I = 0.05 mol dm-”)

protein surface activity in the presence of a lipophilic compound in the system. This is because amylose, like the protein, is able to interact with lipophilic compounds’”14 forming inclusion complexes. Figure 4 shows the effect of amylose on the change with time of interfacial pressure n of ovalbumin adsorbing at the planar aidwater interface. The light scattering data indicate a large positive value of the cross second virial coefficient (Apr-pOl = 13.3 x lo4 m3 mol kg-’) for this biopolymer pair in the aqueous medium. So, evidently, the thermodynamically unfavourable interactions between amylose and ovalbumin (see equation 2) in bulk aqueous solution causes the increase in the protein surface activity. Let us now consider the effect of amylose on the protein surface activity in the presence of the sodium salt of capric acid (Na-caprate). First of all, it was observed that Na-caprate is able to change the protein surface activity significantly. Table 6 shows the change of the interfacial pressure with the Na-caprate concentration in the protein solution. We see that the ovalbumin surface activity can dramatically change in the presence of Na-caprate. To explain the effect observed we have estimated the change in protein conformational stability due to Na-caprate by differential scanning calorimetry. The protein conformational changes have been used as a measure of the extent of protein interactions with the Na-caprate molecules. Figure 5(a) and (b) show the strong decrease of the thermodynamic parameters of the protein heat denaturation process, AdH and A&, with increasing Na-caprate concentration. Thus, sodium caprate strongly denatures

253

A . S . Antipova, M . G . Semenova, and A . P. Gauthier-Jacques

a) 26

I

I

I

I

-

-

14 -

-

24

. 6 30

T

16

-

12.10

-

-

8 -

6 4

0

I

1

I

1

5

10

I5

20

Na-caprate concentration. m M b)

4

o,oo

2

4

6

8

10

12

Na-caprate concentration,m M

Figure 5

Change of the thermodynamic parameters of the protein heat denaturatior process (25"C, pH 7.0, I = 0.05 mol dm-3) with concentration of Nu, caprate: (a) AnH; (b) A&,,


254

Table 6

Changes of interfacial pressure 7~ f o r 0.001 % wl v ovalbumin solution under influence of Nacaprate after 3 hours at the plane air-water interface (25 "C, p H 7.0, I = 0.05 mol dm-3) Sample

ovalbumin ovalbumin ovalbumin ovalbumin ovalbumin

+ 1.55 mM Na-caprate + 4.55 mM Na-caprate + 7 mM Na-caprate + 20 mM Na-caprate

T l m N m-' 9.9 13 13 19

20

ovalbumin due to its amphiphilic nature. The hydrophobic chains penetrate into the inner part of the protein, and the additional charged head group evidently enhances protein denaturation due to strengthening of the electrostatic repulsion. The rise of the protein surface hydrophobicity causes significant increase in the protein surface activity (see Table 6). Before considering the effect of arnylose on the protein surface activity in the presence of Na-caprate it is necessary to confirm the reliability of the method of the amylose addition to the protein solution. So, for ternary solutions, a solution of the polysaccharide of twice the concentration was added to a doubly concentrated solution of protein/Na-caprate mixture. After mixing the protein concentration in the system was equal to 0.5% w/v, Na-caprate - 7mM and amylose varied in the range (O.lYo - YO). All solutions were shaken for 45 minutes at 30 "C before being diluted by buffer (approximately 500 times) before being used for tensiometry. Under such a low final concentration, neither the amylose nor the Na-caprate was found to exhibit any surface activity . Table 7 shows the effect of amylose concentration on the protein surface activity in the presence of Na-caprate. The addition of arnylose to the protein

Table 7

Change of interfacial pressure IT f o r 0.001 YO wlv ovalbumin solution in the presence of 7mM Na-caprate under influence of amylose after 3 hours at the plane air-water interface (25 "C, p H 7.0, I = 0.05 mol d m -")

Amylose concentrationlmM 0 0.1 0.8 1

T l m N m-' 19 20 16 13.5

255

A . S. Antipova, M . G. Semenova, and A. P . Gauthier-Jacques

a)

Na-caprate concentration, m M

12

-

I

I

1

I

1

Ovalbumin, 0.8% w/v Amylose and Na-caprate Ovalbumin and Na-caprate

0

A

-

-

13-

.

03-

7

n.

$!!

-

0.q
2 mm the deposition of both types of particles was at the mass transport controlled rate. This was deduced from the observation that the radial variation of scattered light intensity (proportional to the particle flux to the surface) varied with ?I4as predicted by equation (1). This means that the deposition was not affected by repulsive electrostatic interactions. The effect of an electrostatic repulsion would be to flatten out the volcano curve and hence to reduce the deposition rate.' The observed behaviour of the protein-coated particles therefore cannot be explained by this. The results suggest that the presence of protein on a particle reduces its ability to stick to the I T 0 surface when compared to a bare particle. Deposition of polystyrene latex particles is thought to involve protruding polymer segments on a particle that are able to bridge to a surface forming bonds, even in the presence of a barrier.I7 The presence of an adsorbed layer of plactoglobulin (thickness -2 nmI8) may therefore act as a steric barrier preventing the close approach of the particle to the surface and so reducing its ability to bridge. Given the compact nature of globular proteins, the coated surface should itself be less hairy and this would also tend to reduce any bridging. The reason that less protein-covered particles stick is that the shear-rate, and hence the hydrodynamic force F H ,a particle experiences is greater as the radial distance decreases. (The radial velocity component varies as r-"14 in a walljet"). Whether or not a particle becomes deposited will depend on the balance between the hydrodynamic force and the forces acting to immobilize it. The latter will be dependent on surface characteristics of both particle and surface. For r > 2 mm the effect of FH is minimal. An increase in shear-rate also causes a reduction in the contact time between the particle and the surface. The decreased contact time may become less than the time required to form a bond. l 7

Particle Release Negligible particle release was observed on rinsing the surface with the original NaCl solution, since the scattered light intensity remained constant. However, in all cases, there was a decrease in scattered light intensity upon rinsing with distilled-deionized water or with 15 mmol dm-3 SDS. This was due to release of particles. Release of protein-coated particles was on average greater under corresponding conditions than release of bare particles. In all cases there was a notable fraction of particles which remained fixed to the surface. Results from the particle release experiments are summarized in Table 1. Release of particles can be explained by the increase in electrostatic repulsion between the particles and the ITO. This can be inferred from the electrokinetic results. Release in distilled-deionized water is consistent with the I T 0 surface being negatively charged. We have no data at present on

273

S. Joscelyne and C . Tragdrdh

Table 1

Fraction of particles released under different solution conditions Release conditions

Type of particle

0.05 mol

Distilleddeionized water

0.015 mol

0.015 mol

dm--' Na CI

dmP3 SDS

dm-3 NaCl

negligible negligible

0.12 It 0.08 0.51 f 0.14

0.23 f 0.11 0.55 f 0.04

negligible 0.05 0.03

-

Bare Protein-covered

+

whether SDS adsorbs to the ITO. However, Bos et al.19 have found that the adsorption of SDS and a cationic surfactant to an I T 0 electrode surface was very dependent on its potential. So we shall assume it is small. If the strength of the repulsion between particles and surface is proportional to the product of the respective 1) were found still to be detectable immediately upon cessation of shearing, but with an average diameter approximately one quarter of that of the original droplets (Figure 4). These results follow the prediction** that, when the viscosity ratio is extrapolated to give corresponding Capillary numbers, the gelatin-in-maltodextrin emulsion would exhibit resistance to breakup, under the shear conditions used. Therefore, the equilibrium droplet-size distribution seems to be dominated by the break-up mechanism, adding to the evidence that coalescence is hindered. The agar-in-gelatin emulsion has an extremely high viscosity ratio (vi/vc = 35). Grace22predicts that no droplet break-up occurs above a value of 4. We found that when shearing was stopped the agar droplets were unaltered both in size and appearance. There was no indication of shear induced d i ~ t o r t i o n , ~ ~ which is predicted by Walstraz3 to be due to rotation of the droplet in the shear field. Conversely, the gelatin-in-agar emulsion, which has a low viscosity ratio

T. J . Foster et al.

Figure 5

353

Micrographs showing emulsion of typegelatin-in-agar (a) immediately after cessation of shearing and (b) after 1 minute of relaxation. Bar = 100 pm

(qi/qc= 0.03), is also resistant to breakup, due to viscous dissipation of the shear energy in the continuous phase. Figure 5a shows that striations of gelatin in the agar are formed upon shearing, indicating that there is enough shear energy to distort the droplet, but not to promote break-up. When the shear is stopped, the system does then relax back to form droplets (Figure 5b). We observe, however, that many of the resulting droplets are larger than those in the freshly prepared emulsion, suggesting that shearing of the gelatin-in-agar system has induced some coalescence. Upon shearing the maltodextrin-in-gelatin emulsion ( q i / v c> 1) the droplets become too small to detect by CSLM (i.e. submicron) when viewed immediately after cessation of shearing. We cannot be sure whether the droplets are still discrete entities, or are just very small, or that the system has become miscible. With time the droplets do become visible but this cannot be explained further.

Phase Inversion It is known that in oil-water emulsions the phase continuity is determined by the phase volume, with inversion commonly occurring at 5050, but also that phase inversion can be induced at a variety of phase volumes as a consequence of an engulfment process which forms drops-within-drops when sheared.26 This process only occurs with low levels of solids at the interface. Similarly for biopolymer emulsions the phase sense is determined by phase volume.9 So, when mixtures are allowed to reach thermodynamic equilibrium, they do not exist with an included phase volume greater than ca. 0.45 (between 0.45 and 0.55 the phase sense changes from area to area within the sample). However, dispersed phase volumes greater than 0.5 can be achieved if the biopolymers in the mixture is gelled under shear.8 We have investigated whether the input of mechanical energy, in the form of shear, can cause phase inversion of biopolymer emulsions, where the two biopolymers are liquid and at an included phase volume of 0.25. This is not

354

Emulsion Behaviour of Non-Gelled Biopolymer Mixtures

uncommon in conventional emulsions, because high shear fields (with the correct cocktail of emulsifiers) are used to make very low fat spreads (20% fat) which are fat continuous (although solid fat is required to aid this process27). The evidence from this work is that, upon shearing biopolymer emulsion droplets, break-up dominates over coalescence and therefore phase inversion should not take place in the non-gelled liquid-liquid state. Increasing the collision efficiency by approaching the temperature of gelation of the dispersed phase would be expected to increase the probability of phase inversion by changing the hardness of the droplets. However, unlike oil-water emulsions, the rigidity of the droplet exists not just at the interface, but throughout its entirety. In the systems under study, this was not a problem as inversion could be induced if the continuous phase was the first gelling biopolymer.

4 Conclusions Biopolymer emulsions are governed by the same stability principles as for oilwater emulsions, and can be treated as such both theoretically and practically. A number of general statements can be made

(i) The creaminghedimentation rates follow Stokes’ Law. (ii) Coalescence rates of droplets are phase dependent, which implicates the interfacial properties of the droplets, providing a stabilization effect. (iii) When sheared, the extent of droplet break-up is determined by the ratio of the shear viscosities of the internal droplet phase and the continuous phase. The equilibrium droplet size is determined by break-up, as the nature of the droplets does not allow efficient collision for coalescence. (iv) Flocculation and phase inversion are only apparent when the dispersed phase and continuous phase, respectively, are allowed to gel under shear.

Acknowledgements The authors would like to thank Mrs Penny Knight, Mr Andrew Hilliard and Mr Alex Aldred for the technical assistance, and Dr Eddie Pelan for many interesting and helpful discussions.

References 1. P. Cairns, M. J. Miles, V. J. Morris, and G. J. Brownsey, Carbohydr. Res., 1987, 160,411. 2. V. B. Tolstoguzov, Food Hydrocolloids, 1991,4,429. 3. Y. A. Antonov, N. V. Losinkaya, V. Y. Grinberg, V. T. Dianova, and V. B. Tolstoguzov, Colloid Polym. Sci.,1979, 257, 1159. 4. V. B. Tolstoguzov and E. E. Braudo, J. Texture Stud., 1983, 14, 183. 5. A. H. Clark, R. K. Richardson, S. B. Ross-Murphy, and J. M. Stubbs, Macromolecules, 1983, 16, 1367. 6. S. Kasapis, E. R. Morris, I. T. Norton, and C. R. T. Brown, Carbohydr. Polym., 1993, 21,261.

T. J . Foster et al.

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7. A. S. Medin and J.-C. Janson, Carbohydr. Polym., 1993,22, 127. 8. C. R. T. Brown, T. J. Foster, I. T. Norton, and J. Underdown, in ‘Biopolymer Mixtures’, ed. S. E. Harding, S. E. Hill and J. R. Mitchell. Nottingham University Press, Nottingham, 1995, p. 65. 9. T. J . Foster, C. R. T. Brown, and I. T. Norton, in ‘Gums and Stabilisers for the Food Industry’, ed. G. 0. Phillips, P. A. Williams and D. J. Wedlock, IRL Press, Oxford, 1996, vol. 8, p. 297. 10. V. B. Tolstoguzov, in ‘Functional Properties of Food Macromolecules’, ed. J. R. Mitchell and D. A. Ledward, Elsevier Applied Science, London, 1986, p. 385. 11. A. Syrbe, P. B. Fernandes, F. Dannenberg, W. Bauer, and H. Klostermeyer, in ‘Food Macromolecules and Colloids’, ed. E. Dickinson and D. Lorient, Royal Society of Chemistry, Cambridge, 1995, p. 328. 12. T. J. Foster and I. T. Norton, unpublished data. 13. P. Becher, ‘EmulsionsTheory and Practice’, 2nd edn, Reinhold, New York, 1965, chap. 5. 14. E. Dickinson, ‘An Introduction to Food Colloids’, Oxford University Press, Oxford. 1992, chap. 4. 15. A. Fillery-Travis, D. Clark, and M. Robins, Food Sci. Technol. Today, 1990,4(2), 89-93. 16. G . G. Stokes, Trans. Cambridge Phil. SOC., 1851,9. 17. A. C. Hilliard, unpublished data. 18. S. Kasapis, S. Alevisopoulos, R. Abeysekera, P. Manoj, I. S. Chronakis, and M. Papageorgiou, in ‘Gums and Stabilisers for the Food Industry’, ed. G. 0. Phillips, P. A. Williams and D. J. Wedlock, IRL Press, Oxford, 1996, vol. 8, p. 195. 19. C. M. Durrani, D. A. Prystupa, A. M. Donald, and A. H. Clark, Macromolecules, 1993,26,981. 20. M. A. K. Williams, E. R. Keenan, H. Zhong, T. K. Halstead, and D. M. Goodall, submitted for publication. 21. E. Dickinson, J. G. Ma, V. J. Pinfield, and M. J. W. Povey, in ‘Food Macromolecules and Colloids’, ed. E. Dickinson and D. Lorient, Royal Society of Chemistry, Cambridge, 1995, p. 223. 22. H. P. Grace, Chem. Eng. Commun., 1982, 14,225. 23. P. Walstra, Chem. Eng. Sci., 1993,48,333. 24. W. Leo, S. R. Moore, I. Campbell, W. Morley, and P. Ayazi Shamlou, Tratzs. 1. Chem. E . , 1994,72C, 24. 25. E. Dickinson, in ‘Controlled Particle, Droplet and Bubble Formation’, ed. D. J. Wedlock, Butterworth-Heinemann, Oxford, 1994, chap. 7. 26. I. J. Campbell, I. T. Norton, and W. Morley, Neth. Milk Dairy J . , 1996, 50, 167. 27. I. J. Campbell, in ‘Food Colloids’, ed. R. D. Bee, P. Richmond and J. Mingins, Royal Society of Chemistry, Cambridge, 1989, p. 272.

Structural Properties of Heat-Set Whey Protein Gels By Marleen Verheul and Sebastianus P.F.M. Roefs NETHERLANDS INSTITUTE FOR DAIRY RESEARCH (NIZO), P.O. BOX 20, 6710 BA EDE, T H E NETHERLANDS

1 Introduction Whey proteins possess useful gelling properties which are of intercst for the food industry. The major proteins in whey are P-lactoglobulin (P-lg), alactalbumin (a-la), bovine serum albumin (BSA) and immunoglobulins (Ig). Heating induces denaturation of the proteins. As a result of the conformational changes, non-polar and reactive thiol groups are exposed and these can cause aggregation of the proteins via non-covalent interactions and covalent disulphide bonds.' This aggregation process may eventually result in the formation of a gel. Different types of gel will be formed, depending on the extent of the conformational changes, the type and kinetics of the aggregation process, and the nature of the interactions.2,' Transparent gels with a fine-stranded structure are formed under conditions of strong electrostatic repulsion, whereas they are white, opaque, and with a particulate structure, under conditions of weak electrostatic At neutral pH, where the main whey proteins are all negatively charged, fine-stranded gels are formed at low ionic strength (NaCl concentrations of 0.05 M or less) and the appearance of t h e gels changes from transparent to white (opaque) if the ionic strength is increased to 0.2 M NaCl. The changes in appearance (and other properties) of the gel are due to the formation of coarse and particulate structures. This paper introduces permeability measurements as a very sensitive technique to investigate the properties of heat-set protein gels. Permeability measurements have been done before o n casein gels,'-' but, as far as we know, they have not been reported for heat-set (whey) protein gels. We have investigated heat-set whey protein gels formed at neutral pH as a function of NaCl concentration and whey protein concentration.

356

351

M. Verheul and S.P. F. M. Roefs

2 Materials and Methods Preparation of WPI Dispersions and Gels Whey protein isolate (WPI) powder with the trade name Bipro, produced by Davisco International Inc. (USA) and purchased from Domo Food Ingredients, Beilen (The Netherlands), was used for the experiments. The powder contained approximately 89% (w/w) protein (including 70% (w/w) P-lg, 11% (w/w) a-la, 5% (w/w) Ig and 4% (w/w) BSA), 2% (w/w) ash, less than 1% lactose, and 4% (w/w) water. WPI dispersions were prepared by dissolving the powder in 0.1-3 M NaCl solutions, made with double-distilled water to total protein concentrations of 35-89 g/l. The dispersions were stirred for at least 2 hours, centrifuged for 10 min at 20,000 g and filtered using a 0.45 pm nonprotein-adsorbing filter to remove insoluble materials. The pH of the WPI dispersions was 6.9 f 0.2. WPI gels were made by heating the WPI dispersions at 68.5 "Cfor 20 hours.

Scanning Electron Microscopy The microstructure of a WPI gel containing 35.6 g/I protein and 0.4 M NaCl was studied using scanning electron microscopy (SEM). After heat treatment small pieces (approximately 1 x 1 X 1 mm) of the WPI gel were fixed with 2% (v/v) glutaraldehyde in water for 1 hour and then washed twice with water. The pieces of gel were put on a filter paper and air-dried. After 'freezing-in' using freon, they were freeze-dried. The dried samples were fractured and coated with approximately 25 nm gold in a Baltzer sputter coater. Micrographs were made with a JEOL - JEM1200EX in the scanning mode at an acceleration voltage of 60 kV.

Permeability Measurements Liquid permeation is the flow of solvent through a fixed matrix and its rate depends on the geometry, scale and spatial distribution of the percolated matrix." For a laminar flow through a homogeneous fixed matrix, the liquid flux v obeys Darcy's law. In one direction this flow is given by

-B v=-VP,

v

where v is the liquid flux (volume flow ratekross-sectional area (m s-')), B is the permeability coefficient (m2), 17 is the dynamic viscosity of the flowing liquid (Pa s) and VP is the pressure gradient over the fixed matrix (Pa m-'). Permeability measurements were made according to the method developed by van Dijk.' Gels were made in glass tubes with an inner diameter of 3.7 mm and a length of approximately 25 cm, which were open at both ends. The tubes were placed in a glass cylinder which was filled with the desired WPI dispersion.

358

Figure 1

Structural Properties of Heat-Set Whey Protein Gels

Schematic representation of apparatus used for making a permeability measurement; the reference tube is on the left, and the measuring tube is on the right. For symbols, see text

Unless otherwise stated the height of the dispersion in the tubes was about 8 cm. The cylinder was covered with a glass stopper and put in a waterbath at 68.5 "Cfor 20 hours. After heat treatment the cylinder was cooled in ice-water. The tubes were cleaned on the outside and 12 tubes together with 5 reference tubes were placed in a thermostatted (20 "C)measuring vat made of plexiglass (Figure 1). The vat was filled with the NaCl solution used for dissolving the WPI. Approximately 0.4 ml of the same NaCl solution was put on top of each WPI gel. In one series of measurements the height of NaCl solution put on top of the gel was varied. The level of the NaCl solution on top of each gel was read at regular time intervals with the help of a cathetometer. The time interval between the readings depended on the permeability of the gel matrix. The quantity V P has only one component in the flowing x-direction, and so the pressure gradient at any moment is given by

where h ( w ) and h(t)are the heights of the liquid levels in the reference tube and in the gel tube respectively, H is the height of the WPI gel and g is the gravitational acceleration (see Figure 1). The liquid flux is given by

The initial pressure gradient was -9 x lo3 Pa m-'. The liquid flux varied from

M . Verheul and S.P . F. M . Roefs

359

approximately to m s-l and was sufficiently low to ensure laminar flow." Substituting equations (2) and ( 3 ) in equation (1) (Darcy's law) and integrating from t = 0 to t = t leads to:

Bgel is the permeability coefficient of the gel (m2) and v is the kinematic viscosity of the flowing liquid (m2 s). To calculate B,,], the right-hand side of equation (4), indicated as r(t),was plotted versus the measuring time for each tube and the mean permeability coefficient of the 12 tubes measured was determined. The BgeIvalues given in the following section are mean values and the quoted errors represent the standard deviation of the mean.

3 Results and Discussion Figure 2 shows an electron micrograph of a WPI gel made in 0.4 M NaCl, containing 35.6 g/l protein. It shows a particulate gel structure. As for other particulate gel structures, the gel was macroscopically white.- The size of the building particles that can be observed in the gel is of the order of 100 nm,

Figure 2

SEM picture of 35.6 gll W P l gel in 0.4 M NaCl heated at 68.5 "C for 20 holm

360

Structural Properties of Heat-Set Whey Protein Gels

v)

1.5

E 0

1 .o

L

0.5 0.0

0

3

6 time

Figure 3

9

12

15

(lo3 s)

Determination of Bgelfrom the experimental readings at 20 "C for 44.5g / l WPI gels heated at 68.5 "Cfor 20 hours in NaCl solutions: V,0.2 M ; a,0.5 M;+,lM

which is much larger than the molecular size of the native whey proteins ( la

d=

2yWeCL VCG

* VCG

5 irreversible, 107-15 kinetics of, 109-12, 177 of polysaccharide, 297-301 of protein, 7, 11, 31, 32, 37, 116, 128-35,235, 331, 360, 362,399 reaction-controlled, 110-15, 363 reversible, 107-10, 116, 119-21, 123 simulation of, 112-9 types of, 107-126 Air cells in ice-cream, 55,61-5 Albumin, see bovine serum albumin, a-lactalbumin, erc. Algae, types of, 279-82 Alginate, gelation of, 279-93 Alginic acid gels, 283,291-2 Allegra-Hawley theory, 147, 148 40 1

402 Amide I region, 68, 72-4 Amphiphiles, see surfactants Amylopectin, 317 Amylose, 250-6 I-Anilinonaphthalene-8-sulfonate (ANS), 129, 130,394,396-7 Anisotropy under shear, 305,313,315 Artificial foods, 287 Association of emulsifiers, 45-54 of polysaccharide helices, 297-302 Associative phase separation, 303, 316, 317 Attenuation, ultrasonic, 140, 145-7, 148,150-2, 15643 Attributes, textural, 21, 31 Bacteria, 127,266, 279 Bacterial alginates, 280 Bakery products, 52 Baking creams, 285 Bancroft’s rule, 377 Bernouilli’s law, 380 Bernoullian distribution, 280 Beverages, acidified, 244 Bilayer(s), 46-9, 72-3, 188, 202, 203, 207-10 Bile salts, 46 Biopolymer mixtures, emulsion behaviour of, 346-55 Birefringence, 51 Blade homogenizer, 394,396, 398 Block copolymers, alginates as, 280 Bohlin Rheometer, 20, 32,259, 295, 336 Bond formation, time of, 272 Bovine serum albumin (BSA), 127-36, 190-2, 196, 197,258-64, 304-15, 356,357 Breaking load, gel, 290 Breaking stress, bond, 173-5 Bridging flocculation, 123,226,258-61, 350 Bridging polymer during deposition, 272 Brij35, 138 Brittleness, 168, 280, 285 I-Bromo-hexadecane, 138, 141,142 Brownian dynamics simulation, 115-18

Subject Index Brownian motion, 107 Bubbles characterization of, 150 deformation of, 380 disruption of, 367-81 formation of, see foam making stability of, see foam stability Bulk modulus, 162 Burst(ing) membrane, 383-5, 394 Butter oil, 55-61, 191 Calcium alginate gels, 282, 284-90, 292 Calcium ions, 227,236,242-3,284, 285,288, 290 Calorimetry, 78-81,130,247,256 (see also differential scanning calorimetry (DSC)) Cannon viscometer, 237 Capillary numbers, 352 Capillary pressure, 207 Capric acid, 78-90,252 Carbohydrate(s), see polysaccharides, starch, sucrose, sugars, etc. Carbohydrate structure, effect on protein surface activity, 245-57 Carbonyl stretching region, 71 Carboxymethylceilulose (CMC), 30415 Carrageenan, 292 1-carrageenan, 316-25 K-carrageenan, 236,237,240-3, 294302,319 Casein(ate)(s), 67-76, 121, 122, 15260, 192,217,229-44,25&60,261, 336-43,371 a,-/a,,-casein, 67-75, 190, 191, 196, 197,231,233,319, 321 /3-casein, 67-75, 189-91, 197,201, 217-28,231, 233, 319, 321,379 K-casein, 190, 191,236,243, 319 Casein gel(s), 112-3, 335-45, 356 Casein micelles, 57, 107, 113, 162-5, 236 Casein particles/submicelles, 112, 121, 162, 163,237,335-8, 341,343 Casein strands, 335, @ 43Cavitation, 386 Centrifugation, 56, 198,230,268,319, 347,348,357,399

Subject Index

Charge neutralization, 226,270 Cheese texture, 18 Chelating agent, 288 Chewing, perception during, 21 Chitosan, 202 Churning, 63 Chymosin, 112 a-Chymotrypsin, 229 Circular dichroism (CD), 68, 129, 130, 229,394,396 Circulation inside drop, 375 Cluster-cluster aggregation, 112-5, 123 Clustering in mixed films, 103 of protein particles, 24-7, 335-8 Coagulation, see aggregation Coalescence, 11 of air cells, 63-5 of droplets in emulsions, 121,236, 346,3504,383 during emulsionlfoam formation, 376-80,382-92,398 Coarsening of gel, 118 Coaxial cylinders, see concentric cylinder apparatus Coil-helix transition, 297, 321 Cold setting, 283, 292 Collapse point/pressure, monolayer, 93,95,97 Collision efficiency/frequency,351, 352 Colloid mill, 368, 386-8, 392 Colloidal particle, types of 107 Competitive adsorption between proteins, 67, 188,383 between proteins and emulsifiers, 55, 57-8,67,69,74, 188 Complex(es) associative, 317 inclusion, 252,256 protein+mulsifier, 333 protein-polysaccharide, 23644,25863 protein-surfactant, 68, 69-70 Complex shear modulus, 259,261-3, 332 Computer simulation, 108, 112-8, 123, 173 Concentric cylinder apparatus, 169, 170,306,321

403 Conductance, anomalous, 269 Conductivity of emulsions, 7, 12 Configurational entropy, 120 Confocal scanning laser microscopy (CSLM), 33740,347,353 Conformational stability, protein, 78-90 Conglomerates, protein particle, 24,25 Connective tissue, 29,30,31 Connectivity, chain, 220 Consumer panel, 19 Contact angle, 61 Contrast-matched water, 219 Controlled release, 285 Cooperative binding, 283,291 Correlation functions, 237 Correlation length, 116, 118,305,306 Couette geometry, 295,304-6 Cream, whipping of, 63 Cream layers, 348,350 Cream liqueurs, 59 Creaminess, 21,23 Creaming, 119, 121, 123, 13743, 145, 34650,354 (see also sedimentation) Critical gelation concentration, 115 Critical micelle concentration (CMC), 45, 188, 191,213,385 Critical overlap concentration, 304-6 Critical polymer concentration, 303 Critical Weber number, 6, 368, 369, 374 Cross-linking of alginate, 283,285 of polysaccharides, 310 of proteins, 32634 Crossed cylinders, 204,205 Crystallization in emulsions, 57,59-61, 193 of fat, 168-80 Crystals at droplet interface, 351 of emulsifier, 47 of fat, 59,61, 167-80 Cubic mesophase, 48-51,59 Cumulants, 237 Curvature, radius of, 367 Cutting, sensory perception after, 21

404 Cygnus ultrasound velocity meter, 151, 152 Cystcine, 130

Subject Index

Dimerization, protein, 133

Dimethyl(dodecy1)phosphine oxide, 208,211,212

Dimyristoylphosphatidylglyccrol Darcy’s law, 338,35740,363 Debye length, 361 Deformation of aggregates, 115 of bubblesldrops, 367 Degree of polymerization (DP), 290 Denaturation, see protein(s), denaturation Density of biopolymer phases, 347, 348 Depletion flocculation, 119-21, 137, 138, 152, 161,260 Depletion force(s), 120, 303, 310-13 Depletion stabilization, 120, 123 Dephosphorylated p-casein, 219,2214 Depolymerization, 280, 285 Deposition of bacteria, 266 of particles, 266-75 Derjaguin approximation, 205, 213 Desorption, 99, 103 Desserts, 53 Destabiliza tion of emulsions, see emulsion stability of fat, 63, 65 of monolayers, 99-103 Destructive tests, 27 Detergency, 273 Dextran, 119 Dextran sulphate, 2 5 M 4 Dextrin, 248-51 Dialysis, 192, 219, 230 Djffcrential scanning calorimetry (DSC), 56, 59-61,79, 88,90, 129, 169, 180, 193,229, 252,256, 394 Diffusion of emulsifier to interface, 384 from monolayer, 99 Diffusion coefficient particle, 269 rotational, 177 Diffusion-limited (cluster) aggregation (DLCA), 109, 112-5, 123, 172 Diffusion setting of alginate gel, 285-8, 289 Diglycerides, 45-8, 56,68,70

(DMPG), 72

Dioleoylphosphatidylcholine (DOPC), interactions of, 67-76 Disjoining pressure, 206, 377 Dispersing processes, 4 1 7 Dispersing zone, 382,387-92 Disruption of drops/bubbles, 367-82 (see also droplet break-up) Distearin, 97 Disulfide bonds/linkages, 78,82, 128, 130, 133,217,356 Distilled monoglycerides, 48-50, 53 Dithiothreitol, 328 DLVO theory, 108-9,207 Dodecyl alcohol-10-glycolether (LEOlo), 385-90 Double-layer force(s), 201, 214 Dough, 52 Droplet break-up, 351-4, 367-82, 38792 Droplet distortion, 352,353, 371, 375 Droplet size effect of emulsifying agent on, 189, 371,374,375-7 effect of energy density on, 382,38792 effect of nature of oil phase on, 189 effect of oil content on, 23&1 effect of processing conditions on, 7-9,262, 264, 352,368-70,3765, 38742,3974 effect of protein concentration on, 371 effect of shear flow on, 368-70 effect of viscosity on, 382, 387, 389, 398 influcnce on creaming/ sedimentation, 347-50 time-dependent changes in, 68 Droplet-size distribution, 7, 19, 57, 68, 138, 139, 141,230,259,261-2, 347,352, 370, 377, 387, 396-7 Drug release, 294 Dynamic light scattering (DLS), 21821,237,238, 267

Subject Index

Dynamic surface properties, 98,375-80 (see also surface diffusion, surface dilational modulus, surface dilational viscosity, surface shear viscosity) Eddies, 368, 376 Egg lecithin, 48-52,67,333 Egg white protein(s), 77, 127, 128 Egg yolk, as emulsifier, 385,386,390, 39 1 Egg yolk protein, 6,7, 11, 16 Elastic modulus, 162, 168, 282 Elasticity, monolayer, 98 Electron microscopy of ice-cream, 56,62,63 of liquid crystalline structures, 51, 52 of polysaccharide networks, 294-302 of protein gel networks, 19-20,25, 26,357,359 Electron spin resonance (ESR), 194-99 Electrophoresis, 269-70 Electrophoretic mobility, 259, 268 Electrostatic interactions in adsorbed layer, 220,261 alginate-protein, 284, 285 during emulsification, 377 effect on particle deposition/release, 272-4 in gels, 326,328, 361, 363 protein-polysaccharide, 261,309-13, 317,321 protein-protein, 307 Electrostatic screeningkhielding, 227, 261,306,309,361,363 Electrostatic stabilization, 108-9 Electrostriction, 263 Ellman’s reagent, 394 Elongational flow of casein strand, 341 during emulsion/foam making, 368, 376 Emulsification, 367-400 (see also homogenization) Emulsification machine construction, 4-5,382-92,394-5 Emulsifier(s) (see also surfactants) association of, 45-54 effect on emulsion gel rheology, 333

405 protein displacement by, 55,5743, 63,75,333 a-tending, 53, 58 Emulsifying activity (index), 190 Emulsifying efficiency, protein, 128, 134 Emulsion, definition of, 346 Emulsion behaviour of biopolymer mixtures, 346-55 Emulsion gel(s), 113, 119, 123, 152-7, 260-1,326-34 Emulsion making, 617,138,230,237, 259,351,354,367-400 Emulsion rheology, 9-11, 121, 155, 25845,326-34 Emulsion sausages, 29-42 Emulsion stability to acidification, 237-43 determination of, 7, 11-12,56,68, 13740,346 effect of adsorbed protein on, 217, 229 effect of interparticle forces on, 201 effect of polydispersity on, 123 effect of polysaccharides on, 119, 140-3,152-7,159,236-44,258-65

effects of salt on, 377 effect of emulsifier/surfactant on, 5566,69,74, 186 in flow, 11-12,58,59,351-4 by liquid crystals, 51, 58 ultrasound studies of, 136-66 Endothermic peaks, 129, 130 Energy density during emulsification, 382,387-92 Energy dissipation during emulsion/ foam making, 368 Energy input during processing, 7-13, 16,17,373,398 Engineering aspects of colloid formulation, 3-17 Entanglements, 6, 16,296,310,311, 332 Enthalpy of denaturation, 79-89,394,396 of fusion, 169, 180 Entropic contribution to mixing, 250,303 to interparticle forces, 201

406 Enzymic activity, effect of high pressure on, 127, 128 Enzymic cross-linking of protein, 32633 Enzymic hydrolysis (techniques), 112, 223,229 Epimerization, 280,282 Ethanol effect on adsorbed layers, 92-104 effect on exchange between droplets, 193 Ethylenediaminetetraacetic acid (EDTA), 288 Ethylene oxide surfactants, 21 1 Evanescent wave microscopy, 266 Exchange, kinetics of, 188, 192-4 Excluded volume, 115, 116,220,221 Expert panel, 20,31 Extractable fat, 5 6 9 , 61, 63,65 Factorial design, 19 Falling apart, sensory perception of, 21-3,24 Fat crystals aggregates of, 168-81 B-crystals, 49, 50 growth of, 168 Fat extraction technique, 56 Fat morphology in cheese, 18 Fat particles in emulsion sausages, 30,32, 37-8 in ice-cream, 55-65 Fatty acids (see aIso free fatty acids) adsorption of, 187, 195 interaction with protein, 77-90, 192, 197,198 spin probes of, 194-9 Film(s) black, 214 disjoining pressure of, 206, 214 drainage of, 351,352 elasticity of, 98-9 force between interfaces of, 206-7, 214 mixed, 92-104 rupture of, 351, 379-80 stability/structure of, 92-104 stretched, 379-80 thinning of,377

Subject Index Filtering of protein solutions, 131 Fish-eyes, 285 Flavour assessment, 31, 34, 39 Flexibility of interface, 215 of protein, 229 Flexible bonding, 115-16 Flocculation, 109-11 (see also aggregation) bridging, 123,25841,350 by caseinate particles, 121 depletion, 119-21, 137,138, 152, 161,260 effect on creaming, 137-67 effect of shear on, 261,315 of emulsions, 119, 121, 13767,207, 25845,346,351,354 of fat crystals, 170 kinetics of, l W l 1 by polysaccharide, 236, 307-10,313 of protein, 309-15 Florisil-treated oil, 393, 394 Flory-Huggins parameters, 220, 227 Flour, 52 Flow-induced structuring, 3 Ffuoresence measurements, 130, 347, 394,396 Foam lamella, 206 Foam making, 4-5,16, 129,367-81 Foam stability, 64-5, 133-5, 259 Foam structure, 65 Foaming of protein(s), 78, 128, 133-5 Forces between lipid-coated interfaces, 201-16 Form factor, 175 Fouling deposits, 266 Fourier transform infrared (FTIR), 6875,229 Fourier transform ultrasonic technique, 137, 139-40, 159, 161 Fractal aggregates/clusters/flocs, 109, 112-5, 172430,336,340, 362-3 Fractal dimension(ality), 112-8, 17280,336,338,362 Fractal structure, 112-23, 172, 335, 362-3 Fracture of strands in gel, 34&3 Fracture force, 335, 341,343 Fracture properties, 23-4, 341

Subject Index Fracture stresdstrain, 32, 35, 36, 3841,341,342 Fracture test, 20 Free fatty acids, 51 Freezing, 51, 56 Fringes of equal chromatic order (FECO) ,204,205 Fruit juices, 127 Functional properties, effect of high pressure on, 127-35 Galactomannan mixed systems, 294302 Gap geometries of dispersing machines, 4-17 Gas bubbles, see bubbles Gas-liquid transition, 119 Gauche conformation of ethylene oxide chain, 211 of phospholipid, 71-2 Gel(s) ageing of, 285,322,340-3 alginate, 280, 283-92 carrageenan, 321-3 casein, 112-3,3354,356 chemical, 331,332 cold setting, 283,292 emulsion, 113, 123, 152-7,260-1, 326-34 fat crystal, 170 flow through, 338,357-9 fracture of, 23-4, 341 gelatin, 321-3 heat-set, 18-28,32&-33,35&64 melting of, 321-3 milk, 335 mixed, 27,284,285,321-3 particle/particulate, 18-28, 112-23, 332,335-45,359,362,363 permeability of, 336,338,340 phase-separated(-ing), 116, 336 physical, 331 polymer, 331, 332 polysaccharide, 279-302 protein, 18-42, 116,32645,35644 rearrangements in, 113, 115-19,335 -45 reinforcement of, 327, 329 swelling of, 288 thermoreversible, 292

407 weak, 152, 162,263,285 whey protein, 356-64 Gel (permeation) chromatography, 16, 68, 129,131,318 (a-)Gel phase, 47-53,58,59,202,203, 207-10 Gel(ation) point, 113 Gel-sol transition, see sol-gel transition Gel strength, 280,290,291 Gelatin, 292,316-25, 347-53 Gelation of alginate, 279-93 of alginic acid, 283,291-2 of egg white, 78, 128 of gelatin, 351 kinetics of, 285-8 of meat protein, 29-30,38 of milk, 164 of mixed biopolymers, 27,284,285, 294-302 under shear, 353 of whey protein, 326 Gibbs adsorption isotherm, 99 Gibbs elasticity, 378 Gibbs-Marangoni effectlmechanism, 377-80 Glass membrane, see membrane, micro-porous glass Globular protein(s), surface activity of, 245-57,378-9 11s Globulin, 77-90,245-51 D-Glucono-d-lactone (GDL), 284, 288-91,335,336 Gluco-mannan, 300 Glucose, effect on protein surface activity, 245-8 ,L?-1,4-Glycans,299 Glycerol lactopalmitate, 58 Glycerol monopalmitate, 48,56 Glycolipids, 45,46, 52 Gouy-Chapman theory, 269 Grainy/gritty texture, 21, 23, 27 Groundnut oil, 121, 122 Guinier analysis, 222, 224 (a-L-)Guluronic acid, 279,280,289-92 Hairy particles, 269 Hamaker constant, 175 Heat capacity, protein, 79-89

408 Heat (induced) denaturation, see thermal denaturation Heat-set gels, 18-28, 326-33,35644 Hedonic test, 7 a-Helical content, 129, 130,396 Helices K-carrageenan, 298-301 gelatin, 322-4 xanthan, 296,297 Helix-coil transition, 297, 321, 323 n-Hexadecane, 192, 193 Hexagonal mesophase, 48-51 High-pressure homogenizer(s), 369, 370, 374, 382, 386-92, 394-7 High-pressure processing, 127-36, 258, 259 Hildebrandt equation, 169, 180 High-methoxyl pectin, 236 Hofmeister ion series, 187 Homogenization, 56,122, 128, 198,230, 238,259, 369-70,386-92, 394-7 Hooke’s law, 205 Hydration force, 47,209-15, 273 Hydrodynamic diameterlradius. 221, 226,237,239-43 Hydrodynamic forcehnteraction, 174, 272 Hydrodynamic layer thickness, 218-27 Hydrogen bonding, 128, 186-7, 191, 210, 247, 326,328 Hydrogenated palm oil, 169 Hydrolysis, alginatc, 285 Hydrophilic-lipophilic balance (HLB), 188 Hydrophobic-hydrophilic balance, protein, 247,248,256 Hydrophobic interaction(s), 71, 128, 213,254, 326, 328 Hydrophobicity, protein, 79-82, 130, 133, 190,227,229,233, 396 Hydrophobicized mica surface(s), 212, 213 Hydroxyethyl cellulose (HEC), 119, 138, 1 4 1 4 , 146, 304-15 Hyperbolic flow, 368,369 Hyperfine structure, 194 Ice-cream ernulsion/(pre)mix, 53, 5565,67, 198

Subject Index

Ideal mixing, 169 Image analysis, 20, 32 Immiscibility of mixed films, 93-5 Immobilization technique, 287 Immunoglobulins, 356,357 Inclusion complex, 252,256 Incompatibility, see thermodynamic incompatibility Inertial forces, 368,370, 374, 376 Infrared spectra, 68-75 Instron, 32 Insulin, 190,201 Interactions alginate-ion, 283 alginate-protein, 285 associative, 316,318, 319, 323-4 biopolymer, 346 bubble-bubble, 377 casein-carrageenan, 236, 240-3, 319 casein-pectin, 236-40 cluster-cluster, 113-5 droplet-droplet, 123,201, 377 of emulsifiers/surfactants, 45-53, 92104 film-sub-phase, 98 in galactomannan + K-carrageenan mixtures, 299 in galactomannan xanthan mixtures, 299 in gelatin + L-carrageenan mixtures, 3 16-25 globulin-dextran, 250 hetero-polymer, 300, 301 of lipid-coated surfaces, 201-16 ovalbumin-amylose, 252 of particles, 107-126, 148, 173, 336, 343,351 particle-polymer, 120 particle-surface, 272 polymer-polymer, 120,260 of polysaccharides in emulsions, 23644 polysaccharide-polysaccharide, 297, 299 protein-lipid, 67-76,77, 185-200, 229,252-6 protein-polysaccharide, 23644,245, 249,250,258-65,285,309-13, 316-25

+

409

Subject Index Interactions (Continued) protein-protein, 81,90,249,307, 310,335 protein-surface, 190-1 protein-surfactant, 67,69-70 segment-segment, 220 segment-surface, 220,221 segregative, 316, 318 time-dependent, 110-11 Interfacial complexation, 67, 69-70, 75, 237,239,259,260 Interfacial instability, 380 Interfacial pressure, 78-90,245-54 Interfacial shear viscosity, see surface shear viscosity Interfacial tension during emulsification/foaming, 371, 373-5,398 gradients of, 372-80 measurement of, 3834,393-6 in water-in-water emulsions, 352 Interferometric surface force technique, 201,2034,212 Internal setting of alginate gel, 285, 288-90 Interparticle potential(s), 108-11, 112, 123, 173,303,351 Interpenetration of aggregates, 113, 115 Intrinsic viscosity, 305 Inversion, emulsion, 11 Ion binding to alginate, 283,290 to K-carrageenan, 298 Ion-exchange chromatography, 218 Ion pairs, 128 Ionic cross-linking, 283, 285 Isoelectric focusing (IEF), 129, 131 Isoelectric point (pl), 18,29, 191,23643,307,317 Isotropic turbulence, 368, 376 Jet homogenizer, 259 Juiciness, 31, 34, 39 Junction zones in gelation, 290 Kolmogorov theory, 368 Kozeny-Carman equation, 363 Krafft pointltemperature, 47,49,50 Krieger-Dougherty relation, 174

a-Lactalbumin, 237,356,357 Lactic acid bacteria, 127 Lactic acid esters of monoglycerides, 53 ,%Lactoglobulin, 18-27,70,107, 12736, 189-91, 197,237,25840,26674,32633,356,357 Lactylated monoglyceride, 58 Lag phase, 111 Lamella, forces in, 206 Lamellar mesophase, 47-51,59,202, 203,207-10 Laminar flow, 368,369,388 Langmuir trough technique, 92,93 Laplace pressure, 367,369,375 Latex particles deposition of, 26675 flocculation of, 119 protein adsorbed on, 219,221,226 Lattice model(s), 123, 218, 219-20,227 Lauda balance, 93 Lecithin(s), 45,48-51,56,61,67-76, 189,333 Leguminous plants, 77 Lennard-Jones particles, 118 Light microscopy, use of, 7, 19-24,3& 3,52, 121, 122, 138, 139,206-7, 267-9,347 Light scattering, 16, 56, 78, 81.90, 168, 170, 175-7, 180, 189, 198,218-21, 229,237,249,252,267-74,316 analogy with ultrasound scattering, 160-1 Line widths, NMR, 230,234-5 Linear (viscoelastic) region, 170, 295, 32 1 Linoleate-bound ovalbumin, 190 Lipid(s), adsorption of, 186-8 Lipid-coated interfaces, 201-16 Lipid-protein interactions, see proteinlipid interactions Liposome(s), 50,73 Liquid crystalline phases of lipids, 4553

forces in, 213,214 at oil-water interface, 51, 52 Liquid-like order, 116,309-15 Liquid-liquid phase separation, 119, 303,304 Locust bean gum (LBG). 294-302

410 Longitudinal wave, 379 Loop-train-tail model, 218 Low-fat cakes, 51, 52 Low-fat foods, 51, 53 Low-fat mayonnaise, 6 Low-fat spreads, 354 Lowry method, 268,393 Lyotropic mesomorphism, 45-9 Lysine residues, 326 Lysolecithin(s), 45, 72 Lysozyme, 201,378,379 Maltodextrin, 248,249, 317,347-53 Mannoselgalactose ratio, 295-301 @-D-)Mannuronic acid, 279,280,292 Marangoni effect, 99,373,377,379 Marine algae, 279 Markov approximation, 220 Markovian statistics, 280 Mass fractals, 112 Mastersizer, 7,56, 68,69, 138-43, 148, 198,259,387 Mastication, 18, 21 Mayonnaise, 3 , 6 , 7,52 Mean field approach, 173 Meat fibres, 29,30 Meat protein functionality, 29-41 Mechanical spectroscopy, see oscillatory rheology Melting of fat crystals, 180 Melting resistance of ice-cream, 55, 635 Membrane(s) biological, 185, 188, 194, 201 burst(ing), 383-5, 394 micro-porous glass, 393-99 Mesh size, 306, 307, 311 Mesophases of lipid-water systems, 4553 Metmyoglobin, 128 Mica sandwich technique, 295,298 Mica sheetslsurfaces, 204, 205, 212, 213,295 Micelles, 45-8, 119, 161-3, 188 Microfluidizer, 68, 238,386, 389-92 Microorganisms, see bacteria Micro-phase separation, 307-10 Micro-porous glass (MPG) method of emulsion preparation, 393-400

Subject Index Microscopy, see CSLM, electron microscopy, light microscopy, Microstructure, see electron microscopy, light microscopy, structure Milk, ultrasound studies of, 162, 164 Milk dialysate, surface activity of, 192 Milk gels, 335,340 Milk powder, 198, 199 Milk processing, 127 Milk protein emulsions aerated, 5 5 4 6 monodisperse, 123 Milk protein gel, 326 Mineral oil, 153 Miscibility of mixed monolayers, 91, 92-7, 103 Miscibility of polymers, 250 Mixed biopolymer systems, 27, 34655 Mixed films, monoglyceride, 92-104 Mobility in adsorbed protein layer, 232, 233 of amphiphilic molecules, 188 electrophoretic, 259, 268 Mode 11in g of adsorbed layer, 217-28 of aggregation, 107-23, 148 of creaming, 123, 137,152 of gelation, 112-23 Molecular weight of polysaccharide(s), 280,285,287, 305, 318 of protein(s), 81, 131, 133, 318, 394, 396,399 I-Mono-decanoyl-glycerol, 80-3 Monodisperse colloid(s)/emulsjons, 116, 123, 148 Monoglyceride(s) (see also glycerol monopalmitate, etc.) adsorption of, 187 complexes of, 68,69-70 forces between layers of, 207-1 1 in ice-cream, 56-65 interaction with protein, 77-90, 190 mesophases of, 45-53, 58-9 mixed films of, 92-104 Monolayer(s), 45, 59, 92-104, 188,225, 298,299 Monoolein, 48,92-104,208,209

Subject Index Monopalmitin, 48,49,207-9,212-14 1-Mono-palmitoyl-glycerol, 88 Monostearin, 92-104 Mouthfeel, 7,21 Multilayers, 58,65, 191 Multiple scattering, 137, 140, 147 Multivariate regression, 19, 24,25,37 Myelin basic protein, 72 Myofibrillar proteins, 29 Myosin, 29,30 Net test, 31 Network(s) of aggregated particles/droplets, 11223,143-5,32633,351 of casein, 341 coupled, 324,346 of fat crystals, 168-80 of globular protein, 18,326-33 interpenetrating, 324, 346 of meat protein, 29,30,32 of mixed biopolymers, 323-4,346 of polysaccharide, 294-302 of starch, 11, 13, 16 Networking ability, macromolecular, 16 Neutron reflectivity, 218-27 Neutron scattering, 303-15 Newton black film, 214 Nitrogen gas, sparging with, 129, 134, 135 Nitroxide moiety, 194-7 Non-destructive tests, 27, 150 Non-invasive probe, 137 Nuclear magnetic resonance (NMR), 229-35,280 Nucleation fat crystal, 168 in gelation, 290 in monolayer(s), 93, 103, 104 n-Octadecane, 193 Octyl-B-glucoside, 208,213, 214 Oiling-off, 168 Oligomeric proteins, 128 Olive oil, 177, 178 Opalescence, 160 Optical microscopy, see light microscopy

411 Orthokinetic stability, 56,58,65 Oscillatory rheology bulk, 9-16,20,26-7,32, 170,259-63, 288,289,295,297-300,327-33, 336,340-2 surface, 99 Osmotic compressibility, 303 Osmotic pressure, 119, 120,202 Osmotic stress technique, 202-3, 207, 212,213 Ovalbumin, 77-8,82-90, 128, 190,250, 252-6 Overflowing cylinder apparatus, 375 Overprocesssing of emulsions, 12, 16 Overrun, 56,63 Overwhipping, 63 Packed bed, 350 Palm (kernel) oil, 169 Palmitic acid, 77, 86-90 monoglyceride of, 49,86,88 Paraffin oil, 45 Paramagnetic species, 194, 198 cis-Parinaric acid (CPA), 394, 396 Particle deposition, 266-75 Particle interactions, 108-1 1, 148, 263 Particle packing, 116 Particle-size distribution, see dropletsize distribution Particulate gels, see gels, particle/ particulate Partitioning, protein, 311 Pasteurization, 51, 56, 127 Peanut butter, 19 Pectin, 236-40.243-4 Peptide bonds, 227 Percolating network, 115-21,351 Periodic boundary conditions, 115 Permeability (coefficient), 336, 338. 340,356-63 Permeation pressure, 399 Persistence length, 305,306 Phase diagrams of emulsifier-water systems, 49-51 of mixed polymer systems, 316-8. 348 Phase angle, 32.35.36.297.299 Phase inversion, 11,346,353-4

412 Phase separation during aggregatiodgelation, 118, 336 induced by gravity, 123, 141-3,348 liquid-liquid, 119, 303,304 in mixed biopolymer systems, 316-7, 347 in protein-polysaccharide mixture, 304,307-10,324 Phase transition, monoglyceride, 59,210 Phosphatidylcholine, 49,67-75,210 Phosphatidylethanolamine, 210,211 Phospholipids, 45-52, 67-76, 190,20711,229 (see also lecithin(s)) Phosphoproteins, 227 Phosphorylation, 218 Phosphoserines in casein, 220,231, 233,235 Pickering stabilization, 61 Plant proteins, 77 Polarimetry, 322, 323 Polarizing microscopy, 48, 52 Polydispersity, 123,267,273 Polydispersity index, 280 Polyethyleneglycol (PEG), 387, 388 Polyglycerol esters, 51 Polyguluronic acid (poly-guluronate), 28&3 Polymannuronic acid (polymannuronate), 280-3,291 Polymer-depleted layer, 120, 351 Polymorphs, triglyceride, 169, 174-9 Polysaccharide(s), see carrageenan, chitosan, dextran, pectin, starch, etc. Polysaccharide networks, 295-302 Polysorbates, see Tween(s) Polystyrene latex, see latex particles Pores flow through, 168,338,361 membrane, 394,397,398 Pore-size distribution, 19-27, 116, 361, 362 Porosity, 25 Potassium ions, effect on K-carrageenan gels, 298 Potential of mean force, 108 Power density, 368, 369, 379,387,392 Power input during processing, 7, 13-17 Power number, 5

Subject Index Precipitation alginate, 283, 291 complexes, 318, 324 protein, 116, 128 Preference rating, 19 Premix emulsions, ice-cream, 56-61 Pressure fluctuations during emulsion/ foam making, 368,380 Pressure gradient(s), 338, 343, 357-63 Pressure transducer, 206 Primary maximum/minimum, 109, 111 Primary structure, 219 Principal component analysis, 19, 21, 33,38-9 Processing, high pressure, 127-36,25844 influence on structure-rheologytexture relationships, 3-17 thermal, 25843, 326-33 Prolate ellipsoid, 307 Proline turns, 227 Propylene glycol monostearate, 53 Protein(s) amphiphilicity of,190-2, 218, 247-8 coagulation of, 7, 11 conformation stability of, 252, 256 denaturation of, 16,29,79-90, 12733,252-5,326,327,333, 356,362, 393,394,397 displacement of, 55-9, 194,333 in emulsionlfoam formation, 133-5, 371,374-5, 378-9, 393-9 high pressure processing of, 126-135 polymerization of, 326 solubility of, 29, 188, 191-2, 397, 399 subunits of, 128 surface activity of, 77-91, 186-91, 24557,398 surface diffusion of, 379 Protein adsorbed layer(s), see adsorbed protein layer(s) Protein gels, 18-42, 116,32645,35664 Protein-lipid interactions, 67-91, 185200,229,252-6 Protein load, 56, 189, 192 Protein-plysaccharide interactions, 236-44,245,249,250,258-65,285, 309-13,31625

Subject Index

Protein-polysaccharide mixtures, 24865,303-25, 346-55 Protein-protein bonds, 326, 335,340-1 Protein-surfactant interactions, 67,69-70 Proteinase, 229 Proteoheparan sulphate, 201 Protrusion forces, 209-15 Prout-Tompkins equation, 93, 100, 102 Radial distribution function, 116 Radius of gyration, 120 Raman spectroscopy, 68 Random close packing, 174 Random coil (structure), 256,296,298, 379 Random walks, 112 Reaction-limited (cluster) aggregation (RLCA), 110, 112-5, 123.363 Rearrangements in gels, 113, 115-19, 335-45 Recoalescence during emulsion/foam formation, 372,390,391 avoidance/prevention of, 376-81, 387-8 Reducing agent, 328-9 Relaxation, monolayer, 93,99, 100, 103-4 Relaxation times, NMR, 230, 234 Release of deposited particles, 266-75 Rennet (casein) gel(s), 164-5,335, 344 Repulsive forces between bilayers, 48 Restructuring of aggregate(s)/gel(s), 113, 118 of foods, 287 Reversed hexagonal phase, 51 Reynolds number, 5,368,369 Rheology (see also viscosity) of concentrated colloids, 123 of emulsions, 9-11, 121, 155,258-65, 326-34 of emulsion gels, 326-34 of fat crystal dispersions, 168-80 of globular protein gels, 19, 20, 23, 26-7 influence of processing on, 3-17 of polysaccharide networks, 294-302 of protein gels, 326-34 of protein + polysaccharide mixtures, 304,305,311,312,317,321-4

413 relationship to creaming, 121, 155 relationship to structurehexture, 317 of sausages, 35-6 of starch suspensions, 12-16 from ultrasound measurements, 1626 Rheometrics rheometer, 67,305,321 Rigidity modulus, 162 Rotational correlation time, 195 Rotational diffusion coefficient, 177 Rotor-stator system(s)/unit(s), 4-5, 7, 16,382,386,388-91 Rubber, natural, 119 Rubberiness, 34, 39-41 Salad dressing, 13, 52 Salting-out effect, 362 Saponins, 46 Sarcoplasmic proteins, 29 Sausage batter, 30-2 Sausages, emulsion, 29-42 Scanning electron microscopy (SEM), 19-20,25,26, 56,62,63, 357, 359, 362 Scattering, see light scattering, neutron scattering, ultrasound scattering Scheludko interferometric equation, 207 Scheutjens-Fleer theory, 218,219 Screening, see electrostatic screening Second-derivative (infrared) spectra, 71-4

Second virial coefficient, 81, 246-52 Secondary layer, 225 Secondary minimum, 109, 111 Secondary structure, 68,72-4,75,217, 396 Sediment layer, 350 Sedimentation, 123,347-50,354 (see also creaming) Segment density profile(s), 219-27 Segregative phase separation, 303, 316, 317,324 Self-assembled structures, 45-9, 188, 297-301 Self-consistent-field modelling, 21728 Self-similarity, 112

414 Semi-dilute solution, 305-7,311 Sensory analysislperception (see also texture) of emulsion sausages, 2 9 4 2 of mayonnaise, 7, 12, 13 of particulate gels, 18-28 Serum proteins, see whey proteins Serum separation, 152-9, 335, 348 (see also creaming) Sharp(ed) edge valve, 389-91 B-Sheet(s), 72-5 Shear (flow) bubble/droplet disruption in, 368-81 effect on emulsion stability, 11-12, 56,3514 effect on scattering behaviour, 304, 3114 Shear plane, 269 Shelf-life, 168, 236 Shielding of charge, see electrostatic screening Short-range order peaks, 307-10 Signal processing, 140 Silica particles, 119 Sintered fat crystals, 170 Size-exclusion chromatography, 394, 399 Skim(med) milk powder, 55,57 Small-angle neutron scattering (SANS), 303-15 Smoluchowski equation, 268 Smoothness, 19 Soap-water mesophases, 45 Sodium azide, 138,237, 295 Sodium caprate, 78-89,252-6 Sodium carboxymethyl cellulose, 119 Sodium caseinate, see casein(ate)(s) Sodium dodecyl sulphate (SDS), 67, 70,26674,372,377-9,394 SDS-PAGE, 69 Sodium palmitate, 84-9 Sodium salt of fatty acids, 77-90 Sodium stearate, 48 Sodium stearoyl-2-lactylate (SSL), 50-1 Sol-gel transition, 288, 321 (see also gelation) Solid fat contentlfraction, 56, 57, 150, 168, 180 Solvation layers, 209

Subject Index

Solvent extraction technique, 56 Solubility, protein, 29, 188, 191-2, 397, 399 Sonication, 69,72,78, 139 Sorbitan ester(s), see Tween(s) Soy(a)(bean) lecithin, 56,58,67,333 (see also lecithin(s)) Soya (soybean) oil, 52,68,189,238-42, 333,393,394 Soy(bean) protein, 128,371 Sparging, 129, 134, 135 Specific heat (capacity), 79, 140 Specific (droplet) surface area, 56, 193, 230,231,371 Specular neutron reflectance, 218 Spin-echo method, 230 Spin-lattice relaxation time, 230 Spin probes, 194-8 Spinodal decomposition, 118 Spoon test, 12, 13 Spoonability, 7 Spray-dried emulsions, 58 Spray-dried WPC, 385 Spread monolayers, 92-104 Spreadability, 19, 168 Spreading of proteins, 379 Stability of aerated emulsions, 55-66 conformational, 79-90 of emulsions, see emulsion stability of monoglyceride mixed films, 92104 orthokinetic, 56, 58, 65 Star volume, 20-3 Starch emulsifier interaction with, 51,53 as stabilizer, 7, 8, 11 suspensions of, 12-16 waxy maize, 6 Statistical mechanics, 108 Stearic acid monoglyceride of, 49 spin probes of, 194-7 Stearoyl-lactylates, 46 Steric barrier to deposition, 272 Steric forces, 201, 209-15,304 Steric hindrance, 350,352 Steric stabilization, 108, 109, 120, 217, 260,272,274,350

Subject Index Sterilization, 51, 127 Sticky spheres, 123 Sticky texture, 21 Stokes' Law, 347,354 Stokes radii, 237 Stokes velocities, 139 Storage/loss moduli, see oscillatory rheology Strand characteristics, 20, 24-7, 2 9 6 302,340,343 Stress carrying chaindstrands, 177-80 Stress overshoot, 170 Stresses during emulsion/foam processing, 5 4 Stretching bands, infrared, 68,70-2 Structure of adsorbed layer(s), 185-99.217-28 of aggregated systems, 107-126, 150, 175-80 of alginate gel, 287-90 of alginate molecule, 280-2 effect of polydispersity on, 123 effect of processing on, 3-17 effect on sensory perception, 18-42 effect of shearing on, 351-4 of emulsifier mesophases, 45-53 of emulsion gels, 327,329,351 of fat crystal aggregates, 168-81 of ice-cream, 55,56,61-3 of monoglyceride mixed films, 92104 of polysaccharide gels, 285,294-302 of polysaccharides in solution, 305, 306 of protein(s) in solution, 68-75, 12833 of protein gels, 18-42, 112-3,356-64 of protein-polysaccharide mixtures, 303-15 rearrangement of, 110, 111 relationship to function, 279 relationship to particle interactions, 107-26,356 relationship to rheologyltexture, 342 relationship to water-holding, 29-42 supermolecular, 296301 Structure factor, 175 Structuring agents, 53

415 Structuring by flow, 3 Sucrose, effect on protein properties, 57,2454 Sucrose esters, 70 Sugars, effect on protein surface activity, 2454,256 Sugar (based) surfactant, 213,214 Sulfhydryl (SH)groups, 128, 130,3937 Sunflower oil, 169 Supersaturation, 169-80 Surface activity, 77-91,185-200,24557,396 (see also adsorption, surface tension) Surface coverage of deposited particles, 270 in emulsions, 57-8 on latex particles, 268, 273 at macroscopic interface, 222-5 Surface diffusion, 379 Surface dilational modulus, 373,375 Surface dilational viscosity, 3 7 5 4 Surface excess concentration, 187 Surface force experiments, 108,201-15 Surface fouling, 266 Surface free energy, 374 Surface hydrophilicity, 248,256 Surface hydrophobicity, 82,84, 88. 129,130-5, 190, 197,248,254, 256,394,397 Surface pressure, 92-103,245,378,379 Surface shear viscosity, 259 Surface tension (see also adsorption, interfacial tension, surface activity) effect on droplet/bubble break-up, 6 of stretched film, 380 of water, 187 Surfactants adsorption of, 1 8 6 8 , 3 8 5 4 displacement of protein by, 55-9,67, 194 effect on emulsion stability, 55-66, 119 monolayers of, 45,46 role during emulsion/foam formation, 370-81 Swelling of alginate gels, 288 of lipids in water, 45-51.53.202-3

416 Swelling pressure, 202,203,209-12 Syneresis, 285,289,291,294,335-44 Synergistic polysaccharide networks, 294-302 T-test, 33 Tail-train-loop model, 218 Tartaric acid esters, 51 Tensile testing, 19,20, 31-2, 35,3841, 342 Tensiometry, 78, 88,254, 383 n-Tetradecane, 154,158,160, 189, 192, 258,327-33 Texture of cheese, 18 of emulsion sausages, 29-42 of heat-set protein gels, 18-28 of mayonnaise, 7, 12, 13 of meat products, 30 of peanut butter, 19 relationship to structurehheology, 342 types of, 21,346 Thermal conductivity, 140 Thermal denaturation, 190,252-6,326, 331,333,356,394,396 Thermal processing, 25&63,32633 Thermodynamic incompatibility, 303, 324,346 Thermodynamics of adsorption, 186-8 of biopolymer solution, 246-9 of colloidal aggregation, 119 of crystallization, 169 of mutual exclusion, 249,250 of oil exchange between droplets, 193 of protein association, 81,90 of protein denaturation, 79-90 in study of food colloids, 185 Thermosetting systems, 292 Thermotropic mesomorphism, 46 Thickener, 279 Thickness, impression of, 12, 13 Thin film balance, 2067,214 Tie-lines, 347, 348 Time-scales during emulsification/ foaming, 372, 375 Tip streaming, 375

Subject Index Tooth disc dispersing machine(s), 389, 39 1,392 Toppings, 53, 58 Train-loop-tail model, 218 trans conformation of ethylene oxide chain, 211 of phospholipid, 71-2 Transducers, ultrasonic, 13940, 151, 152 Transglutaminase (TGase), 326-34 Transmission electron microscopy (TEM), 19,20 Tricaprin, 78-83 Triglycerides, properties of, 45-9, 7890 Triglyceride-water interface, 189, 192, 333 Triolein (trioleoylglycerol), 50, 192, 196, 198,229,234 Tristearate, 177, 178 Trypsin, 229 Turbidity, 318, 324,372 Turbulent flow, 5-6, 368,369,374, 376, 3869,393 P-Turns, 73 Tween(s), 46,48,51, 56, 57,67, 152, 153, 161-3,189,198, 199,333, 394,396 Tyrosinase, 128 Ultrasonic attenuation, 140, 145-52, 15643 Ultrasonic homogenizer, 230 Ultrasonic relaxation, 162 Ultrasonic spectroscopy, 137, 148 Ultrasonication, see sonication Ultrasonics, characterization of aggregation/floccuIation by, 13767 Ultrasound profiler, 151, 156 Ultrasound scattering, 137, 140, 147, 151, 159,161 analogy with light scattering, 160-1 Ultrasound velocity scanning, 138, 151 Ultra-turrax (UT), 370 Underprocessing of emulsions, 12, 16 Unfolding, protein, 81, 84, 90, 128 Urick equation, 151, 161

Subject Index

Van der Waals attraction between bilayers, 48, 53 between fat crystals, 168,170 between particle and surface, 273 between protein molecules, 307 between spherical particles/droplets, 1745,207 between surfactant-coated surfaces, 212,213 in thin film, 214 Vegetable oil, 385-93 Vegetables, 127 Velocity dispersion, ultrasound, 145-7, 148 Vesicles, 50, 51 Vibrational spectra, 70-4 Video, use of, 139,143,206,207 Viscoelasticity, see rheology Viscosity (see also rheology) dynamic, 357 effect on creamingkedimentation, 347-50 effect on emulsion droplet size, 382, 387,389,398 of fat crystal dispersions, 168-5 intrinsic, 305 kinematic, 359 of polysaccharide solutions, 237 of proteidpolysaccharide solutions, 260,311,312 of starch suspensions, 13-16 Voluminosity, 338 Wall-jet cellkystem, 266-9, 272 Waring blender, 138 Water activity, 202,203 Water binding to adsorbed protein, 227 Water-holding of emulsion sausages, 2942 of whey protein gels, 361

417 Water-in-water emulsions, 346, 349, 350 Water release from newly cut surface, 21 Water-soluble polymers, flocculation by, 119 Water structure, 247 Wave vector, 1754,306,307 Weber number, 6,368,369 Weissenberg number, 12 Wetta bili ty , 267 Wheat lipids, 52 Whey protein(s), see bovine serum albumin (BSA), a-lactalbumin, P-lactoglobulin Whey protein concentrate (WPC), 128, 133,198,199,385,393-9 Whey protein gels, 18-28, 326, 35664 Whey protein isolate (WPI), 6, 193, 194,237,240-3,326,357-64 Whippable emulsions, 53, 58 Whipping properties, 78 (see also foam making) Wilhelmy plate, 93, 383-5,394 X-ray crystallography/diffraction, 45-8, 72,202,203 X-ray scattering, 227,301 Xanthan (gum), 119, 152-7, 159,294-7, 301,302 Yielding behaviour, 170,340, 341 Yoghurts, set, 335 Young’s modulus, 291, 292 Zeta potential, 268-70 Zetasizer, 268

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