Gums and Stabilisers for the Food Industry 9 (Pt. 9)

Gums and Stabilisers for the Food Industry 9

Gums and Stabilisers for the Food Industry 9

Edited by Peter A. Williams North East Wales Institute, Wrexhum, UK

Glyn 0.Phillips Newtech Innovation Centre, Wrexham, UK

THE ROYAL CHEMISTRY Information Services

The proceedings of the Ninth Gums and Stabilisers for the Food Industry Conference-Functional Aspects held at Plas Coch College, The North East Wales Institute, W ~ x h a mon 7-1 1 July 1997.

The front cover illustration is taken from the contribution by V.J. Moms, p. 361

Special Publication No. 2 I8 ISBN 0-85404-708-5 A catalogue record for this book is available from the British Library 0 The Royal Society of Chemistry 1998

All rights reserved. Apart from any fair dealing for the purpose of research or private study, or criticism or review as permitted under the terms of the UK Copyright, Designs and Patents Act, 1988, this publication may not be reproduced, stored or transmitted, in any form or by any means, without the prior permission in writing of The Royal Society of Chemistry, or in the case of reprographic reproduction only in accordance with the terms of the licences issued by the Copyright Licensing Agency in the UK, or in accordance with the terms of the licences issued by the appropriate Reproduction Rights Organization outside the UK. Enquiries concerning reproduction outside the terms stated here should be sent to The Royal Society of Chemistry at the address printed on this page. Published by The Royal Society of Chemistry, Thomas Graham House, Science Park,Milton Road, Cambridge CB4 4WF, UK For further information see our web site at www.rsc.org Printed and bound by Bookcraft (Bath) Ltd

Preface Once again I have the privilege to introduce Volume 9 of the Gums and Stabilisers Series, which has been so well received by the target group: industrial producers of hydrocolloids, users and academics with special interest in the subject. The present volume provides eloquent testimony to the progress made in this field, and the contributions represent the cutting edge of new developments. Several key themes are emphasised. The Volume includes the PILNIK LECTURE, Structural Features of Native and Commercially Extracted Pectins, presented by Dr H Schols, a disciple of Professor Pilnik. We were all disappointed that Professor Pilnik could not be with us at the Conference. His contribution to the development of pectin chemistry and applications has been outstanding, and we are all delighted that his name is now associated with the Gums and Stabilisers Wrexham Conference, and this Volume. Also included are a number of contributions concerned with the structure-function relationships of various pol ysaccharides and protein systems including microparticulated cellulose, enzyme-modified xyloglucan and marine algae proteins. Considerable progress is also reported in our understanding of interactions in mixed biopolymer systems which have received so much interest in recent years. The most rapidly expanding area of application of hydrocolloids is presently in the health sector. Low calorie, fat replacement and controlled emulsification call for new materials which are now emerging in the starch, cellulose and protein areas. The elegant contribution by Dr Ian Norton illustrates that preparations in the form of new microgels can overcome shortcomings of individual chemical components in the field of fat replacement. The input from the Physics Departments at the Universities of Swansea and Bristol emphasise that clever new methods are required to quantify the properties of such microgels, and bring the physicist into partnership with the starch chemist - unthinkable a few years ago even. The age-old Gum Arabic does not qualify as a ‘new material’ but still survives and expands in new applications, and remains a target for replacement. Nutritional aspects are also addressed. Nutraceuticals - many prefer the term functional foods for this area of food product - have been designed to improve health by using dietary fibre hydrocolloids, which function via the colon rather than the stomach. The contributions illustrate an underlying danger in going ‘over the top’ with health claims not clinically proven. Such excesses could lead to greater regulatory control, and possibly prevent the introduction of desirable and healthy products. The Tenth Conference will be held in Wrexham on 5-9 July 1999. Please note that date NOW. We aim to make this a celebratory event. It will be a pleasure to welcome you all again.

Glyn 0. Phillips Chairman, Food Hydrocolloids Trust

Members of the Gums and Stabilisers for the Food Industry Conferences Organising Committee

The Ninth Gums and Stabilisers for the Food Industry Conference owed its success to the enthusiasm and dedication of the members of the Conference Organising Committee:

Mr G A Barber

Honorary Treasurer

Mr P Cowburn

National Starch and Chemical Co

Dr T Foster

Unilever Research

Mr D Gregory

David Gregory Associates

Ms S Hills

Danisco Ingredients

Dr I Hodgson (Vice Chairman)

The NutraSweet Kelco Co

Mr H Hughes (Secretariat)

The North East Wales Institute

Dr R G Morley

Delphi Consultant Services, USA

Prof J R Mitchell

University of Nottingham

Prof E R Morris

Cranfield University

Dr J C F Murray

Hercules Ltd

Miss L Patterson

Dalgety Ltd

Prof G 0 Phillips (Chairman)

The Food Hydrocolloids Trust

Mr A Procter

Cerestar (UK) Ltd

Ms V Sharpe

Groupe RhBne Poulenc

Dr S Whitehouse

Nest16 Ltd

Prof P A Williams (Secretariat)

The North East Wales Institute

Contents

1. Structural and Functional Properties of Polysaccharides

Structural Features of Native and Commercially Extracted Pectins. H A Schols, J M Ros, P J H Daas, E J Bakx and A G J Voragen

3

Rheological Studies of Aqueous Dispersions of Microparticulated Cellulose. K Nishinari, E Miyoshi and T Takaya.

16

Rheological and DSC Studies of Aqueous Dispersions and Gels of Curdlan. K Nishinari, M Hirashima, E Miyoshi and T Takaya

*26

Unique Gelling Properties of Non-starch Polysaccharides from Pre-processed Wheat Bran. W Cui, P J Wood, J Weisz and J Mullin.

34

Viscoelastic studies of Locust Bean Gum Hydrogel Prepared by Freezing and Thawing. Interaction between Polymer Molecules in Locust Bean Gum - Water Systems During Cooking and Freezing Processes. R Tanaka, T Hatakeyama and H Hatakeyama

43

Studies on the rheological behaviour of the CV-70 polysaccharide aqueous solutions. A R P Scamparini, D M Mariuzzo and J D S Gutierrez

48

Effects of Salts and Saccharides on the Rheological Properties and Pulsed NMR of Rice Starch during Gelatinization and Retrogradation Processes. K Katsuta

59

F A 0 Regional Project on Gum Arabic Specifications and Quality Control. E Casadei

69

Gum Arabic Replacement in Confectionery Applications. P Lawson, M Gonze, F Van Der Schueren and K Rosenplenter.

76

PrimacelTMFood Cellulose. J M Evans, R C Clark and N Morrison

84

...

Vlll

Gums and Stabilisers for the Food Industry

Rheological and Thermal Studies on the Sol-Gel Transition of Aqueous Solutions of Enzymatically Modified Xyloglucan. M Shirakawa, Y Uno, K Yamatoya and K Nishinari

94

2. Protein Systems Phase-separated Mixed Gelatin-Milk Protein Systems AM Hermansson A Altskar and E Jordansson

107

ESR Spin Probe Study of Legumin and Ovalbumin Interactions with Lipophilic Compounds in Solutions. L A Wasserman, M Le Meste, M V Motiakin, V P Yuryev and A M Wasserman

117

A Study of the Hydration Behaviour of Rennet Caseins in Calcium Chelating Salt Solution using a Rheological Approach. M P Ennis, M M O'Sullivan and D M Mulvihill

127

Gelation Properties of Smooth and Skeletal Muscle Myosin Proteins. P G Somers and N K Howell

136

Thermal Stability and Sol-Gel Transitions of Whey and Egg White Proteins. P Relkin, T Meylheuc, B Launay and K Raynal

145

Marine Algae Proteins: A Study on the Extraction, Thermal Denaturation and Functionality of Spiroulina paczjica. I S Chronakis and A C Sanchez

I54

3. Functional Interactions in Mixed Biopolymer Systems Functional Interactions in Multicomponent Polysaccharide-Containing Systems. E E Braudo

169

Gels made of Wheat Starch and Ethyl(hydroxyethy1)cellulose (EHEC): Rheological Behaviour: H Larsson and A-C Eliasson

184

Solution and Gelation Properties of Protein-Polysaccharide Mixtures: Signature by Small-angle Neutron Scattering and Rheology. D Renard, F Boue' and J Lefebvre

189

Influence of Milk Proteins on the Gel Transition Temperature and the Mechanical Properties of Weak k-carrageenan Gels. A Tziboula and D S Home

202

Contents

ix

Milk Gels with Low Methoxyl Pectins. D Oakenfull and A Scott

212

Depletion Flocculation of Casein Micelles Induced by the EPS of a Lactic Acid Bacterium. R Tuinier and C G de Kruif

222

Phase Separation of Aqueous StarchlGalactomannan Systems: Influence of P-Lactoglobulin Addition. C B Closs, V B Tolstoguzov, B Conde-Petit and F Escher

23 1

Novel Gels from Xanthan-Locust Bean Gum by Addition of Electrolyte. C Gomes, M P Gongalves and P A Williams

239

The Use of Confocal Laser Scanning Microscopy in Studying Mixed Biopolymer Systems. C Gamier. S Bourriot and J-L Doublier.

247

4. Processing developments The Science and Technology of Fluid Gels. I T Norton, T Foster and R Brown

259

Developments in Process Technology - and their Potential Uses. P J Wallin

269

Tiger Striping in Injected Poultry - Causes and Cures. K Philp, Z DeFreitus, D Nicholson and R Hoffinan

276

Effect of Stabilisers and Water Content on the Rheological Properties of Prefermented Frozen Wheat Doughs. J Rasanen and K Autio

286

The Mechanism of Functionality of Potato Starch in Meat Products. E Tornberg, K Andersson and I Asplund

295

Processing and Functional Behaviour of Low Tannin Mesquite Gum. 305 F M Goycoolea, A M Calderbn de la Barca, J R Balderram, J R Valenzuela and G Hema'ndez Protein-Polysaccharide Interactions in Emulsions Containing High Pressure Treated Protein. E Dickinson and K Pawlowsky

3 14

Liquid-core Hydrocolloid-Oil Capsules A Nussinovitch and A Solomon

323

X

Gums and Stabilisers for the Food Industry

The Transformation from Enthalpic Gels to Entropic Rubbery and Glass-like States in High Sugar Biopolymer Systems. V Evageliou, S Kasapis and G Sworn

333

The Stability of Carrageenans to Processing. W M Murrs

345

5. New Methodologies Applications of Atomic Force Microscopy in Food Science. V J Morris

36 1

Microrheology of Swollen Starch Granules. S P Carrington, L R Fisher and J A Ode11

37 1

A Combined Pulse-Resonance Rheometer for Viscoelastic Gels. S T Beckett, I D Roberts, A S Whitehouse and P R Williams

38 1

Molecular Images of Gels Formed under Shear using Atomic Force Microscopy. D Power, I Larson, P Hartley D E Dunstan and D V Boger

388

6. Nutritional Aspects Functional Foods - Factors Critical to Success. J Young

397

Rheological Characterization of Inulin. I E Bishay

403

Properties and Applications of Chicory Inulin. A Franck

409

The Use of Acacia Gum as a Source of Soluble Dietary Fibre. T P Kravtchenko

413

List of Participants

42 1

Subject Index

428

STRUCTURAL FEATURES OF NATIVE AND COMMERCIALLY EXTRACTED PECTINS

H. A. Schols,* J. M. Ros, P. J. H. Daas, E. J. Bakx and A. G. J. Voragen Wageningen Agricultural University, Department of Food Technology and Nutritional Sciences, Div. Food Science, Chemistry group, Bomenweg 2, 6703 HD Wageningen.

1 INTRODUCTION

Pectin is defined as a mixture of heteropolysaccharidesconsisting predominantly of partfly methoxylated galacturonic acid residues.Pectinsare present in relatively large proportions in plant tissue, where they are a part of the cell wall, playing a role in cell growth control and e.g. in the defknce against invasions of micro-organisms’. Pectins also play an important role in the physical and sensorial properties of fiesh h i t and vegetables (ripeness, texture) and their processing characteristics (canned fhits and vegetables, purees, juices, etc.)*. On an industrial scale, pectins extracted fiom agricultural plant materials (by-products) l i e apple pomace i d lemon/lime peel are used in food applications for their ability to form gels under specific conditions or to increase the viscosity of liquids. They are also widely applied as stabilisers in acid milk product^'.^. Increased interest for pectins fiom a nutritional, medical and pharmaceutical point of view originates fiom the (claimed) role of pectin as e.g. a dietary fibre, cholesterol lowering compound and anti-tumour agent4. 1.1 Chemical Structure of Pectin

The main building block of pectin is a galacturonicacid residue, while another important feature of pectin is based on the ability of the galacturonic acid residues to form methyl esters. The DM (Degree of Methyl esterification) is defined as the moles of methoxyl groups per 100 moles of galacturonosyl residues. Pectins are referred to as ’high-methoxyl pectins’ when the DM is 50% or higher, while ‘low- methoxyl pectins’ have a DM value below 30%’. Notwithstanding the high content in galacturonic acid residues, pectins always contain (small) amounts of other sugar moieties like rhamnose, arabinose and galactose. Pectins are considered to consist of rather pure homogalactururonan parts (polymers of a - ( l 4 ) linked galacturonosyl residues) in addition to more complex ‘subunits’. Examples of such complex segments of pectic molecules are rhamnogalacturonan I and I1 (RG-I, RG-11) as reviewed by the group of Alber~heim’~~. Food scientists often refer to the highly branched parts of the pectin molecule as ‘haiky’ or ramified regions, in contrast to the homogalacturonanor ‘smooth’ regions’. Following the work of Barrett and Northcote’, De

4

Gums and Stabilisersfor the Food Industry

Figure 1 Schematic structure of apple pectin’ including rhamnogalacturonan segments of the backbone or smooth regions (SR) and ramified or hairy regions (HR) Vries et al’ studied pectin fractions isolated from apple tissue by mild extraction procedures. Their research resulted in a model in which ca. 10% of all galacturonosyl residues made up the ramified regions containing almost all neutral sugars present in pectin (Figure 1, kom ref ’). Although the characteristics of the hairy regions (e.g. sugar composition, sugar linkage composition) compare to those published for Rhamnogalacturonan I, it is suggested that they are certainly not identical. The backbone of RG-I is considered to consist of a strictly alternating sequence of rhamnose and galacturonic acid, where the ramified regions found by De Vries et a1* and many other researchers (examples given refo3”)seemed to consist of rhamnose and galacturonic acid in a rather different ratio (0.1-0.9). We have dedicated substantial research towards the ramified regions of pectins fiom various sources, isolated by using commercially available mixes of cell wall degrading enzymes (pectinases, hemicellulases and cellulases) as well as with pure and well defined enzymes like polygalacturonase and pectin esterase. For this reason, a short summary of the conventional pectolytic enzymes will be given, next to a description of a completely new class of pectolytic enzymes. 1.2 Enzymes able to degrade Pectins

Pectic enzymes are classified according to their mode of attack on the galacturonan part of the pectin molecule”. Many reviews have been published discussing the mode of action and characteristics of the common pectic enzymes and they will therefore only be discussed briefly. Pectin methylesterase (PE) is able to de-esterify high-methoxyl pectin resulting in low-methoxyl pectins and released methanol. PE may act blockwise (plant PE) or in a more random fashion (fungal PE). Polygalacturonase (PG), pectin lyase (PL) and pectate lyase (PAL) all belong to the class of depolymerases. PG cleaves the linkage between two adjacent unesterified galacturonic acid residues in a hydrolytic way, where PL and PAL split this linkage by a mechanism of 13-elimination resulting in a 4,5-unsaturated galacturonosyl residue at the non-reducing end of the degradation product. PL needs highmethoxyl pectins to act on, while PAL splits next to unesterilied galacturonosyl moieties. After the isolation and partial characterisation of the Hairy Regions of apple pectins, Schols et all0 described the presence of an enzyme initially termed rhamnogalacturonase, able to hydrolyse the linkage between galacturonic acid and rhamnose in alternating sequences of rhamnose and galacturonic acid. Now this enzyme is referred to as rhamnogalacturonan (RG) hydrolase since Mutter et all3 recently described the presence of an RG lyase able to split the same molecule between a rhamnose and a galacturonic acid residue in a 13e l i i t i v e way”*’5. Subsequent research resulted in the discovery of an acetylesterase which specifically removes acetyl groups from the ramified regions of pectinI6, while also

Structural and Functional Properties of Polysaccharides

5

RG rha-ase

RG GalA-ase Figure 2 Degradation of Rhamnogalacturonans (RG) and oligomers thereof by RG specific enzymes (galA, ga:a[acturonicacid; rha, rhamnose; gal, galactose; Ac, acetyl group)

an RG rhamnohydrolase and an RG galacturonohydrolase, specific towards rhamnogalacturonan oligomers, were re~ognised'~.In addition, a D-galactosidase was isolated which acted in synergy with the RG rhamnohydrolase. These findings resulted in the statement by Mutter et al" that 'in analogy to the enzymic degradation of the homogalacturonan regions of pectins, a whole array of enzymes specific for the degradation of pectic hairy regions, is present in nature'. The sites of attack of these enzymes are illustrated in Figure 2. It should be mentioned that D-galactosidase is also able to release galactose from other substrates and therefore is considered as being not RG-specific. The availability of the different enzymes in purified forms has accelerated the structural characterisation of specific pectin fractions as will be illustrated below. 2 APPLE PECTINS

Apple tissue and pectins extracted from apple pomace have been studied for decades by many r e s e a r c h e r ~ ' ~ ~Our ~ ' ~interest - ~ ~ . in pectin research was triggered in a new direction by the results of a study in which we compared various methods to produce apple juice with or without cell wall degrading enzymes2'. Especially the manufacture of apple juice by means of the enzymic liquefaction process resulted in the presence of high amounts of pectins in the juice'o22'. Due to the presence of various types of pectic (e.g. PG, PE, PL), hemicellulosic and cellulosic enzymes in the enzyme mixture, insoluble cell wall pectins as well as soluble pectins present in the apple tissue were almost completely solubilised and degraded leaving a soluble enzyme-resistant pectin fraction. 2.1 Pectic Hairy Regions from Apple Juice A fist characterisation of the pectic material which was isolated fiom apple juice produced by the liquefaction process by ultrafiltration (cut-off 60 kDa) showed that the

6

Gums and Stabilisersfor the Food Industry

fraction was relatively low in galacturonic acid (21 mol%) and rich in arabinose (55 mo1%) in addition to rhamnose (6 mol%), xylose (8 mol%), and galactose (9 mol%). Surprisingly, ca. 60 moles of acetyl groups were found per 100 moles of galacturonic acid, while the degree of methyl esterification (DM ) was only 42%". The characteristics of this fraction resemble those found before for the h a i i regions of apple pectin by De Vries et a?. Since it could not be ruled out that OUT fraction had been modified by the liquefaction enzymes, it was termed Modified Hairy Regions. High-performance size-exclusion chromatography (HPSEC) revealed that MHR was quite heterogeneous and consisted of about 70% of a fraction with an estimated molecular weight of 90 kDa, while two other populations of lower molecular weights were also observed. The prevailing high-Mw population was relatively rich in xylose and acetyl groups, the ratio between rhamnose and galacturonic acid was quite similar for the three populations. After screening of more than sixty different enzyme preparations, only one commercial enzyme mixture (Ultra SP, Novo Nordisk) was found to be able to degrade the pectic molecules in the backbone22.After isolation and characterisation of the responsible enzyme named rhamnogalacturonan hydrolase, it was shown that the enzyme catalyses the hydrolysis of the linkage between galacturonic acid and rhamnose by an endo-mechanism. Oligomers having a backbone length of 4-6 residues of an alternating sequence of rhamnose and galacturonic acid with or without single unit galactose substitution on 0-4 of the rhamnose moiety were Since MHR was shown to be a mixture of various populations, SEC was used to obtain the three individual populations, but only rather small variations were found in the sugar composition. Of the

Pectin

n

Homogalacturonan

s

o 0 L).

methyl ester galacturonic acid rhamnose galactose arabinose

Figure 3 Hypothetical structure of apple pectin and of the prevailing population of the hairy regions isolated from apple juice manufactured by the liquejaction process. Subunit I, xylogalacturonan; subunit II, stubs of the backbone rich in arabinan side chains: subunit Ill, rhamnogalacturonase oligomers. The exact distribution of acetyl groups is not presented; it is assumed that the major part of acevl groups is located within subunit 111

Structural and Functional Properties of Polysaccharides

I

three populations, only the two high Mw h t i o n s could be degraded by RG hydrolase. To examine both the high and low Mw fragments in the RG hydrolase digest of the population of MHR having the highest Mw, the degradation products after incubation with RG hydrolase and RG acetyl esterase were fiactionated. Chromatographyover a SEC column revealed the presence of a xylose-rich galacturonan fraction (cu. 30 kDa) in addition to an arabmose-rich fiaction (cu. 10 kDa) and a h t i o n representing the above mentioned RG hydrolase oligomers. Anionexchange chromatography of the xylose-rich hction resulted in three xylogalacturonan populationsz4 dif€ering in methyl esterilkation of the galacturonosyl residues. The arabinose-rich W i o n (>80 mol% arabinose) still contained some rhamnose and galacturonic acid and is supposed to represent some stubs of the backbone having relatively long ambinan side chains. From this work9923,a model is presented in Figure 3. Since this model is only based on the structure and enzymic degradation of pectin originating fiom apple tissue, pectin fbctions isolated from other plant material were also studied to decide whether this model is also valid for pectins fiom other origins. 2.2 Hairy Regions from various Sources

Plant tissue originating fiom dfirent fruits and vegetables (e.g. leek, onion, potato, carrot, pear) was treated with commercial liquefsction enzymes and the corresponding MHR fiactions were isolated and ~haracterised’~”.Although the isolated MHR hctions varied (slightly) in their arabinose and xylose content, all M H R molecules were quite similar with respect to molecular weight distribution, sugar (linkage) composition and degree of esterification. Also the oligomeric degradation products after RG hydrolase treatment were identical to the ones isolated fiom a digest of apple MHR. Only small difkences in relative amounts of the various oligomers were found, while the xylogalacturonan segment (Figure 3, subunit I) seemed to be absent in the MHR samples originating fiom carrot, leek and potato. Another consideration which has been made is that the M H R hctions isolated after the liquefsction process may originate fiom pectins differently located in the cell wall having different structures. For this reason, pectins were isolated fiom apple tissue by sequential extraction including extractants l i e cold and hot buffer, chelating agents and diluted alkali at low temperaturesz5.All pectins were characterised and the ramified regions were isolated fiom the pectins by incubation with a pure polygalacturonase and pectin esterase. In general, it could be concluded that a higher level of ramified regions rich in neutral sugars were obtained fiom pectins extracted under more severe conditions. Per same amount of hairy regions, higher amounts of RG oligomers, which were also more branched with galactose moieties, could be released by RG hydrolase when these hairy regions originated fiom pectins extracted under harsh conditionse.g. diluted alkali. From all these investigations, it was concluded that the model as presented in Figure 3 could also be valid for pectins from various origins although variations in the relative amounts of the subunits will be present. Depending on the plant m a t e d , arabmose-rich side chains (apple, pear) could be replaced by hairy regions in which galactose units were predominantly present (carrot, onion). The xylogalacturonan subunit is suggested to have a specific h c t i o n associated with storage tissues of reproductive organs, as the cell walls of peas, soybeans, watermelon and pine pollen also have been shown to contain xylogalacturonans6.

8

Gums and Stabilisers for ihe Food Industry

3 LEMON PECTINS

Lemon and lime peels are of increasing importance as a source for the industrial manufacture of pectins. Also during the processing of oranges, lemons, mandarins etc. to e.g. canned peeled h i t segments pectins may play a key role. For this reason and to understand the differences and similarities between pectins extracted on an industrial scale and on lab-scale (using rather mild conditions), lemon pectins were studied in more detail. 3.1 The Effect of Extraction Method on the Characteristics of Lemon Pectin

For the extraction of lemon pectins, flavedo was removed manually from intact lemons, followed by the recovery of the albedo. The segments were pressed to obtain the lemon juice which was dialysed to yield lemon juice pectins. The albedo was extracted with aqueous 70% ethanol to obtain the alcohol insoluble solids (AIS), which were then subsequently extracted by chelating agents and diluted alkali at low temperature (ChSS and ASS fractions). The sugar composition of these fractions are shown in Table 1, together with data reported for industrially extracted pectins“. It can be seen that the ratio arabinose to galactose is completely different for the two industrially extracted pectins. The sugar composition as well as the degree of methyl esterification and acetylation of the ChSS pectins resemble those of the industrial lemon pectin preparation while the alkali extracted pectins contained much more neutral sugars. The relative amounts of the neutral sugars were quite different. Similar findings were obtained for the pectins extracted fiom the juice where the content of galactose predominates over the arabinose content. These findings suggested that the various pectins are different with respect to their h a i i regions. This was further substantiated by incubation of the pectins with RG hydrolase or with PG (Figure 4). It can be seen that PG digestion resulted in an almost complete degradation of the ChSS and ASS pectins, although for the latter pectin somewhat higher amounts of high Mw enzyme-resistant material (i.e. h a i i regions) were found”. Incubation of both pectins with RG hydrolase released the typical oligomers ( ret. times ca. 32 min) as discussed above in Figure 3.The HPSEC patterns also showed that RG hydrolase action did not result in a shill to lower molecular weights for the ChSS pectin as it did for the ASS pectin. This suggests that the hairy regions present in ChSS pectin are located at the extremities of the molecules, whereas the hairy regions of ASS pectin are presumably distributed more randomly over the pectin molecule. Apart from the sugar composition, also the degradability of the industrially extracted lemon pectin by RG hydrolase resembles that of the ChSS pectin (Kravtchenko, personal communication). sugar

Apple”

Lemon”

___ RhalllnOSe

2

2

kabinosc Xylosc Mannose Galactose Glucose GalAcid DM DA

9

3 tr tr 6 1 88 72

1

tr 6 16 66

74 5

1

Lemon Juice 2 10

2 2 15

7 61 91 5

Albedo AlbedoASSZ6 ChSSZ6 11 20 2 tr

1 4

tr I 2 2

5 4 51

YO

79 2

__ -_

Table 1 Sugar composition of pectins extractedfrom various sources

9

Structural and Functional Properties of Polysaccharides

I

al

blank

blank

r -

n

1.

sa

. I

I

a4

Retention time Figure 4 HPSEC elution patterns of saponified lemon albedo ChSS (a) and ASS (6) pectins before and after degradation by polygalacturonase or RG hydrolase 3.2 Lemon Pectin Modified Hairy Regions

Some characteristics of the so called Modified Hairy Regions obtained fiom lemon albedo and lemon juice are presented in Table 2, together with those found for apple M H R as discussed above. Lemon albedo MHR is almost free of xylose and has relatively low amounts of methyl esters and acetyl groups. Still quite some similarities are found with apple MHR: both MHR preparations were rich in arabinose, while the ratio of rhamnose to galacturonic acid is within the same order of magnitude. These findings are in good agreement with the data published by Ralet & Thibault for pectin hctions from commercial lemon dietary fibre”. The MHR fiom lemon juice however differed significantly having a rhamnose to galacturonic acid ratio of ca. 1:10. The galactose and arabinose content also differed as was expected from the sugar composition of the intact pectins. Enzymic degradation of the lemon albedo MHR and fractions therefrom is described in more detail elsewhereI6. It was concluded that the majority of the oligomeric degradation products released by RG hydrolase were lacking in galactose substitution (see also Figure 3), although this differed slightly for the various MHR populations. sugar

Apple

Lemon

Lemon

Albedo

Juice

___l_-l-_____~-I__-__

Rhamnose Arabinose Xylose Mannose Galaaose Glucose GalAcid DM DA

6 55 8 0 9

5 56

5 10

1 1

6

I

8 I 28

1

21 42 60

25 6 46 17 25

39

13

Table 2 Sugar composition of Lemon and Apple Modified Hairy Regions obtained after enzymic liquefaction

10

Gums and Stabilisers for the Food Industry

3.3 Lemon Pectin Hairy Regions

As a consequence of the isolation procedure resulting in an almost complete liquefaction of lemon tissue, the MHR fraction originates from various populations of pectins present in the plant cell wall. Therefore we also studied ramified regions as obtained from the sequentially, mildly extracted pectins after PGPE treatment28. The sugar composition of these HR fractions is presented in Table 3. The fractions HR-I and HR-I1 eluted differently from the SEC column and differed in molecular weight (ca. 90 kDa versus ca. 30 kDa) while the Residue HR fraction represents the material which was solubilised from the cellulose-rich Residue (remaining after the isolation of ChSS and ASS pectins) by the action of PG and PE. It can be seen that the sugar composition of the HR fractions differed for the various molecular weight fractions and for hairy regions of different origin. Most remarkable was the high xylose content in the ChSS-I1 and ASS-I1 HR fractions. Although ChSS-I1 seemed to represent a rather complex mixture of polymers, ASS-I1 comprises for almost 60% of xylose and galacturonic acid. These findings suggest that, despite the very low xylose content of lemon pectin, a xylogalacturonan (as has been identified from apple pectic material) is present in lemon as well. Treatment of the h a i i regions by RG hydrolase again resulted in a shift to lower molecular weights (Figure 5). It can be seen that only the highest Mw fractions (ChSS-I, ASS-I, R e d ) are degraded, while the HR fractions having lower Mw values are not affected at all. RG hydrolase digestion also resulted in the release of the typical oligomers (Figure 6 ) , although it is observed that the amount of oligomers per mg HR as well as the relative amounts per oligomer (branched versus linear) again seemed to depend on the extraction method of the corresponding pectin. It can concluded that commercial lemon pectins have more in common with the easy-to-extract (calciumbound) pectins than with pectins which could only be extracted by akali treatment. The Hairy Region fractions obtained from the various isolated lemon pectins showed some dissimilarities with each other with respect to the sugar composition, the enzymic degradability by RG hydrolase and the amount and type of oligomers released.

ChSS-I

ChSS-I1

ASS - 1

ASS - I1

RhalIUlOSe

4

3

Arabinose Xylose Mannose Galactose Glucose GalAcid IIM DA

59

5 23 7 13 11 9 35 55

5 28 28 1

sugar

I 1 21

I 15

25 22

58

5 1 19

Residue 2 53 2

tr

8 1

tr 33 1

16

29

10

41

Table 3 Sugar composition of the Hairy Regionsfrom lemon pectins obtained afrer degradation by PG and PE

Structural and Functional Properties of Polysaccharides -__ __ _____ -- -___ - -- I

11 --

P

I' 18 Retention time (min)

Figure 5 HPSEC elution patterns of the pectic Haity Regions obtained from various mild@ extracted lemon albedo pectin fractions before (thick line) and after (thin line) RG hydrolase digestion: ChSS HR-I (a); ChSS HR-I1 (b); ASS HR-I (c); ASS HR-I1 (d); RES HR-I (e)

,~

._

1

/

-1 w4

1

id E l

.

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-1

0

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A h

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I

I

41

--~c 15

20

25

30

35

Racmtion time (min)

Figure 6 HPAEC elution profles of saponij?ed- ChSS-HR-I (a), ASS-HR-I (b), and ResidueHR-I (c) after treatment with RG hydrolase. The structures of the oligomers are described4: I , RhazGalAZ,4, RhazGalAzGaiz;6,RhajGalA,; 8, RhajGalAJGalz 4 METHYL ESTER DISTRIBUTION OVER THE PECTIN MOLECULE

From the comparison of industrially extracted pectins and pectins extracted using mild conditions, it can be concluded that during the industrial extraction process some glycosidic linkages are split. In general, this results in relatively high galacturonic acid contents since part of the neutral sugar side chains within the hairy regions will be removed. Also some of the relatively weak linkages between rhamnose and galacturonic acid residues may have been split resulting in a reduced molecular weight. In general, it is thought that the properties of industrial pectins in e.g. food applicationsmainly depend on the homogalactur o w regions within the backbone and attention has been focused on the distribution of methyl esters over the galacturonosyl residues to explain variations in the behaviour of difFerent pectins. Based on degradation studies using purified pectin lyase and pectate lyase, De Vries suggested that the methoxyl groups were randomly distributed over the galacturonan chain within native apple p e ~ t i n ~ ~For ' ~ 'commercially . extracted lemon pectin using polygalacturonases followed by subsequential analysis of the oligomers released, Kravtchenko et a13' found indications that small blocks of unesterified galacturonosyl

12

Gums and Srabilisers for the Food Industry

residues were present. Also Kiyohara and Yamada3’ found great differences in the methyl ester distribution in pectins from the roots of Angelica acutiloba Kitagawa having the same DM. Chemical methods to determine the distribution of methyl esters over the pectin molecules include the reduction of methyl esterified galacturonic acid moieties prior to specific hydrolysis of the formed galactose linkages by hydrofluoric acid33.Also the use of advanced spectrometric methods like NMR and MS have been d e m ~ n s t r a t e d ~ ~ To” ~be . able to examine enzyme digests obtained by e.g. PG treatment for the presence of oligomeric fragments varying in methyl esterification, a method has been developed to analyse partially methyl esterified oligogalacturonides by HPLC. 4.1 Low pH High-Performance Anion-Exchange Chromatography One important breakthrough of the last years was the development of high-performance anion-exchange chromatography at high pH followed by pulsed amperometric detection This same separation and its use in the analysis of oligogalacturonic method has been adapted to elution conditions at lower pH (5-7)to increase the number of oligomers to be separated4’ or to improve the separation between pectin- and pectate lyase degradation products4*. To ensure PAD detection, post-column alkali addition was used. Until now, this method was not reported for the examination of methyl estersed galacturonosyl oligomers. The slightly modified methodd3of Hotchkiss and Hicks4‘ was used to separate a PG digest of a low-methoxyl pectin (DM=30) using a CarboPac PA1 column (Dionex). A complex elution pattern was found (Figure 7, line a). In addition to the series of nonesterified galacturonic acid oligomers (1, 2, 3, etc.) other compounds were also eluted in a regular pattern. The PG digest was saponified under mild conditions to remove all ester groups and re-analysed again under the same conditions. The resulting elution pattern is shown in Figure 7 line b. It can be seen that after removal of all ester groups, a homologous series of galacturonic acid oligomers up to a DP=12 can be recognised. For further characterisation of the methyl esterified oligomers, Matrix Assisted Laser Desorptioflonisation Time of Flight Mass spectroscopy (Maldi-Tof MS) was used43.From the mass spectrum (Figure 8) a whole range of oligomers up to masses of ca. 2200 Da (DP

0

10

20

30

40

50

60 70 80 Retention time (min)

Figure 7 HPAEC elution profile at pH 5 on a CarboPac PA1 column ofa PG digest of a DM=30 Pectin before (a) and after (b) saponijicationprior to injection

Structural and Functional Properties of Polysaccharides

30

13

I’

569

3’ 583

I

ST

oJ

,

. . ..

.

.. .

.,

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. .

. .-

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.

.

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Figure 8 Maldi-Tof mass spectrum of the PG digest DM=30pectin (dhb matrix). The size of the oligomers (DP value) and the number of methoxyl groups per oligomer (in superscript) are indicated

= 12) can be recognised. Next to the mass of a sodiated unesterified trimer of galacturonic acid ( d z 569), a second mass peak differing by 14 units could be detected, indicating the presence of an additional methyl ester. In analogy, higher oligomers also carrying one or more methyl esters could be identified. The absence of higher unesterikd oligomers can easily be explained by fact that PG is able to degrade these oligomer~’~~~~. The DP value of the oligomers and the number of methoxyl groups present (in superscript) are indicated in the figure. Since e.g. octamers with only one or two methoxyl groups were not found, it is suggested that PG is not hindered by a low methoxyl substitution. The exact elution mechanism of the various methyl esterified oligomers (influence of number of methyl esters and DP on elution behaviour) has been revealed after preparative anion-exchange chromatographyover Source lSQ (Pharmacia) and subsequentialanalysis of all fractions by Maldi-Tof MS. These results will be published in detail elsewhere4’. Unfortunately, so far no information is available on the exact location of a methoxyl group within a certain oligomer and as a consequence it is not known whether e.g. the octamer with three methyl esters represents one or more isomers. Since this information is of vital importance for revealing the methyl ester distribution over the galacturonosyl residues, attempts are being made to separate these isoforms to enable recognitionand quantification. 5 CONCLUSIONS

it can be concluded that substantial new information has become available with respect to the chemical fine structure of pectins. Recently, more information has become available on the structure of pectic h a i i regions. It has been p r ~ p o s e dthat ~ ’ ~pectins are built up of different subunits (e.g. HGA, RG-I,RG-11, apiogalacturonan,xylogalacturonan)which may vary in relative amounts; depending on the origin of the pectin. Some units may even be absent in certain sources. This has been illustrated by the discovery of xylogalacturonan segments in pectins from apple, which are also found in small amounts in lemon pectins. The exact chemical structure of the various subunits and their distribution over the hairy

14

Gums and Sfabilisersfor the Food Industry

regions and the distribution of the different hairy regions over the pectin molecule is still unknown. The same is true for the precise distribution of methyl esters over the homogalacturonan backbone. Recently, Glahn and R0lh-1~~ reported on the large scale separation of calcium-sensitive and non-calcium sensitive pectin which enables a more detailed study of the individual pectin molecules. In addition to their findings, we are convinced that next to long (calcium reactive) stretches of unesterified galacturonic acid residues, also a more subtle distribution of smaller blocks, not sensitive towards calcium, may exist. Methods are now available to study such methoxyl group distributions which, to our opinion, should be used in combination with specific pectic enzymes l i e PG and PL. The same methods can also be applied to study the physicochemical and catalytic properties of pectic enzymes resolving their mode of action in much more detail. The aim of the described research was to make a contribution to the basic knowledge of pectic cell wall polysaccharides and of industrially extracted pectins. Such knowledge is essential in understanding the relation between chemical structure of pectins and their h c tional properties. When more information becomes available about the desired structure of pectin (e.g. distribution of methyl esters, absence or presence of calcium-sensitive blocks or ramified regions, presence of acetyl groups) with respect to specific applications, new strategies for the extraction and the modification of industrial pectins may lead to newly formulated pectin preparations. Also other (food) applications of pectins may be recognised. The obtained knowledge may also contribute to improve the processing conditions of h i t and vegetables and may lead to a better use of agricultural by-products. Acknowledgement

The authors want to thank dr. P.W. Arisz of the Hercules European Research Center B.V. in Bameveld, The Netherlands for recording the Maldi-Tof MS spectra. Part of this work was financially supported by Hercules Incorporated via its subsidiaries Hercules European Research Center (Bameveld, The Netherlands) and Copenhagen Pectin A/S (Lille Skensved, Denmark). The Commission of the European Union is acknowledged for the financial support to J.M.R. as a fellow researcher within the 'Human Capital and Mobility' programme. References 1. 2. 3. 4. 5. 6.

7. 8. 9.

A. Bacic, P.J. Harris, and B.A. Stone. The Biochemistry of Plants, Vol 14, Carbohydrates, Academic Press, London, 1988,297-369. A.G.J. Voragen, W. Pilnik, J-F. Thibault. M.A.V. Axelos, C.M.G.C. Renard. In: A.M. Stephen (Ed) Foodpolysaccharides and their applications, Marcel Dekker Inc., New York, 1995,287-339. C. Rollin and J. De Vries. In: P. Harris (ed)Food Gels, Elsevier, Amsterdam, 1996, 401-434. K.W. Waldron and RR Selvendran. In: K.W. Waldron, I.T. Johnson and G.R. Fenwick (eds) Food and Cancer Prevention: Chemical and biological aspects. Royal Soc. Chem. 1993, 307-326 M. O"eil1, P. Albersheim, and A.G. Darvill. In: P.M. Dey (Ed.) Methods in Plant Biochemistry, Vol. 2, Carbohydrates, Academic, London, 1990,415-441. P. Albersheim, A.G. Darvill, M.A. O'Neill, H.A. Schols, A.G.J. Voragen. In: J. Visser and A.G.J. Voragen ( 4 s ) Progress in Eiotechnology 14: Pectins and pectinases, Elsevier, Amsterdam. 1996,4755 A.J. Barrett, and D.H. Northcote. Eiochem. J., 94 (1965) 617-627. J.A. De Vries, F.M. Rombouts, A.G.J. Voragen, and W. Pilnik. Carbohydr. Polym., 2 (1982) 25-33. H.A. Schols, A.G.J. Voragen. In: J. Visser and A.G.J. Voragen (eds) Progress in Biotechnology 14: Pectins andpectinases, Elsevier, Amsterdam. 1996, 3-19.

Struchual and Functional Properties of Polysaccharides

15

LO. H.A. Schols, M.A. Posthumus, and A.G.J. Voragen. Carbohydr.Res., 206 (1990) 1 17-129. 1I . H.A. Schols and A.G.J. Voragen. Carbohydr. Res., 256 (194) 83-95. 12. W. Pilnik, and F.M. Rombouts. In: G.G. Birch, N. Blakebrough, and K.J.Parker (Eds) Enqyms and F o o d h e s s i n g , Applied Science Publishas LTD, London, 1981, pp. 105-128. 13. M. Mutter, I.J. Colquhoun, HA. Schols, G. Beldman, A.G.J. Voragen. Plant Physiol. 110 (1 996) 7377. 14. G. Beldman, M. Mutter, M.J.F. Searlevan Leeuv.cn, L.A.M. van den Broek, HA. Schols, A.G.J. Voragen. Progress in Biotechnologv 14: Pectins andpectinases. J. Visser and A.G.J. Voragen (eds), Elsevier 1996,231-245. 15. M. Mutter, C.M.G.C. Renard, G. Beldman, HA. Schols, A.O.J. Voragen. In: J. Visser and A.G.J. Voragen (eds) Progress in Biorechnologv 14: Pectins and pectinases, Elsevier, Amsterdam. 1996, 263-274. 16. M.J.F. Searle-van Iauv.cn, L.A.M. van den Rroek, HA. Schols, G. Beldman, and A.G.J. Voragen. Appl. Microbiol. Biotechnol., 38 (1992) 347-349. 17. M. Mutter, O. Beldman,H.A. Schols, andA.G.J. Vmgen. PlantPhysiol.. 106(1994)241-250. 18. Renard, J.-F. Thihult, A.G.J. Voragen, L.A.M. van den Broek and W. Pilnik. Carbohydr. Polym. 22 (1993) 203-210. 19. T.P. Kractchenko, A.G.J. Voragen and W. Pilnik. Carbohydr. Polym..,l8(1992)17-25. 20. M.Knee, Phytochemistty, 17(1978) 1257-1260. 21. HA. Schols, P.H. in 't Veld, W. van Deelen, and A.G.J. Voragen. Z Lebensm. Unters. Forsch., 192 (1991) 142-148. 22. KA. Schols. C.C.J.M. Gem& M.J.F. Searlevan h w e n , F.J.M. Kormeling, and A.G.J. Voragen. Carbohydr. &s., 206(1990) 105-115. 23. H.A. Schols, A.G.I. Voragen, and I.J. Colquham. Carbo@dr. Res., 256 (1994) 97-1 11. 24. H.A. Schols, E.J. Balac, D. Schipper, and A.G.J. Voragen. Carbohydr.Res., 279 (1995) 265-279. 25. H A . Schols. E. Vierhuis, E.J. Bakx,and A.G.J. Voragen. Carhhydr. Res., 275 (1995) 343-360. 26. J.M.Ros, HA. Schols, A.G.J. Voragen. Carbohydr. Res. 282 (1996) 271-284 a a l and J.-F. Thibault. C a r b o w . Rer. 260 (1994) 283-2%. 27. M.C. R 28. J.M.Rm, HA. Schols, A.G.J. V~ragen.Carbohydr. Polym , submitted. 29. J.A. Lk Vries, F.M. Rombaas, A.G.J. Voragen, and W. Pilnik. Carbohydr.Polym.,3 (1983) 245-258. 30. J.A. De Vries, M. Hansen, J. Soderberg, P.E. Glahn,and J.K. Pedmsen. Curbohyak. Polym.,6 (1986) 165-176. 31. T.P. Kravtchenko, M. Penci, A.G.J. Voragen, and W. Pilnik. Carbohydr. Polym., 20 (1993) 195. 32. H. Kiyohara and H. Yamada. Carbohydr. Polym. 25 (1994) 117-122. 33. A.J. Mort, F. Qiu, N.O. Maness. Curbohydr. Res. 247 (1993) 21-35. 34. H.Grasdalen, A.K.A n d a m and B. Larsen. Carbohydr. R ~ s .289 (1996) 105-114. 35. E. Westerlmd, P. Aman, RE. A n d e w and R Andasson. Carbohydr. P ~ l y m14 . (1991) 179-187. 36. R.E. Aries, C.S. Gutteridge, W.A. Laurie, J.J. Boon and G.B. Eijkel. Anal.Chem., 60 (1988) 14991502. 37. Y.C. Lee. Anal. Biochem. 189 (1990) 151-162. 38. Y.C.Lee. J. Chromatogr.A , 720(1996) 137-149. 39. N.A. Shanley, L.A.M. van den Broek,-A.G.J. Voragen and M.P.Coughlan. J. Biotechn. 28 (1993) 179- 197. 40. N.A. Shanley, L.A.M. van den Broek, A.G.J. Voragen and M.P. Coughlan. J. Biotechn., 28 (1993) 199-218. 41. A.T. Hotchkiss, Jr. and K.B.Hicks. Cwbohydr. Res. 247 (1993) 1-7. 42. H.P. Lieker, K.Thielecke, K.Buchholz and P.J. Reilly. C0bohydr. Res. 238 (1993) 307-31 I . 43. P.J.H. Daas, P.W.Arisz, H.A. Schols, G.A. de Ruiter and A.G.J. Voragen. Anal. Biochem. submitted 44. Glahn and C. Rolh. In: G.O. Philips, D.J. Wedlock, and P.A. Williams (Eds) Gwns and Stubilizers for the Food Industry 8, IN- Press, Oxford, 1996.393402.

*Correspondence: [email protected]. WAU.NL

RHEOLOGICAL STUDIES OF AQUEOUS DISPERSIONS OF MICROPARTICULATED CELLULOSE

K. Nishinari, E. Miyoshi’ and T. Takaya Department of Food and Nutrition, Faculty of Life Science, Osaka City University, Sumiyoshi, Osaka 558, Japan +Presentaffi1iation:Division of Development and Environmental Studies, Osaka University of Foreign Studies, Osaka 562, Japan

ABSTRACT Storage and loss shear moduli of aqueous dispersions of microparticulated cellulose with different sizes ( l . l p m , 1 . 8 p m , 3.2pm, 6.5pm) increased with increasing temperature. Although the dispersions are not true gels, they showed a plateau in the frequency dependence of storage and loss moduli. The storage modulus of dispersions of microparticulated cellulose as a function of concentration increased by a power law with an exponent of three. Flow curves of dispersions showed hysteresis; shear stress observed during the increase in shear rate is larger than that observed during the decrease in shear rate. The hysteresis was more pronounced at higher temperatures. The storage modulus was separated into entropic and energetic contributions, and the entropic contribution was found to be positive for the temperature range from 5 to 70°C and to increase with increasing temperature. It is suggested that a tenuous network is formed in the dispersion, which becomes more solid-like at higher temperatures.

INTRODUCTION Excessive intake of fat causes obesity, heart attack, and high blood pressure. Many investigations have been carried out to make fat substitutes by using non-calorie carbohydrate or dietary fibre. Lean meat is sometimes not acceptable for its mouthfeel, and texture modifiers with high water holding capacity have been sought. The unique properties of microcystalline cellulose as a potential stabiliser and a fat substitute have been studied by many investigators’. A cream-like dispersion of microparticulated cellulose has been prepared recently, and it has attracted much attention because of its unique rheological properties and the possibility of being used as a new texture modifie?. It has a cream-like appearance, a smooth mouthfeel and zero calorific value. It has possible use as a fat substitute for dressings, mayonnaises, whipcreams, fat spreads, ice cream, yogurt and so on. Since the spinnability of the dispersion of microparticulated cellulose is far smaller than that of gelatinised starch dispersion, it is suitable for preparing foods of “short” texture. It may be used as dietary fibres in biscuits, breads, cakes and noodles’. It has an advantage that it is not chemically treated but produced only by mechanical degradation by high shear.

Srrucrural and Functional Properties of Polysaccharides

17

viscoelastic behaviour of dilute cellulose fibre suspensions has been studied3, and the results were interpreted by Rouse’s theory for dilute polymer solutions4. However, the viscoelastic behaviour of concentrated cellulose fibre dispersions with very small particle sizes has never been studied as far as the authors are aware. Rheological properties of concentrated cream-like dispersions of microparticulated cellulose are described in the present work.

MATERIALS AND METHODS

Materials Cream-like 10%dispersions of microparticulated cellulose with different median sizes (1.1 ,u m, 1.8,u m, 3.2 p m, 6.5 ,um) were kindly given by Asahi Chemical Industries Ud.(Tokyo, Japan). They consist of 10% crystalline cellulose and 90% water. Dispersions of various concentrations were prepared by diluting 10% dispersions with distilled water using a magnetic stirrer. P

S

llle storage shear modulus G ’ and the loss shear modulus G ” of dispersions of microparticulated cellulose were measured as a function of frequency at a constant temperature or as a function of temperature at a constant frequency in the linear viscoelastic regime ( shear strain less than 4%) using a Fluids Spectrometer RFSII or a Dynamic Stress Rheometer from Rheometrics Ltd.(NJ, USA). Ther temperature was controlled within fO.l“C, and the dispersion was covered by silicone oil to prevent the evaporation of water. The details of the dynamic viscoelastic measurements have been described previou~ly~*~. viscosity measurements Steady -shear viscosity of dispersions of microparticulated cellulose was measured at various temperatures by a Fluids Spectrometer RFSII from Rheometrics Ltd.(NJ, USA) using a cone and plate geometry of 25 mm diameter, over a shear-rate range from 0.01 s-’ to lOOs-’. The shear rate was changed stepwise from 0.01 s-’to 100 s.’ for 10 min.

Steady-*

D -

Particle size was measured by a Particle Size Analyzer LA-500 from Horiba Co.Ud., Kyoto.

RESULTS AND DISCUSSION Figure 1 shows the particle size distribution of dispersions of microparticulated cellulose, which are used in the present study, determined by Asahi Chemical Industries Ud. using a Particle Size Analyzer LA-500 from Horiba Co.Ltd., Kyoto. The median size d, which divides the distribution evenly (50 per cent of the material is smaller than d, ) ’ is shown at the upper-left corner of each figure; 1.1pm, 1.8pm, 3.2,um, 6.5pm. The cumulative frequency curves is represented by a solid curve which shows the cumulative percentage equal to or smaller than a given size, per cent undersize. The fraction of the smallest median size ( l.l,um) seems to have a bimodal distribution. Although the distribution is not so narrow, the values of the median sizes are sufficiently different. Figure 2 shows the frequency dependence of storage and loss shear moduli G’ and G” for aqueous dispersions of microparticulated cellulose of 1.8 p m with different concentrations at 25 “c and at 60 “c . At both temperatures and for all concentrations G ’

18

Gums and Stabilisers for the Food Industry

U%

F?!

Particle Size / ~ r n Figure 1. Frequency histogram of microparticulated cellulose. F, frequency; U, undersize. shows a plateau and is larger than G" at all the frequencies accessible, which is characteristic of solid-like behaviour. As is well-known4, dilute solutions of flexible polymers show liquid-like behaviour i.e., G" predominates G' at all the frequencies accessible, and both moduli are strongly frequency dependent. The solid-like mechanical spectra shown in Figure 2 suggest the presence of some network structure. The value of storage modulus decreased by three decades with decreasing concentration by five times. The concentration dependence of the storage modulus will be discussed later. Although G' shows a plateau at 60°C and is larger than the value at 25"c, it became more frequency dependent. Since both storage and loss shear moduli of the dispersion of microparticulated cellulose did not change when kept at 60°C for 60 min, the dispersion was in a thermal equilibrium state and the evaporation of water could be neglected. The reason why both moduli became more frequency dependent at a higher temperature in spite of the increase of these values is not clear at present. Figure 3 shows the temperature dependence of G' and G"of storage and loss shear moduli G' and G" for aqueous dispersions of microparticulated cellulose at a constant frequency of lrad/s and at a scan rate of l"C/min. All the systems showed a remarkable hysteresis; both G' and G" were larger in the heating process than in the cooling process although this hysteresis became almost negligible when the scanning rate changed from l"C/min to 0.2"C/min as will be shown later in Figures 6 and 7. The fact that G' for a dispersion once heated is larger than G' for non-heated dispersion, however, suggests that some ordered structure is formed on heating. However, the ordered structure which is formed on heating seems to be tenuous and reverted to the initial state on cooling, i.e.

19

Structural and Functional Properties of Polysaccharides

completely thermoreversible. The phenomenon that the elastic modulus increases with increasing temperature is well known for aqueous solutions of methyl cellulose*-'o, of xyloglucan from which some galadose residues are and of curdlan,which all form gels on However, the cream-like appearance of the present dispersion of microparticulated cellulose suggests that it is somewhat different from gels of methyl cellulose, xyloglucan or curdlan. In fact, it flows when it is subjected to a large deformation whilst a true gel is found to break under a large deformation. This is therefore a so-called weak gel". The temperature dependence of elastic modulus will be discussed later. Flow curves for aqueous dispersions of microparticlated cellulose are shown in Figure 4, and show shear-thinning behaviour. m e hysteresis was more pronounced at higher temperatures than at lower temperatures,indicating that ordered structures formed at higher temperatures were broken down to a greater extent. Aqueous dispersions of microparticulated cellulose showed a larger elastic modulus at higher temperatures than at lower temperatures, but as the structure formed at higher temperatures is fragile, it breaks.at a lower shear rate than the structure formed at lower temperatures.

105

(a) 1.8-25'C

(b) 1.8-6O'C

I

5%

3%

25% 2%

101

Id0

I 10

Figure 2. Frequency dependence of storage shear modulus G' and loss shear modulus G" for 2,2.5, 5 , 6 , 8 and 10 % dispersionsof microparticulated cellulose (size 1.8p m) at 25°C and at 60°C.

20

Gums and Stabilisers for the Food Industry

The concentration dependence of storage modulus at lradls for dispersions of microparticulated cellulose with various sizes from 1.1 to 6.5 p m can be represented by G '=K(C - C,)" , where C, is the critical concentration below which storage modulus cannot be detected, and n is the exponent (Figure 5). The concentration dependence of elastic modulus of gels is often described by a so-called square power law, i.e. n=2 l 6 or n =2.25 according to a recent scaling treatment for true elastic gels. In low concentration gels, however, the exponent is found sometimes far larger than two as predicted by cascade theoryI6 or by a modified theory of rubber elasticity 18. Systems consisting of carbon black dispersed in concentrated solutions of polystyrene in diethyl phthalate were found to exhibit a second plateau at lower frequencies in addition to the well-known rubbery plateau '. It was suggested that the existence of solid particles

15()oo

1.8-10Vo loo00

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io

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-

00

10

20

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40

50

80

70

Temperature / ' C Figure 3.

Temperature dependence of storage shear modulus G' and loss shear modulus G" for 10, 6 , and 2.5 % dispersions of microparticulated cellulose (size 3.2 ,!L m and 1.8 ,!L m). Cooling and heating rate, l.O"C/min. Angular frequency. l.Orad/s.

in polymer solutions makes the relaxation time much longer than the longest relaxation time of the polymer molecules, and that the interaction between solid particles or the interaction between solid particles and polymer chains causes the second plateau at lower frequencies

21

Structural and Functional Properties of Polysaccharides

than the rubbery plateau. The exponent in the concentration dependence of the storage modulus at the rubbery plateau and at a second plateau were found to be 3 and 5 respectively. For our dispersions of microparticulated cellulose, the exponent was found to be about three,

110%-5'C 1.1 1.8,3.2.6.5

300 200

0

20

do

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1

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1

Shear rate I s -l Figure 4. Flow curves of 10%dispersions of microparticulated cellulose of various sizes (1.1,1.8, 3.2 and 6.5 p m) at 5, 25 and 60°C.

22

Gums and Stubilisers for the Food Industry

irrespective of the size of microparticles, suggesting that the network structure is weaker than that of true gels. This is perhaps due to the weak molecular forces which make the network structure. Since the modulus increases with increasing temperature, the molecular forces responsible for building up an ordered structure may be hydrophobic rather than hydrogen bonding. 5000

I

5000

6.5 G'= 2.939(C-C 0)2.994

4000i

4000

C0=0.0917

3.2 G' = 2.105( C-C 0)S.m C0=0.325

I

3000 2000 1000

-z

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4

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c3 5000

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Figure 5.

/

G' =3.0 19(C - C O ) ' . ~ ~ ~ C0=0.2625

2

4

6

8

1

0

%

Concentration dependence of storage modulus of dispersions of microparticulated cellulose of various sizes (1.1, 1.8, 3.2 and 6.5 p m) at l.Orad/s and at 25°C.

Figure 6 shows the storage shear modulus of 2% dispersions of microparticulated cellulose with different sizes (1.1, 1.8, 3.2 and 6 . 5 p m ) at lradh as a function of temperature. When the scan rate was slow enough <0.2"c/min, the thermal hysteresis was almost negligible i.e. the heating curve and cooling curve coincided. The tendency of increasing G' with increasing temperature was most apparent for a 2% dispersion of microparticulated cellulose with 1 . 1 m ~ and the least for a 2% dispersion of microparticulated cellulose with 3.2 p m. Figure 7 shows the storage shear modulus of dispersions of microparticulated cellulose of various concentrations (2, 5, and 10%) with 6.5 p m at lradh as a function of temperature. The most concentrated dispersion showed the most pronounced increase in G' with increasing temperature.

Structural and Functional Properties of Polysaccharides

23

so00

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---lo00

D

Figure 6. Storage modulus G' of 2% dispersions of microparticulated cellulose of various sizes (6.5,um, 3.2pm, 1.8pm and l . l p m ) as a function of temperature, and the separation into entropic G, and energetic G, contributions. Angular frequency, l.Orad/s. Scan rate, 0.2Vmin. Elastic modulus can be separated to entropic and energetic contributions as has been done for agarose gels m. G

G,=G,tG,

Gums and Stabilisersfor the Food Industry

24

-1-4 O

P

4

0

0

6

0

Figure 7. Storage modulus G' of dispersions of microparticulated cellulose (size 6.5 p m) of various concentrations (lo%, 5% and 2%) as a function of temperature, and the separation into entropic G,and energetic G, contributions. Angular frequency: l.Orad/s. Scan rate, 0.2"C/min. where G and r are the shear modulus and the shear strain, respectively. The quantities A& and AUv represent the change in entropy density and internal energy density for the deformation at a constant volume. The entropic contribution G, and the energetic oontriiution G, for dispersions of microparticulated cellulose are shown by the broken curve and dotted-line together with the observed (total) storage modulus G (EG,) (thick solid curve) in Figure 6 and Figure 7 respectively. G , was always positive and increased whilst G , decreased and became negative above a certain temperature with increasing temperature. For G , >O the entropy decreases by deformation, and there will appear resistance against it; for G, O indicates that if the energy increases by deformation , there will be resistance against it, whereas G ,
Structural and Functional Properties of Polysaccharides

REFERENCES 1. A.P.Imeson and W.Humphreys, In ‘Thickening and Gelling Agents for Food’, 2nd Ed., A.Imeson Ed., Blackie Academic & Professional, London, 1997p.180. 2. Y. Komuro, Shokuhin to kaihatsu,1992,27, 47. 3. A. Attanasio, U. Bernini and G. Segre, J. Colloid Interface.Sci., 1969,29, 81. 4. J.D. Ferry, ‘Viscoelastic Properties of Polymers’, 3rd Ed., John Wiley, New York, 1980. 5. E. Miyoshi, T. Takaya and K. Nishinari, Food Hydrocoll., 1994,8, 505. 6. E. Miyoshi, T. Takaya and K. Nishinari, Zhermochim. Acfa., 1995, 267, 269. 7. R. J. Hunter, ‘Foundations of Colloid Science’, Vol.1, Clarendon Press, Oxford, 1991, p.119. 8. A. Haque and E.R. Morris, Carbohydr.Polym., 1993,22, 161. 9. M. Vigouret, M. Rinaudo and J. Desbrieres, J.Chim.Phys., 1996,93, 858. 10. K. Nishinari, K.E. Hofmann, H. Moritaka, K. Kohyama and N. Nishinari, Macromol. Chem. Phys., 1997,198, 1217. 11. M.Shirakawa, Y. Uno, K. Yamatoya and K. Nishinari, This volume. 12. J. S. G. Reid, H. Edwards and I. C. M. Dea, In ‘Gums and Stabilisers for the Food Industry 4’, G. 0. Phillips, D. J. Wedlock and P. A. Williams Eds., IRL Press , Oxford, 1988,p.398. 13. A. Konno, Y. Azechi and H. Kimura,Agric.Biol.Chem., 1979,43, 101. 14. T. Harada, K. Okuyama, A. Konno, AKoreeda and A. Harada, Carbohydr.Polym., 1994,24, 101. 15. A. Konno, K. Okuyama, A. Koreeda, A. Harada, Y. Kanzawa and T. Harada, In ‘Food Hydrocolloids: Structures, F’roperties,and Functions’, Nishinari,K. and Doi, E. Eds., Plenum, New York, 1994, p.113. 16. A.H. Clark and S.B. Ross-Murphy, Adv.Polym.Sci., 1987,83, 57. 17. P.G. de Gennes, ‘Scaling Concepts in Polymer Physics’, Cornell Univ. Press, Ithaca, N.Y. 1979. 18. D. Oakenful1,J. FoodSci., 1984,49, 1103. 19. S. Onogi, T. Masuda and T. Matsumoto, Tram. SOC.Rheol., 1970,14, 275. 20. K. Nishinari, M. Watase and K. Ogino, Makromol.Chem., 1985,185, 2663. 21. A. Kose, M. Ozaki, K. Takano, Y. Kobayashi and S . Hachisu, J. Colloid Interface Sci., 1973,44, 330. 22. K. Kamide and M. Saito, Adv. Polymer Sci., 1987,83, 1.

25

RHEOLOGICAL AND DSC STUDIES OF AQUEOUS DISPERSIONS AND GELS OF CURDLAN

K.Nishinari, M.Hirashima, E.Miyoshi+,T.Takaya Department of Food and Nutrition, Faculty of Human Life Science, Osaka City University, Sumiyoshi, Osaka 558, Japan

'Present affiliation: Division of Development and Environmental Studies, Osaka University of Foreign Studies, Osaka 562, Japan

ABSTRACT Mechanical spectra of aqueous dispersions of curdlan showed solid-like behaviour both at 40°C and at 70"C, and the mechanical loss tangent for the dispersions decreased with increasing temperature. 2% dispersions heated at temperatures higher than 120°C did not show any endothermic peak in the second DSC run heating curves, indicating that structures formed at higher temperatures than 120°C are stable and thenno-irreversible. However, 2% dispersions prepared by heating in the temperature range from 55 to 115°C were thermo-reversible. Stress-strain curves for cylindrical gels of curdlan prepared at 90°C were determined.

INTRODUCTION Curdlan is a microbial polysaccharide, which consists of Dglucosyl residues connected by fi -(1+3)-linkages, produced by Alculigenesfaeculis vur. myxogenes 10C3 lo'. When a dispersion of curdlan is heated, it forms a gel. If it is heated around 60"c, and then cooled it forms a so-called low-set gel which is thermo-reversible '. If it is heated above 80"C, it is said that it fonns a thermo-irreversible gel '. Konno and his coworkers showed that the gel formed by dispersions heated in the temperature range from 60 to 120°C was thermo-reversible, and that gels formed by heating above 145°C were thermoirreversible. In spite of these tremendous efforts, the gelation mechanism of curdlan aqueous dispersions still remains unclear. Curdlan has been used for controlling the texture of noodles, surimi, hams and sausages, tofu and many other foods6. It has successfully been used for spherical jelly containing fruit juices; when dispersions of curdlan containing fruit juices are heated, gel

Structural and Functional Properties of Polysaccharides

27

formation occurs from the outer surface of the spheres. From tofu containing curdlan has a smooth texture. The measurements of dynamic viscoelasticity and stress-strain relationships, and differentialscanning calorimetry (DSC) were wried out to shed some light upon the relation between the structure and properties for the further development in the industrial utiliisation m the present work.

MATERIALS AND METHODS Powdered curdlan sample (Lot.DL15A) was kindly given by Takeda Chemical Industries Ltd.(oSaka, Japan). Powders were dispersed in distilled water by using a homogenizer at 1200 rpm and at O"c, then transferred into a vial which was soaked in icewater, and sonicated for 10 mm. The dispersion was stirred by using a magnetic stirrer at 40°C overnight, and used for dynamic viscoelasticmeasurement, yield stress measurement and differential scanning calorimetry. The dispersions were poured into a teflon mould with 2Omm diameter and 20 mm height, and hcated at 90°C for 6Omin. Then, these were kept at 40°C for 24 hours, and then at 5°C for 8 hours to make cylindrical gels for uniaxial compression tests. DSC measurements were undertaken using a sensitive DSC apparatus, PTClOD,8240A (Rigaku Denki Ltd., Tokyo) or a sensitive micro DSCIII from Setaram (Caluire, France). Powdered curdlan sample was sealed into a silver pan of 70 &t Q (Seiko Elecfmnics, Ltd., Tokyo) or into a stainless steel cell of lo00 &t Q from Setaram with distilled water. The sealed pan was left overnight before heating to attain equilibrium. Distilled water was used as a reference material. The storage shear modulus G' and the loss shear modulus G" of dispersions of polysaccharides were observed as a function of frequency at a constant temperature, and/or as a function of time at a constant frequency and temperature, and/or as a function of temperature at a constant frequency in the linear viscoelastic regime ( shear strain less than 4%) using a Fluids Spectrometer RFSII or a Dynamic Stress Rheometer DSR from Rheometrics Ltd.(NJ, USA). m e details of the dynamic viscoelastic measurements were described previously '-*. Yield stress of dispersions was determined using the RFSII or D!SR by two methods: 1)The measurement of shear stress as a function of shear rate and the extrapolationof shear rate to zero, 2) The measurement of shear stress and shear strain. m e strcss was applied at different time intervals, 6Os, 120s and 180s. The yield s m s was determined as the stress at which the relation bttwecn strcss and strain deviated from the linearity'.

Sms-straincuryes for gels of curdlan were measured at 25 f 1°C by uniaxial compression of a cylindrical gel (2Omm diameter and 2Omm height) at a compression speed of lOmm/s using a Rheoner RE3305 of Yamaden Co.(Tokyo, Japan). The plunger used was of teflon plate with a smooth surface (diameter:5(hnm).

28

Gums and Stabilisers for the Food Industry

Measurements of syneresis of gels Cylindrical gels of curdlan prepared by the method described above were cut into thin slices of 2.Omm thickness. These disks were put into aluminium cups, and weighed. These were dried at 30'C for 5 hours, and at 135'C for 2 hours. Then, these were put into a desiccator and left at room temperature to be weighed. The difference between the water content in gels measured just after preparation and in gels kept at 30'C for 5 hours is taken as exuded water.

RESULTS AND DISCUSSION Rheology The frequency dependence of storage and loss shear moduli, G' and G" for aqueous dispersions of curdlan of 2-4% at 40'C is shown in Figure 1A G' is larger than G" at all the frequencies and shows a plateau region even at lower frequencies. This is a typical behaviour seen in true elastic gels. The similar rheological behaviour for dispersions of colloidal particles which are not soluble in water has been also observed for dispersions of monodisperse polystyrene lattices 10 and xanthan-konjacmannan II. The difference in the mechanical spectra of "true" gels and the dispersions cannot be distinguished only by the measurement of the frequency dependence in the small deformation range 12. These dispersions have been shown to have a yield stress, and to flow under the shear higher than this yield stress whilst the true gels break beyond a certain deformation. 1000-,--------------,

10000-,-------------,

A

B tJ.tJ.tJ.tJ.tJ.l::.l:.f':,/''i tJ. ;> ~tJ.tJ.tJ.tJ.tJ.tJ.tJ.~~ooooooooooo 000

1000 00000

.

JDDDDDDDDDDDDDDDDDDDJ [

0 • • • • • • • • • • • • • • • • • • • 1• 100

••••••••••••••••••••••• 1-t----.------r-----,.-------1 0.01 0.1 1 10 100

10 -t-----r-----,.----.--------j 0.01 01 1 10 100

wlrad/s

wlrad/s

Figure 1

Frequency dependence of storage shear modulus G' and loss shear modulus G" for 2, 3 and 4 % aqueous dispersions of curdlan at 40'C(A) and at 70'C(B). O(G'), .(G',), 2%; O(G'), e(G"), 3%; 6(G'), .&(G"), 4%.

Figure 1B shows the frequency dependence of G' and G" for 2-4 % aqueous dispersions of curdlan at 70'C. Both moduli increased remarkably on heating, and tan = G"/G' is far smaller (about 0.1) for dispersions at 70'C than for dispersions at 40'C (about 0.3-0.4) indicating that the dispersions changed into a more solid-like state on heating; these gels formed at 70'C show fracture behaviour beyond a certain deformation. Figure 2 shows the shear strain for 2, 3 and 4% aqueous dispersions of curdlan at

a

29

Structural and Functional Properties of Polysaccharia'es

40°C as a function of stress which was applied at different time intervals (60s, 120s and 180s). All experimental strain values induced by stresses applied at different time intervals

coincide at low stress where stress and strain are linear indicating the elastic response, but a deviation was observed above a certain stress which is the yield stress '. The dipersion behaved as an elastic solid below the yield stress, and it began to flow above the yield stress. The values of yield stress determined in this way were O.lOPa, 0.12Pa and 1.2Pa for 2%, 3%and 4% dispersions respectively.

5

t /Pa

--

-I-601

-+-

1208

1-

Pr-

t/Pa

-D-

6or

-+-

1201 1801

t /Pa

Figure 2. Shear stress-strain curves for 2% (A), 3% (B) and 4% (C) aqueous dispersions of curdlan at 40°C.

30

Gum and Stabilisers for the Food Industry

The frequency dependence of storage and loss shear moduli, G’ and G”, solutions of curdlan in DMSO with concentrations 1, 2 and 3% at 4O‘c: showed a behaviour characteristicof a concentrated polymer ~olution’~ : G”predominates at lower frequencies but G’ is h - e r than G” at higher frequencia because molecular chains can disentangle during long periodsof oscillation at lower frequencies whilst the entanglement points play the role of a temporary crosslink during short periods of oscillation at higher frequencies. The cross-over frequency of G’and G” shifted to lower frequencies as has becn observed for many polymer solutio11s~~, including bhpolymer solutionssuch as hyaluronic acid solutions1s and gellan gum solutions 16. The behaviour of curdlan in DMSO is quite different from that in water because it is not soluble in water whilst it dissolves in DMSO. The yield stress of DMSO solutions of curdlan was negligiibly small in comparison to that of aqueous dispemions of curdlan. The elastic modulus of gels formed by curdlan-DMSO-water was shown to be maximum at around 0.277mole fraction DMSO at the temperature range from 15 to 85°C ”. Figure 3 shows the stress-strain curves of 2, 3 and 4% cylindrically moulded gels prepaed by heating aqueous dispersions of curdlan at 90°C for 6Omin. The elastic modulus E determined from the initial slope, breaking stress and breaking strain increased with increasing concentration C of curdlan. It is well known that the concentration dependence of the elastic modulus of gels can be represented by a power law E = AC‘ for sufficiently concentrated gels, and the exponent n has been shown to be about 2 for many gels. The exponent n for our curdlan gels was 1.75, and k was 30.02. These values are not so different from those for gels of kappaamageenan 1 8 . 0

.

---t 2% --t 3%

+ 4% 6 -

k

--. 4’

2-

0.0

0.1

0.2

0.3

0.4

0.5

strain

Figure 3. Stress-straincurves of aqueous curdlan gels of various concentrations prepared by heating at 90°C for 60 min. Compression speed, lOmm/s; Measurement temperature, 25°C. Differential Scannine Calorimetrv (DSC) Heating D!X curves for 5, 7.5 and 10 wt% curdlan dispersions showed an endothermic peak at about 5 8 C , which shifted very slightly to higher temperatures and became sharper with increasing concentration of curdlan. The appearance of an endothermic peak on heating should be attributed to the disordering of structure, or swelling of curdlan

31

Structural and Functional Properties of Polysaccharides

partides as is observed for the gelatinisation of starch ''.''. The endothermic enthalpy per lmg curdlan AH was 12.5mJ, which did not depend so much on the concentration in the concentration range examined. Figure 4 shows cooling DSC curves for 2% curdlan dispersions which had been heated at various temperatures for 30 min. from 50°C to 115°C. Cooling DSC curves for the solutions heated at temperatures lower than 50°C did not show any peak, while those heated higher than 55°C showed double exothermic peaks at 38°C and at 31°C. These exothermic peaks were attiiiuted to structural ordering due to the formation of hydrogen bonds, and the appearance of the two exothermic peaks suggests that the formation of gels occu~sby two steps or the formation of different structures. The exothermic enthalpy was maximum for the dispersion which was heated at 70°C. Since the curdlan suspension forms a gel on heating above the temperature range from 70 to 8O"C, the main molecular forces responsible for the formation of junction zones are considered as hydrophobic interactions. The dispersions which are heated above 120°C showed no exothermic peak in cooling DSC curves,indicating that gels formed on heating above 120°C become thermoirreversible and molecular rearrangements by the formation of hydrogen bonds on cooling are inhibited. The critical heating temperature 120°C above which the formed gels become thermo-irreversible is lower than 145°C reported by Konno et al ', and the reason for this may be attriiuted to the difference in molecular weight and concentration of curdlan. Junction zones formed on heating at temperatures higher than 120°C seem to be of a permanent character. The network structure formed at higher temperatures by heating is stabilised mainly by hydrophobic i n t d o n . Although hydrophobic interaction is a weak interaction with comparison to hydrogen bonding or covalent bonding, the accumulation of interacting sites by hydrophobic interaction may form a rigid structure in which the molecular motions are restricted. Then, the rearrangement of molecular chains is inhibited, and therefore the formation of hydrogen bonds on cooling is also prevented for these systems which have been heated above 120°C. The heating at higher temperatures may be necessary to swell and rearrange the curdlan molecules so that they may form a more stable

55 60

70 80 90

Temperature/c Figure 4 DSC cooling curves of 2% aqueous dispersions of curdlan kept at various temperatures for 30 min. Cooling mte:l"C/min. The numbers beside each curve represent the tempemlure in "C at which the dispersion was kept.

32

Gums and Stabilisersfor the Food Industry

associated structure. Although the dispersions heated at temperatures lower than 115°C showed exothermic peaks in cooling DSC curves which should be attributed to the structural ordering mainly by the formation of hydrogen bonds, dispersions heated at temperatures higher than 120°C may form junction mnes of a permanent character and further gel formation by hydrogen bonding on cooling does not occur for these dispersions. A structure formed by heating a 2% dispersion of curdlan at temperatures ranging from 55 to 115°C is partially thermo-reversible because a dispersion heated at these temperatures showed exothermic peaks in the cooling DSC curves and endothermic peaks in the second run heating DSC curves. This thermo-reversible network is formed by weakly bound molecular chains, and can be strengthened by hydrogen bonds formed on Cooling. The thermo-irreversible gels formed by heating at temperatures higher than 120°C seemed to have more rigid structure, and did not show any exothermic peak on cooling because the more rigid structure does not allow the rearrangement of molecular chains by the formation of hydrogen bonds on cooling. Figure 5 shows the degree of thermo-irreversible gelation dG which is defined as dG = l - A H , , / A H , for 10%aqueous dispersion of curdlan, where AH,, is the endothermic enthalpy determined from the endothermic peak in the s m n d run DSC heating curve for a curdlan aqueous dispersion kept at temperature T for 60 min., and AH , is the endothermic enthalpy in the first run heating. If gels formed by heating are completely thermoirreversible, no endothermic peak should appear in the second run DSC heating curve. As is seen clearly from this figure, the degree of thermo-irreversible gelation increases steeply in the temperature range from 50 to 70"C, and then continues to increase gradually with increasing temperature. It seems that aqueous dispersions of curdlan heated in the temperature range from 50 to 70°C form a partially irreversible gel, and tend to become almost irreversible gels above 120°C. The temperature range at which dG increases steeply should shift to lower temperatures with increasing concentration of curdlan. How fast dG increases should depend not only on the concentration but also on the molecular weight of curdlan, and this should be explored in the future.

,

0.2

-*o.o 40

60

80

100

1 0

Temperature/"c Figure 5 The degree of thermo-irreversible gelation dG = 1- AH, / A H ,for a 10% aqueous dispersion of curdlan as a function of heating temperature T,where AH 2T is the endothermic enthalpy determined from the endothermic peak in the second run DSC heating curve for a 10% aqueous dispersion of curdlan kept at temperature T for 60 min., and AH is the endothermic enthalpy in the first run heating.

Structural and Functional Properties of Polysaccharides

33

Svneresis When the concentrations of curdlan gels just after the preparation were 2, 3, 4, and 5wt%, the concentrations of curdlan gels kept at 30°C for 5 hours became 2.362, 3.255, 4.148, and 5.174wt%. Then, the syneresis ratios determined as the exuded water content were 15.627, 7.956, 3.696 and 3.528% indicating the syneresis was more conspicuous in dilute gels than in concentrated gels. When cornstarch was added to curdlan gels, the syneresis was inhibited. When the concentrations of corn starch added were 1 and 2wt% for 2wt% curdlan gels, the syneresis ratios were 6.605 and 2.919%. In conclusion, curdlan aqueous dispersions form thermo-reversible gels by heating in the temperature range from 55 to 115 “C, and a further heating at higher temperatures leads to the formation of thermo-irreversible gels. The aitical transition temperature should, however, depend on the concentration, and molecular weight of curdlan. Further structural studies should be performed. All these results are consistent with previous findings’” that gels formed at higher temperatures are thermo-irreversible, and that gels formed at lower temperatures are thermo-reversible although 120°C is far higher than a previously reported temperature (60-80°C) and lower than the recently reported temperature (145°C) ?he syneresis of curdlan gels can be prevented by adding corn starch.

’.

REFERENCES 1. T. Harada, Trends in Glycoscience and Glycotechnology, 1992,4, 309. 2. T. Harada, K. Okuyama, A. Konno, A. Koreeda and A. Harada, Carbohydr.Polym., 1994,24, 101. 3. A. Konno and T. Harada, Food Hydrocoll., 1991,5, 427. 4. Y. Kanzawa, T. Harada, A. Koreeda, A. Harada and K. Okuyama, Carbohydr.Polym., 1989,10, 299. 5. A. Konno, K. Okuyama, A. Koreeda, A. Harada, Y. Kanzawa and T. Harada, In K. Nishinari and E. Doi, (eds.), ‘Food Hydrocolloids: Structures, Properties, and Functions’, Plenum, New York, 1994, pp.113-118. 6. M. Miwa, Y. Nakao and K. Nara: ibid, pp.119-124. 7. E. Miyoshi, T. Takaya and K. Nishinari, Food Hydrocoll ., 1994,8, 505. 8. E. Miyoshi, T. Takaya and K. Nishinari, Macromol. Symp., 1995,99, 83. 9. H.Hoffmann, C. Thunig, P. Schmiedel and U. Munkert, Faraahy Discuss., 1995, 101, 319. 10. T. Matsumoto and T. Okubo, J. Rheol., 1991,35, 135. 11. K. P. Shatwell, I. W. Sutherland, S. B. Ross-Murphy and I. C. M. Dea, Carbohydr. Polym., 1991,14, 131. 12. A. H. Clark and S . B. Ross-Murphy, Adv. Polym. Sci., 1987,83,57. 13. M.Hirashima, T. Takaya and K. Nishinari , Zhermochim. Acta, in press. 14. W. W. Graessley, Adv. Polym. Sci., 1974, 16, 1. 15. Y. Kobayashi, A. Okamoto and K. Nishinari, Biorheology, 1994,31, 235. 16. E. Miyoshi, T. Takaya and K. Nishinari, Carbohydr.Polym., 1996,30, 109. 17. M. Watase and K. Nishinari, In K. Nishinari and E. Doi, (eds.) , ‘Food Hydrocolloids:Structures, Properties, and Functions’, Plenum, New York, 1994, pp. 125-129. 18. M. Watase and K. Nishinari: Colloid Polymer Sci., 1982,260, 971. 19. K. Kohyama and K. Nishinari, J. Agric. Food Chem., 1991,39, 1406. 20. J. Lelievre and H. Liu, ZRermochirn. Acta , 1994,246, 309.

Unique Gelling Properties of Non-starch Polysaccharides from Pre-processed Wheat Bran

W. (Steve) Cui, and Peter J. Wood, J. Weisz and J. Mullin Food Processing and Quality Improvement Program Agriculture and Agri-Food Canada 95 Stone Road West Guelph, Ontario, N1H 8J7, Canada FPQIP SO02 ABSTRACT Non-starch polysaccharides (NSP), extracted with 0.5M NaOH from pre-processed wheat bran, contained arabinoxylans ( 77%) and P-D-glucan (23%). Water-soluble pentosans have been reported to form gels by an oxidative process; this is believed to be an important functional component of bread quality. In contrast to the water-soluble pentosans extracted from wheat flour, NSP from pre-processed wheat bran formed gels upon cooling. A strong gel was obtained when 2.0% of NSP was cooled at 4°C for 16 hr. The gel structure changed as the temperature increased from 4°C to 70°C with an apparent structure breakdown temperature at about 40°C. Significant hysteresis was also observed between the cooling and heating curves of NSP gels.

INTRODUCTION Wheat pre-processing is a recently developed technology which removes bran gradually from the outer layers of wheat before milling (Dexter and Wood, 1996). The potential advantages of debranning before milling are (i) increased milling capacity and (ii) improved flour quality. An additional advantage of the gradual debranning technology is that it produces novel dietary fibre enriched bran fractions. Preliminary experiments carried out in our laboratory indicated that one of the bran fractions obtained by the pre-process technology (fraction B) is rich in alkali-extractable non-starch polysaccharides (mostly pentosans and P-D-glum). It is well known that water-soluble pentosans isolated from wheat flour can form irreversible gels upon oxidation (Hoseney and Faubion, 1981, Izydorczyk et al., 1991a). However, we observed that the non-starch polysaccharides extracted from wheat bran can form gels by cooling, and the gel structure changes with temperature. This phenomenon has not been reported in the literature for pentosans extracted from wheat. Therefore, the objectives of the present study were: (i) to extract and characterize the non-starch polysaccharides from preprocessed wheat bran, (ii) to study the unique gelling properties of NSP by rheological measurements.

Srructural and Functional Properties of Polysaccharides

35

MATERJALS AND METHODS Samples and Materials Commercial pre-processed wheat bran samples were obtained from E. Timm & Son Ltd, (Goole, UK). Thermal stable a-amylase (Bacillus subtilis) was purchased from Calbiochem (La Jolla, California, USA). All other chemicals were of reagent grade unless otherwise specified.

Extraction of Non-Starch Polysaccharides Pre-processed wheat bran samples were refluxed with 70% ethanol at 70°C in a water bath for 3hr. The ethanol extract was separated from the residue by centrifugation (20Oog, 10 min at room temperature). The residue was washed twice with 70% ethanol. The washings were combined with the ethanol extract as an ethanol soluble fraction, which was analysed for phenolics (Collins and D'Attilio, 1996). The ethanol extracted residue was first air dried at room temperature and then vacuum dried at 70°C for 4hr, which is noted as Residue A (Fig. 1). Residue A (log) was dispersed and heated in 20Oml water (PH 6.5-7) to 95"C, then pre-heated (95°C) a-amylase (3,000 unit) was added. The system was kept at 90-95°C for 30min then allowed to cool to room temperature. The a-amylase soluble fraction was separated from the residue by centrifugation (50008) for 15 min. The residue was washed twice with 50 ml of water. The a-amylase soluble fraction was analyzed for pentosans and PD-glum while the residue was vacuum dried at 70°C for 4hr as Residue B. Residue B was extracted with 0.5M NaOH for 2 hr at room temperature then neutralized with 2N HCl. The mixture was centrifuged at 5 OOOg at room temperature for 20 min to separate the supernatant from the residue. The supematant was precipitated in 2 vol of 95% ethanol, separated by centrifugation. The precipitate was washed twice with 95% ethanol then freeze dried.

Chemical and Monosaccharide Analyses Ash and moisture were determined according to AOAC methods (AOAC, 1980). Protein was determined by Lowry's method Gowry et al., 1954). Monosaccharide analysis of the non-starch polysaccharides was carried out by hydrolysing the polysaccharides in 1M HzSO~at 100°C for 2 hrs. The hydrolysates were diluted and filtered, then analysed by a Dionex system as described by Wood et al. (1994).

Rheological Measurements and Sample Preparation 2.00/0 (w/w) of NSP was dissolved in distilled water at 80°C for 30 min, then cooled to 25°C. Centrifugation was used to remove air bubbles. Gelling was at 4°C for 16hr. Dynamic rheological tests were performed on a Bohlin CVO stress controlled rheometer (Bohlim Instruments, N.J. USA) with a cone-plate geometry (4" angle and 40 mm diameter).

36

Gums and Stabilisers for the Food Industry

RESULTS AND DISCUSSION Chemical Composition of Pre-processed Wheat Bran Proximate analysis of the bran fractions obtained from wheat pre-processing is summarized in Table I. The bran fractions removed by the debranning process were -10% of the starting wheat. Of the bran fractions, fraction A accounted for 40% which was mainly composed of insoluble fibre (77.2%) with only 1.6% soluble fibre. A small amount of starch (4.3%) was also detected in this fraction together with 0.4% of f3-Dglucan. Fraction C had the highest level of starch. However, the contents of f3-D-glucan, soluble and insoluble dietary fibre were comparable to that of fraction B on a starch-free basis. Since fraction B had the highest level of f3-D-glucan (2.6%, compared to 0.5% in the wheat and 0.4% in fraction A) and soluble dietary fibre (8.5%) with a relatively low level of starch contaminant, an extraction of alkali-soluble non-starch polysaccharides (NSP) was done using this fraction. Table I: Proximate analysis (dry weight basis) of commercially produced pre-processed wheat fractions. Sample

Yield'

Starch

f3-D-Glucan

(%)

(%)

(%)

IDFD SDP" TDFD (%)

(%)

(%)

Starting wheat

100

59.6

0.5

8.4

3.0

11.4

Pre-processed wheat

90

67.5

0.6

5.1

3.0

8.0

Finished flour

---

75.2

0.2

1.4

2.3

3.7

Bran Fraction A"

4.0

4.3

0.4

77.2

1.6

78.8

Bran Fraction Be

3.0

13.4

2.6

29.2

8.5

37.6

Bran Fraction CO

3.0

31.9

1.9

15.7

6.8

22.5

a: yield was estimated from commercial production. b: IDF, SDF, TDF; insoluble, soluble and total dietary fibre, respectively. c: Fraction A, B, and C; friction, 1sl abrasion and 2nd abrasion products respectively (Dexter and Wood, 1996). Extraction of Non-Starch Polysaccharides (NSP) from Wheat Bran Fraction B As shown in Figure 1, wheat bran fraction B was first treated with 70% ethanol at 70nC for 3hr to remove the ethanol soluble materials, such as fat, free sugars, amino acids and some phenolics. The residue from ethanol extraction, Residue A, was then digested by thermal stable a-amylase to eliminate starch which might contaminate the NSP in a later stage. There was no arabinose and xylose detected in the a-amylase treated liquid phase upon acid hydrolysis, nor f3-D-glucan. This result suggests that there is no l3-glucan that is soluble in hot water and the starch in Residue A can be removed without losing the alkali-extractable non-starch polysaccharides. The a-amylase treated

Structural and Functional Properties of Polysaccharides

37

residue, Residue B, was then extracted with 0.5MNaOH at room temperature for 2 hr. There were no starch contaminates found in the extracted NSP.

I Pre-Processed Wheat Bran I Ethanol Soluble

1

I ResidueA I a-Amylase and Hot Water Soluble

I Residue B I G"tralk3

I

NSP (Non-Starch Poly saccharides)

Figure 1: Processing flow chart for the extraction of non-starch polysaccharides from pre-processed wheat bran.

The yield of NSP was 18% of Residue B, accounting for about 9%of the original bran (fraction B). The NSP was not extractable by hot water and required alkali for extraction. It appeared that alkali broke down the barrier which kept NSP in the cell wall. This might be explained by the presence of phenolics in wheat bran, e.g. ferulic acids. These phenolics might be involved in cross-linking with the pentosans, which would prevent these polysaccharides being solubilised in water. Closely associated and entangled, but non-cross-linked polysaccharides,might also be rendered water insoluble. The NaOH by breaking down the cross-linking, would enable the solubilisation of NSP in aqueous solutions. The extracted NSP is easily dispersed in cold water; however, heat is necessary to bring the polysaccharides into solution.

38

Gums and Stabilisersfor the Food Industry

Chemical and Monosaccharide Compositions of NSP NSP contained about 6% proteins, 4.9% ash and 5.6% moisture. By difference, the carbohydrate content was 83.5%. The total neutral sugar content of NSP was estimated (following acid hydrolysis) about 58% (unpublished data), which suggested that then might be substantial amounts of uronic acid and/or phenolic acid associated with the extracted NSP. The relative monosaccharide composition of NSP is presented in Table 2. Arabinose and xylose accounted for 77% of the total sugar, and glucose 22.9% with only a trace amount of galactose. The ratio of xylosdarabinose in the pentosans was 3 which differed significantly from the value of 1.1 to 2 of pentosans extracted from wheat flour (Izydorczyk and Biliaderis, 1994). This structural difference might be responsible for the unique properties of NSP from pre-processed wheat bran described later, although it contains a substantial amount of P-D-glucan. Table 2. Relative neutral monosaccharide composition of NSP

NSP

Arabinose

Xylose

G1ucose

Galactose

19.0%

58.0%

22.9%

0.1%

Gelling Properties of Non-Starch Polysaccharides (NSP) Solutions/Dispersions Most water-soluble pentosans from wheat flour exhibited Newtonian flow behavior up to 1.5% polymer concentration, except pentosans extracted from the cultivar Katepwa, which exhibited shear thinning flow behavior at 1% polymer concentration at high shear rate, but Newtonian flow behavior was reported at 0.75% polymer concentration (Izydorczyk et al., 1991b). Water-soluble pentosans from wheat flour can also form irreversible gels via covalent cross-linking of phenolic acid substituents (Geissmann and Neukom, 1973). NSP extracted from pre-processed wheat bran (fraction B) exhibited a weak gel property at room temperature, where the storage modulus G'was greater than the loss modulus G" over a wide range of frequency, as shown in Figure 2. When 2.0% (w/w) NSP sample was kept at 4°C overnight (-16hr), a strong gel was formed. The mechanical spectrum of this gel at 4°C is shown in Figure 3. The storage modulus G' was almost independent of frequency; in contrast to G', the loss modulus G" increased substantially with the increase of frequency. When the same sample was warmed to room temperature, the gel became weaker as the storage modulus G' was more frequency dependent (Figure 4) compared to the mechanical spectrum at 4°C (Figure 3).

39

Structural and Functional Properties of Polysaccharides

100

10

1

Frequency (Hz) Figure 2. Mechanical spectrum of 2.0% NSP in water at 25°C.

10

-

0.01

0.1

1

10

Frequency (Hz) Figure 3. Mechanical spectrum of 2.0%NSP in water at 4°C after cooling at 4°C for 16 hr.

40

Gums and Stabilisers.for the Food Industry A

Frequency (Hz) Figure 4. Mechanical spectrum of 2.0% (w/w) NSP in water at 25°C after cooling at 4°C for 16 hr.

n ([I

50-

cn

3

40-

U

2

g

302 0 -

([I L

0

CI

10 -

v, 0

10

20

30

40

50

60

70

80

Temperature OC Figure 5. Heating and cooling curves of 2.0%(w/w) NSP in water at 0.1 Hz.

Structural and Functional Properties of Polysaccharides

41

Figure 5 represents the heating and cooling curves of 2.0% (w/w) NSP in water. In the heating curve, G' decreased slightly with temperature at the lower temperature end, then approached a plateau until 30-35°C. A steady decrease of G' was then observed from 40 to 70°C. On the cooling curve, G' increased slowly when the sample started to cool from 70°C. A sharp increase of G was observed when the temperature was below 20°C. This sharp increase in storage modulus G indicates the development of an ordered structure. Between the cooling and heating curves, significant hysteresis was observed which suggests that a substantial amount of energy is required to destroy the gel structure. It is worth noting that heating the sample at the preparation stage is crucial to obtain a gel. If the sample is not heated, there is no gel development upon cooling. This observation suggests that there might be some synergistic interactions between the arabinoxylans and P-D-glucans present in NSP, since energy was needed to unfold the ordered structure of the individual polysaccharides in order to form a more ordered gel structure upon cooling. However, further research is required to fully understand the mechanisms involved in the gelling process of NSP.

CONCLUSIONS From this study it is found that pre-processed wheat bran fraction B is rich in non-starch polysaccharides (NSP). It is a potential source of both soluble (8.5%) and insoluble (29.2%) dietary fibres. The alkali extractable NSP was a mixture of pentosans (-77%) and P-D-glucans (-23%) and exhibited unique gelling properties upon cooling. This gelling property is different from water-soluble pentosans isolated from wheat flour, which only form irreversible gels by oxidative gelation.

ACKNOWLEDGMENT The authors thank E. Timm & Son Ltd, (Goole, UK) for the pre-processed wheat bran samples. REFERENCES AOAC. 1980. Official methods of analysis. 13"'. Ed. Ed. Association of Official Analytical Chemists, Washington, D.C. Collins, F.W. and D' Attilio, R. 1996. Phenolics of preprocessed wheat: Isolation, structure and quantitative analysis of major bran constituents. AACC Annual Meeting, September 15-19. Pp 249. Dexter, J.E. and Wood, P.J. 1996. Recent applications of debranning of wheat before milling. Trends in Food Sci. & Techn. 7:35-41. Geissmann, T. and Neukom, K.1973. On the composition of the water-soluble wheat flour pentosans and their oxidative gelation. Lebensm. Wiss. Technol. 659.

42

Gums and Stabilisersfor the Food Industry

Hoseney, R.C. and Faubion, J.M. 1981. A mechanism for the oxidative gelation of wheat flour water-soluble pentosans. Cereal Chem. 58:421-424. Izydorczyk, M., Biliaderis, C.G. and Bushuk, W. 1991a. Oxidative gelation studies of water-soluble pentosans from wheat. J. Cereal Sci. 11: 153-169. Izydorczyk, M., Biliaderis, C.G. and Bushuk, W. 1991b. Physical properties of watersoluble pentosans from different wheat varieties. Cereal Chem. 68: 145-150. Izydorczyk, M. and Biliaderis, C.G. and Bushuk, W. 1994. Studies on the structure of wheat-endosperm arabinoxylans. Carbohydr. Polym., 24:61-71. Lowry, 0.1.,Rosebrough, N.J., Farr, A.L. and Randall, R.J. 1951. Protein measurement with the folin phenol reagent. J. Biol. Chem., 193:265-275. Wood, P.J., Weisz and Blackwell, B.A. 1994. Structural studies of (1-’3),(1-’4)-fl-Dglucans by C”-nuclear magnetic resonance spectroscopy and by rapid analysis of cellulose-like regions using high-performance anion-exchange chromatography of oligosaccharides released by lichenase. Cereal Chem. 71:301-307. Wood, P.J., Weisz, J., Fedec, P., Burrows, V.B. 1989. Large scale preparation and properties of oat fractions enriched in (1+3),(1+4)-P-D-glucan. Cereal Chem. 66197-103.

INTERACTION BETWEEN POLYMER MOLECULES IN LOCUST BEAN GUM-WATER SYSTEMS DURING COOLING AND FREEZING PROCESSES Ryohei Tanakal, Tatsuko Hatakeyama' and Hyoe Hatakeyama3 Forestry and Forest Products Research Institute, Tsukuba Norin Kenkyu Danchi-nai, P.O.Box 16, Ibaraki 305,Japan National Institute of Materials and Chemical Research, 1-1 Higashi, Tsukuba, Ibaraki 305,Japan Fukui Institute of Technology, 3-6-1 Gakuen, Fukui 910, Japan

1. INTRODUCTION

Aqueous solutions of locust bean gum (LBG) do not form a hydrogel by ordinary gel-forming conditions when heated to near boiling point of water and then cooled in a refrigerator (ca. SOC). However, when an aqueous solution of LBG is frozen and thawed, a hydrogel is formed. Research on this type of hydrogel from galactomannan polysaccharides including LBG has been carried out by Dea et ul. in the 198O's'*'. Investigation of the gelation mechanism and properties of the hydrogels is still continuing. In the previous study, we have reported that a hydrogel from a LBG solution shows syneresis by r e p t i n g a freezing-thawing treatment (Figure 1) and that the gel strength increases with the number of freezing-thawing cycles (Figure 2)'. It was found that gelation conditions such as freezing-thawing cycles and cooling rate affect the interaction between polymer molecules, resulting in the variation in the gel strength.

solution

hydrogel

f

Y "

0

Flgure 1 Physical appearance of 1 % LBG hydrogel

1 2 3 4 Number of freezing-thawing cycles

5

Figure 2 Vkcosity of 3% LBG hydrogel afer each freezing-thawing cycle

Gums and Stabilisers for the Food Industry

44

In this paper, further investigation of the formation of junction zones in LBG water systems including hydrogels and solutions has been carried out by viscoelastic studies. The behaviour of LBG - water systems in these studies is compared with those of other galactomannan polysaccharide - water systems.

2. MATERIALS AND METHODS LBG was purchased from Sigma Chemical Co., U.S.A. with molecular weight of 3. lx105as reported. The molecular structure is shown in Figure 3. Tara gum and guar gum consist of the same mannose backbone and galactose side chains with different galactose / mannose ratios, which are 1:3 for tara gum and 1:2 for guar gum. Those samples were lundly supplied by Fuji Kagaku Kogyo Co., Ltd., Japan and by Daiichi Kogyo Seiyaku Co., Ltd., Japan under the trade names of Snow Gum F-80 and DKS Fine Gum G-270, respectively. An appropriate amount of a polymer sample was placed in a 20ml glass container with approximately log of deionised water and heated at 105°C for 30 min. The heated solution was then cooled to room temperature before rheological measurements. An Advanced Rheometnc Expansion System (ARES) (Rheometric Scientific Inc., U.S.A.), fixed with either a 25mm or a 40mm parallel plate measuring system, was used for all rheological measurements in this study. Operation mode was a dynamic frequency sweep over the frequency range of 0.1-100 radlsec (= 0.016 16 Hz) at a fixed temperature of 2 5 T . Freezing-thawing and cooling-heating treatments of polymer solutions could be carried out in the sample holder of the instrument, as the temperature was controlled by a furnace fitted with a liquid nitrogen supplier. When a sample solution was cooled on the ARFS, the cooling rate was l"C/min and then heated at S"C/min immediately after cooling. The temperature at which the sample was cooled is defined as T,. Accordingly, T, of a frozen sample is the freezing point of the polymer solution, and T, of an unfrozen sample is the minimum temperature during cooling .

-

HO

Figure 3 Molecular structure of locust bean gum

Structural and Functional Properties of Polysaccharides

45

3 . RESULTS AND DISCUSSION Figure 4 shows frequency sweeps of the storage modulus (G’) and the loss modulus ( G ) before and after cooling to Tx= 4.493 (a), T,= -5.393 (b) and Tx= -14.2“C (c). At 4.493, the aqueous sample was not frozen and there were no changes in G’ and G measured after heating to 25°C. When the sample was frozen at -14.2“c, a hydrogel was formed and the G’- and G”- frequency curves show a typical gel pattern, i.e. both moduli are independent of frequency. At -5.393, the sample was not frozen, so gelation did not occur. However, there are slight deviationsin the G’ and G” curves Observed as shown in Figure 4-(b). Figure 5 shows G’ ratio (G’cyl I G’cyo) plotted against T,. G’qois the G’at 3.16 radlsec (= 0.5 Hz) measured before cooling (cycle 0) and GIcyl is that measured after cooling (cycle 1). When T, was below -6.593, LBG solutions were frozen and gels were formed. This temperature range represents a ‘frozen area’. In this area, the G’ ratio increased as T, decreased. The reason for this phenomenon is still not clear. However, we have found that the size of ice crystals is reduced when the cooling rate at freezing increases, resulting in an increase in the strength of LBG hydroge13. Therefore, the variation in the G’ ratio at different T,’s below -6.593 may also be explained by the size of ice crystals formed during freezing. Tx’s between -6.5Tand -2.593 are categorised as a ‘transitional temperature area’, where the aqueous solution is not frozen but a marginal increase in G’ is observed. This increase in G’ suggests that LBG molecules interact with each other in aqueous solution. The freezing process, i.e. the formation of ice crystals between LBG molecules, seems to be essential for gel formation. However, the presence of the transitional temperature area suggests that the interaction between the polymer molecules occurs at a temperature higher than the freezing point. When LBG solution was stored at a temperature above a freezing point of water for several days, a non-equilibrium state of the solution was also observed. Figure 6 shows transitions of G’ and G” of a 3% LBG solution during storage at 5°C for 13 days. Compared with Figure 4, the spectrum patterns of the solution even after 13 days of storage are not gel-like and the physicai apparance of the solution was of a viscous fluid. The ratios of G’ (frequency = 3.16 radlsec) of each measuring day to that of day 0 are plotted as shown in Figure 7. The ratios of G’ of 3% LBG solution stored at cu. 23°C (room temperature) and those of 2% tara gum and 5% guar gum stored at 5°C are also plotted. G’ of the LBG solution was 1.5 times higher after storing at 5°C for 13 days. When the LBG solution was stored at 2393, G’ did not change for the first 7 days and showed a slight increase after 13 days. These facts suggest that LBG solution is in a nonequilibrium state at both 593 and 23°C. On the other hand, the tara gum and p a r gum solutions did not show such increases in G’ after storing at 593. Instead, the viscoelasticity of these polymer solutions decreased steadily during storage, probably because of the increase in the solubility of polymers in water. As reported previouslg, tara gum and guar gum do not form hydrogels by freezingthawing. The formation of hydrogels is dependent on the distribution of galactose side chains on the mannose backbone, as suggested by Dea el d.l,’ They concluded that blocks

Gums and Srabilisers for rhe Food Indusrry

46

A A dtcw cwllng

1000 . h

a b b

. . . . . 1.

10

10' .1

100

10

1

Frequency (radk)

100

Frequency (rad/s)

Figure 4-(a) Frequency sweeps of G' (open symbols) and G" (closed symbolr) of 3% LBG solution before and after cooling: T, = 4.4"C

Figure 44b) Frequency sweeps of G' (open symbols) and G " (closed symbols) of 3% LBG solution before and after cooling: T, = -53°C

2.51

.

.

.

.

.

.

.

2.0 -

%

El

c

: 1.5

2

. :

0 1.0

.1

..................... 1

10

FROZEN

100

Frequency (rad/s)

Figure 44c) Frequency sweeps of G' (open symbols) and G" (closed symbols) of 3% LBG solution before and after cooling: T, = -14.2"C

Figure 5 An effect of T, on G'of 3% LBG solution f hydrogel

47

Structural and Functional Properties of Polysaccharides

.1

1

10

Frequency (radls)

Figure 6 Frequency sweeps of G’ and G” of 3% LBG solution stored at 5°C

100

0

5 10 Days of storage (n)

15

ngUre 7 EffecrS of storage on G’s of galactomannan plysaccharide solutions

containing only mannose units in different polymer molecules were attached side by side to form the gel structure. The interaction between LBG molecules during storage can be explained in the same manner, as the interaction did not occur in the tara and guar gum solutions. However, the interaction is too weak to form a gel and only a viscous fluid can be formed. 4. CONCLUSIONS

The strength of a locust bean gum hydrogel prepared by freezing-thawing is dependent on the temperature reached on freezing. At temperatures above the freezing point of water, there is a transitional temperature range where a LBG solution does not become a gel, but the solution viscosity increases. Viscosity of a LBG solution also increases after storage at an appropriate temperature. The increasing rate of G’ is higher at a lower temperature than at a higher temperature. This fact indicates that the viscoelasticity of LBG solution depends on temperature and time. References 1. I.C.M.Dea, E.R.Morris, D.A.Rees, and EJ.Welsh, Carbohydr.Res., 1977.57, 249. 2. I.C.M.Dea, ‘Industrial Polysaccharides: Genetic Engineering, Structure I Property Relations and Applications’, ed.M.Yalpani, Elsevier Science Publishers B.V.,

Amsterdam, 1987,p207. 3. R.Tanaka, T.Hatakeyama and H.Hatakeyama, Polymer International, in press.

STUDIES ON THE RHEOLOGICAL BEHAVIOUR POLYSACCHARIDE AQUEOUS SOLUTIONS

OF

THE

CV-70

A. R. P. Scamparini, D. M. Mariuzzo and J. D. S. Gutierrez Department of Food Science, Food Engineering Faculty State University of Campinas, P.O.Box:6121, CEP 13081-970, Campinas, SiTo Paulo, Brazil

ABSTRACT The CV-70 gum is a water-soluble polysaccharide produced by bacteria Beijerinckia sp. found in soil cultivated with sugar cane. The aim of this work was the study of the rheological behaviour of CV-70 aqueous solutions using purified (P) and non-purified (NP) CV-70 gum. After the fermentation period, the CV-70 gum was purified using enzymatic hydrolysis with papain solutions (0.05 g/ml), at pH 6.5, 65"C, for 3 h. After dialysis, the gum was re-precipitated, dried, and powdered. CV-70 aqueous solutions were prepared at the original pH (6.5 and 6.4, for P and NP, respectively), and the rheological measurements carried out at 15, 25 and 45OC. Changes in the CV-70 solutions pH were made, to pH 2.0 and 12.0. Results showed that the protein present in the CV-70 NP solutions had altered the rheological behaviour when compared with CV70 P solutions, giving smaller apparent viscosity values. pH changes were capable of affecting the rheological characteristics of both, CV-70 P and NP solutions. Temperature increase led to a decrease in the solution's apparent viscosity. All CV-70 solutions showed non-Newtonian behaviour, fitting a pseudoplastic model, where the apparent viscosity decreases with the shear rate increase.

1 INTRODUCTION

Microbial polysaccharides, known as biopolymers, are produced by almost all microorganisms but, from the point of view of commercial production, fungi and bacteria are the easiest to use. Such microorganisms have the capability to grow in pure cultures, in large-scale bath fermentations.' Furthermore, :they produce biopolymers with potential applications in a variety of industrial sectors. In the food industry, there are numerous applications for biopolymers, which are used as thickening agents and suspending or gelling polymers. The industrial interests are centered on extracellular polysaccharides, due to the ease of their extraction and purification, in a good yield. If a

Structural and Functional Properties of Polysaccharides

49

biopolymer is to have a large range of applications, it must possess rheological properties, whereby their viscosity decreases in the presence of a shear stress. Also, these properties must be continuous during temperature, pH and ionic strength changes. For future industrial applications, the knowledge of the above properties of biopolymers is important and affects the end-product characteristic^.^,^ Polysaccharide production, purification and characterization has been studied for many years, and a lot of purification techniques were developed. Nevertheless, almost of them are very timeconsuming and expensive. The aim of this work was the study of procedures for polysaccharide purification, using for this, the proteolytic enzyme papain, and comparing its effects on the rheological behaviour of CV-70 polysaccharide. CV-70 polysaccharide was produced by bacteria Beijerinckia sp. isolated fiom soil cultivated with sugar cane, in Ribeiriio Preto, S.P., Brazil. Beijerinckia sp. is a genus of gram-negative rods, aerobic bacteria, usually found in sugar cane root, capable of producing polysaccharide under cellular stress conditions. The CV-70 polysaccharide was produced in a fermentation medium containing 5% sucrose as the carbon source, tryptose and salts, at 25°C. The biopolymer structure is not completely defined, but previous studies showed it is composed of glucose, galactose and fucose in 3: 1:3 ratio? Primary rheological studies on CV-70 polysaccharide showed that this biopolymer has strong pseudoplastic characteristics and the existence of a high yield stress and high apparent viscosity at low shear rate. The dependence of ap arent viscosity of CV-70 aqueous solutions on temperature changes was confirmed too.

P

2 METHODS 2.1 CV-70 Polysaccharide Production A strain of Beijerinckia sp. isolated from sugar cane root was used to produce the CV-70 polysaccharide under aerobic fermentation. The fermentation medium containing 5% sucrose as carbon source, 0.05% MgS04, 0.01% K2HPO4, 0.05% KH,PO, and 0.5% tryptose was sterilized at 121°C for 15 min. Fermentation conditions were 200 rpm for 72 h, at 25°C. After this period, the fermentation broth was centrifuged at 11SO0 x g, 30 min and the polysaccharide was precipitated with 80% ethanol, yielding 7-9 g/l. The CV-70 polysaccharide was dried at 55°C under vacuum and powdered.

2.2 Sample Purification and Preparation In order to obtain the purified polysaccharide (P), a 1% aqueous solution was treated with a solution of papain (0.05 g/ml) at pH 6.5 and 65°C for 3 h, until protein presence was not detected, then dialysed against distilled water for 3 days, as previously studied4. 1% CV-70 polysaccharide (P and NP) aqueous solution were prepared with ultrapure water (NANOPURE), with magnetic stirring at 5PC. pH changes were made with 2N HCl and 50% (w/v) NaOH.Al1samples were sonicated before measurements in order to eliminate bubbles.

Gums and Stabilisersfor the Food Industry

50

8.00

i

15 "C

6.00

v) v)

4.00 v)

2.00

0.00 0.00

100.00

200.00

300.00

Shear Rate [1/s]

Figure 1 Flow curvesfiom CV-70 P (1%) aqueous solution, pH 6.5, at 15, 25 and 45°C

51

Structural and Functional Properties of Polysaccharides

15 "C

10.00

12.00

v) v)

0

8.00

4.00

0.00

0.00

100.00

200.00

300.00

Shear Rate [lls]

Figure 2 Flow curvesfiom CV-70NP (1%) aqueous solution, pH 6.4, at 15,25 and 45 "C

Gums and Stabilisers for the Food Industry

52

2.00 1

15 "C 1.60 I

v!

a

.-h

1.20

. I -

v)

0

0 v)

5 S E a

I

. I -

0.80

P

'

2

0.40

0.00

I

I

I

Figure 3 Apparent viscosity curves from CV-70 P (1%) aqueous solution, pH 6.5, at 1.5, 25 and 45°C

53

Structural and Functional Properties of Polysaccharides

8.00

-

'I< I

45 "C I

I

I

I

Figure 4 Apparent viscosity curvesfrom CV-70 N P (I%) aqueous solution, p H 6.4, at 1 5 2 5 and 45 'C

54

Gums and Stabilisersfor the Food Industry

6.00

i

pH = 6.5

i

, /

, , '

-

, ,

, x

pH=2

4.00

Q

B

2.00

0.00 0.00

100.00 200.00 Shear Rate [l/s]

300.00

Figure 5 Flow curvesfrom CV-70 P (1%) aqueous solution, T = 25 "C,atpHs 2, 6.5 and 12

55

Structural and Functional Properties of Polysaccharides

16.00

1 i

12.00

u) v)

cn

8.00

4.00

0.00 0.00

100.00

200.00

300.00

Shear Rate [l/s]

Figure 6 Flow curvesfiom CV-70NP ( I %) aqueous solution, T = 25 'C,atpHs 2, 6.4 and 12

Gums and Stabilisers for the Food Industry

56

0.80

pH = 6.5

0.60

-

i

v!

(d

L .-c fn 0

8

0.40

5 c C

!z

CTI

pH=2

Q

2 I

0.20

0.00

I

0.00

I

I

100.00

200.00

300.00

Shear Rate [l/s]

Figure 7 Apparent viscosity curves from CV-70 P ( I %) aqueous solution, T = 25 "C, at pHs 2, 6.5 and 12

57

Structuraland Functional Properties of Polysaccharides

2.50 -, , pH = 6.4

, , ,

2.00

(jj'

eli

s, >-

1.50

.~

In

0

s

c:

~

, ,

, , ,

(J

In

-

, , , , , , , , , , ,

,

1.00

11l

, , ,

,

Co Co

, ,

-c

',pH =2

0.50

pH = 12 ~~-~~=~-----~-----0.00

100.00

200.00

300.00

Shear Rate [1/5]

Figure 8 Apparent viscosity curves from CV-70 NP (1 %) aqueous solution, T pHs 2,6.4 and 12

=25 ·C. at

Gum and Stabilisersfor the Food Industry

58

2.3 CV-70Gums Rheological Measurements

A Haake Rheometer, model CV2ON, plus Rotovisco 303 software was used. It is a rotational rheometer, equipped with a coaxial cylinder sensor. The inner cylinder was held at rest and the outer was subjected to a defined shear stress. Fluids were placed in the annular gap. The rheometer was adjusted to produce a shear rate from 0 to 300 s-', following the return to 0 s-'. Sample temperatureswere kept at 15, 25 and 45°C using a constant temperaturethermal liquid bath and circulator.

3 RESULTS AND CONCLUSION All the CV-70 aqueous solutions showed typical properties of pseudoplastic fluids as can be noted in Figures 1, 2, 5 and 6. Figures 3, 4, 7 and 8 showed that for all CV-70 aqueous solutions the apparent viscosities decreased strongly with the increase of shear rate. Despite the fact that the hysteresis curves were not totally overlapped, they were considered to be, due their small variation. It was possible to observe a small yield point in P solutions at 45°C (Figure l), and at 25"C, pH 12 (Figure 5). At pH 12, and 25"C, NP solution showed an initial shear stress, too (Figure 6). CV-70 P solutions showed an apparent viscosity three folds smaller than the CV-70 NP solutions as can be noted in Figures 3, 4, 7 and 8. It is, therefore, possible to say that the protein present in the biopolymer increased its apparent viscosity. Temperature changes affected the apparent viscosity of P and NP solutions in the same way. On increasing the temperature the apparent viscosity of solutions decreased. pH changes of CV-70 polysaccharide solutions showed a larger apparent viscosity at their natural pH (pH 6.5 to P and 6.4 to NP). A smaller apparent viscosity was observed for solutions in strongly basic (pH 12) solutions for P and NP. Strongly acid solutions showed less change but, P and NP solutions showed a decrease in apparent viscosity values. In conclusion, CV-70 polysaccharide purification using papain solutions did not affect the rheological behaviour of purified related to non-purified CV-70 aqueous solutions and this process could be used to purify this polysaccharide. Further studies should take place, using different polysaccharides, rheometers and conditions as pH, temperature, shear rate, shear stress and frequence variation to confirm this.

References 1 . I. W. Sutherland, 'Biotechnology', H. Dellweg, Verlag Chemie, Weinhein, 1983,3, 3, 531. 2. M. Rinaudo, J Appl. Polymer Sci.: Applied Polymer Symposium, 1993,52,11. 3. M. Milas, M. Rinaudo, M. h i p p e r and J. L. Schuppser, Macromolecules, 1990,23, 2506. 4. D. M. Mariuzzo, Msc Thesis, State University of Campinas, 1996. 5. C. T. Vendruscolo, PhD Thesis, State University of Campinas, 1995.

Effects of Salts and Saccharides on Rheological Properties and Pulsed NMR of Rice Starch during Gelatinization and Retrogradation Processes

K. KATSUTA Department of Food Science & Nutrition, Nara Women’sUniversity Kitauoyanishi-Machi, Nara 630, Japan

ABSTRACT Changes in the dynamic viscoelastic properties of rice starch suspension (paste) during heating (gelatinization process) at rate of 1“c /min and changes in the static viscoelasticity (creep parameter) of starch gels during storage (retrogradationprocess) were observed. The effects of some salts (NaCI, KCI, CaC12, BaCL and Mgc12) and saccharides (maltose) on their properties were investigated. ’Ihe pulsed NMR measurements for the starch gels were also carried out. When salts were added to the starch-water system, the onset temperature of gelatinization for starch was increased, but the behavior of temperature dependence of G’ for the starch-water-saltssystems after passing the maximum peaks was similar to that for the starch-water system. The decreasing rate of creep compliance of starch gels with storage time was innuend by salts and saccharides, and the rate constant, i.e., hardening rate (k) ,was controlled by them. This shows that salts as well as saccharides prevent the retrogradation of starch. ’The spin-spin relaxation time (Tz) obtained by pulsed NMR of stored starch gels became shor&erwith addition of salts and saccharides, suggesting that salts and saccharides impede the mobility of water and flexibility of starch chains in the gels.

INTRODUCTION

Rice is the most important food stuff for the Japanese. Rheological studies of rice starch and rice produds have been carried out’”. However, there are no reports on the kinetic treatment of changes in the rheological properties of rice starch gels during retrogradation except that by Katsuta et a f 9 . Katsuta and co-workers studied the retrogradation of rice starch gels by. kinetic treatments on the basis of viscoelastic parameters and reported that saccharides stabilized the gel matrices in the starch-water system.

60

Gums and Stabilisers for the Food Industry

‘Ihe effects of saccharides on the gelatinization of starches have been extensively studied and reported. Many workers have stated that saccharides increase the gelatinizing Hansen and ~o-workers’~ classified the mechanisms into three types; 1) competition between the saccharides and starch for available ~ a t e r ; ’ ~ 2) . ’ ~inhibition of the swelling of the starch gran~les;‘~’’’~’~ 3) penetration to the starch granule and interaction in the amorphous area”. In our previous papers, we reported that the ability of saccharides to stabilize the gel structure was found to be closely related to the stereo-chemical conformation of saccharide molecules7’. That is, saccharides stabilized the water structure in the starch-water system, hence the gel matrices were strengthened and the flexibility of the starch chains was reduced. If the ability of saccharides in impeding the retrogradation of starch can be explained by the structure-making effect for water, some ions will possess similar effects. In the present study, therefore, the effects of some salts on the retrogradation and gelatinization of rice starch were investigated by static and dynamic viscoelastic measurements. Additionally, the spin-spin relaxation times of retrograded starch gels containing the salts were compared to those with saccharides.

MATERIALS AND METHODS Materials Non-glutinous rice starch was obtained from Shimada Kagaku Kogyo Co. Ltd. (Niigata, Japan). Reagent grade saccharides and salts were used without further purification in this study. Preparation of starch paste (sol) Rice starch was weighed into a flask and degassed under vacuum to remove the air for 15 min. Then degassed,doubly distilled water was added to the flask and degassed again while stirring for 60 min, and salts andlor saccharides were added and stirred again for 30 min. The starch suspension was heated at 55 “c for 3 rnin while stirring to obtain a homogeneous sol state. The sol (paste) was studied by dynamic viscoelastic measurements and gels prepared. Preparation of starch gels The starch sol mentioned above was poured into glass tubes precoated with coating reagent, Sigma-coat (Sigma Co., St Louis, MO, USA), and heated at 6 5 c for 5 rnin to obtain the fixed sample. After centrifugation at 700 X g for 20 rnin to remove air bubbles, the tubes were heated at 95°C for 90 rnin for complete gelatinization, and then cooled and stored for different periods of time at 0°C. The stored sample was carefully removed from the tube, cut into cylindrical blocks (11 4 X 10 mm) using an Ultrasonic Sample-Cutter (USC-3305, Yamaden Co., Tokyo, Japan) to produce a smooth surface and submitted to creep measurements. Dynamic viscoelastic measurements A rheometer (MR-3001500 Trial Model by Katsuta, Rheology, Co., Kyoto, Japan) was used for the dynamic viscoelastic measurements of starch paste.

Struciural and Functional Properties of Polysaccharides

61

An aliquot of starch paste was placed in the gap (50 nm and a revised gap for increasing temperature) between the cone (5.295 deg, 3.196 cm diameter) and plate. The exposed sample surface betwem the mne and plate was covered with silicone oil, and then test fixture, cone-and-plate, were wholly covered by a bob-comb type case filled with silicone oil to prevent the evaporation of water from the sample during testing. Sinusoidal strain with a frequency of 0.16 Hz ( o =1rad/sec) and with the angle of oscillation of 0.2 deg (7=0.038)was applied.

Static viscoelastic (Creep) measurements A Creep Meter (Rheoner RE-3305/33005, Yamaden Co., Tokyo, Japan) was used for the static viscoelastic (creep) measurements. Measurementswere carried out under uniaxial compression at 25°C for 300 sec. Nuclear magnetic resonance (NMRI measurements A pulsed NMR spectrometer (JNM-MU 25A Model, JEOL, Tokyo, Japan) was used to measure the spin-spin (Tz)relaxation times. The measurements of TZwere made using the Gill-Meihom modification of Carr-Purcell pulsed sequence (CPMG method) at 25 MHz . The starch pastes alone and containing salts and/or saccharides were carefully transferred to the NMR tube and bubbles were removed by centrifigation. After gelatinization at 95 “c , the tubes were stored at 0 “c for 24 hrs and used for the experiments.

RESULTS A N D DISCUSSION Effects of salts on the retrogradation kinetics In our previous works6-9’21,the creep tests were carried out and the creep compliance value was defined as follows, J =J (300)-t

m/

D

(1)

where J (300) is the creep compliance value at 300 sec, t the observation time and 71 the steady flow viscosity, and the changes in the J value of rice starch gels with storage time could be expressed by a first-order reaction equation. However, as shown in Figure 1, the plots of l o g J versus storage time t for 30% rice starch gel containing 200 mM KCI is not straight line but a little curvilinear. The result indicates that the reaction governing the retrogradation process of starch is the n-th order reaction, hence the decreasing rate of J with time t can be represented by following equation.

62

Gums and Stabilisers for rhe Food Indusrry

Integrationof Equation (2) in cases of n =1and n Z 1gives J /Jo=exp (-k t )

n =1

(3)

nfl

(4)

and

J / J ~ = [l+(n-l)k t respectively. Here, J Equation (4) gives

also

1-"('4

is the creep compliance value at t = 0. In the case of n =2,

J/J0=1+kt

(5 )

1/J = l / J o + ( l / J , ) k t

(6)

-4.1 -

- 8 -4.2 - 8 n

T

g

-4.3

-4.5

-

' 0

30% Rice Starch + 200mM KCI

8

'

R I

I

1000 2000 Storage Time (min)

3000

Figure 1. Logarithmic values of creep complianceJ against storage time t for 30%rice starch gel containing200 mM KCI.

4

+200mMKCI

c3 d X

a b 5 2 1 30% Rice Starch

1 0

1000 2000 StorageTime (min)

Figure 2. 1/J versus t plots for rice starch gels.

3000

Structural and Functional Properties of Polysaccharides

63

In Figure 2, the plots of 1/ J versus t for the rice starch gels are presented. Since the plots for both gels with and without KCI were almost straight lines, the retrogradation process of rice starch could be expressed by the second order reaction. Retrogradation rate k of the control is larger and initial creep complianceJ, is smaller than the gels containing KCI, indicating that KCI strengthens the gel matrix of rice starch and impedes the retrogradation of starch (Table I). Table 1 Effects of salts on retrogradationrate k and initial creep compliance J, for 30%rice starch gels. J, (Pa", X 10-4) Control +200mM KCI +200mM NaCl +lo0 mM BaCL, +loo mM CaCh +lo0 mM MgC1,

9.22f0.51 7.25 0.37 7.11 f0.33 6.39 f0.31 6.36 f0.30 6.06 f 0.30

*

k (min",

X

10-3

6.27 f 0.32 4.29f0.21 4.07 0.19 5.53f0.22 5.68f0.22 5.97 f 0.24

*

The values show the average obtained from 5 to 7 series experiments. The results in Figures 1and 2 show one series data. The ability of KCI in impeding the starch retrogradation is almost similar to NaCI, though NaCl is more effective than KCl. The sodium ions have a structure-makingeffect for water, but potassium ions do not. When salts were added to starch gels, their ability to impede the retrogradation of starch could not be explained only by the structure-making effect for water. Another possibility should be explored in the future.

Gelation and fusion profile of rice starch during the gelatinization process The changes in storage shear modulus (G') with temperature, i.e., temperature dependence,for 10%(W/W) rice starch paste was previously observed and classified into five stages and shown as gelation and fusion profiles as in Figure 3"*". In stage I , the value of G' rapidly increases, resulting from the expansion of the volume fraction of the solid layer (starch) due to the absorption of water. The value of G' either remains constant or increases or decreases slightly in stage fl because of the interaction between expansion of the volume fraction and the Nling-in between particles. In stage IlI , the value of G' increases and reaches the peak. After passing the peak, the value of G' decreases. This stage obviously corresponds to the disappearance of the particulate structure. This is the fusion process and a major event taking place during this stage IV is the melting of crystallites. At the point of the minimal peak of G', the fusion of crystallites is concluded. The value of G' increases again after passing the minimum peak. This stage seems to involve development of mechanical strength and the starch chains probably develop a better "knitting" structure. In this stagev , however, evaporation of water from the knitting structuremust occur. On the other hand, the temperature dependence of G' for 30% star& paste was different from the profile for 10%paste. The development of G' with temperature for 30% paste could be classified into four stages (Figure 4).

64

Gums and Stabilisers for the Food Industry

10 6 10

n

2

y

10 10

10 10 10 10 10 50

60

70

80 90 100 110 Temperature ( g= )

120

Figure 3. Gelation and fusion profiles for the gelatinization process of 10%rice starch paste.

10

lo5 n

10 4 10 3

5

102

co

i5 10' 10 O 10 -l 10 -2 50

60

70

80 90 100 Temperature ("C )

110

120

Figure 4. Gelation and fusion profile for 30% rice starch paste. The value of G' for 30% starch rapidly increases by several orders of magnitude to about lo4 Pa. The onset temperature, To, corresponds to the temperature for the commencement of the gelatinization of starch. This increase of G' value reflects the expansion of the volume fraction of the starch granule as for 10% starch paste, but the were maximum value is greater than that for 10% starch (lo2 Pa). The stages and distinguishable in the profile for 10% starch, but not in that for 30% starch. In the case of

65

Structural and Functional Properties of Polysaccharides

30% starch paste, starch particles and/or granules are closely packed at Tp, hence the effect of filling-in between granules might become dominant rather than the expansion of volume fraction. This behavior in stage fl in Figure 4 is characteristic of the 30%, i.e., concentrated, starch paste which is opposite to 10% (dilute) paste. At the fusion temperature, Tf, the melting of crystallites begins and it is completed at T,.

Effects of salts on the gelation and fusion profile of rice starch

10

30% Rice Starch

n

Q

jj:/

0

e5 10 2

3

o Control 0 +200mM NaCl

10 10 O

1Wmin

10 -l

0

10 -2

Q

60

50

.

t

70

.

t

.

1

.

1

.

90 100 Temperature (“c ) 80

1

.

110

120

Figure 5. Effect of NaCl on the temperature. dependence of G’ for 30% rice starch paste.

KCI NaCl Control 30

40

50

60 70 Temperature (T)

80

Figure 6. Effects of salts on the onset temperature To and peak temperature Tp. In Figure 5, the temperature dependence of G’ for 30% rice starch paste containing 200 mM NaCl is shown and cornpard to the control. When NaCl was added to the

Gums and Stabilisers for the Food Industry

66

starch-water system, the onset temperature To shifted to higher temperatures, and the temperature of the end of melting of crystallites, T, , shifted to lower temperature, but G' value for melted starch-water-NaC1 system was found slightly greater than the control. 1 0 5 ~

; 30% Rice Starch

50

60

90 100 Temperature ("c)

70

80

8

110

120

Figure 7. Effect of BaCh on the temperature dependence of G ' for 30% rice starch paste. The results indicate that NaCl delays the commencement of the gelatinization and strengthens the structure of starch-water system. The similar temperature dependencies of G' were observed from the other starch-water-salts systems (Figure 6),except systems containing BaCIz (Figure 7), although the profiles for the other starch-water-salts systems were not shown.

The spin-spin (Tz) relaxation time of starch gels The spin-spin relaxation lines of retrograded starch gels with and without salts were linear (Figure 8). When 200 mM KCI was added to the starch-water system, the mobility of the system decreased. The T2 values, calculated from the lines in Figure 8 and the results obtained from the other temperatures, are plotted against measuring temperature in Figure 9. At any temperatures, the TZvalues for starch-water-salts system were smaller than those for the starch-water system. This means that salts impede the mobility of water in gels as well as saccharides. Divalent ions, Mg2+,reduce the Tz value rather than monovalent ions. However, adding monovalent ions to starch-water system, similar TZ values were observed for Na' and K'. Sodium ion has structure-makingeffects for water as well as saccharides and Mg", but potassium ion has structure-breaking effects. In the case of ions, the ability to stabilize the starch-water system might be different from saccharides. The clathrate compound with starch chain and ions, such as amylose-iodine complex, might be introduced to consider the effects of salts on retrogradation of starch.

67

Structural and Functional Properties of Polysaccharides

1

0 n c)

sM

-1 +200mMKCI

-2

100

0

200

300

Time (ms) Figure 8. The spin-spin relaxation lines for rice starch gels. The gels were stored at 0°C for 24 hrs, and the signal amplitude, M(t), was observed at 30°C by the CPGM method.

1 n

1.9

0

0

W

2

8

0

-

2

I

30% Rice starch gels

0

1.8

-

0

0

0

A

d

n

0

0

A

A

l A

1.7 I 20

0

I

I

30

40

I

50 Temperature ('C)

I

60

70

Figure 9. Effects of salts and maltose on TZvalues measured at various temperature. Control (30%rice starch gel : and 30%rice starch gels containing 200 mM NaCl 200 mM KCl (A), 100 mM M g C l z (A) and 30 mM Maltose (0).

(a),

o),

CONCLUSION When salts were added to the starch-water system, the mobility of water in the system decreased, hence the association of the starch chains was retarded. 'Ihis is the

68

Gums and Stabilisers for the Food Industry

reason why salts and saccharides impede the retrogradation of starches. However, the species of ions among monovalent (and among divalent), did not significantly affect the gelatinization and retrogradation of starch. It should be considered that not only the interaction between ions and water but also the formation of compound between ions and starch chain, such as amylose helix and/or double helix of amylopectin, in starch-water system contributes to the stabilization of the starch-water system.

REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9.

10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24.

H. Horiuchi:Agric. Biol. Chem., 1980,44, 1231. R. Chinnaswamy, K.R. Unnikrishnan and K.R. Bhattacharya: Starch, 1985,37, 99. S . Tsuji: J. Jpn. SOC.Food Sci., Technol., 1988,35, 742. M. Takagi, L. Zhang, M. Kawai, N. Morita and S. Matsumoto:J. Jpn. SOC.Starch Sci., 1989,36, 239. S. Nagashima, S. Sawayama, A. Kawabata and M. Nakamura: . Jpn. SOC.Starch Sci., 1990,37, 21. K. Katsuta, M. Miura and A. Nishimura: Food Hydrocoll., 1992,6, 187. K. Katsuta, A. Nishimura and M. Miura: Food Hydrocoll., 1992,6, 387. K . Katsuta, A. Nishimura and M. Miura: Food Hydrocoll., 1992, 6, 399. K . Katsuta, A. Nishimura and M. Miura: "Food Hydrocolloids: Structure, Properties, and Functions", eds. by K. Nishinari and E. Doi, Plenum Press, New York, 1994, p.175. M.N. Bean and W. YamazakkCerealChem., 1978,55, 936. K. Kospel and R.C. Hoseney: Cereal Chem., 1980,57, 49. A. Chungcharoen and D.B. Lund: CerealChem., 1987,64, 240. L.M. Hansen, C.S. Setser and J.V. Paukstelis: CerealChem., 1989, 66, 411. R.I. Derby, B.S. Miller, B.F. Miller and H.B. Trimbo: Cereal Chem., 1975,52, 702. R.C. Hoseney, W.A. Atwell and D.R. Lineback: Cereal Foods World, 1977,22, 56. L. Slade and H. Levine: "Industrial Polysaccharides" eds. by S.S. Stivala, V. Crescenzi and I.C.M. Dea, Gordon and Breach Science, New York, 1987, p.387. B.L. D'Appolonia: CerealChem., 1972, 49, 532. H.L. Savage and E.M. Osman: Cereal Chem., 1978, 55,447. M. Wooton and A. Bamunuarachchi: Starch, 1980,32, 126. R.D. Spies and R.C. Hoseney: Cereal Chem., 1982,59, 128. M. Miura, A. Nishimura and K. Katsuta: Food Structure, 1992,11, 225. N. Nakajima and D.W. Ward: J. Appl. Polym. Sci., 1983,28, 807. N. Nakajimaand M.R. Sadeghi: Int. Polym. Processing, 1989, 4(1), 16. K. Katsuta: J . Appl. Glycosci., 1996, 43, 541.

F A 0 Regional Project on Gum Arabic Specifications and Quality Control

E. Casadei Food Quality and Standards Service Food and Nutrition Division FA0 Rome Italy

1

Introduction

As defined by the joint FAOWHO Expert Committee on Food Additives and Contaminants (JECFA), Gum Arabic, also called Acacia Gum, is a dried exudation obtained from the stems and branches of Acacia senegal (L.) Willdenow or closely related species. It consists mainly of a complex, slightly acidic polysaccharide and their calcium, magnesium and potassium salts, which on hydrolysis yields arabinose, galactose, rhamnose and glucuronic acid. The precise chemical and molecular structure differs according to the botanical origin of the gum, and these differences are reflected in some of the analytical properties of the gum. As a result, the functional properties, uses to which gum arabic is put, and its commercial value are also very dependent on its origin. Unground gum arabic occurs as white or yellowish white spheroidal tears of varying size or as angular fragments. It is also available commercially in the form of white to yellowish white flakes, granules, powder or spraydried. Gum arabic has been used in a wide range of food products for many years. It is a unique multi-functional food additive being emulsifier, flavouring agent, humectant, stabilizer, thickener, surface-finishing agent, and in addition, retards sugar crystallisation, which is an important property in typical applications. The trend which has enhanced the growth of gum productioduse over the past decade has been the increasing consumption of convenience foods. As in most other sectors of the additives industry, increasing health-consciousness has tended to fuel growth for thickeners of natural origin. Irrespective of their intrinsic qualities, however, “two disadvantages” of natural exudate gums - their irregularity of supply and consequently wide fluctuating prices - have caused them to lose market share in favour of their competitors, modified cellulose, modified starches and biosynthetic gums. Towards the end of the 1960s total world gum arabic production was around 70 OOO tonnes. Events in the 1970s and ‘80s led to fluctuations in both the supply and price of gum arabic and, as a consequence, to changes in demand. The severe Sahelian drought of 1973/74 resulted in a world shortage of gum arabic and high prices. A low point of approximately 20 OOO tonnes of Sudanese exports was reached in 1975, which recovered to around 40 OOO tonnes during 1979. A further drought in 1982-84 saw levels of exports fall to below 20 OOO tonnes in some years in the midllate 1980s and early 1990s.

70

Gums and Stabilisers for the Food Industry

Uses of gum arabic fall in three categories; food, pharmaceutical and technical. Typical applications in food are the uses in beverages, candies, chewing gum, confectionery, dairy products, fats, fillings, frostings, nut products, nuts, puddings, snack foods and tinned vegetables. Gum arabic is a valuable export product which has all the potentials to provide employment and income for thousands of people in gum producing countries and elsewhere, if the collection, processing, quality control and marketing is adequately organized. Therefore, the strengthening of gum arabic production by stabilizing the supply and improving the quality of the gum to comply with the requirements of the gum arabic identity and purity specifications of the Joint FAO/WHO Expert Committee on Food Additives (JECFA), and of the users, is an absolute necessity. The latest JECFA gum arabic specifications for identity and purity, prepared by the Expert Committee in 1995, refers to gum arabic as the “dried exudation obtained from the stems and branches of Acacia senegal (L.) Willdenow or closely related species of Acacia (fam. Leguminosae)”; the previous JECFA specification (FA0 1990) also used the term “closely related species”. It is not clear which species can be considered “closely related species”. The recent JECFA specification (FA0 1995) has dropped the use of specific rotation and nitrogen as purity criteria, facilitating the commercialisation of mixtures of different gums under the generic name of gum arabic. The specifications developed by the Committee had three purposes: to identify the substance that has been subject to toxicological testing; to ensure that the substance is of the quality required for safe use in food; and to reflect and encourage good manufacturing practices. In addition to the above three purposes, the JECFA specifications should also reflect the product in commerce. The specifications of this important natural gum will be reconsidered again by the committee as soon as major data for revaluation become available.

FA0 regional project under Technical Co-operation Programme on “Quality Control of gum arabic” TCPIRAF14557 2

In view of the regional nature of gum arabic production and related problems, and in order to meet the needs of African producers whilst at the same time trying to resolve some of the ambiguities referred to above, FA0 formulated the above project which had the following objectives: a) to acquire information on all aspects of gum arabic production and quality control, and on the basis of recommendations for improvements, assist producer countries in their efforts to improve the quality of their products so as to meet international specifications. b) to generate the necessary botanical and analytical data on gum arabic produced by different countries, and traded in international markets, for use by JECFA in developing international specifications for food-grade gum arabic.

Structural and Functional Properties of Polysaccharides

71

The project was elaborated as a regional project, covering the producing countries of the so-called gum belt of Sub-Saharan Africa. The belt stretches as a broad band from Mauritania, Senegal and Mali in the west, through Burkina Faso, northern Benin, Niger, northern parts of Nigeria, Cameroon and Chad, northern Central African Republic to Sudan, Eritrea, Ethiopia and Somalia in the Horn of Africa. Sudan is the world’s biggest producer of gum arabic, and since very little is consumed domestically it is also the main source of gum international trade. Until recently Nigeria was the second biggest producer and exporter. Of the other producers, Chad is the next most important after Sudan and Nigeria. Although some of the gum exported from Chad, as well as Central African Republic is believed to originate from Sudan through illegal cross-border trade, most of the increase in imports from Chad is a result of increasing local production.

In view of the information available on gum arabic producing countries, the regional project was elaborated including twelve African countries, eleven from the gum belt of Sub-Saharan Africa and Zimbabwe, because of its production of commercial gum arabic from Acacia karrao, a species most widespread in Southern Africa. The interested countries were: Burkina Faso, Chad, Ethiopia, Ghana, Kenya, Mali, Mauritania, Niger, Nigeria, Senegal, Sudan and Zimbabwe The expected outputs of the project were: a) the production of documented data on botanical sources, characteristics and properties of gum arabic, produced and traded by the 12 African countries b) reviews available on gum arabic production and quality control systems and on identity and purity specifications applied in each of the above countries with proposals for actions to improve the quality of their products so as to meet international specifications 2.1

WorkDlan

Gum arabic international specialists, under the technical supervision of the Food Quality and Standards Service (ESNS) and in close collaboration with the national counterparts made field visits to gum arabic producing countries, to review production and quality control systems and applied specifications in each country. A plan for sample collection was elaborated during the briefing session and adopted after consultation with other members of the mission. The plan required obtaining two types of samples from each country:

- authentic samples from the main gum producing species collected by consultant botanists or national experts. These samples were used as reference for each species; - typical commercial samples offered on the market but with known background i.e. indicating commercial name and geographical origin.

72

Gums and Stabilisers for the Food Industry

A total of 101 samples of gums representing the main commercial plant gums produced in Sub-Saharan Africa were collected from 12 countries. The samples were analysed using the main analytical methods of physico-chemical, carbohydrate and cationic composition. Representative samples were also analysed for amino acids and molecular profiles.

3

Results and conclusion

3.1

Botanical sources and management asDects

A total of 17 Acacia species were identifie1 in the twelve African countries as producing gum collected by local communities - either for export or for domestic use. Acacia senegal, A . seyal and A . polyacantha have widespread distribution within the gum belt. Other gum yielding Acacia species have a limited regional distribution. For instance, A. karoo is confined to Southern Africa, A.drepanolobium and A . paoli to eastern Africa and the Horn of Africa, while A . laeta and A . dudgeoni are confined to West Africa. A . gournensis, A . macrostachya and A. macrothyrsa have even more restricted distribution in West Africa. 3.2

Production, quality and markets

The project estimated production level for gum arabic and the main species producing Acacia gum in the twelve countries. It illustrates the wide variation in the scale of production, although Sudan, Nigeria and Chad are very much bigger producers than the rest. Sudan is pre-eminent not only because it accounts for the majority of gum arabic in the world trade but because it sets the standards by which others are judged in terms of quality. Nigeria has the benefits of the same botanical resources as Sudan but has a poor reputation for the quality of its gum; its best is a good as Sudanese gum but much of it is of variable and inferior quality. Chad has recently increased production and quality of its gum arabic is comparable to Sudanese. The quality of gum from the other countries is variable. Table 1 shows estimated production level for gum arabic and the main species producing Acacia gum in the 12 countries. 3.3

ComDosition. identity and Duritv

The data generated from the analysis of 101 samples were evaluated by three methods; comparison of the analytical data based on aggregate parameters, use of molecular profiles and multivariate statistical analysis known as chemometrics. Acacia senegal and A . seyal were confirmed as the main sources of gum arabic of commerce accounting for up to 95% of total gum produced. A. senegal contributes about 70% and A . seyal 1525%. The remaining comes from A . polyacanrha and A. laeta that are often sold in admixture with A . senegal gums among West African producing countries. The mean values[physicochemical, carbohydrate and amino acid composition] for gum from A. senegal and A . seyal were consistent with published data and typical of each type of gum irrespective of source i.e. country or locality.

73

Structural and Functional Properties of Polysaccharides

Table 1 Summary of gum arabic data for 12 African producing countries (botanical source, production, imports into EC, USA and Japan, and main European markets) Annual imports to main European

Country

Main botanic source

Annual productiona

Annual imports to EC, USA, Japanb

Sudan

A. senegalvar. senegal A . seyal

17,100 3.900

EC USA Japan

12,200 3,800 1,750

EC USA Japan

4,500 300 3

Germany France

2,500 1,300 650

A . senegal var. senegal } A. seyal

Nigeria

6,OOO-lO,OOO?

1

France

4,900

UK

2,400 2,300 1,300

Italy Germany

UK

Ethiopia

A . senegal A . seyal

250-300 50-100

EC USA Japan

80

Germany

80

Kenya

A . senegal var. Rerensis) A . senegal var. senegal }

200-500?

EC USA Japan

40

Italy UK

25 10

Zimbabwe

A . karroo

<30

EC USA Japan

Ghana

A . polyacantha A . sieberana


EC USA Japan

UK

50

A . senegal

200 -300

Burkina Faso

A.

30 2

50

Seyal

A . Senegal A . Seyal

3,500 1,500

EC

Mali

A . senegal A . Seyal

500

EC

140

Mauritania

A . senegal

400

EC

180

Niger

A . senegal A . seyal

300

EC

SCnCgal

A . senegal

700

EC

Chad

Notes:

3,500

UK

2,800 600

France

45

150

France

115

450

France UK

300 130

a Estimates except for Sudan which are 7-year annual averages (1988-94) b Annual averages fromtrade statistics (EC and Japan 1988-93: USA 1991-94)

Source FAO, A Review of Production, Markets and Quality Control of Gum Arabic in Africa, by B.N. Chikamai, Rome.1996

France

74

Gums and Stabilisersfor the Food Industry

It was clearly demonstrated that though related i.e. possessing the same chemical species, the two gums could be distinguished from each other by all the three methods. This supports the idea of producing and marketing the two gums separately if future improvements in quality and quality control are to be attained. It was shown further that A. laeta and A. polyacantha are closely related to A. senegal while A. karroo is closely related to A. seyal. An evaluation of the methods revealed that chemometrics when applied to the analytical data obtained in the investigation is a powerful method of characterising the gum arabic of commerce, by identifying individual species of Acacia and those gums which would be adulterants within the terms of the JECFA definition of gum arabic. The conventional analytical methods are capable of differentiating between the two main sub-genera on the basis of optical rotation and the relative proportions of nitrogen (hence protein) content, viscosity. composition of individual sugars and uronic acid. Such sugar analyses would also show the presence of non-Acacia gums, particularly the presence of other non-permitted sugars such as mannose, galacturonic acid or xylose. While the method of molecular characterisation based on gel permeation chromatography (GPC) can distinguish different gums on the basis of the shape of elution profiles, it seems to be influenced greatly by the equipment and skill of the operator. These factors can affect interpretation on the results and hence specification of gum arabic. 4

Recommendations

The project recommended that further assistance be provided to gum arabic producing countries to improve the quality of their products so as to meet international specifications: (i) the preparation and publication of a manual or technical profile, covering all aspects of gum arabic production, from collection from the tree to the point of export of the product; (ii) a package of training initiatives be developed to promote “good manufacturing practice” and “quality consciousness” among producers and traders of gum arabic at all levels with the manual serving as one of the main tools for training; (iii) priority be given to undertaking resources survey in all the countries producing gum arabic (with the exception of Sudan and Chad) and improving the resource base. itself. The project has also elaborated specific recommendations, based on botanical and analytical data generated from the project, to JECFA in developing international specifications for food-grade gum arabic as follows: the term gum arabic in the present JECFA specifications should be revised (i) to include A. senegal and A. seyaf and “closely related” species. The words

Structural and Functional Properties of Polysaccharides

75

"closely related" should apply to those species established in the study to be chemically closely related to A. Senegal and A . seyal respectively; (ii) in view of the observed analytical differences between the two main gums, they should be produced and marketed separately so as to minimise variability and improve aspects of quality and quality control. Data generated in the study should be used in drawing up the revised specification; (iii) the general methods by JECFA need to be updated to take into account newer methods e.g. chemometrics in specifying gum arabic of commerce. This is in addition to the current methods (FA0 1995).

Note: The present paper was extmcted from the following reports:

-

TCP/RAF/4557 Report of a review of production, markets and quality control of E. Casadei, J:J. W. Coppen, H.O. Ab&l Now, D. Cesareo) FA0 1996

gum ambic in Africa (B.N Chikumai,

-

TCP/RAF/4557 Report of the Chemical Analysis for the clranrcterisation and specification of gum ambic (B.N. Chikamai, G.O. Phillips, E. caradei) FA0 1996

GUM ARABIC REPLACEMENT IN CONFECTIONERY APPLICATIONS

P.Lawson, M. Gonze, F. Van Der Schueren and K. Rosenplenter Cerestar Application Centre Food Havenstraat 84, B- 1800 Vilvoarde, Belgium

1 ABSTRACT

Gum Arabic is a natural product obtained mainly from trees grown in the Sudan. As the climate of this area is subject to dramatic changes, the quality has been variable. In addition, the availability has been dependent on political factors and the price is higher than other polysaccharides. For these reasons, the Starch Industry has been striving to develop a replacement The emphasis has been on matching the properties and not the structure of Gum Arabic. This paper describes the breakthrough by Cerestar in developing a modified starch which not only mimics the properties of Gum Arabic in certain confectionery applications, but offers additional advantages. This modified starch is C*AraTex. It is based on tapioca starch, modiEed by acid treatment to reduce viscosity and by hydroxypropylation to provide stability. C*AraTex is successfully used to match the hard, brittle texture of confectionery gums and the sealing properties in coated sweets, giving equivalent excellent shelf-life. In both application areas, advantages such as greater flexibility in textures, lower viscosity, lack of lump formation using low speedflow shear mixers, easier water evaporation during stoving and faster coating times are found with C*AraTex. When combined with maltitol syrup, good quality sugar-free gums can be formulated. Details of the properties and applicationsof C*AraTex are described. 2 INTRODUCTION

Gum Arabic is the dried exudate obtained from various species of trees of the genus Acacia. About 500 species of Acacia can be found over Africa, Asia, Australia and Central America, but only the varieties from the republic of Sudan and its neighbowing countries in West-Africa are commerually important The exudate is collected by hand and sorted in central collection stations, before being exported, further processed and graded. The colour of the gums in the natural exudate form varies from almost colourless through shades of yellow, amber and orange to dark brown. Gum Arabic is a polysaccharide with a relatively high molecular mass and a complex structure’ (as shown in Figure 1). It is a branched molecule with a protein content of about 2.0 2.5 56. The molecule consists of a p-(1+3) linked galactopyrawse backbone chain with numerous branches linked through p-(1+6) galactopyranose residues and containing arabinopyranose, arabinofuranose, rhamnopyranose, glucuronic acid and 4-O-methyl-glucuronic acid (the compositionis shown in Table 1).

77

Structural and Functional Properties of Polysaccharides

Gum Arabic can be separated into three principal fractions, namely an arabinogalactan, an arabinogalactan-protein complex and a glycopotein. Table 1 Chemical Composition of Gum Arabic

Compound

Galactose Arabinase Rhamrose

Glucuronic acid Protein

Abbreviation

Galp.

Araf. Rhap. GIcA. n.a.

% in Gum

36.0 30.0 12.6 19.2 2.2

As Gum Arabic is a natural product, its quality is variable because the g e c m a s of production are subject to dramatic climatic changes. There have been availability problems due to political turbulence in these regions and the price is expensive compared with otfier polysaccharides. Despite these problems, Gum Arabic is used in food. non-food and pharmaceutical industries. In the confectionery industry, it is a traditional gelling agent for gums or pastilles and as a sealing agent in coated sweets. How&er, as technical innovation in equipment and formulations has imeased drastically, a consistent quality, Iowa cost, readily available altemative, which matches or i m p v e s the application properties, has been sought. This challenge has been met with C*AraTex, a modified starch.

78

Gums and Stabilisersfor the Food Industry

3 C*ARATEX C*AraTex is a tapioca starch which has been modified by the processes of acid thinning and etherillcation (see Figure 2). These processes are approved by European food legislation.

[Etherification by hydroxypropylation

I

in

Figure 2 Starch Chemical Modifications

d ‘cn

The treatment is carefully controlled to give a well defined d e w of substitution and degradation that mimics the functional properties of Gum Arabic. The use of tapioca starch as raw material offers several advantages in the production of modified food starch, such as a bland taste, good stability, and good clarity due to its high amylopectin content (approx. 83 %). However, the hot viscosity of pasted native tapioca starch is far too high for confectionery use. To reduce this, acid thinning with either hydrochloric or sulphuric acid is carried out below the gelatinisation temperature of the tapioca starch. The a-1, 4 bonds are more prone to hydrolysis than the a-1, 6 bonds. Acid modification occurs by preferential attack of the acid on the amorphous section of the granule. The level was selected so that the hot viscosity was very similar to that of Gum Arabic. As a consequence, retrogradation is increased as more molecules are in the optimum molecular size range for association : DP80 DP100. However, there was a much faster viscosity increase on cooling (set-back). To control this tendency of retrogradation, a second modification was applied. This was treatment with ppylene oxide which introduces mono-functional side groups onto the starch molecule. The reaction is known as etherification and was carried out under strong alkaline conditions,giving high stabdity towards hydrolysis (higher than by introducing an acetyl group). The hydroxypmpyl groups caused a weakening and disruption of the associative forces between neighbwring starch molecules (thus reducing the gelatinisation temperature) and gave a higher transparency. Only a low level of substitution is required (one hydroxypropyl group per ten anhydroglucose units) to achieve a very high stability. The resulting product has a similar peak viscosity and low set-back to Gum Arabic, as can be seen on the Brabender profile (Figure 3). The comparative Brabenders were carried out using 30% d. b. slurries, prepared at pH 5.5, which were heated under controlled stirring from 50°C to

Structural and Functional Properties of Polysaccharides

79

Figure 3 Brabender Viscosiv Profiles 95°C (15°C. per minute), held at 95°C for 30 minutes and finally oooled to 50°C over a 30 minuteperiod. The test predicted that the perfamance in confectionery applications, w h m hearing and cooling profiles were employed e.g. in the production of gums & jellies, would be similar to gum arabic. Evaluations under pilot plant conditions with equipment typical of full scale manufacture were carried Out as described in section 4. 4 APPLICATIONS OF C*ARATEX IN GUMS & JELLIES2

C*AraTex can successfully replace gum arabic in both traditional glucuse/suaoseand sugar-free gums. It was found, however, that during shelf-life testing, a slight skidcrust formabion was found. The addition of 1-296 of hydrolysed cdlagen (a mixture of water-soluble proteim of high average molecular mass) to the C*AraTex, improved the perfarmane without affecting the workability and the gum texture. 4.1 Traditional Glucose / Sucrose products

Gum Arabic has been traditionally used in the formulation of gums with a very hard, brittle texture. C*AraTex completely matches the clarity and mouthfeel of such gums and is very similar in texture (with the incorponrtion of collagen. the texture during shelf life is identical). It also provides additional benefits of lack of stickiness, flexibility of texture from very hard to medium/soft and improved procesSing characteristics than the Gum Arabic standard. It also has a bland non "starchy" flavour allowing good flavour release from the gums. "here is also evidence that a percentage of the gum arabic can be replaced by C*AraTex in sugar-free gums without affecting the non-cariogenicity of the product (in contrast to aadirional starches).

4.1.1 Qpical formulations of gums based on C* AraTex The levels of C*AraTex to achieve hard, medium and soft textures are shown in Table 2. Whilst the glucose syrup used is high maltose syrup (H.M.S.), equally good products can be made with traditional 42 D.E. syrup nr the nPw r * P w w l fvninc lwhirh allnw c i i m w rpnlaFpmpntc nf a h n v ~ Iul%\

80

Gums and Stabilisers for the Food Industry

Table 2 Gum Recipes Gum Texture

soft

Medium

Hard

C*AraTex Glucose syrup: H.M.S. Sucrose Water Citric acid: 50% solution Flavour Colour Final dry substance: %

23 29 19 29 2 q.s. q.s. 87

25 25 19 31 2 q.s. q.s. 85

28 19 19 34 2 q.s. q.s. 85

AU the gums can be successfully manufactured by the usual manufacturing processes used in the confectionery industry : open pan, jet-cooking or tubular cooking. C*AraTex offers, due to its excellent dispersibility and very low viscosity, several workability advantages in comparison with Gum Arabic. 4.1.2 Gum manufacture, with C*AraTex A pre-mix is ma& by adding the sucrose/C*AraTex to warm water at approx. 50°C followed by addition of the glucose (at approx. 40°C ). The advantages over Gum Arabic at this stage are good dispersibility, avoiding possible lump formation, and the requirement for only low shear mixing. The slurry is cooked, using open pan, jet or tubular equipment, to a temperature in the range 117-119°C (the microscope is used to ensure an optimum cook of 5-9 uncooked starch granules in a field of vision), and a dry substance of approx. 74%. Gum Arabic slurry, in comparison, can only be mked to approx. 68% d.s. due to the higher viscosity. After cooking, the mass is allowed to stand for 20 minutes at 90-95°C to let the air bubbles diffuse out of the solution (vacuum treatment is not required), before the acid, colour and flavour are added. Then the mass is deposited in moulding starch at typically 75-80 "C. The final step is the stove drying of the gums in the moulds at 55°C. As the liquor is at higher solids and the water evaporation is easier, shorter drying times result compared with gum arabic. Texture analysis and sensorial evaluation have shown that the properties of the C*AraTex gums are very similar to Gum Arabic gums. Shelf-lifetesting has indicated no deformation of the gums after 6 months storage at 20 T and 65 % relative humidity. Addition of citric acid (up to 3 %) or Salmiak (NH,Cl, up to 4 % for typical Benelux and Scandanavianproducts ) has no effect on the properties and stability of C*AraTex gums. 4.2 Sugar-Free Gums

C*AraTex can, in addition to use in traditional glucosdsucrose sweets, also be used in sugarFree gums by combining with a polyol such as maltitol syrup (Maltidex 110, with a maltitol content of 75% and d.s. of 83%). The hard Gum Arabic textuie can be matched at the same usage level as well as medium and soft textures (see Table 3). Similar processing benefits are found, as with glucosdsucrose gums, when replacing the Gum Arabic by C*AraTex i.e. improved dispersibility, cooking to higher d.s. which negates the need for vacuum treatment of the cooked liquor, and shorter drying times. Excellent storage stability is found with these sugar-free gums.

Structural and Functional Properties of Polysaccharides

81

Table 3 Sugar Free Cwns with C*AraTex Gum Texture C*AraTex Maltitol Syrup Moulding ds. Moulding Viscosity 70°C. (mPas) Drying Temp."C./Time hrs.

Medium Soft

Medium Hard

Hard

Very Hard

39 61

43 57

47.5 52.5

53 47

1520

1624

2810

3120

70 60

1 6 0

AU polyol based sugar-& products are lower in calories than their su(xoseJgucoGe counterparts (in Europe the values are 2.4 kcal/g. for polyols compared with 4.0 W g . for "sugars"-in other world-wide markets different values are assigned) and are suitable for diabetics. Control of usage, by guidance on the product label, is advised due to the laxative nature of the polyols. There is a new polyol, Erythritol, currently undergoing approval assessment in the E C , which does not lead to a laxation effect when eaten. A further aspect of sugar free products is the need to be noncariogenic. Caries is a disease of the tooth that occurs as the result of a complex mechanism in which oral bacteria, especially Streptococcus mutans,and fermentable carbchydrates play important roles. When oral bacteria come into contact with food that contains fermentable carbohydrates, they will convert them into organic acids, and lower the pH. Since bacteria in the mouth are mainly located on the surface of the teeth, this local high acid concentration causes Ca" ions to be released from the hydroxyapatite of the teeth. thereby weakening the moth matrix. By the enzymatic action of the salivary a-amylase, the carbohydrates are easily degraded, and the substrates become even more easily available to the bacteria. The requirement for non-cariogenicity is that the pH is not reduced below the value of 5.7. It has been demonstrated that traditional starches including native tapioca, the base for C*AraTex, are degraded and the pH falls below 5.7. However, the highly specific acid thinning and hydroxypropylation reactions confer a lower cariogenic potential to C*AraTex. Current tests show that a 5050 combination of C*AraTex and gum arabic gives gums which meet the non-cariogenic claim. Conformation by the Mtihlemann Test (an in vivo test measuring the pH drop on the tooth surface of volunteers during chewing of the test products) is required. 5 APPLICATIONS OF C*ARATEX IN COATING 5.1 Introduction

Coating is a very traditional, but effective, way to deliver a better appearance and a better shelf-life to confectionery products. Although the technology of the panning process has been upgraded from the laboratory pan to the larger scale drum-coater. the requirements for good film forming ingredients have largely stayed the same. C*AraTex provides a breakthmugh in starch technology to produce a modified starch that delivers the same coating properties as the traditionally used gum arabic. However, this modified starch offers a number of advantages. The addition of hydrolysed collagen is not required to achieve the functionality. 5.2 Pre-Coating

&-coating is used to overcome the problem of a final coat not adhering to the centre due to the presence of too much oil or fat as in the case of nuts. In addition,.this pre-coating prevents

82

Gums and Stabilisersfor the Food Industry

the appearance of dark patches on the surfaces of the product. Precoating also protects centres such as chewing gum from the influence of the environment i.e. temperature and humidity. preventing stickiness. In general. between 2 and 10 layers of pre-coatings were applied to the centres. depending on the cOmpOSition of the centres and of the preaating material. The function of the pre-coating material is to All up the pcxes and form a thin protective fflm around the centre which then adhens to the coating material. Gum Arabic has been traditionally used. However. the lump material has to be ground to and the resulting solution has to be cleared afterwards. 'Ihis process may take up to 24 hours. A precleaned and spraydried gum arabic has partially resolved this, but at an even higher cost to the conkctiomq manufacturer. A disadvantage of gum arabic is that at relatively low concenrration e.g. 30%. suspemlo~of these pmducts have a high viscosity. and do not easily flow around the centre. Also, at this low concenaation, mcxe layers are required, which leads to longer processing times.

0

0

5.2.1 Manyfacturing process A solution of C*AraTex is prepared at a concentration in the range 30-50% by dispersing in water at 50°C to 60°C. 'Ihe solution is cooled and held between 30°C and 50°C. The pre-dried centres are coated with 0.8 to 1.3% w/w of C*AraTex solution and rotated unril an even layer has been formed (at room temperature). The process is repeated until the desired number of layers has been applied (normally bemeen two and ten, &pending on the dry substance of the coating solution). Between the application of each layer, the centres are revolved for 2J3 minutes (without dry air blowing). The c e n m are allowed to completely dry on open trays, preferably overnight, before final coating.

5.2.2 Comparison between C*AraTex and Gum Arabic. Precoating trials of sugar-free chewing gum were canied out with 30% solutions of C*AraTex and gum arabic. The chewing gum centres were coated with 5 and 10 layers of each material prior to final coating with sorbitol solution (seeTable 4). No significant difference in terms of chewing gum hardness and shelf-life was observed between both products. Generally, it was also seen that 10 layers gives less sticky, mare m c h y end products. Sensorial evaluationshave confirmedthese findings.

Table 4 Recotlting Sugar-Free Chewing Gum with C*AraTex Hardness g. Hardnessg. 1 1 week

Hardness g. 3 weeks storage at 70% R.H./25"C.

Centres precoatedwith 5 layers of: C*AraTex Gum Arabic

2200 1950

2300 2100

1700 1900

Centres precoated with 10 layers of: C*AraTex Gum Arabic

2350 2500

2700 2600

2100 2150

C*AraTex is, due to its good dispersibility and low viscosity. is easier to handle than gum arabic. A 50% solution of C*AraTex has a similar viscosity to a 30% solution of gum arabic, which meam the total amount of material applied ih a single layer is much higher compared with

Structural and Functional Properties of Polysaccharides

83

gum arabic. A total number of 2/3 layers is sufficient for the pre-mating of chewing centres. compared with 5 layers of gum arabic. ‘Ihe result is a considerable reduction of the coating time. 5.3 Find coatlng of Nuts with C*AraTex

C*AraTex can be used to coat the outer surface of nuts, such as almonds and provide a film which prevents the migration of fat onto the outer surface.

0

0

0 0

5.3.1 Method A 40% or 50% solution of C*AraTex solution is preparedby dissolving the powder in warm water at 3040°C. 7 laym 300 g of C*AraTex are applied to 20 lcg of almonds. allowing 5 minutes drying with the pan rotating,between layers. the final layer, the almonds are left in the pan for 20 minutes. ’Ihe final step is to store the coated almonds for 24hours at room tempemlure.

5.3.2 Advantages compared with Gum Ambic. Additional laym ace requid with Gum Arabic as only a m m dilute solution of 30% concenmtlon can be prepad Also, it was f d that the almonds coated with gum arabic required storage for 10 days to allow the coating to harden suf’ficiently. 6 CONCLUSION

C*AraTex, an acid binned hydmypropylated tapioca starch, can replace Gum Arabic on a weight to weight basis without comptomising the final product properties in terms of texture, film forming and organoleptic properties. C*AraTex is also &ex to use and povides a reliable supply at constant quality and competitive price. compared to Gum Arabic and is now being used successfully in the Confectionery Industry. REFERENCES 1. A. M.Stephen, “Structure and properties of Exudate Gums”, Gums & Stabilisers for the Food Industry 5. Oxford University Ress, 1990, p. 3. 2. V. De Coninck, F. Van Der Schumn, “Starch Composition”, European Patent, 0679340, 1994.

PRIMACELm FOOD CELLULOSE

J.M. Evans. R.C. Clark and N.A. Morrison The NutraSweet Kelco Company Tadworth, Surrey, KT20 5HQ or 8225 Aero Dr., San Diego, CA 92123-1718

1 INTRODUCTION PrimaCelTMis a new family of food cellulose products made by the NutraSweet Kelco Company (NKC) It is a GRAS ingredient in the US and is awaiting approval for food use As a new food ingredient, its nature and functionality and the varied food in the EC applications where it offers benefits are still being discovered 4.5,6. This paper will cover the basic properties of PrimaCel and will discuss some of its features and potential applications. The PrimaCel family of rheological modifiers are composed of fermentation derived cellulose in combination with various co-agents. The level and type of co-agent used in PrimaCel may differ depending on the needs of the application. The co-agents function to keep the cellulose from aggregating and ensure a uniform dispersion The bacterial cellulose used in PrimaCel is produced by the aerobic fermentation of Acetobacter xylinum 8,9. This cellulose is chemically identical to plant sources of cellulose, but is more efficient in use, due to the morphology of the fibres as well as their networked structure lo. This results in stronger and more fibre-fibre interactions for the bacterial cellulose. The cellulose in PrimaCel functions like a continuous reticulated network of ultrafine fibres which can suspend particles, and viscosify food systems. Figure 1. sho

’.

’.

Figure 1 Scanning Electron Micrograph of a PrimaCel Dispersion

Structural and Functional Properties of Polysaccharides

85

1.1 Food Cellulosics and PrimaCel Some food cellulosics are polymeric thickeners e.g. carboxymethylcellulose (CMC), methylcellulose (MC) and hydroxypropylcellulose (HPC). These water-soluble polymers are produced by chemical modification of cotton or wood pulp cellulose 11J2. They function by the hydration of individual molecules into well solvated flexible chains. In solution, each of these molecules occupies a given hydrodynamic volume and thus results in increased viscosity of the aqueous phase. These water-soluble cellulose derivatives have moderately pseudoplastic rheological properties 13, and will have inherent impaired stability due to their water-solubility. Degradation of even a single p 1-3-4linkage making up the polymer backbone, will reduce its molecular weight and therefore its viscosity. Other food cellulosics may be referred to as particulate thickeners. PrimaCel and Microcrystalline cellulose (MCC), fit into this category. These are insoluble because the bundles of cellulose chains in them are not chemically modified. Generally, particulate thickeners are more pseudoplastic than water-soluble polymers, and have a yield stress i.e. a certain force must be exceeded to cause the system to flow. The viscosity imparted by these cellulose based particulate thickeners results from interactions between individual cellulose fibres. The bacterial cellulose in PrimaCel is a bundle of many cellulose chains, which form microfibres. There are important differences in the way these two types of thickeners function. The viscosity induced by particulate thickeners is due to interactions between the solid particles, whereas in polymeric thickeners the viscosity is due to the well solvated, flexible chains. The long-term stability of polymeric thickeners is determined by hydrolysis i.e. the breaking down of the polymer itself, rather than the breaking down of the interactions between the particles in the case of particulate thickeners. In order to make polymeric thickeners functional, there must be sufficient shear to prevent lumping of the powder, so that the polymer is completely solubilized, whereas particulate thickeners require far greater shear, because in this case, shear is required to disperse the particles. Finally, the rheological properties are dominated by rate dependent effects for polymeric thickeners and time dependent effects for particulate thickeners. 2 PREPARATION OF PRIMACEL PrimaCel is provided in the form of a dry, white powder. It first needs to be dispersed in liquid with relatively mild mixing (e.g.with a propeller mixer), and then requires activation. This is achieved by passing the dispersed material through a pressure drop homogeniser or colloid mill. The activation step is required in order to pull apart the individual fibres, to make the material functional. Addition of salts and acidic ingredients should be delayed until after the material has been activated. Such materials may diminish the effectiveness of the co-agent and result in less effectively dispersed cellulose.

2.1 Preparation and Measurement of dispersions used for this study The PrimaCel used for this study was a spray dried composition containing cellulose, CMC and sucrose. The dry powder was added to a simulated tap water and mixed for 15 minutes to disperse. It was then activated using a specially designed extensional homogenizer at 1,500 psi. A conventional APV Gaulin type homogenizer at a pressure of 3,000 to 4,000 psi would be comparable and equally effective. After activation, the dispersions were allowed to remain at room temperature for 24 to 48 hours, before being tested. This ensured that the sample had reached an equilibrium. Three different rheological instruments were used for testing the samples. Unless stated otherwise, dispersions were prepared and tested at a 0.5% concentration at room temperature (21 to 23°C). Yield stress was measured with a Rheometric Scientific SR-200 controlled stress rheometer equipped with a parallel plate tool 50 mm in diameter and spaced 1.O mm apart. A stress ramp test run over a 90 second period of time was used to find the yield

86

Gums and Stabilisersfor ihe Food Industry

stress. The sample was allowed to remain in the instrument for 5 minutes to recover its structure prior to running the test. Most of the other tests were run with a Rheometric Scientific ARES instrument equipped with a 50 mm 0.02 radian cone. Details of each test are given in the description of the test results. Like the stress ramp test, an equilibration period of 5minutes was allowed before the tests were run. Thermal stability was determined with a Rheometric Scientific Pressure rheometer (RPR7800) equipped with a Couette test fixture. Heating was done at a rate of 2°C per minute to each temperature tested. Once at temperature, a 5 minute equilibration was allowed before testing started. 3 FEATURES OF PRIMACEL 3.1 Introduction PrimaCel is a family of products containing fermentation derived cellulose in combination with various co-agents. Different applications will require different co-agents. PrimaCel can be used as a highly efficient thickener, stabiliser and film former. It can be used as a starch extender and, in addition, can be used to modify texture to produce a more creamy mouthfeel in low-fat food systems. It is highly resistant to physical and chemical degradation, and its viscosity is nearly temperature independent. It has excellent tolerance to pH, salts and moderate levels of oxidising and reducing agents. In addition, it is capable of thickening non-aqueous media such as glycerine and corn syrups. 3.2 Yield Stress PrimaCel has a practical yield stress i.e. within the time-scale appropriate in food systems there is a yield stress. This is shown in Figure 2.

2000

1500

* 1000 E

.-C-

z

500

0

0

10

20

40 50 Stress, T (dynes/cm*)

30

60

Figure 2 Stress Sweep for a 0.35% PrimaCel Dispersion

70

87

Structural and Functional Properties of Polysaccharides

Figure 2 shows clearly that there is a definite stress which causes the material to suddenly flow. This is the practical yield stress, and for the concentration given above is about 58 dynes/cm2. As the concentration of PrimaCel is increased, so the practical yield stress will increase, although this relationship is not directly proportional. This is shown in Figure 3. 1o3

1oo

0.1

PrimaCel Concentration, (%)

1

Figure 3 Eflect of Concentration of a PrimaCel Dispersion on Yield Stress PrimaCel excels in its performance, not just by its practical yield stress, but because it has a high surface area and high aspect ratio. The high aspect ratio of the ultra-fine fibres has been measured by atomic force microscopy (AFM) and scanning electron microscopy (SEM)I4. 3.3 Pseudoplasticity Dispersions of PrimaCel have a high degree of pseudoplasticity, i.e. they have a rate dependent decrease in viscosity. Therefore as the shear rate is increased, so the apparent viscosity decreases. This is shown for various PrimaCel dispersions, at different concentrations in Figure 4. As the concentration of PrimaCel dispersion is increased, so the viscosity is increased. This characteristic pseudoplastic behaviour can be useful during processing, making the material easy to pump around the plant, but still provide the necessary viscosity in the final product.

Gums and Stabilisersfor the Food Indusrry

88

.

lo7 p . .

I

I

I

I

I

1

I0

100

3lo6 m

Q L E

lo5

0

F

10' 0.01

0.1

1 Shear rate (S')

Figure 4 Viscosity and Shear Rate for Various PrimaCeI Dispersion Concentrations 3.4 Thixotropy

In addition to being pseudoplastic, PrimaCel dispersions are also thixotropic. This means that they have a time dependent decrease in viscosity, i.e. if the material is subjected to a constant shear rate for a period of time, the apparent viscosity will decrease. It also means that the original viscosity is fully recovered after a period of time. These characteristics can be useful for various applications where athixotropic behaviour is expected - for example in a ketchup-type product. Figure 5 shows a situation where the PrimaCel dispersion is subjected to an initial shear rate of Is-'. During this period the apparent viscosity drops slightly. The material is then subjected to a high shear rate of 1000s-I for about 10 seconds. During this short time period, the viscosity remains relatively constant. When the shear rate is then reduced to 1s-l again, it can be seen that the majority of the viscosity is recovered very quickly, but the final part takes time to recover. After about 30 minutes full recovery is achieved. 3.5 Temperature Stability Many hydrocolloids lose apparent viscosity as the temperature is increased. PrimaCel has a stable viscosity across a wide temperature range (Figure 6). The stability can be controlled by the co-agent composition. This could be useful, for example, if a product needed to be heated, with the sauce on top, without the sauce running down the sides, or if suspension of solid particles throughout a severe heating process was necessary - for exampie in a chocolate milk.

lo4

I

. . , . .

I

I

, . . .

v i t i a l 1 S' shear rate

s

h

a

I

lo3

I

, . .

. . ,

Complete recovery : after 30 minutes .

-

Final 1 s-' shear rate

0

B

v

F

.is102 v)

8

-

v)

5 10'

1d

1000 6' shear rate I

.

.

.

.

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3.6 pH Stability Figure 7 shows how the viscosity of a PrimaCel dispersion remains almost constant across a very wide pH range. This is because PrimaCel is insoluble in water.

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3.7 Salt Stability PrimaCel dispersions are stable in high salt levels, provided that the PrimaCel is dispersed and activated before adding the salt. Again this property is due to the fact that PrimaCel is insoluble in water (Figure 8).

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91

Structural and Functional Properties of Polysaccharides

3.8 Basic Rheological Characteristics PrimaCel dispersions can be examined by small-scale deformation rheological equipment to determine their fundamental characteristics. If a strain sweep is made on a 0.35% PrimaCel dispersion (Figure 9), it can be seen that at low percentage strain, the storage modulus (G') is higher than the loss modulus (G'). This indicates that at these strain levels the material behaves like a solid. At higher strain levels the material breaks down and begins to flow like a liquid. Compared to many particulate thickeners, PrimaCel dispersions can tolerate relatively high strain values before their structure is broken down.

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Strain, y (%) Figure 9 Strain Sweep of a PrimaCel Dispersion When a frequency sweep is performed on a PrimaCel dispersion, within the linear viscoelastic region (strain values < 25%) defined in Figure 9, the mechanical spectrum is typical of gel-like behaviour. That is, the elastic modulus, G' is higher than G (loss modulus), and the G' versus frequency curve is relatively flat at low frequency values. This is shown in Figure 10 . 3.9 Other Properties PrimaCel will stabilise foams. It does this by two mechanisms. Firstly, it tends to migrate to the aidliquid interface and thus stabilise the air bubble size by helping to reduce coalescence, and, in addition, the yield stress of the bulk aqueous phase will help to prevent movement of the air bubbles. PrimaCel is similar to starch in two important aspects. Both starch and PrimaCel build viscosity through particle interactions (Section 1.1) - the difference being that starch is in granular form, whereas PrimaCel is fibrous; and they both have important thixotropic properties. However, PrimaCel is non-calorific, is not heavy or pasty, and does not mask flavours. In addition, PrimaCel can be used at lower usage levels.

Gums and Stabilisersfor the Food Industry

92

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Figure 10 Frequency Sweep of a PrimaCel Dispersion PrimaCel can prevent ice crystal growth on frozen storage by migrating to the surface of the ice crystals and minimising their growth. In addition, it builds up the viscosity of the aqueous phase, which also slows the rate of growth of the crystals. One of PrimaCel’s characteristics which has been of tremendous interest to food manufacturers, has been its textural role, especially in low-fat or fat-free foods. It has been shown to impart a creaminess and body in foods such as yogurt, fromage frais and chocolate milk. One of the important rheological characteristics of a fat or fat-like material is its combination of pseudoplastic (rate dependent) and thixotropic (time dependent) rheological properties. By tailoring the type of co-agent used, and its level, PrimaCel dispersions can be made to mimic the rheological properties of fat. PrimaCel is known to be an excellent film former. Cellulose is used to create one of the most widely used and strong films known, paper. The film formation properties of PrimaCel dispersions will be of interest and benefit for fat or moisture barriers in food.

4. APPLICATIONS OF PRIMACEL The properties of PrimaCel which have been discussed are important to many food applications Is, including: Dressings: to suspend herbs etc, and add mouthfeel and body. Sauces andgravies:to add viscosity which does not decrease as the temperature is increased. Soups: to add body, creaminess and mouthfeel and to suspend solids. Frozen desserts: to prevent ice crystal growth on storage. Aeratedproducts:to stabilise the foam structure. Margarine spreaddcheese spreads: less resistance to shear during application followed by recovery of structure when shear is removed. Confections: to add viscosity. Syrups and toppings: to add viscosity and maintain this viscosity at high temperature, and to modify flow properties. Chocolate milk: to suspend cocoa solids and contribute to a full, creamy mouthfeel. Yogurt andji-omageji-ais: to give body and creaminess and decrease syneresis.

Structural and Functional Properties of Polysacchrides

93

References

1. Cuthill, I., (1995), Int. Food Marketing & Technology., 9, (4), 56-59. 2. Gorski, D., (1997), Dairy Foods., June edition. 3. Schmitt, D.F., Frankos, V.H., Westland, J., and Zoetis, T., (1991), J. h e r . College Technology., lo,(5), 541. 4. Kent, R.A., Stephens, R.S., and Westland, J.A., (1991), Food Technology., 45, (6) 108. 5. Messenger, R., (1997), Food Processing., May edition. 6. Deis, R.C., (1997), Food Product Design., March edition, 33. 7. McGinley, E.J., (1981), U.S Patent, 4,263,334. 8. Ben-Bassat, A., Coddington, K.D., and Johnson, D.C., (1992), U.S Patent, 5,114,849. 9. Embuscado, M.E., Marks, J.S., and BeMiller, J.N., (1994), Food Hydrocolloids., (5), 407iDoelker, E., (1990), Stud. Polymer Sci., 8, 125. 10. Black, N.P., (1990), Tappi Journal., Sept edition. 11. Doelker, E., (1990), Stud. Polymer Sci., 8, 125. 12. Mombellet, H., and Bale, P., (1988), Manufacturing Chemist., Nov edition, 47. 13. Mitchell, J.R., (1979)., Polysaccharides in Foods. Editors - Blanshard, J.M.V., and Mitchell, J.R., 5 1. 14. Clark, R.C., Morrison, N. and Chen, Y.L. (1997), Unpublished Studies. 15. Shanahan,C., Zdanis, D.A, Clark, R.C., Evans, J.M., and Comfort, S.F., (1997), U.S Patent Application No 8,668,439.

s,

Rheological and Thermal Studies on the Sol-Gel Transition of Aqueous Solutions of Enzymatically Modified Xyloglucan

Mayumi SHIRAKAWA, Yoichiro UNO, Kazuhiko YAMATOYA

and Katsuyoshi NISHINARI

'

Food a d Food Addrtrves Research Laborutorres. Darnppon Pharmaceutical Co.. Ltd, Suita. Osaka. 564 Japun * Department of Food and Nutrition. Faculty of Human Lfe Ycience, Osaka City Wnrversiv Sumryoshi, Osaka, 558 Jupan.

.

ABSTRACT Aqueous solutions of galactose-removed xyloglucan have unique thermal properties. They have two sol-gel transition temperatures. At the low-temperature transition, a gel is formed on heating and melts on cooling. On the other hand, at the high-temperature transition, the gel melts on heating. These sol-gel transitions are completely thermo-reversible. Thermal properties of xyloglucan with a 43% galactose removal-ratio were studied by differential scanning calorimetry (DSC). DSC curves for the 1% xyloglucan solution showed small peaks. The heating curve showed two small endothermic peaks at 31 "c and 97 "c . The cooling curve showed two small exothermic peaks at 93 "C and 9 "c . These peaks are induced by a structural change in the sol-gel transition. The rheological measurements of xyloglucan with a 43% galactose removal-ratio were performed for the low-temperature transition. The kinetic analysis at a constant temperature showed that the gelling rate increased with increasing concentration of xyloglucan. The 1% xyloglucan solution changes from a sol to weak gel between 15 and 20 "c , and changes from a weak gel to strong gel between 25 and 30 "c . The gelation mechanism of xyloglucan at the low-temperature transition is similar to that of methylcellulose, suggesting that hydrophobic interactions are important.

MTRODlJCTION Seeds of the tamarind tree (Tumurindw indica L.) contain large proportions of xyloglucan as a storage polysaccharide. It is heat resistant, and stable in the presence of acidsalt. It is less shear-thinning than other thickeners (1). Therefore, the tamarind seed xyloglucan is widely used in the food industry in Japan and some Asian countries as a thickening and stabilizing agent.

Structural and Functional Properties of Polysaccharides

95

Recently, many investigations on the relationship between the polysaccharide structure and rheological properties have been carried out (2-4). The tailoring of the polysaccharide structure can produce novel properties (5). As the first step in molecular modelling, we investigated the tailoring of xyloglucan properties by enzymatic modification. In this study, we tried to remove the galactose residues from the tamarind seed xyloglucan with microbial B -galactosidase. The tamarind seed xyloglucan is composed of three units of xyloglucan oligomer, heptasaccharide (Figure 1 (a)), octasaccharide (Figure 1 (b)) and nonasaccharide (Figure 1 (c)), respectively. These units are different in their number and position of galactose residues (6). Xyloglucan has a linear B -(1,4) glucan backbone which carries a! -D-xylopyranosyl and B -D-galactopyranosyl-( 1 2)- a! -D-xylopyranosyl side chains. The tamarind seed xyloglucan alone does not form a gel. It is necessary to add a large amount of sugar or alcohol to induce the gelation of xyloglucan (1). It has been reported that gelation occurs when about 50% of the galactose residues are released from xyloglucan by B -galactosidase from nasturtium cotyledons (7). However, little is known about this phenomenon. We have studied the effects of the galactose removal-ratio and temperature on the gelling behavior of xyloglucan (8-9). Furthermore rheological and DSC measurements were carried out to clarifL the sol-gel transition in the present work. +

MATERIALS AND METHODS

The galactose-removed xyloglucan was prepared by enzymatic modification from native xyloglucan. Tamarind seed xyloglucan (Glyroid, Dainippon Pharmaceutical Co. Ltd., Osaka, Japan) was used as the substrate, and B -galactosidase from Bacillus

(a) heptasaccharide (b) octasaccharide (b-1) (b-2)

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Figure 1 The unit structures of oligosaccharides from tamarind xyloglucan. (Glu : glucose, Xyl : xylose, Gal : galactose)

96

Gums and Stabilisers for the Food Industry

crrculuns (Biolacta N5, Daiwa Kasei K.K., Osaka, Japan) was used for removing galactose residues. The reaction was done at pH 6.0 and at 50 "C. The galactose removal-ratio was estimated from the composition of three xyloglucan oligomer-units shown in Figure 1. The compositions of xyloglucan oligomer-units were measured by HPLC. Differential scanning calorimetry (DSC) measurements were carried out using a Setaram micro DSC- JII calorimeter (Setaram, Caluire, France) at a heating and cooling rate of 0.5 "Cimin. The thermal scanning rheology and frequency dependence of the storage shear modulus (G') and the loss shear modulus ( G " ) were performed with a Rheometrics Fluids Spectrometer RFS II (Rheometrics Co. Ltd., NJ, USA) at a heating rate of 0.5 "CImin.

RESULTS AND DISCIJSSION Figure 2 shows the sol-gel transition temperature of a 2% xyloglucan solution as a function of the galactose removal-ratio. Gelation occurred at 50 "c when the removalratio exceeded about 35%. Xyloglucan from which galactose was removed showed two sol-gel transition points : one at a low-temperature and the other at a high-temperature .

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Structural and Functional Properties of Polysaccharides

91

In xyloglucan with a galactose removal-ratio of 35%, sol was transformed into gel on heating at about 40 "C and reverted to sol again at about 80 "C, while xyloglucan with a galactose removal-ratio of 50% transformed from a sol to gel at 5 "c . Although the sample remained a gel at 100 C , it was transformed to sol again on heating at 110 "C . These sol-gel transitions were completely thermo-reversible. When the galactose removal-ratio exceeds 30%, the gel strength increases, and the temperature range at which the gel is formed becomes broader. Figure 3 shows the heating and cooling DSC curves of a 1% xyloglucan solution with a 43% galactose removal-ratio. The heating curve shows two small and broad endothermic peaks at 31 "C and 97 "C . The cooling curve shows two small exothermic peaks, one at 93 "C which is sharp and the other at 9 "C which is broad. These peaks may be induced by a conformational change of xyloglucan molecules accompanying the sol-gel transition. At the low temperature, the gel set at 31 "c on heating and melted at 9 "C on cooling.

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Figure 3 Differential scanning calorimetry (DSC) curves of a 1% xyloglucan solution with a 43% galactose removal-ratio. (heating curve ; (a), cooling curve : (b), heating or cooling rate : 0.5 "C/min)

98

Gums and Stabilisers for the Food Industry

At the high temperatures, the gel melted at 97 "C on heating and set at 93 "c on cooling. This confirms that the sol-gel transition is thermo-reversible. Since gel is formed on heating, the molecular forces which are responsible for gel formation are hydrophobic interaction and not hydrogen bonds. Table I shows the enthalpy of the sol-gel transition on heating for various gelling agents. The network structure of thermo-reversible gels such as gelatin (1 0), K -carrageenan (11) and agarose (12) are formed mainly by hydrogen bonds. While gels of methylcellulose ( 13) and methylhydroxypropylcellulose ( 14) are formed mainly by hydrophobic interaction. The energy for hydrophobic interaction is estimated about a quarter of the energy for hydrogen bonds (15). The fact that the enthalpy accompanying the sol-gel transition for galactose-removed xyloglucan is very small in comparison to that for gelatin, K -carrageenan and agarose is in good agreement with the abovementioned statement that galactose-removed xy loglucan forms a gel by hydrophobic interaction. Figure 4 shows the temperature dependence of G' and G" during the heating process for xyloglucan with a 43% galactose removal-ratio at various concentrations. For a 1% xyloglucan solution, in the lower temperature range below 20 "c , G' was much smaller than G" and both moduli decreased gradually with increasing temperature as is often observed in polymer solutions (16). 'Then G' began to increase steeply at ahout 20 "C , and became greater than G" at around 77 "c . G' and G" increaqed and the cross-over temperature shifted to a lower temperature with increasing concentration. These cross-over temperatures are plotted against the xyloglucan concentration in Figure 5. The cross-over temperature shifted to lower temperatures with increasing concentration, indicating that concentrated solutions begin to form a gel at a lower temperature than dilute solutions on heating.

TABLE I

Enthalpy of sol-gel transition on heating for various gelling agents

AH gel to sol ge I at i n K-carrageenan agarose sol to gel methyl ce I Iu I ose methylhydroxypropylcel lulose xy I og I ucan with a 43% ga I actose remova I -rat i o

(J/g)

reference

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(1 0) (1 1)

16.0 6.9 4.4

(1 3) (1 4)

(1 2)

Present work

Structural and Functional Properties of Polysaccharides

99

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Figure 4 The temperature dependence of G' and G" on heating for aqueous solutions of xyloglucan (with a 43% galactose removal-ratio) at various concentrations. ( w ; 1.0 rad/s, strain : lo%, heatingrate : 0.5"c/min)

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Figure 5 Cross-over temperature of xyloglucan with a 43% galactose removal-ratio as a function of xyloglucan concentration. (frequency : 1.0 mils, strain : lo%, heating rate ; 0.5 "c /min)

Gums and Stabilisersfor the Food Industry

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Figure 6 shows G' for solutions of xyloglucan with a 43% galactose removal-ratio at various concentrations at 50 "c as a function of time. The observed gelation curves were approximated well by the equation for the first order reaction

G'(t) = C'

-

-[ 1-

exp { - k ( t - t o ) } ]

where G' is the saturated storage shear modulus G, t o is the gelation time and k is the rate constant of gelation. G' was determined from the observed gelation curve, k and I o were estimated by curve fitting using the least squares method. In Figure 6, the solid line shows the best curve fit. The gelling rate increased with increasing xyloglucan concentration. It is consistent with the results shown in Figure 5. Figure 7 shows the frequency dependence of G' and G" for a 1% solution of xyloglucan with a 43% galactose removal-ratio at various temperatures. The viscoelastic behavior at 15 "C was typical of a dilute polymer solution. G' was smaller than G" throughout the accessible frequency range, and both moduli were strongly frequency dependent. At any temperature below 15 "C, the viscoelastic behaviour of this sample was typical of a dilute polymer solution (data not shown). G' was larger than G" throughout the frequency range at 20°C. G' showed a plateau in lower frequencies, but was slightly frequency dependent at higher frequencies. On the other hand, G" was frequency dependent, which is characteristic of a weak gel.

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Structural and Functional Properties of Polysaccharides

101

The behavior at 25 “C was of a weak gel, too (data not shown). G’ was much larger than G throughout the frequency range at 30 ”c . G’ was frequency independent, but G” was just slightly frequency dependent, and showed a shallow dip around 1 rad/s which has been observed in many elastic gels (17-19). Figure 8 shows the comparison of DSC and thermal scanning rheological measurements at low-temperature gel formation. It was suggested that the temperature at which G’ drastically changes should correspond to the transition temperature. In DSC measurements, the onset temperature corresponds to the transition temperature. On the other hand, the peak temperature of the DSC curve roughly coincides with cross-over temperature and transition temperature from weak gel to strong gel. By the tailoring of xyloglucan using microbial B -galactosidase, gelation occurred when 35% of the galactose residues had been removed. Gelation is never observed in native xyloglucan. However, solutions of the galactose-removed xyloglucan shows two sol-gel transition temperatures, and at the low-temperature transition, the gel is formed on heating and converted to a sol state on cooling. Furthermore this transition was thermo-reversible. This unique property is not recognized for other polysacchaxide gels.

n

1

Figure 7 The frequency dependence of G’ and G“ for a 1% xyloglucan solution (with a 43% galactose removal-ratio) at various temperatures. (strain : 10%)

Gums and Stabilisersfor the Food Industry

102

Reid et al. reported the gelation mechanism of xyloglucan from which galactose residues were removed (7). They indicated that these galactose residues are the major structural features determining the water solubility of xyloglucan molecules. They concluded that the removal of these residues from xyloglucan increases the polymer-polymer interaction leading to gelation. Small-angle X-ray scattering experiments have shown that association of the main chain occurs, and the lateral stacking of thin rods was observed upon gelation with the removal of the galactose residues from xyloglucan (9). With regard to the galactose-removed xyloglucan, the gelation on heating at the low-temperature transition suggests that the contribution of hydrophobic interactions are important as in methylcellulose (13). Below the lower transition temperature, it shows a dilute polymer solution with a rigid chain and a flat structure. The hydrophobic interaction between the /3 -glucan backbone becomes stronger with increasing temperature. Therefore the network structure is formed gradually. On further heating,

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Structural and Functional Properties of Polysaccharides

103

a strong crosslinking network is formed by hydrophobic association. It has strong gel characteristics. Furthermore by heating above the higher transition temperature, the molecular movement is enhanced breaking down the hydrophobic network. The gel again reverts to sol. We are going to study the relationship between galactose removal-ratio and rheological behavior in future. Hopefully, we would like to clarify the high-temperature transition.

REFERENCES (1) M. Glicksman, in ‘Food Hydrocolloids’, M. Glicksman Ed., CRC Press, Boca Raton, Florida, 1986. Vol. IU, p.191 (2) G. 0. Phillips, P. A. Williams and D. J. Wedlock Eds., ‘Gums and Stabilisers for the Food Industry 8’. IRL Press, Oxford, 1996 (3) K. Nishinari and E. Doi Eds., ‘Food Hydrocolloids : Structures, Properties and Functions’, Plenum Press, New York, 1994 (4) A. Stephen Ed., ‘Food Polysaccharides and Their Applications’, Marcel Dekker Inc., New York, 1995 ( 5 ) I. C. M. Dea, Pure &Appl. <,’hem., 1989, 61, 1315 (6) W. S. York, H. van Halbeek, A. G. Darvill and P. Albersheim, Curbohydr. Res., 1990,200,9 (7) J. S. G. Reid, M. Edwards, and 1. C. M. Dea, in ‘Gums and Stabilisers for the Food Industry 4’. G. 0. Phillips, P. A. Williams and D. J. Wedlock Eds., IRL Press, Oxford, 1988, p.391 (8) M. Shirakawa, K. Yamatoya and K. Nishinari, Food Hydrocoll., in press (9) Y. Yuguchi, M. Mimura, H. Urakawa, M. Shirakawa, K. Yamatoya, S. Kitamura and K. Kajiwara, in ‘Proceedings of the International Workshop on Green Polymers’, H. T. Adisesha, S. T. Sudirjo, P. R. Pangpabean, J. Arda and C. W. Soetrono Eds., Indonesian Polymer Association, Indonesia, 1997, p.306 (10) K. Gekko, X. Li and S. Makino, Biosci. Biotech. Riochem., 1992, 56, 1279 (11) M. Watase and K. Nishinari, Mukromol. (‘hem. 1987, 188, 2213 (12) M. Watase and K. Nishinari, Mukromol. (‘hem. 1987, 188, 1177 (13) A. Haque and E. R. Moms, Curbohydr. Polym., 1993, 22, 161 (14) Y. Yuguchi, H. Urakawa, S. Kitamura, S. Ohno and K. Kajiwara, Food Hydmcoll., 1995, 9, 173 (15) R. J. Hunter, ‘Foundations of Colloid Science’, Clarendon Press, Oxford, 1991, Vol. 1, Chapter 10 (16) J. D. Ferry, ‘Viscoelastic Properties of Polymers’, 3rd Ed., John Wiley, New York, 1980 (17) K. Nishinari, Jpn, J. Appl. P&. 1976. 15, 1263 (18) T. Nagano, T. Akasaka and K. Nishinari, Riopolvmers, 1994, 34, 1303 (19) K. S . Zaner and T. P. Stossel, J. Riol. Chem., 1983, 258, 11004

PHASE-SEPARATED MIXED GELATIN-MILK PROTEIN SYSTEMS

A.M. Hermansson, A. A l t s h and E. Jordansson SIK, the Swedish Institute for Food and Biotechnology, P.0 Box 5401, S-40229 Gothenburg, Sweden

1 INTRODUCTION

There is an increasing demand for knowledge about mixed biopolymer systems in order to impart the desired textural properties and stability to food products. This is especially the case for low-fat products, where the fat content is to be reduced without changing the characteristicproperties of the food product. Incompatibility between food biopolymers in solution has recently been reviewed by Tolstugumv and c o ~ o r k e r s 'Bulk ~ ~ . phase separation makes it possible to establish phase diagrams to describe phase composition in equilibrium at a given temperature. Phase separation can also occur fiom a homogeneous solution due to gel formation of one or both bi~polymers'*~". In such cases the kinetics during gel formation play an important role for the distribution of phases, and process conditions will influence the mixed structure and related properties. Phase inversion can be induced by shear if the continuous phase is the first gelling polymer7**.If temperature treatment is combined with high pressure, the route of gel formation can be changed, and it has recently been demonstrated that the phase behaviour of mixed whey proteidgelatin gels can form phase-separated bicontinuous, phase-separated discontinuous, as well as coacervate structures, by varying the process conditions9. Mixtures of gelatin and milk powder are commonly used in the aqueous phase of spreads. In spite of this, information about the phase behaviour of gelatidmilk protein mixtures is scarce. As mentioned above, gelatidwhey protein mixtures have been studied especially with regard to the gel formation of whey protein in relation to that of gelatin. In the case of gelatidmilk powder mixtures, gelatin alone is the gel-forming biopolymer. In a previous study, we found indications of demixing of gelatin and milk proteins within the aqueous phase of some low-fat spreads". In this paper, we will present results from a Nordfood project showing that phase separation takes place in gelatidmilk protein mixtures. Confocal laser scanning microscopy (CLSM) makes it possible to follow the development of phase separation during cooling. This technique has been used in combination with electron microscopy to characterise the nature of the phase-separated systems. We will demonstrate how the composition and the cooling conditions affect the distribution of phases. Examples will also be given of the behaviour of the two components in the aqueous phase of spreads.

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108

2 EXPERIMENTAL 2.1 Sample preparation The gelatin used was acid-processed with a nominal strength of 250 Bloom and a mean molecular weight of 118 kD. It was produced by Extraco, Klippan . The skim milk protein powder containing 35.5% protein, was supplied by Norske Meierier. The aqueous model systems were prepared by mixing gelatin (1.5-3.0%) and skim milk powder (2-18%) in distilled water to which 0.01% rhodamin 6G was added. The mixtures were heated to 50"C, and 1% NaCl was added to the warm solutions. Spreads with 40% fat and a 60% aqueous phase were prepared on a pilot plant scale by Norske Meierier. The aqueous phase was composed of 3% gelatin and 14% skim milk powder. The salt concentration, emulsifier composition, flow rate and temperature at the inlet of the cooling system were varied according to a factorial design. Only results referring to the structure of the aqueous phase will be referred to in this paper. 2.2 Microscopy Warm samples were transferred to the Linkam temperature table placed on the CLSM stage. The sample holder was tempered to 28, 24, 20, 15 10 and 5°C and the changes in the structure were directly studied in the CLSM (Leica TCS 4D) at an excitation wavelength of 488 nm. Samples were also quenched to 1"C in an ice bath and studied in the CLSM. Rhodamin makes the milk protein more strongly fluorescent than the gelatin, which makes it possible to differentiate between the two phases. Rhodamin was used as a stain both for the aqueous model systems and for the aqueous phase of the spreads. Transmission electron microscopy (TEM) was used to obtain information about the tine structure and verify the distribution of phases. Two different preparation techniques were used'land the aqueous gels were prepared by chemical fixation, dehydration, polymerisation and thin-sectioning; the spread samples were prepared by rapid freezing, freeze etching and replication. Thin sections and replicas were evaluated in a JEOL 1OOCX-I1microscope at 80 kV.

3 RESULTS AND DISCUSSION

3.1 Aqueous mixed systems Confocal laser scanning microscopy makes it possible to follow the dynamics of phase separation on cooling. Warm solutions of gelatidmilk proteins were homogeneous, and phase separation was observed when gelatin started to form a gel. The effects of the two components on the distribution of the phases were investigated. Mixed systems were studied at 1.5 and 3% gelatin. The milk powder concentration was varied from 2 to 18% (0.7 and 6.4% protein). Phase separation was observed in the whole concentration regime and all systems were gelatin continuous. Gel formation was slower at the lower than at the higher gelatin concentration, and the kinetic trapping of the dispersed phase was more obvious with decreasing temperature at both concentrations.

109

Protein Systems

2%

4%

6%

14%

1Opm ~.

Figure 1. CLSM images of phase-separated gels containing 1.5% gelatin and 2, 4, 6 or 14% skim milkpowder at 15 "C. Figure 1 shows the effects of increasing concentrations of milk protein at 1.5% gelatin, when the phase separation has been hlly developed at 15OC and the structure did not change significantly with time. The structural changes were monitored as a function of time at each concentration. Phase separation took place even at the lowest milk protein concentration, and small inclusions of milk proteins could already be observed when the sample had reached 15°C , The structure did not change with time even after storage overnight. At 4%,bigger droplets started to form after 90 min, and after prolonged storage a mixture of small and bigger droplets can be seen, as illustrated in Figure 1. At higher concentrations, the bigger droplets are predominant in the fully developed structure and they formed earlier with increasing concentrations of milk protein. At a concentration of 10-18% skim milk powder the final structure was developed within 10 minutes.

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2%

4%

6%

14%

1Opm -~

Figure 2. CLSM images of phase-separated gels containing 3% gelatin and 2, 4, 6 or 14% skim milkpowder at I5 "C. When the same experiment was performed at the higher gelatin concentration, the distribution of the milk protein phase changed, as illustrated in Figure 2. At the lowest milk protein concentration, the situation was the same as for the lower gelatin concentration. Small inclusions were formed that did not change with time. At 4Yhbigger droplets started to form after only 5 minutes at 15"C, and there was a tendency towards flocculation and partial coalescence that was not observed at the -lower gelatin concentration. This tendency was increased with increasing milk powder concentration, and a more and more bicontinuous structure was observed. The particle size of the milk protein inclusions was smaller than at the lower gelatin concentration. The results show that the speed of phase separation is influenced by the milk protein concentration, but that the way the milk protein phase is dispersed in the gelatin phase is determined by the kinetics of gel formation and the gelatin concentration.

111

Protein Systems

20°C

15°C

5°C

1 "C

1 Opm

Figure 3. CLSM images ofphase-separated gels containing 1.5% gelatin and 14% skim milk powder quenched and studied at diyerent cooling temperatures.

The kinetics of gel formation is also influenced by the cooling temperature. This was studied by quenching warm solutions with 14% skim milk powder and 1.5 or 3% gelatin to different temperatures from 28°C to 1°C and observing the development of phase separation in the CLSM. Results from studies at the lower gelatin concentration are shown in Figure 3. At temperatures close to the gelation temperature at 20-24OC, phase separation occurred slowly and big spherical droplets were formed. The droplet size decreased with decreasing temperature, but the appearance was fairly similar in the range 5-1 5 O C . At the lowest temperature, 1 "C, a bicontinuous structure of aggregated droplets was formed. This structure is similar in character to that formed at the higher gelatin concentration.

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112

20°C

5°C

1"C

1Opm

Figure 4 . CLSM images of phase-separated gels containing 3% gelatin and 14% skim milk powder quenched and studied at different cooling temperatures

The results obtained at 3% gelatin and 14% skim milk powder are shown in Figure 4. Droplets fused together are observed at all temperatures. The results show clearly that the droplet size and the dimensions of the milk protein phase decrease with decreasing temperature. The interpretation of the CLSM images is based on the difference in intensity of the fluorescent signal between the milk protein and the gelatin. Detailed information about the structure of the various phases was obtained by TEM. The fine structures of four samples were compared, where the gelatin concentration and the cooling temperature were varied. Figure 5 shows the structure of gels composed of 1.5% gelatin and 14% milk powder and gels composed of 3% gelatin and 14% milk powder cooled to 15°C and 1°C. The micrographs show the dispersed phase with spherical casein micelles. The fine gelatin network is forming the continuous phase surrounding the dispersed milk protein phase. In the gels with the higher gelatin concentration, the

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Figure 5. TEM micrographs obtained by thin sectioning showing the dispersed milk protein phase in the continuous gelatin phase a) 1.5% gelatin and 14% milk powder cooled to 15 "C. b) 1.5% gelatin and 14% milk powder cooled to 1 "C. c) 3% gelatin and 14% milkpowder cooled to 15 T.d) 3% gelatin and 14% milkpowder cooled to 1 "C. irregular shape of the dispersed phase reflects partial coalescence of droplets in agreement with the observation made with the CLSM images. If the mixed gels obtained at the two cooling temperatures are compared, we can see that the gel formed at the lowest cooling temperature contains more small inclusions of milk proteins in the gelatin phase than the gel formed at 15°C. This is most evident in the gels obtained at the lower

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gelatin concentration. The results indicate that the kinetic trapping at 1OC is so quick that the phase separation is less complete than at the higher temperature. Another interesting difference between the samples cooled to different temperatures is that the milk protein phase seems to be less dense at the lower cooling temperature. The higher magnification in Figure 6 reveals details of the gelatin network surrounding the milk protein droplet. The difference in density in the aqueous droplet is also clearly seen. This observation can be an indication of differences in water partitioning and efficient concentrations due to differences in the processing conditions. CLSM provides a powerful tool to evaluate the dynamics of phase separation. The results show that the kinetics of gelation determine the nature of the mixed structure. A high gelatin concentration and a low cooling temperature promote the formation of a bicontinuous structure where droplets with milk protein partly fuse together and are trapped within the gelatin network. At the lower gelatin concentration, a dispersed structure with relatively big milk protein droplets was formed except at the lowest cooling temperature. An increase in the milk protein concentration increased the kinetics of phase separation at both gelatin concentrations, but not the nature of the dispersed phase. Electron microscopy provides clear evidence of the difference in structure between the two phases, and the results obtained by TEM and CLSM are in full agreement. The detailed information provided by electron microscopy is of importance in M e r i n g understanding of the mechanisms involved in phase separation.

Figure 6. Details of an milk protein domain surrounded be the gelatin network of 1.5% gelatin and 14% milk powder gels cooled to a) 15 " C and b) 1 "C.

Protein Systems

115

3.2 Spreads Gelatidmilk powder mixtures are commonly used in the aqueous phase of low fat spreads. It was therefore of interest to evaluate the microstructure in spreads with one of the compositionsused in the model system. Spreads with 3% gelatin and 14% skim milk powder with and without the addition of salt were produced under a number of processing conditions. Demixing within the aqueous phase was observed in all samples. Figure 7 shows one example, where we can see big aqueous domains as well as small spherical droplets dispersed in the unstained dark fat phase. The milk protein is more fluorescent and shows up brighter than the gelatin. In the bigger domains, aggregates of gelatin “droplets” are surrounded by milk protein, and there are small inclusions of milk proteins within the gelatin phase. The appearance of the phase-separated aqueous phase is different from that in the model systems, and the gelatin seems to be the dispersed phase. This difference can be due to several factors. The spreads have undergone severe processing and it has been reported that the first gelling biopolymer is likely to form the dispersed phase in a system under shear’.’. Preliminary studies of the aqueous model systems did not show phase inversion due to shear, but further studies might be needed.

I Opm

0.2 pm

~

Figure 7. CLSM image of the aqueous domain in a low fat spread

Figure 8. Freeze etched replica showing casein micelles at the outer surface and gelatin in the interior of a droplet in a spread

Another factor of importance in the complex spreads is the contribution of the fat phase to the distribution within the aqueous phase. As can be seen from Figure 7, milk protein surrounds the gelatin not only in the bigger domains but also in the small spherical droplets. Figure 8 shows the fine structure of such a droplet obtained by freezeetching and transmission electron microscopy. The fracture plane shows the structure at the outer surface towards the fat phase, as well as the structure in the interior of the

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Gums and Stabilisers for the Food Industry

droplet. Here we can clearly see that spherical casein micelles are located at the outer surface towards the fat phase, and that gelatin makes up the structure within the droplet. The results give evidence that phase separation takes place within the aqueous phase in spreads. We also have indications that processing conditions affect the distribution of biopolymers within the aqueous phase, but further studies are needed to sort out main effects as well as complex interaction effects in such a complex structure-forming process.

4 ACKNOWLEDGEMENTS

This work forms part of the Nordfood Project, No 93 183, “Structures in fat continuous foods”. The financial support from the Nordic Industrial Fund, National Funds and supporting industries is gratefully acknowledged.

5 REFERENCES 1. V.Ya. Grinberg and V.B. Tolstuguzov, Food Hydrocolloids, 1997, 11, 145. 2. D.V. Zasypkin, E.E. Braudo and V.B. Tolstuguzov, Food Hydrocolloids, 1997, 11, 159. 3 . V.I. Polyakov, V.Ya. Grinberg and V.B. Tolstuguzov, Food Hydrocolloids, 1997, 11, 171. 4. A.H. Clark, R.K. Richardson, G. Robinson, S.B. Ross-Murphy and A.C. Weaver, Prog. Fd.Nutr.Sci.,1982,6, 149. 5. A.H. Clark. In ‘Biopolymer mixtures’, Eds S.E. Harding, S.E. Hill and J.R. Mitchell, Nottingham University Press, 1995,37. 6. P. Walkenstrom and A.M. Hermansson, Food Hydrocolloids, 1997, 11, 195. 7. C.R.T. Brown, T.J. Foster, I.T. Norton and J. Underdown, In ‘Biopolymer mixtures’, Eds S.E. Harding, S.E. Hill and J.R. Mitchell, Nottingham University Press, 1995,65. 8. T.J. Foster, J. Underdown, C.R.T. Brown, D.P. Ferdinand0 and I.T. Norton, In ‘Food Colloids, Proteins, Lipids and Polysaccharides’, Eds E. Dickinson and B. Bergensal, The Royal Society of Chemistry, 1997,346. 9. P. Walkenstram and A.M. Hermansson, Food Hydrocolloids, 1997, in press. 10. A.M. Hermansson and E. Jordansson, In ‘Gums and Stabilisers for the Food Industry’, Eds G.O. Phillips, P.A. Williams and D.J. Wedlock, IRL Press, Oxford, 1992, 6,409. 11. A.M. Hermansson and M. Langton, In ‘Physical Techniques for the Study of Food Biopolyrner’ Ed. S.B. Ross-Murphy, Blackie, Chapman & Hall 1993,277.

Protein Systems

PHASE-SEPARATED MIXED GELATIN-MILK PROTEIN SYSTEMS

A.M. Hermansson, A. A l t s h and E. Jordansson SIK, the Swedish Institute for Food and Biotechnology, P.0 Box 5401, S-40229 Gothenburg, Sweden

1 INTRODUCTION

There is an increasing demand for knowledge about mixed biopolymer systems in order to impart the desired textural properties and stability to food products. This is especially the case for low-fat products, where the fat content is to be reduced without changing the characteristicproperties of the food product. Incompatibility between food biopolymers in solution has recently been reviewed by Tolstugumv and c o ~ o r k e r s 'Bulk ~ ~ . phase separation makes it possible to establish phase diagrams to describe phase composition in equilibrium at a given temperature. Phase separation can also occur fiom a homogeneous solution due to gel formation of one or both bi~polymers'*~". In such cases the kinetics during gel formation play an important role for the distribution of phases, and process conditions will influence the mixed structure and related properties. Phase inversion can be induced by shear if the continuous phase is the first gelling polymer7**.If temperature treatment is combined with high pressure, the route of gel formation can be changed, and it has recently been demonstrated that the phase behaviour of mixed whey proteidgelatin gels can form phase-separated bicontinuous, phase-separated discontinuous, as well as coacervate structures, by varying the process conditions9. Mixtures of gelatin and milk powder are commonly used in the aqueous phase of spreads. In spite of this, information about the phase behaviour of gelatidmilk protein mixtures is scarce. As mentioned above, gelatidwhey protein mixtures have been studied especially with regard to the gel formation of whey protein in relation to that of gelatin. In the case of gelatidmilk powder mixtures, gelatin alone is the gel-forming biopolymer. In a previous study, we found indications of demixing of gelatin and milk proteins within the aqueous phase of some low-fat spreads". In this paper, we will present results from a Nordfood project showing that phase separation takes place in gelatidmilk protein mixtures. Confocal laser scanning microscopy (CLSM) makes it possible to follow the development of phase separation during cooling. This technique has been used in combination with electron microscopy to characterise the nature of the phase-separated systems. We will demonstrate how the composition and the cooling conditions affect the distribution of phases. Examples will also be given of the behaviour of the two components in the aqueous phase of spreads.

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2 EXPERIMENTAL 2.1 Sample preparation The gelatin used was acid-processed with a nominal strength of 250 Bloom and a mean molecular weight of 118 kD. It was produced by Extraco, Klippan . The skim milk protein powder containing 35.5% protein, was supplied by Norske Meierier. The aqueous model systems were prepared by mixing gelatin (1.5-3.0%) and skim milk powder (2-18%) in distilled water to which 0.01% rhodamin 6G was added. The mixtures were heated to 50"C, and 1% NaCl was added to the warm solutions. Spreads with 40% fat and a 60% aqueous phase were prepared on a pilot plant scale by Norske Meierier. The aqueous phase was composed of 3% gelatin and 14% skim milk powder. The salt concentration, emulsifier composition, flow rate and temperature at the inlet of the cooling system were varied according to a factorial design. Only results referring to the structure of the aqueous phase will be referred to in this paper. 2.2 Microscopy Warm samples were transferred to the Linkam temperature table placed on the CLSM stage. The sample holder was tempered to 28, 24, 20, 15 10 and 5°C and the changes in the structure were directly studied in the CLSM (Leica TCS 4D) at an excitation wavelength of 488 nm. Samples were also quenched to 1"C in an ice bath and studied in the CLSM. Rhodamin makes the milk protein more strongly fluorescent than the gelatin, which makes it possible to differentiate between the two phases. Rhodamin was used as a stain both for the aqueous model systems and for the aqueous phase of the spreads. Transmission electron microscopy (TEM) was used to obtain information about the tine structure and verify the distribution of phases. Two different preparation techniques were used'land the aqueous gels were prepared by chemical fixation, dehydration, polymerisation and thin-sectioning; the spread samples were prepared by rapid freezing, freeze etching and replication. Thin sections and replicas were evaluated in a JEOL 1OOCX-I1microscope at 80 kV.

3 RESULTS AND DISCUSSION

3.1 Aqueous mixed systems Confocal laser scanning microscopy makes it possible to follow the dynamics of phase separation on cooling. Warm solutions of gelatidmilk proteins were homogeneous, and phase separation was observed when gelatin started to form a gel. The effects of the two components on the distribution of the phases were investigated. Mixed systems were studied at 1.5 and 3% gelatin. The milk powder concentration was varied from 2 to 18% (0.7 and 6.4% protein). Phase separation was observed in the whole concentration regime and all systems were gelatin continuous. Gel formation was slower at the lower than at the higher gelatin concentration, and the kinetic trapping of the dispersed phase was more obvious with decreasing temperature at both concentrations.

109

Protein Systems

2%

4%

6%

14%

1Opm ~.

Figure 1. CLSM images of phase-separated gels containing 1.5% gelatin and 2, 4, 6 or 14% skim milkpowder at 15 "C. Figure 1 shows the effects of increasing concentrations of milk protein at 1.5% gelatin, when the phase separation has been hlly developed at 15OC and the structure did not change significantly with time. The structural changes were monitored as a function of time at each concentration. Phase separation took place even at the lowest milk protein concentration, and small inclusions of milk proteins could already be observed when the sample had reached 15°C , The structure did not change with time even after storage overnight. At 4%,bigger droplets started to form after 90 min, and after prolonged storage a mixture of small and bigger droplets can be seen, as illustrated in Figure 1. At higher concentrations, the bigger droplets are predominant in the fully developed structure and they formed earlier with increasing concentrations of milk protein. At a concentration of 10-18% skim milk powder the final structure was developed within 10 minutes.

110

Gum and Srabilisers for the Food Industry

2%

4%

6%

14%

1Opm -~

Figure 2. CLSM images of phase-separated gels containing 3% gelatin and 2, 4, 6 or 14% skim milkpowder at I5 "C. When the same experiment was performed at the higher gelatin concentration, the distribution of the milk protein phase changed, as illustrated in Figure 2. At the lowest milk protein concentration, the situation was the same as for the lower gelatin concentration. Small inclusions were formed that did not change with time. At 4Yhbigger droplets started to form after only 5 minutes at 15"C, and there was a tendency towards flocculation and partial coalescence that was not observed at the -lower gelatin concentration. This tendency was increased with increasing milk powder concentration, and a more and more bicontinuous structure was observed. The particle size of the milk protein inclusions was smaller than at the lower gelatin concentration. The results show that the speed of phase separation is influenced by the milk protein concentration, but that the way the milk protein phase is dispersed in the gelatin phase is determined by the kinetics of gel formation and the gelatin concentration.

111

Protein Systems

20°C

15°C

5°C

1 "C

1 Opm

Figure 3. CLSM images ofphase-separated gels containing 1.5% gelatin and 14% skim milk powder quenched and studied at diyerent cooling temperatures.

The kinetics of gel formation is also influenced by the cooling temperature. This was studied by quenching warm solutions with 14% skim milk powder and 1.5 or 3% gelatin to different temperatures from 28°C to 1°C and observing the development of phase separation in the CLSM. Results from studies at the lower gelatin concentration are shown in Figure 3. At temperatures close to the gelation temperature at 20-24OC, phase separation occurred slowly and big spherical droplets were formed. The droplet size decreased with decreasing temperature, but the appearance was fairly similar in the range 5-1 5 O C . At the lowest temperature, 1 "C, a bicontinuous structure of aggregated droplets was formed. This structure is similar in character to that formed at the higher gelatin concentration.

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112

20°C

5°C

1"C

1Opm

Figure 4 . CLSM images of phase-separated gels containing 3% gelatin and 14% skim milk powder quenched and studied at different cooling temperatures

The results obtained at 3% gelatin and 14% skim milk powder are shown in Figure 4. Droplets fused together are observed at all temperatures. The results show clearly that the droplet size and the dimensions of the milk protein phase decrease with decreasing temperature. The interpretation of the CLSM images is based on the difference in intensity of the fluorescent signal between the milk protein and the gelatin. Detailed information about the structure of the various phases was obtained by TEM. The fine structures of four samples were compared, where the gelatin concentration and the cooling temperature were varied. Figure 5 shows the structure of gels composed of 1.5% gelatin and 14% milk powder and gels composed of 3% gelatin and 14% milk powder cooled to 15°C and 1°C. The micrographs show the dispersed phase with spherical casein micelles. The fine gelatin network is forming the continuous phase surrounding the dispersed milk protein phase. In the gels with the higher gelatin concentration, the

Protein System

113

Figure 5. TEM micrographs obtained by thin sectioning showing the dispersed milk protein phase in the continuous gelatin phase a) 1.5% gelatin and 14% milk powder cooled to 15 "C. b) 1.5% gelatin and 14% milk powder cooled to 1 "C. c) 3% gelatin and 14% milkpowder cooled to 15 T.d) 3% gelatin and 14% milkpowder cooled to 1 "C. irregular shape of the dispersed phase reflects partial coalescence of droplets in agreement with the observation made with the CLSM images. If the mixed gels obtained at the two cooling temperatures are compared, we can see that the gel formed at the lowest cooling temperature contains more small inclusions of milk proteins in the gelatin phase than the gel formed at 15°C. This is most evident in the gels obtained at the lower

114

Gums and Srabilisers for the Food Industry

gelatin concentration. The results indicate that the kinetic trapping at 1OC is so quick that the phase separation is less complete than at the higher temperature. Another interesting difference between the samples cooled to different temperatures is that the milk protein phase seems to be less dense at the lower cooling temperature. The higher magnification in Figure 6 reveals details of the gelatin network surrounding the milk protein droplet. The difference in density in the aqueous droplet is also clearly seen. This observation can be an indication of differences in water partitioning and efficient concentrations due to differences in the processing conditions. CLSM provides a powerful tool to evaluate the dynamics of phase separation. The results show that the kinetics of gelation determine the nature of the mixed structure. A high gelatin concentration and a low cooling temperature promote the formation of a bicontinuous structure where droplets with milk protein partly fuse together and are trapped within the gelatin network. At the lower gelatin concentration, a dispersed structure with relatively big milk protein droplets was formed except at the lowest cooling temperature. An increase in the milk protein concentration increased the kinetics of phase separation at both gelatin concentrations, but not the nature of the dispersed phase. Electron microscopy provides clear evidence of the difference in structure between the two phases, and the results obtained by TEM and CLSM are in full agreement. The detailed information provided by electron microscopy is of importance in M e r i n g understanding of the mechanisms involved in phase separation.

Figure 6. Details of an milk protein domain surrounded be the gelatin network of 1.5% gelatin and 14% milk powder gels cooled to a) 15 " C and b) 1 "C.

Protein Systems

115

3.2 Spreads Gelatidmilk powder mixtures are commonly used in the aqueous phase of low fat spreads. It was therefore of interest to evaluate the microstructure in spreads with one of the compositionsused in the model system. Spreads with 3% gelatin and 14% skim milk powder with and without the addition of salt were produced under a number of processing conditions. Demixing within the aqueous phase was observed in all samples. Figure 7 shows one example, where we can see big aqueous domains as well as small spherical droplets dispersed in the unstained dark fat phase. The milk protein is more fluorescent and shows up brighter than the gelatin. In the bigger domains, aggregates of gelatin “droplets” are surrounded by milk protein, and there are small inclusions of milk proteins within the gelatin phase. The appearance of the phase-separated aqueous phase is different from that in the model systems, and the gelatin seems to be the dispersed phase. This difference can be due to several factors. The spreads have undergone severe processing and it has been reported that the first gelling biopolymer is likely to form the dispersed phase in a system under shear’.’. Preliminary studies of the aqueous model systems did not show phase inversion due to shear, but further studies might be needed.

I Opm

0.2 pm

~

Figure 7. CLSM image of the aqueous domain in a low fat spread

Figure 8. Freeze etched replica showing casein micelles at the outer surface and gelatin in the interior of a droplet in a spread

Another factor of importance in the complex spreads is the contribution of the fat phase to the distribution within the aqueous phase. As can be seen from Figure 7, milk protein surrounds the gelatin not only in the bigger domains but also in the small spherical droplets. Figure 8 shows the fine structure of such a droplet obtained by freezeetching and transmission electron microscopy. The fracture plane shows the structure at the outer surface towards the fat phase, as well as the structure in the interior of the

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droplet. Here we can clearly see that spherical casein micelles are located at the outer surface towards the fat phase, and that gelatin makes up the structure within the droplet. The results give evidence that phase separation takes place within the aqueous phase in spreads. We also have indications that processing conditions affect the distribution of biopolymers within the aqueous phase, but further studies are needed to sort out main effects as well as complex interaction effects in such a complex structure-forming process.

4 ACKNOWLEDGEMENTS

This work forms part of the Nordfood Project, No 93 183, “Structures in fat continuous foods”. The financial support from the Nordic Industrial Fund, National Funds and supporting industries is gratefully acknowledged.

5 REFERENCES 1. V.Ya. Grinberg and V.B. Tolstuguzov, Food Hydrocolloids, 1997, 11, 145. 2. D.V. Zasypkin, E.E. Braudo and V.B. Tolstuguzov, Food Hydrocolloids, 1997, 11, 159. 3 . V.I. Polyakov, V.Ya. Grinberg and V.B. Tolstuguzov, Food Hydrocolloids, 1997, 11, 171. 4. A.H. Clark, R.K. Richardson, G. Robinson, S.B. Ross-Murphy and A.C. Weaver, Prog. Fd.Nutr.Sci.,1982,6, 149. 5. A.H. Clark. In ‘Biopolymer mixtures’, Eds S.E. Harding, S.E. Hill and J.R. Mitchell, Nottingham University Press, 1995,37. 6. P. Walkenstrom and A.M. Hermansson, Food Hydrocolloids, 1997, 11, 195. 7. C.R.T. Brown, T.J. Foster, I.T. Norton and J. Underdown, In ‘Biopolymer mixtures’, Eds S.E. Harding, S.E. Hill and J.R. Mitchell, Nottingham University Press, 1995,65. 8. T.J. Foster, J. Underdown, C.R.T. Brown, D.P. Ferdinand0 and I.T. Norton, In ‘Food Colloids, Proteins, Lipids and Polysaccharides’, Eds E. Dickinson and B. Bergensal, The Royal Society of Chemistry, 1997,346. 9. P. Walkenstram and A.M. Hermansson, Food Hydrocolloids, 1997, in press. 10. A.M. Hermansson and E. Jordansson, In ‘Gums and Stabilisers for the Food Industry’, Eds G.O. Phillips, P.A. Williams and D.J. Wedlock, IRL Press, Oxford, 1992, 6,409. 11. A.M. Hermansson and M. Langton, In ‘Physical Techniques for the Study of Food Biopolyrner’ Ed. S.B. Ross-Murphy, Blackie, Chapman & Hall 1993,277.

ESR SPIN PROBE STUDY OF LEGUMIN AND OVALBUMIN INTERACTIONS WITH LIPOPHILIC COMPOUNDS IN SOLUTIONS

L. A. Wasserman*, M. Le Meste**, M. V. Motialun***, V. P. Yuryev* and A. M. Wasserman*** *Emanuel’s Institute of Biochemical Physics, RAS, 4 Kosygin str., Moscow, 117334, Russia **ENSBANA, 1Esplanade Erasme, 21000 Dijon, France ***Semenov’s Institute of Chemical Physics, RAS, 4 Kosygin str., Moscow, 117334Russia

1 INTRODUCTION The features of interactions of proteins with organic compounds reflect the physical and chemical properties of proteins in solutions’ and their biological actions’.*. The results concerning the mechanism of interactions of proteins with organic compounds are important for fUndamental researches and applications. Complex formation of the so-called ‘nutriment’ proteins with organic cornpounds defines the multiple hnctional properties of proteins, such as their ability to be processed into nutriment products. Usually, nutriment products represent multicomponent systems, consisting of proteins and various additives (lipids, sugars, aromatic compounds, e t ~ . ) ~ ’ ~ . The spin probe technique gives unique information about the mechanism of interaction of low molecular weight organic molecules with proteins5. It has been shown6 that binding of spin probes (one simulating an aromatic amino acid molecule, and the another a molecule of fatty acid) to bovine serum albumin results from hydrophobic interactions. A single type of adsorption center on the surface of the protein was observed for the first probe, while the second probe interacted with two types of centers. Spin-labelled stearic acids were also observed to interact with as-casein as revealed by the dramatic decrease in their rotational mobility7. It was shown previously that lipophilic compounds affect thermodynamic properties and surface activity of legumin and ovalbumin in a solution*. However, the mechanism of interaction of lipophilic compounds with these proteins cannot be considered clarified yet. The aim of the present work is to clear up the features of the interaction mechanism of legumin and ovalbumin with some lipophilic compounds in aqueous solutions using the spin probe technique. Spin-labelled steak acids were chosen as model lipophilic compounds. They differ by the disposition of a doxy1 group on the hydrocarbon chain relatively to the carboxyl group. Different other stable nitroxyl radicals were also used. 2 EXPERIMENTAL METHODS 11s vicia Fubu (legumin) was extracted according to the methods described in ’. Egg albumin (ovalbumin), five-fold recrystallized (Biline, Lietuva), was used without additional purification. Molecular weight of proteins, as determined by the light scattering technique”, were found

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118

to be 350 kDa for legumin and 44 kDa for ovalbumin, respectively. Protein globule radius is R = 50 A for legumin", R = 30 A for ovalbumin'. Rotational correlation times of protein globules in aqueous solution at 2OoC,calculated with the StokesEinstein equation using values of radii mentioned above, equal 1.3 x lo=]s and 2.7 x lo4 s, respectively. Spin probes, whose structures are shown below, were used:

doxy1 stearic acids (Sigma Co., USA) n = 5 (SDSA,l), n = 12 (12-DSA,2), n = 16 (16-DSA,3) CHzCHzOC l2H25

5-doxyldecane (4) (Sigma Co., USA)

I

2,2,6,6-tetramethyl-4-(2-dodecyloxyethyl)-pyperidineN -oxyl(S)

CH2COOH

I

2,2,6,6-tetramethyl-4-(carboxymethyl)-pypendine-N -oxyl(6)

a

C

A.

O

O

H

2,2,5,5-tetramethyl-3-carboxypyrrolineN -oxyl(7) Spin probes 5-7 were synthesized at the Institute of Academy of Sciences, by L . k Krinitskaya.

Chemical

Physics, Russian

Protein Systems

119

Spin-labelled stearic acids are not very soluble in water. Two techniques were used to prepare solutions of these compounds in buffer (0.1 M phosphate buffer pH 7.0). According to the first technique, a known weight of the spin probe was introduced into buffer (0.25 mglml at complete solubility the concentration would be 6 . 5 ~ 1 M). 0 ~ The system was sonkated at a 22 kHz frequency for 2.5 min and then mixed intensively for 1 hour at a temperature of 4550 "C (a similar technique was used in * for preparing solutions of lipophilic compounds). In the second technique, spin-labelled stearic acids were introduced into phosphate buffer as solutions in ethanol (similar to works 6*12). In this case, the concentration of spin-labelled stearic acids was found to be 1 . 3 ~ 1 M. 0 ~ Spin probe 5 was also introduced into the buffer, as a solution in ethanol (concentration in the buffer was 2 . 8 ~ 1 0M). - ~ The alcohol concentration in the buffer did not exceed 2 weight %. Spin probes 4,6,7 are better soluble in water than doxylstearic acids and probe 5. Solutions of these spin probes in the buffer were prepared by the usual technique.Theconcentration of probe 4 was 1 x lo4 M, those of probes 6 and 7,7 x 10" M. Proteins were dissolved in solutions of spin probes in the buffer (concentrations of legumin and ovalbumin in the solution were 1.4 x lo4 M and 1.1 x lo3 M, respectively). Lipophilic compounds (palmitic acid, sodium palminate and sodium decanoate, Sigma Co., USA, without additional purification) were dissolved in phosphate buffer similarly to spinlabelled stearic acids (by the first technique). These solutions were then added to protein solutions. The concentration of non-paramagnetic lipophilic compounds in solutions of proteins was 10 mM. ESR spectra were recorded with an ESR spectrometer Radiopan (Poland) at room temperature under conditions far fiom saturation. Spectra were recorded two hours after the preparation of solutions. Control experiments showed that ESR spectra, recorded two hours &er and a day after the preparation of solutionswere practically the same. 3 RESULTS AND DISCUSSION

ESR spectra of 5-DSA solutions in the buffer, and those in the presence of proteins and lipophilic compounds are shown in Figure I. The ESR spectrum of 5-DSA in buffer (probe introduced by the first technique, see Figure la) corresponds to spin probes with a 'fast r~tation'~ and is characterized by a rotational correlation time z = 3x 10'' s. The ESR spectrum of 5-DSA, introduced into the buffer by the second technique, does not sigruficantlydiffer fiom that shown in Figure la, so it is not shown here. The ESR spectrum of the spin probe changes sigmficantly after adding protein to the solution. This result indicates that an intensive interaction between the spin probe and the protein globule takes place. However,this interaction is implemented in different ways for the two proteins used. The rotational correlation time of 5-DSA (z I1x lo4 s) in a solution containing legurnin (the probe was introduced by the first technique, see Figure lb) is nearly two orders of magnitude higher than the correlation time of the probe rotation in buffer, i.e. the rotational mobility of the spin probe is reduced sharply. Introduction of non-labelled lipophilic compounds into the solution causes no modification of the spectrum of 5-DSA adsorbed on a macromolecule of legumin. Figure l c shows the spectrum of 5-DSA,adsorbed on the legurnin globule, in the presence of sodium decanoate. Similar spectra were obtained in the presence of palmitic acid and sodium palminate. Thus, the presence of lipophilic compounds in solution, at the concentrationused in this study, causes no apparent effect on 5-DSA adsorption on the

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Gums and Stabilisers for the Food Industry

Figure 1 ESR p c t r a of 5-DSA: a) in phoqhate buffer (PH 7.0, 0.I M); b) and (i) in legurnin solution; c) in legurnin solution with decanoate sodium (I0 mM); e) and S;, in ovalbumin solution;fl in ovalbumin solution with decanoate s d u m (I0 mM). In the m e of a), b), c), e), fl 5-DSA was i n t r d c e d into the buffer by the j r s t technique; in the case of (i) and& - by the second technique (via dissolving in ethanol).

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legumin globule. The ESR spectrum of 5-DSA adsorbed on ovalbumin is more complicated than that of 5-DSA adsorbed on legumin (see Figure le, the probe was introduced into the buffer by the first technique). A badly resolved singlet exists in the central part of the spectrum. Apparently, this spectrum is a superposition of spectra of ‘isolated’ probes (lines corresponding to these probes are marked by arrows in Figure le) and probes, adsorbed on close adsorption centers, or on centers capable for binding several molecules of 5-DSA. In the latter case,dipole and exchange interactions of unpaired electrons of spin probe paramagnetic fragments are displayed in the spectrum. When lipophilic compounds (sodium decanoate, palmitic acid or sodium palminate) were present in the solution, they were adsorbed together with spin-labelled stearic acid, and the probability for several spin-labelled molecules being adsorbed on neighbouring centers decreased. In this case, a spectrum (see Figure If) of adsorbed radicals, which do not interact with each other was observed. It was similar to the spectrum shown in Figure Ic. ESR spectra of 5-DSA (introduced into the buffer by the second technique, from solution in ethanol) in solutions of legumin and ovalbumin are shown in Figures Id and 1% respectively. Changing the sample preparation technique affects weakly the ESR spectrum of 5-DSA in legumin solutions, but significantly changes the ESR spectrum of 5-DSA in solutions of ovalbumin. In the latter case, the spectrum observed is the superposition of spectra of adsorbed and ‘free’ spin probes. Apparently, the presence of ethanol in solution causes a weak effect on the binding constant of 5-DSA to legumin, but noticeably reduces its binding constant to ovalbumin (the solubility of the probes in water should be higher in the presence of alcohol and thus the partition between the water phase and the protein environment should be different). Figures 2 and 3 show the ESR spectra of 12-DSA and 16-DSA respectively, in the buffer and in solutions of proteins. The ESR spectra display a weak dependence on the way of their introduction into solution. The results obtained for each protein on the example of probes introduced into the solution by the first technique are now discussed. The ESR spectra of 12-DSA and 16-DSA in legumin solutions (Figures 2b and 3b) are superpositions of spectra of ‘fast’ and ‘slow’ rotating radicals. It is natural to suggest that ‘fast’ rotating radicals belong to the ‘free’ spin probes, whereas ‘slow’ rotating probes belong to adsorbed doxylstearic acids. We have calculated a theoretical spectrum as a superposition of spectra of ‘fast’ and ‘slow’ rotating radicals. The calculations were made according to the model of isotropic rotation and the following values for the electron-spin parameters of a radical were used: & = 2.0088, g, = 2.0061, = 2.0027; A, = 6.3 Gs, A, = 5.8 Gs, A, = 33.6 Gs 13. Simulation ofthe spectra was performed by the program of V.P. Tim~feev’~. It has been found that the calculated spectrum (Figure 2b) satisfactorily correlates with the experimental spectrum of 12-DSA in legumin solution, if it is accepted that the proportion of ‘free’ radicals does not exceed 3%, and the correlation times of ‘slow’ and ‘fast’ spectra are ‘51% lx10*sandr2= 1 ~ 1 O ’ ~ respectively. s, The experimental spectrum of 16-DSA in legumin solution correlates satisfactorily with the calculated one, if the part of ‘free’ radicals does not exceed 2% (Figure 3b), and the correlation t i e s are TI = 1 x 10” s and r2 = 2x 10” s, respectively. Two important circumstances should be mentioned. First of all, correlation times of adsorbed spin-labelled stearic acids S-DSA,lZDSA and 16-DSA (T 21 1x10‘ s) were essentially independent on the position of the doxyl group. The latter result suggests a model of stearic acid adsorption on legumin globule, according to which the whole (or most of) the hydrocarbon chain of stearic acid interacts with hydrophobic parts of the protein globule. Then, introduction of a doxyl group into positions 12 or 16 of the hydrocarbon chain caused a reduction of the binding constant of the stearic acid to protein (in comparison with 5-DSA).

’,

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Gums and Stabilisers for rhe Food Industry

a )

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.

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Figure 2 ESR spectra of I2-DsA: a) in phosphate bufler (pH 7.0, 0.1 M); b) in legtimin solution; c) in owlbumin solution. Dolled line w k s the theoretical spectrum, caIculaed ar a suppsition 0fspecb.a with correhion times rl = I x I Q8 s (molar purt is 0.97) and r~= 1 ~ 1 0 " (molarpart ~s is 0.03). It should be mentioned, however, that the part of 12-DSA and 16-DSA not adsorbed by legurnin remained quite small. The ESR spectra of 12-DSA and 16-DSA in ovalbumin solutions (Figures 2c and 3c) were similar to the spectra of these radicals in the buffer. This result could be due to the fact that the introduction of a doxyl group into positions 12 and 16 of hydrocarbonic chain reduces the binding constant of stearic acid to ovalburnin so significantly that adsorbed 12-DSA and 16DSA are not observed in the ESR spectra, or it could suggest that the interactions occur mainly through the polar head of the fatty acid and do not affect significantly the mobility of the hydrocarbon chain. Thus, it was of great interest to establish whether proteins in solution interact with compounds containing a hydrocarbon chain, but having no carboxyl group, and with compounds containing a mboxyl group, but having no hydrocarbon chain. To answer this question spin probes 4-7 were used. 5-doxyldecane (4) was chosen as a model compound of a decane molecule (a lipophilic compound without a carboxyl group). However, it should be taken into account that a doxyl

123

Protein Systems

1 0 . 0

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Figure 3 ESR spectra of I&DSA: a) in p h a p h t e buffer @H 7.0, 0.1 M);b) in legumin solution; c) in owlbwnin solution. Dotted line mark the theoretical spectrum, calcuhted as superposition of spectra with correlation times q = 1 x 1 s~( m o b part is 0.9%)and z j = 2 x 1 ~ " s( m o I a r p t is 0.02).

moiety modifies the decane molecule and can affect its capabiity for being adsorbed by proteins. The ESR spectrum of 5doxyldecane in the buffer (Figure 4a) differs fiom the ESR spectrum of this radical in legumin solution (Figure 4b). Apparently, the latter spectrum is a superposition of spectra of 'free' and adsorbed probes. However, differences in spectra shown in Figures 4a and 4b are small. It is impossible to determine quantitatively the correlation times and propoxtion of adsorbed radicals fiom the spectra in Figure 4b. The ESR spectrum of Sdoxyldecane in ovalbumin solution (Figure 4c) is S i a r to the spectrum of this radical in the buffer. Apparently, 5doxyldecaneinteracts weakly (or does not interact at all) with ovalbumin. The spin probe 5 also simulates a lipophilic compound, which contains no carbony1 group. This probe possesses a nitroxyl radical and a hydrocarbon chain (-CuHZ).separated by a CH2CH20 group. The spectra are shown in Figure 5. The correlation time of probe 5 increased in the presence of proteins: 'I= 0 . 5 10"s ~ in the buffer, 'I= 9x 1OIos in the legUmin solution, and 'I= 2 x lO-'Os in the ovalbumin solution. It is evident that in both casesthe spin probe is

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Gums and Stabilisers for the Food Industry

Figure 4 ESR spectra of 5-doqdakcane: a) in phosphate buffer (PH 7.0, 0.1 M); b) in legumin solution; c) in ovalbuminsolution. adsorbed on the protein molecules. However, the paramagnetic moiety was disposed at a remote distance from the hydrocarbon chain, by which adsorption, apparently, occurred, and it could rotate because of the flexibility of the radical chain (rotational correlation times of protein globules are much higher than those of the adsorbed spin probe 5 ) . Adsorption on a legumin molecule changed the rotational mobility of the spin probe much more significantly than adsorption on an ovalbumin molecule. The reason for this result could be that the paramagnetic moiety in the spin probe, adsorbed by legumin, is closer to the adsorption center than in the spin probe adsorbed by ovalbumin. Spin probes 6 and 7, which contain no long hydrocarbonic chain (and are much more water soluble), are not adsorbed by molecules of legumin and ovalbumin. ESR spectra of these probes in the buffer and in solutions of proteins are quite similar. The results of the study performed indicate the following. Hydrophobic interactions of legumin and ovalbumin with spin-labelled stearic acids cause adsorption of the stearic acids on the protein globules. These interactions occur in different ways for the two proteins used and they are more extended for legumin than for ovalbumin. The whole (or the most part 09 the hydrocarbon chain of a stearic acid seems to be bound to sites on the legumin globule surface. In this case, molecules of stearic acid are adsorbed on separate adsorption centers of legumin, disposed on ‘remote’ distances from each other (the distances, on which dipole and exchange interactions of nitroxyl radicals are not observed). ‘Close’ adsorption centers or centers

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Figure 5 ESR spectra of the spin probe V: a) in phosphute bufer (PH 7.0, 0.1 M); b) in legumin solution; c) in ovalbuminsolution. I is the signal of quartz ampoule.

capable of binding several molecules can participate in the adsorption process of spin-labelled stearic acid (5-DSA) with ovalbumin. Palmitic acid, sodium palminate and sodium decanoate appear to be adsorbed by ovalbumin together with 5-DSA. Introduction of a doxy1 group into positions 12 and 16 of the hydrocarbon chain reduces the binding constant of the fatty acid to legumin and ovalbumin. Both legumin and ovalbumin adsorb organic compounds, which contain a rather long hydrocarbonic chain, but they do not adsorb compounds, which contain no lipophilic groups. Results obtained with probes 1-4 (spin labelled stearic acids and 5Doxyldecane) support that the carboxylic group of the fatty acid apparently also contributes to the interaction with proteins.

References 1 . C. Tanford, ‘PhysicalChemisny of Macromolecules’, Wiley and Sons, New York and London, 1961 2. ‘The’Proteins’, Eds. N. Neurath and L. K. Beiley, Acad. Press, New York, 1965.

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Gums and Stabilisers fur the Fuod Industry

3. V. B. Tolstoguzov, In ‘Gumsand Stabiilisers for Food Industry’, Eds. G. 0. Phillips, D. I. Wedlock, P. A. Williams,IRL Press, Oxford, 1992, vol. 6, p. 241. 4. V. B. Tolstoguzov, ‘Artifid Foodstuffs’, Nauka, Moscow, 1978 (in Russian). 5. A. N.Kunetsov, ‘Method of spin probe’, Nauka, Moscow, 1976 ( in Russian). 6. A. N. Kuznetsov, B. Ebert, G. Lassrnan, A. B. Shapiro, Biochim. Biophys. Acta, 1975, 379, 139. 7. M. Le Meste,

P. Tainturier, J.-L. Gelin, In ‘Food colloids - proteins, lipids and polysaccharides’, Eds. E. Dickinson, B. Bergenstahl, RSC, 1997, p. 185. 8. L. A. Wasserman, M. G. Sernenova, In ‘Food colloids - proteins, Lipids and polysaccharides’, Eds. E. Dickinson, B. Bergenstahl, RSC, 1997, p. 77. 9. I. A. Popello, V. V. Suchkov, V. Ya. Grinberg, V. B. Tolstoguzov, Prikl. Biochem. Microbiol., 1988, 24, 50 (in Russian). 10. L. A. Wasserman, M. G. Sernenova, FoodH@ocolloiak, 1997,11,3 19. 11. P. Plietz, G. Damaschun, Sfud Biophysica, 1986,116, 153. 12. V. A. Livshits, V. A. Kuuletsov, Molec. Biol., 1980, 14, 182 (inRussian). 13. ‘Spin Labeling. Theory and applications’, Ed. L. J. Berliner, Acad. Press, New York, 1976. 14. V. Tirnofeev, B. Samarinov, J Chem.

Soc. Perhn Trans 2,

1995, 1345.

A STUDY OF THE HYDRATION BEHAVIOUR OF RENNET CASEINS IN CALCIUM CHELATING SALT SOLUTION USING A RHEOLOGICAL APPROACH

Michael P. Ennis, Michele M. OSullivan and Daniel M. Mulvihill Department of Food Chemistry, National Food Biotechnology Centre, University College, Cork, Ireland.

1 ABSTRACT Rennet casein is insoluble in water but can be hydrated in warm calcium-chelating salt solutions. Once hydrated the casein can emulsify fats or oils and when cooled the mix sets to give a matrix similar in texture to cheese and which is referred to as analogue cheese. Considerable variation has been observed in the performance of commercial rennet caseins in the manufacture of cheese U U ~ O ~ U ~Commercial S. caseins exhibiting a range of functional performances have been obtained and are being examined to establish possible causes of this variation in functional performance. A simplitled model system of the analogue cheese making process has been devised; this involves hydrating a rennet casein in a solution of a chelating salt at 55 OC with stimng. The rheological changes that occur as the rennet casein hydrates have been followed using a controlled shear rate rheometer fitted with a purpose designed paddle stirrer. Hydration of the rennet casein particles takes place in a number of stages including swelling, aggregation, network formation and breakdown. Considerable variation was observed between casein samples in swelling times, time taken to reach maximum viscosity, maximum viscosity reached and stability .of the hydrated caseins. The rheological characteristics of the rennet caseins during hydration were strongly influenced by the concentration of chelating salt in the hydrating solution.

2 INTRODUCTION Rennet casein is regarded as the protein of choice in the manufacture of Mozzarella cheese analogues that have good melt and stretch properties.’ The rennet casein can be stored for long periods without spoilage or developing off-flavours.2 it can be used as the sole protein component in the analogues and it has a bland flavour and suitable colour. These properties make rennet casein superior to other caseins and caseinates in the manufacture of analogue cheeses. An analogue or imitation cheese is essentially an oil-in-water emulsion where the fat phase is dispersed in a continuous proteinhqueous matrix, as in processed cheeses.” The emulsion is stabilised by the amphipathic protein molecules reducing the interfacial free energy of the dispersed oil droplets and by increasing the viscosity of the proteidaqueous phase. Performance characteristics of the cheese analogue (shreddability, stretchability, elasticity etc) arc strongly influenced by the nature of the protein in the aqueous phase. In the manufacture of rennet casein based analogue cheeses, calcium-chelating salts (also called emulsifying salts) are used to disrupt the calcium mediated cross-bridges in the rennet casein and thus allow the protein to hydrate.’ By increasing the ability of the protein to hydrate, the emulsifying ability of the protein is enhanced. Increased water binding by the protein leads to 211increase in the aqueous phase viscosity and improved stabilising of the

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Gums and Stubilisers for the Food Industry

oil-in-watcr emulsion. A wide range ofchelating salts are suitable for use i n the manufacture of cheese analogues.4-6Other ingredients such as acidulants and hydrocolloids may also be used in cheese analogues.6 Rennet caseins from dilkrent manufacturers and even different hatches of rennet casein from thc same nianufacturer can exhibit considerable variation in performance in the manufacture of cheese analogues. Problems encountered include unsatisfactory hydration of the rennet casein and consequent failure to emulsify the oil phase, over emulsification of the oil phuse and vaiiahility in the performance characteristics of the checse analogue produced. This study reports on the wide variation in rheological behaviour of rennet caseins as they hydrate in a simple model system comprising rennet casein. chelating salt and water’.

3 MATERIALS AND METHODS Thirty six samples o l rennet caseins produced under nominally identical processing conditioiis were obtained from a local manufacturer and stored under ambient conditions. The nitrogen content of each rennet casein was determined in triplicate by the macroKjeldahl method.’

Figure 1 Rheologicnl ~ / i i u l y . w .were s c m Y e d out rising (I Haakr M I 0 heatl.fitted with u piirjmse huilt .stirsing ptidille und MVI cup. The sttiinless steel pmriiile was constructed by uttcrching to ( I .shaft t n o , f l c i t 36 iiim x 18 i?ii?i plates etrch pei:fortirerl by T w o 7 i?ii?i diurneter hole.\. ( I S .shown. Rheological analyses were cairied out as follows. A stock solution of the chelating salt, disodium orthophosphate (Na2HP04) in deionised distilled water was prepared. Rennet casein was sicved using a SO0 pm mesh-size sicvc and sufficient rennet casein to give 6 g protein was weighed out. This quantity of rennet casein was added to pre-warmed (55 (C) solution prepared from the stock chelating salt solution and water so that the final weight of dispersion was 7 5 g and the concentration of chelating salt was from 0.0 to 1.0%. w/w. (The final dispersion was 8%, w/w, protein.) The dispersion was stirred at 600 rpm

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immediately using a Haake M 10 controlled shear rate rheometer head fitted with a purposebuilt paddle stirrer (Figure I) and M V l cup. The temperature was maintained at 55 O C throughout the test and all determinations were canied out in triplicate.

4 RESULTS Hydration of rennet caseins in the solution of chelating salt was found to occur in a number of stages as shown in Figure 2. Wetting and swelling of the individual particles occurred first (a), followed by aggregation of the swollen particles (b). Aggregation and swelling of the particles continued and formation of a network of particles commenced (c). Network formation and swelling continued until all of the available liquid was taken up and a macroscopically uniform very viscous dispersion was formed (d). On continued shearing the viscosity decreased (e) and a low viscosity fluid, similar in appearance to milk, eventually formed (f). The time taken for the first peak (c) in the rheological profile to appear is defined as the swell time and t!ie swell viscosity is defined as the viscosity index at that time. The hydration time is defined as the time taken to reach the maximum viscosity (d) and the hydrated viscosity is the viscosity index at that time. Stability is defined as the time taken for the viscosity index to decrease from the maximum (d) to half of the maximum value (e).

Figure 2 H\Itlstirion pivfile of reiiiier casein in (1.4%, w/w. soliirion qf chelating salt tlisotlilim orthol’ho9’hore.

Thc rheological behaviour of any of the rennet caseins during hydration was found to be strongly dependent on the concentration of chelating salt in the hydrating solution. At low chelating salt concentrations (<0.3%,w/w) the rennet casein particles were observed to swell slightly due to imhibation of the surrounding solution, and showed a small increase in viscosity (Figure 3a). However, even after prolonged periods of shear rennet casein was present as discrete swollen particles.

130

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Protein Systems

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resulted in small decreases in swell viscosity, while the maximum viscosity index attained as the rennet casein hydrated increased dramatically as the chelating salt concentration was increased up to OS%, w/w, (Figure 3c) and a macroscopically homogeneous viscous dispersion formed. Further increasing the chelating salt concentration. to 0.7%. wlw, decreased the maximum viscosity attained on hydration of the rennet caseins and further increased the swell time and hydration time (Figure 3d). At chelating salt concentrations w/w, no distinct peaks in the rheological profiles were obvious and higher than 0.841, discrete particles of rennet casein were present even after prolonged periods of shearing. The effect of chelating salt concentration on the hydration behaviour of six rennet caseins was studied in detail. The viscosity index attained by each rennet casein increased as the salt concentration wa9 increased to 0.5%J, w/w, and subsequently decreased as the salt concentration was increased further (Figure 4a). The stability of the rennet caseins showed a marked increase as the chelating salt concentration was increased from 0.4%. w/w, with maximal stability at 0.5%. w/w (Figure 4b). Hydration time increased as the concentration of the chelating salt increased throughout the range 0.2%, w/w, to 0.7%. w/w, (Figure k). During hydration in O.S%, w/w, chelating salt solution the majority of the rennet casein samples showed a maximum viscosity index of between 75 and 95 compared to a range of 20 to 80 when hydrated in 0.4%. w/w. chelating salt solution (Figure 5). The hydration times in O.S%, w/w, chelating salt were from 42 to 77 min while in 0.4%. w/w, chelating salt hydration was generally faster with hydration times between 18 and 65 min. Rennet caseins exhibitcd much greater stability when hydrated in US%, w/w, chelating salt solution with values hctween 1.9 and 62 min, compared to values of 1.2 to 15.3 min when hydrated in 0.4%. w/w, chclating salt (Figure 6). S DISCUSSION

Chelation of calcium present in the rennet casein by the sodium phosphate led to a decrease in the extent of calcium mediated protein-protein cross-linking. The substitution of sodium for calcium, and the binding of phosphate groups to the protein increased the solubility of the protein. At low concentrations of chelating salt the protein became partly hydrated but sufficient calcium mediated cross-linking remained for he rennet casein particles to maintain their integrity and limited swelling of the particles occurred. As the concentration of the chclating salt was increased to about 0.5%. w/w. the extent of protein hydration increased while the amount of calcium mediated protein-protein cross-linking decreased. Swelling of the particles was greatly increased and. more importantly. interaction between proteins in different particles was facilitated leading to the observed aggregation. network formation and eventual loss of the discrete particulate nature of the rennet casein dispersion. The nature and extent of the interparticle interactions played a major role in determining the overall viscosity of the dispersion. Soluhilisation of protein as sodium caseinate also contiihuted to the increased viscosity. The subsequent decrease in viscosity on continued shearing of the hydrated rennet casein was prohahly due 10 the combined effect of shear and the greater conformational freedom of the proteins, which led to exposure of hydrophohic sites on the proteins and thereby faditatcd increased hydrophobic protein-protein interactions, which resulted in reduced solubility of the proteins, a greater tendency to form colloidal protein aggregates similar to micelles and the ultimate collapse of the gel-like network structure that had previously formed. Such changes are consistent with the milk-like appearance of the samples following prolonged shearing. The increase in hydration time as the chelating salt concentration was increased may be explained as follows. Rapid hydration of the protein at the surface of rennet casein particles may have led to the formation of a gel-like outer layer, making the surface poorly permeable to the chelating salt solution and so retarding the chelation of calcium in the interior of the particles. This increased the time taken for the particles to swell, aggregate. form a network and become fully hydrated.

132

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134

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135

The hydration hehaviour of the rennet caseins in 0.4%J,,w/w, chelating salt solution were L'ound to he related to how the rennet caseins performed during the manufacture of cheese analogues. Rennet caseins with fast hydration times and high viscosities had very good emulsifying ahility and were generally the most satisfactory in producing analogue cheeses. However. in certain cheese analogue manufacturing processes the use of these rennet caseins led to over-emulsification of the oil phase in the analogue. This results in a product with unsatisfactory melting characteristics.* Rennet caseins with long hydration times in 0.4%. w/w, chelating salt solution were found to hydrate poorly during cheese analogue manufacture antl those that showed low hydrated viscosities in 0.4%. w/w, chelating salt solution also pcrl'ormetl poorly in the manufacture of analogues. Intermediate stability when hydrated i n O.S%, w/w, chelating salt solution coincided with good performance in the manufacture of cheese analogues. Rennet caseins with very low viscosities and low stab es when hydrated in the model system may he unahle to maintain the oil phase as a suitable emulsion during cheese analogue manufactuie, and so fail to produce a satisfactory cheese analogue. Rennet caseins cxhihiting very high stability perl'onn poorly duiing chcese analogue manufacture, showing \cry slow incorporation of the oil phase. The successful manufacture of a cheese analogue. and the functional properties of the chccse such as shretldahility, nieltability and izlease of free oil on heating are influenced by the functionality of the rennet casein, making the selection of a suitable rennet casein an important factor i n producing a satisfactory cheese analogue. Rheological analysis of hydration hehaviour may provide a basis for selecting the most suitable rennet caseins for particular applicaLions. 6 ACKNOWLEDGEMENT

M.P.E. antl M.M.O'S. are supported hy grant aid under the Food Sub-programme of the Operational Programme for Industrial Developinent which is administered by the Department 01' Agriculture. Food and Forestry. Dublin and supported by national and EU fu ntls. 7 REFERENCES

I . W. R. Aimutis, Food Trchnology Eiiropc, 1995, 2:2, 30. 2 . B. E. Rybinski, L. E. Dawson, D. G. Bixby and L. E. Woodford, U.S. Patent No. 5 244 687, 1993.

3. L. A. Shimp, Food Technology, 1985, 5 ( 5 ) , 63.

4.M. Caric and hl. Kalah. "Cheese: chemistry, physics & microhiobgy. (2nd edn.)" (P. F. Fox cd.). Elscvicr Science Puhlishcrs, London, 1993. p467. 5. S. K. G u p ~ a C. . K;irAi:iclian antl R. C. Lindsay, J. Duii?JSci., 1984, 67, 764. 6. M. P. Ennis and D. M. Mulvihill, "Proceedings of the 5th Cheese Symposium"

(T. M. Cogan, P. F. Fox and R. P. Ross cds.), Teagasc, Duhlin, Ireland, 1996. 7. International Dairy Fcdcration. "IDF Standard 92: 1979. Caseins and caseinatesdctcrminnlion 01' prolein content.", Brussels. 1979. X. D. P. Neville and D. M. Mulvihill, "Proceedings of the 25th Annual Food Science & Technology Research Conference", University College Cork, Ireland, 1995.

GELATION PROPERTIES OF SMOOTH AND SKELETAL MUSCLE MYOSIN PROTEINS

Peter G. Somers and Nazlin K. Howell School of Biological Sciences University of Surrey Guildford Surrey GU2 5XH.

1 INTRODUCTION The objectives of this study were: 0 To obtain a detailed knowledge of the mechanisms of gelation of smooth muscle myosin proteins and to compare them with skeletal myosin proteins and To use the knowledge obtained from the above studies in order to enhance the gelation of smooth muscle. Smooth muscle comprises a diverse range of biological tissues including stomach, lung and intestines, which are major by-products of the meat industry. The physiological function of smooth muscle far exceeds that of the skeletal system in vivo; yet the functional performance of such a diverse muscle group in comminuted food products is poorly understood. It is included, when permitted, as a ‘filler’ meat 1 but contributes little to the binding of the system. This paper attempts to explain, at the molecular level, the reason for the poor gelation properties and discusses factors affecting the thermal aggregation properties of smooth muscle proteins, compared to skeletal muscle proteins. The main muscle protein myosin is responsible for the excellent gelling and emulsifying properties of skeletal meat proteins in comminuted meat products and myosin was therefore selected for study. It is interesting to note that characterisation of skeletal myosins from fish, mammals and birds indicates that heat gelation properties are not only species specific but also tissue specific e.g. avian slow twitch leg muscle myosin has heat gelation properties approximately a third compared with fast twitch breast muscle The differences in the structure of myosin therefore contribute to the differing gelation properties.

’.

Myosin comprises about 55% of the muscle proteins. It is a large molecular weight protein of over 500,000 daltons. Myosin can be divided enzymatically into subfragments. S1 is the globular head region which contains the sites for ATPase activity whereas the rod fragment is a fibrous a-helical coiled-coil protein -(heptad repeat) which confers polymerisation competence to the myosin molecule under physiological ionic conditions. Low molecular weight proteins known as light chains are bound to the S1 region. (Figure 1.)

Protein Systems

137

This paper summarises studies undertaken on the gelation of smooth and skeletal muscle proteins, details of which are being submitted for publication elsewhere by the authors.

s1 4

~

~

s2

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LMM

b

b

Rod

b

Abbreviations S1; Subfragment 1. S2; Subfragment 2. LMM; Light meromyosin.

HMM; Heavy meromyosin. LC; Light chains

Figure 1. Proteolytic substructure of the myosin molecule 2 MATERIALS AND METHODS 2.1 Materials

Smooth muscles,chicken gizzard, pig aorta, pig stomach and pig oesophagus (mixed skeletal/smooth),were obtained from a commercial abattoir 30 mins post mortem and frozen at -2OOC. Chicken breast skeletal muscle was used as a control. Myosin was prepared according to a modified method of Sobieszek and Bremn~el.~’’ In addition, proteolytically derived subfragments were isolated from pig stomach smooth muscle as well as a control myosin isolated from avian skeletal muscle according to Cross et al.6

2.2 Methods

The purity of isolated myosin isoforms was tested by ATPase measurement and polyacrylamide gel electrophoresis; the nativity was assessed by electron microscopy. The isolated proteins were studied using a range of ionic strengths (0.05, 0.15 and 0.6M NaC1) and at pH 6 , 7 and 8 using the following techniques:

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138

Rheological Evaluation Small deformation rheology was undertaken using either 1% (w/v) myosin or 0.5% subfragment protein samples on a Rheometrics controlled stress rheometer using a 40mm cone and plate geometry with a 0.3mm gap. Temperature sweeps from 359OoC -2OOC at 2 Wmin at frequency 1 rad/sec as well as frequency sweeps (1-100

rad/sec) at 2OoC were undertaken. High Sensitivily Scanning Microcalorimetry (HSDSC) High sensitivity differential scanning microcalorimetry was undertaken on a Microcal HSDSC using approximately 0.1 - 0.4% protein solutions and scan rate of 60 kh. Transmission electron microscopy (TEM) Samples were studied by negative staining using 1% uranyl acetate. A Siemens 102 TEM operating at 100 KV was used for image generation. All proteins were studied at a protein concentration of 0.2 mg/ml. Turbidity meusurements Turbidity measurements were made in a Perkin Elmer Lambda-2 uv/vis spectrophotometer as a function of time and a data point was recorded every second. The temperature reading on the thermometer corresponding to a given time measurement was recorded and the time ordinate converted to temperature. Protein concentration was 1 mg/ml and scan rate 60k/hr. Sodium Dodecyl Sulphate-Polyacrylamide Gel Electrophoresis (SDS-Page) Dissociating, discontinuous gradient sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-Page) using 1Opg/ml protein samples was performed according to Laemmli

’.

3 RESULTS AND DISCUSSION

Characterisation of the myosin isoforms The myosin isoforms were successfully isolated as shown by the purity of the extracts by SDS-polyacrylamide gel electrophoresis (SDS-Page) which indicated discrete heavy and light meromyosin bands (Figure 2). In addition transmission electron micrographs (Figure 3) showed that all isoforms formed coherent filaments at low ionic strength. Moreover, all isoforms liberated about 0.14 pM Pi/mg/min which was lower than that of skeletal myosin (0.6 pM Pi/mg/min) but indicated that they were not denatured.

139

Protein Systems

Lane 1

MHC

2

3

4

-

Light chains

Lane

4 = Standards 1 = Smooth muscle myosin

2 = Chicken breast myosin ~

3 = Pigoesophagusmyosin

Figure 2. Separation of myosin isoforms by SDS-polyacrylamide gel electrophoresis

Figure 3. Transmission electron micrographs of undenatured pig slomach myosin isoform (magnificationx 28,000).

140

Gums and Stabilisers for the Food Industry

RheoIogicaI properties

Typical rheological curves obtained from temperature sweeps (20-90' C) of the smooth and skeletal muscle myosins in 0.15M NaCl and pH 6.0 are shown in Figure 4. All myosin isoforms except aorta showed a characteristic transition at 50-60' C. The G' values increased both on heating and cooling. However the smooth muscle myosins were some 90% lower than those obtained for avian breast skeletal muscle. The gelation of myosin as a function of pH is also shown in Figure 4; this indicated that gelation was greatest at low pH values. The ionic strength also influenced the gelation properties of myosin at pH6 as indicated by Figure 5. Smooth muscle myosin was more soluble at lower ionic strength values and indicated higher gel strength and G'values.

10000 j

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10 !Breast myosin, pH 81

1 11

1

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0.1 20

40

60

80

100

Temperature ("C) Figure 4. Temperature sweeps showing the influence of pH on the thermal gelation properties of pig stomach and chicken breast myosin.

141

Protein Systems

%G oesophagus myosin Pig stomach myosin

OM NaCl

0.6M NaCl 150mM NaCl50mM NaCl

Figure 5 . Comparison of the thermal gelation properties of the myosin isoforms relative to the control isoform (breast) as a function of ionic strength at pH6.

i

100

Pig Stomach myosin, pH7,8

80

1 7 Breast myosin,

60

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1

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.

i

50

.

i

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.

70

l

80

Temperature ("C) Figure 6 . Turbidity profiles ofpig stomach and chicken breast myosin as a j k c t i o n of PH

Gums and Stabilisersfor the Food Industry

142

Turbidity Aggregation was observed for skeletal muscle followed by pig and chicken gizzard but not for aorta myosin (Figure 6). On heating, at pH6, aggregation was higher compared with samples tested at pH7 or 8, thus reflecting the higher gelling properties at pH6. Dijierential scanning calorimetry Differences were observed in the transitions for skeletal and smooth muscle myosins. The DSC spectra for pig stomach myosin are shown in Figure 7. The main peak observed was confirmed to be LMM (Somers, 1997).9 In the case of smooth muscle the LMM transition was found to be partially reversible at pH 7,8. However,at pH 6 pig myosin indicated aggregation at 45OC which was not reversible on re-scanning. These results confirm findings by the turbidity and rheological techniques that aggregation and gelation properties were higher at pH 6 compared with pH 7 or 8.

Deconvoluted native spectra, pH 8 30

40

50

60

Native spectra, pH 6 30

70

40

50

60

70 1

1OO(

CP

aoo 600

KJMol" K-' 400 200

0

I 000

800

600

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40

50

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70

250

- 250

200 150

'

O0

50

50

0 -50

-50

30

40

50

60

70

Temperature ("C)

Figure I. Dijierential scanning calorimetry spectra for pig stomach myosins at pH 8 and 6 (Somers et al, 1997).

Electron microscopy Transmission electron microscopy indicated that the native smooth myosin molecules did not aggregate at 20°C. However, on heating to 90°C aggregation of the S1 heads was evident, in a similar way to that observed for skeletal myosin.8 In contrast to the skeletal myosin, however, the oligomers thus formed were soluble and not capable of gelation via the LMM region. This is represented diagramatically in Figure 8. The above evidence together with additional DSC and circular dichroism experiments point to the differences in LMM behaviour of smooth muscle compared to skeletal muscle and may be the key to the differences in gelation properties of the two types of myosins (Somers et al, 1997).

143

Protein Systems k t i v e monomeric myosin molecuks

I

1 when heated hut the oligomers solubk and do not gel

*200c+

!mC+

cool

Figure 8. Schematic model of the aggregation of smooth muscle myosin

4 CONCLUSIONS The studies indicate that the thermal gelation properties of smooth muscle myosin are dramatically less competent than the skeletal isoform. Rheological and DSC evaluation c o n f m that skeletal breast myosin has superior gelling under all ionic conditions tested; oesophagus skeletal myosin has intermediate gelling properties and pig and chicken stomach muscle are significantly less functional but could be improved by either lowering the ionic strength or pH by altering the charge. In addition, the functional disparity between these two proteins has been assigned to the light meromyosin region; this fragment, isolated from skeletal myosin, is capable of thermal gelation whereas the smooth muscle equivalent remains completely soluble and free of aggregation when heated under identical ionic conditions. The contrasting behaviour of smooth and skeletal light meromyosin subfragments, has, for the first time been interpreted at the protein sequence level and is attributed to a predominance of glutamic acid in the latter protein which disrupts water structure at the protein-solvent interface (Somers and Howell, 1997 submitted). Restructuring this hydration water induces gelation both in the light meromyosin fragment and the intact protein and thus offers potential means of improving smooth muscle gelation in comminuted meat systems.

144

Gums and Stabilisers for the Food Industry

References 1. R. A. Lawrie, ‘Meat Science’ 3rd Edition Pergamon Press, Oxford, 1979. 2. A. Asghar, J. Morita, K. Samejima, and T. Yasui,. Agric. Biol. Chem., 48,2217, 1984. 3. W.F. Harrington,. In ‘The Proteins’ Vol4,276 Eds. H. Neurath, R.L.Hil1, 1979. 4. A. Sobieszek and R. D. Bremel, Eu. J. Biochem., 55,49-60, 1975. 5. A. Sobieszek and J. V. Small,. J. Mol. Biol., 101, 75-92, 1976 . 6. R.A. Cross and J. Vandekerckhove, Febs Letters, 200 (2), 355-360, 1986. 7. U. K. Laemmli, Nature (Lond), 227,680-685, 1970. 8. A.Sharpe, and G . Offer, J. Sci. Food Agric., 58,63-73, 1992. 9. P.G. Somers, PhD Thesis, University of Surrey, 1997.

Acknowledgements

The authors would like to thank Dr. B. Chowdhry, Greenwich University for the use of the HSDSC.

THERMAL STABILITY AND SOL-GEL TRANSITIONS OF WHEY AND EGG WHITE PROTEINS

P. Relkinl*, T. Meylheucz, B. Launayl and K. Raynall lLaboratoire de Biophysique des Mattriaux Alimentaires Dtpartement Science de 1'Aliment Ekole Nationale Sugrieure des Industries Alimentaires 2Laboratoire de Gtnie de 1'Hygihne et des Proc&d&s Alimentaires 1, Avenue des Olympiades, 9 1 744 Massy-France.

ABSTRACT Thermal behaviour of whey and egg white proteins were studied by differential scanning calorimetry, DSC, in various buffers and at concentrations that favour protein-protein interactions. Characteristic parameters, such as temperature of maximal peak deviation, Tmm, and heat of reactions, AHcal, were extracted from the DSC signals. Diagrams of heat-induced sol-gel state transitions obtained with various protein solutions were discussed in terms of characteristic temperatures of protein conformational changes. The temperature dependence of the gel-time showed two regimes on each side of a critical pre-heating temperature,Tcr, the temperature sensitivity being higher in the low temperature range. For mixtures of whey proteins and egg white proteins Tcr Tmax, the maximal DSC peak deviation of the major protein component and of the less abundant ones, respectively. The supramolecular structures of some gels, obtained after heating at T 2 Tmm, were observed by scanning electron microscopy. The micrographs were discussed in terms of molecular conformationalchanges as a function of the buffer content .

-

1 INTRODUCTION The native tertiary structure of globular proteins is stabilized by non covalent (ionic, hydrogen and hydrophobic) and covalent (disulfide) bonds. Both intra-molecular structural changes and intermolecular interactions have been shown (1-5) to be dependent on the protein type and concentration, and also on extrinsic physicochemical parameters (pH, type and concentration of co-solutes or co-solvants, time-temperature treatment...). Tombs (6) suggested that differences in turbidity reflect differences in the arrangement of heat-denatured globular proteins. More recent studies (7- 14) showed that mechanical and structural properties of thermally-induced globular protein gels depend strcngly on the conditions of formation. Clark et al. (3) interpreted the network formation in the heat-induced aggregation process of globular proteins, in terms of a "strings of beads", where the homogeneity over long distances correlates with changes in gel opacity caused by alteration in pH and ionic strength. They suggested that thermal aggregation of globular proteins required some degree of unfolding (exposure to the aqueous medium of initialy buried amino acid groups) for enhancing intermolecular hydrogen bonding, ionic pairing and hydrophobic interactions. DSC, with small volume samples and high scan rates, has been applied to quantify the thermal stability of concentrated proteins (10, 15-25).The results showed that the extent of intermolecular

146

Gums and Stabilisers for the Food Industry

interactions and the level of reversibility, when considered (16, 17) are specific not only on the protein (type and concentration) but also on the physicochemical conditions including pH, composition of buffers (nature and concentration of salt ions) and heating rates. Recently, we combined DSC, sol-gel state diagrams and electron microscopic observations to compare the thermal behaviour of globular proteins and the structurepatterns of their heat-induced gels formed in different chemical conditions (19,20). Our results indicated that the times needed for sol-gel transitions were highly temperaturedependent, when formed at T I Tmax (peak temperature) of either the major protein component (whey system) or to a minor one or their mixture (egg white mixture). In the present work we applied a similar experimental approach to various solutions of whey and egg white proteins heated in sodium phosphate buffer or milk ultrafltrate permeate.

2 EXPERIMENTAL The whey protein isolate (WPI) and P-lactoglobulin (p-lg) samples were kindly supplied by the "Laboratoirede TechnologiesLaititres", INRA-Rennes (26) and BesnierBridel-France, respectively. They were obtained by membrane micro- and diafiltration of whey prepared from skim milk after previous extraction of micellar casein. Egg white protein concentrates (EWP) were kindly supplied by Ovonor-Diana (France). Ovalbumin, lysozyme. bovine serum albumin (BSA) and a-lactalbumin (a-la) samples were supplied by Sigma. All the protein samples were dispersed in sodium phosphate buffer (SPB, pH7, 0.1 M). p-lg and whey protein isolate were dispersed also in milk ultrafiltrate permeate (MUP). MUP, in powdered form was dissolved at 0.6wt% and its pH adjusted to 6.65. The protein concentrations (8wt%) were determined by the Folin-Lowry's (whey proteins) or biuret method's (egg white proteins) with bovin serum albumin as a standard. The pH of all solutions was adjusted with NaOH (IM)or HCI (1 M). The overall heat-induced structural changes (denaturation+aggregation)of protein molecules were monitored by differential scanning calorimetry (DSC7, Perkin Elmer) using stainless-steel pans. The protein solutions were submitted to a first heating run from 20 to 110 "C at 10 "C min-l, followed by a quenching step to 20 "C and a reheating. The second thermogram, used as a sample baseline, was subtracted from the first one except for lysozyme and a-la that showed peak transition reversibility. Calorimetric parameters, such as peak temperature, Tmax, and enthalpy changes, AHcal, were extracted from the DSC signals, after correction from the furnace and sample thermal-lag and using a straight sample baseline between the onset and the end of the heat flow deviation, respectively (17). To study the aggregation properties of pre-denatured proteins, protein solutions were heated, at -10 "C min-1, in sealed tubes (1 ml) from room temperature to various temperatures lying between the initial and final peak temperatures and then cooled to 4 "C. The time-temperature values of sol-gel transitions evaluated, by tilting the test tubes were used to draw sol-gel state-diagrams. Microstructures of heat-induced gels were observed by scanning electron microscopy (SEM, Jeol-model JSM-2500) after a double glutaraldhehyde (1.5 %) and osmium tetroxide (1 %) fixation, followed by dehydration in a series of ethanol solutions (30 2 100%) and gold metallisation ( 10 mA, 450 s).

3 RESULTS AND DISCUSSION

3.1 Thermal Stability

147

Protein Systems

Figure 1 shows examples of DSC thermograms obtained by heating from 20 to 110°C at 10 "C min-1 the ovalbumin and lysozyme mixture and egg white proteins dispersed (8wt%) in SPB ( 0.1 M, pH7). Figure 2 shows DSC signals of p-lg and WPI dispersed (8wt%) in MUP. The heat flow signals extended on a large temperature range from 55 "C to 90 "C for p-lg and egg white proteins and to 100 "C, for WPI mixture. They exhibited shoulders corresponding to heat transitions of minor components such as conalbumin, lysozyme and ovomucin for the EWP mixture (21-23) and to alactalbumin, lactoferrine and BSA for WPI (24). For the EWP mixture, the major peak (80 "C ) and the shoulder (-85 "C) corresponded to the transition of ovalbumin and of its intermediate-form,respectively. The DSC trace did not present a transition at 90 "C, that would correspond to S-ovalbumin molecules. The development of these molecular species (more heat stable conformers) is favoured by a long storage time, as seen in the thennogram (fig. 1) of the ovalbumin / lysozyme mixture (shoulder and peak at approx. 85 "C and 88 "C, respectively) which has been prepared with Sigma samples stored at -20 "C for several months.

-

-

Temperature ("C)

Fig.1 Thermograms of EWP and ovalbumin / lysozyme and EWP mixtures in SPB.

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6 0

7 0

8 0

9 0

100

Temperature ("C)

Fig.2 Thennograms of p-lg and WPI mixtures in MUP.

The calorimetric parameters extracted from the signals obtained from various protein solutions are reported in Table 1. Peak temperatures, T m a , which represent temperatures of the maximum rate of conformationalchange of protein molecules, are in agreement with the published values obtained in similar conditions of heating rate, protein concentration and buffer composition. &al values result from dissociation and denaturation (endothermic) processes, on which an aggregation (exothermic) phenomenon could be superimposed during the time course of DSC measurements. The overall AHcal values obtained in WPI and EWP (15 and 10 J.g-1, respectively) are lower than those expected from a linear combination of the contributions of their components. The values reported in Table 1 show also that the temperature of heat transition of p-lg molecules was lower in SPB than in MUP. This could be due to the presence of lactose and of kosmotropic ions such as chloride, citrate, tartrate, sulfate and phosphate that could lead to an increase of the heat stability of p-lg dimers (27). This hypothesis is confirmed by the dissymetrical shape of the thermogram: the shoulder on the left part of the major peak might be due to the dissociation of dimers before denaturation at a higher temperature.

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--------------------------------------------------------------------------------------------------------------------------------------------------------------- ~ ""'l Tmax is the temperature of maximal peak deviation, L\Hcal is the calorimetric heat of reaction, (*) temperatures of shoulders, (a) ~ SPB:sodium phosphate buffer, MUP: milk ultrafiltrate permeate (b) ovalbuminllyzozyme in 5:1 weight ratio, (c) WPI, whey protein ~ isolate, EWP, egg white proteins (d) calculated values. &.

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Literature

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74.9 78 65.2 64.5 62.1*,73.5 60*, 72*,77.4

7 6.65 7 7 7 6.65

pH

P-Ig (SPB) P-Ig (MUP) n-la (SPB BSA (SPB) WPI (SPB) WPI(MUP)

Sample (bufferj'' (8wt%)

Table 1. Characteristic temperatures and heat of reactions obtained by DSC

~

00

Protein Systems

149

3.2 Gelation of Pre-Denatured Proteins Sol-gel state transitions with the pre-denatured molecules were determined by a simple tilting test. The Arrhenius plots of p-lg and WPI mixture in MUP and of ovalbumidlysozyme and EWP mixtures in SPB are reported in Figures 3 and 4.

2.7

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Fig.3 Arrhenius plots of gel times for p-lg (diamond) and WPI (circles) in MUP.

-

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Fig.4 Arrhenius plots of gel times forovalbumidlysozyme (circles) and EWP mixtures (triangles) in SPB.

These curves show that when pre-heating was made at a given temperature, times needed to reach the sol-gel state transitions are highly dependent on this temperature. On the high temperature range, they became much less temperature-dependent. The fitting of the experimental points by an Arrhenius model gives two plots intersecting at a temperature, Tcr. This temperature was approximatively equal to Tmm (fig. 2) of p-lg in pure solution or in mixture in WPI (pH6.65). Tcr (fig.4) is very close to that of the lowest DSC transition for the EWP and to that of the lysozyme transition in ovalbumidlysozyme mixture (fig. 1). The first peak transition in EWP is assymetrical; it could correspond to a mixture of minor protein components such as lysozyme (69.8 "C), conalbumin (63 "C), ovomucoid (77 "C)or their associated complexes: at pH7, lysozyme (PI= 10.7) and ovomucoid (pI=4.1) molecules might be associated by electrostatic forces. Previously (28), the formation of heat-induced gels of BSA solutions (9% protein concentration and pH 6.5) was studied with a mechanical testing procedure. The best fitting of the time-temperature results corresponding to the sol-gel transitions was also obtained using two successive Arrhenius plots intersecting at a temperature, Tcr, close to the onset of denaturation, as determined by spectropolarimetry. This difference could be due either to differences in the methodologies used to determine both denaturation and aggregation mechanisms, or to the protein itself. However, our results obtained with various globular proteins, indicated that both conformational changes and sol-gel transitions seemed to be specific not only on the native protein structure but also on the physicochemical conditions.The methodology used in the present study indicates that for whey proteins, Tcr occurs when the maximal rate of conformational change of the major protein (p-lg), is reached. However, for egg white proteins in mixtures this change seems to be associated with the heat conformational changes of minor protein components. Recently (20), we showed that intermolecular SWS-S reactions in p-lg at neutral pH

150

Gums and Stubilisers for the Food Industry

were not involved during the time course of the DSC experiments at 10 "Cmin-1. For egg white proteins at a neutral pH, formation of stable intermolecular p-sheet structures was detected by Raman (29) and circular dichroism spectroscopy (30), suggesting the formation of cross-linked intermolecular P-sheet structures from partially unfolded molecules with more surface exposed hydrophobic groups. A first increase in the pform of egg white proteins (2% at pH9.5, in distilled water) after pre-heating at 2°C min-1 from 20 "C to various temperatures, was observed to occur between 60 and 70 "C. The temperature values corresponding to the minor peak transition (fig.1) and to Tcr (fig. 4) are both in this temperature range, suggesting that the first step of the aggregation kinetics might be controlled by conformational changes of the minor protein components. The formation of S-S cross-linking reactions in egg white proteins in mixtures was demonstrated (30) to start at a much higher temperature (-80 "C).By heating the whey protein solutions at a temperature close to the DSC peak temperature, hydrophobic interactions within the core of protein molecules could be irreversibly broken (1, 2), leading, on cooling, to an increased surface exposure of hydrophobic residues. Intermolecular p-structures are also supposed to be involved in heat-induced aggregation of p-lg (3 1). At higher temperatures, intermolecular SWS-S oxidative reactions could play a major role, explaining why the gel time is less temperaturedependent (20). For all the protein solutions investigated in this study, the drop in temperature-dependency of the gel time at T 2 Tmax may be interpreted, following the hypothesis of Richardson and Ross-Murphy (28), as due to different mechanisms of heat-induced structural changes. They suggested that thermal aggregation of globular proteins required some degree of unfolding (exposure to the aqueous medium of initially buried amino acid groups), enhancing intermolecular hydrogen bonding, ionic pairing and hydrophobic interactions. Our DSC data show that when the maximal rate of the heat denaturation process of a key protein component is attained, the gel time becomes less temperature sensitive.

3.3 Microstructures of gels Microscopic observations by SEM were made on gels of p-lg and WPI (in MUP or SPB) and on gels of lysozyme, lysozyme/ovalbuminmixture and EWP (in 0.1 M SPB;pformed by heating from 20 "Cto 80 "C(- 10 "C min-l), holding for 2 min and quenching to 4 "C. The network microstructures, observed at the same magnification, showed different patterns in MUP or in SPB. In SPB, the surface structure of gels obtained with both whey and EWP (fig. 5 ) seems to be much more homogeneous than for whey protein in MUP (fig.6). In order to explain our DSC data, the structure stabilizing effects of MUP ionic salts and lactose could be responsible for the corresponding network microstructure, constituted by large aggregates in MUP, while in SPB surface gels seem to be filamentous. The aggregates of pure p-lg gels are larger (fig.6 a) than those of WPI (fig.6b). This could arise from an association mechanism between p-lg molecules or between them and other whey proteins such as a-la and BSA which have lower denaturation temperatures (Table 1). The strands, constituted by many aggregates are thicker in lysozyme when alone (fig. 5a) than in mixture with ovalbumin (fig. 5b). The network microstructure of EWP (fig.%) seems to be as homogeneous as those given by p-lg (fig. 5d) and WPI (fig. 5e) when heated in SPB. Again, the structure stabilizing effect of buffer containing sodium phosphate ions (SPB) is lower than that of the one containing chloride, citrate and sulfates, calcium, magnesium ions and lactose (MUP).

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5a Pure lysozyme

5d Pure P-lactoglobulin

5b Ovalbumidlysozymemixture

5e Whey Protein Isolate mixture

Fig 5 Microstructure patterns of gels obtained in monosodium phosphate buffer (bar = 1 p for all the images, except for ovalbumidlysozyme mixture, where it corresponds to 1.33 p).

6a Whey Protein Isolate mixture

6b Pure &lactoglobulin

Fig. 6 Microstructural patterns of gels obtained in milk-ultrafine permeate, (bar = 1m).

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4 CONCLUSION In the absence of high electrostatic repulsive forces due to the screening of protein surface charges by ionic salts, molecular conformational states are stabilized more in MUP than in SPB. This result is reflected by a lower heat transition temperature of p-lg molecules in SPB (74.9 "C) than in MUP (78°C). For whey protein mixtures, the temperature of the major peak transition corresponds to the one of p-lg but it did not signicantly change upon mixing in SPB or in MUP. However, the shoulders in the DSC signals indicate heat conformationalchanges of the other protein components. For EWP, the major peak of heat transition corresponds also to the major protein component but the overall heat of reaction is much lower than the total heat of reaction expected from additional contributions of the other components: this indicates increased intermolecular associations during the course of DSC measurements. When the maximal rate of heat conformational change of at least one protein component in the mixture is reached (peak temperature), the gel times become hardly sensitive to the temperature of pre-heating. In whey protein mixtures, p-lg heat conformational changes seem to be the rate-limiting process for heat-induced aggregation but in the case of egg white protein mixtures, the denaturation process of lysozyme, conalbumin, ovomucoid or their associated complexes, could be the rate-limiting step. The microstructure of heat-induced gel networks is granular in MUP and filamentous in SPB, which may be associated to the higher stabilizing effect of MUP ions, compared to SPB ones. However, granular structures are also observed in gels of pure lysozyme and of ovalbumidlysozyme mixtures, due probably to the thermal stability of lysozyme molecules. In EWP, the dense gel matrix results from more denatured protein complexes. Other studies are in progress to understand the possible relationships between calorimetric and microscopic data.

Acknowledgements "Minist&rede l'Agriculture, de la P&cheet de 1'Alimentation" is acknowledged for financial support ,program "Aliment Demain (R 94/51) 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15.

References P.L Privalov and D.L.Potekhin, Methods Enzymol., 1986, 1 3 1 , 4 . K.A. Dill, Biochemistry, 1990,29,7133. A.H. Clark, F.J. Judje, J.B. Richards, J.M. Stubbs, and A. Sugget, Int. J. Peptide Protein Res. , 1981, 17, 380. J. Lefebvre and P. Relkin,'Surface Activity of Proteins. Chemical and Physicochemical Modifications.' M. Decker, New-York-Basel-Hong Kong, 1996. Tolstogouzov, 'Gums and Stabilisers in Food Industry 7', IRL Press, OxfordNew York, Tokio, 1994. M.P. Tomb, Faraday Discuss. Chem. SOC., 1974, 57, 158. V.R. Harwalkar and M. Kalab, Milchwinschaf,1985,40, 313. D.M. Mulvihill and J.E. Kinsella J. Food Sci., 1988, 53, 231. M. Stading. PhD Thesis, The Swedish Institute for Food Research, 1993. T-X. Liu, PhD Thesis, ENSIA-France, 1993. D. Renard, PhD Thesis, UniversitC de Nantes, 1994. P. Aymard, PhD Thesis, UniversitC du Maine, 1995. I. Heertje and F.S.M. van Kleef, Food Microstructure, 1986,5, 91. E. Doi. Trends Food Sci. Technol.. 1993.4. 1. J.N. De Wit and G.A.M. Swinkels, Biochim. Biophys. Acta , 1980,624,40.

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16. I.V. Sochava ,T.V. Belopolskaya and 0.1. Smirnova, Biophys. Chem., 1985, 22, 323. 17 P. Relkin, Critical Reviews in Food Sci. Nutrition, 1996,36,565. 18. N. Hagolle, PhD Thesis, ENSIA-France, 1997. 19. P. Relkin, T. Meylheuc, B. Launay and K. Raynal, J. Thermal Analysis (in press). 20. P. Relkin, B. Launay and T-X. Liu, Thermochim. Acta (in press). 21. J. W. Donovan and C. J. Mapes, J. Sci. Food Agric., 1976,27, 197. 22. S. Barbut and C.J. Findlay, 'Thermal Analysis of Foods', Elsevier Applied Science, London and New-York, 1990. 23. M. Rossi and A. Schiraldi, Thermochim. Acta, 1992,199, 115. 24. M. Paulsson and P. Dejmek, J. Dairy Sci., 1990, 73,45. 25. V. Bernal and P. Jelen, J. Dairy Sci., 1985.68, 2852. 26. J. Fauquant, J. L. Maubois and A. Pierre, Technol. Laititre, 1988,1028,21. 27. N. K. D. Kella and J.E. Kinsella, Int. J. Peptide Prot. Res., 1988, 32, 396. 28. R.K Richardson and S. B. Ross-Murphy, Int. J. Biol. Macromol., 1981,3, 315. 29. P. C. Painter and J. L. Koening, Biopolymers, 1976, 15, 2155. 30. Y. Mine , T. Noutomi T. and N. Haga ,J. Agric. Food Chem., 1990, 38,2122. 3 1. J.E. Matsuura and M.C. Manning, J. Agric. Food Chem., 1994,42, 1650.

.

* Correspondence: [email protected]

MARINE ALGAE PROTEINS: A STUDY ON THE EXTRACTION, THERMAL DENATURATION AND FUNCTIONALITY OF SPZROULZNA PACZFZCA

Ioannis S. Chronakis

'

and Ana Cristina Sanchez

I Physical Chemistry 1, Center for Chemistry and Chemical Engineering, Lund University, 221 00 Lund, Sweden. Food Technology, Faculty of Veterinary Science, University of Zaragoza, 50013 Zaragoza, Spain.

*

1 ALGAE AS SEA VEGETABLES The algae are a diverse group of cryptogamic plants, which, morphologically can be found in the form of single cells, colonies of physiologically-independent cells or as large complex thalli. Two main categories could be classified depending on whether they are basically unicellular (micro-algae) or thalloid (macro-algae). Some common features among varieties of algae are that all algae are autotrophic, and none are differentiated into true roots, stems or leaves, therefore the simplicity of their tissues is an advantage in enabling many of the algae to grow more rapidly than conventional agricultural crops.' Sea vegetables are rich in polysaccharides (as known cf alginates, carrageenans etc.), which have traditionally been used by the food industry, have a competitive content of minerals, certain vitamins, and a low lipid content. Above all their protein content, particularly in most of the red seaweeds and blue-green classes, is very high. In some red seaweed proteins it can represent 35 to 47% and in some blue-green algae up to 50 and 70% of the dry matter, comparable to those found in high-protein vegetables (cf. soybeans).'**Furthermore, it is well recognised there is limited space to grow plants and there is potential for the use of non-conventional sources for feeding humans (and animal). In this way, algae could be the centre of further attention, considering in particular their high levels of productivity in comparison with the conventional crops. 1.1 Spiroulina Alga

Spiroulina is blue-green alga (Cyanobacterium) belonging to the family of Oscillatoriaceae and forms unbranched, multicellular helicoidal filaments with a length of about 200-300pm and 5-10pm broad.3 The alga cells are filled with a system of membranes called photosynthetic thylakoid. The cells are about 5 p broad and 2pm long separated from each other by distinct but thin septa. Spiroulina has a high protein content 60-70% by weight and contains a rich source of vitamins, specially vitamin B12, pro-vitamin A @-carotene), minerals, especially iron, and is one of the few sources of dietary y-linolenic acid!

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The amino acid composition of Spiroulina is generally well balanced, reflecting its potential as human food, animal feed and as a source of natural products. Typical amino acid concentrations shows it is low in sulphur-containing amino acids and in tryptophan and have been reported as (g (16g N)-'):Glutamic acid 12.7, Aspartic acid 9.1, Leucine 8.7, Arginine 7.6, Alanine 7.9, Valine 7.5, Lysine 5.1, Isoleucine 5.7, Serine 5.3, Phenylalanine 5.0, Tyrosine4.6, Proline 4.1, Methionine 2.6 .....Tryptophan 1.5.5*6 Spiroufina alga is already successfully commercialised in the health food market (annual production of food-grade totals 800 OOOkg), where it is widely used as a dietetic product, i.e. to help people to lose weight without being hungry or suffering malnutrition. Recently, more attention has been given to the study of the therapeutic effects of Spiroulina? Many preclinical studies and a few clinical studies suggest several therapeutic effects ranging from reduction of cholesterol and cancer to enhancing the immune system, increasing intestinal luctobacifli, reducing nephrotoxicity by heavy metals, drugs and radiation protection. Despite that, Spiroufina protein could be a technological important resource for food products, there are no studies on the physicochemical properties, thermal denaturation, aggregation and thermogelation of Spiroulina protein and a l p proteins in general. These protein transitions are of great interest for industrial food processing aiding toward the understanding and control of the hctionality and quality of food products.

2 SOME PHYSICOCHEMICAL PROPERTIES OF SPIROULINA PROTEIN

2.1 Extraction Procedure of the Spiroulina Protein Spray dried algal powder of Spiroulina Pacifica was kindly donated from Cyanotech Corporation (Hawaii). The composition analysis is mainly reported as 60% protein, 19% carbohydrates, 6% lipids, 8% minerals and 7% moisture.*The ratio of acidic (aspartic and glutamic acid) to the basic (aqinine, lysine) residues present in the protein -reveals that Spiroulina is mildly acidic and carries a negative charge at neutral pH (isoelectric point at pH 3). The absence of a cellulosic cell wall and the presence of only a thin cell wall membrane may account for the easier extractability of this protein. The spray dried powder was dissolved in Millipore water at pH 10 where previous studies have shown greater solubility at alkaline solutions.' The procedure used for extraction of the protein from the cells is shown in Figure 1. The freeze dried protein was dissolved in Millipore water, dialysed for two days at 5°C against Millipore water in order to eliminate the presence of salt, centrifuged at 6000rpm for 30 min to remove any particulate matter, then the pH was adjusted to 7 and freeze dried again. The freeze dried powder was defatted with n-hexane (3 times over a period of 5 hours, 5"C), dried under vacuum in a desiccator and stored at 5°C for further analysis. The nitrogen content of Spiroulinawas determined by the micro-Kjedhal method (a factor of 6.25 was used to convert nitrogen to protein).Spiroufina alga and the extracted protein contain 59.1 and 78.6% protein, respectively. However, this is approximate since the amount of non-protein nitrogen is unestimated. Due to this the protein concentration will be referred as protein flour received after the extractionprocedure.

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The protein was highly soluble near neutral pH even at high protein concentrations and solubility was shown to depend on pH and ionic strength. In the presence of salts, the turbidity increased and the solubility of alga protein in water decreased, unlike that of other proteins. This is probably due to the presence of the pigment-protein complexes present in the algae.7*8*9 As the proteins are already in the form of complexes, the force of attraction between the protein ion and the salt is less and this accounts for its low solubility. Spiroulina Pacijka Alga Powder Aqueous Extraction A)

1

1/20

(PowderMillipore water pH 10, Homogenisation Ultraturrax 15min, 16000rpm, 25°C)

Centrifugation (7000rpm, 30 min, 25"C), Collect Supernatant

1

B)

1/10

(PelletMillipore water pH 10, Homogenisation Ultraturrax 15min, 16000rpm, 25°C)

Centrifugation (7000rpm, 30 min, 25"C), Collect Supernatant

1

A ) + B ) supernatant 0.1 M HCl, pH 3

Centrifugation (7000 rpm for 30 min, 25°C)

1

Pellet Neutralisation (0.05N NaOH),

Freeze Drying

Figure 1 Extraction of Spiroulina Alga Protein

2.2 Electrophoretic Profile of Spiroulina Pacijica Protein Dissociation of the water-soluble protein subunits and pigment-protein complexes is possible under the action of sodium dodecly sulfate and beta-mercaptoethanol as denaturing and reducing agents, respectively. The mobilities from SDS-PAGE electrophoresis (silver staining method with gradient gel 8-25 media) indicate that Spiroulina is composed of 4 major bands (data not shown). The molecular weights estimated on the electrophoresis were 21.3, 16.8, 13.2 and 11.7 kDa. The first two bands dominated most strongly. These results are correlated with the ultracentrihge data where two major groups with S20,w values of 2.6 S and 4.75 S identified in Spiroulina Plantensis p r ~ t e i n Furthermore, .~ the range of molecular weight of the protein in the reduced form was estimated between 10 kDa and 67 kDa.

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In general, algae have been reported to contain several low molecular weight proteins such as cytochrome c, ferridoxin, and flavopr~tein.’*~*’~ Spiroulina protein contain as well pigments of chlorophyll, carotenoids, phycocyanin and allophycocyanin bound to the proteins in the form of complexes. Nevertheless, the protein components can be found both in a free and in a pigment-bound state as observed from the minor bands on the electropherogram with different molecular weights.

2.3 Absorbance Spectrum of Spiroulina Pacifica The absorption spectrum (25OC) of the protein is characterised by a maximum at 286 nm in W which typically corresponds to aromatic chromophores of tyrosine, phenylalanine and tryptophan. However, maximum absorption was observed as well at 410 nm, 620 nm and 674 nm in the visible region. As discussed above, the major part of the alga protein usually exists in the form of pigment-protein complexes. Therefore, the maximum of absorbance at 674nm may be due to the presence of chlorophyll-protein pigment while at 620 and 410 may arises from phycocyanobilin and allophycocyanin pigments existing in the algae (Figure 2). Divalent ions (0.02M CaC12) increase the turbidity of the protein solution, alter the intensity and increase the absorbance but do not significantly produce conformational changes or modify the environment of the aromatic amino acids. Therefore, the increased light scattering when salt was added is probably due to the presence of aggregates. When the pH of the unheated solutions was adjusted to 12 or 4M urea was added a similar spectra with lower absorbance values was obtained (however, the maximum absorbance at 620nm was diminished) suggesting complete denaturation of the protein at high alkaline environments (i.e. pH > 11) and at high molar concentration of protein denaturing agents.

1.2 9)

eifi 0.8 s 2 0.4

0 350

450

550

650

750

nrn

Figure 2 Absorbance Spectrum of to.Smgiml Spiroulina Pacifica protein at 25 “C, in Tris 0.I M HCI bufler pH 7 and in 0.02MCaCl2 x 2H20 in Tris 0.1M HCl bufer pH 7.

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3 THERMAL DENATURATION - CALORIMETRIC STUDIES Spiroulina protein belong to the proteins with a co-operative narrow range of thermal transitions. Two main endothermic peaks were observed with denaturation temperatures at 867 and 5109°C in 0.1M Tris HCl buffer at pH 7 (Figure 3). When the protein was reheated, a broad endothermic peak was observed which covers the above transitions denoting a partially denatured state and absence of reversibility. These thermal transitions may originate h m the two major components with Sz0,w values of 2.6 S and 4.75 S detected from the sedimentation velocity pattern in Spiroulina Plantensis.' At pH 4.5, in the same buffer, almost no differences were observed for the midpoint transition temperatures while the enthalpy is decreased probably due to protein aggregation as the solubility was decreased (aggregation can be considered as an exothermic reaction). The thermal denaturation temperature was decreased by almost 7 degrees at alkaliie pH. Hence, it seems likely that the solubility profile of the protein introduced from the pH changes affect the denaturation state of the protein. When salt (0.004M or 0.02 CaC12) was added both peak transition midpoint temperatures followed the same dependence with pH as without salt, nevertheless, were progressively increased. At pH 9 the denaturation transition changes were influenced most (62 and 63"C, respectively). Therefore, salt stabilises the quaternary structure against dissociation and denaturation and shifts the transitions to higher temperaturesthan in the case of water. At higher salt concentrations(0.02M) a small transition was obtained above 125°C but there are no clear conclusions from these structural states of the protein.

Nevertheless, it is known that polyhydric cosolvents such as sugars stabilise the denaturation of the protein and strengthen the hydrophobic interactions. Two types of hydrophobic interactions, which are influenced in different ways by added solutes, have been proposed. Pairwise interaction in which hydrophobic groups associate but otherwise remain surrounded by water and complete transfer of a hydrophobic group from an aqueous to a nonpolar environment. Sucrose was found to reduce the tendency for a complete transfer of hydrophobic groups from an aqueous to a nonpolar environment." As shown in Figure 4, the magnitude of the -bilking effect varies progressively with the amount of sucrose added. The first transitions were shifted to higher temperatures (from 66.9 to 75.1 and 85.9OC, with 0, 30 and 50% w/w sucrose, respectively). Nevertheless, the second transition temperatures were unexpectedly decreased. It is probable that the second transition is a consequence of the reaction between protein and a reducing sugar (Maillard reaction). The Maillard reaction is in principle a second-order chemical reaction with an activation energy 100-200kJ/m01.12Thus,a significant effect of the heating rate on the midpoint temperature of the second transition could explain the effect of sugars at high temperatures. The details of the protein structure involving contacts between non polar side chains may also be important when sucrose was added. Finally, preliminary results show an increased thermostability of Spiroulina protein at low moisture content (i.e. <30%) and protection against irreversible thermal transitions. One main endothermic peak at substantiallyhigher temperatures was detected implying a different physicochemicalmechanism of thermal denaturation. This may have importance for the drying procedure during the production of Spiroulina protein isolates.

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Protein Systems

67.1C

40.0

I

I

60.0

80.0

I

I

100.0 120.0 lelp. c

I1 40.0

140.0 40.0

I

I

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Tew. C

140.0

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I

I

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Tea C

140.0

Figure 3 Thermograms of the denaturation of Spiroulina Pacificaprotein; in Tris 0.1MHCl bufer; in 0.004M CaClz x 2H20 and 0.02M CaClz x 2Hfl in Tris 0.I M HCl; at pHs 4.5, 7 and 9.Heating rate 10 Wmin. Scale in 0.05mW.

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Gums and Stabilisers for the Food Industry

J

50.0

60.0

70.0

80.0

90.0 lW.O 110.0 120.0

Tew. C Figure 4 Thermogram of the denaturation of SpiroulinaPacificaprotein; in Tris O.IM HCI buffer pH 7 and at 0, 30 and 50% of sucrose. Heating rate I0 OC/min. Scale in 0. Im W.

4 FUNCTIONAL PROPERTIES 4.1 Protein Gelation Spiroulino protein is sensitive to heating between 40-90°C.Like in many other proteins, upon heating intramolecular and intermolecular conformational changes and reactions occur which are generally denoted as initial denaturation, unfolding of the native protein and aggregate formation (Figure 5). The general effect of temperature suggests an important role of hydrophobic interactions (as discussed below) modified by the charged state of the protein molecules. The network development was not increased markedly beyond 75°C indicating further an insufficientinvolvement of sulfydryl-disulfide covalent interactions stiffening the performed aggregates (in accordance with the low content of sulphur-containing amino acids). During long curing at 90°C (and mainly at pH 9) the inter-aggregate adherence decreased which can be attributed to the stability of the hydrophobic forces. Furthermore, on cooling a progressive increase in elasticity was observed, usually attributed to the formation of multiple hydrogen bonds causing a marked increase in the rigidity of aggregates. In the presence of calcium chloride Spiroulina gelation exhibited a decrease in elastic modulus presumably as a result of the lower solubility and perturbation of the electrostatic interactions of protein in the presence of salt. Decrease of gelation temperature (G'>G'') with salt concentrations was even observed, for example at 43°C and 32°C in water and calcium chloride for 6%w/w protein, respectively. It is interesting to note that at high native protein concentrations the water binding was too high and the protein has the ability to swell, therefore, a colloidal dispersion was observed. In the Figure 5d is shown a frequency sweep of a highly viscous protein dispersion in water and in salt before any heat treatment. This probably means that the

Protein Systems

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,

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Frequency (Hz) Figure 5 Llynamic oscillatory measurements of 8 % w h Spiroulina protein at lffz, 2% strain and 1 W m i n in Tris O.lM HCl bufferand in 0.02MCaCl2 x 2HN in Tris O.lMHC1 bufler andpH 7, as afirnction of temperature a) during heating, b) during cooling, c) as afunction offiequencyof oscillation at SOC and d ) as afwction ofji.equencyof oscillationfor 14% w h native Sphdina protein at 30OC. G' (-), G" (-) in Tris baifer; G' (O), G" (0) in salt.

high solubility of native protein may increase the total surface a m of the protein molecules which results in increased bound water. Exposure of hydrophobic substituents to the aqueous environment during swelling, would be expected to lead to formation of structure water enclosures and swollen bundles. During heating to 55°C the network was temperature-independent which may reflect the relative stability of molecular association within and between the swollen bundles. The difference between water and CaC12 may arise h m the lower solubility of the alga protein in the presence of salts. Unlike globular proteins, general fibrous proteins tend to swell in the s01vent.l~

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The critical gelling concentration (Figure 6) of heated and cooled preparations is of the order of 1.5% and 2.5%w/w in water and in CaC12, respectively, values fairly low for the thermal gelation of proteins. 4

3 n

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.

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,

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6

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,

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.

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Concentration (YOw/w) Figure 6 Concentration dependence of elastic modulifor SpiroulinaPacifica protein in Tris 0.1MHCl buffer pH 7 at 90°C (0)and at 5 “c (0); and in 0.02M CaC12x 2HtO in Tris 0.1MHCl bufferpH 7 at 90°C (0) and 5°C (H).

4.2 Molecular Forces of Thermal Association and Gelation in Spiroulina Protein The significant weakening of the network (Figure 7a), while the gelation process remains unchanged, induced by sodium thiocyanate is a phenomenon attributed to the Thus, hydrophobic weakening of hydrophobic interactions by the SCN- i~n.’~*’’*’~ interactions contribute substantially to the facilitation of molecular association in the course of gelation as well as to the stabilisation of the gel structure in Spiroulina protein. The proposed interpretation assumes that the hydrophobic interactions originally help to stabilise the native structure and participate in intermolecular associations due to conformationalchanges of the protein under heating. Therefore,the temperature at which the maximum storage modulus is reached must be also related to the maximum stability of the hydrophobic bonds involved, which are known to increase with temperature. However, it has been estimated that the maximum intensity of hydrophobic interactions in protein-protein interactions reached at different temperatures as a function of the amino acids involved. Brandts classes the side chains of the amino acids in 6 groups and reports that hydrophobicity and maximum stability of the interactions of side chains of the amino acids alanine and arginine is reached at 60°C and of the amino acids leucine, isoleucine, proline and valine, methionine and lysine near 75OC.I’ Propylene glycol is known to enhance protein-protein electrostatic interactions by decreasing the dielectric constant of water, to favour hydrogen bonding and to hinder

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Figure 7 Cooling and heating projlesfiom dynamic oscillatory measurements of 6 % w h Spiroulina Pacificaprotein in Tris O.1MHCI bufferpH 7 as a control sample (0) and in: a) 0.2M and 0.4M NaSCN and 20% Propylene Glycol; b) 0.02M and 0.2MN-ethlymaleimide and 0.4M 2-mercaptoethanol;c) 0.JSM and JM urea; d) 20% and 50% sucrose. All solutions in Tris 0.lM HCI bufferpH 7. Dotted lines represent the temperature history, 1Hz, 2% strain, I V m i n . hydrophobic interactions. The drastic decrease of the modulus on heating agnes with the diminish of hydrophobic interactionsthat originally stabilise the network, while on cooling, further decrease of the network rigidity supports the involvement of hydrogen bonds. However, it seems that hydrogen bonds alone are not sufficient to form and complement the network structure. (G' values of 6% protein with 20% and 50% propylene glycol arc 42.5 and 16.4 Pa). Addition of an amphiphilic cosolute, such as ethylene glycol, was found to increase the modulus for the dispersion state during initial heating to 62°C but caused a dramatic decrease and complete dissociation of the structure on further heating. These results are

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consistent with the suggestion that increasing solubilization of the initial aggregates, in the presence of co-solute, inhibits any hrther structure formation and network re-establishment on further heating-cooling (data are not shown). The presence of 0.02M N-ethylmaleimide (NEM) reduces the elastic modulus on heating (while the gelation temperature remain unchanged, i.e. 43°C); however, by lowering the temperature the modulus of the protein gel was reinforced (Figure 7b). At 0.2M NEM, the gelation temperature was at 38"C, while the modulus was fiuther increased, probably due to the charge neutralisation effects. Moreover, by the addition of 0.4M 2-mercaptoethanol, formation and maintenance of a network structure was also created. Therefore, intermolecular sulfydryl and disulfide bonds were not substantially involved in the establishment and structure of the strands,I6 leadiig to formation of the network structure of Spiroulina protein gels. Further, breakage of most of the S-Sbridges would not hinder hydrophobic interactions but would diminish cross linking density. Presumably, the disulphide reduction will also enhance the hydrophobic groups exposition and their subsequent interaction during heating. Finally, the reduced contribution of disulfide bonds may be responsible for the relatively low elasticity of the protein. Urea favours the gelation of some proteins, nevertheless, more frequently results in the destabilisation of the gel structure. The urea effects are due to its interference with protein-protein hydrogen bonding or to increase the solubility of hydrophobic groups. The last action would result in weakening of the hydrophobic interactions on increasing the temperature and decreasing the modulus on M e r cooling. Both types of action towards the denaturation and destabilisation enhancement are shown in the Figure 7c. Effective dissociation of the subunits requires treatment with high concentrations of urea (i.e. 4M) or strong detergents like 4M 2-mercaptoethanol, or alkaline pH (>11.0). Addition of sucrose as co-solvent enhances the overall structure of water which indirectly strengthens hydrophobic interactions and network association. The magnitude of the stabilising effect and the network rigidity increases with the amount of sucrose added (Figure 7d). 4.2.1 The eflecf of Calcium Chloride. As illustrated in Figure 8 the protein association was followed by the higher elastic modulus in the heating direction, either at neutral or alkaline pH (9) when low salt concentrations were present. The decrease of elastic modulus in 0.02M calcium chloride can be attributed to charge neutralisation effects and may reflect the involvement of electrostatic contributions in the three-dimensional network and their perturbation by added salt. Thus, salt affects protein-protein interactions due to electrostatic shielding and to interference with hydrophobic bonds. On further cooling, reduction of the network rigidity at pH 9 (independent of salt concentration), denotes that the increased solubility of hydrophobic groups diminishes the crosslinked network arrangement. Therefore, when the protein is more charged it develops a weaker network. At 5°C the tan 6 (G"/G') follows the order 0.02M pH9 > 0.004M pH9 > 0.004 pH 7 > 0.02 pH 7, hence, the crossslinked density was reduced at higher alkaline pH while the stabilisation and elasticity of the gels were favoured by higher salt concentrations at neutral pH.

165

Protein Systems 1' 100

I

600 80

Gi3

/

400/

/

200

60

/

/

/

/

ue

n

g

s

c,

e

/

40

/ -'

E

F 20

0, 0

3000

6000

r0 9000

Time (sec) Figure 8 Cooling and heating profiles from dynamic oscillatory measurements of 6%w/w Spiroulina Pacifica protein in 0.004Mand 0.02M CaCl2 x 2H20 at pH 7 and 9 in Tris 0.1MHCI bufer. Dotted line represents the temperature history, lHz, 2% strain, 1 Wmin.

5 CONCLUSIONS

The three-dimensionalstructure for Spiroulina protein can be presented with the polar amino acid residues located on the proteins surface and nonpolar hydrophobic side chains buried in the interior of the molecule. Spiroulina Pacifica contains a relatively high proportion of hydrophobic amino acids. The non-polar side chain of these amino acids can interact with each other, especially in an aqueous environment to form hydrophobic bonds which play a significant role in Spiroulinaprotein gelation. On the other hand it contains a relatively high proportion of amino acids with acidic or basic side groups hence its high solubility in aqueous solvents. The solubility of the protein is lower in the presence of salt than in water mainly due to the protein-pigment complexes present in algae proteins. Furthermore, the ionic strength seems to have an effect on solubility of the protein and subsequently on network association. When the protein is more charged (at alkaline pH) it develops a weaker network structure. Denaturation reached with or without divalent cations lead to quite different aggregation properties particularly at low protein concentrations since the gelation of Spiroulina denatured with these ions is controlled as well by electrostatic repulsions. Intermolecular disulfide bonds are not important in connecting the molecules but affects the physical properties of the gels. The reduced contribution of disulfide bonds may contribute to the low elasticity of the protein. Nevertheless, the critical gelling concentration is particular low for the thermal association of proteins and at high concentrations the protein has the ability to swell in

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aqueous solvents. Electrophoresis data show multiple bands with two major groups; however, is not clear how the molecular weight and charge distributionare important in the native conformation, subunit interactions and functional properties of the protein. Finally, blue-green alga Spiroulina PaciJca is a promising source of protein for food products.

References 1. 2. 3. 4.

A.Wood, D.F. Toerien and R. K. Robinson, In: Developments in Food Proteins -7 ed. B.J. F. Hudson, Elsevier Applied Science, London 1991. E.W. Becker, in:Micro-Algae biotechnoloay, ed. M.A. Borowitzka, L.J. Borowitzka, Cambridge University Press, 1989. G. Hedenskog and A. Hofsten, Physiologia Plantarum, 1970,23,209. A. Belay, Y.Ota,K. Miyakawa and H. Shimamatsu, J. Applied Phycology, 1993, 5,235.

5. 6. 7. 8. 9. 10. 11. 12.

13. 14. 15. 16. 17.

A. Richmond in: Micro-algal biotechnology, ed. M.A. Borowitzka, L.J.Borowitzka, Cambridge University Press, 1989. C. Paoletti, G. Florenzano, R. Materassi and G. Galdini. Sciema e rechnologia GegliAlimenti, 1973,3,171. M. Anusuya Devi, G. Subbulakshmi, K. Madhavi Devi and L.V. Venkataraman, J. Agric. Food Chem., 1981,29,522. J.P. Thronber, Methods Enzymol., 1971,23,682. H.L. Crespi and U. Smith, J.J. Katz, Biochemistry, 1968,7,223. S.S. Voronkova, N.P. Sadkova, O.N. Albitkaya, S.A. Pavlova and S.V. Rogozhin. Biokhimiya IMicrobiologiya, 1980,16,363. J.F. Back, D. Oakenfull, M.B. Smith, Biochemistty, 1979,18,5191. H.A.C Thijssen and P.J.A.M. Kerkhof, In Physical, Chemical and Biological Changes in Food caused by Thermal Processing. ed. T.Hoyem and 0. Kvale, Applied Science, London, 1977. J.M. Gosline and C. French, Biopolymers, 1979,18,2091. T. Nagano, H. Mori and K. Nishinari, J. Agric. Food Chem., 1994,42,14 15. B. Zheng, Y.Matsumura and T. Mori, J. Food Sci., 1992,57,423. S . Utsumi S. and J.E. Kinsell4.I FoodSci., 1985,50,1278. J.P. Brandts, in: Thermobiologv, Academic Press, New York, 1967.

FUNCTIONAL INTERACTIONS IN MULTICOMPONENT POLYSACCHARIDECONTAINING SYSTEMS

E.E.Braudo Institute of Biochemical Physics Russian Academy of Sciences Vavilov str.28, Moscow, 117813, Russia

ABSTRACT Two approaches to the analysis of the phase state of solutions of biopolymer mixtures, in particular gels, are discussed. The first one proceeds from the physicochemical analysis, i.e. the determination of compositiodproperties relationships. The second, quasi-the-odynamic approach consists of the determination of pair interaction parameters and prediction of the phase state of a system. The separation of the second virial coefficient of a ternary solution into components characterizing pair interactions gives insight into the nature of structure-forming processes in the solution and allows prediction of the phase state of concentrated systems. Some mechanisms of the formation of non-coulombic proteidpolysaccharidecomplexes are discussed, and the contribution of complex formation between proteins and starch polysaccharides into properties of multicomponent systems is emphasized. 1. INTRODUCTION Mixtures of biopolymers has become one of the most popular objects in food science in the last decades, and research in the field is most advanced in Great Britain. The great impetus for studies of multicomponent systems was given by the development of new food technologies that are manipulated with biopolymers in order to tailor products with desired composition and properties. Many of these products are so-called "chewy ge1s"l. Naturally, polymer-polymer interactions play the key role in the formation of such gels as well as other food dispersed sy~tems2,~. One of the first examples of the implementation of the new technological approach was the production of man-made caviar and meat substitutes in the 1960~4.5. Roughly at that time there were formulated ideas about filled gels which contain either active or inert 6llers6 as well as mixed and complex gels formed by mixtures of gelforming polymer^^,^^^^*. This is rather similar to the classification proposed by V.Moms9. The importance of the problem of thermodynamic compatibility of proteins and polysaccharides for the tailoring of food systems was stressed by V.B.T~lstoguu>v~~~~~~~*~~~. Clearly, the phase state of multicomponent food systems to a large extent determines their hctional properties. In its turn, the phase state is determined by the whole set of intermolecular interactions in the system. It is worth noting that it was a

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mixture of aqueous solutions of two food biopolymers, namely gelatin and soluble starch, with which the phenomenon of thermodynamic incompatibility was discovered by M.W.Beijerinck more than 100 years ago12. The problem of structure formation in solutions containing starch polysaccharides and proteins, in particular gelatin, is topical up to the present day, and it is briefly discussed below. There are two alternative methodologies of the analysis of phase states of multicomponent systems. The most fiindamental one consists in the prediction of phase equilibria from the parameters of interactions between all components of the system13. Regretably, in the case of gels the possibilities of thermodynamic analysis are limited by the absence of information about the effects of gelation and temperature changes below the gelation point on the phase equilibrium.For this reason the approach based on physicochemical analysis is used 14.

2 PHYSICOCHEMICAL ANALYSIS

The physicochemical analysis developed by N.S.Kurnakovin the second decade of the century is directed to the determination of relations between the composition and properties of equilibrium systemsi5. As an example in the Figure 1 are shown effects of the composition of mixtures of gelatin and agarose on the elasticity modulus of geld6. This system was investigated by some a u t h o r ~ ' ~The - ~ ~addition . of agarose to gelatin at concentrations which do not exceed the limit of compatibility results in an increase of the elasticity modulus of single-phase gels due to the excluded-volume effect25. This effect causes also the decrease of the minimum concentration for gelation of the both polymer components in mixed systems in comparison to solutions of individual gelling agents (Figure 2). The fiirther increase in the fraction of agarose leads to the transition from a single-phase to a two-phase gel. This phenomenon was observed earlier20.

2

L

6

0

Agorose. YO.ww Gelatin, YO,w/w

Figure 1 Compositiondependences of the conventionally instant Young modulus of gelatinlagarose gels, pH 4.7, 20 @I6. Ageing at l o @ afrer gelation: , I day; 0 , 7 +s. Dotted lines, Young modulus of the corresponding agarose or gelatin gels. Solvent: (a) water; (b) 0.1 A4 NaCl.

171

Functional Interactions in Mixed Biopolymer Systems

The position of the maximum of the elasticity modulus of mixed gels at low agarose concentrations does not depend on gel ageing fiom 1 to 7 days (Figure I). This reflects the equihbrium nature of the phase transition. At the same time the second peak disappears after more than 1 day storage, which testifies its non-equilibrium nature. This peak apparently is a result of a superpositionof two effects: the decrease of the gel modulus due to the disruption of gel network by dispersed particles25 and the increase of gel modulus resulting fiom the initial deswelling of the continuous gelatin-enriched phase. The main minimum of the elasticity modulus corresponds to the phase inversion. This is continned by the changes in the temperature of gel melting (Figure 3). a8

t

I

0.0s

I

a1 0.15 Agarose, K, w/w

a2

Agarose, %,w/w Gelatin, %,w/w

Figure 3 Compositiondependence Figure 2 Minimal concentrationfor of the meltingpoint of gelatidagarose gekztion versus composition of mixed W o g e l s , pH 4.7, 20 T16. agarose/geIatinsolutions at 20 ~ 1 6 . 0, water, pH 4.7; 0, 0.1A4 NaCl, pH 4.7; Ageing at 10 T @er gelation, 1 dby. Dotted lines, plotsfor binary systems. 4 0.05MNa2HPO4, pH 6.9.

Similar dependences are observed in the case of gelatin gels which contain additions of methylcellulose. The latter does not form gels under the conditions of the experiment. Thus, only the gelatin-based gels could be investigated. It is shown that gelatin and methylcellulose are incompatible in solution above the melting temperatures of gelatin gels26. As is seen in the Figure 4, small additions of methylcellulose change mechanical properties of gelatin gels similar to the additions of agarose. Comparison of the positions of the maxima in Figure 4 and the position of the binodal on the phase diagram of the water gelatin - methylcellulose system (Figure 5 ) shows an expansion of the incompatibility area after gelatin gelation. In specific cases a composition-propertyplot can give information about the structure of a surface layer in dispersed systems. Thus, the emulsifying capacity and stabilizing action of methylcellulose changes non-monotonously with the increase in the polymer concentration (Figures 6,7). This reflects the transition &om dilute to semi-dilute solution and respectively fiom the adsorption of isolated macromolecules to the adsorption of associations of macromolecules. Contrary to methylcellulose, the emulsifjmg capacity of gelatin depends monotonously on the concentration (Figure 6). Apparently gelatin macromolecules in the helical conformation are associated in the whole range of concentrations studied. The similarity of the concentration dependences of emulsiing capacity of gelatin and mixed gelatidmethylcellulose solutions shows that the mixed adsorptionlayer is enriched with gelatin.

-

172

Gums and Srabilisersfor the Food Indusrry

I

I

I

I

I

I

I

a5

0.3

0.1

t

I

Methy\cellulose, %,

I

0.1 Q3 Methylcellulose, yo, w/w

W/W

0.5

Figure 4 Effect of methylcellulose additions on the mechanical properties of gelatin gels, pH 3.0, 20 @I4. (a) Conventionally instant Young modulus, (6) Initial Newtonian viscosity. I. 3% w h of gelatin; 2. 5% whv of gelatin. Ageing at 4 T after gelation, 7 &s.

-1

-? 10 5

10 Gelatin, 91. w/w

Figure 5 Phase diagram of the water/gelatin/methylcellulosesystem, pH 3.0,35 T Z 6 . 0,phase separation threshold, q criticalpoint, x, point corresponding to the maxima on the Figure 4, curve 2.

a4

12 20 Concent ra t ion,%, w m

Figure 6 Dependences of the emulsijjing capacity on the polymer concentration, pH 6.8, 25@27. I . gelatin; 2. methylcellulose; 3.gelaiidmethylcellulose I : I w h mixture.

Figures 6,8,9 demonstrate a synergistic effect of gelatidmethylcellulose mixtures in the formation and stabilization of emulsions and foams. This is typical of proteidpolysaccharide mixtures and is explained by the formation of a stronger gel-like stabilizing layer around dispersed particles due to the exclusion-volume effect or complexation10.29-32,

Functional Interactions in Mixed Biopolymer Systems

173

1,00

0

U

-r

eX 96 u

73 C

0 "

'

a&

"

' ' " ' 0.8 1.2 1.6 Methylcellulose, %, w/w

I

Figure 7 Dependence of the emulsion stability on the concentrationof methylcellulose,pH 6.8, 25 T, volume fraction of the oil phase (corn oil) - 65Y$8.

2

Ce la+in, %, w/w

20

Me+hylcellulose, %, w/w

Figure 8 Dependence of the stability of emulsions on the composition of the emulsifier, pH 6.8, 25 '17, volume fraction of the oil phase (corn oil) - 65Y67

10

6 Time, h

Figure 9 Volume of foam versus storage time, pH 3.0, 25 T 1 4 , I . 2% w h gelatin solution; 2. 0.5% w h methylcellulose solution; 3. mixture of the solutions I : I w h . It should be stressed that the value of the information which is obtained from the physicochemical analysis can be enhanced substantially, if theoretical approaches based on modeling are additionally used. Such approaches are being developed successllly in this country by A.H.Clark,E.R.Morris, S.Kasapis, I.T.Norton, S . B . R o s s - M ~ r p h yand ~ ~ other -~~ researchers. It should be only remarked that some problems can arise from the fact that experimentally determined values of elasticity modulus of physical gels deviate strongly from equilibrium values39. Thus, one should be careful by the direct correlation of the elasticity modulus with molecular parameters of gel networks in the framework of theories of high elasticity.

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3. DETERMINATION OF PAIR INTERACTION PARAMETERS

A quasi-thermodynamic approach to the analysis of multicomponent systems is based on the determination of Gibbs energy of mixing of polymer components in solution and the revealing of contributions of pair interactions between all components. If we know these parameters, we can predict with the great probability the phase state of concentrated solutions (thermodynamic incompatibility or simple coacervation, formation of soluble complexes and formation of insoluble complexes, including liquid-liquid phase separation or complex coacervation).In other words, we can predict the segregative or associative phase separation, according to the classification proposed by L.Piculel1and his colleagues13~40, as well as the single-phasestate of a system. It is well known that thermodynamic incompatibility of two polymers in a common solvent occurs at least by one of two conditions41'5 - the polymer interaction parameter ( ~ 2 4is) substantially positive and - the difference between the interaction parameters of each of the polymers with the solvent ( ~ 1 2and ~ 1 4 is) sufficientlylarge ('AX-effect'). The parameter ~ 2 is 4 determined by the contributions of both the enthalpy of interactions between the polymers and the non-configurational entropy to the Gibbs energy of polymer mixing in solution. Specific interactions between the polymers, i.e.the formation of interpolymer complexes, reduce ~ 2 4resulting , in the transition from a segregative phase separation to the formation of a single-phase system. Extremely strong interactions between the polymers and low affinity of the complexes to the solvent cause the associative phase separation. A simple way to the determination of pair interaction parameters consists in the measurement of the second virial coefficients of solutions of a polymer mixture - A2(24) and separate polymer components in the same solvent - A2(2) and Az(4). The second virial coefficient of the solution of a polymer mixture can be separated into components which characterize interactions between the two polymers - A24 as well as interactions between each polymer and the solvent - A12 and A14. To do this we have to introduce a non-thermodynamic assumption that the components of the second virial coefficient which characterize interactions of polymer components with the solvent are equal to the second vifid coefficients of the corresponding binary solutions, i.e. A12 = A2(2) and A14 = A2(4). This method is used in polymer chemist@. Flory-Huggins pair interaction parameters can be calculated from the components of the second virial coefficient using Krigbaum-Flory theory in the lattice-model approximation47,4*, It can be sh0wn49-5~that the necessary and sufficient conditions for a single-phase state at any composition of a three-component system (the full stability with respect to diffusion) are

A12 > 0

A12,A14 A242

21

175

Functionul Interactions in Mixed Biopolymer Systems

Condition (3) shows that the phase state of a three-component system is determined by a subtle balance of pair interactions between all components. In particular, the transition fiom a segregated two-phase system to a single-phase one can arise not only due to the formation of interpolymer complexes but also as a result of an increase of the afEnity of the polymer componentsto the solvent51.This is illustrated by the Table 1.

Table 1

Protein

The efect of limited trypsin-inducedproteolysis on the characteristics of the system 0.1 M NaCl (I) - broad bean legumin (2) ficoll (4), 1egumin:ficoll ratio, 1:I w h , pH 7.2, 25 c(751.

-

AI2.

A14*

A249

A\2.A14 A22

I0-3.rn3.rnol.kg-2

Native

-0.3M.23

0.5M.05

i ' ? Phase ~ state o f a system containing 17% legurnin and 7% ficoll

-1.5k1.2 two-phase

1 .lM.ll Modified

0.6M.09

0.4M.04

5.0f1.0 singlephase

If phase separation occurs, the position of the spinodal can be calculated either on the basis of Flory-Huggins parameters or directly fiom the values of second virial coefficients of binary and ternary solutions50. The prediction of the phase state of Concentrated systems can be limited through the possible concentrationdependences of pair interaction parameters or contributions of triple interactions. Thus, these predictions should be checked experimentally. Nevertheless, the second virial coefficient approximation allowed us to describe the phase state of concentrated solutions of mixtures of broad bean legumin and ficoll (Table 1). The method of light-scattering allows also a direct determination of binary and triple interaction parameters in concentrated solutions52-54. An interesting problem for a chemist is the nature of complex formation between the polymers in solution. The most investigated case is the formation of complexes between oppositely charged polyelectrolytes.The driving force for the complexation in this case can be not only the formation of ionic pairs, but also the increase of the entropy due to the desolvation of charged groups. We call this type of complex formation the coulombic one. If short-range interactions, such as van derwaals interactions or hydrogen bonding55, are developed between oppositely charged macromolecules brought into proximity due to wulombic interactions, the complex formation would be irreversible. This means, in particular, that the complexes would not dissociate by the increase in ionic strength or changes in pH of the solution56.Thus, by the inclusion of enzymes into non-soluble coulombic complexes formed by anionic polysaccharides and polycations with long-chain alkyl or aryl side groups there were obtained, without covdent binding, preparations stable at ionic strengths up to 1 h157.58.

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The formation of polymer complexes in solution can be also the result of interactions other than coulombic. It is so-called non-coulombic complex f ~ r m a t i o nContrary ~~. to reversibly formed coulombic complexes, non-coulombic complexes are stable at high ionic strengths. Some authors even distinguish between polyelectrolyte (coulombic) complexes and interpolymer complexes, the latter being formed by hydrogen bond@. Well-known examples of such complexes are complexes between polycarbonic acids and poly (ethylene glycol) or poly (vinyl alcohol). Complexes of high-esterified pectinates with gelatin formed at pH values above its isoelectric point and high ionic strengths6] and complexes of highesterified pectinates with alginates formed in acid media62 probably arise through hydrogen bonds, which involve ester groups of pectic substances. Besides hydrogen bonds, there are other interactions, such as charge-transfer interactions or the formation of interpolymer bridges through polycoordination metal ions63, that can account for the complex formation. A universal factor promoting non-coulombic complex formation is hydrophobic interactions. There are not only protein molecules but also molecules of carbohydrates that contain hydrophobic fragments exposed to the solvent and, thus, are capable of intermolecular hydrophobic i n t e r a c t i o n ~ ~ ~These , ~ 5 . latter are traditionally assigned to entropydriven interactions&, although an alternative viewpoint exists67. Desolvation processes play a decisive role in entropy-driven interactions. As an example interactions between pectic substances and human serum albumin (HSA) at ionic strength 0.1 and pH values higher than the isoelectric point of the protein68will be concerned. The temperature dependences of interaction parameters of HSA and a sodium pectinate with the degree of esterification (DE) 76% in the range 10-30°C are shown in Figure 10. The quasi-thermodynamic parameters of mixing are calculated according to the relations50.69.70 -2

AGljM= 0.5.10-3RTM~A1jm

(i = 2,4)

(4)

A

AG14M = 10-3RTM1A24m

(5)

*

A H I =~0.5.1 ~ 0-3RTM1[GA12m/GT-1]w

(6)

A

AH14M = 0.5.10-3RTM1[GAi4m/GT-']m4

(7)

Here the components of the second virial coefficient are expressed in the molal concentration scale and the thermodynamic parameters are related to the molal concentration of the polymers m2 = m4 = 1 The values of quasi-thermodynamic parameters which characterize pair interactions in the mixed HSNpectinate solution are shown in the Table 2. It is seen that, contrary to interactions between single-type polymers, interactions between HSA and pectinate are favorable. These interactions are endothermic and are accompanied by the increase of the entropy of the system. The endothermic nature of interactions was confirmed by dilution calorimetry68. Interaction parameters of HSA with a series of pectic substances are shown in the Table 3. Negative values of A24 point to the complexation of HSA with all pectic sub-

Functional Interactions in Mired BiopoIymer System

177

stances studied. The interaction parameter does not increase in absolute value with increase in DE. Thus, it can be suggested that hydrophobic interactions are not the driving force of complex formation. We proposed that complexation between HSA and pectic

-0.2

'

Figure 10 Temperature dependences of the second virial coeflcient components of the system 0.1 M NaCI (1) - human serum albumin (2) - sodium pectinate, degree of esterijication - 76%, (4), pH 7.0, albumidpectinate ratio - 3:2 whv, pH 7.068. MoIaI concentrationscaIe. substances proceeds through interactions between polar groups followed by their desolvation. The latter process determinesthe signs of the changes of both enthalpy and entropy of the system.

Table 2 Quasi-thermodynamic parameters which characterize interactions between components of a system 0.I M NaCI (1) - human serum albumin (2) - sodium pectinate, a'egree of ester$cation - 76% (4), albumidpectinate ratio - 3:2 w h , pH 7.0, 25 't2 68. Parameter, I @, M.kgz.mob3

1-2

1-4

2-4

0.0H.01

-3.7iO. 04

6.2iO.60

-0.2iO.03

-5.2iO.21

7.339.77

0.2H.02

1.5H.18

-1.liO.17

4

AGii

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Gum and Srabilisersfor the Food Industry

Table 3

Components of the second virial coeffr'cientfor the systems 0.I M NaCl (I) - human serum albumin (2) - sodium pectinate (4), albumidpectinate ratio - 312 whv, pH 7.0, 25 T68. A 1 2 = 0.8.10-3 m3.mol.kg2

Degree of esterlfication of pectinate,

A141

A24p A12.A14 A22

YO 0 45 47 50 57 70 76 89 98

11.0 3.6 3.2 4.1 3.0 2.8 1.9 1.5 1.5

-4.3 -3.8 -3.5 -1.9 -1.1 -0.8 -0.7 -0.9 -1.6

0.48 0.20 0.21 0.91 1.98 3.50 3.10 1.48 0.47

The application of the conditions for the fill stwility of a system in respect to di fusion shows that the systems studied are either homogeneous in the whole range of compositions or that separation of interpolymer complexes from the solution occurs.

4.COMPLEXES BETWEEN PROTEINS AND a-D-GLUCANS A class of interpolymer complexes important both in food chemistry and biological chemistry are complexes of proteins with a-D-glucans. The latter include both starch polysaccharides and glycogen. Complexes of proteins with a-glucans were investigated before World War I1 , mainly by St.J.von Przylecky and his coworkers71-74. These authors discriminate between two types of complexes (or simplexes, according to their terminology). Complexes of the fist type arise due to coulombic interactions between positively charged protein molecules and negatively charged molecules of glucans. These complexes dissociate by an increase in ionic strength. Coulombic complexes with proteins are formed, for example, by potato amylopectin at pH values below the isoelectric point of the protein75. It is well known that potato amylopectin contains phosphate groups. However, the same type of interactions was ascribed to rice76977 and wheat78 starches. The second, the most intriguing type of complexes are those of phosphorus-fiee polysaccharides as well as complexes of phosphorylated polysaccharides formed at pH values exceeding the isoelectric point of the protein. In the last case complexes arise only in the presence of salts71.73*79980. The composition of these complexes and their strength vary over a wide range80,*1.

Functwnal Interactions in Mixed Biopolymer Systems

179

The nature of interactions leading to the formation of complexes of the second type is unknown. However St.J.von Przylecki and his coworkers showed that the guanidine group of arginine residues and the hydroxyl group of tyrosine residues provide the complexation73982-85. Tyrosine residues even form 0-glycosyl bonds with g l y ~ o g e n ~ ? ~ ~ . E.L.Rozenfeld and E.G.Plyshevskayashowed that the ability to form complexes in neutral medium depends on the structure both of the protein and the polysaccharide88. Fibrillar proteins bind polysaccharides stronger than globular, and the denaturation of globular proteins enhances their binding ability89,90*91. The feature of polysaccharide structure critical for complexation is the occurrence of branches containing no less than five anhydroglucose units90.92993. The complexing ability of dextran is much less than that of glycogen or amylopectin94. Complex formation with proteins effects the enzymic hydrolysis of a-glucans. Thus, St.J.von Przylecki and B.Philipowicz showed that the rate of hydrolysis of starch and glycogen with a-amylase in the pH range 6.6-7.6decreases due to their interactions with myosin95. S.E.Karpiak and coworkers reported about the reduction of digestibility of glycogen with muscle a-amylase by the increase in the content of muscle proteins%. They suggested that the regulation of the activity of a-amylase in animal organs proceeds mainly through the complexation of glycogen with proteins. In contrast to this, E.L.Rozenfeld did not observe effects of proteins on the hydrolysis of glycogen catalyzed by the muscle a - a m ~ l a s e ~ ~ . At the same time she reported about the increase in the activity of phosphorylasedue to the complexation of glycogen with n1yosin97-9~. V.Ya.Grinberg and V.B.Tolstoguzov systematically investigated interrelations between the segregative phase separation and the complexation of waxy maize amylopectin and gelatin or human serum albuminl~JOl.Segregated two-phase systems are formed at the low ionic strength and pH value (4.7) equal to the isoelectric point of a protein (the same is true for r n a l t o d e x t M ~ ~Increase ~ ~ ~ ~ in ~ ~the) .ionic strength or the lowering of pH resulted in the formation of single-phase system, however,an increase in the ionic strength at pH values below the isoelectric point of the protein led again to segregative phase separation. The interplay of segregative and associative processes, probably, determines the shape of the phase diagram of the system 0.1 M NaCl - globulin fraction of sofiean proteins - potato amylopectin (Figure 11)1". At low concentrations of both polymeric components segregative processes prevail, but at higher concentrationsthe contribution of a weak complexation becomes noticeable. In other words, the increase in the total polymer concentration is accompanied with the decrease of the parameter ~ 2 4 However . one should take in mind that annular phase diagrams can also arise by certain relations between concentration-independentpair interaction ~arameters41~42. This sketchy outline of proteinla-glucan interactions is incomplete. Some important aspects have not been discussed,in particular the problem of proteidstarch interactions in cereals. These interactions determine to a great extent functional and technological properties of cereals, in particular the baking quality of wheat f l o ~ r s ~ The ~ ~ aim , ~ of ~ .this report was only to show that properties of multicomponent systems which contain proteins and starch polysaccharides are determined both by segregative and associative interactions.

Gums and Stabilisersfor the Food Industry

8-

.

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j4 -

-n 0

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0'

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12

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Figure 11 Phase diagram of the system 0.1 M NaCl - soybean ghJbUllFiS - potato amylopectin, pH 6.9; 25 7:. Coordinates of the criticalpoint: cprOr,= 3.6%; ca,,,, = 1.9%. References 1. M.L.Anson. In 'Processed Plant Protein Foodstuffs' (A.M.Altshu1,ed.), Academic

Press, New York, 1958, p.282. 2. V.B.Tolstoguzov and E.E.Braudo, J. Texture Stud., 1983, 14, 183. 3. V.B.Tolstoguzov.In 'Food Structure - Its Creation and Evaluation' (J.M.V.Blanshard and JRMitchell, eds.), Butterworths, London, 1988, p. 181. 4. V.B.Tolstoguzov, 'Artificial Foodstuffs', Nauka Publ., Moscow, 1978,231 pp.(Russ.). 5. V.B.Tolstoguzov, Food Hydrocoll., 1995,9, 3 17. 6. E.E.Braudo and V.B.Tolstoguzov, Nahrung/Food, 1974, 18, 173. 7. V.B.Tolstoguzov, NahrungiFood, 1974, 18, 523. 8. V.B.Tolstoguzov, EEBraudo and E.S.Wainerman,NahrungFood, 1975,19,973. 159. 9. V.J.Moms, Chem.Ind(London), 1985, (9, 10. V.B.Tolstoguzov. In 'Food Hydrocolloids. Structure, Properties and Functions' (K.Nishinari and E.Doi, eds.), Plenum Press, New York and London, 1993, p.327. 1 1. V.Ya.Grinberg and V.B.Tolstoguzov, Food Hydrocoll., 1997, 11, 145 12. M.W.Beijerinck,Centralbl. Bakteriol. Abt.2, 1896,2,627. 13. L.Piculel1, K.Bergfeldt and S.Nilsson. In 'Biopolymer Mixtures' (S.E.Harding, S.E.Hilland J.R.Mitchel1, eds.), Nottingham University Press, Nottingham, 1995, p.13. 14 D.V.Zasypkin, EEBraudo and V.B.Tolstoguzov,FoodHydrocoll., 1997, 11, 159. 15. V.Ya.Anosov, M.I.Ozerova and Yu.Ya.Fialkov, 'Fundamentals of Physicochemical Analysis', Nauka Publ., Moscow, 1978, p.7 (Russ.). 16. A.M.Gotlieb, I.G.Plashchina,E.E.Braudo, E.F.Titova, E.M.Belavtseva and V.B.Tolstoguzov, NahrungIFood, 1988,32,927. 17. H.Moritaka, K.Nishinari, H.Horiuchi and M.Watase, J. Texture Stud, 1980, 11,257. 18. M.Watase and K.Nishinari,HheoLActa, 1980, 19,220. 19. M.Watase and K.Nishinari,Biorheol., 1983,20,495. 20. A.H.Clark, R.K.Richardson, S.B.Ross-Murphyand J.M.Stubbs, Macromol., 1983, 16, 1367 21. H.McEvoy, S.B.Ross-Murphyand A.H.Clark. In 'Gums and Stabilizers for the Food Industry-2' (G.O.Phillips,D.J.Wedlock and P.A.Williams,eds.), Pergamon Press, Oxford, 1984, p.111.

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22. M.Kobayashiand N.Nakahama,J. Texture Stud, 1986, 17, 161, 23. Y.Shiinoki and T.Yano, FoodHydrocoll., 1986, 1, 153. 24. H.Horiuchi and J.Sugiyama,Agric.and Biol.Chem., 1987,51,2171. 25. V.B.Tolstoguzov,V.P. Belkina, V.Ya.Gulov, V.Ya.Grinberg,E.F.Titova and E.M.Belavtseva,Sfurk, 1974,26, 130. 26. E.V.Grishchenkova,Yu.A.Antonov, E.E.Braudo and V.B.Tolstoguzov, Nahrung/Foai, 1984,28, 15. 27. E.E.Braudo, A.M.Gotlieb,1.G.Plashchinaand V.B.TolstoguzovNahrung/Food, 1986 30,355. 28. E.V.Grishchenkova,PhD Thesis, G.V.PlekhanovMoscow High School of Economics, 1984 (Russ.). 29. V.B.Tolstoguzovand E.E.Braudo, JDispersion Sci.and Technol.,1985,6, 575. 30. E.Dickinson and S.R.Euston. In 'Food Polymers, Gels and Colloids' (E.Dickinson, ed.), Royal Society of Chemistry, Cambridge, 1991, p. 132. 31. E.Dickinson and M.G.Semenova,J.Chem.Soc.FaradayTrans., 1992,88,849. 32. E.Dickinson. In 'Biopolymer Mixtures' (S.E.Harding, S.E.Hil1and J.R.Mitchel1,eds.), Nottingham University Press, Nottingham, 1995, p.349. 33. A.H.Clark and S.B.Ross-Murphy,BrifishPolym. J., 1985 17, 164. 34. A.H.Clark.In 'Food Structure and Behaviour' (J.M.V.Blanshardand P.Lillford, eds.), Academic Press, London., 1987, p. 13. 35. S.B.Ross-Murpy.In 'Viscoelasticity of Biomaterials' W.Glasser and H.Hatakeyama, eds.), American Chemical Society Symposium Series 489, American Chemical Society, Washington, D.C., 1992, p.204. 36. E.R.Morris, Carbohydr.Polym., 1992,17,65. 37. S.Kasapis,E.R.Morris, I.T.Norton and A.H.Clark, Carbohya'r. Polym., 1993,21, 269. 38. S.Kasapis. In Biopolymer Mixtures' (S.E.Harding, S.E.Hil1and J.R.Mitchel1,eds.), Nottingham University Press, Nottingham, 1995, p. 193, 39. E.E.Braudo and 1.G.Plashchina.In 'Food Macromolecules and Colloids' (E.Dickinson and Dhrient, eds.), The Royal Society of Chemistry, Cambridge, 1995, p.480. 40. L.PiculeH and B.Lindman, Adv.ColUnferjSci.,1992,41, 149. 41. L.Zeman and D.Patterson,Macromol., 1972,5,5 13. 42. C.C.Hsu and J.M.Prausnitz,Macromol., 1974, 7,320. 43, A.Robard, D.Patterson and G.Delmas, Macromol., 1977, 10, 706. 44. A.Robard and D.Patterson Macromol., 1977, 10, 1021. 45. D.Patterson, Polymer Eng.and Sci., 1982,22,64. 46. P.Kratochvi1and L.O.SudeloCAcfaPharm.Suec., 1986,23,31. 47. W.R.Kriegbaumand P.J.Flory,J.Chem.Phys., 1952,20,873. 48. A.J.Hyde and A.G.Tanner,J. Coll.InfeijXci., 1968,28, 179. 49.I.Prigogine and R.Defay, 'Treatise on ThermodynamicsBased on the Methods of Gibbs and DeDonder. Vol. 1. Chemical Thermodynamics',Longmans, Green and Co., London, New York, Toronto, 1954, 509 pp. 50. A.G.Ogston,Arch.Biochem.Biophys.,Suppl. I, 1962,39. 5 1. A.N.Dadenko, V.Yu.Vetrov, A.P.Dmitrochenko,A.L.Leontiev,E.E.Braudo and V.B.Tolstoguzov,Nahrung/Fd, 1992,36, 105. 52. Th.G.Scholte, Eur.Polym.J., 1970,6, 1063. 1972, No 39,281. 53. Th.G.Scholte,J.Polym.Sci.C., 54. J.Lipatov, V.Chornaya, A.Nesterov and T.Todosiichuk.Polymer Bull., 1984,12,49. 55. GStainsbv. FoaiChemisfrv. 1980.6.3. 56. E.E.Braudo, L.P.Sologub, K.D.Schwenke, Yu.1.Chimirovand V.B.Tolstoguzov, NahrundFd. 1978.22.531. 57.Z.A.Strdtsov<E.E.Braudoand V.B.Tolstoguzov.In 'Abstracts of Reports on the 1st All-Union Symposium Preparation and Uses of Immobilized Enzymes', Tallin, 1974, p.34 (Russ.). 58. A.A.Klyosov, V.K.Shvyadas, P.S.Nys, E.M.Savitskaya, Z.A.Streltsova, E.E.Braudo and V.B.Tolsioguzov. Ibid, p.8 1 (Russ.).

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59.E.E.Braudoand Yu.A.Antonov.In 'Food Proteins. Structure and Functionality' (K.D.Schwenkeand R.Mothes, eds.), VCH, Weinheim, New York, Basel, Cambridge, Tokyo, 1993,p.210. 60.V.V.Rodin,A.V.Kharenko,V.A.Kemenova,Kolloidny Zhurnal, 1996,58,659 (Russ.). 61.Yu.A.Antonov,N.P.Lashko, Yu.K.Glotova,A.Malovikovaand O.Markovich,Food H*ocoII., 1996,10, 1. 62.K.Tofl. In 'Progr.Fd.Nutr.Sci.Vol.6. Gums and Stabilizersfor the Food Industry. Interactionsof Hydrocolloids'(G.O.Phillips,D.J.Wedlock and P.A.Williams, eds.). Pergamon Press, Oxford., 1984,p.89. 63.A.Y.Sheris,A.N.Gurov and V.B.Tolstoguzov,[email protected]., 1989,10,87. 64. Yu.Yano and M.Yanada,Kyoto Joshi Daigaku Shokumatsu Gakkaishi, 1994,49,1. 65.N.Garti and D.Reichman, Food Hydrocoll., 1994,8,155-173. 66.W.Kauzmann, AdxProtein Chem., 1959,14,1. 67.P.L.Privalovand S .J.Gill, Pure and AppLChem., 1989,61,1097. 68.M.G.Semenova,V.S.Bolotina, A.P.Dmitrochenko,A.L.Leontiev, V.I.Polyakov, E.E.Braudoand V.B.Tolstoguzov,Curbohyak Polym., 1991,15,367. 69.E.Edmond and A.G.Ogston,BiochernJ, 1968,109,569. 70.V.I.Polyakov,I.A.Popello, V.Ya.Grinberg and V.B.Tolstoguzov, Nahrung/Food, 1986,30,81. 71.St.J.vonPrzy1eckiand R.Majmin, Biochem.Z., 1934,271,168. 72.St.J.vonPrzylecki, K.Kasprzykand H.Rafalowska,Biochem.Z., 1936,286,360. 73.St.J.vonPrzylecki, Monatsh. $:hem., 1936,69,243. 74.St.J.von Przylecki, Proc.Roy.Soc.(London) A, 1939,127,26. 75.MSamec and A.Durjava, Kolloid-Beih., 1934,40,449;Chem.Abstr., 1935,29,5331. 76.I.Takeuchi,K.Shimada and SNakamura, Nippon nogei kagaku kaishi, 1968,42, 294. 77.I.Takeuchi, Cereal Chem., 1969,46,570. 78.L.K.Dahle, CerealChem., 1971,48,706. 79.St.Bartuszek,Biochem.Z., 1932,253,279. 80. St.J.vonPrzylecki and R.Majmin, Biochem.Z., 1934,273,262. 81.St.J.vonPrzylecki and R.Majmin, Biochem.Z., 1935,277,1. 82.N.Giedroyc, J.Cichockaand E.Mystowski,Biochem.Z., 1935,281,420. 83.St.J.vonPrzylecki, J.Cichocka and H.Rafalowska,Biochem.Z., 1936,284,169. 84.St.J.von Przylecki, Sprawozdania Posiedzen Towaryst.Nauk Warzwskie, Widzialu 4, 1936;Chem.Absp., 1939,33,8638. 85.St.J.vonPrzylecki and M.Kolazkowska,Biochem.Z., 1937,291,76. 86.M.A.Aon and J.A.Curtino, Bi0chem.J., 1985,229,269. 87.R.J.Rodriguezand W.J.Whelan,Biochem.andBiophys.Res.Commun., 1985,132, 829. 88.E.L.Rozenfeld and E.G.Plyshevskaya,Uspekhi Sovremennoy Biologii, 1958,46,130 (Russ.). 89.E.G.Plyshevskayaand E.L.Rozenfeld,Doklady Akademii Nauk SSSR, 1954,94, 1141 (Russ.). 90.E.L.Rozenfeldand E.G.Plyshevskaya,Biokhimiya, 1954,19,161 (Russ.). 91.E.L.Rozenfeldand E.G.Plyshevskaya,Biofizika, 1956,1,143 (Russ.). 92.E.L.Rozenfeldand E.G.Plyshevskaya,Doklady Akademii Nauk SSSR, 1952,85,615 (Russ.). 93.E.G.Plyshevskaya,E.L.Rozenfeldand V.F.Gachkovski,Doklady Akademii Nauk SSSR, 1952,86,63(Russ.). 94.E.L.Rozenfeldand E.G.Plyshevskaya,Doklady Akademii Nauk SSSR, 1954,95,333 (Russ.). 95.St.J.von Przylecki and B.Filipowicz,Biochem.Z., 1935,275,62. 96.S.E.Karpiak,K.A.Sobiech, J.Jakielaszyk and E.Gandyra,Comp.Biochem.Physiol., 1982,72B,317. 97.E.L.Rozenfeld,Biokhimiya, 1950,15,272(Russ).

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98. E.L.Rozenfeld,Doklady Akademii Nauk SSSR, 1948,62,373 (Russ.). 99. E.L.Rozenfeldand E.G.Plyshevskaya,Biokhimiyu, 1955,20,205 (Russ.). 100. V.Ya.Grinbergand V.B.Tolstoguov,Izvestiya Akademii Nauk SSSR,Seriya Khimicheskqa, 1970, 1423 (Russ.). 101. V.Ya.Grinbergand V.B.Tolstoguzov,Carbohyab:Res., 1972,25,3 13. 102. S.Kasapis,E.R.Moms, 1.T.Nortonand M.J.Gidley, Carbohyri.Polym., 1993,21, 249. 103. S.Kasapis,E.R.Morris,1.T.Nortonand R.T.Brown, Carbohydr.Polym.,1993,21, 261. 104. V.I.Polyakov,1.B.Likhodzievskayaand E.E.Braudo, unpublished. 105. E.E.Braudo,In 'The 9th International Cereal and Bread Congress. Paris, 1-5 June 1992. Abstracts of Technical Sessions', p.25. 106. W.E.Marshalland J.Chrasti1. In Biochemistry of Food Proteins' @.J.F.Hudson,ed.), Elsevier AppLSci., London and New York, 1992, p.75.

Functional Interactions in Mixed Biopolymer Systems

FUNCTIONAL INTERACTIONS IN MULTICOMPONENT POLYSACCHARIDECONTAINING SYSTEMS

E.E.Braudo Institute of Biochemical Physics Russian Academy of Sciences Vavilov str.28, Moscow, 117813, Russia

ABSTRACT Two approaches to the analysis of the phase state of solutions of biopolymer mixtures, in particular gels, are discussed. The first one proceeds from the physicochemical analysis, i.e. the determination of compositiodproperties relationships. The second, quasi-the-odynamic approach consists of the determination of pair interaction parameters and prediction of the phase state of a system. The separation of the second virial coefficient of a ternary solution into components characterizing pair interactions gives insight into the nature of structure-forming processes in the solution and allows prediction of the phase state of concentrated systems. Some mechanisms of the formation of non-coulombic proteidpolysaccharidecomplexes are discussed, and the contribution of complex formation between proteins and starch polysaccharides into properties of multicomponent systems is emphasized. 1. INTRODUCTION Mixtures of biopolymers has become one of the most popular objects in food science in the last decades, and research in the field is most advanced in Great Britain. The great impetus for studies of multicomponent systems was given by the development of new food technologies that are manipulated with biopolymers in order to tailor products with desired composition and properties. Many of these products are so-called "chewy ge1s"l. Naturally, polymer-polymer interactions play the key role in the formation of such gels as well as other food dispersed sy~tems2,~. One of the first examples of the implementation of the new technological approach was the production of man-made caviar and meat substitutes in the 1960~4.5. Roughly at that time there were formulated ideas about filled gels which contain either active or inert 6llers6 as well as mixed and complex gels formed by mixtures of gelforming polymer^^,^^^^*. This is rather similar to the classification proposed by V.Moms9. The importance of the problem of thermodynamic compatibility of proteins and polysaccharides for the tailoring of food systems was stressed by V.B.T~lstoguu>v~~~~~~~*~~~. Clearly, the phase state of multicomponent food systems to a large extent determines their hctional properties. In its turn, the phase state is determined by the whole set of intermolecular interactions in the system. It is worth noting that it was a

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mixture of aqueous solutions of two food biopolymers, namely gelatin and soluble starch, with which the phenomenon of thermodynamic incompatibility was discovered by M.W.Beijerinck more than 100 years ago12. The problem of structure formation in solutions containing starch polysaccharides and proteins, in particular gelatin, is topical up to the present day, and it is briefly discussed below. There are two alternative methodologies of the analysis of phase states of multicomponent systems. The most fiindamental one consists in the prediction of phase equilibria from the parameters of interactions between all components of the system13. Regretably, in the case of gels the possibilities of thermodynamic analysis are limited by the absence of information about the effects of gelation and temperature changes below the gelation point on the phase equilibrium.For this reason the approach based on physicochemical analysis is used 14.

2 PHYSICOCHEMICAL ANALYSIS

The physicochemical analysis developed by N.S.Kurnakovin the second decade of the century is directed to the determination of relations between the composition and properties of equilibrium systemsi5. As an example in the Figure 1 are shown effects of the composition of mixtures of gelatin and agarose on the elasticity modulus of geld6. This system was investigated by some a u t h o r ~ ' ~The - ~ ~addition . of agarose to gelatin at concentrations which do not exceed the limit of compatibility results in an increase of the elasticity modulus of single-phase gels due to the excluded-volume effect25. This effect causes also the decrease of the minimum concentration for gelation of the both polymer components in mixed systems in comparison to solutions of individual gelling agents (Figure 2). The fiirther increase in the fraction of agarose leads to the transition from a single-phase to a two-phase gel. This phenomenon was observed earlier20.

2

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Figure 1 Compositiondependences of the conventionally instant Young modulus of gelatinlagarose gels, pH 4.7, 20 @I6. Ageing at l o @ afrer gelation: , I day; 0 , 7 +s. Dotted lines, Young modulus of the corresponding agarose or gelatin gels. Solvent: (a) water; (b) 0.1 A4 NaCl.

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The position of the maximum of the elasticity modulus of mixed gels at low agarose concentrations does not depend on gel ageing fiom 1 to 7 days (Figure I). This reflects the equihbrium nature of the phase transition. At the same time the second peak disappears after more than 1 day storage, which testifies its non-equilibrium nature. This peak apparently is a result of a superpositionof two effects: the decrease of the gel modulus due to the disruption of gel network by dispersed particles25 and the increase of gel modulus resulting fiom the initial deswelling of the continuous gelatin-enriched phase. The main minimum of the elasticity modulus corresponds to the phase inversion. This is continned by the changes in the temperature of gel melting (Figure 3). a8

t

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Agarose, %,w/w Gelatin, %,w/w

Figure 3 Compositiondependence Figure 2 Minimal concentrationfor of the meltingpoint of gelatidagarose gekztion versus composition of mixed W o g e l s , pH 4.7, 20 T16. agarose/geIatinsolutions at 20 ~ 1 6 . 0, water, pH 4.7; 0, 0.1A4 NaCl, pH 4.7; Ageing at 10 T @er gelation, 1 dby. Dotted lines, plotsfor binary systems. 4 0.05MNa2HPO4, pH 6.9.

Similar dependences are observed in the case of gelatin gels which contain additions of methylcellulose. The latter does not form gels under the conditions of the experiment. Thus, only the gelatin-based gels could be investigated. It is shown that gelatin and methylcellulose are incompatible in solution above the melting temperatures of gelatin gels26. As is seen in the Figure 4, small additions of methylcellulose change mechanical properties of gelatin gels similar to the additions of agarose. Comparison of the positions of the maxima in Figure 4 and the position of the binodal on the phase diagram of the water gelatin - methylcellulose system (Figure 5 ) shows an expansion of the incompatibility area after gelatin gelation. In specific cases a composition-propertyplot can give information about the structure of a surface layer in dispersed systems. Thus, the emulsifying capacity and stabilizing action of methylcellulose changes non-monotonously with the increase in the polymer concentration (Figures 6,7). This reflects the transition &om dilute to semi-dilute solution and respectively fiom the adsorption of isolated macromolecules to the adsorption of associations of macromolecules. Contrary to methylcellulose, the emulsifjmg capacity of gelatin depends monotonously on the concentration (Figure 6). Apparently gelatin macromolecules in the helical conformation are associated in the whole range of concentrations studied. The similarity of the concentration dependences of emulsiing capacity of gelatin and mixed gelatidmethylcellulose solutions shows that the mixed adsorptionlayer is enriched with gelatin.

-

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Figure 4 Effect of methylcellulose additions on the mechanical properties of gelatin gels, pH 3.0, 20 @I4. (a) Conventionally instant Young modulus, (6) Initial Newtonian viscosity. I. 3% w h of gelatin; 2. 5% whv of gelatin. Ageing at 4 T after gelation, 7 &s.

-1

-? 10 5

10 Gelatin, 91. w/w

Figure 5 Phase diagram of the water/gelatin/methylcellulosesystem, pH 3.0,35 T Z 6 . 0,phase separation threshold, q criticalpoint, x, point corresponding to the maxima on the Figure 4, curve 2.

a4

12 20 Concent ra t ion,%, w m

Figure 6 Dependences of the emulsijjing capacity on the polymer concentration, pH 6.8, 25@27. I . gelatin; 2. methylcellulose; 3.gelaiidmethylcellulose I : I w h mixture.

Figures 6,8,9 demonstrate a synergistic effect of gelatidmethylcellulose mixtures in the formation and stabilization of emulsions and foams. This is typical of proteidpolysaccharide mixtures and is explained by the formation of a stronger gel-like stabilizing layer around dispersed particles due to the exclusion-volume effect or complexation10.29-32,

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1,00

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Figure 7 Dependence of the emulsion stability on the concentrationof methylcellulose,pH 6.8, 25 T, volume fraction of the oil phase (corn oil) - 65Y$8.

2

Ce la+in, %, w/w

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Me+hylcellulose, %, w/w

Figure 8 Dependence of the stability of emulsions on the composition of the emulsifier, pH 6.8, 25 '17, volume fraction of the oil phase (corn oil) - 65Y67

10

6 Time, h

Figure 9 Volume of foam versus storage time, pH 3.0, 25 T 1 4 , I . 2% w h gelatin solution; 2. 0.5% w h methylcellulose solution; 3. mixture of the solutions I : I w h . It should be stressed that the value of the information which is obtained from the physicochemical analysis can be enhanced substantially, if theoretical approaches based on modeling are additionally used. Such approaches are being developed successllly in this country by A.H.Clark,E.R.Morris, S.Kasapis, I.T.Norton, S . B . R o s s - M ~ r p h yand ~ ~ other -~~ researchers. It should be only remarked that some problems can arise from the fact that experimentally determined values of elasticity modulus of physical gels deviate strongly from equilibrium values39. Thus, one should be careful by the direct correlation of the elasticity modulus with molecular parameters of gel networks in the framework of theories of high elasticity.

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174

3. DETERMINATION OF PAIR INTERACTION PARAMETERS

A quasi-thermodynamic approach to the analysis of multicomponent systems is based on the determination of Gibbs energy of mixing of polymer components in solution and the revealing of contributions of pair interactions between all components. If we know these parameters, we can predict with the great probability the phase state of concentrated solutions (thermodynamic incompatibility or simple coacervation, formation of soluble complexes and formation of insoluble complexes, including liquid-liquid phase separation or complex coacervation).In other words, we can predict the segregative or associative phase separation, according to the classification proposed by L.Piculel1and his colleagues13~40, as well as the single-phasestate of a system. It is well known that thermodynamic incompatibility of two polymers in a common solvent occurs at least by one of two conditions41'5 - the polymer interaction parameter ( ~ 2 4is) substantially positive and - the difference between the interaction parameters of each of the polymers with the solvent ( ~ 1 2and ~ 1 4 is) sufficientlylarge ('AX-effect'). The parameter ~ 2 is 4 determined by the contributions of both the enthalpy of interactions between the polymers and the non-configurational entropy to the Gibbs energy of polymer mixing in solution. Specific interactions between the polymers, i.e.the formation of interpolymer complexes, reduce ~ 2 4resulting , in the transition from a segregative phase separation to the formation of a single-phase system. Extremely strong interactions between the polymers and low affinity of the complexes to the solvent cause the associative phase separation. A simple way to the determination of pair interaction parameters consists in the measurement of the second virial coefficients of solutions of a polymer mixture - A2(24) and separate polymer components in the same solvent - A2(2) and Az(4). The second virial coefficient of the solution of a polymer mixture can be separated into components which characterize interactions between the two polymers - A24 as well as interactions between each polymer and the solvent - A12 and A14. To do this we have to introduce a non-thermodynamic assumption that the components of the second virial coefficient which characterize interactions of polymer components with the solvent are equal to the second vifid coefficients of the corresponding binary solutions, i.e. A12 = A2(2) and A14 = A2(4). This method is used in polymer chemist@. Flory-Huggins pair interaction parameters can be calculated from the components of the second virial coefficient using Krigbaum-Flory theory in the lattice-model approximation47,4*, It can be sh0wn49-5~that the necessary and sufficient conditions for a single-phase state at any composition of a three-component system (the full stability with respect to diffusion) are

A12 > 0

A12,A14 A242

21

175

Functionul Interactions in Mixed Biopolymer Systems

Condition (3) shows that the phase state of a three-component system is determined by a subtle balance of pair interactions between all components. In particular, the transition fiom a segregated two-phase system to a single-phase one can arise not only due to the formation of interpolymer complexes but also as a result of an increase of the afEnity of the polymer componentsto the solvent51.This is illustrated by the Table 1.

Table 1

Protein

The efect of limited trypsin-inducedproteolysis on the characteristics of the system 0.1 M NaCl (I) - broad bean legumin (2) ficoll (4), 1egumin:ficoll ratio, 1:I w h , pH 7.2, 25 c(751.

-

AI2.

A14*

A249

A\2.A14 A22

I0-3.rn3.rnol.kg-2

Native

-0.3M.23

0.5M.05

i ' ? Phase ~ state o f a system containing 17% legurnin and 7% ficoll

-1.5k1.2 two-phase

1 .lM.ll Modified

0.6M.09

0.4M.04

5.0f1.0 singlephase

If phase separation occurs, the position of the spinodal can be calculated either on the basis of Flory-Huggins parameters or directly fiom the values of second virial coefficients of binary and ternary solutions50. The prediction of the phase state of Concentrated systems can be limited through the possible concentrationdependences of pair interaction parameters or contributions of triple interactions. Thus, these predictions should be checked experimentally. Nevertheless, the second virial coefficient approximation allowed us to describe the phase state of concentrated solutions of mixtures of broad bean legumin and ficoll (Table 1). The method of light-scattering allows also a direct determination of binary and triple interaction parameters in concentrated solutions52-54. An interesting problem for a chemist is the nature of complex formation between the polymers in solution. The most investigated case is the formation of complexes between oppositely charged polyelectrolytes.The driving force for the complexation in this case can be not only the formation of ionic pairs, but also the increase of the entropy due to the desolvation of charged groups. We call this type of complex formation the coulombic one. If short-range interactions, such as van derwaals interactions or hydrogen bonding55, are developed between oppositely charged macromolecules brought into proximity due to wulombic interactions, the complex formation would be irreversible. This means, in particular, that the complexes would not dissociate by the increase in ionic strength or changes in pH of the solution56.Thus, by the inclusion of enzymes into non-soluble coulombic complexes formed by anionic polysaccharides and polycations with long-chain alkyl or aryl side groups there were obtained, without covdent binding, preparations stable at ionic strengths up to 1 h157.58.

Gums and Srabilisers for the Food Indusrry

176

The formation of polymer complexes in solution can be also the result of interactions other than coulombic. It is so-called non-coulombic complex f ~ r m a t i o nContrary ~~. to reversibly formed coulombic complexes, non-coulombic complexes are stable at high ionic strengths. Some authors even distinguish between polyelectrolyte (coulombic) complexes and interpolymer complexes, the latter being formed by hydrogen bond@. Well-known examples of such complexes are complexes between polycarbonic acids and poly (ethylene glycol) or poly (vinyl alcohol). Complexes of high-esterified pectinates with gelatin formed at pH values above its isoelectric point and high ionic strengths6] and complexes of highesterified pectinates with alginates formed in acid media62 probably arise through hydrogen bonds, which involve ester groups of pectic substances. Besides hydrogen bonds, there are other interactions, such as charge-transfer interactions or the formation of interpolymer bridges through polycoordination metal ions63, that can account for the complex formation. A universal factor promoting non-coulombic complex formation is hydrophobic interactions. There are not only protein molecules but also molecules of carbohydrates that contain hydrophobic fragments exposed to the solvent and, thus, are capable of intermolecular hydrophobic i n t e r a c t i o n ~ ~ ~These , ~ 5 . latter are traditionally assigned to entropydriven interactions&, although an alternative viewpoint exists67. Desolvation processes play a decisive role in entropy-driven interactions. As an example interactions between pectic substances and human serum albumin (HSA) at ionic strength 0.1 and pH values higher than the isoelectric point of the protein68will be concerned. The temperature dependences of interaction parameters of HSA and a sodium pectinate with the degree of esterification (DE) 76% in the range 10-30°C are shown in Figure 10. The quasi-thermodynamic parameters of mixing are calculated according to the relations50.69.70 -2

AGljM= 0.5.10-3RTM~A1jm

(i = 2,4)

(4)

A

AG14M = 10-3RTM1A24m

(5)

*

A H I =~0.5.1 ~ 0-3RTM1[GA12m/GT-1]w

(6)

A

AH14M = 0.5.10-3RTM1[GAi4m/GT-']m4

(7)

Here the components of the second virial coefficient are expressed in the molal concentration scale and the thermodynamic parameters are related to the molal concentration of the polymers m2 = m4 = 1 The values of quasi-thermodynamic parameters which characterize pair interactions in the mixed HSNpectinate solution are shown in the Table 2. It is seen that, contrary to interactions between single-type polymers, interactions between HSA and pectinate are favorable. These interactions are endothermic and are accompanied by the increase of the entropy of the system. The endothermic nature of interactions was confirmed by dilution calorimetry68. Interaction parameters of HSA with a series of pectic substances are shown in the Table 3. Negative values of A24 point to the complexation of HSA with all pectic sub-

Functional Interactions in Mired BiopoIymer System

177

stances studied. The interaction parameter does not increase in absolute value with increase in DE. Thus, it can be suggested that hydrophobic interactions are not the driving force of complex formation. We proposed that complexation between HSA and pectic

-0.2

'

Figure 10 Temperature dependences of the second virial coeflcient components of the system 0.1 M NaCI (1) - human serum albumin (2) - sodium pectinate, degree of esterijication - 76%, (4), pH 7.0, albumidpectinate ratio - 3:2 whv, pH 7.068. MoIaI concentrationscaIe. substances proceeds through interactions between polar groups followed by their desolvation. The latter process determinesthe signs of the changes of both enthalpy and entropy of the system.

Table 2 Quasi-thermodynamic parameters which characterize interactions between components of a system 0.I M NaCI (1) - human serum albumin (2) - sodium pectinate, a'egree of ester$cation - 76% (4), albumidpectinate ratio - 3:2 w h , pH 7.0, 25 't2 68. Parameter, I @, M.kgz.mob3

1-2

1-4

2-4

0.0H.01

-3.7iO. 04

6.2iO.60

-0.2iO.03

-5.2iO.21

7.339.77

0.2H.02

1.5H.18

-1.liO.17

4

AGii

178

Gum and Srabilisersfor the Food Industry

Table 3

Components of the second virial coeffr'cientfor the systems 0.I M NaCl (I) - human serum albumin (2) - sodium pectinate (4), albumidpectinate ratio - 312 whv, pH 7.0, 25 T68. A 1 2 = 0.8.10-3 m3.mol.kg2

Degree of esterlfication of pectinate,

A141

A24p A12.A14 A22

YO 0 45 47 50 57 70 76 89 98

11.0 3.6 3.2 4.1 3.0 2.8 1.9 1.5 1.5

-4.3 -3.8 -3.5 -1.9 -1.1 -0.8 -0.7 -0.9 -1.6

0.48 0.20 0.21 0.91 1.98 3.50 3.10 1.48 0.47

The application of the conditions for the fill stwility of a system in respect to di fusion shows that the systems studied are either homogeneous in the whole range of compositions or that separation of interpolymer complexes from the solution occurs.

4.COMPLEXES BETWEEN PROTEINS AND a-D-GLUCANS A class of interpolymer complexes important both in food chemistry and biological chemistry are complexes of proteins with a-D-glucans. The latter include both starch polysaccharides and glycogen. Complexes of proteins with a-glucans were investigated before World War I1 , mainly by St.J.von Przylecky and his coworkers71-74. These authors discriminate between two types of complexes (or simplexes, according to their terminology). Complexes of the fist type arise due to coulombic interactions between positively charged protein molecules and negatively charged molecules of glucans. These complexes dissociate by an increase in ionic strength. Coulombic complexes with proteins are formed, for example, by potato amylopectin at pH values below the isoelectric point of the protein75. It is well known that potato amylopectin contains phosphate groups. However, the same type of interactions was ascribed to rice76977 and wheat78 starches. The second, the most intriguing type of complexes are those of phosphorus-fiee polysaccharides as well as complexes of phosphorylated polysaccharides formed at pH values exceeding the isoelectric point of the protein. In the last case complexes arise only in the presence of salts71.73*79980. The composition of these complexes and their strength vary over a wide range80,*1.

Functwnal Interactions in Mixed Biopolymer Systems

179

The nature of interactions leading to the formation of complexes of the second type is unknown. However St.J.von Przylecki and his coworkers showed that the guanidine group of arginine residues and the hydroxyl group of tyrosine residues provide the complexation73982-85. Tyrosine residues even form 0-glycosyl bonds with g l y ~ o g e n ~ ? ~ ~ . E.L.Rozenfeld and E.G.Plyshevskayashowed that the ability to form complexes in neutral medium depends on the structure both of the protein and the polysaccharide88. Fibrillar proteins bind polysaccharides stronger than globular, and the denaturation of globular proteins enhances their binding ability89,90*91. The feature of polysaccharide structure critical for complexation is the occurrence of branches containing no less than five anhydroglucose units90.92993. The complexing ability of dextran is much less than that of glycogen or amylopectin94. Complex formation with proteins effects the enzymic hydrolysis of a-glucans. Thus, St.J.von Przylecki and B.Philipowicz showed that the rate of hydrolysis of starch and glycogen with a-amylase in the pH range 6.6-7.6decreases due to their interactions with myosin95. S.E.Karpiak and coworkers reported about the reduction of digestibility of glycogen with muscle a-amylase by the increase in the content of muscle proteins%. They suggested that the regulation of the activity of a-amylase in animal organs proceeds mainly through the complexation of glycogen with proteins. In contrast to this, E.L.Rozenfeld did not observe effects of proteins on the hydrolysis of glycogen catalyzed by the muscle a - a m ~ l a s e ~ ~ . At the same time she reported about the increase in the activity of phosphorylasedue to the complexation of glycogen with n1yosin97-9~. V.Ya.Grinberg and V.B.Tolstoguzov systematically investigated interrelations between the segregative phase separation and the complexation of waxy maize amylopectin and gelatin or human serum albuminl~JOl.Segregated two-phase systems are formed at the low ionic strength and pH value (4.7) equal to the isoelectric point of a protein (the same is true for r n a l t o d e x t M ~ ~Increase ~ ~ ~ ~ in ~ ~the) .ionic strength or the lowering of pH resulted in the formation of single-phase system, however,an increase in the ionic strength at pH values below the isoelectric point of the protein led again to segregative phase separation. The interplay of segregative and associative processes, probably, determines the shape of the phase diagram of the system 0.1 M NaCl - globulin fraction of sofiean proteins - potato amylopectin (Figure 11)1". At low concentrations of both polymeric components segregative processes prevail, but at higher concentrationsthe contribution of a weak complexation becomes noticeable. In other words, the increase in the total polymer concentration is accompanied with the decrease of the parameter ~ 2 4 However . one should take in mind that annular phase diagrams can also arise by certain relations between concentration-independentpair interaction ~arameters41~42. This sketchy outline of proteinla-glucan interactions is incomplete. Some important aspects have not been discussed,in particular the problem of proteidstarch interactions in cereals. These interactions determine to a great extent functional and technological properties of cereals, in particular the baking quality of wheat f l o ~ r s ~ The ~ ~ aim , ~ of ~ .this report was only to show that properties of multicomponent systems which contain proteins and starch polysaccharides are determined both by segregative and associative interactions.

Gums and Stabilisersfor the Food Industry

8-

.

.$ C

j4 -

-n 0

E"-

a

0'

1

4

1

I

L

I

8 Soybean globulins, '10

12

16

Figure 11 Phase diagram of the system 0.1 M NaCl - soybean ghJbUllFiS - potato amylopectin, pH 6.9; 25 7:. Coordinates of the criticalpoint: cprOr,= 3.6%; ca,,,, = 1.9%. References 1. M.L.Anson. In 'Processed Plant Protein Foodstuffs' (A.M.Altshu1,ed.), Academic

Press, New York, 1958, p.282. 2. V.B.Tolstoguzov and E.E.Braudo, J. Texture Stud., 1983, 14, 183. 3. V.B.Tolstoguzov.In 'Food Structure - Its Creation and Evaluation' (J.M.V.Blanshard and JRMitchell, eds.), Butterworths, London, 1988, p. 181. 4. V.B.Tolstoguzov, 'Artificial Foodstuffs', Nauka Publ., Moscow, 1978,231 pp.(Russ.). 5. V.B.Tolstoguzov, Food Hydrocoll., 1995,9, 3 17. 6. E.E.Braudo and V.B.Tolstoguzov, Nahrung/Food, 1974, 18, 173. 7. V.B.Tolstoguzov, NahrungiFood, 1974, 18, 523. 8. V.B.Tolstoguzov, EEBraudo and E.S.Wainerman,NahrungFood, 1975,19,973. 159. 9. V.J.Moms, Chem.Ind(London), 1985, (9, 10. V.B.Tolstoguzov. In 'Food Hydrocolloids. Structure, Properties and Functions' (K.Nishinari and E.Doi, eds.), Plenum Press, New York and London, 1993, p.327. 1 1. V.Ya.Grinberg and V.B.Tolstoguzov, Food Hydrocoll., 1997, 11, 145 12. M.W.Beijerinck,Centralbl. Bakteriol. Abt.2, 1896,2,627. 13. L.Piculel1, K.Bergfeldt and S.Nilsson. In 'Biopolymer Mixtures' (S.E.Harding, S.E.Hilland J.R.Mitchel1, eds.), Nottingham University Press, Nottingham, 1995, p.13. 14 D.V.Zasypkin, EEBraudo and V.B.Tolstoguzov,FoodHydrocoll., 1997, 11, 159. 15. V.Ya.Anosov, M.I.Ozerova and Yu.Ya.Fialkov, 'Fundamentals of Physicochemical Analysis', Nauka Publ., Moscow, 1978, p.7 (Russ.). 16. A.M.Gotlieb, I.G.Plashchina,E.E.Braudo, E.F.Titova, E.M.Belavtseva and V.B.Tolstoguzov, NahrungIFood, 1988,32,927. 17. H.Moritaka, K.Nishinari, H.Horiuchi and M.Watase, J. Texture Stud, 1980, 11,257. 18. M.Watase and K.Nishinari,HheoLActa, 1980, 19,220. 19. M.Watase and K.Nishinari,Biorheol., 1983,20,495. 20. A.H.Clark, R.K.Richardson, S.B.Ross-Murphyand J.M.Stubbs, Macromol., 1983, 16, 1367 21. H.McEvoy, S.B.Ross-Murphyand A.H.Clark. In 'Gums and Stabilizers for the Food Industry-2' (G.O.Phillips,D.J.Wedlock and P.A.Williams,eds.), Pergamon Press, Oxford, 1984, p.111.

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181

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GELS MADE OF WHEAT STARCH AND ETHYL(HYDR0XYETHYL)CELLULOSE (EHEC): RHEOLOGICAL BEHAVIOUR

Helena Larsson and Ann-Charlotte Eliasson Dept of Food Technology University of Lund Box 124, S-22100 Lund, Sweden

1 INTRODUCTION Starch is a component of many food systems, where the consistency is important. In such systems starch may be used to improve or manipulate the rheological properties of the system. When diluted starch pastes are stored the influence on the rheological properties (gel formation) is initially related to the gelation of amylose but also, to some extent, to crystallisation within the amylose matrix. At longer storage times the increase in gel rigidity is attributed to the development of more rigid granules, i.e. crystallisation of amylopectin within the granules.' The most important factors determining the mechanical properties at small deformations are the amylose gel strength and the deformability, the volume fraction and the shape of the granules.* When starch is mixed with a hydrocolloid the continuous phase is modified. The predominant effect is a concentration of the polymers in solution (amylose and the added hydrocolloid), resulting in increased viscosity. However, the mixing of dissimilar polymers may give different types of network^.^ Compatibility between the polymers is important for the formation of the networks. A reduced gel strength at elevated dextran concentrations was shown for amylose-dextran gels. This was explained as an enrichment of dextran in droplets, resulting from polymer incompatibility .5 The phase separation of amylose-amylopectin blends has also been ascribed to incompatibility phenomena.6 Similarly, the complex rheological behaviour of blends including maize starch and hydrocolloids (guar, locust bean and xanthan gum) was suggested to result from a phase separation caused by immiscibility of the polymer^.^ In the present study the influence of ethyl(hydroxyethy1)cellulose (EHEC) on the gel behaviour of starch has been investigated. EHEC results from the process where cellulose has been made water soluble by chemically incorporating small amounts of more hydrophobic ethyl and relatively hydrophilic ethylene oxide groups into the hydrophilic cellulose backbone. Through this modification EHEC takes on amphiphilic properties in water solution. One property of EHEC is that its solubility in water is dependent on temperature. At high temperatures the hydrophobic chains aggregate to hide from the surrounding water, and a thermoreversible phase separation is observed. The temperature at which the solution demixes and becomes cloudy is referred to as the cloud point (T,p).8 The present investigation describes the gel properties of wheat starch in the presence of three EHECs, differing in molecular weights and all having a cloud point below the temperature at which most of the amylose goes into solution.

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2 MATERIALS AND METHODS The gelling of native wheat starch (Lyckeby SttirkelsenAB, Kristianstad, Sweden) in the presence of ethyl(hydroxyethy1)cellulose (EHEC) was studied for three EHECs with increasing molecular weights in the order: El, E2 and E3 (80,000.170,000 and 350,000). The cloud point (Tcp) was approximatelythe same for the three EHECs, i.e. 61.1-61.9"C. Both the molecular weight estimations and the cloud points were given by the manufacturer (AkzoNobel Surface Chemistry AB, Stenungsund, Sweden). Gelatinisation was allowed to take place by heating the mixtures of wheat starch (6 wt %) and distilled water or EHEC-solution (1.0 wt %) in sealed test-tubes at 90°C. Dynamic rheological measurements were performed (in the linear viscoelastic region, at 25°C and after a resting period of 340 minutes) in the Bohlin VOR rheometer (Metric Analys, Stockholm, S ~ e d e n )Confocal .~ laser scanning was performed in reflected light (Molecular Dynamics, Sunnyvale, CA, USA) on the gelatinised samples after approximately 12 hours storage at room temperature.

3 RESULTS AND DISCUSSION The mechanical spectra of wheat starch (6%) and a mixed gel of wheat starch (6%) and EHEC (E2, 1%) at 25°C are shown in Figure 1. The spectrum for starch is consistent with that from a strong gel." When EHEC was introduced the mechanical response was completely changed. Although the accessible frequency window was limited, two viscoelastic regions, separated in time, were indicated. One EHEC of a lower molecular weight (El) and one EHEC of a higher molecular weight (E3) were used to accentuate the difference in the moduli of these two time regions (Figure 2 a and b, respectively). The elastic plateau where G is greater than G" at low frequencies, is indicated with E l (Figure 2 a). As the storage modulus was almost independent of time, and greater than G at the lower frequencies, a network with junction zones of longer life-time compared with the time of observation existed. It is suggested that this plateau at low frequencies represents the gel strength of the mixed system. In Figure 2 b the spectrum beyond where G = G" is shown with the blend including E3. The time dependency of G and G" increased at lower frequencies, but levelled off, with G > G" at frequenciesbeyond 0.05 Hz. In this time region, the contribution from dynamic entanglements is suggested to strongly influence the rheological behaviour of the mixed system. The influence from dynamic entanglements at a certain frequency can be compared with the behaviour of a semi dilute polymer solution, where G > G at low frequencies and G < G at high frequencieswith a "cross-over'' ( G = G ) at a certain frequency. This was observed for the EHECs (3 % wt) at 25"C9

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k

>

u u

1

0.01

0.1 Frequency/&

1

10

Figure 1. The mechanical spectra of wheat starch (6%)and a mixed gel of wheat starch (6%)and EHEC (E2, 1%) at 25°C (after 340 minutes storage). Starch gel: G' (@) and G" (0). Starch and EHEC: G' ( A) and G" ( A). (Resultsfrom Larsson and Eliasson').

Figure 2. The mechanical spectra of mixed gels of wheat starch (6%)and EHEC (1%)at 25°C. El (a)and E3 (b). G' (0)and G" (0). (Resultsfrom Larsson and Eliasson').

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Figure 3. Confocal laser scanning micrographs of starch (top)and starch +I 96 EHEC, E2 (bottom) after approximately 12 hours storage at room temperature.

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In Figure 3, micrographs of the starch gel and the blend including 1% E2 are shown. Due to the fact that the modified cellulose did not reflect light, i.e. appeared black in the micrographs, two more or less discontinuous phases were indicated when EHEC was included. The starch phase is indicated by the deformed starch granules (dark grey), located in the brighter amylose matrix. The EHECs studied passed through a thermoreversible phase separation at a temperature around 61"C, which was below the temperature at which the most of the amylose goes into solution. This suggests that amylose was allowed to leach out from the granules into EHEC-deficient regions. EHEC may then be described as acting as a partially continuous soft filling material, which breaks up the stiffer starch gel. Thus the two viscoelastic regions separated in time (Figure 2 a-b) were suggested to illustrate a phase separation of starch and EHEC. This means that two (three) phases were present in the blends, where one was enriched in starch (amylose and granules) and the other in EHEC. Consequently, one of the most important conclusions of the study was that the influence of time (frequency) is extremely important for studies on starch gelling in blends. This means that rheological studies on mixed gels must be accompanied by determination of the mechanical spectra. ACKNOWLEDGEMENTS We would like to thank Katarina Lindell for valuable discussions on EHEC and Peter Ekstrom for professional help with the confocal laser scanning microscope. REFERENCES

1. Miles, M. J., V. J. Morris, P. D. Orford, S. G. Ring. CarbohydrateResearch, 1985, 135,271-278. 2. Ring, S. G.. G. Stainsby. Prog. Food Nutr. Sci., 1982,6,323-329. 3. Sajjan, U. S., M. R. R. Rao. Carbohydr. Polym., 1987,7,395-402. 4. Cairns, P., M. J. Miles, V. J. Moms, G. J. Brownsey. Carbohydr. Res., 1987,160, 41 1-423. 5. Kalichevsky, M. T., P. D. Orford, S. G. Ring. Carbohydr. Polym., 1986.6, 145-154. 6. Kalichevsky, M. T., S . G. Ring. Curbohydr.Res., 1987,162,323-328. 7. Alloncle, M., J.-L. Doublier. Food Hydrocolloids, 1991,5,455-467. 8. Lindell, K., Thesis, Lund University, Sweden, 1996. 9. Larsson, H., A.-C. Eliasson. Carbohydr. Polym., 1996, submitted. 10. Clark, A. H., S. B. Ross-Murphy. Adv. Polym. Sci., 1987,83,57-192.

Solution and Gelation Properties of Protein-PolysaccharideMixtures: Signature by Small-Angle Neutron Scattering and Rheology

D. Renard, F. BouC' and J. Lefebvre INRA, Centre de Recherches Agro-Alimentaires, Laboratoire de Physico-Chimie des Macromoltcules, BP 71627,44316 Nantes Cedex 3, France * Laboratoire Lton Brillouin, CE-Saclay, 91 191 Gif-sur-Yvette Cedex, France

ABSTRACT The aim of this study was to understand the competition between phase separation and gelation during thermal treatment for BSA - HEC or CMC mixtures located in the onephase region of the state diagram at 20 "C. We used dynamical rheological and smallangle neutron scattering (SANS) experiments to characterize the structure of the biopolymer mixtures in both sol and gel states. In the sol state, according to SANS and rheological measurements, the structure of the mixtures located in the one-phase region was heterogeneous and could be compared to colloidal suspensions with incipient flocculation. In the gel state, drastic changes occurred in the structure and the rheological behaviour of the gels obtained from the mixtures as compared to pure BSA, depending on the pH and temperature conditions but also the polysacchwide used. In particular, the addition of CMC to BSA solution led to the formation of gels with different macroscopic aspects ranging from a milky to a transparent character depending on the polymer concentration; moreover, competition between gelation and phase separation occurred depending on the pH and the temperature applied to the system. 1 INTRODUCTION Proteins and polysaccharides are present together in many kinds of food systems, and both types of food macromolecule contribute to the structure, texture and stability through their thickening or gelation behaviour.' Owing to the application of new physico-chemical techniques,*.' much is now known at the molecular level about the functional properties of individual biopolymers. Nevertheless, our knowledge of the role of biopolymer-biopolymer interactions, especially protein-polysaccharide interactions, in relation to functionality in complex multiphasic systems, such as food mixed solutions or gels, is still rather limited.4 Investigations of hydrocolloid interactions in aqueous solutions and gels are necessary for modelling and improvement of conventional foods,

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the development of novel formulated foods and for controlling functional properties of food systems by added hydro colloid^.^ Many studies showed that aqueous solutions of mixed proteidpolysaccharide are susceptible to phase separation, the low entropy of mixing leading to two basic types of liquid-liquid phase separation, associative and segregative.’. The former refers to an association usually pictured in terms of a mixed polysaccharide/protein complex concentrating both components in one phase, whereas the latter refers to a polysaccharide/protein segregation thought of as a micro-phase separation driven by an ‘incompatibility’ of the unlike constituents. When a mixed biopolymer solution containing gel-forming polymer(s) is heated above (or cooled below) the gel temperature of either or both components, phase separation and gelation become competing processes and several alternative gel microstructures are possible. The resultant structure depends not only on the nature of the polymers themselves but also on the degree of miscibility of polymers in solution, the relative time scales of phase separation and gelation, and the thermal history to which the mixture is subjected.’ Studies on protein-polysaccharide mixtures have been rarely focused on the onephase region of the phase diagram, described classically as the domain where there is a molecular-scale coexistence of the two unlike macromolecules. The aim of this study was to investigate the structure of the systems Bovine Serum Albumin (BSA)+HydroxyEthylCellulose (HEC) and BSA+CarboxyMethylCellulose (CMC) mixed solutions in water in the one-phase region. We finally report the structural features of BSA gels in the presence of non-gelling polysaccharides and try to elucidate the competition between phase separation and gelation in these systems. We have used small-angle neutron scattering ( S A N S ) as well as dynamical rheological measurements. We give particular attention to the balance between attractive and repulsive forces in these systems by varying the polymer nature (neutral type HEC or polyelectrolyte type CMC) and the pH of the solutions.

2 MATERIALS AND METHODS BSA was from ICN Biomedicals (UK) and was used without further purification. Stock solutions (20% w/w) were prepared in water at pH 5.2 (initial pH value of the stock solution) and 7 (by adjusting the pH value with 1.0 M NaOH). NaCMC sample denoted CMC 7LXF (degree of substitution 0.71) was supplied by Hercules SA (France) and HEC sample, namely HEC LV (‘low viscosity’; molar substitution: 1.65), was obtained from Polysciences (UK). Molecular characteristics determined for these polysaccharides are collected in Table 1. We prepared protein+polysaccharide mixtures in the one-phase region of the phase diagrams by mixing protein and polysaccharide stock solutions in a 50/50 w/w ratio at pH values of 5.2 and 7 in water. The final composition of these mixtures was either 0.1/10% or l/lO% w/w in both cases of mixed solutions and gels. The polysaccharide concentrations were chosen in order to be below or above the critical overlap concentration C’ (Table 1). We checked that the pH values of the mixtures were identical to those of the protein stock solution. S A N S measurements were made at a wavelength k10 fi using the PAXY multidetector instrument of the Laboratoire LCon Brillouin, Saclay (France); the sampleto-detector distance used was 3.2 m. Using pure H,O as the solvent, the signal from the

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polysaccharide solutions was negligible and without upturn at low q values, whereas the contrast BSNwater was sufficient to obtain a good signal-to-noise ratio. The intensity from the polysaccharide was subtracted from the intensity obtained for the proteinpolysaccharide mixtures.

Table 1. Molecular characteristics of HEC and CMC in NaCl O.lM and 20 "Cand correlation length 5 in semi-dilute solution at C = l % w h

[rl I (m1.g.')

HEC LV

CMC 7LxF

162

289

75000

119200

23.5

21.5

2.30

4.85

8.9

19.8

10.5'

23.0'

(Intrinsic viscosity)

(g.moP) (Viscosimetric average molecular weight)

R i b ) (Radius of gyration)

c*(g.1') (Critical overlap concentration)

5 (nm)

(Correlation length)

LP (nm)

(Persistence length in water)

[ql ,Mv and C' are taken from ref. (8); 'Calculated from C' using the equation 0 C' = 3MvI (47tN,RS3);'from ref. (9); from ref. (10).

We thus were very close to observing protein-protein correlations only. The data were converted in intensity versus the wave vector q (= 41dh sine, where 28 is the scattering angle) and normalized for transmission and sample path length. Then, in order to obtain an absolute intensity scale, the data were normalized to a water run. The intensity was finally expressed in terms of molecular mass (g.mol-I) as the density and the contrast variation of BSA in H,O are known." Measurements were performed at ambient temperature for the mixed solutions. For the mixed gels, the samples were directly heated in the quartz cuvettes 2h at 80 "C and were analysed by S A N S after standing for 24 h at room temperature. In order to avoid evaporation, mixed solutions were topped in the cuvettes with a thin layer of paraffin oil before heating. Rheological measurements were performed at 25 "C using a Rheometrics RFS II instrument with a Couette device (gap width of 1 mm) in the case of the mixed solutions (see ref. 11 for experimental details). Small-amplitude oscillatory shear experiments were performed on heat-set mixed gels with a Carri-Med CS 100 rheometer using the cone-and-plate geometry (cone diameter 2 cm; angle 4"). The gels (2h 80 "C) were formed in-situ in the rheometer and the kinetics of gelation were followed at 1 Hz under 0.01 strain amplitude. After quenching at 25 "C and one hour rest (we previously checked that the moduli G' and G" at 1 Hz kept constant after quenching at 25 "C), the mechanical spectra were recorded between loT3and 10 Hz under 0.03 strain amplitude.

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3 RESULTS AND DISCUSSION

3.1 Characteristics of the polysaccharides Table 1 gives some structural characteristics of the polysaccharides used in this work. These polymers are classically described as semi-rigid chains as revealed by their persistence length values (Lp-10 nm). In the case of the CMC molecule, the polyelectrolyte effect makes the chain expansion and rigidity increase (due to intramolecular electrostatic repulsions). The polysaccharide solutions were prepared below and above the critical overlap concentration C' delimitating the dilute / semi-dilute regime and the chains are characterized by their radius of gyration Rg below C' and their correlation length 6 above C' (which corresponds to the mesh size of a semi-dilute polymer solution). Theoretical predictions on colloidal polymer-induced attraction (depletion flocculation) suggest that the depletion width is of the order of the polymer radius of gyration in the dilute regime and decreases to the order of the correlation length in the semi-dilute regime where the polymer chains interpenetrate.'*

3.2 Structural behaviour of the mixed solutions 3.2. I Polysaccharide-protein mixtures in the semi-dilute regime. To summarize our previous results obtained on BSA mixed with polysaccharide solutions in the semi-dilute regime, according to combined SANS and rheological measurements, the structures of the mixtures located in the one-phase region of the phase diagram were heterogeneous and could be compared to colloidal suspensions where the beginning of flocculation is evident.''' I 3 According to the distances between neighboring particles, the structures of these systems appear to fall into two classes. (i) In the situation where no attraction is established between the polysaccharide and the protein (i.e. BSA/HEC pH 5.2 in water, BSNCMC pH 7 in water, BSNCMC pH 5.2 in 0.1M NaCl), flocculation of BSA particles via depletion forces or electrostatic repulsions between protein and polysaccharide is important. (ii) In the case where an attraction exists between the protein and segments of the polysaccharide coils (i.e. BSNCMC pH 5.2 in water) or a preferential distance between charged BSA particles is maintained (i.e. BSA/HEC pH 7 in water or 0.1M NaCI), the extent of flocculation via depletion or electrostatic forces is considerably reduced. These two types of structural features were independent of the molecular weight of the polysaccharide. This agreed with the idea that, in the semi-dilute regime, the mesh size 6 does not depend on the molecular weight of the polymer. 3.2.2 Influence of polysaccharide concentration. The effect of increasing the polysaccharide concentration (ranging from dilute to semi-dilute regime) was to induce more concentration fluctuations between particles at large scale, i.e. the degree of flocculation of BSA particles became greater. This arose from the fact that the simplest way to minimize the free energy of mixing was to separate protein molecules from the entangled polysaccharide chains (the elevation of osmotic pressure caused by the increase of polysaccharide chains in the medium forced the BSA particles to come close to each other). Independently of the heterogeneous structures developing at large scale, an attraction between the polysaccharide and the protein may occur. An illustration of the non-specific attractive interaction between the polysaccharide and the protein molecules is given in

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Figure 1 where the scattering intensity I (g.mo1’) is displayed as a function of the wave vector q (nm”) for BSNCMC mixtures in water at pH 5.2. For BSA solution near the isoelectric point (IEP = 4.8), a large increase of intensity at low q values corresponding to an attractive interaction (usually assumed to be van der Waals attraction) indicates incipient precipitation of the protein. For the BSNCMC mixtures, we observe surprisingly the appearance of a correlation peak (maximum in the scattered intensity corresponding to a wave vector value qm,) in the scattering diagram whereas this peak does not exist in the scattering profile of the BSA solution. Moreover, the position of the peak shifts towards higher q values as CMC concentration increases.

I 2,ox1o5t

I --?

< 02 0,4 0,6 03

0,o 0,o

q (nm-’)

Figure 1. Scattering profiles I ( q ) (g.mot‘)versus q (nm-’)for BSA solution (10% w/w) and BSAKMC mixtures in water at pH 5.2: BSA (-); BSAKMC 10/0.1% w h (a; 10/0.48% w/w (*); 10/1% w/w (0) The position of the peak gives a mean distance (2nlq,,) between BSA particles ranging from 34.9 nm in the dilute regime to 12.5 nm in the semi-dilute regime. The existence of this liquid-like short-range order between BSA molecules could result from an attraction between the anionic polysaccharide and the protein. The BSA particles would be adsorbed onto the individual polymer coil segments in dilute regime or entrapped in the crosslinks of the entangled polymer coils network in the semi-dilute regime, and hence assume a preferential distance of separation in the mixture. This distance decreases as polysaccharide concentration increases. The consequence is that flocculation may still occur in these systems, but to a lower extent than in the cases where there is no attraction because a fraction of BSA molecules is adsorbed onto the polymer chain or entrapped within the entangled polysaccharide coils. Similar conclusions relating to the attraction of protein molecules to polymer coil segments were given to explain protein partitioning in two-phase aqueous polymer ~ystems.’~ Other authors gave evidence of the formation of soluble polyion-protein complexes even when the net charge on the protein was identical in sign to the charge on the p01yelectrolyte.l~ This finding was seen as evidence for the predominant role of (< charge patches D on the protein in the formation of complexes with polyelectrolytes. Finally, the nature of the polysaccharide influences considerably the structure of the solutions when the particles are charged. Indeed, the addition to BSA solutions of neutral polysaccharide has no effect on the preferential distances existing between charged BSA

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molecules whatever the polysaccharide concentration used. On the contrary, the increase of charged polysaccharide concentration leads to the vanishing of the short-range order peak due to the strong charge fluctuations existing between the two charged components of same sign.

3.3 Structural behaviour of the mixed gels The structural features of BSA gels in the presence of non-gelling polysaccharide HEC or CMC were followed by S A N S and rheology. Gelation of globular proteins is the result of an aggregation process, which is generally triggered by a conformational change of the protein induced by a modification of solvent conditions.I6Heat-set globular protein gels will vary in structure as the balance between repulsive and attractive forces in the medium change; this will lead to structures ranging from linear aggregates to large compact clusters with a certain fractality defining the type of aggregation process involved (diffusion-limited or reaction-limited cluster-cluster aggregation).16'17. '* Since gel network formation may be a diffusion-controlled process, the increase of viscosity by adding polysaccharide to the medium may be an influential parameter." Moreover, the charge borne by the polysaccharide may influence the size and the shape of the aggregates and the level of heterogeneity in the final network. Finally, gelation will lead to changes in the thermodynamics of mixing. As gelation proceeds, both the effective molecular mass of the BSA and the width of the molecular mass distribution will increase; thus the entropy of mixing will decrease. In addition, there will be an effect of the network elasticity on the free energy of mixing: gels with a low elasticity can be expected to tolerate only a limited degree of phase separation from a second polymer species.'" The time at which the elasticity effect becomes significant will depend on the relative rates of the gelation and phase separation. 3.3.1 BSA gels obtained in presence of non-gelling neutral polysaccharide. Scattering profiles of BSA gels alone (2h 80 "C) or mixed with HEC (0.1 or 1% w/w) in water at pH 5.2 (a) or pH 7 (b) are displayed on Figure 2. All the systems at pH 5.2 showed at macroscopic scale a milky opacity after heating whereas those formed at pH 7 were quite clear. These visual observations lead to the first conclusion that the degree of heterogeneity in the mixed BSA / HEC gels formed near the isoelectric point was pronounced. These heterogeneities are reflected on a mesoscopic scale in the S A N S curves. The scattered intensity of BSA gels formed at pH 5.2 with or without HEC followed a power law over one decade in q (nm-I) with an exponent of 4.0, suggesting the existence of very large compact clusters with smooth surfaces (if the boundary between the scattering objects and the matrix is sharp, the scattering varies as q4 and must be proportional to the total interface between the two homogeneous media; in addition, three-dimensional objects with smooth surfaces give a fractal exponent of 4). If we assume that the clusters have a spherical shape, a mean diameter for BSA aggregates of 3 +/- 0.1 pm is found. The q4 power law dependence is followed on the scattering curves by a correlation peak at high q values, indicating that preferential distances between BSA particles inside the cluster are maintained. The position of the peak gives an interparticle distance of 10 nm (centre-to-centre distance of ca. 1.4 BSA diameters). The physical picture we may suggest of these systems is that very large clusters loosely connected between them are formed upon heating; these clusters are composed of individual BSA monomers arranged

Functional Interactions in Mixed Biopolymer Systems

o

e;

S

195

oE 10 .9

10

•

10 10 0,1

Log q (nm")

0,1

Log q (nm')

Figure 2 Scattering profiles /(q) (g.mol'] versus q (nm') for BSA and BSAlHEC mixed gels in water at pH 5.2 (a) or pH 7 (b): BSA 10% w/w (-); BSAlHEC 10/0.1% w/w (_); BSAlHEC 10/1% w/w (e)

inside the cluster at very close distances from each other, giving rise to a quasicrystalline organization (or a glassy solid state) inside the aggregate. The structure of the gel seems to be not affected by the presence of HEC as revealed in Figure 2a. Prolonged thermal treatment on these systems leads to gelation without macroscopic phase separation. For the gels formed at pH 7 where electrostatic repulsions between BSA particles dominate (Figure 2b), a single peak is observed which shifts slightly to larger q values as the polysaccharide concentration increases. The fact that long-range correlations are still present after thermal treatment was previously demonstrated on ~-lactoglobulin gels and was interpreted as the existence of preferential distances between charged aggregates." A mean interaggregate distance of 24 nm is found in the mixed gels. A decrease of intensity in the peak region and a displacement of the peak position towards higher q values is observed as the HEC concentration increases. This means that BSA aggregates formed with adding polysaccharide are lower in size and at closer distances. In addition, the upturn of scattered intensity observed at low q values for the 10/1% BSAlHEC case suggests that heterogeneities may be present at large scale (i.e. concentration fluctuations are still present in the gel state). The neutral polysaccharide HEC would prevent the aggregation process of charged BSA particles without promoting nevertheless a phase separation process (a prolonged thermal treatment did not affect the system). As the preferential distances between BSA particles are maintained both in the sol and gel states, we may assume that HEC molecules would not reduce the repulsive potential between charged proteins but would act only as a steric barrier (by purely excluded volume effects) in the development of the aggregation process; a fraction of BSA monomers would not consequently participate to the formation of the network. 3.3.2 BSA gels obtained in presence of non-gelling charged polysaccharide. Scattering profiles of BSA gels alone (2h 80°C) or mixed with CMC (0.1 or 1% w/w) in water at pH 5.2 (a) or pH 7 (b) are shown on Figure 3. The gels formed at pH 5.2 appeared with a milky opacity excep~ the one formed with the highest CMC

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concentration which was opalescent. The inverse tendency was observed for the systems at pH 7 where a strong turbidity developed in the mixed gel formed with the highest polyelectrolyte concentration. The insert in figure 3a shows the scattering curves obtained for mixed gels (10/1% w/w) formed at a lower temperature (55 "C) and two heating times. These systems were completely transparent or slightly opalescent on a macroscopic scale. For pH 7 at 55 "C (insert in figure 3b), phase separation occurred coupled with gelation of the two separated phases as heating time was prolonged. From these first visual observations, we may say that the polyelectrolyte chains modified completely the microstructure of BSA gels; the high level of heterogeneous structures developing in the gels near the isoelectric point was considerably reduced whereas a competition between gelation and phase separation took place at a pH value well above the isoelectric point. The scattering intensity for BSA gel mixed with CMC 0.1% w/w at pH 5.2 (Figure 3a) followed a power law with an exponent value of 4.0over the entire q range explored as for BSA gel alone. The existence of large compact clusters with smooth surfaces is again demonstrated and these aggregates formed in the presence of CMC have bigger sizes as revealed by the higher scattered intensities obtained. The new feature in these mixed gels is that the correlation peak at high q values disappeared, indicating that the preferential distances between BSA particles inside the aggregates are now lost at this observation scale. In the case where the polyelectrolyte concentration is increased (with CMC 1% w/w), the structure of the mixed gel changes completely. A fractal regime seems to appear at low q values over less than one decade with an apparent fractal dimension value of 2.05 (Fig. 3a), value consistent with a reaction limited cluster-cluster aggregation model (RLCA).*'

10

,

. 0:1 .

Log q (nrn-')

I

1

Figure 3. Scattering profiles I ( q ) (g.moT') versus q (nm-')for BSA and BSNCMC mixed gels (2h 80 "C)in water at pH 5.2 ( a ) or pH 7 (b); the slopes delimit the fractal regime of the structures. BSA 10% w h (-); BSNCMC 10/0.1% w h (W); BSNCMC 10/1% w h (a).Insert: BSMCMC 1O/I% w/w mixed gels in water at pH 5.2 ( a ) or pH 7 ( b ) obtained for a thermal treatment of (0)3h 55 "C and (0) 9h 55 "C

Functional Interactions in Mixed Biopolymer Systems

197

This finding would mean that the aggregation process of BSA particles would be slowed down in the presence of a semi-dilute polyelectrolyte solution as very less compact structures are formed. Moreover, the limiting agent of the process would be of chemical nature as revealed by the fractal dimension value consistent with a reactional (and not diffusional) limited process. This hypothesis has to be related to the experimental observations done at lower temperature (insert in Figure 3a). In these conditions (55 "C), the scattered intensities for the aggregates follow a power law over near one decade with an exponent of 1.O irrespective of heating time, suggesting the development of fractal structures for both heating times, and that these structures are linear at this observation scale. How can we explain the linear shape of the aggregates formed at low temperature? The experimental observations done for the mixed solutions clearly demonstrated an adsorption process of BSA particles onto the polysaccharide chains (i.e. formation of soluble complexes). Based on these results, the physical picture we could give at the S A N S scale of the gelation process which takes place at low temperature would be that the linear fractal aggregates would grow between the entangled polyelectrolyte chains in the semi-dilute regime (no linear aggregates were found in the dilute regime; see Figure 3a). These stiff small aggregates forming the network would be frozen in the entangled CMC coils. With an elevation of temperature (from 55 to 80 "C), an enhancement of the molecular flexibility of the CMC chains would allow an increase of branching of the protein aggregates as revealed by the higher apparent exponent value found on the scattering curve (d,= -2.05; see Figure 3a). The action of CMC on heat-induced gelation of BSA at pH 5.2 appeared mainly due to << chemical >> interactions rather than physical entanglement. Similar conclusions were given recently on a system based of egg white gels mixed with dextran ~u1fate.I~ These results would suggest that a control of gelation by adding polyelectrolyte to a globular protein solution (provided that specific physicochemical conditions and molar ratio are selected) can be exerted and novel structures could be developed. For the gels formed at pH 7 (Figure 3b), a single correlation peak is observed which shifts towards higher q values (i.e. lower distances) with increasing polysaccharide concentration. The mean interaggregate distance ranges from 25 nm for BSA gel alone to 21.5 nm for BSAICMC 1011% wlw mixed gel. Moreover, the short-range order peak tends to vanish with increasing polyelectrolyte concentration. The consequence is a depression of the intensity in the peak region and a subsequent increase of the scattered intensity at low q values. The same features were observed in the scattering curves of the mixed BSAICMC solutions and were interpreted in terms of a flocculation phenomenon arising from strongly repulsive electrostatic interactions between the two charged components of same sign." In the case of mixed gels, the results could be interpreted in terms of a demixing phenomenon as gelation of charged BSA particles proceeds. The demixing process competes with gelation as temperature is lowered or as the CMC concentration is increased due to strong charge fluctuations between the anionic polysaccharide and the negatively charged protein. This idea is confirmed by the results obtained at 55 "C (insert in figure 3b). In these conditions, turbidity (with a density gradient indicative of a beginning of demixing) developed in the mixed system which remained in solution at this temperature and a phase-separated mixed gel was observed for a prolonged heating time (the upper phase was a transparent gel whereas the lower phase appeared as a turbid gel). From a scattering viewpoint, these features are characterized by what it seems to be fractal regimes at low q values with apparent fractal dimension near 1.0 for the two systems, indicating the formation of linear aggregates at

198

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the SANS scale. Moreover, a correlation peak appears in the intermediate q range when the mixed system is heated for a long period, giving a mean interaggregate distance of 19.4 nm. Nevertheless, these stiff small aggregates formed at low temperature are not able to coexist with the semi-dilute polyelectrolyte solution and a macro-phase separation occurs after a certain time; the large charge fluctuations between the clusters and the polysaccharide chains tend to maximize the free energy of mixing (i.e. increase the repulsions between the charged components), leading to a demixing on a macroscopic scale. As these mixed systems are of segregative type from a phase separation viewpoint, enough BSA particles are present in each phase to ensure the formation of a network. This leads to the formation of a homogeneous network in the upper phase as BSA is present at a lower volumic fraction and to that of a turbid gel in the BSA particle enriched lower phase. At higher temperature, the interplay between phase separation and gelation leads to a nonequilibrium phase-separated morphology which is frozen by the onset of gelation. This condition tends to the limit of extremely fast BSA particles aggregation compared to the rate of phase separation, this effect leading to micro-phase separation (as revealed in Figure 3b by the upturn of intensity at low q values). Other scattering patterns are given in the literature for systems which gel and phase separate at comparable rates.2"

3.4 Rheological behaviour of the mixed gels In this section we try to relate the observed mesostructures of the mixed gels at the SANS scale to the elasticity of the network obtained at the macroscopic scale by means of small deformation dynamical rheological measurements. The rheological properties of the mixed solutions are reported elsewhere and the main conclusions are that the increase of both dynamic moduli G' and G" and apparent viscosities qapp of these solutions as compared to the values obtained for the polysaccharide solutions clearly indicated the possible formation of BSA flocs in these systems." We shall focus on the gelation kinetic aspects since the mechanical spectra of the resulting gels were not so informative (the dependence of moduli on the frequency was slight within the experimental window). Figure 4 displays typical traces of the storage modulus G' (f = 1 Hz; y = 0.01) against time as a BSA 10% w/w or a BSA/HEC (BSNCMC) 10/1% w/w solutions are heated at 80 "C. The same profiles with lower values were obtained for the loss modulus G". The kinetics of gelation of BSA alone at pH 5.2 follows a two-step process (Figure 4a) with a first visible plateau near 4500 s; no second plateau is evidenced at the time scale explored. The BSA gels formed near the isoelectric point are of particle type, developing heterogeneous structures loosely connected between them as the temperature increases. A similar kinetic profile is obtained when HEC is added to the BSA solution except that the process is slowed down. The first plateau is less evident and appears at a longer time; moreover, the shear moduli (G' and G ) are reduced by 20% at the end of the kinetics. The HEC molecules would act only by excluded volume effects on the aggregation process as the kinetics of gelation is slightly slowed down in the mixed gels and as the structure of the clusters of BSA particles did not change at the SANS scale. For BSA solution at pH 7 (Figure 4a), the aggregation process is much more << ordered D as compared to pH 5.2 as the repulsive barriers between charged particles have to be levelled off. The consequence is that shear moduli have high values and tend to reach a plateau as the increase of organization in the network formation gives a higher elasticity; the storage modulus after 2h at 80 "C is 4 fold higher at pH 7 than at pH 5.2.

199

Functional Interactions in Mixed Biopolymer Systems

100

80 150

60 100 40 50

20

2

4

6

i

0

0 0

2

4

6

Figure 4. Evolution of G' (Pa) as a fwtction of time (s) at 80 "C and 1 Hz under 0.01 strain amplitude for BSA 10% w h and BSA/HEC 10/1% w h (a) or BSAKMC 10/1% w h (b) gels at pH 5.2 (open symbols) and pH 7 cfilled symbols). BSA pH 5.2 ( 0 ) ; BSA/HEC or BSMCMC pH 5.2 (0);BSA pH 7 (m); BSA/HEC or BSAICMC pH 7 (e) Many authors suggested that this highly viscoelastic character was related to the formation of intermolecular disulfide bonds; SWS-S interchange reactions are susceptible to occur at neutral or alkaline pH values. The increase of elasticity is nevertheless always encountered in the case where strong repulsive interactions are present (i.e. even at acidic pH values where the ionization of the -SH groups is rather limited). The addition of HEC increases both the kinetic process and the shear moduli values (70% higher than for BSA alone). The increase of the aggregation process can be related to the S A N S results where it was found that preferential distances between aggregated species were maintained ( N ordered D character of the network) and heterogeneities developed at large scale (giving rise to a higher sensitivity in the viscoelastic response) in the presence of HEC. The traces of the storage moduli of BSA gels and BSNCMC mixed gels at pH 5.2 and pH 7 are displayed on Figure 4b. The addition of CMC at pH 5.2 causes the first part of the kinetic profile observed for BSA alone to vanish and leads to a linear increase of the storage modulus as a function of time (G' reaches a 230% higher value as compare to BSA gel at the end of the kinetic). When CMC concentration is lowered (0.1% w/w), the first part of the kinetic profile with a pseudo-plateau remains observable and the increase of G' is also important (results not presented). S A N S results brought into evidence that non-specific interactions occurred between the CMC coils and the BSA particles leading to a radical change in the aggregate structure; linear and stiff aggregates more or less branched as the temperature was increased were observed. These observations are reflected in the highly viscoelastic character of the mixed gel as compared to BSA gel alone during its formation at 80 "C. The polyelectrolyte chains would act as a repulsive potential in the gelation process; the consequence would be that a u directed D aggregation of protein molecules would take place (a transition from particle gel to homogeneous network is observed).

zoo

Gums and Stabilisers for the Food Industry

When the mixed BSAlCMC solution is heated at pH 7, a large increase of the storage modulus is observed (170% higher than for BSA gel alone) without changes in the kinetic profile. This result has to be related again with the preferential distances observed in neutron scattering between the charged aggregates indicating an organized structure at local scale and the large heterogeneities revealed at large scale. An interesting feature is observed after quenching the sample to 25°C: the moduli measured at I Hz are 360% higher than those obtained for BSA gel alone. In addition, the spectrum of the mixed gel is characteristic of a viscoelastic liquid with a decrease of G' at low frequencies. These results could be related to the micro-phase separation process which seemed to occur in these systems as revealed by SANS by the upturn of scattered intensity observed at low q values. Table 2 summarizes the influence of added polysaccharide to the BSA gel characteristics in terms of the storage modulus at 1 Hz at the end of the gelation kinetic at 80°C and after quenching to 25 "C. Table 2. Storage modulus G' (f = 1 Hz) obtained at 80°C (end of kinetic) and 25 °C (mechanical spectrum) for BSA 10% w/w gels and BSAlHEC or BSAlCMC 10/1% w/w mixed gels formed at pH 5.2 and pH 7 G' (* 10-3 Pa)

BSA

BSAlHEC

BSAlCMC

T=80°C

pH 5.2

18.1

15.1

60.1

T=25°C

pH7 pH 5.2

73.1 38.6

124.2 30.6

196.6 136.7

pH7

124

262.3

572.6

These results clearly demonstrate that an increase of the viscoelastic behaviour of globular protein gels can be obtained by addition of polysaccharide and more particularly of a polyion. The reinforcement of the structures comes from a non-specific interaction between the polysaccharide chains and the globular protein surface (maybe of electrostatic type). Further experiments would be needed in order to understand clearly the nature of these interactions.

REFERENCES 1. E. Dickinson and K. Pawlowsky, in Gums and Stabilisers for the Food 1ndustry, G.O. Phillips, P.A. Williams and DJ. Wedlock (eds), Oxford University Press, Oxford, 1996, 8, 181. 2. S.B. Ross-Murphy (ed.) Physical Techniques for the Study of Food Biopolymers, Chapman & Hall, London, 1994. 3. E. Dickinson (ed.) New Physico-Chemical Techniques for the Characterization of Complex Food Systems, Chapman & Hall, London, 1995. 4. E. Dickinson and DJ. McClements (eds) Advances in Food Colloids, Chapman & Hall, London, 1995. 5. V.Ya. Grinberg and V.B. Tolstoguzov, Food Hydrocoll., 1997,11,145. 6. S.E. Harding, S.E. Hill and 1.R. Mitchell (eds) Biopolymer Mixtures, Nottingham University Press, 1995.

Functional Interactions in Mixed Biopolymer Systems

20 1

7. C.M. Durrani and A.M. Donald, Macromolecules, 1994,27, 1 10. 8. C. Castelain, J.L. Doublier and J. Lefebvre, Carbohydr. Polym., 1987,7, 1. 9. W. Brown, D. Henley and J. Ohman, Makromol. Chem., 1963,64,49. 10. W. Brown and D. Henley, Makromol. Chem., 1964,79,68. 11. D. Renard, F. BouC and J. Lefebvre, in Food Colloids - Proteins, Lipids and Polysaccharides, E. Dickinson and B. BergenstAhl (eds), Royal Society of Chemistry, Cambridge, 1997,303. 12. P.G. de Gennes, Scaling Concepts in Polymer Physics, Cornell University Press, Ithaca, New York, 1979. 13. D. Renard, F. BouC and J. Lefebvre, Physica B, 1997, in press. 14. N.L. Abbott, D. Blankschtein and T.A. Hatton, Macromolecules, 1992,25,5192. 15. J.M. Park, B.B. Muhoberac, P.L. Dubin and J.Xia, Macromolecules, 1992,25,290. 16. D. Renard, M.A.V. Axelos and J. Lefebvre, in Food Macromolecules and Colloids, E. Dickinson and D. Lorient (eds), Royal Society of Chemistry, Cambridge, 1995,390. 17. D. Renard, M.A.V. Axelos, F. Bout5 and J. Lefebvre, Biopolymers, 1996, 39, 149. 18. J-C. Gimel, D. Durand and T. Nicolai, Macromolecules, 1994,27,583. 19. D.V. Zasypkin, E.E. Braudo and V.B. Tolstoguzov, Food Hydrocoll., 1997,11, 159. 20. R.H. Tromp, A.R. Rennie and R.A.L. Jones, Macromolecules, 1995,28,4129. 21. D. Renard, M.A.V. Axelos, F. BouB and J. Lefebvre, J. Chim. Phys., 1996,93,998. 22. P. Meakin, Phys. Rev. Lett., 1983,51, 1119. 23. N. Matsudomi, K. Tomonobu, E. Moriyoshi and C. Hasegawa, J. Agric. Food Chem., 1997,45,546.

INFLUENCE OF MILK PROTEINS ON THE GEL TRANSITION TEMPERATURE AND THE MECHANICAL PROPERTIES OF WEAK K-CARRAGEENAN GELS

A. Tziboula & D.S. Home Hannah Research Institute Ayr, KA6 5HL,UK

ABSTRACT The gelation of milk protein-carrageenan mixtures was investigated using oscillatory techniques. In the presence of milk proteins gelation occurs at relatively low Kcarrageenan concentrations and temperatures below the carrageenan helix-to-coil transition temperature. From work to date it appears the carrageenans interact speciflcalb with K-casein to form a complex but evidence for a similar interaction between K-carrageenan and micellar caseins in milk is less clear-cut. In this p p e r we focus on the importance of milk proteins on gelation at very low K-carrageenan concentrations (i.e. below 0.02%). At the same carrageenan levels gelation was also evident in the absence of milkproteins, i.e. in milk ultrafiltrate. It was alsofound that the cooling procedure of the carrageenan mixtures influenced the gelation temperature and jinal gel strength. Rapid cooling rates slowed the rate of conformational transition of carrageenan and it took longer to achieve critical levels where gelation could proceed. This effect was a finction of the milk componentspresent in the mixture. With entire milk the conformational transition was rapid and spontaneous aggregation occurred; with milk ultraflltrate there was a slow transition which was greatly influenced by the cooling procedure, followed ultimately by gelation. In the presence of serum proteins the effect was similar to that of entire milk. Overall, the paths of gel formation differed in the milk ultrajiltrate-, milk-, or serum-, carrageenan mixtures. These results led to the conclusion that milk proteins intedere with gel formation possibb via electrostatic interactions between carrageenan molecules and K-casein on the sudace of the micelles. A synergistic effect has also been observed between carrageenan and serum proteins which resulted in much stronger gels. Thus a specific interaction between serum proteins and carrageenan cannot be ruled out. 1 INTRODUCTION

Amongst the most important industrial applications of carrageenans is their use as thickeners and stabilisers in milk products. The carrageenans are linear polymers of alternating a-1,4and p-1,3 linked anhydrogalactose residues with varying amounts (1040%) of sulphate half ester groups. In aqueous solution and in the presence of cations

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203

carrageenans form thermoreversiblegels. When aqueous dispersions of carrageenans are heated at temperatures above 6OoC, the polymer hydrates and adopts a random coil conformation. Gelation occurs on cooling at a critical temperature, the sol-gel transition temperaturqand is the result of a two-stage reaction involving helix formation followed by aggregation (1-6). The carrageenan fbnctionality in milk has been ascribed to an electrostatic interaction with K-casein to form a complex which aggregates into a threedimensional network (7-10). Although such an interaction is possible between carrageenans and isolated K-casein, evidence for a similar interaction between native casein micelles and carrageenans is not available. Research was initiated in our laboratory to study carrageenan-milk protein interactions using rheological measurements. Using low frequency oscillation at the lowest applicable stress is a favourable technique for in situ studies of the kinetics of gel formation because it causes minimal structure damage during the gelling process. Carrageenan gelation in the presence of milk proteins is complex and multifactorial (1 1). It was shown that the relative importance of milk proteins to gelation varied with carrageenan concentration. At levels of carrageenan greater than 0.1% gelation was the result of association of K-carrageenan helices to produce self-supporting gels. The mechanism of gelation and mechanical properties of the gels were influenced mainly by the concentration of carrageenan, ionic environment and pH. At these levels of carrageenan, milk proteins had little effect on gelation and any interactions between carrageenan and milk proteins were masked by the predominating carrageenad carrageenan interactions (1 1). It was obvious that, in order to study interactions between carrageenan and milk proteins, the carrageenan concentration had to be reduced to the minimum levels required for gelation. These levels were found to differ with the type of milk protein present in the mixtures. Lower carrageenan concentrations were required for gelation in the presence of serum proteins than in milk ultrafiltrate and even higher carrageenan amounts were required for gelation in the presence of casein micelles (12). A second full factorial design was employed to study the relative importance of the milk proteins to gelation at carrageenan levels below 0.018% w/w. It was found that at a given ionic strength doubling the milk protein content suppressed the gelation temperature or prevented gelation. This effect was more pronounced with the micellar caseins whilst whey proteins were effective only at low carrageenan concentration (ie 0.012% w/w). When gelation did occur, the gel transition temperature was negatively correlated with the protein level (11). However, in this work the path of gel development was followed during cooling from 6O-2O0C.Examination of the rheological profiles revealed that in some cases gelation would have occurred, had the mixtures been cooled to lower temperatures. Preliminary work also showed that whilst the rate of cooling of the mixtures during the rheological test had little effect on the gelation temperature at high carrageenan or ion concentrations, it became important at low carrageenan and ion concentrations. In this paper we examine the influence of the cooling procedure of the carrageenanmilk mixtures, at low carrageenan concentrations and constant ionic strength, on the kinetics of gelation and the mechanical properties of the gels.

Gums and Stabilisers for the Food Industry

204

2 MATERIALS AND METHODS

2.1 Milk Fractions. Bulk milk from the Institute herd was skimmed by centrihgation at 1200g for 30 minutes at 4OC (Mistral 6000 centrifuge; Sanyo Gallenkamp plc., Leicester, UK). From the skimmed milk the following fractions were prepared: i) Skim milk ultrafiltrate (UF) using a pressure filter equipped with a membrane of molecular weight cut-off 10 kDa (Amicon Ltd, Danvers, MA). ii) Serum fractions from skim milk by centrihgation at 43000g for 120 minutes (Sorval RC 5B; Du Pont Instruments, Newtown, CT. The supernatant layer obtained during the preparation of micellar caseins was designated as the serum fraction. This comprised mainly the serum protein, soluble casein (15-20% total casein) and the soluble phase of milk. The serum fraction was hrther purified by isoelectric precipitation of the soluble caseins at pH 4.6 followed by exhaustive dialysis against bulk milk for 48hrs (serum to milk ratio 1:250, two milk changes) to permit ionic equilibria to be attained. The final pH of the serum fraction was 6.75-6.80. 2.2 K-carrageenan: K-Carrageenan from Euchema cottonoii was converted to the sodium form using cation exchange resin (Amberlite IR120 cation exchange resin in the Na form, BDH Ltd, Poole, UK). The polysaccharide was recovered by filtration and freeze dried. The extent of conversion of the carrageenan to the sodium form was evaluated using atomic absorption spectroscopy. The sodium content was estimated to be 5.67% WIW. 2.3 Milk Protein-CarrageenanMixtures: K-carrageenan was added to the appropriate milk fraction while stirring at room temperature. The mixture was heated to 7OoC for 20 min to hydrate the carrageenan and then transferred to the rheometer 2.4 Rheologzcal studies: Low frequency oscillation ( O . O S H z , applied stress 5mPa) was performed on a controlled stress rheometer (CVO; Bohlin Instruments, Gloucestershire, UK), using the double gap measuring geometry. Although the instrument calculates the phase angle (6) at which the strain response lags behind the applied stress, allowing the calculation of storage modulus (G), viscous modulus (G') and complex viscosity (q*), only the complex modulus (G*)is considered here A temperature gradient from 6O-1O0C, at various cooling rates was employed during the oscillation test. The gelation temperature ofthe mixtures was taken as the point where tan 6 = l . 3 RESULTS

AND DISCUSSION

3.1 Influence of cooling rate on the gelation of K-carrageenan.

Carrageenan was added to either milk or milk ultrafiltrate at concentrations 0.014 & 0.017% and in serum at 0.008% wlw respectively . A lower carrageenan level was used in serum because it was found that serum-carrageenan gels were much stronger than milkor UF- carrageenan gels (12). The carrageenan concentration of 0.008% was chosen because it produced gels of comparable strength to those obtained at the higher levels of carrageenan (ie 0.014- 0.017% w/w) employed in milk or milk ultrafiltrate mixtures. The gelation process was followed as the carrageenan mixtures were cooled from 60 OC to 10 0 C at cooling rates of 1.30, 0.26, 0.13, 0.065 C I minute.

205

Functional Interactions in Mixed Biopolymer Systems

Typical rheological profiles of the logarithm of G* plotted against temperature in UF, milk, or serum fraction are shown in Figure 1. The arrows mark the gelation temperature (GT) where the phase angle is 45'; at this point the contributions to structure from the viscous and elastic components became equal (tan 6= G"/G=l). Cooling procedure influenced the gelation temperature of the mixtures. The faster the cooling rate during the oscillation test the lower the GT. This effect was more pronounced in milk ultrafiltrate than in milk. The rate of cooling also influenced the gelation temperature in serum, albeit 2.5

1.5

0.5 n

a"

2.0

E

"'\-

+ 0.017% K-carrageenan Milk

a

v

*

0 en

1.0

0

I4

0 .o I

3.O

I

Serum + 0.008% K-carrageenan

2 .o 1.o 0 .o I

10

20

I

30

I

40

I

50

I

60

Temperature (OC) Figure 1. Plots of complex shear modulus (G*) development during cooling of UF-, milk-, serum- carrageenan mixtures from 60-lO0C; Cooling rates: a, 1.3; b, 0.26; c, 0.13; d, 0.065 'Clminute respectively. The arrows mark the gelation temperature (GT).

Gums and Stabilisers for the Food Industry

206

at lower carrageenan concentrations. In fact a rapid rate of cooling inhibited gelation in the presence of serum proteins (Figure 1, serum; curve a). Figure 2 illustrates the effect of cooling procedure on GT; plots are the mean gelation temperatures for the two levels of carrageenan studied in milk and milk ultrafiltrate. For example, at the cooling rate 1.3 OC /minute there was a difference of 15 degrees in the gelation temperatures observed between 40 milk and milk ultrafiltrate (Figure 2). This difference became smaller the slower the 30 cooling rate of the mixtures until at the slowest rate of cooling the gelation temperature was identical in both media, around a0 36OC. These results emphasise the complexity of the carrageenanmilk protein interactions and the lo need to optimise experimental Qo 05 LO 15 conditions in order to make meaninghl comparisons and Rate of cooling (OCIminute) conclusions. The shapes of the gelation curves in the different media Figure 2. EfJ'ect of cooling rate on the gelation were clearly different, indicative temperature of carrageenan mixtures. Mean values of different gelation paths for 0.014 and 0.017 % w h carrageenan in: depending on the composition of milk; - - - - - milk ultrafiltrate. Bars indicate the aqueous phase of the system standard deviation (SO). (i.e. presence of serum proteins and casein micelles). Furthermore, in all cases there was an- early rapid increask in the complex modulus (G*) in the range 39-36OC, designated (T,), which did not always correspond to the rheological GT. The gelation of K-carrageenan is a two-stage process involving helix formation followed by aggregation and is influenced primarily by the ionic environment (ie the amount, valency and the identity of the ions present). Nevertheless, for any ionic form of the carrageenan the inverse of coil-to-helix transition temperature is a linear hnction of the total cation concentration CT (c~=c.+yc~).where c, is the concentration of cations in the system, c, is the concentration of counterions originating from the carrageenan salt and y is a mean activity coefficient to account for the electrostatic attraction between the counterions and the carrageenan molecules (15 , 16). Milk proteins having an overall negative charge also contributed to a hrther lowering of y. For the carrageenan concentration studied here, CT was fixed at levels equivalent to that of skim milk hence coil-helix transition temperature was also invariant under the conditions employed. As the initial inflexion in G* occurred at similar temperatures in all systems studied it was reasonable to suggest that this was the point of the conformational transition of the carrageenan molecules (T,). It was also noteworthy that T, was observed even when rheological GT was not attained (as was the case with the carrageenan-serum mixtures at the cooling rate .l.3°C/minute). Comparison of the

-

Functional Interactions in Mixed Biopolyrner Systems

207

progress in the development of G* showed that in milk ultrafiltrate the gelation of carrageenan took place in two distinct stages (Figure 1, UF).After the initial increase in G* there was a temperature range during which G* increased at a slower rate. This lag phase was the result of either a slow gradual conformational transition of the carrageenan molecules (1S), or of cation mediated aggregation at supermolecular level (4). It is likely that at rapid cooling rates as the mixtures pass too quickly through Tt, , the rate of the conformational transition of carrageenan is slowed and it takes longer to achieve critical levels where gelation can proceed. Beyond this, there was a second sharp inflexion in the growth of the shear modulus. This point corresponded to the GT (indicated by the arrows in Figure l), where G=G". The temperature differential (AT) between the two inflexions on the G* progress increased with the cooling rate of the mixtures and was linearly related to the square root of the cooling rate (Figure 3). In UF, AT was minimal (approximately 3OC) at the slowest rates of cooling and maximum (approximately 23OC) at the fastest. In milk Tt, coincided with GT irrespective of the cooling rate (Figure 1; milk); the transition temperature was sharper indicating faster conformational transitions and the elimination of lag phase indicated a spontaneous initiation of aggregation. The differences in the shape of the G* development profile showed clearly that milk proteins altered the path of gel formation possibly via an interaction between K-casein on the micellar surface and carrageenan molecules. Finally, in serumcarrageenan mixtures Tt, occurred Figure 3. Temperature dflerential between Ksharply at temperatures between 36-39OC which again differed from carrageenan conformational transition temperature (Ttr) and rheological gelation the rheological GT. In the presence of serum proteins the initial temperature (GT), in milk ultrajiltrateconformational change of the carrageenan mixtures;0.014%; -, carrageenan was accompanied by 0.017% w h carrageenan respectively, fast increase in structure in the sample. At the gelation point where the contributions to the mechanical properties of the gels from the viscous and elastic components were equal, there was a decrease in the growth of the shear modulus. This decrease extended over a narrow temperature range and was possibly the result of structural damage because of the weak nature of the network formed. Yet again the shape of the curve differed from that of the UF-carrageenan or milk-carrageenan mixtures. Carrageenan formed stronger gels when serum proteins were included; even at the lower carrageenan concentration in serum, gels were stronger than in the milk- or UFcarrageenan mixtures. These results were consistent with previous observations over a

208

Gums and Stabilisers for the Food Industry

range of carrageenan concentrations and suggested a synergistic behaviour between carrageenan and the serum proteins (12). Furthermore, the final gel strength was inversely related to the rate of cooling of the mixtures. The effect of cooling rate on the G* at 10°C for the two levels of carrageenan studied in milk or UF is shown on Figure 4 . Decreasing the cooling rate from 1.3 to 0.065 0 Clminute resulted in a fourfold increase in G*. Similar findings have been previously reported by Hermansson et al. who found large effects of the cooling procedure on the observed G of K-carrageenan gels. For a 1% sample in 0.1M KCl the value of G’ obtained at a cooling rate of O.SoC/rninute was found to be three times larger than that obtained at 1.S°C/minute (16). These results indicate that the effect of cooling rate on GT and G* development were merely reflecting the time-dependent progress of the aggregation reaction. The presence of milk proteins clearly influences the 0.0 0.5 1.0 1.5 aggregation mechanism perhaps by providing the substrate on Cooling rate (OC/minute) which aggregation can readily proceed. Figure 4. Plots of the mean G* at 10°C for the In studies of rennet gelation dvferent rates of cooling during carrageenan Home (17, 18) has shown that gelation in milk or milk ultrajltrate. Data are mean the gelation profiles show a values for 0.014 and 0.017% carrageenan in: -------scaling behaviour involving only milk; milk ultrajiltrate. Bars indicate k SD two independent parameters, gel strength at infinite time and gelation time. When we tried to apply the same approach to the carrageenan gels we found that the G* curves were ordered in a parallel fashion according to their individual rate of cooling. Replotting each profile as a function of its own reduced time scale t/tg where t, = gelation time we obtained a series of curves apparently ordered according to their gel strength which again was a hnction of the cooling procedure (Figure 6.1). The similarity of the shapes of the curves suggested that the shear modulus axis could be normalised against its individual value at tit, = 1.3, an arbitrarily chosen value of reduced time. We see that within the time scale of these experiments, the individual gelation profiles reduce to a master curve (Figure 6.2). The curves of gel strength development during carrageenan gelation are therefore demonstrating a simple scaling behaviour. This scaling behaviour was the same irrespective of the effect of the cooling procedure of the mixtures. Scaling behaviour was observed in all three media studied, namely UF-, milk- and serumcarrageenan mixtures (results not shown). However, the slopes of the normalised curves differed in the presence of micellar caseins or serum proteins because milk proteins interfere with gel formation. ~

Functional Interactions in Mixed Biopolymer Systems

'2 0•10

209

c

~ -Ie

~ 0.05 0.00

...J

t::.:~==::::%:===~L-

o

5

10

15

Time (hrs) Figure 5. Gel firming kinetics of UF-carrageenan mixtures at different cooling rates; Level of x-carrageenan 0.014% wlw; Cooling rates: a, 1.3; b. 0.26; c, 0.13; d; 0.065 °C/minute respectively.

d

(1)

~ 0.10 -Ie

~

0.05

::l 3.0

...

(2)

-Ie

~ 2.0

~ ~

1.0 0.0

o

0.5

1.0

1.5

Reduced time tltg Figure 6. (1) The data of Figure 5 replotted as a junction of reduced time (t/tg), where tg is the gelation time of the individual run; (2) G* for each curve normalised to the value of the modulus attained at reduced time t/tg = 1.3 on that curve.

Gums and Stabilisers for the Food Industry

210

The presence of scaling behaviour has several implications. It suggests that the growth of gel strength is a function of two independent parameters, G* at infinite time and gelation time itself Selectingthe rate of firming as a parameter to characterise the reaction is of little value since the rate of firming depends on both ultimate gel strength and t,. 4 CONCLUSIONS

These studies have shown that the cooling procedure influenced the gelation temperature of carrageenan-milk protein mixtures. Rapid cooling rates slowed the rate of the coil-tohelix transition of carrageenan; consequently it took longer to achieve critical levels where gelation could proceed. This effect was a hnction of the milk components present in the mixture. With entire milk there was a marginal effect; with milk ultrafiltrate there was a slow transition before gelation occurred; with serum proteins the effect of cooling rate was similar to milk. It is possible that milk proteins assist gelation by forming a template on which aggregation can proceed. Irrespective of the rate of cooling, the shear modulus exhibited a simple scaling behaviour. The different rates of cooling simply influence the rate of conformational transition of carrageenan and hence further aggregation. When there is sufficient time for network formation to proceed then the observed differences in GTs are small. When log G* was plotted against temperature, it was revealed that the path of gel formation was different in the UF-, milk-, or serum- carrageenan mixtures. Furthermore, serum-carrageenangels had significantly higher shear modulus. These results indicate that milk proteins interfere with gel formation. A synergistic effect has been also observed between carrageenan and serum proteins which resulted in much stronger gels, and a specific interaction between serum proteins and carrageenan cannot be ruled out. Acknowledgements

The authors wish to thank D. Lammerhirt and S . Ziegler for their technical assistance. This research was funded by the Scottish Office Agriculture, Environment and Fisheries Department. References 1. D. A. Rees, British FoodMamfacturing Industries Research, Symposium Proceedings No 13. London, 1972,7 2. E.R. Moms, D. A. Rees, G. Robinson,J. Mol. Biol., 1980, 138, 349. 3. I.T. Norton, M.D. Goodall, E.R. Moms, D. A. Rees, J. Chem. Soc.FarTrans., 1983, 79, 2501. 4. A.M. Hermansson, Curbohyak Polym., 1989, 10, 163. 5. J. Sugiyama, C. Rochas, T., Turquois, F. Taravel, H. Chanzy, Carbohydr.Polym., 1994,23,26 1. 6. J. Borgstrom, L. Piculell, C. Viebke, Y . Talmon, Int. J. Biol, Macromol., 1996, 18, 223. 7. J. Grinrod, T.A. Nickerson, J. Dairy Sci., 1968, 51, 834. 8. T.A.J. Payens, J. DairySci., 1972,51, 141.

Functional Interactions in Mixed Biopolymer Systems

211

9. T.H.M. Snoeren, PhD Thesis, Nederlands Institute voor Zuivelondenoek, Ede, The Netherlands 1976. 10. T.H.M. Snoeren, T.A.J. Payens, J. Jeurnink, P. Both, Neth. Milk Duiry J., 1975, 30, 132. 1 1. D. D. Drohan, A. Tziboula, D. McNulty, D.S.Home, Food H+ocoll., 1997, 11, 101. 12. A. Tziboula & D.S. Home, HRI Symposium 1997, Caseins & Caseinutes:Structures, Networks and Interactions, Prmeedngs, 1997, (to be published). 13. D.A.Rees A&. Curbohyak Chem. Biochem., 1969, 21,267. 14. C. Rochas and M. Rinaudo, 1980, Biopolymers, 19,2165. 15. L. Picullel, Food Sci. Technol. 1995, 67,205. 16. A.-M. Hermansson, E. Emksson, E. Jordansson, Curbohydr.Polym., 1991,16, 297. 17. D.S Home, J. Chim. Phys. 1996, 93, 977. 18. D.S Home, “Food Macromolecules and Colloids”, (E. Dickinson and D. Lorient, eds), Royal Society of Chemisty, 1995, p. 456.

MILK GELS WITH LOW METHOXYL PECTINS

David Oakenfull and Alan Scott Food Ingredients Program, CSIRO Division of Food Science and Technology, P.O.Box 52, North Ryde, NSW 2113, and Hydrocolloids Research, 195 Fox Valley Road, Wahroonga, NSW 2076, AUSTRALIA

1 ABSTRACT

Low methoxyl pectins can form gels when added to milk. Presumably gelation occurs through the calcium-ions present in the milk - in free solution and associated with casein micelles. But the mechanism remains unclear, and casein, and other proteins, may be active in the gelation process. Gels were formed by mixing skimmed milk powder (SMP) and a low methoxyl pectin (26% DE). Gel strength was measured as the absolute shear modulus (G) at 15°C. With fixed concentrations of SMP (5, 10 and 15% w/w), G increased with the square of the pectin concentration; with a fixed concentration of pectin (1 96 w/w), G increased linearly with increasing concentration of SMP (3-1596 wlw). In a second type of experiment, pectin (1% w/w) was sealed in dialysis tubing and equilibrated with different concentrations of SMP (2-1596 w/w). Under these conditions, the gel strength was greater than when the components were intimately mixed. Thus there is no direct interaction between pectin and casein micelles, analogous to the interaction between K-carrageenan and casein micelles. The mixed gel appears to consist of a LM pectin matrix in which the milk protein is embedded - but the mixed gel is weaker than would be expected from the shear modulus of gels formed from the LM pectin alone, probably a consequence of thermodynamic incompatibility between the protein and the poly saccharide. 2 INTRODUCTION Pectins are a class of polysaccharides with structures based on 1,4-linked a-Dgalacturonic acid, interrupted by L-rhamnose residues carrying some neutral sugars as side chains (mainly &galactose and L-arabinose) - the detailed structure depending on the plant source and the extraction procedure.' The carboxylic acid groups are partly esterified with methanol and the degree of esterification WE) varies with the age and type of plant tissue from which the material was extracted. The gelling characteristics depend on the DE, and for this purpose pectins can be divided into two classes - high methoxyl (HM) pectins with degrees of esterification typically within the range 5580% and low methoxyl (LM) pectins with less than 50% esterification. HM pectins form gels at low pH (below about 3.6) when a cosolute is present (typically sucrose at

Functional Interactions in Mixed Biopolymer Systems

213

a concentration of greater than 55% by weight);* LM pectins, with more free carboxylic acid groups, form gels only in the presence of calci~m-ions,~ as shown in Figure 1. H H

Figure 1

nK negg-boxnmade1 for junction wnes in gels of LM pectin. The linkage geometry introduces a buckled sheet structure to the polysacchnde chaim and the junction wnes are stabilised by calcium ions (e). l?w upper structure shows an approximation to the chelation of a calcium-ion by a single chain.

LM pectins can also form gels when added to milk.' Whole milk typically contains about 13 mM total calcium,' much of which, as calcium phosphate, is intimately involved with the structure and stabilisation of casein micelles.' However, some of this calcium is also present in free solution and Augustin and Clarke' have found that the activity of calcium-ions in reconstituted dried skim milk (9% total solids) ranges from 0.6 to 2 mM (depending primarily on pH). Thus it is not unreasonable to suppose that in milk, gelation of LM pectins occurs through the calcium-ions naturally present.' But the mechanism remains unclear, and casein, and other proteins, may be active in the gelation process. The possibility of direct interaction between low methoxyl pectins and casein is suggested by the fact that these pectins stabilise the protein in low-pH milk systems'" and that carrageenans, which are also negatively charged polysaccharides, associate strongly with casein micelles.' 3 EXPERIMENTAL Low heat skimmed milk powder (SMP) and the LM pectin (Sanofi 'pectin red 3g'; 18-3096 DE) were analysed for calcium, sodium and potassium by ICP-AES (carried out by the CSIRO Division of Exploration and Mining) with the results given in Table 1. The pectin's DE was estimated by titrimetry" and found to be 26%. The LM pectin and skim milk powder were separately dissolved in deionised water. Appropriate mixtures were sealed in glass tubes and held at 15°C for 18 hours. The gel strength was then measured as the absolute shear modulus (G) by the method of Oakenfull, Parker and Tanner." Gels were formed from the LM pectin in the absence of SMP by slowly injecting aqueous calcium chloride (0.1 M) into a hot solution of the pectin (1% w/w; 90OC). The solutions were then held at 15°C for 18 hours and the shear modulus measured as described previously.

214

Gums and Stabilisers for the Food Industry

Table 1

Metal ions present in skim milk powder and LM pectin (mg/g) as determined by ICP-LES Ca

Na

K

Skim milk powder

13.9

4.4

17.5

LM pectin

1.3

29

1.4

Gels were also formed from LM pectin (1 96 w/w) sealed in dialysis tubing and equilibrated with different concentrations of SMP (2-1596 w/w) at 15°C for 72 hours (a small concentration of sodium azide was added to prevent microbiological activity). The gels formed in the dialysis tubing were transferred to glass tubes for measurement of G. Calcium-ion activities were measured with a Radiometer ion-selective electrode standardised against calcium chloride (10 and 0.1 mM). Activity coefficients were calculated from the Debye-Hiickel equation.I2 4 RESULTS AND DISCUSSION 4.1 Gelation of

LM Pectin with Calcium Chloride

Gels were formed from a fixed concentration of the LM pectin (1% w/w) by adding different levels of calcium as calcium chloride. We found the shear modulus (G) to increase with the activity of added calcium-ion (2-10 mM) as shown in Figure 2. The calcium-ion activities shown were calculated from analytical concentrations of calcium-ions; the equilibrium activities after gelation would be lower from loss of frec calcium by chelation to the pectin. On this basis, and within the range of concentfations shown, G appears to increase with the square of the calcium-ion activity.

1000

9

0

5

10

calcium ion activity (mM) Figure 2

Shear moduIus 0)of gek formed from a jixed concentratjon of LM pectin (1% whv) with added calcium chlori& at 15°C. Calcium concentration is plotted as the calcium-ion activity.

215

Functional Interactions in Mixed Biopolymer Systems

4.2 Gelation of LM Pectin witb S M P

4.2.1 Fixed Concenrretions of SMP. With fixed concentrations of S M P (5, 10 and 15% w/w), the shear modulus increased with the concentration of pectin (c), as shown in Figure 3. (To a good approximation, G increased with 2,typical of polysaccharide gels at relatively low concentration^.^^)

0

5

10

pectin (% w/w) Figure 3

(G) of gels fonned at 15°C with LM pectin and SMP added at 10% (A)and 15% @) (wh).

Shear modJus

4.2.2 Fixed Concentration of LM Pectin. With a tixed concentration of pectin (1% w/w), the shear modulus increased linearly with increasing concentration of S M P (3-1596 w/w), as shown in Figure 4. The calcium-ion activity in solutions of S M P is "buffered" and varies little between 5 and 20% SMP. For example, Figure 5 shows Augustin and Clarke's measurements of calcium-ion activity in two S M P solutions (9% and 19.6%) at different P H . ~The calcium-ion activity is sensitive to pH, but not to the concentration of SMP. Thus the strength of the gels appears to increase with the magnitude of the total pool of available calcium (most of it attached to casein micelles) rather than the activity of free calcium-ions. For comparison, Figure 4 includes data showing the effect of S M P on the shear modulus of gels of 5% (w/w) gelatin. The modulus increases very little with concentration of S M P compared with the corresponding results for LM pectin. Thus filler reinforcement by the casein micelles" and any modulus-enhancing effect of the lactose" (that constitutes about 52% of the S W ) are unlikely to be making significant contributions to the strength of the LM pectin gels.

4.2.3 Calcium-Zon Activity Varied by Aausting pH. Augustin and Clarke's data6 (Figure 5) show that the calcium-ion activity in a solution of S M P can be varied by adjusting the pH. In Figure 6, we show the effect of varying the calcium-ion activity in this way on the modulus of gels formed from 1% (w/w) pectin and 5% (w/w) SMP. The pH was varied between 6 and 7 by adding very small amounts of either 1M hydrochloric acid or 1M sodium hydroxide. The modulus appears to increase linearly with calcium-ion activity.

216

Gums and Stabilisers for the Food Industry

500

I

I

n

0

4

8

12

16

SMP (% w/w) Figure 4

Efect of SMP on the shear modulus of LM pectin gels (1% w/w; A) and gelatin gels (5% w h ; 0 ) at 15°C.

6.00

6.50

7.00

PH Figure 5

Calcium-ion activity in two solutions of SMP (9% A; 19.6% H) with natural and adjusted pH, measured at 25°C. (From Augustin and Clarke.').

This result can be explained in principle by a simple mass-action approach. The classical theory of rubber elasticity gives the following relationship between the shear modulus (G) and the concentration of the gel-forming polymer ( c ) : ' ~

217

Functional Interactions in Mixed Biopolymer Systems

RTc G =Mc

where R is the gas constant, T the absolute temperature and M, the number average molecular weight of the “active chains“, i.e. the segments of polymer chain joining adjacent cross-links. Neglecting non-random distribution of free carboxylic groups along the polysaccharide chain, and disruptions to the regular structure by rhamnose residues, M, is proportional to the number of unoccupied binding sites (in moledlitre) for calcium-ions on the polysaccharide ([d).Neglecting any Cooperativity,” and assuming that there is a single association constant (KA)for calcium-ions binding to the polysaccharide chain, gives the relationship:

where [Ca.B] is the number of occupied binding sites. Mass balance then gives the relationship:

where [B], is the total number of binding sites (proportional to the concentration of polysaccharide). Thus equations (1) and (3) indicate that G should increase linearly 2 OOO 1 mol-’. with calcium-ion activity, and from the data shown in Figure 6, KA

-

600 *.---

n

a

a

*.-*

400 .

+..-A’ ..-*

W

c!J

200

A

A

..--I

$-&’* ..--

A

:/+-

calcium ion activity (mM) Figure 6

Efect of calcium-ion activity (vaned by adjusmru of pH) on the shear modulus (G) of gels f o m d by LM pectin (1 % w h ) and SMP 5 % (wh) at 15°C.

Gums and Stabilisers for the Food Industry

218

4.3 Gelation of LM Pectin with S M P Separated by a Dialysis Membrane In a second type of experiment, LM pectin (1 96 w/w) was sealed in dialysis tubing and equilibrated with different concentrations of S M P (2-1596 w/w). The LM pectin formed gels within the dialysis membrane. As only small molecules and ions can pass through the membrane, gehtion presumably occurs through wcapture" of calcium-ions from the S M P . Solutions of S M P have a small equilibrium concentration of free calcium-ions, as shown in Figure 4. These ions are free to diffuse through the membrane and bind to pectin; equilibrium is restored by loss of calcium from the w i n miwlles. Figure 7 shows the shear modulus of gels prepared by dialysis compared with gels prepared under equivalent conditions, but with the components intimately mixed. The gel strength was always greater when the pectin and SMP were separated by a dialysis membrane.

n

(d

a

(3

300 0 0

5

10

15

SMP (%) -re

7

Shear modulus (G)of gels formed at lS°Cfrom LM pectin (1 96 w/w) dialysed against SMP @) compared with gels prepared under equivalent conditions, but with the components intimately mixed (A).

The results shown in Figure 7 suggest that there is interaction between the protein and polysaccharide in the mixed system - but one that weakens the gel. Proteins and polysaccharides are frequently thermodynamically incompatible. l8 Mixtures can form two phases, one rich in protein and the other rich in plysaccharide, and such behaviour has been reported for mixtures of HM pectin and SMP." The greater electrostatic charge of LM pectin would be expected to further enhance thermodynamic incompatibility.1s The shear modulus for such two-phase systems can be calculated from the polymer blending laws and the modulus of the composite must always lie between -the moduli of the separate 'component~.''.~ For a LM pectin-rich gel matrix in which is embedded a much softer (probably liquid)

219

Functional Interactions in Mixed Biopolymer Systems

protein-rich phase, the isostrain model is appropriate." The modulus of the composite (4.3 is then related to the moduli of the two components (GIand GJ by the equation: CI.2 =

4G

+

42Gr

(4)

where 4 and are the respective volume fractions of the two phases. This model accounts qualitatitively for the results shown in Figure 7. As the concentration of S M P increases, the volume fraction of the protein-rich phase must increase. Thus the greater the concentration of SMP, the greater the divergence between the modulus of gels prepared by dialysis and the modulus of gels prepared from the equivalent mixture of LM pectin and S M P , as seen in Figure 7.

(a

4.4 Gelation of LM Pectin with S M P Compared with r-carrage-enan

It is instructive to compare the gels formed from LM pectin and S M P with the gels formed from carrageenan and S M P under equivalent conditions. ~-eenan interacts strongly with casein,9 specifically forming a stable complex with casein?'*^^ It is believed that in rr-carrageenan-milk gels, casein micelles are linked to form a gel network by interaction of free loops and tails of bound carra rag- molecules; below the carrageenan's helixail transition temperature (these free ~ - c a r r a g e e ~ segments link by forming double helices).23 In Figure 8, shear modulus values for gels formed from S M P and LM pectin and are compared with gels formed from S M P and carrageenan under equivalent conditions. rr-carrageenan forms gels at much lower concentrations of polysaccharide than LM pectin - and the rr-carrageenan gels are vastly stronger than the gels formed by LM pectin for the same concentration of polysaccharide. This strongly suggests that there is no direct interaction between the milk proteins and LM pectin, and that the gel network is formed by LM pectin molecules only.

0.00

0.50

1 .oo

polysaccharide (% w/w) Figure 8

Shear modulus (G) of gels fbnncd at 15°C from SMP (5 96 w h ) and either rr-carmgeenan (A)or LM pectin p).

220

Gums and Stabilisers for the Food Industry

5 CONCLUSIONS

There appears to be no direct interaction between LM pectin and casein micelles - as occurs in the gelation of milk with K-carrageenan. The casein appears primarily to be a source of the calcium-ions that enable LM pectin molecules to crosslink and form a gel network. The mixed gel would then consist of a LM pectin matrix in which the milk proteins are embedded. Nonetheless, casein is more than simply a source of calcium-ions. The milk proteins have a strong influence on the gelation process and the mixed gel is weaker than would be expected from the shear modulus of the LM pectin alone. This result can be explained by thermodynamic incompatibility between the protein and the polysaccharide causing some degree of separation into protein-rich and polysaccharide-rich phases - but, as yet, we have no independent evidence for this.

Acknowledgement We thank the Australian Dairy Research and Development Corporation for their financial support and permission to publish this work.

References 1. 2.

3. 4. 5.

6. 7. 8. 9. 10. 11. 12. 13. 14.

C. Rolin, in ‘Industrial Gums’, eds. R.L. Whistler and J.N. BeMiller, Academic Press, San Diego, 1993, Chapter 10, p. 257. D. Oakenfull, in ‘The Chemistry and Technology of Pectin’, ed. R.H. Walter, Academic Press, San Diego, 1991, Chapter 5 , p. 87. M.A.V. Axelos and J.-F. Thibault, in ‘The Chemistry and Technology of Pectin’, ed. R.H. Walter, Academic Press, San Diego, 1991, Chapter 6, p. 109. R.E. Hargrove and J.A. Alford, in ‘Fundamentals of Dairy Chemistry’, eds. B.H. Webb, A.H. Johnson and J.A. Alford, AVI Publishing Co., Westport, 1974, Chapter 2, p. 58. P.F. Fox and D.M. Mulvihill, in ‘Food Gels’, ed. P. Hanis, Elsevier, London, 1990, Chapter 4, p. 121. M.-A. Augustin and P.T. Clarke, J. Dairy Res., 1991, 58, 219. A.C. Hoefler, in ‘The Chemistry and Technology of Pectin’, ed. R.H. Walter, Academic Press, San Diego, 1991, Chapter 3, p. 51. H.C. Ambjerg Pedersen and B.B. Jmgensen, Food Hydrocolloids, 1991, 5 , 323. D.G. Dalgleish and E.R. Morris, Food Hydrocolloids, 1988, 2 , 311. T.H. Schulz, Methodr Carbohydr. G e m , 1965, 5 , 189. D.G. Oakenfull, N.S. Parker and R.I. Tanner, in ‘Gums and Stabilisers for the Food Industry 4’, eds. G.O. Phillips, D.J. Wedlock and P.A. Williams, IRL Press, Oxford, 1988, p.231. R.A. Robinson and R.H. Stokes, ‘Electrolyte Solutions’, Butterworths, London, 1959, p.229. D. Oakenfull, CRC Crit. Rev. Food Sci. Nutr., 1987, 26, 1. S. Ring and G. Stainsby, Prog. Food Nutr. Sci., 1982, 6 , 323.

Functional Interactions in Mixed Biopolymer Systems

15. 16.

221

K. Nishinari, M. Watase, E. Miyoshi, T. Takaya, and D. Oakenfull, Food Technol., 1995 (October), 90. L.R.G. Treloar, ‘The Physics of Rubber Elasticity’, Clarendon Press, Oxford,

1975. 17. 18. 19. 20. 21. 22.

J.F. Thibault and M. Rimaudo, Biopolymers, 1986,25, 455. V.B. Tolstoguzov, in ‘Food Proteins and their Applications’, eds. S. Damodaran and A. Paraf, Marcel Dekker, New York, 1997,p. 171. L.H. Sperling, ‘Introdwrion to Physical Polymer Science’, John Wiley and Sons, New York, 1992,p. 464. A.H. Clark, R.K. Richardson, S.B. Ross-Murphy and J.M. Stubbs, Macromolecules, 1983,16, 1367. J. Grinsrod and T.A. Nickerson, J. Dairy Sci., 1968,51, 834. T.H.M. Snoeren, T.A.J. Payens, J. Jeunink and P. Both, Milchwissenschfl,

1975,30,393. 23.

S.Y. Xu, D.W. Stanley, H.D. Goff, V.J. Davidson and M. Le Maguer, J . Food Sci., 1992, 57, 96.

DEPLETION FLOCCULATION OF CASEIN MICELLES INDUCED BY THE EPS OF A LACTIC ACID BACTERIUM

R. Tuinier” and C.G. de Kruif‘ ‘NIZO, Netherlands Institute for Dairy Research, P.O.Box 20,671 0 BA Ede, The Netherlands. ’Department of Physical and Colloid Chemistry, Wageningen Agricultural University, Dreijenplein 6,6703 HI3 Wageningen, The Netherlands.

Abstract Casein micelles in skim milk become effectively attractive when an exocellular polysaccharide(EPS) produced by Lactococcus lactis subsp. cremoris NIZO B40 is added. This results in a phase separation of the colloidal gas-liquid type.We determined a phase diagram which describes the demixing of EPS and casein micelles in skim milk. Furthermore, dynamic light scattering measurements were made to determine the effect of the EPS concentration on the self-diffusion coefficients of casein micelles in skim milk. This showed that casein micelles become more attractive upon increasing the B40 EPS concentration. From these experiments the Baxter parameter TB was determined. By using the condition for spinodal decomposition we calculated the volume fraction of casein micelles at the spinodal. This results in a phase diagram which agrees quite well with the observed phase boundaries. 1 Introduction

Solutions containing both polysaccharidesand milk proteins may exhibit phase separation

.I, as was reported for example by Antonov et al. [2] and Tolstoguzov [3]. The phenomenon of relevance to dairy products to which polysacchkides are added as a thickener or as a stabilizer. Even more important is the ‘in-situ’ production of exocellular polysaccharides (EPSs) by lactic acid bacteria in products such as yogurt and viilli. The EPS seems to determine the rheological properties typical of these products. The EPSs are responsible for the thread-like behaviour on pouring some types of yogurt. This behaviour is also referred to as ‘long behaviour’ (see van Marle et u1.[5]).In previous studies we have characterized physical properties of an EPS produced by Lactococcus lactis subsp. cremoris NIZO B40, which has a number-averaged molar mass of 1.47.106 g/mol and a number-averagedradius of gyration of 86 nm [4]. The polydispersity index of this polymer ( = M A n ; the ratio of the weight- and number-averaged molar masses respectively)is 1.13 and this polysaccharide has interesting thickening capacities in comparison with other polysaccharides. Furthermore, we analysed the viscosity and the shear thinning behaviour as a h c t i o n of the polymer concentration [4]. The next step was to study the physical properties of systems containing both this B40 EPS and milk proteins.

Functional Interactions in Mixed Biopolymer Systems

223

We chose low-heat skim milk as a milk protein model system, since it is, compared to other commercial dairy products, a relatively simple system. From a colloid physics point of view skim milk can be considered as a dispersion of casein micelles (about 100 nm radius and a volume fraction +ill) in a continuous phase containing water, salts,lactose and small (< 5 nm) globular proteins. The casein micelles are, strictly speaking,association colloids of four different casein proteins and calcium phosphate. These casein micelles are quite stable since one can boil milk or add kitchen salt without inducing flocculation. Adding B40 EPS to skim milk and exceedinga certain concentrationinduces phase separationas will be shown in section 4. When the continuous phase of skim milk is mixed with B40 EPS nothing occurs, except perhaps at very high concentrations.This indicates that the interactionsbetween casein micelles and I340 EPS are responsible for the observed phenomena. The phase separation is caused by the so-called depletion type of interaction. The physics of this can be described as follows. On mixing (spherical) colloidal particles with polymer molecules these polymers are excluded from the surface of the ‘solid’ object by a distance equal to the average radius of the polymer molecule. In the case of sphericalprotein particles, such as casein micelles in milk, this depletion layer is also present when a polysaccharide does not adsorb onto the casein micelle. In the depletion layer the partial osmotic pressure @ of the polymer I )is smaller than in the bulk due to a lower polymer concentration in that layer. Brownian motion of the casein micelles occasionally causes overlap of two depletion layers. This overlap volume (Vovdlap) of two depleted layers increasesthe bulk volume availableto the polymers and thereby decreases the free energy of the system by an amount ll*V,dq The result is that the colloidal particles tend to stick together. One may say they are pushed together by the unbalanced osmotic pressure difference. This kind of flocculation is called depletion flocculation.Asakura and Oosawa [6] were the first to describethe origin of the depletion interactionbetween colloidal particles and non-adsorbing polymers. They stated that the attractive force between the particles is proportional to the osmotic pressure difference between the polymer solution and the solvent. An early example of the practical use of the depletion phenomenon is the concentrationof natural latex by plant polysaccharides as described by Vester [7]. Later, Vrij [8] developed a theoretical model which describes the depletion mechanism. De Hek and Vrij [9,10] observed phase separation in mixtures of polystyrene polymers and colloidal silica spheres and the theory of Vrij [8] describesthe limiting polymer concentration for this system. More recently, Lekkerkerker el ul. [111 and Poon and Pusey [121 extended and refined the theoretical description. Here, we will show that B40 is a non-adsorbing polymer for the casein micelles and induces depletion flocculationin skim milk. In the followingtheoretical section we will explain our approach to describe the experimental results which are presented subsequently. 2 Theory

2.1 Adhesive hard sphere model For skim milk de Kruif [13-15] showed that casein micelles can be treated as hard spheres, under the conditionsrelevant for this study. When hard spheres become sufficientlyattractive a (colloidal) gas-liquid or gas-solid condensation takes place which can be regarded as a phase separation.Casein micelles become attractive when B40 EPS is added to skim milk.

224

Gums and Stabilisers for the Food Industry

A way to simplify the interaction potential profile for adhesive spheres is to use a discrete interaction potential U(r). For mathematical convenience Baxter [ 161 used a square well potential with an infinitely narrow gap (lim A-0, where A is the range of the attraction) which is described in the following equation :

h [ l 2 t B A / ((3 + A)]

0

(3 < r < ((3 + A), ( o + A ) < r,

1

where G equals the sphere diameter, the Baxter parameter TB, the temperature T and Boltzmann’s constant kB. The Baxter parameter is allowed to take values o< oB< 00 and the inverse of TB reflects the strength of the attractive force (adhesiveness) between the spheres. Using Baxter’s approach Watts et al. [171 showed that TB can be directly related to $*, the volume fraction at which spinodal demixing takes place. For a volume fraction (4) lower than 0.12 Penders and Vrij [ 181 simplified the expressions of Watts et al. [ 171 to :

2 For higher 4, the PY theory can be used [181. Equation 2 allows us to calculate the volume fraction at which demixing takes place at a certain adhesiveness. So, when tg is determined the spinodal can be calculated. It must be remarked here that the attractive potential induced by the EPS is certainly not short ranged. The range of the potential, its width, is of the order of the radius of gyration of EPS [8]. Nevertheless we apply the Baxter model since it provides a theoretical framework which allows us to describe the phase separation quantitatively. Experimentally, TB can be measured by means of various experimental techniques. The way TB is related to the self-diffusion of (adhesive) hard sphere suspensions is treated in the next section. 2.2 Self-diffusion De Kruif et al. [ 191 have shown that tg can be determined experimentally by dynamic light scattering. With this technique we will determine the short-time self-diffusion coefficients of casein micelles as a function of the B40 EPS concentration. Self-diffusion is related to the dynamics of a single particle in a system with a homogeneous density. Due to the Brownian motion of colloidal particles the intensity of the scattered light fluctuates. The scattered light intensity auto-correlation function g(t) depends on the decay time t of these fluctuations which are thus coupled to Brownian diffusion. For polydisperse systems ln(g(r)1) can be expanded in a series of cumulants :

1 In[g(z)-l]= In p - r,.t+ --T2.s 2!

2

-...+-

rnTn n!

3

where rl, l-2 and r, are the first-, second- and nth-order cumulants and p is a material constant. Usually distributions that are not too broad are analysed by a second-order cumulant fit. Then from rl(=Q2Ds,with Q the scattering wave vector (Q44xn sin(B/2)/hL, where n is the refractive index, 8 the scattering angle and h the wavelength of the incoming light beam)), the self-diffusion coefficient can be calculated. In dilute colloidal suspensions containing

225

Functional Interactions in Mixed Biopolymer Systems

spherical particles, the hydrodynamic radius of the diffusing sphere aH can be calculated from D, by using the Stokes-Einstein equation : 4

with the solvent viscosity qsand Boltzmann’s constant kg. For a suspension of colloidal hard spheres Batchelor [20] and Felderhof [21] calculated the following relation between D,, the short-time self-diffusion coefficient, and Q : _ Ds - 1- kl4

5

DO where Do is the diffusion coefficient at idinite dilution and kl is a constant which has a positive value.This dependence can be understood since self-diffusion will be hydrodynamicallyhindered more strongly when there are more particles per unit volume. If in the derivation of Eq.(5) it is assumed that the diffusing particle is surrounded by non-adhesive Brownian hard spheres with fixed positions the value of kl=l.832 [20,21]. When relaxation of the surrounding particles is taken into account kl is 2.06 [22]. When the particles become adhesive, the probability of two particles being near to one another is larger than for hard spheres. This will apparently give larger particles resulting in a smaller diffusion coefficient. Cichocki and Felderhof [23] extended the equations of motions of Felderhof to those for adhesive spheres and their result for the diffusion coefficient is :

DS _ -

DO

0.295 1-(1.832+-)+

6

TB

which shows that D, indeed does decrease with increasing adhesiveness (TB becoming smaller). Equation (6) gives us the opportunityto calculate TB by measuring D, for adhesive hard spheres. In a colloidal suspension of skim milk with casein micelles the partial osmotic pressure (n)is larger at higher EPS concentration. This means that at higher EPS concentration the adhesiveness increases when the concentration of non-adsorbing EPS increases. 3 Experimental methods

3.1 Material

- NIZO B40 EPS NIZO B40 EPS was used as described elsewhere [4]. A Lactococcus lactis subsp. cremoris NIZO B40 was used to inoculate a whey permeate medium. After EPS production EPS was isolated using various filtration steps [4]. This isolate was freeze-dried and used as such in this study. - Skim milk Reconstituted skim milk was prepared by mixing 10.45 grams of skim milk powder in 100 grams of demineralized water at 4OOC. The suspension was stirred and kept at this temperature for 45 minutes. Afterwards, the milk was centrifuged at 10000 r.p.m for 20 minutes in order to remove a little undissolved milk powder left in the solution. Considering casein micelles as hard spheres, measurements of the relative viscosity as a function of the casein micelle concentration allowed Jeurnink and De Kruif [24] to calculate a volume fraction of 0.130 for casein micelles in (low-heat) skim milk.

226

Gums and Stabilisers for the Food Industry

-Permeate Skim milk permeate was prepared from skim milk by a membrane filtration process. An Amicon hollow-cartridge HIMPO 1-43 membrane with a cut-off of 0.1 pm was used. The pH of the permeate was the same as that of the skim milk (6.60 k 0.10). - Mixtures The mixtures were prepared by dissolving B40 EPS diluted in permeate and mixing this B40 EPS-skim milk permeate solution with skim milk. All mixtures were studied at room temperature. - Preservative Since phase separation was sometimes observed only after a few weeks we used antimicrobial agents breservatives) in order to prevent growth of micro-organismsduring the experiments. We used 0.02 %(m/m) sodium ethylmercurithiosalicylate (CzHsHgSCfiCOONa - thiomersal, BDH Chemicals), with which we could not observe any bacterial growth or any pH changes for more than 6 weeks. Without adding EPS, skim milk and permeate containing thiomersal were stable for months. 3.2 Dynamic Light Scattering Dynamic light scattering experiments were performed to determine the diffusion coefficientsof the casein micelles with Malvern Hi-C particle size analysis equipment, which operates in a back-scattering mode. Under these conditions the wave vector has a maximum value at fixed wavelength. This is important since different diffusion processes can be recognized, i.e. self-diffusion and collective diffusion. For the relevant time scales ts and tc of both processes one can derive [ 191 : t

2 tD

- (Qa)*

7

where a represents the radius of the colloidal sphere. For Qa << 1 the diffusion process observed in DLS is the collective diffusion which is driven by a macroscopic concentration gradient. For Qa >> 1 the distance over which a particle moves is small with respect to its own radius, which is the condition for short time self-diffusion. For our set-up, the experimental conditions give a Qm2.2. Therefore, we concluded that self-diffusion of casein micelles is studied with the Hi-C equipment. Measurements were made at 298 K.

4 Results and discussion

This section contains two parts. Firstly, a description including a phase diagram of the skim milWpermeateh340 EPS mixtures will be given in section 4.1. In 4.2 the results from the dynamic light scattering experiments will be presented followed by calculations of the phase boundaries using the theoretical approach explained in section 2 and comparison with the observed phase line. 4.1 Observations We mixed B40 EPS with (low-heat skim milk) permeate (skim milk without casein micelles). Since B40 EPS is not soluble (on a practical time scale) at concentrations > 10 g/l this was the highest B40 EPS concentration studied (it should be noted here that lactic acid

221

Functional Interactions in Mixed Biopolymer Systems

bacteria produce EPSs up to concentrations of 0.5 gA). In these permeate solutions no phase separation could be observed for months. This leads to the conclusion that a skim milk permeate4340 EPS solution does not demix when practical amounts of whey proteins and B40 EPS are used. Therefore we used permeate to dissolve B40 EPS after which a EM0 EPS permeate solution was mixed with skim milk. In this way the required EPS 'end' concentration (the concentration in the final mixture) as well as the volume hction of casein micelles (further referred to as $) could easily be adjusted (0
2.2

2.0

2.0

1.5

t;

...

1.o

n v)

* <

-w

... .. ... ....

Ea

Y

d 1.6

*

0.5

1.8

0

0.0

0.00

I

I

0.05

0.1 0

L

0.15

Figure I Visually observedphase diagramfor the EPS concentration (cP, m afirnction of the volume fiaction of casein micelles (4. Indicated are the phase boundnries (squares).stable mirtures (open circler) (ind unstable mirtures tfilled circles).

1.4 0.00

0.05

0.10

0.1 5

Figure 2 The selfdl@ksion coeficient of casein niicelles as a function of their volumefraction.

With time the upper layer becomes translucent. After one week the upper phase has the same appearance as skim milk permeate. This indicates that the bottom layer is concentrated in casein micelles. Furthermore, we determined the B40 EPS concentration in the upper layer. For instance at b0.07, samples with 0.75, 1.OO and 1.25 g/l had an EPS concentration in the upper layer of 0.87, 1.03 and 1.33 gA respectively. Systematically, there is a slight increase in

228

Gums and Stabilisers for the Food Industry

the B40 EPS concentration in the upper translucent layer. Thus, there is a B40 EPS concentrated phase in the upper layer. 4.2 Self-diffusion The diffusion coefficient of the casein micelles in milk and several solutions of milk diluted with permeate was measured at EPS concentrations below the phase boundary to illustrate the hard sphere behaviour of casein rnicelles. The diffusion coefficient is given as a fhction of the volume fraction of the casein micelles in permeate in figure 2 . This selfdiffusion coefficient has a linear dependence on the volume fraction. Consequently, we can extrapolate its value to $=O and we find a Do value of 2.09.10-12m2.s.'. According to equation (4) this gives a z-average hydrodynamic radius of the casein micelles of 1 17 nm. This is consistent with literature values [25]. The volume fraction dependence in permeate can be described rather well with equation (5), when instead of 1.83 the factor of 2.0 is used. When relaxation of the spheres surrounding the self-diffusing casein micelles is taken into account, the theoretical value is 2.06 [ 2 2 ] . Next, the self-difiion of the casein micelles was studied as a function of the B40 EPS concentration. Some indicative results are plotted in figure 3 for $=O. 1 1. Firstly, it is shown that increasing the EPS concentration involves a decrease of the self diffusion coefficient. We assume that for EPS concentrations above the phase boundary no representative Baxter parameter can be determined. Secondly, it can be noticed that at very low EPS concentrations there is hardly any effect on the diffusion coefficient. When B40 EPS adsorbs onto the casein micelles a thick layer of about 80-90 nm (the radius of gyration is a reasonable measure of the hydrodynamic layer thickness of adsorbed polymers) could be expected even at very low polymer concentrations. This would then strongly decrease the diffusion coefficient of the casein micelles; their hydrodynamic size would increase by a factor of about 1.7. The independence of D, on B40 EPS at very low concentrations gives us a strong indication that B40 EPS indeed is a non-adsorbing polymer for casein micelles.

1

2.5 I

2.0

I

2.0 1.5

-

n

c:

1.5

c

CI)

W

0" 1 .o

1 .o

0.5

0.5 0.0

0.4 cp

0.8

1.2

(g/U

Figure 3 Self-diffiision coeffjcients measured in diluted skim milk (@=O I I ) for various B10 EPS concentrations Measurements are given helow /men triunglrs)and ahow (/2ied mangles) the phuce line

0.0 0.00

0.1 0

0.05

0.15

+ Figure 4 Phase diagram with visital[y obsrr-rcd

phase boundaries (open circles) and phase boundaries calculatedfrom self-difision measurement^ (using Eqm /2)&16))

Functional Interactions in Mixed Biopolymer Systems

229

The diffision coefficients were determined for various series of polymer concentrations at various volume fractions. From these data the Baxter parameter was calculated with Eq.(6). Using Eq.(2) the corresponding 0 ' was calculated. In this way the B40 EPS concentration can be related to 0'. The phase diagram which was obtained from the self-diffusion measurements is given in Hgure 4, in which we have also plotted the experimental phase boundaries from Hgure 1. Both sets of data are consistent although the phase boundary derived from the selfdiffusion coefficient measurements is lower. The discrepancy may originate in the fact that phase separationjust above the phase boundary is extremely slow (and hence is not observable). Another point is that the application of Baxter's sticky sphere model is hardly justified in view of the range of the depletion potential. Nevertheless, its use helps us to understand the phase behaviour.

5 Conclusions Thermodynamic incompatibilityor depletion flocculation is found when B40 EPS is mixed with casein micelles. B40 EPS is a non-adsorbing polymer when it is in solution with casein micelles. Volume restriction and osmotic effects are the driving forces for the depletion mechanism. We were able to calculate a phase diagram from dynamic light scattering experimentswhich is comparable with observations. The adhesive hard sphere model can describe the phase behaviour of casein micelles mixed with B40 EPS. Acknowledgement

This work was financially supported by the Netherlands Association of Biotechnology Centres (ABON). Cyril Renaud is thanked for performing many experiments and his pleasant co-operation. Professor Lekkerkerker (Van't Hoff laboratory, Utrecht University) is thanked for encouraging discussion and bringing references 17 and 18 to our attention. Dr. Zoon and Dr. Roefs from NIZO and Professors Cohen Stuart and Fleer from the Department of Physical and Colloid Chemistry at Wageningen University are thanked for stimulating discussions. References [11 Tolstoguzov, V.B., Food Hydrocolloids 4 (1991) 429. [2] Antonov, Yu.A., Soshinskii, A.A., Glotova, Yu.K., Appl. Biochem. Microbiol.,30 (1994) 760. [3] Tolstoguzov, V.B., in : Food Colloids andpolymers :stability and mechanical properties, E.Dickinson, P.Walstra, Eds., Royal SOC.Chem., 113, 1992. [4] Tuinier, R. et al., to be published. [5] Marle, M.E. van, et al., to be published. [6] Asakura, S., Oosawa, F., J Pol. Sci. 33 (1958) 183. [7] Vester, C.F., KolloidZ. 84 (1938) 63. [8] Vrij, A,, Pure & App. Chem. 48 (1 976) 471. [9] De Hek, H., Vrij, A., J. Colloid Interface Sci. 70 (1979) 592. [101 De Hek, H., Vrij, A., J Colloid interface Sci. 84 (1981) 409.

230

Gums and Stabilisersfor the Food Industry

[ l l ] Lekkerkerker, H.N.W., Poon, W.C.K., Pusey, P.N., Stroobants, A., Warren, P.B., EurophyxLetf.20 (1992) 559. [12] Poon, W.C.K., Pusey, P.N., in : Observation andSimulation ofphase Transitions in Complex Fluids, M. Baus, L.F.Rull, J-P.Ryckaert (Ed.) (1995) 3. [131 de Kruif, C.G., Jeumink, Th.J.M., P. Zoon, Nefh. Milk Dairy J 46 (1992) 123. [14] de Kruif, C.G., Langmuir 8 (1992) 2932. [15] de Kruif, C.G., J Colloidlnterfae Sci. 156 (1993) 38. [16] Baxter, R.J., J. Chem. Phys. 49 (1968) 2770. [171 Watts, R.O., Henderson, D., Baxter, R.J., A h . Chem. Phys. 21 (1971) 42 1. [18] Penders, M.H.G., Vrij, A., A h . Colloidlnterface Sci. 36 (1991) 185. [191 de Kruif, C.G., Jansen, J.W., Vrij, A., Symp. Complexfluids p.3 15-343,1985. [20] Batchelor, G.K., J Fluid Mech. 74 (1976) 1. [21] Felderhof, B.U., J Phys. A 11 (1978) 929. [22] Rallison, J.M., Hineh,E.J.,J. FluidMech. 167 (1986) 131. [23] Cichocki, B., Felderhof, B.U., J Chem. Phys. 93 (1990) 442. [24] Jeumink, Th.J.M., De Kruif, C.G.,J Dairy Rex 60 (1993) 139. [25] Hansen, S., Bauer, R., Lomholt, S.B., Quist, K.B., Pedersen, J.S., Mortensen, K., Eur. Biophys. J. 24 (1996) 143.

Phase Separation of Aqueous Starch/GalactomannanSystems: Influence of P-Lactoglobulin Addition C.B. Closs, V.B. Tolstoguzov, B. Conde-Petit', F. Escher' Nest16 Research Centre, Vers-chez-les-Blanc, CH- 1000 Lausanne, Switzerland Swiss Federal Institute of Technology, Dept. of Food Science, CH-8092 Ziirich

ABSTRACT Phase behaviour (at 25°C and 60"C), kinetics of phase separation and rheological properties of aqueous amylopectidgalactomannan mixtures were investigated. The influence of P-lactoglobulin addition on the aforementioned parameters was studied. Binodals typical of this system are located towards the concentration axis of the lower molecular weight polysaccharide, i.e. amylopectin. The addition of 1% (w/w) plactoglobulin to the amylopectidgalactomannansystem resulted in a shift of the binodal towards lower polysaccharide concentrations. The P-lactoglobulin was partitioned equally between the two polysaccharide phases. Phase separation kinetics in the bulk of a 5 ml sample determined from Vertical Scan Near Infrared Analysis indicate a completion of phase separation after 8 hours.

1 INTRODUCTION

Starchlnon-starch hydrocolloid systems are important in the control of the texture of many food products. It is well known that the combination of starch and non-starch hydrocolloids (named hydrocolloids hereafter) can lead to a considerable viscosity increase. However, the mechanism of the interaction between starch and hydrocolloids has not yet been fully explained. Previous studies have focused on starch solubles or dispersions approaching complete starch granule disintegration I,'. Alloncle et al. included starch granules in their work and described the starchhydrocolloid system as a suspension of swollen starch particles dispersed in a solution of hydrocolloid. The existence of thermodynamic incompatibility between the starch polysaccharides and the hydrocolloid biopolymer has been suggested Phase diagrams describing thermodynamic incompatibility have been established for the two starch polysaccharides amylose and amylopectin and numerous other polysaccharides but, 2,43s.

232

Gums and Stabilisers for the Food Industry

in spite of the industrial importance of starchhydrocolloid blends, their phase behaviour has not been thoroughly studied. Whey proteins, with P- lactoglobulin being the major component, find increasing use in many different food applications, including products containing starchhydrocolloid blends *. The partitioning of proteins between the coexisting phases of two-phase aqueous polymers has been the subject of numerous studies focusing on the purification of proteins from synthesis media 9*'0. The distribution of proteins between the phases of a two-phase aqueous polymer system is conveniently characterised by a partition coefficient,

where C, and C , are the protein concentrations in the top and the bottom phases, respectively '. The partition coefficient is influenced by the size of the protein, its surface properties and molecular weight as well as by the composition of the coexisting phases and the molecular weight of the phase-forming polymers ". Knowledge of the distribution of proteins between the coexisting phases of separating polysaccharide blends being scarce, advances in this field could be of great use for food formulation, where systems always contain a mixture of polysaccharides and proteins and microphase separation is frequently present.

2 MATERIALS AND METHODS Water-soluble (partially degraded) amylopectin from potato was supplied by Roquette Frkres, and pure guar galactomannan was kindly provided by Systems Bio Industries. P-Lactoglobulin from sweet whey, purchased from Besnier Bridel, was further purified by precipitation at pH 5.2 and subsequent freeze-drying.

2.1 Sample preparation Concentrated amylopectin solutions were prepared on a weight percentage basis by dissolving the amylopectin in distilled water at 80°C. Guar galactomannan stock solutions were prepared in distilled water and heated to 50°C for 15 min. Polysaccharide blends were made by weighing out the appropriate masses of amylopectin solution, guar solution and distilled water into a beaker. 1% (w/w) P-lactoglobulin was added to the blends and the samples were vigorously mixed for a minimum of 2 hours with a magnetic stirring bar at room temperature (25°C).

2.2 Determination of relative molecular weight distribution Gel permeation chromatography was performed on a Pharmacia HR 1/60 column packed with Sephacryl S-1000 gel (Pharmacia). The columns were eluted with degassed

Functional Interactions in Mixed Biopolymer Systems

233

0.01 M NaOH. Photometric detection (Gilson Holochrome) was carried out after postcolumn orcine-sulphuric acid derivatisation I*.

2.3 Phase diagrams The blends were transferred to plastic centrifuge tubes and were left to bulk phase separate overnight at room temperature (25°C) or in a waterbath at 60°C. To complete phase separation, the samples were centrifuged at 15Oxg for 3 hours. The tube was sliced at the interface, thereby allowing the isolation of the individual upper and lower phases. Polymer concentrations in both the initial blends and the separated phases were determined by High Performance Anion Exchange Chromatography in combination with Pulsed Amperometric Detection (HPAE-PAD, equipment by Dionex) after subjection to weak hydrolysis in 0.5 M H2S04at 130°C for 75 min 13. 2.4 Kinetics of phase separation

The kinetics of liquid-liquid phase separation were determined at room temperature (25°C) from Vertical Scan Near Infrared (NIR) Analysis using a Turbiscan MA 1000 vertical scan macroscopic analyser. Aliquots of 5 g were filled into glass tubes of 12 mm diameter and backscattered, as well as transmitted light at a wavelength of 860 nm was measured for the entire height of the sample (- 50 mm). The polysaccharide blends were measured directly after mixing and subsequent measurements were performed periodically thereafter for up to 12 hours. 2.5 Determination of protein concentration

The concentration of P-lactoglobulin in the blends and in the individual phases was measured by bicinchoninic acid (BCA) assay (Pierce), based on the method of Smith ef al. The samples were diluted 20x; 2 ml of BCA reagent were added to 1 0 0 ~ 1of diluted sample in a tube and the contents of the tube were vigorously mixed prior to incubation in a waterbath at 37°C for 30 min. The absorbance was measured in a Phillips PU8620 spectrophotometerat 562 nm immediately thereafter. 3 RESULTS AND DISCUSSION Blends of initially translucent amylopectin and guar galactomannan solutions become cloudy immediately after mixing, providing a first indication of the immiscibility of the two solutions. The polysaccharide blends were found to separate into two visually distinct phases within a period of 8 hours. Phase diagrams of mixed aqueous biopolymer systems containing amylopectin and galactomman are given in Figure 1. Bulk phase separation occurred, resulting in a galactomman-rich upper phase and an amylopectin-rich lower phase.

234

Gums and Stabilisersfor the Food Industry

2.00 1.80

-E 5C 5

2-E 8

1.60 1.40 1.20

1.00

0.80

0.60 0.40

0.20 0.00 0.00 1.00 2.00 3.00 4.00 5.00

6.00 7.00 8.00 9.00 10.00 11.00

Amylopectin [%I Fig 1

Phase diagrams of amylopectidgalactomannan blends at 25°C and 60°C (tie lines, ---- water distribution lines, initial blends, o bulkphases)

The points on the curves represent the polysaccharide weight concentrations as determined by HPAE-PAD. The binodals in the amylopectidgalactomannan phase diagrams in Figure 1 are not symmetrical, but located towards the abscissa (representing the amylopectin concentration) and the slope of the tie lines is not parallel with the slope of the water distribution lines. In these polysaccharide blends the amylopectin has a lower molecular weight than the galactomannan, resulting in two distinct peaks on the gel permeation chromatogram (Figure 2). Binodals computed from the Flory-Huggins theory for typical flexible chain polymers are shifted towards the lower molecular weight polymer with decreasing molecular weight of one of the polymers 14. The shift of the binodals in Figure 1 towards the abscissa indicates that the theory developed for flexible chain polymers can be applied to more rigid polysaccharides. The binodal branch representing the total concentration of the galactomannan-rich upper phase shows a slant towards higher amylopectin concentrations. This is due to the galactomannan enrichment in the upper phase, leading to an approximately sixfold increase in galactomannan concentration (i.e. -1.8%) in the upper phase for the highest blend concentration (7.5% amylopectid0.3% galactomannan). As a result of the increased galactomannan concentration the upper phase is gel-like. This is in agreement with the work of Dea er al. Is, who found galactomannans to form weak gels at concentrations >0.75%. The gelled upper phase does not permit complete phase separation by locking in remainders of the lower amylopectin-rich phase. This means

235

Functional Interactions in Mixed Biopolymer Systems

Arbitrary Units

4 .00-

3.50-

3.00-

2.50-

2.00I

0.00

60.00

I

I

I

I

I

I

100.00

150.00

200.00

250.00

300.00

350.00

Retention Time [s]

Fig 2

Gel permeation chromatogram representing relative molecular weights of amylopectin and p a r galactomannan

that the phase diagrams presented here are non-equilibrium by nature and can be regarded as diagrams of state, showing the critical gelation concentration of the galactomannan-rich upper phase at -0.8% galactomannan. The phase diagram established at 60°C (Figure 1) showed a displacement of the b i n d towards higher polysaccharide concentrations with a corresponding increase of the single phase region. The addition of 1% ( d w ) P-lactoglobulin resulted in a decrease of both the singlephase region and the gradient of the tie lines (Figure 3). This alteration of phase diagrams upon the addition of protein has been described in the works of Albertsson and Baskir 16, who found that protein concentrations >0.1% (w/w) could influence the two-phase equilibrium associated with the phase forming polymer solutions and that at higher protein concentrations the proteins may even have to be counted as “phaseforming” components. The P-lactoglobulin is distributed equally between both polysaccharide phases, i.e. the partition coefficients are around 1 (Table 1). Generally, high molecular weight substances tend to distribute more unilaterally between the two coexisting phases than lower molecular weight substances like inorganic salts and amino acids 17. However, Sasakawa and Walter l7 found partition coefficients around 1 for proteins below a molecular weight of 20,000 Daltons in the two-phase system polyethylene oxide (PEO)/dextran.

’

236

Gums and Stabilisers for the Food Industry

0.00 1.00 2.00 3.00 4.00

5.00 6.00 7.00 8.00 9.00 1o.00 11.00 Amylopectin [%I

Fig 3

Phase diagrams of amylopectidgaluctomannan blends with and without 1% Plactoglobulin (tie lines, ---- water distribution lines, initial blends, obulkphases)

The molecular weight of P-lactoglobulin falls into this category, hence the partition coefficients of around I found here are in agreement with these data. Vertical scan NIR analysis of biphasic systems in the process of phase separation was employed to study the phase separation kinetics of the amylopectin/galactomannan systems. The difference in turbidity between the initial biphasic blends and the separated phases makes this method sensitive to the changes occurring during separation. Figure 4 shows that first remarkable changes in turbidity have been registered after 1 hour and constant values were reached 8 hours after preparation of the initial blends. Once separation is complete, the amylopectin-rich lower phase becomes translucent and allows transmission of up to 59% of light at NIR wavelengths The transparency of the galactomannan-rich upper phase is considerably lower and transmission of light through this phase does not exceed 5%. From these measurements it can be seen that liquid-liquid bulk phase separation is complete after 8 hours, resulting in two discrete phases separated by a well defined interface with almost no meniscus due to the low interfacial tension between the two chemically and physically similar phases. The addition of P-lactoglobulin did not influence the kinetics of phase separation as measured by NIR (data not shown).

231

Functional Interactions in Mixed Biopolymer Systems

Table 1 PLactoglobulin partition coeficientsfor the two-phase system amylopectidgalactomannan Initial Blend Concentration Amylopectin I Galactomman K [%I [%] 0.3 1.08 7.5 0.3 1.04 6.0 0.3 1.03 5.0 0.3 1.05 4.0 0.3 0.96 3.5

I

Transmission 80%

("/I

60% 40%

-

20%

0% -20%

-40%

I

0 hours ___

~

0

10

20

30

40

50

Sample height [mm]

Fig 4

Changes in NIR transmission through an amylopectidgalactomannan blend over time

4 CONCLUSIONS

The results presented in this study reveal that the theories established for flexible chain polymers are applicable to liquid-liquid phase separating blends of polysaccharides. The addition of P-lactoglobulin to the polysaccharide blends investigated in this work did not result in unilateral partitioning of the protein between the two coexisting phases, but in an even distribution of the P-lactoglobulin after phase separation. NIR analysis proved to be a useful tool in determining the kinetics of bulk phase separation. Further work is in progress to investigate the influence of salts and pH on the system.

238

Gums and Stabilisersfor rhe Food Industry

5 REFERENCES 1. 2. 3. 4. 5. 6. 7.

8. 9. 10. 1 1. 12. 13. 14. 15.

16. 17. 18.

D.D. Christianson, J.E. Hodge, D. Osborne and R.W. Detroy, Cereal Chem., 1981, 58,513. W.-M. Kulicke, D. Eidam, F. Kath, M. Kix and A.H. Kull, Starch, 1996, 48 (3), 105. M. Alloncle, J. Lefebvre, G. Llamas and J.L. Doublier, Cereal Chem., 1989,66,90. M. Alloncle and J.L. Doublier, Food Hydrocoll., 1991. 5,455. B. Conde-Petit, A. Pfirter and F. Escher, Food Hydrocoll., 1997, in press. M.T. Kalichevsky and S.G. Ring, Carbohydrate Rex, 1987,162,323. Y.A. Antonov, M.G. Pletenko and V.B. Tolstoguzov, Vysokomolek. Soed, 1987, A29,2477. J.E. Kinsella and D.M. Whitehead, A h . FoodandNutrition Res., 1990,33,343. P.A. Albertsson, ‘Partition of Cell Particles and Macromolecules’, Wiley, New York, 197 1 . M.R. Kula, K.H. Kroner and H. Hustedt, Adv. Biochem. Eng., 1982,24,73. N.L. Abbott, D. Blankschtein and A.T. Hatton, Bioseparation, 1990,1, 19 1. M.T. Tollier and J.P. Robin, Ann. de technologie agricole, 1979,28, 1. N.O.G. Jsrgensen and R.E. Jensen, FEMSMicrobiologv Ecologv, 1994,14,79. C.C. Hsu and J.M. Prausnitz, Macromolecules , 1974,7,320. I.C.M. Dea, E.R. Moms, D.A. Rees, E.J. Welsh, H.A. Barnes and J. Price, Carbohydrate Rex, 1977,57,249. J.N. Baskir, PhD Thesis, Massachusetts Institute of Technology, 1988. S. Sasakawa and H. Walter, Biochemistry, 1972, 11,2760. Smith, P.K., Krohn, R.I., Hermansson, G.T., Mallia, A.K., Gartener, F.H., Provenzano, M.D., Fujimoto, E.K., Goeke, N.M., Olson, B.J. & Klenk, D.C. 1985. Measurement of Protein Using Bicinchoninic Acid. Anal. Biochem., 150, 76.

Novel Gels from Xanthan-Locust Bean Gum by Addition of Electrolyte

C.Gomes', M.P.Gongalves', P.A.Williamsz 1.CEQUP I Faculdade de Engenharia da Universidade do Porto. Rua dos Bragas, 4099 Porto Codex, Portugal 2.Centre for Water Soluble Polymers, The North East Wales Institute, Plas Coch, Mold Road, Wrexham, LLI 1 2AW U.K.

ABSTRACT Studies have been undertaken to investigate the effect of various electrolytes (NaCI, N(CH3)&1 (TMA) and CaClz) on the solution behaviour of xanthan and xanthanlocust bean gum mixtures using small deformation oscillatory rheometry and differential scanning calorimetry (DSC). Xanthan solutions (1.0% wlw) in water and at low concentration of electrolyte exhibited thermal hysteresis and a maximum in G was obtained at a given temperature (coolingheating cycles), which depended on the electrolyte present. For higher electrolyte concentrations G increased with decreasing temperature and increasing concentration of electrolyte. In CaCIz, the curves of G were superimposable for [~a*'p~m~. For xanthan5BG mixtures (0.9/0.1% - 0.610.4%, wlw), the cross-over of G and G ' did not differ very much from that of xanthan alone, but at 10°C G was much higher for the mixtures. G was found to depend on the type of electrolyte: Na'>N(CH,)4'>CaZ'>without electrolyte. Rheological experiments were carried out on mixtures in the presence of 25 mM NaCl varying the temperature of mixing. It was found that the cooling curves (to 20°C) of the two polysaccharides mixed at 20°C and then heated to 50°C [i.e. below the disorder to order transition (75"C)I was virtually the same as the cooling curve for samples mixed at 80°C (i.e. above the disorder to order transition). The heating curves (from 20°C) were also similar. For the mixtures prepared at 20°C, G was very much lower, in the heating curve. This behaviour has been explained in terms of self aggregation of the xanthan molecules.

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WTRODUCTION Xanthan and locust bean (LBG) gums are thickeners which form thermoreversible gels on mixing. These synergistic effects have been known for a long time and have many technological applications, particularly in the food industry (Rocks, 1971; Kovacs, 1973). LBG is a plant seed galactomannan; its chemical structure is based on a p(1-4) linked mannan backbone C6-substituted by a-galactosyl residues. In aqueous solution, this neutral macromolecule adopts a rather extended random coil conformation (Sabater de Sabates, 1979). The average degree of substitution is about one quarter, but the distribution of galactose along the mannan chain is irregular, with unsubstituted and completely substituted regions. This block structure has been assumed to be responsible for gelling interactions with xanthan or carrageenan (Dea and Morrisson, 1975; Dea et al., 1977; Morris et al., 1980). Xanthan is a charged bacterial polysaccharide consisting of a p- 1,Clinked glucopyranose backbone with a trisaccharide side chain linked to every second glucose residue. The side chain consists of two mannose units separated by a glucuronic acid residue. The mannose residue attached to the cellulosic backbone is variably acetylated, and the terminal mannose may contain a pyruvate group. The proportion of acetyl and pyruvate substituents is determined by the culture and postfermentation conditions. Xanthan gum undergoes a temperature induced conformational transition from an ordered helical structure, where the side chains are folded in and associated with the backbone, to a disordered structure, where the side chains project away from the backbone. The transition is dependent on the ionic strength of the solution, the pH, and the nature of the electrolyte (Holzwarth, 1976). At present, there is still some debate as to the nature of the ordered form, with arguments presented for both single and double stranded structures. Some debate also exists regarding the .xanthan-LBG interaction. Some authors argue that LBG must be present when the disordered xanthan conformation is available for interaction, and before the xanthan chains self-associate (Cheetham et al., 1988; Brownsey et al., 1988). Other results suggest that LBG can interact with xanthan at temperatures lower than the temperature of the conformational transition, that is, with the ordered conformation of xanthan (Lopes, et al., 1992; Morris, et al., 1977). The objectives of this paper are to show the influence of different electrolytes on the rheological behaviour of xanthan and x a n t h d B G mixtures and the influence of the thermal treatments applied to one mixed system.

MATERIALS AND METHODS Materials A sample of Locust Bean Gum Extract RefLA-22 (LBG) was kindly supplied by Systems Bio-Industries (SBI), France. The mannosdgalactose ratio obtained by gas chromatography was 3.85fo.17. The intrinsic viscosity was found to be 11.73 dL.g-' in distilled water at 25°C as determined by capillary flow viscometry. The viscosimetric molecular weight was found to be 1 . 3 5 ~ 1 Daltons. 0~

(z)

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A sample of xanthan gum (Ref.2226) was also kindly supplied by Systems BioIndustries (SBI), France. To perform thermal treatments another xanthan sample supplied by Kelco International was used.

Samde preparation Xanthan gum solutions (l%, w/w) were prepared in distilled water or in the presence of electrolyte (NaC1, N(CH&Cl (TMA) or CaC12) solution and stirred at room temperature for 30 minutes and then heated to 80°C and stirred for 1 hour in a stoppered flask. LBG solution (1%, w/w) was prepared in distilled water or in electrolyte solution and stirred at room temperature for 1 hour and then heated to 90°C and stirred for 30 minutes in a stoppered flask. Solutions of concentration lower than 1% were prepared using the stock solution (1%). The dilutions were carried out in a stoppered flask stirred at 80°C for 15 minutes. Xanthan - LBG blends were prepared by mixing the 1% solutions of xanthan and LBG in the appropriate ratios. The mixtures were heated and stirred at 80°C for 30 minutes in a stoppered flask. Rheological Measurements Rheological measurements were performed on a controlled stress rheometer CarriMed CSL. A cone-plate geometry was used (cone diameter:4.0 cm; angle: 2"; truncation: SSpm). The strain input for all the dynamic measurements was 5% (within the linear viscoelastic range). Cooling-heating cycles were performed at 0.5"C per minute, between 8O-10-8O0C, at 6.28 rad*s-'. Mechanical spectra were obtained at 20°C after a kinetic experiment at this temperature, within the range 0.6283 - 62.83 rad*s''. Kinetic experiments were performed at 6.28 rad*i' and 20"C, for three hours. Differential Scanning Calorimetrv Measurements Measurements were carried out using the Setaram Micro DSC. The samples were prepared in the same way as for the rheological measurements. A program cycle was followed in which the sample was cooled between 85°C and 20°C at O.S"C/min, followed by heating back to 80°C. RESULTS AND DISCUSSION The value of G , at a fixed frequency of 6.28 rad*i', on cooling and heating for xanthan solutions with different concentrations of NaCl or CaC12 is presented in figures l a and lb, respectively. For xanthan solutions in TMA (data not shown) the results were very similar to those obtained with NaCl. For concentrations of NaCl2 25mM, G'>G' in the range of temperatures studied. The same was observed for TMA concentrations 2 40mM and CaCl2 concentrations > 1mM. When the electrolyte was absent or present in low concentration, besides thermal hysteresis, a maximum in G' was observed at a given temperature (-5O"C, in water, -38°C in NaCl, -40°C in TMA and -1 8°C in CaCh). Under these conditions, the electrostatic repulsions involving the

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glucuronic and pyruvic groups are high and the xanthan molecules are known to adopt partially ordered conformations below -60°C in water and -80°C in 40mM NaCl (Annable et al., 1994). For lower ionic strengths G' reaches a maximum and then 50

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Figure la: Coolingheatingcycles of xanthan solutions [ 1% (w/w)] at d i f f m t concentratiolls of NaCl - lmM, IOmM, 25&, 4OmM and in water. Frequency: 6.28 rad.s-'. Cooling curyes solid symbols; in lmh4 NaC1; A in lOmh4 NaCl; in 25mM hating curves open symbols. in water; NaCl; 0- in 40mM NaCl.

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Figure lb: Cooling/heating cycles of xanthan solutions [(1% (w/w)] at different concentrations of CaClz lmM, 5mM, lOmM, 20mM and in water. Frequency: 6.28 rad.i'. Cooling curves solid symbols;heating curves open symbols. - in water; in ImM CaC12; A in 5mM CaC12; in lOmM CaC12; 0- in 20mM CaC12.

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drops at lower temperatures. At higher concentrations of electrolyte, the electrostatic repulsions are less important due to charge screening and G was found to increase monotonously with decreasing temperature. In NaCl (Rgla) and TMA, the values of G', at a given temperature, are higher the higher the electrolyte concentration. But in CaC12, for CaCI22 5mM the curves of G superimposewith each other (Figure lb). Since Ca2', is a divalent cation, it can promote crosslinking through the carboxyl groups on the side chains of the xanthan molecules. For our xanthan sample a Ca" concentration of -7mM is required to neutralise the carboxyl groups present and this is close to the value of S m M obtained in our experiments for the maximum in G . Xanthan/LBG mixtures (0.9/0.1% - 0.6/0.4%, w/w) were subjected to the same treatments. The cross-over of the G' and the G ' of the mixtures did not differ very much fiom that of xanthan alone; however, G was much higher for the mixtures (Figure2). The maximum value of G at lVC,was obtained for a xanthan/LBG ratio of 60140, in 25mM NaCl and lOmM CaC12. Gelation occ~rredat higher temperatures in the presence of monovalent ions compared to divalents ions. The G value found at 10°C was dependent on the electrolyte type and showed the following trend: Nd>N(CH,),'>Ca''> without electrolyte. 600

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Thermal treatment Experiments were carried out in order to gain an understanding of the effect of the temperature of mixing on the rheological properties of the xanthan/LBG (60/40) mixtures at 1% (w/w), in 25mM NaCI. At this electrolyte concentration, DSC showed that the order-disorder transition occurred at 75°C. It was found that the cooling curves (to 20°C) of the two polysaccharides mixed at 20°C and then heated to 50°C (i.e. below the disorder to order transition) were virtually the same as the cooling cume for samples mixed at 80°C (i.e. above the disorder to order transition) or mixed at 20°C and then heated to 80°C. The heating curves (from 20°C) were also similar

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(Fig. 3). However, for the mixtures prepared at 2OoC or at 40T,G’ was much lower, in the heating curves.

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Figure 3: Heating curve of xanthan/LBG (60/40), 1% (w/w),25mM NaCI, Sppm NaN3. G’ - solid symbols; G” open symbols. - solutions mixed hot at 8OOC; A - solutions mixed cold then heated to 80OC; - solutions mixed cold then heated to 50T; - solutions mixed cold then heated to 40°C; 0 solutions mixed cold at 20°C.

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Figure 4: Mechanical spectra of xanthadLBG (60/40), 1% (w/w).25mM NaCl, 5ppm NaN3. G‘ solid symbols; G” - open symbols. - solutions mixed hot at 80OC; A solutions mixed cold then heated to 80°C; - solutions mixed cold then heated to 50°C; 0 - solutions mixed cold then heated to 40OC; solutions mixed cold at 20OC.

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Mechanical spectra, obtained at 2OoC after holding the gel at this temperature, for 3 hours, confirmed the above results. Gels obtained by mixing LBG and xanthan

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solutions at 20 or 40°C were less elastic and more frequency dependent than gels obtained in the other conditions (Figure 4). Apparently, from these results, it is not necessary to completely disorder the xanthan molecules to obtain maximum interaction with LBG. However it was necessary to increase the temperature up to 50°C. This corresponds to the temperature at which the interaction of xanthan with LBG was detectable in the coolingheating experiments (Figure 5).

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The reduced interaction at 20°C or 40°C compared to that at higher temperatures could be due to the fact that there is self association of the ordered xanthan molecules resulting in lesser tendency for the LBG to interact with xanthan. It has previously been argued that the driving force for the interaction is the inherent tendency for the xanthan molecules to wish to reduce polymer-solvent contacts (Annable at al.). As the temperature of mixing increases,the xanthan-xanthan self association is reduced and xanthan-LBG interactions become more competitive, since in the latter there would be no electrostatic repulsion between interacting molecules. For T>50"C the xanthanLBG interactions become predominant and no differences are detectable in the rheological measurements. ACKNOWLEDGEMENTS One of us (CG) thanks the Portuguese Program PRAXIS XXI / BM / 2278194 for a support grant through the Master Degree Studies. MPG and PAW are gratefil for a JNICT/British Council award.

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REFERENCES -Annable,P., Williams,P.A.& Nishinari, K. 1994. Macromolecules, 27, 4204-421 1. -Brownsey, G.J., Cairns, P., Miles, M.J., and Morris,V.J., 1988. In Gums and Stabilisers for the Food Industry, 4, eds G.O.Phillips, D.J.Wedlock & P.A. Williams. IRL Press, Oxford, UK. -Cheetham,N.W.H. & Mashimba,E.N.M.1988.Carbohydr. Polymer., 9, 195-212. -Dea,I.C.M. & Morrison,A. 1975. Adv. Cub. Chem. Biochem., 31,241-312. -Dea,I.C.M., Morris,E.R., Rees,D.A., Welsh,E.J., Barnes,H.A. & Price,J. 1977. Carbohydr.Res.,57, 249-272. -Holzwarth, G., 1976. Biochemistry, 15,4333. -Kovacs,P.,1973. Food Technol., 27, 26. -Lopes,L., Andrade,C.T., Milas,M. & Rinaudo,M. 1992. Carbohydr. Polym., 17, 121126. -Moms,E.R., Rees, D.A., Robinson, G. & Young,G.A., 1980. J.Mol.Bio1. 138, 363. -Rocks, J.K., 1971. Food Technol., 25,476 -Morris,E.R., Rees,D.A., Young,G., Walkinshaw,M.D. & Darke,A. 1977. J. Mol. Biol., 110, 1-16. -Sabater de Sabates, 1979. These de Doctorat-Ingenieur - ENSIA - Universites Paris VII et Paris XI.

THE USE OF CONFOCAL LASER SCANNING MICROSCOPY IN STUDYING MIXED BIPOLYMER SYSTEMS

Catherine GARNIER, Sophie BOURRIOT and Jean-Louis DOUBLIER INRA, Laboratoire de Physico-Chimie des Macromolhcules, Rue de la GeraudiBre, BP 71 627,44 316 Nantes Cedex 03, France

ABSTRACT The ultra-structure of polysaccharide/polysaccharide (dextrdocust bean gum and xanthdocust bean gum) and polysacchariddprotein (dextrdmicellar casein and guar gum/micellar casein) mixtures has been studied by confocal laser scanning microscopy. Polysaccharides were covalently coupled with rhodamine iso-thiocyanate (RITC), which is fluorescent at a wavelength of 543 nm. Proteins were labelled with 1,8-anilinonaphthalene sulfonate (ANS), which is fluorescent in UV light. Hence, phase separation processes can be evidenced in the different systems observed, due to either thermodynamic incompatibility between the different species or to depletion-flocculation phenomena in the case of micellar caseins. Results from the microscopic observations were correlated with the rheological behaviour and the macroscopic state of the mixed systems. 1 INTRODUCTION Mixed biopolymer systems are widely used in the food industry because of the synergistic properties they develop, which improve the texture of products. They ocyur very often in dairy products in which casein constitutes the major protein component . However, for many of these systems, the molecular mechanisms underlying the dramatic changes in the textural properties are not yet well understood. Xanthan is a high molecular weight extracellular polysaccharide produced by fermentation from the bacterium Xunthornonus Cumpestris. The polysaccharide has a backbone constituted of (1-4)-P-D-glucose residues. Every two residues, the backbone is substituted by a charged trisaccharidic side chain consisting of two mannose units separated by a glucuronic acid residue. The mannose residue close to the backbone can be acetylated and the terminal one can be pyruvated *. The polymer undergoes a conformational transition from an ordered helical structure at low temperature to a disordered one at higher temperature. The transition temperature is highly dependent on the salt content, the stability of the ordered conformation being shifted towards higher temperature as the ionic strength increases '. Xanthan gum, which does not gel by itself, is employed by the food industry as a thickening and stabilizing agent. Galactomannans (GM) are seed polysaccharides. They have a linear backbone of (14) linked P-D-mannose residues substituted with sidechains constituted by a single (1-6)-aD-galactose residue. Locust bean gum (LBG) has a mannose to galactose ratio of about 3 3 , while for guar gum (GG), this ratio is about 1.8. Galactomannans are used in the food industry as thickeners. Dextran is a microbial polysaccharide. The synthesis of dextran from sucrose is catalyzed by the extracellular enzyme dextransucranase isolated from - Leuconosroc mesenteroides. Dextran, owing to its high percentage of (1,6)-a-D-glycosidic linkages, is a very flexible polysaccharide with a high solubility and low solution viscosity 4* '. Moreover,

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dextran does not adopt any specific conformation; it remains as a random coil in solution '. Dextran is not used in the food industry because of its negligible contribution to solution viscosity and its inability to form gels'. Casein micelles have a relatively large and rather complex structure (diameter = 20600 nm) *. This colloidal assembly is a supramolecular association of individual casein subunits: a s l , a S 2 , 0 and K caseins. These fractions are organized within the micelle according to their hydrophobic and hydrophilic groups, yielding submicelles; K-casein is thought to be mainly present on the surface of the micelle, providing a sterically stabilizing outer layer '. The submicelles are held together by colloidal calcium phos hate lo. This implies that the micelle is in dynamic equilibrium with its ionic environment 9 . E . XanthadGalactomannan mixtures, containing two non-gelling agents, lead to gel formation. It is generally considered that the gel properties arise from specific interactions between the two polymeric species 1 2 . Recently, an alternative model was proposed, suggesting that the gel was formed by adsorption of the galactomannan onto the surface of xanthan superstrands 1 3 . Although these models are plausible in the case of locust bean gum due to the presence of smooth zones along the mannan backbone, they are less probable for guar gum. In this study, dextradlocust bean gum mixtures were studied as a model system to obtain information about the behaviour of locust bean gum in presence of other polysaccharides, and to compare their behaviour with that of xanthan/locust bean gum systems. In parallel, the effect of time on the behaviour of a locust bean gum solution was also studied. In the same way, a dextrdmicellar casein system was studied as a model system for the general case of polysaccharide/casein mixtures. Guar gum was also used in mixture with casein micelles as a food polysaccharide. Phase diagrams were established when the system exhibited a macroscopic phase separation. The ultrastucture of the mixed systems was determined by confocal laser scanning microscopy after suitable labelling of the biopolymer with fluorescent dyes, and the structural information was correlated with the rheological behaviour of the systems. 2 MATERIALS AND METHODS

2.1 Materials Xanthan sample was kindly supplied by Rh6ne-Poulenc (France). The pyruvate content was 4.5% (70% pyruvated) and the acetate content was 5.2% (fully acetylated). Its intrinsic viscosity in 0.13 M KC1 was 65 dl/g. Dextran T500 was provided by Pharmacia LKB Biotechnology AB (Sweden). Its weight average molecular weight was 519 000, as determined using light scattering by the supplier. Its intrinsic viscosity was 0.5 1 dug in water. Locust bean gum was obtained from Meyhall Chemical (Switzerland). Its intrinsic viscosity in water was 14.5 dl/g. Guar gum was obtained from Systems Bio-Industries (France) and had an intrinsic viscosity of 12.9 dug in water. Locust bean gum and dextran were covalently labelled with rhodamine isothiocyanate (RITC) by modifying slightly the procedure of de Belder and Granath 14. Polysaccharides (2 g) were dispersed in 40 ml of DMSO for 15 min at room temperature. Pyridine (1 ml),RITC (0.15 g) and dibutyltin dilaurate (50 mg) were added, and the mixture was heated for 2 h at 65°C. After filtration, the resulting powder was washed several times with ethanol 95" to remove free dye, once with absolute ethanol, and once with acetone. The RITC-labelled polysaccharides were then dried overnight in an oven at 40°C. The intrinsic viscosity value obtained for RITC-locust bean gum in water was 12.6 dl/g, showing a very slight depolymerization due to the reaction. Micellar casein was a native calcium phosphocaseinate sample purified by ultrafiltration and then freeze-dried. This was kindly supplied by Laboratoire de Recherches et de Technologie Laitikre (INRA Rennes, France) and was prepared by P. Schuck. It had the following characteristics : total protein content 90.7%; non casein protein 5.0%; lactose 0.5%; salts 8.3%.

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2.2 Preparation Xanthan gum was dispersed in 0.013 M KCl at room temperature by stimng at 900 tr/min for 15 min. Galactomannans were dispersed in the solvent (0.013 M KCl or water for locust bean gum and 0.25 M NaCl for guar gum) at room temperature under magnetic stirring for 30 min, and then heated to 80°C for 30 min. Xanthdocust bean gum blends were at 0.5% total concentration. They were prepared by mixing appropriate amounts of xanthan and galactomannan solutions at 25°C. The blends were then heated at 80°C under magnetic stirring for 30 min. at 20°C in water or in 0.25 M NaCl Dextran solutions (0.1% to 20%) were prepared . under magnetic stirring during 2h30. Micellar casein (0.1% to 20%) was dispersed in 0.25M NaC1, at 20"C, pH 7, by stirring with a paddle at 1300tr/min for 5 min and then sonicated for 8 min at 50 Watts. The particle size distribution was checked using a Malvem Mastersizer IP laser granulometer. The average diameter was 0.32 Fm, which was in agreement with literature data 2.3 Methods Rheological measurements were performed using a controlled 2.3.I Rheology. strain rheometer (Rheometrics Fluid Spectrometer RFS 11) in oscillatory shear with coneplate geometry (diameter 5 cm, angle 0.04 rad, gap 50 pm) at 25°C. The frequency range was between 0.01 and 100 rad/s. The value of the applied strain changed with the nature of the samples, but it was verified that this value was within the linearity limits of viscoelasticity. Confocal Laser Scanning Microscopy (CSLM) 2.3.2 Confocal Microscopy. was performed with a Zeiss LSM 410 Axiovert microscope. Excitation of RITC was performed at 543 nm and the emission was recorded above 570 nm. The excitation of ANS using the UV laser was performed at 364 nm and the emission was recorded above 397 nm. Blends were placed between a preheated concaved slide and a coverslip and sealed to prevent evaporation. They were examined at 25°C with a water immersed x40 objective. By generating optical sections, confocal laser scanning microscopy is a powerful technique enabling greater insight into the ultrastructure of systems without any heavy treatment of the sample, such as physical section for conventional microscopy. This technique offers many advantages over the standard fluorescent microscope, enabling irradiation of discrete regions within the sample and generating images with a high degree of spatial resolution without interference from out of focus fluorescence. 2.3.3 Phase Diagrams. The phase diagram of dextradmicellar casein mixtures was established according to the polymer concentrations in 0.25M NaCl, at pH 7 and at 20°C and that of the dextrdocust bean gum mixed system in water and at 20°C. The mixtures were centrifugated at 3500 g after 48 hours. For dextradcasein systems, the upper phase was a dextran rich phase while micellar casein was more concentrated in the lower phase. For the upper phase, the casein content was obtained by measuring absorbance at 277 nm, the dextran concentration being determined with a refractometer. The composition of the lower phase was obtained by calculating the concentrations from the volumes. For dextradlocust bean gum systems, high pressure size exclusion chromatography coupled on line with an Erma Optical differential refractometer was used to determine the polymer distribution and the concentration in the two phases : the lower phase contained only dextran whereas the upper phase contained both polysaccharides 15. 3 RESULTS AND DISCUSSION

3.1 Polysaccharide-Polysaccharide Mixtures 3.1.1 Dextradhcust Bean Gum Mixtures Figure 1 shows the phase diagram of the DextranILBG mixed system. The binodal curve was drawn by checking the occurrence of phase separation in the mixtures, due to thermodynamic incompatibility between the

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polysaccharides, and the tie-lines were determined from the polymer concentration in each phase. The binodal separates the monophasic region at low concentrations and the biphasic zone, at higher concentrations in both polysaccharides. As can be seen from the figure, the binodal is not symetrical, but is shifted towards the phase containing the biopolymer of lower molecular weight, i.e. dextran. Furthermore, the upper phase is highly concentrated in galactomannan. It must be noted that it is impossible to prepare directly LBG solutions above a concentration of 2%because of the very high viscosity. On the other hand, it can be seen that the binodal shows a strong curvature when the LBG concentration in the upper phase increases.

[Dextran] %

Figure 1 Phase diagram of the dextran/ZBG mixture. Triangle :composition of the initial mixture. Circles: composition of the upper phase. Squares :composition of the lower phase.

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Figure 2 Rheological behaviour and microscopic structure (A = 543 nm) of the upper phase of a dextradR1TC-LBG system. [Dextran] = 2.4%, [RITC-LBG] = 2.9%. Figure 2 shows the rheological behaviour and the microstructure of an upper phase obtained from an initial mixture containing 0.7% of RITC-labelled LBG and 4.7% dextran. After phase separation, the upper phase obtained had a composition of 2.4% dextran and 2.9%RITC-LBG. It can clearly be seen from the mechanical spectra (Figure 2a) that G' is above G" over all the frequency range and that both moduli are only slightly dependent upon the frequency. Furthermore, the plateau reached by G' at low frequency clearly indicates that the behaviour is solid-like. Information about the microstructure of the system was obtained by observation of the upper phase by confocal microscopy using a laser wavelength of 543

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nm (Figure 2b). This wavelength corresponds to the excitation of RITC, and as this fluorochrome is covalently bound to LBG, clear zones on the photograph locate LBG in the system whereas dextran, which is not labelled, does not fluoresce and hence appears dark. It can be seen that LBG is organized in the system as a network, containing dextran into its meshes. This LBG network would then be responsible for the gel behaviour observed in rheology. One can assume that the network is formed by aggregation of the chains in the unsubstituted zones of the galactomannan backbone. The unusual curvature of the phase diagram could thus be explained by the fact that dextran can not be fully expelled from the upper phase by the centrifugation process because of the gelation of the system.

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cycles ‘. Figure 3 shows the rheological behaviour and the microstructure of a freshly prepared 1% LBG solution. The mechanical spectrum shown in Figure 3a is that of a typical macromolecular solution, with G’ higher than G” at high frequency, and G’ lower than G” below a cross-over frequency , G’ varying as o2and G” as o in the low frequency region. In Figure 3b, it is seen that fluorescence is distributed all over the picture, indicating no structure formation. Figure 4 shows the rheological behaviour and the microstructure of a 15 day-aged 1% LBG solution. After 2 weeks, the mechanical spectrum of the system (Figure 4a) has changed dramatically. Indeed, G’ is higher than G” over all the frequency range and G’ shows a plateau at low frequency, indicating a gel structure, close to that observed in Figure 2a. The same type of behaviour was observed for a more concentrated LBG solution after two weeks (1.85%) Is. By using confocal microscopy, it can be seen in Figure 4b that fluorescence is now not distributed over all the picture, but that a very fleecy fluorescent network can be observed, in agreement with the rheological behaviour obtained for the same system. LBG seems able to form a network at this concentration without lowering the temperature. 3.1.2 XanthadLBG Mixtures. Figures 5a, 5b and 5c show xanthanIRlTCLBG mixtures at a total polysaccharide concentration of 0.5 % and at mixing ratio of 20/80, 50/50, and 90/10, respectively, in 0.013 M KCI. The three pictures show small fluorescent inclusions corresponding to zones enriched in LBG. In addition, at high LBG content (Figure 5a), these inclusions seemed to be included in a fleecy fluorescent network distributed all over the picture. As the content in LBG in the mixture decreased, the size of the inclusions seemed to decrease and their number increased. It appears then that the wellknown synergism occurring between xanthan and LBG could be due to a phase separation process promoted by thermodynamic incompatibility between polysaccharides and leading to concentration of xanthan in dark zones and to a mutual concentration of LBG in clear zonesi6.17. These observations confirm the results obtained by polarization microscopy, where it was seen that xanthan molecules organized themselves as liquid crystalline mesophases in definite enriched xanthan areas ”. However, they are in contradiction with the general model proposing the existence of a coupled network trough specific junction zones between both polymers. At a high LBG content, the viscoelastic properties of concentrated xanthan could be reinforced by those of self-associated LBG (see above). Two interpenetrating networks would then be formed, and the phase separation process would be stopped by the gelation of the system. a b C

Figure 5 20/80 (a), 50/50 (b), 90/10 ( c ) xanthadRITC-LBG mixtures at 0.5% in 0.013 M KCI. 3.2 Polysaccharide-Protein Mixtures 3.2.1 DextradMicellar Casein Mixtures. Figure 6 shows the phase diagram of the dextrdmicellar casein system. As for dextran/LBG systems, dextradmicellar casein mixtures exhibit macroscopic phase separation above a critical polymer concentration,

Functional Interactions in Mixed Biopolymer Systems

253

leading to an upper phase enriched in dextran (circles) and to a lower phase enriched in casein (squares). Figure 7 shows a focal plane of a system containing 6.6% dextran and 0.5% casein labelled with ANS and thus situated in the monophasic region of the phase diagram. The fluorescence is homogeneous all over the picture :the system, macroscopically situated in the one-phase region, is also monophasic at the microscopic scale.

Figure 6 Phase diagram of the dextrad micellar casein system in 0.25 M NaCl, pH 7and20"C

Figure 7 Microstructure of a dextrard micelhr casein system containing 6.6% dextran and 0.5% casein+ANS. A= 364 nm.

Figure 8 shows the microstructure of a system containing 6.6 % dextran, covalently labelled with RITC, and 3.3% casein, labelled with A N S . This system, situated in the biphasic zone of the phase diagram, was observed before the occurrence of the macroscopic phase separation. Figure 8a corresponds to the picture obtained by illumination at 364 nm and clearly exhibits an inhomogeneous partitioning of the fluorescence : indeed, dark and clear zones are visible, the latter corresponding to zones highly enriched in micellar casein. Figure 8b corresponds to the picture obtained by illumination at 543 nm. In this case, clear areas correspond to the fluorescence of RITC, and as the fluorochrome is covalently bound to dextran, clear areas locate dextran in the system. In contrast, dark zones correspond here to casein enriched zones. It must be noticed that clear areas in Figure 8a correspond to dark zones in Figure 8b, and vice versa. The system is then heterogeneous at the microscopic scale even before the macroscopic phase separation. a b

Figure 8 Microscopic structure of a dextrdcmein system containing 6.6% RITC-dextran and 3.3% casein + ANS. a ) 1 = 364 nm b) 1 = 543 nm.

254

Gums and Stabilisersfor the Food Industry

Casein micelles appear then to be concentrated in zones of large sizes (10-100 pm) while the average size of the micelle is around 0.3 pm. Demixing in this system would then be ascribed to aggregation of the casein micelles. A depletion-floculation me~hanism'~'*~ could be at the origin of the phenomenon. This arises when dextran molecules are excluded from the space between the casein particles. The difference of osmotic pressure existing between the depletion zone and the bulk solution containing dextran molecules forces the micellar casein together inducing aggregation. Figure 9 shows the rheological behaviour of a dextran solution at 6.6%, a micellar casein suspension at 3.3% and a mixture containing 6.6%dextran and 3.3% casein, and thus having the same composition as that observed in confocal microscopy. For the dextran solution and the casein suspension, G' and G" are highly dependent upon the frequency and both systems behave as typical macromolecular solutions. The mixture of the two components exhibited sharply different viscoelastic properties : indeed, G' and G" were much less dependent upon the frequency, particularly at low frequency, and G' was higher than G" over all the frequency range. However, no plateau at low frequency was obtained for G', indicating that the admixture of the two components did not lead to a gel, but to a highly structured system. Indeed, the values of the moduli were higher for the mixture than for the solutions. From the microscopic observations, it is possible to ascribe the structure of the solution to the aggregation of micellar caseins. 10'

1oo h

Q

!5

10-1

i3 i3

10-2

n

lo3 1 0 4 t 102

Casein 3.3%

........ . I

10.'

I

A A

...l.lll

........ .......d

1oo o (rad/s)

I

1o2

10'

Figure 9 Viscoelastic properties of a dextran solution (Dx), a casein suspension, and a mixture dextradcasein in 0.25 M NaCl at 20°C. G' :filled symbol, G" :empty symbol.

a

b

o (rad/s)

Figure 10 : Rheological behaviour and microscopic structure ( A = 364 nm) of guurkasein system containing 0.2 % guar and 3 % casein + ANS.

Functional Interactions in Mixed Biopolymr Systems

255

The same general trends were displayed 3.2.2 GuurMicellur Casein Mixtures. for guartcasein mixed systems. Indeed, they exhibited also a macroscopic phase separation leading to an upper phase enriched in galactomannan and to a lower phase enriched in proteins, Figure 10 shows the rheological behaviour and the microscopic structure of a mixture containing 0.2% guar gum and 3% casein labelled with A N S and observed at a wavelength of 364 nm. This system is located in the biphasic zone of the phase diagram. Figure 10a shows that the one component systems behaves here again as typical macromolecular solutions whereas the mixture shows less pronounced frequency dependence for G’ as for G”. In this case, G’ was lower than G” over all the frequency range but the storage modulus tended to level off at low frequency. Here again the system was structured but no gel was obtained. The microscopic structure clearly shows that the fluorescence distribution is very heterogeneous leading to the existence of large clear zones (>lo0 pm) containing aggregated casein micelles (Figure lob). As for the case of dextrdcasein, the involvement of a depletion-flocculation mechanism promoted by the addition of guar molecules to a casein suspension could explain the aggregation of the micelles and then the structuring of the system.

4 CONCLUSION By using confocal laser scanning microscopy, phase separation processes have been evidenced both in polysacchariddpolysaccharide and in polysaccharide/prokin systems. This technique is a powerful tool for studying biopolymer mixtures. Indeed, the sample preparation is simple, consisting of labelling which did not modify too much either the intrinsic properties of the components or their behaviour in mixture. Optical sectioning is produced instead of physical sectioning, which avoids any artefact on the structure of the sample. The images generated are of a high degree of spatial resolution, as no interference from out of focus fluorescence is present. Furthermore, by imaging the sample at different focal planes and superimposition of the different images obtained, the three dimensional structure of the mixture can also be obtained. Moreover, it has been shown that the information obtained can be correlated both with the rheological behaviour of the mixed system and with the macroscopical state.

References 1. H.E. Swaisgood, ‘Developments in Dairy Chemistry’, Fox P.F. (ed.), Applied Science Publishers, 1982, 1, 1. 2. P.E. Jansson, L. Kenne and B. Lindberg, Curbohydr. Res., 1975.45, 275. 3. M. Milas and M. Rinaudo, Curbohydr. Res., 1979,76, 189. 4. I. Tvaroska, S. Perez and R.H. Marchessault, Curbohydr. Res., 1978,61,97. 5 . B.A. Burton and D.A. Brant, Biopolym., 1983,22, 1769. 6. I.C.M. Dea, E.R. Morris, D.A. Rees, J. Welsh, H.A. Barnes and J. Price Curbohydr. Res., 1977, 57, 249. 7. R.D. McCurdy, H.D. Goff, D.W. Stanley and A.P. Stone, Food Hydrocoll., 1994, 8, 609. 8. D.G. Schmidt, ‘Developments in Dairy Chemistry’, Fox P.F. (ed.), Applied Science Publishers, 1982, 1, 60. 9. H. Visser ‘Protein Interactions’, Visser H. (ed), VCH, 1992, 135. 10. H.J.M. Van Dijk, Neth. Milk Dairy J., 1990, 44, 65. 11. C. Holt, ‘Advances in Protein Chemistry’, Anfisen C.B., Edsall J.T., Richards F.M., Eisenberg D.S. (eds), Academic Press Inc., 43, 1992, 63. 12. P. Cairns, M.J. Miles, V.J. Morris and G.J. Brownsey, Curbohydr. Res., 1987, 160, 411. 13. L. Lundin and A.-M. Hermansson, Curbohydr. Polym., 1995,26, 129. 14. A.N. De Belder and K. Granath, Curbohydr. Res., 1973,30, 375. 15. C. Garnier, C. Schorsch and J.-L. Doublier, Curbohydr. Polym., 1995,28, 313.

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Gums and Stabilisers for the Food Industry

16. C. Garnier, C. Schorsch and J.-L. Doublier, ‘Proceedings of the 1’‘ International Symposium on Food Rheology and Structure’, Windhab E.J. and Wolf B . (eds),Vincentz Verlag, 1997, 155. 17. C. Schorsch, C. Garnier and J.-L. Doublier, Curbohydr. Polym., 1997, in press. 18. C. Schorsch, C. Garnier and J.-L. Doublier, Curbohydr. Polym., 1995, 28, 319. 19. S. Asakura and F. Oosawa, J. Chem. Phys. , 1954, 22, 1255. 20. S. Asakura and F. Oosawa, J. Polymer Sci., 1958,33, 183.

Processing Developments

The Science and Technology of Fluid Gels.

Ian Norton,

T i m

Foster and Rupert Brown.

Unilever Research, Colworth House, Shambrook, Bedfordshire, England.

1 INTRODUCTION

Traditionally hydrocolloids are used in the Food Industry to give gelling andor thickenin properties and thus to impart textures and breakdown properties to manufactured foods'u23' Typical gel forming hydrocolloids (eg. agar, pectin, alginate, carrageenan, gelatin, gellan) have a common feature in that the cause of gelation is via an aggregation step. This can be ion mediated or thermally induced, but the resultant gel is a product of extensive aggregation of ordered polymer chain?. The temperature range between ordering initiatiOn and completion is dependent on the hydrocolloid used eg. kappa carrageenan6 = 8"C, gellan' = 20"C,agar* = 25°C. With the recent consumer demands for high quality, healthy (low fat) foods it has been shown that none of the traditional hydrocolloids give the desired emulsion-like properties. Thus both the Food Manufacturers and Ingredient Suppliers have sought ways to modify the material properties of hydrocolloids in order to impart fat/emulsion like behaviour. This has been partially achieved by producing de-watered protein precipitates9, carbohydrate based particulateslo'l 1.12 , and fibre based ~ubstitutes'~ all of which fail to provide optimum in-mouth performance. An alternative way to do this has been to use particulated gels produced by or by producing the gel and then breaking it into either forming the gel in shear 14~15~16 particles by subsequent shearing or chopping. Such materials are commonly called fluid gels as they are pourable liquids under conditions were the materials would normally have solid like (gel) properties. Fluid gels can be successfully produced for a number of hydrocolloids eg. ~arrageenans'~, agars14,agarosel4, alginates", pectins14, gellans'6 etc. (figure 1 shows typical fluid gel particles produced from a 3% agar solution). The final properties of fluid gels are dependent on the hydrocolloid used and particle size (which can be modified at fixed shear rate by the hydrocolloid type and initial concentration or by varying the shear conditions at constant polymer concentration and cooling rate). Figure 1 shows that the particle Size has been reduced from -30pm at a shear rate of 40s' to less than 10pm at a shear rate of 750s-I. This gives particles in the size range of oil droplets found in typical emulsion products. The problem is that there is currently little knowledge on why certain hydrocolloids form stable fluid gels or how the physical properties of the fluid gels are dependent on the microstructureand colloidal interactions of the particles. This poses key scientific questions: (i) why do only some hydrocolloids produce stable particles while others produce

B.

260

Gums and Stabilisersfor the Food Industry

Figure 1. Fluid gel particles of agar produced at average shear rates of 40s' (left) and 750s-' (right). 25pm. space-filling gels within the process or particles that heal back to the quiescent gel state and (ii) what are the differences in physical properties between gels and fluid gels, both in bulk properties and at the molecular level. In this paper we develop a hypothesis to explain how and why fluid gels are produced and remain stable. 2 MATERIALS AND METHODS The materials used were agar (Luxara 1253, Arthur Branwell & Co Ltd),agarose (Type 1A, Sigma), iota carrageenan (Genuvisco X0908), sodium kappa carrageenan (Genugel Xo909)(both Hercules UK Ltd.), gelatin (Limed Hide (Type B), Bloom 270 (Croda Chemicals). 2.1 Fluid Gel Formation.

Sheared gel particles have. been produced using a variety of shearing devices, from the well defined geometries of the cone and plate viscometer and the more complex flow of a couette system, to the batch stirred pot (a laboratory scale scraped surface heat exchanger). All geometries have been characterised using viscosity standards to show that the flow is predominantly laminar. 2.1.1 Couette Flow. Hydrocolloids were dissolved with stirring (Silverson Lab. mixer) in water or salt solutions and heated to 95°C (60°C for gelatin). A concentric cylinder (couette flow) device (modified with an insert to eliminate the low shear region at the base of the geometry) was used to make fluid gels by quench cooling from 95°C to 5°C at various shear rates. 2.1.2 Jacketed Stirring Vessel. Hot solutions of hydrocolloids (as above) were m l e d while being sheared in a jacketed shear device (figure 6) operated at various shaft speeds. Stimng continued for 120min. after the system reached the lower temperature.

Processing Developments

26 1

2.2 Fluid gel characterisation.

2.2. I Microscopy. Sheared agar/agarose dispersions (100~1)were placed in the well of a microscope slide, treated with aqueous Rhodamine solution (2pl,O.OS%w/w)) and an optical slice (0.7pm thick, 50pm under the surface) examined with a Confocal Scanning Light Microscope (BioRad MRC 600) using a x 60 objective and video recorder (Video Mag. x2000) and laser excitation wavelength of 488nm. 2.2.2 Culorimerry (OSC). Fluid gel dispersions (-800mg.) were loaded into the sample holder of a Setaram MicroDSC II calorimeter at 22°C The samples were first heated to above the melting temperature of the dispersions, then cooled to 5°C and re-heated immediately to 95 "C. In this way the melting temperature of the fluid gel dispersions and the corresponding quiescently formed gels were compared. Alternatively fluid gels were heated to certain temperatures, below Tmr, and immediately recooled. This allows assessment of disordering during the initial heating cycle. 2.2.3 Rhcology. A Canimed CSI500 Rheometer fitted with parallel plate geometry coated with emery paper ( P a grade) was used to perform large and small deformation tests: (i) Stress sweeps of fluid gels, carried out using applied stresses that generated a range (ii) Temperature dependence of dynamic modulus was of shear rates up to 100 S'. measured over the range 10°C to 100°C using a stress of lOPa at 1Hz. Strain dependence of dynamic modulus (G)measured using the Rheometrics Dynamic Analyzer RDA I1 over the strain range 0.001 to 1.0 units. 2.2.4 NMR. Relaxation measurements were used to estimate the amount of free solvent water present in fluid gel dispersions of agar and agarose. Proton spin-spin relaxation Q was used to quantify the amount of water expressed from fluid-gel dispersions subjected to 2000 x g for 10 min. The measurements were made by a Resonance Instruments 'Maran' spectrometer operated at 20 MHz and ambient temperature using a Carr-Purcell-MeiboomGill pulse sequence with an interpulse spacing of 2 0 0 p The resulting signal envelope was resolved into its constituent relaxation time responses (up to two processes) using a nonlinear regression analysis package. 3 RESULTS AND DISCUSSION 3.1 Why some hydrocolloids form stable fluid gels.

Stable fluid gels can be produced from hydrocolloids that gel by extensive aggregation of ordered chains e.g. calcium pectate, calcium alginate, potassium kappacarrageenan, gellan, agarose and agar. If the amount of aggregation is reduced then stable fluid gels are not produced. The particles are stable and fluid gels are produced if shearing is continued throughout the ordering transition of the biopolymer. An example is that of agar (and agarose) which forms stable fluid gel dispersions if cooled to at least 30°C below the onset of ordering and subsequent aggregation. As the final quench temperature is raised an increasing amount of agar(ose) can be shown to aggregate after shearing has ceased, resulting in 'healing' of the originally fluid dispersion. Hydrocolloids which form stable fluid gels are the systems that want to de-water and effectively precipitate from solution (ie. there is phase separation (de-mixing) into polymerrich and polymer-poor regions). At common cooling rates, in the absence of shear, the

262

Gums and Stabilisers for the Food Industry

system becomes kinetically trapped to form a three dimensional structure. It has recently been concluded" that the phase separation event in the formation of agarose gels takes place by a spinodal decomposition mechanism. It is still not clear whether this is the correct mechanism. It is evident from a number of techniques that the ordering of agarose into the double helix conformation, which is required for aggregation, is under kinetic control, and is also probably very much governed by polymer fine structure. Therefore, a nucleation and growth mechanism can not be ruled out. Whichever mechanism is responsible for the initial stages of demixing, it appears that particles start to form in the early stages of the aggregation process, during which the nuclei are subjected to shear forces and will appear to behave as water-in-water emulsion droplets. The droplets will therefore be undergoing coalescence and break-up, which will produce an equilibrium droplet size. The extent of coalescence and break-up in classic emulsions is governed by the interfacial tension of the droplets and the nature of the interface. In recent work we have shown that in gelling systems of phase separated biopolymers, the balance of coalescence to break-up is also dependent on the elastic modulus of the droplet". These droplets become more resistant to break-up as gelation proceeds, due to increases in their internal elastic modulus, and so more shear stress is required to break-up the droplet/particle. When the shear stress is insufficient to promote droplet break-up the equilibrium droplet size will increase until the droplets are too elastic to coalesce, and so control the final droplet size. Experimental results indicate that the temperature at which coalescence ceases (for agar 1253 at a concentration of 3% and a cooling rate of lS"Cmin-') is between 37SoC-35"C. At this temperature approximately 60% of the chains have still to undergo the disorder-order transition. This would indicate that disordered hydrocolloid chains at or below this temperature are present within the already formed particles which upon further cooling would then increase the strength of those particles. If shearing is stopped at a temperature above that of complete ordering, but below 35"C, then the resultant structure is not that of sheared gel, but is one in which the particles are strongly bonded to each other. This gives rise to a gel which is paste-like and fails at quite low distance scales. In support of the hypothesis that the disordered chains are situated inside the preformed particles it is found that if the shear rate is altered (increased or decreased), but not removed at that same temperature in a repeat experiment, the resulting particle size is not affected. Upon cessation of shearing the particles become connected together by aggregation. Conversely, if the shear rate is changed above 35"C, then a proportion of particles are formed whose size is governed by the new shear flow conditions. If shearing is stopped at such temperatures, then upon further cooling a structure is formed which has the properties of a quiescent gel. The origin of this structure is presumed to be a developing connectivity between the pre-formed particles driven by the potential for nucleation in the ungelled matrix. It appears that in a shear field the demixing into polymer-rich and polymer-poor regions is taken to its extreme, and the polymer-poor region acts as a depleted layer between the particles. This layer imparts long term stability to the system, giving a barrier to interaction of polymer chains of separate particles. The extent of 'phase separation' has been investigated using N M R relaxation time measurements. Spin-spin (TJ relaxation times are sensitive to molecular motions of the water molecules in carbohydrate gels. In general, the mobility depends on the polymer concentration and therefore N M R relaxation measurements can distinguish free water from water in gel particles (fluid gels). Knowing that the rate of diffusion of water is D = 2.5. lo" m2.s1and the typical T2 values for proton, the domain

263

Processing Developments

sizes that can be discriminated by NMR relaxation are calculated from the diffusion equation: a2 = 2Dt where a = distance, t = relaxation time. In practice, by gently centrifuging agar fluid gels in NMR tubes, under conditions where no liquid layer could be separated from the quiescent gels, it was possible to separate a low viscosity upper layer from a high viscosity lower layer that contained the gel particles. N M R measurements on the tubes from the centrifuge showed that the proton signals could be analyzed in terms of two relaxation processes (Table 1). The rapidly decaying signals were the same as those observed for the quiescent gels while the longer T2values show that the low viscosity liquid observed on the surface of the fluid gels is a low concentration polymer solution. Given that the T2value for pure water at room temperature is about 2 seconds, we can calculate that the low viscosity liquid contains about 0.1% of Agar in the 1% fluid gel and about 0.2% of Agar in the 3% fluid gel. In contrast to agar, the liquid phase separated by centrifugation from agarose fluid gel was identified to be pure water by its relaxation time. These results show that the fluid gels are heterogeneous in nature such that some of the water exists in regions which are polymer poor, probably in the interstitial spaces between the particles. Stability of fluid gel particles is also dependent upon storing at temperatures which are well below the equilibrium melting temperature (T,,J. It is difficult to form stable gelatin fluid gels for this reason. Figure 2 shows that when gelatin is shear m l e d to 2"C, and stored at this temperature there is evidence of a re-healing process, but the structure only has an elastic modulus 1/3rd that of a quiescently cooled gel held at the same temperature for a period of 16 hours. However when the same fluid gel is cured at 15°C it very quickly adopts the same gel properties as the equivalent quiescent gel. For hydrocolloids whose equilibrium melting temperatures are higher than that of gelatin eg. agar and kappa carrageenan (due to the increased extent and length of aggregates), only small increases in elastic modulus are seen when heated to temperatures close to Tmhand subsequently re-cooled (Table 2). The amount of increase in G' is linearly related to the amount of helical melting of the agar, and indicates that the elastic modulus of the fluid gel is simply related to the amount of network formed between the particles. The value is close to that expected when the values of G' for the quiescently cooled gels are compared.

Table 1 :Proton relaxation NMR T2 measurements of Agarjluid-gel.

I

T, (msec).

Proportion of signal 70 %

[Agar] 1

30 % I

118 (polymer rich)

I I

834 (polymer poor)

264

Gums and Stabilisers for the Food Industry

Table 2: Efect of heating on the stability offluid gels (Shear-cooled at 500 rpm and at 2"Clmin).

Quiescent Gel

60

6.85

273

2188

8.01

(G' = 51600) k carrageenan (G' = 6700)

70

18.53

347

5432

15.65

50

6.23

765

1656

2.16

G' ( P a )

*'O0O

I

1,500

1.000

500

20,000

0

40,000 Time (s)

60,000

80,000

Figure 2. 3 % gelatin gels shear-cooled @lied symbols) and quiescently cooled (open symbols) cured at 2°C (.:El) and 15°C (a:O).

""0

20

40 60 80 Temperature (C).

100

120

Figure 3. Calorimetric heating curves (0.S"Clmin) for 3% agar gels produced quiescently (0)and at 750s.' (a).

Processing Developments

265

3.2 The difference between gels and fluid gels. 3.2.1 At the molecular level. The total amount of ordering in the fluid gel is unaffected by the shear field. The extent of molecular ordering determined by DSC measurements was found to be the same for both quiescent and fluid gels. For example, the melting enthalpies for 3% Agar were 10.6 & 0.6 and 10.1 0.7 kl mole residues-' for the fluid gel after shearing at 40s'' and 750s'' respectively and was 9.8 f 0.5 kJ mole residues' for the quiescent gel. As the enthalpy of the transition is dependent on the amount of helix formed and not on the extent of the aggregation process this shows that shearing has not affected the coil to helix process. The shape of the peaks (figure 3) indicates that although the overall helix content is the same for both samples the fluid gel has a significant fraction of ordered helices which start to melt at lower temperatures ie. around 30°C, and the overall melting is about 2°C lower. This shows that the entropy change for the transition is less negative for the fluid gel (ie. there is more disorder in the fluid gel than in the quiescent gel) which is consistent with fewer or smaller aggregates and suggests that the shearing process has limited the side by side packing of the helices. This would be expected if the chain interaction has been limited by the shear such that perfect side by side alignment can only occur within a particle and not between particles. 3.2.2 Bulkproperrics. As expected, the elastic modulus (G') for the fluid gel is lower than for the quiescent gel (figure 4) by about two orders of magnitude. However, at low strains the fluid gel has solid like character, but then fails as the strain is increased to between 5 and 10%. Thus the fluid gels exhibit yield stress behaviour. This was demonstrated by studying the flow curves (figure 5) in which all concentrations of agar between 1 and 5 % show an apparent yield stress which increases with concentration, followed by a transition region and finally, at higher shear rates (> -20i1),the materials have a Bingham viscosity which increases with concentration of the agar. The effect has been shown to be reversible with the yield stress recovering back to its original values after a few minutes in the absence of shear. These rheological findings imply that as we stress the systems initially the particles have weak interactions and are capable of storing energy and resisting the applied forces. As the strain or stresses are increased the particle/particle interactions are disrupted and the system flows (ie. behaves as a liquid). Upon re-heating the 3 % agar fluid gels the G' increases at temperatures above 40°C (figure 6) before decreasing as melting of the ordered molecules occurs at the normal temperatures ie. '90°C. Such an increase in G' seems to be a consequence of the particles swelling with temperature as the hydrocolloid becomes more soluble. This would lead to the system forming a greater phase volume of particles rather than relaxing towards the quiescent gel state arising from an increase in the extent of aggregation between the particles. However, as discussed above, if the systems are subsequently re-cooled, then particle-particle aggregation is possible if the disordered coils are spatially arranged such that re-ordering and aggregation takes place between particles. Thus the most likely explanation is that the system becomes more dynamic resulting in some molecular ordering/ aggregation of helices occurring between the particles.

266

Gums and Stabilisers for rhe Food Industry

...

100.000 .

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.

.

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.

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0

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.

,

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Figure 4. Strain dependence of Dynamic moduli of I . 75 % agar gels. (G' =filled symbols: G" = open symbols). Fluid gels (0:O) and quiescent gels p:O). S h e a r stress ( N / m * 2 ) 300 . x

250

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261

Processing Developments

The dependence of bulk properties of fluid gels on the particle interactions is further supported by comparing different concentrations of agar/agarose sheared at the same shear rate. We find that as the concentration of polymer is decreased the number of particles that are produced also decreases and therefore the phase volume of the depleted layer increases. If the particles are diluted, free water is observed spontaneously below 60%phase volume of particles ie. at phase volumes below the packing fraction, and the G' value falls by two orders of magnitude upon similar dilution. The irregular shape of the particles contributes to the bulk roperties as upon dilution they do not follow the Hertz relation~hip'~ (agar spheres do ) and can be fitted by the Kreiger-Doherty equation*', but with a [ ~ exponent l]+ greater ~than that for model spheres.

Po

4 CONCLUSIONS AND CURRENT MODEL

4.1 Formation of fluid gels.

Hydrocolloids that form gels via extensive aggregation of ordered chains and are stored at temperatures that favour the ordered state produce stable fluid gels if shearing is continued through the whole of the ordering process. If either gelation does not involve aggregation or the system is close to the equilibrium for the disordering transition then unstable particles are produced that rapidly loose their identity and the system relaxes back to the quiescent gel once shearing is stopped. Thus fluid gels are a consequence of aggregate formation by hydrocolloids that are de-watering and trying to precipitate from solution (the hydrocolloid is "trapped" during de-mixing into polymer rich and polymer poor regions). 4.2 Stability of fluid gels. Under quiescent conditions the system is kinetically trapped to form a three dimensional structure. This produces a gel rather than allowing the biopolymer to precipitate. When the polymer chains are forced to order and aggregate while being sheared then helix formation and aggregation are forced to occur within small areas of space, determined by the shear fields, thus giving rise to gel particles. Within this disruption of the ordering process small amounts of solution low in polymer concentration are produced and less extensive alignment and aggregation of the helices occurs. As the hydrocolloids are trying to de-water the polymer poor solution in the interstitial space between the particles acts as a depleted layer. This then imparts long term stability to the fluid gels (i.e. it stops the polymer chains from separate particles becoming aggregated together which, with time, would lead to relaxation of the system back to the quiescent gel structure). As there will be a limited number of contacts between the particles and chain misalignment between the aggregates from the different particles we would expect there to be weak particlelparticle interactions which can be disrupted at low shear rates and shear stresses, thus leading to the weak gel nature of the materials. The overall rheology/viscosityof the system is a consequence of particlelparticle interactions caused by the high phase volume produced from the shear device. If the fluid gels are warmed

Gums and Stabilisers for the Food Industry

268

and the biopolymer becomes more soluble the particles swell giving an increased phase volume of particles and stronger particle/particle interactions.

Acknowledgements We would like to thank S.Ablett, A.Darke, W.J.Frith and P.Knight for carrying out the experimental work. References 1, 'Industrial Gums - Polysaccharides and Their Derivatives', eds. R.L. Whistler and J.N. BeMiller, Academic Press, 1959. 2. M. Glicksman, 'Gum Technology in the Food Industry', Academic Press, 1969. 3. 'Food Structure - It's Creation and Evaluation', eds. J.M.V. Blanshard and J.R. Mitchell, Butterworths, London, 1988. 4. 'Food Gels', ed. P. Harris, Elsevier Applied Science, London, 1990. 5. D.A Rees, Polysaccharide conformation, In 'Carbohydrates', MTP International Review of Science, Organic Chemistry, Series 1, ed. G . 0 Aspinall, Butterworths, London, 1973. 6. F.M. Goycoolea, T.J. Foster, R.K. Richardson, E.R. Morris and M.J. Gidley, In 'Gums and Stabilisers for the Food Industry 7 ' , eds. G.O. Phillips, P.A. Williams and D.J. Wedlock, IRL Press, Oxford, 1994, p333. 7. K. Nishinari, In 'Gums and Stabilisers for the Food Industry 8', eds. G.O. Phillips, P.A. Williams and D.J. Wedlock, IRL Press, Oxford, 1996, p371 8. M.J. Gidley, N.D. Hedges, R.A. Hoffmann and C.P. Dawes, In 'Gums and Stabilisers for the Food Industry 7', eds. G.O. Phillips, P.A. Williams and D.J. Wedlock, IRL Press, Oxford, 1994, p3. 9. N.S. Singer, S. Yamamoto and J. Latella, European Patent No: EP 0250623, 1986. 10. F.C. Bing, Journal of the American Dietetic Association, 1976, 69(5), 498-505. 11. A.C. Hoefler, J.A. Sleap and J.E. Trudso, European Patent No: 0501758 A l , 1992. 12. F. Schierbaum, B. Kettlitz, S. Radosta, F. Reuther, M. Richter and W. Vorwerg, Acta - Alimentaria - Polonica, 1984, 10(1/2), 69-99. 13. R.D. M'Cormick, Food Product Developmcw, 4(4), 19-20, 22 & 26. 14. C.R.T. Brown and I.T. Norton, European Patent No: EP 355908, 1991. 15. N.D. Hedges and I.T. Norton, European Patent No: EP 432835, 1991. 16. W.F. Chalupa, US Patent Application: US 94-265524, 1994. 17. A. Emanuele and M.B. Palma-Vittorelli, Physical Review Letters, 1992, 69, 81-84. 18. T.J. Foster, J. Underdown, C.R.T. Brown, D.P. Ferdinand0 and I.T. Norton, In 'Food Colloids - Proteins, Lipids and Polysaccharides', eds. E. Dickinson and B. Bergensml, The Royal Society of Chemistry, Cambridge, 1997, p346-356. 19. H. Hertz, Gesammebe Werke, Leipzig, 1895. 20. 1.M Kreiger, Adv. Colloid and Inreface Sci., 1972, 3, 111. 21. W.J. Frith and A. Lips, In 'Proc. XIIth Congr. on Rheology', eds. A. Ait-Kadi, J.M. Dealy, D.F. James and M.C. Williams, Canadian Rheology Group, 1996.

The Science and Technology of Fluid Gels.

Ian Norton,

T i m

Foster and Rupert Brown.

Unilever Research, Colworth House, Shambrook, Bedfordshire, England.

1 INTRODUCTION

Traditionally hydrocolloids are used in the Food Industry to give gelling andor thickenin properties and thus to impart textures and breakdown properties to manufactured foods'u23' Typical gel forming hydrocolloids (eg. agar, pectin, alginate, carrageenan, gelatin, gellan) have a common feature in that the cause of gelation is via an aggregation step. This can be ion mediated or thermally induced, but the resultant gel is a product of extensive aggregation of ordered polymer chain?. The temperature range between ordering initiatiOn and completion is dependent on the hydrocolloid used eg. kappa carrageenan6 = 8"C, gellan' = 20"C,agar* = 25°C. With the recent consumer demands for high quality, healthy (low fat) foods it has been shown that none of the traditional hydrocolloids give the desired emulsion-like properties. Thus both the Food Manufacturers and Ingredient Suppliers have sought ways to modify the material properties of hydrocolloids in order to impart fat/emulsion like behaviour. This has been partially achieved by producing de-watered protein precipitates9, carbohydrate based particulateslo'l 1.12 , and fibre based ~ubstitutes'~ all of which fail to provide optimum in-mouth performance. An alternative way to do this has been to use particulated gels produced by or by producing the gel and then breaking it into either forming the gel in shear 14~15~16 particles by subsequent shearing or chopping. Such materials are commonly called fluid gels as they are pourable liquids under conditions were the materials would normally have solid like (gel) properties. Fluid gels can be successfully produced for a number of hydrocolloids eg. ~arrageenans'~, agars14,agarosel4, alginates", pectins14, gellans'6 etc. (figure 1 shows typical fluid gel particles produced from a 3% agar solution). The final properties of fluid gels are dependent on the hydrocolloid used and particle size (which can be modified at fixed shear rate by the hydrocolloid type and initial concentration or by varying the shear conditions at constant polymer concentration and cooling rate). Figure 1 shows that the particle Size has been reduced from -30pm at a shear rate of 40s' to less than 10pm at a shear rate of 750s-I. This gives particles in the size range of oil droplets found in typical emulsion products. The problem is that there is currently little knowledge on why certain hydrocolloids form stable fluid gels or how the physical properties of the fluid gels are dependent on the microstructureand colloidal interactions of the particles. This poses key scientific questions: (i) why do only some hydrocolloids produce stable particles while others produce

B.

260

Gums and Stabilisersfor the Food Industry

Figure 1. Fluid gel particles of agar produced at average shear rates of 40s' (left) and 750s-' (right). 25pm. space-filling gels within the process or particles that heal back to the quiescent gel state and (ii) what are the differences in physical properties between gels and fluid gels, both in bulk properties and at the molecular level. In this paper we develop a hypothesis to explain how and why fluid gels are produced and remain stable. 2 MATERIALS AND METHODS The materials used were agar (Luxara 1253, Arthur Branwell & Co Ltd),agarose (Type 1A, Sigma), iota carrageenan (Genuvisco X0908), sodium kappa carrageenan (Genugel Xo909)(both Hercules UK Ltd.), gelatin (Limed Hide (Type B), Bloom 270 (Croda Chemicals). 2.1 Fluid Gel Formation.

Sheared gel particles have. been produced using a variety of shearing devices, from the well defined geometries of the cone and plate viscometer and the more complex flow of a couette system, to the batch stirred pot (a laboratory scale scraped surface heat exchanger). All geometries have been characterised using viscosity standards to show that the flow is predominantly laminar. 2.1.1 Couette Flow. Hydrocolloids were dissolved with stirring (Silverson Lab. mixer) in water or salt solutions and heated to 95°C (60°C for gelatin). A concentric cylinder (couette flow) device (modified with an insert to eliminate the low shear region at the base of the geometry) was used to make fluid gels by quench cooling from 95°C to 5°C at various shear rates. 2.1.2 Jacketed Stirring Vessel. Hot solutions of hydrocolloids (as above) were m l e d while being sheared in a jacketed shear device (figure 6) operated at various shaft speeds. Stimng continued for 120min. after the system reached the lower temperature.

Processing Developments

26 1

2.2 Fluid gel characterisation.

2.2. I Microscopy. Sheared agar/agarose dispersions (100~1)were placed in the well of a microscope slide, treated with aqueous Rhodamine solution (2pl,O.OS%w/w)) and an optical slice (0.7pm thick, 50pm under the surface) examined with a Confocal Scanning Light Microscope (BioRad MRC 600) using a x 60 objective and video recorder (Video Mag. x2000) and laser excitation wavelength of 488nm. 2.2.2 Culorimerry (OSC). Fluid gel dispersions (-800mg.) were loaded into the sample holder of a Setaram MicroDSC II calorimeter at 22°C The samples were first heated to above the melting temperature of the dispersions, then cooled to 5°C and re-heated immediately to 95 "C. In this way the melting temperature of the fluid gel dispersions and the corresponding quiescently formed gels were compared. Alternatively fluid gels were heated to certain temperatures, below Tmr, and immediately recooled. This allows assessment of disordering during the initial heating cycle. 2.2.3 Rhcology. A Canimed CSI500 Rheometer fitted with parallel plate geometry coated with emery paper ( P a grade) was used to perform large and small deformation tests: (i) Stress sweeps of fluid gels, carried out using applied stresses that generated a range (ii) Temperature dependence of dynamic modulus was of shear rates up to 100 S'. measured over the range 10°C to 100°C using a stress of lOPa at 1Hz. Strain dependence of dynamic modulus (G)measured using the Rheometrics Dynamic Analyzer RDA I1 over the strain range 0.001 to 1.0 units. 2.2.4 NMR. Relaxation measurements were used to estimate the amount of free solvent water present in fluid gel dispersions of agar and agarose. Proton spin-spin relaxation Q was used to quantify the amount of water expressed from fluid-gel dispersions subjected to 2000 x g for 10 min. The measurements were made by a Resonance Instruments 'Maran' spectrometer operated at 20 MHz and ambient temperature using a Carr-Purcell-MeiboomGill pulse sequence with an interpulse spacing of 2 0 0 p The resulting signal envelope was resolved into its constituent relaxation time responses (up to two processes) using a nonlinear regression analysis package. 3 RESULTS AND DISCUSSION 3.1 Why some hydrocolloids form stable fluid gels.

Stable fluid gels can be produced from hydrocolloids that gel by extensive aggregation of ordered chains e.g. calcium pectate, calcium alginate, potassium kappacarrageenan, gellan, agarose and agar. If the amount of aggregation is reduced then stable fluid gels are not produced. The particles are stable and fluid gels are produced if shearing is continued throughout the ordering transition of the biopolymer. An example is that of agar (and agarose) which forms stable fluid gel dispersions if cooled to at least 30°C below the onset of ordering and subsequent aggregation. As the final quench temperature is raised an increasing amount of agar(ose) can be shown to aggregate after shearing has ceased, resulting in 'healing' of the originally fluid dispersion. Hydrocolloids which form stable fluid gels are the systems that want to de-water and effectively precipitate from solution (ie. there is phase separation (de-mixing) into polymerrich and polymer-poor regions). At common cooling rates, in the absence of shear, the

262

Gums and Stabilisers for the Food Industry

system becomes kinetically trapped to form a three dimensional structure. It has recently been concluded" that the phase separation event in the formation of agarose gels takes place by a spinodal decomposition mechanism. It is still not clear whether this is the correct mechanism. It is evident from a number of techniques that the ordering of agarose into the double helix conformation, which is required for aggregation, is under kinetic control, and is also probably very much governed by polymer fine structure. Therefore, a nucleation and growth mechanism can not be ruled out. Whichever mechanism is responsible for the initial stages of demixing, it appears that particles start to form in the early stages of the aggregation process, during which the nuclei are subjected to shear forces and will appear to behave as water-in-water emulsion droplets. The droplets will therefore be undergoing coalescence and break-up, which will produce an equilibrium droplet size. The extent of coalescence and break-up in classic emulsions is governed by the interfacial tension of the droplets and the nature of the interface. In recent work we have shown that in gelling systems of phase separated biopolymers, the balance of coalescence to break-up is also dependent on the elastic modulus of the droplet". These droplets become more resistant to break-up as gelation proceeds, due to increases in their internal elastic modulus, and so more shear stress is required to break-up the droplet/particle. When the shear stress is insufficient to promote droplet break-up the equilibrium droplet size will increase until the droplets are too elastic to coalesce, and so control the final droplet size. Experimental results indicate that the temperature at which coalescence ceases (for agar 1253 at a concentration of 3% and a cooling rate of lS"Cmin-') is between 37SoC-35"C. At this temperature approximately 60% of the chains have still to undergo the disorder-order transition. This would indicate that disordered hydrocolloid chains at or below this temperature are present within the already formed particles which upon further cooling would then increase the strength of those particles. If shearing is stopped at a temperature above that of complete ordering, but below 35"C, then the resultant structure is not that of sheared gel, but is one in which the particles are strongly bonded to each other. This gives rise to a gel which is paste-like and fails at quite low distance scales. In support of the hypothesis that the disordered chains are situated inside the preformed particles it is found that if the shear rate is altered (increased or decreased), but not removed at that same temperature in a repeat experiment, the resulting particle size is not affected. Upon cessation of shearing the particles become connected together by aggregation. Conversely, if the shear rate is changed above 35"C, then a proportion of particles are formed whose size is governed by the new shear flow conditions. If shearing is stopped at such temperatures, then upon further cooling a structure is formed which has the properties of a quiescent gel. The origin of this structure is presumed to be a developing connectivity between the pre-formed particles driven by the potential for nucleation in the ungelled matrix. It appears that in a shear field the demixing into polymer-rich and polymer-poor regions is taken to its extreme, and the polymer-poor region acts as a depleted layer between the particles. This layer imparts long term stability to the system, giving a barrier to interaction of polymer chains of separate particles. The extent of 'phase separation' has been investigated using N M R relaxation time measurements. Spin-spin (TJ relaxation times are sensitive to molecular motions of the water molecules in carbohydrate gels. In general, the mobility depends on the polymer concentration and therefore N M R relaxation measurements can distinguish free water from water in gel particles (fluid gels). Knowing that the rate of diffusion of water is D = 2.5. lo" m2.s1and the typical T2 values for proton, the domain

263

Processing Developments

sizes that can be discriminated by NMR relaxation are calculated from the diffusion equation: a2 = 2Dt where a = distance, t = relaxation time. In practice, by gently centrifuging agar fluid gels in NMR tubes, under conditions where no liquid layer could be separated from the quiescent gels, it was possible to separate a low viscosity upper layer from a high viscosity lower layer that contained the gel particles. N M R measurements on the tubes from the centrifuge showed that the proton signals could be analyzed in terms of two relaxation processes (Table 1). The rapidly decaying signals were the same as those observed for the quiescent gels while the longer T2values show that the low viscosity liquid observed on the surface of the fluid gels is a low concentration polymer solution. Given that the T2value for pure water at room temperature is about 2 seconds, we can calculate that the low viscosity liquid contains about 0.1% of Agar in the 1% fluid gel and about 0.2% of Agar in the 3% fluid gel. In contrast to agar, the liquid phase separated by centrifugation from agarose fluid gel was identified to be pure water by its relaxation time. These results show that the fluid gels are heterogeneous in nature such that some of the water exists in regions which are polymer poor, probably in the interstitial spaces between the particles. Stability of fluid gel particles is also dependent upon storing at temperatures which are well below the equilibrium melting temperature (T,,J. It is difficult to form stable gelatin fluid gels for this reason. Figure 2 shows that when gelatin is shear m l e d to 2"C, and stored at this temperature there is evidence of a re-healing process, but the structure only has an elastic modulus 1/3rd that of a quiescently cooled gel held at the same temperature for a period of 16 hours. However when the same fluid gel is cured at 15°C it very quickly adopts the same gel properties as the equivalent quiescent gel. For hydrocolloids whose equilibrium melting temperatures are higher than that of gelatin eg. agar and kappa carrageenan (due to the increased extent and length of aggregates), only small increases in elastic modulus are seen when heated to temperatures close to Tmhand subsequently re-cooled (Table 2). The amount of increase in G' is linearly related to the amount of helical melting of the agar, and indicates that the elastic modulus of the fluid gel is simply related to the amount of network formed between the particles. The value is close to that expected when the values of G' for the quiescently cooled gels are compared.

Table 1 :Proton relaxation NMR T2 measurements of Agarjluid-gel.

I

T, (msec).

Proportion of signal 70 %

[Agar] 1

30 % I

118 (polymer rich)

I I

834 (polymer poor)

264

Gums and Stabilisers for the Food Industry

Table 2: Efect of heating on the stability offluid gels (Shear-cooled at 500 rpm and at 2"Clmin).

Quiescent Gel

60

6.85

273

2188

8.01

(G' = 51600) k carrageenan (G' = 6700)

70

18.53

347

5432

15.65

50

6.23

765

1656

2.16

G' ( P a )

*'O0O

I

1,500

1.000

500

20,000

0

40,000 Time (s)

60,000

80,000

Figure 2. 3 % gelatin gels shear-cooled @lied symbols) and quiescently cooled (open symbols) cured at 2°C (.:El) and 15°C (a:O).

""0

20

40 60 80 Temperature (C).

100

120

Figure 3. Calorimetric heating curves (0.S"Clmin) for 3% agar gels produced quiescently (0)and at 750s.' (a).

Processing Developments

265

3.2 The difference between gels and fluid gels. 3.2.1 At the molecular level. The total amount of ordering in the fluid gel is unaffected by the shear field. The extent of molecular ordering determined by DSC measurements was found to be the same for both quiescent and fluid gels. For example, the melting enthalpies for 3% Agar were 10.6 & 0.6 and 10.1 0.7 kl mole residues-' for the fluid gel after shearing at 40s'' and 750s'' respectively and was 9.8 f 0.5 kJ mole residues' for the quiescent gel. As the enthalpy of the transition is dependent on the amount of helix formed and not on the extent of the aggregation process this shows that shearing has not affected the coil to helix process. The shape of the peaks (figure 3) indicates that although the overall helix content is the same for both samples the fluid gel has a significant fraction of ordered helices which start to melt at lower temperatures ie. around 30°C, and the overall melting is about 2°C lower. This shows that the entropy change for the transition is less negative for the fluid gel (ie. there is more disorder in the fluid gel than in the quiescent gel) which is consistent with fewer or smaller aggregates and suggests that the shearing process has limited the side by side packing of the helices. This would be expected if the chain interaction has been limited by the shear such that perfect side by side alignment can only occur within a particle and not between particles. 3.2.2 Bulkproperrics. As expected, the elastic modulus (G') for the fluid gel is lower than for the quiescent gel (figure 4) by about two orders of magnitude. However, at low strains the fluid gel has solid like character, but then fails as the strain is increased to between 5 and 10%. Thus the fluid gels exhibit yield stress behaviour. This was demonstrated by studying the flow curves (figure 5) in which all concentrations of agar between 1 and 5 % show an apparent yield stress which increases with concentration, followed by a transition region and finally, at higher shear rates (> -20i1),the materials have a Bingham viscosity which increases with concentration of the agar. The effect has been shown to be reversible with the yield stress recovering back to its original values after a few minutes in the absence of shear. These rheological findings imply that as we stress the systems initially the particles have weak interactions and are capable of storing energy and resisting the applied forces. As the strain or stresses are increased the particle/particle interactions are disrupted and the system flows (ie. behaves as a liquid). Upon re-heating the 3 % agar fluid gels the G' increases at temperatures above 40°C (figure 6) before decreasing as melting of the ordered molecules occurs at the normal temperatures ie. '90°C. Such an increase in G' seems to be a consequence of the particles swelling with temperature as the hydrocolloid becomes more soluble. This would lead to the system forming a greater phase volume of particles rather than relaxing towards the quiescent gel state arising from an increase in the extent of aggregation between the particles. However, as discussed above, if the systems are subsequently re-cooled, then particle-particle aggregation is possible if the disordered coils are spatially arranged such that re-ordering and aggregation takes place between particles. Thus the most likely explanation is that the system becomes more dynamic resulting in some molecular ordering/ aggregation of helices occurring between the particles.

266

Gums and Stabilisers for rhe Food Industry

...

100.000 .

I

.

.

I

.

0

0

0

0

.

,

r,

10,000 ;

1,000~0

100

10

0

.

r 7

0

0

C'

0

0

O

.

I

"

O

1

,

8

-

-,

Figure 4. Strain dependence of Dynamic moduli of I . 75 % agar gels. (G' =filled symbols: G" = open symbols). Fluid gels (0:O) and quiescent gels p:O). S h e a r stress ( N / m * 2 ) 300 . x

250

.

x

x

n

0

'

bx

150 100 :

0

20

0

O

40

D

o

60

0

100

80

0 0 0 0

1 0 ,0 0 0 :

1,000 r

0

......'. ...-..

100 r 10; 1

O

I

I = =1.111.1=

I =

D

0

0

1 1 1

261

Processing Developments

The dependence of bulk properties of fluid gels on the particle interactions is further supported by comparing different concentrations of agar/agarose sheared at the same shear rate. We find that as the concentration of polymer is decreased the number of particles that are produced also decreases and therefore the phase volume of the depleted layer increases. If the particles are diluted, free water is observed spontaneously below 60%phase volume of particles ie. at phase volumes below the packing fraction, and the G' value falls by two orders of magnitude upon similar dilution. The irregular shape of the particles contributes to the bulk roperties as upon dilution they do not follow the Hertz relation~hip'~ (agar spheres do ) and can be fitted by the Kreiger-Doherty equation*', but with a [ ~ exponent l]+ greater ~than that for model spheres.

Po

4 CONCLUSIONS AND CURRENT MODEL

4.1 Formation of fluid gels.

Hydrocolloids that form gels via extensive aggregation of ordered chains and are stored at temperatures that favour the ordered state produce stable fluid gels if shearing is continued through the whole of the ordering process. If either gelation does not involve aggregation or the system is close to the equilibrium for the disordering transition then unstable particles are produced that rapidly loose their identity and the system relaxes back to the quiescent gel once shearing is stopped. Thus fluid gels are a consequence of aggregate formation by hydrocolloids that are de-watering and trying to precipitate from solution (the hydrocolloid is "trapped" during de-mixing into polymer rich and polymer poor regions). 4.2 Stability of fluid gels. Under quiescent conditions the system is kinetically trapped to form a three dimensional structure. This produces a gel rather than allowing the biopolymer to precipitate. When the polymer chains are forced to order and aggregate while being sheared then helix formation and aggregation are forced to occur within small areas of space, determined by the shear fields, thus giving rise to gel particles. Within this disruption of the ordering process small amounts of solution low in polymer concentration are produced and less extensive alignment and aggregation of the helices occurs. As the hydrocolloids are trying to de-water the polymer poor solution in the interstitial space between the particles acts as a depleted layer. This then imparts long term stability to the fluid gels (i.e. it stops the polymer chains from separate particles becoming aggregated together which, with time, would lead to relaxation of the system back to the quiescent gel structure). As there will be a limited number of contacts between the particles and chain misalignment between the aggregates from the different particles we would expect there to be weak particlelparticle interactions which can be disrupted at low shear rates and shear stresses, thus leading to the weak gel nature of the materials. The overall rheology/viscosityof the system is a consequence of particlelparticle interactions caused by the high phase volume produced from the shear device. If the fluid gels are warmed

Gums and Stabilisers for the Food Industry

268

and the biopolymer becomes more soluble the particles swell giving an increased phase volume of particles and stronger particle/particle interactions.

Acknowledgements We would like to thank S.Ablett, A.Darke, W.J.Frith and P.Knight for carrying out the experimental work. References 1, 'Industrial Gums - Polysaccharides and Their Derivatives', eds. R.L. Whistler and J.N. BeMiller, Academic Press, 1959. 2. M. Glicksman, 'Gum Technology in the Food Industry', Academic Press, 1969. 3. 'Food Structure - It's Creation and Evaluation', eds. J.M.V. Blanshard and J.R. Mitchell, Butterworths, London, 1988. 4. 'Food Gels', ed. P. Harris, Elsevier Applied Science, London, 1990. 5. D.A Rees, Polysaccharide conformation, In 'Carbohydrates', MTP International Review of Science, Organic Chemistry, Series 1, ed. G . 0 Aspinall, Butterworths, London, 1973. 6. F.M. Goycoolea, T.J. Foster, R.K. Richardson, E.R. Morris and M.J. Gidley, In 'Gums and Stabilisers for the Food Industry 7 ' , eds. G.O. Phillips, P.A. Williams and D.J. Wedlock, IRL Press, Oxford, 1994, p333. 7. K. Nishinari, In 'Gums and Stabilisers for the Food Industry 8', eds. G.O. Phillips, P.A. Williams and D.J. Wedlock, IRL Press, Oxford, 1996, p371 8. M.J. Gidley, N.D. Hedges, R.A. Hoffmann and C.P. Dawes, In 'Gums and Stabilisers for the Food Industry 7', eds. G.O. Phillips, P.A. Williams and D.J. Wedlock, IRL Press, Oxford, 1994, p3. 9. N.S. Singer, S. Yamamoto and J. Latella, European Patent No: EP 0250623, 1986. 10. F.C. Bing, Journal of the American Dietetic Association, 1976, 69(5), 498-505. 11. A.C. Hoefler, J.A. Sleap and J.E. Trudso, European Patent No: 0501758 A l , 1992. 12. F. Schierbaum, B. Kettlitz, S. Radosta, F. Reuther, M. Richter and W. Vorwerg, Acta - Alimentaria - Polonica, 1984, 10(1/2), 69-99. 13. R.D. M'Cormick, Food Product Developmcw, 4(4), 19-20, 22 & 26. 14. C.R.T. Brown and I.T. Norton, European Patent No: EP 355908, 1991. 15. N.D. Hedges and I.T. Norton, European Patent No: EP 432835, 1991. 16. W.F. Chalupa, US Patent Application: US 94-265524, 1994. 17. A. Emanuele and M.B. Palma-Vittorelli, Physical Review Letters, 1992, 69, 81-84. 18. T.J. Foster, J. Underdown, C.R.T. Brown, D.P. Ferdinand0 and I.T. Norton, In 'Food Colloids - Proteins, Lipids and Polysaccharides', eds. E. Dickinson and B. Bergensml, The Royal Society of Chemistry, Cambridge, 1997, p346-356. 19. H. Hertz, Gesammebe Werke, Leipzig, 1895. 20. 1.M Kreiger, Adv. Colloid and Inreface Sci., 1972, 3, 111. 21. W.J. Frith and A. Lips, In 'Proc. XIIth Congr. on Rheology', eds. A. Ait-Kadi, J.M. Dealy, D.F. James and M.C. Williams, Canadian Rheology Group, 1996.

DEVELOPMENTS IN PROCESS TECHNOLOGY AND THEIR POTENTIAL USES

-

Dr. Peter J. Wallin Dalgety plc, Station Road, Cambridge, CB 1 2JN, UK TelOl223 359181, Fax 01223 360712

1 INTRODUCTION Generally the food manufacturing industry is conservative and slow to adopt new process technologies. Some of the technologies used today are the same as they were more than 50 years ago as illustrated in Table 1.

soups Stetilisation Cannin& retorted products Fermentation Beers, milk products Meats, pasta, biscuits and hit Dehydration Salt, pickles and other preservatives V&US foods cooking Meats, fish, vegetables, etc. Fruit, vegetables, ready meals, meats and fish Freezing Meats, pasta, etc. Modified atmosphere packaging Table 1. Typical Traditional Food Processes Obviously, there are some reasons for this and there have been some improvements, in particular with respect to computer controls, hygiene and safety. There is now however a move by food companies to explore some of the newer emerging technologies in order to gain a competitive edge either by cost reduction or through the production of new and novel products. The more outward approach towards alternative processing methods has been partly driven by market trends and consumer requirements and preferences for products such as:Healthy and more natural foods Convenience foods - ready meals and snacks Functional foods Nutraceuticals

Gums and Stabilisers for the Food Industry

270

0

Improved quality of cooked or chilled foods New novel food products

-

There is, therefore, a requirement to investigate applications and utilise new process technologies in order to overcome some of the shortcomings of the more traditional methods of food processing and provide more ‘added value’. Figure 1 indicates the positioning of some typical food products and the move by some food companies towards more innovative and protected products. There are a number of new, or relatively new, food processing technologies which are being used as a means of providing ‘added value’ to products.

< u > i

COMMODITY Figure 1. Positioning of Products The type of food products which companies are looking to gain a technological advantage in are those which can provide differentiation, such as:0

0 0 0

0

Unique or different texture Control of appearance - colour New and natural designer aromas Flavour Functional ingredients

Below are some specific areas of process technology which are beginning to have an impact and are replacing and challenging the existing traditional technologies. The technologies below have the advantage of providing some of those differentiating attributes listed above. Preservation and cooking Microwavetradio frequency

Processing Developments

27 1

High pressure Pulsed light and high energy fields New Functionality Biochemical Separation technologies New products Cryogenics Extrusion Robotics

2 NEW TECHNOLOGIES The technologies are reviewed below along with how they could potentially be utilised to produce novel and innovative functional food ingredients thus enabling companies to ‘add value’ and potentially provide protection for their products. 2.1 Preservation and Cooking

2. I . I Microwaves. Although by no means a new technology’ most households now have a microwave to defrost, reheat or cook foods in the home. However, in the factory there are few companies utilising the technology. Early systems did have shortfalls as they had uneven fluxes which created hot or cold spots and therefore uneven cooking. Commercial manufacturing.systems have been developed to enable - the tempering of fruit and butter, pasteurisation and cooking of pate, drying of fruit and vegetables, ready meal processing, sauces and blanching of vegetables. Where there could be more interest is in the area of how microwaves interact with specific food ingredients and how such knowledge could then be used to create new food products. The improved control of heat input could be utilised along with the bulk heating effect to provide an alternative cooking method. 2.1.2 Radio-Frequency. This technology has been utilised for specific applications such as the drying or baking of food products like wafers and other biscuits. Equipment companies such as Petrie Technologies Ltd. have combined radio frequency baking with conventional convection heating (ARFA).

A more recent development by APV Pasilac AS has been that of cooking ham rolls.

This was carried out by passing them through a RF cooking system using a new pumping system. It is claimed that the process improves the taste, structure and water absorption ability. Similar systems have been developed by Procter in the USA. 2.1.3 High Pressure Processing. High pressure processing is an interesting development first proposed in 1899* and then being exploited by the Japanese through a technology transfer programme from the ceramics industry. The process has the ability

272

Gums and Stabilisers for the Food Industry

to produce texture and flavour changes in products along with some microbial kill in a one step process. A number of interesting effects such as the gelling of proteins have been reported3. The technology has the advantage of being able to process products in the pack but it does have a high capital and operating cost hence its use to date for niche products such as avocado and more recently premium fresh citrus juices4. It would be interesting for further developments to take place so as to allow similar effects to be produced but using a continuous system and at a lower capital investment. 2.1.4 Pulsed Light and High Energy Fields. There has always been an interest in being able to sterilise or treat chilled foods quickly and easily, at a low cost, so as to extend their shelf life. Radiation is very effective in achieving this and is permitted for use to treat certain food products. Other technologies which are being developed include the use of pulsed light’ and high energy fields6. The pulsed light system used an intensely bright light source and the food to be treated passes under the light and the spoilage micro-organisms are killed by the radiant energy emitted from the intense light source. The technology does work to some degree depending on the nature of the food and the level of microbial growth. There is a drawback in that shadowing can affect how much of the surface is treated. 2.2 New Functionality

Most food ingredients companies are looking at ways in which their products can be differentiated. The differentiation can take place in many ways; however, this paper focuses on the process and methods by which products are produced. It is interesting to note that the approach of many food ingredients companies is now becoming similar to that of the pharmaceutical and petrochemical industries in that chemical and biochemical types of processes are being employed and reactions specifically controlled to deliver product streams which can then be purified. In some ways it is similar to an oil refinery’s fractionating column. In most processes the raw materials are very cheap but significant value can be added by separating the raw materials into specific fractions, compounds, components, etc. Through the extraction and refining of components many thousands of times the price per ton can be added to the raw material value thus transforming a commodity into a speciality product. The major processes that are being utilised are those of fermentation or other biotechnological methods. This type of main process produces a mixed product and an effluent stream which require the use of advanced downstream processing. The further processing required is in the form of unit operations such as distillation, sedimentation, flocculation or filtration. Following this, a refinement or concentration stage is required to arrive at the product. Many new ingredients are either extracted, by the method described above, from vegetable based raw materials or synthetically processed.

2.2.1 Biochemical. The use of biotechnology for the processing of foods is not new but its application has become more widespread as more sophisticated and functional ingredients have been required by the major food sectors such as confectionery and snack foods.

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273

From an equipment and processing point of view the equipment used is fairly standard but it is the control and knowledge of the processes required that has moved forward. More advanced controls and process monitoring assists to control the process more accurately. The downstream processing has developed significantly and the developments in separation using membranes has made an major impact. The production of ceramic materials and composite materials has allows one to accurately control the pore sizes of the filter enabling separation even down to specific molecular weights.

2.2.2 Separation technologies. The dry separation technologies used in the food industry have been very traditional such as the milling process. There have been some advances and improvements in the design of roller mills but the fundamentals have remained the same. More recently there have been some significant developments such as the Satake’s PeriTec mill7 for the debranning of wheat. There have been a number of improvements in the design of air classification systems through the improved control of key operating parameters such as feed rate and air velocities. Another major advance in separation technology’ has been the use of membranes. This technology has allowed the selective separation of components even at a molecular level. New membranes are being developed to reduce costs, to increase efficiency of separation and to reduce blinding. There are also developments in dry separation which allow the fractions from a milling process to be segregated according to their shape and density. These air classifiers have been improved through an improved understanding of the process and operating conditions. Modelling tools have allowed flow visualisation to assist and improve knowledge.

2.3 New Products The technologies detailed in this section have been specifically selected because they illustrate how new technologies can be utilised to produce new products.

2.3.1 Cryogenics. Over recent years there have been some major developments in freezing technology. Now many freezers use liquid nitrogen’ rather than mechanical freezing systems. This type of system allows a more rapid freezing of products which can provide a ‘fresher’ product. It also allows the freezing of intricate and more delicate shapes and food structures.

2.3.2 Extrusion. Extrusion technology is one of the oldest forms of processing systems and yet it is still one that holds many opportunities for new product development and innovation. A number of equipment companies have developed advanced co-extrusion systems that allow fillings to be encapsulated within a crunching outer shell. Other systems allow a multi-laminate to be formed with an extruded filling and then profiled through a forming die.

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Gums and Stabilisersfor the Food Industry

2.3.3 Robotics. Although, when robotics are mentioned one does not immediately think of food ingredients or hydrocolloids, the development of applications has lead to the robotic icing of cakes", and this does lead one to think about the rheological behaviour of fondant or creams. When these types of materials are pumped under pressure they can display some quite interesting characteristics when forced out through a shaped nozzle. Other applications have been for handling meat, packaging of assorted chocolates and the portioning and trimming of fish and chicken fillets. 2.4 Other Process Developments

There are other technological developments" some of which are carried out by entrepreneurs or equipment companies. A small company, Revtech, have designed an 'Ohmic' type drying system for solids. There are also new technologies which can be utilised to treat or process materials to improve the product quality. New drying processes have been developed to allow retention of flavours or the control of texture when re-hydrated. The technology of encapsulation for food ingredients is an interesting technology but it is to some extent still in its infancy and it utilises mostly existing technology from spray drying and coating. New technologies could be employed to produce coated products in which the coating has functional properties such as osmotic or selective adsorption properties. Encapsulation is used in the pharmaceutical industry to affect the slow release of drugs into the blood stream. The same type of process could be used in food processing to ensure the release or reaction of ingredients or flavours at the right time within the process. This could have a significant effect on the product's attributes such as the colour, texture or aroma.

3 CONCLUSIONS Within the food industry new product development tends to concentrate on the use of new ingredients and on recipe or packaging changes and it is seldom that the processing technology is seen as an appropriate driving force. Process technology does open up the door to allow greater differentiation of products and the potential to protect them. However, the effort of developing a new machine or technology can take a considerable time and expense, often 5-10 years, at a cost of several E millions and there is some risk. Therefore, very few food companies can afford to embark on such projects. A lower cost and quicker route to technology development could be utilising technologies from other industries and adapting or re-using it. This then provides a quicker and lower risk option. It is important with any new technology that more is known about the equipment design and how it interacts with the product or ingredients. This field is usually

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275

unexplored but could hold the key to how some of the newer technologies could be successfully applied so as to enable product differentiation and a cost benefit. References 1. Sperber, ‘Microwaves pasteurize refrigerated entrees, Food Proc., 1988, Dec, 156-

162. 2. B.H. Hite, West Virginia Agricultural Experimental Station, Bulletin, 1899,58, 15. 3. D. Knorr, ‘Hydrostatic pressure treatment of food’, New Methods of Food Preservation, Edited by G.W. Gould, Blackie Academic and Professional, London, 1995, ~ ~ 1 5 9 - 1 7 5 . 4. Anon, ‘High-pressure Juice’, Food Eng. Znt., 1997, June, 22,3. 5. Dunn, T. Ott and W. Clark, ‘Pulsed-Light Treatment of Food and packaging’, Food Technology, 1995, Sept., 95-98. 6. ‘FDA clears Coolpure cold-pasteurisationprocess’, Food Eng., 1995, Oct, 32-34. 7. Satake U.K. Ltd., ‘Technical profile: wheat debranning process’, World Grain, 1996 8. ‘Separation processes in the food and biotechnology industries’, A.S. Grandison and M.J. Lewis, Woodhead Publishing Limited, Cambridge, 1996. 9. Anon, ‘Food Freezing doubles production’, Processing, 1997, May, 13-14. 1 O.Anon, ‘Robots in the Bakery’, Control Systems, 1997, June, 17. 11. ‘Food Processing - Recent Developments’, A.G. Gaonkar, Elsevier Science bv, Amsterdam, 1995.

-

TIGER STRIPING IN INJECTED POULTRY ITS CAUSES AND CURES

K.Philp', Z.DeFreitus2, D.Nicholson3, R.Hoffman4. I

Quest International, Kilnagleary, Carrigaline, Co Cork, Ireland. Quest International, 2402 7th street, NW, Rochester, MN 55901, USA. 3Quest International, NABTC, 5 1 15 Sedge Blvd, Hoffman Estates, IL 60192, USA. 4 Unilever Research, Colworth House, Shambrook, Bedford, MK44 1LQ, UK. 2

1 ABSTRACT

Optical and X-ray microscopy were used to determine that the common product defect found in injected poultry known as tiger striping, zebra striping, feathering or striations is due to swollen particles of hydrocolloid binder forcing the meat fibres apart. It has been shown that the swelling properties of carrageenans produced from different manufacturing processes had significantly different swelling profiles. Semi refined carrageenan was noted to have a greatly reduced ability to swell in an aqueous system and this was attributed to the fact that the material is processed in a suspension and the original cellulosic matrix is left intact and is not removed as in a traditional refined carrageenan process. The semi refined carrageenan was then shown to have an improved performance in injected poultry over traditionally refined carrageenan by reducing the incidence of tiger striping without reducing the purge control.

2 INTRODUCTION Carrageenans are sulphated linear polysaccharides extracted from red seaweed of the class Rhodophyceae. In the seaweed itself the carrageenan performs many functions including acting as a water reservoir to prevent desiccation, acting as a cation-exchange barrier and providing a degree of general mechanical protection. Carrageenan has been used in food preparation for many hundreds of years and traditionally Chondrus Crispus has been collected from the shores of Ireland for use in a local milk pudding. A tradition that is still carried on today. It has been regularly asserted that the name carrageenan derives from the Irish county Carraghen, as there is no such county this can only be a myth. The name is more likely to be connected to the Irish diminutive for rock or pebble. Commercially, carrageenans are extracted from several species of red seaweed, each specie will yield a different ratio of the three basic carrageenan types; kappa, iota and lambda. Traditional processing technology involved the dissolution and conversion of the carrageenan in hot alkali, filtering, concentrating, precipitating via various means and then drying and grinding. More recently a different technology has evolved in which the

271

Processing Developments

carrageenan is treated in a solid slurry rather than in solution. This technology produces a carrageenan sold as E407a or ‘processed eucheuma seaweed’ in markets that follow European legislation but simply as carrageenan in the USA and markets that follow FDA legislation. Hydrocolloids are commonly used in the meat industry. Their incorporation into meat products helps to modify the texture of products, improve the sliceability, increase the yield and reduce the purge. However the use of hydrocolloids can also have some drawbacks, under some processing conditions hydrocolloids are known to cause tiger striping. Tiger striping is characterised by broad dark striations running parallel with the meat fibres. The striations usually appear as voids in the meat filled with gel. The aim of this research was to identify the causes of tiger striping and determine how they could be minimised by the correct selection of binder and/or conditions.

3 MATERIALS AND METHODS 3.1 Brine preparation An amount of water was stirred with a high shear mixer and the phosphate added and stirred for one minute. Next the salt and dextrose were added and the mixture stirred for a further minute followed by the carrageenan. The whole mixture was stirred for two minutes and then enough ice was added to reduce the temperature to near O°C.

Typical brine recipe:

Water Sodium tripolyphosphate Salt Dextrose Carrageenan Ice

74.0 1.3 6.9 6.9 2.2 8.7

3.2 Production of turkey hams

The turkey breasts were injected to the required level by a two pass injection method. Two breasts were then macerated to help protein extraction and vacuum tumbled for 1 hour. The breasts were then placed in ‘cook-in bags’ and heat shrunk in boiling water for three seconds. The turkey breasts were then cooked in a controlled humidity oven through a cycle of 52OC .for 1 hour, 6OoC for 1 hour, 68OC for 1 hour and then 77OC until the internal temperature of the breast has reached 7loC. After cooking the breasts were cooled and stored in ice overnight. 3.3 Quantifying ‘tiger striping’ The individual turkey breasts were sliced into 1” slices and alternate slices taken and each face stained with 0.1% methylene blue in 50% iso-propanol which highlighted the areas of carrageenan concentration. Each breast would give eight faces for analysis and

278

Gums and Srabilisers for the Food Indusrry

each breast was duplicated. The percent of tiger striping was calculated as the striped area in the meat over the total area.

3.4 Microscopy of turkey breasts Slices of the turkey breast were stained with 0.05% toluidine blue to highlight the carrageenan concentrations for optical microscopy and stained with rhodamine to enhance the fluorescence for confocal microscopy.

4 RESULTS AND DISCUSSION

4.1 The cause of tiger striping Tiger striping is a common product defect in the USA and upon examination of injected poultry products from a variety of regions it clearly occurs world-wide. To define what causes tiger striping we first need to identify the composition of a given tiger stripe.

Figure 1. Photograph of partly stained turkey breast showing substantial tiger striping. One tiger striped area is labelled for identification (labelled A). It is clear from the turkey slice (figure 1) that the heavily striped area of meat is concentrated in the outer layers of the breast. The areas with greater levels of striping also appear to have higher concentrations of carrageenan, recognised by the greater level of staining. Optical microscopy of the stained turkey sections shows (figure 2) that the tiger stripe does not consist of an homogenous region of gel but rather a densely packed region of swollen binder particles. Confocal microscopy showed the particles to be packed in

Processing Developments

279

between bundles of the meat fibres. X-ray SEM confirms the presence of carrageenan in the striped area from the autofluorescence of the high concentration of sulphur atoms (figure 5).

Figure 2. Photographs of striped area of turkey breast. Toluidine blue stained tiger stripe is labelled A. If the swelling of the binder particles in between the meat fibres forcesthe meat fibres apart this could create a local pocket of high carrageenan concentration. Furthermore it implies the potential for striping of a binder is related directly to its swelling properties in a typical cooking cycle. 4.2 Particle swelling

In the preparation of brine for injected products carrageenans are added to the brine after solubilizationof the phosphate, salt and dextrose. This sequence is used to decrease the solubility of the carrageenan. Consequently,carrageenans are only dispersed upon injection and only during thermal processing will they swell, solubilize and finally gel. However, different types of carrageenan present different swelling profiles. To measure this the sluny viscosity was determined during a heating cycle. As a particle swells in a suspension, the measurable viscosity of the slurry increases. Therefore, one can measure and compare the swellingprofiles of carrageenans under differentconditions. Two different forms of traditionally refined carrageenan were tested alongside the new ‘semi-refined’ technology. Alcohol precipitated grade carrageenan is isolated by precipitating the carrageenan from aqueous solution with iso-propanol. This leads to a very open structure which gives the carrageenan the ability to swell readily, even in a cold brine. From the graph (figure 3) it can be seen that the alcohol precipitated grade starts to swell very early compared to the other grades and generates a very high viscosity suggesting that very large swollen particles are formed prior to dissolution. Carrageenan from a roller dried process is concentrated and then directly dried on a hot roller rather than by precipitation. This process tends to make denser particles which are less prone to swelling in brine. The roller dried material is inhibited from swelling initially but during the cooking cycle it swells to form particles as large as those from the alcohol precipitated

Gums and Stabilisers for the Food Industry

280

grade. The semi refined carrageenan shows very different behaviour. The swelling is greatly inhibited and a much higher temperature is required to obtain any swelling at all. Even at higher temperatures the semi refined particles never swell to the size of either of the traditionally refined materials. Viscosity (Brabender RVF(SNU))

600

4% carrageenan in 0.1M KC)

n

500 400 300

\\

,Semi

refined

200 100

0

20

35

50

65

80

95

Temperature ("C)

Figure 3. Swelling profile of carrageenans produced by different processing routes. The semi refined carrageenan is processed suspended in KOH solution and the carrageenan molecule never fully dissolves. One of the by-products of this processing is that a small amount of residual seaweed cellulose remains in the carrageenan particle. This cellulose is removed in processes that dissolve the carrageenan as a route to refined carrageenan. Processes that use an alcohol suspension to prepare 'refined' carrageenan coincidentally normally process weeds that inherently have very low levels of residual cellulose. The cellulose remaining in semi refined carrageenan forms a three dimensional matrix which restricts the swelling of the particle. The carrageenan is still functional because upon heating the carrageenan partially dissolves and leaches out from the cellulose matrix.

4.3 Salts concentrations and particle swelling The effect of sodium chloride concentration on the swelling profile was determined for a roller dried carrageenan sample. During heating the samples swell to a peak viscosity and then the particle begins to break up and dissolve and the viscosity drops. The addition of salt reduces the peak viscosity due to the swelling of the carrageenan and also increases the temperature required for peak swelling. What is important is that with a standard brine we have say 6.9% sodium chloride, sugars and the phosphate which is likely to give us a

Processing Developments

28 1

peak swelling of around 65’C to 75’C which unfortunately is close to the peak cooking temperature for products such as turkey hams. Hence the typical processing temperature for turkey hams exacerbates the tiger striping problem by providing conditions close to optimal for swelling, (Figure 4).

Viscosity (SNU) 250

;-

-

.

-----

~-

_ _ -

0%

-1I i

200 150 100

50 0

25

42

55 65 85 Temperature (“C)

95

4% suspension in various % salt

Figure 4. The effect of sodium chloride concentration on the swelling profile of a traditionally processed carrageenan. In the turkey meat itself the salts are not necessarily evenly distributed. Using electron microscopy (figure 5) the autofluorescence for sodium across a freeze dried tiger stripe showed the sodium ions to be evenly distributed across both the tiger striped area and the muscle tissue itself. The sulphur autofluorescence clearly shows a greater concentration of sulphur atoms in the tiger stripe area rather than in the muscle itself. This indicates that the carrageenan is not evenly distributed throughout the meat sample but has concentrated areas in the stripe. The potassium atoms are interesting in that they seem to remain associated with the carrageenan and do not diffuse throughout the meat protein. This is an important result because of the effect potassium has on the gelling properties of the carrageenan. The carrageenan effectively seeing a higher concentration of potassium than the average will swell less than may be expected if it was assumed all the ingredients were evenly distributed around the meat sample.

Gums and Stabilisersfor the Food Industry

282

Figure 5. X-ray micrograph of the edge of a tiger stripe. Top left going clockwise are electron micrograph, sodium autofluorescence, sulphur autofluorescence and potassium autofluorescence. 4.4 Concentration and carrageenan type A factorial combination of three differently processed carrageenans (alcohol precipitated, roller dried and semi refined) and three different concentrations (0.3, 0.4 and 0.5%) were evaluated to determine how concentration affects carrageenan striping for the different manufacturing processes, (Figure 6). Striped area (%)

w Roller dried W Semi refined

Carrageenan concentration (YO)

Figure 6

The effect of concentration and manufacturing process on tiger striping.

Processing Developments

283

It is clear that roller dried and alcohol precipitatedcarrageenanboth have very similar performances and in increasing the concentration from 0.3% upwards causes an increase in the level of tiger striping observed. However the semi refined carrageenan showed a much lower level of tiger striping at all concentrations and furthermore the level of tiger striping was not greatly increased with increased concentration of carrageenan. This result means that semi refined carrageenan can be treated in formulations in a more robust manner without risking a significant increase in striping level.

4.5 Cooking temperature A factorial experiment of two carrageenan types (roller dried and semi refined) and four internal temperatures (50, 60, 70 and 7 7 C ) was used to determine the effect of cooking temperature on tiger striping. All products were run at 0.5% carrageenan concentration,(Figure 7). Degree of striping (%)

-" I

I

15

10

5 n

50

60 70 Internal temperature ("C)

77

Figure 7 Effect of cooking temperature and carrageenan type on the level of tiger striping. At 5OoCthe carrageenan does not appear to have swelled significantly and has yet to force any of the meat fibres apart. However at 6OoCthe particles have reached their peak swelling and the level of visible tiger striping has increased to a maximum. At higher temperatures there is a slight decrease in the striping level, most probably due to some of the carrageenan solubilising and leaching away from the larger gel pockets. 4.6 Purge control

Obviously of crucial importance to the meat processor is how well the carrageenan performs in the reduction of purge from the products. To assess this carrageenan from three different types of processing was studied at three levels, 0.3% which is below the

Gums and Stabilisers for the Food Industry

284

normal usage level and 0.4% and 0.5% which represent more typical usage levels in industry, (Figure 8). Purge loss (%) 12 10

IRoller dried 0 Semi refined

8 6 4 2

0

0.3 0.4 0.5 C a r ra g e e n an con cent rat io n (YO )

Figure& Purge loss from turkey hams made with different levels and types of carrageenan.

At very low levels the semi refined and alcohol precipitated grade both perform poorly. The roller dried grade appears to give a better performance. At 0.4% usage levels the semi refined material actually performs better than either of the traditionally refined grades. At the higher levels both roller dried and semi refined provide better purge control than a traditionally refined product.

4.7 Model for tiger striping formation Most carrageenan types for injection have similar particle sizes in the powder form, usually below 75p.However what is important to a meat processor, from a point of view of potential needle blockage, is the particle size going through the needle. In the case of alcohol precipitated grades this is invariably greater than 75p due to the fact the material swells in cold brine to a certain extent. Neither roller dried or semi refined swell in cold brine and are therefore excellent grades to prevent needle blockage, (Figure 9).

285

Processing Developments

Roller dried

I

lcohol precipitated

Semi refined

Powder

In Brine

0

0

In Cooked meat

0

0

0

*

Figure 9. Representation of the swelling of different carrageenan types during a meat processing cycle.

During injection the particles are forced into areas between the meat fibres and are packed tight. The level of tiger striping is related to the amount of swelling from this point to the end of cooking. The alcohol precipitated grade has already swollen to a limited extent but during cooking swells much more and this can produce striping. The roller dried grade is very similar because it will swell substantially upon cooking and force the meat fibres apart. Semi refined carrageenan is very different in that swelling is almost non existent during the cooking cycle so there is little to force the fibres apart. This coupled with the lack of needle blockage points to the fact that the ultra fine 75p material commonly used in meat injection applications is unnecessary and a slightly coarser grade could be as functional and prove more cost effective.

5 CONCLUSIONS A model mechanism is proposed to explain the formation of tiger striping in injected meat products. The mechanism depends on the swelling nature of carrageenan particles and the relative degree of swelling from the moment of injection to the final product. Clearly from this mechanism semi refined carrageenan with its greatly restricted ability to swell in aqueous systems should, and does, show a greatly reduced level of tiger striping. Experimental evidence shows that in injected poultry systems semi refined carrageenan offers better purge protection than alcohol precipitated carrageenan. The lack of swelling in semi refined carrageenans greatly reduces the problem of needle blockage and allows the use of a coarser grade which enhances the cost effectiveness nature of semi refined carrageenan further. The combination of excellent purge protection and no tiger striping problems combined with the price advantage of semi refined carrageenan makes semi refined carrageenan the binder of choice for the cost and quality conscious meat processor.

EFFECT OF STABILISERS AND WATER CONTENT ON THE RHEOLOGICAL PROPERTIES OF PREFERMENTED FROZEN WHEAT DOUGHS

J. RWnen and K. Autio

v?T Biotechnology and Food Research PO Box 1500 FIN-02044 VTT, Finland

1 INTRODUCTION Frozen doughs are widely used in industrial bakeries to make baking more profitable. Transportation is facilitated and labour costs are reduced as fresh breads are baked in bakeoffstations when needed by consumers. However, loaf volumes are usually smaller and quality is poorer for breads baked from frozen doughs, especially in those with low fat content. The major changes in the baking quality of frozen doughs are caused by the loss of yeast viability and decreased gas-holding capacity. When doughs have been fermented before freezing, the need for gas production after thawing is reduced. Prefermented doughs are easier to handle than unfermented, because products will freeze and thaw faster than dense dough. as air transfers heat better. Bake-off stations will also be cheaper to construct without proofing ovens. On the other hand, the extended gluten network is very sensitive to ice crystal damage resulting in poorer breads. In earlier studies we have observed that the baking quality of prefermented frozen doughs can be greatly improved by reducing prefermentation time' and water content2. The explanation has been found in the more even pore size distribution of gas cells. containing more small bubbles'". The amount of "free" and freezable water also has an important role in frozen doughs'-'. However, all the changes cannot be explained by the pore structure or amount of "free" water. Thus, the aim of this paper is to concentrate on the rheological properties of yeasted frozen dough. Yeasted doughs are ditlicult to study because the dimensions and physical properties of dough change with time6.'. Thus, the major studies in dough rheology have been carried out with non-yeasted dough, the rheological properties of which differ greatly from those of yeasted doughs. Several instruments are available for continuous measurement of yeasted dough stability: maturograph', oven riser recorder', gasograph" and rheofermentometer". All the above mentioned instruments are empirical and simulate some part of the baking process (fermentation or oven rise). Probably the only experiments of fundamental rheology on yeasted dough have been carried out by Kaufmann and coworkers".

287

Processing Developments

In this work the rheological properties of prefermented frozen doughs were analysed with empirical (farinograph and maturograph) and fundamental methods after I4 days frozen storage. The effects of S-kimo (additive from Puratos, Belgium), prefermentation time. water content and hydrocolloids (six different types) on rheology were examined. 2 MATERIALS AND METHODS 2.1 Baking test

The experiments were carried out with a commercial flour, which had an 11.8% protein content and 56.5% water absorption ability. The dough formulation was 100% flour, 49 yeast (compressed, Finnish Yeast Ltd.), 4% shortening, 4% S-kimo (additive from Puratos. Belgium), 2% sugar and 1.5% salt, as in the earlier studieslJ. Optimal water content was determined with a farinograph to obtain 500 BU con~istency'~. AU dough-handling processes followed the conditions described in the earlier studies: prefermentation time', maturograph and reduced water content' and fundamental rheology'. In the studies six different hydrocolloids were included in doughs. Table 1 describes the manufacturers and main components of these samples. Hydrocolloids were added at two different levels: 0.5% and 1.5% on a flour basis. In all cases the optimal water absorption was determined for different additive levels with a farinograph (Table 1). Hydrocolloids were hydrated in part of the water to form a uniform gel, and mixed with the dry ingredients. After this yeast was suspended in the rest of the water and added into the dough. Hydrocolloids were analysed at two different water levels: reduced with 2 and 4 percentage units from optimal. AU the analyses were performed after 14 days frozen storage at - 18°C at least twice. Table 1

Hvdrocolloids cmd Water Absorption Ability

Hydrocolloid. Manufacturer (Main Component)

Amount

Optimal Absorption

P I

[%I

A Stabilizer XC-8444. TIC GUMS (Blend. standardiscd with dextrin)

0 0.5 1.5

56.5 59.0 63.5

B Colloid 1023 T.TIC GUMS

0.5 1.5

62.5 66.0

C Cekol 700 P. MetsP-Serla (CMCJ

0.5 1.5

61 .O 67.5

D Cckol 30000 P. Metsi-Scrla

0.5 I .5

64.5 71.0

0.5 1.5

61.0 67.5

(Blend)

(CMCJ

E Alginate FD 155. Danisco lngred. (Sodium Alginatc)

288

Gums and Stabilisers for the Food Industry

2.2 Fundamental rheology A StressTech rheometer (ReoLogica Instruments AB, Sweden) was used to measure the fundamental rheological parameters of doughs. The frozen doughs were thawed for Yz hour before samples were taken. Circular samples, diameter 20 mm and thickness 2.0 mm (non-yeasted dough) or 3.0 mm (yeasted dough), were prepared from dough sheets and measured with a parallel plate system. The samples were compressed with the upper plate, the normal force being less than 2.0 N during compression. Before the measurements, which took 80 s, the normal force was 1.0 N or smaller. Drying of the samples, when being measured by the StressTech, was prevented by using silicon oil. The measurements were made at 25°C and with a constant stress of 10.0 Pa (corresponds to a strain 1.5 * Non-yeasted doughs were measured with constant gap, but an autotension, limit of normal force 0.1 N, was used with yeasted doughs. Thus, for yeasted doughs the change in gap during the measurement was also monitored in addition to measuring storage (G’) modulus. The tests were repeated at least four times.

-

2.3 Empirical rheology 2.3.1 Farinograph The mixing tolerance of fermented doughs was analysed with a Brabender’s farinograph. The sample weight was 400 g before fermentation, thus being smaller than usually used in measuring water absorption (300 g flour + -60% water as flour basis). Figure 1 presents an example of a farinogram with fermented dough. The lowest point occurred after 1.5 min mixing, after which the dough becomes stronger because of water redistribution and

Figure 1 A Sample Farinogram, Fermented Dough.

Figure 2 Maturogruph Principle and a Sample Curve.

Processing Developments

289

forming new bonds. The lowest point was used to analyse the rheological properties of samples, which were thawed for an hour at 34°C and 80%RH before measuring. 2.3.2 Maturograph The maturograph measurements were carried out according to Seibel and Cromment~yn'~~'' and MettlerI6. Maturograph is designed to analyse the fermentation properties of dough and the measurement is based on two steps which are repeated until the peak of the curve can be observed (Fig. 2). In the first step dough is expanding and the plunger, which is in contact with the dough, is moving upwards. After 2 min expanding the plunger is forced down. Dough level (height of the highest peak) is established according to Figure 2. Temperature and humidity in the maturograph were adjusted to 34°C and 80% RH,as in the baking tests. The fresh control doughs (150 g pieces) were measured immediately after flooring and moulding, whereas the prefermented frozen doughs were measured after an hour of thawing at 34"C, 80% RH. The doughs were thawed in the measuring pan, because partly or wholly thawed doughs could not be transferred to the pan without causing the dough structure to collapse. All measurements were repeated from two to four times. 3 RESULTS AND DISCUSSION

3.1 Rheological properties and baking quality Figure 3 summarises the progress of baking quality during our project on frozen prefermented doughs'-'. All the presented conditions (S-kimo, prefermentation time and water content) had a favourable effect for more attractive bread. However, they were all needed together to minimise the difference between bread baked from 14 days frozen dough and fresh bread (Figs. 3a and 3e). The effect of the above mentioned conditions on the rheological properties of dough will be discussed below.

Figure 3 Buking Quality afrer 14 Days Frozen Storage: Fresh Reference (A), without Additives (B), with S-Kim0 (C), Shorter Prefermentation (0)and Shorter Prefermentation + Reduced Water Content (E).

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Gums and Stabiiisers for the Food Industry

3.1.1 Fwidumentul rheology The raw materials greatly changed the rheological properties of the dough (Fig. 4).Figure 4 shows that S-kimo and shortening had a similar etYect. resulting in a softer dough. This was seen as the greatest decrease in G’ values of water-tlour mixtures. S-kimo contains emulsifier (DATEM) and amylases, which are known to make dough softer. On the other hand, the main components of S-kimo are wheat flour and gluten. which have the opposite properties. Increasing all the ingredients (including yeast) further decreased G’ values from that with S-kimo or shortening. However, warmer water, salt or sugar did not have any statistical effect. Effect of S-kimo, prefermentation time (40 or 25 min) and water content on fermented doughs were studied as G’ values, as shown in Figure 5a. S-kimo resulted in softer dough as in the case of water-flour mixtures (Fig. 4). The rigidity of dough increased as prefermentation time and water content were reduced. However, frozen storage did not seem to have any statistical effect on dough rigidity. When water-flour mixtures were examined the result was totally the opposite: frozen storage greatly decreased the rigidity, thus explaining the lower gas-holding capacity of frozen doughs (Fig. 6). This kind of deterioration could be prevented by reducing water content. A StressTech rheometer (ReoLogica Instruments AB, Sweden) was equipped with a gas cylinder. which facilitated the measurement of change in gap during monitoring of

........................................ ............... ..........................................

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l4

01714

0 1714

01714

0 1 714

01714

0 1 714 0 1 7 1 4 warn staag. n m [q

0 1 714

hmm 81mg. Tlm [dl

Figure 4 Effect of Ingredients on G’ Vulues of Fresh Wuter-Florrr Mixtures. Legends Designute the Ingredient Added to the Previous Sumpie. Except Thut “Slrortening” Does Not Include S-kitno.

Figure 5 Effect ofFrozen Sroruge und Different Conditions on (A) G ’ Vdues und ( B ) AGup of Fermented Doriglrs. The Time Irulicures Prefermentution Time.

Processing Developments

29 1

14

P o I I I I I I I 0

2

4

6

0

10

(2

12

10

.I 8

‘-t B,

4 0

1

7

1

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4

Figure 6 Effect of Water Content and Frozen Storage on the Storage Modidits (GO)in WaterFlour Mixtures. Figure 7 Effect of Prefermentation Time (40 or 25 min) and Water Content (Optimal or Reduced) on (A) Farinograms and (B) Maturograms of Yeasted Doughs.

storage (G’) modulus. This expansion potential (AGap) is presented under the same conditions as G’ above (Fig. 5b). When no S-kimo was included freezing decreased the expansion potential, which further decreased during longer frozen storage. A response similar to that with fresh dough was observed on AGap when dough had a lower water content and shorter prefermentation time. In general, frozen storage from 7 to 14 days did not have any statistical effect, which was also seen in the earlier baking tests’”. The expansion potential of yeasted doughs (AGap) had a better correlation with baking results (Fig. 3) than the small deformation measurement (G’ values). This can be explained by the fact that the rheological changes in baking occur at larger deformations. 3.1.2 Empirical rheology Empirical experiments were carried out with Brabender’s farinograph (miXing tolerance) and maturograph (fermentation stability) after 14 days frozen storage (Figs. 7a and 7b, respectively). Farinograms presented an increased elasticity as prefermentation time and water content were reduced. A similar result was also seen in maturograms. The low level of maturograms resulted from the fact that the plunger (1.5 g), which forced the dough down after 2 min breaks. resulted in the escape of gases (Fig. 7b). Thus, the gas-holding capacity of dough was improved by shorter prefermentation and reduced water content. This was also observed in mixing tolerance and expansion potential (Figs. 7a and 5b, respectively).

Gums and Stabilisers for the Food Industry

292

3.2 Hydrocolloids in frozen doughs Reduced water content seemed to have a great effect on the baking quality and rheological properties of frozen dough (discussed above). However, this will increase the manufacturing costs as the dough includes more flour. Thus, we studied how the water content of dough could be increased by hydrocolloids without changing the amount of “free” water and baking properties of frozen doughs after 14 days storage. Hydrocolloids also have the potential to improve the gas-holding capacity of dough”, which is the most critical property for high value prefermented frozen doughs. Dietary fibre content of baked products will also be increased by hydrocolloids as they are classified as soluble dietary fibres. 3.2.1 Water absorption Hydrocolloids are well known for their high water absorption capability. In these experiments six different hydrocolloids were used at two different levels (0.5% and 1.5% as flour basis). The increase in water absorption is seen in Table 1. When a higher amount of hydrocolloids was used, the water absorption as well as dough development time increased in all cases (result for dough development time not shown). 3.2.2 Empirical rheology Figure 8 summarises the effects of the hydrocolloids used on the rheological properties of dough after 14 days frozen storage. In all the conditions studied, the total water content of the dough was higher than in the reference (Fig. 7). Stabilizer XC-8444 and Alginate FD 155 always resulted in the strongest doughs and Cekol 3oooO P in the softest. The amount of hydrocolloid added did not have any clear effect on the rheological properties, because the effect could be adjusted by the water content. Baking quality after 14 days frozen storage for Cekol 700 P is shown in Table 2. It is obvious that the baking quality is equal to the reference even though the added water content of dough increased. The water amounts added were in Table 2: (A) 54.5% (as the reference), (B) 59.0%, (C) 57.0%, (D) 65.5% and (E) 63.5%. Figure 8c shows that Cekol 700 P at an amount of 0.5% was much stronger than at the 1.5% level. Table 2

Baking Quality after 14 Days Frozen Storage for Cekol700 P

Hydrocolloid

Water’

[”/.I

Loaf Volume

Form Ratio

[mil

*

f

A

0

-2

573

9

0.55

0.01

B C

0.5

-2 -4

590 600

14

22

0.53 0.60

0.02 0.01

-2 -4

660 720

22 8

0.46 0.47

0.01 0.01

0.5

D

I .5

E

1.5

percentage units

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Processing Developments

hlaturogram

Farinngram A

400 650

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onn ---

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E

-5

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3 360

-*

m

E320

550

p 450 -1

s g280

3

C

O240

350 250 150

1M

0

2

4 6 8 MlxlngTlma [mln]

10

12

o

20

40 m 80 100 Fernnt.llon Tlma [mlnl

120

140

Figure 8 Furinogrums and Mutrrrogrurns uJer 14 Days Frozen S t o m p with Six Different Hvdrocolloids (A - E. CIS Described in Tllble I ) . The Amount of Hydrocolloids and Reduced Wuter Content (Percentuge Units): A = 0.5% -2, A = 0.5% -4, 0 = 1.5% -2 and 0 = 1.5% -4.

294

Gums and Stabilisersfor the Food Industry

This was also observed as higher form ratios (measure of maximum heighvwidth) and smaller loaf volumes. In both cases smaller water content resulted in a better form ratio. Although hydrocolloids at the 1.5% level resulted in softer doughs, the gas-holding properties of the dough improved relative to the reference, which was seen as larger loaf volumes (Table 2, D and E).

4 CONCLUSIONS The baking quality of prefermented frozen wheat doughs was well predicted by the expansion potential of yeasted doughs (AGap during viscoelastic measurements). However, the small deformation rheology presented almost no correlation to baking quality. This can be explained by the fact that the rheological changes during fermentation occur at a greater deformation than the 1.5 * 10” used in the small deformation studies. Brabender’s farinograph and maturograph also proved to be valuable and quick methods for analysing the baking properties of yeasted dough. In all experiments shorter prefermentation and reduced water content showed results similar to those with fresh doughs. Hydrocolloids facilitated higher total dough water content without changing baking properties. There were no obvious differences between analysed samples or added hydrocolloid levels. Thus the baking properties are related to the correct balance between dough elasticity, amount of “free” water and gassing power after frozen storage.

References 1. 2. 3.

J. Rashen, H. Hiirkonen and K. Autio, Cereal Chem., 1995,72,637. J. Rasanen, T. Laurikainen and K. Autio, Cereal Chem., 1997,74,56. J. Rastinen, J.M.V. Blanshard, J.R. Mitchell and K. Autio, Journal of Cereal Science, 1997, submitted. 4. J. Rashen, J.M.V. Blanshard, W. Derbyshire and K. Autio, Journal of Cereal Science, 1997, submitted. 5 . J. Rashen, J.M.V. Blanshard, M. Siitari-Kauppi and K. Autio, Cereal Chem., 1997, submitted. 6. A.S. Szczesniak, Cereal Foods World, 1988,33,841. 7. A.H. Bloksma, Cereal Foods World, 1990,35237. 8. W. Seibel, Baker’s Dig., 1968,42,44. 9. C.J. Marek and W. Bushuk, Cereal Chem., 1967,44,300. 10. G.L. Rubenthaler, P.L. Finney, D.E. Demaray and K.F. Finney, Cereal Chem., 1980,57,212. 1 1 . Z . Czuchajowska and Y. Pomeranz, Cereal Foods World, 1993,38,499. 12. B. Kaufmann, Kulmbach and M. Kuhn, GefreideMehl und Brot, 1994,48,50. 13. The American Association of Cereal Chemists. Approved Methods of the AACC, 9th ed. Method 54-21, approved April 1961, revised October 1994, St. Paul, MN, USA, 1995. 14. W. Seibel and A. Crommentuyn, Brot Gebuck, 1963a 17, 139. 15. W. Seibel and A. Crommentuyn, Brot Gebuck, 1963b, 17, 145. 16. E. Mettler, Dissertation, Rheinische Friedrich-Wilhelms-Universitat:Bonn, 1990. 17. D.M.W. Anderson and S.A. Andon, Cereal Foods World, 1988,33,844.

THE MECHANISM OF FUNCTIONALITY OF POTATO STARCH IN MEAT PRODUCTS

E. Tornberg and K. Anderson Swedish Meat Research Institute, Sweden I. Asplund Lyckeby StaerkelsenFood and Fibre AB,Sweden

1 INTRODUCTION

We can roughly divide meat products, in which additives like potato starch are mostly used, into three categories: a) ham-type products b) coarsely comminuted products such as hamburgers and meat loaves and c) finely comminuted products, called emulsion sausages. Typical examples of the latter products are the Frankfkrter and Bologna types of sausages. In the manufacture of comminuted meat products (b and c), the content of the meat raw material can vary from 25 to loo%, depending on the type of product. Ice, water, salt and additives are usually also added to the product. Important features of meat products, besides sensory attributes such as consistency, taste, appearance and juiciness, are water and fat holding. These properties of the meat product are very dependent upon the formation of a meat protein matrix, a gel. This is especially pronounced in the emulsion sausage, which is the type of meat product that will be focused upon in this paper. As discussed in a previous paper', the quality of this meat protein gel is influenced by a number of factors, such as the amount and type of protein extracted into the aqueous phase (the most important protein being myosin), the conformation and the degree of aggregation of the soluble proteins, the amount and form of the insoluble compounds and the heating conditions during gel formation. The main benefits of using potato starch in emulsion sausages are improved waterholding and consistency in the product, without causing any off-flavours, at moderate concentrations.The added level of potato starch is normally between 4 and 12% in products of both a high and a low percentage of meat. Among the different starch products, potato starch is unique in the sense that the granules, where the starch is kept, are relatively large. Not only is it the size of the granule that is unique for potato starch, but also the behaviour on heating and cooling (gelatinization), in comparison with other starches. The native potato starch granule starts to swell at 55OC and at about 75°C it obtains a maximum degree of swelling, which is much larger than other starches. On fbrther heating, the swollen starch granules begin to disrupt and amylose leaks and a colloidal dispersion of granule fragments, hydrated starch aggregates and dissolved amylose molecules is formed. This viscous dispersion is called starch paste and, when allowed to stand, a phenomenon called retrogradation can take place. Then the amylose molecules start to crystallise, forming a precipitate at low concentrations and a gel at high concentrations. Through modification of the potato starch the gelatinization behaviour of the starch can be changed in accordance with an optimum application.

296

Gums and Stabilisers for the Food Industry

Although there is extensive experience of using potato starch as an additive in meat products little information is available to explain the fknctionality of potato starch, i.e. the nature of the relationship between the structure of the potato starch and the heat-induced meat protein gel in the finished product. The only recent extensive study on these aspects in myosin-based products is the one by Verrez-Bagnis et al., 1993' on the relationship between starch granule structure and the textural properties of heat-induced surimi gels. The ongoing discussion regarding the explanation of added starch enhancing the gel strength of a surimi-based product, could thus be formulated: Either the swollen starch granules embedded in the meat protein gel act as a reinforcement of the gel or part of the starchy material that has leaked out of the starch granules (amylose) forms intergranular connections and thereby a gel. The study of Verrez-Bagnis et al., 1993' suggests the former mechanism more, as they found that the higher the gelatinization temperature of the starch, the lower the textural properties of the surimi product and the lower the degree of swelling of the starch granules. In this study, we have used a somewhat different approach than Verrez-Bagnis el al.2 since not only the end products, with different added starches, have been compared, with regard to textural and structural properties, but also the products during the heating procedure. It has been shown by Svegmark and Hermansson, 19913 that the diffusion of amylose out of the swollen granules is dependent on the amount of water phase outside the granules. Therefore, it is important to register the temperature at which water is expelled from the denatured meat proteins and how that coincides with the swelling of the granules and the leaking of amylose from the granules. The aim is to study these phenomena in the present investigation, using potato starches of different gelatinization temperatures, in emulsion sausages. Moreover, the importance of the amylose in contributing to improved gel strength and water holding of the sausages has been verified by comparing starches with and without amylose (amylopectin starches). The properties of the sausages investigated have been the microstructure, as revealed by light microscopy, cooking losses and viscoelastic properties. 2 MATERIALS AND METHODS

2.1 Materials One native, three modified potato starches and two amylopectin starches from potato and barley were supplied by Lyckeby Staerkelsen Food and Fibre AB, Kristianstad, Sweden and Amylogene HB, Svaloev, Sweden, respectively. The raw material for the sausage production was pork and beef meat of standardised fat content (20%), rindless pork fat, nitrite salt, water and the different starches given above. All meat raw material was delivered 4 days post-mortem and was stored for 2-4 days at +4"C before sausage production. The chemical composition (water, protein, fat and connective tissue content) of the meat raw material was determined in accordance with the procedure given by Hertzman et al., 19934.

2.2 Preparation of Samples In the recipes for the sausages, the fat and the starch content were kept constant at 15 and 6.4%0,respectively. A reference was made without any starch added. It is important to keep the waterlprotein (w/p) -ratio the same in all sausages (including the reference), if a comparison between the different starches regarding the water-holding capacity (WHC) of

Processing Developments

297

sausages is to be made, as the w/p-ratio is a strong factor controlling WHC. In this study, the w/p-ratio was set to 7.8. The chemical composition of the sausage batters was calculated from the chemical composition of the raw material. The sausage batters were made in a 20 1 Muller bowl chopper with 6 knives at a speed of 1400/2800 rpm, The batch size was 7 kg. The ingredients were added and disintegrated at low speed in the chopper in the following order, meat (20 s), nitrite salt (10 s), watedice (30 s), starch (30 s) and fat (20 s). The speed of the chopper was then increased and comminution was continued for 130 s to a final temperature of 8-14°C. The pH of the batter was analysed and varied from 5.6 to 5.8. The batters were made in triplicate for the native and the modified potato starches and in duplicate for the amylopectin starches. 2.3 Cooking Losses

The sausage batter was stuffed into 30 mm (0)heat-resistant plastic casings. They were heat-treated in a water-bath with a temperature-gradient of 1.O"C/min. Samples were collected every 5th degree, when heating from 40 to 7SoC, and cooled directly in ice-water to room temperature before weighing. The cooking loss was determined as the percentage loss based on the initial total weight. The determination was made with an average accuracy of *l%. 2.4 Rheological Measurements

The pasting curves of a 10% (w/w) starch dispersion in 2.5% NaCl solution were determined using a Bohlin VOR Rheometer. The complex modulus, G*, was registered as a function of the heating temperature. The temperature gradient was l.S"C/min. up to 60"C, whereafter heating was performed at a rate of O.TC/min. until the end temperature of 80°C. Registration was carried out once per minute. Continuous evaluation (every 2nd minute) of the viscoelastic properties of the sausage batters was followed during thermal processing at a temperature-gradient of 1.O"C/min. The batters were subjected to a sinusoidal shear at 1 Hz (Bohlin Reometer System, Sweden). The sample cell consisted of a parallel plate, the thickness of the sample was 56 mm and the diameter about 30 mm. All measurements were performed in the linear viscoelastic region checked in a strain sweep, whereafter the strain was set constant at 0.0125. The sample was surrounded by a 2.5% NaCl solution to avoid desiccation during heating. The rheological behaviour was monitored in terms of the storage modulus (G). The lag between the temperature in the sample and the temperature in the surrounding salt solution was corrected for (e5"C). The shrinkage or swelling of the sausage samples during cooking was also followed continuously as percentage change in height, using a pneumatically controlled device attached to the system maintaining the same low compression pressure on the sample. The reproducibility of the G and swelling measurements were on average *1.5 Wa and *1.5%, respectively. 2.5 Microstructure

Samples of the different raw sausage batters were frozen in liquid nitrogen and cryosectioned using a Leitz Cryostat 1720 Digital. The sections, 8 pm thick, were stained with Lugol's solution (for one and a half minutes and then rinsed with water) and orange G (0.10% orange G for 5 minutes followed by rinsing in distilled water for 1 minute). Using this technique, the starch-amylose became blue-violet and the proteins yellow, while the fat was unstained. The sections were examined placed on a heating table (Sensortek, TS-4)

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Gums and Stabilisersfor the Food Industry

under the light-microscope (Nikon Optiphot). Photographs were taken continuously during heating at a rate of l.O"C/min. at a magnification of 134X. The gelatinization temperature of the starch granules was determined in this way, whereas the microstructure of the sausage batters at 75°C was made by cryo-sectioning the samples from the cooking loss determinations. 2.6 Statistical Analyses

Non-linear regression was performed using SYSTAT for Windows (SYSTAT, Inc., Evanston, Illinois, USA). 3 RESULTS 3.1 Starch Dispersion Rheology

Figure 1 The complex modulus G* (Pa) of starch dispersions (10%) as afinction of the heating temperature for native (2) diflerently modijedpotato starches (3, 4 and 5) and amylopectin starchesfrom barley (6) and potato (7) The different starch dispersions (10% w/w) were, in this investigation, characterised by viscoelastic measurements in sinusoidal shear and the complex modulus, G*, is plotted against heating temperature from 50 to 80°C in Figure 1. In these types of studies, the native potato starch (2) starts to swell at about 65°C and reaches a plateau value from 75"C, having a modulus around 400 Pa. All the three modified potato starches used in this study start to swell at a lower temperature than the native one and the degree of swelling at the maximum value is also substantially larger. The modified potato starch with the lowest swelling temperature ( 5 ) was accompanied by the greatest swelling, expressed in a maximum modulus around 1200 Pa, i.e. three times the swelling of the native. It can also be observed, for the modified starches, that there is a decrease in the modulus at temperatures above 60-65°C. This phenomenon suggests that the starch granules have started to fall apart and a starch paste has been formed for the modified starches. On the contrary, the amylopectin starches from barley (6) and potato (7) have less swelling than the native potato starch, only reaching a modulus around 100 and 200 Pa, respectively. For the amylopectin potato starch, the temperature of swelling is the same as

299

Processing Developments

for the native potato starch, whereas the barley amylopectin starch does not start to swell until temperatures above 70°C. 3.2 Sausage Batter Rheology

How these six different starches influence the rheology of a sausage batter, when added at a concentration of 6.4% (dry weight), can be seen in Figure 2. As a reference (1). a sausage batter without any additives, but with the same w/p-ratio (7.8), has been chosen.

0 20

30

40

50

60

70

1 80

Heating Temperature (OC)

Figure 2 The elastic modulus, GI,of sausage batters as afunction of heating temperature without (I) and with acidtives, such as native (2) and three modifiedpotato starches (3, 4 and 5) and amylopectin starchesjiom barley (6) andpotato (7)

According to the reference (1) in Figure 2, the gelation of the meat proteins starts at around 55"C, and at 75°C the batter has solidified to such an extent that an elasticity of about 28 kPa is obtained. When the native potato starch is added (2) to the batter, the commencement of gelling at 55°C does not change, but the elasticity at 75°C is enhanced to a value of about 35 kPa. The modified potato starch starts to swell at 60°C (3 in Figure 1) and the gelling point of the batter is shifted to a temperature 5°C lower than that of native potato starch according to Figure 2. The maximum elasticity, around 28 @a, of this modified starch is the same as that of the reference, but lower than the one with native potato starch added. There is also a tendency for the modified potato starch to obtain a lower batter elasticity at the highest temperatures. This tendency is even more pronounced in the modified potato starch number 5.For modified starches numbers 4 and 5, the starting point of gelling is shifted even hrther down to 4 0 4 ° C . This is in accordance with a lower temperature, when the starch granules start to swell (Figure 1). The amylopectin potato starch (7) has a similar influence on the rheology of the batter as the native one. Surprisingly, the batter with added amylopectin starch fiom barley obtains a lower starting point for gelling (a50°C), compared to the amylopectin potato starches, although it does not start to swell at about 7OoC, according to Figure I. The end-point elasticity, however, is more or less the same as both amylopectin and native potato starch.

Gums and Stabilisers for the Food Industry

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3.3 Swelling and Shrinkage of Sausage Batters During rheological measurements on the sausage batters, a continuous recording of the change in height of the sample during heating was achieved. These registrations can be viewed in Figure 3 for the sausage batters without and with the 6 types of starches added. For the sausage batter without any additives (I), the least ultimate swelling is obtained (=12%), It is interesting to note that there seems to be a shrinkage up to about 50°C for all samples. This effect is probably due to the floating of the batter under the weight of the upper plate, before the meat gel is set. During gelling, however, an expansion is observed and this is more pronounced, when some potato starch or amylopectin barley starch is added to the batter. In this respect, the native (2) and the amylopectin potato starch (7) seem to be the most efficient, as they give rise to a swelling from 30 to 35% at 75°C.When the modified potato starches (3-5) are added to the sausage batters, they end up in a degree of swelling between 18 to 28%. The amylopectin starch from barley (6) comes closer to a swelling of 23%.

20

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Heating Temperature (OC)

Figure 3 7he swelling and shrinkage of sausage batters as a function of heating temperature without (I) and with aditives, such as native (2) and three modrfied potato starches (3, 4 and 5) and amylopectin starchesfrom barley (6) andpotato (7) 3.4 Microstructure of Sausage Batters Some important characteristics, such as the temperature of granule swelling and the microstructure of the starch granules at the end-point temperature 7S"C, are enumerated in Table 1. Modified potato starch (5) swells at the lowest and amylopectin barley starch at the highest temperature in the sausage batter, which is in accordance with the ranking order, as observed in Figure 1, for starch dispersions. However, the swelling temperature, as registered under the light microscope, is lower than the one measured as the modulus of starch dispersion. This is likely to be so, since the prerequisite for detecting any rise in the rheological parameters is the granules starting to touch each other, which can only occur after some degree of swelling. Therefore the swelling temperature of the granules, as observed under the light microscope, can be considered to be more "realistic" than the one obtained fiom the rheological measurements.

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Table 1 Characteristics of the microstnrcture of sausage batters with different types of starches added Temperature ("C) at the start Microstructure of the granules at 75°C of swelling of the granules 55 Swollen, but still largely stained.

Type of starch added Native potato starch (2)

Modified potato starch (3) Modified potato starch

49

Swollen, but less stained than the native.

46

Swollen granules, slightly stained. More of the blue-violet in the water phase. The structure of the granules has more or less vanished. Stained blue-violet in the water phase. Smaller granules than in potato. Somewhat blue-violet staining in the granules. Large swollen granules. Somewhat stained.

(4)

Modified potato starch

45

(5)

Amylopectin barley starch (6) Amylopectin potato Istarch (7)

61

53

I

I

I

The microstructure of the different starches in the sausage batter at 75OC reveals that the modified potato starches are overswollen, i.e. the granules are disrupted and the amylose has started to leak out. This is more pronounced compared to the native potato starch and the more so, the lower the swelling temperature of the modified starch. Amylopectin starch fiom potato behaves similarly to the native one, except for the fact that the former has a much lower content of amylose, which is evident in the micrographs as much less stained granules. The largest difference in microstructure between amylopectin starches from barley and potato is the size of the granules, which are much smaller in the case of barley. 3.5 Cooking Losses in Sausage Batters

J

4 0 5 0 8 0 7 0 8 0

Heating Temperature ("C)

Figure 4 Cooking losses in sausage batters as ahnction of heating temperature without (1) and with additives, such as native (2) and three modfled potato starches (3, 4 and 5) and amylopectin starchesfrom barley (6) and potato (7) The cooking losses in the different sausage batters during the heating process are of great importance and have been plotted as a function of heating temperature in Figure 4.

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The sausage without any additives (1) shows the largest cooking losses, especially at temperatures above 55"C,which is the temperature where the meat proteins start to gel (Figure 2). When adding native potato starch (2) at 6.4%, a substantial reduction in cooking losses is obtained, resulting in losses of only 1-2% in the whole temperature range Of 50-75°C. The modified potato starches 3, 4 and 5 are as good in water holding as the native potato starch up to 55°C; whereafter the water is less firmly held, resulting in cooking losses of about 7-10% at the end-point temperature. Still, a lot of improvement in the WHC, compared to the meat protein system, is however achieved using the modified starches. Amylopectin potato starch (7) is almost as good as native potato starch in the entire temperature region, whereas the amylopectin barley starch (6)does not take care of the water expelled from the meat protein system until after 55"C,and even more so after 60°C. This is in accordance with the swelling of the amylopectin barley starch granules, that does not start until about 60°C (Table 1).

4 DISCUSSION

-1

Table 2 An overview of all the characteristics of the sausage batters that were observed at 75°C. Whengiven, mean std.

*

Type of sausage batter Without (1) Native potato starch added (2) Mod. potato starch added (3) Mod. potato starch added(4)

Granule swelling Degree of swelling Cooking of starch granules losses temp. (light microscopy) (light microscopy) (%)

55

Largely swollen, stained

49 46

Mod. potato starch added (5)

45

Amylopectin starch from barley (6) Amylopectin starch from potato (7)

61

53

23 f 2 2 f 0.5

mi

Swollen, less 7 f 3 26fl stained Swollen, slightly 10 f 3 stained. Blue-violet in the water phase Disrupted 7 f 3 29f2 granules, water phase blue-violet Small granules, 7 f 0.5 relatively unstained Large swollen 2 f 0.5 granules, relatively unstained

28f1

In order to summarize the results obtained in this investigation, these have been tabulated in Table 2. Firstly, it can be stated that the starches added influence the W H C of the sausage batters more strongly than the consistency, in this case measured as G'. Verrez-Bagnis et a1.' only discuss starch as a textural additive in surimi-products, but for a long time our experience of using starch as an additive in emulsion sausages has been that it primarily acts as a water binder. Reference to the ongoing discussion as to whether starchy material (mainly amylose) leaking out of the granule forms intergranular connections and thereby enhances both the WHC and the textural properties of the sausages, does not seem to be applicable to this study. On the contrary, the modified starches where amylose seemed to have leaked out of

303

Processing Developments

the granule to the greatest extent (4 and 5 , where the water phase was mostly stained blueviolet), were the least effective in water holding. Moreover, amylopectin potato starch containing very small amounts of amylose was as effective in water holding as the native potato starch. According to Figure 1, the degree of swelling of the granules seems to be larger for the modified starches, even at the end-point temperature of 7S°C,but swelling of the sausage batters is greatest in the sausages with the native and amylopectin potato starch added. Verrez-Bagnis et a1.2also noticed there was no correlation between the texture properties of the surimi and the volume fraction of partially swollen starch granules. For optional water holding, the ultimate swelling of the sausage batter and the swelling temperature (gelatinization temperature) of the starch granule seem to go together. However, when plotting cooking losses at 75OC as a function of the gelatinization temperature (Figure 5), it is not a linear relationship, rather a quadratic one (r=O.67**). This Figure tells us it is optimal, with regard to water holding in sausages, to have a starch that starts to gelatinize at temperatures of about 5S0C,which is the case for both native and amylopectin potato starch. The results presented in Figbres 2 and 4 suggest that the mechanism for the relationship observed in Figure 5 is that the starch granules start to swell directly after the gelling of the meat protein system starts (at 55OC according to Figure 2). Then the starch granule can immediately take care of the water that is expelled fiom the meat system (Figure 4). When the gelatinization temperature is lower, the starch granules start to disrupt and cannot hold water to the same extent as the intact and swollen starch granules at higher temperatures. If the gelatinization temperature of the starch granule is too high, water losses will have already occurred fiom the system before the swelling starch granule can take care of the water, hence poorer water holding.

a

a

3

m10

c 0

-

5-

4

04 4 0 4 5 5 0 5 5 8 0 8 5

Swelling Temperature ("C)

Figure 5 Cooking losses in sausage batters when heated to 75OC, as afunction of the starting temperaturefor gelatinization (judgedusing light microscopy) of diflerent starches: ( 2 : ~ native ) potato starch, matifid potato starches (3x, 4:A and5:o)andmylopectin starchesfrom barley (6:@)andpotato (7:w. (r=O.67* *)

304

Gums and Stabilisers for the Food Industry

References 1 . Anderson, K., Anderson, A. & Tornberg, E. Food Colloids-Proteins, Lipids and Polysaccharides, 1997. E. Dickinson & B. BergenstAhl, Eds. Royal Society of Chemistry, Cambridge, p. 29-42. 2. Verrez-Bagnis, V., Bouchet, B. & Gallant, D.J. 1993. Food Structure, vol. 12, pp. 309320. 3 . Svegmark, K. & Hermansson, A.-M. 1991. Food Structure 10, 117-129. 4. Hertzman, C., Olsson, U. & Tornberg, E. 1993. Meat Science 35, 119-141.

PROCESSING AND FUNCTIONAL BEHAVIOUR OF LOW-TANNIN MESQUITE GUM

F.M. Goycoolea, A.M. Calderon de la Barca, J.R. Balderrama, J.R.Valenzuela and G. Hernhdez Centro de Investigation en Alimentacion y Desarrollo, A.C. P.O. Box 1735 Hermosillo, Sonora C.P. 83000 Mexico 1 ABSTRACT The identified presence of tannins along with the lack of toxicological testing on mesquite gum from Prosopis spp. and other exudate gums, have hitherto been the leading arguments to regard their intake as potentially hazardous, hence currently they are not permitted food additives. Mean average tannin concentrations in mesquite gum samples collected in the plains of Sonoran Desert were 0.46 f 0.02%, whereas dark gum ‘tears’ and commercial batches had consistently high tannin levels (1.9 f 0.06 %) jeopardising its quality. The aim of this investigation was to evaluate the technical feasibility of using ultrafiltration-diafiltration (UF-DF) technology in order to remove the naturally occurring tannins of mesquite gum. To this end, mesquite gum solutions (-4.5% w/* were concentrated -3-fold on hollow fibre polysulphone membranes (total area 0.45 m ) of 10 or 50 kDa molecular weight cut-off ,(MWCO) sizes at constant temperature (-55 “C) and pressure gradient (1.05 kgcm- ). Tannin contents were reduced to 38 and 30 YOof the original concentration respectively in the 10 and 50 kDa MWCO membranes with similar exponential functions of time. Protein contents were reduced during UF-DF, particularly in the 50 kDa MWCO membrane and during the diafiltration washing cycles. The citrus oil encapsulating capacity of gum arabic was slightly greater than that of both native and UF-DF treated mesquite gum, as evaluated by spray drying freshly prepared o/w emulsions of orange peel oil in gum with modified corn starch carrier matrices. The loss of low molecular weight glycoproteic fractions along with changes in the fine balance of such fractions during UF-DF could account for the slightly reduced functionality found for low tannin mesquite gum in comparison with gum arabic. Yet UFDF appears to be a feasible technology to treat this material so as to render it suitable for use in the food industry. 2 INTRODUCTION Mexico imports over ca. 80% of the industrial hydrocolloid market shafe of gums used by the country, with an approximate size by value of $28 million USD. These are chiefly polysaccharide hydrocolloids from plant and seaweed sources. Rational and sustainable use of low-input agriculture plants as sources of high added-value food gums, could represent a potential benefit and source of income in desert rural zones of Mexico and Latin America. One such botanical source is mesquite (Prosopis spp) a N-fixing legume tree widely distributed in arid and semi-arid regions of Latin America and the world. The exudate polysaccharide material of the bark of mesquite, hereof regarded to as mesquite gum, is also locally known in Sonora Mexico as ‘chricutu’. The gum is biosynthesised as a secondary metabolite and stored in the cavities of the xylem ayd phloem as a response to mechanical wounding and heat and water physiological stress. In the past, the exudate

306

Gums and Stabilisersfor the Food Industry

gum of mesquite3tree was used in folk medicine by the native indians of Sonora and southern Arizona. Currently, mesquite gum still has domestic uses in Sonora, chiefly in folk medicine and people commonly chew the gum tears directly claiming that it has a pleasant taste. Although the carbohydrale-iesidue chemical composition of mesquite gum it has only recently become apparent that its has been known fyr over two decades, solution rheology, emulsion behaviour 899 and possibly its tertiary structure are very similar to those of gum arabic from Acacia senegal (L.) WiZZd. The mesquite gum primary structure is a highly branched complex polysaccharide, b e y r g a protein component, whose overall contents varies with botanical origin (1.2-5.8%). ’ As a result of this, the molecule is highly soluble in water, adopts a compact structure in solution, and fiepce it contributes little to the solution viscosity (i.e. intrinsic viscosity 0.11 d1.g’ ). The mesquite gum primary structure has been described as a core of P-D-galactose residues, comprising a (1-+3)-linked backbone with (1-+6)-linked branches, bearing L-arabinose (pyranose and h a n o se ring forms), L-rhamnose, P-D-glucuronfie and 4-O-Me-P-Dglucoronate as a single residue or oligosaccharide side chains. ’’6 A closely re&efi primary structure has been identified in the polysq!charide fraction of gum arabic, ’ including as well a protein component (- 2.3 %). A ‘wattle blossom’ model has been proposf. 4 describe the tertiary structure of the highest molecular mass fractiop of gum arabic, by virtue of which several polysaccharide domains of M,-2 x 10 are held together by a peptide chain. The existence of such assemblies in gum mesquite has not yet been demonstrated. Nevertheless, the hydrodynamic size of native mesquite g v molecule resembles that of the arabinogalactan (AG) major fraction of gum arabic. Further evidence of similarity between both gum materials has been documented in a recent study, in which polyclonal antibodies against each gum were used to show that the carbohydrate-rich components with slow mobility in two-dimensional immyqelectrophoresis gels had close immunological identity with the faster ones of gum arabic. Among the unique functional features which many industrial applications of gum arabic rely on, is the ability &I stabilise o/w emulsions and encapsulate citrus essential oils The emulsifying and stabilising performance of mesquite in powdered flavourings. gum in o/yl9sFulsions and in encapsulating orange citrus oil has also been documented. ’ ’ The close similarity of chemical properties and functional behaviour have led to consider mesquite gum a suitable replacement of gum arabic in food applications. However, while gum arabic is a firmly established food additive awarded ‘AD1 not specified’ and GRAS status respectively by JECFA (European Community, official gditive number E 414) and FDA (U.S.A.), mesquite gum is a no-permitted food additive. The leading argument so far put forward is the lack of toxicological tests. Moreover, the presence of tannin compounds as well as high Mn levels (up to 1680 ppm) have been identified in mesquite gum sampl52 of Prosopis spp from varying botanical origins and in Mexican commercial samplfj. These two associated factors have been associated with potential mutagenic effects. The presence of high tannin levels (-1.9%) has recently been confirmed in Prosopis exhdate gum samples, but only in dark gum ‘tears’ from well identified individual trees. The use of ultrafiltration ,pd cross-flow microfiltration has been reported in order to clarify gum arabic solutions, so It was the aim of this study to assess the feasibility of using such technology in order to remove tannins from mesquite gum solutions as well as to study the effect of such processes on the overall functionality.

-

’’

*’

3 MATERIALS AND METHODS

3.1 Materials A batch (-8 kg) of commercial mesquite gum was pur-hased from a local outlet; a batch (-5 kg) of corn acid-modified starch Amiogum 23 was a gift from American Maize Products Co. (Hammond, IN, USA); a sample (-750 ml) of cold-pressed orange

Processing Developments

307

peel essential oil was donated by Esencitricos S. de R.L, Tlalnepantla, Mexico. Gum arabic from Acacia senegal was from Sigma Chemicals Co. (St Louis, MO, USA ). 3.2 Ultrafiltration-Diafiltration (UF-DF) Mesquite gum tears (- 4.5% w/w) were gently stirred in water until they were completely dissolved, the solution was filtered with a cloth before being loaded into the tank of the ultrafiltration unit (Romicon Mod. HFLAB 5/RF, Wobum MA). The UF modulf was a top-bench unit fitted with hollow fibre membrane cartridges (total area 0.45m ) of nominal MWCO 10 or 50 kDa. Ultrdiltration was conducled at a constant temperature of -55 "C, with a transmembrane pressure of 1.05 kg'cm- , to 35% of the original volume (i.e. 12.6% w/w total solids on both runs). During dialfiltration, the UF retentates were washed with distilled water, 1 or 3 times respectively, for the 10 or 50 kDa membranes and re-concentrated to the same extent as during UF. The permeate stream was sampled periodically and the flux was determined with a stop watch. Retentate was also sampled periodically and its concentration determined by refkactometry using a prepared calibration curve and the overall processing time on both membranes was 160 Tin. The retentate samples v#re reconstituted to the original concentration for tannin and protein content analysis. 3.3 Emulsions Formulation and Preparation The emulsions base formulae had the@followingcomposition (% w/w): cold-press orange peel essential oil 5.0; Amiogum 23 5.0; water 82.5, and either mesquite gum or gum arabic 7.5. Emulsions were prepared by stirring the solutions with an Ultraturrax T25 (Janke and Kunkel Gmbh, Staufen, Germany) fitted with a microshaft operated at a speed setting of ca. 20000 rpm for 1 min. 3.4 Spray Drying The freshly prepared emulsions were fed into a laboratory spray drier unit (Mod. Anhydro Laboratory Spray Dryer No. 1, APV Anhydro A/S, S~borg,Denmark) at a rate of 1.97 f 0.05 kg hr- . Atomigation was achieved on a Bosch atomiser (Mod. 1210) running at a speed -34500 min- . Temperatures of the drying air in the cabinet and outlet of the recovery tower were respectively, 220 "C and 80 "C. The yield of recovered powders was 87.3 k 4.4 % and the average moisture content was 4.9 & 0.8%. The water evaporation rate was 1.6 f 0.04 kg h . The amount of citrus oil retained by the gumstarch carrier matrix during spray drying was determined by steam distillation of a powder sample (ca. 5g) previously dissolved in water. The distilled oil was separated from the water with a mixture of chloroform:methanol (1:l) in a separation funnel. The solvent was distilled in a Goldfish unit for ca. 2h and the remainder evaporated in a convection oven. The quantity of oil extracted was referred to the original sample weight. The percentage oil recovered (i.e. encapsulated oil) was obtained as the ratio between the quantity of the oil recovered after steam distillation-extractionand the calculated expected quantity (i.e. 28.6% w/w). 3.5 Immunization and purification of antibodies Specific rabbit antibodies to or against commercial gum arabic (Anti-A) %d to mesquite gum (Anti-M) collected during the spri% season of 1995 were raised and purified by chromatography on a Protein A column. 3.6 Crossed Immunoelectrophoresis Electrophoresis in 1% agarose gels was performed as described el~ewhere.~' Electrophoresis buffer was 0.1 M glycine, 26 mM Tris, pH 8.6. Seven p1 of native arabic (A) or mesquite gums (M) and retentate (Mr) and permeate (Mp) UF-DF treated mesquite

308

Gums and Stabilisers for the Food Industry

gum were run initially at 10 Vlcm, 1:30 h in the first dimension gels. The second dimension was run at 2-3 V/cm overnight, gels contained 125 p1 of Anti-A plus 125 p1 of Anti-M gum rabbit antibodies. Plate size was 5 x 6.3 cm. 4 RESULTS AND DISCUSSION Tannin contents previously found in Prosopis velutina gum exudates analysed in our laboratory are shown in Table 1. For comparison, data for commercial samples used on the UF-DF trials have also been included. It follows that tannin contents in light-coloured mesquite gum tears collected from an identified tree in 1995 and 1996 were almost identical (- 0.43%). While a sample of dark coloured nodules (also from the same tree), 0.06), than did the collected in 1996, had much higher tannin contents (1.9% corresponding light tears (0.43% f 0.03). The tannin contents found on the commercial sample batch used on the UF-DF trials was similar togreviously reported values for hightannin commercial mesquite gum of Mexican origin. Although high tannin levels were found particularly in well differentiated ‘dark tears’ in some tree specimens, the present analysis also confirms that Mexican commercial mesquite gum consistently shows high tannin levels to be a problem. This led us to study the feasibility of using ultrafiltration technology as a means to eliminate such compounds. In Figures l a and l b are illustrated respectively, the permeate flux and total soluble solids concentration time profiles, during the filtration operations in the two different pore size membranes. The UF-DF permeate flux curves in Figure l a show that during both operations, there is a constant decrease in the rate of filtration with time. Moreover, markedly lower gradients were recorded for the 10 kDa than for the 50 kDa MWCO membrane, as a result of the greater amount of permeate removed through the greater pore size membrane (Figure 1b) leading to a great$; extent of fouling per unit time as observed elsewhere during gum arabic ultrafiltration. Note that in both cases, the addition of water during diafiltration, temporarily disrupts the impregnation of solids into the membrane pores, thus effectively increasing the overs]! process flux, as previously documented during the treatment of milk in the same unit.

*

Table 1 Tannin Contents in Mesquite and Arabic Gum Exudates Sample

Tannin (%)

Gum Arabic (Sigma Chem.) a

0.49 f 0.04

Mesquite Gum, (4 sites, 1995)

0.46 f 0.02

Mesquite Gum from a Single Tree: 1995 - ‘light tears’ a 1996 - ‘light tears’ a 1996 - ‘dark tears’ a

0.44 + 0.05 0.43 0.03 1.90 f 0.06

Commercial Mesquite Gum

2.1 1 - 2.27

a Mean

*

average values and standard deviation from triplicate analysis; data from Ref. 19 Sample used in this study

Processing Developments

0

309

20

40

60

80

100

120

140

160

100

120

140

160

time (min)

I 0

20

40

60

80 time (min)

Figure 1 Profiles of permeate flux (a) and retained total solids (b), during ultrafiltration and diafiltration of mesquite gum in hollow fibre membranes of lOkDa ( 0 ) and 5OkDa (A) molecular weight cut-off (initial mesquiy gum concentration 4.5% (w/w); 55OC; membrane area 0.45 m ). Figure 2a shows tannin concentrations of the retentates as a function of overall processing time, during UF-DF operations for the two different MWCO membranes tested. Inspection of the curves shows that in both molecular size regimes, tannins are removed as exponential functions of overall processing time with no signs of discontinuities in the curves between the UF process and DF washes. In both cases a steady value is reached, beyond which tannins are no longer removed by diafiltration. In the 50 kDa MWCO membrane pore size the rate of tannins removal is faster as evidenced by the steeper slope during the UF than through the 10 kDa membrane. It is also evident that the overall amount of removed tannin increases, the larger the pore size (2.11 to 0.64% and 2.27 to 0.85% respectively for 50 and 10 kDa MWCO membranes). Notice that in the 10 kDa MWCO membrane, most of the tannins compounds are removed during the ultrafiltration operation with a slight further decrease achieved during diafiltration. In the 50 MWCO membrane, at least one diafiltration washing cycle seems necessary in order to reduce the tannin content close to the steady value corresponding to - 70% of removed tannin. Quantitative analysis of removed tannins, indicates that 70% of the total amount of such polyphenolic compounds originally present in mesquite gum, are associated with gum fractions of equivalent size lower than 50 ma, 62% with fractions smaller than 10 kDa and the remaining 8% is associated with fractions between 10 and 50 kDa.

310

Gums and Stabilisersfor the Food Industry

3.0 2.5

1

(a) 0

1.5

0 A A

j? 1.0 0.5

0 . O I 0 20 '

"

'

'

,

1

40

" " '

60

"

80

100

120

140

160

time (min)

6 ,

I

DF 1 0 k D

I

.t 0

20

40

60

80

100

120

140

160

time (rnin)

Figure 2 Profiles of tannins (a) and protein (b) retention, during ultrafiltration and diafiltration of mesquite gum in hollow fibre membranes of lOkDa ( 0 ) and 5OkDa (A) molecular weight cut-off (initial mesquite guy concentration 4.5% (w/w); 55°C; membrane area 0.45 m ). Figure 2b illustrates the retention pattern of protein fractions on both MWCO sizes tested. From the 10 kDa MWCO trace, notice that there is a progressive subtle loss of protein-containing compounds as the processing time proceeds. By contrast, in the 50 kDa membrane the protein content of the retentate, initially decreases to 4% during UF. As the process continues, a sudden decrease in protein content is observed when the washing diafiltration cycles are applied. Hence, the UF-DF process on both membranes, yields two low tannin gums with low and high protein contents. The immunological identity of low tannin processed mesquite gum was investigated and compared to native mesquite gum and gum arabic as well as to the separated fraction as permeate. Figure 3 presents tandem immunoelectrophoresisplates obtained in order to identify antigenic components present in mesquite gum treated by UF-DF through the 50 kDa membrane and reconstituted to the original concentration (4.5% wlw) with water. Peak b in plate 1, corresponds to a fused precipitats of close identity between both gums, which is in good agreement with previous results. Left and right shoulders observed in peak b correspond respectively, to gum arabic and mesquite gum. After UF-DF treatment, the identity component from mesquite gum grows in the retentate (right shoulder, plate 3)

-

311

Processing Developments

Figure 3 Tandem immunoelectrophoresis of mesquite gum (A@ and its fractions obtained by UF-DF (50 kDa MWCO membrane) (Mp = mesquite gum permeate, Mr = mesquite gum retentate) as compared to commercial gum arabic (A). Seven ml of each sample were separated by electrophoresis in I% agarose gels in the Jirst dimension. All the plates contained 125 ml of anti-A + 125 of anti-M antibodies, mixed with the agarose gel in the second dimension.

30 25

20

15 10

GA

MG

MG 10

MG50

Figure 4 Amount of encapsulated oil in spray-dried powders of emulsions formulated (&w) as: 82.5% water; 5% orange peel essential oil; 5% Amiogum 23 ;and 7.5 % gum arabic (GA) or either native mesquite gum (MG) or low-tannin mesquite gum ultraJiltrated in hollow jibre membranes of molecular weight cut-off 10 kDa (MG 10) or 50 kDa (MG 50).

312

Gums and Stabilisers for the Food Industry

while other minor non-identical components are lost in the permeate (plate 2) and can be distinguished as the features distinguishable on top of the GA peaks. Such,&ractions correspond to glycoproteins of low molecular mass identified previously. Similar overall qualitative behaviour was observed for the mesquite gum retentate and permeate 10 kDa MWCO membrane (results not shown). The functional performance of processed low tannin mesquite gum, was tested in a typical applicffion where gum arabic is deemed to be unique, namely in citrus oil encapsulation. Figure 4 shows the results of oil retention in the four spray dried powders analysed. Notice that gum arabic had superior capacity to retain oil in the powder emulsions, as compared to both native and low tannin gum mesquite products. Moreover, the 50 kDa MWCO membrane had lower oil retention than did the 10 kDa MWCO treated sample. From these results, the importance of glycoproteic fractions of intermediate molecular mass between 10 and 50 kDa is clear. Such fractions seem to be lost in the permeate during UF-DF, thus effectively affecting the rapid adsorption intY3the o/w interface and short term stabilisation of the emulsion before spray drying. A mixture of low and high molecular mass glycoproteic fractions of gum arabic has been suggested to be best for a rapid adsorption at the interfq$e, small droplet size formation and further long-term stabilisation of o/w emulsions. In our study, where freshly prepared emulsions were spray-dried and functionality assessed as the amount of retained oil in the powders, the role of small glycoproteic fractions (> 50 kDa) of mesquite gum along with their net N-content, in emulsion formation, seems to be magnified. The gradual decrease in functionality as the amount and size of the different glycoproteic fractions removed from native mesquite gum increases (cf % encapsulated oil for native mesquite gum > UF-DF 10 kDa > UF-DF5O kDa), could be explained in terms of the disruption in the fine balance of the different molecular fractions varying in molecular mass and N-content. From the immunoelectrophoresis experiments and functionality trials of the treated products, it appears that the identity components between native gum arabic and mesquite gum do not correspond to the ones responsible for the functional property evaluated. Based on the tests conducted, it appears that ultrafiltration is a feasible technology to reduce tannin levels from mesquite gum exudate by at least 60%. Although the process certainly can be further optimised, two run protocols can be recommended. In the 50 kDa MWCO membrane, only the UF treatment may suffice to reduce tannins by more than 50% of the original contents, without major loss of glycoproteic fractions that may compromise its functionality (Figure 2b). Such treatment would have the intrinsic advantage of reducing the overall processing time to -50 minutes, and would result in important savings in the volume of consumed water. The functional quality of a sample treated under such conditions remains to be evaluated. This process would be recommended for instance to treat gum mesquite batches bearing moderate tannin levels (i.e. -1%). Use of the 10 kDa MWCO membrane combining ultrafiltration with one diafiltration washing cycle, seems adequate to eliminate up to 62% of polyphenolic compounds. These processing conditions do not reduce significantly the amount of small glycoproteic fractions and the oil encapsulating capacity of mesquite gum persists after UF-DF treatment and it is still acceptable when compared to gum arabic performance. Ultrafiltration combined with previous hand sorting of mesquite gum from the Sonoran Desert, in order to eliminate ‘dark tears’ and large bark pieces, seem to be suitable operations so as to reduce the amount of naturally occurring tannin compounds, bound to be problematic in applying for clearance for use in food. Acknowledgements

Help from Mr. Germ& Cumplido, Mr. Luis Robles and Mrs. Adriana Bolaiios during experimental work is gratefully acknowledged.

Processing Developments

313

References

1. Instituto Nacional de Estadistica, Geografia e Informacion ‘Tablas de Estadisticas de Comercio Exterior’, Aguascalientes, Mexico, 1994. 2. Greenwood and P. Morey, Bot. Gaz., 1979,140,32. 3. R. S. Felger, ‘Mesquite Its Biology in Two Desert Scrub Ecosystems’, ed. B. B. Simpson, Dowden Hutchinson and Ross Inc., Pensylvania, 1977, p.150. 4. G. 0. Aspinall and C. C. Whitehead, Can J. Chem., 1970,48,3840. 5. G. 0.Aspinall and C. C. Whitehead, CanJ. Chem., 1970,48,3850. 6. S . C. Churms, E. H. Merrifield and A. M. Stephen, Carbohydr Res., 1981,90,261. 7. F. M. Goycoolea, E. R. Morris, R. K. Richardson and A. E. Bell, Carbohydr. Polym., 1995,27,37. 8. E. J. Vernon-Carter and P. Sherman, J. Texture Stud., 1980, 11,351. 9. C. Beristain, E. m a , H. S. Garcia and E. J. Vernon-Carter, Int. J. Food Sci. Tech.,1996,31,379. 10. D. M. W. Anderson and J. G. K. Farquhar, In?. Tree Crops J., 1982,2, 15. 11. P. Jurasek and G. O.Phillips, Food Hydrocolloids, 1993,7,337. 12. C. A. Street and D. M. W. Anderson, Talanta, 1983’30,887. 13. S . C. Churms, E. H. Memfield and A. M. Stephen, Carbohydr.Rex, 1983,90,261. 14. J. Defaye and E. Wong, Carbohydr.Res., 1986,150,221. 15. D. M. W. Anderson, D. M. Douglas, N. A. Momson and W. Weiping, Food Addit. Contam., 1990,3,303. 16. S. Connolly, J. C. Fenyo and M. C.Vandevelde, FoodHydrocolloids, 1987,516,477. 17. R. C. Randall, G. 0. Phillips and P. A.Williams, Food Hydrocolloids, 1989,3,65. 18. S . Connolly, J. C. Fenyo and M. C.Vandevelde, Carbohydr.Res., 1988,8,23. 19. F. M. Goycoolea, A. M.Calderon de la Barca, J. R. Balderrama and J. R.Valenzuela, Int. J. Biol. Macrom., 1997,21,29. 20. R. L. Whistler and J. N. BeMiller, ‘Industrial Gums. Polysaccarides and their Derivatives’ 3rd edition, Academic Press Inc., San Diego, USA, 1993, p. 309. 21. F. Thevenet,‘Flavour Encapsulation’,ed. S. J. Risch and G. A. Reineccius, American Chemical Society, Washington, 1988, Symposium Series No. 370. p. 37. 22. E. J. Vernon Carter and P. Sherman, J. Disp. Sci. Tech., 1981,2,349. 23. D. M. W. Anderson and W. Weiping, Food Hydrocolloids,1989,3,235. 24. H. F. Stich, ‘Carcinogens and Mutagens in the Environment, Food Products’, CRC Press, Boca Raton, Florida,1982, Vol.1, p. 135. 25. M. Decloux, M. Dornier and LGratius, In?. J. FoodSci. Tech.,1996,31, 153. 26. D. M. W. Anderson and N. A. Momson, Food Hydrocolloids, 1989,3,57. 27. 0 .H. Lowry, N. J. Rosebrough, A. L. Farr and R. J. Randall, J. Biol. Chem., 1951, 193,265. 28. R. D. Jurd and T. C. Berg-Hansen,‘Gel Electrophoresis of Proteins, a Practical Approach’, ed B. D. Hames and D. Rickwood, IRL Press, Oxford, 1990, 2nd ed., p.366. 29. Pharmacia. ‘Monoclonal antibody purification’, Handbook Pharmacia Biotech., Upssala, Sweden, 1994, p.53. 30. N. H. H. Heegaard and T. C. Bsg-Hansen, Applied and Theoretical Electrophoresis, 1990,1,249. 31. I. Higuera-Ciapara, M. Esqueda-Valle, and J. Nieblas, J. FoodSci., 1995,60,645. 32. Ray A.K., Bird P.B., Iacobucci G.A. and Clark B.C., Food Hydrocolloids, 1995, 9, 123.

PROTEIN-POLYSACCHARIDE INTERACTIONS IN EMULSIONS CONTAINING HIGH PRESSURE TREATED PROTEIN

Eric Dickinson and Karin Pawlowsky Procter Department of Food Science University of Leeds Leeds LS2 9JT

1 INTRODUCTION Protein-polysaccharide interactions have an important influence on the structure, stability and rheology of food colloids.1 The properties of an oil-in-water emulsion containing both protein and polysaccharide depend on the strength and nature of the mixed biopolymer interactions in the bulk aqueous phase and at the surface of the droplets.Z4 When the added polysaccharide concentration is low and the protein-polysaccharide interaction is net attractive, the emulsion may become flocculated due to the presence of polysaccharide bridges between protein-coated droplets. Based on small-deformation shear rheological measurements, it has been shown5 that this type of bridging flocculation is induced in an emulsion stabilized by bovine serum albumin (BSA) by addition of dextran sulphate (DS) at pH 7 and low ionic strength. Similar behaviour has also been found6 on addition of tcarrageenan (t-CAR) to a BSA-stabilized emulsion at pH 6 and low ionic strength. The behaviour in both cases can be attributeds-7 to interfacial electrostatic complex formation between the adsorbed globular protein and the anionic polysaccharide. Treatment at high isostatic pressures of a few thousand atmospheres produces changes in the conformational structure of globular proteins-leading to denaturation, aggregation or gelation.S*O Such high pressure processing would also be expected to influence the interactions of globular proteins with polysaccharides, with consequences for the stability and rheology of emulsion systems containing these mixed biopolymers. In this paper new results are presented on the effect of high pressure treatment of protein on the properties of emulsions containing BSA + t-CAR, and a comparison is made with the results previously reported'] for emulsions containing BSA + DS.

2 EXPERIMENTAL The food-grade t-carrageenan was kindly donated by Systems Bio Industries (Carentan, France). Bovine serum albumin (lot no. 14H9350) and n-tetradecane were purchased from Sigma Chemicals (St. Louis, MO). All other reagents were AnalaR grade. Buffer solutions ( 5 mh4 imidazole) were prepared with double-distilled water; 0.02 wt% sodium azide was added as antimicrobial agent for the stability storage experiments. Polysaccharide solutions were prepared by dispersing the powder in buffer solution and stirring continuously for 30

Processing Developments

315

minutes at 70 "C. Protein was dissolved in buffer solution at the desired concentration,and the pH readjusted to 6.0 thereafter. High pressure processing was carried out using a Stansted Mark I1 apparatus (Stansted Fluid Power, Essex, UK). Protein solution was transferred into polythenehylon bags which were vacuum-sealed and immersed in the pressurizing fluid. Processing conditions were controlled as described previouslyll to give a pressurization temperature of 30 f 1 "C at a set pressure in the range 200-700 MPa for a dwell time of 30 minutes. Emulsions were prepared from 45 vol% oil + 55 vol% aqueous phase (containing 4.6 wt% BSA) using a laboratory-scale jet homogenizer. Polysaccharide solutions of the appropriate concentrations were mixed with freshly prepared stock emulsions (45 ~01%)to give emulsion samples (20 vol% oil, 1.7 wt% BSA) containing various concentrationsof ICAR in the range 04.22 wto?. Half of each emulsion was stored quiescently in a sealed glass tube at 25 "C. The time-dependent change in thickness of any (semi-)transparent serum layer at the bottom of the tube was monitored visually. The other half was kept in a separate tube at 25 "C with periodic gentle agitation for the purpose of monitoring timedependent changes in droplet size distribution. The average volume-surface diameter d32 = Zp,d?IXp&, where ni is the number of droplets of diameter di, was recorded using a Malvem Mastersizer S2.01. Particle electrophoretic mobilities were determined using a Malvem Zetasizer 4 laser Doppler apparatus. A single droplet of the BSA-stabilized stock emulsion (45 vol% oil) was introduced into a set of buffered solutions of +CAR (10-4 to 10-2 W ? )prepared by serial dilution, and the highly diluted emulsion samples were injected into the quartz capillary cell. The complex shear modulus G* of concentrated emulsions was determined at 1 Hz using a controlled stress Bohlin CS-50 rheometer with a concentric cylindrical cell. The stock BSA-stabilized emulsion (45 vol% oil) was diluted with appropriate I-CARsolutions to give concentrated polysaccharide containing emulsions (40 vol% oil, 2.7 wt% BSA). Dynamic oscillatory measurements were made at 30 "C in the linear viscoelastic regime (0.5% maximum shear strain). The solutions for surface tension measurement were prepared containing 0.25 w-t% BSA + 0 or 1 wt% t-CAR adjusted to pH 6.0. Following high pressure treatment the solutions were diluted to wt%. Time-dependent surface tension at 25 "C was monitored by the static Wilhelmy plate method using a Kriiss digital tensiometer KlOST with a platinum plate. Before the start of the experiment the surface of the sample solution was 'sucked' to remove any already adsorbed molecules. 3 RESULTS In the absence of added polysaccharide, the oil-in-water emulsions made with native or pressure-treated protein showed no evidence of flocculation or coalescence over the experimental time-scale. This was indicated by constancy of the average diameter of the BSA-coated droplets at d32 = 0.55 k 0.01 pm over a period of up to 10 days quiescent storage at 25 "C. Irreversible flocculation due to added I-CARwas indicated as shown in Figure 1 by the large increase in apparent particle diameter d32*(the asterisk denoting that the quantity refers to the average floc size and not the size of individual droplets). For the emulsion made with the native (untreated) BSA, small I-CAR additions do not have any significant effect on d32*, but at a polysaccharide concentration of c = 0.005 wt% there

316

Gums and Stubilisersfor the Food Industry

was found to be a large increase in the apparent droplet size to d32* > 10 pm; this can be attributed to extensive flocculation by polymer bridging.' 1 At much higher t-CAR contents (c = 0.1 wt%) the average droplet size falls to lower values (d32* < 2 pm) indicating some restabilization by the adsorbed polysaccharide saturating the surface of the BSA-coated droplets. We note that the highest t-CAR contents considered here are below those leading to polysaccharide gel formation in the aqueous phase. Low-stress apparent viscosities of BSA + t-CAR solutions at the same concentrations as in the aqueous phase of the 20 vol% emulsions were found6 to be < 10 mPa s. Experiments were carried out on emulsions containing protein that had been subjected to high pressure processing before the emulsion preparation and polysaccharide addition. Figure 1 indicates that the emulsion made with BSA pressure-treated at 400 MPa has a similar dependence of d32* on t-CAR content as that for the untreated BSA system or that treated at 200 MPa (latter not shown). However, with a treatment pressure of 2 500 MPa, there is a substantial change in flocculation behaviour. The maximum in d32*(c) is clearly shifted to a considerably higher t-CAR content, i. e. cmax= 0.1 wt% in the 500 MPa treated system, as compared with cmax= 0.02 wt% in the 400 MPa treated system.

20

d3;

bm)

f

15

10

5

0.0001

0.001

0.01

0.1

1

c (wt%)

Figure 1 Effect of added t-CAR concentration c on the apparent average droplet diameter d32* of BSA-stabilized emulsions (20 vol% oil, 1.7 wt% protein, 5 mM imidazole, pH 6.0) stored for 9 days at 25 "C. High pressure treatment: 0,none; A, 400 MPa; m, 500 MPa.

317

Processing Developments

80

--

60

--

40

--

20

--

H (%I

o ! 0.0001

I

0.001

0.01

0.1

1

c (wt%)

Figure 2 Effect of added t-CAR concentration c on the serum layer thickness H (expressed as percentage of total sample height) of BSA-stabilized emulsions (20 vol% oil, I . 7 wt% protein, 5 mM imidazole, pH 6.0) stored for 9 days at 25 "C. High pressure treatment: 0,none; A, 400 MPa; 500 MPa.

.,

Figure 2 compares the effect of polysaccharide on the extent of serum separation in emulsions made with untreated BSA (20 vol% oil, 1.7 wt% protein) with that in the equivalent emulsions made with BSA treated at 400 or 500 MPa. While the general effect of the +CAR addition is to increase the extent of serum separation after 9 days storage, the consequence of the high pressure treatment is to shift this effect to higher polysaccharide contents. For the emulsion made with protein treated at 200 MPa the creaming behaviour (not shown) is essentially the same as that for the emulsion made with untreated protein. Increasing the treatment pressure from 400 to 500 MPa leads to a large increase in the extent of serum separation at the higher levels of t-CAR addition. Confirmation of the presence of a substantial net attractive interaction between t-CAR and BSA in bulk solution at pH 6 is provided by the time-dependent surface tension data in Figure 3. Addition of 0.004 wt% I-CAR to a 0.001 wt% native BSA system leads to a very much slower rate of decrease in the tension, with the steady-state value for the mixed biopolymer system (after several hours) remaining over 10 mN m-1 higher than that for the pure BSA system.

Gums and Stabilisers for the Food Industry

318

While the rate of tension decrease for BSA pre-treated at 600 MPa is much greater than that for untreated protein, the t-CAR + pressure-treated BSA mixture is rather similar in behaviour to the t-CAR + native BSA mixture. This indicates that the t-CAR complexes with the protein either in its native or pressure-denatured form. Figure 3 confirms that the presence of t-CAR in the BSA solution prior to pressurization also gives the same effect. When the time-dependent tension measurements were made in the presence of 0.1 M sodium chloride, it was found6 that results for pure BSA and BSA + t-CAR were identical within the experimental error. This confirms the predominantly electrostatic character of the BSA-I-CAR interaction at pH 6. On addition of sufficient electrolyte to screen the local molecular charges, the complexation disappears. Table 1 records the measured electrophoretic mobility p of protein-coated droplets in solutions of varying polysaccharide concentration c relative to mobility po in absence of polysaccharide (c = 0). The increase in p/po with increasing c is consistent with the increased net negative charge on the droplets due to complexation of t-CAR with the BSA adsorbed layer at the oil-water interface. The lower values of p/po at low c for emulsions made with pressure-treated protein may be indicative of a less strong interaction between tCAR and adsorbed pressure-treated BSA than with adsorbed native BSA.

75

t

70

2

65

E

C

.-0cn C c"

60

55

50

! 0

100

200

300

400

500

Adsorption time ( m i d

Figure 3 Time-dependent surface tensions of biopolymer solutions (5 mM imidazole, pH 6.0, 25 "C): O,O.OOI Wt% BSA; 4 0.001Wt% BSA + 0.004 wt% \-CAR; 8, 0.001 Wt% BSA treated at 600 MPa; A, 0.001 wt% BSA treated at 600 MPa + 0.004 wt% t-CAR; 0.001 wt% BSA + 0.004 wt% t-CAR treated together at 600 MPa.

*,

319

Processing Developments

Table 1

Efect of high pressure treatment ofprotein before emulsification on electrophoretic mobility p at 25 "C of BSA-coated emulsion droplets in solutions ofpH 6 at I-CAR concentration c (relative to po at c = 0)

P/PO a c / wtY0 0

10-4 10-3 10-2

OMPa

200MPa

1.o 1.35 1.65 1.70

1.o 1.18 1.27 1.41

300MPa 400MPa 1.o 1.19 1.33 1.57

1.o 1.24 1.23 1.55

500MPa

700MPa

1.o 1.06 1.31 1.75

1.o 1.03 1.28 1.37

a Estimated experimental error f 0.07

We turn now to the effect of high pressure treatment of BSA on the small-deformation rheology of concentrated emulsions (40 vol% oil, 2.7 wt% BSA). Figures 4 and 5 show the dependence of the complex shear modulus G* (1 Hz) as a function of the polysaccharide content c. For the emulsions made with the native protein, there is a distinct maximum in G*(c) at cmax= 0.04 wt%. This behaviour is consistent with bridging flocculation at a low added polymer level followed by restabilization at higher polymer contents.

200

t

G* (Pa) 100

0 0

0.1

0.2

Figure 4 Effect of added I-CAR concentration c on complex shear modulus G* at 1 Hz and 30 "Cfor BSA-stabilized emulsions (40 vol% oil, 2.7 wt% protein, 5 mM imidazole, pH 6.0). High pressure treatment: *, none: 0, 200 MPa; 4 300 MPa.

Gums and Stabilisers for the Food Industry

320

150

G* (Pa) 100

50

0

0.1

0.2 c (wt%)

Figure 5 Effect of added t-CAR concentration c on complex shear modulus G* at 1 Hz and 30 "Cfor BSA-stabilized emulsions (40 vol% oil, 2.7 wt% protein, 5 mM imidazole, pH 6.0). High pressure treatment: - - - -, none; 0,300 MPa; A, 500 MPa; a, 700 MPa.

Figure 4 shows that the same distinct maximum in G*(c) occurs also in measurements on emulsions made with BSA pre-treated at pressures of 200 MPa and 300 MPa, although the peak shape appears slightly narrower in the pressure-treated systems. Figure 5 confirms the persistence of the bridging flocculation peak in the data for the emulsion made with BSA treated at 400 MPa, but its disappearance in the data for emulsions made with protein pre-treated at 500 MPa or above. This sharp change in emulsion properties at around 400500 MPa processing pressure is mirrored by the stability data in Figures 1 and 2.

4 DISCUSSION We have demonstrated that the anionic polysaccharide t-CAR forms a soluble complex with native and pressure-treated BSA at pH 6 and low ionic strength. In protein-stabilized emulsion systems this protein-polysaccharide interaction leads to bridging flocculation of native BSA-coated droplets at relatively low added t-CAR concentrations as demonstrated by changes in droplet-size distributions, creaming behaviour, and small-deformation rheology. Whereas pressure treatment of BSA at 200-400 MPa prior to emulsion formation was found not to have any substantial influence on the emulsion properties, treatment at 500 MPa and above was found to disrupt the bridging effect and to shift the flocculation behaviour to much higher added polysaccharide contents.

321

Processing Developments

3

E

.-i X il c.

a

0.: 0.6

I

,+'*

:*

.,U. ..

2.5

5

,,

2

*

"!

0

0

200

300

400

500

700

Figure 6 Comparison of effects of high pressure treatment on the rheology of BSAstabilized emulsions containing added +CAR (open symbols) or DS filled symbols). Relative complex shear modulus G*/Go* at 1 Hz and 30 "C is plotted against treatment pressure p for two differentpolysaccharide concentrations: O, lefr scale, concentration of maximum bridgingflocculation cmm; A, A, right scale, c = 0.24 wt%.

.,

Rather similar behaviour to that reported here was found previously in our laboratory' 1 with BSA-stabilized emulsions containing anionic dextran sulphate (DS) at pH 7. That is, the addition of a low concentration of the polysaccharide gave a bridging flocculation maximum in the complex shear modulus which was absent from data for emulsions made with BSA previously subjected to intense high pressure processing (-400 MPa). The qualitative similarity in the rheological behaviour obtained with these two protein + polysaccharide emulsion systems is illustrated in Figure 6. Plotted as a function of the treatment pressure of the protein prior to emulsification are the relative changes in the values of G* for (a) the polysaccharide content of cmaxcorresponding to the maximum bridging flocculation (0.04 wt% for L-CAR,0.08 wt% for DS) and (b) the (arbitrary) high polysaccharide content of c = 0.24 wt%. The striking closeness in the trends of behaviour suggests that the same physico-chemical mechanism is occurring in both systems. Some similarities in their interactions with proteins might be expected for 1-CAR and DS because both polysaccharides have a high density of sulphate groups and are of similar weight-average molecular weight ( 5 x lo5 daltons). It has been shown previously4.577 that, at neutral pH and low ionic strength, DS forms an electrostatic complex with BSA in the dissolved or adsorbed state under solution conditions where both the protein and the polysaccharide carry a net negative charge. The BSA-t-CAR interaction is different from

322

Gums and Stabilisersfor the Food Industry

the BSA-DS interaction, however, in that its consequences only become properly apparent at pH 6,6 whereas the BSA-DS interaction is clearly strong also at pH 7.5t7 This may be reasonably attributed to some substantial differences in local charge density or molecular flexibility of the two anionic polysaccharides. So the key conclusions are: (i) the addition of either t-CAR or DS gives bridging flocculation of droplets of an electrostatic character in BSA-stabilized emulsions, and (ii) the processing of BSA at high pressure (> 400 MPa) prior to emulsion formation eliminates this bridging flocculation effect by changing the nature of the protein-polysaccharide interactions. In summary, these results indicate that high pressure processing of protein emulsifier can have a very considerable influence on interfacial protein-polysaccharide interactions in the resulting emulsion, with important implications for stability and texture.

ACKNOWLEDGEMENT E.D. acknowledges receipt of a ROPA Award (for K.P.) from the Biotechnology and Biological Sciences Research Council.

References

1. 2. 3. 4. 5.

6. 7.

8. 9. 10.

11.

E. Dickinson and D. J. McClements, ‘Advances in Food Colloids’, Blackie, Glasgow, 1995, chap. 3. E. Dickinson and S. R. Euston, in ‘Food Polymers, Gels and Colloids’ (ed. E. Dickinson), Royal Society of Chemistry, Cambridge, 1991, p. 132. E. Dickinson, in ‘Food Polysaccharides and their Applications’ (ed. A. M. Stephen), Marcel Dekker, New York, 1995, p. 501. E. Dickinson, in ‘Biopolymer Mixtures’ (eds. S. E. Harding, S. E. Hill and J. R. Mitchell), Nottingham University Press, Nottingham, 1995, p. 349. 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. E. Dickinson and K. Pawlowsky, J. Agric. Food Chem., 1997, accepted for publication. E. Dickinson and V. B. Galazka, in ‘Gums and Stabilisers for the Food Industry’ (eds. G. 0. Phillips, P. A. Williams and D. J. Wedlock), IRL Press, Oxford, 1992, vol. 6, p. 351. K. Heremans, in ‘High Pressure Processing of Foods’ (eds. D. A. Ledward, D. E. Johnston, R. G. Eamshaw and A. P. M. Hasting), Nottingham University Press, Nottingham, 1995, p. 81. K. Heremans, J. Van Camp and A. Huyghebaert, in ‘Food Proteins and their Applications’ (ed. S . Damodaran and A. Paraf), Marcel Dekker, New York, 1997, p. 473. W. Messens, J. Van Camp and A. Huyghebaert, Trends Food Sci. Technol., 1997, 8, 107. E. Dickinson and K. Paw1owsky;J. Agric. Food Chem., 1996,442992.

LIQUID-CORE HYDROCOLLOID-OIL CAPSULES

A. Nussinovitch and A. Solomon

Institute of Biochemistry, Food Science and Nutrition The Hebrew University of Jerusalem Faculty of Agriculture, Food and Environmental Quality Sciences P.O. Box 12. Rehovot 76100 Israel 1 ABSTRACT Liquid-core hydrocolloid-oilcapsules were produced in a single step, an advantage relative to the other, multistage methods of fluid-core-capsule production. The contents of the capsule consisted of either 90% glycerol and 10% CaC12 solution, or combinations of 40 to 65% glycerol with 2540% soybean oil, and 10% CaC12 or BaC12. The capsule was made spherical by keeping the density of the liquid medium in the bead higher than that of the alginate solution used to create the membrane. Mechanical properties of the liquid-core capsules were determined using an lnstron universal testing machine, and Ba2+ was found to contribute more to the strength of the capsule than Ca2+. Moreover, the former capsules were stiffer. No significant differenceswere found between the engineering strains at failure of the two kinds of capsules. The higher the oil content within the capsule, the weaker it was. The oil content within the capsules was changed by induced shrinkage and the velocity at which the capsule floated to the surface of the cross-linking solution was determined. The weight loss of the liquid core immersed in specified solutions was described in terms of a two-parameter nonexponential model that had been previously proposed for other physical accumulation and decay phenomena. The model fmed the experimental data well and enabled a calculation of the initial weight loss rate, which was found to be faster with the Ba2+ system. 2 INTRODUCTION Liquid-core hydrocolloid capsules are liquids encapsulated in a spherical polymer membrane1. Lim and Sun1, were the first to describe a method of producing alginatepolylysine liquid-core microcapsules to encapsulate pancreatic islets. Production of these capsules included suspending cells in a sodium-alginate solution, forming small spherical calcium-alginate beads by crosslinking with calcium salt, and reacting with polylysine to create a polylysine-alginate membrane around the bead. In the final stage the bead's core, composed of calcium-alginate gel, was solubilized, thus forming a liquid- core microcapsule containing cells. Until recently liquid-core hydrocolloid capsules were produced in several stages. A onestep technique to form and modify the properties of liquid-core hydrocolloid capsules was first described by our groug. Proteins with molecular weights of 2500-205 000 were entrapped within liquid-core alginate. alginate-chitosan or alginate-polylysine capsules. The ratio, R(t), between protein concentration of a particular protein in the external fluid into which capsules were immersed at a given time and the equilibrium-evaluated concentration

324

Gums and Stabilisersfor the Food Industry

of that same protein in the external fluid was calculated and plotted against time. These diffusion results were compared to diffusion from 'whole' alginate beads. Improved slowrelease properties were achieved when polylysine or chitosan were used to change the permeability of the alginate membrane. These chemical treatments also strengthened the membrane and affected its b r i t t l e n e ~ s ~ - ~ . Recently, the properties of temperature-stable liquid-core hydrocolloid capsules were described5. The contents of the capsule consisted of either distilled water or a 2, 5 or 30% sucrose solution, although other viscous liquids can be used. Mechanical properties of various capsules were studied after incubation at 25, 37, 45, 55 and 85°C for 5, 30 and 60 min, respectively; those of the liquid-core water capsules were studied for a further 2 weeks at 25°C. Capsules having a membrane with a higher hydrocolloid concentration displayed more stress at failure and less brittleness than those with lower solid membrane contents5. The weakest capsules were those with water contents incubated for longer times at higher temperatures. That manuscript presented the temperature-stability relationship of liquid-core capsules. Projections of three-dimensional curves of mechanical property vs. time, temperature or percent sugar, offer a convenient way of examining the desired mechanical properties and their dependence on liquid-core composition and incubation conditions5. While information on liquid-core beads can be found elsewhere, the inclusion of oil within a capsule is less widespread. In fact, the only report on such an inclusion is a Japanese patent discussing a method to prepare a fish egg analog. This consists of a liquid-core capsule including a drop of colored oil within a viscous medium. The slimy flavored content was designed to simulate caviar. The capsules were relatively big, having a diameter of about 6 mm6. Inclusion of oil within a liquid-core bead can be advantageous, if oil soluble ingredients such as vitamins, colors, flavors,etc. are to be included within the formulation. Thus a macroscopic simulation of oil microencapsulation by spray-drying or another method may be conceived. This manuscript aims at providing information on how to create a spherical liquid-core hydrocolloid oil capsule; to check the limits of oil addition to the formulation; to study the mechanical properties of such beads, and to investigate how to strengthen the beads, as well as to follow changes in the content of such beads after their shrinkage. 3 MATERIALS AND METHODS 3.1 Preparation of Liquid-Core Hydrocolloid-Oil Beads

Various liquid-core capsules were produced (Figure 1) using a method previously described by Nussinovitch et a1.3-5. A solution of either 10% (wh) CaC12 (Merck Company, Darmstadt Germany), or BaC12, also containing 40, 65 or 90% (w/w) glycerol, 0, 25 or 50% (w/w) soybean oil and 0.1% (w/w) Tween 80, was homogenized for 5 min at room temperature, at 18 000 rpm using an Ultra turax. The solution was cooled to 4°C before it was dropped, with continuous and gentle stirring, into 500 ml of a 0.2% (w/w) LV sodiumalginate (Sigma, St. Louis, MO, USA) solution (mol. wt. 70 000-80 000, 61% mannuronic acid and 39% guluronic acid ). Spherical beads resulted via a rapid cross-linking reaction between the divalent Ca or Ba ions and the alginate. The resultant capsules were lefl in the alginate solution for 5 min for additional strengthening of the membrane, and were later transferred to a bath of 2% (w/w) sodium alginate for 1 min followed by further immersion in 10% (w/w) CaC12 or BaCI2 for additional cross-linking.

325

Processing Developments

<. Glycerol

>

~

Homogenize 5 min/2S0 C 18000 rpm t

Cooling AW

solution 0.2% rw/w) (“mi;/250~~~+ Liquid-core capsules

,

,

Capsule removal

,

Physical and chemical property analyses * Varying concentrations

Figure 1 Production of liquid-core hydrocolloid-oil capsules 3.2 Bead Measurements Bead diameters were measured e0.03 mm) by digital calliper (Mitutoyo, Japan) prior to the mechanical tests. The thickness of the membrane was estimated by light microscopy. 3.3 Sphericity Determinations

The sphericity of the liquid-core beads was determined by dividing the diameter of a sphere of the same volume as the object being analyzed by the diameter of the smallest circumscribing sphere or, usually, the longest diameter of the object. This expression for sphericity expresses the shape character of the solid relative to that of a sphere of the same volume. The ratio between the two-already mentioned diameters is the aspect ratio. The Feret diameter, the longest diameter of the object, surface area, average volume and some idea of surface smoothness were derived from image-processing of pictures taken with a camera, using the W-cue soflware of the Galai Company (Migdal HaEmek, Israel). 3.4 Mechanical Measurements

The mechanical properties of the liquid-core capsules were determined by uniaxial compression to failure between lubricated plates, i n an lnstron universal testing machine (model 1100, lnstron Corporation, Canton, MA). The lnstron was card-interfacedwith a 486compatible IBM personal computer. A special program enabled data collection of the Instron’s voltage vs. time measurements into digitized force-deformation, force-time, stressstrain or stress-time files, using any desired definition of stress and strain. The force versus

326

Gums and Stabilisers for the Food Industry

time data was converted to a "pseudo-stress" versus engineering strain relationship according to the following substitutions: cr = (F/Ao)

(1)

where o is the 'pseudo-stress', F the force needed to burst the capsule, and Ao the crosssectional area of the original capsule,and, EE = (~D/Do)

(2)

where EE is the dimensionless engineering strain, ~D the total deformation, and Do the original diameter of the bead. Ten specimens of each bead type were examined. 3.5 Densities of Liquid-Core-Capsule Contents

Densities of glycerol and soybean oil were taken from a standard textbook. The density of the mixture was found by summing the component densities of the continuous and dispersed phases: (3)

where V is the volume fraction. The subscripts 1 and 2 are the continuous phase and dispersed phase, respectively. Calculated densities were confirmed by pycnometer measurement. 4 RESULTS AND DISCUSSION Liquid-core hydrocolloid-oil beads (4.2-5.0 mm diameter with 70 to 90-f.lm thick membranes) were produced instantly in a one-step operation (Figure 1). Table 1 demonstrates the different compositions of the liquid-core beads produced which varied in their internal fluid content and density. Table 1 Compositions ofliquid-core capsules Type of capsule

Glycerol content %(w/w)

Soybean content % (w/w)

oil

CaCI/BaCI2 solution %(w/w)

1

90

0

10

2

65

25

10

3

40

50

10

Pm at ,f'C (stlml) 1.221 *1.224 1.136 *1.137 1.040 *1.042

*Pm - solution with Ba2+ Three basic types of beads were produced, containing 40 to 90% glycerol, 0 to 50% soybean oil and 10% of either CaCI2 or BaCI2 (Figure 2).

Processing Developments

327

Figure 2 Liquid-core hydrocolloid-oil capsule consisting 40% glycerol and 50% soybean oil Membranes were first strengthened after capsule formation with Ca or Ba cations, followed by later immersion in 2% sodium alginate and strengthening of this alginate layer with either CaC12 or BaC12. Six types of beads were produced and tested. The beads strengthened by immersion in the BaCl2 bath had different membrane properties and composition than those immersed in the CaC12 bath. The production flow chart is summarized in Figure 1. To achieve spherically shaped liquid-core beads, the density of the bead contents had to be at least slightly higher than that of the 0.2% alginate solution (-1 .O glml), explaining the high concentrations of cross-linking agent used. The density of the 90% glycerol capsule was highest, either 1.221 glml when strengthened with Ca2+ or 1.224 glml with Ba2+. The higher the oil content within the liquid composing the capsule, the lower its liquid medium's density. The lowest density values were recorded for a liquid-medium composition of 40% glycerol and 50% oil: 1.040 and 1.042 glml for Ca2+ and Ba2+, respectively. In all the capsules containing entrapped oil, deviation from sphericity was minimal (Table 2).

Gums and Stabilisers for the Food Industry

328

Table 2 Image-processing results ofliquid-core hydrocolloid oil capsules Type ofcapsule

Max. projection area (mm')

Equivalent diameter (mm)

(mm)

(-)

(mm')

Ba'+ - 25% oil

15.35

4.42

4.44

095

45.77

Ba L+ - 50% Oil

16.78

4.62

4.64

0.95

52.40

CaL+ - 25% Oil

58.90

Average Feret's

Aspect ratio

Average volume

1811

4.80

4.83

0.94

Ca'f - 50% oil

20.65

5])

5.15

0.96

71.61

"Ba '+ - 25% Oil

])65

4.17

4.20

0.93

38.71

"Ba L+ - 50% Oil

15.49

4.44

4.47

0.91

46.83

"Ca '+ - 25% Oil

1861

4.87

4.90

0.97

61.47

_ 50% Oil

19.43

4.97

5.00

0.96

65.38

"Ca

'+

Tested either immediately or I day

* after production

Although the aspect ratio ranged from 0.91 to 0.97, most capsules had a value of -0.95, reflecting their high degree of sphericity. The smallest liquid-core volume was recorded for the capsules with the least included oil, and for those which underwent further cross-linking and contained 25% included oil. The maximum projected area was smallest with lowest oil content and Sa cross-linking, and was as a function of time. The longer the time, the smaller the capsule. The influence of production conditions on the mechanical properties of the capsules is demonstrated in Table 3.

different

Table 3 Mechanical properties ofliquid-core hydrocolloid-oil capsules Type of cation

Oil content % (w/w)

Ca2+

8a 2 +

0 25 50 0 25 50

a (kPa) B6± 15 66 ± 7 40 ± 5 185 ± 19 97 ± 10 64 ± 5

B

(-)

0.76 0.73 0.70 0.78 0.77 0.77

± 0.03 ± 0.04 ± 0.02 ± 0.02 ± 0.02 ± 0.03

Deformahility modulus (kPa) 22 ± 2 16 ± 2 12 ± 1 25 ± 2 21 ± 2 15 ± 2

R2 of rr vs

B

0.936 0.922 0.902 0.975 0.925 0.884

Each result is the average of at least 10 determinations ± S.D.

The properties are expressed as 'pseudo stress' and the engineering strain at failure. Typical stress-strain relationships for Ca2+ and 8a 2+ are presented in Figure 3, and for liquid-core hydrocolloid-oil capsule after compression and bursting in Figure 4.

329

Processing Developments 200

-

-180 160 ~-

-~

Ba2’-cross linked

o% oil

3

140 -120 -100 --

t 5

25% oil

80 --

60 --

40 ~-

50% oil

20 --

0 0.2

0.4

0.6

0.8

1

02

0.4

06

0.8

1

100 80

0

Strain (-)

Figure 3 Stress-strain relationships of liquid-core hydrocolloid capsules

Figure 4 Liquid-core hydrocolloid-oil cap4wleafter compression and bursting

Gums and Stabilisers for the Food Industry

330

Membrane composition, and especially the type of cation used as a cross-linker, influenced the results. In general, the higher the oil content within the capsule, the less strong it was. Membrane strengthened with Ba2+ was always stronger than that strengthened with Ca2+. With Ba2+ capsules, respective stresses at failure were 185, 97 and 64 kPa for 0, 25 and 50% included oil (Table 3). With Ca2+ liquid-core oil beads, strengths were 136, 66 and 40 kPa for 0, 25 and 50% included oil respectively. None of the capsules were brittle, their engineering strain at failure deviating between 0.79 to 0.78. The deformability modulus calculated at a strain value of about 0.12 (when the highest R2 of S I vs. E was achieved) followed the same trend as detected for strength (Table 4 and Figure 5).

Table 4 Deformahility modulii qfCa2+and Bd' cross-linked liquid-core capsules

Type o f cation

oil content % (w/w)

Ca2+

0

Ba"

25 50 0 2s 50

Deformability modulus (kPa) 22 f 2 16 f 2 12f1 25f2 21 f 2 1Sf2

2.5

'

2

8 b

1.5

ofavs & 0.936 0.922 0.902 0.975 0.925 0.884

%oil

h

m

R2

l

v)

0.5 0 0

0.05

0.1

0.15

Strain (-)

Figure 5 Stress-strain relationships ofBa2+cross-linked,for dejimnahility modulus calculations In other words, capsules were stiffer when membranes were strengthened with Ba2+, vs. Ca2+. Moreover the higher the oil content within the capsule, the less stiff were the membrane and the capsule (Figure 5).

331

Processing Developments

Upon transfer to Ca2+ or Ba2+ solutions for extra strengthening, the Iiquid- core beads shrank. Presumably, glycerol diffused out through the capsule membrane. Evidence for this assumption was provided by the identification of glycerol residues in the solution in which the capsules were immersed. A reduction reaction of C,s+ to Cr+3 and changes in the color of the bath from orange to green reflected the fact that the alcohol groups of the glycerol had been oxidized to aldehydes and ketones in an acidic environment. As a consequence of these cross-linking solutions, it was possible to achieve liquid-core beads with an oil content as high as 66% (Table 5).

Table 5 Oil content after capsule shrinkage Type of cation

Initial oil content

Final oil content

% (w/w)

% (w/w)

Ca2+

25 50 25 50

33 ± 1 61 ± 2 35 ±0.5 66 ±2

Ba2+

Eachresultis the average of at least 10determinations ± S.D. Since the oil is entrapped within the capsule, it is assumed that capsules with even higher oil content can be achieved by changing the cross-linking solution or using other liquids that could further shrink the capsule, such as acetone. In parallel to capsule strengthening and changes in intemal oil concentration, an interesting phenomenon was observed. Because the liquid contents within the bead changed density, the capsules began to float to the surface of the solution. The time taken and velocities at which the capsules rose to the surface are given in Table 6.

Table 6 Floating time and velocity ofshrunken liquid-core hydrocolloid oil capsules Type ofcapsule

Ca2+ -25%oil ea2+ -50%oil Ba2+ -25% oil Ba2+ -50% oil

Time (min) 35±l 9±l 32±1 7±l

Floating velocity (mm/sec)

1.0 2.6 1.4 2.8

Eachresultis the average of 10determinations ± S.D. Ca2+-strengthened capsules contalnlrq 25% oil floated to the surface in -35 min at a velocity of 1 mm/s. Ca2+-strengthened capsules with 50% oil were floating after 9 min, rising at a velocity of 2.6 mm/s.

5 CONCLUSION A method to produce liquid-core hydrocolloid-oil capsules is presented. Their shrinkage enabled the production of capsules with as high as 66% included oil. Liquid-core hydrocolloid-oil capsules can be used for food and non-food purposes, when oil-soluble ingredients are to be included.

332

Gums and Stabilisers for the Food Industry

REFERENCES

F. Lim. and A.M. Sun, Science, 1980, 210, 908. A. Nussinovitch, Z.Gershon, Z. and M . Nussinovitch, Food Hydrocolloids, 1996, 10,21. A. Nussinovitch, 'Thermostable liquid cells'. Israeli patent application no. 103,354, 1992. A. Nussinovitch, 'Thermostable liquid cells'. American patent application no. 08/320,755, 1994. 5. A. Nussinovitch, 2.Gershon, and M. Nussinovitch, Food Hydrocolloids, 1997, 11, 209. 6.T. Ueda, United States patent no. 4.702,921, 1987. 1. 2. 3. 4.

THE TRANSFORMATION FROM ENTHALPIC GELS TO ENTROPIC RUBBERY

AND GLASS-LIKE STATES IN HIGH SUGAR BIOPOLYMER SYSTEMS

VASILW EVAGELIOU, STEFAN KASAPIS and GRAHAM SWORN'

Agricultural and Biosystems Engineering Department, Cradield University, S h e College, S h e , Bedfordshire MK45 4DT, UK 'The NutraSweet Kelco Company, Waterfield, Tadworth, Surrey KT20 5HQ,UK

ABSTRACT We present evidence that low molecular weight co-solutes can cause massive changes m the nature and temperaturedependence of the structures formed by commercially important gelling biopolymers at normal levels of use. Modest concentrations of sugars (< 40%) promote conventional disorder to order transitions and reinforce the rigday of deacylated gellan and K-carrageenan gels. By contrast, at intermediate levels of co-solute (40 to 70%) there is a drop in the network strength but structures can now form at high temperatures (even at 90°C). Calorimetry demonstrates that the rise in network strength is accompanied by more pronounced enthalpic events, but the change m enthalpic order declines m accordance with the reduction in storage modulus at the intermediate range of sugar. Over 70% co-solute, systems show a progressive fiedecade increase m shear moduli with G" overtaking G', as also seen in the entropic transition fkom rubbery to glassy consistency in amorphous synthetic polymers. Gelatin and high methoxy pectin also show this dramatic behaviour. Vitrification of the high sugar biopolymers was followed using the time temperature superposition principle and the combined WiUiams-Landel-Ferry/fiee volume theory thus providing an objective approach for the prediction and control of the textural properties of confectionery products. INTRODUCTION The last four decades have seen the proliferation of research on the hctional properties of low solids biopolymer systems leading to well established relationships between their conformational characteristics and structural behaviour in aqueous solutions and gels.' High solids foods have always been popular but development work was left traditionally to the skilled artisan. Only m the last ten years or so has the importance of the glass transition and the glassy state to the understanding of the texture of high solids systems started to become widely appreciated.2 Food studies close to the glass transition temperature have focused on four types of low water content systems: (i) sugars, (ii) starch hydrolysates of various molecular weights, (iii) biopolymers, in particular starch and gluten and (iv) biopolymers plasticized by relatively low concentrations of sugars (biopolymer:sugar ratio of 1: 1 or By far the most extensive investigations looked at the structural

Gums and Stabilisers for the Food Industry

334

properties and associated relaxations of systems at the glass transition using differential scanning calorimetry (DSC).5 Mechanical measurements are limited, confined to Dynamic Mechanical Thermal Analysis and only a few have been made to determine viscoelastic properties over a wide temperature or fiequency range as has been so successll for syntheticpolymers.6 In synthetic polymer science the viscoelastic behaviour fiom a low Viscosity melt to a brittle glass is modelled with the so-called composite (or master) m e . ' As illustrated in Figure 1 this is given as a fimction of fiequency (i.e. time of measurement) or temperature and comprises four distinct parts. At low fiequencies molecular flow takes place, the applied energy mainly dissipates and the viscous element of the system dominates (I). With increasing fiequency, topological entanglements increasingly appear as stable physical interactions leading to a crossing over of moduli and the occurrence of a rubbery plateau (II). In the case of permanent cross-links (e.g. lightly vulcanised rubber), of course, networks display a low-fiequency equ%%riummodulus and the flow region is not observed. Further reduction in the experimentaltime sees the advent of the glass transition where the viscous component becomes again dominant and this is related to the immobilisation of the polymeric backbone (III).Finally at extremely high fiequencies the moduli cross over for a third time and the system enters the glassy state where only stretching and bending of chemical bonds is allowed (IV)."he interplay of moduli is also seen in their phase lag (tan 6) which thrice acquires the value of 1. Similar developmentsm viscoelasticity are recorded on reducing the temperature, increasing the molecular weight of the material, and increasing the polymnc concentration in diluted systems. Clearly a possible description of the structural properties of high solids biopolymers in the form of Figure 1 is of theoretical interest. This is also of considerable practical importance since the rheology m the rubbery region as the glass transition is approached determiuesthe processing, storage and ultjmately the textural properties of many foods.

1

I1

:

I11

: IV

i

Low

High

Frequency Temperature

High Low

Figure 1 Composite m e of (3, G" and tan 6 (G/G') for amorphous synthetic polymers as a function of fiequency or temperature illustrating the four viscoelastic regions: flow (I), rubbery (n),glass transition (III),and glassy state (IV).

Processing Developments

335

EXPERIMENTAL EVIDENCE FOR THE EVOLUTION OF BIOPOLYMER NETWORK BEHAVIOUR WITH INCREASING LEVELS OF CO-SOLUTE

The Eflect on Mechanical and Thermal Stability at Modest Levels of Co-solute Early observations on the behaviour of polysaccharide systems at relatively low levels of sugar mcluded work by Nkhhwi, O a k d and co-workers.8 "hey prepared cylindrical disks of concentrated K-carrageenan (1-6%) and agarose (2-12%) gels with up to 45% sucrose or glucose. Samples were aged at ragerator temperature and then heated and subjected to small deformation longitudiualwhtions. It was found that increasing levels of sugar produced stronger systems which required higher temperatures for loss of cohesion and the distortion of cylindrical gels. This was rationalised on the basis of additional polymer-polymer associations since sugar competeswith macromoleculesfor water-polymer hydrogen bonding. Polysaccharidepreparations at normal levels of use (< 1%) are solutions at high temperature and cooling runs can unveil u& insights into the mechanism of structure formation. Figure 2 reproduces mechanical profiles for a couple of gelling polysaccharides obtained during controlled cooling at l"C/miu (ref 9 and unpublished results). Thus a gellan solution is indistinguishable fiom water at high temperatures and upon cooling converts into a brittle gel over a narrow temperature range. Beyond a few degrees below the gel pomt the gel has nearly constant Wdily.Addition of 30% sucrose shifts the onset of gelation to higher temperatures and creates substantially stronger networks. In the case of K-carrageenan, glucose syrup was used which is a polydisperse medium with an average dextrose equivalent of 42. As for gellau, addition of 20% glucose syrup (dry solids) results m more thermally stable and reinforced K-carrageenan gels. This, of course, is congruent with earlier arguments for additional ordered associations in a sugar environment.8 However, at 40% glucose syrup the transition becomes broader and networks develop continuouslythroughout the temperature range (absence of plateau) thus presaging a diEerent mechanism of structure formation at intermediatelevels of co-solute. 5

----------

\

3

4

n

s"

s" 1

2

0

1 0

20

40

60

Temperature ("C) Figure 2 Development of G' for 0.5%warrageenan at 0.01 M KCl (-;

first y axis) and

0.5% low acyl gellan with 7 mN added CaCL, (---; second y axis) at levels of co-solute shown by the individual traces (scan rate of l"C/min; fiequacy of 1.6 Hz = l o rads).

336

Gums and Srabilisers for the Food Industry

The Merging of Two Digerent Processes at Intermediate Levels of Co-solute Small deformation measurements on gellan samples document the changing nature of modulus development and thennal stability at higher co-solute levels. As shown m Figure 3a cooling of the polysaccharide in the presence of 45% sucrose reveals a leap in thermal stability with structures forming at about 80"C.9 A low-gradient ascent of modulus then follows which merges with what appears to be, m terms of temperature band and overall shape, the sharp transition of gellan systems in Figure 2. Addition of an extra 5% co-solute further consolidatesthe solid-like response of the early process which is now recorded at the highest accessible temperature of 90°C. By contrast the low temperature step loses out as seen by its broadening and the formation of a weaker structure. At 60% sucrose the transformation is complete, the sigmoidal transition is extinct and the storage modulus follows a monotonic rise with decreasing temperature. The graph also includes the cooling spectnun of gellan at 70% co-solute which comprises 50% sucrose and 20% glucose syrup; the latter being added to prevent sucrose crystallisation. There is an order of magnitude increase in network strength whose build up appears to accelerate at temperatures below 40°C. This feature develops further at higher levels of co-solute with the 70% composition broadly defining the lower bound of its range. An alternative way of demonstrating the changing pattern of viscoelasticity at the intermediaterange of co-solute is to examine its dependence on fiequency. Figure 3b depicts mechanical spectra of storage modulus at the end of cooling runs (5°C) for the gelladsugar systemgAs we would expect fiom the cooling profiles of Figure 2, addition of sucrose fiom 0% to 30% results m stronger structures which yield mechanical spectra with the flat fiequency dependence typical of hydrogels.'O The appearance of the high temperature mechanism m Figure 3s is associated with a U-turn and an incessant weakening of networks which at 60% sucrose are almost three orders of magnitude below the maximum v h e s of 0 at 30% co-solute. Furthermore, the partial loss of structure is accompanied with increasing fiequency dependence of the solid-like component m systems. The structural knots of gellan networks are multichain aggregates which are stabilised by a surrounding hydration layer," and this arrangement can support the additional order at the lower range of sucrose (up to 30%). It appears, however, that the shortage of water with increasing levels of co-solute and its efficient hydrogen bonding with sugarsI2reduce the availability of aggregate-stabilising water molecules and as a re& the network strength plummets at intermediate levels of sucrose (30-60%). We have now verilied that the extent of structural loss at 60% co-solute follows the sequence h c t o s e < glucose < This relates to the increasing number of adjacent equatorial -OH per molecule in the series, whose separation matches the characteristic size of water spaces (0.486 nm) m an ice lattice.I2 At 60% co-solute systems remain homogeneous and transparent so there is no question that the drop m storage modulus is due to the formation of a macroassembly of precipitated aggregates. In addition, the deformation at the breaking pomt of a network soars fiom about 0.25 to 0.6units of strain in an aqueous environment and at 60% co-solute, respectively." Therefore the survivhg cross-links join flexiile chams that require substantial stretching before the yield strain is reached. As shown m Figure 3b for gellan preparations with 70% and 85% co-solute (and discussed m the following subheading), this type of network exhibits an entirely different behaviour fiom the conventionalviscoelasticity of hydrogels; see the resurgence in storage modulus and its strong fiequency dependence. At the intermediate level of co-solute (30-60%), however, it appears that both types of structure, the partially cross-linked at the high temperature end and the aggregated enthalpic at lower temperatures, co-exist thus effectively creating a composite system with a single structuring agent.

337

Processing Developments

3.0

2.5 n

G g 2.0 s"

0

o%, 40.h

W

M J

3

3.5 -

1.5

1.o

0

20

40

80

60

-2.5

100

-1.5

Temperature ("C)

-0.5

0.5

1.5

(fiequency/Hz)

4.0

d

3.5

4.0

$, 3.0 3

3.0

ca

h

8

W

s" 2.0

2.5

2.0

1.o

I

I

0.0 -20

0

20

40

60

80

Temperature("C)

Figure 3 Dynamic oscillatory measurements of a) G' as a function of temperature for 0.5% low acyl gellan plus 3.5 m M added CaCL, with 45%, 50%, 60% sucrose, and 50% sucrose + 20% glucose syrup (cooling rate: l"C/miu; fiequency: 1.6 Hz);b) G' as a

function of fiequency at the end of the cooling runs (5°C) for the above gellan sample with 0%, lo%, 20%, 30%, 40%,50%, 60% sucrose, 50% sucrose + 20% glucose syrup, and 50% sucrose + 35% glucose syrup (the spectnun at 40% sucrose is shown as open squares); c) G' as a hction of fiequency of oscillation for 0.5% K-carrageenan with 10 mM added KCl at levels of glucose syrup shown by the mdividual traces (5°C); d) both moduli for the above K-carrageenan sample at 65% (bottom spectrum) and 75% (top spectrum) glucose syrup with cooling and heating traces being depicted as solid and dashed lines respectively.

338

Gums and Stabilisersfor the Food Industry

As seen for gellan, increasing mounts of co-solute induce significant changes in the structural properties of a K-carrageenan geL Figure 3c illustrates corresponding mechanical spectra for the polysaccharide at the end of the cooling runs of Figure 2 (unpublished results). At low levels of glucose syrup (up to 40%) the values of storage modulus remain independent of the fiequency of oscillation. The rise in network strength persists at 60% co-solute which also shows signs of an impending fiequency dependence. Gratifymgly, an extra 5% glucose syrup causes the network to weaken by half an order of magnitude and to develop further its frequency dependence. The values of G' recover parthlly at 70% co-solute and accelerate rapidly over the experimental fiequency range in the 75% formulation. All m all, a structural transformation occurs at intermediate levels of glucose syrup (around 60%) as seen for the gellan preparations. Clearly, the variation of the viscous component of networks comes into question. To examiue this,we have plotted the temperature profiles of both moduli for K-camageenan systems moving fiom the intermediate to the upper range of co-solute (Figure 3d). Cooling induces a disorder to order transition which for the 65% sample occurs at ca. 42°C judged fiom the crossing over of the moduli traces (fiequency of 1.6 Hz). The unusual course of G" which rises continuously and converges on the G' prompted us to extend the cooling run to -20°C. Indeed, at subzero temperatures water CrystaIIisation and reinforcement of the solid-like character is not observed, but instead the moduli cross over once more and the viscous component becomes dominant. Heating of the sample shows that the second transition is pdectly reversible wheieas values depart fiom the cooling trace at higher temperatures, the network Surviving up to 60°C. T h d hysteresis is also evident at 75% glucose syrup but the system as a whole is more thermally stable with structure formation and melting occurring at about 56 and 71°C respectively. Further, the increase in co-solute content accelerates the onset of the second transition and the development of a very viscous element (values of G" are in excess of lo5 Pa at -20°C for the 75% formulation). This outcome will now be explored both qualitativelyand theoretically. The Vitrifkation of Biopol'er Networks at High Levels of C*solute To bring into view the entirety of the event observed m Figure 3d we prepared samples with 85% glucose syrup (unpublished K-carrageenan results). Textures were rubbery at high temperatures and oscillatory measurements recorded a predominant elastic response with a high tan 6 value (ca. 0.56 at 80°C in Figure 4a). In agreement with data in Figure 3 4 cooling mainly contriiutes to the development of the liquid-like component which now overtakes the G' trace much earlier than the cross-point at 75% glucose syrup (temperatures of 60 and 9°C respectively). Following this, the viscoelasticity mounts up four orders of magnitude as seen for the glass transitions of amorphous synthetic p o i y m ~ ~ sThe . * ~rSle of co-solute is that of an antiplasticizer (as compared with water) with increasing amounts accelerating the advent of vitrification in the K-carrageenan network. Finally, the modulus traces cross over for a second time and the solid-like response becomes dominant at temperatures below -10°C. Thus the system advanced through a significant part of the master curve in Figure 1, namely: fiom the rubbery plateau and the glass transition zone to the beginning of the glassy state. Glass transitions were also noted for low acyl gellan and high methoxy pectin, where the co-solute was a mixture of 50% sucrose plus 35% glucose syrup.I5 In the absence of polymeric material, the same co-solute composition shows a converse pattern as Sucrose padally crystallises on cooling and overall a solid-like behaviour arises. Therefore, it appears to be a reciprocal inhibition of co-solute crystallisation and extensive biopoiymer orderiag in the high solids system which facilitates vitrification of the whole. Reduced macro-

339

Processing Developments

8

7 n

$ 6

8

W

35 4

I

3

-20

I

I

I

I

0

20

40

60

80

Temperature ("C)

40

b I .

.. .

.. 10

! 0

I

20

1

I

60 Glucose Syrup ( % ) 40

I

80

100

Figure4 a) Overlappmg cooling and heating profiles of storage and loss modulus for 0.5% K-carrageenan at 10 mM KCI m the presence of 85% glucose syrup (scan rate: l"C/min; frequency: 1.6 Hz). b) hthalpy changes as a h a i o n of mcrasing concentration of glucose syrup obtained during cooling of the above wxrrageenan sample at a scan rate of O.l"C/min.

340

Gums and Stabilisers for the Food Industry

molecular order was suggested to lie behind the drop in storage modulus in Figures 3b and 3c which was accompanied by the formation of clear gels with elastic texture (hgh yield strain). In the case of K-carrageenan we had the opportunity to acquire additional evidence for this event since its high solids structures melt out below the boiling point and can be handled on a slow scanning calorimeter operating between 5 and 95°C. Figure 4b reproduces changes in the enthalpic content (AH) of exotherms obtained during cooling of the polysaccharide with increasing amounts of glucose syrup. It was not possible to parameterise accurately the aqueous gel of K-carrageenan because a significant part of the transition takes place at the low temperature end, but work by Goycoolea et al. suggested that the value of AH for this system is well below 29 J/g.16 The reinforcement of rigidity at modest concentrations of co-solute m Figures 2 and 3c is reflected in the values of enthalpy which rise fiom = 26 to 34 J/g at 10% and 50% glucose syrup, respectively. Further, the drop in storage mo&s at intermediate levels of glucose syrup in Figure 3c is accompanied . . . . g order with the AH for the 85% formulation being 13.8 J/g. We, therefore, by propose that in a high solids regime the polysaccharide/sugar interactions can not stabilise extensive intermolecular order as seen for the conformative hydrogen bonding between water molecules and a polysaccharide. Reduced cross-linking imparts flexibility to the chain segments which are now capable of undergoing a rubber-to-glass transition. Calorimetricwork by Gidley et ul. in high solids polysaccharidewater systems documented that the (1+6) glycosidic linkages act as loci of conformational disorder and allow vitrification of macromolecular chains." On the present evidence, non ( 1 4 6 ) linked polysaccharides show glass transition phenomena provided that molecular order is curtailed in a high sugar environment.

-

THEORETICAL CONSIDERATIONS OF THE VISCOELASTICITY IN A HIGH SUGAR BIOPOLYMER SYSTEM Molecular flexiiility and the lack of crystallisation allows modulus development in the form depicted in Figure 4a. This, of course, is the joint (and complicated) outcome of both temperature and fiequency effects on structural behaviour. In an elegant treatment of this problem, Ferry and co-workers disentangled the two parameters in a constitutive equation that follows the vitrification of amorphous synthetic pol~mers.'~The requirement for a successll analysis is that of an unchanged internal structure or in other words the relaxation times of processes should have the same response to experimentalvariation. In this case the timetemperature superposition priuciple (TTS) is applicable which dictates that a viscoelastic parameter at any point in a temperature run is associated with a response at a reference temperature as long as a fiequency related adjustment (the so-called shift factor) is implemented.ls We have now tested the applicability of 'ITS to biopolymer glasses which is typically discussed for high sugar K-carrageenan samples. They were cooled at l"C/min fiom 60 to -20°C and every ten degrees mechanical spectra for storage and loss modulus were recorded (Figures 5a and 5b, respectively). The starting point of the cooling run was taken as the reference temperature and data were shifted horizontally until a good superposition over a substantial part of adjacent curves was achieved (Figure 5c). Experimental data are fitted in Figure 5d which also shows the variation in their viscoelastic ratio. Clearly, a low gradient G' trace and the tan 6 remaining below the value of one define the rubbery region at the low fiequency range. In agreement with the temperature profile of Figure 4a, higher fiequencies

34 1

Processing Developments

8.5 7.5

n

6.5

G

g

5.5

4.5 3.5

2

,

-1

-2

1

0

'

1.1

C

8.5 7.5

. 1.0

h

-

6.5

1

m

E 5.5

r+

'0 0

B

W

M

?

- 0.9

I 0.8

- 2.5 4 -2

0

2

4

6

Log (fi.equency/Hz)

8 - 2

0

2

4

6

8

m3 (fiequencym

Figure 5 Mechanical spectra of a) storage and b) loss modulus for 0.5% K-carrageenan (10 mM KC1) in the presence of 85% glucose syrup taken during controlled cooling (l"C/mb) at 60 (w), 50 (a), 40 (A), 30 (A), 20 (O), 10 (o),0 (+),-10 (0)and -20°C (x). C) Horizontal superposition of the fiequency sweeps at the reference temperature of 60°C and d) plotting of the best fit and the damping factor (tan 6 = G"/G').

Gums and Stabilisers for the Food Industry

342

0

1.8 1.4 1.0 0.6 0.2

-0.2 ! 0

I

I

I

I

I

I

1

2

3

4

5

6

7

log aT 10

0.1

Temperature ("C) Figure 6 a) Shift factors derived from the simultaneous superposition of storage and loss modulus curves for high methoxy pectin (+), low acyl gellan (A) and K-carrageenan (0,O) at 85% co-solute, with straight lines following the WLF fit m the T , form. b) Cooling profiles of G' (B), G" ( 0 ) and tan 6 (0) for 9.7% gelatin plus 60.3% glucose syrup (fiequency: 1.6 Hz;scan rate: l°C/miu).

343

Processing Developments

unveil the glass transition region which is then followed by the onset of the glassy state (G' overtakes G" at fiequencies > lo7 Hz).Meanwhile, the values of tan 6 become higher than 1, reach a maximum, level off around 1, and then drop reflecthg the transition fiom a liquidto a solid-like consistency, Overall the 'ITS allowed the construction of a composite curve that spans ten decades of fiequency and six orders of magnitude of Viscoelasticity. As mentioned above, the spectacular increase m moduli during vitrification is followed by the W b h d e l - F e r r y (WLF) equation which can be given m the following log a,= CP(T - To)/(Tm- T) where a, is the horizontal shift tictor, To is the reference temperature and T, is the Vogel temperature19 which m this exercise is equal to To C;. The WLF equation was used m combmation with the fiee volume theory which advocates that the total specific volume of a polymer is the sum of the occupied volume (van der Waals radii, vibrations of individual residues) and the fiee volume of packing irregularities or long string-like movements of macromolecules. Further, the thermal expansion coefficient of the fieevohune undergoes a discontinuity at the glass transition temperature (Tg) suggesting the collapse of the fiee volume in comparison to the total volume. It follows that the parameters C; and C i can be written as a function of the fiactional increase m fiee volume and its thermal expansion co-efficient used to characterise the physical state of a high solids systemzoFor samples that undergo vitrification a linear relationship should be obtained that allows calculation of the WLF parameters (Figure 6a). Thus the Tgs for high solids pectin, gellan and K-carrageenan were found to be -53, -26 and -7°C respectively. In the fist two polysaccharides the co-solute was a mixture of 50% sucrose plus 35% gh~cosesyrup, and the discrepancyin the vitrification temperatures is a reflection of the gellan network which is supported by a surrounding hydration layer whose shortage at high levels of co-solute leads to rapid vitrification. On the other hand, pectin uses sugar as an integral part of its network supported by a subtle balance ofhydrophilic and hydrophobic interactions, and this requires more extreme conditions for the structural transformation into a glassy state. In the case of K-carrageenan, sucrose was replaced by glucose syrup (85%) leading to an earlier vitrification due to a stronger antiplasticizingeffect with increasing molecular weight of the co-solute. This allows us to capture the beginning of the glassy state which is also demarcated by the gradual deviation of shift factors fiom the linear WLF fit m Figure 6a. For this case, the supeqosition of relaxation processes at subzero temperatures (the glassy state) has been shown to follow Arrhenius kinetics. The Tg predicted fiom the WLF analysis (-7°C) is very close to the second cross over of moduli in Figure 4a (FJ -10°C) and together argue that the iuitial complicated relationship of vitrification has been correctly split into a fiequency and a temperature dependence ofviscoelasticity(Figures 5d and 6a, respectively). Finayl, we have caught a glimpse of interesting phenomena m high sugar gelatin systems that merit fiuther research. As discussed in this work, sugar-saturated polysaccharide chains can form thermally stable networks below the boiling pomt which upon cooling undergo a rubber-to-glass transformation. As shown m Figure 6b, however, the antiplasticizing action of co-solute on the gelatin network does not impart similar thermal stabhty to intermolecular associations.z' Thus viscous solutions are obtained for preparations of 70% solids at temperatures down to 35OC. Further cooling induces a disorder-to-order transition and a rubbery gelatin network is formed with a high tan 6 value (R 0.43 at 20°C). At subzero temperatures the viscoelastic ratio becomes greater than one with both moduli developing an increasing temperature dependence. Together, they span the composite curve of Figure 1 fiom the terminal relaxation region to the glass transition and offer the opportunity of molecular modelling in a continuum of low and high

-

Gums and Stabilisers for the Food Industry

344

fiequency/temperature contributions. Almost certainly, a +g theory that relates relaxation processes fiom entanglement points in solution to local motions of the vitrified backbone will provide fundamental understanding as historically exploited to great effect in synthetic polymer science.

ACKNOWLEDGEMENTS The authors are gratefd to Professor J.R Mitchell of the University of Nottingham for stimulating discussionson the glassy state of biopolymers.

REFERENCES 1. 2. 3. 4. 5. 6. 7.

8. 9. 10.

11. 12. 13. 14.

15. 16. 17. 18. 19. 20. 21.

D.A. Rees, Biochem. .I,1972,126,257. L. Slade and H. Levine, Crit. Rev. FoodSci. Nutr., 1991, 30, 115. L. Slade and H. Levine, in: The Glassy State in F o d , ed. by J.M.V. Blanshard and P.J. Lillford, Nottingham University Press, 1993, p. 35. M.T. Kalichewsky, E.M. Jaroskiewicz and J.M.V. Blanshard, Polymer, 1993,34,346. H. Levine and L. Slade, in: Food Structure its Creation and Evaluation, ed. by J.M.V. Blanshard and J.R Mitchell, Butterworths, London, 1988, p. 149. M.T. Kalichevsky, J.M.V. Blanshard and RD.L. Marsh, in: The Glassy State in F&, ed. J.M.V. Blanshard and P.J. Lillford, Nottingham University Press, 1933, p. 133. I.M. Ward and D.W. Hadley, in: An Introduction to the Mechanical Properties of Solid Polymers, John Wiley & Sons, Chichester, 1993, p. 84. K Nishina~i,M. Watase, E. Miyoshi, T. Takaya and D. Oakenfull, Food Technology, 1995, October issue, 90. L.E. Whittaker, I.M. Al-Ruqaie, S. Kasapis, and RK Richardson, Carbohydrate Polymers, 1997, 33, 39. E.R Moms, in: G u m and Stabilisers for the Food Industry 2, ed. by G.O. Phillips, P.A. Williams and D.J. Wedlock, Pergamon Press, Oxford, 1984, p. 57. R. Chandrasekaran and A. Radha, Trends in Food Science h Technology, 1995, 6, 143. M.J. T i t , A. Suggett, F. Franks, S. Ablett and P.A. Quickenden, Journal of Solution Chemistry, 1972,1, 131. G. Sworn and S. Kasapis, FoodHydrocolloids, 1997, in press. M.L. Williams, RF. Landel and J.D. Ferry, Journal of the American Chemical h i e @ , 1955,77,3701. I.M. Al-Ruqaie, S. Kasapis, RK Richardson and G. Mitchell, Polymer, 1997, in press. F.M. Coycoolea, RK Richardson, E.R. Morris and M.J. Gidley, Biopolymers, 1995, 36, 643. M.J. Gidley, D. Cooke and S. Ward-Smith, m: The Glassy State in Foods, ed. by J.M.V. Blanshard and P.J. Lillford, Nottingham University Press, 1993, p. 303. A.V. Tobolsky,J. Appl. Phys., 1956,27,673. H. Vogel, Phys. Z., 1921,22; 645. J.D. Ferry, in: Viscoelastic Properties of Polymers, John Wdey & Sons, New York, 1980, p. 264. M.H. Ong, A.S. Whitehouse, R Abeysekera, LM. Al-Ruqaie and S. Kasapis, Food Hydrocolloids, 1997, in press.

THE STABILITY OF CARRAGEENANS TO PROCESSING

WMMARRS FOOD INGREDIENTS DEPARTMENT LEATHERHEAD FOOD RESEARCH ASSOCIATION RANDALLS RD,LEATHERHEAD SURREY KT22 7RY

1. INTRODUCTION

The carrageenans are linear sulphated galactans extracted from a variety of red seaweeds (Rhodophyta). Their Qssacchande repeating unit is based on a P-D-galacto~ranosylsugar unit linked through its 1,3 positions to an a-D-galactopyranosyl unit through positions 1,4. The various types of carcageenan differ primarily in the content and position of ester sulphate substituent groups and in the extent to which the aD-galactose units are converted to the 3,6 anhydro bridge form which stabilises the C1 chair conformation and promotes helical structure in the chain. Association of helical chains forms the mechanism for gelation. The most highly sulphated h-carragsnan is devoid of 3,6 anhydro bridges and cannot form gel structures. The K- and L- carrageenans,with one and two sulphate groups per disaccharide repeating unit respectively, are partially converted to the 3,6 anhydro form and are capable of forming gels in the presence of suitable cations. The t-carrageenan forms weak gels in the presence of calcium ions whilst Kcarrageenan forms much stronger gels with potassium ions. All three types of carrageenan find application as functional ingredients for food products, but the gelling carrageenans have the widest range of applications. The carrageenans have a variety of uses in non-food applications and have been used as a food ingredient for at least two hundred years. A great deal is known about their fine structure, their solution and gelling properties and their hnctionality in foods.' However, one area which remains controversial and which has generated considerable interest over the past twenty years relates to the question of the possible toxicity of low molecular weight carrageenam and therefore their safety as food ingredients. This arose because of published accounts of certain low molecular weight degraded carrageenam causing colonic ulceration in guinea pigs and rabbits. As a result of these reports, the FDA authorised additional tests in 1972 and concluded that the Food Additive Regulations for carrageenan should be strengthened to ensure that low molecular weight degraded material should be absent from food grade, native carrageenan.2 It was accepted that the term "low molecular weight'' described polysaccharide having a molecular weight less than 1OOkD.

The current specification for food grade carrageenan in both the USA and in Europe includes a minimum viscosity requirement of 5 centipoise for a 1.5% solution at 75OC.' This was intended to ensure that degraded carrageenan did not enter the food chain. However, it has always been recOgnised that a viscosity speafication alone cannot ensure the absence of low molecular weight material and carrageenans meeting the viscosity specification could contain significant amounts of material having molecular weights less than 1ookD. After a lengthy period of enquiry and experimental investigation, the status of carrageenanwas reviewed by the Food Advisory Committee in 1992.4 They considered the results of a number of new studies on the

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Gums and Stabilisersfor the Food Industry

safety of food grade carrageem. In some animal studies, small amounts of carrageenan were absorbed by the gut. This suggested that the food grade carrageenan used may have contained small amounts of low molecular weight material since there was no evidence that gut bacteria could break down carrageenan. Furthermore, the effect of carrageenan absorption on the immune system was not known. The Committee recommended that carrageenan be reclassified into Group B pending further studies of the effects of camageenan on the immune system within a period of two years. It was also proposed that the specification for carrageenan should include a requirement that native carrageenan should not contain more than 1oo/o material with a molecular weight less than 1OOkD. After a hrther period of critical examination, the European Commission issued draft Wty specifications for a number of additives including carrageenan in 19%'. These included the long-standing viscosity specification and an additional requirement that the average molecular weight be not less than 1OOkD. This, together with the explicit requirement that carrageenan be not hydrolysed or chemically degraded, was intended to ensure that low molecular weight carrageenam should not enter the food chain. The precise definition of average molecular weight was not specified in this draft document.

The possibility that carrageenans might be degraded during the processing of food products and also in the gastrointestinaltract has prompted several studies into these aspects of carrageenan stability. Studies of the effect of heat processing on carrageenan in milk-salt systems have shown that some changes in moIecular structure do occuf as a result of heating.' A milk-salt solution was u ~ e dto repduce the ionic environment of milk in the absence of milk protein which might interfere with the electrophoretic characterisation of the camageenan component. The electrophoretic mobility of ~ m g e e n a nwas increased after heating at 122°C for 30 minutes but was not af€ected by heating at this temperature for 5 minutes. Later work on milk-salt systems, using end-group analysis, solution viscosity, sedimentation analysis and agarose gel electrophoresis to measure changes in molecular weight, confirmed the reduction in carrageenan molecular weight with heating time at l22OC.' Electrophoresis of carrageenan on cellulose acetate strip does not provide accurate information relating to molecular weight since the mobility is determined by chargdweight ratio rather than molecular size. It is now known that native polysaccharides of high molecular weight have a restricted mobility on cellulose acetate strip due.to steric hindrance as the large macromolecules pass through the surface layer of the strip.' A Limited depolymerisation reduces this restriction with the result that the polysaccharides move more rapidly as a m o w band according to their chargdweight ratio. This &ect was observed in the milk-salt systemsbut no accurateinformation on molecular weight could be expected using this method. Much more useful information on the fate of carrageenan in processed foods and in the gastrointestinal tract can be obtained using gel permeation or size exclusion chromatography to characterise the molecular weight distribution of carrageenan samples. This technique enables an accurate quantification of the various molecular weight species to be made. Studies of native carrageenans have shown that K-, I- and Atypes contain a broad distribution of molecular weights with a low-molecular weight tail extending down to 2OkD. The commercial carrageenam examined were found to have less than 3% material with a m o k d a r weight 10- than 2oml3. The hydrolysis of K- and i-geenam under conditions designed to mimic the gastrointestinal environment has been studied using GPC on Sepharose CL4B and light scattering methods.' The molecular weight characterisation was carried out in 0.2M LiCl at 60°C which was above the helix-coil transition temperature for this solution. Both types of carrageenan were found to depolymerise in acid solution at 3 p C , but K-carrageenan was more rapidly broken down than i-carrageenan. It was also found that native carrageenans contained a broad distribution of molecular weights including material below 1OOkD. The reason for the carrageenan being more susceptible to acid hydrolysis is probably the lack of helix structure at 3 p C . Further studies of K-carrageenans showed that, in 8 commercial samples, 25% of the carrageenan on average had material below IOOkD and that the depolymerisation of carrageenan in simulated gastric juice was considerabIe." These studies were designed to indicate the extent of carrageenan degradation in the stomach but it should be emphasised that the carrageenans were in their sodium ion form and not in their gelled form as they normally would be in food products.

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347

The stability of carrageenansin their gelled form in a simulated gastric environment has been studied under physiologicaIIy realistic conditions." he stability of car rage en an^ was determined using size exclusion chromatography linked in line with laser light scattering photometry. K-car~ageenanin the presence of potassium ions is comparatively stable after 6 hours at pH 1.2 andat 37T, the most were conditionsinvestigated, The weight average molecular weight remained in excess of 2OOkD and only 20% of the carrageenan had a molecular weight less than 1OOkD.1-Carrageenan was even more stable than carr rage en an. In simulated gastric juice, only ~ WofO~ c ~ n a g e e n ahad n a molecular weight less than 1OOkD.

Clearly, carrageenan in the presence if co-gelling cations is much more stable than carrageenan in sodium ion form at 3 7 T . Howwer, at higher temperatures the carrageenan is in the random coil state and is more swceptible to acid degradation. Studies of the stability of r-carrageenan in the presence of potassiumions have shown that acid-catalysed hydrolysis occurs at t e m p e m between 55OC and 95OC.'* LkgmWon was described as a lirstader random hydrolysis process. A 25% reduction in molecular weight was produced at pH 3 after 1.4 hoursat 5OOC andafter only 28 seconds at 90°C. At pH 4, a similar reduction in molecular weight was recorded after 8 hours at 50°C and after I5 minutes at 9ooc. The rate of depolymerisation is strongly influenced by both pH and temperature.

This raises the important question as to the stability of carrageem to processing at elevated temperatures in food ProQCts containing acids. There is very little information available concerning the fate of carrageenan in model food systems which have been subpcted to standard food processing conditions. These typically range from pasteurisation to ultra-high-bmpmhm-short-time processes. The aim of this project was to determine the effect of various heat processes on the molecular weight distribution of carrageenans in simple water-based systems including a model water dessert jelly formulation. The approach to this study was based on the need to process carrageenans in a standard gelling formulation using standard Wtemperature regimes. The processing conditions were qmduced in the laboratory for prelimimy studies of simple water-based gel systems. Pilot plant was employed to process dessert jellies at higher temperatures ( I l 5 T - 14OT)for short times. The carrageenans were erbr;saed from the formulation after processingby nondestmme ' methods and characterised using sizeexclusion chromatography. The chromatography system was calibrated using carrageenan standardp of predetermined molecular weight. The molecular weight distributions of the processed ou~ageenanswere collstNctedby applying the calibration curve to the elution profile data. The chromatographic c o n d i t i m for the e!Wive separation of carrageenan molecules on the basis of size have been widely investigated, Most workers in the field agree that a column temperature of 5OOC60°C is necessary to ensure that the carrageenan molecules are in the unassociated,ranQm coil state during fractionation. Succesdul molecular weight characterisations have been made using TSK PW columns with lithium chloride as eluentI3, Lichrospher loo0 DIOL columns with sodium sulphate eluent", TSK PW columnswith lithium nitrate eluent", Sepaarose CL4B and CL-2B columns with sodium sulphate eluentI5* PSS HEMA BIO columns with sodium iodide eluentI6,and Shodex OH-Pakcolumns with lithium chloride eluent". In this study, molecular weight distributions were determined using TSK PW columns with sodium dphate as eluent. This research was sponsored by the Ministry of Agnculture, Food and Fisheries (MAFF) and was designed to pwide furtherinformation about the susceptibility of carrageenans to hydrolytic breakQwnin simulated food prooesses. The information provided by this work has contributed to the discussion of the purity StanQrdr and specifications for carrageenans to be adopted by the member states of the Eumpean Community. As a result of these discussions, the European Commission has published a revision of the 19% dmft proposals. The revision document'* states that carrageenan shall not be hydrolysedor OtheMiSe chemically degraded, and must comply with the original viscosity specification (1.5% solution must have a viscosity not less than 5 mPa.s at 75T)but contains no molecular weight specification of any kind. These proposals will go before a Standing Committee for final ratification towards the end of 1997.

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Gums and Stabilisersfor the Food Industry

2. EXPERIMENTALPROCEDURE

All aspects of the experimental approach to this research, including materials selection, industrystandard food formulations, processing conditions, and methods for column calibration and molecular weight determination, were discussed in detail with a sub-committee of MARINALG representing the major carrageenan manufacturers namely, Copenhagen Pectin A/S @enmark), FMC Marine Colloids (USA), Litex/FMC (Denmark), Sanofi Bio-Industries (France) and Hercules Inc.(USA).

In particular, this committee organised the preparation and molecular weight characterisation of a range of carrageenan fractions which were used to calibrate the size exclusion chromatographic system. The carrageenan fractions were prepared in the laboratories of Copenhagen Pectin NS and FMC Marine Colloids. These fractions, ten in all, were characterised in the laboratories of Sanofi Bio-Industries using size exclusion chromatography coupled to low angle laser light scattering photometry (HPSECLLALS). 2.1 Size exclusion chromatography: calibration of the system

The HPSEC chromatographic system consisted of a Waters Model 5 10 pump driving eluant via a Waters 712 WISP Autosampler through a TSK PW 50000/5000/4000 triple column set, maintained at 5OoC, to a Waters 410 Differential Refractometer maintained at 50°C. The eluent, 0.1M sodium sulphate, was maintained at 7OoC and continuously purged by a flow of helium to minimise the dissolved gas content. Carrageenan samples at a concentration of 0.05% were eluted at a flow rate of OSmVmin. The output from the differential refractometer was collected and processed using Waters Millennium 2010 s o h a r e . The carrageenan molecular weight standards together with molecular weight data are shown in Table I Table I : Carrageenan molecular weight standards

Type

Weight average (kD)

Number average (kD) 190 158 122 122 73 17 126

K-

810 600 510 410 240 23 470 250 100

K-

60

11-

1111-

K-

K-

105

54 37

The carrageenan standards were dispersed in 0.1M sodium sulphate and heated to 60°C to give 0.2% solutions. These were filtered through 0.45 micron membranes and diluted to 0.05% with eluent. The samples were then eluted from the HPSEC system and the response data processed to provide a molecular weightlelution volume calibration curve which best fitted the input molecular weight data. This calibration curve is shown in Figure 1. The equation which defines the calibration curve is as follows:

-

log Mw = 21.1 - 1.9*V+ 0.081*V2 000124*V3

(regression coefficient = 0.9904)

349

Processing Developments 6.50-1

I_i

5.504

i$

sm

. 5.00-

4.00-L 16100

18100

20100

22100

24100

26:OO

28100

3C 00

Volume (ml)

Figure 1. HPSEC calibration curve for carrageenans

2.2 pWstability p d d e s of native carrageenans

The stability of native carrageenans in aqueous solution over a range of pH was established for various types of carrageenan extracted from single seaweed sources. The canageenans were in their sodium ion form without co-gelling cations. The carrageenans included type from Eucheuma cottonii, r-type from Eucheuma spinosum, a d h mixture extracted from Chondrus crispus and a d h hybnd carrageenan from G i g a r t i ~radula. The carrageenans were dispersed in distilled water and dissolved at 75T. All cations were exchanged for sodium ion by adding a cationexchange resin in sodium ion form (Amberlite IR-120) to the hot solution. The canageenan solutions were decanted from the resin beads and the m g e e n a n s were recovered by precipitation with propan-2-0l followed by drymg.

The carrageenanswere then dissolved in sodium citratelcitricacid buffer with pH values from 3.5 to 6.0 to a concentration of 1%. These solutions were held at 75OC for 30 minutes, cooled to ambient and the processed carrageenans precipitated and m e r e d as before. The carrageenan samples were dissolved in 0.1M sodium sulphate to 0.2%, dialysed against 0. IM sodium dphate to remove other electrolytes, filtered thrcriigh 0.45 micron membranes and diluted with eluent to 0.05%. They were then characterised by HBEC to produce elution profiles from which the molecular weight distribution curves were calculated using the calibrationrelationship. The elution profiles (not shown) recorded the decrease in molecular weight as the carrageenans were depolymerised at the various pH values. In all cases except one, the elution profile was a single broad peak tailing off at the low molecular weight end The exception was carrageenan from Giga~tinaradula which developed a bi-modal distribution as depolymerisation progressxi h4ate.rial around IOOkD molecular weight eluted around the mid point of the elution curve.

Gums and Stabilisersfor the Food Industry

350

A typical molecular weight distribution calculated from the elution data is shown in Figure 2. The fractional distribution, cumulative bstribution and lOOkD marker line are recorded on these profiles.

I

I

6.00

5.00

10.3

4.00

m

Figure 2. Typical molecular weight distribution profile for carrageenan

The Millennium 2010 sofhvare calculates the percentage of material with a molecular weight less than IOOkD whch is represented by a vertical line on the molecular weight distribution profile. This fraction was recorded for each of the processed carrageenans and is shown plotted against solution pH in Figure 3.

0 0

80 V

3

I

6 3

w

o

G radula

t

Ccrispus

PH o

Enpinosum

Figure 3. Effect ofpH on carrageenan stability at 75V.for 30 minutes

G

Ecottonii

Processing Developments

351

These results show that the different types of carrageenan have reasonable stability to heating at 75OC down to pH 4 and that the rate of depolymerisationincreases dramatically as the pH decreases from 4 to 3. The tarrageenan is the mast stable form whilst the ~arrageenanhas the greatest sweptibility to acid hydrolysis. The carrageenans from Gigartina radula and ChonQus crispus have intermediate stability. It is also clear that the undegraded material contains significant amounts of material, between 5% and 25% having a molecular weight less than 1OOkD.

This illustrates the susceptibility of carrageenans, particularly KarragemaU, to acid hydrows at elevated temperature in formulations having a pH below 4. The carrageenan manufacturers are aware of this fact and make clear in their instructions to users of their carrageenan products the importance of pH control. For example, the recommended pH for a water dessertjelly formulation is 4.1. Consequently, the stability of carragemans to acid hydrolysis has been examined in model systems hfkred at pH 4 and processed using a variety of timdtemperature regimes. 2.3 Stability of carrageenans in industry-standardprsteurisedwater deraert jelly formulation

The stability of carragemans in a pasteurised water dessert jelly formulation was assessed using 5 single-weed carrageenan extracts and 8 commercial carrageenan products. The single-weed extracts were obtained from Eucheuma cottonii (K) and Eucheuma spinosum (I) and were designated K-I, K-2, K-3, 1-1 and I-2. The commercial CarrageenanS Were designated K-4, K-5, K-6, 1-3, 1-4, 1-5, a - 1 and dh-2, the symbols indicating the principle carrageenan type in each product.

The industry-standard water jellies were made by dissolving carrageenan in water and adding sucrose, potassium citrate and citric acid. The final composition was carrageenan 1Y' sucrose 18%. potassium citrate 0.32% and citric acid 0.35%. This gave a pH around 4 in the final product. In one set of jellies, the citric acid was added with the other ingm&ents to the water. In a second set. the acid was ad&d at 80°C before pasteurisation thus minimising the exposure of the carrageenan to low pH. In a third set of jellies, a little more acid was added to the product to give a final pH around 3.6 instead of the recommended 4.0 All jelly samples were processed in the laboratory at 8OOC for I5 minutes. The samples were then neutralised with sodium hydroxide and cations were exchanged for sodium ions using ion exchange resin at SOT. The solutions were then cooled and dnlysed against 0.1M sodium sulphate for 24 hours. This was followed by a second dialysis against fresh eluent for 24 hours. Experiments with carrageenan solutions showed that this dialysis procedure did not alter the measured molecular weight distrihtion (results not shown). Samples were diluted with eluent to give a final carrageenan concentration of 0.05% ready for HPSEC analysis. After filtration through 0.45 micron membranes, the samples were characterised by HPSEC and the percentage of material having a molecular weight less than IOOkD was recorded The values OMained for the singleweed carrageenan extracts are displayed in Figure 4. The unprocessed native carrageenanscontained between 5% and 12% of material less than 1OOkD. This fraction increased by a few percentage points after pasteurisation in the water jelly product but did not exceed 20%. The late addition of the citric acid component just before pasteurisation had the beneficial effect of reducing the limited hydrolysis of carrageenan. The importance of pH control was emphasised by the effect of allowing the pH of the jelly to fall to 3.6 instead of the recommended 4.1. The proportion of material less than lOOkD in the pH 3.6 samples increased dramatically in the case of ~arrageenanto levels far outside the range associated with native carrageenans and therefore outside the range acceptable for food grade carrageenans. The iarrageenans were much more stable to hydrolysis than the warragemans and fractions less than lOOkD did not exceed 25% in these jellies. These results illustratethe importance of pH control,particularly for products containing Karrageenan.

As the thermal processing becomes more severe, so the sensitivity of hydrolysis to pH wuld be expected to increase making it more difficult to minimise hydrolysis of the carrageenan component.

352

Gums and Stabilisersfor the Food Industry 40

35

30 25

20

b

I 8

15

10

5

0 kappa 1 parent

iota 1

kappa 2

pH4.0 acid later

i

kappa 3

iota 2

pH3.6 - 3.7

pH4.0

a

pH3.6 - 3.7

Figure 4. Stability of K- and i-carrageenans in a pasteurised water dessert jelly

The behaviour of K- and i-carrageenans was reflected in water jellies made with commercial carrageenan products. The stability of these carrageenans after pasteurisation in the same water jelly formulation is illustrated in Figure 5. This shows the percentage of the fraction less than 1OOM) in the unprocessed carrageenans (in duplicate), in the pH 4 product and in the pH 3.6 product. 40

35

30 25

E! V

20

b

I R

15

10

5

0 kappa4

I

iota 3 parent

parent

iota4

1

kappa 5 pH4.0

kapjlaml

I

kappa 6 pH3.6 - 3.7

iota5

I

kap/lam 2 pH3.6 - 3.7

Figure 5. Stability of commercial carrageenans in a pasteurised water dessert jelly

Processing Developments

353

The stability characteristics of the commercial carrageenans were similar to those of the single-weed Carrageenans. The K-W carrageenans were much less stable to hydrolysis than the 1-type carrageenam, parbcularly in the pH 3.6 jellies. All carrageenans showed a limited degree of hydrolysis at pH 4 which fell carrageenans were well outside the within the range acceptable for food grade carrageenan. The K-@ acceptable limits at pH 3.6. It can be concluded from these results that the limited hydrolysis of carrageenan which occurs during pasteurisation of water jellies does not sigmticantly extend the low molecular weight content beyond the range commonly found in native food grade carrageenans. It is also clear that cana age en an is the most susceptible to acid hydrolysis and may be more extensively degraded at higher tempahues. It was therefore decided to investigate the stability of kcarrageenan in the standard water jelly formulation processed at higher temperatures for reduced times. 2.4 Stability of K-carrageenan in water jellies processed at higher temperatures

The industry-standard water dessert jelly formulation was prepared using the commercial type carrageenan designated ~ 4The . product was prepared in 200 litre batches and processed through the pilot plant shown in Figure 6 .

Static mmers

-2zZEEm

J

Holding tube 1.8 m, 0.5 min at 90 Wh

Relief valve

Figure 6. Pilot pant assemblyfor the processing of waterjelly product

The carrageenan,sugar and potassium citrate componentswere Mended as dry powders using a Gardner Gloucester Ribbon Blender, added to the water in the Liqukerter with stirring and heated to 70°C to dissolve. The solution was then transferred to the second holding tank and maintained at 35°C 40°C. The citric acid was added to the product just before processing through the system.

-

The product was subjected to various timdtemperatureregimes by pmping through static mixers into a pre-heater, through the tubular heat exchanger and out via a cooling unit to be collected in sample jars. The jars contained an equivalent amount of sodium hydroxide solution to neutralise the pr-duct as it entered the sample jar. This procedure ensured that no degradation of the carrageenan took place after

Gums and Stabilisersfor the Food Industry

354

exchanger. The ~-~rrageenan in each of the samples was then subjected to cation exchange, dialysis and characterisation by HPSEC as before. The tubular heat exchanger was equilibrated at a chosen temperature and the product passed through at a controlled flow rate which defined the residence time at that temperature. In this way, a wide range of timdtemperature conditions was achieved The water jelly formulation was processed at temperatures in the range 85T to 115°C at 5T intervals for 30 seconds residence time (Figure 7). It was also subjected to l l 5 T and 12OOC for 15 seconds (Figure 8), and at l20T to 140°C for 10 seconds (Figure 9).

70

60 50

40

30 20

10

0 85

95

105

115

temperature ICI holding tank treated

Figure 7. Stability of k-carrageenan in waterjellies processed at 85oC - I 1 5 T for 30 seconds

70

6o

II

I

?

2ol

40 30

10

0 -.

-

temperature ICI holding tank

treated

Figure 8. Stability ofk-carageenan in waterjellies at I l5Oc and l2ODCfor 15 seconds

355

Processing Developments

50-

fR

40

temperatwe I CI holding tank

heated

Figure 9. Stabiliv of K-carrageenan in waterjellies processed at 120oC-140OCfor 10 seconds

The data presented in these Figures show the ef€ect of processing temperature on the camgeenaa fraction 4 O O k D molecular weight. At each temperature, the carragenan before processing ("holding tank") is compared with the carrageenan in the same sample after processing ("treated"). The. fraction 4 O O k D in the original carrageenan is also shown for comparison. In the temperature range of 85OC-115OC and at 30 seconds residence time, there was only a small increase in the hction 4OOkD (Figure 7). Compared with the native carrageenan, the material afler processing at ll5OC contained around 18% fraction
The p i t y specifications for food grade carrageenans and other additives have been under review for some years. The view has been expressed that, in addition to the current viscosity stamlard, a molecular weight specification for carrageenans would be required to ensure the exclusion of low molecular weight material from food products. The term "low molecular weight" in this context refers to the carrageenan fraction having a molecular weight less than 1OOkD. The introduction of a molecular weight specification for carrageenan would generate a number of for the industry. Any specification would need to be consistent with the fact that native food grade carrageenans contain varying amounts of low molecular weight fraction which might be as high as 25%. If a limit less than 25% on the permitted quantity of this fractionwere to be imposed, then many food grade carrageenans would become illegal. It would also be very difficult to police such legislation unless internationally agreed protocols for the molecular weight characterisation of carragemamwere in place.

-ems

The need for molecular weight restrictions would have to be basedon evidence that carrageenans in food products might contain unaooep(ablyhigh levels of low molecular weight fraction and this evidence seems to be lacking. The safety of native carrageenam is not in question following extensive toxicity studies. This study has shown that carrageenans should not be sigtuficantly hydrolysed when heat-pmcesed in simple water-based gel systems provided that the industry-recommendedformulations and processing conditions are adhered to. The limited hydrolysis that does take place falls within the range normally assocjated with native food grade carrageenans.

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356

Of course, it is important that food manufacturers using carrageenans should adhere strictly to the recommended pH and processing parameters since these determine the degree of hydrolysis of the carrageenan component. Only at very high temperatures might degradation become a problem if processing times are not strictly controlled. The stability of carrageenans in water-gel systems is likely to reflect the stability of carrageenans in more complex food formulations. For example, in milk systems or meat products, the carrageenan component is stabilised not only by interaction with co-gelling cations but also by interaction with proteins, both of which would be expected to generate secondary and tertiary structures with relatively high resistance to acid hydrolysis The fate of carrageenans in the digestive system does not raise any new problems since carrageenans in the presence of co-gelling cations have been shown to be stable to hydrolysis in the gastric environment. Carrageenam without co-gelling cations would have no function in foods and questions as to the stability of carragenans in sodium salt form therefore have little relevance in framing food legislation. These findings indicate that there is no reason to suppose that extensive degradation of carrageenan takes place during the processing of foods. This, together with published information on the acid-stability of carrageenan in gelled form, suggests that there is no compelling reason for introducing a specified limit on the low molecular weight fraction for carrageenans in food products. The European Commission evidently endorses this view and the most recent review of the draft proposals on purity specifications for carrageenans'* does not include a molecular weight specification. 4. Conclusions

Native food grade carrageenam of the I-, K- and dh- types have broad molecular weight distributions in which the low molecular weight fraction with molecular weight <1OOkD can constitute as much as 25% of the total polysaccharide. These carrageenans have acceptable stability to hydrolysis in a pasteurised water jelly formulation at pH levels down to 4, the pH specified by carrageenan manufacturers for this type of product. The type carrageenan is markedly less stable than either wanageenan or the Klh hybrid carrageenan. This stability has been demonstrated in a wide range of carrageenans from identified single seaweed sources and in blended commercial products. The susceptibility of carrageenans to hydrolysis at pH levels significantly lower than 4 emphasises the importance of adhering strictly to the pH values recommended by the carrageenan manufactureres for heatprocessed products.

K-(hmgeenan, the least stable of the carrageenan types, has stability to hydrolysis when processed in an industry-standard water jelly formulation at higher temperatures for shorter times. Hydrolysis is not sigmiicant when the product is heated at 120°C for 15 seconds. However, hydrolysis becomes sipficant, and the low molecular weight fraction increases to levels well beyond 25%, when the product is heated to 135°C 140°C for 10 seconds. Clearly, at these very high temperatures, residence times must be carefully controlled to keep the degree of hydrolysis of the carrageenan within acceptable limits.

-

5. Acknowledgments

We would like to express our thanks to the Ministry of Agriculture, Fisheries and Food for sponsoring this work We would also like to thank Gerard Brigand (Sanofi Bio-Industries), Arne GrafT Andersen (Copenhagen Pectin NS),Dimitri Stancioff (FMC Marine Colloids), John Rystedt ( L i t e m C ) , Alexandra Hadjiianni (FMC Brussels) and Rodney Gray (Hercules Inc) for their advice and help in providing materials and establishing the most appropriate analytical and processing methods. In particular, we thank h e G& Andersen and Dimitri Stancioff for preparing the carrageenan molecular weight standards and Gerard Brigand for measuring their molecular weight distributions.

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6. References

1. L.Picullel “Food Polysaccharides and Their Applications”Marcel Dekker, 1995, 205

2. Institute of Food TechnologistsExpert Panel on Food Safety and NutritioR J.Food Sci., 1973.38, 367 3. European Commission, The Miscellaneous Food Additives Regulations, 1995, Council Directive 78/663/EEC;amended by Commission Directive 90/612/EEC 4. MAFF Food Advisory Committee, Review of Emul.yi$ers and Stabilisers in Food Regulations, HMSO, 1992. FdXCfllEPfll, 43 5 . European Commission, Drap CommissionDirective, 1996,111/5343/96EN 6. P.M.T.Hansen, M.W.Renoll, J.Food Sci., 1974.39. 103

7. S.Badui, N.Desai,&P.M.T.Hansen, J.Agnc.FoodChem., 1978, 26, (3). 675 8. W.M.Marrs, & RJ.Hart, Agro-Food-lndustri High-Tech., 1993, 3, (6). 24 9. L-G.Ekstriim, J.Kuivinen & G.Johansson, Carb.Res., 1983. 116, 89 10. L-G.Ekstr(lm, Carb.Res., 1985, 135, 283 11. I.Capron, M.Yvon & GMuller, Food Hydrocolloids, 1996, 10, (2). 239 12. D.A.Myslabodski, DStancioff & Ruth A.Heckert, Carb.Polym.. 1996. 31. 83

13. D.Lecacheux, R.Panaras, GBrigand, & GMartin, Carb.Polym., 1985, 5, 423 14. GSworn, W.M.Mam&RJ.Hart, J.Chromat., 1987. 403, 307 15. W.M.Mam, ‘‘Gums and Stabilisersfor the Foodlndustry 4”IRL Press, 1988, 399 16. S.J.Chambers,M.J.Langdon, P.A.Williams & RJ.White, ‘‘Gums and Stabilisersfor the Food Industry 7”IRLPress, 1994, 37

17. S.K.Singh & S.P.Jacobsson, Carb.Polp., 1994, 23, 89 18. European Commission, Draj? CommissionDirective, 1997, IIl/5343/96 EN Rev. I

APPLICATIONS OF ATOMIC FORCE MICROSCOPY IN FOOD SCIENCE

V.J. Moms

Institute of Food Research, Nonwich Laboratory, Norwich Research Park, Colney, Norwich NR4 7UA, UK. 1 ABSTRACT In recent years methodolgy has been developed which allows reliable imaging of certain types of biological materials by Atomic Force Microscopy (AFM). Use of AFM is still not routine and, although considerable effort is still required in optimising specimen preparation for given applications, it is possible to use AFM to tackle problems in food science. Examples will be given in the use of the technique to study biopolymers, mixed biopolymer systems, biopolymer association and gelation, and the study of proteins at interfaces. Present evidence suggests that AFM will join the growing family of microscopic methods which have proved invaluable for investigating complex food systems. 2 INTRODUCTION

Probe microscopes are imaging devices which image by feeling the surface of materials, rather than viewing the sample by transmission or reflection of radiation, as with conventional microscopes1 . The resolution of conventional microscopes is limited by the wavelength of the radiation used to illuminate the specimen. In order to achieve molecular or sub-molecular resolution it is necessary to use high energy electrons. The electron microscope requires the sample to be imaged under vacuum and elegant methodology has had to be developed in order to eliminate beam damage, and to preserve the 'native' state of the biological material. These problems do not arise with probe microscopes. The most usehl probe microscope for biological applicationsis the Atomic Force Microscope (AFM). This feels the surface with a sharp probe which senses changes in force between the probe and the surface as they are scanned relative to each other. The resolution of the AFM is determined by the sharpness'of the probe and the accuracy with which the probe can be positioned relative to the surface. Modern instruments can achieve sub- molecular resolution in gaseous and liquid environments offering the possibility of studying systems with minimum sample preparation*. Early studies using probe microscopes to image biological materials were plagued with problems of irreproducibilityand the observation of a number of artifacts. The origins of such dficulties are now fairly well understood and it is possible to obtain reliable images of biological systems2. This means that it is becoming possible to apply AFM to investigate real problems rather than just carrying out studies to validate the method. Most new microscopic methods have proved invaluable in food science. The following is intended to illustrate applications of AFM and suggests that this technique will join the arsenal of microscopicmethods widely used in food science. 3 MATERIALS AND METHODS

The AFM used in this study was designed and manufactured by Ea t Coast Scientific (Cambridge, UK). Its design and operation are described elsewhere . Direct contact (repulsive force) imaging was carried out under constant force conditions. Short narrow Nanoprobe cantilevers (Digital Instruments, Santa Barbara, CA, USA) were used with a quoted force constant of 0.38 Nm-1. Samples were imaged after deposition onto

f

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freshly cleaved mica. Individual biopolymers, biopolymer networks, gels and interfacial layers were imaged under butanol. Plant cell wall fragments were imaged in a hydrated state in air. Interfacial films were deposited from a flat interface by pulling the mica through the interface. 4 RESULTS AM)DISCUSSION 4.1 Individual Polysaccharides

Elimination of adhesion problems allows reproducible AFM imaging of polysaccharides4.Figure la shows images of the stiff bacterial polysaccharide xanthan gum,

a

b

Figure 1. A M images of individual polysaccharides. (a) Entangled network of xanthan macromolecules: image size 140Ox 1400 nm. (3) Multibranched pectin molecule from a sodium carbonate extract from green tomato cell wall material: image size 28Ox 280 nm. Both are ak constantforce imagesfor deposits on mica imaged under butanol. obtained after deposition from a dilute aqueous solution onto freshly cleaved mica, and then imaged under butanol. Such images permit determinationof molecular length and molecular length distributions. Under appropriate conditions it is possible to obtain higher resolution images of such polysaccharidesillustrating the helical structure of the polymer5. There are a variety of methods available for studying regular helical structures of polysaccharides and techniques such as X- ray diffraction of oriented fibres can provide structural information at atomic resolution. Thus, such high resolution AFM studies of regular polysaccharides will only allow one to confirm conclusions drawn from other methods. AFM studies on heterogeneous populations of irregular polysaccharides offer the possibility for obtaining new information on molecular structure. Studies on pectic polysaccharides selectively and sequentially extracted from green tomatoes illustrate this point. CDTA extraction releases pectic polysaccharides crosslinked within the cell wall by divalent cations. A subsequent extraction with sodium carbonate releases ester-linked pectic polysaccharides. Pectic polysaccharides are generally considered to be essentially linear polymers, consisting predominately of a polygalacturonic acid backbone (containing blocks of free acid residues and methyl esterified residues), interspersed by short rhamnogalacturon regions (RG regions) which are decorated with short neutral sugar sidechains6. Our present AFM studies suggest that the extracted pectic polysaccharidesare actually a complex mixture of linear,

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single branched, double branched and even multiple branched structures7. An example of a complex multiple branched 'sodium carbonate' pectin is shown in figure lb. Present chemical information on such extracts6 suggests that these branches are probably polygalacturonic acid rather than neutral sugars. This needs to be confirmed, as does the site of the linkage (RG region or normal backbone). Analysis of such images for populations of the polysaccharides is allowing determination of distributions of contour length (molecular weight), branch length and number of branches per molecule. Such physical measurements complement chemical analysis and can be used to probe natural processes such as ripening, or to investigate genetic manipulation of plant cell wall structure. The present studies are of marked heterogeneities (large branches). The use of AFM coupled with antibody labelling, or more selective labels, will allow more subtle changes, such as distribution of ester residues, short neutral sugar branches or RG regions to be located in fiture studies. 4.2 Polysaccharideorganisation AFM can also be used to stud organised structures such as the association of cellulose fibres within plant cell wallsl9. At present, in order to reveal the molecular structure of the cell wall, it is necessary to homogenise the plant tissue and to isolate p d e d fragments of the cell wall. These fragments are deposited onto mica and imaged in air. Although imaged in air the wall fiagxnents retain water and it is possible to image the cell wall in the hydrated state. Imaging in air makes it difficult to control the imaging force: thus at present it is possible to visualise the stiffer cellulose based network but not possible to see the weaker pectin based network. Figure 2a shows an image of Chinese water chestnut cell wall obtained by imaging under constant force conditions. The image shows bright and dark regions with some fibrous structure visible in the brighter regions of the image. The bright and dark regions occur because the cell wall is curved (rough) and a large number of grey levels are needed to represent the image. The eye can only discriminate a limited range of grey levels and thus can only perceive molecular structure in certain areas (bright parts of the image). In the dark regions mol ular information is still available but cannot be perceived. Previously it has been shown that the molecular structure can be emphasised usin the method of 'error signal mode imaging'. In this method the image is constructed by ollowing the instantaneous responses of the probe cantilever assembly to changes in force as the probe and sample are scanned relative to one another. Although this method reveals molecular structure throughout the entire image, the height information corresponds to changes in force and is not a true representation of the size of the molecular structures imaged. To obtain real topographical images of the molecular structure it is necessary to subtract away the curved surface of the cell wall: effectively projecting the molecular structure onto a plane so that the grey levels in the image are used to maximum effect in visualising the molecular networkg. This can be achieved by locally averaging the topographical data (fig. 2a) at each point in the image, thus removing the 'high frequency' molecular information, and leaving an image of the 'low fiequency' curved background structure of the cell wall, which can then be subtracted from the original topographical image, to produce the planar projection of the molecular structure (fig 2b). Here it is possible to see clearly the layered structure of the cell wall. Although comparable images of the cell wall can be obtained by TEMIO, the present images are of hydrated cell walls with minimal sample preparation. Thus the present data validates previous TEM studies and the methods developed to preserve the 'native' structure. However, the major advantage of the present approach is the opportunity to study in real time processes such as the enzymatic breakdown of the cell wall.

r

f

Another source of cellulose is bacterial cellulose produced by Acetobacter xylium

This material is the primary component of the Philippino fermented food product Nata. The extracted cellulose, produced under the trade name Cellulon (produced by Weyerhaeuser Company), has had a generally recognised as safe status (GRAS) from the FDA since 1992.

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a

b

C

d

Figure 2. AFM images of complex molecular structures. (a) Cellulose based layered structures present in hydrated Chinese water chestnut cell wall extracts: image size 200Ox 2000 nm. Topographical image 0) Processed version of the image in jig. 2a corresponding to a projection of the molecular structure onto a flat plane. (c) Processed topographical image of bacterial cellulose as produced by Acetobacter qdinum. Image size 8000x 8000 nm. (4 Topographical image of PNG carrageenan showing a mixed iota carrageenan and cellulose network. Image size 700x 700 nm. AN topographical images were obtained under dc constantforce (repulsive) conditions. Detailed sample preparation conditions are as referenced in the text.

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a

b

C

d

Figure 3. AFM images of pobsaccharide networks (a) iota carrageenan. Image size 1OOOx 1000 nm. (b) kappa carrageenan. Image size lOOOx I000 nm. (c) potassium geilan. Image size I600 x I600nm. (4 potassium gellan. Image size 800 x 8OOnm.

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Recently Nutrasweet- Kelco have acquired the rights to this material and have marketed the first (PrimaCel) of a number of possible ingredients based on this material. Figure 2c shows an AFM image of a cellulose pellicle produced by A. xylinum . The image has been enhanced by the method described previously for cell walls9 and shows the cellulose network surrounding the bacterial cells. The cellulose appears as flat bands, presumably composed of individual fibres, rather than the complete layers of fibres observed with the plant cell walls (fig 2b). Because different polysaccharides have different shapes and sizes it is also possible to visualise individual polysaccharides within mixed

b a Figure A AFM images of polysaccharide networks andgelprecursers. (a) Iu/1gellan, Image size 150Ox I500 nm. (6) precursers of potassium gellan network. Image size 800 x 800nm. polymer systems. An example is PNG carrageenan. The milder extraction process used to prepare PNG carrageenan1 means that the carrageenan is not completely extracted from the algal cell wall, and that the final product contains remnants of the cellulose based algal cell wall. This is shown in figure 2d for a sample of PNG iota carrageenan. The two polysaccharides can be distinguished in the mixture by their different shapes and sues. The stiffer cellulose structure also appears to exhibit different contrast; appearing brighter in the image. Imaging by AFM will involve some compression of the molecule as the probe scans across it: molecules with differing stiffness will be compressed to different extents and this may account for the different contrast in the image. The image reveals the more extended cellulose network interpenetrating through the flexible iota carrageenan network.

*

4.3 Gels and Networks

As well as looking at natural polysaccharide networks, it is also possible to study induced molecular association in networks and gels. If a dilute solution of a polysaccharide is deposited onto mica, and then air dried, then this results in a concentration of the polysaccharide solution during drying. For non-gelling polysaccharides entangled networks are formed4. In such networks the polymers crossover one another and such crossover points are easily recognisable because of a doubling of height in the image4. For a gelling polysaccharide, concentration during drying leads to the formation of a permanent network, and a flat hydrated film4912. These films provide a model for studying the structure of gels. Films formed from different polysaccharides (fig. 3,4) show common features but also

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marked differences in structure. A common feature of the networks shown in figs. 3 & 4 is the fibrous nature of the network. This fibrous structure is in marked contrast to the conventionally accepted picture of gels as micellar networks with highly ordered junction zones linked by flexible disordered molecular chains. Despite the chemical similarity between iota and kappa carrageenan there are clear differences in the appearance of the networks formed under similar conditions (figs. 34b). The fibrous structure of the iota network (fig.3a) is constant in thickness and more flexible than the kappa network (fig. 3b), which shows a wider variation in thickness and stiffer strands. The gellan network (fig. 3c,d) is more similar in appearance to the kappa network (fig. 3b) than to the iota network (fig.3a). Investigation of these films can be used to test models of gelation and to explain the observed differences in appearance. This is best illustrated for the model gelling polysaccharide gellan. In the presence of bulky counterions, such as tetramethyl ammonium (TMA) ions, gellan undergoes a coil-helix transition o cooling, shows some degree of association, but does not form a permanent gel network13. Addition of common counterion cations (eg Na, Ca, Mg etc), or acidification, leads to the formation of a permanent network s t r u c t u b . These situations can be simulated in the films by varying the preparation conditions for AFM studies14. Figures 3c,d show networks formed for the predominately potassium salt form of gellan. Figure 4a shows the equivalent situation for the TMA salt form. In the TMA form no permanent network is formed: the image shows a heterogeneous population of branched aggregates all of the same thickness. By contrast the "coils"

temperature

temperature

crystalline junction zone

Figure 5. Fibrous model for gelkm gum gelation.

images in figs 3c,d are of a continuous branched fibrous network. The fibre thickness is variable. Some of the fibres contain short thin stubs, which appear to be embryonic branches which failed to grow and develop as part of the crosslinked network. If the stock solution concentration used to prepare the samples imaged in figs 3c,d is further diluted, then it is possible to ensure that concentration upon air drying fails to exceed the threshold concentration for gelation, and the deposit on the mica consists of gel precursors (fig. 4b). These structures are also a highly heterogeneous population of branched aggregates. The thickness of the fibres varies from aggregate to aggregate. These findings are consist nt with a fibrous model for gelation (fig.5) proposed on the basis of light scattering studies1 4.

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Studies on films provide a means of testing models for gelation of polysaccharides. They represent a bridge between the conclusions drawn from physical chemical studies on gels and sols and the results of X- ray studies on oriented fibres. The films are the starting point for the preparation of fibres. The AFM data also complement studies by TEM or cry0 TEM methods. It would be desirable to demonstrate that the structures seen in the films are representative of the structures formed in the real gels. AFM offers the possibility of imaging hydrated gels14. A problem in attempting to image gels is the distortion of the gel

a

b

Figure 6. AFM images of aqueous gellan gels. (a) 1.2% w/w acid set gellan gel. Image size 2x 2 microns. @) 0.3% w/w gel containing 0.3M KCI. Image size 5x 5 micronsprocessed error signal mode image. All images obtained after deposition onto mica and imaged (dc constant force) under butanol. structure by the probe during imaging. As the probe is scanned relative to the surface it will deform the gel blumng the image. To overcome this problem studies have been made on gellan gels with high shear moduli. A 1.2% potassium gellan gel has a modulus of about 200 Pa. AFM images of such gels are poor, showing bright and dark regions with little evidence for molecular structure. If a 1.5% potassium gellan gel is melted, D- gluconic acid lactone added to a concentration of 0.3%, and the sol set on cooling, then the acid set gel has a substantially higher modulus of about 1000 Pa. Figure 6a shows an image of such a el The branched fibrous structure seen in the films is clearly present in the hydrated gel 4. The modulus of the gel can also be raised by the addition of excess potassium ions allowing imaging of the network structure of the gel. This is illustrated in fig. 6b which shows images of a 0.3% potassium gellan gel prepared in the presence of 0.3M KCl. This gel is still quite weak but structure has been revealed by scanning the gel rapidly.

K

4.4 Interfaces An exciting application for AFM is the study of the molecular structure of layers formed at oil- water or air- water interfaces, and responsible for the stabilisation of emulsions and foams16. Figure 7a shows an early example of such studies: a BSA monola er formed at an oil- water interface and then deposited onto mica for imaging under butanolY6.The height measurements on the image between light and dark regions of the

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New Methodologies

image confirm that the film is one molecule thick and that the bright objects in the image are individual proteins. It is also possible, by isolating structures from the interface as a function of time, to observe structural changes at the interfaces responsible for changes in interfacial properties. Under certain circumstances gelatin will aggregate at air - water interfaces forming a network structure. Initially the gelatin appears as a flexible 'coil': the contrast in these images is poor. With time rod-like structures appear and the number density of such structures increases with time. At longer times larger and thicker rods can

a

b

Figure 7. A M images of interfacial films. (a) BSA network formed at an oil- water interface. Image size 20Ox 200 nm. (5) Gelatin aggregate of tvpe formed at air- water interface. Image size 80Ox 800 nm. be observed. Some of these features are illustrated in fig. 7b: the image shows an equilibrium between the ill-defined 'coil' structures and thin rods (of dimensions which are believed to be individual helices) and a larger rod, believed to be formed by lateral association of the smaller (helical) structures. Such studies illustrate the potential of AFM for probing interfacial behaviour.

5 CONCLUSIONS In the last few years it has become possible to obtain reproducible AFM images of biopolymers. AFM studies can achieve molecular andor sub- molecular resolution on biopolymers in aqueous or gaseous environments. The emphasis is changing from simple validation of the use of AFM to the application of the technique to study biophysical problems. The methods can be used to study biopolmer structure, molecular heterogeneity, molecular association and gelation, and molecular behaviour at interfaces. With time further applicationsin food science will be developed. However, even at present, AFM is providing new insights into problems in food science. 6 ACKNOWLEDGEMENTS VJM wishes to acknowledge the experimental skills of A Patrick Gunning and Andrew R Kirby in the biological applicationsof AFM. The studies on cell walls have relied on the expertise of Keith Waldron and Annie Ng. The early studies on proteins at interfaces

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were made with David Clark and Pete Wilde. The unpublished work on gelatin at air- water interfaces was carried out via a collaboration between Alan Mackie and Patrick Gunning. The present research has been supported by the BBSRC through the CSG support grant for

IFR.

7 REFERENCES 1. V.J. Moms, Prog. Biophys. & Mol. Biol., (1994) 6 1,13 1. 2. H.G. Hansma and J.H. Hoh, Ann. Rev. Biophys. Biomol. Structure. (1994) 23,115. 3. T.M.H. Wong, PhD Thesis, University of Cambridge, UK. (1991). 4. A.R. Kirby, A.P. Gunning and V.J. Moms, Biopolymers. (1996) 38,355. 5. A.R. Kirby, A.P. Gunning, V.J. Moms and M.J. Ridout, Biophys. J. (1995) 68,359. 6. A.G.J. Voragen, W. Pilnik, J-F. Thibault, M.A.V. Axelos and C.M.G.C. Renard, 'Food Polysaccharides and Their Applications', ed. A.M.Stephen, Marcel Dekker, Inc., London, Basel, Hong Kong, 1995, Chapter 10, p. 287. 7. A.N. Round, A.J. McDougall, S.G. Ring and V.J. Moms, Carbohydr. Res. (1997) in press. 8. A.R. Kirby, A.P. Gunning, K.W. Waldron, V.J. Moms and A. Ng, Biophys. J. (1996) 70, 1138. 9. A. Round, A.R. Kirby and V.J. Moms, Microscopy & Analysis (1996) 55,33. 10. M.C. McCann, B. Wells and K. Roberts, J. Cell Sci. (1990) 96,323. 11. G.O. Phillips, 'Gums and Stabilisers for the Food Industry, 8', eds. G.O. Phillips, D.J. Wedlock and P.A. Williams, IRL Press, Oxford, 1996, p. 403. 12. A.P. Gunning, A.R. Kirby, M.J. Ridout, G.J. Brownsey and V.J. Moms, Macromolecules ( 1996) 29,679 1. 13. V.J. Morris, 'Food Polysaccharides and Their Applications', ed. A.M. Stephen, Marcel Dekker Inc., London, Basel, Hong Kong, 1995, Chapter 1 1, p. 341. 14. A.P. Gunning, A.R. Kirby, M.J. Ridout, G.J. Brownsey and V.J. Morris, Macromolecules (1 996) 29,679 1 [corr. Macromolecules (1 997) 30, 163-41. 15. A.P. Gunning and V.J. Morris, Int. J. Biol. Macromol. (1990), 12,338. 16. A.P. Gunning, P.J. Wilde, D.C. Clark, V.J. Morris, M.L. Parker and P.A. Gunning, J. Colloid & Interface Sci. (1996) 183,600.

MICRORHEOLOGY OF SWOLLEN STARCH GRANULES

S.P. Carrington,L.R. Fisher and J.A. Ode11

H.H.Wills Physics Laboratory University of Bristol Tyndall Avenue, Bristol BS8 1TL

1 INTRODUCTION Suspensions of swollen (gelatinised) starch granules are an important component of many processed foods. Other workers have attempted to model the rheological behaviour of such suspensions under processing conditions, the models being based on the assumed viscoelastic properties of individual swollen granules.' Actual measurements of such properties to date have been rather limited.' Techniques recently developed in this laboratory now allow a more detailed assessment of the mechanical properties of individual granules to be made.' These techniques are based on direct measurements of deformation (or strain) as a function of applied force for individual swollen granules, using an adapted flexible micropipette apparatu~.~The results and observations from compression and indentation tests on potato starch granules point to the existence of a mechanically effective boundary membrane. There can be a considerable contribution from the tensile stress in the membrane to the apparent stiffness of the granule, whose mechanical properties are thus more complex than would be the case if the granule were simply an elastic amylopectin gel particle, as has been previously assumed.' We report here the results from a new technique, developed as a further adaptation of the flexible micropipette apparatus, which subjects individual swollen granules to a defined strain as the applied stress is varied.

2 MATERIALS AND METHODS All measurements refer to potato starch (supplied by Sigma) in distilled water at room temperature (25 "C) after pre-swelling at 65 "C. Figure 1 shows the conformation of a selection of swollen potato starch granules under water at room temperature. Typical features of swollen starch granules of this type are that one end of the granule is quite smooth, while the remainder is more highly convoluted. Deformation measurements have previously been performed using flexible micropipettes both to hold the particles and to apply a deforming force, with the magnitude of the force being calculated from the bending of one pipette.' In the adaptation of the apparatus described here, a micropipette is used to position a swollen granule as

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required, but the subsequent deformation is applied by using hydrostatic pressure to suck the granule along a tapering capillary tube.

Figure 1 A videomicrograph of swollen potato starch granules on a microscope slide, under water at room temperature. The apparatus is shown schematically in Figure 2 . Actual videomicrographs showing the experimental procedure are given in Figure 3(A-D). The manipulation is carried out under water at all times, and involves picking up a swollen granule from a microscope slide using a micromanipulator controlled micropipette and gentle suction. Figure 1 shows the initial contact with a granule on the microscope slide, and Figure 3(A) shows the subsequent lifting of the granule off the slide. Pressure control via hydrostatic pressure head

Conical caDillarv

Micropipette used to pick up and r>lace starch granules in in the canillarv

/ k

/

& Granule position in the capillary with increasing suction pressure from L to R

Figure 2 Schematic of the apparatus used to deform the swollen starch granules.

New Methodologies

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Figure 3 (A): Pick up of a swollen granule from the microscope slide using a micropipette (see also Figure 1). (B) Introduction of a swollen granule into the tapering capillary. (C): Ejection of a swollen granule into the capillary (D): Conformational change of a swollen granule as it is sucked into the capillary using a hydrostatic pressure head. The swollen granule is then introduced into a capillary tube of tapering internal crosssection, which has been mounted onto the same microscope. slide (Figure 3(B)). The tapering capillary has been hand-pulled using a gas microflame. The granule is then ejected from the holding micropipette at the entrance of the tapering capillary, using slight positive pressure (Figure 3(C)). The holding pipette can now be withdrawn. The other end of the tapering capillary is attached by flexible tubing to a variable height (cathetometer mounted) water head. A hydrostatic pressure can therefore be created to drive the granule towards the narrower part of the capillary (Figure 3(D)), or indeed in the reverse direction. A “zero” position for the pressure head is found such that there is no flow and the granule sits undisturbed but stretched sufficiently to fill the Capillary. Whilst we take this as our zero position, it is important to note that there is a pressure difference across the granule maintaining this initial deformation. From this position, the pressure head is incrementally reduced in order to deform the granule with the cone profile, subjecting it to

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a defined variation in strain. The granule displacement and equilibrium conformation at given pressures are recorded by videomicroscopy. The extent to which the deformation is reversible can be evaluated by raising the pressure head back to the “zero” position and observing any permanent change of shape of the granule. The videomicrographs are imported into a Silicon Graphics workstation, using a frame grabber to capture the appropriate video images. These captured frames allow an analysis of the cone profile, and hence the strain variation, to be made, along with the change in conformation of the granule as the pressure head is altered.

3 RESULTS AND ANALYSIS Figure 4(A-F) shows a swollen granule under increasing deformation in the tapering capillary, as the pressure head is gradually lowered. Each of the single frames corresponds to an equilibrium granule conformation at a given setting of the pressure head. Figure 4(A) is the initial conformation of the granule for this particular experiment i.e. the “zero” pressure, or zero strain starting conformation. In this conformation, the swollen granule appears as if it is just starting to fill the cone, but has not sealed it completely. There are visible creases or convolutions running along the length of the granule which have not sealed to the capillary boundary, and which allow flow of liquid past the granule, as observed by the passage of minute particles. As the deformation process continues and the granule seals completely, the creases are seen to stretch and diminish, and particles can no longer pass. At low strains, friction between the granule and the glass capillary is negligible. This can be demonstrated by gently tapping the connecting tube between the capillary and the pressure head, to provide a very slight pressure fluctuation. The granule oscillates in the capillary but settles at the same equilibrium position. At higher strains, the effect of friction seems to increase, probably as a result of higher normal forces. This can be overcome by the same gentle tapping of the connecting tube, to ensure that the granule has reached a true equilibrium position at the given setting of the pressure head. The raw data obtained for a typical granule, taken from the series of frames including those given in figure 4(A-F), is presented in Figure 5. This is a plot of pressure, in terms of mm of water from the pressure head, against the length of the granule in the capillary. The granule stiffens as its state of deformation is increased. The raw data can be transformed into a stress-strain curve by using a work balance analysis. The work done in moving the granule by the pressure head is the pressure multiplied by the displaced volume. In the absence of friction, this work may be equated to the effective tensile force on the granule multiplied by the increased length of the granule, thus yielding the tensile force. Figure 6 gives the results of this analysis from the raw data presented in Figure 5. The plot shows a linear region to approximately 30% strain, after which there is significant stiffening. The apparent modulus for the initial deformation is 640 Pa, and the stiffening at higher strains increases this to 9500 Pa. Examination of the granule conformations prior to the stiffening suggests that the initial deformation involves the stretching and pulling out of the creases and wrinkles which exist on the granule surface. After the surface convolutions have been pulled out, the boundary layer then becomes stretched and tensioned itself, leading to the increased apparent modulus beyond 30% strain. The inference of this is that the surface layer (or boundary membrane) provides the most significant contribution to the apparent stiffness of the swollen potato starch granule.

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Figure 4 (A-F):Progressive deformation of a swollen granule in a tapering capillary. The above are equilibrium conformationsfor a given water pressure head setting. Figure 4(A) is “Zero”pressure. (B) 0.2mm. (C) 0.4mm. ( D ) 1 . 4 ~ . (E) 6.4mm. (F) I1.4mm. Field of view changes from Figures 4(D) to 4(E). Granule boundaries have been drawn in to enhance visibility.

316

Gums and Stabilisers for the Food Industry 18

L

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b

L

m 10

%

E E

-t 8 u)

z

6

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0 125

135

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length Nrn

Figure 5 Plot of pressure (mm water) against length (pm)for the deformation of a typical granule along the tapering capillary. The initial conformation corresponds to that in Figure 4(A).

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Figure 6 Plot of stress against strain for the deformation of u typical granule along the tapering capillary. The zero strain conformation corresponds to that in Figure 4(A).

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The above is in agreement with the results of compression and indentation mechanical tests carried out previou~ly.~ There is, however, an apparent difference between the magnitude of the moduli obtained from the compression test and the tapering capillary deformation. The compression test provided force-displacement curves which were of the same shape as Figure 5 , but the initial modulus for small compressions was 17 Pa, which increased to 233 Pa at large compressions. It appears that the capillary deformation provides an initial modulus which is in line with that from large compressions between two essentially flat surfaces. The difference between the two techniques is in their effectiveness in pulling out convolutions in the surface of the granule. It is clear from visual observations that initial deformation in the tapering capillary involves the stretching and pulling out of the surface creases, whereas this is only accomplished in the compression experiments at high deformations (and to a lesser overall extent). Thus, the initial tapering capillary deformations are in a similar regime to that which is obtained at high deformations in the compression test. An initial analysis shows that the rate of surface area increase for deformation of a sphere to an oblate spheroid (approximating to the compression experiment) is substantially lower than the stretching of a cylinder with hemispherical end caps (as for the tapering capillary experiment) for the same strain. Indeed, the initial rate of change of surface area with deformation for the compression experiment is zero, and therefore the very soft response at low deformations in this geometry may be attributed to the modulus of the gel interior alone.

Figure 7 (A,B): Videomicrographs showing plastic deformation in the tapering capillary experiment. (A) Granule conformation prior to straining. (B) Granule conformation after straining. The reversibility of the deformation in the tapering capillary can be studied by taking the pressure head back to its “zero” position after each deformation process, and observing the change in granule conformation during this cycle. There is reversible (elastic) deformation up to a certain point, after which there is permanent (plastic) deformation of the granule. This is illustrated in Figure 7, which shows a swollen granule in the capillary entrance before straining (A) and after straining (B). The resultant change in shape is marked. The level of strain required before permanent deformation occurs is around 35%.

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Figure 8 shows a pressure versus length curve for a starch granule taken well beyond the elastic limit. In this case, the granule experiences a strain of almost 100%. There is a clear yield point at 35% strain, followed by plastic deformation.

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8 A 7 -

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=

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e

3 2 1 0 170

150

190

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250

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270

I*ngm (rm)

Figure 8 Plot of pressure (mm water) against length (p) for the deformation of a swollen potato starch granule taken beyond its elastic limit. MIume

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*

4 4

1

1.05

11

115

1.2

1.25

1.3

4

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,

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Strain

Figure 9 Plot of volume against strain for the deformation of a granule in the tapering capillary experiment.

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The volume of the swollen starch granule can be calculated as it deforms within the constraints of the tapering capillary from simple geometry. A plot of volume against strain is shown in Figure 9. There is a reduction in volume as the deformation process proceeds. The initial volumes are subject to the highest error because the granule shape does not approximate well to a cylinder (see Figure 4(A)), but even when allowing for this, the apparent volume is reduced to about half the initial value. We are not at this stage in a position to say whether the volume change is due to compression of surface convolutions, to loss of water or to a combination of both of these factors.

4 CONCLUSIONS We have demonstrated a new technique based on a further adaptation of the flexible micropipette apparatus, which is capable of measuring the mechanical properties of individual swollen starch granules. The potato starch granules tested show reversible elastic behaviour up to a certain strain limit (approximately 30%), with an associated apparent modulus of 640 Pa. The granules then exhibit permanent (plastic) deformation beyond this limit and further stiffening.The apparent modulus at higher strains is 9500 Pa. The deformation process in the tapering capillary involves a significant reduction in granule volume. Previous results from compression tests of individual swollen potato starch granules showed reversible elastic behaviour, but gave an apparent initial modulus of 17 Pa, which increased to 233 Pa at high deformation^.^ The results of further mechanical tests in this study were consistent with the presence of an effective surface membrane enclosing the particle. The difference in the results from the deformation in the tapering capillary and the compression test cannot be explained if the starch granule is simply considered to be an elastic gel particle. The results from the two tests can be reconciled if the surface membrane provides a considerable contribution to the stiffness of the deforming granule. The deformation regime at low strains in the tapering capillary involves the stretching and pulling out of surface convolutions. In the compression test, this regime is only reached at high deformations, and to a lesser overall extent. The comparison of apparent moduli in both of these cases is found to be consistent. The mechanical tests show that the contribution from the tensile stress in the membrane can dominate the apparent stiffness behaviour of the swollen potato starch granule. As a consequence, a more complex model would be required for the interpretation of the deformation properties of individual granules, than would arise from an elastic gel particle. Such a model should in turn lead to a better understanding of the viscoelastic behaviour of starch suspensions, which is of great significance for the properties and processing of food.

Acknowledgements The financial support of the BBSRC and the Royal Society is gratefully acknowledged.

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References 1. 2.

I.D. Evans and A. Lips, J. Texture Studies, 1992,23,69. I.D. Evans, D.R. Haisman, E.L. Elson, C. Pasternak and W.B. McConnaughey, J. Sci. Food. Agric., 1986,37,573. 3. “Deformation Mechanics of Individual Swollen Starch Granules”, L.R. Fisher, S.P. Canington and J.A. Odell, in Starch: Structure and Function, eds. A.M. Donald, P. Frazier and P. Richmond, RSC Cambridge, (1997). 4. L.R. Fisher, J. Chem. SOC. Faraday Trans., 1993,89,2567.

A COMBINED PULSE-RESONANCERHEOMETER FOR VISCOELASTIC GELS

ST Beckett3, ID Roberts’, AS Whitehouse3 and PR Williams2 1. Nest16 Research Centre, Vers-Chez-les-Blanc, CH-1000 Lausanne 26, Switzerland 2. Department of Chemical Engineering, University of Wales, Swansea, Singleton Park Swansea, SA2 8PP, UK. 3. Nestle R&D Centre, York, PO Box 204, York YO1 lXY, UK Keywords: Thermoreversiblegelation, viscoelasticity, shearwave dispersion 1 SYNOPSIS The viscoelastic properties of ‘critical-gels’ at the gel-point (GP) are of wide scientific and engineering interest’. Although the inherent rapidity and small-strains associated with wave-based rheometry are appropriate to the time-dependent, strainsensitive critical gels, wave dispersion poses potential problems in the analysis of measurements. Herein we describe a rheometer which combines ‘pulse’- and ‘resonance’- techniques, and show how dispersion-induced errors are eliminated at GP. 2 THEORETICAL 2.1 Shear wave propagation

In plane harmonic shearwave propagation in a linear viscoelastic medium of density p, the complex shear modulus, G*, is given by2 :

where r (= [ l/x, + i2n/h]) is a constant in which x, is the exponential damping length, w is the angular frequency and h is the wavelength. G* has components G’ and G”, the storage and loss moduli respectively, where G”/G’ = tan& The phase velocity, v, is given by ok, where k is the angular wavenumber (2dh). For known w and p, G’ and G” are obtained from measurements of v and 6. In the characterisation of transient, weakly structured systems it is desirable to obtain v and 6 using a fixed pathlength. This may be achieved by combining pulse-and resonance-techniques. 2.2 Resonance bandwidth and pulse propagation measurements

We consider wave propagation in a gap of width x between two parallel plates, one undergoing forced displacement with amplitude a = hcos(wt), the other being fixed. As w or x are varied, resonances occur, providing a method of measuring 6. The procedure may be illustrated with reference to the ‘power resonance function’, PXw), given by:

382

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P,(o)

= w2Z2/((o,Z-

02)2

+ o2Z2)

(11)

Here Z is a damping constant and oois the resonant frequency. Tan6 is given by Ao/oo, where do = (02 - 01)in which 01 and 0 2 are the half-power points of the resonance curve. In practice the resonant amplitude bandwidth is recorded, a reduction in amplitude by a factor 2’” corresponding to a power reduction by a factor 2. The amplitude range wherein ooremains constant establishes the linear viscoelastic limit. In pulse propagation measurements, a ‘wave velocity’, vwr derived from the ‘time-of-flight’ of a disturbance propagating over a known distance through a viscoelastic medium provides a shear modulus, G (= p v, 2). The pulse frequency components travel at different phase velocities and v, can be identified with the ‘groupvelocity’, U ( = do/dk ). In viscoelastic systems U > v, thus errors arise in the determination of G, compounded by omission of an appropriate correction for attenuation.

2.3 Wave dispersion and the critical-gel The analyses invoked in the techniques described above assume negligible wave dispersion, but in viscoelastic systems and tan6 are (generally) frequency dependent, giving rise to anornolous dispersion-induced errors. However, for a system at GP, G* is given by the Gel Equation’ where; G’(o) = G”(o)/(tan a d 2 )

(111)

and in which n is the viscoelastic exponent (0 < a < l), tan6 (= a7d2) and hence Z is independent of frequency and dispersion-induced errors in the resonance bandwidth analysis disappear. Recognising that at GP a controls the frequency dependence of G’ and G” which, in turn, determine the value of U/v, Eqns.(I) and (111) may be used to calculate U/v for all critical gel states. The GP wave dispersion is bounded by the elastic solid (U/v = 1) and Newtonian fluid (U/v = 2) conditions respectively. For a in the range 0 < a < 1, U/v has a characteristic value, independent of frequency. Thus, knowing 6 and U/v at GP, vwand G* are found without dispersion-induced error. 3 EXPERIMENTAL

3.1 The apparatus A schematic of the instrument is shown in Figure 1. One of the two plates (A) undergoes forced harmonic displacement in its own plane in response to the distortion of a piezo crystal, resulting in the propagation of plane shear waves, within the viscoelastic medium between the plates in the cylindrical sample reservoir. These are reflected at plate B, resulting in wave interference between A and B. A voltage is produced, from a piezo crystal at B, in response to the impingement and reflection of waves at its surface. Disc positioning is effected by micrometer. A microcomputer (HP P5/75) incorporating a Data Acquisition Processor (DAP

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Figure 1. Schematic Representation of the Pulse-Resonance Rheometer 2400-hereafter DAP) performs waveform generation, data collection, and analysis. The DAP incorporates two microprocessors; a 20MHz DSP56001 and a l6MHz 80C186, giving a 235 kHz sampling rate, with real-time data processing. The DAP was programmed using DAPL Interpreter v.3.30 (Microstar, Tx). The DAP was programmed to generate a sweep fiequency modulated waveform for digital to analogue conversion (DAC) and output. A second DAP generated the squarewave used for pulse measurements which was stored in a FIFO buffer prior to DAC and output, the output being updated every lops. The output repetition rate was set so as to prevent interference between outgoing and reflected pulses. This programme recorded both the output squarewave and corresponding pulse waveform for analysis. The latter procedure scaled the captured data for display, and the time delay between the input square wave and resulting pulse was read, using on-screen software cursors. With the plates in contact the instrument’s response time, K, was found fiom the delay between the leading edge of the squarewave and the peak of the rheometer response. The DAP was also programmed to implement a Fast Fourier Transform on successive 8192point blocks of data, which provided the power spectrum of the waveforms. The sweepand pulse-function were performed sequentially, the former involving a measurement time of 1second, the square wave analysis taking 3 seconds. Before each test, K, the instrument’s resonant response was determined. Tests established that ‘sample’ resonances were in the range 150-250Hz. By adjustment of the plates, the instrument’s (peak) response minst was shifted to >> 250 Hz. An example is shown in Figure 2; the instrument response extends over a range of 200Hz (from 320Hz to 480Hz) and does not coincide with the range within which bandwidth measurements were made. In this range (70Hz to 310Hz) the instrument’s response is of negligible amplitude ( < 20mV). The upper limit of the bandwidth was usually 260Hz.

-

-

-

-

3.2 The gel system To demonstrate the foregoing principles, measurements were made in an aqueous dispersion of gelatin, which forms thermoreversible gels. The gelatin powder (Sanofi Bio Industry, Fra.) was dispersed at 25OC in deionised water, heated to 6OoCand held for 45

Gums and Stabilisers for the Food Industry

3 84

Amplitude /mV 500 450 L 400 L

'\

I

50

--0

100

200

300

Frequency /Hz

:

3

500

)O

Figure 2. Instrument resonance response; measured with plates in contact. The regions of peak amplitude and resonant bandwidth response experienced for gelatin during experimentation are indicated. minutes prior to testing. Experiments were performed on 10% w/w samples, these being transferred to the rheometer and the development of structure monitored as the sample cooled naturally to ambient temperature (= 22OC). 4 RESULTS AND DISCUSSION The data displayed in Figures 3 and 4 were obtained using a 0.5mm plate separation. The instrument's response was characterised, giving K = 1.65ms and Winst = 360Hz. The resonant sample response, ma, was = 180Hz, and the maximum upper bandwidth frequency, 0 2 . was 220Hz. The first measurement, after 2 minutes (temperature T =46' C), is indicative of an extremely weak mechanical structure, with v and 6 of 0.18mi' and 28' respectively, the corresponding values of G' and G" being lo2 Pa and 54 Pa respectively. In the first 10 minutes, T falls from 5OoC to 35OC. During this time the decrease in 6 from 28' to 18.2', and corresponding increase in v from 0.18 ms-' to 0.55 ms'' indicates the increasing mechanical strength of the sample; 6 attains a constant value after 7 minutes, which is maintained for a further 70 minutes with little variation (from 18.4 to 19.2'). The velocity is constant (0.58mi') between 5 and 50 minutes, giving values of G' and G" of

New Methodologies

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61’

v / mi‘

30

3

25

2.5 4

20

’

t ’

,

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I

‘

2

15

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10

1

5

0.5 0

Y

0

1

100

10

0

1000 -

Time /mins Figure 3. Phase angle (diamonds) and wave velocity (circles) versus time for 10% w/w gelatin. The region in which the sample temperature corresponded to the published gelation temperatures for this system is indicated.

= 350 Pa and = 110 Pa respectively. It is interesting to note that the rate of cooling is markedly decreased during this time, with T falling only 3OC in 43 minutes. As T falls to the gelation temperature (28-30’C) v rises, from 0.66ms-’ to 0.72ms-’. G’ and G”, increase as the temperature falls from 30.5’ to 29’C, with G’ increasing from 500 Pa to 750 Pa and G” from 160 Pa to 250 Pa. This indicates a rapid increase in mechanical strength. Thereafter, as T continues to decrease, the strengthening of the mechanical structure is evident with 6 decreasing to 6’ (indicative of a highly elastic system) and v increases to 2.4ms-’ after 1000 minutes. G’ and G” increase with time, reaching 6.1 O3 Pa and 6.102Pa respectively after 100 minutes but, in agreement with the work of others4.’ no equilibrium plateau values of the moduli were recorded.

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G’, G” I Pa

Temperature I OC

loo00

50 45

1000

40 35

100

30 25

10

1

100

10

1000

Time /mins Figure 4. G’ (squares), G” (triangles) and temperature (crosses) versus time for 10% wlw gelatin. Calculated from the experimental data displayed in Figure 3. 5 CONCLUSIONS

A high-frequency rheometer which exploits wave interference has been developed to study the evolution of G* in a system undergoing gelation. The wide range of dynamic rigidity encountered (-lo2 Pa to >5.104Pa) and the avoidance of dispersioninduced errors at GP emphasise its potential for characterising systems of process engineering concern. An in-line version is under development. 6 ACKNOWLEDGEMENTS PRW is supported by an EPSRC Advanced Fellowship (1 990-98)

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7 REFERENCES 1. Winter, H.H. and Chambon, F., 1986, Analysis of linear viscoelasticity of a crosslinkingpolymer at the gel point, J. Rheol., 30(2): 367-382. 2. Ferry, J.D., Viscoelastic Properties of Polymers (Wiley, New York, 1980). 3. Williams, P.R. and Williams, D.J.A., 1994, The influence of wave dispersion

characteristicson the determination of mechanical relaxation spectra, J. Rheol., 38(4): 1211-1226. 4. Camali, J.O., 1992, Gelation of physically associating biopolymer systems, Rheol. Acfa, 3 1:399-412. 5 . Djabourov, M., Maquet, J., Thevaneau, H., Leblond, J. and Papon, P., 1985, Kinetics of gelation of aqueous gelatin solutions,Br. Polym.J., 17: 169-174.

MOLECULAR IMAGES OF GELS FORMED UNDER SHEAR USING ATOMIC FORCE MICROSCOPY

David Powerl, Ian Larsonl, Patrick Hartleyl, Dave E. Dunstan2 and David V. Boger2. Advanced Minerals Product Centre, 2CRC for Industrial Plant Biopolymers Department of Chemical Engineering University of Melbourne, Parkville, 3052, Australia.

ABSTRACT A method for measuring the dynamic rheological properties of gels formed after the application of controlled pre-shear has been developed. Uniform shear at differing rates is applied to the fluid in the rheometer during the gel formation process for a fixed period of ten minutes. Measurements of the dynamic rheological properties of the resultant gels have been conducted for a range of pre-shear rates. The model gel system of hydroxypropyl guar using borate ions as the cross linking agent has been used in this study. The final strength of the gelling system, as indicated by the storage modulus, is strongly dependent on the pre-shear history. An apparent maximum in the equilibrium storage modulus with increasing pre-shear rate is observed for constant pre-shear time. In order to understand the molecular conformational changes induced in these systems by the pre-shear, atomic force microscope images of gel samples on mica have been obtained. With no pre-shear, the gels formed appear random in their structure. Gels formed under pre-shear rates of 200 s-1 show a microscopic structure of fibril type aggregates of aligned polymer strands while at the higher shear rate of 1000s-1 globular type structures are observed. INTRODUCTION

A considerable body of work exists on the rheological measurement of gels(1). Given the importance of gels to industry and life this is not surprising. The importance of the gel state to the food industry is substantial. Significant debate in the literature has been conducted as to the nature and definition of the gel state(2).

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This work describes results obtained for one gel system exposed to differing amounts of controlled pre-shear. Hydroxypropyl guar (HPG) is a semi-flexible polymer of large molecular weight in which the gel strength and properties may be systematically varied using different borate cross linker concentrations. The HPGhorate cross linker system is used as a hydraulic fracture fluid in the petroleum industry and as such has been the subject of considerable study(1). Atomic force microscopy (AFM) has been used to obtain images of the gels at the molecular level to enable the rheological properties of the gels to be interpreted from the microscopic structure of the gels formed. Insight into the orientation of polymers in shear fields is gained in this work as the molecules in the gel are effectively locked in place by the gelling process. Prior experimental work has focused on light scattering from dilute polymer solutions in shear fields to measure conformational and orientational changes induced in the solutions by application the of shear(3). The use of the AFM enables direct observation of the changes induced by the shear field and avoids the difficulties encountered with scattering techniques in both measurement and interpretation of the data. The rheological data obtained in this study are interpreted in view of the AFM images and discussed with reference to our understanding of the alignment of polymers in shear fields. EXPERIMENTAL, SECTION A method for measuring the dynamic rheological properties of gels formed after application of controlled pre-shear has been developed(1). This has involved modification of a controlled shear rate Weissenberg rheogoniometer to enable injection of the polymer and cross linker into the cone and plate geometry simultaneously as is shown in Figure 1. The cone-and-plate geometry enables the gelling solution to be exposed to a uniform shear field(4). Polymer solution

Crosslinker

i r

Figure 1. Diagram showing the cross sectional view of the rheometer geometry used in the experiments. Injection of the sample is made through the central hole in the cone and plate geometry.

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The gel system chosen is hydroxypropyl guar with borate ions as the cross linker. The degree of cross linking in the gel for a given polymer concentration is controlled by buffering the pH of a boric acid solution to approximately 11 where the dominant species is the borate ion. The HPG was obtained from Dowell Schlumberger while the boric acid and sodium hydroxide wefe AR reagent obtained from BDH and used without further purification. The concentration of HPG used in all experiments described herein was 0.48 wt% with 0.36g/l boric acid and 0.26 g/l NaOH. The HPGhoric acid mixture and the NaOH solution held in separate syringes are simultaneously injected into the cone-and-plate geometry of the rheometer and a controlled shear rate applied for ten minutes. The shear was then ceased and dynamic oscillatory measurements performed to measure the storage and loss moduli with time. The sample was subjected to a sinusoidally varying strain to determine the storage and loss moduli respectively. Prior measurement ensured that the displacements were such that all measurements were conducted in the linear visco-elastic region(1). Figure 2 shows the development of the storage modulus, G , as a function of time for samples exposed to different controlled pre-shear rates for ten minutes. Upon completion of the rheological measurement samples were removed from the cone-and-plate geometry and thin slices placed on freshly cleaved mica sections in a manner similar to that used by Kirby et al.(5). The sample preparation procedure is described as follows. The HPG gel samples were placed on freshly cleaved mica and dried in air for 24 hours. This sample preparation was performed with as little sample deformation and disruption as possible. The samples prepared in this way may not be absolutely representative of the gel system in that drying of the samples must lead to dilation of the structure as water is lost from the gel network. The AFM used was a Nanoscope IIIa ( Digital Instruments, Santa Barbara, California ). The clearest images, shown in this work, were obtained using Tappingmodem in air.

RESULTS AND DISCUSSION Results showing the increase in the storage modulus with time are shown in Figure 2. The storage modulus, G , is used as a measure of the gel strength. A complete set of rheological data has been obtained for these systems and is reported elsewhere(l).The values of G, at a fixed time after the cessation of the pre-shear, show a maximum with increasing shear rate. The highest steady state value of G’ is observed at a pre-shear rate of 200 s-1 and decreases with increasing pre-shear rate thereafter. In order to understand the molecular conformational and hence structural changes induced in the gel network by the pre-shear, AFM images of gel samples were obtained. While care must be taken in attributing absolute dimensions to the length scales seen in the AFM images, the general structural features Observed should be representative of those in the gel network. Both dilation of the samples upon drying and possible

391

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70

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.

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40

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600 800 1000

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2000 4000 6000 8000 10000

Time (s) Figure 2. The development of the Storage Modulus (G‘) as a function of time after the cessation of ten minutes of controlled pre-shear at the pre-shear rates indicated by the symbols.

.10.0

100.0

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50.0

-5.0

0.0

nu

IIY

nu

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.n

Figure 3. Atomic Force Microscope image of a HPG/Borate/NaOH gel with no pre-shear. The gel has been placed on a mica sheet for imaging. The image is taken after sample preparation as described in the text.

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distortion during preparation for AFM imaging introduce potential artefacts in the interpretation of the images. Fortunately however, the images obtained are consistent with the observed rheological behaviour as will be discussed. Figure 3 shows a typical sample for the unsheared gel as dried on mica. The random nature of the gel network is evident with individual polymer molecules being clearly distinguishable. Both the polymer molecules and the junction zones appear to be randomly distributed throughout the gel network. Unfortunately, no absolute numbers may be attributed to the polymer chain dimensions as the height and not the width of the image must be used(1). However, the polymer does appear to be single molecular strands between the junction zones. A typical image of the gel formed with 200 s-1 pre-shear is shown in Figure 4. Significant structural differences to those observed for the unsheared case are evident. The formation of fibril type structures can be seen combined with a general orientation of the bundles of these fibres. The fibrils appear to be bundles of the polymer cross linked along the backbone of the molecule and are hrther aggregated into larger filaments. The primary structures appear to be bundles of several polymer molecules. The images are strong evidence that the application of shear aligns the polymer molecules in the shear plane. Care must be taken in absolute interpretation of the data with reference to dilute polymer solution behaviour as a chemical driving force is present due to the cross linker combined with the shear force in this gelling system. As such, the gel system is more complex than simple dilute solutions of polymers. However, the shear field must impose directional order on the unformed gel as the non pre-sheared system appears to be isotropic as seen in Figure 3. The advantage in using the gelling system is that the perturbation to the random structure by the applied shear is locked in place by the cross linking reaction. We believe this work to be the first direct measurement of the orientation and deformation of semi-dilute solutions of polymer molecules in shear fields. It should be noted that the HPG is a semi-flexible polymer in solution whose chemical structure is described in detail elsewhere(1). The direction of orientation cannot be unequivocally assigned to an axis of the shear field as the sample was removed from the rheometer. Future work will investigate the orientation induced in these systems. The supposition that the polymer molecules deform and align with the shear field is supported by these observations. At higher shear rates the fundamental polymer structure is not as long range as that shown in Figure 5 and appears to be generally less ordered than the structure observed after 200 s-1 pre-shear. At 1000 pre-shear the fibril structures are no longer present and a more globular structure is observed as seen in Figure 5. There does also appear to be evidence of a network type structure present around the globular structures. The high shear field does not allow the formation of the long strand like bundles of the polymer but more ball like structures combined with some residual type network structure.

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Presumably at the onset of gelation the effectively larger molecular weight species are unstable in the high shear fields and tumble to form the globular structures observed. The above observations are intuitively consistent with the observed rheological properties for the differing pre-shear rates where the strongest structure is found for the ordered fibre type gel with the weakest structure observed at the higher pre-shear rates. Interesting data on the anisotropy of the rheological properties should be observed at the intermediate shear rates.

1.50

5 0 . 0 nn

I

2 5 . 0 nu

1.00

0 . 0 nu

0.50

n 0

0.50

1.50-

1.00

JJM

Figure 4.Atomic Force Microscope image of a HPG/Borate/NaOH gel pre-sheared at 200 U s for 10 minutes. Sample preparation is as described in the text.

6 CONCLUSIONS In conclusion, the optimal structural properties namely the elasticity of gelled materials appear to be formed in moderate shear fields. Furthermore, this work shows that polymer molecules are aligned in shear fields, albeit with an added chemical driving force of cross linking. The consequences of this work to optimal polymer processing are immense as optimal structural properties are produced at intermediate shear fields; an unexpected result. Further work will pursue the effect of molecular weight and polymer flexibility.

Gums and Stabilisers $or the Food Industry

3 94

100.0 nn

2.00 5 0 . 0 nu

0 . 0 nn

1.00

0 0

1.00

2.00 L)n

Figure 5. Atomic Force Microscope image of a HPG/Borate/NaOH gel pre-sheared for 10 minutes at 1000 1/ s . The sample was prepared as described in the text. ACKNOWLEDGMENTS. The authors would like to thank Phillipa Scholl for technical assistance with sample preparation and Dr. Franz Grieser for the use of the AFM facilities, as well as acknowledge the Advanced Mineral Products Special Research Centre and the Australian Research Council for financial support provided through a Special Researchers grant to Professor David V. Boger. REFERENCES. 1. D. Power, Ph. D. Thesis, Rheological Characterisation of Hydraulic Fracturing Fluids, University of Melbourne, 1997. 2. K. Almdal, J. D y e , S. Hvidt and 0. Kramer, Polymer Gels and Networks, 1,5 , 1992. 3. A Link and J. Springer, Macromolecules, 26,464, 1993. 4. K. Walters, Rheometry, Wiley, New York, 1975.

5. A. R. Kirby, A. P. Gunning and V. J. Moms, Trends in Food Science and Technology, 6 , 359, 1995.

Nutritional Aspects

FUNCTIONAL FOODS - FACTORS CRITICAL TO SUCCESS

John Young Leatherhead Food Research Association Randalls Road Leatherhead Surrey KT22 7RY

INTRODUCTION Before considering those factors that are critical to the success of functional foods, it is important to define the term functional foods. While this would still appear to be a topic for debate, the general consensus is that they are everyday foodstuffs that are expected to have a specific effect on health due to the relevant constituent(s) of the food or drink and for which claims can be made regarding the specific beneficial effect on health to be expected. Thus it is the potential to associate a claim with the food or drink that makes it "functional". While the terms functional food and nutraceutical are often mentioned in the same breath, there is a clear distinction. While the term functional food describes everyday foodstuffs and therefore by definition excludes products in pill or potion form, the term nutraceutical does not. Originating in the US, the term nutraceutical covers a broader range of products and includes, in addition to traditional foods, isolated nutrients, dietary supplements and diets, herbal products, etc. However, for the purpose of this paper comments are restricted to functional foods, which, to reiterate, are recognisable as everyday foodstuffs. Rarely has there been as much debate, controversy and uncertainty surrounding a new development in the food industry. However, that this is the case for functional foods is perhaps not surprising as this concept is all about establishing a role for food that goes beyond that of just pure nutrition. Increasingly, through advances in understanding of the links between diet and health, it is recognised that functional foods could have a significant role to play in reducing the risk of a whole range of disease conditions, including cardiovascular disease, cancer and osteoporosis. Food producers, food ingredients suppliers and food retailers, in attempting to allay the fears of consumer pressure groups and regulators alike about this additional role, are keen to promote functional foods as just normal, everyday foods that have a health benefit claim. In essence, the distinction between functional foods and traditional health

398

Gums and Stubilisers for the Food Industry

products is the science underpinning their development, with functional foods in general considered to be based on stronger scientific principles. Seen as a potential problem by consumer pressure groups and to some extent by nutritionists is the view that widespread availability of functional foods could undermine the long established and widely supported position that the best route to effecting improvements in health through diet is to increase consumption of certain products such as h i t s and vegetables and fish and decrease intake of fatty foods, in particular. Indeed, it could be argued that the recent introduction into the UK of fat-based functional spreads could lead to further consumer confhion on what constitutes a healthy diet. On the one hand, the Government and nutritionists are supporting a decrease in consumption of fatty foods, while industry is advocating consumption of health-promoting fat-based foods. Clearly, it is desirable, if not essential, to support the introduction of such products with information that minimises consumer misunderstanding and confusion. From an analysis of the performance of recently introduced functional foods, it is possible to identify factors that are critical to success. While the success of all food and drink products is influenced by such factors as advertising, product quality, pricing and market positioning, manufacturers of functional foods and drinks face additional challenges, mainly as a result of the health claims and/or inferences being made for such products. These place a heavy demand on the whole consumer communication process, i.e. advertising, consumer education, public relations, etc. In this context, it is also important to recognise that the full potential of functional foods may not be realised if regulatory change is not forthcoming that allows the link between certain dietary components and selected diseases to be made on the pack. Some thoughts and perspectives on factors critical to success of functional foods follow. FACTORS CRITICAL TO SUCCESS Food Safety From a safety point of view, functional foods should not be considered differently from any other food. It is also important to realise that certain new functional foods should be viewed as potential novel foods. With this in mind, the evaluation should include a consideration of novel raw materials, the application of novel processes, and the potential novel use or role in the diet. It is recommended that the novelty of the functional food in any of these aspects should trigger a more formal safety assessment, both at company and government level. This should be along the lines required for other types of novel foods, with some emphasis placed on the consequences of the new functionality. Owing to their diversity, novel foods, and by association functional foods, are considered by the International Life Sciences Institute (ILSI) to require a case-bycase evaluation of safety, which addresses both nutritional and toxicological considerations. In future, nutritional assessments of novel foods will need to be carried out against a background of rapidly advancing knowledge on the role of diet in the causation and

Nutritional Aspects

399

prevention of many diseases. Consequently, it is to be recommended that an integral element of a company’s functional food strategy should be the preparation of a comprehensive scientific dossier covering both health claims and nutritional safety. Proof of efficacy

While it is critical to the future success and credibility of functional foods that any associated claims are supported by a strong scientific dossier, the nature and scope of this support are still topics of much debate. The recent criticism of the clinical trial used to support the yoghurt-stylefbnctional food Gaio from MD Foods served to raise doubts and questions about the scale of research required to substantiate claims. Central to the criticism was the limited sue of the trial, a view that is strongly challenged by MD Foods. Nevertheless, there is clear evidence that the food industry recognises the need to substantiate the claims associated with functional foods with research, including human studies. While, by pharmaceutical industry standards, clinical studies supporting recent functional foods have been modest, there is a view that the burden of proof for products that offer a potential reduction of disease, i.e. fbnctional foods, should not be as great as it is for those that claim to prevent or cure disease, i.e. pharmaceuticals. Only time will tell if this is a practical way forward. Remembering that a food (and not a drug) is being evaluated, it is also critical that developers, regulators, law enforcers, consumer groups, etc., are aware that food has distinct characeristics that may make the establishment of a relationship with disease difficult. Therefore, when interpreting the experimental data, it must be remembered that foods are often complex systems, where the physiologically active ingredients are not in pure form, and the physiological effect may be due to the synergy between several compounds. In addition, the interpretation of clinical trials must recognise that the addition of a food or food constituent to the diet cannot be interpreted as a single controlled interventionbecause of the complexity of food systems. However, what this does serve to highhght is the strategic importance of securing access to the appropriate skills necessary to develop a coherent and well planned approach to the efficacy of functional food. Without this, manufacturers of functional foods could find themselves vulnerable. Finally, there is the view that the sustained credibility of and necessity for functional foods will only come from follow-up studies indicating that they can effect demonstrable reductions in certain disease conditions. This raises the delicate issue of where the responsibility lies for instigating and fbnding such research initiatives.

Consumer education

If fbnctional foods are to realise their full potential, the consumer will need to be educated about their various health propositions and how they should be used within the context of a normal dietary pattern. While both the UK Government and the European Commission are giving increasing emphasis to improving the consumer’s understanding

400

Gums and Stabilisers for the Food Industry

of the role of food and other lifestyle factors on general health and well-being, the ultimate responsibility still rests with the manufacturer and/or retailer. However, this does not mean that Government does not have an important role to play. It can assist by creating an environment that not only ensures consumer safety as a first priority, but also lends credence to the role of certain dietary constituents in disease prevention. This is precisely what happened in the US following the introduction of the Nutrition Labelling and Education Act in 1990. At present there is still a lack of knowledge, and some confusion and misunderstanding within the media, law enforcement agencies, the medical profession and consumer groups, which implies the need for more broadly based communication strategies.

Market positioning It has already been indicated that industry is keen to position functional foods as normal foodstuffs carrying a health claim; it follows that they should be positioned in store alongside traditional products, where possible. This may well prove more problematic for products that may appear as new concepts to the consumer. An example is the fermented milk drink Yakult. This is currently sited with the drinking milks in the UK, but is equally well suited to placement with yoghurts. Consumer research is seen as a particularly effective route to identifying the best in-store location for such products.

Price To charge too high a premium for products whose intended role is to provide a common health benefit could lay the food industry open to criticism. This is particularly true now that growing attention is being given to the comparatively poor diet of the lower socio-economic groups. Clearly, any company that adopts a pricing structure/policy that puts functional foods out of the reach of such groups could be criticised for creating a class of elitist foods. However, in most cases, manufacturers/retailers are pursuing a prudent pricing policy. Indeed, price premiums are generally in line with those of other ‘healthy’ products, e.g. reduced-fat lines. While the consumer will probably tolerate slightly higher prices for functional foods, the failure of Ribena Juice & Fibre has shown that there is a limit; this product was launched at nearly twice the price of standard Ribena. Price was not necessarily the only reason for its failure, but it is not unreasonable to suggest that the consumer was not prepared to pay such a high price premium for a variant that already had a strong health profile, i.e. standard ready-to-drink Ribena. The failure of Ribena Juice & Fibre is also notable in that it highlights that a strong brand name combined with a strong health claim does not guarantee success. However, it is important to note that certain markets appear more tolerant of high price premiums for certain functional foods, Finland being a case in point. Raisio

Nutritional Aspects

40 1

recently launched the cholesterol-lowering spread Benecol, which sells extremely well, even though it is six times the price of a normal margarine. Health claims strategy

A number of early entrants into the European functional foods market were supported by overt health claims, particularly in the UK. Notable entrants in this respect were Gaio yoghurt (MD Foods), and Ribena Juice & Fibre (SmithKline Beecham), both of which claimed, both on-pack and in advertising, to be of benefit in lowering blood cholesterol levels and (by inference) heart disease. These claims, and the associated advertising, received a swift rebuke from the Advertising Standards Authority (ASA), forcing both companies to reconsider health claims and associated advertising. While MD Foods has responded by taking a bullish stance, the publicity surrounding the ASA ruling led to a lot of adverse media coverage for the Company and for other functional foods on the market. Indeed, such was the concern of consumer pressure groups that they were instrumental in persuading the Ministry of Agriculture, Fisheries and Food (MAFF) in the UK to undertake a joint consultation exercise with all appropriate parties (i.e. consumer groups, manufacturers, retailers, research organisations and trading standards authorities), the outcome of which was reported to ministers. In consequence, the Food Advisory Committee is in the process of reviewing the appropriateness of its 1990 Report on Food Labelling. While there are those that feel that additional regulation is the only effective route to controlling misleading health claims, an industry code of practice is widely considered to be a more practical approach. Central to the ongoing debate is the need for systems and communication strategies that lead to a more informed consumer, which at the same time allay the fears of pressure groups that the industry is trying to exploit consumer gullibility with regard to health claims. However, with the science underpinning many of the current functional foods on the market being far from fully understood, a prudent approach on health claims is recommended for the foreseeable future, especially if manufacturers wish to avoid adverse publicity. This potential for adverse publicity creates a dilemma, as barriers to conveying health benefits associated with functional foods may well hold back consumer acceptance and thereby market development. However, closer examination of health claim strategies across Europe does indicate that it is possible to convey a health benefit to consumers without running the risk of adverse media publicity or intervention by the regulatory authorities. In recognition of the problems associated with health claims, some suppliers of functional ingredients, notably ORAFTI, have proposed a number of nutritional concepts that, while specifically related to the use of the prebiotics inulin and oligofiuctose, nevertheless have some general applicability to other functional ingredients/functional foods. Clearly, if innovation and creativity in food manufacture are not going to be stifled, it is important that a common position be reached among all interested parties.

402

Gums and Srabilisersfor the Food Industry

Summary Despite some early product failures and an uncertain regulatory environment, there are still major opportunities for innovation of added-value food and drinks products that have the potential to improve health and reduce risk of some important diseases. However, to realise this, manufacturerswill need to consider the complexity of the issues and the new disciplines and business operationdstructures they will require.

FUNCTIONAL FOODS - FACTORS CRITICAL TO SUCCESS

John Young Leatherhead Food Research Association Randalls Road Leatherhead Surrey KT22 7RY

INTRODUCTION Before considering those factors that are critical to the success of functional foods, it is important to define the term functional foods. While this would still appear to be a topic for debate, the general consensus is that they are everyday foodstuffs that are expected to have a specific effect on health due to the relevant constituent(s) of the food or drink and for which claims can be made regarding the specific beneficial effect on health to be expected. Thus it is the potential to associate a claim with the food or drink that makes it "functional". While the terms functional food and nutraceutical are often mentioned in the same breath, there is a clear distinction. While the term functional food describes everyday foodstuffs and therefore by definition excludes products in pill or potion form, the term nutraceutical does not. Originating in the US, the term nutraceutical covers a broader range of products and includes, in addition to traditional foods, isolated nutrients, dietary supplements and diets, herbal products, etc. However, for the purpose of this paper comments are restricted to functional foods, which, to reiterate, are recognisable as everyday foodstuffs. Rarely has there been as much debate, controversy and uncertainty surrounding a new development in the food industry. However, that this is the case for functional foods is perhaps not surprising as this concept is all about establishing a role for food that goes beyond that of just pure nutrition. Increasingly, through advances in understanding of the links between diet and health, it is recognised that functional foods could have a significant role to play in reducing the risk of a whole range of disease conditions, including cardiovascular disease, cancer and osteoporosis. Food producers, food ingredients suppliers and food retailers, in attempting to allay the fears of consumer pressure groups and regulators alike about this additional role, are keen to promote functional foods as just normal, everyday foods that have a health benefit claim. In essence, the distinction between functional foods and traditional health

398

Gums and Stubilisers for the Food Industry

products is the science underpinning their development, with functional foods in general considered to be based on stronger scientific principles. Seen as a potential problem by consumer pressure groups and to some extent by nutritionists is the view that widespread availability of functional foods could undermine the long established and widely supported position that the best route to effecting improvements in health through diet is to increase consumption of certain products such as h i t s and vegetables and fish and decrease intake of fatty foods, in particular. Indeed, it could be argued that the recent introduction into the UK of fat-based functional spreads could lead to further consumer confhion on what constitutes a healthy diet. On the one hand, the Government and nutritionists are supporting a decrease in consumption of fatty foods, while industry is advocating consumption of health-promoting fat-based foods. Clearly, it is desirable, if not essential, to support the introduction of such products with information that minimises consumer misunderstanding and confusion. From an analysis of the performance of recently introduced functional foods, it is possible to identify factors that are critical to success. While the success of all food and drink products is influenced by such factors as advertising, product quality, pricing and market positioning, manufacturers of functional foods and drinks face additional challenges, mainly as a result of the health claims and/or inferences being made for such products. These place a heavy demand on the whole consumer communication process, i.e. advertising, consumer education, public relations, etc. In this context, it is also important to recognise that the full potential of functional foods may not be realised if regulatory change is not forthcoming that allows the link between certain dietary components and selected diseases to be made on the pack. Some thoughts and perspectives on factors critical to success of functional foods follow. FACTORS CRITICAL TO SUCCESS Food Safety From a safety point of view, functional foods should not be considered differently from any other food. It is also important to realise that certain new functional foods should be viewed as potential novel foods. With this in mind, the evaluation should include a consideration of novel raw materials, the application of novel processes, and the potential novel use or role in the diet. It is recommended that the novelty of the functional food in any of these aspects should trigger a more formal safety assessment, both at company and government level. This should be along the lines required for other types of novel foods, with some emphasis placed on the consequences of the new functionality. Owing to their diversity, novel foods, and by association functional foods, are considered by the International Life Sciences Institute (ILSI) to require a case-bycase evaluation of safety, which addresses both nutritional and toxicological considerations. In future, nutritional assessments of novel foods will need to be carried out against a background of rapidly advancing knowledge on the role of diet in the causation and

Nutritional Aspects

399

prevention of many diseases. Consequently, it is to be recommended that an integral element of a company’s functional food strategy should be the preparation of a comprehensive scientific dossier covering both health claims and nutritional safety. Proof of efficacy

While it is critical to the future success and credibility of functional foods that any associated claims are supported by a strong scientific dossier, the nature and scope of this support are still topics of much debate. The recent criticism of the clinical trial used to support the yoghurt-stylefbnctional food Gaio from MD Foods served to raise doubts and questions about the scale of research required to substantiate claims. Central to the criticism was the limited sue of the trial, a view that is strongly challenged by MD Foods. Nevertheless, there is clear evidence that the food industry recognises the need to substantiate the claims associated with functional foods with research, including human studies. While, by pharmaceutical industry standards, clinical studies supporting recent functional foods have been modest, there is a view that the burden of proof for products that offer a potential reduction of disease, i.e. fbnctional foods, should not be as great as it is for those that claim to prevent or cure disease, i.e. pharmaceuticals. Only time will tell if this is a practical way forward. Remembering that a food (and not a drug) is being evaluated, it is also critical that developers, regulators, law enforcers, consumer groups, etc., are aware that food has distinct characeristics that may make the establishment of a relationship with disease difficult. Therefore, when interpreting the experimental data, it must be remembered that foods are often complex systems, where the physiologically active ingredients are not in pure form, and the physiological effect may be due to the synergy between several compounds. In addition, the interpretation of clinical trials must recognise that the addition of a food or food constituent to the diet cannot be interpreted as a single controlled interventionbecause of the complexity of food systems. However, what this does serve to highhght is the strategic importance of securing access to the appropriate skills necessary to develop a coherent and well planned approach to the efficacy of functional food. Without this, manufacturers of functional foods could find themselves vulnerable. Finally, there is the view that the sustained credibility of and necessity for functional foods will only come from follow-up studies indicating that they can effect demonstrable reductions in certain disease conditions. This raises the delicate issue of where the responsibility lies for instigating and fbnding such research initiatives.

Consumer education

If fbnctional foods are to realise their full potential, the consumer will need to be educated about their various health propositions and how they should be used within the context of a normal dietary pattern. While both the UK Government and the European Commission are giving increasing emphasis to improving the consumer’s understanding

400

Gums and Stabilisers for the Food Industry

of the role of food and other lifestyle factors on general health and well-being, the ultimate responsibility still rests with the manufacturer and/or retailer. However, this does not mean that Government does not have an important role to play. It can assist by creating an environment that not only ensures consumer safety as a first priority, but also lends credence to the role of certain dietary constituents in disease prevention. This is precisely what happened in the US following the introduction of the Nutrition Labelling and Education Act in 1990. At present there is still a lack of knowledge, and some confusion and misunderstanding within the media, law enforcement agencies, the medical profession and consumer groups, which implies the need for more broadly based communication strategies.

Market positioning It has already been indicated that industry is keen to position functional foods as normal foodstuffs carrying a health claim; it follows that they should be positioned in store alongside traditional products, where possible. This may well prove more problematic for products that may appear as new concepts to the consumer. An example is the fermented milk drink Yakult. This is currently sited with the drinking milks in the UK, but is equally well suited to placement with yoghurts. Consumer research is seen as a particularly effective route to identifying the best in-store location for such products.

Price To charge too high a premium for products whose intended role is to provide a common health benefit could lay the food industry open to criticism. This is particularly true now that growing attention is being given to the comparatively poor diet of the lower socio-economic groups. Clearly, any company that adopts a pricing structure/policy that puts functional foods out of the reach of such groups could be criticised for creating a class of elitist foods. However, in most cases, manufacturers/retailers are pursuing a prudent pricing policy. Indeed, price premiums are generally in line with those of other ‘healthy’ products, e.g. reduced-fat lines. While the consumer will probably tolerate slightly higher prices for functional foods, the failure of Ribena Juice & Fibre has shown that there is a limit; this product was launched at nearly twice the price of standard Ribena. Price was not necessarily the only reason for its failure, but it is not unreasonable to suggest that the consumer was not prepared to pay such a high price premium for a variant that already had a strong health profile, i.e. standard ready-to-drink Ribena. The failure of Ribena Juice & Fibre is also notable in that it highlights that a strong brand name combined with a strong health claim does not guarantee success. However, it is important to note that certain markets appear more tolerant of high price premiums for certain functional foods, Finland being a case in point. Raisio

Nutritional Aspects

40 1

recently launched the cholesterol-lowering spread Benecol, which sells extremely well, even though it is six times the price of a normal margarine. Health claims strategy

A number of early entrants into the European functional foods market were supported by overt health claims, particularly in the UK. Notable entrants in this respect were Gaio yoghurt (MD Foods), and Ribena Juice & Fibre (SmithKline Beecham), both of which claimed, both on-pack and in advertising, to be of benefit in lowering blood cholesterol levels and (by inference) heart disease. These claims, and the associated advertising, received a swift rebuke from the Advertising Standards Authority (ASA), forcing both companies to reconsider health claims and associated advertising. While MD Foods has responded by taking a bullish stance, the publicity surrounding the ASA ruling led to a lot of adverse media coverage for the Company and for other functional foods on the market. Indeed, such was the concern of consumer pressure groups that they were instrumental in persuading the Ministry of Agriculture, Fisheries and Food (MAFF) in the UK to undertake a joint consultation exercise with all appropriate parties (i.e. consumer groups, manufacturers, retailers, research organisations and trading standards authorities), the outcome of which was reported to ministers. In consequence, the Food Advisory Committee is in the process of reviewing the appropriateness of its 1990 Report on Food Labelling. While there are those that feel that additional regulation is the only effective route to controlling misleading health claims, an industry code of practice is widely considered to be a more practical approach. Central to the ongoing debate is the need for systems and communication strategies that lead to a more informed consumer, which at the same time allay the fears of pressure groups that the industry is trying to exploit consumer gullibility with regard to health claims. However, with the science underpinning many of the current functional foods on the market being far from fully understood, a prudent approach on health claims is recommended for the foreseeable future, especially if manufacturers wish to avoid adverse publicity. This potential for adverse publicity creates a dilemma, as barriers to conveying health benefits associated with functional foods may well hold back consumer acceptance and thereby market development. However, closer examination of health claim strategies across Europe does indicate that it is possible to convey a health benefit to consumers without running the risk of adverse media publicity or intervention by the regulatory authorities. In recognition of the problems associated with health claims, some suppliers of functional ingredients, notably ORAFTI, have proposed a number of nutritional concepts that, while specifically related to the use of the prebiotics inulin and oligofiuctose, nevertheless have some general applicability to other functional ingredients/functional foods. Clearly, if innovation and creativity in food manufacture are not going to be stifled, it is important that a common position be reached among all interested parties.

402

Gums and Srabilisersfor the Food Industry

Summary Despite some early product failures and an uncertain regulatory environment, there are still major opportunities for innovation of added-value food and drinks products that have the potential to improve health and reduce risk of some important diseases. However, to realise this, manufacturerswill need to consider the complexity of the issues and the new disciplines and business operationdstructures they will require.

RHEOLOGICAL CHARACTERIZATION OF INULIN

I. E. Bishay Monsanto Life Sciences Co., Nutrition and Consumer Products Sector, Discovery Research, 601 East Kensington Road, Mt. Prospect, Illinois 60056.

1 INTRODUCTION Inulin is a polysaccharide composed of a sucrose disaccharide (glucose (G) and fructose (F))with additional fructose units attached by a p- 1 4 2 linkage to give the general formula of GF,, where n can range from 2 to over 60. Short chain inulins below about n=7 are called oligofructose. Much like starch, inulin is a storage carbohydrate for plants. It is found in nature in such products as Jerusalem artichoke, chicory, garlic and onions. It is estimated that the average daily per capita inulin consumption in the US diet is between 1 to 4 g.’ The human digestive system does not have the necessary enzymes to cleave the p- 1 4 2 linkage between the fructose units. Therefore inulin is a reduced calorie ingredient, estimated at 1.5 to 2.3 kcal/g (6.3 to 9.5 kJ/g).2 The energy value of inulin will depend on the molecular weight distribution, and quantity of mono- and disaccharides present. Inulin is fermented by the microbial flora of the colon causing the formation of secondary metabolites including volatile fatty acids.’ Studies have shown inulin to have a favorable influence on the level of bifidus flora in the colon. Furthermore, studies indicate that inulin decreases blood cholesterol, increases HDULDL ratio and results in a decrease in serum lipids.4 Inulin is an approved food ingredient in many countries and meets the definition of dietary fiber? 2 EXPERIMENTAL METHODS 2.1 Materials

Inulin was obtained from Rh6ne -Poulenc Inc., Cranbury, New Jersey, and was an unfractionated inulin obtained from chicory, with an average degree of polymerization of 10 and about 8% glucose, fructose and sucrose (Raftiline ST)! Xanthan (Keltrol RD) and Alginate (Kelgin MV/F) were obtained from the NutraSweet Kelco Co., Deerfield, Illinois. Unmodified dent corn starch (Melojel) was obtained from National Starch & Chemical Co., Bridgewater, New Jersey. Sodium carboxymethylcellulose was obtained from Hercules, Inc., Wilmington, Delaware. Microcrystalline cellulose (Avicel PH-101) was obtained from FMC Corp., Philadelphia, Pennsylvania. Calcium alginate gel was formed by adding 0.25% CaC1;2Hz0 to 1% Alginate.

Gums and Stabilisers for the Food Industry

404

2.2 Methods All samples were received as powders and rehydrated at 80°C for about 30 minutes, and then allowed to cool slowly to room temperature. Rheological methods were performed on a Bohlin CS Controlled Stress Rheometer, Bohlin Instruments, Inc., Cranbury, New Jersey; equipped with a circulating water bath for temperature control. Samples were placed in a serrated C14 cup-and-bob assembly, where the serration is necessary to limit slippage. Very low viscosity samples were instead measured in a double gap concentric cylinder, to provide increased surface area and improved data quality.

3 RESULTS & DISCUSSION

3.1 Effect of Concentration Viscosity versus shear rate measurements were conducted for inulin at 5, 10, 15, 20, 25, 30, 35 and 40% solids. Figure 1 displays the results of these experiments.

loo

-3

-.n b

r---------1

10

1

ul

8

._

0.1

>

0 01 0 001 01

1

10

100

1000

Shear Rate (Vsec)

Figure 1 Viscosity of inulin at various concentrations (25°C) A convenient way to quantify the rheological flow of fluids is the power law equation:

where o is the shear stress, K is a consistency index, y is the shear rate, and n is a dimensionless number that quantifies the closeness to Newtonian flow; with n .= I indicating Newtonian flow; n > 1 for dilatant, or shear thickening, flow; and n < I for pseudoplastic, or shear thinning, flow. Table 1 lists the result of the power law fits for inulin at various concentrations.

405

Nutritional Aspects

Table 1: Power law f i t resultsfor various inulin concentrations. Inulin Concentration (wt %) 5% 10 % 15 % 20 % 25 % 30 % 35 % 40 %

Exponent

Consistency Index (K) 0.00073 0.00079 0.0014 0.0171 0. I45 0.438 1 1.97 15.55

0 1.17 1.18 1.11 0.924 0.692 0.667 0.307 0.226

Inulin is only soluble at room temperature up to about 15% solids. At 15% solids and below, inulin displays Newtonian behavior typical of soluble, low molecular weight systems. Even at 20% solids, where most of the inulin is in solution, the small amount of suspended inulin does not alter the Newtonian behavior. However, at 25% solids and above, inulin begins to display strong shear thinning behavior. Furthermore, above 15% solids, there begins a dramatic increase in the viscosity dependence on concentration, as can be seen by the increase in the consistency index as well as in the viscosity versus concentration plot in Figure 2.

0

10

20

30

40

Concentration (W%)

Figure 2 Viscosity of inulin at 25°C and 25 sec-' as a function of concentration. Furthermore, at 35% and 40% solids there is a yield stress of about 15 Pa. All this information suggests formation of a weak particle gel, comprised of the 15% soluble inulin with the remaining quantity in a stable suspension. This particle gel is relatively strong at low shear rates, but is easily disrupted by shearing. The rapid increase in viscosity tapers off above 35% solids. Although increasing the inulin content at this stage further strengthens the gel strength, the increase is much less dramatic.

3.2 Temperature Effect on the Particle Gel In order to assess the temperature effects on the particle gel, a 40% solids gel was placed in the rheometer at 25°C that then heated at 1°C per minute while oscillating the rheometer cup at 1 Hz, and low amplitude, so as not to disrupt the gel with the shear of the instrument.

Gums and Stabilisers for the Food Industry

406

350

-

-I-Heat

300 -250

--

-b

a200 --

n

150 -100 --

50 -0 0

20

40

60

80

100

Temperature (deg C)

Figure 3 Temperature dependence of inulin gels (35% inulin, I Hz oscillation, I”C/min heatkooling) The heating curve (Figure 3 ) displays a “melting” of the gel beginning at about 35°C and progressing rapidly from 40°C to 55°C. This is most likely the region during which the greatest portion of the inulin is going into solution. By the end of the temperature scan, all the inulin gel has dissolved. Thus, the “melting” of the gel is really a dissolution of the inulin suspension. Upon re-cooling, (cooling rate of 1°C per minute) a portion of the dissolved inulin begins to fall out of solution and re-form the particle gel. The reformation of the gel is delayed to about 45”C, probably due to a delay in the precipitation of the inulin, forming a supersaturated solution, before the rapid formation of the particle gel. Once the transition in complete, at about 30”C, a firmer gel has formed as compared to the initial gel (340 Pa versus 250 Pa). This is probably a result of forming the gel under the static conditions of the rheometer, as opposed to the initial gel, which was also formed under static conditions, but was disturbed by placing it in the rheometer. Even though an equilibrium time of 60 minutes was used to reform the gel, this was not sufficient, requiring an even longer time, or a reheating and cooling step.

3.3 Interaction with Other Hydrocolloids Interaction of 35% inulin gel with 1 % xanthan, 1% sodium carboxymethylcellulose (CMC), 1% microcrystalline cellulose (MCC), 10% unmodified starch gel, I % sodium alginate, and 1% calcium alginate gel was studied. Of these systems, the soluble hydrocolloids, such as xanthan, sodium alginate, sodium carboxymethylcellulose, do not show any significant interaction with inulin. Microcrystalline cellulose, the only particulate suspension studied, also does not show any interaction with inulin. However, the two gel systems both show an interaction: starch displays a negative synergy with inulin, while calcium alginate gel shows a strong positive synergy with inulin.

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Table 2: Power law fit resultsfor inulin interactions with various hydrocolloids. Hydrocolloid Svstem 35 % Inulin 1 % Xanthan 1 % Xanthan + 35 % Inulin 1 %CMC 1 % CMC + 35% Inulin 1 %MCC 1 % MCC + 35 % Inulin 10 % Starch 10 % Starch + 35 % Inulin 1 % NaAlginate 1 % NaAlginate + 35 % Inulin 1 % CaAlginate 1 % CaAlginate + 35 % Inulin

Consistency Index (K) 11.97 4.86 27.7 0.159 8.80 0.00457 8.86 204.5 24.97 0.277 13.98 10.12 21.88

Exponent (n) 0.307 0.223 0.310 0.906 0.451 1.007 0.393 0.237 0.213 0.854 0.549 0.345 0.209

The negative synergy between inulin and starch may be due to water affinity. When the starch gel is formed, it binds and immobilizes the majority of the water in the system. Upon the addition of inulin, the latter has a greater affinity for the water than the starch polysaccharides. The water that is now bound to the inulin chains is more mobile than when it was bound to the starch, this is simply due to the inulin having shorter, more mobile molecules. With the water now bound to shorter, faster moving inulin chains, the result is a lower overall viscosity and weaker gel strength. Another possibility is that inulin is acting as an antiplasticizerthat raises the glass transition temperature of the starch, as is seen when some other low molecular weight ingredients were added to starch,’(see Figure 4). -0-

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Figure 4 Viscosityplots displaying the positive synergy between inulin and alginate gel and the negative synergy between inulin and starch.

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The synergy between inulin and calcium alginate is not due to Ca2+ion interaction with inulin, as Ca2’ ions have little effect on inulin rheology (for example at 25°C and 25 sec-’ shear rate 35% inulin has a viscosity of 1.343 Pa’sec, while 35% inulin with 0.25% CaC12’2H20has a viscosity of 1.357 Pa’sec). This synergy may be due to either the inulin colloidal suspension, or the dissolved inulin, reinforcing the alginate gel network by taking part in the alginate junction zones that are already formed by Ca2+ion bridging between two alginate guluronic acid residues, the so-called “egg-box’’ model. The fact that there is no synergy seen between inulin and sodium alginate, suggests that simple entanglement is not responsible for the inulin-calcium alginate synergy, but a model involving the alginate gel junction zones is probably needed to explain the inulin-calcium alginate synergy.

4 REFERENCES 1 . J.V. Loo, P. Coussement, L. De Leenheer, H. Hoebregs and G. Smits; Critical Rev. in Food Sci. and Nutrition, 1995, 35(6):525-552. 2 . (a) N. Hosoya, B. Dhorrannintra and H. Hidaka; J. Clin. Biochem. Nutr.,1988, 5, 677 4 ; and (b) C. Molis, B. Flourie, F. Ouarne, M.-F. Gailing, S. Lartigue, A. Guibert, F. Bornet and J.-P. Galmiche; Am. J. Clin. Nutr.,l996, 64(3):324-8.

3. H. Hidaka, T. Eida, T. Takizawa, T. Tokanaga and Y. Tashiro; Bifidobacteria Microflora,1986, 5( l ) , 37-50.

4. ( 1 ) R.C. McKellar and H.W. Modler; Appl. Microbiol. Biotechnol.,l986, 31, 537541; ( 2 )T. Mitsuoka, H. Hidaka and T. Eida; Die Nuhrung,1987,31,427-436;(3) H. Hidaka, T. Eida, T. Takizawa, T. Tokanaga and Y. Tashiro; Bifdobacteriu Microflora, 1986, 5( l ) , 37-50; ( 4 )T. Sano; Presented at 3rd Neosugar Res. Conf., Tokyo, Nov. 8 , 1986; (5) Y. Takahashi; Presented at 3rd Neosugar Res. Conf., Tokyo, Nov. 8,1986; ( 6 ) T. Mitsuoka; Presented at 3rd Neosugar Res. Conf., Tokyo, Nov. 8, 1986.

5. J.E. Spiegel, R. Rose, P. Karabell, V.H. Frankos and D.F. Schmitt; Food Technology, 1994,48( I), 85-89; and RhBne-Poulenc - FDA communication documents. 6 . RhBne-Poulenc technical bulletins and ingredient specifications.

7. L. Slade and H. Levine, in Industrial Polysaccharides. The Impact of Biotechnology and Advanced Methodologies ( S . Stivala, V. Crescenzi and I.C.M. Dea, eds.), Gorden and Breach, New York, 1987, p. 387.

PROPERTIES AND APPLICATIONS OF CHICORY INULIN

Dr. A. Franck ORAFTI Research & Quality Aandorenstraat 1 3300 Tienen Belgium

1 INTRODUCTION

Inulin belongs to the group of fructans which are naturally occuring in more than 30,000 kinds of plants and which are, after starch, the most abundant non-structural carbohydrates.1Inulin is found in many vegetables, fruits and cereals commonly used in the diet, such as onion, garlic, artichoke, leek, asparagus, banana and wheat. It has therefore always been a part of the human diet. In Europe and US, its average daily intake has been estimated to be at least 2g per inhabitant.' Inulin is industrially obtained from chicory roots, which are also used in some countries for the production of a coffee substitute, and which look a little bit like sugar beets. The production process of inulin, which is sold as a white powder under the tradename RAFITLINE@( O m , Belgium), includes the extraction of the naturally occuring inulin by diffusion in hot water, followed by refining and spray-drying. Inulin has been confirmed as a food ingredient all over Europe and in the US it has a Self-Affirmed GRAS status. Inulin is used in most developed countries of the world in a variety of food products. Chemically speaking, chicory inulin is a mixture of oligo- and polysaccharides of which almost all (>98%)are linear chains of fructose having the structure GFn (with G=glucose, F=fructose and n=number of fructose units linked to one another)? In native chicory inulin the number of fructose units linked together ranges from 2 to more than 60,with an average degree of polymerisation (DP) of the order of 10. Inulin from which the lower DP-fraction has been physically removed and having an average DP of about 25 is also available (RAFTILINEmHP)for high performance fat replacement. In inulin the fructose units are linked together by 8(2-1) bonds which make it indigestible for humans and all higher animals. 2 UNIQUE NUTRITIONAL BENEFITS Thanks to its specific chemical structure, which our digestive enzymes cannot hydrolyse, inulin passes through the mouth, the stomach and the small intestine without undergoing any significant change and without being metabolised. This has been confirmed in human studies with ileostomy volunteers.3 Inulin then enters the large intestine where it is easily and totally fermented by the microbial flora, mainly by the beneficial bacteria. Inulin is consequently transformed into bacterial mass, short chain fatty acids (SCFA) and some gases.* Due to this particular metabolic pathway, inulin is a low-calorie ingredient. Its caloric value is indeed limited to the one contributed by SCFA produced by fermentation and partially resorbed. Energy value studies have shown that chicory inulin has a caloric value of about 1kcaVg.S Thanks to its indigestibility inulin can furthermore be used in the diet of diabetics because its ingestion does not affect glycemia, nor glucagon or insulin secretion.6 Inulin on the other hand is a soluble dietary fibre. It is indeed not hydrolysed

410

Gums and Stabihers for rhe Food Industry

by the human digestive enzymes, as specified in the definition of dietary fibres. But inulin also induces typical fibre effects on the gut function, such as a reduction of the stool pH, a relief of constipation and an increase in stool weight or bulking effect (1-2g increase in fresh stool weight per g of inulin ingested, which is comparable to the effect of other soluble fibres such as pectin and guar gum).' In the case of the ingestion of large amounts, typical side effects usually associated with dietary fibres (e.g. mild flatulence) can also be observed. Rat studies showed furthermore a very significative reduction (40%) in serum and liver triglycerides as well as an improvement of the HDULDL cholesterol ratio on ingestion of inulin.* An improvement of the lipid metabolism has also been observed in a first human study with slightly hypercholesterolemic volunteer^.^ Furthermore, the ingestion of inulin stimulates the specific growth of beneficial intestinal bacteria, mainly Bifidobacteria, while inhibiting harmful micro-organisms such as Clostridia, in the human colon.IOJ1 Inulin is therefore a prebiotic food ingredient.'* This specific promoting effect on the bifidus population in the large intestine is usually regarded as beneficial to the well-being and health of the host. Its potential implications in the prevention and treatment of intestinal disorders and infections are further studied. In rats the ingestion of inulin also has been shown to increase significantly the absorption of important minerals such as calcium and magnesium,13an effect which is currently under evaluation in humans. Inulin furthermore decreases the incidence of pre-cancerous colonic lesions (aberrant crypt foci) induced in experimental animals and could therefore significantly contribute to the prevention of colon cancer.14 More research is running in this very interesting area. As more and more scientific data become available, the nutritional benefits of inulin keep surprising researchers and nutritionists. Chicory inulin therefore is often taken as a practical illustration of an active food ingredient for 'functional foods'.

3 INTERESTING TECHNOLOGICAL PROPERTIES Consumers are no longer willing to compromise on taste and texture, even if they want better-balanced and healthier foods. The incorporation of inulin in the formulation allows to improve both the flavour release and the mouthfeel in a wide range of food applications. Chicory inulin is available as white powders with a high purity and a well-known chemical composition. The taste is neutral (slightly sweet or not sweet at all depending on the chain length), without any off-flavour or after taste, and it combines easily with other ingredients. Inulin behaves like a bulk ingredient, contributing to body and mouthfeel.Is It can be used in recipes sweetened with intense sweeteners such as aspartame and acesulfame K to provide a rounder mouthfeel, a better sustained (fruit) flavour with reduced after taste and an improved stability. Inulin is moderately soluble in water (maximum 10%at room temperature), which allows its incorporation in watery systems where the precipitation of other fibres could be a problem. This is particularly interesting in table spreads, dairy products and drinks. Inulin also can be used to upgrade the appearance and eating quality of other high fibre products such as extruded cereals and snacks with more crispiness and expansion or baked goods with an excellent texture and good moisture retention properties. Inulin furthermore improves the stability of foams and emulsions, such as chocolate and fruit mousses, ice creams, table spreads and sauces. It can also partially replace gelatin, sodium caseinate and other stabilisers in different food products. In very acid conditions (pH<4), it can be partially hydrolysed into fructose, when it is incorporated in non-refrigerated food products with a long ~helflife.'~ 4 SURPRISING FAT REPLACEMENT Inulin has a remarkable capacity to replace fat thanks to a patented technology called [email protected] When inulin (at a high concentration, e.g. 25%) is thoroughly mixed with water or another aqueous liquid, using a shearing device such as a homogeniser or a rotor-stator mixer, a creamy structure (or inulin creme) results which can easily be incorporated into foods to replace fat. In some cases, this gel can even be

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produced directly in situ by submitting a mixture of inulin, water and the other ingredients of the food product to an appropriate mixing device included in the processing lime. The particle gel of inulin in water imitates fat extremely well, it provides a smooth fatty mouthfeel, as well as a glossy aspect and a well-balanced round flavour. It shows a short texture affecting positively the spreadability of foods such as table spreads, cheese products, liver paste and other meat or fish based spreads. Delicate aromas and flavours, such as milk notes, remain unaffected. When using an inulin gel, food products showing better organoleptical characteristics can be obtained. Furthermore it enables replacement of fat by a low-calorie soluble dietary fibre and so fulfils three dietary guidelines at once. Inulin has already been successfully applied in fat-reduced table spreads, cheese products, meat products, frozen desserts, fillings, sauces and meal replacers. As far as fat Rplacement is concerned high performance inulin (RAFTILINEWP) shows about twice the functionality compared to standard chicory inulin (RAFT'ILINE@ST),allowing for lower dosage levels and ingredient costs. A special instant quality which does not require shearing to give a stable gel also has been developed using a specific spray-drying process (RAFIILINE@ST-Gel). The gel strength obtained depends on the inulin concentration and total dry substance content, on the shearing parameters such as temperature, time, speed or pressure and also on the type of shearing device used.16 The gel strength increases furthermore during the first hours after shearing to become constant after about 24 hours. Electron cryomicroscopy has shown that such an inulin creme is composed of a tridimensional network of insoluble sub-micron inulin particles in water. These particles of about lOOnm in size aggregate to form larger clusters having a diameter of 1 to 5 microns. Large amounts of water are immobilised in that network, as confirmed by N M R experiments. The X-ray diffraction pattern confirmed furthermore the crystalline nature of the creme whereas the starting inulin powder is essentially amorphous. An inulin gel exhibits a visco-elastic rheological behaviour characterised by a certain elasticity as well as a viscosity. The gel also shows shear-thinning and thixotropic properties and is characterised by a relatively low yield stress. This indicates a reversible breakdown of the gel's structure under stress. Oscillatory rheological experiments have confirmed the weak gel properties. When submitted to an increasing deformation, it loses gradually its solid properties: the elasticity modulus decreases whereas the fluid properties - thus the viscosity modulus - which is initially lower, increases more and more. Submitted to an increasing deformation the gel behaves thus more and more like a suspension. The evolution of the elasticity modulus during a strain sweep allows furthermore the comparison of the inulin creme with other materials: it shows in fact a dynamic behaviour quite similar to a margarine-type product, but rather different from a typical polymer gel such as a starch one for instance. 5 SUCCESSFULFOOD APPLICATIONS

Inulin can be used for either its nutritional or its technological advantages, but it is often applied to offer the consumer a double benefit: an improved organoleptical quality and a nutritional 'plus'. All this makes it much easier and efficient to add dietary fibres to food products than in the past. It is also possible to go further and to add other nutritional messages such as a prebiotic (bifidus stimulation) claim. Specific marketing concepts have been built around inulin and some are already applied in different foodstuffs. Inulin also has permitted the launch of low-fat and low-calorie foods without compromising on taste and texture. Inulin has become a key ingredient offering new opportunities to the food industry looking for the well-balanced and yet better-tasting products of the future.

References 1.

2.

J. Van Loo, P. Coussement, L. De Leenheer, H. Hoebregs and G. Smits, Crit. Rev. Food Sc. & Nutr., 1995.35, 525. L. De Leenheer and H. Hoebregs, SturcWSUirke, 1994,46, 193.

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3. 4.

5. 6. 7. 8.

9. 10. 11. 12. 13. 14. 15. 16.

Gums and Stabilisers for the Food Industry

L. Ellegard, H. Anderson and I. Bosaeus, Eur. J. Clin. Nutr., 1997, 51, 1. N. Delzenne and M. Roberfroid, Lebensm-Wiss. u.-Technol., 1994, 27, 1. M. Roberfroid, G.R. Gibson and N. Delzenne, Nutr. Rev., 1993,51, 137. H.B. Lewis, J.A.M.A., 1912, April 20, 1176. M. Roberfroid, Crit. Rev. Food Sc. & Nutr., 1993, 33, 103. N. Kok, M. Roberfroid, A. Robert and N. Delzenne, Brit. J. Nutr., 1996, 76, 881. M. Davidson, C. Synecki, K. Maki and K. Drennan, Proc. First ORAF77 Res. Con$, 1995. G.R. Gibson, E.R. Beatty, X. Wang and J.H. Cummings, Gastroenterology, 1995, 108, 975. M. Roberfroid, J. Van Loo and G.R. Gibson, J. Nutr., 1997 (in press). G.R. Gibson and M. Roberfroid, J. Nutr., 1995, 125, 1401. N. Delzenne, J. Aertssens, H. Verplaetse, M. Roccaro and M. Roberfroid, Life Sc., 1995, 57, 1579. B.S. Reddy, R. Hamid and C.V. Rao, Curcinogenesis, 1997 (in press). P. Coussement, Eur. Dairy Mag., 1995,3,22. A. Franck, Proc. 1992 FIE Con$, 1993.

The Use of Acacia Gum as a Source of Soluble Dietary Fibre

T.P. KRAVTCHENKO Colloides Naturels International, I29 chemin de Croisset, B.P. 4151, 76723 Rouen, France

ABSTRACT The demand of consumers for a healthier diet is not anymore to be demonstrated. This new trend has led to the development of the so-called ‘functional foods’ among which belong foodstuffs fortified with dietary fibre. Acacia gum is a natural dietary fibre produced by Acacia trees. Resistant to human digestive enzymes, Acacia gum is not digested in the upper intestinal tract but is fermented in the large gut. Its fermentation leads to stimulation of the endogenous microflora (prebiotic effect) and the production of short-chain fatty acids. Health benefits are particularly well documented in the scientific literature. Thanks to its high molecular weight and its complicated molecular structure (highly branched arabinogalactan), Acacia gum does not exhibit the nutritional side-effects (laxative effect and/or flatulence) generally associated with other bulking agents or low molecular weight fibre sources. From a technological point of view, thanks to its very high solubility associated with a low viscosity and the absence of taste and odour, Acacia gum can be incorporated in large amounts to foodstuffs without disturbance of their organoleptic properties. In addition, Acacia gum, which has long been used for its functional properties (texturisation, stabilisation of emulsions, formation of edible films, compression...), can be used to combine the addition of dietary fibre with the performance of a technologicalfunction. INTRODUCTION Acacia gum, also known as gum arabic, is one of the oldest food ingredients. Despite periodical attempts to replace it, acacia gum is still used widely for its very typical texturising, film-forming, emulsifylng or stabilising properties. Traditional crude gums have nowadays been replaced by ‘HighTech’ acacia gums, designed to fulfil all requirements of the modern food industry. More recently, the food industry has seen the emergence of the new concept of ‘functionalfoods’ which are designed for actively promoting health. Recognition of the low level of fibre in Western countries’diets has led to the development of foods fortified with dietary fibre. From a marketing point of view, most consumers are already convinced of the beneficial effects of dietary fibre. Problems rather come fiom the formulation of such fibrefortified foods. Indeed, if consumers expect their food to be healthy, they do not want to eat without pleasure: the fibre-fortified foods must have the same organoleptic qualities as the conventional foods. In practice, the addition of significant amounts of dietary fibre is often limited by side-effectssuch as sandy texture, increase in viscosity or bad tastdodour While other low molecular weight fibre types, which have been designed to overcome textural limitations, may provoke some digestive disagreements, acacia gum has been known for many years by the scientific community as a potential source of dietary fibre. Its

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nutritional properties have been well demonstrated. In addition, acacia gum can be added in very large amounts while maintaining the original taste and texture of the food in which it is incorporated. And last but not least, because of its highly complex polymeric structure, acacia gum does not show any nutritional side-effects.

ACACIA GUM, A NATURAL FOOD INGREDIENT Acacia gum is defined in the Pharmacopoeiasas 'the gummy exudate flowing naturally or obtained by incision of the trunk and branches of Acacia senegal and other species of M c a n origin'. There are many Acacia species (some 700) of which few can provide gum volumes of industrial interest. M e r harvest, which is carried out during the dry season (December-May in the sub-Saharian area), acacia gum is purified by a simple physical process (centrihgation and tiltration) without chemical or enzymatic modification. A simple sterilisation procedure ensures bacterial safety.Finally, highly water-soluble powders are obtained by spray-drying and/or granulation (figure 1).

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415

Nutritional Aspects

From a chemical point of view, acacia gum is made of a galactan main chain carrying heavily branched galactodarabinose side-chains. Some rhamnose and/or glucuronic acid may be present as side-chain terminations (figure 2). The overall chemical composition varies from one Acacia species to another and for individual species in relation to their geographical origin. Because of its highly branched stmctwe, acacia gum occupies a very low hydrodynamic volume and thus develops a very low viscosity (figure 3). From a regulatory point of view, it is recognised as a food additive by the joint FAOWHO committee on food additives (JECFA) and by the EU regulation, with no AD1 (acceptable daily intake) specified. In the USA, F D A recognises acacia gum as GRAS for already many years and it is registered in the US Food Chemical Codex under the reference 1841330. In the EU, acacia gum is also compatible with foods conforming with the organic mode of production (Reg. 2092/91).

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Figure 3. Influence of concentration on the viscosity of an acacia gum solution NUTRITIONAL PROPERTIES OF ACACIA GUM

Dietary fibre is a general term covering a wide variety of substances which are not digested in the upper part of the human digestive tract. Several definitions have been proposed but one of the most relevant is that proposed by Trowell and Burkitt (2) and retained by the Association of Official Analytical Chemists (AOAC): 'the remnants of plant cells resistant to hydrolysis by the alimentary enzymes of man. It is composed of cellulose, hemicellulose, oligosaccharides,pectins, gums, waxes and lignin'. The passage of dietary fibres through the digestive tract results in several physiological effects which may be profitable for human health. Acacia gum is a low-viscosity soluble dietary fibre which mainly affects the human physiology by systemic effects via its fermentationby the intestinal flora. Mechanical contributionto transit time As compared with insoluble or highly viscous fibres, acacia gum only plays a very limited role in the upper gastrointestinaltract, probably because of its low viscosity (3). Contrary to other low-viscosity fibres, such as oligo-saccharides, acacia gum does not exhibit laxative side-effects. Indeed, large amounts of undigested low-MW molecules increase the intraluminal osmotic pressure, stimulating the migration of water from the body to the intestinal content and therefore provoking diarrhoea by excess of water. Thanks to its polymeric nature (Mw higher than 300,000 daltons) acacia gum does not

416

Gums and Srabilisersfor the Food Industty

disturb the osmotic pressure and thus does not exhibit laxative side-effects. Clinical tests with human subjects showed good tolerance towards acacia gum at dosages up to 50 grams per day (4). Prebiotic effects In the large bowel acacia gum is readily fermented by the endogenous bacterial flora. Mc Lean-Ross et al. showed that no acacia gum could be recovered from the faeces of humans (5) or rats (6) fed acacia gum, thus indicating that it is completely fermented by the human colonic flora. Indeed, fermentable acacia gum promotes the growth of resident gut microbes; however, such a fermentation requires an adaptation period of a few days (7, 8). The human colon contains a very complex bacterial ecosystem where at least 400 distinct species coexist in equilibrium with their human host (9). Dietary fibre represents an important source of carbon nutrient and enrichment of the diet with acacia gum promotes the increase of the number of leaving bacterial cells (4, 10, 1 I), thus increasing the stool weight and facilitating the expulsion of faeces. However, among the several hundreds of different bacterial species, those belonging to the genus Bacteroides, Bifidobacterium and Lactobacillus are particularly stimulated. Wyatt et al. (8) observed that the proportion of the faecal flora able to degrade acacia gum (i.e. Bifidobacterium, Bacteroides and Lactobacillus genera) rose from an initial level of 6.5% to more than 50% during acacia gum diet. This effect is called bifidogenic or prebiotic (12), i.e. stimulation of the endogenous beneficial bacterial strains. This change in the species composition of the colonic microflora may be explained by the fact that not all bacterial species possess the necessary enzymes to degrade fibre molecules (1 3), but other reasons such as the change of pH may be taken into account. The beneficial effects of the intestinal flora have been described extensively elsewhere (e.g. 14) and will not be rediscussed in this review. A large part of the metabolic effects of soluble dietary fibre may be ascribed to its fermentation by the colonic flora (14, 15). Gas production The fermentation of acacia gum leads to the formation of hydrogen, carbon dioxide, and methane (5,6,11). Gases are final metabolites which are normally expulsed with faeces or breath. However, in the presence of large amounts of highly fermentable fibre, fermentation is very rapid in the caecum and the gas production may provoke flatulence (16). Because of its highly branched chemical structure which is hardly degraded by bacterial enzymes, the fermentation of acacia gum is very slow. Gas production is therefore delayed in time and displaced all along the large gut without provoking the feeling of bloating. Production of short chain fatty acids The fermentation of acacia gum also leads to the formation of SCFA as it has been evidenced in rats (6, 7, 10, 11, 17, 18, 19, 20), in vitro tests using human faecal inoculates (21,22,23,24) and even in living humans (5). The first observable effect of the SCFA production is a drop of the intraluminal pH (6, 7, 13, 18) which is usually considered as a beneficial factor for the health of the colon. Lowering the intraluminal pH also contributes to modulate the distribution among the various bacterial species (prebiotic effect). SCFA are efficiently absorbed from the colonic lumen and are metabolised by various tissues. Butyrate is preferably metabolised by colonocytes, propionate is almost

417

Nutritional Aspects

quantitatively taken up by the liver and may modulate hepatic carbohydrate and lipid metabolism (25) whereas acetate largely escapes colonic and hepatic metabolisms and probably serves primarily as a fie1 for peripheral tissues such as muscles. As compared with other fibre sources, acacia gum elicits the formation of butyrate and propionate at the expense of acetate, thus promoting several metabolic effects. A high proportion of butyrate and propionate may be due to the complexity of the acacia gum molecules (26).

Acacia gum

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Colonic flora

Trophic effect on large gut

Energy for mumsa Modulation of cell differentiation Absorption of waterlsalts Energy for peripheral tissues Hepatic metabolism

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Cancer prevention

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Dislestibilitv of nutrients Improved nutrition Synthesis of vitamins Stimulation of the immunity system Inhibition of pathogenic bacteria Metabolism of carcinogens Cancer prevention

Nitrogen excretion Detoxification Figure 4. Synoptic summary of the nutritional properties of acacia gum There is some evidence that acacia gum stimulates intestinal epithelial cell turnover (27), thus contributing to the maintenance of the mucosal wall. Several studies on rats (7, 10, 11, 20) showed that acacia gum also elicits a marked enlargement of the caecal lumen and atrophic effect on the caecal wall in rats. Dietary fibre has often proved to reduce blood lipids. In rats fed hypercholesterolemic diets and acacia gum, Annison et ul. (17) did not find any effect on the concentration of plasma and liver cholesterol, whereas Kiriyama et ul. (28) found an appreciable depressing activity on cholesterol levels. In man, Haskell et ul. (29) and Jensen et ul. (30) did not find any effect of acacia gum on any plasma lipid parameter. By feeding higher quantities of acacia gum (at least 25 grams per day), Mc Lean-Ross et ul. (S), Sharma (31) and Eastwood et ul. (32) observed a significant decrease of the plasma cholesterol concentration, especially LDL and VLDL fractions. As compared with other hypocholesterolemic fibres, acacia gum has almost no effect on the viscosity of the intestinal content and does not alter faecal bile acid excretion (5, 10, 32). Moreover, the hypocholesterolemic effect of acacia gum requires a diet adaptation period of a few days, reinforcing the idea that it is mediated by the SCFA produced by the colonic microflora. Feeding acacia gum increases urea flux from blood to caecum and increases net nitrogen retention in the caecum, resulting in a significant decrease of the blood urea content (4,20,23, 33).

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Modulation of the supply of the different SCFA by feeding acacia gum is thus of great significance for promoting health. However, because of the complexity of the mechanisms involved, many conclusionsrelative to acacia gum remain to be confirmed.

APPLICATIONS OF ACACIA GUM AS A DIETARY FIBRE IN FOOD PRODUCTS Thanks to its low viscosity (figure 3) and its absence of taste and odour, acacia gum can be added in large quantities without disturbing the organoleptic properties of the food

product in which it is incorporated. In order to guarantee a high technological neutrality, CNI has developed a new range of selected acacia gums called FIBREGUMTM. Traditional confectionery gums may contain more than 40% acacia gum. Other conventional food products, processed ingredients or even dietetic specialities have been formulated in order to compensate the lack of dietary fibre in usual Western diets. They usually contains 2-5% acacia gum, providing 2- 15g dietary fibre per serving dose.

Hiah stability in acidic conditions

As compared with most other polysaccharides,acacia gum is extremely resistant to various physico-chemical treatments, especially in acidic conditions. For example, acacia gum does not show any loss of dosable dietary fibre during the pasteurisation of h i t juices (table 1). Table 1. Resistance of FIBREGUM (6%) to a heat treatment in orange juice (pH 3.2) Residual DF (%) 1.53 1.64 Blank 1.62 6.91 Control 6.88 6.94 7.02 Heated for 10 min. at 100°C 6.73 7.06

Acacia a m . a bi-functional inaredient It must kept in mind that acacia gum is traditionally used as a food additive for many different applications (34, 35). It is thus possible to combine fibre-enrichment (nutritional value) with a given functional property. In slimming products, since it is not digested (calorific value below than 2 kcdg), acacia gum has long been used as a bulking agent. In cereal bars, FIBREGUM TX provides sticking properties as well as good moisture stability on storage. In instant powdered beverages, FIBREGUM INSTANT may be used to encapsulate natural flavours andor vitamins to protect them against oxidation and minerals to limit their oxidising power. In dietetic tablets, FIBREGUM INSTANT serves as cohesive agent which can used for direct compression. In salad dressings, FIBREGUM AS can replace conventional emulsifier/stabiliser combinations without affecting the overall product stability. The possibilities for incorporation of acacia gum into food products are almost unlimited. Some examples of the application of acacia gum as a dietary fibre are given in Table 2.

Nutritional Aspects

419

Table 2. Some examples of application of acacia gum as a source of dietary fibre yoghurts, cheese, acid milk drinks, dairy desserts Dairy products: soft gums, hard gums, marshmallows, wine gums,nougat, Confectionery: chocolate jams, compotes, fruit preparations Processed h i t : bread, cakes, biscuits, cereal bars, extruded cereals Bakery: ice creams, sorbets, frozen yoghurt Frozen desserts: Beverages: fruithegetablejuices soft drinks instant powdered drinks emulsified meat, cured meat Meat products: Dietetics: meal substitutes, nutritive supplements REFERENCES 1. 2. 3. 4.

Street C.A., AndersonD.M.W., (1983)Talanta, 30(11), 887. Trowell H., Burkitt D., (1986) Bol. Assoc. Med. P. Rico, dec. 1986,541. Adiotomre J., Eastwood M.A., Edwards C.A., Brydon W.G., (1990) Am. J. Clin. Nutr., 52, 128. Bliss D.Z., Stein T.P., Schleifer C.R., Settle R.G., (1996) Am. J. Clin. Nutr., 63, 392.

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Mc Lean-Ross A.H., Eastwood M.A,, Anderson J.R., Anderson D.M.W., ( 1983) Am. J. Clin. Nutr., 37, 368. Mc Lean-Ross A.H., Eastwood M.A., Brydon W.G., Busuttil A,, Mc Kay L.F., Anderson D.M W., (1984) Brit. J. Nutr., 51,47. Tulung B., Remesy C., Demigne C., (1987) J. Nutr., 117, 1556. Wyatt G.M., Bayliss C.E., Holcroft J.D., (1 986) Brit. J. Nutr., 5 5 2 6 1. Gournier-Chateau N., (1994) in Lesprobiotiques en alimentation animale et himaine, Technique et Documentation, Lavoisier, Paris, p. 9. Walter D.J., Eastwood M.A., Brydon W.G., Elton R.A., (1986) Brit. J. Nutr., 55, 465.

11.

Walter D.J., Eastwood M.A., Brydon W.G., Elton R.A., (1988) Brit. J. Nutr., 60, 225.

12. 13.

Gibson G.R., Roberfroid M.B., (1995) J. Nutr., 125, 1401. Salyers A.A., Kuritza A.P., McCarthy R.E., (1985) Proc. SOC.Exp. Biol. Med.,

14.

Roberfroid M.B., Bornet F., Bouley C., Cummings J.H., (1 995) Nutr. Rev., 53(5),

15. 16.

Hoverstad T., (1986) Scand. J. Gastroenterol., 19 (suppl. 93), 89. Stevens J, Levitsky D.A., Van Soest P.J., Robertson J.B., KalkwarfK.J., Roe D.A., (1987) Am. J. Clin. Nutr., 46,812. Annison G., Trimble R.P., Topping D.L., (1995) J. Nutr., 125(2), 283. Storer G.B., Illman R.J., Trimble R.P., Snoswell A.MP., Topping D.L., (1984) Nutr. Res., 4, 701. Topping D.L., Illman R.J., Trimble R.P., (1985) Nutr. Rep. Int., 32(4), 809. Younes H., Garleb K.A., Behr S.R., Remesy C., Demigne C. (1 994) FASEB J., 8,

180,415. 127.

17. 18. 19. 20.

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Gums and Stabilisers for the Food Industry

21.

Bourquin L.D., Titmeyer E.C., Fahey G.C., Garleb K.A., (1 993) Scand. J. Gastroenterol., 28(3), 249. May T., Mackie R.I., Fahey G.C., Cremin J.C., Garleb K.A., (1 994) Scand. J. Gastroenterol., 29(10), 916. Mortensen P.B., Hove H., Clausen M.R., Holtung K., ( 1991) Scand. J. Gastroenterol., 26, 1285. Titmeyer E.C., Bourquin L.D., Fahey G.C., Garleb K.A., (1 991) Am J. Clin. Nutr., 53, 1418. Demigne C., Remesy C., (1991) Report of the tenth Ross conference on medical research, Columbus, Ohio, Ross laboratories, p. 17. Remesy C., Demigne C., Chartier F., (1980) Reprod. Nutr. Develop., 20, 1339. Howard M.D., Gordon D.T., Garleb K.A,, Kerley M.S., ( 1 995) J. Nutr., 125, 2604. Kiriyama S., Okazaki Y., Yoshida A,, (1969) J. Nutr., 97, 382. Haskell W.L., Spiller G.A., Jensen C.D., Ellis B.K.,Gates J.E., (1 992) Am. J. Cardiol., 69(5), 433. Jensen C.D., Spiller G.A., Gates J.E., Miller A.AF, Whittam J.H., (1993) J. Am. Coll. Nutr., 12(2), 147. SharmaR.D., (1985) Nutr. Res., 5, 1321 Eastwood M.A., Brydon W.G., Anderson D.M.W.,(1986) Am. J. Clin. Nutr., 44, 51. Assimon S.A., Stein T.P., (1 994) Nutr., 10(6), 544. Imeson A., (1 992) in Thickening and gelling agentsfor food, Blackie Academic & Professional, London, p. 66. Thevenet F., (1995) in Encapmlation and controlled release of food ingredients (eds Risch S.J. & Reineccius G.A.), ACS Symposium Series, American Chemical Society, Washington DC, p. 5 1,

22. 23. 24. 25. 26. 27. 28. 29. 30.

31. 32. 33. 34.

35.

List of Participants ANDERSON C.

Reading Scientific Services Ltd, Whiteknights, PO Box 234, Reading RG6 6LA, UK.

ASAI I.

San-Ei Gen FFI Inc, 1-11 Sanwa-cho 1 chome, Toyonaka City, Osaka 561, Japan.

AYMARD P.

Unilever Research, Colworth House, Sharnbrook, Bedfordshire MK44 7LQ, UK.

BARBER G.A

17 Harvey Avenue, Nantwich, Cheshire CW5 6LE, UK.

BARCLAY F.

University of Nottingham, ABFS(F), Sutton Bonnington Campus, Loughborough, Leics LEI2 5RD. UK.

BEST E.

NestlC, 809 Collins Avenue, Marysville, Ohio 43040-4002, USA.

BISHAY 1.E

Monsanto, 601 E Kensington Road, Mount Prospect, IL 60056, USA.

BLAKE T.

Firmenich SA, Route des Jeunes I , CH-1211 Geneva, Switzerland.

BOURRIOT S.

INRA-LPCM, Rue de la GCraudiBre, BP 71627,44316 Nantes Cedex 03, France.

BOWLER P.

RHM Technology Ltd, The Lord Rank Centre, Lincoln Road, High Wycombe, Bucks HP12 3QR, UK.

BOYAR M.M.

Spillers Petfoods, PO Box 238, Bridge Road, Southall, Middlesex, UK.

BRAUDO E.E

Institute of Biochemical Physics, Russia Academy of Sciences, Vavilov str. 28, Moscow 117813, Russia.

BROOKS M.

Quest International, Kilnagleary, Carrigaline, Co. Cork, Ireland.

CARRINGTON S.P.

H.H Wills Physics Laboratory, University of Bristol, Royal Fort, Tyndall Avenue, Bristol BS8 ITL, UK.

CASADEI E.

Food and Nutrition Division, Food and Agriculture Organisation of the UN, Wale delle Terme di Caracalla, 1 00100 Roma, Italy.

CHOUARD G

SIAS-MPA, 17 Av de 8 Mai 1945,77295 Mitry Mory, France

CHRONAKIS I S .

Physical Chemistry I , Lund University, Box 124, S-22100 Lund, Sweden.

CLARK B.C.

The Coca Cola Company, PO Drawer 1734, Atlanta, Georgia 30301, USA.

CLOSS C.B.

NestlC Research Centre, PO Box 44, CH-1000, Lausanne 26, Switzerland.

COSTEUX S

ESPCI, 10 rue Vauquelia, 75231 Paris Cedex 03, France

422

Gums and Stabilisersfor the Food Industry

COMFORT S.

The NutraSweet Kelco Co, Waterfield, Tadworth, Surrey KT20 5HQ, UK.

COOPER G.

Cerestar UK Ltd, Trafford Park, Manchester MI7 IPA, UK.

COWBURN P.

National Starch and Chemical, Prestbury Court, Greencourts Business Park, 333 Styal Road, Manchester M22 5LW, UK.

CRUTTENDEN N.A.

The NutraSweet Kelco Co, Waterfield, Tadworth, Surrey KT20 5HQ, UK.

CUI w.

Centre for Food and Animal Research, Agriculture and Agri-Food Canada, Ottawa, Ontario, KIA OC6, Canada.

CUNIN C

National Starch and Chemical, IM Altenschemel55, D-67435 Nevstadt, Germany Wageningen Agricultural University, Dept of Food Science, Bomenweg 2, P 0 Box 8129,6700 EV Wageningen, The Netherlands.

DAAS PJH De COCK P

Eridania Beghin Say, Havenstraat 84, Vilvoorde, B- 1800, Belgium

DE'ITMAR P.

Reckitt and Colman, Dansom Lane, Kingston-upon-Hull HU8 7DS, UK.

DICKINSON E.

Procter Dept of Food Science, University of Leeds, Leeds LS2 9JT, UK.

DJABOUROV M.

ESPCI, Lab Physique Thermique, 10 rue Vanguelia, 75231 Paris, Cedex 03. France.

DRAGET K.I.

NOBIPOL, Dept of Biotechnology, Norwegian Institute of Science and Technology, San Saelands vei 6/8, N7034 Trondheim, Norway.

DUNSTAN D.E

Dept of Chemical Engineering, Melbourne University, Parkville, Victoria 3052, Australia.

EGGERT-MAHYOU U. Meyhall AG, Postfach 862, 8280 Kreuzlingen, Switzerland. EKHART P.

TNO Nutrition and Food Research, PO Box 360,3700 A J Zeist, The Netherlands.

ELIASSON A-C.

Dept of Food Technology, University of Lund, PO Box 124, S22100 Lund, Sweden.

ENNIS M.P.

Dept of Food Chemistry, University of Cork, Ireland

EVANS J.M

The NutraSweet Kelco Co., Waterfield, Tadworth, Surrey KT20 5HR, UK.

FERNANDEZ P.

Friskies R&D Centre Amiens, BP 47, 80800 Aubigny, France.

FISZMAN S.

Instituto de Agroqumicia y Tecnologia de Alimentos. CSIC, Apartado de Correos 73, 46100 Burjarist, Valencia, Spain.

423

List of Participants

FOSTER T.

Unilever Research, Colworth House, Sharnbrook, Bedfordshire MK44 7LQ, UK.

FOX J.E.

G.C. Hahn & Co., Aegidienstrasse 22,23552 Lubeck, Germany.

FRANCK A.

Orafii S.A., Aandorenstraat 1,3300 Tienen, Belgium.

GARNIER C.

INRA-LPCM, Rue de la GQandiBre, BP 71627,44316 Nantes Cedex 03, France.

GOETHALS M.

Nutricia Research, PO Box 1,2700 MA Zoetemeer, The Netherlands.

GONTALVES M.P.

Faculdade de Engenharia, Universidade di Porto, Rua dos Bragas, 4099 Porto Codex, Portugal.

GOYCOOLEA F.M.

Centro de Investigaci6n en Alimentaci6n y Desarrollo AC, Km 0.6 Carretera a la Victoria, PO Box 1735, Hermosillo, Sonora 83000, M6xico.

GREGORY D.

David Gregory Associates, The Old School, Denston, Newmarket, Suffolk CB8 8PP, UK.

GREGSON C.

University of Nottingham, ABFS(F) Sutton Bonnington Campus, Loughborough, Leicestershire LEI 2 5RD, UK.

HELGERUD T

Pronova Biopolymer AS, PO Box 494, N-3002 Drammen, Norway.

HERMANSSON A-M.

SIK Swedish Institute for Food Research, Postbox 5401,40229 Goteborg, Sweden.

HODGSON I.

The NutraSweet Kelco Co., Waterfield, Tadworth, Surrey KT20 5HQ, UK.

HOLLAND J

Carbery Milk Products, Ballineen, Co Cork, Ireland

HOWELL N.K.

School of Biological Sciences, University of Surrey, Guildford, Surrey GU2 5XH. UK.

HUGHES H.

The North East Wales Institute, Plas Coch, Mold Road, Wrexham LL1 2AW, UK.

IMESON A.

FMC Corporation (UK) Ltd, Unit 3c, Harcourt Way, Meridian Business Park, Leicester LE3 2WP, UK.

IZZARD M.

Unilever Research, Colworth House, Sharnbrook, Bedfordshire MK44 7LQ, UK.

KANEGAE M

Meiji Seika Kaisha Ltd, R & D Labs, 5-3-1 Chiyoda, Sakado-shi, Saitama 350-02, Japan Wolff and Olsen, gr Backerstr. 13, D 20095 Hamburg, Germany.

KARG S. KASAPIS S.

Dept of Food Science, Cranfield University, Silsoe, Bedfordshire MK45 4DT, UK.

KATZBAUER B.

Jungbunzlauer GmbH, Pernhofen 1, A-2064 Wulzeshofen, Austria.

424

Gums and Stabilisers for the Food Industry

KATSUTA K.

Dept of Food Science and Nutrition, Nara Women’s University, KitanoyanishiMachi, Nara 630, Japan.

KEISER A

Creaderm SA, Z I Pra de Plan, CH-1618 Chatel-St-Denis, Switzerland

KERRISON B

Food Spectrum Pty Ltd, P 0 Box 147, Goodna, Queensland 4300, Australia

KETTLITZ B.

Cerestar R+D, Havenstraat 84, Vilvoorde, B- 1800, Belgium

KOK M.S.

University of Nottingham, ABFS(F), Sutton Bonnington Campus, Loughborough, Leicestershire LE12 5RD, UK.

KOKELAAR A.

Quest International, PO Box 2, 1400 CA Bussum, The Netherlands.

KRAVTCHENKO T.P. Colloids Naturels International, BP 4151, F76723 Rouen Cedex, France. LARSSON H.

Dept Food Industry, University of Lund, PO Box 124, S-22100 Lund, Sweden.

LAURSEN K.

Danisco Ingredients, Edwin Rahrs Vej 38, DK 8220 Braband, Denmark.

LAWSON M

FMC Corporation, Food Ingeredients Division, Route 1 and Plainsboro Road, Box 8, Princeton, Nj 08543, USA

LAWSON P.

Cerestar UK Ltd, Trafford Park, Manchester MI7 lP, UK.

LUNDIN L.

Unilever Research, Colworth House, Sharnbrook, Bedfordshire MK44 7LQ, UK.

LYONS H J

Kerry Algae Ltd, Sackville, Ardfert, Co Kerry, Ireland

MADSEN F

Danisco Ingrewdients, Edwin Rahrs Vej 38, Dk 8220 Braband, Denmark

MANOJ P.

Institute of Food Research, Norwich Laboratory, Norwich Research Park, Colney, Norwich NR4 7UA, UK.

MARRS W. M.

Leatherhead Food RA, Randalls Road, Leatherhead, Surrey KT22 7AY, UK.

MAY C.

Citrus Colloids Ltd, Pomona Place, Hereford HR4 ODA, UK.

MAZOYER J.

Systems Bio-Industries, Centre de Recherch, 50500 Baupste, France

MEYER D

Herbafood Nahrungsmittel GmbH, Phobener Chaussee, D- 14542 WerderEIavel, Germany

MITCHELL J R

Faculty of Agricultural and Food Sciences, University of Nottingham, Sutton Bonnington, Loughborough LEI2 5RD, UK

MODLISZEWSKI J

FMC Corporation, 523 Midwood Drive, Brick, New Jersey, USA

MOLLER F.

Copenhagen Pectin AS, Ved Banen 16, DK-4623 Lille Skensved, Denmark.

MOREAU S.

Setaram, 7 rue de I’Oratoire, 69300 Caluire, France

List of Participants

425

MORGAN K.

Industrial Research Ltd, Gracefield Research Centre, Gracefield Road, PO Box 31-310, Lower Hutt, New Zealand.

MORRIS V.J.

Institute of Food Research, Norwich Laboratory, Norwich Research Park, Colney, Norwich NR4 7UA, UK.

MORRIS E.R.

Dept of Food Science, Cranfield University, Silsoe, Bedfordshire MK45 4DT, UK.

MORRISON N.

The NutraSweet Kelco Co., 8355 Aero Drive, San Diego, CA 92123-1718, USA.

MULLER G.

UNR CNRS 6522 ‘Polymeres, Biopolymeres, Membranes’, Universite de Rouen, 76521 Mont Saint Aignan Cedex, France.

MULVIHILL D

Dept of Food Chemistry, University College of Cork, Cork, Ireland

MURRAY J.C.F.

Hercules Ltd, 31 London Street, Reigate, Surrey RH2 9YA.

NEISER S.

NOBIPOL, University of Trondheim, Sem Saelands vei 6/8, Trondheim N-7034, Norway.

NISHINARI .K

Dept of Food and Nutrition, Faculty of Human Life Sciences, Osaka City University, Sumiyoshi, Osaka 558, Japan.

NOBLE 0.

Danone, CIRDC, 15 Avenue Galilee, 92350 Le Plessis Robinson, France.

NORTON I.

Unilever Research, Colworth House, Sharnbrook, Bedfordshire MK44 ILQ, UK.

NUSSINOVITCH A.

The Hebrew University of Jerusalem, Faculty of Agriculture, Food and Environmental Quality Sciences, PO Box 12, Rehovot 76100, Israel.

OAKENFULL D

Hydrocolloids Research, 195 Fox Valley Road, Wahroonga, NSW 2076, Australia.

OLESEN P.

Copenhagen Pectin AS, Ved Banen 16, DK 4623, Lille Skensved, Denmark.

OMOTO T.

San-Ei Gen FFI Inc, 1-11 Sanwa-chome, Toyonaka City, Osaka 561, Japan.

ONG M

Nest16 R+D, P 0 Box 204, Haxby Road,York, YO1 IXY, UK

ONSOYEN E.

Pronova Biopolymer AS, PO Box 494, N-3002 Drammen, Norway.

OSMAN M-E.

The Gum Arabic Company, Khartoum, Sudan.

PATTERSON L.

Dalgety FTC, Station Road, Cambridge CBI 2JW, UK.

PHILLIPS G.O.

Croesnewydd Hall, Wrexham Technology Park, Wrexham LL13 7YP, UK.

PHILP K.

Quest International, Kilnagleary, Carrigaline, Co. Cork, Ireland.

PICULELL L.

Physical Chemistry 1, Lund University, Box 124, S-22100 Lund, Sweden.

426

Gums and Stabilisersfor the Food Industry

PONNE C.T.

ATO-DLO, PO Box 17,6700 AA Wageningen, The Netherlands.

PROCTER A.

Cerestar, Trafford Park, Manchester M17 IPA, UK.

PYBUS R

Dept of Chemical Engineering, University of Bradford, Bradford, West Yorkshire, BD7 IDP, UK

QIN Y.

Innovative Technologies Ltd, Winsford Industrial Estate, Winsford, Cheshire CW7 3PD, UK.

RASANEN J.

V l T Biotechnology and Food Research, PO Box 1500,02044 V?T, Finland.

RELKIN P.

ENSIA, 1 Avenche des Olympiades, 91744 Massy, France.

RENARD D.

INRA-LPCM, BP 71627,44316 Nantes Cedex 03, France.

ROBERTS I.D.

Nestle Research Centre, Vers-chez-les-Blanc, PO Box 44, CH- 1000 Lausanne 26, Switzerland.

SCAMPARINI A.

PO Box 6121, CEP 13081-970, Campinas-SP, Brazil.

SCHLOSSER E.E.

Unilever Research Lab, POBOX 114, 3130 AC Vlaardingen, The Netherlands

SCHOLS H.A.

Dept of Food Science, Wageningen Agricultural University, Bomenweg 2, 6703 HD Wageningen, The Netherlands.

SCHORSCH C.

Unilever Research, Colworth House, Sharnbrook. Bedfordshire MK44 ILR, UK.

SHARPE V.

RhBne Poulenc Chemicals UK, Poleacre Lane, Woodley, Stockport, UK.

SIMONET F.

INRA-LPCM, Rue de la GCraudiire, BP 71627,44316 Nantes Cedex 03, France

SHIRAKAWA M.

Dainippon Pharmaceutical Co. Ltd. 33-94 Enoki-cho, Sinta, Osaka 564, Japan.

SLADE R.

SALTLIC, Roboan House, Dauntsey, Chippenham, Wilts, SN15 4HR, UK

SMORENBURG H

Unilever Research, P 0 Box 114, 3130 AC Vlaardingen, The Netherlands

SOMERS P.G.

School of Biological Sciences, University of Surrey, Guildford, Surrey GU2 5XH, UK.

SONDERGAARD K.M. Danisco Ingredients, Edwin Rahrs Vej 38, DK 8220 Braband, Denmark. SOUSA I.

Inst. Sp. Agronomia, Univ Tecnicade Lisboa, Tapadadao a Juda, 1399 Lisboa Codex, Portugal.

SPIERS C.

Pedigree Petfoods, Mill Street, Melton Mowbray, Leics LEI3 IBB, UK

STOLLE-SMITS T.

ATO-DLO, PO Box 17,6700 AA Wageningen, The Netherlands

List of Participants

427

SWENSON P

Bunge Foods, 3582 McCall Place, NE Atlanta, GA 30340, USA

SWORN G.

The NutraSweet Kelco Co, Waterfield, Tadworth, Surrey KT20 5HQ. UK.

TANAKA R.

Forestry and Forest Products Research Institute, PO Box 16, Tsukuba Norin Kenkyu Danchinai, Ibaraki, Japan.

THERKELEN G.

Copenhagen Pectin AS, Ved Banen 16, DK-4623 Lille Skensved, Denmark.

TO K-M.

The NutraSweet Kelco Co, Waterfield, Tadworth, Surrey KT20 SHQ, UK.

TORNBERG E.

Swedish Meat Research Institute, PO Box 504, S-24424 Kavlinge, Sweden.

TUINIER R.

Dept of Physical and Colloid Chemistry. Wageningen Agricultural University, Wageningen, The Netherlands.

TURQUOIS T.

Nest16 Research Centre, P 0 Box 44,CH-1000, Lausanne 26, Switzerland

TZIBOULA A.

Hannah Research Institute, Ayr, Scotland KA6 5HL, UK.

VAN CASTEREN W.

Dept of Food Science, Wageningen Agricultural University, Bomenweg 2, 6703 HD Wageningen, The Netherlands.

VREEKER R.

Unilever Research, P 0 Box 114,3130 AC Vlaardingen. The Netherlands

WALLIN P.J.

Dalgety FIG, Station Road, Cambridge CBI 2JW, UK.

WAREING M.

Arthur Bramwell and Co Ltd, 58-62 High Street, Epping, Essex, UK.

WASSERMAN L.A.

Institute of Biochemical Physics, RAS, 4 Kosygin Street, Moscow 117334, Russia.

WHITE R.

Optokem Instruments Ltd, Nercwys, Flintshire CH7 4EW, UK.

WICHMANN J.

Danisco Ingredients, Edwin Rahrs Vej 38, DK-8220 Braband. Denmark.

WILLIAMS P.A.

The North East Wales Institute, PIas Coch, Mold Road, Wrexham LL11 2AW. UK.

WILLIAMS R.

CSIRO, Division of Food Science and Technology, Melbourne Laboratory, PO Box 20, Highett, Victoria 3190, Australia.

WINWOOD R.

ADM Food Additives Division, Church Manorway. Erith, Kent DA8 IDL, UK.

YOSHIMURA M.

Dept of Food Science and Nutrition, Himeji College of Hyogo, Himeji, Hyogo 670, Japan.

YOUNG J.

Leatherhead Food RA, Randalls Road, Leatherhead, Surrey, KT22 7RY. UK

ZORBA M.

Ege University, 1388 Sokak 4/4, 35220 Alsancak-Izmir, Turkey.

ZUBER U.

Unipektin AG. CH-8264 Eschenz. Switzerland

Subject Index

1,8 Anilinonaphthalene 247-255 Acacia gum (see also Gum arabic) nutritional properties 4 15,416 source of soluble fibre 4 13-4 19 chemical structure 414 Acacia karoo 72-75 Acacia polyacantha 72-75 Acacia senegal 70-75 Acacia seyal72-75 Acetobacrer xylinium 84 Agar fluid gels 260-268 Agarose enthalpy of sol-gel transition 98 Agaroselgelatin gels 170,17 1 Aggregation of myosin 143 Albedo 8,9 Alga spiroulina 154-165 Alginate synergy with inulin 408 Alginate beads 324 Amylopectin - galactomannan mixtures 23 1-237 Anion exchange chromatography of pectins 12 Antibodies 307 Apple pectin 4-6 Arrhenius plots 149 ATPase activity 136,137 Atomic force microscopy 361-369, 388-394 Bacterial cellulose AFM images of 364 Bacterial Polysaccharide 240 Baking quality 289-292 Baking test 287 Beijerinckia sp 48,49 Binodals 234 Biopolymer mixtures 169-181 Bovine serum albumin 146, 314-322 Bovine serum albumin mixtures 190.201 Brabender viscosity of carrageenan 280.28 1 Brabender viscosity 78,79

C*AraTex modified starch 76-83 Calcium ion activity 214-220 Calcium phosphocaseinate 248 Calcium-chelating 127- 135 Calorimetric studies 158 Calorimetry of fluid gels 261 Capsules 323-331 Carrageenan (see also Kappa and Iota carrageenan) AFM images of 365-368 Brabender viscosity 280,28 1 extraction 276, 277 enthalpy of sol-gel transition 98 stability to processing 345-356 specification for 345,346 size exclusion chromatography of 348-356 Tiger Striping using 276-285 Carboxymethyl cellulose/BSA mixtures 190-20 1 Casein micelles 115,212-220.222-229,247-255 Caseins rennet 127-135 Cellulose bacterial, AFh4 images of 364 microparticulated 16-25 PrimaCelTM84-93 Cheeses processed 127 Chewing gum 82 Chicken breast myosin 139 Chicory inulin 409-41 1 Cloud point 184,185 Coalescence 262 Complex modulus of BSA-stabilised emulsions 320 of starch dispersions 298 Confectionery gum arabic replacement in 76-83 Confocal laser scanning micrographs of starch 187 Confocal laser scanning microscopy 107-1 16,247-255 to study mixed systems Convenience foods 269 Correlation length 191,192 Creep compliance 61-63 Creep measurements on rice starch 60-68

Subject Index Critical'overlap concentration 19 I , 192 Crossed immunoelectrophoresis 307-3 1 1 Cryogenics 273 Curdlan DSC studies on 26-33 rheological studies on 26-33 CV-70 polysaccharide 48-58

429 Enzymatically modified xyloglucan 94-103 Enzymes pectin 4 ESR spectra 119-125 ESR spin probe 117-126 Esterification degree of 212 Ethyl hydroxyethyl cellulose gels, with starch 184-188 Eucheuma seaweed 277 Exclusion volume effect 172 Extrusion 273

Degree of esterification 212 Dehydration 269 Demixing 262 Denaturation 149,154,158,159 Depletion flocculation 222-229, 247 Dessert jelly formulations 35 1-355 Dextranllocust bean gum mixtures 247-255 Dextranlmicellar casein mixtures 247-255 Dietary Fibre soluble 413-419 Differential scanning calorimetry of curdlan 26-33 of myosin protein 142 of proteins 147 of xanthadlocust bean gum 241-245 of xyloglucan 96,97 Diffusion coefficient 224-229 Dough wheat 286-294 5-Doxyldecane 122 5-Doxy1 stearic acid 119 Dynamic light scattering 224-226 Dynamic modulus of agar gels 266 Dynamic rheological properties 388-394 Dynamic viscoelastic measurements 17 of curdlan 27 of microparticulate cellulose 17 of rice starch 60-68

FA0 gum arabic project 69-75 Farinograph 288, 293 Fat replacement 410-41 1 Fermentation 269 Fermented doughs 286-294 First order reaction 99 Flavedo 8 Flory-Huggins parameters 174-176 Fluid gels 259-268 Foams stabilisation of 172, 173 Freeze etching I15 Freeze-thaw treatment 43 Freezing 269 Frequency dependence of xyloglucan 101 Frequency sweeps of LBG solutions 46 of PrimaCel 9 1,92 Frozen doughs 286-294 Functional foods 269, 397-402 Fusion profile 64

Egg albumin 117 Egg box model 2 13 Egg white proteins 145-152 Elastic modulus 23 of fluid gels 265 of sausage batters 299 Electrophoretic mobility 3 18 Emulsions 315-322 oil in water 127,128 stabilisation of 172,173 Emulsion formulation 307 Encapsulated oil 3 11 Enthalpic contribution 16 Enthalpic gels 333-334 Enthalpy of sol-gel transition 98 Entropic contribution 16 Entropic rubbery state 333

Galactomannan 43,240,247 - starch systems 231-237 Galacturonic acid 3 P-Galactosidase 5,95,101 Gel permeation chromatography 232,233,235 Gelatinlagarose gels 170,171 Gelatin/methylcellulose mixtures 172 Gelatinisation of rice starch 59-68 Gelation enthalpy of sol-gel transition 98 of LM Pectin 212-220 of milk protein carrageenan mixtures 202-210 of milk protein systems 107-1 16 of muscle protein 136- 144 of protein-polysaccharide mixtures 189-201 of rice starch 63-68 protein 145-152, 160-162

430 Gelation curves 99 Gelatin gels 383-386 shear-cooled 264 Gelation mechanism of curdlan 26 thermo-irreversible 32 Gellan gum 335-343 Gelling properties of wheat bran non-starch polysaccarides 34-42 Gels AFM images of 366-368,388-394 agarose/gelatin 170.17 1 curdlan 26,33 enthalpic 333-334 fluid 259-268 gelation 264 heat induced 149 inulin 405-408 kappa carrageenan 202-2 10 LM pectin 212-220 microstructure of 150 milk 212-220 mixed 184-188, 194-201 phase separation 109 quiescent 264-268 rice starch 59-68 syneresis of 28.33 thermoreversible 203 viscoelastic 381-386 xanthan-locust bean gum 239-245 Gibbs energy of mixing 174,175 Glass transition 334 Glass-like 333 Glassy state 334 Globular proteins 145 a-D-Glucans 178 P-D-Glucans 34.42 Guar gudmicellar casein mixtures 247-255 Gum arabic (see also Acacia gum) composition of 77 F A 0 project 69-75 production 69-75 replacement for 76-83 representative segment of 77 source of soluble fibre 4 13-419 specifications 69-75 tannin content 308 Hairy regions 4-7.10 Hard sphere model 224-227 Heat of reactions 148 Heat-induced gels 149 Helical structure 240 High methoxy pectins 3,212 High pressure 270-272 High pressure processing 3 15

Gums and Stubilisersfor the Food Industry Hydration of rennet caseins 127-135 Hydration profile of rennet casein 129 Hydrogel of locust bean gum 43-47 Hydroxypropylation of starch 78 Hydroxyethyl celluloseiBSA mixtures 190-201

IIS Vicia Faba 117 Immiscibility 233 Incompatibility 107, 174, 184 Interaction parameter 174- 176 Interfacial films AFM images of 369 Inulin chicory 409-41 1 rheological characterisation 403-408 synergy with alginate 408 Iota carrageenan (see also Carrageenan) in emulsions 314-322 Isostrain model 2 19 Jellies 79 Junction zones 31.44, 185,213 Kappa carrageenan (see also Carrageenan) gelation 219 gel transition temperature of 202-210 gels 202-210 influence of milk proteins on 202-210 mechanical properties of 202-210 storage modulus of 335 Kinetics of gel formation 109-1 16 of phase separation 231-237 of retrogradation 61,63 Lactic acid bacterium 222-229 Lactococcus luctis 222 P Lactoglobulin 146,231-237 Legumin 117-126 Lemon pectins 8 Lipophilic compounds 117-126 Liquid-core hydrocolloid capsules 323-33 1 Liquid-liquid separation 236 Loaf volume 292 Locust bean gum gels with xanthan 239-245 hydrogels of 43-47 structure of 44 Locust bean guddextran mixtures 247-255 Locust bean gudxanthan mixtures 247-255 Loss modulus of amorphous polymers 334

Subject Index of curdlan 26-33 of locust bean gum 45.46 of microparticulated cellulose 16-25 of wheat bran non-starch polysaccharide 39.40 of xanthan solutions 24 1-245 of xyloglucan 99-102 Low methoxyl pectins 3,212-220 Lysozyme 146-151 Marine algae proteins 154-165 Maturograph 288,289 Meat Turkey 276-277 Meat products starch in 295-303 Mechanical spectra of wheat starch 186 of xanthan/LBG 244 Meromyosin 137 Mesquite gum 305-312 Methyl cellulose enthalpy of sol-gel transition 98 Methyl ester distribution 11 Methylcellulose/gelatinmixtures 172 Micellar caseiddextran mixtures 247-255 Micellar caseidguar gum mixtures 247-255 Microbial polysaccharide 26,48, 247 Microparticulated cellulose 16-25 Microrheology of starch granules 371-379 Microscopy of fluid gels 26 I of turkey breast 278 Microstructure of gels 150,151 of sausage batters 297,300,301 Microwave frequency 270-272 Milk gels 2212-220 Milk protein systems 107-116.202-210,222 Mixed biopolymer systems 107,247-255 Mixed gels 194-201 Mixtures biopolymer 169-181 Molecular images by AFM 388-394 Molecular weight distribution for carrageenan 350-355 Mozzarella cheese 127-135 Multicomponent systems 169-181 Muscle protein 136-144 Myosin proteins 136-144 NIZO B40 EPS NMR relaxation 26 1 Non-coulombic complex I76 Non-starch polysaccharides from wheat bran 34-42

43 1 Nutraceuticals 269 Nutritional properties ofacaciagum415,416 Oil capsules 323-331 Oil in water emulsions 127.128, 315-322 Oligosaccharides from tamarind xyloglucan 95 Ovalbumin 117-126 Pair interaction parameters 174-176 Particle size distribution of microparticulated cellulose 17,lE Partition coefficient 232, 236, 237 Pasteurisation 269 Pectate lyase 4 Pectin enzymes 4 Pectins anion exchange chromatography I1,12 apple 4 hairy regions 4,5,6,7,10 high methoxyl3-15,212 low methyl 3 low methoxyl212-220 methylester distribution 11 size exclusion chromatographyof 9,11,12 structural features 3-15 Pentosans 34 Persistence length 191,192 Phase diagrams 227,233,234,236 of dextranlmicellar casein 253 of dextranLBG 250 Phase inversion 107 Phase separated gels 107-116 Phase separation 109,223 of starcMgalactomannansystems 23 1-237 kinetics 23 1-237 Phase-separated systems 107-I 16 Phosphorylated polysaccharides 178 Pig oesophagus myosin 139 Polyacrylamidegel eletrophoresis 137,138 Polygalacturonase4 Polysaccharide protein mixtures 189-201 Pol ysaccharide-polysaccharide mixtures 249-252 Potato starch 295-303 microrheology of 37 1-379 Poultry 276-285 Pseudoplasticity of PrimaCel87 PrimaCeIrM food cellulose 84-93 Process technology 269-275 Proteolytic substructure 137 Prosopis spp 305 Protein high pressure processing 3 I5

432 milk 107-116, 202-210 myosin 136-144 Protein -polysaccharide interactions 252-255, 3 14-322 Protein complexes 178 Protein concentration determination of 233 Protein gelation 160,162 Protein-polysaccharide mixtures 189-201 Proteins egg white 145-152 interactions of 117 marine algae 154-165 thermal stability 145 whey 145-152 Pulse propagation 38 I Pulse-resonance rheometer 38 1-386 Pulsed light 270-272 Pulsed NMR of rice starch 59-68 Quiescent gels 264-268 Radio frequency 270, 27 I Radius of gyration 19I , 192 Relaxation time 20,66,263 Replacement for gum arabic 76-83 Reticulated network 84 Retrogradation of rice starch 59-68 rate 63 Rheological analyses of rennet caseins 127- 135 Rheological behaviour of CV-70 polusaccharides 48-50 of mixed gels 198-201 Rheological characteristics of PrimaCel91 Rheological characterisation of inulin 403-408 Rheological measurements of sausage batter 299 of starch 297-299 on xanthadlocust bean gum 241-245 Rheological properties dynamic 388-394 of amylopectidgalactomannan mixtures 23 1-237 of myosin 140 of rice starch 59-68 of wheat doughs 286-294 Rheological studies of microparticulated cellulose 16-25 of xyloglucan 94-103 Rheological behaviour of dextran/LBG 250-252 of guadcasein 254

Gums and Stabilisers for the Food Industry Rheology of BSA-stabilised emulsions 321 of fluid gels 261 Rheometer combined pulse-resonance 38 1-386 Rhodamine iso-thiocyanate 247-255 Rice starch 59-68 Robotics 273 Rotational correlation time 119,123 Rubbery plateau 21 Rubbery state 333 Salts effect of gelation 59-68 Sausage batters 296-303 Scanning electron micrograph of PrimaCel 84 Scanning electron microscopy high sensisitivity 138 of gels 146,150 Second virial coefficient 177 Separation technologies 273 Serum separation 317 Shear modulus of carrageenan mixtures 205 of pectin gels 2 14-220 Shear wave propagation 38 I Shift factors 342 Size exclusion chromatography of carrageenan 348-350 of pectins 9 , l l Skeletal muscle protein 136-144 Skim milk 225 Small angle neutron scattering 189-20 I Smooth muscle protein 136-144 Sodium decanoate 120 Sol-gel transition of xyloglucan 94-103 Sol-gel transitions of protein 145-152 Sphericity determinations 325 Spin probe 117-126 Spin-spin relaxation time 66 Spiroulina pacifica 154- 165 Spray drying 307 Spreads lowfat 107-116 Starch - galactomannan systems 23 1-237 hydroxyproplylation 78 modified 76-83 potato 295-303 rice 59-68 Starch dispersion rheology 298 Starch gels wheat 184- 188

SubjectIndex

Starch granules microrheology of 371-379 Steady shear viscosity measurements 17 Stearic acids spin labelled 119-124 Sterilisation 269 Storage moduli of BSA gels 199,200 Storage modulus of amorphous polymers 334 of curdlan 26-33 of hydroxypropylguar 390-394 of kappa carageenan 335-339 of low acyl gellan gum 335-337 of locust bean gum 45,46 of microparticulated cellulose 16-25 of rice starch 63.64 of wheat bran non-starch polysaccharide 39,40 of xanthan solutions 241-245 of xyloglucan 99-102 Stress sweep for PrimaCel86 Stress-strain curves 27-30 sugar high, systems 333-344 Sugar free gums 80.81 Surface tension 318 Syneresis of gels 28,33 Tamarind seed xyloglucan 94,95 Tannin in mesquite gum 305-312 Tapioca starch modified 76-83 Thermal association 162 Thermal denaturation 158 Thermal gelation 140,141,143 Thermal stability of proteins 145-152 Thermal studies on xyloglucan 94-103 Thermal treatment 243 Thermo-irreversiblegelation 32.33 Thermodynamic incompatibility 247 Thermograms of denaturation 159,160

433 Thermoreversible gels 203,240 Thixotropy of PrimaCel88.89 Tiger striping 276-285 Transmission electron microscopy 113,114, 138, 139, 142 Trypsin-induced proteolysis 175 Turkey hams 277

Ultrafiltration-diafiltration307-3 12 Video micrograph 372,377 Viennetta 270 Viscoelastic behaviour of xyloglucan 100,101 Viscoelastic gels 381-386 Viscosity of inulin 404-408 Viscosity index 129, 130, 133 Viscosity-shear rate of PrimaCel88 Water absorption ability 287-292 Water content of wheat doughs 286-294 Weak gel 100 Wheat bran non-starch polysaccharides 34-42 Wheat doughs 286-294 Wheat starch gels 184-188 Whey proteins 145-152 Williams-Landel-Ferry equation 343 X-ray scanning electron microscopy 279-282 Xanthanilocustbean gum mixtures 247-255 Xanthan - locust bean gum gels 239-245 Xanthan macromolecules AFM images of 362 Xyloglucan enzymaticallymodified 94- 103 Yield stress of curdian dispersions 27-29 of PrimaCel86,87 Yoghurt 270 Young’s modulus of gelatinlagarose gels 170,171