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

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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


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


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


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


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


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

.

3!$ I

il*

-1

0

I

A h

5

10

I

- b

~h

T ~ .-I

1

Z:lI

6

Il

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


30

13

I’

569

3’ 583

I

ST

oJ

,

. . ..

.

.. .

.,

i”’

. . .,m

. .

. .-

. . . , l ~

.

.

.-

.

4z...

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


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


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


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

5000

o

io

20

30

do

50

0

sb io

2500

io

zo

I

30 do 60 sb io

120

1

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o

I1.8-25%

2000 1500 lo00

500 0

0

io

20

30 do

$0

60

io

-

00

10

20

30

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


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

60

80

1

1.1 3.2 1.8

400

. ff

In

300

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v)

v)

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100

00

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20

40

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300

200

1.1 32 65

100

1.8

- T

0

20

do

~

80

7 -

80

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

0

0

2

4

6

8

c3 5000

1.1

4ooo3000

3000-

2000]

2000-

1000

10000, 0

(C-CO)

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 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 , ~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


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


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.


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


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


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


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


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.


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


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


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


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


.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


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.


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


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


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


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


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


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


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


‘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


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


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


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


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


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


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


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.


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


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


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

3000

1

I600

60

1200

a, 400 B

800

200

400

n

a

0

0

0

3000

6Ooo

Time (sec)

9000

0

3000

6000

9000

Time (sec)

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

164


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


166

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

Gum and Stabilisersfor the F w d Industry

170

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,


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.


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.


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.

Functional Interactions in Mixed Biopolymer Sysrem

181

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.).

182


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 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

Gum and Stabilisersfor the F w d Industry

170

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


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,


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.


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.


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.


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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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.

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.


185

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

186


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').


187

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


188

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

194


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


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

196


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


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 > 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


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 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


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 (0N(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.


240

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)


241

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


242

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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Functiona~Interactions in Mixed Biopolymer Systems

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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Temperature ("C) Figure 2: Cooling curves of xanthan/LBG mixtures (1% (w/w)at Werent ratios xanthan/LBG) in 25mM NaCl. Frequency: 6.28 rad.d.G solid symbols; G ' open symbols. 90/10; 80/20; A 70/30; 60140.

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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


244

(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

245


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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Temperature("C) Figure 5: Cooling w e . F6.28rad*i'. 0 - xanthan/LBG (0.6/0.4), lo/o(w/w) in 25mM NaCl; 0 xanthan, 0.6% in 25mM NaC1.

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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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G u m and Stabilisersfor the Food Industry

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,


248

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%.


249

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

250


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

25 1


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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o (rad/s) Figure 4 Rheological behaviour and microscopic structure ( A = 543 nm) of a 15 day-aged 1% RITC-LBG solution. In parallel, the effect of ageing time on the structure of a 1% LBG solution has also been studied. Indeed, it is well known that LBG can form a gel following freeze-thaw

252


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,


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.

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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'

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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

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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.

256


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


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.


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


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


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


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).


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


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


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


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.


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


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


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


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).


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,

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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

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'

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261


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


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


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


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


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.


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.

274


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

Processing Developmenrs

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


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


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


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


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


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.


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.


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


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


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


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


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.


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).

290

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

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

r

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.


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

0

1

rmzmstawn n

4

m [q

7

1

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).


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

293


hlaturogram

Farinngram A

400 650

360

550

320

450

280

350

240

250 150

200 0

4

2

6

10

8

12

B m

40

80

80

100

120

140

0

2 0 4 0

80

8 0 1 0 0 1 2 0 1 4 0

0

20

40

50

80

100

120

140

0

20

40

60

80

100

120

140

550

320

450

280

350

240

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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.

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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.

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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


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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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


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.

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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.


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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%.

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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

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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

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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* *)

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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

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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


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


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


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


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


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


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.


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


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


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.


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


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.


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


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


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


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


~

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


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).


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).


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


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


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


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


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

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:

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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).


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

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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.

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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


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-

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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


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.

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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


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Log (fi.equency/Hz)

8 - 2

0

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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').


342

0

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-0.2 ! 0

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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


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

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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

346


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.


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.

348


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.


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


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


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


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


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

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