Properties of water in foods in relation to quality and stability

Duckworth who organized the symposium entitled: Water Relations of Foods, held at the University of Strathclyde Scotland in September 1974, under the auspices of Internatioral Union of F

Properties of Water in Foods

NATO ASI Series

Advanced Science Institutes Series

A Series presenting the results of activities sponsored by the NATO Science

Committee, which aims at the dissemination of advanced scientific and technological knowledge, with a view to strengthening links between scientific communities

The Series is published by an international board of publishers in conjunction with the NATO Scientific Affairs Division

A Life Sciences Plenum Publishing Corporation

C Mathematical and D Reidel Publishing Company

Physical Sciences Dordrecht and Boston

D Behavioural and Martinus Nijhoff Publishers

Social Sciences Dordrecht/Boston/Lancaster

E Applied Sciences

F Computer and Springer-Verlag

Systems Sciences Berlin/Heidelberg/New York

G Ecological Sciences

Series E: Applied Sciences - No 90

1985 Martinus Nijhoff Publishers

Dordrecht I Boston I Lancaster

Published in cooperation with NATO Scientific Affairs Division

Proceedings of the NATO Advanced Research Workshop on Influence of Water on Food Quality and Stability (ISOPOW III), Beaune, France, September 11-16, 1983 Library of Congress Catalog in Publication Data

NATO Advanced Research Workshop on Influence of

Water on Food Quality and Stability (1983

Beaune, France)

(NATO ASI series Series E, Applied sciences

no 90)

"Proceedings of the NATO Advanced Research Workshop

on Influence of Water on Food Qualtiy and Stability

(Isopow III), Beaune, France, September 11-16,

1983" "Published in cooperation with NATO Scientific

Affairs Division."

Includes bibliographical references and index

1 Food Water activity Congresses I Simatos, D

II Multon, J L (Jean Louis),

1938-III North Atlantic Treaty Organization Scientific

Affairs Division IV Title ~ Series

Martinus Nijhoff Publishers, P.O Box 163, 3300 AD Dordrecht, The Netherlands Copyright © 1985 by Martinus Nijhoff Publishers, Dordrecht

Softcover reprint of the hardcover 1 st edition 1985

Division of Food Research CSIRO - North Ryde - N.S.W - Australia

Department of Bioscience and Biotechnology University of Strathclyde - Glasgow - Scotland

- U.K

Department of Botany University of Cambridge - Cambridge - U.K

Unilever Research - Colworth Laboratory Bedford - U.K

U.S Army Natick Research and Development Center Natick - Mass - U.S.A

Department of Nutrition and Food Science Massachusetts Institute of Technology Cambridge - Mass - U.S.A

Campbell Soup Company Camden - New Jersey - U.S.A

Food Science Department University of Alberta - Edmonton - Alberta - Canada Food Science Research Center

Chapman College - Orange - Cal - U.S.A

VII

Preface

Water is recognized as being an important factor in numerous mena connected with the quality of food For instance, it plays a part

pheno-in the textural properties of several commodities Moreover, water

is an essential parameter determining the behaviour of food products

in the course of many processing operations : on water, will depend the amount of energy necessary for freezing or dehydrating the product; water will strongly influence the evolution of physical, chemical and biochemical phenomena taking place in the product during processing operations such as heating, drying, etc Water will also influence the same reactions, as well as the activity of microorganisms, during the storage of food products under various conditions As a result, all aspects

of quality - sensory, nutritional and hygienic properties of the food

- will be affected

In all these circumstances, the water content of a product is obviously

an important factor, but equally important may be the physical properties

of this water, such as its thermodynamic activity and its mobility

Actual-ly, the concept of water activity (a ) is now widely used by the food industry and in the legislation of sever')¥l countries

The idea of a small, international meeting devoted to a synthetic review and discussion of knowledge on these various matters, was first developed by Dr R.B Duckworth who organized the symposium entitled: Water Relations of Foods, held at the University of Strathclyde (Scotland)

in September 1974, under the auspices of Internatioral Union of Food Science and Technology (IUFoS T) The proceedings , edited by R.B Duckworth, still serve as a useful source of reference material for any food scientist or technologist concerned with the subject

2

A second international conference (ISOPOW II), also sponsored by IUFoS T, was organized to complement and supplement information repor-ted in Glasgow and was held in Osaka (Japan) in September 1978 Contri-butions from Japanese scientists, who have had a traditional interest

in the preservation of foods through control of their water content and water activity, introduced valuable new information The proceedings3 were edited by L.B Rockland and G.F Stewart

The third symposium of the series (ISOPOW III) was held in Beaune (France) in September 1983 As for the previous meetings, the underlying

VIII

objective in designing the programme was to examine the relevance

of recent advances in fundamental knowledge on the subject to practical problems, as they appear in the industrial processing of foods

A very large amount of work has been devoted to the physical ties of water in foods, or more generally in biological systems These properties are commonly considered to play a major, even somewhat magic, role in the structure and functioning of these materials The now classical concept of "bound water" can, however, now be replaced

proper-by a more precise description of the state of water thanks to the various physico-chemical techniques presently available The first objective then of ISOPOW III was to review the more recently acquired knowledge concerning the properties of water in food materials and to reevaluate the roles of water in phenomena of interest to the food scientist

Sorption phenomena still receive special attention and it was priate to consider recently developed isotherm equations and some novel experimental data on sorption hysteresis

appro-A better understanding of the effects of water on various phase tions and reactions which can take place in food products at low and intermediate moisture is highly desirable; The importance of the mobility that water may impart to solutes and the effects of other possible mecha-nisms were discussed Several non-equilibrium phenomena of great signifi-cance for the food industry were considered: superficial water activity, diffusion of solutes and retention of aroma, rheology of hygroscopic powders, effectiveness of packaging

transi-Although it may be argued that regarding "the cell as a simple meter" is a "gross over-simplification", food microbiologists find the water activity concept extremely valuable as a determining factor for microbial activity It was thus appropriate to again review the present state of knowledge on the ways water may control the activity of micro-organisms Then, the combined effects of a and other environmental factors on survival, growth and activity of s'i¥veral types of microorga-nisms were described, this knowledge being the basis for the promising concept of "hurdles technology" which recently appeared and is being increasingly more widely employed

osmo-Intermediate moisture foods (IMF) and other water actIvIty - or moisture - adjusted foods receive an increasing interest from the food industry and a session of ISOPOW III was devoted to various practical aspects of this question: evaluation of the water binding power of macro-molecules, new humectants, manufacturing aspects

Influences of water on quality in certain specific commodity groups were treated: diffusion of water and solutes during processing and storage

of fish ; the water binding capacity of meat ; relationships between composition, texture, microbial growth and a in cheese ; prediction

of a in confectionery and sugar products w w

IX The freezing behaviour of water is a matter of prime importance

in a meeting like ISOPOW : the behaviour of water at low temperatures

is generally illuminating with regard to the state of water in the system considered ; a better knowledge of this behaviour will also permit optimi-zation of the freezing process for the preservation of foods and also for the less well-known but interesting technique of freeze-texturing Finally, the important practical problems of moisture determination was approached with a comparison of sensors for the measurement of air humidity and a presentation of the results obtained in the course

of a collaborative European project (COST 90) aimed at developing a standard method of isotherm determination

Water Relations of Foods, 1975, R.B Duckworth, ed., Acad Press,

716 p

2 International Symposium on the Properties of Water

3 Water Activity : Influences on Food Quality, 1981 L.B Rockland and G.F Steward, eds., Acad Press, 921 p

Acknow ledgments

The symposium was held under the auspices of the International Union

of Food Science and Technology (IUFoST) and of the Commission tionale des Industries Alimentaires It was allocated a grant from NATO,

Interna-as an Advanced Research Workshop

Financial support was received from the following French agencies: Ministere de l'Education Nationale, Ministere de l'Industrie et de la Recherche, Direction des Industries Alimentaires et Agricoles, Institut National de la Recherche Agronomique, Conseil Regional de Bourgogne The following companies also financially contributed to the organization

of, ISOPOW III : Allied Grocery Products, Arnott's Biscuits Pty Ltd,

¥s'~ Boake Allen Australia Ltd, Cadbury Schweppes Pty Ltd, Cottee's Gener~l Foods Ltd, CSR Ltd, Sugar Division, H.J Heinz Co Australia Ltd, <Nestle Australia Ltd, Quaker Products Australia Ltd (Australia) Biscuiterie Nantaise, Bridel, B.S.N Gervais Danone, Lesieur-Cotelle, Mars Alimentaire, Rowntree-Mackintosh S.A., Royal Canin, SOPAD-Nestle, Societe des Produits du MaYs (France) Maizena Gesellschaft mbH (Germany) Ajinomoto Co., Ajinomoto-General Foods Co., Coca Cola Japan, Eizai Co., Fuji Seiyu Co., Hasegawa Koryo Co., Kagome Co., Kaneegafuchi Kagaku Kogyo Co., Kikkoman Co., Kirin Beer Co., Knorr

x

Shokuhin Co., Kureha Kagaku Kogyo Co., Lion Co., Meiji Nyugyo Co., Meiji Seika Co., Morinaga Seika Co., NIhon Reizo Co., Nisshin Seifun Co., Shiseido Co., Takara Shuzo Co., Takeda Yakuhin Kogyo Co., Toyo Seikan Co., Yamasu Shoyu Co., Yukikrushi Nyugyo Co (Japan) C.P.e Nederland B.V (Nederland) Cadbury Schweppes, Devro, Finlay, Londreco, Mars, Rank Hovis, Unilever, United Biscuits (United Kingdom) Beckman Instruments, Bristol-Myers, General Mills Inc., Gerber Foods Inc., Hershey Foods Corp., ITT Continental Baking Co., e.H Knorr, Lipton, The Pillsbury Company, Mc Cormick and Company Inc, Proctor and Gamble (U.S.A.) The editors wish to acknowledge the kind and efficient efforts in organizing and promoting ISOPOW III of L.B Rockland, President of the Permanent Executive Committee of ISOPOW-IUFoST and of all the members of the ISOPOW III Coordinating Committee : S Arai, J.e Cheftel, B Colas, J Dardenne, J.F Diehl, R.B Duckworth, M Fujimaki,

S Gal, A.e Hersom, M Karel, J Kefford, T.P Labuza, G Lavoue,

M Le Meste, D Lorient, F Leistner, e van den Berg, E von Sydow The editors also express their thanks to all who have contributed

to the quality of this book : authors for careful preparation of their manuscripts, members of the editorial committee for pertinent review

of the submitted papers The efforts of B Colas, M Le Meste and M.L Bettenfeld, who have kindly collaborated in the final editing work are gratefully acknowledged, as are those of N Desbois for secretarial services and for the typing of the whole proceedings

Other Participants in the Symposium

Session 1 Fundamentals: high moisture systems

Water and aqueous solutions : recent advances

F Franks

Phase separation in protein - water systems and the formation

XI

V VII

IX

XV XIX

M.P Tombs

The dipalmitoylphosphatidylcholine (DPPC) - water system 37

R Perron

An enzymatically· modified protein as a new surfactant

and its function to interact with water and oil in an emulsion

system

S Arai and M Watanabe

Session 2 Fundamentals : low and intermediate

moisture systems

The influence of soluble components on water sorption

hyste-49

K.A Johnston and R.B Duckworth

Some facts concerning water vapour sorption hysteresis

H Bizot, A Buleon, N Mouhous-Riou and J.L Multon

Influence of temperature on sorption equilibria 95

H Weisser

Development of B.E.T.-like models for sorption of water

C van den Berg

Solution thermodynamics and the starch-water system 133

M Le Maguer

XII

Session 3 Physico-chemical and enzymatic changes

Effects of water activity and water content on mobility

of food components, and their effects on phase transitions

M Karel

Enzyme activity as a function of water activity 171

R Drapron

The influence of water content and temperature on the

formation of Maillard reaction intermediates during the

K Eichner, R Laible and W Wolf

Polysaccharide-water interactions thermal behavior of

T.J Maurice, L Slade, R.R Sirett and C.M Page

Session 4 Microbiology and water activity

Present state of knowledge of a effects on microorganisms 229

D Richard-Molard, L Lesage and b "';ahagnier

The antimicrobial activity of sugar against pathogens of

S Selwyn and J Durodie

Hurdle technology applied to meat products of the shelf

stable product and intermediate moisture food types 309

L Leistner

Session 5 Technology : diffusion properties and

non equilibrium states Fundamentals of diffusion of water and rate of approach

T Roth and fK Loncin

Diffusivity of sorbic acid in food gels at high and intermediate

S Guilbert, A Giannakopoulos and J.C Cheftel

XIII

Aroma diffusion : the influence of water activity and of

A Voilley and M Le Meste

Choice of packages for foods with specific consideration

C Mannheim and N Passy

The role of water in the rheology of hygroscopic food powders 393

M Peleg

Session 6 Technology: humectants and new intermediate

moisture foods (IMF)

A pragmatic approach to the development of new

Polyglycerols and poly glycerol esters as potential water

activity reducing agents Chemistry and sensory analysis 481

J.G Kapsalis, D.H Ball, D.M Alabran and A.V Cardello

Session 7 Freezing and low temperature phenomena

Complex aqueous systems at subzero temperatures 1j.97

F Franks

The freezing of biological cells in aqueous solutions

C Korber, K Wollhover and M.W Scheiwe

Freezing in polymer - water systems and properties of

Computed instrumental analysis of the behavior of water

N Nagashima and E Suzuki

XIV

Session 8 Commodities : properties of water and technology

Water in fish: its effects on quality and processing 573

M Kent

The effect of water on the quality of meat and meat products:

xv

Contributors

Alabran D.M., U.S Army Natick Research and Development Center,

Natick, Massachusetts 01760, U.S.A

Arai S., Department of Agricultural Chemistry, The University of

Tokyo, Bunkyo-ku, Tokyo 113, Japan

Ball D.H., U.S Army Natick Research and Development Center, Natick,

Massachusetts 01760, U.S.A

Van den Berg c., Department of Food Science, Section Food and neering, Agricultural University Wageningen, Netherlands

Bioengi-Bizot H., Laboratoire de Stockage et Conservation des Denrees

Alimen-taires, I.N.R.A., Centre de Recherches de Nantes, Rue de la

Geraudiere, 44072 Nantes Cedex, France

Blond G., Laboratoire de Biologie Physico-Chimique, Ecole Nationale

Super ieure de Biologie Appliquee a la Nutrition et a I I Alimentation, Universite de Dijon, France

Brimelow C.J.B., Londreco Limited, Hayes, Middlesex, United Kingdom Buleon A., Laboratoire de Stockage et Conservation des Denrees Alimen-

taires, I.N.R.A., Centre de Recherches de Nantes, Rue de la

Geraudiere, 44072 Nantes Cedex, France

Bussiere G., Roquette, 62136 Lestrem, France

Cahagnier B., Laboratoire de Stockage et Conservation des Denrees

Alimentaires, I.N.R.A., Centre de Recherches de Nantes, Rue de

la Geraudiere, 44072 Nantes Cedex, France

Cardello A.V., U.S Army Natick Research and Development Center,

Natick, Massachusetts 01760, U.S.A

Cheftel J.C., Laboratoire de Biochimie et Technologie Alimentaires,

Universite des Sciences et Techniques, 34060 Montpellier, France

Drapron R., Station de Technologie Alimentaire, I.N.R.A., 1 avo des

Olympiades, 91305 Massy, France

Duckworth R.B., Division of Food Science, Department of Bioscience

and Biotechnology, University of Strathclyde, Glasgow, Scotland

Durodie J., Department of Medical Microbiology, Westminster Medical

School, University of London, Horseferry Road, London S W 1 P 2AR, United Kingdom

Hamm R., Federal Center for Meat Research, E.C Baumann Strasse

20, D 8650 Kulmbach, Fed Rep of Germany

Jakobsen M., Alfred Jorgensen Laboratory for Fermentation Ltd., Frydendalsvej 30, DK 1809 Copenhagen V, Denmark

Johnston K.A., Division of Food Science, Department of Bioscience and Biotechnology, University of Strathclyde, Glasgow, Scotland, United Kingdom

Jung G., Federal Research Centre for Nutrition, Karlsruhe, Fed Rep

Kent M., Torry Research Station, 135 Abbey Road, Aberdeen, Scotland Kervinen R., Department of Chemistry, Laboratory of Biochemistry and Food Technology, Helsinki University of Technology, 02150 Espoo 15, Finland

Korber C., Helmholtz-Institut fur Biomedizinische Technik an der RWTH Aachen, Goethestr 27-29, D 1500 Aachen, Fed Rep of Germany

Labuza T.P., Department of Food Science and Nutrition, University

of Minnesota, Saint Paul, Minnesota 55108, U.S.A

Laible R., Fraunhofer-Institut fUr Lebensmitteltechnologie und packung, Schragenhofstrasse 35, D 8000 Munchen 50, Fed Rep

Ver-of Germany

Ledward D.A., Department of Applied Biochemistry and Food Science, School of Agriculture, University of Nottingham, Sutton Bonington, Loughborough, LE12 5RD, United Kingdom

XVII Leistner L., Federal Centre for Meat Research, 8650 Kulmbach, Fed Rep

Lesage L., Laboratoire de Stockage et Conservation des Denrees taires, I.N.R.A., Centre de Recherches de Nantes, Rue de la

Alimen-Geraudiere, 44072 Nantes Cedex, France

LiHford P.J., Unilever Central Resources, Colworth House Laboratory, Sharnbrook, Bedford, MK44 lLQ, United Kingdom

Linko P., Department of Chemistry, Laboratory of Biochemistry, and Food Technology, University of Technology, 02150 Espoo 15, Finland Loncin M., Department of Food Engineering of the University, D 7500 Karlsruhe, Fed Rep of Germany

Mannheim C., Department of Food Engineering and Biotechnology, Technion, Israel Institute of Technology, Haifa 32000, Israel

Maurice T.J., General Foods Inc., Cobourg, Ontario, Canada

Mouhous-Riou N., Laboratoire de Stockage et Conservation des Denrees Alimentaires, I.N.R.A., Centre de Recherches de Nantes, Rue de

la Geraudiere, 44072 Nantes Cedex, France

Multon J.L., Laboratoire de Stockage et Conservation des Denrees Alimentaires, I.N.R.A., Centre de Recherches de Nantes, Rue de

la Geraudiere, 44072 Nantes Cedex, France

Nagashima N., Central Research Laboratories, Ajinomoto c., Inc Suzuki-cho, Kawasaki-ku, Kawasaki 210, Japan

Page C.M., General Foods Inc., Cobourg, Ontario, Canada

Passy N., Department of Food Engineering and Biotechnology, Technion, Isarel Institute of Technology, Haifa 32000, Israel

Peleg M., Department of Food Engineering, Agricultural Engineering Building, University of Massachusetts, Amherst MA 01003, U.S.A Perron R., C.N.R.S., 2 a 8 rue H Dunant, 94320 Thiais, France

Rautalinna E.K., Department of Chemistry, Laboratory of Biochemistry and Food Technology, University of Technology, 02150 Espoo 15, Finland

Richard-Molard D., Laboratoire de Stockage et Conservation des Denrees Alimentaires, I.N.R.A., Centre de Recherches de Nantes, Rue de

la Geraudiere, 44072 Nantes Cedex, France

Roth T., Department of Food Engineering of the University, D 7500 Karlsruhe, Fed Rep of Germany

Serpelloni M., Roquette, 62136 Lestrem, France

Sirett R.R., General Foods Inc., Cobourg, Ontario, Canada

Slade L., General Foods Corp., Tarrytown, N.Y., U.S.A

Suzuki E., Central Research Laboratories, Ajinomoto Co., Inc cho, Kawasaki-ku, Kawasaki 210, Japan

Suzuki-Spiess W.E.L., Federal Research Centre for Nutrition, Karlsruhe, Fed Rep

of Germany

Tombs M.P., Unilever Research Laboratory, Sharnbrook, United Kingdom Troller J.A., Procter and Gamble Co., Cincinnati, Ohio 1+5222, U.S.A Vainonpaa J., Food Research Laboratory, Technical Research Centre

of Finland, 02150 ESPOO 15, Finland

Voilley A., Laboratoire de Biologie Physico-Chimique, Ecole Nationale Superieure de Biologie Appliquee a la Nutrition et a l'Alimentation, Universite de Dijon, France

Watanabe M., Department of Agricultural Chemistry, University of Tokyo, Bunkyo-ku, Tokyo 113, Japan

Weisser H., Institute of Food Process Engineering, Kaiserstrasse 12,

D 7500 Karlsruhe 1, Fed Rep of Germany

Wolf W., Bundesforschungsanstalt fUr Ernahrung, Engesser Strasse

20, D 7500 Karlsruhe 1, Fed Rep of Geq:nany

Wollhover K., Helmholtz-Institut fUr Biomedizinische Technik an der RWTH Aachen, Goethestr 27-29, D 1500 Aachen, Fed Rep of Germany

XIX

Other participants in the symposium

Beltzung P., Merck - Chimie, 5, rue Anquetil, 94-130 Nogent S/Marne, France

Bigalli G., Hershey Foods Co., 1025 Reese Avenue, B.P 805, Hershey

Bousset J., Laboratoire de Recherches sur la Viande, INRA,

78350 Jouy en Josas, France

Buckle K., School of Food Technology, University of New South Wales, P.O Box 1, Kensington, NS W 2033, Australia

Bureau Go, A.D.R.I.A.C., Universite de Reims, B.P 34-7,

51062 Reims Cedex, France

Buyukkoksal Go, Marmara Research Institute, P.O Box 21, Gebze, Kocaeli, Turkey

Catherin Co, Mars Alimentaire, Route de Bitche, 67500 Haguenau,

France

Caurie Mo, Federal Polytechnic, P.O Box 50, Haro, Nigeria

Chicherio A.E., Novasina AG, Thurgauerstr 50, 8050 Zurich, Suisse Christian J., Division of Food Research C.S.I.R.O., B.P 52,

2113 North Ryde N.S.W., Australia

Colas Bo, Laboratoire de Biologie Physico-Chimique, ENS.BANA,

Universite de Dijon, 21100 Dijon, France

Combes Do, Departement de Genie Biochimique, I.N.S.A., Avenue

de Rangueil, 31077 Toulouse Cedex, France

xx

Condliffe W.F., H.J Heinz Co Ltd, Hayes Park, Hayes,

Midd UB4 8AL, United Kingdom

Corry J., Ministry of Agriculture Fisheries and Food,

65 Romney Stret, London 5 WIP 3 RD, United Kingdom

Cowley K.M., United Biscuit, St Peter's Road, Furze platt,

Maidenhead, Berkshire SL6 7QU, United Kingdom

Dame M., Laboratoire Soredab, La Boissiere Ecole, 78120 Rambouillet, France

Daniau G., Vandamme - Pie qui Chante, 300 rue Clemenceau,

59139 Wattignies, France

Davidson P., Hygrodynamics Inc., 949 Selim road, Sdverspring,

'MD 20910, U.S.A

Demeaux M., Laboratoire de Biochimie Alimentaire, ENS.BANA,

Universite de Dijon, 21100 Dijon, France

Qeveau J., Compagnie des Salins du Midi et des Salines de l'Est,

24 route d'Aulnay, 93140 Bondy, France

Ericsson B., Department of Food Technology, University of Lund, B.P 740, 5 - 220 07 Lund, Sweden

Gal S., Haco Ltd, 3073 Gumlingen, Suisse

GaugazM., Societe d' Assistance Technique pour les Produits Nestle S.A., B.P 88, 1814 La Tour de Peilz, Suisse

Gellf G., Universite de Technologie de Compiegne, B.P 233,

60206 Compiegne Cedex, France

Getchell R., MC Cormick & Co., 202 Wight Avenue, Hunt Valley,

Glittenberg D., MaTzena G.mbH, 1 Knorrstrasse, B.P 2760,

o 7100 Heilbronn, Fed Rep of Germany

Harding W., Mars LTD, Dundee Rd., Slough SLl 4JX, United Kingdom Henry M., C.N.R.S., 2 rue Henry Dunant, 94320 Thiais, France

Hermansson A.M., SIK, The Swedish Food Institute, B.P 5401,

XXI Kimizuka A., Central Research Laboratories - Ajinomoto Co., Inc 1-1 Suzuki-cho - 210 Kawasaki-ku, Kawasaki, Japan

Kirk J., Campbell Soup Company, Camden, New Jersey, U.S.A

Klaarenbeek T., Smiths Food Group bv, Westelijke Randweg 5,

1721 CH Broek op Langedijk, Netherland

Lacout J.M., Rowntree Mackintosh, Rue de Cluj, B.P 30,

21019 Dijon Cedex, France

Lang K., General Foods Research Department, 520 William Street, Cobourg, Ontario K9A 4L4, Canada

Lavoue G, Laboratoire de Biochimie Alimentaire, ENS.BANA, Universite

de Dijon, 21100 Dijon, France

Leblanc M., Societe Jules Richard & Pekly, 116 Quai de Bezons,

95102 Argenteuil, France

Legenhausen R.F., Beckman Instruments, 89 Commerce Road,

Cedar Grove, NJ 07009, U.S.A

Le Meste M., Laboratoire de Biologie Physico-Chimique, ENS.BANA, Universite de Dijon, 21100 DIJON, France

Le Mestre G., Rutter' Instrumentation, 126 rue du General Leclerc,

94360 Bry S/Marne, France

Lerici C.R., University of Bologna, 7 via S Giacomo, 40126 Bologna, Italia

Loisel c., E.N.I.T.I.A.A., rue de la Geraudiere, 44072 Nantes Cedex, France

Lorient D., Laboratoire de Biochimie Alimentaire, ENS.BANA, Universite

de Dijon, 21100 Dijon, France

Makishima S., Meiji Seika Co., 580 Horikawacho, Saiwaiku 210,

Kawasaki, Japan

Masteil M., Institut Scientifique et Technique des Peches Maritimes, B.P 1049, 44034 Nantes Cedex, France

Mathlouthi M., I.U.T., Universite de Dijon, B.P 510,

21014 Dijon Cedex, France

Motarjemy Y., Lunds University, B.P 50, 230 53 Alnarp, Sweden

Musso F., Francereco, 3 rue Charles Tellier, 60000 Beauvais, France Nagashima N., Central Research Laboratory, Ajinomoto Co.,

Suzuki-cho, Kawasaki-ku, Kawasaki, Japan

Ollivon M., C.N.R.S., 2 rue Henry Dunant, 94320 Thiais, France

Pascat B., A.D.R.I.A.C., Universite de Reims, B.P 347,

51062 Reims Cedex, France

Pelaez J., Mc Cormick & Co., 202 Wight Avenue, Hunt Valley,

Maryland 21031, U.S.A

Rieunier F., Biscuiterie Nantaise, rue Lotz-Cosse, B.P 5X,

44040 Nantes Cedex, France

Rockland L., Food Science Research Center, Chapman College,

333 GLassel Street, Orange CA 92666, U.S.A

Slinde E., Norvegian Food Research Institute, As - NLH N 1432,

Norway

Switka J., Department of Catering, Merchant Navy Academy,

Czerwonich Kosynierow, Gdynia 81-225, Poland

Thibeaudeau P., Royal Canin, B.P 234, 56006 Vannes Cedex, France

White C., Campden Food Preservation Research Assoc., Chipping

Campden, Gloucestershire GL55 6LD, United Kingdom

Yigit V., Marmara Research Institute, PO Box 21, Gebze, Kocaeli, Turkey

WAT ER AND AQUEOUS SOLU TI ONS RECENT ADVA NCES

F Franks

The past five years have wi tnessed some s i gnificant advances in t he physics , phys i cal chemistry and biochemis t ry of water and processes involving water They extend from the es timat ions of re l iable poten-tial func t ions to descr ibe water- water interactions t o the basic mechanisms tha t underlie the phenomena associated wi t h the sur vival

of l i ving organisms under conditions of extreme physiological water

s tress This review can only provide keywords and signposts which the i nt eres t ed r eade r should follow by consulting recent review publ i cations cited at t he end of t his chapter

I shall deal with the various topics in increasing order of conce tual comp l exi t y This is not to say t hat concep t ual simplicity goes hand in hand with s i mplicity in execu t ion Conceptually nothing could

p-be much simpler or mo r e impor t ant than t he calculat i on of the action energy between two isolated water molecules ; this is however

inter-a tinter-ask which s ti ll confounds theoreticiinter-ans The problems thinter-at inter-a r ise

if a thi r d molecule is included in such calculations have yet to be solved On the other hand , the appl i cations of advanced diffract i on

t ec hniques have led to the deta i led desc r iption of conceptually more complex systems , such as the hydration shell of an ion in solution

or t he distribution of wa t e r molecu l es surrounding native pr~ins

2, THE INTERACTIONS BETWEEN TW O ISO L.,b.TE D WATER ~iO L E C U LE S

AS A BASIS FOR LIQ UI D S TATE CALCULAT I ONS

The molecular pa i r potential f unc tion Uij(r , Q) i s probably the most fundamental quantity required fo r the development of a molecular

D Simalos and J.L Multon (editors), Properties of Waler in Foods_ ISBN 97(H4·01()'8756-8

© 1985, Martinus Nijhoff Publishers, Dordracht

2 F Franks

description of a liquid Here Uij describes the potential energy

between interacting molecules i and j as function of the distance of separation r and the angles describing the mutual orientation of the two molecules, D In the case of water the calculation of Uij is made difficult because of : 1) the almost tetrahedrally disposed charges

on the H20 molecule and 2) uncertainty about the exact description

of the water-water hydrogen bond Even if the interaction energy between two molecules could be calculated exactly, the question must arise whether the macroscopic properties of a liquid can be obtained

by the pairwise addition of these energies, or whether effects due

to cooperativity (interactions between 3 or more molecules) need to

be taken into account

Several recent computer simulation approaches are on record for the calculation of Uij The much used ST2 is a pairwise additive potential based on the water molecules as regular tetrahedra with four point charges placed at the vertices It was optimized against the known properties of liquid water and it is therefore an "effec-tive" pair potential Clementi et al devised a pair potential (CL) based on high level quantum mechanical calculations and using a three point charge model for H20 More recently Finney and his colleagues have attempted to include cooperativity effects by introducing a dipole polarizability term into their calculations which are not based on the assumption of molecular tetrahedrality

A comparison of the three models against the experimental ties of water shows up their respective advantages and shortcomings For instance, Figure I is such a comparison for the temperature

proper-dependency of the 2nd virial coefficient, B(T) of water vapour

Essentially B(T) measures the deviation from ideal gas behaviour As expected, ST2, being a model optimized for the liquid state, fails, whilst F performs well Testing the water models against the measured radial distribution functions, g(r) as obtained from X-ray and neutron scattering by liquid water, allows detailed analyses to be made Thus, X-ray data refer mainly to oxygen atom distributions, goo(r), while for neutrons the scattering cross sections for 0 and D are of the same order of magnitude Information on local molecular orienta-tions is therefore obtainable It seems that none of the models can closely match the details of the various g(r) functions

The consistency of the three models can also be explored by

calculations of the potential energy at constant distance of tion of the oxygen atoms, but at varying orientations Effectively this corresponds to the distortion of the hydrogen bond by rotating one of the two molecules Figure 2 shows such a potential energy diagram ; position A is the equilibrium configuration that exists in ice-I By performing a trans-cis transformation, we arrive at position

separa-B where the resultant molecular dipole are aligned It is seen that the ST2 and F models indicate double minima, but the CL model only exhibits a broad minimum Major discrepancies between the two models

Water and aqueous solutions

ST2

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also arise in the repulsive part of the interaction, corresponding

to H-H crowding along the 0-0 line of centres Here the ST2 and F water models appear to predict insufficient repulsion (compared to the best quantum mechanical calculations) whereas the CL model is too repulsive by approximately 50 kJ mol- i (N.B kT = 2 kJ mol-i)

3

At the second level of approximation the structure of liquid water

lS now understood in terms of a more or less randomly hydrogen bonded network, but this level is inadequate to account for some of the subtle water mediated biological effects, e.g the detailed role of

hydration in maintaining macromolecular native states To make further progress we require better molecular potential functions and it is likely that these will require an improved treatment of cooperative effects as well as a more realistic model of the H20 molecule itself

of electrolytes and in the interactions of (hydrated) ions with macromolecules

Until recently the behaviour of the ion lin solution could only be treated by assigning to the solvent various averaged bulk properties, such as a dielectric permittivity or a viscosity This type of

approach formed the basis of the Born equation which describes the

Water and aqueous solutions 5 free energy of transferring an ion from the gas phase to an infinitely dilute solution only in terms of some notional ionic radius, the ionic charge and the permittivity of the solvent The Born approach is able

to account for aqueous solutions only when some quite unrealistic correction terms are added Similarly, the Debye-Huckel theory of ionic interactions and the Onsager-Fuoss theory of ion conductance treat the solvent as a continuum whose only properties are a bulk permittivity or viscosity

The advent of neutron diffraction has made possible the detailed study of the "structure" of at least the primary ion hydration shell Its advantages over the use of X-ray diffraction are that : 1) the scattering length depends on the isotopic state of the scattering nucleus and 2) the total radial distribution function G(r), obtained from the experimental scattering cross section, is the linear sum of atomic pair distribution functions gij (r) For an aqueous solution

of a simple electrolyte M+X-, G(r) is then the sum of ten terms, each

describing a particular gij (r) The ]0 i-j pairs are shown in Table

I For technical reasons D20 is used as solvent in preference to H20

In principle the ten individual gij (r) functions can be isolated from the total scattering curve, but In practice this is not a useful procedure because : 1) up to 90 % of the total scattering is due to D20 and 2) the deconvolution of the total scattering curve into ten contributing curves is subject to much uncertainty It is here that the advantage of neutron methods becomes apparent By using difference scattering techniques it is possible to reduce the number of gij(r) terms from ten to four This is achieved by performing the experiment with two isotopes of M+ (or X-) and taking differences All the gij (r)

6 UVlCUOVi.J.J that cOVl~bute to the e.xp~me.Vltaf J., Catt~Vlg LVlte.Vi.J.J.{;tlj

Atom-atom pair First order difference

distribution function Substitution Substitution

+

6 F Franks

terms that do not contain M+ (or X-) cancel out (see Table I), making the deconvolution of G(r) much less uncertain Hydration is described

by gM-O(r) and gM-D(r), whereas gM-M(r) and gM-X(r) provide details

of the ionic distribution The latter may be even better defined by

a second order difference experiment in which double isotope ments of M+ and X- are combined, with the elimination of the hydration contributions

experi-The results so far available indicate that Ni Z+, CaZ+, Na+ and K+ all possess hydration shells in which the ° atoms are disposed

octahedrally about the cation, but that the DZO resultant molecular dipole points in a direction that is not collinear with M+-O, but inclined at an angle that appears to decrease to 0° at infinite

dilution

Similarly, the CI- ion hydration shell in aqueous NaCI and CaClz consists of six DZO molecules disposed octahedrally but asymmetrically about the anion, as shown in Figure 3

The neutron diffraction experiments performed so far have led to the following interesting results :

I Monatomic ions (with the exception of Li+) possess the same primary hydration shell, although the structural detail gets less well defined when the ionic radius increases

Z Even at quite high concentrations the ions do not appear to penetrate one another's hydration shells

3 In the case of NiCIZ, Ni-Ni correlations have been identified, but only at separations in excess of 0.5 nm The gCI-CI(r) function

is structureless, so that ion ordering does not appear to take place

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Water and aqueous solutions 7 even at a concentration of 4.4 m

Neutron diffraction is also being applied to more detailed tural investigations of water itself In line with the above

struc-discussion of ion hydration, the total scattering function provides

a G(r) which is the linear sum of go-o(r), gO-D(r) and gD-D(r), the first of which is almost directly comparable to the X-ray radial distribution function Preliminary neutron results suggest that the computer simulation models of the water molecule overestimate the tetrahedral order in liquid water, but also fail to give sufficient structure at longer range, typically in the region of 0.3 - 0.5 nm

As is to be expected, temperature and pressure have a profound effect

on the spatial and orientational molecular order in the liquid

FUNCTIONING OF PROTEINS

During the past decade the view that water plays a vital role in the manifold functions of proteins has gained general acceptance, but the details of this involvement are still unclear Under ~n v~vo conditions proteins fulfil well defined functions, e.g enzymes, hormones, transport vehicles, defence The functions which are of interest to the food processor are of a different nature and require

a partial or complete reconstruction of the native state of the

protein, in order to render it capable of gelation, surface activity, fibre forming, etc If we have made some headway in elucidating the role played by water in the ~n v~vo functioning of proteins, then the same cannot be claimed for the technological functions

The stability of a native protein relative to some unfolded,

denatured state is highly marginal ; it rarely exceeds the sum total energy of three or four hydrogen bonds, whereas the folded structure contains hundreds of such bonds This indicates that the net free energy of stabilization is made up'of several contributions, some of which have a net destabilizing effect Several of these contributions involve solvation interactions

It is generally held that the knowledge of structure is a good starting point for speculations regarding function Most protein crystals contain upwards of 50 % water which suggests that the packing

of the protein molecules in the crystal are sensitive to their

hydration environment Recent improvements in X-ray and neutron

scattering have made possible the identification of discrete water molecules within protein structures, albeit in the crystalline state

It has thus been possible to assign spatial coordinates to water molecules with a reasonable degree of confidence Here again it must

be borne in mind that X-ray data usually provide information only about the position of oxygen atoms, although some recent very high resolution experiments have actually yielded water hydrogen positions

8 F Franks

~n crystals of some small ~lobular proteins The level of resolution

is of particular importance because water molecules, when in protein crystals, have diffusional freedom, even to the extent that molecular exchange can take place

The proteins for which water coordinates are available number approximately fifty, sufficient for definite water locations to be identified Thus, water is often present as a ligand of a metal ion, e.g water molecules occupy one site in the Zn 2+ coordination shells

of carbonic anhydrase, carboxypeptidase-A and horse liver alcohol dehydrogenase, and two sites of the Mn++ coordination shell in

concanavalin-A Many other similar metal-water ligands have been suggested on the basis of N.M.R studies and the possible involvement

of water in the catalytic roles of the enzymes has been discussed The majority of charged amino acid residues are usually found on the periphery of the protein, exposed to the solvent, and the few charges residues located in the interior form salt bridges with oppositly charged groups On a few occasions, however, unpaired charged residues exist in the centre of-the protein; these are usually hydrated Sometimes water mediated salt bridges are found,

as shown in Figure 4 for papain, where the salt bridge is linked to

a water network which provides an efficient proton transfer chain to the exterior solvent medium

As might be expected, most of those water molecules that can be located by diffraction methods are found linking main chain >c=o

groups to N-H groups which are too well separated from one another for direct hydrogen bonds Many diverse water arrangements have been identified linking main chain polar groups ; sometimes single water molecules may be hydrogen bonded to three or four different residues

In other cases the hydrogen bond bridge between distant polar groups may consist of two or even three water molecules While the predomi-nant hydrogen bond lengths lie between 0.20 and 0.30 nm (as they also do in liquid water), peptide-water bridges lying outside this

F ~gUJLe 4 A wa.:tVl me.cU a.:te.d Mli bJUdg I' ~n pap~n

po~~~o~ anI' ~hown Adap.:te.d 6~om BVl.e.n~e.n (5) only oxyge.n

Water and aqueous solutions

hgUJ1 e 5 A wa.teJt blLidge UYlk.iYlg cU.otant peptide boruu ~Yl

a-ehymotJtyp~~Yl Adapted nJtom F~YlYley (6)

9

range have also been reported An analysis of the water molecule distributions in proteins shows a large variety of geometrical

arrangements with severe.1y distorted hydrogen bonds Apart from

linking main chain polar groups, water molecules are also found to link main chain atoms to side chain hydrogen bond donors or acceptors, e.g in phe, ser, met, tyro

Water molecules interacting with polar groups nearer the periphery

of a protein tend to be less well localized, although a first tion shell can often be identified in which water forms bridges

hydra-between adjacent exposed residues Of course these adjacent residues are not necessarily consecutive in the amino acid sequence For

instance, in a-chymotrypsin a water molecule forms two links each with val 3 and ser 119, as shown in Figure 5

One important function of water appears to be in the stabilization

of reverse S-turns, i.e in regions where there are few intrapeptide hydrogen bonds Despite such hydration, the regions of the reverse S-turns still possess a considerable degree of diffusional freedom

So far the expected clathrate type hydration of exposed apolar residues has not been detected, but this may well be due to a lack

of spatial localization of water molecules Computer simulation

studies of the hydration of apolar molecules suggest that water

molecules can form a large variety of cavity hydration structures which are energetically almost equivalent, so that their detection

by scattering methods would be almost impossible On the other hand,

"ordered" water is often found in the hydrophobic clefts of enzymes and at substrate binding sites Such ordering is of a spatial nature only, because the water molecules involved can exchange rapidly with the external solvent

Finally, water also forms well defined bridges between amino acid residues belonging to different protein molecules in a cDystal, or

to different subunits belonging to the same protein Figure 6 shows the water arrangement between two molecules of a-chymotrypsin, indi-cating how the symmetry that exists between the protein molecules is

'-' t2-COVld hydltauon '-' ht2-li, '-'~VlCt2- :tht2-y on!y havt2- wcttt2-lt VLt2-Mt2-'-':t

maintained in the water network Refinements of multisubunit tures have also led to the identification of many new water mediated intersubunit hydrogen bond bridges which enhance the overall stability

struc-of the oligomer Thus, by including solvent positions in the ment of the alSI haemoglobin contacts, the number of possible hydrogen bonds is found to increase from 5 to 17 or 19

refine-The above summary of solvent involvement in the stabilization of specific conformation of complex macromolecules emphasizes how

marginal such stability is and how easily it is perturbed by what appear to be minor changes in the nature of the solvent One must conclude that theoretical calculations on protein folding/unfolding mechanisms that do not take account of the solvent contributions are,

at best, academically stimulating exercises The very fact that the net free energy of stabilisation of a native protein is equivalent

to no more than 3-5 hydrogen bonds clearly demonstrates that the solvent plays a central, rather than a peripheral role in protein functions

The condensation of a vapour or the crystallization of a liquid

Water and aqueous solutions 11 requires a nucleation event, otherwise the original mother phase will persist as metastable state below the equilibrium condensation point

or freezing point respectively A nucleus capable of initiating the growth of a daughter phase results from a random density fluctuation which brings together enough molecules in the right cluster configu-ration to resemble the molecular arrangement in the daughter phase The probability of nucleation increases with decreasing temperature and approaches unity (for a given volume) at the so-called nucleation temperature, Tn'

The physical properties of water, from its equilibrium freezing point, Tf , to Tn have recently been investigated in detail Iri the absence of crystallization, the properties do not exhibit disconti-nuities at Tf However, if the physical properties of liquid water

in the temperature range O-IOO°C are considered abnormal, then such abnormalities of the liquid become progressively more marked as the temperature approaches Tn' This is demonstrated for the density and specific heat, Figures 7 and 8 respectively It has been suggested that a singularity exists at -4SoC where many of the properties of water diverge This type of behaviour has been discussed in terms of

a physicial spinodal instability

Just as Tf of water is lowered by pressure or the presence of solutes, so Tn is affected in a similar fashion, as shown in Figures

9 and 10 Tn(P) follows Tf(P), with Tn(O) ~ -40°C With increasing pressure the triple point ice-I/ice-III/liquid is reached at -22°C, beyond which Tf increases with increasing pressure, a behaviour also reflected in Tn(P) This suggests that the lowest temperature to which pure water can be undercooled is approx -90°C (at 200 kPa) The effects of solutes on Tn can be represented in the form of a van't Hoff plot Although second order solute specific effects are apparent in Figure 10, the similarity between In x (l/Tf) and In x (l/Tn) is striking In principle, substantial degrees of undercooling can be achieved at high concentrations, provided that the solute does not crystallize but yields a supersaturated solution However, with increasing solute concentration and decreasing temperature the

viscosity of the solution can rise steeply and may reach and exceed the viscosity (IOISp) normally associated with the glass transition,

Tg, at which ice crystallization becomes very slow (measured in centuries per cm crystal growth) The supersaturated solution then exists in a glassy state, mechanically a solid, but a homogeneous mixture

Figure II summarises the measured diffusional properties of liquid water down to -90°C in the form of an Arrhenius plot It is evident that with the extended temperature range of almost 200°C (correspon-ding to ~(I/T) ~ 1.8xI0-3K-I) the viscosity and diffusion coefficient

of water cannot be fitted with a constant activation energy Indeed, using the normally quoted value for the activation energy, referred

(77)

to room temperature, leads to an error of almost two orders of

magnitude in T when extrapolated to -90°C

Studies of undercooled water and supersaturated aqueous solutions are of special importance in processes where ice crystallization and morphology are to be controlled and they may also turn out to be essential in the development of more advanced techniques of low

temperature preservation of labile materials These aspects of aqueous solutions are also of relevance to cold acclimatization, such as is

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SOLUTI ONS

During the past few years the study of the thermodynamic properties

of dilute aqueous solutions of non-electrolytes has revived, mainly because refined experimental methods for the direct and indirect

measurements of enthalpies, heat capacities and volumetric properties have become available, but also because developments in statistical thermodynamics and computer simulation have made possible the analysis

of interactions of solute molecules with one another and with water The measurements have been extended to ternary solutions (water + two solutes) of various types A rigorous thermodynamic treatment has been provided for experimental studies of multi-component systems, such as water + protein + salt + neutral solute (sugar), of importance

in the elucidation of solute effects on the conformational stability

Water and aqueous solutions 15

of the macromolecules

Most of the experimental studies are based on the interpretation

of thermodynamic excess properties, e.g GE, as a series expansion

in solute concentration

(1)

where m2 is the molal concentration of component 2 and the cients g22 and g222 represent the molecular pair and triplet inter-action free energies of solute molecules For the more general case

coeffi-of a ternary solution which contains solutes 2 and 3, Eq (1) takes the form

in Eq (2) which are related to the osmotic virial coefficients and hence to the potential of mean force between molecules, the most fundamental property of the molecular systems

Similar measurements of excess enthalpies, heat capacities, volumes and compressibilities of mixing provide a more complete picture of the interactions Table II is a summary of some results, taken from several sources The data have been selected to demonstrate certain important features of aqueous solutions containing different types

of molecules

The following generalizations demonstrate the subtleties and

diversities of aqueous solutions

I g23 < 0 indicates net attractive interactions between solutes molecules In the case of urea the attractions arise fram favourable (exothermic) enthalpic effects, presumably due to intersolute hydrogen bonding However, for alcohols, tetrahydrofuran and other types of hydrophobic solutes, the net attractions are due to a large positive TS23 term, indicative of hydration effects and hydrophobic interac-tions The negative g23 is then the resultant of two repulsions

rather than an intrinsic attraction

2 Subtle stereospecific effects are at work in solutions of

polyhydroxy compounds ; g23 appears to be positive, except for

inositol which has a cyclic configuration The enthalpy coefficients h23 are particularly sensitive to minor stereochemical changes, with

endothermic mixing as the rule, but as the number of -OH groups

increases, so h23 appears to become negative (exothermic mixing) Interactions between hydroxylic and hydrophobic solutes appear to

be dominated by the destructive interference of incompatible hydration shells Incidentally, this is also true for the interactions of

polyhydroxy compounds with globular proteins and probably lies at the basis of the stabilizing (salting-out) effect such compounds have on the native states of the proteins

So far few heat capacity interaction coefficients have been

determined experimentally, but they are likely to be more informative for comparison than h23 coefficients, since the latter relate to a single temperature only, usually 25°C Comparisons of different

solutes at one arbitrary temperature can be quite misleading Volume (density) measurements also tend to be made at a single temperature and the same arguments apply The temperature and pressure derivatives

of volume will eventually shed more light on the role of hydration shells in governing the interactions of molecules and ions in aqueous solutions

The concept of "bound" water figures largely in the biological literature and in many technologies and is commonly advanced to

Water and aqueous solutions 17

explain deviations from some predicted behaviour of aqueous systems Solute molecules or certain functional groups on solid substrates are assigned water binding properties which permit a certain number of water molecules to be bound so that they can be treated as distinguis-hable from water molecules in the bulk Distinctions are even made between strongly and weakly bound water, although quantitative

descriptions of strong and weak are rare and no indications are

provided as to the type of interaction that might provide a binding stronger than the hydrogen bond between two water molecules The net result of such hypothetical binding is then an apparent increase in the solute concentration or a reduction in the water activity The properties of bound water are said to differ from those of the normal aqueous medium, and it does not contribute to the colligative

behaviour of solutions and therefore does not exhibit the normal freezing characteristics of water ; in fact, it does not appear to freeze at all

It is commonly postulated that binding results from interactions between water and specific surface sites on polar solids, presumably those capable in participating in hydrogen bond formation On the other hand it has also been claimed that molecular solutes, in

particular polyhydroxy compounds, are able to bind water, and

stoichiometric hydration numbers have been assigned to a wide range

of molecules, e.g carbohydrates and am~no acids

Water binding is commonly treated in terms of equilibrium dynamics and chemical stoichiometry Such approaches can lead to chemical paradoxes regarding the stability/instability of such

thermo-hydrated solute species For instance, the analysis of the liquidus curve of water/ethylene glycol (EG) mixtures by postulating that deviations of the freezing point depression from the "ideal" value are due to an equilibrium of the type

leads to the unacceptable result that the concentration of the

dihydrate increases with increasing temperature and that the

equilibrium is shifted almost completely to the right as the rature approaches the boiling point of water !

tempe-Even more important is the fact that in low and intermediate moisture systems, water sorption is subject to marked hysteresis effects The treatment of such systems in terms of equilibrium

thermodynamics is quite inappropriate The calculation of binding constants from sorption data must be largely meaningless, bearing

in mind the defined properties of thermodynamics functions of state, such as G, H, Sand Cp and the assumptions which lie at the basis of the various equilibrium sorption models

One cannot deny that concentrated and supersaturated solutions,

18 F Franks

low moisture solids, aqueous slurries, undercooled liquids, and

hydrated amorphous solids are of great importance, or that the

quantitative study of such systems, experimentally as well as

theoretically, presents major problems However, science and logy are not served by facile approaches which gloss over the real factors involved, in favour of some notional quasi-chemical reaction

techno-of water with polar molecules

Fortunately the past few years have witnessed several useful

developments in studies of the properties of liquid water in confined spaces, in supersaturated solutions and of low moisture « 50 %

water) systems Such rigorous investigations are beginning to lay the foundations of a proper understanding of complex aqueous systems Israelachvili and his colleagues have produced powerful experimen-tal verification of hydration forces between surfaces, thus confirming the previous theoretical predictions by Ninham of the necessity of such structural forces which become important as the distance of separation between surfaces is reduced to ~ 5 nm The refined

experimental technique which combines the measurement of distance and force has been applied to a variety of solid surfaces and aqueous media Unlike the electrostatic double layer forces and van der Waals forces, the repulsive hydration forces decay exponentially and are observed only in the presence of cations (other than H+) adsorbed at negatively charged surfaces The decay length is of the order of

1 nm, giving the force an effective range of 3 nm Similar repulsive forces have been observed between phospholipid bilayers and can be inferred to exist in clay suspensions, silica dispersions and in stacked biological membrane systems (thylakoids)

Studies of the effects of sequential hydration on the internal dynamics and functioning of previously dried proteins have shed light

on hydration interactions in low moisture systems The results for lysozyme are summarized in Table III The estimated proportion of unfreezable water, 0.3 gig, appears to correlate well with the

complete monolayer coverage of all polar groups It is again necessary

to emphasize the dynamic nature of these interactions : even at very low levels of hydration, a substantial fraction of peptide protons are able to exchange with D20

Detailed N.M.R relaxation results on water sorbed on a wide

variety of substrates confirm the complex dynamics of water molecules under such conditions The water diffusion both parallel and perpen-dicular to the substrate surface, and the chemical exchange rates are markedly affected, as is also the rotational tumbling of water molecules Under the influence of the substrate surface such rotation tends to become anisotropic It is important, however, to emphasize that such perturbed diffusion is observed exclusively in low moisture systems As more water is added, eventually to yield a solution, so the dynamic properties of water become more "normal" It is inappro-

Water and aqueous solutions 19

Tabl~ III Ev~n~ a~~ompanying ~h~ ~eque~al hy~~on 06 ~y

ly~ozyme P.L Poole and J.L Finn~Yl unpub~hed d~a

acid residues saturated side chain polar group saturation

peptide NH saturation

saturation polar group monolayer coverage

apolar surface coverage 0.4 complete monolayer

coverage

Protein

proton redistribution, S-S ordering, residues assume normal pK values Protein loosens up Side chain/backbone conformational shifts

enzyme activity begins

enzyme activity increases

priate, therefore, to advance the notion of a hydration sphere of well defined dimensions within which water is in a distinguishably different state from that characteristic of water molecules in the bulk It is more helpful to treat low and intermediate moisture

systems, viscous supersaturated solutions and aqueous glasses as metastable stationary states in which the attainment of the true equilibrium state is inhibited by kinetic barriers Such an approach can account for the observed phenomena of hysteresis, unfreezable water, etc

It seems sensible to assume that any interactions involving water are of the hydrogen bonding type, since water is able to participate both as a bifunctional donor and acceptor Hydration interactions are of particular interest N.M.R on undercooled solutions is a powerful device for monitoring subtle differences between chemically very similar molecules and between -OH groups in different positions

on a given molecule With the aid of undercooled solutions it is possible to resolve hydroxyl proton shifts, even in H20 solutions They appear downfield from the solvent signal Line width measurements have made possible the determination of proton exchange rates and have revealed subtle stereochemical effects on exchange and diffu-sional dynamics of polyhydroxy compounds in solution The observed