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Water content, water activity, water structure and the stability of foodstuffs
Food Control 12 (2001) 409±417
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Water content, water activity, water structure and the stability of foodstus Mohamed Mathlouthi * Laboratoire de Chimie Physique Industrielle, Facult e des Sciences, Universit e de Reims Champagne-Ardenne, B.P. 1039±51687 Reims C edex 2, France Received 11 December 2000; received in revised form 7 March 2001; accepted 8 March 2001
Abstract Determination of water content, whatever the accuracy of the analytical method, is not suciently informative in relation to the stability of the investigated food product. Water activity aw brings a supplement of information as it accounts for the availability of water for degradation reactions. The understanding of why certain products are more stable than others at the same aw needs an elucidation of water structure. Of particular importance are the interactions (hydrophilic, hydrophobic) between water and the components of the foodstu and the eect of the soluble molecules of the food on the hydrogen bonding in solvent water. Studying water in foods should start with an anlytical determination of water content for commercial and legal reasons which are evident. This has to be completed with the measurement of the thermodynamic activity of water in the food. Such a parameter aw should hold an important place in the identi®cation of the food product, especially as regards its shelf life. A further step in unveiling the behaviour of water in foods consists in determining water molecules in the molecules in the studied food matrix. The tripartite (analytical, thermodynamical and structural) approach to water in foods will be examined based on examples of sugars and sugar rich products. Ó 2001 Elsevier Science Ltd. All rights reserved.
1. Introduction Water is omnipresent in foodstus and the surrounding atmosphere. Determination of water content is one of the most frequent analysis in the laboratory of a food industry. However, depending on the method of analysis, it is not the same type of moisture content, which is measured. Even the term ``moisture'' itself is questionable as this might include other liquids than water, wetting the product (Isengard, 1995). The most common method of water analysis usually accepted at a commercial level is the desiccation method. This method is based on the mass loss after drying of the sample. However, part of mass loss might originate from the volatility of other gases than water. Not only the desiccation or ``oven drying'' is criticisable, but also all methods of determination of water contents have their drawbacks. The sole value of ``water content'' in a food is not inform about the nature of water, if it is ``bound'' or
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``free'', ``inherent'' or ``occluded'', etc. The knowledge of each of these fractions is important as it helps in understanding the process which is at the origin of each fraction. Beside measuring water content in the foodstu, many laboratories are now equipped with devices which allow determination of water activity aw . However, depending on the technique of water activity measurement, the result may be dierent. It is necessary for a slow equilibrium between the product and air surrounding it that this equilibrium is reached. The obtaining of equilibrium is asymptotic. One has to inform about the time needed to reach such an equilibrium. Rapid measurement of aw using the dew point technique only gives the surface equilibrium relative humidity (ERH) of the product, which may not be sucient for shelf-life prediction for example. To account for the behaviour of a food product in the whole range of relative humidities to which it may be submitted during storage, there is a need of establishing the water vapour sorption isotherm, preferentially at storage temperature. The shape of sorption isotherm may change depending on the type of product and its anity for water. Brunauer, Emmett, and Teller (1938) have described ®ve
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types of water vapour sorption isotherms. Soluble foods like sugar adopt an asymptotic shape of isotherm when relative humidity tends towards 100%, and heterogeneous complex foods show a sigmoid curve. Analysis of the shape of sorption isotherms shows that they may be divided into three zones, respectively, corresponding to monolayer strongly bound water, linear region of less rigidly and capillary adsorbed water and the third region of solvent or free water. This description of the three species of water is a rough accounting of the dierences in the structure of water. Indeed, equilibrium of hydration of a food generally described by water activity aw depends on the structure of food components, their eect on the solvent (water) and other properties like the surface activity of the product. Direct analysis of the structure of water in a food system is dicult to achieve. Utmost, one can have an insight into ``free'' and ``bound'' water from NMR or other techniques like thermal analysis of frozen systems. Amid the water properties that may be used for the elucidation of its structure, the behaviour as a solvent and the interactions with dierent classes of molecules are of great importance. 2. Water content Determination of water content in foodstus can be performed using either direct or indirect methods. Direct determinations may be based on some physical separation techniques like distillation, drying or chemical reactions producing gases like H2 or C2 H2 which are measured using speci®c techniques. Indirect determinations rely on the spectroscopic properties of water molecules. It is the case for NMR, infrared and Raman spectroscopy which are non-destructive techniques, as well as microwave spectroscopy or microwave resonator methods. Some properties of the foodstus especially sugar solutions and fruit juices allow obtaining of the dry substance (concentration) in the medium. These properties are either optical (refractometry, polarimetry), gravimetric (density) or electrical (conductivity) and allow indirect determination of water content (as a dierence). A rapid survey of these methods is given together with the limits and disadvantages they might have. Their suitability to the prediction of food product stability is also reported. 2.1. Direct determination 2.1.1. Physical separation Oven drying for a standardised period and a conventional temperature (3 h at 105°C for sugar) is very often the legal method of determination of water content. The period of drying is speci®ed for each type of
product. Dierent sources of errors may be found in oven drying. These are, for example, the incomplete removal of water, the loss of other volatiles than water during the drying period, the formation of a crust at the surface of the product which slows down the escape of water, the decomposition of the product and Maillard reaction which produces water. Vacuum-oven drying takes place at lower temperatures (70°C) for longer periods (6 h). Such conditions may be less destructive for heat sensitive samples. Nevertheless, duration of drying may not be sucient to allow the food to come to steady state. Size of particles in¯uences both oven and vacuum-oven drying. Solvent extraction may be used to extract water form food with an organic solvent prior to its analysis using a chemical titration. Distillation which cannot be used for the analysis of traces of water has a relatively long duration (P1 h) (Isengard, 1995). 2.1.2. Chemical reactions Dierent quantitative chemical reactions, which produce gas, exist and can be used to quantify water content provided that the released gas is accurately analysed. Amid these reactions, we ®nd H2 O CaH2 ! CaO H2 O H2 O CaC2 ! CaO C2 H2
1 2
Reaction (2) is still used in sugar lump workshops to have a rapid (10±20 min) tool of controlling the rate of moisturising the sugar prior to pressing and drying the cubes. Volume of C2 H2 , which is directly linked to water content, might change as a function of temperature in the workshop. Karl Fischer titration: Karl Fischer initiated this method in 1935, who introduced a reagent speci®c of water which contains pyridine, methanol, sulphur dioxide and iodine. Later, it was shown (Verhoef & Barendrecht, 1976) that pyridine is not necessary and could be replaced by other bases with higher basicity whereas methanol which takes part in the reaction cannot be replaced by other alcohols (Isengard, 1995). Using imidazole (Z) as a base, we have a two-step reaction at the basis of water titration CH3 OH SO2 Z ! ZH CH3 OSO2 ZH CH3 OSO2 I2 H2 O 2Z ! 3ZH CH3 OSO3 2I
The titrating reagent is I2 , which reacts stoichiometrically with water. The ®rst excess of I2 indicates the end-point of reaction. Indication of end-point is obtained through an abrupt drop of voltage due to the presence of the redox couple I2 =I at the polarised platinum electrodes. Karl Fischer titration proves to be the most accurate method, which can be used for all
M. Mathlouthi / Food Control 12 (2001) 409±417
values of water content from traces to high levels. This method is now computerised and is more and more used especially as sugars contain only traces of water. 2.1.3. Chromatography Gas chromatography has been applied to the determination of water content in freeze-dried vaccines (Bervelt, 1975). However, there is a need that water is extracted by organic solvent prior to analysis and that the sample is homogeneous. Extraction solvent should have a high anity for water and be protected from the surrounding atmosphere humidity. Using methanol or DMF together with molecular sieves as in Karl Fischer titration, proves to be an ecient method of extraction. Generally, Porapaq-Q is used to ®ll the column. This method is relatively rapid providing that the solvent or other impurities do not have peaks that obscure the peak of water. 2.1.4. Spectroscopy Interaction of the water molecule with electromagnetic radiation may be used in the analysis of water content. The fact that hydrogen atoms in water have nuclei which possess magnetic properties allowing them to behave as small bar magnets is exploited in proton NMR for the determination of water in foods (Troller & Christian, 1978). NMR spectroscopy is informative on hydrogen atoms. These are more easily detected in a liquid environment. So that this technique is more adapted to distinguish between free water and bound water (crystallisation water for example) than the accurate determination of water content. Moreover, there is a need of obtaining precise calibration speci®c of the analysed product based on a good reference method. Near infrared (NIR) absorption of water occurs at dierent wavelengths (1950, 1450 and 977 nm). The ratios of the intensities of the bands at 1950 and 1450 nm are used as a measure of water content (Vornhof & Thomas, 1970). It is also possible to extract water from the food with a solvent (methanol, dimethylformamide) prior to analysis by NIR spectroscopy. Computerised NIR spectrometers are used in dierent food industries for the determination of water content and other food constituents. This method requires a speci®c calibration for the food analysed and the use of statistical methods of exploitation of results which are included in the software. The colour, particle size, thickness and texture of the product in¯uence results. The re¯ectance technique allows detection of surface water and might not be representative of the whole product if it is not homogeneous. Microwave spectroscopy uses the dipolar character of water molecules. The shift in wavelength and the attenuation of the amplitude of the waves when a sample is placed between the emitter and receiver of microwaves
411
are used to determine the amount of water content (Isengard, 1995). Parameters such as water concentration, density and thickness of the analysed sample may have an eect on the result. Mobile water can be measured more easily than bound or crystallisation water. A calibration is needed. The method may be used for on-line measurements provided that the thickness of sample is known. Another application of microwaves to the determination of water in foods is that of the resonator. If a sample containing water is put in the resonator chamber, the resonance frequency of microwaves shifts under the eect of water as well as the height of the resonance peak. Again a speci®c calibration is needed. 2.1.5. Physical properties related to water content Refractometers are used to determine the percentage of sugar in sugar syrups or fruit juices. By dierence to 100, the water content is deduced. Polarimetry is also used for pure sugar solutions or technical sugar solutions after defecation and ®ltration. These optical methods only determine optically active substances and the water content is only determined by dierence. This might be sucient for a rapid analysis of syrups. Thermal analysis using dierential thermal analysis (DTA) or dierential scanning calorimetry (DSC) may be used during the heating of a frozen sample to determine freezable water, which is approximately the fraction of water, considered as mobile or ``free''. Such techniques are also informative on the glassy state of water in foodstus, which might help in the interpretation of the behaviour of the product during drying. 3. Water activity and sorption isotherms Most of the methods used for the determination of water activity or ERH of foods were originally set for the measurement of relative humidity in atmospheric air by the meteorologists (Troller & Christian, 1978). When water activity aw is measured it is generally required to know if the product has reached the critical zone where spoilage reactions may occur or not. That is why accuracy within 0.01aw unit is sucient for most food-related applications. Water activity is de®ned as the ratio of partial pressure of water vapour in the product (p) to that in presence of pure water po : p aw : po If there was no dierence in the interaction between water and water on the one hand and water and solute on the other, the determination of water activity would have been easy and its expression n2 aw Xw n1 n2
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directly obtained from the mole fraction Xw of water molecules n2 to total molecules in the solutions. For real solutions Aw c Xw , where c is an activity coecient. The higher the modi®cation of water binding by the solute, the more c coecient is dierent from 1. 3.1. Manometry As water vapour pressure is given in tables for different temperatures, a direct measurement of water vapour pressure in the food should give the best direct tool of determination of aw . To achieve this measurement there is a need of establishing vacuum and working at temperatures as low as )80°C. Working at zero pressure in one side of the oil manometer with the freeze trap of moisture and leaving the sample on the other side release its vapour permits accurate measurement of aw . This method needs uniform accurate temperature measurement and the device is extremely fragile (Troller & Christian, 1978). 3.2. Electric hygrometer The sample is placed in a tight measuring chamber at a controlled stable temperature. During equilibration the sample releases humidity and the same relative humidity at the level of the electrical sensor and in the sample is observed. The sensing element may be either a conducting polymer or an electrolytic element like saturated LiCl solution. Another type of electric hygrometer uses the measurement of electrical conductivity or capacitance of a hygroscopic substance like LiCl crystal or the anodised surface of an aluminium rod (Troller & Christian, 1978). Filters that adsorb contaminants are often needed. The sensors need frequent calibration with saturated salt solutions. Except in the extremes of the water activity range (below 0.15 or above 0.95), the accuracy of the electric hygrometer is satisfactory. Among the drawbacks of this type of aw -meter are the hysteresis observed and the sensitivity of sensor to high relative humidities (near saturation).
though this type of measurement can be rapid and precise, it only accounts for surface aw . The surface of the mirror should be clean and non-contaminated and the amount of condensed water negligible if it is desired to have the measured relative humidity equal to ERH of the food. 3.4. Other methods of measurement of relative humidity Other methods of measurement of relative humidity may be used to determine aw . It is the case for the wet bulb-dry bulb thermometric technique, the hair hygrometer, the freezing point depression or the aptitude of certain chemical compounds to change colour at a given relative humidity. These methods may be used to give an approximate value of aw during the storage of a food product. 3.5. Water vapour sorption isotherms 3.5.1. General aspect and meaning A water vapour sorption isotherm represents the variation of water content as a function of water activity at a given temperature. The general aspect of a sorption isotherm usually observed for a food product is sigmoõd (Fig. 1).
3.3. Dew point Dew point measurement is also used to determine aw . The temperature at which saturation of water is observed and the beginning of condensation which is obtained at the cooled surface of a mirror is related to vapour pressure and consequently to aw . This is well known from the psychrometric chart of humid air. Photodetection of the condensation on the cooled (Peltier eect) mirror together with the precise measurement of mirror allows deducing of relative humidity in the cell where the sample of food was disposed. However, al-
Fig. 1. Three regions of water vapour sorption curve and the BET method of determination of monolayer water.
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Table 1 Saturated salt solution
aw
LiCIH2 O CH3 COOK MgCl2 6H2 O K2 CO3 Mg NO3 2 6H2 O NaCl NH4 2 SO4 CdCl2 Li2 SO4 K2 CrO4 KNO3 K2 SO4 Na2 HPO4
0.12 0.23 0.33 0.44 0.52 0.75 0.79 0.82 0.85 0.88 0.94 0.97 0.98
This curve is established using a microclimate method or an electrical hygrometer equipped with the sorption accessory. In the microclimate method a small amount of sample is disposed in a cupel as a thin layer. The cupel is placed in a gas-tight jar where relative humidity is ®xed with saturated salt solutions (Table 1). Evolution of product water content, which should be determined prior to submitting the samples to water activity equilibration, is followed by weighing. When equilibrium (constant mass) is reached, by sorption or desorption, the water content at equilibrium is represented as a function of ERH or water activity aw ERH=100. The water vapour sorption curve obtained is composed of three regions A, B and C. Region A corresponds to hydration monolayer where water molecules are bonded to the product by strong H-bonds. The amount of monolayer water may be determined using BET (Brunauer, Emmett and Teller) isotherm method (Brunauer et al., 1938) (Fig. 1). The BET isotherm is expressed by aw 1 c 1 aw ; mo c 1 aw m mo c where aw is the water activity; m the water content (dry basis); mo the water content in the monolayer and c is the constant related to adsorption heat. This relation is applicable for aw 6 0:55. From a= 1 am f aw , it is possible to determine graphically mo
1 ordonate at the origin slope
and c
1 eQs=RT ordonate at the origin mo
with Qs the heat capacity of the monolayer. Region B corresponds to the linear part of sorption isotherm (Fig. 1). Water is adsorbed as multilayers of
Fig. 2. Hysteresis of water vapour sorption isotherms.
molecules of hydrogen bonded to the monolayer, or entrapped in the food by capillarity, Van der Waals forces, etc. Region C is that of the so-called ``free'' or solvent water. Water molecules in this region are much less strongly bound than in regions A and B. This fraction of water is available for mould growth or dissolving of soluble solutes. Sorption of water vapour by solid foodstus shows very often a hysteresis phenomenon. Adsorption and desorption curves are not superposed (Fig. 2). The origin of hysteresis is linked to the type and strength of the bonds between water and product. It is well known that ®lling and emptying of capillaries in the product does not follow the same kinetics. This is one reason for the dierence between adsorption and desorption isotherms. Another reason is the change in the structure of some food constituents (starch for example) when they are submitted to various aw . Hysteresis is generally observed at water contents above monolayer. Hysteresis of sorption isotherms has some consequences as regards chemical and microbial stability of food products. The fact that water content is higher for desorption than for adsorption at the same aw leads to degradations such as the loss in vitamin C for a model food system prepared by desorption in the range of aw from 0.32 to 0.93 (Lee & Labuza, 1975). 3.5.2. Application of sorption isotherms Sorption isotherms are important for more than one reason. From the point of view of thermodynamics, they are informative on sorption and desorption enthalpies and the type of binding of water to dry matter. On a structural level, they can help in understanding the role of particle size, amorphous state or speci®c area in water vapour sorption. Concerning the technological aspect, water vapour sorption isotherms are useful in the prediction of shelf-life, the control of drying and the
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release of water which dissolves more sugar initiating a caking process. If the impurities at the surface of sucrose crystals involve other sugars more hygroscopic like glucose or fructose, then the instability of sugar is observed at lower relative humidities.
Fig. 3. Water vapour sorption isotherm of crystalline sugar.
prevention of such accidents as caking and sticking of food powders. 3.5.2.1. Storage stability sugar. If sugar for example, if crystalline is stable when aw is maintained below 0.83 (Fig. 3). Stability can be reached easily at ordinary temperature and relative humidity for pure sucrose well crystallised. If some impurities are remaining at the surface in the thin ®lm of saturated syrup surrounding the crystals, the behaviour of sugar towards water vapour may change. As a general rule, impurities in sugar syrup increase sugar solubility, which leads to a lowered water activity of saturated solution. This means that problems such as caking or stickiness may occur at relative humidities below 80%. The same type of instability occurs when the size of crystals is small or when amorphous sugar obtained by the breakage of crystals or by a rapid drying at high temperature is present at the surface of sugar crystals. Recrystallisation of amorphous sugar may occur at relative humidities as low as 38% at 20°C (Fig. 4) with
3.5.2.2. Shelf-life and water activity of intermediate moisture foods. Water availability for microbial spoilage in intermediate moisture foods (IMF) can be minimised through formulation. For sponge cake, taken as an example of IMF, usually water activity is around 90%. Adding water activity depressors to the formula of the cake combined with modi®ed atmosphere packaging (MAP) (less than 1% of O2 ) proves to be an ecient method for extending the shelf-life from few days to several months. Change in water availability during storage of sponge cake may be manifested by a hysteresis between adsorption and desorption isotherms. To minimise sorption hysteresis and depress signi®cantly (from 0.90 to 0.84) the value of aw of sponge cake, the addition of 0.5±1% of soya protein was found ecient (Guinot, 1988). Moreover, eciency of water retention is increased if additive is added to ``creaming'' which is treated thermally by heating at 45°C for 10 min. Such a treatment allows the globular structure of protein to unfold and sites of ®xation of water molecules by Hbonds to become available. Combining this formulation modi®cation with MAP 50%CO2 50%N2 and storing the product at 4°C allows the increase of shelflife of sponge cake up to 10 weeks without microbial spoilage. Water activity depressors prove to be useful in increasing the stability of baked goods. If sucrose is taken as a reference standard with humectancy (aw depressor ability) equal to 1, then it may be observed that dierent ingredients used to prepare cakes might be more or less good humectants (Table 2). Table 2 Comparison of humectancies of cake ingredients with sugar taken as reference standard (humectancy 1)
Fig. 4. Water intake by amorphous sugar.
Product
Humectancy
Flour butter Fat, egg Skin milk powder Raisins Leavening powder Salt Tartaric and citric acids Glucose syrup 42 DE (DS) Glucose syrup 64 DE (DS) Glucose, fructose, invert Sorbitol Glycerol
0.2 0 1.2 0.9 3 11 3 0.7 0.9 1.4 2 4
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However, glycerol although a good water activity depressor might not be the ideal ingredient to protect against bacterial growth. Water although retained around glycerol molecules is mobile enough to allow the growth of bacteria (Sperber, 1983). It is also the case for fructose, which is also known to have much mobile hydration water around it than sucrose or glucose.
4. Water structure Most of the reactions occurring during the storage of a foodstu, like lipid oxidation, enzyme degradation, Maillard reaction may ®nd an interpretation related to the structure of water. Likewise the speci®c interactions of the dry substance with water, namely hydrophilic or hydrophobic interactions can be helpful to understand the behaviour of the product towards water vapour during its preservation. A brief recall of the structure of pure liquid water will be reported before the description of solute±solvent interactions in foods and their consequences on the stability of food products. 4.1. Liquid water structure The chemical structure of H2 O, where an atom of oxygen occupies the centre of a regular tetrahedron, two vertices of which are the H atoms and the two others the lone pairs of electrons of the oxygen atom, is at the origin of its 3D structure. Indeed, such a con®guration allows each water molecule to be associated to four molecules by H-bonds. Moreover, hydrogen bonds are sensitive to charge transfer from donors to acceptors in a sequence which produces an eect called ``cooperativity'' (Frank, 1974). Other liquids like alcohols form chains of H-bonds because they have only one hydroxyl group whereas water forms tetrahedral clusters. Water structure was one of the most studied subjects these last years using either computer models or experimental spectroscopic techniques. However, no theory or conclusions drawn from experimental results can account for its particular physical properties. Computer models for water structure generally describe this liquid as a continuous network of H-bonds randomly arranged. These models have two disadvantages: the lack of details and a static character. Experimental techniques such as neutron scattering, FTIR and Raman spectroscopy were used to investigate structure and dynamics of the hydrogen bonding in liquid water. Decomposition of the experimental Raman band in four components assigned to four species of clusters with Hbonds diering in strength and number was proposed by
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Luu, Luu, Rull, and Sopron (1982). Such a model may be adopted as a reference for comparative studies of the eect of solutes on water structure. 4.2. Hydrophilic interactions Cations of interest in the food industry like Na ; Ca2 or Mg2 are known to immobilise several layers of hydration water. Generally such ions are known to have three layers of water around them. A ®rst layer strongly bound to the ion, a second layer of perturbed water because it is submitted to the attraction due to electrical ®eld of the ion on the one hand and to the ``cooperativity'' of H-bonds with water molecules of the third layer where water is less in¯uenced by the ion. Beside this type of attraction of water molecules around a hydrophilic solute, the hydration of small non-electrolytes and biopolymers is generally considered as hydrophilic. Small hydrophilic molecules like sugars have a sphere of hydration characterised by a number of hydration (®ve for sucrose) and a ``structure maker'' eect as they have a long-range eect of orientation of bulk water molecules. The hydration of food polymers is sensitive to their conformations and this can be measured by dierential scanning calorimetry (Silvonen, Lindberg, Seppala, Evasti, & Hautahoo, 1982). It is also important to take into account the eect of the phase (gel, emulsion,) in which the biomolecules are embedded, which may change their conformation. In particular globular proteins may change conformation as concentration is increased and temperature raised to expose their hydrophobic backbone to water and establish intramolecular H-bonds. Interactions of water with the hydrophobic surface of folded proteins corresponds to the so-called hydrophobic hydration. 4.3. Hydrophobic interactions Hydrophobic interactions are due to the hydration of apolar groups like methyl groups. Interaction between such a group and water is weaker than water±water hydrogen bonding, so that, when several water molecules surround an apolar group, they do not have a direct contact with the group and hence reinforce their water±water bonds. This layer of water is much less mobile than bulk water. Its enthalpy is decreased and its entropy is increased. Hydrophobicity of proteins may be at the origin of the stability of emulsions. Processed meat products may be considered as emulsions where hydrophobic hydration of the proteins together with the hydrophilicity of salt and glycerol tends to immobilise a maximum of water and to increase the shelf-life (Lacroix & Castaigne, 1984).
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Beside the structure of water near hydrophobic biopolymers, it is needed to know the type of organisation of water molecules in the vicinity of solid interfaces to interpret their sorption±desorption behaviour, which very often exhibits hysteresis phenomenon. 4.4. Interfacial water and sorption hysteresis Physical properties of interfacial water seem to be dierent from those of bulk water. Water molecules in the vicinity of adsorption sites at the surface of a solid are more rigidly organised. The kinetics of adsorptoin of water by proteins at air±water interface is very slow (Terminassian-Saraga, 1981), and protein denaturation is found to depend on surface tension of the adsorbed ®lm of water. Water vapour sorption by foods is sensitive to surface tension. This was demonstrated (Labuza & Rutman, 1968) by use of tensioactive agents in model systems. Increasing the surfactant in the formula induces a decrease in water content. Surfactants also decrease the hysteresis amplitude. Moistening by the tensioactive agent reduces to 0 the dierences between contact angles during sorption and desorption processes. Moreover, an ``ink bottle'' model for capillaries seems to be convenient and supports the hypothesis of surface tension decrease by the surfactant, which changes the mechanism of capillary ®lling. 4.5. Water structure and food preservation Extending the shelf-life of a food product consists in preventing its degradation by biochemical reactions or microbial growth. Prevention of degradation means prevention of availability of water for degradation reactions (Mathlouthi, 1986). To achieve such an objective, it is necessary to reinforce the hydrogen bonding of water and to reduce its mobility. Such a result may be obtained through the formulation of the product and the control of the type of hydration by establishing water vapour sorption curves. It is also useful to know the thermal history of the product (thermal treatment, storage temperature) in order to interpret the change in mobility of water. For the inhibition of growth of micro-organisms, it is needed to lower aw . However solutes like glycerol, although good water activity depressors, may enter the bacterial cell without causing an osmotic stress, so that bacteria grow in presence of glycerol at low aw values. It seems that viability of pathogen bacteria in foodstus is controlled by osmotic pressure regulation with dierent solutes (Sperber, 1983). So that retention of water by an additive is not the sole parameter in formulation. It is also needed to know the eect of solute on water mobility.
5. Conclusion The sole determination of water content, even using accurate methods like Karl Fischer titration, is not sucient to predict their shelf-life. Water activity proves to be a good indicator of the preponderance of one or another of the degradation reactions that might occur during storage of foodstus. For example, lipid oxidation occurs below aw 0:30 and Maillard reaction exhibits an optimum for aw 0:65. To understand the mechanisms of degradation reactions and why some foods are stable whereas others are not stable at the same aw , there is a need of knowledge of the nature and concentration of the dierent components and of water structure. Mobility of water and its availability for biochemical reactions depend on the type of interaction it might have with solutes. Approaching water in foodstus, especially in view of studying storage stability and shelf-life should rely on three types of information: analytical determination of water content, thermodynamical activity of water and the unveiling of water structure in presence of the soluble constituents of the foodstu. References Bervelt, E. (1975). Dosage de l'humidite residuelle dans les vaccins lyophilises. Journal of Biological Standardisation, 3, 321±330. Brunauer, S., Emmett, P., & Teller, E. (1938). Adsorption of gases in multimolecular layers. Journal of American Chemical Society, 60, 309±319. Frank, H. S. (1974). In W. A. P. Luck (Ed.), Structure of water and aqueous solutions (pp. 10±47). Weinheim: Verlag Chemie. Guinot, P. (1988). Contribution a la prolongation de la duree de vie d'une genoise industrielle. Ph.D. Thesis, Dijon. Isengard, H.-D. (1995). Rapid water determination in foodstus. Trends in Food Science and Technology, 6, 155±162. Labuza, T. P., & Rutman, M. (1968). The eect of surface active agents on sorption isotherms of a model food system. Canadian Journal of Chemical Engineering, 46, 364±368. Lacroix, C., & Castaigne, F. (1984). Emulsi®cation des viandes: r^ oles et actions de certains parametres de composition: proteines vegetales, sel, sucre et glycerol. Science Aliments, 4, 505±511. Lee, S., & Labuza, T. P. (1975). Destruction of ascorbic acid as a function of water activity. Journal of Food Science, 40, 370±373. Luu, C., Luu, D. V., Rull, F., & Sopron, C. E. (1982). Etude par eet raman de la perturbation de l'eau liquide par une substance etrangere: I ± Modele d'association de l'eau liquide. Journal of Molecular Structuring, 81, 1±10. Mathlouthi, M. (1986). In M. Mathlouthi (Ed.), Food packaging and preservation, theory and practice (pp. 137±164). London: Elsevier. Silvonen, R., Lindberg, J. J., Seppala, C., Evasti, M., & Hautahoo, C. (1982). Studies on water retention and water release from some protein systems. Journal of Thermal Analysis, 25, 101±105. Sperber, W. H. (1983). In¯uence of water activity on foodborne bacteria ± a review. Journal of Food Protection, 46, 142±145. Terminassian-Saraga, L. (1981). Protein denaturation on adsorption and water activity at interfaces: an analysis and suggestion. Journal of Colloid and Interface Science, 80, 393±401. Troller, J. A., & Christian, J. H. B. (1978). Water activity and food (pp. 13±47). New York: Academic Press.
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Vornhof, D. W., & Thomas, J. H. (1970). Determination of moisture in starch hydrolysates by near-infrared and infrared spectrophotometry. Analytical Chemistry, 42, 1230.
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Water content, water activity, water structure and the stability of foodstus Mohamed Mathlouthi * Laboratoire de Chimie Physique Industrielle, Facult e des Sciences, Universit e de Reims Champagne-Ardenne, B.P. 1039±51687 Reims C edex 2, France Received 11 December 2000; received in revised form 7 March 2001; accepted 8 March 2001
Abstract Determination of water content, whatever the accuracy of the analytical method, is not suciently informative in relation to the stability of the investigated food product. Water activity aw brings a supplement of information as it accounts for the availability of water for degradation reactions. The understanding of why certain products are more stable than others at the same aw needs an elucidation of water structure. Of particular importance are the interactions (hydrophilic, hydrophobic) between water and the components of the foodstu and the eect of the soluble molecules of the food on the hydrogen bonding in solvent water. Studying water in foods should start with an anlytical determination of water content for commercial and legal reasons which are evident. This has to be completed with the measurement of the thermodynamic activity of water in the food. Such a parameter aw should hold an important place in the identi®cation of the food product, especially as regards its shelf life. A further step in unveiling the behaviour of water in foods consists in determining water molecules in the molecules in the studied food matrix. The tripartite (analytical, thermodynamical and structural) approach to water in foods will be examined based on examples of sugars and sugar rich products. Ó 2001 Elsevier Science Ltd. All rights reserved.
1. Introduction Water is omnipresent in foodstus and the surrounding atmosphere. Determination of water content is one of the most frequent analysis in the laboratory of a food industry. However, depending on the method of analysis, it is not the same type of moisture content, which is measured. Even the term ``moisture'' itself is questionable as this might include other liquids than water, wetting the product (Isengard, 1995). The most common method of water analysis usually accepted at a commercial level is the desiccation method. This method is based on the mass loss after drying of the sample. However, part of mass loss might originate from the volatility of other gases than water. Not only the desiccation or ``oven drying'' is criticisable, but also all methods of determination of water contents have their drawbacks. The sole value of ``water content'' in a food is not inform about the nature of water, if it is ``bound'' or
*
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``free'', ``inherent'' or ``occluded'', etc. The knowledge of each of these fractions is important as it helps in understanding the process which is at the origin of each fraction. Beside measuring water content in the foodstu, many laboratories are now equipped with devices which allow determination of water activity aw . However, depending on the technique of water activity measurement, the result may be dierent. It is necessary for a slow equilibrium between the product and air surrounding it that this equilibrium is reached. The obtaining of equilibrium is asymptotic. One has to inform about the time needed to reach such an equilibrium. Rapid measurement of aw using the dew point technique only gives the surface equilibrium relative humidity (ERH) of the product, which may not be sucient for shelf-life prediction for example. To account for the behaviour of a food product in the whole range of relative humidities to which it may be submitted during storage, there is a need of establishing the water vapour sorption isotherm, preferentially at storage temperature. The shape of sorption isotherm may change depending on the type of product and its anity for water. Brunauer, Emmett, and Teller (1938) have described ®ve
0956-7135/01/$ - see front matter Ó 2001 Elsevier Science Ltd. All rights reserved. PII: S 0 9 5 6 - 7 1 3 5 ( 0 1 ) 0 0 0 3 2 - 9
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types of water vapour sorption isotherms. Soluble foods like sugar adopt an asymptotic shape of isotherm when relative humidity tends towards 100%, and heterogeneous complex foods show a sigmoid curve. Analysis of the shape of sorption isotherms shows that they may be divided into three zones, respectively, corresponding to monolayer strongly bound water, linear region of less rigidly and capillary adsorbed water and the third region of solvent or free water. This description of the three species of water is a rough accounting of the dierences in the structure of water. Indeed, equilibrium of hydration of a food generally described by water activity aw depends on the structure of food components, their eect on the solvent (water) and other properties like the surface activity of the product. Direct analysis of the structure of water in a food system is dicult to achieve. Utmost, one can have an insight into ``free'' and ``bound'' water from NMR or other techniques like thermal analysis of frozen systems. Amid the water properties that may be used for the elucidation of its structure, the behaviour as a solvent and the interactions with dierent classes of molecules are of great importance. 2. Water content Determination of water content in foodstus can be performed using either direct or indirect methods. Direct determinations may be based on some physical separation techniques like distillation, drying or chemical reactions producing gases like H2 or C2 H2 which are measured using speci®c techniques. Indirect determinations rely on the spectroscopic properties of water molecules. It is the case for NMR, infrared and Raman spectroscopy which are non-destructive techniques, as well as microwave spectroscopy or microwave resonator methods. Some properties of the foodstus especially sugar solutions and fruit juices allow obtaining of the dry substance (concentration) in the medium. These properties are either optical (refractometry, polarimetry), gravimetric (density) or electrical (conductivity) and allow indirect determination of water content (as a dierence). A rapid survey of these methods is given together with the limits and disadvantages they might have. Their suitability to the prediction of food product stability is also reported. 2.1. Direct determination 2.1.1. Physical separation Oven drying for a standardised period and a conventional temperature (3 h at 105°C for sugar) is very often the legal method of determination of water content. The period of drying is speci®ed for each type of
product. Dierent sources of errors may be found in oven drying. These are, for example, the incomplete removal of water, the loss of other volatiles than water during the drying period, the formation of a crust at the surface of the product which slows down the escape of water, the decomposition of the product and Maillard reaction which produces water. Vacuum-oven drying takes place at lower temperatures (70°C) for longer periods (6 h). Such conditions may be less destructive for heat sensitive samples. Nevertheless, duration of drying may not be sucient to allow the food to come to steady state. Size of particles in¯uences both oven and vacuum-oven drying. Solvent extraction may be used to extract water form food with an organic solvent prior to its analysis using a chemical titration. Distillation which cannot be used for the analysis of traces of water has a relatively long duration (P1 h) (Isengard, 1995). 2.1.2. Chemical reactions Dierent quantitative chemical reactions, which produce gas, exist and can be used to quantify water content provided that the released gas is accurately analysed. Amid these reactions, we ®nd H2 O CaH2 ! CaO H2 O H2 O CaC2 ! CaO C2 H2
1 2
Reaction (2) is still used in sugar lump workshops to have a rapid (10±20 min) tool of controlling the rate of moisturising the sugar prior to pressing and drying the cubes. Volume of C2 H2 , which is directly linked to water content, might change as a function of temperature in the workshop. Karl Fischer titration: Karl Fischer initiated this method in 1935, who introduced a reagent speci®c of water which contains pyridine, methanol, sulphur dioxide and iodine. Later, it was shown (Verhoef & Barendrecht, 1976) that pyridine is not necessary and could be replaced by other bases with higher basicity whereas methanol which takes part in the reaction cannot be replaced by other alcohols (Isengard, 1995). Using imidazole (Z) as a base, we have a two-step reaction at the basis of water titration CH3 OH SO2 Z ! ZH CH3 OSO2 ZH CH3 OSO2 I2 H2 O 2Z ! 3ZH CH3 OSO3 2I
The titrating reagent is I2 , which reacts stoichiometrically with water. The ®rst excess of I2 indicates the end-point of reaction. Indication of end-point is obtained through an abrupt drop of voltage due to the presence of the redox couple I2 =I at the polarised platinum electrodes. Karl Fischer titration proves to be the most accurate method, which can be used for all
M. Mathlouthi / Food Control 12 (2001) 409±417
values of water content from traces to high levels. This method is now computerised and is more and more used especially as sugars contain only traces of water. 2.1.3. Chromatography Gas chromatography has been applied to the determination of water content in freeze-dried vaccines (Bervelt, 1975). However, there is a need that water is extracted by organic solvent prior to analysis and that the sample is homogeneous. Extraction solvent should have a high anity for water and be protected from the surrounding atmosphere humidity. Using methanol or DMF together with molecular sieves as in Karl Fischer titration, proves to be an ecient method of extraction. Generally, Porapaq-Q is used to ®ll the column. This method is relatively rapid providing that the solvent or other impurities do not have peaks that obscure the peak of water. 2.1.4. Spectroscopy Interaction of the water molecule with electromagnetic radiation may be used in the analysis of water content. The fact that hydrogen atoms in water have nuclei which possess magnetic properties allowing them to behave as small bar magnets is exploited in proton NMR for the determination of water in foods (Troller & Christian, 1978). NMR spectroscopy is informative on hydrogen atoms. These are more easily detected in a liquid environment. So that this technique is more adapted to distinguish between free water and bound water (crystallisation water for example) than the accurate determination of water content. Moreover, there is a need of obtaining precise calibration speci®c of the analysed product based on a good reference method. Near infrared (NIR) absorption of water occurs at dierent wavelengths (1950, 1450 and 977 nm). The ratios of the intensities of the bands at 1950 and 1450 nm are used as a measure of water content (Vornhof & Thomas, 1970). It is also possible to extract water from the food with a solvent (methanol, dimethylformamide) prior to analysis by NIR spectroscopy. Computerised NIR spectrometers are used in dierent food industries for the determination of water content and other food constituents. This method requires a speci®c calibration for the food analysed and the use of statistical methods of exploitation of results which are included in the software. The colour, particle size, thickness and texture of the product in¯uence results. The re¯ectance technique allows detection of surface water and might not be representative of the whole product if it is not homogeneous. Microwave spectroscopy uses the dipolar character of water molecules. The shift in wavelength and the attenuation of the amplitude of the waves when a sample is placed between the emitter and receiver of microwaves
411
are used to determine the amount of water content (Isengard, 1995). Parameters such as water concentration, density and thickness of the analysed sample may have an eect on the result. Mobile water can be measured more easily than bound or crystallisation water. A calibration is needed. The method may be used for on-line measurements provided that the thickness of sample is known. Another application of microwaves to the determination of water in foods is that of the resonator. If a sample containing water is put in the resonator chamber, the resonance frequency of microwaves shifts under the eect of water as well as the height of the resonance peak. Again a speci®c calibration is needed. 2.1.5. Physical properties related to water content Refractometers are used to determine the percentage of sugar in sugar syrups or fruit juices. By dierence to 100, the water content is deduced. Polarimetry is also used for pure sugar solutions or technical sugar solutions after defecation and ®ltration. These optical methods only determine optically active substances and the water content is only determined by dierence. This might be sucient for a rapid analysis of syrups. Thermal analysis using dierential thermal analysis (DTA) or dierential scanning calorimetry (DSC) may be used during the heating of a frozen sample to determine freezable water, which is approximately the fraction of water, considered as mobile or ``free''. Such techniques are also informative on the glassy state of water in foodstus, which might help in the interpretation of the behaviour of the product during drying. 3. Water activity and sorption isotherms Most of the methods used for the determination of water activity or ERH of foods were originally set for the measurement of relative humidity in atmospheric air by the meteorologists (Troller & Christian, 1978). When water activity aw is measured it is generally required to know if the product has reached the critical zone where spoilage reactions may occur or not. That is why accuracy within 0.01aw unit is sucient for most food-related applications. Water activity is de®ned as the ratio of partial pressure of water vapour in the product (p) to that in presence of pure water po : p aw : po If there was no dierence in the interaction between water and water on the one hand and water and solute on the other, the determination of water activity would have been easy and its expression n2 aw Xw n1 n2
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directly obtained from the mole fraction Xw of water molecules n2 to total molecules in the solutions. For real solutions Aw c Xw , where c is an activity coecient. The higher the modi®cation of water binding by the solute, the more c coecient is dierent from 1. 3.1. Manometry As water vapour pressure is given in tables for different temperatures, a direct measurement of water vapour pressure in the food should give the best direct tool of determination of aw . To achieve this measurement there is a need of establishing vacuum and working at temperatures as low as )80°C. Working at zero pressure in one side of the oil manometer with the freeze trap of moisture and leaving the sample on the other side release its vapour permits accurate measurement of aw . This method needs uniform accurate temperature measurement and the device is extremely fragile (Troller & Christian, 1978). 3.2. Electric hygrometer The sample is placed in a tight measuring chamber at a controlled stable temperature. During equilibration the sample releases humidity and the same relative humidity at the level of the electrical sensor and in the sample is observed. The sensing element may be either a conducting polymer or an electrolytic element like saturated LiCl solution. Another type of electric hygrometer uses the measurement of electrical conductivity or capacitance of a hygroscopic substance like LiCl crystal or the anodised surface of an aluminium rod (Troller & Christian, 1978). Filters that adsorb contaminants are often needed. The sensors need frequent calibration with saturated salt solutions. Except in the extremes of the water activity range (below 0.15 or above 0.95), the accuracy of the electric hygrometer is satisfactory. Among the drawbacks of this type of aw -meter are the hysteresis observed and the sensitivity of sensor to high relative humidities (near saturation).
though this type of measurement can be rapid and precise, it only accounts for surface aw . The surface of the mirror should be clean and non-contaminated and the amount of condensed water negligible if it is desired to have the measured relative humidity equal to ERH of the food. 3.4. Other methods of measurement of relative humidity Other methods of measurement of relative humidity may be used to determine aw . It is the case for the wet bulb-dry bulb thermometric technique, the hair hygrometer, the freezing point depression or the aptitude of certain chemical compounds to change colour at a given relative humidity. These methods may be used to give an approximate value of aw during the storage of a food product. 3.5. Water vapour sorption isotherms 3.5.1. General aspect and meaning A water vapour sorption isotherm represents the variation of water content as a function of water activity at a given temperature. The general aspect of a sorption isotherm usually observed for a food product is sigmoõd (Fig. 1).
3.3. Dew point Dew point measurement is also used to determine aw . The temperature at which saturation of water is observed and the beginning of condensation which is obtained at the cooled surface of a mirror is related to vapour pressure and consequently to aw . This is well known from the psychrometric chart of humid air. Photodetection of the condensation on the cooled (Peltier eect) mirror together with the precise measurement of mirror allows deducing of relative humidity in the cell where the sample of food was disposed. However, al-
Fig. 1. Three regions of water vapour sorption curve and the BET method of determination of monolayer water.
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Table 1 Saturated salt solution
aw
LiCIH2 O CH3 COOK MgCl2 6H2 O K2 CO3 Mg NO3 2 6H2 O NaCl NH4 2 SO4 CdCl2 Li2 SO4 K2 CrO4 KNO3 K2 SO4 Na2 HPO4
0.12 0.23 0.33 0.44 0.52 0.75 0.79 0.82 0.85 0.88 0.94 0.97 0.98
This curve is established using a microclimate method or an electrical hygrometer equipped with the sorption accessory. In the microclimate method a small amount of sample is disposed in a cupel as a thin layer. The cupel is placed in a gas-tight jar where relative humidity is ®xed with saturated salt solutions (Table 1). Evolution of product water content, which should be determined prior to submitting the samples to water activity equilibration, is followed by weighing. When equilibrium (constant mass) is reached, by sorption or desorption, the water content at equilibrium is represented as a function of ERH or water activity aw ERH=100. The water vapour sorption curve obtained is composed of three regions A, B and C. Region A corresponds to hydration monolayer where water molecules are bonded to the product by strong H-bonds. The amount of monolayer water may be determined using BET (Brunauer, Emmett and Teller) isotherm method (Brunauer et al., 1938) (Fig. 1). The BET isotherm is expressed by aw 1 c 1 aw ; mo c 1 aw m mo c where aw is the water activity; m the water content (dry basis); mo the water content in the monolayer and c is the constant related to adsorption heat. This relation is applicable for aw 6 0:55. From a= 1 am f aw , it is possible to determine graphically mo
1 ordonate at the origin slope
and c
1 eQs=RT ordonate at the origin mo
with Qs the heat capacity of the monolayer. Region B corresponds to the linear part of sorption isotherm (Fig. 1). Water is adsorbed as multilayers of
Fig. 2. Hysteresis of water vapour sorption isotherms.
molecules of hydrogen bonded to the monolayer, or entrapped in the food by capillarity, Van der Waals forces, etc. Region C is that of the so-called ``free'' or solvent water. Water molecules in this region are much less strongly bound than in regions A and B. This fraction of water is available for mould growth or dissolving of soluble solutes. Sorption of water vapour by solid foodstus shows very often a hysteresis phenomenon. Adsorption and desorption curves are not superposed (Fig. 2). The origin of hysteresis is linked to the type and strength of the bonds between water and product. It is well known that ®lling and emptying of capillaries in the product does not follow the same kinetics. This is one reason for the dierence between adsorption and desorption isotherms. Another reason is the change in the structure of some food constituents (starch for example) when they are submitted to various aw . Hysteresis is generally observed at water contents above monolayer. Hysteresis of sorption isotherms has some consequences as regards chemical and microbial stability of food products. The fact that water content is higher for desorption than for adsorption at the same aw leads to degradations such as the loss in vitamin C for a model food system prepared by desorption in the range of aw from 0.32 to 0.93 (Lee & Labuza, 1975). 3.5.2. Application of sorption isotherms Sorption isotherms are important for more than one reason. From the point of view of thermodynamics, they are informative on sorption and desorption enthalpies and the type of binding of water to dry matter. On a structural level, they can help in understanding the role of particle size, amorphous state or speci®c area in water vapour sorption. Concerning the technological aspect, water vapour sorption isotherms are useful in the prediction of shelf-life, the control of drying and the
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release of water which dissolves more sugar initiating a caking process. If the impurities at the surface of sucrose crystals involve other sugars more hygroscopic like glucose or fructose, then the instability of sugar is observed at lower relative humidities.
Fig. 3. Water vapour sorption isotherm of crystalline sugar.
prevention of such accidents as caking and sticking of food powders. 3.5.2.1. Storage stability sugar. If sugar for example, if crystalline is stable when aw is maintained below 0.83 (Fig. 3). Stability can be reached easily at ordinary temperature and relative humidity for pure sucrose well crystallised. If some impurities are remaining at the surface in the thin ®lm of saturated syrup surrounding the crystals, the behaviour of sugar towards water vapour may change. As a general rule, impurities in sugar syrup increase sugar solubility, which leads to a lowered water activity of saturated solution. This means that problems such as caking or stickiness may occur at relative humidities below 80%. The same type of instability occurs when the size of crystals is small or when amorphous sugar obtained by the breakage of crystals or by a rapid drying at high temperature is present at the surface of sugar crystals. Recrystallisation of amorphous sugar may occur at relative humidities as low as 38% at 20°C (Fig. 4) with
3.5.2.2. Shelf-life and water activity of intermediate moisture foods. Water availability for microbial spoilage in intermediate moisture foods (IMF) can be minimised through formulation. For sponge cake, taken as an example of IMF, usually water activity is around 90%. Adding water activity depressors to the formula of the cake combined with modi®ed atmosphere packaging (MAP) (less than 1% of O2 ) proves to be an ecient method for extending the shelf-life from few days to several months. Change in water availability during storage of sponge cake may be manifested by a hysteresis between adsorption and desorption isotherms. To minimise sorption hysteresis and depress signi®cantly (from 0.90 to 0.84) the value of aw of sponge cake, the addition of 0.5±1% of soya protein was found ecient (Guinot, 1988). Moreover, eciency of water retention is increased if additive is added to ``creaming'' which is treated thermally by heating at 45°C for 10 min. Such a treatment allows the globular structure of protein to unfold and sites of ®xation of water molecules by Hbonds to become available. Combining this formulation modi®cation with MAP 50%CO2 50%N2 and storing the product at 4°C allows the increase of shelflife of sponge cake up to 10 weeks without microbial spoilage. Water activity depressors prove to be useful in increasing the stability of baked goods. If sucrose is taken as a reference standard with humectancy (aw depressor ability) equal to 1, then it may be observed that dierent ingredients used to prepare cakes might be more or less good humectants (Table 2). Table 2 Comparison of humectancies of cake ingredients with sugar taken as reference standard (humectancy 1)
Fig. 4. Water intake by amorphous sugar.
Product
Humectancy
Flour butter Fat, egg Skin milk powder Raisins Leavening powder Salt Tartaric and citric acids Glucose syrup 42 DE (DS) Glucose syrup 64 DE (DS) Glucose, fructose, invert Sorbitol Glycerol
0.2 0 1.2 0.9 3 11 3 0.7 0.9 1.4 2 4
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However, glycerol although a good water activity depressor might not be the ideal ingredient to protect against bacterial growth. Water although retained around glycerol molecules is mobile enough to allow the growth of bacteria (Sperber, 1983). It is also the case for fructose, which is also known to have much mobile hydration water around it than sucrose or glucose.
4. Water structure Most of the reactions occurring during the storage of a foodstu, like lipid oxidation, enzyme degradation, Maillard reaction may ®nd an interpretation related to the structure of water. Likewise the speci®c interactions of the dry substance with water, namely hydrophilic or hydrophobic interactions can be helpful to understand the behaviour of the product towards water vapour during its preservation. A brief recall of the structure of pure liquid water will be reported before the description of solute±solvent interactions in foods and their consequences on the stability of food products. 4.1. Liquid water structure The chemical structure of H2 O, where an atom of oxygen occupies the centre of a regular tetrahedron, two vertices of which are the H atoms and the two others the lone pairs of electrons of the oxygen atom, is at the origin of its 3D structure. Indeed, such a con®guration allows each water molecule to be associated to four molecules by H-bonds. Moreover, hydrogen bonds are sensitive to charge transfer from donors to acceptors in a sequence which produces an eect called ``cooperativity'' (Frank, 1974). Other liquids like alcohols form chains of H-bonds because they have only one hydroxyl group whereas water forms tetrahedral clusters. Water structure was one of the most studied subjects these last years using either computer models or experimental spectroscopic techniques. However, no theory or conclusions drawn from experimental results can account for its particular physical properties. Computer models for water structure generally describe this liquid as a continuous network of H-bonds randomly arranged. These models have two disadvantages: the lack of details and a static character. Experimental techniques such as neutron scattering, FTIR and Raman spectroscopy were used to investigate structure and dynamics of the hydrogen bonding in liquid water. Decomposition of the experimental Raman band in four components assigned to four species of clusters with Hbonds diering in strength and number was proposed by
415
Luu, Luu, Rull, and Sopron (1982). Such a model may be adopted as a reference for comparative studies of the eect of solutes on water structure. 4.2. Hydrophilic interactions Cations of interest in the food industry like Na ; Ca2 or Mg2 are known to immobilise several layers of hydration water. Generally such ions are known to have three layers of water around them. A ®rst layer strongly bound to the ion, a second layer of perturbed water because it is submitted to the attraction due to electrical ®eld of the ion on the one hand and to the ``cooperativity'' of H-bonds with water molecules of the third layer where water is less in¯uenced by the ion. Beside this type of attraction of water molecules around a hydrophilic solute, the hydration of small non-electrolytes and biopolymers is generally considered as hydrophilic. Small hydrophilic molecules like sugars have a sphere of hydration characterised by a number of hydration (®ve for sucrose) and a ``structure maker'' eect as they have a long-range eect of orientation of bulk water molecules. The hydration of food polymers is sensitive to their conformations and this can be measured by dierential scanning calorimetry (Silvonen, Lindberg, Seppala, Evasti, & Hautahoo, 1982). It is also important to take into account the eect of the phase (gel, emulsion,) in which the biomolecules are embedded, which may change their conformation. In particular globular proteins may change conformation as concentration is increased and temperature raised to expose their hydrophobic backbone to water and establish intramolecular H-bonds. Interactions of water with the hydrophobic surface of folded proteins corresponds to the so-called hydrophobic hydration. 4.3. Hydrophobic interactions Hydrophobic interactions are due to the hydration of apolar groups like methyl groups. Interaction between such a group and water is weaker than water±water hydrogen bonding, so that, when several water molecules surround an apolar group, they do not have a direct contact with the group and hence reinforce their water±water bonds. This layer of water is much less mobile than bulk water. Its enthalpy is decreased and its entropy is increased. Hydrophobicity of proteins may be at the origin of the stability of emulsions. Processed meat products may be considered as emulsions where hydrophobic hydration of the proteins together with the hydrophilicity of salt and glycerol tends to immobilise a maximum of water and to increase the shelf-life (Lacroix & Castaigne, 1984).
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Beside the structure of water near hydrophobic biopolymers, it is needed to know the type of organisation of water molecules in the vicinity of solid interfaces to interpret their sorption±desorption behaviour, which very often exhibits hysteresis phenomenon. 4.4. Interfacial water and sorption hysteresis Physical properties of interfacial water seem to be dierent from those of bulk water. Water molecules in the vicinity of adsorption sites at the surface of a solid are more rigidly organised. The kinetics of adsorptoin of water by proteins at air±water interface is very slow (Terminassian-Saraga, 1981), and protein denaturation is found to depend on surface tension of the adsorbed ®lm of water. Water vapour sorption by foods is sensitive to surface tension. This was demonstrated (Labuza & Rutman, 1968) by use of tensioactive agents in model systems. Increasing the surfactant in the formula induces a decrease in water content. Surfactants also decrease the hysteresis amplitude. Moistening by the tensioactive agent reduces to 0 the dierences between contact angles during sorption and desorption processes. Moreover, an ``ink bottle'' model for capillaries seems to be convenient and supports the hypothesis of surface tension decrease by the surfactant, which changes the mechanism of capillary ®lling. 4.5. Water structure and food preservation Extending the shelf-life of a food product consists in preventing its degradation by biochemical reactions or microbial growth. Prevention of degradation means prevention of availability of water for degradation reactions (Mathlouthi, 1986). To achieve such an objective, it is necessary to reinforce the hydrogen bonding of water and to reduce its mobility. Such a result may be obtained through the formulation of the product and the control of the type of hydration by establishing water vapour sorption curves. It is also useful to know the thermal history of the product (thermal treatment, storage temperature) in order to interpret the change in mobility of water. For the inhibition of growth of micro-organisms, it is needed to lower aw . However solutes like glycerol, although good water activity depressors, may enter the bacterial cell without causing an osmotic stress, so that bacteria grow in presence of glycerol at low aw values. It seems that viability of pathogen bacteria in foodstus is controlled by osmotic pressure regulation with dierent solutes (Sperber, 1983). So that retention of water by an additive is not the sole parameter in formulation. It is also needed to know the eect of solute on water mobility.
5. Conclusion The sole determination of water content, even using accurate methods like Karl Fischer titration, is not sucient to predict their shelf-life. Water activity proves to be a good indicator of the preponderance of one or another of the degradation reactions that might occur during storage of foodstus. For example, lipid oxidation occurs below aw 0:30 and Maillard reaction exhibits an optimum for aw 0:65. To understand the mechanisms of degradation reactions and why some foods are stable whereas others are not stable at the same aw , there is a need of knowledge of the nature and concentration of the dierent components and of water structure. Mobility of water and its availability for biochemical reactions depend on the type of interaction it might have with solutes. Approaching water in foodstus, especially in view of studying storage stability and shelf-life should rely on three types of information: analytical determination of water content, thermodynamical activity of water and the unveiling of water structure in presence of the soluble constituents of the foodstu. References Bervelt, E. (1975). Dosage de l'humidite residuelle dans les vaccins lyophilises. Journal of Biological Standardisation, 3, 321±330. Brunauer, S., Emmett, P., & Teller, E. (1938). Adsorption of gases in multimolecular layers. Journal of American Chemical Society, 60, 309±319. Frank, H. S. (1974). In W. A. P. Luck (Ed.), Structure of water and aqueous solutions (pp. 10±47). Weinheim: Verlag Chemie. Guinot, P. (1988). Contribution a la prolongation de la duree de vie d'une genoise industrielle. Ph.D. Thesis, Dijon. Isengard, H.-D. (1995). Rapid water determination in foodstus. Trends in Food Science and Technology, 6, 155±162. Labuza, T. P., & Rutman, M. (1968). The eect of surface active agents on sorption isotherms of a model food system. Canadian Journal of Chemical Engineering, 46, 364±368. Lacroix, C., & Castaigne, F. (1984). Emulsi®cation des viandes: r^ oles et actions de certains parametres de composition: proteines vegetales, sel, sucre et glycerol. Science Aliments, 4, 505±511. Lee, S., & Labuza, T. P. (1975). Destruction of ascorbic acid as a function of water activity. Journal of Food Science, 40, 370±373. Luu, C., Luu, D. V., Rull, F., & Sopron, C. E. (1982). Etude par eet raman de la perturbation de l'eau liquide par une substance etrangere: I ± Modele d'association de l'eau liquide. Journal of Molecular Structuring, 81, 1±10. Mathlouthi, M. (1986). In M. Mathlouthi (Ed.), Food packaging and preservation, theory and practice (pp. 137±164). London: Elsevier. Silvonen, R., Lindberg, J. J., Seppala, C., Evasti, M., & Hautahoo, C. (1982). Studies on water retention and water release from some protein systems. Journal of Thermal Analysis, 25, 101±105. Sperber, W. H. (1983). In¯uence of water activity on foodborne bacteria ± a review. Journal of Food Protection, 46, 142±145. Terminassian-Saraga, L. (1981). Protein denaturation on adsorption and water activity at interfaces: an analysis and suggestion. Journal of Colloid and Interface Science, 80, 393±401. Troller, J. A., & Christian, J. H. B. (1978). Water activity and food (pp. 13±47). New York: Academic Press.
M. Mathlouthi / Food Control 12 (2001) 409±417 Verhoef, J. C., & Barendrecht, E. (1976). Mechanism and reaction rate of the Karl Fischer titration reaction ± Part I: Potentiometric measurements. Journal of Electroanalytical Chemistry, 71, 305.
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Vornhof, D. W., & Thomas, J. H. (1970). Determination of moisture in starch hydrolysates by near-infrared and infrared spectrophotometry. Analytical Chemistry, 42, 1230.