Food Structure Food Structure

Volume 12 Number 3 Article 3

1993

Water-holding Properties of Milk Protein Products - A Review Water-holding Properties of Milk Protein Products - A Review

W. Kneifel

A. Seiler

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Recommended Citation Recommended Citation Kneifel, W. and Seiler, A. (1993) "Water-holding Properties of Milk Protein Products - A Review," Food Structure: Vol. 12 : No. 3 , Article 3. Available at: https://digitalcommons.usu.edu/foodmicrostructure/vol12/iss3/3

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Food Structure, Vol. 12, 1993 (Pages 297-308) 1046-705X/93$5.00 + .00 Scanning Microscopy International, Chicago (AMF O'Hare), IL 60666 USA

WATER-HOLDING PROPERTIES OF MILK PROTEIN PRODUCTS- A REVIEW

W. Kneife11 and A. Seile.-2

1Department of Dairy Research and Bacteriology, Universi ty of Agriculture, Gregor Mendel-Sir. 33, A-I 180 Vienna, Austria

2Nestle Research Center, Vers-chez-les-Bianc, CH-1000 Lausanne 26, Switzerland

Abstract

Water-holding properties have been well recognized by food technologists among !he diversity of functional properties attributed to milk protein products. In gener­al , water-holding is accomplished by a complex ity of in­teractions between water and milk proteins. Besides the term water-holding, synonyms such as water retention, imbibing and hydration have been used to describe this phenomenon. This paper provides a clearer understand­ing of this parameter by considering some fundamentals of both the molecular structure of milk proteins and the physical in terrelationships between water and milk pro­tein powder particles. Differences in water-holding properties of milk protein products are frequently ob­served and may be due to the nature of the protein and to technological influences. Methods measuring water absorption and methods tha t measure water retention are applied for the examination of water-holding. By fo l­lowing this distinction, the principles of various methods (e.g., the Baumann method, the absorption capacity test, Farinographic procedures, net tests, filtration tests, modem instrumental techniques) are reviewed.

Key Words: Functional properties, milk proteins, water holding.

Paper received June 7, 1993 Manuscript received September 17, I 993 Direct inquiries to W. Kneifel Telephone number: 43 I 7654 6100 FAX number: 43 I 3 199193

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Introduction

Milk proteins exhibit a multitude of properties such as digestibility , high nutritive value, 'GRAS ' ('generally regarded as safe') conformity and compatibility with other ingredients in food formulations (Kinsella, 1988; Morr, 1989, 1991). In addition, the application of cer­tain powdered dairy products (e.g., some types of case­in , caseinates, coprecipi tates, and UF proteins and pro­tein hydrolyzates) can improve significantly the texture of many food products. There are typical features at­tributed to milk proteins which can be characterized as 'functional properties' (Table 1). Among these, water holding of milk protein products has been recogni zed to be of particular importance in food technology and re­search. Milk proteins can replace many functional in­gredients in a broad variety of foods ranging from dairy products, breads , biscuits , confectionery, meat products, pastries, soups, ice cream, infant foods, margarine, low fat spreads, etc. (for comprehensive surveys see Kirkpatrick and Fenwick, 1987; Modler, 1985; Morr, 1985; De Wit , 1984, 1985; Nienhaus and Reimerdes, 1987). Furthermore, wide-spread use o f milk protein produc\s in the non-food area has been promoted (e.g. , Harwalkar and Brown, 1989; Souihward , 1991; Veerman and Hutten, 1991 ).

The term 'water holding' covers a variety o f proper­ties (Fig. 1) which have been used as synonyms in the literature. Different theories of the principles of water­protein interac tions have been reported in the past. Ac­cording to Kinsella et a/. ( 1989), six basic forms of water associated wi th proteins can be distinguished : (1) Structural water, which is unavailable for chemical reac­tions; (2) Hydrophobic hydration water, wh ich surrounds apolar residues in a cage-like structure; (3) Monolayer water, which represents the first absorbed water bonded to protein groups and may be available for certain reac­tions; (4) Unfreezable water, which includes all water that does not freeze at the sharp transition temperature, depending on the content of polar side chains and on the amino acid composition; (5) Capillary water, which is mechanically held by surface forces in the protein mole­cule; (6) Hyd rodynamic hydration wa\er, which ' loosely'

W. Kneifel and A. Seiler

Figure 1. Parameters used for describing the 'water­holding ' properties.

gobu/ar fissured

agglomerate hollow

Figure 2. Kinds of particle structures of protein powders.

Table 1. Functional properties as frequently attributed to milk proteins

Wettability Solubility

Water-holding Surface activity

Emulsifying capacity Emulsifying stability

Viscosity Colloidal stabi lity

Foaming Organoleptic properties

surrounds the protein. Other models of protein-water interaction were described by many authors (e.g., Her­mansson, 1986; Mohsenin , 1986; Geurts er al., 1974). The amount of water associated with protein depends on factors such as the amino acid composition, the number of exposed polar groups, surface hydrophobicity, pH value, ionic composition and strength, temperature, and concentration (Kinsella el al., 1989).

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ln order to simplify the rather complex mechani sms of protein and water interactions in food systems, two

categories have been proposed (Kneifel er al., 1991): (I) the part of water that is bound to the molecule and is no longer available as a solvent ('absorbed water'), and (2) the part of water that is entrapped in the protein matrix ('retained water').

Several factors may affect the interactions between a powdered product and water irrespective if these inter­actions take place with pure water or water present in a food matrix . The wetting process is influenced mainly by the moisture and the structure of the powder. In this context, the contact angle between the powder particles and water plays an important role. The rate and extent of rehydration depend on mechanical influences (e.g., mixing and stirring). The chemical nature of milk pro­teins determines the nature of the system (sol ution, sus­pension, or dispersion). Subsequently, physical md chemlcal reactions take place, and a three-dimensiQnal network is formed that takes up and holds the water. This mixture also shows so-called swelling propert ies and is capable of holding/binding/entrapping water.

Milk protein products act differently in different food product applications. The differences are caused by variations in pH, salt concentration and surface ten­sion as well as by the processing conditions governed by temperature and mechanical effects. For instance, most milk powders show low dispersibility helow 40°C, whereas an optimum can be obseJVed at 60°C (Lascelles and Baldwin, 1976). The dispersibility of a milk pow­der is influenced by its particle dimensions. According to Singh and Newstead (1992) , the optimum particle di­ameter for well-soluble milk powders normally rar1ges from 150 to 200 f'm and is determined by processing condi tions. Increasing the particle size gradually im­proves both the dispersibility and the so-called 'instant' behavior of milk powders. Moreover, the properties of a co-matrix may play a dominant role in the dispersion process. A co-matrix may enable gel formation via trap­ping of water (Kneifel er al., 1991).

As described above, structure as well as porosity of the powder largely influence the initial phase of wetting. In general, six main forms of .structures are common with powder products (Fig. 2). Dried dairy products can be assigned either to the ' globular' , 'porous', ·fis­sured', 'agglomerate' or 'crude' type. Most spray-dried milk protein powders exhibit a typical porous to globular porosity , while roller-dried powders exhibit irregular structures. Micrographs of sodium caseinate powder particles from two different producers are presented in Figures 3a and b. Although both are spray-dried prod­ucts of comparable particle dimensions, significant dif­ferences in their surface st ructure can be observed. Compared to the powder shown in Fig. 3a, that in Fig.

Water-holding of milk protein products

Figures 3a,h. Scanning electron micrographs of two different sodi um caseinates, visuali zing the differences in powder structure.

Table 2. Average wate r-binding capaci ty of some milk proteins (Kinsella et al., 1989) .

Component Bound water (g/100 g product)

Caseinate (dry) 5.6

Caseinate ("w = 0.9) 40

11-Lactoglobul in (dry) 6. 7

11-Lactoglobulin ("w = 0.9)

Native casein micelle

32

200- 400

3b exhibits improved water-holding and dispersibili ty properties due to its pronounced porosi ty.

Different mechanisms govern the water sorption characteristics of a protein powder particle. A porous powder granule has different micro- and macrocapillar structures which determine its behavior in a fluid medi­um. Thus, several theories of sorption behavior are based on whether the structure of the sorbent is porous

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o r non-porous (Aguilera and Stanley, 1990). The area and charge of the surface as well as the shape of the capillaries influence directly water absorption and de­sorption (Jensen and Nielsen, 1982; Sanderson, 1978). The effect of these properties can be described graphi­cally as a hysteresis curve (Aguilera and Stanley, 1990).

Water-binding Characteristics of Milk Proteins

In contrast to globular proteins , intact casein micel­les are capable of binding relative large amounts of wa­ter. Water entrapment in the native micellar structure is partly accomplished by the colloidal calcium phosphate, and also by the hydrophilic nature of <-casein and its posi tion in the submicelles. As demonstrated by Hardy and Steinberg (1984) with casein , the balance within the ' triangle' water-protein-solutes is of particular impor­tance for its physical stability. Globular proteins such as 11-lactoglobulin display varying degrees of hydration, de­pending on denaturation , aggregation , and interaction with other proteins (De Wit , 1984 , Kinsella, 1984) . As a consequence of heat treatment, the protein is unfolded and may exhibit an increased water-binding capacity. While there is still some disagreement, most researchers have reported a sl ight increase in bound water as the protein denatures. The amount of water bound in dena­tured milk protein depends on the nature of the protein and the dry matter content (Bech , 1980). Furthermore, the firmness of the heat-induced network largely deter­mines water-holding , because water is entrapped more effectively in a firm structure than in softer gels (Plock and Kessler, 1992). Average water-binding capacities of selected dairy proteins are presented in Table 2.

It has been demonstrated that progressive preheating of milk fo r the manufacture of sodium caseinate im­proves the water-holding capacity of the products (Kneifel eta/. , 1990) . When the milk was heated at a rate of 120°C/min, the amount of water held by theca­seinate was more than 5-fold the amount held by the non-heated control caseinate (Knei fel et al., 1990). Increased water-holding propert ies were also observed with polymerized (Korolczuk, 1984) or chemically modi­fied (Canton and Mulvihill , 1983 ; Kroll et al. , 1984) ca­seins, high-calcium coprecipitates (Thomas et a/., 1974; Vattula et a/., 1979), and neutralized coprecipitates (Southward, 1985). On an average, skimmed milk pow­ders did not show different water-holding capacit ies as a function of heat treatment (Knightbridge and Goldman, 1975). These au thors tested doughs enriched wi th sever­al milk protein products. Based on the findings, they made a distinction between ' highly absorptive' (sodium caseinate, soluble coprecipitates), ' medium absorptive' (calcium caseinate, dispersible coprecipitates, insoluble coprecipitates), and 'low absorptive' (skim milk powder,

W . Kneifel and A. Sei ler

2 3 4 5 6 Degree of hydrolysis (o/o)

Figure 4. influence of enzymatic hydrolysis on the change of water-holding capacity (WHC) of caseins {Abert and Kneifel , unpublished). Casein suspensions were hydrolyzed with Bromelain (E.C. 3.4.22.4) (Biocon, Rosenheim, Germany) and Corolase PN (E. C. 3.4.24.4) (Roehm, Darmstadt, Germany) at pH 6.7 and at SS "C, followed by beat inactivation. Hydrolyzed products were dried using a Buechi 190 Mini laboratory spray drier (Buechi, Flawil , Switzerland), before examination of their WHC.

Tahle 3 . Methods for the assessment of water-hold ing properties (Kneifel et a/., 1991 ).

Measurement of water absorption

Baumann appamtus

Absorption capacity test

water retention

Net test and modifications

Centrifugation tests

Spcctrophotom. rehydration test Capillary suction method Viscosity strength measurement Pressure methods Farinographic methods Filtration tests

Cryoscopic osmometry Special instrumenta l tech-

Sorption isothenns niqucs (DSC, NMR, etc .)

casein, lactalbumin) products. According to Delaney ( 1976) , the water-holding capacity of whey protein con­centrate lies within the same order of magnitude as of skim milk powders.

In a recent study (Abert and Knei fel , unpublished) it was shown that some casein hydrolyzates obtained by enzymatic modification exhibited improved water-hold­ing capacity in comparison to non-treated products (Fig. 4). The results depended on the nature of the enzyme used, on whether the milk was preheated or not before casein production, and on the pH conditions during hy­drolysis. Negative statistical relationships (P < 0.001) between water-holding capacity and nitrogen solubility as well as emulsion activity index were evident. Other researchers (Mietscb er al. , 1989) used Alcalase" or

300

Neutrase ~ for enzyme treatment of milk proteins and ob­tained a relatively low water-holding capacity in the treated products.

Methods for Water-holding Capacity Measurement

According to Chou and Morr (1979), the term 'water-holding capaci ty ' can be defined as a quantitative indication of the amount of water retained within a pro­tein matrix under certain defined conditions. ft usually also includes entrapped water. fn this context , ' hydra­tion' must also be considered. It is expressed in grams of water associated with or occluded by I gram (dry weight) of protein (Mulvihill and Fox, 1989) and is closely related with the so-<:alled voluminosity (Swaisgood, 1982; Walstra, 1979) according to the following equation:

( I)

where V Voluminosity (cm3/g prote in) v? Specific vol ume of pure water v2 Specific volume of the dry protein d1 Hydration (g water/g protein)

Different methods that are usually applied for esti­mat ing the water-holding properti es of food proteins have been described by Kneifel eta/ . ( 1991 ). ln gener­al, the testing procedures are either tests unde r model conditions or tests in actual food systems (Harper, 1984). Many of the methods used in laboratories are ar­bitrary and empirical and are app lied as so-called inter­nal methods. Because of the lacking comparability of these tests due to different measuring principles, stand­ardized procedures that enable a speci fi c and reliable characterization of the milk prote in products should be developed. Another problem arises from the fact that many testing procedures were o riginally developed for substances other than milk protei ns and had , therefore, to be modified and adapted.

Methods for the assessment of the water-holding ca­pacity are based mainly on the application of e ither an external force such as pressure, centrifugation, and cap­illary suction of a porous material being in contact with the sample, or on the evaluation of swelling when meas­uring the fluid uptake or the amount of water released during filtration. In these methods, the amount of water released or held by the sample is examined . Despite the difficulty of evaluating the actual mechani sm associated with a give~ method , an attempt has been made to dis­tingui sh between the procedures that measure water ab­sorption and those that register water retention (Table 3). In the following section, the different testing princi­ples will be described according to this di stinction.

Water-holding of milk protein products

a)

c)

Circulating powder solution

Peristaltic pump

e)

Graduated capillary

Perspex chamber

b)

d)

f)

Powder sample

Water

~~~~ Prep.,e 12,. powder suspension and transfer in to MPS-1 devices

t ~ ~ Centrifuge for 10 min

..-. at 1, 100 rpm (ambient temp.)

~u~~

(4 replicates per sample)

~ Collect filtrate cups

~ Weigh filtrate cups

and determine

~ !the amount of released fluid !

~ ~: I ~ Suspend 4.5 g powder ~ §n 150 mL water of ao ·c

~ ~ Stir at 120 rpm for 2 h in a water bath at so•c

't Mix with a Jab homogenizer for2min Cool down to ambient temperature

t l'~ Ptpe t 50 mL mto a funnel

~ Collect ftltrate

ill I dunng 5 mm

11

= m a measurmg cylmder

WHC (ml) • 50 - F1ltrate Vol

Figure 5. Principles of some methods for the examination of water-holding properties o f milk protein products: a : the Baumann apparatus; b: the Absorption Capaci ty Test; c: the Spectrophotometric Rehydration Test; d: the Modified Net Test ; e: the Capi llary Suction Test; and f: the Filtration Test.

Water Absorption Measurement

Baumann apparatus

This is a c lassical device for testing the water uptake of a powdered product (Baumann, 1967). It consists of a thermostatized funnel connected to a horizontal gradu­ated capillary attached to the top of the funn el (Fig . Sa).

30 1

The powder is dusted onto a wetted filter paper that is placed on a fritted glass filter set on the top of the fun­nel. The water uptake by the sample at equilibrium is read off from the graduated capillary in milliliters and is ex pressed on a dry basis. Although a glass lid is used to minimize losses due to evaporation, a blank value should be determined and considered in the calculation.

W. Kneifel and A. Seiler

One drawback of this method arises from the fact that only small amounts (usually 20-500 mg) o f powder are tested. Due to the principle of this procedure, the results reflect the wettability properties rather than the water-holding characteristics. Thus, the procedure is useful primarily for studying the very first steps of water uptake of a prevailingly non-soluble powder when brought into contact with water. Hermansson ( 1972) monitored the water-uptake of different food proteins by means of the Bauman apparatus and found that soybean protein was distinctly superior to sodium caseinate and to a whey protein concentrate in terms of water-holding. Different times ranging from about 20 minutes (whey protein concentrate) to about 150 minutes (soybean pro­tein) were needed by the samples to reach an equilibri­um. The Baumann method is of advantage for pred ict­ing the water-binding capacity of hydrocolloids (Wallingford and Labuza, 1983).

Absorption capacity test

This method was developed by Seiler (unpublished) and is derived from the Baumann apparatus testing prin­ciple. It was applied for the first time by Kneifel et al. (1992). A plastic tube to which a paper membrane is at­tached at the lower end is filled with the powder sample and put on a fril (porosity 0) for a chosen time (Fig. Sb). The frit itself is in contact with water, at ambient temperature , which passes through the frit and the mem­brane into the powder cylinder. Weighing is carried out before and after water absorption and also after the wet and dry phases have been separated . These procedures allow determination o f the following parameters: ( I) the vertical propagation height of water in to the powder (mm); (2) the amount o f wetted powder (g) per area (cm2); (3) the amount of water (g) absorbed by I cm3 of dry powder; (4) the amount of water (g) absorbed by I g of dry powder.

With this method , the coeffi cient of variation of pa­rameter (4) lies within the range of 2 .4 and 12.6% (Kneifel et al., 1992) . In analogy to the Baumann appa­ratus method, the absorpt ion capacity test characterizes mainly the initial phase of water uptake, depending on factors such as wettability , capillarity, particle size, and dissolution effects. The rehydration initiation phenome­non of a powdered sample can , therefore , be studied by using this procedure. However, this method may be af­fected by the solubility of the les t material in water, be­cause highly soluble powders may diffuse back into the water via the membrane and the frit.

Spectrophotometric rehydration test

This method is based on continuous spectrophoto­metric measurements of the change in transmission den­sity of the dispersed powder as a function of time (De Wit and Klarenbeek, 1986). The device consists of a

302

cylindrical tube with a fritted glass bottom onto which a known amount of the sample is placed (Fig. 5c). The glass tube is connected to a spectrophotometer (set at 600 nm) equipped with a flow-through cell and an X-Y recorder. A defined volume of water is circulated by means of a peristaltic pump. An optical index that de­fines a kinetic relationship between the reconstitution properties and the absorption characteristics of the powder is then calculated.

More recently, a modification of this method has been introduced. Samples of protein-water mixtures are removed from the tube at two different times and are measured in a separate spectrophotometer (De Wit. 1989) . The rehydration behavior is estimated based on the differences between the two readings. As reported by those authors using this rehydration method , the pro­cedure has shown an acceptable reproducibility when a defined set-up of the testing assembly is used .

Viscosity strength measurement

Intrinsic viscosity of a protein-water mixture in­creases by the same facto r by which the volume fraction is increased during hydration. Based on this criterion, rotation viscometers have been used for calculating the hydration capacities of casein micelles (Dewan et al., 1973), whey protein concentrates (McDonough et al. , 1974), casein solutions (Korolczuk , 1982a), ac idic milk protein concentrates and caseins (Korolczuk, 1982b), as well as polymerized casein derivatives (Korolczuk, 1984). Cross-linked caseins of high viscosity exhibited higher hydration levels than untreated proteins. Korolczuk ( 1982a) used a defined fo rmula for calculat­ing the water-holding capacity from the data obtained by viscosity measurements. The equation has experimental­ly been proven valid fo r 68 casein samples. In contrast to models described earlier, thi s eq uation was found to be valid for a relatively broad range of protein concen­trations (Korolczuk , 1982a).

Farinographic methods

The Brabender farinograph technique has been mainly used for measuring the water absorption by flours, doughs and soybean products. Only a few modi­fi ed methods have been reported regarding its use in measuring the water absorption characteristics of flour and protein blends. Knightbridge and Goldman ( 1975) studied factors affecting the water-absorption capacity of dried milk products in doughs. Based on this technique, milk protein products have been classified by their suita­bility as dough ingredients. Heat-precipitated whey pro­teins , e .g. , take up between 70 and 147 g of water per 100 g of powder (Short, 1980). A relationship between the water-holding capacity and the protein content of the whey protein powders was observed. Other reports on farinographic testing (Delaney, 1976; Guy et al . , 1974)

Water-holding of milk protein products

describe the water-holding behavior of different whey protein concentrates used in doughs. In analogy to the procedures used for routine examination of dough, the constant-flour-weight (300 g) or constant-dough-weight (480 g) methods have been applied by most users of this method.

Cryoscopic osmometry

This technique has been chosen to test thickening agents such as hydrocolloids and only in one case to test dairy products (Tarodo de Ia Fuente and Alais, 1975). These authors used this method to monitor the solvation behavior of casein in milk . In principle, cryoscopic os­mometry measures a colligative property, related to the ability of a substance to depress the freezing point. The freezing point is then converted into an effective osmotic concentration expressed as milliosmoles per kilogram of water (Rey and Labuza, 1981 ).

Monitoring of sorption isothenns

Basically , water absorption characteristics of pow­ders in an atmosphere of defi ned relative humidi ty can be described by means of typical sorption isotherms (Aguilera and Stanley , 1990; Chou and Morr, 1979; Mulvihill , 1992; Ozimek eta/., 1992). The weight up­take after ex posing the powder sample to an atmosphere of given water activi ties (e.g., over saturated salt solu­tions) can serve as a measure for water absorp ti on. Corresponding values are expressed as g of water/ 100 g of protei n product (G rufferty and Mulvihill, 1990). In many proteins , a moisture equi libri um wi ll be obtained within 24 hours (Hagenmaier, 1972). However. 4 days were needed to equ il ibra te a para-casein preparation (Geurts et lll. , 1974). Whey powders usually exhibit differences in adsorption of moisture due to lactose re­crystralli z.ation. Lactose-free skim milk powders often give isotherms of sig moid shape (Kinsella, 1984).

Methods for Water Retention Measurement

Net test and moditications

The net te~t combines filtrati on and centrifuga tion procedures and is carried out using a special plexiglass equipment (Hermansson and Lucisano, 1982; Wierbicki et a/., 1957) . The assembly introduced by the former authors consists of a tube in which the gel is formed, a filter paper to be placed on a net (200-l'm mesh) fi xed between the upper and the bottom tube (inner diameter of II nun) . Following gel format ion in the upper tube (closed with a rubber stopper at the lower end) , the gel is cooled and the stopper is removed. Then the upper tube is connected with the lower part and the whole as­sembly is centrifuged at 790 g. Moisture loss of the gel is assessed by weighing the gel before ancl after centri fu-

303

gation. The result must be corrected for the water up­take by the filter paper. One advantage of th is method is that the low speed centrifugation used usually limits structural breakdown of the gel. Thus, only weakly re­ta ined or non-retained water will be separated .

A modification of this testing principle was de­scribed by Kneifel et al. (1992) and was applied for the examination of several milk protein powders. In this study , the Amicon Micropartition System MPS-1 (Ami­con Corp., Danvers, MA) , available commercially, was used to centrifuge the mixture. Previously, this device was also applied for the assessment of the amount of water-soluble nitrogen substances in caseinates (Kneifel and Beurel, 1990). The system consists of two plastic tube units with a filter in between and connected by clips. The filter has to be punched out from a Schlei­cher & Schuell fi lter paper sheet no. 287 (this type was found to be opti mal based on the results of preliminary trials). A 12 % powder suspension in dist illed water is stirred with 500 rpm for I hour at 80'C (Fig. 5d). The mixture is then made up to the initial weight with water to compensate for the amount of evaporated water. The sample reservoir is filled up to the mark wi th the mix­ture. Four devices are placed into a conventional Ger­ber centrifuge and centrifuged at ambient temperature for I 0 minutes at I, I 00 rpm. The mean weight of the four filtrates is used as a measure for the water-holding capacity. In a comparison wi th two other methods for the characterization of water-holding properties of a variety of milk protein powders, relatively low variation coeffi cients ranging from 0.5 to 5.7% were calculated (Kneifel eta/., 1992). In general, net tests can be as­signed more or less to the category of so-called applied tests, since they enable a simulat ion of actual food systems.

Other centrifugation tests

Many centrifugation methods have been described; the tests are based on high-speed or low-speed centrifu­ga tion of protein-water mixtures that are prepared under defined conditions (e.g., Hermansson and Lucisano, 1982; Luther eta/., 1983; Sollars, 1973; Sternberg et a/ . , 1976; Thompson et a/. , 1969). In principle, the protein-water mixture is centrifuged in a tube and ei ther the amount of the liquid released or the protein with the remaining water is weighed and the supernatant is dis­carded. One important drawback of high-speed tech­niques is the possible damage of the network structure. Thus, methods which apply relatively lower centrifuga­tion g-values are preferably used because they prevent structural changes.

Capillary suction methods

In addition to the so-called capillary volumeter (Hofmann , 1975), which has been developed primarily

W. Kneifel and A. Seiler

for the examination o f meat, a special device measuring the capillary suction potential has been described by Labuza and Lewicki ( 1978) and used for testing gelatin , starch and carrageenan gels. A diagram of this assem­bly is shown in Figure Se. The gel to be measured is placed in a polypropylene cup, layered with filter paper with a predetermined moisture content , sealed with a rubber stopper pierced by a glass capillary with a bo re of 0 .3 mm. This capillary avoids pressure formati on during the time when the cup is closed . After storing the cup with the sample at 6 °C for 72 hours, the equilib­rium water content of the filter paper is determined . A large contact surface and a thin gel layer should be en­sured to achieve fast movement of water from the gel in­to the filter paper. The measurement is affected by the initial gel concentration, the moisture of the fi lter paper, and the temperature of the equilibration experiment. A variation coeffi cient as low as 2.5% is reported for th is method (Labuza and Lewicki , 1978).

Pressure methods

ln analogy to the capillary suction test , pressur~ procedures are used mainly for the assessment of meat products (e.g. , Lee and Patel , 1984). A specimen is placed in a Universal Testing Machine for compression along the vertical axis and the released nuid is collected on prewe ighed dry filter paper sheets. The amount o f the expressed fluid is calculated from the weight guin .

Another pressure method was described for testing milk proteins (Kabus, 1972). The sample is weighed on a filter paper and pressed between two solid plates under defined conditions. The whole assembly is covt! red with aluminum foi l to prevent water evaporation du ri ng the procedure .

filtration tests

As schematically shown in Figure Sf, a dispersion or a solution of a powdered sample in wate r is prepared according to a standardized stirring and mixing proce­dure (Kneifel et al. , 1990; Rustad and Nesse, 1983). After a given equilibration time, the volume of water re­leased from an aliquot of the mixture is measured. This method has been used mostly to screen caseinates to be incorporated in processed cheese as additives. When us­ing this method , the water-uptake of the filter as well as the ability of the protein gel to clog the pores o f the fil ­ter paper have to be considered. The coeffi cient of va ri ­ation of this method is relatively high (Kneifel et a/., 1992).

Another filtrati on test was described by De Wit ( 1988) who estimated water binding in yogurt samples containing whey proteins as gelling additives. In that study , the amount o f fluid drained from 150 ml yogurt duri ng I hour was measured with graduated tubes, after storing the samples for 25 hours at 4 °C.

304

Special instrumental techniques

During the last decade, differential the rmal analysis systems have become a powerful tool for an in-depth study o f physical changes in food systems . The general purpose of these measurements is to monitor the differ­ence between enthalpy changes that occur in a sample and in some inert reference materia l during heating. Generally , the methods used to accomplish this objective may be divided into th ree types: (1) Differential thermal analysis (DTA) , (2) the Boersma DTA , and (3) Differ­ential scanning calorimetry (DSC). Details of these techniques have been extensively reviewed by Lund ( 1983).

DSC has been used mostly to s tudy protein denatur­at ion (e. g . , De Wit , 1988) and starch gelatinization (e.g., Wootton er al., 1974). However, it has also been demonstrated by some authors (Ruegg er a /., 1974; Ruegg and Blanc, 1976) that this technique is suitable for observing the hydration pheno menon in mi lk pro­teins. Berlin eta/. ( 1973) used DSC for the estimation of the amount of freezable water in whey protein con­centrates.

Nuclear magnetic resonance (NMR) has also been shown to be a valuable technique for investigating kinet­ic properties of water as well as protein-water interac­tions of milk proteins (Di Nola and Brosio, 1983; Farrell et a/. . 1989: Kuntz. 197 1 ). Lelievre and Creamer ( 1978) used NMR in a s tudy of protein-water interactions during the setting of curd , and Callaghan et a/. ( 1983) used it to characteri ze the state of water in cheese. Methods such as infrared and Raman spectros­copy are mainly of academic interest but may also con­tribute to the understanding of the behavior of non­freezable water (Schnepf, 1989).

X-ray and neut ron scauering techniques have made it possible to identi fy water molecules within protein structures. One limitation of thi s procedure is that the protein must be in a crystalline state and only the posi­tion of the oxygen atom can be determined (Schnepf, 1989).

Conclus ions

Water-holding properties of a protein are the result of a broad array of factors governing pro tein-water in­teractions in food systems.

Although there are several theories de..10eribing water-holding characteri stics o f prote ins, it has not been possible to define exactly the term 'water-holding ' . Thus, it is useful to use terms such as ' bound ', ' free' , o r 'structural' water speci fi cally in context with the measuring technique and the environmental condi tions employed. There are many methods avai lable that pro­vide information on the mechani sms of hydration and

Water-holding of milk protein products

water binding. However, many of them measure the complexity of chemical, physical, and mechanical parameters of the powder solution , dispersion, or sus­pension. This implies that each method should be viewed as one piece of a complex puzzle and a sufficient characterization o f the water-holding property can only be achieved when based on a set of different tests. The modified net test seems to offer some advantages over other measuring principles such as filtration and absorp­tion capaci ty tests in terms of precision.

Many of the important functional properties of pro­teins in foods are related to the interaction of water with food proteins. Milk proteins offer a considerable poten­tial in mediating and promoting functional effects. It has, therefore, become an important challenge for the food scientists to develop, improve, and suffic iently characteri ze the functionality of these substances.

Acknowledgments

Thanks are due to Mr. V. Fryder from Nestle Re­search for taking micrographs of the caseinate samples.

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Discussion with Reviewers

J. N. De Wit: What is meant by 'water not available as a solvent'? Is that water absorbed because of steric ex­el uSion of other solutes, or is this highly structured water around polar or apolar groups? Which techniques are used to characterize that type of water? Authors: As mentioned in the text , the division into two categories of 'bound water' was primarily chosen in order to simplify the complexity of protein-water inter­actions. Depending on the protein product to be exam-

W. Kneifel and A. Seiler

ined, 'water not available as a solvent ' may cover both the steric exclusion and the highly structured water. To elucidate the nature of that types of water in a gel, it would therefore be necessary first to consider the nature of the protein (e.g . , whether the protein may bind water ionically , through hydrogen bond reinforcement or rath­er excludes it because of containing hydrophobic side chains) (see Labuza and Busk , 1979) and its physical properties (e.g . , solubility , dispersibility , etc .), and then to chose the methodology (several methods will be nec­essary in most ~ases) to be applied.

P. S. Kindstedt : The 'water-holding' of natural cheese can vary considerably depending on the type and age of cheese. I believe that the state(s) of water in cheese plays an important role in determining textural and rheo­logical characteristics, therefore a better understanding of how water exists in cheese is needed. Which analyti ­cal strategies would you suggest to use in order to char­acteri ze the water phase and its state of ' boundness' in

cheese? Authors : Water-holding properties o f cheese are strongly affected by pH and salt content as well as by technological conditions (e.g., pressure, temperature). Geurts er al. (1974) (text ref.) reported that several methods (water non-solvent for various solutes, sorption isotherms, isopiestic methods) would be necessary for a sufficient characterization of water bindi ng properties in cheese. Additionally, NM R can serve as a useful tool. Based on NMR , it has been concluded (Callaghan era/. , 1983, tex t re f. ) that there is strong evidence that wa ter diffusion in cheese is confined to surfaces within the protein matri x. In general , if cheese is considered, a suggestive methodological strategy should not only focus the final product, but also the curd and the 'young' cheese. In this context , the survey on physical prope r­ties of curd syneresis given by Walstra et al. ( 1987) is recommended for reading.

308

P. S. Kindstedt: Dried grated Italian cheeses such as Parmesan and Romano have been popular in the U .S. for many years . Recently , a growing market has devel­oped for dried grated cheeses that traditionally have not been available in grated form , such as Cheddar. The hygroscopic propert ies of such cheeses can be quite dif­ferent than those of Parmesan and Romano, leading to problems with functionality . Which analytical strategies would you suggest to use to evaluate the water-binding characteristics of dried grated cheese? Authors: In order to evaluate such products, a rather simple method used for assessing the hygroscopicity properties of whey powders (comment: with this catego­ry of products , hygroscopicity problems are also fre­quently observed) might be adopted. This method has been described in a booklet by NIRO Atomi zer ( 1978) and is based on equilibrating a certa in amount of product on a frit , under an atmosphere o f de fined humidity ac­complished by a saturated NH4CI solution . A special glassware equipment is necessary.

Additional References

Labuza TP, Busk GC (1979) . Analysis of the water binding in gels. J. Food Sci. 44, 1379-1385.

NIRO Atomizer AIS ( 1978). Determination of hygroscopicity. Method Al4a. In: Analyrical Merhods for Dried Milk Products. NIRO Research Laboratory , Copenhagen, Denmark (in German).

Walstra P, van Dijk HJM , Geurts TJ (1987). The syneresis o f curd . In : Cheese: Chemistry, Physics and Microbiology - Vol I. General Aspecrs. Fox PF (ed.). Elsevie r Applied Science, London, 135- 177.