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shear history on the thermal gelation of whey protein concentrates
Food Hydrocolloids Vol.S no.I pp.45-61, 1994
The effect of temperature/shear history on the thermal gelation of whey protein concentrates S.M.Taylor and P.l.Fryer Department of Chemical Engineering, University of Cambridge, Pembroke Street, Cambridge CE2 3RA, UK Abstract. In many food processes proteins are heated and sheared prior to gelation. Experiments were conducted in which protein concentrate solutions at pH 7.0 and 5.2 were sheared for different times prior to monitoring subsequent quiescent gelation using a Rheometries dynamic spectrometer. A systematic method for the analysis of particle sizes in a protein gel has been developed. At pH 5.2, where denaturation controls gelation, shear during the initial stages of heating increased the gel strength, probably due to shear preventing rapid aggregation into weak networks, but long durations of shear decreased the gel strength. In contrast, the gelation of pH 7.0 protein solutions is aggregation limited. Aggregation is enhanced by shear; this is shown both by the formation of protein aggregates visible microscopically and an increase in the initial gel strength after shear. Breakage of the protein network as a result of shear has also been noted. The two processes act together; for pH 7.0 solutions the final gel strength is a more complex function of the time of shear before gelation than for pH 5.2. It is suggested that the rheology of a sheared gel might be described in terms of effective rather than actual protein concentration, i.e. that shear induces aggregation that reduces the amount of protein available to form a gel network. This method was successfully applied to gelation at pH 5.2, but was not as successful at pH 7.0.
Introduction
Whey protein concentrates (WPCs) are by-products of the dairy industry resulting from the manufacture of cheese or the separation of casein from skim milk. WPCs are used in the food industry for their functional properties, which include emulsifying, whipping and fat/flavour binding ability (1). In addition, whey proteins can form strong gels upon heating, and so can be used to give desirable textural properties to foods. Processing to give desired functional properties is difficult; protein and gel properties vary both with their physicochemical environment and processing history. The relationship between the processing which the proteins receive and the properties they impart to the final product is unclear. The thermal gelation of whey protein concentrates is generally accepted to involve the initial denaturation of the protein (unfolding of the native structure), followed by aggregation of the protein into networks (2). Three distinct stages of this process have been used (3,4): n ---,) d (denaturation), 2d ---,) d2 (initiation), d + d, ---,) dx + 1 (propagation), when n is native protein, d id denatured protein, and d, is an aggregate of x denatured protein molecules. Initiation and propagation are two separate stages in the formation of protein aggregates. Either of the three reaction steps could be rate-limiting for the thermal gelation of whey protein concentrates (4). Considerable work has been carried out to establish the effect of the physicochemical environment on whey protein gelation. Factors known to affect gelation include pH, ionic concentration, temperature and protein concentration (4,5). The rate-limiting step in the formation of gels from WPCs has been 45
S.M.Taylor and P.J.Fryer
identified at a range of solution pH and heating temperatures (3,4). Between 63 and 85°C gelation at pH 7.0 the rate-limiting step is propagation, the addition of denatured protein molecules to an aggregate. At pH 5.2, between 65 and 73°C gelation is limited by the initiation step, while it is denaturation limited between 75 and 85°C (4). Commercially, gels may well undergo shear during both production and subsequent processing; often solutions of WPCs, or products containing them, must be pumped during processing, often at elevated temperatures, such as during pasteurisation. Static gelation is controlled by chemical reactions. When solutions of proteins are sheared the overall aggregation process can be promoted by increasing the rate of collisions between particles. However, shear can also break up aggregates formed by reaction and mass transfer. It is difficult to predict which process will predominate in a given situation. Quantitative data on the variation of product properties with process conditions is of obvious concern to process engineers in the food industry. This paper studies the effect of processing whey protein solutions, prior to gelation, on their subsequent gel properties. Although the material is not as well characterised as a single protein would be, the conclusions are of industrial relevance, and the mechanisms found are of general interest. The paper first reviews work on (i) the effect of shear on protein below the temperature at which reactions occur, (ii) the effect of shear on collision-controlled aggregation and (iii) the effect of shearing protein before gelation. Experiments have been carried out to develop an understanding both of the processes which occur and the variations in product rheology which result. The eventual aim of the work is to quantify the sensitivity of gelation to shear and to produce equations which can be incorporated into design calculations; however, at this preliminary stage, only a semi-quantitative approach has been adopted. The experiments determine whether gel strength changes substantially with shear rate and duration and attempts to correlate the variation in strength with the structure of the gel observed microscopically.
Shear effect on protein structure at low temperatures Protein thermal denaturation and aggregation is slow below 60°C (6), so the effect of shear on protein structure can be separated from that of temperature. At low concentrations the effect of shear depends on shear rate. Both Steventon (6) and Qingnong et al. (7) found that WPC solutions with protein concentrations ::;20% (w/w) did not show time dependent behaviour at shear rates :::;3000 S-I. The protein structure is unaffected by these shear rates. However, at significantly higher rates (>5500 S-I) the protein is denatured (8,9,10), which will affect its subsequent heat gelation properties. At higher protein concentrations, the volume fraction taken up by the protein is such that aggregates of molecules in solution are common; Steventon (6), Qingnong et al. (7) and Pradipasena and Rha (8) have found evidence of the breakup of aggregates during shear at high concentration. Mechanisms for aggregate breakup are discussed below. 46
Temperature/shear history in protein gelation
Effect of shear
Oil
the growth and breakup of aggregates
Mass transfer as well as reaction can affect the rate of aggregation. The effect of shear on mass transfer controlled aggregation has been well studied for both WPC and other proteins . Mass transfer controlled aggregation under hydrodynamic shear is a two stage process: diffusion controlled (perikinetic) growth followed by hydrodynamic shear controlled (orthokinetic) growth (II). Proteins initially aggregate at a rate determined by diffusion controlled collisions to form primary aggregates (6) of - 0. 1- 1 urn (6,11,12 ,13) . These primary aggregates are large enough for fluid motion to become important in promoting collisions, so aggregation switches from peri kinetic to orthokinetic control (II). Classical theories for such aggregation are based on the equations for the rates of both peri kinetic and orthokinetic aggregation developed by Smoluchowski (14). These equations predict that particles can grow indefinitely during shear, which is clearly unreal istic due to particle breakage . Maximum aggregate sizes in shear have been observed by several workers (15,16,17). Although no maximum size was detected, Steven ton (6) found that WPC aggregates sheared for the same time at 1476 s" J were smaller than those formed at 288 s" J . The net growth rate and size of any protein aggregates formed under shear will depend on the balance between growth and shear-controlled breakage (15,17). Parker et al. (18) proposed the following mechanisms for breakage: (i) deformation and rupture of particles by fluctuating dynamic pressures; (ii) erosion of primary aggregates from the surface of aggregates by either or all of aggregate-aggregate and aggregate-solid surface collisions, and by shear stresses generated by the fluid motion ; (iii) fragmentation of aggregates . Bell and Dunnill (16) found the dominant mechanism of soya protein aggregate breakup was fragmentation, i.e. mechanism (iii), with erosion occurring to a lesser extent: fragmentation occurred first, reducing irregularlyshaped aggregates to more regular shapes, and erosion then became the dominant mechanism . Twineham et al. (19) found the breakup of a soya protein precipitate was due to the removal of fragments by particle-particle and particle-surface collision, rather than directly by hydrodynamic shear. Modifications to the Smoluchowski analysis to include breakage (e.g. 19,20) have been successfully used to model the isoelectric precipitation of soy protein (19,21). Effect of pre-gelation shear on gelation Since many gelation reactions occur by aggregation reactions between denatured proteins, it would be expected that if proteins were denatured by the shear or gelation was aggregation reaction controlled, the gelation of protein solutions which have been previously heated and sheared would be different from unsheared protein solutions. The effect of high shear rates prior to the heat gelation of whey proteins was studied by Ker and Toledo (10) . Protein suspensions were sheared at 5800 s I for an unspecified length of time at both 25 and 70°C, and then gelled at 70°C. Sheared protein suspensions both gelled more rapidly than unsheared, and produced stronger gels (10).
47
S.:\1.Taylor and P.J.l'ryer
The above work is relevant to extrusion or homogenisation because of the high shear rate. The effect of temperature and lower shea r rates prior to quiescent gelation. such as the effect of flow through a pasteuriser holding tube before gelation , is indu stri ally rele vant but has not been studied . Under the se circumstances. the effect of she ar ma y be complex: as shown above . shear can (i) cause denaturation at high shear rates. (ii) enhance aggregation by promoting collisions, or (iii) enhance breakup of protein aggregates . All three processes might occur simultaneously; however. it is very unlikely th at aggregates would form at shear rates large enough to cause denaturation . Re alistically, mechanisms (ii) and (iii) will be the two competing ones in process equipment other than in homogenisers. Although shear can incre ase both aggregation and breakage , it is unclear which process will predominate . An experimental programme was thus co nd ucted to investigate th e effect of shear on gelation of WPC at two pH values where different mechanisms control the overall process.
Materials and methods Solutions of a commercial whey protein concentrate containing 75% protein (C arbery Milk Products. Eire; composition in Table I) were made up to a protein concentration of 14% (w/w) in distilled water. The pH was adjusted to 5.2 or 7.0 by adding 1 mol/drrr' NaOH or 1 mol/drn' HCI, and allowed to equilibrate for 2 h while stirring a t room temperature . Aliquots (6 ml) or prepared whe y protein solutions were placed in the 12.5 mm cone and plate test fixture (0.1 radian cone angle) of a Rheometries RDS II dynamic spectrometer (Rheometries Inc. , Union. NJ) which was preheated in a watcrbath to 85 ± O.l °C and subjected to a steady shear of 0-40
Table I. Composition of the whe y prote in conce ntra te used Co mpo nent
Fracti on of powder (% w/w )
13-lactoglobulin a-l act albumin Bovine serum albumin Immunoglobulins Proteose pcptones
37.50 11.25 3.75 6.00 16.50
Total protein Lactose Fat
75.0
Ca' .
B.O 9.0
Na ' K'
0.35 0.26 0.18 0.65
Total ash Moisture
4.0 4.0
PO}-
48
Temperature/shear history in protein gelation
for times between 0 and 240 s; the samples took <20 s to reach the final temperature. Solutions were then allowed to gel in situ at 85°C. All conditions of pH, shear rate and duration were carried out in duplicate, with the control conditions (shear at 0 S-I) repeated four times. The rheology of the gelling protein solutions were followed in oscillatory mode at a strain amplitude of 0.5% and a frequency of 1 rad.s -1. These test conditions were chosen to fit within the linear viscoelastic region of the material, where G' and Gil are not functions of frequency, as shown in Figure 1. G', the storage modulus, is an order of magnitude larger than Gil, and was thus used here to quantify the structure of the gel; the gel strength was taken as G' after 45 min (5). The maximum rate of increase of G' with time (dG'/dt m ax ) was taken as a measure of the rate of development of the gel fraction. The maximum rate was measured by fitting a straight line to G' data for the initial stages of gelation, where G' was rapidly increasing with time. The region where the increase was greatest was found by linear regression, and taken as dG' /dt m ax (5). The time interval over which data was fitted was sufficiently small to allow an of at least 0.90. In addition, the first measurement of G' after completing the shear, G'o, was taken as a measure of initial gel structure since G' measures the sample elasticity. The rheological data reflect the effect of shearing the sample during its gelation. Shear rates were chosen to represent those likely to be experienced during flow in a pipe, as well as being easily attainable in the Rheometries. The application of shear led to the formation of gels made up of visible aggregates of particles bound together within a gel matrix. Conventional particle sizing techniques, such as the Malvern Mastersizer used by Steventon (6), which rely on the particles being separable so that they can be individually sized, could thus not be used here. Aggregate sizes were estimated by the following process: S-I
,2
(i)
after completion of the time sweep, gels were carefully removed from the text fixture of the rheometer and stored in sealed plastic bags at 4°C to prevent deterioration. (ii) photographs were taken of thin regions of the sheared gels through a light microscope. More than one photograph was taken of each gel so that 1ססOO-.------------,
00000000000000000000000000000
~
1000
~
100
~
10
~ 1+----,--------,------j 0.1 1.0 10.0 100.0 Frequency (.. ')
Fig. 1. Frequency sweep for 14% (w/w) WPC gel formed at 85°C. Strain amplitude = 1%. G' (0), Gil (0).
49
S.M.Taylor and P.J.Fryer
representative samples were obtained. The magnification obtained for 135 mm x 90 mm prints was 50x. (iii) the sizes of the aggregates making up the gel were then measured using image analysis of the photographs. using an Optomax V image analyser (Analytical Measuring Systems. Saffron Walden, UK). Preliminary selection of aggregates was done using the Optomax and visually; if they lacked enough contrast to be detected by the image analyser, lines were drawn around particles with a marker pen. Areas of particles were then measured, and equivalent circular diameters calculated by the image analyser to obtain the particle size distributions. The data from all photographs of the same gel were pooled to give more accurate results. (iv) the arithmetic mean of the particle size distribution was used for further data analysis. This method is illustrated by Figure 2: the photograph of the sheared protein gel (a) did not have sufficient contrast to allow image analysis. so particles were highlighted in (b). Image analysis of the highlighted particles gave rise to the particle size distribution shown in (c). This procedure is obviously approximate and selection of aggregate size is partially subjective. However. since the method is systematic. it is felt that trends in the data will reflect actual changes in aggregate size. and enable differences to be determined. It is emphasised that the diameters calculated from the image analysis are not equivalent spherical diameters. The growth of large particles was limited in the vertical direction by the dimensions of the rheometer test geometry. However, it was felt that this would not seriously affect the growth of the smaller particles. Results
Effect of shear at pH 5.2 on gel strength Figure 3 shows the effect of the application of shear rates of 10-40 s 1 for durations of 15-240 s on the gelation of WPC at pH 5.2. The two horizontal lines in Figure 3(a) represent the range of gel strengths of the control (unsheared) material. Figure 3(a) shows a clear relationship between shear and gel strength. For <90 s of shear, the final gel strength increases, especially at a shear rate of 20 S-I. There is an approximately linear decrease in the gel strength with increasing duration of shear, until after 180 s all the gels are weaker by a factor of -2 than unsheared material. A similar effect can be seen in Figure 3(b), which shows the effect of shear on dG'ldt m ax , again compared with un sheared material. Figure 3(c) plots the first measurement of G' after the end of shear (G'o) against the time of that measurement; i.e. for material presheared for 120 s, G' 0 points are seen at 120 s on the horizontal axis. The lines plotted on Figure 3(c) are the time sweeps for the unsheared protein solutions. To determine the effect of the shear on the network structure. the presheared data should be compared with the unshearcd data at the same time. The sheared data lie consistently at or below the unsheared controls. 50
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Fig. 2. Extraction of particle sizes from photographs of gels of sheared WPC75 solutions. (a) Original photograph of gel formed from 14% (w/w) pH 7.0 protein solution sheared for 180s at 40 S-I and 8Ye. (b) Particles highlighted prior to image analysis of the photograph. (c) Particle size distribution from image analysis.
51
S.M.Taylor and P.J.Fryer
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Fig. 3. Effect of shear at rates of 10 (0), 20 (.) and 40 S-I (+) on gel rheology at pH 5.2: (a) gel strength; (b) dG'/dtm ax ; (c) G'i; The horizontal lines indicate the range of G' and dG'/dtm a x measured for gels formed from the unsheared protein solutions, while the lines in (c) are the time sweeps for un sheared protein solutions.
52
Temperature/shear history in protein gelation
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Fig. 4. Effect of shear at rates of 10 (0), 15 (A), 20 (_), 30 (x ) and 40 S-1 (+) on gel rheology at pH 7.0: (a) gel strength; (b) dG'/dtm ax ; (c) G'(; The horizontal lines indicate the range of G' and dG'/ dt m a x measured for gels formed from the unsheared protein solutions, while the lines in (c) are the time sweeps for unsheared protein solutions.
53
S.M.Ta)lor and P.J.Fryer
Effect of shear at p H 7.0 on gel strength Figure 4( a) shows the effect of the application of shear rates of 10-40 s-) for 15240 s on the strength of WPC gels at pH 7.0 and R5°C. Unlike the data for pH 5.2. which showed a clear trend, the scatter in the sheared data is considerably greater than the control data , i.e. the range between the lines. It is thus immediately obvious that shear does affect gel strength. However, since the spread of the data is considerable. there is no clear change in gel strength with shear history. The data presented in Figure 4(a) suggests the following trends for more than 60 s of shear. although no claim can be made for statistical significance: (i) protein gels formed after shear at 10, 30 and 40 S-1 are generally weaker than unshcarcd protein gels; (ii) protein gels formed after shear at 15 and 20 s I are on the whole stronger than unshcared protein gels. However. some gels. particularly at longer shear durations. were much weaker. Figure 4(b) shows the variation in dG'4/dt m " , as a function of shear rate and duration in comparison with the rate for unsheared material. The application of shear obviously affects the rate of formation of gel structure, and trends can be detected after -60 s of shear:
(i) dC' /dt m a x is essentially unaffected by shear at the lowest and highest shear rates used , i.e . 10 and 40 s "' ; (ii) dG'/dt m ax increased for shear rates of 15, 20 and 30 s 1. Figure 4(c) plots G'o against time for pH 7.0; as in Figure 3(c). data for unshcared controls are also given. This shows that nearly all of the sheared protein gels had a higher initial gel strength than the controls. i.c . that there is more network structure in the sheared gel, reflected in the elasticity of the system .
Effect of shear on aggregate formation The application of shear at pH 7.0 for at least 60 s lead to the eventual formation of visible particles in the gel matrix which were not present in the unsheared gels. Particles were not visible through an optical microscope in gels formed at pH 5.2, even at magnifications x 100, 2.5 times greater than that necessary to recognise particles in the pH 7.0 gels. Data extracted from photographs of the gels using the image analysis technique are given in Figure 5. Statistically significant differences were seen between the sizes of particles formed at different rates and durations of shear:
(i) 54
in gels sheared at rates of 10 and 15 S-I, the size of particles increased significantly (95% level) with the duration of shear;
Temperature/shear history in protein gelation
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(ii) however, the size of particles in gels sheared at higher rates of 20, 30 and 40 S-1 did not change with shear duration (90% level); (iii) for a duration of shear >60 s (the gelation time at 85°C, 22), the size of particles varied with shear rate. Gels sheared at 15 s-] had the largest particle size by at least a factor of two. 10 s-] came next, followed by 20 and 30 s -1 (not significantly different), then 40 s-]. Where differences were seen, they were in all cases significant at the 95% level. There was no direct correlation between particle size and rheological properties of the gels. Discussion The effect of temperature/shear history has been studied experimentally at two pH values at 85°C, one (pH 5.2) at which denaturation is controlling, and one (pH 7.0) at which aggregation controls the gelation process (4). The pH 5.2 G' and dG'/dtm a x results suggest that two effects are occurring: (i) the initial increase in final gel strength with duration of shear must reflect an increase either in bond strength or the number of bonds brought about by 55
S.M.Taylor and P.J.Fryer
shear. Steventon (6) noted that at pH 5. aggregation rapidly formed an irregular structure of loosely packed aggregates. due to rapid aggregation after denaturation . as shown by Taylor et al. (4) . The application of shear over short periods of time (30 or 60 s) probably pre vents these loose aggregates forming, making a larger number of bonds available for the network, and preventing rapid aggregation into weak networks. (ii) as the time for which the material is sheared increases , however, the final gel strength decreases. During shear, when protein molecules denature , they will form aggregates within the fluid. These particles will bond less strongly to other aggregates during subsequent quiescent gelation . The G'lI measurements again demonstrate that shear leads to weaker structures; proteins which have aggregated in the bulk form weaker bonds to other aggregates once shear is complete. The effect of shear at pH 7.0 on the gelation of WPCs is more complex; there is a large variation in the gel strength. These results suggest that shear is enhancing aggregation (leading to increased gel strength) as well as causing breakup of aggregates (leading to weaker gels). G'i, for the sheared protein solutions were higher than the unsheared solutions, suggesting that aggregation is enhanced by shear. This is consistent with the results of Taylor et al. (4), who found that gelation was aggregation limited at pH 7.0. At pH 5.2 no aggregates were visible through the optical microscope , even at magnifications of x 1(}() (2.5 times that used for the pH 7.0 gels). This is to be expected since the gelation process of pH 5.2 solutions at 85°C has been found to be denaturation limited (4) ; the increased frequency of collisions due to shear would not lead to an increase in the aggregation rate , so visible particles do not form. However , the formation of visible particles as a result of shear during the early stages of gelation shows that aggregation of whey proteins at pH 7.0 is enhanced by shear. The increase of particle size with shear duration at 10 and 15 S-I , coupled with the fact that no particles are visible in unsheared gels, also suggests that enhancement of aggregation is occurring as a result of shear. At higher shear rates (15-40 S-I) particle size decreases as shown in Figure 5; since the physical conditions, temperature and pH are the same, this clearly indicates that aggregate breakup is occurring . The particle size increase with duration of shear at 10 and 15 s -1 suggests that a limiting particle size had not been reached over these shear durations. Particle size varies with shear rate and time at pH 7.0. No correlation was observed between particle size and rheology of the gels; the data were clustered according to shear rate. Shear rate and time thus appear to determine the gel structure directly, rather than producing particles of a given size which then determine the mechanical properties of the gel.
Calculation of effective concentration of sheared protein gel matrices Data suggests that during shear prior to quiescent gelation, denaturation and 56
Temperature/shear history in protein gelation
aggregation of the protein will occur to form discrete particles. This decreases th e amount of protein available to react and form a network during subsequent gelation . The change in the mechanical properties of the pre-sheared gels ob served above is as a result of reactions during shear. Thi s might be expressed as a change in the 'effective concentration', Ceff , of the material; i.e. if, from a 10% (w/w) whey protein solution , 20% of the protein aggregates during the preshearing process, the material may subsequently behave as if it were an 8% (w/w) solution. This hypothesis has been tested below. The concentration dependence of G' and dG'ldt m ax at pH 5.2 and 7.0 was obtained (22). Best-fit lines were fitted to this data and rearranged to find the effective concentration which produces a given G' or dG' ldt m ax : pH 5.2, 10-18% (w/w) protein solutions: Cc ff
=
(53.48
G,)1 /4.46
(1)
pH 7.0, 10-16% (w/w) protein solutions:
Cef f
= (G' + 7780)/820
(2)
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=
(465 x 105 dG'ldt ma x )1 15 .26 ·
(3)
pH 7.0, 10-20% (w/w) protein solutions: Ceff
=
(1 ' 78 x 107 dG' ldt max ) 116.80
(4)
Equations (1)-(4) were used to calculate the effective concentration as a function of the length of shear before quiescent gelation, and hence determine whether it is a useful concept. For each combination of shear rate and length of shear, G' or dG'ldt m ax was found experimentally. The appropriate equation was then used to find Ceff . Measures of effective concentration can be found for each gel from both G' and dG'ldt m ax using equations (1,2) and (3,4). Figure 6 shows the variation with time of shear of the effective concentration of gels formed at pH 7.0. The effective concentrations suggested by dG'ldtm ax are higher than the real concentration, whilst that found using the final gel strength is generally less. The effect of the duration of shear also appears different; Ceff calculated from G' decreases, and that from dG'ldt m ax increases with time . This shows that effective concentration is not a useful concept in modelling the effect of shear on the gelation of pH 7.0 solutions of WPC; if it were , the two measures by which it can be estimated should be similar. Figure 7 shows the variation of effective concentration formed from sheared pH 5.2 solutions. In contrast to pH 7.0, the data from G' is similar to that from dG'ldt m ax ' Unlike pH 7.0 , Figure 7 shows that at pH 5.2, the effective 57
S.M.Taylor and P.J.Fryer
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Fig. 6. Effective concentration of protein in pH 7.0 gel matrices formed from shear at rates of (a) 10, (b) 20, and (c) 40 S-I. Effective concentration calculated from the G' (0) using equation (3) and dG'/dtm ax ( . ) using equation (5).
58
Temperature/shear history in protein gelation
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Duration of shear (s)
Fig. 7. Effective concentration of protein in pH 5.2 gels calculated from G' (0) using equation (4) and dG'/dtm ax ( . ) using equation (6). Gels formed during shear at rates of 10 (a), 20 (b) and 40 S-I
(c).
59
S.M.Taylor and P.J.Fryer
concentration calculated by both methods increases over the first 60 s of shear, and subsequently decreases. This is probably due to shear over the initial periods of heating preventing rapid aggregation of protein into disordered particles. However, shearing during the period that the gel would normally be forming (times greater than 60 s; 22) leads to a decrease in the effective gel strength because the crosslinks between aggregates cannot form. Here the concept of effective concentrations appears to work well; the effect of shear on the strength of pH 5.2 whey protein gels could be explained by the effect of the shear on the amount of protein available for network formation. More work will be needed to determine the usefulness of this idea. Generality of results As discussed above, the gelation properties of whey proteins are known to vary with composition. The material used in this work is a commercial WPC; it would be expected that variations would be seen between different' batches and between WPC produced from different sources. Table lshows that the material is similar to that studied by other workers (23); changes in the mineral level will affect gelation, either through direct interactions with the protein (addition of calcium) or by changing the ionic strength of the solution and thus the forces between proteins [addition of sodium (24)]. Whilst the numerical values of the data will vary, it is expected that as the WPC composition used here is typical, the trends seen will be common to other samples. Conclusions
Experiments have been carried out to characterise the behaviour of WPC solutions heated and sheared prior to gelation. The gelation of pH 5.2 protein solutions is denaturation limited. Shear for <60 s leads to an increase in gel strength due to the prevention of rapid aggregation of denatured protein. However, longer periods of shear lead to a decrease in gel strength as protein aggregates are disrupted by the shear. The gelation of pH 7.0 protein solutions is aggregation limited. When shear is applied prior to gelation, large protein aggregates form and the initial gel strength after shear increases, suggesting that aggregation is enhanced by shear. The final gel strength decreases with increasing time of shear, suggesting that shear has a secondary effect in breaking up protein networks, similar to that seen for pH 5.2 solutions. These two processes act together; data for the final gel strength is a more complex function of the time of shear before gelation for pH 7.0 solutions than for pH 5.2. Owing to the lack of optical contrast between particles and the medium, it was only possible to carry out a semi-quantitative analysis of the sizes of aggregates seen under the optical microscope. However, the protocol developed allowed the range of particle sizes measured at different shear rates and durations to be measured in a systematic way. The analysis also suggested that shear-induced breakup occurred at pH 7.0. 60
Temperature/shear history in protein gelation
It can be concluded that the application of she ar to whey protein solutions while the y are heated can gre atl y affect the strength and structure of the subsequent gels. Thi s suggests that processing equipment a nd procedures need to be car efull y controlled to avoid the disruption of the gelation process. An attempt has been made to express changes in the gel properties in terms of cha nges in effective protein con centration . This appears promising. More work will, howe ver , be needed to characterise the processes see n her e. In addition to work on industrial fluid s, there is a need for future work on purer syste ms, such as 13-lactoglobulin. Acknowledgements Dr M .R .Mackley is thanked for making available the Rheometries RDS II. Mr R .T .J.Marshall is also thanked for his assistance in collecting rheological data. PJF wishes to thank the staff of the Intensive Coronary Care Unit, Queen Elizabeth Medical Centre, Birmingham. The authors would like to acknowledge the financial support of the Cambridge Commonwealth Trust and Express Foods (Europe) Ltd. References 1. de Wit.J .N. (1984) Neth. Milk Dairy J. , 38, 71-89. 2. Ferry,J .D . ( 1948) Ad v. Protein Chem . 4 , 1-78. 3. Steve nto n,A J .. Gladd en ,L.F. and Fryer ,PJ . ( 1991) J. Texture Stud . , 22, 201-218. 4. Taylor ,S.M ., Gladden ,LF. and Fryer ,P .l. (1993) J. Dairy Res. , in press. 5. Tay lor,S.M . and Fryer ,P .l. ( 1992) Entropie , 168, 57-64 . 6. Stevento n,A J . (1993) Thermal aggregation of whey prot eins. PhD thesis, Un iversity of Cambridge . 7. Oin gnong ,T., Munro ,P.A . and McCarth y,a .l. (1990) Proceedings of the 18th Australasian Chem ical Engineering Confe rence. pp . 666-674 . 8. Pradip asen a ,P. and Rh a ,C. (1977) J. Textur e Stud. , 8, 311- 325. 9. Harri s.Ll., ; Pecar.MiA . and Pearce ,R.J . (1989) Aust. 1. Dairy Tech.. 44, 78-81. 10. Ker ,Y.C. and TOledo,R .T. (1992) J. Food . Sci. , 57, 82- 85. 11. Bell.D i.l. . Ho are,M. and Dunnill,P . (1983) A d~' . Biochem . Engng ; 26,1-72. 12. Chan,M.Y .Y., Hoare ,M. and Dunnill ,P . (1986) Biotech. Bioengng ; 28, 387-393. 13. Fisher ,R .R . and Glatz,C.E. (1988) Biotech, Bioengng ; 32, 777-785. 14. Smoluchowski ,M . ( 1917) Z. Physik . Chem ., 92, 129-1 68. 15. Virk ar ,P .D ., Hoare ,M ., Chan,M.Y.Y. and Dunnill.P. (1982) Biotech. Bioengng; 24, 871-887. 16. Bell,D .J . and Dunnill ,P . (1982) Biotech. Bioengng, 24,1 271-1285. 17. Hoare .M , (1982) Trans. I. Chem . Engng , 60, 79-87. 18. Parker,D.S. , Kaufman .W .l. and Jenkins ,D. (1972) J. Sanit. Eng. Div. Proc. Am. Soc. Civ. Eng. , 98 (SAl), 79-99. 19. Twineham,M., Hoare,M. and Bell,D .J . (1984) Chem. Eng. ss., 39, 509-513. 20. Harris,H.S., Kaufman,W.J . and Kron e,R.B. (1966) J. Sanit. Eng. Div, Proc. Am. Soc. Civ. Eng ., 92 (SA6), 95-111. 21. Fisher ,R .R . and Glatz ,C.E . (1988) Biotech. Bioengng , 32,786-796. 22. Taylor ,S.M. (1993) Rheology and biochem istry of whey protein gels. PhD thesis, University of Ca mbridge . 23. Jost.R. (1993) Trends Food Sci. Techn ., 4, 283-288. 24. Reynard ,D . and Lefeb vre,J . ( 1992) Int. J. Bioi. Macromol. , 14,287-291.
Received on September I , 1993; accepted on December 10, 1993
61
The effect of temperature/shear history on the thermal gelation of whey protein concentrates S.M.Taylor and P.l.Fryer Department of Chemical Engineering, University of Cambridge, Pembroke Street, Cambridge CE2 3RA, UK Abstract. In many food processes proteins are heated and sheared prior to gelation. Experiments were conducted in which protein concentrate solutions at pH 7.0 and 5.2 were sheared for different times prior to monitoring subsequent quiescent gelation using a Rheometries dynamic spectrometer. A systematic method for the analysis of particle sizes in a protein gel has been developed. At pH 5.2, where denaturation controls gelation, shear during the initial stages of heating increased the gel strength, probably due to shear preventing rapid aggregation into weak networks, but long durations of shear decreased the gel strength. In contrast, the gelation of pH 7.0 protein solutions is aggregation limited. Aggregation is enhanced by shear; this is shown both by the formation of protein aggregates visible microscopically and an increase in the initial gel strength after shear. Breakage of the protein network as a result of shear has also been noted. The two processes act together; for pH 7.0 solutions the final gel strength is a more complex function of the time of shear before gelation than for pH 5.2. It is suggested that the rheology of a sheared gel might be described in terms of effective rather than actual protein concentration, i.e. that shear induces aggregation that reduces the amount of protein available to form a gel network. This method was successfully applied to gelation at pH 5.2, but was not as successful at pH 7.0.
Introduction
Whey protein concentrates (WPCs) are by-products of the dairy industry resulting from the manufacture of cheese or the separation of casein from skim milk. WPCs are used in the food industry for their functional properties, which include emulsifying, whipping and fat/flavour binding ability (1). In addition, whey proteins can form strong gels upon heating, and so can be used to give desirable textural properties to foods. Processing to give desired functional properties is difficult; protein and gel properties vary both with their physicochemical environment and processing history. The relationship between the processing which the proteins receive and the properties they impart to the final product is unclear. The thermal gelation of whey protein concentrates is generally accepted to involve the initial denaturation of the protein (unfolding of the native structure), followed by aggregation of the protein into networks (2). Three distinct stages of this process have been used (3,4): n ---,) d (denaturation), 2d ---,) d2 (initiation), d + d, ---,) dx + 1 (propagation), when n is native protein, d id denatured protein, and d, is an aggregate of x denatured protein molecules. Initiation and propagation are two separate stages in the formation of protein aggregates. Either of the three reaction steps could be rate-limiting for the thermal gelation of whey protein concentrates (4). Considerable work has been carried out to establish the effect of the physicochemical environment on whey protein gelation. Factors known to affect gelation include pH, ionic concentration, temperature and protein concentration (4,5). The rate-limiting step in the formation of gels from WPCs has been 45
S.M.Taylor and P.J.Fryer
identified at a range of solution pH and heating temperatures (3,4). Between 63 and 85°C gelation at pH 7.0 the rate-limiting step is propagation, the addition of denatured protein molecules to an aggregate. At pH 5.2, between 65 and 73°C gelation is limited by the initiation step, while it is denaturation limited between 75 and 85°C (4). Commercially, gels may well undergo shear during both production and subsequent processing; often solutions of WPCs, or products containing them, must be pumped during processing, often at elevated temperatures, such as during pasteurisation. Static gelation is controlled by chemical reactions. When solutions of proteins are sheared the overall aggregation process can be promoted by increasing the rate of collisions between particles. However, shear can also break up aggregates formed by reaction and mass transfer. It is difficult to predict which process will predominate in a given situation. Quantitative data on the variation of product properties with process conditions is of obvious concern to process engineers in the food industry. This paper studies the effect of processing whey protein solutions, prior to gelation, on their subsequent gel properties. Although the material is not as well characterised as a single protein would be, the conclusions are of industrial relevance, and the mechanisms found are of general interest. The paper first reviews work on (i) the effect of shear on protein below the temperature at which reactions occur, (ii) the effect of shear on collision-controlled aggregation and (iii) the effect of shearing protein before gelation. Experiments have been carried out to develop an understanding both of the processes which occur and the variations in product rheology which result. The eventual aim of the work is to quantify the sensitivity of gelation to shear and to produce equations which can be incorporated into design calculations; however, at this preliminary stage, only a semi-quantitative approach has been adopted. The experiments determine whether gel strength changes substantially with shear rate and duration and attempts to correlate the variation in strength with the structure of the gel observed microscopically.
Shear effect on protein structure at low temperatures Protein thermal denaturation and aggregation is slow below 60°C (6), so the effect of shear on protein structure can be separated from that of temperature. At low concentrations the effect of shear depends on shear rate. Both Steventon (6) and Qingnong et al. (7) found that WPC solutions with protein concentrations ::;20% (w/w) did not show time dependent behaviour at shear rates :::;3000 S-I. The protein structure is unaffected by these shear rates. However, at significantly higher rates (>5500 S-I) the protein is denatured (8,9,10), which will affect its subsequent heat gelation properties. At higher protein concentrations, the volume fraction taken up by the protein is such that aggregates of molecules in solution are common; Steventon (6), Qingnong et al. (7) and Pradipasena and Rha (8) have found evidence of the breakup of aggregates during shear at high concentration. Mechanisms for aggregate breakup are discussed below. 46
Temperature/shear history in protein gelation
Effect of shear
Oil
the growth and breakup of aggregates
Mass transfer as well as reaction can affect the rate of aggregation. The effect of shear on mass transfer controlled aggregation has been well studied for both WPC and other proteins . Mass transfer controlled aggregation under hydrodynamic shear is a two stage process: diffusion controlled (perikinetic) growth followed by hydrodynamic shear controlled (orthokinetic) growth (II). Proteins initially aggregate at a rate determined by diffusion controlled collisions to form primary aggregates (6) of - 0. 1- 1 urn (6,11,12 ,13) . These primary aggregates are large enough for fluid motion to become important in promoting collisions, so aggregation switches from peri kinetic to orthokinetic control (II). Classical theories for such aggregation are based on the equations for the rates of both peri kinetic and orthokinetic aggregation developed by Smoluchowski (14). These equations predict that particles can grow indefinitely during shear, which is clearly unreal istic due to particle breakage . Maximum aggregate sizes in shear have been observed by several workers (15,16,17). Although no maximum size was detected, Steven ton (6) found that WPC aggregates sheared for the same time at 1476 s" J were smaller than those formed at 288 s" J . The net growth rate and size of any protein aggregates formed under shear will depend on the balance between growth and shear-controlled breakage (15,17). Parker et al. (18) proposed the following mechanisms for breakage: (i) deformation and rupture of particles by fluctuating dynamic pressures; (ii) erosion of primary aggregates from the surface of aggregates by either or all of aggregate-aggregate and aggregate-solid surface collisions, and by shear stresses generated by the fluid motion ; (iii) fragmentation of aggregates . Bell and Dunnill (16) found the dominant mechanism of soya protein aggregate breakup was fragmentation, i.e. mechanism (iii), with erosion occurring to a lesser extent: fragmentation occurred first, reducing irregularlyshaped aggregates to more regular shapes, and erosion then became the dominant mechanism . Twineham et al. (19) found the breakup of a soya protein precipitate was due to the removal of fragments by particle-particle and particle-surface collision, rather than directly by hydrodynamic shear. Modifications to the Smoluchowski analysis to include breakage (e.g. 19,20) have been successfully used to model the isoelectric precipitation of soy protein (19,21). Effect of pre-gelation shear on gelation Since many gelation reactions occur by aggregation reactions between denatured proteins, it would be expected that if proteins were denatured by the shear or gelation was aggregation reaction controlled, the gelation of protein solutions which have been previously heated and sheared would be different from unsheared protein solutions. The effect of high shear rates prior to the heat gelation of whey proteins was studied by Ker and Toledo (10) . Protein suspensions were sheared at 5800 s I for an unspecified length of time at both 25 and 70°C, and then gelled at 70°C. Sheared protein suspensions both gelled more rapidly than unsheared, and produced stronger gels (10).
47
S.:\1.Taylor and P.J.l'ryer
The above work is relevant to extrusion or homogenisation because of the high shear rate. The effect of temperature and lower shea r rates prior to quiescent gelation. such as the effect of flow through a pasteuriser holding tube before gelation , is indu stri ally rele vant but has not been studied . Under the se circumstances. the effect of she ar ma y be complex: as shown above . shear can (i) cause denaturation at high shear rates. (ii) enhance aggregation by promoting collisions, or (iii) enhance breakup of protein aggregates . All three processes might occur simultaneously; however. it is very unlikely th at aggregates would form at shear rates large enough to cause denaturation . Re alistically, mechanisms (ii) and (iii) will be the two competing ones in process equipment other than in homogenisers. Although shear can incre ase both aggregation and breakage , it is unclear which process will predominate . An experimental programme was thus co nd ucted to investigate th e effect of shear on gelation of WPC at two pH values where different mechanisms control the overall process.
Materials and methods Solutions of a commercial whey protein concentrate containing 75% protein (C arbery Milk Products. Eire; composition in Table I) were made up to a protein concentration of 14% (w/w) in distilled water. The pH was adjusted to 5.2 or 7.0 by adding 1 mol/drrr' NaOH or 1 mol/drn' HCI, and allowed to equilibrate for 2 h while stirring a t room temperature . Aliquots (6 ml) or prepared whe y protein solutions were placed in the 12.5 mm cone and plate test fixture (0.1 radian cone angle) of a Rheometries RDS II dynamic spectrometer (Rheometries Inc. , Union. NJ) which was preheated in a watcrbath to 85 ± O.l °C and subjected to a steady shear of 0-40
Table I. Composition of the whe y prote in conce ntra te used Co mpo nent
Fracti on of powder (% w/w )
13-lactoglobulin a-l act albumin Bovine serum albumin Immunoglobulins Proteose pcptones
37.50 11.25 3.75 6.00 16.50
Total protein Lactose Fat
75.0
Ca' .
B.O 9.0
Na ' K'
0.35 0.26 0.18 0.65
Total ash Moisture
4.0 4.0
PO}-
48
Temperature/shear history in protein gelation
for times between 0 and 240 s; the samples took <20 s to reach the final temperature. Solutions were then allowed to gel in situ at 85°C. All conditions of pH, shear rate and duration were carried out in duplicate, with the control conditions (shear at 0 S-I) repeated four times. The rheology of the gelling protein solutions were followed in oscillatory mode at a strain amplitude of 0.5% and a frequency of 1 rad.s -1. These test conditions were chosen to fit within the linear viscoelastic region of the material, where G' and Gil are not functions of frequency, as shown in Figure 1. G', the storage modulus, is an order of magnitude larger than Gil, and was thus used here to quantify the structure of the gel; the gel strength was taken as G' after 45 min (5). The maximum rate of increase of G' with time (dG'/dt m ax ) was taken as a measure of the rate of development of the gel fraction. The maximum rate was measured by fitting a straight line to G' data for the initial stages of gelation, where G' was rapidly increasing with time. The region where the increase was greatest was found by linear regression, and taken as dG' /dt m ax (5). The time interval over which data was fitted was sufficiently small to allow an of at least 0.90. In addition, the first measurement of G' after completing the shear, G'o, was taken as a measure of initial gel structure since G' measures the sample elasticity. The rheological data reflect the effect of shearing the sample during its gelation. Shear rates were chosen to represent those likely to be experienced during flow in a pipe, as well as being easily attainable in the Rheometries. The application of shear led to the formation of gels made up of visible aggregates of particles bound together within a gel matrix. Conventional particle sizing techniques, such as the Malvern Mastersizer used by Steventon (6), which rely on the particles being separable so that they can be individually sized, could thus not be used here. Aggregate sizes were estimated by the following process: S-I
,2
(i)
after completion of the time sweep, gels were carefully removed from the text fixture of the rheometer and stored in sealed plastic bags at 4°C to prevent deterioration. (ii) photographs were taken of thin regions of the sheared gels through a light microscope. More than one photograph was taken of each gel so that 1ססOO-.------------,
00000000000000000000000000000
~
1000
~
100
~
10
~ 1+----,--------,------j 0.1 1.0 10.0 100.0 Frequency (.. ')
Fig. 1. Frequency sweep for 14% (w/w) WPC gel formed at 85°C. Strain amplitude = 1%. G' (0), Gil (0).
49
S.M.Taylor and P.J.Fryer
representative samples were obtained. The magnification obtained for 135 mm x 90 mm prints was 50x. (iii) the sizes of the aggregates making up the gel were then measured using image analysis of the photographs. using an Optomax V image analyser (Analytical Measuring Systems. Saffron Walden, UK). Preliminary selection of aggregates was done using the Optomax and visually; if they lacked enough contrast to be detected by the image analyser, lines were drawn around particles with a marker pen. Areas of particles were then measured, and equivalent circular diameters calculated by the image analyser to obtain the particle size distributions. The data from all photographs of the same gel were pooled to give more accurate results. (iv) the arithmetic mean of the particle size distribution was used for further data analysis. This method is illustrated by Figure 2: the photograph of the sheared protein gel (a) did not have sufficient contrast to allow image analysis. so particles were highlighted in (b). Image analysis of the highlighted particles gave rise to the particle size distribution shown in (c). This procedure is obviously approximate and selection of aggregate size is partially subjective. However. since the method is systematic. it is felt that trends in the data will reflect actual changes in aggregate size. and enable differences to be determined. It is emphasised that the diameters calculated from the image analysis are not equivalent spherical diameters. The growth of large particles was limited in the vertical direction by the dimensions of the rheometer test geometry. However, it was felt that this would not seriously affect the growth of the smaller particles. Results
Effect of shear at pH 5.2 on gel strength Figure 3 shows the effect of the application of shear rates of 10-40 s 1 for durations of 15-240 s on the gelation of WPC at pH 5.2. The two horizontal lines in Figure 3(a) represent the range of gel strengths of the control (unsheared) material. Figure 3(a) shows a clear relationship between shear and gel strength. For <90 s of shear, the final gel strength increases, especially at a shear rate of 20 S-I. There is an approximately linear decrease in the gel strength with increasing duration of shear, until after 180 s all the gels are weaker by a factor of -2 than unsheared material. A similar effect can be seen in Figure 3(b), which shows the effect of shear on dG'ldt m ax , again compared with un sheared material. Figure 3(c) plots the first measurement of G' after the end of shear (G'o) against the time of that measurement; i.e. for material presheared for 120 s, G' 0 points are seen at 120 s on the horizontal axis. The lines plotted on Figure 3(c) are the time sweeps for the unsheared protein solutions. To determine the effect of the shear on the network structure. the presheared data should be compared with the unshearcd data at the same time. The sheared data lie consistently at or below the unsheared controls. 50
Temperature/shear history in protein gelatlon Scale: I mm =
_
(a)
') "\
,.
,
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~
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Fig. 2. Extraction of particle sizes from photographs of gels of sheared WPC75 solutions. (a) Original photograph of gel formed from 14% (w/w) pH 7.0 protein solution sheared for 180s at 40 S-I and 8Ye. (b) Particles highlighted prior to image analysis of the photograph. (c) Particle size distribution from image analysis.
51
S.M.Taylor and P.J.Fryer
(a) 5000
..
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Ii
~
:.
ljl
~
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Fig. 3. Effect of shear at rates of 10 (0), 20 (.) and 40 S-I (+) on gel rheology at pH 5.2: (a) gel strength; (b) dG'/dtm ax ; (c) G'i; The horizontal lines indicate the range of G' and dG'/dtm a x measured for gels formed from the unsheared protein solutions, while the lines in (c) are the time sweeps for un sheared protein solutions.
52
Temperature/shear history in protein gelation
(a) 5000
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Fig. 4. Effect of shear at rates of 10 (0), 15 (A), 20 (_), 30 (x ) and 40 S-1 (+) on gel rheology at pH 7.0: (a) gel strength; (b) dG'/dtm ax ; (c) G'(; The horizontal lines indicate the range of G' and dG'/ dt m a x measured for gels formed from the unsheared protein solutions, while the lines in (c) are the time sweeps for unsheared protein solutions.
53
S.M.Ta)lor and P.J.Fryer
Effect of shear at p H 7.0 on gel strength Figure 4( a) shows the effect of the application of shear rates of 10-40 s-) for 15240 s on the strength of WPC gels at pH 7.0 and R5°C. Unlike the data for pH 5.2. which showed a clear trend, the scatter in the sheared data is considerably greater than the control data , i.e. the range between the lines. It is thus immediately obvious that shear does affect gel strength. However, since the spread of the data is considerable. there is no clear change in gel strength with shear history. The data presented in Figure 4(a) suggests the following trends for more than 60 s of shear. although no claim can be made for statistical significance: (i) protein gels formed after shear at 10, 30 and 40 S-1 are generally weaker than unshcarcd protein gels; (ii) protein gels formed after shear at 15 and 20 s I are on the whole stronger than unshcared protein gels. However. some gels. particularly at longer shear durations. were much weaker. Figure 4(b) shows the variation in dG'4/dt m " , as a function of shear rate and duration in comparison with the rate for unsheared material. The application of shear obviously affects the rate of formation of gel structure, and trends can be detected after -60 s of shear:
(i) dC' /dt m a x is essentially unaffected by shear at the lowest and highest shear rates used , i.e . 10 and 40 s "' ; (ii) dG'/dt m ax increased for shear rates of 15, 20 and 30 s 1. Figure 4(c) plots G'o against time for pH 7.0; as in Figure 3(c). data for unshcared controls are also given. This shows that nearly all of the sheared protein gels had a higher initial gel strength than the controls. i.c . that there is more network structure in the sheared gel, reflected in the elasticity of the system .
Effect of shear on aggregate formation The application of shear at pH 7.0 for at least 60 s lead to the eventual formation of visible particles in the gel matrix which were not present in the unsheared gels. Particles were not visible through an optical microscope in gels formed at pH 5.2, even at magnifications x 100, 2.5 times greater than that necessary to recognise particles in the pH 7.0 gels. Data extracted from photographs of the gels using the image analysis technique are given in Figure 5. Statistically significant differences were seen between the sizes of particles formed at different rates and durations of shear:
(i) 54
in gels sheared at rates of 10 and 15 S-I, the size of particles increased significantly (95% level) with the duration of shear;
Temperature/shear history in protein gelation
(a)
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i
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Fig. 5. Particle sizes for sheared pH 7.0 protein samples. Shear rates of 10 (0), 15 CA.), 20 (.), 30 (x ) and 40 S-1 (+); (a) shows all data, (b) shows only 10, 20, 30 and 40 S-1 data.
(ii) however, the size of particles in gels sheared at higher rates of 20, 30 and 40 S-1 did not change with shear duration (90% level); (iii) for a duration of shear >60 s (the gelation time at 85°C, 22), the size of particles varied with shear rate. Gels sheared at 15 s-] had the largest particle size by at least a factor of two. 10 s-] came next, followed by 20 and 30 s -1 (not significantly different), then 40 s-]. Where differences were seen, they were in all cases significant at the 95% level. There was no direct correlation between particle size and rheological properties of the gels. Discussion The effect of temperature/shear history has been studied experimentally at two pH values at 85°C, one (pH 5.2) at which denaturation is controlling, and one (pH 7.0) at which aggregation controls the gelation process (4). The pH 5.2 G' and dG'/dtm a x results suggest that two effects are occurring: (i) the initial increase in final gel strength with duration of shear must reflect an increase either in bond strength or the number of bonds brought about by 55
S.M.Taylor and P.J.Fryer
shear. Steventon (6) noted that at pH 5. aggregation rapidly formed an irregular structure of loosely packed aggregates. due to rapid aggregation after denaturation . as shown by Taylor et al. (4) . The application of shear over short periods of time (30 or 60 s) probably pre vents these loose aggregates forming, making a larger number of bonds available for the network, and preventing rapid aggregation into weak networks. (ii) as the time for which the material is sheared increases , however, the final gel strength decreases. During shear, when protein molecules denature , they will form aggregates within the fluid. These particles will bond less strongly to other aggregates during subsequent quiescent gelation . The G'lI measurements again demonstrate that shear leads to weaker structures; proteins which have aggregated in the bulk form weaker bonds to other aggregates once shear is complete. The effect of shear at pH 7.0 on the gelation of WPCs is more complex; there is a large variation in the gel strength. These results suggest that shear is enhancing aggregation (leading to increased gel strength) as well as causing breakup of aggregates (leading to weaker gels). G'i, for the sheared protein solutions were higher than the unsheared solutions, suggesting that aggregation is enhanced by shear. This is consistent with the results of Taylor et al. (4), who found that gelation was aggregation limited at pH 7.0. At pH 5.2 no aggregates were visible through the optical microscope , even at magnifications of x 1(}() (2.5 times that used for the pH 7.0 gels). This is to be expected since the gelation process of pH 5.2 solutions at 85°C has been found to be denaturation limited (4) ; the increased frequency of collisions due to shear would not lead to an increase in the aggregation rate , so visible particles do not form. However , the formation of visible particles as a result of shear during the early stages of gelation shows that aggregation of whey proteins at pH 7.0 is enhanced by shear. The increase of particle size with shear duration at 10 and 15 S-I , coupled with the fact that no particles are visible in unsheared gels, also suggests that enhancement of aggregation is occurring as a result of shear. At higher shear rates (15-40 S-I) particle size decreases as shown in Figure 5; since the physical conditions, temperature and pH are the same, this clearly indicates that aggregate breakup is occurring . The particle size increase with duration of shear at 10 and 15 s -1 suggests that a limiting particle size had not been reached over these shear durations. Particle size varies with shear rate and time at pH 7.0. No correlation was observed between particle size and rheology of the gels; the data were clustered according to shear rate. Shear rate and time thus appear to determine the gel structure directly, rather than producing particles of a given size which then determine the mechanical properties of the gel.
Calculation of effective concentration of sheared protein gel matrices Data suggests that during shear prior to quiescent gelation, denaturation and 56
Temperature/shear history in protein gelation
aggregation of the protein will occur to form discrete particles. This decreases th e amount of protein available to react and form a network during subsequent gelation . The change in the mechanical properties of the pre-sheared gels ob served above is as a result of reactions during shear. Thi s might be expressed as a change in the 'effective concentration', Ceff , of the material; i.e. if, from a 10% (w/w) whey protein solution , 20% of the protein aggregates during the preshearing process, the material may subsequently behave as if it were an 8% (w/w) solution. This hypothesis has been tested below. The concentration dependence of G' and dG'ldt m ax at pH 5.2 and 7.0 was obtained (22). Best-fit lines were fitted to this data and rearranged to find the effective concentration which produces a given G' or dG' ldt m ax : pH 5.2, 10-18% (w/w) protein solutions: Cc ff
=
(53.48
G,)1 /4.46
(1)
pH 7.0, 10-16% (w/w) protein solutions:
Cef f
= (G' + 7780)/820
(2)
pH 5.2, 10-18% (w/w) protein solutions: Ce ff
=
(465 x 105 dG'ldt ma x )1 15 .26 ·
(3)
pH 7.0, 10-20% (w/w) protein solutions: Ceff
=
(1 ' 78 x 107 dG' ldt max ) 116.80
(4)
Equations (1)-(4) were used to calculate the effective concentration as a function of the length of shear before quiescent gelation, and hence determine whether it is a useful concept. For each combination of shear rate and length of shear, G' or dG'ldt m ax was found experimentally. The appropriate equation was then used to find Ceff . Measures of effective concentration can be found for each gel from both G' and dG'ldt m ax using equations (1,2) and (3,4). Figure 6 shows the variation with time of shear of the effective concentration of gels formed at pH 7.0. The effective concentrations suggested by dG'ldtm ax are higher than the real concentration, whilst that found using the final gel strength is generally less. The effect of the duration of shear also appears different; Ceff calculated from G' decreases, and that from dG'ldt m ax increases with time . This shows that effective concentration is not a useful concept in modelling the effect of shear on the gelation of pH 7.0 solutions of WPC; if it were , the two measures by which it can be estimated should be similar. Figure 7 shows the variation of effective concentration formed from sheared pH 5.2 solutions. In contrast to pH 7.0, the data from G' is similar to that from dG'ldt m ax ' Unlike pH 7.0 , Figure 7 shows that at pH 5.2, the effective 57
S.M.Taylor and P.J.Fryer
(a) 17 16
-;
~ ~
~ ~
u·
15
•
•
14
• •• • 0
0
0
13
8
0
•
•
0
• 0
0
12 0
0
11 10 0
100
50
150
200
250
Duration of shear (s)
(b) 17
15
••
• 8
~
I 0
0
III
"
0
0
~
14
~
13
0
12
0
~
••
• •Ii•
16
0
0
U':
0
11
10 0
100
50
150
200
Duration of shear (s) (c) 17 16 0
15
. •• • • 8
~
~ ~ u':
14
!
•
•-
0
0
0
0
13
e
0
12
0 0
11
10 0
50
100
150
200
Duration of shear (s)
Fig. 6. Effective concentration of protein in pH 7.0 gel matrices formed from shear at rates of (a) 10, (b) 20, and (c) 40 S-I. Effective concentration calculated from the G' (0) using equation (3) and dG'/dtm ax ( . ) using equation (5).
58
Temperature/shear history in protein gelation
(a) 16
15
g
~
~
~ U
·
0 0
••
•
14
0
~ 0
•
13
0
•
12 0
50
100
150
200
Duration of shear (s) (b) 16 0
15
~ ~
~ U
·
I!!I
•
14
0
-. i
0
0
i
• ~
13
0
••
12 50
0
150
100
200
Duration of shear (s) (c) 15 0
14
~ ~ ~
·
0
• •
~
• 13
Ii
•
~ ~
U
0 0
•
• ~
12
0
11
0
50
100
150
200
Duration of shear (s)
Fig. 7. Effective concentration of protein in pH 5.2 gels calculated from G' (0) using equation (4) and dG'/dtm ax ( . ) using equation (6). Gels formed during shear at rates of 10 (a), 20 (b) and 40 S-I
(c).
59
S.M.Taylor and P.J.Fryer
concentration calculated by both methods increases over the first 60 s of shear, and subsequently decreases. This is probably due to shear over the initial periods of heating preventing rapid aggregation of protein into disordered particles. However, shearing during the period that the gel would normally be forming (times greater than 60 s; 22) leads to a decrease in the effective gel strength because the crosslinks between aggregates cannot form. Here the concept of effective concentrations appears to work well; the effect of shear on the strength of pH 5.2 whey protein gels could be explained by the effect of the shear on the amount of protein available for network formation. More work will be needed to determine the usefulness of this idea. Generality of results As discussed above, the gelation properties of whey proteins are known to vary with composition. The material used in this work is a commercial WPC; it would be expected that variations would be seen between different' batches and between WPC produced from different sources. Table lshows that the material is similar to that studied by other workers (23); changes in the mineral level will affect gelation, either through direct interactions with the protein (addition of calcium) or by changing the ionic strength of the solution and thus the forces between proteins [addition of sodium (24)]. Whilst the numerical values of the data will vary, it is expected that as the WPC composition used here is typical, the trends seen will be common to other samples. Conclusions
Experiments have been carried out to characterise the behaviour of WPC solutions heated and sheared prior to gelation. The gelation of pH 5.2 protein solutions is denaturation limited. Shear for <60 s leads to an increase in gel strength due to the prevention of rapid aggregation of denatured protein. However, longer periods of shear lead to a decrease in gel strength as protein aggregates are disrupted by the shear. The gelation of pH 7.0 protein solutions is aggregation limited. When shear is applied prior to gelation, large protein aggregates form and the initial gel strength after shear increases, suggesting that aggregation is enhanced by shear. The final gel strength decreases with increasing time of shear, suggesting that shear has a secondary effect in breaking up protein networks, similar to that seen for pH 5.2 solutions. These two processes act together; data for the final gel strength is a more complex function of the time of shear before gelation for pH 7.0 solutions than for pH 5.2. Owing to the lack of optical contrast between particles and the medium, it was only possible to carry out a semi-quantitative analysis of the sizes of aggregates seen under the optical microscope. However, the protocol developed allowed the range of particle sizes measured at different shear rates and durations to be measured in a systematic way. The analysis also suggested that shear-induced breakup occurred at pH 7.0. 60
Temperature/shear history in protein gelation
It can be concluded that the application of she ar to whey protein solutions while the y are heated can gre atl y affect the strength and structure of the subsequent gels. Thi s suggests that processing equipment a nd procedures need to be car efull y controlled to avoid the disruption of the gelation process. An attempt has been made to express changes in the gel properties in terms of cha nges in effective protein con centration . This appears promising. More work will, howe ver , be needed to characterise the processes see n her e. In addition to work on industrial fluid s, there is a need for future work on purer syste ms, such as 13-lactoglobulin. Acknowledgements Dr M .R .Mackley is thanked for making available the Rheometries RDS II. Mr R .T .J.Marshall is also thanked for his assistance in collecting rheological data. PJF wishes to thank the staff of the Intensive Coronary Care Unit, Queen Elizabeth Medical Centre, Birmingham. The authors would like to acknowledge the financial support of the Cambridge Commonwealth Trust and Express Foods (Europe) Ltd. References 1. de Wit.J .N. (1984) Neth. Milk Dairy J. , 38, 71-89. 2. Ferry,J .D . ( 1948) Ad v. Protein Chem . 4 , 1-78. 3. Steve nto n,A J .. Gladd en ,L.F. and Fryer ,PJ . ( 1991) J. Texture Stud . , 22, 201-218. 4. Taylor ,S.M ., Gladden ,LF. and Fryer ,P .l. (1993) J. Dairy Res. , in press. 5. Tay lor,S.M . and Fryer ,P .l. ( 1992) Entropie , 168, 57-64 . 6. Stevento n,A J . (1993) Thermal aggregation of whey prot eins. PhD thesis, Un iversity of Cambridge . 7. Oin gnong ,T., Munro ,P.A . and McCarth y,a .l. (1990) Proceedings of the 18th Australasian Chem ical Engineering Confe rence. pp . 666-674 . 8. Pradip asen a ,P. and Rh a ,C. (1977) J. Textur e Stud. , 8, 311- 325. 9. Harri s.Ll., ; Pecar.MiA . and Pearce ,R.J . (1989) Aust. 1. Dairy Tech.. 44, 78-81. 10. Ker ,Y.C. and TOledo,R .T. (1992) J. Food . Sci. , 57, 82- 85. 11. Bell.D i.l. . Ho are,M. and Dunnill,P . (1983) A d~' . Biochem . Engng ; 26,1-72. 12. Chan,M.Y .Y., Hoare ,M. and Dunnill ,P . (1986) Biotech. Bioengng ; 28, 387-393. 13. Fisher ,R .R . and Glatz,C.E. (1988) Biotech, Bioengng ; 32, 777-785. 14. Smoluchowski ,M . ( 1917) Z. Physik . Chem ., 92, 129-1 68. 15. Virk ar ,P .D ., Hoare ,M ., Chan,M.Y.Y. and Dunnill.P. (1982) Biotech. Bioengng; 24, 871-887. 16. Bell,D .J . and Dunnill ,P . (1982) Biotech. Bioengng, 24,1 271-1285. 17. Hoare .M , (1982) Trans. I. Chem . Engng , 60, 79-87. 18. Parker,D.S. , Kaufman .W .l. and Jenkins ,D. (1972) J. Sanit. Eng. Div. Proc. Am. Soc. Civ. Eng. , 98 (SAl), 79-99. 19. Twineham,M., Hoare,M. and Bell,D .J . (1984) Chem. Eng. ss., 39, 509-513. 20. Harris,H.S., Kaufman,W.J . and Kron e,R.B. (1966) J. Sanit. Eng. Div, Proc. Am. Soc. Civ. Eng ., 92 (SA6), 95-111. 21. Fisher ,R .R . and Glatz ,C.E . (1988) Biotech. Bioengng , 32,786-796. 22. Taylor ,S.M. (1993) Rheology and biochem istry of whey protein gels. PhD thesis, University of Ca mbridge . 23. Jost.R. (1993) Trends Food Sci. Techn ., 4, 283-288. 24. Reynard ,D . and Lefeb vre,J . ( 1992) Int. J. Bioi. Macromol. , 14,287-291.
Received on September I , 1993; accepted on December 10, 1993
61