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Influence of whey protein denaturation on κ-carrageenan gelation
Colloids and Surfaces B: Biointerfaces 12 (1999) 299 – 308
Influence of whey protein denaturation on k-carrageenan gelation A. Tziboula *, D.S Horne Hannah Research Institute, Ayr, KA6 5HL, Scotland, UK
Abstract Response surface methodology was applied to study the effect of different heating temperature/time treatments on whey protein denaturation and its effect on k-carrageenan gelation in milk. The path of gel formation was followed using small deformation rheology and the extent of whey protein denaturation was determined by gel permeation chromatography. k-Carrageenan did not influence the rate of whey protein denaturation and it was unlikely that whey protein denaturation played a significant role on k-carrageenan gelation in milk. In skim milk serum or skim milk ultrafiltrate the path of gel formation and gel strength were not influenced by the severity of heat treatment but increasing the concentration of whey proteins enhanced the gel strength. Heat treatment became important for carrageenan gelation in skim or recombined milks (i.e. in the presence of casein micelles) by influencing the gelation temperature and gel strength. Increasing the concentration of whey proteins in the recombined milks had a beneficial effect on gel strength. © 1999 Elsevier Science B.V. All rights reserved. Keywords: Whey protein; k-Carrageenan; Gel permeation chromatography
1. Introduction The carrageenans are sulphated linear polysaccharides of alternating b-1,3- and a-1,4-linked galactose residues that are used to modify texture and stability in dairy foods. In the presence of cations they form thermoreversible gels. It is generally accepted that carrageenans interact specifically with the caseins (mainly k-casein but also as1- and b-caseins) to form a complex which
* Corresponding author.
aggregates into a three-dimensional network [1– 4]. The ability of carrageenans to stabilize dairy products such as chocolate milk and cream has been demonstrated for polymer concentrations as low as 50 ppm [3]. Despite, the economic importance of carrageenans in dairy applications, few studies of their behaviour in milk have been published. Carrageenan gelation in the presence of serum proteins is of particular interest. We found that the serum-carrageenan gels were stronger than the corresponding milk gels [5–7]. Lower concentrations of carrageenan were required for gelation in
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serum rather than in milk [7]. Heating the carrageenan dispersions at temperatures above 60°C is necessary for the hydration of the polymer. Such treatments take the milks into temperature ranges where whey protein denaturation is observed [8]. Such denaturation could influence gelation. The main objective in this work was to investigate the effect of whey protein denaturation on k-carrageenan gelation, in polymer dispersions with or without casein micelles. The carrageenan concentration was fixed at 200 ppm. At these concentrations the carrageenan gels are weak allowing the effects from the interactions with caseins and/or whey proteins to be discerned. Otherwise carrageenan gelation swamps the interaction effects. Gelation was followed during cooling of the milk – carrageenan mixtures from 59 to 5°C, at a rate of 1°C 15 min − 1. Admittently, this slow cooling rate is not practical in most industrial applications. Nevertheless, it has been shown that the rate of cooling had an enormous effect on the gelation temperature and the mechanical properties of the gels [6]. Response surface methodology was used to study the effect of three variables on k-carrageenan gelation: (1) heating temperature (i.e. from 60 to 90°C); (2) duration of heating (i.e. from 1–10 min); the temperature/time combinations determined the extent of heat-denaturation of the various whey proteins [8]; and (3) whey protein content. Using these variables, two designs were generated: in the first, casein micelles were not included in the carrageenan mixtures. The whey protein content was varied from 0 to 100% (i.e. complete absence of whey proteins to their normal concentration found in skim milk). So in the first design k-carrageenan (200 ppm) was added to skim milk ultrafiltrate (SMUF), skim milk serum/SMUF solutions at a dilution ratio 1:1 and skim milk serum. The ionic composition was maintained equivalent to that in skim milk. In the second, casein micelles were also included in the mixtures at constant concentration equivalent to that in skim milk. The extent of whey protein denaturation was measured by gel permeation chromatography and
the path of carrageenan gelation was studied using low frequency oscillation.
2. Materials and methods
2.1. Response surface methods Response surface methodology is used in the empirical study of relationships between one or more measured responses and a number of variables. Their advantage over the one-variable-at-atime approach is that they provide information on what might happen if the variables were changed together. The two designs employed were selected from the library of experimental designs implemented in Minitab version 11.2. They were two central composite designs of three variables, each set at two levels. These designs allowed efficient fitting and checking of second-degree polynomial models: y= b0 + b1x1 + b2x2 + b3x3 + b11x 21 + b22x 22 + b33x 23 + b12x1x2 + b13x1x3 + b23x2x3 where bii are the coefficients and x1, whey protein content; x2, heating temperature (60–90°C); x3, heating time (1–10 min). The k-carrageenan concentration was fixed at 200 ppm. In the first design, the whey protein content in the solutions varied from nil (i.e. SMUF) to 100% (skim milk serum). Casein micelles were not included in the mixtures. In the second design casein micelles were also present in the mixtures. The whey protein content was adjusted as in the first design. Casein micelles were resuspended in the solutions prior to carrageenan addition. The amount of resuspended caseins was kept constant equivalent to that in skim milk. In the extremes of treatments k-carrageenan was dispersed in SMUF (absence of whey proteins and casein micelles) and skim milk (whey proteins and casein micelles present). Table 1 identifies the variables and specifies their levels for each run. The repeatability of the measurements, was estimated by the standard error of the mean (SEM) in the central points of the designs.
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2.2. Preparation of milk fractions Bulk milk from the Institute herd was skimmed by centrifugation at 1200×g for 30 min at 4°C (Mistral 6000 centrifuge; Sanyo Gallenkamp, Leicester, UK). Skim milk ultrafiltrate (SMUF) was prepared from skim milk using an ultrafiltration cell (TCF10A, Amicon, Danvers, MA) equipped with a membrane of molecular weight cut-off of 10 kDa. Micellar caseins were pelleted from skim milk by centrifugation at 43 000× g for 60 min (Sorval RC 5B; Du Pont Instruments, Newtown, CT). The micellar caseins were resuspended, by overnight stirring at 4°C, in SMUF-serum solutions according to the design requirements listed in Table 1. These were designated recombined milks.
Table 1 Description of the variables and specification of the levels for the response surface designs Whey protein concentration (% of total in milk)
Temperature (°C) Time (min)
301
Skim milk serum was the supernatant layer obtained during the preparation of micellar caseins. This comprised mainly the serum protein, soluble casein (15–20% total casein) and the soluble phase in milk. The soluble casein was removed by isoelectric precipitation at pH 4.6, followed by centrifugation and filtration. The serum fraction was then dialysed at 4°C against skim milk, for ionic equilibration until the final pH of the serum fraction was 6.779 0.02 (48 h; three skim milk changes; serum to skim milk ratio 1:250). The serum fraction was diluted with the appropriate amount of SMUF to adjust the whey protein level (Table 1).
2.3. Carrageenan k-Carrageenan from Euchema cottonoii (Aldrich, Gillingham, Dorset, UK) was converted to the sodium form using cation exchange resin (Amberlite IR120 cation exchange resin in the Na form, BDH, Poole, UK). The polysaccharide was recovered by filtration to remove resin and freeze drying of the filtrate. The sodium content was determined to be 5.67% w/ w by atomic absorption spectroscopy.
a,b
0 0 0 0 0 50 50 50 50 50 50
60 60 75 90 90 60 75 75 75 75 90
1.0 10.0 5.5 1.0 10.0 5.5 5.5 1.0 5.5 10.0 5.5
100 100 100 100 100
60 60 75 90 90
1.0 10.0 5.5 1.0 10.0
a Design 1: the whey protein content was varied by diluting skim milk serum with SMUF; 0%, SMUF; 50%, skim milk serum/SMUF 1:1; 100%, Skim milk serum. b Design 2: whey protein content was varied as for design 1. Casein micelles were added in SMUF or skim milk serum/ SMUF 1:1, at concentrations equal to skim milk.
2.4. Preparation of k-carrageenan dispersions k-Carrageenan was added to the appropriate milk fraction (skim milk, skim milk serum, SMUF, SMUF-serum solutions, recombined milks) while stirring at room temperature for at least 1 h. Complete dispersion of the carrageenan was achieved by homogenisation for 2 min, at standard setting (Silverson laboratory mixer emulsifier, Silverson machines, Waterside, Chesham, Bucks). The mixture were heated to the required temperatures in a boiling water bath and immediately transferred to another thermostated controlled water bath, set at the required temperature, where they continued to be heated for the appropriate time according to the temperature/time combinations in Table 1. The mixtures were then transferred from that state to the rheometer which was sitting at 59°C.
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2.5. Dynamic rheology Low frequency oscillation (0.08 Hz, applied stress 5 mPa) was performed on the milk-carrageenan mixtures as they were cooled from 59 to 5°C, at a cooling rate 1°C 15 min − 1, using a controlled stress rheometer (CVO; Bohlin Instruments, Gloucestershire, UK), fitted with the double gap measuring geometry. The geometry consists of a hollow cylinder (diameter 45 mm) which is lowered into a cylindrical groove in an outer cylinder (diameter 50 mm). Although the instrument calculates the phase angle (d), the angle by which the strain response lags behind the applied stress and this allows the derivation of the storage (G%), viscous (G¦), complex (G*) moduli and complex viscosity (h*), only the complex modulus G* =( G% 2 + G¦ 2)1/2 of the carrageenan gels is considered here. The gelation temperature of the gels was taken as the temperature where tan d =1. Because of the long duration of each run, prevention of evaporation was achieved by fitting the solvent trap on the double gap geometry. The applied stress 5 mPa was, as always, a compromise between attempting to operate in the linear viscoelastic region of the structures created and of maintaining a measurable response on gelation. The stress used here, 5 mPa, is just above the bottom end of the stress range permissible with the double-gap geometry in the Bohlin Instrument. The strain response varied with the cooling cycle and viscoelastic properties of the milk-carrageenan mixtures. The initial strain response (at 59°C) was constant at 1.42 9 0.01 whilst at 5°C it ranged from 0.4 for the weakest structures to 0.002 for stronger gels.
2.6. Gel permeation fast protein liquid chromatography (GPC) The extent of whey protein denaturation was measured in a separate experiment by gel permeation chromatography. Samples were prepared with and without k-carrageenan according to the conditions in Table 1. After heat treatment as specified in Table 1, the samples were cooled at a
rate of 1°C 15 min − 1 to simulate the cooling profile of the rheological test. The samples containing whey proteins were then filtered through 0.2 mm filter to remove the aggregated denatured whey proteins and k-carrageenan. In the samples containing micellar caseins, the denatured whey proteins were precipitated together with the casein by adjusting the pH to 4.6, followed by filtration. The whey protein composition of the filtrates was assessed by gel permeation chromatography on a Superdex 75 column HR 10/30 (Pharmacia, Biotech, Milton Keynes, UK) eluted with a Tris–HCl buffer (pH 7.0, 100 mM Tris, 0.5 M NaCl) at a flow rate 0.5 ml min − 1 [9]. The extent of denaturation of the whey proteins was calculated from the relative decrease in the area of their elution peaks compared with the corresponding areas in filtrates from unheated skim milk or serum.
3. Results and discussion
3.1. Design 1: influence of whey protein denaturation on k-carrageenan gelation in dispersions without casein micelles Typical rheological profiles of the development of the complex moduli (G*) during cooling of the various carrageenan mixtures from 59–5°C, at a rate of 1°C 15 min − 1, are shown in Fig. 1. The panels show the results for the extremes of treatments. Thus, k-carrageenan was dispersed: Fig. 1(a), in skim milk (casein micelles and whey proteins present); Fig. 1(b), in recombined milk (i.e. casein micelles resuspended in milk ultrafiltrate); Fig. 1(c), in skim milk serum which does not contain casein micelles; and Fig. 1(d), in SMUF which does not contain either whey proteins or casein micelles. The slow cooling rate was chosen because of its great effect on the gelation temperature and the mechanical properties of the gels. Gelation studies in carrageenan-milk and carrageenan-SMUF mixtures showed that a reduction in the rate of cooling from 1°C 45 s − 1 to 1°C 15 min − 1 resulted in an increase in the gelation temperature by 10 and 25°C respectively [6]. Fast cooling rates also resulted in weaker gels. This means that
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Fig. 1. Influence of heat treatment on k-carrageenan gelation: (a) skim milk; (b) recombined milk; (c) milk serum; (d) skim milk ultrafiltrate (SMUF); (e) indicates no gelation; —— , 90°C for 10 min; – – – – , 90°C for 1 min; — – — – — – 75°C for 5.5 min; -------, 60°C for 10 min; ······ 60°C for 1.0 min; TTR, transition temperature; GT, gelation temperature.
although the cooling rate does not influence the temperature of the conformational transition of carrageenan and the mechanism of gel formation, it affects the perceived rheogical gelation times and gel strengths in short experiments and can lead to erroneous conclusions. In all samples, there was an initial lag period during which the G* was very low and the mixture remained fluid. Below the gelation point the lack of structure in the material was reflected by the low G% values (in the range 10 − 4 – 10 − 5 Pa). These extremely low values are simply indicative of fluid samples. The onset of carrageenan gelation was signaled by a sharp rise in G* at the carrageenan coil-to-helix transition temperature (TTR, ca. 38°C). This point reflects the first ‘real’ measurement of the viscoelastic contributions to the structure of the samples. At this point the strain dropped to 0.006 for the weakest gels and 1.2 for the strongest changing rapidly as the gel develops. Thereafter the path of gel formation
was influenced by the protein composition of the mixtures. A detailed account of the effect of milk proteins on the path of carrageenan gelation has been given elsewhere [7]. In the absence of casein micelles (Fig. 1(c–d) heat treatment did not influence either the gelation temperature or the rate of gel strength development. However, the path of gel formation differed: in SMUF gelation took place into distinct steps, the second sharp rise in G* corresponding to the rheological gelation temperature (GT, Fig. 1(d)). In SMUF, the GT was :5°C lower than the coil to helix transition temperature. In skim milk serum which includes whey proteins, k-carrageenan gelation proceeded in a single step but the initial rise in G* was followed by a region of loss of structure, though recovery of structure on further cooling was almost immediate (see inset Fig. 1(c)). This anomaly in the built up of structure has been noticed previously [7] and has been attributed to thermoreversible
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(Table 2). Thus, it was confirmed that the whey protein content had a positive effect on gel strength (pB 0.005, Table 2). Whilst the heating temperature did not have a significant effect, the duration of heat treatment seemed to influence the development of G*. This effect was more pronounced at maximum concentrations of whey proteins (Fig. 2; PB 0.05, Table 2). The contour plots in Fig. 2, the estimated plots of G* as a function of whey protein content and heating time combinations, were calculated for a model where the square terms, temperature, whey protein× temperature and heating time× temperature interactions were omitted. The repeatability of the measurements was estimated by the SEM of the central points of the designs. SEM for GT and G* were 1.35°C and 10 mPa respectively. Comparing the GPC data on the extent of whey protein denaturation, with and without k-carrageenan, it was concluded that k-carrageenan, at the levels employed here, did not influence the rate of denaturation of the individual whey proteins. As was anticipated, b-lactoglobulin (blg) and a-lactalbumin (a-la) denaturation were
aggregation of whey proteins possibly ogether with k-carrageenan. As the gel strength and gelation path were not influenced by heat treatment it was concluded that whey protein denaturation did not play a significant role in k-carrageenan gelation in mixtures of varying whey protein content. However, comparison of the k-carrageenan – SMUF rheological profiles (Fig. 1(d)) with the corresponding situations in serum (Fig. 1(c)), showed that carrageenan formed stronger gels in the presence of whey proteins. Analysis of variance of the full quadratic model, showed that the linear and interaction terms of the regression function were the most important whilst the second degree terms were not statistically significant. The data was remodeled using only these statistically significant terms because the reduced function represented more accurately our data. From the linear terms, temperature and its interaction with the other terms were also omitted from the regression because these two were identified as not significant in the initial analysis. The re-fitted model explained : 85.9% of the variance in the data
Table 2 Response surface regression: analysis of variance and the estimated regression coefficients of the effect of whey protein, heating temperature and heating time, on the complex modulus at 5°C (G*) of carrageenan gels in the presence or absence of casein micellesa Analysis of variance
Design 1: absence of casein micelles
Design 2: presence of casein micelles
r
r
P
r 2 =91.3%
*** *** NS *
Regression equation Linear terms Square terms Interaction
r 2 = 87.7%
Terms
Coefficients
Constant Whey protein Temperature Heating time Whey×temp. Whey×heat. time Heat. time×temp.
1245.0 552.4 100.9 290.0 141.4 274.9 146.4
a
*** NS *
r: The correlation coefficient of the regression equation. The terms were excluded from the respective models. * PB0.05. ** PB0.01. *** PB0.005.
b
P
Coefficients *** *** Nsb * NSb * NSb
287.1 154.0 452.0 207.3 179.8 49.9 254.7
NS NS ** * * NSb *
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played a significant role in k-carrageenan gelation in serum. Finally, from the gelling behaviour of k-carrageenan in SMUF, it was concluded that the different heat treatments had little effect on the extent of hydration of the polymer.
3.2. Design 2: influence of whey protein denaturation on k-carrageenan gelation in the presence of casein micelles
Fig. 2. Design 1: contour plots of the estimated complex modulus (G*) of carragean gels at 5°C, as a function of whey protein content and heating time. Casein micelles were not present in the mixtures; k-carregeenan concentration fixed at 200 ppm. Isobars indicate gel strength (Pa).
controlled by the temperature/ heating time combinations (Table 3). Of the other remaining whey proteins, immunoglobulins were the most heat sensitive and relatively mild heat treatments (60°C) resulted in complete denaturation. Serum albumin and lactoferrin were denatured at temperatures above 75°C (results not shown). The combined effect of heating temperature and duration of heating on the amounts of denatured b-lg and a-la, expressed as percentage of total, are shown in Fig. 3(a – b). These plots were generated by fitting the reduced second-order polynomial model for the statistically significant terms shown in Table 3. The regression equations were highly significant and accounted for 95.9% and 89.5% of the variance in the data for b-lg and a-la respectively. b-Lg was more heat sensitive than a-la (compare Fig. 3(a,b)). Furthermore, whilst the effect of duration of heat treatment was insignificant at 60°C it became important at higher temperatures. This effect was more pronounced with a-lactalbumin, the most heat resistant of the whey proteins. These results were in agreement with previously published data [8]. Overall, it was concluded that k-carrageenan did not influence the rate of denaturation of the individual whey proteins, as measured by aggregate formation in the whey protein mix, and it was unlikely that whey protein denaturation
From the gelation profiles shown in Fig. 1(a,b) it is apparent that the various heat treatments Table 3 Response surface regression: analysis of variance and estimated regression coefficients of the effect of casein micelles, heating temperature, heating time on the extent of whey protein denaturationa Analysis of variance
Regression equation Linear terms
b-lactoglobulin
a-lactalbumin
r
P
r
P
r 2 =95.9%
****
r 2 =89.5%
****
****
****
Square terms
****
***
Interaction
***
****
Term
Coefficients for blg
Constant
309.75
***
351.07
***
Casein micelles Temperature
1.36 −9.75
NSb
−4.40 −9.68
** ***
Heating time
−5.50
NS
−12.66
****
(Temp)2
0.08
****
0.07
***
Temp×heat. Time
0.09
***
0.20
****
a
****
Coefficients for a-la
r: The correlation coefficient of the regression equation. The term was excluded from the refitted model for b-lactoglobulin denaturation. ** PB0.01. *** PB0.005. **** PB0.001. b
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Fig. 3. Contour plots of the estimated effects of heating temperature and duration of heat treatment on (a) b-lactoglobulin and (b) a-lactalbumin denaturation in a whey protein mixture from skim milk. Isobars indicate extent of denaturation (% of total).
become important for k-carrageenan gelation in mixtures containing casein micelles. Thus, gelation did not take place following mild heat treatments (60°C for 1 – 10 min). Furthermore, the rate of gel strength development increased with the severity of heat treatment. This effect was attributed to the presence of casein micelles in the carrageenan mixtures, because it was consistently observed irrespective of the whey protein content in the samples. Gelation took place in a single step (both in skim milk-carrageenan and recombined milk- carrageenan mixtures), signaled by a sharp rise in G* at 38°C, followed by fast aggre-
gation and gelation. The sharp rise in G* was also observed in the non-gelling samples (Fig. 1(a,b)), which indicated conformational change of the polymer but lack of capability for further network formation. Fitting the full second order model to the data showed that the second order terms, some main terms and two way interactions were not statistically significant. Therefore, the data was remodeled using only the statistically significant terms and the results are shown in Table 2. In mixtures containing casein micelles, the strength of the carrageenan gels (taken as G* at 5°C) was influenced mainly by the heating temperature/time combinations (PB0.05; Table 2). The contour plot of the estimated G* as a function of heating treatment is shown in Fig. 4(a). It can be seen that maximum gel strength is associated with high heating temperature/time combinations. As with the first design an increase in the whey protein content also had a beneficial effect on G*. This effect was more pronounced at high heating temperatures (PB 0.05, Table 2). The effect of whey protein content and heating temperature on gel strength is shown in Fig. 4(b). Both plots in Fig. 4 were generated using only the statistically significant terms in Table 2. These results imply that k-carrageenan interacts with the casein micelles, thus reducing the polymer availability for a gelation role. In other words, we always observe only carrageenan gelation. Interaction with casein micelles reduces kcarrageenan available for gel formation and this weakens the gel produced. Such an interaction between carrageenan and casein micelles was also shown in electrophoretic mobility studies in solutions containing diluted milk and concentrations of carrageenans of up to 0.2 mg ml − 1, although no compensation was made for viscosity effects [10]. What we see in this work is a heat sensitive situation where the casein–k-carrageenan complex content is reduced by heating, allowing stronger gels to be formed. When whey proteins are present in company with casein micelles, we also get stronger gels. In this context, we suggest that whey proteins interact with micelles and reduce their ability to interact with k-carrageenan, hence the increase in gel strength.
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These results do not agree with previously published studies on i-carrageenan gelation [11]. Langendorff et al. [11] showed there, that milk-i-carrageenan gels were significantly stronger than SMUF – carrageenan gels. However, fast cooling rates were applied in their rheological studies and we have shown these to influence both the rheological gelation time and mechanical properties of the gels [6]. This effect of cooling rate differs depending on the milk components present in the system, being more pronounced in SMUF–car-
307
rageenan than in milk–carrageenan mixtures [6]. Finally, Langendorff et al. [11] show anomalously high initial G¦ values (i.e. at 65°C) in milk, a factor of ten greater than their measurements in SMUF. In our studies initial G¦ were around 0.4–1.4 mPa in SMUF– and milk–carrageenan mixtures respectively, at a concentration of carrageenan 0.1% w/w [5]. That the effects of heat treatment on carrageenan gelation in milk are related to differences in the extent of hydration of the carrageenan molecule can be ruled out because similar observations were made when carrageenan was added to preheated milk. These results are the subject of another publication. Nevertheless, other heat induced changes in milk could influence k-carrageenan gelation. For example, changes in the solubility of inorganic calcium phosphate and a decrease in pH influence the protein charge and hence the interaction potential with other polymers. Side chains of some aminoacids, especially that of lysine also become very reactive on heating and it is possible that they also influence k-carrageenan gelation. The extent of heat induced denaturation of the individual whey proteins in skim or recombined milk was similar to that in the serum (Table 3). As with the first design k-carrageenan was not found to have a significant effect on the rate of whey protein denaturation. As a result of this observation and because the effect of heat treatment on the path of k-carrageenan gelation was associated with the casein fraction rather than the whey protein fraction in the mixtures, it was concluded that whey protein denaturation did not play a significant role on carrageenan gelation. However, it could not be ruled out that whey protein interactions with casein micelles have a minor influence on gel strength.
4. Conclusions
Fig. 4. Design 2: contour plots of the estimated complex modulus (G*) of skim milk carrageenan gels at 5°C as a function of (a) heat treatment and (b) whey protein content and temperature. Casein micelles were included in the mixtures; k-carragean concentration 200 ppm. Isobars indicate gel strength in Pa.
Response surface methodology was employed to study the effect of whey protein denaturation on k-carrageenan gelation in milk. It was concluded that k-carrageenan did not influence the rate of heat denaturation of the individual whey proteins and it was unlikely that whey protein denaturation played a significant role in k-carrageenan gelation.
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In serum and skim milk ultrafiltrate, carrageenan gelation proceeded irrespective of the heat treatment of the mixtures and the gel strength was enhanced with the whey protein content. In the presence of casein micelles the path of gel formation was greatly influenced by the heating temperature/time combinations. In the presence of casein micelles gelation did not occur with mild heat treatments (60°C for 1 – 10 min) at this low level of carrageenan employed. The presence of whey proteins together with the caseins enhanced gel strength. On the other hand, for the same whey protein content the milk – carrageenan gels were weaker than the respective serum–carrageenan gels. These results suggested that in the absence of caseins, carrageenan interacted with the serum proteins and this interaction had a synergistic effect on gel strength. k-Carrageenan interacted also with the caseins but this interaction had an inhibitory effect on gelation. Nevertheless, heat induced denaturation of the whey proteins and their interaction with the casein micelles did not interfere with the gelation mechanism to any major extent.
Acknowledgements The authors wish to thank Dr W. Steele for expert technical support. This research was
.
funded by the Scottish Office Agriculture, Environment and Fisheries Department.
References [1] C.-F. Lin, in: H.D. Graham (Ed.), Food Colloids, Avi Publishing, Westport, CT, 1977, p. 320. [2] P.M.T. Hansen, in: G.O. Phillips, D.J. Wedlock, P.A. Williams (Eds.), Gums and Stabilisers for the Food Industry-1, Proceedings of the 1st International Conference, Wrexham, July 1981, Pergamon Press, Oxford, 1982, p. 127. [3] H.M. Snoeren, k-carrageenan. A study on its physicochemical properties, sol-gel transition and interaction with milk proteins. Thesis, Nederlands Instituut voor Zuivelonderzoek, Ede, The Netherlands, 1976. [4] G. Stainsby, Food Chem. 6 (1980) 3. [5] D.D. Drohan, A. Tziboula, D.D. McNulty, D.S Horne, Food Hydrocoll. 1 (1997) 101. [6] A. Tziboula, D.S. Horne, in: G.O. Phillips, D.J. Wedlock, P.A. Williams (Eds.), Gums and Stabilisers for the Food Industry, vol. 9, Proceedings of the 9th International conference, Wrexham, July 1997, IRL Press at Oxford University Press, Oxford, New York, Tokyo, 1998, p. 202. [7] A. Tziboula, D.S. Horne, Int. Dairy J. 8 (1998) (in press). [8] A.J.R. Law, J.M. Banks, D.S. Horne, J. Leaver, I.G. West, Milchwissenschaft 49 (1994) 63. [9] A.J.R. Law, J. Leaver, J.M. Banks, D.S. Horne, Milchwissenschaft 48 (1993) 663. [10] D.G. Dalgleish, E.R. Morris, Food Hydrocoll. 2 (1988) 311. [11] V. Langendorff, B. Cuvelier, B. Launay, A. Parker, Food Hydrocoll. 11 (1997) 35.
Influence of whey protein denaturation on k-carrageenan gelation A. Tziboula *, D.S Horne Hannah Research Institute, Ayr, KA6 5HL, Scotland, UK
Abstract Response surface methodology was applied to study the effect of different heating temperature/time treatments on whey protein denaturation and its effect on k-carrageenan gelation in milk. The path of gel formation was followed using small deformation rheology and the extent of whey protein denaturation was determined by gel permeation chromatography. k-Carrageenan did not influence the rate of whey protein denaturation and it was unlikely that whey protein denaturation played a significant role on k-carrageenan gelation in milk. In skim milk serum or skim milk ultrafiltrate the path of gel formation and gel strength were not influenced by the severity of heat treatment but increasing the concentration of whey proteins enhanced the gel strength. Heat treatment became important for carrageenan gelation in skim or recombined milks (i.e. in the presence of casein micelles) by influencing the gelation temperature and gel strength. Increasing the concentration of whey proteins in the recombined milks had a beneficial effect on gel strength. © 1999 Elsevier Science B.V. All rights reserved. Keywords: Whey protein; k-Carrageenan; Gel permeation chromatography
1. Introduction The carrageenans are sulphated linear polysaccharides of alternating b-1,3- and a-1,4-linked galactose residues that are used to modify texture and stability in dairy foods. In the presence of cations they form thermoreversible gels. It is generally accepted that carrageenans interact specifically with the caseins (mainly k-casein but also as1- and b-caseins) to form a complex which
* Corresponding author.
aggregates into a three-dimensional network [1– 4]. The ability of carrageenans to stabilize dairy products such as chocolate milk and cream has been demonstrated for polymer concentrations as low as 50 ppm [3]. Despite, the economic importance of carrageenans in dairy applications, few studies of their behaviour in milk have been published. Carrageenan gelation in the presence of serum proteins is of particular interest. We found that the serum-carrageenan gels were stronger than the corresponding milk gels [5–7]. Lower concentrations of carrageenan were required for gelation in
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serum rather than in milk [7]. Heating the carrageenan dispersions at temperatures above 60°C is necessary for the hydration of the polymer. Such treatments take the milks into temperature ranges where whey protein denaturation is observed [8]. Such denaturation could influence gelation. The main objective in this work was to investigate the effect of whey protein denaturation on k-carrageenan gelation, in polymer dispersions with or without casein micelles. The carrageenan concentration was fixed at 200 ppm. At these concentrations the carrageenan gels are weak allowing the effects from the interactions with caseins and/or whey proteins to be discerned. Otherwise carrageenan gelation swamps the interaction effects. Gelation was followed during cooling of the milk – carrageenan mixtures from 59 to 5°C, at a rate of 1°C 15 min − 1. Admittently, this slow cooling rate is not practical in most industrial applications. Nevertheless, it has been shown that the rate of cooling had an enormous effect on the gelation temperature and the mechanical properties of the gels [6]. Response surface methodology was used to study the effect of three variables on k-carrageenan gelation: (1) heating temperature (i.e. from 60 to 90°C); (2) duration of heating (i.e. from 1–10 min); the temperature/time combinations determined the extent of heat-denaturation of the various whey proteins [8]; and (3) whey protein content. Using these variables, two designs were generated: in the first, casein micelles were not included in the carrageenan mixtures. The whey protein content was varied from 0 to 100% (i.e. complete absence of whey proteins to their normal concentration found in skim milk). So in the first design k-carrageenan (200 ppm) was added to skim milk ultrafiltrate (SMUF), skim milk serum/SMUF solutions at a dilution ratio 1:1 and skim milk serum. The ionic composition was maintained equivalent to that in skim milk. In the second, casein micelles were also included in the mixtures at constant concentration equivalent to that in skim milk. The extent of whey protein denaturation was measured by gel permeation chromatography and
the path of carrageenan gelation was studied using low frequency oscillation.
2. Materials and methods
2.1. Response surface methods Response surface methodology is used in the empirical study of relationships between one or more measured responses and a number of variables. Their advantage over the one-variable-at-atime approach is that they provide information on what might happen if the variables were changed together. The two designs employed were selected from the library of experimental designs implemented in Minitab version 11.2. They were two central composite designs of three variables, each set at two levels. These designs allowed efficient fitting and checking of second-degree polynomial models: y= b0 + b1x1 + b2x2 + b3x3 + b11x 21 + b22x 22 + b33x 23 + b12x1x2 + b13x1x3 + b23x2x3 where bii are the coefficients and x1, whey protein content; x2, heating temperature (60–90°C); x3, heating time (1–10 min). The k-carrageenan concentration was fixed at 200 ppm. In the first design, the whey protein content in the solutions varied from nil (i.e. SMUF) to 100% (skim milk serum). Casein micelles were not included in the mixtures. In the second design casein micelles were also present in the mixtures. The whey protein content was adjusted as in the first design. Casein micelles were resuspended in the solutions prior to carrageenan addition. The amount of resuspended caseins was kept constant equivalent to that in skim milk. In the extremes of treatments k-carrageenan was dispersed in SMUF (absence of whey proteins and casein micelles) and skim milk (whey proteins and casein micelles present). Table 1 identifies the variables and specifies their levels for each run. The repeatability of the measurements, was estimated by the standard error of the mean (SEM) in the central points of the designs.
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2.2. Preparation of milk fractions Bulk milk from the Institute herd was skimmed by centrifugation at 1200×g for 30 min at 4°C (Mistral 6000 centrifuge; Sanyo Gallenkamp, Leicester, UK). Skim milk ultrafiltrate (SMUF) was prepared from skim milk using an ultrafiltration cell (TCF10A, Amicon, Danvers, MA) equipped with a membrane of molecular weight cut-off of 10 kDa. Micellar caseins were pelleted from skim milk by centrifugation at 43 000× g for 60 min (Sorval RC 5B; Du Pont Instruments, Newtown, CT). The micellar caseins were resuspended, by overnight stirring at 4°C, in SMUF-serum solutions according to the design requirements listed in Table 1. These were designated recombined milks.
Table 1 Description of the variables and specification of the levels for the response surface designs Whey protein concentration (% of total in milk)
Temperature (°C) Time (min)
301
Skim milk serum was the supernatant layer obtained during the preparation of micellar caseins. This comprised mainly the serum protein, soluble casein (15–20% total casein) and the soluble phase in milk. The soluble casein was removed by isoelectric precipitation at pH 4.6, followed by centrifugation and filtration. The serum fraction was then dialysed at 4°C against skim milk, for ionic equilibration until the final pH of the serum fraction was 6.779 0.02 (48 h; three skim milk changes; serum to skim milk ratio 1:250). The serum fraction was diluted with the appropriate amount of SMUF to adjust the whey protein level (Table 1).
2.3. Carrageenan k-Carrageenan from Euchema cottonoii (Aldrich, Gillingham, Dorset, UK) was converted to the sodium form using cation exchange resin (Amberlite IR120 cation exchange resin in the Na form, BDH, Poole, UK). The polysaccharide was recovered by filtration to remove resin and freeze drying of the filtrate. The sodium content was determined to be 5.67% w/ w by atomic absorption spectroscopy.
a,b
0 0 0 0 0 50 50 50 50 50 50
60 60 75 90 90 60 75 75 75 75 90
1.0 10.0 5.5 1.0 10.0 5.5 5.5 1.0 5.5 10.0 5.5
100 100 100 100 100
60 60 75 90 90
1.0 10.0 5.5 1.0 10.0
a Design 1: the whey protein content was varied by diluting skim milk serum with SMUF; 0%, SMUF; 50%, skim milk serum/SMUF 1:1; 100%, Skim milk serum. b Design 2: whey protein content was varied as for design 1. Casein micelles were added in SMUF or skim milk serum/ SMUF 1:1, at concentrations equal to skim milk.
2.4. Preparation of k-carrageenan dispersions k-Carrageenan was added to the appropriate milk fraction (skim milk, skim milk serum, SMUF, SMUF-serum solutions, recombined milks) while stirring at room temperature for at least 1 h. Complete dispersion of the carrageenan was achieved by homogenisation for 2 min, at standard setting (Silverson laboratory mixer emulsifier, Silverson machines, Waterside, Chesham, Bucks). The mixture were heated to the required temperatures in a boiling water bath and immediately transferred to another thermostated controlled water bath, set at the required temperature, where they continued to be heated for the appropriate time according to the temperature/time combinations in Table 1. The mixtures were then transferred from that state to the rheometer which was sitting at 59°C.
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2.5. Dynamic rheology Low frequency oscillation (0.08 Hz, applied stress 5 mPa) was performed on the milk-carrageenan mixtures as they were cooled from 59 to 5°C, at a cooling rate 1°C 15 min − 1, using a controlled stress rheometer (CVO; Bohlin Instruments, Gloucestershire, UK), fitted with the double gap measuring geometry. The geometry consists of a hollow cylinder (diameter 45 mm) which is lowered into a cylindrical groove in an outer cylinder (diameter 50 mm). Although the instrument calculates the phase angle (d), the angle by which the strain response lags behind the applied stress and this allows the derivation of the storage (G%), viscous (G¦), complex (G*) moduli and complex viscosity (h*), only the complex modulus G* =( G% 2 + G¦ 2)1/2 of the carrageenan gels is considered here. The gelation temperature of the gels was taken as the temperature where tan d =1. Because of the long duration of each run, prevention of evaporation was achieved by fitting the solvent trap on the double gap geometry. The applied stress 5 mPa was, as always, a compromise between attempting to operate in the linear viscoelastic region of the structures created and of maintaining a measurable response on gelation. The stress used here, 5 mPa, is just above the bottom end of the stress range permissible with the double-gap geometry in the Bohlin Instrument. The strain response varied with the cooling cycle and viscoelastic properties of the milk-carrageenan mixtures. The initial strain response (at 59°C) was constant at 1.42 9 0.01 whilst at 5°C it ranged from 0.4 for the weakest structures to 0.002 for stronger gels.
2.6. Gel permeation fast protein liquid chromatography (GPC) The extent of whey protein denaturation was measured in a separate experiment by gel permeation chromatography. Samples were prepared with and without k-carrageenan according to the conditions in Table 1. After heat treatment as specified in Table 1, the samples were cooled at a
rate of 1°C 15 min − 1 to simulate the cooling profile of the rheological test. The samples containing whey proteins were then filtered through 0.2 mm filter to remove the aggregated denatured whey proteins and k-carrageenan. In the samples containing micellar caseins, the denatured whey proteins were precipitated together with the casein by adjusting the pH to 4.6, followed by filtration. The whey protein composition of the filtrates was assessed by gel permeation chromatography on a Superdex 75 column HR 10/30 (Pharmacia, Biotech, Milton Keynes, UK) eluted with a Tris–HCl buffer (pH 7.0, 100 mM Tris, 0.5 M NaCl) at a flow rate 0.5 ml min − 1 [9]. The extent of denaturation of the whey proteins was calculated from the relative decrease in the area of their elution peaks compared with the corresponding areas in filtrates from unheated skim milk or serum.
3. Results and discussion
3.1. Design 1: influence of whey protein denaturation on k-carrageenan gelation in dispersions without casein micelles Typical rheological profiles of the development of the complex moduli (G*) during cooling of the various carrageenan mixtures from 59–5°C, at a rate of 1°C 15 min − 1, are shown in Fig. 1. The panels show the results for the extremes of treatments. Thus, k-carrageenan was dispersed: Fig. 1(a), in skim milk (casein micelles and whey proteins present); Fig. 1(b), in recombined milk (i.e. casein micelles resuspended in milk ultrafiltrate); Fig. 1(c), in skim milk serum which does not contain casein micelles; and Fig. 1(d), in SMUF which does not contain either whey proteins or casein micelles. The slow cooling rate was chosen because of its great effect on the gelation temperature and the mechanical properties of the gels. Gelation studies in carrageenan-milk and carrageenan-SMUF mixtures showed that a reduction in the rate of cooling from 1°C 45 s − 1 to 1°C 15 min − 1 resulted in an increase in the gelation temperature by 10 and 25°C respectively [6]. Fast cooling rates also resulted in weaker gels. This means that
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303
Fig. 1. Influence of heat treatment on k-carrageenan gelation: (a) skim milk; (b) recombined milk; (c) milk serum; (d) skim milk ultrafiltrate (SMUF); (e) indicates no gelation; —— , 90°C for 10 min; – – – – , 90°C for 1 min; — – — – — – 75°C for 5.5 min; -------, 60°C for 10 min; ······ 60°C for 1.0 min; TTR, transition temperature; GT, gelation temperature.
although the cooling rate does not influence the temperature of the conformational transition of carrageenan and the mechanism of gel formation, it affects the perceived rheogical gelation times and gel strengths in short experiments and can lead to erroneous conclusions. In all samples, there was an initial lag period during which the G* was very low and the mixture remained fluid. Below the gelation point the lack of structure in the material was reflected by the low G% values (in the range 10 − 4 – 10 − 5 Pa). These extremely low values are simply indicative of fluid samples. The onset of carrageenan gelation was signaled by a sharp rise in G* at the carrageenan coil-to-helix transition temperature (TTR, ca. 38°C). This point reflects the first ‘real’ measurement of the viscoelastic contributions to the structure of the samples. At this point the strain dropped to 0.006 for the weakest gels and 1.2 for the strongest changing rapidly as the gel develops. Thereafter the path of gel formation
was influenced by the protein composition of the mixtures. A detailed account of the effect of milk proteins on the path of carrageenan gelation has been given elsewhere [7]. In the absence of casein micelles (Fig. 1(c–d) heat treatment did not influence either the gelation temperature or the rate of gel strength development. However, the path of gel formation differed: in SMUF gelation took place into distinct steps, the second sharp rise in G* corresponding to the rheological gelation temperature (GT, Fig. 1(d)). In SMUF, the GT was :5°C lower than the coil to helix transition temperature. In skim milk serum which includes whey proteins, k-carrageenan gelation proceeded in a single step but the initial rise in G* was followed by a region of loss of structure, though recovery of structure on further cooling was almost immediate (see inset Fig. 1(c)). This anomaly in the built up of structure has been noticed previously [7] and has been attributed to thermoreversible
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(Table 2). Thus, it was confirmed that the whey protein content had a positive effect on gel strength (pB 0.005, Table 2). Whilst the heating temperature did not have a significant effect, the duration of heat treatment seemed to influence the development of G*. This effect was more pronounced at maximum concentrations of whey proteins (Fig. 2; PB 0.05, Table 2). The contour plots in Fig. 2, the estimated plots of G* as a function of whey protein content and heating time combinations, were calculated for a model where the square terms, temperature, whey protein× temperature and heating time× temperature interactions were omitted. The repeatability of the measurements was estimated by the SEM of the central points of the designs. SEM for GT and G* were 1.35°C and 10 mPa respectively. Comparing the GPC data on the extent of whey protein denaturation, with and without k-carrageenan, it was concluded that k-carrageenan, at the levels employed here, did not influence the rate of denaturation of the individual whey proteins. As was anticipated, b-lactoglobulin (blg) and a-lactalbumin (a-la) denaturation were
aggregation of whey proteins possibly ogether with k-carrageenan. As the gel strength and gelation path were not influenced by heat treatment it was concluded that whey protein denaturation did not play a significant role in k-carrageenan gelation in mixtures of varying whey protein content. However, comparison of the k-carrageenan – SMUF rheological profiles (Fig. 1(d)) with the corresponding situations in serum (Fig. 1(c)), showed that carrageenan formed stronger gels in the presence of whey proteins. Analysis of variance of the full quadratic model, showed that the linear and interaction terms of the regression function were the most important whilst the second degree terms were not statistically significant. The data was remodeled using only these statistically significant terms because the reduced function represented more accurately our data. From the linear terms, temperature and its interaction with the other terms were also omitted from the regression because these two were identified as not significant in the initial analysis. The re-fitted model explained : 85.9% of the variance in the data
Table 2 Response surface regression: analysis of variance and the estimated regression coefficients of the effect of whey protein, heating temperature and heating time, on the complex modulus at 5°C (G*) of carrageenan gels in the presence or absence of casein micellesa Analysis of variance
Design 1: absence of casein micelles
Design 2: presence of casein micelles
r
r
P
r 2 =91.3%
*** *** NS *
Regression equation Linear terms Square terms Interaction
r 2 = 87.7%
Terms
Coefficients
Constant Whey protein Temperature Heating time Whey×temp. Whey×heat. time Heat. time×temp.
1245.0 552.4 100.9 290.0 141.4 274.9 146.4
a
*** NS *
r: The correlation coefficient of the regression equation. The terms were excluded from the respective models. * PB0.05. ** PB0.01. *** PB0.005.
b
P
Coefficients *** *** Nsb * NSb * NSb
287.1 154.0 452.0 207.3 179.8 49.9 254.7
NS NS ** * * NSb *
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played a significant role in k-carrageenan gelation in serum. Finally, from the gelling behaviour of k-carrageenan in SMUF, it was concluded that the different heat treatments had little effect on the extent of hydration of the polymer.
3.2. Design 2: influence of whey protein denaturation on k-carrageenan gelation in the presence of casein micelles
Fig. 2. Design 1: contour plots of the estimated complex modulus (G*) of carragean gels at 5°C, as a function of whey protein content and heating time. Casein micelles were not present in the mixtures; k-carregeenan concentration fixed at 200 ppm. Isobars indicate gel strength (Pa).
controlled by the temperature/ heating time combinations (Table 3). Of the other remaining whey proteins, immunoglobulins were the most heat sensitive and relatively mild heat treatments (60°C) resulted in complete denaturation. Serum albumin and lactoferrin were denatured at temperatures above 75°C (results not shown). The combined effect of heating temperature and duration of heating on the amounts of denatured b-lg and a-la, expressed as percentage of total, are shown in Fig. 3(a – b). These plots were generated by fitting the reduced second-order polynomial model for the statistically significant terms shown in Table 3. The regression equations were highly significant and accounted for 95.9% and 89.5% of the variance in the data for b-lg and a-la respectively. b-Lg was more heat sensitive than a-la (compare Fig. 3(a,b)). Furthermore, whilst the effect of duration of heat treatment was insignificant at 60°C it became important at higher temperatures. This effect was more pronounced with a-lactalbumin, the most heat resistant of the whey proteins. These results were in agreement with previously published data [8]. Overall, it was concluded that k-carrageenan did not influence the rate of denaturation of the individual whey proteins, as measured by aggregate formation in the whey protein mix, and it was unlikely that whey protein denaturation
From the gelation profiles shown in Fig. 1(a,b) it is apparent that the various heat treatments Table 3 Response surface regression: analysis of variance and estimated regression coefficients of the effect of casein micelles, heating temperature, heating time on the extent of whey protein denaturationa Analysis of variance
Regression equation Linear terms
b-lactoglobulin
a-lactalbumin
r
P
r
P
r 2 =95.9%
****
r 2 =89.5%
****
****
****
Square terms
****
***
Interaction
***
****
Term
Coefficients for blg
Constant
309.75
***
351.07
***
Casein micelles Temperature
1.36 −9.75
NSb
−4.40 −9.68
** ***
Heating time
−5.50
NS
−12.66
****
(Temp)2
0.08
****
0.07
***
Temp×heat. Time
0.09
***
0.20
****
a
****
Coefficients for a-la
r: The correlation coefficient of the regression equation. The term was excluded from the refitted model for b-lactoglobulin denaturation. ** PB0.01. *** PB0.005. **** PB0.001. b
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Fig. 3. Contour plots of the estimated effects of heating temperature and duration of heat treatment on (a) b-lactoglobulin and (b) a-lactalbumin denaturation in a whey protein mixture from skim milk. Isobars indicate extent of denaturation (% of total).
become important for k-carrageenan gelation in mixtures containing casein micelles. Thus, gelation did not take place following mild heat treatments (60°C for 1 – 10 min). Furthermore, the rate of gel strength development increased with the severity of heat treatment. This effect was attributed to the presence of casein micelles in the carrageenan mixtures, because it was consistently observed irrespective of the whey protein content in the samples. Gelation took place in a single step (both in skim milk-carrageenan and recombined milk- carrageenan mixtures), signaled by a sharp rise in G* at 38°C, followed by fast aggre-
gation and gelation. The sharp rise in G* was also observed in the non-gelling samples (Fig. 1(a,b)), which indicated conformational change of the polymer but lack of capability for further network formation. Fitting the full second order model to the data showed that the second order terms, some main terms and two way interactions were not statistically significant. Therefore, the data was remodeled using only the statistically significant terms and the results are shown in Table 2. In mixtures containing casein micelles, the strength of the carrageenan gels (taken as G* at 5°C) was influenced mainly by the heating temperature/time combinations (PB0.05; Table 2). The contour plot of the estimated G* as a function of heating treatment is shown in Fig. 4(a). It can be seen that maximum gel strength is associated with high heating temperature/time combinations. As with the first design an increase in the whey protein content also had a beneficial effect on G*. This effect was more pronounced at high heating temperatures (PB 0.05, Table 2). The effect of whey protein content and heating temperature on gel strength is shown in Fig. 4(b). Both plots in Fig. 4 were generated using only the statistically significant terms in Table 2. These results imply that k-carrageenan interacts with the casein micelles, thus reducing the polymer availability for a gelation role. In other words, we always observe only carrageenan gelation. Interaction with casein micelles reduces kcarrageenan available for gel formation and this weakens the gel produced. Such an interaction between carrageenan and casein micelles was also shown in electrophoretic mobility studies in solutions containing diluted milk and concentrations of carrageenans of up to 0.2 mg ml − 1, although no compensation was made for viscosity effects [10]. What we see in this work is a heat sensitive situation where the casein–k-carrageenan complex content is reduced by heating, allowing stronger gels to be formed. When whey proteins are present in company with casein micelles, we also get stronger gels. In this context, we suggest that whey proteins interact with micelles and reduce their ability to interact with k-carrageenan, hence the increase in gel strength.
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These results do not agree with previously published studies on i-carrageenan gelation [11]. Langendorff et al. [11] showed there, that milk-i-carrageenan gels were significantly stronger than SMUF – carrageenan gels. However, fast cooling rates were applied in their rheological studies and we have shown these to influence both the rheological gelation time and mechanical properties of the gels [6]. This effect of cooling rate differs depending on the milk components present in the system, being more pronounced in SMUF–car-
307
rageenan than in milk–carrageenan mixtures [6]. Finally, Langendorff et al. [11] show anomalously high initial G¦ values (i.e. at 65°C) in milk, a factor of ten greater than their measurements in SMUF. In our studies initial G¦ were around 0.4–1.4 mPa in SMUF– and milk–carrageenan mixtures respectively, at a concentration of carrageenan 0.1% w/w [5]. That the effects of heat treatment on carrageenan gelation in milk are related to differences in the extent of hydration of the carrageenan molecule can be ruled out because similar observations were made when carrageenan was added to preheated milk. These results are the subject of another publication. Nevertheless, other heat induced changes in milk could influence k-carrageenan gelation. For example, changes in the solubility of inorganic calcium phosphate and a decrease in pH influence the protein charge and hence the interaction potential with other polymers. Side chains of some aminoacids, especially that of lysine also become very reactive on heating and it is possible that they also influence k-carrageenan gelation. The extent of heat induced denaturation of the individual whey proteins in skim or recombined milk was similar to that in the serum (Table 3). As with the first design k-carrageenan was not found to have a significant effect on the rate of whey protein denaturation. As a result of this observation and because the effect of heat treatment on the path of k-carrageenan gelation was associated with the casein fraction rather than the whey protein fraction in the mixtures, it was concluded that whey protein denaturation did not play a significant role on carrageenan gelation. However, it could not be ruled out that whey protein interactions with casein micelles have a minor influence on gel strength.
4. Conclusions
Fig. 4. Design 2: contour plots of the estimated complex modulus (G*) of skim milk carrageenan gels at 5°C as a function of (a) heat treatment and (b) whey protein content and temperature. Casein micelles were included in the mixtures; k-carragean concentration 200 ppm. Isobars indicate gel strength in Pa.
Response surface methodology was employed to study the effect of whey protein denaturation on k-carrageenan gelation in milk. It was concluded that k-carrageenan did not influence the rate of heat denaturation of the individual whey proteins and it was unlikely that whey protein denaturation played a significant role in k-carrageenan gelation.
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In serum and skim milk ultrafiltrate, carrageenan gelation proceeded irrespective of the heat treatment of the mixtures and the gel strength was enhanced with the whey protein content. In the presence of casein micelles the path of gel formation was greatly influenced by the heating temperature/time combinations. In the presence of casein micelles gelation did not occur with mild heat treatments (60°C for 1 – 10 min) at this low level of carrageenan employed. The presence of whey proteins together with the caseins enhanced gel strength. On the other hand, for the same whey protein content the milk – carrageenan gels were weaker than the respective serum–carrageenan gels. These results suggested that in the absence of caseins, carrageenan interacted with the serum proteins and this interaction had a synergistic effect on gel strength. k-Carrageenan interacted also with the caseins but this interaction had an inhibitory effect on gelation. Nevertheless, heat induced denaturation of the whey proteins and their interaction with the casein micelles did not interfere with the gelation mechanism to any major extent.
Acknowledgements The authors wish to thank Dr W. Steele for expert technical support. This research was
.
funded by the Scottish Office Agriculture, Environment and Fisheries Department.
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