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Effect of individual whey proteins on the rheological properties of acid gels prepared from heated skim milk
International Dairy Journal 13 (2003) 401–408
Effect of individual whey proteins on the rheological properties of acid gels prepared from heated skim milk Johanna F. Graveland-Bikker1, Skelte G. Anema* Food Science Section, Fonterra Research Centre, Private Bag 11029, Palmerston North, New Zealand Received 2 July 2002; accepted 21 November 2002
Abstract The effect of fortification of whey-protein-depleted (WPD) milk with different levels of b-lactoglobulin (b-LG), a-lactalbumin (a-lac) or mixtures of b-LG and a-lac on the rheological properties of acid skim milk gels was investigated. Milk samples were heated at 80 C for 30 min before acidification with glucono-d-lactone. Acid gels prepared from heated WPD milk had lower G0 values, longer gelation times and lower gelation pHs than those prepared from heated skim milk. The G0 values of acid gels from the WPD milk were not as low as those observed for unheated milk, which may be attributed to the residual whey proteins in the WPD milk. Addition of increasing levels of a-lac to the WPD milk prior to heat treatment caused only small increases in the G0 values of the acid gels, and had a small effect on the gelation time and gelation pH. Similarly, the addition of a-lac to WPD milk with added b-LG caused small increases in the G0 value of the acid gels when compared with that of the WPD/b-LG milk sample regardless of the genetic variant of b-LG. The addition of increasing levels of b-LG to the WPD milk prior to heat treatment and acidification caused marked increases in the G0 value and the gelation pH and a reduction in the gelation time. This effect was observed whether the b-LG was added by itself or in combination with a-lac. There were some differences in behaviour between the different b-LG variants. The addition of increasing levels of b-LG B or b-LG C to the WPD milk prior to heating and acidification caused an almost linear increase in the G0 value. In contrast, the addition of b-LG A caused a progressive increase in the G0 value with added protein levels up to about 0.9% (w/w) but little further change at higher addition levels. r 2003 Elsevier Science Ltd. All rights reserved. Keywords: Acid gels; Whey proteins; a-Lactalbumin; b-Lactoglobulin; Rheological properties
1. Introduction The slow acidification of milk from its natural pH (about pH 6.7) to about pH 4.6 causes the milk to form a gel. Heat treatment of milk at temperatures above about 70 C prior to acidification is a common method of increasing the gel firmness and reducing the level of syneresis. This effect is a consequence of whey protein denaturation during the heat treatment as these denatured proteins are more susceptible to inter-protein aggregation with other denatured whey proteins or with the casein micelles. Dannenberg and Kessler (1988a, b) *Corresponding author. Tel.: +64-6-350-4649; fax: +64-6-3561476. E-mail address: [email protected] (S.G. Anema). 1 Present Address: Food Physics Group, Department of Agrotechnology and Food Science, Wageningen University, P.O. Box 8129, 6700 EV Wageningen, The Netherlands.
showed that the firmness of set non-fat yoghurt increased and the amount of syneresis decreased as the level of whey protein denaturation was increased. Lucey, Tamehana, Singh, and Munro (1998) showed that the specific association of denatured whey proteins with the casein micelles is important for structure formation and, in particular, for the marked increase in firmness of acid gels made from heated milk. The fortification of milk with whey protein concentrate (WPC) can further modify the textural and rheological properties of acid gels. The addition of WPC to milk prior to heating increases the firmness and reduces the syneresis of the resultant acid gels (Robinson & Tamime, 1986) and reduces the gelation time, increases the pH of gelation and markedly increases the G0 value of the gels (Lucey, Munro, & Singh, 1999). Interestingly, Bikker, Anema, Li, and Hill (2000) have shown that the addition of whey protein mixtures containing b-lactoglobulin (b-LG) A progressively
0958-6946/03/$ - see front matter r 2003 Elsevier Science Ltd. All rights reserved. doi:10.1016/S0958-6946(02)00190-5
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increased the firmness of the acid gels with added protein levels up to about 0.7% (w/w) but there was little further change at higher addition levels. In contrast, the addition of increasing levels of whey protein mixtures containing b-LG B or b-LG C caused an almost linear increase in the firmness of the acid gels at all addition levels. This behaviour was considered to be due to the differences in the aggregation behaviour between the different genetic variants of b-LG. Although there have been a few studies on the effect of adding whey proteins to milk prior to heat treatment and acidification on the rheological properties of acid gels, these have been on whey protein mixtures, such as WPC or combinations of purified proteins. In addition, these whey proteins have been additional to those already found in the milk at the natural levels. There have been no studies on the effect of the individual whey proteins, particularly b-LG and a-lactalbumin (a-lac), on the properties of acid gels. This study examined the effect of purified b-LG (the A, B and C variants) and purified a-lac, or mixtures of purified b-LG and a-lac, on the rheological properties of acid skim milk gels. Wheyprotein-depleted (WPD) milk was used as the milk source as this reduced the complications from the naturally occurring whey proteins in the skim milk.
2.3. Sample preparation, heat treatment and gel formation The required quantities of the purified b-LG and a-lac were added directly to the WPD skim milk. The milk was allowed to stir for about 1 h at ambient temperature and then placed in a refrigerator set at 4.5 C for at least 12 h before use. WPD skim milk samples with the added whey proteins (30 mL) were allowed to equilibrate to room temperature for 6 h. The milks were adjusted to pH 6.65 then heated at 80 C for 30 min in a thermostatically controlled oil bath preset to 80 C. After heat treatment, the milk samples were cooled by immersion in ice water until the temperature was below 30 C. The milk samples were acidified using glucono-dlactone (GDL; Sigma Chemical Co., St. Louis, MO, USA) at a 2% addition level. In selected samples, the pH change with time was monitored and was shown to be similar for all milk samples, regardless of the presence or absence of the whey proteins. This indicates that the casein and milk salts dominated the pH buffering of the milk, and that the whey depletion process or the addition of whey protein components had only a very small effect on the pH versus time curves. 2.4. Rheological measurements
2. Materials and methods 2.1. Milk supply, preparation of b-LG and preparation of a-lac Details on obtaining the fresh milk, on the preparation of the reconstituted skim milk samples, and on the separation and purification of b-LG and a-lac have been reported previously (Bikker et al., 2000).
The rheological properties of the acidified milks were monitored with time using low amplitude dynamic oscillation as has been described previously (Bikker et al., 2000). The changes in the rheological properties during acidification were monitored. In addition the effects of the time scale of the applied strain (0.01– 10 Hz) and the effect of temperature (30 C to 5 C) on the rheological properties of the set gels were examined. All experiments were performed in duplicate.
2.2. Preparation of WPD milk WPD milk was prepared by subjecting milk to a combination of ultrafiltration and microfiltration techniques. A large volume (about 25 L) of reconstituted skim milk was ultrafiltered using a 10,000 Da (nominal) hollow fibre membrane cartridge and the associated pumping equipment (Amicon, Inc., Beverly, MA, USA) until about 3 L of milk permeate was collected. A separate sample of milk (0.5 L) was microfiltered using a membrane cartridge with a 0.1 mm pore size. This membrane allows the passage of a-lac and b-LG but retains the casein and some of the higher molecular weight whey proteins. The volume of microfiltrate was measured periodically and an equivalent volume of the ultrafiltrate was added back to the milk. This procedure was continued until the desired level of whey protein depletion was achieved. Electrophoretic analysis indicated that the WPD milk contained less than 5% of the original b-LG and only traces of the original a-lac.
3. Results All G0 versus time curves for the acid gelation of milk were similar in shape to those reported previously (Lucey, Teo, Munro, & Singh, 1997; Lucey et al., 1998, 1999; Bikker et al., 2000). The G0 used in the discussion refers to the final G0 value measured after 6 h, whereas the gelation pH and gelation time refer to the pH and time at which G0 became X1 Pa. Fig. 1 shows the gelation curves with time for the heated milk, heated WPD milk and heated WPD milk with 0.3% b-LG and 0.15% a-lac added. A curve for the unheated milk is included for comparison. The unheated WPD milk was virtually indistinguishable from that of the unheated skim milk. Key gelation parameters are summarised in Table 1. Acid gels prepared from unheated skim milk had very low G0 values, long gelation times and low gelation pHs. Samples prepared
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from heated (80 C/30 min) milk had markedly higher G0 values, reduced gelation times and increased gelation pHs (Fig. 1, Table 1). These observations are in accord with literature reports (van Vliet & Keetels, 1995; Lucey et al., 1997, 1998, 1999; Bikker et al., 2000). The WPD milk had a G0 value about one-third that of the heated milk, but still markedly higher than that of the unheated milk. In addition, the gelation time and gelation pH were intermediate between those of the unheated and heated skim milk samples. Analysis has shown that the WPD milk had similar mineral and casein content to the original skim milk. In addition, the pH change on acidification was similar for all milk samples regardless of whether whey proteins were present or absent. Electrophoretic analysis indicated that only traces of initial a-lac and approximately 5% of
Fig. 1. Storage modulus, G0 , as a function of time for acid milk gels made at 30 C with GDL: () unheated skim milk; (J) heated skim milk; (.) heated WPD milk; and (X) heated WPD milk + 0.3% bLG+0.15% a-lac.
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the initial b-LG remained in the WPD milk. However, the minor whey proteins, in particular bovine serum albumin, immunoglobulin G and lactoferrin are only partially removed during the depletion process. These residual whey proteins, although at relatively low concentration, will denature and aggregate during heat treatment and this may account for the increase in G0 and gelation pH and the decrease in gelation time when WPD milk is compared with the unheated milk. The addition of a-lac and b-LG to WPD milk at approximately natural levels produced a similar G0 versus time curve to that of the original skim milk. This indicates that the whey protein depletion process of the skim milk and the purification processes for b-LG and a-lac did not markedly change the acid gelation properties of the milk as long as the whey protein composition was similar to its initial state. The effect of adding a-lac to the WPD milk prior to heat treatment and acidification, and the effect of adding a-lac to WPD milk that also contained 0.3% b-LG B is shown in Fig. 2. Similar results (not shown) were obtained for b-LG A and C. Key gelation parameters are summarised in Table 1. The addition of 0.3% 0.6% and 0.9% a-lac increased the G0 of the acid gels by about 20, 50 and 100 Pa, respectively. When compared with the heated WPD milk, the gelation pH increased slightly and the gelation time decreased slightly on addition of a-lac. For all samples, the final G0 and the gelation pH were markedly lower and the gelation time was longer than those of the heated skim milk, even though the sample with 0.9% added a-lac had a total whey protein level about twice that of the original milk.
Table 1 Gelation properties of unheated and heated skim milk samples, and heated WPD milk samples with various levels of added a-lac or various levels of added a-lac with 0.3% added b-LG A, B or C. Samples were acidified with GDL at 30 C Sample
Gelation time (min)
Gelation pH
Final G0 at 30 C (Pa)
Unheated skim milk/unheated WPD milk Heated skim milk Heated WPD milk
97 35 67
4.76 5.29 4.95
22 373 152
47 804 297
55 52 52
5.04 5.08 5.08
167 212 249
320 425 478
WPD milk+0.3% a-lac +0.3% b-LG A WPD milk+0.6% a-lac +0.3% b-LG A WPD milk+0.9% a-lac +0.3% b-LG A
32 33 33
5.33 5.31 5.31
376 434 460
752 929 1042
WPD milk+0.3% a-lac +0.3% b-LG B WPD milk+0.6% a-lac +0.3% b-LG B WPD milk+0.9% a-lac +0.3% b-LG B
30 31 31
5.35 5.34 5.34
399 451 497
784 931 1173
WPD milk+0.3% a-lac +0.3% b-LG C WPD milk+0.6% a-lac +0.3% b-LG C WPD milk+0.9% a-lac +0.3% b-LG C
31 31 30
5.34 5.34 5.35
418 455 474
886 969 1097
WPD milk+0.3% a-lac WPD milk+0.6% a-lac WPD milk+0.9% a-lac
Final G0 at 5 C (Pa)
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Fig. 2. Storage modulus, G0 , as a function of time for acid milk gels made at 30 C with GDL. WPD milk with various levels of added a-lac or WPD milk 0.3% added b-LG B and various levels of added a-lac: () WPD milk; (J) WPD milk+0.3% added a-lac; (.) WPD milk+0.6% added a-lac; (,) WPD milk+0.9% added a-lac; (’) WPD milk+0.3% b-LG; (&) WPD milk+0.3% b-LG and 0.3% added a-lac; (m) WPD milk+0.3% b-LG and 0.6% added a-lac; and (n) WPD milk+0.3% b-LG and 0.9% added a-lac.
The addition of 0.3% b-LG to the WPD milk markedly increased the G0 of the acid gels. The addition of a-lac had a slightly greater effect on the G0 of the acid gels when added in the presence of 0.3% b-LG, compared with milk that was free of b-LG. The observed increase in G0 , when compared with the samples with only b-LG added, were about 75, 140 and 175 Pa on the addition of 0.3% 0.6% and 0.9% a-lac, respectively. The addition of increasing levels of a-lac to the WPD milk with 0.3% added b-LG had little effect on the gelation pH and the gelation time when compared with the milk with added b-LG only. The effect of adding the different b-LG variants to the WPD milk on the final G0 is shown in Fig. 3A, whereas the effect of adding b-LG and a-lac at a 2:1 ratio (i.e. the approximate ratio in which these proteins are found in skim milk) on the final G0 values is shown in Fig. 3B. Key gelation parameters are summarised in Tables 2 and 3. Interestingly the effect of adding increasing levels of the different variants of b-LG were similar regardless of whether they were added in the presence or absence of a-lac (Fig. 3A). The addition of increasing levels of protein containing b-LG B and b-LG C to the WPD milk caused a progressive increase in the G0 value throughout the protein addition range. In contrast, the addition of increasing levels of b-LG A to the WPD milk caused a progressive increase in the G0 value with added protein levels up to about 0.6–0.9% (w/w), whereas at higher addition levels little further change was observed. These results are very similar to those observed when model whey protein concentrates with different b-LG variants were added directly to skim milk (Bikker et al., 2000). In all samples, the G0 of the set gels at 5 C was approximately twice that observed at 30 C, so that
Fig. 3. (A) Final storage modulus, G0 , as a function of added b-LG for set acid milk gels made at 30 C with GDL: () WPD milk+various levels of added b-LG A; (J) WPD milk+various levels of added bLG B; (.) WPD milk+various levels of added b-LG C. (B) Final storage modulus, G0 , as a function of added protein (2:1 ratio of b-LG A to a-lac) for set acid milk gels made at 30 C with GDL: () WPD milk+added b-LG A and a-lac; (J) WPD milk+added b-LG B a-lac; and (.) WPD milk+added b-LG C and a-lac.
the observed effects of the added whey proteins were similar at the two temperatures (Tables 1–3). This effect of temperature on the G0 value of acid gels is in agreement with literature reports (Roefs & van Vliet, 1990; Lucey et al., 1997; Bikker et al., 2000). At both 30 C and 5 C and for all samples, plots of the logarithm of frequency against the logarithm of G0 produced straight lines with slopes of about 0.15 (results not shown), which is similar to previous reports for acid skim milk gels (Lucey et al., 1997, 1998, 1999; Bikker et al., 2000). At both 30 C and 5 C, the plots of tan d against the logarithm of frequency were curved with a minimum at a frequency of about 0.3 Hz and higher tan d at higher and lower frequency (Fig. 4). This is in agreement with literature reports for other types of acid milk protein gel systems (Chen, Dickinson, & Edwards, 1999; Bikker et al., 2000). At any particular frequency, the tan d increased very slightly when a-lac was added to the WPD milk with 0.3% added b-LG (Fig. 4A). These results indicate that the acid gels had a slightly more
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Table 2 Gelation properties of heated WPD milk samples with various levels of added b-LG A, B or C. Samples were acidified with GDL at 30 C Sample
Gelation time (min)
Gelation pH
Final G0 at 30 C (Pa)
Final G0 at 5 C (Pa)
WPD WPD WPD WPD WPD
milk+0.3% milk+0.6% milk+0.9% milk+1.2% milk+1.5%
b-LG b-LG b-LG b-LG b-LG
A A A A A
30 25 22 19 18
5.34 5.40 5.43 5.47 5.48
312 411 538 512 576
560 717 856 830 889
WPD WPD WPD WPD WPD
milk+0.3% milk+0.6% milk+0.9% milk+1.2% milk+1.5%
b-LG b-LG b-LG b-LG b-LG
B B B B B
29 24 21 18 17
5.35 5.41 5.45 5.48 5.49
293 487 574 709 798
560 880 950 1150 1308
WPD WPD WPD WPD WPD
milk+0.3% milk+0.6% milk+0.9% milk+1.2% milk+1.5%
b-LG b-LG b-LG b-LG b-LG
C C C C C
30 26 22 19 18
5.34 5.39 5.43 5.47 5.48
351 466 580 699 851
650 814 996 1228 1390
Table 3 Gelation properties of heated WPD milk samples with various levels of added a-lac and b-LG A, B or C. Samples were acidified with GDL at 30 C Sample
Gelation time (min)
Gelation pH
Final G0 at 30 C (Pa)
WPD WPD WPD WPD WPD WPD
milk+0.075% a-lac +0.15% b-LG A milk+0.15% a-lac +0.3% b-LG A milk+0.225% a-lac +0.45% b-LG A milk+0.3% a-lac +0.6% b-LG A milk+0.45% a-lac +0.9% b-LG A milk+0.6% a-lac +1.2% b-LG A
41 33 27 25 21 18
5.22 5.31 5.38 5.40 5.45 5.48
288 375 475 487 493 481
535 580 862 943 936 974
WPD WPD WPD WPD WPD WPD
milk+0.075% a-lac +0.15% b-LG B milk+0.15% a-lac +0.3% b-LG B milk+0.225% a-lac +0.45% b-LG B milk+0.3% a-lac +0.6% b-LG B milk+0.45% a-lac +0.9% b-LG B milk+0.6% a-lac +1.2% b-LG B
41 35 28 23 22 18
5.22 5.29 5.37 5.42 5.43 5.48
268 348 487 567 631 705
505 689 871 938 1188 1373
WPD WPD WPD WPD WPD WPD
milk+0.075% a-lac +0.15% b-LG C milk+0.15% a-lac +0.3% b-LG C milk+0.225% a-lac +0.45% b-LG C milk+0.3% a-lac +0.6% b-LG C milk+0.45% a-lac +0.9% b-LG C milk+0.6% a-lac +1.2% b-LG C
40 33 27 22 20 17
5.23 5.31 5.38 5.43 5.46 5.49
275 370 459 496 583 728
513 685 875 909 1166 1551
viscous character as the a-lac concentration was increased. In contrast to the effect of a-lac, at any particular frequency, the tan d decreased markedly as the level of added b-LG was increased. This effect was observed when only the b-LG concentration was increased (Fig. 4B) and when both the b-LG and a-lac concentrations were increased (results not shown). This indicates that the acid gels from these milks had a more elastic character as the b-LG concentration was increased, and that the effect of b-LG addition dominated when a-lac was also present. Only small differences in tan d were observed between b-LG A,
Final G0 at 5 C (Pa)
b-LG B and b-LG C at any particular whey protein addition level (results not shown).
4. Discussion Numerous changes occur to milk on slow acidification (Robinson & Tamime, 1986, 1993; Mulvihill & Grufferty, 1995; Lucey & Singh, 1998). Of particular importance to acid gelation is the reduction in protein solubility as the milk pH is reduced to levels close to the isoelectric point of the protein constituents. For
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Fig. 4. Final loss tangent, tan d, as a function of frequency for acid milk gels when measured at 30 C: (A) WPD milk with 0.3% added bLG C and various levels of added a-lac: () WPD milk; (J) WPD milk+0.3% b-LG C; (.) WPD milk+0.3% b-LG C and 0.3% a-lac; (X) WPD milk +0.3% b-LG C and 0.6% added a-lac; (’) WPD milk+0.3% b-LG C and 0.9% added a-lac. (B) WPD milk with various levels of added b-LG C; () WPD milk; (J) WPD milk+0.3% b-LG C; (.) WPD milk+0.6% b-LG C; (X) WPD milk+0.9% b-LG C; (’) WPD milk+1.2% b-LG C; and (&) WPD milk+1.5% b-LG C.
unheated milk, gelation occurred at about pH 4.8 (Table 1) which is close to the isoelectric point of casein (about pH 4.6). Native whey proteins remain soluble and therefore play no part in the structure formation in acid gels prepared from unheated milk. There are several reports outlining the changes to milk on heating (Singh & Creamer, 1992; Holt, 1995; Singh, 1995). Of interest to acid gelation is the denaturation and aggregation of the whey proteins which occurs when milk is heated at temperatures above about 70 C (Dannenberg & Kessler, 1988c). Unlike the native whey proteins, denatured whey proteins are insoluble at their isoelectric points. As the pH of the heated WPD milk with added b-LG or added b-LG and a-lac approaches the isoelectric point of the b-LG (about pH 5.3), the denatured whey proteins aggregate and contribute to the acid gel structure. As a consequence, the gelation pH increased from about pH 4.8 in unheated milk to about pH 5.35 in the heated WPD milk with 0.3% added b-LG or 0.3% added b-LG and 0.15% added a-lac. The gelation pH increased further on addition of higher levels of b-LG, either with or without a-lac (Tables 2 and 3). No measurable
difference between the A, B and C variants of b-LG was observed. Similarly, very little difference in gelation time or gelation pH could be observed between milks with just b-LG added, or both b-LG and a-lac added. The pH of gelation increased from about pH 4.8 in unheated milk to about pH 5.0–5.1 in the heated WPD milk with added a-lac. This increase was observed at all concentrations of added a-lac. The experimentally determined isoelectric point for a-lac is reported to be at pH 4.8 (Swaisgood, 1982), which is only slightly higher than that of casein. This small difference in isoelectric pH probably accounts for the small change in gelation pH for the milk samples with added a-lac. The addition of increasing levels of b-LG to the WPD milk prior to heat treatment caused marked increases in the G0 values of the acid gels (Fig. 3A). This effect was observed regardless of whether a-lac was present or absent (compare Figs. 3A and B). In contrast, the addition of increasing levels of a-lac to the WPD milk prior to heat treatment and acidification had only a small effect on the G0 value of the acid gels regardless of whether b-LG was present or not (Fig. 2). The rigidity of gels and their resistance to deformation is dependent on the number and the strength of contact points between aggregated particles (van Vliet & Keetels, 1995). Therefore, these results suggest that there are a larger number of interconnections of higher strength in the samples with added b-LG when compared with those with low levels of b-LG or with added a-lac. An important feature of the primary structure of b-LG is the presence of a free sulphydryl group and two disulphide linkages. In the native conformation, the sulphydryl group is buried within the protein tertiary structure and is relatively unreactive. However, on heating, this group is exposed and is able to undergo thiol–disulphide exchange reactions with other denatured whey proteins or with k-casein (and possibly aS2casein) within the casein micelle. These exchange reactions introduce new intermolecular covalent bonds between the different protein species, and in particular between the denatured b-LG and the casein micelles. Lucey et al. (1998) showed that the increased disulphide cross-linking between the denatured whey proteins and the casein micelles plays a dominant role in increasing the G0 value of acid gels made from heated milk. In the absence of other whey proteins (e.g. b-LG, bovine serum albumin, immunoglobulin G), the denaturation of a-lac is substantially reversible (Ruegg, Moor, & Blanc, 1977). This may account for the smaller effect of a-lac in the WPD milk when compared with the effect in the presence of b-LG (Fig. 2). However, there may be sufficient bovine serum albumin and immunoglobulin G present in the WPD milk to allow irreversible aggregation of a-lac as only relatively low levels of proteins with reactive free sulphydryl groups are required (Gezimati, Creamer, & Singh, 1997). The
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observation that high levels of a-lac have only a small effect even in the presence of b-LG indicates that denatured a-lac does not incorporate into the acid gel structure in the same way as denatured b-LG. The primary structure of a-lac contains four disulphide bonds but no free thiol groups. As a consequence, denatured a-lac can be incorporated into aggregate structures only via thiol–disulphide exchange reactions with denatured proteins that contain free sulphydryl groups (e.g. b-LG) or through non-disulphide bonding (e.g. hydrophobic bonding). Therefore, although a-lac can be incorporated into aggregate structures, it does not increase the number of free sulphydryl groups available to form disulphide cross-links. If a-lac is added to milk, larger aggregates incorporating the a-lac can be formed but these will contain fewer disulphide crosslinks; therefore, a weaker structure may be expected when compared with the structures formed from the addition of b-LG. This difference in aggregation behaviour may account for the differences in acid gel strength when a-lac and b-LG are added to WPD milk. The results in Fig. 3 show that addition of the B and C variants of b-LG to skim milk prior to heating and acidification caused an almost linear increase in the G0 value of the resultant acid gels, whereas the addition of the A variant increased the G0 value up to a certain level of added protein (0.6–0.9%) but had little further effect at higher addition levels. These results are very similar to those observed when model WPC containing b-LG A, B C were added to skim milk prior to heating and acidification (Bikker et al., 2000). These differences in the effect of the b-LG variants were considered to be due to reported differences in denaturation and aggregation behaviour of the denatured proteins during heating (Manderson, Hardman, & Creamer, 1998; Manderson, Creamer, & Hardman, 1999). This in turn would contribute to the differences in the properties of the milk during subsequent acidification and gel formation (Bikker et al., 2000).
5. Conclusions This study showed that the addition of a-lac to WPD milk prior to heat treatment and acidification had only a small effect on the G0 value of the acid gels, regardless of whether b-LG was present or not. In contrast, the addition of b-LG to the WPD milk caused marked increases in the G0 value of the acid gel, with similar effects in the presence or absence of a-lac. These differences are probably related to differences in the aggregation behaviour between these protein species and, in particular, the fact that a-lac can be incorporated into aggregate structures only if other denatured whey proteins with free sulphydryl groups are available. As reported previously, the addition of increasing levels of
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b-LG B and C to the WPD milk caused a progressive increase in the final G0 value at all added protein levels, whereas the addition of b-LG A had an effect at addition levels up to 0.9% with little further effect at higher addition levels. This effect is probably related to the differences in aggregation behaviour between the different b-LG variants.
Acknowledgements The authors would like to thank Siew Kim Lee for training on the Bohlin rheometer, Andrew Rogers for assistance with the pH measurements during acidification, Yuming Li for technical assistance, Mike Boland, Lawrie Creamer and Jeremy Hill for useful discussions and Claire Woodhall for proofreading the manuscript. This work was supported by the New Zealand Foundation for Research, Science and Technology, contract nos. DRI0001 and DRIX0201.
References Bikker, J. F., Anema, S. G., Li, Y., & Hill, J. P. (2000). Rheological properties of acid gels prepared from heated milk fortified with whey protein mixtures containing the A, B and C variants of b-lactoglobulin. International Dairy Journal, 10, 723–732. Chen, J., Dickinson, E., & Edwards, M. (1999). Rheology of acidinduced sodium caseinate stabilized emulsion gels. Journal of Texture Studies, 30, 377–396. Dannenberg, F., & Kessler, H.-G. (1988a). Effect of denaturation of b-lactoglobulin on texture properties of set-style nonfat yoghurt. 1. Syneresis. Milchwissenschaft, 43, 632–635. Dannenberg, F., & Kessler, H.-G. (1988b). Effect of denaturation of b–lactoglobulin on texture properties of set-style nonfat yoghurt. 2. Firmness and flow properties. Milchwissenschaft, 43, 700–704. Dannenberg, F., & Kessler, H.-G. (1988c). Reaction kinetics of the denaturation of whey proteins in milk. Journal of Food Science, 53, 258–263. Gezimati, J., Creamer, L. K., & Singh, H. (1997). Heat-induced interactions and gelation of mixtures of b-lactoglobulin and alactalbumin. Journal of Agricultural and Food Chemistry, 45, 1130–1136. Holt, C. (1995). Effect of heating and cooling on the milk salts and their interaction with casein. In P. F. Fox (Ed.), International Dairy Federation Special Issue No. 9501. Heat-induced changes in milk (2nd ed.). (pp. 105–133). Brussels: International Dairy Federation. Lucey, J. A., & Singh, H. (1998). Formation and physical properties of acid milk gels: A review. Food Research International, 30, 529–542. Lucey, J. A., Teo, C. T., Munro, P. A., & Singh, H. (1997). Rheological properties at small (dynamic) and large (yield) deformations of acid gels made from heated milk. Journal of Dairy Research, 64, 591–600. Lucey, J. A., Tamehana, M., Singh, H., & Munro, P. A. (1998). Effect of interactions between denatured whey proteins and casein micelles on the formation and rheological properties of acid skim milk gels. Journal of Dairy Research, 65, 555–567. Lucey, J. A., Munro, P. A., & Singh, H. (1999). Effect of heat treatments and whey protein addition on the rheological properties and structure of acid skim milk gels. International Dairy Journal, 9, 275–279.
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Manderson, G. A., Hardman, M. J., & Creamer, L. K. (1998). Effect of heat treatment on the conformation and aggregation of b-lactoglobulin A, B, and C. Journal of Agricultural and Food Chemistry, 46, 5052–5061. Manderson, G. A., Creamer, L. K., & Hardman, M. J. (1999). Effect of heat treatment on the circular dichroism spectra of bovine b-lactoglobulin A, B, and C. Journal of Agricultural and Food Chemistry, 47, 4557–4567. Mulvihill, D. M., & Grufferty, M. B. (1995). Effect of thermal processing on the coagulability of milk by acid. In P. F. Fox (Ed.), International Dairy Federation Special Issue No. 9501 Heat-induced changes in milk (2nd ed.) (pp. 188–205). Brussels: International Dairy Federation. Robinson, R. K., & Tamime, A. Y. (1986). The role of protein in yoghurt. In B. J. F. Hudson (Ed.), Developments in food proteins., Vol. 4. (pp. 1–35). London: Elsevier Applied Science. Robinson, R. K., & Tamime, A. Y. (1993). Manufacture of yoghurt and other fermented milks. In: R. K. Robinson (Ed.), Modern dairy technology, Vol. 2, Advances in milk products (2nd ed.) (pp. 1–48). London: Elsevier Applied Science.
Roefs, S. P. F. M., & van Vliet, T. (1990). Structure of acid casein gels. 2. Dynamic measurements and interaction forces. Colloids and Surfaces, 50, 161–175. Ruegg, M., Moor, U., & Blanc, B. (1977). A calorimetric study of the thermal denaturation of whey proteins in simulated milk ultrafiltrate. Journal of Dairy Research, 44, 509–520. Singh, H. (1995). Heat-induced changes in casein, including interactions with whey proteins. In P. F. Fox (Ed.), International Dairy Federation Special Issue No. 9501. Heat-induced changes in milk (2nd ed.) (pp. 86–104). Brussels: International Dairy Federation. Singh, H., & Creamer, L. K. (1992). Heat stability of milk. In P. K. Fox (Ed.), Advanced dairy chemistry, Vol. 1. (pp. 621–656). London: Elsevier Science Publishers. Swaisgood, H. E. (1982). Chemistry of milk protein. In P. F. Fox (Ed.), Developments in dairy chemistry, Vol. 1. (pp. 1–59). London: Elsevier Science Publishers. van Vliet, T., & Keetels, C. J. A. M. (1995). Effect of preheating of milk on the structure of acidified milk gels. Netherlands Milk and Dairy Journal, 49, 27–35.
Effect of individual whey proteins on the rheological properties of acid gels prepared from heated skim milk Johanna F. Graveland-Bikker1, Skelte G. Anema* Food Science Section, Fonterra Research Centre, Private Bag 11029, Palmerston North, New Zealand Received 2 July 2002; accepted 21 November 2002
Abstract The effect of fortification of whey-protein-depleted (WPD) milk with different levels of b-lactoglobulin (b-LG), a-lactalbumin (a-lac) or mixtures of b-LG and a-lac on the rheological properties of acid skim milk gels was investigated. Milk samples were heated at 80 C for 30 min before acidification with glucono-d-lactone. Acid gels prepared from heated WPD milk had lower G0 values, longer gelation times and lower gelation pHs than those prepared from heated skim milk. The G0 values of acid gels from the WPD milk were not as low as those observed for unheated milk, which may be attributed to the residual whey proteins in the WPD milk. Addition of increasing levels of a-lac to the WPD milk prior to heat treatment caused only small increases in the G0 values of the acid gels, and had a small effect on the gelation time and gelation pH. Similarly, the addition of a-lac to WPD milk with added b-LG caused small increases in the G0 value of the acid gels when compared with that of the WPD/b-LG milk sample regardless of the genetic variant of b-LG. The addition of increasing levels of b-LG to the WPD milk prior to heat treatment and acidification caused marked increases in the G0 value and the gelation pH and a reduction in the gelation time. This effect was observed whether the b-LG was added by itself or in combination with a-lac. There were some differences in behaviour between the different b-LG variants. The addition of increasing levels of b-LG B or b-LG C to the WPD milk prior to heating and acidification caused an almost linear increase in the G0 value. In contrast, the addition of b-LG A caused a progressive increase in the G0 value with added protein levels up to about 0.9% (w/w) but little further change at higher addition levels. r 2003 Elsevier Science Ltd. All rights reserved. Keywords: Acid gels; Whey proteins; a-Lactalbumin; b-Lactoglobulin; Rheological properties
1. Introduction The slow acidification of milk from its natural pH (about pH 6.7) to about pH 4.6 causes the milk to form a gel. Heat treatment of milk at temperatures above about 70 C prior to acidification is a common method of increasing the gel firmness and reducing the level of syneresis. This effect is a consequence of whey protein denaturation during the heat treatment as these denatured proteins are more susceptible to inter-protein aggregation with other denatured whey proteins or with the casein micelles. Dannenberg and Kessler (1988a, b) *Corresponding author. Tel.: +64-6-350-4649; fax: +64-6-3561476. E-mail address: [email protected] (S.G. Anema). 1 Present Address: Food Physics Group, Department of Agrotechnology and Food Science, Wageningen University, P.O. Box 8129, 6700 EV Wageningen, The Netherlands.
showed that the firmness of set non-fat yoghurt increased and the amount of syneresis decreased as the level of whey protein denaturation was increased. Lucey, Tamehana, Singh, and Munro (1998) showed that the specific association of denatured whey proteins with the casein micelles is important for structure formation and, in particular, for the marked increase in firmness of acid gels made from heated milk. The fortification of milk with whey protein concentrate (WPC) can further modify the textural and rheological properties of acid gels. The addition of WPC to milk prior to heating increases the firmness and reduces the syneresis of the resultant acid gels (Robinson & Tamime, 1986) and reduces the gelation time, increases the pH of gelation and markedly increases the G0 value of the gels (Lucey, Munro, & Singh, 1999). Interestingly, Bikker, Anema, Li, and Hill (2000) have shown that the addition of whey protein mixtures containing b-lactoglobulin (b-LG) A progressively
0958-6946/03/$ - see front matter r 2003 Elsevier Science Ltd. All rights reserved. doi:10.1016/S0958-6946(02)00190-5
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increased the firmness of the acid gels with added protein levels up to about 0.7% (w/w) but there was little further change at higher addition levels. In contrast, the addition of increasing levels of whey protein mixtures containing b-LG B or b-LG C caused an almost linear increase in the firmness of the acid gels at all addition levels. This behaviour was considered to be due to the differences in the aggregation behaviour between the different genetic variants of b-LG. Although there have been a few studies on the effect of adding whey proteins to milk prior to heat treatment and acidification on the rheological properties of acid gels, these have been on whey protein mixtures, such as WPC or combinations of purified proteins. In addition, these whey proteins have been additional to those already found in the milk at the natural levels. There have been no studies on the effect of the individual whey proteins, particularly b-LG and a-lactalbumin (a-lac), on the properties of acid gels. This study examined the effect of purified b-LG (the A, B and C variants) and purified a-lac, or mixtures of purified b-LG and a-lac, on the rheological properties of acid skim milk gels. Wheyprotein-depleted (WPD) milk was used as the milk source as this reduced the complications from the naturally occurring whey proteins in the skim milk.
2.3. Sample preparation, heat treatment and gel formation The required quantities of the purified b-LG and a-lac were added directly to the WPD skim milk. The milk was allowed to stir for about 1 h at ambient temperature and then placed in a refrigerator set at 4.5 C for at least 12 h before use. WPD skim milk samples with the added whey proteins (30 mL) were allowed to equilibrate to room temperature for 6 h. The milks were adjusted to pH 6.65 then heated at 80 C for 30 min in a thermostatically controlled oil bath preset to 80 C. After heat treatment, the milk samples were cooled by immersion in ice water until the temperature was below 30 C. The milk samples were acidified using glucono-dlactone (GDL; Sigma Chemical Co., St. Louis, MO, USA) at a 2% addition level. In selected samples, the pH change with time was monitored and was shown to be similar for all milk samples, regardless of the presence or absence of the whey proteins. This indicates that the casein and milk salts dominated the pH buffering of the milk, and that the whey depletion process or the addition of whey protein components had only a very small effect on the pH versus time curves. 2.4. Rheological measurements
2. Materials and methods 2.1. Milk supply, preparation of b-LG and preparation of a-lac Details on obtaining the fresh milk, on the preparation of the reconstituted skim milk samples, and on the separation and purification of b-LG and a-lac have been reported previously (Bikker et al., 2000).
The rheological properties of the acidified milks were monitored with time using low amplitude dynamic oscillation as has been described previously (Bikker et al., 2000). The changes in the rheological properties during acidification were monitored. In addition the effects of the time scale of the applied strain (0.01– 10 Hz) and the effect of temperature (30 C to 5 C) on the rheological properties of the set gels were examined. All experiments were performed in duplicate.
2.2. Preparation of WPD milk WPD milk was prepared by subjecting milk to a combination of ultrafiltration and microfiltration techniques. A large volume (about 25 L) of reconstituted skim milk was ultrafiltered using a 10,000 Da (nominal) hollow fibre membrane cartridge and the associated pumping equipment (Amicon, Inc., Beverly, MA, USA) until about 3 L of milk permeate was collected. A separate sample of milk (0.5 L) was microfiltered using a membrane cartridge with a 0.1 mm pore size. This membrane allows the passage of a-lac and b-LG but retains the casein and some of the higher molecular weight whey proteins. The volume of microfiltrate was measured periodically and an equivalent volume of the ultrafiltrate was added back to the milk. This procedure was continued until the desired level of whey protein depletion was achieved. Electrophoretic analysis indicated that the WPD milk contained less than 5% of the original b-LG and only traces of the original a-lac.
3. Results All G0 versus time curves for the acid gelation of milk were similar in shape to those reported previously (Lucey, Teo, Munro, & Singh, 1997; Lucey et al., 1998, 1999; Bikker et al., 2000). The G0 used in the discussion refers to the final G0 value measured after 6 h, whereas the gelation pH and gelation time refer to the pH and time at which G0 became X1 Pa. Fig. 1 shows the gelation curves with time for the heated milk, heated WPD milk and heated WPD milk with 0.3% b-LG and 0.15% a-lac added. A curve for the unheated milk is included for comparison. The unheated WPD milk was virtually indistinguishable from that of the unheated skim milk. Key gelation parameters are summarised in Table 1. Acid gels prepared from unheated skim milk had very low G0 values, long gelation times and low gelation pHs. Samples prepared
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from heated (80 C/30 min) milk had markedly higher G0 values, reduced gelation times and increased gelation pHs (Fig. 1, Table 1). These observations are in accord with literature reports (van Vliet & Keetels, 1995; Lucey et al., 1997, 1998, 1999; Bikker et al., 2000). The WPD milk had a G0 value about one-third that of the heated milk, but still markedly higher than that of the unheated milk. In addition, the gelation time and gelation pH were intermediate between those of the unheated and heated skim milk samples. Analysis has shown that the WPD milk had similar mineral and casein content to the original skim milk. In addition, the pH change on acidification was similar for all milk samples regardless of whether whey proteins were present or absent. Electrophoretic analysis indicated that only traces of initial a-lac and approximately 5% of
Fig. 1. Storage modulus, G0 , as a function of time for acid milk gels made at 30 C with GDL: () unheated skim milk; (J) heated skim milk; (.) heated WPD milk; and (X) heated WPD milk + 0.3% bLG+0.15% a-lac.
403
the initial b-LG remained in the WPD milk. However, the minor whey proteins, in particular bovine serum albumin, immunoglobulin G and lactoferrin are only partially removed during the depletion process. These residual whey proteins, although at relatively low concentration, will denature and aggregate during heat treatment and this may account for the increase in G0 and gelation pH and the decrease in gelation time when WPD milk is compared with the unheated milk. The addition of a-lac and b-LG to WPD milk at approximately natural levels produced a similar G0 versus time curve to that of the original skim milk. This indicates that the whey protein depletion process of the skim milk and the purification processes for b-LG and a-lac did not markedly change the acid gelation properties of the milk as long as the whey protein composition was similar to its initial state. The effect of adding a-lac to the WPD milk prior to heat treatment and acidification, and the effect of adding a-lac to WPD milk that also contained 0.3% b-LG B is shown in Fig. 2. Similar results (not shown) were obtained for b-LG A and C. Key gelation parameters are summarised in Table 1. The addition of 0.3% 0.6% and 0.9% a-lac increased the G0 of the acid gels by about 20, 50 and 100 Pa, respectively. When compared with the heated WPD milk, the gelation pH increased slightly and the gelation time decreased slightly on addition of a-lac. For all samples, the final G0 and the gelation pH were markedly lower and the gelation time was longer than those of the heated skim milk, even though the sample with 0.9% added a-lac had a total whey protein level about twice that of the original milk.
Table 1 Gelation properties of unheated and heated skim milk samples, and heated WPD milk samples with various levels of added a-lac or various levels of added a-lac with 0.3% added b-LG A, B or C. Samples were acidified with GDL at 30 C Sample
Gelation time (min)
Gelation pH
Final G0 at 30 C (Pa)
Unheated skim milk/unheated WPD milk Heated skim milk Heated WPD milk
97 35 67
4.76 5.29 4.95
22 373 152
47 804 297
55 52 52
5.04 5.08 5.08
167 212 249
320 425 478
WPD milk+0.3% a-lac +0.3% b-LG A WPD milk+0.6% a-lac +0.3% b-LG A WPD milk+0.9% a-lac +0.3% b-LG A
32 33 33
5.33 5.31 5.31
376 434 460
752 929 1042
WPD milk+0.3% a-lac +0.3% b-LG B WPD milk+0.6% a-lac +0.3% b-LG B WPD milk+0.9% a-lac +0.3% b-LG B
30 31 31
5.35 5.34 5.34
399 451 497
784 931 1173
WPD milk+0.3% a-lac +0.3% b-LG C WPD milk+0.6% a-lac +0.3% b-LG C WPD milk+0.9% a-lac +0.3% b-LG C
31 31 30
5.34 5.34 5.35
418 455 474
886 969 1097
WPD milk+0.3% a-lac WPD milk+0.6% a-lac WPD milk+0.9% a-lac
Final G0 at 5 C (Pa)
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Fig. 2. Storage modulus, G0 , as a function of time for acid milk gels made at 30 C with GDL. WPD milk with various levels of added a-lac or WPD milk 0.3% added b-LG B and various levels of added a-lac: () WPD milk; (J) WPD milk+0.3% added a-lac; (.) WPD milk+0.6% added a-lac; (,) WPD milk+0.9% added a-lac; (’) WPD milk+0.3% b-LG; (&) WPD milk+0.3% b-LG and 0.3% added a-lac; (m) WPD milk+0.3% b-LG and 0.6% added a-lac; and (n) WPD milk+0.3% b-LG and 0.9% added a-lac.
The addition of 0.3% b-LG to the WPD milk markedly increased the G0 of the acid gels. The addition of a-lac had a slightly greater effect on the G0 of the acid gels when added in the presence of 0.3% b-LG, compared with milk that was free of b-LG. The observed increase in G0 , when compared with the samples with only b-LG added, were about 75, 140 and 175 Pa on the addition of 0.3% 0.6% and 0.9% a-lac, respectively. The addition of increasing levels of a-lac to the WPD milk with 0.3% added b-LG had little effect on the gelation pH and the gelation time when compared with the milk with added b-LG only. The effect of adding the different b-LG variants to the WPD milk on the final G0 is shown in Fig. 3A, whereas the effect of adding b-LG and a-lac at a 2:1 ratio (i.e. the approximate ratio in which these proteins are found in skim milk) on the final G0 values is shown in Fig. 3B. Key gelation parameters are summarised in Tables 2 and 3. Interestingly the effect of adding increasing levels of the different variants of b-LG were similar regardless of whether they were added in the presence or absence of a-lac (Fig. 3A). The addition of increasing levels of protein containing b-LG B and b-LG C to the WPD milk caused a progressive increase in the G0 value throughout the protein addition range. In contrast, the addition of increasing levels of b-LG A to the WPD milk caused a progressive increase in the G0 value with added protein levels up to about 0.6–0.9% (w/w), whereas at higher addition levels little further change was observed. These results are very similar to those observed when model whey protein concentrates with different b-LG variants were added directly to skim milk (Bikker et al., 2000). In all samples, the G0 of the set gels at 5 C was approximately twice that observed at 30 C, so that
Fig. 3. (A) Final storage modulus, G0 , as a function of added b-LG for set acid milk gels made at 30 C with GDL: () WPD milk+various levels of added b-LG A; (J) WPD milk+various levels of added bLG B; (.) WPD milk+various levels of added b-LG C. (B) Final storage modulus, G0 , as a function of added protein (2:1 ratio of b-LG A to a-lac) for set acid milk gels made at 30 C with GDL: () WPD milk+added b-LG A and a-lac; (J) WPD milk+added b-LG B a-lac; and (.) WPD milk+added b-LG C and a-lac.
the observed effects of the added whey proteins were similar at the two temperatures (Tables 1–3). This effect of temperature on the G0 value of acid gels is in agreement with literature reports (Roefs & van Vliet, 1990; Lucey et al., 1997; Bikker et al., 2000). At both 30 C and 5 C and for all samples, plots of the logarithm of frequency against the logarithm of G0 produced straight lines with slopes of about 0.15 (results not shown), which is similar to previous reports for acid skim milk gels (Lucey et al., 1997, 1998, 1999; Bikker et al., 2000). At both 30 C and 5 C, the plots of tan d against the logarithm of frequency were curved with a minimum at a frequency of about 0.3 Hz and higher tan d at higher and lower frequency (Fig. 4). This is in agreement with literature reports for other types of acid milk protein gel systems (Chen, Dickinson, & Edwards, 1999; Bikker et al., 2000). At any particular frequency, the tan d increased very slightly when a-lac was added to the WPD milk with 0.3% added b-LG (Fig. 4A). These results indicate that the acid gels had a slightly more
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Table 2 Gelation properties of heated WPD milk samples with various levels of added b-LG A, B or C. Samples were acidified with GDL at 30 C Sample
Gelation time (min)
Gelation pH
Final G0 at 30 C (Pa)
Final G0 at 5 C (Pa)
WPD WPD WPD WPD WPD
milk+0.3% milk+0.6% milk+0.9% milk+1.2% milk+1.5%
b-LG b-LG b-LG b-LG b-LG
A A A A A
30 25 22 19 18
5.34 5.40 5.43 5.47 5.48
312 411 538 512 576
560 717 856 830 889
WPD WPD WPD WPD WPD
milk+0.3% milk+0.6% milk+0.9% milk+1.2% milk+1.5%
b-LG b-LG b-LG b-LG b-LG
B B B B B
29 24 21 18 17
5.35 5.41 5.45 5.48 5.49
293 487 574 709 798
560 880 950 1150 1308
WPD WPD WPD WPD WPD
milk+0.3% milk+0.6% milk+0.9% milk+1.2% milk+1.5%
b-LG b-LG b-LG b-LG b-LG
C C C C C
30 26 22 19 18
5.34 5.39 5.43 5.47 5.48
351 466 580 699 851
650 814 996 1228 1390
Table 3 Gelation properties of heated WPD milk samples with various levels of added a-lac and b-LG A, B or C. Samples were acidified with GDL at 30 C Sample
Gelation time (min)
Gelation pH
Final G0 at 30 C (Pa)
WPD WPD WPD WPD WPD WPD
milk+0.075% a-lac +0.15% b-LG A milk+0.15% a-lac +0.3% b-LG A milk+0.225% a-lac +0.45% b-LG A milk+0.3% a-lac +0.6% b-LG A milk+0.45% a-lac +0.9% b-LG A milk+0.6% a-lac +1.2% b-LG A
41 33 27 25 21 18
5.22 5.31 5.38 5.40 5.45 5.48
288 375 475 487 493 481
535 580 862 943 936 974
WPD WPD WPD WPD WPD WPD
milk+0.075% a-lac +0.15% b-LG B milk+0.15% a-lac +0.3% b-LG B milk+0.225% a-lac +0.45% b-LG B milk+0.3% a-lac +0.6% b-LG B milk+0.45% a-lac +0.9% b-LG B milk+0.6% a-lac +1.2% b-LG B
41 35 28 23 22 18
5.22 5.29 5.37 5.42 5.43 5.48
268 348 487 567 631 705
505 689 871 938 1188 1373
WPD WPD WPD WPD WPD WPD
milk+0.075% a-lac +0.15% b-LG C milk+0.15% a-lac +0.3% b-LG C milk+0.225% a-lac +0.45% b-LG C milk+0.3% a-lac +0.6% b-LG C milk+0.45% a-lac +0.9% b-LG C milk+0.6% a-lac +1.2% b-LG C
40 33 27 22 20 17
5.23 5.31 5.38 5.43 5.46 5.49
275 370 459 496 583 728
513 685 875 909 1166 1551
viscous character as the a-lac concentration was increased. In contrast to the effect of a-lac, at any particular frequency, the tan d decreased markedly as the level of added b-LG was increased. This effect was observed when only the b-LG concentration was increased (Fig. 4B) and when both the b-LG and a-lac concentrations were increased (results not shown). This indicates that the acid gels from these milks had a more elastic character as the b-LG concentration was increased, and that the effect of b-LG addition dominated when a-lac was also present. Only small differences in tan d were observed between b-LG A,
Final G0 at 5 C (Pa)
b-LG B and b-LG C at any particular whey protein addition level (results not shown).
4. Discussion Numerous changes occur to milk on slow acidification (Robinson & Tamime, 1986, 1993; Mulvihill & Grufferty, 1995; Lucey & Singh, 1998). Of particular importance to acid gelation is the reduction in protein solubility as the milk pH is reduced to levels close to the isoelectric point of the protein constituents. For
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Fig. 4. Final loss tangent, tan d, as a function of frequency for acid milk gels when measured at 30 C: (A) WPD milk with 0.3% added bLG C and various levels of added a-lac: () WPD milk; (J) WPD milk+0.3% b-LG C; (.) WPD milk+0.3% b-LG C and 0.3% a-lac; (X) WPD milk +0.3% b-LG C and 0.6% added a-lac; (’) WPD milk+0.3% b-LG C and 0.9% added a-lac. (B) WPD milk with various levels of added b-LG C; () WPD milk; (J) WPD milk+0.3% b-LG C; (.) WPD milk+0.6% b-LG C; (X) WPD milk+0.9% b-LG C; (’) WPD milk+1.2% b-LG C; and (&) WPD milk+1.5% b-LG C.
unheated milk, gelation occurred at about pH 4.8 (Table 1) which is close to the isoelectric point of casein (about pH 4.6). Native whey proteins remain soluble and therefore play no part in the structure formation in acid gels prepared from unheated milk. There are several reports outlining the changes to milk on heating (Singh & Creamer, 1992; Holt, 1995; Singh, 1995). Of interest to acid gelation is the denaturation and aggregation of the whey proteins which occurs when milk is heated at temperatures above about 70 C (Dannenberg & Kessler, 1988c). Unlike the native whey proteins, denatured whey proteins are insoluble at their isoelectric points. As the pH of the heated WPD milk with added b-LG or added b-LG and a-lac approaches the isoelectric point of the b-LG (about pH 5.3), the denatured whey proteins aggregate and contribute to the acid gel structure. As a consequence, the gelation pH increased from about pH 4.8 in unheated milk to about pH 5.35 in the heated WPD milk with 0.3% added b-LG or 0.3% added b-LG and 0.15% added a-lac. The gelation pH increased further on addition of higher levels of b-LG, either with or without a-lac (Tables 2 and 3). No measurable
difference between the A, B and C variants of b-LG was observed. Similarly, very little difference in gelation time or gelation pH could be observed between milks with just b-LG added, or both b-LG and a-lac added. The pH of gelation increased from about pH 4.8 in unheated milk to about pH 5.0–5.1 in the heated WPD milk with added a-lac. This increase was observed at all concentrations of added a-lac. The experimentally determined isoelectric point for a-lac is reported to be at pH 4.8 (Swaisgood, 1982), which is only slightly higher than that of casein. This small difference in isoelectric pH probably accounts for the small change in gelation pH for the milk samples with added a-lac. The addition of increasing levels of b-LG to the WPD milk prior to heat treatment caused marked increases in the G0 values of the acid gels (Fig. 3A). This effect was observed regardless of whether a-lac was present or absent (compare Figs. 3A and B). In contrast, the addition of increasing levels of a-lac to the WPD milk prior to heat treatment and acidification had only a small effect on the G0 value of the acid gels regardless of whether b-LG was present or not (Fig. 2). The rigidity of gels and their resistance to deformation is dependent on the number and the strength of contact points between aggregated particles (van Vliet & Keetels, 1995). Therefore, these results suggest that there are a larger number of interconnections of higher strength in the samples with added b-LG when compared with those with low levels of b-LG or with added a-lac. An important feature of the primary structure of b-LG is the presence of a free sulphydryl group and two disulphide linkages. In the native conformation, the sulphydryl group is buried within the protein tertiary structure and is relatively unreactive. However, on heating, this group is exposed and is able to undergo thiol–disulphide exchange reactions with other denatured whey proteins or with k-casein (and possibly aS2casein) within the casein micelle. These exchange reactions introduce new intermolecular covalent bonds between the different protein species, and in particular between the denatured b-LG and the casein micelles. Lucey et al. (1998) showed that the increased disulphide cross-linking between the denatured whey proteins and the casein micelles plays a dominant role in increasing the G0 value of acid gels made from heated milk. In the absence of other whey proteins (e.g. b-LG, bovine serum albumin, immunoglobulin G), the denaturation of a-lac is substantially reversible (Ruegg, Moor, & Blanc, 1977). This may account for the smaller effect of a-lac in the WPD milk when compared with the effect in the presence of b-LG (Fig. 2). However, there may be sufficient bovine serum albumin and immunoglobulin G present in the WPD milk to allow irreversible aggregation of a-lac as only relatively low levels of proteins with reactive free sulphydryl groups are required (Gezimati, Creamer, & Singh, 1997). The
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observation that high levels of a-lac have only a small effect even in the presence of b-LG indicates that denatured a-lac does not incorporate into the acid gel structure in the same way as denatured b-LG. The primary structure of a-lac contains four disulphide bonds but no free thiol groups. As a consequence, denatured a-lac can be incorporated into aggregate structures only via thiol–disulphide exchange reactions with denatured proteins that contain free sulphydryl groups (e.g. b-LG) or through non-disulphide bonding (e.g. hydrophobic bonding). Therefore, although a-lac can be incorporated into aggregate structures, it does not increase the number of free sulphydryl groups available to form disulphide cross-links. If a-lac is added to milk, larger aggregates incorporating the a-lac can be formed but these will contain fewer disulphide crosslinks; therefore, a weaker structure may be expected when compared with the structures formed from the addition of b-LG. This difference in aggregation behaviour may account for the differences in acid gel strength when a-lac and b-LG are added to WPD milk. The results in Fig. 3 show that addition of the B and C variants of b-LG to skim milk prior to heating and acidification caused an almost linear increase in the G0 value of the resultant acid gels, whereas the addition of the A variant increased the G0 value up to a certain level of added protein (0.6–0.9%) but had little further effect at higher addition levels. These results are very similar to those observed when model WPC containing b-LG A, B C were added to skim milk prior to heating and acidification (Bikker et al., 2000). These differences in the effect of the b-LG variants were considered to be due to reported differences in denaturation and aggregation behaviour of the denatured proteins during heating (Manderson, Hardman, & Creamer, 1998; Manderson, Creamer, & Hardman, 1999). This in turn would contribute to the differences in the properties of the milk during subsequent acidification and gel formation (Bikker et al., 2000).
5. Conclusions This study showed that the addition of a-lac to WPD milk prior to heat treatment and acidification had only a small effect on the G0 value of the acid gels, regardless of whether b-LG was present or not. In contrast, the addition of b-LG to the WPD milk caused marked increases in the G0 value of the acid gel, with similar effects in the presence or absence of a-lac. These differences are probably related to differences in the aggregation behaviour between these protein species and, in particular, the fact that a-lac can be incorporated into aggregate structures only if other denatured whey proteins with free sulphydryl groups are available. As reported previously, the addition of increasing levels of
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b-LG B and C to the WPD milk caused a progressive increase in the final G0 value at all added protein levels, whereas the addition of b-LG A had an effect at addition levels up to 0.9% with little further effect at higher addition levels. This effect is probably related to the differences in aggregation behaviour between the different b-LG variants.
Acknowledgements The authors would like to thank Siew Kim Lee for training on the Bohlin rheometer, Andrew Rogers for assistance with the pH measurements during acidification, Yuming Li for technical assistance, Mike Boland, Lawrie Creamer and Jeremy Hill for useful discussions and Claire Woodhall for proofreading the manuscript. This work was supported by the New Zealand Foundation for Research, Science and Technology, contract nos. DRI0001 and DRIX0201.
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