Factors affecting the hydrolytic action of plasmin in milk

ARTICLE IN PRESS

International Dairy Journal 15 (2005) 305–313 www.elsevier.com/locate/idairyj

Factors affecting the hydrolytic action of plasmin in milk Anthony Crudden, Patrick, F. Fox, Alan L. Kelly Department of Food and Nutritional Sciences, University College Cork, Cork, Ireland Received 31 October 2003; accepted 13 August 2004

Abstract The indigenous milk proteolytic enzyme, plasmin, is of technical importance as its hydrolytic action on casein may affect the quality of dairy products. The objective of this study was to examine the factors that affect plasmin-induced proteolysis during storage of milk and how such proteolysis may affect some properties of the casein micelles. Two competing mechanisms affected plasmin activity during storage at different temperatures, i.e., autolysis (self-hydrolysis) and plasminogen activation; at 5 1C, the former mechanism was more significant than the latter, while at 37 1C, activation predominated during the early stages of storage. At the latter temperature, however, 65% of plasmin activity was lost after 10 d, due to autolysis. Changing plasmin activity influenced the rate of hydrolysis of b-casein; at 37 1C, hydrolysis of b-casein ceased when 40% of the original b-casein remained. In addition, the temperature of storage significantly affected the composition of the casein micelles. Dissociation of b-casein during storage at 5 1C was significantly increased by preheating of milk (90 1C for 10 min); at 37 1C, dissociated b-casein in the milk serum was readily hydrolysed by plasmin. While pre-heating milk to 90 1C for 10 min greatly reduced plasmin activity and proteolysis, addition of exogenous plasmin to heated milk appeared to promote the release of b-lactoglobulin/k-casein complexes into milk serum. Overall, storage conditions had a considerable effect on the plasmin system and its subsequent action on casein micelles. r 2004 Elsevier Ltd. All rights reserved. Keywords: Milk; Plasmin; b-casein; Autolysis; Hydrolysis

1. Introduction Approximately 80% of the protein in bovine milk consists of a mixture of four phosphoproteins, aS1 -, aS2 -, b- and k-caseins, which exist as micelles in association with calcium phosphate (‘micellar’ or ‘colloidal’ calcium phosphate). The relative amounts of these proteins in the casein micelles are influenced by environmental factors; for example, during cold storage, extensive dissociation of b-casein occurs, with a concomitant increase in the level of b-casein in ultracentrifugal supernatants (Davies & Law, 1983; Dalgleish & Law, 1989). Roefs, Walstra, Dalgleish, and Horne (1985) and Dalgleish and Law (1988) reported solubilisation of other caseins during storage at 4 1C, particularly at Corresponding author. Tel.: +353 21 4903405; +353 21 4270001. E-mail address: [email protected] (A.L. Kelly).

fax:

0958-6946/$ - see front matter r 2004 Elsevier Ltd. All rights reserved. doi:10.1016/j.idairyj.2004.08.008

lower pH values. Pasteurisation or warming of milk partly reversed these changes (Ali, Andrews, & Cheeseman, 1980; Davies & Law, 1983) by increasing or strengthening hydrophobic interactions between the caseins and restoring micellar structure (De la Fuente, 1998). Levels of caseins in milk may be affected by the action of enzymes. Plasmin, the principal indigenous proteinase in milk, is optimally active at pH 7.5 and 37 1C, and is the primary agent of proteolysis in good quality milk (for reviews see Grufferty & Fox, 1988a; Bastian & Brown, 1996; Kelly & McSweeney, 2003). Plasmin is the active constituent of a complex enzyme system and is closely associated with the casein micelles, as is its zymogen, plasminogen (Politis, Barbano, & Gorewit, 1992). Plasminogen activators (PAs) are serine proteinases responsible for the conversion of plasminogen to plasmin (Lu & Nielsen, 1993; Heegaard, Rasmussen, & Andreasen, 1994; White et al., 1995). The caseins act as

ARTICLE IN PRESS 306

A. Crudden et al. / International Dairy Journal 15 (2005) 305–313

an immobilisation matrix for plasminogen activation (Baer, Ryba, & Collin, 1994; Politis, White, Zativon, Goldberg, Gou, & Kinstedt, 1995). The close proximity of plasmin to its substrate ensures that hydrolysis is an efficient process (Korycka-Dahl, Ridadeau Dumas, Chene, & Martal, 1983). The activity of plasmin in milk is regulated by a series of inhibitors, both of plasmin and PAs; such inhibitors are in the serum phase of milk (Weber & Nielsen, 1991; Politis, 1996). Plasmin preferentially cleaves polypeptide chains after a lysine or, to a lesser extent, an arginine residue (Ueshima, Okada, & Matsuo, 1996). aS1 -Casein is susceptible to proteolysis by plasmin (Eigel, 1977; Andrews & Alichanidis, 1983; McSweeney, Olson, Fox, Healy, & Højrup, 1993) and the l-caseins are products of this hydrolysis (Aimutis & Eigel, 1982; O’Flaherty, 1997). However, b-casein is the preferred substrate for plasmin and its hydrolysis results in the production of g-caseins and proteose-peptones (Andrews, 1983; Recio, Amigo, Ramos, & Lopez-Fandino, 1997; Trujillo, Guamis, & Carretero, 1998). aS2 -Casein is also readily hydrolysed by plasmin; to date, about eight cleavage sites for plasmin on this protein have been identified (Visser, Slangen, Alting, & Vreeman, 1989; Le Bars & Gripon, 1989). However, k-casein is resistant to proteolysis by plasmin (Diaz, Gouldsworthy, & Leaver, 1996). Plasmin has been implicated in a number of defects in dairy products, including reductions in curd strength and curd yield (Mara, Roupie, Duffy, & Kelly, 1998), age gelation of UHT milk (Kelly & Foley, 1997) and the formation of bitter off-flavours (Harwalkar, Cholette, McKellar, & Emmons, 1993). However, the hydrolysis of caseins by plasmin is considered to be important in the ripening of many cheese varieties (Farkye & Fox, 1992). While much of the work on plasmin has focussed on factors that affect its activity in fresh milk, and on the proteolytic products of its action on caseins, surprisingly little information is available on the factors that affect proteolysis in milk during storage. The objective of this study was to examine the influence of storage at 5 1C, which is the typical storage temperature for raw milk, at 20 1C, the temperature at which ultra-high temperature (UHT) milk is commonly stored, or at 37 1C, which is the optimum temperature for plasmin action, on proteolysis in milk and some properties of the casein micelles.

2. Materials and methods 2.1. Milk supply and storage conditions Skim milk (SM) was prepared by defatting raw whole milk (CMP Dairies, Cork) by centrifugation at 2000g

for 30 min at 20 1C and filtering through glass wool to remove any suspended fat particles. Sodium azide (NaN3, 0.05%, w/v) was added to prevent microbial growth. In some cases, SM was heated to 90 1C for 10 min (heated skim milk, HSM), subsequently cooled in iced-water to o5 1C and NaN3 added. Samples of SM and HSM were stored at 5 1C for 30 d or at 20 or 37 1C for 10 d. Throughout storage, all milk samples were monitored for changes in casein micelle size (Huppertz, Fox, & Kelly, 2004a), lightness (L*value), using a Minolta CR300 calorimeter (Osaka, Japan; Huppertz, Grosman, Fox, & Kelly, 2004c) and pH. All treatments were repeated three times, using different samples of milk. 2.2. Assay for plasmin activity Samples of SM or HSM were mixed in a 3:1 ratio (milk:citrate) with 0.4 M trisodium citrate (Richardson & Pearce, 1981) and centrifuged at 15,800g for 30 min before assaying the supernatant for plasmin activity. Plasmin activity was determined as described by Richardson and Pearce (1981), using the non-fluorescent substrate, N-succinyl-L-alanyl-L-phenylalanyl-L-lysyl-7amido-4-methyl coumarin (Sigma Chemical Co., St. Louis, MO 63103, USA), which is cleaved by plasmin to yield a fluorescent product, 7-amido-4-methylcoumarin (AMC). Plasmin activity was expressed in AMC units (nanomoles of AMC released per minute) mL 1 of milk. 2.3. Electrophoretic analysis of hydrolysis of casein Samples of SM and HSM were diluted in a 1:4 ratio (milk:buffer) with single-strength sample buffer before analysis by urea polyacrylamide gel electrophoresis (urea–PAGE; Andrews, 1983). Gels were stained by the method of Blakesley and Boezi (1977) and destained in distilled water. The band corresponding to b-casein was identified using a sodium caseinate standard and protein bands were quantified by densitometric analysis using Total Lab V1.10 software (Nonlinear Dynamics, Newcastle-upon-Tyne, UK). 2.4. Measurement of dissociation behaviour of b-casein in milk Samples of SM or HSM, stored for different times at 5, 20 or 37 1C, were ultracentrifuged at 100,000g for 60 min at 20 1C. The level of b-casein in the serum phase was determined by diluting the ultracentrifugal supernatants in a 1:4 ratio with single-strength sample buffer, and analysing by urea–PAGE (Andrews, 1983); similarly, ultracentrifugal pellets were redispersed in lactosefree synthetic milk ultrafiltrate (Jenness & Koops, 1962) to the original volume using a mortar and pestle, filtered through glass wool, and subsequently diluted 1:4 with

ARTICLE IN PRESS A. Crudden et al. / International Dairy Journal 15 (2005) 305–313

single-strength sample buffer before analysis by urea– PAGE.

HSM was incubated at 37 or 5 1C for up to 72 h in the absence or presence of 6 mg L 1 purified porcine plasmin (Sigma Chemical Co., St. Louis, MO, USA). The specificity of porcine plasmin on b-casein is identical to that of bovine plasmin (Lane and Fox, 1999) and the level added was shown in preliminary experiments to give significant and progressive hydrolysis over this time period. After different periods of storage, sub-samples of the HSM were ultracentrifuged at 100,000g for 60 min at 20 1C. SDS–PAGE, as described by Laemmli (1970), was performed on the supernatants under both dissociating-non-reducing (NRSDS–PAGE) and dissociating-reducing conditions (RSDS–PAGE). In RSDS–PAGE, the sample buffer contains 2-mercaptoethanol (10%, v/v), which was added to reduce disulphide bonds formed between kcasein and b-lactoglobulin (b-lg) during heating (Cho, Singh, & Creamer, 2003). Samples stored at 37 1C in the presence of plasmin were removed after shorter times than samples without plasmin, as the high level of exogenous plasmin led to a more rapid rate of proteolysis. 2.6. Statistical analysis All experiments were repeated on three occasions, using different milk samples. The effects of variables such as storage temperature or duration of storage on measured parameters (e.g., plasmin activity, dissociation of b-casein) were examined by one-way Analysis of Variance (ANOVA), using Minitab statistical software (version 12; Minitab Ltd., Coventry, UK). Comparisons of means were performed using Tukey’s pairwise comparisons, at the 95% level of significance.

3. Results 3.1. Effect of storage temperature on the stability of plasmin activity in milk Storage temperature greatly affected plasmin activity in incubated SM (Fig. 1). In SM stored at 37 1C, plasmin activity increased significantly (5%; Po0:05) during the first 2 d of storage, but decreased gradually to 34% of the original activity after 10 d; the effect of storage time at 37 1C on plasmin activity was highly significant overall (Po0:001), with activity after 2, 6 or 10 d being significantly different from that in the initial milk. However, during storage at 20 1C, a distinctly different

110 Plasmin activity (% of control)

2.5. Measurement of dissociation of b-lactoglobulin/kcasein complexes from casein micelles

307

100 90 80 70 60 50 40 30 0

0

2

5

4 6 8 Storage at 37 or 20°C (d) 10

15 20 Storage at 5°C (d)

25

10

30

12

35

Fig. 1. Relative plasmin activity in raw bovine milk (expressed as % of that in fresh milk at d 0) during storage at 37 (), 20 (’) or 5 (m) 1C. Results shown are means and standard deviations of data from triplicate trials.

pattern was apparent; an initial decrease in plasmin activity (by 12% on d 2) was followed by a recovery to a level similar to that in the control sample; the effect of storage time at 20 1C on plasmin activity was also highly significant (Po0.001). Throughout storage at 5 1C, there was a progressive decrease in plasmin activity (effect of storage time, Po0.001); most of the decrease (20%) occurred during the first 5 d of storage. Heating milk at 90 1C for 10 min reduced the plasmin activity in HSM by 75% and no further changes in plasmin activity were observed during storage at 5, 20 or 37 1C (data not shown). To ascertain if plasmin was released from casein micelles during storage, activity in the ultracentrifugal supernatants of SM stored at 20 1C was determined (Fig. 2). This temperature was used as total plasmin activity changed least during storage, compared to 5 or 37 1C. Plasmin activity in the serum phase approximately doubled during 10 d storage, from 0.038 at d 0 to 0.078 AMC units mL 1 by d 10. While the magnitude of these changes was relatively small, the effect was highly significant (Po0.001), with activity after 2 days of storage being significantly higher for all samples than in the unincubated sample. Throughout storage at 5, 20 or 37 1C there were no significant changes in casein micelle size, pH or colour, in either SM or HSM (data not shown).

3.2. Effect of storage temperature on the hydrolysis of b-casein Because of the differences observed in plasmin activity in SM stored at 5, 20 or 37 1C, the resultant rate of

ARTICLE IN PRESS A. Crudden et al. / International Dairy Journal 15 (2005) 305–313

308

plasmin activity after 5 d of storage, examined using one-way ANOVA, was highly significant (Po0.001), with means at each temperature differing from all others using a Tukey’s comparison test.

0.08 0.07

3.3. Effect of storage temperature on the level of b-casein in milk serum

0.06 0.05 0.04 0.03 0

2

4

6 8 Storage at 20°C (d)

10

12

Fig. 2. Plasmin activity (expressed as AMC units mL 1) in ultracentrifugal supernatants from skim milk stored at 201C. Results shown are means and standard deviations of data from triplicate trials.

Residual β-casein (% of control)

120

100

80

60

40

20 0

0

2

5

4 8 6 Storage at 37 or 20°C (d) 10

15 20 Storage at 5°C (d)

25

10

30

12

35

Fig. 3. Hydrolysis of b-casein in skim milk (expressed as % of the level of b-casein in fresh milk at d 0) during storage at 37 (), 20 (’) or 5 (m) 1C. Results shown are means and standard deviations of data from triplicate trials.

hydrolysis of b-casein at each temperature was studied (Fig. 3); at all three temperatures, the effect of storage time was highly significant (Po0.001). As was the case for plasmin activity, temperature significantly influenced the rate and extent of hydrolysis of b-casein. SM samples stored at 37 1C underwent the greatest hydrolysis, with most (56%) hydrolysis of b-casein occurring during the first 4 d of storage. At 20 1C, the decrease in b-casein was generally linear throughout storage, with 30% of b-casein being hydrolysed after 10 d (Fig. 3). At 5 1C, limited hydrolysis of b-casein was observed. Very little hydrolysis of b-casein was observed in HSM during storage at 5 1C for 30 d, or at 20 or 37 1C for 10 d (data not shown). The effect of storage temperature on

The effect of storage time on the level of b-casein in ultracentrifugal supernatants (serum) of SM differed for each temperature studied (Fig. 4); the effect of storage time on dissociation of b-casein was, however, significant (Po0.001) at each temperature. At 37 1C, the level of non-sedimentable b-casein decreased by 87% during the first 6 d and remained constant thereafter. During storage at 20 1C, the level of non-sedimentable b-casein remained constant during the first 8 d of storage but then decreased by 25% during the remaining 2 d. At 5 1C, the level of b-casein in SM serum approximately doubled during the first 5 d of storage; this was followed by a subsequent decrease up to d 10, with little further change over the remaining 20 d of storage. The effect of storage temperature on level of non-sedimentable bcasein, after 5 d of storage was highly significant (Po0.001) when examined using one-way ANOVA, with means at each temperature differing (Po0.05) from all others. Urea–PAGE analysis of the ultracentrifugal pellets (micelles) from skim milk stored at 37 1C showed a progressive reduction in the level of b-casein with a concomitant increase in levels of g-caseins (Fig. 5). Pre-heating milk to 90 1C for 10 min had a significant impact on the level of non-sedimentable b-casein during Level of β-casein in supernatant (% of control)

Plasmin activity (AMC units mL-1)

0.09

250

200

150

100

50

0 0

0

2

5

4 6 8 Storage at 37 or 20°C (d) 10

15 20 Storage at 5°C (d)

25

10

30

12

35

Fig. 4. Level of b-casein in ultracentrifugal supernatants from skim milk stored for different time periods at 37 (), 20 (’) or 5 (m) 1C, expressed as a % of non-sedimentable b-casein in fresh milk at d 0. Results shown are means and standard deviations of data from triplicate trials.

ARTICLE IN PRESS 1

2

3

4

5 γ2-casein γ1-casein

γ3-casein β-casein

αS2-casein αS1-casein

Non-sedimentable β-casein in heated milk (% of control)

A. Crudden et al. / International Dairy Journal 15 (2005) 305–313

309

450 400 350 300 250 200 150 100 50 0

2

4

6

8

10

12

Storage at 37 or 20°C (d)

0

5

10

15

20

25

30

35

Storage at 5°C (d)

Proteose Peptones

Fig. 6. Level of non-sediment able (100,000g for 60 min at 20 1C) bcasein in skim milk heated to 90 1C for 10 min followed by storage at 37 (), 20 (’) or 5 (m) 1C, expressed as % of b-casein in the ultracentrifugal supernatant of fresh milk at d 0. Results shown are means and standard deviations of data from triplicate trials.

Fig. 5. Urea–PAGE electrophoretograms (pH 8.9, 12.5% T, 4.5% C) of ultracentrifugal pellets from skim milk stored at 37 1C for 0 (lane 1), 1 (lane 2), 2 (lane 3), 6 (lane 4) or 8 (lane 5) d.

subsequent storage at 5 1C (Po0.001); however, the effect of storage time was less significant at 37 1C (Po0.05) and non-significant at 20 1C (Fig. 6). When samples of SM were ultracentrifuged immediately after heating there was a 19% increase in the level of b-casein in the supernatants relative to the unheated samples. However, further storage of HSM at 5 1C resulted in a rapid increase in the level of b-casein in the supernatant during the first 5 d of storage, followed by a slight decrease by 10 d and a subsequent slow further increase. By comparison, changes at 20 or 37 1C were very small. 3.4. Effect of plasmin on the dissociation of heat-induced k-casein/b-lactoglobulin complexes from the casein micelles The level of k-casein in the ultracentrifugal supernatant of HSM without added plasmin did not change during incubation for up to 72 h at 5 or 37 1C, as determined by SDS–PAGE under reducing conditions (Fig. 7a; lanes 4–6 or 10–12, respectively). In HSM containing 6 mg L 1 plasmin, the level of k-casein in the ultracentrifugal supernatant increased throughout storage at either 5 or 37 1C (Fig. 7a; lanes 7–9 or 13–15,

respectively); the increase in the level of non-sedimentable k-casein was more pronounced at the latter temperature. k-Casein was not evident on non-reducing gels, which contained no 2-mercaptoethanol (Fig. 7b). On both RSDS–PAGE (Fig. 7a) and NRSDS–PAGE (Fig. 7b), the levels of caseins in the ultracentrifugal supernatants of HSM stored at 5 1C were clearly higher than those in supernatants from HSM samples stored at 37 1C. The dissociation of caseins was also increased by the presence of plasmin.

4. Discussion 4.1. Effect of storage conditions on plasmin activity and subsequent hydrolysis of b-casein This study focused on aspects of the plasmin system in milk and was concerned particularly with the effects of time and temperature of storage on plasmin activity. Proteolysis in good quality milk, especially hydrolysis of b-casein, is due primarily to plasmin (Kelly & McSweeney, 2003). In this study, SM stored at 37 1C showed extensive degradation of b-casein (Fig. 3), with most hydrolysis occurring during the first 4 d of storage. This initial phase of extensive proteolysis was concurrent with an increase in plasmin activity (Fig. 1), which could be attributed to the activation of the zymogen, plasminogen, by the action of PA (Richardson, 1983; Korycka-Dahl et al., 1983) and the fact that 37 1C

ARTICLE IN PRESS A. Crudden et al. / International Dairy Journal 15 (2005) 305–313

310

1

2

3

4

5

6

7

8

9 10 11 12 13 14 15

66 kDa 45 kDa

αS-caseins

36 kDa β-casein

29 kDa 24 kDa

κ-casein β-lactoglobulin

20 kDa

α-lactalbumin

14.2 kDa (a) 1 2

3

4

5

6

7

8

9 10 11 12 13 14 15

66 kDa 45 kDa 36 kDa

29 kDa 24 kDa

αS-caseins β-casein

20 kDa 14.2 kDa

α-lactalbumin

(b) Fig. 7. (a) Reducing SDS–PAGE and (b) non-reducing SDS–PAGE electrophoretograms showing molecular weight makers (lane 1), fresh skim milk (lane 2) and ultracentrifugal supernatants of fresh skim milk (lane 3) or HSM stored at 5 1C for 24, 48, 72 h in the absence (lanes 4–6) or presence (lanes 7–9) of 6 mg mL 1 plasmin or stored at 37 1C for 24, 48, 72 h (lanes 10–12) in the absence of plasmin or for 8, 24, 48 h (lanes 13–15) in the presence of 6 mg mL 1 plasmin.

is the optimum temperature for plasmin activity (Kaminogawa, Mizobuchi, & Yamauchi, 1972). However, the rate of proteolysis at 37 1C was significantly reduced in the later days of storage, which corresponded to a decrease in plasmin activity (Fig. 1). This decrease could be attributed to the autolysis (self-hydrolysis) of plasmin, as observed by Ueshima et al. (1996) and Huppertz, Fox, and Kelly (2004b). On storage of SM at 5 1C, there was a progressive but less extensive decrease in plasmin activity (Fig. 1), as was also observed by Guinot-Thomas, Al Ammoury, Le

Roux, and Laurent (1995) and Ueshima et al. (1996), who suggested that this may also result from autolysis of plasmin. Decreasing plasmin activity at 5 1C, possibly as a result of autolysis, and storage of SM at a temperature far from the optimum for plasmin activity probably contributed to the relatively low extent of hydrolysis of b-casein observed (Fig. 3). At 37 1C, activation and autolysis appeared to occur in sequence; however, at 5 1C only autolysis was apparent, indicating that little activation of plasminogen occurs at a low temperature. The effect of temperature on the activation

ARTICLE IN PRESS A. Crudden et al. / International Dairy Journal 15 (2005) 305–313

of plasminogen has not, to the best of our knowledge, been previously studied. Heating milk for 10 min at 90 1C significantly reduced plasmin activity (results not shown), as reported by Grufferty and Fox (1988b), Kennedy and Kelly (1997) and Saint-Denis, Humbert, and Gaillard (2001). Two possible mechanisms may have contributed to the loss of plasmin activity. Firstly, plasmin may have been irreversibly thermally denatured (Metwalli, De Jongh, and van Boekel, 1998; Saint-Denis et al., 2001). Secondly, exposure of the free-SH group during denaturation of b-lg may inactivate plasmin through thiol-disulphide interchange reactions (Kennedy & Kelly, 1997; Saint-Denis et al., 2001). The reduction in proteolysis observed in heated milk may have been due to both the decreased plasmin activity and formation of b-lg/k-casein complexes which hinder the access of plasmin to susceptible peptide bonds on b-casein (Enright & Kelly, 1999). Storage of SM at 20 1C resulted in the release of plasmin into the serum (Fig. 2). It is possible that the hydrolysis of b-casein by plasmin during storage not only resulted in the degradation of b-casein but that also, perhaps, hydrolysis in the region of anchor points between b-casein and plasmin (i.e., lysine residues), resulted in the release of plasmin from the micelles into the serum phase of milk.

311

reassociation of this protein with the micelles during storage. It appears that g-caseins partitioned between serum and micellar phases; some of these products were present in both fractions. While storage of skim milk at 5 1C resulted in the release of b-casein into the serum of milk, pre-heating milk at 90 1C for 10 min greatly increased this dissociation (Figs. 6,7a, b); this effect was also observed by Law (1996) following pre-heating of milk at 85 1C for 10 min. It was also observed in this study that preheating milk at 90 1C for 10 min resulted in an elevated level of k-casein in serum relative to the unheated control (Fig. 7a; lanes 4 and 3, respectively). Holt (1992) proposed that a significant proportion of b-casein is bound to k-casein and may be involved in the internal cohesion of the casein micelles, and several authors have reported that pre-heating milk at 80 1C results in the release of a significant amount of k-casein into the serum (Singh & Fox, 1985; Law, 1996; Anema & Klostermeyer, 1997; Anema, 1998; Anema & Li, 2000). Therefore, the elevated level of b-casein in the supernatant of heated milk stored at 5 1C may result from a combination of the dissociation of b-casein during cold storage, and its release with k-casein during pre-heating. 4.3. Effect of plasmin action on the level of nonsedimentable k-casein in SM

4.2. Factors affecting the level of b-casein in SM serum The effects of temperature and storage time on the level of b-casein in the serum were examined by ultracentrifugation and urea–PAGE. The phenomena responsible for the solubilisation of caseins, in particular b-casein, during cold storage have been well documented (Davies & Law, 1983; Roefs et al., 1985; Dalgleish & Law, 1988, 1989). Ali et al. (1980) reported that the dissociation of b-casein into the serum phase of milk reached a maximum after 48 h of storage at 4 1C and that, on further storage, there was a partial reversal of this process, as also observed in this study (Fig. 4). Ali et al. (1980) concluded that the equilibrium between colloidal and soluble calcium phosphate affected the equilibrium of casein constituents in milk serum and their degree of association with casein micelles. At 37 1C, the optimum temperature for plasmin activity (Kaminogawa et al., 1972), the decrease in the level of b-casein in the supernatant is presumably due to plasmin activity, as increases in the levels of g-caseins in the ultracentrifugal supernatants (results not shown) during storage at 37 1C were clearly indicative of hydrolysis of b-casein by plasmin (Andrews, 1983; Recio et al., 1997; Trujillo et al., 1998). Analysis of the ultracentrifugal pellet showed (Fig. 5) a progressive decrease in the level of b-casein, thus precluding

SDS electrophoretograms of ultracentrifugal supernatants of HSM in the presence of the reducing agent, 2mercaptoethanol (Fig. 7a) showed that the level of nonsedimentable k-casein increased only slightly during storage at either 5 or 37 1C (Fig. 7a; lanes 4–6 or 10–12, respectively) in the absence of plasmin. However, in the presence of plasmin, there was a greater increase in the level of non-sedimentable k-casein when milk was stored at either 5 or 37 1C (Fig. 7a; lanes 7–9 or 13–15, respectively). This may suggest that hydrolysis of caseins by plasmin releases the k-casein/b-lg complex into the serum. A number of authors (Kohlmann, Nielsen, & Ladisch, 1991; Venekatachalam, McMahon, & Savello, 1993; Auldist, Coats, Sutherland, Hardham, McDowell, & Rogers, 1996; Enright, Bland, Needs & Kelly, 1999) have commented on a possible role of plasmin in the age gelation of UHT milk. A mechanism for this was proposed by McMahon (1995), who suggested that age gelation was initiated by the release of the b-lg/k-casein complex, formed during heating, into milk serum, possibly by the hydrolysis around ‘anchor points’ by plasmin. The results of this study may support the hypothesis that plasmin plays a role in the age gelation of UHT milk. However, the mechanism of age gelation is inherently complex and is influenced by several factors (for review, see Datta & Deeth, 2001).

ARTICLE IN PRESS 312

A. Crudden et al. / International Dairy Journal 15 (2005) 305–313

5. Conclusion During storage of milk at 5, 20 or 37 1C, considerable autolysis of plasmin occurred, which significantly influenced the rate and extent of hydrolysis of b-casein. Furthermore, the temperature of storage had a significant effect on the composition of the casein micelles. Dissociation of b-casein was most pronounced at 5 1C, a process which was significantly enhanced by preheating. The addition of plasmin to heated milk indicated that plasmin may be responsible for the release of the kcasein/b-lg complex, which is thought to be a preliminary step in the phenomenon of age-gelation observed in UHT milk. The results of this study show that the temperature of storage strongly affects the activity of plasmin and suggest that milk should be cooled rapidly and kept cold throughout all stages of the supply chain and used as quickly as processing operations allow, to minimise the hydrolysis of casein by plasmin and thereby preserve the functionality of the milk. References Aimutis, W. R., & Eigel, W. N. (1982). Identification of l-casein as plasmin-derived fragments of bovine as1-casein. Journal of Dairy Science, 65, 175–181. Ali, A. E., Andrews, A. T., & Cheeseman, G. C. (1980). Influence of storage of milk on casein distribution between the micellar and soluble phases and its relationship to cheese-making parameters. Journal of Dairy Research, 47, 371–382. Andrews, A. T. (1983). Proteinases in normal bovine milk and their action on the caseins. Journal of Dairy Research, 50, 45–55. Andrews, A. T., & Alichanidis, E. (1983). Proteolysis of caseins and the proteose-peptone fraction of bovine milk. Journal of Dairy Research, 50, 275–290. Anema, S. G. (1998). Effect of milk concentration on heat-induced, pH-dependent dissociation of casein from micelles in reconstituted skim milk at temperatures between 20 and 120 1C. Journal of Agricultural and Food Chemistry, 46, 2299–2305. Anema, S. G., & Klostermeyer, H. (1997). Heat-induced, pHdependent dissociation of casein micelles on heating reconstituted skim milk at temperatures below 100 1C. Journal of Agricultural and Food Chemistry, 45, 1108–1115. Anema, S. G., & Li, Y. (2000). Further studies on the heat-induced, pH-dependent dissociation of casein from micelles in reconstituted skim milk. Lebensmittel-Wissenschaft und Technologie, 33, 335–343. Auldist, M. J., Coats, S. J., Sutherland, B. J., Hardham, J. F., McDowell, G. H., & Rogers, G. L. (1996). Effect of somatic cell count and stage of lactation on the quality and storage life of ultra high temperature milk. Journal of Dairy Research, 63, 377–386. Baer, A., Ryba, I., & Collin, J. C. (1994). Binding of bovine plasminogen to immobilised casein and its activation thereon. International Dairy Journal, 4, 597–616. Bastian, E. D., & Brown, R. J. (1996). Plasmin in milk and dairy products: an update. International Dairy Journal, 6, 435–457. Blakesley, R. W., & Boezi, J. A. (1977). A new staining technique for proteins in polyacrylamide gels using Coomassie Brilliant Blue G250. Analytical Biochemistry, 82, 580–581. Cho, Y., Singh, H., & Creamer, L. K. (2003). Heat-induced interactions of b-lactoglobulin A and k-casein B in a model system. Journal of Dairy Research, 70, 61–71.

Dalgleish, D. G., & Law, A. J. R. (1988). pH-induced dissociation of bovine casein micelles. I. Analysis of liberated caseins. Journal of Dairy Research, 55, 529–538. Dalgleish, D. G., & Law, A. J. R. (1989). pH-induced dissociation of bovine casein micelles. II. Mineral solubilisation and its relation to casein release. Journal of Dairy Research, 56, 727–735. Datta, N., & Deeth, H. C. (2001). Age gelation of UHT milk: a review. Food and Bioproducts Processing, 74, 197–210. Davies, D. T., & Law, A. J. R. (1983). Variation in the protein composition of bovine casein micelles and serum casein in relation to micellar size and milk temperature. Journal of Dairy Research, 50, 67–75. De la Fuente, M. A. (1998). Changes in the mineral balance of milk submitted to technological treatments. Trends in Food Science and Technology, 9, 281–288. Diaz, O., Gouldsworthy, A. M., & Leaver, J. (1996). Identification of peptides released from casein micelles by limited trypsinolysis. Journal of Agriculture and Food Chemistry, 44, 2517–2522. Eigel, N. (1977). Effect of bovine plasmin on as1-B and k-A caseins. Journal of Dairy Science, 60, 1399–1406. Enright, E., Bland, A. P., Needs, E. C., & Kelly, A. L. (1999). Proteolysis and physicochemical changes in milk on storage as affected by UHT treatment, plasmin activity and KIO3 addition. International Dairy Journal, 9, 581–591. Enright, E., & Kelly, A. L. (1999). The influence of heat treatment of milk on susceptibility of casein to proteolytic attack by plasmin. Milchwissenschaft, 54, 491–493. Farkye, N. Y., & Fox, P. F. (1992). Contribution of plasmin to Cheddar cheese ripening: Effect of added plasmin. Journal of Dairy Research, 59, 209–216. Grufferty, M. B., & Fox, P. F. (1988a). Milk alkaline proteinase. Journal of Dairy Research, 55, 609–630. Grufferty, M. B., & Fox, P. F. (1988b). Heat stability of the plasmin system in milk and casein systems. New Zealand Journal of Dairy Science and Technology, 23, 143–152. Guinot-Thomas, P., Al Ammoury, M., Le Roux, Y., & Laurent, F. (1995). Study of proteolysis during storage of raw milk at 4 1C: effect of plasmin and microbial proteinases. International Dairy Journal, 5, 658–697. Harwalkar, V. R., Cholette, H., McKellar, R. C., & Emmons, D. B. (1993). Relation between proteolysis and astringent off-flavor in milk. Journal of Dairy Science, 76, 2521–2527. Heegaard, C. W., Rasmussen, L. K., & Andreasen, P. A. (1994). The plasminogen activation system in bovine milk: differential localization of tissue-type plasminogen activator and urokinase in milk fractions is caused by binding to casein and urokinase receptor. Biochimica et Biophysica Acta, 1222, 45–55. Holt, C. (1992). Structure and stability of bovine casein micelles. Advances in Protein Chemistry, 43, 63–151. Huppertz, T., Fox, P. F., & Kelly, A. L. (2004a). High pressure treatment of bovine milk: effects on casein micelles and whey proteins. Journal of Dairy Research, 71, 98–106. Huppertz, T., Fox, P. F., & Kelly, A. L. (2004b). Plasmin activity and proteolysis in high pressure-treated bovine milk. Le Lait, 84, 297–304. Huppertz, T., Grosman, S., Fox, P. F., & Kelly, A. L. (2004c). Heat and ethanol stabilities of high pressure-treated bovine milk. International Dairy Journal, 14, 125–133. Jenness, R., & Koops, J. (1962). Preparation and properties of a salt solution which simulates milk ultrafiltrate. Netherlands Milk and Dairy Journal, 16, 154–164. Kaminogawa, S., Mizobuchi, H., & Yamauchi, K. (1972). Comparison of bovine milk protease with plasmin. Journal of Agricultural and Biological Chemistry, 36, 2163–2167. Kelly, A. L., & Foley, J. F. (1997). Proteolysis and storage stability of UHT milk as influenced by milk plasmin activity, plasmin/

ARTICLE IN PRESS A. Crudden et al. / International Dairy Journal 15 (2005) 305–313 b-lactoglobulin complexation, plasminogen activation and somatic cell count. International Dairy Journal, 7, 411–420. Kelly, A. L., & McSweeney, P. L. H. (2003). Indigenous proteinases in milk. In P. F. Fox, P. L. McSweeney (Eds.), Advanced dairy chemistry, Volume A: Proteins, (3rd ed.) (pp. 495–521). New York: Kluwer Academic/Plenum Publishers. Kennedy, A., & Kelly, A. L. (1997). The influence of somatic cell count on the heat stability of bovine milk plasmin activity. International Dairy Journal, 7, 717–721. Kohlmann, K. L., Nielsen, S. S., & Ladisch, M. R. (1991). Effects of a low concentration of added plasmin on ultra-high temperature processed milk. Journal of Dairy Science, 74, 1151–1156. Korycka-Dahl, M., Ribadeau Dumas, B., Chene, N., & Martal, J. (1983). Plasmin activity in milk. Journal of Dairy Science, 66, 704–711. Laemmli, U. K. (1970). Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature, 227, 678–685. Lane, C. N., & Fox, P. F. (1999). The individual or combined action of chymosin and plasmin on sodium caseinate or b-casein in solution: Effect of NaCl and pH. Le Lait, 79, 423–434. Law, A. J. R. (1996). Effects of heat treatment and acidification on the dissociation of bovine casein micelles. Journal of Dairy Research, 63, 35–48. Le Bars, D., & Gripon, J. C. (1989). Specificity of plasmin towards bovine as2-casein. Journal of Dairy Research, 56, 817–821. Lu, D. D., & Nielsen, S. S. (1993). Isolation and characterization of native bovine milk plasminogen activators. Journal of Dairy Science, 76, 3369–3383. Mara, O., Roupie, C., Duffy, A., & Kelly, A. L. (1998). The curdforming properties of milk as affected by the action of plasmin. International Dairy Journal, 8, 807–812. McMahon, D. J. (1995). Age gelation of UHT milk: Changes that occur during storage, their effect on shelflife and the mechanism by which age-gelation occurs. In . Heat Treatments and Alternative Methods (pp. 315–326). Brussels, Belgium: International Dairy Federation. McSweeney, P. L. H., Olson, N. F., Fox, P. F., Healy, A., & Højrup, P. (1993). Proteolytic specificity of plasmin on bovine as1-casein. Food Biotechnology, 7, 143–158. Metwalli, A. A. M., De Jongh, H. H. J., & Van Boekel, M. A. J. S. (1998). Heat inactivation of bovine plasmin. International Dairy Journal, 8, 47–56. O’Flaherty, F. (1997). Characterisation of some minor caseins and proteose peptones of bovine milk. M.Sc. Thesis, National University of Ireland, Cork. Politis, I. (1996). Plasminogen activator system: implications for mammary cell growth and involution. Journal of Dairy Science, 79, 1097–1107.

313

Politis, I., Barbano, D. M., & Gorewit, R. C. (1992). Distribution of plasminogen and plasmin in fractions of bovine milk. Journal of Dairy Science, 75, 1402–1410. Politis, I., White, J. H., Zavizon, B., Goldberg, J. J., Gou, M. R., & Kinstedt, P. (1995). Effect of individual caseins on plasminogen activation by bovine urokinase-type and tissue-type plasminogen activators. Journal of Dairy Science, 78, 484–490. Recio, I., Amigo, L., Ramos, M., & Lopez-Fandino, R. (1997). Application of capillary electrophoresis to the study of proteolysis of caseins. Journal of Dairy Research, 64, 221–230. Richardson, B. C. (1983). The proteinases of bovine milk and the effect of pasteurization on their activity. New Zealand Journal of Dairy Science and Technology, 18, 233–245. Richardson, B. C., & Pearce, K. N. (1981). The determination of plasmin in dairy products. New Zealand Journal of Dairy Science and Technology, 16, 209–220. Roefs, S. P. F. M., Walstra, P., Dalgleish, D. G., & Horne, D. S. (1985). Preliminary note on the change in casein micelles caused by acidification. Netherlands Milk and Dairy Journal, 39, 119–122. Saint Denis, T., Humbert, G., & Gaillard, J. L. (2001). Heat inactivation of native plasmin, plasminogen and plasminogen activators in bovine milk: a revisited study. Le Lait, 81, 715–729. Singh, H., & Fox, P. F. (1985). Heat stability of milk: pH-dependent dissociation of micellar k-casein on heating milk at ultra high temperatures. Journal of Dairy Research, 52, 529–538. Trujillo, A. J., Guamis, B., & Carretero, C. (1998). Hydrolysis of bovine and caprine caseins by rennet and plasmin in model systems. Journal of Agriculture and Food Chemistry, 46, 3066–3072. Ueshima, S., Okada, K., & Matsuo, O. (1996). Stabilisation of plasmin by lysine derivatives. Clinica et Chimica Acta, 245, 7–18. Venkatachalam, N., McMahon, D. J., & Savello, P. A. (1993). Role of protein and lactose interactions in the age gelation of ultra-high temperature processed concentrated skim milk. Journal of Dairy Science, 76, 1882–1894. Visser, S., Slangen, K. J., Alting, A. C., & Vreeman, H. J. (1989). Specificity of bovine plasmin in its action on bovine as2-casein. Milchwissenschaft, 44, 335–339. Weber, B. A., & Nielsen, S. S. (1991). Isolation and partial characterisation of a native serine-type protease inhibitor from bovine milk. Journal of Dairy Science, 74, 717–764. White, G. H., Zavizion, B., O’Hare, K., Gilmore, J., Guo, M. R., Kinstedt, P., & Politis, I. (1995). Distribution of plasminogen activator in different fractions of bovine milk. Journal of Dairy Research, 62, 115–122.