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The Curd-forming Properties of Milk as Affected by the Action of Plasmin
PII : S0958-6946(98)00118-6
Int. Dairy Journal 8 (1998) 807—812 1999 Elsevier Science Ltd. All rights reserved Printed in Great Britain 0958-6946/99/$ — see front matter
The Curd-forming Properties of Milk as Affected by the Action of Plasmin Orla Maraa, Christophe Roupiea, Arthur Duffyb and Alan L. Kellya * ? Department of Food Technology, University College Cork, Cork, Ireland @ Dairy Products Research Centre, Moorepark, Fermoy, Co. Cork, Ireland (Received 13 July 1998; accepted 20 October 1998) ABSTRACT Purified plasmin was added to skim milk at a range of levels (0.1—10 mg L\) and the samples subsequently incubated to allow hydrolysis of casein by the enzyme. Milk clotting properties (as measured by Formagraph) and curd yield properties of the milk were measured after incubation. Increasing plasmin addition, with concomitantly increased levels of hydrolysis of casein, resulted in increased curd firming time and cutting time and reduced firming rate and final curd firmness (all P(0.001). Rennet clotting time was only increased with extensive hydrolysis by plasmin. Curd yield was reduced with increasing plasmin addition, with concomitantly increased losses of protein in whey. With low levels of enzyme addition, however, the magnitude of most effects was small. Curd moisture was unaffected by plasmin addition to milk at any level of enzyme addition. It may be concluded that extensive hydrolysis of casein by plasmin, independent of any other alterations in milk quality, has a significant influence on some milk cheesemaking properties. However, it appears that plasmin may not be responsible for changes in cheese moisture in, for example, late lactation milk. 1999 Elsevier Science Ltd. All rights reserved Keywords: plasmin; milk; cheese; coagulation
INTRODUCTION
moisture content is the expense of adding this enzyme to large quantities of milk. Recently, however, a small-scale centrifugation method has been reported to provide a good assessment of fresh cheese yield, unadjusted for moisture content (Macheboeuf et al., 1993; Lopez-Fandino et al., 1996). The Formagraph (Bastian et al., 1991) also may be used to study the coagulation properties of milk. The objective of this study was to use the method of Lopez-Fandino et al. (1996) to investigate the effects of plasmin hydrolysis of milk protein on the yield and moisture of renneted curds subsequently prepared from that milk. Also, the rennet clotting properties of milk hydrolysed by plasmin were studied using a Formagraph. An advantage to this experimental approach was that the hydrolytic action of plasmin on the casein micelles and the influence of this action on subsequent coagulation of milk could be studied in isolation from other changes in milk (such as those associated with late lactation or mastitis concomitant with elevated milk plasmin activity) to determine the exact influence of the action of this enzyme on milk cheesemaking properties.
Plasmin, the principal alkaline proteinase in bovine milk, readily hydrolyses b-casein and a -casein, and to a lesser extent a -casein, in bovine milk (Bastian and Brown, 1996). It has been proposed that increasing levels of plasmin, for example in late lactation milk, and the subsequent action of this enzyme on the caseins, may be linked to poor cheesemaking properties of such milk (Okigbo et al., 1985). However, it has been shown that a normal coagulum can form and syneresis proceed as usual even after extensive degradation of casein by plasmin (Pearse et al., 1986), while Bastian et al. (1991) found no correlation between plasmin activity and milk clotting parameters on studying a large number of milks of different breeds, stages and numbers of lactation and in different seasons. The effects of plasmin hydrolysis of casein on cheese yield and moisture content are less well described, and are of particular interest, both from an economical and quality perspective (Pearse et al., 1986). Previous studies have associated plasmin action in milk, particularly in late lactation and high SCC milks, with increased moisture content of cheese made from such milk (Donnelly et al., 1984; O’Keeffe, 1984; Barbano et al., 1991). A factor in designing experiments to determine the influence of enzymes such as plasmin on cheese yield and
MATERIALS AND METHODS Plasmin hydrolysis of milk In each experiment porcine plasmin (P8644, Sigma Chemical Co., Poole, Dorest, UK) was added to samples of pasteurised bovine skim milk from a local dairy
*Corresponding author. Tel.: 00 353 21 903405; Fax: 00 353 21 270213; E-mail: [email protected] 807
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O. Mara et al.
company at levels from 0.1—10 mg L \. Porcine plasmin has been shown to have similar specificity to bovine plasmin in cheesemaking trials (Farkye and Landkammer, 1992). Samples with no added plasmin were incubated in each experiment as controls. Plasmin was typically dissolved in distilled water at 1 mg mL\ concentration and the appropriate amount of this concentrated stock solution added to portions of milk for incubation. Sodium azide (0.05% w/v) was added to all milks before incubation to prevent bacterial contamination. All incubations were carried out at 37°C for exactly 5 h before renneting, unless otherwise specified. Preparation of renneted gels
RESULTS Influence of plasmin hydrolysis over time on milk coagulation The yield of rennet curds prepared from milk to which no plasmin was added (control milk) did not vary over 5 h storage at 37°C (Fig. 1). During this time the moisture content of the control curds was also relatively constant, and there was a slight increase in protein content of the control whey. Milk which received 8 mg L\ plasmin, however, showed decreased yields as the hydrolysis proceeded, and had elevated losses of protein in whey. Curd moisture in the plasmin-treated milk was decreased initially by plasmin digestion but increased after 2 h, with
The procedure of Machebeouf et al. (1993), as modified by Lopez-Fandino et al. (1996), was followed. Each 50 mL sample of hydrolysed or unhydrolysed milk was mixed with 1 mL rennet solution (Hansen’s Standard Plus 200 rennet, 1.5 g in 100 mL 0.05 M acetate buffer, pH 5.4). The milk samples were allowed to set at 32°C for 40 min, cut, allowed stand for 10 min, and centrifuged at 13,000 rpm for 15 min at 4°C. Whey was then carefully removed and yields calculated. Analysis Protein contents of whey samples were determined by the Kjeldahl method. Moisture of curds was determined after drying to constant weight at 105°C. Rennet clotting properties of the milk were measured by Formagraph (Foss Electric, DK-3400, Hiller+d, Denmark) according to the method of McMahon and Brown (1982). Milk (10 mL) was renneted at 35°C using 32 kL of rennet. Clotting time (RCT), curd firmness 60 min after rennet addition (A60) and time required to achieve a curd firmness of 20 mm (K20) were determined for the milks at their natural pH. Curd firming rate and cutting time were also calculated, as described by Bastian et al. (1991). Proteolysis of caseins was examined using polyacrylamide gel electrophoresis, by the method of Andrews (1983). Gels were stained by the method of Blakesley and Boezi (1977). Statistical analysis The effects of plasmin content of milk and subsequent digestion of caseins on milk cheesemaking properties were examined by analysis of variance (ANOVA), where the various levels of plasmin added in the experiments performed were divided into 4 groups (0, 0.25—1.0, 2.0—5.0 and '5.0 mg plasmin added per litre of milk). Analysis of duplicate measurements for each parameter listed, with the exception of curd yield, was then performed for the data from 4, 10, 6 and 4 independent trials, respectively. Means of parameters at different plasmin levels were compared using Tukey’s test (at P(0.05). Relationships between amount of plasmin added and clotting and yield properties of milks were also examined using Pearson’s regression analysis. All analyses were performed using the Minitab statistical analysis package (Version 10.0, Minitab Ltd., Coventry, CV4 8HX, UK).
Fig. 1. Change in curd yield, % protein content in whey and curd moisture (%) on storage of control milk at 37°C (whole line) and milk with 8 mg L\ plasmin added (dotted line). Each point shows the mean and standard deviation of 4 separate experiments.
809
Plasmin and cheesemaking
the level at the end of incubation being similar to that at the start of the incubation. Plasmin content and curd yield For the next series of experiments, incubation time was held constant at 5 h and the level of plasmin added varied in the range 0.1—10.0 mg plasmin L\ milk, with subsequent measurement of coagulation and cheese yield properties of the milk. The extent of casein degradation resulting from this incubation is shown in Fig. 2. bCasein breakdown increased with plasmin level and, at levels above 0.75 mg L\, extensive hydrolysis was seen after 5 h incubation.
Fig. 2. Urea polyacrylamide gel electrophoretogram (12.5% T, 4% C, pH 8.9) of milk after 5 h digestion at 37°C with varying amounts of plasmin added. Lane 1, no plasmin added. Lanes 2—8, 0.25, 0.5, 0.75, 2.5, 5.0, 7.5 and 10.0 mg plasmin added L\ milk, respectively.
Curd yield and adjusted yield decreased with increasing milk plasmin content (Table 1), but curd moisture was unaffected by plasmin. Losses of protein in whey were increased with plasmin addition at levels above 1.0 mg L\. Rennet clotting time of milk decreased at low levels of plasmin addition ((5.0 mg L\) but was increased at levels '5.0 mg L\. The parameters K20 and A60 were unaffected by plasmin contents of (1.0 mg L\ but increased and decreased, respectively, at higher plasmin levels. Curd firming rate decreased at added plasmin levels '1.0 mg L\, while cutting time was significantly increased at plasmin levels '5.0 mg L\. When analysis of relationships between measured parameters in the above experiments was carried out using regression analysis, the level of plasmin addition to milk was weakly negatively correlated with yield (r"!0.451, P(0.05) and strongly positively correlated with whey protein losses (r"0.783, P(0.001), while there was no correlation with curd moisture content (Table 2). Strong correlations were taken to suggest a linear relationship between plasmin addition (and hence degree of hydrolysis of casein by the enzyme) and the measured parameter, while less significant correlations were seen for parameters which were either only affected by high levels of plasmin addition or where other factors may have influenced the relationship between the parameters. For example, overall RCT was positively correlated with amount of plasmin added (r"0.524, P(0.01), although this was probably due to the large influence of the increased RCT of samples at levels of plasmin addition '5.0 mg L\. Amount of plasmin added was strongly negatively correlated with both curd firming rate (K20, r"!0.926, P(0.001), and final curd firmness (A60, r"!0.962, P(0.001). The amount of plasmin added was also strongly positively correlated with cutting time (r"0.850, P(0.001) and strongly negatively correlated with firming rate (r"!0.878, P(0.001). In terms of other relationships between experimental parameters, yield was weakly correlated with A60 (r"0.395, P(0.05), and whey protein losses were positively and negatively correlated with K20 (r"0.619,
Table 1. Milk Coagulation and Cheese-yielding Properties after Treatment with Increasing Levels of Plasmin Plasmin added (mg L\)
Significance
0
0.25—1.0
2.0—5.0
'5.0
n
4
10
6
4
Curd yield Curd moisture (%) Adjusted yield Whey protein (%) Rennet clotting time (RCT, min) Firming time (K20, min) Curd firmness A60 (mm) Cutting time (min) Firming rate
12.0 72.1 11.2 1.03 25.3 13.3 38.8 38.7 0.66
10.4 72.8 9.4 1.07 24.1 13.4 38.7 37.5 0.65
9.9 72.0 9.3 1.22 23.5 17.5 30.1 41.0 0.58
8.6 72.8 7.8 1.43 27.8 24.8 19.6 51.9 0.52
g 100 mL\ milk. Adjusted to a constant 70% moisture. * P(0.05; ** P(0.01; *** P(0.001; NS, not significant. Values in a row followed by the same letter are not significantly different (P(0.05).
* NS ** *** ** *** *** *** ***
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O. Mara et al.
Table 2. Correlation Matrix of the Relationships between Rennet Coagulation Properties (Measured by Formagraph) of Milk, Amount of Added Plasmin and the Yield and Moisture Content of Rennet Curds Prepared from such Milk after Plasmin Hydrolysis
Yield Moisture P RCT K20 A60
Plasmin
Yield
Moisture
!0.451* NS 0.783*** 0.524** 0.926*** !0.962***
— NS NS NS NS 0.395*
— — NS NS NS NS
P — — — NS 0.619*** !0.850***
RCT
K20
A60
— — — — NS !0.481**
— — — — — !0.944***
— — — — — —
n"24 for all correlations. *** P(0.001; ** P(0.01; * P(0.05; NS, not significant. mg plasmin added L\ milk. Protein level in whey. Rennet clotting time (McMahon and Brown, 1982). Time from start of gel development until a width of 20 mm is reached on the Formagraph chart (McMahon and Brown, 1982). Width of Formagraph plot 60 min after rennet addition (McMahon and Brown, 1982).
Table 3. Correlation Matrix of Factors Affecting the Yield and Moisture Content of Curds Prepared from Milk and the Amount of Protein Lost in Whey (P ) after Incubation with Added Plasmin Plasmin Yield !0.329** Moisture NS Whey protein 0.412***
Yield
Moisture
P
— 0.522*** NS
— — 0.346**
— — —
n"56 for all correlations. *** P(0.001; ** P(0.01; * P(0.05; NS, not significant. mg plasmin added L\ milk.
P(0.001) and A60 (r"!0.850, P(0.001), respectively. RCT was weakly negatively correlated with A60 (r"!0.481, P(0.01), while K20 and A60 were significantly negatively correlated (r"!0.944, P(0.001). In a further series of trials, similar incubation conditions were used to prepare hydrolysed samples for further investigation of the relationship between plasmin added to milk and curd-yielding properties (Table 3). In these experiments, the amount of active plasmin added to milk was again negatively correlated with curd yield (r"!0.329, P(0.01) and positively correlated with losses of protein (r"0.412, P(0.001) in whey. There was, again, no correlation between amount of plasmin added and curd moisture content.
DISCUSSION Low levels of plasmin activity (as resulted from the addition of (1.0 mg L\ exogenous plasmin) had no significant effect on curd yield, whey protein losses, or rennet clotting properties of milk. Higher levels ('1.0 mg L\), however, resulted in decreased yields, slower curd firming and softer final curds, longer times from renneting to cutting, and (at plasmin concentrations '5.0 mg L\), increased milk rennet clotting times. There was, consistently, no relationship between plasmin action and curd moisture content.
In general, these results are in contrast to the findings of Bastian et al. (1991), among others, who found no correlation between plasmin activity and milk clotting properties. Two possible explanations can be offered for this. Firstly, in studies of naturally occurring variation in milk plasmin activity and clotting properties, it is likely that other factors, such as milk pH and protein content, cow breed and season have a greater effect in determining milk clotting properties than does plasmin activity. However, in the current study milk taken from a commercial supply over a short period of time (1 month) was used as a base for standardisation of experiments, the sole variable in which was hence the degree of plasmin hydrolysis of casein (assuming a relatively constant baseline plasmin activity in all samples). By varying the amount of plasmin added prior to milk incubation, the sole experimental variable was extent of casein hydrolysis due to the action of plasmin. Secondly, the range of plasmin levels used in these experiments was larger than would be found in natural milk samples. Direct comparison of added plasmin levels to levels found in the study of Bastian et al. (1991) are difficult, due to the disparity between units used to indicate plasmin activities. However, levels of native plasmin activity found by Benslimane et al. (1990) and Richardson and Pearce (1981) were of the order of 0.1—0.5 mg L\, which are in the lower range of levels of plasmin added here, in which range only small effects on curd-forming properties were noted. This may explain why many studies have found little effect of plasmin activity on milk clotting properties, but shows that high levels of hydrolysis of casein by plasmin will certainly impair milk curdforming properties. This may be the case when very high levels of plasmin activity are found, such as mastitic milk (Bastian and Brown, 1996), or where temperature abuse or excessive holding of milk creates the opportunity for the enzyme to cause significant hydrolysis. Overall, it may be stated that, in the model system used in this study, high levels of plasmin activity exerted significant effects on many cheesemaking properties of milk. The reduction in RCT reported by Pearse et al. (1986) at early stages of casein hydrolysis by plasmin, with a rise in RCT at high degrees of hydrolysis, was apparent in the present study. The level of plasmin added in that study (32 mg L\) was higher than the highest level used in the present work, but it is interesting to note that after 5 h
Plasmin and cheesemaking
incubation with this level of plasmin in the earlier study, the RCT was still considerably lower than that of control milk, in contrast to the present study, where 5 h incubation with 5—10 mg L\ plasmin resulted in an increase in RCT relative to the control. This may be due to differences between the choice of artificial micelle milk, as used by Pearse et al. (1986), or skim milk as a model system. Politis and Ng-Kwai-Hang (1988) found that RCT was not significantly influenced by b-casein level in milk, while curd firmness and firming rate were related to b-casein level, which is consistent with the role of plasmin shown here. This is also in agreement with the findings of Yun et al. (1982), who suggested that b-casein may be essential for curd hardening and firmness. It has been suggested that plasmin action could affect the process of rennet coagulation in two opposing ways: by increasing accessibility of i-casein molecules, and by interference of degradation products with aggregation and coagulation mechanisms (Pearse et al., 1986). It is possible, judging from the results of the present study, that at low levels of plasmin action, the former activity predominates, while more extensive hydrolysis results in poor clotting properties, due to a combination of interference mechanisms and a decrease in available protein for curd formation. The finding that curd moisture content is unrelated to plasmin action, even at high levels of degradation, is surprising, however, as plasmin has been suggested as a possible cause of high moisture content of cheese made from late lactation milk (Donnelly et al., 1984). The significant correlation between plasmin activity and yield in the current experiments is, as would be expected, due to the high losses of protein in whey, and demonstrates that plasmin action, in itself, has a significant negative influence on cheese yield. It appears, however, that the residual intact casein after plasmin hydrolysis forms a curd which, while weaker than normal, retains water in a manner similar to control curds. Pearse et al. (1986) found that quite extensive hydrolysis of caseins by plasmin did not affect greatly the curd syneresis. The results of the current study extend this to show that similar final moisture contents will then arise in curds prepared from control and plasmin-hydrolysed milk. Hence, plasmin activity should not be considered a cause of high moisture content of cheese made from milk of high plasmin activity, such as late lactation milk. High moisture contents of cheese made from such milks must instead be linked to factors such as alteration of the pH or salts balance of milk. The same argument may be proposed for cheese made from mastitic milk of high somatic cell count (SCC), and concomitantly high plasmin activity, which is associated with reduced yields, increased losses of protein in whey and increased cheese moisture (Barbano et al., 1991). The results of the current study suggest that, while the reduced yields and increased losses associated with cheese made from high SCC milk may be due to plasmin activity, the increased moisture content of such cheese is not due to plasmin action, and hence must be due to other factors, such as the activity of non-plasmin enzymes on the caseins, or the composition and mineral balance of the milk. CONCLUSION Plasmin-mediated hydrolysis of casein in milk, over a wide range of plasmin levels, was linked to reduced
811
curd yields and increased losses of protein in whey, although at levels similar to those found in many studies of natural variations in plasmin activity in milk, effects were relatively minor. Milk extensively hydrolysed by plasmin had increased curd firming time and cutting time, and reduced firming rate and final curd firmness. However, milk rennet clotting time was less sensitive to plasmin action, while curd moisture content was not affected by plasmin action, leading to the conclusion that the high moisture content of cheese made from milk with high plasmin levels, such as late lactation or mastitic milk, is not due to the action of plasmin.
ACKNOWLEDGEMENTS This research was partly funded by grant-aid under the Food Sub-Programme of the Operation Programme for Industrial Capital Development which is administered by the Department of Agriculture, Food and Forestry and supported by national and EU funds.
REFERENCES Andrews, A. T. (1983) Proteinases in nomal bovine milk and their action on caseins. Journal of Dairy Research 50, 45—55. Barbano, D. M., Rasmussen, R. R. and Lynch, J. M. (1991) Influence of milk somatic cell count and milk age on cheese yield. Journal of Dairy Science 74, 369—388. Bastian, E. D., Brown, R. J. and Ernstrom, C. A. (1991) Plasmin activity and milk coagulation. Journal of Dairy Science 74, 3677—3685. Bastian, E. D., and Brown, R. J. (1996) Plasmin in milk and dairy products: an update. International Dairy Journal 6, 435—457. Benslimane, S., Dognin-Bergeret, M. J., Berdague, J.-L. and Guademer, Y. (1990) Variation with season and lactation of plasmin and plasminogen in Montbeliard cows’ milk. Journal of Dairy Research 57, 423—435. Blakesley, R. W. and Boezi, J. A. (1977) A new staining procedure for proteins in polyacrylamide gels using Coomassie Brilliant Blue G250. Analytical Biochemistry 82, 580—581. Donnelly, W. J., Barry J. G. and Bucheim, W. (1984) Casein micelle compositional and syneretic properties of late lactation milk. Irish Journal of Food Science and ¹echnology 8, 121—130. Farkye, N. Y. and Landkammer, C. F. (1992) Milk plasmin activity influence on Cheddar cheese quality during ripening. Journal of Food Science 57, 622—625. Lopez-Fandino, R., Carrascosa, A. V. and Olano, A. (1996) The effects of high pressure on whey protein denaturation and cheese-making properties of milk. Journal of Dairy Science 79, 929—936. McMahon, D. J. and Brown, R. J. (1982) Evaluation of Formagraph for comparing rennet solutions. Journal of Dairy Science 65, 1639—1642. Macheboeuf, D., Coulon, J.-B. and D’Hour, P. (1993) Effect of breed, protein genetic variants and feeding on cows’ milk coagulation properties. Journal of Dairy Research 60, 43—54. O’Keeffe, A. M. (1984) Seasonal and lactational influences on moisture content of Cheddar cheese. Irish Journal of Food Science and ¹echnology 8, 27—37. Okigbo, L. M., Richardson, G. H., Brown, R. J. and Ernstrom, C. A. (1985) Casein composition of cows milk of different chymosin coagulation properties. Journal of Dairy Science 68, 1887—1982.
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Pearse, M. J., Linklater, P. M., Hall, R. J. and MacKinlay, A. G. (1986) Extensive degradation of casein by plasmin does not impede subsequent curd formation and syneresis. Journal of Dairy Research 53, 477—480. Politis, I. and Ng-Kwai-Hang, K. F. (1988) Effects of somatic cell count and milk composition on the coagulating properties of milk. Journal of Dairy Science 71, 1740—1746.
Richardson, B. C. and Pearce, K. N. (1981) The determination of plasmin in dairy products. New Zealand Journal of Dairy Science and ¹echnology 16, 209—220. Yun, S.-E., Ohmiya, K. and Shimizu, S. (1982) Role of b-casein in milk curdling. Agricultural and Biological Chemistry 46, 443—449.
Int. Dairy Journal 8 (1998) 807—812 1999 Elsevier Science Ltd. All rights reserved Printed in Great Britain 0958-6946/99/$ — see front matter
The Curd-forming Properties of Milk as Affected by the Action of Plasmin Orla Maraa, Christophe Roupiea, Arthur Duffyb and Alan L. Kellya * ? Department of Food Technology, University College Cork, Cork, Ireland @ Dairy Products Research Centre, Moorepark, Fermoy, Co. Cork, Ireland (Received 13 July 1998; accepted 20 October 1998) ABSTRACT Purified plasmin was added to skim milk at a range of levels (0.1—10 mg L\) and the samples subsequently incubated to allow hydrolysis of casein by the enzyme. Milk clotting properties (as measured by Formagraph) and curd yield properties of the milk were measured after incubation. Increasing plasmin addition, with concomitantly increased levels of hydrolysis of casein, resulted in increased curd firming time and cutting time and reduced firming rate and final curd firmness (all P(0.001). Rennet clotting time was only increased with extensive hydrolysis by plasmin. Curd yield was reduced with increasing plasmin addition, with concomitantly increased losses of protein in whey. With low levels of enzyme addition, however, the magnitude of most effects was small. Curd moisture was unaffected by plasmin addition to milk at any level of enzyme addition. It may be concluded that extensive hydrolysis of casein by plasmin, independent of any other alterations in milk quality, has a significant influence on some milk cheesemaking properties. However, it appears that plasmin may not be responsible for changes in cheese moisture in, for example, late lactation milk. 1999 Elsevier Science Ltd. All rights reserved Keywords: plasmin; milk; cheese; coagulation
INTRODUCTION
moisture content is the expense of adding this enzyme to large quantities of milk. Recently, however, a small-scale centrifugation method has been reported to provide a good assessment of fresh cheese yield, unadjusted for moisture content (Macheboeuf et al., 1993; Lopez-Fandino et al., 1996). The Formagraph (Bastian et al., 1991) also may be used to study the coagulation properties of milk. The objective of this study was to use the method of Lopez-Fandino et al. (1996) to investigate the effects of plasmin hydrolysis of milk protein on the yield and moisture of renneted curds subsequently prepared from that milk. Also, the rennet clotting properties of milk hydrolysed by plasmin were studied using a Formagraph. An advantage to this experimental approach was that the hydrolytic action of plasmin on the casein micelles and the influence of this action on subsequent coagulation of milk could be studied in isolation from other changes in milk (such as those associated with late lactation or mastitis concomitant with elevated milk plasmin activity) to determine the exact influence of the action of this enzyme on milk cheesemaking properties.
Plasmin, the principal alkaline proteinase in bovine milk, readily hydrolyses b-casein and a -casein, and to a lesser extent a -casein, in bovine milk (Bastian and Brown, 1996). It has been proposed that increasing levels of plasmin, for example in late lactation milk, and the subsequent action of this enzyme on the caseins, may be linked to poor cheesemaking properties of such milk (Okigbo et al., 1985). However, it has been shown that a normal coagulum can form and syneresis proceed as usual even after extensive degradation of casein by plasmin (Pearse et al., 1986), while Bastian et al. (1991) found no correlation between plasmin activity and milk clotting parameters on studying a large number of milks of different breeds, stages and numbers of lactation and in different seasons. The effects of plasmin hydrolysis of casein on cheese yield and moisture content are less well described, and are of particular interest, both from an economical and quality perspective (Pearse et al., 1986). Previous studies have associated plasmin action in milk, particularly in late lactation and high SCC milks, with increased moisture content of cheese made from such milk (Donnelly et al., 1984; O’Keeffe, 1984; Barbano et al., 1991). A factor in designing experiments to determine the influence of enzymes such as plasmin on cheese yield and
MATERIALS AND METHODS Plasmin hydrolysis of milk In each experiment porcine plasmin (P8644, Sigma Chemical Co., Poole, Dorest, UK) was added to samples of pasteurised bovine skim milk from a local dairy
*Corresponding author. Tel.: 00 353 21 903405; Fax: 00 353 21 270213; E-mail: [email protected] 807
808
O. Mara et al.
company at levels from 0.1—10 mg L \. Porcine plasmin has been shown to have similar specificity to bovine plasmin in cheesemaking trials (Farkye and Landkammer, 1992). Samples with no added plasmin were incubated in each experiment as controls. Plasmin was typically dissolved in distilled water at 1 mg mL\ concentration and the appropriate amount of this concentrated stock solution added to portions of milk for incubation. Sodium azide (0.05% w/v) was added to all milks before incubation to prevent bacterial contamination. All incubations were carried out at 37°C for exactly 5 h before renneting, unless otherwise specified. Preparation of renneted gels
RESULTS Influence of plasmin hydrolysis over time on milk coagulation The yield of rennet curds prepared from milk to which no plasmin was added (control milk) did not vary over 5 h storage at 37°C (Fig. 1). During this time the moisture content of the control curds was also relatively constant, and there was a slight increase in protein content of the control whey. Milk which received 8 mg L\ plasmin, however, showed decreased yields as the hydrolysis proceeded, and had elevated losses of protein in whey. Curd moisture in the plasmin-treated milk was decreased initially by plasmin digestion but increased after 2 h, with
The procedure of Machebeouf et al. (1993), as modified by Lopez-Fandino et al. (1996), was followed. Each 50 mL sample of hydrolysed or unhydrolysed milk was mixed with 1 mL rennet solution (Hansen’s Standard Plus 200 rennet, 1.5 g in 100 mL 0.05 M acetate buffer, pH 5.4). The milk samples were allowed to set at 32°C for 40 min, cut, allowed stand for 10 min, and centrifuged at 13,000 rpm for 15 min at 4°C. Whey was then carefully removed and yields calculated. Analysis Protein contents of whey samples were determined by the Kjeldahl method. Moisture of curds was determined after drying to constant weight at 105°C. Rennet clotting properties of the milk were measured by Formagraph (Foss Electric, DK-3400, Hiller+d, Denmark) according to the method of McMahon and Brown (1982). Milk (10 mL) was renneted at 35°C using 32 kL of rennet. Clotting time (RCT), curd firmness 60 min after rennet addition (A60) and time required to achieve a curd firmness of 20 mm (K20) were determined for the milks at their natural pH. Curd firming rate and cutting time were also calculated, as described by Bastian et al. (1991). Proteolysis of caseins was examined using polyacrylamide gel electrophoresis, by the method of Andrews (1983). Gels were stained by the method of Blakesley and Boezi (1977). Statistical analysis The effects of plasmin content of milk and subsequent digestion of caseins on milk cheesemaking properties were examined by analysis of variance (ANOVA), where the various levels of plasmin added in the experiments performed were divided into 4 groups (0, 0.25—1.0, 2.0—5.0 and '5.0 mg plasmin added per litre of milk). Analysis of duplicate measurements for each parameter listed, with the exception of curd yield, was then performed for the data from 4, 10, 6 and 4 independent trials, respectively. Means of parameters at different plasmin levels were compared using Tukey’s test (at P(0.05). Relationships between amount of plasmin added and clotting and yield properties of milks were also examined using Pearson’s regression analysis. All analyses were performed using the Minitab statistical analysis package (Version 10.0, Minitab Ltd., Coventry, CV4 8HX, UK).
Fig. 1. Change in curd yield, % protein content in whey and curd moisture (%) on storage of control milk at 37°C (whole line) and milk with 8 mg L\ plasmin added (dotted line). Each point shows the mean and standard deviation of 4 separate experiments.
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Plasmin and cheesemaking
the level at the end of incubation being similar to that at the start of the incubation. Plasmin content and curd yield For the next series of experiments, incubation time was held constant at 5 h and the level of plasmin added varied in the range 0.1—10.0 mg plasmin L\ milk, with subsequent measurement of coagulation and cheese yield properties of the milk. The extent of casein degradation resulting from this incubation is shown in Fig. 2. bCasein breakdown increased with plasmin level and, at levels above 0.75 mg L\, extensive hydrolysis was seen after 5 h incubation.
Fig. 2. Urea polyacrylamide gel electrophoretogram (12.5% T, 4% C, pH 8.9) of milk after 5 h digestion at 37°C with varying amounts of plasmin added. Lane 1, no plasmin added. Lanes 2—8, 0.25, 0.5, 0.75, 2.5, 5.0, 7.5 and 10.0 mg plasmin added L\ milk, respectively.
Curd yield and adjusted yield decreased with increasing milk plasmin content (Table 1), but curd moisture was unaffected by plasmin. Losses of protein in whey were increased with plasmin addition at levels above 1.0 mg L\. Rennet clotting time of milk decreased at low levels of plasmin addition ((5.0 mg L\) but was increased at levels '5.0 mg L\. The parameters K20 and A60 were unaffected by plasmin contents of (1.0 mg L\ but increased and decreased, respectively, at higher plasmin levels. Curd firming rate decreased at added plasmin levels '1.0 mg L\, while cutting time was significantly increased at plasmin levels '5.0 mg L\. When analysis of relationships between measured parameters in the above experiments was carried out using regression analysis, the level of plasmin addition to milk was weakly negatively correlated with yield (r"!0.451, P(0.05) and strongly positively correlated with whey protein losses (r"0.783, P(0.001), while there was no correlation with curd moisture content (Table 2). Strong correlations were taken to suggest a linear relationship between plasmin addition (and hence degree of hydrolysis of casein by the enzyme) and the measured parameter, while less significant correlations were seen for parameters which were either only affected by high levels of plasmin addition or where other factors may have influenced the relationship between the parameters. For example, overall RCT was positively correlated with amount of plasmin added (r"0.524, P(0.01), although this was probably due to the large influence of the increased RCT of samples at levels of plasmin addition '5.0 mg L\. Amount of plasmin added was strongly negatively correlated with both curd firming rate (K20, r"!0.926, P(0.001), and final curd firmness (A60, r"!0.962, P(0.001). The amount of plasmin added was also strongly positively correlated with cutting time (r"0.850, P(0.001) and strongly negatively correlated with firming rate (r"!0.878, P(0.001). In terms of other relationships between experimental parameters, yield was weakly correlated with A60 (r"0.395, P(0.05), and whey protein losses were positively and negatively correlated with K20 (r"0.619,
Table 1. Milk Coagulation and Cheese-yielding Properties after Treatment with Increasing Levels of Plasmin Plasmin added (mg L\)
Significance
0
0.25—1.0
2.0—5.0
'5.0
n
4
10
6
4
Curd yield Curd moisture (%) Adjusted yield Whey protein (%) Rennet clotting time (RCT, min) Firming time (K20, min) Curd firmness A60 (mm) Cutting time (min) Firming rate
12.0 72.1 11.2 1.03 25.3 13.3 38.8 38.7 0.66
10.4 72.8 9.4 1.07 24.1 13.4 38.7 37.5 0.65
9.9 72.0 9.3 1.22 23.5 17.5 30.1 41.0 0.58
8.6 72.8 7.8 1.43 27.8 24.8 19.6 51.9 0.52
g 100 mL\ milk. Adjusted to a constant 70% moisture. * P(0.05; ** P(0.01; *** P(0.001; NS, not significant. Values in a row followed by the same letter are not significantly different (P(0.05).
* NS ** *** ** *** *** *** ***
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Table 2. Correlation Matrix of the Relationships between Rennet Coagulation Properties (Measured by Formagraph) of Milk, Amount of Added Plasmin and the Yield and Moisture Content of Rennet Curds Prepared from such Milk after Plasmin Hydrolysis
Yield Moisture P RCT K20 A60
Plasmin
Yield
Moisture
!0.451* NS 0.783*** 0.524** 0.926*** !0.962***
— NS NS NS NS 0.395*
— — NS NS NS NS
P — — — NS 0.619*** !0.850***
RCT
K20
A60
— — — — NS !0.481**
— — — — — !0.944***
— — — — — —
n"24 for all correlations. *** P(0.001; ** P(0.01; * P(0.05; NS, not significant. mg plasmin added L\ milk. Protein level in whey. Rennet clotting time (McMahon and Brown, 1982). Time from start of gel development until a width of 20 mm is reached on the Formagraph chart (McMahon and Brown, 1982). Width of Formagraph plot 60 min after rennet addition (McMahon and Brown, 1982).
Table 3. Correlation Matrix of Factors Affecting the Yield and Moisture Content of Curds Prepared from Milk and the Amount of Protein Lost in Whey (P ) after Incubation with Added Plasmin Plasmin Yield !0.329** Moisture NS Whey protein 0.412***
Yield
Moisture
P
— 0.522*** NS
— — 0.346**
— — —
n"56 for all correlations. *** P(0.001; ** P(0.01; * P(0.05; NS, not significant. mg plasmin added L\ milk.
P(0.001) and A60 (r"!0.850, P(0.001), respectively. RCT was weakly negatively correlated with A60 (r"!0.481, P(0.01), while K20 and A60 were significantly negatively correlated (r"!0.944, P(0.001). In a further series of trials, similar incubation conditions were used to prepare hydrolysed samples for further investigation of the relationship between plasmin added to milk and curd-yielding properties (Table 3). In these experiments, the amount of active plasmin added to milk was again negatively correlated with curd yield (r"!0.329, P(0.01) and positively correlated with losses of protein (r"0.412, P(0.001) in whey. There was, again, no correlation between amount of plasmin added and curd moisture content.
DISCUSSION Low levels of plasmin activity (as resulted from the addition of (1.0 mg L\ exogenous plasmin) had no significant effect on curd yield, whey protein losses, or rennet clotting properties of milk. Higher levels ('1.0 mg L\), however, resulted in decreased yields, slower curd firming and softer final curds, longer times from renneting to cutting, and (at plasmin concentrations '5.0 mg L\), increased milk rennet clotting times. There was, consistently, no relationship between plasmin action and curd moisture content.
In general, these results are in contrast to the findings of Bastian et al. (1991), among others, who found no correlation between plasmin activity and milk clotting properties. Two possible explanations can be offered for this. Firstly, in studies of naturally occurring variation in milk plasmin activity and clotting properties, it is likely that other factors, such as milk pH and protein content, cow breed and season have a greater effect in determining milk clotting properties than does plasmin activity. However, in the current study milk taken from a commercial supply over a short period of time (1 month) was used as a base for standardisation of experiments, the sole variable in which was hence the degree of plasmin hydrolysis of casein (assuming a relatively constant baseline plasmin activity in all samples). By varying the amount of plasmin added prior to milk incubation, the sole experimental variable was extent of casein hydrolysis due to the action of plasmin. Secondly, the range of plasmin levels used in these experiments was larger than would be found in natural milk samples. Direct comparison of added plasmin levels to levels found in the study of Bastian et al. (1991) are difficult, due to the disparity between units used to indicate plasmin activities. However, levels of native plasmin activity found by Benslimane et al. (1990) and Richardson and Pearce (1981) were of the order of 0.1—0.5 mg L\, which are in the lower range of levels of plasmin added here, in which range only small effects on curd-forming properties were noted. This may explain why many studies have found little effect of plasmin activity on milk clotting properties, but shows that high levels of hydrolysis of casein by plasmin will certainly impair milk curdforming properties. This may be the case when very high levels of plasmin activity are found, such as mastitic milk (Bastian and Brown, 1996), or where temperature abuse or excessive holding of milk creates the opportunity for the enzyme to cause significant hydrolysis. Overall, it may be stated that, in the model system used in this study, high levels of plasmin activity exerted significant effects on many cheesemaking properties of milk. The reduction in RCT reported by Pearse et al. (1986) at early stages of casein hydrolysis by plasmin, with a rise in RCT at high degrees of hydrolysis, was apparent in the present study. The level of plasmin added in that study (32 mg L\) was higher than the highest level used in the present work, but it is interesting to note that after 5 h
Plasmin and cheesemaking
incubation with this level of plasmin in the earlier study, the RCT was still considerably lower than that of control milk, in contrast to the present study, where 5 h incubation with 5—10 mg L\ plasmin resulted in an increase in RCT relative to the control. This may be due to differences between the choice of artificial micelle milk, as used by Pearse et al. (1986), or skim milk as a model system. Politis and Ng-Kwai-Hang (1988) found that RCT was not significantly influenced by b-casein level in milk, while curd firmness and firming rate were related to b-casein level, which is consistent with the role of plasmin shown here. This is also in agreement with the findings of Yun et al. (1982), who suggested that b-casein may be essential for curd hardening and firmness. It has been suggested that plasmin action could affect the process of rennet coagulation in two opposing ways: by increasing accessibility of i-casein molecules, and by interference of degradation products with aggregation and coagulation mechanisms (Pearse et al., 1986). It is possible, judging from the results of the present study, that at low levels of plasmin action, the former activity predominates, while more extensive hydrolysis results in poor clotting properties, due to a combination of interference mechanisms and a decrease in available protein for curd formation. The finding that curd moisture content is unrelated to plasmin action, even at high levels of degradation, is surprising, however, as plasmin has been suggested as a possible cause of high moisture content of cheese made from late lactation milk (Donnelly et al., 1984). The significant correlation between plasmin activity and yield in the current experiments is, as would be expected, due to the high losses of protein in whey, and demonstrates that plasmin action, in itself, has a significant negative influence on cheese yield. It appears, however, that the residual intact casein after plasmin hydrolysis forms a curd which, while weaker than normal, retains water in a manner similar to control curds. Pearse et al. (1986) found that quite extensive hydrolysis of caseins by plasmin did not affect greatly the curd syneresis. The results of the current study extend this to show that similar final moisture contents will then arise in curds prepared from control and plasmin-hydrolysed milk. Hence, plasmin activity should not be considered a cause of high moisture content of cheese made from milk of high plasmin activity, such as late lactation milk. High moisture contents of cheese made from such milks must instead be linked to factors such as alteration of the pH or salts balance of milk. The same argument may be proposed for cheese made from mastitic milk of high somatic cell count (SCC), and concomitantly high plasmin activity, which is associated with reduced yields, increased losses of protein in whey and increased cheese moisture (Barbano et al., 1991). The results of the current study suggest that, while the reduced yields and increased losses associated with cheese made from high SCC milk may be due to plasmin activity, the increased moisture content of such cheese is not due to plasmin action, and hence must be due to other factors, such as the activity of non-plasmin enzymes on the caseins, or the composition and mineral balance of the milk. CONCLUSION Plasmin-mediated hydrolysis of casein in milk, over a wide range of plasmin levels, was linked to reduced
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curd yields and increased losses of protein in whey, although at levels similar to those found in many studies of natural variations in plasmin activity in milk, effects were relatively minor. Milk extensively hydrolysed by plasmin had increased curd firming time and cutting time, and reduced firming rate and final curd firmness. However, milk rennet clotting time was less sensitive to plasmin action, while curd moisture content was not affected by plasmin action, leading to the conclusion that the high moisture content of cheese made from milk with high plasmin levels, such as late lactation or mastitic milk, is not due to the action of plasmin.
ACKNOWLEDGEMENTS This research was partly funded by grant-aid under the Food Sub-Programme of the Operation Programme for Industrial Capital Development which is administered by the Department of Agriculture, Food and Forestry and supported by national and EU funds.
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Pearse, M. J., Linklater, P. M., Hall, R. J. and MacKinlay, A. G. (1986) Extensive degradation of casein by plasmin does not impede subsequent curd formation and syneresis. Journal of Dairy Research 53, 477—480. Politis, I. and Ng-Kwai-Hang, K. F. (1988) Effects of somatic cell count and milk composition on the coagulating properties of milk. Journal of Dairy Science 71, 1740—1746.
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