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* MN-SD Dairy Foods Research Center, Department of Food Science and Nutrition, University of Minnesota, St. Paul 55108
Department of Food Science, Purdue University, West Lafayette, IN 47906
1 Corresponding author: lmetzger{at}umn.edu
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONCONCLUSIONSACKNOWLEDGEMENTSREFERENCES |
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Key Words: proteolysis • chymosin • plasmin • lactic acid bacteria
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONCONCLUSIONSACKNOWLEDGEMENTSREFERENCES |
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Calcium and P contribute to casein micellar structure and removal of Ca and P from caseins causes disaggregation of casein micelles (Hsu and Shipe, 1986). This disaggregation exposes a larger surface area of proteins to proteinases and leads to an increase in enzyme–substrate interactions. Therefore, cheeses with decreased Ca and P exhibit higher proteolysis (Marcos et al., 1976; Lawrence et al., 1987). However, studies that investigate the influence of Ca on proteolysis in cheese generally use modifications in set and drain pH to obtain cheeses with different levels of Ca. These modifications also control the proportion of residual chymosin in the cheese (Stadhouders, 1962; Holmes et al., 1977). A higher level of chymosin will obviously increase the rate of proteolysis. In addition, Lawrence et al. (1987) proposed that pH at draining influences plasmin retention in cheese. However, Farkye and Fox (1990) did not observe any influence of drain pH on residual plasmin activity in cheese.
Relatively few studies have reported the influence of lactose content of cheeses on proteolysis during ripening. Residual lactose content influences the extent of acid production in cheese and should influence bacterial growth and pH-dependent activity of enzymes. Shakeel-Ur-Rehman et al. (2004) demonstrated that cheeses with higher lactose had lower total amino acids concentration after 180 d of ripening. Peichevski and Petrova (1979) reported that washing of cheese curds using water up to 20% before scalding and after cutting increased the biochemical processes in Vitosha cheese. An increase in proteolysis in washed curd cheeses was also observed by Stadhouders (1962), who attributed it to a higher retention of chymosin in these cheeses.
The S/M of cheese markedly influences its rate of proteolysis (Marcos et al., 1976; Thomas and Pearce, 1981; Lane and Fox, 1999). A lower S/M increases proteolysis of 
S1- and ß-casein (Thomas and Pearce, 1981; Schroeder et al., 1988; Lane and Fox, 1999). Grufferty and Fox (1988) found that the addition of NaCl to milk caused a release of plasmin from casein micelles. Therefore, a higher rate of salting may lower plasmin retention in cheese, and should decrease the extent or rate of proteolysis. In addition, S/M influences water activity (aw) of cheese (Marcos et al., 1981) and therefore influences growth and protein metabolism of starter or NSLAB (Dawson and Feagan, 1957). The aw of cheese can also modify the activity of other proteolytic enzymes present in cheese.
Although the above mentioned studies investigated the influence of one or more factors on proteolysis in cheese or model systems, no study, to our knowledge, has examined influence of all the 3 factors in unison. The objective of this study was to investigate the concomitant influence of Ca and P, residual lactose, and S/M on proteolysis in Cheddar cheese through its influence on indigenous milk enzymes (plasmin) and externally added milk-clotting enzymes (chymosin), and number of live starter and NSLAB.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONCONCLUSIONSACKNOWLEDGEMENTSREFERENCES |
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. The cheeses were ripened for 48 wk, and changes in starter and NSLAB counts, and pH 4.6-soluble N were monitored during ripening. Cheeses at d 1 were also analyzed for residual activity of chymosin, plasmin, and plasminogen.
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Microbiological Analyses
Starter bacteria and NSLAB counts were measured in cheese curds before salting, and cheeses at d 1, and wk 4, 8, 16, 32, and 48. Starter count was analyzed using Bacto-Elliker agar (Difco Elliker broth, Becton, Dickinson and Company, Sparks, MD) and NSLAB using LBS agar (adjusted to pH 5.4 using glacial acetic acid). A 10-g sample of cheese was homogenized with 90 mL of 2% trisodium citrate buffer for 2 min using a stomacher (Colworth Stomacher 400, AJ Seward, London, UK) at room temperature. Serial dilutions were prepared using 2% sodium citrate buffer and samples were inoculated into sterile disposable Petri plates and pour-plated with respective media. The plates were then incubated at 32°C for 48 h, under aerobic conditions for starter bacteria; and anaerobically (BBL Gas-Pak Anaerobic System; Becton, Dickinson) for NSLAB. In addition, cheeses were analyzed to identify the type of lactococci present; that is, Lactococcus lactis ssp. lactis vs. Lactococcus lactis ssp. cremoris. To identify the presence of cremoris vs. lactis, the cheese dilutions were plated on Bacto-Elliker agar, and incubated at different temperatures (30 and 40°C). The presence of similar number of colonies of starter bacteria in the plates incubated at 30 and 40°C indicated that the starter bacteria used was primarily L. lactis ssp. lactis, which can grow at both temperatures (Sandine et al., 1972). The possibility of contamination of these plates with NSLAB (lactobacilli) was checked by morphological (cocci vs. rods) examination of cells from the various colonies using a microscope.
Residual Chymosin Activity
Residual chymosin activity in cheeses at d 1 was determined by using a method similar to that of Hurley et al. (1999). In this procedure, approximately 1 g of cheese was homogenized with 20 mL of 0.1 M trisodium citrate using a high-shear Omni mixer-homogenizer (model 17105, Omni International, Waterbury, CT). About 210 µL of cheese homogenate was transferred to a 1.5-mL microcentrifuge tube (Fisher Scientific), and mixed with 90 µL of 1 mg/mL aqueous solution of synthetic heptapeptide substrate (Pro-Thr-Glu-Phe-[NO2-Phe]-Arg-Leu, Bachem Bioscience Inc., King of Prussia, PA). To this mixture, 600 µL of 100 mM sodium formate buffer (containing 0.05% sodium azide, adjusted to pH 3.2) was also added. The microcentrifuge tubes were then incubated at 37°C for 24 h. The reaction was terminated by heating the tubes at 70°C for 10 min using a heating block (Temp-Blok Equatherm, Laboratory-Line Instruments, Inc., Melrose Park, IL). The tubes were then centrifuged (Biofuge 15, Heraeus Sepatech GmbH, West Germany) at 16,000 x g for 10 min to remove the precipitated proteins and other insoluble matter. The supernatant was filtered through 0.22-µm syringe filters (Millex-GV, Millipore Corporation, Bedford, MA) and 175 µL of the filtrate was injected into the HPLC. The conditions for HPLC analysis were similar to those suggested by Hurley et al. (1999).
Peak areas of the product were used for quantifying the residual activity of chymosin in the cheeses. Results for chymosin activity were expressed in terms of International Milk Clotting Units (IMCU) per gram using a standard curve. The standard curve was prepared using appropriate dilutions of double-strength chymosin (Chymax, Chr. Hansen, Inc., Milwaukee, WI), which had an activity of 976 IMCU/mL.
Residual Plasmin and Plasminogen Activity
Residual plasmin and plasminogen activity in cheeses at d 1 were measured using a method described by Fajardo-Lira et al. (2000).
Statistical Analyses
A 2 x 2 x 2 factorial model with 3 replications was used for statistical analysis (Table 2). Changes in starter and NSLAB counts, and pH 4.6 soluble N during ripening were analyzed using a repeated measures design. The PROC GLM procedure of SAS, which involved 3 factors (Ca and P, residual lactose, and S/M) as class variables, was used for the data analyses (SAS Institute, 1990).
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RESULTS AND DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONCONCLUSIONSACKNOWLEDGEMENTSREFERENCES |
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). Cheeses with high Ca and P had a mean residual chymosin activity of 0.26 IMCU/g, which corresponded to about 26% of the total chymosin added to the milk during cheese manufacture (Table 3). In contrast, cheeses with low Ca and P had a residual chymosin activity of 0.40 IMCU/g that corresponded to about 41% of the total chymosin added to the milk during cheese manufacture (Table 3). Previous researchers have reported that about 6 to 7% of the chymosin added to the milk is retained in the curd (Holmes et al., 1977). These differences in absolute values of chymosin retention can be attributed to use of different analytical methods to quantify residual chymosin. Our results, however, are similar to those of Nelson (2005), who used a similar method for analyzing cheeses for residual chymosin activity.
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Residual Plasmin and Plasminogen Activity
The plasmin content of the cheeses was significantly (P < 0.05) influenced by Ca and P; and marginally (P = 0.06) by lactose (Table 2). Mean plasmin content in our cheeses ranged from 0.007 to 0.013 µg/mg of cheese (Table 3), with higher plasmin concentration in the high Ca and P treatments than in the low Ca and P treatments. The plasminogen content of the cheeses was also significantly (P < 0.05) influenced by Ca and P; with high Ca and P treatments having higher plasminogen concentration as compared with the low Ca and P treatments. The mean plasminogen content of the cheeses ranged from 0.21 to 0.35 µg/mg of cheese (Table 3).
The differences in residual plasmin and plasminogen content between high vs. low Ca and P treatments can be attributed to the higher set and drain pH used in the high Ca and P treatments. The influence of drain pH on plasmin retention has also been suggested by Lawrence et al. (1987). They proposed that the higher plasmin activity in Swiss-type cheeses, compared with Cheddar cheeses, is related to the higher drain pH. However, Farkye and Fox (1990) did not observe an effect of drain pH on plasmin retention in Cheddar cheeses.
Our results show that there is a lower concentration of plasmin compared with plasminogen in the cheeses (Table 3). This could be due to the presence of active inhibitors of plasminogen activators in milk, which were still active in Cheddar cheese. In contrast, manufacture of Swiss or Mozzarella-type cheeses involves extreme heat treatment, which can inactivate inhibitors of plasminogen activators, which increases plasmin activity in Swiss or Mozzarella-type cheeses (Bastian and Brown, 1996).
Starter Counts During Ripening
Average starter counts were significantly (P < 0.05) influenced by Ca and P and time, whereas the interaction of time x S/M was marginally (P = 0.06) significant (Table 4). As shown in Figure 1, the number of starter bacteria in all treatments decreased during ripening. However the decrease was slightly larger in the high S/M treatments compared with their low S/M counterparts. Cheeses with high Ca and P had lower number of starter bacteria throughout ripening compared with their low Ca and P counterparts (HHH vs. LHH, HHL vs. LHL, HLH vs. LLH, and HLL vs. LLL).
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Our results indicate that a significant number of starter bacteria (~106 cfu/g) were still present in Cheddar cheese at the end of 48 wk of ripening. Similar levels of viable starter counts have been observed by other researchers (Dawson and Feagan, 1957; Broadbent et al., 2003). The presence of starter bacteria at high levels that are comparable with NSLAB during cheese ripening, even at 48 wk, indicates their possible role in influencing cheese characteristics during ripening. Although lysis of dead starter bacterial cells is known to contribute to proteolysis in cheese, the potential contribution from surviving starter bacteria cannot be ignored.
NSLAB Counts During Ripening
Nonstarter lactic acid bacteria counts in cheeses were significantly (P < 0.05) affected by time, and the interaction of time x S/M; and marginally (P = 0.08) influenced by Ca and P (Table 4). As shown in Figure 1, the number of NSLAB in cheese at the beginning of ripening was low and gradually increased during ripening. The rate of increase of NSLAB population in cheese during ripening was influenced by S/M. As is apparent from Figure 1, cheeses with low S/M showed a larger increase in NSLAB counts compared with their high S/M counterparts (HHH vs. HHL, HLH vs. HLL, LHH vs. LHL, LLH vs. LLL). An increase in the number of NSLAB in Cheddar cheese during ripening is expected because of their ability to grow under cheese ripening conditions (Mistry and Kasperson, 1998; Crow et al., 2001; Broadbent et al., 2003). A larger increase in NSLAB for low S/M treatments might be due to a more conducive growth environment in terms of ionic strength and aw in cheeses with low S/M compared with high S/M. A similar effect of salt content on growth of L. helveticus was observed by Roy (1991).
As shown in Figure 1, there was substantial variability among the replicates for NSLAB counts. This variation signifies that the occurrence and growth of NSLAB in cheese is largely contributed by the initial bacterial load of milk, and adventitious bacterial contamination of milk during cheese manufacture, yet the growth and selection of NSLAB in cheeses will be influenced by the conditions prevailing in the cheese including its chemical composition. Similar observations have been made by Crow et al. (2001), and a recent study by Broadbent et al. (2003) compared growth of adjunct bacteria in a Cheddar and Colby-type cheese microenvironment. They observed a greater degree of species heterogeneity in Colby cheeses compared with Cheddar cheeses.
Changes in pH 4.6-Soluble N during Ripening
Changes in pH 4.6-soluble N in cheeses were significantly (P < 0.05) influenced by Ca and P, residual lactose, S/M, time, and the interactions of time x Ca and P, time x lactose, and time x S/M (Table 4). The level of soluble N increased in all treatments during ripening, leading to a 3- to 5-fold increase in soluble N by the end of ripening (Figure 2). This increase was largest in early ripening (until 16 wk), after which the increase was smaller. The level of pH 4.6-soluble N is an index of primary proteolysis in cheese. Primary proteolysis is more prominent during the first few weeks of ripening and involves hydrolysis of caseins by residual chymosin, and to a lesser extent by plasmin, resulting in the formation of large and intermediate-sized peptides. Subsequent hydrolysis of these peptides by chymosin and enzymes from the starter and NSLAB results in the formation of smaller peptides and free amino acids during prolonged ripening and does not significantly influence pH 4.6-soluble N (Fox and McSweeney, 1996; Sousa et al., 2001). Hence, larger changes in pH 4.6-soluble N are observed during the initial stages of ripening compared with the later stages of ripening.
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). A similar effect of Ca and P on proteolysis in cheeses has been observed by other researchers (Marcos et al., 1976; Hsu and Shipe, 1986; Lawrence et al., 1987). The observed differences may be attributed to differences in residual chymosin activity or the state of the paracasein network in low Ca and P cheeses. Treatments with low Ca and P had a higher level of residual chymosin activity (Table 3) compared with treatments with high Ca and P. A higher level of chymosin would be expected to increase the rate of proteolysis. In addition, colloidal calcium phosphate acts as a cementing material within casein micelles (Hsu and Shipe, 1986). Depletion of the colloidal calcium phosphates from milk to obtain low Ca and P cheeses may also result in a reduction in Ca and P crosslinking in the paracasein network of cheese. A reduction in crosslinking may expose a larger surface area of proteins to proteinases and may lead to an increase in enzyme–substrate interactions, and in proteolysis. In addition, differences in moisture content between high and low Ca and P cheeses (Table 1) might have a partial influence on proteolysis in cheeses.
In contrast to chymosin, plasmin activity was higher (P < 0.05) in the high Ca and P treatments than in low Ca and P treatments (Table 2). However, the influence of high plasmin activity on proteolysis of high Ca and P cheeses was not apparent. This can be attributed to the relatively low proteolytic activity of plasmin compared with chymosin in Cheddar cheese. The low plasmin activity in Cheddar cheese is thought to be due to the presence of inhibitors of plasminogen activators in Cheddar cheese. The presence of inhibitors will prevent the conversion of plasminogen to its active form, plasmin, which can cause proteolysis (Bastian and Brown, 1996). This is also supported by the fact that a major fraction of plasminogen in our cheeses still existed as plasminogen, and was not converted to its active form (Table 3).
Effect of Residual Lactose.
Cheeses with low lactose exhibited more proteolysis than cheeses with high lactose (P < 0.05). In addition, a significant interaction effect of time with lactose (P < 0.05) indicates that the rate of change of pH 4.6-soluble N was different in cheeses with low lactose compared with high lactose (Figure 2). A similar effect of lactose on proteolysis was observed by Shakeel-Ur-Rehman et al. (2004) and Stadhouders (1962). Stadhouders (1962) observed that cheeses that were manufactured by washing the curds for 20 min had about 1.7 times more rennet than the cheeses that were not washed. The modifications used in our study to manufacture cheeses with different levels of lactose also showed partial influence on residual chymosin activity (Table 2), with low lactose treatments having higher chymosin activity compared with high lactose treatments (Table 2). These differences in residual chymosin might have partially influenced proteolysis in cheeses with high vs. low lactose. However, a complete explanation for differences in proteolysis between high vs. low lactose cheeses is not known. These differences in proteolysis can be partially related to differences in moisture between low and high lactose treatments (Table 1).
Effect of S/M.
Cheeses with a low S/M exhibited higher proteolysis compared with cheeses with high S/M (P < 0.05). In addition, the significance of the interaction effect of time x S/M (P < 0.05) indicates that the rate of proteolysis was different in the low S/M cheese compared with their high S/M counterparts. A similar influence of S/M on proteolysis has been observed by other researchers (Marcos et al., 1976; Thomas and Pearce, 1981; Mistry and Kasperson, 1998; Lane and Fox, 1999).
The differences in proteolysis due to S/M can be attributed to changes in protein conformation, or bacterial proteolytic activity. Varying the salt content in cheese influences the ionic strength and may result in conformational changes not only in the enzymes responsible for proteolysis but also in the caseins, which are hydrolyzed (Kristiansen et al., 1999). In aqueous solutions, salt in low concentrations reduced the ability of chymosin to degrade ß-casein, but did not reduce its ability to degrade S1-casein (Kristiansen et al., 1999). Similarly, Thomas and Pearce (1981) observed that a change in S/M in Cheddar cheese from 4 to 8% increased the level of unhydrolyzed S1-casein content from 5 to 60% at 1 mo of ripening at 10°C, whereas the level of unhydrolyzed ß-casein increased from 50 to 95%. Cheeses with a low S/M also supported a higher population of starter bacteria and exhibited a more rapid increase in NSLAB counts during ripening. This indicates that cheeses with high S/M had lower activity of lactic acid bacteria compared with low S/M cheeses. A lower bacterial proteolytic activity might have contributed to the lower levels of proteolysis in high S/M cheeses. Gobbetti et al. (1999) also reported that increasing NaCl from 2.5 to 7.5% had a negative effect on the proteinase activity of cetrain lactococcus strains, and attributed it to a decrease in aw.
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CONCLUSIONS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONCONCLUSIONSACKNOWLEDGEMENTSREFERENCES |
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ACKNOWLEDGEMENTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONCONCLUSIONSACKNOWLEDGEMENTSREFERENCES |
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Received for publication August 15, 2005. Accepted for publication September 26, 2005.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONCONCLUSIONSACKNOWLEDGEMENTSREFERENCES |
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) lactic acid bacteria counts during ripening of the 8 cheeses (mean of 3 replicates). Treatments: HHH = high Ca and P, high lactose, and high salt-to-moisture (S/M); HHL = high Ca and P, high lactose, and low S/M; HLH = high Ca and P, low lactose, and high S/M; HLL = high Ca and P, low lactose, and low S/M; LHH = low Ca and P, high lactose, and high S/M; LHL = low Ca and P, high lactose, and low S/M; LLH = low Ca and P, low lactose, and high S/M; LLL = low Ca and P, low lactose, and low S/M. Data for cheese curds before salting are not shown.