Thermal inactivation of chymosin during cheese manufacture

The aspartic proteinase, chymosin (EC 3.4.23.4) is the principal milk clotting enzyme used in cheese production and is one of the principal proteolytic agents involved in cheese ripening. Varietal differences in chymosin activity, due to factors such as cheese cooking temperature, fundamentally influence cheese characteristics. Furthermore, much chymosin is lost in whey, and further processing of this by-product may require efficient inactivation of this enzyme, with minimal effects on whey proteins. In the first part of this study, the thermal inactivation kinetics of Maxiren 15 (a recombinant chymosin preparation) were studied in skim milk ultrafiltration permeate, whole milk whey and skim milk whey. Inactivation of chymosin in these systems (at pH 6.64) followed first order kinetics with a D45.5 value of 100 +/- 21 min and a z-value of 5.9 +/- 0.3 degrees C. D-Values increased linearly with decreasing pH from 6.64 to 6.2, while z-values decreased as pH decreased from 6.64 to 6.4, but were similar at pH 6.4 and 6.2. Subsequent determination of chymosin activity during manufacture of Cheddar and Swiss-type cheese showed good correlations between predicted and experimental values for thermal inactivation of chymosin in whey. However, both types of cheese curd exhibited relatively constant residual chymosin activity throughout manufacture, despite the higher cooking temperature applied in the manufacture of Swiss cheese. Electrophoretic analysis of slurries made from Cheddar and Swiss cheese indicated decreased proteolysis due to chymosin activity during storage of the Swiss cheese slurry, but hydrolysis of sodium caseinate by coagulant extracted from both cheese types indicated similar levels of residual chymosin activity. This may suggest that some form of conformational change other than irreversible thermal denaturation of chymisin takes place in cheese curd during cooking, or that some other physico-chemical difference between Swiss and Cheddar cheese controls the activity of chymosin during ripening.     

Isolation,purification and characterization of chymosin from riverine buffalo (Bubalos bubalis)

Chymosin, an aspartyl proteinase, is used for curdling of milk and manufacture of cheese. We report the purification and the physicochemical properties of chymosin isolated from the abomasal tissue of buffalo calves. The enzyme preparation extracted from buffalo abomasal tissues could be purified 29-fold using anion exchange and gel filtration chromatography. The molecular weight of the purified enzyme was 35.6 kDa on SDS-PAGE. Partial N-terminal amino acid sequence of the first eight amino acid sequences of buffalo chymosin was identical to the first eight amino acid sequences of cattle chymosin. Buffalo chymosin exhibited a skewed bell-shaped stability profile as a function of temperature with maximum activity near 55 degrees C. Milk clotting activity decreased gradually as pH increased. The enzyme became completely inactive, however, above pH 7.0. The ratio of milk clotting to proteolytic activity was 3.03. When compared with cattle chymosin, there were subtle differences in the stability and relative proteolytic activity of buffalo chymosin.     

微小毛霉与米黑毛霉凝乳酶的制备及其凝乳质构特性研究

将经乙醇分步沉淀所制得的微小毛霉和米黑毛霉凝乳酶以小牛皱胃酶为对照在不同温度、不同时间和不同pH值下凝乳,以下压过程的平均力为衡量标准,对其物性进行了比较,得出在相同条件下,米黑毛霉凝乳酶的凝乳要好于微小毛霉凝乳酶,并在高温和酸性环境要比微小毛霉凝乳酶稳定。     

Influence of pH on retention of camel chymosin in curd

Retained coagulant in cheese initiates casein breakdown and influences cheese structure and flavour formation. This study investigated the influence of milk pH on retention of camel chymosin in curd and compared it with bovine chymosin. Milk at five different pH levels was coagulated with same coagulant activity of each chymosin and centrifuged. Chymosin activity in whey was determined using the synthetic peptide Pro-Thr-Glu-Phe-(NO₂-Phe)-Arg-Leu as substrate and HPLC analysis of the resulting product. Camel chymosin had 2.7 times lower activity in milk than bovine chymosin at the same coagulation activity. The retention of camel chymosin in curd was rather constant at ∼20% between pH 6.65 and 6.00, while it increased almost linear from 2 to 21% for bovine chymosin. The lower pH dependence for retention of camel chymosin than of bovine chymosin may be explained by a lower negative charge of the camel chymosin molecule.     

Evaluation of Thiol Activated Proteases from Clam Viscera as a Rennet Substitute for Cheese-Making

Clam rennet, which is a crude enzyme preparation of cathepsin B-like protease from clam viscera was characterized and compared to porcine pepsin and calf rennet for its suitability as a milk coagulant in cheesemaking. Clam rennet was more proteolytic and produced a softer curd than the other two coagulants. However, influences of the pH and temperature on milk clotting with clam rennet were very similar to those of calf rennet. The cheddar cheese made from clam rennet was not inferior to the Cheddar cheese made from calf rennet. Quality enhancement occurred despite the view that high ratio of proteolytic to clotting activity is generally considered to be unfavorable for cheese-making. The higher proteolytic activity appeared to accelerate the ripening process. A small yield loss occurred as a result of excessive proteolysis during cheese-making.     

Estimation of Residual Pepsin and Chymosin Activity in Curd,

Pasteurized milk (225 g) adjusted to pH 6.2 was set with 3.5 milk clotting units of chymosin (EC 3.4.23.4). The same amount of milk at pH 5.8 was set with 3.5 milk clotting units of porcine pepsin (EC 3.4.23.1). Fifteen minutes after clotting, the curd was broken, and curd and whey were separated by centrifugation at 3500 × g for 20 min. The curd (30 g) was extracted at pH 6.8 in 450 ml water or at pH 6.2 (chymosin) or 5.8 (pepsin) in 450 ml 1 M sodium chloride.Chymosin was completely released from the curd and accounted for by both methods of extraction. Pepsin was completely released and accounted for after extraction in 1 M sodium chloride at pH 5.8 but was partly inactivated during extraction at pH 6.8.Assay of curd extracts and whey by a linear agar diffusion test accounted for 102 ± 6% of the pepsin activity added to milk when the curd was extracted in 1 M sodium chloride. Extraction at pH 6.8 allowed recovery of only 63% of the activity. Chymosin recovery was 100 ± 5% by both methods of curd extraction.     

Variation in Coagulation Properties of Milk from Individual Cows,

Milk samples from 50 Holstein cows were tested monthly for 10 mo for total protein, casein, fat, somatic cells, and pH. A Formagraph was used to measure chymosin coagulation properties. Significant variations in coagulation time and curd firmness were observed in relation to period of lactation, individual cows, and milk pH. A high negative correlation coefficient (?.86) was observed between coagulation time and curd firmness measured 30 min after addition of chymosin. The mean coagulation time generally increased as lactation progressed and milk yield decreased. Curd firmness was generally greatest in midlactation samples.Milk from 38% of the cows did not coagulate in 30 min 1 mo prior to their dry periods. The frequency of failure to coagulate was 68% in winter and 32% in fall. Milk pH was the most significant factor that affected coagulation time and curd firmness. Simulated cheese making procedures were utilized to estimate recovery of fat and protein in curd. Curd yield calculated from the recovery data ranged from 5.4 to 14% with a mean of 9.2%.     

Effect of rennet coagulation time on composition, yield, and quality of reduced-fat cheddar cheese

This study compared the effect of coagulum firmness at cutting on composition of 50% reduced-fat Cheddar cheese. Coagulum firmness was determined by subjective evaluation by the cheese maker. Three firmness levels were tested, and these corresponded to average times of coagulant addition to cutting the curd of 25, 48, and 65 min. A slow acid-producing culture was used, and ripening times were altered to give similar curd pH values throughout cheese making. A longer rennet coagulation time (firmer coagulum at cutting) resulted in an increase in cheese moisture as well as an increase in cheese yield. The percentages of fat recovered in the cheese decreased with increasing curd firmness. The percentage of nitrogen recovered in the cheese was similar among the treatments. The amount of whey collected from the curd after milling increased as the coagulum firmness at cutting increased. Higher moisture content and lower pH of cheese made from the firmer curd at cutting contributed to softer, smoother-bodied cheeses, but the Cheddar flavor intensity was not affected.     

Culture Filtrates from a Fermented Rice Product (Lao-Chao) and Their Effects on Milk Curd Firmness

To develop a new Oriental-style dairy product, the characteristics of culture filtrate from lao-chao with Rhizopus oryzae, used as a milk-clotting agent, and factors (heat-treatments, calcium, sucrose, and curdling temperature) affecting curd firmness were determined. The optimal conditions for proteolytic activity were around 40°C and pH 3. No activity was detected over the range of pH 8 to 11. After high-heat treatment (121°C, 15 min) on skim milk, no clotting was observed. Sucrose resulted in the retardation of milk-clotting. Ca⁺⁺ could be used to increase curd firmness which also increased from 29. 2–2.8g to 80. 3–4.7g when the curdling temperature was increased from 25°C to 45°C.     

Effects of pH, calcium chloride, and chymosin concentration on coagulation properties of abnormal and normal milk

Individual Holstein cow milk samples were selected for good and poor chymosin-coagulation characteristics. The effect of pH adjustment, addition of .02% calcium chloride, and variation in chymosin concentration on coagulation properties of good and poor coagulating samples was evaluated. Pooling 50% good and 50% poor coagulating samples did not improve the average coagulation properties of the poor samples. Reducing milk pH to 6.3 caused a significant decrease in coagulation time but a less marked increase in curd firmness. The greatest increase in curd firmness was obtained by a combination of reducing milk pH, addition of .02% calcium chloride, and reducing chymosin concentration. High-chymosin concentration at reduced pH decreased coagulation time without substantially increasing curd firmness. Curd disintegration was more apparent at high-chymosin concentration in the poor coagulating samples.     

Survival of Listeria monocytogenes during the manufacture and ripening of Swiss cheese.

Rindless Swiss cheese was made from a mixture of pasteurized whole and skim milk that was inoculated to contain 10(4) to 10(5) cfu of Listeria monocytogenes (strain Ohio, California, or V7)/ml. During clotting of milk, numbers of L. monocytogenes remained nearly unchanged. When the curd was heated gradually to attain the cooking temperature (50 degrees C), numbers of L. monocytogenes increased by approximately 40 to 45% over those in inoculated milk. Cooking curd at 50 degrees C (122 degrees F) for 30 to 40 min resulted in resilient curd having a pH of 6.40 to 6.45 and decreased L. monocytogenes by 48% compared with numbers of the pathogen in inoculated milk. After curd was pressed under whey, numbers of L. monocytogenes increased by approximately 52% over those in inoculated milk and reached their maxima at the end of this stage. A sharp decrease in numbers of L. monocytogenes occurred during brining of cheese blocks (7 degrees C for 30 h). The population of L. monocytogenes continued to decrease during cheese ripening. Average D values for strains California, Ohio, and V7 were 29.2, 24, and 22.5 d, respectively. Listeria was not detected (direct plating, and cold enrichment) after 80, 77, and 66 d of ripening of Swiss cheese made from milk inoculated with strains California, Ohio, and V7, respectively. Thus, Swiss cheese made in this study did not permit extended survival of L. monocytogenes.     

Onopordum acanthium L. (Asteraceae) flowers as coagulating agent for cheesemaking

By trituration in liquid nitrogen, homogenization in 0.1 mol/L citric acid–sodium citrate buffer (pH 3.0) containing 1.0 mmol/L EDTA, and centrifugation (10000 × g) of fresh Onopordum acanthium L. (Asteraceae) flowers, a proteolytic preparation (“onopordosin”) was obtained (pI 4.4), which showed proteolytic and clotting activity (0.546 ± 0.004 Rennet Units/g flowers and 0.090 ± 0.003 Endopeptidase Units/g flowers, respectively) at acid pH values, conditions that favor its use in cheese manufacturing. Hydrolytic behavior (SDS-Tricine PAGE) of onopordosin was compared with that of chymosin in bovine milk clotting assays. Onopordosin was used as vegetal coagulant with pasteurized bovine milk to manufacture a semi-hard type cheese which was analyzed by sensory panelists at the Experimental Superior Institute of Food Technology in Argentina, who evaluated different parameters and concluded that the quality of the cheese made with onopordosin was similar to that of commercial cheeses.     

Effect of camel chymosin on the texture,functionality, and sensory properties of low-moisture,part-skim Mozzarella cheese

The objective of this study was to compare the effect of coagulant (bovine calf chymosin, BCC, or camel chymosin, CC), on the functional and sensory properties and performance shelf-life of low-moisture, part-skim (LMPS) Mozzarella. Both chymosins were used at 2 levels [0.05 and 0.037 international milk clotting units (IMCU)/mL], and clotting temperature was varied to achieve similar gelation times for each treatment (as this also affects cheese properties). Functionality was assessed at various cheese ages using dynamic low-amplitude oscillatory rheology and performance of baked cheese on pizza. Cheese composition was not significantly different between treatments. The level of total calcium or insoluble (INSOL) calcium did not differ significantly among the cheeses initially or during ripening. Proteolysis in cheese made with BCC was higher than in cheeses made with CC. At 84 d of ripening, maximum loss tangent values were not significantly different in the cheeses, suggesting that these cheeses had similar melt characteristics. After 14 d of cheese ripening, the crossover temperature (loss tangent = 1 or melting temperature) was higher when CC was used as coagulant. This was due to lower proteolysis in the CC cheeses compared with those made with BCC because the pH and INSOL calcium levels were similar in all cheeses. Cheeses made with CC maintained higher hardness values over 84 d of ripening compared with BCC and maintained higher sensory firmness values and adhesiveness of mass scores during ripening. When melted on pizzas, cheese made with CC had lower blister quantity and the cheeses were firmer and chewier. Because the 2 types of cheeses had similar moisture contents, pH values, and INSOL Ca levels, differences in proteolysis were responsible for the firmer and chewier texture of CC cheeses. When cheese performance on baked pizza was analyzed, properties such as blister quantity, strand thickness, hardness, and chewiness were maintained for a longer ripening time than cheeses made with BCC, indicating that use of CC could help to extend the performance shelf-life of LMPS Mozzarella.     

The influence of milk pasteurization temperature and pH at curd milling on the composition, texture and maturation of reduced fat cheddar cheese

Reduced fat milks were pasteurized, for 15 s, at temperatures ranging from 72 to 88°C to give levels of whey protein denaturation varying from ˜ 3 to 35%. The milks were converted into reduced fat cheddar cheese (16–18% fat) in 500 litre cheese vats; the resultant cheese curds were milled at pH values of 5.75 and 5.35. Raising the milk pasteurization temperature resulted in impaired rennet coagulation properties, longer set-to-cut times during cheese manufacture, higher cheese moisture and moisture in the non-fat cheese substance, lower levels of protein and calcium and lower cheese firmness. Increasing the pH at curd milling from 5.35 to 5.75 affected cheese composition and firmness, during ripening, in a manner similar to that of increasing milk pasteurization temperature. Despite their effects on cheese composition and rheology, pasteurization temperature and pH at curd milling had little influence on proteolysis or on the grading scores awarded by commercial graders during ripening over 303 days .     

Interactions of Calcium,pH, Temperature,and Chymosin During Milk Coagulation,

Holstein milk samples with good and poor chymosin-coagulation characteristics were coagulated in the Formagraph using different combinations of five levels of chymosin, three pH, and three temperatures in the presence and absence of .02% added calcium chloride.All the main factors significantly altered both coagulation time and curd firmness. Multiple comparisons of mean coagulation times showed that lower concentrations of chymosin (.01, .02, and .03 rennin units/ml milk) were significantly different in altering coagulation time and were different from higher concentrations (.04 and .05 rennin units/ml milk). The three pH produced significantly different mean coagulation times. Addition of more than .02 rennin units/ml milk was not necessary for adequate curd firmness in 30 min after chymosin addition where the pH of the milk was 6.4 or lower. Addition of .02% calcium chloride to milk was not necessary for adequate curd firmness 30 min after chymosin addition if other milk coagulation factors were adequately adjusted (pH ≤ 6.4; chymosin concentration = .02 rennin unit/ml milk; temperature = 37°C).     

Interaction between sodium chloride and texture in semi-hard Danish cheese as affected by brining time,dl-starter culture,chymosin type and cheese ripening

Reduced NaCl in semi-hard cheeses greatly affects textural and sensory properties. The interaction between cheese NaCl concentration and texture was affected by brining time (0–28 h), dl-starter cultures (C1, C2, and C3), chymosin type (bovine or camel), and ripening time (1–12 weeks). Cheese NaCl levels ranged from <0.15 to 1.90% (w/w). NaCl distribution changed during ripening; migration from cheese edge to core led to a more homogeneous NaCl distribution after 12 weeks. As ripening time increased, cheese firmness decreased. Cheeses with reduced NaCl were less firm and more compressible. Cheeses produced with C2 were significantly firmer than those produced with C1; cheeses produced with C3 had higher firmness and compressibility. In NaCl reduced cheese, use of camel chymosin as coagulant resulted in significantly higher firmness than that given using bovine chymosin. Overall, cheese NaCl content is reducible without significant textural impact using well-defined starter cultures and camel chymosin.     

Effects of Somatic Cell Counts and Milk Composition on the Coagulating Properties of Milk

Forty-two Holstein cows were selected to provide monthly milk samples with varying SCC for 1 yr. Coagulating properties of samples measured as rennet clotting time, rate of curd firming, and curd firmness at cutting were determined by a formagraph. Milk samples were analyzed for fat, protein, lactose, total solids, casein, individual caseins, urea, SCC, and pH. Least squares analyses of data, after adjustments were made for the effect of milk composition, indicated that elevated SCC were associated with a significant increase in rennet clotting time and slower rate of curd firming. An increase of SCC from 100,000 to 500,000 SCC/ml resulted in an increase of approximately 2.1 and 2.2% in RCT and K20, respectively. A further increase of SCC to above 1,000,000/ml resulted in an overall increase of 20.7 and 13.84% in RCT and K20, respectively. Regression analyses indicated that K20 was decreased by 5.42 min and curd firmness at cutting was increased by 12.92 mm for every percentage in milk casein. Rennet clotting time, rate of curd firming, and curd firmness at cutting were increased by 3.52, 3.41, min and decreased by 9.45 mm, respectively, for every unit increase in milk pH.     

Effect of Heat-Stable Proteases on the Kinetic Parameters of Milk Clotting by Chymosin

The milk clotting per unit casein hydrolytic activities of proteases from 14 psychrotrophic pseudomonad isolates of raw milk ranged from 0.77 to 9.97 at 30°C. The milk clotting activities of chymodn and T16 protease were not completely additive, especially at high chymosin concentration when clotting time was relatively fast. The T16 protease was not effective in catalyzing the enzymic step of milk clbtting at O°C in the time expected on the basis of its milk clotting at 30°C. Milk incubated with the T25 motease for 8 days at 4°C and then clotted with chymosin at 30°C exhibited weak curd consistency.     

Estimation of genetic parameters for cheese-making traits in Spanish Churra sheep

The global production of sheep milk is growing, and the main industrial use of sheep milk is cheese making. The Spanish Churra sheep breed is one of the most important native dairy breeds in Spain. The present study aimed to estimate genetic parameters for a wide range of traits influencing the cheese-making ability of Churra sheep milk. Using a total of 1,049 Churra ewes, we studied the following cheese-making traits: 4 traits related to milk coagulation properties (rennet coagulation time, curd-firming time, and curd firmness at 30 and 60 min after addition of rennet), 2 traits related to cheese yield (individual laboratory cheese yield and individual laboratory dried curd yield), and 3 traits measuring curd firmness over time (maximum curd firmness, time to attain maximum curd firmness, and syneresis). In addition, a list of milk traits, including the native pH of the milk and several milk production and composition traits (milk yield; the fat, protein, and dried extract percentages; and the somatic cell count), were also analyzed for the studied animals. After discarding the noncoagulating samples (only 3.7%), data of 1,010 ewes were analyzed with multiple-trait animal models by using the restricted maximum likelihood method to estimate (co)variance components, heritabilities, and genetic correlations. In general, the heritability estimates were low to moderate, ranging from 0.08 (for the individual laboratory dried curd yield trait) to 0.42 (for the fat percentage trait). High genetic correlations were found within pairs of related traits (i.e., 0.93 between fat and dried extract percentages, ?0.93 between the log of the curd-firming time and curd firmness at 30 min, 0.70 between individual laboratory cheese yield and individual laboratory dried curd yield, and ?0.94 between time to attain maximum curd firmness and syneresis). Considering all the information provided here, we suggest that in addition to the current consideration of the protein percentage trait for improving cheese yield traits, the inclusion of the pH of milk as a measured trait in the Churra dairy breeding program would represent an efficient strategy for improving the cheese-making ability of milk from this breed.     

Primary proteolysis studied in a cast cheese made from microfiltered milk

Primary proteolysis was studied in a starter-free cheese model made from microfiltered (MF) milk (19.0% casein,<0.2% whey proteins). Specificity of plasmin and chymosin activity was investigated in the pH range 5.0–6.0, by analysis of peptide composition using high-performance liquid chromatography and liquid chromatography–mass spectrometry. Hydrolysis experiments with purified caseins were performed to aid identification of peptides released by specific activities. Plasmin had no activity in cheese below pH 5.4, while its activity increased from pH 5.4 to 6.0. Chymosin activity on the Phe₂₃–Phe₂₄ bond of α_S1-casein had an optimum pH around 5.3, while release of the bitter peptide β-casein (f193–209), effected by chymosin, was highest at pH 6.0. At pH <5.3, the specificity of chymosin on α_S1-casein changed, and the peptide bond Leu₂₀–Leu₂₁ was cleaved at an increasing rate with decreasing pH. Demineralisation of the MF retentate generally increased proteolytic activity.