• Asian-Australasian Journal of Animal Sciences
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• v.23 no.10
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• pp.1369-1379
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• 2010

This study investigated purification and milk-clotting activity of the enzymes produced by Bacillus subtilis var, natto and Rhizopus oligosporus compared with that of commercial rennet. The clotting time, viscosity, tension and microstructure of the curd and electrophoretic patterns of milk proteins were determined. The milk-clotting activity/proteolytic activity ratios (MCA/PA ratio) of B. subtilis, R. oligosporus and commercial rennet were also compared. The results revealed that the curd formed by the commercial rennet had the highest viscosity and curd tension and the shortest clotting time among the three enzymes. However, curd produced by Rhizopus enzymes was ranked as second. From the MCA/PA ratio and electrophoretogram analyses it could be concluded that the enzyme produced by B. subtilis had the highest proteolytic activity, while the commercial rennet had the highest milk-clotting activity. Observations of microstructures of SEM showed that the three-dimensional network for curd formed by commercial rennet was denser, firmer and more smooth. The milk-clotting activity, specific activity, purification ratio and recovery of the purified enzymes produced by both the tested organisms were also determined with ion exchange chromatography and gel filtration.

• Journal of Animal Science and Technology
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• v.44 no.6
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• pp.801-808
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• 2002

Proteolytic activities of some commercial milk clotting enzymes(rennet, trypsin, pepsin, papain W-40, neutrase 1.5 and protease S) in bovine skim milk containing 0.02% $CaCl_2$ were determined by measuring DH(Degree of Hydrolysis), NPN(Non Protein Nitrogen) and by comparing patterns of SDS-PAGE(Sodium Dodecyl Sulphate Polyacrylamide Gel Electrophoresis). The DH of microbial enzymes(neutrase 1.5 and protease S) and trypsin in bovine skim milk were higher than those of pepsin and papain W-40. The amounts of NPN in the milk treated with trypsin and the other animal enzymes(rennet and pepsin) showed the highest and lowest degrees of proteolysis, respectively. SDS-PAGE showed that trypsin and protease S hydrolyzed $\alpha$-lactalbumin and papain W-40 hydrolyzed $\beta$-lactoglobulin slightly, while neutrase 1.5 hydrolyzed both $\alpha$-lactalbumin and $\beta$-lactoglobulin after treating for 90 min. Trypsin and protease S easily hydrolyzed ${\alpha}_s$-casein and $\beta$-casein, which were not hydrolyzed by rennet. Papain W-40 hydrolyzed $\kappa$-casein more than rennet as shown in SDS-PAGE. Based on the results of the experiments, the DH and NPN of trypsin, neutrase 1.5 and protease S were shown to be higher than those of the other enzymes. The SDS-PAGE patterns of papain W-40 and neutrase 1.5 were similar with that of rennet.

• Korean Journal of Agricultural Science
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• v.7 no.2
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• pp.190-201
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• 1980

This experiment was designed to optimize the process of manufacturing the soybean cheeses and to elucidate the chemical changes during ripening when the chemical changes during ripening when the milk components and enzyme preparations were added to the raw materials. Conditions for extracting soybean protein such as temperature, duration and amount of water added were determined; various coagulaters were compared by checking the curd texture and yield; starters from S. thermophilus, S. lactis MLB and S. cremoris EB-9 were tested as single- or multi-stain combinations; and the effects of skim milk and/or rennins-both microbial and calf origin-addition upon the process of manufacturing and ripening were studied. The results obtained were as follows. 1. optimal conditions for soybean extraction were found to be: temperature $100^{\circ}C$, duration 10 minutes, and amount of water added 9-fold, as considered the extraction rate of solids and proteins, and curd yield. 2. Sodium gluconate was the most effective among the coagulators tested, and 5% of single-strain starter from S. thermophilus was appered to be adequate inoculum for curd formation. 3. The effects of skim milk and/or rennins addition on the process of manufacturing and ripening of soybean cheeses were: 1) The addition of rennins resulted in fast formation of curd, especially with skim milk it was so. And Hansen rennet extracts brought better results in curd formation than Meito rennet extracts did. 2) No significant effect was observed on the changes in moisture content during ripening, however the levels of moisture contents in the products were higher in case of using Meito rennet extracts. 3) Effect on pH changes during ripening was also not significant in general, while levels of pH were decrease markedly during manufacturing and the initial stage of ripening. 4) The levels of bacterial counts were much higher in case of skim milk addition throughtout the ripening period. In general the numbers were reached to approximately $10^8cells/g$ during manufacturing, then decreased gradually to below $10^2cells/g$ in 8 weeks of ripening. 5) The addition of skim milk and/or rennin resulted in higher ripening index, and skim milk plus Meito rennet extracts was appeared to be best combination for the ripening index.

• Korean Journal of Food Science and Technology
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• v.20 no.2
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• pp.245-251
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• 1988

Thermolysin was immobilized on Dowex MWA-1 with 10% glutaraldehyde and incorpo rated into a fluidized-bed continuous coagulation scheme to make Cheddar type cheese. The activity yield of thermolysin was 25%. The immobillized thermolysin was stable at $60^{\circ}C$ in the presence of 1/200M calcium ions and the half-life value is 16 days at the temperature. Raw milk alkalified to pH 7.0 was passed through a column of thermolysin beads at $55^{\circ}C$, cultivated with Streptococcus cremoris and allowed to coagulate. A typical milk curd was formed to make Cheddar type cheese, avoiding troublesome microbial contamination successfully during continuous hydrolysis process. During ripening of this cheese for 6 months at $10^{\circ}C$, its ripening ratio and taste were similar to those of cheese prepared by the traditional method.