Comparison of Milk-clotting Activity of Proteinase Produced by Bacillus Subtilis var, natto and Rhizopus oligosporus with Commercial Rennet

  • Chen, Ming Tsao (Department of Bioindustry Technology, Da Yeh University) ;
  • Lu, Ying Yu (Department of Bioindustry Technology, Da Yeh University) ;
  • Weng, Tien Man (Department of Bioindustry Technology, Da Yeh University)
  • Received : 2008.12.17
  • Accepted : 2009.07.11
  • Published : 2010.10.01


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.


  1. Arima, K., J. Yu and S. Iwasaki. 1970. Milk-clotting enzyme from Mucor pursillus var. Lindt. In: Methods in enzymology, Vol. 19, (Ed. G. E. Perlmann and L. Lorand). pp. 446-459. Academic Press, New York.
  2. Babbar, I. J., R. A. Scrinivasan, S. C. Chakravorty and A. T. Dudani. 1965. Microbial rennet substitutes-a review. Indian J. Dairy Sci. 18:89-95.
  3. Berkowitz-Hundert, R., J. Leibowitz and J. Ilany-Feigenbaum. 1964. Researches on milk-clotting enzymes from Palestinian plant sources. Enzymologia 27:332-342.
  4. Bernard, F. G., Z. Alexandre, M. Robert and M. Catherine. 2004. Production and characterization of bioactive peptides from soy hydrolysate and soy-fermented food. Food Res. Intern. 37:123-131.
  5. Bradford, M. M. 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72:248-254.
  6. Cheryan, M., P. J. van Wyk, N. F. Olson and T. Richardson. 1975. Secondary phase and mechanism of enzymatic milk coagulation. J. Dairy Sci. 58:477-481.
  7. Dalgleish, D. G. and A. J. R. Law. 1988. pH-induced dissociation of bovine casein micelles. I. Analysis of liberated caseins. J. Dairy Res. 55:529-538.
  8. Harlow, E. and D. Lane. 1988. Antibodies. pp. 636-69, pp. 685. Cold Spring Harbor Laboratory, New Yor.k, USA.
  9. Huang, M. H. and M. Y. Chen. 2006. Effects of addition of activated carbon and chitosan on characteristics of yoghurt. J. Chin. Soc. Anim. Sci. 35:187-200.
  10. Knight, S. G. 1966. Production of a rennin-like enzyme by molds. Can. J. Microbiol. 12:420-422.
  11. Labropoulos, A. E., W. F. Collins and W. K. Stone. 1984. Effects of ultra-high temperature and vat processes on heat-induced rheological properties of yogurt. J. Dairy Sci. 67:405-409.
  12. Lagoueyte, N., J. Lablee and B. Tarodo de la Fuente. 1994. Temperature affects microstructure of renneted milk gel. J. Food Sci. 59:956-959.
  13. McMahon, J. D. and R. J. Brown. 1984. Enzymatic coagulation of casein micelles: a review. J. Dairy Sci. 67:919-929.
  14. Modler, H. W., M. E. Larmond, C. S. Lin, D. Froehich and B. Emmons. 1983. Physical and sensory properties of yogurt stabilized with milk proteins. J. Dairy Sci. 66:422-429.
  15. Parnll-Clunies, E. M. Y. Kauda and J. M. Deman. 1986. Influence of heat treatment of milk on the flow properties of yogurt. J. Food Sci. 51:1459-1462.
  16. Preetha, S. and R. Boopathy. 1997. Purification and characterization of a milk clotting protease from Rhizomucor miehei. World J. Microbiol. Biotechnol. 13:573-578.
  17. Puhan, Z. and D. M. Irvine. 1973. Reduction of proteolytic activity of B. subtilis proteases by acidification of milk cheddar cheese manufacture. J. Dairy Sci. 56:323-327.
  18. Rao, L. Krishna and D. K. Mathur. 1979. Assessment of purified bacterial milk clotting enzyme from bacillus subtilis K-26 for cheddar cheese making. J. Dairy Sci. 62:378-383.
  19. Richardson, G. H., J. H. Nelson, R. E. Lubnow and R. L. Schwarberg. 1967. Rennin-like enzyme from Mucor pusillus for cheese manufacture. J. Dairy Sci. 50:1066-1072.
  20. Sardinas, J. L. 1969. Milk-curdling enzyme elaborated by Endothia parasitica. US. Plant 316:275-453.
  21. Sardinas, J. L. 1972. Microbial rennets. Adv. Appl. Microl. 15:39-73.
  22. Seker, S., H. Beyenal and A. Tanyolac. 1999. Modeling milk clotting activity in the continuous production of microbial rennet from Mucor miehei. J. Food Sci. 3:525-529.
  23. Sushil Kumara, Neeru S. Sharmaa, Mukh R. Saharanb and Randhir Singh. 2005. Extracellular acid protease from Rhizopus oryzae: purification and characterization. Process Biochem. 40:1701-1705.
  24. Wang, H. L., D. I. Ruttle and C. W. Hesseltine. 1969. Milk-clotting activeity of proteinases produced by Rhizopus. Can. J. Microbiol. 15:99-104.

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