Biotechnology and Bioprocess Engineering

, Volume 14, Issue 6, pp 788–794 | Cite as

Production of milk clotting protease by a local isolate of Mucor circinelloides under SSF using agro-industrial wastes

  • R. Sathya
  • B. V. Pradeep
  • J. Angayarkanni
  • M. Palaniswamy


Agro-industrial residues, a cheap source of energy have high potential in the area of fermentation for the production of enzymes. Twenty agro-industrial residues were evaluated to check the possibility of potential utilization of substrates in SSF for milk clotting enzyme protease production by Mucor circinelloides. In this study, dhal husk holds the greatest promise for cost effective production of the milk clotting enzyme. The dhal husk supported maximum milk clotting protease production, and yield was improved with the supplementation of sucrose and yeast extract as carbon and nitrogen source, respectively. Among all the physico-chemical parameters tested, the best results were obtained in a medium having moisture content of 20% at pH 7.0, when inoculated with 30% of spore suspension and incubated at 30°C for 5 days. The activity was increased further on addition of Ca2+, Cu2+, and Mg2+ ions. The purified milk-clotting protease obtained from M. circinelloides was successfully applied and compared with commercial rennet in the manufacture of a cheddar cheese.


dhal husk agro-industrial residues milk-clotting protease solid-state fermentation cheese production 


Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.


  1. 1.
    Rao, M. B., A. M. Tanksale, M. S. Ghatge, and V. V. Deshpande (1998) Molecular and biotechnological aspects of microbial proteases. Microbiol. Mol. Biol. Rev. 62: 597–635.Google Scholar
  2. 2.
    Manachini, P. L. and M. G. Fortina (1998) Production in sea-water of thermostable alkaline protease by a halotolerant strain of Bacillus licheniformis. Biotechnol. Lett. 20: 565–568.CrossRefGoogle Scholar
  3. 3.
    Moreira, K. A., T. S. Porto, M. F. S. Teixeira, A. L. F. Porto, and J. L. Lima Filho (2003) New alkaline protease from Nocardiopsis sp.: partial purification and characterization. Process Biochem. 39: 67–72.CrossRefGoogle Scholar
  4. 4.
    Tunga, R., B. Shrivastava, and R. Banerjee (2003) Purification and characterization of protease from solid state culture of Aspergillus parasiticus. Process Biochem. 38: 1553–1558.CrossRefGoogle Scholar
  5. 5.
    Jarai, G., H. van den Hombergh, and F. B. Buxton (1994) Cloning and characterization of the pepE gene of Aspergillus niger encoding a new aspartic protease and regulation of pepE and pepC. Gene 145: 171–178.CrossRefGoogle Scholar
  6. 6.
    Young, J. W., A. Wadeson, D. J. Glover, R. V. Quincey, M. J. Butlin, and E. A. Kamei (1996) The extracellular acid protease of Yarrowia lipolytica: sequence and pH-regulated transcription. Microbiol. 142: 2913–2921.CrossRefGoogle Scholar
  7. 7.
    vankuyk, P. A., B. F. Cheetham, and M. E. Kate (2000) Analysis of two Aspergillus nidulans genes encoding extracellular proteases. Fungal Genet. Biol. 29: 201–210.CrossRefGoogle Scholar
  8. 8.
    Sousa, M. J. and F. X. Malcata (2002) Advances in the role of a plant coagulant (Cynara cardunculus) in vitro and during ripening of cheeses from several milk species. Lait 82: 151–170.CrossRefGoogle Scholar
  9. 9.
    Tsugo, T., U. Yoshino, K. Taniguchi, A. Ozawa, Y. Miki, S. Iwasaki, and K. Arima (1964) Cheese-making by using the milk-clotting enzyme of Mucor pusillus. Lindt. 1. Rennet properties of the enzyme. Jap. J. Zootech. Sci. 35: 221–228.Google Scholar
  10. 10.
    Abdel-Fattah, A. F., A. S. Ismail, and S. A. El Aassar (1984) Production of rennin like enzyme by Absidia cylindrospora. Agric. Wastes. 11: 125–131.CrossRefGoogle Scholar
  11. 11.
    Ismail, A. M. S., S. A. El Aassar, and A. F. Abdel-Fattah (1984) Production of milk clotting and proteolytic enzymes by fungi. Agric. Wastes. 10: 95–102.CrossRefGoogle Scholar
  12. 12.
    Hashem, A. M. (2000) Purification and properties of a milk-clotting enzyme produced by Penicillium oxalicum. Bioresour. Technol. 75: 219–222.CrossRefGoogle Scholar
  13. 13.
    Cavalcanti, M. T. H., M. F. S. Teixeira, J. L. Lima Filho, and A. L. F. Porto (2004) Partial purification of new milk-clotting enzyme produced by Nocardiopsis sp. Bioresour. Technol. 93: 29–35.CrossRefGoogle Scholar
  14. 14.
    Tubesha, Z. A. and K. S. Al Delaimy (2003) Renin like milk coagulant enzyme produced by a local isolate of Mucor. Int. J. Dairy Technol. 56: 237–341.CrossRefGoogle Scholar
  15. 15.
    Couto, S. R. and M. A. Sanroman (2006) Application of solid-state fermentation to food industry-a review. J. Food Eng. 76: 291–302.CrossRefGoogle Scholar
  16. 16.
    Holker, U. and J. Lenz (2005) Solid-state fermentation — are there any biotechnological advantages? Cur. Opin. Microbiol. 8: 301–306.CrossRefGoogle Scholar
  17. 17.
    Krishna, C. (2005) Solid-state fermentation systems-an overview. Cri. Rev. Biotechnol. 25: 1–30.CrossRefGoogle Scholar
  18. 18.
    Nagampoothiri, K. M. and A. Pandey (1996) Solid state fermentation for L-glutamic acid production using Brevibacterium sp. Biotechnol. Lett. 18: 199–204.CrossRefGoogle Scholar
  19. 19.
    Roukas, T. (1999) Citric acid production from pod by solid-state fermentation. Enz. Microbiol. Biotechnol. 24: 54–59.CrossRefGoogle Scholar
  20. 20.
    Vanderberghe, L. P. S., C. R. Soccol, A. Pandey, and J. M. Lebeault (2000) Citric acid production by Aspergillus niger in solid state fermentation. Bioresour. Technol. 74: 175–178.CrossRefGoogle Scholar
  21. 21.
    Pandey, A., P. Selvakumar, C. R. Soccol, and P. Nigam (1999) Solid state fermentation for the production of industrial enzymes. Cur. Sci. 77: 149–162.Google Scholar
  22. 22.
    Selvakumar, P. and A. Pandey (1999) Solid state fermentation for the synthesis of inulinase from Staphylococcus sp. and Kluyveromyces marxianus. Process Biochem. 34: 851–855.CrossRefGoogle Scholar
  23. 23.
    Balakrishna, K. and A. Pandey (1996) Production of biologically active secondary metabolites in solid state fermentation. J. Sci. Ind. Res. 55: 365–372.Google Scholar
  24. 24.
    Ohno, A., T. Ano, and M. Shoda (1996) Use of soybean curd residue, okara, for the solid state substrate in the production of lipopeptide antibiotic, iturin A by Bacillus subtilis NB 22. Process Biochem. 31: 801–806.CrossRefGoogle Scholar
  25. 25.
    Yang, S. S. and W. J. Swei (1996) Oxytetracycline production by Streptomyces rimosus in solid state fermentation of corn-cob. W. J. Microbiol. Biotechnol. 12: 43–46.CrossRefGoogle Scholar
  26. 26.
    Kota, K. P. and P. Sridhar (1999) Solid state cultivation of Streptomyces clavuligerus for cephamycin C production. Process Biochem. 34: 325–328.CrossRefGoogle Scholar
  27. 27.
    Sekhar, C. and K. Balaraman (1998) Optimization studies on the production of cyclosporin A by solid state fermentation. Bioprocess Eng. 18: 293–296.CrossRefGoogle Scholar
  28. 28.
    Ramana Murthy, M. V., E. V. S. Mohan, and A. K. Sadhukhan (1999) Cyclosporin A production by Tolypocladium inflatum using solid state fermentation. Process Biochem. 34: 269–280.CrossRefGoogle Scholar
  29. 29.
    Tunga, R., R. Banarjee, and B. C. Bhattacharyya (1998) Optimizing some factors affecting protease production under solid state fermentation. Bioprocess Eng. 19: 187–190.CrossRefGoogle Scholar
  30. 30.
    Elibol, M. and A. R. Moreira (2005) Optimizing some factors affecting alkaline protease production by a marine bacterium Teredinobacter turnirae under solid substrate fermentation. Process Biochem. 40: 1951–1956.CrossRefGoogle Scholar
  31. 31.
    Prakasham, R. S., C. H. Subba Rao, and P. N. Sarma (2006) Green gram husk-an inexpensive substrate for alkaline protease production by Bacillus sp. in solid-state fermentation. Bioresour. Technol. 97: 1449–1454.CrossRefGoogle Scholar
  32. 32.
    Palaniswamy, M., B. V. Pradeep, R. Sathya, and J. Angayarkanni (2008) Isolation, identification, and screening of potential xylanolytic enzyme from litter degrading fungi. Afri. J. Biotech. 7: 1978–1982.Google Scholar
  33. 33.
    Arima, K., J. Yu, and S. Iwasaki (1970) Methods in Enzymology. pp. 446–459. In: G. Perlmann, L. Lorand (eds.). Milk clotting enzyme from Mucor pusillus var. Lindt. Academy Press, NY, USA.Google Scholar
  34. 34.
    Pandey, A., C. R. Soccol, P. Nigam, D. Brand, R. Mohan, and S. Roussos (2000) Biotechnological potential of coffee pulp and coffee husk for bioprocesses. Biochem Eng. J. 6: 153–162.CrossRefGoogle Scholar
  35. 35.
    Shata, H. M. A. (2005) Extraction of Milk- clotting enzyme produced by solid state fermentation of Aspergillus oryzae. Polish J. Microbiol. 54: 241–247.Google Scholar
  36. 36.
    Preetha, S. and R. Boopathy (1994) Influence of culture conditions on the production of milk clotting enzyme from Rhizomucor. W. J. Microbiol. Biotech. 10: 527–530.CrossRefGoogle Scholar
  37. 37.
    Sannabhadti, S. S. and R. A. Srinivasan (1977) Milk clotting enzymes from Abisidia ramosa. Part 1. Factors influencing production. Ind. J. Dairy Sci. 30: 331–335.Google Scholar
  38. 38.
    Thakur, M. S., N. G. Karanth, and N. Krishna (1990) Production of fungal rennet by Mucor meiehei using solid state fermentation. Appl. Microbiol. Biotechnol. 32: 409–413.CrossRefGoogle Scholar
  39. 39.
    Nehra, K. S., S. Dhillon, K. Chaudhary, and S. Randir (2002) Production of alkaline protease by Aspergillus sp. under submerged and solid substrate fermentation. Ind. J. Microbiol. 42: 43–47.Google Scholar
  40. 40.
    Moon, S. H. and S. J. Parulekar (1991) A parametric study of protease production in batch and fed batch cultures of Bacillus firmus. Biotechnol. Bioeng. 37: 467–483.CrossRefGoogle Scholar
  41. 41.
    Yu, P. J. and C. C. Chou (2005) Factors affecting the growth and production of milk clotting enzyme by Amylomyces rouxii in rice liquid medium. Food Technol. Biotechnol. 43: 283–288.Google Scholar
  42. 42.
    D’souza, T. M. and L. Pereira (1982) Production and immobilization of a bacterial milk clotting enzyme. J. Dairy Sci. 65: 2074–2081.CrossRefGoogle Scholar
  43. 43.
    Ghareib, M., H. S. Hamdy, and A. A. Khalil (2001) Production of intracellular milk clotting enzyme in submerged cultures of Fusarium subglutinans. Acta Microbiol. Polon. 50: 139–147.Google Scholar
  44. 44.
    Su, Y. C. and W. P. Chen (1970) Studies on milk clotting enzymes from microorganisms. Part 1. Screening tests and production of the enzymes. J. Chin. Agric. Chem. Soc. 8: 73–83.Google Scholar
  45. 45.
    Hung, Y. C. and C. C. Chou (1997) Growth and milk clotting enzyme production in submerged culture of Amylomyces rouxii. J. Chin. Agric. Chem. Soc. 35: 422–432.Google Scholar
  46. 46.
    Nigam, J. M., K. R. Pillai, and J. N. Baruah (1981) Effect of carbon and nitrogen sources on neutral proteinase production by Pseudomonas aeruginosa. Folia Microbiol. 26: 358–363.CrossRefGoogle Scholar
  47. 47.
    Zandrazil, F. and H. Brunert (1981) Investigation of physical parameters important for solid-state fermentation of straw by white rot fungi. Eur. J. Appl. Microbiol. Biotechnol. 11: 183–188.CrossRefGoogle Scholar
  48. 48.
    Lekha, P. K. and B. K. Lonsane (1994) Comparative titres, location, and properties of tannin acyl hydrolase produced by Aspergillus niger PKL104 in solid state, liquid surface, and submerged fermentation. Process Biochem. 29: 497–503.CrossRefGoogle Scholar
  49. 49.
    Chu, I. M., C. Lee, and T. S. Li (1992) Production and degradation of alkaline protease in batch cultures of Bacillus subtilis ATCC 14416. Enz. Microbiol. Technol. 14: 755–761.CrossRefGoogle Scholar
  50. 50.
    Gupta, R., Q. K. Beg, S. Khan, and B. Chauhan (2002) An overview on fermentation, downstream processing and properties of microbial proteases. Appl. Microbiol. Biotechnol. 60: 381–395.CrossRefGoogle Scholar
  51. 51.
    Hesseltine, C. W., M. Smith, and H. L. Wang (1976) Product of fungal spore as inocula for oriental fermented food. Dev. Ind. Microbiol. 17: 101–115.Google Scholar
  52. 52.
    Hashem, A. M. (1999) Optimization of milk-clotting enzyme productivity by Penicillium oxalicum. Bioresour. Technol. 70: 203–207.CrossRefGoogle Scholar
  53. 53.
    Abdel-Fattah, A. F. and S. A. Saleh (1979) Production and isolation of milk clotting enzyme from Aspergillus versicolor. Zbl. Bakt. II Abt. 134: 547–550.Google Scholar
  54. 54.
    Kobayashi, F., M. Yabuki, K. Hoshino, and M. Sakamoto (1975) Isolation and characterization of Trametes ostreiformis K-1, and purification and properties of milk clotting enzyme produced by fungus. J. Agric. Chem. Soc. Jpn. 49: 81–92.Google Scholar

Copyright information

© The Korean Society for Biotechnology and Bioengineering and Springer-Verlag Berlin Heidelberg 2009

Authors and Affiliations

  • R. Sathya
    • 1
  • B. V. Pradeep
    • 1
  • J. Angayarkanni
    • 2
  • M. Palaniswamy
    • 1
  1. 1.Department of Microbiology, School of Life SciencesKarpagam UniversityCoimbatoreIndia
  2. 2.Department of Microbial BiotechnologyBharathiar UniversityCoimbatoreIndia

Personalised recommendations