ISSN 2308-4057 (Печать),
ISSN 2310-9599 (Онлайн)


Due to the scarcity of natural rennet, in this study we have considered an option of using in cheesemaking a milk-clotting enzyme produced by the Irpex lacteus fungus. We describe main properties of I. lacteus coagulant: milk-clotting activity (МА), overall proteolytic activity (PA), thermal stability, MА dependence on рН level and calcium ions content. Partially purified preparations of I. lacteus milk-clotting enzyme was obtained by salting out and gel-filtration. Technological properties of the I. lacteus coagulant were compared to natural calf rennet and cow pepsin (CP). МА of I. lacteus enzyme amounted to 29.1 ± 0.7 RU/ml, with protein content of 23 mg/ml. The coagulant was completely inactivated at 60○С. Thermal stability of I. lacteus milk-clotting enzyme, MA sensitivity to pH variations and Ca2+ content were comparable to respective parameters of calf rennet and CP. Overall PA of the I. lacteus coagulant exceeded CP and calf rennet activity by 33 and 220 times, respectively. As for enzymatic specificity, the following order was observed: calf rennet (100%) > CP (14.9%) > I. lacteus coagulant (0.5%). These findings suggest that there is a need to increase the MA of I. lacteus coagulant in order to be able to use it in cheesemaking. We have considered chemical, biochemical, and genetic corrective actions applicable to technological properties of microfungal milk coagulants.
Ключевые слова
milk-clotting enzymes, rennet, rennet substitutes, microfungal coagulants, milk-clotting activity, proteolytic activity, thermal stability, cheesemaking, mucorpepsins
  1. Birkkjaer H. and Jonk P. Technological suitability of calf rennet substitutes. International Dairy Federation Bulletin, 1985, no. 194, pp. 8-13.
  2. Feijoo-Siota L., Blasco L., Rodríguez-Rama J.L., et al. Recent patents on microbial proteases for the dairy industry. Recent Advances in DNA and Gene Sequences, 2014, vol. 8, no. 1, pp. 44-55. DOI: 10.2174/ 2352092208666141013231720.
  3. Harboe M., Broe M.L., and Qvist K.B. In: Technology of Cheesemaking. Law B.A. and Tamime A.Y. (eds.), John Wiley & Sons., Ch. 3. The Production, Action and Application of Rennet and Coagulants, 2010, pp. 98-129. DOI: 10.1002/9781444323740.ch3.
  4. Jacob M., Jaros D., and Rhom H. Recent advances in milk clotting enzymes. International Journal of Dairy Technology, 2011, vol. 64, no. 1, pp. 14-33. DOI: 10.1111/j.1471-0307.2010.00633.x.
  5. Flamm E.L. How FDA approved chymosin: a case history. Bio/Technology, 1991, vol. 9, no 4. pp. 349-351.
  6. Kappeler S.R., van den Brink H.(J.)M., Rahbek-Nielsen H., et al. Characterization of recombinant camel chymosin reveals superior properties for the coagulation of bovine and camel milk. Biochemical and Biophysical Research Communications, 2006, no. 342, vol. 2, pp. 647-654. DOI: 10.1016/j.bbrc.2006.02.014.
  7. Kimberlin R.H. Bovine spongiform encephalopathy and public health: some problems and solutions in assessing the risk. 3rd International Symposium on Transmissible Subacute Spongiform Encephalopathies: Prion Diseases, March 18-20, Paris, Elsevier, 1996, pp. 487-502.
  8. Nasr A.I.A.M., Ahmed I.A.M., and Hamid O.I.A. Characterization of partially purified milk-clotting Enzyme from sunflower (Helianthus annuus) seeds. Food Science and Nutrition, Early View (Online Version of Record published before inclusion in an issue), First published online: 15.01.2016 ( DOI: 10.1002/fsn3.338.
  9. Shah M.A., Mir S.A., and Paray M.A. Plant proteases as milk-clotting enzymes in cheesemaking: a review. Dairy Science and Technology, 2014, vol. 94, no. 1, pp. 5-16. DOI: 10.1007/s13594-013-0144-3.
  10. Jacob M., Jaros D., and Rhom H. Recent advances in milk clotting enzymes. International Journal of Dairy Technology, 2011, vol. 64, no. 1, pp. 14-33. DOI: 10.1111/j.1471-0307.2010.00633.x.
  11. Isselnante S., Boudjenah-Haroun S., Nouani A., et al. Molecular characterization of crude enzymatic extract from Algerian camel abomasum (Camelus dromedarius). Emirates Journal of Food and Agriculture. 2016, vol. 28, no. 3, pp. 217-223. DOI: 10.9755/ejfa.2015-07-490.
  12. Shamtsyan M., Dmitriyeva T., Kolesnikov B., and Denisova N. Novel milk-clotting enzyme produced by Coprinus lagopides basidial mushroom. LWT-Food Science and Technology, 2014, vol. 58, no. 2, pp. 313-672. DOI: 10.1016/ j.lwt.2013.10.009.
  13. Tanhua L.Y., Da L., Zhoulin L., et al. Screening and characterization of a mutant fungal aspartic proteinase from Mucor pusillus. The Open Biotechnology Journal, 2015, vol. 9, pp. 119-126. DOI: 10.2174/ 1874070701509010119.
  14. Elchaninov V.V. Milk-clotting enzyme from reindeer abomasum. Cheesemaking and Buttermaking, 2006, no. 4, pp. 42-44. (In Russian).
  15. Kobayashi H., Kusakabe I., and Murakami K. Purification and characterization of two milk-clotting enzymes from Irpex lacteus. Agricultural and biological chemistry, 1983, vol. 47, no. 3, pp. 551-558.
  16. Kikuchi E., Kobayashi H., Kusakabe I., and Murakami K. Fiber-structured cheese making with Irpex lacteus milk-clotting enzyme. Agricultural and biological chemistry, 1988, vol. 52, no.5, pp. 1277-1278.
  17. Kobayashi H., Sekibata S., Shibuya H., Yoshida S., Kusakabe I. and Murakami K. Cloning and sequence analysis of cDNA for Irpex lacteus aspartic proteinase. Agricultural and biological chemistry, 1989, vol. 53, no. 7, pp. 1927-1933.
  18. Kikuchi E., Kobayashi H., Kusakabe I., Murakami K. Suitability of milk-clotting enzyme from Irpex lacteus for Gouda cheese manufacture. Japanese Journal of Zootechnical Science, 1988, vol. 59, pp. 532-540.
  19. Zagnitko Yu.P. Some physical and chemical properties of enzymic preparations derived from strain V-02 of Irpex lacteus FR. Immunology, Allergology, Infectiology (Section: Fingal biotechnologies in medicine and industry), 2010, no. 1, pp. 249-250. (In Russian).
  20. Boyko M.I. and Kuznetsova I.A. Search for the best source of carbon nutrition for strains of Irpex lacteus, producers of milk-clotting exoproteinases. Immunopathology, Allergology, Infectiology (Section: Research in morphology, physiology, and biocgemistry. Fungi under stress), 2010, no. 1, pp. 18. (In Russian).
  21. Boyko S.М. and Ivanov I.N. Kul'tury bazidial'nogo griba Irpex lacteus kak perspektivnye produtsenty fermentov sychuzhnogo deystviya. [Cultures of a basidial fungus Irpex lacteus as promising producers of enzymes with rennet activity]. Materialy Mezhdunarodnoy nauchno-prakticheskiy konferentsii «Perspektivy i problemy razvitiya biotekhnologii v ramkakh edinogo ekonomicheskogo prostranstva stran Sodruzhestva» [Proc. of the Intern. Sci. and Prac. Conf. “Perspectives and challenges of biotechnological development within the united economic space of the Commonwealth countries”], Мinsk, 25-28 May, 2005, pp. 21-22.
  22. Teplyakova T.V., Psurtseva N.V., Kosogova T.A., et al. Antiviral Activity of Polyporoid Mushrooms (Higher Basidiomycetes) from Altai Mountains (Russia). International Journal of Medicinal mushrooms, 2012, vol. 14, no. 1, pp. 37-45. DOI: 10.1615/IntJMedMushr.v14.i1.40.
  23. Kriger А.V. and Belov А.N. Metody issledovaniya molokosvertyvayushchikh fermentnykh preparatov [Methods of study for milk-clotting enzymic preparations]. In: Vliyanie fermentnykh preparatov na protsess sozrevaniya i kachestvo syrov [Influence of enzymic preparations on the ripening process and quality of cheese], LAP LAMBERT Academic Publishing Gmbh & Co KG, 2011, pp. 46-49.
  24. Cornelius D.A. Process for decreasing the thermal stability of microbial rennet. Patent US, no. 4348482, 1982.
  25. Asmus C.V., Cornelius D.A., and Sternberg M.M. Acylation of Mucor pusillus microbial rennet enzyme. Patent US, no. 4362818, 1982.
  26. Higashi T., Iwasaki S., and Kobayashi Y. Microbial rennet having increased milk coagulating activity and method for production thereof. Patent US, no. 4530906, 1985.
  27. Havera H.J. and Humphreys J.D. Method for increasing the milk clotting activity of thermolabile Rhizomucor pusillus rennet. Patent EP, no. 230231, 1987.
  28. Harboe M.K. Advances in Experimental Medicine and Biology. James M.N.G., Ed. Springer, 1998, vol. 436, Ch. Aspartic Proteinases. Rhizomucor miehei aspartic proteinases having improved properties. pp. 293-296. DOI: 10.1007/978-1-4615-5373-1_40.
  29. Harboe M.K. and Kristensen P.B. Microbially derived enzymes having enhanced milk clotting activity and method producing the same. Patent US, no. 6127142, 2000.
Как цитировать?
Lebedev L.R., Kosogova Т.А., Teplyakova Т.V., Kriger A.V., Elchaninov V.V., Belov А.N., and Коval' А.D. Study of technological properties of milk-clotting enzyme from Irpex lacteus (Irpex lacteus (Fr.) Fr.). Foods and Raw Materials, 2016, vol. 4, no. 2, pp. 58–65. DOI: 10.21179/2308-4057-2016-2-58-65.
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