Enzymatic proteolysis during the conversion of milk into cheese

The transformation of milk into cheese occurs under the influence of many physicochemical, biochemical and microbiological processes, among which proteolysis plays a very important role. Proteolysis belongs to the most complex type of irreversible post-translational modification of proteins. Enzymatic proteolysis catalysts at different stages of cheese production are native milk enzymes, exo- and endopeptidases of starter and non-starter microorganisms, and milk-clotting enzymes. The article presents a brief overview of modern ideas about the properties, mechanism of action and specificity of the main representatives of enzymes that hydrolyze milk proteins at the stages of preparing milk for coagulation, during rennet coagulation and subsequent maturation of cheeses. These include the plasmin system of milk, enzymes of psychrotrophic bacteria and lactic acid microorganisms that enter milk both accidentally (non-starter microflora) and planned in the form of starter cultures from specially selected strains. Milk-clotting enzymes, having fulfilled their main function — milk coagulation — partially pass into cheese and, along with enzymes of starter microorganisms and plasmin, participate in proteolytic processes during cheese ripening. It is generally accepted that proteolysis in ripening cheeses is the most significant biochemical process that affects the formation of taste, aroma and texture along with lipolysis and glycolysis. The combination of proteolysis products (peptides, amino acids, amines, etc.) is individual for different types of cheese and varies depending on the technological parameters of production, including the duration of maturation. Proteolysis in cheeses has been studied by many scientists in various aspects. This review supplements the known information with new information, without claiming to be comprehensive.

1. Thompson, А., Boland, M., Singh, Н. (2009). Milk proteins: from expression to food. Academic Press, 2009. https://doi.org/10.1016/B978–0–12–374039–7.X0001–3

2. D’Ambrosio, C., Arena, S., Salzano, A. M., Renzone, G., Ledda, L., Scaloni, A. (2008). A proteomic characterization of water buffalo milk fractions describing PTM of major species and the identification of minor components involved in nutrient delivery and defense against pathogens. Proteomics, 8(17), 3657–3666. https://doi.org/10.1002/pmic.200701148

3. Rout, Р.К., Verma, М. (2021). Post translational modifications of milk proteins in geographically diverse goat breeds. Scientific Reports, 11, Article 5619. https://doi.org/10.1038/s41598–021–85094–9

4. Baptista, D.P., Gigante, M.L. (2021). Bioactive peptides in ripened cheeses: release during technological processes and resistance to the gastrointestinal tract. Journal of the Science of Food and Agriculture, 101(10), 4010–4017. https://doi.org/10.1002/jsfa.11143

5. Goulding, D.A., Fox, P.F., O’Mahony, J.A. (2020). Milk proteins: An overview. Chapter in a book: Milk Proteins: From Expression to Food. Amsterdam: Elsevier, 2020. https://doi.org/10.1016/b978–0–12–815251–5.00002–5

6. Fox, P.F., Uniacke-Lowe, Т., McSweeney, P.L.H., O’Mahony, J.A. (2015). Milk proteins. Chapter in a book: Dairy Chemistry and Biochemistry. Springer International Publishing Switzerland, 2015. https://doi.org/10.1007/978–3–319–14892–2

7. Gorbatova, K.K., Gunkova, P.I. (2010). Biochemistry of milk and dairy products. Saint-Petersburg: GIORD, 2010. (In Russian)

8. Holland, J.W. (2008). Post-translational modifications of caseins. Chapter in a book: Milk Proteins: From Expression to Food. Amsterdam: Elsevier, 2008. https://doi.org/10.1016/B978–0–12–374039–7.00004–0

9. O’Mahony, J. A., Fox, P. F. (2012). Milk Proteins: Introduction and Historical Aspects. Chapter in a book: Advanced Dairy Chemistry. Springer, Boston, 2012. https://doi.org/10.1007/978–1–4614–4714–6_2

10. Guerin, J., Burgain, J., Gomand, F., Scher, J., Gaiani, C. (2017). Milk fat globule membrane glycoproteins: Valuable ingredients for lactic acid bacteria encapsulation? Critical Reviews in Food Science and Nutrition, 59(4), 639–651. https://doi.org/10.1080/10408398.2017.1386158

11. Lopez, C., Cauty, C., Guyomarc’h, F. (2018). Unravelling the complexity of milk fat globules to tailor bioinspired emulsions providing health benefits: the key role played by the biological membrane. European Journal of Lipid Science and Technology, 121(10), Article 1800201. https://doi.org/10.1002/ejlt.201800201

12. Elchaninov, V.V. (2019). The proteins of milk fat globule membrane. 1. Genesis and structure of the milk fat globule, nomenclature of proteins of milk fat globule membrane. Dairy Industry, 7, 24–27. (In Russian)

13. Rogers, L. D., Overall, C. M. (2013). Proteolytic post-translational modification of P\proteins: Proteomic tools and methodology. Molecular and Cellular Proteomics, 12(12), 3532–3542. https://doi.org/10.1074/mcp.M113.031310

14. Sousa, M.J., Ardö, Y., McSweeney, P.L.H. (2001). Advances in the study of proteolysis during cheese ripening. International Dairy Journal, 11(4–7), 327–345. https://doi.org/10.1016/S0958–6946(01)00062–0

15. Ward, O.P. (2011). 3.49 — Proteases. Comprehensive Biotechnology, 571–582. PMCID: PMC7152071. https://doi:10.1016/b978–0–08–088504–9.00222–1

16. Ardö, Y. (2021). Enzymes in cheese ripening. Chapter in a book: Agents of Change. Enzymes in Milk and Dairy Products. Switzerland: Springer, Cham., 2021. https://doi.org/10.1007/978–3–030–55482–8_15

17. Webb, E.C. (1992). Enzyme nomenclature 1992. Recommendations of the Nomenclature Committee of the International Union of Biochemistry and Molecular Biology on the Nomenclature and Classification of Enzymes. San Diego: Academic Press, 1992.

18. France, T.C., O’Mahony, J.A., Kelly, A.L. (2021). The plasmin system in milk and dairy products. Chapter in a book: Agents of Change. Food Engineering Series. Springer, Cham., 2021. https://doi.org/10.1007/978–3–030–5548–8_2

19. Nielsen, S.S. (2002). Plasmin system and microbial proteases in milk: Characteristics, roles, and relationship. Journal of Agricultural and Food Chemistry, 50(22), 6628–6634. https://doi.org/10.1021/jf0201881

20. Crudden, A., Kelly, A.L. (2003). Studies of plasmin activity in whey. International Dairy Journal, 13(12), 987–993. https://doi.org/10.1016/s0958–6946(03)00140–7

21. Srinivasan, M., Lucey, J.A. (2002). Effects of added plasmin on the formation and rheological properties of rennet-induced skim milk gels. Journal of Dairy Science, 85(5), 1070–1078. https://doi.org/10.3168/jds.s0022–0302(02)74167–2

22. Borda, D., Indrawati, Smout, C., Van Loey, A., Hendrickx, M. (2004). High pressure thermal inactivation kinetics of a plasmin system. Journal of Dairy Sciеnse, 87(8), 2351–2358. https://doi.org/10.3168/jds.s0022–0302(04)73357–3

23. Budkevich, R.O., Eremina, A.I., Evdokimov, I.A., Fedortsov, N.M., Martak, A.A., Budkevich, E.V. (2018). The physical properties of the casein in solution: effect of ultra-high pressure. Food Systems, 1(3), 4–12. https://doi.org/10.21323/2618–9771–2018–1–3–4–12

24. Villalobos, J.C., Sigler, A.I.G., Oliete, B., Sánchez, R.A., Jiménez, L., Sánchez, N.N. et al. (2015). Relationship of somatic cell count and composition and coagulation properties of ewe’s milk. Mljekarstvo, 65(2), 138–143. https://doi.org/10.15567/mljekarstvo.2015.0208

25. Villalobos, J.С., Garzón, A.I., Martínez Marín, A.L., Arias, R., Ciocia, F., McSweeney, P.L.H. (2018). Plasmin activity in Manchega ewe milk: The effect of lactation, parity and health of the udder, and its influence on milk composition and rennet coagulation. Small Ruminant Research, 158, 57–61. https://doi.org/10.1016/j.smallrumres.2017.10.005

26. Sánchez, A.F., Muñoz, J.P., Villalobos, J.C., Sánchez, R.A., Garzón, A., de Pedro, E.A.S. (2021). Coagulation process in Manchega sheep milk from Spain: A path analysis approach. Journal of Dairy Science, 104(7), 7544– 7554. https://doi.org/10.3168/jds.2020–19187

27. Somers, J.M., Guinee, T. P., Kelly, A.L. (2002). The effect of plasmin activity and cold storage of cheese milk on the composition, ripening and functionality of mozzarella-type cheese. International Journal of Dairy Technology, 55(1), 5–11. https://doi.org/10.1046/j.1471–0307.2002.00030.x

28. Mironenko, I.M. (2021). Functions of ionic calcium and native milk proteases in the process of rennet clotting. Cheesemaking and Buttermaking, 1, 25–28. https://doi.org/10.31515/2073–4018–2021–25–28 (In Russian)

29. Mironenko, I.M. (2019). Probable participants in the process of rennet coagulation of milk. Cheesemaking and Buttermaking, 4, 20–23. (In Russian)

30. Ardö, Y., McSweeney, P.L.H, Magboul, A.A., Upadhyay, V.K., Fox, P.F. (2017). Biochemistry of cheese ripening: Proteolysis. Chapter in a book: Cheese. Chemistry, Physics and Microbiology. Academic Press. https://doi.org/10.1016/B978–0–12–417012–4.00018_1

31. Ozer, В.В., Akdemir-Evrendilek, G. (2014). Microbiology of Raw Milk. Chapter in a book: Dairy Microbiology and Biochemistry. Boca-Raton: CRC Press, 2017.

32. Glück, С., Stressler, Т., Fischer, L. (2021). Heat-stable microbial peptidases associated with the microbiota of raw milk. Chapter in a book: Agents of Change. Enzymes in Milk and Dairy Products. Switzerland: Springer, Cham., 2021. https://doi.org/10.1007/978–3–030–55482–8_11

33. Pukančíková, L., Lipničanová, S., Kačániová, M., Chmelová, D., Ondrejovič, M. (2016). Natural microflora of raw cow milk and their enzymatic spoilage potential. Nova Biotechnologica et Chimica, 15(2), 142– 155. https://doi.org/10.1515/nbec‑2016–0015

34. Baur, C., Krewinkel, M., Kranz, B., von Neubeck, M., Wenning, M., Scherer, S. et al. (2015). Quantification of the proteolytic and lipolytic activity of microorganisms isolated from raw milk. International Dairy Journal, 49, 23–29. https://doi.org/10.1016/j.idairyj.2015.04.005

35. Datta, N, Deeth, H.C. (2003). Diagnosing the cause of proteolysis in UHT milk. LWT — Food Science and Technology, 36(2), 173–182. https://doi.org/10.1016/S0023–6438(02)00214–1

36. Kelly, A.L., Larsen, L.B. (2021). Agents of change: Enzymes in milk and dairy products. Springer Cham, 2021. https://doi.org/10.1007/978–3–030–55482–8

37. Settanni, L., Moschetti, G. (2010). Non-starter lactic acid bacteria used to improve cheese quality and provide health benefits. Food Microbiology, 27(6), 691–697. https://doi.org/10.1016/j.fm.2010.05.023

38. Pogačić, T., Mancini, A., Santarelli, M., Bottari, B., Lazzi, C., Neviani, E. et al. (2013). Diversity and dynamic of lactic acid bacteria strains during aging of a long ripened hard cheese produced from raw milk and undefined natural starter. Food Microbiology, 36(2), 207–215. https://doi.org/10.1016/j.fm.2013.05.009

39. Wouters, J. T.M., Ayad, E.H.E., Hugenholtz, J., Smit, G. (2002) Microbes from raw milk for fermented dairy products. International Dairy Journal, 12(2–3), 91–109. https://doi.org/10.1016/s0958–6946(01)00151–0

40. Bluma, A., Ciprovica, I. (2015). Diversity of lactic acid bacteria in raw milk. Research for Rural Development, 1, 157–161.

41. Hernandez-Valdes, J.A.; van Gestel, J.; Kuipers, O.P. (2020). A riboswitch gives rise to multi-generational phenotypic heterogeneity in an auxotrophic bacterium. Nature Communications, 11(1), e00133. https://doi:10.1038/s41467–020–15017–1

42. Tagliazucchi, D., Martini, S., Solieri, L. (2019). Bioprospecting for bioactive peptide production by lactic acid bacteria isolated from fermented dairy food. Fermentation, 5(96), Article 96.. https://doi.org/10.3390/fermentation5040096

43. Parente, E., Cogan, T.M., Powell, I.B. (2017). Starter cultures: General aspects. Chapter in a book: Cheese. Chemistry, Physics and Microbiology. Academic Press, 2017. https://doi.org/10.1016/B978–0–12–417012–4.00008_1

44. Kitaevskaya, S.V., Ponomarev, V.Y., Reshetnik, O.A. (2022). Evaluation of the proteolytic activity of new cryoresistant lactobacillus strains. Proceedings of Universities. Applied Chemistry and Biotechnology, 12(1), 76–86. https://doi.org/10.21285/2227–2925–2022–12–1–76–86 (In Russian)

45. Kieliszek, M, Pobiega, K, Piwowarek, K, Kot, AM. (2021). Characteristics of the proteolytic enzymes produced by lactic acid bacteria. Molecules, 26(7), Article 1858. https://doi.org/10.3390/molecules26071858

46. Korhonen, H., Pihlanto, A. (2003). Food — derived bioactive peptidesopportunities for designing future foods. Current Pharmaceutical Design, 9(16), 1297–1308. https://doi.org/10.2174/1381612033454892

47. Halavach, T.N., Zhabanos, N.K., Furik, N.N., Kurchenko, V.P., Rizevsky, S.V. (2013). Substrate specificity and enzymatic activity level in the cleavage of milk protein fractions with probiotic microorganisms. Topical Issues of Processing of Meat and Milk Raw Materials, 8, 130–142. (In Russian)

48. Odegov, N.I., Grishkova, A.V., Belov, A.N. (2019). To the question of directed regulation of proteolytic processes in cheeses. Cheesemaking and Buttermaking, 5, 24–26. https://doi.org/10.31515/2073–4018–2019–5–24–26 (In Russian)

49. Myagkonosov, D.S., Mordvinova, V.A., Abramov, D.V., Delitskaya, I.N. (2014). Special features of proteolysis in different groups of cheese types. Cheesemaking and Buttermaking, 2, 24–27. (In Russian)

50. Franco, I., Prieto, B., Urdiales, R., Fresno, J.М., Carballo, J. (2001). Study of the biochemical changes during ripening of Ahumado de Áliva cheese: a Spanish traditional variety. Food Chemistry, 74(4), 463–469. https://doi:10.1016/s0308–8146(01)00164–9

51. Rampilli, M., Larsen, R., Harboe, M. (2005). Natural heterogeneity of chymosin and pepsin in extracts of bovine stomachs. International Dairy Journal, 15(11), 1130–1137. https://doi.org/10.1016/j.idairyj.2004.10.003

52. Horne, D.S., Lucey, J.A. (2017). Rennet-induced coagulation of milk. Chapter in a book: Cheese. Chemistry, Physics and Microbiology. Academic Press, 2017. https://doi.org/10.1016/b978–0–12–417012–4.00005–3

53. Britten, M., Giroux, H.J. (2021). Rennet coagulation of heated milk: A review. International Dairy Journal, 124, Article 105179. https://doi.org/10.1016/j.idairyj.2021.105179

54. Fox, P.F., Guinee, T.P., Cogan, T.M., McSweeney, P.L.H. (2016). Enzymatic coagulation of milk. Chapter in a book: Fundamentals of Cheese Science, New York: Springer, 2016. https://doi.org/10.1007/978–1–4899–7681–9_7

55. Jaros, D., Rohm, H. (2017). Rennets: Applied aspects. Chapter in a book: Cheese. Chemistry, Physics and Microbiology. Academic Press, 2017. https://doi.org/10.1016/B978–0–12–417012–4.00003_1

56. Kriger, A.V., Belov, A.N. (2010). Effect of enzyme compositions on cheese proteolysis. Cheesemaking and Buttermaking, 3, 38–40. (In Russian)

57. Ye, А., Cui, J., Singh, H. (2011). Proteolysis of milk fat globule membrane proteins during in vitro gastric digestion of milk. Journal of Dairy Science, 94(6), 2762–2770. https://doi.org/10.3168/jds.2010–4099

58. Abramov, D.V., Myagkonosov, D.S., Delitskaya, I.N., Mordvinova, V.A., Municheva, T.E., Ovchinnikova E. G. (2019). Perspectives of using complex milk-clotting enzyme preparations for ripening rennet cheeses production. Cheesemaking and Buttermaking, 1, 24–26. https://doi.org/10.31515/2073–4018–2019–1–24–26 (In Russian)

59. Myagkonosov, D.S., Abramov, D.V., Ovchinnikova, E.G., Municheva, T.E. (2019). Prospects of using microbial substitutes of chymosin in cheesemaking. Cheesemaking and Buttermaking, 4, 14–17. (In Russian)

60. Amira, A. B., Besbes, S., Attia, H., Blecker, C. (2017). Milk-clotting properties of plant rennets and their enzymatic, rheological, and sensory role in cheese making: A review. International Journal of Food Properties, 20(sup1), S76–S93. https://doi.org/10.1080/10942912.2017.1289959

61. Myagkonosov, D. S., Abramov, D. V., Delitskaya, I. N., Ovchinnikova, E. G. (2022). Proteolytic activity of milk-clotting enzymes of different origin. Food Systems, 5(1), 47–54. https://doi.org/10.21323/2618–9771–2022–5–1–47–54 (In Russian)

62. Khattab, A. R., Guirguis, H. A., Tawfik, S. M., Farag, M. A. (2019). Cheese ripening: A review on modern technologies towards flavor enhancement, process acceleration and improved quality assessment. Trends in Food Science and Technology, 88, 343–360. https://doi.org/10.1016/j.tifs.2019.03.009

63. Fox, P.F. (1989). Proteolysis during cheese manufacture and ripening. Journal of Dairy Science, 72(6), 1379–1400. https://doi.org/10.3168/jds.S0022–0302(89)79246–8