Dairy Science & Technology

, Volume 94, Issue 1, pp 5–16 | Cite as

Plant proteases as milk-clotting enzymes in cheesemaking: a review

  • Manzoor Ahmad ShahEmail author
  • Shabir Ahmad Mir
  • Mohd Amir Paray
Review Paper


Plant proteases used as milk coagulants in cheesemaking are reviewed in this paper. Plant proteases have been used as milk coagulants in cheesemaking for centuries either as crude extracts or in purified form. These coagulants are an alternative to the calf rennet due to the limited availability and high price of rennet, religious factors, diet or ban on recombinant calf rennet in some countries. These enzymes are found in almost all kinds of plant tissues and can be obtained from their natural source or through in vitro culture to ensure a continuous supply of plant proteases. Almost all the enzymes used as milk coagulants belong to aspartic proteases, but enzymes from other groups such as cysteine and serine proteases have also been reported and possess the ability to clot milk under proper conditions. The excessive proteolytic nature of most plant coagulants has limited their use in cheese manufacturing due to lower yields of cheese, bitter flavors and texture defects. The search for new potential milk-clotting enzymes from plants still continues in order to meet the increasing global demand for diversified and good quality cheese production.


Plant protease Milk-clotting activity Cheese Casein Vegetable coagulant 


  1. Agboola SO, Chan HH, Zhao J, Rehman A (2009) Can the use of Australian cardoon (Cynara cardunculus L.) coagulant overcome the quality problems associated with cheese made from ultrafiltrated milk? LWT Food Sci Technol 42:1352–1359CrossRefGoogle Scholar
  2. Ahmed IAM, Morishima I, Babiker EE, Mori N (2009a) Characterisation of partially purified milk-clotting enzyme from Solanum dubium Fresen seeds. Food Chem 116:395–400CrossRefGoogle Scholar
  3. Ahmed IAM, Morishima I, Babiker EE, Mori N (2009b) Dubiumin, a chymotrypsin-like serine protease from the seeds of Solanum dubium Fresen. Phytochem 70:483–491CrossRefGoogle Scholar
  4. Ahmed IAM, Babiker EE, Mori N (2010) pH stability and influence of salts on activity of a milk-clotting enzyme from Solanum dubium seeds and its enzymatic action on bovine caseins. LWT Food Sci Technol 43:759–764CrossRefGoogle Scholar
  5. Asakura T, Watanabe H, Keiko A, Soichi A (1997) Oryzasin as an aspartic proteinase occurring in rice seeds: purification, characterization, and application to milk-clotting. J Agric Food Chem 45:1070–1075CrossRefGoogle Scholar
  6. Bah S, Paulsen BS, Diallo D, Johansen HT (2006) Characterization of cysteine proteases in Malian medicinal plants. J Ethnopharmacol 107:189–198CrossRefGoogle Scholar
  7. Barros RM, Ferreira CA, Silva SV, Malcata FX (2001) Quantitative studies on the enzymatic hydrolysis of milk proteins brought about by cardosins precipitated by ammonium sulfate. Enzyme Microb Technol 29:541–547CrossRefGoogle Scholar
  8. Barros RM, Extremina CI, Goncalves IC, Braga BO, Balcao VM, Malcata FX (2003) Hydrolysis of alpha-lactalbumin by cardosin A immobilized on highly activated supports. Enzyme Microb Technol 33:908–916CrossRefGoogle Scholar
  9. Bruno MA, Pardo MF, Caffini NO, López LMI (2002) Purification of a new endopeptidase isolated from fruits of Bromelia hieronymi Mez (Bromeliaceae). Acta Farm Bonaer 21:51–56Google Scholar
  10. Bruno MA, Trejo SA, Aviles XF, Caffini NO, Lopez LMI (2006) Isolation and characterization of hyeronymain II, another peptidase isolated from fruits of Bromelia hieronymi Mez (Bromeliaceae). Protein J 25:224–231CrossRefGoogle Scholar
  11. Bruno MA, Lazza CM, Errasti ME, Lόpez LMI, Caffini NO, Pardo MF (2010) Milk clotting and proteolytic activity of an enzyme preparation from Bromelia hieronymi fruits. LWT Food Sci Technol 43:695–701CrossRefGoogle Scholar
  12. Brutti CB, Pardo MF, Caffini NO, Natalucci CL (2012) Onopordum acanthium L. (Asteraceae) flowers as coagulating agent for cheesemaking. LWT Food Sci Technol 45:172–179CrossRefGoogle Scholar
  13. Chazarra S, Sidrach L, Lόpez-Molina D, Rodríguez- Lόpez JN (2007) Characterization of the milk-clotting properties of extracts from artichoke (Cynara scolymus, L) flowers. Int Dairy J 17:1393–1400CrossRefGoogle Scholar
  14. Cimino C, Cavalli SV, Spina F, Natalucci C, Priolo N (2006) Callus culture for biomass production of milk thistle as a potential source of milk-clotting peptidases. Electron J Biotechnol 9:237–240CrossRefGoogle Scholar
  15. Cordeiro MC, Pais MS, Brodelius PE (1998) In: Bajaj YPS (ed) Biotechnology in agriculture and forestry. Springer, HeidelbergGoogle Scholar
  16. Devaraj KB, Gowda LR, Prakash V (2008) An unusual thermostable aspartic protease from the latex of Ficus racemosa (L.). Phytochem 69:647–655CrossRefGoogle Scholar
  17. Domingos A, Miguel G, Clemente A, Pais MSS (1992) Production of clotting enzymes by in vivo plants and cell suspension cultures of Centaurea calcitrapa (Compositae). NATOASI series. Kluwer, NetherlandsGoogle Scholar
  18. Domingos A, Cardos PC, Xue ZT, Clemente A, Brodelius PE, Pais MS (2000) Purification, cloning and autoproteolytic processing of an aspartic proteinase from Centaurea calcitrapa. Eur J Biochem 267:6824–6831CrossRefGoogle Scholar
  19. Egito AS, Girardet JM, Laguna LE, Poirson C, Mollé D, Miclo L, Humbert G, Gaillard JL (2007) Milk-clotting activity of enzyme extracts from sunflower and albizia seeds and specific hydrolysis of bovine κ-casein. Int Dairy J 17:816–825CrossRefGoogle Scholar
  20. Esteves CLC, Lucey JA, Pires EMV (2002) Rheological properties of milk gels made using coagulants of plant origin and chymosin. Int Dairy J 12:427–434CrossRefGoogle Scholar
  21. Esteves CLC, Lucey JA, Hyslop DB, Pires EMV (2003) Effect of gelation temperature on the properties of skim milk gels made from plant coagulants and chymosin. Int Dairy J 13:877–885CrossRefGoogle Scholar
  22. Faccia M, Picariello G, Trani A, Loizzo P, Gambacorta G, Lamacchia C, Di Luccia A (2012) Proteolysis of Cacioricotta cheese made from goat milk coagulated with caprifig (Ficus carica sylvestris) or calf rennet. Eur Food Res Technol 234:527–533CrossRefGoogle Scholar
  23. Fernández-Salguero J, Sanjuán E (1999) Influence of vegetable and animal rennet on proteolysis during ripening in ewes’ milk cheese. Food Chem 64:177–183CrossRefGoogle Scholar
  24. Fernández-Salguero J, Sanjuán E, Montero E (1991) A preliminary study of the chemical composition of Guía cheese. J Food Compos Anal 4:262–269CrossRefGoogle Scholar
  25. Fox PF (1989) Proteolysis during cheese manufacture and ripening. J Dairy Sci 72:1379–1400CrossRefGoogle Scholar
  26. Gonzalez-Rabade N, Badillo-Corona JA, Aranda-Barradas JS, Oliver-Salvador MC (2011) Production of plant proteases in vivo and in vitro—a review. Biotechnol Adv 29:983–996CrossRefGoogle Scholar
  27. Harboe M, Broe ML, Qvist KB (2010) In: Law BA, Tamime AY (eds) Technology of cheese making. Wiley-Blackwell, LondonGoogle Scholar
  28. Hashim MM, Mingsheng D, Iqbal MF, Xiaohong C (2011) Ginger rhizome as a potential source of milk coagulating cysteine protease. Phytochem 72:458–464CrossRefGoogle Scholar
  29. Heimgartner U, Pietrzak M, Geertsen R, Brodelius P, Figueiredo AC, Pais MSS (1990) Purification and partial characterization of milk-clotting proteases from flowers of Cynara cardunculus. Phytochem 29:1405–1410CrossRefGoogle Scholar
  30. Jacob M, Jaros D, Rohm H (2011) Recent advances in milk clotting enzymes. Int J Dairy Technol 64:14–33CrossRefGoogle Scholar
  31. Katsaros GI, Tavantzis G, Taoukis PS (2010) Production of novel dairy products using actinidin and high pressure as enzyme activity regulator. Innov Food Sci Emerg Technol 11:47–51CrossRefGoogle Scholar
  32. Kumari M, Sharma A, Jagannadham MV (2010) Decolorization of crude latex by activated charcoal, purification and physico-chemical characterization of Religiosin, a milk-clotting serine protease from the latex of Ficus religiosa. J Agric Food Chem 58:8027–8034CrossRefGoogle Scholar
  33. Kumari M, Sharma A, Jagannadham MV (2012) Religiosin B, a milk-clotting serine protease from Ficus religiosa. Food Chem 131:1295–1303CrossRefGoogle Scholar
  34. Lima-Costa ME, Van Gulik WM, Ten Hoopen HJG, Pais MSS, Cabral JMS (1996) Protease and phenol production of Cynara cardunculus L. cell suspension in a chemostat. Enzyme Microb Technol 19:493–500CrossRefGoogle Scholar
  35. Llorente BE, Brutti CB, Natalucci CL, Caffini NO (1997) Partial characterization of a milk clotting proteinase isolated from artichoke (Cynara scolymus L., Asteraceae). Acta Farm Bonaer 16:37–42Google Scholar
  36. Llorente BE, Brutti CB, Caffini NO (2004) Purification and characterization of a milk-clotting aspartic proteinase from globe artichoke (Cynara scolymus L.). J Agric Food Chem 52:8182–8189CrossRefGoogle Scholar
  37. Lo Piero AR, Puglisi I, Petrone G (2002) Characterization of “lettucine”, a serine-like protease from Lactuca sativa leaves, as a novel enzyme for milk clotting. J Agric Food Chem 50:2439–2443CrossRefGoogle Scholar
  38. Lourenco PML, de Castro S, Martins TM, Clemente A, Domingos A (2002) Growth and proteolytic activity of hairy roots from Centaurea calcitrapa: effect of nitrogen and sucrose. Enzyme Microb Technol 31:242–249CrossRefGoogle Scholar
  39. Low YH, Agboola S, Zhao J, Lim MY (2006) Clotting and proteolytic properties of plant coagulants in regular and ultrafiltered bovine skim milk. Int Dairy J 16:335–343CrossRefGoogle Scholar
  40. Lufrano D, Faro R, Castanheira P, Parisi G, Veríssimo P, Vairo-Cavalli S, Simões I, Faro C (2012) Molecular cloning and characterization of procirsin, an active aspartic protease precursor from Cirsium vulgare (Asteraceae). Phytochem 81:7–18CrossRefGoogle Scholar
  41. Macedo IQ, Faro CJ, Pires EM (1993) Specificity and kinetics of the milk-clotting enzyme from Cardoon (Cynara cardunculus L.) toward bovine κ-casein. J Agric Food Chem 41:1537–1540CrossRefGoogle Scholar
  42. Néstor GM, Rubí CGD, Héctor JC (2012) Exploring the milk-clotting properties of a plant coagulant from the berries of S. elaeagnifoliun var. Cavanillies. J Food Sci 71:89–94CrossRefGoogle Scholar
  43. Oliveira A, Perira C, Soares da Costa D, Teixeira J, Fidalgo F, Pereira S, Pissarra J (2010) Characterization of aspartic proteinases in C. cardunculus L. callus tissue for its prospective transformation. Plant Sci 178:140–146CrossRefGoogle Scholar
  44. Ordiales E, Martín A, Benito MJ, Hernández A, Ruiz-Moyano S, Cόrdoba MG (2012) Technological characterisation by free zone capillary electrophoresis (FZCE) of the vegetable rennet (Cynara cardunculus) used in “Torta del Casar” cheese-making. Food Chem 133:227–235CrossRefGoogle Scholar
  45. Otani H, Matsumori M, Hosono A (1991) Purification and some properties of a milk clotting protease from the young seeds of Albizia julibrissin. Anim Sci Technol 62:424–432Google Scholar
  46. Pino A, Prados F, Galán E, McSweeney PLH, Fernández-Salguero J (2009) Proteolysis during the ripening of goats’ milk cheese made with plant coagulant or calf rennet. Food Res Int 42:324–330CrossRefGoogle Scholar
  47. Pontual EV, Carvalho BEA, Bezerra RS, Coelho LCBB, Napoleão TH, Paiva PMG (2012) Caseinolytic and milk-clotting activities from Moringa oleifera flowers. Food Chem 135:1848–1854CrossRefGoogle Scholar
  48. Raposo S, Domingos A (2008) Purification and characterization milk-clotting aspartic proteases from Centaurea calcitrapa cell suspension cultures. Process Biochem 43:139–144CrossRefGoogle Scholar
  49. Raposo S, Lima-Costa ME (2006) Rheology and shear stress of Centaurea calcitrapa cell suspension cultures grown in bioreactor. Biotechnol Lett 28:431–438CrossRefGoogle Scholar
  50. Rawlings ND, Barrett AJ (2004) Families of serine peptidases. Methods Enzymol 244:19–61CrossRefGoogle Scholar
  51. Reis PM, Lourenço PL, Domingos A, Clemente AF, Pais MS, Malcata FX (2000) Applicability of extracts from Centaurea calcitrapa in ripening of bovine cheese. Int Dairy J 10:775–780CrossRefGoogle Scholar
  52. Renner E, Abd El-Salam MH (1991) Application of ultrafiltration in the dairy industry. Elsevier, LondonGoogle Scholar
  53. Roa I, Lόpez MB, Mendiola FJ (1999) Residual clotting activity and ripening properties of vegetable rennet from Cynara cardunculus in La Serena cheese. Food Res Int 32:413–419CrossRefGoogle Scholar
  54. Roseiro LB, Barbosa M, Ames JM, Wilbey RA (2003) Cheesemaking with vegetable coagulants—the use of Cynara L. for the production of ovine cheeses. Int J Dairy Technol 56:76–85CrossRefGoogle Scholar
  55. Salvador SM, Novo C, Domingos A (2006) Evaluation of the presence of aspartic proteases from Centaurea calcitrapa during seed germination. Enzyme Microb Technol 38:893–898CrossRefGoogle Scholar
  56. Sanjuán E, Millán R, Saavedra P, Carmona MA, Gόmez R, Fernández-Salguero J (2002) Influence of animal and vegetable rennet on the physiochemical characteristics of Los Pedroches cheese during ripening. Food Chem 78:281–289CrossRefGoogle Scholar
  57. Schaller A (2004) A cut above the rest: the regulatory function of plant proteases. Planta 220:183–197CrossRefGoogle Scholar
  58. Sharma A, Kumari M, Jagannadham MV (2012) Religiosin C, a cucumisin-like serine protease from Ficus religiosa. Process Biochem 47:914–921CrossRefGoogle Scholar
  59. Sidrach L, Garcia-Canovas F, Tudela J, Rodríguez-Lόpez JN (2005) Purification of cynarases fromtichoke (Cynara scolymus L.): enzymatic properties of cynarase A. Phytochem 66:41–49CrossRefGoogle Scholar
  60. Silva SV, Malcata FX (2005) Studies pertaining to coagulant and proteolytic activities of plant proteases from Cynara cardunculus. Food Chem 89:19–26CrossRefGoogle Scholar
  61. Silva SV, Allmere T, Malcata FX, Andrén A (2003) Comparative studies on the gelling properties of cardosins extracted from Cynara cardunculus and chymosin on cow’s skim milk. Int Dairy J 13:558–564CrossRefGoogle Scholar
  62. Sousa MJ, Malcata FX (2002) Advances in the role of a plant coagulant (Cynara cardunculus) in vitro and during ripening of cheese from several milk species. Lait 82:151–170CrossRefGoogle Scholar
  63. Tamer MI (1993) Identification and partial purification of a novel milk-clotting enzyme from Onopordum turcicum. Biotechnol Lett 13:427–432CrossRefGoogle Scholar
  64. Tamer MI, Mavituna F (1997) Protease from freely suspended and immobilized Mirabilis jalapa. Process Biochem 32:195–200CrossRefGoogle Scholar
  65. Tripathi P, Tomar R, Jagannadham MV (2011) Purification and biochemical characterisation of a novel protease streblin. Food Chem 125:1005–1012CrossRefGoogle Scholar
  66. Uchikoba T, Kaneda M (1996) Milk-clotting activity of cucumisin, a plant serine protease from melon fruit. Appl Biochem Biotechnol 56:325–330CrossRefGoogle Scholar
  67. Vairo-Cavalli S, Claver S, Priolo N, Natalucci C (2005) Extraction and partial characterization of a coagulant preparation from Silybum marianum flowers. Its action on bovine caseinate. J Dairy Res 72:271–275CrossRefGoogle Scholar
  68. Vairo-Cavalli S, Silva SV, Cimino C, Malcata FX, Priolo N (2008) Hydrolysis of caprine and ovine milk proteins, brought about by aspartic peptidases from Silybum marianum flowers. Food Chem 106:997–1003CrossRefGoogle Scholar
  69. Verissimo P, Esteves C, Faro C, Pires E (1995) The vegetable rennet of Cynara cardunculus L. contains two proteinases with chymosin and pepsin-like specificities. Biotechnol Lett 17:621–626CrossRefGoogle Scholar
  70. Verissimo P, Faro C, Moir AJG, Lin Y, Tang J, Pires E (1996) Purification, characterization and partial amino acid sequencing of two new aspartic proteinases from fresh flowers of Cynara cardunculus L. Eur J Biochem 235:762–768CrossRefGoogle Scholar
  71. Yadav RP, Patel AK, Jagannadham MV (2011) Purification and biochemical characterization of a chymotrypsin-like serine protease from Euphorbia neriifolia Linn. Process Biochem 46:1654–1662CrossRefGoogle Scholar
  72. Yadav RP, Patel AK, Jagannadham MV (2012) Neriifolin S, a dimeric serine protease from Euphorbia neriifolia Linn.: purification and biochemical characterisation. Food Chem 132:1296–1304CrossRefGoogle Scholar
  73. Yegin S, Dekker P (2013) Progress in the field of aspartic proteinases in cheese manufacturing: structures, functions, catalytic mechanism, inhibition, and engineering. Dairy Sci Technol. doi: 10.1007/s13594-013-0137-2 CrossRefGoogle Scholar

Copyright information

© The Author(s) 2013

Open Access This article is distributed under the terms of the Creative Commons Attribution License which permits any use, distribution, and reproduction in any medium, provided the original author(s) and the source are credited.

The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder.

To view a copy of this license, visit (

Authors and Affiliations

  • Manzoor Ahmad Shah
    • 1
    Email author
  • Shabir Ahmad Mir
    • 1
  • Mohd Amir Paray
    • 1
  1. 1.Department of Food TechnologyIslamic University of Science and TechnologyAwantiporaIndia

Personalised recommendations