Journal of Food and Nutrition Research
ISSN (Print): 2333-1119 ISSN (Online): 2333-1240 Website: Editor-in-chief: Prabhat Kumar Mandal
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Journal of Food and Nutrition Research. 2019, 7(3), 244-254
DOI: 10.12691/jfnr-7-3-10
Open AccessArticle

Isolate and Extract for Milk Clotting Enzymes from the leaves of Moringa Oleifera, Carica Papaya and Mangifera Indica and Use in Cheese Making: The Case of Western Hararage Region, Ethiopia

Abebe Getu Derso1, and Getachew Gashaw Dagnew2

1Department of Food Science and Nutrition, Oda Bultum University, Chiro, Ethiopia

2Department of Biology, Oda Bultum University, Chiro, Ethiopia

Pub. Date: March 26, 2019

Cite this paper:
Abebe Getu Derso and Getachew Gashaw Dagnew. Isolate and Extract for Milk Clotting Enzymes from the leaves of Moringa Oleifera, Carica Papaya and Mangifera Indica and Use in Cheese Making: The Case of Western Hararage Region, Ethiopia. Journal of Food and Nutrition Research. 2019; 7(3):244-254. doi: 10.12691/jfnr-7-3-10


Milk-clotting activity was screened from leaves of Carica papaya, Mangifera indica and Moringa oleifera in order to use the leaf with the highest milk-clotting activity as a source of the enzyme. The results of the present study showed that milk-clotting activity was only detected in the leaf extracts of Carica papaya and Moringa oleifera and the leaf extract from the leaf of Mangifera indica showed no activity. Ammonium sulfate precipitation was used in this study and the results showed that the highest milk-clotting activity was obtained with 40 % ammonium sulfate saturation. Maximum temperature for the crude extract of Carica papaya and Moringa olifera showed milk clotting activity at 70°C and 65°C respectively and also showed the highest clotting activity at a pH of 7.5 and 7 respectively. There was more than 80% retention of the milk-clotting activity of the crude extract of Carica papaya and Moringa olifera after 1h incubation at 60°C and 55°C respectively and the result also indicated that the crude extract retained more than 80% of its activity between pH 5 to pH 7.5 and pH 5 to pH 6 respectively. Finally the crude extract of Carica papaya and Moringa oleifera has a highest enzymatic activity at a concentration of 70 and 90 grams respectively.

activity milk clotting enzyme Carica papaya and Moringa oleifera

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[1]  Jacob, M., Jaros, D. & Rohm, H. 2011. Recent advances in milk clotting enzymes. International journal of dairy technology, 64, 14-33.
[2]  Lopez A, Teixeira G, Liberato MC, Pais M S and Clemente A (1998) New vegetal sources for milk clotting enzymes. Journal of Molecular Catalysis B: Enzymatic, 83-181.
[3]  Roseiro LB, Barbosa M, Ames JM, Wilbey RA (2003). Cheese making with vegetable coagulants-the use of Cynara L. for the production of ovine milk cheeses. Int. J. Dairy Technol. 56(2): 76-85.
[4]  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: 335-343.
[5]  Egito AS, Girardet JM, Laguna LE, Poirson C, Mollé D, Miclo L, Humbert G, Gaillard JL. (2007). Milkclotting activity of enzyme extracts from sunflower and albizia seeds and specific hydrolysis of bovine κ-casein. Int Dairy J 17: 816-825.
[6]  Duarte, A.R., Duarte, D.M.R., Moreira, K.A., Cavalcanti, M.T.H., Lima-Filho, J.L.D. and Porto, A.L F. (2009): Jacaratiacorumbensis O. Kuntze a new vegetable source for milk-clotting enzymes. Brazilian Archives of Biology and Technology, 52(1): 1-9.
[7]  Silva SV, Malcata FX. (2005). Studies pertaining to coagulant and proteolytic activities of plant proteases from Cynara cardunculus. Food Chem 89:19-26.
[8]  Uchikoba T, Kaneda M. (1996). Milk-clotting activity of cucumisin, a plant serine protease from melon fruit. Appl Biochem Biotechnol 56: 325-330.
[9]  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: 1070-1075.
[10]  Lo Piero AR, Puglisi I, Petrone G (2002) Characterization of “lettucine”, a serine-like protease from Lactuca sativa leave, as a novel enzyme for milk clotting. J Agric Food Chem 50:2439-2443.
[11]  Omotosho, O.E., Oboh, G. and Iweala, E.E.J. (2011), “Comparative effects of local coagulants on the nutritive value, in vitro digestibility and sensory properties of ‘wara’ cheese”, International Journal of Dairy Science, Vol. 6 No.1, pp. 58-65.
[12]  Arima.K, J. Yu, S. Iwasaki. (2007), Milk-clotting enzyme from Mucor pusillus var. Lindt, Methods in Enzymology 19; 446-459.
[13]  Elmazar M. M. E., El-Sayed S. T. & Al-Azzouny R. A. 2012. Screening Some Local Egyptian Seeds Extract for Milk-Clotting Activity and Physicochemical Characterization of Brassica Napus Seed Extract. Journal of Agriculture and Food Technology, 2(2), 28-34.
[14]  Mohamed Ahmed, I. A., Morishima, I., Babiker, E. E. & Mori, N. 2009a. Characterization of partially purified milk-clotting enzyme from Solanum dubium Fresen seeds. Food Chemistry, 116 (2), 395-400.
[15]  Mohamed Ahmed, I. A., Morishima, I., Babiker, E. E. & Mori, N. 2009b. Dubiumin, a chymotrypsin-like serine protease from the seeds of Solanum dubium Fresen. Phytochemistry, 70 (4), 483-491.
[16]  Nestor, G.-M., Rubi, C.-G. D. & Hector, J.-C. 2012. Exploring the MilkClotting Properties of a Plant Coagulant from the Berries of S. elaeagnifolium var. Cavanilles. Journal of Food Science, 71(1), C89-C94.
[17]  Anusha, R., SingH, M. K. & Bindhu O.S. 2014. Characterization of potential milk coagulants from Calotropis gigantean plant parts and their hydrolytic pattern of bovine casein. European Food Research and Technology, 238, 997-1006.
[18]  Guiama V. D., Libouga D. G., Ngah E. & Mbofung C. M. 2010. Milkclotting activity of berries extracts from nine Solanum plants. African Journal of Biotechnology, 9(25), 3911-3918.
[19]  Yousif, B. H., Mcmahon, J. D. & Shammet, M. K., 1996. Milk-clotting Enzyme from Solanum dobium Plant. International Dairy Journal, 6, 637-644.
[20]  Mazorra-Manzano, M. A., Perea-Gutiérrez, T. C., Lugosánchez, M. E., Ramirez-Suarez, J. C., Torres-Llanez, M. J., González-Córdova, A. F. & Vallejo-Cordoba, B. 2013. Comparison of the milk-clotting properties of three plant extracts. Food Chemistry, 141, 1902-1907.
[21]  Najera, A. I., De renobales, M. & Barron, L. J. R. 2003. Effects of pH, temperature, CaCl2 and enzyme concentrations on the rennet-clotting properties of milk: a multifactorial study. Food Chemistry, 80, 345-352.
[22]  Shehata, A.E., Fayet, E. A., Isamil, A. A. & Salem, M. M. 1996. Production and characterization of bacterial coagulants as calf rennet replace for Egyptian cheese making. Egyptian Journal of Food Science, 24 (3), 417-449.
[23]  Gupta, R.; Beg, q. K.; Lorenz, p. Bacterial alkaline proteases: molecular approaches and industrial applications. Applied Microbiology and Biotechnology, v.  59, p.  15-32, 2002. PMid: 12073127.
[24]  Tripathi, P.; Tomar, R.; Jagannadham, M. V. Purification and biochemical characterization of a novel protease streblin. Food Chemistry, v. 125, p. 1005-1012, 2011.
[25]  Devaraj, k. B.; Gowda, l. R.; Prakash, v. An unusual thermostable aspartic protease from the latex of Ficus racemosa (L.). Phytochemistry, v. 69, p. 647-655, 2008.
[26]  Demir, Y.; Güngör, A.; Duran, E.D.; Demir, 2008 N. Cysteine protease (capparin) from capsules of caper (Capparis spinosa). Food Technol. Biotechnol., 46, 286-291.
[27]  Harboe, M. K.; Budtz, P. The production, action and application of rennet and coagulants. Technology of Cheese making. Sheffield: Academic Press, 1999. p. 33-65.
[28]  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 32:413-419.