Production of milk coagulating proteases by amazonian edible mushroom of the Polyporaceae family (Basidiomycota)
Resumo
Cheese is a dairy product that plays a fundamental role in the global economy. However, factors such as high rennet prices and religious and ethnic motivations against the use of animal-derived enzymes encourage the search for new coagulation agents. In this context, edible mushrooms have been emerging as promising sources of coagulant proteases for cheese production. The aim of this research was to evaluate the production of coagulant proteases and determine the parameters that influence the synthesis of these biocatalysts by Lentinus crinitus DPUA 1693. For enzyme production in liquid medium, the mushroom was cultivated in Potato Extract (PDA), Sabouraud Extract (SAB), Corn Meal (CMA), supplemented with yeast extract (YE) 0.5% (w/v) and Glucose, Yeast Extract, and Peptone (MGYP+YE). The proteolytic and coagulant activities were determined using 1% (w/v) azocasein solution and 10% (w/v) skimmed milk, respectively, as substrate. The parameters that interfere in the coagulant production were evaluated, such as inoculum size, fermentation time, and inoculum age. The protein profile of the crude extract and the zymogram to confirm the proteolytic action of the extract were determined by non-denaturing polyacrylamide gel electrophoresis (SDS-PAGE). The significant values of coagulant activity (18.21 U mL-1) in SAB and MGYP, as well as protease activity (1.64 U mL-1 and 1.66 U mL-1), respectively, with the ratio and strong clot. The significant coagulant production was verified under the following conditions: inoculum size (20%), fermentation time (10 days), and inoculum age (16 days). The significant values of activity and coagulant ratio, as well as the evidence of proteolytic action in zymograms, indicate the technological quality of the application of enzymes in the industrial sector. Lentinus crinitus DPUA 1693 is a natural source of coagulant with characteristics for the application of enzymes in the dairy sector.
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Referências
Aljammas, H. A., Yazji, S., & Azizieh, A. (2022). Optimization of protease production from Rhizomucor miehei Rm4 isolate under solid-state fermentation. Journal of Genetic Engineering and Biotechnology, 20(1), 1-13. https://doi.org/10.1186/s43141-022-00358-9
Arima, K., Yu, J., & Iwasaki, S. (1970). Milk-Clotting enzyme from Mucor pussilus var. Lindt. Methods in Enzimology, 19, 446-459. https://doi.org/10.1016/0076-6879(70)19033-1
Bakr, A., Ibrahim, O., El-Ghandour, A. E.-S., & El-Deeb, N. (2022). Purification and characterization of milk clotting enzyme from edible mushroom (Pleurotus florida). Letters in Applied NanoBioScience, 11(2), 3362-3373. https://doi.org/10.33263/LIANBS112.33623373
Batista, S. C. P., Prado, F. B., Brito, A. K. P., Coelho, M. P. S. L. V., Castillo, T. A., Martim, S. R., & Teixeira, M. F. S. (2021). Residual biomass from Amazon’s horticultural residues processing for mycelial growth and production of proteases by an edible mushroom specie. Research, Society and Development, 10(3), 1-11. https://doi.org/10.33448/rsd-v10i3.13393
Bensmail, S., Mechakra, A., & Fazouane-Naimi, F. (2015). Optimization of milk-clotting protease production by a local isolate of Aspergillus niger FFB1 in solid-state fermentation. Journal of Microbiology, Biotechnology and Food Sciences, 4(5), 467-472. https://doi.org/10.15414/jmbfs.2015.4.5.467-472
Braga, R. S. B., Brito, E. C. M., Souza, R. A. T., Teixeira, F. S., & Martim, S. R. (2020). Lentinus villosus Klotzsch (1833) AM 169: a natural and renewable source of alkaline protease. Brazilian Journal of Development, 6(11), 85867-85883. https://doi.org/10.34117/bjdv6n11-127
El-Baky, H. A., Linke, D., Nimtz, M., & Berger, R. G. (2011). PsoP1, a milk-clotting aspartic peptidase from the basidiomycete fungus Piptoporus soloniensis. Journal of Agricultural and Food Chemistry, 59(18), 10311-10316. https://doi.org/10.1021/jf2021495
Lebedeva, G. V., & Proskuryakov, M. T. (2009). Purification and characterization of milk- clotting enzymes from oyster mushroom (Pleurotus ostreatus (Fr.) Kumm). Applied Biochemistry and Microbiology, 45(6), 623-625. https://doi.org/10.1134/S0003683809060088
Leighton. T. J., Doi, R. H.,Warren, R. A. J., & Kelln, R. A. (1973). The relationship of serine protease activity to RNA polymerase modification and sporulation in Bacillus subtilis. Journal Molecular Biology, 76(1), 103-122. https://doi.org/10.1016/0022-2836(73)90083-1
Magalhães, A. A. S., Silva, T. A., Teixeira, M. F.S., Cruz Filho, R. F., Silva, S. D., Gomes, D. M. D., & Pereira, J. O. (2019). Production and characterization of proteolytic enzymes from Lentinus crinitus (L.) Fr. 1825 DPUA 1693 from the Amazon biome (Polyporaceae). Boletim do Museu Paraense Emílio Goeldi. Ciências Naturais, 14(3), 453-461. https://doi.org/10.46357/bcnaturais.v14i3.231
Majumder, R., Banik, S. P., & Khowala, S. (2015). Purification and characterisation of κ-casein specific milk-clotting metalloprotease from Termitomyces clypeatus MTCC 5091. Food Chemistry, 173, 441-448. https://doi.org/10.1016/j.foodchem.2014.10.027
Mamo, J., Kangwa, M., Fernandez-Lahore, H. M., & Assefa, F. (2020). Optimization of media composition and growth conditions for production of milk-clotting protease (MCP) from Aspergillus oryzae DRDFS13 under solid-state fermentation. Brazilian Journal of Microbiology, 51(2), 571–584. https://doi.org/10.1007/s42770-020-00243-y
Martim, S. R., Silva, L. S. C., Souza, L. B. Carmo, E. J., Alecrim, M. M., Vasconcellos, M. C., Oliveira, I. M. A., & Teixeira, M. F. S. (2017). Pleurotus albidus: A new source of milk-clotting proteases. African Jornal of Microbiology Research, 11(17), 660-667. https://doi.org/10.5897/AJMR2017.8520
Martim, S. R., Silva, L. S. C., Alecrim, M. M., Teixeira, L. S., & Teixeira, M. F. S. (2021). Milk-clotting proteases from Pleurotus albidus: an innovative alternative for the production of Minas frescal cheese. Acta Scientiarum. Biological Sciences, 43(1), 1-12. https://doi.org/10.4025/actascibiolsci.v43i1.57275
Minitab LLC (2017). Minitab statistical software: version 18.0 [software]. Minitab LLC. https://www.minitab.com/pt-br/
Ning, Y., Yang, H., Weng, P., & Wu, Z. (2021). Zymogram analysis and identification of the extracellular proteases from Bacillus velezensis SW5. Applied Biochemistry and Microbiology, 57(1), 27-37. https://doi.org/10.1134/S0003683821100082
Nolli, M. M., Contato, A. G., Brugnari ,T., Buzzo, A. J. R., Aranha, G. M., Inácio, F. D., Peralta, R. M., & Souza, C. G. M. (2022). Evaluation of the milk clotting potential and characterization of proteases from Aspergillus sp. and Pleurotus albidus. Acta Scientiarum. Technology, 44(2), 1-10. https://doi.org/10.4025/actascitechnol.v44i2.57766
Okamura-Matsui, T., Takemura, K., Sera, M., Takeno, T., Noda, H., Fukuda, S., & Ohsugi, M. (2001). Characteristics of a cheese-like food produced by fermentation of the mushroom Schizophyllum commune. Journal of Bioscience and Bioengineering, 92(1), 30-32. https://doi.org/10.1016/s1389-1723(01)80194-8
Pacifico, S., Caputo, E., Piccolella, S., & Mandrich, L. (2024). Exploring new fruit- and vegetable-derived rennet for cheese making. Applied Sciences, 14(6), 1-14. https://doi.org/10.3390/app14062257
Qasim, F., Diercks-Horn, S., Gerlach, D., Schneider, A., & Fernandez-Lahore, H. M. (2022). Production of a novel milk-clotting enzyme from solid-substrate Mucor spp. culture. Journal of Food Science, 87(10), 4348-4362. https://doi.org/10.1111/1750-3841.16307
Ravikumar, G., Gomathi, D., Kalaiselvi, M., & Uma, C. (2012). A protease from the medicinal mushroom Pleurotus sajor-caju; production, purification and partial characterization. Asian Pacific Journal of Tropical Biomedicine, 2(1), 411-417. https://doi.org/10.1016/S2221-1691(12)60198-1
Roohi, Zaheer, M. R., & Gupta, A. (2019). Current development and future perspectives of microbial enzymes in the dairy industry. In Enzymes in food biotechnology: Production, applications, and future prospects (pp. 287-302). Academic Press. https://doi.org/10.1016/B978-0-12-813280-7.00017-7
Sathya, R., Pradeep, B. V., Angayarkanni, J., Palaniswamy, M. (2009). Production of milk clotting protease by a local isolate of Mucor circinelloides under SSF using agro-industrial wastes. Biotechnology and Bioprocess Engineering, 14, 788-794. https://doi.org/10.1007/s12257-008-0304-0
Shamtsyan, M., Dmitriyeva, T., Kolesnikov, B., Denisova, N. (2014). Novel milk-clotting enzyme produced by Coprinus lagopides basidial mushroom. LWT-Food Science and Technology, 58(2), 343-347. https://doi.org/10.1016/j.lwt.2013.10.009
Shata, H. M. (2005). Extraction of milk-clotting enzyme produced by solid state fermentation of Aspergillus oryzae. Polish Journal of Microbiology, 54(3), 241-247.
Song, P., Zhang, X., Wang, S., Xu, W., Wang, F., Fu, R., & Wei, F. (2023). Microbial proteases and their applications. Frontiers in Microbiology, 14, 1-24. https://doi.org/10.3389/fmicb.2023.1236368
Zhang, Y., Wang, J., He, J., Liu, X., Sun, U., Song, X., & Wu, Y. (2023). Characteristics and application in cheese making of newly isolated milk-clotting enzyme from Bacillus megaterium LY114. Food Research International, 172, 113202. https://doi.org/10.1016/j.foodres.2023.113202
Copyright (c) 2025 Samara Claudia Picanço Batista, Ana Kezia Pimentel de Brito, Laynah Pimenta, Elliza Emily Perrone Barbosa, Salomão Rocha Martim, Tiara Sousa Cabral , Edson Junior do Carmo, Maria Francisca Simas Teixeira (Autor)
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