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Milk-Clotting and Proteolytic Properties of a Partially Purified Pepsin from Yellowfin Tuna (Thunnus albacares) and its Potential for Cheesemaking

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The dairy industry traditionally uses aspartic proteases from bovine and microbial sources for cheese processing, but there is interest in alternative enzyme sources to supply the large global demand for rennet. This study investigated the biochemical characterization of a partially purified pepsin of yellowfin tuna with milk-clotting activity (MCA), in addition to evaluating its potential use in the manufacture of fresh cheese through texturometric and organoleptic characterization. The molecular weight of tuna pepsin was 36 kDa. The optimal temperature of MCA was recorded at 60 °C. Caseinolytic activity and MCA of tuna pepsin peaked at pH 6. A high MCA was recorded when 0.018% CaCl2 was added to the reaction mixture. The electrophoretic profile of casein hydrolyzed by tuna pepsin showed a 14.8 kDa fragment, a molecular weight like that of para-κ-casein. Cheeses made from commercial chymosin and tuna pepsin showed a similar protein content (11% of total wet weight); however, cheese produced with pepsin had a higher fat content. The cohesiveness and chewiness of cheese made with partially purified pepsin were significantly higher than those made with chymosin. There were no differences in the sensory analysis between cheese made with both clotting agents; however, 60% of panelists preferred cheese made with chymosin. Pepsin extracted from yellowfin tuna stomach showed interesting milk-clotting properties with potential biotechnological use, which would contribute to the reusing and revalorization of byproducts derived from the tuna processing industry.

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Data Availability

The datasets generated during and/or analyzed during the current study are available from the corresponding author on reasonable request.


  • Abebe, B., & Emire, S. (2020). Manufacture of fresh cheese using east African Calotropis procera leaves extract crude enzyme as milk coagulant. Food Science & Nutrition, 8(9), 4831–4842.

    Article  CAS  Google Scholar 

  • Belenkaya, S. V., Rudometov, A. P., Shcherbakov, D. N., Balabova, D. V., Kriger, A. V., Belov, A. N., Koval, A.D., & Elchaninov, V. V. (2018). Biochemical properties of recombinant chymosin in alpaca (Vicugna pacos L.). Applied Biochemistry and Microbiology, 54(6), 569–576.

  • Ben Amira, A., 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.

    Article  CAS  Google Scholar 

  • Bradford, M. M. (1976). A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Analytical Biochemistry, 72(1–2), 248–254.

    Article  CAS  PubMed  Google Scholar 

  • Brewer, P., Helbig, N., & Haard, N. F. (1984). Atlantic cod pepsin—Characterization and use as a rennet substitute. Canadian Institute of Food Science and Technology Journal, 17(1), 38–43.

    Article  CAS  Google Scholar 

  • Burgess, R. R. (2009). Protein precipitation techniques. In R. R. Burgess & M. P. Deutscher (Eds.), Methods in enzymology (pp. 331–342). Academic Press.

    Google Scholar 

  • Calzada, J., del Olmo, A., Picon, A., Gaya, P., & Nuñez, M. (2015). Effect of high-pressure processing on the microbiology, proteolysis, biogenic amines and flavour of cheese made from unpasteurized milk. Food and Bioprocess Technology, 8, 319–332.

  • Caro, I., Soto, S., Fuentes, L., Gutiérrez-Méndez, N., García-Islas, B., Monroy-Gayosso, K. E., & Mateo, J. (2014). Compositional, functional and sensory characteristics of selected Mexican cheeses. Food and Nutrition Sciences, 5, 366–375.

    Article  Google Scholar 

  • Dagostin, J. L. A., Carpine, D., & Masson, M. L. (2013). Influence of acidification method on composition, texture, psychrotrophs, and lactic acid bacteria in minas frescal cheese. Food and Bioprocess Technology, 6, 3017–3028.

    Article  CAS  Google Scholar 

  • De la Parra, A. M., Rosas, A., Lazo, J. P., & Viana, M. T. (2007). Partial characterization of the digestive enzymes of Pacific bluefin tuna Thunnus orientalis under culture conditions. Fish Physiology and Biochemistry, 33(3), 223–231.

    Article  CAS  Google Scholar 

  • Drake, M. D., & Delahunty, C. M. (2017). Chapter 20 - sensory character of cheese and its evaluation. In Editor(s): P. L. H. McSweeney, P. F. Fox, P. D. Cotter, & D. W. Everett (Eds.), Cheese (4th ed., pp. 517–545), Academic Press.

  • Egito, A. S., Girardet, J. M., Laguna, L. E., Poirson, C., Mollé, D., Miclo, L., Humbert, G., & Gaillard, J. L. (2007). Milk-clotting activity of enzyme extracts from sunflower and albizia seeds and specific hydrolysis of bovine κ-casein. International Dairy Journal, 17(7), 816–825.

    Article  CAS  Google Scholar 

  • Feijoo-Siota, L., & Villa, T. G. (2011). Native and biotechnologically engineered plant proteases with industrial applications. Food and Bioprocess Technology, 4, 1066–1088.

    Article  CAS  Google Scholar 

  • Foegeding, E. A., & Drake, M. A. (2007). Invited review: Sensory and mechanical properties of cheese texture. Journal of Dairy Science, 90(4), 1611–1624.

    Article  CAS  PubMed  Google Scholar 

  • García-Gómez, B., Vázquez-Odériz, L., Muñoz-Ferreiro, N., Romero-Rodríguez, Á., & Vázquez, M. (2020). Rennet type and microbial transglutaminase in cheese: Effect on sensory properties. European Food Research and Technology, 246(3), 513–526.

    Article  Google Scholar 

  • González-Córdova, A. F., Yescas, C., Ortiz-Estrada, Á. M., Hernández-Mendoza, A., & Vallejo-Cordoba, B. (2016). Invited review: Artisanal Mexican cheeses. Journal of Dairy Science, 99(5), 3250–3262.

    Article  PubMed  Google Scholar 

  • González-Velázquez, D. A., Mazorra-Manzano, M. A., Martínez-Porchas, M., Huerta-Ocampo, J. A., Vallejo-Córdoba, B., Mora-Cortes, W. G., Moreno-Hernández, J. M., & Ramírez-Suarez, J. C. (2021). Exploring the milk-clotting and proteolytic activities in different tissues of Vallesia glabra: A new source of plant proteolytic enzymes. Applied Biochemistry and Biotechnology, 193(2), 389–404.

    Article  PubMed  Google Scholar 

  • Gutiérrez-Méndez, N., Troncoso-Reyes, N., & Leal-Ramos, M. Y. (2013). Texture profile analysis of Fresh cheese and Chihuahua cheese using miniature cheese models. Tecnociencia Chihuahua, 7(2), 65–74.

    Google Scholar 

  • Guerard, F., & Le Gal, Y. (1987). Characterization of a chymosin-like pepsin from the dogfish Scyliorhinus canicula. Comparative Biochemistry and Physiology. B, Comparative Biochemistry, 88(3), 823–827.

  • Guinee, T. P. (2007). 33 Why is CaCl2 often added to cheesemilk?. 69. In P. L. H. McSweeney (ED.), Cheese problems solved. CRC Press.

  • Hayaloglu, A. A., Karatekin, B., & Gurkan, H. (2014). Thermal stability of chymosin or microbial coagulant in the manufacture of Malatya, a Halloumi type cheese: Proteolysis, microstructure and functional properties. International Dairy Journal, 38(2), 136–144.

    Article  CAS  Google Scholar 

  • Homaei, A., Lavajoo, F., & Sariri, R. (2016). Development of marine biotechnology as a resource for novel proteases and their role in modern biotechnology. International Journal of Biological Macromolecules, 88, 542–552.

    Article  CAS  PubMed  Google Scholar 

  • Ismail, B., Mohammed, H., & Nair, A. J. (2019). Influence of proteases on functional properties of food. In B. Parameswaran, S. Varjani (Eds.), Green Bio-processes. Energy, Environment, and Sustainability. Springer.

  • Jay, J. M. (1998). High-temperature food preservation and characteristics of thermophilic microorganisms. In: J. M. Jay (ED.), Modern Food Microbiology. Food Science Texts Series. Springer.

  • Khanal, B. K. S., Bhandari, B., Prakash, S., Liu, D., Zhou, P., & Bansal, N. (2018). Modifying textural and microstructural properties of low fat Cheddar cheese using sodium alginate. Food Hydrocolloids, 83, 97–108.

    Article  CAS  Google Scholar 

  • Khanal, B. K. S., Budiman, C., Hodson, M. P., Plan, M. R., Prakash, S., Bhandari, B., & Bansal, N. (2019). Physico-chemical and biochemical properties of low fat cheddar cheese made from micron to nano sized milk fat emulsions. Journal of Food Engineering, 242, 94–105.

    Article  Google Scholar 

  • Laemmli, U. K. (1970). Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature, 227(5259), 680–685.

    Article  CAS  PubMed  Google Scholar 

  • Mazorra-Manzano, M. A., Moreno-Hernández, J. M., Ramírez-Suarez, J. C., de Jesús Torres-Llanez, M., González-Córdova, A. F., & Vallejo-Córdoba, B. (2013a). Sour orange Citrus aurantium L. flowers: A new vegetable source of milk-clotting proteases. LWT-Food Science and Technology, 54(2), 325–330.

  • Mazorra-Manzano, M. A., Perea-Gutiérrez, T. C., Lugo-Sánchez, M. E., Ramirez-Suarez, J. C., Torres-Llanez, M. J., González-Córdova, A. F., & Vallejo-Cordoba, B. (2013b). Comparison of the milk-clotting properties of three plant extracts. Food Chemistry, 141(3), 1902–1907.

    Article  CAS  PubMed  Google Scholar 

  • Mazorra-Manzano, M. A., Ramírez-Suarez, J. C., & Yada, R. Y. (2018a). Plant proteases for bioactive peptides release: A review. Critical Reviews in Food Science and Nutrition, 58(13), 2147–2163.

    Article  CAS  PubMed  Google Scholar 

  • Mazorra-Manzano, M. A., Moreno-Hernández, J. M., Ramírez-Suarez, J. C. (2018b). Milk-Clotting Plant Proteases for Cheesemaking. In: M. G. Guevara, & G. R. Daleo (Eds.), Biotechnological applications of plant proteolytic enzymes. Springer, Cham.

  • Moreno-Hernández, J. M., Pérez, M. D. J. B., Osuna-Ruiz, I., Salazar-Leyva, J. A., Ramirez-Suarez, J. C., & Mazorra-Manzano, M. Á. (2017). Exploring the milk-clotting properties of extracts from Bromelia pinguin fruit. Journal of Microbiology, Biotechnology and Food Sciences, 7(1), 62–66.

    Article  Google Scholar 

  • Nájera, 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(3), 345–352.

    Article  Google Scholar 

  • Nájera-Domínguez, C., Gutiérrez-Méndez, N., Carballo-Carballo, D. E., Peralta-Pérez, M. R., Sánchez-Ramírez, B., Nevarez-Moorillón, G. V., Quintero-Ramos, A., García-Triana, A., & Delgado, E. (2022). Milk-gelling properties of proteases extracted from the fruits of solanum Elaeagnifolium cavanilles. International Journal of Food Science.

  • Nalinanon, S., Benjakul, S., & Kishimura, H. (2010a). Biochemical properties of pepsinogen and pepsin from the stomach of albacore tuna (Thunnus alalunga). Food Chemistry, 121(1), 49–55.

    Article  CAS  Google Scholar 

  • Nalinanon, S., Benjakul, S., & Kishimura, H. (2010b). Purification and biochemical properties of pepsins from the stomach of skipjack tuna (Katsuwonus pelamis). European Food Research and Technology, 231(2), 259–269.

    Article  CAS  Google Scholar 

  • Nicosia, F. D., Puglisi, I., Pino, A., Caggia, C., & Randazzo, C. L. (2022). Plant Milk-Clotting Enzymes for Cheesemaking. Foods, 11(6), 871.

    CAS  PubMed  Google Scholar 

  • Norris, E. R., & Mathies, J. C. (1953). Preparation, properties, and crystallization of tuna pepsin. Journal of Biological Chemistry, 204(2), 673–680.

    Article  CAS  PubMed  Google Scholar 

  • Osuna-Ruiz, I., Espinoza-Marroquin, M. F., Salazar-Leyva, J. A., Peña, E., Álvarez-González, C. A., Bañuelos-Vargas, I., & Martínez-Montano, E. (2019). Biochemical characterization of a semi-purified aspartic protease from sea catfish Bagre panamensis with milk-clotting activity. Food Science and Biotechnology, 28(6), 1785–1793.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Pedrero, F. D. L., Pangborn, R. M. (1989) Evaluación sensorial de los alimentos: Métodos analíticos. Alhambra Mexicana.

  • Pereira, N. D. L. A., & Fernández‐Gimenez, A. V. (2017a). An alternative coagulant in cheese manufacturing. In M. H. F. Henriques & C. J. D. Pereira (Eds.), Cheese production, consumption and health benefits. Nova Science Publishers, Inc.

  • Pereira, N. D. L. A., & Fernández-Gimenez, A. V. (2017b). Exogenous enzymes in dairy technology: Acidic proteases from processing discards of shrimp Pleoticus muelleri and their use as milk-clotting enzymes for cheese manufacture. International Journal of Food Science & Technology, 52(2), 341–347.

    Article  CAS  Google Scholar 

  • Picon, A., Alonso, R., Gaya, P., & Nuñez, M. (2013). High-pressure treatment and freezing of raw goat milk curd for cheese manufacture: Effects on cheese characteristics. Food and Bioprocess Technology, 6, 2820–2830.

  • Rios-Herrera, G. D., Ruiz, I. O., Hernández, C., Valdez-Ortiz, A., Sandoval-Gallardo, J. M., Martínez-Montaño, E., Ramírez-Pérez, J. S., & Salazar-Leyva, J. A. (2019). Chihuil sea catfish Bagre panamensis viscera as a new source of serine proteases: Semi-purification, biochemical characterization and application for protein hydrolysates production. Waste and Biomass Valorization, 11, 5821–5833.

    Article  Google Scholar 

  • Rossano, R., Larocca, M., Lamaina, A., Viggiani, S., & Riccio, P. (2011). The hepatopancreas enzymes of the crustaceans Munida and their potential application in cheese biotechnology. LWT-Food Science and Technology, 44(1), 173–180.

    Article  CAS  Google Scholar 

  • Rossano, R., Piraino, P., D’Ambrosio, A., O’Connell, O. F., Ungaro, N., McSweeney, P. L. H., & Riccio, P. (2005). Proteolysis in miniature cheddar-type cheeses manufactured using extracts from the crustacean Munida as coagulant. Journal of Biotechnology, 120(2), 220–227.

    Article  CAS  PubMed  Google Scholar 

  • Sato, K., Goto, K., Suzuki, A., Miura, T., Endo, M., Nakamura, K., & Tanimoto, M. (2018). Characterization of a milk-clotting enzyme from Hericium erinaceum and its proteolytic action on bovine caseins. Food Science and Technology Research, 24(4), 669–676.

    Article  CAS  Google Scholar 

  • Shamsuzzaman, K., & Haard, N. F. (1983). Evaluation of harp seal gastric protease as a rennet substitute for Cheddar cheese. Journal of Food Science, 48(1), 179–182.

    Article  CAS  Google Scholar 

  • Shan, J., Zhang, Y., Liang, J., & Wang, X. (2020). Characterization of the processing conditions upon textural profile analysis (tpa) parameters of processed cheese using near-infrared hyperspectral imaging. Analytical Letters, 53(8), 1190–1203.

    Article  CAS  Google Scholar 

  • Simmons, M., Ru, G., Casalone, C., Iulini, B., Cassar, C., & Seuberlich, T. (2018). Discontools: Identifying gaps in controlling bovine spongiform encephalopathy. Transboundary and Emerging Diseases, 65, 9–21.

    Article  PubMed  Google Scholar 

  • Slamani, R., Labadi, R., Brahim Errahmani, M., & Bellal, M. M. (2018). Purification and characterisation of milk-clotting and caseinolytic activities of pepsin isolated from adult sheep abomasa. International Journal of Dairy Technology, 71(3), 764–770.

    Article  CAS  Google Scholar 

  • Soodam, K., Ong, L., Powell, I. B., Kentish, S. E., & Gras, S. L. (2014). The effect of milk protein concentration on the microstructure and textural properties of full fat cheddar cheese during ripening. Food and Bioprocess Technology, 7(10), 2912–2922.

    Article  CAS  Google Scholar 

  • Sun-Waterhouse, D., Zhao, M., & Waterhouse, G. I. (2014). Protein modification during ingredient preparation and food processing: Approaches to improve food processability and nutrition. Food and Bioprocess Technology, 7(7), 1853–1893.

    Article  CAS  Google Scholar 

  • Tavares, J. F. P., Baptista, J. A. B., & Marcone, M. F. (1997). Milk-coagulating enzymes of tuna fish waste as a rennet substitute. International Journal of Food Sciences and Nutrition, 48(3), 169–176.

    Article  CAS  PubMed  Google Scholar 

  • Torres-González, J. D., González-Morelos, K. J., & Acevedo-Correa, D. (2015). Análisis del perfil de textura en frutas, productos cárnicos y quesos. ReCiTeIA, Revisiones De La Ciencia, Tecnología e Ingeniería De Los Alimentos, 14(2), 63–75.

    Google Scholar 

  • Tunick, M. H., & Van Hekken, D. L. (2010). Rheology and texture of commercial queso fresco cheeses made from raw and pasteurized milk. Journal of Food Quality, 33, 204–215.

    Article  Google Scholar 

  • Uniacke-Lowe, T., & Fox, P. F. (2017). Chapter 4 - Chymosin, pepsins and other aspartyl proteinases: Structures, functions, catalytic mechanism and milk-clotting properties. In P. L. H. McSweeney, P. F. Fox, P. D. Cotter & D. W. Everett (Eds.), Cheese (pp. 69–113). Academic Press.

  • Walter, H. E. (1984). Proteinases: Methods with haemoglobin, casein and azocoll as substrates. In H. U. Bergmeyer (Ed.), Methods of enzymatic analysis (pp. 270–277). Verlag Chemie, Weinheim.

  • Zhao, L., Budge, S. M., Ghaly, A. E., Brooks, M. S., & Dave, D. (2011). Extraction, purification and characterization of fish pepsin: A critical review. Journal of Food Processing and Technology, 2(6), 2–6.

    Article  Google Scholar 

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The authors thank to Ingeniería Industrial del Pacífico, S.A. (IIPSA) for their kind donation of fishery by products. Also, the authors thank María Elena Sánchez Salazar for her editorial work in English.

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Emmanuel Martínez-Montaño: conceptualization, investigation, experimentation, and original draft preparation; Idalia Osuna-Ruíz: conceptualization, experimentation, and original draft preparation; Jesús Aarón Salazar-Leyva: conceptualization, manuscript reviewing, and resource management; Reyna Tiznado Garzón: experimentation and data analysis; María de Lourdes García-Magaña: experimentation, data analysis, and original draft preparation; Israel Benítez García: manuscript reviewing; Jesús Martín Moreno-Hernández: manuscript reviewing; Isaura Bañuelos-Vargas: experimentation and data analysis; Crisantema Hernández: experimentation and data analysis.

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Correspondence to Emmanuel Martínez-Montaño.

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Osuna-Ruíz, I., Tiznado-Garzón, R., Salazar-Leyva, J.A. et al. Milk-Clotting and Proteolytic Properties of a Partially Purified Pepsin from Yellowfin Tuna (Thunnus albacares) and its Potential for Cheesemaking. Food Bioprocess Technol 16, 1769–1780 (2023).

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