Thermostable trypsin‐like protease by Penicillium roqueforti secreted in cocoa shell fermentation: Production optimization, characterization, and application in milk clotting

• Published in: Biotechnology and applied biochemistry Vol. 69; no. 5; pp. 2069 - 2080
• Main Authors: Nogueira, Laísa Santana, Tavares, Iasnaia Maria de Carvalho, Santana, Nívio Batista, Ferrão, Sibelli Passini Barbosa, Teixeira, Jabson Meneses, Costa, Floriatan Santos, Silva, Tatielle Pereira, Pereira, Hugo Juarez Vieira, Irfan, Muhammad, Bilal, Muhammad, Oliveira, Julieta Rangel, Franco, Marcelo
• Format: Journal Article
• Language: English
• Published: Calgary Wiley Subscription Services, Inc 01-10-2022
• Subjects: Biochemical characteristics; biochemical characterization; Biochemistry; Carbon dioxide; Cheese; Clotting; Coagulation; Cobalt; Cocoa; Dairy products; Design of experiments; Doehlert design; Enzymatic activity; Ethanol; Experimental design; Fabrication; Fermentation; Food industry; Fungi; Hexanes; Milk; milk clotting; Optimization; Penicillium roqueforti; Protease; Proteinase; Substrates; Trypsin; trypsin‐like protease
• ISSN: 0885-4513; 1470-8744
• DOI: 10.1002/bab.2268

The increased demand for cheese and the limited availability of calf rennet justifies the search for milk‐clotting enzymes from alternative sources. Trypsin‐like protease by Penicillium roqueforti was produced by solid‐state fermentation using cocoa shell waste as substrate. The production of a crude enzyme extract that is rich in this enzyme was optimized using a Doehlert‐type multivariate experimental design. The biochemical characterization showed that the enzyme has excellent activity and stability at alkaline pH (10–12) and an optimum temperature of 80°C, being stable at temperatures above 60°C. Enzymatic activity was maximized in the presence of Na+ (192%), Co2+ (187%), methanol (153%), ethanol (141%), and hexane (128%). Considering the biochemical characteristics obtained and the milk coagulation activity, trypsin‐like protease can be applied in the food industry, such as in milk clotting and in the fabrication of cheeses.
The increased demand for cheese and the limited availability of calf rennet justifies the search for milk‐clotting enzymes from alternative sources.