Fabricação de produtos lácteos com proteases ácidas sintetizadas por lentinula da floresta amazônica

Abstract

The dairy sector is of great importance to the global economy, providing foods that are highly rich in proteins. By-products of the dairy industry, such as whey, are sources of bioactive peptides and are used in the production of foods with high nutritional quality. The need for milk-clotting enzymes has increasingly driven research toward the search for renewable sources. Several mushroom species are potential alternatives for the production of milk-clotting enzymes, as they naturally synthesize these biocatalysts with biochemical characteristics suitable for the development of dairy products. Therefore, the objective of this study was to evaluate the cultivation conditions for the synthesis of high levels of milk-clotting enzymes by Lentinus crinitus DPUA 1693 under standardized conditions and their application in the development of innovative dairy products. In this study, a mycelial strain of Lentinus crinitus DPUA 1693, provided by the DPUA Culture Collection of the Federal University of Amazonas, Manaus, Amazonas, Brazil, was used. The bioprocess was conducted using submerged fermentation with Potato Dextrose Agar (PDA+YE), Sabouraud Agar (SAB+YE), Corn Extract (MIL+YE), supplemented with 0.5% (w/v) yeast extract (YE), and Malt, Glucose, Yeast Extract, Peptone (MGYP) medium. Fermentation was conducted at 150 rpm and 30 °C for 8 days. To obtain an enzymatic extract with a high milk-clotting activity-to-proteolytic activity ratio, bioprocess parameters influencing milk coagulation were evaluated, including inoculum age, fermentation time, inoculum size, and carbon and nitrogen source concentrations, followed by scale-up. Proteolytic and milk-clotting activities were determined using 1% (w/v) azocasein solution and 10% (w/v) skim milk as substrates, respectively. Subsequently, a type of cheese was produced using the standardized L. crinitus milk clotting enzymes, as well as a functional dairy beverage containing whey and mycelial biomass of the edible mushroom. These food products were evaluated for their proximate composition and microbiological characteristics. The highest milk-clotting activity (67.94 U/mL), accompanied by low proteolytic activity (3.58 U/mL), was obtained using sucrose and meat peptone as nutritional sources, resulting in a high milk-clotting activity to-proteolytic activity ratio (18.97), which is considered essential for efficient milk coagulation. The most effective parameters for milk-clotting enzyme production were an inoculum age of 16 days, an inoculum size of 20.0%, and a fermentation time of 10 days. Proteolytic activity was particularly favored under the conditions of a 20.0% inoculum size, 16-day-old inoculum, and 14 days of fermentation. The enzymes showed optimum activity at pH 5.0 and a temperature of 50 °C. Their application in cheese and dairy beverage production demonstrated technological feasibility, with the final products presenting adequate nutritional composition, microbiological safety, and promising functional characteristics.