Produção, otimização e caracterização de coagulantes produzidos por espécies de Aspergillus

Abstract

The rennet extracted from calf, a source of coagulants in bovine milk, in recent years has not met the requirements of some consumer groups, due to various causes, such as changing eating habits and religious beliefs. Worldwide, several researches are being carried out in the search for new sources of enzymes that can meet the needs of the cheese market. Based on the industrial importance of fungi, this study aimed to evaluate species of the genus Aspergillus to select a strain that produces coagulant enzymes from bovine milk and to investigate regulatory mechanisms of coagulant synthesis to identify characteristics for industrial application. The strains: A. clavato flavus DPUA 929, A. fischeri DPUA 653, A. flavo furcatis DPUA 1451, A. flavo furcatis DPUA 1455, A. japonicus DPUA 542, A. japonicus DPUA 1727, A. melleus DPUA 323, A. niger DPUA 398, A. niger DPUA 399, A. ostianus DPUA 315, A. oryzae DPUA 541, A. oryzae DPUA 1624 and A. wentii DPUA 1725, are the 13 representatives of the genus Aspergillus. In the first step, the lines were authenticated based on macromorphological and micromorphological characteristics. Pure and viable cultures were used in the selection of a species that produced proteases and coagulants by submerged fermentation under standardized conditions. The enzyme extract recovered from fermentation was used to determine the activity of proteases and later partial characterization regarding the effect of pH and temperature on the activity and stability and effect of metal ions and inhibitors on the enzyme activity. And to identify the nutritional requirements, they were selected as a source of carbon, starch and as a source of nitrogen, casein, associated with three different incubation times, and for the synthesis of coagulants by Aspergillus, in a complete factorial design 23 , with 3 central points, totaling 11 experiments. The extract with the highest coagulant ratio was characterized as to the effect of pH and temperature on enzyme activity and stability. The effect of inhibitors and metal ions on clotting activity was also evaluated, in addition to the evaluation of in vitro cytotoxicity using human fibroblasts. Most of the tested strains produced proteases in liquid medium. Aspergillus flavo furcatis DPUA 1451 was identified as a significant source of serine proteases and metalloproteases that express significant catalytic activity at pH 5.0 and 50 °C, with stability at pH 7 to 9, up to 40 °C, in 60 minutes, which demonstrates the potential use of these biocatalysts in the food industry, mainly for cheese production.