Optimization of the production of a new milk clotting enzyme by Aspergillus flavus thermophile on agro-industrial residues medium and characterization of the enzyme

Abstract

This study focused on the optimization of the production of a milk clotting protease by Aspergillus flavus, isolated from a thermal station. The fermentation was carried out on wheat bran as a medium, followed by a partial characterization of the enzyme. The fungal isolate was screened for protease production based on clear zone hydrolysis on skim milk agar, and then it was identified as belonging to the genus Aspergillus based on its morphological characteristics. This identification was confirmed by molecular analyzes with 100% similarity with the A. flavus strain. After 7 days of fermentation, the crude extract is recovered by simple filtration. The production and optimization of the milk coagulant protease using wheat bran and agro-industrial waste as a medium was carried out using a statistical method of experimental design based on the Plackett and Burman matrices. The results showed that the optimal fermentation medium consisted of wheat bran (4% w/w), incubated at a temperature of 30 °C, with a pH of 5.0 after 6 days of fermentation. The enzyme was partially purified by ammonium sulfate precipitation, yielding an estimated proteolytic activity of 2,985 U. The partially purified protease exhibits optimal activity at pH 5 with hemoglobin as a substrate and an optimal temperature of 50 °C. The Lineweaver-Burk plot showed a KM value of 4.83 g/L and a Vm of 1,560 U. This enzyme causes very rapid coagulation of fresh cow’s milk with an optimum at 45 °C, a pH 7 and a concentration of 50 mM of calcium.