AP-04: Aspergillus niveus Protease as coagulant agent. Extraction and characterization of milk clotting activity

Abstract

Subject description: In the cheese industry, aspartyl proteases are used as a coagulating agent and rennet was the first to be used. It is an enzyme extracted from the stomach of young ruminants. Indeed, it is relatively expensive and faces the constraint of sacrificing these animals. This situation has given impetus to research in order to replace this clotting agent with other less expensive proteases such as microbial one. Objectives: An acid protease produced by a fungal species isolated from an extreme environment, a thermal soil in the region of Teleghma (Mila), is studied as a source of enzymes to be used in cheese making. Methods: Phenotypic and molecular identification showed that it is Aspergillus niveus. This strain of fungus exhibited significant proteolytic activity on milk agar. A fermentation is carried out on a liquid medium based on wheat bran enriched with the composition gram per liter 5g of (NH4) 2SO4; 2g of KH2PO4; 1g of K2HPO4 and 0.5g MgSO4, at 30°C and pH 6 with stirring at 150rpm/min) . After 7 days of incubation, the partially purified enzymatic extract is recovered by simple filtration followed by fractionated precipitation with ammonium sulphate. Results and discussion: The partial purification shows a proteolytic activity of 2598UI with a coagulant activity of 0.221 UP/ml under standard conditions. This activity was strongly dependent on the pH and temperature of the milk; it showed optimal milk coagulation at pH 5 and 60°C. The RS rennet strength of this extract increased hyperbolically with increasing calcium concentrations and the concentration was saturated at 50 mM. Indeed, a concentration of 50mM NaCl is accompanied by a decrease in coagulant activity. Conclusion: these results are encouraging; the use of a cheap and available substrate such as wheat bran saves the cost price of the production of a milk coagulating protease on an industrial scale