Breeding Of Aspergillus Oryzae With High Acid Protease Activity For Soy Sauce Fermentation

Soy sauce, a kind of nutritious seasoning, is widely used all over the world. During its fermentation the pH value of soy sauce mash drops fast with the accumulation of organic acids, and the activities of neutral and alkaline proteases decline rapidly in the later fermentation, which restrain the decomposition of proteins and the quality of soy sauce. For this reason, the breeding of high acid protease activity strain is of vital importance for soy sauce fermentation industry.In this dessertation, a high acid protease producing strain, mutant B-2 (CGMCC 8199), was obtained by the Atmospheric and Room Temperature Plasma (ARTP) treatment of Aspergillus oryzae 3.042 (CGMCC 3.00951) spores, and the fermentation conditions of B-2 were optimized. Mutant B-2 was also used for the lab-scale production of low-salt solid-state soy sauce. The main contents and results were as follows:(1) Spores of A. oryzae 3.042 were mutated, and mutant B-2 was obtained by pre-screening and re-screening. The activity of acid protease was measured in the subculture. The differences between mutant B-2 and A. oryzae 3.042 were compared, such as colonial morphology, spore morphology, spore germination ratio and strain growth curve. Besides, the variation of alkaline, neutral and acid protease was compared between the two strains.(2) In order to improve the acid protease activity, the culture conditions and fermentation conditions were optimized. The optimized culture conditions were determined: bran was carbon source, bean pulp was nitrogen source, bran:bean pulp=7:3, NaCl was 0.1%, Tween-80 was 1%, water addition was 36%, temperature was 30℃, fermentation period was 48h, inoculum size was 1×106 spores/35g koji.(3) The ammoniacal nitrogen, total nitrogen, reducing sugar, total acid and soluble salt-free solid contents were compared between A. oryzae 3.042 and mutant B-2. The total nitrogen increased 16%, and soluble salt-free solid increased 8% in soy sauce fermented by mutant B-2.(4) Aspergillus niger FND-A47 was mixed with A. oryzae 3.042 or mutant B-2 for koji-making and the amino acid composition of soy sauce was measured. The contents of flavour amino acid, essential amino acid and total amino acid were significantly improved in soy sauce fermented by A. oryzae 3.042 and A. niger FND-A47 mixture.The physical and chemical parameters of lab-scale prepared soy sauce were analysed. It indicated that mutant B-2 which was provided with improved acid protease activity played a positive role in soy sauce fermentation, and the mixed culture of mutant B-2 and A. niger FND-A47 was efficient for the quality of soy sauce.