Mutation Breeding Of Aspergillus Oryzae With High-yielding Protease And Optimizing Of Culture Conditions

Protease is biological catalyst produced by living cells, and it uses widely. Aspergillus oryzae has widely protease series, it is one of safety microbial strains announced by the US Food And Drug Administration and American Association Of Feed Companies, it is also the main strains of fermented foods such as soy sauce in China. For this reason, improve protease's vitality of Aspergillus oryzae has important significance for developing light industry and improving product quality.In this thesis, we selected Aspergillus oryzae shanghai brewing 3.042 as original strain. After ultraviolet mutagenesis, a strain with higher neutral protease was obtained, named Aspergillus oryzae UV-16. The genetic stability of this strain was well, and its neutral protease was increased from 1996.5 U·g^-1 to 2368.0 U·g^-1 which was inhanced 18.6% compared with original strain. Other enzyme of the strain was investigated, and we found that enzyme system of this mutant strain did not change basically.We studied the influence of substrate's compositon and fermentation conditions for Aspergillus oryzae UV-16. Though the single factor experiment, we taked bran as carbon source and soybean meal as nitrogen source, the additive amount of soybean meal was 20%, KNO₃ was 0.8%, MgSO₄ was 0.07%, Na₂HPO₄ was 0.14%, inoculation was 1×10⁸ spore of Aspergillus oryzae per 10g substrate, temperature 28℃, water added was 85%, fermentation time was 70h, natural pH, FeSO₄ has inhibitory action to produce enzyme. After single factor expperiment, enzyme activity was improved to 2707.8 U·g^-1 which was inhanced 14.3% compared with mutant strain.After that, we made response surface experiment including water added, temperature, fermentation time, soybean meal additives, KNO₃ and MgSO₄. We found fermentation time, soybean meal additives were the two main influencing factors after PB experiment, and deterined the center point of response surface through the steepest ascent. And then, the best cultivation condition was obtained through the central composite experiment: water added was 87%, the additive amount of soybean meal was 23.37%, KNO₃ was 0.7%, MgSO₄ was 0.08%, Na₂HPO₄ was 0.14%, inoculation was 1×10⁸ spore of Aspergillus oryzae per 10g substrate, temperature 29℃, fermentation time was 67h.. Enzyme activity of Aspergillus oryzae UV-16 was improved to 2824.9 U·g^-1 which was inhanced 4.3%.Aspergillus oryzae UV-16 used in sauce brewing process, founding the strain had srong propagate ablity and not easy to contaminate. The soy sauce did not have peculiar smell and with good flavor, its amino acids was 7.7% higher than the one made by Aspergillus oryzae shanghai brewing 3.042. Other indexes was in accord with the national standards. Therefore, Aspergillus oryzae UV-16 was suitable for industrial production.