| Sufu is a kind of traditional fermented soybean food in China, however, sufu is at a disadvantage in a competitive market because of its extensive production process. Under the background, the strains which can produce high activity protease were screened from commonly used fermented soybean food strains and fermentation conditions for producing proteinase were studied. High protease-producing strain for sufu was treated by UV. Optimal strain, as auxiliary strain, was used in the process of sufu fermentation via flour cake Qu and mixed with other strains in order to improve sufu quality.Medium compositions and fermentation conditions for producing neutral protease through the cultivation of Bacillus natto23413, Aspergillus oryzae2035and40214, Aspergillus niger2395, Aspergillus flavus2405were optimized. Through single factor trial and orthogonal experiment, the optimal medium composition and fermentation conditions of strain Bacillus natto23413were determined as sucrose6.0%, NH4Cl4.0%and NaCl0.5%, culture temperature37℃, the initial pH6.5and cultivation time48h, the activity of neutral protease was enhanced to756.90U/mL, increased to198.26%; Aspergillus oryzae2035were determined as malt sugar10.0%, soybean meal4.0%and KNO30.15%, culture temperature32℃, the initial pH7.0, the addition ratio of water was80.0%and cultivation time84h, the activity of neutral protease was enhanced to2395.73U/mL, increased to145.89%; Aspergillus oryzae40214were determined as malt sugar8.0%, yeast extract4.0%and Na2HPO40.05%, culture temperature28℃, the initial pH6.5and cultivation time96h, the addition ratio of water was60.0%, the activity of neutral protease was enhanced to2845.87U/mL, increased to165.89%; Aspergillus niger2395were determined as malt sugar10.0%, soybean power4.0%and MgSO4·7H2O0.15%, culture temperature28℃, the initial pH6.0, the addition ratio of water was70.0%and cultivation time84h, the activity of neutral protease was enhanced to2472.30U/mL, increased to161.88%; Aspergillus flavus2405were determined as glucose8.0%, peptone4.0%and KH2PO40.15%, culture temperature32℃, the initial pH6.5, the addition ratio of water was80.0%and cultivation time72h, the activity of neutral protease was enhanced to1688.85U/mL, increased to149.19%.The further research on optimal strain for flour cake Qu and basic material of sufu fermentation was in order to improve protease production capacity. Bacillus natto23413, Aspergillus oryzae40214and Aspergillus niger2395were treated by UV. The strain was finally obtained through the first screening with casein hydrolysis circle and the second screening with protease activity. The genetic stability was stdudied. The mutation UV-010 which protease activity was enhanced to106.15%compared with original strain, the mutation UV-9which protease activity was enhanced to106.36%compared with original strain and the mutation UV-99which protease activity was enhanced to111.28%compared with original strain. They all have better genetic stability.The study revealed the impact factors to enzyme action, a mutation UV-010:the optimal temperature was60℃, fine thermal stability for the range of temperature40℃～50℃, fine pH stability for the range of pH6.0～9.0, morever, Ca2+、Mg2+、Mn2+、Cu2+、Zn2+. Fe3+、Al3+have an inhibitory effect, and Na+、K+showed some activation; a mutation UV-9:the optimal temperature was50℃, fine thermal stability for the range of temperature40℃～50℃, fine pH stability for the range of pH6.0～7.0, morever, Mg2+、Mn2+、Cu2+、Zn2+、Fe3+、Al3+have an inhibitory effect, and Na+、K+、Ca2+showed some activation.In order to ensure the activity of enzyme, we choose the1st medium formulations without NaCl, adding80.0%of wheat bran, adding20.0%of wheat flour, the amount of water is80.0%. We choose soybean juice which was similar to tufu component as a main component of basic material in order to get high activity protease. The addition of soybean juice was determined to be15.0%through the study. Under this condition, protease production and accumulation were better. |
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