Studies On Breeding Of Good Protease Of Aspergillus Oryzae That Applies To Brewing For Buckwheat And The Solid-state Fermentation

In this study,a higher protease of Aspergillus oryzae enzymolsis protein of buckwheat production strain HUV-2 was obtained by the way of Chemical mutagenesis and Physical mutagenesis,and the(On the base,the cultivation conditions of koji(protease of Aspergillus oryzae) and some enzymatic characteristics protease of Aspergillus oryzae were studied.The low -salt solid-stata fermentation conditions were optimized by orthogonal tests.The results showed that the amount of water had the most influence on soy sauce,followed by culture tempreture and NaCl addition,and the C:N had the least influence.Finally,strain HUV-2 was applied in the production process of buckwheat vinegar and buckwheat miso,which provide some application values in production.The results are summarized as follows:1.At first,the original strain Aspergillus oryzae J were obtained by screening from Xi'an brewing company,whose enzyme productivity is 1631.7U/g.Then it was mutated by HNO₂, UV and UV+LiCl.The optimal mutation effect were obtained by HNO₂ 0.30mol/L,and 70s UV+1%LiCl;we use a rapid method for choosing the higher production strain,also the stability of the mutants were studied,finally the mutant HUV-2 was obtained and its enzyme productivity was 5659.2U/g,and it is 3.05 higher than that of original strain.2.The optimal fermentation conditions for protease of Aspergillus oryzae production were determined by orthogonal experiment.The compositions of fermentation medium were bran 25%,buckwheat 12.5%,caramel 2.5%,soybean flour 10%,water 50%;The incubation temperature is 32℃;amount of inoculum is 0.3%.Obtained crude enzyme powder by Saturated ammonium sulfate method and freeze-drying method.3.Effect of different factors on protease activity in three generations of koji was studied. The studies of the characteristics of protease of Aspergillus oryzae showed that the optimum temperature is 55℃.But protease activity remained 108.5%,at 40℃and 120 minutes under the conditions of treatment in protease thermal stability test;the optimum pH is 7.2,and it's the best stability;Co²⁺ showed inhibition for this enzyme,the remaining plasma did neither obvious activation and did not significantly inhibition.It can be seen that protease of HUV-2 can resist impact of some ions on protease activity in production environment.4.In fermentation,the discipline was studied of HUV-2,at the same time the fermentation conditions were optimized.Amino nitrogen content as protease enzyme capacity indicators was determinated in the fermentation process.The best culture temperature is 40℃, carbon and nitrogen ratio is 4:1,moisture is 65%and salt is 10%.Amino nitrogen content is 0.85g/100mL,total acid is 2.09g/100mL,taotal nitrogen1.81g/100mL,soluble salt-free solids 18.71 g/100mL,chloride 9.79g/100mL,reducing sugarin4.78g/100mL in buckwheat sauce by HUV-2 strain.5.Under laboratory conditions,HUV-2 was added to three generations koji when brewed buckwheat vinegar,buckwheat miso and buckwheat paste.Sensory index and physical and chemical indicators as evaluation criteria,the results showed that total acid is 4.30g/100mL,amino nitrogen content is 0.30g/100mL,reducing sugarin0.81 g/100mL, chloride 3.11 g/100mL,soluble salt-free solids 1.68g/100mL and non-valatile acid 1.17g/mL in buckwheat vinegar.Total acid is 1.40g/100g,amino nitrogen content is 0.48g/100g, reducing sugarin 6.61 g/100g,chloride 1.32 g/100g,soluble salt-free solids 53.19g/100g and water 38.2%in buckwheat miso.Total acid is 1.60g/100g,amino nitrogen content is 0.34g/100g,reducing sugarin25.84g/100g,chloride7.86g/100g,soluble salt-free solids 42.48g/100g and water 45.9%in buckwheat paste.