Screening Of Protease-producing Bacteria In Liquor Pit Mud And Its Application In Liquor Fermentation

Chinese liquor is a unique distillation of wine,in liquor brewing process,the appropriate amount of protease can effectively hydrolyze the protein of the material,and promote cell growth and reproduction,and improve utilization of raw materials to produce a variety of flavoring substances,protease-producing bacteria plays an important role in wine production.The old pit mud of Fu Yu Lao Jiao limited company was taken as the experimental material,skimmed milk as a selective medium,through isolation and purification of bacteria,protease-producing bacteria were got.The diversity were analysized by morphological,physiological and biochemical.The high-yield protease strains were screened by transparent circle method and protease activity.By molecular identification techniques,the species relationship of strains were determined.The three strains of high-yield protease were combined to complex strains.The culture conditions of complex strains were optimized to improve the enzyme activity,and used to liquor fermentation.After screening protease-producing bacteria,fungi are identified by ITS sequence,AF6 is Penicillium chrysogenum,BF4 is Aspergillus oryzae.Bacteria is identified by16SrDNA,HA30 is Bacillus subtilis,HB29 is Bacillus amyloliquefaciens,HC32 is Bacillus thuringiensis.Based on bacteria breeding fast and short fermentation period,three strains with high activity endopeptidase,aminopeptidase,carboxypeptidase were selected,HA30aminopeptidase vitality is 84.269 U/mL,HB29 peptide enzyme activity is 104.516U/mL,HC32 carboxypeptidase activity is 73.538 U/mL,The three strains were combined to complex strains.The protease activity of complex strains were higher than single strains.The optimal single factors of complex strains are incubation time 16h,shaking speed 140r/min,culture temperature 37?,inoculation 2%.The fermentation medium components were optimized by response surface methodology,the significant factors and concentration are glucose 3.611g/100mL,KH₂PO₄ 0.263g/100mL,CaCl₂0.064g/100mL,and the bacteria concentration is 5.152×10¹⁰cfu/mL.After optimizing the enzyme activities of endopeptidase,aminopeptidase,carboxypeptidase are113.574U/mL,92.337U/mL,88.269U/mL,and increased by 8.148%,6.780%and17.290%respectively.The optimization of complex strains are used in the laboratory liquor liquid and solid-state fermentation,the productivity was increased by 5.547%and5.547%,the utilization rate of raw materials were increased by 6.588%and 7.511%respectively.The liquor were more lightly fragrance,and reached the national secondary standard of Chinese liquor.