Optimization of Fermentation and Application of Amylase-producing Bacillus velezensis A8-1

WU Shuangshuang; ZHANG Zekun; YE Changwen; YAN Jianying; WANG Qingyue; SUN Zhikang; LI Liqun; HAO Jie; LIU Dandan

doi:10.15933/j.cnki.1004-3268.2026.02.017

Abstract：The amylase-producing strain Bacillus velezensis A8-1 preserved in the early stage of the experiment was used as the test strain,and its metabolic capacity and sensitivity to different carbon sources and chemical substances were analyzed by Biolog technology.The enzyme production process was optimized through single-factor experiments,Placket-Burman experiments and Box-Behnken experiments.The amplification effect of the process was verified by fermentation in a 1 000 L tank,and the change characteristics of chemical composition and sensory quality of tobacco leaves after the application of amylase produced by this amylase-producing strain in tobacco leaf fermentation were explored.The results indicated that among the 70 different carbon sources,Bacillus velezensis A8-1 had metabolic capacity for 65 of them and was intolerant to most of the bacteriostatic agents.The optimal shaking flask fermentation conditions after fermentation optimization were as follows:corn flour 20 g/L,soybean cake flour 80 g/L,K2HPO4 1 g/L,fermentation time 36 h,temperature 37℃,pH value 7,inoculation amount 3%,and liquid loading volume 50 mL.Under these conditions,the enzyme activity could reach up to 133.3 U/mL at most.In a 1 000 L fermenter,the enzyme activity was 161.0 U/mL.After applying this amylase to the fermentation of tobacco leaves,the starch content of the tobacco leaves decreased by 15.4%,while the contents of reducing sugar and total sugar increased by 5.6%and 8.5%respectively.The contents of polyphenolic compounds,amino acids and Amadori compounds all increased to varying degrees,indicating that it had certain improvement effects on the chemical composition of tobacco leaves.The sensory evaluation results showed that the fermented tobacco leaves improved to varying degrees in terms of comfort,smoke characteristics and aroma style,with the total sensory evaluation score increasing by 0.64 points.In conclusion,the industrial fermentation production of the amylase-producing strain Bacillus velezensis A8-1 is feasible,and the application of the produced amylase in tobacco leaf fermentation can effectively improve the quality of tobacco leaves and has good practical application prospects and promotion value.