Functional Analysis Of Bacillus Amyloliquefaciens JY06 And Its Mutants For Soy Sauce Fermentation

Ethyl carbamate(EC)is a carcinogen,and the presence of it in fermented foods probably cause food safety issues.Generally reduction of EC in fermented foods is achieved by elimination of its precursors due to the fine thermal stability of EC.It was reported that EC could be reduced in soy sauce by microbial interference.It is of great importance to further reduce or eliminated ethyl carbamate in soy sauce.This study aimed on characterizition and mutagensis of B.amyloliquefaciens JY06,and evaluation of capability in citrlline consumption of mutants when added in soy sauce.The results of this study include:(1)The single factor experiment was conducted to optimize the culture conditions of B.amyloliquefaciens JY06,and the optimal culture conditions were verified to be: dextrin 40 g/L,yeast extract 10 g/L,tryptone 10 g/L,NaCl10 g/L,pH 7.0,inoculum concentration 4% in a 3 L fermenter,the concentration of strain JY06 reached 1.1×1011 CFU/m L,which was 3 orders of magnitude higher than of the control.(2)Atmospheric and room temperature plasma(ARTP),directional domestication,and UV mutagenesis methods were used to mutate strain B.amyloliquefaciens JY06.Then a high-throughput screening approach was employed to characterize mutants with high arginine utilization capability.Comparing with B.amyloliquefaciens JY06,the arginine utilization capability of mutants C12,B1 and E6 improved 18%,17.4% and 17.1%.Under the conditions of ethanol and fatty acids,the arginine utilization capability of mutants C12,B1 and E6 improved 72%,33% and 31% when comparing with JY06.And under the conditions of high-salt,the arginine utilization capability of mutants C12,B1 and E6 is 1.26,1.2 and 1.23 times as B.amyloliquefaciens JY06.(3)B.amyloliquefaciens JY06 and its mutants were investigated following addition to soy sauce moromi during fermentation to initiate reduction of citrulline and EC.Compared with the JY06 parent strain,the addition of C12 or E6 to the moromi during soy sauce fermentation decreased the citrulline content in soy sauce by 15.6% and 14.7%,respectively,and reduced EC by 19.3% and 13.1%,respectively.(4)The presence of key genes encoding ADI(Arginine deiminase)and OTC(Omithine transcarbamylase)in the genome of JY06 was confirmed by amplification of arcA and arcB,and presence of arcC was decided by detection of CK(Carbamate kinase)activity.Transcriptional analysis of arcA and arcB in JY06 and the mutants upon salt stress and other factors(presence of ethanol and fatty acids)revealed that,transcription level of arcA in mutants C12 and E6 was higher than that in JY06,the ratio of arcA and arcB was less than 1 in mutants(except for C12 cultivated in medium containing ethanol and fatty acids).The results indicated an enhanced capability in argininine consumption and citrulline elimination of mutants.