Screening And Functional Research On The Flavor Genes About Lobster Or Soya Sauce Of Bacillus Subtilis

Lobster-flavor food(such as liquor)fermented by Bacillus subtilis at high temperature and soya sauce flavor food(such as Douchi)fermented by Bacillus subtilis at medium-low temperature are very popular,but the mechanism of lobster-flavor and soy sauce flavor is still unclear.Through the transcriptome sequencing and enrichment analysis of the soybean industrial production strain Bacillus subtilis BJ3-2 at medium temperature(45°C)and medium-low temperature(37°C),19 differentially significant genes were screened out.5genes that have significant differences were chosen as candidate genes and verification test was done by real-time fluorescence quantitative PCR.Finally,rocF(arginase)was selected for homologous recombination and its physiological function on the lobster(soya sauce)flavor was verified by double exchange knockout.The main research contents and results are as follows:1.The second generation of the transcriptome sequencing of the bacillus subtilis BJ3-2was done at medium temperature(45?)and low temperature(37?).58 genes with significant differences were enriched in 27 metabolic pathways by expression analysis,GO,KEGG annotation and enrichment analysis of the differentially expressed genes,among which pyrimidine metabolism,arginine and proline metabolism,alanine,aspartate and glutamate metabolism were the most significant.The screening conditions were set as Fold-change?2 and FDR?0.05,19 significant differential genes were finally screened out.Combined with literature reports and comprehensive analysis,selected rocF(arginase),rocG(glutamatedehydrogenase),uxaC(Glucuronicacidisomerase),uxuA(mannitol dehydrogenase),and pckA(phosphoenolpyruvate carboxylase)are chosen as candidate genes for lobster(soya sauce)flavor.2.The five lobster(soya sauce)flavor candidate genes screened by the transcriptome were verified by real-time fluorescence quantitative PCR.Using 16S rRNA as the internal reference gene,the dissolution curves and amplification curves of the five candidate genes were determined,and the differences among gene expressions were calculated.Combined with transcriptome differences,it was found that the expression trends of rocF,rocG,uxuA and pckA were consistent with the results of transcriptome sequencing except for uxaC.3.RocF double exchange knockout vector,pUC18+HLarm+CmR+HRarm,was constructed.Using pUC18 as the initial vector,restriction restriction sites of Sac I,BamH I,Pst I,Hind III were used to bind the left arm(HLarm),right arm(HRarm)of BJ3-2 genome and chloramphenicol gene(CmR)from pHT01cas9-p43 vector,and confirmed by colony PCR,plasmidPCR,doubleenzymedigestionandsequencing.Finally,pUC18+HLarm+CmR+HRarm recombinant exchange vector was obtained.4.The double-crossing plasmid vector was transformed into BJ3-2 with a transformation efficiency of 5×10~2 cfu/?g,and verified by Gram stain,RCR verification and sequencing,and BJ3-2?rocF knockout strain was successfully screened out finally.5.BJ3-2?rocF and BJ3-2 were used to ferment soybeans,and sensory evaluation showed:when BJ3-2?rocF fermented soybean was compared with BJ3-2 fermented soybean,color,soya sauce-flavor,lobster-flavor,texture and ammonia smell were no obvious change,only the viscosity of the silk was slightly reduced at 37°C;the degree of browning of the color was reduced,the viscosity was enhanced,the lobster-flavor was more prominent,and the ammonia taste was reduced by 112.9 ppm at 45°C.The experimental results show that:?rocF does not affect the normal growth of Bacilus subtilis BJ3-2,and the sensory changes of BJ3-2?rocF and BJ3-2 37°C fermented soybean meal are not significant,which indicates that there is no significant relationship between rocF and sauce flavor at 37°C.Traditionally,browning was positively correlated with sauce,but at 45°C,the browning degree of BJ3-2?rocF fermented soybean meal was weakened,which indicates that there was no correlation between browning and sauce flavor,and the enhancement of sauce aroma indicated that rocF is important for sauce flavor.The negative regulation gene,the ammonia smell decreased,indicating that rocF is positively correlated with the formation of ammonia by arginine metabolism.By successfully knocking out rocF,the study provides a method for studying the function of the flavor of lobster(soya sauce).By means of transcriptome sequencing,real-time fluorescence quantitative PCR verification and gene knockout,a theoretical basis was provided for exploring the formation mechanism of flavor in lobster-flavor and soya sauce-flavor products.