The Preliminary Research On The Molecular Mechanism Of Mixed Fermentation With Lactobacillus Plantarum And Bacillus Subtilis To Improve Flavor Of Douchi

Douchi which combined nutrient value with health function is a microbe-fermented soybean food.Flavor,to some extent,determines directly the quality of douchi and acceptance by consumers.Fermented douche with a pure Bacillus sp.will make douchi of single flavor and have ammoniacal odour at different degrees,add lactic acid bacteria(LAB)to fermented douchi with a pure Bacillus sp.as mixed-fermentation will improve significantly the flavor of douchi.In this study,combined with transcriptome technology and functional genomics analysis methods,with Lb.plantarum YM-4-3 and B.subtilis SP-8-5 as study material,we plan to compare and analyze the changes condition of volatile compounds and difference of gene expression between Lb.plantarum YM-4-3 separate fermentation and YM-4-3—SP-8-5 mixed-fermentation,and to screen candidate genes which probably related to flavor form from genome of YM-4-3,and to study the biological functions of partial key-candidate genes using gene knock-out technology.These studies will provide theoretical foundation for building pathway of synthesizing and douchi flavor compounds,and interpreting the molecular mechanism of improving douchi flavor with mixed-fermentation,and making douchi have both tasty and benefit health functions.The main contents and results are as follows:1.Obtained finished douchi that fermented by Lb.plantarum YM-4-3,B.subtilis SP-8-5 separately and Lb.plantarum YM-4-3—Bacillus subtilis SP-8-5 mixed,By optimizing the fermentation conditions to obtain a better sensory evaluation douchi samples.The results show that the mixed fermented douchi get the highest score,27.7 points.2.Through headspace solid phase micro extraction(HS-SPME)and GC(GC-MS)method for the separation and identification of the main volatile compounds.Lb.plantarum YM-4-3 and B.subtilis SP-8-5 alone fermented douchi respectively obtained 27 and 34 kinds of odor components,Lb.plantarum YM-4-3—B.Subtilis SP-8-5 mixed fermentation of the 41 kinds of odor components.3.Using RNA-seq to determine the gene transcriptional changes in Lb.plantarum YM-4-3 separate fermentation and YM-4-3—SP-8-5 mixed-fermentation,and evaluate the differential expressed genes' functionand probable metabolic pathway involved by bioinformatics analysis.The results show that the presence of amino acid metabolic pathways significantly enriched in the mixed fermentation process,deduced that amino acid metabolic pathways to effectively improve the flavor of douchi.4.Real-time quantitative PCR(RT-PCR)was used to detect Lb.plantarum YM-4-3 in a separate fermentation and the B.subtilis SP-8-5 when mixed fermentation gene expression,the qPCR results highly consistent with transcriptome data,It means the accuracy of transcriptome data.5.According to the results of flavor compounds identified,acetoin and 2,3-butanediol compound may be the key flavor compounds of mixed fermentation douchi,in acetoin as a first object of study.Selecting the gene which is coding the enzyme of acetoin formed directly associated,a-acetolactate decarboxylase,acetolactate synthase and 2,3-butanediol dehydrogenase,construct an expression vector and verify its functionality.