Screening Of High Diacetyl-Producing,Aroma Characteristics And Function Gene-expression Of Lactic Acid Bacteria

With the pursuit of nature and health benefits of customers,the endogenous production which has become a development trend.And diacetyl is a compound that contributes significantly to the flavor of milk.Because lactic acid bacteria with high-yield of diactyl is scarce and the metabolic pathway of lactic acid bacteria has not been studied clearly,the endogenous production cannot fully meet the needs of consumers.In current study,high-throughput screening of lactic acid bacteria with high-yield diacetyl was carried out by the coupling of microplate-microplate reader from traditional productions.And the growth characteristics,aroma characteristics and metabolic pathways of the selected flavoring bacteria were analyzed to provide a basis for the development of new fermented yogurt products.The mainly conclusions of this study are as follows:（1）High-throughput screening of lactic acid bacteria with high-yield diacetyl was carried out by the coupling of microplate-microplate reader.The results show that the regression equation of the standard curve established by this method is y=0.0149 x+0.0932,R²=0.9966,showing a good linear relationship;the standard deviation and the recovery rate were good.And the correlation among UV spectrophotometry and gas chromatography showed a good linear relationship.A high diacetyl-producing Lactobacillus casei was selected from the traditional fermented yogurt used this method and named L.casei TCS.The optimum growth temperature of the strain was 37°C,entered the logarithmic growth phase at2 h,and entered the stationary phase at about 12 h.When used for yoghurt fermentation,the acidity began to change significantly at 4 h,reached the end of fermentation at 10 h,and the fermentation stopped.（2）The aroma compounds and seven sensory attributes of four different Lactobacillus casei fermented yoghurts were evaluated by GC-MS and sensory evaluation.The results showed that the content of diacetyl in the L.casei TCS fermented yoghurt（9.54 mg/L）was much higher than that of the other three fermented yoghurts（L.casei Zhang:4.56±0.23 mg/L、BD-II:3.78±0.28 mg/L、LC2W:2.05±0.17 mg/L）.And there were significant differences between the three sensory attributes of typical yogurt,creamy taste and overall acceptability（P<0.05）.（3）Complete genome sequencing was performed on L.casei TCS using high-throughput sequencing technology.The results show that the L.casei TCS genome consists of a chromosome and a plasmid.Both were ring-shaped,with lengths of 3,077,701 bp and 66,782 bp;with an average G+C content of 46.28%and 44.79%,containing 3092 and 71CDSs,respectively.3,163 genes,60 tRNA sequences and 15 rRNAs were identified in the L.casei TCS genome.（4）The yield of diacetyl is increased by optimizing fermentation conditions and adding appropriate precursors to the fermentation mechanism,and the expression levels of key genes（α-als,α-aldB,ldh）are studied.The results showed that when the initial pH was 6.5,the inoculum was 4%,and the fermentation temperature was 37°C,L.casei TCS was more likely to metabolize producing diacetyl in the fermentation process.When the proper amount of glucose,serine,Mg²⁺and sodium citrate were added to the fermentation mechanism,the yield of diacetyl can be increased to some extent.There was a significant positive correlation with the content of diacetyl in different fermentation time;the content of diacetyl was significantly positively correlated with the key functional genesα-als and ldh2664,but not related to the expression of other genes.