Preliminary Analysis And Application Of The Synthesis Mechanism Of Diacetyl In Lactobacillus Casei

Diacetyl is one of the important flavor compounds that contributes significantly to the flavor of fermented milk.We usually enhance diacetyl content by exogenous addition to improve the flavor of the fermented milk.However,this is contrary to the current trend of the development of green and pure natural products in the food industry.Therefore,increasing the endogenous production of diacetyl is a hot research topic of fermented milk.In this paper,we analyzed the metabolic pathways of lactic acid bacteria to synthesize diacetyl,searching for key genes affecting diacetyl synthesis,and then used genetic engineering to carry out gene overexpression and gene knockout transformation of Lactobacillus casei TCS to analyze the mechanism of the synthesis of diacetyl.The flavor analysis technique such as gas chromatography-mass spectrometry was used to study the fermented milk flavor of recombinant strains with high yield of diacetyl.The main conclusions of this paper are as follows:(1)Through genetic modification: knockout α-acetolactate decarboxylase gene alsD,NADH oxidase gene nox overexpressing,α-acetolactate synthase gene als S overexpressing,we obtained mutant strains and determined the concentration of diacetyl and acetoin in yogurt,which fermented by mutant strains.The results showed that the three single-gene effects could lead to an increase in the metabolic flux of α-acetolactate in the diacetyl or acetoin direction of Lactobacillus casei TCS,indicating that these three genes are the key genes for the synthesis of diacetyl in Lactobacillus casei TCS.(2)The yield of diacetyl and acetoin in the fermented yogurt,which fermented by mutant strains with gene nox or als S overexpressing based on the knockout gene als D except the recombinant strain TCSN4 with nox inducible overexpression,was significantly higher than that of the single gene.That is,the double genes modification is significantly higher than that of the single gene.Among them,the diacetyl yield of the yogurt fermented by the strain TCSDNP with gene nox constructived overexpression based on the gene als D knockout and TCSN4 was the most significant,about 2.46 times and 2.1 times higher than TCS.(3)The aroma compounds of the yogurt fermented by the strains with or without genetic modification were analyzed by GC-MS.The number and total concentration of aroma compounds of the yogurt fermented by TCSD with knockout gene als D and TCSN4 were significantly higher than the original strain TCS.The genetic modification have significant effects on the content of ketones,alcohols and aromatics in yogurt.The recombinant strains TCSD and TCSN4 significantly improved the contentration of ketones and alcohols in yogurt,increasing the milky of yogurt and enriching yogurt aroma,and enhancing the overall aroma quality of the yogurt.