Effect Of Threonine On Maotai Flavor

The composition of Maotai flavor and its production mechanism have always been the hot research content of Maotai flavor liquor.At present,there have been many reports on the precursor of pyrazine,but there are few studies on its precursor ammonia.The function of amino acids in microbial fermentation is not clear.Therefore,in this paper,a strain of Bacillus subtilis with strong maotai flavor was used to simulate the fermentation of maotai flavor simulated koji,and the components of free amino acids in the fermentation were detected and analyzed.Through the analysis of the synthetic pathway of 2,5-Dimethylpyrazine precursor amino acid with threonine,gene knockout was carried out on the key enzyme in the metabolism of threonine.In order to verify its effect on the formation of pyrazines in the process of microbial fermentation,and then understand the effect of threonine on the formation of maotai flavor,the main research contents and results are as follows:1 Five strains preserved in the microbiology laboratory of Guizhou University were used to ferment the soybean fermentation medium,which was proved to be able to produce strong Maotai flavor by experiments.After the fermentation,the laboratory researchers smelled the odor of the fermentation and observed the degree of browning,and screened out the strain Bacillus subtilis which could produce strong Maotai flavor E20 as the experimental strain,and Bacillus subtilis 168 as the control strain.2 The maotai-flavored strain Bacillus subtilis E20 and the non-protruding maotai-flavored Bacillus subtilis 168 were used to simulate maotai flavor fermentation on soybean fermentation medium and sorghum wheat 1:1 mixture fermentation medium,respectively,and determination of free amino acids in fermentation substance by OPA-FMOC pre-column derivatization.Sixteen amino acids were detected in all fermented products,but cystine was not detected.Due to the different fermentation substrates,the composition and difference of free amino acids in the fermentation product were large.The free amino acid composition of the fermented product is also different after the same fermentation medium is subjected to different treatments and simulated fermentation.3 The threonine dehydrogenase gene(tdh)involved in the synthesis of 2,5-dimethylpyrazine from threonine metabolism was cloned.Using Bacillus subtilis E20 DNA as a template,the E20 tdh gene was amplified by designing specific amplification primers and then ligated with the cloning vector p MD18-T and transformed into E.coli DH5? cells to obtain a tdh gene cloning strain.According to the sequencing results,the total length of TDH gene was 1044 bp,encoding 347 amino acids.The similarity between TDH gene and TDH gene from Bacillus subtilis 168(NCBI Accession:CP053102)was 99.04%.The similarity between TDH protein encoded by E20 tdh gene and TDH protein of Bacillus subtilis(NCBI accession number: WP?128441634)was 99.42%.4 Relevant bioinformatics analysis of the threonine dehydrogenase protein(TDH)of E20 strain showed that the molecular weight of E20-TDH protein is about 37 KDa,the theoretical value of p I is5.94,and the instability index is 31.27.It is a stable soluble hydrophobin and does not contain the signal peptide is not transported across the cell membrane and is very likely to be located in the periplasm.The protein catalyzes threonine and NAD to produce ethyl L-2-aminoacetate and NADH.The active site of the enzyme may be located in the 67-81 amino acid sequence with four conserved domains.The secondary structure prediction shows that the ?-helix accounts for 31.99%,the ?-turn accounts for 9.51%,the extended chain accounts for 22.48%,and the random coil accounts for36.02%.Compared with the TDH protein of strain 168,there were differences in protein molecular weight,theoretical p I,instability coefficient,secondary structure and tertiary structure.5 Using Bacillus subtilis E20 DNA as template,design primers containing restriction sites for PCR amplification,ligate with expression vector p ET-22b(+)and transform into E.coli BL21(DE3)competent cells to obtain tdh gene-containing The expression strain E.coli BL21/p ET22b-tdh.After induced by different culture time,different culture temperature and different concentration of inducer(IPTG),the crude enzyme solution was extracted for SDS-PAGE detection and the gray value of electrophoresis strips were analyzed.The results showed that the most suitable induction conditions were: temperature 25?,concentration of inducer IPTG 0.5mmol/L,induction time 12 h,the prokaryotic expression strain E.coli BL21/p ET22b-tdh was induced under this induction condition,and the target protein was detected by SDS-PAGE mainly in the form of inclusion bodies.6 Based on the p UC18 vector,primers containing restriction sites were designed to specifically amplify the cat gene from Bacillus subtilis 1S101 DNA as a resistance screening marker gene.At the same time,two pairs of primers containing restriction sites were designed,and the homologous left arm and homologous right arm were amplified by PCR using the DNA of the target strain E20 as the template.The tdh gene knockout plasmid p UC18-HL-CAT-HR was successfully constructed by restriction enzyme digestion and ligation.7 The tdh knockout plasmid p UC18-HL-CAT-HR was transformed into the competent cells of Bacillus subtilis E20 by chemical transformation and the tdh knockout E20 strain was obtained,which was named as E20-?tdh.The tdh gene knock-out strain was used to carry out the simulated fermentation of maotai flavor.Through sensory evaluation,it was found that compared with the wild strain E20,the fermentation product of E20-?tdh strain browned the same and both have a certain viscosity.The maotai flavor in the fermentation products of E20-?tdh strain became lighter,and had a strong charred flavor compared with E20.HS-SPME-GC/MS was used to detect the volatile flavor components of soybean fermented products.The results showed that the relative content of pyrazines in soybean fermented products was greatly reduced after tdh gene knockout,and the content of tetramethyl pyrazine was more than that in wild strain E20 fermented products.The content of2,5-dimethylpyrazine was high in the fermentation products of wild-type strain E20,but not detected in the fermentation products of E20-?tdh strain.It is speculated that 2,5-dimethylpyrazine is the main pyrazine which affects the flavor of Maotai flavor.It was further explained that threonine mainly affected the formation of Maotai flavor by metabolizing to 2,5-dimethylpyrazine,and the relative content of 2,5-dimethylpyrazine was positively correlated with the flavor of Maotai.