Study Of High-yield Tetramethylpyrazine Microbial Screening From Liangzhou Fumigated Vinegar Traditional Brewing And Its Preliminary Metabolism Mechanism

Liangzhou fumigated vinegar has been famous for its distinct flavor in China. How to enhance flavor in traditional fermented vinegar has attracted a large interest in recent years. Acetoin(ACT) is a precursor of tetramethylpyrazine(TTMP), a popular food flavor found in different traditional fermented foods. In our study, we isolated the function microbes to produce acid from the substrate of liangzhou fumigated vinegar during its traditional brewing by classic microbial isolating method. The Voges-Proskauer(V-P) chromogenic reaction method and GC-FID were used to obtain the high- yield ACT strain. It was identified by physiological and biochemical characteristics and 16 S r RNA gene sequence similarit ies. The fermentation condition for producing ACT was optimized by the response surface methodology. Using GC-FID and some regular physical and chemical indicators measuring methods, the fermentation characteristics of the strain and the relationship between ACT and Tetramethylpyrazine during the liquid fermentation process were determined.At the same time, the dynamic change of volatile component within 7 days of fermentation was determined by GC-MS and combined with the mathematical statistics and the rational analysis, a preliminary metabolic pathway and mechanism of the strain to produce tetramethylpyrazine has been work out, and we also got the product spectrum of flavor substances. The aim was to perfect the vinegar brewing process, improve the quality of vinegar with the screened strains, the main research results were as follows:1. Strains were isolated from the substrates collected during the acetic acid fermentation stage in the process of liangzhou fumigated vinegar. Based on the isolated strains, 51 strains including 8 strains of Bacillus, 9 strains of lactic acid bacteria, 6 strains of yeasts and 28 strains of acetic acid bacteria were found, which have the ability to produce the acetoin by the V-P chromogenic reaction method. A high ACT yield of 14.02g/L and TTMP yield of 2.77g/L strain C92 was isolated by GC-FID and it was identified as Acetobacter pasteurianus by physiological and biochemical, combined with 16 s r RNA.2. The factors influencing the ACT yield of the strain C92 were stud ied, including temperature, inoculated quantity, p H and rotating speed. The fermentation condition of the high- yield strain of A. pasteurianus C92 was optimized by single factor design and response surface methodology(RSM). The optimum fermentation conditions were temperature of 31.31 ° C, inoculated quantity of 8.27 %, p H 6.55, shaking at 209 r/min. The ACT and TTMP yield could reach 19.04 g/L and 3.82g/L at these conditions, which was 35.8 % and 37.9% higher than the original fermentation production respectly.3. Through dynamic tracing the fermentation of A. pasteurianus C92, we find that its process to produce ACT was a classic growth-associated fermentation. ACT was a primary sugar metabolism product. Sugar was the original energy and it was converted into the ACT, while arriving at a certain concentration, the ACT was transform into C5H9 NS, a key metabolite from ACT to TTMP, and at last two molecular C5H9 NS merged into one molecular tetramethyl pyrazine.4. The product spectrum of the strain C92 was examined by GC-MS. We found that the strain produced the most abundant fermentation flavor substances at the 6th day, and reached 68 kinds, including 13 pyrazines,accounting for 9.71% of the total volatile components. In additional, there were 10 kinds of acids, 4 ketones, 11 kinds of esters, 6 kinds of aldehydes, 14 kinds of alcohols, 5 kinds of phenols, and 5 kinds of others, each was accounted for 12.99%,0.74%, 4.46%, 0.71%, 4.46%, 28.21% and 1.75% of total composition, respectively.Considering the all results above, the acetic acid bacterium plays a major role and priority to the acetic fermentation process of liangzhou fumigated vinegar and it builds a system with other microbes during the brewing state. The acetic acid bacteria are the base and key of forming the vinegar main common components in the vinegar brewing system. The Acetobacter pasteurianus C92 could produce a variety of flavor compounds in the fermentation, and its characteristics of aroma components make direct effect to the vinegar quality. The special function microbe existing in the process of brewing plays an indispensable role for forming Liangzhou fumigated vinegar unique flavor.