Biosynthesis Of Tetramethylpyrazine Precursors In The Acetic Acid Fermentation Of Zhenjiang Aromatic Vinegar

Zhenjiang aromatic vinegar is commonly used as condiment. It contains not only flavor compounds but also a variety of nutrients which contribute to its health care function such as tetramethylpyrazine. This research focused on the formation mechanism of tetramethylpyrazine and its precursors(mainly acetoin) during the acetic acid fermentation stage of Zhenjiang aromatic vinegar. It expanded new ways for investigating formation mechanisms of functional compounds in vinegar and provided theoretical basis of fermentation technology, biosynthesis of tetramethylpyrazine and health care products development rich in tetramethylpyrazine.(1) During the acetic acid fermentation stage, the contents of acetoin and diacetyl showed an increasing tendency during the 0-20 th day and 7-16 th day respectively. Tetramethylpyrazine was detected and kept on the rise since the 15 th day. The result of precursors(pyruvate, diammonium phosphate, leucine, acetoin and diacetyl) addition into the vinegar Pei verified the biopathway of tetramethylpyrazine and its precursors in the microbial community. It also indicated that acetoin, diacetyl and ammonium ion were important precursors of tetramethylpyrazine indeed and the acetoin formation from diacetyl catalyzed by DR(diacetyl reductase) was weak in the vinegar Pei.(2) The genes diversity of three key enzymes in the acetoin pathway in the vinegar Pei microbial community were investigated by clone libraries. Degenerate primers were designed using COnsensus-DEgenerate-Hybrid Oligonucleotide Primers strategy and validated to be of high specificity and compatibility. Gene fragments were amplified, cloned and sequenced using genome DNA from vinegar Pei as template and then clone libraries were established. Coverage C and rarefaction curves showed that ALS(acetolactate synthase) library and DR library could farely reflect the gene diversity in the microbial community, however ALDC(acetolactate decarboxylase) library was less diverse. Cluster analysis and phylogenetic analysis revealed that the formation of acetoin was mainly accomplished through the pathway pyruvate �' acetolactate �' acetoin by Acetobacter and Lactobacillus cooperatively. The genetic complementation between these two genera may contribute to functional complementation. Moreover, DR library result also indicated that the formation of acetoin from diacetyl catalyzed by DR, which was mainly accomplished by Bifidobacterium indicum, L. fermentum and a few microbes of low abundance, was weak.(3) Six functional bacteria strains isolated from vinegar Pei were selected according to the clone library results and cultivated in situ and in vitro. A. pasteurianus G3-2 produced no or trace of acetoin when mono-cultured both in situ and in vitro. In vitro, L. buchneri F2-5, L. casei 10M1-5 and L. reuteri 26M17-3 produced more acetoin when co-cultured with A. pasteurianus G3-2 than that when they were monocultured. In situ, L. fermentum 37M10-3 produced more acetoin when co-cultured with A. pasteurianus G3-2 than that when it was monocultured. The fermentation results revealed that several different Lactobacillus species could produce more acetoin when co-cultured with A. pasteurianus under certain fermentation condition which provided evidence to the functional complementation theory raised above.