Study On The Screening And Fermentation Characteristics Of Tetramethylpyrazine-producing Strains In Traditional Fermented Douchiba

Traditional charachteristic Douchiba,a condiment with full aroma and delicious taste,is produced from high-quality soybeans by microbial natural fermentation.Douchiba is rich in numerous nutrients and physiologically active compounds.Tetramethylyrazine,a key flavor component and health function factor,endowed Douchiba with characteristic flavor and physiological activities,including the inhibition of platelet aggregation and thrombosis.Therefore,taking Dafang Douchiba as the research subject in this study,the characteristic flavor compounds and the flora structure were analyzed,and the correlation between tetramethylyrazine and dominant microorganisms was clarified.Furthermore,the fine strains producing tetramethylyrazine were isolated and identified.In addition,the pre-fermentation process parameters optimization and the quality evaluation of single-strain and mixed-strain fermented Douchiba were carried out,which could provide scientific basis for the high value development of Douchiba.The main research results are as follows:1.There was no obvious difference in the types of flavor substances in traditional Douchiba samples.HPLC analysis showed that succinic acid was the highest organic acid content in Douchiba,and the taste free amino acids were mainly sweet and bitter,and the tetramethylyrazine content ranged from 83.32 ～ 190.01 μg/g.Total 71 volatile compounds were identified by HS-SPME-GC-MS,mainly including acids,pyrazines and esters.Furthermore,the 28 characteristic flavor compounds with VIP > 1(p < 0.05)were mainly tetramethylyrazine,tartaric acid,polypeptide,serine,isovaleric acid,butyric acid,propionic acid,acetic acid,methyl acetate,and 2,3,5-trimethyl.2.The diversity and abundance of bacteria were higher than that of fungi in Douchiba samples.Bacillus,Tetragenococcus and Anaerosalibacter were the most abundant bacteria,especially Bacillus with 23.04% to 73.16%.Fungi were mainly Ascomycetes(83.89%),mainly including Aspergillus,Issatchenkia,Mucor,Debaryomyces,Saccharomycopsis and Fusarium.The characteristic flavor compounds,sunch as tetramethylyrazine,succinic acid,and butyric acid,were significantly correlated with the abundance of bacteria or fungi(p < 0.05).Interestingly,tetramethylyrazine had a positive correlation with 9 species of bacteria(Pseudogracilibacillus,Bacillus,Anaerosalibacter,Oceanobacillus,Ligilactobacillus,Atopostipes,Leucobacter,Corynebacterium and Sinibacillus)and 5 species of fungi(Aspergillus,Issatchenkia,Dipodascus,Microascus and unidentified).3.Through the preliminary screening,re-screening and safety experiments,two strains S2-2 and S6-J1 with good safety and producing tetramethylyrazine were identified,namely Bacillus subtilis S2-2 and Hyphopichia sp.Strain S6-J1,respectively.4.Combined single-factor and orthogonal tests,the prefermented process parameters of single and mixed microorganism were optimized.When the fermentation conditions of S2-2were 50 g of soybean raw material,2 % inoculum,45 ℃ fermentation temperature and 4 d fermentation time,the tetramethylyrazine content was 305.11 μg/g,which was 1.15 times higher than that of unoptimized sample.When the fermentation conditions of S2-2 and S6-J1 were 60 g of soybean raw material,6 % inoculum(strain ratio of 7:1),30 ℃ fermentation temperature and 4 d fermentation time,the tetramethylyrazine content was 340.11 μg/g,which was 1.26 times higher than thatof unoptimized sample.5.The quality characteristics of pre-fermented douchiba samples which mixed with salt and fermented at room temperature for 15 days were evaluated.The tetramethylyrazine contents in MG(Bacillus subtilis strain S2-2 + Hypericum sp.strain S6-J1)and SG(Bacillus subtilis strain S2-2)were 312.73 and 286.31 μg/g,respectively,which were 2.95 times and2.15 times higher than those of natural fermentation samples,respectively.Meanwhile,the contents of amino acid nitrogen,soluble protein,polypeptide,total organic acid and fatty acid are the highest in MG,and the contents of total acid,reducing sugar and total amino acid are the highest in SG.Total 103 volatile compounds were detected by GC-IMS,mainly including aldehydes,esters,ketones and pyrazines.Among pyrazines,the relative content of tetramethylyrazine is the highest,especially in MG sample.In conclusion,tetramethylyrazine showed significantly positive correlation with Bacillus and yeast in Douchiba,and the tetramethylyrazine content and the flavor compounds were improved by mixed fermentation of Bacillus subtilis S2-2 and Hypericum sp.strain S6-J1.