Formation Of Tetramethylpyrazine In Sesame-flavored Baijiu And Its Brewing Technology With High-yield TTMP

Sesame-flavored baijiu is one of the twelve types of baijiu,and its unique brewing technology creates a typical style of the liquor.Tetramethylpyrazine(TTMP)is an important flavor compound and a functional ingredient in sesame-flavored baijiu.However,there is little research on the formation of TTMP in sesame-flavored baijiu and its related brewing processes.Using Xuanjiu,a typical representative of sesame-flavored baijiu in Anhui as the research material,the formation of TTMP and process improvement measures in sesame-flavored baijiu,the screening of strains with high-yield TTMP,the preparation of functional bacterial cultures,the effects on the in-situ brewing microbes of sesame-flavored baijiu and its product flavors using the functional bacteria,the preparation process of the functional fuqu with high-yield TTMP and the effect of TTMP in ethanol-water system on mouse liver have been studied in this work.These works initially revealed the formation mechanism of TTMP in sesame-flavored baijiu and provided a scientific basis for obtaining the best brewing process with high-yield TTMP.(1)Based on GC-MS and HPLC-MS/MS,the change of TTMP in the whole industrial production process of Xuanjiu sesame-flavored baijiu was analyzed.Stack fermentation,solid-state fermentation and the distillation were the key processes for TTMP production in fermented grains(zaopei)during the sesame-flavored baijiu fermentation.The higher TTMP existed in bacterial fuqu.TTMP was not generated in fresh liquor during the storage process.High temperature could accelerate the formation of TTMP in zaopei,and the effective process strategies for improving TTMP in sesame-flavored baijiu had been proposed.(2)A functional strain with high-yield TTMP was screened in the micro-ecosystem of sesame-flavored baijiu using the protease and acetoin(ACT)as the screening markers.The strain was identified and named as Bacillus amyloliquefaciens XJB-104 using the identification of the physiological and biochemical,electron microscope,and molecular biological.The fermentation process of the functional bacterial cultures had been obtained using single factor and orthogonal experiments,and was as follows:inoculation amount 8%;medium: sucrose 60 g/L,yeast extract 25 g/L,(NH4)2HPO4 30g/L,Na H2PO4 17.5 g/L,p H 7.0.The two-stage fermentation process was performed to verify the TTMP producing ability of B.amyloliquefaciens XJB-104,and the yield ofTTMP reached 11.43 g/L in 40 h.The fuqu medium was fermented using B.amyloliquefaciens XJB-104 to produce TTMP,and the TTMP yield was 202.54 mg/kg,which reached the higher level of the reported starting strain.(3)The effects on baijiu fermentation and its product quality after inculcation with B.amyloliquefaciens XJB-104 were studied by monitoring the important physical and chemical parameters,the number and structure succession of brewing microorganisms during baijiu fermentation,and the sensory evaluation of baijiu.The inoculation of functional bacterial showed little impact on the key physical and chemical parameters of the fermentation and the diversity of brewing microorganisms of sesame-flavored baijiu.Bacillus was an absolutely dominant microorganism in stack fermentation and the early stage of solid-state fermentation,and Lactobacillus was the dominant microorganism in the later stage of solid-state fermentation.The relative abundance of Bacillus in the inoculated group(88.47%)was higher than that in the control group(82.14%).And the abundance of Bacillus was also higher than that of the control group during whole baijiu fermentation.After inculcation with the functional bacteria,the content of TTMP in zaopei and fresh liquor were significantly increased to 1.90 mg/kg and 1.39 mg/L respectively,which were 2714.29% and 2316.67% higher than its in control group.After inculcation,the quality of baijiu was significantly improved.The bacterial XJB-104 still showed the excellent characteristics of high-yield TTMP in the natural brewing system.In addition,the positive correlation was shown in TTMP,temperature and Bacillus abundance.High temperature flavored the formation of TTMP in zaopei.The TTMP increasement in zaopei of the experimental group was caused by the increased abundance of Bacillus in the fermentation microbiota using the directional perturbation of the functional bacteria B.amyloliquefaciens XJB-104.At the same time,it was also confirmed that TTMP in baijiu was mainly produced by the brewing microorganisms with biological pathway.(4)The functional fuqu of B.amyloliquefaciens XJB-104 was obtained using the single factor and BBD response surface optimization,and its prepared process was as follows: medium: 700 g of bran,300 g of soybean meal,1.85 g of Na OH,1.33 L of water;inoculation amount was 8%;the content of TTMP in fuqu reached 607.58 mg/kg on 71 h,which was about three times than its original yield.And distillers' grains(DGS)was used as the raw material for producing the functional fuqu.The prepared process of DGS functional fuqu was as follows: the medium contained bran 700 g,soybean meal300 g,DGS 367 g,Na OH 11.2 g,water 1.2 L;inoculation amount 8%;the yield ofTTMP was 1.28×103mg/kg at 3.5 d,which was about 6.3 times than its original yield and higher than any previous results.(5)The TTMP fermentation of B.amyloliquefaciens XJB-104 was optimized by single factor experiment using DGS as the raw material,and its product was purified.In 40 h,the yield of TTMP reached 3.18 g/L.The 1.85 g of purified TTMP was obtained from the fermentation broth(1 L)with a yield of 58.18%,and its purity was >99%.The main impurity was trimethylpyrazine.Using mice as materials,it was confirmed that TTMP in the ethanol-water system could improve liver tissue pathology,and moderately regulate the biochemical indicators related to liver injury(ALT,AST,AKP,and LDH),antioxidant reactions(T-SOD,CAT,MDA,and GSH)and inflammatory stress(NF-?B,TNF-?,IL-1?,IL-6,MCP-1,i NOS and COX-2)to exert hepatoprotective activity,and its mechanism may be related to the improvement of the antioxidant defense system of liver tissue and the suppression of inflammatory response.