Study On The Application Of Bacillus Sp.with High Pyrazine Production In Sesame-flavored White Wine

Pyrazines are six-membered heterocyclic compounds containing two nitrogen atoms at the 1,4 positions of the benzene ring.There are a wide variety of compounds,some of which have a wide range of applications in medicine,agriculture and food.Among them,pyrazines detected in white wine can have a certain influence on the flavor and taste of white wine,and are one of the key components with unique aroma in white wine.In addition,pyrazines are also of wide interest as a class of health factors.In this project,a series of studies were conducted to analyze the types,functions and specific enhancement methods of pyrazines in sesame-flavored baijiu with regard to the detection methods,biological functions and their applications in sesame-flavored baijiu,and the results obtained are as follows:（1）A liquid-liquid extraction direct injection-gas chromatography-mass spectrometry method was developed for the determination of pyrazine flavor components in white wine and its spirits.The results showed that the linear correlation coefficients of the eight pyrazines were greater than 0.999,the minimum detection limit was 10μg/L,the spiked recoveries were 87%～116%,and the relative standard deviations were 3%～10%,indicating that the method is accurate and precise,and can be used for the routine determination of pyrazine flavor components in sesame-flavored white wine and wine grains.The method was used for the routine detection of pyrazine flavor components in sesame-flavored white wine and wine spirits.The method was optimized for the determination of pyrazine compounds in wine spirits,and single-factor experiments were conducted on the key factors of pretreatment,such as extractant concentration,extraction temperature and extraction time.The results showed that the extraction of pyrazine compounds in wine spirits was most effective when 12%ethanol was used as the extractant,the extraction temperature was 50℃and the extraction time was 30 min.（2）The pyrazines currently contained in white wine were studied by means of biological evaluation techniques.Firstly,cells were treated with different concentrations of pyrazines to observe their effects on cell survival.The results showed that 2,3,5-trimethylpyrazine,2,3-dimethylpyrazine and 2-methylpyrazine had an inverse effect on cell survival,and the higher the concentration,the greater the effect,while tetramethylpyrazine had almost no effect on cell survival,and still had more than 50%survival at high concentrations.Based on the changes in cellular autophagic activity,a high-throughput cellular autophagy marker platform was established to study the effects of pyrazines on cellular autophagy.It was shown that tetramethylpyrazine had a good promotion effect on cellular autophagy,and other pyrazines had no significant effect.Finally,the antioxidant effect of tetramethylpyrazine was investigated by treating normal hepatocytes with H₂O₂and establishing an oxidative stress model of hepatocytes.The results showed that tetramethylpyrazine alleviated the oxidative damage of hepatocytes to some extent and proved that it could maintain the normal survival of cells.（3）Combined with the traditional brewing process of sesame-flavored liquor,Bacillus subtilis,which is high in pyrazine compounds,was used as a functional strain and cultured into a functional bacterial solution under a specific fermentation process,which was first added to the wine grains at the stacking stage to carry out synergistic fermentation with traditional macrophytes and gluten bran,thereby increasing the content of pyrazine compounds in the original sesame-flavored liquor.The results showed that the spirits with the addition of functional bacterial liquid had a certain degree of increase in its temperature and moisture content during the stacking process,and the content of pyrazine compounds in the spirits with the addition of functional bacterial liquid during the stacking process was 3232μg/L.The content of pyrazine compounds in the experimental group with the addition of functional bacterial liquid at the end of fermentation was as high as 6007μg/L,which was 2.87 times higher than that in the group without the addition of functional bacterial liquid,and the total amount of pyrazine compounds in the original wine of the experimental group with the addition of functional bacterial liquid was 3776μg/L.In addition,by simulating distillation,the fermentation broth of functional bacteria was added to the distillation pot for string distillation experiment,and the total amount of pyrazine compounds in the original wine obtained from the distillation of the experimental group was significantly higher than that of the control group,and the concentration of pyrazine compounds in the control group was about 898μg/L,while the experimental group could reach about 3266μg/L,which increased the concentration by 2.6 times.（4）Based on the traditional production process of sesame-flavored liquor producers,the functional branquat obtained by mixing and culturing functional bacteria high in pyrazine compounds with bran was added to the stacking process and fermented synergistically with traditional branquat and daguerreotype,and the production process of pyrazine compounds-producing functional branquat was investigated by single-factor test method,and the effects of five factors,namely inoculum amount,bran addition,water addition,incubation temperature,incubation The effects of five factors on the production of pyrazine compounds from functional bacterial bran were investigated,and the optimal preparation process of functional bran was optimized by response surface methodology.The results showed that the optimal conditions for pyrazine production from bacterial bran were 55 g of bran addition,45 g of water addition,14 h of incubation time,12%of inoculum,and 37℃of incubation temperature,and the total amount of pyrazine compounds was 3397μg/L.The key physicochemical factors of wine grains stacking process were investigated,and the results showed that the number of bacteria in wine grains increased from 8.6×10⁸to9.2×10⁸,while yeasts and molds did not change much.The temperature and moisture increased to some extent during the stacking process,while the acidity did not change significantly and the consumption rate of starch and reducing sugar increased.The total amount of pyrazine compounds in the spirits of the functional bran group was5.06 times higher than that of the control group;in the sensory evaluation of the original wine,the sesame-flavored original wine produced by the functional bran experimental group had a more typical sesame-flavored style with a strong burnt aroma,a long finish and a full-bodied wine,indicating that the functional wine spirits had a positive effect on the improvement of the wine body.