Application Of Food-derived Phenolic Compounds To The Process Of Chinese Yellow Rice Wine Fermentation For Ethyl Carbamate Regulation And Its Mitigation Mechanism

Ethyl carbamate(EC)is usually formed by the spontaneous reaction between ethanol and by-products produced by incomplete metabolism of nitrogen-containing compounds during the fermentation of food.EC is a potential genotoxic and carcinogenic to humans.However,EC content of traditional Chinese yellow rice wine is significantly higher than other alcoholic beverages,which gives a potential threat to the health of consumers.Therefore,EC reduction is of great importance to the sustainable development of the rice wine industry.It was found that gallic acid and protocatechuic acid could influence arginine catabolism.In this study,the effect of food-derived phenolic compounds on EC formation and its mitigation mechanism in Chinese yellow rice wine fermentation were further studied.The main findings are as follows:1.Effect of key food-derived phenolic compounds on EC formation and the hedonic quality of Chinese yellow rice wine were studied.It was found that GC and PC could significantly inhibit the formation of EC in Chinese yellow rice wine fermented with Saccharomyces cerevisiae(S.cerevisiae).In this study,the mixed-culture with S.cerevisiae and Lactobacillus brevis(L.brevis)could significantly inhibit the formation of EC during Chinese yellow rice wine fermentation,when contrasted to S.cerevisiae.EC concentration could be further reduced by 68.4%at most through adding 1×10~8 CFU/L L.brevis during S.cerevisiae fermentation,which was the optimal mixed-culture fermentation method.Unfortunately,the coexistence of gallic acid and L.brevis attenuated their inhibitory effect on EC formation.Bamboo leaves extract(BLE)could be used as a natural inhibitor of EC in multi-microbial fermentation systems.And 500 mg/L BLE had the most significant inhibitory effect on EC formation in mixed-culture fermentation system with S.cerevisiae and L.brevis.In the meanwhile,GC,PC and BLE addition could enrich amino acids and improve hedonic quality of Chinese yellow rice wine to a certain extent.2.Chinese yellow rice wine,fermented with S.cerevisiae and L.brevis,was suited to be stored in a low temperature and dark environment,which was helpful to reduce the formation of EC and enhance the flavor of Chinese yellow rice wine.During the storage period,BLE addition could also inhibit the formation of EC in the rice wine,have a beneficial influence on the hedonic quality of yellow rice wine.However,the storage temperature should be stable for the Chinese yellow rice wine with BLE addition.3.In Chinese yellow rice wine simulation system,the effects of key food-derived phenolic compounds on EC precursors,enzymes,and arginine metabolic pathways were studied at the metabolic,enzymology and transcription levels,respectively.It was found that BLE could reduce the content of urea and citrulline in the simulation culture system.The results of RT-q PCR showed that BLE had influence on arginine transport in S.cerevisiae,which significantly up-regulated arginine uptake gene in vacuole(VBA2 gene)so that inhibited arginine metabolism and reduced the contents of urea in the simulation culture system.The results of validation experiment by VBA2?proved that VBA2 gene was the target of BLE.Knocking out VBA2 gene could significantly up-regulate arginine and urea degradation genes CAR1 and DUR1,2 in S.cerevisiae,and finally reduce the urea content in the fermentation system.4.The transcriptional expression regulation mechanism of BLE to S.cerevisiae was studied by RNA-Sequencing in multi-microbial fermentation systems.The results showed that BLE could affect the autophagy(ATG family)and membrane fusion(VPS family)processes of S.cerevisiae,promote the formation of volatile esters(ATF1,ATF2)during fermentation,affect the transport of aspartic acid(ODC2)which may affect arginine metabolism of S.cerevisiae during Chinese yellow rice wine brewing.