Study On Citrulline Accumulation And Reduction By Lactic Acid Bacteria Isolated From Chinese Rice Wine Fermentation Liquor

Ethyl carbamate?EC?,a carcinogenic compound in Chinese rice wine,mainly derived from spontaneous reaction of ethanol with carbamoyl compounds,such as urea and citrulline.Previous study showed that besides the well-known urea,citrulline was also an important precursor of EC in Chinese rice wine.Much effort has made to decrease urea content in Chinese rice wine,but little literature was available on formation mechanism and control strategy of citrulline.In this study,the capability and influencing factors of fermenting lactic acid bacteria?LAB?to accumulate citrulline were determined.Finally,the application potential of a LAB strain with special citrulline assimilating feature in decreasing EC content was analyzed.The dynamics of bacteria community structure during Chinese rice wine production process were analyzed via polymerase chain reaction-denaturing gradient gel electrophoresis?PCR-DGGE?,which proved that LAB especially lactobacillus was the predominant bacteria.Meanwhile,from the fermenting liquor,a total of 25 LAB strains were isolated,all of which exhibited strain specific capability to degrade arginine and accumulate citrulline(63-579mg·L^-1)during exponential growth phase.Unlike other strains,the strain Lactobacillus brevis2-34 did not accumulate any citrulline,which was attributed to its citruline re-uptake ability.The arc gene clusters that encoded enzymes involved in arginine deiminase pathway?ADI pathway?of LAB strains were sequenced and analyzed.Among species L.brevis,Lactobacillus fermentum,Lactococcus lactis and Weissella cibaria,arc gene clusters were somewhat different in gene composition and sort order.However,all the clusters contained sequence conserved arcABCD genes,which encoded the key enzymes of ADI pathway.The arcAB genes,which correlated with arginine degrading ability,showed high similarity among different species.Therefore,the degenerate primers were designed to detect arcAB genes in other LAB strains,therefore to determine their arginine degrading capability.The environmental conditions during fermentation were simulated,by adjusting concentrations of glucose and alcohol,as well as initial pH,to investigate factors that influence citrulline accumulation by LAB strains.The low glucose content repressed bacterial growth while high glucose content repressed transcription of arc genes.As initial pH increased,the arginine was degraded faster,and more citrulline was accumulated(maximum 285 mg·L^-1).When the alcohol degree was less than 8%vol,citrulline formation was not affected.The strain L.brevis 2-34 could degrade arginine and excrete citrulline in early exponential growth phase,but reutilized extracellular citrulline later,before arginine completely degraded.This strain also showed strong capability to utilize citrulline when cultured with citrulline as the only nitrogen source,or co-cultured with LAB strain that excreted a large amount of citrulline.When inoculating L.brevis 2-34 into Chinese rice wine fermentation,the contents of citrulline and EC were decreased by 46.4%and 41.0%,respectively,comparing to that using L.fermentum 2-36 as inoculators.These results indicated a great application potential of L.brevis 2-34 for decreasing EC content in Chinse rice wine.