Formation Mechanism And Elimination Strategy Of Ethyl Carbamate In Soy Sauce

Ethyl carbamate(EC)is a group 2A carcinogen generated from the reaction of a few precursors and ethanol in many fermented foods and alcoholic beverages. Soy sauce fermentation is a mixed fermentation process in which the main EC precursor and its formation pathway has not been resolved, resulting in the failure to develop an effective strategy to eliminate the EC. Therefore, to investigate the key microbes associated with the formation of EC precursors, explain the relevance of key microbes and the production of EC precursors, and clarify the EC formation pathway, is very important to seek a way to control or reduce the EC content in soy sauce. In this paper, the formation mechanism of EC and its elimination strategy was investigated by studying EC precursor of soy sauce and its formation patterns. The main results of this study are as follows:(1) EC content in soy sauce samples with different fermentation processes was investigated, the results demonstrated that the EC formation was mainly occurred during the sterilization process, especially in the soy sauce samples with the addition of yeast; The main precursor of EC is citrulline with the simulation experiment. Citrulline was accumulated during lactic acid fermentation and alcoholic fermentation. The lactic acid fermentation period is the most important citrulline accumulation period during soy sauce fermentation. About 1.38 g?L-1 citrulline was accumulated during this period, accounting for 69% of the total accumulations. A high-throughput screening method with specific orientation was designed based on citrulline accumulation period and the characteristic of citrulline accumulation. Ten citrulline accumulation strains were effectively separated from both koji and moromi mash; We also found that the exogenous addition of Zygosaccharomyces rouxii ZQ01 was related to the accumulation of citrulline during alcoholic fermentation, and the exogenous addition of Z. rouxii ZQ02 was related to the reduction of citrulline during lactic acid fermentation.(2) The two Z. rouxii strains which used in the soy sauce fermentation process did not utilize arginine, and thus will not accumulate citrulline and urea with the degradation of arginine. Glycine, alanine and asparagine were identified as preferred nitrogen sources of Z. rouxii ZQ01 and Z. rouxii ZQ02. The addition of preferred nitrogen sources(alanine and glycine) in the medium when cultivated Z. rouxii ZQ02 stimulated the consumption of both urea and citrulline. However, utilization of citrulline and urea by Z. rouxii ZQ02 was strongly inhibited under saline stress, resulting in the accumulation of ethyl carbamate precursors produced by other microorganisms during soy sauce fermentation; The genotype and salt tolerance of two soy sauce yeasts Z. rouxii ZQ01(to produce alcohol) and Z. rouxii ZQ02(to produce aroma compounds) were compared. Z. rouxii ZQ01 was an allodiploid and Z. rouxii ZQ02 was an amphidiploid. Z. rouxii ZQ02 showed higher resistance of osmatic stress than ZQ01.(3) Pediococcus acidilactici was the main citrulline accumulation strain during the lactic acid fermentation based on the analysis of citrulline accumulation capabilities of the isolates. 18% NaCl is the main cause of citrulline accumulation by P. acidilactici through the arginine deiminase(ADI) pathway, 18% NaCl induced the unequal proportional reduction of arcA(coding arginine deiminase, ADI) and arcB(coding ornithine carbamoyl transferase, OTC), resulting in the relative increase of arcA gene expression. The relatively increase of arcA gene expression may cause the increase of intracellular ADI / OTC enzymatic activity ratio, resulting in the rate of citrulline synthesis greater than the rate of citrulline decomposition.(4) Staphylococcus was determined as the main arginine consumption and citrulline accumulation genus during alcoholic fermentation with the analysis of citrulline producing capabilities of the isolates. However, the conversion ratio from arginine to citrulline of Staphylococcus in arginine consumption medium was far below the conversion ratio in moromi mash sample from alcoholic fermentation. Through further research, the enhanced solubility of free fatty acids in the moromi mash induced by ethanol stimulated the accumulation of citrulline during the alcoholic fermentation in soy sauce production.(5) Based on the study above, the method of addition of a strain with the ability to utilize arginine in soy sauce and did not accumulate citrulline was established to make global disturbance of the microorganisms in soy sauce, and thereby eliminate EC production. To avoid secondary pollution, a salt tolerant strain Bacillus amyloliquefaciens JY06 with high arginine consumption ability and low citrulline accumulation capacity was isolated from the moromi mash of late lactic acid fermentation. The concentration of citrulline was dramatically decreased from 26.83 mM to 5.13 mM(reduced by 81%), and ethyl carbamate(EC) in soy after sterilization was decreased from 97.4 μg?kg-1 to 16.8 μg?kg-1(reduced by 83%) compared to the control, when B. amyloliquefaciens JY06 was employed in soy sauce fermentation process. Meanwhile, the aroma of soy sauce was improved by increasing the ester content with the addition of B. amyloliquefaciens. EC precursors were successfully eliminated by using the microbiao perturbation method, and thereby reducing the generation of EC...