| Ethyl Carbamate(EC),a group 2A carcinogen,commonly found in fermented foods such as Chinese rice wine,sufu and soy sauce.Soy sauce is widely consumed and highly appreciated in Asia,EC level in some fermented soy sauce was as high as 128.9μg/L,which made soy sauce a potential harm source for residents,particularly for children.EC determination is mainly based on the instrumental detection technology of GC-MS for now,hence cannot meet with the demand of low-cost and rapid detection for the determination of large-scale sample;besides,the production of fermented soy sauce needs long-term and complicated procedures,the formation path and mechanism of EC in soy sauce are not clear enough,hence it is difficult to propose feasible EC-controlling strategies.In this paper,the EC determination methods are developed and the targeted strategies for EC controlling are proposed through exploration of the EC formation mechanism in fermented soy sauce.The main results are as follows:(1)Two kinds of EC determination methods were established.(1)An indirect competitive ELISA for EC determination was developed.By introducing a spacer arm containing a phenyl group to directly link to the amide group of EC,after coupling with protein,the prepared immunogen can induce animal’s immune response which produces antibody targeting EC molecule.Then the phenyl group was replaced by linear aliphatic linker with approximately euclidean length in the design of heterologous coated antigen so that the recognition of antibody against phenyl group in spacer arm is eliminated.Finally,a specific and high-affinity monoclonal antibody against EC was obtained,and the heterologous ic-ELISA based on it can determine EC specifically with LOD of 1.3 mg/L,the developed ic-ELISA cannot satisfy for the determination of low levels of EC in soy sauce.However,after purification and concentration by solid phase extraction,the recoveries in spiked sample with above 400μg/L EC of rice wine were 109.2%,with CV of17.1%,indicating it can be used in screening of high contaminated samples.(2)A high-performance liquid chromatography was developed for EC determination in soy sauce based on the rapid reaction between EC and 9-xanthydrol at room temperature.After systematical optimization of extraction,derivatization,fluorescence spectrum and chromatographic conditions,the method can be applied to EC determination in soy sauce.The LOD and recovery of EC determination were respectively 3.91μg/L,13.03μg/L and81.5%-95.4%,with coefficient of variation less than 10%.The results obtained by proposed method in accordance with that by GC-MS,indicating a suitable EC determination method for soy sauce.(2)The formation rules of EC in fermented soy sauce were clarified.(1)Through analyzing EC content in fermented foods in Guangzhou,except for sufu,it was founded that the detecting rate of EC in high-salt liquid fermented soy sauce was 100%,and EC contents in high-salt liquid fermented soy sauce were significantly higher than that in low-salt solid fermented soy sauce as well as most of other fermented soybean foods.EC level showed the positive correlation with ethanol content(R~2=0.875),and it was also correlated to total arginine and free ornithine content in high-salt liquid fermented soy sauce products.(2)EC and its precursors were not accumulated at the stage of moji-making,besides,EC contents were lower than 8μg/L during moromi fermentation.The critical formation stage of EC was the heating treatment stage,whose EC accumulation accounted for 54.8%to 83.5%of the total EC content.During heating treatment,through adding potential EC precursors of the same amount as that in raw soy sauce,we found that EC formed by adding citrulline and ethanol far exceed that of urea,indicating ethanol and citrulline were two critical EC precursors in main EC accumulation stage.The increase of EC in raw soy sauce with p H of 6 was significantly lower than that in raw soy sauce with p H of 4.6-5.(3)Ethanol content exhibited significant correlation with EC content throughout the whole production,according with the result of the simulative fermentation in laboratory.Moreover,the low fermentation temperature was conducive to slow down the decrease of p H and to delay the alcoholic fermentation,finally resulting in reduction of EC.(4)P.acidilactici and W.confusa isolated from moromi mash with ADI pathway gene were confirmed the main strains accumulating citrulline.Through analysis,it was found that salt stress was the main reason for P.acidilactici and W.confusa’s citrulline accumulation.The ADI gene expression was reduced significantly under salt stress,the proportion of arc A/arc B expression in P.acidilactici increased resulting in low rate of citrulline decomposition,while it have no significant differences in W.confuse.The conversion ratio of arginine to citrulline by these two strains could be reduced at low temperature and high p H under salt stress.(3)Two feasible EC-controlling strategies were developed.(1)A direct EC reduction method was proposed for preventing EC formation during heat treatment process.Through analysis of the main factors affecting EC formation during heating treatment,it was concluded that metal ions and arginine content had a little influence on EC formation.EC formation during heating treatment can be significantly inhibited by ornithine and quercetin.Besides,Kinetic study has indicated that ornithine and quercetin could reduce formation rate of EC,and the final EC content could be reduced by quercetin.Via addition of optimized amount of ornithine and quercetin gained from central composite design,EC in soy sauce was reduced 42.1%and 47.2%when temperature of heating treatment was set at80oC and 90oC respectively.Besides,there were no remarkable changes in aroma profile and color of soy sauce.(2)A method for EC and citrulline mitigation was developed during soy sauce fermented process.Two kinds of polyphenols,quercetin and gallic acid,that can reduce the citrulline accumulated capacity of P.acidilactici and W.confusa,Enterococcus faecium that greatly consume arginine but did not accumulate citrulline,and a good-flavour strain of Enterobacter sp.were added in moromi fermentation,the citrulline content in moromi was dramatically decreased from 2.23 mg/m L to 1.61 mg/m L.2%ethanol content were added into raw soy sauce,after heat treatment,EC in soy sauce was significantly decreased from 36.49μg/L to 9.82μg/L(reduced by 69.5%).Meanwhile,the addition of polyphenols demonstrated little influence on aroma profile of soy sauce,and the addition of the two strains increased the relative content of 2-methylbutanal,3-methylbutanal,benzencacctaldehyde and ethanol,hence improved the flavor of soy sauce.Finally,the EC content in soy sauce was successfully reduced to 20 g/L through microbial perturbation,and the quality of soy sauce products satisfied the national requirements.The EC mitigation technologies were established for heat treatment process and fermentation process respectively.These results provided a theoretical basis for reducing EC level in soy sauce and improving the quality of soy sauce. |
