Detection Of Genes Related To Biogenic Amines And Ethyl Carbamate In Oenococcus Oeni And Study Of Influencing Factors On Ethyl Carbamate Production

Ethyl Carbamate (EC) and biogenic amines (BAs) are widely found in fermented foods including wine. They decrease the safety of wine, for EC is a carcinogen and excessive BAs cause allergic reaction. So, the producing mechanism, influencing factors and testing methods, also the detection of EC- and BA-producing bacterium in fermented foods were studied. With the development of science and technology, molecular biological techniques play an important role in the identification of EC- and BA-producing bacterium. Screening and utilizing malolactic bacterium strains that do not possess genes related to EC and BA producing in wine brewing can improve the quality and safety.50 Oenococcus oeni (O. oeni) strains selected from China's major grape and wine region were detected for the genes related to EC and BA: arginine deiminase (arcA), ornithine transcarbamylase (arcB), carbamate kinase (arcC), histidine decarboxylase (hdc), ornithine decarboxylase (odc), tyrosine decarboxylase (tdc), in addition, the effect of ethanol, SO2, arginine and pH on EC forming was observed. The main results are listed as follows:1 Testing of genes related to ethyl carbamateGene arcA, arcB and arcC in O. oeni were amplified by primer sets arcAF/arcAR, arcBF/arcBR and CK5'/CK3', respectively. The results showed that 50 O. oeni strains possess the arcA, arcB and arcC gene after sequencing the amplified single bright band and comparison with the gene sequence in Genebank, indicating all the strains has the potential to produce EC.The PCR amplification system (20μL) was standard. The reaction conditions were: initial denaturation temperature, 95℃, 5min; 30 cycles, denaturation temperature, 95℃, 30s; annealing time 30s; extension temperature, 72℃, 30s; the final extension temperature 72℃, 5min.2 Testing of genes relevant to biogenic aminesSix pairs of primer sets were synthesized in the experiment: Primer sets CL1mod/JV17HC and PHDC1/PHDC2 for hdc gene test, primers sets P1-rev/P2-for and TD2/TD5 for tdc gene, primers sets 3/16 and odcf/odcr for odc gene. After comparing the amplified effects of the six pairs of primers at different annealing temperature, primers CL1mod/JV17HC, P1-rev/P2-for, 3/16 were chosen for hdc gene, tdc gene and odc gene detection. The results showed that all the 50 O. oeni strains do not have histidine decarboxylase gene, tyrosine decarboxylase gene and ornithine decarboxylase gene.The PCR amplification system (20μL) is standard. The reaction conditions were: initial denaturation temperature, 95℃, 5min; 30 cycles, denaturation temperature, 95℃, 30s; annealing time 30s; extension temperature, 72℃, 30s; the final extension temperature 72℃, 5min.3 Effect of influencing factors on EC formingThe effect of alcohol, arginine, pH and SO2 on EC production was studied in the model wine via solid-liquid extraction and GC-MS method. The results showed that increased alcohol content promoted the formation of EC, also at the low concentration of arginine, but the formation was inhibited at high concentration of arginine; while EC yeild did not change significantly at the experimental pH and SO2 levels.