The Interaction Effect Of Five Pesticides With Yeast Microorganism And The Fate Of Pesticide Residues During Wine Fermentation

Wine,as the representative of typical grape processed product,is produced by grape and fermentative microorganisms through alcohol fermentation.During the brewing process,the pesticide residues on the surface of grapes will be gradually transferred to the fermentation system along with each processing step,and interact with microorganisms,such as saccharomyces cerevisiae.Pesticide residues may affect the growth and fermentation of yeast.At the same time,various changes may occur under the action of fermentative microorganisms,such as adsorption,metabolism and degradation.Furthermore,the pesticide residues will eventually remain in processed products,and cause potential harm to the quality and safety of grape processed products.This paper explored the differences in growth toxicity of five pesticides on saccharomyces cerevisiae,clarified the interaction between saccharomyces cerevisiae and five target pesticides in fermentation environment,summarized and analyzed the fate of pesticide residues during wine fermentation through aseptic cultured and processing experiments.This study will provide data reference for the rational selection,application and risk assessment of pesticides on grapes.The main conclusions are as follows:Firstly,an analytical method was established for determination of six compounds（difenoconazole,pyraclostrobin,tebuconazole,azoxystrobin,thiamethoxam,clothianidin）in grape,grape juice,wine and grape pomace by ultra-high performance liquid chromatography tandem mass spectrometry（UPLC-MS-MS）.The modified Qu ECh ERS method was used to pretreat the samples.Acetonitrile was used as extractant.PSA was used as sorbents,and quantification was performed by external standard method.The recoveries of the six compounds in various matrices were ranged from 72.02%to 112.81%,with relative standard deviations（RSD）in the range of 0.75%to 13.19%,and the limit of quantitation（LOQ）was 0.01 mg/kg.The results meet the requirements of residue detection,and could be used for quantitative determination of six compounds in grape and its processed products.Secondly,saccharomyces cerevisiae were cultured in the medium under sterile condition.The effects of pesticides on the growth of saccharomyces cerevisiae were determined by setting different pesticide stress conditions.The results showed that the inhibitory effects of pesticides on the growth of yeast were different with pesticides,and the inhibitory effects of five pesticides were successively as follows:difenoconazole>tebuconazole>pyraclostrobin>azoxystrobin and thiamethoxam.Two triazole fungicides,difenoconazole and tebuconazole,had significant inhibitory effects on the growth of yeast,and the higher the concentration or relative amountwas,the more obvious the inhibition effect was observed in exposure test.Two methoxyacrylate fungicides,pyraclostrobin and azoxystrobin,had slight or no effect on the growth of yeast at different concentrations.Thiamethoxam,as a neonicotinoid insecticide,had no obvious effect at three spiked levels.In addition,mixed exposures had no obvious synergistic toxicity to yeast growth.Thirdly,the fermentation process was simulated by model synthetic medium,and the interaction between saccharomyces cerevisiae and five target pesticides in the fermentative environment was determined.Specifically,the saccharomyces cerevisiae used in the experiments had no obviously degradative and metabolic activity to the five pesticides in the fermentation process.The effects of pesticides on the growth of saccharomyces cerevisiae were different in the fermentation environment,and two triazole fungicides,difenoconazole and tebuconazole,had obviously inhibitory effects.Fourthly,through the wine processing experiments,the differences in the change and distribution of the five pesticides during the wine making process were identified.Under different fermentation modes,the processing factors（PFs）of the five pesticides in the whole process were between0.030-0.236（spontaneous fermentation）or 0.032-0.234（inoculated fermentation）,and the process could greatly decreased the levels of pesticide residues in processed products.In different processing steps,fermentation and clarification processes can significantly reduce the levels of most pesticide residues in wine,and the degree was affected by the chemical properties such as K_OWof pesticide itself.In addition,the distribution of pesticides in each processing product showed obviously difference.The target compounds can be enriched in grape pomace and other by-products.The enrichment ratios of the target compounds were between 0.38 and 37.82,and the enrichment ratio were positively correlated with log K_OWof pesticides.