典型手性杀菌剂在葡萄种植-家酿制酒过程中的立体选择性降解和迁移规律研究

Chiral fungicides have been used widely for treating plasmopara viticola, botrytis cinerea, sphaceloma ampelimum, coniella diplodiella and so on in grape planting. The residue in table grape has been paid attention. However, it is short of the research on dynamic change of fungicide residues during the process of winemaking and evaluation on the levels of fungicide residues in wine. The risk of chiral pesticide residue to human health is generally evaluated with racemate. Considering the possibility of different bioactivity and toxicity between enantiomers, the risk evaluation for chiral pesticide with racemate may not be accurate. In this project, three typical chiral fungicides including myclobutanil, propiconazole and difenoconazole will be sprayed on grape in field and the ripening grape after spraying fungicides will be made to wine by home winemaking. The degradation process of their enantiomer residues will be analyzed by enantiomeric LC-MS/MS and UPC2-MS/MS analysis technology. Then the enantiomer residues in grape pomace and fermentation must during wine homemaking process will be analyzed. The transformation between enantiomers of each three fungicides during home winemaking process and the effect of added yeast on the enantoselective degradation of chiral fungicide will also be studied. The enantioselective degradation of three fungicides during grape planting and transferring pattern of them from grape to homemaking wine will be figured out. The absolute configuration for each enantiomer of three fungicides will be identified by VCD and computer associated calculation. It will provide scientific data for evaluating risks of the chiral fungicide residues in grape and homemaking wine.

葡萄种植中经常使用杀菌剂防治霜霉病、黑痘病和白粉病等病菌。鲜食葡萄上的杀菌剂残留问题已引起重视，但较少研究杀菌剂在葡萄加工酿酒过程中的残留动态变化和酒中残留水平。目前手性三唑类杀菌剂在葡萄生产中应用广泛，但评估它们对人类健康的风险通常不区分对映体差别，而往往对映体生物活性和毒性差异悬殊，易导致手性杀菌剂在葡萄及葡萄酒残留的风险评估不够准确。本项目拟通过大田种植葡萄杀菌剂喷施试验和葡萄家酿制酒过程，借助实验、计算振动圆二色光谱和多种手性分析手段，明确三种典型三唑类手性杀菌剂（腈菌唑、丙环唑、苯醚甲环唑）对映体在葡萄上的动态降解，并进一步阐明葡萄家酿制酒过程中发酵汁和葡萄渣中对映体残留的动态迁移变化、对映体是否存在转换以及添加酵母对对映体迁移的影响，明晰它们在葡萄种植-家酿制酒过程中的降解和迁移规律，揭示其立体选择性机制，为更清晰的评估葡萄及家酿葡萄酒中手性杀菌剂残留的安全性提供科学数据支持。

食品安全日渐成为各阶层关注的焦点，葡萄和家酿葡萄酒中潜在的农药残留风险也不容忽视；同时手性农药对映异构体的生物活性、毒性以及在植物和环境中的降解等方面可能存在差异。因此，对典型手性杀菌剂在葡萄种植过程中立体选择性降解规律及其在葡萄家酿过程中残留追踪分析具有至关重要的意义。本文研究了葡萄种植施药后五种手性杀菌剂（腈菌唑、噁唑菌酮、双炔酰菌胺、丙环唑和苯醚甲环唑）的立体选择性降解，同时测定葡萄家酿制酒过程中五种目标杀菌剂的迁移-降解研究及添加酿酒酵母对其过程的影响。在项目实施中，主要取得如下研究成果：（1）建立了腈菌唑、噁唑菌酮、双炔酰菌胺、丙环唑和苯醚甲环唑在葡萄及葡萄酒基质中的HPLC-MS/MS和超临界流体色谱串联质谱（SFC-MS/MS）的检测方法，同时确定了腈菌唑和噁唑菌酮单体的绝对构型（R/S）及其在色谱柱上的流出顺序。（2）在葡萄种植过程中腈菌唑、噁唑菌酮、丙环唑和苯醚甲环唑存在着显著的立体选择性降解，S-腈菌唑、S-噁唑菌酮、丙环唑-A2、丙环唑-B1、苯醚甲环唑-A2和苯醚甲环唑-B1在葡萄中优先降解；双炔酰菌胺不存在立体选择性行为。（3）添加酵母菌不会明显影响5种目标杀菌剂在葡萄家酿制酒过程中的降解规律，但会加快发酵过程，致使更多目标杀菌剂从葡萄果肉中转移至酒汁中，也加快杀菌剂的降解作用。目标杀菌剂在葡萄酿造过程其残留规律不仅与酿造工艺有关，且还与杀菌剂的理化性质密切相关。本研究提供了家酿葡萄酒过程中5种常见杀菌剂残留水平，为中国家酿葡萄酒杀菌剂残留风险评估提供基础数据。部分成果已在相关领域发表SCI收录学术期刊论文4篇、国内相关领域核心期刊1篇；同时，受项目资助，培养硕士研究生1名，于2018年提交硕士论文。