戊唑醇对映体发酵过程选择性行为及其干扰葡萄酒风味品质机制研究

Tebuconazole, a class of chiral fungicide, is used for the control of a wide range of diseases in grapes. The enantiomers of tebuconazole are markedly different in their toxicity and activity. However, due to their complex chemical structure and physiological activity, the research has only been confined to racemic compounds. In addition, there has generally been lack of research on their selective degradation behavior during wine processing. In a previous study, we found that there is stereoselective degradation of the chiral tebuconazole during fermentation and aging processes. However, the key factors which impact on the stereoselectivity of enantiomers and the mechanism of how tebuconazole residue affects flavor and quality of wines are not clear. Consequently, in the present study, the effect of different parameters of fermentation and aging process on chiral pesticide tebuconazole enantioselective degradation behavior will be studied. And the study will be based on metabolomic concepts and strategies, using the GC and UPLC-MS/MS and UPLC/GC-Q-TOF/MS for determination, the different metabolites and the stereoselective metabolism mechanism of tebuconazole. In addition, E-nose, E-tongue and SPME-GC-MS will be used for quantitative and qualitative analysis of flavor including higher alcohols and esters in wine which will be treated with different tebuconazole enantiomers. Furthermore, transcriptomics and metabonomics will be used to study the interference of Saccharomyces cerevisiae metabolic pathway by different tebuconazole enantiomers. In conclusion, it is envisaged that our findings for the enantioselectivity in fermentation and aging processes and the molecular mechanism of tebuconazole enantiomers on wine quality and flavor may have some implications for better risk assessment of chiral pesticides.

戊唑醇是葡萄上常用的手性杀菌剂，其对映体间毒性及活性存在显著差异，但选择性残留行为尚不清楚，安全评价研究仍停留在消旋体水平上，给消费者带来严重安全隐患。前期通过戊唑醇对映体残留分析发现，葡萄酒发酵及陈酿过程是影响对映体选择性行为的关键加工环节，但对映体间的选择性行为及其影响风味品质机制不清。本项目通过研究发酵及陈酿过程中戊唑醇对映体残留行为规律，揭示其选择性降解、转化和富集机制；利用质谱、核磁等方法定性定量分析戊唑醇对映体代谢产物，明确发酵及陈酿过程中代谢途径；应用电子鼻/舌、固相微萃取-气相色谱质谱联用技术分析葡萄酒中高级醇、酯类等香气成分差异；基于转录组学和代谢组学技术研究戊唑醇对映体干扰酿酒酵母代谢途径机制，初步阐明戊唑醇对映体干扰葡萄酒风味品质的分子机理。完成上述研究将有助于揭示戊唑醇对映体间选择性行为及其干扰葡萄酒风味品质机制，同时为其对映体的安全评价研究提供理论依据。

葡萄种植过程中化学农药使用造成农药残留时有发生，对其主要加工品葡萄酒风味品质及食品安全带来安全隐患。本项目以手性杀菌剂戊唑醇为研究对象，建立了基于超临界流体色谱-质谱联用技术（SFC-MS/MS）葡萄、葡萄酒等基质中戊唑醇高效、灵敏、快速的对映体分离及定量分析方法。明确了手性农药戊唑醇在葡萄酒酿造过程中的降解规律，研究结果表明，戊唑醇对映体在葡萄酒发酵过程存在选择性降解行为，戊唑醇在酒精发酵阶段降解最为明显，并且(-)-R-戊唑醇降解速率快于(+)-S-戊唑醇，导致(+)-S-戊唑醇相对富集。进一步利用Q-TOF/MS对葡萄酒中戊唑醇代谢物进行筛查确证，初步明确了戊唑醇在葡萄酒酿造过程中降解生成戊唑醇-5-酮基（Tebuconazole-5-keto）、三唑丙氨酸（Triazole alanine）、戊唑醇谷氨酰胺（tebuconazole-deschloro-OH-glutamate）、三唑乙酸（Triazole acetic acid）、戊唑醇-1-羟基-5-酮基（tebuconazole-1-hydroxy-5-keto）等代谢产物。戊唑醇残留带来葡萄酒安全问题的同时会影响其风味品质。利用电子鼻、电子舌、电子眼、SPME-GC-MS、GC-IMS等技术对戊唑醇处理后酿造的葡萄酒风味品质进行评价，发现戊唑醇显著影响葡萄酒的感官品质，且(-)-R-戊唑醇起主要干扰作用。进一步利用高通量测序技术研究了戊唑醇对映体对葡萄酒发酵微生物选择性干扰机制，结果表明戊唑醇对映体对葡萄酒微生物的影响在不同发酵阶段表现出显著差异。结果表明戊唑醇通过影响微生物的氨基酸合成、糖代谢等代谢途径，影响葡萄酒脂肪族化合物合成，从而造成葡萄酒风味品质的改变。研究结果为手性三唑类杀菌剂在葡萄酒加工过程中的安全评价和风险评估提供科学数据支撑，为指导企业生产提供了理论指导；本项目培养博士研究生1名，硕士研究生3名，以第一或通讯作者发表学术论文9篇，其中SCI论文7篇。