紫苏醛对食品储藏中黄曲霉的抑制作用及其机理研究

Food spoilage caused by Aspergillus flavus is becoming a major food safety issue of global concern. As a natural green safe compound, perillaldehyde (PEE) showed significant antifungal activities, however, its mechanism of action has not yet been ascertained. Therefore, the present project will evaluate the antifungal activities of PEE through measuring various in vitro markers, such as mycelial growth, and also assess in vivo antifungal activities by exposing it to PEE vapor. On top of that, systematic study on the mechanism of antifungal action of PEE against A. glavus will be conducted. 1) To determine the exact target of PEE, at the cellular level, the effects of PEE on inhibition of ergoterol synthesis, lesion in the plasma membrane and release of cellular material will be investigated; at the subcellular level, the effects of PEE on mitochondrial ATPase, succinate dehydrogenase (SDH), malate dehydrogenase (MDH), membrane potential and ROS accumulation will be assessed; at the molecular level, the effects of PEE on the damage of DNA and RNA will be evaluated. 2) Based on the mitochondrial lesion, DAPI staining, Annexin V staining, metacaspase activity and release of cytochrome c (Cyt C) will be investigated to identify an apoptosis pathway in A. flavus. 3) Based on the DNA or RNA lesion, PEE-induced cell cycle arrest will be further verified through the changes in expression levels of cell-cycle genes. In this project, by elucidation of the antifungal activities and mechanisms of PEE against A. flavus, we can provide a very important theoretical basis for the application and development of new natural food preservatives.

黄曲霉引起的食品腐败变质已为全球关注的重大食品安全问题。紫苏醛（PEE）作为绿色安全化合物其抗真菌活性显著，但作用机理尚未阐明。本项目拟通过研究PEE对菌丝生长等体外指标评价抗黄曲霉活性，同时利用气相防腐技术评价体内抗黄曲霉活性，在此基础上系统进行作用机理研究：1）明确PEE抗菌靶标，分析PEE在细胞水平上对麦角固醇合成、细胞膜损伤、细胞核物质泄漏的影响，在亚细胞水平上对线粒体ATP酶、SDH酶、MDH酶、膜电位及ROS累积的影响，在分子水平上对DNA和RNA损伤的影响；2）以线粒体损伤为切入点，通过DAPI染色、Annexin V染色、Caspase活性、Cyt C的释放等研究揭示其凋亡途径；3）基于PEE对核酸分子损伤，通过周期蛋白基因表达水平的变化以探明PEE对细胞周期的阻滞阶段。本项目通过以上研究阐明PEE抑制黄曲霉的作用及机理，将为新型天然食品防腐剂的应用与开发提供理论依据。

黄曲霉引起的食品腐败变质已为全球关注的重大食品安全问题。紫苏醛（PAE）作为绿色安全化合物其抗真菌活性显著，但作用机理尚未阐明。本项目通过研究PAE在紫苏挥发油中的含量以及不同地区的含量变化，继而对菌丝生长等体外指标评价抗黄曲霉活性，同时利用气相防腐技术评价体内抗黄曲霉活性。结果发现，PAE是紫苏挥发油中的主要成分，PAE体外抗黄曲霉活性显著（最低抑制菌丝生长的接触实验浓度为0.5 μL/mL，挥发实验则浓度达到了0.08 μL/mL air，在0.4 μL/mL浓度时完全地抑制了黄曲霉毒素的产生），对樱桃西红柿中常见的腐败菌显示出了非常好的防腐效果（PAE在浓度为0.05 μL/mL air时效果最佳）。在体内外PAE抗黄曲霉活性的研究基础上，系统地研究了PAE对黄曲霉的作用机理。结果发现，通过流式细胞仪检测细胞膜损伤以及细胞膜成分麦角固醇含量的影响以评价PAE对细胞膜有明显地抑制作用，验证了PAE是以黄曲霉细胞膜为其中的一个生物靶标来发挥抗真菌作用的。此外，我们通过透射电镜观察、流式细胞仪进行胞质钙离子水平检测、线粒体膜电位的变化、DNA碎片化和凝集、形态学观察、磷脂酰丝氨酸外翻、ROS 水平检测、细胞色素C释放、Caspase活性检测等指标进行PAE诱导黄曲霉细胞凋亡的研究，本项目验证了PAE最终是通过介导metacaspase途径诱导黄曲霉细胞凋亡而发挥抗真菌作用。本项目的实施对推动和指导PAE作为新型食品防腐剂的应用有着重要的理论价值，同时为抗菌剂的开发提供新的思路。