解脂亚罗酵母控制葡萄赭曲霉毒素A产生与降解的机制研究

Ochratoxin A (OTA) is a toxin fungal secondary metabolite, which is mainly produced by several species of Penicillium and Aspergillus, OTA has teratogenicity, carcinogenicity and mutagenicity, so it exits potential hazard to human and animals. OTA has been identified in various fruits and fruit products all over the world, especially in grape and its products. Using antagonistic yeast to control OTA in grape and its products has represented a good prospect, but the related research is still in its infancy, especially the mechanism of controlling OTA synthesizing and biodegradation in grape and its products by antagonistic yeast is not clear yet, which seriously influenced the application of biological control technology in the control of mycotoxins on postharvest fruits. In this study, antagonistic yeast Yarrowia lipolytica which was isolated by our research team and Penicilluim rubens which could produce OTA isolated from grape, were chosen to the research object, in vivo and in vitro conditions, the control efficacy of Y. lipolytica against postharvest blue mould decay of grape caused by P. rubens, the mechanisms of controlling the growth of P. rubens and OTA synthesizing on grape by Y. lipolytica were investigated, so as to elucidate the possible mechanism of controlling and biodegradation of OTA on grape by antagonistic yeast, which can provide theoretical basis for the practical application of antagonistic yeast in postharvest fruits.

赭曲霉毒素A（OTA）是一种有毒的真菌次级代谢产物，主要有曲霉属和青霉属产生，具有致畸、致癌、致突变等多种毒性，对人和动物的健康存在潜在的危害。OTA对水果及水果制品的污染在全球范围内都比较严重，尤其是葡萄及其制品。采用拮抗酵母菌控制葡萄及其制品中OTA的方法展现出了良好的应用前景，但其相关研究目前尚处于起步阶段，特别是拮抗酵母菌控制葡萄OTA产生和降解已经存在的OTA的机制尚不明确，严重影响了生物防治技术在水果采后真菌毒素控制方面的应用。本项目拟以课题组前期筛选得到的解脂亚罗酵母和OTA产生菌Penicillium rubens为研究对象，在葡萄活体和离体条件下，研究解脂亚罗酵母对P. rubens生长和产OTA的影响及其控制和降解葡萄中P. rubens产生OTA的机制，从而阐明拮抗酵母控制水果OTA产生和降解的可能机制，为拮抗酵母控制水果真菌毒素的实际应用提供一定的理论依据。

本项目以葡萄为实验材料，以课题组前期分离筛选的拮抗酵母菌解脂亚罗酵母和葡萄上产OTA的菌株P. rubens为研究对象，研究解脂亚罗酵母对葡萄P. rubens的抑制作用和对P. rubens产OTA的影响，以及解脂亚罗酵母控制和降解葡萄中由P. rubens产生OTA的机制，进而阐明拮抗酵母控制葡萄OTA产生和降解的可能机制，为拮抗酵母控制水果真菌毒素的实际应用提供一定的理论依据。主要结论如下：.（1）Y. lipolytica能够显著控制葡萄采后由P. rubens引起的青霉病，且Y. lipolytica浓度越高，伤口处的腐烂率、腐烂直径越小；Y. lipolytica能够显著降低葡萄伤口处由P. rubens产生OTA的含量；体外试验表明，Y. lipolytica能够显著抑制P. rubens的孢子萌发及芽管长度。.（2）无论是20°C或4°C，Y. lipolytica均能够快速定殖在葡萄表皮保持较高酵母数量；Y. lipolytica能够增强葡萄体内抗性相关酶（PPO，POD，CAT，PAL，APX，GLU）活性并且能显著增强编码相应酶的基因表达量。.（3）Y. lipolytica胞内酶能够降解OTA，且55%-65%饱和度硫酸铵分级沉淀物对OTA的降解效果最好。Y. lipolytica胞内酶55%-65%饱和度硫酸铵分级沉淀物中包括羧肽酶、与能量代谢、氧化应激等相关的蛋白；推测羧肽酶直接参与OTA的降解，其它蛋白在OTA降解的过程中参与细胞对OTA引起的氧化胁迫的响应。.（4）利用薄层层析（TLC）、高效液相色谱（HPLC-FLD）和液质联用（HPLC-PDA-MS）检测技术，对Y. lipolytica胞内酶降解OTA的产物进行鉴定，并考察了产物对HEK293细胞的毒性。Y. lipolytica胞内酶降解OTA的主要产物为OTα，产物OTα对HEK293细胞的毒性低于OTA。