甘草影响赭曲霉毒素A产生菌增殖及产毒的机制研究

Liquorice is one of the most frequently used herbs in Traditional Chinese Medicine. In addition, liquorice is widely used in food product, beverage and other fields. Compared to other herbs, liquorice is more easily contaminated by ochratoxin A (OTA). OTA is a toxic metabolite produced by several fungi of the genera Aspergillus and Penicillium, with strong carcinogenic, teratogenic and mutagenic effects. Contamination of fungi not only decreases the quality of medicinal herbs, but also represents a special hazard to consumers. Our previous study found that the OTA producing fungi associated with liquorice were special, we hypothesize that both the chemical characteristics of liquorice and environmental factors contribute to the proliferation and OTA production of fungi, which result in the susceptibility of liquorice to OTA. This project is performed to 1) analyze the OTA producing fungi associated with liquorice during harvesting, processing and marketing using morphological observation combined with multiple sequence alignment; 2) analyze the effects of liquorice chemical composition on fungal proliferation and OTA production under different environmental factors; 3) found the molecular mechanisms behind this phenomenon using differential disply reverse transcription polymerase chain reaction technology. The results can not only guide the scientific prevention of OTA contamination in liquorice, but also enrich the OTA synthesis and regulation theory.

甘草是中药中应用最广泛的药材之一，还广泛应用于食品、饮料等领域。与其他药材相比，甘草极易受赭曲霉毒素A（OTA）污染。OTA是由青霉及曲霉属真菌产生的有毒次生代谢产物，具有极强的致癌、致畸、致突变作用，受污染的甘草不仅质量下降，还具有重大安全隐患。课题组前期研究发现，甘草上具有独特的OTA产毒菌群，甘草易感OTA的可能机制是其化学成分与环境因子协同作用，促进了这些OTA产毒菌的增殖及OTA合成。本研究针对这一假说开展如下研究：1）系统分析采收、加工以及储藏等环节污染甘草的OTA产毒菌类群，探明我国甘草上OTA污染发生的生物基础；2）通过多因子分析，考察不同环境条件下甘草化学成分对OTA产毒菌增殖及产毒能力的影响；3）应用差异显示反转录聚合酶链式反应技术研究甘草中单体化合物促进目标菌株OTA合成的分子机制。研究结果不仅能从源头指导甘草中OTA污染的科学预防，还能丰富OTA合成调控的理论。

甘草是中药中应用最广泛的药材之一，还广泛应用于食品、饮料等领域。与其他药材相比，甘草极易受赭曲霉毒素A（OTA）污染。OTA是由青霉及曲霉属真菌产生的有毒次生代谢产物，具有极强的致癌、致畸、致突变作用，受污染的甘草不仅质量下降，还具有重大安全隐患。本项目从乌拉尔甘草的主产地：新疆，甘肃，宁夏，内蒙古以及河北安国及广西玉林药材市场采集甘草药材，系统分析了采收、加工以及储藏等环节污染甘草的OTA产毒菌类群，结果发现Penicillium chrysogenum， P. glycyrrhizacola，P. polonicum，P. verrucosum，Aspergillus ochraceus，A. westerdijkiae是甘草上主要的OTA产生菌，甘草根际土壤是污染菌的主要来源；采用多基因系统发育分析，形态学观察，生理特性观察以及次生代谢产物谱分析对主要污染菌青霉属，曲霉属及其有性型属进行了物种分类，系统发育，次生代谢产物分析的系统研究，澄请了这些类群的物种概念以及其产生的真菌毒素谱，为阐明OTA污染的生物基础提供理论依据；以Penicillium verrucosum以及Aspergillus westerdijkiae这两种最重要的OTA产毒菌为出发材料，分析了甘草提取物及主要化合物对OTA产毒菌生长及产毒的影响，发现甘草醇提物（1%）对产毒菌生长影响较大，甘草多糖（0.25 mg/ml）对P. verrucosum的 OTA产量具有显著促进作用，0.075 mg/ml的甘草苷对P. verrucosum的OTA产量具有显著抑制作用。甘草苷以及甘草多糖均能提高A. westerdijkiae的OTA产量，但差异不显著；采用转录组分析了甘草苷，甘草多糖对P. verrucosum转录基因的调控，发现甘草苷刺激243个基因上调，甘草多糖刺激79个基因上调；将Penicillium verrucosum 以及 Aspergillus westerdijkiae接种经辐照灭菌的甘草药材，测定了甘草内在质量的变化。相关研究取得较好的预期，发表SCI收录论文6篇，其中项目主持人为第一作者或通讯作者的论文4篇。