六堡茶后发酵过程中真菌多样性及其桔霉素产生机制研究

Liupao tea, one of the typical Chinese brick tea, geographical indication product protected by government in China, is belong to the full fermented dark tea. Studies have shown that citrinin is a hepato-nephrotoxin in a wide range of species which mainly produced by Penicillium and Aspergillus, and these fungi are main microorganism which present in Liupao tea and other dark teas. In our preliminary reserch, Penicillium citrinum and Aspergillus oryzae were isolated from Liupao tea and the residue of citrinin was detected in part of tea samples, which implyed there is a risk of contamination by citrinin in Liupao tea. However, at home and abroad,there was no research reports about citrinin, as well as the source of citrinin in Liupao tea and other dark teas. In our study, fungal community of producing citrinin, the key synthetic gene and of them changes in processing and storage in Liupao tea in species level will be analyzed by 18S hypervariable region of microbial diversity combine with high-throughput genome sequencing. Meanwhlie, the residue of citrinin will be detedcted by HPLC/MS through simulation the processing condition to produced Liupao tea by pure culture fermentation , and the fungi proudeced citrinin will be isolated, identificated, and validated by toxicity test through inoculation. Meanwhile , it will be explained the formation mechanism of citrinin on the tea substrate. The key scientific problem of where citrinin come from will be clarified by the technology of pure culture combine with culture-independent approach, and the toxin-producing strains will be obtained. This study is expected to lay the theoretical foundation for prevention and control of citrinin contamination in Liupao tea and other dark teas.

广西梧州六堡茶是我国传统名茶，已获国家地理标志产品保护，属于后发酵型黑茶。研究证明，桔霉素是一种具有肾脏毒性的真菌毒素,青霉和曲霉是产生桔霉素的主要真菌之一，也是存在于六堡茶及其他黑茶中的主要微生物。本课题组从六堡茶中分离出了桔青霉、米曲霉等菌株，并在部分茶样中检测出了桔霉素残留，提示六堡茶存在桔霉素污染的风险。但目前国内外未见六堡茶等黑茶中桔霉素残留及其来源的研究报道。本课题拟通过宏基因组高通量测序技术和18S高变区的微生物多样性分析方法，在对微生物分离纯化的基础上，从种水平上解析六堡茶中产桔霉素的微生物群落结构、关键合成基因及其在加工贮藏中的变化；同时，模拟六堡茶加工条件进行霉菌纯培养，通过HPLC/MS示踪并筛选产桔霉素霉菌，开展菌种产毒能力、产毒基因表达及在茶叶基质上形成桔霉素机理的研究。本课题通过免培养和纯培养技术将阐明六堡茶中桔霉素污染风险，为黑茶中桔霉素污染防控奠定理论基础。

广西梧州六堡茶是我国传统名茶，已获国家地理标志产品保护。本课题高通量测序技术，结合纯培养方法，分析了六堡茶后发酵过程的微生物多样性。在渥堆过程初期细菌以鞘氨醇单胞菌、甲基杆菌属、金黄杆菌属等为优势菌，渥堆结束后期以葡萄球菌、考克氏菌属、肠杆菌科、微球菌科等；真菌渥堆初期以曲霉属、分枝孢子霉属、青霉属，Blastobotrys为主，渥堆结束后以曲霉属，Trichomonascus ，Rasamsonia，德巴利酵母属为主；在陈化过程真菌占主导地位以曲霉属和散囊菌属为优势菌属，以青霉属和酵母为次要菌属。利用纯培养技术已分离鉴定并保存菌株约830株，其中细菌共30个属、79个种；真菌共33个属、80个种，放线菌约共7个属、13个种。桔青霉普遍存在六堡茶中，在不同的厂家，不同的生产工艺段都有存在。.模拟六堡茶加工条件进行霉菌纯培养，探讨了菌种产毒能力、产毒基因表达及在茶叶基质上形成桔霉素机理。在茶叶上，桔霉素的最大积累量为0.29 μg/mL。利用Baranyi & Roberts初级模型拟合了桔青霉在茶叶上的生长规律，桔青霉在茶叶上产桔霉素的最适温度为25～30℃，最适水分活度为0.90。各个培养条件下桔霉素含量在16天左右达到最大值。通过产生桔霉素的青霉属菌群结构及其变化分析，发现青霉属是原料带来的，且可能含有桔青霉，桔青霉在后发酵过程检出率50%左右，产毒性分析结果显示桔青霉和Penicillium gerundense均可以产生桔霉素。ctnA和orf5基因都是桔霉素生产的正向调节因子。通过桔霉素生物合成相关7个基因(ctnD、orf1、ctnA、pksCT、orf5、orf7、ctnG)，分析了六堡茶不同生产阶段样品中桔霉素污染风险。在模拟自然高湿储藏过程中，成品茶样品储藏21天中检测出桔霉素。本课题通过免培养和纯培养技术将阐明六堡茶中桔霉素污染风险，为黑茶中桔霉素污染防控奠定理论基础。本课题已经发表论文相关论文10篇，其中SCI收录5篇，EI收录1篇。申请相关专利2项。已经培养博士生1人，研究生6人。