新疆地区红曲霉种质资源调查及其代谢产物分析

Xinjiang province which located in the hinterland of the Eurasian continent is a typical arid inland area. Diversified and special microorganism resources is able to found in Xinjiang due to its special variety of ecological environment. Monascus spp. has the ability to produce esters, pigment, lovastatin which inhibiting cholesterin synthesis, citrinin and other metabolites. At present, the investigation into Xinjiang Monascus resources has not yet been carried on. In addition, there is a paucity of further information on the research of Monascus metabolites. Therefore, culture-independent 18S rDNA gene sequence analysis will be performed in this study to investigate the microbial diversity of Monascus in Xinjiang, and thus some endemic Monascus species will be revealed. Isolation of these Monascus species will then be conducted, their fermentation products will be analyzed in order to screening and breeding Monascus strains with enduring high-lysine-concentration, non-citrinin and high yield of lovastatin and esters. The relationship between gene expression and cell metabolism in Monascus will be established by revealing the mechanisms of genetic regulation and control of these metabolic production pathways. The results and outcomes in this study will provide valuable data and theory foundation for the utility of Monascus spp. in Xinjiang.

新疆位于欧亚大陆腹地，为典型的干旱内陆地区，其特殊多样的生态环境孕育了特殊多样的微生物资源。红曲霉是一类发酵过程可产生酯类、色素、lovastatin(洛伐他汀，一种抑制胆固醇合成的物质)、和桔霉素等代谢产物的微生物。我国目前尚未对新疆地区红曲霉资源进行调查，对其代谢产物的研究也欠深入。项目采用免培养法18S rDNA 基因测序方法对新疆地区富集到的红曲霉菌落进行多样性分析，与已有红曲霉菌种对比，确定新疆地区特有红曲霉菌种；采用培养法分离菌落中红曲霉并进行发酵产物分析，探寻产光稳定性红曲色素、不产桔霉素、高产洛伐他汀和酯类物质的红曲霉菌种，对比发酵产物和不同红曲霉种间基因差异，初步建立红曲霉基因与代谢产物之间关联，为红曲霉基因育种提供有价值的依据。

本项目在新疆南北疆具有特征气候的八个地区进行红曲霉种质资源调查及采样292个，采用培养法分离纯化，分别从新疆白酒曲、腐乳中分离纯化得到2株红曲霉菌，富集得到1株红曲霉菌，从北京、天津、吉林、广东等地区腐乳中分离得到9株红曲霉菌。新疆红曲霉菌落与其它地区红曲霉比有明显不同，10种红曲霉菌丝体大体相似。针对现有红曲霉富集技术中存在的技术缺陷，设计发明出“一种利用回生淀粉富集红曲霉的方法”：利用回生淀粉富集红曲霉菌能够减少酵母菌和细菌繁殖而允许红曲霉孢子萌发和生长，水分蒸发量少、可自动加湿的红曲霉菌富集培养基及装置。对筛选出的13株红曲霉菌进行液态或固态培养，采用双波长紫外分光光度计、质谱分析仪等对培养基中的洛伐他汀、红曲色素、酯类及桔霉素等代谢产物进行测定分析比较。研究结果：蛋白胨+P葡萄糖培养基最适于新疆地区红曲霉，所产红曲色素含量最高6.809×10-2mg/mL，在所有发酵液中新疆地区红曲霉均未检测出桔霉素。确定红曲霉固体发酵产洛伐他汀的条件以及从酒曲中分离的红曲霉液态发酵生产酯类化合物的条件。研究结果：当高粱：麸皮=7:3，温度 35℃，时间7 d时，红曲霉产酯最高达222.52mg/L。为解决红曲霉光褪色问题发明了“一种回生淀粉在降低红曲红色素光褪色方面的应用”方法。对新疆地区腐乳中与其它4个地区腐乳中分离纯化后的10种红曲霉菌进行分子生物学遗传差异性分析。研究结果：对红曲霉菌色素基因的DNA扩增片段测序，长度为1024～1121bp，通过多序列比对分析发现，10种红曲霉菌的红曲色素基因在3’端和5’端存在明显的序列差异；该基因的系统发育树表明ZBX-新疆与9M-北京之间的遗传关系较近，将为红曲霉基因育种提供有价值的依据。本项目发表论文5篇，其中4篇SCI，授权发明专利2项。