基于多组学技术的广陈皮特征风味组分及其陈化微生物代谢机制研究

Empirically, when Pericarpium Citri Reticulatae (in Chinese Chenpi) is aged and stored for a longer period, it has better smell and taste, and features greater health benefit as in the proverb “older is better”. However, the systemic analysis of metabolite changes during its aging is limited and no study is reported about its aging mechanism so far. Pericarpium Citri Reticulatae cv. Chachiensis (in Chinese Guangchenpi) is the dried and aged peel of Citrus reticulata Blanco cv. Chachiensis. It is listed as a premium Pericarpium Citri Reticulatae in the Chinese Pharmacopoeia due to its outstanding medicinal and health benefits. Its production practice implies that microbial fermentation plays a role in the aging. Before this application, we have performed preliminary survey of the microbial community of Guangchenpi and identified rich microbiota. We also observed the accumulation of phenolics and flavonoid during aging of Guangchenpi. Based on these preparatory works, we raise this application to characterize the microbial succession and its contribution to the metabolite changes during aging of Guangchenpi through meta-omics analysis. Metabolomics analysis such as chromatography and mass spectra will be used to investigate the characteristic metabolites and their fluctuation during aging of Guangchenpi. The microbial composition and succession during aging of Guangchenpi will be explored by multiple approaches, such as amplicon analysis, isolation and cultivation of microbial strains, as well as quantitative PCR. The microbial succession will be correlated with the metabolite change during aging to identify the putative microbial contributor. The metagenomic approach will be applied to investigate the functional gene and predict possible metabolic pathways involved in aging. Further metatranscriptomic analysis of active transcripts will reveal the actively expressed genes and identify differentially expressed genes during aging. Mapping these genes in database, such as KEGG, will indicate which pathways they are involved and how these pathways might contribute to the biosynthesis of the characteristic phytochemicals or metabolite of aged Guangchenpi. Meanwhile, the microbial species and novel functional genes identified in Guangchenpi will be the treasure for future utilization.

陈皮素有“陈久者良”的说法，但缺乏陈化过程代谢物变化的系统分析，陈化机理研究尚属空白。广陈皮是新会茶枝柑果皮经干燥、多年陈化而成的道地陈皮，其生产实践提示，微生物发酵参与了陈化过程。在已初步探测到广陈皮丰富微生物菌群和多酚成分变化的基础上，本项目拟运用多组学技术研究广陈皮特征风味组分及其陈化微生物代谢机制。应用代谢组学系统探明广陈皮陈化过程主要食品化学变化及特征风味组分。融合扩增子测序生物多样性分析、微生物分离纯培养和定量PCR，揭示广陈皮陈化过程微生物菌群的群落结构特征和演替变化规律。宏基因组解析菌群功能基因和代谢通路。宏转录组关注活跃表达功能基因，筛选差异表达基因，通过关联相对应样品的风味组分代谢组数据，探索对特征风味组分形成有贡献的活跃表达基因和代谢通路，解析菌群代谢对广陈皮特征风味形成的贡献与机制。同时挖掘微生物种质资源和新功能基因资源。本研究将为广陈皮陈化机理提供基础和新的见解。

陈皮素有“陈久者良”的说法，但缺乏陈化过程代谢物变化的系统分析，微生物参与其陈化机理的研究在本项目开展前尚属空白。本项目以道地陈皮-广陈皮为研究对象，运用多组学技术深入研究其特征风味组分及陈化微生物代谢机理。主要研究内容与结果：1）扩增子测序生物多样性分析揭示陈化过程微生物菌群群落结构特征和演替变化规律。16S rRNA基因扩增子测序检测不同厂家样品中的细菌菌群OTU，揭示出广陈皮拥有共同的核心细菌群落，如芽孢杆菌属和乳球菌属。分析不同陈化年份广陈皮与非广陈皮样品细菌菌群OTU，提示陈化最初两年菌群演替对陈化的重要性。2）联合代谢组、宏基因组与分离菌株解析菌群代谢对广陈皮特征风味形成的贡献与机制。GC-MS系统解析陈化过程中精油代谢物变化规律，广陈皮与其他地区陈皮精油组分及含量存在明显差异：如2-（甲氨基）苯甲酸甲酯（MMA）、α-合金欢烯等，且陈化的最初两年是广陈皮特定风味物质形成的关键点，是研究陈化机理的重要时期。LC-MS代谢组分析检测到1036个代谢物，有机杂环化合物和脂类及类脂化合物的含量在陈化过程逐渐上升，而有机酸及其衍生物的总含量下降。随陈化而增加的如亚麻酸、异棕榈酸、油酸、Queuine等。宏基因组检测检测到细菌、真菌、病毒和古细菌基因，与饱和或不饱和脂肪酸生物合成相关基因，和推测参与代谢橙皮苷、Queuine等的基因。从广陈皮分离菌株中，筛选获得能转化广陈皮特征植物化合物橙皮苷等物质的菌株。3）挖掘广陈皮微生物种质资源和新功能基因资源。从广陈皮分离获得细菌菌株，从益生菌特性和抗菌活性两角度挖掘出潜在的功能菌株。4）发现广陈皮特有的MMA可抑制铜绿假单胞菌群体感应并解析其作用机制，为进一步研究MMA对广陈皮陈化过程微生物菌群的影响提供了新的思路。本项目研究结果为微生物参与广陈皮陈化提供了依据和新见解，为解析广陈皮“陈久者良”和进一步品质改良奠定基础。