酸菜发酵生态系统中细菌群落与代谢物组特征耦合机制

Microbial ecosystem, which is composed of microbial communities and the environment, is an ideal model to understand the interactions between species and the environment. Metabolites are the final response of cells to environmental changes. Therefore, metabolomes involved in communities directly represent the structure and function of the ecosystem. Firstly, this present work constructs the metabolic network of heterofermentative L. paracasei HD1.7. Secondly, this study takes the L. paracasei HD1.7 starter culture fermentation of pickle Chinese cabbage as research model as well as the natural fermentation of pickle Chinese cabbage as control model. Metabolomic techniques, including stable isotope labeling, nuclear magnetic resonance and chromatogram-mass spectrum, are applied to investigate the metabolomes features. Genomic techniques such as quantitative real-time PCR and barcoded pyrosequencing are adopted to research the variation of structure and quantity of bacterial communities. Subsequently, this research aims to figure out the dynamic regular pattern between bacterial communities and metabolomes. Finally, based on the comparative analysis on bacterial communities and motabolomes features (1) between L. paracasei HD1.7 and research model; (2) between research model and control model, the final goal is to explore (1) coupling mechanism between bacterial communities and metabolomes features and (2) response mechanism of L. paracasei HD1.7 to environmental variation on metabolome level. This research work not only expands the metabilomics applications in microbial ecology, but also is practically supervised to improve the production process and build up the quality protocol in the filed of pickle Chinese cabbage production.

微生物群落与环境构成的生态系统是研究物种及其与环境相互作用机制的理想模型。代谢物是细胞对环境变化的终端响应，因此群落代谢物组是生态系统结构和功能的直接表征。本项目拟构建供试菌L. paracasei HD1.7异型乳酸发酵代谢网络，并以其为发酵剂构建的酸菜发酵生态系统为研究模型，采用稳定性同位素标记、核磁共振和液相-质谱联用等代谢组学技术，结合实时定量PCR和条码焦磷酸测序等基因组学技术，研究细菌群落数量、结构和代谢物组动态变化，探讨细菌群落演替和代谢物组变化的一般规律。通过比较分析供试菌单独发酵与研究模型、研究模型与对照模型中的细菌群落与代谢物组特征，探索（1）细菌群落与代谢物组特征耦合机制和（2）L. paracasei HD1.7对环境因素的代谢物组水平应答机制。本研究拓展了代谢组学在微生物生态学中的研究范畴，对酸菜发酵生产工艺的完善和质量检测体系的建立也具有实践指导意义。

项目背景：东北酸菜(pickle Chinese cabbage)（以下简称酸菜）是利用乳酸菌发酵生产的腌渍食品，因营养丰富、风味独特而备受青睐。发酵过程中，微生物尤其是乳酸菌是优势菌群，乳酸菌内部及其与非乳酸菌之间此消彼长，它们产生多种代谢产物，赋予酸菜特殊的风味。酸菜发酵过程中生物与非生物因素间的相互作用十分复杂，是微生物生态学与系统生物学研究的理想模型。研究内容：本项目利用代谢组学技术与基因组学技术，以分离自酸菜发酵液的土著乳酸细菌副干酪乳杆菌（Lactobacillus paracasei）HD1.7为供试菌株，在考察异型乳酸代谢情况的基础上，以该菌株为发酵剂构建了酸菜发酵系统，研究了细菌群落演替与代谢物组特征的耦合关系。重要结果：L. paracasei HD1.7作为发酵剂，改变细菌群落的结构和数量，即促进优势菌群生长，降低细菌多样性；对代谢物的影响包括增加多种营养物质含量，降低亚硝酸盐含量，促进风味物质生成等。关键数据：从菌群演替角度看，在发酵前期，对照组中菌体数量较少，乳球菌属（Lactococcus）占相对优势；试验组中，L. paracasei HD1.7作为优势菌在发酵系统中占主导地位。随着发酵的进行，两体系中细菌大量繁殖，物种丰度逐渐升高。对照组中，菌群多样性不断升高，乳杆菌属（Lactobacillus）逐渐成为优势菌，Lactococcus及其它有害菌的数量减少。试验组中，菌群的多样性先下降后升高，Lactobacillus始终保持优势地位。从代谢物水平看，同一系统不同发酵阶段及同一发酵阶段不同系统之间，代谢产物的种类及丰度存在显著差异。发酵开始后，发酵液中有机酸含量逐渐增加，且试验组高于对照组。其中，在发酵末期，乳酸的含量分别为8.55±0.29 g/L（对照组）和9.15±0.04 g/L（试验组）。维生素C含量随着发酵时间的延长呈下降趋势，在发酵末期试验组VC含量为455.93±2.82 mg/kg显著高于对照组（293.97±1.63 mg/kg）。对照组在发酵第3 d时出现亚硝酸盐峰，峰值为19.02±0.52 mg/kg，试验组亚硝酸盐含量一直在1.00 mg/kg以下。在发酵25 d时，试验组中游离氨基酸的总量为116.71±0.15 mg/kg，显著高于对照组（104.35±0.12 mg/kg）。在整个发酵过程中共检测出