榨菜腌制过程中微生物群落结构演变与风味物质组成之间的相关性研究

Fuling Zhacai (pickled mustard tuber) is one of the three famous pickled vegetables in the world. Its production process includes alternate steps of three salting and three squeezing. Microorganisms thrive in all three salting steps, producing complex flavor components through their metabolic activities, giving the Zhacai a unique and strong flavor. However, it is not clear that the core functional microbiota which are closely related to the complex flavor components. Therefore, the control of fermentation process is mainly based on experience, lacking effective measures to improve the stability and reproducibility of flavor quality..This project plans to study the correlation relationship between the dynamic variation of microbial community structure and flavor components during the pickling process of Zhacai. Samples of Zhacai and its brine will be collected at each stage of the whole pickling process. Firstly, the total metagenomic DNA of microorganisms will be extracted from each Zhacai brine sample, the amplification products of 16S rRNA gene (total length, about 1500 bp) and ITS gene will be sequenced by PacBio SMRT sequencing platform, resulting in the most complete and in-depth data of microbial community structure and its dynamic variation. Secondly, in simultaneous, the four most critical flavor components of Zhacai, including organic acids, low molecular sugars, free amino acids and volatile flavor components, will be determined with HPLC, HPLC-ELSD (evaporative light scattering detector), automatic amino acid analyzer and GC-MS, respectively. Thirdly, all data generated from abovementioned two aspects will be subject to the correlation analysis with the methods of multivariate statistics, such as O2PLS (bidirectional orthogonal partial least squares), PLSR (partial least squares regression), RDA/CCA (canonical correlation analysis), PCA/PCoA (principal component analysis /principal coordinate analysis) and correlation coefficient heat map, so as to determine the core functional microbiota which have a significant correlation with most compositions of flavor compounds. Finally, two methods will be used to verify the function of the core functional microbiota: the function prediction analysis with the use of PICRUSt (phylogenetic investigation of communities by reconstruction of unobserved states) for bacteria or FUNGuild (fungi functional guild) for yeast and mold, and the traditional methods of screening active strains and analyzing flavor components when pure strain is inoculated in the fermentation process. This work will provide the most critical theoretical data for the establishment of controllable fermentation system, so as to improve the stability and reproducibility of the flavor quality of Zhacai.

榨菜腌制过程中经过微生物的发酵作用产生独特的风味品质，目前不清楚与风味物质组成高度相关的核心功能菌群，缺少有效的过程控制以提高风味品质的稳定性和重现性。本项目拟在榨菜腌制不同阶段采集样品，用PacBio SMRT 对微生物宏基因组DNA的16S rRNA(全长1500 bp)和ITS基因测序，深度解析微生物群落结构演变过程，并且与糖类、有机酸、氨基酸、挥发性成分组成变化过程的数据结合起来，应用多元统计法(O2PLS、PLSR、RDA/CCA、PCA/PCoA、Correlation Coefficient Heat Map)分析两者之间的相关性，找出在腌制过程中稳定存在、相对丰度较高、与风味物质组成高度相关的核心功能菌群，并且用PICRUSt/FUNGuild预测和纯菌株接种发酵两种方法验证核心功能菌群的功能。本项目的研究将为建立可控的发酵体系、提高榨菜风味品质稳定性和重现性提供理论依据。

榨菜在腌制过程中经过微生物的发酵作用形成独特的风味品质，本项目拟通过微生物群落结构和风味物质组成变化过程、以及它们之间相关性的研究，揭示对风味物质组成高度相关的核心功能菌群。本项目按照原计划实施。课题组于2018年11月至2019年3月、 2019年11月至2020年3月两次跟踪榨菜腌制过程。在第一次榨菜腌制过程的研究中，运用Illumina MiSeq对细菌16S rRNA基因和真菌ITS基因测序，分析检测了基础生化指标（酸度、盐度、亚硝酸盐、还原糖、氨基酸态氮），采用O2PLS（bidirectional orthogonal partial least squares）和相关性系数热图（correlation coefficient heat map）分析细菌和真菌群落结构与生化指标的相关性，得出与生化指标高度相关的核心功能菌群。用PICRUSt（phylogenetic investigation of communities by reconstruction of unobserved states）预测细菌和真菌群落的功能。.在第二次榨菜腌制过程的研究中，采用PacBio Sequel平台对细菌16S rRNA基因和真菌ITS基因测序，分别达到1400 和1200 bp的测序长度，覆盖基因的全长，同时分析检测了基础生化指标（酸度、盐度、亚硝酸盐）和风味成分（有机酸、糖类、挥发性风味成分、氨基酸），运用相关性分析方法(O2PLS、correlation coefficient heat map)分析得出与风味成分数据高度相关的核心功能菌群。用PICRUSt预测细菌和真菌群落的功能。分析探讨了与风味成分数据高度相关的菌属。.此外，课题组采用培养法分离纯化榨菜腌制汁液中的微生物，筛选出了2株对芥子苷降解活性强、对酸/盐耐受性好的菌株，有待运用于榨菜腌制发酵菌剂。.本课题研究的意义和价值：（1）明确了筛选优良菌株开发榨菜腌制发酵菌剂的方向。（2）榨菜是一种富含γ-氨基丁酸（γ-aminobutyric acid，GABA）的食品，有开发前景。（3）提出了减盐腌制的构想。

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