间接消减型发酵剂抑制腌鱼中生物胺积累的分子机制

Biogenic amines (BAs) can accumulate in high concentration in salted fish due to microbial activity and cause toxic effects in consumers. Utilization of starters is a good solution to eliminate the biogenic amines in the products. Starters such as lactic acid bacteria (LAB) can regulate the growth and metabolic activities of the microbial community in fermented food, thus helping eliminate the biogenic amines. However, the molecular mechanism of the phenomena is largely unknown. Metaproteomic methods are perfect tools to study spatial and temporal patterns of gene expression in microbial ecosystems, helping to unveil which species contribute BAs accmulation and how starters downregulate BAs metabolic activities. .In this project, the microbiome samples in the salted Scomberomorus niphonius (group 1), as well as the microbiome samples in the salted S. niphonius inoculated with Lactobacillus plantarum (group 2), will be chosen as the objects to investigate the metabolic dynamics of the BAs-related microbes (BAMs) and the interaction mechanism between L. plantarum and the BAMs, by way of label-free quantitative metaproteomic methods, imaging mass spectrometry tools, real-time PCR assays and Western blot methods. In details, two approaches will be carried out: (1) the quantification analysis of proteins expressed by group 1 and group 2, referring to KEGG pathway information, to distinguish the key BAs-related proteins and the microbes; (2) the investigation of pair-wise interactions mechanism between L. plantarum and BAMs, by way of in situ characterization of extracellular metabolites networks and BAs related metabolic pathways regulation by L. plantarum. The goal of this project is to illustrate the molecular regulation mechanism of L. plantarum on the BAMs. It is anticipated that the result of this project will lead not only to a better understanding of the BAs elimination mechanism of Lactobacillales, but also to methods that allow increased control over, and will enable the rational development of new and more-effective starter culture systems.

接种发酵剂是降低腌鱼等自然发酵食品中微生物有害代谢产物生物胺积累的重要方式。间接消减型发酵剂通过调控复杂微生物群落的代谢活性以抑制生物胺产生,而其分子机制尚不清晰，对其深入研究是提高发酵剂的筛选效率和稳定性，进而推动发酵剂商业化的关键。基于蛋白表达水平是反映微生物群落代谢特性的直接证据，本项目将在前期优化宏蛋白质组样品前处理方法、提高蛋白鉴定数量的基础上，以自然发酵腌鱼、接种间接消减型发酵剂的腌鱼中微生物群落为研究对象，利用宏蛋白质组技术研究腌鱼加工中微生物群落蛋白表达的动态变化规律，结合微生物群落的生物胺代谢动力学特性，挖掘腌鱼微生物群落中参与生物胺代谢酶系及相应目标菌株信息；进而采用发酵剂-目标菌株共培养方式，利用原位质谱成像及分子生物学技术开展共培养体系胞外代谢物的鉴定和胞内关键酶的表达分析两方面研究，从分子层面阐明间接消减型发酵剂对目标菌株的生长和生物胺代谢调控机制。

腌鱼等自然发酵食品中微生物会产生有害代谢产物生物胺。接种发酵剂是降低腌鱼生物胺积累的重要方式。间接消减型发酵剂可以抑制生物胺产生,而其分子机制尚不清晰。针对该问题，本项目研究内容如下：一、固相发酵食品微生物群落代谢不均，因此对生物胺精细分布的研究需要建立针对小量样品的分析方法，本项目建立了基于固相微萃取小柱制备的鱼肉前处理方法，该方法可以同时检测鱼肉中组胺等六种生物胺，与之前的生物胺检测方法相比，本项目所建立前处理方法将样品需求量缩小至50毫克，并减少了有机溶剂的消耗量。该法无需样品衍生化处理，与之前文献相比，所检测生物胺均具有更低的检测限和定量限。二、筛选了十余株可以显著降低不同腌鱼体系中生物胺积累的乳酸菌，并将其中3株抑制生物胺积累能力强的植物乳杆菌保藏为专利菌株，可以降低不同腌鱼中超过96%组胺积累，降低53-97%的生物胺积累。三、以自然发酵腌鱼、接种植物乳杆菌的腌鱼中微生物群落为研究对象，通过对微生物群落的组学分析结合代谢物动力学分析等技术，研究了腌鱼加工中微生物群落的演替规律，挖掘出腌鱼微生物群落中参与生物胺代谢的目标菌株信息，包括摩根氏菌、肠球菌等十几个种属，并构建模拟体系分析并明确植物乳杆菌通过代谢产物抑制目标菌属积累生物胺。四、在此基础上，本项目从腌鱼中筛选了上述目标菌株，即以摩氏摩根菌为代表的25株产生物胺菌株。采用植物乳杆菌-摩氏摩根菌共培养方式，通过基因组重测序、微生物生长动力学和代谢物分析等技术挖掘了植物乳杆菌基因组中产植物乳杆菌素相关的基因及其产生的植物乳杆菌素的类型，同时明确了植物乳杆菌的产物对腌鱼中产生物胺菌株生长和生物胺积累的抑制规律。综上，本项目开发了检测发酵食品中生物胺精细分布的方法，获得了增强腌鱼安全性的植物乳杆菌发酵剂，明确了腌鱼中产生物胺的目标菌株，阐述了植物乳杆菌发酵剂对产生物胺菌株的生长和代谢抑制规律。本项目的开展为腌鱼的安全生产作出了贡献。