云南回族牛干巴中生物胺的形成与累积机制

Biogenic amines are organic bases commonly present in fermented meat product, and they can be produced in high amounts by microorganisms through the activity of amino acid decarboxylases. Dried cured beef of Hui nationality in Yunnan is a traditional meat product which processed by natural fermenting. Our previous studies have shown that high amounts of biogenic amine were present in dried cured beef of Hui nationality, and excessive consumption of these amines can be of health concern. However, there is still not a clear-cut mechanism of formation and accumulation. The aim of this project is to study changes of biogenic amines and microflora in the processing of dried cured beef of Hui nationality combinating modern instrument analysis, molecular biology technique and traditional microbiological methods to isolate and identify dominant microorganisms which produced amino acid decarboxylase and biogenic amine oxidase. The growth characteristics and properties of enzyme producing in different conditions will be subsequently study, and the interaction among the dominant microorganisms producing amino acid decarboxylase, dominant microorganisms producing biogenic amine oxidase will be analysis. Finally, the existence of quorum sensing signal molecules among the microorganisms which produced amino acid decarboxylase will be detect, and the changes of dominant microorganisms producing amino acid decarboxylase and dominant microorganisms producing biogenic amine oxidase will be study during the process, including the influence on the changes of biogenic amines. The results of this study will provide a scientific theoretical basis for the quality control of dried cured beef of Hui nationality in Yunnan.

生物胺是由微生物氨基酸脱羧酶催化氨基酸脱羧产生的，在发酵肉制品中广泛存在的对人体有潜在危害的一类有机化合物。云南回族牛干巴是一种自然发酵的传统民族肉制品，我们前期研究发现，其生物胺含量较高，存在安全隐患，但其形成与累积机制尚不清楚。本项目拟利用传统微生物研究手段结合现代仪器分析与分子生物学技术，研究云南回族牛干巴在加工过程中各工艺点生物胺与菌相变化，分离鉴定其产氨基酸脱羧酶优势微生物和产生物胺氧化酶优势微生物，探讨它们在不同条件下的生长特性和产酶特性；分析产氨基酸脱羧酶优势微生物之间、产生物胺氧化酶优势微生物之间及这两类微生物之间对生物胺形成和累积的交互作用；检测产氨基酸脱羧酶微生物之间是否存在群体感应的信号分子，并在其加工过程中研究产氨基酸脱羧酶优势微生物和产生物胺氧化酶优势微生物变化及其对生物胺消长变化的影响，阐明云南回族牛干巴中生物胺形成和累积机制，为生物胺控制提供科学理论依据。

为研究云南回族牛干巴中生物胺的形成与累积机制，本项目利用传统微生物研究手段结合现代仪器分析与分子生物学技术，利用传统工艺制作牛干巴，分析牛干巴加工过程中理化指标和微生物种类及含量的变化，通过菌相构成确定了乳酸菌为牛干巴加工过程中的优势菌，利用离子色谱法测得牛干巴在不同加工阶段中精胺含量最高，其次为酪胺和腐胺。从成熟牛干巴样品中分离出3株乳酸球菌均为明串珠菌属，并未发现其他菌属，其中1株为肉明串珠菌，1株为乳明串珠菌，1株为肠膜明串珠菌葡聚糖亚种。通过PCR-DGGE法和HPLC法分析确定肠膜明串珠菌葡聚糖亚种和肉明串珠菌作为后期产氨基酸脱羧酶优势菌。此外，通过探讨不同条件下的肠膜明串珠菌葡聚糖亚种生长特性和产酶特性发现pH和温度对肠膜明串珠菌葡聚糖亚种的生长有显著影响且对精胺和酪胺存在交互影响。在牛干巴加工过程中，体系内两株乳酸球菌（肠膜明串珠菌葡聚糖亚种、肉明串珠菌）存在交互作用，且两株菌株以不同的比例作为强化接种剂时，体系中存在协同作用。而产生物胺氧化酶优势菌的分离鉴定及其特性有待进一步研究。此外本项目还研究了产氨基酸脱羧酶微生物的群体感应现象，结果表明肉明串珠菌和肠膜明串珠菌葡聚糖亚种可分泌C4-HSL、C6-HSL、C8-HSL类型的信号分子，产生物胺菌在28℃、37℃条件下均能够形成生物被膜，温度会影响菌株产生物胺量及产生物胺菌株分泌AHLs，试验结果初步揭示了云南回族牛干巴中生物胺形成和累积机制，为生物胺控制提供科学理论依据。