三川火腿中生物胺累积分子机理研究

Biogenic amines is known to be worldwide as the potential substances impact on human health, which are widely present in fermented meat products. The vast number of consumers are deeply affected by Sanchuan ham with natural fermentation due to the unique processing, color, flavor and taste. However, we found the higher levels of biogenic amines in Sanchuan ham, which will harmful to human health. Biogenic amines production and the molecular mechanism of accumulation are still blank. This project uses HPLC, molecular biology and traditional microbiological culture methods to track the changes of the biogenic amines and bacterial content, isolate and identify the amino acid decarboxylase strains and amine oxidase microbes, and explore their growth and enzymatic properties. The interaction between the dominant bacteria produced biogenic amines was analyzed by Real-time PCR and RP-HPLC. Quorum sensing between the amino acid decarboxylase was assayed by using HPLC-MS-MS combined with flatbed color method. This project will reveal the dynamic changes of biogenic amines and microflora, get the effect of dominant bacteria on the generation and accumulation of biogenic amines, and explain the molecular mechanism of accumulation of biogenic amines, and which will provide the theoretical basis and scientific foundation to control the biogenic amines formation and accumulation in Sanchuan ham, and also provide new ideas for the biogenic amines formation and accumulation mechanism in other foods.

生物胺是世界公认对人体健康有潜在影响的物质，其广泛存在于发酵肉制品中。三川火腿采用自然发酵，因加工独特，色香味俱全深受消费者喜爱。但我们前期发现其生物胺含量较高，存在安全隐患，且影响生物胺产生和累积分子机理的研究目前仍是空白。本项目采用HPLC、分子生物学和传统微生物培养方法追踪三川火腿加工过程中生物胺和菌相变化，分离鉴定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，并探究其生长特性和产酶特性；运用Real-time PCR和RP-HPLC分析产酶优势微生物之间对生物胺的形成和累积的交互作用。运用HPLC-MS-MS结合平板显示法检测产氨基酸脱羧酶微生物之间是否存在群体感应。通过研究探明生物胺与菌相的动态变化规律，揭示优势产酶微生物对生物胺产生和累积的影响，阐释生物胺累积的分子机理，为三川火腿的品质控制提供理论基础和科学依据，也为其它食品中生物胺的形成及累积机制的诠释提供新的思路。

本项目采用HPLC、分子生物学和传统微生物培养方法追踪三川火腿加工过程中生物胺和微生物变化，在加工过程中发现苯乙胺、尸胺、腐胺、组胺、酪胺、亚精胺及精胺等7种生物胺；生物胺总量呈先上升后平稳再下降趋势。三川火腿中主要由葡萄球菌属、乳杆菌属、嗜冷菌属和放线多孢菌属组成。火腿表面葡萄球菌属、乳杆菌属、嗜冷菌属和放线多孢菌属占火腿表面菌群百分比分别为25.54%、7.7%、6.91%和6.02%，占火腿内部菌群百分比分别为28.28%、8.53%、7.65%和6.66%。利用双层显色培养基法、氧化酶试验、HPLC法检测及16SrDNA对产氨基酸脱羧酶菌和产生物胺氧化酶菌株进行鉴定。筛选出9株产氨基酸脱羧酶菌和5株产生物胺氧化酶菌，其中屎肠球菌、粪肠球菌和清酒乳杆菌高产酪胺。三种菌在24h内是生长呈先快速上升后趋于平稳，酪胺含量呈先快速上升后不断平衡状态。在15℃后酪胺呈快速上升趋势。在不同NaCl含量培养三种菌24h发现酪胺含量呈下降趋势。在两两混合发酵模拟体系中，混合菌产酪胺的含量在15℃后呈快速上升趋势尤其在25℃时酪胺含量显著增加。研究发现产优势产生物胺菌株可分泌信号分子AI-2心当细菌生长进入稳定期时，AI-2活性呈明显递增趋势。菌群密度达到一定数量时，AI-2积累量也达到一定浓度阈值随后降低。三川火腿成熟阶段，样品中的微生物具有QS特性。接种胺氧化菌的生物胺含量均低于空白组，混合胺氧化菌发酵剂显著降低尸胺和酪胺且降解效果优于单一发酵剂。本项目研究结果为提高三川火腿安全性提供一定的保障。