腐乳中生物胺形成的微生物机制研究

Sufu is a kind of traditionally fermented soybean food, which is made from soybean and produced mainly by pre-fermentation, salting and post-fermentation process on tofu blank. It has been widely consumed as an appetizer for centuries and has been loved deeply by Chinese people. However, it is prone to form excessive biogenic amines by incomplete metabolism of nitrogenous compounds during fermentation..Biogenic amines are serious harmful to human health, especially histamine and tyramine. In preliminary studies, the applicants found that the contents of histamine and tyramine were the highest among biogenic amines in some sufu samples investigated, whose average contents were 4.1 and 3.8 times of the American FDA limits, respectively. Biogenic amines contents could be reduced using suitable fermentation microorganisms, but the mechanism was unclear. This study was intended to investigate biogenic amines types and contents in sufu with different types and production stages using RP-HPLC column derivatization method. The relationship among amino acids, enzymes activity and biogenic amines were also surveyed to proven the formation law of biogenic amines. The genome sequencing technology was used to study the microbial flora structure, distribution and evolution law during sufu production process, then advantage bacteriums of biogenic amines production were separated and identificated to illustrate the microbial way of biogenic amine formation. PCR technique was used to detect amino acid decarboxylase genes of advantage bacteriums of biogenic amine production, meanwhile, amino acid decarboxylase production characteristics and interactions of strains, and influences on the formation of biogenic amines were also studied. GC-MS method was used to detect quorum sensing signal molecules of advantage bacteriums of biogenic amine production to clear the influence of quorum sensing on biogenic amine formation.These studies would illustrate the formation and accumulation mechanism of sufu biogenic amine and provide a theoretical basis for the ascension of sufu safety and industrial level.

腐乳是大豆经磨浆、成坯、前酵、腌制、后酵等过程制作的传统发酵豆制品，发酵产生的组胺和酪胺等生物胺对人体健康造成危害。申请人前期研究发现，组胺和酪胺是腐乳中含量较高的生物胺，平均含量是FDA限量标准的4.1和3.8倍，采用适宜发酵微生物可降低生物胺含量，但其形成机制不清。本项目拟以生物胺含量最高的青方腐乳为代表样品，使用RP-HPLC法测定腐乳生物胺种类和含量，研究氨基酸、脱羧酶、胺氧化酶与生物胺形成的关系，探明生物胺形成规律；利用宏基因组测序技术研究腐乳微生物菌群结构、分布及演变规律，并分离鉴定产生物胺优势菌，阐明腐乳生物胺形成的微生物途径；使用PCR法检测产生物胺优势菌氨基酸脱羧酶基因，研究菌株产酶特性、交互作用及对生物胺形成的影响，并检测产生物胺优势菌株群体感应信号分子，明确群体感应对菌株生物胺形成的影响，阐明腐乳生物胺形成的微生物机制，为提升腐乳食用安全性和工业化水平提供理论依据。

腐乳是大豆经磨浆、成坯、前酵、腌制、后酵等过程制作的传统发酵豆制品，已有一千多年加工历史，其风味独特、营养丰富，深受喜爱，但腐乳加工工艺粗放，且氨基酸丰富，易形成过量生物胺，对人体健康造成危害。项目主要研究内容和结果如下： .1、研究了腐乳生物胺产生规律及影响因素.研究发现，市售64种腐乳平均生物胺含量为277.59 ~ 1372.57 mg/kg，青方腐乳生物胺含量最高；豆坯未检测到生物胺，前酵生物胺增长不多，毛坯和盐坯主要生物胺是苯乙胺和尸胺，后酵生物胺含量增加，出现色胺和亚精胺；发酵第28 d检测到组胺，为105.86 mg/kg，发酵结束达210.52 mg/kg，为主要的生物胺；发酵菌种影响腐乳前酵生物胺，其中雅致放射毛霉发酵腐乳组胺、酪胺含量最低。.2、研究了腐乳发酵过程中微生物群落结构的演替变化.高通量测序结果显示，白坯的优势细菌属为蓝细菌和库特氏菌属，接种五通桥毛霉后，不动杆菌属的相对丰度增加，成为盐坯优势菌，后酵过程不动杆菌属、肠球菌属、链球菌属为主要菌；白坯优势真菌为毛孢子菌属、曲霉菌属和放射毛霉属，且放射毛霉在毛坯中占44.74 %；后酵曲霉菌属和红曲霉属占比达95%以上，成为后酵阶段的优势真菌属。.3、分析了腐乳生物胺与细菌群落及主要成分的相关性.PLSR和Pearson相关性分析表明，细菌与生物胺存在相关性，如组胺与短波单胞菌属和未分类的肠杆菌属相关性较好；苯乙胺、色胺与肠球菌属、葡萄球菌属和链球菌属相关；亚精胺与链球菌属相关，生物胺与多肽、氨基态氮、中性蛋白酶活性有极显著正相性，相关性系数均在0.8以上。.4、研究了腐乳中产生物胺菌及其发酵特性.采用平板显色结合色谱法，筛选到产生物胺菌59株，测定了高产生物胺菌在不同温度、pH、氨基酸、碳源、乙醇和盐含量下产胺能力，并进行发酵试验，发现接种产胺菌发酵组生物胺含量显著高于对照组，其中热带芽孢杆菌接种组主要产组胺、苯乙胺和尸胺，而枯草芽孢杆菌接种组产色胺较多。.研究结果可揭示腐乳生物胺产生途径，为提升腐乳食用安全性和工业化水平提供理论依据。