高糖介导乳酸乳球菌Agu酶家族表达机理研究

Lactococcus lactis is widely used in dairy industry, it could produce putrescine catalyzed by Agu enzyme family from agmatine via the agmatine deiminase (AGDI) pathway. Putrescine is potential hazardous biogenic amine in the fermentation food. In the present study, Lactococcus lactis was treated by different concentration of glucose and lactose, the expression level of catabolite control protein CcpA, Agu enzyme family were analyzed by proteomics, the expression level of AGDI cluster was detected by qPCR, and the up-regulated or down-regulated expression level of protein was monitored, in order to get the regulation of expression level of AGDI cluster controlled by CcpA and the variable role of glucose and lactose concentration; The correlations between the above and the regulation of the biogenic amines accumlation, the nutrition factors consume as well as PH and other physical factors were also calculated in order to realize ‘fermentation parameter → catabolite control protein expression → enzyme expression → biogenic amines control’. This study could help develop control theory of putrescine regulated by fermentation parameter of glucose and lactose in Lactococcus lactis, hich is of great insignificance and provide the evidence for provention and control of hazardous substances in the fermentation food.

乳酸乳球菌广泛应用于乳品发酵行业，能够通过胍基丁胺脱亚胺酶代谢途径（AGDI）中Agu酶家族催化生成腐胺。腐胺是一种存在于发酵食品中的潜在危害性生物胺。本课题通过蛋白组学和实时定量PCR技术研究乳酸乳球菌在不同糖组分、浓度下分解代谢物控制蛋白（CcpA）和Agu酶家族差异表达谱，以及AGDI基因簇转录表达谱，发现CcpA调控AGDI基因簇转录变化规律和调控酶蛋白表达上调或下调的作用变量。结合代谢组学技术检测生物胺积累、基质成分、pH值等物理化学因子的动力学变化规律，进一步量化分析各参数和变量之间的相关性，得到发酵过程糖调控参数，实现“发酵参数诱导→分解代谢物控制蛋白表达→合成酶系表达→生物胺调控”。从生物胺产生菌乳酸乳球菌自身代谢特点出发，调节发酵过程糖参数来消减产品中的生物胺。本研究完善乳酸乳球菌发酵糖参数对腐胺合成调控技术理论，对发酵食品中危害物质预防调控具有重要意义。

腐胺是一种存在于发酵食品中的潜在危害性生物胺。本课题通过ELISA和实时定量PCR技术，研究乳酸乳球菌在不同葡萄糖培养下的分解代谢物控制蛋白（CcpA）和Agu酶家族差异表达谱，以及AGDI基因簇转录表达谱，结果表明随着葡萄糖浓度的升高，CcpA与cre位点结合，抑制了AGDI基因簇转录表达，继而减少腐胺生成途径的Agu酶家族蛋白的含量，降低了乳酸乳球菌生成的腐胺含量，120mM葡萄糖培养下的乳酸乳球菌腐胺生成量仅为0.1899 mg/g CDW。构建乳酸乳球菌蛋白差异表达图谱，蛋白组学和代谢组学结合进一步分析，高糖能够下调乳酸乳球菌氨基酸跨膜转运体活性，羟甲基、甲酰及相关转移酶活性，能够抑制乳酸乳球菌腐胺、组胺等生物胺的生成；高糖能够上调乳酸乳球菌氧化还原酶活性，过氧物酶活性，脱氨酶活性等，上调苯丙氨酸代谢通路蛋白表达，增强内酰胺、阳离子抗菌肽等抗生素生物合成途径；高糖能够上调有关于丙酮酸代谢、三羧酸循环途径，能够增加短链脂肪酸的生成量；高糖增加乳酸乳球菌的碳代谢流量（糖酵解途径，果糖和甘露糖代谢途径、麦芽糖合成途径），减少氮代谢流量（苯丙氨酸代谢和色氨酸代谢流通量、半胱氨酸和蛋氨酸代谢，丙氨酸、天门冬氨酸和谷氨酸代谢通路）。本研究完善乳酸乳球菌发酵糖参数对腐胺合成调控技术理论，对发酵食品中危害物质预防调控具有重要意义。