格氏乳球菌素KS对食源性致病菌抑制机理研究

Pollution and reproduction of foodborne pathogens have been one of the critical issues that threaten the safety for human beings. In general, the traditional way to solve the residue and contamination of foodborne pathogens includes heat treatments, frozen storage, adjusting pH and adding preservatives. However, the traditional processing method not only destroys the unique flavor of food, but also may cause the quality of seafood and eventually affect the value of food. According to the above problems, the project, based on previous studies, utilizes a new broad spectrum bacteriocin garvicin KS to treat foodborne pathogenic bacteria. By using its characteristic of combination with the specificity receptor of foodborne pathogenic bacteria, the resistance mutants to garvicin KS were first constructed. In addition, point mutation, created by PCR, was used to eliminate the polar amino acids of the antibacterial peptide chain and construct a new antibacterial peptide chain of garvicin KS. By comparing the antibacterial effect of the peptide chain, previously and afterwards, how garvicin KS connects to receptor was determined. Finally, according to the identification of garvicin KS receptor protein and its binding with the receptor, the mechanism of garvicin KS inhibition of foodborne pathogenic bacteria was clarified. The results would provide the basic evidence for the development and application of Class IIe bacteriocins, such as garvicin KS, in the future.

食源性致病菌污染和繁殖一直是威胁食品安全的首要问题之一。抑制食源性致病菌污染和增殖的传统方法有热处理、冷冻冷藏、调节pH和添加防腐剂等。然而传统方法可能造成食品风味和品质下降，影响食品的价值。针对上述问题，该项目应用新型广谱格氏乳球菌素（garvicin KS）作用于食源性致病菌，利用其与食源性致病菌受体特异性结合的特点，构建耐garvicin KS突变体，通过全基因组测序技术，对比食源性致病菌野生型和突变体的基因，筛选其潜在结合受体，利用基因敲除和回补技术确定garvicin KS结合受体。另外，应用点突变技术替代抑菌肽链的极性氨基酸，构建新的抑菌肽链。通过对比抑菌肽链前后抑菌效应变化，确定garvicin KS连接受体方式。根据识别garvicin KS受体蛋白及其与受体结合方式，阐明garvicin KS抑制食源性致病菌作用机理。为IIe类乳酸菌细菌素发展及其应用提供可靠的基础依据。

乳酸菌细菌素在多种抗菌应用中具有巨大潜力，例如作为天然食品防腐剂，以及感染治疗中的抗菌剂。然而，穷人对各种细菌素作用方式的理解阻碍了它们的发展和应用应用。为了更好地了解广谱细菌素，我们制作了乳酸乳球菌IL1403的garvicin KS抗性突变体。13株耐garvicin KS的乳酸乳球菌IL1403分离突变体并测序。值得注意的是，所有的突变体都显示出同一基因的截断突变，编码一种应激反应的噬菌体休克蛋白C（PspC）蛋白质pspC完全敲除的乳酸乳杆菌菌株表现出更强的耐药性。.为了研究garvicin KS、LcnG和其他细菌素是否使用相同的靶点，我们进行了细菌素与细胞的交叉结合试验。结果表明，与野生型相比，抗garvicin KS突变体与garvicin KS和Lcn G的结合较少，与nisin的结合在野生型和突变体之间没有差异，但当PspC被免疫沉淀拉低时，没有观察到结合。此外，通过免疫沉淀检查与两种细菌素的结合，如完全细菌素或以单个亚单位的形式，证明PspC可以作为它们的受体。PspC的角色似乎抓住了18多肽细菌素浓度较低，可能有助于促进单个肽之间的相互作用，并可能将细菌素转移到其真正的靶点。