莓实假单胞菌aprD基因对其致腐作用的调控机制

Pseudomonas fragi is favored by the chill chain applied to the fresh meat distribution and storage. These species constitute the main spoilage microorganism of chilled meat. Previous studies have found that P. fragi displayed strong spoilage potential, and its spoilage potential was weakened when the aprD gene was deleted. This result demonstrated that aprD plays a significant role in the bacterial spoilage associated with P. fragi. However, the action pattern and regulatory mechanism were still unclear. In the present study, P. fragi NMC25, an isolate with strong spoilage potential, and its aprD deletion mutant are tested as the target bacteria, chilled chicken and standard growth medium (TSB) are used as the growth substrate. The characteristic bacterial phenotypes such as proteolytic capacity and cell membrane property are analyzed. Differential metabolites are identified by metabolomics technologies. On the basis of these metabolites, the potential spoilage biomarker and specific signal pathway are screened. Evaluating the function of aprD in the chilled meat spoilage associated with P. fragi. Through these investigations related above, the regulation of aprD on the spoilage effect of P. fragi will be revealed, and the spoilage characteristics will be linked with gene function and related metabolic pathways. Our research will perfect the scientific theory of the P. fragi spoilage mechanism, and also provide the theoretical basis for developing effective preservation system and ensuring the safety of chilled meat.

莓实假单胞菌极易在鲜肉冷链运输和储藏过程中生长，是导致冷鲜肉腐败的重要腐败菌。前期研究发现，莓实假单胞菌有较强的致腐能力，且当aprD基因缺失后该菌的致腐能力显著减弱，表明aprD基因在莓实假单胞菌致腐过程中起到关键作用，但其调控机制尚不明确。本研究拟以前期分离到的一株强致腐莓实假单胞菌NMC25及其aprD基因缺失株为研究对象，以标准培养基TSB和冷鲜鸡肉为生长基质，分析aprD对细菌分解蛋白能力、细胞膜特性等生物学特征的影响；利用代谢组学技术分析差异代谢产物，寻求潜在的腐败标志物并鉴定aprD调控细菌腐败能力的关键信号通路；评价aprD对莓实假单胞菌在冷鲜肉腐败过程中的作用。本研究将全面揭示莓实假单胞菌aprD基因对该菌致腐效应的调控作用，将腐败特征与基因功能及相关代谢通路联系起来。研究成果有助于完善莓实假单胞菌致腐机制，为进一步优化冷鲜肉保鲜体系，保障冷鲜肉安全性提供理论指导。

莓实假单胞菌是导致有氧条件下储存冷藏肉腐败的重要优势腐败菌，它的细胞外蛋白酶有助于肉的变质。AprD被认为是参与假单胞菌碱性胞外蛋白酶分泌的重要成分。由此可见，aprD很可能在莓实假单胞菌的致腐过程中起重要作用。本研究拟以前期分离到的一株强致腐莓实假单胞菌NMC25及其aprD缺失株为研究对象，以标准培养基TSB和冷鲜鸡肉为生长基质，（1） 分析aprD对细菌分解蛋白能力、生物被膜形成等生物学特征的影响。研究发现aprD对于表面相关行为非常重要，例如ΔaprD缺失株改变了自聚性和泳动性，在生物膜中形成相对无序的松散聚集，导致结构较薄，死亡细胞较多。同时，细胞外碳水化合物和蛋白质含量也发生了显著变化；（2）利用代谢组学技术分析差异代谢产物，寻求潜在的腐败标志物并鉴定aprD调控细菌腐败能力的关键信号通路。细胞内代谢组学分析显示aprD参与了几种细胞代谢途径，主要包括碳水化合物途径、氨基酸途径和核苷酸途径，而细胞外代谢的特征阐明了腐败相关代谢物（如肌酸、IMP、精胺、脂肪酸、氨基酸和寡肽）的变化可能与aprD缺失高度相关；（3）评价aprD对莓实假单胞菌在冷鲜肉腐败过程中的作用。结果表明，aprD通过影响胞外蛋白酶活性、TVBN的产生、pH和肉样品的感官性状来促进莓实假单胞菌的腐败活性。综上，本研究强调了aprD介导的细胞代谢和生理行为在调节莓实假单胞菌致腐效应中的作用。与此同时，研究也揭示了呼吸链上nuoB对莓实假单胞菌致腐效应的调控机制。研究成果有助于完善莓实假单胞菌致腐机制，为进一步优化冷鲜肉保鲜体系，保障冷鲜肉安全性提供理论指导。