【摘要】 为探究肉类优势腐败菌群的混合生物被膜互作规律，采用结晶紫法、苯酚硫酸法、二喹啉甲酸法和激光扫描共聚焦显微镜（CLSM）观察等分析在15℃和25℃肉源分离株隆德假单胞菌（PL）与3种致腐菌的混合生物被膜特性，及对蛋白酶和消毒剂耐受性的影响。结果表明，从腐败牛肉中分离鉴定的23株菌中，假单胞菌属占43.5%，其次为热杀索丝菌、不动杆菌属和类香味菌属。将优势腐败菌隆德假单胞菌与约翰逊不动杆菌、热杀索丝菌及类香味菌共培养，发现低温显著促进双菌混合生物被膜的形成。PL、AJ、BT在15℃时的生物被膜形成量均显著高于25℃时的，混合生物被膜在15℃时的形成量显著高于PL单培养的。PL与AJ共培养时，被膜菌数目比单培养时高0.75～0.78 lg(CFU/cm~2),15℃共培养时，胞外多糖分泌量增加15.67～22.01μg/mL，与AJ、BT共培养的胞外蛋白分泌量分别增加7.35,6.70μg/mL。CLSM观察发现PL与MP或AJ混合生物被膜聚集团聚，形成不均一结构，被膜厚度增至48.99～49.22μm。此外，混合生物被膜的形成，显著提高假单胞菌的蛋白酶活性和化学耐受性。经0.06%次氯酸钠处理后，15℃时PL与AJ、BT混合被膜残留率高达95.61%,96.16%，显著高于PL单培养。结论：肉源致腐菌PL与3种致腐菌形成结构复杂的混合被膜，在低温环境下被膜菌增强对次氯酸钠的抵抗性，为探究肉源腐败微生物混合被膜的污染和控制奠定良好的基础。更多还原

【Abstract】 The aim of the study is to explore the mixed biofilm interaction among dominant spoilage bacteria isolated from meat. The dual-biofilm characteristics of Pseudomonas lundensis and three spoilers were evaluated using by crystal violet staining, phenol sulfuric acid assay, bicinchoninic acid assay and confocal laser scanning microscope(CLSM) observation at 15 ℃ and 25 ℃, as well as protease activity and disinfectant tolerance. The results showed that Pseudomonas spp. accounted for 43.5%, followed by Brochothrix thermosphacta, Acinetobacter spp. and Myriodes spp. among 23 spoilers isolated from beef. The amount of biofilm formation of PL, AJ and BT at 15 ℃ was significantly higher than that at 25 ℃, and levels of dual-biofilms at 15 ℃ was significantly more than single culture, indicating that low temperature significantly promoted the formation of mixed biofilm. The number of cells in biofilm co-cultured with PL and AJ was0.75-0.78 lg(CFU/cm~2), which were higher than that of single culture. Compared to PL single biofilm, the secretion of extracellular polysaccharides increased by 15.67-22.01 μg/mL, and the production of extracellular protein co-cultured with AJ or BT rose by 7.35 μg/mL and 6.70 μg/mL respectively. CLSM observation also revealed that the dual-biofilms of PL and AJ or MP formed clusters with heterogeneous structures, and their thickness of these biofilm increased to 48.99-49.22 μm. In addition, the co-culture of PL and three strains significantly enhanced its protease activity and chemical tolerance. After treatment with 0.06% sodium hypochlorite, the residual rate of dual-biofilms of PL, AJ or BT was 95.61% and 96.16% at 15 ℃, which was significantly higher than that of PL single biofilm. Thus, it was revealed that meat-derived spoilage bacteria PL and three strains formed a complex dual-biofilm with strains-dependence, and the PL cells as dual-biofilm formed at low temperature enhanced its resistance to sodium hypochlorite. This study provided a good foundation for exploring the contamination and control of mixed biofilms from meat-derived spoilage microorganisms.更多还原