肉源隆德假单胞菌和致腐菌的混合生物被膜形成特征

Characterizations of Dual-biofilms Forming by Pseudomonas lundensis and Spoilage Bacteria from Meat

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/cm2),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。

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 was 0.75-0.78 lg(CFU/cm2),which were higher than that of single culture.Compared to PL single biofilm,the secretion of extracellular polysaccharides increased by 15.67-22.01μg/mL,and the production of extracellular protein co-cultured with AJ or BT rose by 7.35μg/mL and 6.70μg/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 48.99-49.22μm.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.