Objective To investigate the effect of Lactobacillus delbrueckii fermented supernatant on Candida albicans biofilm and explore its possible mechanism and effective components.Methods Lactobacillus delbrueckii was isolated from vaginal Lactobacillus capsules (Ding Junsheng) and identified by mass spectrometer and sequencing.Lactobacillus delbrueckii fermented supernatant was prepared.The effect of Lactobacillus delbrueckii fermented supernatant on Candida albicans biofilm was tested by microplate crystal violet staining method and catheterization tube modeling method.Biofilm morphological changes were analyzed by crystal violet staining and Live/ Dead fluorescent dyes.Exopolysaccharides of Candida albicans biofilm was measured by the sulphruic acid-phenol method.The effect of Lactobacillus delbrueckii fermented supernatant on the adherence of Candida albicans biofilm was tested by the method of colony counting on plate.The fermented supernatant was treated with NaOH,catalase,typsin and proteinase K respectively.Then the treated fermented supernatant's effect on Candida albicans biofilm formation was detected.One-way ANOVA or Wilcoxon Rank Sum Test was used as statistical analysis for quantitative data.Results Lactobacillus delbrueckii was successful ly isolated and identified from Ding Junsheng.Lactobacillus delbrueckii fermented supernatant could significantly and dose-dependantly inhibited the formation of Candida albicans biofilm(F=10.804,P=0.000).The morphological changes showed that the fermented supernatant could decrease the formation of biofilm.When treated with Lactobacillus delbrueckii fermented supernatant,Candida albicans biofilm expolysaccharides was significantly inhibited (F=17.140,P=0.000) and adherence ability was reduced.Compared to untreated fermented supernatant (A490=0.486±0.112),the biofilm inhibition effect of fermented supernatant treated with NaOH (A490=0.675 ± 0.095),catalase(A490=0.577 ± 0.118),typsin(A490=0.600 ± 0.044) and proteinase K (A490-0.495±0.084)only decreased slightly,but when compared to the control group (A490=1.079 ± 0.158),the treated fermented supernatant still showed significant biofilm inhibition effect (x2=26.052,P=0.000),which suggested that the effective components of fermented supernatant were organic acid and bacteriocin-like substance.Conclusions Lactobacillus delbrueckii fermented supernatant can effectively inhibit Candida albicans biofilm formation.The action mechanisms are related with expolysaccharides decreasement and adherence reduction.The function components of fermented supernatant are possibly organic acids and bacteriocin-like substance.%目的 研究从定君生胶囊分离出的德氏乳杆菌发酵上清在白念珠菌生物膜形成中的作用及机制,并探索发酵上清中可能的效应成分.方法 从阴道用乳杆菌活菌胶囊定君生中分离培养出德氏乳杆菌,并用质谱仪和基因测序的方法进行鉴定.采用微量孔板结晶紫染色法和导尿管建模法检测发酵上清对白念珠菌生物膜形成的影响,并用Live/Dead荧光染料染色,显微镜拍照观察白念珠菌生物膜的形态学变化.用浓硫酸-苯酚法检测发酵上清对白念珠菌生物膜胞外多糖的影响,平板培养计数法检测发酵上清对白念珠菌生物膜起始黏附作用的影响.将发酵上清进行碱中和、过氧化氢酶处理、胰蛋白酶处理和蛋白酶K处理后,再检测其对白念珠菌生物膜形成的作用,探索发酵上清中可能起作用的成分.采用单因素方差分析或秩和检验对计量数据进行统计分析.结果 成功从定君生中分离鉴定出德氏乳杆菌.德氏乳杆菌发酵上清可以显著抑制白念珠菌生物膜的形成(F=10.804,P=0.000),并呈现量效关系.形态学检测结果显示发酵上清液处理组的白念珠菌生物膜量明显减少.发酵上清可以降低白念珠菌生物膜胞外多糖的含量(F=17.140,P=0.000),并显著抑制白念珠菌的黏附.碱中和后的发酵上清(A490=0.675±0.095)、过氧化氢酶处理(A490=0.577±0.118)、胰蛋白酶处理(A490=0.600±0.044)和蛋白酶K处理(A490=0.495±0.084)后的发酵上清比未处理的发酵上清(A490=0.486±0.112)抑膜效果略下降,但与对照组(A4=1.079±0.158)相比,仍然有显著抑制作用(x2=26.052,P=0.000),提示发酵上清中起作用的成分主要是有机酸和类细菌素.结论 德氏乳杆菌发酵上清可以显著抑制白念珠菌生物膜的形成,作用机制与降低胞外多糖含量和抑制起始黏附有关,发酵上清中起作用的成分可能为有机酸和类细菌素.
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