低pH处理对两歧双歧杆菌KLDS2.0603黏附能力的影响

【目的】确定低pH处理对两歧双歧杆菌KLDS2.0603黏附能力及其表面物理化学性质的影响。【方法】将两歧双歧杆菌KLDS2.0603菌体在不同低pH的PBS溶液中处理一定时间后,采用平板菌落计数法和直接镜检法,测定其经历不同pH的酸性环境后的黏附能力,及其表面疏水性和自动聚集能力。【结果】不同pH的PBS溶液处理后的双歧杆菌菌体,其黏附能力均不同程度下降,除pH 5.0的处理组外,其余处理组均显著低于空白组。此外,经不同pH的PBS溶液处理后,仅pH 3.0和3.5的两处理组,双歧杆菌表面疏水性显著提高。除pH 1.0、1.5和5.0的处理组外,其余处理组的自动聚集能力均显著下降。【结论】低pH的酸性环境会降低两歧双歧杆菌KLDS2.0603的黏附能力,并且双歧杆菌的自动聚集能力和表面疏水性也发生相应变化。除pH 3.0和3.5的处理组外,三者之间呈现一定的正相关性。     

应用实时荧光定量PCR方法定量检测双歧杆菌对Caco-2细胞的黏附

【目的】采用实时荧光定量PCR的方法定量分析黏附于Caco-2细胞的双歧杆菌,并建立一种快速有效分离黏附于细胞的细菌的方法。【方法】采用Triton X-100溶液处理黏附于Caco-2细胞上的菌体,确定获得最佳分离效果的处理时间;建立实时荧光定量PCR定量检测双歧杆菌的方法,获得标准曲线,进行特异性、灵敏度、重复性评价;应用建立的方法分析11株双歧杆菌对Caco-2细胞的黏附能力。【结果】Triton X-100处理黏附于Caco-2细胞的双歧杆菌的最佳作用时间为10 min。实时荧光定量PCR定量检测双歧杆菌的方法重复性好、特异性强、灵敏度高;起始模板浓度范围在104?108 CFU/mL之间具有良好的线形关系,相关系数>99%,在该浓度范围线性方程为：y=?3.345 2x+37.637 0。应用建立的方法定量分析双歧杆菌的黏附能力,与直接镜检法相比差异不显著(P>0.05),检测时间由48 h缩短至4 h。【结论】Triton X-100分离处理结合实时荧光定量PCR方法是一种快速、有效的检测双歧杆菌对Caco-2细胞黏附能力的方法。     

婴幼儿粪便中双歧杆菌的分离及其菌株特性研究

为筛选具有潜在益生功能的双歧杆菌,本研究采用改良MRS培养基,从婴幼儿粪便中分离到5株菌株:AR499,AR667,AR668,AR669和AR610。通过16S r DNA测序分别鉴定为两歧双歧杆菌,短双歧杆菌,动物双歧杆菌,长双歧杆菌和假小链双歧杆菌。对其耐酸耐胆盐能力和黏附性能进行测试,结果表明,菌株AR499耐酸性较好,菌株AR610有较强的耐胆盐能力,菌株AR499的自动聚集能力和细胞表面疏水性都较高。实验表明,菌株AR499可作为一株耐受性和黏附性能较好的益生菌进一步深入研究,以期应用于优良双歧杆菌产品的开发。     

干酪乳杆菌LC2W表面黏附相关蛋白的提取与鉴定

目的提取和鉴定干酪乳杆菌LC2W表面黏附相关蛋白,初步探索LC2W对胃癌细胞MKN-45细胞的黏附机制。方法LiCl处理、Sephadex G-75柱层析分离提取LC2W的表面蛋白,用黏附试验、电镜观察和SDS-PAGE电泳进行黏附相关蛋白的鉴定。结果LC2W经LiCl处理后,扫描电镜结果发现菌体表面粗糙但仍完整,黏附试验表明其对MKN-45细胞的黏附能力显著降低。提取到的表面蛋白的分子量分别为41.6、63.5、66.2 kDa。粗提物经柱层析后发现分子量为41.6 kDa的组分可以明显增强经LiCl处理过的菌体的黏附,而与未经处理的菌体黏附情况类似。结论表面蛋白参与了LC2W对MKN-45细胞的黏附,其主要活性成分的分子量为41.6 kDa。     

双歧杆菌的粘附特性及其对肠道致病菌的体外拮抗作用

本文采用体外细胞培养法, 从实验室现有的20株不同生境来源的双歧杆菌中筛选具有较强粘附能力的菌株, 并通过混合培养和牛津杯方法, 研究了具有粘附特性的双歧杆菌对肠道致病菌的体外拮抗作用。结果显示, 长寿老人源菌株A03和I06的粘附能力最强, 其菌液对金黄色葡萄球菌及大肠杆菌均具有显著的抑制性, 但是菌体及中和后的发酵液均没有抑菌性, 说明受试菌主要通过其代谢产物中的有机酸来发挥其抑菌性能; 此外, 通过分析比较受试菌和肠道致病菌分别与Caco-2细胞粘附后释放的乳酸脱氢酶量, 证实双歧杆菌与致病菌对细胞的作用具有本质上的区别, 双歧杆菌的粘附能减缓致病菌对细胞所造成的损害。     

呼吸道潜在益生菌D-19表面疏水性及自动聚集能力的研究

目的 从健康儿童口咽部筛选出多株具有抑菌作用的链球菌D-19,探究链球菌D-19表面疏水性及自动聚集能力的影响因素.方法 利用菌体对碳氢化合物的黏附测定多株链球菌D-19的表面疏水率,紫外分光光度法测定D-19的自动聚集能力,并采用不同环境条件和蛋白酶处理菌体表面来确定菌体表面疏水率及自动聚集能力的影响因素.结果 链球...     

一株石油烃降解菌的细胞疏水性及其乳化性质

【目的】从新疆油田石油污染土壤中分离到一株在25 °C条件下利用烃类产生生物表面活性剂的菌株红球菌(Rhodococcus sp.) HL-6, 对其菌体细胞疏水性及所产表面活性剂进行研究。【方法】通过细胞粘附性、表面张力及乳化活性测定对菌株所产表面活性剂进行性质研究。【结果】菌株HL-6在亲水性和疏水性基质中均能产生生物表面活性剂, 在疏水性基质中可以将培养液表面张力由初始的62.487 mN/m降到30.667 mN/m, 培养液在pH 6?9及NaCl浓度1%?5%范围内乳化效果良好, 在4 °C到55 °C范围内乳化效果均为100%, 菌株对柴油的耐受能力很高, 在30%柴油浓度下依然生长良好并且有44%的乳化活性。【结论】HL-6菌株的细胞表面具有很强的疏水性, 这有助于菌体细胞对烃类的摄取。该菌株能够利用烃类基质生产生物表面活性剂, 可以明显降低培养液表面张力并且对石油烃具有良好的乳化作用。说明菌株HL-6能够适应海洋滩涂石油污染的环境, 并可用于严重石油污染区域的生物修复。     

弯曲广布乳杆菌 HFS9 基因组分析及益生特性

【背景】近年来,弯曲广布乳杆菌的健康益处受到广泛关注;基因组分析结合表型实验的方法为益生菌开发提供了新思路。【目的】探索分离自健康人粪便的弯曲广布乳杆菌HFS9基因组特征及益生潜力。【方法】通过泛基因组分析对弯曲广布乳杆菌基因组特征进行描述,基于功能数据库注释并分析HFS9益生相关基因;通过耐酸耐胆盐实验、自聚集、疏水性和细胞黏附实验、抗生素敏感性试验、自由基清除实验及抑菌试验等对HFS9的体外益生特性进行评估;构建小鼠结肠炎模型初步评估HFS9的体内抗炎作用。【结果】弯曲广布乳杆菌具有开放的泛基因组和保守的核心基因组。HFS9基因组大小为1.97 Mb,GC含量为41.86%,蛋白质编码区数目为2 050个;含乳杆菌噬菌体PLE3编码基因及与细胞黏附、酸耐受、胆盐耐受和抗氧化等相关的益生基因。HFS9在酸和胆盐环境中的存活率分别为62.42%和92.92%;自聚集能力和疏水性分别为63.33%和75.00%,对人结肠癌细胞(HT-29细胞)的黏附率为12.97%;对选用的大多数抗生素敏感;对6种常见人体致病菌均具有抑制作用;具有清除1,1-二苯基-2-三硝基苯肼[1,1-diphen...     

新生婴儿源植物乳杆菌HLPL03的环境耐受性及其代谢功能低聚糖的生物学活性

【目的】解析健康新生婴儿胎便中植物乳杆菌HLPL03的益生功能,评价其环境耐受性及代谢功能低聚糖的生物学活性。【方法】通过耐受胃肠道条件、过氧化氢和抗生素试验,评估植物乳杆菌HLPL03对极端环境的耐受性;利用改良培养基,评价植物乳杆菌HLPL03代谢功能低聚糖的能力;同时,探究功能低聚糖对植物乳杆菌HLPL03抑菌活性、疏水性和黏附能力的影响。【结果】植物乳杆菌HLPL03在pH 2.5条件下培养3 h后,活菌数仍在10⁴ CFU/mL以上;在0.30%胆盐中培养6 h后,活菌数接近10⁷ CFU/mL;在1.0 mmol/L H₂O₂强氧化剂条件下培养6 h,活菌数显著升高(P<0.001);除低聚木糖外,植物乳杆菌HLPL03能代谢多种功能低聚糖,并对常见食源性致病菌具有较强的抑制能力;棉子糖是改善植物乳杆菌HLPL03生物学活性的最佳低聚糖,其能提高菌株表面疏水性达36.1%,且促进菌株在Caco-2细胞上的黏附率由16.78%提高至42.11%。【结论】健康新生婴儿源植物乳杆菌HLPL03具有良好的抗环境胁迫能力,且其生物学活性能被棉子糖等功能低聚糖有效促进,可作为特色乳酸菌进行研究和开发。     

藤椒精油对腐败解淀粉芽孢杆菌DY1a生物被膜的抑制作用

【背景】芽孢杆菌是豆制品的重要腐败菌,在气液界面形成生物膜,对产品生产带来持续污染。【目的】探讨藤椒精油(Zanthoxylum armatum DC.essential oil,ZA-EO)对腐败解淀粉芽孢杆菌DY1a菌体及生物被膜的抑制作用与机制。【方法】采用气相色谱-质谱(gas chromatography-mass spectrometer,GC-MS)分析藤椒精油主要成分与相对含量,通过二倍稀释法测定藤椒精油对菌株的最低抑菌浓度(minimum inhibitory concentration,MIC)和最低杀菌浓度(minimum bactericidal concentration,MBC),并分析精油对腐败菌胞外蛋白酶活性、腐败菌生物被膜形成抑制及成熟生物被膜的清除作用,采用扫描电镜结合三维光学显微镜分析生物被膜形貌结构变化,测定生物被膜胞外聚合物(extracellular polymeric substance,EPS)多糖与蛋白质含量变化;并通过细菌运动能力、细胞黏附及自聚集能力、细胞表面疏水性和Zeta电位来初步探讨藤椒精油对生物被膜的抑制机理。【结果】藤椒精...     

Influence of cell surface properties on adhesion ability of bifidobacteria

Adhesion ability of bifidobacteria to the intestinal mucosa is considered to be the prerequisite for colonization of bifidobacteria and can protect against gastrointestinal pathogens infection. The aim of this study was to investigate bifidobacterial surface traits related to adhesion ability in vitro and characterize the cell surface substances that may be involved in the adhesion process of bifidobacteria. Twelve strains of Bifidobacterium spp. were studied for the correlation among their adhesion ability, autoaggregation ability and surface hydrophobicity. The strain that exhibited good adhesion ability also showed high degree of hydrophobicity and strong autoaggregation ability. Pepsin treatment had negative effect on the surface traits and adhesion ability of B. bifidum KLDS2.0603 (P < 0.01), it revealed that hydrophobicity, autoaggregation and adhesion process maybe mediated by proteinaceous components on the surface of cell. Moreover, the adhesion and autoaggregation ability decreased after extraction of B. bifidum KLDS2.0603 with 5 mol l⁻¹ LiCl, and an unreported 50-kDa surface protein which can bind to Caco-2 cell was observed by western blotting. Our results indicated that surface hydrophobicity and autoaggregation ability can be used together for preliminary screening the strains with high adhesion ability, and the present of the surface proteinaceous components would contribute to understand the interactions between bifidobacteria and human intestinal mucosa.     

In vitro effects of bovine lactoferrin on autoaggregation ability and surface hydrophobicity of bifidobacteria

Changes in autoaggregation ability and surface hydrophobicity of bifidobacteria with addition of bovine lactoferrin in liquid media were investigated. Lactoferrin addition caused loss of autoaggregation ability, disappearance of microscopic clusters and produced consistent turbidity in the cultured medium compared with control. Similar outcomes with addition of bovine lactoferrin hydrolysates (pepsin), bovine transferrin or ovotransferrin suggested that the effect is not lactoferrin-specific. On the other hand, addition of proteins, except bovine transferrin, did not alter surface hydrophobicity. These results indicate that one or more surface components involved in autoaggregation of bifidobacteria are proteins.     

Autoaggregation and surface hydrophobicity of bifidobacteria

Thirteen strains of four different Bifidobacterium spp. were observed for their autoaggregation ability and surface hydrophobicity, and correlation between these two traits was determined. Bifidobacteria were classified into high, medium and low autoaggregation strains according to autoaggregation ratio measured from changes in absorbance of media. High autoaggregation strains showed microscopic clustering of cells, whereas low and medium autoaggregation strains showed no such clustering. Autoaggregation ability decreased in high autoaggregation strains but increased in medium and low autoaggregation strains when the assay was performed at higher temperature (37°C compared with 25 and 10°C). Bacterial strains belonging to the high, medium or low autoaggregation group were correlated in terms of their surface hydrophobicity and evaluated based on changes in absorbance of the bacterial suspension before and after extraction with xylene. These results indicate that autoaggregation ability, together with surface hydrophobicity and microscopic image could be used for evaluating the adhesion ability of potential probiotic bifidobacterial strains. Moreover, a synergistic effect of pH and media may be involved in autoaggregation.     

Conditions affecting cell surface properties of human intestinal bifidobacteria

The cell surface properties of human intestinal bifidobacteria have been characterized for 30 strains isolated from a fecal sample. Strain identification to the species level was obtained by restriction analysis of the amplified 16S rRNA gene and confirmed by DNA/DNA reassociation experiments. The isolates were grouped in four genetically homogeneous clusters whose members belonged to Bifidobacterium bifidum, Bifidobacterium adolescentis, Bifidobacterium longum and Bifidobacterium pseudocatenulatum species. Cell surface properties of Bifidobacterium strains were evaluated by determining the level of hydrophobicity, adhesion to hydrocarbons and contact angle measurements, and their autoaggregation ability. The results showed high and homogeneous level of hydrophobicity in all tested strains when contact angle measurements values were considered. On the contrary, autoaggregation assays and bacterial adhesion to hydrocarbons detected interesting differences in cell surface properties among the tested Bifidobacterium strains. The highest levels of autoaggregation, detected in B. bifidum and B. adolescentis strains, were strictly dependent on the pH of the medium. Moreover, protease treatment experiments suggested that proteins had a key role in the autoaggregating ability of B. bifidum and B. adolescentis strains.     

Adhesion and aggregation ability of probiotic strain Lactobacillus acidophilus M92

AIMS: To investigate aggregation and adhesiveness of Lactobacillus acidophilus M92 to porcine ileal epithelial cells in vitro, and the influence of cell surface proteins on autoaggregation and adhesiveness of this strain. METHODS AND RESULTS: Lactobacillus acidophilus M92 exhibits a strong autoaggregating phenotype and manifests a high degree of hydrophobicity determined by microbial adhesion to xylene. Aggregation and hydrophobicity were abolished upon exposure of the cells to pronase and pepsin. Sodium dodecyl sulphate polyacrylamide gel electrophoresis of cell surface proteins revealed the presence of potential surface layer (S-layer) proteins, approximated at 45 kDa, in L. acidophilus M92. The relationship between autoaggregation and adhesiveness to intestinal tissue was investigated by observing the adhesiveness of L. acidophilus M92 to porcine ileal epithelial cells. Removal of the S-layer proteins by extraction with 5 mol l-1 LiCl reduced autoaggregation and in vitro adhesion of this strain. CONCLUSIONS: These results demonstrate that there is relationship between autoaggregation and adhesiveness ability of L. acidophilus M92, mediated by proteinaceous components on the cell surface. SIGNIFICANCE AND IMPACT OF THE STUDY: This investigation has shown that L. acidophilus M92 has the ability to establish in the human gastrointestinal tract, which is an important determinant in the choice of probiotic strains.     

Auto-aggregation and Co-aggregation ability in bifidobacteria and clostridia

A total of 142 human and 88 calf bifidobacteria were isolated and identified; approximately 12 % of all isolated strains exhibited auto-aggregation (Agg) phenotype (Agg+). Properties considered to be predicting for their adhesion to intestine, i.e. auto-aggregation, and hydrophobicity were determined by xylene extraction in 18 human and 8 calf origin bifidobacteria. Co-aggregation of 8 human bifidobacteria with 8 clostridia was also evaluated. Agg varied between 16.3 and 96.4 %, hydrophobicity values ranged from 0 to 82.8 %. The strongest Agg and hydrophobicity were observed in B. bifidum and B. merycicum isolates. However, there were no statistically significant correlations between these two properties. Variability in the percentage of Agg and hydrophobicity was observed after cultivation of bifidobacteria on different carbon sources. All bifidobacteria showed co-aggregation ability with clostridia tested but there were remarkable differences depending on specific combinations of strains. The bifidobacterial strains with the highest ability to co-aggregate with clostridia were B. bifidum I4 and B. longum I10 isolated from infants; these strains gave also high values of Agg. Agg properties together with co-aggregation ability with potential pathogen can be used for preliminary selection of probiotic bacteria.     

Adhesion, autoaggregation and hydrophobicity of 13 strains of Bifidobacterium longum

To identify bacterial traits related to adhesion ability in human bifidobacteria, 13 strains of Bifidobacterium longum isolated from human gastric juice and intestine were studied. Strains were tested for their capability to adhere to Caco-2 cells and classified as adhesive (Adh+) or non-adhesive (Adh-). Adh+ and Adh- strains were then investigated for their autoaggregation ability and surface hydrophobicity. Comparing the properties of Adh+ and Adh-, we observed that strains were able to adhere to cell monolayers if they autoaggregate and manifest a good degree of hydrophobicity as determined by microbial adhesion to hydrocarbons. These two traits could be used for preliminary screening to identify potentially adherent isolates.     

The correlation between surface hydrophobicity and adherence of Bifidobacterium strains from centenarians' faeces

To develop food-grade bifidobacteria micro-ecologics, screening for Bifidobacteria strains which can adhere to intestinal epithelial cells was finished. Twenty-three bifidobacterial strains tested were isolated from centenarians in Bama country, the fifth long-lived district in the world. Surface hydrophobicity and adherence capability to intestinal epithelial cells in vitro of bifidobacteria were simultaneously investigated for the first time. It has been demonstrated that all the strains exhibited adhesive properties to some extent using intestinal Caco-2 cell line in in vitro model. It could be conclude that the higher hydrophobic strains the stronger adhesive capability. The highest value of hydrophobicity (37.24+/-1.45% and 32.06+/-1.21%) was obtained for strains H-10 and I-6, respectively; correspondingly, the strongest adherence ability (49.47+/-4.88/cell and 47.33+/-2.72/cell) was achieved, respectively. Correlation between surface hydrophobicity and adherence ability of different Bifidobacterium strains including polynomial regression equation (R2=0.78) had been achieved. The present study provided a liable and effective method for screening bifidobacteria with the ability to adhere to intestinal epithelial cells.