植物乳杆菌表面性质及对Caco-2细胞的黏附

通过对10 株不同来源的植物乳杆菌进行自聚集能力、表面疏水性以及体外黏附Caco-2细胞能力的测定,探究植物乳杆菌表面性质与黏附能力之间的关系,利用LiCl对植物乳杆菌进行处理,探究参与植物乳杆菌对细胞黏附过程的物质。结果表明：植物乳杆菌3-4对Caco-2细胞的黏附能力最强。所选的不同植物乳杆菌之间自聚集能力、表面疏水性以及对Caco-2细胞的黏附能力存在差异性;对细胞的黏附能力与表面疏水性存在着显著的相关性（P＜0.05）,因此,自聚集能力和疏水性可以作为筛选具有高黏附细胞能力的植物乳杆菌的参考指标。同时LiCl处理前后,植物乳杆菌自聚集能力和对细胞的黏附能力均有所下降,表明菌株表面蛋白类物质及其他大分子物质均参与自聚集和黏附过程。     

大黄鱼源腐败菌的黏附特性与生物膜特性分析

以分离纯化自冰鲜大黄鱼的3 株希瓦氏菌（MA1-5、MA1-7、MA1-13）和3 株假单胞菌（R3-1、R3-2、R3-5）为供试菌株,研究鱼源腐败菌的表面疏水性、自凝聚能力、生物膜和腐败菌对鱼体（鱼肠、鱼鳃和体表）黏液的黏附能力,探明黏附特性与不同部位黏附能力的相关性,并进一步研究生物膜在不同因素下的形成特性。研究表明,希瓦氏菌具有更强的表面疏水性和自凝聚能力,对鱼体黏液的黏附性强,并具有更强的生物膜形成能力。主成分分析和聚类分析说明黏附能力在属间存在差异性,属内具有相似性。腐败菌对鱼肠和鱼鳃的黏附能力与自凝聚能力和生物膜形成能力具有较高的相关性,对体表黏附能力只与生物膜形成能力有关。腐败菌生物膜的形成受初始菌浓度、培养时间、温度、pH值、盐含量等多种环境因素影响。希瓦氏菌在初始菌浓度106 CFU/mL以上、37 ℃、pH 7～8、0.8% NaCl时形成的生物膜量最多;生物膜在12～24 h期间快速形成,并在36 h达到峰值后,随培养时间延长而逐渐下降。经鱼体黏液包被后,生物膜形成量受到显著影响,鱼鳃黏液促进生物膜形成,鱼肠黏液抑制生物膜形成。     

双歧杆菌在不同材料上形成生物膜的研究

目的:评价双歧杆菌生物膜生成能力.方法:采用结晶紫染色和刚果红染色等方法对几种不同双歧杆菌形成生物膜的能力进行测定和鉴定.结果:采用不同途径分离的9株双歧杆菌分别在亲水性材料和疏水性材料上展现不同的生物膜形成能力.所有菌株在聚丙烯材料上均表现出弱的生物膜形成能力,而在聚苯乙烯和硼硅酸盐材料表面均表现出不同程度的生物膜形成能力.利用盖玻片对生物膜的显微观察表明生物膜含有多糖成分.细胞表面疏水性研究表明双歧杆菌中生物膜的形成与细胞表面疏水性未见相关性.结论:双歧杆菌具有一定的生物膜生成能力.     

辣木籽抗菌肽对金黄色葡萄球菌生物膜的影响

辣木籽抗菌肽MOp2、MOp3对金黄色葡萄球菌具有良好的抑菌效果,但是其对生物膜是否产生影响尚未清楚。采用结晶紫半定量法、荧光显微镜观察、酶解实验和分子对接技术等探讨从辣木籽蛋白水解物中分离鉴定到的新型抗菌肽MOp2、MOp3对金黄色葡萄球菌生物膜的影响。结果表明:MOp2、MOp3的最小抑制生物膜质量浓度(MBIC)和最小清除生物膜质量浓度均为4mg/mL和8mg/mL;1×MBIC下MOp2与MOp3对金黄色葡萄球菌生物膜清除率分别为63.28%和67.90%;荧光显微镜发现,MOp2、MOp3处理后的生物膜黏附减少,细菌数量减少;细菌初期黏附率在1×MBIC的MOp2、MOp3处理后分别降低44.19%和50.77%,且表面疏水性降低;酶解实验表明,金黄色葡萄球菌生物膜胞外聚合物中含量最多的为胞外蛋白(质量分数49.01%),其次为胞外多糖和胞外DNA,且MOp2、MOp3会抑制胞外聚合物中这3种成分的分泌合成;分子对接结果显示,MOp2、MOp3均可以与生物膜形成关键蛋白AgrA、CshA、LuxS和SarA结合,这可能对金黄色葡萄球菌群体感应系统造成一定影响,从而抑制生物膜形成。研究旨在为辣木籽抗菌肽在食品工业中的防腐保鲜应用提供理论依据。     

枯草芽孢杆菌在鱼片保鲜中的应用

目的:为以生物膜形成能力为筛选依据的枯草芽孢杆菌生物保鲜剂开发提供新思路。方法:测定枯草芽孢杆菌的疏水能力、自凝聚率、共凝聚率及生物膜形成能力，并利用枯草芽孢杆菌对冰鲜罗非鱼片进行保鲜，测定罗非鱼片的鲜度和挥发性风味物质。结果:枯草芽孢杆菌的生物膜形成能力越强，对罗非鱼肉的保鲜效果最佳。枯草芽孢杆菌可显著降低鱼肉中的挥发性盐基氮含量，使鱼肉在贮藏第3天的TVB-N含量为24.43 mg/100 g，低于对照组的34.58 mg/100 g；并且显著抑制鱼肉中醇类、酚类、酯类和酸类，如异戊醇、苯酚、吲哚、乙酸等挥发性风味成分的产生。同时，较生物膜形成能力而言，枯草芽孢杆菌的抑菌能力并未体现出与保鲜能力的相关性。结论:枯草芽孢杆菌的自凝聚和生物膜形成能力与其对罗非鱼片的保鲜能力最为相关，可作为保鲜用枯草芽孢杆菌筛选的快速指标。     

乳酸杆菌的表面特性及其黏附能力的研究

乳酸杆菌是肠道益生菌,主要通过其表面的黏附因子定殖于肠道而发挥益生作用。为了研究乳酸杆菌表面性质与黏附能力之间的关系,选择五种乳酸杆菌,分别进行自聚集能力、表面疏水性及与致病菌大肠杆菌ATCC25922和枯草芽孢杆菌K的共聚能力测定。同时,利用氯化锂和高碘酸钠分别处理乳酸杆菌后再与致病菌进行共聚作用,研究乳酸杆菌表面参与黏附的活性物质。结果表明,约氏乳酸杆菌F0421和副干酪乳酸杆菌M5-L具有良好的表面性质和黏附特性。乳酸杆菌与枯草芽孢杆菌K的共聚作用较好。同时,氯化锂和高碘酸钠处理前后,乳酸杆菌对致病菌的聚集能力有所下降,表明菌株表面蛋白及多糖参与了黏附过程。     

莲子抗性淀粉与双歧杆菌的黏附作用

莲子抗性淀粉（lotus seed resistant starch,LRS）是一种可以促进双歧杆菌增殖的良好碳源,其可在被利用前与菌发生物理性黏附接触,这种黏附接触可能会促进双歧杆菌增殖进而使二者黏附体系在人体中发挥益生功效,然而目前对二者黏附能力的影响因素及黏附机理尚不清楚。因此该研究以微波法制备Ⅲ型莲子抗性淀粉（LRS3）,并通过不同的酶、胆盐浓度、pH值条件、生长介质、温度的处理以及测定二者的疏水性和表面电荷,研究LRS3与双歧杆菌黏附的影响因素。结果表明LRS3与双歧杆菌黏附体系在通过胃肠道过程中损失较大,静电相互作用对其存在一定的影响,疏水相互作用对二者黏附体系的影响存在菌株差异性。推测该黏附作用机理可能涉及一类特异性的细胞表面蛋白。     

克罗诺杆菌的生物膜检测和药敏性分析

目的：了解38 株主要来源于我国婴幼儿食品克罗诺杆菌分离株的生物膜形成能力和药敏性。方法：首先用fusA序列分析,对43 株克罗诺杆菌进行准确鉴定;采用半定量黏附实验（结晶紫染色法）测定分离株的生物膜形成能力,并选用八大类11 种抗生素测定其抗生素敏感性。结果：鉴定出34 株阪崎克罗诺杆菌、7 株丙二酸盐克罗诺杆菌和2 株都柏林克罗诺杆菌。这些克罗诺杆菌分离株均有一定生物膜成膜能力,其中丙二酸盐克罗诺杆菌的成膜能力较强;菌株普遍对四环素和万古霉素耐药性较高,共有14 个耐药谱;四环素的耐药性与生物膜形成能力有显著相关性。结论：实验结果表明克罗诺杆菌的成膜性和耐药性都较强,对相关食品企业的消杀措施和临床治疗提出了挑战。     

类植物乳杆菌L-ZS9黏附特性研究及胃肠道环境对其黏附力的影响

为评价类植物乳杆菌L-ZS9的黏附特性及胃肠道环境对其黏附力的影响,以HT-29细胞为黏附模型,测定类植物乳杆菌L-ZS9的黏附率,考察菌株的表面疏水性、自聚集能力;以胰蛋白酶、牛血清白蛋白、过碘酸钠处理菌株,分析类植物乳杆菌L-ZS9的黏附素类型;进一步在体外模拟胃肠道环境,考察胃肠道环境对类植物乳杆菌L-ZS9黏附能力的影响。整个研究以黏附力较高的益生菌鼠李糖乳杆菌LGG作为阳性对照菌株。研究结果表明:L-ZS9的黏附率为23.33%、表面疏水率为49.86%,静置2 h自聚集百分比为2.63%,显著高于对照菌株鼠李糖乳杆菌LGG。过碘酸钠处理对L-ZS9的黏附能力没有影响,牛血清白蛋白和胰蛋白酶处理使菌株黏附数分别从927个细菌/100个细胞降低到244个细菌/100个细胞和211个细菌/100个细胞。表明脂磷壁酸和菌体表面蛋白是L-ZS9的主要黏附因子。经过胃肠道逆环境处理后,类植物乳杆菌L-ZS9的黏附数从4.06×108 cfu/m L降低到3×106 cfu/m L,但高于经过相同处理的鼠李糖乳杆菌LGG。研究结果证明类植物乳杆菌L-ZS9是一株具有强黏附能力的潜在益生菌。     

副溶血弧菌生物膜的形成及壳聚糖的抑制作用

考察了不同来源的副溶血弧菌生物膜的形成能力,分析了不同培养条件对副溶血弧菌生物膜形成的影响,并进一步研究了壳聚糖对生物膜形成的抑制作用。采用结晶紫法测定生物膜形成量,XTT法检测生物膜的代谢活性,苯酚-硫酸法测定生物膜的胞外多糖,分析不同浓度壳聚糖对副溶血弧菌生物膜的抑制效果。结果表明,不同菌株生物膜形成条件不同,成膜能力不同,在蟹壳上更易形成生物膜。壳聚糖表现出了较好的抑制副溶血弧菌生物膜形成的能力,最低抑菌浓度下对生物膜形成的抑制率可达70.98%,胞外多糖的减少率为78.04%,代谢活性抑制率为46.83%。     

粪肠球菌Z096对副溶血弧菌生物被膜及群体感应的抑制作用

为了研究粪肠球菌Z096对副溶血弧菌生物被膜和群体感应（quorum sensing,QS）系统的抑制作用,采用竞争、清除、排阻三种方式模拟Z096与副溶血弧菌在微菌落环境中的相互作用,并进一步探究了Z096的提取物（Z096-E）对副溶血弧菌生物被膜形成、成熟生物被膜清除、细胞表面疏水性、自聚性、QS信号分子AI-2活性、群集泳动能力以及胞外多糖和蛋白合成的影响。结果表明:Z096可通过竞争、清除、排阻的方式与副溶血弧菌相互作用,降低浮游和生物被膜状态的副溶血弧菌细胞数量,干扰副溶血弧菌在载体表面的粘附,且Z096-E能够显著抑制副溶血弧菌生物被膜形成,有效清除成熟生物被膜,1.6 mg/mL的Z096-E处理12 h,副溶血弧菌生物被膜抑制率为70.43%,代谢活性减少率为84.15%;12.8 mg/mL的Z096-E处理副溶血弧菌成熟生物被膜4 h,生物被膜清除率为58.21%,代谢活性减少率为69.84%。而且1.6 mg/mL的Z096-E对副溶血弧菌群集和泳动能力、细胞表面疏水性和自聚性、胞外多糖和蛋白合成的抑制率分别为47.26%、53.56%、63.37%、89.38%、77.65%和51.91%,抑制效果具有浓度依赖性。此外,Z096-E可使副溶血弧菌QS信号分子AI-2活性减弱,表明Z096-E是一种AI-2类群体感应抑制剂,其可通过干扰QS系统,从而影响副溶血弧菌的生理特性。因此,本研究发现了一株能够抑制副溶血弧菌生物被膜的乳酸菌,其提取物Z096-E能作为一种防控副溶血弧菌生物被膜的新型乳酸菌生物制剂,这对消除致病菌生物被膜污染以及开发新型抗菌剂具有积极的作用。     

六株人源益生菌的表面性质与黏附性能研究

通过测定六株人源益生菌的自凝集及疏水性能力,并以人结肠腺癌细胞系Caco-2和HT-29作为体外细胞黏附模型评价其黏附能力,最后探究生长阶段、菌体浓度和孵育时间对发酵乳杆菌CECT5716黏附能力的影响。结果表明嗜酸乳杆菌F-1的自凝集率最高,为69.92%;疏水性最强的是鼠李糖乳杆菌MP-108,达到90.10%;乳双歧杆菌AD011对Caco-2和HT-29的黏附性均为最强,分别为(42.80±0.68)、(47.01±0.20) CFU/cell;不同益生菌的自凝集能力、疏水性及黏附能力均存在菌株特异性,其自凝集能力、疏水性及黏附能力间无明显相关性。此外,发酵乳杆菌CECT5716黏附能力随菌体浓度增大而显著(p<0.05)增加,当菌体浓度达108 CFU/mL以上时黏附能力不再显著提高;随着共孵育的时间延长,其黏附能力也增强,在共孵育2 h后不再提升;且菌体生长稳定期时表现出的黏附能力最强,达到(3.61±0.20) CFU/cell。所考察的益生菌大部分都具有较强的潜在黏附能力,其中以乳双歧杆菌AD011最强。这些益生菌具有可在人体肠道定殖生长的潜在益生功能,在食品和药品等行业中具有良好的应用前景。     

Biofilm formation characteristics of bacterial isolates retrieved from a reverse osmosis membrane

High-quality water purification systems using reverse osmosis (RO) membrane separation have faced a major challenge related to biofilm formation on the membrane surface, or biofouling. To understand this issue, the biofilm formation characteristics of four bacterial isolates previously retrieved from an RO membrane treating potable water were investigated. Biofilm formation of all four isolates occurred to different extents in microtiter plates and could be related to one or more cell properties (hydrophobicity, surface charge, and motility). For Dermacoccus sp. strain RO12 and Microbacterium sp. strain RO18, bacterial adhesion was facilitated by cell surface hydrophobicity, and for Rhodopseudomonas sp. strain RO3, adhesion was assisted by its low surface charge. Sphingomonas sp. strain RO2 possessed both twitching and swarming motilities, which could be important in mediating surface colonization. Further, strains RO2, RO3, and RO12 did not exhibit swimming motility, suggesting that they could be transported to RO membrane surfaces by other mechanisms such as convective permeate flow. The biofilm formation of RO2 was further tested on different RO membranes made of cellulose acetate, polyamide, and thin film composite in continuous flow cell systems. The resultant RO2 biofilms were independent of membrane surface properties and this was probably related to the ex-opolysaccharides secreted bythe biofilm cells. These results suggested that RO2 could colonize RO membranes effectively and could be a potential fouling organism in RO membranes for freshwater purification.     

Beneficial biofilm formation by industrial bacteria Bacillus subtilis and related species

Biofilms are densely packed multicellular communities of microorganisms attached to a surface or interface. Bacteria seem to initiate biofilm formation in response to specific environmental cues, such as nutrient and oxygen availability. Biofilms undergo dynamic changes during their transition from free-living organisms to sessile biofilm cells, including the specific production of secondary metabolites and a significant increase in the resistivity to biological, chemical, and physical assaults. Bacillus subtilis is an industrially important bacterium exhibiting developmental stages. It forms rough biofilms at the air-liquid interface rather than on the surface of a solid phase in a liquid, due to the aerotaxis of the cells. Biofilm formation by B. subtilis and related species permits the control of infection caused by plant pathogens, the reduction of mild steel corrosion, and the exploration of novel compounds. Although it is obviously important to control harmful biofilm formation, the exploitation of beneficial biofilms formed by such industrial bacteria may lead to a new biotechnology.     

冷却猪肉及托盘表面细菌生物被膜分析和肉源荧光假单胞菌的鉴定与被膜研究

为研究冷却猪肉及其接触面中细菌生物被膜能力并探讨肉类特定腐败菌的生物被膜特征,分析了腐败的冷却肉和销售托盘表面的细菌生物被膜形成能力;利用形态学观察、16S r DNA分析和VITEK2微生物鉴定系统鉴定了冷却肉中一株生物被膜能力较强的荧光假单胞菌(Pseudomonas fluorescens);测定了该菌在培养基和猪肉浸提液中的生物被膜能力;用激光共聚焦和扫描电子显微镜观测了其生物被膜的结构特征。结果发现:冷却肉中及其销售托盘表面的细菌能够形成生物被膜的比例较高,37%和45%的细菌具有较强生物被膜形成能力。肉源性荧光假单胞菌能够在培养基和肉液中形成大量生物被膜,具有较强的生物被膜能力。显微成像结果表明荧光假单胞菌在培养6 h后有明显黏附,18 h后多糖分泌增多、菌体堆积并形成具有生物功能的立体生物被膜。这些特点可能有利于荧光假单胞菌在冷却肉表面黏附并成为优势腐败菌。     

Comparison of adhesion characteristics of common dairy sporeformers and their spores on unmodified and modified stainless steel contact surfaces

The attachment of aerobic spore-forming bacteria and their spores to the surfaces of dairy processing equipment leads to biofilm formation. Although sporeformers may differ in the degree of attachment, various surface modifications are being studied in order to develop a surface that is least vulnerable to attachment. This study was conducted to compare the extent of adhesion of spores and vegetative cells of the thermotolerant sporeformer Bacillus licheniformis and the high-heat-resistant sporeformers Geobacillus stearothermophilus and Bacillus sporothermodurans on both native and modified stainless steel surfaces. We studied the effect of contact surface and cell surface properties (including surface energy, surface hydrophobicity, cell surface hydrophobicity, and zeta potential) on the adhesion tendency of both types of sporeformers and their spores. Attachment to native and modified (Ni-P-polytetrafluoroethylene, Ni-P-PTFE) stainless steel surfaces was determined by allowing interaction between the respective contact surface and vegetative cells or spores for 1 h at ambient temperature. The hydrophobicity of vegetative cells and spores of aerobic spore-forming bacteria was determined using the hexadecane assay, and zeta potential was determined using the Zeta sizer Nano series instrument (Malvern Panalytical, Malvern, UK). The results indicated a higher adhesion tendency of spores over vegetative cells for both thermotolerant and high-heat-resistant sporeformers. On comparing the sporeformers, B. sporothermodurans demonstrated the highest adhesion tendency followed by G. stearothermophilus; B. licheniformis exhibited minimal attachment on both surfaces. The tendency to adhere varied with cell surface properties, decreasing with lower cell surface hydrophobicity and higher cell surface charge. On the other hand, modifying contact surface properties for higher surface hydrophobicity and lower surface energy decreased attachment.     

多孔钛片表面ZnO薄膜的表征及其对腐败希瓦氏菌生物被膜的抑制性能

为研究ZnO薄膜微观形貌对其抑制腐败希瓦氏菌生物被膜性能的影响,采用水热合成法制备了ZnO薄膜,并对其进行X射线衍射(XRD)、扫描电子显微镜(SEM)和水接触角表征分析。结果表明,ZnO为六方纤锌矿晶体结构,水热反应时间影响ZnO薄膜的微观形貌。延长水热反应时间,薄膜表面ZnO均匀堆积,其附着量不断增加。多孔钛片呈疏水性,水热合成ZnO薄膜呈亲水性。ZnO薄膜表面的生物被膜附着量随菌体培养时间延长而增加。0~2 h,ZnO薄膜表面生物被膜粘附率和被膜菌总数上升很快,菌体在生物被膜内迅速繁殖;2~12 h,生物被膜粘附率和被膜菌总数持续增长,生物被膜进入生长期;12~36 h,被膜粘附率及被膜菌菌落总数变化较小,生物被膜进入成熟期;36 h后,生物被膜粘附率和被膜菌总数下降,生物被膜进入衰退期。由于ZnO具有杀菌性能,ZnO薄膜对生物被膜的抑制性能增强,且制备ZnO薄膜的水热反应时间越长,样品对生物被膜的抑制性能越优。ZnO杀菌性能对材料表面抗生物被膜粘附性能的贡献远优于材料表面的疏水性能。     

The adherence of Salmonella Enteritidis PT4 to stainless steel: the importance of the air-liquid interface and nutrient availability

Biofilm formation on stainless steel by Salmonella enterica serovar Enteritidis PT4 during growth in three different nutritious conditions was studied. The ability of micro-organisms to generate biofilms on the stainless steel surfaces was studied for a total period of 18 days at 20 degrees C, under three different experimental treatments: (i) growth medium (tryptone soy broth) was not refreshed (no further nutrients were provided) during the incubation period, (ii) growth medium was renewed every 2 days and (iii) growth medium was renewed every 2 days and at the same time the planktonic cells from the old medium were transferred to the new fresh medium. It was found that biofilms developed better and a higher number of adherent cells (ca. 10(7) cfu/cm(2)) were recovered when the organism was grown in periodically renewed nutrient medium than when the growth medium was not refreshed. Regardless of the availability of nutrients, biofilm development was better (range 2-3 logs greater) when coupons were not totally covered by the growth medium and part of the surface was exposed to the air-liquid interface, than when coupons were submerged in the medium. The results suggest that existence of air-liquid interface and adequate nutrient conditions provide the best environment for Salmonella Enteritidis PT4 biofilm formation on stainless steel. The possible role of stationary phase planktonic cells in biofilm development by sessile/attached microbial cells is also discussed.