溶藻弧菌生物被膜形成能力及特性分析

采用改良的微孔板法对114株溶藻弧菌的生物被膜形成能力强弱进行测定,挑选其中1株最强被膜形成能力的菌株进行不同环境因素下的特性分析,并用荧光显微镜观察被膜形成情况。结果表明:测定菌株中有112株具有被膜形成能力(98.25%),其中具有强或中等强度被膜形成能力的有65株(57.02%)。28℃条件下,溶藻弧菌在含3%Na Cl、p H为6.00~8.00的TSB中培养16 h时生物被膜的形成量最大;而添加一定浓度的Mn2+、Cu2+、Ca2+、Mg2+后则不同程度地抑制被膜的形成,并且抑制效果依次降低;菌株在接触亲水性表面(不锈钢和玻璃)时被膜形成量显著高于疏水性表面(聚苯乙烯)(p0.05),且在不锈钢表面形成量最大;荧光显微镜下的观察结果与微孔板法一致。不同溶藻弧菌菌株生物被膜的形成能力具有很大差异且受不同环境因素的影响。研究结果可以为有效控制溶藻弧菌被膜形成提供理论支持。     

实验用金黄色葡萄球菌生物被膜体外模型的建立

以96孔板为载体,通过改良微孔板法研究细菌接种浓度、培养基pH、培养基浓度、培养温度、培养时间5个单因素对金黄色葡萄球菌生物被膜生长量的影响。在单因素试验基础上,选择培养基pH、培养温度、培养时间3个因素,设计二次回归正交旋转组合试验,建立金黄色葡萄球菌生物被膜实验模型。结果表明,生物被膜成膜最佳条件为细菌接种浓度109CFU/mL、培养基pH7.3、培养基质量分数6%、培养温度37.5℃、培养时间为64.9h,此时96孔板所测光密度理论优化值为3.113 8。     

培养条件对蜡样芽孢杆菌生物被膜生长的影响

以常见的食源性致病菌蜡样芽孢杆菌为研究对象,采用超声平板计数法对其生物被膜形成过程中的不同影响因素进行了研究。结果表明:在37℃、富营养环境、中性及弱碱性条件下,蜡样芽孢杆菌易形成大量生物被膜;添加低浓度的NaCl和葡萄糖均可促进生物被膜的形成,但促进作用不明显;添加高浓度的NaCl和葡萄糖对蜡样芽孢杆菌生物被膜生长有显著抑制作用。     

环境条件对蜂房哈夫尼亚菌群体感应信号分子活性及其形成生物被膜能力的影响

以腐败大菱鲆中分离的蜂房哈夫尼亚菌(Hafniaalvei,Ha-01)为研究对象,采用报告平板打孔法和96微孔板法分别探究环境因素(碳源、NaCl浓度、pH及培养温度)对Ha-01的N-酰基高丝氨酸内酯(AHLs)生成量及生物被膜形成的影响。通过添加外源信号分子标准品研究Ha-01群体感应系统与其生物被膜形成之间的关系。结果表明,碳源对Ha-01分泌AHLs的影响能力由高到低为木糖葡糖糖果糖麦芽糖乳糖蔗糖,以木糖为碳源时生物被膜产生量较高;低NaCl质量浓度能够促进Ha-01的生长,增强其AHLs分泌能力,当NaCl质量浓度为2g/(100mL)时,其生物被膜产生量最高;Ha-01在碱性环境中的AHLs活性较低,pH6.0时AHLs活性较大,pH7时生物被膜产生量较大;Ha-01在低温或高温的胁迫环境下,其AHLs产生量较大,而较低或较高温度均能抑制其生物被膜产生,25℃时其生物被膜产生量最大。当添加外源信号分子标准品时,其生物被膜的产生量均显著增加。蜂房哈夫尼亚菌的AHLs分泌及生物被膜形成均受环境条件的影响,且群体感应现象能够调控其生物被膜的产生。     

噬菌体vB_SauM_RS对乳源金黄色葡萄球菌生物被膜的清除作用

目的：以金黄色葡萄球菌为宿主菌，分离裂解性噬菌体vB_SauM_RS，测定其生物学特性，并探究其在两种牛奶中的抑菌效果及对生物被膜的清除作用。方法：将噬菌体与金黄色葡萄球菌以1∶100的比例（菌体数量比）分别接种于脱脂牛奶和全脂牛奶中，分别在4 ℃和25 ℃环境下处理，测定不同时间脱脂牛奶和全脂牛奶中细菌浓度和噬菌体效价，分析噬菌体在两种牛奶中的抑菌效果；之后通过结晶紫染色、平板计数法和双层平板法测定经噬菌体处理后不同时间膜内细菌浓度和噬菌体效价，分析vB_SauM_RS对不同材料表面生物被膜的清除效果，并通过荧光显微镜观察生物被膜清除前后膜内细菌数量的变化。结果：vB_SauM_RS形成的噬菌斑清晰透亮、外有晕环；透射电子显微镜观察其属于肌尾噬菌体科；最佳感染复数为0.01、潜伏期为20 min、平均裂解量为39 PFU/cell，其在20～50 ℃和pH 4.0～11.0条件下稳定，具有高效的裂解效率；vB_SauM_RS在牛奶中抑菌作用明显，与对照组相比，在低温（4 ℃）条件下24 h使脱脂牛奶和全脂牛奶中细菌浓度分别下降1.079（lg（CFU/mL））和1.021（lg（CFU/mL）），在室温（25 ℃）条件下24 h使脱脂牛奶和全脂牛奶中细菌浓度分别下降5.418（lg（CFU/mL））和5.740（lg（CFU/mL））；通过平板计数法和荧光显微镜观察显示vB_SauM_RS对生物被膜内细菌的清除效果明显，对不同材料表面生物被膜均有良好的清除效果。结论：vB_SauM_RS不仅对牛奶中金黄色葡萄球菌有良好的裂解效果，对生物被膜也有很好的清除效果，具有作为金黄色葡萄球菌生物抗菌剂的应用潜力。     

薰衣草精油对不同温度下形成的副溶血弧菌成熟生物被膜的清除作用

该文研究了副溶血弧菌在32、10℃下生物被膜的形成,并采用结晶紫染色法、四唑鎓盐(XTT)法、叠氮溴化丙锭与荧光定量PCR结合技术、激光共聚焦显微镜观察的手段以评估不同浓度的薰衣草精油和4种常用抗菌剂对副溶血弧菌成熟生物被膜的清除效果,进一步分析了2种温度下形成的生物被膜对不同浓度的薰衣草精油的抗性。研究结果表明,副溶血弧菌在两种温度下均能形成稳定的生物被膜,32℃下形成的生物被膜量显著高于10℃;低温生物被膜对抗菌剂的抗性更强;在5种抗菌剂中,薰衣草精油对副溶血弧菌生物被膜的清除效果最佳。1/2最低抑菌浓度(minimum inhibitory concentration, MIC)～4 MIC的薰衣草精油对副溶血弧菌32℃下形成的常温生物被膜和10℃下形成的低温生物被膜的清除率分别为36.39%～72.38%和58.20%～67.62%,使代谢活性分别下降35.72%～71.38%和42.06%～50.48%。激光共聚焦显微镜图像显示,经薰衣草精油处理的生物被膜结构稀疏,胞外多糖减少,死菌数量增加。此外,薰衣草精油对成熟生物被膜细胞具有良好的杀伤效果,随着其处理浓度的增加,生物被膜...     

环境因素促进单增李斯特菌生物被膜的形成

本实验研究了在营养正常和贫瘠状态下,环境因素包括p H值、氯化钠、常见碳水化合物及危险罗尔斯通氏菌对单增李斯特菌生物被膜形成的影响,并使用激光扫描共聚焦显微镜(Confocal laser scanning microscope,CLSM)观察单增李斯特菌和危险罗尔斯通氏菌单、双菌种生物被膜。结果表明:弱酸、碱性条件、低浓度的氯化钠以及危险罗尔斯通式菌能显著促进单增李斯特菌形成生物被膜,但与营养正常状态相比,营养贫瘠状态下的生物被膜形成量降低。几种常见碳水化合物(葡萄糖、蔗糖、木糖醇、半乳糖及魔芋精粉)也有一定的促进作用,特别是木糖醇和魔芋精粉,引起的增长率分别高达75.00%和96.67%。CLSM结果表明,单增李斯特菌和危险罗尔斯通氏菌生物被膜的空间结构不同,但活菌数都比较多。致病菌生物被膜的形成会对食品安全构成巨大威胁,而很多环境因素又有增强生物被膜形成的作用,应引起食品生产等行业的关注。     

酒酒球菌在青梅汁中的生长及苹果酸乳酸发酵特性的研究

以青梅汁为原料,利用酒酒球菌的苹果酸-乳酸发酵(MLF)对青梅汁进行生物降酸的研究。结果表明:接种量为2.0×108CFU/mL,青梅汁的pH≥3.4时,MLF能正常进行,并使样品的酸度降低61.35%以上;接种量为2.6×107CFU/mL时,青梅汁的pH为3.6才能触发MLF,样品的酸度降低了77.60%;接种量为2.3×108CFU/mL,在pH3.6和4.0的青梅汁中,添加1.0g/100g的葡萄糖的样品,与不添加葡萄糖的样品相比,其酸度分别降低了27.04%和34.63%;在柠檬酸-柠檬酸钠缓冲体系中,酒酒球菌使体系酸度降低了26.40%,证实酒酒球菌可利用柠檬酸作为碳源,进行MLF。     

不同食源微生物生物被膜形成特征及亚致死浓度消毒剂对菌体成膜的影响

研究4种细菌在体外不同培养条件下形成生物膜的能力,并探索了亚致死质量浓度消毒剂对细菌生物成膜的影响。在不同营养物质浓度和培养条件下以及不同质量浓度的季铵盐下,以微孔板法检测菌体成膜性。高温、低浓度的葡萄糖和NaCl促进菌体形成生物膜,高质量浓度葡萄糖和NaCl、较高的培养温度,成膜受到抑制。菌体成膜受到多种因素的影响,合理控制食源微生物生物被膜的形成,是促进食品安全和卫生的重要手段。     

植物乳杆菌SCP53生物膜的形成条件

研究了不同状态下植物乳杆菌SPC53生物膜形成能力的情况。通过改变其生长的环境条件与营养状况,采用结晶紫染色法和扫描电镜(scanning electron microscope,SEM)观察,定量及定性分析其生物膜的形成。结果表明:接种量5×10~7CFU/mL,培养时间36 h,培养温度42℃,pH值5.8是植物乳杆菌SCP53生物膜形成的最适培养条件,在MRS肉汤中依次添加200 mg/mL NaCl、75 mg/mL葡萄糖、7.5 mg/mL果胶、75 mg/mL全脂乳粉,可以促进植物乳杆菌生物膜的形成,形成后的生物膜A_(490)值为4.492 3。对照组A_(490)值为0.136 5,扫描电镜结果显示生物膜与浮游菌体有明显差异。     

不同培养条件及群体感应信号分子对蜂房哈夫尼亚菌生物被膜的影响

为研究蜂房哈夫尼亚菌(Hafnia alvei)Ha-01生物被膜形成的过程及不同培养条件(碳源、p H值、Na Cl质量分数、黏附材料)对其生物被膜形成的影响,采用超声波平板法及扫描电镜法研究不同培养条件下菌株Ha-01生物被膜活菌数及生长情况,并通过添加外源标准群体感应信号分子(N-酰基高丝氨酸内酯(N-acyl-homoserine lactones,AHLs))中的C6-HSL研究了AHLs与其生物被膜形成的关系。结果显示,菌株Ha-01生物被膜的形成与培养时间密切相关;在不同碳源的培养条件下形成生物被膜能力不同,其中在以木糖为培养基时形成量最大,达到7.51(lg(CFU/cm~2)),与在LB培养基中相比增加10.28%;在中性培养条件下更利于其生物被膜的形成,活菌数为7.77(lg(CFU/cm~2));在Na Cl质量分数为2%时,其生物被膜产生量最大,活菌数为7.18(lg(CFU/cm~2));在不同黏附材料上生物被膜形成能力从大到小依次为铝片、锌片、玻璃片,活菌数分别为7.22、6.48、6.11(lg(CFU/cm~2));且添加C6-HSL量越多,其生物被膜产生能力越强。研究表明,培养条件能够影响菌株Ha-01生物被膜形成,且AHLs可以调控其生物被膜形成。     

保加利亚乳杆菌和嗜热链球菌生物膜形成研究

采用置片法和菌落计数法对嗜热链球菌(Streptococcus thermophilus)和保加利亚乳杆菌(Lactobacillus bulgaricus)在不同载体表面(椰果粒、不锈钢网布、塑料片、陶瓷片和玻璃片)上形成生物膜的能力进行研究。结果表明：椰果粒和不锈钢网布是适宜乳酸菌单菌生物膜形成的载体,培养7d椰果粒和不锈钢网布上的单菌生物膜初始菌密度可达107CFU/cm2,并且在后续培养中能稳定在3.2×106CFU/cm2左右;混菌生物膜菌密度连续7d稳定在1×107CFU/cm2左右。利用扫描电镜对S. thermophilus和L. bulgaricus形成的混菌生物膜进行观察,结果表明在椰果粒和不锈钢网布表面上S. thermophilus和L. bulgaricus形成典型的混菌生物膜结构。     

培养条件对奇异变形杆菌生物膜生长的影响

采用改良微孔板培养,结晶紫染色、平板计数以及电镜扫描方法研究不同初始菌浓度、培养环境(温度、气体环境)、培养基成分(pH、氯化钠浓度、碳源种类和浓度),培养基用量及接触材料对奇异变形杆菌(Proteus mirabilis,P. mirabilis)生物膜生长的影响。经过24 h培养,初始菌浓度10 CFU/cm2时,生物膜形成量较低,初始菌浓度102~108 CFU/cm2时,生物膜形成量明显;37 ℃有氧条件适宜P. mirabilis生物膜形成;培养基组成(2.0% NaCl,1.0%麦芽糖,pH8.0)和加倍用量对P. mirabilis生物膜形成具有明显的促进作用;粗糙的木制表面最易使P. mirabilis粘附并形成生物膜,硅胶次之,聚丙烯塑料和盖玻片表面比较光滑,菌体不易粘附,生物膜难以形成;不同接触材料形成的P. mirabilis生物膜结构和形态不同,硅胶上生物膜为蘑菇状,盖玻片上生物膜成扁平状。本文研究结果可为食品生产加工过程中P. mirabilis污染防控提供理论依据和技术支撑。     

Effect of temperature, pH, and water activity on biofilm formation by Salmonella enterica enteritidis PT4 on stainless steel surfaces as indicated by the bead vortexing method and conductance measurements

An assay was developed in an effort to elucidate the effect of important environmental parameters (temperature, pH, and water activity [aw]) on Salmonella Enteritidis biofilm formation on stainless steel surfaces. To achieve this, a modified microbiological technique used for biofilm studying (the bead vortexing method) and a rapid method based on conductivity measurements were used. The ability of the microorganism to generate biofilm on the stainless surfaces was studied at three temperatures (5, 20, and 37 degrees C), four pH values (4.5, 5.5, 6.5, and 7.4), and four aw values (0.5, 1.5, 5.5, and 10.5% NaCl). Results obtained by the bead vortexing method show that maximum numbers of adherent bacteria per square centimeter (106 CFU/cm2) were attained in 6 days at 20 degrees C. Biofilm formation after 7 days of incubation at 20 degrees C was found to be independent of the pH value. In addition, the high concentration of sodium chloride (10.5% NaCl, aw = 0.94) clearly inhibited the adherence of cells to the coupons. Conductance measurements were used as a supplementary tool to measure indirectly the attachment and biofilm formation of bacterial cells on stainless steel surfaces via their metabolic activity (i.e., changes in the conductance of the growth medium due to microbial growth or metabolism). Results obtained by conductance measurements corresponded well to those of the bead vortexing method. Furthermore, we were able to detect cells that remained attached on the metal surfaces even after vortexing via their metabolic activity. The results, except for demonstrating environmental-dependent Salmonella Enteritidis biofilm formation, indicated that traditional vortexing with beads did not remove completely biofilm cells from stainless steel; hence, conductance measurements seem to provide a more sensitive test capable to detect down to one single viable organism.     

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。     

基于非水溶性大豆纤维的双歧杆菌生物膜成膜条件优化研究

为了提高双歧杆菌的抗逆性，该试验建立基于非水溶性膳食纤维的成膜体系，并优化了双歧杆菌生物膜成膜条件。以大豆纤维（添加量10 g/L）为成膜基质，以成膜活菌数为响应值，通过单因素试验研究碳源、氮源、培养温度、初始pH值以及NaCl添加量对双歧杆菌成膜的影响。结果表明，最显著的影响因素为初始pH值，培养温度和NaCl添加量。Box-Behnken响应曲面分析得到最优的成膜条件为：培养基碳源为葡萄糖、氮源为胰蛋白胨、额外添加NaCl 0.35%、培养温度37 ℃，初始pH值7.0。在此最优条件下制备的双歧杆菌成膜活菌数最多为1.22×109 CFU/mL，为实现双歧杆菌生物膜的可控成膜，特别是工业规模的发酵制备提供了理论基础和实验依据。     

Transfer of surface-dried Listeria monocytogenes from stainless steel knife blades to roast turkey breast

Listeria contamination of food contact surfaces can lead to cross-contamination of ready-to-eat foods in delicatessens. Recognizing that variations in Listeria biofilm-forming ability exist, the goal of this study was to determine whether these differences in biofilm formation would affect the Listeria transfer rate during slicing of delicatessen turkey meat. In this study, six previously identified strong and weak biofilm-forming strains of Listeria monocytogenes were grown at 22 degrees C for 48 h on Trypticase soy agar containing 0.6% yeast extract and harvested in 0.1% peptone. Thereafter, the strains were combined to obtain two 3-strain cocktails, resuspended in turkey slurry, and inoculated onto flame-sterilized AISI grade 304 stainless steel knife blades that were subjected to 6 and 24 h of ambient storage at approximately 78% relative humidity. After mounting on an Instron Universal Testing Machine, these blades were used to obtain 16 slices of retail roast turkey breast. Based on an analysis of the slices by direct plating, Listeria populations decreased 3 to 5 log CFU per slice after 16 slices. Overall, total transfer to turkey was significantly greater for strong (4.4 log CFU total) as opposed to weak (3.5 log CFU total; P < 0.05) biofilm formers. In addition, significantly more cells were transferred at 6 (4.6 log CFU total) than at 24 h (3.3 log CFU total; P < 0.05) with Listeria quantifiable to the 16th slice, regardless of the inoculation level. Increased survival by the strong biofilm formers, as evidenced by viability staining, suggests that these strains are better adapted to survive stressful conditions than their weak biofilm-forming counterparts.     

Salmonella enterica Enteritidis biofilm formation and viability on regular and triclosan-impregnated bench cover materials

Contamination of food contact surfaces by microbes such as Salmonella is directly associated with substantial industry costs and severe foodborne disease outbreaks. Several approaches have been developed to control microbial attachment; one approach is the development of food contact materials incorporating antimicrobial compounds. In the present study, Salmonella enterica Enteritidis adhesion and biofilm formation on regular and triclosan-impregnated kitchen bench stones (silestones) were assessed, as was cellular viability within biofilms. Enumeration of adhered cells on granite, marble, stainless steel, and silestones revealed that all materials were prone to bacterial colonization (4 to 5 log CFU/cm(2)), and no significant effect of triclosan was found. Conversely, results concerning biofilm formation highlighted a possible bacteriostatic activity of triclosan; smaller amounts of Salmonella Enteritidis biofilms were formed on impregnated silestones, and significantly lower numbers of viable cells (1 × 10(5) to 1 × 10(6) CFU/cm(2)) were found in these biofilms than in those on the other materials (1 × 10(7) CFU/cm(2)). All surfaces tested failed to promote food safety, and careful utilization with appropriate sanitation of these surfaces is critical in food processing environments. Nevertheless, because of its bacteriostatic activity, triclosan incorporated into silestones confers some advantage for controlling microbial contamination.     

培养条件对阪崎克罗诺杆菌食品分离菌株生物被膜形成的影响

目的研究不同培养条件对阪崎克罗诺杆菌食品分离菌株生物被膜形成的影响。方法从23株阪崎克罗诺杆菌食品分离菌株中筛选5株菌株作为研究对象,利用试管法和96孔微板法检测不同培养条件下阪崎克罗诺杆菌的菌膜形成情况。结果 5株菌株在胰蛋白胨大豆肉汤培养基(triptych soy broth,TSB)中均具有较强的成膜能力;培养基中添加不同浓度的营养因子对阪崎克罗诺杆菌生物被膜的形成影响不同,葡萄糖对5株阪崎克罗诺杆菌食品分离菌株的成膜能力有明显促进作用,乳糖的添加对阪崎克罗诺杆菌生物被膜形成有一定影响,但蔗糖的添加对阪崎克罗诺杆菌生物被膜形成影响不明显;培养基中添加一定浓度的氯化钠可以有效抑制阪崎克罗诺杆菌生物被膜形成;p H对阪崎克罗诺杆菌的成膜能力也有一定影响,中性环境有利于阪崎克罗诺杆菌的被膜形成。结论阪崎克罗诺杆菌食品分离菌株具有形成细菌生物被膜的能力,并且不同培养条件对阪崎克罗诺杆菌生物被膜的形成影响不同。     

Influence of peroxyacetic acid and nisin and coculture with Enterococcus faecium on Listeria monocytogenes biofilm formation

Biofilm formation is a matter of concern in food industries because biofilms facilitate the survival of pathogenic bacteria such as Listeria monocytogenes, which may contaminate food-processing equipment and products. In this study, nisin and two Enterococcus faecium strains were evaluated for their effect on biofilm formation by L. monocytogenes cultured in brain heart infusion broth and on stainless steel coupons. Elimination of preformed L. monocytogenes biofilms by peroxyacetic acid also was tested. Adhesion control experiments were performed with pure cultures of L. monocytogenes after swab collection of adhered cells, which were then enumerated on PALCAM agar plates and visualized by scanning electron microscopy. Formation of a biofilm was recorded when the number of adhered cells was at least 10(3) CFU/cm2. When L. monocytogenes was cocultured with E. faecium bac-, the number of adhered L. monocytogenes cells was 2.5 log lower (P = 0.002) when initially compared with the control culture, but after 6 h of incubation a biofilm was again detected. However, in coculture on stainless steel coupons, E. faecium bac+ inhibited L. monocytogenes adherence and did not allow biofilm formation for up to 48 h (P < 0.001). In the presence of nisin or after treatment with peroxyacetic acid, bacterial growth was reduced (P < 0.001) up to 4.6 and 5.6 log CFU/cm2, respectively, when compared with L. monocytogenes cultures on untreated coupons. However, after these treatments, cells were still present, and after 24 h of incubation, a renewed biofilm was detected in L. monocytogenes cultures treated with nisin. Although all tested conditions reduced L. monocytogenes growth to some extent, only coculture with E. faecium bac+ efficiently reduced biofilm formation, suggesting a potential control strategy for this pathogen.