副干酪乳杆菌发酵液中抗菌肽的筛选及对副溶血性弧菌的抑菌作用

副溶血性弧菌是一种常见的食源性致病菌,严重威胁食品安全和人体健康。本研究利用超高效液相色谱-质谱联用技术鉴定副干酪乳杆菌发酵液中的多肽序列,并通过生物信息学筛选出可能的抗菌序列（RQQAENLAKFAKKG）,命名为Yt9z。研究结果表明其对副溶血性弧菌的最低杀菌质量浓度（MBC）为125μg/mL,可在3 h内将细菌完全杀死。通过膜通透、透射电镜、DNA凝胶阻滞分析、圆二色谱等试验探究其抑菌机制,结果在不同溶液环境下,抗菌肽Yt9z能改变自身的二级结构,进而增加细菌细胞膜通透性,并穿透细胞膜与DNA结合使其死亡。这些发现为抗菌肽Yt9z应用于副溶血性弧菌污染提供了理论参考。     

反式肉桂醛对副溶血性弧菌的抑制作用

本研究通过测定TC对副溶血性弧菌的最小抑菌浓度(Minimum Inhibitory Concentration,MIC)评价其抑菌效果;随后通过测定TC对副溶血性弧菌生长曲线、生长动力模型、细胞膜完整性及细胞形态的影响探究其可能的抑菌机理;最后,构建副溶血型弧菌污染的鲜虾模型,评价TC对鲜虾中副溶血性弧菌的控制作用。结果表明,TC对副溶血性弧菌的MIC为50～70μg/mL;TC可降低副溶血性弧菌最大生长速率、延长其生长延滞期;TC可使副溶血性弧菌细胞膜完整性显著降低,并使副溶血性弧菌细胞形态干瘪、皱缩;在鲜虾模型中,体积分数0.4%的TC在1 h(4℃)使鲜虾中的副溶血性弧菌降低至检出限以下。研究结果表明,TC有潜力作为天然的抗菌物质应用于鲜虾及其他海产品中有效控制副溶血性弧菌。     

抗菌肽Met-Ala-Lys对铜绿假单胞菌细胞膜的作用机制

以抗菌肽Met-Ala-Lys(MAK)和铜绿假单胞菌为研究对象,采用NPN荧光探针法,圆二色谱法以及原子吸收光谱测定测定铜绿假单胞菌细胞膜结构的变化,探讨MAK对目标菌生长的影响作用机制。结果表明:随着MAK对铜绿假单胞菌作用时间的增加,MAK的二级结构尤其是无规则卷曲结构发生变化明显,细胞内膜(吸附速率从54.03%降至27.33%)和外膜(荧光强度在前30 min持续增强,30 min后基本稳定在45左右)受到影响,通透性明显增强,导致钾离子(从处理10 min时迅速增加)和蛋白质泄漏量(从4.65μg/m L到10.25μg/m L)以及细胞膜的疏水性随之增加,细胞表面膜电位发生变化。表明MAK能改变铜绿假单胞菌的表面特性,抑制其生长,进而起到抗菌作用。本研究为抗菌肽MAK的抗菌机理制研究奠定一定的理论基础。     

创伤弧菌控制措施与风险评估的研究进展

创伤弧菌为革兰氏阴性嗜盐菌,天然存在于世界各地温暖的河口与海洋环境中,是一种重要的食源性致病菌,人类感染往往与食用受污染的海产品有关。食源性创伤弧菌感染最常见的临床症状是原发性败血症,病死率超过50%。文中介绍了创伤弧菌生物学特性、控制措施及风险评估的研究进展。     

柠檬醛对副溶血性弧菌的抑制作用

有效预防和控制副溶血性弧菌对食品的污染对于保障公众健康具有重要意义。本研究首先利用琼脂稀释法测定7?种植物源活性物质（丁香酸、阿魏酸、绿原酸、硫辛酸、原儿茶酸、原儿茶醛和柠檬醛）对副溶血性弧菌的最小抑菌浓度,在此基础上选择柠檬醛进行后续实验;通过检测经柠檬醛处理后的副溶血性弧菌生长曲线、细胞膜电位、胞内ATP浓度、细胞膜完整性以及细胞形态的变化,探究柠檬醛对副溶血性弧菌的抑制作用及可能的作用机理。结果表明：柠檬醛相比于其他6?种植物源活性物质对副溶血性弧菌有更好的抑菌效果,其对两株标准菌株和8?株分离菌株的最小抑菌浓度在0.10～0.60?mg/mL范围内;柠檬醛能够引起副溶血性弧菌细胞膜电位去极化、胞内ATP浓度降低及细胞膜完整性下降,同时可使细胞皱缩变形。本研究结果表明柠檬醛具有良好的抑菌功效,并有潜力作为天然的抗菌物质应用于食品工业。     

水飞蓟素对副溶血性弧菌的抑制作用

目的 以两种副溶血性弧菌为研究对象,探究水飞蓟素对于副溶血性弧菌的抑制及其作用机制。方法 通过琼脂板稀释法和肉汤稀释法确定水飞蓟素对两株菌的最小抑制浓度（MIC）,然后进一步通过研究水飞蓟素在不同培养基上处理后的副溶血性弧菌的生长曲线、生物膜形成情况、细胞膜完整性及钾离子外流情况的变化,探究水飞蓟素在不同培养基上对副溶血性弧菌的抑制作用及可能的作用机制。结果 水飞蓟素能够抑制生物膜的形成,损害细胞膜,导致细胞变形,显著增加钾离子外流,来抑制副溶血性弧菌的生长,并且不同培养基的抑制作用是不一样的。结论 水飞蓟素能够对副溶血性弧菌产生较强的抑制作用,有作为天然的抗菌物质应用于食品工业的潜力。     

香辛料提取物对副溶血弧菌生物膜的抑制作用

对大蒜、肉桂等6种天然的香辛料分别采用水和80%乙醇进行提取并筛选出对副溶血弧菌抑菌能力较强的提取物,探究这些香辛料提取物对副溶血弧菌生物膜的抑制作用。研究结果表明大蒜、肉桂、丁香具有较强的抗菌作用,而花椒、小茴香、迷迭香则相对较弱。肉桂和大蒜的乙醇提取物对副溶血弧菌的最低抑菌浓度(Minimum inhibitory concentration,MIC)皆为6.25 mg/m L。亚抑菌浓度的提取物除了能够抑制副溶血弧菌生物膜的形成,还能抑制生物膜内细菌的代谢活性,减少细菌胞外多糖的分泌。激光共聚焦扫描显微镜(Confocal Laser Scanning Microscope,CLSM)观察发现,处理后死细胞的数量明显增多,且生物膜内多糖的含量明显变少。      

大肠杆菌细胞膜固定相萃取辣木籽抗菌肽

制备大肠杆菌细胞膜固定相(E.coli cell membrane stationary phase,ECMSP),研究其对辣木籽抗菌肽的萃取效果。研究超声时间、超声功率对大肠杆菌破壁效果的影响,分析大肠杆菌细胞膜与大孔硅胶的吸附特征及ECMSP对辣木籽抗菌肽的吸附效果,明确萃取前后抑菌活力变化及反相-高效液相色谱（RP-HPLC）差异峰。结果表明：在超声功率500 W、超声时间30 min条件下,大肠杆菌破壁效果较好;大孔硅胶对大肠杆菌细胞膜的饱和吸附值（Csmax）为9.98 μg/mg、吸附常数（K*）为171.02 μg/mL;萃取前后抗菌肽对大肠杆菌和金黄色葡萄球菌的抑制效果差异明显,RP-HPLC分析表明萃取前后出现了3个差异峰。研究结果可为辣木籽抗菌肽的后续开发利用提供理论基础和应用参考。     

水产品中创伤弧菌的快速检测与分离

目的:对水产品中创伤弧菌的快速检测与分离技术进行研究。方法:蛤蜊肉经高速匀浆处理后,在含有多粘菌数B的碱性蛋白胨水中增菌培养(35～37℃,12～16h),然后用PCR技术检测样品。PCR检测的目的基因是创伤弧菌的溶细胞毒素(hemolysin/cytolysin)基因,扩增产物为222bp。对PCR结果呈阳性的样品做原位杂交实验,用碱磷酶标记的寡核苷酸探针VVAP来分离创伤弧菌。通过对购买于青岛丹东路小市场的蛤蜊进行检验,分离出了2株创伤弧菌。结果表明:应用PCR、原位杂交技术能快速检测和分离水产品中存在的创伤弧菌。     

东南沿海地区零售海产品中创伤弧菌的监测

目的了解我国东南沿海地区零售海产品中创伤弧菌的污染状况。方法采用3%氯化钠碱性蛋白胨水增菌,改良纤维二糖-多粘菌素B-多粘菌素E(mCPC)琼脂和纤维二糖-多粘菌素E(CC)琼脂分离海产品试样中的创伤弧菌,可疑菌落的鉴定采用生化试验或PCR法。采用最可能数(MPN)法和PCR法检测牡蛎试样的创伤弧菌污染水平。结果天然污染海产品试样创伤弧菌的检出率为19.8%(20/101)。牡蛎试样中45%(55/122)创伤弧菌密度低于3MPN/g的最低检出限。牡蛎中创伤弧菌的污染水平存在季节性差异。结论未来应加强对我国海产品中创伤弧菌污染状况的监测,尤其是在温暖的季节。     

低温贮藏条件下创伤弧菌和副溶血性弧菌失活模型的建立

为掌握对虾中创伤弧菌和副溶血性弧菌在5℃和一18~C低温贮藏条件下失活动力学特征,分别采用线性模型、Weibull模型、Logistic模型对创伤弧菌和副溶血性弧菌的失活曲线进行拟合。研究结果表明,在5~C条件下,创伤弧茵VvHB09的耐冷力较强;．18℃条件下,副溶血性弧菌ATCC17802和创伤弧菌VvSH09的耐冷力较强。线性模型比weibull模型、Logistic模型更适合拟合创伤弧菌和副溶血性弧菌的失活特征。     

Temperature Effects on the Depuration of Vibrio parahaemolyticus and Vibrio vulnificus from the American Oyster (Crassostrea virginica)

ABSTRACT:  This study investigated temperature effects on depuration for reducing Vibrio parahaemolyticus and Vibrio vulnificus in American oyster ( Crassostrea virginica ). Raw oysters were inoculated with 5-strain cocktail of V. parahaemolyticus or V. vulnificus to levels of 10⁴ to 10⁵ MPN (most probable number)/g and depurated in artificial seawater (ASW) at 22, 15, 10, and 5 °C. Depuration of oysters at 22 °C had limited effects on reducing V. parahaemolyticus or V. vulnificus in the oysters. Populations of V. parahaemolyticus and V. vulnificus were reduced by 1.2 and 2.0 log MPN/g, respectively, after 48 h of depuration at 22 °C. Decreasing water temperature to 15 °C increased the efficacy of depuration in reducing V. parahaemolyticus and V. vulnificus in oysters. Reductions of V. parahaemolyticus and V. vulnificus in oysters increased to 2.1 and 2.9 log MPN/g, respectively, after 48 h of depuration at 15 °C. However, depurations at 10 and 5 °C were less effective than at 15 °C in reducing the Vibrio spp. in oysters. Extended depuration at 15 °C for 96 h increased reductions of V. parahaemolyticus and V. vulnificus in oysters to 2.6 and 3.3 log MPN/g, respectively.     

Two-stage Nested PCR Effectiveness for Direct Detection of Vibrio vulnificus in Natural Samples

The nested primers designed to amplify a 222-base pair portion of the hemolysin gene, vvhA, were specific for all V. vulnificus strains tested. The nested PCR amplification, coupled with direct extraction of template DNA, revealed improved sensitivity sufficient for detection of 1 to 10 CFU V. vulnificus in 1 mL of seafood homogenates, and eliminated the need for enrichment culturing. Thereby, the nested PCR method achieved a broader applicability, making it effective for extensive use in identification of the pathogen in natural samples such as raw seafoods, seawater and sediments.     

2018年广州市水产品中创伤弧菌和河弧菌检测分析

目的 了解为期一年的监测中广州市水产品创伤弧菌和河弧菌污染水平、菌株携带毒力基因和分子分型情况。方法 对广州市水产品中创伤弧菌和河弧菌进行分离鉴定,毒力基因鉴定和肠道细菌基因间重复序列聚合酶链式反应(Eric-PCR)。结果 创伤弧菌阳性率为10.4%(31/298),河弧菌为5.0%(15/298);用PCR法检测创伤弧菌毒力溶血素基因vvhA全部阳性,vcgC/E和16S rRNA A/B分型结果显示,共有CB型、EA型和CA型3种基因型别。河弧菌毒力相关基因vfh、toxR全部阳性,hupO携带率为60.0%(9/15)、vfp携带率为80.0%(12/15);Eric-PCR扩增出8～14条100～2 000 bp之间的条带,将15株河弧菌在相似系数为0.8处分为5个群11个类型。结论 广州市水产品中创伤弧菌和河弧菌污染情况较严重,大部分菌株携带毒力基因,Eric-PCR结果显示15株河弧菌在亲缘关系上具有相关性,应加强防控。     

创伤弧菌的优化培养及快速检测

研究了创伤弧菌优化培养的方案,用以提高该菌的检出率。利用Design-Expert软件中的Box-Behnken中心组合实验原理,设计一组3因素3水平实验,通过响应曲面分析,得到优化培养参数为:含盐量3.65%,pH6.75,培养温度37.00℃,在该条件下培养液的OD595nm为0.520。利用创伤弧菌优化培养条件对市售海产样品进行了培养,并通过聚合酶链式反应（Polymerase Chain Reaction,PCR）对样品进行快速检测,且该菌的检出率高达23.3%。结果表明:应用本实验的优化培养方案、PCR法能快速有效检测水产品中存在的创伤弧菌。      

创伤弧菌致病性及致病机制研究进展

创伤弧菌广泛存在于海水和牡蛎等海产品中,具有很强的细胞毒性和溶血性,被美国疾病预防控制中心(CDC)列为三大致病性弧菌之一。本文就创伤弧菌在食品中的污染状况,人类感染途径及临床表现,致病性、致病机制和检测方法等进行综述,以期为该菌感染的预防和治疗提供理论依据。     

Improving Detection of Vibrio vulnificus in Octopus variabilis by PCR

PCR methods can detect foodborne pathogenic bacteria with simplicity, specificity and speed. In order to improve sensitivity and speed of PCR methods for detection of Vibrio vulnificus in small octopus homogenate, several media and culture conditions were compared. Modified brain heart infusion media containing 2% NaCl and adjusted to pH 8.0 and 30°C was most effective for enrichment of the bacteria. Procedures affecting the efficiency of template DNA extraction and target DNA amplification were also optimized. By these combined efforts, a PCR procedure capable of detecting V. vulnificus as low as 10 cells/mL within 10h was developed.     

Detection and Enumeration of Vibrio vulnificus by Direct Colony Immunoblot

ABSTRACT:  A direct colony immunoblot method (DCI) for the enumeration of Vibrio vulnificus was developed. Bacterial colonies were transferred from agar plates to membranes, which were then dried and blocked with bovine serum albumin. Subsequently, the membranes were treated with anti- V. vulnificus H antibodies, washed and incubated with peroxidase-conjugated goat anti-rabbit IgG. After a final wash, the membranes were exposed to a substrate mixture containing H₂O₂ which resulted in the development of a purple color by V. vulnificus colonies. The DCI detected all clinical and environmental V. vulnificus strains tested and did not cross-react with other Vibrio species including V. cholerae , V. parahaemolyticus , or V. fluvialis . The DCI was then compared to the DNA hybridization procedure (DNAH) using V. vulnificus agar plates inoculated with mixed cultures of V. vulnificus and V. parahaemolyticus and V. vulnificus -seeded oyster homogenates. Both DCI and DNAH detected 1 to 2 log colony forming units (CFU)/mL V. vulnificus mixed with 4 log CFU/mL V. parahaemolyticus . Both methods were comparable and demonstrated no significant statistical differences when enumerating V. vulnificus in mixed cultures or in oyster homogenates seeded with levels of V. vulnificus from 2 to 6 log CFU/mL. The DCI demonstrated clearer color development and was less time consuming than the DNAH.