食品副溶血性弧菌检验方法的合作研究

比较食品副溶血性弧菌检验国家标准GB/T4789.7-2003和修订法的检测效果。采用两种方法对3类食品(牛肉馅、冻鳕鱼和牡蛎肉)检测3个染菌水平,染菌试样共213份。结果显示,国标法和修订法对染菌试样的检测阳性数分别为47和83。对于3类食品的3个染菌水平,统计学检验结果提示,修订法的检测效果等同或优于国标法。     

抗副溶血弧菌IgY的制备及活性检测

副溶血性弧菌(Vibrio parahaemololyticus,Vp)是我国微生物食源性疾病的主要病因,且是海水养殖业中的常见病原菌,造成巨大的经济损失。本研究制备副溶血弧菌免疫原,并以5×107、5×108CFU/ml两种剂量分别免疫母鸡,获得特异性的抗副溶血弧菌IgY;并建立ELISA方法检测免疫鸡蛋中的抗体效价,高剂量免疫组的抗体效价可达1:57600,低剂量免疫组的抗体效价达1:28800;低剂量组抗体效价可维持250d,高剂量组抗体效价可维持300d以上。     

乙醇促进副溶血性弧菌直接耐热溶血素的基因表达

研究乙醇对副溶血性弧菌(Vibrio parahaemolyticus)直接耐热溶血素(thermostable direct hemolysin,TDH)产生和它的编码基因tdh表达的影响。以0.5%、1.0%、2.0%、4.0%、8.0%乙醇处理2株带有tdh副溶血性弧菌ATCC33847和SZ32,研究对副溶血性弧菌有氧或者无氧生长的影响。选取1.0%乙醇处理来研究TDH产量和tdh表达变化。使用TDH抗血清试剂盒测定副溶血性弧菌培养液上清中TDH水平;使用荧光定量PCR方法分析tdh基因表达状况。低浓度(0.5%、1.0%、2.0%)乙醇存在时,副溶血弧菌菌株的生长未受到显著影响,乙醇浓度4.0%时副溶血性弧菌生长受到明显抑制,8%时未见有细菌生长。1.0%乙醇处理副溶血性弧菌培养液上清中TDH水平较未处理显著上升。胞内tdh表达水平升高,ATCC33847在有氧和无氧时分别升高到6.6倍和5.7倍,SZ32在有氧和无氧时分别升高到5.9倍和8.6倍。乙醇能够促进tdh基因表达从而使得TDH蛋白产量升高。本研究还比较了甲醇、乙醇、正丙醇对tdh表达的影响,发现它们对tdh表达均具有促进作用但相互之间没有明显差异。     

丁酸钠对副溶血性弧菌毒性的影响及机制研究

目的 探究丁酸钠对副溶血性弧菌毒性的影响及机制。方法 通过测定丁酸钠对副溶血性弧菌生物膜形成能力及黏附宿主细胞能力, 评估其对副溶血性弧菌毒性的影响; 此外基于傅里叶变换红外光谱法和拉曼光谱法检测胞外成分变化;利用RT-qPCR探究毒力基因转录水平变化。结果 亚致死浓度的丁酸钠基本不影响副溶血性弧菌生长,但对生物膜形成有显著地抑制作用; 丁酸钠处理后,副溶血性弧菌对Caco-2细胞的黏附率分别降低28%、41%和61%; 傅里叶变换红外光谱(FT-IR)和拉曼光谱表明丁酸钠能影响副溶血性弧菌脂肪酸及蛋白质疏水性以及降低核酸、碳水化合物和蛋白质的峰面积; RT-qPCR分析显示丁酸钠可显著下调与副溶血性弧菌毒力相关基因的转录表达。结论 丁酸钠具有一定的抑制副溶血性弧菌毒性的能力, 有潜力用于预防及控制副溶血性弧菌造成的食源性疾病。     

两种弧菌显色培养基在疑似副溶血性弧菌鉴定中的效果比较

为了比较环凯和科玛嘉弧菌显色培养基对食品中可疑菌株的初步鉴定作用,本文按国标(GB/T4789.7-2008)程序,以环凯和科玛嘉弧菌显色培养基初步鉴定硫代硫酸盐-柠檬酸盐-胆盐-蔗糖琼脂(TCBS)的副溶血性弧菌可疑菌株,最后用API和MID鉴定证实。在97份食品样品的定性与定量检测中,TCBS疑似菌株有109株,对应样品有31份。环凯和科玛嘉弧菌显色培养基对109株TCBS可疑菌株初步鉴定的结果相同,均检出64株显紫色的可疑菌株,对应样品22份。经API和MID确认,阳性菌株60株,阳性样品为22份,阳性率为22.68%。两种培养基初步鉴定TCBS可疑菌株的敏感性、特异性和符合性均分别为100.00%、91.84%和96.33%。环凯和科玛嘉弧菌显色培养基对TCBS的可疑菌株具有较好的初步鉴定效果。     

副溶血弧菌TDH的原核表达及单克隆抗体的制备

为实现副溶血性弧菌tdh基因的原核表达,采用TCBS选择培养基从贝类中筛选副溶血性弧菌疑似菌株;根据Gen Bank上已有的tdh基因序列,设计并人工合成引物,通过PCR技术鉴定副溶血性弧菌并扩增tdh基因;酶切后定向插入到p ET-28a表达载体中,构建重组表达质粒p ET-28a-tdh,转入E.coli Rosetta中,在IPTG诱导下进行TDH蛋白表达。为制备耐热直接溶血毒素TDH单克隆抗体,用纯化的蛋白作为免疫原免疫BALB/c小鼠,成功用原核载体表达的TDH蛋白为免疫原,制得一株能稳定分泌单克隆抗体的杂交瘤细胞,命名为T9N10。获取腹水并经Ni-NTA Resin亲和柱纯化,其稳定分泌的单克隆抗体经鉴定为Ig G1,相对分子质量约为146 000,并表现出较强的特异性。本研究为开发副溶血弧菌免疫学快速检测和深入的研究奠定良好的物质基础。     

4种防腐剂对副溶血弧菌生物膜形成的抑制作用

在确定4种防腐剂壳聚糖、山梨酸钾、脱氢乙酸钠和ε-聚赖氨酸对副溶血弧菌(Vibro parahaemolyticus)的最小抑菌浓度(minimum inhibitory concentration,MIC)的基础上,研究其对副溶血弧菌生物膜的抑制作用。采用结晶紫染色法测生物膜形成量,XTT法测生物膜代谢活性,硫酸-苯酚法测定生物膜中胞外多糖的分泌量。结果表明,壳聚糖的MIC最小,为1.25 mg/m L。4种防腐剂在MIC以及亚抑菌浓度条件下对副溶血弧菌生物膜均有明显的抑制作用,不仅抑制生物膜的形成,而且能显著降低细菌的代谢活性,减少胞外多糖的分泌,其中壳聚糖对副溶血弧菌生物膜的抑制作用最强。     

柠檬草精油对副溶血性弧菌的抑菌活性及机制

目的:探究柠檬草精油(LG-EO)对副溶血性弧菌的抑菌活性和机理。方法:通过测定LG-EO对副溶血性弧菌的最低抑制浓度(MIC)、时间-杀灭分析、上清液中AKP活性、电导率和蛋白质泄漏量、细胞形态、膜电位、呼吸链脱氢酶活力及胞内DNA的含量和结构等,研究LG-EO对副溶血性弧菌的抑菌活性和机制。结果:LG-EO对副溶血性弧菌的MIC范围80~120 μg/mL,在80~280 μg/mL处理浓度范围,其杀灭效果随浓度和时间的增加而提高,240 μg/mL LG-EO处理10 min即可灭活全部副溶血弧菌。与高于MIC的LG-EO共培养,副溶血弧菌细胞壁、膜的完整性被破坏,导致上清液中AKP活性、蛋白质含量和电导率上升,细胞边界模糊和内容物流失。菌体细胞膜电位、呼吸链脱氢酶和ATP酶活性等显著降低。基因组DNA的核酸电泳和紫外吸收光谱提示,DNA在LG-EO作用下流失严重并与主要成分柠檬醛发生互作。结论:柠檬草精油对副溶血性弧菌有较强的抑菌作用,LG-EO通过破坏细胞壁、膜构造,干扰细胞能量代谢,造成DNA流失或变性等快速杀灭副溶血性弧菌。本研究为探明LG-EO的抑制致病性弧菌机理奠定良好的基础,在水产品加工业中具有广泛的应用潜力。     

肉骨粉中沙门氏菌及副溶血性弧菌的分离与鉴定

采用传统生化鉴定及血清学方法,联合半自动细菌鉴定仪及噬菌体方法,从FEPAS测试样品肉骨粉中成功分离鉴定出沙门氏菌和副溶血性弧菌,并结合食品微生物学检验实践,对分离鉴定过程进行了总结分析。     

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。     

食品中副溶血性弧菌的危害分析、检测与预防控制

副溶血性弧菌是一种广泛分布于近岸海水、海底沉积物和海产品中的嗜盐性细菌，也是引起我国特别是沿海地区细菌性食物中毒危害的首要食源性致病菌。为了提高人们对副溶血性弧菌的重视程度，降低食源性疾病的风险，本文就副溶血性弧菌的生物特性、毒力因子、污染途径、食物中毒特点、检测方法以及预防措施作简要综述。     

2008—2010年上海市夏秋季市售海产品中副溶血性弧菌污染监测结果分析

目的 了解上海市夏秋季节市售海产品中副溶血性弧菌的污染水平和特征.方法 2008-2010年5-10月,采用GB/T 4789.7-2008《食品卫生微生物学检验副溶血性孤菌检验》方法,对上海市批发市场、集贸市场、卖场超市和餐饮单位等污染物监测点的市售海产品进行副溶血性弧菌的定性和定量检测.结果 共监测市售海产品941件,副溶血性弧菌总体检出率为13.2％,不同种类、不同监测月份和不同采样地点的海产品,其副溶血性弧菌检出率和样品几何平均浓度总体上差异有统计学意义(P＜0.05).其中,海产虾类副溶血性弧菌检出率(25.0％)和样品几何平均浓度(5.0 MPN/g)显著高于其他类海产品(P＜0.05);8月份海产品副溶血性弧菌检出率(27.4％)和样品几何平均浓度(3.3 MPN/g)显著高于其他监测月份(P＜0.05);集贸市场副溶血性弧菌检出率(28.5％)和批发市场样品几何平均浓度(3.9 MPN/g)显著高于其他采样地点(P＜0.05).结论 上海市市售海产品中副溶血性孤菌的污聚率较高,应进一步开展海产品中副溶血性弧菌的风险监测和评估,并针对副溶血性弧菌污染的高风险环节开展监管.     

副溶血性弧菌整合接合元件核心基因表达对温度变化的响应

采用实时荧光定量聚合酶链式反应方法分析在15～ 4 2 ℃ 温度范围内副溶血性弧菌（Vi b r i oparahaemolyticus）CHN25整合接合元件（integrative and conjugative elements,ICEs）ICEVpaChn1核心基因表达对温度变化的响应。结果表明：温度介导ICEVpaChn1元件保守模块核心基因表达发生显著变化,其中,编码进入排斥蛋白Eex（entry exclusion）的基因对温度变化最为敏感,低于或高于37 ℃的温度条件均强烈抑制eex基因表达（＞10 倍）。此外,在15～37 ℃范围内,温度的升高显著激活编码整合酶Int（integrase）、接合转移蛋白TraI（transfer protein I）和TraG、DNA修复蛋白RumA基因的表达,且在37 ℃达到最大值;与其他检测基因明显不同,温度升高抑制转录抑制子SetR基因的表达,促进int等基因转录激活子SetC和SetD的积累,进而刺激切离,促进ICEVpaChn1元件的接合转移。实验结果揭示了环境温度对ICEs元件核心基因表达的影响,发现低温（＜15 ℃）和高温（＞37 ℃）条件均可能阻遏ICEVpaChn1元件及其携带基因信息在不同种属细菌间的接合转移。     

Incidence and Enumeration of Vibrio parahaemolyticus in Shellfish from Two Retail Sources and the Genetic Diversity of Isolates as Determined by RAPD-PCR Analysis

A total of 83 shellfish samples from two local retail sources (A and B) yielded 38 samples positive for the presence of Vibrio parahaemolyticus based on 3 tube MPN enrichments and isolation from thiosulfate-citrate-bile salts-sucrose agar (TCBS) and biochemical tests. The 38 positive samples yielded 133 biochemically presumptive isolates of V. parahaemolyticus. Among these 133 presumptive isolates, 104 were confirmed by the polymerase chain reaction (PCR), which yielded more reliable identification results than the biochemical tests. The 38 biochemically presumptive samples yielded 29 samples that were confirmed by PCR to be positive for the presence of V. parahaemolyticus. RAPD analysis with three random primers was performed to examine the genetic diversity of 64 strains among the PCR confirmed V. parahaemolyticus isolates from both retail sources. 52 of 56 composite RAPD types consisted of single strains, indicating that most of the V. parahaemolyticus isolates were genetically quite heterogeneous. No strains representing the same RAPD type occurred in both retail outlets, implying that contamination of the shellfish by V. parahaemolyticus from the 2 retail sources was from different environmental locals and shellfish harvesting areas. Eight genomic clusters were generated at the 25% similarity level in a dendrogram based on RAPD profiles. With few exception, isolates with close genetic relationships grouping into an individual cluster tended to be derived from the same retail source.     

Incidence and Enumeration of Vibrio parahaemolyticus in Shellfish from Two Retail Sources and the Genetic Diversity of Isolates as Determined by RAPD-PCR Analysis

A total of 83 shellfish samples from two local retail sources (A and B) yielded 38 samples positive for the presence of Vibrio parahaemolyticus based on 3 tube MPN enrichments and isolation from thiosulfate-citrate-bile salts-sucrose agar (TCBS) and biochemical tests. The 38 positive samples yielded 133 biochemically presumptive isolates of V. parahaemolyticus. Among these 133 presumptive isolates, 104 were confirmed by the polymerase chain reaction (PCR), which yielded more reliable identification results than the biochemical tests. The 38 biochemically presumptive samples yielded 29 samples that were confirmed by PCR to be positive for the presence of V. parahaemolyticus. RAPD analysis with three random primers was performed to examine the genetic diversity of 64 strains among the PCR confirmed V. parahaemolyticus isolates from both retail sources. 52 of 56 composite RAPD types consisted of single strains, indicating that most of the V. parahaemolyticus isolates were genetically quite heterogeneous. No strains representing the same RAPD type occurred in both retail outlets, implying that contamination of the shellfish by V. parahaemolyticus from the 2 retail sources was from different environmental locals and shellfish harvesting areas. Eight genomic clusters were generated at the 25% similarity level in a dendrogram based on RAPD profiles. With few exception, isolates with close genetic relationships grouping into an individual cluster tended to be derived from the same retail source.     

A Critical Review about the Health Risk Assessment of PAHs and Their Metabolites in Foods

Polycyclic aromatic hydrocarbons (PAHs) are a family of toxicants that are ubiquitous in the environment. These contaminants generate considerable interest, because some of them are highly carcinogenic in laboratory animals and have been implicated in breast, lung, and colon cancers in humans. Dietary intake of PAHs constitutes a major source of exposure in humans. Factors affecting the accumulation of PAHs in the diet, their absorption following ingestion, and strategies to assess risk from exposure to these hydrocarbons following ingestion have received very little attention. This review, therefore, focuses on concentrations of PAHs in widely consumed dietary ingredients along with gastrointestinal absorption rates in humans. Metabolism and bioavailability of PAHs in animal models and the processes, which influence the disposition of these chemicals, are discussed. Finally, based on intake, disposition, and tumorigenesis data, the exposure risk to PAHs from diet is presented. This information is expected to provide a framework for refinements in risk assessment of PAHs.