【作者】 姬华；

【导师】 刘秀梅；

【作者基本信息】 江南大学 ， 食品营养与安全， 2012， 博士

【摘要】 创伤弧菌（Vibrio vulnificus）和副溶血性弧菌（Vibrio parahaemolyticus）,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,是两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%。食源性副溶血性弧菌感染主要的临床症状是胃肠炎。国家食源性疾病监测网的数据显示,副溶血性弧菌是我国上报食源性疾病暴发事件的首要致病因素。本文建立肉汤和海产对虾中创伤弧菌和副溶血性弧菌的生长模型,研究低温条件下创伤弧菌和副溶血性弧菌的失活模型、超高压作用下副溶血性弧菌的致死模型。研究我国零售对虾中创伤弧菌的污染状况,对创伤弧菌分离株进行了毒力分型和耐药试验。利用创伤弧菌的预测模型和监测数据,建立高温季节对虾中创伤弧菌的定量风险评估模型。本文提出控制创伤弧菌和副溶血性弧菌的措施。首先通过单因素实验得到适宜创伤弧菌VvFJ04在胰蛋白胨大豆肉汤中生长的pH值、温度和盐度。运用响应面分析法优化VvFJ04的培养条件,获得创伤弧菌VvFJ04二级响应面生长模型:Y=0.42+0.00451x₁+0.03x₂+0.005671x₃+0.026x₁x₂+0.004875x₁x₃-0.002875x₂x₃+0.001078x₁²-0.051x₂²-0.013x₃²。VvFJ04的最优培养条件为pH值8.29、温度32℃和盐度2.38%。VvFJ04二级响应面生长模型适用于分析和预测不同试验条件下VvFJ04的生长情况。再次利用单因素实验,确定适宜副溶血性弧菌ATCC 17802生长的温度、盐度、pH值。在此基础上,应用响应面分析法研究不同温度（28.59℃～45.41℃）、盐度（1.32%～4.68%）、pH值（5.32～8.68）对副溶血性弧菌生长速率的影响。Baranyi一级生长模型拟合不同试验条件下副溶血性弧菌的生长曲线,利用响应面法构建副溶血性弧菌生长速率的预测模型,对构建的模型进行验证,并利用计算预测标准差（SEP）、平方根误差（RMSE）、准确性因子（A_f）和偏差因子（B_f）对响应面模型进行数学检验,验证指标均在合理范围内。为了进一步了解创伤弧菌VvFJ04、VvHB09、VvSH09和副溶血性弧菌ATCC 17802在2% NaCl胰蛋白胨大豆肉汤和南美白对虾中的生长特点,运用修正Gompertz方程、修正Logistic方程、Baranyi方程拟合15℃～40℃条件下创伤弧菌和副溶血性弧菌的一级生长模型。结果表明,三种方程均能很好地拟合4株弧菌的生长,决定系数（R²）值均大于0.91,并计算4株弧菌的生长速率、迟滞期和最大细菌浓度。在15℃条件下,副溶血性弧菌ATCC 17802和创伤弧菌VvFJ04均能在2% NaCl胰蛋白胨大豆肉汤中缓慢生长,而创伤弧菌VvHB09和VvSH09均不能在肉汤中生长;在15℃条件下,副溶血性弧菌ATCC 17802能在对虾中生长,而3株创伤弧菌均不能在对虾中生长。35℃～40℃条件下,两种弧菌的生长速率最大。结合修正Gompertz方程、Ratkowsky方程和Davey方程构建了不同温度条件下创伤弧菌和副溶血性弧菌的生长速率、迟滞期的二级模型。为了掌握对虾中创伤弧菌和副溶血性弧菌在5℃和–18℃低温贮藏条件下的失活动力学特征。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℃条件下,创伤弧菌VvHB09的耐冷力较强;–18℃条件下,副溶血性弧菌ATCC 17802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。为了研究超高压对副溶血性弧菌ATCC 17802的杀灭效果,利用Design Ex_pert软件,借助Box_-Behnken试验设计方法,考察压力、温度和保压时间协同超高压对副溶血性弧菌的杀菌作用,建立了副溶血性弧菌致死模型: Y = 4 .34+0.69x₁ +2.48x₂+0.97x₃+0.6x₁x₂+0.22x₁x₃+0.39x₂x₃- 0 .27x₁ 2 + 0.18x₂²+0.29x₃,通过模型计算超高压杀灭6个数量级副溶血性弧菌的工艺参数:温度为25.2℃,压力为230.3 MPa,保压时间为13.6 min。研究超高压处理对副溶血性弧菌超微结构的影响,利用透射电子显微镜观察,超高压处理后副溶血性弧菌细胞断裂、出现缺口,胞浆泄露。为了确定零售对虾中创伤弧菌的污染率和污染浓度,从中国10个省份采集零售对虾样品,结合最可能数（MPN）法和PCR技术检测创伤弧菌溶血素A基因。研究结果表明,239份对虾样品中有140份对虾检出创伤弧菌,创伤弧菌的几何平均浓度为153.3 MPN/g。不同地区零售对虾中创伤弧菌的污染状况存在统计学差异（P<0.05）,南方样品的污染浓度（179.6 MPN/g）明显高于北方样品的污染浓度（7.6 MPN/g）。128份检出创伤弧菌的对虾样品中,79份对虾样品携带临床株,8份携带环境株,7份对虾样品分离的创伤弧菌无法分型,34份对虾样品既携带临床株又携带环境株。耐药试验发现,对于169株创伤弧菌分离株,绝大多数菌株对测试的12种抗生素都敏感,部分菌株对阿米卡星、氨苄西林、庆大霉素和四环素的敏感性降低。研究结果表明,中国零售海产对虾中创伤弧菌的污染很普遍,大多数对虾样品携带临床型创伤弧菌,极有可能对人群健康构成威胁。微生物风险评估主要评估食品中的病原微生物可能对人群引起的潜在危害,以指导风险管理者制定相应的管理措施。本文开展对虾中创伤弧菌的定量风险评估。评估遵循广泛认可的风险评估程序,涉及危害识别、危害特征描述、暴露评估和风险特征描述。使用零售对虾中创伤弧菌的污染率和污染浓度数据、人群对虾的消费频率和消费量数据、预测微生物学模型,建立暴露评估模块。结合暴露评估模块的结果与贝塔-泊松剂量反应模型,推测消费者因食用对虾而发生创伤弧菌感染的概率为1.977×10^-5,并提出减少创伤弧菌危害的风险管理措施。本文的研究结果可以为海产品生产和消费过程中有效降低这两种弧菌的危害提供技术支持,为制定旨在预防和控制由这两种弧菌引起的食源性疾病的风险管理措施提供基础数据。更多还原

【Abstract】 Vibrio vulnificus and Vibrio parahaemolyticus are gram-negative halophilic bacteria that naturally occur in warm estuarine water and marine environment worldwide. They are recognized as the important cause of human foodborne disease associated with the consumption of contaminated seafood. The most common clinical presentation of foodborne V. vulnificus infection is primary septicemia, with an average mortality rate exceeding 50%. V. parahaemolyticus accounted for the largest number of outbreaks and cases in China, and the most common clinical presentation of V. parahaemolyticus infection is gastroenteritis. We also studied the growth kinetics model of V. vulnificus and V. parahaemolyticus in broth and shrimp. The survival rates of V. vulnificus and V. parahaemolyticus inoculated into shrimp samples were measured at low temperature, their survival curves were plotted respectively. The model of V. parahaemolyticus was inactivated by the Ultra High Pressure （UHP） was studied. In the present study, we determined not only the prevalence and levels of V. vulnificus in retail shrimps collected in warmer months, but also the virulence characteristics and antibiotic sensitivity of V. vulnificus isolated from the shrimps. Using data of V. vulnificus predictive model and surveillance, we carried out the quantitative risk assessment of V. vulnificus in shrimp in warmer season. This paper suggests measures to control V. vulnificus and V. parahaemolyticus.At first, the optimum pH, temperature and salinity for V. vulnificus VvFJ04 growth in tryptic soy broth （TSB） were determined by single-factor analysis respectively. The response surface model was applied to study optimization of the growth parameters for VvFJ04, Y=0.42+0.00451x₁+0.03x₂+0.005671x₃+0.026x₁x₂ +0.004875x₁x₃-0.002875x₂x₃+0.001078x₁²-0.051x₂²-0.013x₃². The results showed that the optimum conditions for germination as follows: pH was 8.29, temperature was 32°C and salinity was 2.38%. The experimental results indicated that the proposed model can be used for describing and predicting the growth feature of VvFJ04 at different environmental factors.The optimum temperature, salinity, and pH for V. parahaemolyticus ATCC 17802 growth were determined by single-factor analysis respectively. The effects of different temperatures （28.59°C–45.41°C）, concentrations of sodium chloride （NaCl, 1.32%–4.68%） and pH values （5.32–8.68） on the growth rates of V. parahaemolyticus were studied. The primary growth curves were fitted using the Baranyi equation. A response surface model （RSM） was prepared, predictions of V. parahaemolyticus growth rates could be obtained from growth curves. Mathematical evaluation demonstrated that the standard error of prediction （%SEP）, the root-mean-squares error （RMSE）, accuracy factor （Af） and bias factor （Bf） were within acceptable range.The future study was to understand the growth kinetics characteristics of V. vulnificus strains VvFJ04, VvHB09, VvSH09 and V. parahaemolyticus ATCC 17802 in 2% NaCl TSB and Pacific white shrimp. The primary growth models of V. vulnificus and V. parahaemolyticus were fitted well by the modified Gompertz equation, modified Logistic equation, Baranyi equation at from 15°C to 40°C. The determination coefficient R² values were greater than 0.91. The lag time, growth rate, and maximum population density of each primary model were compared. At 15°C, V. parahaemolyticus and V. vulnificus VvFJ04 grew very slowly in 2% NaCl TSB broth, VvHB09 and VvSH09 did not grow in TSB. Three V. vulnificus did not grow in shrimp at 15°C, only V. parahaemolyticus grew slowly in shrimp at 15°C. The growth rates of V. vulnificus and V. parahaemolyticus were maximum at 35°C and 40°C. The Gompertz equation, Ratkowsky equation and Davey equation were used to predict the secondary models of the growth rate and lag time for V. vulnificus and V. parahaemolyticus.In order to study the variation of inactive V. vulnificus and V. parahaemolyticus in shrimp under 5°C and–18°C. Then Linear, Weibull and Logistic models were applied to predict curves of inactive V. vulnificus and V. parahaemolyticus, respectively. At 5°C, VvHB09 showed more resistance to cool pressure. At–18°C, V. parahaemolyticus strain ATCC 17802 and VvSH09 showed more resistance to cold pressure. Moreover, Linear model was proved to be more accurate and useful in fitting V. vulnificus and V. parahaemolyticus inactivation curves based on mathematical evaluation than nonlinear models, Weibull and Logistic model.In order to study the effect of inactive V. parahaemolyticus ATCC 17802 by the Ultra High Pressure, recurring to Design Expert and the Box-Behnken experiment method to design and study the effect of the UHP cooperated with different temperature and time on the deadly ratio of V. parahaemolyticus, the response surface model of V. parahaemolyticus sterilized by the UHP was built, Y=4.34+0.69x₁+2.48x₂ +0.97x₃+0.6x₁x₂+0.22x₁x₃+0.39x₂x₃–0.27x₁²+0.18x₂²+0.29x₃². The optimum process parameters for inactivation of six logarithms V. parahaemolyticus were obtained as: temperature was 25.2°C, pressure was 230.3 MPa, and pressure holding time was 13.6 min. The effect of UHP on the microstructure of V. parahaemolyticus ATCC17802 was investigated by Transmission Electron Microscope （TEM）. The results showed that the cell wall of V. parahaemolyticus was fractured, breached and leaked out cytoplasm. To quantify the prevalence and the levels of V. vulnificus in shrimps, the shrimp samples were collected from ten cities in China. The most probable number （MPN） method was used combining with the polymerase chain reaction （PCR） which was used to classify the isolates by detecting V. vulnificus hemolysin gene （vvhA）. One hundred and forty out of 239 samples were positive for V. vulnificus, with the geometric mean level at 153.3 MPN/g. The prevalence and levels of V. vulnificus in shrimps varied in different geographic areas （P<0.05）, with the level values for the samples in the South cities （179.6 MPN/g） remarkably higher than that in the North cities （7.6 MPN/g）. In addition, we found that 79 of 128 V. vulnificus positive samples contained C-type （Clinical） strains and 8 contained E-type （Environmental） strains. Seven shrimp samples contained untypeable V. vulnificus and 34 samples had both C-type and E-type strains. The antimicrobial susceptibility of the isolates to 12 antibiotics was tested as well. Most of the 169 isolates remained susceptible to the majority of antimicrobials tested, whereas some strains tend to be less sensitive to amikacin, ampicillin, gentamicin and tetracycline. In conclusion, V. vulnificus is commonly found in retail marine shrimp in Chinese seafood market, and most of the shrimp samples contained C-type strains, which may pose a potential threat to human health.A microbiological risk assessment is to assess the potential hazard caused by pathogens in food which may affect public health, in order to guide risk managers to establish related measures. A quantitative microbial risk assessment of V. vulnificus in shrimp was undertaken. The assessment was conducted in accordance with the widely accepted procedures for risk assessment, which involoves hazard identification, hazard characterization, exposure assessment, and risk characterization. Using the data on the prevalence and levels of V. vulnificus in shrimp, the frequency and quantity of shrimp consumption and predictive model, exposure assessment model was established. The outcome of the exposure assessment model was combined with a beta-poisson dose-response model to estimate the probability of disease. Simulation results showed that the probability of V. vulnificus infection was 1.977×10^-5. It provided measure to reduce the risk of V. vulnificus.The results of this paper provide technical support to reduce the risk of two bacteria at seafood harvest and consumption period. The basic data of this research build the risk management to prevent and control foodborne illness causing by V. vulnificus and V. parahaemolyticus.更多还原