【作者】 汪雯；

【导师】 李延斌；

【作者基本信息】 浙江大学 ， 生物系统工程， 2013， 博士

【摘要】 副溶血性弧菌(Vibrio parahaemolyticus, VP)是革兰氏阴性菌,兼性厌氧,嗜盐,为多形态杆菌或稍弯曲弧菌,可引起肠胃炎,主要分布于海水及海产品中,是造成沿海地区海产品食源性疾病爆发的主要病原菌之一。目前,国内外学者已针对海产品中副溶血弧菌的杀菌技术开展了广泛研究,根据加工条件,海产品种类及各地区消费者饮食习惯等因素,各国使用的海产品杀菌方法有所差异,该领域的总体研究趋势为在保证杀菌效果的同时兼顾海产品风味、色泽、口感及营养成分。传统的热杀菌方法可在较短时间内取得良好的杀菌效果,但极易对海产品感官品质造成破坏。因此,近年来化学杀菌方法及低温杀菌法在海产品加工中较受欢迎。微生物定量风险评估(quantitative microbial risk assessment, QMRA)可通过数学模型对食品生产、加工、运输、零售和消费环节中的微生物污染水平进行定量估计,预测病原菌引发的疾病风险。通过风险评估的手段可确定食品生产至消费过程中重要的风险因子,并定量的评价各种风险控制措施。本课题综合利用农业与食品科学、微生物学、预测微生物学、化学、统计学、工程学等多领域知识,以虾仁中副溶血弧菌为研究对象,以可行性及消费者可接受为前提,研究三种可能应用于虾仁加工中清洗环节的灭菌方法,并以微生物预测模型为工具,评价各杀菌因素对杀菌效果的影响。同时,本研究对冷冻虾仁自原料捕获至消费环节的副溶血弧菌污染建立定量微生物风险评估模型,利用文献回顾、专家意见及合理假设的方法对模型中参数进行分布拟合,预测虾仁中副溶血弧菌污染引发疾病的风险概率,并以此为手段,定量的评价虾仁清洗环节中三种杀菌方法对风险控制的效果。本课题主要内容及结论如下：1、基于Box-Behnken实验设计,利用乳酸(1%,2%,3%,v/v)及壳聚糖(0.4%,1%,1.6%,w/v)的混合溶液对生鲜虾仁进行振荡(90,110,130rpm)清洗(10,20,30分钟),对杀菌结果进行数学建模。结果表明乳酸与壳聚糖的混合液清洗可有效减少虾仁中的副溶血弧菌(本实验中取得的细菌死亡量为2.17～4.32log CFU/g),且未对虾仁感官品质造成不利影响。基于响应曲面设计建立的预测模型回归系数(R2)为0.92,标准误差平方根(RMSE)为0.196,失拟检验结果不显著(p>0.05),此外,验证实验结果显示模型准确度(Bf)及精确度(Af)分别为1.01和1.05,表明该模型可用于清洗中细菌死亡量的预测。杀菌因素中乳酸、壳聚糖及二者的交互作用对灭菌效果影响显著(p<0.05),其中乳酸起主导作用。模型预测结果表明在最优因素组合条件下,即利用3%乳酸结合1.6%壳聚糖对虾仁进行振荡转速为110rpm的清洗10分钟,可减少副溶血弧菌4.5log CFU/g。2、本实验在温热的水浴(47,50,53℃)中结合超声波(96,150,204W)对人工接种了副溶血弧菌的生鲜虾仁进行清洗,对灭菌效果进行数学建模。利用Weibull模型作为一级模型拟合细菌致死量与时间的关系曲线,基于最小二乘法回归细菌死亡量数据,固定模型中形状因子(shape parameter)以降低参数间自相关性,然后以Weibull中尺度因子为响应量建立响应曲面方程(二级模型),研究其与温度及超声波功率间的定量关系。实验发现温热的水浴及超声波分别对虾仁进行清洗时副溶血弧菌死亡量十分有限,二者联合使用可显著提高杀菌效果,在温水中进行超声波清洗可在短时间内减少副溶血弧菌超过4log CFU/g。模型拟合结果发现固定曲线形状因子可以减小一级模型参数间的自相关性,并且不会对模型的回归系数(R2)造成太大影响。固定形状因子后,Weibull模型中的尺度因子(scale parameter)与灭菌温度和超声波功率间的关系可由响应曲面模型拟合,回归系数(R2)高达0.99,标准误差平方根(RMSE)为0.17。验证实验显示模型的准确度因子(Bf)及精确度因子(Af)分别为0.97和1.03,表明该二级模型可较准确的拟合一级模型参数。一级模型与二级模型结合可得到虾仁中副溶血弧菌死亡量与加热温度、超声波功率及清洗时间三因素间关系的预测方程。模型分析结果显示温度、超声波功率对细菌致死作用影响显著,且二者间交互作用显著。研究表明热超声杀菌技术有望应用于虾捕获后的清洗环节,有效减少副溶血弧菌的污染,本研究中建立的预测模型可用于虾仁清洗过程中副溶血弧菌致死量的预测。3、采用Box-Behnken实验设计方法,应用弱酸性电解水与乳酸的混合液对虾仁进行清洗,调查清洗液对虾仁中副溶血弧菌的杀灭效果,并以细菌死亡量为响应量建立预测模型。结果表明弱酸性电解水单独使用对虾仁中副溶血弧菌的杀灭作用十分有限,与适当浓度的乳酸结合,可以在保证虾仁感官品质的同时提高杀菌效率。FDA要求海产品加工企业应用的杀菌技术应至少杀灭副溶血弧菌3.52个对数单位,在本研究中发现,pH值为5.18,有效氯浓度为73.0mg/L,氧化还原电位为1018.4mV的弱酸性电解水与3%(v/v)的乳酸以130rpm的转速对虾仁清洗10分钟,副溶血弧菌死亡量达到4.36log CFU/g,符合FDA标准。响应曲面方程可用于细菌致死量与电解水比例、乳酸浓度及清洗转速等杀菌因子的数学拟合,预测模型的回归系数(R2)为0.98,均方根误差为0.104,失拟检验结果不显著(p>0.05),验证实验结果显示该模型的准确度因子(Bf)及精确度因子(Af)分别为1.01和1.09,表明该预测模型可用于此杀菌方法的杀菌效果预测。4、本研究对副溶血弧菌对人类健康的危害进行识别,并为虾自捕获至消费环节建立暴露评估模型,结合FDA颁布的剂量效应模型及校正因子,对冷冻虾仁引发的副溶血弧菌中毒风险进行预测。在大量文献调查、专家意见及科学假设的基础上,本研究以随机分布函数作为风险评估模型的输入参数,以蒙特卡洛分析方法进行模型拟合。预测结果显示冷冻虾仁中副溶血弧菌污染引发疾病的风险概率平均值为2.5×10-9/人/餐,我国因冷冻虾仁中副溶血弧菌污染造成的年致病人数约为344.7人。敏感性分析结果表明暴露途径中重要的风险因子依次为捕获期温度、家庭贮藏温度和时间、初始污染浓度、捕获期未冷藏时间及未充分烹饪概率。烹饪为最有效的风险缓解因子,充分加热对减小风险具有重要意义。此外,情景分析结果表明本研究中采用的乳酸结合壳聚糖清洗、超声波结合温热处理及弱酸性电解水结合乳酸清洗方法可使年发病率减小至基线模型的2.5%,24.0%及2.6%,由此可见,虾仁加工中采取适当的清洗方法可有效缓解食源性疾病的发生风险。更多还原

【Abstract】 Vibrio parahaemolyticus is a natural inhabitant in estuarine marine water and seafood has been recognized as the most frequent vehicle for this pathogenic bacterium. Along with the increase of supply and consumption of seafood worldwide, the illnesses outbreaks associated with V. parahaemolyticus have aroused concerns over the public health. Various intervention methods for reducing the bacteria in seafood products have been investigated and practiced. Each method has distinct advantages and disadvantages depending on the properties of products, the conditions of processing, and the preference of consumers. The successful application of appropriate intervention strategies could effectively reduce the contamination of V. parahaemolyticus in seafood, and consequently contribute to the reduction of public health risk. The principal aim of quantitative microbial risk assessment (QMRA) is to quantify the level of risk in order to prevent unacceptable exposures and to design improved production, processing, distribution, and preparation systems that would help in reducing exposures to acceptable limits. The risk assessment process helps to identify any of those stages from production to consumption that contribute to an increased risk of foodborne illnesses. QMRA has been recognized as a predictive and decision-making tool in organizing available data, and assessing the impact resulting from exposure to different pathogens.The main contents and resultsof this study are summarized as follows.(1) Lactic acid and chitosan are natural antimicrobials for food decontamination in the washing process of seafood. In this research, a4-factor response surface model based on the Box-Behnken experimental design was developed to evaluate the effects of lactic acid (1%,2%, and3%, v/v), chitosan (0.4%,1%, and1.6%, w/v), rotational rate (90,110, and130rpm) and washing time (10,20, and30min) on reduction of V. parahaemolyticus inoculated in raw peeled shrimps. These treatments achieved2.2to4.3log10CFU/g reduction of V. parahaemolyticus in shrimps. Stepwise stratification led to a simplified model that has satisfactory performance as evidenced by statistical indices (R2=0.92; p<0.0001; RMSE=0.196) and external validation parameters [bias factor (Bf)=1.01; accuracy factor (Af)=1.05]. The model generated an optimum treatment combination (3%lactic acid,1.6%chitosan, and rotational rate at110rpm) that could achieve greatest bacterial reduction of4.5log10CFU/g. Among the four factors, lactic acid and chitosan were the major contributors for bacterial decontamination. Analysis of variances showed a significant interactive inactivation effect (p<0.05) from combined use of lactic acid and chitosan. The treatments did not have adverse effects on the quality attributes such as color and pH of the shrimps.(2) The effect of thermo-ultrsound treatment on the survival of V. parahaemolyticus in raw peeled shrimps was investigated in this study as an alternative inactivation method in seafood postharvest washing process. Raw peeled shrimps inoculated with V. parahaemolyticus were treated with mild heat (47,50, and53℃) combined with ultrasound (0.96,150, and204W) based on a3×4full factorial design and the bacterial survival curves were fitted with a Weibull model. Due to the high correlations of the shape parameter "n" and the scale parameter "n" in the Weibull model, an overall n-value was estimated from the whole set of bacterial inactivation data and n-values were estimated for each set of inactivation curve accordingly. A response surface model was generated to describe the scale parameter as a function of temperature and ultrasonic power. The results indicated that the Weibull model with the overall n-value could be used to describe the bacterial reduction with the time of exposure to the thermo-ultrasound treatments, which is well evidenced by the small values of root mean square errors (RMSE) and the high values of correlation of determination (R2). The quadratic model was validated with independent experiments within the prediction range. Statistical indices (R2=0.99; p<0.0001; RMSE=0.17) and validation parameters (Bf=0.97; Af-1.03) showed a satisfied performance of the quadratic model. The results indicated that the thermo-ultrasound treatment was effective, simple, and cost effective for inactivation of V. parahaemolyticus in shrimps during postharvest washing process.(3) Weakly acidic electrolyzed water (AEW) was used in combined with lactic aicd to reduce V. parahaemolyticus in raw peeled shrimps. A3-factor response surface model based on the Box-Behnken experimental design was developed to evaluate the antimicrobial effects of AEW (1/3,2/3and3/3, ratio, v/v), lactic acid (1%,2%, and3%, v/v), and rotational rate (90,110, and130rpm). The investigation results showed that the treatments achieved1.36～4.02log10CFU/g reduction of V. parahaemolyticus in shrimps. The developed model proved a satisfied prediction performance as evidenced by statistical indices (R2=0.99; p<0.0001; RMSEO.104) and external validation parameters (Bf=1.01; Af=1.09). The model generated an optimum treatment combination (100%AEW,3%chitosan, and rotational rate at130rpm) that could achieve greatest bacterial reduction of4.36log10CFU/g, which met the guideline value (3.52log10CFU/g) suggested by FDA for industrial invention method to remove Ⅴ. parahaemolyticus from seafood. Among the four factors, lactic acid and AEW were the major contributors for bacterial decontamination. Analysis of variances showed a significant interactive inactivation effect (p<0.05) from combined use of AEW and lactic acid.(4) In this study, a quantitative risk assessment model comprising initial bacterial contamination, post-harvest washing, transport to market, retail storage, transport to household, home storage and consumption was developed to evaluate the relative effectiveness of different post-harvest washing treatments on reducing the risk reduction by Ⅴ. parahaemolyticus in frozen peeled shrmps. Three treatments were lactic acid combined with chitosan (LA+CH), thermo-ultrasound treatment (TH+UL), and weak acid electrolyzed water combined with lactic acid (AEW+LA). Data obtained from laboratory experiment, scientific literatures, professional suggestion and assumption were used to parameterize the input variables and the growth/survival kinetics of Ⅴ. parahaemolyticus at different processing stages. The model was run using Monte Carlo simulation with100,000iterations. The simulated results showed that the risk of illness was2.5×10-9/year/person the number of illnesses per year should be344.7in China. The result of our study is reasonable as compared with the epidemiological data and results of previous risk assessment reports. Sensitivity analysis identified that the temperature and time of harvest were the most significant risk factors. Scenario analysis showed that LA+CH, TH+UL AEW+LA treatments reduced the risk of illness to2.5%,24.0%and2.6%as compared with the baseline model, respectively, suggesting the intervention method could potentially reduce the risk of Ⅴ. parahaemolyticus in frozen peeled raw shrimps. The risk model can assist seafood industry in adopting appropriate risk mitigation strategies to reduce seafood-borne illness caused by Ⅴ. parahaemolyticus.更多还原