基于预测模型的冷却猪肉中气单胞菌风险评估研究

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摘要

本课题以冷却猪肉为对象，对冷却猪肉中优势有害菌进行分离鉴定，并对冷

却猪肉中菌落总数和优势有害菌的生长情况进行研究，将分离得到的优势有害菌

分组接种于冷却猪肉中，对冷却猪肉的腐败情况进行感官评定；将气单胞菌接种

至表面灭菌的冷却猪肉上，研究不同培养温度和不同实验条件下气单胞菌在猪肉

中的生长规律，并根据所得数据拟合一级和二级预测微生物学模型；对上海市冷

却猪肉中气单胞菌的初始污染量进行检测，以冷却猪肉开始销售为起点，消费者

最终食用为终点，根据所得模型，估计最终食用时冷却猪肉中气单胞菌的含量，

评估消费者因食用冷却猪肉而发生气单胞菌食物中毒的风险。具体研究结果如下：

第一，通过对不同的选择性培养基上典型菌落进行初步分离鉴定，得出假单

胞菌、肠杆菌、热杀索丝菌、气单胞菌是冷却猪肉的优势有害菌。从冷却猪肉在 4℃

冷藏温度下菌落总数与各类细菌的生长可知，菌落总数和优势有害菌在第 5d 后开

始显著增长，其中肠杆菌增长最快，热杀索丝菌相对最慢。由感官评定结果可知，

随着肉中微生物繁殖，冷却猪肉在颜色、质地和气味上都产生了显著变化，气单

胞菌无法单独对冷却猪肉产生致腐能力，需要与其他菌协同作用。

第二，将气单胞菌接种到经过 80℃无菌水灭菌的冷却猪肉中，构建 Gompertz

和Baranyi 一级模型来描述气单胞菌在不同温度下的生长状况，结果表明，修正的

Gompertz 模型对气单胞菌生长曲线的拟合效果优于 Baranyi 模型，R2均在 0.97 以

上。应用平方根模型和 Arrhenius 模型对由修正的 Gompertz 模型得出的最大比生

长速率进行拟合，所得平方根模型拟合效果略优于 Arrhenius 模型。用平方根模型

对由修正的 Gompertz 模型得出的迟滞期进行拟合，结果表明所建二级模型能真实

快速有效地预测 0~35℃贮藏条件下猪肉中气单胞菌的生长状况。

第三，采用响应曲面模型(RSM)研究温度、pH 值、初始菌浓度对猪肉中气单

胞菌生长的影响。应用 Gompertz 模型对不同试验条件下气单胞菌的生长曲线进行

拟合，生长曲线的参数生长率(U)和迟滞期(LPD)采用 RSM 方法建立冷却猪肉中气

单胞菌的生长预测模型。然后随机选择试验组合对建立的方程进行验证,并应用计

算均方误差（MSE）、准确因子(AF)和偏差因子(BF)的方法对建立的生长预测方程

进行数学检验。结果表明，温度、pH 值和初始菌浓度对气单胞菌生长影响显著,

用RSM 方法建立的生长预测模型可以较好的模拟猪肉中气单胞菌在不同试验条件

下的生长情况。

第四、运用所得预测模型，并结合调查数据，采用Monte Carlo分析模拟了气

单胞菌从加工后的销售、运输以及储藏过程中会产生的数量变化和食用后可能造

成危害的概率分布。对模型进行敏感性分析结果显示，初始污染水平是影响气单

胞菌危害性的最重要因素，其次家庭储藏时间和储藏温度也是影响气单胞菌致病

风险的重要因素。因此要建议生产者要加强生产过程中的卫生管理，并且建议消

费者缩短储藏时间，降低储藏温度。

关键词：气单胞菌冷却猪肉预测微生物学模型风险评估

Risk Assessment of Aeromonas spp.in Chilled Pork Based

on Predictive Model

ABSTRACT

Harmful bacteria in chilled pork were separated and identified in this research, and

the variations of total aerobic and specific spoilage bacteria of chilled pork during cold

storage were also studied in this paper. Different combinations of the specific spoilage

bacteria were inoculated into the chilled pork to detect the perishable condition by sense

evaluation. The strain of Aeromonas spp. was inoculated into chilled pork, and then

fitted by primary models and secondary models to predict the growth of Aeromonas spp.

at different storage temperatures and different experimental conditions. Aeromonas spp.

Contamination situation of commercial chilled pork has been carried out in Shanghai.

Meanwhile, growth model was used to predict Aeromonas spp. growth under storage

and transportation. The risk assessment report was obtained for infection of Aeromonas

spp. in chilled pork by human consumption. The results were showed as follows:

Firstly, this study indicated that Pseudomonas spp.,Enterobacteriaceae spp.,

Brochothrix thermosphacta, and Aeromonas spp. were initial harmful bacteria of chilled

pork through analysis of harmful bacteria of chilled pork. From the analysis of total

plate count and the growth of harmful bacteria, it showed that specific spoilage bacteria

had been increasing significantly after the 5 day cold storage. Enterobacteriaceae spp.

grew faster than others. Furthermore, Brochothrix thermosphacta grew slowest. The

results of sensory evaluation showed that the color, aroma, and texture of the chilled

pork had significant changed. The strain of Aeromonas spp. might be not the only

comtamination factor of chilled pork, and it must coordinate with other specific spoilage

bacteria.

Secondly, chilled pork was inoculated with Aeromonas spp. after disinfected by

80℃sterile water, and primary models (Gompertz and Baranyi) were developed in

order to find the best-fitting model to describe the growth kinetic of Aeromonas spp..

By a comparison, the fitting effect of modified Gompertz model on growth curve of

Aeromonas spp. was better than that of Baranyi model, with all the value of R2beyond

0.97. The maximum specific growth rate that obtained from modified Gompertz model

was fitted by square-root model and Arrhenius model, respectively, and the fitting effect

of square-root model was better than that of Arrhenius model. The lag phase that

obtained from modified Gompertz model was fitted by Square-root model.The results

indicated that the secondary model could be a useful tool for predicting growth of

Aeromonas spp. in chilled pork during 0 to 35 ℃really and rapidly.

Thirdly, effects of temperature, pH value and inoculation volume on the

parameters of Aeromonas spp. from chilled pork under laboratory conditions were

investigated in this paper. The curves generated within different conditions were fitted

using Gompetz function. Then two parameters (growth rate and lag-time) of the growth

curves were modeled using a quadratic polynomial equation of response surface model

(RSM). Additional combined conditions were randomly selected for model validation,

and mathematical testing were applied for the developed models, including the mean

square error (MSE), accuracy factor (AF) and bias factor (BF). It indicated that the

temperature, pH and inoculation volume on the growth of Aeromonas spp. were

significant, and RSM provided a useful and accurate method of predicting the growth

parameters of Aeromonas spp..

Finally, Monte Carlo simulation technique, combinated with the mathematical

model and some survey data, was used to simulate the influence of environmental

factors on the number of Aeromonas spp. during storage, transportation and

consumption. The mathematical distribution of cell concentration at the end of

consumption was determined. Through sensitivity analysis, inoculation volume was

identified as the factor that has the largest effect on the number of Aeromonas spp., and

the second is home storage temperature and time.

Key Words: Aeromonas spp.; Chilled Pork; Predictive Models; Risk

Assessment

摘要

ABSTRACT

第一章绪论 .............................................................................................................12

§1.1 冷却肉概况及存在的质量安全问题 .........................................................2

§1.1.1 冷却肉概况 ..............................................................................................2

§1.1.2 冷却肉存在的质量安全问题 ..................................................................2

§1.2 预测微生物学及其在风险评估中的应用 .....................................................2

§1.2.1 预测微生物学模型简介 ..........................................................................2

§1.2.2 模型验证 .................................................................................................4

§1.2.3 预测微生物学在风险评估中的应用 ......................................................4

§1.3 课题研究意义及主要内容 .............................................................................6

§1.3.1 研究意义 .................................................................................................6

§1.3.2 研究内容 .................................................................................................6

第二章冷却猪肉中优势有害菌的分离鉴定 .............................................................6

§2.1 引言 ................................................................................................................7

§2.2 材料与方法 ....................................................................................................7

§2.2.1 材料与设备 ..............................................................................................7

§2.2.2 菌落总数测定 ..........................................................................................7

§2.2.3 优势有害菌测定 .....................................................................................8

§2.2.4 冷却猪肉中优势有害菌的分离鉴定 .....................................................8

§2.2.5 冷却猪肉冷藏过程中感官评定 ..............................................................8

§2.3 结果与讨论 .....................................................................................................8

§2.3.1 冷却猪肉中菌落总数和优势有害菌的变化 .........................................8

§2.3.2 冷却猪肉中优势有害菌的鉴定 .............................................................9

§2.3.3 冷却猪肉冷藏过程中的感官评定 ..........................................................9

§2.4 本章小结 ......................................................................................................10

第三章冷却猪肉中气单胞菌生长预测模型的建立和检验 .................................11

§3.1 前言 ...............................................................................................................11

§3.2 材料与方法 ...................................................................................................12

§3.2.1 材料与设备 ............................................................................................12

§3.2.2 菌种的选用及活化 ................................................................................12

§3.2.3 样品接种和贮藏 ....................................................................................12

§3.2.4 微生物计数 ............................................................................................12

§3.2.5 建模理论 ...............................................................................................12

§3.2.6 模型拟合软件 .......................................................................................14

§3.3 结果与讨论 ...................................................................................................14

§3.3.1 气单胞菌一级模型的构建 ....................................................................14

§3.3.2 气单胞菌二级模型的构建 ....................................................................18

§3.3.3 模型的检验 ...........................................................................................19

§3.4 本章小结 ......................................................................................................22

第四章不同实验条件下猪肉中气单胞菌生长预测模型的建立和验证 ...............23

§4.1 引言 ..............................................................................................................23

§4.2 材料与方法 ...................................................................................................24

§4.2.1 材料与设备 ............................................................................................24

§4.2.2 菌种的选用及活化 ................................................................................24

§4.2.3 样品接种和贮藏 ....................................................................................24

§4.2.4 微生物计数 ............................................................................................24

§4.2.5 实验设计 ...............................................................................................24

§4.2.6 建模理论 ...............................................................................................25

§4.2.7 模型拟合软件 .......................................................................................25

§4.3 结果与讨论 ...................................................................................................26

§4.3.1 生长曲线的拟合与生长参数的观测 ...................................................26

§4.3.2 RSM 生长预测方程的建立和验证 ...................................................... 26

§4.3.3 预测方程的数学检验 ............................................................................26

§4.4 本章小结 ..................................................................................................29

第五章冷却猪肉中气单胞菌的定量风险评估 .......................................................30

§5.1 引言 ...............................................................................................................30

§5.2 材料与方法 ..................................................................................................31

§5.2.1 冷却猪肉中气单胞菌检测 ....................................................................31

§5.2.2 冷却猪肉中气单胞菌的定量风险评估 ................................................31

§5.2.3 敏感度分析 ...........................................................................................33

§5.3 结果与分析 ..................................................................................................33

§5.3.1 上海市冷却猪肉中气单胞菌检测结果 ...............................................33

§5.3.2 危害识别 ................................................................................................33

§5.3.3 危害特征描述 ........................................................................................36

§5.3.4 暴露评估 ...............................................................................................37

§5.3.6 敏感度分析 ...........................................................................................45

§5.3.7 讨论 ........................................................................................................46

§5.4 本章小结 ...................................................................................................47

第六章结论与展望 ...................................................................................................48

§6.1 研究结论 .......................................................................................................48

§6.2 建议 ..............................................................................................................48

附录 I .......................................................................................................................... 49

参考文献 .....................................................................................................................54

在读期间公开发表的论文和承担科研项目及取得成果 .........................................58

致谢 .........................................................................................................................58

第一章绪论

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摘要：

基于预测模型的冷却猪肉中气单胞菌风险评估研究摘要本课题以冷却猪肉为对象，对冷却猪肉中优势有害菌进行分离鉴定，并对冷却猪肉中菌落总数和优势有害菌的生长情况进行研究，将分离得到的优势有害菌分组接种于冷却猪肉中，对冷却猪肉的腐败情况进行感官评定；将气单胞菌接种至表面灭菌的冷却猪肉上，研究不同培养温度和不同实验条件下气单胞菌在猪肉中的生长规律，并根据所得数据拟合一级和二级预测微生物学模型；对上海市冷却猪肉中气单胞菌的初始污染量进行检测，以冷却猪肉开始销售为起点，消费者最终食用为终点，根据所得模型，估计最终食用时冷却猪肉中气单胞菌的含量，评估消费者因食用冷却猪肉而发生气单胞菌食物中毒的风险。具体研究结果...

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