运用Real-time PCR方法建立大肠杆菌O157:H7生长预测模型

运用Real-time PCR方法建立大肠杆菌O157:H7不同温度(10,15,20,25,30℃)条件下的初级生长动力模型,包括修正Gompertz模型、Logistic模型。结果表明,两种模型均能很好拟合两种计数方法,得到大肠杆菌O157:H7在不同温度条件下生长曲线;每条曲线存在明显的迟滞期、对数期和稳定期,且温度对微生物的生长速率和迟滞期有明显影响,随温度的升高生长速率显著增加而迟滞期明显缩短。通过相关系数(R2)、均方根误差(MSE)、偏差因子(Bf)和准确因子(Af)等评估模型的准确性,R2均≥0.99;MSE值均小于0.1 lg cfu/m L,模型拟合程度较好。偏差因子Bf值和准确因子Af值均在可接受的范围内,模型准确性高。通过与传统培养计数得到的预测模型进行比较,运用Real-time PCR方法建立的模型与传统方法建立模型无显著差别,能较好地预测大肠杆菌O157:H7的生长情况,解决了传统预测模型耗时、耗力等问题。     

不同贮藏温度下鸡肉中大肠杆菌O157:H7生长预测模型的建立

目的 建立大肠杆菌在鸡胸肉的生长预测模型,为其加工、贮藏保鲜等过程的温度控制提供参考。方法 以大肠杆菌（Escherichia coli O157:H7）为研究对象,监测其在4、8、10、12、16、20、28、37℃贮藏条件下熟鸡胸肉上的生长情况。采用Gompert模型建立熟鸡胸肉上大肠杆菌的一级生长模型,并将一级生长模型拟合得到的数据代入Ratkowsky方程建立二级模型,综合考虑准确因子Af、偏差因子Bf和均方根误差R2并检验模型的可行性。结果 10、12、16、20、28和37℃条件下拟合的一级生长模型相关系数均在0.94以上,说明能够用来预测该温度范围内大肠杆菌在熟鸡胸肉上的生长动态。在4℃和8℃条件下,大肠杆菌在熟鸡胸肉上呈现先上升后下降再上升的趋势,而在10、12、16、20、28和37℃下,大肠杆菌先进入延滞期,后进入快速增长的指数期,且温度越高,指数期越短。结论 在10～37℃下可以用Gompert模型拟合鸡胸肉中E. coli O157:H7的生长预测模型,且效果较好。     

基于预测微生物学的冷却牛肉货架期预测模型的建立

为实时监测冷却牛肉贮藏期间的品质变化与货架期,分别测定冷却牛肉0、4、7、10 ℃贮藏过程中假单胞菌菌数、总挥发性盐基氮含量和感官评分。利用修正的Gompertz方程建立不同贮藏温度下假单胞菌生长的动力学模型,以Belehradek方程为二级模型,描述贮藏温度对假单胞菌生长的影响,并利用2、5、8 ℃条件下贮藏的冷却牛肉验证货架期预测模型的准确性。结果表明：所建立的Gompertz模型拟合相关系数均在0.99以上,预测结果准确度在1.013～1.126之间,偏差度在0.926～1.057之间,贮藏温度与μmax 1/2和（1/λ）1/2均呈良好的线性关系,说明建立的一级和二级模型能够真实、有效地预测0～10 ℃贮藏条件下冷却牛肉中假单胞菌的生长情况;货架期的预测值与实测值相对误差在±10%以内,该货架期模型可有效预测冷却牛肉在0～10 ℃贮藏条件下任意温度下的货架期。     

气调包装冷却牛肉特定腐败菌生长模型的建立

以气调包装冷却牛肉为研究对象,研究储藏在0、5、10℃温度条件下气调包装冷却牛肉中特定腐败菌(假单胞菌)的生长变化规律。通过Matlab7.0 软件拟合不同温度条件下假单胞菌的生长情况,得到假单胞菌生长的Gompertz 模型参数;利用响应面方程建立假单胞菌生长的二级模型;由平方根方程建立了温度对假单胞菌生长动力学影响的数学模型。进而验证0～10℃低温条件下假单胞菌的生长动力学模型。结果表明：建立的Gompertz 方程拟合相关系数均在0.99 以上,二级模型偏差度和准确度均在0.90～1.05 之间,说明建立的一级和二级模型能够真实有效地预测0～10℃条件下气调包装冷却牛肉中假单胞菌的生长情况。     

草鱼鱼糜中沙门氏菌生长预测模型的建立

为建立鱼糜中沙门氏菌生长预测模型,选用新鲜草鱼鱼糜和鼠伤寒沙门氏菌作为研究对象,比较了4、10、20、28、37℃条件下鱼糜中鼠伤寒沙门氏菌的生长情况,分别采用Huang模型,Baranyi模型和修正的Gompertz模型进行拟合,建立鱼糜中鼠伤寒沙门氏菌一级生长动力学模型。并用平方根模型方程描述温度与比生长速率和延滞期的关系,得到鼠伤寒沙门氏菌生长二级模型。使用判定系数R2,准确因子(Af),偏差因子(Bf)和均方误差(MSE)对一级和二级模型可靠性进行评价,结果表明修正的Gompertz模型更适合于描述4~37℃条件下鱼糜中鼠伤寒沙门氏菌的生长变化,二级平方根模型可用于描述鱼糜中鼠伤寒沙门氏菌的生长参数,能够为鱼糜中沙门氏菌的监测提供一定的参考依据。     

冰温冷藏鲜肉的菌落总数动态变化预测模型拟合验证

采用Huang模型、Baranyi模型和修正的Gompertz模型对猪肉、鸡胸肉和牛肉在不同温度条件下冷藏时的菌落总数进行拟合,通过比较拟合效果筛选出能够准确预测鲜肉在冰温条件下贮藏时菌落总数的预测模型,进一步采用平方根模型来描述贮藏温度对菌落总数的影响,并用偏差因子和准确因子进行预测模型验证。结果表明：采用Huang模型对猪肉、鸡胸肉和牛肉分别在－1.0、－1.0、2.5 ℃条件下贮藏过程中菌落总数拟合的效果优于Baranyi模型和修正的Gompertz模型,Huang模型可以更好地预测不同冰温条件下贮藏鲜肉的菌落总数变化;通过平方根模型拟合得到的牛肉贮藏温度与最大比生长速率和迟滞期的判定系数R2Adj分别为0.999和0.985,这表明牛肉贮藏温度与最大比生长速率和迟滞期之间存在良好的线性关系;预测模型的准确因子和偏差因子平均值分别为1.164和0.998,尤其是在－2.5 ℃贮藏条件下,准确因子和偏差因子分别为1.009和1.000,均接近于1.000,预测模型可靠性高,说明Huang模型能较好地预测冰温贮藏鲜肉菌落总数的动态变化。     

冷却牛肉中沙门氏菌生长预测模型的建立和验证

建立了恒温条件下牛肉中沙门氏菌的生长动力学模型,准确掌握并及时预报沙门氏菌的生长情况,为冷却牛肉生产过程中该菌的控制提供有效的手段。将不同血清型的沙门氏菌混合菌株(以下简称沙门氏菌)接种到无污染的冷却牛肉表面,分别置于0、4、7、10、15和20℃恒温条件下贮藏并计数,采用Linear方程或修正的Gompertz方程拟合一级模型,并采用Ratkowsy平方根方程拟合二级模型。最后分别在恒温和波动温度下对模型进行验证。修正的Gompertz方程能较好地描述沙门氏菌在4~20℃贮藏条件下的生长动态(R2均大于0.99),而在0℃下则适用Linear方程拟合。采用平方根模型构建的沙门氏菌预测二级模型,表明温度对最大比生长速率(μmax)和迟滞期(λ)均呈现良好的线性关系(R2均大于0.96)。用贮藏在9℃、12℃和波动温度下沙门氏菌的生长实验值进行验证,偏差度分别为0.913、0.997、0.889,准确度分别为1.118、1.019、1.147。该预测模型能有效预测4~20℃条件下沙门氏菌在冷却牛肉中的生长情况。     

不同温度下椰汁中金黄色葡萄球菌的生长动力学模型比较

探讨不同温度下椰汁中金黄色葡萄球菌的生长预测模型。将菌悬液接种到椰汁中,测定不同温度(20、25、30、36℃)下的生长数据。使用Matlab软件拟合得到修正Gompertz(MGompertz)、修正Logistic(MLloistic)和Baranyi模型,比较残差和拟合度选择最优一级模型,并拟合出生长参数。用平方根和二次多项式方程建立二级模型,通过相关系数、偏差因子和准确因子对二级模型进行检验。在20~36℃下,Baranyi模型拟合出的各个拟合度最优,Baranyi模型适宜作为模拟金黄色葡萄球菌在椰汁中生长的一级预测模型。二次多项式相较于平方根模型可以更好地表达温度与最大比生长速率及延滞期的关系。因此选择Baranyi模型和二次多项式模型描述不同温度下椰汁中金黄色葡萄球菌的生长。     

温度生长预测模型在大肠杆菌O157:H7控制中的应用

大肠杆菌O157:H7是严重危害人类健康的肠道致病菌。本实验研究了该菌在温度、pH、NaCl浓度影响下的生长繁殖规律,确定了大肠杆菌O157:H7的最适生长条件。建立了在pH、NaCl浓度最佳情况下鸡肉和肉汤中该菌的温度生长预测模型。以模型为依据,提出了出口分割鸡肉中控制大肠杆菌O157:H7的措施。     

模拟蟹肉中副溶血性弧菌预测生长预测模型的建立

为了控制模拟蟹肉中副溶血性弧菌的危害,运用预测食品微生物学的方法,本文分别研究了在温度10～37℃条件下模拟蟹肉中副溶血性弧菌的生长规律,建立了一级和二级预测模型。结果表明,副溶血性弧菌的生长曲线在此温度范围内都呈典型的S型,故本文采用Gompertz模型拟合,二级模型采用常用的平方根模型拟合,拟合方程为U=0·01578(T+7·294)。通过模型验证,预测值和实测值的残差在±0·04范围内,偏差因子和精准因子均在可接受范围内,模型具有可靠性。     

Growth rates of Salmonella and Escherichia coli O157:H7 in irradiated beef.

Ground beef was irradiated to 0, 2, or 4 kGy and then inoculated with a mixed culture of four serotypes of salmonellae or five strains of Escherichia coli O157:H7. The ground beef was stored at either 15 or 25 degrees C, and the growth of the inoculated bacteria was monitored over time. Growth parameters were determined for both the salmonellae and the E. coli O157:H7 using the Gompertz equation. There was no significant difference in lag phase duration or generation time, irrespective of the dose to which the ground beef had previously been exposed. Furthermore, the lag phase durations and generation times determined in this study did not differ significantly from previously published values. This suggests that, although irradiation eliminates a significant portion of the spoilage microflora in ground beef, the absence of this microflora provides no competitive advantage to the growth of salmonellae or E. coli O157:H7 in ground beef.     

Effects of salt, sodium pyrophosphate, and sodium lactate on the probability of growth of Escherichia coli O157:H7 in ground beef

Ground beef products are susceptible to contamination with Escherichia coli O157:H7. The objective of this study was to examine the effect of salt, sodium pyrophosphate (SPP), and sodium lactate on the probability of growth of E. coli O157:H7 in ground beef under a temperature abuse condition. Ground beef containing 0 to 2.25% salt, 0 to 0.5% SPP, and 0 to 3% lactate was inoculated with a four-strain mixture of E. coli O157:H7, vacuum packaged, and stored at 10°C for 15 days. A total of 25 combinations of the three additives, each with 20 samples, were tested. A logistic regression was used to model the probability of growth of E. coli O157:H7 (with a 1.0-log CFU/g increase during storage) as a function of salt, SPP, and lactate. The resultant probability model indicated that lactate at higher concentrations decreased the probability of growth of E. coli O157:H7 in ground beef, and the effect was more pronounced at higher salt concentrations. At salt concentrations below 1.3%, the increase of SPP concentration marginally increased the growth probabilities of E. coli O157:H7. The model illustrated the effect of salt, SPP, and lactate on the growth probabilities and growth or no-growth behavior of E. coli O157:H7 in ground beef and can be used to improve the microbial food safety of ground beef products.     

贮藏过程中冷却牛肉微生物模型的建立

冷却牛肉贮藏过程中易受微生物的污染,通过建立微生物数学模型,可以预测冷却牛肉的污染情况。实验以自然污染下的冷却牛肉为介质,对0、4、7、10、15、20℃恒温条件下的菌落总数进行计数,利用SAS软件拟合Modified Compertz方程,并对比生长率和时间拟合了平方根二级模型,最后在波动温度下对模型进行了验证。结果显示,Modified Compertz方程能很好地反映冷却牛肉中菌落总数的污染情况,恒定温度下的模型拟合度都在0.95以上,对波动温度条件下的菌落总数的预测值与实测值进行比较,偏差度为0.90,准确度为1.12,表明菌落总数生长模型可以可靠地预测冷却牛肉的微生物污染情况。     

Empirical Distribution Models for Escherichia coli 57:H7 in Ground Beef Produced by a Mid-size Commercial Grinder

ABSTRACT: The purpose of this research was to develop empirical models that describe the amount and distribution of ground beef contaminated with Escherichia coli O157:H7 when a contaminated beef trim is introduced into a batch of uncontaminated beef before processing in a mid-size commercial grinder (34 g/s). A beef trim was inoculated with a rifampacin-resistant strain of E. coli O157:H7 and added to a batch of noncontaminated trims at the grinding step. To study the distribution of the E. coli O157:H7^rif in the ground beef, 6 treatments with different inoculum levels (1 to 6 log₁₀ colony-forming units [CFU]) were tested. Removal or pick up of the residual contamination with E. coli O157:H7^rif left in the grinder was evaluated. E. coli O157:H7^rif was detected in 9% to 86% of the total ground beef for the 1 to 6 log₁₀ CFU inoculum levels, respectively. E. coli O157:H7^rif contamination was detected in the collar that fixes the grinder's die and blade to the hub. An exponential algorithm described the relationship between the quantities of ground beef containing E. coli O157:H7^rif and the inoculum level ( R ²= 0.82). Distribution models based on a Chi-squared algorithm were developed for each inoculum level describing the contamination level as a function of the batch fraction processed ( R ²= 0.81 to 0.99). The results of this study corroborate that when beef processors test for pathogenic contamination in a mid-scale grinder, they should test the beef residues in the collar that fixes the grinder's die and blade to the hub.     

食品中大肠杆菌O157的PCR检测与wzy基因的测序分析

大肠杆菌O157:H7 和O157:H- 是重要食品致病菌。从食品中分离得到一株O157:H7 菌株EC5。将该菌株PCR 扩增wzy 基因全长并克隆测序。结果表明,wzy 基因序列与大肠杆菌O157:H7 标准株EDL933 有100% 同源性。从NCBI GenBank 数据库下载O157 菌株wzy 基因序列,针对保守序列设计引物并扩增O157 菌株和非O157 菌株。PCR 结果表明,wzy 基因特异性强,可作为检测O157 菌株的标志基因。本研究为开发大肠杆菌O157 的分子检测技术提供新的研究思路。     

Evaluation of Escherichia coli O157:H7 growth media for use in test-and-hold procedures for ground beef processing

Since the mid-1990s, the beef industry has used a process called test and hold, wherein beef trim and ground beef are tested to keep products contaminated with Escherichia coli O157:H7 out of commerce. Current O157:H7 detection methods rely on a threshold level of bacterial growth for detection, which is dependent on the growth medium used. Twelve media were examined for growth and doubling time: buffered peptone water (BPW), SOC (which contains tryptone, yeast extract, KCl, MgCl2, and glucose), buffered peptone water plus SOC (BPW-SOC), Bacto-NZYM, RapidChek E. coli O157:H7 medium, BioControl EHEC8 culture medium, Neogen Reveal for E. coli O157:H7--Eight Hour medium (Neogen Reveal 8), BAX System medium for E. coli O157:H7 (BAX) BAX System medium for E. coli O157:H7 MP (BAX-MP), modified E. coli broth, nutrient medium, and tryptic soy broth (TSB). All media were tested at 37 or 42 degrees C under static or shaking conditions. The eight media with the highest total CFU per milliliter and most rapid doubling times were BPW-SOC, NZYM, RapidChek, EHEC8, Neogen Reveal 8, BAX, BAX-MP, and TSB. The ability of these eight media to enrich E. coli O157:H7 in ground beef was further evaluated through time-course experiments using immunomagnetic separation. Of these media, TSB was the easiest to prepare, had a wide application base, and was the least expensive. In the test-and-hold process, the normal ratio of medium to product is 1:10. In this study, a 1:3 ratio worked as well as a 1:10 ratio. Processors using test-and-hold procedures could use 1 liter of TSB to enrich for E. coli O157:H7 in a 375-g sample instead of the usual 3.375 liters, thus saving reagents, time, and labor while maintaining accuracy.     

Growth of Escherichia coli O157:H7 in raw ground beef stored at 10 degrees C and the influence of competitive bacterial flora,strain variation,and fat level

Pure-culture broth-based models of the growth of Escherichia coli O157:H7 have been used to estimate its behavior in ground beef, even though these models have not been adequately validated for this food product. This situation limits accurate estimates of the behavior of E. coli O157:H7 in ground beef and introduces uncertainties in risk assessments. In the present study, the growth of single and multiple strains of E. coli O157:H7 were measured in retail ground beef stored at 10 degrees C for up to 12 days, and the results were compared with estimates generated by the U.S. Department of Agriculture's Pathogen Modeling Program (PMP; version 5.1). At pH 5.9, the PMP predicted a maximum population density (MPD) of 9.13 log10 CFU/g, an exponential growth rate (EGR) of 0.052 log10 CFU/h, and a lag time of 56.3 h. Similar parameter values were observed for sterilized ground beef; however, no lag phase was observed. In contrast, the mean MPD and EGR for retail ground beef were 5.09 log10 CFU/g and 0.019 log10 CFU/h, respectively, and no lag phase was observed. Both the EGR and the MPD increased with decreasing fat levels. There was low variation in the MPD and EGR parameters for the nine E. coli O157:H7 ground beef isolates. Two isolates of competitive native flora were separately added to sterilized ground beef, and the EGR and MPD decreased as the ratio of competitive flora to E. coli O157:H7 increased. For one strain, at ratios of 1:1, 10:1, and 100:1, the EGRs were 0.033, 0.025, and 0.018 log10 CFU/h, respectively, and the MPDs were 6.14, 5.08, and 4.84 log10 CFU/g, respectively. These results demonstrate that existing broth-based models for E coli O157:H7 must be validated for food and that models should consider the effects of the food matrix, the competitive microflora, and potential pathogen strain variation.