Validation of predictive models describing the growth of Listeria monocytogenes

In this study, predictions for growth rate of Listeria on food products were evaluated by both general applicable models and specific growth models. Literature values, obtained from a large number of publications, for growth rates in/on a variety of foods were compared by graphical and mathematical analysis with predictions given by various models. Apart for the great advantage of being generally applicable, the general models performed best. However, only small differences between the various models were observed. Model predictions were accurate within a factor of about two to four, depending on the type of product. The predictions should therefore not be considered as absolute; it is important to understand the limitations of the performance of models. All results and all assumptions should be criticised, but in many cases the accuracy will be sufficient to use these types of models as a tool in management decisions.     

A predictive model for heat inactivation of Listeria monocytogenes biofilm on stainless steel

Heat treatment of potential biofilm-forming sites is sometimes used for control of Listeria monocytogenes in food processing plants. However, little information is available on the heat treatment required to kill L. monocytogenes present in biofilms. The purpose of this study was to develop a predictive model for the heat inactivation of L. monocytogenes in monoculture biofilms (strains Scott A and 3990) and in biofilms with competing bacteria (Pseudomonas sp. and Pantoea agglomerans) formed on stainless steel in the presence of food-derived soil. Biofilms were produced on stainless steel coupons with diluted tryptic soy broth incubated for 48 h at 25 degrees C. Duplicate biofilm samples were heat treated for 1, 3, 5, and 15 min at 70, 72, 75, 77, and 80 degrees C and tested for survivors using enrichment culture. The experiment was repeated six times. A predictive model was developed using logistic regression analysis of the fraction negative data. Plots showing the probability of L. monocytogenes inactivation in biofilms after heat treatment were generated from the predictive equation. The predictive model revealed that hot water sanitation of stainless steel can be effective for inactivating L. monocytogenes in a biofilm on stainless steel if time and temperature are controlled. For example, to obtain a 75% probability of total inactivation of L. monocytogenes 3990 biofilm, a heat treatment of 80 degrees C for 11.7 min is required. The model provides processors with a risk management tool that provides predicted probabilities of L. monocytogenes inactivation and allows a choice of three heat resistance assumptions. The predictive model was validated using a five-strain cocktail of L. monocytogenes in the presence of food soil.     

Modelling the growth of Listeria monocytogenes in dynamic conditions

A recurrent neural network for the prediction of Listeria monocytogenes growth under pH and a(w) variable conditions was developed. The use of this model offered the possibility to take into account the consequences of the variations of the factors on L. monocytogenes growth. The effects of solutions, such as NaCl, acetic acid and NaOH, and their interactions on the response of L. monocytogenes cells were studied. Furthermore, the results showed the capacity of the recurrent neural network to predict growths carried out in different experimental conditions without using those used for its elaboration.     

A predictive model to determine the effects of pH, milkfat, and temperature on thermal inactivation of Listeria monocytogenes.

Listeria monocytogenes is a foodborne pathogen of significance because of its comparatively high heat resistance, zero tolerance in ready-to-eat foods, and growth at refrigeration temperatures. A 3 x 3 x 3 factorial study was done to determine the effects of milkfat (0%, 2.5%, 5.0%), pH (5.0, 6.0, 7.0), and processing temperature (55 degrees C, 60 degrees C, 65 degrees C) on the thermal resistance of L. monocytogenes in a formulated and homogenized milk system. Data were fit to a modified Gompertz equation where parameter estimates characterized three regions of a survival curve: the shoulder, maximum slope, and tail. Statistical analysis was done for each of the 27 individual treatment sets to visualize individual effects on parameter estimates and to evaluate how well the Gompertz equation represented the data. A regression model for the Gompertz equation was generated to predict the logarithmic surviving fraction of L. monocytogenes based on all 27 treatments and their single and interactive effects. The shoulder region of the survival curve was affected by pH; however, the maximum slope was affected by temperature, milkfat, and the interaction of temperature x milkfat. Validation of the model suggests that the predictions are best suited for processing above 62 degrees C. Trends over time for a 4-log reduction in cells (4D values) were evaluated using results from the 27 individual treatment sets, the regression model for the Gompertz equation, and a linear equation. At lower temperatures, 4D values by the three methods varied by twofold. At higher temperatures, all methods gave similar 4D values, suggesting that death became more linear. Based on this study all three factors affect heat resistance for specific regions of a survival curve, and a predictive model was developed that can be used as a preliminary estimate for L. monocytogenes inactivation.     

Listeria monocytogenes在经高氧化还原电位酸性水处理的鲜食莴苣上预测模型的建立

采用高氧化还原电位酸性水(EOW)对接种过单核细胞增生李斯特菌的鲜食莴苣进行处理,研究残留菌在不同温度下的保存期限。建立Gompertz,Logistic和Baranyi初级模型,描述单增李斯特菌在不同温度下的生长情况,对比结果表明,Gompertz模型的判定系数R2=0.9913,能够更好地拟合李斯特菌在各个温度下的生长状况,并得到李斯特菌生长的Gompertz模型生长参数(SGR,LT,MPD)。利用平方根模型对其的最大比生长速率的平方根-温度进行拟合,得到莴苣上单核细胞增生李斯特菌生长的二级模型:SGR=0.015T+0.069。使用判定系数(R2)、均方误差(MSE)、偏差因子(BF)和准确因子(AF)对模型进行验证,结果表明,本研究得出的二级模型能够很好地预测单核细胞增生李斯特菌在相应环境下的生长状况。     

Effect of prior growth conditions on the thermal inactivation of 13 strains of Listeria monocytogenes in two heating menstrua

The thermal tolerance of 13 Listeria monocytogenes strains was tested using a submerged heating coil apparatus. The strains were grown individually for 18 h at 37 degrees C in acidogenic tryptic soy broth (without dextrose) supplemented with 1% glucose and 1% glutamine (TSB+G) or nonacidogenic tryptic soy broth supplemented with 1% glutamine but containing no glucose (dextrose) (TSB-G). The former medium results in cells induced for pH-dependent, stationary-phase acid resistance, whereas the latter medium allows L. monocytogenes to grow to high numbers in the absence of glucose, yielding cells that are not induced for pH-dependent, stationary-phase acid resistance. The average final pH values of the 18-h TSB+G and the TSB-G cultures were 4.7 and 6.7, respectively. The cells grown in the acid resistance-inducing and non-acid resistance-inducing media were then tested in two heating menstrua that consisted of brain heart infusion broth adjusted to pH 3.0 and water activity (a(w)) of 0.987 or pH 7.0 and a(w) 0.970. In 14 of the 26 menstruum-strain combinations tested, the acid resistance-induced strains were more heat resistant then the equivalent noninduced cultures. No difference in the pattern of thermal resistance in response to induction of acid resistance was apparent among the different serovars tested. The results suggest that the ability of prior induction of acid resistanceto enhance thermal resistance can vary substantially among L. monocytogenes strains.     

Formation of biofilms by Listeria monocytogenes under various growth conditions

Eight strains of Listeria monocytogenes (7644, 19112, 15313, Scott A, LCDC, 10403S, SLCC, and 1370) produce biofilms when grown on polyvinyl chloride microtiter well plates. The growth medium (tryptic soy broth [TSB] or modified Welshimer's broth [MWB] at 32 degrees C) influenced the amount of biofilm formed; maximum biofilms were formed in MWB by six strains and in TSB by the remaining two strains. This result suggests that the growth medium is critical in development of L. monocytogenes biofilm. This organism also produced biofilms on stainless steel chips. Biofilm formation on these chips was observed following growth in TSB at 4, 20, and 37 degrees C. After 20 h of incubation at 20 or 37 degrees C, the cell density was approximately 10(6) CFU per chip, and after 4 days incubation at 4 degrees C, the cell density was 10(5) CFU per chip. L. monocytogenes strain Scott A biofilm formation on stainless steel chips was visualized using scanning electron microscopy, which revealed dense aggregates of cells held together by meshlike webbing.     

Heat resistance of Listeria monocytogenes

The heat resistance data on Listeria monocytogenes in culture media and foods are summarized. Most heat resistance data for foods have been obtained in dairy, meat, poultry, and egg products. Limited data have been published on seafood, fruits, and vegetables. The methodologies employed have evolved over time; hence data from earlier experiments are not directly comparable to more recent studies. Many factors influence the heat resistance of L. monocytogenes. Variation exists among different strains in their ability to withstand heat treatment. In addition, heat resistance is influenced by age of the culture, growth conditions, recovery media, and characteristics of foods such as salt content, a(w), acidity, and the presence of other inhibitors. Listeriae are more heat resistant than most other nonspore-forming foodborne pathogens, and thus, processing recommendations based on data from experiments with Salmonella spp. or pathogenic Escherichia coli may not be sufficient to eliminate similar numbers of L. monocytogenes. The data provided in this review may prove useful for food processors in determining appropriate times and temperatures for producing foods free of vegetative pathogens.     

Survival, growth, and thermal resistance of Listeria monocytogenes in products containing peanut and chocolate

Outbreaks of listeriosis associated with the consumption of ready-to-eat foods have raised interest in determining growth, survival, and inactivation characteristics of Listeria monocytogenes in a wide range of products. A study was undertaken to determine the thermal tolerance of L. monocytogenes in a peanut-based beverage (3.1% fat), whole-fat (3.5%) milk, wholefat (4.0%) and reduced-fat (1.0%) chocolate milk, a chocolate-peanut spread (39% fat), and peanut butter (53% fat). The D60 degrees C value (decimal reduction time at 60 degrees C) in peanut beverage (3.2 min) was not significantly different (P > 0.05) than the D60 degrees C value in whole-fat milk (3.3 min) or whole-fat chocolate milk (4.5 min) but significantly lower (P < or = 0.05) than the D60 degrees C value in reduced-fat chocolate milk (5.9 min). The pathogen was significantly more resistant to heat when enmeshed in chocolate-peanut spread (water activity [aw] of 0.46; D60 degrees C = 37.5 min) and peanut butter (aw of 0.32; D60 degrees C = 26.0 min) than in liquid products. At 10 degrees C, the pathogen grew most rapidly in whole-fat chocolate milk and slowest in peanut beverage. At 22 degrees C, populations increased significantly within 12 and 16 h in whole-fat milk and reduced-fat chocolate milk, respectively, and within 8 h in whole-fat chocolate milk and peanut beverage. Initial populations (3.37 to 4.42 log CFU/g) of L. monocytogenes in chocolate-peanut spread and peanut butter adjusted to an aw of 0.33 and 0.65 declined, but the pathogen was not eliminated during a 24-week period at 20 degrees C. Survival was enhanced at reduced aw. Results indicate that a pasteurization process similar to that used for full-fat milk would be adequate to ensure the destruction of L. monocytogenes in peanut beverage. The pathogen survives for at least 24 weeks in chocolate-peanut spread and peanut butter at an aw range that encompasses that found in these products.     

A model describing the effect of temperature history on lag time for Listeria monocytogenes

A model was built to describe the influence of the temperature and the duration of pre-incubation on the lag time for regrowth of Listeria monocytogenes at low temperature. This model is consistent with the usual procedure used to calculate lag times of cultures growing under fluctuating temperatures. It also describes the effect of prolonged starvation conditions on the regrowth lag time and takes into account the influence of the physiological state of inocula in predictive models.     

培养条件对单增李斯特菌生长的影响

以纯度为99.97%的单增李斯特菌为研究对象,通过测定菌株在各培养条件下菌液的OD600值,分析了不同的NaCl浓度、pH、温度及NaCl浓度为3%～8%(梯度为1%),pH为6～9(梯度为1),温度为0～40℃(梯度为10℃)的交互作用条件下单增李斯特菌的生长状况。结果表明:菌株的对数增长期为8～14h,稳定期为14～18h,18h以后进入衰亡期;菌株在NaCl浓度为0.5%～3.5%范围内生长良好,其最适生长pH为7.5,最适生长温度为37℃。各因素的交互作用通过SPSS软件进行方差分析,得到其p<0.01,即各因素的交互效应极显著。      

A predictive model for the influence of food components on survival of Listeria monocytogenes LM 54004 under high hydrostatic pressure and mild heat conditions

The combination of high hydrostatic pressure with mild temperature was explored to achieve a predictive model of microbial inactivation in food matrix processing. The pressure processing conditions were fixed at 448 MPa for 11 min at the treatment temperature of 41 degrees C, which have been determined as the optimum processing conditions considering six log-cycle reductions of Listeria monocytogenes. Based on the results, response surface methodology (RSM) was performed in the present work, the influence of food components like soybean protein (0-5.00%), sucrose (0.25-13.25%), bean oil (0-10.00%), and pH (4-10) of the food matrix on survival of L. monocytogenes by high pressure and mild heat was studied, and a quadratic predictive model for the influence of food components and pH of food matrix on L. monocytogenes reduction by high pressure and mild heat was built with RSM accurately. The experimental results showed that the efficiency of L. monocytogenes reduction in milk buffer and food matrix designed in the present work, under the HPP treatment process parameters described above, were different. The soybean protein (P=0.0086), sucrose (P<0.0001), and pH (P=0.0136) significantly affected reduction of L. monocytogenes, but the effect of bean oil on reduction of L. monocytogenes was not significant (P=0.1028). The predictive model is significant since the level of significance was P<0.0001 and the calculated F value (11.53) is much greater than the tabulated F value (F(0.01 (14, 5))=9.77). Moreover, the adequacy of the predictive model equation for predicting the level of L. monocytogenes reduction was verified effectively by the validation data.     

A predictive model for the effect of temperature and predrying treatments in reducing Listeria monocytogenes populations during drying of beef jerky

The objective of this study was to model the effect of drying temperatures (52, 57, and 63 degrees C) and predrying treatments on the inactivation of Listeria monocytogenes on beef jerky. Before drying, beef slices were inoculated with a 10-strain composite of L. monocytogenes and then treated with the following: (i) nothing (C), (ii) traditional marinade (M), or (iii) dipping in 5% acetic acid solution for 10 min, followed by M (AM). In addition, sequential stresses (exposure to 10% NaCl, followed by an adjustment of the pH to 5.0 and, subsequently, a water bath at 45 degrees C) were applied to the inocula before beef contamination and drying at 63 degrees C. Surviving L. monocytogenes were determined on tryptic soy agar plus 0.6% yeast extract (TSAYE) and on PALCAM agar at 0, 2, 4, 6, 8, and 10 h during drying. Data were modeled by a linear regression (treatment AM) and a logistic-based equation capable of fitting biphasic inactivation curves without initial shoulder (treatments C and M). The total log reductions expressed as the CFU per square centimeter of L. monocytogenes (3.9 to 5.1) for the samples treated with M (3.5 to 5.4) when compared with C were similar, whereas AM-treated samples had higher (6.1 to 6.8) reductions. All survival curves were characterized by an initial rapid decrease in populations within the first 2 h, which was followed by a secondary death phase at a lower rate. No significant (P > or = 0.05) differences in inactivation were observed due to drying temperatures in the range (52 to 63 degrees C) tested. Inactivation differences between recovered counts of stressed and unstressed cells were significant (P < 0.05) in PALCAM but not in TSAYE. The acidified predrying treatment (AM) had higher pathogen inactivation during drying than other treatments, regardless of drying temperature. The models developed may be useful in designing effective drying processes for beef jerky.     

Microbial competition: effect of Pseudomonas fluorescens on the growth of Listeria monocytogenes

Listeria monocytogenes Scott A was cultured alone and in coculture with Pseudomonas fluorescens ATCC 33231 to characterize quantitatively the effects of microbial competition on the growth of this psychrotrophic pathogen. The bacteria were cultured in brain–heart infusion broth (BHI), using a 3×3×3×2 complete factorial design to assess the impact of temperature (4, 12, 19°C), initial pH (5·0, 6·0, 7·0), and sodium chloride content (5, 25, 45 gl⁻¹) on the interaction between the two micro-organisms. Samples were periodically plated on BHI agar and Vogel Johnson agar to obtain total counts and L. monocytogenes counts, respectively. Growth curves were generated by fitting the data to the Gompertz equation, and the derived growth kinetics were compared. WhenP. fluorescens did influence the growth of L. monocytogenes, the primary effect was a suppression of the maximum population density (MPD) reached by the pathogen. Suppression of L. monocytogenes was generally associated with low incubation temperatures (4°C) and sodium chloride levels (5 and 25 gl⁻¹). Slight increases (<1·0 log cfu ml⁻¹) in the MPD attained by L. monocytogenes were observed when grown in the presence of P. fluorescens at higher temperatures (12 and 19°C) and sodium chloride levels (25 and 45 gl⁻¹) when the pH was 5·0. The current study supports earlier work that indicates that reliance on microbial competition as a barrier to control L. monocytogenes in refrigerated foods will require detailed knowledge of how the interaction between the pathogen and the microflora is affected by environmental and food characteristics such as storage temperature, pH, and water activity.     

A predictive model to determine the effects of temperature,sodium pyrophosphate,and sodium chloride on thermal inactivation of starved Listeria monocytogenes in pork slurry

The effects and interactions of 27 combinations of heating temperature (57.5 to 62.5 degrees C), sodium pyrophosphate (SPP) level (0 to 0.5%, wt/vol), and salt (NaCl) level (0 to 6%, wt/vol) on the thermal inactivation of starved Listeria monocytogenes ATCC 19116 in pork slurry were investigated. A split-split plot experimental design was used to compare all 27 combinations. L. monocytogenes survivors were enumerated on tryptic soy agar supplemented with 0.6% yeast extract. The natural logarithm (loge) of the means of decimal reduction times (D-values) were modeled as a function of temperature, SPP level, and NaCl level. Increasing concentrations of SPP or NaCl protected starved L. monocytogenes from the destructive effect of heat. For example, D-values for the pathogen at 57.5 degrees C in pork slurry with 0, 3, and 6% NaCl were 2.79, 7.75, and 14.59 min, respectively. All three variables interacted to affect the thermal inactivation of L. monocytogenes. A mathematical model describing the combined effect of temperature, SPP level, and NaCl level on the thermal inactivation of starved L. monocytogenes was developed. There was strong correlation (R2 = 0.97) between loge D-values predicted by the model and those observed experimentally. The model can predict D-values for any combination of variables that falls within the range of those tested. This predictive model can be used to assist food processors in designing thermal processes that include an adequate margin of safety for the control of L. monocytogenes in processed meats.     

Searching for new mathematical growth model approaches for Listeria monocytogenes

ABSTRACT:  Different secondary modeling approaches for the estimation of Listeria monocytogenes growth rate as a function of temperature (4 to 30 °C), citric acid (0% to 0.4% w/v), and ascorbic acid (0% to 0.4% w/v) are presented. Response surface (RS) and square-root (SR) models are proposed together with different artificial neural networks (ANN) based on product functions units (PU), sigmoidal functions units (SU), and a novel approach based on the use of hybrid functions units (PSU), which results from a combination of PU and SU. In this study, a significantly better goodness-of-fit was obtained in the case of the ANN models presented, reflected by the lower SEP values obtained (< 24.23 for both training and generalization datasets). Among these models, the SU model provided the best generalization capacity, displaying lower RMSE and SEP values, with fewer parameters compared to the PU and PSU models. The bias factor (B_f) and accuracy factor (A_f) of the mathematical validation dataset were above 1 in all cases, providing fail-safe predictions. The balance between generalization properties and the ease of use is the main consideration when applying secondary modeling approaches to achieve accurate predictions about the behavior of microorganisms.     

鲜切结球莴苣中单增李斯特菌生长预测模型的建立

为建立不同温度下鲜切结球莴苣中单增李斯特菌生长模型,将单增李斯特菌接种到鲜切结球莴苣表面,并于不同温度下贮藏,获得其在4、8、16、24和32℃下的生长数据,选用Gompertz模型进行拟合,建立初级生长模型。在此基础上建立二级模型研究温度对初级模型中单增李斯特菌生长动力学参数的影响,并进行数学检验。结果表明,对最大比生长速率和延滞时间建立平方根模型,结果呈良好的线性关系,相关系数R2分别为0.977 2和0.984 7,所建立的预测模型能很好地描述不同温度下单增李斯特菌的生长动态。     

On the different growth conditions affecting silver antimicrobial efficacy on Listeria monocytogenes and Salmonella enterica

Silver is known to inhibit microorganisms and therefore it is an ideal candidate for its incorporation in a wide variety of materials for food applications. However, there is still a need for understanding how silver prolonged exposure to bacterial contamination affects the bioavailability of the active silver species. In the present study, growth curves of Listeria monocytogenes and Salmonella enterica were performed for 3-5days in Tryptic Soy Broth (TSB) and M9 minimal medium (M9) in the presence of silver ions and silver solutions previously in contact with the growth media. The cultivability of the bacteria under these conditions was correlated with the viability of the bacterial populations as measured by flow cytometry analysis (FC) using a LIVE/DEAD BacLight kit. It was found that, after a period where viable counts were not detected, bacterial populations recovered and were able to proliferate in most cases. The resuscitation of the cultures was explained by both the existence of a resilient fraction of bacteria in a compromised state and the parallel inactivation of the silver species. This inactivation was found to be highly influenced by time dependant chemical reactions taking place in the environment of exposure, producing differences of at least 3 fold between results for nutrient rich environments and results for limiting environments. This study points out the need for understanding these chemical interactions and bacterial mechanisms of adaptation and may have relevance in the design of silver-based antimicrobial systems for food-related applications.     

Application of predictive models to estimate Listeria monocytogenes growth on frankfurters treated with organic acid salts

Organic acid salts including sodium lactate, sodium diacetate, potassium benzoate, potassium sorbate, and their combinations were assessed as potential inhibitors of Listeria monocytogenes growth on frankfurters. Predictive models for L. monocytogenes growth on frankfurters treated with these salts were compared to select a proper L. monocytogenes growth curve model under these conditions. Sigmoidal equations, including logistic and Gompertz equations, are widely used to describe bacterial growth. In this study, the reparameterized Gompertz model provided a better fit to the L. monocytogenes growth data compared with the other models that were included in this study. Rather than a fixed value for the maximum number of organisms, the reparameterized Gompertz model allows this quantity to be estimated from the data to determine the effect, if any, of the treatments on maximum population density. This information is expected to improve practical methodology for hazard characterization of microbial pathogens on ready-to-eat meat products.     

接种量对单增李斯特菌生长期及生长界面的影响

为了初步了解接种量对单增李斯特菌生长状况及生长/非生长界面的影响,本实验对单增李斯特菌在0、4、10、25℃下通过培养菌液在600nm下的吸光光度值对其生长周期曲线分别进行了测定,并分析了不同接种量的单增李斯特菌菌液在25℃下的生长周期状况,探讨了纯培养条件下不同盐度和pH下,接种量对单增李斯特菌生长/非生长状况的影响。结果表明:不同的温度下,单增李斯特菌的生长周期有很大的差别;而在相同温度下,接种量对单增李斯特菌的生长周期有较大的影响,随着接种水平的降低,菌种生长所需的延滞时间越长,接种量为107CFU/mL时,其生长延滞期为0~4h,而当接种量减少为10CFU/mL时,其生长延滞期为0~16h;而对于单增李斯特菌的生长/非生长界面而言,接种量对其也有一定的影响,但其作用机制还有待进一步深入的研究。