Reduced detectability of Listeria monocytogenes in the presence of Listeria innocua

Recent foodborne crises have demonstrated the importance of monitoring food safety. In terms of microbiological criteria, food safety requires the reliable detection of pathogens such as Listeria monocytogenes along the food chain by appropriate analytical methods. However, indications exist that accompanying Listeria innocua strains suppress the growth of L. monocytogenes during selective enrichment, which may cause reduced or even inhibited detection. To study these effects, the limit of detection of L. monocytogenes was investigated in the presence of L. innocua using the International Organization for Standardization standard method ISO 11290-1 and the VIDAS LDUO system, an automated method based on enzyme-linked fluorescence technology. The challenge was to provide low initial Listeria concentrations at sufficient precision to quantify the influence on the probability of detection of L. monocytogenes. The application of reference materials appropriate for quantitative test methods and a standardized dilution procedure were necessary to ensure accurate CFU levels of defined proportions of mixtures of both Listeria species. During selective enrichment, overgrowth of L. monocytogenes by L. innocua could be confirmed, leading to high rates of false-negative results. Moreover, with both methods, a significant decrease in the detectability of L. monocytogenes could be quantified at ratios of 2:1 at very low concentrations representative of natural contamination levels often found in foods and environments. It is concluded that there is a need to improve existing procedures with respect to selective enrichment, as well as the detection techniques.     

Factors influencing resuscitation and growth of heat injured Listeria monocytogenes 13-249 in sous vide cooked beef

The growth of Listeria monocytogenes 13-249 in vacuum-packed, minced beef was investigated as a function of degree of heat injury (including no injury i.e. uncooked beef), growth phase (logarithmic and late stationary phase), pH (5.6 and 6.2), and storage temperature (3, 10 and 20 degrees C) during a storage period of 30 days. Late logarithmic and late stationary phase cultures of L. monocytogenes 13-249 showed similar growth in refrigerated, vacuum-packed, raw minced beef with a high pH (6.2). In normal pH (5.6) beef there was no growth at 3 degrees C while growth at 10 and 20 degrees C was only observed for logarithmic phase cultures. Heat injured late stationary phase cultures with 95-99.9% injured cells in the surviving population (as measured by differential plating on enriched vs. selective media after sous vide cooking) did not grow or repair sublethal injuries in sous vide cooked beef at 3 degrees C while repair and growth took place at 10 as well as at 20 degrees C. In logarithmic phase cultures heat injury occurred very rapidly and > or = 99.9% heat injury was observed in all trials in spite of much lower pasteurization values and fewer log10 reductions compared with late stationary phase cultures. Regardless of growth phase, all cultures where a high degree of heat injury (> or = 99.9%) was observed, did not subsequently grow in the beef product at 3 or 10 degrees C within 30 days. Growth of heat injured cultures preexposed to heat shock (46 degrees C, 30 min) or slowly rising temperatures (0.3 degrees C min(-1)) before heat injury was also investigated. Heat shocked or heat adapted cultures generally responded in the same manner as non-stressed cultures (no growth at 3 degrees C) except that a longer lag phase was observed in beef processed at slowly rising temperatures and in normal pH beef at 10 degrees C. Although processing at slowly rising temperatures may slightly increase the survival of L. monocytogenes 13-249 in cooked beef, there seem to be no indication of an increase in subsequent growth potential of the surviving cells.     

Predicting heat inactivation of Listeria monocytogenes under nonisothermal treatments

The aim of this study was to find a model that accurately predicts the heat inactivation of Listeria monocytogenes (ATCC 15313) at constantly rising heating rates (0.5 to 9 degrees C/min) in media of different pH values (4.0 to 7.4). Survival curves of L. monocytogenes obtained under isothermal treatments at any temperature were nearly linear. Estimations of survival curves under nonisothermal treatments obtained from heat resistance parameters of isothermal treatments adequately fit experimental values obtained at pH 4.0. On the contrary, survivors were much higher than estimations at pH 5.5 and 7.4. The slower the heating rate and the longer the treatment time, the greater the differences between the experimental and estimated values. An equation based on the Weibullian-like distribution, log S(t) = (t/delta)p, accurately described survival curves of L. monocytogenes obtained under nonisothermal conditions within the range of heating rates investigated. A nonlinear relationship was observed between the scale parameter (delta) and the heating rate, which allowed the development of an equation capable of predicting the inactivation rate of L. monocytogenes under nonisothermal treatments at pH 5.5 and 7.4. The model predictions were a good fit to the measured data independent of the magnitude of the thermotolerance increase. This work might contribute to the increase in safety of those food products that require long heating lag phases during the pasteurization process.     

Behavior of Listeria monocytogenes in the presence of sodium propionate

Survival or growth of Listeria monocytogenes in Tryptose Broth supplemented with 0, 0.05, 0.1, 0.15, 0.2, 0.25 or 0.3% sodium propionate was determined when the pH of the medium was 5.0 or 5.6 and incubation was at 4, 13, 21 and 35 degrees C. The pathogen grew in all controls, propionate-free broth, except at 4 degrees C and pH 5.0. At pH 5.6 and 4, 13, 21 and 35 degrees C the bacterium grew in the presence of all propionate concentrations used in this study. The higher concentrations permitted only minimal growth with smallest ultimate populations and longest generation times. Reducing the pH to 5.0 served to minimize growth further at 13, 21 and 35 degrees C than that observed at the same temperatures but at pH 5.6. The extent of growth was directly proportional to the propionate concentrations; at high concentrations, propionate caused a gradual decrease in populations and/or prolonged the lag phase. At 35 degrees C, a concentration of 0.25% did not allow growth, whereas 0.3% caused inactivation of the pathogen after 80 h of incubation. At 4 degrees C and pH 5.0, all concentrations of sodium propionate caused a gradual decrease in populations during the incubation period.     

Survival of Listeria monocytogenes on sliced cooked sausage after treatment with pediocin AcH

A preparation with pediocin AcH bound to its heat-killed producer cells Lactobacillus plantarum WHE 92 (starter culture ALC01, Wisby, Denmark) by adjusting the pH of the preparation to 6.0 was studied for its effects against Listeria monocytogenes ATCC 7644 and (spoilage) lactic acid bacteria on sliced cooked sausage. The pediocin AcH preparation or 0.9% (w/w) NaCl dilution (as a control) were randomly distributed dropwise on the surface of the slices. Treated slices were vacuum-packed and stored at 6 degrees C. Microbiological analysis and determination of pH values were performed after 3, 6, 9, 14 and 21 days of storage. Flavour of the sausages was evaluated after 7 and 11 days of storage. The pediocin preparation had effect (p > 0.05) neither on the growth of lactic acid bacteria, on the pH value nor on the flavour of vacuum-packed sliced sausage during 21 days of storage compared to control. However, during 6 days of storage, the number of L. monocytogenes decreased from the initial level of 2.7 log cfu/g sausage to < 2 log cfu/g, while on the control sausages the number of L. monocytogenes remained at the inoculated level. The numbers of L. monocytogenes remained at those levels to the end of storage period (21 days). However, the treated samples were determined to be Listeria positive, which indicates that the pediocin preparation was not efficient enough to kill all L. monocytogenes.     

Viability of Clostridium perfringens,Escherichia coli,and Listeria monocytogenes surviving mild heat or aqueous ozone treatment on beef followed by heat,alkali, or salt stress

The threat of pathogen survival following ozone treatment of meat necessitates careful evaluation of the microorganisms surviving under such circumstances. The objective of this study was to determine whether sublethal aqueous ozone treatment (3 ppm of O3 for 5 min) of microorganisms on beef surfaces would result in increased or decreased survival with respect to subsequent heat, alkali, or NaCl stress. A mild heat treatment (55 degrees C for 30 min) was used for comparison. Reductions in three-strain cocktails of Clostridium perfringens, Escherichia coli O157:H7, and Listeria monocytogenes on beef following the heat treatment were 0.14, 0.77, and 1.47 log10 CFU/g, respectively, whereas reductions following ozone treatment were 1.28, 0.85, and 1.09 log10 CFU/g, respectively. C. perfringens cells exhibited elevated heat resistance at 60 degrees C (D60 [time at 60 degrees C required to reduce the viable cell population by 1 log10 units or 90%] = 17.76 min) following heat treatment of beef (55 degrees C for 30 min) but exhibited reduced viability at 60 degrees C following ozone treatment (D60 = 7.64 min) compared with the viability of untreated control cells (D60 = 13.84 min). The D60-values for L. monocytogenes and E. coli O157:H7 following heat and ozone exposures were not significantly different (P > 0.05). C. perfringens cells that survived ozone treatment did not exhibit increased resistance to pH (pH 6 to 12) relative to non-ozone-treated cells when grown at 37 degrees C for 24 h. The heat treatment also resulted in decreased numbers of surviving cells above and below neutral pH values for both E. coli O157:H7 and L. monocytogenes relative to those of non-heat-treated cells grown at 37 degrees C for 24 h. There were significant differences (P < 0.05) in C. perfringens reductions with increasing NaCl concentrations. The effects of NaCl were less apparent for E. coli and L. monocytogenes survivors. It is concluded that pathogens surviving ozone treatment of beef are less likely to endanger food safety than are those surviving sublethal heat treatments.     

Microbial heat resistance of Listeria monocytogenes and the impact on ready-to-eat meat quality after post-package pasteurization

Several methods using bactericides, hydrostatic pressure, and post-package pasteurization technologies to control Listeria monocytogenes (LM) in ready-to-eat meats have been attempted. In addition to controlling LM contamination, any newly developed technology must have minimal effects on organoleptic properties. The objectives of this study were to: (1) determine the heat resistance of LM in two brands (A and B) of bologna differing in formulations, and, (2) evaluate the effects of post-package pasteurization on product quality. Fat content did not affect LM heat resistance in bologna at 55, 60, and 65 °C; however, Brand B bologna had a numerically lower inactivation rate. Microbial heat resistance differed (P < 0.05) with changes in pasteurization temperature. Time and temperature affected (P < 0.05) cook-loss and L^∗ Hunter color value for both bologna brands. These data show that post-package pasteurization is effective but suggest that meat formulations may need modification to prevent development of negative quality characteristics.     

Recoverability of Listeria monocytogenes after coculture with Tetrahymena pyriformis

Bactivory by protozoa is a major factor that limits the number of bacteria in nature and may control the presence of Listeria monocytogenes. The effectiveness of Tetrahymena pyriformis destruction of L. monocytogenes was measured. Within 1 hr, 35–40 T. pyriformis cells ingested an average of 1,219 CFU of L. monocytogenes. Gentamicin was then added to kill un-ingested Listeria. In 24 hr, the recoverable bacteria were reduced at an exponential rate to undetectable levels (<1 per culture). A genetically diverse set of L. monocytogenes cultures all reduced Listeria recovery by the same degree. In assays without addition of gentamicin, numbers of attached L. monocytogenes cells were lessened from an average of log 6.5 CFU/2 ml culture to log 4.7 CFU/2 ml culture. T. pyriformis was capable of lowering numbers of both free-swimming and attached L. monocytogenes. This technology may have applications to control L. monocytogenes in food processing environments.     

Power ultrasound treatment of Listeria monocytogenes in apple cider

Inactivation experiments with Listeria monocytogenes 10403S, an ultrasound-resistant strain, were conducted at sublethal (20, 30, and 40 degrees C) and lethal (50, 55, and 60 degrees C) temperatures in saline solution (pH 7.0), acidified saline solution (pH 3.4), and apple cider (pH 3.4) with and without application of ultrasound (20 kHz, 457 mW.ml(-l)). The survival of recoverable L. monocytogenes 10403S in apple cider was evaluated, and the effects of temperature, ultrasound, pH, and food matrix on inactivation were studied. Application of ultrasound increased the inactivation rate at both sublethal and lethal temperatures. Additional death of L. monocytogenes 10403S was due to low acidity at the lethal temperatures. The reduction in surviving L. monocytogenes 10403S followed first order kinetics at sublethal temperatures, but at lethal temperatures, a two-section linear model described the inactivation behavior. The bactericidal effect of thermosonication was additive in apple cider. The survival tests of L. monocytogenes 10403S in apple cider indicated the possibility of using a mild treatment condition in combination with ultrasound to achieve a 5-log reduction in number of listerial cells.     

Effects of CO2 on the resuscitation of Listeria monocytogenes injured by various bactericidal treatments

To assure the microbiological safety and quality of a food product, a combination of preservation hurdles is often used. Therefore, the effects of carbon dioxide at concentrations of 0, 20, 40 and 60% in modified atmospheres on the resuscitation of Listeria monocytogenes cells injured by mild bactericidal treatments during storage at 7 degrees C were examined. The bactericidal treatments were intense light pulses (ILP), chlorine dioxide (ClO(2)), lactic acid (LA) and heat. The results indicated additional bactericidal effects of CO(2) on cultures treated with LA, ClO(2) and ILP, with additional reductions in viable L. monocytogenes of 0.5-1.0 log cfu/ml. Lag phase duration was significantly different between the different treatments, with non-treated cells having the shortest lag phase, followed by that of heat, intense light pulses, lactic acid and finally ClO(2) treated cells. Maximum growth rate was also estimated and results showed a negative correlation with increasing CO(2) concentrations. A relationship was found between the amount of sub-lethally damaged cells after a mild inactivation treatment and the lag phase duration in the CO(2) environment. Current findings demonstrate the possibility that combining mild decontamination treatments and packaging in a CO(2) enriched environment could reduce the risk of L. monocytogenes infections in food due to an extension of the lag phase.     

Estimation of the survival curve of Listeria monocytogenes during non-isothermal heat treatments

Published survival curves of Listeria monocytogenes under several constant temperatures in the range of 50–65°C could be described by the model Log₁₀[S(t)]=−b(T)t^n(T), where S(t) is the momentary survival ratio, and b(T) and n(T) coefficients whose temperature dependence was expressed by empirical models. When the temperature history T(t) is also expressed algebraically, b(T) and n(T) are transformed into time dependent terms, b[T(t)] and n[T(t)] respectively. If there is no growth and damage repair during the heating process, and the momentary inactivation rate only depends on the momentary temperature and survival ratio, then the solution of the differential equation dLog₁₀[S(t)]/dt=−b[T(t)]*n[T(t)]*{−Log₁₀[S(t)]/b[T(t)]}^∧{(n[T(t)]−1)/n[T(t)]} provides the survival curve under the specified non-isothermal conditions. The validity of this model is demonstrated by the agreement of its predictions, calculated numerically using Mathematica®, to reported survival data of Listeria during heating at a constant and varying rates. Unlike in the traditional calculation methods of microbial survival, the one employed here does not require that microbial mortality be a process following a first or any other order kinetics model.     

Viability of Listeria monocytogenes and Salmonella Typhimurium after isochoric freezing

Isochoric freezing, different from isobaric (conventional) freezing, allows for storage below freezing temperatures without significant damage from ice formation. While several types of tissues have been successfully stored in sub-zero isochoric conditions, it is unknown how isochoric freezing affects pathogenic microorganisms. Thus, the objective of this study was to investigate the survival of Salmonella Typhimurium and Listeria monocytogenes at below freezing storage (<0°C) in isochoric conditions. Tested conditions included storage at −4, −7, and −15°C for 24 hr and at −15°C for 1, 2, 3, 6, 12, and 24 hr. A comparison of bacterial survival during isobaric freezing was included with every trial. Additionally, bacterial cells were examined for morphological damage using transmission electron and field-emission scanning electron microscopes. Isochoric freezing at −15°C for 24 hr reduced both species of bacteria down to unrecoverable levels and maximum efficacy achieved after the 6 hr timepoint for L. monocytogenes and the 12 hr timepoint for S. Typhimurium. When viewed using electron microscopy, S. Typhimurium cells were noticeably disfigured with regions of cytosol separated from the cell wall. The results of this study demonstrate that isochoric freezing is capable of substantial levels of pathogen reduction. Unlike conventional nonthermal interventions, isochoric freezing does not require additional devices such as elevated pressure machines or pulsed electric fields and can be achieved with simple, inexpensive, rigid closed volume containers such as household freezers or commercial cold storage facilities.     

超声波处理对单核细胞增生性李斯特菌生物被膜的清除效果研究

以单增李斯特菌为具体研究对象,构建单菌种生物被膜研究模型,通过电镜定性检测生物被膜的形成,并在此基础上研究了超声清除技术对生物被膜的分离效果。实验结果表明,超声处理的温度,时间和功率对生物被膜的清除率均呈正增长。通过响应面的双因素交互影响分析和标准回归曲线一次项的偏回归方程系数分析,可以得出三个因素对清除率的影响效果顺序为功率>时间>温度。通过单因素及响应面分析优化工艺,得出最优化的反应方案为:超声功率为250W、温度35℃和时间7min,在此条件下进行验证实验,被膜清除率为96.34%。      

超声波处理对单核细胞增生性李斯特菌生物被膜的清除效果研究

以单增李斯特菌为具体研究对象,构建单菌种生物被膜研究模型,通过电镜定性检测生物被膜的形成,并在此基础上研究了超声清除技术对生物被膜的分离效果。实验结果表明,超声处理的温度,时间和功率对生物被膜的清除率均呈正增长。通过响应面的双因素交互影响分析和标准回归曲线一次项的偏回归方程系数分析,可以得出三个因素对清除率的影响效果顺序为功率时间温度。通过单因素及响应面分析优化工艺,得出最优化的反应方案为:超声功率为250W、温度35℃和时间7min,在此条件下进行验证实验,被膜清除率为96.34%。     

Elimination of Listeria monocytogenes on hotdogs by infrared surface treatment

ABSTRACT:  The objective of this research was to develop an infrared pasteurization process with automatic temperature control for inactivation of surface-contaminated Listeria monocytogenes on ready-to-eat meats such as hotdogs. The pasteurization system contained 4 basic elements: an infrared emitter, a hotdog roller, an infrared sensor, and a temperature controller. The infrared sensor was used to monitor the surface temperature of hotdogs while the infrared emitter, modulated by a power controller, was used as a heating source. The surface temperature of hotdogs was increased to set points (70, 75, 80, or 85 °C), and maintained for bacterial kill. The infrared surface pasteurization was evaluated using hotdogs that were surface-inoculated with a 4-strain L. monocytogenes cocktail to an average initial inoculum of 7.32 log (CFU/g). On the average 1.0, 2.1, 3.0, or 5.3 log-reduction in L . monocytogenes was observed after the surface temperature of hotdogs was increased to 70, 75, 80, or 85 °C, respectively. Holding the sample temperature led to additional bacterial inactivation. With a 3 min holding at 80 °C or 2 min at 85 °C, a total of 6.4 or 6.7 logs of L. monocytogenes were inactivated. This study demonstrated that the infrared surface pasteurization was effective in inactivating L. monocytogenes in RTE meats.     

Modeling the combined effect of high hydrostatic pressure and mild heat on the inactivation kinetics of Listeria monocytogenes Scott A in whole milk

The survival curves of Listeria monocytogenes Scott A inactivated by high hydrostatic pressure were obtained at four temperatures (22, 40, 45 and 50 °C) and two pressure levels (400 and 500 MPa) in UHT whole milk. Elevated temperatures substantially promoted the pressure inactivation of L. monocytogenes. A 5-min treatment of 500 MPa at 50 °C resulted in a more than 8-log₁₀ reduction of L. monocytogenes, while at 22 °C a 35-min treatment was needed to obtain the same level of inactivation. Tailing was observed in all survival curves, indicating that the linear model was not adequate for describing these curves. The log-logistic model consistently produced best fits to all survival curves and the modified Gompertz model the poorest. The Weibull model produced fits as good as the log-logistic model at the temperature range of 40–50 °C. The Weibull model provided reasonable predictions of inactivation of L. monocytogenes at temperature levels other than the experimental temperatures; however, the log-logistic model was found to be inferior at predicting inactivation.     

Evaluation of surface contamination and the presence of Listeria monocytogenes in fish processing factories

The main objective of this study was to determine the level of surface contamination in fish processing factories and the presence of Listeria in the factory environment and products. Another objective was evaluation of the different hygiene-monitoring methods. Total aerobic heterotrophic and enterobacteria, yeast and mold samples were collected and ATP levels measured in 28 factories. The number of well or adequately washed and disinfected factories was small (2 of 28), in terms of total aerobic heterotrophic bacterial counts on the surfaces. Most surfaces contaminated with bacteria were heavily contaminated. Results of the ATP and the total bacteria contact agar slide methods were poorly correlated (r = 0.21) although 68% of the samples were categorized as good to moderate or unacceptable with both methods. The Listeria-positive surface samples usually contained increased numbers of total bacteria (70.9%). The contamination of products and raw fish together with Listeria spp. was 45% and with Listeria monocytogenes 12%. Cold smoked fish was the most contaminated, with 75% Listeria spp. and cold salted fish with 20% L. monocytogenes. Listeria innocua was found in the samples more than twice as often as L. monocytogenes.     

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.     

Reduction and survival of Listeria monocytogenes in ready-to-eat meats after irradiation

A five-strain Listeria monocytogenes culture was inoculated onto six different types of ready-to-eat (RTE) meats (frankfurters, ham, roast beef, bologna, smoked turkey with lactate, and smoked turkey without lactate). The meats were vacuum packed and stored at 4 degrees C for 24 h prior to irradiation. Populations of L. monocytogenes were recovered by surface plating on nonselective and selective media. The margins of safety studied include 3-log (3D) and 5-log (5D) reduction of pathogenic bacteria to achieve an optimal level of reduction while retaining organoleptic qualities of the meats. A 3-log reduction of L. monocytogenes was obtained at 1.5 kGy when nonselective plating medium was used. The dosages for 3-log reduction were 1.5 kGy for bologna, roast beef, and both types of turkey and 2.0 kGy for frankfurters and ham on the basis of use of selective medium. The D10-values ranged from 0.42 to 0.44 kGy. A 5-log reduction of L. monocytogenes was obtained at 2.5 kGy with nonselective medium. With selective medium, the dosages were 2.5 kGy for bologna, roast beef, and both types of turkey and 3.0 kGy for frankfurters and ham. Survival of L. monocytogenes in the same RTE meat types after irradiation was also studied. Meats were inoculated with 5 log L. monocytogenes per g and irradiated at doses of 2.0 and 4.0 kGy. Recovery of the surviving organisms was observed during storage at temperatures of 4 and 10 degrees C for 12 weeks. Preliminary results showed no growth in meats irradiated at 4.0 kGy. Survivors were observed for irradiated meats at 2.0 kGy stored at 10 degrees C after the second week. No growth was observed in samples irradiated at 2.0 kGy stored at 4 degrees C until the fifth week.