The resistance ofListeria monocytogenesto high hydrostatic pressure in foods

Two strains ofListeria monocytogenes(NCTC 11994, Lm1 and a poultry isolate, Lm2) were exposed to high pressure (375MPa) in ultra high temperature (UHT) treated milk, raw chicken mince, raw beef mince, cooked chicken mince and cooked beef mince for up to 30min at ambient temperature. Survival of the two strains in UHT milk was significantly greater than previously observed in non-nutrient phosphate buffered saline (PBS). Survival in cooked chicken mince and cooked beef mince was only marginally greater than survival in PBS, whilst survival in raw chicken and raw beef mince was generally less than observed in PBS. In all food substrates, the proportion of survivors sustaining injury increased with increasing duration of compression.The two strains were also treated at 375MPa in cooked beef mince for up to 30min at 45°C. Inactivation of both strains was greater at 45°C than at ambient temperature. Treatment at 375MPa for 5min at 45°C resulted in approximately 4.5log₈-cycle reduction; however extending the treatment time resulted in only a slight further decrease in cell counts. The effects of temperature (25, 35, 45 and 55°C) on survival at 200 and 375MPa (15min) in UHT milk was also assessed. Increasing the temperature resulted in increasing levels of inactivation.     

Detection and identification ofListeria monocytogenesin food by PCR and oligonucleotide-specific capture plate hybridization

This paper reports on an ELISA-based detection method for PCR-amplifiedListeria monocytogenesiap gene fragment. During PCR, a label (digoxigenin-11-dUTP) is incorporated in the amplicon. After amplification, the product obtained is hybridized in streptavidin-coated microtitre plates prepared with biotinylated-specific oligonucleotide as a DNA probe, and then an enzyme immunoassay reveals the specifically bound complex, which permits identification ofL. monocytogenes. A total of 48 food samples were tested to validate the method involved. The PCR-oligonucleotide specific capture plate hybridization (OSCPH) is easily applicable and much faster than traditional detection ofL. monocytogenesin food. The hybridization in microtitre plates is also more sensitive than routine agarose gel electrophoresis.     

Modelling combined effects of temperature and pH on the heat resistance of spores ofBacillus cereus

The development of relationships between the pH of a heating medium and the thermal resistance of contaminant micro-organisms is important and of public health significance. A number of mathematical models have been presented in recent years, including that of Mafart and Leguérinel (1998). However, in this model the effect of possible interactions between temperature and pH onD-values was not assessed. The consequences of ignoring interaction terms in models have been assessed, and a comparison with Mafart's model that includes an interaction term showed that interaction terms can be neglected and that Mafart's model can be used in thermal process calculations. It appears possible to adopt a standard value ofZ_pH, for example the 3.6 value, and the conventional concept of biological destruction value L(T) (ratio of the sterilization value and the exposure time at a fixed heating temperature) may then be extended to L(T,pH) (the same ratio at a fixed temperature with a fixed pH of the heating menstruum).     

Modelling the effect of a temperature shift on the lag phase duration of Listeria monocytogenes

The aim of this work is to study and model the effect of a temperature shift on h₀, the product of the growth rate by the lag phase duration (μλ). Our work is based on the data of Whiting and Bagi [Int. J. Food Microbiol. 73 (2002) 291], who studied the influence of both the pre-incubation temperature (T_prior) and the growth temperature (T_growth) on λ values of Listeria monocytogenes. We introduce a new model to describe the evolution of the parameter h₀ as a function of T_prior and T_growth, and compare it to Whiting and Bagi's published polynomial model that describes the influence of T_prior and T_growth on λ independently of μ. For exponential as well as stationary phase cells, h₀ increases almost linearly with the magnitude of the temperature shift. A simple linear model of h₀ turns out to be more suitable to predict λ values than a polynomial model of λ.     

Testing matrix, inoculum size, and incubation temperature affect monolaurin activity againstListeria monocytogenes

Current literature reports conflicting minimal inhibitory concentrations (MIC) (range 10–96 g ml⁻¹) of glycerol monolaurate (monolaurin) againstListeria monocytogenes. To resolve these disagreements, the individual MIC of monolaurin against eightL. monocytogenesstrains was determined using the standard agar dilution technique on three commercial media (trypticase soy agar, plate count agar, and Oxford modified medium), and a catfish-based medium using three inocula sizes and three incubation temperatures. Mean MIC was 16 g ml⁻¹on commercial media at 25 and 35°C. Endpoints at 15°C were twofold lower (8 g ml⁻¹) than at 25 and 35°C. On catfish-based medium, MIC values were four- to eightfold higher (64–128 g ml⁻¹) compared with commercial media and depended on incubation temperature and size of inoculum tested. Poor solubility of monolaurin (85 g ml⁻¹) in aqueous solutions and its reduced activity in the presence of food components limits its application as an antimicrobial agent in foods.     

Model of the influence of time and mild temperature on Listeria monocytogenes nonlinear survival curves

Heat treatment has long been regarded as one of the most widely used and most effective means of destroying pathogens in food. Up to now the linear relationship between the death rate and the temperature has been used when choosing the best heat treatment to apply. However, the information given by this linear relationship is no longer sufficient when nonlinear survival curves are observed. Consequently, the agri-food industry needs a tool to choose the best mild heat treatment to apply in the case of nonlinear survival curves. This study deals with the temperature-induced death of Listeria monocytogenes CIP 7831 in the stationary phase of growth. Eleven temperatures were tested. With the proposed primary and secondary models good fits of our data were obtained. A model describing both the effect of the duration of treatment and the temperature on the logarithm of the number of survivors was then built. A clear increase in the precision of the estimation of the parameters was obtained with this model. Moreover, with this model a new graphical strategy to choose a mild heat increase regarding a maximal survivor number has been proposed.     

Modelling the individual cell lag phase: effect of temperature and pH on the individual cell lag distribution of Listeria monocytogenes

The individual-based approach of the lag phase is gaining interest, especially for pathogens that initially contaminate food products in low amounts. In this paper, the effect of temperature (30, 10, 7, 4 and 2 °C) and pH (7.4, 6.1, 5.5, 5.0, 4.7 and 4.4) on the individual cell lag phase of Listeria monocytogenes was examined in a factorial design, using OD measurements. Individual lag phases of about 100 individual cells per condition were examined and calculated using a linear extrapolation method. Generation times were calculated out of the slope.The obtained data were analyzed at three different levels: in a first approach, the mean values were calculated for each set of environmental conditions and compared to predictions made by the USDA's Pathogen Modeling Program (PMP) for analogous growth conditions. The PMP predictions of the generation times were in the same order of magnitude as the obtained data, although a persistent underestimation could be observed. The observed individual cell lag data differed from lag phase predictions by PMP. Possible reasons for this discrepancy are discussed.Secondly, histograms of individual lag phase measurements were constructed for the different temperature–pH combinations. In this way, the influence of both factors on the variability of individual lag phases could be estimated. At low stress levels, most individual cells showed a short lag phase resulting in a compression of the histograms at the zero-lag level, while, at high stress levels, the histograms shifted to longer lag phases with a significant increase in variability.Thirdly, 37 different distribution types were fitted to the datasets to reveal the distributions that fitted best the obtained data. The gamma distribution was preferred at moderate stress levels, while the Weibull distribution was chosen for harsher growth conditions.     

Evaluation of lactic acid and monolaurin to controlListeria monocytogeneson Stracchino cheese

The behaviour ofListeria monocytogeneswas evaluated during storage of Italian Stracchino cheese dipped in lactic acid (1.4%) or surface treated with (1)-monolauroyl-(rac)-glycerol (monolaurin, 200 μg cm⁻²). The cheese was surface inoculated with approximately 5×10²cfu cm⁻²ofL. monocytogenes, and stored under vacuum at 5°C for 12 days. The lactic acid treatment resulted in lower counts (P<0.05) ofL. monocytogenescompared with counts on untreated cheese washed with water. When lactic-treated cheese was stored at 5°C, levels ofL. monocytogenesdid not change appreciably. Treating cheese with monolaurin also significantly reduced the number ofL. monocytogenes. Furthermore, 12 day counts were less than the untreated control.     

Modelling the influence of the sporulation temperature upon the bacterial spore heat resistance, application to heating process calculation

Environmental conditions of sporulation influence bacterial heat resistance. For different Bacillus species a linear Bigelow type relationship between the logarithm of D values determined at constant heating temperature and the temperature of sporulation was observed. The absence of interaction between sporulation and heating temperatures allows the combination of this new relationship with the classical Bigelow model. The parameters zT and zT(spo) of this global model were fitted to different sets of data regarding different Bacillus species: B. cereus, B. subtilis, B. licheniformis, B. coagulans and B. stearothermophilus. The origin of raw products or food process conditions before a heat treatment can lead to warm temperature conditions of sporulation and to a dramatic increase of the heat resistance of the generated spores. In this case, provided that the temperature of sporulation can be assessed, this model can be easily implemented to rectify F values on account of possible increase of thermal resistance of spores and to ensure the sterilisation efficacy.     

The influence of commonly used selective agents on the growth of Listeria monocytogenes

The effects, upon addition of various selective agents to a rich growth medium (BHIEM) on the growth of Listeria monocytogenes and non-Listeria were studied in microtitre wells. A combination of nalidixic acid (0.1 g/l), acriflavine (0.02 g/l) and fosfomycin (0.1 g/l) added to the medium (BHIEM_NAF) was found to be the least inhibitory to Listeria while at the same time, effectively inhibitory to non-Listeria organisms. Recovery of heat injured Listeria monocytogenes in BHIEM, BHIEM_NAF and the two traditional Listeria-media, UVM I and II, was compared. Recovery in BHIEM was obtained within 24 h of incubation at 30°C, whereas recovery in BHIEM_NAF took up to 48 h. Growth and recovery in UVM I and II never reached the same level as in BHIEM or BHIEM_NAF.     

A predictive model for heat inactivation of Listeria monocytogenes biofilm on buna-N rubber

The purpose of this study was to develop a predictive model for the heat inactivation of Listeria monocytogenes in monoculture (strains Scott A and 3990) and with competing bacteria (Pseudomonas sp. and Pantoea agglomerans) formed on buna-N rubber with and without the presence of food-derived soil. Biofilms were produced on rubber disks in dilute Tryptic Soy broth (dTSB) with incubation for 48 h at 25 °C. Duplicate biofilm samples were heat treated for 1, 3, 5, and 15 min at 70, 72, 75, 77 and 80 °C and tested for survivors using enrichment media. The experiment was repeated six times. A predictive model was developed and plots were generated showing the percent probability of L. monocytogenes inactivation in biofilms after heat treatment. For example, to achieve a 95% probability level of complete inactivation required heat treatment of 76 °C for 6 min. The predicted model was validated using a five-strain cocktail of L. monocytogenes. The validated prediction model indicates that with proper maintenance of the time/temperature controls L. monocytogenes in biofilms on rubber surfaces will be inactivated. This model can be used as a tool in the selection of hot water sanitation processes for rubber surfaces.     

Partial characterization of an antagonistic substance produced byStaphylococcus xylosus1E and determination of the effectiveness of the producer strain to inhibitListeria monocytogenesin Italian sausages

Staphylococcus xylosus1E, isolated from Italian sausages, produced a substance with antagonistic activity againstListeria monocytogenesand other gram-positive bacteria during growth on a solid medium. Inhibitory activity in cell-free supernatant fluids of cultures in various broths was unsuccessfully researched. An active preparation of the inhibitory substance was obtained from the cells of the producer strain by desorption with 9moll⁻¹urea. The inhibitor was not affected by autoclaving (121°C for 15 min), whereas 50% activity remained after pronase and esterase treatment. SDS-PAGE allowed a diffuse smear of stainable material with inhibitory activity to be detected. The 1E inhibitor exhibited a bactericidal mode of action through adsorption to the cells and leakage of nucleic acids. The inhibition ofL. monocytogenesbyS. xylosus1E was also observed in Naples-type sausages. After 21 days of maturation, in salami in whichL. monocytogeneswas challenged withS. xylosus1E, the viable counts of the pathogen were reduced by approximately 2 log cycles, whereas 0.5 log reduction occurred in sausages with the pathogen alone. NoL. monocytogeneswere recovered after 75 days in sausages inoculated withS. xylosus1E, while the pathogen was still present at this time in control sausages.     

Modelling the growth of Listeria monocytogenes in fresh green coconut (Cocos nucifera L.) water

The behaviour of Listeria monocytogenes in the fresh coconut water stored at 4 °C, 10 °C and 35 °C was studied. The coconut water was aseptically extracted from green coconuts (Cocos nucifera L.) and samples were inoculated in triplicate with a mixture of 5 strains of L. monocytogenes with a mean population of approximately 3 log₁₀ CFU/mL. The kinetic parameters of the bacteria were estimated from the Baranyi model, and compared with predictions of the Pathogen Modelling Program so as to predict its behaviour in the beverage. The results demonstrated that fresh green coconut water was a beverage propitious for the survival and growth of L. monocytogenes and that refrigeration at 10 °C or 4 °C retarded, but did not inhibit, growth of this bacterium. Temperature abuse at 35 °C considerably reduced the lagtimes. The study shows that L. monocytogenes growth in fresh green coconut water is controlled for several days by storage at low temperature, mainly at 4 °C. Thus, for risk population this product should only be drunk directly from the coconut or despite the sensorial alterations should be consumed pasteurized.     

Prediction of time to growth of Listeria monocytogenes using Monte Carlo simulation or regression analysis,influenced by sublethal heat and recovery conditions

Stochastic models, including the variability in extent and probability of microbial growth, are useful for estimating the risk of foodborne illness (i.e. Nauta, 2000). Risk assessment typically has to embrace all sources of variability. In this paper, a stochastic approach to evaluate growth of heat damaged Listeria monocytogenes cells influenced by different stresses (pH and presence of eugenol) was performed, using an individual-based approach of growth through OD measurements. Both the lag phase duration and the “work to be done” (h₀ parameter) were derived from the growth curves obtained. From results obtained histograms of the lag phase were generated and distributions were fitted. Histograms showed a shift to longer lag phases and an increase in variability with high stress levels. Using the distributions fitted, predictions of time to unacceptable growth (10² cfu/g) of L. monocytogenes were established by Monte Carlo simulation and they were compared with results from statistical methods. It was evidenced that both methods (Monte Carlo and regression analysis) gave a good indication of the probability of a certain level of growth other than the average. Tornado plots were obtained to establish a sensitivity analysis of the influence of the conditions tested (heat, pH, eugenol) applied to the microorganism and their combinations.     

Evaluation of the effect of defrosting practices of ground beef on the heat tolerance of Listeria monocytogenes and Salmonella Enteritidis

The effect of common defrosting practices of ground beef, including (i) defrosting in the refrigerator (5 °C for 15 h), (ii) defrosting at room temperature (25 °C for 12 h) and (iii) defrosting in the microwave, on the heat tolerance of artificially inoculated Listeria monocytogenes and Salmonella Enteritidis, was studied. The thermal inactivation of S. Enteritidis was not, overall, affected by defrosting practices. In contrast, defrosting at room temperature resulted, overall, in an increased heat tolerance of L. monocytogenes compared to the rest tested defrosting practices. Inactivation kinetics of the two pathogens for the different defrosting practices were determined by fitting the data to the Weibull model. The δ parameter of the Weibull model (heat challenge time (min) required for the first 1-log reduction) for S. Enteritidis and for defrosting at 25 °C, microwave defrosting, defrosting at 5 °C and for the control (fresh ground beef inoculated with the pathogens just before the heat challenge trials) was 1.13, 1.62, 1.60 and 0.96, respectively, while the corresponding values for L. monocytogenes were 20.13, 10.82, 9.95 and 9.47, respectively. The findings of this study should be useful in risk assessments and in developing food handling guidelines for the consumers.     

Modelling of the growth of populations of Listeria monocytogenes and a bacteriocin-producing strain ofLactobacillus in pure and mixed cultures

The cell growth biokinetics of Listeria monocytogenes and a bacteriocin-producing strain ofLactobacillus sake were studied. The organisms were grown both separately and together in a broth system. It was observed that Lact. sake was better suited to growth at low pH than L. monocytogenes. The presence of bacteriocin impacted on L. monocytogenes, by quickly reducing the number of these microbes in mixed populations. A model for the growth ofL. monocytogenes and Lact. sake populations grown separately and in mixed culture is proposed. The model includes a novel approach for better describing the effect of pH change, the ability of Lact. sake to produce bacteriocin and the effect of bacteriocin on L. monocytogenes in the broth culture. Additionally, terms are introduced that describe the competitive effect of one population on the other. The model is fitted to data collected for the growth of each population, either grown alone or together. There were no significant differences between the parameter estimates for each replicate, supporting the hypothesis that the proposed model is a useful representation of the growth and interaction of the two microbial populations.     

Comparing predicting models for heat inactivation of Listeria monocytogenes and Pseudomonas aeruginosa at different pH

Under the same experimental conditions it has been demonstrated that whereas survival curves of Listeria monocytogenes in the range of temperatures from 54 to 62 °C followed a first-order kinetic, those of Pseudomonas aeruginosa in the range of temperatures from 50 to 56 °C were not linear showing a shoulder followed by a linear region. The first order kinetic model did not describe survival curves of P. aeruginosa. A model based on the Weibull distribution (Log₁₀(N_t/N₀)=(1/−2.303)*(t/b)ⁿ)) accurately described the inactivation kinetics of both microorganisms at the three pHs of 4, 5.5, 7.4 investigated. For both microorganisms, the b value depended on the treatment temperature and the pH of the treatment medium. Whereas for L. monocytogenes the n value was independent of the treatment conditions, for P. aeruginosa the n value depended on the pH of the treatment medium.The model based on the Weibull distribution was capable of accurately predicting the treatment time to inactivate five Log₁₀ cycles of both microorganisms at the three pHs investigated.     

胡椒单萜类化合物对单增李斯特菌抑菌机理的研究

本文研究了胡椒单萜类化合物对单增李斯特菌(L.monocytogenes)的抑菌机制,通过分析单增李斯特菌差异蛋白、呼吸链复合体以及三磷酸腺苷酶(ATP酶)等指标,根据同源建模与分子对接技术探索其作用靶点。添加胡椒单萜类化合物可显著抑制单增李斯特菌的生长(p<0.05),同时Na⁺-K⁺-ATPase、Ca²⁺-ATPase、呼吸链复合体I~V活力均显著低于对照组(p<0.05),其中复合体V在蛋白水平显著下调。胡椒单萜类化合物可使单增李斯特菌细胞膜通透性发生变化,抑制ATPase和呼吸链复合体活性,使得ATP合成减少,从而导致菌体衰亡。这为胡椒单萜类化合物应用于食品保鲜提供理论基础。     

Mathematically modeling the repair of heat-injured Listeria monocytogenes as affected by temperature, pH, and salt concentration

Heat-injured cells of Listeria monocytogenes were inoculated into Listeria repair broth (LRB) adjusted to various pH levels (4.2, 5.0, 6.6, 8.0 and 9.6) and salt concentrations (0.5%, 2.5%, 5.0%, 7.5% and 10.0% w/v) at controlled temperatures (4, 10, 22, 37 and 43 °C) in a complete factorial manner (5³). Repair of the injured microorganisms was evaluated using selective and non-selective plating media. The Gompertz parameters, which were generated by fitting the equation with the bacterial counts, were used to calculate the repair percentage as a function of time from which the repair time was estimated. All growth curves fit the Gompertz equation well (R² ≥ 0.972). A first-order model described the repair trend closely (R² = 0.989 ± 0.011). Heat-injured Listeria could fully repair in LRB only under 63 of 125 conditions tested during 21 days of incubation. Refrigeration temperature was the most effective means to prevent the repair of heat-injured Listeria. The minimum temperature required for repair increased with an increase in NaCl concentration. The pH ranges at which the repair could occur were narrower at 4 and 10 °C than those at higher temperature. The repair was observed in media containing 10% NaCl between temperatures of 22 and 43 °C at pH 6.6.