Inactivation kinetics of three Listeria monocytogenes strains under high hydrostatic pressure

High hydrostatic pressure (HHP) inactivation of three Listeria monocytogenes strains (EGDe, LO28, and Scott A) subjected to 350 MPa at 20 degrees C in ACES buffer resulted in survival curves with significant tailing for all three strains. A biphasic linear model could be fitted to the inactivation data, indicating the presence of an HHP-sensitive and an HHP-resistant fraction, which both showed inactivation according to first-order kinetics. Inactivation parameters of these subpopulations of the three strains were quantified in detail. EGDe showed the highest D-values for the sensitive and resistant fraction, whereas LO28 and Scott A showed lower HHP resistance for both fractions. Survivors isolated from the tail of LO28 and EGDe were analyzed, and it was revealed that the higher resistance of LO28 was a stable feature for 24% (24 of 102) of the resistant fraction. These HHP-resistant variants were 10 to 600,000 times more resistant than wild type when exposed to 350 MPa at 20 degrees C for 20 min. Contrary to these results, no stable HHP-resistant isolates were found for EGDe (0 of 102). The possible effect of HHP survival capacity of stress-resistant genotypic and phenotypic variants of L. monocytogenes on the safety of HHP-processed foods is discussed.     

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.     

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.     

Physiological damages of Listeria monocytogenes treated by high hydrostatic pressure

A fluorescent glucose analogue, 2-[N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl) amino]-2-deoxy-D-glucose (2-NBDG), which had been developed previously for the analysis of glucose uptake activity by living cells, was investigated to evaluate its applicability for assaying the viability of yeasts. Fluorescence intensities of the yeast population were measured by fluorescence spectrophotometry upon exposure to antifungal agents after staining with 2-NBDG and were compared to the number of colony forming units (CFU). A good correlation was obtained between the yeast viability, determined by the CFU, and the accumulation of 2-NBDG by yeast cells (correlation constant: r=0.98). Susceptibility testing of amphotericin B and miconazole against yeast strains by plate count and 2-NBDG fluorescence method yielded corresponding results. In conclusion, we found that staining with 2-NBDG is a rapid and sensitive method for the assessment of yeast cell viability.     

Model for Listeria monocytogenes inactivation on dry-cured ham by high hydrostatic pressure processing

The aim of the work was to develop and validate a model of the inactivation of Listeria monocytogenes on dry-cured ham by high hydrostatic pressure (HHP) processing, as a function of the technological parameters: intensity, length and fluid temperature. Dry-cured ham inoculated with L. monocytogenes was treated at different HHP conditions (at 347-852 MPa; for 2.3 to 15.75 min; at 7.6 to 24.4 °C) following a central composite design. Bacterial inactivation was assessed in terms of logarithmic reductions of L. monocytogenes counts on selective media. According to the best fitting and most significant polynomial equation, pressure and time were the most important factors determining the inactivation extent. The significance of the quadratic term of pressure and time indicated that little effect was observed below 450 MPa, whereas holding time longer than 10 min did not result in a meaningful reduction of L. monocytogenes counts. Temperature did not show significant influence at the range assayed. The model was validated with results obtained from further experiments and bibliographical data within the range of the experimental domain. The accuracy factor and bias factor were within the proposed acceptable values indicating the suitability of the model for predictive purposes, such as prediction of the process criteria to meet the Food Safety Objectives. The results of this work may help food processors to select optimum processing conditions of HHP.     

Effect of hydrostatic pressure and antimicrobials on survival of Listeria monocytogenes and enterohaemorrhagic Escherichia coli in beef

The effects of marinades on Listeria monocytogenes and enterohaemorrhagic Escherichia coli were investigated in pressure treated beef steaks. Meat was treated with 600 MPa or 450 MPa. Marinades did not enhance pressure inactivation of E. coli in beef steaks and marinades prevented pressure-induced sublethal injury in L. monocytogenes. Membrane-active essential oils carvacrol and thymol, and thiol-reactive allyl-isothiocyanate (AITC) and cinnamaldehyde were selected to investigate potential synergistic activity of clean label antimicrobials with pressure. Carvacrol accelerated pressure inactivation of E. coli in beef steaks; however, carvacrol increased pressure resistance of E. coli in buffer, and had no effect on survival of E. coli in ground beef. Thymol had no effect in either buffer or meat. AITC and cinnamaldehyde exhibited synergistic activity with pressure on E. coli in buffer; however, cinnamaldehyde did not affect survival of E. coli after pressure treatment of meat. Synergistic inactivation of AITC with pressure was observed only at concentrations that are negatively affect meat quality. AITC and carvacrol may be practically applied for enhancing the bacterial inactivation and extending the shelf life of beef steaks.     

A predictive model that evaluates the effect of growth conditions on the thermal resistance of Listeria monocytogenes

A predictive model for Listeria monocytogenes was developed using cells grown in different pH and milkfat levels before subsequent thermal inactivation in identical pH and milkfat conditions. Inactivation of the cells used combinations of temperature (55, 60, 65 degrees C), pH (5.0, 6.0, 7.0), and milkfat (0%, 2.5%, 5.0%) in a complete 3 x 3 x 3 factorial design with each test done in triplicate. A modified Gompertz equation was used to model nonlinear survival curves with the following three parameter estimates: A for the shouldering region, B for the maximum death rate, and C for the tailing region. All treatment sets were analyzed together in a regression model using the modified Gompertz equation. There was good confidence in the overall model when it was used to predict values for the entire data set. The correlation of determination, R2, between the observed log surviving fraction (LSF) of cells from each of the conditions studied in the experiment, for the overall model was 0.811. For the A and B parameter estimates, temperature or milkfat alone, and the interaction of temperature and milkfat significantly (p < 0.05) affected the shouldering region and maximum death rate of a survival curve, respectively. These results were compared to a previously published predictive model, generated for cells grown under optimum conditions (pH 7.0, 0% milkfat), where pH was the only significant (p < 0.05) factor affecting the shoulder region. These results suggested that the conditions of the growth environment had an important impact on survival curve shape and the estimates of the predictive model. Specifically, there were more factor interactions involving temperature and milkfat level. These growth factors affected the shoulder region and maximum rate of death of the survival curve when cells were grown in identical medium conditions to which they were heated. Differences related to shouldering and inactivation rates for cells grown in different conditions may have important and practical importance for estimating inactivation of L. monocytogenes. This study provides some evidence on the importance of growing conditions when evaluating microbial heat resistance.     

Reduction of counts of Listeria monocytogenes in cheese by means of high hydrostatic pressure

Inactivation of Listeria monocytogenes (strains NCTC 11994 and Scott A) was evaluated in model cheeses submitted to 10 min HHP treatments of 300, 400 or 500 MPa at 5 or 20 degrees C. Counts were measured immediately after high hydrostatic pressure (HHP) treatment (day 1) and after 2, 15 and 30 days of storage at 8 degrees C. Both strains behaved significantly different after 400 and 500 MPa, being NCTC 11994 more sensitive. Scarce differences were found among final values at both HHP treatment temperatures. Initial reductions (log cfu/g) for 400 MPa at 20 degrees C were 2.9 +/- 0.2 for strain NCTC 11994 and 1.5 +/- 0.2 for Scott A. They reached after 30-day storage 5.3 +/- 0.2 and 4.6 +/- 0.4 log cfu/g for NCTC 11994 and Scott A, respectively. For 500 MPa treatments, day-1 reductions of both strains were around 5-log cfu/g, and counts fell below quantification limit after 30 days. Injured cells (around 0.8-log cfu/g) were mostly observed in 400 MPa treated samples on days 1 and 2. Starter cells suffered higher inactivation and injury. For 20 degrees C treatments, its final counts (log cfu/g) at 300, 400 and 500 MPa were: 8.5 +/- 0.2, 5.4 +/- 0.3 and 2.5 +/- 0.1, respectively. These figures evidence the HHP potential to improve safety of cheese products.     

Application of high hydrostatic pressure to eliminate Listeria monocytogenes from fresh pork sausage.

Ground pork patties were inoculated separately with 10(9) CFU/g each of three strains of Listeria monocytogenes obtained from the National Animal Disease Center (NADC). Inoculated patties were packaged under vacuum and treated at 414 megapascals (60,000 lb/in2) for up to 60 min by high hydrostatic pressure (HHP). Survivors were determined by surface plating onto modified Oxford agar and trypticase soy agar with yeast extract, as well as by the most probable number method using Listeria enrichment broth. Average D values ranged from 1.89 to 4.17 min, depending on the strain, with the most virulent strain (reported by the NADC) having the highest D value. We tested the usefulness of applying a mild heat treatment at 50 degrees C, simultaneously with HHP, to lower these values. Average D values ranged from 0.37 to 0.63 min, depending on the strain. Thus, a 10-log10 reduction could be achieved even in the most pressure-resistant strain of L. monocytogenes by a 6-min application of heat and HHP. Shelf life studies were also conducted, with spoilage levels reached after 5 days of storage at 4 degrees C for controls versus 28 days for treated samples. Sensory evaluation of uninoculated grilled patties showed that panelists could not distinguish between those treated by heat and HHP and untreated controls (P<0.05). Thus, treatment by HHP in combination with mild heating can be used successfully to produce safer, longer-lasting fresh pork without affecting quality.     

Modeling survival of high hydrostatic pressure treated stationary- and exponential-phase Listeria innocua cells

High Hydrostatic Pressure (HHP) inactivation (325–400 MPa; 0–20 min; maximum temperature 30 °C) of cells of Listeria innocua CECT 910 was studied in two different growth phases (exponential and stationary), and the corresponding survival curves were obtained for each case. The curves were fitted to two nonlinear models, the modified Gompertz equation and the Baranyi model. The kinetic constants calculated for both models, µ_max and k_max, indicated that cells in exponential growth phase were more sensitive to pressure than those in stationary phase. Both mathematical models were suitable for describing L. innocua HHP survival curves, rendering kinetic constants that increased with increasing pressure. When considering the experimental models validation, both Gompertz and Baranyi predicted in a similar way, however Baranyi had slightly lower A_f (Accuracy factor) and B_f (Bias factor) values, which indicated better prediction values. In summary, both mathematical models were perfectly valid for describing L. innocua inactivation kinetics under HHP treatment.Industrial relevanceThe mathematical models for inactivation and growth of microorganisms are the foundation of predictive microbiology and are used in risk assessments procedures as part of the food safety management system. Besides, these models together with those applied to inactivation of enzymes and destruction of quality factors are essential to optimize processes and thus to lay the foundations for industrial processing. It is therefore necessary to identify generally applicable kinetic models that will produce primary and secondary kinetic parameters and are statistically reliable as a key tool to predict the behaviour of microorganisms, enzymes and quality factors after processing.     

Effects of nisin and acid on the inactivation and recovery of Listeria monocytogenes biofilms treated by high hydrostatic pressure

Food Science and Biotechnology - This study was designed to evaluate the inactivation, detachment, and recovery properties of Listeria monocytogenes biofilms treated by the combined high...     

pH and solute concentration of suspension media affect the outcome of high hydrostatic pressure treatment of Listeria monocytogenes

The effect of pH and solute concentration of suspension media on high hydrostatic pressure (HHP) induced inactivation of Listeria monocytogenes (approximate 10(8) CFU/ml) was investigated by the using treatment between 300 MPa and 600 MPa at 25 degrees C for 10 min. The suspension media used in this study represented different concentrations (0.1% to 10%) of buffered peptone water (BPW) with an adjusted pH of 4 to 7. An increase in the concentration of BPW resulted in a decreased HHP-induced inactivation of L. monocytogenes that was dependent on the pH of the medium. HHP-treatment at 300 MPa showed no bactericidal effect at neutral pH regardless of the BPW concentration. When the pH of BPW (0.1% to 5%) was reduced to 4, L. monocytogenes was completely inactivated (more than an 8 log cycle reduction) with a HHP-treatment of at least 300 MPa. HHP-treatment above 400 MPa completely inactivated L. monocytogenes in a relatively dilute BPW (0.1% and 1%) with an adjusted pH below 6. While only a 2 log cycle reduction was observed in 10% BPW at the pH ranging from 5 to 7 after treatment with 600 MPa, L. monocytogenes in 10% BPW at pH 4 was completely inactivated. Even though a significant bactericidal effect of HHP-treatment was not observed when applied with a low pressure such as 300 MPa or suspended in higher BPW at neutral pH, a reduction of the pH greatly affected the HHP-induced inactivation of L. monocytogenes. These results indicated that information concerning the pH of food or media would greatly assist an optimization of HHP-treatment for the inactivation of bacteria.     

Inhibition of Listeria monocytogenes and Salmonella by natural antimicrobials and high hydrostatic pressure in sliced cooked ham

The effectiveness of nisin, lactate salts, and high hydrostatic pressure to inhibit the growth of Listeria monocytogenes and Salmonella in sliced cooked ham was studied through a combination of PCR-based detection methods, most probable number, and classical microbial enumeration techniques (International Organization for Standardization protocols). A synergistic effect to inhibit a cocktail of Listeria monocytogenes CTC1010, CTC1011, and CTC1034 was observed between potassium lactate, high hydrostatic pressure (400 MPa, 17 degrees C, 10 min), and low storage temperature when sliced cooked ham was stored for 84 days at 1 degrees C. The high hydrostatic pressure treatment also proved to be useful to inhibit a cocktail of Salmonella enterica serotypes London CTC1003, Schwarzengrund CTC1015, and Derby CTC1022.     

Effects of ultra high hydrostatic pressure on Listeria monocytogenes and natural flora in broth, milk and fruit juices

Listeria monocytogenes was subjected to ultra high hydrostatic pressure (UHHP) treatments from 200 to 700 MPa at 25 °C in broth, raw milk, peach juice and orange juice. Survivor curves showed that cell death increased as pressure increased. After 10 min pressure treatment at 400 MPa reductions of about 2.09 and 2.76 log CFU mL^?1 in aerobic bacteria and L. monocytogenes, respectively, were produced in raw milk, this increased to 5.09 and 6.47 log CFU mL^?1, respectively, at 600 MPa. Death of bacteria at UHHP treatment was greater in orange juice than peach juice, and in peach juice than milk. Listeria monocytogenes was more sensitive to increased pressure than increased pressurization time. Injury of L. monocytogenes occurred from 0 to 100%. Factors effecting the rate of microbial inactivation are: pressure, age of cell, composition of medium, and pressurization time. UHHP inactivation can be used to extend shelf life and increase food quality during storage, and may also contribute to inactivation of L. monocytogenes.     

High hydrostatic pressure processing of sliced fermented sausages: A quantitative exposure assessment for Listeria monocytogenes

Fermented sausages have traditionally been considered to be safe products from a microbiological point of view, mainly due to nitrite addition, their low a_w and reduced pH. However, post-process contamination during slicing and packaging operations may increase microbial concentration and prevalence on final products. A stochastic simulation modelling approach was conducted to determine the extent of Listeria monocytogenes survival on sliced chorizo submitted or not to high hydrostatic pressure (HHP) treatments after post-process contamination (i.e., cross-contamination during slicing). A probabilistic model comprising nine steps from mixing of raw materials to consumption was constructed. The effects of various initial levels of L. monocytogenes in the meat batter (−1.43–3 log cfu/g), HHP treatments (400–600 MPa/18 °C for 0–12 min) and nitrite concentrations (0–150 ppm) on L. monocytogenes distribution were assessed by means of the application of predictive models, literature information and data obtained experimentally. Once implemented, the probabilistic model was simulated by using Monte Carlo analysis. The probability distribution of L. monocytogenes contamination levels was determined for various scenarios. Model outputs showed that cross-contamination during slicing was an important source contributing to increase pathogen prevalence and concentration on final products, with transferred levels equal to 0.59 ± 0.48 log cfu/g. Under all simulated scenarios, formulation and storage conditions, the level of L. monocytogenes on sliced vacuum-packed chorizo at the consumption phase was estimated to be lower than 100 cfu/g and pressure treatments at 600 MPa for 10–12 min would result in non-contaminated packs. Overall, the probabilistic model developed in this study from raw material reception up to the end of the shelf-life (i.e., 90 days) of sliced fermented sausages is proposed as a suitable tool to determine combinations of HHP treatments and nitrite concentrations ensuring the compliance with microbiological criteria.     

Predictive model for inactivation of Campylobacter spp. by heat and high hydrostatic pressure

Campylobacter represents one of the leading causes of foodborne enteritis. Poultry and its products frequently transmit the pathogen. The objective of the present study was to model predictively the short-term inactivation of Campylobacter in a ready-to-eat poultry product to develop an economic high-pressure treatment. We inactivated baroresistant strains of Campylobacter jejuni and Campylobacter coli, grown to stationary phase on nutrient agar and inoculated in poultry meat slurry, by heat and high hydrostatic pressure. Incubation at ambient pressure at 70 degrees C for 1 min and at 450 MPa at 15 degrees C for 30 s inactivated more than 6 log CFU of this foodborne pathogen per ml of poultry meat slurry. Thermal and pressure inactivation kinetics of C. coli and C. jejuni in poultry meat slurry were accurately described by a first-order kinetic model. A mathematical model was developed from 10 to 65 degrees C and from ambient to 500 MPa that predicts the reduction in numbers of Campylobacter in response to the combination of temperature, pressure, and treatment time. We suggest the high-pressure treatment of foods to avoid health risks caused by Campylobacter. The nonthermal short-term treatment of the examined food model system represents a successful step to an economic high-pressure procedure.     

Susceptibility of Listeria monocytogenes to high pressure processing: A review

Listeria monocytogenes has been regarded as an emerging food pathogen responsible for listeriosis, a serious disease given its high mortality rate. The need for better food processing methods has led to an increased interest in high pressure processing (HPP), a novel nonthermal method presented as “producer” of safer food products. This review provides an overview of the effects of HPP on Listeria monocytogenes and on L. innocua, with the latter often used as an amenable surrogate for the pathogenic species. The factors that affect the susceptibility of listeriae to HPP, as well as the long-term implications of postprocessing recovery, are discussed in the perspective of the use of HPP to improve the safety of potential food vehicles.     

A model describing the relationship between regrowth lag time and mild temperature increase for Listeria monocytogenes

In order to comply with the consumer demand for ready-to-eat and look 'fresh' products, mild heat treatment will be used more and more in the agrofood industry. Nonetheless there is no tool to define the most appropriate mild heat treatment. In order to build this tool, it is necessary to study and describe the response of a bacterial population to a mild increase in temperature, from the dynamic point of view. The response to a mild increase in temperature, defined by stress duration and temperature, consisted in a mortality phase followed by the lag time of the survivors and their exponential growth. The effect of the mild increase in temperature on the mortality phase was described in a previous paper (Bréand et al., Int. J. Food Microbiol., in press). The effect of the stress duration on the lag was presented in a previous paper (Bréand et al., Int. J. Food Microbiol. 38 (1997) 157-167). In particular, the biphasic relationship between the lag and the stress duration was observed and modelled with a four parameter nonlinear model: the primary model (Bréand et al., Int. J. Food Microbiol. 38 (1997) 157-167). The study presented in this paper deals with the effect of the stress temperature on the biphasic relationship between the lag time and the stress duration. The secondary models describing the effect of the stress temperature on this biphasic relationship, were empirically built from our experimental data concerning Listeria monocytogenes. This work pointed out that the higher the stress temperature, the narrower the range of stress duration for which the lag time increased. Since the primary and the secondary models of the lag time were available, the global model describing the effect of the mild increase duration and temperature directly on the lag was fitted. This model allowed an improvement of the parameter estimator precision. The potential contribution in mild heat treatment optimization of this global model and the one built for the mortality phase (Bréand et al., Int. J. Food Microbiol., in press) is discussed.     

High hydrostatic pressure and biopreservation of dry-cured ham to meet the Food Safety Objectives for Listeria monocytogenes

This work aimed to evaluate the effect of nisin application (biopreservation) combined with high hydrostatic pressure processing (HHP) on the behavior of Listeria monocytogenes CTC1034 intentionally inoculated (at ca. 10(7)cells/g) onto the surface of ready-to-eat (RTE) sliced dry-cured ham. Two types of dry-cured ham, which had different water activities and fat contents were studied (a(w) of 0.92 and 14.25% fat and a(w) of 0.88 and 33.26% fat). Three batches were prepared for each type of product: (C) control, without nisin; (N) nisin directly applied (200 AU/cm(2)) and (F) nisin applied through active packaging, polyvinyl alcohol films with 200 AU/cm(2). Half of the samples were pressurized at 600 MPa for 5min. Counts of L. monocytogenes were periodically monitored throughout 60 days of storage at 8°C. The physico-chemical characteristics of the products enabled the survival of L. monocytogenes, but it was significantly reduced by the presence of nisin. The effect of biopreservation was greater when applied directly to the surface and in the product with lower water activity in comparison with the active packaging and the high water activity products, respectively. The immediate inactivation of L. monocytogenes by HHP ranged from 1.82 to 3.85 Log units, depending on the type of dry-cured ham. The lower the water activity, the less was the inactivation induced by HHP, both immediately and during storage. The reduction of L. monocytogenes immediately after HHP and during storage was more evident in batches with nisin applied directly to the surface of the product. The pathogen was not detected in some samples from day 5 of storage in the product with higher water activity. The effect of nisin applied through active packaging was lower than the direct application. The results of the present study indicated that HHP, as post-processing listericidal treatment, is more effective (both immediately and long term) than the use of nisin as an antimicrobial measure. However, the both hurdles combined (i.e. biopreservation and HHP) provided a wider margin of safety in the control of L. monocytogenes during the storage of RTE cured meat products.