Non-Thermal Inactivation Models for Listeria monocytogenes

The effects of temperature, lactic acid (or pH), sodium chloride, and sodium nitrite on the non-thermal inactivation of a three strain mixture of Listeria monocytogenes were examined in brain heart infusion broth. A total of 249 survivor curves representing 157 combinations of the four variables were generated. The survivor curves were described mathematically by fitting data using linear and nonlinear primary models. Supplemental studies demonstrated that (1) preculturing the microorganism in an acidic environment or in media containing glucose increased acid tolerance, (2) survivor curve tailing was not due to the presence of a more resistant subpopulation, and (3) the rate of non-thermal inactivation was independent of initial population density. Response surface models were developed for predicting the effects and interactions of the four independent variables on the inactivation of Listeria monocytogenes under adverse environmental conditions.     

Inactivation of Listeria monocytogenes/Pseudomonas biofilms by peracid sanitizers.

The ability of peracetic acid and peroctanoic acid sanitizers to inactivate mixed-culture biofilms of a Pseudomonas sp. and Listeria monocytogenes on stainless steel was investigated. Types of biofilms tested included a 4-h attachment of the mixed-cell suspension and a 48-h biofilm of mixed culture formed in skim milk or tryptic soy broth. Biofilm-containing coupons were immersed in solutions of hypochlorite, peracetic acid, and peroctanoic acid either with or without organic challenge. Organic challenge consisted of either coating the biofilms with milk that were then allowed to dry, or adding milk to the sanitizing solution to achieve a 5% concentration. Surviving cells were enumerated by pouring differential agar directly on the treated surfaces. The peracid sanitizers were more effective than chlorine for inactivating biofilm in the presence of organic challenge. The 48-h mixed-culture biofilm grown in milk was reduced to less than 3 CFU/cm2 by 160 ppm of peracid sanitizer after 1 min of exposure. Peroctanoic acid was more effective than peracetic acid against biofilm cells under conditions of organic challenge. Pseudomonas and L. monocytogenes were inactivated to similar levels by the sanitizer treatments, even though Pseudomonas predominated in the initial biofilm population.     

Inactivation and injury of Listeria monocytogenes as affected by heating and freezing

The effects of heating and freezing Listeria monocytogenes in tryptose phosphate broth an inactivation and injury were investigate. Four strains (Scott A, Brie-1, LCDC 81–861, and DA-3) were examined. Only slight decreases in the viable populations pf all four strains were detected after 75 min at 50°C. Populations of strains Scott A, LCDC 81–861, and DA-3 were reduced to non-detectable levels after 10 min at 60°C while viable cells of strain Brie-1 were recovered after heating at 60°C for 30 min. Viable populations of all four strains declined steadily at 52,54, and 56°C; similar inactivation rates were observed for strains Scott A, LcDC 81–861, and DA-3. Strain Brie-1 was consistently the most heat resistant. Substantial populations of viable cells of all strains heated at 52, 54, and 56°C were injured as evidenced by increased sensitivity to NaCl when plated on tryptose phosphate recover agar. Strain DA-3 was the most susceptible strain to sublethal heat injury. Viable populations of all four test strains were not appreciably reduced after 14 days at −18°C, although strain LCDC 81–861 lost viability at a more rapid rate at this temperature than did the other test strains. About 72, 82, 73, and 80% of Scott A, Brie-1, LCDC 81–861, and DA-3 cells, respectively, held at −18°C for 14 days were not receovered on tryptose phosphate agar (TPA) supplemented with 8% NaCl compared to populations recovered on TPA not supplemented with NaCl.     

Inactivation of Listeria innocua on Frankfurters by Ultraviolet Light and Flash Pasteurization

ABSTRACT:  Listeria monocytogenes , a psychrotrophic foodborne pathogen, is a recurring postprocess contaminant on ready-to-eat meat (RTE) products including frankfurters. Flash (Steam) Pasteurization (FP) and ultraviolet light (254 nm-UVC) has been shown to reduce levels of L. monocytogenes and L. innocua on frankfurters. In this study, the use of UVC light followed by FP to inactivate L. innocua, a nonpathogenic surrogate for L.monocytogenes , on frankfurters that contained sodium diacetate and potassium lactate (SDA/PL) in a pilot-plant setting was investigated. Application of UVC (1.0 J/cm²), followed by FP (0.75 s steam/121 °C) resulted in inactivation of 3.19 log L. innocua , while application of UVC (4.0 J/cm²), followed by FP (3 s steam/121 °C) resulted in inactivation of 3.89 log of L. innocua . A refrigerated storage study (8 °C) of frankfurters that contained SDA/PL that were treated with UVC followed by FP revealed the growth of L. innocua was inhibited for approximately 8 wk following application of the interventions. The use of UVC in combination with FP had little effect on frankfurter color and texture. The combination of UVC, FP, and SDA/PL was found to be an effective hurdle process for decontamination of frankfurter surfaces.     

Ultraviolet Light (254 nm) Inactivation of Listeria monocytogenes on Frankfurters That Contain Potassium Lactate and Sodium Diacetate

ABSTRACT:  Listeria monocytogenes , a psychrotrophic foodborne pathogen, is an occasional postprocess contaminant on ready-to-eat meat (RTE) products including frankfurters. Ultraviolet C light (UVC) is an FDA-approved technology for the decontamination of food surfaces. In this study, the ability of UVC to inactivate L. monocytogenes on frankfurters that contained potassium lactate (PL) and sodium diacetate (SDA), either before or after packaging, was investigated. UVC irradiation of frankfurters that were surface-inoculated with L. monocytogenes resulted in a 1.31, 1.49, and 1.93 log reduction at doses of 1, 2, and 4 J/cm², respectively. UVC treatment had no effect on frankfurter color or texture at UVC doses up to 4 J/cm². Frankfurter meat treated with UVC doses up to 16 J/cm² did not increase mutagenesis in bacterial or human cells, either with or without exogenous metabolic activation. UVC treatment of single-layer frankfurter packs at a dose of 2 J/cm² resulted in a 0.97 (± 0.14) log reduction of L. monocytogenes . Following 8 wk of refrigerated storage L. monocytogenes levels decreased by only 0.65 log in non-UVC-treated frankfurter packs compared with 2.5 log in the UVC-treated packs. Because the numbers of L. monocytogenes associated with contaminations of ready-to-eat meats are typically very low, the use of UVC in combination with potassium lactate and sodium diacetate has the potential to reduce the number of frankfurter recalls and foodborne illness outbreaks.     

Inactivation Kinetics of Listeria monocytogenes by High-Pressure Processing: Pressure and Temperature Variation

The enhanced quasi-chemical kinetics (EQCK) model is presented as a methodology to evaluate the nonlinear inactivation kinetics of baro-resistant Listeria monocytogenes in a surrogate protein food system by high-pressure processing (HPP) for various combinations of pressure (P= 207 to 414 MPa) and temperature (T= 20 to 50 °C). The EQCK model is based on ordinary differential equations derived from 6 "quasi-chemical reaction" steps. The EQCK model continuously fits the conventional stages of the microbial lifecycle: lag, growth, stationary phase, and death; and tailing. Depending on the conditions, the inactivation kinetics of L. monocytogenes by HPP show a lag, inactivation, and tailing. Accordingly, we developed a customized, 4-step subset version of the EQCK model sufficient to evaluate the HPP inactivation kinetics of L. monocytogenes and obtain values for the model parameters of lag (λ), inactivation rate (μ), rate constants (k), and "processing time" (tp). This latter parameter was developed uniquely to evaluate kinetics data showing tailing. Secondary models are developed by interrelating the fitting parameters with experimental parameters, and Monte Carlo simulations are used to evaluate parameter reproducibility. This 4-step model is also compared with the empirical Weibull and Polylog models. The success of the EQCK model (as its 4-step subset) for the HPP inactivation kinetics of baro-resistant L. monocytogenes showing tailing establishes several advantages of the EQCK modeling approach for investigating nonlinear microbial inactivation kinetics, and it has implications for determining mechanisms of bacterial spore inactivation by HPP. Practical Application: Results of this study will be useful to the many segments of the food processing industry (ready-to-eat meats, fresh produce, seafood, dairy) concerned with ensuring the safety of consumers from the health hazards of Listeria monocytogenes, particularly through the use of emerging food preservation technologies such as high-pressure processing.     

Inactivation of Listeria monocytogenes Scott A on artificially contaminated frankfurters by high-pressure processing

Vacuum-packaged frankfurters, inoculated with 24-h cultures of Listeria monocytogenes Scott A (approximately 10(9) CFU/ml) by injection into the packages, were held at pressures of 300, 500, and 700 MPa for up to 9 min. L. monocytogenes were washed from the surface of the frankfurter and plated onto brain heart infusion agar. During the time to achieve 300, 500, and 700 MPa (come-up time), L. monocytogenes populations decreased by 1, >3, and >5 logs, respectively. Additional inactivation of L. monocytogenes occurred while the samples were held at 300 and 500 MPa. A 5-log reduction in bacterial population was possible at all pressure treatments; however, pressurization at 700 MPa showed the fastest inactivation with L. monocytogenes reduced from 10(8) to 10(2) CFU/package during the come-up time. These results show that high-pressure processing may be a viable method for controlling foodborne pathogens in postprocessed, packaged frankfurters.     

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.     

Inactivation of barotolerant Listeria monocytogenes in sausage by combination of high-pressure processing and food-grade additives

Food-grade additives were used to enhance the efficacy of high-pressure processing (HPP) against barotolerant Listeria monocytogenes. Three strains of L. monocytogenes (Scott A, OSY-8578, and OSY-328) were compared for their sensitivity to HPP, nisin, tert-butylhydroquinone (TBHQ), and their combination. Inactivation of these strains was evaluated in 0.2 M sodium phosphate buffer (pH 7.0) and commercially sterile sausage. A cell suspension of L. monocytogenes in buffer (10(9) CFU/ml) was treated with TBHQ at 100 ppm, nisin at 100 IU/ml, HPP at 400 MPa for 5 min, and combinations of these treatments. Populations of strains Scott A, OSY-8578, and OSY-328 decreased 3.9, 2.7, and 1.3 log with HPP alone and 6.4, 5.2, and 1.9 log with the HPP-TBHQ combination, respectively. Commercially sterile sausage was inoculated with the three L. monocytogenes strains (10(6) to 10(7) CFU/g) and treated with selected combinations of TBHQ (100 to 300 ppm), nisin (100 and 200 ppm), and HPP (600 MPa, 28 degrees C, 5 min). Samples were enriched to detect the viability of the pathogen after the treatments. Most of the samples treated with nisin, TBHQ, or their combination were positive for L. monocytogenes. HPP alone resulted in a modest decrease in the number of positive samples. L. monocytogenes was not detected in any of the inoculated commercial sausage samples after treatment with HPP-TBHQ or HPP-TBHQ-nisin combinations. These results suggest that addition of TBHQ or TBHQ plus nisin to sausage followed by in-package pressurization is a promising method for producing Listeria-free ready-to-eat products.     

Inactivation of Listeria monocytogenes on frankfurters by monocaprylin alone or in combination with acetic acid

The antilisterial activity of monocaprylin (MC) and its combination with acetic acid (AA) on frankfurters was investigated. Each frankfurter was surface inoculated with a three-strain mixture of Listeria monocytogenes to obtain an inoculation level of 4.0 log CFU per frankfurter, and then dipped for 35 s in sterile deionized water (45 or 50 degrees C) containing 1% ethanol (control), 50 mM MC plus 1% ethanol, 1% AA plus 1% ethanol, or 50 mM MC plus 1% AA plus 1% ethanol. Samples were vacuum packaged, stored at 4 degrees C for 77 days, and analyzed for L. monocytogenes. Sensory odor and color of frankfurters were evaluated using a 9-point hedonic scale. Color was also objectively measured using the Minolta Chroma Meter. From day 0 to day 77, population counts of L. monocytogenes on frankfurters dipped in antimicrobial solutions at 50 degrees C were consistently lower than the control counts. Similar results were observed for samples treated at 45 degrees C. However, L. monocytogenes grew readily on control samples at both temperatures. Dipping of frankfurters in antimicrobial solutions (45 or 50 degrees C) significantly reduced (P < 0.05) the populations of L. monocytogenes. After 70 days of storage, L. monocytogenes was completely killed in samples dipped in MC+AA solution at 50 degrees C. The antimicrobial treatments did not affect the odor or color of the samples (P > 0.05). Overall, results indicated that dipping of frankfurters with MC reduced L. monocytogenes, and inclusion of AA further enhanced MC antilisterial activity, without any negative effect on odor or color.     

Inactivation of Listeria monocytogenes on agar and processed meat surfaces by atmospheric pressure plasma jets

An apparatus for generating atmospheric pressure plasma (APP) jet was used to investigate the inactivation of Listeria monocytogenes on the surface of agar plates and slices of cooked chicken breast and ham. He, N₂ (both 7 L/min), and mixtures of each with O₂ (0.07 L/min) were used to produce the plasma jets. After treatment for 2 min with APP jets of He, He + O₂, N₂, or N₂ + O₂, the numbers of L. monocytogenes on agar plates were reduced by 0.87, 4.19, 4.26, and 7.59 log units, respectively. Similar treatments reduced the L. monocytogenes inoculated onto sliced chicken breast and ham by 1.37 to 4.73 and 1.94 to 6.52 log units, respectively, according to the input gas used with the N₂ + O₂ mixture being the most effective. Most APP jets reduced the numbers of aerobic bacteria on the meat surfaces to <10² CFU/g, and the numbers remained below that level of detection after storage at 10 °C for 7 days. The results indicate that APP jets are effective for the inactivation of L. monocytogenes on sliced meats and for prolonging the shelf-life of such foods.     

Inactivation of Listeria monocytogenes Scott A 49594 in apple juice supplemented with cinnamon

Normal (pH 3.7) and adjusted (pH 5.0) pasteurized apple juice containing cinnamon (0, 0.1, 0.2, and 0.3%) was inoculated with Listeria monocytogenes Scott A 49594 at 10(4) CFU/ml and stored at 5 and 20 degrees C for 7 days. Counts on tryptic soy agar (TSA), modified Oxford (MOX) medium, and thin agar layer (TAL) were determined at 1 h and 1, 3, and 7 days. The TAL method (MOX medium overlaid with TSA) was used for the recovery of injured cells. In apple juice, both at normal and adjusted pH, with any doses of cinnamon, no L. monocytogenes (a 4.6-log CFU/ml reduction) was detected after 1 h of storage at both temperatures, and no growth occurred at any points of storage. Therefore, cinnamon by itself (regardless of pH) had a pronounced killing effect. A further enrichment step with brain heart infusion agar showed that L monocytogenes was completely inactivated in apple juice stored at 20 degrees C, except in pH 5.0 samples with 0.1% of cinnamon. The TAL method was as effective as TSA in recovering injured cells of L. monocytogenes. Cinnamon considerably inactivates L. monocytogenes in apple juice and thus enhances the safety of this product.     

Inactivation of Listeria monocytogenes and Escherichia coli O157:H7 biofilms by micelle-encapsulated eugenol and carvacrol

Carvacrol and eugenol were encapsulated in micellar nonionic surfactant solutions to increase active component concentrations in the aqueous phase and used to treat two strains of Listeria monocytogenes (Scott A and 101) and two strains of Escherichia coli O157:H7 (4388 and 43895) grown as biofilms in a Centers for Disease Control and Prevention reactor. L. monocytogenes biofilms were grown in two different growth media, 1:20 TSB and Modified Welshimer's broth (MWB), while E. coli O157:H7 was grown in M9. In general, L. monocytogenes strains were more resistant to both micelle-encapsulated antimicrobials than E. coli O157:H7 strains. The two antimicrobials were equally effective against both strains of E. coli O157:H7, decreasing viable counts by 3.5 to 4.8 log CFU/cm(2) within 20 min. For both bacteria, most of the bactericidal activity took place in the first 10 min of antimicrobial exposure. Biofilm morphology and viability were assessed by the BacLight RedoxSensor CTC Vitality kit and confocal scanning laser microscopy, revealing an increasing number of dead cells when biofilms were treated with sufficiently high concentrations of carvacrol- or eugenol-loaded micelles. This study demonstrates the effectiveness of the application of surfactant-encapsulated essential oil components on two pathogen biofilm formers such as E. coli O157:H7 and L. monocytogenes grown on stainless steel coupons.     

Inactivation of Listeria monocytogenes by high hydrostatic pressure: effects and interactions of treatment variables studied by analysis of variance

High hydrostatic pressure is regarded as possible alternative method for food preservation. One of the primary considerations for industrial applications is the ability of this method to eradicate pathogenic micro-organisms. This study subjected L. monocytogenes suspensions, in a phosphate buffer (pH 7·0) or in a citrate phosphate buffer (pH 5·6), to high hydrostatic pressure treatments relative to the following variables: pressure (200–600 MPa), treatment time (3, 10 and 20 min), temperature (4, 20 and 40°C) and the pH of the suspension medium (5·6 and 7·0). An optimal design of 40 runs was obtained using the Fedorov algorithm, and responses were studied by analysis of variance in terms of cell survival on plate count agar. Efficiency was determined by log₁₀comparisons of the numbers of live cells before and after treatment. A statistically significant relationship was found between the four variables considered (pressure, pH, treatment time and temperature), their interactions (treatment time vs pressure, pH vs treatment time, pH vs pressure, pressure vs temperature, treatment time vs temperature) and the inactivation of L. monocytogenes. R -squared statistical analysis indicated that the linear model used accounted for more than 98·5% of the variability in the inactivation ofL. monocytogenes .     

Inactivation and Injury of Listeria monocytogenes in a Minimal Medium as Affected by Benzoic Acid and Incubation Temperature

The population of Listeria monocytogenes in a minimal medium (lacking a nitrogen source) at pH 5.5 decreased logarithmically during incubation. Rate of death of the pathogen, indicated by D-values, was greatly affected by changes in the temperature of incubation (4–35°C), but to a much lesser extent by the presence of benzoic acid (3000 ppm). Injury of L. monocytogenes during incubation was equally detectable on tryptose agar containing 6% salt and McBride Listeria Agar containing 0.5% lithium chloride. A greater degree of injury was detected at the lower than the higher temperatures of incubation, but presence of benzoic acid (3000 ppm) did not seem to affect the extent of injury.     

Assessing biofilm formation by Listeria monocytogenes strains

When a microtitre plate assay was used to quantify biofilm production by Listeria monocytogenes strains following growth in Tryptone Soy Broth (TSB) for 48 h at 20 degrees C, 127 of 138 strains (92.0%) were classified as weak, 9 of 138 strains (6.5%) as moderate and only 2 of 138 strains (1.5%) as strong biofilm formers. The strains included environmental, animal, food (persistent and sporadic strains) and clinical isolates previously typed using esterase electrophoresis (ESE) and multi-locus enzyme electrophoresis (MEE). Strains from different sources produced similar quantities of biofilm, whereas biofilm production by ESE type II strains, irrespective of source, was greater than that observed for other ESE types. No correlation between MEE type and biofilm production was observed. A Petri dish assay which allowed parallel quantification and microscopic examination of biofilms was used to examine biofilm formation by selected L. monocytogenes strains during growth in TSB for 14 days at 20 degrees C. Results from these assays showed that following prolonged incubation, some L. monocytogenes strains categorized as weak biofilm formers by the 48 h microtitre assay, were able to form biofilms similar in terms of quantity and structure to those produced by strains classified as strong or medium biofilm formers. Results from 14-day Petri dish assays confirmed 48 h microtitre assays regarding greater biofilm production by ESE type II strains compared to other ESE types of L. monocytogenes. Biofilm production was similar for ESE type II persistent and sporadic food isolates but reduced for ESE type II clinical strains.