Inactivation of Salmonella,E. coli and Listeria monocytogenes in phosphate-buffered saline and apple juice by ultraviolet and heat treatments

Two strains of Escherichia coli (K-12 and O157:H7), Salmonella (enteritidis and typhimurium) and Listeria monocytogenes (AS-1 and M24-1) were individually suspended in phosphate-buffered saline (PBS) and apple juice prior to exposure to UV radiation (220–300 nm) and heating at 55 °C. The calculated decimal reduction times (D value, min) varied with suspending medium and mode of inactivation. The AS-1 and M24-1 strains of L. monocytogenes were found to be most resistant to UV in PBS (0.28–0.29 min) while the AS-1 strain was most resistant in juice (1.26 min). The AS-1 strain of L. monocytogenes and E. coli O157:H7 were most heat resistant when suspended in PBS (4.41 min) and juice (4.43 min), respectively. Results obtained from this study may be used by apple juice processors in selecting appropriate organisms for UV irradiation or heat treatment lethality validations.     

Inactivation of Listeria monocytogenes inoculated on disposable plastic tray,aluminum foil,and paper cup by atmospheric pressure plasma

The objective of this study was to investigate the effect of atmospheric pressure plasma (APP) on Listeria monocytogenes inoculated onto disposable food containers including disposable plastic trays, aluminum foil, and paper cups. The parameters considered in APP processing were input power (75, 100, 125, and 150 W) and exposure time (60, 90, and 120 s). The bacterial reduction in the disposable plastic trays, aluminum foil, and paper cups was associated with increased input power and exposure time of APP. The D₁₀ values were calculated as 49.3, 47.7, 36.2, and 17.9 s in disposable plastic trays, 133, 111, 76.9, and 31.6 s in aluminum foil and 526, 65.8, 51.8, and 41.7 s in paper cups at 75, 100, 125, and 150 W of input power, respectively. There were no viable cells detected after 90 and 120 s of APP treatment at 150 W in disposable plastic trays. However, only three decimal reductions of viable cells were achieved in aluminum foil and paper cups at 150 W for 120 s. These results demonstrate that APP treatment is effective for inactivation of L. monocytogenes and applicable for disposable food containers. However, the type of material is crucial and appropriate treatment conditions should be considered for achieving satisfactory inactivation level.     

Inactivation of Listeria monocytogenes and Listeria innocua in yogurt drink applying combination of high pressure processing and mint essential oils

Influences of high pressure processing (HPP) on some physical properties and on inactivation of Listeria monocytogenes and Listeria innocua in a yogurt drink (ayran) were quantified with or without addition of mint essential oil. Pressure treatment alone or combined with mint essential oil did not cause significant changes in pH, water activity, color and serum protein separation (p > 0.05). HPP of ayran samples at 600 MPa for treatment time of 300 s reduced L. monocytogenes and L. innocua by more than 5-log units (p ≤ 0.05) at ambient temperature. Addition of mint essential oil further enhanced inactivation of both bacteria by more than 1 log cfu mL^?1. Combination of mint essential oil with HPP provided a reduction in pressure treatment severity by 100–300 MPa or by 210 s to achieve the same amount of inactivation relative to HPP alone. Both Weibull distribution and log-logistic models were fitted to survival data. High pressure-processing combined with mint essential oil appeared to be a promising technique for preserving microbiologically-safe ayran with no significant impacts to product quality.     

Inactivation of Staphylococcus spp. strains in whole milk and orange juice using ultra high pressure homogenisation at inlet temperatures of 6 and 20 °C

The objective of this work was to evaluate the inactivation induced by ultra high pressure homogenisation (UHPH) of Staphylococcus aureus ATCC 13565 and Staphylococcus carnosus CECT 4491 inoculated into milk and orange juice considering the effect of inlet temperature of the sample (6 and 20 °C) on the lethality values and on the production of sublethal injuries. Samples of UHT whole milk and UHT orange juice were inoculated at a concentration of approximately 7.0 log (CFU/ml) and pressurized with a dual valve UHPH machine at 300 MPa at the primary homogenising valve and at 30 MPa on the secondary valve. Viable and injured bacterial counts were measured 2 h after UHPH treatment and after 3, 6, and 9 days of storage at 4 °C for milk, and after 3, 6, 9, 12, and 15 days of storage at 4 °C for orange juice. The inlet temperature, the food matrix and the kind of strain influenced significantly (P < 0.05) the lethality level, which was higher for S. aureus in whole milk at an inlet temperature of 20 °C. No sublethal injuries were detected after treatments. The change over time of viable counts for both strains showed a very strong decreasing tendency during the storage at 4 °C for orange juice, while the strain S. carnosus showed a low decreasing tendency and greater resistance when inoculated in milk and pressurized at 6 °C.     

Simple and rapid detection of Listeria monocytogenes in fruit juice by real-time PCR without enrichment culture

We developed a simple, rapid procedure for the detection of Listeria monocytogenes in unpasteurized fruit juice using real-time PCR without enrichment culture. PCR inhibitors were removed from fruit juices using Chelex resin followed by gel filtration with a Sephadex column. The purification step can be completed in 20 min, and purified juice samples can be used directly as PCR templates without further dilution. PCR conditions were optimized and maximum sensitivity (ca. 1 cell/reaction) was achieved. This convenient method should prove useful for high-throughput surveillance of L. monocytogenes as well as other food-borne pathogens that may contaminate fruit juices.     

Quantification of persistence of the food-borne pathogens Listeria monocytogenes and Salmonella enterica during manufacture of Italian fermented sausages

The objective was to evaluate the survival capability and quantify the persistence of the food-borne pathogens Listeria monocytogenes and Salmonella enterica in the Cacciatore, Felino and Milano type salami preparation. The batter of each sausage was inoculated with a five-strain cocktail of L. monocytogenes or S. enterica (ca. 10⁴–10⁵ CFU/g) and their progression was monitored at specific time intervals during preparation of the sausages. Four different batches were prepared at different times for each sausage/pathogen combination. Different models were used to fit the experimental data and to calculate the kinetic parameters. The best model was chosen based on statistical comparisons. S. enterica proved to be more sensitive susceptible to fermentation and ripening processes than L. monocytogenes. Both pathogens, however, survived relatively well as the result of the conditions (pH, a_w and fermentation temperature) prevailing during fermentation and ripening of the sausages. Water activity proved to be a key factor in the survival of the microorganisms. The statistical analysis of quantitative data gathered from challenge tests is useful for the food business operators as it can provide practical information on the process parameters combinations that could lead to a better control of the pathogens.     

Ralstonia insidiosa serves as bridges in biofilm formation by foodborne pathogens Listeria monocytogenes,Salmonella enterica,and Enterohemorrhagic Escherichia coli

Biofilm formation on abiotic surfaces in fresh produce processing facilities may play a role in foodborne outbreaks by providing protective microniches for pathogenic bacteria. Our previous study showed that a strain of Ralstonia insidiosa isolated from a fresh produce processing plant could enhance the incorporation of Escherichia coli O15:H7 in biofilms under various environmental conditions. These results raised the concern that R. insidiosa might have the ability to incorporate other foodborne pathogens and promote their survival and growth in biofilms. To test this hypothesis, 6 strains of Shiga toxin producing E. coli, 2 strains of Salmonella, and 6 strains of Listeria monocytogenes were examined for dual-species biofilm formation with R. insidiosa. A significant increase in biomass formation was observed in 7 of the 14 R. insidiosa-pathogen combinations, while significantly enhanced incorporation of pathogenic cells into biofilms was seen in 12 of the 14 R. insidiosa-pathogen combinations. The synergistic interactions between R. insidiosa and the tested foodborne pathogens seemed dependent on intimate cellular contact between the two strains. Overall, this study showed that R. insidiosa could enhance the incorporation of biofilms of different types of foodborne pathogenic bacteria and should be considered a bridging bacterium for biofilm formation in various food processing environments.     

Effects of high pressure homogenization on the activity of lysozyme and lactoferrin against Listeria monocytogenes

The effects of high pressure homogenization treatment at 100 MPa (HPH), in comparison to different heat treatments, 70 °C for 30 s, 70 °C for 5 min or 100 °C for 5 min, on the activity of lysozyme and lactoferrin, were studied. The antimicrobial activities of lysozyme and lactoferrin were tested on Listeria monocytogenes inoculated in milk or cultural medium.The results indicated that antimicrobial activities of lactoferrin and lysozyme were enhanced and/or accelerated by HPH treatment. Particularly, the highest immediate inactivation values were recorded when L. monocytogenes cells were added to HPH-treated lactoferrin, processed simultaneously or separately with the target microorganism. Although to a lesser extent than HPH treatment the heat treatments applied also were able to increase the antimicrobial activity of lysozyme.     

Characterisation of the resistance and the growth variability of Listeria monocytogenes after high hydrostatic pressure treatments

The use of non-thermal methods for food preservation is due to consumer demands for microbiological safe products, without changes in the sensory and nutritional qualities of the product. High hydrostatic pressure (HHP) has emerged as an alternative to traditional thermal processing methods for foods.Listeria monocytogenes CECT 5672 was treated under HHPs (350, 400 and 450 MPa) for 3, 16 and 23 min. The effects of pH (5, 6 and 7) and sodium chloride concentration (0, 0.5 and 1.0) of the recovery medium were studied on L. monocytogenes. The kinetic parameters were estimated by the method described by Métris, George, and Baranyi (2006). From results obtained, histograms of the lag phase were generated and distributions were fitted. The duration of the lag phase of HHP damaged cells increased with the application of additional stresses. Histograms showed a shift to longer lag phases and an increase in variability with high stress levels in the recovery medium.Using a primary model together with Monte Carlo simulation, predictions of time to growth (100 cfu/g) of L. monocytogenes were established and they were compared with deterministic predictions. It was evidenced that deterministic predictions do not give a good indication of the probability of a certain level of growth.     

Reduction of Escherichia coli O157, Listeria monocytogenes and Salmonella enterica sv. Typhimurium populations on fresh-cut bell pepper using gaseous ozone

Bacterial contamination is the main cause of food poisoning which can lead to diarrhoea, abdominal cramp, vomiting as well as death. Bacterial contamination can potentially be controlled by application of gaseous ozone due to its antibacterial activity. Therefore, the objective of this study was to investigate the efficacy of gaseous ozone for reducing food-borne pathogens such as Escherichia coli O157, Listeria monocytogenes and Salmonella enterica sv. Typhimurium on fresh-cut bell pepper. Efficacy of gaseous ozone to reduce bacterial populations was investigated in vitro and in vivo. Results showed that optimum effect of ozone on reducing bacterial populations was achieved with short term exposures of less than 6 h. Ozone reduced the bacterial population by disrupting bacterial cell structure, which lead to cellular death. Results also showed that bacterial cells have different resistance to ozone where ozone was more effective against L. monocytogenes, followed by E. coli O157 and Salmonella Typhimurium. Optimal reduction of the bacterial population on fresh-cut bell pepper was achieved with exposure to 9 ppm ozone for 6 h. This treatment reduced 2.89, 2.56 and 3.06 log of E. coli O157, Salmonella Typhimurium and L. monocytogenes populations, respectively. In conclusion, exposure to 9 ppm ozone for 6 h helps in reducing food-borne pathogen on fresh-cut bell pepper and has high potential to be an alternative sanitization treatment to reduce pathogen population on fruit.     

Effects of changes in pH and temperature on the inhibition of Salmonella and Listeria monocytogenes by Allyl isothiocyanate

The minimum inhibitory (MIC) and minimum bactericidal concentrations (MBC) of mustard allyl isothiocyanate (AITC) against five strains each of Salmonella and Listeria monocytogenes individually, and when combined by genus were studied in Mueller-Hinton broth at 21 or 37 °C as well as the interactive effects of pH (5.0–9.0) at temperatures of 4–21 °C on cell viability when held under these conditions for up to 10 d. The stability of 200 ppm AITC was monitored during these trials. The MIC and MBC values of AITC ranged from 60 to 100 ppm and 120–180 ppm, respectively, at 37 °C and ranged from 10 to 40 ppm and 200–600 ppm, respectively, at 21 °C against both pathogens. AITC had no antimicrobial activity at low temperatures (4 or 10 °C) and alkaline pH over 10 d, but at neutral pH, AITC reduced L. monocytogenes by 4.14 and 8.45 log₁₀ CFU/ml at 4 or 10 °C, respectively. At acidic pH, AITC was more effective against Salmonella which was reduced by 2.56 and 6.48 log₁₀ CFU/ml at 4 and 10 °C, respectively. However, AITC was more effective at combinations of 21 °C and neutral pH against L. monocytogenes (cells were not detected at and beyond 3 d) and at combinations of the higher temperature and acidic pH against Salmonella (cells were not detected at and beyond 6 d). Mustard AITC was more stable at low pH and temperature indicating that it can be an effective antimicrobial at combinations of low or neutral pH values and room or refrigerator temperatures (4–10 °C) against these foodborne pathogens.     

Evaluation of Zataria multiflora Boiss. essential oil activity against Escherichia coli O157:H7, Salmonella enterica and Listeria monocytogenes by propidium monoazide quantitative PCR in vegetables

Essential oils (EOs) have long been applied as flavoring agents in foods, and due to their content in antimicrobial compounds, they have potential as natural agents for food preservation. Recently, real-time PCR in combination with PMA has successfully been applied to discriminate between live Escherichia coli O157:H7 and dead bacteria killed by cumin, clove, oregano and cinnamon EOs. In this study, initial experiments were performed in order to elucidate the minimum bactericidal concentration of Zataria multiflora EOs on E. coli O157:H7, Salmonella enterica and Listeria monocytogenes. Thereafter PMA-qPCR was applied in order to selectively quantify life cells within a bacterial population treated with Z. multiflora EO. Inactivation was obtained at EO concentrations of 0.02, 0.035, 0.045 for L. monocytogenes, E. coli O157:H7 and S. enterica, respectively. L. monocytogenes were totally killed in 30 min while it took 1 h 30 min for the gram negative pathogens. As a conclusion Z. multiflora EO has potential as natural food additive or biopreservative since it was able to irreversibly inactivate the three pathogens tested, at lower concentrations than other EOs and short exposition times. In addition, the PMA-qPCR approach proved efficient to selectively detect live pathogenic bacteria in vegetables following inactivation with Z. multiflora EO.     

The determination of ready-to-eat foods into Listeria monocytogenes growth and no growth categories by challenge tests

To assess the potential for a food to allow the growth of Listeria monocytogenes throughout its shelf life and be compliant with European Commission regulations, a challenge test was designed by the EU Community Reference Laboratory. The procedure for the determination of the growth potential includes the following: product characteristics, shelf-life of the product, number of batches, choice of the strain(s), preparation of the inoculum, preparation and inoculation of the test units, storage conditions, measurement of physico-chemical characteristics of the food and microbiological analysis to measure any increase in the pathogen load. The results are then used to assign a ready-to-eat food into a growth or no growth category, and if the food is able to support the growth of L. monocytogenes, to quantify the behavior of this bacteria in a food between production and consumption.     

Simultaneous detection of Escherichia coli O175:H7, Salmonella spp., and Listeria monocytogenes by multiplex PCR

The wide application of nucleic acid amplification techniques and the increasing industrial interest toward rapid methods has led to the development and application of PCR based methods for the detection of microbial pathogens in food. In the present paper we describe the development of a multiplex PCR method for simultaneous detection of Salmonella enterica serovar Typhimurium, Listeria monocytogenes and Escherichia coli O157:H7 in a complex food matrix (liquid whole egg).Four different DNA extraction procedures were evaluated for their application on food and, among these, Chelex resin combined with a DNA purification step were found to better perform on the food system considered.A multiplex PCR system was developed, based on the evaluation and combination of published primer sets, and applied to the simultaneous detection of the target pathogens plus an internal amplification control, both in culture media and in a model food system.The overall system proposed, based on an overnight enrichment step followed by DNA isolation and multiplex PCR, was satisfactorily tested for its specificity and sensitivity and allowed the detection of the presence of bacterial DNA and the identification of the target pathogens down to 10 cells/25 g liquid whole egg.     

Modelling the effect of pH and pectin concentration on the PEF inactivation of Salmonella enterica serovar Typhimurium by using the Monte Carlo simulation

The effects of pH (3.5, 4 and 4.5), electric field (15, 25, 35 and 40 kV/cm) and pectin concentration (0.1, 0.3 and 0.6%) on the inactivation kinetics of Salmonella typhimurium in an orange juice-milk beverage treated by pulsed electric fields (PEF) were studied. A secondary model, based on Weibull distribution function, was used together with a Monte Carlo simulation to establish the most influential factors on the final number of Salmonella cells after PEF treatment. The Monte Carlo simulation can be a useful and practical tool for the industry to predict how the process parameters and product formulation will influence the food safety of the product.     

Inactivation of Escherichia coli O157:H7 by high pressure carbon dioxide combined with nisin in physiological saline,phosphate-buffered saline and carrot juice

The individual and combined effects of high pressure carbon dioxide (HPCD) and nisin (200 IU/mL) on the inactivation of Escherichia coli O157:H7 suspended in physiological saline (PS, pH 5.60), phosphate-buffered saline (PBS, pH 5.60 or 7.00) or carrot juice (pH 6.80) were evaluated. The pressure in this study was 5 and 8 MPa, the temperature was 25 °C–45 °C, and the treatment time was 5–65 min. Inactivation of cells in PS (pH 5.60) by HPCD followed first order kinetics, the k (the inactivation rates) increased while the D (decimal reduction time) decreased in the presence of nisin, however, the acid solution dissolving nisin rather than nisin itself played a prominent role in this combination effect with HPCD in PS buffer. The inactivation kinetics of cells in PBS (pH 5.60 or 7.00) and carrot juice (pH 6.80) by HPCD followed slow-to-fast two-stage kinetics and was fitted by the modified Gompertz equation. The M (the time at which the absolute death rate is maximum) significantly decreased in the presence of nisin. HPCD enhanced the sensitization of E. coli to nisin and the time for the complete inactivation was shortened by 2.5–5 min in PBS buffer and carrot juice by combination of HPCD and nisin (HPCD + nisin) than by HPCD alone. Regression coefficients (R²) and mean square error (MSE) were used to evaluate the model performance, indicating that the models could provide a good fitting to the experimental data.     

Inactivation of food poisoning bacteria and Geobacillus stearothermophilus spores by high pressure carbon dioxide treatment

Inactivation of the vegetative foodborne pathogens by high pressure carbon dioxide treatment (HPCT) was investigated. Pseudomonas aeruginosa IFO3445, Aeromonas hydrophila IFO13286, Salmonella enteritidis JCM3313, Salmonella typhimurium IID1000, Yersinia enterocolitica JCM1677, Escherichia coli O157:H7 ATCC35150, E. coli O157:H7 ATCC43889, Staphylococcus aureus FDA209P and Listeria monocytogenes 1/2a were used in this study. In all cases, HPCT at 35 °C, 10 MPa for 1 min reduced initial numbers by approximately 6 logs. In addition HPCT inactivation of 9 pathogenic strains of Bacillus cereus (mixture of spores and vegetative cells) was investigated. HPCT at 75 °C, 10 MPa for 120 min resulted in significant inactivation in all the strains. The effect of HPCT (30 MPa at 95 °C for up to 120 min) on the inactivation of Geobacillus stearothermophilus spores in the presence of sodium chloride (3% and 6% w/v), glucose (6% and 12% w/v) and ethanol (10% and 20% v/v) was also studied. Both sodium chloride and glucose had a protective effect and the level of inactivation was reduced. The effect was in proportion to the solute concentration. However, 10% and 20% ethanol did not significantly affect the level of inactivation.     

Modeling the inactivation of Neosartorya fischeri ascospores in apple juice by high pressure,power ultrasound and thermal processing

Neosartorya fischeri is a mould that spoils acid foods and can produce mycotoxins. In this work, the efficacy of high pressure processing (HPP, 600 MPa) and power ultrasound (24 kHz, 0.33 W/mL) in combination with 75 °C for the inactivation of four week old N. fischeri ascospores in apple juice was investigated and compared with 75 °C thermal processing alone. The HPP-75 °C process was the most effective technique for inactivating N. fischeri spores, resulting in 3.3 log reductions after 10 min vs. no inactivation for thermosonication (TS) and thermal processing. Unexpectedly, activation shoulders were observed during the TS process. Then, the effect of different temperatures on the ascospore inactivation in apple juice by HPP-thermal, TS and thermal processing was investigated, and the log survivors vs. time were modeled. Faster inactivation was achieved at higher temperatures for all the technologies tested, indicating the significant role of temperature for the spore inactivation, alone or combined with other processes. The Weibull model described the spore inactivation better by 600 MPa HPP-thermal (50, 60, 75 °C) and thermal (85, 90 °C), whereas Lorentzian was more appropriate for the TS treatment (65, 70, 75 °C). In conclusion, HPP is the best food preservation technology due to higher spore inactivation in apple juice at the same temperature.     

Inactivation of 3-strain cocktail pathogens inoculated into Bajirak jeotkal,salted, seasoned,and fermented short-necked clam (Tapes pilippinarum), by gamma and electron beam irradiation

The objective of this study was to determine the efficacy of gamma and electron beam irradiation of the food-borne pathogens including 3-strain cocktail of Listeria monocytogenes (ATCC 19114, 19115, and 19111), Staphylococcus aureus (ATCC 6538, 25923, and 29213), and Vibrio parahaemolyticus (ATCC 17802, 33844, and 27969) in Bajirak jeotkal (8% salt), salted, seasoned and fermented short-necked clam, commercially available in the market. Irradiation (0.5, 1, 2, and 5 kGy) significantly reduced the initial microbial level not only immediately after irradiation but also during storage at 10 °C for 4 weeks (P < 0.05). No viable cells were detected at 5 kGy of irradiation at a detection limit of 10¹ CFU/g. Gamma irradiation was more effective than electron beam irradiation, and yielded D₁₀ values of 0.64, 0.63, and 0.29 kGy for L. monocytogenes, S. aureus, and V. parahaemolyticus, and those of electron beam irradiation were 0.79, 0.81, and 0.36 kGy, respectively. Results suggest that a low dose irradiation can improve the microbial quality and reduce the risk by the food-borne pathogens of Bajirak jeotkal, which has limited alternative sterilization methods due to the temperature characteristics of the products. Furthermore, in practical application, the irradiation source should be considered to obtain an effective dose for decontamination.