Moderate electric fields can inactivate Escherichia coli at room temperature

The inactivation of Escherichia coli using moderate electric fields (MEF) below 25 °C, was investigated. Keeping the temperature always below 25 °C demonstrated that electric fields are involved in the inactivation of E. coli, without possible synergistic temperature effects. Electric fields above 220 V cm⁻¹ promoted death rates of 3 log₁₀ cycles of E. coli in less than 6 min, and even higher rates at greater electric fields, while presumably overcoming the thermal degradation caused by conventional high temperature treatments. A non-thermal model was proposed that successfully describes the E. coli death kinetics under this treatment. SEM observations of E. coli cells after the exposure to the MEF treatment, revealed changes at the cell membrane level, indicating a possible cause for the cell death rates. These results show that this treatment holds potential for sterilization of thermolabile products (e.g. serum and other physiological fluids, food products), by itself or as a complement of the traditional heat-dependent techniques.     

A numerical approach for predicting volumetric inactivation of food borne microorganisms in liquid substrates by pulsed light treatment

Pulsed light (PL) technology is able to effectively destroy a wide variety of food spoilage and pathogenic microorganisms. However, the effectiveness of PL treatment depends on direct exposure of the target microorganisms to the short, high energy pulses of light. The complex physical and chemical properties of foods affect the way light propagates through a given food substrate, and thus there is a real potential for insufficiently or non-uniformly treated products. The objective of this work was to develop a method for predicting levels and spatial distribution of microbial inactivation in PL treatment of liquid substrates, and to validate the predictions with experimental data. Three liquids with different composition and optical properties (BPB, TSB, apple juice) were inoculated with either Escherichia coli ATCC 25922 or Listeria innocua FSL C2-008 and treated with PL, in two different geometries. The Weibull model was used to describe the microbial inactivation kinetics for each organism. The kinetic equations were coupled with previously determined equations describing either the total fluence (F_total) or UV fluence (F_UV) distribution in each of the liquids, for either cylindrical or rectangular prismatic geometries. COMSOL simulation software was used to generate maps of spatial distribution of microbial inactivation and to predict the average volumetric inactivation for each substrate. The model that used F_total provided gross over-estimations for microbial inactivation, while using F_UV as the treatment dose yielded reasonably good predictions of microbial inactivation, especially for the more opaque and turbid substrates. This approach can help processors determine which substrates would be suitable for PL treatment, and to design highly effective and uniform PL treatments.     

Chemical and biological characteristics of Cuminum cyminum and Rosmarinus officinalis essential oils

Essential oils extracted by hydrodistillation from Cuminum cyminum and Rosmarinus officinalis were characterized by means of GC and GC–MS. C. cyminum and R. officinalis contained α-pinene (29.1%, 14.9%), 1,8-cineole (17.9%, 7.43%) and linalool (10.4%, 14.9%), respectively, as the major compounds. C. cyminum oil exhibited stronger antimicrobial activity than did R. officinalis oil against E. coli, S. aureus and L. monocytogenes. Complete death time on exposure to Cuminum cyminum L. and Rosmarinus officinalis L. oils were 20 and 25 min 180 and 240 min and 90 and 120 min for E. coli, S. aureus and L. monocytogenes, respectively. Radical-scavenging and antioxidant properties were tested by means of 1,1-diphenyl-2-picrylhydrazyl (DPPH) assay and the β-carotene bleaching test. These properties were compared to those of Thymus x-porlock essential oil, used as a reference ingredient. The radical scavenging performance of the rosemary oil was better than that of C. cyminum. Results from the antioxidant test were better than those provided by the radical-scavenging activity. C. cyminum and R. officinalis essential oils may be considered as potent agents in food preservation.     

Variation of microbial load and visual quality of ready-to-eat salads by vegetable type,season, processor and retailer

Microbial components and visual quality were determined on 1158 consumer units of ready-to-eat salads from several processors, two per each of 579 process lots, with residual shelf-life varying around a mode of five days, collected over 19 months in the years 2006–2008 from retail stores of two Italian cities close to a major producing and processing area. The salads were mainly baby leaf of single species (lettuce, arugula, spinach, lamb’s lettuce), with approximately 10% of the lots made up by mixes of 2–4 species. One unit per lot was analyzed on the day of collection and the other at the consume-by date. No Salmonella or Listeria monocytogenes was found (detection limit: presence in 25 g). Escherichia coli was detected in 27% of the lots (detection limit: 5 cfu/g), with probability of occurrence and counts highest in Autumn and for lettuce and arugula. Average visual quality was higher and other components of the microbial load were lower in Winter and Spring compared to Summer and Autumn (−0.6 log cfu/g of total aerobic counts, −1.3 log cfu/g of coliforms, −0.6 log cfu/g of yeasts and moulds). Lactic acid bacteria were detected more frequently in Spring and Summer (up to 50% of the lots). The rate of increase of microbial populations during shelf life was not affected by the level of initial contamination. At the consume-by date total aerobic count exceeded 7.2 log cfu/g for 50% of the lots and 7.7 log cfu/g for 25%. Salads from the biggest processor and retailer showed slightly higher visual quality scores, lower odds of E. coli occurrence and lower microbial loads. Visual quality scores showed significant negative relationships with the levels of lactic acid bacteria, coliforms and total viable counts.     

Effect of preinoculation growth media and fat levels on thermal inactivation of a serotype 4b strain of Listeria monocytogenes in frankfurter slurries

Preinoculation growth conditions and fat levels were evaluated for effects on the heat resistance of Listeria monocytogenes strain MFS 102 in formulated frankfurter slurries and on frankfurter surfaces. Comparison of linear inactivation rates (D-values) for cells heated in frankfurter slurry showed that growth conditions were significant (P<0.05) factors affecting subsequent thermal resistance. The average D(60 degrees C)-values for the five preinoculation growth media tested from most resistant to least heat resistant were: tryptic soy broth with 0.6% yeast extract (TSBYE) (2.2 min) and 8.5% fat slurry (2.2 min), followed by 23% fat slurry (1.7 min) and 11% fat slurry (1.7 min), and then TSYBE with quaternary ammonium compounds added (TSBYE+Q) (1 min). The fat level in the frankfurter heating media also had a significant (P<0.05) effect on the thermal death rate of L. monocytogenes. Cells heated in 8.5% fat slurry had a significantly higher (P<0.05) D(60 degrees C)-value (2.2 min) than those heated in 11% fat (1.0 min) and 23% fat slurry (0.9 min). Growth media (TSBYE, 8.5% fat slurry, and TSBYE+Q), and fat level (15% and 20%), however, were not significant factors (P>0.05) affecting thermal inactivation rates on frankfurter surfaces. Heat inactivation rates were consistently higher on frankfurter surfaces compared to similar treatments done in frankfurter slurry. On frankfurter surfaces, a 2.3- to 5.1-log(10) reduction was achieved after 15 min depending on frankfurter surface type. The time necessary to achieve a 3-log(10) reduction using post-processing pasteurization of frankfurters in a hot water-bath at 60 degrees C almost doubled for cells grown in TSBYE and heated in 23% fat frankfurter slurry (19.6 min) versus cells grown and heated in 8.5% fat frankfurter slurry (10.8 min).     

Reduction of bacteria on spinach, lettuce, and surfaces in food service areas using neutral electrolyzed oxidizing water

Food safety issues and increases in food borne illnesses have promulgated the development of new sanitation methods to eliminate pathogenic organisms on foods and surfaces in food service areas. Electrolyzed oxidizing water (EO water) shows promise as an environmentally friendly broad spectrum microbial decontamination agent. EO water is generated by the passage of a dilute salt solution ( approximately 1% NaCl) through an electrochemical cell. This electrolytic process converts chloride ions and water molecules into chlorine oxidants (Cl(2), HOCl/ClO(-)). At a near-neutral pH (pH 6.3-6.5), the predominant chemical species is the highly biocidal hypochlorous acid species (HOCl) with the oxidation reduction potential (ORP) of the solution ranging from 800 to 900mV. The biocidal activity of near-neutral EO water was evaluated at 25 degrees C using pure cultures of Escherichia coli, Salmonella typhimurium, Staphylococcus aureus, Listeria monocytogenes, and Enterococcus faecalis. Treatment of these organisms, in pure culture, with EO water at concentrations of 20, 50, 100, and 120ppm total residual chlorine (TRC) and 10min of contact time resulted in 100% inactivation of all five organisms (reduction of 6.1-6.7log(10)CFU/mL). Spray treatment of surfaces in food service areas with EO water containing 278-310ppm TRC (pH 6.38) resulted in a 79-100% reduction of microbial growth. Dip (10min) treatment of spinach at 100 and 120ppm TRC resulted in a 4.0-5.0log(10)CFU/mL reduction of bacterial counts for all organisms tested. Dipping (10min) of lettuce at 100 and 120ppm TRC reduced bacterial counts of E. coli by 0.24-0.25log(10)CFU/mL and reduced all other organisms by 2.43-3.81log(10)CFU/mL.     

Lack of growth of Listeria monocytogenes and Staphylococcus aureus in temperature abuse of E-beam treated ready-to-eat (RTE) cooked ham

The behaviour of Listeria monocytogenes and Staphylococcus aureus in vacuum-packed cooked ham slices treated with an electron beam and stored at 4, 7 and 10 °C was investigated. Cooked ham slices were inoculated with L. monocytogenes and S. aureus and electron beam treated at 2 and 3 kGy. After treatment, a long temperature-dependent death phase was observed, followed by growth at a slower rate than in untreated samples. Assuming a hypothetical load of 10 cells/g or cm² of L. monocytogenes and S. aureus as an original contamination in an industrial situation, an E-beam treatment of vacuum-packed cooked ham slices at 2 kGy guarantees the microbiological safety of the product along its shelf life, even if a noticeable temperature (10 °C) abuse occur during its storage period. Likewise, the E-beam treatment gave rise to a substantial increase of the RTE cooked ham shelf life off-sensory features associated to the spoilage only were detected in non-treated samples (controls) after 8 and 18 days of storage at 10 °C and 7 °C, respectively.     

Modeling inactivation kinetics of food borne pathogens at a constant temperature

Four food borne pathogens (Listeria monocytogenes CA and Ohio₂, Salmonella enteritidis FDA and Salmonella typhimurium E21274, Escherichia coli O157:H7 931 and 933, Staphylococcus aureus 485 and 765) were inactivated under mild temperature (60 °C) and their survival curves determined at selected time intervals. Tailing was observed in all survival curves as a monotonic upward concavity. The resulting survival curves were either described by the Weibull or traditional first-order model and goodness of fit of these models was investigated. Regression coefficients (R²), root mean square error (RMSE) and correlation plots suggested that Weibull model produced a better fit to the data than the traditional model. Hazard plots suggested that the Weibull model was fully appropriate for the data being analysed. Although more studies should be carried out to evaluate the applicability of the nonlinear models, the present study has shown that thermal process calculations should most probably be reconsidered. This could lead to a reduction in under- and over-processing of thermally treated foods     

Inhibition of Listeria monocytogenes and Escherichia coli O157:H7 in liquid broth medium and during processing of fermented sausage using autochthonous starter cultures

The antimicrobial effect of two autochthonous starter cultures of Lactobacillus sakei was evaluated in vitro (in liquid broth medium) and in situ assays. The inactivation of foodborne pathogens Listeria monocytogenes (serotype 4ab No 10) and Escherichia coli O157:H7 ATCC 43888 was investigated during the production of fermented sausage according to a typical Greek recipe using L. sakei strains as starter cultures. The inactivation kinetics were modeled using GInaFiT, a freeware tool to assess microbial survival curves. By the end of the ripening period, the inhibition of L. monocytogenes was significant in treatments with L. sakei 8416 and L. sakei 4413 compared to the control treatment. A 2.2-log reduction of the population of E. coli O157:H7 resulted from the autochthonous starter culture L. sakei 4413 during sausage processing. The use of the autochthonous starter cultures constitutes an additional improvement to the microbial safety by reducing foodborne pathogens.     

Inactivation of Escherichia coli O157:H7, Listeria monocytogenes, Salmonella enterica and Shigella flexneri on spinach leaves by X-ray

Several recent foodborne disease outbreaks associated with leafy green vegetables, including spinach, have been reported. X-ray is a non-thermal technology that has shown promise for reducing pathogenic and spoilage bacteria on spinach leaves. Inactivation of inoculated Escherichia coli O157:H7, Listeria monocytogenes, Salmonella enterica and Shigella flexneri on spinach leaves using X-ray at different doses (0.1, 0.2, 0.3, 0.5, 0.75, 1.0, 1.5 and 2.0 kGy) was studied. The effect of X-ray on color quality and microflora counts (mesophilic counts, psychrotrophic counts and yeast and mold counts) of untreated and treated spinach was also determined. A mixture of three strains of each tested organism was spot inoculated (100 μl) onto the surface of spinach leaves (approximately 8–9 log ml⁻¹), separately, and air-dried, followed by treatment with X-ray at 22 °C and 55–60% relative humidity. Surviving bacterial populations on spinach leaves were evaluated using a nonselective medium (tryptic soy agar) with a selective medium overlay for each bacteria; E. coli O157:H7 (CT-SMAC agar), L. monocytogenes (MOA), and S. enterica and S. flexneri (XLD). More than a 5 log CFU reduction/leaf was achieved with 2.0 kGy X-ray for all tested pathogens. Furthermore, treatment with X-ray significantly reduced the initial inherent microflora on spinach leaves and inherent levels were significantly (p < 0.05) lower than the control sample throughout refrigerated storage for 30 days. Treatment with X-ray did not significantly affect the color of spinach leaves, even when the maximum dose (2.0 kGy) was used.     

The effects of acid adaptation on Escherichia coli inactivation using power ultrasound

Inactivation of Escherichia coli in liquids was carried out using power ultrasound. Parameters examined included amplitude levels (0.4 µm, 7.5 µm, 37.5 µm), treatment time, cell condition (non-adapted cells, acid adapted cells), liquid media (TSB, model orange juice and model apple juice) and E. coli strain (ATCC 25922, NCTC 12900). The efficacy of ultrasound treatment was found to be a function of amplitude level, treatment time and media (p < 0.05). The kinetics of inactivation followed zero order kinetics (R > 0.95), with the highest inactivation achieved using an amplitude of 37.5 µm. The D-values of E. coli 25922 at all amplitudes in model orange juice were not significantly different than in TSB media. However, at 0.4 µm and 37.5 µm amplitude D-values of E. coli 12900 were significantly different in model orange juice compared to TSB media. When efficacy of ultrasound was assessed in model apple juice and phosphate buffered saline treatment times were significantly reduced by comparison with TSB. Inactivation of E. coli was found to be influenced by strain, prior acid adaptation and suspension liquid, but the effect was negated at the higher amplitude levels.

Industrial relevance

To facilitate the preservation of unstable nutrients many juice processors have investigated alternatives to thermal pasteurisation, including un-pasteurised short shelf life juices with high retail value. This trend has continued within the European Union. However within the US recent regulations by the FDA have required processors to achieve a 5-log reduction in the numbers of the most resistant pathogens in their finished products. This rule comes after a rise in the number of food borne illness outbreaks and consumer illnesses associated with consumption of untreated juice products. Pathogenic E. coli may survive in acid environments such as fruit juices for long periods. Ultrasound has been identified as one possible non-thermal technology to meet the required microbial log reduction. However it is important to determine if conditions such as acid adaptation and pathogen strain influence ultrasound efficacy, if the technology is to be adopted by industry.     

Effects of different sanitizing treatments on biofilms and attachment of Escherichia coli and Listeria monocytogenes on green leaf lettuce

The effects of ozone (2 mg/L), chlorine (100 mg/L) and organic acid (0.25 g/100 g citric acid plus 0.50 g/100 g ascorbic acid) treatments at 10 °C for 2 min on the removal of Escherichia coli and Listeria monocytogenes cells embedded inside biofilms on the surface of lettuce leaves were studied. None of the sanitizing treatments were found effective in removing the bacterial biofilms. Initiation of biofilms was observed after 24 h of incubation. Bacterial cells appeared as individual cells, rather than clusters after 6 h incubation, thus 99.9% reductions in both E. coli and L. monocytogenes counts were achieved with all the three treatments. However, after 48 h incubation, none of the treatments resulted in higher than 90% reduction in microbial counts. Biofilm formation was demonstrated for the 48 h incubated samples with SEM images.     

Inactivation kinetics of pulsed electric field-resistant strains of Listeria monocytogenes and Staphylococcus aureus in media of different pH

A study of the effect of pulsed electric fields (PEF) on the inactivation of Listeria monocytogenes STCC 5672 and Staphylococcus aureus STCC 4459 in McIlvaine buffer covering a range from pH 3.5 to 7.0 was conducted. Mathematical models based on the Weibull distribution were developed to describe the influence of the electric field strength, treatment time and pH of the treatment medium on the lethality of both Gram positive pathogenic bacteria after PEF treatments. Both microorganisms were more sensitive to PEF in media of low pH, although the influence of the pH on the PEF resistance was more significant in S. aureus. In the best cases scenario, the highest inactivation levels achieved were 3.3 and 6.1 log₁₀ cycles for L. monocytogenes and S. aureus respectively in pH 3.5 after 500 μs of 35 kV/cm. Based on these results and those observed in literature, L. monocytogenes STCC 5672 at any pH investigated has been shown as one of the most PEF resistant microorganism. Therefore, this microorganism should be considered as a possible target microorganism to define process criterion for PEF pasteurization.     

The hygiene practices of three systems of game meat production in South Africa in terms of animal class and health compliance

Three game meat production systems used on game ranches in South Africa are reported on. System one is applied in the game export market and conforms to the hygiene requirements of the European Union (EU). System two and three entail game meat available on the local market not subjected to any regulation. System 2 however, implemented basic meat hygiene values.     

Sporulation boundaries and spore formation kinetics of Bacillus spp. as a function of temperature, pH and a(w)

Sporulation niches in the food chain are considered as a source of hazard and are not clearly identified. Determining the sporulation environmental boundaries could contribute to identify potential sporulation niches. Spore formation was determined in a Sporulation Mineral Buffer. The effect of incubation temperature, pH and water activity on time to one spore per mL, maximum sporulation rate and final spore concentration was investigated for a Bacillus weihenstephanensis and a Bacillus licheniformis strain.     

Inactivation of Escherichia coli, Listeria innocua and Saccharomyces cerevisiae by UV-C light: Study of cell injury by flow cytometry

Flow cytometry (FCM) is a powerful tool for analyzing physiological characteristics of microorganisms on a single-cell basis and identifying heterogeneities within population. This work analyzed the UV-C induced damage on Escherichia coli ATCC 11229; Listeria innocua ATCC 33090 and Saccharomyces cerevisiae KE162 cells by applying flow cytometry technique. The UV-C doses, obtained by altering the exposure time and measured by the iodide-iodate chemical actinometer, ranged between 0 and 5 kJ/m². E. coli; L. innocua and S. cerevisiae populations were quantified by plate count technique. For flow cytometry studies, cells were labeled with fluorescein diacetate (FDA) for detecting membrane integrity and esterase activity, and with propidium iodide (PI) for monitoring membrane integrity. The results showed that mechanisms of cellular damage differed according to time of exposure to ultraviolet radiation and the organism tested. E. coli and S. cerevisiae sub-populations with PI increased within the first minutes of UV-C treatment, without much change afterwards. On the contrary, FCM was used to detect the inactivation of those L. innocua sub-populations of viable microorganisms (maintaining metabolic activity) which were non-culturable due to membrane rupture and thus not detectable by viable plate count technique.     

Effect of high-pressure processing on reduction of Listeria monocytogenes in packaged Queso Fresco

The effect of high-hydrostatic-pressure processing (HPP) on the survival of a 5-strain rifampicin-resistant cocktail of Listeria monocytogenes in Queso Fresco (QF) was evaluated as a postpackaging intervention. Queso Fresco was made using pasteurized, homogenized milk, and was starter-free and not pressed. In phase 1, QF slices (12.7 × 7.6 × 1 cm), weighing from 52 to 66 g, were surface inoculated with L. monocytogenes (ca. 5.0 log₁₀ cfu/g) and individually double vacuum packaged. The slices were then warmed to either 20 or 40°C and HPP treated at 200, 400, and 600 MPa for hold times of 5, 10, 15, or 20 min. Treatment at 600 MPa was most effective in reducing L. monocytogenes to below the detection level of 0.91 log₁₀ cfu/g at all hold times and temperatures. High-hydrostatic-pressure processing at 40°C, 400 MPa, and hold time ≥15 min was effective but resulted in wheying-off and textural changes. In phase 2, L. monocytogenes was inoculated either on the slices (ca. 5.0 log₁₀ cfu/g; ON) or in the curds (ca. 7.0 log₁₀ cfu/g; IN) before the cheese block was formed and sliced. The slices were treated at 20°C and 600 MPa at hold times of 3, 10, and 20 min, and then stored at 4 and 10°C for 60 d. For both treatments, L. monocytogenes became less resistant to pressure as hold time increased, with greater percentages of injured cells at 3 and 10 min than at 20 min, at which the lethality of the process increased. For the IN treatment, with hold times of 3 and 10 min, growth of L. monocytogenes increased the first week of storage, but was delayed for 1 wk, with a hold time of 20 min. Longer lag times in growth of L. monocytogenes during storage at 4°C were observed for the ON treatment at hold times of 10 and 20 min, indicating that the IN treatment may have provided a more protective environment with less injury to the cells than the ON treatment. Similarly, HPP treatment for 10 min followed by storage at 4°C was the best method for suppressing the growth of the endogenous microflora with bacterial counts remaining below the level of detection for 2 out of the 3 QF samples for up to 84 d. Lag times in growth were not observed during storage of QF at 10°C. Although HPP reduced L. monocytogenes immediately after processing, a second preservation technique is necessary to control growth of L. monocytogenes during cold storage. However, the results also showed that HPP would be effective for slowing the growth of microorganisms that can shorten the shelf life of QF.     

Water activity and temperature effects on mycotoxin production by Alternaria alternata on a synthetic tomato medium

Alternaria spp. have been reported to be the most frequent fungal species invading tomatoes. Certain species, in particular the most common one, A. alternata, are capable of producing several mycotoxins in infected plants and in agricultural commodities. Alternariol (AOH), alternariol monomethyl ether (AME), and tenuazonic acid (TA) are some of the main Alternaria mycotoxins that can be found as contaminants of food. The objective of this study was to determine the effect of water activity (a_w, 0.904, 0.922, 0.954, and 0.982) and temperature (6, 15, 21 and 35 °C) on mycotoxin production on a synthetic tomato medium of a cocktail inoculum of five strains of A. alternata isolated from tomato fruits affected by Blackmould. The optimum AOH production occurred at 0.954 a_w after 28 days of incubation at 21 °C. A temperature of 21 °C was the most favourable for AOH synthesis at all a_w levels. The maximum concentration of AME was determined at 0.954 a_w and 35 °C. The optimum conditions for TA accumulation were 0.982 a_w and 21 °C. At the 0.904 a_w no growth or germination was registered at 6 °C and 15 °C over the whole incubation period. At 21 °C and 35 °C growth occurred slowly but none of the toxins were detected at this a_w level. In general, high a_w levels were favourable for mycotoxin production. None of the other toxins was detected at quantifiable levels at 6 °C after the whole incubation period. A storage temperature of 6 °C or below could be considered as safe for tomato fruits and high moisture tomato products (a_w > 0.95), in relation with Alternaria toxins. The results obtained here could be extrapolated to evaluate the risk of spoilage in tomato fruits and tomato products caused by this pathogen.     

Microbiological quality and potential public health risks of export meat from springbok (Antidorcas marsupialis) in Namibia

To assess the microbiological quality and safety of export game meat; i) a total of 80 pooled meat samples for aerobic plate count (APC) and Enterobacteriaceae ii) water used in harvesting and processing for microbiological quality and iii) meat and rectal contents for Salmonella spp. and Shiga toxin Escherichia coli (STEC) were evaluated in 2009 and 2010. No differences (p > 0.05) in the APCs were observed between the years, but the mean Enterobacteriaceae count for 2009 was 1.33 ± 0.69 log₁₀ cfu/cm² compared to 2.93 ± 1.50 log₁₀ cfu/cm² for 2010. Insignificant Heterotrophic Plate Count (HPC) levels were detected in 9/23 field water samples, while fecal bacterial (coliforms, Clostridium perfringens and enterococci) were absent in all samples. No Salmonella spp. was isolated and all E. coli isolates from meat were negative for STEC virulence genes (stx1, stx2, eae and hlyA), suggesting a negligible role by springbok in the epidemiology of STEC and Salmonella.