Growth of Listeria monocytogenes on vacuum-packaged horsemeat for human consumption

In order to investigate the likelihood of Listeria monocytogenes (serotype 4b, ATCC 19115) growth on vacuum-packaged horsemeat at refrigeration temperature, fourteen horsemeat surface/volume homogeneous 150 g weight pieces were superficially inoculated with serotype 4b L. monocytogenes and vacuum packaged. The samples were stored at 4 ± 1 °C. Two pieces (one for pH determination and one for L. monocytogenes counts) were examined at days 0, 7, 14, 21, 28, 35 and 42. Surface pH did not show significant variations during the experiment. The average L. monocytogenes initial contamination level was 1.77log₁₀ CFU/g. A lag phase of 7 days was recorded. The exponential growth rate between day 7 to day 35 was 0.125log₁₀ CFU/day, corresponding to 3.51log₁₀ CFU/g in 28 days. At the end of the experiment the mean L. monocytogenes log₁₀ CFU/g was 5.78.     

Antibacterial activity of extracts from some edible plants commonly consumed in Asia

Extracts of edible plants (26 species) from China, Japan, Thailand and Yemen were screened for their antibacterial activity against Bacillus cereus, Staphylococcus aureus, Listeria monocytogenes, Escherichia coli and Salmonella infantis. Buffered methanol (80% methanol and 20% PBS) and acetone extracted inhibitory substances against tested bacteria from 16 plants, as revealed by the disc assay. The minimum inhibitory concentrations (MICs) of extracts determined by the agar dilution method ranged from 165 to 2640 mg l⁻¹. The most sensitive microorganism to extracts from Azadirachta indica, Cinnamomum cassia, Rumex nervosus, Ruta graveolens, Thymus serpyllum and Zingiber officinale was B. cereus, with MIC of 165 to 660 mg l⁻¹. E. coli and S. infantis were only inhibited by Cinnamomum cassia extracts at the highest MIC (2640 mg l⁻¹). L. monocytogenes (Tottori) was more resistant than the ATCC 7644 strain to extracts from Ruta chalepensis, Artemisia absinthium and Cissus spp. EDTA (0.85 mM) reduced the MICs of Cinnamomum cassia and Cissus rotundifolia by at least 50% when tested against E. coli, S. infantis, S. aureus and L. monocytogenes.     

Microbiological quality of 18 degrees C ready-to-eat food products sold in Taiwan

A total of 164 samples of 18 °C ready-to-eat (RTE) food products, purchased in 1999–2000 from convenience stores and supermarkets in central Taiwan, were examined to determine the microbiological quality of these products. The 18 °C RTE food products, manufactured by 16 factories, were divided into groups based on the type of food and their major ingredients. Aerobic plate count, coliforms, Escherichia coli, Bacillus cereus, Staphylococcus aureus and psychrotrophic Pseudomonas spp. were evaluated. The incidence of E. coli and coliforms in these 18 °C RTE food products was 7.9% and 75.0%, respectively, while 49.8% and 17.9% of the samples were found to contain B. cereus and S. aureus, respectively. Among the samples tested, 1.3% of the food products contained more than 10⁵ CFU g⁻¹ of B. cereus and 0.7% contained more than 10⁵ CFU g⁻¹ of S. aureus. The pH values of the samples were all below 7.0, except for cold noodles, which had pH values ranging from 5.18 to 8.20. Among the five types of 18 °C food products tested, the highest incidence of E. coli (16%) and Pseudomonas spp. (64.0%) were detected in hand-rolled sushi in a cone shape. On the other hand, the highest incidence rate of coliforms, B. cereus, and S. aureus were found in sandwiches (88%), cold noodles (66.7%) and rice balls rolled in seaweed (25.0%), respectively. Food products made of ham contained the highest incidence of coliforms (88.0%) and E. coli (16.0%), while food products containing meat and ham as the major ingredients had the highest incidence rates of B. cereus (62.5%) and S. aureus (26.1%), respectively. For coliforms, E. coli, B. cereus and S. aureus, the percentage of 18 °C RTE food products exceeding the microbiological standards for RTE food accepted by Republic of China was 75.0%, 7.9%, 49.8% and 17.9%, respectively.     

Prediction of Listeria spp. growth as affected by various levels of chemicals, pH, temperature and storage time in a model broth

The effects of concentration of NaCl (0.5 to 12.5%), methyl paraben (0.0 to 0.2%), sodium propionate (0.3%), sodium benzoate (0.1%), potassium sorbate (0.3%), pH (>5.9) temperature (4 to 30°C), storage time (up to 58 d) and inoculum (>10⁵ to >10⁻² per ml) on the log₁₀ probability percentage of one cell of Listeria spp. to initiate growth in a broth system were evaluated in a factorial design study. At pH 5.96 and temperature ranging from 4 to 30°C the concentrations of sodium propionate, potassium sorbate, and sodium benzoate examined allowed growth of L. monocytogenes with lag phases at 4°C of 18, 27 and 21 days, respectively. For 0.1 and 0.2% methyl paraben growth of all Listeria spp. was initiated at 8°C and 30°C, respectively. At pH 6, concentration of 12% NaCl supported the growth of L. monocytogenes at 8 to 30°C, whereas 12.5% inhibited all Listeria species. Four regression equations were derived relating probability of growth initiation to temperature, concentrations of NaCl and preservatives storage time, and Listeria species specific effects. From these equations, the number of cells needed for growth initiation can be calculated. The impact of this type of quantitative study and its possible application on the development of microbial standards for foods is discussed.     

Microbiological quality of ayib, a traditional Ethiopian cottage cheese

One-hundred samples of ayib, a traditional Ethiopian cottage cheese, were purchased from Awassa market and analysed for their aerobic mesophilic counts, psychrotropic counts, yeasts and molds, coliforms, spore-formers, enterococci, lactic acid bacteria, Listeria monocytogenes, staphylococci and Bacillus cereus. The majority of the samples showed counts of mesophilic aerobic bacteria, yeasts and enterococci of 10⁸, 10⁷ and 10⁷ cfu/g. About 55% of the samples were positive for coliforms and faecal coliforms. Listeria spp. were not detected in any of the samples. B. cereus and S. aureus were isolated at varying frequencies but at low numbers (10²–10³). The pH value of the samples varied between 3.3 and 4.6 with about 40% having pH lower than 3.7.     

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

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

The model based on the Weibull distribution was capable of accurately predicting the treatment time to inactivate five Log₁₀ cycles of both microorganisms at the three pHs investigated.     

Effect of temperature and growth media on the attachment of Listeria monocytogenes to stainless steel

Listeria monocytogenes ATCC 19111 cultivated in nutrient-rich medium (brain heart infusion, BHI) or starved in minimal medium (10% filter sterilized pond water and 90% sterilized distilled water) were investigated for their initial attachment to austenitic stainless steel No. 4 with satin finish at 4 °C, 20 °C, 30 °C, 37 °C, or 42 °C. A droplet (10 μl) containing  10⁷ CFU/ml of L. monocytogenes suspended in BHI or minimal medium was placed on the stainless steel surface. After holding in saturated humidity for 3 h at the desired temperature the surface was washed and prepared for scanning electron microscopy (SEM). Using SEM, attachment of L. monocytogenes was determined by counting cells remaining on the surface. When L. monocytogenes cultivated in BHI were used, with the exception of the number of attached cells being lower at 42 °C than at 37 °C and 30 °C, the number of attached cells increased with increasing temperature (P < 0.05). When L. monocytogenes starved in minimal medium were used, the number of attached cells also increased with increasing attachment temperature (P < 0.05), but the number of attached cells at 42 °C was lower than that at the other temperatures. The attachment of L. monocytogenes to stainless steel surface was greater when cultivated in rich medium of BHI vs starved in the minimal medium.     

Effects of combined exposure of micrococcus luteus to nisin and pulsed electric fields

Death and injury following exposure of Micrococcus luteus to nisin and pulsed electric field (PEF) treatment were investigated in phosphate buffer (pH 6.8, σ=4.8 ms/cm at 20°C). Four types of experiment were carried out, a single treatment with nisin (100 IU/ml at 20°C for 2 h), a single PEF treatment, a PEF treatment followed by incubation with nisin (as before) and addition of nisin to the bacterial suspension prior to the PEF treatment. The application of nisin clearly enhanced the lethal effect of PEF treatment. The bactericidal effect of nisin reduced viable counts by 1.4 log₁₀ units. Treatment with PEF (50 pulses at 33 kV/cm) resulted in a reduction of 2.4 log₁₀ units. PEF treatment followed by nisin caused a reduction of 5.2 log₁₀ units in comparison with a 4.9 log₁₀ units reduction obtained with nisin followed by PEF. Injury of surviving cells was investigated using media with different concentrations of salt. Sublethally damaged cells of M. luteus could not be detected by this means, following PEF treatment.     

Inhibition of Listeria monocytogenes by a bacteriocinogenic Lactobacillus sake strain in modified atmosphere-packaged Brazilian sausage

Lactobacillus sake 2a is a bacteriocinogenic strain isolated from “lingüiça frescal”, a Brazilian sausage. The combined effect of modified-atmosphere (MA) packaging (100% CO₂ and 50% CO₂/50% N₂) and addition of L. sake 2a on inhibition of growth of Listeria monocytogenes was evaluated in “lingüiça” stored at 6 °C. By the end of the first week, the inhibition of L. monocytogenes due to MA was significant (P0.05) while the presence of L. sake 2a did not influence significantly the growth of the pathogen. After 14 days, a reduction of 1.3–1.4 log in counts of L. monocytogenes was observed in samples containing L. sake 2a only or MA packaged only, while a reduction of 3.5 log was detected in those submitted to both treatments. Results indicate that inhibition of L. monocytogenes in “lingüiça frescal” by the bacteriocinogenic L. sake 2a is enhanced by the packaging of the product in MA.     

Antilisterial activity of lactic acid bacteria inoculated on cooked ham

This study was conducted to evaluate the ability of Lactobacillus sakei 1, a bacteriocin-producing (bac⁺) lactic acid bacterium (LAB), isolated from Brazilian fresh pork sausage to inhibit two Listeria monocytogenes strains (serotypes 4b and 1/2a) on cooked, sliced vacuum-packaged ham. L. sakei ATCC 15521 was used as a non-bacteriocin producer (bac⁻). L. monocytogenes (ca. 2 log CFU/mL) and LAB (ca. 6 log CFU/ml) were inoculated on the sterilized ham, vacuum-sealed and incubated at 8 °C for 10 days. A treatment with the bacteriocin Chrisin (UI/ml) was included. Both L. monocytogenes strains were significantly inhibited in the presence of either bac⁺ and bac⁻ LAB in comparison to the control (L. monocytogenes alone). Using a bacteriocinogenic strain of LAB did not offer an additional barrier to listerial growth in the studied meat system. The application of Chrisin did not affect at all the growth of L. monocytogenes.     

The occurrence of Listeria monocytogenes in cheese from a manufacturer associated with a case of listeriosis

A case of listeriosis was associated with the consumption of a soft cheese produced in England. Goats cheese and other products from the same food manufacturer were examined for the presence of Listeria over the following 11 months. Listeria monocytogenes was isolated from 16 of 25 cheese samples on retail sale, 12 of 24 cheese samples obtained directly from the factory, and from shelving within the plant. Phage-typing of 68 isolates of L. monocytogenes from cheese samples and the factory showed that 66 (97%) were indistinguishable from the strain isolated from the patient's cerebrospinal fluid and stool. L. monocytogenes was not isolated from seven goats milk or two yoghurt samples. Listeria innocua was isolated from 10 cheese samples, two of which contained no other species of Listeria. Levels of L. monocytogenes shortly after production were low (<10/g), but were higher (10⁵–10⁷ cfu/g) in six of the 16 cheese samples obtained from retail outlets. Multiplication of L. monocytogenes was demonstrated in cheeses contaminated at the factory and held at 4°C in the laboratory.     

Antilisterial activity of lactic acid bacteria isolated from "Alheiras" (traditional Portuguese fermented sausages): In situ assays

A total of 226 lactic acid bacteria (LAB) isolated from “Alheira”, a traditional Portuguese fermented sausage, were screened for antagonistic activity against some pathogenic microorganisms, including Listeria monocytogenes. The objective was to isolate LAB with antibacterial activity from “Alheiras” and to select strains that could be used in “Alheira” production. Isolates displaying antibacterial activity against Listeria innocua and L. monocytogenes were investigated for the nature of the antibacterial compounds active against these microorganisms. Results showed that two LAB cultures retained activity in the supernatants after neutralization and catalase treatment. These two strains were both identified as Pediococcus pentosaceus. The final aim of this work was to test the antilisterial activity of these two strains during storage of “Alheira mass” (sterilized), at 4 °C. The growth of L. innocua population was significantly suppressed in the paste of “Alheira” when the samples were co-inoculated with the LAB strains, in comparison with the paste only inoculated with L. innocua or co-inoculated with a bacteriocin negative strain of Ped. pentosaceus (ca. 1 × 10⁷ CFU/g after 28 days of incubation).     

Bacteriological quality assurance (BQA) of mechanically deboned meat (MDM)

Adequacy of bacteriological quality assurance during the commercial production of mechanically deboned meat (MDM) was assessed. Lax standards of hygiene during production were observed, resulting in high numbers of Staphylococcus aureus, viz. 10⁴ to 10⁵ cfu g⁻¹, and severe contamination with Enterobacteriaceae: 10⁵ to 10⁶ cfu g⁻¹. These data indicate that measures of hygiene observed during boning of carcasses and during collection, storage and transport of bones or poultry parts should be markedly tightened, while conditions of refrigerated storage of raw materials and MDM should be improved. Use of bones of poor sensory quality (discoloration, abnormal smell) generally resulted in MDM of inferior bacteriological quality.

Phage typing, biotyping and assessment of enterotoxin production was carried out with 136 St. aureus cultures, isolates from mechanically deboned pork produced at one plant. Fifty-five per cent of the isolates was not typable, 28% was typable with human phages, 8% with bovine phages. The majority of the strains could not be explicitly assigned to any Meyer and/or Hájek and Mar álek types. Applying the simplified system of Devriese to eighteen strains isolated in our investigation, ten were found to belong to the poultry ecovar, one to the bovine ecovar, while seven strains were non-host specific. None of the isolates produced enterotoxins A–E.

Microbiological inspection of end products is recommended as part of an integrated quality assurance system. The following reference values for the final product (maximal colony counts to be expected under GMP conditions expressed as 95th percentile) were calculated: Pig MDM: log₁₀ mesophilic colony count 6·8 and log₁₀ cfu mesophilic Enterobacteriaceae g⁻¹ 4·8; Poultry MDM: log₁₀ mesophilic colony count 6·6 and log₁₀ cfu mesophilic Enterobacteriaceae g⁻¹ 4·7.     

Characterization of the Effect of Sprouting or Solid-State Bioprocessing by Dietary Fungus on the Antibacterial Activity of Soybean Extracts Against Listeria monocytogenes

Listeria monocytogenes is one of the most severe food-borne bacterial infections causing Listeriosis. As L. monocytogenes can survive harsh adverse conditions - such as low pH, high NaCl, and refrigeration temperatures - as well as resist current antimicrobial measures such as the use of disinfectants and antibiotics, there is a need for alternative anti-Listeria strategies. In the search for new antimicrobial agents, much recent research has focused on the potential of dietary phenolic compounds. In this study, soybean extracts enriched for phenolic content via dark-germination sprouting or solid-state bioprocessing by the dietary fungus Rhizopus oligosporus or Lentinus edodes were investigated for in vitro antibacterial activity against L. monocytogenes.L. monocytogenes growth was inhibited most effectively by R. oligosporus bioprocessed soybean extracts, which showed anti-Listeria activity at total phenolic concentrations as low as 10 µg 100 µL^-1. In both sprouted soybean extract and L. edodes-bioprocessed soybean extract the anti-Listeria activity was not observed until at least 200 µg total phenolic content 100 µL^-1 was used. Anti-Listeria activity by soybean extract was associated with phenolic mobilization but not with antioxidant activity. Further, R. oligosporus bioprocessed soybean extracts were shown to inhibit the growth of L. monocytogenes in fish and meat systems at refrigeration temperatures. The potential involvement of mobilization of antimicrobial versus non-antimicrobial phenolics during sprouting and solid-state bioprocessing was hypothesized and discussed.     

Bacterial cells exposed to nanosecond pulsed electric fields show lethal and sublethal effects

Cell suspensions of Escherichia coli K12 and Salmonella typhimurium were exposed to electrical pulses of 32 ns duration at a field intensity of 100 kV/cm and a repetition rate of 30 pulses per second for a total of 300 s. Treated cells were plated onto Tryptone Soya Agar (TSA) and TSA supplemented with NaCl, and cell counts were monitored daily for 3 days. The concentrations of NaCl used were 3 and 4% (w/v) for E. coli and 4 and 5% (w/v) for S. typhimurium. Treatment under these conditions resulted in a 2 log₁₀ reduction for E. coli and approximately a single log₁₀ reduction for S. typhimurium. For both species of bacteria it was discovered that the surviving population was composed of only 1% of uninjured cells. Moreover, the proportion of sublethally injured cells increased more rapidly than the total recoverable population suggesting a process of injury accumulation culminating in death rather than an ‘all or nothing’ mechanism. Sublethal injury manifested itself in a proportion of the injured population of both species by an extended lag phase at longer treatment times. Finally, possible mechanisms by which nanosecond electric pulses inactivate bacteria are discussed.     

The storage life of chilled pork packaged under carbon dioxide

Pork cuts of longissimus dorsi muscle with overlaying fat and skin were packed under vacuum in film of low oxygen transmission rate, or under CO₂ in gas impermeable aluminium foil laminate. Cuts were stored at +3 or −1·5°C. Vacuum packaged cuts were grossly spoiled by Brochothrix thermosphacta after 2 weeks' storage at 3°C and after 5 weeks at −1·5°C. Cuts packaged under CO₂ were grossly spoiled by B. thermosphacta after 5·5 weeks' storage at 3°C. Growth of B. thermosphacta was suppressed when CO₂ packaged cuts were stored at −1·5°C. At that temperature, slow growth of enterobacteria was detected after a lag of about 18 weeks. The enterobacteria caused gross spoilage of an increasing proportion of cuts between 18 and 26 weeks. Muscle tissue with pale, soft, exudative (PSE) characteristics tended to lose colour after long storage periods, apparently because of loss of myogglobin with exudate. Until spoilage, the eating qualities of pork appeared little affected by prolonged storage.     

Traditional dry fermented sausages produced in small-scale processing units in Mediterranean countries and Slovakia. 1: Microbial ecosystems of processing environments

Microbial ecosystems were surveyed in 314 environmental samples from 54 Southern and Eastern European small-scale processing units (PUs) manufacturing traditional dry fermented sausages. The residual microflora contaminating the surfaces and the equipment were analysed after cleaning and disinfection procedures. All the PU environments were colonised at various levels by spoilage and technological microflora with excessive contamination levels in some of the PUs. Sporadic contamination by pathogenic microflora was recorded. Salmonella and Listeria monocytogenes were detected in 4.8% and 6.7% of the samples, respectively, and Staphylococcus aureus was enumerated in 6.1% of the samples. Several critical points were identified, such as the machines for S. aureus and the tables and the knives for L. monocytogenes; this knowledge is crucial for the improvement of hygiene control systems in small and traditional meat processing industries. The variability of the residual contamination emphasized the different cleaning, disinfecting and manufacturing practices routinely followed by these small-scale processing units.     

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

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

The influence of oxygen on the differentiation of temperature-sensitive and temperature-insensitive Lactococcus lactis subsp. cremoris starter strains

The differentiation of temperature-insensitive and temperature-sensitive strains of Lactococcus lactis subsp. cremoris by using a modified sodium-β-glycerophosphate/milk medium is described (temperature-insensitive strains are defined as those that continue to grow at 38°C and temperature-sensitive strains as those that do not grow, or grow poorly, at 38°C). The physiological basis for the differentiation assay was examined by using L. lactis subsp. cremoris strains 2146 (temperature-insensitive) and 2182 (temperature-sensitive) as test strains. After aerobic incubation on the medium, strain 2146 formed uniform colonies, 0·5 mm in diameter, while strain 2182 formed larger colonies, 1·0–1·5 mm in diameter. The differential was dependent on the medium constituents, on an aerobic gas phase, and on the effects of H₂O₂ generated within the colonies. The addition of 0·5% (w/v) pyruvate to the medium facilitated the growth of colonies of strain 2146 to 3-mm diameter, while the colony size of strain 2182 remained at 1-mm diameter, and thus the colony-size differential between strains was reversed. The growth of both strains was inhibited at 0·4–0·8% air saturation during suspension culture in sodium-β-glycerophosphate/milk medium. The inclusion of catalase in the cultures overcame the growth inhibition. There was no observable difference between the two strains in their oxygen sensitivity or NADH oxidase/peroxidase enzymology.     

Listeria innocua and aerobic mesophiles during chill storage of inoculated mechanically recovered poultry meat treated with high hydrostatic pressure

Mechanically recovered poultry meat (MRPM) was inoculated with Listeria innocua 910 CECT at a level of approximately 10⁸ CFU g⁻¹. Vacuum-packaged samples were treated by combinations of pressure (350, 400, 450 and 500 MPa), time (5, 10, 15 and 30 min) and temperature (2, 10 and 20°C) and later stored at 2°C for 2 months. Counts of L. innocua and aerobic mesophilic bacteria were determined 1, 4, 7, 15, 30 and 60 days after pressurisation. For mesophiles, in most treatments, pressurization at 2°C gave the significantly best results. High pressure caused a marked bactericidal effect on L. innocua: reductions higher than 7.5 log units were achieved in several cases. Some cells were just sublethally injured by pressure. Samples treated at 500 MPa for 30 min at 2°C had counts of only 2.3 log units after 60 days of chill storage. Noninoculated pressurised MRPM did not show Listeria growth throughout storage. These results suggest that high pressure processing can enhance the microbiological quality of MRPM.