Influence of temperature and surfactant on Escherichia coli inactivation in aqueous suspensions treated by moderate pulsed electric fields

This research employed a conductometric technique to estimate the inactivation kinetics of Escherichia coli cells in aqueous suspensions (1 wt.%) during simultaneous pulsed electric fields (PEF) and thermal treatments. The electric field strength was E = 5 kV/cm, the effective PEF treatment time t_PEF was within 0–0.2 s, the pulse duration t_i was 10^− 3 s, the medium temperature was 30–50 °C, and the time of thermal treatment t_T was within 0–7000 s. The damage of E. coli was accompanied by cell size decrease and release of intracellular components. The synergy between PEF and thermal treatments on E. coli inactivation was clearly present. The non-ionic surfactant Triton X-100 additionally improved its inactivation. The characteristic damage time followed the Arrhenius law within the temperature range 30–50 °C with activation energies W = 94 ± 2 kJ mol^− 1 and W = 103 ± 5 kJ mol^− 1 with and without the presence of surfactant, respectively. Relations between cell aggregation, cell ζ-potentials and presence of surfactant were analysed.     

Effect of water activity on inactivation of Listeria monocytogenes and lactate dehydrogenase during high pressure processing

The aim of this study was to investigate the effect of water activity (a_w) on the inactivation of Listeria monocytogenes and lactate dehydrogenase (LDH) during high pressure processing (HPP). For microbial inactivation lyophilized cells of L. monocytogenes 19,115 were left dry or were suspended in 10 ml of 0.1% peptone water, 10 ml of glycerol, or mixtures of glycerol and peptone water. All samples of various a_ws were high pressure (HP) processed at ambient temperature at 600 MPa for 300 s. Following HPP, samples were serially diluted in 0.1% peptone and spread-plated on Tryptic Soy agar supplemented with Yeast Extract. For enzyme inactivation, 4.2 mg of lyophilized LDH was suspended in 2 ml of 100 mM phosphate buffer (pH 7.4), 2 ml of peptone water or glycerol, or in 2 ml mixtures of glycerol and peptone water. A lyophilized sample with no added liquid was also included. All enzyme samples were subjected to HPP as described above. After HPP, LDH was diluted to 0.28 μg/ml in 100 mM phosphate buffer (pH 7.4). LDH activity was assessed by measuring the change in concentration of β-NADH as a function of time. Dynamic light scattering analysis (DLS) was performed to examine the size distribution, polydispersity, and hydrodynamic radius of LDH before and after HPP. No significant difference in CFU/g was observed between lyophilized cells not subjected to HPP and lyophilized cells subjected to 600 MPa for 300 s (P < 0.05). However, lyophilized cells that were suspended in 100% to 60% peptone water showed a ~ 7.5-log₁₀ reduction when subjected to HPP. Survival of L. monocytogenes following HPP significantly increased (P < 0.05) when the peptone water concentration was decreased below 60% (a_w ~ 0.8). DLS results revealed that LDH suspended in buffer underwent aggregation following HPP (600 MPa, 300 s). Inactivation rate constants obtained using a first-order kinetic model indicated that untreated and HP processed lyophilized LDH had similar activities. When LDH was subject to HPP in solutions containing glycerol, enzyme activity decreased as the water content increased (r² = 0.95). Lyophilization completely protected L. monocytogenes and LDH from inactivation by high pressure. Furthermore, enzyme activity and cell survival increased as water activity was decreased. We postulate low a_w results in protein stabilization, which prevents protein denaturation and cell death during HPP.     

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.     

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.     

Survival of pathogenic microorganisms in an egg-nog-like product containing 7% ethanol

A liquor consisting of whole egg, saccharose (25% w/v) and ethanol (7.0% w/v) was artificially contaminated with Salmonella enteritidis, S. typhimurium, Staphylococcus aureus (three different strains), Bacillus cereus and Listeria monocytogenes. After 3 weeks of incubation at 22°C the numbers of Salmonella, S. aureus and L. monocytogenes decreased more than 3 log₁₀ units. Under such conditions, however, the total number of microorganisms increased 3 log₁₀ units. At 4°C the decrease of pathogenic microorganisms was much slower and a decrease of 3 log₁₀ units was observed only after 7 weeks of incubation. Egg-nog, without ethanol, incubated at 22°C allowed growth of Salmonella and S. aureus, while the numbers of B. cereus spores remained unchanged. Vegetative cells of B. cereus as well as L. monocytogenes decreased in numbers. However, after prolonged incubation the numbers of L. monocytogenes increased significantly.     

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).     

Exopolysaccharides production as affected by lactic acid bacteria and fermentation time

The aim of this work was to examine the ropiness of Lactobacillus helveticus BCRC14030, L. helveticus BCRC14076, and Streptococcus thermophilus BCRC14085, and evaluate the effect of fermentation time on exopolysaccharides (EPS) production by the ropy strain. Each of the three strains was inoculated in skim milk medium and incubated in a fermenter for 0–84 h at pH 5, 37 °C. The fermented samples, containing a net volume of 300 ml skim milk, were withdrawn at intervals of 0, 12, 16, 24, 32, 36, 48, 60, 72, and 84 h for determinations of ropy condition, EPS yield, and molecular mass. EPS with ropiness values of 11.3–21.0 mm, produced from L. helveticus BCRC14030 at 32–60 h demonstrated the ropy nature of the strain. Those EPS were composed of high molecular mass of 26,500 kDa. The highest EPS yield of 0.73 g l⁻¹ from this strain was observed (P < 0.05) at less favourable fermentation condition of 60 h. In addition, a relationship between the presence of high molecular mass and the ropiness of EPS from L. helveticus BCRC14030 was revealed.     

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.     

Screening of lactic acid bacteria isolates from dairy and cereal products for exopolysaccharide production and genes involved

Scanning electron microscopy (SEM) studies revealed that exposure to 4lethal alkaline stress induced statistically significant (P < 0.05) changes in mean cell length, radius and volume in Listeria monocytogenes and a derived σ^B deficient mutant. Bacterial morphology was altered at pH values above 9.0, to include single filamentous or elongated chain forms. Such filamentation and chain formation was observed in the parent strain and in the σ^B deficient strain, and in buffered and non-buffered media. Giemsa staining revealed that the filaments were multi-nucleate, with nucleoids spaced along the length of the atypical cells. In buffered media, longer alkaline exposure was associated with increases in the frequency and length of filamentation. In non-buffered medium, longer exposure was associated with gradual decline in length and the frequency of observation of filaments. Transfer of alkaline treated cells to neutral conditions was associated with the formation of septa within filaments, cell division, and a rapid return to normal morphology, i.e. within 3 h. The observed effects, and their reversibility, may be important in increasing the alkaline tolerance of this pathogen during phagocytosis within the innate human immune system response, and in adaptation/survival in food environments treated with alkali detergents and/or sanitisers. Such atypical cells may be associated with increased survival of L. monocytogenes in adverse environments and may also contribute to qualitative and quantitative underestimation of this important pathogen in food processing environments, with potential implications in public health.     

Physiological properties of Lactobacillus paracasei, L. danicus and L. curvatus strains isolated from Estonian semi-hard cheese

Growth and survival of Lactobacillus paracasei (six strains), L. danicus sp. nov. (four isolates, two strains) and L. curvatus (two strains) from semi-hard Estonian cheeses were comparatively studied in different environmental conditions of relevance for their growth in cheese and survival in gastric environment. Maximum specific growth rates for L. paracasei strains varied between 0.40 and 0.57 h⁻¹, and all strains were tolerant to low water activities, heating at 60 °C for 30 min and pH 3. The newly discovered genetically distinct species L. danicus was characterized by low maximum growth rates (0.26–0.38 h⁻¹) and low temperature optimum (<30 °C). It was acid and heat sensitive and inhibited at salt concentrations from 4% and water activities below 0.93. L. curvatus was characterized by the highest growth rates (0.65–0.70 h⁻¹), tolerance to high NaCl concentrations, but sensitivity to heating, bile salts and low pH. The study showed that genetically different LAB species isolated from cheese could be distinguished by simple cultivation experiments.     

Effect of environmental stresses on the mean and distribution of individual cell lag times of Escherichia coli O157:H7

The effect of starvation, heat or acid stress on duration of individual cell lag time (τ) and standard deviation (SD) of τ was investigated using Escherichia coli O157:H7. Cells were stressed by exposure to acid (pH 3.5), heat (50 °C), or starvation in either glucose-free mineral medium (MOPS), tryptic soy broth (TSB) or Luria broth (LB). Stressed cells were then diluted into wells of a Bioscreen plate to obtain single cells per well. Replicate time to detection (t_d) values were obtained using the Bioscreen and used to calculate the τ and SD. Significant (P ≤ 0.05) increases in τ over untreated controls were found for the following treatments: 14 days in acid; 2 h of heating; 3 days starvation in MOPS; and 2 days starvation in either TSB or LB. The largest increase in τ was > 2-fold from 2.5 to 5.6 h observed with the heat treatment. MOPS starvation was more detrimental to the cells than was acid treatment over the same time period. A significant increase in SD was found with 21 days acid treatment, and 2 days starvation in either TSB or LB. No significant increase in SD was found for MOPS starvation or heat treatment. Lognormal, Gamma, ExtremeValue and Weibull distributions were fitted to the τ data using BestFit. The results suggest that the Lognormal distribution is suitable for fitting τ data from either stressed or unstressed cells.     

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.     

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.     

Temperature shows greater impact on bovine Longissimus dorsi muscle glycogen debranching enzyme activity than does salt concentration

The degradation of glycogen progresses by the co-operation of two enzymes: glycogen phosphorylase (phosphorylase) and glycogen debranching enzyme (GDE). We studied the effect of temperature (4–42 °C) and salt concentration (0–3% NaCl) on bovine M. longissimus dorsi GDE activity. GDE activity (n = 4) decreased significantly with decreasing temperature from about 40–4 °C. GDE exhibited 52% activity at 25 °C and 11% at 4 °C compared to its optimum activity measured at 39 °C. In rapidly chilled meat, the reduction in GDE activity may substantially delay the rate of glycolysis. However, residual GDE activity at 4 °C seems sufficient to enable the attainment of normal ultimate pH if the available time is long enough. An increase in salt concentration from 0% to 2% and to 3% induced a significant (P < 0.001) increase in the ultimate pH of ground bovine meat (n = 6), but showed no effect on GDE activity.     

A method for simultaneous fluorometry and rheology of connective tissue in bulk meat

A probe tipped with optical fibres was mounted on the load cell of a compression tester and pushed into well-aged beef rib roasts (Canada Grade AAA, n = 6, 33 ± 3.6 days post-mortem). Fluorescence (F; excitation 365 nm, emission >420 nm) and reflectance (R; 365 nm) were measured through single optical fibres. Diffuse R was measured using different fibres for illumination and detection, thus responding to tissue between the two fibres. Replication was by a matrix pattern of penetrations on single roasts. For example, in a typical roast, F was correlated with the force of penetration (mean r = 0.86 ± 0.06, n = 20, all P < 0.001). R was less (P < 0.001) strongly correlated with penetration force (mean r = 0.46 ± 0.10, n = 20, all P < 0.001). F signals from connective tissue contained less peaks than R signals from both connective and adipose tissue (respectively, 2.75 ± 0.43 versus 5.57 ± 0.67 peaks cm⁻¹, P < 0.001, n = 20 pairs) and F peaks were wider than R peaks (respectively, 3.54 ± 0.88 versus 1.38 ± 0.19 mm, P < 0.001, n = 20 pairs). For the spinales dorsi aponeurosis, the depth at which peak force was reached was strongly correlated with the depths at which both peak F and peak R were reached (r = 0.98, P < 0.001, n = 20 for both). Diffuse R was only weakly correlated with penetration force (mean r = 0.29 ± 0.12 with only 5/10 correlations significant P < 0.001). This new method showed the primary resistance to dorso-ventral penetrometry of well-aged beef rib roasts originated from connective tissue.     

Effect of temperature and water activity on growth and ochratoxin A production by Australian Aspergillus carbonarius and A. niger isolates on a simulated grape juice medium

The effect of water activity (0.92, 0.95, 0.965 and 0.98) and temperature (15 °C, 25 °C, 30 °C and 35 °C) on growth rate and ochratoxin A (OA) production by five strains of Aspergillus carbonarius and two strains of A. niger isolated from Australian vineyards was characterised on a synthetic grape juice medium. Maximum growth for A. carbonarius occurred at ca 0.965 a_w and 30 °C, and for A. niger, at ca 0.98 a_w and 35 °C. The optimum temperature for OA production was 15 °C and little was produced above 25 °C. The optimum a_w for toxin production was 0.95–0.98 for A. carbonarius and 0.95 for A. niger. Toxin was produced in young colonies after and, typically, did not continue to accumulate the entire surface area of the plate was colonised. Rather, the amount decreased as colonies aged. Trends for growth and OA production were similar among Australian isolates and those from European grapes, as reported in the literature.     

Thermal inactivation of the frozen thawed traditional meat starter culture, Pediococcus pentosaceus, in a meat model system

The equation, y_(t) = y₍₀₎e^kt, was fitted (R = 0.9281, 0.9220 and 0.9117, respectively) to thermal inactivation data (55, 60 and 65 °C) of the traditional meat starter culture Pediococcus pentosaceus (10⁷ cfu/ml) in a meat model system. The population reduction constant (‘k’) increased (about 2.5- and 3-fold) with an increase in the treatment temperature (from 55 to 60 °C and from 60 to 65 °C, respectively). The Q₁₀ (55–65 °C) for ‘k’ was 7.63. Thermal treatments of 19.1, 9.0 and 3.1 min (55, 60 and 65 °C, respectively) reduced the population of P. pentosaceus by 2.0 logs. The value of ‘k’ and the duration of the thermal treatment played an important role in the extent of the inactivation of the culture. The “zero inactivation” temperature (T₀) for P. pentosaceus was 49.9 °C. About 5 logs of the culture would be destroyed at 63 and 68 °C within about 15.5 and 6.5 min, respectively.     

Physical meat quality characteristics of hot-deboned ostrich (Struthio camelus var. domesticus) Muscularis gastrocnemius, pars interna during post-mortem aging

There is a risk of shortening and toughening with hot-deboning of muscles. However, with refrigerated aging this phenomenon may be negated. Vacuum-packed hot and cold-deboned ostrich Muscularis gastrocnemius, pars interna were stored for 21 d at 4 °C to investigate the effects of hot-deboning on quality characteristics of ostrich meat during refrigerated storage. Muscle pH did not differ (P > 0.05) between hot and cold-deboned muscles during storage. Hot-deboning caused (P < 0.0001) more purge in the vacuum packages of the hot-deboned muscles (1.83 ± 1.31%) than in the cold-deboned muscles (0.67 ± 075%) during the 21-d aging period. Hot-deboned muscles were tougher (P < 0.05) than cold-deboned muscles from 24 h up to 5 d. Although hot-deboning caused muscles to be tougher than cold-deboned muscles, with aging at 4 °C beyond 5 d this toughness was found to be insignificant.     

Inhibition of Listeria monocytogenes by Elite Clonal Extracts of Oregano (Origanum vulgare)

Food safety continues to be a major concern for the food industry in recent years. One of the industry's top priorities has been to find alternative ways to preserve their newly developed foods while satisfying the increasing consumer demand to produce safe, all-natural products. In order to achieve this “clean label”, much research has been devoted to the use of effective plant-based antimicrobials, such as those from herbs and spices, to replace chemical preservatives. However, due to the cross-pollination character of herbs and spices, there is a lot of genetic heterogeneity among different batches of the same plant species. This poses a problem for the routine use of plants, and their extracts, as a barrier towards microbial growth. In order to combat this, a unique tissue-culture-based selection strategy was used to isolate an elite phenolic phytochemical-producing clonal line of oregano (Origanum vulgare). Ethanol extracts of this elite clonal line of oregano were then used to study its inhibitory action against Listeria monocytogenes in both broth and meat systems. Thymol and carvacrol, two of the main phenolic constituents of oregano extracts, were also tested in both systems to evaluate their activity against that of the whole oregano extract.

Results indicate that thymol, carvacrol, and the clonal oregano line were all effective in inhibiting the growth of L. monocytogenes in both systems. Approximately 150-200 ppm of pure carvacrol or thymol was needed in order to significantly inhibit the growth of L. monocytogenes in broth, while at least 1200 ppm (corresponding to 27.8 μg phenolics/ml) of the elite clonal oregano extract was needed to do the same. Inconclusive results were obtained when the clonal line was compared to store-brand samples of oregano. In meat systems, 800 ppm of the oregano extract was able to significantly inhibit the growth of the pathogen more so than 800 ppm of carvacrol. A possible explanation for this is that the oregano extract was able to work more effectively at the interface of the lipid and water-soluble portions of the meat than the carvacrol. These results are promising for the food industry since we have now developed an approach for a highly consistent and reliable natural source of antimicrobial activity for future studies.