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).     

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.     

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.     

Behaviour of Listeria monocytogenes in raw sausages (merguez) in presence of a bacteriocin-producing lactococcal strain as a protective culture

The effectiveness of a bacteriocin produced by Lactococcus lactis subsp. lactis M in reducing population level and growth of Listeria monocytogenes ATCC 7644 in fermented merguez sausage was examined. Two different formulas (with or without added nitrites) were assayed and predetermined numbers of Listeria (ca 10⁶ cfu g⁻¹) were added to sausage mixture. The effect of in situ production of the bacteriocin by Lactococcus lactis M on Listeria monocytogenes ATCC 7644 during fermentation and storage of merguez sausages at room (ca 22 °C) or at refrigeration (ca 7 °C) temperature was tested. Results indicated that counts of Listeria monocytogenes were decreased during fermentation of merguez samples fermented with either the bacteriocin-producing Lactococcus lactis M (Bac⁺) or a nonbacteriocin-producing Lactococcus lactis J (Bac⁻). However, reduction in Listeria cfu's was greater in samples fermented with the Bac⁺ than in those fermented with the Bac⁻ starter. In merguez sausage made without nitrites addition, the Bac⁺ starter induced further decrease in Listeria counts by 1.5 log cycles compared with that induced by the Bac⁻ starter. While in merguez samples with added nitrites (0.4%), the effect of the bacteriocin produced in situ was less important than in those made without nitrites addition.     

Isolation,characterisation and identification of lactic acid bacteria from bushera: a Ugandan traditional fermented beverage

One hundred and thirteen strains of lactic acid bacteria (LAB) were selected from 351 isolates from 15 samples of traditionally fermented household bushera from Uganda and also from laboratory-prepared bushera. Isolates were phenotypically characterised by their ability to ferment 49 carbohydrates using API 50 CHL kits and additional biochemical tests. Coliforms, yeasts and LAB were enumerated in bushera. The pH, volatile organic compounds and organic acids were also determined.

The LAB counts in household bushera varied between 7.1 and 9.4 log cfu ml⁻¹. The coliform counts varied between <1 and 5.2 log cfu ml⁻¹. The pH of bushera ranged from 3.7 to 4.5. Ethanol (max, 0.27%) was the major volatile organic compound while lactic acid (max, 0.52%) was identified as the dominant organic acid in household bushera.

The initial numbers of LAB and coliforms in laboratory-fermented bushera were similar; however, the LAB numbers increased faster during the first 24 h. LAB counts increased from 5.5 to 9.0 log cfu ml⁻¹ during the laboratory fermentation. Coliform counts increased from 5.9 to 7.8 log cfu ml⁻¹ at 24 h, but after 48 h, counts were less 4 log cfu ml⁻¹. Yeasts increased from 4.3 to 7.7 log cfu ml⁻¹ at 48 h, but thereafter decreased slightly. The pH declined from 7.0 to around 4.0. Lactic acid and ethanol increased from zero to 0.75% and 0.20%, respectively.

Lactic acid bacteria isolated from household bushera belonged to Lactobacillus, Streptococcus and Enterococcus genera. Tentatively, Lactobacillus isolates were identified as Lactobacillus plantarum, L. paracasei subsp. paracasei, L. fermentum, L. brevis and L. delbrueckii subsp. delbrueckii. Streptococcus thermophilus strains were also identified in household bushera. LAB isolated from bushera produced in the laboratory belonged to five genera (Lactococcus, Leuconostoc, Lactobacillus, Weissella and Enterococcus. Eight isolates were able to produce acid from starch and were identified as Lactococcus lactis subsp. lactis (four strains), Leuconostoc mesenteroides subsp. mesenteroides (one strain), Leuconostoc mesenteroides subsp. dextranicum (one strain), Weissella confusa (one strain) and L. plantarum (one strain).     

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.     

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.     

Antagonistic effect on Listeria monocytogenes and L. innocua of a bacteriocin-like metabolite produced by lactic acid bacteria isolated from sucuk

Two Lactobacilli and four Pediococci strains producing bacteriocin-like metabolities isolated from sucuk were tested with agar spot tests and well diffusion assays for their inhibitory activity against 16 Listeria strains, also isolated from sucuk. The production of organic acids and hydrogen peroxide limited, L. sake Lb 706 (used as a bacteriocin producer strain) and the isolated lactic acid bacteria (LAB) showed inhibitory activity against all of the Listeria strains, while L. sake Lb 706-A (used as a bacteriocin non-producer mutant) had the same effects against only two Listeria monocytogenes strains (51, 52) in agar spot tests. In the well diffusion assays, while L sake Lb 706 and four Pediococci isolates (413, 416, 419, 446) exhibited inhibitory activity against all of the Listeria strains tested, L. sake Lb 706-A and two of the Lactobacilli isolates (77, 116) showed no effect on the Listeria strains tested.     

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.     

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.     

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.     

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.     

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.     

Characterization of Listeria spp. isolated from ready-to-eat products in Argentina using SDS–PAGE and restriction endonuclease

Listeria spp. are considered of interest in public health since their presence indicates the potencial existence of L. monocytogenes. Total cellular proteins and DNA from four strains of L. monocytogenes serotype 4, four strains of L. monocytogenes belonging to serotype 1, twelve strains of L. innocua, four strains of L. seeligeri and two strains of L. welshimeri isolated from ready–to–eat food were studied by SDS–PAGE and restriction endonuclease digestion. SDS–PAGE protein profiles obtained were species specific and could be evaluated by visual comparison. Enzyme for restriction endonuclease analysis was EcoRI, discriminating L. monocytogenes from other Listeria spp. These methodologies might be a helpful tool and a good alternative for epidemiological tracking of listeriosis in laboratories, where other methods are not available.     

Selective enumeration and survival of bifidobacteria in fresh cheese

Five species of bifidobacteria (15 strains), two strains of Lactococcus lactis ssp. lactis, two strains of L. lactis ssp. cremoris, and one strain of L. lactis ssp. lactis var diacetylactis were included in a study to develop a selective medium for enumeration of bifidobacteria from fresh cheese. Viable counts of bifidobacteria or lactococci on modified Columbia agar base (CAB with 0.05% cysteine-HCl) plus raffinose (0.5%) containing various selective agents were compared with non-selective media. The mCAB plus raffinose with lithium chloride (2 g L⁻¹) and sodium propionate (3 g L⁻¹) with pH adjusted to 5.1 was used successfully as a selective medium for the enumeration of bifidobacteria from fresh cheese. Using this medium, it was determined that bifidobacteria could survive up to 15 days at a level higher than 10⁶ cfu g⁻¹ in a fresh cheese stored at 4 or 12°C. The decrease in the viable counts of bifidobacteria was faster during storage at 4°C than at 12°C.     

Some characteristics of lactic acid bacteria present in commercial sucuk samples

A total of 10 sucuk samples, obtained from Denizli, Turkey were analysed for some physical, chemical and microbiological characteristics. In addition, lactic acid bacteria (LAB) strains producing bacteriocin-like metabolites were isolated and identified. The production of some typical metabolites of the cultures isolated was investigated. At the end of the research, the average values of the pH, water and fat content were 5.1, and 37.2% and 30.5%, respectively. Microbiological analyses results were determined: average 8.34 log CFU/g TAMB, 8.91 log CFU/g LAB (at the MRS agar) and average 8.25 log CFU/g LAB (at the Elliker's lactic agar). The average counts of yeast-mould, coliform and Enterobacteriaceae were found to be 5.0 log CFU/g, 3.28 log CFU/g and 3.27 log CFU/g, respectively. In this study, counts of yeast-mould in the two samples, coliform counts in the five samples, and Enterobacteriaceae counts in the three samples were < 1.0 log CFU/g. A total of 6 of 100 LAB isolates obtained from the sucuk samples were found as a strain producing bacteriocin-like metabolites. These 6 strains were identified as follows; 3 strains Lactobacillus plantarum and 3 strains Pediococcus pentosaceus. According to the findings, these strains have the potential to be used as a sucuk starter culture. Additionally, acid and flavour compounds, other undesirable metabolite-producing activities of the strains, were determined in the model system. From these results it was concluded, after the determination of the toxicological properties, that the 4 strains of LAB identified (L. plantarum 13 P. pentosaceus 15 P. pentosaceus 74 and P. pentosaceus 75) would be useful as the starter and protective culture in the processing of the sucuk and similar fermented products.     

Incorporation of Lactobacillus acidophilus in Minas fresh cheese and its implications for textural and sensorial properties during storage

The effect of Lactobacillus acidophilus on instrumental texture profile and related properties of Minas fresh cheese during storage at 5 °C and on sensory performance was investigated. Four cheese-making trials were prepared, two supplemented with a mesophilic type O culture (T1, T2) and two with lactic acid (T3, T4). L. acidophilus was added in T2 and T3. The viability of L. acidophilus, instrumental texture profile analysis and related properties were monitored during storage for up to 21 days. Probiotic cheeses T3 were firmer by the end of storage, due to higher values of pH and hardness. Differences detected were attributed to the starter, rather than to L. acidophilus. Viability of L. acidophilus during storage ranged from 6.04 to 6.93 for T2 and from 5.46 to 6.53 log cfu g⁻¹ for T3, which performed better in sensory evaluation. Minas fresh cheese is a suitable food system for the delivery of L. acidophilus.     

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.     

Effects of dietary vitamin E supplementation and packaging on the colour stability of sliced pasteurized beef ham

The effect of supplementation of vitamin E (2025 IU animal⁻¹ day⁻¹) in the diet of beef bulls on the colour stability of pasteurized beef ham was studied. Control and enriched diets were provided for the last 136 days before slaughter. Pasteurized hams were manufactured from Mm. semitendinosus from eight animals per dietary group. Half of the samples of sliced ham from control (CON) and supplemented (SUP) bulls were packaged under vacuum (VAC) and half in low-oxygen modified atmosphere packs (FOG, gas mixture: CO₂/N₂=50/50). The packages were kept under constant illumination for 28 days at 8°C. During storage, the number of colony-forming units (CFU) reached a maximum of 5x10⁷ g⁻¹. The microflora was dominated by lactic acid bacteria. The supplementation with vitamin E showed no effect on microbial growth. Lipid oxidation was stable during storage. A significant difference between both dietary groups was detected for the decrease in the redness values during storage. Redness values of CON vacuum-packaged samples decreased (P < 0.01) with time, whereas those for the SUP products only tended to decrease. The redness values of FOG-packed ham were higher than those of VAC-packed ham at the end of the display period, irrespective of the dietary group. Overall, colour appeared to be more stable in the FOG-packed products than in the VAC-packed products. It can be concluded that dietary supplementation of bulls with vitamin E appears to offer only a minor improvement in colour stability over current feeding regimens when the Mm. semitendinosus are used to make cured, pasteurized ham-type products.     

Detection and Rapid Purification of Internalin B as a Protein Marker in Listeria monocytogenes

Clinical and food strains of Listeria monocytogenes have been found to express InternalinB (InlB) without polymorphism. InlB, which is a 67-kDa surface protein, behaves as an invasion and adhesion protein of the bacterium into cells. Thus, InlB could be a good candidate as a protein marker to detect L. monocytogenes. Three strains of L. monocytogenes (ATCC 19115, serotype 4b, ATCC 19111, serotype 1/2a, ATCC 7644, serotype 1/2c) were tested to detect and purify InlB. L. grayii (ATCC 25400) and L. innocua (isolated from soft cheese) were used as controls that did not express InlB due to the absence of its gene on the chromosome. InlB was quantitatively extracted in a solubilized form by treatment of L. monocytogenes with 1 M Tris-Cl at pH 7.5. Immunoblot analysis using anti-InlB polyclonal antibody revealed that L. monocytogenes 19115 had the lowest expression, which required enrichment of InlB for its detection. Simple spin-ion exchange chromatography with strong acidic cation exchanger was used to enrich the InlB protein that is strongly basic. Most impurities in the column were washed with 25 mM sodium acetate whereas the InlB protein was only protein retained in the column and can be eluted by 1 M NaCl. The data presented here showed that spin-ion exchange chromatography was found to be a simple and rapid method to enrich and purify InlB within 20 min.