The molecular characterisation and antimicrobial properties of amidated bovine β-lactoglobulin

Amidation of bovine β-lactoglobulin (β-Lg) imparted antimicrobial properties to this protein. Amidated β-Lg was strongly bactericidal against resting cells of Pseudomonas fluorescens, Pseudomonas fragi and Bacillus subtilis, but had a much weaker effect against Escherichia coli, Enterococcus faecalis, Salmonella typhimurium and Listeria monocytogenes. Neither native nor amidated β-Lg was effective against the yeast Saccharomyces cerevisiae and the mould Penicillium candidum. Mass spectrometric analysis demonstrated that amidation of β-Lg converted aspartyl and glutamyl residues to asparaginyl and glutaminyl residues, respectively, and that the amidation reaction did not occur to the same extent on every β-Lg molecule. The charge change was also confirmed by SDS–PAGE, ion exchange chromatography and the change in isoionic point of β-Lg. Reverse-phase chromatography showed that amidation also led to alterations in hydrophobicity of the β-Lg molecule. The antibacterial properties of the amidated β-Lg appear to be dependent on the net positive charge and charge distribution on the molecule.     

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.     

Modelling the effect of a temperature shift on the lag phase duration of Listeria monocytogenes

The aim of this work is to study and model the effect of a temperature shift on h₀, the product of the growth rate by the lag phase duration (μλ). Our work is based on the data of Whiting and Bagi [Int. J. Food Microbiol. 73 (2002) 291], who studied the influence of both the pre-incubation temperature (T_prior) and the growth temperature (T_growth) on λ values of Listeria monocytogenes. We introduce a new model to describe the evolution of the parameter h₀ as a function of T_prior and T_growth, and compare it to Whiting and Bagi's published polynomial model that describes the influence of T_prior and T_growth on λ independently of μ. For exponential as well as stationary phase cells, h₀ increases almost linearly with the magnitude of the temperature shift. A simple linear model of h₀ turns out to be more suitable to predict λ values than a polynomial model of λ.     

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.     

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.     

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.     

Filament formation by Salmonella spp. inoculated into liquid food matrices at refrigeration temperatures,and growth patterns when warmed

In this study, the formation of multicellular filamentous Salmonella cells in response to low temperatures was investigated by using isolates of Salmonella enterica serovar Enteritidis PT4 and S. enterica serovar Typhimurium DT104 as the inocula. The formation of filamentous cells in two liquid food matrices at the recommended maximum temperature for refrigeration (8 degrees C) was monitored and compared with that in tryptone soya broth. Giemsa staining was performed to locate nuclear material within the filaments. Single filaments were warmed on agar at 37 degrees C, and the subsequent rate of septation was quantified. For all strains tested, > 70% of the Salmonella cells inoculated had become filamentous after 4 days in media at 8 degrees C, indicating that filamentation could occur during the shelf life of most refrigerated foods. Strains with impaired RpoS expression were able to form filaments at 8 degrees C, although these filaments tended to be shorter and less numerous. All strains also formed filamentous cells at 8 degrees C in retail milk or chicken meat extract. Filaments often exceeded 100 microm in length and appeared straight-sided under the microscope in media and in foods, and Giemsa staining demonstrated that regularly spaced nucleoids were present. This phenotype indicates that an early block in cell septation is probably responsible for filamentation. When filaments were warmed on agar at 37 degrees C, there was a rapid completion of septation, and for one filament, a >200-fold increase in cell number was observed within 4 h. There are clear public health implications associated with the filamentation of Salmonella in contaminated foods at refrigeration temperatures, especially when the possibility of rapid septation of filamentous cells upon warming is considered.     

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.     

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.     

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.     

Models and mechanisms for bacteriocin action and application

There is considerable research on bacteriocin genetics, purification, and properties. Less is known about the mechanism(s) by which bacteriocins kill pathogens, the physical chemistry of the bacteriocin/pathogen interaction, and of the variables which influence bacteriocins' efficacy in foods. Such knowledge is prerequisite to the wider applications of bacteriocins and to increasing their efficacy by genetic engineering. Mechanistic studies using spores as bacteriocin targets are relatively few. Empirical challenge studies in a variety of foods have had mixed results. Working with well defined model foods, we have determined that increasing protein or phospholipid concentrations decrease nisin 's effectiveness against Clostridium botulinum growth from spore inocula. Nisin is also less effective at abuse compared to refrigerated temperatures. This may be a general characteristic of bacteriocins since increasing temperature decreases many bacteriocins' inhibition of Listeria monocytogenes in foods. L. monocytogenes vegetative cells provide a better target for bacteriocin action than do C. botulinum spores. Bacteriocins dissipate proton motive force (PMF) in L. monocytogenes, C. sporogenes and vegetative cells of other sensitive species. The cytoplasmic membrane is generally considered to be the site at which bacteriocins act. We have adopted fluorescence spectroscopy to characterize the interaction of bacteriocins with liposomes comprised of lipids extracted from L. monocytogenes membranes. The regulatory status of bacteriocins, various models for bacteriocin action, and future prospects for their application are also discussed.     

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.     

Coding repeat instability in the FLO11 gene of Saccharomyces yeasts

In Saccharomyces yeasts, the FLO11 gene encodes an adhesin involved in filamentation, invasive growth, flocculation and adherence to solid surfaces. In wild Saccharomyces flor yeasts, a particularly expanded FLO11 allele also confers to these yeasts the ability to float under stressing liquid environments. We report here that, under optimal laboratory conditions, the repeats domain of the FLO11 gene in these wild yeasts is extremely unstable. Changes in length in the FLO11 coding repeats domain affected Flo11p‐associated functions but, interestingly, some of these functions were affected more than others. Therefore, length variations in this single gene provide a combinatorial diversity, which may contribute to a very rapid adaptation to fluctuating environments. Functional analysis of contracted alleles indicated that buoyancy was not associated to FLO11 length. In contrast, this property depended on the different types of repetitive units found in this gene. Thus, not only variations in the number of intragenic repeats but also the abundance and/or distribution of the different repetitive units may have phenotypic and evolutionary implications. Copyright © 2009 John Wiley & Sons, Ltd.     

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.     

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.     

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.     

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.     

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.