Bacteriocin Concentration Research Articles

Growth-inhibition of hiochi bacteria in namazake (raw sake) by bacteriocins from lactic acid bacteria

The bacteriocins produced by Lactococcus lactis subsp. lactis C101910 (C101910) and NBRC 12007 (NBRC 12007) were used to prevent the growth of sake spoiling hiochi bacteria (Lactobacillus hilgardii, Lactobacillus fructivorans, and Lactobacillus paracasei) in namazake, which is raw (unpasteurized) sake. The bacteriocin concentrations required for decreasing the viable cell concentrations of L. hilgardii and L. fructivorans below the detection limit (1.0 x 10(2) cells/ml) in 24 h from the initial concentration of 4.0-9.5 x 10(5) cells/ml in the namazake at pH 4.5 and at 4 degrees C, were 18-35 U/ml and 5.6 U/ml for the bacteriocin from C101910 and NBRC 12007, respectively. To decrease the viable cell concentration of L. paracasei from the initial concentration of 7.5 x 10(5) cells/ml to below the detection limit (1.0 x 10(2) cells/ml) in 24 h, 350 U/ml bacteriocin from C101910 and 140 U/ml bacteriocin from NBRC 12007 were required. In experiments using McIlvaine buffer (pH 4.5) with 15% ethanol instead of namazake as the medium, the viable cell concentrations of L. hilgardii and L. paracasei decreased to less than 1.0 x 10(2) cells/ml, whereas those of L. fructivorans decreased to less than 1.0 x 10(3) cells/ml, when bacteriocins were added at the concentrations that had proven effective in namazake. The membrane depolarization assay using a fluorescent probe showed that the presence of ethanol stimulated the collapse of the membrane potential induced by bacteriocins. The ethanol induced collapse of the membrane potential suggests that the application of bacteriocins at the storage stage of namazake is more beneficial than when used in other stages of the sake brewing process.

Effect of enterocin EJ97 against Geobacillus stearothermophilus vegetative cells and endospores in canned foods and beverages

Geobacillus stearothermophilus is a thermophilic bacterium typically responsible for the flat-sour spoilage of low-acid canned food with high water activity. Control of vegetative cells and spores of G. stearothermophilus strains CECT 48 and CECT 49 by enterocin EJ97 produced by Enterococcus faecalis EJ97 is described. Both strains were highly sensitive to EJ97 in a culture medium. In samples from canned foods inoculated with a cocktail of vegetative cells or endospores of the two strains and stored at 45 °C for 30 days, viable cell counts were reduced below detection levels. The time course of microbial inactivation depended on the food sample and bacteriocin concentration. Dormant endospores were resistant to EJ97 short-time treatments (5 min), but endospores activated to germinate by heat became bacteriocin sensitive. The simultaneous application of enterocin EJ97 and heat treatments (90 and 95 °C) on dormant endospores had an increased antimicrobial effect that depended both on the bacteriocin concentration and the heat temperature. Results from this study strengthen the potential of enterocin EJ97 for biopreservation against G. stearothermophilus in canned vegetable foods and drinks.

Response of Bacillus cereus ATCC 14579 to challenges with sublethal concentrations of enterocin AS-48

BackgroundEnterocin AS-48 is produced by Enterococcus faecalis S48 to compete with other bacteria in their environment. Due to its activity against various Gram positive and some Gram negative bacteria it has clear potential for use as a food preservative. Here, we studied the effect of enterocin AS-48 challenges on vegetative cells of Bacillus cereus ATCC 14579 by use of transcriptome analysis.ResultsOf the 5200 genes analysed, expression of 24 genes was found to change significantly after a 30 min treatment with a subinhibitory bacteriocin concentration of 0.5 μg/ml. Most of up-regulated genes encode membrane-associated or secreted proteins with putative transmembrane segments or signal sequences, respectively. One operon involved in arginine metabolism was significantly downregulated. The BC4206-BC4207 operon was found to be the most upregulated target in our experiments. BC4206 codes for a PadR type transcriptional regulator, while BC4207 codes for a hypothetical membrane protein. The operon structure and genes are conserved in B. cereus and B. thuringiensis species, but are not present in B. anthracis and B. subtilis. Using real-time qPCR, we show that these genes are upregulated when we treated the cells with AS-48, but not upon nisin treatment. Upon overexpression of BC4207 in B. cereus, we observed an increased resistance against AS-48. Expression of BC4207 in B. subtilis 168, which lacks this operon also showed increased resistance against AS-48.ConclusionBC4207 membrane protein is involved in the resistance mechanism of B. cereus cells against AS-48.

INACTIVATION OF VEROCYTOTOXIGENIC ESCHERICHIA COLI AND LISTERIA MONOCYTOGENES CO‐CULTURED WITH LACTOBACILLUS SAKEI IN A SIMULATED MEAT FERMENTATION MEDIUM

ABSTRACT The effect of bacteriocin producing Lactobacillus sakei 4413 (an isolate from a Greek dry‐fermented sausage) on inhibition of co‐cultured verocytotoxigenic Escherichia coli or Listeria monocytogenes strains was investigated. In the simulated meat fermentation medium (containing a mixture of nitrogen sources, glucose, sodium chloride and curing agents, with initial pH 6.5) incubated at 25C, L. sakei 4413 produced the highest concentration of bacteriocin in the exponential growth phase, reducing the pH of the medium to 4.3 and producing 0.54% w/v lactic acid. Three primary models (i.e., the Gompertz model, the Baranyi model and the Whiting‐Buchanan model) were evaluated for modeling survival curves of different verocytotoxigenic E. coli and L. monocytogenes strains co‐cultured with L. sakei 4413. The inactivation models evaluated indicated an acceptable degree of goodness‐of‐fit. However, the Whiting‐Buchanan model seems more accurate for fitting microbial inactivation curves, showing the highest R2values and lowest root mean square values in co‐cultures of L. sakei and E. coli or L. monocytogenes. Using the Whiting‐Buchanan model, a 4‐D (99.99%) inactivation of E. coli and L. monocytogenes strains was achieved after 50.9 and 48.7 h, respectively, in co‐cultures with L. sakei 4413 in the simulated meat fermentation medium incubated at 25C. In a synthetic medium containing a mixture of different nitrogen sources (i.e., peptone from meat, meat extract, yeast extract) the effect of temperature and glucose on the inhibitory action of L. sakei 4413 against E. coli O157 EDL‐932 was studied by central composite response surface methodology. The results indicate that the minimum time for a 4‐log decline (11.5 h) for E. coli O157 EDL‐932 in co‐culture with L. sakei 4413 can be achieved at 26C and 2% w/v glucose. The results indicate that L. sakei 4413 could be used as bio‐preservative or starter adjunct in meat fermentations in order to minimize the risk associated with the survival of the above foodborne pathogens.PRACTICAL APPLICATIONSThe development of indigenous starters is very promising in order to produce fermented sausages with high sanitary and sensory qualities. This work contributes to the development of a starter adjunct from an indigenous L. sakei (isolated from a traditional sausage manufactured by a Greek producer). L. sakei added in a model system inactivates verocytotoxigenic E. coli and L. monocytogenes. As L. sakei strain is well adapted to the meat environment, it will lead to sausages that do not present a sanitary risk.

Assay of enterocin AS-48 for inhibition of foodborne pathogens in desserts.

Enterocin AS-48 was tested against Staphylococcus aureus, Bacillus cereus, and Listeria monocytogenes in different kinds of desserts. The highest activity against S. aureus was detected in baker cream. However, in yogurt-type soy-based desserts and in gelatin pudding, AS-48 (175 arbitrary units [AU]/g) reduced viable cell counts of S. aureus by only 1.5 to 1.8 log units at most. The efficacy of AS-48 in puddings greatly depended on inoculum size, and viable S. aureus counts decreased below detection levels within 24 h for inocula lower than 4 to 5.5 log CFU/g. For L. monocytogenes, bacteriocin concentrations of 52.5 to 87.5 AU/g reduced viable counts below detection levels and avoided regrowth of survivors. The lowest activity was detected in yogurt-type desserts. For B. cereus, viable cell counts were reduced below detection levels for bacteriocin concentrations of 52.5 AU/g in instant pudding without soy or by 175 AU/g in the soy pudding. In gelatin pudding, AS-48 (175 AU/g) reduced viable cell counts of B. cereus below detection levels after 8 h at 10 degrees C or after 48 h at 22 degrees C. Bacteriocin addition also inhibited gelatin liquefaction caused by the proteolytic activity of B. cereus.

Effect of enterocin AS-48 in combination with high-intensity pulsed-electric field treatment against the spoilage bacterium Lactobacillus diolivorans in apple juice

Enterocin AS-48 was tested in apple juice against the cider-spoilage, exopolysaccharide-producing strain Lactobacillus diolivorans 29 in combination with high-intensity pulsed-electric field (HIPEF) treatment (35 kV/cm, 150 Hz, 4 μs and bipolar mode). A response surface methodology was applied to study the bactericidal effects of the combined treatment, with AS-48 concentration and HIPEF treatment time as process variables. At subinhibitory bacteriocin concentrations, microbial inactivation by the combined treatment increased as the bacteriocin concentration and the HIPEF treatment time increased (from 0.5 to 2.0 μg/ml and from 100 to 1000 μs, respectively). Highest inactivation (4.87 logs) was achieved by 1000 μs HIPEF treatment in combination with 2.0 μg/ml AS-48. While application of treatments separately did not protect juice from survivors during storage, survivors to the combined treatment were inactivated within the following 24 h of storage, and the treated samples remained free from detectable lactobacilli for at least 15 days at temperatures of 4 °C as well as 22 °C. The combined treatment could be useful for inactivation of exopolysaccharide-producing L. diolivorans in apple juice.

Further characterization of a bacteriocin produced byLactobacillus paracaseiHL32

Purification, identification and partial characterization of bacteriocin produced by Lactobacillus paracasei HL32. It has been shown to have activity against Porphyromonas sp. The purification of bacteriocin consisting of gel exclusion followed by anion exchange chromatography produced a single band upon an electrophoresis gel with a molecular weight corresponding to 56 kDa. The isolated protein contained 171 amino acids and the first 151 were sequenced. The bacteriocin contained a high percentage of cationic amino acids near the N-terminus, hydrophobic amino acids in the central region (Leu, Ile, Val, Phe, Trp and Gly) and hydrophilic residues (Ser, Asn and Gln) at the C-terminus. This structure did not match with that of previously reported bacteriocins. The antimicrobial activity of the bacteriocin was determined against some pathogens and normal microbiota (P. gingivalis, P. intermedia, T. forsythensis, S. salivarius and S. sanguinis) found in saliva and crevicular fluid. The bacteriocin was found to inhibit P. gingivalis at the minimum bactericidal concentration (MBC) of 0.14 mmol l(-1), but was found not to inhibit the other oral micro-organisms. The bacteriocin was found from transmission electron microscopy studies to cause pore formation in the cytoplasmic membranes of P. gingivalis at the pole and induce potassium efflux. Bacteriocin concentrations of two to four times of MBC were shown to induce haemolysis. The bacteriocin was heat-stable, surviving at 110 degrees C under pressure and possessed activity over a pH range of 6.8-8.5. Only a small reduction of activity was found to occur after incubation in biological fluids (saliva and crevicular fluid). A novel bacteriocin has been identified that has selective activity against Porphyromonas sp. associated with periodontal disease. The findings of this work gained the knowledge of specific antibacterial activity of bacteriocin against Porphyromonas gingivalis.

Effect of replacing glutamic residues upon the biological activity and stability of the circular enterocin AS-48

Bacteriocins are antimicrobial peptides produced by bacteria and have a relatively narrow range of activity against closely related strains. AS-48 is a circular bacteriocin produced by Enterococcus faecalis that acts against many gram-positive and some gram-negative bacteria, and could well serve as a natural food preservative and antimicrobial agent. The structure of AS-48 is a five-helix bundle in which a hypothetical plane containing the C(alpha) atoms of E4, E20, E49 and E58 segregates a patch of positively charged residues from the rest of the hydrophobic or uncharged surface residues. The aim of this study is to investigate the significance of the four glutamic residues with regard to the potency, stability and functionality of enterocin AS-48. Four genetically engineered variants of AS-48 were obtained by replacing each glutamic residue with alanine by site-directed mutagenesis. Each mutant peptide was purified from E. faecalis cultures. The activity of highly concentrated samples and the MIC were determined against nine bacterial strains by the spot-assay method. Structural studies were made with circular dichroism (CD) spectroscopy. Occasional alterations to the net charge of AS-48 did not significantly affect its activity when high concentrations of bacteriocin were used. Nevertheless, according to the MIC values, three of the four mutated peptides showed weaker activity against the majority of the gram-positive bacteria tested. CD spectroscopy showed that the derivatives were well structured, in a similar way to those of the native molecule, with no modifications in their helix content. The spatial location of the Glu residues rather than their negative charge played a critical role in AS-48 target-cell specificity and bactericidal activity, because the replacement of Glu with Ala modify the interactions between neighbouring residues through their side chains and the interaction to the solvent affecting the protein stability and causing variations in the activity levels against identical organisms.

Inactivation of exopolysaccharide and 3-hydroxypropionaldehyde-producing lactic acid bacteria in apple juice and apple cider by enterocin AS-48

The bacteriocin enterocin AS-48 was tested against exopolysaccharide producing lactic acid bacteria (LAB) strains of Lactobacillus collinoides, Lactobacillus dioliovorans and Pediococcus parvulus as well as two 3-hydroxypropionaldehyde (3-HPA)-producing Lb. collinoides strains causing apple cider spoilage. In fresh-made apple juice, a bacteriocin concentration of 2.5 μg/ml reduced the LAB viable cell counts below detection levels during the course of incubation at 10 and 22 °C for most strains tested, except for Lb. collinoides 5 and Lb. dioliovorans 29. These two strains were significantly inhibited at 10 °C by 5 μg/ml AS-48 or completely inactivated at 22 °C. In a commercial Basque apple cider, the added bacteriocin (2.5 μg/ml for Lb. collinoides strains 9 and 10, and 5 μg/ml for the rest of strains) completely inactivated all LAB strains tested during storage at 10 as well as 22 °C. In the commercial Asturian apple cider tested the LAB strains showed a poor capacity for survival, but the added bacteriocin was equally effective in reducing the numbers of survivors. When a cocktail of the five LAB strains was tested in commercial Basque apple cider, viable cell counts were reduced below detection levels after 2 days for a bacteriocin concentration of 12.5 μg/ml regardless of storage temperature. Comparison of RAPD-PCR profiles revealed that strain Lb. dioliovorans 29 was always the predominant survivor detected in bacteriocin-treated samples.

Semi-preparative scale purification of enterococcal bacteriocin enterocin EJ97, and evaluation of substrates for its production

The influence of substrate composition on the production of enterocin EJ97 and the conditions for semi-preparative bacteriocin recovery have been studied. Final bacteriocin concentrations of 12.5 or 15.6 mg/l were obtained in the commercial media brain heart infusion broth (BHI) and tryptic soya broth, respectively. The bacteriocin was also produced in the complex medium CM (8.75 mg/l), in which the vitamin supplement was essential for production. Some combinations of meat peptone and yeast extract plus either soy peptone or BHI also supported bacteriocin production, at concentrations of 6.25-7.5 mg/l. In cow milk (whole, half-skimmed, and skimmed), the final bacteriocin concentrations obtained ranged from 7.5 to 11.25 mg/l. Highest bacteriocin activity was obtained by using pasteurised milk whey as growth substrate (up to 25 mg/l), suggesting that this bacteriocin can be obtained on a large scale by using this cheap food-grade industrial by-product. Highest bacteriocin titres were always obtained after 8 h of incubation at 37 degrees C. Semi-preparative concentration and purification of enterocin EJ97 produced in a complex medium was achieved by bulk cation exchange chromatography without previous cell separation, followed by reversed-phase chromatography. This two-step procedure allowed preparation of milligram quantities of purified bacteriocin, which is an improvement compared to purification procedures established for most other bacteriocins (35). The availability of purified enterocin EJ97 will facilitate other studies such as the elucidation of its molecular structure and its interaction with target bacteria.

Incorporation of the Antilisterial Bacteriocin-like Inhibitory Substance from Pediococcus parvulus VKMX133 into Film-forming Protein Matrices with Different Hydrophobicity

The antilisterial bacteriocin-like inhibitory substance (BLIS) produced by Pediococcus parvulus VKMX133 was incorporated into protein film matrices of ethanol-soluble corn zein (CZ), and water-soluble whey protein isolate (WPI). Various BLIS concentrations were added to film-forming solutions (FFS), cast, dried, and cut in circular sections (28.27 mm2). Antimicrobial activity of films was evaluated by measuring inhibition zones against Listeria innocua ATCC 33090 on tryptic soy broth (TSB) agar. BLIS released from films into water at 10 °C was determined. Film effectiveness was evaluated by measuring the reduction of L. innocua population (108 colony-forming units [CFU]/mL) in peptone water where film sections were immersed. Film topography was analyzed by scanning electron microscopy (SEM). The minimum BLIS concentration in FFS to generate films with antimicrobial activity was 833 and 3333 arbitrary units per milliliter (AU/mL) for CZ and WPI, respectively. BLIS released into water was detected only for CZ films. Antimicrobial CZ films were more effective in reducing L. innocua population than WPI films at the same BLIS concentrations. SEM showed that surface topography was porous for CZ and more closed and compact for WPI films. BLIS can be entrapped into film protein matrices to produce edible antimicrobial packaging. However, BLIS inhibitory action against L. innocua and release were dependent on film nature and topography, and probably on hydrophobic and electrostatic interactions arising between the protein matrices and BLIS. High concentration of bacteriocin in films does not necessarily improve their effectiveness against L. innocua.

Inhibition of Bacillus licheniformis LMG 19409 from ropy cider by enterocin AS-48

To determine the activity of enterocin AS-48 against ropy-forming Bacillus licheniformis from cider. Enterocin AS-48 was tested on B. licheniformis LMG 19409 from ropy cider in MRS-G broth, fresh-made apple juice and in two commercial apple ciders (A and B). Bacillus licheniformis was rapidly inactivated in MRS-G by 0.5 microg ml(-1)AS-48 and in fresh-made apple juice by 3 microg ml(-1). Concentration-dependent inactivation of this bacterium in two commercial apple ciders (A and B) stored at 4, 15 and 30 degrees C for 15 days was also demonstrated. Counts from heat-activated endospores in cider A plus AS-48 decreased very slowly. Application of combined treatments of heat (95 degrees C) and enterocin AS-48 reduced the time required to achieved complete inactivation of intact spores in cider A to 4 min for 6 microg ml(-1) and to 1 min for 12 microg ml(-1). D and z values also decreased as the bacteriocin concentration increased. Enterocin AS-48 can inhibit ropy-forming B. licheniformis in apple cider and increase the heat sensitivity of spores. Results from this study support the potential use of enterocin AS-48 to control B. licheniformis in apple cider.

In vitro antibacterial activity of the peptide PsVP-10 against Streptococcus mutans and Streptococcus sobrinus with and without glycocalyx

The antibacterial activity of the peptide PsVP-10 obtained from Pseudomonas sp. R10 against Streptococcus mutans and Streptococcus sobrinus was investigated. One hundred and twenty strains of S. mutans and 120 strains of S. sobrinus with and without glycocalyx were isolated from saliva samples in trypticase–yeast–cysteine–sucrose–bacitracin (TYCSB) agar. Bacterial identification was made by polymerase chain reaction. Glycocalyx production was observed in modified TYCSB agar and confirmed with a modified version of the microplate adherence assay. The minimum inhibitory concentration (MIC) of PsVP-10 bacteriocin was determined by means of the agar dilution method, and the time of bacterial death was calculated by means of colony-forming unit counts. The MIC of the bacteriocin PsVP-10 for both bacterial species with and without glycocalyx was <2 mg/L and the time of bacterial death was less than 240 s for all the studied bacterial strains. Thus, bacteriocin PsVP-10 could be an interesting possibility to combat these cariogenic bacterial species.

Determination of bacteriocin activity with bioassays carried out on solid and liquid substrates: assessing the factor "indicator microorganism"

BackgroundSuccessful application of growth inhibition techniques for quantitative determination of bacteriocins relies on the sensitivity of the applied indicator microorganism to the bacteriocin to which is exposed. However, information on indicator microorganisms' performance and comparisons in bacteriocin determination with bioassays is almost non-existing in the literature. The aim of the present work was to evaluate the parameter "indicator microorganism" in bioassays carried out on solid -agar diffusion assay- and liquid -turbidometric assay- substrates, applied in the quantification of the most studied bacteriocin nisin.ResultsThe performance of characterized microorganisms of known sources, belonging to the genera of Lactobacillus, Pediococcus, Micrococcus and Leuconostoc, has been assessed in this work in the assays of plate agar diffusion and turbidometry. Dose responses and sensitivities were examined and compared over a range of assay variables in standard bacteriocin solutions, fermentation broth filtrates and processed food samples. Measurements on inhibition zones produced on agar plates were made by means of digital image analysis. The data produced were analyzed statistically using the ANOVA technique and pairwise comparisons tests. Sensitivity limits and linearity of responses to bacteriocin varied significantly among different test-microorganisms in both applied methods, the lower sensitivity limits depending on both the test-microorganism and the applied method. In both methods, however, only two of the nine tested microorganisms (Lactobacillus curvatus ATCC 51436 and Pediococcus acidilactici ATCC 25740) were sensitive to very low concentrations of the bacteriocin and produced a linear-type of response in all kinds of samples used in this work. In all cases, very low bacteriocin concentrations, e.g. 1 IU/ml nisin, were more accurately determined in the turbidometric assay.ConclusionThe present work shows that in growth inhibition techniques used in bacteriocin quantification, the choice of the indicator microorganism is critical. Evaluation of sensitivity levels and type of produced responses showed that they can vary widely among different test-microorganisms and different applied methods, indicating that not all microorganisms can be used successfully as indicators and that measurements of growth inhibition in liquid media produce more reliable results.

Inhibition of toxicogenic Bacillus cereus in rice-based foods by enterocin AS-48

The antimicrobial effect of the broad-spectrum bacteriocin enterocin AS-48 against the toxicogenic psychrotrophic strain Bacillus cereus LWL1 has been investigated in a model food system consisting of boiled rice and in a commercial infant rice-based gruel dissolved in whole milk stored at temperatures of 37 °C, 15 °C and 6 °C. In food samples supplemented with enterocin AS-48 (in a concentration range of 20–35 μg/ml), viable cell counts decreased rapidly over incubation time, depending on the bacteriocin concentration, the temperature of incubation and the food sample. Enterotoxin production at 37 °C was also inhibited. Heat sensitivity of endospores increased markedly in food samples supplemented with enterocin AS-48: inactivation of endospores was achieved by heating for 1 min at 90 °C in boiled rice or at 95 °C in rice-based gruel. Activity of enterocin AS-48 in rice gruel was potentiated by sodium lactate in a concentration-dependent way.

Stability of Enterocin AS-48 in Fruit and Vegetable Juices

Enterocin AS-48 is a candidate bacteriocin for food biopreservation. Before addressing application of AS-48 to vegetable-based foods, the interaction between AS-48 and vegetable food components and the stability of AS-48 were studied. Enterocin AS-48 had variable interactions with fruit and vegetable juices, with complete, partial, or negligible loss of activity. For some juices, loss of activity was ameliorated by increasing the bacteriocin concentration, diluting the juice, or applying a heat pretreatment. In juices obtained from cabbage, cauliflower, lettuce, green beans, celery, and avocado, AS-48 was very stable for the first 24 to 48 h of storage under refrigeration, and decay of activity was markedly influenced by storage temperature. In fresh-made fruit juices (orange, apple, grapefruit, pear, pineapple, and kiwi) and juice mixtures, AS-48 was very stable for at least 15 days at 4°C, and bacteriocin activity was still detectable after 30 days of storage. Gradual and variable loss of activity occurred in juices stored at 15 and 28°C; inactivation was faster at higher temperatures. In commercial fruit juices (orange, apple, peach, and pineapple) stored at 4°C, the bacteriocin was completely stable for up to 120 days, and over 60% of initial activity was still present in juices stored at 15°C for the same period. Commercial fruit juices stored at 28°C for 120 days retained between 31.5% (apple) and 67.71% (peach) of their initial bacteriocin activity. Solutions of AS-48 in sterile distilled water were stable (120 days at 4 to 28°C). Limited loss of activity was observed after mixing AS-48 with some food-grade dyes and thickening agents. Enterocin AS-48 added to lettuce juice incubated at 15°C reduced viable counts of Listeria monocytogenes CECT 4032 and Bacillus cereus LWL1 to below detection limits and markedly reduced viable counts of Staphylococcus aureus CECT 976.

Control of Alicyclobacillus acidoterrestris in fruit juices by enterocin AS-48

Alicyclobacillus acidoterrestris is a spoilage-causing bacterium in fruit juices. Control of this bacterium by enterocin AS-48 from Enterococcus faecalis A-48-32 is described. Enterocin AS-48 was active against one A. acidocaldarius and three strains of A. acidoterrestris tested. In natural orange and apple juices incubated at 37 °C, vegetative cells of A. acidoterrestris DSMZ 2498 were inactivated by enterocin AS-48 (2.5 μg/ml) and no growth was observed in 14 days. In commercial fruit juices added of AS-48 (2.5 μg/ml) and inoculated with vegetative cells or with endospores of strain DSMZ 2498, no viable cells were detected during 90 days of incubation at temperatures of 37 °C, 15 °C or 4 °C, except for apple, peach and grapefruit juices inoculated with vegetative cells and incubated at 37 °C which were protected efficiently for up to 60 days. Remarkably, in all commercial fruit juices tested, no viable cells were detected as early as 15 min after incubation with the bacteriocin. Endospores incubated for a very short time (1 min) with increasing bacteriocin concentrations were inactivated by 2.5 μg/ml AS-48. Electron microscopy examination of vegetative cells and endospores treated with enterocin AS-48 revealed substantial cell damage and bacterial lysis as well as disorganization of endospore structure.

Control of Listeria monocytogenes in model sausages by enterocin AS-48

In this work we describe the control of Listeria monocytogenes CECT 4032 in sausage by adding the enterocin AS-48 producer strains Enterococcus faecalis A-48-32 and Enterococcus faecium S-32-81, and also by adding a semi-purified preparation of the bacteriocin. Addition of preformed AS-48 caused a significant decrease ( P<0.01) in the number of viable listeria even at the lowest bacteriocin concentration tested (112 AU/g). At a higher concentration (225 AU/g) listeria were below the detection level (1.99 log units/g) in meat at 3 days of incubation but growth of listeria was observed again after 9 days. For an AS-48 concentration of 450 AU/g, no viable listeria were detected after 6 and 9 days of incubation. When E. faecalis A-48-32 was used as inoculum at approximately 10 7 cfu/g, listeria counts decreased progressively from start of experiment, being below detection level at day 9. The best results were obtained with E. faecium S-32-81, since listeria were undetectable at 6 days of incubation. Bacteriocin concentrations in samples reached concentrations of 60 and 80 AU/g for strains A-48-32 and S-32-81, respectively. These results clearly indicate that AS-48 can be used in the control of L. monocytogenes in sausages.

Comparative Study on Antibacterial Effect of Pediocin ACCEL and Nisin Against Fluorescencestained Listeria monocytogenes BCRC 14845

sity in stained cells and increase in efflux were observed with the increase in bacteriocin concentrations up to sity in stained cells and increase in efflux were observed with the increase in bacteriocin concentrations up to sity in stained cells and increase in efflux were observed with the increase in bacteriocin concentrations up to sity in stained cells and increase in efflux were observed with the increase in bacteriocin concentrations up to 12.5 12.5 12.5 12.5 � g/mL. No further decreases in fluorescence intensity and cell numbers were obtained when the concentrag/mL. No further decreases in fluorescence intensity and cell numbers were obtained when the concentrag/mL. No further decreases in fluorescence intensity and cell numbers were obtained when the concentrag/mL. No further decreases in fluorescence intensity and cell numbers were obtained when the concentrag/mL. No further decreases in fluorescence intensity and cell numbers were obtained when the concentration of bacteriocins was over 12.5 tion of bacteriocins was over 12.5 tion of bacteriocins was over 12.5 tion of bacteriocins was over 12.5 tion of bacteriocins was over 12.5�

Comparison of the sensitivity of commercial strains and infant isolates of bifidobacteria to antibiotics and bacteriocins

Abstract Eighteen bifidobacteria, six isolated from infant feces and 12 commercial strains (of which 10 were purchased from the American Type Culture Collection), were tested for sensitivity to 14 antibiotics and four bacteriocins including nisin A, nisin Z, pediocin PA-1 and mutacin B-Ny266. All bacteria were resistant to vancomycin, kanamycin, neomycin and streptomycin. Infant isolates were more sensitive than commercial strains to cloxacillin, ampicillin, chloramphenicol, tetracycline, rifampicin and novobiocin. Sensitivity to bacteriocins determined by a microplate assay varied widely. Generally, nisin A was the most effective bacteriocin followed by nisin Z and mutacin B-Ny266. Pediocin PA-1 appeared to have no inhibition at 70 μg mL −1 . Commercial strains showed relatively variable sensitivity to bacteriocins compared to infant isolates, which were inhibited within a narrow range of bacteriocin concentration. Bacteriocin tolerance could be easily gained by prolonging the exposure time. Death–time curves and transmission electron microscopy (TEM) of logarithmic and stationary cells of two strains ( Bifidobacterium adolescentis ATCC 15704 and infant isolate Bifidobacterium sp. RBL67) incubated with twice the minimum inhibitory concentration of nisin A and nisin Z for 3 h revealed that log-phase cells were more sensitive than stationary-phase cells. TEM showed that the cell membrane is the most likely site of bactericidal effects of nisin.