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.     

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 degrees C, vegetative cells of A. acidoterrestris DSMZ 2,498 were inactivated by enterocin AS-48 (2.5 microg/ml) and no growth was observed in 14 days. In commercial fruit juices added of AS-48 (2.5 microg/ml) and inoculated with vegetative cells or with endospores of strain DSMZ 2,498, no viable cells were detected during 90 days of incubation at temperatures of 37 degrees C, 15 degrees C or 4 degrees C, except for apple, peach and grapefruit juices inoculated with vegetative cells and incubated at 37 degrees 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 microg/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.     

Treatment of vegetable sauces with enterocin AS-48 alone or in combination with phenolic compounds to inhibit proliferation of Staphylococcus aureus

The antimicrobial activity of enterocin AS-48 against Staphylococcus aureus was tested in vegetable sauces, alone and in combination with phenolic compounds. When added alone at 25 microg/ml, AS-48 inactivated all detectable staphylococci in napoletana and pesto sauces stored at 22 degrees C, but it only caused limited growth inhibition when these sauces were stored at 10 degrees C, as well as in other sauces such as carbonara and green sauce for fish. At 80 microg/ml, AS-48 eliminated all detectable staphylococci in napoletana, pesto, and green sauce for fish regardless of storage temperature, but it still had much more limited effect in carbonara sauce. Antistaphylococcal activity was potentiated significantly when AS-48 was used in combination with the phenolic compounds carvacrol, geraniol, eugenol, terpineol, caffeic acid, p-coumaric acid, citral, and hydrocinnamic acid. The efficacy of the combined treatments depended both on the phenolic compound and the type of sauce. In carbonara sauce stored at 22 degrees C, the combinations of 80 microg/ml AS-48 and 20 mM hydrocinnamic acid or 126 mM carvacrol reduced viable counts of staphylococci below detection limits for up to 30 days.     

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 degrees C, 15 degrees C and 6 degrees C. In food samples supplemented with enterocin AS-48 (in a concentration range of 20-35 mug/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 degrees 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 degrees C in boiled rice or at 95 degrees 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 degrees 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 degrees C; inactivation was faster at higher temperatures. In commercial fruit juices (orange, apple, peach, and pineapple) stored at 4 degrees 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 degrees C for the same period. Commercial fruit juices stored at 28 degrees 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 degrees 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 degrees 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.     

Combined effect of enterocin AS-48 and high hydrostatic pressure to control food-borne pathogens inoculated in low acid fermented sausages

The single and combined effects of enterocin AS-48 and high hydrostatic pressure (HHP) on Listeria monocytogenes, Salmonellaenterica, and Staphylococcus aureus was investigated in fuet (a low acid fermented sausage) during ripening and storage at 7 °C or at room temperature. AS-48 (148 AU g⁻¹) caused a drastic 5.5 log cfu g⁻¹ decrease in L. monocytogenes (P < 0.001) and a significant (P < 0.01) inhibition (1.79 logs) for Salmonella at the end of ripening (10 d). After pressurization (400 MPa) and storage Listeria counts remained below 5 cfu g⁻¹ in all fuets containing AS-48 (pressurized or not). HHP alone had no anti-Listeria effect. HHP treatment significantly reduced Salmonella counts, with lowest levels in pressurized fuets with AS-48. S. aureus showed similar growth for all treatments and storage conditions. These results indicate that AS-48 can be applied alone to control L. monocytogenes and combined with HHP treatment to control Salmonella in fuets.     

Evaluation of an enterocin AS-48 enriched bioactive powder obtained by spray drying

Enterocin AS-48 is a cationic cyclic bacteriocin produced by Enterococcus faecalis with broad bactericidal activity. Currently we are assaying the efficacy of AS-48 as biopreservative in foods. In this work we have applied the spray drying process to different AS-48 liquid samples to obtain active dried preparations. We have also assayed different methods, heat, UV irradiation and filtration, to inactivate/remove the AS-48 producer cells from the samples. Best results were obtained for the sample from CM-25 cation exchange, for which it was also possible to completely eliminate/inactivate the producer cells by heat or UV irradiation without loss of activity. When added at 0.016% or 5% to Brain Heart Infusion broth or to skim milk, respectively, the AS-48 powder caused early and complete inactivation of Listeria monocytogenes. A partial inhibition of Staphylococcus aureus was achieved in broth and in skim milk supplemented with 2.5% and 10% AS-48 powder, respectively.     

Microbial diversity changes in soybean sprouts treated with enterocin AS-48

Seed sprouts may act as vehicles for foodborne pathogenic bacteria. In the present study, the effect of washing treatment with the enterococcal bacteriocin enterocin AS-48 on the microbiota of two batches of soybean sprouts was studied by culture-dependent and independent methods throughout storage at 10 °C. Viable cell counts of bacteriocin-treated samples revealed some modifications only for lactic acid bacteria and enterococci during storage. In the control samples from batch 1, the culture-independent DGGE analysis revealed species from genera Rahnella and Serratia as the predominant bacteria at early stages. Several bands corresponding to other genera (two Pantoea bands, one Escherichia band, and five Enterobacter bands) were also detected during storage of control samples, especially at days 3 and 5, while one Rahnella band disappeared. By contrast, some of the enterobacteria (Pantoea Escherichia and Enterobacter) were not detected or showed very faint bands in batch 1 bacteriocin-treated samples, in which two new and intense bands corresponding to genera Enterococcus and Leuconostoc were detected. Batch 2 showed a more homogeneous bacterial population, composed mainly by species of genus Enterobacter together with Pantoea. The major modifications detected in the bacteriocin-treated samples from batch 2 included the loss of one genus Enterobacter band at days 3, 5 and 7, and the detection of a new band corresponding to genus Leuconostoc at days 5 and 7. These results suggest that bacteriocin treatment disturbs the microbial balance in sprouts, producing changes in the microbial profile that cannot be detected by culture-dependent methods. The results also encourage the use of culture-independent methods to gain more insights into the global effects of bacteriocins in food systems.     

Efficacy of enterocin AS-48 against bacilli in ready-to-eat vegetable soups and purees

The broad-spectrum bacteriocin enterocin AS-48 was tested for biopreservation of ready-to-eat vegetable foods (soups and purees) against aerobic mesophilic endospore-forming bacteria. By adding AS-48 (10 microg/ml), Bacillus cereus LWL1 was completely inhibited in all six vegetable products tested (natural vegetable cream, asparagus cream, traditional soup, homemade-style traditional soup, vegetable soup, and vichyssoise) for up to 30 days at 6, 15, and 22 degrees C. A collection of strains isolated from spoiled purees showed slightly higher resistance to AS-48 in the order Paenibacillus sp. > Bacillus macroides > B. cereus, although they were also completely inhibited in natural vegetable cream by AS-48 at 10 microg/ml. However, cocktails of five or eight strains composed of B. cereus (three strains), B. macroides (two strains), and Paenibacillus sp., Paenibacillus polymyxa, and Paenibacillus amylolyticus showed higher bacteriocin resistance with AS-48 of up to 50 microg/ml required for complete inactivation in natural vegetable cream stored at 22 degrees C. Repetitive extragenic palindromic sequence-based PCR (REP-PCR) analysis showed that paenibacilli (along with some B. cereus) was the predominant survivor in the cocktails after bacteriocin treatment. To increase the effectiveness of enterocin AS-48, the bacteriocin was tested (at 20 microg/ml) against the eight-strain cocktail in natural vegetable cream in combination with other antimicrobials. The combination of AS-48 and nisin had a slight but significant additive effect. Bactericidal activity was greatly enhanced by phenolic compounds (carvacrol, eugenol, geraniol, and hydrocinnamic acid), achieving a rapid and complete inactivation of bacilli in the tested puree at 22 degrees C.     

Biocontrol of psychrotrophic enterotoxigenic Bacillus cereus in a nonfat hard cheese by an enterococcal strain-producing enterocin AS-48

Bacillus cereus is a food poisoning bacterium of great concern, especially in milk products. In this study, we describe the efficient control of the psychrotrophic and toxigenic strain B. cereus LWL1 in milk and in a nonfat hard cow's cheese by the enterocin AS-48 producer strain Enterococcus faecalis A-48-32 (Bac+). No viable B. cereus cells were detected after 72 h incubation in milk coinoculated with the AS-48-producing strain and B. cereus. Diarrheic toxin production was also markedly inhibited by the Bac+ strain to eightfold lower levels compared with control cultures of B. cereus. In cheeses manufactured by inoculation with a commercial starter (about 6.8 log CFU/ml) and B. cereus (about 4 log CFU/ml), the latter reached 6.27 log CFU/g after 5 days of maturation, and approximately 8 log CFU/g after 15 days. However, in cheeses made from milk inoculated with the starter along with a mixture of E. faecalis-B. cereus (2/1 ratio), counts of B. cereus decreased by approximately 1.0, 2.0, 4.32, and 5.6 log units with respect to control cheeses after 5, 10, 15, and 30 days of ripening, respectively. Growth of E. faecalis A-48-32 was associated with enterocin AS-48 production and persistence in cheese. Interestingly, growth of starter cultures was not affected by the Bac+ strain, and neither was lactic acid production. These results clearly indicate that E. faecalis A-48-32 produced satisfactory amounts of bacteriocin in cheese and support the potential use of AS-48-producing strains as culture adjuncts to inhibit B. cereus during cheese manufacture and ripening.     

Effect of enterocin AS-48 in combination with biocides on planktonic and sessile Listeria monocytogenes

Enterocin AS-48 was tested on a cocktail of Listeria monocytogenes strains in planktonic and sessile states, singly or in combination with biocides benzalkonium chloride, cetrimide, hexadecylpyridinium chloride, didecyldimethylammonium bromide, triclosan, poly-(hexamethylen guanidinium) hydrochloride, chlorhexidine, hexachlorophene, and the commercial sanitizers P3 oxonia and P3 topax 66. Combinations of sub-inhibitory bacteriocin concentrations and biocide concentrations 4 to 10-fold lower than their minimum inhibitory concentrations (MIC) completely inhibited growth of the planktonic listeriae. Inactivation of Listeria in biofilms formed on polystyrene microtiter plates required concentrations of enterocin AS-48 greater than 50 μg/ml, and biocide concentrations ten to 100-fold higher. In combination with enterocin AS-48 (25 or 50 μg/ml), microbial inactivation increased remarkably for all biocides except P3 oxonia and P3 topax 66 solutions. Polystyrene microtiter plates conditioned with enterocin solutions (0.5-25 μg/ml) decreased the adherence and biofilm formation of the L. monocytogenes cell cocktail, avoiding biofilm formation for at least 24 h at a bacteriocin concentration of 25 μg/ml.     

Inactivation of Geobacillus stearothermophilus in canned food and coconut milk samples by addition of enterocin AS-48

The cyclic bacteriocin enterocin AS-48 was tested on a cocktail of two Geobacillus stearothermophilus strains in canned food samples (corn and peas), and in coconut milk. AS-48 (7 μg/g) reduced viable cell counts below detection levels in samples from canned corn and peas stored at 45 °C for 30 days. In coconut milk, bacterial inactivation by AS-48 (1.75 μg/ml) was even faster. In all canned food and drink samples inoculated with intact G. stearothermophilus endospores, bacteriocin addition (1.75 μg per g or ml of food sample) rapidly reduced viable cell counts below detection levels and avoided regrowth during storage. After a short-time bacteriocin treatment of endospores, trypsin addition markedly increased G. stearothermophilus survival, supporting the effect of residual bacteriocin on the observed loss of viability for endospores. Results from this study support the potential of enterocin AS-48 as a biopreservative against G. stearothermophilus.     

Effect of different activated coatings containing enterocin AS-48 against Listeria monocytogenes on apple cubes

Enterocin AS-48 is a circular bacteriocin with strong anti-Listeria activity. The purpose of the present study was to evaluate the effect of the bacteriocin incorporated into different coating solutions on a cocktail of five L. monocytogenes strains previously inoculated on apple cubes. Coating solutions were made with chitosan, caseinate, alginate, k-carrageenate, xanthan gum, pectin, starch, carboxymethyl cellulose or methyl cellulose. Coatings were applied singly or combined with enterocin AS-48 at 20 or 40 μg/ml. Samples were stored at 4 °C for 7 days. The single application of coatings had almost no effect (as in alginate and methyl cellulose) or had a low effect on Listeria viability (< 2.0 log cycles), with the exception of chitosan coating which showed a strong anti-Listeria activity (up to 3.7 log cycles at day 7). Coatings dosed with 20-μg/ml enterocin AS-48 reduced viable Listeria counts gradually during storage in most cases, achieving significant reductions (p < 0.05) of 1.0 to 1.9 log cycles after 7 days for k-carrageenate, xanthan gum, pectin, starch, carboxymethyl cellulose and methyl cellulose compared to the single coating. At 40 μg/ml, enterocin AS-48 significantly reduced viable counts (p < 0.05) for most coatings (by 1.4 to 3.3 log cycles, depending on the coating) compared with coatings without bacteriocin (except for chitosan). Chitosan, pectin, xanthan gum and carboxymethyl cellulose coatings, supplemented or not with 40 μg/ml AS-48 were further investigated in combination with 20 mM EDTA or with 2.0% sodium lactate. The single addition of sodium lactate showed the greatest effects at day 7, where it reduced viable counts significantly (p < 0.05) by 1.1 to 2.2 log cycles compared to the single coatings (except for chitosan), whereas the combination of sodium lactate and AS-48 reduced viable counts below detection levels also at day 7 for all coatings. The combination of EDTA and AS-48 was much more effective, reducing Listeria counts below detection levels from day 1 for most of the coatings tested. The combination of EDTA and AS-48 was also the most effective at time 0, achieving reductions of viable counts between 2.0 and 2.7 log cycles depending on the coating immediately after treatment compared with single coatings.Industrial relevanceResults from the present study suggest the potential of edible coatings containing enterocin AS-48 and EDTA for inactivation of L. monocytogenes on apple surfaces. Since edible coatings are widely used on fruit surfaces, coatings activated with enterocin AS-48 and EDTA could find application as a hurdle against L. monocytogenes in fresh-cut apple pieces.     

Comparative proteomic analysis of Listeria monocytogenes exposed to enterocin AS-48 in planktonic and sessile states

Enterocin AS-48 is a cyclic peptide of great interest for application in food preservation and sanitation. In the present study, the proteome response of Listeria monocytogenes to purified enterocin AS-48 was studied under two different conditions: planktonic cells and sessile cells grown on polystyrene plates. Ten different proteins were differentially expressed in planktonic L. monocytogenes cells treated with 0.1 μg/ml enterocin AS-48 compared to the untreated controls. Overexpressed proteins were related to stress response (DnaK) or carbohydrate transport and metabolism, while underexpressed and unexpressed proteins were related to metabolism (such as glyceraldehyde-3-phosphate dehydrogenase, pyruvate oxidase, glutamate dehydrogenase or glutamate decarboxylase) or stress (GroEL). In the sessile state, L. monocytogenes cells tolerated up to 10 μg/ml bacteriocin, and the treated biofilm cells overexpressed a set of 11 proteins, some of which could be related to stress response (DnaK, GroEL), protein synthesis and carbohydrate metabolism, while glyceraldehyde-3-phosphate dehydrogenase was the only unexpressed protein. Some of the overexpressed proteins (such as elongation factor Tu and GroEL) could also be implicated in cell adhesion. These results suggest different cell responses of L. monocytogenes to enterocin AS-48 in the planktonic and in the sessile state, including stress response and cell metabolism proteins. While in the planktonic state the bacterium may tend to compensate for the cytoplasmic cell permeability changes induced by AS-48 by reinforcing carbohydrate transport and metabolism, sessile cells seem to respond by shifting carbohydrate metabolism and reinforcing protein synthesis. Stress response proteins also seem to be important in the response to AS-48, but the stress response seems to be different in planktonic and in sessile cells.     

Enhanced bactericidal effect of enterocin AS-48 in combination with high-intensity pulsed-electric field treatment against Salmonella enterica in apple juice

The effect of the broad spectrum cyclic antimicrobial peptide enterocin AS-48 combination with high-intensity pulsed-electric field (HIPEF) treatment (35 kV/cm, 150 Hz, 4 micros and bipolar mode) was tested on Salmonella enterica CECT 915 in apple juice. A response surface methodology was applied to study the bactericidal effects of the combined treatment. The process variables were AS-48 concentration, temperature, and HIPEF treatment time. While treatment with enterocin AS-48 alone up to 60 microg/ml had no effect on the viability of S. enterica in apple juice, an increased bactericidal activity was observed in combination with HIPEF treatments. Survival fraction was affected by treatment time, enterocin AS48 concentration and treatment temperature. The combination of 100 micros of HIPEF treatment, 30 microg/ml of AS-48, and temperature of 20 degrees C resulted in the lowest inactivation, with only a 1.2-log reduction. The maximum inactivation of 4.5-log cycles was achieved with HIPEF treatment for 1000 micros in combination with 60 microg/ml of AS-48 and a treatment temperature of 40 degrees C. Synergism between enterocin AS-48 and HIPEF treatment depended on the sequence order application, since it was observed only when HIPEF was applied in the presence of previously-added bacteriocin. The combined treatment could improve the safety of freshly-made apple juice against S. enterica transmission.     

Inhibition of Bacillus cereus and Bacillus weihenstephanensis in raw vegetables by application of washing solutions containing enterocin AS-48 alone and in combination with other antimicrobials

Enterocin AS-48 is a broad-spectrum cyclic antimicrobial peptide produced by Enterococcus faecalis. In the present study, the bacteriocin was tested alone and in combination with other antimicrobials for decontamination of Bacillus inoculated on alfalfa, soybean sprouts and green asparagus. Washing with enterocin AS-48 solutions reduced viable cell counts of Bacillus cereus and Bacillus weihenstephanensis by 1.0–1.5 and by 1.5–2.38 log units right after application of treatment, respectively. In both cases, the bacteriocin was effective in reducing the remaining viable population below detection levels during further storage of the samples at 6 °C, but failed to prevent regrowth in samples stored at 15 or 22 °C. Application of washing treatments containing enterocin AS-48 in combination with several other antimicrobials and sanitizers (cinnamic and hydrocinnamic acids, carvacrol, polyphosphoric acid, peracetic acid, hexadecylpyridinium chloride and sodium hypochlorite) greatly enhanced the bactericidal effects. The combinations of AS-48 and sodium hypochlorite, peracetic acid or hexadecylpyridinium chloride provided the best results. After application of the combined treatments on alfalfa sprouts contaminated with B. cereus or with B. weihenstephanensis, viable bacilli were not detected or remained at very low concentrations in the treated samples during a 1-week storage period at 15 °C. Inhibition of B. cereus by in situ produced bacteriocin was tested by cocultivation with the AS-48 producer strain E. faecalis A-48-32 inoculated on soybean sprouts. Strain A-48-32 was able to grow and produce bacteriocin on sprouts both at 15 and 22 °C. At 15 °C, growth of B. cereus was completely inhibited in the cocultures, while a much more limited effect was observed at 22 °C. The results obtained for washing treatments are very encouraging for the application of enterocin AS-48 in the decontamination of sprouts. Application of washing treatments containing AS-48 alone can serve to reduce viable cell counts of bacilli in samples stored under refrigeration, while application of combined treatments should be recommended to avoid proliferation of the surviving bacilli under temperature-abuse conditions.     

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.     

Enterococcus faecium P21: a strain occurring naturally in dry-fermented sausages producing the class II bacteriocins enterocin A and enterocin B

Enterococcus faecium P21 isolated from a Spanish dry-fermented sausage shows a narrow antimicrobial activity against closely related lactic acid bacteria and strong antimicrobial activity against spoilage and foodborne Gram-positive pathogenic bacteria such as Listeria monocytogenes, Staphylococcus aureus, Clostridium perfringens and Clostridium botulinum. The antimicrobial activity is produced during growth in MRS broth at 32°C; it is heat resistant (20 min at 80–100°C) and abolished by protease treatment, and withstands exposure to pH 2–11, freeze-thawing, lyophilization and long term storage at −20°C. Purification of the antimicrobial activity by ammonium sulphate precipitation, gel filtration, and cation-exchange, hydrophobic interaction and reverse-phase chromatographies showed that the broad antimicrobial spectrum exerted by E. faecium P21 was indeed due to two peptide bacteriocins. Studies on theirN -terminal amino acid sequences and PCR and DNA analyses revealed that these bacteriocins are identical to the class II bacteriocins enterocin A and enterocin B. The genetic organization of the enterocin A (EntA) operon in E. faecium P21 shows that the bacteriocin structural gene (entA), the immunity gene (entiA) and the induction peptide gene (entF) are clustered and colinearly arranged. Strikingly, this organization was structurally different to that reported for the EntA-producer E. faecium CTC492.     

Inactivation of Staphylococcus aureus in Oat and Soya Drinks by Enterocin AS‐48 in Combination with Other Antimicrobials

The presence of toxicogenic Staphylococcus aureus in foods and the dissemination of methicillin‐resistant S. aureus (MRSA) in the food chain are matters of concern. In the present study, the circular bacteriocin enterocin AS‐48, applied singly or in combination with phenolic compounds (carvacrol, eugenol, geraniol, and citral) or with 2‐nitro‐1‐propanol (2NPOH), was investigated in the control of a cocktail made from 1 methicillin‐sensitive and 1 MRSA strains inoculated on commercial oat and soya drinks. Enterocin AS‐48 exhibited low bactericidal activity against staphylococci in the drinks investigated when applied singly. The combinations of sub‐inhibitory concentrations of enterocin AS‐48 (25 μg/mL) and phenolic compounds or 2NPOH caused complete inactivation of staphylococci in the drinks within 24 h of incubation at 22 °C. When tested in oat and soya drinks stored for 7 d at 10 °C, enterocin AS‐48 (25 μg/mL) in combination with 2NPOH (5.5 mM) reduced viable counts rapidly in the case of oat drink (4.2 log cycles after 12 h) or slowly in soya drink (3.8 log cycles after 3 d). The same combined treatment applied on drinks stored at 22 °C achieved a fast inactivation of staphylococci within 12 to 24 h in both drinks, and no viable staphylococci were detected for up to 7 d of storage. Results from the study highlight the potential of enterocin AS‐48 in combination with 2NPOH for inactivation of staphylococci.