Effect of polythene film activated with enterocin EJ97 in combination with EDTA against Bacillus coagulans

Polythene films coated with the enterococcal bacteriocin enterocin EJ97 alone or in combination with EDTA were tested against Bacillus coagulans CECT 12. Bacteriocin activity was clearly enhanced by EDTA, as shown by viable staining and epifluorescence microscopy observation of treated cells. Activated films were tested in liquids from canned corn and canned peas inoculated with B. coagulans cell suspensions and stored at 4 °C and 20 °C for 24 h. The bacteriocin alone showed highest activity in samples stored at 4 °C, while the maximum performance of EDTA was observed at 20 °C. Films activated with a combination of both antimicrobials showed highest bactericidal activity at 4 °C. In liquid from canned corn and peas stored at 4 °C, the combined treatment reduced the concentrations of viable cells progressively over incubation time. Viable staining revealed an increase in the percentage of dead cells at 20 °C, avoiding proliferation of the bacilli. The bactericidal effect of the combined antimicrobial agents was higher in the liquid of canned peas than that of canned corn. The combined use of viable staining and classical viable cell counts allowed a better estimation of cell damage caused by the antimicrobial treatments.     

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.     

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.     

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.     

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.     

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.     

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.     

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.     

Control of Staphylococcus aureus in sausages by enterocin AS-48

Results presented here are the first contribution on the anti-staphylococcal activity of bacteriocin AS-48 in a model meat sausage system. We have examined bacteriocin application, by inoculation with the enterocin AS-48 producer strain Enterococcus faecalis A-48-32 or by adding a semi-purified bacteriocin preparation. AS-48 inhibits proliferation of Staphylococcus aureus in sausages when added at concentrations of 30 or 40μg/g, achieving a significant reduction of 2 and 5.31 log units, respectively, in viable counts (CFU/g) of staphylococci with respect to the untreated control. The presence of bacteriocin also had a moderate negative effect on total lactic acid bacteria. AS-48(+) strain was developed well in the meat mixture, producing sufficient amounts of AS-48 (to a maximum of 76-88 arbitrary units/g) to control growth of staphylococci. The best result was achieved with a bacteriocinogenic strain inoculum of 10(7)CFU/g.     

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.     

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.     

The effect of adding antimicrobial peptides to milk inoculated with Staphylococcus aureus and processed by high-intensity pulsed-electric field

The use of high-intensity pulsed-electric field (HIPEF) and antimicrobial substances of natural origin, such as enterocin AS-48 (AS-48), nisin, and lysozyme, are among the most important nonthermal preservation methods. Thus, the purpose of this study was to evaluate the combined effect on milk inoculated with Staphylococcus aureus of the addition of AS-48 with nisin or lysozyme, or both, together with the use of HIPEF. Synergy was observed in the reduction of Staph. aureus counts with the following combination methods: i) addition of AS-48 and nisin, ii) addition of AS-48 plus use of HIPEF, and iii) addition of AS-48 and nisin plus use of HIPEF. Specifically, when 28 arbitrary units/mL of AS-48 and 20 IU/mL of nisin were added to the milk, and it was treated with HIPEF for 800 μs, over 6 log reductions were observed in the microorganism. In general, Staph. aureus inactivation was dependent on HIPEF treatment time, antimicrobial doses, and medium pH. During storage of the treated milk, survivor population was related to peptide concentration and temperature. Final cell viability was influenced by the sequence in which the treatments were applied: the addition of AS-48 or AS-48 and nisin was more effective before than after HIPEF treatment. The results obtained indicate that the combination of HIPEF and antimicrobials could be of great interest to the dairy industry, although it is necessary to study further the way in which the combined treatments act.     

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.     

Evaluation of high-intensity ultrasonication for the inactivation of endospores of 3 bacillus species in nonfat milk

Endospores of Bacillus licheniformis [American Type Culture Collection (ATCC) 6634], Bacillus coagulans (ATCC 12245), and Geobacillus stearothermophilus (ATCC 15952) were spiked in sterile nonfat milk, and subjected to high intensity batch ultrasonication treatment at different amplitudes (80 or 100%) and durations (1 to 10 min). Increasing the amplitude from 80 to 100% did not result in enhanced inactivation of G. stearothermophilus endospores. However, an increase in the duration of ultrasonication from 1 to 10 min significantly increased the inactivation of endospores of all 3 species. About 48.96% of the G. stearothermophilus endospores were inactivated by ultrasonication alone, whereas ultrasonication and pasteurization combined increased the inactivation to 65.74%. Inactivation of endospores could be further enhanced to 75.32% by ultrasonication and higher heat (80°C/1 min) combination. Endospores of B. licheniformis and B. coagulans were inactivated to a lesser extent compared with G. stearothermophilus spores. Ultrasonicated B. licheniformis endospores germinated in higher numbers when compared with untreated endospores resulting in their greater inactivation during the combined treatment. During microstructure imaging of ultrasonicated endospores, although no structural damage was noticed, they showed irregular shrinkage and wrinkles with surface coarseness. This may also have contributed to their reduced thermal resistance, in addition to sporulation.     

Analysis of mycotoxin fumonisins in corn products by high-performance liquid chromatography coupled with evaporative light scattering detection

Fumonisins are mycotoxins produced by the fungus Fusarium verticillioides, which is a widespread pathogen of corn. The mycotoxins are known to cause fatal diseases in some domestic animals and have been linked to human esophageal cancer in China and South Africa. Here, we describe a simple method for direct and quantitative analysis of the toxins in food products. The method is based on high-performance liquid chromatography coupled with an evaporative laser scattering detector (HPLC–ELSD) without any prior derivatization of the samples. Using this method, we have analyzed corn-based food samples from central markets in eastern China. The results showed that FB₁ was the main contaminant in the samples. The overall level of fumonisin contamination was relatively low, with a range of 0.25–1.80 μg/g (mean 0.74 μg/g) in 66.7% (16 of 24) of corn samples from Middle-eastern Area, 0.21–0.29 μg/g (mean 0.24 μg/g) in 28.6% (6 of 21) of corn samples from Northeastern Area, and 0.30–3.13 μg/g (mean 0.47 μg/g) in 30.0% (6 of 20) of corn samples from Southeastern Area.     

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.     

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.     

Corn oil and milk enhance the absorption of orally administered allyl isothiocyanate in rats

Allyl isothiocyanate, a chief component of mustard oil, exhibits anticancer effects in both cultured cancer cells and animal models. The accumulation of the N-acetylcysteine conjugate of allyl isothiocyanate, the final metabolite of allyl isothiocyanate, in urine was evaluated in rats that were orally coadministered allyl isothiocyanate with fluids (e.g., water, green tea, milk, and 10% ethanol) or corn oil. The N-acetylcysteine conjugate of allyl isothiocyanate content in urine when allyl isothiocyanate (2 or 4 μmol) was coadministered with corn oil or milk showed a greater increase (1.4 ± 0.22 or 2.7 ± 0.34 μmol or 1.2 ± 0.32 or 2.5 ± 0.36 μmol, 1.6- to 1.8-fold or 1.5-fold, respectively) than when allyl isothiocyanate (2 or 4 μmol) was coadministered with water (0.78 ± 0.10 or 1.7 ± 0.17 μmol). This result demonstrates that corn oil and milk enhance the absorption of allyl isothiocyanate in rats.     

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.     

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.