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Microbiota recovered from fresh produce (Romaine lettuce, endives and cucumbers) was allowed to form biofilms on stainless steel coupons. The formed biofilms were treated with benzalkonium chloride (BC) at three different concentrations (0.01, 0.1, and 1.0 g/l), enterocin AS-48 (50 μg/ml) and combinations of BC and enterocin AS-48. The single treatment with bacteriocin had no effect on viability of sessile bacteria. A high concentration of BC (1.0 g/l) was required to achieve 4.1 logs reduction of viable cell counts. The combination of BC (1.0 g/l) and enterocin AS-48 reduced viable cell counts below detectable levels. High-throughput sequencing analysis revealed that the formed biofilms were composed mainly by Proteobacteria of the genera Pseudomonas and Stenotrophomonas . Treatments with enterocin AS-48 and BC at sub-inhibitory concentrations only induced minor changes in the relative abundance of the different bacterial groups associated with biofilms.

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 ( 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 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 relevance Results 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.

Natural antimicrobials have gained interest as possible inhibitors of biofilm formation. The aim of the present study was to determine the efficacy of antimicrobials derived from essential oils (carvacrol, thymol) plus bacteriocin AS-48 immobilized on two plastic supports (low density polyethylene and polyethylene&ndash, polyamide films) on bacterial inactivation. The polyethylene&ndash, polyamide vacuum-packaging plastic film activated with a combination of thymol plus enterocin AS-48 was the most effective in reducing the concentrations of viable planktonic and sessile cells for Listeria innocua, Lactobacillus fructivorans, Bacillus coagulans, and Bacillus licheniformis. Results from the study highlight the potential of polyethylene&ndash, polyamide film activated with thymol plus enterocin AS-48 for reducing the viable cell concentrations of spoilage Gram-positive bacteria and Listeria in both planktonic and sessile states.

A cocktail of Leuconostoc mesenteroides, Leuconostoc gasicomitatum and Leuconostoc gelidum inoculated on autoclaved cherimoya pulp were treated by high hydrostatic pressure (HHP; 400, 500 or 600 MPa for 8 min) and with enterocin AS-48 (35 μg/g), singly or in combination. After treatments, samples were stored at 4 °C for 30 days or at 22 °C for 10 days. HHP treatments at 400 or 500 MPa reduced viable cell concentrations by 4.3 and 4.9 log cycles, respectively. No survivors were detected at 600 MPa. Bacteriocin addition in combination with HHP achieved an additional reduction of 0.6–0.9 log cycles after treatments at 400 or 500 MPa, and also reduced viable counts for most treatments during storage. In samples treated by HHP, regrowth of leuconostocs was delayed to days 15 (400 or 500 MPa) or 30 (600 MPa) of refrigeration storage, or day 3 at 22 °C (all treatments). Results from the study suggest that treatments at 600 MPa (8 min) can be useful to inactivate leuconostocs in cherimoya pulp.

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.

Bacteriophages have attracted great attention for application in food biopreservation. Lytic bacteriophages specific for human pathogenic bacteria can be isolated from natural sources such as animal feces or industrial wastes where the target bacteria inhabit. Lytic bacteriophages have been tested in different food systems for inactivation of main food-borne pathogens including Listeria monocytogenes, Staphylococcus aureus, Escherichia coli O157:H7, Salmonella enterica, Shigella spp., Campylobacter jejuni and Cronobacter sakazkii, and also for control of spoilage bacteria. Application of lytic bacteriophages could selectively control host populations of concern without interfering with the remaining food microbiota. Bacteriophages could also be applied for inactivation of bacteria attached to food contact surfaces or grown as biofilms. Bacteriophages may receive a generally recognized as safe status based on their lack of toxicity and other detrimental effects to human health. Phage preparations specific for L. monocytogenes, E. coli O157:H7 and S. enterica serotypes have been commercialized and approved for application in foods or as part of surface decontamination protocols. Phage endolysins have a broader host specificity compared to lytic bacteriophages. Cloned endolysins could be used as natural preservatives, singly or in combination with other antimicrobials such as bacteriocins.

The aim of the present study was to determine the efficacy of films activated with enterocin AS-48 plus thymol singly, or in combination with high-hydrostatic pressure (HHP) on the inactivation of Listeria innocua in sea bream fillets and in fruit puree stored under refrigeration for 10 days. L. innocua proliferated in control fish fillets during storage. The activated film reduced viable Listeria counts in fillets by 1.76 log cycles and prevented growth of survivors until mid-storage. Application of HHP treatment to fillets packed in films without antimicrobials reduced Listeria counts by 1.83 log cycles, but did not prevent the growth of survivors during storage. The combined treatment reduced viable counts by 1.88 log cycles and delayed growth of survivors during the whole storage period. L. innocua survived in puree during storage. The activated film reduced Listeria counts by 1.80 and 2.0 log cycles at days 0 and 3. After that point, Listeria were below the detection limit. No viable Listeria were detected in the purees after application of HHP treatment singly, or in combination with the activated film. Results from the study indicate that the efficacy of activated films against Listeria is markedly influenced by the food type.

The aim of this study was to determine the impact of activated plastic films with thymol and enterocin AS-48 and high-hydrostatic pressure (HP) treatment on the bacterial load and bacterial diversity of vacuum-packaged sea bream fillets under refrigerated storage for 10 days. The activated film and the HP treatment reduced aerobic mesophiles viable counts by 1.46 and 2.36 log cycles, respectively, while the combined treatment achieved a reduction of 4.13 log cycles. HP and combined treatments resulted in longer delays in bacterial growth. Proteobacteria were the dominant phyla in sea bream fillets. The relative abundance of Firmicutes increased by the end of storage both in controls and in samples treated by HP singly or in combination with the activated films. The predominant operational taxonomic units (OTUs) found at time 0 in control samples (Listeria, Acinetobacter, Pseudomonas, Enterobacteriaceae, Chryseobacterium) rapidly changed during storage (with an increase of Vibrio, Photobacterium, and Shewanella together with Cloacibacterium and Lactobacillales by the end of storage). The activated film and the HP treatment induced drastic changes in bacterial diversity right after treatments (with Comamonadaceae, Methylobacterium, Acidovorax, and Sphingomonas as main OTUs) and also induced further modifications during storage. Bacterial diversity in activated film samples was quite homogeneous during storage (with Vibrio, Photobacterium, and Shewanella as main OTUs) and approached control samples. HP treatments (singly or in combination with activated films) determined a high relative abundance of Acinetobacter (followed by Pseudomonas and Shewanella) during early storage as well as a higher relative abundance of lactic acid bacteria by the end of storage. The results indicate that the complex dynamics of bacterial populations in the refrigerated sea bream fillets are markedly influenced by treatment and antimicrobials applied.

Enterocin AS-48 is a circular bacteriocin produced by Enterococcus. It contains a 70 amino acid-residue chain circularized by a head-to-tail peptide bond. The conformation of enterocin AS-48 is arranged into five alpha-helices with a compact globular structure. Enterocin AS-48 has a wide inhibitory spectrum on Gram-positive bacteria. Sensitivity of Gram-negative bacteria increases in combination with outer-membrane permeabilizing treatments. Eukaryotic cells are bacteriocin-resistant. This cationic peptide inserts into bacterial membranes and causes membrane permeabilization, leading ultimately to cell death. Microarray analysis revealed sets of up-regulated and down-regulated genes in Bacillus cereus cells treated with sublethal bacteriocin concentration. Enterocin AS-48 can be purified in two steps or prepared as lyophilized powder from cultures in whey-based substrates. The potential applications of enterocin AS-48 as a food biopreservative have been corroborated against foodborne pathogens and/or toxigenic bacteria (Listeria monocytogenes, Bacillus cereus, Staphylococcus aureus, Escherichia coli, Salmonella enterica) and spoilage bacteria (Alicyclobacillus acidoterrestris, Bacillus spp., Paenibacillus spp., Geobacillus stearothermophilus, Brochothrix thermosphacta, Staphylococcus carnosus, Lactobacillus sakei and other spoilage lactic acid bacteria). The efficacy of enterocin AS-48 in food systems increases greatly in combination with chemical preservatives, essential oils, phenolic compounds, and physico-chemical treatments such as sublethal heat, high-intensity pulsed-electric fields or high hydrostatic pressure.

Enterocin AS-48 was tested singly or in combination with biocides on a cocktail of six Bacillus cereus strains in planktonic state and in biofilms formed on polystyrene microtiter plates. The biocides tested were benzalkonium chloride, cetrimide, hexadecylpyridinium chloride, triclosan, chlorhexidine, polyhexamethylen guanidinium chloride and commercial sanitizers P3 oxonia and P3 topax 66. The numbers of survivors were determined after 60 min incubation with biocides or the biocide-bacteriocin combinations. Addition of enterocin AS-48 (25 mg/l) increased the inactivation of planktonic cells by the quaternary ammonium compounds, bisphenols and biguanines tested in a range of biocide concentrations from 0.25 to 2.5 g/l, and by 4 g/l polyguanine. Increased inactivation of the bacilli was also observed for the combination of enterocin AS-48 with 2.5% P3 oxonia, but not by P3 topax 66. In the sessile state, the bacilli were more resistant to biocides and also to the bacteriocin-biocide combinations. Hexadecylpyridium chloride was the most active biocide on biofilms in the single treatments. In the combined treatments with 50 mg/l bacteriocin, hexadecylpyridinium (2.5 g/l), polyhexamethylen guanidinium chloride (4 g/l) and P3 oxonia (2.5%) achieved complete inactivation of bacilli populations. P3 topax 66 showed the lowest performance among all treatments tested, either singly or in combination with bacteriocin. A cocktail of endospores was challenged with biocides and enterocin AS-48 for 60 min at temperatures of 22 °C, 40 °C, and 60 °C. Enterocin AS-48 did not significantly ( p > 0.05) reduce viable counts or increase the lethal effect of biocides. However, treatments with 5 g/l benzalkonium chloride at 60 °C, 2.5 g/l hexadecypyridinium at 60 °C or P3 oxonia at 0.025% and 60 °C or at 0.25% at 22–60 °C achieved complete inactivation of bacterial endospores, both singly and in combination with bacteriocin. Significant reductions of viable counts (1–2 log cycles) were also obtained for some treatments with cetrimide, triclosan or polyhexamethylen guanidinium chloride, but not for chlorhexidine (up to 5 g/l) or P3 topax 66 (up to 1%). Polystyrene surfaces dosed with enterocin AS-48 (25 or 50 mg/l) remained free of detectable bacilli from 2 to 24 h after being inoculated with a cocktail of endospores, but stainless steel surfaces dosed with 50 mg/l bacteriocin did not prevent bacterial growth from endospores. Results from this study suggest that enterocin AS-48 could be applied as enhancer of biocide activity against planktonic and sessile B. cereus cells.

Enterocin AS-48 was tested against planktonic Salmonella enterica UJ3197 in combination with polymyxin B and/or biocides. Enterocin AS-48 added at 50 mg/l or 100 mg/l in combination with 2 mg/l polymyxin B caused partial or complete growth inhibition of S. enterica UJ3197 at 24 h incubation. At 2 mg/l, polymyxin B did not enhance significantly the inhibitory effect of biocides against strain UJ3197, except for 25 mg/l cetrimide. The combination of polymyxin B (2 mg/l) and enterocin AS-48 (25 mg/l) significantly inhibited growth of strain UJ3197 in combination with 2.5 or 25 mg/l cetrimide, 25 mg/l hexadecylpyridinium chloride, 500 mg/l chlorhexidine, and also with 0.16 or 1.6 mg/l poly-(hexamethylen guanidinium) hydrochloride. Biofilms formed by four Salmonella strains (S62, S64, UJ3197, UJ3198) on polystyrene microtiter plates were treated with biocides singly or in combinations with polymyxin B (2 mg/l), enterocin AS-48 (25 or 50 mg/l) or both. Sessile salmonellae showed an increased tolerance to all biocides tested compared to planktonic cells. At the concentration tested, polymyxin B did not enhance the bactericidal activity for any of the biocides on sessile salmonellae. Enterocin AS-48 improved the activity of most biocides, but only for some of the strains and only for the highest bacteriocin concentration tested in most cases. The combinations of enterocin AS-48 and polymyxin B improved inactivation of sessile salmonellae for all biocides tested, although the degree of inactivation was highly dependent on strain and biocide. The combination of polymyxin B (2 mg/l) and enterocin AS-48 (50 mg/l) improved significantly (p

Inactivation of Staphylococcus aureus strains by high hydrostatic pressure (HHP) treatments applied singly or in combination with natural antimicrobials (nisin, enterocin AS-48, cinnamon oil and clove oil) was investigated in rice pudding. Treatments at 600 MPa for 10 min reduced initial populations of staphylococci (7.9 log CFU/g) below detectable levels of 1 log CFU/g in the puddings. Treatments at 500 MPa for 5 min (achieving a 2.9-log reduction of viable counts) were investigated singly or in combination with nisin (200 and 500 IU/g), enterocin AS-48 (25 and 50 μg/g), cinnamon oil (0.2%, vol/wt) or clove oil (0.25% vol/wt). The combined treatment of enterocin AS-48 (50 μg/g) and HHP caused a non-significant reduction of 0.4–0.6 log cycles compared to HHP alone. Additional reductions of 0.87, 1.3 and 1.8 log cycles were recorded for the combined HHP treatments with nisin (500 IU/g), cinnamon oil (0.2%) and clove oil (0.25%), respectively. During refrigeration storage for one week, viable counts in puddings from combined treatments were significantly lower compared to the single HHP treatments, e.g. 1.5–2.7 log cycles for HHP-nisin (500 IU/g), 1.1–1.3 log cycles for HHP-AS-48 (50 μg/g) or approx. 1.5 log cycles for HHP-cinnamon oil (0.2%). These results suggest that the time and intensity of HHP treatments required for inactivation of S. aureus in puddings can be reduced when HHP is applied in combination with selected natural antimicrobials.

Staphylococcus aureus was challenged with the cyclic bacteriocin AS-48 in four commercial sauces singly and in combination with sublethal heat treatments (50–60 °C, for 5 min) and with 2-nitro-1-propanol (2NPOH). Heating at 60 °C in combination with AS-48 (25 μg/ml) reduced initial counts (4.1–4.5 log CFU/ml) below detection limit (1.0 log CFU/ml), while a combined treatment at 55 °C inhibited proliferation of staphylococci in sauces for 24 h at 22 °C. In BHI broth, enterocin AS-48 (0–15 μg/ml) acted synergistically with 2NPOH (0–220 mM), reducing the concentrations of both antimicrobials required for inactivation of S. aureus. In sauces, enterocin AS-48 (25 μg/ml) plus 2NPOH (25 mM) reduced initial cell concentrations (4.1–5.0 log CFU/ml) below detectable levels within 8–24 h, depending on the sauce. The combinations of enterocin AS-48 and 2NPOH or sublethal heat are interesting for testing as part of hurdle technology aimed at reducing the risks for proliferation of S. aureus in sauces.

The plantaricin-producing Lactobacillus plantarum strain EC52 isolated from poto-poto, an ethnic maize fermented food, showed a good capacity to grow in a meat sausage model system. In meat mixtures, this strain caused strong acidification both in single culture and in coculture with inoculated pathogenic bacteria: Listeria monocytogenes and Escherichia coli O157:H7. L. plantarum EC52 had a strong inhibitory effect on L. monocytogenes in meat. The observed antagonism depended on the inoculum concentration of the pathogen, resulting in logarithmic reductions of up to 2.4 log cycles after 15 days of storage at 22°C. Strain EC52 was also able to inhibit growth of Escherichia coli O157:H7 for at least 6 days. These results suggest that strain EC52 could be added in meat mixtures such as those used in the manufacture of fermented sausages as a protective culture to reduce the levels of L. monocytogenes and to inhibit proliferation of E. coli. Key words: Biocontrol, sausage model system, Lactobacillus, Listeria, Escherichia.

Enterocin AS-48 was tested against rope-forming Bacillus subtilis CECT 4002 and Bacillus licheniformis CECT 20, as well as on Bacillus cereus and Bacillus pumilus strains in broth and in experimental dough from wheat flour. Vegetative B. subtilis cells in liquid broth were rapidly inactivated by AS-48 (7 AU/ml). In wheat dough, higher bacteriocin concentrations of 14 and 23 AU/g were required for inactivation of B. subtilis vegetative cells and endospores activated to germinate, respectively. B. cereus LWL1 and B. licheniformis CECT 20 were inactivated by AS-48 (14 AU/g) in doughs stored at 22 °C much faster compared to doughs stored at 10 °C. Strains of Bacillus pumilus were partially inactivated in dough by bacteriocin addition (14 AU/g). Results from this study indicate that enterocin AS-48 can reduce the populations of spoilage and potentially-toxigenic bacilli in wheat dough, decreasing the risks for spoilage and food intoxication.

Quantitative real-time PCR may be a rapid and automated procedure for detection of bacterial pathogens from food samples. Nevertheless, when testing the effects of antimicrobials on the viability of bacterial pathogens in foods, we found that DNA from dead cells interfered greatly in the detection of viable Listeria monocytogenes after treatment with the broad-spectrum bacteriocin enterocin AS-48. To overcome this problem, a quantitative real-time PCR (qRT-PCR) assay based on bacterial mRNA was adapted to quantify viable L. monocytogenes in food after bacteriocin treatments. The procedure allowed a better and faster estimation of viable cells compared to PALCAM viable cell counts when the threshold level was 2 log units/g of food, while PALCAM viable count allowed detection of one log unit/g. This procedure may be useful to verify the efficacy of bacteriocins against L. monocytogenes in foods.

Application of natural antimicrobial substances (such as bacteriocins) combined with novel technologies provides new opportunities for the control of pathogenic bacteria, improving food safety and quality. Bacteriocin-activated films and/or in combination with food processing technologies (high-hydrostatic pressure, high-pressure homogenization, in-package pasteurization, food irradiation, pulsed electric fields, or pulsed light) may increase microbial inactivation and avoid food cross-contamination. Bacteriocin variants developed by genetic engineering and novel bacteriocins with broader inhibitory spectra offer new biotechnological opportunities. In-farm application of bacteriocins, bacterial protective cultures, or bacteriophages, can decrease the incidence of food-borne pathogens in livestock, animal products and fresh produce items, reducing the risks for transmission through the food chain. Biocontrol of fungi, parasitic protozoa and viruses is still a pending issue.

The cyclic peptide bacteriocin enterocin AS-48 was tested (at final concentrations of 0.175, 0.613, and 1.05 AU/ml) against the exopolysaccharide-producing cider spoilage strain Pediococcus parvulus 48 in apple juice in combination with high-intensity pulsed electric field (HIPEF) treatment (35 kV/cm and 150 Hz for 4 mus and bipolar mode). The effect of the combined treatments was studied by surface response methodology, with AS-48 concentration and HIPEF treatment time as process variables. A bacteriocin concentration of 0.613 AU/ml in combination with HIPEF treatment time of 1,000 micros reduced the population of pediococci by 6.6 log cycles in apple juice and yielded an apple juice that was free from pediococci during a 30-day storage period at 4 and 22 degrees Celsius. In contrast, application of HIPEF treatment alone had no effect on the surviving pediococci during storage of juice at 22 degrees Celsius. The combined treatment significantly improved the stability of the juice against spoilage by exopolysaccharide-producing P. parvulus.

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.

Bacteriocins are antimicrobial peptides produced by bacteria. Among them, the enterococcal bacteriocin (enterocin) AS-48 stands for its peculiar characteristics and broad-spectrum antimicrobial activity. AS-48 belongs to the class of circular bacteriocins and has been studied in depth in several aspects: peptide structure, genetic determinants, and mode of action. Recently, a wealth of knowledge has accumulated on the antibacterial activity of this bacteriocin against foodborne pathogenic and spoilage bacteria in food systems, especially in vegetable foods and drinks. This work provides a general overview on the results from tests carried out with AS-48 in different vegetable food categories (such as fruit juices, ciders, sport and energy drinks, fresh fruits and vegetables, pre-cooked ready to eat foods, canned vegetables, and bakery products). Depending on the food substrate, the bacteriocin has been tested alone or as part of hurdle technology, in combination with physico-chemical treatments (such as mild heat treatments or high-intensity pulsed electric fields) and other antimicrobial substances (such as essential oils, phenolic compounds, and chemical preservatives). Since the work carried out on bacteriocins in preservation of vegetable foods and drinks is much more limited compared to meat and dairy products, the results reported for AS-48 may open new possibilities in the field of bacteriocin applications.

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 degrees 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.

A collection of 26 enterococci isolated from dairy and meat products were tested for antimicrobial and proteolytic activity. Enterococcus faecium and E. faecalis were the most frequent species among tested enterococci, and 11 isolates produced antimicrobial compounds. Results revealed that 10 out of 11 enterococci synthesized enterocins showing antimicrobial activity against food-born pathogen such as Listeria monocytogenes and Staphylococcus aureus. The broadest spectrum of antimicrobial activity was detected in E. faecalis BGPT1-10P and BGPT1-78. E. faecalis BG221 showed antimicrobial activity that was not related to production of enterocin, H2O2 or organic acid. Twenty-five enterococci showed strong or moderate proteolytic activity towards β-casein. Two isolates, BGPT1-10P and BGPT1-78, showed the most intense hydrolysis of αs1-, β-, κ-casein fractions, total casein as well as gelatin. Extracellular BGPT1-10P and BGPT1-78 proteinases have a molecular mass of about 29 kDa. Bacteriocin production and proteinase activity of natural isolates of enterococci may be of technological interest in dairy and meat-fermented products.

The inhibitory effect of enterocin AS-48 against Staphylococcus aureus was investigated in various types of bakery ingredients. Antibacterial activity greatly depended on the food substrate, ranging from complete inactivation of S. aureus in liquid caramel (in which the bacterium survived poorly) to no significant inhibition (as in vanilla or chocolate creams). Significant reductions of viable counts in the range of 1.8 to 2.7 log units (P < 0.05) were achieved in substrates like pumpkin confiture or diluted almond cream stored at temperatures of 10 or 22 degrees C. Given the very low activity detected in chocolate substrates, enterocin AS-48 was tested in combination with other antimicrobials. Bactericidal activity increased markedly for the combinations of AS-48 and 0.1% eugenol (v/v), 0.5% 2-nitropropanol (v/v), or 3% Nisaplin (w/v). Enterocin AS-48 could be applied in combination with other antimicrobials for preservation of bakery ingredients against S. aureus.

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.

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 microg/g) reduced viable cell counts below detection levels in samples from canned corn and peas stored at 45 degrees C for 30 days. In coconut milk, bacterial inactivation by AS-48 (1.75 microg/ml) was even faster. In all canned food and drink samples inoculated with intact G. stearothermophilus endospores, bacteriocin addition (1.75 microg 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.

A collection of 25 isolates from foods of animal origin (including mainly milk and cheese, together with meat and ham) was studied. Enterococci were identified at species levels as E. faecalis (9 isolates) and E. faecium (16 isolates). Investigation of virulence factors by PCR amplification revealed incomplete sets of cytolysin genes both in E. faecalis and E. faecium isolates. Among E. faecalis, PCR amplification revealed a high incidence of genes encoding for enterococcal surface protein esp (7/9 isolates), enterococcal antigen efaAfs (6/9), aggregation substance agg (2/9) and sex-pheromone encoding genes ccf, cob, cpd (which were detected in 9, 5 and 3 out of 9 isolates, respectively). By contrast, only esp (7/16 isolates) and efaAfm (10/16) were detected among E. faecium. Antibiotic resistance detected at higher frequencies included rifampicin, nitrofurantoin, ciprofloxacin and levofloxacin. Vancomycin resistance was also detected among E. faecalis and E. faecium. E. faecalis isolates showed decarboxylating activity mostly for tyrosine (5/9 isolates), while E. faecium isolates showed a broader decarboxylating capacity, involving tyrosine (11/16 isolates) ornithine (6/16), lysine (4/16) and histidine (3/16). Six isolates produced bacteriocins, and genes encoding for enterocins A, B, P, L50, and 1071 were detected. Many isolates tested positive for several of the traits investigated, which raises concerns about their possible role as reservoirs for dissemination of antibiotic resistance and virulence traits in foods.

The cyclic antibacterial peptide enterocin AS-48 acted synergistically with p-hydroxybenzoic acid methyl ester (PHBME) and with 2-nitropropanol against Salmonella enterica serovar Enteritidis CECT 4300 in Russian salad. In challenge tests on a cocktail of Salmonella strains (S. enterica ssp. enterica serotype Typhi CECT 409, S. enterica ssp. enterica serovar Choleraesuis CECT 915, S. enterica ssp. enterica serovar Enteritidis CECT 4300, S. enterica ssp. arizonae serovar Arizonae CECT 4395, and S. enterica ssp. salamae CECT 4000) in salad at 10°C, the combinations of PHBME and AS-48 (80 μg/g) or 2-nitropropanol and AS-48 (40 μg/g) reduced the concentrations of viable Salmonella from 4.27 to 4.75 log CFU/g down to the limit of detection for 7 days. Salmonella populations did not increase in control samples (without any antimicrobials or in the presence of AS-48 alone) probably due to the low pH of this type of salad and low temperature of incubation, but retained over 85% viability after 1 week. This work opens new possibilities to expand the spectrum of inhibition of enterocin AS-48 against Salmonella enterica.

This study demonstrates the optimization of AS-48 production by strain Enterococcus faecalis A-48-32 using a whey-derived substrate, Esprion-300 (E-300). To optimise AS-48 production, various factors such as E-300 concentration, glucose, pH and inoculum size were adjusted. The following optimal conditions were established: a growth medium comprising 5% E-300 and 1% glucose, inoculated with 8% producer strain and incubated at 28 °C with a controlled pH of 6.55. Under these conditions a maximum activity of 360 AU mL −1 was attained after 18 h and remained relatively constant for 24 h. The critical factors for optimal AS-48 production in this medium were the pH stabilization at 6.55, in addition to a 1% glucose concentration.

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 degrees C, but failed to prevent regrowth in samples stored at 15 or 22 degrees 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 degrees 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 degrees C. At 15 degrees C, growth of B. cereus was completely inhibited in the cocultures, while a much more limited effect was observed at 22 degrees 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.

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 microg/ml reduced the LAB viable cell counts below detection levels during the course of incubation at 10 and 22 degrees C for most strains tested, except for Lb. collinoides 5 and Lb. dioliovorans 29. These two strains were significantly inhibited at 10 degrees C by 5 microg/ml AS-48 or completely inactivated at 22 degrees C. In a commercial Basque apple cider, the added bacteriocin (2.5 microg/ml for Lb. collinoides strains 9 and 10, and 5 microg/ml for the rest of strains) completely inactivated all LAB strains tested during storage at 10 as well as 22 degrees 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 microg/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.

A bacteriocin-producing lactic acid bacterium (strain 2.5) isolated from cow’s milk used in cheese production from Northern Morocco was selected for its strong anti-listerial activity. The producer strain was identified as Enterococcus faecalis by molecular methods. Strain 2.5 carried the genetic determinants for the two-peptide enterococcal bacteriocin enterocin 1071, and the active bacteriocin was purified to homogeneity by reversed-phase chromatography from culture broths of the producer strain. Strain 2.5 carried two plasmids (of ∼7 and 40 kb). Characterization of strain 2.5 at biosafety level indicated that this strain is non-haemolytic, and lacks the genetic determinants for most of the virulence factors described in enterococci (cylB, cylM, gelE, ace and agg) although it carried the genetic determinants cylA, efaAfs as well as determinants for the sex pheromone peptides cpd, cob, and ccf. Strain 2.5 was resistant to tetracycline, rifampicin, and ciprofloxacin, but it was sensitive to penicillin, ampicillin, vancomycin, and teicoplanin. Results from the present study support the potential role of strain 2.5 as an anti-listerial agent to be tested in traditional fermented foods.

Aims: To determine the effectiveness of enterocin AS-48 on Staphylococcus aureus CECT 976 in combination with chemical preservatives at acidic and neutral pH. Methods and Results: At pH 4·5, the activity of AS-48 increases in the presence of lactic acid (1·0%), acetic acid (0·5% and 1·0%), and citric acid (0·3% and 0·6%)· This synergistic effect has also been observed during the first 8 h of incubation with benzoate (0·06% and 0·12%) and sorbate (2% and 3%)· Interestingly, at pH 7, lactate (1%) increases the inhibitory effect of AS-48, reducing the S. aureus population by 6 log units compared with the control culture. At neutral pH, combinations of AS-48 and sodium tripolyphosphate, STPP (0·3% and 0·5%) also eliminate this pathogen after 24 h. Conclusions: These results indicate that enterocin AS-48 could be applied in combination with a range of chemical preservatives in order to increase its efficacy in inhibiting S. aureus. Significance and Impact of the Study: This study supports the potential use of enterocin AS-48 as a biopreservative to control S. aureus in combination with other food-grade chemical hurdles.

The efficacy of enterocin AS-48 to control Staphylococcus aureus in skimmed milk and in fresh cheese has been assayed. In milk, added AS-48 inhibited S. aureus in a concentration-dependent mode. Bacteriocinogenic strain Enterococcus faecalis A-48-32 produced high amounts of AS-48 in milk (18–20 mm inhibition zones) and controlled S. aureus when co-cultivated at different enterococci/staphylococci ratios (approximately 100/1, 1/1, and 1/100). AS-48, produced ex situ or in situ, in combination with moderate heat treatment (65 °C, 5 min), had a strong anti-staphylococci synergistic effect, with a concentration of 20 μg mL −1 being sufficient to eliminate staphylococci after 6-h incubation. The efficacy of bacteriocinogenic strains, E. faecalis A-48-32 and E. faecium UJA32-81, was lower in unripened cheese, but staphylococci counts in treated cheeses remained at least 1 log cfu g −1 below controls throughout storage. Highest inhibition (4–2 log cfu g −1 below controls) was obtained within the first storage week.

Enterococci are lactic acid bacteria of importance in food, public health and medical microbiology. Many strains produce bacteriocins, some of which have been well characterized. This review describes the structural and genetic characteristics of enterocins, the bacteriocins produced by enterococci. Some of these can be grouped with typical bacteriocins produced by lactic acid bacteria according to traditional classification, whereas others are atypical and structurally distinct from the general classes of bacteriocins. These atypical enterocins recently played an important role in and prompted reclassification of the class II bacteriocins into a new scheme. In this review, a more simplified classification scheme for enterocins based on amino acid sequence homologies is proposed. Enterocins are of interest for their diversity and potential for use as food biopreservatives. The emergence of multiple antibiotic-resistant enterococci among agents of nosocomial disease and the presence of virulence factors among food isolates requires a careful safety evaluation of isolates intended for potential biotechnical use. Nevertheless, enterococcal bacteriocins produced by heterologous hosts or added as cell-free preparations may still be attractive for application in food preservation.

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.

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 40 μg/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.

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 (P0.01) 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.

The bacteriocinogenic strain RJ16 isolated from goat cheese has been identified as Enterococcus faecium by species-specific PCR, DNA–rRNA hybridization and rDNA sequencing. Purified bacteriocin from strain RJ16 is a carboxypeptidase A-resistant peptide with a molecular mass (7125 Da) very close to the cyclic peptide enterocin AS-48. Bacteriocin from strain RJ16 and AS-48 show identical antibacterial spectra, although the former is slightly less active on strains of Listeria monocytogenes and Bacillus cereus. Producer strains show cross-immunity. PCR amplification of total DNA from strain RJ16 with primers for the AS-48 structural gene and sequencing of the amplified fragment revealed an almost identical sequence (99.5%), except for a single mutation that predicts the change of Glu residue at position 20 of AS-48 to Val. Therefore, bacteriocin produced by E. faecium RJ16 should be considered a variant of AS-48, which we call AS-48RJ. PCR amplification revealed that strain RJ16 contains the complete as-48 gene cluster. Hybridization with probes for as-48 gene cluster revealed a chromosomal location of as-48 genes in strain RJ16, being the first example of a chromosomal location of this bacteriocin trait. Strain RJ16 produced enzymes of interest in food processing (esterase, esterase lipase and phytase activities), and did not decarboxylate amino acids precursors for biogenic amines. Strain RJ16 did not exhibit haemolytic or gelatinase activities, and PCR amplification revealed the lack of genes encoding for known virulence determinants (aggregation substance, collagen adhesin, enterococcal surface protein, endocarditis antigens, as well as haemolysin and gelatinase production). Strain RJ16 was resistant to ciprofloxacin ( MIC > 2 mg l - 1 ) and levofloxacin ( MIC > 4 mg l - 1 ) and showed intermediate resistance to nitrofurantoin and erythromycin, but was sensitive to ampicillin, penicillin, streptomycin, gentamicin, rifampicin, chloramphenicol, tetracycline, quinupristin/dalfopristin, vancomycin and teicoplanin. Altogether, results from this study suggest that this broad-spectrum bacteriocin-producing strain may have a potential use in food preservation.

After the discovery of bacteriocin AS-48, a 70-residue cyclic peptide produced by Enterococcus faecalis subsp. liquefaciens, some naturally-occurring cyclic proteins from bacteria have been reported. AS-48 is encoded by the 68-kb pheromone-responsive plasmid pMB2, and the gene cluster involved in production and immunity has been identified and sequenced. This peptide exerts a bactericidal action on sensitive cells (most of the Gram-positive and some Gram-negative bacteria). Its target is the cytoplasmic membrane, in which it opens pores, leading to the dissipation of the proton motive force and cell death, a mechanism similar to that proposed for the action of defensins or, most generally, cationic antibacterial peptides. This fact, together with its remarkable stability and solubility over a wide pH range, suggest that this bacteriocin could be a good candidate as a natural food preservative. The amino acid composition of purified AS-48 shows the absence of modified or dehydrated residues, making it clearly different from lantibiotics. Bacteriocin AS-48 also differs from defensins in that it does not contain cysteines and consequently no disulfide bridges, which makes is high stability even more remarkable. Composition analysis of AS-48 shows a high proportion of basic to acidic amino acids, conferring to this peptide a strong basic character, with an isoelectric point close to 10.5. Determination of the AS-48 structural gene DNA sequence, together with the sequences of AS-48 protease digestion fragments and mass spectrometry determinations, allowed us to determine unambiguously the cyclic structure of the molecule, being the first example of a posttranslational modification in which a cyclic structure arises from a "head-to-tail" linkage. We have solved the three-dimensional structure of AS-48 in solution, and it consists of a globular arrangement of five alpha-helices enclosing a compact hydrophobic core. Interestingly, the head-to-tail peptide link between Trp-70 and Met-1 lies in the middle of alpha-helix 5, which is shown to have a pronounced effect on the stability of the three-dimensional structure. Analysis of structure-function relationship allowed us to propose models to understand the aspects of the molecular function of AS-48. The purpose of this work is to review recent developments in our understanding about the biochemical and biological characteristics and structure of this unusual type of bacteriocin.

Aims: Activity of the bacteriocin EJ97 produced by Enterococcus faecalis EJ97 against strains of ‘Bacillus macroides/B. maroccanus’ isolated from spoiled zucchini puree was investigated. Methods and Results: The influence of several factors like bacteriocin concentration, incubation temperature, pH, growth medium and chemical perservatives on bacteriocin activity was investigated. Enterocin EJ97 [2 arbitrary units (AU) per millilitre] had a marked bactericidal effect on strain INRA P53-2 after 4 h of incubation at 37°C, 24 h at 15°C or 48 h at 4°C. Activity was markedly reduced at pH values of 5·0 and 9·0, but was potentiated by sodium nitrite, sodium benzoate, sodium lactate and sodium tripolyphosphate. Inhibition of strain INRA P53-2 in a commercial vegetable puree required a 10-fold higher bacteriocin concentration. Strain EJ97 was able to grow and produce bacteriocin on vegetable puree, but no inhibition of strain INRA P53-2 was detected. Conclusions: The concentration-dependent bactericidal activity of enterocin EJ97 against strain INRA P53-2 was higher at 37°C and neutral pH, and was potentiated by chemical preservatives. Although enterocin EJ97 was less active in vegetable puree, the concentrations providing bactericidal activity in this food matrix are practical for commercial use. Significance and Impact of the Study: Enterocin EJ97 may have a potential for use in the prevention of food spoilage caused by ‘B. macroides/B. maroccanus’.

Enterocin EJ97 showed a concentration-dependent bactericidal activity against vegetative cells of Bacillus coagulans CECT 12. Highest activity was obtained at incubation temperatures of 37°C and 15°C, but almost no activity was detected during incubation of cells at 4°C. Enterocin EJ97 showed highest activity against B. coagulans CECT 12 at pH 7.0. Some decrease of activity was detected at pH 5.0, and a very low activity was detected during incubation of cells at pH 9.0. Enterocin activity was not modified by sodium benzoate or by sodium chloride, but it was enhanced to some extent by sodium tripolyphosphate, and was highly potentiated by sodium nitrite at concentrations above 50 μg/ml.

In the present study, pulp obtained from cherimoya pulp (Annona cherimola) was inoculated with epiphytic microbiota collected from cherimoya fruits, and supplemented or not with the circular bacteriocin enterocin AS-48 (50 μg/g) and then packed under vacuum. Samples supplemented or not with enterocin were treated by high hydrostatic pressure (600 MPa, 8 min) and then stored at 5 °C for 30 days. The single AS-48 treatment only delayed microbial growth non-significantly (p > 0.05). HHP treatment reduced microbial counts by five log cycles, but it did not prevent further growth of survivors by day 7. The combined treatment (AS-48 + HHP) was the most effective, keeping bacterial cell densities at ≤ 1.5 log CFU/g for up to 15 days. 16S rRNA gene pyrosequencing analysis was done on amplicon libraries from the growth on TSA plates seeded with ten-fold dilutions of pulp suspensions and incubated at 22 °C for 24 h. The results obtained are limited by the experimental conditions used in the study, and only concern the bacterial fraction that was selected by the TSA and growth conditions used. Pantoea (Pantoea agglomerans, Pantoea vagans) were the operational taxonomic units (OTUs) detected at highest relative abundance in bacterial biomass grown from control samples for the first 7 days of storage, followed by Enterococcus gallinarum and Leuconostoc mesenteroides during late storage. The single HHP treatment significantly reduced the relative abundance of OTUs belonging to Pantoea and strongly increased that of endosporeformers (mainly Bacillus firmus and Bacillus stratosphericus) early after treatment, although Pantoea became again the predominant OTUs during storage. Samples singly treated with enterocin AS-48 revealed a strong inhibition of E. gallinarum as well as an early decrease in the relative abundance of Pantoea and an increased relative abundance of OTUs belonging to other Gram-negative species (mainly from genera Serratia and Pseudomonas). The strong microbial inactivation achieved by the combined treatment with enterocin and HHP reduced the levels of viable cells below detectable limits at days 0 and 1, and survivors recovered on TSA at day 7 were represented in > 99% by B. firmus OTU. OTUs from endosporeformers were no longer detected during prolonged incubation, displaced by Pantoea spp., Erwinia billingiae and leuconostocs. Results from the present study indicate that HHP in combination with enterocin AS-48 is more effective in preserving the microbiological quality of cherimoya pulp during storage than the single HHP treatment.

The bacteriocinogenic strain of Enterococcus faecalis EJ97 has been isolated from municipal waste water. It produces a cationic bacteriocin (enterocin EJ97) of low molecular mass (5,340 Da) that is very stable under mild heat conditions and is sensitive to proteolytic enzymes. The amino acid sequence of the first 18 N-terminal residues of enterocin EJ97 indicates that it is different from other known protein sequences. Enterocin EJ97 is active on several gram-positive bacteria including enterococci, several species of Bacillus, Listeria, and Staphylococcus aureus. The producer strain is immune to bacteriocin. Enterocin EJ97 has a concentration-dependent bactericidal and bacteriolytic effect on E. faecalis S-47.

Milk may act as vehicle for human pathogenic bacteria (reviewed by Claeys et al. 2013). Pasteurization of milk before human consumption or for the manufacture of dairy products is often required or recommended. Pasteurizarion will decrease the background spoilage microbiota, but it will not yield a sterile product. Some traditional, highly appreciated fermented dairy foods are still made from raw milk, and there is an ongoing debate on the benefits of consuming raw milk versus pasteurized milk (Claeys et al. 2013). According to foodborne disease reports from different industrialized countries, milk and milk products are implicated in 1–5 % of the total bacterial foodborne outbreaks, with 39.1 % attributed to milk, 53.1 % to cheese and 7.8 % to other milk products (De Buyser et al. 2001; Claeys et al. 2013). Bacteriocins seem an attractive approach to improve the safety of milk and dairy products (especially in those made from raw milk), and at the same time may offer some potential technological applications such as in acceleration of cheese ripening (Table 5.1). The antimicrobial effects of bacteriocins and/or their produced strains have been investigated both in raw milks and in several types of dairy products.

Microbes and/or their natural products have played key roles in the preservation of foods during mankind history (Ross et al. 2002). The rational exploitation of microbial antagonism based on scientific knowledge has been possible after the discovery of the biochemical nature of the antimicrobial substances produced by microorganisms. Bacteriocins produced by the lactic acid bacteria (LAB) have several features that still make them attractive for food preservation: (1) LAB have a long history of safe use in foods; (2) LAB and their cell products—including bacteriocins—are generally recognised as safe; (3) LAB bacteriocins are not active and non-toxic on eukaryotic cells, and (4) due to their proteinaceous nature, bacteriocins are expected to become inactivated by digestive proteases and not exert significant effects on gut microbiota at the concentrations ingested with the food. In addition, LAB bacteriocins may be suitable as preservatives, given (1) their sometimes broad antimicrobial spectrum, including food poisoning and spoilage bacteria, (2) their synergistic activity with other antimicrobials, (3) a bactericidal mode of action exerted at membrane level, which avoids cross resistance with antibiotics of clinical use, (4) stability under the heat and pH conditions achieved under processing of many foods, and (5) their genetic determinants are usually plasmid-encoded, which facilitates genetic manipulation and development of producer strains with improved technological properties. Bacteriocin-encoding plasmids may be transferred to other strains by natural processes, but at the same time there is a risk for loss of the plasmid together with the bacteriocin production capacity.

Fresh produce products can become contaminated with human pathogenic bacteria from different sources (such as manure, irrigation water, insects, and during harvesting and other process operations), and have been implicated in a number of outbreaks (Lynch et al. 2009). Several bacteriocin preparations (such as nisin, pediocin, or enterocin AS-48) have been assayed for inactivation of foodborne pathogenic or toxinogenic bacteria (such as Listeria monocytogenes, Bacillus cereus, and Bacillus weihenstephanensis, Escherichia coli, Salmonella and other enterobacteria) on the surfaces of fresh-cut vegetables and on sprouted seeds (Galvez et al. 2008; Randazzo et al. 2009a, b; Abriouel et al. 2010) (Table 8.1). Bacteriocin treatments have also been proposed for decontamination of whole fruit surfaces, and to avoid transmission of pathogenic bacteria from fruit surfaces to processed fruits (Ukuku et al. 2005; Silveira et al. 2008), and to decrease bacterial survival of bacteria on sliced fruit surfaces during storage.

A few studies have investigated the preservation of egg and egg products by application of bacteriocins (Table 6.1). The commercial use of liquid whole egg requires processing in order to prolong its shelf-life and to inactivate foodborne pathogens. As an alternative to conventional pasteurization, an ultrapasteurization processes (i.e., heating at >60 °C for