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Sweet cherry (Prunus avium L.) is a tree widely cultivated in temperate regions for its tasty and healing fruits. Pruning works on the tree give each year considerable amounts of woody wastes that hardly have any utility. The aims of this work were to detect the most active antioxidants present in a cherry pruning wood sample, to isolate and characterize them, and study the antimicrobial and antibiofilm activities of components found in the wood ethyl acetate extract against a selection of foodborne microorganisms. The online HPLC–DPPH technique allowed the detection of two active antioxidant peaks that, after being isolated by a combination of countercurrent chromatography (FCPC) and conventional preparative techniques, and subsequent structural characterization by NMR, MS and polarimetry, resulted to be (‒)-catechin (1) and (‒)-taxifolin (4). Other components of the cherry wood extract were also isolated, among which compounds 1, 4, and (+)-dihydrowogonin (12) have never been reported in P. avium. A selection of the isolated flavonoids was submitted to antimicrobial and antibiofilm activity evaluations against strains from type culture collections, as well as on multi-resistant strains previously isolated in our laboratory. Those compounds with antimicrobial activity detected in preliminary screenings by standard agar diffusion tests ‒the flavan-3-ol 1, the flavanonols 4 and (+)-aromadendrin (5), the flavanone (+)-pinocembrin (15), and the flavone tectochrysin (17)‒ were subjected to the minimal inhibitory concentration (MIC) test, showing all of them MIC values of 100 μg/mL. Compound 4 also induced a significative inhibition on the formation of biofilms by Enterobacter sp. UJA37p at a concentration of 1 μg/mL and a significative disruption of preformed biofilm by this strain at 0.1 µg/mL. Similar results on biofilm disruption were observed with compound 17.

S- and Fe-cycling bacteria can decisively affect the crystallization of Fe-bearing minerals in sediments from fluvial environments. We have studied the relationships between the Fe-bearing mineral assemblage and the bacterial community composition in the sediments rich in organic matter from the upper Chicamocha river basin (Colombia). Rapid flowing sections of the river contain sediments that have a high redox potential, are poor in organic matter and are enriched in kaolinite and quartz. On the other hand, the mineral assemblage of the sediments deposited in the La Playa dam with a high content in organic matter is enriched in Fe-bearing minerals: (a) vivianite and pyrite in the permanently flooded sediments of the dam and (b) pyrite and goethite in the periodically emerged sediments. The bacterial community composition of these sediments reveals anthropic organic matter pollution processes and biodegradation associated with eutrophication. Moreover, periodically emerged sediments in the La Playa dam contain bacterial groups adapted to the alternation of dry and wet periods under oxic or anoxic conditions. Cell-shaped aggregates with a pyritic composition suggest that sulfate-reducing bacteria (SRB) communities were involved in the precipitation of Fe-sulfides. The precipitation of vivianite in the flooded sediments was favored by a greater availability of Fe(II), which promoted the iron-reducing bacteria (IRB) enrichment of the sediments. The presence of sulfur-oxidizing bacteria (SOB) in the flooded sediments and the activity of iron-oxidizing bacteria (IOB) in the periodically emerged sediments favored both pyrite crystallization under a high sulfide availability and the oxidation of microbially precipitated monosulfides. Moreover, IOB enhanced goethite formation in the periodically emerged sediments.

The aim of the present study was to determine the effect of high hydrostatic pressure (HP) and films activated with a combination of enterocin AS-48 and thymol (AF) on the microbial load and bacterial diversity of fruit puree (banana, apple, pear). HP and AF were applied singly, or in combination (AFHP). Samples were chill-stored. HP and AFHP treatments reduced viable counts significantly (P ≤ 0.05). The predominant bacterial groups in the puree were fam. Comamonadaceae (23.17%) and genera Methylobacterium (21.46%), Acidovorax (8.70%) and Sphingomonas (6.63%). Pseudomonas became relevant by the end of storage (10.50%). Most of the AF samples had higher relative abundances of Comamonadaceae, Methylobacterium, Acidovorax and Sphingomonas and lower relative abundance of Pseudomonas. Application of HP treatment markedly reduced the relative abundances of Comamonadaceae, Methylobacterium, Acidovorax and Sphingomonas. Lactobacillales increased in relative abundance upon application of HP treatment but not during storage, while Pseudomonas increased towards the end of storage. The combined treatment (AFHP) achieved greatest reduction in the relative abundances of Comamonadaceae and Methylobacterium. Results indicate that AFHP treatment had greatest effects on the bacterial diversity of the fruit puree and was also the most effective in keeping total aerobic mesophiles and Enterobacteriaceae below detectable levels.

We studied the relationships between the trace element concentration in sediments from a saline lake at a tropical latitude (Sochagota Lake, Colombia) containing hydrothermal and anthropic inputs with the organic matter content, the mineral assemblage composition and the activity of the bacterial communities of the sediments. Organic matter-poor sediments (TOC

Guacamole is an avocado sauce highly appreciated for its pleasant taste and nutritional value. The present study addressed the impact of high-hydrostatic pressure (HP) treatments on the product safety and bacterial diversity. Four HP treatments, 5 min each, were applied: (A) 450 megapascals (MPa) at 22 &deg, C, (B) 450 MPa at 50 &deg, (C) 600 MPa at 22 &deg, (D) 600 MPa at 50 &deg, C. Controls and treated samples were refrigerated stored for 50 days. The residual surviving fraction was lowest for the 600 MPa treatment at 50 &deg, C. Bacterial growth on media supplemented with antibiotics (cefotaxime and imipenem) or the biocide benzalkonium chloride was detected only from control samples but not from HP-treated samples. High throughput sequencing analysis indicated that the bacterial diversity of control samples was dominated by members of Fam. Enterobacteriaceae, but it changed to a lactic acid microbiota during storage. HP-treated samples showed reduced relative abundances of Enterobacteriaceae and lactic acid bacteria and higher abundances of Pantoea, Ralstonia and Methylobacterium. Results from the study indicate that HP treatments of guacamole at 50 &deg, C show higher microbial inactivation compared to 22 &deg, C. However, all treatments reduced the levels of Enterobacteriaceae and penem-tolerant bacteria and provided product stability against acidification by lactic acid bacteria.

European plum (Prunus domestica L.) is a tree cultivated in many countries for its edible fruits, and necessarily certain amounts of useless woody residues are generated every year during pruning works. The objectives of this research were to recover the phenolics present on that wood wastes and study their antimicrobial and antibiofilm activities against a selection of foodborne microorganisms, including multi-resistant strains. The isolation of components was carried out by combining fast centrifugal partition chromatography (FCPC) and semi-preparative high-performance liquid chromatography (HPLC), and the identification was achieved by conventional techniques. Nine compounds were identified: 2 flavan-3-ols [catechin (1) and epicatechin (2)]; 1 phenolic glucoside [annphenone (3)]; and 6 dimeric A-type proanthocyanidins (4–9). All of them have been reported in Prunus domestica by the first time in this work, with the exception of 1 and 2. Extracts, fractions and purified phenolics were submitted to antimicrobial and antibiofilm activity evaluations. The most significative antimicrobial effect was detected on the strain Enterobacter sp. UJA37p, previously isolated from organic tomato and with high tolerance to different biocides. All pure compounds showed MIC values of 100 μg/mL against this resistant bacterium. On the other hand, when bacteria were previously allowed to attach and form biofilm before treatments, we found interesting results in disruption of preformed biofilms with compound 7 [(+)-epiafzelechin-(2β→O→7,4β→8)-epicatechin], followed by compound 6 [(−)-ent-epiafzelechin-(2α→O→7,4α→8)-catechin] and compound 3 at a concentration of 0.1 μg/mL.

Natural A-type procyanidins have shown very interesting biological activities, such as their proven antiadherence properties against pathogenic bacteria. In order to find the structural features responsible for their activities, we describe herein the design and synthesis of six A-type procyanidin analogues and the evaluation of their antimicrobial and antibiofilm properties against 12 resistant bacteria, both Gram positive and Gram negative, isolated from organic foods. The natural A-type procyanidin A-2, which had known antiadherence activity, was also tested as a reference compound for the comparative studies. Within the series, analogue 4, which had a NO2 group on ring A, showed the highest antimicrobial activity (MIC of 10 μg/mL) and was one of the best molecules at preventing biofilm formation (up to 40% decreases at 100 μg/mL) and disrupting preformed biofilms (up to 40% reductions at 0.1 μg/mL). Structure–activity relationships are also analyzed.

Persistence of antibiotic-resistant Salmonella in the food chain may depend on strain tolerance to other antimicrobials and also on biofilm formation capacity. Yet, there is limited information on sensitivity of antibiotic-resistant Salmonella to other antimicrobials, such as phenolic compounds, chemical preservatives, or antimicrobial peptides. This study aimed at correlating antimicrobial resistance and biofilm formation capacity in antibiotic-resistant, biocide-tolerant Salmonella strains from hen eggshells. A collection of 21 strains previously selected according to their antibiotic resistance and biocide tolerance phenotypes were used for the present study. Strains were inspected for their biofilm formation capacity and for their sensitivity to (i) phenolic compounds (carvacrol, thymol), (ii) chemical preservatives (sodium lactate, trisodium phosphate), and (iii) cationic antimicrobials (polymyxin B, lysozyme-EDTA). Biofilm formation capacity was not correlated with antimicrobial resistances of the planktonic Salmonella. Polymyxin B and the lysozyme-EDTA combinations showed significant ( P < 0.05) positive correlations to each other and to sodium lactate. Significant ( P < 0.05) positive correlations were also observed for benzalkonium chloride and cetrimide with carvacrol, thymol, and trisodium phosphate, or between hexadecylpyridinium chloride and carvacrol. Antibiotic resistance also correlated positively with a higher tolerance to other antimicrobials (cefotaxime, ceftazidime, and ciprofloxacin with carvacrol, thymol, and trisodium phosphate; netilmicin with thymol and trisodium phosphate; tetracycline with carvacrol and thymol). These results must be taken into consideration to ensure a proper use of antimicrobials in the poultry industry, at concentrations that do not allow coselection of biocide-tolerant, antibiotic-resistant Salmonella.

Fat type in diet is responsible for specific changes in gut microbiota (GM). Extra virgin olive oil (EVOO) has been shown to be beneficial for blood pressure and to produce effects on GM. To analyze the cause-effect relationship between intestinal microbial changes and blood pressure, we studied the effect of EVOO on fecal microbiota and systolic blood pressure (SBP) levels in spontaneously hypertensive rats (SHR). SHR were fed either an enriched EVOO diet or a standard diet for a period of 12 weeks. At the end of the experimental period, the microbial profiles in the feces were studied in both groups by using PCR-denaturing gradient gel electrophoresis. Real-time PCR was used to quantify the selected bacterial groups. The results demonstrated significant differences when using Lactobacillus (p

A collection of 38 biocide-adapted strains with significant increases in their tolerance to biocides after step-wise exposure to these compounds were screened for sensitivity to essential oils and chemical preservatives. Several strains adapted to quaternary ammonium compounds (benzalkonium chloride, hexadecylpyridinium chloride or cetrimide) showed a generalized increase in the sensitivity to preservatives. Similar results were found among hexachlorophene- or chlorhexidine-adapted strains. Moreover, tolerance to hexadecylpyridinium chloride showed a very strong positive correlation with 4-hydroxybenzoic acid, thyme oil and sodium nitrite increased sensitivity, as well as a strong correlation with clove oil, potassium sorbate and potassium nitrate increased sensitivity. On the contrary, an increase in tolerance to preservatives was detected among triclosan-adapted strains. Results from this study suggest that exposure of bacteria from foods to biocides is not always associated with co-selection for other antimicrobial resistances, especially against essential oils or chemical preservatives used in the food industry.

The continued agricultural exposure of bacteria to metals such as copper and zinc may result in an increased copper tolerance through the food chain. The aim of this study was to determine the Cu and Zn tolerance of bacteria from fresh produce (cucumber, zucchini, green pepper, tomato, lettuce, vegetable salad, broccoli, cabbage, carrot, green onion, onion, and mango). Isolates (506 aerobic mesophiles) from 12 different food produce products were tested for growth in a range of Cu and Zn concentrations. Selected isolates were identified using 16S rDNA sequencing, and the presence of metal resistance genes was studied using PCR amplification. More than 50% of the isolates had MICs for copper sulfate greater than 16 mM, and more than 40% had MICs greater than 4 mM for zinc chloride. Isolates with high levels of tolerance to Cu and Zn were detected in all the produce products investigated. A selection of 51 isolates with high MICs for both Cu and Zn were identified as belonging to the genera Pseudomonas (28), Enterobacter (7), Serratia (4), Leclercia (1), Bacillus (10), and Paenibacillus (1). A study of the genetic determinants of resistance in the selected gram-negative isolates revealed a high incidence of genes from the pco multicopper oxidase cluster, from the sil cluster involved in Cu and silver resistance, and from the chromate resistance gene chrB. A high percentage carried both pco and sil. The results suggest that Cu and Zn tolerance, as well as metal resistance genes, is widespread in bacteria from fresh produce.

In the present study, a collection of 76 biocide-sensitive bacterial strains isolated from organically produced food were adapted by repeated exposure to increasing concentrations of the quaternary ammonium compounds (QACs) benzalkonium chloride (BC) and hexadecylpyridinium chloride (HDP). The sensitivity of both wildtype strains and their corresponding QAC-adapted strains to other biocides and to antibiotics was studied. QAC tolerance increased in 88.2% of strains for BC and in 30.3% of strains for HDP, with increases in minimum inhibitory concentrations between 2 and over 100 fold. Adaptive resistance was stable after 20 subcultures in biocide-free medium for 7 and 5 of the BC- and HDP-adapted strains, respectively. Adaptation to BC and HDP also reduced the susceptibility to other biocides, mainly hexachlorophene (CF), didecyldimethylammonium bromide (AB), triclosan (TC) and chlorhexidine (CH). BC-adapted strains showed increased antibiotic resistance to ampicillin (AM) followed by sulfamethoxazol (SXT) and cefotaxime (CTX), and some showed increased sensitivity to ceftazidime (CAZ), CTX, AM and STX. Changes in antibiotic resistance in HDP-adapted strains were more heterogeneous and strain-dependent. Main efflux pump genes detected in QAC-adapted strains were acrB, sugE, norC, qacE and qacH, as well as antibiotic resistance genes aac(6_)-Ie-aph(2_)-Ia, aph(2_)-Ic, ant(4_)-Ia, lsa, mrsA/B, ereA, ermB and cat. Membrane anisotropy experiments revealed that QAC adaptation induced an increase in membrane rigidity in the case of BC, while response to HDP was more heterogeneous and strain-dependent. Growth capacity was significantly higher in some QAC-adapted strains and strain-dependent changes in heat tolerance were also detected in QAC-adapted strains. Gastric acid or bile resistances do not seem to be influenced by QAC adaptation.

A collection of 120 bacterial isolates from small medium enterprises involved in the production of cow milk and the manufacture of goat cheese were screened for sensitivity to biocides benzalkonium chloride (BC), cetrimide (CT), hexadecylpyridinium chloride (HDP), triclosan (TC), hexachlorophene (CF) and poly-(hexamethylen guanidinium) hydrochloride (PHMG). Nineteen isolates were selected according to biocide tolerance and identified by 16S rDNA sequencing as Lactococcus sp. (6) Enterococcus sp. (1), Lactobacillus sp. (4), Bacillus sp. (1) Escherichia sp. (5), Enterobacter sp. (1) and Helicobacter sp. (1). These were further characterised regarding antimicrobial resistance phenotype and genotype. Several isolates were multiply (3 or more) tolerant to biocides or resistant to antibiotics, but only two Escherichia sp. isolates and Enterobacter sp. were multiply resistant to biocides and antibiotics. Statistical analysis of biocide tolerance and antibiotic resistance revealed significant positive correlations between different biocides and between biocides and antibiotics. The biocide tolerance genes most frequently found were qacEΔ1 and qacA/B. The sulfonamide resistance gene sul1 was found in two Escherichia sp. isolates and in Enterobacter sp., all of which also carried qacEΔ1. Beta-lactam (blaCTX-M, blaPSE) and tetracycline resistance genes [tet(A), tet(C) and tet(D)] were detected. Efflux pump genes acrB and mdfA were found in most Gram-negative isolates. Results from the study suggest that exposure to biocides can indirectly select for antibiotic resistance.

Summary This study aimed to evaluate the antimicrobial and antibiofilm activities of two procyanidins isolated from an ethyl acetate extract of laurel wood against a selection of foodborne pathogens. The analysis of the extract by HPLC–DAD/ESI–MS allowed us to detect the presence of two procyanidins, which were selectively isolated and identified by chromatographic and spectroscopic means as cinnamtannin B-1 (1) and procyanidin B-2 (2). Procyanidins 1 and 2 exhibited two biological activities: inhibition of bacterial growth at high concentrations and prevention of biofilm formation at lower concentrations. Synergistic effect was also detected when both compounds were tested in combination against Listeria monocytogenes. Significant effects were also detected on disruption of preformed biofilm. The ability of procyanidins to inhibit microbial growth and biofilm formation and to synergistically work with each other may stimulate a market as natural food preservatives, and/or natural sanitisers for processing equipment where foodborne pathogens reside.

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.

We evaluated the efficacy of a new disinfectant product, HLE, to inhibit multiple species of planktonic and biofilm bacterial cultures. The HLE disinfectant comprised of EDTA, lactic acid and hydrogen peroxide, and our data indicated that the disinfectant had effective antimicrobial and anti-biofilm activity even at low concentrations (0.15% to 0.4% HLE, v/v). Furthermore, the HLE disinfectant destabilized biofilm structures eradicated them due to the synergistic effect of EDTA and both antimicrobials (lactic acid and hydrogen peroxide), as revealed by confocal laser scanning microscopy. Additionally, sub-inhibitory concentrations of HLE disinfectant, with EDTA as an efflux pump inhibitor, inhibited the expression of multidrug EfrAB, NorE and MexCD efflux pumps in both planktonic and biofilm cultures. This could provide an alternative way to disinfect surfaces to avoid spreading multi-drug resistant strains in the food chain and the environment by decreasing efflux pump expression and consequently reducing the antibiotic selective pressure caused by systemic antibiotics and disinfectant use.

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.

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.

Salmonella serovars sampled from meat products in Southern Spain (Andalucía) during the period 2002-2007 were analyzed in this study. The serovars most frequently detected (in order) were Typhimurium, Enteritidis, Derby, Anatum and Rissen. Isolates (n = 43) were tested for sensitivity to biocides, including the quaternary ammonium compounds benzalkonium chloride (BC), cetrimide (CT) and hexadecylpyridinium chloride (HDP), and the bisphenols triclosan (TC) and hexachlorophene (CF). The minimum inhibitory concentration (MIC) for the quaternary ammonium compounds was in the range of 25 to 50 mg/L for most isolates, although a few isolates required much higher concentrations, up to 250 mg/L. Bisphenols showed higher inhibitory activity, with a MIC of 2.5 to 25 mg/L. A few isolates showed a “non-wildtype” MIC for TC of up to 250 mg/L. These results indicate a low incidence of tolerance towards quaternary ammonium compounds and triclosan among Salmonella from meats and meat products.

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.

The type of fat in the diet determinates the characteristics of gut microbiota, exerting a major role in the development of metabolic syndrome. We hypothesize that a diet enriched with extra virgin olive oil (EVOO) has a distinctive effect on the intestinal microbiome in comparison with an enriched butter diet (BT) and this effect is related to the physiological benefits exerted by EVOO. Swiss Webster mice were fed standard (SD) or two high fat diets enriched with EVOO or butter. Hormonal, physiological and metabolic parameters were evaluated. At the end of the feeding period, DNA was extracted from faeces and the 16S rRNA genes were pyrosequenced. Among the main significant differences found, BT triggered the highest values of systolic blood pressure, correlating positively with the percentage of Desulfovibrio sequences in faeces, which in turn showed significantly higher values in BT than in EVOO. EVOO had the lowest values of plasmatic insulin, correlating inversely with Desulfovibrio, and had the lowest plasmatic values of leptin which correlated inversely with Sutterellaceae, Marispirillum and Mucilaginibacter dageonensis, the three showing significantly higher percentages in EVOO. The lowest total cholesterol levels in plasma were detected in SD, correlating positively with Prevotella and Fusicatenibacter, both taxa with significantly greater presence in SD. These results may be indicative of a link between specific diets, certain physiological parameters and the prevalence of some taxa, supporting the possibility that in some of the proposed effects of virgin olive oil the modulation of intestinal microbiota could be involved.

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.

High hydrostatic pressure (HHP) treatments (singly or in combination with natural antimicrobials) were tested for stabilization of Manzanilla Alorena seasoned olives stored at 25 °C. HHP (5 min) was highly effective on yeast populations at 300 MPa or higher. No viable yeasts were detected in samples treated at 400 MPa for up to three months. Low levels of endospore-forming bacteria were always detected after HHP treatments. Addition of nisin to the brines reduced bacterial counts by 1.4 log cycles but it had no effect on yeasts when tested singly or in combination with HHP treatment (400 MPa, 5 min). Thyme oil had almost no effect on yeast concentrations, but rosemary oil reduced yeast viable counts progressively during storage. Essential oils in combination with HHP (400 MPa, 5 min) significantly (p

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.

A collection of 31 Lactobacillus pentosus strains isolated from naturally fermented Aloreña green table olives were screened in depth in the present study for their probiotic potential. Several strains could be considered promising probiotic candidates since they showed good growth capacity and survival under simulated gastro-intestinal conditions (acidic pH of 1.5, up to 4% of bile salts and 5 mM of nitrate), good ability to auto-aggregate which may facilitate their adhesion to host cells as multiple aggregates and the subsequent displacement of pathogens. Moreover, co-aggregation of lactobacilli with pathogenic bacteria was shown with Listeria innocua, Staphylococcus aureus, Escherichia coli, and Salmonella Enteritidis as good defense strategy against gut and food pathogens. Furthermore, they exhibited adherence to intestinal and vaginal cell lines, such property could be reinforced by their capacity of biofilm formation which is also important in food matrices such as the olive surface. Their antagonistic activity against pathogenic bacteria by means of acids and plantaricins, and also their different functional properties may determine their efficacy not only in the gastro-intestinal tract but also in food matrices. Besides their ability to ferment several prebiotics, the new evidence in the present study was their capacity to ferment lactose which reinforces their use in different food matrices including dairy as a dietary adjunct to improve lactose digestibility. Lactobacillus pentosus CF2-10N was selected to have the best probiotic profile being of great interest in further studies. In conclusion, spontaneous fermented Aloreña table olives are considered a natural source of potential probiotic L. pentosus to be included as adjunct functional cultures in different fermented foods.

Manzanilla Aloreña (or Aloreña) table olives are naturally fermented traditional green olives with a denomination of protection (DOP). The aim of this study was to search for lactic acid bacteria (LAB) with technological properties of interest for possible inclusion in a starter or protective culture preparation or also as probiotics. A collection of 144 LAB obtained from Aloreña green table olives naturally-fermented by four small-medium enterprises (SMEs) from Málaga (Spain), including lactobacilli (81.94%), leuconostocs (10.42%) and pediococci (7.64%) were studied. REP-PCR clustering and further identification of strains by sequencing of phes and rpo genes revealed that all lactobacilli from the different SMEs were Lactobacillus pentosus. Pediococci were identified as Pediococcus parvulus (SME1) and leuconostocs as Leuconostoc pseudomesenteroides (SME1 and SME4). Genotyping revealed that strains were not clonally related and exhibited a considerable degree of genomic diversity specially for lactobacilli and also for leuconostocs. Some strains exhibit useful technological properties such as production of antimicrobial substances active against pathogenic bacteria such as Listeria monocytogenes, Bacillus cereus, Staphylococcus aureus, Streptococcus mutans and Salmonella enterica, utilization of raffinose and stachyose, production of bile salt hydrolase, phytase and haeme-dependent catalase activities, growth at 10 °C and in the presence of 6.5% NaCl, good acidifying capacity and also resistance to freezing. However, none of the isolates showed protease or amylase activity, and also did not exhibit biogenic amine production from histidine, ornithine, cysteine or tyrosine. On the basis of data obtained, selected strains with potential traits were tested for their survival at low pH and their tolerance to bile salts, and the survival capacity demonstrated by some of the analysed strains are encouraging to further study their potential as probiotics.

The aim of this study was to select autochthonous microorganisms that are able to biosorb heavy metals in aqueous systems. Forty-eight strains, included fungi, yeasts and bacteria, were isolated from water and sludge samples from urban and industrial wastewater treatment plants in the province of Jaen, southern Spain. Purified isolates were screened on the basis of their resistance to Pb(II), Zn(II) and Ag(I). Ten strains were selected and identified by molecular techniques. Tolerance index and minimum inhibitory concentration (MIC) for the three metals were also determined. It can be noted that tolerance of the strains appears to be associated with the sites of their isolation. The best results were obtained for the yeast Trichosporon sp. 4S3 which showed the following MICs: 32–35 mM for lead, 16–18 mM for zinc and 3–5 mM for silver. The use of native strains can be an interesting alternative for bioremediation processes of heavy metals. This study increases the number of microbial strains that have the ability to resist relatively higher concentrations of metals, which may make them recommendable as potentially low-cost biosorbents. The nucleotide sequences of all the selected isolates were deposited in GenBank, which could be of great interest for other investigators as reference strains in bioremediation processes of heavy metals. 2012 Elsevier B.V. All rights reserved.

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.

The effect of high hydrostatic pressure treatments on a cocktail of four Salmonella Enteritidis strains inoculated in commercial Spanish potato omelette (tortilla de patatas) was studied. Inactivation of Salmonella was influenced by treatment time and pressure. Treatments at 600 MPa for 5 or 8 min reduced the counts of Salmonella on selective medium (XLD agar) by 5.9 and 6.5 log cycles, respectively. Investigation of sublethally injured cells by growth on triple agar layer (TAL) plates or in non-selective medium (TSA) revealed that the population of sublethally injured cells able to grow on TSA could be 2.5 to 3.0 log cycles higher (depending on treatment) compared to non-injured cells. The TAL method only allowed recovery of a fraction of sublethally injured cells, suggesting different degrees of cell damage. In addition, tailings were observed in survival curves for treatments above 500 MPa both for selective and non-selective determinations. Omelette samples inoculated with Salmonella and treated at 500 MPa (5 min) or 680 MPa (8 min) were stored at 6 °C for 15 days. Counts of survivors on selective medium increased at day 3 of storage for the less intense HHP treatment (suggesting repair of sublethally injured cells), but not until day 10 for the 680 MPa treatment. Temperature abuse of the treated omelettes (6 h at 22 °C) applied at day 3 of storage increased the concentrations of cells able to grow on non-selective as well as on selective media, an effect that was more pronounced in the samples treated at 500 MPa for 5 min. The results suggest that temperature abuse may compromise the efficacy of HHP treatments against Salmonella in omelettes. Industrial relevance The efficacy of HHP treatments depends greatly on the food substrate and microorganisms. Therefore, the degree of protection afforded by HHP treatments for each foodborne pathogen and food system need to be determined experimentally. So far, there are no previous studies addressing the efficacy of HHP treatments on inactivation of Salmonella in Spanish omelettes. This type of RTE food is widely consumed not only in Spain, but also in many other countries, especially as an appetizer. Industrial application of HHP treatments on omelettes could improve their safety while at the same time enhancing the perception of this food product as a safer food.

The effect of enterocin AS-48 in controlling Lactobacillus sakei, Brochothrix thermosphacta , and Staphylococcus carnosus in a cooked ham system has been studied. AS-48 alone showed activity against Lactobacillus , with 60 μg g −1 reducing lactobacilli below detection levels from the beginning to end of storage at 5 °C. Combinations of 40 μg g −1 AS-48 with nitrate/nitrite, pentasodium tripolyphosphate, sodium pyrophosphate, sodium acetate, and sodium lactate reduced L. sakei below detection levels from the beginning to end of storage. Even 20 μg g −1 of enterocin combined with tripolyphosphate permanently eliminated L. sakei. Enterocin AS-48 (40 μg g −1 ) was also active against B. thermosphacta and S. carnosus , reducing both bacteria by more than 3 log in the cooked ham. Modified atmosphere (40% CO 2 /60% N 2 ) packaging affected neither bacterial growth nor AS-48 activity against any of the three bacteria. In contrast, storage under vacuum remarkably increased the growth and the inhibitory activity of the enterocin against B. thermosphacta and especially against L. sakei .

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.

Enterocin AS-48 was tested alone or in combination with chemical preservatives and/or heat against Listeria monocytogenes and Staphylococcus aureus in a cooked ham model system. AS-48 (20, 40 and 60 μg g −1 ) alone was active against L. monocytogenes at 5 and 15 °C, but it was not sufficient to avoid regrowth of Listeria during the 60 days storage. Combination of AS-48 (40 μg g −1 ) with nitrite/nitrate, pentasodium tripolyphosphate, sodium benzoate or potassium sorbate improved the anti-listeria effect during storage at 5 °C. The most effective combination was AS-48-nitrite/nitrate (0.007%) that reduced listeria below detection level from the beginning to end of storage. Although much more resistant, S. aureus was also inhibited by AS-48 alone at 5 °C, and especially in combinations with nitrite/nitrate, pentasodium tripolyphosphate, sodium lactate and sodium acetate. Best results against both pathogens were obtained when sodium pyrophosphate was applied in combination with 60 μg g −1 AS-48. Sub-lethal heat (60 °C, 2 min) clearly increased AS-48 activity against both Listeria and Staphylococcus .

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.

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.

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 (30-60 microg/g) significantly reduced viable counts of Listeria monocytogenes in Russian-type salad during one week storage at 10 degrees C. Antilisterial activity of AS-48 (30 microg/g) in salad was strongly enhanced by essential oils (thyme verbena, thyme red, Spanish oregano, ajowan, tea tree, clove, and sage oils tested at 1%, as well as with 2% rosemary oil). Antilisterial activity also increased in combination with bioactive components from essential oils and plant extracts, with other related antimicrobials of natural origin or derived from chemical synthesis (carvacrol, eugenol, thymol, terpineol, tyrosol, hydroxytyrosol, caffeic, ferulic and vanillic acid, luteolin, geranyl butyrate, geranyl phenylacetate, pyrocatechol, hydrocinnamic acid, tert butylhydroquinone, phenylphosphate, isopropyl methyl phenol, coumaric acid, and 2-nitropropanol), and with food preservatives (citric and lactic acid, sucrose palmitate, sucrose stearate, p-hydroxybenzoic methylester acid -- PHBME, and Nisaplin). AS-48 acted synergistically with citric, lactic acid, and PHBME. A mixed population of two L. monocytogenes strains was markedly reduced for one week in salads treated with AS-48 (30 microg/g) in combination with lactic acid, PHBME or Nisaplin. The increased bactericidal activity of these combinations is interesting to improve protection against L. monocytogenes during salad storage.

Detersive processes are complex systems involving a great number of variables. To determine the effect of these variables on the washing of hard surfaces and fatty soils is the object of this work. The statistical design of experiments has been used to evaluate the influence of factors such as temperature, soil concentration and surfactant concentration on detergency. The experimental trials have been made in a continuous-flow device where the soiling agent is confined in a column filled with borosilicate glass spheres. Solutions of the commercial surfactant Berol© LFG61 (a mixture of alkylpolyglucosides and fatty-alcohol ethoxylates) have been employed as the wash bath. Both the design of experiments and the continuous experimental system used proved to be an effective tool for detecting the key variables in the cleaning process. Expressions were developed to simulate detergency levels as a function of the variables assayed, always inside the experimental domain. In the trials with oleic acid as the soiling agent, it was found that the temperature and soil concentration were the most important variables to take into account, while the surfactant concentration was not a significant variable. When a semi-solid mixture of different fatty acids was employed, all the variables assayed proved significant, with high detergency values being reached by combining temperature and surfactant concentrations. Results clearly show that the effectiveness of the surfactant used is influenced by the type and concentration of the soil and thus the intended application of the product being developed should be taken into account when designing detergent formulas.

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.

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 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.

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.