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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.

In an ecological study, a collection of Gram-positive bacteria were isolated from Gueddid, an artisanal Tunisian fermented meat. From these, 24 strains showed antimicrobial activity and identified as Enterococcus faecium using molecular methods. The enterococci strains were further investigated regarding their safety aspects and functional properties. All the isolates produce bacteriocins with inhibitory activity against several food spoilage bacteria and food borne pathogens, including Listeria spp. Enterococcus spp. and Staphylococcus aureus. One isolate was active against Escherichia coli CECT 877. The majority of the isolates tested positive upon PCR amplification of structural genes for enterocins A, B and P. Investigation of virulence factors by PCR amplification revealed the presence of genes encoding for gelatinase (gelE), enterococcal antigen (efaAfm), sex pheromone (cpd and ccf) and expression of cytolysin (the haemolytic component cylB) whereas, other presumed virulence genes encoding for (agg, esp, cylM, cylA, and cob) were not detected. The isolates were mostly resistant to erythromycin, rifampicin, ciprofloxacin, lavofloxacin, and nitrofurantoin. All of them showed sensitivity to several antibiotics (ampicillin, penicillin, vancomycin, chloramphenicol, teicoplanin, gentamicin, streptomycin, and quinupristin/dalfopristin). Tyrosine, lysine, ornithine and histidine were not decarboxylated by any enterococcal isolate. Nine of the antagonistic enterococci tested did not show any virulence traits or produced biogenic amines, and still had important technological properties. The safety aspects of these selected strains should be studied in deeper details in order to evaluate their potential for biotechnological applications.

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.

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.

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.

A collection of 16 isolates of Enterococcus faecalis from different vegetable foods were characterized by multilocus sequence typing (MLST). One isolate belonged to sequence type (ST) 9 of the previously described clonal complex 9, which is frequently associated with hospital environments. The rest of the isolates were grouped into two new STs named 168 and 169. ST168 represented a singleton clone that included 14 isolates and seemed to be the predominant type among E. faecalis from vegetable samples. ST168 was closely related to ST72, differing only by one allele type. Singleton ST169 was not related to any of the previously described STs.

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.

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 bacteriocinogenic strain Lactobacillus plantarum 2.9 isolated from the traditional pearl millet-based African fermented food ben saalga was tested for inhibition of food poisoning and pathogenic bacteria in MRS broth and in a malted millet flour slurry. In MRS broth, strain 2.9 completely eliminated Bacillus cereus, Escherichia coli O157:H7 and Salmonella enterica cells within 48 h incubation at 22–30 °C. A much lower inhibition was observed at 15 °C. The inhibitory effect of strain 2.9 on the above-mentioned target bacteria was corroborated in the malted millet flour slurry, reducing viable cell counts below detection levels after 8 h storage for B. cereus or after 24 h for S. enterica and 48 h for E. coli. An Enterobacter aerogenes strain was only moderately inhibited in the slurry. Results from the present study suggest that strain 2.9 could be used as starter culture to improve the microbiological safety of fermented ben saalga.

A collection of enterococci isolated from meat, dairy and vegetable foods from Morocco including 23 Enterococus faecalis and 15 Enterococcus faecium isolates was studied. All isolates were sensitive to ampicillin, penicillin, and gentamicin. Many E. faecalis isolates were resistant to tetracycline (86.95%), followed by rifampicin (78.26% ciprofloxacin (60.87%), quinupristin/dalfopristin (56.52%), nitrofurantoin (43.47%), levofloxacin (39.13%), erythromycin (21.73%), streptomycin (17.39%), chloramphenicol (8.69%), vancomycin (8.69%), and teicoplanin (4.34%). E. faecium isolates showed a different antibiotic resistance profile: a high percentage were resistant to nitrofurantoin (73.33%), followed by erythromycin (66.60%), ciprofloxacin (66.66%), levofloxacin (60.00%), and rifampicin (26.66%), and only a very low percentage were resistant to tetracycline (6.66%). One isolate was resistant to vancomycin and teicoplanin. The incidence of virulence factors was much higher among E. faecalis isolates, especially for genes encoding for sex pheromones, collagen adhesin, enterococcal endocarditis antigen, and enterococcal surface protein. Isolates with multiple factors (both antibiotic resistance and virulence traits) were also more frequent among E. faecalis isolates, in which one isolate cumulated up to 15 traits. By contrast, several isolates of E. faecium had only very few unwanted traits as compared to only two isolates in E. faecalis. The high abundance of isolates carrying virulence factors and antibiotic resistance traits suggests that the sanitary quality of foods should be improved in order to decrease the incidence of enterococci.

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.

A collection comprising 14 isolates of Pediococcus pentosaceus and one Pediococcus acidilactici from the fermentation of caper fruits was studied. All isolates showed very similar fermentation profiles and produced a limited number of exoenzymes. All isolates carried large plasmids of diverse sizes between 20 and 55 kb, while some also contained smaller plasmids between 10 and 16 kb. Cluster analysis of plasmid profiles revealed four main groups with various degrees of similarities. All amino acid decarboxylation tests were negative, suggesting that pediococci are not involved in generation of biogenic amines. None of the isolates showed hemolytic activity. Antimicrobial resistance tests revealed that all isolates were sensitive to 11 different antimicrobials while being resistant to ciprofloxacin (MIC > or =2 mg/liter) and intrinsically resistant to vancomycin (MIC > or =16 mg/liter) and teicoplanin (MIC > or =16 mg/liter).

The production of antimicrobial substances was studied among 195 bacterial isolates from retail table olives. A total 86 isolates tested positive, and they clustered in 10 groups according to their inhibitory spectra. Many isolates (38.37%) produced strong inhibition against all bacteria tested (Listeria innocua, Lactococcus lactis, Bacillus cereus, B. megaterium, Staphylococcus aureus, Micrococcus luteus, Enterococcus faecalis, and Escherichia coli). The selected bacterial isolates were Gram-positive bacteria with rod morphology (62.67%), short rods (26.65%) or cocci (10.67%). Isolates producing antimicrobial substances may be useful as starters to enhance control of table olive fermentation and improve the safety of retail table olives.

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.

Fermentation of capers (the fruits of Capparis sp.) was studied by molecular and culture-independent methods. A lactic acid fermentation occurred following immersion of caper berries in water, resulting in fast acidification and development of the organoleptic properties typical of this fermented food. A collection of 133 isolates obtained at different times of fermentation was reduced to 75 after randomly amplified polymorphic DNA (RAPD)-PCR analysis. Isolates were identified by PCR or 16S rRNA gene sequencing as Lactobacillus plantarum (37 isolates), Lactobacillus paraplantarum (1 isolate), Lactobacillus pentosus (5 isolates), Lactobacillus brevis (9 isolates), Lactobacillus fermentum (6 isolates), Pediococcus pentosaceus (14 isolates), Pediococcus acidilactici (1 isolate), and Enterococcus faecium (2 isolates). Cluster analysis of RAPD-PCR patterns revealed a high degree of diversity among lactobacilli (with four major groups and five subgroups), while pediococci clustered in two closely related groups. A culture-independent analysis of fermentation samples by temporal temperature gradient electrophoresis (TTGE) also indicated that L. plantarum is the predominant species in this fermentation, in agreement with culture-dependent results. The distribution of L. brevis and L. fermentum in samples was also determined by TTGE, but identification of Pediococcus at the species level was not possible. TTGE also allowed a more precise estimation of the distribution of E. faecium , and the detection of Enterococcus casseliflavus (which was not revealed by the culture-dependent analysis). Results from this study indicate that complementary data from molecular and culture-dependent analysis provide a more accurate determination of the microbial community dynamics during caper fermentation.

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.

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’.

Summary The incidence and diversity of enterococci in retail food samples of meat, dairy and vegetable origin was investigated. Enterococci were present, at concentrations of 10 1 to 10 4 CFU/g. Fifty selected isolates from food samples grouped in two separate clusters by RAPD analysis. Cluster G1 (72% of the isolates) contained the E. faecium CECT 410 T type strain, and also showed a high degree of genetic diversity. Cluster G2 (28% of the isolates) contained the E. faecalis CECT 481 T type strain and was genetically more homogeneous. Virulence traits (haemolysin, gelatinase or DNAse activities, or the presence of structural genes cylL , ace, asa1 and esp ) were not detected. All isolates were sensitive to the antibiotics ampicillin, penicillin, gentamicin, streptomycin and chloramphenicol. A high pecentage of isolates were resistant to erythromycin and rifampicin. Many isolates showed intermediate sensitivity to several antibiotics (tetracycline, ciprofloxacin, levofloxacin, or quinupristin/dalfopristin). Vancomycin and teicoplanin resistance was detected in one strain, but vanA, vanB, vanC1, vanC2 or vanC3 genes were not detected. Many of the isolates showed functional properties of food or health relevance. Production of antimicrobial substances was detected in 17 of the isolates, and 14 of them carried structural genes for enterocins A, B and/or P.

Production of laccase activity by the hyphomycete Chalara (syn. Thielaviopsis ) paradoxa CH32 reached highest levels at the idiophase, but was not enhanced by putative laccase inducers. Laccase activity from the extracellular fluid was purified and characterised, consisting of a single protein of 67 kDa, sensitive to heat and to acidic pH. Laccase activity showed an optimum pH of 6.5 for most of the substrates, while some substrates were oxidised only at pH 4.5. The optimum temperature for enzyme activity was 30 °C. Laccase activity was inhibited by metal cations, especially by Hg 2+ . It was also inhibited by reducing agents, EDTA, potassium cyanide and sodium azide. The enzyme retained a high percentage of activity in the presence of some organic solvents (methanol, ethanol and iso -propyl alcohol). The substrate specificity of the purified laccase was greatly influenced by the nature and the position of the substituting groups in the phenolic ring.

It is an already well established and experimentally proven fact that bacterial structures can participate in extra or intracellular accumulation of inorganic material. Myxoccocus coralloides and M. xanthus cells from cultures of 48 h or more presented signs of globular deformities. Further study of these deformities through transmission electron microscopy indicated that small crystalline forms of struvite were present but were sometimes destroyed by the mere impact of the electron beam.

The biosorption for La2+, Co2+, Mn2+, UO2(2+), Pb2+, Ag+, Zn2+, Cd2+ and Cr2+ by wet and dry biomass form Myxococcus xanthus obtained from laboratory cultures and Saccharomyces cerevisiae from the brewing industry has been studied. M. xanthus biomass was found to be the most efficient biosorbent for all of the metals assayed. However, due to the fact that S. cerevisiae is a low cost residual by-product from the brewing industry, and at the same time yields good levels of biosorption, it is considered in this work to be of great interest for use as a detoxifier of heavy metals contaminated waters. In addition, the use of sodium carbonate as a desorbent agent is discussed where it was possible to recover up to 94,53% of UO2(2+) by both M. xanthus and S. cerevisiae biomass.

In this work we have proved that struvite and calcite crystals can be obtained in the presence of the cellular membrane fraction of Myxococcus xanthus, when appropriate supersaturated solutions are used. Probably, the negative charged points of the external side of the cellular structures could reduce the metastability field of struvite and calcite, acting as heterogeneous nuclei of crystallization.

We studied the morphological diversity of struvite crystals produced by Myxococcus coralloides and Myxococcus xanthus in different culture conditions. We discussed the similarities of these crystals with the struvite morphology studied previously following the theory of the periodic bond chains.

Research on antimicrobial peptides is continuously growing because of the possibilities of applications they offer in different domains including food safety, human medicine, and plant biocontrol (phytosanitary).

We studied the possibility of struvite formation using killed cells of Myxococcus xanthus. Cells were killed by heat, UV light and sonication. In all cases, we show that struvite crystallization occurs and we propose that the dead cells or cell debris can act by themselves as heterogeneous crystallization nuclei. We also show that the slime, produced by Myxococcus in a large quantities, is not involved in this process.

The formation under laboratory conditions of newberyite, schertelite, and taylorite in conjunction with struvite by the bacterium Myxococcus coralloides D is reported for the first time. The presence of these syngenetic minerals with struvite was only detected in static liquid cultures.

This chapter deals with food applications of bacteriocins. Regulatory issues on the different possibilities for incorporating bacteriocins as bioprotectants are discussed. Specific applications of bacteriocins or bacteriocin-producing strains are described for main food categories, including milk and dairy products, raw meats, ready-to-eat meat and poultry products, fermented meats, fish and fish products or fermented fish. The last section of the chapter deals with applications in foods and beverages derived from plant materials, such as raw vegetable foods, fruits and fruit juices, cooked food products, fermented vegetable foods and ­fermented beverages. Results obtained for application of bacteriocins in combination with other hurdles are also discussed for each specific case, with a special emphasis on novel food packaging and food-processing technologies, such as irradiation, pulsed electric field treatments or high hydrostatic pressure treatment.

Cereal doughs are an important part of human diet, but at the same time can act as vehicles for the transmission of human pathogenic bacteria. In the present study, four pathogenic or toxinogenic bacteria (Escherichia coli O157:H7, Salmonella enterica serovar Enteritidis, Bacillus cereus and Staphylococcus aureus) were inoculated in a dough made from corn flour in combination with the single antimicrobial compounds carvacrol and 2-Nitro-1-propanol (2NPOH). Survival of single and mixed populations in the treated doughs incubated at 37 degrees C was followed by culture-dependent and independent methods (TTGE). All strains were completely inactivated within 24 h by the tested compounds at 5% final concentration, but showed variable inhibition at lower concentrations of 0.5% and 2%. Sensitivity to antimicrobial compounds was in general modified when strains were tested in cocultures compared with single cultures. B. cereus was more sensitive to carvacrol (minimum bactericidal concentration, MBC, 0.5%) in coculture with S. aureus. It was also more sensitive to 2NPOH in cocultures with S. aureus and with S. enterica (MBC, 2% in both cases). S. aureus was more resistant to carvacrol (MBC, 5%) in cocultures with B. cereus, E. coli, as well as S. enterica. However, sensitivity to 2NPOH was not modified in any of the coculture experiments (MBC, 2%). E. coli was more resistant to carvacrol (MBC, 5%) in cocultures with S. aureus and with S. enterica. Resistance of E. coli to 2NPOH also increased in cocultures with B. cereus (MBC, 5%) and with S. aureus (MBC, 2%), but not with S. enterica (MBC, 0.5%). S. enterica was more resistant to carvacrol (MBC, 5%) in cocultures with E. coli, but it was more sensitive to 2NPOH in cocultures with B. cereus as well as with S. aureus (MBC, 2% in both cases). TTGE analysis of survivors from cocultures treated with 2NPOH or carvacrol allowed a good estimation of the identity of survivors according to their DNA band patterns. Results from this study indicate that the efficacy of antimicrobials such as carvacrol and 2NPOH is greatly influenced by the complexity of the microbial populations under target and the relationships between individual populations.

A collection of enterococci from clinical samples, fruits and vegetables, water and soil was investigated for the presence of endocarditis- and biofilm-associated pilus (ebp) genes by PCR amplification and also by colony hybridisation. A high percentage of Enterococcus faecalis clinical isolates (94.59%) as well as all isolates from fruits and vegetables and two of the three isolates from water and soil carried ebpA, ebpB and ebpC genes. For E. faecium, PCR amplification revealed that over 81.81% of isolates from water and soil carried the three genes, compared to the lower incidence detected among isolates from vegetables (68.42%) and clinical samples (33.33%). The remaining E. faecium isolates tested negative both in the PCR and hybridisation tests. This is the first report of ebp detection among non-clinical E. faecium. Comparative sequence analysis of ebp genes revealed a higher similarity of clinical isolate E. faecalis EH17 with E. faecalis V583, while the non-clinical isolates E. faecalis V30C2 and E. faecium A9C4 grouped in a separate cluster. Greatest differences among clusters were observed for ebpC gene. Results from the present study suggest that enterococcal populations adapted to specific environments may also differ in the sequences of pili-encoding genes. Such variations may be important to produce functional pili and may have direct implications on the adhesion and colonisation capacity of isolates.

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

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

Thirty one bacteriocin-producing Lactobacillus isolates were identified among 135 lactobacilli isolated from the Congolese fermented maize product poto poto, during the preparation and from the finished product. Using species-specific PCR and 16S rRNA gene sequencing, 28 and 3 isolates were identified as L. plantarum and L. fermentum, respectively. Cluster analysis of RAPD-PCR fingerprints revealed two main groups (G1 and G2) plus the L. fermentum isolate C4-13. Group G1 contained 23 isolates with a similarity coefficient > 74.5%, and could be divided in two subgroups (G1-1, G1-2) each with several branches, plus the L. plantarum isolate C11. Group G2 contained 8 isolates with a similarity coefficient > 86%, with two main branches. Using PCR amplification with specific primers, several genes of the plantaricin cluster found in L. plantarum C11 were identified in the isolates. The number of genes that were detected varied between the strains. The L. fermentum isolate EC11 also contained the plnDEFG genes. PCR amplification of DNA from isolates with primers directed to the upstream and downstream region of the plantaricin cluster generated an amplicon identical to that obtained with DNA from the control strain L. plantarum WCFS1. Amplification products from the positive strains were used for restriction analysis with HindIII, EcoRI and KpnI in separate reactions. Cluster analysis of restriction profiles revealed high similarities for EcoRI and HindII digest profiles, and an identical profile for all KpnI digests. The L. fermentum EC11 isolate clustered with L. plantarum strains in a group with a high correlation coefficient. The results suggest a low degree of diversity in the plantarincin gene cluster. However, other strains that tested positive for individual plantaricin genes may present great heterogeneity in the plantaricin operons. Because of their broad spectra of inhibition (including Escherichia coli, Salmonella enterica, Enterobacter aerogenes, Bacillus cereus, Staphylococcus aureus, Listeria monocytogenes, and Enterococcus faecalis), isolates from the present study could be used to improve the safety and storage stability of poto poto.

A comparative study was carried out among enterococci isolated from fruits and vegetable foods, water and soil, and clinical samples. Results indicate strong differences in the numbers of enfterococcal species found in different environments as well as their abundance. While Enterococcus faecalis was clearly the predominant species in clinical samples, Enterococcus faecium predominated in vegetables, and it slightly outnumbered E. faecalis in water samples. Other species (Enterococcus hirae, Enterococcus mundtii, Enterococcus durans, Enterococcus gallinarum and Enterococcus casseliflavus) were found more frequently in vegetables, water, and specially in soil. Isolates from vegetable foods showed a lower incidence of antibiotic resistance compared to clinical isolates for most antimicrobials tested, especially erythromycin, tetracycline, chloramphenicol, ciprofloxacin, levofloxacin, gentamicin and streptomycin for E. faecalis, and quinupristin/dalfopristin, ampicillin, penicillin, ciprofloxacin, levofloxacin, rifampicin, choramphenicol, gentamicin and nitrofurantoin for E. faecium. E. faecium isolates from vegetable foods and water showed an average lower number of antibiotic resistance traits (2.95 and 3.09 traits for vegetable and water isolates, respectively) compared to clinical samples (7.5 traits). Multi-resistant strains were also frequent among clinical E. faecalis isolates (5.46 traits on average). None of E. faecalis or E. faecium isolates from vegetable foods, water and soil showed beta-haemolytic activity, while 25.64% of clinical E. faecalis did. A 51.28% of E. faecalis clinical isolates tested positive for the cylA, cylB, cylM set of genes, while some or all of these genes were missing in the rest of isolates. In clinical E. faecalis and E. faecium isolates, the genetic determinants for the enterococcal surface protein gene (esp), the collagen adhesin gene (ace) and the sex pheromone gene ccf (as well as cob in E. faecalis) showed a clearly higher incidence compared to isolates from other sources. E. faecalis isolates from vegetable foods and water had much lower average numbers of virulence genetic determinants per strain (4.23 and 4.0, respectively) compared to clinical isolates (8.71). Similarly, among E. faecium the lowest average number of traits per strain occurred in vegetable food isolates (1.72) followed by water (3.9) and clinical isolates (4.73). Length heterogeneity (LH)-PCR typing with espF–aceF–ccfF and espF–ccfF primers revealed genomic groups that clearly differentiated clinical isolates from those of vegetable foods, water and soil (except for two clinical isolates). The large differences found in the incidence of antibiotic resistance and virulence factors and in the genetic fingerprints determined by LH-PCR suggest a clear separation of hospital-adapted populations of enterococci from those found in open environments.

Poto poto (a maize sourdough) and degue (a pearl millet-based food) are two traditional African fermented foods. The molecular biology of toxigenic and pathogenic bacteria found in those foods is largely unknown. The purpose of this study was to study the phylogenetic relatedness and toxigenic potential of 26 Bacillus cereus group isolates from these traditional fermented foods. The relatedness of the isolates was evaluated with repetitive element sequence-based PCR (REP-PCR) and 16S rDNA sequencing analysis. A multiplex real-time PCR assay targeting the lef and capC genes of B. anthracis pXO1 and pXO2 plasmids and the sspE chromosomal gene of B. cereus and B. anthracis also was carried out. Melting curve analysis of the sspE amplification product was used to differentiate B. cereus from B. anthracis, and the presence of the B. cereus enterotoxin genes was determined with PCR amplification. Isolates had 15 different REP-PCR profiles, according to which they could be clustered into four groups. 16S rDNA sequencing analysis identified 23 isolates as B. cereus or B. anthracis and three isolates as B. cereus or Bacillus sp. Multiplex real-time PCR amplification indicated the absence of the lef and capC genes of B. anthracis pXO 1 and pXO2 plasmids, and melting curve analysis revealed amplification of the 71-bp sspE product typical of B. cereus in all isolates instead of the 188-bp amplicon of B. anthracis, confirming the identity of these isolates as B. cereus. Four isolates had amylolytic activity. All isolates had lecithinase activity and beta-hemolytic activity. Enterotoxin production was detected in two isolates. The emetic toxin gene was not detected in any isolate. The nheB toxin gene was detected in 19 isolates by PCR amplification; one of these isolates also contained the hblD (L1) gene. The cytotoxin K cytK-1 gene was not detected, but the cytK-2 gene was clearly detected in six isolates.

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

Bacteriocins are ribosomally-synthesized peptides or proteins with antimicrobial activity, produced by different groups of bacteria. Many lactic acid bacteria (LAB) produce bacteriocins with rather broad spectra of inhibition. Several LAB bacteriocins offer potential applications in food preservation, and the use of bacteriocins in the food industry can help to reduce the addition of chemical preservatives as well as the intensity of heat treatments, resulting in foods which are more naturally preserved and richer in organoleptic and nutritional properties. This can be an alternative to satisfy the increasing consumers demands for safe, fresh-tasting, ready-to-eat, minimally-processed foods and also to develop “novel” food products (e.g. less acidic, or with a lower salt content). In addition to the available commercial preparations of nisin and pediocin PA-1/AcH, other bacteriocins (like for example lacticin 3147, enterocin AS-48 or variacin) also offer promising perspectives. Broad-spectrum bacteriocins present potential wider uses, while narrow-spectrum bacteriocins can be used more specifically to selectively inhibit certain high-risk bacteria in foods like Listeria monocytogenes without affecting harmless microbiota. Bacteriocins can be added to foods in the form of concentrated preparations as food preservatives, shelf-life extenders, additives or ingredients, or they can be produced in situ by bacteriocinogenic starters, adjunct or protective cultures. Immobilized bacteriocins can also find application for development of bioactive food packaging. In recent years, application of bacteriocins as part of hurdle technology has gained great attention. Several bacteriocins show additive or synergistic effects when used in combination with other antimicrobial agents, including chemical preservatives, natural phenolic compounds, as well as other antimicrobial proteins. This, as well as the combined use of different bacteriocins may also be an attractive approach to avoid development of resistant strains. The combination of bacteriocins and physical treatments like high pressure processing or pulsed electric fields also offer good opportunities for more effective preservation of foods, providing an additional barrier to more refractile forms like bacterial endospores as well. The effectiveness of bacteriocins is often dictated by environmental factors like pH, temperature, food composition and structure, as well as the food microbiota. Foods must be considered as complex ecosystems in which microbial interactions may have a great influence on the microbial balance and proliferation of beneficial or harmful bacteria. Recent developments in molecular microbial ecology can help to better understand the global effects of bacteriocins in food ecosystems, and the study of bacterial genomes may reveal new sources of bacteriocins.

The microbial composition of the traditional fermented foods poto poto (a maize dough from the Rep. of Congo) and degue (a millet dough from Burkina Faso) was studied by a culture-independent approach using TTGE to separate the amplified target V3 region of the 16S rRNA gene from total microbial community, followed by DNA sequencing and homology search. Three different extraction methods were used. Guanidium thiocyanate-based DNA extraction provided better performance regarding purity and DNA yield, allowing the detection of a higher number of DNA bands by TTGE in poto poto. By contrast, all three methods yielded similar results for degue samples, indicating that the performance of the DNA extraction method largely depends on the food composition. Sequencing of DNA bands from TTGE gels corresponding to poto poto samples revealed the presence of Lactobacillus gasseri, Enterococcus sp., Escherichia coli, Lactobacillus plantarum/paraplantarum, Lactobacillus acidophilus, Lactobacillus delbrueckii, Bacillus sp., Lactobacillus reuteri and Lactobacillus casei. The following bacteria were identified in degue: L. gasseri, Enterococcus sp., E. coli, Lactobacillus fermentum, Lactobacillus brevis, and L. casei.

A collection of lactic acid bacteria isolated from ben saalga, a traditional fermented gruel from Burkina Faso, was screened for bacteriocin production. Seven isolates were selected for their broad antimicrobial spectra, which overall included strains of Bacillus cereus, Bacillus licheniformis, Enterococcus faecalis, Listeria innocua, Listeria monocytogenes, Staphylococcus aureus, Escherichia coli and Salmonella enterica. Cluster analysis of RAPD-PCR patterns revealed that six of the isolates represent different strains. The six selected strains were identified as Lactobacillus plantarum by 16S rDNA sequencing, species-specific PCR and multiplex PCR of the recA gene. PCR amplification revealed the presence of genes of the plantaricin cluster described in L. plantarum C11. Among them, strain 5.2.2 carried the largest number of genes from this cluster. (c) 2006 Elsevier B.V. All rights reserved.

A collection of lactobacilli comprising species of Lactobacillus plantarum (43 isolates), Lactobacillus brevis (9 isolates) and Lactobacillus fermentum (6 isolates) obtained from spontaneous fermentations of capers (the fruits of Capparis spinosa) were investigated for resistance to antimicrobial agents. All isolates were resistant to vancomycin and teicoplanin (MIC > 16 microg/ml). Resistance to ciprofloxacin (MIC > 2 microg/ml) was detected in all isolates of L. brevis and L. fermentum as well as in most isolates of L. plantarum, whilst resistance to levofloxacin showed a much lower incidence. Among L. plantarum and L. brevis isolates, low levels of resistance to tetracycline and/or nitrofurantoin were detected. Higher resistance levels were also detected in some isolates. Resistance to penicillin and rifampicin were also detected among L. plantarum isolates. All isolates were sensitive to ampicillin, erythromycin, chloramphenicol, gentamicin, streptomycin, and quinupristin/dalfopristin.