Increased inactivation of exopolysaccharide-producing Pediococcus parvulus in apple juice by combined treatment with enterocin AS-48 and high-intensity pulsed electric field.

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.

Assay of enterocin AS-48 for inhibition of foodborne pathogens in desserts.

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.

Effect of enterocin AS-48 in combination with high-intensity pulsed-electric field treatment against the spoilage bacterium Lactobacillus diolivorans in apple juice.

Enterocin AS-48 was tested in apple juice against the cider-spoilage, exopolysaccharide-producing strain Lactobacillus diolivorans 29 in combination with high-intensity pulsed-electric field (HIPEF) treatment (35 kV/cm, 150 Hz, 4 micros and bipolar mode). A response surface methodology was applied to study the bactericidal effects of the combined treatment, with AS-48 concentration and HIPEF treatment time as process variables. At subinhibitory bacteriocin concentrations, microbial inactivation by the combined treatment increased as the bacteriocin concentration and the HIPEF treatment time increased (from 0.5 to 2.0 microg/ml and from 100 to 1000 micros, respectively). Highest inactivation (4.87 logs) was achieved by 1000 micros HIPEF treatment in combination with 2.0 microg/ml AS-48. While application of treatments separately did not protect juice from survivors during storage, survivors to the combined treatment were inactivated within the following 24 h of storage, and the treated samples remained free from detectable lactobacilli for at least 15 days at temperatures of 4 degrees C as well as 22 degrees C. The combined treatment could be useful for inactivation of exopolysaccharide-producing L. diolivorans in apple juice.

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

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

Inactivation of exopolysaccharide and 3-hydroxypropionaldehyde-producing lactic acid bacteria in apple juice and apple cider by enterocin AS-48.

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.

Comparative analysis of genetic diversity and incidence of virulence factors and antibiotic resistance among enterococcal populations from raw fruit and vegetable foods, water and soil, and clinical samples.

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 enterococcal 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.