An oxidation resistant pediocin PA-1 derivative and penocin A display effective anti-Listeria activity in a model human gut environment.

Pediocin PA-1 is a class IIa bacteriocin that is particularly effective against the foodborne pathogen Listeria monocytogenes. The loss of activity of PA-1 pediocin due to methionine oxidation is one of the challenges that limit the wider application of the bacteriocin. In this study, we heterologously expressed an oxidation resistant form of pediocin PA-1, i.e., pediocin M31L, and compared its activity to that of native pediocin PA-1 and to penocin A, a pediocin-like bacteriocin that displays a narrower antimicrobial spectrum. Minimal inhibitory concentration assays revealed that pediocin M31L was as effective as PA-1 and more effective than synthetic penocin A against Listeria with negligible activity against a range of obligate anaerobic commensal gut bacterial species. The anti-Listeria activity of these pediocins was also assessed in a simulated human distal colon model assay using the L. monocytogenes, spiked at 6.5 ± 0.13 Log CFU/mL, as a bioindicator. At 24 h, pediocin M31L and penocin A (2.6 µM) reduced Listeria counts to 3.5 ± 0.4 and 3.64 ± 0.62 Log CFU/mL, respectively, whereas Listeria counts were considerably higher, i.e. 7.75 ± 0.43 Log CFU/mL, in the non-bacteriocin-containing control. Ultimately, it was established that synthetic penocin A and the stable pediocin M31L derivative, heterologously produced, display effective anti-Listeria activity in a human gut environment.

Exploring the cytotoxic mechanisms of Pediocin PA-1 towards HeLa and HT29 cells by comparison to known bacteriocins: Microcin E492, enterocin heterodimer and Divercin V41.

The purpose of this study was to explore potential mechanisms of cytotoxicity towards HeLa and HT29 cells displayed by Pediocin PA-1. We did this by carrying out sequence alignments and 3D modelling of related bacteriocins which have been studied in greater detail: Microcin E492, Enterocin AB heterodimer and Divercin V41. Microcin E492 interacts with Toll-Like Receptor 4 in order to activate an apoptosis reaction, sequence alignment showed a high homology between Pediocin PA-1 and Microcin E492 whereas 3D modelling showed Pediocin PA-1 interacting with TLR-4 in a way reminiscent of Microcin E492. Furthermore, Pediocin PA-1 had the highest homology with the Enterocin heterodimer, particularly chain A; Enterocin has also shown to cause an apoptotic response in cancer cells. Based on this we are led to strongly believe Pediocin PA-1 interacts with TLRs in order to cause cell death. If this is the case, it would explain the difference in cytotoxicity towards HeLa over HT29 cells, due to difference in expression of particular TLRs. Overall, we believe Pediocin PA-1 exhibits a dual effect which is dose dependant, like that of Microcin. Unfortunately, due to the COVID-19 pandemic, we were unable to carry out experiments in the lab, and the unavailability of important data meant we were unable to provide and validate out solid conclusions, but rather suggestions. However, bioinformatic analysis is still able to provide information regarding structure and sequence analysis to draw plausible and evidence based conclusions. We have been able to highlight interesting findings and how these could be translated into future research and therapeutics in order to improve the quality of treatment and life of cancer patients.

Encapsulation of Purified Pediocin of Pediococcus pentosaceus into Liposome Based Nanovesicles and its Antilisterial Effect.

AIMS: To encapsulate a purified bacteriocin into a nanovesicles and check its antibacterial effect. BACKGROUND: Although the use of nano-encapsulated bacteriocins in food matrices is poorly reported, encapsulated nisin can reduce L. monocytogenes counts in whole and skimmed milk and in soft cheese. OBJECTIVE: The present study deals with the extraction and purification of a bacteriocin from an isolated strain Pediococcus pentosaceus KC692718. A comparative study of the effect of free pediocin and liposome encapsulated pediocin against Listeria sp. was performed. METHODS: The purification of the extracted cell free supernatant was subjected to ammonium sulphate precipitation, cation exchange chromatography followed by gel permeation chromatography. The bacteriocin activity and protein concentration were determined using Lowry's method. The characterization of the pure pediocin was done. Liposome like nanovesicle was constructed and the stability of the liposome encapsulated pediocin was checked. Finally, the antibacterial effect was comparatively studied of the free pediocin, liposome, and liposome encapsulated pediocin simultaneously. RESULTS: The pediocin of 3.6kDa was purified with a specific activity of 898.8. AU/mg. It remained stable from pH 2.0-8.0 was found to be moderately stable above 80°C and remain stable for one month when stored at -20°C. The encapsulated pediocin showed stability since it retained 50% of its initial activity. The encapsulated pediocin showed 89% of encapsulation efficiency. CONCLUSION: The encapsulated pediocin not only improved pediocin stability but also enhanced the controlled release of the antimicrobial substances, enough for inhibiting the foodborne pathogen L. monocytogenes.

Application of yeast surface display system in expression of recombinant pediocin PA-1 in Saccharomyces cerevisiae.

Pediocin PA-1 is a bacteriocin that shows strongly anti-microbial activity against some Gram-positive pathogens such as Listeria monocytogenes, Staphylococcus aureus, and Enterococcus faecalis. With the broad inhibitory spectrum as well as high-temperature stability, pediocin has a potential application in the food preservation and pharmaceutical industry. Pediocin has been studied to express in many heterologous expression systems such as Escherichia coli, Saccharomyces cerevisiae, and Pichia pastoris as a free peptide. Here we showed in this study a new strategy by using yeast surface display system to produce the anchored pediocin PA-1 on the cell surface of Saccharomyces cerevisiae, which could be used directly as a pediocin resource. We had successfully constructed a recombinant S. cerevisiae W303 strain that could express pediocin PA-1 on the cell surface. The pediocin-expressing yeast could inhibit the growth of Shigella boydii and Shigella flexneri, which have never been reported before for pediocin activity. Besides, the pediocin expression level of the recombinant S. cerevisiae strain was also evaluated in three different media: synthetic defined (SD), basic medium (BM), and fermentation medium (FM). BM medium was shown to give the highest production yield of the recombinant yeast (4.75 ± 0.75 g dry cell weight per 1 L of culture) with the ratio number of the pediocin-expressing cells of 93.46 ± 2.45%. Taken together, the results clearly showed that pediocin can be displayed on yeast cell surface as anchored protein. The application of yeast cell surface system enables a new door of pediocin application on either food or feed industries. Graphical abstract.

Synergistic inhibition mechanism of pediocin PA-1 and L-lactic acid against Aeromonas hydrophila.

Pediocin PA-1 (PA-1) is a membrane-targeting bacteriocin from lactic acid bacteria, which shows antimicrobial activity against a wide range of Gram-positive pathogens. However, the outer membrane of Gram-negative bacteria does not allow pediocin access to its target. In this work, the synergistic inhibitory mechanism of PA-1 with L-lactic acid against Gram-negative aquaculture and food pathogen Aeromonas hydrophila (A. hydrophila) was analyzed. The combined treatment of 3.5 mmol/L L-lactic acid and 50 µmol/L (or 30 µmol/L) PA-1 had strong bacteriostatic and bactericidal activity against A. hydrophila. Full wavelength scanning and ELISA assay revealed the release of lipopolysaccharide (LPS) from the outer membrane of A. hydrophila caused by L-lactic acid treatment. Laser confocal microscopic imaging of A. hydrophila with FITC-labeled pediocin PA-1 proved the accumulation of PA-1 on lactic acid-treated bacterial cells. PA-1 then caused a rapid dissipation of membrane potential (Δψ) and a proton gradient difference (ΔpH) in lactic acid-treated A. hydrophila. Pediocin PA-1 also caused an increase in the extracellular ATP level. Morphology revealed by SEM and TEM showed that combined treating with lactic acid and PA-1 induced vesicles on the cell surface, the outer and inner membrane disruption, and even cytoplasm leakage and cell lysis. The results proved a potential mechanism of the synergistic inhibition of lactic acid and PA-1 against A. hydrophila, by which L-lactic acid released the outer membrane LPS, making it possible for PA-1 to contact the plasma membrane of A. hydrophila, resulting in the dissipation of proton-motive force in the inner membrane and cell death.

Phenotypic comparison and DNA sequencing analysis of a wild-type and a pediocin-resistant mutant of Listeria ivanovii.

Listeria ivanovii is one of the two pathogenic species within the genus Listeria, the other being Listeria monocytogenes. In this study, we generated a stable pediocin resistant mutant Liv-r1 of a L. ivanovii strain, compared phenotypic differences between the wild-type and the mutant, localised the pediocin-induced mutations in the chromosome, and analysed the mechanisms behind the bacteriocin resistance. In addition to pediocin resistance, Liv-r1 was also less sensitive to nisin. The growth of Liv-r1 was significantly reduced with glucose and mannose, but less with cellobiose. The cells of Liv-r1 adsorbed less pediocin than the wild-type cells. Consequently, with less pediocin on the cell surface, the mutant was also less leaky, as shown as the release of intracellular lactate dehydrogenase to the supernatant. The surface of the mutant cells was more hydrophobic than that of the wild-type. Whole genome sequencing revealed numerous changes in the Liv-r1 chromosome. The mutations were found e.g., in genes encoding sigma-54-dependent transcription regulator and internalin B, as well as in genes involved in metabolism of carbohydrates such as glucose and cellobiose. Genetic differences observed in the mutant may be responsible for resistance to pediocin but no direct evidence is provided.

Biopreservation approaches to reduce Listeria monocytogenes in fresh vegetables.

Two biopreservation approaches for fresh lettuce, rocket salad, parsley and spinach were studied. The potential of Pediococcus pentosaceus DT016, as a protective culture, to suppress Listeria monocytogenes in vegetables during storage was evaluated. The pathogen numbers in the vegetables inoculated with P. pentosaceus DT016 were significantly (p < 0.01) lower throughout the storage period and, at the last storage day, a minimum difference of 1.4 log CFU/g was reported when compared with the vegetables without the protective culture. Moreover, by using two levels of L. monocytogenes (about 6 and 4 log CFU/g), it was observed that the antagonist effect of P. pentosaceus was higher for the lower pathogen numbers. The second approach evaluated a pediocin DT016 solution to inactivate and control L. monocytogenes proliferation. The pathogen load was studied after washing with: water, chlorine and the pediocin solution and along storage at 4  °C. Comparing the various washing solutions, the vegetables washed with pediocin presented significantly (p < 0.01) lower pathogen numbers throughout storage, by a minimum of 3.2 and 2.7 log CFU/g, than in vegetables washed with water and chlorine, respectively. The proposed methodologies are promising alternatives to maintain the safety of fresh vegetables during extended storage at refrigeration temperature.

Non-thermal approach to Listeria monocytogenes inactivation in milk: The combined effect of high pressure, pediocin PA-1 and bacteriophage P100.

Non-thermal food processing and replacement of chemical additives by natural antimicrobials are promising trends in the food industry. The objective of the present work was to evaluate the effect of a process which combines mild high hydrostatic pressure - HHP (200 and 300 MPa, 5 min, 10 °C), phage Listex™ P100 and the bacteriocin pediocin PA-1 as a new non-thermal process for destruction of Listeria monocytogenes (104 CFU mL-1 or 107 CFU mL-1) in milk. For inoculum levels of 104 CFU mL-1, HHP combined with phage P100 eliminated L. monocytogenes immediately after pressurization. When L. monocytogenes was inoculated at levels of 107 CFU mL-1, a synergistic effect between phage P100, pediocin PA-1 and HHP (300 MPa) on the inactivation of L. monocytogenes was observed during storage of milk at 4 °C. For non-pressure treated samples inoculated with phage or pediocin or both, L. monocytogenes counts decreased immediately after biocontrol application, but regrowth was observed in a few samples during storage. Phage particles were stable during refrigerated storage for seven days while pediocin PA-1 remained stable only during three days. Further studies will have to be performed to validate the findings of this work in specific applications (e.g. production of raw milk cheese).

Unveiling the potentials of bacteriocin (Pediocin L50) from Pediococcus acidilactici with antagonist spectrum in a Caenorhabditis elegans model.

Human-milk-based probiotics play a major role in the early colonization and protection of infants against gastrointestinal infection. We investigated potential probiotics in human milk. Among 41 Lactic acid bacteria (LAB) strains, four strains showed high antimicrobial activity against Escherichia coli 0157:H7, Listeria monocytogenes ATCC 15313, Bacillus cereus ATCC 14576, Staphylococcus aureus ATCC 19095, and Helicobacter pylori. The selected LAB strains were tested in simulated gastrointestinal conditions for their survival. Four LAB strains showed high resistance to pepsin (82%-99%), bile with pancreatine stability (96%-100%), and low pH (80%-94%). They showed moderate cell surface hydrophobicity (22%-46%), auto-aggregation abilities (12%-34%), and 70%-80% co-aggregation abilities against L. monocytogenes ATCC 15313, S. aureus ATCC 19095, B. cereus ATCC 14576, and E. coli 0157:H7. All four selected isolates were resistant to gentamicin, imipenem, novobiocin, tetracycline, clindamycin, meropenem, ampicillin, and penicillin. The results show that Pediococcus acidilatici is likely an efficient probiotic strain to produce < 3 Kda pediocin-based antimicrobial peptides, confirmed by applying amino acid sequences), using liquid chromatography mass spectrometry and HPLC with the corresponding sequences from class 2 bacteriocin, and based on the molecular docking, the mode of action of pediocin was determined on LipoX complex, further the 13C nuclear magnetic resonance structural analysis, which confirmed the antimicrobial peptide as pediocin.

The case for class II bacteriocins: A biophysical approach using "suicide probes" in receptor-free hosts to study their mechanism of action.

Class II bacteriocins are unmodified membrane-active peptides that act over a narrow spectrum of target bacteria. They bind a specific receptor protein on the membrane to form a pore, leading to membrane permeabilization and cell death. However, little is known about the molecular events triggering the pore formation after the bacteriocin recognizes the receptor. It is not clear yet if the pore is the same receptor forced into an open conformation or if the pore results from the bacteriocin insertion and oligomeric assembly in the lipid bilayer. In order to reveal which model is more suitable to explain the toxicity mechanism, in this work we use chimeric peptides, resulting from the fusion of the bitopic membrane protein EtpM with different class II bacteriocins: enterocin CRL35, pediocin PA-1 and microcin V. E. coli strains lacking the specific receptors for these bacteriocins were chosen as expression hosts. As these constructs display a lethal effect when they are heterologously expressed, they are called "suicide probes". The results suggest that, indeed, the specific receptor would act as a docking molecule more than as a structural piece of the pore, as long as the bacteriocin is somehow anchored to the membrane. These set of chimeric peptides also represent an in vivo system that allows to study the interaction of the bacteriocins with real bacterial membranes, instead of model membranes. Hence, the effects of these suicide probes in membrane fluidity and transmembrane potential were also assessed, using fluorescence spectroscopy. The data show that the different suicide probes are able to increase phospholipid order and depolarize the membranes of receptor-free bacterial cells.

Pediocin-Like Antimicrobial Peptides of Bacteria.

Bacteriocins are bacterial antimicrobial peptides that, unlike classical peptide antibiotics, are products of ribosomal synthesis and usually have a narrow spectrum of antibacterial activity against species closely related to the producers. Pediocin-like bacteriocins (PLBs) belong to the class IIa of the bacteriocins of Gram-positive bacteria. PLBs possess high activity against pathogenic bacteria from Listeria and Enterococcus genera. Molecular target for PLBs is a membrane protein complex - bacterial mannose-phosphotransferase. PLBs can be synthesized by components of symbiotic microflora and participate in the maintenance of homeostasis in various compartments of the digestive tract and on the surface of epithelial tissues contacting the external environment. PLBs could give a rise to a new group of antibiotics of narrow spectrum of activity.

Purification and characterization of pediocin from probiotic Pediococcus pentosaceus GS4, MTCC 12683.

Pediococcus pentosaceus GS4 (MTCC 12683), a probiotic lactic acid bacterium (LAB), was found to produce bacteriocin in spent culture. Antibacterial and antagonistic potential of this bacteriocin against reference strains of Staphylococcus aureus (ATCC 25923), Escherichia coli (ATCC 25922), Pseudomonas aeruginosa (ATCC 25619), and Listeria monocytogenes (ATCC 15313) was proven by double-layer and well diffusion methods wherein nisin and ampicillin were used as positive controls. Bacteriocin in supernatant was purified and analyzed by SDS-PAGE, RP-HPLC, and circular dichroism (CD). The physico-chemical properties of purified bacteriocin were characterized being treated at different temperatures (30 to 110 °C), pH (3.0 to 12.0), with different enzymes (α-amylase, pepsin, and lysozyme), and organic solvents (hexane, ethanol, methanol, and acetone) respectively. The molar mass of bacteriocin (named pediocin GS4) was determined as 9.57 kDa. The single peak appears at the retention time of 2.403 with area amounting to 25.02% with nisin as positive control in RP-HPLC. CD analysis reveals that the compound appears to have the helix ratio of 40.2% with no beta sheet. The antibacterial activity of pediocin GS4 was optimum at 50 °C and at pH 5.0 and 7.0. The pediocin GS4 was not denatured by the treatment of amylase and lysozyme but was not active in the presence of organic solvents. This novel bacteriocin thus m ay be useful in food and health care industry.

Bacteriocins: perspective for the development of novel anticancer drugs.

Antimicrobial peptides (AMPs) from prokaryotic source also known as bacteriocins are ribosomally synthesized by bacteria belonging to different eubacterial taxonomic branches. Most of these AMPs are low molecular weight cationic membrane active peptides that disrupt membrane by forming pores in target cell membranes resulting in cell death. While these peptides known to exhibit broad-spectrum antimicrobial activity, including antibacterial and antifungal, they displayed minimal cytotoxicity to the host cells. Their antimicrobial efficacy has been demonstrated in vivo using diverse animal infection models. Therefore, we have discussed some of the promising peptides for their ability towards potential therapeutic applications. Further, some of these bacteriocins have also been reported to exhibit significant biological activity against various types of cancer cells in different experimental studies. In fact, differential cytotoxicity towards cancer cells as compared to normal cells by certain bacteriocins directs for a much focused research to utilize these compounds as novel therapeutic agents. In this review, bacteriocins that demonstrated antitumor activity against diverse cancer cell lines have been discussed emphasizing their biochemical features, selectivity against extra targets and molecular mechanisms of action.

Synthesis, antimicrobial activity and conformational analysis of the class IIa bacteriocin pediocin PA-1 and analogs thereof.

The antimicrobial peptide pediocin PA-1 is a class IIa bacteriocin that inhibits several clinically relevant pathogens including Listeria spp. Here we report the synthesis and characterization of whole pediocin PA-1 and novel analogs thereof using a combination of solid- and solution-phase strategies to overcome difficulties due to instability and undesired reactions. Pediocin PA-1 thus synthesized was a potent inhibitor of Listeria monocytogenes (MIC = 6.8 nM), similar to the bacteriocin produced naturally by Pediococcus acidilactici. Of particular interest is that linear analogs lacking both of the disulfide bridges characterizing pediocin PA-1 were as potent. One linear analog was also a strong inhibitor of Clostridium perfringens, another important food-borne pathogen. These results are discussed in light of conformational information derived from circular dichroism, solution NMR spectroscopy and structure-activity relationship studies.

A new way of producing pediocin in Pediococcus acidilactici through intracellular stimulation by internalized inulin nanoparticles.

One of the most challenging aspects of probiotics as a replacement for antibiotics is to enhance their antimicrobial activity against pathogens. Given that prebiotics stimulate the growth and/or activity of probiotics, we developed phthalyl inulin nanoparticles (PINs) as prebiotics and observed their effects on the cellular and antimicrobial activities of Pediococcus acidilactici (PA). First, we assessed the internalization of PINs into PA. The internalization of PINs was largely regulated by glucose transporters in PA, and the process was energy-dependent. Once internalized, PINs induced PA to produce substantial amounts of antimicrobial peptide (pediocin), which is effective against both Gram-positive (Salmonella Gallinarum) and Gram-negative (Listeria monocytogenes) pathogens. When treated with small-sized PINs, PA witnessed a nine-fold increase in antimicrobial activity. The rise in pediocin activity in PA treated with PINs was accompanied by enhanced expression of stress response genes (groEL, groES, dnaK) and pediocin biosynthesis genes (pedA, pedD). Although the mechanism is not clear, it appears that the internalization of PINs by PA causes mild stress to activate the PA defense system, leading to increased production of pediocin. Overall, we identified a prebiotic in nanoparticle form for intracellular stimulation of probiotics, demonstrating a new avenue for the biological production of antimicrobial peptides.

A New Method of Producing a Natural Antibacterial Peptide by Encapsulated Probiotics Internalized with Inulin Nanoparticles as Prebiotics.

Synbiotics are a combination of probiotics and prebiotics, which lead to synergistic benefits in host welfare. Probiotics have been used as an alternative to antibiotics. Among the probiotics, Pediococcus acidilactici (PA) has shown excellent antimicrobial activity against Salmonella Gallinarum (SG) as a major poultry pathogen and has improved the production performances of animals. Inulin is widely used as a prebiotic for the improvement of animal health and growth. The main aim of this study is to investigate the effect of the antimicrobial activity of inulin nanoparticles (INs)-internalized PA encapsulated into alginate/chitosan/alginate (ACA) microcapsules (MCs) in future in vivo application. The prepared phthalyl INs (PINs) were characterized by DLS and FE-SEM. The contents of phthal groups in phthalyl inulin were estimated by ¹H-NMR measurement as 25.1 mol.-%. The sizes of the PINs measured by DLS were approximately 203 nm. Internalization into PA was confirmed by confocal microscopy and flow cytometry. Antimicrobial activity of PIN-internalized probiotics encapsulated into ACA MCs was measured by co-culture antimicrobial assays on SG. PIN-internalized probiotics had a higher antimicrobial ability than that of ACA MCs loaded with PA/inulin or PA. Interestingly, when PINs were treated with PA and encapsulated into ACA MCs, as a natural antimicrobial peptide, pediocin was produced much more in the culture medium compared with other groups inulin-loaded ACA MCs and PA-encapsulated into ACA MCs.

Controlled functional expression of the bacteriocins pediocin PA-1 and bactofencin A in Escherichia coli.

The bacteriocins bactofencin A (class IId) and pediocin PA-1 (class IIa) are encoded by operons with a similarly clustered gene organization including a structural peptide, an immunity protein, an ABC transporter and accessory bacteriocin transporter protein. Cloning of these operons in E. coli TunerTM (DE3) on a pETcoco-2 derived vector resulted in successful secretion of both bacteriocins. A corresponding approach, involving the construction of vectors containing different combinations of these genes, revealed that the structural and the transporter genes alone are sufficient to permit heterologous production and secretion in this host. Even though the accessory protein, usually associated with optimal disulfide bond formation, was not required for bacteriocin synthesis, its presence did result in greater pediocin PA-1 production. The simplicity of the system and the fact that the associated bacteriocins could be recovered from the extracellular medium provides an opportunity to facilitate protein engineering and the overproduction of biologically-active bacteriocins at industrial scale. Additionally, this system could enable the characterization of new bacteriocin operons where genetic tools are not available for the native producers.

Phosphotransferase systems in Enterococcus faecalis OG1RF enhance anti-stress capacity in vitro and in vivo.

Phosphotransferase systems are common and essential in bacteria, which are in charge of sugar transportation and phosphorylation. However, phosphotransferase systems were found in recent years to be associated with environmental stress factors. This study investigated the role of the mannose/fructose/sorbose phosphotransferase systems in Enterococcus faecalis OG1RF in adaption to harsh environments by construction of pts mutants. More than one mannose/fructose/sorbose phosphotransferase system was found in E. faecalis OG1RF, and the elimination of pts gene at different loci generated different after-effects corresponding to different ambiences. An in vitro study showed that the presence of intact phosphotransferase systems in E. faecalis OG1RF promoted resistance to hydrogen peroxide and acid and enhanced susceptibility to pediocin. In vivo study demonstrated that the presence of intact phosphotransferase systems induced more hazardous substances like superoxide dismutase (SOD) in Caenorhabditis elegans and enhanced bacterial infection and survival in macrophages J774A.1 and BMM. In addition, phosphotransferase systems regulated transcription of antioxidant and catabolite genes such as katA, gor, lysR, hypR, rex, hprK and tpx to different extents (-6.3- to 3.5-fold). It is therefore suggested that pts genes are regulatory factors promoting adaption of E. faecalis OG1RF to stressful conditions, thereby enhancing the possibility of bacterial survival and infectivity.

High hydrostatic pressure effects on Listeria monocytogenes and L. innocua: Evidence for variability in inactivation behaviour and in resistance to pediocin bacHA-6111-2.

The effect of high hydrostatic pressure (HHP) on the survival of 14 strains of Listeria monocytogenes from food or clinical origins, selected to represent different pheno and genotypes, was evaluated. Stationary phase cells were submitted to 300, 400 and 500 MPa at 10 °C, for 5 min. A high variability in the resistance of L. monocytogenes to pressure was observed, and particularly two strains isolated from food were significantly more baroresistant than the rest. Strains of L. monocytogenes resistant to one or more antibiotics exhibited significantly higher levels of survival after the high pressure treatment at 400 MPa. No correlation was found between strains' origin or thermal tolerance and resistance to HHP. The suitability of two strains of L. innocua as surrogates of L. monocytogenes, was also investigated. These exhibited significantly higher sensitivities to HHP than observed for some L. monocytogenes. The antimicrobial effect of the antilisterial bacteriocin (bacHA-6111-2) increased after L. monocytogenes cells had been exposed to pressure. The data obtained underlines the importance of strain selection for studies aiming to evaluate HHP efficacy to ensure safety of HHP-treated foods.

Pediocin SA-1: A selective bacteriocin for controlling Listeria monocytogenes in maize silages.

In this study, we assessed the potential as silage additive of a bacteriocin produced by Pediococcus acidilactici Northern Regional Research Laboratory (NRRL) B-5627 (pediocin SA-1). Maize was inoculated either with a bacterial starter alone (I) or in combination with the bacteriocin (IP), and untreated silage served as control. We monitored the products of fermentation (ethanol, and lactic and acetic acids), the microbial population, and the presence of the indicator strain Listeria monocytogenes Colección Española de Cultivos Tipo (CECT) 4032 (1×10(5) cfu/g) after 1, 2, 5, 8, 16, and 30d of ensiling. Our results indicated antilisterial activity of the bacteriocin, anticipating the disappearance of L. monocytogenes in IP compared with I and control silages. The PCR-denaturing gradient gel electrophoresis analysis revealed the addition of the bacteriocin did not affect the bacterial communities of the spontaneous fermentation, and the inoculant-containing bacteria (Lactobacillus plantarum, Lactobacillus buchneri, and Enterococcus faecium) were found in addition to the bacterial communities of untreated maize silages in I and IP silages. Both treatments increased the concentration of antimicrobial compounds (acetic acid, ethanol, and 1,2-propanodiol) and led to lower residual sugar contents compared with the control, which would provide enhanced aerobic stability. The fact that the identified species L. plantarum, L. buchneri, and E. faecium produce some of these inhibitory compounds, together with their persistence throughout the 30d of fermentation, suggest these bacteria could actively participate in the ensiling process. According to these results, pediocin SA-1 could be used as an additive to control the presence of L. monocytogenes in maize silages selectively, while improving their fermentative quality and eventually their aerobic stability.