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Pediocin (Ped) is a 4.6kDa bacteriocin effective against Listeria monocytogenes and several Gram-positive bacteria. To produce purified Ped with high yield and apply it to food preservation, this study expresses and purifies recombinant Ped (rPed) from Pichia pastoris X33::ped, and preliminary applies it in shrimp preservation. The results showed that methanol 1% is optimal for inducing the expression of rPed from P. pastoris X33::ped, achieving 6,400 AU/ml of rPed after 72 hours of yeast culture on a BSM medium. The rPed purification process was successfully established with a sample loading rate of 155.9 cm/h. This process yielded rPed with 91.7% purity and 11.0 mg per liter of culture. This rPed could preserve shrimp at 4°C for 07 days. Therefore, these results demonstrate the potential of producing rPed from Pichia pastoris X33::ped and applying it in food preservation.

Simple Summary: The consumption and appreciation of traditional foods have grown worldwide in the last century, valuing their cultural heritage, natural-associated content, and manufacturing process. Nonetheless, the increase in foodborne outbreaks associated with the consumption of these types of foods has become a concern. High-pressure processing (HPP)-assisted biocontrol is a non-thermal, multi-hurdle technology based on a cocktail of environmentally friendly antimicrobials that can be applied in synergy with mild HPP at low temperatures (≤10 °C) to promote the inactivation of target pathogenic bacteria and provide a sustainable alternative to conventional thermal food decontamination. Beyond the preservation of the physicochemical and organoleptic properties of foods, HPP-assisted biocontrol scarcely influences the endogenous microbiota. Traditional foods are increasingly valued by consumers, whose attention and purchase willingness are highly influenced by other claims such as 'natural', 'sustainable', and 'clean label'. The purpose of the present study was to evaluate the impact of a novel non-thermal food processing method (i.e., HPP-assisted biocontrol combining mild high hydrostatic pressure, listeriophage Listex, and pediocin PA-1 producing Pediococcus acidilactici) on the succession of bacterial communities and quality of a fermented sausage model. A comparative analysis of instrumental color, texture, and lipid peroxidation revealed no significant differences (p > 0.05) in these quality parameters between non- and minimally processed fermented sausages throughout 60-day refrigerated storage (4 °C). The microbiota dynamics of biotreated and untreated fermented sausages were assessed by 16S rRNA next-generation sequencing, and the alpha and beta diversity analyses revealed no dissimilarity in the structure and composition of the bacterial communities over the analyzed period. The innovative multi-hurdle technology proposed herein holds valuable potential for the manufacture of traditional fermented sausages while preserving their unique intrinsic characteristics. [ABSTRACT FROM AUTHOR]

Abstract Background Pediocin PA-1 is a bacteriocin of recognized value with applications in food bio-preservation and the medical sector for the prevention of infection. To date, industrial manufacturing of pediocin PA-1 is limited by high cost and low-performance. The recent establishment of the biotechnological workhorse Corynebacterium glutamicum as recombinant host for pediocin PA-1 synthesis displays a promising starting point towards more efficient production. Results Here, we optimized the fermentative production process. Following successful simplification of the production medium, we carefully investigated the impact of dissolved oxygen, pH value, and the presence of bivalent calcium ions on pediocin production. It turned out that the formation of the peptide was strongly supported by an acidic pH of 5.7 and microaerobic conditions at a dissolved oxygen level of 2.5%. Furthermore, elevated levels of CaCl2 boosted production. The IPTG-inducible producer C . glutamicum CR099 pXMJ19 P tac pedACD Cg provided 66 mg L−1 of pediocin PA-1 in a two-phase batch process using the optimized set-up. In addition, the novel constitutive strain P tuf pedACD Cg allowed successful production without the need for IPTG. Conclusions The achieved pediocin titer surpasses previous efforts in various microbes up to almost seven-fold, providing a valuable step to further explore and develop this important bacteriocin. In addition to its high biosynthetic performance C. glutamicum proved to be highly robust under the demanding producing conditions, suggesting its further use as host for bacteriocin production.

Background: Pediocin PA-1 is a bacteriocin of recognized value with applications in food bio-preservation and the medical sector for the prevention of infection. To date, industrial manufacturing of pediocin PA-1 is limited by high cost and low-performance. The recent establishment of the biotechnological workhorse Corynebacterium glutamicum as recombinant host for pediocin PA-1 synthesis displays a promising starting point towards more efficient production. Results: Here, we optimized the fermentative production process. Following successful simplification of the production medium, we carefully investigated the impact of dissolved oxygen, pH value, and the presence of bivalent calcium ions on pediocin production. It turned out that the formation of the peptide was strongly supported by an acidic pH of 5.7 and microaerobic conditions at a dissolved oxygen level of 2.5%. Furthermore, elevated levels of CaCl2 boosted production. The IPTG-inducible producer C. glutamicum CR099 pXMJ19 Ptac pedACDCg provided 66 mg L−1 of pediocin PA-1 in a two-phase batch process using the optimized set-up. In addition, the novel constitutive strain Ptuf pedACDCg allowed successful production without the need for IPTG. Conclusions: The achieved pediocin titer surpasses previous efforts in various microbes up to almost seven-fold, providing a valuable step to further explore and develop this important bacteriocin. In addition to its high biosynthetic performance C. glutamicum proved to be highly robust under the demanding producing conditions, suggesting its further use as host for bacteriocin production. [ABSTRACT FROM AUTHOR]

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 $$ \pm {\rm{ }}$$ 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 $$ \pm $$ 0.4 and 3.64 $$ \pm $$ 0.62 Log CFU/mL, respectively, whereas Listeria counts were considerably higher, i.e. 7.75 $$ \pm {\rm{ }}$$ 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.

Bacteriocins are ribosomally synthesized bacterial antimicrobial peptides that have a narrow spectrum of antibacterial activity against species closely related to the producers. Pediocin-like (or class IIa) bacteriocins (PLBs) exhibit antibacterial activity against several Gram-positive bacterial strains by forming pores in the cytoplasmic membrane of target cells with a specific receptor, the mannose phosphotransferase system (man-PTS). In this study, we report the cryo-electron microscopy structures of man-PTS from Listeria monocytogenes alone and its complex with pediocin PA-1, the first and most extensively studied representative PLB, at resolutions of 3.12 and 2.45 Å, respectively. The structures revealed that the binding of pediocin PA-1 opens the Core domain of man-PTS away from its Vmotif domain, creating a pore through the cytoplasmic membranes of target cells. During this process, the N-terminal β-sheet region of pediocin PA-1 can specifically attach to the extracellular surface of the man-PTS Core domain, whereas the C-terminal half penetrates the membrane and cracks the man-PTS like a wedge. Thus, our findings shed light on a design of novel PLBs that can kill the target pathogenic bacteria. IMPORTANCE Listeria monocytogenes is a ubiquitous microorganism responsible for listeriosis, a rare but severe disease in humans, who become infected by ingesting contaminated food products (i.e., dairy, meat, fish, and vegetables): the disease has a fatality rate of 33%. Pediocin PA-1 is an important commercial additive used in food production to inhibit Listeria species. The mannose phosphotransferase system (manPTS) is responsible for the sensitivity of Listeria monocytogenes to pediocin PA-1. In this study, we report the cryo-EM structures of man-PTS from Listeria monocytogenes alone and its complex with pediocin PA-1 at resolutions of 3.12 and 2.45 Å, respectively. Our results facilitate the understanding of the mode of action of class IIa bacteriocins as an alternative to antibiotics. [ABSTRACT FROM AUTHOR]

A search for multidrug-resistant bacteria was conducted in 1,143 clinical samples in Morocco from which 244 isolates were tested against 29 different antibiotics. This showed a prevalence of resistant strains of 68.4%. The spectra of resistance ranged from 1 to 21. Ampicillin, Ticarcilin, Amoxicillin, Piperacillin and Ciproflaxin were the most frequently resisted. Eight antibiotics were tested against two selected strains, S. aureus 400037 from conjunctive mucosa and A. baumannii 280291 urine, which showed resistance to concentrations up to 240 βg.ml-1. A. baumannii 280291 also resisted the two bacteriocins nisin A and pediocin PA-1, while S. aureus 400037 showed relative sensitivity to nisin A with a MIC of 125 βg.ml-1 but was insensitive to pediocin PA-1. However, the combined use of antibiotics and bacteriocins showed potentiating effects of bacteriocins on antibiotics or synergistic effects between the two antimicrobials. Combinations with nisin A resulted for instance in a sensitisation of S. aureus 400037 to penicillin G, amoxicillin, ampicillin, cefaclor and ciprofloxacin with MIC values as low as 0.19; 0.78; 0.31; 3.75 and 0.62 ìg.ml-1 respectively. Against A. baumannii 280291, the MIC value of gentamycin decreased from 60 to 3.75 βg.ml-1, in the presence of nisin. Similar results were obtained when antibiotics were combined with pediocin PA-1 and such effects were even stronger when both bacteriocins were used along with antibiotics. The present study shows that pathogenic bacteria that developed high levels of resistance to a wide array of antibiotics can be rendered sensitive to the same antibiotics simply by combining them with bacteriocins. [ABSTRACT FROM AUTHOR]

Enterococcus faecium E16, a strain isolated from goats' cheese made from unpasteurized milk, showed antibacterial activity, in particular against Listeria monocytogenes strains. The active compound was produced at high amounts in de Man Rogosa and Sharpe broth at 37 °C. It was purified to homogenity from culture supernatant by using a simple method consisting of cation-exchange and reversed-phase chromatographies. Mass spectrometry showed that the antibacterial activity was due to a 4 621.32 Da peptide. Its amino acid sequence was analysed using Edman degradation an it showed that the peptide consisted of 44 unmodified amino acids with two disulphide bonds linking cysteine residues at positions 9, 14 and at positions 24, 44. This structure was shown to be identical to pediocin PA-1, a class IIa bacteriocin. The antimicrobial activity of the produced pediocin PA-1 covered a range of bacteria, with strong activity against many species of Gram-positive bacteria, especially the food-borne pathogen Listeria monocytogenes, but no activity against Gram-negative bacteria. This study represents the first reported case of natural pediocin PA-1 production by an Enterococcus species. [ABSTRACT FROM AUTHOR]

The present study was carried out to enhance shelf life of raw buffalo milk by the use of pediocin PA-1 containing fermented cheese whey (PCFCW) as bio-preservative. Ten distinct genera of milk microbes could be identified as Streptococcus, Enterococcus, Salmonella, Campylobacter, Lactobacillus, Enterobacter, E. coli, Shigella, Candida and Aspergillus. For the production of PCFCW, supplemented cheese whey medium was fermented with Pediococcus pentosaceous NCDC 273 for 24 h. Partially purified PCFCW was prepared using ammonium sulphate precipitation followed by membrane ultrafiltration with 10 kDa and 3 kDa membrane (Millipore) respectively. The fraction between 10 and 3 kDa was used as bio-preservative. This bio-preservative was found to contain pediocin PA-1 at the level of 2.04 × 10 5 AU/ml. In view of the wide antibacterial spectrum of pediocin, the PCFCW formulation was added at 1%, 5%, and 10% (v/v) concentrations to raw buffalo milk to reduce total microbial load. A significant decrease (p˂0.05) was observed in total viable count on TGYEA, Staphylococcus aureus count on BPA, and lactic acid bacterial count on MRSA. However, the reduction in coliform count on VRBA and yeast-mould count on PDA was non-significant (p˂0.05). Therefore, it can be concluded that partially purified PCFCW was effective in reducing total microbial load in raw buffalo milk. [ABSTRACT FROM AUTHOR]

Class IIa bacteriocin pediocin PA-1 has broad-spectrum activity and is a well-characterized candidate food biopreservative. Here, a simple approach is designed to extend the application of pediocin PA-1 in improving the antibacterial activity of electrospun poly(caprolactone) (PCL) grafts through combining PA-1 with HGFI, which is a self-assembled protein with characteristics allowing the modulation of surface properties of other materials originated from Grifola frondosa . Saccharomyces cerevisiae was used as the host for expression of fusion protein PA-1-linker-HGFI (pH) and his-tag purification was used to purify recombinant protein pH. An antibacterial activity assay showed the fusion protein pH retained the biological property of native PA-1. Water contact angle, X-ray photoelectron spectroscopy, immunofluorescence assay and atomic force microscopy indicated the surface properties of HGFI were greatly preserved by the fusion protein pH. Finally, antibacterial activity of pH-modified PCL substrate measurements implied the fusion protein significantly improved the bacterial-resistance of the PCL film through dressing the PCL fibers with the recombinant pH protein. This work presents a new perspective on the application of hydrophobin and pediocin PA-1 in antibacterial medical devices. [ABSTRACT FROM AUTHOR]

The antimicrobial activity of lactic acid bacteria (LAB) isolated from traditional ripened foods against various foodborne pathogens was evaluated, and the bacteriocins produced by the selected LAB of interest were characterized. For this, 33 bacteria were initially selected for their ability to produce proteinaceous compounds with antimicrobial activity. Subsequently, only three isolates were selected since they did not have virulence factors or were resistant to antibiotics. Due to the greater bacteriocin activity spectrum, Lactilactobacillus sakei 205 was selected, and its bacteriocin was characterized. The highest activity level (6400 AU/mL) was observed against Enterococcus faecalis ATCC 29212 during the growth of Lb. sakei 205 for 18–20 h. The bacteriocin, with an approximate molecular size of 24 kDa, remained stable at temperatures ranging from 25 °C to 60 °C, at pH values between 4.0 and 6.0 and was sensitive to all the detergents and inactivated with the proteolytic enzyme proteinase K, confirming its proteinaceous nature. Furthermore, it affected the viability of all the studied pathogens and repressed their growth for at least 12 h. In this preliminary study, the identified properties of Lb. sakei 205 strain and its produced bacteriocin indicate its potential application as a biopreservative in traditional ripened foods. • Lactilactobacillus sakei 205 isolated from dry-cured fermented sausage was selected. • Lb. sakei 205 produced proteinaceous compounds with antimicrobial activity. • Lb. sakei 205 bacteriocin(s) remained stable at environmental factors. • Lb. sakei 205 bacteriocin(s) (∼24 kDa) repressed the growth of foodborne pathogens for at least 12 h. • Lb. sakei 205 strain is a potential biopreservative in the food industry. [ABSTRACT FROM AUTHOR]

The current study was performed to test the ability of the Pediococcus pentosaceus NCDC 273 strain for rapid utilization of lactose from paneer whey. We optimized the lactose utilization by monitoring the constitutive expression of the β-gal gene using real-time RT-PCR and also quantified lactic acid production using high-performance liquid chromatography (HPLC). Screening of β-gal gene for lactose utilization efficiency in P. pentosaceus NCDC 273 strain efficiency showed a significant (P < 0.0001) expression of the β-gal gene in the supplemented paneer whey medium (SPWM; lactose rich) compared to commercially available Deman-Rogosa-Sharpe (MRS; d -glucose rich) broth medium. In addition, with the growth of P. pentosaceus NCDC 273 in SPWM, the expression of the β-gal gene was found significantly (P < 0.001) higher at 6h of fermentation during the exponential growth phase and declining no significantly at stationary phase. After 48 h of incubation, HPLC data revealed a high lactose conversion rate (42.12%) to lactic acid production (14.50 ± 0.48 g L−1). Moreover, pediocin PA-1 activity in fermented SPWM showed almost contamination-free lactic acid production. Hence, our results showed the sustainable utilization of dairy waste paneer whey for lactic acid production by P. pentosaceus NCDC 273 at a lower disposal cost. [Display omitted] • Potential of Pediococcus pentosaceus NCDC 273 for the sustainable utilization of dairy waste paneer whey. • Highest lactose conversion (42.12%) was observed to produce lactic acid (14.50 ± 0.48 g L−1) after an incubation period of 48 h. • Pediocin PA-1 activity showed almost contamination free lactic acid production. [ABSTRACT FROM AUTHOR]

Due to innate and acquired resistance in Enterococcus faecalis against most antibiotics, identification of new alternatives has increased interest in diverse populations of potent cationic antimicrobial peptides (CAMPs) for treatment and natural food biopreservation. The CAMPs, after crossing the cell wall to the periplasmic space, kill their target strain by forming pores in the cell membrane. However, reports of resistance against these CAMPs necessitated the understanding of step(s) interfered with while acquiring this resistance, for designing effective CAMP analogs. In this direction, we selected stable and gradual dose-dependent pediocin PA-1 single exposure resistant (Ped) mutants of E. faecalis, which conferred cross-protection to diverse CAMPs, viz., HNP-1, nisin and alamethicin but not to polymyxin B, lysozyme and vancomycin. With these Ped mutants of E. faecalis there was: a gradual neutralization in cell wall surface charge involving D-alanylation of wall teichoic acids (WTA) and lipoteichoic acids (LTA), increase in cell-surface hydrophobicity, increased cell aggregation and biofilm formation and ultra-structural changes in the cell wall, and a reduction of periplasmic space. In addition, a gradual decrease in expression of mannose PTS two ( mpt) operon was also observed with distinct changes in growth rate achieving the same biomass production during the stationary phase. These results show that resistance to these CAMPs is not due to mpt directly acting as a docking molecule but due to changes in the cell wall, which increased the permeability barrier to CAMPs diffusion to reach the periplasmic space. [ABSTRACT FROM AUTHOR]

The present study was carried out to screen the pediocin producer strains viz., Pediococcus pentosaceous NCDC 273, Pediococcus acidilactici NCDC 252 P. pentosaceous NCDC 35, Enterococcus faecium NCDC 124 and Lactobacillus plantarum NCDC 20 for their ability to utilize lactose, to optimize pediocin production using response surface methodology (RSM) and to purify pediocin using aqueous-two phase system (ATPS). Screening of five pediocin-producer strains for lactose utilizing capability showed that P . pentosaceous NCDC 273 and P . acidilactici NCDC 252 express β- gal gene. Response surface data showed maximum pediocin PA-1 production of 2.61 × 10 4 AU/ml at an initial pH of 7.0,temperature 29.8 °C, and cheese whey concentration 82.8 g l − 1 from P. pentosaceous NCDC 273 and 3.22 × 10 3 AU/ml at an initial pH of 6.95, temperature 28.2 °C, and CWC 77.79 g l − 1 from P. acidilactici NCDC 252. The pediocin PA-1 produced by both the strains was purified using the ATPS and characterized for purity and activity. PEG/potassium phosphate system was found to be efficient in purifying the pediocin PA-1, which was then ultrafiltered and lyophilized. Our results showed the efficient production of purified pediocin PA-1 at reduced cost. [ABSTRACT FROM AUTHOR]

Eighteen dairy starter cultures, spoilage and food-borne pathogenic strains were analyzed for susceptibility to antimicrobial peptides pediocin PA-1 (PedPA-1) and nisin, through the individual 50 % inhibitory concentrations (IC) determination. The IC of purified PedPA-1 was found to be more potent than nisin against five spoilage and food-borne pathogenic strains, i.e., Bacillus cereus NCDC 240, Enterococcus faecalis NCDC 114, Enterococcus faecium NCDC 124, Streptococcus agalactiae NCDC 208 and Staphylococcus aureus NCDC 110. The IC of PedPA-1 and nisin ranged from 6.58 to 0.29 µM and 18.91 to 0.03 µM, respectively. Further, PedPA-1 producing Pediococcus pentosaceus NCDC 273 and Pediococcus acidilactici NCDC 252 strains were evaluated for potential probiotic attributes by in vitro studies. Both pediococci strains were able to survive at low pH and 2 % bile with a good bile salt hydrolase activity, cell surface hydrophobicity and β-galactosidase activity that makes them potentially good candidates for probiotics. These strains with proven promising probiotic attributes are good candidates for further investigation through in vivo studies to elucidate their potential health benefits. [ABSTRACT FROM AUTHOR]

This work describes an effective combined ultrafiltration (UF)-nanofiltration (NF) membrane process for the separation and concentration of pediocin PA-1 from fermentation broth. Three polysulfone (PS) membranes with MWCOs of 5, 10, and 30 kDa were tested for ultrafiltration effectiveness. The 10 kDa membrane was selected because it displayed high permeability, good pediocin PA-1 recovery and reduced fouling. When the volume concentration factor (VCF) of UF reached 2.5, continuous diafiltration (DF) was carried out. The optimal diafiltration factor (DFF) was 1. The permeate obtained from UF was then concentrated by NF. When the VCF of NF reached 4.5, pediocin PA-1 recovery loss was only 10.5%. This two-stage membrane process improved the loading solutions by 4.5-fold, allowing up to 71.6% recovery of pediocin PA-1. After the membrane process, the NF retentate could be concentrated and subjected to preparative chromatography to obtain purified pediocin PA-1, or it could be dried to obtain a rich pediocin PA-1 preservative. [ABSTRACT FROM AUTHOR]

The significance of positively charged residues for the target cell binding of pediocin PA-1 bacteriocins was studied by site-directed mutagenesis. Most of the charged residues are located in the N-terminal half of the peptide, which is thought to mediate the initial binding of these bacteriocins to their target cells through electrostatic interactions. Mutated peptides in which the positively charged residues were substituted or increased in number were constructed, and some of these peptides exhibited a twofold increase in the bacteriostatic activity. The greatest enhancement was achieved by introduced the positive charges at position 13, their results show the benefits of introducing an additional cationic residue within this patch in the middle of the N-terminal half of pediocin PA-1 bacteriocins. Thus, the presence of additional cationic residues in the N-terminal half influenced the electrostatic binding of this bacteriocin to its target cells and increased the potency of the peptide on the potency of Micrococcus luteus and Staphylococcus aureus. Significance and Impact of the Study No previous work has systematically examined the N-terminal cationic residues of the pediocin PA-1 for their functional importance or redundancy. In this study, we examined the structure-function relationships of pediocin PA-1 by site-directed mutagenesis. Mutated peptides in which the positively charged residues were substituted and increased in number exhibited a twofold increase in the bacteriostatic activity. This study demonstrated the importance of the cationic patch in the N-terminal half of pediocin PA-1. The cationic residues influenced the electrostatic binding of the bacteriocin to the target cells and had a greater effect on the potency of the peptide towards Micrococcus luteus and Staphylococcus aureus. [ABSTRACT FROM AUTHOR]

To evaluate the presence of mobile genetic elements (MGEs) in intergeneric and interspecific pediocin producing lactic acid bacteria (LAB) the flanking regions of the pediocin PA-1/AcH (pediocin PA-1) operon was characterized. In Enterococcus faecium Acr4 and Lactobacillus plantarum Acr2 a variation in the amplicon size in the downstream region of the operon was identified, suggesting a deletion in this region. Beyond that, in pediocin PA-1 encoding plasmids MGEs such as IS Lpl1 and mobilization regions were detected by Southern hybridization analysis. Phylogenetic analyses of the E. faecium Acr4 IS Lpl1 gene sequence suggested the gene transfer from lactobacilli in the environment. The tyrosine recombinase detected in pediocin plasmids of Pediococcus acidilactici H and K7 indicate a possible transfer of the entire operon among LAB. Since these elements are known to be associated with transfer of genes linked to the bacteriocin production, antibiotic resistance, and sugar utilization, we suggest similar mechanism for natural spread of pediocin PA-1 operon among different bacterial species. [ABSTRACT FROM AUTHOR]

Pediocin PA-1, mainly produced from Pediococcus spp., is a well-known class IIa bacteriocin showing strong anti-listerial activity. Pediococcus acidilactici K10 producing the bacteriocin was transformed with pLR5cat(S)_PSAB in which the pediocin PA-1 structural gene ( pedA) was translationally fused with a deduced signal sequence of the bifidobacterial α-amylase gene for secretion. The P. acidilactici K10 transformant presented increased bacteriocin activity when compared with that of native P. acidilactici K10. The P. acidilactici K10 mutant (K10M), in which the plasmid for pediocin PA-1 production is cured, also showed bacteriocin activity when transformed with pLR5cat(S)_PSAB, indicating that the pediocin gene was expressed in hosts. An increase in pedA gene transcription level and the detection of both transcripts from the pediocin operon and PSAB were also confirmed. The P. acidilactici K10 [pLR5cat(S)_PSAB] transformant was more inhibitory against Listeria monocytogenes than that of the P. acidilactici K10 wild type in co-cultures. [ABSTRACT FROM AUTHOR]

Listeria monocytogenes is responsible for severe foodborne infections, which can be life-threatening especially for infants and elderly populations. The emergence of antibiotic-resistant pathogens has stimulated the search for new strategies, such as the use of bacteriocins, to prevent or cure foodborne infectious diseases in the intestine. In this study, we evaluated the efficacy of the bacteriocin pediocin PA-1 from Pediococcus acidilactici UL5 to inhibit Listeria ivanovii, used as a surrogate for L. monocytogenes, under physiological conditions of the terminal ileum, simulated in a continuous in vitro fermentation model. A fecal sample from a healthy adult was immobilized and propagated for 30 days in a continuous stirred tank reactor, fed with a nutritive medium simulating the ileal chime (pH 7.5). After reaching a pseudo-steady state, the reactor was inoculated five times with L. ivanovii to reach a final concentration of 10 CFU/ml within the reactor. Two spikes of L. ivanovii without adjunction of pediocin PA-1 served as control assays, and three other spikes were done to test the effects of three concentrations of pediocin PA-1 corresponding to 2, 3, and 5× the minimum inhibitory concentration (MIC) active against L. ivanovii. The concentration of L. ivanovii in the reactor was followed for 8 h using the PALCAM selective medium. The different groups of commensal bacteria were enumerated on selective medium or using fluorescence in situ hybridization. Our data showed that pediocin PA-1 is stable in the ileum conditions and that it is able to exert its inhibition activity against L. ivanovii in a dose-dependent manner. The addition of pediocin PA-1 at 5 × MIC induced a complete disappearance of L. ivanovii (5 log reduction) within 5 h, compared to a reduction of 2 logs, corresponding to the washout phenomenon, when no pediocin PA-1 was added. Reduction of 0.8 and 1.3 logs within 8 h was also obtained with the addition of 2 and 3 × MIC, respectively. The same experiment has shown that addition of pediocin-PA1 in the reactor had a negligible effect on the balance of commensal bacteria. [ABSTRACT FROM AUTHOR]

Abstract: The use of pediocins as food additives or drugs requires a simple and rapid method by which large quantities of homogeneous pediocin are produced at industrial level. Two centrifugation steps required during initial stages of purification i.e. separation of cells from fermentation broth and collection of precipitates after ammonium sulphate precipitation are the major bottlenecks for their large scale purification. In the present work, pediocin production by a new a dairy strain, Pediococcus pentosaceous NCDC 273 (identical to pediocin PA-1 at nucleotide sequence level), was found to be optimum at initial pH of 6.0 and 7.0 of basal MRS supplemented with 20g/l of glucose or lactose at 20 and 24h, respectively. Immobilization of cells through entrapment in alginate-xanthan gum gel beads with chitosan coating resulted in negligible cell release during fermentation. Thus, the cell free extract was directly collected through decantation, avoiding the need of centrifugation step at this stage. Subsequent ammonium sulphate precipitation at isoelectric point of pediocin PA-1 (8.85), using magnetic stirrer at high speed (approx. 1200rpm), resulted in forceful deposition of precipitates on the wall of precipitation beaker allowing their collection using a spatula, avoiding centrifugation step at this stage also. Further purification using cation-exchange chromatography resulted in yield of 134.4% with more than 320 fold purification with the specific activity of 19×105 AU/mg. The collection of single peak of pediocin at 41.9min in RP-HPLC, overlapping with standard pediocin PA-1, resulted in yield of 1.15μg from 20μl of sample applied. The overlapping of RP-HPLC peak and SDS-PAGE band corresponding to 4.6kDa, confirmed the purity and identity of pediocin 273 as pediocin PA-1. [Copyright &y& Elsevier]

Abstract: Pediocin PA-1 is an antimicrobial peptide produced by lactic acid bacteria (LAB) that has been sufficiently well characterised to be used in food industry as a biopreservative. Sulphur dioxide is the traditional antimicrobial agent used during the winemaking process to control bacterial growth and wine spoilage. In this study, we describe the effect of pediocin PA-1 alone and in combination with sulphur dioxide and ethanol on the growth of a collection of 53 oenological LAB, 18 acetic acid bacteria and 16 yeast strains; in addition, production of pediocin PA-1 by Pediococcus acidilactici J347-29 in presence of ethanol and grape must is also reported. Inhibitory concentrations (IC) and minimal bactericide concentrations of pediocin PA-1 were determined against LAB, and revealed a bacteriostatic effect. Oenococcus oeni resulted more sensitive to pediocin PA-1 (IC50 = 19 ng/ml) than the other LAB species (IC50 = 312 ng/ml). Cooperative inhibitory effects of pediocin PA-1 and either sulphur dioxide or ethanol were observed on LAB growth. Moreover, the pediocin PA-1 producing P. acidilactici strain J347-29 was able to grow and produce the bacteriocin in presence of ethanol (up to 4% ethanol in the fermentation broth) and grape must (up to 80%), which indicated that pediocin PA-1 can be considered as a potential biopreservative in winemaking. [Copyright &y& Elsevier]

Abstract: Development of resistance could render antimicrobial peptides ineffective as bio-preservatives in food. Variants resistant to nisin A or pediocin PA-1 were developed from Listeria monocytogenes and a variant resistant to polymyxin E was developed from Escherichia coli RR1. Inhibition of these organisms by these agents alone or in combination was assessed using critical dilution microassay and optical density measurement. The combination of pediocin or nisin with polymyxin E was synergistic against all five strains. The polymyxin/nisin combination at 9.3/32, 4.7/62.5 and 0.6/15.6 μg/ml inhibited growth of nisin-resistant L. monocytogenes, pediocin-resistant L. monocytogenes and polymyxin-resistant E. coli, respectively, by factors of 94%, 97% and 74% compared to controls. The pediocin/nisin combination was effective against L. monocytogenes and its variants, but not E. coli or its variant. Polymyxin (0.21 μg/ml) and polymyxin/nisin (0.3/7.8 μg/ml) reduced E. coli growth measured in the log phase by, respectively, 31.25% and 93.54%. L. monocytogenes growth in the logarithmic and stationary phases was reduced, respectively, by 90.46% and 77.52% by polymyxin/pediocin at 4.7/25 μg/ml. Our results suggest that the effective concentration of bacteriocin for control of resistant L. monocytogenes and E. coli variants could be lowered considerably by combination with polymyxin E. This suggests searching for polymyxin-like compounds to increase the efficiency of bacteriocins and slow the emergence of resistant mutants. This could be an important step towards the expanded use of bacteriocins in the medical arena. [Copyright &y& Elsevier]

Two heterologous expression systems using thioredoxin ( trxA) as a gene fusion part in Escherichia coli were developed to produce recombinant pediocin PA-1. Pediocin PA-1 structural gene pedA was isolated from Pediococcus acidilactici PA003 by the method of polymerase chain reaction (PCR), then cloned into vector pET32a(+), and expressed as thioredoxin-PedA fusion protein in the host strain E. coli BL21 (DE3). The fusion protein was in the form of inclusion body and was refolded before purification by nickel-iminodiacetic acid (Ni-IDA) agarose resin column. Biological activity of recombinant pediocin PA-1 was analyzed after cleavage of the fusion protein by enterokinase. Agar diffusion test revealed that 512-arbitrary unit (AU) recombinant pediocin PA-1 was obtained from 1 ml culture medium of E. coli (pPA003PED1) using Listeria monocytogenes as the indicator strain. Thioredoxin-PedA fusion gene was further cloned into pET20b(+). Thioredoxin-PedA fusion protein was detected in both the periplasmic and cytoplasmic spaces. The recombinant pediocin PA-1 from the soluble fraction attained 384 AU from 1 ml culture medium of E. coli (pPA003PED2). Therefore, biologically active pediocin PA-1 could be obtained by these two hybrid gene expression methods. [ABSTRACT FROM AUTHOR]

Abstract: The inhibitory effects of the pediocin PA-1 in frankfurters and the Pediococcus acidilactici MCH14 pediocin-producing strain itself in Spanish dry-fermented sausages were evaluated. The experiments were carried out by in situ assays using Listeria monocytogenes and Clostridium perfringens as target bacteria strains. The inhibition of L. monocytogenes by the P. acidilactici MCH14 pediocin-producing strain was effective in Spanish dry-fermented sausages, being the counts of this pathogen reduced by 2 log cycles compared to the control. In frankfurters, the counts of L. monocytogenes, using 5000 bacteriocin units/ml (BU/ml) of the pediocin PA-1 produced by P. acidilactici MCH14, decreased by 2 and 0.6 log cycles after storage at 4°C for 60days and at 15°C for 30days, respectively, with respect to the control. Similar results were obtained for C. perfringens, with 5000BU/ml the counts of this strain were reduced by 2 and 0.8 log cycles after storage at 10°C for 60days and at 15°C for 30days, respectively, with respect to the control. Both the pediocin PA-1 and the P. acidilactici MCH14 pediocin-producing strain could be a useful alternative for protection against these food-borne pathogens in meat products. [Copyright &y& Elsevier]

Abstract: The bacteriocin pediocin PA-1 has potential use as a food biopreservative, and understanding its effect on the commensal gut microbiota is important for assessment of consumer risks associated with the use of biopreservative cultures. Effects of ingested (i) pediocin PA-1 producing Lactobacillus plantarum DDEN 11007, (ii) the plasmid cured pediocin negative L. plantarum DDEN 12305, or (iii) supernatants of either of these two strains on the composition of the intestinal microbiota of Human Microbiota Associated (HMA) rats were examined by selective cultivation and molecular methods. The culturable microbiota was in all treatments dominated by lactic acid bacteria and coliforms and no changes in the rat commensal microbiota were detected after ingestion of either of the two L. plantarum strains as determined by both culturable methods and molecular methods (DGGE). Both strains were detected in the faeces after ingestion. Pediocin PA-1 did not mediate changes of the rat microbiota, and a biological assay indicated that the bacteriocin was degraded or inactivated during passage through the intestine. [Copyright &y& Elsevier]

Antimicrobial peptides possess cationic and amphipathic properties that allow for interactions with the membrane of living cells. Bacteriocins from lactic acid bacteria, in particular, are currently being studied for their potential use as food preservatives and for applications in health care. However, bacteriocin exploitation is often limited owing to low production yields. Gene cloning and heterologous protein or peptide production is one way to possibly achieve overexpression of bacteriocins to support biochemical studies. In this work, production of recombinant active pediocin PA-1 (PedA) was accomplished in Escherichia coli using a thioredoxin (trx) gene fusion (trx–pedA) expression approach. Trx–PedA itself did not show any biological activity, but upon cleavage by an enterokinase, biologically active pediocin PA-1 was obtained. Recombinant pediocin PA-1 characteristics (molecular mass, biological activity, physicochemical properties) were very similar to those of native pediocin PA-1. In addition, a 4- to 5-fold increase in production yield was obtained, by comparison with the PA-1 produced naturally by Pediococcus acidilactici PAC 1.0. The new production method, although not optimized, offers great potential for supporting further investigations on pediocin PA-1 and as a first-generation process for the production of pediocin PA-1 for high-value applications. [ABSTRACT FROM AUTHOR]

Aims: To evaluate the sensitivity of 21 common intestinal bacteria to six antibiotics and three broad-spectrum bacteriocins (nisins Z and A and pediocin PA-1). Methods and Results: Neutralized cell-free culture supernatants containing active bacteriocins, and antibiotics were tested with the agar diffusion test and the disc-diffusion method, respectively. The tested intestinal strains showed high sensitivity to most antibiotics except for streptomycin and oxacillin. Nisins A and Z (8 μg per well) had similar activity spectra and inhibited all Gram-positive intestinal bacteria at different levels (except Streptococcus salivarius), with bifidobacteria (except Bifidobacterium breve and Bif. catenulatum), Collinsella aerofaciens and Eubacterium biforme being the most sensitive strains, but they were not active against Gram-negative bacteria. Surprisingly, none of the tested strains were inhibited by pediocin PA-1 (16 μg per well). Conclusion: Pediocin PA-1 which is very active against Listeria spp. and other food pathogens did not inhibit major intestinal species in the human intestine in contrast to both nisins A and Z. Significance and Impact of the Study: Our data suggest that pediocin PA-1 has potential to inhibit Listeria within the intestinal microbiota without altering commensal bacteria. [ABSTRACT FROM AUTHOR]

Abstract: In this work, heterologous production of pediocin PA-1 in Lactococcus lactis ESI 153 and ESI 515 (Nis+), two strains selected because of their technological properties for cheesemaking, was achieved after transformation with plasmids pMC117, pRK119 and pCNC1, which contain the complete pediocin operon under the control of the strong P32 promoter. The pediocin production of the L. lactis ESI 153 derivatives containing pRK119 or pCNC1 was higher (approximately 165%) than that achieved by the natural pediocin PA-1 producer Pediococcus acidilactici 347. In the case of the L. lactis ESI 515 derivatives, those containing pRK119 or pCNC1 showed a pediocin production level similar (95–100%) to that of P. acidilactici 347. [Copyright &y& Elsevier]

Abstract: Pediocin PA-1 is a representative class IIa bacteriocin which is small and heat-stable and has a consensus motif, –YGNGV–. The plasmid pQE40PED, encoding pediocin PA-1 fused with His-tagged mouse dihydrofolate reductase (DHFR), was constructed and introduced into Escherichia coli M15 strain. The fusion protein was overexpressed in the strain after induction of isopropyl-β-d-thiogalactopyranoside (IPTG) and purified by nickel-nitrilotriacetic acid (Ni-NTA) metal affinity chromatography. For the recovery of biologically active pediocin PA-1, the purified fusion protein was cleaved by Factor Xa protease and the liberated pediocin PA-1 was finally purified by ultrafiltration with a 75% yield. The molecular mass of the purified recombinant pediocin PA-1 was the same as that of native pediocin PA-1 on an electrophoresis gel. [Copyright &y& Elsevier]

Abstract: Expression of the pedA gene from Pediococcus acidilactici, coding for mature bacteriocin Pediocin PA-1, was investigated using the yeast Pichia pastoris to obtain larger quantities of pediocin to support additional studies, including structure–function research. Following various cloning strategies, a KM71H (Muts) strain was selected. A significant concentration (74μg/ml) of extracellular recombinant pediocin was obtained but the pediocin showed no biological activity. Supernatant fluids from P. pastoris cultures, harboring or not pedA, inhibited the biological activity of natural pediocin PA-1. The recombinant pediocin appeared as a mixture of three main fractions (7–8, 11, 20kDa vs. 4.6kDa for natural pediocin PA-1). The recombinant pediocin was also less hydrophobic and behaved differently when subjected to isoelectric focusing. Strong evidence indicated that some “collagen-like” material was tightly associated, most probably via covalent binding, to the recombinant pediocin. The “collagen-like” material was most probably responsible for the lack of biological activity of the recombinant pediocin and for the differences observed regarding some of the physico-chemical properties. Both the recombinant pediocin and natural pediocin were sensitive to collagenase, suggesting that pediocin PA-1 may possess a somewhat “collagen-like” nature. Interestingly, recombinant pediocin preparations showed the ability to assemble into fibrils. [Copyright &y& Elsevier]

Heterologous production of pediocin PA-1 in nisin and non-nisin-producing Lactococcus lactis strains, which had been previously selected because of their technological properties for cheese making, was investigated. Plasmid pFI2160, which contains a hybrid gene (L-pedA) encoding the fusion between the lactococcin A leader and propediocin PA-1, and also the genes lcnC and lcnD, that encode the lactococcin A secretion apparatus, was introduced into L. lactis ESI 153 and L. lactis ESI 515 (Nis+). The pediocin production level of their respective transformants, L. lactis CL1 and L. lactis CL2 (Nis+), was approximately 600 and 400 ng mL-1, respectively, which represents a 30% and a 20% of the quantity produced by the natural pediocin PA-1 producer Pediococcus acidilactici 347. Transformation of L. lactis ESI 515 with pFI2160 did not affect its ability to produce nisin. Pediocin bioassays showed the stability of pFI2160 in both heterologous hosts under selective and non-selective conditions. [Copyright &y& Elsevier]

A 1 day turbidometric microplate bioassay (TMB) was developed for the rapid, accurate and precise quantification of lactic acid bacteria (LAB) bacteriocins (nisin Z and pediocin PA-1). Parameters such as the concentration of the indicator strains and the incubation time were optimized for each bacteriocin. A high correlation coefficient (r2=0.992±0.004) was obtained for the exponential regression in the nisin Z concentration range of 20–120 ng/ml with 1×107 CFU indicator strain (Pediococcus acidilactici UL5) and an incubation time of 3 h. Using these parameters, the detection limit was estimated at 80 ng/ml (3.2 IU/ml), compared to 300 ng/ml for the agar diffusion assay (ADA). High precision (<7%) and accuracy (10%) were obtained for all nisin Z concentrations tested. Similar results were obtained with pediocin PA-1 with r2=0.993±0.005, a precision (8.2%) and an accuracy lower than 15%. [Copyright &y& Elsevier]

Fourier transform infrared (FTIR) spectroscopy was used to investigate the secondary structure of pediocin PA-1 in different aqueous media in relation to its antimicrobial activity. The experiments were performed at pD (pH meter corrected for deuterium isotope effect) 6, 7, and 8 and during a heating–cooling cycle of 20–80 °C. At pD 6, (i.e. pediocin’s most active form), the FTIR results show that pediocin adopts an unordered structure with a small contribution of β-turn. After a heating–cooling cycle, thermally-induced changes in pediocin are reversed and its activity is maintained. Increasing the pD to 7 and 8 leads to a more ordered secondary structure. For these two pD values, an increase in temperature induces an irreversible aggregation of protein as revealed by the amide I′ band. The analysis of the Tyr region provides more insight into the aggregation process. In fact, it appears to be a two-step process, involving first the C (carboxy)-terminus of pediocin and then the N (amino)-terminus. This study reveals two major points: (1) the preservation of pediocin flexibility is essential for maintaining its activity; and (2) the aggregation of its C-terminus is sufficient to induce a loss of activity, suggesting that this region plays an important role in the activity of pediocin. [Copyright &y& Elsevier]

PCR amplification analysis with pediocin PA-1 (pedA1) specific primers, a gene probe covering the pedA1 structural gene, and an immunological probe based on anti-peptide antibodies with predetermined specificity for pedA1, have been evaluated for pedA1 structural gene detection and quantification of bacteriocin production in Pediococcus acidilactici strains of meat origin. PCR products were obtained from colonies and purified plasmid DNA. The gene probe was used in dot-blot and colony-blot hybridization analysis. The production of pedA1 was evaluated by a non-competitive indirect ELISA (NCI-ELISA), a competive indirect ELISA (CI-ELISA), a competitive direct ELISA (CD-ELISA), and by immunoblotting. The approaches taken in the generation and performance of the reported probes could be useful for the rapid detection and identification of other bacteriocin-producing lactic acid bacteria (LAB) from many substrates and reservoirs and for quantification of bacteriocin production in food and environmental isolates and heterologous hosts. [ABSTRACT FROM AUTHOR]

[Display omitted] • Feasibility of associating phages to bacteriocinogenic LAB proved in fermented food. • L. monocytogenes eliminated in fermented sausage model with HHP-LAB-phage system. • Synergistic listericidal effect between hurdles demonstrated in a solid matrix. • In situ biosynthesis of pediocin PA-1 firstly reported in solid matrix. Consumers' quest for healthier, locally produced foods, renders the demand for these products increasingly prominent. The purpose of the present work was to evaluate the impact of a non-thermal multi-hurdle approach, which associated mild high hydrostatic pressure (HHP, 300 MPa), the bacteriophage Listex™ P100, and the pediocin PA-1 producing Pediococcus acidilactici HA 6111-2, as a novel minimal processing towards Listeria monocytogenes eradication in Alheira (a traditional fermented meat sausage from Northern Portugal). The combination of the three hurdles achieved the USDA-FSIS 5 log reduction (in accordance with the standard guidelines for ready-to-eat foods), being the only treatment to elicit the absence of L. monocytogenes immediately following processing (p < 0.05). The pair association of HHP with Listex™ P100 was unable to eliminate L. monocytogenes , whilst in the HHP-pediocin PA-1 producing P. acidilactici treated samples the eradication was delayed when compared to the three hurdles combination. In addition to the listericidal effect of the HHP-phage-lactic acid bacterium treatment, no significant differences (p > 0.05) in the pH values were observed, and the semi-quantification of the in situ biosynthesized pediocin PA-1 was documented for the first time in a fermented meat sausage model. [ABSTRACT FROM AUTHOR]

Bacteriocins are ribosomally synthesized bacterial antimicrobial peptides that have a narrow spectrum of antibacterial activity against species closely related to the producers. Pediocin-like (or class IIa) bacteriocins (PLBs) exhibit antibacterial activity against several Gram-positive bacterial strains by forming pores in the cytoplasmic membrane of target cells with a specific receptor, the mannose phosphotransferase system (man-PTS). In this study, we report the cryo-electron microscopy structures of man-PTS from Listeria monocytogenes alone and its complex with pediocin PA-1, the first and most extensively studied representative PLB, at resolutions of 3.12 and 2.45 Å, respectively. The structures revealed that the binding of pediocin PA-1 opens the Core domain of man-PTS away from its Vmotif domain, creating a pore through the cytoplasmic membranes of target cells. During this process, the N-terminal β-sheet region of pediocin PA-1 can specifically attach to the extracellular surface of the man-PTS Core domain, whereas the C-terminal half penetrates the membrane and cracks the man-PTS like a wedge. Thus, our findings shed light on a design of novel PLBs that can kill the target pathogenic bacteria. IMPORTANCE Listeria monocytogenes is a ubiquitous microorganism responsible for listeriosis, a rare but severe disease in humans, who become infected by ingesting contaminated food products (i.e., dairy, meat, fish, and vegetables): the disease has a fatality rate of 33%. Pediocin PA-1 is an important commercial additive used in food production to inhibit Listeria species. The mannose phosphotransferase system (man-PTS) is responsible for the sensitivity of Listeria monocytogenes to pediocin PA-1. In this study, we report the cryo-EM structures of man-PTS from Listeria monocytogenes alone and its complex with pediocin PA-1 at resolutions of 3.12 and 2.45 Å, respectively. Our results facilitate the understanding of the mode of action of class IIa bacteriocins as an alternative to antibiotics.

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.

Abstract: Pediocin PA-1 was conjugated with keyhole limpet hemocyanin (KLH) and used to immunize rabbits and mice for the production of polyclonal (PAb) and monoclonal (MAb) antibodies. Titers of PAb and MAb of about 4.7 and 2.9 were obtained after three and six immunizations, respectively. An enzyme linked immunosorbent assay (ELISA) was developed for the detection and quantification of pediocin. [Copyright &y& Elsevier]

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). • Synergistic effect between phage P100, pediocin PA-1 and high pressure was demonstrated. • Listeria monocytogenes (104 CFU mL−1) eliminated in milk by the pressure-phage system, being comparable to HTST pasteurization. • The synergistic system (pressure-phage-pediocin) prevented L. monocytogenes regrowth during refrigerated storage. • Listeria monocytogenes regrowth was not prevented when hurdles were applied individually. • Phage particles were stable in milk during storage at 4 °C. [ABSTRACT FROM AUTHOR]