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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.

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 (10 4  CFU mL -1 or 10 7  CFU mL -1 ) in milk. For inoculum levels of 10 4  CFU mL -1 , HHP combined with phage P100 eliminated L. monocytogenes immediately after pressurization. When L. monocytogenes was inoculated at levels of 10 7  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)., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

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., (Copyright © 2016. Published by Elsevier B.V.)

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]

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]

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]