Immunity gene pedB enhances production of pediocin PA-1 in naturally resistant Lactococcus lactis strains

Heterologous production of the antilisterial bacteriocin pediocin PA-1 in lactococci is an attractive objective to increase the safety of dairy products. In a previous paper, we developed a system for the heterologous production of the bacteriocin pediocin PA-1 in pediocin-resistant lactococcal hosts through a leader exchange strategy. The system was based on 3 genes, 1 encoding the fusion between the lactococcin A leader and propediocin PA-1, and the other 2 encoding the lactococcin A secretion machinery. In this study, we investigated whether the addition of the pediocin PA-1 immunity gene (pedB) to this system has any effect on pediocin production. Introduction of the plasmid(s) carrying the genes described above into nisinproducing and non-nisinproducing lactococcal hosts led to a significant increase in the production of pediocin compared with the equivalent pedB-devoid systems. In addition, we obtained a nisin-producing strain with the ability to secrete pediocin PA-1 at a level equivalent to that of the parental strain Pediococcus acidilactici 347, which represents a notable improvement over our previous systems.     

Detection of pediocin PA-1-producing pediococci by rapid molecular biology techniques

Five bacteriocinogenic lactic acid bacteria strains (347, X13, Z102, A172 and P20) independently isolated from fermented sausages were identified asPediococcus acidilacticiby carbohydrate fermentation patterns and other biochemical characteristics. This fact, together with their activity againstListeria monocytogenes, suggested that they could be pediocin PA-1 producers. Rapid molecular biology techniques were used to detect the pediocin PA-1 operon in these strains. PCR, dot-blot and Southern hybridization, and DNA sequencing results confirmed that all of them had the genetic determinants for pediocin PA-1 biosynthesis encoded in a 9.4 kb plasmid. The bacteriocin produced byP. acidilactici347 has been purified by a procedure that included ammonium sulphate precipitation and cation exchange, hydrophobic-interaction and reverse-phase chromatography. The amino acid sequence of this bacteriocin was identical to pediocin PA-1, confirming the expression of the pediocin PA-1 genes.     

Antimicrobial activity of pediocin PA-1 against Oenococcus oeni and other wine bacteria

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.     

Megaplasmid encoding novel sugar utilizing phenotypes,pediocin production and immunity in Pediococcus acidilactici C20

A bacteriocinogenic strain of Pediococcus acidilactici C20 isolated from fowl intestine was found to bear a single megaplasmid. Strain C20 exhibited an ability to ferment a wide range of sugars including lactose, maltose and melibiose. Curing experiments employing novobiocin and high temperature indicated that the genetic determinants for pediocin production, immunity function and utilization of several sugars resided on the megaplasmid. Pediocin production with different sugars was tested and culture filtrate activity varied from 2500 AU ml⁻¹to 3500 AU ml⁻¹at 1% of carbon source. Molecular and biochemical techniques were used to characterize the bacteriocin produced by strain C20. It was found to be a pediocin AcH/PA-1 like bacteriocin with high antilisterial activity.     

Enhanced production of pediocin PA-1 in wild nisin- and non-nisin-producing Lactococcus lactis strains of dairy origin

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.     

Characterisation of an antiviral pediocin-like bacteriocin produced by Enterococcus faecium

The bacteriocin-producing strain Enterococcus faecium ST5Ha was isolated from smoked salmon and identified by biomolecular techniques. Ent. faecium ST5Ha produces a pediocin-like bacteriocin with activity against several lactic acid bacteria, Listeria spp. and some other human and food pathogens, and remarkably against HSV-1 virus. Bacteriocin ST5Ha was produced at high levels in MRS broth at 30 °C and 37 °C, reaching a maximum production of 1.0 × 10⁹ AU/ml, checked against Listeria ivanovii ATCC19119 as target strain and surrogate of pathogenic strain Listeria monocytogenes. The molecular weight of bacteriocin ST5Ha was estimated to be 4.5 kDa according to tricine-SDS-PAGE data. Ent. faecium ST5Ha harbors a 1.044 kb chromosomal DNA fragment fitting in size to that of pediocin PA-1/AcH. In addition, the sequencing of bacteriocin ST5Ha gene indicated 99% of DNA homology to pediocin PA-1/AcH. The combined application of low levels (below MIC) of ciprofloxacin and bacteriocin ST5Ha resulted in a synergetic effect in the inhibition of target strain L. ivanovii ATCC19119. Bacteriocin ST5Ha displayed antiviral activity against HSV-1, an important human pathogen, with a selectivity index of 173. To the best of our knowledge, this is the first report on Ent. faecium as a potential producer of pediocin-like bacteriocin with antiviral activity.     

Characterization and heterologous expression of a class IIa bacteriocin,plantaricin 423 from Lactobacillus plantarum 423, in Saccharomyces cerevisiae

Lactobacillus plantarum 423 produces a small heat-stable antimicrobial protein designated plantaricin 423. This protein is bactericidal for many Gram-positive foodborne pathogens and spoilage bacteria, including Listeria spp., Staphylococcus spp., Pediococcus spp., Lactobacillus spp., etc. The DNA sequence of the plantaricin 423-encoding region on plasmid pPLA4 revealed a four open reading frame (ORF) operon structure similar to pediocin PA-1/AcH from Pediococcus acidilactici and coagulin from Bacillus coagulans I(4). The first ORF, plaA, encodes a 56-amino acid prepeptide consisting of a 37-amino acid mature molecule, with a 19-amino acid N-terminal leader peptide. The second ORF, plaB, encodes a putative immunity protein with protein sequence similarities to several bacteriocin immunity proteins. The plaC and plaD genes are virtually identical to pedC and pedD of the pediocin PA-1 operon, as well as coaC and coaD of the coagulin operon. Plantaricin 423 was cloned on a shuttle vector under the control of a yeast promoter and heterologously produced in Saccharomyces cerevisiae.     

Production of pediocin PA-1, and coproduction of nisin A and pediocin PA-1, by wild Lactococcus lactis strains of dairy origin

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.     

In vitro evaluation of the probiotic attributes of two pediococci strains producing pediocin PA-1 with selective potency as compared to nisin

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 (IC₅₀) determination. The IC₅₀ 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 IC₅₀ 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.     

乳酸片球菌素PA-1在大肠杆菌中的表达与纯化

为了实现该细菌素的外源表达,本实验首先利用聚合酶链式反应从乳酸片球菌PAF中扩增出乳酸片球菌素PA-1的结构和免疫基因,然后克隆到表达载体pGEX-6p-1,构建了N端含有GST-His-DDDDK标签的重组质粒pGEX/his-pedAB,然后转化进入大肠杆菌Rosetta（DE3）感受态细胞,经异丙基硫代半乳糖苷诱导,重组乳酸片球菌素PA-1在大肠杆菌胞内成功表达。表达的融合蛋白先经过镍亲合层析柱纯化,然后注入谷胱甘肽S-转移酶亲和色谱柱用肠激酶处理,释放出成熟的乳酸片球菌素PA-1。利用高效液相色谱和质谱技术检测乳酸片球菌素PA-1纯度。以单核细胞增生李斯特氏菌CMCC54004为指示菌,利用琼脂扩散法检验乳酸片球菌素PA-1活性。结果表明,携带GST-His-DDDDK标签的融合蛋白无活性,标签切除后其抑菌活性恢复,且其纯度达90%以上。     

High‐Throughput Isolation of Bacteriocin‐Producing Lactic Acid Bacteria,with Potential Application in the Brewing Industry

Lactic acid bacteria (LAB) were isolated from malted cereals by means of a high‐throughput screening approach and investigated for antimicrobial activity against a range of beer‐spoiling bacteria. Putative bacteriocin‐producing strains were identified by 16S rRNA analysis and the inhibitory compounds were partially characterized. Following determination of the inhibitory spectra of the strains, an unspeciated Lactobacillus sp. UCC128, with inhibitory activity against a range of beer‐spoiling strains was subjected to further characterization. A bacteriocin was purified from this strain and analyzed by mass spectrometry to determine the weight of the protein. The result indicated that the bacteriocin was highly similar to pediocin AcH/PA‐1 from Pediococcus acidilactici. The bacteriocin‐producers identified in this study have the potential to be used in the brewing industry to enhance the microbiological stability of beer in conjunction with hurdles already in place in the brewing process.     

Pediocin PA-1 and a pediocin producing Lactobacillus plantarum strain do not change the HMA rat microbiota

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.     

Characterization of two bacteriocins produced by Pediococcus acidilactici isolated from "Alheira", a fermented sausage traditionally produced in Portugal

Lactic acid bacteria were isolated from "Alheira" sausages that have been sampled from different regions in Portugal. The sausages were produced according to different recipes and with traditional starter cultures. Two isolates (HA-6111-2 and HA-5692-3) from different sausages were identified as strains of Pediococcus acidilactici. Each strain produces a bacteriocin, designated as bacHA-6111-2 and bacHA-5692-3. Both bacteriocins are produced at low levels after 18 h of growth in MRS broth (3200 AU/ml against Enterococcus faecium HKLHS and 1600 AU/ml against Listeria innocua N27). BacHA-6111-2 and bacHA-5692-3 are between 3.5 kDa and 6.5 kDa in size, as determined by tricine-SDS-PAGE. Complete inactivation or significant reduction in antimicrobial activity was observed after treatment of cell-free supernatants with proteinase K, pronase and trypsin. No change in activity was recorded when treated with catalase. Both bacteriocins are sensitive to treatment with Triton X-114 and Triton X-100, but resistant to Tween 20, Tween 80, SDS, Oxbile, NaCl, urea and EDTA. The bacteriocins remained stable after 2 h at pH 6.0. A decrease in antibacterial activity was recorded after 60 min at 100 degrees C. After 60 min at 80 degrees C, 60 degrees C and 25 degrees C the antibacterial activity against L. innocua N27 decreased by 25%. Addition of bacHA-6111-2 and bacHA-5692-3 (1600 AU/ml) to a mid-log (5-h-old) culture of L. innocua N27 inhibited growth for 7 h. Addition of the bacteriocins (3200 AU/ml) to a mid-log (5-h-old) culture of E. faecium HKLHS repressed cell growth. The bacteriocins did not adhere to the surface of the producer cells. Both strains contain a 1044 bp DNA fragment corresponding in size to that recorded for pediocin PA-1. Sequencing of the fragments from both bacteriocins revealed homology to large sections of pedA (188 bp), pedB (338 bp) and pedC (524 bp) of pediocin PA-1 and the bacteriocins are considered similar to pediocin PA-1.     

Anti-listeria activity of poly(lactic acid)/sawdust particle biocomposite film impregnated with pediocin PA-1/AcH and its use in raw sliced pork

A novel poly(lactic acid) (PLA)/sawdust particle (SP) biocomposite film with anti-listeria activity was developed by incorporation of pediocin PA-1/AcH (Ped) using diffusion coating method. Sawdust particle played an important role in embedding pediocin into the hydrophobic PLA film. The anti-listeria activity of the PLA/SP biocomposite film incorporated with Ped (PLA/SP + Ped) was detected, while no activity against the tested pathogen was observed for the control PLA films (without SP and/or Ped). Dry-heat treatment of film before coating with Ped resulted in the highest Ped adsorption (11.63 ± 3.07 μg protein/cm²) and the highest anti-listeria activity. A model study of PLA/SP + Ped as a food-contact antimicrobial packaging on raw sliced pork suggests a potential inhibition of Listeria monocytogenes (99% of total listerial population) on raw sliced pork during the chilled storage. This study supports the feasibility of using PLA/SP + Ped film to reduce the initial load of L. monocytogenes on the surface of raw pork.     

Comparative antimicrobial activity of enterocin L50, pediocin PA-1, nisin A and lactocin S against spoilage and foodborne pathogenic bacteria

The present work compares, under the same stated experimental conditions, the antimicrobial activity of crude and purified enterocin L50, pediocin PA-1, nisin A and lactocin S, produced by lactic acid bacteria (LAB) isolated from Spanish dry-fermented sausages. The bacteriocins were purified to homogeneity by ammonium sulphate precipitation, gel filtration (for lactocin S), and cation-exchange, hydrophobic-interaction, and reverse-phase-chromatography; high yields of pure bacteriocins were obtained. Minimal inhibitory concentration (MIC) of pure enterocin L50, pediocin PA-1, nisin A and lactocin S was determined against a broad spectrum of Gram-positive bacteria, including spoilage and foodborne pathogenic bacteria. The purified bacteriocins showed a broad antimicrobial spectrum similar to that exerted by crude bacteriocins. Enterocin L50 and pediocin PA-1 were very active againstListeria monocytogenes, which was quite resistant to nisin A and lactocin S. Enterocin L50 also displayed antimicrobial activity againstStaphylococcus aureus,Clostridium perfringensandClostridium botulinum. However, these pathogens were weakly inhibited, or not at all, by the other pure bacteriocins.     

Hydrostatic pressure and bacteriocin-triggered cell wall lysis of Leuconostoc mesenteroides

Exposure of cell suspensions of Leuconostoc mesenteroides Ly to either high hydrostatic pressure (HP: 345 MPa at 25 °C for 5 min) or pediocin AcH (2000 or 5000 AU/ml) produced cell viability loss over 6 log cycles and reduction in optical density (OD) of approximately 80% in 1 h at 600_nm. The reduction in OD was associated with cell lysis. Time-lapse studies following treatments revealed that cell death and cell lysis are two separate but dependent events. Both HP and pediocin AcH damaged the cell wall, which then probably triggered the autolysin system of the cells to further degrade the cell walls. When cells were pressurized in the presence of pediocin AcH, cell wall degradation was faster and more extensive.     

Factors influencing production of bacteriocins by lactic acid bacteria

Bacteriocins of lactic acid bacteria have potential for use as food biopreservatives to control spoilage and pathogenic bacteria. For economical use in food, the bacteriocins have to be produced in large amounts and preferably by growing the strains in media containing food grade ingredients. The influence of several factors on production of nisin by Lactococcus lactis, pediocin AcH by Pediococcus acidilactici, leuconocin Lcm1 by Leuconostoc carnosum Lm1 and sakacin A by Lactobacillus sake Lb 706 were studied. Among the several strains studied in each species, only some strains produced higher amounts of the bacteriocins. Production of a bacteriocin in a simple medium can be increased by growing the cells at optimum pH and supplementing with nutrients specific for a species/strain. Conditions that provide high cell density favor high bacteriocin production. Growing cells under an optimum environment for 16 h facilitates high bacteriocin production. For high yield of a bacteriocin, these aspects need to be considered.     

Pediocin PA-1 production during repeated-cycle batch culture of immobilized Pediococcus acidilactici UL5 cells

Pediocin PA-1 production by Pediococcus acidilactici UL5 cells immobilized in kappa-carrageenan/locust bean gum gel beads was studied during repeated-cycle batch (RCB) culture with pH control in Man Rogosa and Sharpe (MRS) broth supplemented with 1% glucose and whey permeate (SWP) medium. The pediocin PA-1 production by free P. acidilactici cells pH-controlled batch culture has reached 2048 and 4096 AU ml(-1) after 11 and 12 h of incubation, with volumetric productivities of 187 and 342 AU ml(-1) h(-1) in SWP and MRS media, respectively. In RCB culture, immobilized cells reached a maximum concentration of 7.3+/-0.2 x 10(10) and 4.3+/-0.9 x 10(10) cfu g(-1) of beads in MRS and SWP media, respectively. The maximum pediocin PA-1 activity obtained during RCB fermentation was 4096 AU ml(-1); it was attained after only 0.75 and 2 h of incubation in MRS and SWP media, respectively. The corresponding volumetric productivities were 5461 and 2048 AU ml(-1) h(-1). Pediocin PA-1 production in the RCB culture was highly stable over 12 fermentation cycles carried out over 3 d in SWP media.     

Survival of Listeria monocytogenes on sliced cooked sausage after treatment with pediocin AcH

A preparation with pediocin AcH bound to its heat-killed producer cells Lactobacillus plantarum WHE 92 (starter culture ALC01, Wisby, Denmark) by adjusting the pH of the preparation to 6.0 was studied for its effects against Listeria monocytogenes ATCC 7644 and (spoilage) lactic acid bacteria on sliced cooked sausage. The pediocin AcH preparation or 0.9% (w/w) NaCl dilution (as a control) were randomly distributed dropwise on the surface of the slices. Treated slices were vacuum-packed and stored at 6 degrees C. Microbiological analysis and determination of pH values were performed after 3, 6, 9, 14 and 21 days of storage. Flavour of the sausages was evaluated after 7 and 11 days of storage. The pediocin preparation had effect (p > 0.05) neither on the growth of lactic acid bacteria, on the pH value nor on the flavour of vacuum-packed sliced sausage during 21 days of storage compared to control. However, during 6 days of storage, the number of L. monocytogenes decreased from the initial level of 2.7 log cfu/g sausage to < 2 log cfu/g, while on the control sausages the number of L. monocytogenes remained at the inoculated level. The numbers of L. monocytogenes remained at those levels to the end of storage period (21 days). However, the treated samples were determined to be Listeria positive, which indicates that the pediocin preparation was not efficient enough to kill all L. monocytogenes.