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.     

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.     

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.     

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.     

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.     

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.     

Expression and purification of a fusion-typed pediocin PA-1 in Escherichia coli and recovery of biologically active pediocin PA-1

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-beta-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.     

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.     

PCR-mediated site-directed mutagenesis on PedB gene and HaeIII restriction as a rapid tool for discrimination among pediocin AcH/PA-1 producer strains

Nucleotide sequences of amplified pedB genes from Pediococcus parvulus andLactobacillus plantarum pediocin AcH/PA-1 producer strains were performed. The obtained data were aligned with the published Pediococcus acidilactici pedB sequence, showing a single base substitution in the third codon position of a putative serine. A PCR-mediated site directed mutagenesis on pedB gene, followed by Hae III restriction analysis was then carried out with the aim to rapidly discriminate among pediocin AcH/PA-1 producer strains of different species.     

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。     

Pediocin PA-1, a wide-spectrum bacteriocin from lactic acid bacteria

Pediocin PA-1 is a broad-spectrum lactic acid bacteria bacteriocin that shows a particularly strong activity against Listeria monocytogenes, a foodborne pathogen of special concern among the food industries. This antimicrobial peptide is the most extensively studied class Ila (or pediocin family) bacteriocin, and it has been sufficiently well characterized to be used as a food biopreservative. This review focuses on the progress that have been made in the elucidation of its structure, mode of action, and biosynthesis, and includes an overview of its applications in food systems. The aspects that need further research are also addressed. In the future, protein engineering, genetic engineering and/or chemical synthesis may lead to the development of new antimicrobial peptides with improved properties, based on some features of the pediocin PA-1 molecule.     

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.     

Biodiversity of Bacteriocin-Producing Lactic Acid Bacteria from Mexican Regional Cheeses and their Contribution to Milk Fermentation

The aim of this work was to examine the biodiversity of bacteriocin-producing lactic acid bacteria from homemade cheeses produced in Veracruz (México) and assess their contribution as adjunct cultures in dairy products. Ninety-three presumptive bacteriocinogenic strains were detected by direct antagonism assays and 29 of them were active against Enterococcus faecalis NRRL-B537, Listeria innocua 062 AST, or Listeria monocytogenes ATCC19115 by the well diffusion test using cell-free supernatants, adjusted to pH 6.0 to exclude inhibition by organic acids. Positive isolates were identified by partial sequencing of the 16s rDNA as Pediococcus acidilactici (four isolates), Enterococcus faecium (17 isolates), Lactobacillus plantarum (six isolates) and Lactobacillus fermentum (two isolates). RAPD-PCR discriminated seven groups with a 50% similarity and revealed the presence of the same isolates. The coding genes for the synthesis of plantaricin EF, plantaricin JK, plantaricin N, plantaricin NC8 and the inducing peptide plantaricin A were detected by PCR in L. plantarum. Similarly, enterocin P and pediocin PA-1 genes were amplified from Enterococcus and Pediococcus genomic DNA, respectively. Overall, co-culturing of bacteriocin producing Lactobacillus and Pediococcus strains with the dairy starter Lactococcus lactis IPLA947 did not interfere with milk acidification. Lactose consumption, acidification rate and production of lactic acid were unchanged. Nonetheless, higher levels of acetic acid, ethanol and succinic acid were detected depending on the strain. Our results demonstrate the diversity of bacteriocinogenic species in homemade Mexican cheeses which may be used as adjunct cultures to enhancing safety of this well-appreciated cheese while providing a richer range of metabolites.     

Biochemical,antimicrobial and molecular characterization of a noncytotoxic bacteriocin produced by Lactobacillus plantarum ST71KS

Lactobacillus (Lb.) plantarum ST71KS was isolated from homemade goat feta cheese and identified using biochemical and molecular biology techniques. As shown by Tricine-SDS-PAGE, this lactic acid bacterium produces a bacteriocin (ST71KS) with an estimated molecular weight of 5.0 kDa. Bacteriocin ST71KS was not affected by the presence of α-amylase, catalase and remained stable in a wide range of pH and after treatment with Triton X-100, Triton X-114, Tween 20, Tween 80, NaCl, SDS, urea and EDTA. This bacteriocin also remained active after being heated at 100 °C for 2 h and even after 20 min at 121 °C; however, it was inactivated by proteolitic enzymes. Production of bacteriocin ST71KS reached 6400 AU/mL during stationary growth phase of Lb. plantarum cultivated in MRS at 30 °C and 37 °C. Bacteriocin ST71KS displayed a bactericidal effect against Listeria monocytogenes strains 603 and 607 and did not adsorb to the producer cells. Lb. plantarum ST71KS harbors two bacteriocin genes with homology to plantaricin S and pediocin PA-1. These characteristics indicate that bacteriocin ST71KS is a class IIa bacteriocin. The peptide presented no toxic effect when tested in vitro with kidney Vero cells, indicating safe technological application to control L. monocytogenes in foods.     

The effects on vegetative cells and spores of three bacteriocins from lactic acid bacteria

The sensitivities of vegetative cells of strains ofListeria, Clostridium, Staphylococcus, Lactococcus, Lactobacillus, MicrococcusandPediococcus, and of spores ofClostridiumandBacillusto three broad spectrum bacteriocins (nisin A, nisin Z and pediocin) from lactic acid bacteria were determined by a critical dilution micro-assay. The minimal inhibitory concentrations (MIC) of partially purified bacteriocins, prepared by a pH-dependent adsorption/desorption process, were determined and expressed in arbitrary units ml⁻¹and in μ g ml⁻¹of pure bacteriocin. The MICs of bacteriocins varied considerably between species and even between strains of the same species, as clearly shown for nine strains ofListeria monocytogenes. When bacteriocin activity was expressed in μ g ml⁻¹, pediocin was more effective againstListeria monocytogenesthan nisin A or nisin Z. The latter bacteriocins, in concentrations between 23 and 69 μ g ml⁻¹, prevented outgrowth ofClostridiumandBacillusspores for at least 10 days. Although pediocin at 17 μ g ml⁻¹prevented outgrowth ofB. stearothermophilusandC. butyricumspores for up to 7 days, it apparently activated the germination ofB. subtilisspores.     

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.     

提高乳酸菌细菌素合成量方法的研究进展

细菌素是某些细菌通过核糖体合成机制产生的蛋白质或多肽,能够抑制与其亲源关系相同或相近的微生物,某些细菌素在食品加工和发酵过程中能抑制致病菌和腐败菌。乳酸菌被认为是一般公认安全,其细菌素具有安全性高、稳定性好、抑菌谱广等优点,作为一种新型食品防腐剂备受关注,但商品化的乳酸菌细菌素十分有限,仅限于Nisin和Pediocin PA-1等少数几种,合成量低是细菌素在食品中应用受限的主要原因之一。从不同原料中筛选高产菌株、发酵培养基和发酵条件优化、诱变育种、原生质体融合、基因工程方法、群体感应系统调控六个方面,论述了增加乳酸菌细菌素合成量的方法,以期为实现乳酸菌细菌素的工业化生产提供一定的借鉴。     

A rapid turbidometric microplate bioassay for accurate quantification of lactic acid bacteria bacteriocins

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%.     

Antimicrobial activity of pediocin-producing Lactococcus lactis on Listeria monocytogenes,Staphylococcus aureus and Escherichia coli O157:H7 in cheese

The antimicrobial activity of two pediocin-producing transformants obtained from wild strains of Lactococcus lactis on the survival of Listeria monocytogenes, Staphylococcus aureus and Escherichia coli O157:H7 during cheese ripening was investigated. Cheeses were manufactured from milk inoculated with the three pathogens, each at approximately 6 log cfu mL⁻¹. Pediococcus acidilactici 347 (Ped⁺), Lc. lactis ESI 153, Lc. lactis ESI 515 (Nis⁺) and their respective pediocin-producing transformants Lc. lactis CL1 (Ped⁺) and Lc. lactis CL2 (Nis⁺, Ped⁺) were added at 1% as adjuncts to the starter culture. After 30 d, L. monocytogenes, S. aureus and E. coli O157:H7 counts were 5.30, 5.16 and 4.14 log cfu g⁻¹ in control cheese made without adjunct culture. On day 30, pediocin-producing derivatives Lc. lactis CL1 and Lc. lactis CL2 lowered L. monocytogenes counts by 2.97 and 1.64 log units, S. aureus by 0.98 and 0.40 log units, and E. coli O157:H7 by 0.84 and 1.69 log units with respect to control cheese. All cheeses made with nisin-producing LAB exhibited bacteriocin activity throughout ripening. Pediocin activity was only detected throughout the whole ripening period in cheese with Lc. lactis CL1. Because of the antimicrobial activity of pediocin PA-1, its production in situ by strains of LAB growing efficiently in milk would extend the application of this bacteriocin in cheese manufacture.