Monoclonal antibody-based enzyme immunoassay for pediocins of Pediococcus acidilactici.

Monoclonal antibody (MAb) R2-AR against pediocin RS2 was developed. Mice were immunized for 12 weeks with pediocin RS2 conjugated to a polyacrylamide gel. Two hybridoma fusions yielded an MAb that in Western blots (immunoblots) reacted only with pediocins RS2 and AcH (3 kDa) from Pediococcus acidilactici RS2 and H, respectively, and did not react with any other bacteriocin, including sakacin A from Lactobacillus sake Lb 706, leuconocin LCM1 from Leuconostoc carnosum LM1, nisin from Lactococcus lactis ATCC 11454, and pediocin A from Pediococcus pentosaceus FBB61. Each of the bacteriocin bands on sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels was confirmed to be biologically active by a gel overlay test performed with sensitive indicator organisms. In dot immunoblot assays, the MAb could detect a minimum of 32,000 arbitrary units of pediocin RS2 or AcH per ml. In colony immunoblot assays, the MAb was used successfully to differentiate bac+ and bac- variants of P. acidilactici RS2 strains.     

Isolation and partial characterization of a bacteriocin produced by Pediococcus pentosaceus K23-2 isolated from Kimchi

Aims:  Screening and partial characterization of a bacteriocin produced by Pediococcus pentosaceus K23-2 isolated from Kimchi, a traditional Korean fermented vegetable.
Methods and Results:  A total of 1000 lactic acid bacteria were isolated from various Kimchi samples and screened for the production of bacteriocin. Pediocin K23-2, a bacteriocin produced by the Pediococcus pentosaceus K23-2 strain, showed strong inhibitory activity against Listeria monocytogenes . The bacteriocin activity remained unchanged after 15 min of heat treatment at 121°C or exposure to organic solvents; however, it diminished after treatment with proteolytic enzymes. The bacteriocin was maximally produced at 37°C, when the pH of the culture broth was maintained at 5·0 during the fermentation, although the optimum pH for growth was 7·0. The molecular weight of the bacteriocin was about 5 kDa according to a tricine SDS-PAGE analysis.
Conclusions:  Pediococcus pentosaceus K23-2 isolated from Kimchi produces a bacteriocin, which shares similar characteristics to the Class IIa bacteriocins. The bacteriocin is heat stable and shows wide antimicrobial activity against Gram-positive bacteria, especially L. monocytogenes .
Significance and Impact of the Study:  Pediocin K23-2 and pediocin K23-2-producing P. pentosaceus K23-2 could potentially be used in the food and feed industries as natural biopreservatives, and for probiotic application to humans or livestock.     

Production and stability of pediocin N5p in grape juice medium

The production and stability of pediocin N5p from Pediococcus pentosaceus , isolated from wine, were examined in grape juice medium. Maximum growth and higher titre (4000 U ml^-1) were observed at a initial pH of 7·5 and 30°C. The activity of the inhibitory substance was stable between pH values from 2·0 to 5·0 at 4° and 30°C. At pH 10·0 it was completely inactivated. When submitted to 30 min at 80°, 100° and 115°C, maximal stability was observed at pH 2·0. Ethanol up to 10% did not affect pediocin activity at acid pH, nor did 40–80 mg 1^-1 SO₂, independently or combined with different ethanol concentrations, affect inhibitory activity.     

A Note on the Total Viable Counts and Selective Enumeration of Anaerobic Bacteria in the Caecal Content, Soft and Hard Faeces of Rabbit

Total viable counts, proteolytic, ureolytic, cellulolytic and NH⁺₄ utilizing anaerobic bacteria in caecal contents, soft and hard faeces of rabbits were studied. No significant differences between the bacterial counts from the caecum and soft faeces were observed ( P < 0·05) but there were significant differences between the bacterial counts of the soft and hard faeces ( P < 0·01).     

Plasmid-Associated Bacteriocin Production and Sucrose Fermentation in Pediococcus acidilactici

Production of bacteriocin activity designated pediocin PA-1 was associated with the presence of a 6.2-megadalton plasmid in Pediococcus acidilactici PAC1.0. The bacteriocin exhibited activity against P. acidilactici, P. pentosaceus, Lactobacillus plantarum, L. casei, L. bifermentans, and Leuconostoc mesenteroides subsp. dextranicum. Partial characterization of pediocin PA-1 is described. The molecular weight of pediocin PA-1 was ca. 16,500. Additionally, strain PAC1.0 was found to contain a 23-megadalton plasmid associated with sucrose-fermenting ability.     

Pediocin PD-1, a bactericidal antimicrobial peptide from Pediococcus damnosus NCFB 1832

Pediocin PD-1, produced by Pediococcus damnosus NCFB 1832, is inhibitory to several food spoilage bacteria and food-borne pathogens. However, pediocin PD-1 is not active against other Pediococcus spp. and differs in this respect to other pediocins produced by Pediococcus acidilactici and Pediococcus pentosaceus. Production of pediocin PD-1 starts during early growth and reaches a plateau at the end of exponential growth. Pediocin PD-1 was partially purified and its size was determined by tricine-SDS-PAGE as ≈ 3·5 kDa. The isoelectric point (pI) of pediocin PD-1 is ≈ 3·5, as determined with the Rotofor electrofocusing cell (BioRad). Pediocin PD-1 is heat-resistant (10 min at 121°C) and remains active after 30 min of incubation at pH 2–10. Pediocin PD-1 is resistant to treatment with pepsin, papain, α-chemotrypsin and trypsin, but not Proteinase K. Pediocin PD-1 is bactericidal against sensitive cells of Oenococcus oeni (previously Leuconostoc oenos ).     

Association of a 13.6-Megadalton Plasmid in Pediococcus pentosaceus with Bacteriocin Activity

Pediococcus pentosaceus FBB61 and L7230, isolated from cucumber fermentations, produced a bacteriocin, designated pediocin A, which had identical activity spectra against species of Pediococcus, Clostridium, Staphylococcus, and Streptococcus. Both strains possessed a 13.6-megadalton plasmid (pMD136). Plasmid curing experiments suggested that both bacteriocin immunity and production determinants were encoded by pMD136. Use of pediocin-producing strains in food fermentations is discussed.     

Characterization of nisin-resistant variants of Pediococcus acidilactici UL5, a producer of pediocin

Four spontaneous nisin-resistant variants R1, R1M, T5 and T7 of Pediococcus acidilactici UL5, a pediocin producer, were isolated on a nisin gradient. The minimum inhibitory concentration of Ped. acidilactici UL5 using an agar diffusion test was 0·25 ng, while that of R1, R1M, T5 and T7 were 10, 25 and more than 32·5 μg for the two latter, respectively. Nisin resistance phenotype was stable after 60 generations in MRS nisin-free liquid media and 10 consecutive transfers in solid medium. Pediococcus acidilactici UL5 and its nisin-resistant variants exhibited the same total DNA profile, level of production of pediocin and adsorption of nisin on the cell surface. The specific growth rate (μ) decreased with the level of resistance of the culture. Nisin-resistant variants and parental strain UL5 showed differences in sensitivity to antibiotics in which some act on the cell surfaces. Moreover, the fatty acid composition of the cell wall in nisin-resistant variants, compared with UL5, was different, particularly in C16:1 and C18:1. Results suggest that a change in structure/composition of nisin-resistant variants might be associated with nisin resistance.     

Protein synthesis, nitrogen excretion and long-term growth of juvenile Pleuronectes flesus

This study aimed to measure protein synthesis using a stable isotope method, investigate protein-nitrogen flux in a flatfish Pleuronectes flesus , and use the data to test the hypothesis that individual differences in growth efficiency were related to individual differences in protein-nitrogen flux mediated through differences in protein synthesis and degradation. Three measurements of protein-nitrogen flux via consumption, protein synthesis and nitrogenous excretion were made for individual flounder during a 212-day period and fractional rates of protein-nitrogen flux were scaled for a 50–g flounder to provide mean values for protein consumption (2·11 ± 0·21% day⁻¹), protein synthesis (2·08±0·23% day⁻¹), protein growth (0·71±0·06% day⁻¹) and protein degradation (1·37±0·24% day⁻¹). Mean rates of nitrogenous excretion were 0·142 mg N g⁻¹ day⁻¹ and 0·047 mg N g⁻¹ day⁻¹ for ammonia and urea, respectively. Individual flounder had different protein growth efficiencies and this was correlated negatively and significantly with mean rates of protein synthesis ( r - 0·70; P <0·05) and degradation ( r - 0·67; P < 0·05) and correlated positively and significantly with the efficiency of retaining synthesized protein ( r +0·63, P <0·05). This supported the proposed hypothesis that flounder which grow more efficiently achieve this through adopting a low protein turnover strategy.     

Genetic differentiation among populations of the shortfinned eel Anguilla australis from East Australia and New Zealand

Variability at seven microsatellite loci was used to survey the genetic population structure of the shortfinned eel Anguilla australis . Samples were collected from six estuaries along the east coast of Australia and from three estuaries around New Zealand. Hierarchical analysis of molecular variance of the five loci with good fit to Hardy–Weinberg genotypic proportions detected highly significant differences among samples ( F _ST= 0·016, P < 0·001). The fixation index between countries ( F _CT= 0·012, P < 0·001) was more than double the index among samples within countries ( F _SC= 0·005, P < 0·05). An unweighted pair-group method with arithmetic mean (UPGMA) tree also supported the separation of Australian and New Zealand populations, as did assignment tests, which correctly assigned 80 and 84% of the individuals to Australia and New Zealand, respectively. Isolation-by-distance appeared among samples overall ( r = 0·807, P < 0·001), but not among samples within countries ( r = 0·027, P > 0·05 in Australia; r = 0·762, P > 0·05 in New Zealand). These findings indicate that populations of A. australis in East Australia and in New Zealand may be reproductively isolated from one another. Genetic differentiation among populations of A. australis was two- to 10-fold higher than that among populations of other temperate eels in the North Atlantic Ocean, suggesting that two group of A. australis may reflect sub-species. Anguilla australis in the two countries have different genetic structures and thus require separate management. Genetic isolation between Australian and New Zealand populations indicates that juveniles recruit independently into these two regions from geographically or temporally isolated spawning areas.     

Isolation, purification and partial characterization of plantaricin 423, a bacteriocin produced by Lactobacillus plantarum

Lactobacillus plantarum 423, isolated from sorghum beer, produces a bacteriocin (plantaricin 423) which is inhibitory to several food spoilage bacteria and food-borne pathogens, including Bacillus cereus , Clostridium sporogenes , Enterococcus faecalis , Listeria spp. and Staphylococcus spp. Plantaricin 423 is resistant to treatment at 80 °C, but loses 50% of its activity after 60 min at 100 °C and 75% of its activity after autoclaving (121 °C, 15 min). Plantaricin 423 remains active after incubation at pH 1–10 and is inactivated when treated with pepsin, papain, α-chymotrypsin, trypsin and Proteinase K. Plantaricin 423 was partially purified and its size estimated at 3·5 kDa, as determined by tricine-SDS-PAGE. The mechanism of activity of plantaricin 423 is weakly bactericidal, as determined against Oenococcus oeni (previously Leuconostoc oenos ). High DNA homology was obtained between the plasmid DNA of strain 423 and the pediocin PA-1 operon of Pediococcus acidilactici PAC 1·0, suggesting that plantaricin 423 is plasmid-encoded and related to the pediocin gene cluster.     

Effect of fasting on resting metabolic rate and postprandial metabolic response in Silurus meridionalis

Resting metabolic rate in southern catfish of 2 and 5 day fasting groups were significantly higher than that of the 15 day fasting group ( P  < 0·05). After feeding, peak metabolic rate of specific dynamic action (SDA) of the 15 day fasting group was significantly lower than that of the 2 and 5 day fasting groups ( P  < 0·05). The duration of the SDA of the 15 day fasting group was significantly longer than that of the 2 day fasting group ( P  < 0·05) and the SDA coefficient of the 15 day fasting group was significantly lower than that of the 2 day fasting group ( P  < 0·05).     

Homologues and bioengineered derivatives of LtnJ vary in ability to form D-alanine in the lantibiotic lacticin 3147

Ltnα and Ltnβ are individual components of the two-peptide lantibiotic lacticin 3147 and are unusual in that, although ribosomally synthesized, they contain d-amino acids. These result from the dehydration of l-serine to dehydroalanine by LtnM and subsequent stereospecific hydrogenation to d-alanine by LtnJ. Homologues of LtnJ are rare but have been identified in silico in Staphylococcus aureus C55 (SacJ), Pediococcus pentosaceus FBB61 (PenN), and Nostoc punctiforme PCC73102 (NpnJ, previously called NpunJ [P. D. Cotter et al., Proc. Natl. Acad. Sci. U. S. A. 102:18584-18589, 2005]). Here, the ability of these enzymes to catalyze d-alanine formation in the lacticin 3147 system was assessed through heterologous enzyme production in a ΔltnJ mutant. PenN successfully incorporated d-alanines in both peptides, and SacJ modified Ltnα only, while NpnJ was unable to modify either peptide. Site-directed mutagenesis was also employed to identify residues of key importance in LtnJ. The most surprising outcome from these investigations was the generation of peptides by specific LtnJ mutants which exhibited less bioactivity than those generated by the ΔltnJ strain. We have established that the reduced activity of these peptides is due to the inability of the associated LtnJ enzymes to generate d-alanine residues in a stereospecific manner, resulting in the presence of both d- and l-alanines at the relevant locations in the lacticin 3147 peptides.     

Production of a nisin Z/pediocin mixture by pH-controlled mixed-strain batch cultures in supplemented whey permeate

The conditions for high production of nisin Z and pediocin during pH-controlled, mixed-strain batch cultures in a supplemented whey permeate medium with Lactococcus lactis subsp. lactis biovar. diacetylactis UL719, a nisin Z producer strain, and variant T5 of Pediococcus acidilactici UL5, a pediocin-producing strain resistant to high concentrations of nisin, were studied. Mixed cultures were performed at 37 °C and pH 5·5 by first inoculating with variant T5 and then with L. diacetylactis UL719 after 8 h incubation, and were compared with single-strain batch cultures. High productions of both nisin Z and pediocin were obtained after 18 or 16 h incubation during mixed cultures, with titres of 3000 and 730 AU ml⁻¹, or 1060 and 1360 AU ml⁻¹, respectively, corresponding to approximately 75 and 55, or 25 and 100 mg l⁻¹ of pure nisin Z and pediocin, respectively. In pure cultures, nisin Z and pediocin productions were higher than in mixed cultures, and maximum activities were obtained after 10 h incubation, with approximately 10 000 AU ml⁻¹ (250 mg l⁻¹ pure nisin Z) and 2500 AU ml⁻¹ (190 mg l⁻¹ pure pediocin). During mixed cultures, significant pediocin degradation was observed in the culture supernatant fluid after 16 h incubation, together with a sharp drop in Ped. acidilactici UL5 cell viability. In the test conditions, the feasibility of producing a nisin/pediocin mixture by mixed-strain fermentation was demonstrated. The bacteriocin mixture produced in a supplemented whey permeate medium could be used as a natural food-grade biopreservative with a broad activity spectrum.     

Isolation by distance and selection effects on genetic structure of sardines Sardina pilchardus Walbaum

Allozyme data were used to analyse the genetic structure of Sardina pilchardus populations. Fifty samples from 15 locations between the North Sea and Mauritania, including samples from the Azores, Madeira and the Mediterranean Sea, were surveyed. A weak but significant structure was found between all samples ( F _ST= 0.057, P < 0·001). This structure results from a change in the most common allele of SOD* between the North African and the Azores populations separated by the greatest distance. This locus seemed to be under selective pressure according to the test of neutrality, and the variations in allele frequencies may be explained due to isolation by distance (IBD) of coastal populations (from Mauritania to the North Sea) ( r = 0·86, P < 0·001). When SOD* was removed from the analyses, IBD was not observed in coastal populations ( r = 0·236, P > 0·05) but was observed over the whole range ( r = 0·321, P = 0·05). The genetic structure of S. pilchardus is driven by both IBD and selective processes.     

The effect of temperature, pH and medium in a surface adhesion immunofluorescent technique for detection of Listeria monocytogenes

A rapid surface adhesion-based immunofluorescence technique was used to detect Listeria monocytogenes from inoculated culture systems. The effect of culture type (pure, mixed and meat), pH (7·00, 6·40, 4·76 and 3·13), acids (citric and HCl) and temperature (25°, 30° and 37°C) on the adhesion of Listeria to the polycarbonate membrane used in this technique was determined. It was found that pH had a significant effect ( P < 0·05) with higher numbers of Listeria adhering at low pH values (4·76). Culture type was also important with significantly higher numbers of Listeria ( P < 0·05) adhering to membranes immersed in meat cultures than in pure or mixed cultures. This effect was seen at 30°C but not at 25° or 37°C. The total viable count (TVC) on the membrane was unaffected by pH but temperature had an influence with optimum adhesion occurring at 25°C. The reasons for observed differences and their implications for the surface adhesion immunofluorescent rapid method are discussed.     

Effects of a synbiotic product on blood antioxidative activity in subjects colonized with Helicobacter pylori

Aim:  To evaluate the impact of the consumption of a synbiotic product on the antioxidative activity markers of blood in asymptomatic H. pylori -colonized persons.
Methods and Results:  Fifty-three healthy adult volunteers without gastric symptoms participated in a randomized, double-blind placebo-controlled study. The crossover consumption of the enterocoated capsules containing antioxidative Lactobacillus fermentum ME-3, Lact. paracasei 8700:2 and Bifidobacterium longum 46 with Raftilose P95 lasted for 3 weeks and did not change the H. pylori colonization. In H. pylori -positive subjects the sera values of total antioxidative status (TAS) were significantly lower compared to H. pylori -negative subjects (0·97 vs 1·05 mmol l⁻¹, P  = 0·008). After the consumption of the synbiotic, TAS values (0·97 vs 1·03 mmol l⁻¹, P  = 0·004) increased, while the ratio between oxidized and reduced glutathione (0·035 vs 0·030, P  = 0·016) decreased in H. pylori -positive subjects.
Conclusions:  The consumption of a synbiotic containing an antioxidative probiotic strain improved the reduced systemic antioxidative activity in H. pylori -colonized asymptomatic subjects.
Significance and Impact of the Study:  A synbiotic product containing an antioxidative probiotic strain may be useful in the reduction of systemic oxidative stress in H. pylori infection.     

Survival of Listeria monocytogenes in sea water and effect of exposure on thermal resistance

Survival, recoverability and sublethal injury of two strains of Listeria monocytogenes , Scott A and an environmental strain KM, on exposure to sea water at 12·8 or 20·8 °C was determined using in situ diffusion chambers. Plate counts were used to assess recoverability and injury while 5-cyano-2,3-ditolyl tetrazolium chloride (CTC) reduction was used to determine respiratory activity. T₉₀ values (times for 10-fold decreases in numbers of recoverable cells) on non-selective medium (trypticase soya agar with 0·6% yeast extract) at 12·8 and 20·8 °C were 61·7 and 69·2 h for L. monocytogenes Scott A, and 103·0 and 67·0 h for L. monocytogenes KM, respectively. On selective medium (Oxford agar), T₉₀ values at 12·8 and 20·8 °C were 60·6 and 56·9 h for L. monocytogenes Scott A, and 83·0 and 65·9 h for L. monocytogenes KM, respectively. With Scott A, the percentage of sublethally injured cells at 12·8 and 20·8 °C was 1·7 and 17·7%, respectively, while for KM the values were 19·0 and 1·6%, respectively. The fraction of cells reducing CTC but which were not recoverable on plating progressively increased on exposure to sea water. Listeria monocytogenes KM challenged at 58 °C showed an apparent increase in heat resistance after exposure to sea water at 20·8 °C for 7 d ( D ₅₈= 2·64 min) compared with before exposure ( D ₅₈= 1·24). This increase in thermal resistance was not apparent at temperatures greater than 63 °C, and analysis of the best-fit regression lines fitted to the thermal data obtained from the two cell populations indicated that their thermal resistance was not significantly different ( P > 0·05) over the temperature range tested (58–62 °C).     

Reduction of caecal Listeria monocytogenes in Leghorn chicks following treatment with a competitive exclusion culture (PREEMPT™)

Day-of-hatch Leghorn chicks were treated by oral gavage with PREEMPT^™, a continuous-flow competitive exclusion culture containing broiler caecal bacteria, followed by an oral challenge with Listeria monocytogenes , to determine the effects of PREEMPT^™ on L. monocytogenes caecal colonization. Increased ( P < 0·001) concentrations of caecal propionic acid in control chicks compared with PREEMPT^™-treated chicks at 3 days of age were indicative of the establishment of the PREEMPT^™ bacteria. Caeca from control chicks at 7 days after the oral challenge with L. monocytogenes contained mean 3·4 ± 1·4 log cfu g⁻¹ of caecal content, while caeca from PREEMPT^™-treated chicks contained no detectable Listeria. Enrichment for L. monocytogenes resulted in 100 % of caeca from control chicks testing culture-positive for L. monocytogenes , while none of the caeca from PREEMPT^™-treated chicks were culture-positive. The results indicated that prophylactic treatment of newly hatched chicks with PREEMPT^™ significantly reduced caecal colonization by L. monocytogenes.     

Analysis of the factors related with mate choice and reproductive success in male three-spined sticklebacks

Territorial three-spined sticklebacks moved 5·3 times as far as non-territorial males in 2 min ( P < 0·001) and spent 11·1 times longer in aggression in the pools ( P < 0·001). Territorial males had slightly higher condition factors than non-territorial males. Condition factor was correlated positively with the gonad mass ( P < 0·006), carotenoid concentration ( P < 0·006) and the activity of CS in the axial muscle ( P < 0·05) and lactate dehydrogenase (LDH) in pectoral muscle ( P < 0·003). The male traits best correlated positively with female mate choice were courtship effort ( P < 0·001), coloration ( P < 0·003) and initial condition ( P < 0·025). Courtship behaviour was related to intestine mass ( P < 0·018), axial ( P < 0·028) and pectoral muscle citrate synthase (CS) activity ( P < 0·047); coloration was related to gonad mass ( P < 0·037). These muscle enzymes may be involved in ATP generation for sustained activities or in recuperation between bouts of burst activity. Females that choose to mate with assiduously courting males which bear higher CS levels may be choosing individuals that show honestly their good condition and capacity to accomplish reproductive tasks.