Lactobacillus salivarius: bacteriocin and probiotic activity.

Lactic acid bacteria (LAB) antimicrobial peptides typically exhibit antibacterial activity against food-borne pathogens, as well as spoilage bacteria. Therefore, they have attracted the greatest attention as tools for food biopreservation. In some countries LAB are already extensively used as probiotics in food processing and preservation. LAB derived bacteriocins have been utilized as oral, topical antibiotics or disinfectants. Lactobacillus salivarius is a promising probiotic candidate commonly isolated from human, porcine, and avian gastrointestinal tracts (GIT), many of which are producers of unmodified bacteriocins of sub-classes IIa, IIb and IId. It is a well-characterized bacteriocin producer and probiotic organism. Bacteriocins may facilitate the introduction of a producer into an established niche, directly inhibit the invasion of competing strains or pathogens, or modulate the composition of the microbiota and influence the host immune system. This review gives an up-to-date overview of all L. salivarius strains, isolated from different origins, known as bacteriocin producing and/or potential probiotic.

Optimization of pressure-induced germination of Bacillus sporothermodurans spores in water and milk.

Bacillus sporothermodurans produces highly resistant endospores that can survive ultra-high-temperature treatment in milk. The induction of endospore germination before a heat treatment could be an efficient method to inactivate these bacteria and ensure milk sterility. In this work, the rate of spore germination of B. sporothermodurans LTIS27 was measured in distilled water after high-pressure treatments with varying pressure (50-600 MPa), treatment temperature (20-50 °C), pressure-holding time (5-30 min) and post-pressurization incubation time (30-120 min) at 37 °C or 4 °C. The results showed that pressure-induced germination was maximal (62%) after a treatment at 200 MPa and 20 °C and increased with pressure-holding time and post-pressurization incubation time. Treatment temperature had no significant effect on germination. A central composite experimental design with three factors (pressure, pressure-holding time, and post-pressurization incubation time) using response surface methodology was used to optimize the germination rate in distilled water and in skim milk. No factor interaction was observed. Germination was induced at lower pressure and was faster in milk than in distilled water, but complete germination was not reached. The optimum germination obtained with experimental data was 5.0 log cfu/mL in distilled water and 5.2 log cfu/mL in milk from 5.7 log cfu/mL of spores initially present in the suspension. This study shows the potential of using high hydrostatic pressure to induce the germination of B. sporothermodurans spores in milk before a heat treatment.

In vitro evaluation of the probiotic potential of Lactobacillus salivarius SMXD51.

Lactobacillus salivarius SMXD51 was previously isolated from the cecum of a Tunisian poultry and found to produce a bacteriocin-like substance highly active against the foodborne pathogen Campylobacter jejuni. The aim of this study was to examine some probiotic properties of the strain: acid and bile tolerance, capacity of adhesion, stimulation of immune defences (IL-6, IL-8, IL-10 and ß-defensin 2), and modulation of the barrier integrity. The results showed that L. salivarius SMXD51 can tolerate gastrointestinal conditions (acid and bile), adhere to intestinal cells and stimulate the immune system. The bacterium strengthened the intestinal barrier functions through the increase of the transepithelial electrical resistance (TEER) and reinforcement of the F-actin cytoskeleton. One hour pretreatment with L. salivarius SMXD51 protected against Pseudomonas aeruginosa PAO1-induced decrease of TEER and damage of the F-actin cytoskeleton. Our results highlight that L. salivarius SMXD51 fulfils the principle requirements of an efficient probiotic and may be seen as a reliable candidate for further validation studies in chicken.

Growth and reducing capacity of Listeria monocytogenes under different initial redox potential.

AIMS: To determine the reducing capacity of Listeria monocytogenes and to highlight the effect of redox potential on its growth parameters. METHODS AND RESULTS: The reducing capacity of L. monocytogenes was monitored in Brain Heart Infusion Broth media at different initial redox potential (Eh) and pH at 37 degrees C. The effect of Eh obtained by gas flushing (air, N(2) and N(2)-H(2)) or by adding potassium ferricyanide and dithiotreitol in concentration from 1 to 10 mmol l(-1)on L. monocytogenes growth parameters at pH 6.0, 7.0 and 8.0 was investigated. A total change of 539 mV (+ or - 44 mV) from an initial redox value of +330 + or - 8 mV to a more negative potential in redox curves was observed resulting from L. monocytogenes growth at pH 7.0 at 37 degrees C. A significant influence of pH and redox potential on L. monocytogenes lag phase of growth was shown (P < 0.05). CONCLUSIONS: Listeria monocytogenes exhibited longer lag phase in reducing conditions and at pH 6.0. The method used to modify the redox potential was shown to have no effect on growth parameters at pH 7.0. SIGNIFICANCE AND IMPACT OF THE STUDY: The provided information on the extending lag time and the possible delayed growth of this major pathogen in reducing conditions might be useful for its control in foods.

Psychrotrophic lactic acid bacteria used to improve the safety and quality of vacuum-packaged cooked and peeled tropical shrimp and cold-smoked salmon.

Previously isolated lactic acid bacteria (LAB) from seafood products have been investigated for their capacity to increase the sensory shelf life of vacuum-packaged shrimp and cold-smoked salmon and to inhibit the growth of three pathogenic bacteria. Two different manufactured batches of cooked, peeled, and vacuum-packaged shrimp were inoculated with seven LAB strains separately at an initial level of 5 log CFU g-t, and the spoilage was estimated by sensory analysis after 7 and 28 days of storage at 8 degrees C. Two Leuconostoc gelidum strains greatly extended the shelf life of both batches, two Lactococcus piscium strains had a moderate effect, two bacteria were spoilers (Lactobacillus fuchuensis and Carnobacterium alterfunditum), and the last one (another Leuconostoc gelidum strain) showed highly variable results depending on the batch considered. The four strains showing the best results (two Leuconostoc gelidum and two Lactococcus piscium strains) were selected for the same experiment in cold-smoked salmon. In this product, Lactococcus piscium strains showed better inhibiting capacities, improving the sensory quality significantly at 14 and 28 days of storage. Finally, the inhibiting capacities of two strains (one Leuconostoc gelidum strain and one Lactococcus piscium strain) were tested against three pathogenic bacteria (Vibrio cholerae, Listeria monocytogenes, and Staphylococcus aureus) by challenge tests in shrimp. LAB and pathogenic bacteria were coinoculated in vacuum-packaged shrimp and enumerated during 5 weeks. Lactococcus piscium strain EU2241 was able to reduce significantly the number of Listeria monocytogenes and S. aureus organisms in the product by 2 log throughout the study for Listeria monocytogenes and up to 4 weeks for S. aureus.

Divercin V41 from gene characterization to food applications: 1998-2008, a decade of solved and unsolved questions.

The emergence of an increasing number of antibiotic resistant human clinical bacteria has been a great cause of concern for the last decades. As an example, Staphylococcus aureus isolates in the hospital environment are becoming more and more resistant to antibiotics including vancomycin which is considered as a last line of defence in treatment of Staphylococcus aureus-resistant methicillin. On the other hand, food safety is threatened by development of pathogenic bacteria including Listeria monocytogenes, Campylobacter jejuni, Salmonella enteritidis, Escherichia coli O157:H7 and Staphylococcus aureus. The use of antimicrobial peptides such as glycopeptides, semi-synthetic peptides, bacteriocins including lantibiotics offers a hope to face these clinical and food microbiology concerns. Clinical approval of new chemotherapeutic agents requires a long period of time. Research on bacteriocins has demonstrated potential use to fight against undesired foodborne pathogens but the use industrial use of bacteriocins is limited. To date only lantibiotic nisin and in class IIa bacteriocin Pediocin PA-1 are legally used as food preservative in many countries. The present minireview is focused on divercin V41 (DvnV41), a class IIa bacteriocin naturally produced by Carnobacterium divergens V41. The last decade has been the witness of intensive investigations carried out on this cationic peptide tempting to answer multiple questions covering basic and applied aspects. DvnV41 has shown a wide spectrum of activity either alone or in combination with nisin and/or polymixins (synergistic effect). This outcome indicates that Cb. divergens V41 could potentially be used for safe and efficient prevention of L. monocytogenes growth in cold smoked salmon.

Selection and evaluation of seafood-borne psychrotrophic lactic acid bacteria as inhibitors of pathogenic and spoilage bacteria.

In this study, inhibitory psychrotrophic lactic acid bacteria were isolated and investigated for future use in biopreservation of seafood products. Screening of 5575 colonies isolated from various seafood products resulted in the selection of 132 colonies presenting inhibitory properties. Among them, 52 isolates had characteristics of LAB and showed growth at 15 degrees C but not at 30 degrees C. The inhibition spectrum of these 52 isolates against 14 target strains (Gram-positive and -negative) showed inhibition of typical seafood spoiling and pathogenic bacteria and enabled the formation of seven interesting clusters. Sequencing of the 16S rRNA gene of a representative isolate from each cluster identified three Leuconostoc gelidum, two Lactococcus piscium, one Lactobacillus fuchuensis and one Carnobacterium alterfunditum. Theses strains did not produce histamine nor tyramine, and showed no particular antibiotic resistance profile. Growth rate as a function of temperature was tested for one L. piscium and one L. gelidum isolate and confirmed their psychrotrophic behavior. One out of seven isolates showed bacteriocin-like activity. The inhibition mechanisms of the other isolates are still unknown but may be due to competition for substrate. Absence of a bacteriocin-like component could be a positive point to gain rapid authorization for food application in France. This collection of LAB is now ready for testing on products.

Rapid identification of Lactobacillus nantensis, Lactobacillus spicheri and Lactobacillus hammesii species using species-specific primers.

Based on the 16S-23S ribosomal DNA (rDNA) intergenic spacer region (ISR), an identification tool for rapid differentiation of Lactobacillus nantensis, Lactobacillus spicheri and Lactobacillus hammesii, species isolated recently from French sourdough was developed. The DNA fragments containing ISRs were amplified with primers pairs 16S/p2 and 23S/p7. Clone libraries of the PCR-amplified rDNA with these primers were constructed using a pCR2.1 TA cloning kit and sequenced. The DNA sequences obtained were analyzed and species-specific primers were designed from these sequences. Two PCR amplicons, which were designated small ISR (S-ISR) and large ISR (L-ISR), were obtained for all Lactobacillus species studied. The L-ISR sequence reveale2d the presence of two tRNA genes, tRNAAla and tRNAIle. Species-specific primers designed allowed rapid identification of these species. The specificity of these primers was positively demonstrated as no response was obtained for more than 200 other species tested.

Modelling the effect of the redox potential and pH of heating media on Listeria monocytogenes heat resistance.

AIMS: Study the effect of redox potential and pH of the heating media on Listeria monocytogenes heat resistance and model its action at fixed temperature. METHODS AND RESULTS: The heat resistance of Listeria monocytogenes at 58 degrees C was studied in Brain Heart Infusion broth as a function of pH (from 5.0 to 7.0) and redox potential (E(h7)). The media redox was adjusted with nitrogen gas, potassium ferricyanide and dithiothreitol. A Weibull model was used to fit survival curves. The heat resistance parameter (delta(58 degrees C)) was estimated from each inactivation curve. A major effect of pH was observed. Bigelow model was used to describe the effect of redox potential on the apparent L. monocytogenes heat resistance. The highest delta(58 degrees C) values have been obtained at pH 7.0 and oxidizing conditions. CONCLUSIONS: The developed model indicates that the E(h7) has a significant effect and varied depending on the pH of the heating media. The z(redox) values, calculated from delta(58 degrees C) allowed quantifying the influence of heating media redox potential on L. monocytogenes thermal inactivation. SIGNIFICANCE AND IMPACT OF THE STUDY: The obtained model shows the action of redox potential on L. monocytogenes thermal destruction and might be useful to take into account in food thermal processes.

A one-step reaction for the rapid identification of Lactobacillus mindensis, Lactobacillus panis, Lactobacillus paralimentarius, Lactobacillus pontis and Lactobacillus frumenti using oligonucleotide primers designed from the 16S-23S rRNA intergenic sequences.

AIMS: Species-specific primers targeting the 16S-23S ribosomal DNA (rDNA) intergenic spacer region (ISR) were designed to rapidly discriminate between Lactobacillus mindensis, Lactobacillus panis, Lactobacillus paralimentarius, Lactobacillus pontis and Lactobacillus frumenti species recently isolated from French sourdough. METHODS AND RESULTS: The 16S-23S ISRs were amplified using primers 16S/p2 and 23S/p7, which anneal to positions 1388-1406 of the 16S rRNA gene and to positions 207-189 of the 23S rRNA gene respectively, Escherichia coli numbering (GenBank accession number V00331). Clone libraries of the resulting amplicons were constructed using a pCR2.1 TA cloning kit and sequenced. Species-specific primers were designed based on the sequences obtained and were used to amplify the 16S-23S ISR in the Lactobacillus species considered. For all of them, two PCR amplicons, designated as small ISR (S-ISR) and large ISR (L-ISR), were obtained. The L-ISR is composed of the corresponding S-ISR, interrupted by a sequence containing tRNA(Ile) and tRNA(Ala) genes. Based on these sequences, species-specific primers were designed and proved to identify accurately the species considered among 30 reference Lactobacillus species tested. CONCLUSIONS: Designed species-specific primers enable a rapid and accurate identification of L. mindensis, L. paralimentarius, L. panis, L. pontis and L. frumenti species among other lactobacilli. SIGNIFICANCE AND IMPACT OF THE STUDY: The proposed method provides a powerful and convenient means of rapidly identifying some sourdough lactobacilli, which could be of help in large starter culture surveys.

Response of Listeria monocytogenes to liquid smoke.

AIMS: To investigate the effect of liquid smoke on growth, survival, proteomic pattern and haemolytic potential of Listeria monocytogenes. METHODS AND RESULTS: Growth and survival curves were recorded in brain-heart infusion broth supplemented with three concentrations of liquid smoke. L. monocytogenes growth was inhibited in the presence of 15 microg ml(-1) phenol while a rapid decrease in cell viability occurred in the presence of 30 microg ml(-1) phenol. The proteome of L. monocytogenes cytosoluble proteins was slightly modified after 2-h incubation with 30 microg ml(-1) phenol but no protein already characterized in response to other known stresses was induced, except the protease ClpP. Liquid smoke inhibited the haemolytic potential without affecting hly gene expression, showing a potential inhibition of protein activity or stability. CONCLUSIONS: The presence of liquid smoke in a rich medium strongly affected growth and survival of L. monocytogenes. Brief smoke stress affected the metabolic pathways and inhibited the haemolytic activity of L. monocytogenes. SIGNIFICANCE AND IMPACT OF STUDY: This study is a first step in the investigation of the influence of a smoked product on L. monocytogenes strains.

Identification of lactic acid bacteria from poultry feces: evidence on anti-campylobacter and anti-listeria activities.

Forty-five strains of lactic acid bacteria were isolated from poultry feces sampled from an industrial farm located in the Nantes area (France), and most of them belong to the Streptococcus or Lactobacillus genus according to the results obtained by Galerie API and molecular methods. The most representative lactic acid bacteria strains were screened for antibacterial compound production against 2 indicator organisms (Listeria innocua F and Campylobacter jejuni 11168) by means of the agar diffusion test. Strain S37, identified as Enterococcus faecalis, exhibited a clear anti-listerial activity and a slight anti-Campylobacter activity, whereas strain S42, identified as Lactobacillus reuteri, exhibited only anti-Campylobacter activity. Regarding the results of proteolytic, heat, and neutralizing treatments of supernatants from the aforementioned strains, we can conclude that antagonism observed is attributed to antimicrobial peptide or bacteriocin in the case of strain S37, whereas it is ascribable to a nonbacteriocin, likely a reuterin, in the case of strain S42.

Identification of a new molecular target of class IIa bacteriocins in Listeria monocytogenes EGDe.

It was shown recently (Calvez et al. 2007) that glpQ and pde genes are involved in intermediate resistance of Enterococcus faecalis JH2-2 to DvnV41, a class IIa bacteriocin produced by Carnobacterium divergens V41. The listerial orthologs of lpQ and pde genes might be lmo0052 and lmo1292 genes, respectively. Here, the role of lmo0052 and lmo1292 genes in resistance of Listeria monocytogenes EGDe to DvnV41 and MesY105 was investigated. L. monocytogenes EGDe was inactivated in lmo0052 and/or lmo1292 by homologous recombination. Listerial mutant strain EGDSC02 (inactivated in the putative glpQ gene), was slightly resistant to DvnV41. The listerial mutant strain EGDSC01 (inactivated in the putative pde gene) remained, as the wild-type strain, sensitive to DvnV41, but was affected in growth parameters.

Improvement of enterocin P purification process.

Purification and heterologous expression of enterocin P (EntP), a sec-dependent bacteriocin produced by Enterococcus faecium, in Escherichia coli is described. PCR-amplified product of the enterocin P structural gene entP was cloned into plasmid pET-32b under the control of the inducible T7lac promoter. The neo-synthesized EntP was genetically modified by an addition of 3 extra amino acids, leading to recombinant EntRP. Active EntRP was recovered from the cytoplasmic soluble fraction of E. coli harboring appropriate recombinant plasmid, characterized by ELISA and Western-blot analysis and purified by immunoaffinity chromatography. The use of E. coli as heterologous host and pET-32b as expressing vector offers promising tools for heterologous production of class IIa bacteriocin.

Laser heating of thermoluminescent plates: application to intraoperative radiotherapy.

A new system of radiation dose mapping based on laser heating of thermoluminescent dosimetric plates (TLDP) has been developed. Application of this technique to intraoperative radiotherapy (IORT) has been investigated. Preliminary results show that TLDP are a possible alternative to radiological films and ionization chambers since they provide some advantages such as 2D dose measurements with digital storage, large dynamic dose range, and easy processing.

Effect of chelatants on gellan gel rheological properties and setting temperature for immobilization of living bifidobacteria.

The effect of various concentrations of sequestrants (sodium citrate, sodium metaphosphate, and EDTA) was studied on gellan gel (1.5-2.5% (w/v)) setting temperature and rheological properties. Addition of EDTA between 0 and 0.8% (w/v) led to a progressive decrease of setting temperature. Citrate and metaphosphate decreased this parameter when added up to 0.4 or 0.6%, depending on gellan gum concentration, eventually resulting in the absence of gel formation at room temperature for the 1.5% gellan solution containing 0.4% citrate. This effect was accompanied by a significant decrease of gel strength and stiffness and might be attributed to the binding of the divalent cations required for chain association during gelation by chelatants. With the aim of lowering the gel setting temperature during the cell entrapment process while maintaining high mechanical properties, a gel made of 2.5% gellan gum and 0.2% sodium citrate was used to entrap Bifidobacterium longum ATCC 15707. Ions and pH of the inoculum during the immobilization step influenced the long-term mechanical stability of the gel beads during continuous fermentation in a stirred tank reactor. High stability as well as high biocatalyst activity was obtained when a washed cell suspension was used as the inoculum. Gellan gel produced by dissolving gellan gum in a sodium citrate solution may be a promising entrapment matrix for temperature-sensitive cells such as mesophilic lactic acid bacteria and eukaryotic cells.

Isolation and partial characterization of an antibacterial substance produced by Enterococcus faecium.

A strain of Enterococcus faecium isolated from Bulgarian yellow cheese "kashkaval" produced a bacteriocin-like substance named enterococcin A 2000. The antibacterial substance had a low molar mass (< 2 kDa), was relatively stable toward heat but was sensitive to selected proteolytic enzymes. It was active against Gram-positive bacteria including enterococci, such as Listeria, Bacillus and Streptococcus, and also against Gram-negative E. coli. Production of enterococcin A 2000 has a maximum near the end of the exponential phase of producer growth. The peptide was purified by ammonium sulfate precipitation, butanol extraction, followed by cation-exchange chromatography and reversed-phase chromatography. A partial sequence of purified enterococcin A 2000 indicated that this substance does not belong to the class IIa of bacteriocins presenting the consensus anti-Listeria motif YGNGV.

On the luminescence properties of CaSO4:Ce.

The luminescent properties of cerium doped calcium sulphate are studied: fluorescence and excitation spectra, optical absorption and thermoluminescence (TSL). It is known that, in rare earth doped CaSO4, only cerium induces a strong 400 degrees C TSL peak. In CaSO4:0.2%Ce samples synthesised under oxidising conditions, the recovery step of Ce3+ fluorescence is correlated with the 400 degrees C TSL peak readout, as mentioned by Nair er al. Our results indicate that an oxidation of Ce3+ ion does occur under X-irradiation (Ce3+ --> Ce4+), followed by a complete return to the trivalent state after thermal annealing at about 500 degrees C; our results confirm the hypothesis of Nair et al that Ce3+ ions are oxidised by ionising irradiation. So, a pure redox reaction seems the most probable for the 400 degrees C TSL peak of CaSO4:Ce. Moreover, the use of the 400 degrees C TSL peak for high temperature dosimetry applications is discussed.

Effect of recombinant divercin RV41, structural variants and the activators of potassium channels on Listeria monocytogenes EGDe.

The effect of recombinant divercin RV41 (DvnRV41) and its structural variants on the K-channel formation was determined. The growth of Listeria monocytogenes EGDe (sensitive phenotype) and its isogenic strain (resistant phenotype) was assessed in the presence of DvnRV41 combined or not with pinacidil, NS1619, cromakalim (as K-channel activators), iberiotoxin and glipizide (as K-channel blockers). The combined action of DvnRV41 and K activators permitted formation of ATP-dependent pores. The combination of DvnRV41 and ATP-dependent pore activator cromakalim inhibited the growth of sensitive strain. The antilisterial activity of structural variants was less important than that of DvnRV41 but their mode of action remained overall similar.

Identification of lactic acid bacteria involved in the spoilage of pasteurized "foie gras" products.

The spoiling microflora of a re-packaged French "foie gras" product was studied. A total of 54 isolates, originating from two different factories, were identified using phenotypical and molecular methods (partial 16S rDNA sequencing). Weissella viridescens was the main species detected in the products from factory 1 (64% of the isolates). These products had a low lactic acid concentration and were considered as non-spoiled. The microflora of factory 2 was dominated mainly by the genus Lactobacillus (95% of the isolates), and the high lactic acid concentration of these products was linked with a strong spoilage. Among the 30 Lactobacillus strains, three species were predominant: Lactobacillus sakei (nine isolates), Lactobacillus coryniformis (eight isolates) and Lactobacillus paraplantarum (five isolates). Challenge tests were performed to confirm the involvement of the Lactobacillus strains in the spoilage of the product. Sterile "foie gras" samples were inoculated with 14 LAB strains from the collection. The most acidifying strains belonged to the species L. sakei, Lactobacillus plantarum and L. paraplantarum. This confirmed the role of the strains from the Lactobacillus genus as the main spoilers of "foie gras" products and will be useful to design new quality protocols and extend the shelf-life of these products.