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PubMed2003

Fermentation by Lactobacillus fermentum Ogi E1 of different combinations of carbohydrates occurring naturally in cereals: consequences on growth energetics and alpha-amylase production.

Glucose, fructose, sucrose and starch are naturally present in cereals. Fermentation of different combinations of these carbohydrates by Lactobacillus fermentum Ogi E1, a sourdough heterofermentative lactobacillus, was investigated to determine effects on fermentation kinetics, growth energetics and alpha-amylase production. Irrespective of the substrate combination, the strain was able to simultaneously produce alpha-amylase and consume starch, glucose, fructose and sucrose. In mixtures of starch with either sucrose or fructose or with both fructose and glucose, yields of alpha-amylase from biomass (Y(amy/x)) were similar to those observed for starch. However, for starch and glucose or starch, glucose, fructose and sucrose mixtures, both Y(amy/x) and the specific rate of alpha-amylase production decreased markedly. In fructose- or sucrose-containing mixtures, mannitol was formed stoichiometrically indicating that fructose served as electron acceptor, and acetate was produced at constant yield from biomass (Y(ac/x)) (1 g acetate g biomass(-1)). Acetate production was expected to confer to the strain a competitive advantage during natural fermentation by improving biomass formation and growth through an increase in the ATP gain. Y(ATP) varied depending on the carbohydrate mixture, indicating different effects of substrate mixtures on the efficiency in ATP coupling to biomass formation. Compared to starch fermentation, the highest value of Y(ATP) (29 g biomass mol ATP(-1)) was estimated for the starch/fructose mixture but no increase in mu(max) was observed. The lowest value (16 g biomass mol ATP(-1)) was obtained for the starch, glucose and fructose mixture, whereas for the mixture of all carbohydrates, Y(ATP) was similar to that obtained with starch alone (20 g biomass mol ATP(-1)) and it was intermediary for the starch and sucrose mixture (17 g biomass mol ATP(-1)). It is concluded that competitiveness of the strain cannot be based on expected energy gain in mixed substrate fermentation involving fructose and sucrose with glucose and starch, but rather on its ability to simultaneously use carbohydrates while producing alpha-amylase and to produce acetic acid. Acetic acid production could enhance the strain capacity to inhibit nonacid-tolerant, competitive microflora at the earlier stage of natural fermentation.

Acetic Acid↗
PubMed2003

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.

Amino Acid Sequence↗
PubMed2003

Probiotic and milk technological properties of Lactobacillus brevis.

Two Lactobacillus brevis strains ATCC 8287 and ATCC 14869(T), were evaluated for their applicability as putative probiotics in dairy products. The strains expressed good in vitro adherence to human Caco-2 and Intestine 407 cells and tolerated well low pH, bile acids and pancreatic fluid under in vitro conditions. In antimicrobial activity assays, strain ATCC 8287 showed inhibitory properties toward selected potential harmful microorganisms, particularly against Bacillus cereus. Both L. brevis strains were resistant to vancomycin, which is typical for the genus Lactobacillus. The L. brevis strains were not able to acidify milk to yoghurt but were suitable as supplement strains in yoghurts. This was shown by producing a set of yoghurt products and analysing their rheological and sensory properties during a cold storage period of 28 days. Survival of the strains through human intestine was examined in 1-week feeding trials. Despite its human origin, L. brevis ATCC 14869(T) could not survive through the gastrointestinal (GI) tract, whereas L. brevis ATCC 8287 was detected in the faecal samples taken during and immediately after ingestion of the strain. In conclusion, L. brevis ATCC 8287 is a promising candidate as a probiotic supplement in dairy products.

Animals↗
PubMed2003

Mode of action of lactocin 705, a two-component bacteriocin from Lactobacillus casei CRL705.

Lactocin 705 is a bacteriocin whose activity depends on the complementary action of two peptides (705alpha and 705beta) of 33-amino-acid residues each and is produced by Lactobacillus casei CRL705. Biologically active, synthetic lactocin 705 was used to study the mode of action on sensitive cells of Lactobacillus plantarum CRL691. The addition of 90 nmol l(-1) of lactocin 705 to cells of L. plantarum dissipated both, the membrane potential (DeltaPsi) and the pH gradient (DeltapH). Energized membrane, obtained after the addition of glucose, were more susceptible to lactocin 705 action leading to the immediate release of intracellular K(+) and inorganic phosphate. When the role of various ions on sensitive cells were analyzed, only Ca(2+) ion exhibited a protective effect against lactocin 705. These data suggest that the presence of a proton motive force (PMF) promotes the interaction of the bacteriocin with the cytoplasmic membrane of energized cells, leading to pore formation which allows for the efflux of ions, thereby ensuring efficient killing of target bacteria.

Bacteriocins↗
PubMed1997

Production, purification and characterization of reutericin 6, a bacteriocin with lytic activity produced by Lactobacillus reuteri LA6.

A bacteriocin (Reutericin 6) produced by Lactobacillus reuteri LA6, was purified by hydrophobic chromatography from the modified MRS broth (D'-MRS) with 6180-fold increase in specific activity with 14% recovery. The molecular weight of reutericin 6 was determined to be 2.7 kDa by SDS-PAGE and ESI-MS. By amino acid analysis, reutericin 6 comprised of 67% hydrophobic and polar neutral amino acids. Lanthionine was not detected. The lytic activity against Lactobacillus delbrueckii subsp. bulgaricus JCM 1002T and N1A1 B6 was detected by the decrease of both turbidity and the number of viable cells, and by leaking of beta-galactosidase.

Amino Acids↗
PubMed1997

Ropy slime-producing Lactobacillus sake strains possess a strong competitive ability against a commercial biopreservative.

Aseptically handled Frankfurters were treated with a commercial Lactobacillus alimentarius biopreservative and inoculated with different cell concentrations of four ropy slime-producing Lactobacillus sake strains. The packages were vacuum sealed and kept at 6 degrees C for 28 days, after which the production of ropy slime was evaluated. The inoculation test was controlled by sealing the different control packages containing either aseptically manufactured sausages without any bacterial inoculation, packages containing biopreservative only or packages inoculated only with the four different ropy slime-producing strains. Authenticity of the biopreservative strain after the cold storage period was ascertained by performing EcoRI restriction endonuclease analysis of 30 randomly selected isolates originating from the biopreservative control packages. All patterns were identical to the pattern of the original L. alimentarius biopreservative strain. The biopreservative was found to be ineffective against the four ropy slime-producing L. sake strains. The strongest slime producers inoculated with approximately 1 colony forming units (CFU)/cm2 could compete efficiently with the L. alimentarius inoculated at a level of 10(7) CFU/cm2 on sausage surfaces. This commercial biopreservative failed to occupy the vital niche of the four ropy slime-producing L. sake strains leading to spoilage in almost all packages.

Food Packaging↗
PubMed1998

Molecular characterization of the Lactobacillus community in traditional processing of Mozzarella cheese.

The natural Lactobacillus community involved in traditional Mozzarella cheese production has been investigated. The bacterial associations of whey, curd before stretching and Mozzarella were analyzed using randomly amplified polymorphic DNA (RAPD) to follow growth kinetics, and 16S rDNA sequencing to identify the taxonomical position of isolated strains. Analysis of RAPD fingerprints revealed that the Lactobacillus community was composed of 13 different biotypes and the sequence analysis of 16S rDNA demonstrated that the isolated strains belong to L. plantarum, L. fermentum, L. helveticus and L. casei subsp. casei. In addition, two strains of Weissella hellenica were isolated on selective media for lactobacilli. The four L. casei subsp. casei strains and W. hellenica contained sequences related to the prtP gene coding for proteinase, and the highest proteolytic activity in milk was found in one strain of L. casei subsp.casei.

Carbohydrate Metabolism↗
PubMed1999

Duplication of the beta-galactosidase gene in some Lactobacillus plantarum strains.

Curing of a plasmid that encoded a beta-galactosidase gene (beta-gal) from the Lactobacillus plantarum strain of dairy origin LL441 was not accompanied by complete loss of the lactose utilization phenotype. DNA-DNA hybridization, using an internal fragment of the beta-gal gene as a probe, revealed a second determinant located on the chromosome of the cured derivatives. The chromosomal copy was present in all of a series of beta-Gal+ L. plantarum and Lactobacillus pentosus strains from different origins. In addition, four other L. plantarum strains harboured plasmid encoded beta-gal genes as well. Since both sequences cross-hybridized and present a similar genetic organization, it is postulated that the plasmid copy was generated through gene duplication and, probably, selected by growth of the strains in lactose rich environments.

DNA, Bacterial↗
PubMed1999

Detection and characterization of a novel antibacterial substance produced by Lactobacillus plantarum ST 31 isolated from sourdough.

Lactobacillus plantarum ST31 isolated from sourdough produced an antimicrobial substance inhibiting other strains of the genera Lactobacillus, Leuconostoc, Pediococcus, Streptococcus, Bacillus and some foodborne pathogens including Staphylococcus aureus. This antimicrobial substance was inactivated by proteolytic enzymes. Consequently, it was characterized as a bacteriocin and was designated plantaricin ST31. This bacteriocin was stable in the pH range 3-8 and it was not affected by amylolytic enzymes. Production of plantaricin was pH and temperature dependent, and maximum yields were obtained in MRS broth cultures maintained at pH 6 and incubated at 30 degrees C in the exponential phase to the early stationary growth phase of the producer organism. This bacteriocin was purified by using consecutive ammonium sulfate and reversed-phase chromatography. It is a peptide of 20 amino acid residues with a mass of 2755+/-0.3 Da, as determined by electrospray mass spectrometry. The sequence of Plantaricin ST31 showed no similarity to those of other bacteriocins. Plantaricin ST31 production appeared to be chromosomally encoded.

Adsorption↗
PubMed2002

Recombinant Lactobacillus johnsonii as a mucosal vaccine delivery vehicle.

Lactobacilli are considered to be safe organisms making them attractive as vehicles for oral vaccination. We report that Lactobacillus johnsonii (Lj) partially survived simulated gastric conditions in vitro, suggesting that it could be used as an oral vaccine delivery vehicle. In order to test this approach, we used the cell wall anchored proteinase PrtB, isolated from Lactobacillus delbrueckii subsp. bulgaricus as a model antigen. Using a new vector system, we demonstrated expression of both proteinase PrtB alone and a mimotope peptide derived from tetanus toxin integrated in the sequence of proteinase PrtB (TTmim-PrtB fusion protein) on the surface of Lj. Oral immunisation of mice with recombinant Lj, expressing the TTmim-PrtB fusion protein induced a systemic IgG response against Lj and recombinantly expressed proteinase PrtB but no antibody response against the tetanus toxin mimotope suggesting that the mimotope was not sufficiently immunogenic to induce an immune response. Interestingly, a proteinase PrtB specific fecal IgA response was also induced, indicating that the proteinase PrtB fusion protein expressed as a cell surface protein on Lj can induce both systemic and local mucosal immune responses.

Administration, Oral↗
PubMed1982

Effects of lactobacillus, antacids and antibiotics on the levels of nitrite in the gastro-intestinal tracts of rats fed sodium nitrate.

No nitrite was detected in the tissues or contents of the gastro-intestinal tracts of normal rats but after 2 wk on a diet containing 0 . 5% sodium nitrate the levels of nitrite in the stomach, small intestine and large intestine contents were 0 . 83%, 1 . 64-2 . 07 and 0 . 83 micrograms/g of contents respectively. Concurrent administration of 2% Lactobacillus preparation and 0 . 5% sodium nitrate in the diet for 2 wk further increased the nitrite levels in the intestines and slightly increased the level in the stomach. The elevation of nitrite levels induced by sodium nitrate administration was potentiated considerably by combined treatment with sodium bicarbonate and hetacillin producing nitrite levels of 3 . 16, 2 . 93-5 . 18 and 1 . 96-2 . 34 micrograms/g of the contents of the stomach, small intestine and large intestine respectively. Like hetacillin, minomycin and thiamphenicol also potentiated the nitrite production whereas amikacin (another antibiotic) strongly inhibited the formation of nitrite in the stomach. The different effects of the antibiotics may be due to their selective activities on the various microbes. The results indicate that the levels of nitrite in the gastro-intestinal tract are regulated by the level of nitrate intake, the population of microflora and the gastric pH. The safety of combined medication with antacids, antibiotics and Lactobacillus preparations in man deserves further investigation.

Animals↗
PubMed1998

Safety and tolerance of Lactobacillus reuteri supplementation to a population infected with the human immunodeficiency virus.

Probiotic supplementation may provide health benefits, especially for individuals with an underlying disease state that makes them more susceptible to infections. The purpose of this experiment was to evaluate the safety and tolerance of Lactobacillus reuteri ingestion by subjects infected with the human immunodeficiency virus (HIV). Thirty-nine subjects consumed a freeze-dried preparation of L. reuteri or a placebo for 21 days in a double-masked, parallel design experiment. Serum chemistry, haematology, immune profile, urinalysis, physical examination, gastrointestinal tolerance and faecal microbiota data were collected. No clinically significant changes were noted in any of the safety parameters measured. Overall, tolerance was good in both groups. Consumption of L. reuteri tended to increase faecal levels of L. reuteri on days 7, 14 and 21 of treatment feeding (P < 0.06, P < 0.11 and P = 0.05, respectively). However, faecal levels of L. reuteri and total Lactobacillus species were lower than levels previously observed in healthy male adults. Overall, this study documents that L. reuteri may be fed to HIV-positive individuals at 1 x 10(10) colony forming units/day without any clinically significant safety or tolerance problems.

Adult↗
PubMed1999

Inhibitory effects of freeze-dried milk fermented by selected Lactobacillus bulgaricus strains on carcinogenesis induced by 1,2-dimethylhydrazine in rats and by diethylnitrosamine in hamsters.

Fermented milk products might be used for cancer chemoprevention due to their putative anticarcinogenic and antitumor activities. The diet was supplemented with freeze-dried milk fermented by Lactobacillus bulgaricus strain LBB.B 144 (product FFM.B 144) added throughout the experiment at doses of 1.3 g and 2.5 g per rat, 5 times a week starting 3 weeks before the first carcinogen injection. This treatment significantly inhibited, by 26.2-28.6% and by 34.2%, the total intestinal carcinogenesis induced by 1,2-dimethylhydrazine (DMH, 21 mg/kg, s.c., once per week for 20 weeks) in male and female BD6 rats, respectively. FFM.B144 decreased the tumor incidence and multiplicity in large bowel, caecum, and duodenum. Protective effects were better expressed in female animals, with exception of that observed in duodenum. Supplementation of diet with freeze-dried milk fermented by Lactobacillus bulgaricus strain LBB.B5 (product FFM.B5) inhibited DMH-induced carcinogenesis only in the large bowel, but had no significant protective effect when all intestinal tumors were taken into account. However, both freeze-dried products favorably shifted the differentiation of large bowel tumors by increasing the proportion of benign and highly differentiated malignant tumors and decreasing in parallel the number of poorly differentiated carcinomas without influencing the tumor size. A lower number of cases with visible mesenterial metastasis was also observed in FFM-treated rats. In addition, both FFM.B 144 and FFM.B5 significantly inhibited, by 26-33%, the induction in the same rats of ear-duct tumors. FFM.B144 but not FFM.B5 was also effective in inhibiting the tracheal carcinogenesis induced in Syrian golden hamsters by diethylnitrosamine (DEN, 100 mg/kg, two s.c. injections), the protective effect being better expressed in female animals. The anticarcinogenic potential of some fermented milk products might be exploited in chemoprevention of cancer in humans.

1,2-Dimethylhydrazine↗
PubMed2003

Purification and characterisation of mannitol dehydrogenase from Lactobacillus sanfranciscensis.

Mannitol dehydrogenase (MDH) was purified and characterised from Lactobacillus sanfranciscensis. Two peptide fragments of MDH were N-terminally sequenced for the first time in the genus Lactobacillus. The purified enzyme had an apparent molecular mass of 44 kDa and catalysed both the reduction of fructose to mannitol and the oxidation of mannitol to fructose. The K(m) value for the reduction reaction was 24 mM fructose and that for the oxidation 78 mM mannitol. The optimum temperature was 35 degrees C, the pH optima for the reduction or oxidation were 5.8 and 8, respectively.

Amino Acid Sequence↗
PubMed2003

New integration vector using a cellulase gene as a screening marker for Lactobacillus.

The new integration vector for Lactobacillus, pJC4, was developed using the extracellular endoglucanase A gene (celA) of Clostridium thermocellum as a screening marker. pJC4 was transformed into four Lactobacillus species, Lb. johnsonii, Lb. gasseri, Lb. bulgaricus, and Lb. plantarum. In each species, the pJC4 integrants were easily and accurately detected by the appearance of a clear halo on a cellulase screening plate without any false transformants. Polymerase chain reaction and Southern hybridization indicated that all transformants with clear halos contained pJC4 in their chromosomal DNAs. The celA gene could be a useful screening marker for other lactic acid bacteria.

Cellulase↗
PubMed2003

In vitro conjugal transfer of tetracycline resistance from Lactobacillus isolates to other Gram-positive bacteria.

The ability of 14 Lactobacillus strains, isolated from fermented dry sausages, to transfer tetracycline resistance encoded by tet(M) through conjugation was examined using filter mating experiments. Seven out of 14 tetracycline-resistant Lactobacillus isolates were able to transfer in vitro this resistance to Enterococcus faecalis at frequencies ranging from 10(-4) to 10(-6) transconjugants per recipient. Two of these strains could also transfer their resistance to Lactococcus lactis subsp. lactis, whereas no conjugal transfer to a Staphylococcus aureus recipient was found. These data suggest that meat lactobacilli might be reservoir organisms for acquired resistance genes that can be spread to other lactic acid bacteria. In order to assess the risk of this potential hazard, the magnitude of transfer along the food chain merits further research.

Animals↗
PubMed2003

csp-like genes of Lactobacillus delbrueckii ssp. bulgaricus and their response to cold shock.

The two csp-like genes from the lactic acid bacterium Lactobacillus delbrueckii ssp. bulgaricus were characterized and designated cspA and cspB. The gene cspA has been identified using a polymerase chain reaction (PCR)-based approach with degenerated primers and further characterized using an inverse PCR strategy. cspA encodes a protein of 65 amino acid residues which displays between 81 and 77% identity with proteins CspL and CspP of Lactobacillus plantarum. cspB has been identified as a cspA ortholog using the partial sequence of the L. bulgaricus ATCC11842. cspB encodes a protein of 69 amino acids which has 42% identity with CspA. Northern blot analyses showed that cspA is transcribed as a single gene and that its transcription increased after a temperature downshift from 42 to 25 degrees C. In contrast, cspB is part of an operon transcribed at constant level irrespective of the temperature. These results indicate that cspA encodes the only Csp-like protein of L. bulgaricus induced by a downshift of temperature.

Amino Acid Sequence↗
PubMed2003

Construction of vectors for inducible gene expression in Lactobacillus sakei and L plantarum.

We have constructed vectors for inducible expression of genes in Lactobacillus sakei and Lactobacillus plantarum. The key elements of these vectors are a regulatable promoter involved in the production of the bacteriocins sakacin A and sakacin P and the genes encoding the cognate histidine protein kinase and response regulator that are necessary to activate this promoter upon induction by a peptide pheromone. The vectors are built up of cassettes that permit easy exchange of all parts through restriction enzyme digestion and ligation. Using beta-glucuronidase as a reporter enzyme, variants of these vectors were compared with each other, and with a corresponding system based on genes involved in the production of nisin. Several of the new vectors permitted tightly controlled and efficient expression of beta-glucuronidase in both L. sakei and L. plantarum.

Gene Expression Regulation, Bacterial↗