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PubMed1993

Characterization and sequence analysis of a small cryptic plasmid from Lactobacillus curvatus LTH683 and its use for construction of new Lactobacillus cloning vectors.

Lactobacillus curvatus LTH683, a strain originally isolated from raw sausage, contains the single cryptic plasmid called pLC2. The sequence and genetic organization of the complete 2489-bp plasmid pLC2 was determined and used as the basis for construction of a series of vectors useful in Lactobacillus strains. The major parts of pLC2 nucleotide sequence could be aligned with other plasmids from gram-positive bacteria replicating by a rolling circle mechanism of replication (RCR). Direct evidence for a RCR mechanism was obtained by showing the accumulation of single-stranded plasmid intermediates in the presence of rifampicin. Three protein-coding sequences could be predicted and the corresponding proteins were detected after in vitro transcription/translation of pLC2 plasmid DNA. ORFs 1 and 3 showed minor homologies to plasmids of gram-positive bacteria. The replication protein coded by ORF2 and its corresponding target sequence, the plus origin, were similar to replication regions of other gram-positive bacteria plasmids like pLS1, pWV01, and pE194. Upstream of the ori+ site, in a noncoding region, which was nonessential for replication, strong homology to other Lactobacillus plasmids like pC30i1, pLP1, pLJ1, and pLAB1000 could be detected. A palindromic sequence predicted to be the minus origin of replication was localized there. Small vectors (3213 bp) suitable for cloning in lactobacilli were constructed based on a 1635-bp DNA fragment of pLC2, containing the region necessary for replication, marked with the chloramphenicol resistance gene and a multiple cloning site.

Amino Acid Sequence↗
PubMed1992

Systematics of the Lactobacillus population on rat intestinal mucosa with special reference to Lactobacillus reuteri.

The systematics of the Lactobacillus population of the intestines of 88 different rats was studied; 80 rats had been fed on fermented oat-meal soup (Molin et al. 1992). One-hundred-twenty-two Lactobacillus strains from the intestinal mucosa were phenotypically classified together with twenty-eight reference strains of Lactobacillus and Leuconostoc, using 49 unit characters. Data were examined using Jaccard coefficient, and unweighted pair group algorithm with arithmetic averages. Two major and eleven minor clusters were defined at the 76% SJ-similarity level: Cluster 1 included thirty isolates which could not be identified further, but had resemblance to the type strains of L. jensenii, L. gasseri, L. crispatus, and to some extent to L. acidophilus. Cluster 12 including fifty-four intestinal isolates was identified as L. reuteri; and so was cluster 13 (five isolates). Isolates of the major clusters were found in all parts of the intestines. The genomic homogeneity of the L. reuteri isolates was scrutinized by endonuclease restriction analysis of the chromosomal DNA, and the isolates could be divided into six genomic strains.

Algorithms↗
PubMed2005

Oral administration of two probiotic strains, Lactobacillus gasseri CECT5714 and Lactobacillus coryniformis CECT5711, enhances the intestinal function of healthy adults.

Modifications in gastrointestinal parameters, intestinal colonization and tolerance are some of the main goals claimed for probiotics. However, although healthy people are the common target for these new functional food products, the number of clinical trials analysing the effects of probiotics in gastrointestinal parameters of healthy subjects is very scarce. A randomized, double blind, placebo-controlled human clinical trial involving 30 healthy adults was performed to investigate the effect of a fermented product containing two probiotic strains, Lactobacillus gasseri CECT5714 and Lactobacillus coryniformis CECT5711, on several blood and fecal parameters, most of them related to the host intestinal function. The volunteers were randomly distributed into two groups, one receiving a standard yogurt and the other a similar dairy fermented product in which the Lactobacillus delbreuckii subsp. bulgaricus yogurt strain had been replaced by a combination of the probiotic strains L. gasseri CECT5714 and L. coryniformis CECT5711. The volunteers that received the probiotic strains reported no adverse effects and the strains could be isolated from their feces at a relatively high level. In fact, the concentration of fecal lactic acid bacteria significantly increased in the probiotic group. Additionally, the oral administration of the probiotic strains led to an improvement of parameters such as the production of short chain fatty acids, the fecal moisture and the frequency and volume of the stools. As a result, the volunteers assigned to the probiotic group perceived a clear improvement in their intestinal habits. The study revealed that probiotics may exert a positive effect on healthy adults.

Adult↗
PubMed2004

Intra-specific variation of Lactobacillus plantarum and Lactobacillus pentosus in sensitivity towards various bacteriocins.

Fifty-two strains belonging to the Lactobacillus plantarum species group were identified and typed. They represented 32 clones of Lactobacillus plantarum and 7 clones of Lactobacillus pentosus. Sensitivity of all strains towards bacteriocins of four different producer strains was investigated using a deferred inhibition test (DIT). Substantial intra-specific variation in sensitivity of clones was observed towards bacteriocinogenic lactic acid bacteria producing nisin ( Lactococcus lactis ATCC 11454) or pediocin PA-1 ( Pediococcus acidilactici PAC-1.0), while none of the strains were sensitive towards the two remaining bacteriocin producers. The minimum inhibitory concentration (MIC) of nisin towards selected strains confirmed the DIT results. No correlation between the susceptibility of fourteen selected strains towards nisin and an array of antibiotics was found. The present study indicates that the variation in bacteriocin-sensitivity within target species might be a potential limitation for the application of bacteriocins as biopreservatives.

Anti-Bacterial Agents↗
PubMed2002

Diversity in specificity of the extracellular proteinases in Lactobacillus helveticus and Lactobacillus delbrueckii subsp. bulgaricus.

AIMS: To investigate the diversity in specificity of cell-bound extracellular proteinases in Lactobacillus helveticus and Lactobacillus delbrueckii subsp. bulgaricus. METHODS AND RESULTS: HPLC analysis of whole-cell preparations of 14 Lact. delbrueckii subsp. bulgaricus and eight Lact. helveticus strains incubated with alpha (s1)-casein (f 1-23) detected at least six distinct proteolytic patterns. Differences between groups were found in both the primary and secondary specificity toward alpha(s1)-casein (f 1-23) and its breakdown products. No correlation was found between the o-phthaldialdehyde (OPA) general proteolysis analysis and alpha(s1)-casein (f 1-23) cleavage profiles. CONCLUSIONS, SIGNIFICANCE AND IMPACT OF STUDY: Using the alpha(s1)-CN (f 1-23) method, six patterns of proteolysis were found in the dairy lactobacilli tested. Understanding the influence of Lactobacillus proteinase specificity on casein degradation should facilitate efforts to develop starter cultures that predictably improve the functional properties of Mozzarella cheese.

Chromatography, High Pressure Liquid↗
PubMed1996

Lactobacillus paraplantarum sp. now., a new species related to Lactobacillus plantarum.

Four strains of facultatively heterofermentative lactobacilli isolated from beer and human feces have physiological characteristics similar to those of Lactobacillus plantarum. Unlike 66% of the L. plantarum strains tested (F. Bringel, M.-C. Curk, and J.-C. Hubert, Int. J. Syst. Bacteriol. 46:588-594, 1996), these strains do not catabolize alpha-methyl-D-mannoside. However, because they exhibit little DNA relatedness to L. plantarum and Lactobacillus pentosus, these four strains were classified as members of a new species, Lactobacillus paraplantarum; strain CNRZ 1885 (= CIP 104668) is the type strain.

Base Composition↗
PubMed2002

Lactobacillus equi sp. nov., a predominant intestinal Lactobacillus species of the horse isolated from faeces of healthy horses.

Lactobacillus equi sp. nov. is described on the basis of 18 strains isolated as one of the predominant intestinal lactobacilli from horse faecal specimens. These 18 strains were isolated from 10 horses of 6 different farms out of 20 horses of 10 farms examined. They were gram-positive, facultatively anaerobic, catalase-negative, non-spore-forming, non-motile, lactic-acid-homofermentative rods. The DNA G+C content was 38.9+/-0.8 mol %. DNA-DNA hybridization failed to associate these strains closely with any of the validly described type strains used. Analysis of the 16S rRNA gene sequence of representative strain YIT 0455T revealed that the new isolates represent a new Lactobacillus species, for which the name Lactobacillus equi is proposed. The type strain is YIT 0455T (= ATCC BAA-261T = JCM 10991T).

Animals↗
PubMed2004

Reclassification of Lactobacillus kefirgranum Takizawa et al. 1994 as Lactobacillus kefiranofaciens subsp. kefirgranum subsp. nov. and emended description of L. kefiranofaciens Fujisawa et al. 1988.

Fourteen homofermentative lactic acid bacteria that were isolated from kefir grains and kefir fermented milks were assigned to either Lactobacillus kefiranofaciens or Lactobacillus kefirgranum, based on their characteristic morphotypes, phenotypic features and SDS-PAGE profiles of whole-cell proteins. Further genotypic analyses on representative strains from both taxa demonstrated that L. kefiranofaciens and L. kefirgranum share 100 % 16S rDNA sequence similarity and belong phylogenetically to the Lactobacillus acidophilus species group. DNA-DNA binding values of >79 % and analogous DNA G+C contents of 37-38 mol% showed that the strains studied belonged to one species: L. kefirgranum is a later synonym of L. kefiranofaciens. An emended description is proposed for L. kefiranofaciens. Due to the specific morphological and biochemical characteristics of these taxa in kefir grain formation, it is proposed that L. kefirgranum should be reclassified as L. kefiranofaciens subsp. kefirgranum subsp. nov.

Animals↗
PubMed2006

Production of a heterologous nonheme catalase by Lactobacillus casei: an efficient tool for removal of H2O2 and protection of Lactobacillus bulgaricus from oxidative stress in milk.

Lactic acid bacteria (LAB) are generally sensitive to H2O2, a compound that they can paradoxically produce themselves, as is the case for Lactobacillus bulgaricus. Lactobacillus plantarum ATCC 14431 is one of the very few LAB strains able to degrade H2O2 through the action of a nonheme, manganese-dependent catalase (hereafter called MnKat). The MnKat gene was expressed in three catalase-deficient LAB species: L. bulgaricus ATCC 11842, Lactobacillus casei BL23, and Lactococcus lactis MG1363. While the protein could be detected in all heterologous hosts, enzyme activity was observed only in L. casei. This is probably due to the differences in the Mn contents of the cells, which are reportedly similar in L. plantarum and L. casei but at least 10- and 100-fold lower in Lactococcus lactis and L. bulgaricus, respectively. The expression of the MnKat gene in L. casei conferred enhanced oxidative stress resistance, as measured by an increase in the survival rate after exposure to H2O2, and improved long-term survival in aerated cultures. In mixtures of L. casei producing MnKat and L. bulgaricus, L. casei can eliminate H2O2 from the culture medium, thereby protecting both L. casei and L. bulgaricus from its deleterious effects.

Animals↗
PubMed1991

Complementation of the inability of Lactobacillus strains to utilize D-xylose with D-xylose catabolism-encoding genes of Lactobacillus pentosus.

The inability of two Lactobacillus strains to ferment D-xylose was complemented by the introduction of Lactobacillus pentosus genes encoding D-xylose isomerase, D-xylulose kinase, and a D-xylose catabolism regulatory protein. This result opens the possibility of using D-xylose fermentation as a food-grade selection marker for Lactobacillus spp.

Aldose-Ketose Isomerases↗
PubMed1992

1,3-Propanediol:NAD+ oxidoreductases of Lactobacillus brevis and Lactobacillus buchneri.

In the cofermentation of glycerol with a sugar by Lactobacillus brevis and Lactobacillus buchneri, a 1,3-propanediol:NAD+ oxidoreductase provides an additional method of NADH disposal. The enzyme has been purified from both L. brevis B22 and L. buchneri B190 and found to have properties very similar to those reported for the enzyme from Klebsiella pneumoniae. The enzymes required Mn2+ and are probably octamers with a molecular mass of 350 kDa. Although not absolutely specific for 1,3-propanediol when tested as dehydrogenases, the enzymes have less than 10% activity with glycerol, ethanol, and 1,2-propanediol. These properties contrast sharply with those of a protein isolated from another Lactobacillus species (L. reuteri) that ferments glycerol with glucose and previously designated a 1,3-propanediol dehydrogenase.

Alcohol Dehydrogenase↗
PubMed1998

Molecular cloning and functional expression in lactobacillus plantarum 80 of xylT, encoding the D-xylose-H+ symporter of Lactobacillus brevis.

A 3-kb region, located downstream of the Lactobacillus brevis xylA gene (encoding D-xylose isomerase), was cloned in Escherichia coli TG1. The sequence revealed two open reading frames which could code for the D-xylulose kinase gene (xylB) and another gene (xylT) encoding a protein of 457 amino acids with significant similarity to the D-xylose-H+ symporters of E. coli, XylE (57%), and Bacillus megaterium, XylT (58%), to the D-xylose-Na+ symporter of Tetragenococcus halophila, XylE (57%), and to the L-arabinose-H+ symporter of E. coli, AraE (60%). The L. brevis xylABT genes showed an arrangement similar to that of the B. megaterium xylABT operon and the T. halophila xylABE operon. Southern hybridization performed with the Lactobacillus pentosus xylR gene (encoding the D-xylose repressor protein) as a probe revealed the existence of a xylR homologue in L. brevis which is not located with the xyABT locus. The existence of a functional XylR was further suggested by the presence of xylO sequences upstream of xylA and xylT and by the requirement of D-xylose for the induction of D-xylose isomerase, D-xylulose kinase, and D-xylose transport activities in L. brevis. When L. brevis was cultivated in a mixture of D-glucose and D-xylose, the D-xylose isomerase and D-xylulose kinase activities were reduced fourfold and the D-xylose transport activity was reduced by sixfold, suggesting catabolite repression by D-glucose of D-xylose assimilation. The xylT gene was functionally expressed in Lactobacillus plantarum 80, a strain which lacks proton motive force-linked D-xylose transport activity. The role of the XylT protein was confirmed by the accumulation of D-xylose in L. plantarum 80 cells, and this accumulation was dependent on the proton motive force generated by either malolactic fermentation or by the metabolism of D-glucose. The apparent affinity constant of XylT for D-xylose was approximately 215 microM, and the maximal initial velocity of transport was 35 nmol/min per mg (dry weight). Furthermore, of a number of sugars tested, only 6-deoxy-D-glucose inhibited the transport of D-xylose by XylT competitively, with a Ki of 220 microM.

Amino Acid Sequence↗
PubMed2002

Identification and characterization of novel surface proteins in Lactobacillus johnsonii and Lactobacillus gasseri.

We have identified and sequenced the genes encoding the aggregation-promoting factor (APF) protein from six different strains of Lactobacillus johnsonii and Lactobacillus gasseri. Both species harbor two apf genes, apf1 and apf2, which are in the same orientation and encode proteins of 257 to 326 amino acids. Multiple alignments of the deduced amino acid sequences of these apf genes demonstrate a very strong sequence conservation of all of the genes with the exception of their central regions. Northern blot analysis showed that both genes are transcribed, reaching their maximum expression during the exponential phase. Primer extension analysis revealed that apf1 and apf2 harbor a putative promoter sequence that is conserved in all of the genes. Western blot analysis of the LiCl cell extracts showed that APF proteins are located on the cell surface. Intact cells of L. johnsonii revealed the typical cell wall architecture of S-layer-carrying gram-positive eubacteria, which could be selectively removed with LiCl treatment. In addition, the amino acid composition, physical properties, and genetic organization were found to be quite similar to those of S-layer proteins. These results suggest that APF is a novel surface protein of the Lactobacillus acidophilus B-homology group which might belong to an S-layer-like family.

Amino Acid Sequence↗
PubMed1996

Viability of Lactobacillus acidophilus and Lactobacillus casei in fermented milk products during refrigerated storage.

The viability was investigated of five strains of Lactobacillus acidophilus and one strain of Lactobacillus casei that were added as adjuncts to yogurt and cultured buttermilk during 28 d of refrigerated storage at 5 to 7 degrees C. A modification of LBS (Lactobacillus selection) agar was used for the enumeration of L. acidophilus and L. casei. The medium allowed the colony formation of the adjunct bacteria while preventing colony formation of the traditional yogurt or buttermilk starter cultures. At each sampling period, colonies from the selective agar medium were isolated for confirmation of identity to confirm that only L. acidophilus and L. casei were enumerated, that their characteristics did not change during storage, or both. The strains of L. acidophilus varied in both cultured products. In buttermilk, L. acidophilus MUH-41, O-16, and L-1 exhibited no significant loss in viability, but strains 43121 and La-5 did. No significant loss in viability of L. acidophilus MUH-41 and L-1 occurred in yogurt prepared using culture CM2; however, strains 43121, O-16, and La-5 lost viability. In the yogurt prepared using culture YC-4, L. acidophilus 43121 exhibited no significant loss in viability, but MUH-41, O-16, L-1, and La-5 did. There was no loss in viability of L. casei GG during storage of any of the cultured products.

Animals↗
PubMed1999

Use of modified Lactobacillus selective medium and Bifidobacterium iodoacetate medium for differential enumeration of Lactobacillus acidophilus and Bifidobacterium spp. in powdered nutritional products.

Modified Lactobacillus selective agar (APT agar + sodium acetate and glacial acetic acid; mLBS) was compared to selective modified Lactobacillus selective medium (LBS agar + tomato juice and acetic acid; mLSM) and nonselective de Man Rogosa Sharpe (MRS) agar for the enumeration of Lactobacillus acidophilus in probiotic-containing powdered nutritional products. The mLBS agar was equivalent to MRS agar and superior to the mLSM agar for enumerating L. acidophilus in products stored in sealed cans at 22 degrees C. When samples were analyzed for L. acidophilus concentration after high temperature storage in sealed cans or storage in open cans at high relative humidity, the mLBS and MRS agars were highly correlated (r2 = 0.93). Modified Bifidobacterium iodoacetate medium (12.5 mg iodoacetic acid/liter; mBIM) was compared to MRS agar + bile, cysteine, and dicloxacillin (MRS + BCD) for enumerating Bifidobacterium infantis or Bifidobacterium lactis in the nutritional products. The two media were equivalent for enumerating B. infantis in product stored at 22 degrees C in sealed cans. However, the two media were poorly correlated (r2<0.50) for enumeration of B. infantis and B. lactis in products stored in sealed cans at high temperature or in open cans at high relative humidity. The mLBS medium has potential industry application as a relatively inexpensive, convenient differential enumeration method for L. acidophilus. The mBIM medium cannot be recommended as a sole medium for enumeration of probiotic Bifidobacterium spp. in powdered nutritional products stored under high temperature and/or high relative humidity conditions.

Agar↗
PubMed1993

Antimicrobial compounds from Lactobacillus casei and Lactobacillus helveticus.

Three strains of Lactobacillus casei and one of Lactobacillus helveticus were examined for antagonistic activity toward twenty five indicator strains of different species. Under conditions eliminating the effects of organic acid and hydrogen peroxide, culture supernatants of all the Lactobacillus strains exhibited a wide spectrum of inhibitory activity toward microorganisms of different genera. The inhibitory compound secreted by one strain of L. casei was active against Clostridium tyrobutyricum. The active components were insensitive to proteolytic enzyme and heat treatment.

Anti-Bacterial Agents↗
PubMed2006

Augmentation of antimicrobial metronidazole therapy of bacterial vaginosis with oral probiotic Lactobacillus rhamnosus GR-1 and Lactobacillus reuteri RC-14: randomized, double-blind, placebo controlled trial.

This study enrolled 125 premenopausal women diagnosed with bacterial vaginosis (BV) by presence of vaginal irritation, discharge and 'fishy' odor, and Nugent criteria and detection of sialidase enzyme. The subjects were treated with oral metronidazole (500 mg) twice daily from days 1 to 7, and randomized to receive oral Lactobacillus rhamnosus GR-1 (1 x 10(9)) and Lactobacillus reuteri RC-14 (1 x 10(9)) or placebo twice daily from days 1 to 30. Primary outcome was cure of BV as determined by normal Nugent score, negative sialidase test and no symptoms or signs of BV at day 30. A total of 106 subjects returned for 30-day follow-up, of which 88% were cured in the antibiotic/probiotic group compared to 40% in the antibiotic/placebo group (p<0.001). Of the remaining subjects, 30% subjects in the placebo group and none in the probiotic group had BV, while 30% in the placebo and 12% in the probiotic group fell into the intermediate category based upon Nugent score, sialidase result and clinical findings. High counts of Lactobacillus sp. (>10(5) CFU/ml) were recovered from the vagina of 96% probiotic-treated subjects compared to 53% controls at day 30. In summary, this study showed efficacious use of lactobacilli and antibiotic in the eradication of BV in black African women.

Adolescent↗