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PubMed2002

Isolation, characterization and inhibition by acarbose of the alpha-amylase from Lactobacillus fermentum: comparison with Lb. manihotivorans and Lb. plantarum amylases.

Extracellular alpha-amylase from Lactobacillus fermentum (FERMENTA) was purified by glycogen precipitation and ion exchange chromatography. The purification was approximately 28-fold with a 27% yield. The FERMENTA molecular mass (106,000 Da) is in the same range as the ones determined for L. amylovorus (AMYLOA), L. plantarum (PLANTAA) and L. manihotivorans (MANIHOA) alpha-amylases. The amino acid composition of FERMENTA differs from the other lactobacilli considered here, but however, indicates that the peptidic sequence contains two equal parts: the N-terminal catalytic part; and the C-terminal repeats. The isoelectric point of FERMENTA, PLANTAA, MANIHOA are approximately the same (3.6). The FERMENTA optimum pH (5.0) is slightly more acidic and the optimum temperature is lower (40 degrees C). Raw starch hydrolysis catalyzed by all three amylases liberates maltotriose and maltotretaose. Maltose is also produced by FERMENTA and MANIHOA. Maltohexaose FERMENTA catalyzed hydrolysis produces maltose and maltotriose. Finally, kinetics of FERMENTA, PLANTAA and MANIHOA using amylose as a substrate and acarbose as an inhibitor, were carried out. Statistical analysis of kinetic data, expressed using a general velocity equation and assuming rapid equilibrium, showed that: (1) in the absence of inhibitor k(cat)/Km are, respectively, 1x10(9), 12.6x10(9) and 3.2x10(9) s(-1) M(-1); and (2) the inhibition of FERMENTA is of the mixed non-competitive type (K(1i)=5.27 microM; L(1i)=1.73 microM) while the inhibition of PLANTAA and MANIHOA is of the uncompetitive type (L(1i)=1.93 microM and 1.52 microM, respectively). Whatever the inhibition type, acarbose is a strong inhibitor of these Lactobacillus amylases. These results indicate that, as found in porcine and barley amylases, Lactobacillus amylases contain in addition to the active site, a soluble carbohydrate (substrate or product) binding site.

Acarbose↗
PubMed2002

Use of Lactobacillus-GG in paediatric Crohn's disease.

The potential role of luminal bacteria in initiating the abnormal immune response seen in inflammatory bowel disease is stressed by many observations. A defect in mucosal barrier function could allow luminal bacterial antigens to initiate the chronic relapsing inflammation in Crohn's disease. The potential role of luminal bacteria in initiating the abnormal immune response seen in inflammatory bowel disease is stressed by many observations. A pilot study to investigate the possible effect of Lactobacillus GG in children with active Crohn's disease was conducted. Four male patients were enrolled, median age 14.5 years (range 10-18). In terms of clinical outcome, the patients showed significant improvement. In three patients on Lactobacillus GG, it was possible to taper the dose of steroids. Thus, although our data are obviously very preliminary, Lactobacillus GG appears to be effective in improving the clinical status of children with Crohn's disease. A multicentre study is currently being carried out in 7 US University centres in a randomized, double-blind, placebo-controlled fashion to establish the efficacy of this probiotic in children with Crohn's disease.

Adolescent↗
PubMed2002

Screening and selection of Lactobacillus strains for use as adjunct cultures in production of semi-hard cheese.

Thirty-three Lactobacillus strains were tested as adjuncts in a cheese model system. Eighteen strains originated from cheese (nine Lactobacillus spp. and nine Lb. paracasei/casei) and 15 from human intestinal mucosa (11 Lb. rhamnosus; three Lb. paracasei; one Lb. plantarum). Model cheeses weighing 120 g were made of cheese grains from full-scale production of washed curd semi-hard cheese (Herrgård). The model system was reproducible and similar to full-scale production with respect to moisture, salt content, pH and microbial flora. The model cheeses were sampled for aerobic and anaerobic plate count and viable counts of Lactobacillus and Lactococcus. The presence of adjuncts in the model cheeses was confirmed by typing isolates with Randomly Amplified Polymorphic DNA (RAPD). The sensory properties of model cheeses were described. In a first trial 23 of the 33 adjuncts were re-isolated from the corresponding model cheeses after 9 or 13 weeks. Adjuncts of Lb. paracasei were re-isolated more frequently than adjuncts of Lb. rhamnosus. Nine strains were selected, on the basis of their ability to grow and be a dominating part of the microflora of model cheese with interesting sensory properties. These strains were further studied together with two commercial cultures. The sensory influences on model cheeses of six of the adjuncts were confirmed, and flavour scores were in the range of 2.9-7.1 for model cheeses with different adjuncts while the control had a flavour score of 5.6 (0-10 scale). Survival and growth of seven out of the nine strains correlated with the results of the first trial. Growth and influence on flavour of four adjunct cultures were confirmed in experimental cheese manufactured in a 400-1 open vat.

Bacterial Typing Techniques↗
PubMed2004

Amino acid transamination in permeabilised cells of Lactobacillus helveticus, Lb. paracasei and Lb. danicus.

Aminotransferase (AT) activity against 18 amino acids was studied in ten strains of three species of Lactobacillus. A method for permeabilisation of cells was developed using toluene and ethanol combined with mechanical treatment. It was found that the AT activities in the washed permeabilised cells (W-PC) corresponded well to that in cell-free extracts (CFE). The AT specificity pattern was species as well as strain dependant. Strains of Lb. helveticus had high specificity for aromatic amino acids (ArAA) and lower activity against branched-chain amino acids (BcAA) and Asp, while strains of Lb. paracasei subsp. paracasei degraded BcAA and Asp, but had a lower and variable specificity against ArAA. One of the Lb. paracasei strains was characterised by having very high AT activity against all three BcAA (Ile, Leu, Val) compared with any of the other Lb. paracasei strains tested. Strains of Lb. danicus, which is a newly discovered Lactobacillus species isolated from cheese, had up to about 20 times higher AT activity against Leu than Lb. paracasei and Lb. helveticus. The permeabilised cells of Lb. danicus had also considerably higher AT activity against ArAA than Lb. paracasei and Lb. helveticus strains, and also higher AT activity against Asp. All Lactobacillus strains tested had AT activity against Met, but at a much lower rate than against other amino acids. Results of this study also demonstrated a chemical reaction between alpha-ketoglutaric acid and Asp that was catalysed by pyridoxal-5-phosphate without any AT present.

Amino Acids↗
PubMed2003

The bactericidal effects of Lactobacillus acidophilus, garcinol and Protykin compared to clarithromycin, on Helicobacter pylori.

Chronic infection with Helicobacter pylori causes peptic ulcers, gastric cancer and lymphoma. We evaluated the inhibitory effects of the probiotic Lactobacillus acidophilus DDS-1J, the antibiotic clarithromycin and the natural antioxidants garcinol and Protykin (containing 50% trans-resveratrol) on Helicobacter pylori strain ATCC 49503. The findings of this study indicate that Lactobacillus acidophilus DDS-1J exerts a growth inhibitory effect on H. pylori at a ratio of 1:1 or higher in vitro. In the case of clarithromycin, garcinol and resveratrol, the bactericidal effect is time and concentration dependent. Clarithromycin completely inhibited growth at > or = 62.5 microg/ml at 6 h and at > or = 31.5 microg/ml at 12 h. For garcinol the highest concentration needed for complete inhibition was 31.5 microg/ml at 6 h and 3.9 microg/ml after 12 h incubation. For resveratrol, significant inhibition was noted at 1000 microg/ml at 12 h only. The bactericidal effect of garcinol was reduced by the addition of resveratrol at all concentrations < or = 125 microg/ml at 6 and 12 h. We conclude from this study that Lactobacillus acidophilus DDS-1J inhibits H. pylori at 1:1 and higher ratios. Also, between the two antioxidants, garcinol is much more potent than resveratrol as a bactericidal agent against H. pylori, and that resveratrol may antagonize this effect. Finally, our study showed equivalent or better bactericidal activity of garcinol compared to clarithromycin against H. pylori at 6 and 12 h incubation, indicating a potential role for this antioxidant in treatment for H. pylori infection.

Anti-Bacterial Agents↗
PubMed2001

Lactobacillus acidophilus sepsis in a neonate.

Lactobacillus species are non-spore-forming, anaerobic, gram-positive rods that cause disease in immunocompromised adults. Few cases have been described in children. We present the case of a 2-month-old infant who apparently developed Lactobacillus acidophilus sepsis from an infected central venous catheter. Physicians should be aware that although Lactobacillus species rarely cause disease in children, they should be considered a possible pathogen when isolated from the blood of a newborn infant.

Adult↗
PubMed1999

Heat resistance of Lactobacillus spp. isolated from Cheddar cheese.

Mesophilic Lactobacillus spp. are the dominant organisms in mature Cheddar cheese. The heat resistance of broth grown cultures of Lactobacillus plantarum DPC1919 at temperatures between 50 and 57.5 degrees C, Lact. plantarum DPC2102 at temperatures between 48 and 56 degrees C and Lact. paracasei DPC2103 at temperatures between 50 and 67.5 degrees C was determined. The z-values for Lact. plantarum DPC1919, Lact. Plantarum DPC2102 and Lact. paracasei DPC2103 were 6.7 degrees C, 6.2 degrees C and 5.3 degrees C, respectively. Lactobacillus paracasei DPC2103 showed evidence of injury and recovery, especially at higher temperatures. Milk grown cultures of strains DPC2102 and DPC2103 showed greater heat resistance than broth grown cultures, tailing of the death curves and a nonlinear z-curve. Of the three strains, Lact. paracasei DPC2103 had the potential to survive pasteurization temperatures, whether grown in milk or broth.

Animals↗
PubMed2000

Iron requirement of Lactobacillus spp. in completely chemically defined growth media.

A completely chemically-defined growth medium, containing guanine, thymine, cytidine, 2'-deoxyadenosine and 2'-deoxyuridine as DNA precursors, was developed for Lactobacillus johnsonii, on the basis of statistically designed techniques suitable for other lactobacilli. Particular focus was given to the nucleotide composition of different defined media, and to the specific nucleotide requirements of Lact. johnsonii. Most of the lactobacilli tested grew in a medium containing five free bases, four ribonucleosides or five deoxyribonucleosides. Adenine and guanine were replaceable by inosine. The requirement for thymine and cytosine was satisfied with uracil. The presence of inosine and uracil was identified as being essential for the growth of different Lactobacillus species, displaying their inability to synthesize purines and pyrimidines de novo. Defined recipes with different nucleotide composition were used to investigate iron requirements of lactobacilli. Only marginal differences in growth were observed in iron-depleted media supplemented with five free bases, four ribonucleosides or five deoxyribonucleosides; iron depletion had a greater effect on growth when inosine and uracil were supplied as the only nucleotide sources. The results suggest that iron plays a role in the pyrimidine and purine metabolism of lactobacilli. Lactobacillus spp., particularly Lact. johnsonii, require iron under particular environmental conditions with limited or specific nucleotide sources.

Culture Media↗
PubMed2000

Functional analysis of promoters involved in quorum sensing-based regulation of bacteriocin production in Lactobacillus.

Bacteriocin production in Lactobacillus sake LTH673 involves at least four operons: a regulatory operon (sppIPKR); two operons encoding bacteriocins and their immunity proteins (sppAiA and orfX); and an operon needed for secretion (sppTE). We show here that the response regulator encoded by sppR in L. sake LTH673, as well as the homologous response regulators encoded by plnC and plnD in Lactobacillus plantarum C11, bind to characteristic repeats found in the -80 to -40 regions of spp operons. The promoters controlling bacteriocin operons are strictly regulated, and their activity is increased more than 1000-fold upon activation. Constitutive expression for the regulatory and transport operons is driven, at least in part, by promoters upstream of the -80 to -40 regions. Peak promoter activity of the regulatory and transporter operons precedes that of the two bacteriocin operons. The results reveal how promoters involved in quorum sensing-based regulation of bacteriocin production in Lactobacillus differ in strength, leakiness and timing of their activity.

Bacteriocins↗
PubMed1998

A method for the selective enumeration and isolation of ruminal Lactobacillus and Streptococcus.

Ruminal lactic acid-producing bacteria were selectively isolated and enumerated using a one hour aerobic exposure prior to incubation on a semi-selective Lactobacillus medium, MRS, under anaerobic conditions. The technique allowed growth of pure cultures of ruminal Lactobacillus spp. and Streptococcus bovis without supporting the growth of pure cultures of any of the prominent ruminal bacterial species. In mixed cultures, the one hour aerobic pre-incubation inhibited the growth of the obligate anaerobic ruminal bacteria which can otherwise grow on the MRS medium, and the subsequent anaerobic incubation permitted maximal recovery of the weakly aerotolerant ruminal lactic acid-producing Lactobacillus spp. and Streptococcus spp. The efficacy of this technique in selecting exclusively for the lactic acid-producing bacteria was also demonstrated from populations of rumen bacteria from mixed culture end-point in vitro fermentation, continuous in vitro culture and isolations from fresh ruminal samples.

Animals↗
PubMed1998

Acid and bile tolerance of Lactobacillus isolated from chicken intestine.

Twelve Lactobacillus strains isolated from chicken intestine were used to investigate acid and bile tolerance in vitro. Ten out of the 12 strains were slightly affected by 0.3% bile salts, showing a delay of growth (d) of 0.6-37.2 min compared with growth in control cultures. Two strains were not affected by the bile salts. Of the 12 strains, seven could be arbitrarily classified as resistant (d < 15 min) and five as tolerant (15 min < d < or = 40 min). Lactobacillus strains from the caecum showed better tolerance to acid than those from the ileum. Generally, the survival of the ileal strains was very low at pH 1.0 and 2.0, and moderate at pH 3.0. In contrast, caecal Lactobacillus strains could survive at pH 1.0 for up to 2 h of incubation; growth was moderate at pH 2.0 and good at pH 3.0 and 4.0.

Animals↗
PubMed2000

Development of a rapid method for the identification of Lactobacillus spp. isolated from naturally fermented italian sausages using a polymerase chain reaction-temperature gradient gel electrophoresis.

A rapid method for the identification of Lactobacillus spp. isolated from naturally fermented Italian sausages was developed. It is based on the amplification of a small fragment from the 16S rRNA gene followed by temperature gradient gel electrophoresis (TGGE). Lactobacillus sakei, L. curvatus, L. alimentarius, L. casei, L. plantarum and L. brevis, obtained from International Collections, were used to optimize the method. Thiry-nine strains of Lactobacillus spp. were isolated from naturally fermented sausages and, after traditional identification, were tested by the PCR-TGGE protocol developed. No differences were observed comparing the results obtained, apart from five strains identified as L. curvatus that showed a PCR-TGGE profile identical to L. sakei.

Bacterial Typing Techniques↗
PubMed2001

Combined use of Lactobacillus reuteri and soygerm powder as food supplement.

AIMS: The survival of Lactobacillus reuteri when challenged with glycodeoxycholic acid (GDCA), deoxycholic acid (DCA) and soygerm powder was investigated. Moreover, the impact of Lact. reuteri on the bioavailability of isoflavones present in soygerm powder was examined. METHODS AND RESULTS: The strain experienced a die-off when adding 2 or 3 mmol l-1 bile salts, with more pronounced effects in the case of DCA. By means of a haemolysis test it was shown that toxicity could be due to membrane damage. When 4 g l-1 soygerm powder was added, the Lactobacillus strain survived the bile salt burden better (P < or = 0.05) and the membrane damage in the haemolysis test decreased (P < or = 0.05). The Lact. strain cleaved beta-glycosidic isoflavones during fermentation of milk supplemented with soygerm powder. CONCLUSIONS, SIGNIFICANCE AND IMPACT OF THE STUDY: The interactions between the Lactobacillus strain and soygerm powder suggest that combining both in fermented milk can exhibit advantageous probiotic effects. The relevance of the combination of the strain and the soygerm powder should be studied under more relevant physiological conditions.

Animals↗
PubMed2003

Effect of probiotic Lactobacillus strains in children with atopic dermatitis.

BACKGROUND: Recent studies suggest that oral bacteriotherapy with probiotics might be useful in the management of atopic dermatitis (AD). OBJECTIVE: The purpose of this investigation was to evaluate the clinical and anti-inflammatory effect of probiotic supplementation in children with AD. METHODS: In a double-blind, placebo-controlled, crossover study, 2 probiotic Lactobacillus strains (lyophilized Lactobacillus rhamnosus 19070-2 and Lactobacillus reuteri DSM 122460) were given in combination for 6 weeks to 1- to 13-year-old children with AD. The patients' evaluations were registered after each intervention (ie, better, unchanged, or worse). The clinical severity of the eczema was evaluated by using the scoring atopic dermatitis (SCORAD) score. As inflammatory markers, eosinophil cationic protein in serum and cytokine production by PBMCs were measured. RESULTS: After active treatment, 56% of the patients experienced improvement of the eczema, whereas only 15% believed their symptoms had improved after placebo (P =.001). The total SCORAD index, however, did not change significantly. The extent of the eczema decreased during active treatment from a mean of 18.2% to 13.7% (P =.02). The treatment response was more pronounced in allergic patients (at least one positive skin prick test response and elevated IgE levels), and in this group the SCORAD score decreased (P =.02 compared with nonallergic patients). During active treatment, serum eosinophil cationic protein levels decreased (P =.03). No significant changes in the production of the cytokines IL-2, IL-4, IL-10, or IFN-gamma were found. CONCLUSIONS: A combination of L rhamnosus 19070-2 and L reuteri DSM 122460 was beneficial in the management of AD. The effect was more pronounced in patients with a positive skin prick test response and increased IgE levels.

Administration, Topical↗
PubMed2001

Polyphasic investigation of the diversity within Lactobacillus plantarum related strains revealed two L. plantarum subgroups.

The diversity of 140 strains related to Lactobacillus plantarum was investigated using a polyphasic approach combining two molecular techniques: randomly amplified polymorphic DNA fingerprinting (RAPD) and Southern hybridisation with a pyr probe on BglI digests of chromosomal DNA, as well as phenotypic characterization. The RAPD technique allowed us to classify a subset of 60 representative strains into four groups. One group belonged to Lactobacillus paraplantarum, the second to Lactobacillus pentosus and the two remaining groups to L. plantarum (G(L)p1 and G(L)p2). The Southern hybridisation technique (F. Bringel, M.-C. Curk and J.-C. Hubert, Int. J. Syst. Bacteriol. 46: 588-594, 1996) revealed nine groups of profiles (I to IX). Results indicated an excellent convergence between RAPD and hybridisation classifications for more than 93% (56/60) of the strains studied. When we compared the fermentation patterns of the L. plantarum strains, three differences were found. Melezitose fermentation was not fermented by the G(L)p2 RAPD group, unlike the G(L)p1 RAPD group which included L. plantarum type strain NCIMB11974T. Second, alpha-methyl-D-mannoside was fermented by a majority of the strains of the G(L)p1 RAPD group but by none of the strains in the G(L)p2 RAPD group. Third, dulcitol was catabolized by nearly half of the strains of the G(L)p2 RAPD group but by none of the strains in the G(L)p1 RAPD group. Molecular diversity within L. plantarum was confirmed using Southern profiles, PCR amplification and subsequent sequencing of these PCR products. A 773 bp sequence overlapping the pyrDF genes showed high homology: at least 97% identical in L. plantarum strains (V to IX) and 99.9% identical in hybridisation groups VII and VIII. The same G-T transversion which destroyed the pyrF BglI site was found in 11 strains (hybridisation groups VI, VII and VIII). DNA rearrangements were identified downstream from the pyr genes, by PCR amplification and Southern hybridisation profile analysis in three strains of hybridisation groups VIII and IX, two of which also harboured the G-T transversion.

Blotting, Southern↗
PubMed2001

Differentiation of Lactobacillus plantarum, L. pentosus and L. paraplantarum species by RAPD-PCR and AFLP.

Two high-resolution genotypic techniques (RAPD-PCR and AFLP) were evaluated for their possibility to discriminate the species Lactobacillus plantarum, Lactobacillus pentosus and Lactobacillus paraplantarum and to type these taxa at the infra-species level. In total 23 strains of L. plantarum, three strains of L. pentosus, two strains of L. paraplantarum and two related strains for which the species assignment was not clear, were studied. For RAPD-PCR, suitable oligonucleotides and amplification conditions were selected and tested. For AFLP, a double digest of total genomic DNA was used and a subset of restriction fragments was selectively amplified and visualised using different primer combinations. Both methodologies generated, species-specific electrophoretic profiles. Moreover, the presence of distinct subgroups was revealed within the species L. plantarum.

Cluster Analysis↗
PubMed2002

Removal of common Fusarium toxins in vitro by strains of Lactobacillus and Propionibacterium.

This study was conducted to examine the ability of selected strains of Lactobacillus and Propionibacterium to remove common Fusarium toxins, trichothecenes, from liquid media. The trichothecenes studied were deoxynivalenol (DON), 3-acetyldeoxynivalenol (3-AcDON), nivalenol (NIV), fusarenon (FX), diacetoxyscirpenol (DAS), T-2 toxin (T-2) and HT-2 toxin (HT-2). The Lactobacillus rhamnosus strain GG (LGG), Lactobacillus rhamnosus strain LC-705 (LC-705) and Propionibacterium freudenreichii ssp. shermanii JS (PJS) were incubated in PBS buffer containing 20 microg toxin ml(-1) for 1h at 37 degrees C, and after centrifugation the concentration of the toxins was measured in the supernatant fraction. Both viable and heat-killed forms of LGG and PJS were more efficient than LC-705 in removing the toxins from the liquid media. LGG and PJS removed four of the seven tested toxins (the removal varying from 18 to 93%) and LC-705 two toxins (10-64%). Of the toxins, 3-AcDON was not removed by any of the bacteria; HT-2 was removed by the non-viable LGG and also slightly by non-viable LC-705; DAS was removed by all three bacteria tested. Binding is postulated as the possible mechanism of the removal, since no difference was observed between the ability of viable and heat-killed bacteria in removing the trichothecenes, and no degradation products of the toxins were detected by gas chromatography (GC)-mass spectrometry (MS) analysis. It is concluded that significant differences exist in the ability of the bacteria to bind trichothecenes in vitro.

Decontamination↗
PubMed1997

Effect of maduramicin and monensin on survival of Lactobacillus salivarius 51R administered in the crop and caeca of young chickens.

A rifampicin-resistant Lactobacillus salivarius 51R was administered orally to newly hatched broiler chickens. The resistance to rifampicin enabled us to differentiate the organism administered from indigenous strains. One day after inoculation, Lactobacillus salivarius 51R dominated among lactobacilli in the crop and caeca of all inoculated chickens, even in those ones receiving maduramicin and monensin at 5 and 100 mg per kg of feed mixture, respectively. Coliform counts in both crop and caeca of inoculated chickens were significantly lowered on the first day after treatment. Also, counts of the crop enterococci were decreased in inoculated chickens. Rifampicin-resistant lactobacilli were still present in high numbers in the crop and caecal contents of inoculated chickens sampled 5 days after inoculation. Differences in counts of total lactobacilli, coliform bacteria, and enterococci were mostly nonsignificant in these samples. Our results demonstrate that (i) bacterial counts in the chicken gut were influenced by probiotic Lactobacillus administration, and (ii) chicken lactobacilli are resistant to ionophore coccidiostats under in vivo conditions.

Administration, Oral↗