Role of erythrosine in the inhibition of adhesion of Lactobacillus fermentum strain 737 to mouse stomach tissue

The mechanism by which the food colour erythrosine inhibits the adhesion of Lactobacillus sp. to squamous epithelium in the mouse stomach was investigated using an in vitro adhesion assay. Inhibition of adhesion occurred only after growth of L. fermentum in erythrosine which bound to the bacterial cell surface. Erythrosine did not interfere with the receptor on the epithelial cell surface. Growth, but not the ATP content per cell, was affected by the presence of erythrosine in the growth medium. No consistent correlation between hydrophobicity and growth in two different broths was noted when erythrosine was present. Analyses of phenol/water extracts and transmission electron micrographs revealed no reduction in extracellular polysaccharide after growth in the presence of erythrosine. It was concluded that erythrosine affects bacterial metabolism thereby preventing production of the bacterial adhesin which is not the extracellular polysaccharide.     

Adhesion to porcine squamous epithelium of saccharide and protein moieties of Lactobacillus fermentum strain 104-S.

The mechanism by which Lactobacillus fermentum strain 104-S adheres to porcine squamous epithelium was investigated by studying the adsorption to epithelial cells, and control surfaces, of radioactively labelled material released from the bacterial cells by water extraction. The released material was fractionated by gel filtration and the adsorption of pronase-sensitive and -resistant material in the various fractions to porcine gastric tissue and the control surfaces of polystyrene and immobilized bovine serum albumin (BSA) was determined. The fraction with affinity for the epithelium was characterized by enzymic degradation, periodate oxidation, lipid extraction, and protein and carbohydrate analyses. The adsorption pattern of radioactively labelled crude released material mimicked the adhesion of whole labelled cells to polystyrene and to gastric squamous tissue pieces. On fractionation, the pattern of adsorption to polystyrene and BSA was different from that obtained for the tissue pieces. Considerably less labelled pronase-stable material bound to surfaces of polystyrene and BSA, as compared with the tissue, suggesting that the pronase-resistant component has a tissue-specific affinity. After pronase treatment of the fraction of M(r) about 20,000 (20 K) containing labelled components with affinity for the epithelium, only saccharides were detected. Radioactivity was lost after hydrolysis with HCl, and therefore this pronase-resistant labelled component must be a saccharide. It is concluded that protein moieties in the extract have an affinity for several surfaces, including polystyrene, and that saccharide moieties have a specific affinity for the gastric squamous epithelium.     

Characteristics of the adhesion of PCC Lactobacillus fermentum VRI 003 to Peyer's patches

The characteristics of the adhesion of PCC Lactobacillus fermentum VRI 003 to Peyer's patches was studied in vitro. The adhesion of L. fermentum 003 was strongly inhibited in the presence of d-mannose and methyl-alpha-d-mannoside although other carbohydrates tested, such as N-acetyl-glucosamine, d-galactose, d-glucose and l-fucose, did not affect the adhesion. Lactobacillus fermentum 003 was shown to strongly attach to mannose immobilized on a surface using BSA, suggesting that L. fermentum 003 specifically adhered to mannose-containing molecule(s). Pretreatment of L. fermentum 003 with proteinase K and trypsin decreased the adhesive capacity and bacterial surface extracts diminished adhesion of L. fermentum 003 indicating that cell surface proteins are involved in adhesion to Peyer's patches. It was concluded that a mannose-specific protein mediated adhesion of L. fermentum 003 to the Peyer's patches.     

Application of potential probiotic Lactobacillus fermentum AD1 strain in healthy dogs

Probiotic utilization is becoming increasingly popular in veterinary medicine. However, only few probiotic products are available commercially for use in dogs in our market. Therefore, the aim of our study was to determine the properties of new potential probiotic Lactobacillus fermentum AD1 strain-own canine isolate and to investigate its effect on several microbiological and biochemical parameters in healthy dogs. The strain expressed in vitro survival by pH 3.0 after 3h (86.8%) and in the presence of 1% bile (75.4%). The AD1 strain adhered to the canine and human intestinal mucus. It was sensitive to commonly used antimicrobials. Fifteen healthy dogs were supplemented with 10(9)L. fermentum AD1 for 7 days. At the end of AD1 strain application, numbers of faecal lactobacilli and enterococci increased significantly in the canine faeces. Significant increase of total protein and total lipid and significant reduction of glucose in serum of dogs were noted. These data indicate that L. fermentum AD1 survive transit through the canine gastrointestinal tract, and populate the colon and probably increased absorption of some nutrients. Whether longer time of its application lead to the same results as well as its potential to improve immune function in dogs remains to be determined.     

Protoplast fusion of Lactobacillus fermentum

Tetracycline-resistant (Tetr) erythromycin-resistant (Eryr) fusants of Lactobacillus fermentum 604 carrying a 10-megadalton Tetr plasmid and L. fermentum 605 carrying a 38-megadalton Eryr plasmid were obtained by means of polyethylene glycol-induced protoplast fusion.     

Characterization of alpha-galactosidase from Lactobacillus fermentum

α-Galactosidase activity was studied in Lactobacillus fermentum strains. The optimum temperature was found to be 45°C. The enzyme was inactivated at temperatures higher than 55°C, but remained active during storage at low temperatures (0, -30 and -70°C) for 5 months. Enzyme activity was observed within a 5.0–6.5 pH range, while optimum pH was dependent on the particular strain assayed. The addition of Zn²⁺ to the reaction buffer exerted a slight negative effect upon the activity, while Hg²⁺ and p -chloromercuribenzoate produced a strong inhibition. These results would indicate the presence of -SH groups in the catalytic site of the enzyme.     

Protoplast fusion of Lactobacillus fermentum.

Tetracycline-resistant (Tetr) erythromycin-resistant (Eryr) fusants of Lactobacillus fermentum 604 carrying a 10-megadalton Tetr plasmid and L. fermentum 605 carrying a 38-megadalton Eryr plasmid were obtained by means of polyethylene glycol-induced protoplast fusion.     

A note on the use of a plasmid as a DNA probe in the detection of a Lactobacillus fermentum strain in porcine stomach contents

A plasmid (about 50 kb) was used as a DNA probe to enumerate, by colony hybridization, a strain of Lactobacillus fermentum in the stomach contents of eight piglets. The population sizes obtained by colony hybridization were in agreement with estimated levels calculated on the basis of plasmid profiling of colonies isolated at random from the total lactobacillus population.     

Adhesion of Lactobacillus fermentum 104-S to Porcine Stomach Mucus

The adhesion to whole and fractionated porcine gastric mucus of both Lactobacillus fermentum 104-S cells and a saccharide extracted from this strain was investigated. It has been shown previously that this saccharide had affinity for nonsecreting gastric epithelium. The mucus component(s) with affinity the bacterial cells was partly characterized by gel filtration and treatment with protease or metaperiodate. L. fermentum 104-S extracts containing the saccharide were radioactively labeled, fractionated by gel filtration, and tested for affinity for the gastric mucus component showing receptor activity for the whole cells of strain 104-S. The mucus material with affinity for the bacterial cells had a relative molecular weight of 30–70 K. From the results of treatment with protease or metaperiodate, it is proposed that the mucus components(s) that adhered to the whole bacterial cells contained glycoprotein groups. The radioactively labeled saccharide extracted from L. fermentum 104-S cells did not bind to the mucus fraction that had affinity for the whole cells. Conclusively, we suggest that the mechanism by which cells of L. fermentum 104-S adhere to the gastric mucus is different from the mechanism mediating the adhesion of this strain to the nonsecreting gastric epithelium. Cells of L. fermentum 104-S adhere to a glycoproteinaceous mucus component with a relative molecular weight of 30–70 K. Received: 29 August 1995 / Accepted: 26 December 1995     

A note on the use of a plasmid as a DNA probe in the detection of a Lactobacillus fermentum strain in porcine stomach contents.

A plasmid (about 50 kb) was used as a DNA probe to enumerate, by colony hybridization, a strain of Lactobacillus fermentum in the stomach contents of eight piglets. The population sizes obtained by colony hybridization were in agreement with estimated levels calculated on the basis of plasmid profiling of colonies isolated at random from the total lactobacillus population.     

Comparative study of nine Lactobacillus fermentum bacteriophages

AIMS: To investigate the basic properties of six temperate and three virulent phages, active on Lactobacillus fermentum, on the basis of morphology, host ranges, protein composition and genome characterization. METHODS AND RESULTS: All phages belonged to the Siphoviridae family; two of them showed prolate heads. The host ranges of seven phages contained a common group of strains. SDS-PAGE protein profiles, restriction analysis of DNA and Southern blot hybridization revealed a high degree of homology between four temperate phages; partial homologies were also detected among virulent and temperate phages. Clustering derived from host range analysis was not related to the results of the DNA hybridizations. CONCLUSION: The phages investigated have common characteristics with other known phages active on the genus Lactobacillus. Sensitivity to viral infection is apparently enhanced by the presence of a resident prophage. SIGNIFICANCE AND IMPACT OF THE STUDY: These relationships contribute to the explanation for the origin of phage infection in food processes where Lact. fermentum is involved, such as sourdough fermentation.     

Distinct adhesion of probiotic strain Lactobacillus casei ATCC 393 to rat intestinal mucosa

Adhesion to the intestine represents a critical parameter for probiotic action. In this study, the adhesion ability of Lactobacillus casei ATCC 393 to the gastrointestinal tract of Wistar rats was examined after single and daily administration of fermented milk containing either free or immobilized cells on apple pieces. The adhesion of the probiotic cells at the large intestine (cecum and colon) was recorded at levels ≥6 logCFU/g (suggested minimum levels for conferring a probiotic effect) following daily administration for 7 days by combining microbiological and strain-specific multiplex PCR analysis. Single dose administration resulted in slightly reduced counts (5 logCFU/g), while they were lower at the small intestine (duodenum, jejunum, ileum) (≤3 logCFU/g), indicating that adhesion was a targeted process. Of note, the levels of L. casei ATCC 393 were enhanced in the cecal and colon fluids both at single and daily administration of immobilized cells (6 and 7 logCFU/g, respectively). The adhesion of the GI tract was transient and thus daily consumption of probiotic products containing the specific strain is suggested as an important prerequisite for retaining its levels at an effective concentration.     

Riboflavin-overproducing strains of Lactobacillus fermentum for riboflavin-enriched bread

Lactobacillus fermentum isolated from sourdough was able to produce riboflavin. Spontaneous roseoflavin-resistant mutants were obtained by exposing the wild strain (named L. fermentum PBCC11) to increasing concentrations of roseoflavin. Fifteen spontaneous roseoflavin-resistant mutants were isolated, and the level of vitamin B₂ was quantified by HPLC. Seven mutant strains produced concentrations of vitamin B₂ higher than 1 mg L^?1. Interestingly, three mutants were unable to overproduce riboflavin even though they were able to withstand the selective pressure of roseoflavin. Alignment of the rib leader region of PBCC11 and its derivatives showed only point mutations at two neighboring locations of the RFN element. In particular, the highest riboflavin-producing isolates possess an A to G mutation at position 240, while the lowest riboflavin producer carries a T to A substitution at position 236. No mutations were detected in the derivative strains that did not have an overproducing phenotype. The best riboflavin overproducing strain, named L. fermentum PBCC11.5, and its parental strain were used to fortify bread. The effect of two different periods of fermentation on the riboflavin level was compared. Bread produced using the coinoculum yeast and L. fermentum PBCC11.5 led to an approximately twofold increase of final vitamin B₂ content.     

Lysis of modified walls from Lactobacillus fermentum.

The N and O substitution in wall peptidoglycan from Lactobacillus fermentum was studied in relation to growth phase, as well as the lytic activities and the effect of trypsin on them. The N-nonsubstituted sites were determined by dinitrophenylation techniques. The results indicate that an extensive substitution at the O groups takes place as cells go into the stationary growth phase, concomitant with a decrease in their lysozyme sensitivity. N-nonsubstituted residues, mainly glucosamine, occurred in both exponential-phase and stationary-phase walls but not in the corresponding peptidoglycans. Small amounts of N-nonsubstituted muramic acid were detected in walls and peptidoglycan from cells in the stationary growth phase only. N acetylation of isolated walls did not increase their lysozyme sensitivity but rather decreased it. Autolysis of walls was completely inhibited by the chemical modifications used. Trypsin stimulates the lysozyme sensitivity of native walls but has no effect on walls that had been O deacetylated and N acetylated. It is suggested that the effect of trypsin is due to its action as an esterase removing the O acetylation in lysozyme-resistant walls.     

Protein-mediated adhesion of Lactobacillus acidophilus BG2FO4 on human enterocyte and mucus-secreting cell lines in culture.

The adhesion of Lactobacillus acidophilus BG2FO4, a human stool isolate, to two human enterocytelike cell lines (Caco-2 and HT-29) and to the mucus secreted by a subpopulation of mucus-secreting HT29-MTX cells was investigated. Scanning electron microscopy revealed that the bacteria interacted with the well-defined apical microvilli of Caco-2 cells without cell damage and with the mucus secreted by the subpopulation of HT29-MTX cells. The adhesion to Caco-2 cells did not require calcium and involved an adhesion-promoting factor that was present in the spent supernatant of L. acidophilus cultures. This factor promoted adhesion of poorly adhering human Lactobacillus casei GG but did not promote adhesion of L. casei CNRZ 387, a strain of dairy origin. The adherence components on the bacterial cells and in the spent supernatant were partially characterized. Carbohydrates on the bacterial cell wall appeared to be partly responsible for the interaction between the bacteria and the extracellular adhesion-promoting factor. The adhesion-promoting factor was proteinaceous, since trypsin treatment dramatically decreased the adhesion of the L. acidophilus strain. The adhesion-promoting factor may be an important component of Lactobacillus species that colonize the gastrointestinal tract.     

Protein-mediated adhesion of Lactobacillus acidophilus BG2FO4 on human enterocyte and mucus-secreting cell lines in culture.

The adhesion of Lactobacillus acidophilus BG2FO4, a human stool isolate, to two human enterocytelike cell lines (Caco-2 and HT-29) and to the mucus secreted by a subpopulation of mucus-secreting HT29-MTX cells was investigated. Scanning electron microscopy revealed that the bacteria interacted with the well-defined apical microvilli of Caco-2 cells without cell damage and with the mucus secreted by the subpopulation of HT29-MTX cells. The adhesion to Caco-2 cells did not require calcium and involved an adhesion-promoting factor that was present in the spent supernatant of L. acidophilus cultures. This factor promoted adhesion of poorly adhering human Lactobacillus casei GG but did not promote adhesion of L. casei CNRZ 387, a strain of dairy origin. The adherence components on the bacterial cells and in the spent supernatant were partially characterized. Carbohydrates on the bacterial cell wall appeared to be partly responsible for the interaction between the bacteria and the extracellular adhesion-promoting factor. The adhesion-promoting factor was proteinaceous, since trypsin treatment dramatically decreased the adhesion of the L. acidophilus strain. The adhesion-promoting factor may be an important component of Lactobacillus species that colonize the gastrointestinal tract.     

Characteristics of yeast cell flocculation by Lactobacillus fermentum

The effects of different metal ions, carbohydrates, heat and enzymatic treatments on the flocculation of yeast cells caused by a flocculant type of Lactobacillus fermentum were investigated. Calcium ion was required at pH 3.0, 4.5 and 6.2 for complete flocculation. Some flocculation was detected at pH 4.5 even if no calcium was added to the system. Manganese and magnesium ions were capable of partly replacing calcium at pH 6.2. Mannose had an inhibitory effect on flocculation, while other sugars had no effect. Protease is capable of inhibiting the flocculating ability of bacterial cells. Heat treatment of bacterial cells also destroyed the flocculating ability and the effectiveness of this treatment was pH dependent. No effect of protease or heat treatment on yeast cells was found. The results suggest that a cell wall component of L. fermentum, mannan residues of yeast cells and divalent ions were involved in this phenomenon.     

Comparative characterization of spirosomes isolated from Lactobacillus brevis, Lactobacillus fermentum, and Lactobacillus buchneri

Spirosomes, cytoplasmic fine spirals, were isolated and purified from Lactobacillus brevis ATCC 8287, L. fermentum F-1, and L. buchneri ATCC 4005, and their morphological, biochemical, and immunological properties were investigated. The spirosomes of these lactobacilli were morphologically indistinguishable from one another, and they had the same buoyant density of 1.320 g/cm3 in CsCl. All of the spirosomes were composed of a single protein, spirosin, with an apparent molecular weight of about 95,000 for L. brevis and L. fermentum and of about 96,000 for L. buchneri as estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The spirosins from the three lactobacilli were compared by peptide mapping on SDS-PAGE after cleavage with N-chlorosuccinimide and limited proteolysis with Staphylococcus aureus V8 protease. The peptide map of the L. brevis spirosin was identical with that of the L. fermentum spirosin, whereas it was markedly different from the L. buchneri spirosin. The amino acid composition of the L. brevis spirosin was almost similar to that of the L. fermentum spirosin, while it differed appreciably from the L. buchneri spirosin. Using antiserum against the L. brevis spirosin, immunodiffusion test revealed that the antigenicity of the spirosomes from L. brevis was identical with that from L. fermentum, whereas it was partially different from that from L. buchneri.