Antigenic analyses of Lactobacillus fermenti.

Antigenic analyses of Lactobacillus fermenti were carried out by double immunodiffusion in agar using extracts prepared with cold trichloroacetic acid (TCA) or hot dilute hydrochloric acid (HCL). A common antigen of L. fermenti, designated as antigen f by the author, was extracted from whole cells with dilute HCL, but not with TCA. The antigen f was also observed in Lactobacillus casei. In addition, all strains isolated from human saliva contained antigen 6 in their cell walls, while the antigen was not observed in most of the isolates from human feces. Therefore, L. fermenti could be divided into two subgroups based upon the existence of antigen 6. Antigen 7 which was demonstrated in some strains of L. fementi was shared by other species of lactobacilli belonging to the serological groups D and E. The common antigen 3 found in lactobacilli was extracted from all strains of L. fermenti Sugar components of cell walls were mainly galactose, glucose and glucosamine (including N-acetylglucosamine), but a small amount of rhamnose was present in the cell wall of only one strain. Inhibition tests with various sugars showed that the serologically active sugars were galactose for antigen f and glucose for antigen 6.     

Grouping Antigens of Four Lactobacillus Species and Their Characteristics

Antigenic analyses of Lactobacillus bulgaricus, Lactobacillus lactis, Lactobacillus brevis and Lactobacillus buchneri were carried out by double immunodiffusion in agar. Antigens were extracted from whole cells and cell wall preparations with cold trichloroacetic acid. Most strains of the four species possessed antigen 9 in their cell walls. Another antigen, antigen 10, was found in the cell walls of all the strains of L. brevis and L. buchneri, and in some strains of L. lactis, but not in L. bulgaricus. Fractionation of the antigens was attempted using the cell wall extracts of L. lactis L-10 with only antigen 9 and of L. brevis X-1 with both antigens 9 and 10. The partially purified fractions of antigen 9 and of the complex of antigens 9 and 10 were obtained by zone electrophoresis. However, antigen 10 from the complex could not be separated by the same method or gel filtration on Sephadex G-100 since the two antigens 9 and 10 of the complex always behaved together. The fraction of antigen 9 consisted almost entirely of glycerol and glucose as sugar components, the molar ratio being 2:1. The complex of antigens 9 and 10 also consisted of the same sugars, and the molar ratio of glycerol: glucose was 4:1. Inhibition tests indicated that the immunodominant component of antigen 9 was α-methylglucoside (glucose), and most probably the determinant is a glucosylated glycerol teichoic acid. It was considered that the determinant of antigen 10 is a glycerol teichoic acid although glucosamine and galactosamine inhibited effectively the reaction between antigen 10 and its antibody.     

Grouping antigens of four Lactobacillus species and their characteristics.

Antigenic analyses of Lactobacillus bulgaricus, Lactobacillus lactis, Lactobacillus brevis and Lactobacillus buchneri were carried out by double immunodiffusion in agar. Antigens were extracted from whole cells and cell wall preparations with cold trichloroacetic acid. Most strains of the four species possessed antigen 9 in their cell walls. Another antigen, antigen 10, was found in the cell walls of all the strains of L. brevis and L. buchneri, and in some strains of L. lactis, but not in L. bulgaricus. Fractionation of the antigens was attempted using the cell wall extracts of L. lactis L-10 with only antigen 9 and of L. brevis X-1 with both antigens 9 and 10. The partially purified fractions of antigen 9 and of the complex of antigens 9 and 10 were obtained by zone electrophoresis. However, antigen 10 from the complex could not be separated by the same method or gel filtration on Sephadex G-100 since the two antigens 9 and 10 of the complex always behaved together. The fraction of antigen 9 consisted almost entirely of glycerol and glucose as sugar components, the molar ratio being 2: 1. The complex of antigens 9 and 10 also consisted of the same sugars, and the molar ratio of glycerol: glucose was 4: 1. Inhibition tests indicated that the immunodominant component of antigen 9 was a-methylglucoside (glucose), and most probably the determinant is a glycosylated glycerol teichoic acid. It was considered that the determinant of antigen 10 is a glycerol teichoic acid although glucosamine and galactosamine inhibited effectively the reaction between antigen 10 and its antibody.     

Preliminary characterization of wine lactobacilli able to degrade arginine

Lactobacillus strains able to degrade arginine were isolated and characterized from a typical red wine. All the strains were gram-positive, catalase-negative and produced both D- and L-lactate from glucose. Strains L2, L3, L4, and L6 were able to produce CO₂ from glucose; however, production of CO₂ from glucose was not observed in strains L1 and L5, suggesting that they belong to the homofermentative wine lactic acid bacteria (LAB) group. All of the lactobacilli were tested for their ability to ferment 49 carbohydrates. The sugar fermentation profile of strain L1 was unique, suggesting that this strain belonged to Lactococcus lactis ssp. cremoris, a non-typical wine LAB. Furthermore, a preliminary typing was performed by using a random amplified polymorphic DNA analysis (RAPD-PCR analysis).     

Lactobacilli on Plants

The distribution, enumeration, and identification of lactobacilli on vegetable plants were studied in an area described geographically as being subtropical and moist. The lactobacilli were obtained, by means of quantitative enrichment procedures in Rogosa''s SL broth, from 35.3% of all samples incubated at 32 C, and from 15.4% of the samples incubated at 45 C. Less than 10 lactobacilli/g of plant material were enumerated in 54% of all positive samples. The lactobacilli were found much less frequently and in lower numbers than were streptococci or Leuconostoc mesenteroides. The most frequently isolated lactobacillus was very simular to, but not identical with, Lactobacillus fermenti. It was aerogenic, grew well at both 15 and 45 C, fermented arabinose, lactose, and sucrose, and liberated ammonia from arginine. Of the identified species, L. plantarum, L. fermenti, and L. brevis were the most frequently isolated, whereas L. casei, L. viridescens, L. cellobiosus, L. salivarius, and L. buchneri were obtained from small numbers of samples. The widespread but sporadic distribution of lactobacilli in low numbers seems to indicate that these organisms do not normally thrive on plant surfaces. A ternary cycle, beginning with intestinal waste, followed by mechanical distribution to and among plants, and return to the host via the oral cavity, is suggested.     

Lactobacilli on Plants

The distribution, enumeration, and identification of lactobacilli on vegetable plants were studied in an area described geographically as being subtropical and moist. The lactobacilli were obtained, by means of quantitative enrichment procedures in Rogosa's SL broth, from 35.3% of all samples incubated at 32 C, and from 15.4% of the samples incubated at 45 C. Less than 10 lactobacilli/g of plant material were enumerated in 54% of all positive samples. The lactobacilli were found much less frequently and in lower numbers than were streptococci or Leuconostoc mesenteroides. The most frequently isolated lactobacillus was very simular to, but not identical with, Lactobacillus fermenti. It was aerogenic, grew well at both 15 and 45 C, fermented arabinose, lactose, and sucrose, and liberated ammonia from arginine. Of the identified species, L. plantarum, L. fermenti, and L. brevis were the most frequently isolated, whereas L. casei, L. viridescens, L. cellobiosus, L. salivarius, and L. buchneri were obtained from small numbers of samples. The widespread but sporadic distribution of lactobacilli in low numbers seems to indicate that these organisms do not normally thrive on plant surfaces. A ternary cycle, beginning with intestinal waste, followed by mechanical distribution to and among plants, and return to the host via the oral cavity, is suggested.     

Influence of carbohydrates on cell properties of <Emphasis Type="Italic">Lactobacillus rhamnosus</Emphasis>

Lactobacilli represent normal commensals of the human body, particularly in the gut and vagina where they protect these environments from incoming pathogens via a variety of mechanisms. The influence of the carbohydrate source present in reconstituted MRS growth medium on the different cell properties of two Lactobacillus rhamnosus strains were examined. Two human vaginal isolates, BGHV719 and exopolysaccharide producer strain BGHV954 were analyzed. The results demonstrated that unlike in reconstituted MRS with glucose as a carbon source, the presence of fructose, mannose, or rhamnose, significantly reduced cell surface hydrophobicity of both strains. In addition, differences in cell wall protein composition of L. rhamnosus BGHV719 and alterations in colony mucoidity of L. rhamnosus BGHV954 were also demonstrated. Light and SEM microscopy revealed differences on the cellular level when BGHV719 was cultivated in the presence of different sugars. The results of this study point out the importance of complex relationships between growth medium composition and the different aspects of bacterial behavior, and call for more detailed analyses of versatile bacterial responses to the changes in the environment, including vaginal ecosystem. This is especially important since lactobacilli are amongst the most widely used of probiotics.     

Effect of culture media onLactobacillus hydrophobicity and electrophoretic mobility

The hydrophobicity of six strains representing three species ofLactobacillus was measured using dextran-polyethylene glycol contact angle measurements. These ranged from 123.6° forLactobacillus casei douche to 26.2° forL. casei RC-17 under identical growth conditions. The results indicated that the nutritional environment affected bacterial hydrophobicity. Electrophoretic mobilities of the lactobacilli were also determined and found to be negative for all specimens, and to vary with growth media, especially when sugars were added to urine. The electrophoretic mobility histograms showed one main peak for all strains, exceptLactobacillus acidophilus T-13 which had two peaks, suggesting two morphological sizes or types within its population. In addition, strain T-13 was more positively charged than the other five strains after growth in agar, urine, and supplemented urine. The use of contact angle and electrophoretic mobility techniques allows examination of cell surface properties of lactobacilli that may have importance in the colonization of mucosal epithelia.     

Transport and Metabolism of Lactose, Glucose, and Galactose in Homofermentative Lactobacilli

A number of species of lactobacilli were examined for their ability to ferment both the glucose and galactose moieties of lactose. Lactobacillus helveticus strains metabolized both the glucose and galactose moieties, whereas L. bulgaricus, L. lactis, and L. acidophilus strains metabolized only the glucose moiety and released galactose into the growth medium. All four species tested contained β-galactosidase activity, and no significant phospho-β-galactosidase activity was observed. L. bulgaricus and L. helveticus had a phosphoenolpyruvate (PEP):glucose phosphotransferase system for the uptake of glucose, but no evidence for a PEP:lactose phosphotransferase or PEP:galactose phosphotransferase system was obtained.     

(GTG)<Subscript>5</Subscript>-PCR fingerprinting of lactobacilli isolated from cervix of healthy women

A group of lactobacilli isolated from the cervix of 31 healthy women was characterized by (GTG)₅-polymerase chain reaction (PCR) fingerprinting in order to evaluate this method for identification of vaginal lactobacilli. Obtained fingerprints were compared with profiles available in an in-house database of the CCM bacteria collection covering type and reference strains of multiple lactic acid bacteria including lactobacilli. Selected strains representing individual clusters were further identified by pheS gene sequencing. In total, six lactobacillus species were found among lactobacilli isolated from the cervix of healthy women. The (GTG)₅-PCR method identified Lactobacillus gasseri (11 strains), Lactobacillus fermentum (one), and some of the Lactobacillus jensenii strains (eight out of 11), but failed to identify the remaining strains, including the Lactobacillus crispatus (18), Lactobacillus mucosae (one), and Lactobacillus vaginalis (one) species. L. jensenii strains were distributed over two fingerprint clusters. The majority of samples was dominated by one (GTG)₅-PCR type. The rep-PCR fingerprinting using the (GTG)₅ primer allowed straightforward identification of many, but not all, isolates. This method has been shown to be a useful tool for fast screening and grouping of vaginal lactobacilli, but its combination with another identification method is needed to obtain reliable identification results. In addition, Lactobacillus acidophilus was not shown to be the most common inhabitant of the female genital tract as generally assumed.     

Specific antigens of Lactobacillus acidophilus.

Antigens specific for Lactobacillus acidophilus were investigated by double immunodiffusion in agar-gel. Antigenic materials were extracted from whole bacteria and some walls with cold trichloroacetic acid. Antisera were prepared by intravenous injection into rabbits of suspensions of whole organisms in solutions of bovine serum albumin, which had been heated and then washed. Four specific antigens were found as precipitinogens and denoted as antigens 11, 12, 13 and 14. Of 43 strains of L. acidophilus studied, 33 strains possessed antigen 11, six strains antigen 12, two strains antigen 13 and two strains antigen 14. Sugar compositions of wall preparations were analysed in an attempt to characterize the determinants of antigens 11 and 12. The walls contained glucose, galactose, hexosamine and sometimes glycerol, but no rhamnose was found. It was considered that alpha-glucopyranose was the major component of the determinant of antigen 11 since trehalose and maltose significantly inhibited the reaction between antibody 11 and its antigen; the determinant of antigen 12 was not clarified.     

Isolation and Characterization of the Serotype g Carbohydrate Moiety from an Enzyme Lysate of Streptococcus mutans 6715 Cell Walls

The serotype-specific carbohydrate moiety of Streptococcus mutans was isolated by mild degradation of purified cell walls with a cell-wall lytic enzyme. Cell walls of serotype g S. mutans strain 6715 were digested with M1 enzyme, an endo-N-acetylmuramidase purified from culture supernatants of Streptomyces globisporus strain 1829. The enzyme lysate of the cell walls was applied to a CM Sephadex C-25 column to remove the M1 enzyme from the cell wall lysate and then subjected to Sephadex G-100 column chromatography. Carbohydrate antigens with serotype g specificity, designated M1g, and a peptidoglycan—polysaccharide complex lacking serotype specificity (M1PG) were separated. Purified serotype g antigen was also obtained by autoclaving the S. mutans 6715 whole cells in saline at 120 C for 30 min. The extract was applied to a DEAE Sephadex A-25 column to remove nucleic acids and teichoic acids. The unbound peak fraction was concentrated and re-chromatographed on a Bio-Gel P-100 column. The void volume fraction contained serotype g carbohydrate and was designated RRg antigen. M1g and RRg antigens formed a band of identity with anti-serotype g serum by immunodiffusion. These antigens were composed mainly of galactose, glucose, and rhamnose at an approximate weight ratio of 8 : 4 : 1, while constituent sugars of M1PG consisted of rhamnose and glucose, with no detectable galactose. M1g also contained peptidoglycan residues other than threonine, an interpeptide bridge component of the native cell wall peptidoglycan. Marked inhibition of the quantitative precipitin reaction between M1g and anti-serotype g serum was obtained with melibiose and galactose, which suggests that the immunodeterminant of the serotype g carbohydrate is an α-linked galactose-glucose terminal linkage.     

Chemical Studies on the Cell Walls of Leptospira biflexa Strain Urawa and Treponema pallidum Strain Reiter

The preparation and chemical poperties of the cell walls of Leptospira biflexa Urawa and Treponema pallidum Reiter are described. Both cell walls are composed mainly of polysaccharides and peptidoglycans. The data of chemical analysis indicate that the cell wall of L. biflexa Urawa contains rhamnose, arabinose, xylose, mannose, galactose, glucose and unidentified sugars as neutral sugars, and alanine, glutamic acid, α,ε-diaminopimelic acid, glucosamine and muramic acid as major amino acids and amino sugars. As major chemical constituents of the cell wall of T. pallidum Reiter, rhamnose, arabinose, xylose, mannose, galactose, glucose, alanine, glutamic acid, ornithine, glycine, glucosamine and muramic acid have been detected. The chemical properties of protein and polysaccharide fractions prepared from the cells of T. pallidum Reiter were also partially examined.     

Composition of the cell wall of Chlorella fusca

Isolated cell walls of mature Chlorella fusca consisted of about 80% carbohydrate, 7% protein, and 13% unidentified material. Mannose and glucose were present in a ratio of about 2.7:1 and accounted for most of the carbohydrate. Minor components were glucuronic acid, rhamnose, and traces of other sugars; galactose was absent. After treatment with 2 M trifluoroacetic acid or with 80% acetic acid/HNO₃ (10/1, v/v), a residue with a mannose/glucose ratio of 0.3:1 was obtained, probably representing a structural polysaccharide. An X-ray diffraction diagram of the walls showed one diffuse reflection at 0.44 nm and no reflections characteristic of cellulose. Walls from young cells contained about 51% carbohydrate, 12% protein, and 37% unidentified material. Mannose and glucose were also the main sugars; their absolute amounts per wall increased 6–7 fold during cell growth. Walls isolated with omission of a dodecylsulphate/mercaptoethanol/urea extraction step had a higher protein content and, with young walls, a significantly higher glucose and fucose content. These data and other published cell wall analyses show a wide variability in cell wall composition of the members of the genus Chlorella.Abbreviations GLC gas liquid chromatography - TFA trifluoroacetic acid     

Partial characterization and dynamics of synthesis of high molecular mass exopolysaccharides from Lactobacillus delbrueckii ssp. bulgaricus and Streptococcus thermophilus

Exopolysaccharide (EPS) preparations from Lactobacillus delbrueckii ssp. bulgaricus (L. bulgaricus) strains LBB.B26 and LBB.B332 and Streptococcus thermophilus strains LBB.T54 and LBB.T6V were characterized using ion-exchange chromatography and gel filtration. All four preparations contained a neutral EPS with molecular mass in the range of 1.3−1.6 × 10⁶ Da (HMM-EPS). The EPS preparations from the two L. bulgaricus strains also contained an acidic low molecular mass EPS fraction (LMM-EPS) comprising from 10% to 34% of the total EPS yield. HMM-EPS preparations were subjected to High Pressure Liquid Chromatography (HPLC) analysis of monomer sugars after complete hydrolysis. Glucose, galactose and/or rhamnose in different ratios proved to be the principal sugars building the HMM-EPS from all four strains. The chemical composition of HMM-EPS was strictly strain-specific. The LMM-EPS contained galactose. The viscosifying properties of the four different HMM-EPS varied greatly with intrinsic viscosity in the range from 0.26 (strain B26) to 2.38 (strain T6V). For 24 h the two L. bulgaricus strains accumulated more HMM-EPS in milk (>70 mg l⁻¹) than S. thermophilus strains T54 and T6V (<30 mg l⁻¹), but maximal yields were reached earlier with cocci (8 h) than with rods (16–24 h). The contribution of HMM-EPS production to increased viscosity of fermented milk was demonstrated for all of the tested strains grown as monocultures or as mixed yogurt starters compared to non-EPS producing S. thermophilus LBB.A and poor EPS-producer L. bulgaricus LBB.B5. The extent of increased viscosity was strongly dependent on the nature of the produced HMM-EPS, rather than simply on polymer yield.     

Phage Receptor Material in Lactobacillus casei Cell Wall

A trichloroacetic acid (TCA)-soluble fraction, extracted using cold TCA, was derived from the cell wall of Lactobacillus casei strain S-1. It not only inhibited the adsorption of phage J-l but also desorbed these phages, in their active form, which had previously been adsorbed onto the cell walls. l-Rhamnose, one of the components of this TCA-soluble fraction, had an identical activity to this TCA-soluble fraction, on phage adsorption. This suggested that l-rhamnose is a part of phage receptor material in the cell wall of L. casei strain S-l; and the binding of the phage to the cell wall is reversible, even at 37 C.     

Citrate metabolism in Lactobacillus casei and Lactobacillus plantarum

Information on the factors influencing citrate metabolism in lactobacilli is limited and could be useful in understanding the growth of lactobacilli in ripening cheese. Citrate was not used as an energy source by either Lactobacillus casei ATCC 393 or Lact. plantarum 1919 and did not affect the growth rate when co-metabolized with glucose or galactose. In growing cells, metabolism of citrate was minimal at pH 6 but significant at pH 4·5 and was greater in cells co-metabolizing galactose than in those co-metabolizing glucose or lactose. In non-growing cells, optimum utilization of citrate also occurred at pH 4·5 and was not increased substantially by the presence of fermentable sugars. In both growing and non-growing cells, acetate and acetoin were the major products of citrate metabolism; pyruvate was also produced by non-growing cells and was transformed to acetoin once the citrate was exhausted. Citrate was metabolized more rapidly than sugar by non-growing cells; the reverse was true of growing cells. Citrate metabolism by Lact. plantarum 1919 and Lact. casei ATCC 393 increased six- and 22-fold, respectively, when the cells were pre-grown on galactose plus citrate than when pre-grown on galactose only. This was probably due to induction of citrate lyase by growth on citrate plus sugar. These results imply that lactobacilli, if present in large enough numbers, can metabolize citrate in ripening cheese in the absence of an energy source.     

An in vitro protocol for direct isolation of potential probiotic lactobacilli from raw bovine milk and traditional fermented milks

A method for isolating potential probiotic lactobacilli directly from traditional milk-based foods was developed. The novel digestion/enrichment protocol was set up taking care to minimize the protective effect of milk proteins and fats and was validated testing three commercial fermented milks containing well-known probiotic Lactobacillus strains. Only probiotic bacteria claimed in the label were isolated from two out of three commercial fermented milks. The application of the new protocol to 15 raw milk samples and 6 traditional fermented milk samples made it feasible to isolate 11 potential probiotic Lactobacillus strains belonging to Lactobacillus brevis, Lactobacillus fermentum, Lactobacillus gasseri, Lactobacillus johnsonii, Lactobacillus plantarum, Lactobacillus reuteri, and Lactobacillus vaginalis species. Even though further analyses need to ascertain functional properties of these lactobacilli, the novel protocol set-up makes it feasible to isolate quickly potential probiotic strains from traditional milk-based foods reducing the amount of time required by traditional procedures that, in addition, do not allow to isolate microorganisms occurring as sub-dominant populations.     

Distribution and chemical characterization of regular arrays in the cell walls of strains of the genus Lactobacillus

Abstract The presence of regular arrays (RAs) in the cell walls of strains of the genus Lactobacillus was examined by electron microscopy. The RAs were found in 6 species including L. bulgaricus, L. helveticus, L. acidophilus, L. fermentum, L. brevis and L. buchneri . The RAs were composed of a protein with an apparent M _r ranging from about 41000 to 55000, depending on the species upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The amino acid composition of the RA proteins was shown to be acidic and hydrophobic. The antigenicity of the RA protein from L. buchneri appeared to be specific but not common among the RA proteins from the other lactobacilli.     

Distribution of indigenous Lactobacilli in the digestive tract of conventional and gnotobiotic rats.

Distribution of indigenous lactobacilli in the gastrointestinal tracts of rats was investigated at the species level. The indigenous lactobacilli isolated from conventional rats were divided into three groups, Lactobacillus acidophilus and its related strains, L. fermentum, and L. murini. Localization of the Lactobacillus groups in the gastrointestinal tracts could be distinguished clearly based on arabinose and glucose fermentation reaction of isolates from each part of the gastrointestinal tract. Group I (L. acidophilus and the related strains) and Group II (L. fermentum) were the major populations of lactobacilli on the walls of the non-glandular part and in the contents of the stomachs of both conventional and gnotobiotic rats. Group I predominated in all the parts of the digestive tract of conventional rats, whereas Group II was in the minority in the lower part of the gastrointestinal tracts of both groups of rats. Group III (L. murini) was the predominant population of Lactobacillus in the lower part of small intestine of conventional rats and in all parts of the gastrointestinal tracts of gnotobiotic rats except for the wall of the non-glandular part of the stomach.