Characterization of the Lactobacillus casei group and the Lactobacillus acidophilus group by automated ribotyping

A total of 91 type and reference strains of the Lactobacillus casei group and the L acidophilus group were characterized by the automated ribotyping device Riboprinter microbial characterization system. The L. casei group was divided into five (C1-C5) genotypes by ribotyping. Among them, the strain of L. casei ATCC 334 was clustered to the same genotype group as most of L. paracasei strains and L casei JCM 1134T generated a riboprint pattern that was different from the type strain of L. zeae. These results supported the designation of L. casei ATCC 334 as the neotype strain, but were not consistent with the reclassification of L. casei JCM 1134T as L. zeae. The L. acidophilus group was also divided into 14 (A1-A11, B1-B3) genotypes by ribotyping. L. acidophilus, L. amylovorus, L. crispatus and L. gallinarum generated ribotype patterns that were distinct from the patterns produced by L. gasseri and L. johnsonii. This result confirmed previous data that the L. acidophilus group divided to two major clusters. Five strains of L. acidophilus and two strains of L. gasseri were correctly reidentified by ribotyping. Most strains belonging to the L. casei group and the L. acidophilus group were discriminated at the species level by automated ribotyping. Thus this RiboPrinter system yields rapid, accurate and reproducible genetic information for the identification of many strains.     

Culture media for differential isolation of Lactobacillus casei Shirota from oral samples

This study aimed to develop a solid culture medium for differential isolation of the probiotic strain Lactobacillus casei Shirota (LcS) and for selective cultivation of lactobacilli present in oral samples. Type strains of lactobacilli and isolates from commercial probiotic products were inoculated onto modified de Man Rogosa Sharpe agar (termed 'LcS Select'), containing bromophenol blue pH indicator, vancomycin and reducing agent L-cysteine hydrochloride for differential colony morphology development. L. casei Shirota cultured on the novel medium produced distinctive colony morphologies, different from other lactobacilli tested. LcS-characteristic colonies were recovered on LcS Select medium from samples of saliva and tongue plaque following a four-week probiotic intervention study. The viable count of presumptive LcS colonies correlated with those isolated on a non-commercial lactitol-LBS-vancomycin agar (LLV) developed for a selective isolation of LcS from faeces. The novel LcS Select medium proved suitable for differential isolation of the probiotic strain L. casei Shirota from oral samples containing mixed microbial populations. It can also be used for selective growth of vancomycin-resistant lactobacilli. There are few available culture media that are sufficiently selective to enable isolation of probiotic strains from mixed populations. LcS Select medium provides a cheaper, yet effective tool in this context.     

Convergent synthesis of higher-order oligosaccharides corresponding to the cell-wall polysaccharide of the beta-hemolytic Streptococci group A. A branched hexasaccharide hapten.

A convergent synthesis of a hexasaccharide corresponding to the cell-wall polysaccharide of the beta-hemolytic Streptococci Group A is described. The strategy relies on the preparation of a key branched trisaccharide unit alpha-L-Rhap-(1----2)-[beta-D-GlcpNAc-(1----3)]-alpha-L-Rhap which functions both as a glycosyl acceptor and donor. The hexasaccharide is obtained after only three glycosylation reactions. This fully functionalized unit can serve, in turn, as a glycosyl acceptor or donor for the synthesis of higher-order structures. Deprotection gives a hexasaccharide for use as a hapten in immunochemical studies. The characterization of all compounds by high resolution 1H- and 13C-n.m.r. spectroscopy is also described.     

The linkage between the polysaccharide and mucopeptide components of the cell wall of Lactobacillus casei

1. The linkage between the polysaccharide and mucopeptide components of the cell wall of Lactobacillus casei is rapidly hydrolysed under mild acid-hydrolysis conditions. 2. The release of the polysaccharide is accompanied by the hydrolysis of an N-acetylhexosaminide linkage. The N-acetylhexosamine residue readily forms chromogen and it is concluded that it is substituted on C₍₃₎ by the adjacent sugar. 3. Continued heating of the polysaccharide in acid results in a slower release of reactive N-acetylhexosamine due to the hydrolysis of glycosidic linkages within the polysaccharide. 4. After the linkage between the polysaccharide and mucopeptide has been hydrolysed, acid phosphatase will release approx. 40% of the total phosphorus as inorganic phosphate. 5. It is concluded that the polysaccharide component of the cell wall is joined through its reducing end group to a phosphate grouping in the mucopeptide.     

Utilization of natural fucosylated oligosaccharides by three novel alpha-L-fucosidases from a probiotic Lactobacillus casei strain

Three putative α-L-fucosidases encoded in the Lactobacillus casei BL23 genome were cloned and purified. The proteins displayed different abilities to hydrolyze natural fucosyloligosaccharides like 2'-fucosyllactose, H antigen disaccharide, H antigen type II trisaccharide, and 3'-, 4'-, and 6'-fucosyl-GlcNAc. This indicated a possible role in the utilization of oligosaccharides present in human milk and intestinal mucosa.     

Properties of the polysaccharide and mucopeptide components of the cell wall of Lactobacillus casei

1. The polysaccharide and mucopeptide components of the cell wall of Lactobacillus casei have been separated by mild conditions of acid hydrolysis. 2. Removal of the polysaccharide renders the mucopeptide susceptible to lysozyme. 3. The mucopeptide and polysaccharide components have been analysed and the results compared with those obtained previously. 4. The polysaccharides responsible for group specificity have a terminal reducing N-acetylgalactosamine residue substituted on C((3)) by the adjacent sugar; estimation of this component gave an indication of the molecular weight of the polysaccharides. 5. Evidence has been obtained for the presence of rhamnosyl-(1-->3)-N-acetylgalactosamine among the products of acid hydrolysis of the group B polysaccharide.     

Taxonomic and strain-specific identification of the probiotic strain Lactobacillus rhamnosus 35 within the Lactobacillus casei group

Lactobacilli are lactic acid bacteria that are widespread in the environment, including the human diet and gastrointestinal tract. Some Lactobacillus strains are regarded as probiotics because they exhibit beneficial health effects on their host. In this study, the long-used probiotic strain Lactobacillus rhamnosus 35 was characterized at a molecular level and compared with seven reference strains from the Lactobacillus casei group. Analysis of rrn operon sequences confirmed that L. rhamnosus 35 indeed belongs to the L. rhamnosus species, and both temporal temperature gradient gel electrophoresis and ribotyping showed that it is closer to the probiotic strain L. rhamnosus ATCC 53103 (also known as L. rhamnosus GG) than to the species type strain. In addition, L. casei ATCC 334 gathered in a coherent cluster with L. paracasei type strains, unlike L. casei ATCC 393, which was closer to L. zeae; this is evidence of the lack of relatedness between the two L. casei strains. Further characterization of the eight strains by pulsed-field gel electrophoresis repetitive DNA element-based PCR identified distinct patterns for each strain, whereas two isolates of L. rhamnosus 35 sampled 40 years apart could not be distinguished. By subtractive hybridization using the L. rhamnosus GG genome as a driver, we were able to isolate five L. rhamnosus 35-specific sequences, including two phage-related ones. The primer pairs designed to amplify these five regions allowed us to develop rapid and highly specific PCR-based identification methods for the probiotic strain L. rhamnosus 35.     

The glycolipids of Lactobacillus casei A.T.C.C. 7469

1. The lipids were extracted from Lactobacillus casei A.T.C.C. 7469 with chloroform-methanol mixtures. The glycolipids were obtained by chromatography on silicic acid and DEAE-cellulose (acetate form). 2. Hydrolysis of the glycolipids with alkali gave two glycerol glycosides and a mixture of fatty acids. 3. The glycosides were separated and their structures elucidated. The major component was O-alpha-d-galactopyranosyl-(1-->2)-O-alpha-d-glucopyranosyl-(1-->1)-glycerol and the minor component O-alpha-d-glucopyranosyl-(1-->6)-O-alpha-d-galactopyranosyl-(1-->2)-O-alpha-d-glucopyranosyl-(1-->1)-glycerol. 4. Analysis of the fatty acids by gas-liquid chromatography showed that they were predominantly palmitic acid, octadecenoic acid and lactobacillic acid.     

C群脑膜炎球菌多糖纯化方法的改进

将C群脑膜炎球菌粗多糖的纯化改进为用1:1容量冷酚提取的生产工艺。结果表明,改进后的方法去除蛋白质效果同样能够达到《中国药典》2005版(三部)的要求,总体结果优于改进前。同时能扩大处理量而降低了生产成本,适合规模化生产。     

Cell surface characteristics of Lactobacillus casei subsp. casei, Lactobacillus paracasei subsp. paracasei, and Lactobacillus rhamnosus strains.

Hydrophilic and electrostatic cell surface properties of eight Lactobacillus strains were characterized by using the microbial adhesion to solvents method and microelectrophoresis, respectively. All strains appeared relatively hydrophilic. The strong microbial adhesion to chloroform, an acidic solvent, in comparison with microbial adhesion to hexadecane, an apolar n-alkane, demonstrated the particularity of lactobacilli to have an important electron donor and basic character and consequently their potential ability to generate Lewis acid-base interactions with a support. Regardless of their electrophoretic mobility (EM), strains were in general slightly negatively charged at alkaline pH. A pH-dependent behavior concerning cell surface charges was observed. The EM decreased progressively with more acidic pHs for the L. casei subsp. casei and L. paracasei subsp. paracasei strains until the isoelectric point (IEP), i.e., the pH value for which the EM is zero. On the other hand, the EM for the L. rhamnosus strains was stable from pH 8 to pH 3 to 4, at which point there was a shift near the IEP. Both L. casei subsp. casei and L. paracasei subsp. paracasei strains were characterized by an IEP of around 4, whereas L. rhamnosus strains possessed a markedly lower IEP of 2. The present study showed that the cell surface physicochemical properties of lactobacilli seem to be, at least in part and under certain experimental conditions, particular to the bacterial species. Such differences detected between species are likely to be accompanied by some particular changes in cell wall chemical composition.     

The complete primary structure of the allosteric L-lactate dehydrogenase from Lactobacillus casei

The polypeptide chain of the allosteric L-lactate dehydrogenase (EC 1.1.1.27) of Lactobacillus casei consists of 325 amino acid residues. Despite the strikingly different enzymatic characteristics of the allosteric L-lactate dehydrogenase of L. casei and of the non-allosteric vertebrate enzymes, the sequence of the allosteric enzyme shows a distinct homology with that of the non-allosteric vertebrate enzymes (average identity: 37%). An especially high sequence homology can be identified within the active center (average identity: 70%). A clear deviation of the L. casei enzyme from the vertebrate enzyme is the lack of the first 12 amino acid residues at the N terminus and an additional 7 amino acid residues at the C terminus. The localization of the binding site of the allosteric effector D-fructose 1,6-bisphosphate and pH and effector-induced changes of the spectroscopic properties are discussed on the basis of the primary structure.     

Functional multiplicity of phosphoglucose isomerase from Lactobacillus casei

Phosphoglucose isomerase (D-glucose-6-phosphate ketolisomerase, EC 5.3.1.9), purified from Lactobacillus casei, showed multiplicity with respect to electrophoretic mobility, molecular weight, kinetic properties and responses to erythrose 4-phosphate. Among the three forms isolated, one having a dimeric conformation, was specific for glucose 6-phosphate. Erythrose 4-phosphate inhibited this preparation in a sigmoid fashion, while this compound activated the enzyme for isomerization of ribose 5-phosphate. In tetrameric conformation of the similar subunits, the enzyme was more specific for ribose 5-phosphate and the inhibition exerted by erythrose 4-phosphate was hyperbolic. The possible implications of these observations have been discussed.     

Differentiation of Lactobacillus casei, Lactobacillus paracasei and Lactobacillus rhamnosus by polymerase chain reaction

Lactobacillus casei, Lact. paracasei and Lact. rhamnosus form a closely related taxonomic group within the heterofermentative lactobacilli. These three species are difficult to differentiate using traditional fermentation profiles. We have developed polymerase chain reaction primers which are specific for each of these species based on differences in the V1 region of the 16S rRNA gene. Sixty-three Lactobacillus isolates from cheese were identified using these primers. The 12 Lact. rhamnosus and 51 Lact. paracasei identified in this way were also differentiated using a randomly amplified polymorphic DNA (RAPD) primer.