Structure of a viscous exopolysaccharide produced by Lactobacillus helveticus K16

A viscous extracellular polysaccharide produced by Lactobacillus helveticus K16 has been investigated. Sugar and methylation analysis, 1H and 13C NMR spectroscopy revealed that the polysaccharide is composed of a hexasaccharide repeating unit. The sequence of sugar residues was determined by use of two-dimensional nuclear Overhauser effect spectroscopy and heteronuclear multiple bond connectivity experiments. The structure of the repeating unit of the exopolysaccharide from L. helveticus K16 is as follows: carbohydrate sequence [see text].     

Modeling of the structure in aqueous solution of the exopolysaccharide produced by Lactobacillus helveticus 766.

A method is described for constructing a conformational model in water of a heteropolysaccharide built up from repeating units, and is applied to the exopolysaccharide produced by Lactobacillus helveticus 766. The molecular modeling method is based on energy minima, obtained from molecular mechanics calculations of each of the constituting disaccharide fragments of the repeating unit in vacuo, as starting points. Subsequently, adaptive umbrella sampling of the potential of mean force is applied to extract rotamer populations of glycosidic dihedral angles of oligosaccharide fragments in solution. From these analyses, the most probable conformations are constructed for the hexasaccharide-repeating unit of the polysaccharide. After exploring the conformational space of each of the individual structures by molecular dynamics simulations, the different repeating unit conformations are used as building blocks for the generation of oligo- and polysaccharide models, by using a polysaccharide building program. The created models of the exopolysaccharide produced by L. helveticus 766 exhibit a flexible twisted secondary structure and tend to adopt a random coil conformation as tertiary structure.     

Caroteno-protein and exopolysaccharide production by co-cultures of Rhodotorula glutinis and Lactobacillus helveticus

The lactose-negative yeast Rhodotorula glutinis 22P and the homofermentative lactic acid bacterium Lactobacillus helveticus 12A were cultured together in a cheese whey ultrafiltrate containing 42 g L⁻¹ lactose. The chemical composition of the caroteno-protein has been determined. The carotenoid and protein contents are 248  μ g g⁻¹ dry cells and 48.2% dry weight. Carotenoids produced by Rhodotorula glutinis 22P have been identified as β-carotene 15%, torulene 10%, and torularhodin 69%. After separating the cell mass from the microbial association, the exopolysaccharides synthesized by Rhodotorula glutinis 22P were isolated from the supernatant medium in a yield of 9.2 g L⁻¹. The monosaccharide composition of the synthesized biopolymer was predominantly D-mannose (57.5%). Received 08 July 1996/ Accepted in revised form 11 December 1996     

Structural studies of the exopolysaccharide produced by Lactobacillus rhamnosus strain GG (ATCC 53103)

The structure of the extracellular polysaccharide (EPS) from Lactobacillus rhamnosus strain GG has been investigated. In combination with component analysis, NMR spectroscopy shows that the polysaccharide is composed of hexasaccharide repeating units. Sequential information was obtained by two-dimensional (1)H,(1)H-NOESY, and (1)H,(13)C-HMBC NMR techniques. The structure of the repeating unit of the EPS from Lactobacillus rhamnosus strain GG was determined as: [carbohydrate structure: see text]     

Partial characterization of a bacteriocin produced by Lactobacillus helveticus

AIMS: To investigate the antimicrobial activity of a strain of Lactobacillus helveticus. METHODS AND RESULTS: The culture supernatant fluid Lact. helveticus G51 showed antimicrobial activity against thermophilic strains of Lactobacillus. Purification of the active compound was achieved after gel filtration and ion exchange chromatography. As revealed by SDS-PAGE, active fractions were relatively homogeneous, showing a protein with a molecular mass of 12.5 kDa. The antimicrobial compound was heat labile, inactivated by proteolytic enzymes and had a bactericidal mode of action. CONCLUSION: The antimicrobial activity expressed by Lact. helveticus G51 was correlated with the production of a bacteriocin with properties that were different to other helveticins. SIGNIFICANCE AND IMPACT OF THE STUDY: The study has provided further data on Lact. helveticus bacteriocins. The strong activity of the bacteriocin towards various thermophilic lactobacilli warrants further investigation for its potential to obtain attenuated cultures for the enhancement of the cheese-ripening process.     

Structural characterisation of a perdeuteriomethylated exopolysaccharide by NMR spectroscopy: characterisation of the novel exopolysaccharide produced by Lactobacillus delbrueckii subsp. bulgaricus EU23

The exopolysaccharide (EPS) from Lactobacillus delbrueckii subsp. bulgaricus EU23 was perdeuteriomethylated and the perdeuteriomethylated EPS (pdm-EPS) purified by elution from a C(18) Sep-Pak cartridge. Both 1D and 2D NMR spectra were recorded for the pdm-EPS and these were interpreted to provide assignments for the individual 1H and 13C resonances of the sugar residues of the repeating unit. Using a combination of the results from monomer analysis and linkage analysis of the native EPS and the ROESY and HMBC NMR spectra of the pdm-EPS the following structure has been determined for the repeating unit:A process for characterising polysaccharides having low solubility in aqueous solution is reported.     

Structural characterisation of a highly branched exopolysaccharide produced by Lactobacillus delbrueckii subsp. bulgaricus NCFB2074

Lactobacillus delbrueckii subsp. bulgaricus NCFB2074 when grown in skimmed milk secretes a highly branched exopolysaccharide. The exopolysaccharide has a heptasaccharide repeat unit and is composed of glucose and galactose in the molar ratio 3:4. Using chemical techniques and 1D and 2D NMR spectroscopy the polysaccharide has been shown to possess the following repeat unit structure: [carbohydrate structure: see text].     

Characterization of a proteinaceous antimicrobial produced by Lactobacillus helveticus CNRZ450

An antimicrobial substance which resembles a bacteriocin was identified in culture supernatant fluids of Lactobacillus helveticus strain CNRZ450. The bacteriocin was active against a narrow range of strains from closely rested species of homofermentative lactobacilli. Its mode of action appeared to be bacteriostatic. Partial purification of the bacteriocin suggested that it was a complex protein with a mol. wt of between 30 and 50 kDa, although there is some evidence that the polypeptide monomer has a mol. wt of around 17 kDa. There was no evidence indicating an extrachromosomal location for its genetic determinant. PCR generated an amplicon from total DNA from strain CNRZ450 using primers based on the helJ gene sequence. A fragment showing homology to this amplicon was located in an Eco RI digest of total DNA from strain CNRZ450. The pattern obtained was different from that obtained with the helveticin J producer strain NCFB481. It is possible, therefore, that the antimicrobial from strain CNRZ450 is related to helveticin J at the DNA sequence level although the physical properties of the two antimicrobials reveal several differences.     

Structural determination of a neutral exopolysaccharide produced by Lactobacillus delbrueckii ssp. bulgaricus LBB.B332

The neutral exopolysaccharide produced by Lactobacillus delbrueckii ssp. bulgaricus LBB.B332 in skimmed milk was found to be composed of d-glucose, d-galactose, and l-rhamnose in a molar ratio of 1:2:2. Linkage analysis and 1D/2D NMR (1H and 13C) studies carried out on the native polysaccharide as well as on an oligosaccharide generated by a periodate oxidation protocol, showed the polysaccharide to consist of linear pentasaccharide repeating units with the following structure: -->3-alpha-D-Glcp-(1-->3)-alpha-D-Galp-(1-->3)-alpha-L-Rhap-(1-->2)-alpha-L-Rhap-(1-->2)-alpha-D-Galp-(1-->.     

Growth and exopolysaccharide production by Lactobacillus helveticus ATCC 15807 in an adenine-supplemented chemically defined medium

AIMS: To analyse the exopolysaccharide (EPS) production by Lactobacillus helveticus ATCC 15807 in a chemically defined medium (CDM) and the effect of nutrients and stress culture conditions on cell growth and EPS formation. METHODS AND RESULTS: Cultures were conducted in CDM: (i) containing essential and nonessential bases and vitamins; (ii) without nonessential bases and vitamins [Simplified CDM (SCDM)]; (iii) SCDM supplemented individually with vitamins and bases. The influence of carbohydrates, pH and osmotic culture conditions on growth and polymer formation was analysed. Adenine and lactose stimulated both growth and EPS production. Constant pH fermentations (4.5 and 6.2) did not improve EPS synthesis while NaCl and glycerol were detrimental for growth and polymer formation. In all media the EPS monomer composition was glucose and galactose (2.5 : 1). CONCLUSIONS: A SCDM containing adenine and lactose was optimal for cell growth and EPS formation by Lact. helveticus ATCC 15807. Controlled pH (6.2 and 4.5) and osmotic stress culture conditions did not improve polymer production. The EPS characteristics were identical in all media. SIGNIFICANCE AND IMPACT OF THE STUDY: This work provides a better knowledge on EPS synthesis by Lact. helveticus. A CDM to perform regulation studies on EPS production by Lact. helveticus species is now available.     

Heterofermentative pattern and exopolysaccharide production by Lactobacillus helveticus ATCC 15807 in response to environmental pH

AIMS: The objective of this work was to evaluate the fermentation pattern of and the exopolysaccharide (EPS) production by Lactobacillus helveticus ATCC 15807 in milk batch cultures under controlled pH (4.5, 5.0 and 6.2). METHODS AND RESULTS: EPS concentration was estimated by the phenol/sulphuric acid method and the chemical composition of purified EPS by HPLC. Fermentation products and residual sugars were determined by HPLC and enzymatic methods. The micro-organism shifted from a homofermentative to a heterofermentative pattern, producing acetate (9.5 and 5.8 mmol l(-1)) at pH 5.0 and 6.2, respectively, and acetate (7.1 mmol l(-1)) plus succinate (1.2 mmol l(-1)) at pH 4.5. At pH 5.0 and 6.2, acetate derived from citrate while at pH 4.5 it came from both citrate and pyruvate splitting. The EPS has a MW of 10(5)-10(6) and contains phosphate (81% in average), rhamnose (traces), and glucose and galactose in a ratio of 1 : 1 (pH 6.2) and 2 : 1 (pH 4.5 and 5.0). The highest production (549 mg l(-1)) corresponded to pH 5.0 and the lowest (49 mg l(-1)) to pH 6.2. CONCLUSIONS: The heterofermentative pattern of Lact. helveticus ATCC 15807 was linked to alternative pyruvate pathways and/or citrate metabolism according to the environmental pH. The EPS production was improved under low environmental pH conditions. SIGNIFICANCE AND IMPACT OF THE STUDY: This work provides relevant information of the effect of pH on the metabolism of citrate and EPS production by Lact. helveticus. It may contribute to improve technological aspects of ropy and citrate-utilizing lactic acid bacteria.     

Structural analysis of the curdlan-like exopolysaccharide produced by Cellulomonas flavigena KU

Cellulomonas flavigena KU produces large quantities of an insoluble exopolysaccharide (EPS) under certain growth conditions. The EPS has previously been shown to be a glucose polymer and to have solubility properties similar to curdlan, a β-1,3-D-glucan produced by Alcaligenes faecalis var. myxogenes 10C3K. Furthermore, EPS purified by alkaline extraction stains with aniline blue, a dye specific for curdlan-type polysaccharides. However, EPS-producing colonies of C. flavigena KU do not stain on aniline blue agar as do those of curdlan-producing bacteria. These facts prompted a more thorough structural analysis of the EPS. Here we report that purified EPS is indeed identical to curdlan in primary structure, but that the native form of the EPS may differ from curdlan in physical conformation. Journal of Industrial Microbiology & Biotechnology (2002) 29, 200–203 doi:10.1038/sj.jim.7000277 Received 19 February 2002/ Accepted in revised form 20 May 2002     

Structural characterisation of the exopolysaccharide produced by Streptococcus thermophilus EU20

Streptococcus thermophilus EU20 when grown on skimmed milk secretes a high-molecular-weight exopolysaccharide that is composed of glucose, galactose and rhamnose in a molar ratio of 2:3:2. Using chemical techniques and 1D and 2D-NMR spectroscopy (1H and 13C) the polysaccharide has been shown to possess a heptasaccharide repeating unit having the following structure: [chemical structure: see text]. Treatment of the polysaccharide with mild acid (0.5 M TFA, 100 degrees C for 1 h) liberates two oligosaccharides; the components correspond to the repeating unit and a hexasaccharide equivalent to the repeating unit minus the terminal alpha-L-Rhap.     

Structural studies of an exopolysaccharide produced by Streptococcus thermophilus THS

The structure of an extracellular polysaccharide (EPS) from Streptococcus thermophilus THS has been determined. A combination of component analysis, methylation analysis and NMR spectroscopy shows that the polysaccharide is composed of pentasaccharide repeating units. Sequential information was obtained by two-dimensional (1)H,(1)H-NOESY and (1)H,(13)C-HMBC NMR experiments. NMR data indicate different mobility within the EPS with a stiffer backbone and a more flexible side-chain.     

Structural studies of the main exopolysaccharide produced by the deep-sea bacterium Alteromonas infernus

The structure of the extracellular polysaccharide produced by the mesophilic species, Alteromonas infernus, found in deep-sea hydrothermal vents and grown under laboratory conditions, has been investigated using partial depolymerization, methylation analysis, mass spectrometry and NMR spectroscopy. The repeating units of this polysaccharide is a nonasaccharide with the following structure: [carbohydrate: see text].     

Structural study of the exopolysaccharide produced by a clinical isolate of Burkholderia cepacia

The primary structure of the exopolysaccharide produced by a clinical isolate of the bacterium Burkholderia cepacia was studied by means of methylation analysis, selective degradation, NMR spectroscopy, and electrospray mass spectrometry. The resulting data showed that the parent repeating unit of the exopolysaccharide is a highly branched heptasaccharide with the following structure: Two acetyl groups are present per repeating unit, as noncarbohydrate substituents.     

Structural analysis of the Lactobacillus rhamnosus strain KL37C exopolysaccharide

The exopolysaccharide from the lactic acid bacterium Lactobacillus rhamnosus strain KL37C isolated from human intestinal flora was prepared by sonication of bacterial cell mass suspended in water followed by centrifugation and cold ethanol precipitation of the supernatant. The polysaccharide material was purified by gel permeation chromatography on an TSK HW-50 column and characterised using chemical and enzymatic methods. On the basis of sugar and methylation analysis and 1H, 13C, 1D and 2D NMR spectroscopy the exopolysaccharide was shown to be composed of the following pentasaccharide repeating unit:-->3)-alpha-D-Glcp-(1-->2)-beta-D-Galf-(1-->6)-alpha-D-Galp-(1-->6)-alpha-D-Glcp-(1-->3)-beta-D-Galf-(1-->     

Structure and functions of exopolysaccharide produced by gut commensal Lactobacillus reuteri 100-23

Lactobacillus reuteri strain 100-23 together with a Lactobacillus-free mouse model, provides a system with which the molecular traits underpinning bacterial commensalism in vertebrates can be studied. A polysaccharide was extracted from sucrose-containing liquid cultures of strain 100-23. Chemical analysis showed that this exopolysaccharide was a levan (β-2, 6-linked fructan). Mutation of the fructosyl transferase (ftf) gene resulted in loss of exopolysaccharide production. The ftf mutant was able to colonise the murine gastrointestinal tract in the absence of competition, but colonisation was impaired in competition with the wild type. Biofilm formation by the mutant on the forestomach epithelial surface was not impaired and the matrix between cells was indistinguishable from that of the wild type in electron micrographs. Colonisation of the mouse gut by the wild-type strain led to increased proportions of regulatory T cells (Foxp3+) in the spleen, whereas colonisation by the ftf mutant did not. Survival of the mutant in sucrose-containing medium was markedly reduced relative to the wild type. Comparison of the genomic ftf loci of strain 100-23 with other L. reuteri strains suggested that the ftf gene was acquired by lateral gene transfer early in the evolution of the species and subsequently diversified at accelerated rates. Levan production by L. reuteri 100-23 may represent a function acquired by the bacterial species for life in moderate to high-sucrose extra-gastrointestinal environments that has subsequently been diverted to novel uses, including immunomodulation, that aid in colonisation of the murine gut.     

Physicochemical properties of exopolysaccharide produced by Lactobacillus kefiranofaciens ZW3 isolated from Tibet kefir

An exopolysaccharide (EPS) producing strain, ZW3, was isolated from Tibet kefir grain and was identified as Lactobacillus kefiranofaciens. FT-IR spectroscopy revealed the presence of carboxyl, hydroxyl, and amide groups, which correspond to a typical heteropolymeric polysaccharide. The GC analysis of ZW3 EPS revealed that it was glucogalactan in nature. Exopolymer showed similar flocculation stability like xanthan gum but better than guar gum with a melting point of 93.38 degrees C which is lower than xanthan gum (153.4 degrees C) and guar gum (490.11 degrees C). Compared with other commercially available hydrocolloids like xanthan gum, guar gum and locust gum ZW3 EPS showed much better emulsifying capability.