Dataset Information

Small-scale analysis of exopolysaccharides from Streptococcus thermophilus grown in a semi-defined medium.

ABSTRACT: BACKGROUND:Exopolysaccharides (EPSs) produced by lactic acid bacteria are important for the texture of fermented foods and have received a great deal of interest recently. However, the low production levels of EPSs in combination with the complex media used for growth of the bacteria have caused problems in the accurate analysis of the EPS. The purpose of this study was to find a growth medium for physiological studies of the lactic acid bacterium Streptococcus thermophilus, and to develop a simple method for qualitative and quantitative analysis of EPSs produced in this medium. RESULTS:A semi-defined polysaccharide medium was developed and evaluated on six strains of Streptococcus thermophilus. The EPSs were analysed using a novel protocol incorporating ultracentrifugation for the removal of interfering sugars, hydrolysis and analysis of the monomer composition by High Performance Anion-Exchange Chromatography with pulsed amperometric detection. The medium and analysis method allowed accurate quantification and monomer analysis of 0.5 ml samples of EPSs from tube cultures. CONCLUSIONS:The presented medium should be useful for physiological studies of S. thermophilus, and, in combination with the method of analysis of EPS, will allow downscaling of physiological studies and screening for EPSs.

SUBMITTER: Levander F

PROVIDER: S-EPMC57807 | biostudies-literature | 2001

REPOSITORIES: biostudies-literature

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Small-scale analysis of exopolysaccharides from Streptococcus thermophilus grown in a semi-defined medium.

Levander F F Svensson M M Rådström P P

BMC microbiology 20010926

<h4>Background</h4>Exopolysaccharides (EPSs) produced by lactic acid bacteria are important for the texture of fermented foods and have received a great deal of interest recently. However, the low production levels of EPSs in combination with the complex media used for growth of the bacteria have caused problems in the accurate analysis of the EPS. The purpose of this study was to find a growth medium for physiological studies of the lactic acid bacterium Streptococcus thermophilus, and to devel ...[more]

PMID: 11602017

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Project description:BackgroundThe marine thermophilic bacterium Rhodothermus marinus can degrade many polysaccharides which makes it interesting as a future cell factory. Progress using this bacterium has, however, been hampered by limited knowledge on media and conditions for biomass production, often resulting in low cell yields and low productivity, highlighting the need to develop conditions that allow studies of the microbe on molecular level. This study presents development of defined conditions that support growth, combined with evaluation of production of carotenoids and exopolysaccharides (EPSs) by R. marinus strain DSM 16675.ResultsTwo defined media were initially prepared: one including a low addition of yeast extract (modified Wolfe's medium) and one based on specific components (defined medium base, DMB) to which two amino acids (N and Q), were added. Cultivation trials of R. marinus DSM 16675 in shake flasks, resulted in maximum cell densities (OD620 nm) of 2.36 ± 0.057, cell dry weight (CDW) 1.2 ± 0.14 mg/L, total carotenoids 0.59 × 10-3 mg/L, and EPSs 1.72 ± 0.03 mg/L using 2 g/L glucose in DMB. In Wolfe's medium (supplemented by 0.05 g/L yeast extract and 2.5 g/L glucose), maximum OD620 nm was 2.07 ± 0.05, CDW 1.05 ± 0.07 mg/L, total carotenoids 0.39 × 10-3 mg/L, and EPSs 1.74 ± 0.2 mg/L. Growth trials at 5 g/L glucose in these media either failed or resulted in incomplete substrate utilization. To improve reproducibility and increase substrate utilization, a screening of macroelements (e.g. phosphate) in DMB, was combined with use of trace elements and vitamins of the modified Wolfe's medium. The resulting defined minimal R. marinus medium, (DRM), allowed reproducible cultivations to a final OD620nm of 6.6 ± 0.05, CDW 2.85 ± 0.07 mg/L, a maximum specific growth rate (µmax) of 0.26 h-1, total carotenoids 0.77 × 10-3 mg/L and EPSs 3.4 ± 0.17 mg/L in cultivations supplemented with up to 5 g/L glucose.ConclusionA minimal defined medium (DRM) was designed that resulted in reproducible growth and an almost doubled formation of both total carotenoids and EPSs. Such defined conditions, are necessary for systematic studies of metabolic pathways, to determine the specific requirements for growth and fully characterize metabolite production.

Project description:The cariogenic pathogen Streptococcus mutans effectively utilizes dietary sucrose for the synthesis of exopolysaccharide, which act as a scaffold for its biofilm, thus contributing to its pathogenicity, environmental stress tolerance, and antimicrobial resistance. The two-component system VicRK of S. mutans regulates a group of virulence genes that are associated with biofilm matrix synthesis. Knockout of vicX affects biofilm formation, oxidative stress tolerance, and transformation of S. mutans. However, little is known regarding the vicX-modulated structural characteristics of the exopolysaccharides underlying the biofilm formation and the phenotypes of the vicX mutants. Here, we identified the role of vicX in the structural characteristics of the exopolysaccharide matrix and biofilm physiology. The vicX mutant (SmuvicX) biofilms seemingly exhibited "desertification" with architecturally impaired exopolysaccharide-enmeshed cell clusters, compared with the UA159 strain (S. mutans wild type strain). Concomitantly, SmuvicX showed a decrease in water-insoluble glucan (WIG) synthesis and in WIG/water-soluble glucan (WSG) ratio. Gel permeation chromatography (GPC) showed that the WIG isolated from the SmuvicX biofilms had a much lower molecular weight compared with the UA159 strain indicating differences in polysaccharide chain lengths. A monosaccharide composition analysis demonstrated the importance of the vicX gene in the glucose metabolism. We performed metabolite profiling via (1)H nuclear magnetic resonance spectroscopy, which showed that several chemical shifts were absent in both WSG and WIG of SmuvicX biofilms compared with the UA159 strain. Thus, the modulation of structural characteristics of exopolysaccharide by vicX provides new insights into the interaction between the exopolysaccharide structure, gene functions, and cariogenicity. Our results suggest that vicX gene modulates the structural characteristics of exopolysaccharide associated with cariogenicity, which may be explored as a potential target that contributes to dental caries management. Furthermore, the methods used to purify the EPS of S. mutans biofilms and to analyze multiple aspects of its structure (GPC, gas chromatography-mass spectrometry, and (1)H nuclear magnetic resonance spectroscopy) may be useful approaches to determine the roles of other virulence genes for dental caries prevention.

Dataset Information

Small-scale analysis of exopolysaccharides from Streptococcus thermophilus grown in a semi-defined medium.

Publications

Small-scale analysis of exopolysaccharides from Streptococcus thermophilus grown in a semi-defined medium.

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