Small-scale analysis of exopolysaccharides from Streptococcus thermophilus grown in a semi-defined medium
© Levander et al; licensee BioMed Central Ltd. 2001
Received: 23 July 2001
Accepted: 26 September 2001
Published: 26 September 2001
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.
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.
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.
The texture of fermented milk products is often dependent on the exopolysaccharides (EPSs) produced by lactic acid bacteria. The biosynthesis of EPSs has been extensively studied in recent years, and the amount of EPSs produced and their composition have been the focus of a number of studies [for a review see ]. However, many of the studies have been limited by the fact that complex growth media often contain polysaccharides, and that the methods used for quantification of EPSs require large sample volumes while still having considerable standard deviations. Defined or semi-defined media for the production of EPSs have been described for Lacotococcus lactis[2, 3] and Lactobacillus bulgaricus[4, 5]. However, to our knowledge, no such medium has been described for the industrially important Streptococcus thermophilus, which is widely used for yoghurt and mozzarella production.
Traditional methods for purification of EPSs involve removal of the cells by centrifugation, and protein removal by trichloracetic acid (TCA) precipitation or protease digestion . The polysaccharides are then precipitated with ethanol or acetone, and purified by repeated washing and precipitation or dialysis to remove sugars that interfere with the analysis. The EPSs are normally quantified as total sugars using phenol-sulphuric acid  or anthrone , by weight or by refractive index detection after gel-permeation chromatography . The isolation is laborious and, in addition, some polysaccharides may be difficult to dissolve once precipitated. Furthermore, large sample volumes are required for accurate quantification.
Here we describe a semi-defined medium, which does not contain any background polysaccharides, and in which different strains of Streptococcus thermophilus grow rapidly and produce EPSs. Furthermore, a new small-scale analysis method, which enables simultaneous quantification and monomer analysis of low concentrations of EPS is described.
Results and Discussion
Medium for EPS production
Composition of MST.
Amount per litre*
2.5 g (5.0 without pH control)
2.5 g (5.0 without pH control)
Casamino Acids (Difco)
Method of EPS quantification
Evaluation of medium and analysis
Comparison of strains in MST
Composition of repeating
0.93 ± 0.06
2 Gal, 2 Glc
0.44 ± 0.04
0.63 ± 0.03
0.62 ± 0.03
2 Gal, 2 Glc
0.92 ± 0.08
1 GalN, 2 Gal, 1 Glc
0.96 ± 0.01
Another promising approach for the quantification of EPS has been described by Looijesteijn and Hugenholtz . EPSs are separated from monosaccharides in culture supernatants by gel permeation chromatography and are quantified by refractive index detection. However, the high detection limit of refractive index detection makes this method suitable only for quantification of EPSs from strains producing high levels of EPSs. Quantification by PAD may provide some improvement. Unfortunately, we found that the response factor for EPSs of PAD using a quadruple waveform for carbohydrate detection was very low before hydrolysis (data not shown). Therefore, hydrolysis of the EPSs will probably be necessary before this detection method can be used for quantification.
The MST medium described in this paper should be useful for physiological studies of S. thermophilus. In combination with the purification and analysis method for EPS it can be used for small-scale experiments, and will be useful for screening strains of S. thermophilus for EPSs.
Materials and Methods
Bacterial strains, media and culture conditions
The S. thermophilus strains used in this study were obtained from the University of Huddersfield, except for DSM 8713, which was obtained from the German Collection of Microorganisms and Cell Cultures (DSMZ), and LY03 which was obtained from Vrije Universiteit Brussel, Belgium. The strains were routinely cultured at 42°C in Ml 7 (Oxoid) and stored at -80°C. For studies in MST medium (Table 1, for protocol see Additional file: MST.rtf)), an overnight culture in MST was used as inoculum (2% vol/vol). Tube cultures were performed in glass tubes with an initial volume of 10 ml, and growth was monitored in a tube spectrophotometer by measuring the optical density at 620 nm. pH-controlled batch cultivation was performed in a Biostat fermentor (B. Braun). The pH was adjusted to 6.2 by automatic addition of 10 N NaOH, and the stirring speed was set to 100 rpm. Anaerobic conditions were obtained by nitrogen flushing in the head space. Lactose, galactose and lactate were analysed by HPLC as described previously . Cell growth was monitored by measuring the optical density (OD) at 620 nm. Samples with an OD greater than 0.4 were diluted.
EPS isolation and characterisation
For isolation of EPSs, culture samples were centrifuged at 14,000 × g for 5 minutes to remove the cells. To the supernatant, 1/3 volume of 40% TCA was added in order to inactivate EPS-degrading enzymes and to precipitate proteins. The samples were then stored at 4°C until analysis. The samples were centrifuged for 10 minutes at 20,000 × g, 4°C. Supernatant, 0.5 ml, was added to a microcon M-30 ultracentrifugation filter (Millipore) and centrifuged at 14,000 × g for 20 min. The washing was repeated twice, and finally 0.5 ml water was added and the retentate was eluted upside down at 1000 × g for 3 minutes. For quantification and monomer analysis of the EPSs, 0.25 ml of 8 M HCl or 41 μl TFA was added to 0.25 ml of purified sample. The EPSs were hydrolysed at 100°C for 45 minutes in HCl or for 3 h in TFA. The samples were dried in a Speedvac AES2000 (Savant) at low temperature, dissolved in 1 ml of water, and analysed with HPAEC with PAD. The injection volume was 25 μl and the samples were separated on a Carbopac PA1 column (Dionex, Sunnyvale, CA, USA) with a pre-column in 16 mM NaOH at 1 ml/min. Post-column 300 mM NaOH at 0.5 ml/min was added for optimal detection. The standards contained 1, 5 and 10 μM each of rhamnose, galactosamine, glucosamine, galactose and glucose.
This work was supported by the European Community FAIR programme, contract no CT-98-4267.
- De Vuyst L, Degeest B: Heteropolysaccharides from lactic acid bacteria. FEMS Microbiol Rev. 1999, 23: 153-177. 10.1016/S0168-6445(98)00042-4.View ArticlePubMed
- Looijesteijn PJ, Hugenholtz J: Uncoupling of growth and exopolysaccharide production by Lactococcus lactis subsp. cremoris NIZO B40 and optimization of its synthesis. J Biosci Bioeng. 1999, 88: 178-182. 10.1016/S1389-1723(99)80198-4.View ArticlePubMed
- van Niel EWJ, Hahn-Hägerdal B: Nutrient requirements of lactococci in defined growth media. Appl Microbiol Biotechnol. 1999, 52: 617-627. 10.1007/s00253005156910.1007/s002530051569.View Article
- Grobben GJ, Chin-Joe I, Kitzen VA, Boels IC, Boer F, Sikkema J, Smith MR, de Bont JAM: Enhancement of exopolysaccharide production by Lactobacillus delbrueckii subsp. bulgaricus NCFB 2772 with a simplified defined medium. Appl Environ Microbiol. 1998, 64: 1333-1337.PubMed CentralPubMed
- Chervaux C, Ehrlich SD, Maguin E: Physiological study of Lactobacillus delbrueckii subsp. bulgaricus strains in a novel chemically defined medium. Appl Environ Microbiol. 2000, 66: 5306-5311. 10.1128/AEM.66.12.5306-5311.2000.PubMed CentralView ArticlePubMed
- Cerning J: Exocellular polysaccharides produced by lactic acid bacteria. FEMS Microbiol Rev. 1990, 7: 113-130.View ArticlePubMed
- Dubois M, Gilles KA, Hamilton JK, Rebers PA, Smith F: Colorimetric method for determination of sugars and related substances. Anal Chem. 1956, 28: 350-356.View Article
- Morris DL: Quantitative determination of carbohydrates with Dreywood's anthrone reagent. Science. 1948, 107: 254-255.View ArticlePubMed
- van Marle ME, Zoon P: Permeability and rheological properties of microbially and chemically acidified skim-milk gels. Neth Milk Dairy J. 1995, 49: 47-65.
- Deutsch SM, Molle D, Gagnaire V, Piot M, Atlan D, Lortal S: Hydrolysis of sequenced β-casein peptides provides new insight into peptidase activity from thermophilic lactic acid bacteria and highlights intrinsic resistance of phosphopeptides. Appl Environ Microbiol. 2000, 66: 5360-5367. 10.1128/AEM.66.12.5360-5367.2000.PubMed CentralView ArticlePubMed
- Hardy MR, Townsend RR, Lee YC: Monosaccharide analysis of glycoconjugates by anion exchange chromatography with pulsed amperometric detection. Anal Biochem. 1988, 170: 54-62.View ArticlePubMed
- Marshall VM, Laws AP, Gu Y, Levander F, Rådström P, De Vuyst L, Degeest B, Vaningelgem F, Dunn H, Elvin M: Exopolysaccharide-producing strains of thermophilic lactic acid bacteria cluster into groups according to their EPS structure. Lett Appl Microbiol. 2001, 32: 433-437. 10.1046/j.1472-765X.2001.00937.x.View ArticlePubMed
- Degeest B, De Vuyst L: Indication that the nitrogen source influences both amount and size of exopolysaccharides produced by Streptococcus thermophilus LY03 and modelling of the bacterial growth and exopolysaccharide production in a complex medium. Appl Environ Microbiol. 1999, 65: 2863-2870.PubMed CentralPubMed
- De Vuyst L, Vanderveken F, Van de Ven S, Degeest B: Production by and isolation of exopolysaccharides from Streptococcus thermophilus grown in a milk medium and evidence for their growth-associated biosynthesis. J Appl Microbiol. 1998, 84: 1059-1068. 10.1046/j.1365-2672.1998.00445.x.View ArticlePubMed
- Levander F, Råström P: Requirement for phosphoglucomutase in exopolysaccharide biosynthesis in glucose- and lactose-utilizing Streptococcus thermophilus. Appl Environ Microbiol. 2001, 67: 2734-2738. 10.1128/AEM.67.6.2734-2738.2001.PubMed CentralView ArticlePubMed
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