Structural Studies of an Exopolysaccharide Produced by Streptococcus thermophilus THS Eva-Lisa Nordmark, † Zhennai Yang, ‡ Eine Huttunen, ‡ and Go ¨ ran Widmalm* ,† Department of Organic Chemistry, Arrhenius Laboratory, Stockholm University, S-106 91 Stockholm, Sweden, and Department of Food Technology, University of Helsinki, FIN-00014 Helsinki, Finland Received June 16, 2004; Revised Manuscript Received September 16, 2004 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. Introduction Streptococcus thermophilus is a thermophilic species of lactic acid bacteria that is widely used as starters in dairy industry for the manufacture of fermented products. For example, S. thermophilus in combination with Lactobacillus delbrueckii ssp. bulgaricus is used in starters for yogurt production. In the natural starters for producing certain types of hard cheese, S. thermophilus strains are present predomi- nantly together with other lactic acid bacteria. The impor- tance of S. thermophilus in dairy fermentation lies not only in its production of organic acids which decrease pH of the medium, but also in the ability to produce various other beneficial components, one of them being exopolysaccharides (EPS). Several evidence indicate that production of EPS may confer the fermented products with improved rheological properties, e.g., texture and consistency, and decreased susceptibility to syneresis, i.e., separation of the liquid part from the firm mass, in yogurts, as well as enhanced moisture retention and melting properties in cheese. 1-6 The rheological properties of EPS are influenced by chemical composition, monosaccharide linkages, side-chain groups, and polymer length. The EPS of S. thermophilus Sfi12 which contains R-linked sugar residues had more flexible chains than that of the EPS from Lactococcus lactis ssp. cremoris B40 with -linked sugar residues in the backbone, the former showing a slimy texture of the culture. 7,8 Partial removal of the groups in the side-chain of the EPS from Lactococcus lactis ssp. cremoris strains B39 and B891 reduced the thickening effect of the polymers. 9 The EPS of S. thermophilus Sts having higher molecular weight resulted in a more viscous product than that of strain Rs with lower molecular weight. 10 To further understand the structure-function relationship of EPS, structures of the subunits of these polymers, the primary structure of the EPS produced by a number of S. thermophilus strains have been elucidated. The first EPS structure to be determined was that of ropy strains from S. thermophilus CNCMI 733, 734, and 735, all of which consist of a tetrasaccharide repeating units of D-galactose, D-glucose, and N-acetyl-D-galactosamine. 11 Subsequently, strain Sfi6 has been shown to produce an EPS with identical structure to that of the CNCMI strains, 12 and the EPS of Sfi39 also consisted of a tetrasaccharide repeating unit of D-galactose and D-glucose. 7 The EPS of strains Sfi12 (ref 7) and S3 (ref 13) contained hexasaccharide repeating units; the former containing D-galactose and L-rhamnose, and the latter also D-glucose. The EPS of strains Rs and Sts (ref 10) had a similar structure to that of OR 901; 14 all being branched heptasaccharide repeating units of D-galactose and L-rhamnose. Recently, strains EU20 (ref 15) and 8S (ref 16) have been shown to produce EPS with heptasaccharide repeating units; the former containing D-glucose, D-galactose and L-rhamnose, and the latter D-glucose, D-galactose, D-ribose, and N-acetyl-D-galactosamine, as well as an open chain component, viz., 6-O-(3′,9′-dideoxy-D-threo-D-altro- nonoic acid-2′-yl)-R-D-glucopyranose. In search for EPS producing lactic acid bacterial strains with interesting properties, a yogurt strain of S. thermophilus THS has been found to produce a viscous EPS when grown in skim milk. The EPS has been shown to consist of a pentasaccharide repeating unit of D-galactose and D-glucose, which has not been reported earlier for the S. thermophilus species. In this paper, we report the structural determination of the EPS produced by S. thermophilus THS. Materials and Methods Growth of the Microorganism. Cultivation of S. ther- mophilus THS for producing EPS was carried out at Valio Ltd., Research and Development, Helsinki, Finland. The microorganism was isolated and maintained at -80 °C in glass beads. It was subcultured twice in MRS broth at 37 °C before use. The growth medium used for the production * To whom correspondence should be addressed. E-mail: gw@organ.su.se. † Stockholm University. ‡ University of Helsinki. 105 Biomacromolecules 2005, 6, 105-108 10.1021/bm0496514 CCC: $30.25 © 2005 American Chemical Society Published on Web 11/04/2004