Structural Studies of an Exopolysaccharide Produced by

Biomacromolecules 2005, 6, 105-108

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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 1H,1H-NOESY and 1H,13C-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 predominantly together with other lactic acid bacteria. The importance 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 β-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 * To whom correspondence should be addressed. E-mail: [email protected]. † Stockholm University. ‡ University of Helsinki.

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-altrononoic 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. thermophilus 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

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of EPS consisted of 10% skim milk power (Valio) in water, heat treated at 121 °C for 15 min. The growth of the organism was carried out at 37 °C for 20 h with 1% (v/v) inoculum. Isolation of the Exopolysaccharide. After bacterial growth, trichloroacetic acid (Merck, Darmstadt, Germany) was added to the culture (0.5 L) to a final concentration of 4% (w/v) and stirred for 2 h. Cells and precipitated proteins were removed by centrifugation (35 min, 22 000 g, 4 °C). The supernatant was collected and filtered through an AcroCap filter (0.2 µm, Gelman Sciencies, MI). To precipitate the exopolysaccharide, cold EtOH was gradually added to the filtered supernatant up to one and a half volumes. The EPS precipitate was collected by centrifugation, washed, and dissolved in water obtained from an Alpha-Q Reagent Grade Water Purification System (Millipore Co., Milford, MA). The aqueous solution of the EPS was again filtered through an AcroCap filter and lyophilized (24 mg) on a DURA-DRY freeze-dryer (FTS Systems Inc., Stone Ridge, NY). Part of the material (10 mg) was extensively dialyzed (Cellu Sep T3, Membrane Filtration Products Inc., San Antonio, USA) against water overnight at 4 °C, and the EPS solution was again lyophilized and used for structural analysis. Component Analysis. Hydrolysis of the EPS was performed with 2 M trifluoroacetic acid at 120 °C for 2 h. After reduction with sodium borohydride and acetylation, the samples were analyzed by GLC. The absolute configuration of the sugars present was determined essentially as described by Leontein et al.17 by GLC of their acetylated glycosides, using (+)-2-butanol.18 Alditol acetates and acetylated 2-butyl glycosides were separated on a HP-5 fused silica column using a temperature program of 180 °C (1 min) and 3 °C min-1 to 210 °C. Hydrogen was used as carrier gas. The column was fitted to a Hewlett-Packard model 5890 series II gas chromatograph equipped with a flame ionization detector. The methylation analysis was performed according to Hakomori,19 using sodium methylsulfinylmethanide in dimethyl sulfoxide and methyl iodide. The methylated compounds were purified using Sep-Pak C18 cartridges and recovered using acetonitrile and ethanol.20 The purified methylated sample was hydrolyzed as described above, reduced with sodium borodeuteride and converted into partially methylated alditol acetates and analyzed by GLC using a temperature program of 180 °C (1 min) and 1.5 °C min-1 to 210 °C. GLC-MS analysis was performed on a Thermo Quest GCQ plus spectrometer equipped with a CPSIL8CB fused silica column (15 m). A temperature program of 120 °C (4 min) and 7 °C min-1 to 270 °C was used with helium as carrier gas. NMR Spectroscopy. NMR spectra of a polysaccharide solution in D2O (2 mg in 0.7 mL) were recorded at 65 °C using Varian Inova 600 and 800 spectrometers equipped with 5 mm PFG triple-resonance probes and at 57 °C (due to temperature limitations) on a Bruker DRX 500 MHz spectrometer equipped with a 5 mm PFG triple-resonance CryoProbe. Data processing was performed using vendorsupplied software. Chemical shifts are reported in ppm using internal sodium 3-trimethylsilyl-(2,2,3,3-2H4)-propanoate (TSP, δH 0.00) or external 1,4-dioxane in D2O (δH 67.40) as

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Figure 1. 1H NMR spectrum of the EPS from S. thermophilus THS.

references. For assignment of NMR signals, 1H,1H-DQFCOSY21 and 1H,1H-TOCSY22 experiments with mixing times of 30, 60, and 90 ms, gradient selected 13C-decoupled inverse 1H-detected 1H,13C heteronuclear single-quantum coherence (gHSQC),23 13C-coupled gHSQC experiments as well as gHSQC-TOCSY experiments24 with mixing times of 20 and 50 ms were used according to standard pulse sequences. 1H,1H-NOESY experiments25 with mixing times of 10, 25, and 50 ms and a gradient selected 1H,13C heteronuclear multiple-bond correlation (gHMBC)23 with a 40 ms delay for the evolution of long-range couplings were used for sequence information. The chemical shifts were compared to those of the corresponding monosaccharides.26 Results and Discussion S. thermophilus THS was grown in skim milk, and the EPS was isolated as a 60% ethanol precipitate from the culture medium. The material was filtered and subsequently lyophilized. The 1H NMR spectrum of the EPS (Figure 1) revealed five signals in the region for anomeric protons. These were shown to correlate to resonances from anomeric carbons as deduced from a 1H,13C-HSQC spectrum. Thus, the repeating units of the EPS contain five sugar residues. Sugar analysis of the EPS revealed glucose and galactose in the relative proportions 3:2. Determination of the absolute configurations of these sugars as acetylated (+)-2-butyl glycosides by GLC showed D-Glc and D-Gal. Methylation analysis of the EPS showed the presence of 2,3,4,6-tetraO-methyl-D-Gal, 2,3,6-tri-O-methyl-D-Glc, 2,3,4-tri-O-methyl-D-Glc, and 2,6-di-O-methyl-D-Gal in the relative proportions 1:2:1:1. All partially methylated alditol acetates were positively identified using authentic standards. The assignment of 1H and 13C resonances was performed by 1H,1H and 1H,13C correlated two-dimensional NMR experiments. The sugar residues are denoted A-E from decreasing 1H chemical shifts of the resonances of their

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Structural Studies of an Exopolysaccharide Table 1. Chemical Shift (ppm) of the 1H and

13C

Signals in the NMR Spectra of the EPS from S. thermophilus THSa 1H/13C

sugar residue f6)-R-D-Glcp-(1f A

f4)-β-D-Glcp-(1f B

f3,4)-β-D-Galp-(1f C

f4)-β-D-Glcp-(1f D

β-D-Galp(1f E

1

2

3

4

5

6

4.97 [3.4] (-0.26) 100.5 {172} (7.5) 4.71 [7.8] (0.07) 105.4 {164} (8.6) 4.56 [7.8] (0.03) 104.3 {165} (6.9) 4.52 [8.0] (-0.12) 103.7 {163} (6.9) 4.48 [7.9] (-0.05) 104.2 {164} (6.8)

3.54 (0.00) 73.0 (0.5) 3.30 (0.05) 74.5 (-0.7) 3.79 (0.34) 71.9 (-1.1) 3.40 (0.15) 74.1 (-1.1) 3.57 (0.12) 72.2 (-0.8)

3.78 (0.06) 73.8 (0.0) 3.67 (0.17) 75.7 (-1.1) 3.87 (0.28) 82.0 (8.2) 3.67 (0.17) 75.5 (-1.3) 3.67 (0.08) 74.0 (0.2)

3.68 (0.26) 70.1 (-0.6) 3.61 (0.18) 80.7 (10.0) 4.26 (0.37) 76.9 (7.2) 3.67 (0.25) 79.8 (9.1) 3.95 (0.06) 69.8 (0.1)

4.30 (0.46) 71.7 (-0.7) 3.59 (0.13) 75.9 (-0.8) 3.81 (0.16) 76.9 (1.1) 3.60 (0.13) 75.9 (-0.8) 3.73 (0.08) 76.6 (0.7)

3.97, 4.12 69.0 (7.2) 3.85, 3.974 61.5 3.85, 3.90 61.3 3.83, 4.01 61.6 ∼3.79 62.2

a J H1,H2 values are given in Hz in square brackets and JH1,C1 values in Hz in braces. Chemical shift displacements (∆δ) are reported in parentheses, compared to the corresponding hexose residue.

respective anomeric protons. The chemical shifts of all anomeric resonances were well resolved and 13C chemical shifts for the ring carbons indicated, together with the methylation analysis, that the sugars have the pyranoid ring form. The anomeric proton resonance of residue A at δH 4.97 showed JH1,H2 ) 3.4 Hz revealing an R-linked residue. The 1H and 13C chemical shifts supported the presence of a glucosyl residue, which due to the large positive chemical shift displacement of the C-6 resonance (∆δ ) 7.2) must be substituted at O-6. Thus, residue A is f6)-R-D-Glcp-(1f. The remaining sugar residues are all β-linked since JH1,H2 ≈ 8 Hz and JH1,C1 ≈ 164 Hz. Residues B and D are both f4)β-D-Glcp-(1f as interresidue 1H,1H NOEs are observed from H1 to their H3 and H5 resonances as well as the large glycosylation shifts observed for the C4 resonances (Table 1). The galactosyl residues can be differentiated based on the presence of large 13C glycosylation shifts for the C3 and C4 resonances in residue C and the absence of such changes for the corresponding signals in residue E. Thus, C is f3,4)β-D-Galp-(1f and E is β-D-Galp-(1f. The sequential determination of the sugar residues in the repeating unit is based on 1H,1H-NOESY spectroscopy in which protons close in space can be correlated to each other. Besides intraresidue correlations, several interresidue connectivities can be identified in the 1H,1H-NOESY spectrum (Figure 2 and Table 2). The anomeric proton in residue A shows NOEs to H4 as well as both H6 protons in residue C. The NOEs between H1 in D and both H6 protons in A are also readily identified. Thus, a structural element consisting of three sugar residues can be constructed, viz., f4)-β-DGlcp-(1f6)-R-D-Glcp-(1f4)-β-D-Galp-(1f. This result rules out the possibility that residue E should form an open-chain cyclic acetal linkage to residue C, in analogy with that reported recently.27 NOEs from H1 in C to H4 in B and

Figure 2. Part of a 1H,1H-NOESY NMR spectrum (τmix ) 50 ms) of the EPS from S. thermophilus THS with annotated correlations from anomeric protons. Table 2. Interresidue Correlations Observed in the 1H,1H-NOESY spectra of the EPS from S. thermophilus THSa residue A B C D E as

1H

(anomeric) 4.97 4.71 4.56 4.52 4.48

Residue C C B A D

1H

NOE to

4.26 (s), 3.90 (m), 3.85 (w) 3.87 (m) 3.61 (s) 3.97 (s), 4.12 (w) 3.67 (s)

) strong, m ) medium, w ) weak.

from H1 in B to H3 in C define the backbone of the polymer as f3)-β-D-Galp-(1f4)-β-D-Glcp-(1f. The terminal E residue shows from its anomeric proton a cross-peak in the NOESY spectrum at δH 3.67, where several resonances reside. However, since residue D is 4-substituted and remains the only possibility based on the above data, this NOE is consistent with the termination of the side-chain in the

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repeating unit of the EPS. The combined data identify all glycosidic correlations in the repeating unit of the EPS and its structure as

The structure of the repeating unit was further corroborated by correlations observed in the 1H,13C-HMBC spectrum, viz., H1 in E to C4 in D, H1 in D to C6 in A, and C1 in C to H4 in B. Thus, the EPS structure has a backbone consisting of two sugar residues with a side-chain of three sugar residues. It is suggested from the 1H NMR spectrum of the EPS from S. thermophilus THS that the anomeric resonances from residues B and C in the backbone possess shorter T2 relaxation times compared to those from residues A, D and E in the side-chain (by approximately a factor of 2) indicating different mobility with a stiffer backbone and a more flexible side-chain. Since S. thermophilus is routinely used in yoghurt starters, studies on the EPS produced by this species have gained continued interest. Incorporation of EPS producing cultures in fermented products has been shown to increase the viscosity, stability and water binding capacity of the products,28 and these physical properties are closely related to the structure of the EPS produced in situ. Our studies on the EPSs produced by lactic acid bacteria showed a relatively high viscosity of the EPS from S. thermophilus THS compared with other EPS isolated in our previous studies (unpublished results), possibly explained by the branching pattern with a trisaccharide side-chain of this EPS as demonstrated in the present study. Of the structures of the EPS produced by dairy lactic acid bacteria reported to date, the EPS repeating units usually contain a side-chain of only one or two sugar residues.29 The present study also suggests that manipulation of an EPS structure such as side-chain branching patterns, for example, by genetic engineering of the enzymes involved in EPS biosynthesis, i.e., the glucosyltransferases involved in the side-chain construction,30,31 can be a useful tool to obtain an EPS with desired functionalities. Acknowledgment. This work was supported by grants from the Academy of Finland, the Swedish Research Council, and the Swedish Foundation for Strategic Research (GLIBS). The authors thank Annika Ma¨yra¨-Ma¨kinen (Lic. Sc.) and Tarja Suomalainen (M. Sc.) of Valio Ltd, R&D, for providing the cultured media of S. thermophilus THS.

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References and Notes (1) Perry, D. B.; McMahon, D. J.; Oberg, C. J. J. Dairy Sci. 1997, 80, 799-805. (2) Rawson, H. L.; Marshall, V. M. Int. J. Food Sci. Technol. 1997, 32, 213-220. (3) Low, D.; Ahlgren, J. A.; Horne, D.; McMahon, D. J.; Oberg, C. J.; Broadbent, J. R. Appl. EnViron. Microbiol. 1998, 64, 2147-2151. (4) Perry, D. B.; McMahon, D. J.; Oberg, C. J. J. Dairy Sci. 1998, 81, 563-566. (5) Marshall, V. M.; Rawson, H. L. Int. J. Food Sci. Technol. 1999, 34, 137-143. (6) Peterson, B. L.; Dave, R. I.; McMahon, D. J.; Oberg, C. J.; Broadbent, J. R. J. Dairy Sci. 2000, 83, 1952-1956. (7) Lemoine, J.; Chirat, F.; Wieruszeski, J.-M.; Strecker, G.; Favre, N.; Neeser, J.-R. Appl. EnViron. Microbiol. 1997, 63, 3512-3518. (8) van Kranenburg, R.; van Swam, I. I.; Marugg, J. D.; Kleerebezem, M.; de Vos, W. M. J. Bacteriol. 1999, 181, 338-340. (9) Tuinier, R.; van Casteren, W. H. M.; Looijesteijn, P. J.; Schols, H. A.; Voragen A. G. J.; Zoon, P. Biopolymers 2001, 59, 160-166. (10) Faber, E. J.; Zoon, P.; Kamerling, J. P.; Vliegenthart, J. F. G. Carbohydr. Res. 1998, 310, 269-276. (11) Doco, T.; Wieruszeski, J.-M.; Fournet, B.; Carcano, D.; Ramos, P.; Loones, A. Carbohydr. Res. 1990, 198, 313-321. (12) Stingele, F.; Neeser, J.-R.; Mollet, B. J. Bacteriol. 1996, 178, 16801690. (13) Faber, E. J.; van den Haak, M. J.; Kamerling, J. P.; Vliegenthart, J. F. G. Carbohydr. Res. 2001, 331, 173-182. (14) Bubb, W. A.; Urashima, T.; Fujiwara, R.; Shinnai, T.; Ariga, H. Carbohydr. Res. 1997, 301, 41-50. (15) Marshall, V. M.; Dunn, H.; Elvin, M.; McLay, N.; Gu, Y.; Laws A. P. Carbohydr. Res. 2001, 331, 413-422. (16) Faber, E. J.; van Haaster, D. J.; Kamerling, J. P.; Vliegenthart, J. F. G. Eur. J. Biochem. 2002, 269, 5590-5598. (17) Leontein, K.; Lindberg, B.; Lo¨nngren, J. Carbohydr. Res. 1978, 62, 359-362. (18) Gerwig, G. J.; Kamerling, J. P.; Vliegenthart, J. F. G. Carbohydr. Res. 1979, 77, 1-7. (19) Hakomori, S. J. Biochem. (Tokyo) 1964, 55, 205-208. (20) Waeghe, T. J.; Darvill, A. G.; McNeil, M.; Albersheim, P. Carbohydr. Res. 1983, 123, 281-304. (21) Piantini, U., Sørensen, O. W.; Ernst, R. R. J. Am. Chem. Soc. 1982, 104, 6800-6801. (22) Braunschweiler, L.; Ernst, R. R. J. Magn. Reson. 1983, 53, 521528. (23) Willker, W.; Leibfritz, D.; Kerssebaum, R.; Bermel, W. Magn. Reson. Chem. 1993, 31, 287-292. (24) de Beer, T.; van Zuylen, C. W. E. M.; Hård, K.; Boelens, R.; Kaptein, R.; Kamerling, J. P.; Vliegenthart, J. F. G. FEBS Lett. 1994, 348, 1-6. (25) Kumar, A.; Ernst, R. R.; Wu¨thrich, K. Biochem. Biophys. Res. Commun. 1980, 95, 1-6. (26) Jansson, P.-E.; Kenne, L.; Widmalm, G. Carbohydr. Res. 1989, 188, 169-191. (27) Vinogradov, E.; Bock, K. Angew. Chem., Int. Ed. Engl. 1999, 38, 671-674. (28) Broadbent, J. R.; McMahon, D. J.; Welker, D. L.; Oberg, C. J.; Moineau, S. J. Dairy Sci. 2003, 86, 407-423. (29) Laws, A. P.; Marshall, V. M. Int. Dairy J. 2001, 11, 709-721. (30) Betlach, M. R.; Capage, M. A., Doherty, D. H., Hassler, R. A.; Henderson, N. M.; Vanderslice, R. W.; Marrelli, J. D.; Ward. M. B. Prog. Biotechnol. 1987, 3, 35-50. (31) Laws, A. P.; Gu, Y.; Marshall, V. M. Biotechnol. AdV. 2001, 19, 597-625.

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