Structural Studies of the Exopolysaccharide Produced by

Biomacromolecules 2002, 3, 880-884

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Structural Studies of the Exopolysaccharide Produced by Lactobacillus rhamnosus strain GG (ATCC 53103) ¨ ran Widmalm*,† Clas Landersjo ¨ ,† Zhennai Yang,‡ Eine Huttunen,‡ and Go 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 April 2, 2002; Revised Manuscript Received May 14, 2002

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 1H,1HNOESY, and 1H,13C-HMBC NMR techniques. The structure of the repeating unit of the EPS from Lactobacillus rhamnosus strain GG was determined as:

Introduction Human intestinal microflora comprises hundreds of bacterial species. Maintaining the balance of such a complex ecosystem, e.g., by ingestion of viable bacterial preparations, or probiotics, is of importance for the prevention of pathogenic colonization in the intestine and regulation of intestinal transit.1 Lactobacilli and bifidobacteria are indigenous microflora in humans and are the two most important probiotic groups. Consumption of these probiotic cultures positively affects the composition of the intestinal microflora, alleviates symptoms of lactose intolerance, reduces serum cholesterol, prevents diarrhoea, and increases immune responses and anticarcinogenic activities.2 Lactobacillus rhamnosus strain GG (LGG) is a probiotic strain isolated from healthy human intestinal flora. The beneficial effects of LGG have been extensively studied with respect to its preventive treatment of various intestinal disorders.3 Compared to other probiotic strains, LGG showed a better tolerance to conditions in the digestive tract and better survival in functional foods. LGG had the ability to adhere to and colonize intestinal mucosa, beneficial in improving intestinal immune response and preserving intestinal integrity.1 The adhering of bacteria to intestinal epithelium has been regarded as a prerequisite for probiotic strains to exert beneficial health effects.4 Previous studies on bacterial adhesion showed that capsular polysaccharides might promote the adherence of bacteria to biological surfaces, thereby facilitating the * To whom correspondence may be addressed. E-mail: [email protected]. † Stockholm University. ‡ University of Helsinki.

colonization of various ecological niches.5 The bacterial exopolysaccharides (EPS) were found to be present in adherent biofilms6 and might be involved in initial and permanent adhesion.7 In addition, some proteinaceous compounds, glycoproteins, and small molecules were also shown to function probably as adhesion-promoting factors.8-10 To further understand the roles of the EPS in the probiotic effects of LGG, the structure of the EPS produced by LGG has been investigated. We herein report on the structure thereof. Materials and Methods Growth of the Organism. Cultivation of Lactobacillus rhamnosus strain GG (ATCC 53103) for producing EPS was carried out at Valio Ltd., Research and Development, Helsinki, Finland. The organism was maintained at -80 °C in glass beads, and it was subcultured twice in MRS broth11 at 37 °C before use. Heat-treated (121 °C for 15 min) HYLA milk (enzymatically hydrolyzed lactose, Valio Ltd.) was used for the production of EPS. The growth of the organism was carried out at 37 °C for 20 h. Isolation of the Exopolysaccharide. After bacterial growth, trichloroacetic acid (E. Merck, Darmstadt, Germany) was added to the culture to a final concentration of 4% (w/ v), and the mixture stirred for 2 h at a room temperature. Cells and precipitated proteins were removed by centrifugation (35 min, 22000g, 4 °C). The supernatant was collected and filtered through an Acrocap filter (0.2 µm, Gelman Sciences, MI). The polysaccharide was precipitated by gradually adding cold ethanol to 75% (v/v) to the filtered supernatant. The precipitated EPS material was obtained by

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centrifugation. It was 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 filtered again through an Acrocap filter, and EPS (0.08 g/L) was obtained after lyophilization on a DURADRY freeze-dryer (FTS Systems Inc., Stone Ridge, NY). Part of the material (10 mg) for NMR measurements and component analysis was extensively dialyzed against water overnight at 4 °C, and the EPS solution was again lyophilized. The EPS was further purified by gel permeation chromatography (GPC) on a column (2 × 60 cm) of Superdex-30 (Pharmacia, Uppsala, Sweden) fitted to an FPLC system (Pharmacia) and eluted with pyridinium acetate buffer (0.07 M, pH 5.4). Column effluents were monitored using a differential refractometer (Waters 410). The EPS material eluting in the void volume was lyophilized. Component Analysis. Hydrolysis of the EPS was performed with 4 M HCl at 120 °C for 15 min. After reduction with sodium borohydride and acetylation, the samples were analyzed by gas-liquid chromatography (GLC). The absolute configurations of the sugars present in the EPS were determined essentially as described by Leontein et al.12 by GLC of their acetylated glycosides, using (+)-2-butanol.13 Alditol acetates and acetylated 2-butyl glycosides were separated on an HP-5 fused silica column (0.25 mm × 30 m; Hewlett-Packard) 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. NMR Spectroscopy. NMR spectra of a polysaccharide solution in D2O (3.5 mg in 0.7 mL) were recorded at 37 °C using a Varian Inova 600 spectrometer. Data processing was performed using the VNMR software (Varian). 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 (δC 67.40) as references. For assignment of NMR signals, 1H,1H-DQF-COSY,14 1H,1H-TOCSY15 experiments with mixing times of 30, 60, and 90 ms, gradient selected 13C-decoupled inverse 1H-detected 1H,13C heteronuclear single-quantum coherence (gHSQC),16 and 13Ccoupled gHSQC experiments as well as gHSQC-TOCSY experiments17 with mixing times of 20 and 50 ms were used according to standard pulse sequences. For sequence information, the NOESY experiment18 with a mixing time of 100 ms and a gradient version of the 1H,13C-HMBC experiment16,19 with a 60 ms delay for the evolution of long-range connectivities were used. The chemical shifts were compared to those of the corresponding monosaccharides.20,21 Results and Discussion Lactobacillus rhamnosus strain GG was grown in HYLA milk, and the EPS was isolated as a 75% ethanol precipitate from the culture medium. It was further purified by GPC. From the 1H NMR spectrum of the EPS (Figure 1) it was possible to identify seven signals in the region for anomeric protons. Six of these were shown to correlate to resonances from anomeric carbons as deduced from a 13C-decoupled

Figure 1. Part of the 1H NMR spectrum of the EPS from LGG with resonances from anomeric protons annotated.

Figure 2. Part of the 13C-decoupled 1H,13C-HSQC NMR spectrum of the EPS from LGG. 1

H,13C-gHSQC spectrum (Figure 2). Thus, the repeating units of the EPS contains six sugar residues. In the 1H NMR spectrum signals were also observed at δH 2.05 (3H, s) and 1.32 (3H, d, J ) 6.2 Hz). Sugar analysis of the EPS revealed rhamnose/galactose/ 2-deoxy-2-amino-D-glucose in the relative proportions 9:75: 16. Determination of the absolute configurations of the sugar residues as acetylated (+)-2-butyl glycosides by GLC showed L-Rha, D-Gal, and D-GlcN. That the latter residue is Nacetylated was evident from NMR data (vide infra). The subsequent determination of the structure of the repeating unit of the EPS relies solely on information obtained from 1 H-detected NMR experiments. The assignment of 1H and 13C resonances was performed by 1H,1H- and 1H,13C-correlated 2D NMR experiments. The sugar residues are denoted A-F from decreasing 1H chemical shifts of the resonances of their respective anomeric protons. The chemical shifts of the H1 resonances of residues A and F were well-resolved. The corresponding resonances for residues C, D, and E were heavily but not intractably overlapped (Figure 1). The anomeric proton resonance of residue A at δH 5.26 was not resolved and had a width at half-height of 2.9 Hz, indicating a very small JH1,H2 coupling constant. The anomeric carbon resonance was observed at δC 110.2, indicative of a furanosidic ring form. Disentangling

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Table 1. Chemical Shift (ppm) of the Signals in the 1H and

Landersjo ¨ et al. 13C

NMR Spectra of the EPS from LGGa 1H/13C

sugar residue f3)-β-D-Galf-(1f A

f3)-R-D-Galp-(1f B

f4,6)-R-D-GlcpNAc-(1fc C

β-D-Galf-(1f D

f3)-R-L-Rhap-(1f E

f3)-β-D-Galp-(1f F

1

2

3

4

5

5.26 [2.9]b (0.04) 110.2 (8.2) 5.09 [3.9] (-0.13) 100.4 (7.2) 5.06 [3.8] (-0.15) 95.3 (3.5) 5.051 [2.4] (-0.17) 108.4 (6.4) 5.046 {174} (-0.07) 102.9 (8.1) 4.52 [7.8] (-0.01) 103.7 (6.3)

4.43 (0.41) 80.5 (-1.9) 3.97 (0.19) 68.2 (-1.2) 4.02 (0.14) 54.0 (-1.0) 4.15 (0.13) 81.8 (-0.6) 4.18 (0.26) 67.9 (-3.9) 3.66 (0.21) 70.9 (-2.1)

4.09 (-0.01) 85.3 (8.4) 3.92 (0.11) 78.2 (8.1) 3.95 (0.20) 70.4 (-1.3) 4.08 (-0.02) 77.4 (0.5) 3.90 (0.09) 76.8 (5.8) 3.75 (0.16) 80.9 (7.1)

4.27 (0.22) 82.6 (-0.6) 4.09 (0.14) 69.9 (-0.4) 3.87 (0.38) 79.2 (7.9) 4.01 (-0.04) 83.4 (0.2) 3.56 (0.11) 71.1 (-2.1) 4.09 (0.20) 69.4 (-0.3)

3.89 (0.08) 71.5 (-0.2) 4.14 (0.11) 72.2 (0.9) 4.24 (0.38) 70.3 (-2.2) 3.84 (0.03) 71.6 (-0.1) 3.85 (-0.01) 70.0 (0.9) 3.75 (0.10) 76.1 (0.2)

6 ∼3.70 63.5 (-0.2) ∼3.77 61.9 (-0.1) 3.88, 4.04 66.6 (4.8) ∼3.70 63.6 (-0.1) 1.32 17.6 (-0.1) ∼3.76 61.9 (0.1)

a J H1,H2 values are given in hertz in brackets and JH1,C1 values in hertz in braces. Chemical shift displacements (∆δ) are reported in parentheses, compared to the corresponding hexose residue. b Signal width at half-height. c Chemical shifts for NAc are δH 2.05 and δC 22.7 and 175.2.

of the spin-system showed that it belonged to a galactose, and consequently it is a β-D-Galf residue. In support of this, a correlation between H1 and C4 was observed in the 1H,13CHMBC spectrum, a finding previously described for galactofuranosides.22 Carbon-13 glycosylation shifts can be used to determine substitution positions.23 For residue A a large downfield chemical shift displacement ∆δC of 8.4 is observed for C3, and in addition small upfield chemical shift displacements are observed at C2 and C4, known as β-effects. Thus, A is 3-substituted, and the residue is consequently f3)-β-D-Galf-(1f with the 1H and 13C assignments given in Table 1. The anomeric proton resonance of residue B at δH 5.09 showed JH1,H2 ) 3.9 Hz and the C1 resonance at δC 100.4. Analysis of the 1H and 13C spin systems revealed a pyranoid galactose residue which should be R-linked and 3-substituted, since C3 has ∆δC ) 8.1. Thus, B is f3)-R-D-Galp-(1f. The anomeric proton resonance of residue C has δH 5.06 and JH1,H2 ) 3.8 Hz. The chemical shift of C1, 95.3 ppm, is in the upfield region of anomeric carbon resonances for glycosylated sugars with a ∆δC of only 3.5 ppm. That C is an amino sugar is evident from the chemical shift of its C2 resonance at δC 54.0, typical for carbons linked to a nitrogen atom. Significant downfield glycosylation shifts are observed for C4 (∆δC ) 7.9) and C6 (∆δC ) 4.8). Thus, this residue is f4,6)-R-D-GlcpNAc-(1f. The anomeric proton resonance of residue D at δH 5.051 showed JH1,H2 ) 2.4 Hz and the C1 resonance at δC 108.4, again indicating a furanosidic residue. Also in this case a correlation between H1 and C4 was observed in the 1H,13CHMBC spectrum. However, the chemical shifts of C2 to C6 were similar to those of β-D-Galf, 21 and consequently the

sugar is a terminal one. For the anomeric proton resonance of residue E at δH 5.046, it was not possible to resolve the homonuclear spin-spin coupling of the anomeric proton. However, the corresponding heteronuclear coupling, JH1,C1 ) 174 Hz, is indicative of an equatorial proton for a pyranoid sugar residue. Characteristic chemical shifts for the H6 and C6 resonances of this sugar residue (Table 1) revealed it as a 6-deoxy hexose, which according to the above sugar analysis must be rhamnose. The downfield glycosylation shift was observed for the C3 resonance with ∆δC ) 5.8. A significant upfield chemical shift displacement of -3.9 ppm was observed at C2. Residue E is consequently f3)-R-L-Rhap-(1f. Finally, residue F had the anomeric proton resonance at δH 4.52 and showed JH1,H2 ) 7.8 Hz. The C1 resonance was observed at δC 103.7 and analysis of the spin systems revealed a galactose residue in the pyranoid ring form which is 3-substituted since ∆δC ) 7.1 for C3. Thus, F is f3)-β-D-Galp-(1f. The six sugar residues in the EPS have been identified, their absolute configurations determined, the ring forms revealed, their anomeric configurations elucidated, their substitution positions clarified, and their 1H and 13C NMR resonances assigned. The remaining determination of the sequence of sugar residues constituting the repeating unit of the EPS was performed by 1H,1H-NOESY and 1H,13C-HMBC experiments. The former experiment reveals correlations between protons close in space and in particular for sequence determination those straddling the glycosidic linkage. The latter experiment reveals 1H,13C two- and three-bond correlations and in particular for polysaccharides those at glycosidic linkages, i.e., the anomeric proton to the glycosyloxylated carbon and the anomeric carbon to the proton

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Figure 3. Part of the 1H,1H-NOESY NMR spectrum of the EPS from LGG with annotated correlations from anomeric protons (F2 dimension). Table 2. Inter-residue Correlations Observed in the 1H,1H-NOESY and 1H,13C-HMBC Spectra of the EPS from LGG residue

anomeric atom

residue

NOE

A B B C C C D D D D E E F F F

H1 H1 H1 H1 H1 C1 H1 H1 H1 H1 H1 C1 H1 H1 C1

F A A E E E C C F C B B C C C

H3 H3

HMBC

C3 H2 H3 H3 H6a H6b H1 C6 H3 H3 H4 C4 H4

at the glycosyloxylated carbon. However, the observed correlations from one of the experiments may not suffice to unambiguously determine the sequence, and we therefore usually employ both of them for structural studies of polysaccharides.24 The inter-residue correlations identified from the 1H,1HNOESY spectrum (Figure 3) as well as the trans-glycosidic three-bond correlations observed in the 1H,13C-HMBC spectrum are compiled in Table 2. These data unambiguously identify all glycosidic correlations in the repeating unit of the EPS and its structure as

The above-described relatively small glycosylation shift of C1 in residue C and the large upfield chemical shift displacement of C2 in residue E can now be rationalized by a γ-gauche interaction25 between H1 in C and H2 in E producing the observed carbon-13 chemical shift changes. Thus, these protons should be in close proximity in space

which is supported by the nuclear Overhauser effect (NOE) between H1 in C and H2 in E, for which the cross-peak intensity is larger than that for the protons at the glycosidic linkage, i.e., H1 in C and H3 in E (cf. Figure 3). Also, a weak NOE is present between the anomeric protons of residues D and F, both substituting the branching residue C. EPS of lactic acid bacteria most often contain glucose and galactose, but rhamnose as well as N-acetyl-glucosamine and N-acetyl-galactosamine have also been found.26 The EPS of the Lactobacillus rhamnosus strain C83 consists of linear pentasaccharide repeating units with, inter alia, two β-D-Galf residues.27 Since LGG is a well-known probiotic strain with prominent adhering properties, it is of interest to clarify the physiological functions of the EPS in relation to the polysaccharide structure and its adhesion mechanism. The functions of EPS are generally thought to be of a protective nature such as prevention against desiccation, bacteriophage, protozoan attack, antibiotics, and toxic compounds.6,28 Bacterial EPS may also play a role in adhesion and biofilm formation.29 According to the adhesion studies on probiotic strains, LGG was adhesive, but L. rhamnosus LC-705 was not.30 Correspondingly, LGG produced an EPS as demonstrated in this study, but the nonadhesive L. rhamnosus LC705 did not (unpublished results). It seemed that the EPS of LGG might be involved in the adhesiveness of the organism, though the amount of the EPS produced by LGG was small under the growth conditions in this study. To further understand the physiological roles of EPS, future studies should be carried out on the correlation between EPS production and adhesion, verifying whether EPS are involved in the adhesion of probiotic strains. Acknowledgment. This work was supported by grants from the Academy of Finland and the Swedish Research Council. 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 L. rhamnosus strain GG. References and Notes (1) Marchand, J.; Vandenplas, Y. Eur. J. Gastroenterol. Hepatol. 2000, 12, 1077. (2) Klaenhammer, T. D.; Kullen, M. J. Int. J. Food Microbiol. 1999, 50, 45. (3) Saxelin, M. Food ReV. Int. 1997, 13, 293. (4) Blum, S.; Reniero, R.; Schiffrin, E. J.; Crittenden, R.; MattilaSandholm, T.; Ouwehand, A. C.; Salminen, S.; von Wright, A.; Saarela, M.; Saxelin, M.; Collins, K.; Morelli, L. Trends Food Sci. Technol. 1999, 10, 405. (5) Costerton, J. W.; Cheng, K.-J.; Geesey, G. G.; Ladd, T. I.; Nickel, J. C.; Dasgupta, M.; Marrie, T. J. Annu. ReV. Microbiol. 1987, 41, 435. (6) Whitfield, C. Can. J. Microbiol. 1988, 34, 415. (7) Allison, D. G.; Sutherland, I. W. J. Gen. Microbiol. 1987, 133, 1319. (8) Granato, D.; Perotti, F.; Masserey, I.; Rouvet, M.; Golliard, M.; Servin, A.; Brassart, D. Appl. EnViron. Microbiol. 1999, 65, 1071. (9) Greene, J. D.; Klaenhammer, T. R. Appl. EnViron. Microbiol. 1994, 60, 4487. (10) Roberts, I. S. Microbiology 1995, 141, 2023. (11) de Man, J. C.; Rogosa, M.; Sharp, M. E. J. Appl. Bacteriol. 1960, 23, 130. (12) Leontein, K.; Lindberg, B.; Lo¨nngren, J. Carbohydr. Res. 1978, 62, 359. (13) Gerwig, G. J.; Kamerling, J. P.; Vliegenthart, J. F. G. Carbohydr. Res. 1979, 77, 1. (14) Piantini, U., Sørensen, O. W.; Ernst, R. R. J. Am. Chem. Soc. 1982, 104, 6800.

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(15) Braunschweiler, L.; Ernst, R. R. J. Magn. Reson. 1983, 53, 521. (16) Willker, W.; Leibfritz, D.; Kerssebaum, R.; Bermel, W. Magn. Reson. Chem. 1993, 31, 287. (17) 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. (18) Jeener, J.; Meier, B. H.; Bachmann, P.; Ernst, R. R. J. Chem. Phys. 1979, 71, 4546. (19) Bax, A.; Summers, M. F. J. Am. Chem. Soc. 1986, 108, 2093. (20) Jansson, P.-E.; Kenne, L.; Widmalm, G. Carbohydr. Res. 1989, 188, 169. (21) Baumann, H.; Tzianabos, A. O.; Brisson, J.-R.; Kasper, D. L.; Jennings, H. J. Biochemistry 1992, 31, 4081. (22) Linnerborg, M.; Wollin, R.; Widmalm, G. Eur. J. Biochem. 1997, 246, 565-573. (23) So¨derman, P.; Jansson, P.-E.; Widmalm, G. J. Chem. Soc., Perkin Trans 2 1998, 639.

Landersjo ¨ et al. (24) Widmalm, G. In Carbohydrate chemistry; Boons, G.-J., Ed.; Blackie Academic & Professional: London, 1998; p 448. (25) Wehrli, F. W.; Wirthlin, T. Interpretation of carbon-13 NMR spectra; Heyden & Son: London, 1976; p 27. (26) De Vuyst, L.; Degeest, B. FEMS Microbiol. ReV. 1999, 23, 153. (27) Vanhaverbeke, C.; Bosso, C.; Colin-Morel, P.; Cey, C.; GamarNourani, L.; Blondeau, K.; Simonet, J.-M.; Heyraud, A. Carbohydr. Res. 1998, 314, 211. (28) Looijesteijn, P. J.; Trapet, L.; de Vries, E.; Abee, T.; Hugenholtz, J. Int. J. Food Microbiol. 2001, 64, 71. (29) Robert, I. S. Annu. ReV. Microbiol. 1996, 50, 285. (30) Ouwehand, A. C.; Kirjavainen, P. V.; Gro¨nlund, M.-M.; Isolauri E.; Salminen, S. J. Int. Dairy J. 1999, 9, 623.

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