Nestlé Research Center, Vers-chez-les-Blanc, CH-1000 Lausanne 26, Switzerland, and 2Bijvoet Center, Department of Bio-Organic Chemistry, Utrecht University, P.O. Box 80.075, NL-3508 TB Utrecht, The Netherlands
Received on July 13, 2000; accepted on August 29, 2000.
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Abstract |
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Key words: exopolysaccharide/lactic acid bacteria/nuclear magnetic resonance spectroscopy/Streptococcus macedonicus
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Introduction |
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Streptococcus macedonicus Sc136 is a Gram positive LAB that is part of the starter flora present in Greek sheep and goat cheeses. Recently, in a phylogenetic study based on 16S rRNA sequence comparison, it has been described as a new species differing from Streptococcus thermophilus (Tsakalidou et al., 1998). S. macedonicus Sc136 is a strain capable of both mesophilic and thermophilic growth, that is, growth is sustained between 25°C and 42°C. It produces a high-molecular-mass, highly texturizing EPS composed of glucose (Glc), galactose (Gal), and N-acetylglucosamine (GlcNAc). Considering the texturizing properties of the EPS, the analysis of the EPS structure will contribute to the understanding of the relationship between rheological properties of EPSs and their structure. We present the production, isolation, and the structural determination of the EPS produced by S. macedonicus Sc136. The potential of the EPS, which was shown to contain a milk oligosaccharide related fragment, for the development of improved infant nutrition products will be discussed.
According to recent proposals (Editorial, 1997, 1999; Stubbs, 1997
; White, 1997
; Zyzik and Goldmann, 1999
), authorship contributions are provided: S.J.F.V. collected data, coordinated study, managed data, analyzed data, interpreted data, wrote and edited paper; E.J.F. collected data, analyzed data, interpreted data, edited paper; J.R.N. conceived study, selected bacterial strains, secured funding, edited paper; F.S. investigated bacterial strains, collected part of data, advised on the study, edited paper; J.P.K. designed the study, secured funding, coordinated study, interpreted data, edited paper.
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Results |
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Composition analysis of degraded exopolysaccharide probes
Mild acid hydrolysis of n-EPS (0.3 M TFA; 20 min, 100°C) and subsequent fractionation on a size-exclusion chromatography column (Bio-Gel P-2) gave a degraded polysaccharide material (hyd-PS) with a monosaccharide composition of D-Glc, D-Gal, and D-GlcNAc in a molar ratio of 3:1:1.
De-N-acetylation and deamination of n-EPS followed by fractionation on a size-exclusion chromatography column (Bio-Gel P-2) resulted in the isolation of a degraded polysaccharide (deNAc-PS) and an oligosaccharide fraction (deNAc-oligo). Monosaccharide analysis of deNAc-PS showed the presence of D-Glc and D-Gal in a molar ratio of 2:1. Reduction of the deNAc-oligo fraction and subsequent MALDI-TOF measurements yielded two [M+Na]+ pseudomolecular ions at m/z 512 and 350 compatible with the presence of Hex2anhydroHex-ol-1-d and HexanhydroHex-ol-1-d, respectively. Monosaccharide analysis of reduced deNAc-oligo revealed the presence of D-Glc and D-Gal in a molar ratio of 1.7:1 and anhydroHex-ol-1-d, which in view of the presence of D-GlcNAc in n-EPS was identified as being 2,5-anhydroMan-ol-1-d. Combining these results, reduced deNAc-oligo was constituted of (GalGlc)-2,5-anhydroMan-ol-1-d and Glc-2,5-anhydroMan-ol-1-d in a molar ratio of 1:0.7. The formation of the disaccharide compound was due to the acid lability of the GalfGlc linkage (see below).
Methylation analysis
Methylation analyses were performed on n-EPS, hyd-PS, and deNAc-PS (Table I). The methylation analysis data of n-EPS indicated the presence of a branched hexasaccharide repeating unit containing five internal monosaccharides (for a proof of the pyranose rings, see NMR section); the Galp residue forms a branching point, whereas the Galf residue occurs in a terminal position. Comparison of the methylation analysis data of n-EPS and hyd-PS showed the disappearance of terminal Galf and the complete shift of 6-substituted Glcp into terminal Glcp, demonstrating the occurrence of a terminal Galf-(16)-Glcp fragment. Methylation analysis of deNAc-PS suggested the presence of a linear trisaccharide repeating unit built up from two 4-substituted Glcp residues and one 4-substituted Galp residue (for a proof of the pyranose rings, see NMR section), thereby indicating that the GlcNAc residue is part of the sidechain.
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In the 1D 1H NMR spectrum of deNAc-PS (Figure 1c) three separated H-1 resonances with relative integrals 1:1:1 were found. The three monosaccharide units, denoted B, D, and F by analogy with the n-EPS resonances have H-1 values of 4.93, 4.53, and 4.48, respectively (Table III). No N-acetyl methyl signal was observed.
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For residue A (Hexf), the assignments of H-2,3,4 followed from TOCSY experiments; H-5,6a,6b were identified following A(H-2,3,4) traces in the TOCSY spectra and from the HSQC. The 13C chemical shifts of residue A were found to be virtually identical to those of ß-D-Galf1Me (average chemical shift difference <> = 0.5 ± 0.2 ppm) (Bock et al., 1983
). Furthermore, comparison of the complete set of 13C chemical shifts with the chemical shifts from
-D-Galf1Me (<
> = 1.2 ± 1.5 ppm) unambiguously proved the ß anomeric configuration. Therefore, residue A was identified as a terminal ß-D-Galf-(1
unit.
TOCSY measurements yield an assignment for H-2,3,4,5 of residue B (-Hexp), while the assignments of B(H-6a,6b) resulted from B(H-2,3,4) TOCSY traces and HSQC. The 13C chemical shifts for residue B were similar to those of
-D-Glcp1Me (<
> = 0.9 ± 0.6 ppm) (Bock and Pedersen, 1983
), at the exception of the C-4 position (
B(C-4) 79.6,
= 9.0 ppm), indicating that residue B was a
4)-
-D-Glcp-(1
unit.
In the case of residue C (ß-Hexp), TOCSY cross-peaks were observed between C(H-1) and C(H-2,3,4,5,6a,6b). The carbon chemical shifts of C(C-2) (C(C-2) 56.7) and C(C-3) (
C(C-3) 75.3) and the cross-peak between C(H-2) and C(NAc-CH3) in the TOCSY spectra are characteristic for a GlcpNAc residue. The C(C-6) signal (
C(C-6) 70.2) shifted towards low-field by 9.1 ppm when compared to C-6 of ß-D-GlcpNAc1Me (Bock et al., 1983
), indicated that residue C was a
6)-ß-D-GlcpNAc-(1
unit.
For residue D (ß-Hexp), TOCSY transfers were found from D(H-1) towards D(H-2,3,4,5,6a,6b) but the assignment of D(H-5) needed to be confirmed by the presence of a NOESY cross-peak between D(H-1) and D(H-5) as well as HMBC peaks between D(H-4) D(C-5) and D(C-4) D(H-5). The carbon chemical shifts of residue D as compared to those of relevant reference compounds (Bock and Thøgersen, 1982; Bock and Pedersen, 1983
; Bock et al., 1983
) and the NOESY cross-peak pattern (weak D(H-1) D(H-2), strong D(H-1) D(H-3,5)) identified residue D as a ß-Glcp residue. The D(C-4) resonance (
D(C-4) 80.0) shifted by 9.4 ppm towards low-field compared to C-4 of ß-D-Glcp1Me (Bock and Thøgersen, 1982
) defined residue D as a
4)-ß-D-Glcp-(1
unit.
The assignment of the proton signals of residue E (ß-Hexp) followed from TOCSY cross-peaks between E(H-1) and E(H-2,3,4,5,6a,6b). The four non-anomeric ring protons E(H-2) to E(H-5) covered a small 1H chemical shifts range (<> = 3.50 ± 0.13 ppm). The carbon chemical shifts of residue E were almost identical to those of ß-D-Glcp1Me (<
> = 0.2 ± 0.2 ppm) (Bock and Pedersen, 1983
), at the exception of that of E(C-6) (
E(C-6) 68.1,
= 6.3 ppm), defining residue E as a
6)-ß-D-Glcp-(1
unit.
For residue F (ß-Hexp), no TOCSY cross-peaks were found from F(H-1) towards F(H-5,6a,6b). However, the assignments of F(H-5) and F(H-6a,6b) were obtained from NOESY data (cross-peaks between F(H-1) and F(H-5,6a,6b)) and HMBC data (cross-peaks F(H-4) F(C-5) and F(C-4) F(H-5) as well as F(H-5) F(C-6) and F(C-5) F(H-6a,6b)), and by correlating the 1H resonances to the corresponding 13C resonances in the HSQC spectrum. The low-field F(H-4) 1H chemical shift (F(H-4) 4.28) together with the NOESY cross-peak pattern (weak F(H-1) F(H-4,6a,6b), strong F(H-1) F(H-3,5)) indicated a ß-D-Galp rather than a ß-D-Glcp residue (Bock and Thøgersen, 1982
; Bock and Pedersen, 1983
; Bock et al., 1983
). Both the F(C-3) and F(C-4) 13C resonances (
F(C-3) 81.6,
= 6.3 ppm;
F(C-4) 76.3,
= 6.3 ppm) shifted towards low-field with respect to the corresponding signals in ß-D-Galp1Me (Bock and Thøgersen, 1982
), indicated a 3,4-disubstituted residue. Residue F was therefore identified as a
3,4)-ß-D-Galp-(1
branching unit.
The sequence of the monosaccharide residues was deduced from the presence of cross-peaks in the 1H-13C HMBC and in the NOESY spectra. Relevant cross-peaks are summarized in Table IV. In case of ambiguities with the type of glycosidic linkage, the methylation analysis data and the 13C NMR assignments were also used.
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2D NMR spectroscopy of deNAc-PS
For the 1H and 13C NMR elucidation of deNAc-PS, which was found to be composed of trisaccharide repeating units, a similar strategy as described for n-EPS was applied. A speedy assignment was possible as a result of the greatly decreased viscosity, of the disappearance of the overlap between anomeric resonances and of the existing assignment available for n-EPS. The HSQC spectrum (Figure 3b) showed three cross-peaks from the clearly separated anomeric resonances. The 1H and 13C data are given in Table III, demonstrating the occurrence of the 4)-
-D-Glcp-(1
unit B, the
4)-ß-D-Glcp-(1
unit D, and the
4)-ß-D-Galp-(1
unit F. For residue B a virtual equivalence of all chemical shifts in n-EPS and deNAc-PS was observed (<
(1H)> = 0.08 ppm and <
(13C)> = 0.3 ppm). A similar finding occurred in the case of residue D (<
(1H)> = 0.06 ppm and <
(13C)> = 0.2 ppm). For residue F, small deviations in the 1H and 13C chemical shifts from n-EPS versus deNAc-PS were found (<
(1H)> = 0.11 ppm and <
(13C)> = 0.8 ppm). These deviations were induced by the absence of a substituent at O-3 of residue F, reflected by the shift towards high-field of F(C-3) (deNAc-PS
F(C-3) 73.3; n-EPS
F(C-3) 81.6). The NMR data of deNAc-PS entirely supported the conclusions made for the structure of n-EPS. It was interesting to note that the anomeric proton chemical shifts of B and D were identical to the corresponding values in synthetic ß-D-Glcp-(1
4)-
-d-Glcp-(1
4)-d-Gal (Koeman et al., 1993
).
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Discussion |
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Interestingly, the outlined trisaccharide sequence ß-D-GlcpNAc-(13)-ß-D-Galp-(1
4)-ß-D-Glcp is the minimal trisaccharide sequence common to both lacto-N-tetraose and lacto-N-neotetraose units found in human milk oligosaccharides. Human milk oligosaccharides are a complex mixture in which lactose is the main component (around 70 g/l), while larger structures (total around 20 g/l) are based on lactose and tetraoses backbones substituted by fucosyl and sialic acid residues bound by several types of linkages (Kobata, 1977
; Kobata et al., 1978
; Strecker et al., 1989
; Finke et al., 1999
). Many biological functions have been postulated for human milk oligosaccharides, including bifidogenicity and the ability to inhibit the adhesion of pathogenic microbes to the infant gut mucosa. Moreover, the use of lactic acid bacteria to acidify infant formulas in order to improve their nutritional value has been applied for a long time. Thus, the use of a bacterial strain like S. macedonicus Sc136 for infant formula preparation could present the additional advantage to bring a non-digestible complex polysaccharide available for endogenous fermentations in the infants gut, finally releasing carbohydrates similar to the main molecular structures found in human milk oligosaccharides.
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Materials and methods |
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Monosaccharide analysis
Quantitative monosaccharide analyses of poly- and oligosaccharides were performed by GLC after methanolysis followed by trimethylsilylation (Chaplin, 1982; Kamerling and Vliegenthart, 1989
). Absolute configurations were determined by GLC analysis of the trimethylsilylated (-)-2-butyl glycosides as described (Gerwig et al., 1978
, 1979).
Methylation analysis
For methylation analyses, poly- and oligosaccharides were permethylated using CH3I and NaOH in DMSO as described (Chaplin, 1982; Ciucanu and Kerek, 1984
). After treatment with Na2S2O3 (100 mg/ml) and extraction with CHCl3, the permethylated samples were hydrolyzed with 2 M TFA (2 h, 120°C), then reduced with NaB2H4. After neutralization and removal of boric acid by co-evaporation with MeOH, samples were acetylated with acetic anhydride (3 h, 120°C). The resulting partially methylated alditol acetates were extracted with CH2Cl2 and analyzed by GLC-EIMS (Jansson et al., 1976
).
Mild acid hydrolysis
Native S. macedonicus Sc136 EPS (n-EPS, 4 mg) was hydrolyzed in 0.3 M TFA (20 ml; 20 min, 100°C). After lyophilization, the partially hydrolyzed material was fractionated on a size-exclusion chromatography column (Bio-Gel P-2, 200400 mesh, 50 x 2.5 cm; Bio-Rad), eluted with 5 mM NH4HCO3 monitoring the effluent with a differential refractive index detector (LKB Bromma). The degraded polysaccharide (hyd-PS) was collected and lyophilized.
De-N-acetylation and deamination
A solution of n-EPS (3 mg) in anhydrous hydrazine (0.3 ml), containing hydrazine sulfate (15 mg), was stirred under argon for 20 h at 100°C. The solution was concentrated in vacuo and co-evaporated repeatedly with toluene. Then, the residue was dissolved in water and the solution was desalted on a HighTrap Desalting column (Sephadex G-25 Superfine, Pharmacia) eluted with 5 mM NH4HCO3 at a flow rate of 2 ml/min. The resulting preparation was dissolved in a solution containing 33% acetic acid (1 ml), 5% sodium nitrite (1 ml), and water (0.5 ml), and the solution was stirred for 2 h at room temperature, then neutralized with 2 M NH4OH. The resulting mixture was desalted on a cation-exchange column (Dowex AG 50W-X12,100200 mesh, H+-form; Bio-Rad), reduced using NaB2H4, and fractionated on a size-exclusion chromatography column (Bio-Gel P-2) as described above. The degraded polysaccharide (deNAc-PS) and an oligosaccharide fraction (deNAc-oligo) were collected and lyophilized.
Gas-liquid chromatography and mass spectrometry
Gas-liquid chromatography (GLC) analyses were performed on a CP-Sil 5CB fused silica capillary column (Chrompack CP9002, 25 m x 0.32 mm) using a temperature program from 130°C to 230°C at 4°C/min.
Gas-liquid chromatographyelectron ionization mass spectrometry (GLC-EIMS) analyses were carried out on a Fisons MD800/8060 system (electron energy, 70 eV) equipped with a DB-1 fused silica capillary column (J&W Scientific, 30 m x 0.32 mm) using a temperature program from 150°C to 250°C at 4°C/min.
Matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS) experiments were performed using a Voyager-DE mass spectrometer equipped with a nitrogen laser. Samples were prepared by mixing directly on the target 1 µl oligosaccharide solution with 2 µl aqueous 10% 2,5-dihydroxybenzoic acid as matrix solution.
NMR spectroscopy
Samples were exchanged two times in 99.9 atom% 2H2O (Euriso-Top) by successive lyophilization before being dissolved in 99.96 atom% 2H2O (Euriso-Top). All experiments were recorded on a three-channel Bruker DRX 600 MHz spectrometer equipped with an actively shielded pulsed-field z-gradient inverse triple-resonance probe. Chemical shifts are expressed in ppm by reference to the -anomeric signal of external [13C-1]-glucose (
H-1 5.15 and
C-1 92.90).
The following phase-sensitive two-dimensional experiments were recorded using time proportional phase increments (TPPI) (Marion and Wüthrich, 1983): double-quantum filtered correlation spectroscopy (DQF-COSY) (Piantini et al., 1982
; Rance et al., 1983
), total correlation spectroscopy (TOCSY) (Braunschweiler and Ernst, 1983
) with mixing times between 10 ms and 80 ms, nuclear Overhauser effect spectroscopy (NOESY) (Jeener et al., 1979
; Kumar et al., 1980
) with mixing times between 50 ms and 250 ms, and gradient sensitivity-enhanced 1H-13C heteronuclear single-quantum coherence (HSQC) (Kay et al., 1992
). A magnitude mode gradient-filtered 1H-13C heteronuclear multiple-bond correlation (HMBC) (Bax and Summers, 1986
) was recorded with a J-evolution time of 65 ms. The following number of complex points were acquired (F1, F2): 128x512 (TOCSY and HSQC), 256x512 (NOESY), and 128x1024 (HMBC), with averaging over 32 scans (TOCSY and NOESY) or 256 scans (HSQC and HMBC). Spectral widths (
1,
2) of 4000 Hz x 4000 Hz (TOCSY and NOESY), 16350 Hz x 4000 Hz (HSQC), and 12577 Hz x 4000 Hz (HMBC) were used. A 60° shifted square sine-bell was used in all cases, with zero-filling once. All data were processed using Bruker XWINNMR 2.x software.
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Acknowledgments |
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Abbreviations |
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Footnotes |
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References |
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