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Notes Structural Studies of the Exopolysaccharide Produced by Propionibacterium freudenreichii ssp. shermanii JS Eva-Lisa Nordmark,† Zhennai Yang,‡ Eine Huttunen,‡ and Go1 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 7, 2004 Revised Manuscript Received September 15, 2004
Introduction Propionibacteria are traditionally used as dairy starters for Swiss-type cheeses, and they are also used for commercial production of propionic acid. The dairy propionibacteria are generally food-grade, and certain strains, e.g., Propionibacterium freudenreichii ssp. shermanii JS, have been considered as potential probiotic organisms.1 The use of P. freudenreichii ssp. shermanii JS together with Lactobacillus rhamnosus LC705 (Bioprofit, Valio Ltd., Finland) was inhibitory toward moulds and yeasts.2 P. freudenreichii ssp. shermanii JS showed good adherence to Caco-2 cells in similar levels to Lactobacillus rhamnosus strain GG,3 a well-documented probiotic strain which produced an exopolysaccharide (EPS).4 The production of EPS by dairy lactic acid bacteria has been widely reported, and a number of EPS have been structurally characterized.5 However, although there are a few studies concerning the EPS produced by dairy propionibacteria with respect to growth conditions and production kinetics,6-9 no primary structures of these EPS have been reported. Physiologically, EPS functions to protect bacterial cells against unfavorable environmental factors such as dehydration, bacteriophage, protozoa, antibiotics, and lysozyme.10 The possible involvement of EPS in bacterial adhesion and biofilm formation has also been suggested.11 To further understand the functions of EPS in the probiotic effects of propionibacteria, structural studies have been carried out, in particular by NMR spectroscopy, on an EPS produced by P. freudenreichii ssp. shermanii JS. Materials and Methods Growth of the Microorganism. The growth of the microorganism was done at Valio Ltd., Research and Development, Helsinki, Finland. The P. freudenreichii ssp. shermanii JS strain from Valio’s Culture Collection was maintained at -80 °C in glass beads, and it was subcultured twice in YEL broth12 at 30 °C before use. The growth medium used for the production of EPS consisted of 5% * Corresponding author. E-mail:
[email protected]. † Arrhenius Laboratory. ‡ University of Helsinki.
whey permeate (Valio) and 2% casein hydrolysate in water, heat treated at 121 °C for 15 min. The growth of the microorganism was carried out at 30 °C for 72 h with a 1% (v/v) inoculum. Isolation of the Exopolysaccharide. After bacterial growth, trichloroacetic acid (Merck, Darmstadt, Germany) was added to 0.75 L culture 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 slime that appeared in the supernatant was dispersed by handling with NaCl. All supernatants were 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 from one to two volumes of the supernatant, and the EPS was precipitated at two volumes. The EPS was collected by centrifugation, washed with water, and dissolved in water obtained from an Alpha-Q Reagent Grade Water Purification System (Millipore Co., Milford, MA). After filtration through an AcroCap filter, the aqueous solution of the EPS was lyophilized on a DURA-DRY freeze-dryer (FTS Systems Inc., Stone Ridge, NY) with a yield of 75 mg/L. Part of the material (10 mg) was extensively dialyzed (Cellu Sep T3, Membrane Filtration Product Inc., San Antonio, USA; MW cut off 12-14 kDa) against water for two consecutive days at 4 °C with two changes of water per day, and the EPS solution was subsequently lyophilized. Since the 1H NMR spectrum of the EPS did not show extraneous peaks, this lyophilized material of EPS was used for structural analysis without further purification. 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 GLC. The absolute configurations of the sugars present in the EPS were determined essentially as described by Leontein et al.13 by GLC of their acetylated glycosides, using (+)-2-butanol.14 Alditol acetates and acetylated 2-butyl glycosides were separated on a HP-5 fused silica column using temperature programs of 180 °C (1 min) and 3 °C min-1 to 230 °C. Hydrogen was used as carrier gas. The columns were 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 (3.8 mg in 0.65 mL) were recorded at 55 °C using a Bruker DRX 500 spectrometer equipped with a Cryoprobe and Varian Inova 600 and 800 spectrometers. Data processing was performed using vendor-supplied 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 ref-
10.1021/bm0496716 CCC: $30.25 © 2005 American Chemical Society Published on Web 11/04/2004
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Notes
Table 1. Chemical Shift (ppm) of the 1H and 13C Signals in the NMR Spectra of the EPS from P. Freudenreichii ssp. shermanii JSa 1H/13C
sugar residue
1
2
3
4
f2,3)-β-D-Glcp-(1f 5.15 [7.4] (0.51) 100.2 (3.4)
3.94 (0.69) 80.9 (5.7)
4.11 (0.61) 82.4 (5.6)
3.60 (0.18) 68.8 (-1.9)
3.53 3.77, 3.96 (0.07) 76.6 61.7 (-0.2) (0.3)
β-D-Glcp-(1f2)
3.42 (0.17) 74.9 (-0.3)
3.56 (0.06) 76.8 (0.0)
3.41 (-0.01) 71.1 (0.4)
3.46 3.76, 3.95 (0.00) 77.2 62.2 (0.4) (0.4)
4.99 [7.7] (0.35) 103.1 (6.3)
5
6
a J H1,H2 values are given in Hz in square brackets. Chemical shift displacements (∆δ) are reported in parentheses, compared to those of β-D-Glcp.
erences. For assignment of NMR signals, 1H,1H-DQFCOSY15 and 1H,1H-TOCSY16 experiments with mixing times of 30, 60, and 90 ms and a gradient selected 13C-decoupled inverse 1H-detected 1H,13C heteronuclear single-quantum coherence (gHSQC)17 were used according to standard pulse sequences. For sequence information, the following experiments were used 1H,1H-NOESY18 with mixing times of 10, 25, and 50 ms, 1H,13C-gHSQC-1H,1H-NOESY19 with a mixing time of 50 ms, and gradient selected 1H,13C heteronuclear multiple-bond correlation (gHMBC)17 with a 40 ms delay for the evolution of long-range couplings. The chemical shifts were compared to those of the corresponding monosaccharide.20 Results and Discussion P. freudenreichii ssp. shermanii JS strain was grown in a whey-based medium, and the EPS was isolated as a 66% ethanol precipitate from the culture medium. The material was filtered and subsequently lyophilized. Component analysis of the EPS revealed that it contained D-Glc. The 1H NMR spectrum of the EPS showed two signals in the region for anomeric protons. These were shown to correlate to resonances from anomeric carbons in a 1H,13C-HSQC spectrum. Thus, the repeating units of the EPS contain only two sugar residues. The assignments of 1H and 13C resonances were performed by 1H,1H and 1H,13C correlated 2D NMR experiments and are given in Table 1. The information given by JH1,H2 ≈ 7.5 Hz of the two resonances for the anomeric protons together with 13C chemical shifts for the ring carbons reveals that the constituent sugar residues are β-linked and have the pyranoid ring form. For the glucosyl residue having its anomeric carbon at 103.1 ppm the 13C chemical shift displacements of C2-C6 are all
