Characterization of the levan produced by Paenibacillus bovis sp. nov BD3526 and its immunological activity

Carbohydrate Polymers 144 (2016) 178–186

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Characterization of the levan produced by Paenibacillus bovis sp. nov BD3526 and its immunological activity Xiaofen Xu a,b , Caixia Gao a,b , Zhenmin Liu a,b , Jiang Wu a,b , Jin Han a,b , Minghui Yan a,b , Zhengjun Wu a,b,∗ a b

State Key Laboratory of Dairy Biotechnology, Institute of Bright Dairy & Food Co., Ltd., 1518 West Jiangchang Road, Shanghai 200436, PR China Innovative Platform for the Industry of Dairy Product, Synergetic Innovation Center of Food Safety and Nutrition, Shanghai, PR China

a r t i c l e

i n f o

Article history: Received 27 November 2015 Received in revised form 14 February 2016 Accepted 16 February 2016 Available online 18 February 2016 Keywords: Paenibacillus Levan Levansucrase Conformation Cytokines

a b s t r a c t Paenibacillus bovis sp. nov BD3526 synthesizes a large amount of exopolysaccharides (EPSs) (36.25 g/L) in a semi-defined chemical medium containing 20% (w/v) sucrose. The EPSs were extracted from the cultured broth by ethanol precipitation and purified via anion-exchange and gel permeation chromatography. The Fourier transform infrared (FT-IR) and nuclear magnetic resonance (NMR) spectra showed that the primary EPS fraction (F1) was a linear ␤ (2 → 6)-linked levan. The peak molecular weight (Mp ) of the levan exceeded 2.6 × 106 Da based on high-performance size-exclusion chromatography (HPSEC). The levan adopted a spherical conformation in aqueous solution as confirmed by transmission electron microscopy (TEM). The corresponding levansucrase was identified by SDS-PAGE analysis and in situ polymer synthesis. The in vitro assay demonstrated that the levan significantly stimulated the proliferation of spleen cells and induced the expression of TNF-␣, indicating its potential as a natural immunomodulator. © 2016 Elsevier Ltd. All rights reserved.

1. Introduction Microbial exopolysaccharides (EPSs) are biopolymers secreted by microorganisms into their surroundings. EPSs are widely used in the food industry as viscosifying, stabilizing, gelling, or emulsifying agents (De Vuyst & Degeest, 1999; Laws, Gu, & Marshall, 2001), in environmental areas as bioflocculants, bioabsorbents, and heavy metal removal agents (Wang, Ahmed, Feng, Li, & Song, 2008), and in medicine as antitumor, antioxidant, antiviral, immunostimulatory, or drug delivery agents (Lin & Zhang, 2004; Raveendran, Poulose, Yoshida, Maekawa, & Kumar, 2013). Recently, great interest in microbial EPSs had led to the isolation of a wide variety of bacterial strains with the capacity to produce EPSs with novel structures and functions (Sutherland, 2002). Some microbial EPSs have been marketed as commercial products, (e.g., xanthan from Xanthomonas campestris, gellan from Sphingomonas paucimobilis, bacterial alginates synthesized by Pseudomonas species and Azotobacter chrococcum, hyaluronic acid from Streptococcus equi and succinoglycan from Rhizobium spp.) (Sutherland, 2002).

The genus Paenibacillus consists of facultative anaerobic, endospore-forming bacteria, which was originally included within the genus Bacillus and lately reclassified as a separate genus in 1993 (Ash, Priest, & Collins, 1993). Members of the genus Paenibacillus are mostly soil borne bacteria that are important for the medical, industrial, and agricultural fields (Sutherland, 2002). For example, Paenibacillus polymyxa has attracted much attention because it could produce EPSs with diverse physiological and biotechnological functions (Han, 1989; Han & Clarke, 1990; Lee et al., 1997; Liu et al., 2009; Seldin, 2011; Liang & Wang, 2015). Paenibacillus bovis sp. nov BD3526 (CGMCC 8333 = DSM 28815) was newly isolated from raw yak milk samples collected in southwestern China (Gao et al., 2016). This strain synthesized large amounts of levan in a semi-defined chemical medium containing 20% (w/v) sucrose. The aim of this work was to isolate and characterize the levan produced by Paenibacillus spp. BD3526, to identify the corresponding levansucrase, and to investigate the immunomodulatory activity of the levan in vitro. 2. Materials and methods

∗ Corresponding author at: State Key Laboratory of Dairy Biotechnology, Institute of Bright Dairy & Food Co., Ltd., 1518 West Jiangchang Road, Shanghai 200436, PR China. Fax: +86 21 66553536. E-mail address: [email protected] (Z. Wu). http://dx.doi.org/10.1016/j.carbpol.2016.02.049 0144-8617/© 2016 Elsevier Ltd. All rights reserved.

2.1. Microorganism and medium Strain BD3526 was routinely aerobically propagated on TYC agar at 30 ◦ C and stored in TYC broth supplemented with 15% (v/v)

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glycerol at −80 ◦ C. The TYC medium contained (g L−1 ): sucrose 50.0, tryptone 15.0, yeast extract 5.0, l-cysteine 0.2, CH3 COONa 20.0, NaHCO3 2.0, Na2 SO4 0.1, Na2 HPO4 ·12H2 O 2.0, and NaCl 1.0. The semi-defined chemical medium containing 20% (w/v) sucrose was composed of (g L−1 ): sucrose 200, tryptone 10, yeast extract 5, K2 HPO4 5, and CaCl2 0.34. This medium was adopted from the medium used by Liu, Luo et al. (2010) with some modification. The initial pH value of the medium was 6.8. 2.2. Preparation and purification of EPSs Strain BD3526 freshly cultured in TYC broth at 30 ◦ C and 200 rpm for 24 h was inoculated into the semi-defined chemical medium with a ratio of 2% (v/v) and cultured under the same conditions. After 72 h cultivation, the fermented broth was centrifuged at 12,000 × g at 4 ◦ C for 20 min to remove the bacterial cells and coagulated proteins. The supernatant was heated to 100 ◦ C for 10 min to inactive enzymes capable of degrading the EPS. After cooling to room temperature, 3 vol of pre-chilled ethanol were added to precipitate the EPSs. The precipitate was collected by centrifugation at 12,000 × g for 20 min at 4 ◦ C and redissolved in de-ionized water, followed by five rounds of deproteinization with 1/5 vol of the Sevag reagent (CHCl3 –BuOH, v/v = 5/1) (Staub, 1965). The deproteinized solution was then dialyzed intensively using membrane tubes with a MWCO of 14,000 Da against de-ionized water for 48 h. The retentate was freeze-dried to obtain the crude EPSs. No proteins were detected by the Bradford method (1976). A total of 100 mg of crude EPSs was re-dissolved in 5 mL of 0.05 M Tris–HCl buffer (pH 7.6) and loaded onto a DEAESepharose Fast Flow column (D 2.6 cm × 30 cm, GE Health, USA) pre-equilibrated with the same buffer. The loaded column was eluted with 1.5 column volumes of 0.05 M Tris–HCl buffer (pH 7.6) at a flow rate of 3 mL min−1 to obtain the F1 fraction. Two other weak peaks were observed (F2 and F3) after subsequent elution with 1.5 column volumes of 0.05 M Tris–HCl buffer (pH 7.6) containing a NaCl gradient of 0–1.2 mol L−1 . The eluted fractions were assayed for their carbohydrate content by the phenol-sulfuric acid method (Dubois, Gilles, Hamilton, Rebers, & Simith, 1956). The tubes containing the individual fractions were pooled, dialyzed against de-ionized water for 3 days at 4 ◦ C and lyophilized. The combined contents of F2 and F3 were less than 5% of the total EPS and therefore were abandoned. The F1 fraction was further purified on a Sepharose CL-4B gel column (D 1.6 cm × 100 cm) and eluted with 0.15 M NaCl at a flow rate of 0.2 mL min−1 ; a total of 6 mL of eluent was collected in each tube. The eluent containing polysaccharide was pooled, dialyzed against deionized water for 3 days at 4 ◦ C, and finally lyophilized. 2.3. Infrared spectrum of F1 The Fourier-transformed infrared (FT-IR) spectrum of the purified F1 was recorded on a Nicolet 6700 spectrophotometer (Thermo Fisher, USA). The sample was prepared as a KBr pellet and the pellet was scanned against a blank KBr pellet background at the wavelength range of 4000–500 cm−1 with a resolution of 4.0 cm−1 . 2.4. NMR analysis The 1 H NMR and 13 C NMR spectra of the purified F1 in D2 O were recorded on an Avance III 400 MHz spectrometer (Bruker, Germany) at 25 ◦ C. Sodium trimethylsilylpropionate (TSP) was used as the internal standard (0.00 ppm for 1 H and 0.00 ppm for 13 C). The 13 C–1 H-HMBC experiment was conducted on a Bruker DRX Avance 600 MHz spectrometer at 25 ◦ C.

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2.5. Measurement of molecular weight The purity and average molecular weight of the F1 fraction was determined by high performance size-exclusion chromatography (HPSEC). The analysis was performed on a PerkinElmer series 200 Liquid Chromatography with two TSK gel permeation columns, G6000 PWXL and G4000 PWXL connected in series. F1 was dissolved in 0.05 M NaNO3 at a concentration of 1 mg mL−1 and filtered through a 0.22 ␮m Millipore membrane before loading. A total of 50 ␮L of the F1 solution was loaded and eluted with 0.05 M NaNO3 at 0.6 mL min−1 . The eluent was monitored with a Perkin-Elmer series 200 Refractive Index Detector (RID). The molar mass of F1 was estimated using a calibration curve prepared with pullulan standards of peak molecular weight (Mp ) from 6 kDa to 2560 kDa (Sigma, USA). 2.6. Monosaccharide composition analysis The monosaccharide composition of the F1 fraction was analyzed by high-performance anion-exchange chromatography with pulsed amperometric detector (HPAEC-PAD) (ICS 5000, Dionex, USA) (Shao et al., 2014). The complete hydrolysis of F1 to monosaccharides was achieved by slight modification of the procedure described by Marx, Winkler, and Hartmeier. (2000). In brief, 20 mg of the F1 fraction was hydrolyzed in 2 mL of 0.01 M sulfuric acid at 80 ◦ C for 2 h in a sealed bottle. After cooling to room temperature, the hydrolysate was neutralized with 5 M NaOH and filtered through a 0.22 ␮m membrane (Millipore, MA, USA). Then, the hydrolysate was analyzed on a Carbo Pac PA20 column (ID 3 mm × 150 mm) (Dionex, USA) with a pulsed amperometric detector using gradient elution procedure with H2 O—250 mM NaOH—1.0 M NaAC as mobile phase (Shao et al., 2014). The sample was eluted at 0.5 mL min−1 and the injection volume was 20 ␮L. A set of monosaccharides were used as standards, including arabinose, fructose, galactose, galactosamine, glucosamine, glucose, mannose, rhamnose, ribose, and xylose (Sigma, USA). 2.7. Transmission electron microscope (TEM) The molecular morphology of the F1 fraction was assessed with a high-resolution transmission electron microscope (JEM-2100, JEOL, Tokyo, Japan). F1 was dissolved in distilled water at a concentration of 5 mg mL−1 with stirring for 4 h and filtered through a 0.22 ␮m filter. A drop of F1 solution was deposited on a copper grid coated with a thin carbon film. After being dried at room temperature, TEM images of the sample were taken at an accelerating voltage of 200 kV. 2.8. Identification of the levansucrase by SDS-PAGE and in situ polymer synthesis To avoid the interference by the large amount of EPSs synthesized by strain BD3526 in the medium containing high level of sucrose on the precipitation of the enzymes involved in this procedure, the bacterial strain was cultivated in the semi-defined medium supplemented with sucrose or glucose (as a control) at a reduced concentration, (i.e., 2% (w/v) at 30 ◦ C aerobically for 24 h). The cultivated broth was centrifugated at 12,000 × g for 15 min to remove cells. The supernatant was concentrated 2-fold using Centricon tubes (MWCO: 10,000 Da, Amicon Ultra). Solid diammonium sulphate was slowly added to the concentrated supernatant up to 60% saturation under gentle stirring at room temperature. The precipitated proteins were obtained by centrifugation at 15,000 × g for 10 min and dissolved into a minimum amount of deionized water. The protein solution was then mixed with 4x Laemmli Sample Buffer (Bio-Rad, USA) at a ratio of 3:1 (v/v), and incubated at

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37 ◦ C for 1 h. For SDS-PAGE, 10% acrylamide containing 0.1% SDS was used. Proteins in the gel were stained with Coomassie blue G-250 and destained with 10% acetic acid solution. The standard protein markers were purchased from Bio-Rad (USA). For the in situ detection of the levansucrase activity, the SDSPAGE gel was incubated in 50 mM sodium acetate buffer containing 50 g L−1 sucrose buffered at pH 5.6, as described by Dols, RemaudSimeon, Willemot, Vignon, and Monsan (1998). Briefly, the gel was washed three times with 50 mM sodium acetate buffer (pH 5.6) containing 2 mM CaCl2 and 0.1% (vol/vol) Triton X-100 at room temperature to eliminate the SDS and then soaked in the same buffer supplemented with 50 g L−1 of sucrose at room temperature for 24 h. The protein bands capable of EPS synthesis were detected by the appearance of opaque polymers in the gel as described by Miller and Robert (1986). 2.9. Splenic lymphocyte proliferation assay BALB/C mice (6 weeks old and weighing 20.0 ± 2.0 g) were purchased from Shanghai Slac Laboratory Animal Ltd. (Shanghai, China). Single-cell suspensions of spleen lymphocytes were prepared as previously described by Kaspers, Lillehoj, Jenkins, and Pharr (1994). The effect of F1 on spleen lymphocyte proliferation activity was tested according to the method of Liu, Lu et al. (2010). Briefly, mouse splenocytes in RPMI-1640 medium supplemented with 10% fetal calf serum were seeded into a 96-well flat-bottomed microplate at the density of 2 × 105 cells/well and incubated with the F1 fraction of different final concentrations (16, 32, 64, 128, and 256 ␮g mL−1 ) for 44 h at 37 ◦ C under a humidified 5% CO2 atmosphere, using LPS (5 ␮g mL−1 , Sigma) as a positive control and complete RPMI1640 medium as the blank control. Cell proliferation was assayed by the CellTiter 96 Aqueous One Solution cell proliferation kit (Promega, USA) according to the manufacturer’s instructions. The absorbance was measured at 490 nm by an ELISA reader (SpectraMax M5, Molecular Devices, USA). Each substance was evaluated in quintuplicate. Data were analyzed by one-way analysis of variance (ANOVA) and Student’s t test. Differences were considered statistically significant for *p < 0.05 versus the blank control.

including the 16S rRNA sequence similarity and genomic DNA relatedness to other validated members of this genus (Gao et al., 2016). The GenBank/EMBL/DDBJ accession number for the 16S rRNA sequence of this strain is KM978955. For example, the similarities in the 16S rRNA sequences among strain BD3526 and P. polymyxa ATCC842T and P. polymyxa EJS-3 are 93.8% and 94.0%, respectively, based on the BLAST program (http://blast.ncbi.nlm. nih.gov/Blast.cgi). This result indicated that BD3526 can be discriminated from P. polymyxa strains. Strain BD3526 synthesized a large amount of EPSs (up to 36.25 g L−1 ) when cultivated in semi-defined chemical medium containing 20% (w/v) sucrose at 30 ◦ C for 72 h at 200 rpm. The EPS yield was similar to P. polymyxa EJS-3 (Liu, Luo et al., 2010). The EPSs synthesized by strain BD3526 in the culture broth were prepared via ethanol precipitation, deproteination with the Sevag reagent, dialysis and finally freeze-drying. Then, the EPSs were purified by ion-exchange chromatography and three fractions (F1, F2 and F3) were obtained as shown in Fig. 1. F1 was the predominant EPS fraction (95.7%, w/w) and was further purified by gel permeation chromatography. Only one single and symmetrical peak appeared on the elution profile (data not shown), suggesting that F1 was a homogeneous polysaccharide and needed no further purification. In considering that the crude EPSs contained low ratio of the F2 and F3 fractions (less than 5%), strain BD3526 has great potential to produce the F1 fraction in a simple way. 3.2. FT-IR analysis The FT-IR spectrum of F1 is shown in Fig. 2. The strong band at 3423 cm−1 was attributed to the hydroxyl stretching vibration of the polysaccharide while the bands in 2935 and 2887 cm−1 were ascribed to C H stretching vibration. The broad band at 1636 cm−1 was due to the bound water (Park, 1971). The strong absorption at 1068 cm−1 with two shoulders at 1127 and 1022 cm−1 was dominated by ring vibration overlapped with stretching vibration of C OH groups and the glycosidic linkage C O C stretching vibration (Rapala et al., 2015). The appearance of the characteristic absorptions at 927 and 809 cm−1 indicated the presence of the furanoid ring of the F1 molecular chain (Liu, Lu et al., 2010). The FT-IR result indicated that F1 was a polyfructan, namely inulin or levan.

2.10. Measurement of cytokine production by spleen cells 3.3. NMR analysis For cytokine measurement, mouse splenocytes in RPMI1640 medium supplemented with 10% fetal calf serum were seeded in a 96-well flat-bottomed microplate at the density of 4 × 105 cells/well and incubated with different final concentrations of the F1 fraction (64, 128, and 256 ␮g mL−1 ) for 48 h at 37 ◦ C under a humidified 5% CO2 atmosphere. LPS (50 ng mL−1 ) was used as a positive control and complete RPMI-1640 medium was used as the blank control. Cytokines IL-2, IL-4, IL-6, IL-10, IL-17A, IFNg, and TNF-␣ in the culture supernatant were assayed by flow cytometry using BDTM CBA Mouse Th1/Th2/Th17Cytokine kit (BD, USA) according to the manufacturer’s instructions. Each treatment was performed in triplicate and the results were expressed by their means ± SD (standard deviation). The statistical significance of each treatment was determined by one-way analysis of variance (ANOVA) and Student’s t test. Differences were considered statistically significant at *p < 0.05 and **p < 0.01 versus the blank control. 3. Results and discussion 3.1. Preparation and purification of exopolysaccharide Strain BD3526 was proposed as a novel species in the genus Paenibacillus based on its physiological and genotypic properties,

d-Fructofuranosides are reported to be heat-labile and partially decomposed during the process of derivatization to suitable forms in gas–liquid chromatography analysis. In contrast, 13 C NMR is a method more suited to determining the linkage type of a polyfructan (Shimamura et al., 1987). Therefore, further structure confirmation was performed using 13 C NMR, 1 H NMR and 2D NMR (HMBC) analysis. The recognized characteristic differences between the 13 C NMR spectra of inulin and levan are that the primary carbons (C-1 and C6) are more closely grouped in inulin whereas the ring carbons (C-3, C-4, and C-5) are more closely grouped in levan (Barrow, Collins, Rogers, & Smith, 1984; Hammer & Morgenlie, 1990; Han, 1990; Han & Clarke, 1990; Jathore, Bule, Tilay, & Annapure, 2012; Tajima et al., 1997; Van Geel-Schutten et al., 1999). The 13 C NMR spectrum of F1 (Fig. 3(a)) showed only six intense resonances. Although the peak positions deviated a little from the reported values as listed in Table 1, the relative spacing of the C atoms of F1 was more similar to those assigned to levan rather than those for inulin (Han, 1990; Han & Clarke, 1990). Additionally, the ␤ (2 → 6) linkage could be confirmed by the presence of a down field shifted signal at 66.25 ppm (C-6) in the 13 C NMR spectrum. Therefore, the F1 fraction was a levan-type fructan consisting of ␤ (2 → 6)fructofuranoside. The 1 H NMR spectrum of the F1 fraction displayed

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Fig. 1. The elution profile of EPSs produced by Paenibacillus bovis sp. nov BD3526 on a DEAE-Sepharose Fast Flow column.

Fig. 2. The FT-IR spectrum of the F1 fraction produced by Paenibacillus bovis sp. nov BD3526.

Table 1 Chemical shifts in the different sources. Carbon atom

C-1 C-2 C-3 C-4 C-5 C-6 a b c

13

C NMR spectra of the F1 fraction, inulin and levans from

Chemical shifts (ppm) Inulina

Levana

Levanb

Levanc

F1

60.9 103.3 77.0 74.3 81.1 62.2

59.9 104.2 76.3 75.2 80.3 63.4

60.435 104.696 76.77 75.783 80.880 63.978

61.4 105.6 77.8 76.7 81.7 64.8

62.78 107.08 79.17 78.07 83.16 66.25

Assignment cited from Shimamura et al. (1987). Assignment cited from Jathore et al. (2012). Assignment cited from Sims et al. (2011).

almost identical resonance pattern peaks of levans (Barrow et al., 1984; Hammer & Morgenlie, 1990; Han, 1990; Han & Clarke, 1990;

Jathore et al., 2012; Tajima et al., 1997; Van Geel-Schutten et al., 1999), which also confirmed the levan structure of the F1 fraction, and the detailed assignments for each proton are shown in Fig. 3(b). Furthermore, the C-2 signal of the (2, 6)-linked ␤-d-Fruf residues in the anomeric region of the HMBC spectrum (Fig. 4) showed crosspeaks with the H-6 signal of the (2, 6)-linked ␤-d-Fruf residues, which confirmed the ␤ (2 → 6) linkages of F1. Levan is a natural homopolysaccharide composed of D-fructofuranosyl residues joined together by ␤-(2, 6) linkages. In the case of branched levan, ␤-(2, 1) linkages in the branch chains are also present. The 13 C NMR spectroscopy was previously employed to differentiate branched or linear levans from plants (Hammer & ´ Jennings, & Glaudemans, 1978). There Morgenlie, 1990; Tomaˇsic, were only six intense and sharp carbon signals of the fructofuranosyl residues in the 13 C NMR spectrum of a linear levan while spectra of slightly branched bacterial levans showed a weak C-3

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signal about 0.2 ppm downfield from the major levan C-3 signal (Seymour, Knapp, Zweig, & Bishop, 1979). In the 13 C NMR spectrum of F1, there was no obvious signal downfield from either the dominant C-3 signal or the other C signals, indicating that F1 was a linear levan.

3.4. Molecular weight determination of F1

Fig. 3. The 13 C NMR and 1 H NMR spectra of the F1 fraction produced by Paenibacillus bovis sp. nov BD3526.

Microbial levans are produced mainly by bacteria such as Bacillus subtilis, Zymomonas mobilis, P. polymyxa (formerly Bacillus polymyxa), Aerobacter levanicum, Erwinia amylovora, Rhanella aquatilis, and some Pseudomonas spp. The molecular weights of microbial levans vary greatly with producers as well as the cultivation parameters used. B. subtilis (natto) produced both low (11 kDa) and high (1800 kDa) molecular weight levans while the molecular weight of levan derived from B. polymyxa (NRRLB-18475) was approximately 2 × 106 Da (Han, 1990; Shih, Chen, Wang, Wu, & Liaw, 2010). There was only one symmetrically sharp peak in the HPSEC profile of F1 (Fig. 5), revealing that F1 was a highly pure homogeneous polysaccharide. As the retention time of F1 was shorter than that of the pullulan standard with the highest molecular weight employed in this study, the molecular weight of F1 was estimated by extrapolation of the equation built upon the retention times of the pullulan standards versus their molecular weights. The estimated molecular weight of F1 was approximately 4.8 × 106 Da, which was well within the separation range of the columns (1.0 × 103 –5.0 × 107 Da). To be more precise, F1 is a linear levan with Mp exceeding 2.6 × 106 Da.

Fig. 4. The HMBC spectrum of the F1 fracton produced by Paenibacillus bovis sp. nov BD3526.

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of fructose (11.38 min), suggesting that the F1 fraction consisted almost solely of fructose residues. Sucrose has been reported to be the predominantly initial acceptor in levan formation, and thus a terminal glucose residue should be present in the levan chain. However, it is usually hard to determine the presence of the glucose residue in high molecular weight levans (Han, 1990). 3.6. TEM

Fig. 5. The HPSEC profile of the F1 fraction produced by Paenibacillus bovis sp. nov BD3526.

In aqueous solution, the F1 molecules appeared as small black spheroidal or ellipsoidal particles with diameters in the range of 20–50 nm against a dark grey background (Fig. 7), which was similar to the compact structure of levans from Bacillus vulgatus (Ingelman & Siegbahn, 1944) and Streptococcus salivarius (Newbrun, Lacy, & Christie, 1971). Although F1 was a linear levan with high molecular weight, its aqueous solution was non-viscous even at a high concentration (5% (w/v)) (data not shown), which could be attributed to its spheroidal conformation in aqueous solution.

3.5. Monosaccharide composition analysis

3.7. Identification of the levansucrase

The monosaccharide composition of F1 was identified by HPAEC-PAD after acid hydrolysis. As shown in Fig. 6, there was only one peak. The retention time (11.16 min) coincided with that

Levansucrase (EC 2.4.2.10) is a transfructosylase that catalyzes the formation of levan and fructose-oligosaccharides from sucrose. Levansucrase has been found in a wide variety of microorganisms

Fig. 6. HPAEC profiles of standard monosaccharides and the monosaccharide composition of F1.

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glucose, the proteins expressed by strain BD3526 using glucose as carbon source were used as the controls for SDS-PAGE analysis. Compared with protein bands expressed in the glucose medium, there were two obvious differentially expressed bands with molecular weights of around 40 and 87 kDa (Fig. 8(a)) in the cultured broth containing sucrose as the sole carbon source. The levansucrase band was identified by in situ gel biopolymer synthesis. As shown in Fig. 8(b), a white and opaque band occurred at the position of 87 kDa in the unstained SDS-PAGE gel soaked in a 50 g/L sucrose solution for 24 h at room temperature. This result was suggestive of the formation of a polysaccharide and indicated that the protein band at this position possessed levansucrase activity. Accordingly, the relative molecular weight of the levansucrase was approximately estimated to be about 87 kDa, which was consistent with the previous report (Hee et al., 2005). 3.8. Immunological activity of F1

Fig. 7. The TEM image of F1 in distilled water (2.5 mg mL−1 ).

(Avigad, 1968), among which, B. subtilis are the most extensively studied (Coté & Ahlgren, 1993). In this study, the levansucrase secreted by strain BD3526 was identified by SDS-PAGE and in situ gel polymer synthesis. Since no levan could be synthesized from

The immunological activities of micobial levans have been extensively evaluated. Purified levans from variety of sources were reported to induce an immune response in human subjects (Allen & Kabat, 1957), while levan from Aerobacter demonstrated anti-tumor and immunostimulatory activities in human (Leibovici, Susskind, & Wolman, 1980; Stark & Leibovici, 1986). Recently, the levan synthesized by Bacillus licheniformis 8-37-0-1 was reported to significantly stimulate the proliferation of spleen lymphocytes (Liu, Lu et al., 2010). Lymphocyte proliferation is a crucial event in the activation cascade of both cellular and humoral immune responses. The effect of F1 on spleen lymphocyte proliferation was evaluated by the CellTiter 96 Aqueous One Solution (Promega, USA) kit. The MTS [3-(4,5-dimethylthiazol-2-yl)-5(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium] in the CellTiter 96 kit could be reduced by viable cells into a

Fig. 8. Proteins differentially expressed by strain BD3526 in medium containing glucose or sucrose and the detection of levansucrase activity. (a) SDS-PAGE of proteins expressed in medium containing glucose (lane ‘Glc’) or sucrose (lane ‘Suc’). Lane M shows Precision Plus Protein Standard. (b) In situ gel polysaccharides synthesis. The unstained SDS-PAGE gel was soaked in 50 mM sodium acetate buffer (pH 5.6) with 50 g L−1 of sucrose at room temperature for 24 h.

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Absorbance at 490 nm

(a)

185

0.8

0.6

* *

* 0.4

0.2

0.0

Control

16

32

(b)

90

128 64 Levan ( µg/mL)

256

**

80

IL-2 IL-4 IL-6

70

Cytokines (ng/mL)

LPS

IFN-g

60

TNF IL-17a

50

IL-10

40 30 20

*

*

10

*

0

Control

LPS

* 64

*

*

128

256

Levan (µg/mL) Fig. 9. Immunostimulatory activity of the F1 fraction. (a) Effects of F1 on the proliferation of BALB/C mouse spleen cells in vitro. Splenocytes were stimulated with F1 or LPS (5 ␮g mL−1 ) for 44 h. Proliferation was measured by the CellTiter 96 Aqueous One Solution assay kit and expressed as absorbance at 490 nm. (b) Expression of cytokines in spleen cells from BALB/c mice stimulated by F1 with different concentrations or LPS. Data are expressed as means ± SD. *p < 0.05, **p < 0.01 versus the blank control.

formazan product that is measured by absorbance at 490 nm. The amount of formazan is directly proportional to the number of viable cells. Escherichia coli lipopolysaccharide (LPS, Sigma, USA), a B-cell mitogen, was used as the positive control. F1 stimulated the proliferation of spleen cells in a dose-dependent manner at concentrations ranging from 16 ␮g mL−1 to 256 ␮g mL−1 (Fig. 9(a)). Compared with the blank control, the stimulating effect was significant only at concentrations of 128 ␮g mL−1 or higher (p < 0.05). The mitogenic activity of F1 was similar to that of the levan from B. licheniformis 8-37-0-1 (Liu, Lu et al., 2010). The immunomodulatory effect was further tested on cytokine release by the mouse spleen cells. In comparison with the blank, among the tested cytokines (IL-2, IL-4, IL-6, IL-10, IL-17A, IFNg, and TNF-␣), only the expression of TNF-␣ was significantly up-regulated by F1 at concentrations of 64 ␮g mL−1 or higher (p < 0.05), and no obvious influence on the other cytokines was

observed (Fig. 9(b)). In contrast, all cytokines except IL-4 and IL17A were stimulated to a higher level by low concentration of LPS (Fig. 9(b)). The induction of TNF-␣ expression by only a relatively high concentration of F1 indicated its weak inflammatory effect. A similar phenomenon was observed for the levan isolated from B. subtilis fermented soybean mucilage (Xu et al., 2006). Therefore, the levan (F1 fraction) synthesized by strain BD3526 displayed immunostimulatory activities via up-regulating the TNF-␣ expression level, which is usually expressed by Th1 subset cells, NK cells and macrophages in inflammatory reactions. 4. Conclusions The newly isolated P. bovis sp. nov BD3526 synthesized a large amount of EPSs (36.25 g/L). The primary EPS fraction (F1, 95.7%, w/w) was identified as a linear ␤ (2 → 6)-linked levan. The Mp of this

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