Effect of carbon source on production , characterization and bioactivity of exopolysaccharide produced by Phellinus vaninii Ljup

The effect on different three carbon source (i.e. glucose, fructose and sucrose) on production, chemical characterization and antioxidant activity of exopolysaccharide (EPS) produced by Phellinus vaninii Ljup was investigated in this study. Amongst carbon sources examined, glucose and sucrose were favorable for the mycelia growth, while the maximum EPS yield was achieved when sucrose was employed. The predominant carbohydrate compositions in EPSs identified were gluconic acid, glucose, mannose and galactose acid. Then, FT-IR spectral analysis revealed prominent characteristic groups in EPSs. EPSs molecule exist as nearly globular shape form in aqueous solution. The variation also affects antioxidant activities by investigated by using hydroxyl and DPPH radical scavenging assay. Sucrose was best carbon source from the viewpoint of antioxidant activity due to the relatively high contents of galactose in the EPS with moderate molecular weight and polydispersity.

The submerged culture for exopolysaccharide (EPS) production by medicinal fungi are proved to have many properties, such as immunomodulation, anticancer, antioxidant, hypolipidemic and hyperglycemic activities (Mahapatra and Banerjee 2013, Wasser 2002).Many investigators have attempted to obtain the optimal submerged culture for EPS from several fungi (Tang et al. 2008, Xiong et al. 2012).It is demonstrated that the yields and compositions of the EPS were affected by culturing conditions, especially when using different carbon sources which might also affect
the antioxidant property (Fan et al. 2009, Xiong et al. 2012).However, no data are currently available concerning the effect of carbon sources on the change in chemical characterization and bioactivity of exopolysaccharide of fungal polysaccharides.Therefore, the aim of the study was evaluation of effect of three carbon sources (glucose, fructose and sucrose) on features and antioxidant potential of P. vaninii Ljup EPS produced during submerged cultivation.

CHEMICALS
The carbon sources (glucose, fructose and sucrose) and chemicals for medium culture were of reagent grade, while other chemicals were of analytical grade.The culture broth was centrifuged at 9000 rpm for 15 min, and the resulting supernatant was mixed with four vols. of pure ethanol.The precipitated EPS was collected by centrifugation at 9000 rpm for 15 min and then deproteinized using Sevag reagent (1:4 n-butanol/chloroform, v/v).After removing the proteins and Sevag reagent by centrifugation, the aqueous phase was dialyzed against deionized water and lyophilized to yield the crude polysaccharide.The total EPS in the culture medium was determined by the phenol-sulphuric acid method, using glucose as the standard (Dubois et al. 1956).The dry weight of mycelium was measured after washing the mycelial pellet with distilled water three times and drying at 70 o C to a constant weight (Hwang et al. 2003).

PURIFICATION OF EPS
The polysaccharide was re-dissolved in 0.2 M NaCl solution, and applied to a Sepharose CL-6B column (2.4 cm×100 cm, Sigma Chemical Co., St Louis, MO) and eluted with the same solution at a flow rate of 0.6 ml/min.Fractions (5.0 ml/ tube) were collected by a fraction collector (Hwang et al. 2003).The total carbohydrate content in the EPS was also determined by the phenol-sulfuric acid method (Hu et al. 2014).The flow rate was 0.75 mL/min and the injection volume and concentration was 100 mL and 2 mg/mL, respectively.During the calculation of molecular weights of each EPS, the value of dn/dc (specific refractive index increment) was used from the data in literature (Jumel et al. 1996), in which the estimated dn/dc was 0.14 mL/g.Calculations of molecular weight and root mean square (RMS) radius of gyration for each EPS were performed by the Astra 4.72 software (Wyatt Technology).The RMS radii of each polysaccharide were determined from the slope by extrapolation of the first-order Debye plot (Astafieva et al. 1996).The gross conformation of EPS in aqueous solution could be identified from the double logarithmic plot of RMS radius vs. molecular mass of EPS according to the following equations: where r i is the RMS radius of an EPS molecule, M i is the molar mass of EPS, k is the intercept at the Y-axis (RMS radius), and 1/3, 1/2, and 1 are the critical slope values for determining the molecular conformation of each EPS (Hwang et al. 2003, Lim et al. 2005).

MONOSACCHARIDE COMPOSITION ANALYSIS
For the identification and quantification of monosaccharide, EPS fraction (5 mg) was hydrolyzed with 2 mL of 2 M trifluoroacetic acid

EFFECTS OF CARBON SOURCES IN MEDIUM ON BIOMASS AND EPS PRODUCTION
The results of the biomass and EPS of P. vaninii Ljup from the submerged culture in a 5-L stirred-tank with various carbon sources are shown in Figure 1.After a fermentation period of 8 days, glucose and sucrose were the most suitable carbon source for biomass production.However, the highest EPS yield of 1.96 g/L achieved when sucrose was employed.The result indicates that P. vaninii Ljup has the ability to use different carbon sources, all of which have varied degree of stimulatory effect on biomass and EPS production.Both glucose and fructose are hexoses, which can participate in glycolytic and pentose phosphate pathways.
According to biomass and EPS yield in Figure 1, sucrose was shown to sustain high growth rates in cell cultures and carbon conversion efficiency is also high.It has been reported that different carbon sources had different influences of catabolic repression on the secondary metabolism (Tang et al. 2008, Khondkar et al. 2002).Though it is well known that sucrose is a disaccharide combination of the monosaccharides glucose and fructose, the information concerning the metabolic utilisation of sucrose for EPS production is extremely limited.

PURIFICATION AND ANALYSIS OF CARBOHYDRATES
The EPS was obtained from the fermentation broth from submerged culture of P. vaninii Ljup by the method of ethanol precipitation.In gel filtration chromatography of the culture filtrate on Sepharose CL-6B, the main fraction of each EPS produced from three different carbon sources was coeluted.
The detailed monosaccharide compositions from the carbohydrate composition in the EPS fraction could be worked out from trifluoroacetic acid hydrolysis and GC-MS analysis as illustrated in Table I.As expected, the carbohydrates composition within the EPS varies with the different carbon sources.Nevertheless, the predominant sugars in EPS, identified in this study, were gluconic acid, glucose, mannose and galactose acid.Negligible amounts of rhamnose, ribose and xylose were also detected.This indicates that interconversion among glucose, fructose and sucrose in the microorganism give polysaccharides with the similar composition.It is very interesting to note that the highest content of galactose unit was observed in carbohydrate composition when the sucrose was employed as carbon source (Table I).Kai et al. (2003) found that different carbon sources in the media can influence the hetero-polysaccharides synthesis by Pestalotiopsis microspora.When glucose was used as the carbon source, a considerable amount of mannose units were formed.While with xylose as the carbon source, polysaccharides containing large amounts of mannose and galactose units were produced.Smiderle et al. ( 2012) also reported the similar results when glucose, galactose, xylose or arabinose was used as carbon source to produce polysaccharide by Pleurotus pulmonarius.
Analyzing EPS monosaccharide composition, it was only observed similarities for the culture medium using mannose and arabinose, having mannose as the major component in both EPS samples.However, limited information is available possess similar functional groups.There is a broad stretching intense characteristic peak at ca. 3279-3285 cm -1 typical of -OH groups as well as a weak C-H band at ca. 2922-2928 cm -1 .The characteristic band at ca. 1641-1643 cm -1 could be correlated to the stretching vibration of the carbonyl group (C=O) of the polysaccharide.Bands at ca. 1024-1030 cm -1 suggested the presences of C-O type of linkages (Xie et al. 2010).Each particular polysaccharide possessed the specific bands at 807-810 cm -1 and 916-921 cm -1 , indicating both αand β-configurations of the sugar units (Lim et al. 2005).

MOLECULAR AND STRUCTURAL PROPERTIES
The molecular mass values for two eluted fractions were calculated for the portions of peaks, which lie within the peak ranges.The relevant molecular parameters of each EPS are summarized in Table II.In Table II, the molecular weights of EPSs from glucose, fructose and sucrose as carbon source in the medium were estimated to be 6.255 ×10 5 , 3.132 ×10 5 and 2.469 ×10 5 respectively.Zhang et al. (2004) suggested that moderate molecular weight on the improvement of the bioactivities of the polysaccharides is important.The high values of polydispersity ratio for all EPSs (4.72-8.95)concerning the influence on cellular enzymatic system by different carbon source, detailed discussion on the metabolic process for the EPS formation is difficult currently.

FT-IR SPECTROSCOPY
FT-IR spectroscopy was used as an effective analytical tool to characterize functional groups.Typical IR spectra for the EPS fractions with three different carbon sources have been compiled in Figure 2. All samples exhibited similar characteristic peaks, which indicate that they mean that these EPS molecules exist much more dispersed in aqueous solution and could form large aggregates (Hwang et al. 2003).This information is important because the functional properties of polysaccharides can be greatly influenced by the molecular weight distribution.For each of these moments of the distribution, the root mean square (RMS) radii of the EPSs were calculated (Table II).These data provide a measure of the EPS molecular size in terms of the RMS distance from the molecular center of gravity to its edge (Hwang et al. 2003).Compared with the peaks of glucose and sucrose, the RMS radii for the peak of sucrose ranged from 27.8-51.5nm was moderate (Table II).
The slope for EPS in the double logarithmic plots of RMS radius versus molecular mass was shown in Figure 3.The values of slope of EPS indicated 0.16-0.25,which implies that the EPS molecule exists as a nearly globular shape in aqueous solution (see SEC/MALLS ANALYSIS).The results indicates that carbon source has more influence on molecular weight of EPS than their conformation.

ANTIOXIDANT PROPERTIES ANALYSIS
In vitro antioxidant capacities of all EPS fractions produced by submerged cultivation of P. vaninii Ljup in the media with selected carbon sources were subsequently evaluated using different biochemical methods including DPPH and hydroxyl radical scavenging assays.DPPH radical method is widely used as an index to evaluate the antioxidant potential of natural compounds (Cheng et al. 2011).In this experiment, scavenging rates of EPS fractions with various carbon sources depicted in Figure 4a.proved that radical scavenging activity was also concentration dependent.The scavenging activities of EPS with sucrose as carbon source were superior to those observed for other two EPSs irrespective of the concentration of the fraction (p <0.05), reaching a maximum of 89.2% at 5 mg/mL.Hydroxyl   One of the mechanisms involved in antioxidant activity may originate from the hydrogen atomdonating ability of a molecule to a radical, which results in terminating radical chain reactions and converting free radicals to unharmful products (Hu et al. 2000).The antioxidant activity of polysaccharides might be attributed to their hydroxyl groups and other functional groups, such as C=O, -COOH and -O-, which can donate electrons to reduce the radicals to a more stable form or react with the free radicals to terminate the radical chain reaction (Leung et al. 2009).The superior antioxidant activities of EPS fraction with sucrose as carbon source as compared to the other two EPS fractions may be attributed to the differing molecular properties, i.e. monosaccharide compositions and molecular size.The larger content of galactose in EPS fraction with sucrose as carbon source could be of relevance to account for the essentially different antioxidant properties.Chen et al. ( 2006) also investigated the EPS of Tremella mesenterica from submerged cultures with various carbon sources and found that xylose and glucose were better carbon sources from the viewpoint of immunomodulatory activity.

CONCLUSIONS
The effect of three different carbon sources on EPS production from the submerged culture of P. vaninii Ljup was investigated and the three EPSs were purified and characterized by FT-IR spectroscopy, GC and SEC-MALLS analysis and their antioxidant activities subsequently compared.The results demonstrated that the molecular properties and antioxidant activities varies with the different carbon sources.Sucrose is better carbon source from the viewpoint of antioxidant activity due to the relatively high content of galactose in EPS and moderate molecular size.Thus, the alternate carbon-source strategy may be successfully used for regulation of biosynthesis and activity by P. vaninii Ljup.Further works on the full structure elucidation of EPS fractions from different carbon source by one-dimensional and two-dimensional NMR spectroscopy are in progress in our laboratory.

Figure 1 -
Figure 1 -Effect of three different carbon sources on biomass (a) and EPS production (b) by P. vaninii Ljup in a 5-L stirred-tank.Identical letters indicate no significant differences (p < 0.05).Identical letters indicate no significant differences (p < 0.05).

Figure 2 -
Figure 2 -The FT-IR spectra of the EPS fractions produced by submerged cultivation of P. vaninii Ljup in the media with selected three different carbon sources.

Figure 3 -
Figure 3 -The double logarithmic plots of root mean square radius vs molecular mass for exopolysaccharides of produced by submerged cultivation of P. vaninii Ljup in the media with selected three different carbon sources in MALLS analysis.

Figure 4 -
Figure 4 -Antioxidant activity of EPS fractions produced by submerged cultivation of P. vaninii Ljup in the media with selected three different carbon sources.The results represent mean ± S.D. (n = 3).DPPH (a) and OH (b) radical scavenging activity of P. vaninii Ljup EPS.
(TFA) at 110 o C for 2 h.The hydrolyzate was repeatedly co-concentrated with methanol, reduced with NaBH 4 for 30 min at 20 o C and acetylated with acetic anhydride and pyridine at 100 o C for 20 min.The internal standard sugars were prepared and subjected to GC-MS analysis separately in the same way.The alditol acetates of EPS fraction were analyzed by GC-MS (Varian Co., Model: Star 3600 CX, Lexington, MA, USA) fitted with a fused silica capillary column (Na form, 300mm × 0.25 mm, Supelco Inc., Bellefonte, PA, USA) and a flame ionization detector.

TABLE II Relevant molecular parameters of exopolysaccharides of produced by submerged cultivation of P. vaninii Ljup in the media with selected carbon sources in MALLS analysis. Parameters a Carbon sources in the medium
R n , R w , and R z refer number-, weight-, z-average square mean radius of gyration, respectively.
b M w /M n means polydispersity ratio.c