Effect of Tween 80 and Acetone on the Secretion , Structure and Antioxidant Activities of Exopolysaccharides from Lentinus tigrinus

Lentinus tigrinus is a white-rot fungus naturally occurring on rott en hardwood during spring and summer in China (4). It is an edible and medicinal mushroom as it possesses high nutritive and health value with high quantity of amino acids, few sugars and low calories (5). The natural compounds from this mushroom, such as phenolics, are proved to have antioxidant activity (6). However, there are no reports on the bioactivity of polysaccharides from L. tigrinus.


Introduction
Intracellular polysaccharides (IPS) extracted from mycelia and fruiting bodies of mushrooms and exopolysaccharides (EPS) from submerged culture broth have been widely studied and exploited for their multiple pharmacological activities (1,2).Production of EPS in submerged culture has the potential advantages of saving time and space, as well as the possibility of industrial production (3).
Lentinus tigrinus is a white-rot fungus naturally occurring on rott en hardwood during spring and summer in China (4).It is an edible and medicinal mushroom as it possesses high nutritive and health value with high quan-tity of amino acids, few sugars and low calories (5).The natural compounds from this mushroom, such as phenolics, are proved to have antioxidant activity (6).However, there are no reports on the bioactivity of polysaccharides from L. tigrinus.
Stimulating agents, such as surfactants, vegetable oil and organic solvents are reported to mediate cell permeabilization and thus enhance the secretion of intracellular target products (7).On the other hand, the surfactant and organic solvents are inexpensive and may be removed by simple evaporation.Hence, they are used frequently and eff ectively for cell permeabilization (8,9).The representative surfactants that enhance cell membrane permeability are Tween 40, Tween 60 and Tween 80, while commonly used organic solvents are toluene, chloroform, ethanol, ether and dimethyl sulfoxide (7,9).
Stimulating agents are rarely reported to be used to enhance the permeability of fungi and improve the productivity of specifi c metabolites (10).Plant oil and fatt y acids have been used in fungal fermentations to accelerate and enhance the mycelial growth and EPS secretion (11).However, the eff ect of various surfactants and organic solvents on the secretion and structure of EPS in submerged culture of medicinal fungi was not reported.In this study, the eff ects of several surfactants and organic solvents in an att empt to enhance EPS secretion in submerged culture of L. tigrinus were compared.Tween 80 and acetone were selected to enhance EPS secretion.Aft er isolation and purifi cation, the molecular mass and chemical characteristics of EPS obtained with the addition of Tween 80 (EPS-T), acetone (EPS-A) and control (EPS-C, i.e. without the addition of an agent) were determined.Finally, antioxidant and antiproliferative activity of the EPS were also evaluated and compared.

Mushroom isolation and growth conditions
Lentinus tigrinus was obtained by tissue isolation method from naturally occurring fruiting body and identifi ed by Prof Xinsheng He of Southwest University of Science and Technology, Mianyang, PR China.This strain was maintained on potato dextrose agar (PDA) slant (potato 200 g, glucose 20 g, agar 15 g and deionized water 1000 mL) at the Henan Province Microbiological Culture Collection Center (HPMCC no.35481696) (12).The strain was transferred into the seed medium (30 g/L of glucose and 3 g/L of yeast extract) by punching out a disc (5 mm) from the plate culture.The seed culture was grown in a 250-mL fl ask containing 50 mL of th e medium at 26 °C on a rotary shaker incubator (210×g) for four days.

Flask culture and the treatment with surfactant and organic solvent
The fl ask culture experiments were performed in a 500-mL Erlenmeyer fl ask containing 150 mL of the culture medium (lactose 60 g, yeast extract 4 g and distilled water 1000 mL; pH=5.0) at 26 °C for 12 days aft er inoculation with 4 % (by volume) of the seed culture.To fi nd a suitable surfactant and organic solvents for EPS secretion by L. tigrinus, various surfactants (Tween 40, Tween 60 and Tween 80 from Sigma-Aldrich, St. Louis, MO, USA) and organic solvents (chloroform, ethanol, DMSO and acetone from Sigma Chemical Corp., St. Louis, MO, USA) were added at a volume fraction of 0.2 % to the broth aft er 8 days of cultivation and continued to cultivate up to 12 days.All determinations were done in triplicate to ensure the observed trends were reproducible.

Mycelial dry mass and EPS determination
The mycelial pellets and culture broth were separated by fi ltration.The mycelial dry mass was measured after repeated washing and drying of the pellets.Crude EPS were obtained aft er concentration, ethanol precipitation and centrifugation.The total sugar content was deter mined by the phenol-sulfuric acid method by measuring the absorbance at 490 nm using glucose as the standard (13).

Purifi cation and molecular characterization of EPS
The crude EPS were purifi ed by deproteinization, dialysis and gel fi ltration chromatography on Sepharose CL-6B (Sigma Chemical Corp.) (13).Protein concentration was determined by Bradford ( 14) using bovine serum albu min (BSA; Sigma-Aldrich) as the standard.Aft er hydrolyzation, reduction and acetylation, the alditol acetates of refi ned EPS were analyzed by GC-MS (model Star 3600 CX; Varian Co., Lexington, MA, USA).The infrared spectrum of the refi ned EPS was measured through a Bruker Tensor 27 Fourier transform infrared (FTIR) spectrometer (Bruker Optics, Ett lingen, Germany) using KBr disc technique (15).The EPS molecular mass in the solution was determined by size-exclusion chromatography using a Sepharose CL-6B column (Sigma Chemical Corp.).Standard dextran series (D150, 70, 40, and 10 kDa; Sigma Chemical Corp.) were used for determination of molecular mass of EPS.The separation was carried out at 22 °C at a fl ow rate of 0.6 mL/min.The calibration curve of partition coeffi cient (K av ) vs. log molecular mass was made, in which K av was calculated using the following equation: where V e is the elution volume (mL), V 0 is the column void volume (mL) and V t is the total column bed volume (mL).

Helix-coil transition assay
The conformational structure of the EPS in solution was determined by the addition of Congo Red, which forms complexes with EPS.The transition from a triple helix arrangement to the single-stranded conformation was determined by measuring the absorbance at λ max = 400-800 nm of Congo Red and EPS solution aft er the addition of 0 to 0.5 mol/L of NaOH with the UV-Vis spectrophotometer (TU-1800; Beĳ ing Purkinje General Instrument Co. Ltd., Beĳ ing, PR China) according to our previous report (16).

Antiox idant activity assays
The scavenging activity of hydroxyl and DPPH radicals was evaluated to determine the antioxidant activity of the samples according to our previous report (17).In the assays, 1,1-diphenyl-2-picrylhydrazyl (DPPH; Sigma Chemical Co.) and phenanthroline (Sigma Chemical Co.) were used.

Antiproliferative activity assay
The antiproliferative activity of EPS was measured using the previously reported MTT assay (13).Human hepatoma (Hep G2) and human osteoblast-like (MG-63) cells obtained from Cancer Institute of the Fourth Military Medical University (Xi'an, PR China) were employed and the concentrations of EPS of 25, 50, 100, 200 and 400 μg/ mL were tested in this assay.

Statistical analysis
The results were reported as mean value±standard deviation (S.D.).One-way analysis of variance (ANOVA) was carried out for the comparison of mean values, and those at p<0.05 were considered statistically signifi cant.

Eff ect of surfactants and organic solvents on EPS secretion
Among the tested surfactants and organic solvents, Tween 40 and chloroform enhanced the mycelial growth (Table 1).However, the EPS secretion was improved by 22.4 or 107.2 % when Tween 80 or acetone was used, respectively.Similar results were observed by Liu and Wu (18), who found that Tween 80 had a remarkable promoting eff ect on EPS secretion by Cordyceps sinensis Cs-HK1, increasing the EPS yield more than twofold at the mass per volume ratio of 1.5 %.It was interesting to fi nd that Tween 80 and acetone had negative eff ects on the mycelial biomass, which suggested that they had toxic eff ect on Lentinus tigrinus.The results indicated that the surfactants and organic solvents could modify the composition of the mycelial cell membrane, and thus change the permeability of L. tigrinus mycelia and enhance the EPS secretion (19).Hence, the EPS with the higher molecular mass could be secreted when surfactants and organic solvents are added to the culture medium.

Comparis on of molecular and structural properties of EPS
Fig. 1a shows the elution profi le of EPS obtained by the addition of Tween 80 (EPS-T) or acetone (EPS-A), and the control (without the addition of stimulating agents (EPS-C)).FTIR spectra of the three EPS types are shown in Fig. 1b.All samples showed a broad characteristic peak at approx.3200 cm -1 typical of -OH groups, as well as a weak C-H band at approx.2930 cm -1 .The characteristic band at approx.1599-1642 cm -1 could be correlated with the stretching vibration of the carbonyl group (20).A small peak at approx.1403-1414 cm -1 assigned to amino groups was also observed in the FTIR spectra.Bands at approx.1026-1094 cm -1 suggested the presence of C-O type of linkage (21).The results suggest that the addition of Tween 80 and acetone does not aff ect the functional groups of the EPS.
The molecular masses of EPS-T, EPS-A and EPS-C were 22.1, 137 and 12 kDa, respectively (Fig. 2).The EPS-T and EPS-A had higher molecular masses than EPS-C, which indicated that surfactants and organic solvents interacted with the phospholipid bilayer membranes and decreased the cell membrane permeability.The monomer composition of the three EPS types determined by GC-MS analysis is shown in Table 2. Results indicate that glucose and mannose are the major monosaccharides in all EPSs although their fraction is markedly diff erent, while Tween 80 and acetone could alter the internal metabolism by affecting the enzymatic synthesis (7).Congo Red test can rapidly detect the polysaccharide conformation (22).The λ max of all complexes of Congo Red with EPS is shown in Fig. 3.The addition of EPS-C (in a complex with Congo Red) did not cause any notable change in the λ max (Fig. 3a) with the increase of NaOH concentration, as compared to that of Congo Red alone.This showed that the EPS-C had a random coil conformation in the aqueous solution.However, at low concentrations (0-0.2 mol/L), the λ max shift to a longer wavelength can be observed (Figs.3b and c).When the concentration of NaOH was increased to more than 0.2 mol/L, the λ max slowly dropped.This indicated that EPS-A and EPS-T had a highly ordered conformation of triple helix, which remained stable even under strong alkaline conditions, as reported previously (22).

Antioxidant a nd antiproliferative activities
In vitro antioxidant activities of three types of EPS from L. tigrinus were evaluated using hydroxyl and DPPH radical scavenging assays.The EPS showed strong hydroxyl radical scavenging capacities in a concentration--dependent manner; however, they were not notably different (Fig. 4a).The DPPH scavenging rates of the tested EPS (Fig. 4b) confi rmed that the DPPH scavenging activity of EPS-A was bett er than those of EPS-C and EPS-T, reaching a maximum of 63.0 % at mass concentration of 8 mg/mL.The stronger antioxidant activity of EPS-A than those of EPS-C and EPS-T may be att ributed to their difference in molecular mass (23,24).
The growth inhibitory eff ects of the polysaccharides (in the concen tration of 50-400 μg/mL) against Hep G2 and MG-63 cells were examined.The antitumour activity against Hep G2 increased signifi cantly (Fig. 4c) with the corresponding increase in the concentration of EPS (p<0.05).EPS-A exhibited higher antiproliferative activity than EPS-C and EPS-T.Similarly to Hep G2, the antitumour activities against MG-63 were also concentration dependent (Fig. 4d).EPS-A also had higher antiproliferative activity than other EPS samples.The results indicated that the molecular mass may be the main factor determining the antiproliferative activity of fungal EPS.Zhang et al. (25) found that the EPS from the Pleurotus tuber-regium mycelia exhibited higher antitumour activity in the molecular mass range of 400-800 kDa than other fractions with molecular mass below 50 kDa in vivo and in vitro.

Fig. 2 .Fig. 3 .Fig. 4 .
Fig.2.Molecular mass of the purifi ed exopolysaccharides (EPS, closed circles) produced by submerged culture of Lentinus tigrinus using Tween 80 (EPS-T) and acetone (EPS-A), in comparison with control without the addition of stimulating agents (EPS-C), on Sepharose CL-6B column.Elutes were analyzed by measuring the absorbance at 490 nm (A 490 nm ).Open circles represent diff erent dextrans used as control.K av =partition coefficient

Table 1 .
Eff ect of surfactants and organic solvents on mycelial growth and exopolysaccharide (EPS) production by Lentinus tigrinus in shake fl ask cultures