Extraction and in vitro antioxidant activity of exopolysaccharide by Pleurotus eryngii SI-02.

The extraction parameters for Pleurotus eryngii SI-02 exopolysaccharide (EPS) produced during submerged culture were optimized using response surface methodology (RSM). The optimum conditions for EPS extraction were predicted to be, precipitation time 20.24 h, ethanol concentration 89.62% and pH 8.17, and EPS production was estimated at 7.27 g/L. The actual yield of EPS under these conditions was 7.21 g/L. The in vitro antioxidant results of the EPS showed that the inhibition effects of EPS at a dosage of 400 mg/L on hydroxyl, superoxide anion and 1,1-diphenyl-2-picrylhydrazyl (DPPH) radicals were 59.63 ± 3.72%, 38.69 ± 2.59%, and 66.36 ± 4.42%, respectively, which were 12.74 ± 1.03%, 8.01 ± 0.56%, and 12.19 ± 1.05% higher than that of butylated hydroxytoluene (BHT), respectively. The reducing power of EPS of P. eryngii SI-02 was 0.98 ± 0.05, 60.66 ± 5.14% higher than that of BHT. The results provide a reference for large-scale production of EPS by P. eryngii SI-02 in industrial fermentation and the EPS can be used as a potential antioxidant which enhances adaptive immune responses.

Introduction

Pleurotus eryngii, one of the artificially cultivated and precious mushrooms in many countries, contains many biological bioactive materials, such as protein, essential amino acids, dietary fiber, trace elements, vitamins, and carbohydrates (Liu ). Polysaccharides from P. eryngii fruiting bodies have potential antioxidation, antitumor, antivirus, and immunomodulating properties (Stajic ).

Wasser (2002) reported that many kinds of exopolysaccharide (EPS) derived from filamentous fungi had potent anticancer activities and immunoregulatory properties. Compared with the polysaccharides from fruit bodies and mycelia, the EPS from fermentation broth with similar physiological and pharmacological functions is easily obtained. Many reports concerning to the EPS are mainly focused on the cultivation and production conditions in submerged culture by Pleurotus sajor-caju (Confortin ), Tremella fuciformis (Cho ), Collybia maculate (Lim et al., 2004), Cordyceps jiangxiensis (Xiao et al., 2004), and C. militaris (Kim ). Moreover, the extraction conditions of EPS of P. nebrodensis (Jia ), Morchella esculenta (Meng ), Pholiota squarrosa (Wang and Lu, 2004), Coprinus comatus (Hu ), Grifola frondosa (Cui ), Auricularia auricula (Zhang ) and Ganoderma atrum (Ye ) have been reported. However, the extraction parameters of EPS by P. eryngii in submerged culture and its antioxidant activities in vitro have not been studied.

In this work, factors affecting the extraction of EPS of P. eryngii SI-02 were analyzed by Plackett-Burman (PB) experiments, and three significant variables (precipitation time, ethanol concentration and pH) were chosen to optimize the extraction conditions using response surface methodology (RSM). In addition, the in vitro antioxidant activities of EPS were evaluated with the hydroxyl, superoxide anion, 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging assay and reducing power as main index.

Materials and Methods

Chemicals

Butylated hydroxytoluene (BHT), nitroblue tetrazolium (NBT), methionine (MET) and riboflavin (RF) were from Sigma Chemicals Company (St. Louis, USA). All other chemicals used in this experiment were analytical reagent grade and purchased from local chemical suppliers in China.

Microorganism and liquid culture

A strain of P. eryngii SI-02 was provided by our laboratory and maintained on synthetic potato dextrose agar (PDA). The cultures were incubated for 7 days at 25 °C, stored at 4 °C and subcultured every 3 months. Cultivation in liquid media was carried out in 250-mL Erlenmeyer flasks containing 100 mL of (g/L): potato, 200; glucose, 20; yeast extract, 3; peptone, 0.3; KH2PO4, 1.5, and MgSO47H2O, 1 with natural pH. Flasks were inoculated with a 0.5-cm2 mycelial block of P. eryngii SI-02 from the solid media, incubated at 25 °C for 24 h without shaking, and then shaken on a rotary shaker (Anting, Shanghai, China) at 160 rpm for 8 days.

Measurement and preparation of EPS

The P. eryngii SI-02 cultures were centrifuged at 3000 g for 15 min, the supernatant liquid was mixed with 4 volumes of 95% ethanol (v/v), stirred vigorously and kept at 4 °C for 24 h. After centrifugation (3000 g, 15 min), the precipitated EPS was dissolved in distilled water (60 °C) for 1 h, and the EPS content was determined by the phenol-sulfuric acid method, using glucose as the standard (Chaplin and Kennedy, 1994). EPS powder was obtained by quick prefreezing at −35 °C for 1 h and then by vaccum freeeze drying (Labconco, USA) for 6 h, and applied to detect the antioxidant activities in vitro.

PB experiments for EPS extraction

Initial screening of the most significant parameters affecting EPS production by P. eryngii SI-02 was performed by PB design as reported by Plackett and Burman (1946). Seven variables were studied in the experiment including concentration temperature, concentration multiple, ethanol concentration, ethanol multiple, precipitation temperature, precipitation time and pH. In addition, 5 center points were added for the variables that could be assigned numerical values. The experimental design with the name, symbol code, and actual level of the variables is shown in Tables 1 and 2.

Table 1

Levels and codes of variables for Plackett-Burman design.

Variables	Symbol code	−1	Coded levers0	1
Concentration temperature (°C)	A1	70	80	90
Concentration multiple	A2	2	3	4
pH	A3	5	7	9
Ethanol concentration (%)	A4	75	85	95
Ethanol multiple	A5	2	3	4
Precipitation temperature (°C)	A6	4	8	12
Precipitation time (h)	A7	12	18	24

Table 2

Results of Plackett-Burman for EPS extraction by Pleurotus eryngii SI-02.

Runs	A1	A2	A3	A4	A5	A6	A7	EPS yield (g/L)
1	1	1	−1	1	1	1	−1	5.10
2	−1	1	1	−1	1	1	1	5.33
3	1	−1	1	1	−1	1	1	6.15
4	−1	1	−1	1	1	−1	1	4.77
5	−1	−1	1	−1	1	1	−1	3.72
6	−1	−1	−1	1	−1	1	1	5.11
7	1	−1	−1	−1	1	−1	1	3.10
8	1	1	−1	−1	−1	1	−1	4.07
9	1	1	1	−1	−1	−1	1	5.06
10	−1	1	1	1	−1	−1	−1	5.32
11	1	−1	1	1	1	−1	−1	3.56
12	−1	−1	−1	−1	−1	−1	−1	3.22
13	0	0	0	0	0	0	0	5.09
14	0	0	0	0	0	0	0	5.02
15	0	0	0	0	0	0	0	5.17
16	0	0	0	0	0	0	0	5.12
17	0	0	0	0	0	0	0	5.14
Significant			**	*			*

p < 0.05.

p < 0.01.

Response surface optimization for EPS extraction

Precipitation time, ethanol concentration and pH selected by PB tests were taken into consideration for the optimization of EPS production by the Box-Behnken design. The experimental design with name, symbol code, and actual level of the variables is shown in Tables 3 and 4. The test factors were coded according to the following equation:

Table 3

Levels and codes of variables for Box-Behnken design.

Variables	Symbol		Coded	levels
	Uncoded	Coded	−1	0	1
Precipitation time (h)	X1	x1	12	18	24
Ethanol concentration (%)	X2	x2	75	85	95
pH	X3	x3	5	7	9

Table 4

Experimental and predicted values of EPS based on Box-Behnken design.

Runs	x1	x2	x3	EPS yield (g/L)
				Experimental	Predicted
1	−1	−1	0	3.63	3.45
2	1	−1	0	4.36	4.21
3	−1	1	0	5.27	5.41
4	1	1	0	6.84	7.01
5	−1	0	−1	3.66	3.87
6	1	0	−1	4.97	5.15
7	−1	0	1	5.98	5.79
8	1	0	1	7.08	6.87
9	0	−1	−1	3.03	2.98
10	0	1	−1	5.30	4.94
11	0	−1	1	4.03	4.38
12	0	1	1	7.13	7.18
13	0	0	0	6.94	6.35
14	0	0	0	6.16	6.35
15	0	0	0	5.99	6.35
16	0	0	0	6.73	6.35
17	0	0	0	5.92	6.35

where xi is the coded value of an independent variable, Xi is the real value of an independent variable, X0 is the real value of the independent variable at the center point, and ΔXi is the step change value.

To correlate the response variable to the independent variables, the following quadratic polynomial equation was applied to fit the response variable to a quadratic model:

where Y is the predicted response value, β0 is the intercept term, βi is the linear term, βii is the squared term, βij is the interaction term, xi and xj are the coded level of independent variables.

Hydroxyl radical scavenging assay

Hydroxyl radical scavenging activity was measured according to the method of Winterbourn and Sutton (1984). The reaction mixture contained 1 mL of 0.15 M phosphate buffer saline (pH 7.4), 1 mL of 110 μg/mL safranin, 2 mL of 6 mM EDTA-Fe (II), 1 mL of 3% (v/v) H2O2, and 0.5 mL of the EPS (50–400 mg/L). After incubating at 37 °C for 30 min, the absorbance of EPS was measured at 560 nm, using BHT as a positive control. The EC50 value (mg/L) of EPS or BHT is the effective concentration at which the hydroxyl radicals were scavenged by 50%. The hydroxyl radical scavenging activity was expressed as:

where A0 is the absorbance of the control and A1 is the absorbance of EPS/BHT.

Superoxide radical scavenging assay

Superoxide anion radical scavenging activity was determined according to method of Stewar and Beewley (1980). The reaction mixture (3 mL) contained 13 mM methionine (MET), 10 mM RF, 75 μM NBT, 100 mM EDTA, 50 mM phosphate buffer (pH 7.8), and the EPS (50–400 mg/L). After illuminating the reaction mixture with a fluorescent lamp at 25 °C for 30 min, the absorbance of the EPS was measured at 560 nm, using BHT as a positive control. The whole reaction was assembled in a box lined with aluminium foil. The scavenging rate was calculated using the following formula:

where A0 is the absorbance of the blank and A1 is the absorbance of the EPS/BHT.

DPPH scavenging assay

The DPPH scavenging activity of EPS was measured according to the method of Liu and Zhao (2006). The reaction mixture contained 2 mL of 95% ethanol, 0.1 μM DPPH and 2 mL of the EPS (50–400 mg/L). The solution was incubated at 25 °C for 15 min, and the absorbance of EPS was determined at 517 nm. The antioxidant activity of EPS was evaluated according to the following formula:

where A1 was absorbance of EPS and A0 was the absorbance of the DPPH solution.

Determination of reducing power

The reducing power of EPS was evaluated according to the method of Oyaizu (1986) with slightly modification. The reaction mixtures contained 2.5 mL phosphate buffer (pH 6.6, 0.2 M), 2.5 mL potassium ferricyanide (1%, w/v) and the EPS (50–400 mg/L). After incubating at 50 °C for 20 min, 2.5 mL of trichloroacetic acid (10%, w/v) was added to the mixture for terminating the reaction, and then centrifuged at 1200xg for 10 min. An aliquot of 2.5 mL supernatant was collected and mixed with 2.5 mL deionized water and 0.5 mL FeCl3 (0.1%, w/v). After incubating at room temperature for 15 min, the absorbance of the EPS was measured at 700 nm, using BHT as a positive control.

Statistical analysis

All experiments were carried out in triplicates. Data were processed and analyzed using Design Expert software (Version 7.1.3, Stat-Ease. Inc., Minneapolis, USA) including ANOVA.

Results and Discussion

Determination of parameters of EPS extraction

Table 2 showed that the maximum value of EPS extraction was 6.15 g/L, while the optimal extraction parameters were concentration temperature 90 °C, concentration multiple 2, pH 9, ethanol concentration 95%, ethanol multiple 2, precipitation temperature 12 °C and precipitation time 24 h. Precipitation time, ethanol concentration and pH had a highly significant influence on EPS extraction at the 5% or 1% level, and the influence of other parameters was not significant (p > 0.05). Therefore, these three factors were chosen to optimize the process of EPS extraction using RSM.

Response surface optimization of EPS extraction

The experiments were planned to obtain a quadratic model consisting of 12 runs and 5 center points. The range and levels of three independent variables are shown in Table 3, and the Box-Behnken design matrix together with the experimental and predicted EPS data is shown in Table 4, while adequacy and fitness were evaluated by ANOVA (Table 5).

Table 5

ANOVA for the evaluation of the quadratic model.

Source	Coefficients	S.E.	Sum of squares	Mean square	F-value	p
Model	-	-	27.08	3.01	15.57	< 0.001**
Intercept	6.35	0.20	-	-	-	-
x1 (time)	0.59	0.16	2.77	2.77	14.35	0.007**
x2 (concentration)	1.19	0.16	11.26	11.26	58.25	< 0.001**
x3 (pH)	0.91	0.16	6.59	6.59	34.09	< 0.006**
x1x2	0.21	0.22	0.18	0.18	0.91	0.371
x1x3	−0.05	0.22	0.01	0.01	0.06	0.818
x2x3	0.21	0.22	0.17	0.17	0.89	0.377
x12	−0.39	0.21	0.63	0.63	3.27	0.113
x22	−0.94	0.21	3.70	3.70	19.15	0.003**
x32	−0.54	0.21	1.23	1.23	6.35	0.004**
Lack-of-fit			0.52	0.17	3.82	0.545

R2 = 0.9524. adj-R2 = 0.9812. R = 0.9759.

p < 0.05.

p < 0.01.

By using multiple regression analysis, the polynomial model for an empirical relationship between the extraction rate of IPS and test variables in coded units was expressed by Eq. (6):

where YEPS is the predicted response for the yield of EPS (mg/L), and x1, x2 and x3 are the coded test variables for precipitation time (h), ethanol concentration (%) and pH, respectively.

It can be seen from Table 5 that the linear term regression coefficients (x1, x2, x3) and the quadratic coefficients (x22, x32) were significant at the 1% level, indicating that the precipitation time, ethanol concentration and pH are all significantly correlated with the yield of EPS extraction. The model was also significant (p < 0.0001) with a high F-value (15.57). The value of correlation coefficient (R = 0.9759) indicated good agreement between the experimental and predicted values of EPS, and R2 (determinations coefficient) was 0.9524, showing a good agreement between experimental and predicted values which can explain 95.24% variability of the responses. The value of adjusted determinant coefficient (adj-R2) was 0.9812, suggesting that the total variation of 98.12% for EPS is attributed to the independent variables and only nearly 2% of the total variation cannot be explained by the model. The F-value (3.82) and p-value (0.545) of lack-of-fit implied that it was not significant relative to the pure error, which indicated that the model equation was appropriate to predict the yield of EPS extraction under any combination of values.

To determine optimal levels of the test variables for EPS yield, the 3D response surface described by the regression model is presented in Figure 1. The optimal values of the variables affecting the amount of EPS extraction given by the software were precipitation for 20.24 h, ethanol concentration 89.62% and pH 8.17. Under these optimal conditions, the model gave the maximum predicted values of EPS extraction (7.27 g/L). In the view of the operating convenience, the optimal extraction parameters were determined to be precipitation time 20 h, ethanol concentration 90% and pH 8.2, while the predicted value of EPS extraction was 7.18 g/L, slightly lower than that of the maximum predicted value (7.27 g/L).

Figure 1

Response surface plot for the yield of EPS extraction by P. eryngii SI-02 in submerged culture in terms of the effects of (A) ethanol concentration and precipitation time, (B) pH and precipitation time, and (C) pH and ethanol concentration. Factors that were not included in the axes were fixed at their respective optimum levels.

Triplicate experiments were performed under the determined conditions and the value of EPS extraction (7.21 g/L) in agreement with the predicted value (7.27 g/L) was obtained, which was much higher than 2.77 g/L of C. brasiliensis (Yang ), 2.40 g/L of P. nebrodensis (Jia ), 5.32 g/L of M. esculenta (Meng ), 1.05 g/L of P. squarrosa (Wang and Lu, 2004), 1.46 g/L of C. comatus (Hu ), 1.33 g/L of G. frondosa (Cui ), 1.67 g/L of A. auricula (Zhang ), and 5.23 g/L of G. atrum (Ye ), respectively. The results indicated that the model was adequate for EPS extraction process.

Antioxidant activity of EPS

Free radicals are known to be the major cause of various chronic and degenerative diseases, including aging, coronary heart disease, inflammation, stroke, diabetes mellitus and cancer (Cheng ; Slater, 1984). The antioxidant compounds play an important role in preventing and curing chronic inflammation, atherosclerosis, cancer and cardiovascular disorders (Kohen and Nyska, 2002).

Hydroxyl radicals are main reactive oxygen free radicals in living organisms, which are the important reasons for causing the general processes of aging and tissue damage, and could influence the evolution of many degenerative diseases (In ). As shown in Figure 2A, the scavenging rate of EPS of P. eryngii SI-02 reached 59.63 ± 3.72% (p < 0.01) at a dosage of 400 mg/L, which was 12.74 ± 1.03% higher than that of BHT (52.89 ± 3.48%, p < 0.01). It was also higher than 2.3% of G. atrum (Ye ), 33.4% of Agaricus blazei (Zhang ), 3.5% of Boletus edudis (Kan ), 51.2% of P. nebrodensis (Sheng ), 22.3% of P. adiposa (Hu ), 26.4% of Termitomyces albuminosus (Zhou and Xu, 2008), 5.2% of Armillariella tabescens (Ma ), and 23.6% of T. fuciformis (Li ), respectively. The EC50 value of EPS was 261.37 ± 20.43 mg/L (p < 0.01), which was not only 30.31 ± 2.71% lower than that of BHT (375.06 ± 31.15 mg/L, p < 0.05), but also obviously lower than 5.2 g/L of B. edudis (Kan ), 1.9 g/L of P. nebrodensis (Sheng ), 4.6 g/L of P. adiposa (Hu ), 4.1 g/L of T. albuminosus (Zhou and Xu, 2008), and 8.7 g/L of A. tabescens (Ma ), respectively. The results indicated that the EPS of P. eryngii SI-02 significantly affects the scavenging of hydroxyl radical.

Figure 2

Antioxidant activities of EPS of P. eryngii SI-02 in vitro. (A) Scavenging effect of EPS on hydroxyl radical, (B) Scavenging effect of EPS on superoxide anion radical, (C) Scavenging effect of EPS on DPPH, and (D) Reducing power of EPS.

Superoxide anion is one of the precursors of the singlet oxygen and hydroxyl radicals, therefore, it indirectly initiates lipid peroxidation. Apart from that, the presence of superoxide anion can magnify cellular damage because it produces other kinds of free radicals and oxidizing agents (Athukorala ). The results of superoxide anion radical scavenging assay are shown in Figure 2B and the inhibition activities of EPS and BHT were concentration-dependent at the dosage of (50–400 mg/L). The scavenging rate of EPS at 400 mg/L was 38.69 ± 2.59% (p < 0.01), which was 8.01 ± 0.56% higher than that of BHT (35.82 ± 3.01% p < 0.05). Some researchers reported that the inhibition effects of EPS at 400 mg/L on superoxide anion radical were 6.4% of B. edudis (Kan ), 9.7% of P. adiposa (Hu ), 21.1% of T. albuminosus (Zhou and Xu, 2008), 6.4% of A. tabescens (Ma ), and 16.9% of T. fuciformis (Li ), respectively, markedly lower than that of EPS of P. eryngii SI-02 in this experiment. These data showed that the EPS of P. eryngii SI-02 can effectively protect cell from damage and lipid peroxidation.

DPPH is a stable free radical that shows maximum absorbance at 517 nm in ethanol. When DPPH encounters a proton-donating substance such as an antioxidant, the radical would be scavenged and the absorbance is reduced (Shimada ). It can be seen from Figure 2C that the DPPH scavenging ability of EPS at 400 mg/L was 66.36 ± 4.42% (p < 0.05), 12.19 ± 1.05% higher than that of BHT (59.15 ± 3.94%, p < 0.05), which was much higher than 1.7% of G. atrum (Ye ), 31.6% of Marasmius androsaceus (Dong ), 24.5% of T. albuminosus (Zhou and Xu, 2008) and 4.3% of A. tabescens (Ma ). The EC50 value of EPS was 257.21 ± 21.39 mg/L (p < 0.01), which was lower than that of BHT (291.06 ± 24.75 mg/L p < 0.05), 2.6 g/L of T. albuminosus (Zhou and Xu, 2008), and 2.1 g/L of A. tabescens (Ma ), respectively. The DPPH scavenging results revealed that the EPS probably contained substances that were proton donors and could react with free radicals to convert them to stable diamagnetic molecules.

Figure 2D showed that the reducing power (absorbance at 700 nm) of EPS at 400 mg/L was 0.98 ± 0.05 (p < 0.01), 60.66 ± 5.14% higher than that of BHT (0.61 ± 0.03, p < 0.05). It was also much higher than 0.04 of B. edudis (12), 0.62 of P. nebrodensis (Sheng ), and 0.28 of T. albuminosus (Zhou and Xu, 2008), respectively. These results indicated that the EPS of P. eryngii SI-02 in this study has potential antioxidant capacities.

Conclusions

Response surface methodology using second-order regression for a three-factor-three-level Box-Behnken design was a successful tool for extraction optimization of EPS by P. eryngii SI-02 in submerged culture. The EPS showed antioxidant activities in vitro. The results provide a reference for large-scale extraction of EPS by P. eryngii SI-02 in industrial fermentation and the EPS can be used as a potential antioxidant which enhances adaptive immune responses.