International Journal of Biological Macromolecules 77 (2015) 143–150

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Effect of chemicals on production, composition and antioxidant activity of polysaccharides of Inonotus obliquus Xiangqun Xu ∗ , Lili Quan, Mengwei Shen College of Life Sciences, Zhejiang Sci-Tech University, Hangzhou 310018, China

a r t i c l e

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Article history: Received 10 December 2014 Received in revised form 19 February 2015 Accepted 8 March 2015 Available online 19 March 2015 Keywords: Inonotus obliquus Polysaccharides Stimulatory agents

a b s t r a c t Polysaccharides are important secondary metabolites from the medicinal mushroom Inonotus obliquus. Various fatty acids, surfactants and organic solvents as cell membrane-reorganizing chemicals were investigated for their stimulatory effects on the growth of fungal mycelium and production of exopolysaccharides (EPS) and endopolysaccharides (IPS) by submerged fermentation of I. obliquus. After evaluation of 14 chemicals, oleic acid, Tween 80, and TritonX-100 were chosen for optimization of addition concentration and addition time. Among the three chemicals, 0.1% (v/v) Tween 80 gave maximum production of mycelial biomass, EPS, IPS1, and IPS2 with a increase of 16.6, 81.6, 37.7 and 18.1%, respectively, when supplemented at the early growth phase (24 h after inoculation). These EPS, IPS1, and IPS2 had significantly (p < 0.05) stronger scavenging activity against 2,2-diphenyl-1-picrylhydrazyl (DPPH) radicals than those from the control medium. IPS1 from Tween 80-containing medium was the most effective antioxidant, with an estimated IC50 value of 0.74 mg/mL. This might be attributed to that the EPS and IPS from the Tween 80-containing medium had significantly (p < 0.05) higher content of sugar and glucose among the six monosaccharide compositions than those from the control. The simultaneously enhanced accumulation of bioactive EPS and IPS of cultured I. obliquus supplemented with Tween 80 was evident. © 2015 Elsevier B.V. All rights reserved.

1. Introduction In recent years, the edible and medicinal mushroom and their secondary metabolites have been widely studied due to their health-promoting properties and relatively low toxicity as functional foods and sources for the development of drugs and nutraceuticals. Polysaccharides are one of the most important active ingredients in pharmaceutical fungi, which have become a focal point in studies of food and medicine. Many, if not all, higher Basidiomycetes mushrooms contain biologically active polysaccharides in fruit bodies, cultured mycelium, and cultured broth [1,2]. Inonotus obliquus (I. obliquus) is well known as one of the most popular medicinal species for its therapeutic effect, belonging to the family Hymenochaetacea of Basidiomycetes, growing on birches in the cold latitudes of Europe, Asia, and America. I. obliquus polysaccharides (PS) exhibited great biological activities including immunomodulation [3,4], antitumor [5,6], antioxidant [7–9], and anti-inflammation [6] and had no poisonous effect [3]. Because there is a great need to supply the market and the solid culture of mushroom needs to take long time to complete

∗ Corresponding author. Tel.: +86 571 86843228; fax: +86 571 87055836. E-mail address: [email protected] (X. Xu). http://dx.doi.org/10.1016/j.ijbiomac.2015.03.013 0141-8130/© 2015 Elsevier B.V. All rights reserved.

a fruiting body, investigators have recently exerted their efforts to prepare the mushroom from submerged culture for meeting the increasing consumption demand [3,5,9]. Our previous studies have reported that the exopolysacchrides (EPS) from the culture broth and endopolysaccharides (IPS) from the mycelia by liquid cultured I. obliquus were more effective in antioxidant activity and cytokine induction activity than those from the wild sclerotia [9,10]. Furthermore, our studies have shown that the lignocellulose degradation by the white-rot fungus I. obliquus had enhancement effects on the production and antioxidant activity of I. obliquus EPS and IPS under submerged fermentation [10,11]. However, as bioactive secondary metabolites, I. obliquus PS may encounter the problem of product secretion inhibition. Thus, an investigation on the submerged fermentation of I. obliquus is necessary for improving the production and secretion of the polysaccharides. The production of mushroom mycelial, EPS, and IPS by submerged fermentation is subjected to the growth environment. Hence, many investigates have been conducted on the optimization of submerged culture conditions [12,13]. According to previous studies, various chemical agents including plant oils, fatty acids, organic solvents, and surfactants have been used to accelerate mycelial growth in some mushroom species [14–19]. And many of them have been proved to be effective stimulatory agents in the production of useful metabolites in fungi and medicinal

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mushrooms. The stimulatory effect on mycelial growth and biopolymer production in Cordyceps militaris depended on the compositions of fatty acids [20]. The research of the influence of plant oils on EPS yield by Grifola frondosa showed that the increased concentration of olive oil and soybean oil led to a higher EPS production [15]. Tween 80 was found to be the most effective in enhancing the production of bioactive EPS of Pleurotus tuber-regium [21,22] and the highest Antrodin C production of Antrodia camphorata was obtained with addition of Tween 80 [23]. These stimulatory agents were presumed to increase cell permeability by reorganizing the cell membrane and/or directly affecting synthesis of enzymes involved in the formation of target products [23,24]. Although the chemical agents were proved to enhance the mycelial and some secondary metabolite production of these edible mushrooms by submerged culture, the effects of fatty acids, organic solvents, and surfactants on mycelial growth, and simultaneous production of EPS and IPS by submerged fermentation of I. obliquus have not been reported yet. The objectives of this work were to optimize the production and bioactivity of EPS and IPS of I. obliquus in submerged cultures by screening various fatty acids, surfactants, and organic solvents. The effect of the most effective stimulatory agents on the physicochemical characteristics and antioxidant activity of EPS and IPS was investigated. 2. Materials and methods 2.1. Chemicals A total of 14 agents in 3 chemical categories were added into the culture medium on day 0 of fermentation for the first screen experiment. They include (1) organic solvents: methanol, ethanol, acetone, chloroform, and toluene; (2) fatty acids: linoleic acid, oleic acid, palmitic acid, and stearic acid; and (3) surfactants: polyoxyethylene sorbitan monolaurate (Tween 20), polyoxyethylene sorbitan monooleate (Tween 80), 3-[(3-cholamidopropyl) dimethylammonio]-1-propanesulfonate (CHAPS), polyoxyethylene octyl phenyl ether (TritonX-100), and polyethylene glycol 4000 (PEG 4000). All the chemicals were of analytical grade. 2.2. Liquid culture I. obliquus (CBS314.39) was maintained on malt extract agar slants containing (% w/v) malt extract 3, peptone 0.3, and agar 1.5 at pH 5.6 ± 0.2. The fungi were cultivated at 25 ◦ C for about 2 weeks, then stored at 4 ◦ C and sub-cultured every 3 months. Malt extract agar with mycelia was cultured in the medium (g l−1 : glucose 20, peptone 3, yeast extract 2, KH2 PO4 1, MgSO4 1.5, and CaCl2 0.1 for 4–5 days on a rotary shaker at 28 ◦ C with a speed of 150 rpm. The seed culture was added into the control medium or agent-containing medium and incubated at a rate of 9% (v/v). The control medium contained (g l−1 ): corn flour 53, peptone 3, KH2 PO4 1, ZnSO4 ·7H2 O 0.01, K2 HPO4 ·3H2 O 0.5, FeSO4 ·7H2 O 0.05, MgSO4 0.2, CuSO4 ·5H2 O 0.02, CoCl2 ·6H2 O 0.02, MnCl2 ·4H2 O 0.09, pH = 6.0 optimized by the response surface methodology (RSM) in our previous work that demonstrated corn flour and peptone were the best carbon source and nitrogen source [12]. The corn flour suspension was filtered with gauze after having been drastically hydrolyzed by ␣-amylase and glucoamylase into glucose and the filtration was used in the experiment, the purpose of this operation is to minimize the error in the quantitative analysis of the EPS from early fermentation of I. obliquus [12]. In the agent-containing medium, 0.1% (v/v) chemicals were added and all of the other components were the same as the control medium. The flasks were incubated on a rotary shaker at 28 ◦ C, with a speed of 150 rpm for 9 days.

2.3. Analysis 2.3.1. Determination of mycelia biomass The biomass on day 9 was obtained by filtration, washing the precipitated cells for several times with distilled water till colorless and drying at 45 ◦ C for a sufficient time to determine the biomass dry weight. 2.3.2. Extraction and purification of EPS and IPS After the removal of mycelia by filtration, the culture broth on day 9 was concentrated up to 1/4 volume under vacuum, then 95% ethanol was added to the final concentration (4:1, v/v) and stored at 4 ◦ C overnight. The precipitate was repeatedly washed with 95% ethanol and dialysed (molecular weight cut off: 3000 Da) to remove adherent sugar residue and other small molecules, and lyophilized to give the EPS after centrifugation at 6500 × g for 10 min [9]. Two IPS samples were extracted in different temperatures. Firstly, the mycelial masses on day 9 were washed three times with 100–200 mL aliquots of distilled water then suspended in distilled water with the ratio of 1:5. After the ultrasonic wall-breaking the suspension was extracted twice with water for 2 h at 95 ◦ C and then the supernatant was collected for extraction of IPS1 [3]. The residue was resuspended in distilled water and then heated for 1.5 h in an autoclave at 121 ◦ C, followed by filtration and extraction of IPS2 [25]. The two filtrates were then processed in the same way for the EPS extraction to give IPS1 and IPS2. 2.3.3. Compositional analysis of EPS and IPS The EPS production was calculated by the deduction of reducing sugar of total carbohydrate content in the culture broth during fermentation. The reducing sugar concentration of the culture broth was measured by the DNS method [26]. The total carbohydrate content of the culture broth was determined by phenol-sulfuric acid method [27]. The carbohydrate and protein content of the EPS and IPS obtained above were analyzed by the phenol–sulfuric acid method [27] and the Bradford’s method with bovine serum albumin as a standard [28]. Gas chromatography (GC) was used for analysis of monosaccharide compositions. Hydrolysis and acetylated derivation were performed. The alditol acetates were analyzed by a gas chromatograph Techcomp GC-7900 (Techcomp Inc., Shanghai). The acetylated derivatives were loaded into an Agilent SE-50 capillary chromatography column (30 m × 0.25 mm, 0.25-␮m film thickness). The GC operation was performed under the following conditions: injection temperature: 270 ◦ C; detector temperature: 250 ◦ C. The temperature in the oven was programmed as follows: 110 ◦ C in the beginning, maintained for 5 min, and increased to 180 ◦ C at the rate of 3 ◦ C/min and maintained for 5 min. Subsequently, the temperature was increased to 220 ◦ C at 5 ◦ C/min and maintained for 10 min. The monosaccharide components were identified by matching the GC retention time with standards i.e. rhamnose (Rha), arabinose (Ara), xylose (Xyl), mannose (Man), galactose (Gal), and glucose (Glu) [11]. 2.3.4. Scavenging effect on DPPH radicals The DPPH free radical-scavenging activity was determined according to the reporter method [18]. Briefly, the EPS and IPS were dissolved in water in a concentration gradient (0.5–5 mg/mL), then each 2.4 mL of the extract solution was mixed with 0.8 mL of 1 mM DPPH methanol solution separately, followed by mixing each adequately, standing in the dark for 30 min and measuring the absorbance at 517 nm as Ab1. A control sample containing the same amount of methanol and DPPH radicals was measured as Ab0. The absorbance of samples containing the same amount of methanol and the extract solution was recorded as Ab2. This activity was

(1)

IC50 , the half maximal inhibitory concentration, was calculated by using median-effect analysis. 2.4. Statistical analysis Experimental results were expressed as means ± standard deviation (SD) of triple determinations. All statistical analyses were performed by using the software SPSS Statistics 19.0. Tests of significant differences were determined by Duncan’s multiple range tests at p = 0.05 or independent sample t-test (p = 0.05) by one-way analysis of variance of the data (ANOVA). 3. Results and discussion

14

Mycelial Biomass (g/L)

Ab0 − (Ab1 − Ab2 ) × 100 Ab0

12

mycelial biomass EPS IPS 1 IPS 2

b

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* *** B iii

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8

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0.9

40

0.8

36

0.7

32

0.6 0.5

v

0.4

EPS production (g/L)

18

given as DPPH scavenging rate and was calculated according to the following equation: DPPH scavenging rate (%) =

145

28 24 20 16

6

0.3

4

0.2

2

0.1

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0

0.0

0

Control

Linoleic acid Oleic acid Stearic acid Palmitic acid

12

IPS content (mg/g)

X. Xu et al. / International Journal of Biological Macromolecules 77 (2015) 143–150

8

Fig. 2. Effect of 0.1% (v/v) fatty acids on the mycelial biomass and production of EPS and IPS in submerged fermentation of I. obliguus. Each culture was carried out in triplicate at 28 ◦ C for 9 days. The values (mean value ± standard deviation, n = 3) with different letters or symbols, respectively, have significant difference (p < 0.05).

3.1. Effects of organic solvents Fig. 1 shows the influences of 0.1% (v/v) organic solvents (toluene, chloroform, acetone, methanol, and ethanol) added on day 0 on the mycelial biomass, EPS and IPS production in I. obliquus. All the organic solvents had no detrimental effect on cell growth. It is not consistent with the previous results, in which all the organic solvents, particularly chloroform and toluene, inhibited P. tuberregium mycelial growth [21]. On the other hand, all the organic solvents, particularly chloroform and acetone, inhibited the EPS production. When chloroform and acetone were added into the medium, the EPS production was only 76.7% and 75.3%, respectively, compared to that of the control. However, the detrimental effect of all the organic solvents on I. obliquus EPS production was not consistent with the previous studies in which methanol and hexane significantly (p < 0.05) increased the P. tuber-regium EPS production by 17.7% and 15.5%, respectively [21]. Chloroform and toluene increased Collybia maculate TG-1 EPS production and 0.3% (v/v) toluene increased the EPS production by 86% [29]. In contrary to the detrimental effect on the EPS production, chloroform and ethanol significantly increased IPS1 production by 16.9% and 9.6%, and IPS2 production by 17.2% and 15.2%, respectively (Fig. 1). This result indicated that the addition of these organic solvents had no negative effect on mycelia growth, but significantly reduced the EPS production in I. obliquus. The new discovery in the

16

*

ii

iii

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Mycelial Biomass (g/L)

mycelial biomass EPS IPS 1 IPS 2

a

i

12

a ***** i

b

c

a

****** i

c **** iv

0.9

40

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32

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28 24 20 16

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0.0

0

Control Methanol

Ethanol Chloroform Acetone Toluene

12

IPS content (mg/g)

** ***

EPS production (g/L)

18

8

Fig. 1. Effect of 0.1% (v/v) organic solvents on the mycelial biomass and production of EPS and IPS in submerged fermentation of I. obliguus. Each culture was carried out in triplicate at 28 ◦ C for 9 days. The values (mean value ± standard deviation, n = 3) with different letters or symbols, respectively, have significant difference (p < 0.05).

experiment is that chloroform and ethanol significantly increased IPS1 content although they drastically suppressed the concentration of EPS during submerged culture of I. obliquus. Based on the present results and previously reports [21,29], it may be thought that the increased production of I. obliquus IPS is a secretory function of organic solvents. 3.2. Effects of fatty acids Fig. 2 shows the effect of four fatty acids on the mycelial biomass, and production of EPS and IPS. Addition of oleic acid (C18:1), stearic acid (C18:0), and palmitic acid (C16:0) had a stimulatory effect on mycelial biomass. Stearic acid was the most effective, the mycelial biomass significantly (p < 0.05) increased by 27.1%. In contrast, linoleic acid (C18:2) significantly (p < 0.05) inhibited the mycelial growth. This is consistent with the mycelia growth of Ganoderma lucidum [17] and C. militaris supplemented with linoleic acid [20]. These data also indicated the lack of any correlation between growth stimulation and the extent of unsaturation and chain length of fatty acids. On the other hand, the effect of fatty acids on the EPS and IPS production had no correlation with the mycelial growth. Oleic acid showed a better stimulatory effect than stearic acid and palmitic acid on the EPS production with a significant increase of 16.0% compared with the control (Fig. 2). Linoleic acid, which had no stimulatory effect on mycelial biomass, also increased the EPS production by 14.7% (Fig. 2). This is not in agreement with the results in which EPS production by G. Lucidum [17], C. militaris [20], and P. tuber-regium [21] was inhibited with fatty acid of linoleic acid or was remarkably increased by palmitic acid. This could be partially attributed to the different lipid compositions of the mycelium among these mushrooms. The major components in the lipid of the mycelium of I. obliquus are unknown. The relationship between the effect of these fatty acids and the lipid composition of the mycelium of I. obliquus would be worth of further study. In contrast to the stimulatory effect on the EPS production, all the fatty acids decreased the IPS production except oleic acid that had little effect on IPS1 (Fig. 2). Stearic acid and palmitic acid had more harmful effect on the IPS production. In this study, use of oleic acid, stearic acid, and palmitic acid as additives could stimulate the mycelia of I. obliquus growth. Linoleic acid inhibited the mycelia growth but promoted the EPS production. Only oleic acid promoted both the EPS and IPS1 production in I. obliquus.

X. Xu et al. / International Journal of Biological Macromolecules 77 (2015) 143–150

*

14 12

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Tween 20 Tween 80 CHAPS TritonX-100 PEG 4000

35

32

0.7 0.6

40

16 12 8

Fig. 3. Effect of 0.1% (v/v) surfactants on the mycelial Biomass and production of EPS and IPS in submerged fermentation of I. obliguus. Each culture was carried out in triplicate at 28 ◦ C for 9 days. The values (mean value ± standard deviation, n = 3) with different letters or symbols, respectively, have significant difference (p < 0.05).

3.3. Effects of surfactants Addition of surfactants into the culture medium may be an effective strategy to increase the yields of mycelial cells in fermentation processes since this strategy was proved to be successful in the mushroom fermentation, in which addition of surfactants after 7 days could increase the yield of P. tuber-regium by submerged fermentation [21,22]. In this study, five surfactants (0.1%, v/v) added on day 0 were studied and the results of mycelial biomass, EPS and IPS production on day 9 are shown in Fig. 3. The addition of Tween 20 and Tween 80 showed a strong stimulating effect on all the mycelial biomass and production of EPS and IPS (p < 0.05) while CHAPS exhibited a detrimental effect (p < 0.05). The maximal cell concentration (13.06 ± 0.05 g/L) was obtained from PEG 4000 addition while it exhibited a detrimental effect on the EPS production (p < 0.05). In contrary, TritonX-100 decreased cell concentration obviously (Fig. 3), but it was the most effective in the EPS production that rose from 0.52 to 0.74 g/L (p < 0.05). The maximum EPS production obtained with addition of Tween 80 reached 0.750 g/L, which accounted for a significant enhancement of 45.6%. The results were consistent with those reported by Zhang and Cheung [21] in which the addition of 3.0 (g/L) Tween 80 to the liquid culture of P. tuber-regium increased the cellular viability and EPS production with maximum stimulation. Concerning the effect on IPS production, the presence of Tween 20, Tween 80, and TritonX-100 resulted in a significant increase (p < 0.05), IPS1 and IPS2 content rose from 32.69 to 35.69, 36.09, and 35.78 mg/g, and 28.56 to 31.46, 34.35, and 32.05 mg/g, respectively (Fig. 3). In other words, Tween 20, Tween 80, and TritonX-100 improved both the EPS and IPS production. In theory, surfactants are amphiphilic, containing both hydrophobic groups and hydrophilic groups. The fungal cell membrane also consists primarily of a layer of amphiphilic phospholipids. Hence, the surfactants might partially be incorporated into the fungal cell membrane thereby increasing the uptake efficiency of nutrients from the culture medium. In this way, nutrient absorption to cell surface can be greatly enhanced through the aid of surfactants [21,30]. Tween 80 affected mass transfer by changing composition in P. tuber-regium mycelial cell membrane and significantly increased the glucose consumption rate [22]. It was confirmed in Fig. 4 by the fact that the reducing sugar concentration in the Tween 80-containing medium more rapidly decreased than that in the control, reflecting the ability of I. obliquus to uptake the nutrients from the culture broth in the Tween 80-containing medium.

30

Reducing sugar(mg/mL)

Mycelial Biomass (g/L)

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mycelial biomass EPS **** IPS 1 B iii IPS 2

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****

IPS content (mg/g)

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EPS production (g/L)

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Control Tween 80

25 20 15 10 5 0

0

2

4

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8

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12

Time (d) Fig. 4. Changes of reducing sugar concentration in the control (square) and 0.1% (v/v) Tween 80-containing (circle) medium in submerged fermentation of I. obliguus. The culture was carried out in triplicate at 28 ◦ C for 12 days. Data are presented as the mean value ± standard deviation (n = 3).

The DPPH radical-scavenging activity of EPS, IPS1 and IPS2 from all the media in 0.4 mg/mL was comparatively studied shown in Fig. 5. The scavenging activity of EPS and IPS1 from all the organic solvent-containing media except for the acetone group was stronger than the control (p < 0.05) (Fig. 5a and b). Only toluene as an additive in the medium could promote the scavenging activity of IPS2 (Fig. 5c). The scavenging activity of EPS, IPS1 and IPS2 from oleic acid-containing medium was significantly (p < 0.05) stronger than the control and other fatty acid groups. The scavenging activity of IPS1 from the other fatty acids-containing medium was also higher than the control (Fig. 5b), but reduced the scavenging activity of IPS2 (Fig. 5c). Tween 80 and TritonX-100 largely enhanced the DPPH radicals scavenging activity of EPS and IPS1 (p < 0.05) (Fig. 5a and b). In addition, Tween 80 and Tween 20 improved the scavenging activity of IPS2 (p < 0.05) (Fig. 5c). Based on the above results, many additives significantly stimulated either the mycelia growth, or the production and antioxidant activity of EPS and IPS in the submerged culture of I. obliquus. Among these, oleic acid, Tween 20 and Tween 80 showed the best promoting effect not only on the mycelial growth, but also on the ESP and IPS1 production. In addition, Tween 80 and oleic acid largely enhanced the DPPH radical-scavenging activity of EPS and IPS (p < 0.05), while Tween 20 weakened the scavenging activity of EPS and IPS1 compared with oleic acid and Tween 80. TritonX100 not only significantly stimulated the EPS and IPS production in the submerged culture of I. obliquus but also improved the DPPH radical-scavenging activity of EPS and IPS1. Particularly, Tween 80 and TritonX-100 were first found to be the most effective in simultaneously enhancing both the production and antioxidant activity of EPS and IPS. So far, there were few studies on the enhancement of mushroom IPS production. Although chloroform greatly increased the IPS1 content, it was not chosen to be further studied due to the lower DPPH radical-scavenging activity of EPS and IPS1 and its toxicity. Therefore, the three chemicals of oleic acid, Tween 80, and TritonX-100 with stronger effect on the production and antioxidant activity of EPS and IPS were chosen for next optimization. 3.4. Optimal concentration and addition time Table 1 presents the effect of oleic oil, Tween 80, and TritonX100 in the concentrations of 0.05, 0.1, 0.3, and 0.5% (v/v) on the mycelial biomass, and EPS and IPS production. The mycelia biomass and EPS production increased with the increasing concentration of

X. Xu et al. / International Journal of Biological Macromolecules 77 (2015) 143–150

80

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Co ntr Ol ol eic Li no aci d le Pa ic a lm cid iti St c ac ea ric id Tw acid ee n Tw 20 ee n8 CH 0 A Tr ito PS nX PE -100 G Ch 400 lor 0 of or To m lue Ac ne eto n Et e ha n M o eth l an ol

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Scavening rate (%)

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Co ntr ol Ol eic Li a c no lei id c Pa lm acid iti ca St ea cid ric Tw acid ee n2 Tw 0 ee n CH 80 A Tr ito PS nX PE -100 G Ch 400 lor 0 of or To m lue ne Ac eto n Et e ha n o M eth l an ol

0

Fig. 5. The DPPH radical-scavenging activity of (a) EPS, (b) IPS1 and (c) IPS2 of I. obliquus from the chemical-containing and control medium in 0.4 mg/mL. Data presented as the mean value ± standard deviation (n = 3) with different letters or symbols, respectively have significant difference (p < 0.05).

oleic acid and the maximum values in the addition of 0.5% oleic acid significantly (p < 0.05) improved by 23.7% and 26.4%, respectively. For the IPS production, oleic acid only at 0.1% had slight favorable effect on IPS1. TritonX-100 had a concentration-dependent detrimental effect on the mycelia growth (Table 2), but the EPS, IPS1, and IPS2 production increased with the concentration rising up to 0.1%, 0.1%, and 0.05%, respectively, and then decreased. At the

optimal concentrations, the production increased by 55.2%, 9.5%, and 17.3%. The effect of different concentrations of Tween 80 was shown in Table 2. The mycelial biomass increased as the concentration of Tween 80 up to 0.5%, while the EPS production increased with the concentration rising to 0.1% and then decreased. Accordingly, the maximum amounts of mycelial biomass and EPS were obtained when 0.3% and 0.1% Tween 80 was added corresponding

Table 1 Effect of different concentrations of oleic oil, Tween 80, and TritonX-100 on the mycelial biomass and production of EPS and IPS of cultured I. obliquus. Concentration (mL/100 mL)

Mycelial biomass (g/L)

Control Oleic oil-0.05% Oleic oil-0.1% Oleic oil-0.3% Oleic oil-0.5% Tween 80-0.05% Tween 80-0.1% Tween 80-0.3% Tween 80-0.5% TritonX-100-0.05% TritonX-100-0.1% TritonX-100-0.3% TritonX-100-0.5%

12.65 12.87 13.05 14.16 15.65 12.88 12.90 13.13 13.10 10.64 9.38 9.12 9.02

± ± ± ± ± ± ± ± ± ± ± ± ±

0.15a 0.23f 0.33e 0.41c 0.14b 0.23f 0.13f 0.21d 0.40de 0.13g 0.21h 0.17i 0.19j

EPS production (g/L) 0.515 0.599 0.601 0.625 0.651 0.627 0.739 0.690 0.570 0.524 0.784 0.549 0.475

± ± ± ± ± ± ± ± ± ± ± ± ±

0.010a 0.009d 0.006d 0.003d 0.003cd 0.009d 0.005bc 0.004c 0.010a 0.010a 0.009b 0.004a 0.008a

IPS1 content (mg/g) 32.69 31.03 33.20 31.76 28.64 39.80 38.60 36.35 33.42 33.43 35.78 31.72 27.55

± ± ± ± ± ± ± ± ± ± ± ± ±

0.14a 0.18i 0.46g 0.18h 0.29j 0.12b 0.26c 0.15d 0.22f 0.12f 0.11e 0.25h 0.11k

IPS2 content (mg/g) 28.83 27.27 27.59 27.64 27.86 27.90 34.35 31.39 30.40 33.83 32.05 24.16 23.14

± ± ± ± ± ± ± ± ± ± ± ± ±

0.06a 0.07i 0.18h 0.09h 0.08g 0.19g 0.23b 0.22e 0.16f 0.29c 0.13d 0.32j 0.16k

Mycelial biomass, EPS and IPS production (mean value ± standard deviation, n = 3) with different alphabet letters have significant difference (ANOVA test; p < 0.05).

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Table 2 Effect of 0.1% (v/v) oleic oil, Tween 80, and TritonX-100 added at different cell growth stages (day 0, 1, 3, 5) of cultured I. obliquus on the mycelial biomass and production of EPS and IPS. Addition time

Mycelial biomass (g/L)

Control Oleic oil-0 day Oleic oil-1st day Oleic oil-3th day Oleic oil-5th day Tween 80-0 day Tween 80-1st day Tween 80-3th day Tween 80-5th day TritonX-100-0 day TritonX-100-1st day TritonX-100-3th day TritonX-100-5th day

12.65 13.05 13.04 13.15 13.77 12.90 14.75 13.60 13.63 9.38 9.42 10.32 10.76

± ± ± ± ± ± ± ± ± ± ± ± ±

0.15a 0.33f 0.42f 0.31e 0.50c 0.13g 0.42b 0.30d 0.50d 0.21j 0.46j 0.38i 0.69h

EPS production (g/L) 0.515 0.601 0.649 0.504 0.501 0.739 0.935 0.911 0.501 0.784 0.803 0.468 0.457

± ± ± ± ± ± ± ± ± ± ± ± ±

0.010a 0.006e 0.007e 0.006af 0.005af 0.005d 0.005b 0.006b 0.047af 0.009cd 0.009c 0.010f 0.007f

IPS1 content (mg/g) 32.69 33.20 33.45 31.90 31.24 38.60 45.03 35.25 33.85 35.78 34.49 30.56 23.17

± ± ± ± ± ± ± ± ± ± ± ± ±

0.14a 0.46j 0.53h 0.38i 0.49j 0.26c 0.46b 0.23e 0.16g 0.11d 0.14f 0.15k 0.14l

IPS2 content (mg/g) 28.83 27.59 29.87 25.90 24.78 34.35 34.06 33.88 33.65 32.05 30.43 23.75 19.77

± ± ± ± ± ± ± ± ± ± ± ± ±

0.06a 0.02h 0.15a 0.16i 0.14j 0.23b 0.16c 0.18d 0.19e 0.13f 0.13g 0.19k 0.17l

Mycelial biomass, EPS and IPS production (mean value ± standard deviation, n = 3) with different alphabet letters have significant difference (ANOVA test; p < 0.05).

to a significant (p < 0.05) increase of 3.8% and 43.5%, respectively, when compared with the control. Higher concentration (0.5%, v/v) of Tween 80 resulted in the generation of excessive foam, which has a detrimental effect not only on sterile environment but also on mass and heat transfer process in submerged culture [21]. Tween 80 in all the concentrations except for 0.05% in the IPS2 production played a stimulating role. The optimum concentration was 0.05% for the IPS1 production and 0.1% for the IPS2 production, resulting in a 21.7% and 19.1% increase (p < 0.05), respectively. The results showed that the three agents had different optimal concentrations. Oleic acid and Tween 80 at 0.5% (v/v) had the greatest effect on promoting the growth of mycelia when TritonX-100 had a concentration-dependent detrimental effect on the mycelia growth. For the enhancement of EPS and IPS1 production, the optimal concentrations of oleic acid were 0.5% and 0.1%, respectively. The EPS, IPS1, and IPS2 production increased with the concentration of TritonX-100 rising up to 0.1%, 0.1%, and 0.05%, and with the concentration of Tween 80 rising up to 0.1%, 0.05%, and 0.1%, respectively. Therefore, when all of these effects were considered together, 0.1% (v/v) concentration was used in effect of the three agents on addition time. To better compare the effect of the three agents on the mycelial growth, EPS and IPS production, 0.1% (v/v) oleic acid, TritonX-100, or Tween 80 was added on day 0, 1, 3, and 5 of cultivation, which represented the initial, intermediate, and late stages of the exponential growth phase of the mycelia. Addition of oleic acid at the late growth phase had resulted in a significant increase in the mycelial growth compared with the early ones (Table 2). However, addition in the early growth phase was preferred for the enhanced EPS and IPS production compared with the late ones. The highest EPS, IPS1, and IPS2 production was obtained when oleic oil was added to the medium on day 1 (24 h), with a significant (p < 0.05) increase of 26.0%, 2.3%, and 3.6%, respectively. This is not in agreement with the results of Yang et al. [17], in which the EPS production by G. lucidum was improved with oleic acid but the mycelium growth did not increase in parallel with the concentration of oleic oil. The addition of TritonX-100 at all the growth phase significantly reduced the cell concentrations (Table 2). Although the addition in the late growth phase (days 3 and 5) resulted in the less detrimental effect, it significantly decreased the production of EPS and IPS. However, addition of TritonX-100 on day 0 enhanced the production of EPS and IPS1, and IPS2, with a significant (p < 0.05) increase of 52.2%, 9.5%, and 11.2%, respectively. The result was consistent with the report that adding time of TritonX-100 at the beginning of the fermentation was most effective on the production of hypocrellins with Shiraia sp. SUPER-H168 [31]. The available theory considered that TritonX-100 might change the permeability of the membrane to promote the leakage of the fermentation products. It can help

to increase the yield of fermentation products by enhancing the oxygen transfer performance [32]. Addition of Tween 80 at all the growth phase resulted in a significant (p < 0.05) increase in the mycelial growth, EPS and IPS production except for addition on day 5 in the EPS production (Table 2). The optimum results were achieved when 0.1% (v/v) Tween 80 was added on day 1 (24 h), corresponding to 16.6%, 81.6%, 37.7%, and 18.1% (on day 0) increase in the mycelial biomass, EPS, IPS1, and IPS2 production, respectively (Table 2). The results were different from the report that addition of Tween 80 at the late stage of the exponential growth phase resulted in a significant increase in both the mycelial growth and EPS production compared with the early growth stages [21]. Therefore, it is reasonable to assume that addition of Tween 80 to a culture medium of I. obliquus extend the growth of the mycelia for a longer period possibly by maintaining the intact structure of the mycelial pellets and preventing its disintegration due to the shearing forces during the shake-flask experiments [22]. The high concentration of surfactant might damage the cell membrane or interact with other bio-compounds in cell and then resulted in low cell growth [15]. This could be one of the reasons that the addition of lower concentration of Tween 80 on day 1 was more effective. Tween 80 might partially be incorporated into the fungal cell membrane increasing thus the uptake efficiency of nutrients from the culture medium [21,22]. In addition, Tween 80, having an 18C side chain, could be hydrolyzed by microbial enzymes, such as lipase, to release oleic acid (C18:1) [33]. So the effect of Tween 80 might be partially attributed to its convert to oleic acid. Oleic acid was proved to increase the amount of mycelial biomass and EPS in this study. Among the three agents, Tween 80 was proved to be most effective in simultaneous enhancing the production of EPS and IPS and most favorable to the mycelial growth. Therefore, effect of Tween 80 on the EPS and IPS composition and antioxidant activity was further studied. 3.5. Effect of Tween 80 on the EPS and IPS composition Table 3 is a summary of the major chemical contents and monosaccharide compositions of the EPS, IPS1 and IPS2 from both the control and Tween 80-containing medium. The results showed that all the EPS and IPS by liquid cultured I. obliquus in the control and Tween 80-containing medium were polysaccharide-protein conjugates in agreement with our previous studies [9,10,12]. The sugar content of the IPS1 and IPS2 was significantly (p < 0.05) higher than that of the EPS with an inverse result for the protein content from both the media. Tween 80 could significantly increase the sugar content of the EPS and IPS but decrease the protein content compared with the control (p < 0.05), which was not consistent

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149

Table 3 Chemical content and monosaccharide components of EPS and IPS of cultured I. obliquus. Sugar content (a wt%)

Control-EPS Tween 80-EPS Control-IPS1 Tween 80-IPS1 Control-IPS2 Tween 80-IPS2

38.95 49.49 49.60 57.48 53.05 69.87

± ± ± ± ± ±

0.14a 0.36b 0.51b 0.10d 0.14c 0.07e

Protein content (wt%)

19.75 18.18 17.57 16.72 15.91 15.61

± ± ± ± ± ±

0.67f 0.20e 0.14d 0.05c 0.08b 0.10a

Monosaccharide components (b mol%) Rha

Ara

Xyl

Man

Glu

Gal

6.1 2.1 3.1 3.3 3.8 1.4

14.6 6.3 7.2 2.4 6.0 5.2

14.0 3.0 6.0 2.6 15.3 2.1

20.4 20.1 28.0 6.9 30.2 18.3

20.7 45.3 36.7 73.4 27.1 60.9

24.2 23.2 19.0 11.4 17.6 12.1

a

wt% is expressed as the weight percentage. mol% is expressed as the molar percentage. Data with different alphabet letters in the same column are statistically significantly different according to ANOVA (p < 0.05). b

with the results of P. tuber-regium that the EPS composition (protein content) did not significantly change with the addition of Tween 80 [22]. This discrepancy further proved that different fungi had different response to the additives. All the EPS and IPS from both the media were composed of Rha, Ara, Xyl, Man, Glu, and Gal with various molar ratios. Glu, Man, and Gal were the major monosaccharides (Table 3). The EPS had much less amount of Glu than IPS1 and IPS2 from both the media. Glu was the dominant component and greater than 45 mol% in the EPS, IPS1, and IPS2 from the Tween 80 medium, which was approximate 2fold higher than those from the control medium, respectively. The IPS1 from the Tween 80 medium had the highest amount of Glu greater than 73 mol% but the lowest Ara, Man, and Gal among the six extracts (Table 3). 3.6. Effect of Tween 80 on scavenging activity of the EPS and IPS against DPPH radicals Fig. 6 shows the DPPH radical scavenging activity of the EPS and IPS from the control (CT-) and Tween 80 (T80-) medium at all the tested concentrations in a dose-dependent manner (0.5–5.0 mg/mL). The scavenging activity of T80-EPS was significantly stronger than CT-EPS at all the concentrations tested. T80-IPS1 and T80-IPS2 was more effective than CT-IPS1 and CTIPS2, respectively, when the concentration was 0.5–3.5 mg/mL. The antioxidant activity was summarized and expressed as inhibiting effects of 50% (IC50 ) value (mg/mL) for comparison (Table 4). 80

CT-EPS CT-IPS 1 CT-IPS 2 T80-EPS T80-IPS 1 T80-IPS 2

75

Scavening rate (%)

70 65 60 55 50 45 40 35 30

0

1

2

3

4

5

Concentration (mg/mL) Fig. 6. Concentration-dependent DPPH radical-scavenging activity of EPS and IPS of I. obliquus from the control (CT-) and Tween 80-containing (T80-) medium. Data are presented as the mean value ± standard deviation (n = 3).

Table 4 IC50 values of EPS and IPS of I. obliquus from the control and Tween 80-containing medium in scavenging DPPH radicals. IC50 (mg/mL)

Control Tween 80

EPS

IPS1

IPS2

2.30d 0.88b

1.08c 0.74a

2.28d 0.84b

IC50 value is the effective concentration at which DPPH radicals were inhibited by 50%. Data with different alphabet letters are statistically significantly different according to ANOVA (p < 0.05).

The effective concentrations of T80-EPS, T80-IPS1, and T80-IPS2 corresponding to IC50 values were significantly lower (p < 0.05). The DPPH radicals-scavenging effects exhibited the following order: T80-IPS1 > T80-IPS2 ∼ T80-EPS > CT-IPS1 > CT-IPS2 ∼ CT-EPS. The results might be explained by the higher content of sugar and glucose of T80-EPS and T80-IPS than CT-EPS and CT-IPS, respectively (Table 3). The high molar proportion of Glu might contribute to the enhancement of scavenging on DPPH free radicals [34,35]. The T80-IPS1 with the highest content of Glu had the lowest IC50 value of 0.74 mg/mL. 4. Conclusions The feasibility of using fatty acids, surfactants, and organic solvents for enhancing mycelial growth and polysaccharide production of I. obliquus under submerged fermentation was investigated. The effect of different category agents on the cellular growth, and EPS and IPS production was different. Oleic acid and Tween 80 could be favorable for the mycelial growth, and EPS and IPS production. TritonX-100 could be used as an additive for the EPS and IPS production. Addition of Tween 80 at the early growth stage could simultaneously increase the production and antioxidant activity of I. obliquus EPS and IPS and resulted in the higher sugar content, lower protein content, and higher Glu amount of EPS and IPS compared with the control. It was attributed to that Tween 80 could increase the uptake efficiency of nutrients from the culture medium. Tween 80 is widely used in drug and food as an additive without toxicity [36]. Hence, the strategy of supplementation of Tween 80 in this study can be applied in I. obliquus and other medicinal mushroom fermentation processes for enhancing production of bioactive secondary metabolites. Acknowledgements The authors thank the financial support for the study from the Science and Technology Department of Zhejiang Province, China (2012C23075).

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