Synergistic effects of surfactant-assisted biodegradation of wheat straw and production of polysaccharides by Inonotus obliquus under submerged fermentation
Xiangqun Xu*, Pan Wu, Tianzhen Wang, Lulu Yan, Mengmeng Lin, Cui Chen
College of Life Sciences, Zhejiang Sci-Tech University
Corresponding author:
Prof. Dr. Xiangqun Xu
College of Life Sciences
Zhejiang Sci-Tech University
Hangzhou 310018
China
Email: xuxiangqun@zstu.edu.cn
Abstract
Polysaccharides are important secondary metabolites from the medicinal mushroom Inonotus obliquus. Current work proposes an innovative wheat straw biomass utilization strategy that connects efficient lignocellulose biodegradation with exo-polysaccharide (EPS) production in I. obliquus as a white rot fungus under submerged fermentation. The addition of Tween 80 increased the activities of ligninolytic enzymes MnP, LiP and Lac by 1200%, 125% and 39.9%, respectively. When wheat straw lignin recalcitrance and cellulose crystallinity were substantially reduced with the aid of 1.0 g/L Tween 80 during fermentation, I. obliquus was capable of utilizing the substrate and in turn accumulated polysaccharides. The degradation rate of cellulose, hemicellulose and lignin reached 46.1%, 46.4% and 44.1% on Day 9 of growth, respectively. Meanwhile, the maximum mycelial biomass and EPS production increased by 23.3% and 142.9%, respectively. The EPS had comparatively higher contents of sugar, protein, uronic acid, and mannose ratio in monosaccharide compositions, and higher antioxidant activity against DPPH, ABTS•+ and hydroxyl radicals. In conclusion, surfactant-assisted fungal treatment is an effective method for lignocellulosic substrate bioconversion into mushroom bioactive polysaccharides.
Keywords: Inonotus obliquus; polysaccharides; lignocellulose; Tween 80; antioxidant activity; submerged fermentation
1. Introduction
Lignocellulosic residues that mainly consist of cellulose, hemicelluloses and lignin have a great prospective for bioconversion. Delignification, depolymerization and enzymatic hydrolysis (or fermentation) are required for the conversion of lignocellulose to fermentable sugars and other derivatives. Therefore, pretreatment is a decisive stage in bioconversion of straw biomass. White rot fungi represent one of the promising biological pretreatments because these fungi are efficient degraders of the major components by synthesizing ligninase, cellulase and xylanase (Ghaffar, et al., 2015). The problem that persists today in terms of biological pretreatment of straw lignocellulose is the rate of fungi cultivation and the sensitivity to growth parameters (Ghaffar, et al., 2015).
The medicinal mushroom Inonotus obliquus belongs to the family of Hymenochaetacea of Basidiomycetes and is a white rot fungus distributed in Far East of Russia, northeast China and other adjacent countries at latitudes of 45 °N-50 °N. In nature, it usually forms an irregular shape of sclerotial conk instead of a fruiting body on birch. The sclerotia have been used as a folk medicine to treat cancer and many other diseases, such as those of heart, liver and stomach, since the 16th century (Balandaykin & Zmitrovich, 2015). I. obliquus produces a diverse range of bioactive metabolites including polysaccharides, phenolic compounds, triterpenoids, alkaloids, melanin, and other substances (Balandaykin & Zmitrovich, 2015). The polysaccharides from I. obliquus wild sclerotia and cultured mycelia have antioxidant, antitumor and antidiabetic activities (Du et al., 2013; Hu et al., 2016).
Nevertheless, due to host specificity, rarity in nature and slow growth, the sclerotia of I. obliquus are not a reliable source for industrial production of these bioactive metabolites, while submerged fermentation (SmF) of I. obliquus is a promising alternative for efficiently producing bioactive polysaccharides. We have reported that the polysaccharides from liquid cultured I. obliquus were more effective in antioxidant activity and cytokine induction activity than those from the wild sclerotia (Xu et al., 2011; Xu et al., 2014b). In addition, Tween 80 as a stimulatory agent could significantly enhance the bioactive polysaccharide production (Xu et al., 2015). We also found that I. obliquus as a white rot fungus was capable of degrading the crop residues under submerged fermentation and accumulating bioactive polysaccharides with the lignocellulose degradation (Xu et al., 2014a).
Lignocellulose biodegradation is an extracellular oxidative and hydrolytic process which requires the synthesis of ligninolytic and hydrolytic enzymes. The production and activities of these enzymes are highly regulated by physical and chemical factors including surfactants (Bansal et al., 2014; Iqbal et al., 2011). However, it is not clear how the surfactant effect reflects on the extent of lignocellulose degradation by I. obliquus under SmF and of polysaccharide production by the combination effects of surfactant supplementation and lignocellulose degradation.
The aim of this study was to assess the capacity of Tween 80 to enhance the wheat straw (WS) biodegradation and in turn the exopolysaccharide (EPS) production by I. obliquus under SmF. The WS biodegradation efficiency was investigated by analyzing time-course degradation rate, chemical and surface structural changes using Fourier transform infrared spectroscopy (FTIR) and scanning electron microscopy (SEM) (Xu et al., 2017). The evolution of lignocellulolytic enzyme production was determined to explain the biodegradation efficiency affected with the presence of Tween 80. The yield and antioxidant activity of EPS were comparatively evaluated.
2. Materials and methods
2.1. Liquid fermentation
I. obliquus (CBS314.39) was incubated in the following media at 28 °C and 150 rpm in triplicate (Chen et al., 2010).
Control medium (g/L): corn flour 53, peptone 3, KH2PO4 1, ZnSO4·7H2O 0.01, K2HPO4 0.4, FeSO4·7H2O 0.05, MgSO4·7H2O 0.5, CuSO4·5H2O 0.02, CoCl2, 0.01, and MnSO4·H2O 0.08, pH=6.0. The medium was optimized by response surface methodology for I. obliquus bioactive polysaccharide production under submerged fermentation in our previous work (Chen et al., 2010).
WS-containing medium (g/L): wheat straw 30, and all the other chemicals were the same as the control medium, pH=6.0 (Xu et al., 2014a).
WS + Tween 80-containing medium (g/L): Tween 80 1, wheat straw 30, and all the other chemicals were the same as the control medium, pH=6.0. Tween 80 and WS were added into the culture medium on Day 0. All the chemicals were of analytical grade.
2.2. Analysis of WS chemical composition
WS was dried and extracted with ethanol–benzene (1:2, v/v) mixture for 3 h in a Soxhlet extractor in a boiling water bath. Cellulose and hemicellulose were determined according to van Soest et al. (1991). Lignin was determined by a method using 72% H2SO4 to hydrolyze (Xu et al., 2017). The WS residue was harvested at 48-h intervals during fermentation. The dynamic contents of lignin, hemicellulose and cellulose in the residue before and after degradation were measured and the degradation rates were calculated according to the amount loss (Xu et al., 2017).
2.3. Characterization of WS chemical and surface structure
The chemical structures were characterized by FTIR (Nicolet 5700, USA, Thermo Electron Corporation) using the KBr pellet technique. An accumulation of 32 scans were performed ranging from 2000 cm-1 to 500 cm-1 at a spectral resolution of 4 cm-1. Peak height and area of spectra were determined by Omnic software (Amnuaycheewa et al., 2016).
The surface properties and microstructure were analyzed using SEM. Dried samples (60 °C for 5 days) were coated with gold films and observed under JSM-5610LV (Jeol Ltd., Tokyo, Japan) scanning electron microscope (Xu et al., 2017).
2.4. Determination of cellulolytic and ligninolytic enzyme activity
The enzyme activity of culture broth supernatant was analyzed (Xu et al., 2017). Carboxymethyl cellulase (CMCase) activity was assayed by measuring the release of reducing sugars in a reaction mixture containing carboxymethyl cellulose (CMC) incubated at 50 °C for 30 min. The total cellulase activity (filter paper activity, FPase) activity was assayed by measuring the release of reducing sugars in a reaction mixture containing Whatman No.1 filter paper (1× 6 cm ≈ 50.0 mg ) incubated at 50 °C for 60 min. Released reducing sugar in both the assays was estimated by the 3,5- dinitrosalicylic acid (DNS) method at 540 nm as glucose equivalent (Miller, 1959). β-Glucosidase assay was carried out in the reaction mixture containing 4-nitrophenyl β-D-glucopyranoside (pNPG) at 50 °C for 10 min. The liberated p-nitrophenol (pNP) was measured at 405 nm (Kovács et al., 2009).
Lignin peroxidase (LiP) activity was measured by determining the oxidation rate of veratryl alcohol to veratraldehyde at 30 °C at 310 nm, and 1 mmol veratraldehyde formed per minute was defined as the enzyme unit(Pinto et al., 2012). Mn peroxidase (MnP) activity was determined based on the oxidation of Mn2+ to Mn3+ at 240 nm and the enzyme unit was defined as the amount of enzyme that oxidized 1 mmol MnSO4 per minute (Pinto et al., 2012). Laccase (Lac) activity was determined by oxidation of ABTS [2, 2-azinobis-(3-ethylbenzothiazoline-6-sulfonic acid)] at 420 nm. One unit of activity is defined as the amount of enzyme which leads to the transformation of 1 μmol substrate per minute (Kumari and Das, 2016).
The data presented in the figures correspond to mean values with standard errors in triplicate.
2.5. Extraction and compositional analysis of EPS
Ethanol precipitation was used to get EPS samples from the culture broth on Day 9 for chemical analysis and antioxidant activity evaluation. The precipitate was repeatedly washed with 95% ethanol and acetone, dialyzed (molecular weight cut off: 3000 Da) to remove adherent sugar residue and other small molecules, centrifuged at 6,500 ×g for 10 min, and lyophilized to get CT-EPS, WS-EPS, and WS+Tween 80-EPS from the control, WS-containing, and WS+Tween 80 containing media, respectively (Xu et al., 2011).
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 (Miller, 1959). The total carbohydrate content was determined by the phenol-sulfuric acid method using glucose as a standard (Chen et al., 2010). The protein content was analyzed by the Bradford’s method with bovine serum albumin as a standard (Xu et al., 2011). The total uronic acid content was determined using the carbazole-sulfuric acid colorimetry method (Liang et al., 2018).
To determine EPS monosaccharide compositions, after hydrolysis and acetylated derivation, the alditol acetates were analyzed in an Agilent SE-50 capillary chromatography column (30 m × 0.25 mm, 0.25-μm film thickness) by a gas chromatograph (GC) Techcomp GC-7900 (Techcomp Inc., Shanghai) under the following conditions: injection temperature: 270 °C; detector temperature: 250 °C. 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) (Xu et al., 2015).
2.6. Evaluation of antioxidant activity
The EPS samples obtained in 2.5 were dissolved in water in a concentration gradient (0.5-5 mg/mL) for the three antioxidant assays.
DPPH (2, 2 – diphenyl-1-picrylhydrazyl) free radical-scavenging activity was measured according to the method described by Xu et al. (2015). The 3.2 mL mixture [0.8 mL of DPPH (0.4 mmol/L) and 2.4 mL of the tested sample] was determined at 517 nm after standing 30 min in dark at room temperature.
Scavenging rate (%) = [A0 – (Ax –Ax0)] × 100/A0 (1)
A control contained all the reaction reagents except for the samples was prepared and measured as A0. Ax was the result of samples and Ax0 is the absorbance for background without DPPH.
ABTS•+ antioxidant assay was performed according to Guo’s method with some modification (Guo et al., 2012). The working solution was prepared by dilution of stable ABTS+ free radicals. The absorbance of the mixture (30 µL of the tested sample and 3.0 mL ABTS+ working solution) was determined at 734 nm after 6 min of incubation at room temperature.
Scavenging rate (%) = [A0 – (Ax –B)] × 100/A0 (2)
A control contained all the reaction reagents except for the samples was prepared and measured as A0. Ax was the result of samples and B is the absorbance for background without ABTS+ working solution.
Hydroxyl radical-scavenging activity was determined according to the salicylic method (Xu et al., 2011). The 4 mL final volume reaction mixture contained 1 mL tested sample, 1 mL FeSO4 (9 mmol/L), 1 mL salicylic (9 mmol/L) and 1 mL H2O2 (8.8 mmol/L). After 60 min reaction at 37°C, the mixture was centrifuged at 10,000 g for 8 min. The absorbance of the reaction solutions was measured at 510 nm.
Scavenging rate (%) = [A0 – (Ax – C)] × 100/A0 (3)
A control contained all the reaction reagents except for the samples was prepared and measured as A0. Ax was the result of samples and C is the absorbance for background without H2O2.
2.7. Statistical analysis
All the results were expressed as mean ± standard deviation (SD) of three independent determinations. All statistical analyses were performed by using the software SPSS Statistics 19.0. IC50 value of antioxidant activity, which is the concentration of an inhibitor where the response is reduced by half, was calculated by using probit analysis. 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
3.1. Time courses of mycelial growth
Fig. 1 shows that the mycelial yield of I. obliquus from all the three media increased with the fermentation time till Day 9. The combination of WS and Tween 80 showed a synergistic stimulatory effect on the mycelial growth with a significant (P < 0.05) increase of 23.3% compared with the control group, while the individual addition of WS or Tween 80 achieved an increase of 14.1% and 2% (Xu et al., 2015), respectively. Although previous work demonstrated the individual addition of WS (Xu et al., 2014a) or Tween 80 (Xu et al., 2015) increased the yields of I. obliquus in the fermentation processes, the synergistic effect was first found in this study. The effect partly resulted from the surfactant assistance. Tween 80 was found to affect mass transfer by changing composition in P. tuber-regium mycelial cell membrane and significantly increased the glucose consumption rate, resulting in the enhanced growth (Zhang & Cheung, 2011). However, the straw biomass biodegradation as carbon sources for growth requirements (Salvachúa et al., 2011) likely was enhanced with Tween 80 assistance and played a more important role. Therefore, the effect of Tween 80 on WS biodegradation by I. obliquus under SmF was investigated to better understand the synergistic effect.
3.2. Effect of Tween 80 on WS biodegradation
3.2.1. Degradation rates of lignin, cellulose and hemicellulose
The raw WS consists of 19.2±0.09% of lignin, 29.2±0.18% of hemicelluloses, and 37.7±0.22% of cellulose. The degradation rate of the three components increased dramatically with fermentation time of 12 days (Fig. 2). The lignin removal at 27.3 % in WS and 35.7 % in the WS with Tween 80 occurred on Day 4, while the loss of cellulose was relatively low at 18.2 % and 23.8 %, respectively. The results indicated that I. obliquus had the ability to degrade WS lignin selectively rather than to do simultaneously cellulose at the early stage of fermentation. The final lignin degradation rate reached 39.2 % (WS alone) and 44.1 % (WS with Tween 80) on Day 12, respectively (Fig. 2a). The degradation rate of hemicelluloses increased rapidly to 35.2 % (WS alone) and 38.9 % (WS with Tween 80) on Day 6, Day 4; the degradation rate of cellulose accelerated up to 24.5 % (WS alone) and 23.8 % (WS with Tween 80) on Day 8 and Day 4, respectively. The results indicated that Tween 80 significantly shortened the fermentation time to achieve the similar degradation degree of hemicelluloses and cellulose. The contents of hemicelluloses and cellulose in the WS and WS with Tween 80 were reduced by 42.4 %, 46.4 % (Fig. 2b) and 30.8 %, 46.1 % on Day 12 (Fig. 2c), respectively. I. obliquus is a very rapid and highly efficient lignocellulose degrader under SmF and the efficiency could be further enhanced by the addition of Tween 80. The enhancing effect of Tween 80 on cellulose degradation was the most intense among the three components. A series of intermediate products could serve as valuable biosynthetic building blocks (Luterbacher et al., 2014).
3.2.2. Effect of Tween 80 on WS chemical structural changes
FTIR analysis was conducted to examine the lignocellulose structures of the raw and treated WS without and with Tween 80 to gain further insight into the enhanced effect of Tween 80 on the biodegradation by I. obliquus on Day 9. From the FTIR spectra (Fig. 3), a number of peaks were used to observe changes in the chemical structures of cellulose, hemicelluloses, and lignin (Amnuaycheewa et al., 2016). The FTIR spectral patterns of the three samples were similar but the intensities of some peaks were significantly reduced with biodegradation. For comparison among the three substrates, analysis of these spectra in terms of the percent modifications of each treated substrate relative to the non-treated substrate is summarized in Table 1. The decrease in the band at 1509 cm−1 was more pronounced in the WS with Tween 80 than in WS alone, suggesting a more efficient and substantial degradation of WS lignin. This conclusion was corroborated by the larger diminution in 1426 cm-1, 1604 cm-1, 1730 cm-1 bands. The peak intensities at 1459 cm−1, 1374 cm−1, 1319 cm−1 and 1252 cm−1 were more intensely reduced, implying that the addition of Tween 80 was effective in removing the ether linkages between lignin and carbohydrates and lignin deacetylation. The more significant decreases in the bands at 1162 cm-1, 1374 cm-1, and 1730 cm-1 corresponding to hemicelluloses indicated the larger hemicelluloses degradation in the WS with Tween 80. The diminutions in 898 cm-1 (amorphous cellulose) and 1110 cm-1 (crystalline cellulose) bands were clear indications of severer cellulose degradation. Crystallinity is one of the most important factors influencing cellulose enzymatic digestibility. The peak signal at 1426 cm−1 is strong in crystalline cellulose, and weak in amorphous cellulose (Nelson and O’Connor, 1964). The more pronounced diminutions at 1110 cm-1 (crystalline cellulose) and 1426 cm−1 bands in the WS with Tween 80 were also clear indications of more intense crystalline cellulose degradation (Table 1).
These FTIR analysis results were consistent with the chemical composition analysis shown in Fig. 2 to better explain the enhancing effect of Tween 80 on the WS biodegradation.
3.2.3. Effect of Tween 80 on WS surface structural changes
The untreated WS had intact and relatively smooth surfaces (Fig. 4a). Both the WS with or without Tween 80 subjected to the inoculation with I. obliquus exhibited an eroded morphology with emptiness and cracks (Fig. 4b, c). The microscopic analysis (of the appearance of pores) was in agreement with those obtained by the compositional and FTIR analysis; the WS with Tween 80 experienced more effective lignin removal (Table 1, Fig. 2). After the bio-treatment with Tween 80, the WS structure ruptured significantly, resulting in a noticeably rugged surface with more cracks, erosion troughs, and even hores on the surface of WS (Fig. 4c). The cellulose and hemicellulose in the WS residue have been largely decomposed, leaving a fragile framework of lignin, which were in agreement with the losses of 46.4% and 46.1% by the compositional analysis (Fig. 2).
The exposure of cellulose through structural alteration of the substrates is a crucial factor in the hydrolysis of the remaining cellulosic fraction present in the cell wall. The FTIR and SEM results in this study confirmed the efficient structural alteration of straw biomass by the fungal process with the assistance of Tween 80. Although some studies have confirmed that Tween 20 treatment contributed to the structural changes of lignocellulose pretreatment (Seo et al., 2011) and improved conversion of cellulose with Tween 80 was mainly due to the reduction of the adsorbed cellulase deactivation (Yang et al., 2011), it is not clear how Tween 80 treatment affects the WS degradation during the fungal process. Therefore, the evolution of production and activity of lignocellulolytic enzymes by I. obliquus under SmF with the addition of Tween 80 were comparatively investigated.
3.2.4. Effect of Tween 80 on lignocellulolytic enzyme production
I. obliquus produced MnP (Fig. 5a), LiP (Fig. 5b), and Lac (Fig. 5c) with various activities at different fermentation time in the control, WS-, and WS with Tween 80-containing media. High MnP activities of 23.07 IU/mL, 153.84 IU/mL, and 300 IU/mL were detected on Day 8 in the three media, respectively. The LiP activity appeared peaked at 125.0 IU/mL, 223.08 IU /mL, and 282.26 IU /mL on Day 8, respectively. The Lac activity maximized at 2.08 IU/mL, 2.64 IU/mL, and 2.91 IU/mL on Day 10, Day 10 and Day 8, respectively. The production pattern was inconsistent with our previous work that the highest activities of MnP (159.0 IU/mL) and LiP (123.4 IU/mL) in WS culture peaked much earlier on Day 2 and 4, respectively (Xu et al., 2017). The inconsistency was attributed to the different media used, i.e., the WS-containing medium in this study had much higher corn starch content for the optimal EPS production (Chen et al., 2010) rather than for lignocellulose degradation (Xu et al., 2017). One of the primary causes of such delayed production was assumed to be the tight transcriptional control of lignocellulolytic enzymes via carbon catabolite (from both corn starch and WS) repression (Brown et al., 2014).
WS-Tween 80 medium induced 1.3-fold production of LiP (282. 3 IU /mL) and 2–fold production of MnP (300 IU /mL) in comparison to WS cultures; MnP, LiP and Lac activity efficiently increased by 1200%, 125% and 39.9% in comparison to the control cultures. In particular for LiP activity, these results are noticeable due to the difficulties on achieving elevated enzyme production levels with the LiP-expressing white-rot fungus Phanerochaete chrysosporium (Alam et al., 2009). Other authors pointed out that LiP production in SmF by P. chrysosporium was so low that its detection was very difficult (Shi et al., 2009). In WS-Tween 80 culture of I. obliquus, cyclic production pattern for LiP and MnP, and simultaneous production of Lac activity, indicated synergistic catalytic functions of these powerful oxidoreductases on the WS biodegradation (Table 1, Fig. 2) (Floudas et al. 2012).
Tween 80 has been reported to enhance the production of LiP and MnP from P. chrsosporium IBL-03 and Trametes versicolor in solid state fermentation (SSF); surfactants have the potential to enhance microbial growth in SSF by promoting the penetration of water (Asgher et al., 2011; Iqbal et al., 2011). It is proposed that surfactants alter the permeability and fastens the secretion of enzymes (Zhang and Cheung, 2011). To our knowledge, this is the first report on the enhanced production of LiP, MnP, and Lac together in SmF of one I. obliquus strain with the aid of surfactants.
I. obliquus grown under SmF started producing all the cellulases on Day 2 in the control, WS, and WS-Tween 80 cultures. The CMCase production (5.69, 5.72, and 5.64 IU/mL) (Fig. 5d) and FPase production (2.24, 2.37, and 2.26 IU /mL) (Fig. 5e) peaked on Day 4, whereas maximum β-glucosidase (0.32, 0.40, and 0.23 IU/mL) was observed on Day 10, respectively (Fig. 5f). Unlike the enhanced effect of Tween 80 on ligninolytic enzyme production, addition of Tween 80 did not favor any increase in the cellulase production. The result is not in accordance with earlier reports, where increase in cellulase production has been reported in the presence of Tween 80 from Aspergillus niger and Fomitopsis sp. in SSF (Bansal et al., 2014). Extracellular enzyme production depends greatly on the composition of the medium. The result of this study can be attributed to the fact that sufficient carbon in the media might be acting as a repressor for hydrolytic enzyme production. It is well known that, during carbon limitation, lignocellulolytic fungi shift primary metabolism to the utilization of pre-existing carbon sources within the cell and promote the transcription of secreted hydrolytic enzymes (Brown et al., 2014). In other words, it is not crucial for the fungus to secret hydrolytic enzymes to utilize WS in a medium with sufficient carbon source, therefore, WS or WS-Tween 80 did not exhibit any significant effect on the increase of cellulase production. Nevertheless, the WS cellulose was more effectively hydrolyzed in the presence of Tween 80. It is possible that ligninolytic enzymes with higher activity secreted from I. obliquus in the aid of Tween 80 efficiently removed or disrupted lignin and liberated cellulose and hemicellulose from lignin, then the cellulose and hemicellulose more easily hydrolyzed by the cellulase in the course of the fermentation process. On the other hand, Tween 80 adsorbed on the surface of lignin, preventing the non-specific binding of enzyme and lignin, thus reduced the cellulase adsorption onto lignin and enhanced the enzymatic hydrolysis of cellulose (Lou et al., 2018).
3.3. Synergistic effects of Tween 80 and lignocellulose degradation on EPS production and activity
3.3.1. Effect on production and composition of EPS
Fig. 6 shows the dynamic changes of EPS production in SmF of I. obliquus. The production of WS-EPS and WS+Tween 80-EPS peaked at 1.41 g/L on Day 9 and 1.70 g/L on Day 10 respectively, while CT-EPS production was 0.70 g/L on Day 9. Similar to the previous result, WS degradation significantly enhanced WS-EPS production, partly produced from the degraded holocellulose (Xu et al., 2014a). On the other hand, EPS might be one of the compounds produced by I. obliquus in response to oxidative damage when lignin biodegradation occurs (Chen et al., 2011). In this study, the synergistic effect of Tween 80 and lignocellulose degradation on EPS production was evident. The enhancement might be caused by the severe enzymatic hydrolysis of WS lignin and holocellulose induced by Tween 80 (Fig. 2 and Fig. 5); ultimately the substrates from holocellulose for EPS production would be released and EPS against oxidative damage would be stimulatory produced. Thus, the Tween 80-assisted fermentation strategy was successful in enhancing the production of value-added metabolites.
The Tween 80-assisted fermentation not only enhanced EPS production, but also changed EPS chemical contents and monosaccharide compositions. As shown in Table 2, the contents of sugar, protein, and uronic acid of WS+Tween 80-EPS were significantly (p < 0.05) higher than those of CT-EPS and WS-EPS. It is expected that WS+Tween 80-EPS with higher content of uronic acid would be more water-soluble and therefore higher bioactive (Chen et al., 2018). It is the first time to report that the lignocellulose degradation and Tween 80 stimulation synergistically increased the uronic acid content of EPS.
All the three EPS extracts were composed of Rha, Ara, Xyl, Man, Glu, and Gal with various molar ratios. Glu and Man were the major monosaccharides (Table 2). WS+Tween 80-EPS had the highest amount of Man (28.6 mol%) and Rha (6.5 mol%). The results could be explained by the fact that enhanced holocellulose degradation produced more Man serving as substrates with the aid of Tween 80 (Luterbacher et al., 2014).
3.3.2. Effect on antioxidant activity of EPS
Based on the variability in the antioxidant properties and mechanisms, three antioxidant assays were used to clearly demonstrate the real antioxidant activity of the three EPS extracts. Fig. 7 showed the DPPH (a), ABTS•+ (b) and hydroxyl (c) radical-scavenging activities of CT-EPS, WS-EPS, and WS+Tween 80-EPS. All extracts exhibited in a dose-dependent manner (0.5-5.0 mg/L). As expected, WS-EPS had better effects than CT-EPS, which was in accordance with our previous work (Xu et al., 2014a). Furthermore, WS+Tween 80-EPS exhibited significantly stronger antioxidant activity than WS-EPS in the three assays (Fig. 7). The results might be attributed to the enhanced lignocellulose decomposition (Figs. 2-4) stimulated the fungus to produce more EPS with higher antioxidant effect to protect themselves (Chen et al., 2011).
The most potent assay in the analysis of EPS extracts was the hydroxyl radical scavenging assay with IC50 values of CT-EPS, WS-EPS and WS+Tween 80-EPS down to 1.39, 0.82, and 0.55 mg/mL, respectively, whereas the ABTS•+ assay showed the lowest activity with IC50 values of >100 – 4.16 mg/mL (Table 3). Moderate DPPH radical-scavenging activity of EPS extracts was evident with the IC50 values in between (Table 3). Hydroxyl radicals can readily react with most biomolecules including carbohydrates, proteins, lipids, and DNA in cells, and cause tissue damage or cell death. Thus, removing hydroxyl radicals is important for the protection of living systems. The highest hydroxyl radical-scavenging capacity of all the three EPS extracts could be attributed to the culture media that was optimized for antioxidant EPS production using hydroxyl radical-scavenging activity-guided response surface methodology in our previous studies (Chen et al., 2010). WS+Tween 80-EPS exhibited stronger hydroxyl radical-scavenging activity with the IC50 of 0.55 mg/mL than the Pleurotus eryngii crude polysaccharide (IC50 1.02 mg/mL) and refined polysaccharide (IC50 1.19 mg/mL) (He et al., 2016). The scavenging activity on DPPH radical is a widely used index and a quick method to evaluate antioxidant activity. A higher DPPH radical-scavenging activity (IC50 1.65 mg/mL) was found in this study compared to the Cordyceps cicadae EPS (IC50 7.32 mg/mL) (Sharma et al., 2015) and the Ganoderma lucidum EPS (Wang et al., 2018). WS+Tween 80-EPS was also the most active EPS against ABTS•+ with IC50 value of 4.16 mg/mL among the three EPS extracts and better than the Cordyceps cicadae EPS (6.38 mg/mL) (Sharma et al., 2015).
The results showed that Tween 80 supplement and lignocellulose degradation synergistically promoted the antioxidant activity of EPS. Higher contents of sugar (44.3%), protein (20.5%), and uronic acid (7.75%) (Table 2) likely resulted in the higher antioxidant activity of WS+Tween 80-EPS. Our previous results demonstrated that with increase of the protein content, the antioxidant activities of the three polysaccharide-protein conjugates from the wild sclerotia and I. obliquus culture increased (Xu et al., 2011). Those results were also likely linked to the type of monosaccharide and uronic acid content of WS+Tween 80-EPS (Table 2) (Chen et al., 2018; Liang et al., 2018). It was found that polysaccharides with higher uronic acid content exhibited stronger antioxidant ability because the uronic acid can reduce the generation of hydroxyl (Chen et al., 2018).
Conclusion
This study, for the first time, demonstrated the Tween 80-assisted fermentation strategy for efficient lignocellulose biodegradation and enhanced production and bioactivity of polysaccharides by the white rot fungus and medicinal mushroom I. obliquus under submerged fermentation. The fungus produced high-activity-level ligninolytic enzymes and thus significantly changes the chemical and surface structures of wheat straw during fermentation with the aid of Tween 80. It is significant that surfactant effect and lignocellulose degradation synergistically enhanced the production of bioactive polysaccharides from the medicinal mushroom, resulting from the released substrates from the holocellulose. This study provided an approach to solve the problem of highly efficient both lignocellulose biodegradation and bioactive polysaccharide biosynthesis for bioconversion of lignocellulosic biomass into potential macromolecule pharmaceuticals.
Acknowledgements
The authors thank the financial support for the study from Zhejiang Provincial Natural Science Foundation, China under grant LY16B020013.
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