Enhanced production and antioxidant activity of endo-polysaccharides from Phellinus igniarius mutants screened by low power He-Ne laser and ultraviolet induction

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Enhanced production and antioxidant activity of endo-polysaccharides from Phellinus igniarius mutants screened by low power He-Ne laser and ultraviolet induction ⁎

He-Nan Zhanga,b,c, Hai-Le Maa, , Cun-Shan Zhoua, Yang Yanb,c, Xiu-Lian Yina, Jing-Kun Yana, a b c

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School of Food & Biological Engineering, Jiangsu University, Zhenjiang 212013, China National Engineering Research Center of Edible Fungi, Key Laboratory of Edible Fungi Resources and Utilization (South), Ministry of Agriculture, China Institute of Edible Fungi, Shanghai Academy of Agricultural Sciences, Shanghai 201403, China

A R T I C L E I N F O

A BS T RAC T

Keywords: Phellinus igniarius Mutation Endo-polysaccharides Physicochemical properties Antioxidant activities

In this study, a Phellinus igniarius mutant was screened through low power He-Ne laser and ultraviolet (UV) induction. The mutant was then used to improve the production of endo-polysaccharides. In the shake flasks, the dry weight of the mycelial biomass and the production of endo-polysaccharides derived from the screened mutant (JZx) fermentation were 20.715 g/L and 1.428 g/L, respectively, which were 40.31% and 56.58% higher than those of the control strain (CK). In addition, isozyme electropheresis spectrogram analysis results indicated that the genetic materials of the screened mutants were altered. The endo-polysaccharides obtained from the JZx fermentation were mainly composed of D-glucose, L-rhamnose, and D-mannose in a molar ratio of 2.0:16.0:1.0 and with low-molecular weights (MWs) (1.5 kDa, 61%). These endo-polysaccharides exhibited stronger antioxidant activities in vitro, contained stronger hydroxyl radical scavenging capacity, and higher Trolox equivalent antioxidant capacity (TEAC) (195.43 μmol Trolox/g sample) and ferric reducing ability of plasma (FRAP) (20.57 μmol Fe2+/g sample) values compared with those of the CK. Therefore, the mutant screening through low power He-Ne laser and UV-induction could be an efficient and practical method for the development of the Phellinus strains and thus could improve the production and antioxidant activities of their endo-polysaccharides.

1. Introduction Phellinus igniarius is a basidiomycete fungus under the genus Phellinus of the Polyporaceae family. It is well-known for its significant biological activities and medicinal properties (Mizuno, 1999). The fruiting bodies of P. igniarius have been traditionally used as folk medicine for many years in China, Japan and Korea because of their prominent anti-inflammatory and hemostasis functions. In addition, they also invigorate the liver, promote blood circulation, and reinforce the spleen (Dai, Zhou, Cui, Chen, & Decock, 2010). As a major class of bioactive components of P. igniarius, polysaccharides have been important and beneficial because they demonstrate antioxidative, antibacterial, antiviral, antitumor, antimutagenic, and immunomodulatory activities (Li, Yang, Ma, Yan, & Guo, 2015; Song, Lin, Yang, & Hu, 2008; Suabjakyong, Nishimura, Toida, & Van Griensven, 2015). Therefore, these compounds have increasingly attracted attention for their health benefits. They have been used in the food, medical, and cosmetic industries as therapeutic agents because they exhibit low

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toxicity and have minimal side effects. In recent years, wild or natural P. igniarius has become increasingly rare, and its field-cultivation cycle is time consuming. Thus, the development and use of P. igniarius are limited (Chen, Xiao, Li, & Zhang, 2007). Generally, cultivation of a fruiting body of P. igniarius requires six months, and the product quality is difficult to control when it is cultivated in a solid substrate. The product composition also varies among batches (Liu et al., 2009). Notably, liquid or submerged fermentation has some advantages. It increases mycelia and polysaccharide production even at compact space over a short incubation time and availability of convenient control with less chance of contamination (Hwang, Kim, Choi, & Yun, 2003; Suabjakyong et al., 2015). Therefore, submerged fermentation has become a promising alternative that generates highly efficient production of mycelial biomass and polysaccharides. Although natural breeding can maintain the stability of the strains and produce pure strains, the probability of natural variation is extremely low, and the output frequently occurs in the fluctuation

Corresponding author. E-mail addresses: [email protected] (H.-L. Ma), [email protected], [email protected] (J.-K. Yan).

http://dx.doi.org/10.1016/j.bcdf.2016.11.006 Received 10 June 2016; Received in revised form 29 October 2016; Accepted 19 November 2016 Available online xxxx 2212-6198/ © 2016 Published by Elsevier Ltd.

Please cite this article as: Zhang, H-N., Bioactive Carbohydrates and Dietary Fibre (2016), http://dx.doi.org/10.1016/j.bcdf.2016.11.006

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grown in a 250 mL flask containing 100 mL of basal potato medium (PDB) at 26 °C. The screened P. igniarius strain was initially grown on a PDA medium in a petri dish, and then transferred into the seed medium by punching out 5 mm of the agar plate culture using a cutter. The seed culture was grown in a 250 mL flask, which contains 100 mL of PDB basal medium, at 26 °C placed on a rotary shaker incubator (130 rev min−1) for 8 days. The flask-culture experiments were performed in a 250 mL flask containing 100 mL of medium inoculated with 10% (v/v) of the seed culture. The fermentation medium consisted of the following components: maize flour (50 g/L), bran (15 g/L), mulberry shoot powder (10 g/L), KH2PO4 (2 g/L), MgSO4 (1 g/L).

range of biological production. As modern techniques, genetic engineering and metabolic engineering are increasingly considered as effective methods for microorganism breeding. However, these techniques encounter serious impediments because of limited insights into the genetics, physiology, and biochemistry of organisms (Olano, Lombó, Méndez, & Salas, 2008). Until now, traditional mutation is still the most effective strategy for improving the productive capacity of the strains (Khaliq et al., 2009). In this regard, UV is widely used in mutagenesis-selection protocol. The mutagenic and lethal mechanisms of UV radiation have been elucidated in several microorganisms (Alifano, Lorusso, Nassisi, Tala, & Tredici, 2008; Clark, 1996; Ikehata & Ono, 2011; Ravanat, Douki, & Cadet, 2001). However, because of the lower rate of UV on positive variation, researchers have focused on mutation screening through compound mutation in various ways (Yu et al., 2011). A low-power laser irradiation technology has generated considerable interest with regard to microorganism mutation breeding. Based on a homemade XeCl 308 nm excimer laser, an innovative and effective mutagenesis protocol has been developed for antibiotic-producing industrial strains (Alifano et al., 2008). Laser at wavelength of 620 nm has significant growth-stimulating effect, especially He-Ne laser at wavelength of 632.8 nm (Van Breugel & Bär, 1992). However, to the best of our knowledge, reports on the use of low-power He-Ne laser and UV irradiation to induce microorganism mutation are few. Many research findings demonstrated that the antioxidant activity of the polysaccharides and their derivatives mainly rely on separation procedures, physicochemical properties, water solubility, and even their primary structures. Among the processes regarding these polysaccharides, chromatographic purification of actives anionic polysaccharides, oligoglucuronans, and their derivatives are increasingly attracting interest (Petera et al., 2015; Wang et al., 2016). Some studies focused on the relationship between the structure and antioxidant activity of polysaccharides, elucidation of their antioxidant mechanism at the molecular level, and improvement of their various biological activities through molecular modifications, such as sulfation, carboxymethylation, and regioselective oxidation (Delattre et al., 2015; Elboutachfaiti et al., 2011; Wang et al., 2016). For example, Delattre et al. (2015) reported that a xanthouronic acid sodium salt called xanthouronan, which was produced from xanthan through TEMPOmediated oxidation, exhibits stronger antioxidant activities than native xanthan. In addition, physical irradiation treatments, such as ultrasound, microwave, and UV radiation, have been used to modify polysaccharides in order to enhance their physicochemical properties and biological activities (Delattre & Vijayalaksmi, 2009; Drímalová, Velebný, Sasinková, Hromádková, & Ebringerová, 2005). Thus, a novel microbial mutation method combining low power HeNe laser and UV radiation was used to screen P. igniarius strains to increase the production of endo-polysaccharides. Meanwhile, the physicochemical properties, primary structures, and antioxidant activities of endo-polysaccharides of wild and mutant strains were investigated.

2.3. Protoplast generation P. igniarius mycelia were cultivated and statically grown in a flask for 10 days. The mycelia were then harvested by centrifugation at 10,000g for 10 min before they were triturated and homogenized by a sterilized Eppendorf tube. The mycelia were then incubated in an incubator shaker (100 rev min−1) and allowed to undergoenzymatichydrolysis in 1% lywallzyme mixed with 0.25% driselase (Sigma) containing 0.6 mol/L of mannitol. The enzyme solution was filtered through a sterile 0.22-μm millipore filter before use. After incubation, the protoplasts were obtained by centrifuging at 5000g for 10 min and then were washed twice with sterile osmotic stabilizer 0.6 mol/L mannitol to remove the enzymes. Finally, pure protoplasts were obtained by using sterile G3 sand core funnel. 2.4. Mutagenesis and rational screening Each 1 mL of appropriately diluted suspension was placed into a 1.5 mL Eppendorf tube. The fiber of the laser (LJL40-HA, Shanghai Institute of Laser Technology, China) was vertically irradiated through the tube top, which had an output power of 40 mW and transmission efficiency of 80%. The distance between the suspension liquid level and fiber optic terminus was 10 cm, and the spot radius was 10 mm. The exposure time was 30 min. The suspension was then spread over a plate, which had a diameter of 70 mm. The plate was placed 20 cm from the 15-W UV lamp. The UV directly radiated suspension in the plate. To avoid any photo recombination influence, the irradiation was performed in the dark. After the exposure, the irradiated suspension was diluted, and 100 μL of sample from each diluted suspension was plated on screening agar plates, which contained PDA media supplemented with 0.6 mol/L of mannitol. Although all the operations were performed in the dark to prevent light repairs, red light was allowed. The samples wrapped with black paper were placed in an incubator, and the temperature was set to 26 °C. Black paper was removed after 24 h. The protoplast suspension without any physical radiation was used as control. The performances of all the colonies on each screening plate were investigated after 7 days of cultivation at 26 °C. After each run, the positive mutants, which exhibited excellent and stable genetic traits, were successively cultured five times on separate agar plate PDA media. The strain that exhibited faster mycelium growth rate than that of the CK (average growth rate plus the coefficient of variation) was regarded as a positive mutant strain. The fermentation experiment on the selected mutants was used to determine the biomass yield capacity. The mycelia in submerged culture were washed with distilled water and then freeze dried in the vacuum before they were weighed.

2. Materials and methods 2.1. Strain The wild strain of P. igniarius NO.5.95 was purchased from China General Microbiological Culture Collection Center, CGMCC (Beijing, China) and grown in solid slant medium, potato dextrose agar (PDA) slant.

2.5. Extraction of endo-polysaccharides from P. igniarius

2.2. Media and Cultural Conditions

Dried mycelia (100g) ground in a disintegrator and mixed with 95% ethanol statically for 10 times overnight. The mixtures were centrifuged at 10,000g for 10 min, and the precipitate was extracted with 1000 mL of distilled water at 100 °C thrice for 2 h. The supernatant was collected through centrifugation (5000 rpm, 10 min) and then concentrated

The stock culture was maintained on potato dextrose agar (PDA) slants. The slants were incubated at 26 °C for 7 days and then stored at 26 °C. The mycelium culture prepared for the protoplast was statically 2

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2.8. FT-IR spectroscopy

through evaporation under reduced pressure. The supernatant was subsequently precipitated with 4 volumes of 95% ethanol at 4 °C overnight. The precipitate was redissolved in distilled water and then deproteinized by using a Sevag reagent (Staub, 1956), dialyzed (MWCO 8000–12,000 Da, USA) against distilled water for 48 h, and freeze dried to yield partially purified polysaccharides. The total carbohydrate content was determined through the phenol-sulfuric acid method (Qiao et al., 2009).

FT-IR spectra of the polysaccharides were determined using a Nexus 670 FTIR spectrometer (Thermo Nicolet Co., USA) in the wavenumber range from 500 to 4000 cm−1 with KBr pellets and referenced against air. 2.9. Antioxidant activity assays in vitro In this work, the hydroxyl radical scavenging activity, Trolox equivalent antioxidant capacity (TEAC) assay, and ferric reducing ability of plasma (FRAP) assay were used to evaluate the in vitro antioxidant activities of exo-polysaccharides from P. igniarius. Details of the operating conditions and methods were described as previously reports (Leung, Zhao, Ho, & Wu, 2009; Zhang et al., 2014). All treatments and activity assays were performed in triplicate, and the results were represented by their mean ± standard deviation (SD).

2.6. Antagonistic experiments and identification of isozymes The screened mutant strains and CK were inoculated into a PDA medium plate, while maintaining a distance between them. Antagonistic reactions among strains the strains were observed after 21 days. For protein extraction, the mycelium was rinsed with sterile water, and then transferred to 1.5 mL of sterile Eppendorf tubes before weighing. The fungal biomass was stored at −20 °C before use. A total of 1g of frozen mycelium from each strain was powdered in liquid nitrogen in a sterile porcelain mortar, and then mixed with 2 mL of extraction buffer (0.065 mol/L Tris-citric acid, pH 8.2). The resulting powder mixture transferred to sterile 1.5 mL Eppendorf tubes. Protein extraction was performed in a Beckman ultracentrifuge set at 10,000g, 4 °C for 10 min. The supernatant was aliquoted into sterile 1.5 mL Eppendorf tubes, which were then stored at −70 °C for isozyme analysis. Non-denatured gel electrophoresis was performed in a discontinuous system containing a vertical gel slab unit at 4 °C. The system is similar to SDS-PAGE, but the denaturing agents, such as SDS and ßmercaptoethanol, and heat treatment in a water bath were excluded (McDonald, 1997). Esterases (EST; EC 3.1.1.1) were resolved using 10% resolving gel and 4% stacking gel and buffer with 0.2% glycine and 0.62% Tris (Mohammadi, Aminipour, & Banihashemi, 2004). After the electrophoresis of proteins at 100 V for 5 h, the gel was stained according to Loxdale, Castanera, and Brookes (1983). The catalases (CAT; EC 1.11.1.6) were resolved in 7.5% separating gel and 4% stacking gel at 100 V for 3 h, and a buffer with 14.11% glycine and 3% Tris was used. After electrophoresis, the gel was stained accordingly (Wendel & Weeden, 1990).

2.10. Statistical analysis All treatments and activity assays were performed in triplicate, and the results were represented by their mean ± standard deviation (SD) and were analyzed by one way analysis of variance (ANOVA). S-N-K post hoc was applied for the differences between group means and the Duncan's multiple range test was used for means separation. SPSS 17.0 (SPSS Inc., Chicago, IL, USA) was used for all the statistical analyses. P < 0.05 was considered significantly different. 3. Results and discussion 3.1. Screening of P. igniarius strains by compound mutation In this study, the regenerative colonies of P. igniarius grown on the screening dishes after low power He-Ne laser and UV compound irradiation were selected randomly according to their characteristics. The selected regenerated colonies exhibited a faster growth rate compared with that of the control without irradiation. These colonies were round, white, and neat, and exhibited a dense texture without surrounding exudation. The colonies with excellent morphological characteristics were selected and then subcultured. After five generations of subculturing, nine strains with stable genetic traits were screened. Subsequently, the selected mutant strains and wild stain (CK) was subjected to fermentation experiment. Fig. 1 shows the dry weight of the mycelia biomass and endo-polysaccharides content. The trends are shown to be inconsistent. Compared with the CK, the screened mutant strains had increased biomass yield at different levels (p < 0.05). This result suggested that the mutation mechanism of the composite mutagenesis is complex. The mycelium dry weight and endo-polysaccharides production of the mutant JZx were 40.3% and 56.6%, respectively, which were higher than those of the CK. The maximum dry weight of the mycelium (20.7 g/L) and endo-polysaccharides concentration (1.4 g/L) can be obtained from the JZx in the flask culture. Some studies reported that the Vitreoscilla hemoglobin gene (vgb) was expressed through the chromosomal integration in P. igniarius to improve metabolite yields during submerged fermentation. In the shake flasks, the vgb expression enhanced the endo-polysaccharides production 1.33-fold to 1.18 g/L (Zhu, Sun, & Zhang, 2011). The results indicate that this novel mutagenesis method can enhance endo-polysaccharides yield effectively.

2.7. Preliminary properties and compositions of polysaccharides The total carbohydrate content, protein content and uronic acid content of polysaccharides were determined via phenol-sulfuric acid method using D-glucose as a standard, Bradford method using bovine serum albumin (BSA) as a standard, and sulfuric acid-carbazole method using glucuronic acid as a standard, respectively. The monosaccharide compositions of polysaccharides were analyzed by combination of acid hydrolysis and gas chromatography (GC) on an Agilent 7890A instrument (HP-5 fused-silica capillary column, 30 m×0.25 mm ×1 µm) with the conditions as reported before (Yan, Li, Wang, & Wu, 2010). D-Arabinose (D-Ara), D-glucose (D-Glc), D-galactose (D-Gal), Dmannose (D-Man), L-rhamnose (L-Rha), and D-xylose (D-Xyl) (SigmaAldrich Chemical Co., St. Louis, MO, USA) were used as monosaccharide standards. The molecular weight (MW) of polysaccharides was determined by high pressure gel permeation chromatography (HPGPC), which was performance on instruments with a Waters 1515 isocratic pump and a Waters 2414 refractive index detector. The detailed analysis conditions in previous report (Wang, Pei, Ma, Cai, & Yan, 2014). Dextran MW standards were used for calibration ranging from 5.2 to 1482 kDa (Sigma-Aldrich Chemical Co., St. Louis, MO, USA). All data were collected and analyzed by the online Breeze software package (Waters Corp., Milford, MA, USA).

3.2. Antagonistic experiments and identification of isozymes Mushroom hyphae from a certain source can limit the growth and proliferation of those from another source. This antagonistic reaction occurs at the middle of the growth and development stages of the strains. It can be used to determine the relationship among the tested 3

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Fig. 1. Profiles of the biomass yield of the JZx and CK after the fermentation experiment. The data points are the average from three independent experiments. The error bars indicate the standard error of each data point. The data in the columns with different letters are statistically different according to Duncan's multiple range test (p < 0.05).

Fig. 3. Zymograms of (a) esterase and (b) catalase isozymes in the CK and JZx.

with the JZx. Low power He-Ne laser and UV irradiation can change the preliminary physicochemical properties and chemical composition of the polysaccharides. The relevant results are summarized in Table 1. The protein content from the CK and JZx were negligible, indicating that the protein removal procedure was completed. The polysaccharide content from the JZx (83.24%) was higher than that from the CK (80.58%). Similarly, the uronic acids content from JZx (15.00%) was slightly higher than that of the CK (13.69%). The combination of laser and UV irradiation may promote the accumulation of polysaccharides and uronic acids. Furthermore, uronic acid content had a significant effect on the antioxidant activity of the polysaccharides (Elboutachfaiti et al., 2011). The endo-polysaccharides of JZx possibly had better antioxidant activity than the CK. Monosaccharide analysis results showed that JZx polysaccharide mainly comprised D-Glc, D-Man, and L-Rha with a molar ratio of 2.0:1.0:16.0, whereas CK contained the same monosaccharides but at a different molar ratio (9.0: 1.0: 3.0). In addition, the L-Rha content in the JZx was much higher than that of the control, whereas D-Glc content was far lower than that of the CK. This result indicated that the composite irradiation of He-Ne laser and UV caused the monosaccharide composition in the polysaccharide and reduced the D-Glc content and increased the L-Rha content significantly. Molecular weight distribution results showed that the polysaccharide from JZx contained more polysaccharide fragments with low molecular weights (1.5 kDa, 61%) compared with the control. As a result, low power He-Ne laser and UV irradiation caused changes in the molecular weight distribution of the endo-polysaccharide of P. igniarius. Our previous studies showed that lower–MW polysaccharide fractions exhibited stronger antioxidant activity (Yan, Wang, Ma, & Wang, 2016). Based on these results, we speculated that the endo-polysaccharide of the JZx have better antioxidant activity.

Fig. 2. Antagonistic experiments between the CK and JZx.

strains and identify the mutation of the strains (Gloer, 1995). In the present work, the antagonistic reaction was detected between the screened strain and CK. Fig. 2 shows the results of the antagonistic experiments between the CK and JZx strains and antagonistic reaction between the strains was evident. In addition, the growth rate of the JZx exceeded that of the CK significantly. The result indicated that the genetic material of the screened strains was altered in contrast to that of the CK strain. Esterase isozyme pattern (Fig. 3a) and hydrogen peroxide map (Fig. 3b) show that the distribution of the strips for the JZx strains were different from those of the CK strains. Furthermore, the isozyme banding increased, disappeared, or relatively displaced. Two or three of these effects occurred in the strains. The results suggested that the screened strains were mutated strains, which were identified with respect to isozymes. Isozyme analysis further confirmed that the screened strains obtained from the composite mutagenesis of laser and UV were mutants. 3.3. Preliminary physicochemical properties of polysaccharides Recently, some studies have shown that physical irradiation had an important influence on physicochemical properties and biological activities of the polysaccharides (Delattre & Vijayalaksmi, 2009). In the present study, preliminary physicochemical properties and chemical composition of the exo-polysaccharides of the CK were compared 4

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Table 1 Preliminary properties and chemical compositions of the polysaccharides extracted from the cultured P. igniarius (CK and JZx strains) mycelia. Sample

Carbohydrate (wt%)

Protein (wt%)

Uronic acid (wt%)

Sugar composition (mol%) D-Glc

D-Man

L-Rha

MW (kDa)

Area (%)

CK

80.58 ± 0.73

2.54 ± 0.60

13.69 ± 0.31

9.0

1.0

3.0

≥ 25400 3.8

68 32

JZx

83.24 ± 0.53

1.37 ± 0.28

15.00 ± 0.17

2.0

1.0

16.0

≥ 24600 1.5

39 61

The FTIR spectra of polysaccharides from the JZx and CK are presented in Fig. 4. The two samples showed a broad and strong characteristic absorption peak at approximately 3380 cm−1, and this peak can be attributed to the O-H stretching vibration. In addition, a weak absorption peak at around 2930 cm−1 was observed for the C-H stretching vibration. The absorption peaks at 1647 and 1370 cm−1 were ascribed to the absorption of the COO-deprotonated carboxylic group (Manrique & Lajolo, 2002). The results indicated the characteristic FTIR absorption of polysaccharides. The strong extensive absorption peaks of 1150, 1082, and 1036 cm−1 suggested the presence of C-O-C, C-O-H, C-C, and ring vibrations (Yang et al., 2006). The characteristic absorption peak at 931 cm−1 was associated with the β-configuration of the sugar units. The spectrum of JZx and CK were similar, thereby suggesting that no significant difference occurred in the primary chemical structure between them. Notably, the intensity of the absorption peaks at 3380 and 1036 cm−1 in the JZx were stronger than that of the CK, and this result can be ascribed to the degradation of glycosidic linkages, thus increasing the O-H and C-O groups. The laser and UV mutation caused the fracture in the glycosidic bond but did not affect the main chemical structure of the polysaccharide.

the biomolecules, such as DNA, carbohydrates, proteins, lipids in the cells. Therefore, hydroxyl radicals are effectively scavenged to protect living systems (Cheng, Ren, Li, Chang, & Chen, 2002). As shown in Fig. 5a, the hydroxyl radical scavenging effect of the endo-polysaccharides extracted from the JZx was compared with those of the Vc and CK. The scavenging capacity against hydroxyl radicals of all the polysaccharide samples depended on their concentrations to some extent. With the increase of sample concentration, the activities against hydroxyl radical were enhanced. Vc showed the strongest hydroxyl radical scavenging ability. Meanwhile, JZx exhibited higher hydroxyl radical scavenging ability than CK, but its ability was lower than that of Vc at the test dosage range of 0.05–1.5 mg/mL. The radical scavenging ability of the endo-polysaccharide from the JZx irradiated by low power He-Ne laser and UV was improved. Trolox equivalent antioxidant capacity (TEAC) assay is extensively used in food and health products because it can determine the antioxidant capacity of food, beverage, and health products (Kriengsak, Unaroj, Kevin, Luis, & David, 2006; Leung et al., 2009). Fig. 5b shows the total antioxidant capacity of the endo-polysaccharides extracted from the CK and JZx. The trend was similar with the result of the hydroxyl radical scavenging activity. The total antioxidant ability of the JZx was stronger compared with that of the CK. Its TEAC value was 195.43 trolox/g mol, which was 32.44% higher than that of the control. The endo-polysaccharides from the mycelia of the JZx have better antioxidant ability than that of the CK. Free radical formation is often involved in the oxidation, and free radical scavenging generally requires materials with reduction ability in the reaction. In this regard, ferric reducing ability of plasma (FRAP) assay is used to evaluate the antioxidant capacity of thepolysaccharides (You, Yin, Zhang, & Jiang, 2014). Fig. 5c shows the reduction ability of the endo-polysaccharides from the CK and JZx mycelia. Apparently, the JZx exhibited higher Fe3+ reduction ability compared with CK, and its FRAP value was 20.57 mol Fe2+/g, which higher by approximately 22.8% than that of the CK. Our previous study reported that the antioxidant activities of the polysaccharide fragments might be related to their chemical composition and molecular weight distribution (Zhang et al., 2014). These results indicated that the composition and preliminary structural characterization of the endo-polysaccharides might change under low power He-Ne laser and UV irradiation, and thus this condition requires further study. All the results suggested that low-power He-Ne laser and UV induction not only improve the mycelial biomass of the P. igniarius strains but also enhance the antioxidant activity and radical-scavenging capacity of the endo-polysaccharides in vitro.

3.5. Evaluation of antioxidant activities in vitro

4. Conclusions

Previous studies performed screening on the biomass but did not assess antioxidant activity (Alifano et al., 2008). In the current experiment, the in vitro antioxidant activities of the endo-polysaccharides extracted from the JZx and CK fermentation mycelia were evaluated through hydroxyl radical scavenging, TEAC, and FRAP assays. Hydroxyl radicals can easily cross cell membranes and react with

In summary, a novel and efficient physical irradiation method that combines low power He-Ne laser and UV was employed to improve endo-polysaccharides production of P. igniarius mycelial fermentation. The JZx strain was screened and selected after five generations. Its dry mycelium and endo-polysaccharide yields were 20.7 g/L and 1.4 g/L, respectively, which were 40% and 57% higher than those of the CK. Under low-power He-Ne laser and UV irradiation, the polysaccharide

Fig. 4. FTIR spectra of the JZx and CK polysaccharides.

3.4. FT-IR spectroscopy analysis

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Fig. 5. In vitro antioxidant activities of the endo-polysaccharides extracted from the JZx and CK fermentation mycelia by (a) scavenging capacity on hydroxyl radicals, (b) TEAC assay, and (c) FRAP assay. Vc = Vitamin C (or ascorbic acid). Each value is expressed as means ± SD (n =3). Medicine Chemistry, 10, 4067–4073. Clark, A. J. (1996). recA mutants of E. coli K12: A personal turning point. Bioessays, 18, 767–772. Dai, Y. C., Zhou, L. W., Cui, B. K., Chen, Y. Q., & Decock, C. (2010). Current advances in Phellinus sensu lato: Medicinal species, functions, metabolites and mechanisms. Applied Microbiology and Biotechnology, 87, 1587–1593. Delattre, C., Pierre, G., Gardarin, C., Traikia, M., Elboutachfaiti, R., Isogai, A., & Michaud, P. (2015). Antioxidant activities of a polyglucuronic acid sodium salt obtained from TEMPO-mediated oxidation of xanthan. Carbohydrate Polymers, 116, 34–41. Delattre, C., & Vijayalaksmi, M. A. (2009). Monolith enzymatic microreactor at the frontier of glycomic toward a new route for the production of bioactive oligosaccharides. Journal of Molecular Catalysis B: Enzymatic, 60, 97–105. Drímalová, E., Velebný, V., Sasinková, V., Hromádková, Z., & Ebringerová, A. (2005). Degradation of hyaluronan by ultrasonication in comparison to microwave and conventional heating. Carbohydrate Polymers, 61, 420–426. Elboutachfaiti, R., Petit, E., Pillon, M., Courtois, B., Courtois, J., & Delattre, C. (2011). Evaluation of antioxidant capacity of ulvan-like polymer obtained by regioselective oxidation of gellan exopolysaccharide. Food Chemistry, 127, 976–983. Gloer, J. B. (1995). The chemistry of fungal antagonism and defense. Canadian Journal of Botany, 73, 1265–1274. Hwang, H. J., Kim, S. W., Choi, J. W., & Yun, J. W. (2003). Production and characterization of exopolysaccharides from submerged culture of Phellinus linteus KCTC 6190. Enzyme and Microbial Technology, 33, 309–319. Ikehata, H., & Ono, T. (2011). The mechanisms of UV mutagenesis. Journal of Radiation Research, 52, 115–125. Khaliq, S., Akhtar, K., Afzal, Ghauri, M., Iqbal, R., Mukhtar, & Muddassar, M. (2009). Change in colony morphology and kinetics of tylosin production after UV and gamma irradiation mutagenesis of Streptomyces fradiae NRRL-2702. Microbiological Research, 164, 469–477. Kriengsak, T., Unaroj, B., Kevin, C., Luis, C. Z., & David, H. B. (2006). Comparison of ABTS, DPPH, FRAP, and ORAC assays activity from guava fruit extracts. Journal of Food Composition and Analysis, 19, 669–675. Leung, P. H., Zhao, S. N., Ho, K. P., & Wu, J. Y. (2009). Chemical properties and antioxidant activity of exopolysaccharides from mycelial culture of Cordyceps sinensis fungus Cs-HK1. Food Chemistry, 114, 1251–1256. Li, S. C., Yang, X. M., Ma, H. L., Yan, J. K., & Guo, D. Z. (2015). Purification, characterization and antitumor activity pf polysaccharide extracted from Phellinus igniarius mycelia. Carbohydrate Polymers, 133, 24–30. Liu, Q. N., Liu, R. S., Wang, Y. H., Mi, Z. Y., Li, D. S., Zhong, J. J., & Tang, Y. J. (2009). Fed-batch fermentation of Tuber melanosporum for the hyperproduction of mycelia and bioactive Tuber polysaccharides. Bioresource Technology, 100, 3644–3649.

from the JZx had higher carbohydrate and uronic acid contents compared with that from the CK. In addition, the JZx polysaccharides mainly comprised D-Glc, D-Man, and L-Rha with a molar ratio of 2.0:1.0:16.0 and large amount of low-MW fractions (1.5 kDa, 61%). Furthermore, FTIR spectroscopy analysis suggested that low power He-Ne laser and UV mutation caused the fracture of the glycosidic bond but did not affect the basic chemical structure of the polysaccharides. Endo-polysaccharides extracted from the JZx fermentation mycelia showed stronger radical scavenging capacities and antioxidant activities than those of the CK. Thus, the current study might provide an alternative for the enhancement of the metabolites production in other Phellinus species. The polysaccharide of JZx had stronger antioxidant activities, and thus can be explored as a novel natural antioxidant that can be used in food and medicine. This study could be the first step toward the enhancement of the understanding on the molecular mechanisms of low-power He-Ne laser- and UV-induced mutagenesis. Acknowledgements This work was supported by the National Science and Technology Support Program (2012BAD36B05), Jiangsu Province Ordinary University Innovative Research Program (CXLX11_0602). References Alifano, P., Lorusso, A., Nassisi, V., Tala, A., & Tredici, S. (2008). Application of XeCl308 nm excimer laser radiation to mutagenesis of industrial microorganisms. Radiation Effects & Defects in Solids, 163, 299–305. Chen, L. M., Xiao, J., Li, J., & Zhang, C. (2007). Research and development of Phellinus igniarius. Acta Agriculturae Jiangxi, 5, 028. Cheng, Z. Y., Ren, J., Li, Y. Z., Chang, W. B., & Chen, Z. D. (2002). Study on the multiple mechanisms underlying the reaction between hydroxyl radical and phenolic compounds by qualitative structure and activity relationship. Bioorganic &

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