Optimal Liquid Spawn Conditions and Grain Medium Enhanced Hispidin Production by Phellinus linteus in Solid-State Fermentation

This study aimed to screen a highest hispidin production of strain from 12 strains of Phellinus, and to evaluate the effects of liquid spawn conditions and grain medium on this strain’s hispidin production levels after solid-state fermentation. Results showed that the P. linteus 04 led to the highest hispidin production, this strain was then selected to elucidate the optimal liquid spawn conditions and grain medium for hispidin production. Various liquid spawn conditions were evaluated, and the highest hispidin yield, specic productivity of hispidin, and total content of hispidin were found to be optimal at 1 week of liquid spawn age, cultured with potato dextrose borth, and using 10 % inoculum rate, with each condition resulting to 0.350, 0.325, and 0.328 mg/g dry weight of mycelium, 0.352, 0.251, and 0.249 µg/mg week − 1 specic productivity, 57.90, 60.23, and 61.77 mg/kg dry weight of brown rice medium, respectively. These liquid spawn conditions were then used to determine the appropriate grain medium for hispidin production. The highest hispidin yield and total content of hispidin were observed in pearl barley which resulted in 1.107 mg/g dry weight of mycelium and 199.76 mg/kg dry weight of pearl barley, which led to results that were 4.73-fold and 5.35-fold higher than those of control (brown rice medium). Overall, this study shows that P. linteus hispidin production can be enhanced by solid-state fermentation using optimal liquid spawn conditions and the appropriate grain medium.

The fruiting body of Phellinus is scarcely found in nature; moreover it is expensive as it can be used as raw material for medicine. A long period of time is necessary to cultivate the fruiting body of Phellinus in order to obtain hispidin. Compared to submerged fermentation, the process of solid-state fermentation has led to higher yields and productivities, as well as to improve product characteristics and lower costs due to the utilization of grains or agricultural residues [20]. Recent studies have shown that some active compounds in cereals become enhanced when fermented by mushrooms [21], and that these compounds possess some biological activities such as immunomodulatory effect [22], anti-in ammatory effect [23], and the ability of prevent colon cancer metastasis [24].
Little information is available on whether or not altering liquid spawn conditions and grain media could affect hispidin production by the solid-state fermentation of Phellinus strains. Thus, in this study hispidin production of 12 strains of Phellinus were evaluated in fermented grains through solid-state fermentation; moreover, this study aimed to investigate the effects of liquid spawn conditions such as spawn age, liquid spawn culture medium, and inoculum rate, and grain media on hispidin production.

Materials
Potato dextrose agar, potato dextrose broth, yeast extract and malt extract were purchased from Himedia Laboratories (Mumbai, India). Brown rice was purchased from Wu-Wang Grain Co. Ltd. (Chiayi, Taiwan).
Millet, black rice, wheat, buckwheat, pearl barley, oat and adlay were purchased from a local market in Yuanlin (Changhua, Taiwan). Hispidin and ergosterol were purchased from Sigma-Aldrich Chemical Co. (St. Louis, MO, USA). All other chemical reagents were analytical grade.

Microorganisms
Phellinus apiahynus BCRC35468, P. igniarius BCRC 35308, P. pachyphloeus BCRC MU30300 and Phellinus sp. BCRC 36900 were purchased from the Bioresource Collection and Research Center, Food Industry Research and Development Institute, Hsinchu, Taiwan. Phellinus sp. L was isolated from the fruiting body (obtained from Nantou, Taiwan) and was identi ed by the upper Institute. P. linteus 03, P. linteus 04, P. linteus 06, P. linteus Youhao, P. linteus Wang, P. linteus 85105 and P. linteus Zhejiang were stored in our laboratories and identi ed by Tri-I Biotech. Inc., Taichung, Taiwan. The symbols and sources of these twelve strains are presented in Table 1.
Samples of each Phellinus strains were grown on potato dextrose agar (PDA, 200 g/L diced potatoes; 20 g/L glucose; 15 g/L agar) medium at 25°C for regular subculture, and were maintained on PDA slants at 4°C for a maximum of 3 months.

Liquid spawn preparation
Phellinus samples were initially grown on PDA medium at 25°C, and the mycelium was harvested after 14 days. Two mycelial agar discs (1 cm × 1 cm) were obtained using a sterilized knife and transferred into the spawn media, which contained 100 mL of PDB (PDB, 200 g/L diced potatoes; 20 g/L glucose) in 250 mL asks; then, they were incubated in an orbital shaking incubator at 120 rpm at 25°C for 1 week. The liquid products of the mycelial culture were used directly as liquid spawn.

Basal grain medium preparation, inoculation, and incubation
The basal grain medium used was brown rice, which was prepared by lling wide-neck glass bottles with 60 g of grains and 75 mL of water; these were autoclaved at 121°C for 30 min. After the brown rice medium was cooled to room temperature, these were inoculated with 6 mL of liquid spawn.
Inoculated brown rice media were kept in a spawn running room at 25°C under dark conditions. Samples were taken every week to determine hispidin and ergosterol contents during the 8 weeks of incubation.

Effect of liquid spawn conditions and grain media for hispidin production
Our previous study showed that after screening 12 Phellinus strains, P. linteus 04 led to the highest hispidin yield. Various parameters were then selected to examine the effects of liquid spawn conditions, such as spawn age, liquid spawn culture medium, inoculum rate, and grain media on the hispidin production of P. linteus 04. Each factor was analyzed individually as described below. Samples were taken every week to determine hispidin and ergosterol contents during the 8 weeks of incubation.

Effect of spawn age
To determine the optimal spawn age for hispidin production, P. linteus 04 was cultivated in brown rice media inoculated with different ages of liquid spawn (1, 2, and 3 weeks). The different ages of liquid spawn were cultured as previously described.

Effect of liquid spawn culture medium
To evaluate the best liquid spawn culture medium for hispidin production, cultured in potato dextrose broth (PDB), yeast extract glucose broth (YG, 3 g/L Yeast extract; 3 g/L malt extract; 10 g/L glucose; 5 g/L peptone), and malt extract broth (ME, 20 g/L malt extract; 20 g/L glucose; 1 g/L peptone) medium of P. linteus 04 liquid spawn for 1 week of incubation were inoculated on brown rice media, respectively.

Effect of inoculum rate
To evaluate the optimal inoculum rate for hispidin production, P. linteus 04 was cultivated in brown rice media inoculated with different volumes of inoculum: 3 (5 % of inoculum rate, v/w), 6 (10 % of inoculum rate, v/w), or 9 (15 % of inoculum rate, v/w) mL/bottle were used.

Effect of grain media
The basal brown rice grain medium was used as control, and seven different grain media (millet, black rice, wheat, buckwheat, pearl barley, oat, and adlay) were prepared by lling wide-neck glass bottles with 60 g of grains and 75 mL of water; these bottles were autoclaved at 121°C for 30 min. Grain media fermentation was carried out under the following conditions: liquid spawn was cultured for 1 week, inoculated with 6 mL of inoculum, and cultured with liquid spawn in PDB. Cultivations were maintained for 8 weeks after the liquid spawn inoculation. Samples were taken every week to determine hispidin and ergosterol contents during the 8 weeks of incubation.

Samples analysis
Fermented grain media from the different treatments were harvested and then dried at 60°C to a constant weight, after which they were ground into powder using a grinder. Sample powders were then stored in a desiccator until extract preparation.

Determination of ergosterol content
Ergosterol, a major fungal sterol, can indicate the mycelia levels in fungi [25]. In this study, ergosterol was used as a measure of the growth of Phellinus strains in grain media. Ergosterol extraction from fermented grain media was performed according to the method of Matcham et al. [26] with some modi cations. One gram of dry fermented grain powder was vortexed with 4 mL of hexane for 90 s, centrifuged at 1,310 × g for 10 min, and the supernatant (hexane phase) was transferred into a vial. The residue was further extracted twice with 4 mL of hexane. The hexane phases were collected and dried by evaporation under a vacuum at 40°C. The extract was then dissolved in 5 mL of methanol and ltered through a 0.45-μm lter prior to high-performance liquid chromatography (HPLC) analysis.
Ergosterol content was analyzed using HPLC (PU-2080 Plus, JASCO, Japan) with a Kinetex 5µ C18 100A column (Phenomenex, Torrance, CA, USA), and the ow rate of the mobile phase consisting of methanol was set to 1.2 mL/min. Detection was carried out using a refractive index detector (Shodex RI-71, Showa Denko K.K., Kawasaki, Japan). The eluent was analyzed at 282 nm using a UV/Visible Detector (Shimadzu SPA-20A; Shimadzu, Sci. Inst., Kyoto, Japan). Working solution of ergosterol was freshly prepared. Quanti cation was based on the UV signal response of ergosterol using the external standard method and a standard calibration curve were generated with six points with concentrations of 0, 25, 50, 100, 200, and 400 µg/mL.

Determination of hispidin content
Hispidin extraction from fermented grain media was conducted according to the method of Lee and Yun [27], with some modi cations. In brief, 0.2 grams of dry fermented grain powder was extracted with 10 mL 100% of methanol under an ultrasonic cleaner (DC200H, DELTA Ultrasonic Co., Ltd., Taiwan) for 60 min.
The supernatant was separated by centrifugation, which was set to 18,400 × g for 10 min; this was then ltered through a 0.45-μm lter. The ltrate was then assayed for hispidin content. To measure hispidin, the HPLC system was equipped with a Kinetex 5µ C18 100A column (250 × 4.6 mm, 5 micron, Phenomenex, Torrance, CA, USA). Methanol was used as the mobile phase, and ow rate was set at 1.2 mL/min. The eluent was analyzed at 254 nm using a UV/Visible Detector (Shimadzu SPA-20A; Shimadzu, Sci. Inst., Kyoto, Japan). The concentration of hispidin stock solution was 1000 µg/mL, and stored at -40 °C. Quanti cation was based on the UV signal response of hispidin using the external standard method and a standard calibration curve was prepared using 5-50 µg/mL of hispidin.

Statistical analysis
Data are presented as the mean ± standard deviation (n = 3). Differences between the means of individual groups were assessed using one-way ANOVA with Duncan's multiple-range test.

Results And Discussion
Screening of high hispidin production of Phellinus strain Twelve strains of Phellinus were screened for high hispidin production; the strains used were as follows: 1 strain of P. igniarius, 1 strain of P. apiahynus, 1 strain of P. pachyphloeus, 8 strains of P. linteus and 2 strains of P. sp., respectively. Our results showed that for most tested strains, higher hispidin production was observed at 6 or 8 weeks of fermentation ( Table 2). P. linteus 04 had highest hispidin production, followed by P. linteus 03 and P. linteus L, with their corresponding hispidin yields being 0.395, 0.369, and 0.354 mg/g dry weight of mycelium, respectively. However, the hispidin yields of P. linteus 04 and P. linteus 03 were not signi cantly different (P < 0.05). The highest speci c productivity of hispidin was observed in P. linteus L, followed by P. linteus 04 and P. linteus 03, with the corresponding speci c productivities being 0.396, 0.371, and 0.326 µg/mg week -1 , respectively. The highest total content of hispidin in brown rice medium was observed in P. linteus 04 (70.05 mg/kg dry weight of brown rice medium) at 6 weeks of incubation, followed by P. linteus 03 (69.65 mg/kg dry weight of brown rice medium) and P. linteus L (63.37 mg/kg dry weight of brown rice medium) at 6 weeks and 5 weeks of fermentation time, respectively (Fig. 1). The optimal strain for hispidin production was therefore P. linteus 04, due to its higher hispidin yield and total hispidin content compared to the other strains. As such, P. linteus 04 was further evaluated to determine the optimal liquid spawn conditions and grain medium for hispidin production.
In this study, twelve strains of Phellinus were screened to determine which strain led to the highest hispidin yield. P. linteus 04 was the strain with the highest hispidin production, followed by P. linteus 03 and P. linteus L. The respective hispidin yields of these three strains at their peak hispidin production time were 5.41-fold, 5.05-fold, and 4.85-fold higher than the yield of P. linteus 04 grown on liquid cultures using potato dextrose broth medium fermented for 14 days (0.073±0.005 mg/g dry weight of mycelium; data not shown on the table). According to these results, hispidin yield of P. linteus produced by solid state fermentation is higher than the yield from liquid state fermentation. All the Phellinus strains were able to grow on brown rice medium except for P. apiahynus. Mycelial color ranged from greyish white to yellow, but hispidin yields were not correlated to mycelial color, even if hispidin is a yellow pigment. P. linteus strains could produce hispidin; however, P. apiahynus, P. pachyphloeus, and P. sp. were unable to do so. Therefore, we found that not all Phellinus strains are able to produce hispidin. The result is in accordance with studies that indicate hispidin is present in the genera Phellinus and Inonotus [10,28], such as P. linteus [3,11,12,29] and I. xeranticus [12,29]. Though all P. linteus strains could produce hispidin, differences exist between different P. linteus strains. The hispidin yields of some of the tested strains of P. linteus, such as P. linteus 06, P. linteus Youhao, P. linteus Wang, and P. linteus 85105, led to yields below 0.2 mg/g dry weight of mycelium; no prior studies have reported about these results. At 8 weeks of fermentation, hispidin yield of the hispidin-producing strains were gradually enhanced as fermentation time increased; however, when hispidin yield reached its maximum level, it then rapidly dropped for most of the tested strains. Our results were similar to those of Park et al. [11] who reported that the highest P. linteus hispidin yield using liquid state fermentation (2.5 mg/mL) was observed at 10 days of incubation, and then decreased to near 0 mg/mL at 18 days of incubation. This showed that hispidin might be polymerized by the enzymatic oxidative coupling reaction after the 12th day [11].
Effect of spawn age Fig. 2 showed that three different spawn ages in uenced mycelial growth and hispidin production in brown rice fermented by P. linteus 04 during fermentation. The highest hispidin yield, speci c productivity of hispidin, and total content of hispidin were observed at 1 week of liquid spawn age at 6 weeks of fermentation time which indicated signi cantly higher (P <0.05) than those of 2 and 3 weeks of liquid spawn age, with the corresponding results being 0.350 mg/g dry weight of mycelium, 0.352 µg/mg week -1 speci c productivity, and 57.90 mg/kg dry weight of brown rice medium, respectively. These were followed by the use of 2 and 3 weeks of liquid spawn age together with 6 weeks of fermentation, with the corresponding results for hispidin yield being 0.219 and 0.209 mg/g dry weight of mycelium, speci c productivity at 0.215 and 0.199 µg/mg week -1 , and total hispidin content at 37.26 and 36.63 mg/kg dry weight, respectively.
Effect of liquid spawn culture medium Fig. 3 showed that liquid spawn grown in three different culture media in uenced mycelial growth and hispidin production in brown rice fermented by P. linteus 04 during fermentation. The highest hispidin yield, speci c productivity of hispidin, and total hispidin content were observed in cultures with liquid spawn grown in potato dextrose broth at 7 weeks of fermentation, which resulted in 0.325 mg/g dry weight of mycelium, 0.251 µg/mg week -1 speci c productivity, and 60.23 mg/kg dry weight of brown rice medium. This was followed by cultures with liquid spawn grown in yeast extract glucose broth at 8 weeks of fermentation, resulting in 0.238 mg/g dry weight of mycelium, 0.167 µg/mg week -1 speci c productivity, and 42.42 mg/kg dry weight of brown rice medium. The third highest was observed in cultures with liquid spawn grown in malt extract broth at 7 weeks of fermentation, resulting in 0.191 mg/g dry weight of mycelium, 0.165 µg/mg week -1 speci c productivity, and 31.56 mg/kg dry weight of brown rice medium.
Effect of inoculum rate Fig. 4 showed that three different liquid spawn inoculum rates in uenced mycelial growth and hispidin production in brown rice fermented by P. linteus 04 during fermentation. The highest hispidin yield, speci c productivity of hispidin, and total hispidin content were observed at an inoculum rate of 10 % at 7 weeks of fermentation which indicated signi cantly higher (P <0.05) than those of inoculum rate of 5 and 15 %, resulting in 0.328 mg/g dry weight of mycelium, 0.249 µg/mg week -1 speci c productivity, and 61.77 mg/kg dry weight of brown rice medium. This was followed by an inoculum rate of 5 % at 7 weeks of fermentation, which led to 0.219 mg/g dry weight of mycelium, 0.168 µg/mg week -1 speci c productivity, and 40.70 mg/kg dry weight of brown rice medium. Third highest was the use of 15 % inoculum rate at 7 weeks of fermentation, resulting in 0.149 mg/g dry weight of mycelium, 0.106 µg/mg week -1 speci c productivity, and 29.96 mg/kg dry weight of brown rice medium.

Effect of grain media
According to our results, optimal liquid spawn conditions are as follows: 1 week of liquid spawn age, culturing in potato dextrose broth, and the use of 10 % inoculum rate. These liquid spawn conditions were then used to determine an appropriate grain medium for hispidin production. Fig. 5 shows mycelial growth and hispidin production using different types of grain media fermented by P. linteus 04. Eight different grain media were tested, and brown rice medium was used as a control. The medium that led to the highest hispidin yield, speci c productivity of hispidin, and total hispidin content (Table 3) was pearl barley medium at 6 weeks of fermentation, resulting in 1.107 mg/g dry weight of mycelium, 1.022 µg/mg week -1 speci c productivity, and 199.76 mg/kg dry weight of pearl barley medium. This is followed by black rice medium at 6 weeks of fermentation, resulting in 0.819 mg/g dry weight of mycelium, 1.236 µg/mg week -1 speci c productivity, and 90.43 mg/kg dry weight of black rice medium. Third was adlay medium at 7 weeks of fermentation, resulting in 0.815 mg/g dry weight of mycelium, 0.644 µg/mg week -1 speci c productivity, and 147.28 mg/kg dry weight of adlay medium.
The highest hispidin yield, speci c productivity of hispidin, and total hispidin content for grain media fermented by P. linteus 04 was observed in the pearl barley medium, which led to results that were 4.73fold, 4.17-fold, and 5.35-fold higher than those of control (brown rice medium), respectively. This was followed by black rice medium, with results that were 3.50-fold, 5.04-fold, and 2.42-fold higher than those of control. Adlay medium was third highest, with results that were 3.48-fold, 2.63-fold, and 3.95fold higher than those of control, respectively. This shows that these grain media are appropriate for hispidin production via P. linteus 04 fermentation. Notably, hispidin yield, speci c productivity of hispidin, and total hispidin content reached their maximum levels at 6 weeks of fermentation in the pearl barley medium. Although hispidin yield decreased to 0.599 mg/g dry weight of mycelium and the speci c productivity of hispidin decreased to 0.437 µg/mg week -1 at 7 weeks of fermentation, total hispidin content was found to be 117.3 mg/kg dry weight of the pearl barley medium, which is higher than that of brown rice, millet, black rice, wheat, buckwheat, and oat media (Table 3). These indicate that pearl barley is an appropriate alternative medium as it may steadily induce hispidin production. Our results are in accordance with the work of Martins et al. [20], which stated that changes in phenolic acid pro les due to fermentation strongly depend on the type of substrate, fungus used, and fermentation conditions. Some grains fermented by P. linteus exhibit various biological activities. Germinated brown rice fermented by P. linteus was reported to possess immunomodulatory activity [22], anti-in ammatory activity [23], and the ability to suppress metastasis and induce apoptosis of colon cancer cells [24]. Adlay and rice fermented by P. linteus could signi cantly enhance the total content of ascorbic acid, polyphenols, and avonoids when extracted using hot water [30] (Liang et al., 2009). In our study, grains fermented by P. linteus could enhance total hispidin content. We also demonstrated that solid state fermentation can potentially improve the biological functionalities of compounds in various grains [20]. Therefore, whether or not P. linteus-fermented grain products containing hispidin possess improved biological activity is worth to investigating in the future.

Conclusion
In this study, we screened several Phellinus strains and found that P. linteus 04 is a strain that exhibited a high hispidin yields, hispidin speci c productivity, and total hispidin content when fermented in brown rice. We also concluded that optimal P. linteus 04 hispidin production conditions were as follows: 1 week of liquid spawn age, the use of potato dextrose broth inoculation medium, and the use of 10 % inoculum rate. Moreover, using these liquid spawn conditions, 8 grains were investigated to nd an alternative medium for hispidin production, and our results showed that pearl barley serves as an appropriate alternative medium.   Hispidin production in fermented brown rice by different Phellinus strains during fermentation. Liquid spawn conditions and grain medium were as follows. Liquid spawn culture medium: potato dextrose broth; liquid spawn age: 1 week; water content of grain medium: 55%; spawn rate: 10 % (v/w); grain medium: brown rice.

Figure 2
Effect of three different spawn ages in uenced mycelial growth and hispidin production in brown rice fermented by Phellinus linteus 04 during fermentation. Liquid spawn conditions and grain medium were as follows. Liquid spawn culture medium: potato dextrose broth; water content of grain medium: 55%; spawn rate: 10 % (v/w); grain medium: brown rice.

Figure 4
Effect of three different liquid spawn inoculum rates in uenced mycelial growth and hispidin production in brown rice fermented by Phellinus linteus 04 during fermentation. Liquid spawn conditions and grain medium were as follows. Liquid spawn culture medium: potato dextrose broth; liquid spawn age: 1 week; water content of grain medium: 55%; grain medium: brown rice.