The culture conditions on the mycelium, antioxidant cordyceps militaris

The Culture Conditions on the Mycelium Growth, Antioxidant Capacity of Cordyceps militaris (strains AG-1 and PSJ-1) Dang Ngoc Hung PhD1 , Chun – Li Wang2 *and Liang Horng Lay3 1

Department of Tropical Agriculture and International Cooperation, National Pingtung University of Science and Technology, Pingtung, Taiwan R.O.C 91201

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Department of Plant Industry, National Pingtung University of Science and Technology, Pingtung, Taiwan R.O.C 91201

The address for the first author: Mobile phone number: (+886)-978705467 The e-mail address: hungtuaf@gmail.com The address for the corresponding author: Telephone number: (+886-8) 770 3202 # 6341 Mobile phone number: (+886)-986 058 385 Fax number: (+886-8) 774 0362 The e-mail address: lelia@mail.npust.edu.tw The address for the third author: Telephone number: (+886-8) 7703202#6345, 6055 Mobile phone number: (+886) - 935.262.546 Fax number: 886-8-7740415, 7740158 The e-mail address: layhl@mail.npust.edu.tw

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Department of tropical Agriculture and International Cooperation, National Pingtung University of Science and Technology, Pingtung, 91201, Taiwan, The e-mail address: hungtuaf@gmail.com 2* Department of Plant Industry, National Pingtung University of Science and Technology, Pingtung 91201, Taiwan. Tel +886.986058385; Fax: 08 774 0362; The e-mail address: lelia@mail.npust.edu.tw 3 Department of Plant Industry, National Pingtung University of Science and Technology, Pingtung 91201, Taiwan Tel +886.935262546; Fax: 886-8-7740415, 7740158; The e-mail address: layhl@mail.npust.edu.tw

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Abstract Effects of culture conditions on the growth and antioxidant capacity assessment of Cordyceps militaris mycelium strains AG-1 and PSJ-1 were carried out in laboratory during the Autumn-Winter season of 2015. Different temperature regimes and nutrition, vitamins levels in substrate were evaluated for optimization on mycelia growth. The results showed that two strains AG-1 and PSJ-1 can grow the maximum mycelium radial at the temperature of 24 0 C. Mycelium growth was improved by carbon sources such as glucose, dextrose, and maltose at 3% concentration, mycelium colony diameter achieved the highest value. Whereas glucose, dextrose, and sucrose as carbon sources gave the good mycelium growth of strains AG-1, and at 3 % sucrose concentration, mycelium colony diameter of AG1 achieved the maximum value. Vitamins (B1, B3, B6, and B8) concentrations at 0.03 g/L also gave the greatest values in mycelium colony diameter of both strains AG-1 and PSJ-1. The studies depicted that a substrate mixed with different grain sources (rice, corn; wheat) cultured at 24 0 C, for 12 days supported maximum mycelia growth of the two strains AG-1 and PSJ-1 of C. militaris. The effects of culture media on the anti-oxidation activity of mycelia by freeze drying method were assessed by measuring the DPPH radical scavenging activity and Fe 2+ -chelating ability. A proportional variation in DPPH radical scavenging activity and Fe2+ -chelating ability was observed. The results showed that the cultured Cordyceps mycelia had equally strong antioxidant capacity as compared between two strains AG-1 and PSJ-1.

Keywords: Cordyceps militaris; mycelium growth; nutritional condition; temperature; vitamins; antioxidant 1. Introduction The Cordyceps sinensis species, which are known as one of the Chinese medicinal mushroom, are entomopathogenic fungi belonging to Clavicipitaceae and Ascomycotina. They were generally called “Tochukaso” in Japanese, meaning “winter-insect and summer-plant” because of their following behavior: (i) their spores enter into a specific living insect; (ii) they kill the host by feeding on it, and their hyphae grow in it; (iii) they pass the winter inside the host and eventually form fruiting bodies on the surface of the host insect’s cadaver in summer. Cordycepin produced from Cordyceps sinensis has long been used as tonics and stimulants to enhance energy, which exhibited a potential for energy metabolism. Researchers had reported that different culture conditions had notable effects on the yield of mycelium and metabolic substance (Panda & Swain, 2011), (Kim et al., 2005). To our knowledge, most of the fermentation of C. militaris had used biochemical components as the culture medium, and the extra cellular metabolic substances had been taken lots of attention in the researches Cordyceps mushrooms have been used to treat conditions including respiration and pulmonary diseases, renal, liver, cardiovascular diseases, hyposexuality and hyperlipidemia. It is also used in the treatment of immune disorders, and as an adjunct to modern cancer therapies (Das et al., 2010b). At the same time, it is well known that natural medium contains more nutrition for the growth of microorganisms (Lu et al., 2008) and the complex components in the natural medium would make it difficult to purify the metabolic substances in the culture broth. Therefore, the purpose of this research were to study the effects of culture conditions including temperature, media, vitamin, and grains (rice, corn; wheat) on the growth of Cordyceps militaris mycelium 2. Materials and methods The experiments were conducted in Plant Physiology and Value Added Microorganisms Laboratory, Department of Plant Industry, National Pingtung University of Science and Technology (NPUST) in Taiwan during the Autumn-Winter season of 2015 (September to Juan, 2016).

2.1. Sources of materials Two strains of C. militaris (strains AG-1 and PSJ-1) were obtained from Plant Physiology and Value Added Microorganisms Laboratory, Department of Plant Industry, NPUST in Taiwan and maintained in maltose yeast extract peptone sucrose medium (MYPS) (Lübenau, 1906), (Mossel. et al., 1967). The different grains (brown rice purchased in Taiwan; white rice, black glutinous rice Vietnam; wheat; corn-for obtained from the different sources such as a local market in North Vietnam. The campus of National Pingtung University of Science and Technology, some were bought from merchant in 2

Pingtung county, except the black glutinous rice mountains was imported from Vietnam. Carbon sources (glucose, dextrose, fructose, maltose), vitamin B source (thiamine-HCl (B1,Sigma), nicotinic acid (B3, Sigma), pyridoxine-HCl (B6, Sigma), myo-inositol (B8, Sigma)). The different chemical and instruments used were obtained for different companies in Taiwan and some were imported from Japan and Germany, United States.

2.2. Experiment 1: Several trials were designed to evaluated on the mycelium growth Several trials were designed to evaluate factors affecting mycelium growth of C. militaris two strains AG-1 and PSJ-1. For each test, each treatment will be conduct 5 replications; mycelium discs with similar diameter (1 cm) were used. The discs were obtained by using the sterilized punch-hole tool to cut the mycelium and transplant to a new sterilized medium plate. The diameter of the mycelium extension was measured every 3 days and surface mycelium density was observed.

2.2.1. Culture media Three media were used including: (1) MYPS: 4 g maltose, 6 g peptone, 4 g yeast extract, 10 g sucrose, 20 g agar, and 1,000 mL distilled water; (2) C-DOX: 30 g Sucrose, 2 g NaNO3 , 1 g KH2 PO 4 , 0.5 g MgSO 4 .7H2 O, 0.5 g KCl, 20 g agar powder, and 1,000 mL distilled water. (3) PDA: 200 g potatoes, 20 g dextrose, 30 g Sucrose, 20 g agar powder, and 1,000 mL distilled water. Placed in the center all of media adjusted pH 7 with 1 N NaOH or HCl. For each test, each treatment will be conduct 5 replications. The mycelium discs (1 cm diameter) of each C. militaris two strains AG-1 and PSJ-1 were placed in Petri dishes containing each culture medium (20 mL) under aseptic condition and incubated at 24 0 C in darkness. The diameter of the mycelium expansion was measured every 3 days for 12 days.

2.2.1. Temperature The Petri dishes containing MYPS medium (20 mL) were inoculated with C. militaris mycelium discs and incubated in the dark at six levels of temperature (16, 20, 24, 28, 32, and 36 0 C). For each test, each treatment will be conduct 5 replications. The diameter of the mycelium expansion was measured every 3 days for 12 days.

2.2.3. Different carbon sources Basic MYPS medium containing different carbon sources (glucose, dextrose, fructose and maltose) at 3 % concentration were used for the experiment. MYPS medium was used as control treatment. For each test, each treatment will be conduct 5 replications. The mycelium discs (1 cm diameter) of each C. militaris two strains AG-1 and PSJ-1were placed in medium plates (20 mL medium) incubated at 24 0 C in darkness. The diameter of the mycelium expansion was measured every 3 days for 12 days and the colony morphology was observed and recorded.

2.2.4. Different vitamins B sources and concentrations Thiamine-HCl (B1), Nicotinic acid (B3), Pyridoxine-HCl (B6), Myo-inositol (B8) at 0.03 g/L concentration was used. MYPS was used as the control treatment. The mycelium discs (1 cm diameter) of each C. militaris in two strains AG-1 and PSJ-1were placed in medium plates (20 mL medium) and incubated at 24 0 C in darkness. For each test, each treatment will be conduct 5 replications. The diameter of the mycelium expansion was measured every 3 days for 12 days.

2.2.5. Different grain sources The study used grain sources including brown rice, white rice, and wheat, black glutinous rice Vietnam, and corn grain. They were washed in clean water three times to remove chaff, dust, and other particles, then soaked in water for 6 hr for maximum absorption of water. Soaked grains were again washed and cooked by using autoclave at 121 0 C for 10 min. After cooling down to room temperature, each grain was supplemented with 3 % potatoes, 1 % maltose, 2 % KH2 PO4 , 1.5 % MgSO 4 .7H2 O, 0.02 % vitamin thiamine-HCl (B1) and 1,000-mL distilled water. Supplemented grains were filled into spawn Petri dishes with 30 g grain/petri dish and sterilized by autoclaved at 121 0 C, 1.3 kg/cm2 for 2 hr.

After cooling, each petri dish was inoculated with 1 cm mycelium disc of each C. militaris two strains AG-1 and PSJ-1and was incubated at 24 0 C under dark conditions. For each test, each treatment will be conduct 5 replications. The diameter of the mycelium expansion was measured every 3 days for 12 days.

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2.3. Experiment 2: The measurement of antioxidant capacity Two different liquid culture medium (MYPS and PD) were used to evaluate the antioxidant capacity Different liquid culture medium (MYPS, PD) were used including: (1) MYPS: 4 g maltose, 6 g peptone, 4 g yeast extract, 30 g sucrose, 0.03 g B1 powder, and 1,000 mL distilled water; (2) PDA: 200 g potatoes, 20 g dextrose, 30 g Sucrose, 0.03 g B1 powder, and 1,000-mL distilled. The prepared different shake liquid medium (MYPS, PD) were adjusted to pH 7.0 and divided into bottles of 500 ml, each filled with 200 ml liquid medium, then sterilized in an autoclave at 121 °C, 1.3 kg/cm2 for 1 hr.

The liquid media were cultured under dark condition at 24 °C for 48 h and then two strains were in vibration (shake culture) under the light room condition for 25 days at temperature of 24 0 C with a speed of 100 rpm.

2.3.1. Sample preparation and extraction for antioxidant The culture mycelium of each C. militaris two strains AG-1 and PSJ-1were dried by freeze drying method (-40 o C) for 48 hr. One g of dried mushroom samples was extracted by using reflux extraction method with 100 mL of methanol at 74 o C for 2 hr extraction. After sample were extracts and then filtered through Whatman No. 2 filter paper. The residues were then extracted with one additional 100 mL of methanol. The combined extracts were then evaporated using a vacuum concentrator (Heidolph, Laborta 4000) at 40 0 C to dryness. The dried extracts thus obtained were redissolved in methanol to a concentration of 100 mg/mL and stored at 4 0 C prior to analyses of antioxidant attributes.

2.3.2. Scavenging ability on 1.1- deiphenyl-2-picrylhydrazyl (DPPH) radical Each 4mL of extract of different concentrations (0.5, 1, 2.5, 5, 7.5, and 10 mg/mL) in methanol was mixed with 1 mL of methanol solution containing DPPH (Sigma) resulting in a final concentration of 0.001 M DPPH. The mixture was shaken vigorously and left to stand for 30 min in the dark, and the absorbance was measured at 517 nm against a control butylated hydroxyanisole (BHA) (Shimada et al., 1992.) is used for comparison with the samples. The control sample was also prepared as above without any extract and methanol and was used for the baseline correlation. The concentration in μg/mL required to scavenge 50% of the radical inhibitory concentration 50 (IC 50 ) was obtained from the regression equation. The scavenging ability on DPPH radicals were calculated as the inhibition percentage according to the formula: Scavenging ability (%) = (1- [Absorbance (Sample) / Absorbance (Blank)]) x 100 2.3.3. Chelating ability on ferrous ions Chelating ability was determined according to the method by (Dinis et al., 1994). Each 1 mL of extract of different concentration (0.5, 1.0, 2.5, 5.0, 7.5, and 10 mg/mL) in methanol was mixed with 3.7 ml of methanol and 0.1mL of 2 mM ferrous chloride (FeCl2 .4H2 O). The reaction is initiated by the addition of 0.2 mL of 5 mM ferrozine (Sigma). After 10 min at room temperature, the absorbance of mixture was determined at 562 nm against a control. Ethylennediaminetetraacetic acid is used for comparison with the samples. The control sample was also prepared as above without methanol was used for the baseline correlation. Calculated and the regression equation was obtained from which inhibitory concentration 50 (IC 50 ) was calculated. The chelating ability on ferrous ions is calculated according to the formula: Chelating ability (%) = (1- [Absorbance (Sample) / Absorbance (Blank)]) x 100 2.4. Statistical analysis One-way analysis of variance (ANOVA) was conducted with Duncan’s multiple range tests to compare the mean significant differences (p< 0.05) among treatments by using computer software SAS version 9.1 (SAS Institute Inc., Cary, NC, USA). Each value is expressed as mean ± SE (n=5).

3. RESULTS AND DISCUSSION 3.1. Effect of different culture media on the mycelium growth During the investigation, different media, i.e., MYPS, C-DOX, and PDA were used to identify their effect on mycelium growth. The data (Table 1) showed that the observed mycelium diameter of strains AG-1 on three different media significantly differed (p< 0.05) while the mycelium colony diameter of strains PSJ-1 on three media did not significantly differ. Mycelium densities of both C. militaris two 4

strains AG-1 and PSJ-1were compact in all media. The average mycelium diameter of the two strains ranged from 4.90 ± 0.18 (PSJ-1) to 5.78 ± 0.12 cm (AG-1) at 12 days after inoculation. The mycelium growth was different strains AG-1 on MYPS media was better than those on C-DOX, PDA media in 12 days. The mycelium growth levels in different culture media can be ranked as MYPS-AG-1>PDA AG1>C-DOX-AG-1; MYPS-PSJ-1>PDA-PSJ-1>C-DOX-PSJ-1 at longer days range (9 to 12 days). The mycelium growth was similar at 12 day for PSJ-1 but it was different for AG-1 (Fig 1). The results indicated that cultural media are suitable for mycelium growth of two strains AG-1 and PSJ-1, while MYPS media are more suitable for the mycelium growth of strains AG-1. The results of the present research showed that the mycelium of strains AG-1 took a longer time, but in the case of strains PSJ-1, the mycelium took longer time to complete growth in all media (data not shown) (Shim et al., 2005).

When assessed twenty two different types of media and categorized into poor and rich media in nutritional sources by (Shrestha et al., 2006) reported a similar result that PDA and C-DOX developed abundant mycelial density. He suggested that nutritionally rich media produced abundant mycelial growth and this may be because of the sufficiency of all the requirements for the vegetative growth in C. militaris. Similarly, the type of PDA and C-DOX media had no viable results in dark conditions, because no fruiting body was formed in darkness in C. militaris (Sato & Shimazu, 2002). From those results, we chose MYPS media to prepare for next culture of two strains AG-1 and PSJ-1.

3.2. Effect of temperature on mycelium growth of C. militaris two strains AG-1 and PSJ-1 The optimum temperature for mycelium growth of C. militaris in two strains AG-1 and PSJ-1 were similar, where the appropriate temperature for both AG-1 and PSJ-1 was found at 24 0 C followed by 20 0 C and 28 0 C (Fig 2). At 12 days, result was found: AG-1 at 24 0 C (5.76±0.11 cm; 5.37±0.13 cm, 4.85±0.05 cm, 4.6±0.12 cm, 3.85±0.11 cm diameter), and PSJ-1 at 24 0 C (4.90±0.18 cm, 4.6±0.12 cm, 4.32±0.21 cm, 3.80±0.07 cm, 3.36±0.15 cm diameter) for: 24 0 C, 20 0 C, 28 0 C, 16 0 C, 32 0 C and respectively, where the various temperatures can be ranked as: 24 0 C > 20 0 C >28 0 C >16 0 C >32 0 C. At 9 to 12 days, the mycelium growth of strains AG-1 was significantly faster than that of strains PSJ-1 at each temperature tested and PSJ-1 did not seem to grow after 12 days inoculation at 32 0 C (Table 2).

The mycelium density of strains PSJ-1 was very small and thin at 16 and 32 0 C. These findings are similar to those of a study conducted by (Sung et al., 2002) reported that the mycelia growth of C. militaris strains was optimal at 25 0 C. Although the mycelial growth was favorable at the range of 20 0 C -25 0 C, the fungus appeared to be suppressed at the high temperature of 35 0 C. The optimal temperature of 20 0 C and 24 0 C is also reported by (Park et al., 2001), (Kim & Yun, 2005), (Kim et al., 2003), (Hwang et al., 2003),. From those results, we chose 24 0 to for next culture experiments.

3.3. Effect of carbon sources on mycelium growth of C. militaris Carbohydrate plays an important role as the structural compound and stored in the cell. In order to find the optimum carbon, carbon sources including glucose, dextrose, fructose, maltose (3 % concentration), each type of carbon were used added into the basal medium (MYPS medium) at different experiments. In Fig 3 and the result showed that there was a significant difference in mycelium growth of strains AG-1 and PSJ-1 grown on various carbon sources. Amongst four carbon sources tested, glucose, dextrose were favorable to the mycelium growth (mycelium colony diameter and mycelium density) of strains AG-1, while glucose, dextrose also gave good mycelium growth of strains PSJ-1. In Table 3, nine days the highest mycelium colony diameters of strains AG-1 were obtained from the media containing glucose (5.56 ± 0.06 cm), dextrose (5.32 ± 0.05 cm), and maltose (5.11 ± 0.07 cm), while the highest mycelium colony diameters of strains PSJ-1 were recorded from the media containing glucose (4.89 ± 0.07 cm), dextrose (4.70 ± 0.12 cm) and maltose (4.25 ± 0.10 cm) (Mao et al., 2005b). Glucose was an optimal carbon source for cordycepin production of Chinese traditional medicinal mushroom C. militaris. Moreover, glucose was also identified as the best carbon source for cordycepin production of Chinese traditional medicinal mushrooms C. militaris such as (Jayasinghe et al., 2008; Jeong et al., 2005). Among good carbon sources (glucose, dextrose, fructose, and maltose) was used in the study for strains AG-1 and PSJ-1. Therein glucose gave significantly highest mycelium colony diameter for both strains AG-1 and PSJ-1. It was chosen as carbon source in the following test. The results (Tuli et al., 2014 ) are consistent with earlier reports in which maximum 5

yield of cordycepin was obtained in the range of 4 to 6 % dextrose as carbon source from various strains of C. militaris (Mao et al., 2005a). However (Das et al., 2010a) reported that investigated the optimum production of cordycepin from mutated strain of C. militaris at a concentration of 8.62 % dextrose.

3.4. Effect of vitamins sources on mycelium growth of C. militaris When various vitamins were added to the MYPS medium, thiamine HCl (B1) and Nicotinic acid (B3), Piridoxine (B6); Myo inositol (B8) produced excellent growth of the mycelium of C. militaris two strains AG-1 and PSJ-1. After 12 days of cultivation, the diameter of two strains AG-1 and PSJ-1 growth in thiamine-HCl (B1) and piridoxine (B6) were AG-1 (7.25±0.05 cm); PSJ-1 (6.00±0.03 cm) and AG-1 (6.53±0.05 cm); PSJ-1 (6.04±0.10 cm) (Table 4 and Fig 4), respectively.(Wen et al., 2011) (Ting-chi Wen et al., 2014) reported that the optimum culture vitamins of C. militaris CGMCC2459 were thiamine-HCl (B1) and Acid folic (B9). Among good vitamins sources for strains AG-1 and strains PSJ-1. Thiamine-HCl (B1) gave the significantly highest mycelium colony diameter for both strains AG-1 and strains PSJ-1. It was chosen as the carbon source in the following test. Nevertheless, mycelium growth high of the two strains AG-1 and PSJ-1 at concentrations (more than 0.03 g/L for strains AG-1 and PSJ-1) were unable to increase mycelium colony diameter.

3.5. Effect of grain sources on mycelium growth of C. militaris In this study, five different grains including brown rice, white rice, black glutinous rice Vietnam; wheat and corn were used to indicate their effects on spawn production of C. militaris two strains AG-1 and PSJ-1. The results indicated that mycelium extensions of two strains AG-1 and PSJ-1 was significantly slower than that of control treatments. When compared to mycelium extension of strains PSJ-1, the mycelium extension of strains AG-1 was higher. Mycelium densities of both strains AG-1 and PSJ-1 on grain media were compact and somewhat compact. Brown rice was found to be the most favorable to the mycelium growth of AG-1 and PSJ-1. The highest mycelium colony diameters of AG1 and PSJ-1 obtained in black glutinous rice Vietnam medium were 8.01 ± 0.04 cm and 7.10 ± 0.08 cm, respectively. Brown rice, white rice and corn were next to wheat with mean values for mycelium strains diameter of AG-1 being 7.24 ± 0.06 cm; 6.41 ± 0.04 cm and 6.09 ±0.08 cm; 5.90±0.05 cm, respectively. Those values of PSJ-1were 6.35 ± 0.03 cm; 5.79 ± 0.08 cm and 5.64 ± 0.08 cm, 5.20 ± 0.08 cm respectively (Table 5). This may be due to millet grain size being very small when compared to brown rice, wheat, yellow corn. (Tinoco et al., 2001) found that larger surface and pore of substrates were better supports for mycelium growth rate. This could account for the results recorded by brown rice, wheat, corn and glutinous rice. (Sofi et al., 2014; Ting-chi Wen et al., 2014) reported that mycelium colony diameter of C. militaris CGMCC2459 in various grains was significantly affected by substrate style. The authors also indicated that the larger surface area and spore of substrates are responsible for the more mycelium growth rate.

3.6. Scavenging ability on DPPH radicals Due to the differences in the theoretical bases of differences antioxidant measurements, single antioxidant property model can hardly reflect the antioxidant capacity of samples. In this study, two antioxidant models were used to reflect the antioxidant capacities of C. militaris two strains AG-1 and PSJ-1. DPPH, a relatively stable free radical, has been widely used to examine the free radicalscavenging ability. When DPPH radicals are reduced in the model system with an antioxidant compound, the color violet are measured at 517nm. Consequently, the antioxidant activities of both strains shake liquid medium culture methods are given in Fig 5. At 10 mg/mL, the DPPH scavenging activities were 87.91±0.14 %; 80.37 ±0.36 %; 85.31 ±0.11 %; 83.35 ±0.25 % for MYPS-AG-1, MYPSPSJ-1, PDA-AG-1, PDA-PSJ1 and respectively, while the activity can be ranked as MYPS-AG1>MYPS-PSJ-1; PDA-AG-1>PDA-PSJ1. At higher concentration range (5.0 to 10 mg/ml), fractions showed no significant differences. The antioxidant capacity of aqueous extracts from different media both strains were determined by DPPH. The results were presented in Tab 6. DPPH is a relatively stable free radical and has a maximum absorbance at 517 in methanol, but when it encounters hydrogen-donating components, the absorbance will be reduced rapidly (Chattopadhyay et al., 2010) So, the scavenging activities of MYPS-AG-1 and PDA-AG-1 increased significantly with increasing 6

concentrations. Consequently, these results clearly demonstrated that all the sample culture medium possessed DPPH radical scavenging activities, especially the culture medium of MYPS-AG-1 and PDA-AG-1 showed strong activities.

3.7. Chelating effect on ferrous ions culture medium and methanol extracts.

Chelating ferrous ions capacity of methanol extracts from two strains AG-1 and PSJ-1 mycelium grown in different substrates increased as concentration increased from 0.5 mg/mL to 10 mg/mL (Fig 6). Chelating effects of both strains AG-1 and PSJ-1 extracts were more than 71.14-81.88% at 7.5 mg/mL concentration in both cases of liquid culture medium of two strains AG-1 and PSJ-1. In the Table 6, different substrates gave significant difference (P<0.05) in chelating ability of strains AG-1 and PSJ-1. At MYPS–AG-1 10 mg/mL concentration, the chelating abilities of strain AG-1 of shake culture medium (MYPS-1 and PSJ-1) and culture medium achieved the highest values which ranged from 86.53 ± 0.43 % to 80.95 ± 0.41 % (MYPS-AG-1 and MYPS-PSJ-1) and 84.20 ± 0.34 % to 80.48 ±0.07 % (PDA-AG-1 and PDA-PSJ-1), respectively (. Those of strains PSJ-1 also had the highest values at the concentration of 10 mg/mL with the range from 84.20 ±0.34 % to 80.48 ±0.07 % (shake liquid culture medium). Respectively, reported that the estimated IC50 of various Cordyceps in the antioxidation assays were ranged from 0.08 to 5 mg/ml. These values are in line with the dosage of Cordyceps during medication, which ranges from 1 to 10 g per dose (Zhu et al., 1998). The lowest value of Chelating ability with the highest IC 50 of mushroom PSJ-1 at was recorded at substrate MYPSPSJ-1 and PDA-PSJ-1 Acknowledgments We gratefully acknowledged the Plant Physiology and Value Added Microorganisms Laboratory (two strains AG-1 and PSJ-1 of C.militaris), in The Department of Plant Industry, National Pingtung University of Science and Technology, Pingtung, Taiwan R.O.C 91201, for financial support.

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Table 1.Effect of different culture media on the mycelium growth of Cordyceps militaris two strains AG -1 and PSJ-1 Mycelium colony diameter (cm) Culture 3 days 6 days 9 days 12 days media AG-1 PSJ-1 AG-1 PSJ-1 AG-1 PSJ-1 AG-1 PSJ-1

Mycelium density AG-1

PSJ-1

MYPS 2.14±0.11a 1.97±0.06a 3.72±0.12a 3.61±0.04a 4.98±0.04a 4.49±0.05a 5.78±0.12a 4.90±0.18a C C C-DOX 1.37±0.05c 1.2±0.07b 2.6±0.06c 2.17±0.11b 3.26±0.19c 3.11±0.07c 4.18±0.12c 3.94±0.02a C C PDA 1.86±0.08b 1.36±0.07b 2.98±0.02b 2.38±0.06b 3.7±0.07b 3.78±0.05b 4.84±0.05b 4.52±0.07a C C Means within the same column followed by the same letters are not significantly different at p< 0.05. Each value is expressed as mean ± standard error (SE) (n=5). Days, day after inoculation; C, compact. MYPS, Maltose yeast extracts peptone sucrose medium; C-DOX, czapek–dox medium; PDA, potatoes dextrose agar medium Table 2.Effect of various temperatures on the mycelium growth of C.militaris two strains AG-1 and PSJ-1 Mycelium Mycelium colony diameter (cm) Temperature density 3 days 6 days 9 days 12 days (0 C) AG-1 PSJ-1 AG-1 PSJ-1 AG-1 PSJ-1 AG-1 PSJ-1 AG-1 PSJ-1 a c b c bc c c c 16 1.88±0.06 1.14±0.02 3.26±0.11 1.97±0.07 3.94±0.25 2.92±0.24 4.6±0.12 3.80±0.07 C T a b ab b b b b ab 20 1.98±0.04 1.61±0.17 3.52±0.02 2.48±0.05 4.37±0.05 3.74±0.08 5.37±0.13 4.6±0.12 C C a a a a a a a a 24 2.14±0.11 1.97±0.06 3.72±0.12 3.61±0.04 4.98±0.04 4.49±0.05 5.76±0.11 4.90±0.18 C C a b b b c b c b 28 1.86±0.05 1.49±0.10 3.3±0.12 2.36±0.10 3.84±0.12 3.62±0.03 4.85±0.05 4.32±0.21 C SC 32 1.44±0.22b 1.14±0.02c 1.85±0.20c 1.64±0.17d 3.64±0.17c 2.92±0.08c 3.85±0.11d 3.36±0.15c ST T Means within the same column followed by the same letters are not significantly different at p< 0.05. Each value is expressed as mean ±standard error (SE) (n = 5). Days, day after inoculation; C, compact; SC, Somewhat compact; T, thin; ST, somewhat thin. days, day after inoculation; SC, somewhat compact; T, thin; ST, somewhat thin; C, compact Table 3. Effect of carbon sources in MYPS medium on the mycelium growth of C. militaris two strains AG-1 and PSJ-1 Mycelium colony diameter (cm) Mycelium 3 days 6 days 9 days 12 days density Carbon sources AG-1 AG-1 PSJ-1 AG-1 PSJ-1 AG-1 PSJ-1 AG-1 PSJ-1 PSJ-1 MYPS (control) Glucose Dextrose Fructose

2.14±0.11bc 1.97±0.06a 3.72±0.12c 3.61±0.04b 4.98±0.04c 2.46±0.03 a 2.03±0.03a 4.68±0.14a 3.91±0.05a 5.56±0.06a 2.37±0.11ab 2.00±0.10a 4.46±0.04ab 3.71±0.12b 5.32±0.05b 2.06±0.06c 1.91±0.06a 3.78±0.26c 3.03±0.03c 4.96±0.02c

4.49±0.05bc 5.78±0.12ab 4.90±0.18b 4.89±0.07a 6.13±0.10a 5.57±0.04a 4.70±0.12ab 5.86±0.07a 5.11±0.07b 3.89±0.12d 5.42±0.19b 4.97±0.02b

C C C SC

C C C SC 9

Maltose 2.23±0.10abc 1.97±0.10a 4.14±0.07bc 3.54±0.05b 5.11±0.07c 4.25±0.10c 5.90±0.10a 5.06±0.11b C C Means within the same column followed by the same letters are not significantly different at p< 0.05. Each value is expressed as mean ±standard error (SE) (n = 5).

Cordyceps militaris two strains AG-1 and PSJ-1; MYPS, maltose yeast extracts peptone sucrose medium; days, day after inoculation; SC, somewhat compact; T, thin; ST, somewhat thin; C, compact Table 4. Effect of vitamins sources in MYPS medium on the mycelium growth of C.militaris two strains AG -1 and PSJ-1 Mycelium Mycelium colony diameter (cm) density 3day 6 day 9 day 12 day Vitamins AG-1 PSJ-1 AG-1 PSJ-1 AG-1 PSJ-1 AG-1 PSJ-1 AG- 1 PSJ-1 b a c ab c b d c MYPS (control) 2.14±0.11 1.97±0.06 3.72±0.12 3.61±0.04 4.98±0.04 4.49±0.05 5.78±0.12 4.90±0.18 C C a a a a a a a a Thiamine HCl -B1 2.49±0.04 2.29±0.04 4.38±0.04 3.88±0.06 5.83±0.08 4.98±0.07 7.25±0.05 6.00±0.03 C C Nicotinic Acid - B3 2.20±0.11ab 2.00±0.10a 3.89±0.05bc 3.54±0.08ab 5.26±0.07bc 4.68±0.06ab 5.90±0.03cd 5.31±0.09bc SC SC Piridoxine -B6 2.31±0.04ab 2.15±0.12a 4.03±0.03b 3.76±0.11a 5.46±0.07b 4.85±0.17ab 6.53±0.05b 6.04 ±0.10,a C C b a bc b c ab c b Myo-inositol - B8 2.12±0.05 1.99±0.01 3.83±0.04 3.30±0.13 5.08±0.11 4.71±0.05 6.13±0.08 5.54±0.07 SC T Means within the same column followed by the same letters are not significantly different at p< 0.05. Each value is expressed as mean ±standard error (SE) (n = 5).

Cordyceps militaris two strains AG-1 and PSJ-1; MYPS, maltose yeast extracts peptone sucrose medium; days, day after inoculation; SC, somewhat compact; T, thin; ST, somewhat thin; C, compact.

Table 5. Effect of grain sources on the mycelium growth of C.militaris two strains AG-1 and PSJ-1 Mycelium Mycelium colony diameter (cm) density Grain Day 3 Day 6 Day 9 Day 12 sources AJ-1

PSJ-1

AJ-1

PSJ-1

AJ-1

PSJ-1

AJ-1

PSJ-1

AJ-1

PSJ-1

MYPS (control) Brown Rice White Rice Black glutinous rice Vietnam Wheat

2.14±0.11bc 2.33±0.40ab 1.9±0.06cd

1.97±0.06b 2.05±0.37ab 1.45±0.09c

3.72±0.12cd 4.35±0.78b 3.53±0.10cd

3.61±0.04a 3.66±0.66a 2.62±0.05b

4.98±0.04c 5.35±0.97b 4.61±0.06d

4.49±0.05c 4.72±0.85bc 3.76±0.02d

5.78±0.12d 7.24±0.06b 6.41±0.04c

4.90±0.18e 6.35±0.03b 5.79±0.08c

SC C C

SC C ST

2.58±0.06 a 2.28±0.09 a

4.98±0.10 a

3.9±0.05 a

6.03±0.06 a

5.51±0.02 a 8.01±0.04 a

7.10±0.08 a

C

C

1.78±0.09d

1.38±0.07c

3.29±0.12d

2.33±0.11b

5.10±0.05bc

4.95±0.04b

5.90±0.05d

5.20±0.08de

T

T

Corn

2.23±0.06

1.98±0.02

3.84±0.07

3.8±0.10

5.15±0.08

4.56±0.14

6.09±0.08

5.64±0.08

ST

ST

b

b

c

a

bc

c

cd

cd

10

Means within the same column followed by the same letters are not significantly different at p< 0.05. Each value is expressed as mean ± standard error (SE) (n = 5).

Cordyceps militaris two strains AG-1 and PSJ-1; MYPS, Maltose yeast extracts peptone sucrose medium; days, day after inoculation; SC, somewhat compact; T, thin; ST, somewhat thin; C, compact Table 6. Effect of culture medium on DPPH scavenging of C.militaris two strains AG -1 and PSJ-1 Culture edium (Shack) Control MYPS

Extract concentration (mg/mL) 0.5

1.0

2.5

5.0

89.76±0.00a

89.60±0.17a

89.60±0.15a

BHA

89.53±0.45a

AG-1

43.74±0.18 b

55.63 ±0.33b

64.05 ±0.74b

PSJ-1

27.71 ±0.40c

39.29 ±0.10c

53.49 ±0.54c

7.5

Equation

10

89.97±0.33a

91.07±0.07a

Y = 0.2498x + 89.114

73.35±0.81 b

85.82 ±0.49b

87.91±0.14 b

Y = 9.1636x + 36.344

63.02 ±0.62c

74.10 ±0.80c

80.37 ±0.36c

Y= 10.779x + 18.605

Y =13.959x + 3.1046 AG-1 14.47 ±0.38b 30.84 ±0.58b 50.39 ±0.77b 57.48 ±1.86c 73.27 ±0.77b 85.31 ±0.11b c b b b b PSJ-1 10.84 ±0.45 31.22 ±0.14 50.74± 0.62 63.97 ±0.76 72.99± 0.84 83.35 ±0.25c Y =14.316x + 2.0787 Means within the same column followed by the same letters are not significantly different at p< 0.05. Each value is expressed as mean ± standard error (SE) (n = 3).

Table 7. Effect of culture medium on the chelating ferrous ability of C.militaris two strains AG -1 and PSJ-1 PDA

Extract concentration (mg/ml) Culture medium

Control MYPS

PDA

1

2.5

5

BHA

89.00±0.05a

89.23±0.14a

89.69±0.00a

89.80±0.24a

AG-1

27.09± 0.59b 36.34± 0.58b 53.95 ±0.57b 74.91±0.15 b 82.99±0.17 b

86.35 ±0.35b Y=12.994x + 14.635

PSJ-1

19.79±0.57c

80.95±0.42c

AG-1

14.59± 0.21c 40.47± 0.39c 55.95±0.26 b

26.62±0.62c

54.46 ±0.56b

75.33 ±0.35b

7.5 89.87±0.03a

78.20± 0.38c

63.99 ±0.01b 85.17±0.32 b

10

Equation

0.5

89.90±0.00a

Y=0.186x + 88.93

Y=13.754x + 7.7511

84.20 ±0.50b Y=13.114x + 9.7619

PSJ-1 28.02±0.53b 43.12±0.48b 55.90± 0.29b 63.38±0.51b 71.14± 0.19c 80.48±0.45c Y=10.111x + 21.621 Means within the same column followed by the same letters are not significantly different at p< 0.05. Each value is expressed as mean ± standard error (SE) (n = 3).

11

Fig. 1. Mycelium growth of C.militeris two strains AG-1 (A) and PSJ-1(B) grown for 9 days after inoculation at different MYPS, C-DOX; PDA medium at 24 0 C. C-DOX

MYPS

PDA

A

C-DOX

MYPS

PDA

B

Fig. 2. Mycelium growth of C. militeris two strains AG-1(A) and PSJ-1(B) grown on MYPS medium for 9 days after inoculation at different temperatures.

16 0C

20 0C

16 0C

20 0C

24 0C

28 0C

32 0C

A

24 0C

28 0C

32 0C

B

12

Fig. 3. Mycelium growth of C. militeris two strains AG-1(A) and PSJ-1(B) grown on different carbon sources for 9 days after inoculation at 24 0 C. MYPS (control)

Glucose

Dextrose

Fructos e

Maltose

A

MYPS (control)

Glucose

Dextrose

Fructos e

Maltose

B

Fig. 4. Mycelium growth of C.militeris two strains AG-1(A) and PSJ-1(B) grown on different vitamin sources for 12 days after inoculation at 24 0 C. MYPS (control)

Thiamine HCl â&#x20AC;&#x201C;B1

Nicotinic Acid- B3

MYPS (control)

Thiamine HCl â&#x20AC;&#x201C;B1

Nicotinic Acid- B3

Piridoxine - B6

M yo-inositol -B8

A

Piridoxine - B6

M yo-inositol -B8

B

13