Acta agriculturae Slovenica, 116/2, 337–350, Ljubljana 2020 doi:10.14720/aas.2020.116.2.1104 Original research article / izvirni znanstveni članek Impact of different fermentation characteristics on the production of mycelial biomass, extra-cellular polysaccharides, intra-cellular polysac- charides, and on the antioxidant activities of Cordyceps militaris (L.) Fr. (strains AG-1, PSJ-1) Dang Ngoc HUNG 1 , Chun Li W ANG 2 , Liang Horng LAY 2, 3 , Vu Thi PHUONG 4 Received March 30, 2019; accepted November 24, 2020. Delo je prispelo 30. marec 2019, sprejeto 24. november 2020. 1 National Pingtung University of Science and Technology, Department of Tropical Agriculture and International Cooperation, Pingtung, Taiwan 2 National Pingtung University of Science and Technology, Department of Plant Industry, Pingtung, Taiwan 3 Corresponding author, e-mail: layhl@mail.npust.edu.tw 4 Thai Nguyen University of Sciences, Thai Nguyen, 3Faculty of Tourism, Vietnam Impact of different fermentation characteristics on the pro- duction of mycelial biomass, extra-cellular polysaccharides, intra-cellular polysaccharides, and on the antioxidant activi- ties of Cordyceps militaris (L.) Fr. (strains AG-1, PSJ-1) Abstract: The mycelial biomass, antioxidant activity and production of extra- and intra-cellular polysaccharides pro- duction [EPS, IPS] of Cordyceps militaris strains AG-1, PSJ-1 were evaluated under different submerged liquid culture (SLC) conditions. At 24 0 C mycelial biomass and polysaccharide pro- duction of AG-1, PSJ-1 was optimal using PVC media and static culture conditions; (AG-1: 21.85 ± 1.00; PSJ-1: 18.20 ± 1.84 g l -1 ), and oven drying at 40 o C (AG-1: 25.95 ± 0.84, PSJ-1: 23.55 ± 0.69 mg g -1 ) compared with hot water extraction (AG- 1: 7.07 ± 0.15, PSJ-1: 7.39 ± 0.61 mg g -1 ). Maximum biomass, EPS and IPS production were observed when the initial pH was 6.7: AG-1: 12.92 ± 0.33, 209.70 ± 1.56, 32.62 ± 0.87; PSJ-1: 9.03 ± 0.24 g l -1 , 198.16 ± 0.85 mg g -1 , 30.63 ± 1.96 mg g -1 , respec- tively. The use 3.5 % coconut oil improved biomass, EPS, IPS production, which were 8.27 ± 0.09, 8.01 ± 0.01 g l -1 ; 1208.00 ± 8.60, 1110.40 ± 7.20 mg g -1 ; 32.43 ± 0.49, 29.74 ± 0.44, for AG-1 and PSJ-1, respectively. Both culture condition and oven drying methods had significant effects on H 2 O 2 and radical scavenging activity, ABTS .+ radical activity, lipid peroxidation, and also had effects on total flavonoid and, total phenolic contents. The use of crude submerged liquid culture and oven drying on strains AG-1, PSJ-1 led to extracts with potent antioxidant activity, suggesting the therapeutic use of polysaccharides from strains AG-1, PSJ-1. Key words: antioxidant activities; Cordyceps militaris; ex- tra-cellular polysaccharides and intra-cellular polysaccharides; oven drying; pH Vpliv različnih postopkov fermentacije na produkcijo bioma- se micelija, ekstra in itracelularnih polisaharidov in na antio- ksidacijsko aktivnost kokonovega glavatca (Cordyceps milita- ris (L.) Fr., seva AG-1, PSJ-1) Izvleček: Biomasa micelija, antioksidacijska aktivnost in produkcija zunaj- in znotraj celičnih polisaharidov [EPS, IPS] so bile ovrednotene pri kokonovem glavatcu (Cordyceps mili- taris (L.) Fr., seva AG-1, PSJ-1) v razmerah različnih submer- znih tekočih kultur (SLC) . Pri 24 0 C sta bili biomasa micelija in produkcija polisaharidov pri obeh sevih optimalni v gojišču PVC in stabilnih ramerah; (AG-1: 21,85 ± 1,00; PSJ-1: 18,20 ± 1,84 g l -1 ), po sušenju v pečici na 40 o C (AG-1: 25,95 ± 0,84, PSJ-1: 23,55 ± 0,69 mg g -1 ) v primerjavi z ekstrakcijo z vročo vodo (AG-1: 7,07 ± 0,15, PSJ-1: 7,39 ± 0,61 mg g -1 ). Največja biomasa in produkcija obeh tipov polisaharidov (EPS in IPS) sta bili doseženi pri začetnem pH 6,7: AG-1: 12,92 ± 0,33; 209,70 ± 1,56; 32,62 ± 0,87; PSJ-1: 9,03 ± 0,24 g l -1 ; 198,16 ± 0,85 mg g -1 ; 30,63 ± 1,96 mg g -1 . Uporaba kokosovega olja (3,5 %) je povečala biomaso in produkcijo polisaharidov v obeh sevih in sicer: 8,27 ± 0,09; 8,01 ± 0,01 g l -1 ; 1208,00 ± 8,60: 1110,40 ± 7,20 mg g -1 ; 32,43 ± 0,49; 29,74 ± 0,44. Oba načina gojenja in metode sušenja v pečici so imeli značilen vpliv na aktivnost nevtralizacije H 2 O 2 in prostih radikalov, aktivnost ABTS .+ radi- kala in peroksidacijo lipidov. Imeli so tudi vpliv na vsebnost celokupnih flavonoidov in fenolov. Uporaba surove submerzne tekoče culture in sušenje obeh sevov v pečici sta dali izvlečke s potencialno antioksidacijsko aktivnostjo, kar nakazuje tera- pevtsko uporabo polisaharidov iz obeh sevov kokonovega gla- vatca. Ključne besede: antioksidacijska aktivnost; Cordyceps militaris; zunaj- in znotraj celični polisaharidi; sušenje v pečici; pH Acta agriculturae Slovenica, 116/2 – 2020 338 D.N. HUNG et al. 1 INTRODUCTION Cordyceps militaris (L.) Fr. (Ascomycota: Hypo- creales) is an entomopathogenic fungus that is used in traditional Asia medicine; it is a common parasite of lepidopteran larvae (Shih et al., 2007). The medicinal properties of Cordyceps militaris result from its ability to produce bioactive compounds including cordycepin. At present, liquid fermentation is used for biomass pro- duction. However, optimization is required to maximize production of bioactive compounds. The efficacy of ex- tracts production depends mainly on the strain used, nu- trient sources in the culture medium, and cultivation pa- rameters (Dong et al., 2012). During the fermentation, C. militaris grows in liquid suspension, which is thought to be the best way to produce complex organic compounds including cordycepin (Mao et al., 2005). Vegetable oils can enhance growth rates of mycelium in liquid culture (Schisler & Volkoff, 1977) and reports suggest that oils and fatty acids promote production of fungal metabolites (Kojima et al., 1972). In recent years, considerable research attention had focused on natural compounds with hypoglycemic ac- tivity. Polysaccharides extracted from various medicinal fungi species have shown hypoglycemic activity (Kiho et al., 1997). Although many researchers have tried to op- timize submerged liquids culture conditions to promote extra–cellular polysaccharides (EPS) production by fun- gi, the nutritional requirements and medium conditions for submerged cultures are poorly understood, yet the method remains popular (Park et al., 2001). For C. mili- taris in particular, submerged liquid culture has rarely been studied (Nielsen et al., 1995). Currently, antioxidants extracted from field-col- lected C. militaris fruiting bodies, are investigated ex- tensively for their ability to protect organisms and cells from oxidative damage due to aging and cell degenera- tion (Cazzi et al., 1997). Traditionally, field-collected C. militaris is used widely in both food and pharmaceuti- cal preparations (Isildak et al., 2004). Field-collected and cultivated C. militaris is becoming increasingly popular as a functional food (due to antioxidant activity) as well as for its medicinal properties (Elmastas et al., 2007). In this study, we determined the optimal cultivation conditions (pH and type of vegetable oils used) for pro- duction of C. militaris mycelial biomass. Moreover, we quantified the effect of cultivation conditions on biomass production of biologically active ingredients, extra and intra-cellular polysaccharide production, antioxidant ac- tivities, total phenol content, flavonoid content. We also characterized the biological activity of methanol extracts of mycelial and filtrates obtained using the static cultures method. 2 MATERIALS AND METHODS 2.1 FUNGAL STRAINS Two field-collected strains of Codiceps militaris AG-1, and strain PSJ-1 were obtained from the Plant Physiology and Value Added Microorganisms Labora- tory, Department of Plant Industry, National Pingtung University of Science and Technology (NPUST), Taiwan. Mycelia from each of the two strains were cultivated and maintained in the collection on mannitol yolk polymyxin selective agar (MYPS) (Dang et al., 2018) at 24 o C. 2.2 MEDIA Five submerged liquid culture media were used to evaluate the growth and production of secondary metab- olites: (1) MYPS media: 4 g l -1 malt extract powder; 4 g l -1 yeast extract; 6 g l -1 peptone; 10 g l -1 sucrose; 0.3 g l -1 Vi- tamin B1, and 1000 ml distilled water; (2) PVC media: 30 g l -1 glucose; 10 g l -1 corn powder; 1 g l -1 KH 2 PO 4 ; 0.6 g l -1 K 2 HPO 4 ; 0.7 g l -1 MgSO 4 ·7H 2 O; 0.25 g l -1 FeSO 4 ·7H 2 O; 0.5 g l -1 vitamin B1, 6 g l -1 peptone, and 1000 ml distilled wa- ter. (3) PD culture medium: 200 g l -1 potatoes; 20 g l -1 dex- trose; 30 g l -1 sucrose; 0.5 g l -1 Vitamin B1, and 1000 ml distilled water, (4) Malt-extract medium (ME): 5g l -1 malt extract powder; 5g l -1 peptone; 20g l -1 glucose; 0.3 g l -1 Vitamin B1, and 1000 ml distilled water (Atlas, 1993); (5) Czapek-Dox medium (CD): 30 g l -1 Sucrose; 2 g l -1 NaNO 3 , 1 g l -1 KH 2 PO 4 , 0.5 g l -1 MgSO 4 .7H 2 O, 0.5 g l -1 KCl; 0.3 g l -1 vitamin B1 and 1000 ml distilled water (Ste- vens, 1981). Each cultivation attempt was conducted in five (5) repetitions per medium. 2.3 DETERMINING THE EFFECT OF DIFFER- ENT MEDIA ON BIOMASS OF C. militaris AND QUANTITY OF HOT W ATER EXTRACTED Discs (6 mm diameter cut from culture grown on MYPS plates) of mycelia from the two strains of C. mili- taris were inoculated into the liquid culture medium. Five discs were used for 250 ml of liquid media in each 500 ml Erlenmeyer flask. Flasks were incubated at 24 o C in a temperature controlled rotary shaker (Orbital Shaker Model SK-302AB, Sun Kuan Instruments Co., Kaoshung, Taiwan) for 18 days. The shaking function was set ‘static’ for the first 5 days and then to ‘shake’ (93 rpm) for the fol- lowing 13 days) (Park et al., 2002). After the incubation period mycelia were harvested from each flask by filtra- tion through a pre-weighed filter paper (Whatman No. 1, Whatman Ltd.Toyo Roshi Kaisha, Ltd. Japan), weighed Acta agriculturae Slovenica, 116/2 – 2020 339 Impact of different fermentation characteristics ... , and on the antioxidant activities of Cordyceps militaris (L.) Fr. (strains AG-1, PSJ-1) (wet weight) and then vacuum-dried (dry weight per unit volume of media). The filtrate was concentrated un- der reduced pressure (rotary evaporator) at 4 o C. Dried filtrates and mycelia were ground into fine powders (20 mesh) in a mill grinder before a hot water extraction (Jo et al., 2010). 2.4 EFFECT OF DIFFERENT PH ON BIOMASS AND ON YIELD OF EXTRAC-CELLULAR AND INTRA-CELLULAR POLYSACCHARIDES AF- TER SUBMERGED CULTURE OF C. militaris The effect of pH was done in PVC cultivation me- dium with pH adjusted to 4.5, 5.5, 6.7, 7.5, or 8.0. 1N HCl or 1N NaOH were used for adjusting the pH. Strains AG- 1, PSJ-1 of C. militaris in were then grown at each pH in submerged culture; under static conditions for 18 days at 24 ° C. After 18 days the biomass (dry weight) was deter- mined and the extra and intra-cellular polysaccharides extracted and weighed. Each treatment was conducted 5 repetitions. 2.4.1 Extra-cellular polysaccharides (epd) and intra- cellular polysaccharide (ips) Extra-cellular polysaccharides (EPS) were extracted from the culture filtrate from each flask using standard methods with minor modifications (Fang & Zhong, 2002). Specifically, mycelial biomass in the medium was centrifuged at 10,000 g for 20 min. The supernatant ob- tained was mixed with three volumes of pure ethanol and incubated at 4 °C for 24 hours. The resulting precipitate (EPS) was then separated by centrifugation at 8000 g for 10 min, washed with ultrapure water and lyophilized pri- or to weighing. For intra-cellular polysaccharides (IPS), mycelial biomass was subjected to extraction with boiling water for one hour and the mixture was filtered through Whatman No. 1 filter paper. The filtrate was allowed to precipitate with four volumes of 95 % (v/v) ethanol fol- lowing incubation overnight at 4 °C. The resulting pre- cipitate (IPS) was separated by centrifugation at 8000 g for 10 min, washed with ultrapure water and lyophilized before determining dry mass (Sharma et al., 2015). 2.4.2 Extract of oils on biomass and ips yield after submerged culture of C. militaris The effects of adding different types of oils to the culture medium on biomass and production of extra and intra-cellular polysaccharides during submerged cul- ture was done in PVC medium only. The following oils were used: sunflower oil (HALA Taisun Col Ltd., Tai- wan), olive oil (Olitalia sunflower oil, Italia), coconut oil (coconut oil virgin, Viet Delta Industry Co., Ltd, Vietnam), suet volum % (Pingtung City Supermarket, Taiwan), and peanut oil (HACCP, Taiwan) at concen- trations of 1.5 %, 2.5 %, and 3.5 %. One type of oil was used per setup. Fermentation was done at 24 0 C, at an initial pH of 6.7, in static cultivations conditions for 18 days. Biomass (dry weight) and extra-cellular and in- tra-cellular polysaccharide production were measured as described previously. Each oil type and % treatment was conducted in five repetitions. 2.5 ANTIOXIDANT ACTIVITIES OF MYCELIAL EXTRACTS OF C. militaris GROWN UNDER DIFFERENT SUBMERGED CULTURE CON- DITION The antioxidant activity of mycelial extracts of C. militaris was in a submerged culture of three different media (MYPS, PVC, PD) under of three protocoles: (1) shake (shaking regimes for 18 days at 24 0 C); (2) static (for 18 days at 24 0 C); (3) static + shake (stat- ic for the first 5 days and then ‘shake’ at 93 rpm for the following 13 days). After cultivation mycelia were oven dried, methanol extracts were then evaluated for antioxidant activity In vitro. We specifically quantified the total phenolic content and total flavonoid content. For each test, each treatment was conducted in five repetitions. 2.5.1 Mycelial drying method (oven drying) Fresh mycelia were dried using the oven drying (OD) method (40 o C for 72 h at RH = 65 %). Oven drying was done in hot air oven (Rotek Instruments, B & C Industries, Cochin, India). (Mediani et al., 2013). The samples were ground to powder with a grinder (Yuqi, DM-6, Taiwan), and stored at -20 o C prior to methanol extraction. 2.5.2 Hot water extract Approximately 2 g from each dryed and grinded sample was extracted with 200 ml of boiling distilled water for 2 h. The extract was filtered through filter paper (Advantec No. 1, Japan) while the residue was re-extracted twice under same conditions. The fil- trates obtained from the three separate extractions Acta agriculturae Slovenica, 116/2 – 2020 340 D.N. HUNG et al. were combined, concentrated and lyophilized. The lyophilized extracts were weighed and stored at 4  o C prior to use. 2.5.3 Methanol crude extraction from oven-dried mycelia. Approximately 2 g from each dryed and grinded (DM-6, Taiwan) sample was extracted with 200 ml of 95 % methanol at 75 0 C after 2 h using a bath/circu- lator, and filtered through muslin cloth. The extracts were filtered through Whatman No. 2, Whatman Ltd. Toyo Roshi Kaisha, Ltd. Japan) filter paper. The resi- dues were then extracted with an additional 100 ml of methanol. This was then filtered again through 100 mm of filter paper. Extractions from each sample were done twice and the combined filtrates were con- centrated, under conditions of reduced pressure, in a rotary evapotator at 40  0 C until dry. The dried extracts obtained were re-dissolved in methanol to a concen- tration of 100 mg ml -1 and were stored at 4 o C prior to the analyses of antioxidant attributes. For downstream experiments the dried methanol, filtered, evaporated under reduced pressure and vacuum-dried at 40  0 C to get the viscous residue needed for estimation of anti- oxidant activities. 2.5.4 Scavenging of hydroxyl radicals The hydroxyl radical scavenging activity of C. militaris methanol extract was measured according to the method of (Halliwell et al., 1992). Stock solutions of EDTA (1 mM) were prepared in DMSO and FeCl 3 (10 mM), ascorbic acid (1 mM), H 2 O 2 (10 mM) and deoxyribose (10 mM) in distilled deionized water. For each extract the scavenging activity was determined at different concentrations: 0.5, 1, 2.5, 5, 7.5, and 10 mg ml -1 in methanol dissolved in distilled water, 330 µl of phosphate buffer (50 mM, pH 7.5) and 100 µl of ascor- bic acid. For each extract/ concentration 1000 µl were added to a solution made up of 100 µl of EDTA, 10 µl of FeCl 3 , 100 µl of H 2 O 2 , 360 µl of deoxyribose and incubated at 37 °C for 1 h. After this time 1 ml of the incubated mixture was mixed with 1 ml of 10 % TCA and 1 ml of 0.5 % TBA (in 0.025 M NaOH containing 0.025 % butylated hydroxyl anisole) and the develop- ment of pink chromogen was measured spectrophoto- metrically at 532 nm. The hydroxyl radical scaveng- ing activity of each extract was reported as percentage inhibition of deoxyribose degradation and was calcu- lated according to the formula (1): Where A0 was the control absorbance and A1 was the absorbance of the solution containing either the ex- tract or absorbance of the solution containing either the extract or the standard absorbance. 2.5.5 Scavenging of ABTS .+ , a radical cation. The scavenging activity of the extracts was estimat- ed using the ABTS .+ decolorization method (Arumagam et al., 2006; Re et al., 1999). A stock solution for evalua- tion of antioxidant activity was produced my mixing 5 ml of 7 mM ABTS and 88 μl of 140 mM K 2 S 2 O 8 ; this solution was allowed to complete radical generation for 12–16 h in darkness at room temperature. The stock solution was diluted with ethanol and PBS (pH 7.4) to give an absor- bance of 0.75 at 734 nm. The scavenging activity of each strains of C. militaris methanol extract was determined at concentrations: 0.5, 1, 2.5, 5, 7.5, and 10 mg ml -l . For each extract/ concentration combination 1 ml of extract was added to 1 ml of diluted stock solution and, 5 min after the initial mixing, the absorbance was measured at 734 nm, using ethanol as the blank solution. All mea- surements were performed in triplicate. The total anti- oxidant activity (TAA) percentage was calculated using the formula (2): Where A = absorbance of stock solution and As = absorbance of the extract. 2.5.6 Antioxidant activity in relation to lipid peroxi- dantion The antioxidant activity of C. militaris two strains methanol extracts was determined using a 1,3-diethyl- 2-thiobarbituric acid (DETBA) method (Furuta et al., 1997). Extracts were evaluated at different concentra- tions (0.5, 1, 2.5, 5, 7.5, and 10 mg ml -1 in methanol); for each sample was added to 50 ml of linoleic acid emulsion (2 mg ml -1 in 95 % ethanol) and a positive control butyl- ated hydroxytoluene (BHT) (0.1 mg ml -1 ) were used in this study. The mixture was incubated at 80 o C for 60 min; it was then cooled in an ice bath and mixed with 200 ml of 8 % sodium dodecyl sulfate (SDS), 400 ml of deionized water, and 3.2 ml of 12.5 mM DETBA (Aldrich Chemical Co., WI, USA) in sodium phosphate buffer (pH 3.0). After thoroughly mixing it was incubated at 95 o C Acta agriculturae Slovenica, 116/2 – 2020 341 Impact of different fermentation characteristics ... , and on the antioxidant activities of Cordyceps militaris (L.) Fr. (strains AG-1, PSJ-1) for 15 min and then cooled in an ice bath. Ethyl acetate (4.0 ml) was then added to the mixture, the mixture was centrifuged at 1000 g at 20 o C for 15 min. The fluores- cence of the ethyl acetate layer was then measured in a spectrofluorometer with excitation set at 515 nm and emission set at 555 nm. Each value was expressed as the mean of triplicate measurements +/- standard deviation. The percentage of lipid peroxidation was determined against a blank with no sample added (100 %). The an- tioxidant activity was expressed as the inhibition of lipid peroxidation using the formula (3): 2.5.7 IC50 V ALUES IN AN ANTIOXIDANT ACTIV- ITY . The results of antioxidant activity, hydroxyl radicals, scavenging activity of ABTS+ radical cation, lipid per- oxidation, respectively were normalized and expressed as IC50 (mg extract/ml). A lower IC 50 value (mg extract/ ml) corresponds to the higher antioxidant activity of C. militaris mycelial (obtained by submerged cultivation) extract. 2.6 QUANTIFYING ANTIOXIDANTS 2.6.1 Total phenolic content (tpc) of the extracts. T otal phenolic contents of each methanol extract was determined using gallic acid as a standard (Chan et al., 2009). A sample of 0.2 ml from each extract (10 mg ml -1 in methanol) was mixed with 2 ml of Folin-Ciocalteu’s phenol reagent, 2N (Sigma). The mixture was incubated at room temperature for 5 minutes. Then 1.8 ml of 20 % sodium carbonate (Na 2 CO 3 , Nihon Shiyaku) was added, and the mixture centrifuged at 3000 g for 10 min. The mixture was then incubated for 90 min at room tem- perature. The absorbance of each reaction mixture was measured at 735 nm using a spectrophotometer (Hita- chi U-2800, Japan). Gallic acid was used as a standard to create a calibration curve. The TPC was obtained by in- terpolation from linear regression analysis in mg gallic acid equivalents (mg GAE)/g of dry extract. All tests are performed in triplicate. Two strains mycelial concentra- tion of the sample solution was determined based on a standard curve regression equation (4):  A = 0.8533C + 0.0211,r 2 = 0.997 (Where A is the absorbance and C is the concentra- tion). Then, the extraction rate of TPC in the C. militaris sample was calculated. 2.6.2 Total flavonoid content (tfc) of the extracts. The TFC was determined according to the method of Jia et al. (1999). A sample of 0.2 ml from each ex- tract (100 mg ml -1 in methanol) was mixed with 1.5 ml of distilled water. To this 0.1 ml of 10 % aluminium ni- trate [Al(NO3)3] and 0.1 ml of 1 M potassium acetate (CH3COOK) was added. After 40 min at room tempera- ture, absorbance was measured at 415 nm. Quercetin was used as a standard for a calibration curve. Flavonoid contents was determined by interpolation from linear regression analysis in mg quercetin equivalents (QE)/g dry extract. All tests were done in triplicate. Two strains mycelial concentration in the sample solution was deter- mined based on a standard curve regression equation (5):  A = 3.2173A + 0.0618, r 2 = 0.997 (Where A is the absorbance and C is the concen- tration). From, this extraction rate of TFC in C. militaris samples was calculated. 2.7 EXPERIMENTAL DESIGN AND DATA ANALY- SIS All experiments were done at the Department of Plant Industry, NPUST, Taiwan. One-way analysis of variance (ANOV A) was done followed Duncan’s multiple range tests for means comparisons (p ≤ 0.05). All analy- sis was done in SAS Version 9.4 (SAS Institute Inc., Cary, NC, USA). For each test, each value is expressed as mean ± SE and with 5 replicates (n = 5). 3 RESULTS AND DISCUSSION 3.1 EFFECT OF CULTIV ATION MEDIA ON C. mili- taris BIOMASS PRODUCTION Mycelial wet mass, dry mass and extra-cellular polysaccharide content of both strains were all the great- est in cultures grown in PVC media compared with the other media evaluated (Table 1 and Fig. 1). Overall, sig- nificantly more mycelial biomass was produced by strain AG-1 than by strain PSJ-1 in submerged culture. Over- all, the cultivation methods used for both AG-1, PSJ-1 in this study had low or similar quantities of extracts as obtained in other set-ups. For example, studies on C. militaris strain BCC2816 reported 36 mg of extract from mycelium grown in 5 l of potato dextrose broth medium at 25 o C (Rukachaisirikul et al., 2004.), while 19.1 g l -l were collected from mycelia produced in 57 ml medium at temperature 28 o C, and pH of 6.2 (Chunyan et Acta agriculturae Slovenica, 116/2 – 2020 342 D.N. HUNG et al. Media Mycelial Biomass Static culture fresh mass (g l -l ) Oven dried mass (40 0 C) (mg l -l ) Boiling distilled water crude extract (mg l -l ) AG-1 PSJ-1 AG-1 PSJ-1 AG-1 PSJ-1 MYPS 17.88±0.87 ab 15.03±0.71 ab 23.73±1.02 ab 22.74±0.75 ac 7.53±0.32 a 6.87±0.69 ab PD 10.71±1.13 cc 8.92±1.28 ab 22.90±0.06 ac 24.02±0.04 aa 6.10±0.09 bc 5.91±0.44 ac PVC 21.85±1.00 aa 18.20±1.84 aa 25.95±0.84 aa 23.55±0.69 ab 7.07±0.15 ab 7.39±0.61 aa ME 14.45±1.37 bb 13.59±0.78 ac 18.76±0.85 bc 16.96±0.56 b 5.26±0.50 be 5.05±0.43 bc CD 10.17±0.85 cd 8.63±0.77 bb 21.49±0.68 ab 18.31±0.75 c 5.99±0.04 bd 2.80±0.51 c Table 1: Effect of different liquids media and mycelial biomass on static culture fresh mass, oven dries mass, crude hot water extract of strains AG-1 and, PSJ-1 after 18 days of cultivation. Valuea are expressed as mean± standard deviation (n = 5). Means within the same column followed by the same letters are not significantly different from each other at p ≤ 0.05 according to Duncan’s multiple range tests. pH Mycelial biomass (dry mass; g l -l ) Extra- cellular polysaccharides (mg l -l ) Intra-cellular polysaccharides (mg l -l ) AG-1 PSJ-1 AG-1 PSJ-1 AG-1 PSJ-1 5 4.63±0.03 c 3.95±0.06 b 38.9±0.36 d 32.06±0.55 d 13.31±1.10 ad 12.20±0.84 ad 5.5  5.71±0.05 ac 4.88±0.09 b 45.85±0.87 cd 35.06±1.18 d 15.84±0.79 cd 14.69±0.95 cd 6 8.61±0.71 bc 7.88±0.34 ab 168.56±0.94 ab 168.16±0.67 ab 23.68±0.19 bc 22.62±0.12 bc 6.7 12.92±0.3 b 9.03±0.24 a 209.70±1.56 a 198.16±0.85 a 32.62±0.87 b 30.63±1.96 b 7.5 6.70±0.69 ac 7.93±0.52 a 69.50±4.05 bc 57.39±2.18 bc 9.94±0.86 ade 8.66±0.73 ad 8 5.28±0.43 c 5.07±0.43 ba 39.73±2.87 d 35.16±1.73 cd 8.44±0.74 ae 7.83±0.62 d Table 2: Effect of initial pH and between strains AG-1, PSJ-1 strains on the mycelial biomass, extra-cellular and intra-cellular poly- saccharide production after 18 days in static culture in PVC media at 24 0C. Data show the mean±standard deviation (n = 5). Means within the same column followed by the same letters are not significantly different to each other according to Duncan›s multiple range tests (p ≤ 0.05) Figure 1: Effect of difficult submerged culture, fresh mycelial weight extract cordycepin production, MBDW: Mycelial Biomass dry weight (g/L), EPS: exopolysaccharide, IPS: Intracellular polysaccharides (mg/g DW). PVC media. of C. militaris cultured at 18 days. A: PSJ-1 strain and B: AG-1 strain. C: fresh mycelial of PSG-1 on PVC liquid media; D: fresh mycelial and fruiting body of AG-1 on PVC liquid media. Acta agriculturae Slovenica, 116/2 – 2020 343 Impact of different fermentation characteristics ... , and on the antioxidant activities of Cordyceps militaris (L.) Fr. (strains AG-1, PSJ-1) al., 2009). Our results are in agreement with (Ing-Lung Shih et al., 2007), whose studies showed that metabolites and cell growth of 15.5 g l -l (shaking) after 36 days, and 14.0 g l -l (stat- ic) after 30 days. 3.2 EFFECT OF PH ON THE MYCELIAL C. militaris BIOMASS AND EXTRA- AND INTRA-CELLULAR POLYSACCHARIDES PRODUCTION Initial pH affected the growth of mycelial, as well as the production of extra and intra-cellular polysaccharides, for both AG-1, PSJ-1 strains when cultivated using the sub- merged culture method (Table 2). Submerged liquids culture dry mass, EPS, and IPS were the highest when the initial pH ranged from 6.0 to 6.7, and were the lowest at the high- est and the lowest pHs evaluated (5 and 8). Previous reports have suggested that low pH may increase quantities of extra- cellular polysaccharides in continuous production of many types of Basidiomycetes and Ascomycetes, but this may have been due to oil supplementation (Hsieh et al., 2005; Park et al., 2020; Kim et al., 2001). The optimal pH for cordycepin production by different strains of C. militaris has been re- ported to be in the range of 4-7 (Kang et al., 2014; Zhong et al., 2011). Other studies reported that C. militaris strain BCC2816 achieved the highest dry mycelial mass (19.1 g l -l ), and optimal cordycepin yield (1.8 mg l -l ) at 28 °C, pH 6.2 (Chunyan et al., 2009). In general, the effects of pH in static culture of strains AG-1, PSJ-1 in the present study were lower compared with other studies. For instance, C. militaris strain 3936 produced cordycepin in the pH range of 4.5 to 7.0, with the highest levels at pH 5.5 (213 mg l -l ), which was also con- sistent with earlier reports (Leung et al., 2007). 3.3 EFFECT OF DIFFERENT OILS ON BIOMASS AND ON EPS AND IPS YIELD AFTER SUBMERGED CULTURE OF TWO C. militaris STRAINS Addition of oil sources led to significant increased lev- els of mycelial biomass, EPS and IPS (Table. 3). The great- est mycelial dry mass was achieved in coconut oil (Table. 3). Mycelial biomass, EPS and IPS production were higher when media were supplemented with sunflower oil, olive oil, coco- nut oil, suet, and peanut oil; for all oils growth increased as toe % oil increased. The results are in agreement with (Hsieh et al., 2006), who showed that mycelial growth was increased when media were supplemented with > 1 % olive oil; this was also associated with greater IPS production. Previous reports have also shown that production of extra-cellular polysac- Type of oils Mycelial dry biomass (g l -l ) Extra-cellular polysaccharide (mg l -l )) Intracellular polysaccharide (mg g DM -l ) AG-1 PSJ-1 AG-1 PSJ-1 AG-1 PSJ-1 Sunflower oil 1.5 % 3.49±0.04 cdm 3.22±0.01 ik 254.80±12.69 ag 223.00±5.84 gh 6.62±0.20 ci 6.48±0.05 bh 2.5 % 4.17±0.08 ik 3.96±0.03 fg 291.00±3.74 af 245.20±2.49 g 6.75±0.07 ci 6.36±0.03 bh 3.5 % 4.67±0.03 hi 4.21±0.06 ef 348.80±2.40 de 316.20±3.15 de 14.56±0.28 ade 12.85±0.21 ef Oliver oil 1.5 % 3.32±0.07 d 3.12±0.08 m 113.38±2.94 hi 107.10±1.00 ik 6.86±0.05 ci 6.26±0.03 bh 2.5 % 3.68±0.03 cm 3.47±0.07 hi 139.80±1.71 hi 125.00±1.52 I 8.80±0.08 bch 7.45±0.04 bgh 3.5 % 3.90±0.02 ck 3.72±0.03 gh 154.20±2.22 gh 127.60±1.36 hI 5.59±0.10 ci 5.21±0.19 b Coconut oil 1.5 % 5.87±0.02 bg 5.55±0.07 cd 434.00±1.73 cd 406.60±2.87 bc 17.98±0.14 ad 16.54±0.61 e 2.5 % 6.67±0.07 af 6.14±0.02 c 701.20±7.75 bc 683.60±4.95 ab 18.17±1.17 de 17.01±0.97 de 3.5 % 8.27±0.09 E 8.01±0.0 a 1208.00±2.30 b 1110.40±3.16 a 23.61±1.31 cd 20.39±1.55 cd Suet 1.5 % 5.33±0.10 h 5.14±0.05 de 276.40±3.17 afg 246.00±1.64 fg 7.97±0.90 chi 7.00±0.92 bh 2.5 % 6.50±0.13 afg 6.26±0.04 bc 309.00±2.40 ef 294.20±2.03 ef 10.45±1.18 defg 9.58±0.64 agh 3.5 % 6.18±0.05 abg 6.05±0.02 c 411.60±3.75 de 389.00±3.03 cd 12.18±1.04 abf 11.10±0.66 afg Peanut oil 1.5 % 5.19±0.04 hi 5.06±0.02 df 55.48±0.57 m 53.53±0.35 m 9.95±0.12 bhi 9.37±0.07 agh 2.5 % 6.92±0.02 f 6.78±0.01 ab 60.40±0.16 ik 59.53±0.20 m 6.90±0.05 ci 6.72±0.05 bgh 3.5 % 8.46±0.14 ef 8.22±0.05 a 103.60±2.23 i 100.48±1.70 km 46.17±0.81 d 43.51±0.21 c Table 3: Effect of different type of oils and between strains AG-1, PSJ-1 strains on mycelial biomass, extra-cellualar and intra-cellular polysaccharide production of C. militaris after 18 days of cultivation in static culture in PVC media (initial pH 6.5) at 24 °C. Data show the means of five independent experiments±standard deviation (SD). Each value is expressed as mean±standard deviation (n = 5). Within columns the mean values with different capital letters within a row are significantly different (p ≤ 0.05) to each other according to Duncan’s multiple range tests) Acta agriculturae Slovenica, 116/2 – 2020 344 D.N. HUNG et al. Table 4: Hydroxyl radical scavenging ability at different concentrations of C. militaris two strains grown using nine submerged culture methods that incorporated different media and shaking regimes. The concentration causing 50 % inhibition (IC50) is recorded. Mean values with different capital letters within a row are significantly different to each other (p < 0.05). Means within the same column of C. militaris two strains followed by the same letters are not significantly different at p ≤ 0.05 according to Duncan’s multiple range tests. Extracts were oven dried (n = 5). Submerged culture method Sample concentration (mg ml -l ) Strains 0.50 1.00 2.50 5.00 7.50 10.00 IC50 (µg ml -l ) R² MYPS (shake) AG-1 19.16 ± 0.39 b 28.69 ± 1.03 ac 41.46 ± 1.04 a 56.56 ± 1.01 c 66.32 ± 0.61 ad 80.26 ± 0.62 ad 2.30 R² = 0.9966 PSJ-1 8.94 ± 0.86 ad 8.71 ± 0.39 a 45.58 ± 0.34 b 61.21 ± 0.60 b 69.66 ± 0.83 df 77.00 ± 0.41 acd 2.01 R² = 0.9191 PVC (shake) AG-1 4.99 ± 0.63 e 40.77 ± 1.20 b 15.68 ± 0.42 c 69.57 ± 1.51 B 76.77 ± 0.61 b 81.42 ± 1.01 a 2.49 R² = 0.7894 PSJ-1 10.86 ± 1.01 ad 25.44 ± 1.73 c 34.96 ± 0.64 ac 57.03 ± 0.60 bc 76.54 ± 0.62 c 78.16 ± 0.62 ac 2.33 R² = 0.9697 PD (shake) AG-1 10.10 ± 0.39 d 21.49 ± 0.83 d 35.89 ± 0.83 b 47.74 ± 1.07 d 76.07 ± 0.61 b 78.63 ± 0.22 bd 2.16 R² = 0.9694 PSJ-1 8.25 ± 1.39 abcd 38.91 ± 0.63 b 12.19 ± 1.23 e 47.97 ± 0.59 ae 68.41 ± 1.01 bdf 76.54 ± 0.24 ad 1.84 R² = 0.7819 MYPS (static) AG-1 19.16 ± 1.19 b 29.15 ± 0.82 ac 40.53 ± 0.23 a 51.92 ± 0.70 acd 76.54 ± 0.85 b 82.58 ± 0.41 ac 2.34 R² = 0.9752 PSJ-1 26.36 ± 1.26 c 10.80 ± 0.79 ae 34.50 ± 1.19 ac 49.13 ± 0.41 ad 73.52 ± 0.41 ac 79.79 ± 0.41 ac 2.19 R² = 0.8607 PVC (static) AG-1 10.34 ± 0.63 cd 31.01 ± 1.04 bc 43.32 ± 0.62 a 56.79 ± 1.06 c 71.89 ± 0.61 c 84.67 ± 0.40 c 3.57 R² = 0.9941 PSJ-1 8.48 ± 0.26 abcd 17.77 ± 0.42 d 31.48 ± 0.61 ad 45.64 ± 0.83 e 69.80 ± 0.83 adf 82.58 ± 0.41 b 3.01 R² = 0.9825 PD (static) AG-1 13.82 ± 0.59 bc 26.60 ± 1.27 ace 36.59 ± 0.39 b 55.40 ± 1.20 c 68.18 ± 1.01 cd 78.40 ± 0.39 bd 3.04 R² = 0.0772 PSJ-1 5.46 ± 0.59 bd 28.92 ± 0.82 bc 33.10 ± 0.39 a 50.52 ± 0.41 ad 62.83 ± 0.62 e 75.84 ± 0.62 d 2.79 R² = 0.9803 MYPS (static + shake) AG-1 6.39 ± 0.27 ae 23.11 ± 0.64 de 34.50 ± 1.44 b 54.47 ± 0.84 ac 72.59 ± 0.62 bc 77.70 ± 0.41 be 2.46 R² = 0.9842 PSJ-1 4.76 ± 0.60 b 13.36 ± 0.23 de 29.15 ± 0.62 d 56.56 ± 0.61 c 71.43 ± 0.70 abc 78.40 ± 0.41 ac 2.40 R² = 0.9714 PVC (static + shake) AG-1 9.87 ± 0.24 d 25.20 ± 0.23 ade 32.87 ± 0.60 b 52.15 ± 0.60 ac 64.46 ± 0.41 a 80.72 ± 0.23 ad 2.91 R² = 0.9922 PSJ-1 11.50 ± 0.42 cd 26.13 ± 1.19 c 37.51 ± 0.63 bc 56.33 ± 1.02 c 65.85 ± 0.80 bef 73.29 ± 0.47 de 2.65 R² = 0.9847 PD (static + shake) AG-1 9.64 ± 1.00 ad 28.46 ± 1.00 ac 36.35 ± 0.61 b 50.29 ± 0.61 ad 59.58 ± 0.80 e 77.24 ± 0.84 b 2.55 R² = 0.9881 PSJ-1 7.32 ± 0.38 abd 19.40 ± 0.47 d 33.33 ± 0.23 b 52.85 ± 0.46 cd 67.02 ± 0.82 bdF 78.63 ± 0.22 ac 2.40 R² = 0.9948 Control BHT 88.65 ± 0.30 a 88.87 ± 0.04 a 89.01 ± 0.06 a 89.56 ± 0.25 a 89.72 ± 0.14 a 90.16 ± 0.06 a Acta agriculturae Slovenica, 116/2 – 2020 345 Impact of different fermentation characteristics ... , and on the antioxidant activities of Cordyceps militaris (L.) Fr. (strains AG-1, PSJ-1) Table 5: Scavenging activity of ABTS.+ radical cation and inhibition concentration at 50 % (IC50) values of mycelial extracts of C. militaris two strains grown using nine submerged culture methods that incorporated different media and shaking regimes. Mean values with different upper-case letters within a row are significantly different to each other (p < 0.05). Means within the same column of C. militaris two strains followed by the same lower-case letters are not significantly different to each other at p ≤ 0.05 according to Duncan’s multiple range tests. Extracts were oven dried n = 5). Submerged culture method Sample concentration (mg ml -l ) Strains 0.50 1.00 2.50 5.00 7.50 10.00 IC50 (µg ml -l ) R² MYPS (shake) AG-1 5.92 ± 1.15 ee 16.26 ± 1.08 be 40.01 ± 2.14 ac 13.75 ± 1.60 d 65.98 ± 1.47 b 66.26 ± 0.81 a 3.46 R² = 0.7283 PSJ-1 16.26 ± 1.84 acd 41.68 ± 1.14 b 45.03 ± 0.68 ac 35.54 ± 1.41 cd 62.63 ± 0.68 ab 63.46 ± 1.83 c 3.06 R² = 0.7949 PVC (shake) AG-1 8.16 ± 1.57 ab 23.80 ± 2.09 ab 34.14 ± 1.46 a 45.31 ± 1.20 b 57.04 ± 1.67 a 73.53 ± 1.58 cb 4.56 R² = 0.9944 PSJ-1 8.72 ± 1.39 bd 22.97 ± 2.14 c 39.45 ± 1.67 ad 48.10 ± 1.81 ad 61.51 ± 0.93 ab 66.26 ± 0.64 ad 3.10 R² = 0.9927 PD (shake) AG-1 7.32 ± 1.46 b 22.96 ± 1.12 ab 34.42 ± 1.19 a 58.72 ± 1.57 ab 63.75 ± 1.09 ab 66.54 ± 0.93 a 2.58 R² = 0.95 PSJ-1 4.53 ± 1.14 b 22.97 ± 2.37 c 41.96 ± 1.20 acd 50.90 ± 1.01 abd 57.04 ± 1.30 ab 65.98 ± 2.40 ac 2.87 R² = 0.0935 MYPS (static) AG-1 10.95 ± 1.30 ab 22.40 ± 1.86 b 52.29 ± 0.93 b 37.77 ± 1.03 bc 62.85 ± 1.99 ab 76.32 ± 1.20 b 4.09 R² = 0.8763 PSJ-1 16.82 ± 1.19 ac 31.62 ± 1.93a 44.47 ± 1.20 ac 58.44 ± 0.77 b 65.98 ± 1.60 b 71.29 ± 0.90 ab 3.22 R² = 0.9724 PVC (static) AG-1 17.38 ± 1.11 a 36.37 ± 1.19 a 43.08 ± 0.93 bc 51.18 ± 1.74 ab 65.42 ± 1.67 b 76.58 ± 0.60 b 4.57 R² = 0.9821 PSJ-1 9.28 ± 1.40 abcd 24.64 ± 1.74b bc 35.81 ± 1.65 d 45.87 ± 1.47 d 65.98 ± 1.47 b 73.52 ± 1.82 b 4.03 R² = 0.9896 PD (static) AG-1 8.16 ± 0.84 ab 18.21 ± 3.74 bd 34.14 ± 1.46 a 46.71 ± 1.65 a 61.51 ± 1.41 ab 72.13 ± 1.09 ab 3.45 R² = 0.9969 PSJ-1 12.63 ± 0.83 acd 25.20 ± 1.19 c 48.94 ± 1.37 bc 34.98 ± 2.42 c 64.31 ± 1.56 b 71.29 ± 0.81 ab 2.79 R² = 0.8668 MYPS (static + shake) AG-1 6.21 ± 0.80 bd 40.84 ± 1.65 a 14.86 ± 1.35 d 20.17 ± 0.73 cd 63.19 ± 0.84 ab 72.96 ± 1.11 cb 3.87 R² = 0.6346 PSJ-1 15.43 ± 2.88 acd 26.88 ± 1.19 ac 41.12 ± 0.87 ac 53.97 ± 1.29 abd 66.26 ± 1.34 b 69.61 ± 0.81 abc 3.22 R² = 0.9706 Liquid (static + shake) AG-1 6.21 ± 0.32 bd 20.73 ± 1.01 cb 38.33 ± 1.13 ac 59.56 ± 0.73 a 67.94 ± 1.67 b 74.64 ± 2.23 ab 4.49 R² = 0.9706 PSJ-1 9.56 ± 1.13 abcd 34.98 ± 1.46 abc 60.12 ± 2.61 b 48.94 ± 1.59 ad 66.26 ± 1.43 b 70.11 ± 1.02 abc 3.65 R² = 0.811 PD (static + shake) AG-1 10.39 ± 0.56 b 37.21 ± 1.07 a 42.80 ± 0.73 c 49.78 ± 2.12 b 63.19 ± 0.93 ab 72.96 ± 0.17 cb 3.50 R² = 0.9449 PSJ-1 17.10 ± 1.29 c 31.62 ± 2.04 ac 45.87 ± 1.47 ac 57.32 ± 2.20 b 66.26 ± 1.57 b 71.85 ± 1.25 ab 3.42 R² = 0.976 Control BHT 76.00 ± 1.73 a 76.00 ± 1.15 a 77.33 ± 0.88 a 78.33 ± 1.20 a 78.67 ± 0.88 a 79.33 ± 0.67 A Acta agriculturae Slovenica, 116/2 – 2020 346 D.N. HUNG et al. charides and mycelial growth of the C. militaris significantly increased (nearly tripling) following the addition af vegetable oils to the medium (Park et al., 2002). 3.4 SCAVENGING OF HYDROXYL RADICALS In our study, we found that all mycelial extracts were able to scavenge hydroxyl radicals but that the scav- enging activity was affected by concentration (Table 4). At 10 mg ml -1 , both AG-1, PSJ-1 strains had the great- est hydroxyl radical scavenging activities compared with other concentrations; values ranged from: 77.70 ± 0.41 to 84.67 ± 0.40 % AG-1 and 73.29 ± 0.47 to 79.79 ± 0.41 % for PSJ-1. In terms of grown of the strain in dif- ferent shaking regimes, IC 50 values ranged from AG-1: 2.16-3.57 mg ml -1 , PSJ-1: 1.84-3.01 mg ml -1 . The results are similar to previous studies, where 76 % of the highest scavenging capacity of hydroxyl radicals was achieved at a concentration of 3.67 g l -1 (sample/water). (Shen & Shen, 2001). Also in previous studies, boiling water extracts of cul- tured and natural C. sinensis (Berk.) Sacc. mycelia achieved lower radical scavenging activities (IC 50 : 0.96 ± 0.06 mg l -1 and 1.03 ± 0.03 mg ml -l at concentration of 0.25–2.0 mg ml -l , respectively) (Dong & Yaoa, 2007). In contrast, other stud- ies achieved greater hydroxyl radical scavending ability using hot water extractions of mycelium (37.1 ± 3.8 % to 74.2 ± 4.7 %). (Yuxiang et al., 2006). 3.5 SCAVENGING ACTIVITY OF THE CATION RADICAL ABTS .+ . ABTS .+ scavenging activity for mycelial extracts from strains AG-1, PSJ-1 increased as concentrations increased (Table 5), and was greatest at the 10 mg ml -1 concentration, ranging from 66.54 ± 0.93 to 77.15 ± 0.81 for AG-1 and from 65.98 ± 2.40 to 76.58 ± 0.60 for PSJ-1. The ABTS .+ radical scav- enging activity was achieved by a mycelial extract from strain AG-1, grown on PVC without shaking (static); overall IC 50 values for AG-1 ranged from 2.58-4.57 mg ml -1 . For PSJ-1 IC 50 values obtained in the following treatments: MYPS shake (3.06 mg ml -1 ), PVC shake (3.10 mg ml - l ), MYPS static (3.22 mg ml -l ), PD static (2.79 mg ml -l ), MYPS static+shake (3.22 mg ml -l ), PVC static+shake (3.65 mg l -l ); and PD static+shake (3.42 mg ml -l ), as shown in Table 5. In the studies of others, using differ- ent extraction and fermentation methods of Cordyceps militaris SN-18 also exhibited excellent ABTS .+ radical scavenging activity with an EC 50 of: 5.25 ± 0.03 mg ml -l at 4 mg ml -1 (Y u Xiao et al., 2014 ). According to Sapan et al. (2015), EPS and IPS extracts from mycelia of C. cicadae exhibited strong ABTS .+ radical scavenging activity with EC 50 : 6.38 ± 0.12 mg ml -l (EPS) and 5.23 ± 0.25 mg ml -l (IPS), at a concentration of 8.0 mg ml -l . 3.6 INHIBITION OF LIPID PEROXIDATION Lipid peroxidation by extracts from strains AG-1, PSJ-1 increased with increasing concentration. A con- centration of 10 mg ml -1 of strains AG-1, PSJ-1 resulted in mycelial extracts that exhibited the highest antioxi- dant activity compared with the other concentration, and the results were comparable to butylated hydroxyl tolu- ene used as a positive control (BHT) (Table 6). At 10 mg ml -1 there was no significant difference in the inhibition ambition of strains AG-1, PSJ-1 (p < 0.05). Lipid per- oxidation inhibition activity of the mycelia extract from strain AG-1 grown on the PD medium under ‘shake’ conditions was significantly stronger than the other ex- tracts with the lowest IC 50 values (1.88 mg ml -1 ) (Table 6). Based on estimated IC 50 from the inhibition assays of various Cordyceps strain, concentrations ranged from 0.08 to 5 mg ml -1 . These values are in line with the doses of Cordyceps prescribed medicinally, which ranges from 1-10 g per dose, (Zhu et al. 1998). For example, using a concentration of 15.0 mg ml -1 of C. militaris extract, inhibition of lipid peroxidation reached nearly 50 %, at 4.0 mg ml -1 (Chun-Lun Wang et al., 2015). 3.7 TOTAL PHENOLIC CONTENT (TPC). In this study the TPC of mycelial extract from strain AG-1 ranged from 1.75 ± 0.07 to 3.74 ± 0.18 mg g -1 (Ta- ble 7) and were highest when grown in PVC medium under static conditions (3.74 ± 0.18 mg g -1 extract). For AG-1, the TPC was significantly higher in treatments PVC static, MYPS static, PD static, PVC static+shake, MYPS static+shake, PD static+shake, PVC shake, MYPS shake, and PD shake than PD static treatment. For strain PSJ-1 the TPC of mycelial extracts were lower than those from strain AG-1, with values that range from 1.94 ± 0.21 to 3.23 ± 0.10 mg g -1 . The results of AG-1, PSJ-1 were similar to some previous reports: 3.91 ± 0.16 mg GAE/g extract (Chun Lun Wang et al., 2015); 3.9 ± 0.2 mg GAE/g extract (Lee et al., 2013). This confirms that antioxidant potential is positively correlated with TPC (Kaur & Kapoor, 2002 ). The TPC in our study was much higher, and thus incomparable to the results of previous researchers. In one study TPC obtained from methanolic extracts from natural and cultured C. sinen- sis were: 17.07 ± 0.38 mg GAE/g extract while in another they varied from 12.02-12.14 mg GAE/g (Junqiao Wang et al., 2015). Thus, the TPC of extracts from AG-1, PSJ-1 Acta agriculturae Slovenica, 116/2 – 2020 347 Impact of different fermentation characteristics ... , and on the antioxidant activities of Cordyceps militaris (L.) Fr. (strains AG-1, PSJ-1) Table 6: Inhibition of lipid peroxidation and inhibition concentration at 50 % (IC50) values of mycelial extracts of C. militaris two strains grown using nine submerged culture method that incorporated different media and shaking regimes. Mean values with different upper-case letters within a row are significantly different to each other (p < 0.05). Means within the same column of C. militaris two strains followed by the same lower-case letters are not significantly different to each other at p ≤ 0.05 according to Duncan’s multiple range tests. Extracts were oven dried (n = 5). Submerged culture method Sample concentration (mg ml -l ) Strains 0.50 1.0 2.50 5.00 7.50 10.00 IC50 (µg ml -l ) R² MYPS (shake) AG-1 8.33 ± 1.45 abc 14.67 ± 1.76 cd 28.00 ± 1.15 b 37.67 ± 1.86 cd 58.00 ± 1.53 ab 66.33 ± 1.20 ad 1.79 R² = 0.9827 PSJ-1 11.67 ± 1.20 acd 22.00 ± 1.15 a 35.00 ± 1.00 ac 47.33 ± 1.20 b 57.33 ± 1.33 ad 65.33 ± 1.20 abc 1.69 R² = 0.9509 PVC (shake) AG-1 13.33 ± 0.88 ac 30.67 ± 1.20 abc 40.00 ± 1.15 a 50.33 ± 0.88 ac 61.00 ± 1.53 b 70.67 ± 0.67 ac 2.02 R² = 0.944 PSJ-1 18.00 ± 1.15 b 35.33 ± 0.88 c 21.67 ± 1.20 e 47.67 ± 1.45 b 61.00 ± 1.15 ab 68.00 ± 0.58 ab 1.74 R² = 0.9468 PD (shake) AG-1 10.33 ± 0.88 abc 37.67 ± 1.45 b 37.00 ± 1.00 a 57.00 ± 0.58 b 60.00 ± 0.58 b 62.00 ± 0.58 d 1.88 R² = 0.8298 PSJ-1 8.33 ± 0.88 d 14.00 ± 0.58 b 25.33 ± 1.20 bde 36.33 ± 2.33 a 46.00 ± 1.53 cd 58.67 ± 0.67 c 1.51 R² = 0.9898 MYPS (static) AG-1 12.00 ± 1.15 ac 5.33 ± 0.33 e 28.00 ± 1.00 b 48.00 ± 1.00 c 57.33 ± 0.88 ab 68.00 ± 1.15 ac 2.06 R² = 0.9787 PSJ-1 12.33 ± 0.67 acd 21.33 ± 0.67 ae 39.00 ± 1.00 ac 41.33 ± 0.33 ab 58.00 ± 1.00 abd 66.00 ± 1.53 abc 1.67 R² = 0.9319 PVC (static) AG-1 14.67 ± 0.88 c 28.33 ± 1.67 ac 37.00 ± 1.00 Aa 49.67 ± 0.33 ac 60.00 ± 1.15 b 77.67 ± 1.45 b 2.14 R² = 0.983 PSJ-1 12.33 ± 0.33 acd 27.33 ± 1.45 ad 40.33 ± 1.45 c 47.00 ± 1.00 b 58.33 ± 1.20 abd 70.33 ± 0.33 b 1.83 R² = 0.94 PD (static) AG-1 5.80 ± 0.53 b 25.40 ± 0.80 c 39.67 ± 0.33 a 54.33 ± 0.67 ab 61.33 ± 0.67 b 69.00 ±1.00 ac 1.95 R² = 0.8806 PSJ-1 9.33 ± 0.67 cd 19.00 ± 0.58 abe 29.33 ± 0.67 abd 42.00 ± 1.15 ab 63.33 ± 0.88 b 68.67 ± 0.67 ab 1.85 R² = 0.9742 MYPS (static + shake) AG-1 7.67 ± 1.76 ab 33.67 ± 0.33 ab 16.33 ± 0.88 bc 23.00 ± 1.00 e 58.00 ± 0.58 ab 68.67 ± 0.88 ac 1.84 R² = 0.9232 PSJ-1 13.00 ± 1.53 abc 21.67 ± 0.88 ae 36.00 ± 0.58 ac 46.67 ± 1.33 b 53.33 ± 0.88 d 66.00 ± 2.08 abc 1.73 R² = 0.945 Liquid (static + shake) AG-1 11.00 ± 0.58 abc 27.67 ± 1.33 c 41.00 ± 0.58 a 53.93 ± 1.03 ab 60.67 ± 0.88 b 71.33 ± 1.76 bc 2.02 R² = 0.8872 PSJ-1 12.33 ± 0.33 acd 22.00 ± 1.00 ae 34.33 ± 0.33 acd 44.33 ± 1.45 ab 56.33 ± 0.88 ad 69.00 ± 3.21 ab 1.93 R² = 0.9633 PD (static + shake) AG-1 13.00 ± 1.00 ac 6.00 ± 0.58 de 31.00 ± 0.58 ab 50.00 ± 1.15 ac 54.00 ± 0.58 a 65.33 ± 0.67 ad 1.92 R² = 0.9519 PSJ-1 14.67 ± 0.33 ab 24.33 ± 1.45 ad 35.67 ± 1.76 ac 42.33 ± 0.33 ab 53.33 ± 1.33 ad 62.33 ± 0.88 ac 1.67 R² = 0.961 Control BHT 78.27 ± 0.58 a 78.67 ± 0.88 a 78.67 ± 0.33 a 79.67 ± 0.33 a 82 ± 0.58 a 83.00 ± 0.58 a Acta agriculturae Slovenica, 116/2 – 2020 348 D.N. HUNG et al. in this study depended not only on the use of different submerged culture method but also on the mycelial dry- ing methods used. 3.8 TOTAL FLAVONOID CONTENT (TFC). The TFC of mycelial extracts from AG-1 ranged from 4.86 ± 0.07 to 6.01 ± 0.22 mg g -1 (Table 7). Ex- tracts from mycelial grown in PVC medium and static conditions also gave the highest TFC for AG-1 (6.01 ± 0.22 mg g -1 extract). For AG-1 TFC was significantly greater in mycelial growm in the PVC static, MYPS stat- ic, PD static, PVC static+shake, MYPS static+shake, PD static+shake, PVC shake, MYPS shake, and PD shake than in mycelial grown in the PD static treatment. TFC of PSJ-1 mycelial were lower than those of strain AG-1, and rangerd from 4.70 ± 0.07 to 5.70 ± 0.21 mg g -1 (Ta- ble 7). In our study TFC was significantly dependent on the strain used and on the nine different submerged culture strategies. Previous work on strains AG-1, PSJ-1 have already shown that TFC is less dependent on dry- ing method than the indicated TFC of C. militaris waster medium was 4.26 ± 0.05 mg of QE g -1 (distilled water) (Chun Lun Wang et al., 2015), and that while the TPC from the growing medium of C. militaris was 3.91 ± 0.16 mg GAE g -1 the TFC was higher at 4.26 ± 0.05 mg of QE g -1 (Chun Lun Wang et al., 2015). However, other stud- ies have shown that the TFC of mycelial of C. militaris grown on fermented of unfermented wheat were: 7.08 ± 0.46 mg l -1 ; ethanol 80 %: 7.36 ± 0.37 mg l -1 (ethanol extracts); and 6.07 ± 0.19 mg l -1 (water extract) (Yu Xiao et al., 2014). 4 CONCLUSIONS The impact of various submerged culture strat- egies (shaking regime, drying methods, oil supple- ments) on mycelium growth, EPS and IPS production, and antioxidant activity of C. militaris strains AG-1, PSJ-1 in submerged culture are presented. Submerged culture method drying methods and oils played an important role in mycelial biomass growth, EPS pro- duction, IPS production, and antioxidant activity of extracts. Submerged culture methods for optimal pro- duction of mycelial extracts with high levels of poly- saccharides was achieved. The optimal initial pH for mycelial growth and extra-cellular polysaccharide production was 6.7. Of the five different types of oil sources tested, coconut oil, suet, and sunflower oil, all improved the dry mass of mycelia produced and also the production of both EPS and IPS. Specifically, maximum mycelium growth and extra-cellular poly- saccharide concentrations were achieved in the PVC media containing 3.5 % coconut oil. Results from vari- ous original H 2 O 2 radical scavenging activity, ABTS .+ radical activity, and lipid peroxidation assays revealed that extracts of mycelial from AG-1, PSJ-1 had signifi- cant antioxidant activity, and could represent a poten- tial source of antioxidants of great importance for the treatment of disease. The strong antioxidant activity was related to the total flavonoid content (TFC), and total phenolic content (TPC). Future work on the iden- tification, isolation and structural characteristics of the active components will be the target of our follow-up studies, as these compounds have the potential for use as phyto-therapeutic agents. Submerged culture method Total phenolic contents (mg ml -l ) Total flavonoids content (mg ml -l ) AG-1 PSJ-1 AG-1 PSJ-1 MYPS (shake) 1.94 ± 0.10 bd 2.02 ± 0.17 b 4.91 ± 0.08 b 4.86 ± 0.13 b PVC (shake) 3.04 ± 0.41 ab 2.37 ± 0.08 b 5.55 ± 0.14 ba 5.48 ± 0.31 b PD (shake) 1.75 ± 0.07 d 1.94 ± 0.21 b 4.93 ± 0.28 ab 4.80 ± 0.11 b MYPS (static) 2.76 ± 0.10 abc 2.37 ± 0.21 ab 5.43 ± 0.15 ba 5.28 ± 0.11 b PVC (static) 3.74 ± 0.18 a 3.23 ± 0.10 a 6.01 ± 0.22 a 5.70 ± 0.21 b PD (static) 2.72 ± 0.16 bc 2.25 ± 0.10 b 5.33 ± 0.17 ab 5.22 ± 0.18 b MYPS (static + shake) 2.10 ± 0.18 cd 1.98 ± 0.20 b 4.88 ± 0.22 b 4.90 ± 0.13 b PVC (static + shake) 2.33 ± 0.07 bcd 2.25 ± 0.14 b 5.48 ± 0.28 ab 5.05 ± 0.10 b PD (static + shake) 2.02 ± 0.27 bd 1.94 ± 0.17 b 4.86 ± 0.07 b 4.70 ± 0.07 b Table 7: The total phenolic contents and total flavonoids contents of mycelial extracts of C. militaris grown using nine submerged culture method that incorporated different media and shaking regimes. Mean values with different upper-case letters within a row are significantly different to each other (p < 0.05). Means within the same column of C. militaris strains AG-1, PSJ-1 followed by the same lower-case letters are not significantly different to each other at p ≤ 0.05 according to Duncan’s multiple range tests. Extracts were all oven dried (40 °C) (n = 5). Acta agriculturae Slovenica, 116/2 – 2020 349 Impact of different fermentation characteristics ... , and on the antioxidant activities of Cordyceps militaris (L.) Fr. 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