Enhanced Fruiting Body Production and Bioactive Phytochemicals from White Cordyceps militaris by Blending Cordyceps militaris and Using Cold Plasma Jet

The impact of cold plasma jet (CPJ) on fruiting body production and bioactive phytochemicals in white Cordyceps militaris blended with Cordyceps militaris (blended C. militaris) cultured on solid media was evaluated. CPJ was driven by a 12 kV neon transformer with a duty cycle of 50% and a fixed flow rate of 5 L/min. Primarily, reactive oxygen and nitrogen species (RONS) were produced, including nitric oxides, hydroxyl radicals, and atomic oxygen. Before solid media cultivation, the CPJ was activated in liquid media containing potato dextrose agar of blended C. militaris for different treatment times (30–120 s). Changes in fresh and dry weights and cordycepin content of the most sensitive blended C. militaris cultivar increased by 22.49, 22.56, and 25.23%, respectively. CPJ treatment markedly increased antioxidant enzymes, including superoxide dismutase and glutathione S-transferase, by 34.71 and 17.42%, respectively. Moreover, CPJ treatment significantly increased the contents of bioactive compounds, including total phenolic content and total flavonoid content. The changes in antioxidant activities in the fruiting bodies of blended C. militaris were assayed using DPPH and ABTS radical scavenging activities to assess ferric-reducing antioxidant power. Therefore, CPJ treatment can represent a promising nonthermal method to enhance fruiting body production and nutritional value of blended C. militaris, which has implications for human health.


Introduction
Cordyceps militaris (C. militaris), a member of the Cordycipitaceae family, is a species of Cordyceps fungus. C. militaris is broadly used as a traditional medicinal mushroom and additive food due to its immunomodulation that includes microbial, anti-HIV, antidiabetic, anti-angiogenic, antioxidant, and liver-protective properties [1]. The essential bioactive components of C. militaris include cordycepin, adenosine [2][3][4], phenolic compounds [5], superoxide dismutase (SOD) [6], and protein [7]. Due to its various bioactive components and being to culture, it fetches a high price in the market.
Given its advantages for human health, C. militaris has been shown to increase cordycepin content in solid-state fermentation, submerged, and surface cultures. The optimal conditions for improving cordycepin levels of C. militaris in large-scale liquid culture are influenced by the effects of carbon sources, organic or inorganic compounds, nucleoside analogs, and amino acids [4]. Although fruiting body cultivation in solid media is time consuming, several lines of research have shown that increasing cordycepin production is associated with fruiting body development [8]. In addition, carbon and nitrogen sources have been used in solid media to improve cordycepin and fruiting body production in C. militaris [1].
In previous studies, mutant C. militaris generally occurred due to high-frequency mutation of a gene, low isozyme expression [9], and nuclear phase change [6]. Moreover, ion beam irradiation was performed on C. militaris in surface liquid culture to obtain mutants [1]. White C. militaris, a new natural mutation of C. militaris, was discovered in a laboratory in Thailand. The reason for this mutation in cordycpes is unclear. Its bioactive phytochemicals are similar to those of C. militaris, including cordycepin and adenosine. In addition, the activities of SOD, glutathione S-transferase (GST), and β-glucan in white C. militaris were determined. White cordyceps has become a new food additive with medicinal function for human health in Thailand. However, the production of fruiting bodies in white C. militaris is low due to its mutation. To enhance cultivation yields, white C. militaris was blended with C. militaris, and the fruiting body production was slightly increased. Therefore, a primary scientific challenge is to enhance fruiting body production and bioactive phytochemicals in white C. militaris blended with C. militaris.
Cold plasma (CP) is defined as an ionized gas near room temperature that permits the generation of atoms, electrons, and reactive species at ambient temperatures. It is a nonthermal technology and an advanced oxidation process intensively used in seed germination [10], biomedical methods, and other applications [11,12]. An important characteristic of CP is the generation of reactive oxygen and nitrogen species (RONS), including hydroxyl radicals (OH), atomic oxygen (O), hydrogen peroxides (H 2 O 2 ), and nitric oxide (NO). Recently, several studies reported the effect of CP on improving seed germination of wheat [13][14][15][16], pea [17] and antioxidant enzymes in oilseed rape [18] and tomato [19] and stimulating drosera adelae on tissue culture shoot multiplication [20]. CP also enhanced bioactive phytochemicals, bioactive compounds, and antioxidant activities in brown rice [10] and basil plants [21]. For mushroom cultivation, the fruiting body formation of shiitake mushroom (Lentinus edodes) was enhanced by applying high voltages of 50 and 100 kV [22]. The authors mentioned that the physiological factors for improving the fruiting body production included nutrients, hormones, and gaseous conditions. Thus, as the literature review above, the effect of CP on the enhancement of fruiting body production and nutritional values in white C. militaris blended with C. militaris has never been investigated.
Therefore, the purpose of this study was to improve fruiting body production and bioactive phytochemicals, especially the β-glucan content and activity of SOD GST, in white C. militaris by blending with C. militaris (blended C. militaris) and using a cold plasma jet (CPJ) over different treatment times. CPJ was driven using a low-cost neon transformer with a duty cycle of 50% and a fixed airflow rate of 5 L/min. An optical emission spectrometer was used to obtain the reactive species. Cordycepin and adenosine levels were determined using a high-performance liquid chromatographic (HPLC) technique. Changes in antioxidant enzymes, lipid peroxidation, bioactive compounds (total phenolic contents, total flavonoid content), and antioxidant activity were explored.

Samples of C. militaris
White C. militaris and C. militaris samples prepared on potato dextrose agar (PDA) were supported by ACX Herb Co., Ltd, Prachinburi Province, central Thailand.

CPJ Set Up
A schematic of the CPJ apparatus used in this work (Fig. 1a) was created based on our previous work [23]. Briefly, plasma was generated in a quartz tube (3-mm inner diameter) consisting of a power electrode (a wire copper inserted at the center) and a ground electrode (a plated copper wrapped around the end of the tube). The distance between electrodes was ~ 2 cm. A DC air pump (75 W) was used to produce air applied to the quartz tube, and an airflow meter controlled the air rate at 5 L/min. The air plasma was ignited between the gap of two electrodes by applying a neon transformer with an output of 12 kV, 30 mA, and 25 kHz. The discharge voltage signal V(t) was sampled and recorded on the first channel of an oscilloscope with a voltage divider. The discharge current I(t) was measured using a Pearson current monitor (PMC, model 6585, bandwidth up to 250 MHz), which was recorded on the second channel of the oscilloscope. The calculation of discharge voltage using a voltage divider was described in our previous study [23]. The V-I waveforms were displayed by a digital oscilloscope (TDS2014B; Tektronix, Inc., United States). The average discharge power of the CPJ was determined using Eq. (1) [24]:

Measurement of Optical Emission Spectra of the Generated CPJ
The optical emission spectra were measured using a broadband CCD spectrometer (Exemplar LS; BWTEK Inc., United States). The spectrum measurement ranged from 200 to 850 nm, resolved by 600 grooves/mm and 25 μm slit width. The optical emission was measured approximately 1.0 cm away from the nozzle. The experimental setup is shown in Fig. 1a.

CPJ Treatment
Small pieces of PDA of white C. militaris and C. militaris at a ratio of 1:1 were transferred into a glass jar containing 400 mL of a liquid medium (10 g/L yeast extract, 10 g/L peptone, 20 g/L glucose, 0.1 g/L KH 2 PO 4 , 0.2 g/L MgSO 4 7H 2 O, and 0.2 g/L K 2 HPO 4 ·3H 2 O) with a pH of 7.0. CPJ was applied to the liquid medium of blended C. militaris at different times (30 to 120 s), as shown in Fig. 1b. The distance between the plasma nozzle and the surface of the liquid medium was kept constant at 5 cm for all experiments. In this experimental method, untreated PDA was divided into two groups: CK1 and CK2 were the control of white C. militaris and blended C. militaris, respectively. The blended C. militaris samples were treated in triplicate. Then, the samples were incubated in the dark at 20 ± 2 °C and 60% relative humidity (RH) on a 250-rpm shaker for seven days.

Measurement of Solution Characteristics
The pH, conductivity, and temperature of the liquid medium during the CPJ treatment were determined using pH meter (Exstik waterproof) and conductivity meter (Portable Conductivity Meter, HI 99301D, Hanna Instruments), respectively. The nitrate ( NO − 3 ) of the liquid medium treated with CPJ was measured via the Brucine method [25]. Briefly, a 10 mL of the liquid medium were mixed with 2 mL of (30% w/v) NaCl. A 10 mL sulfuric acid solution (prepared by diluted with deionized water with a ratio of 4:1) was added. The mixture was cooled down at room temperature. Then, 0.1 mL of Brucine-Sulfanilic acid solution was added. After incubation at 95 °C in a water bath for 20 min, the absorbance was determined at 410 nm using a microplate reader (model Enspire; PerkinElmer, Inc.). The NO − 3 was used as a reference standard; the concentration of the sample is expressed as milliliter per liter of nitrate (mg/L of NO − 3 ). The nitrite ( NO − 2 ) concentration in the liquid medium was determined via American Public Health Association (APHA) method [25]. Briefly, one milliliter of 25 mM sulphanilamide was added to 50 mL of liquid medium. The solution was vertex and left at room temperature for 2-8 min. One milliliter of n-1-naphthyl ethylenediamine dihydrochloride solution was added to the solution. After 10 min, the absorbance was determined at 543 nm using a microplate reader (model Enspire; PerkinElmer, Inc.). The NO − 2 was used as a standard. The result is expressed as milliliter per liter of nitrite (mg/L of NO − 2 ). All medium liquid measurements were performed in triplicate.

Cultivation of Blended C. militaris
Solid medium cultivation was conducted in a 1000 mL glass jar with a lid. The solid medium was prepared from 20 g of brown rice mixed with 40 mL of deionized water. Then, it was sterilized by autoclaving at 125 °C for 20 min and cooled to room temperature. Then, 5 mL of seed culture was transferred to the solid medium and kept at 18 ± 2 °C for seven days in the dark. Note that fifty solid media repeats were used for each treatment. Cultivation of blended C. militaris was maintained at 18 ± 2 °C with light/dark conditions of 16/8 and 60% RH for 90 days.

Measurement of Fresh and Dry Weights of Blended C. militaris Fruiting Bodies
The average fresh and dry weights of the fruiting bodies of blended C. militaris were determined after 90 days of cultivation. The fresh weight for each glass jar was measured and is expressed in grams. Next, the fruiting bodies of cordyceps were dried at 40 °C for 48 h, whose weights are also expressed as dry weight in grams.

Extraction
Extraction of blended C. militaris was performed following the Jagtap method [26]. Briefly, powdered samples of blended C. militaris extracts were obtained by homogenizing in ethanol, maintaining a blended C. militaris to ethanol ratio of 1:10. The mixture was placed in an orbital shaker at room temperature for 24 h. The homogenates were placed on a centrifuge at 15,000 rpm at 4 °C for 10 min, and the supernatants were calculated for extraction yield and stored at − 20 °C until use.

Measurement of Cordycepin and Adenosine Contents by HPLC
Before extraction, dried cordyceps were meshed into a small size (less than 20 mesh). For extraction, 1 g dry powder was added to 2 mL ethanol (50%) and sonicated for 2 h in an ultrasonic bath (Wisd, WUC-D06H) at 40 kHz and 300 W. The supernatant was filtered through a microporous membrane (0.45 µm). After evaporation, the concentrating process was performed using rotary vacuum, the evaporate was collected to calculate the %yield.
To determine the cordycepin and adenosine levels by HPLC, 0.1 g of crude extract was added to a 10 mL mobile phase solution (water: acetonitrile), and the mixture was centrifuged. Before injection into the HPLC system, the mixture was filtered through a microporous membrane (0.45 µm). Analysis was performed using a reversed-phase column (VertiSep™ UPS C18 5 µm, 4.6 mm × 250 mm, Vertical, USA). Separation was performed using 0.1% trifluoroacetic acid as a mobile phase, with a 1 mL/min flow rate at 30 °C. For analysis, 20 µL extract was sprayed onto an analytical column. The DAD detector was set at 260 nm to acquire chromatograms. Cordycepin and adenosine levels were estimated by calculating the peak area based on standard curves (10, 25, 50, 70, 100, and 150 µg/mL). The relationship between the corresponding retention time and peak area standard was quantified. The results are expressed as mg/100 g of extract.

Measurement of β-Glucan Content
The β-glucan content of cordyceps extract was determined using the Megazyme Yeast and Mushroom Kit (K-YBGL) (Megazyme Ltd., Bray, Co., Wicklow, Ireland). For total glucan measurement, a 100 mg dry sample was added to 1.5 mL HCl (10 M). After incubation at 30 °C for 45 min, 10 mL of distilled water was added. The mixture was incubated at 100 °C for 2 h and cooled to room temperature, and then 10 mL KOH (2 M) was added. The mixture was transferred into a volumetric flask, and the volume was adjusted to 100 mL by adding 200 mM sodium acetate buffer (pH 5.0). The solution was then centrifuged at 8,000 rpm for 10 min at 30 °C. Next, 0.1 mL of supernatants were pipetted into centrifuge tubes, and 0.1 mL of the mixture solution prepared by mixing Exo-1,3-β-glucanase (20 U/mL) and β-glucosidase (4 U/mL) in 200 mM sodium acetate buffer (pH 5.0) was added. After incubation at 40 °C for 60 min, 3.0 mL GOPOD reagent was added, and the solution was incubated at 40 °C for 20 min. Absorbance was measured at 510 nm using a microplate reader (model Enspire; PerkinElmer, Inc.).
To evaluate the α-glucan content, a 100 mg dry sample was added to 2 M KOH (2 mL) and stirred for 20 min. Eight milliliters of 1.2 M sodium acetate buffer (pH 3.8), 0.2 mL amyloglucosidase (1,630 U/mL), and invertase (500 U/mL) were added to the mixture. After incubation at 40 °C for 30 min, the mixture was centrifuged at 8,000 rpm at 20 °C for 10 min. Then, 0.1 mL of the supernatant was pipetted into a centrifuge tube, and 0.3 ml GOPOD reagent was added. The mixture was kept at 40 °C for 20 min. The absorbance of the sample was measured at 510 nm using a microplate reader (model Enspire; PerkinElmer, Inc.). β-Glucan was calculated following Eq. (2). The results are expressed as percentage of the dry weight (%w/w DW).

SOD Activity
SOD activity was assayed by determining the inhibition of the photochemical reduction of nitroblue tetrazolium (NBT) [27]. The assay mixture was composed of 50 mM sodium phosphate buffer (pH 7.6), one mM EDTA, 12 mM L-methionine, 50 μM NBT, ten μM riboflavin, 50 mM sodium carbonate, and 100 μL blended C. militaris extract. Distilled water was added to a final volume of 3.0 mL. The mixture was placed at a distance of 20 cm from a fluorescent lamp. After 15 min of light exposure, SOD activity was monitored by measuring the increase in absorbance at 560 nm in a microplate reader (model Enspire; PerkinElmer, Inc.). The results are expressed in one unit of SOD, defined as the amount of enzyme required to inhibit NBT photoreduction by 50%.

Glutathione S-Transferase (GST) Activity
GST activity was measured as previously described [28]. Briefly, the mixture contained 40 mM 1-chloro-2,4-dinitrobenzene (CDNB), 900 µL of 0.1 M potassium phosphate buffer (pH 6.5), 50 µL of 0.1 M glutathione, and 25 µL of enzyme extract in a final volume of 1 mL. The assay solution was incubated at 35 °C for 3 min, and the absorbance of the resulting solution was measured at 340 nm for 5 min at 10 s intervals using a microplate reader (model Enspire; PerkinElmer, Inc.) GST activity is expressed as nmol s −1 mg of protein −1 .

Measurement of Malondialdehyde Concentration
Malondialdehyde (MDA) levels monitored as lipid peroxidation (a nonenzymatic oxidative stress maker) were measured according to the procedure described by the manufacturer's instructions (MDA test kits from Sigma Aldrich). A 0.1 g dry sample was briefly immersed in 5 mL (100 g/L trichloroacetic acids (TCA)). The mixture was vortexed and centrifuged at 9,000 rpm for 20 min at 4 °C. After centrifugation, 3 mL of supernatant was mixed with 3 mL of 0.67% thiobarbituric acid (TBA). The mixture was incubated at 100 °C for 20 min and then placed on ice and allowed to cool to room temperature. The MDA concentration was determined at 450, 532, and 600 nm using a microplate reader (model Enspire; PerkinElmer, Inc.) and is expressed as nmol/L of dry weight (nmol/L DW).

Measurement of Total Phenolic Content (TPC)
The TPC of mixed C. militaris was estimated using the Folin-Ciocalteu assay, according to a previously described method [26]. Briefly, 125 µL of blended C. militaris extract was added to 1.8 mL Folin-Ciocalteu reagent. After 5 min, 1.2 mL of 15% sodium carbonate solution was added. The mixture was shaken and incubated at room temperature for 90 min. The absorbance of the mixture was measured at 765 nm using a microplate reader (model Enspire; PerkinElmer, Inc.). Gallic acid was used as a standard. Thus, the TPC of mixed C. militaris is expressed as mg of gallic acid equivalents per g of extract (mg GAE/g extract).

Measurement of Total Flavonoid Content (TFC)
The TFC of blended C. militaris was determined using a colorimetric method [26]. First, 0.5 mL of blended C. militaris extract was mixed with 0.15 mL methanol, followed by 0.1 mL of 10% aluminum chloride, 0.1 mL of 1 M potassium acetate, and 2.8 mL of distilled water. The mixture was vortexed and allowed to stand at room temperature for 30 min. Next, the developed color of the mixture was measured at 415 nm using a microplate reader (model Enspire; PerkinElmer, Inc.). Rutin was used as a standard, and the concentration of flavonoids is shown as mg of rutin equivalents per g of extract (mg RE/g extract).

DPPH Radical Scavenging Activity
The DPPH radical scavenging activity of the blended C. militaris was determined according to a previously reported method [26]. Briefly, 24 mg of DPPH dissolved in 100 mL methanol was used to prepare the stock reagent solution, which was stored at − 20 °C until use. Next, 10 mL stock solution was mixed with 45 mL methanol to make the working solution, and an absorbance value of 1.1 ± 0.02 was measured at 517 nm using a microplate reader (model Enspire; PerkinElmer, Inc.). Blended C. militaris extract with different CPJ treatment times was allowed to react with 3 mL of DPPH solution. The mixture was centrifuged strongly in the dark at room temperature for 30 min. Discoloration of the mixture was achieved at 517 nm. The percentage of DPPH radical inhibition was calculated using the following equation: where A c and A s are the absorbance of the control and sample, respectively.
The percentage (%) plot of the free radical scavenging activity of ascorbic acid versus its concentration was used as a standard curve. The results are expressed as mg ascorbic acid equivalent antioxidant capacity in 1 g of extract (mg AEAC/g extract).

Ferric-Reducing Antioxidant Power (FRAP)
The FRAP assay of blended C. militaris was performed as previously described [26]. In brief, the working FRAP reagent was prepared by mixing 300 mM acetate buffer (pH 3.6), ten mM 2,4,6-tripyridyl-s-triazine (TPTZ) in 40 mM HCl, and 20 mM FeCl 3 ·6H 2 O in a 10:1:1 ratio prior to use and incubated at 37 °C in a water bath for 10 min. A 0.1 mg sample extract was mixed with 2.7 mL of the FRAP reagent and 0.2 mL of distilled water. After 30 min in the dark, the absorbance was measured at 593 nm using a microplate reader (model Enspire; PerkinElmer, Inc.). The ferric reducing ability is expressed as mg Trolox equivalent antioxidant capacity g −1 of extract (mg TEAC/g extract).

ABTS Radical Scavenging Activity
The ABTS radical cation scavenging activity of the samples was determined according to a previously described procedure [29]. Briefly, the ABTS reagent was prepared by mixing 8 mg/mL ABTS solution with 1.32 mg/mL potassium persulfate. The mixture was incubated in the dark at room temperature for 12 h. Then, 20 µL of sample extract was added to 200 µL of ABTS working solution. After 10 min in darkness, the absorbance was measured at 734 nm using a microplate reader (model Enspire; PerkinElmer, Inc.). Radical inhibition was calculated using Eq. (3). Trolox was used as a standard. Thus, the results are expressed as mg of Trolox antioxidant capacity g −1 of extract (mg TEAC/g extract).

Statistical Analyses
All experiments were performed in triplicate, and the data are expressed as the mean ± standard deviation (SD). Significant differences in the results were assessed using one-way analysis of variance (ANOVA) with Duncan's multiple range test (p < 0.05) using SPSS software, version 26.0. Figure 2 shows the discharge voltage and current characteristics of the air CPJ operating under a duty cycle of 50% with an airflow rate of 5 L/min. This experiment demonstrated that the voltage and current discharges were presented from peak to peak. The highest peakto-peak voltage and current were ~ 10.2 kV and ~ 50 mA, respectively. A similar waveform was presented in pulse-modulated radio-frequency dielectric barrier discharge [30]. The voltage and current characteristics during discharge ignition and extinguishment phases are depicted in Fig. 3. For the ignition phase, the discharge voltage and current started at 0.5 and 0.75 ms, respectively (Fig. 3a). Discharge voltage took ~ 0.5 ms to get the stable state (from ~ 0.9 to ~ 5 kV), and discharge current took ~ 0.2 ms from ~ 4.5 to ~ 25 mA. For the extinguishment phase, the discharge voltage and current simultaneously diminished to In addition, the average power dissipated by the air CPJ system was 5.08 W. Figure 4 demonstrates the emission bands of the dominant N 2 second positive system [ N 2 (C 3 ∏ u ) → N 2 (B 3 ∏ g ) at 268-546 nm)] and N 2 first negative system [(N + 2 (B 2 Σ + u ) → N + 2 (X 2 Σ + g ) at 286-587 nm)]. The nitrogen ion (N + ) peak was observed at 593.8 nm. As N + at 500.6 nm was detected, this ion was of interest in this experiment because the highest electron energy was required for ionizing the atom and molecule. This ion was also found in a compact pulse-modulated air plasma jet [31]. The occurrence of nitrogen species in the plasma active zone is shown in reactions (R1-R9) ( Table 1). For the generation of the N 2 first negative system, firstly, the N + 2 (B 2 Σ + u ∶ 19eV) and N + 2 (X 2 Σ + g ∶ 15.5eV) can be populated through direct electronic ionization from the ground state N 2 molecule [ N 2 (X 1 Σ + g ) ] (reactions (R1-R2)) and step-wise electronic ionization from the metastable N 2 molecule [ N 2 (A 3 Σ + u ) ] (R3-R4). Then, the N 2 first negative system occurs by the emission of N + 2 (B 2 Σ + u ) to N + 2 (X 2 Σ + g ) , as described in the reaction (R5) [32]. The N 2 (A 3 Σ + u ∶ 6.17 eV, τ rad = 13 s) molecules can be generated via the direct electronic excitation from N 2 (X 1 Σ + g ) molecules (reaction (R6)). The higher excited molecular states N 2 (B 3 ∏ g : 7.35 eV, τ rad = 13 ms) and N 2 (C 3 ∏ u : 11.3 eV, τ rad = 38 ns) are occurred by direct electronic impact (reactions (R7-R8)). These short-life metastable molecules [ N 2 (B 3 ∏ g )] and [(N 2 (C 3 ∏ u )] can be easily populated by the electronic excitation process stepping from the long-life metastable molecules N 2 (A 3 Σ + u ) . During these phases, the higher energy electron N 2 (C 3 ∏ u ) predominantly emits the N 2 second positive system, which leads to N 2 (B 3 ∏ g ) (reaction (R9) [32].

Discharge Voltage and Optical Emission Spectrum Measurement
At room temperature and 60% RH, the ˙OH bands were 306.6 and 309.4 nm, respectively (inset of Fig. 4). The mechanisms of ˙OH bands were described in our previous work [23]. An atomic hydrogen line (H) at 656.8 nm and atomic oxygen lines (O) at 777.7 and 822.7 nm were observed. For O generation, the O 2 in the gas phase is interacted with electrons with an electron energy of 13.6 eV [33], leading to generate excited O(1D) and groud O(3P), as shown in the reaction (R10) [34,35].

Effects of CPJ Treatment on Solution Characteristics
As the experimental results showed the occurrence of NO − 2 and NO − 3 in the liquid medium, the mechanisms were depicted in reactions (R18-R19) [34]. The interaction between NO 2 and H 2 O leads to NO − 3 and H + formation (R18). Similarly, the interaction of NO 2 , NO, and H 2 O generates NO − 2 and (hydrogen) H + (R19). Various studies have reported that air or oxygen cold plasma-activated in a solution can promote the concentration of NO − 2 and NO − 3 which leads to lower acidification and higher conductivity in the solution due to the existence of H + [36].

Effect of CPJ Treatment on Fruiting Body Production, Cordycepin, and Adenosine of Blended C. militaris
The effect of CPJ treatment on the fresh and dry weights of the blended C. militaris fruiting bodies is presented in Table 2. The 30 s CPJ treatment induced the highest fresh weight of the blended C. militaris fruiting bodies, which significantly increased by 22.49% compared to CK2. Nonetheless, there was no significant difference in fresh weight among the CK2, 60, 90, and 120 s CPJ treatments. Additionally, the fresh weight of CK2 was significantly higher than that of CK1. As shown in Table2, the maximum dry weight value of blended C. militaris fruiting bodies was observed in response to the 30 s CPJ treatment, which was increased by 22.56% compared with CK2. Insignificant changes in the dry weight of , and c pH, conductivity, and temperature of the liquid medium 1 3 C. militaris between the CK2 and CPJ treatments from 60 to 120 s CPJ treatments were observed. There was no significant difference between CK1 and CK2. Figure 6 shows the fruiting bodies of blended C. militaris treated with CPJ and untreated. The artificial fruiting bodies of blended C. militaris were white in color, while the brown rice media were yellow in color due to the color of C. militaris. The length of the artificial fruiting bodies of blended C. militaris treated with 30 s CPJ was greater than that of the untreated samples.
The major bioactive components found in C. militaris are cordycepin and adenosine. The cordycepin structure is similar to adenosine, but the 3'-hydroxyl group does not exist. Cordycepin exhibits a variety of biological and pharmacological activities that have health benefits, including anticancer, antifungal, antiviral, and immunomodulatory activities [37,38].
The values of cordycepin and adenosine in the fruiting bodies of blended C. militaris are depicted in Table 2. The cordycepin values of the blended C. militaris samples for the 30 and 60 s CPJ treatments were significantly greater than those of CK2, while the 90 and 120 s CPJ treatments had no significant differences. The 30 s CPJ treatment exhibited the No studies have shown that cold plasma stimulates fruiting body production, cordycepin, and adenosine in Cordyceps fungus. Previous investigations demonstrated that nitrogen sources (NH 4 NO 3 and NaNO 3 ) were prepared in liquid culture to promote dry mycelia weight and cordycepin [4,39,40]. In the present study, nitrogen sources ( NO − 2 and NO − 3 ) were generated using CPJ activated in liquid media before the fermentation process. Increment of NO − 2 and NO − 3 caused the cell growth and metabolic biosynthesis of blended C. militaris to improve during fermentation. Fruiting body development in solid media culture, cell growth, and bioactive components of C. militaris has been correlated with nucleotide, carbohydrate, and amino acid metabolism and could continue development from the liquid media culture to the solid media culture [8]. Generally, the glutamine and glutamate pathways based on the patterns of amino acid metabolism are the essential biosynthesis pathways of C. militaris [41]. The CPJ-activated liquid media culture generated RONS, and electron pairs led to amino acid generation during the fermentation process. Thus, cell growth and metabolic biosynthesis of blended C. militaris were improved. Due to proper RONS generation, the 30 s CPJ treatment represented the optimal conditions for fruiting body production and cordycepin levels in blended C. militaris. In contrast, the 90 and 120 s CPJ treatments might overproduce RONS, leading to decreased fruiting body production and cordycepin/adenosine levels in blended C. militaris.

Changes in β-Glucan Content
β-glucan is a natural molecule readily available in oat, barley, and mushrooms that has widely been added to food due to having many benefits for improving health, such as attenuating insulin resistance, dyslipidemia, hypertension, and obesity. The critical structure of β-glucan comprises β-d-glucopyranose units linked by (1 → 6) glycosidic bonds for fungal sources and (1 → 4) and (1 → 3) glycosidic bonds for cereals [42].
Changes in β-glucan content in the fruiting bodies of blended C. militaris after CPJ treatment are depicted in Fig. 7. The results showed that 30 s CPJ treatment had no significant effect on β-glucan levels in the fruiting bodies of the blended C. militaris compared to CK2. Nevertheless, β-glucan levels of blended C. militaris were diminished by 7.81% (60 s CPJ), 15.94% (90 s CPJ), and 18.80% (120 s CPJ) compared to CK2. In addition, β-glucan content in the fruiting bodies of the blended C. militaris of CK2 was significantly higher than that of CK1, increasing by 39.54%. Several studies have demonstrated that the degradation of β-glucan occurs with ROS formation, especially hydroxyl radicals [43]. The attraction of ˙OH can breakdown β-glucan, leading to degradation [44]. In the present study, the β-glucan content in the fruiting bodies of blended C. militaris at the 30 s CPJ treatment might slightly scavenge ROS due to a small increase in its content. However, the 60 to 120 s CPJ treatment might overdose the system on ˙OH, resulting in the degradation of β-glucan in the fruiting bodies of blended C. militaris.

Changes in SOD and GST Activities and MDA Levels
GST is an essential mediator in oxidative stress responses in living organism cells. GST is part of a large family of detoxifying enzymes, which can play an essential role in drug resistance. GST catalyzes the conjugation of glutathione (GSH) to an electrophilic center of endogenous and exogenous compounds, which results in the formation of GSS conjugates [45].
Changes in GST activity in the fruiting bodies of blended C. militaris are shown in Fig. 8a. The highest GST activity was observed in the 30 s CPJ treatment, which was significantly increased by 17.42% compared to CK2. On the other hand, a slight decrease in GST activity was noted at the 60, 90, and 120 s CPJ treatments, but these treatments were not significantly different compared to the CK2. In addition, no significant change in GST activity between CK1 and CK2 was observed.
SOD, an antioxidant enzyme, is a scavenger of the superoxide anion that functions by catalyzing the free radical to oxygen and hydrogen peroxide. Thus, it is the first line of defense against oxidative stress in living organisms [27]. SOD is widely used in anti-inflammatory, antitumor, radiation protection, and anti-senility applications. As shown in Fig. 8b, the highest SOD activity in the fruiting bodies of blended C. militaris was observed in response to the 30 s CPJ treatment, which was significantly increased by 34.71% compared to CK2. However, the 60, 90, and 120 s CPJ treatments did not exhibit SOD activity. The lowest SOD activity was found in response to 120 s CPJ treatment, which was decreased by 4.01%. There was no significant difference between CK1 and CK2.
The present study monitored oxidative stress in response to CPJ treatment with respect to lipid peroxidation by determining MDA levels. The MDA content in the fruiting bodies of blended C. militaris is illustrated in Fig. 8c. The MDA content increased with increasing CPJ treatment time. The MDA content of the 60, 90, and 120 s CPJ treatments was significantly increased by 42.01, 41.46, and 55.59%, respectively, compared to CK2. However, there was no significant change in the MDA content between the 30 s CPJ treatment and CK2, but it did slightly increase by 6.58%. Additionally, no significant difference was observed between CK1 and CK2.
Air cold plasma-generated ROS (hydroxyl radical, ozone, atomic oxygen, peroxide, and superoxide) are essential in cell death [46]. In contrast, normal cellular aerobic metabolism produces low to moderate levels of ROS. Typically, ROS are be induced via the effects of environmental and physiological stresses, leading to oxidative damage in living organism cells. Thus, to preserve cell structures and biomacromolecules, living organisms can increase the levels of antioxidant enzymes (SOD, CAT, and GST) and nonenzymatic compounds to resist oxidative stress and scavenge free radicals [47]. In our study, the 30 s CPJ treatment significantly increased SOD and GST activities in the fruiting bodies of blended C. militaris. This finding indicates that CPJ treatment plays an essential role in decreasing oxidative damage and maintaining normal physiological and metabolic activities that promote fruiting body production and cordycepin values in blended C. militaris. Similar results previously showed that cold air plasma also stimulated SOD and GST activities in T. castaneum [48].
High concentrations of oxidative stress can damage proteins and membrane lipids, which is indicated by the MDA content. In the present study, the 30 s CPJ treatment did not alter membrane lipid peroxidation damage in the fruiting bodies of blended C. militaris. However, the 90 and 120 s CPJ treatments generated a high MDA concentration, which reduced the values of cordycepin, adenosine, and β-glucan in the fruiting bodies of blended C. militaris. Several studies have shown that plasma treatment improves seed germination and seedlings in tomatoes [49] and oilseed rape under drought stress [18] by increasing antioxidant enzyme activities and reducing MDA concentrations.

Changes in Total Phenolic Content (TPC) and Total Flavonoid Content (TFC)
Phenolic compounds are secondary metabolites that have been widely isolated in various plants, such as phenolic acids, flavonoids, stilbenes, lignans, and tannins. These compounds are free radical scavengers for stabilizing oxidative stress and protecting biological macromolecules from environmental stress conditions. Phenolics are also potential primary nutrition sources with several health benefits, including antibacterial, anti-inflammatory, and antimutagenic activities [50]. Figure 9 shows changes in TPC and TFC in the fruiting bodies of blended C. militaris. As shown in Fig. 9a, the results indicated that the TPC of blended C. militaris treated with CPJ was significantly higher than that of CK2. The TPC increased with increasing CPJ treatment time from 30 to 90 s, while TCP at 120 CPJ treatment slightly declined compared to the 90 s CPJ treatment. The maximum TPC value in the fruiting bodies of blended C. militaris was 75.54 mg GAE/g extract at the 90 s CPJ treatment (increased by 27.10%), followed by 120 60 and 30 s CPJ treatments, at 69.69 mg GAE/g extract (increased by 17.26%), 68.19 mg GAE/g extract (increased by 14.74%), and 61.45 mg GAE/g extract (increased by 3.40%), compared to CK2. In addition, the TPC in CK2 was also significantly different from that in CK1.
Flavonoids, the major group of phenolic compounds, are most plentiful in plants and fungi [51]. Figure 9b shows that CPJ treatment provided the optimal values of TFC in the blended C. militaris fruiting bodies under all treatment conditions, ranging from 0.015 to 0.17 mg RE/g extract. It was found that the CPJ treatments yielded much higher TFC values, approximately 1-to tenfold, than the values obtained from CK2. The highest levels of TFC were observed in response to 60 s CPJ treatment. No significant difference between CK1 and CK2 was found.
In this context, phenolics, including TPC and TFC in blended C. militaris fruiting bodies, were increased in response to CPJ treatments. These results are similar to earlier studies in which CPJ improved TPC in brown rice and TFC in lettuce and basil leaves [21,52]. Cold plasma pretreatment with hot air drying also increased TPC and TFC in shiitake mushrooms [53]. Phenolics act as antioxidants and have an aromatic ring with one or more hydroxyl groups [54]. Therefore, phenolic compounds may increase under oxidative stress, including ROS, UV radiation, or extreme temperature, which protects against potential damage to living organism cells. In addition, different solvent extraction [55] and cultivation conditions were factors that affected the levels of phenolics in Cordyceps.

Changes in Antioxidant Activity
ROS and RNS are typically generated from cellular metabolism, and several environmental conditions are considered primary factors that can disrupt DNA, lipids, and proteins and are linked to disease and health issues. Therefore, antioxidants are created by several mechanisms to scavenge these reactive species. Recently, the antioxidant activity of C. militaris was found to be significantly related to its high levels of TPC and TFC [45]. Our study evaluated changes in antioxidant capacity in blended C. militaris fruiting bodies based on DPPH and ABTS radical scavenging activities and ferric-reducing antioxidant power (FRAP).
The DPPH assay is frequently used to determine free radical scavenging activity based on hydrogen donation. Figure 10a shows that the DPPH free radical scavenging ability in blended C. militaris fruiting bodies under the 60 to 120 CPJ treatment was significantly higher than CK2, which was enhanced by approximately 12 to 17% compared to CK2. The maximum value of DPPH free radical scavenging ability was observed in response to 90 s CPJ treatment. However, no significant changes were observed between CK1 and CK2 with the 30 s CPJ treatments.
Changes in the ABST assay of blended C. militaris extracts are depicted in Fig. 10b. The 60 and 90 s CPJ treatments conveyed the highest scavenging activity in fruiting bodies of mixed C. militaris, with 171.45 mg TEAC/g extract (an increase of 6.82%) and 171.01 mg TEAC/g extract (an increase of 6.54%) compared to CK2. On the other hand, the 120 s CPJ treatment significantly inhibited the ABTS scavenging activity of blended C. militaris with 145.93 mg TEAC/g extract, decreasing by 9.08%. In addition, no significant 1 3 changes in the ABTS scavenging activity of mixed C. militaris were observed among the CK1, CK2, and 30 s CPJ treatments.
The antioxidant activity of blended C. militaris extracts determined by FRAP is demonstrated in Fig. 10c. The ferric reducing power of mixed C. militaris was significant after CPJ treatments (from 30 to 90 s) compared to CK2 The blended C. militaris extract after 60 s CPJ treatment exhibited the highest ability to reduce Fe 3+ to Fe2 + (3089.78 mg TEAC/g extract, increasing by 50.35%), followed by 30 s CPJ treatment (2669.47 mg TEAC/g extract, increasing by 29.90%), and 90 s CPJ treatment (2241.34 mg TEAC/g extract, increasing by 9.07%), compared to CK2. In contrast, a decrease in ferric reducing power was found after 120 s of CPJ treatment (1551.26 mg TEAC/g extract, decreasing by 26.46%) compared to CK2. There was also a significant difference between CK1 and CK2. Various investigations have reported that the antioxidant activity in plants positively corresponds to bioactive compounds [55]. In the present study, Pearson's correlation coefficient was determined to investigate the relationship between the value of bioactive compounds (TPC and TFC) and their bioactive activities (DPPH and ABTS scavenging activities, FRAP), as depicted in Table 3. The results showed that DPPH radical scavenging activity was strongly related to TPC (R = 0.909). FRAP and TFC had a high linear correlation coefficient (0.851), and the ABTS radical scavenging activity was moderately correlated with TFC (R = 0.590). Therefore, it can be supposed that antioxidant activities in blended C. militaris fruiting bodies treated with CPJ could be governed by TPC and TFC. Similar to previous studies [56], the antioxidant activities in C. militaris with different solvent extracts were strongly correlated to the TPC and TFC.
No research has reported the influence of cold plasma treatment on changes in the levels of bioactive compounds or antioxidant assays in C. militaris fruiting bodies. However, several investigations have been conducted regarding the effect of cold plasma on increasing antioxidant activities in brown rice during germination [10] and basil leaves [21]. Thus, CPJ treatment, which can be considered a stress condition that initiates the accumulation of blended C. militaris bioactive compounds (phenolic and flavonoid) and antioxidant activities, would be beneficial for human health.

Conclusion
The present study investigated fruiting body production, bioactive phytochemicals, bioactive compounds, antioxidant enzymes, and antioxidant activities in blended C. militaris using a CPJ operating under a 50% duty cycle of a neon transformer and a fixed airflow rate of 5 L/min. The production of reactive species NO, ˙OH, and O was measured in the gas phase. The interaction of these reactive species with the H 2 O molecule led to the generation of NO − 2 and NO − 3 in the liquid media before the fermentation process of blended C. militaris. For cultivation, the development of fruiting bodies of blended C. militaris was grown on solid media cultured for 90 days. The results showed that fruiting body production in blended C. militaris was improved in response to CPJ treatments as determined by increased cordycepin and antioxidant enzyme activities and the reduced MDA levels. Moreover, the bioactive compounds (TPC and TFC) and antioxidant activities in blended C. militaris fruiting bodies were significantly increased in response to CPJ treatments. Consequently, CPJ could play an essential role as a promising pretreatment technique for enhancing fruiting body production and improving nutritional values in blended C. militaris. Table 3 Pearson's correlation coefficients between the contents of bioactive compounds (TPC and TFC) and antioxidant activities (DPPH and ABTS scavenging activities, FRAP) *Correlation is significant at p < 0.05 (two-tailed). **Correlation is significant at p < 0.01 (two-tailed)