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Optimization of Growth Conditions and Biological Activities of Nepalese Ganoderma lucidum Strain Philippine

Optimization of Growth Conditions and Biological Activities of Nepalese Ganoderma lucidum Strain... Hindawi Advances in Pharmacological and Pharmaceutical Sciences Volume 2021, Article ID 4888979, 7 pages https://doi.org/10.1155/2021/4888979 Research Article Optimization of Growth Conditions and Biological Activities of Nepalese Ganoderma lucidum Strain Philippine 1 1,2 1,3 4 Krishna Subedi, Buddha Bahadur Basnet , Raju Panday , Manisha Neupane , and Giri Raj Tripathi Central Department of Biotechnology, Tribhuvan University, Kirtipur, Kathmandu, Nepal Faculty of Sciences, Nepal Academy of Science and Technology, Khumaltar, Lalitpur, Nepal National Forensic Science Laboratory, Khumaltar, Lalitpur, Nepal Department of Biotechnology, National Institute of Science and Technology, Lainchaur, Kathmandu, Nepal Correspondence should be addressed to Buddha Bahadur Basnet; budbsn.btechnep@gmail.com Received 25 July 2021; Revised 17 September 2021; Accepted 21 September 2021; Published 4 October 2021 Academic Editor: Ghulam Hussain Copyright © 2021 Krishna Subedi et al. (is is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Ganoderma lucidum has been extensively studied for its valuable medicinal importance. In this study, the artificial cultivation of G. lucidum strain Philippine in different culture media, including sawdust substrate, was performed and optimized on the Potato Dextrose Agar (PDA) media. Phytochemical, antibacterial, and antioxidant analyses were performed and compared between the ethanol extracts prepared from two different cultures (fruit from synthetic log culture and mycelia from PDA media culture). Both the 200 mg/mL and 100 mg/mL concentrations of extracts inhibited all the tested bacteria, and the results were promising than the corresponding control using antibiotics. (e fruit extract showed higher antioxidant potential (150.6 ± 56.92 mg ascorbic acid equivalent/g extract) than mycelial extract (144.28± 81.72 mg ascorbic acid equivalent/g extract). (e results indicate that fruiting bodies of G. lucidum cultivated in a complex dust medium possess higher antioxidant properties than mycelia culture, which can be further explored for therapeutic applications. species include polysaccharides, including triterpenoids, 1. Introduction nucleotides, sterols, steroids, fatty acids, and proteins/pep- Ganoderma (Basidiomycota, Polyporales) is one of the well- tides with the most pharmacologically active compounds known medicinal polypore fungi in the family Gano- triterpenoids and polysaccharides [4]. It has been reported dermataceae and is widely used in East Asia, America, and that G. lucidum contains over 400 bioactive compounds, other countries [1]. Among the known, more than 400 which have several medicinal effects, remarkably such as species, only red, white, black, yellow, blue, and purple antitumor and anti-inflammatory, antimicrobial, antifungal, Reishi have been explored for their potential health-bene- antiviral (specifically against herpes and HIV) effects, ficial properties [2]. Of them, red Reishi (G. lucidum) and antioxidative, and radical scavenging effects [5–9]. black Reishi (G. sinensis) have shown the most significant Several reports revealed the successful artificial culti- health-strengthening effects [3]. East Asian countries con- vation on solid substrates utilizing sawdust and agricultural sumed Reishi for their medicinal value rather than nutri- wastes (rice bran, wheat bran, sugarcane bagasse, rice husks, peanut hulls, coconut fiber, banana leaves, etc.) as well as in tional value to extend the life span and increase youthful vigour and vitality [1]. tea waste [3]. Generally, G lucidum is cultivated on solid- G. lucidum, a cosmopolitan mushroom species, is a state fermentation and takes about six months to form the polypore rack mushroom that changes color during the fruiting body [10]. However, the process is time consuming morphogenesis process from orange-white to bright red. (e and difficult to control, dictating other alternatives for major chemical constituents of G. lucidum and related growing the fungi. One such method of choice is the 2 Advances in Pharmacological and Pharmaceutical Sciences submerged cultivation of mycelia to produce bioactive 2.3. Growth Parameter Optimization in Plate Culture compounds, which can be rapid, economically feasible, and 2.3.1. pH Optimization. PDB (100 mL) was prepared and controllable [11]. adjusted to varied pH from 3.5 to 6.75 (0.25 interval). (en, Most human diseases result from the uncontrolled agar powder (1.5 g) was added to each bottle, shaken, and production of reactive oxygen species (ROS), including free autoclaved before pouring into sterile Petri plates in trip- radicals. When unchecked, the endogenous mechanism for licates. A very minute mat with agar was plugged by using a free radical scavenging in living cells can lead to insufficient borer and inoculated into plates, incubated at 28 C for four neutralization of free radicals, resulting in major deterio- days, and their margin was marked. An increase in mycelial ration conditions such as cardiovascular diseases and cancer length was reported every day, and the growth rate was [12]. Molecules derived from natural sources such as fungi calculated. play a significant role in developing and discovering novel drugs for treatment. Currently, many active compounds from G. lucidum are being studied as treatment options [13]. 2.3.2. Temperature Optimization. Laboratory-prepared PBD (is study cultivates the G. lucidum in various media and was adjusted to pH 5.0. After adding agar, the plates were determines the optimum growth parameters (pH, temper- ° ° incubated at different temperatures (20 C to 36 C) in trip- ature, and carbon sources). In addition, the determination of licates and corresponding control for four days. An increase phenolic content, flavonoid content, antioxidant activity, in mycelia length was measured on the subsequent days, and antibacterial activity of extracts from the mycelia and which indicated lengthwise growth per day. fruiting body was performed. 2. Materials and Methods 2.3.3. Carbon Source Optimization. Liquid broth (LB) (100 mL) media were prepared without a carbohydrate 2.1. Strain Used. G. lucidum strain Philippine was purchased source, and pH was adjusted to 5.0. (en, 15 g different from the Centre for Agricultural and Technological Train- carbohydrate sources (sucrose, trehalose, glucose, lactose, ing, Lalitpur, and cultured in Potato Dextrose Agar (PDA) cellulose, maltose, sorbose, and xylose) as labeled in the flask (Hi-Media, Mumbai, India) plates and slants. (e cultured were added into respective flasks and autoclaved. Each plates were kept at 4 C for research purposes. medium was poured into plates in triplicates, inoculated with small mycelium spawn, and incubated for four days at 28 C, and their margin (apex) was marked. Everyday, the 2.2. Mass Culture increase in mycelia length was measured, which indicated lengthwise growth per day. 2.2.1. Culture on Synthetic Logs. A synthetic log was pre- pared by mixing sawdust powder, raw wheat bran, and raw rice bran. Polypropylene bags (12 ×12 cm ) were filled with 200 g of substrate, sealed with a cotton plug, autoclaved, and 2.4. Sample Preparation and Extraction. (e fruiting bodies maintained to 65% moisture. (e prepared synthetic logs from solid culture and upper mycelia culture from lawn were inoculated with fresh spawn and incubated for 60 days culture were harvested and then dried at 60 C until a at 28 C in diffused light with a relative humidity of 90–95%, constant weight was obtained. (e harvested culture was which was maintained with sprinkling sterile water (0.1% powdered using liquid nitrogen (−196 C) and crushing by calcium) twice daily. Few 1 mm pores were punched, and using a mortar and pestle. 10 gm of each powdered sample sterile cotton was loosely plugged for air circulation. was extracted by 400 mL ethanol using Soxhlet’s apparatus at 60 C, and the extract was dried using a rotary vacuum evaporator. Finally, a 100 mg/mL solution was prepared as a 2.2.2. Culture in Potato Dextrose Broth (PDB). PDB was working solution. prepared in the laboratory by boiling potatoes (300 g) in 1 L water and filtering to obtain the potato broth. Glucose (25 g) was added, and the final pH was adjusted to 5.0 before 2.5. Phytochemical Estimation autoclaving. (e sterile media were then inoculated with 2-3 pieces of fresh mycelia taken from potato dextrose agar 2.5.1. Total Phenolic Content Estimation. Total phenolic (PDA) plate and cultured in a shaking incubator (120 rpm) content was determined with the Folin–Ciocalteu (FC) re- at 28 C for 18 days. agent as in [14]. Briefly, 100 μL of each sample was dissolved in 500 μL (1 :10 v/v dilution) FC reagent and 1.5 mL (20%) sodium carbonate. (e mixture was vortexed for 30 s and 2.2.3. Culture on PDA. PDA plates inoculated with fresh incubated for 2 h in the dark at room temperature for color and actively growing mycelia and incubated at 28 C for one development. Absorbance was then measured at 765 nm week to obtain fresh inoculums as well as for one month for using the Genesys UV/Vis spectrophotometer (CA, USA). extraction. PDA plates were maintained at 4 C for long-term Total phenolic content (TPC) was measured as milligrams of storage. (e growth optimization of the culture was carried gallic acid equivalent/g (mgGAE/g) of sample dry weight out for optimal growth and further experiments. using the standard curve and the equation. Advances in Pharmacological and Pharmaceutical Sciences 3 2.5.2. Total Flavonoid Content (TFC) Estimation. Total fla- adding sample extract to 2.5 mL phosphate buffer (0.2 M, pH vonoid content in the extracts was estimated using the alu- 6.6) and 2.5 mL potassium ferricyanide (1%, w/v) and in- minium chloride colorimetric method [14]. Briefly, 0.5 mL of cubated for 30 min at 50 C. (en, 2.5 mL of 10% tri- plant extract (20 mg/mL) was mixed with 0.1 mL of 10% chloroacetic acid was added to the mixture and centrifuged aluminum chloride, 0.1 mL of 1.0 M potassium acetate, and for 10 min at 3000g. 2.5 mL of the supernatant was then 1.3 mL 90% ethanol. Absorbance was measured at 415 nm in diluted with 2.5 mL distilled water and mixed with 0.5 mL the Genesys UV/Vis spectrophotometer after incubating the freshly prepared ferric chloride (0.1%, w/v). (e absorbance mixture for 40 min at room temperature. TFC was then was measured at 700 nm using the Genesys UV Vis spec- calculated using quercetin as a standard and expressed as mg trophotometer after incubating for 30 min. Increased ab- quercetin equivalent/g (mgQE/g) of sample dry weight. sorbance of the reaction mixture indicated increased reducing power. All experiments were performed in trip- licate using butylated hydroxytoluene as a positive control. 2.6. Antibacterial Activity. Six clinical strains of bacteria (Staphylococcus aureus, Pseudomonas aeroginosa, Bacillus 2.7.3. Phospho-Molybdenum Method. (e total antioxidant subtilis, Salmonella typhii, Klebsiella pneumoniae, and Kleb- estimation of Ganoderma extracts was carried out according siella oxytoca) were obtained from Manmohan Memorial to the work in [6]. Briefly, 0.1 mL aqueous extract (100 μg/ Community Hospital, (amel, Kathmandu. (e antibacterial mL) was mixed with 1 mL of the reagent solution (0.6 M activity was determined by the agar well diffusion method H SO , 28 mM sodium phosphate, and 4 mM ammonium 2 4 according to the work in [15]. Briefly, MHA agar plates were molybdate). Capped tubes were then incubated in a water inoculated with bacterial strain under aseptic conditions, and bath at 95 C for 90 min and cooled to room temperature. 6 mm wells were filled with 20 μL of the test samples (mature ° Absorbance was measured at 695 nm in the Genesys UV Vis fruit extract, lawn culture mycelia) and incubated at 37 C for spectrophotometer. Total antioxidant activity was expressed 24 hours. (e diameter of the zone of inhibition was mea- as ascorbic acid equivalent in mg/g of the extract. sured in millimeters. Likewise, 18–24 h-old single bacterial colonies on agar plates were used to prepare the bacterial suspension with the turbidity of 0.5 McFarland standard 2.8. Statistical Analysis. All the assays were carried out in (equivalent to 1.5 ×10 colony-forming units (CFU)/mL). (e triplicates. One-way analysis of variance (ANOVA) followed turbidity of the bacterial suspension was measured at 600 nm. by Tukey’s test (p< 0.05) was performed to test any sig- Ethanol was used as a negative standard, while penicillin and nificant difference among means using Statistical Package erythromycin were used as positive standards. for the Social Sciences (SPSS) v. 20.0 (IBM Corp., NY, USA). 3. Results and Discussion 2.7. In Vitro Antioxidant Assay 3.1. Influence of Culture Media. (e mycelia growth pattern 2.7.1. Free Radical Scavenging Assay (RSA). Free RSA was of G. lucidum on three different culture media was com- measured as described in [14]. Briefly, ethanol (1 mL) pletely different from one another. containing G. lucidum extract in different concentrations was added to 0.4 mL of 0.2 mL DPPH (2,2-diphenyl-1- picrylhydrazyl) solution. Also, ascorbic acid (3.125 μg/ 3.1.1. Growth Pattern in PDA Media. (e mycelium on PDA mL–100 μg/mL) as standard was taken in different test tubes. media initially produced straight, velvety, whitish cottony (e sample volume was adjusted to 1 mL, adding ethanol. intermingled fabric mat-like colonies, which turned into (en, 0.4 mL DPPH was added to these tubes, shaken gently, yellowish-brown patches with time (45 days’ incubation). and allowed to stand for 45 min at room temperature in the Fresh growing apex had straight silken microfibers heading dark. (en, absorbance was measured at 517 nm in the at the top with an interfiber distance of nearly 0.5 mm. Genesys UV Vis spectrophotometer. RSA was expressed as the inhibition percentage and calculated as follows. 3.1.2. Growth Pattern in Synthetic Logs. Saw dust-powered Abs is the absorbance of DPPH radical in meth- control supplement with raw wheat bran and raw rice bran was used anol, and Abs is the absorbance of DPPH radical in sample as the previous investigation on the cultivation of G. lucidum sample extract. IC value, which represents the minimum using different biomasses which showed saw dust is a good inhibitory concentration of extract required to scavenge 50% substrate among different substrates [17]. Polypropylene of the DPPH free radicals, was calculated as bags (200 g substrate) were covered with spawn in 22 days. pi> 50% − 50 conc> 50% IC � EXP􏼠LN(conc> 50%) − 􏼠 ∗ LN􏼒 􏼓􏼡􏼡. However, bags were opened before browning. (e yellowish- pi> 50% − pi< 50% conc< 50% brown color appeared at the top and sides in still closed bags, (1) indicating more oxygen is necessary for sporophore for- mation as lignin degradation takes place only in aerobic conditions. After six days, the crown changed into a thick 2.7.2. Ferric Reducing Antioxidant Assay (FRAP). FRAP was cap with a white margin. Concentric striations were seen on performed similarly to the protocol adopted by Subhashini a flattened cap and deep-brown coloration at the center with et al. [16]. Briefly, reducing power ability was measured by its thickening. According to Sudheer et al., the shape and 4 Advances in Pharmacological and Pharmaceutical Sciences color of fruiting bodies are dependent on the light provided study, Jayasinghe et al. reported the most suitable temper- during the cultivation [9]. Once harvested, second and third ature for mycelia growth at 30 C [16]. fruiting bodies also developed from the synthetic log having enough substrates. To facilitate irrigation and nutrient ab- 3.2.3. Carbon Source. (e carbon source was optimized by sorption from soil for further fruiting, logs were buried in using different carbohydrates in basal minimum (10 g/L). humid soil rich in humus before primordial formation. Usually, fungi prefer glucose over other carbon compounds due to fast metabolization for cellular energy production 3.1.3. Growth Pattern in PDB Media. In shaking culture ° [16]. At the optimized temperature (30 C) and pH (5.0), after 18 days, the biomass remained submerged and round sorbose was found to be the best carbon source for mycelial wet cotton plug like bulk. In contrast, stationary culture growth of G. lucidum, which was closely followed by tre- remained floating and spread fully, forming a disc 8 mm in halose. Other substrates, i.e., glucose, maltose, and starch, thickness outwardly and 4 mm inwardly with a hydrophobic followed them in descending order. Both sorbose and tre- upper surface and jellied underneath. Biomass was filtered, halose yielded 1.08 cm of mycelial growth per day. On the dried, and weighed about 1.08 g/100 mL from shaking cul- contrary, G. lucidum was almost reluctant to utilize cellulose ture. However, Yang et al. reported, on long incubation (45 and lactose as carbon sources (Figure 1(c)). days), the upper surface in stationary culture changed to yellowish-brown. Mycelia from the disc started to grow upward, adhering to the bottle glass wall as the substratum 3.3. Extraction, Phenolic, and Flavonoid Content. with almost no mycelia remaining free in the bulk liquid Extraction yields were the highest for extracts of G. lucidum similar to the immobilized culture with foam sheet [16]. MCE (11.000%) and then FCE (10.625%). (e mycelia ex- Moreover, the medium was shaken for four days and kept tracts differed significantly in their flavonoid and phenolic still for having the most biomass and ganoderic acid ac- contents. (us, total phenol concentrations in mature fruit cumulation. Maturation takes about 90 days for carpo- and lawn mycelia were 5.6 μg GAE/mg and 0.9448 μg GAE/ phores, whereas suitable biomass and specific metabolites mg, while flavonoid content varied from 45.875 μg QE/mg to can be enriched in less than 20 days [18]. 34.375 μg QE/mg (Table 1). Some researchers have reported and quantified flavonoids in G. lucidum [8]. 3.2. Influence of Physical Factors in Lawn Culture. A semi- solid medium was used to optimize variables for the max- 3.4. Bioassay imum mycelial growth. PDA plates were incubated at room temperature in diffused or no light and 90–95% relative 3.4.1. Antibacterial Activity. (e zone of inhibition in the humidity. Ventilation was facilitated to increase fresh air to agar well diffusion method for FCE and MCE at concen- exchange CO gas. trations 200 and 100 μg/mL with standard drug erythromycin (ERM) and penicillin (PNC) is presented in Figure 2. It is observed that both 200 mg/mL and 100 mg/mL inhibited all 3.2.1. pH. Many fungi can grow in acidic to slightly alkaline the bacteria, and the results were more promising than the media, adjusting to various pH adversities within pH 2.0 and corresponding control antibiotics. Antimicrobial compounds 9.0 [8]. (e optimal pH range for promoting mycelia growth in mushrooms, such as terpenes, lectins, and polysaccharides, of Ganoderma species has been reported to be 5.5–6.0 [10]. act on the bacterial cytoplasmic membrane [7]. For However, in this research, higher growth was observed G. lucidum, various extracts are equally effective when between pH 4.5 and 5.5 (Figure 1(a)). About 1.08 cm in- compared with gentamycin sulphate. Quereshi et al. reported crease in mycelial length per day was observed over a pH that the most active antimicrobial components are generally range of 4.5 to 5.5. Higher and sustained mycelial growth water insoluble; it is expected that low-polarity organic sol- was obtained in the alkaline medium than in the acidic vents would yield more active antimicrobial extracts, and so, medium, whereas growth tended to halt at or below pH 3.0. ethanol is suitable to some extent [19]. According to Mishra Similarly, Lee et al. showed that G. lucidum had a broad pH et al., G. lucidum and other species extract combined with range of 5.0–9.0 for growth, and mostly favorable growth chemotherapeutic agents have been used to treat various was found at pH 5.0 [10]. bacterial infections [15]. Triterpenoids and polysaccharides play a key role in the antibacterial activity. However, terpe- 3.2.2. Temperature. Generally, the minimum and maximum noids, isoflavonoids, and tannins have stronger antimicrobial principal temperatures for the mycelial growth and biomass effects than other compounds singly. Smania et al. observed ° ° of G. lucidum were 9 C and 32 C, respectively [16]. Fungi can methyl australate, a derivative from G. lucidum, showing naturally adjust to various fluctuations in temperature as maximum antibacterial activity against E. coli, P. aeruginosa, they can adapt to induce sporulation on adverse conditions and S. aureus while the least inhibition zone was recorded for to protect the germplasm. In the present study, the highest Bacillus species [20]. Moreover, some components such as ° ° growth was observed between 28 C and 32 C at a previously ganomycin, triterpenoids, and aqueous extracts from optimized pH of 5.0 (Figure 1(b)). Lengthwise mycelial G. lucidum species have a broad-spectrum in vitro antibac- growth increased with increasing incubation temperature terial activity against Gram-positive bacteria, Gram-negative and peaked at 30 C, plunging downwards. Like the current bacteria, and Helicobacter pylori. Advances in Pharmacological and Pharmaceutical Sciences 5 1 1.2 0.9 0.8 0.7 0.8 0.6 0.5 0.6 0.4 0.4 0.3 0.2 0.2 0.1 0 0 3 3.5 4 4.5 5 5.5 6 6.5 7 15 20 25 30 35 40 pH Temperature (°C) (a) (b) 0.9 0.8 0.7 0.6 0.5 0.4 0.3 0.2 0.1 Carbon source (c) Figure 1: Growth optimization of G. lucidum using different parameters: (a) pH; (b) temperature; and (c) carbon source in PDA plates. Table 1: Comparative yield and phytochemical composition of the mature fruit and mycelia of Ganoderma lucidum strain Philippine. S. Raw Crude weight Extract yield Yield Phenolic content (mg GAE/gm of Flavonoid content (mg QE/gm of no. material (g) (g) (%) extract)± SD extract)± SD Mature 1 8 0.85 10.625 5.6± 0.080 45.875± 0.060 fruit Lawn 2 5 0.55 11 0.9448± 0.010 34.375± 0.020 mycelia 3.5. In Vitro Antioxidant Assay potential and vice versa [14]. IC values for ascorbic acid, FCE, and MCE were found to be 30.60134 μg/ml, 3.5.1. DPPH Scavenging Assay. (e scavenging activity 176.7767 μg/mL, and 157.4901 μg/mL, respectively. Ascorbic profiles of the FCE and MCE are shown in Figure 2. (e RSA acid is a far more potent antioxidant than G. lucidum extract, increased with the concentrations of mature fruit extract; having far high IC values. FCE showed a little higher IC 50 50 however, the mycelia extract was more consistent with the value than MCE. Ascorbic acid is a pure compound having increasing concentration. (e mature fruit extract scavenging high antioxidant activity. Despite its lower value, G. lucidum effect on DPPH at concentrations ranging from 100 to extracts showing a positive trend with ascorbic acid signified 300 mg/mL was thrice. In particular, there was an increase in potent antioxidant ability. the scavenging effect of mature fruit extracts up to a 300 mg/ mL concentration (60%), beyond which there was little sig- nificant increase, even up to 1000 mg/mL. On the contrary, 3.5.2. Ferrous Reducing Assay (FRA). (e FRA assay is mycelia extract exhibited a little progressive increase in the widely used to estimate the total antioxidant power of 3+ scavenging effect from 10–1000 mg/mL (about 55%). mushrooms by their capacity to convert ferricyanide (Fe ) 2+ Antioxidant capacity is expressed as their IC is cal- to ferrocyanide (Fe ) [14]. In the present study, the fruiting culated and compared with standards such as ascorbic acid. body had higher reducing power than the mycelia. (e (e lower the IC , the higher will be its antioxidant average reducing power of 13.58± 3.18 mg eqv BHT/g and Length (cm) Length (cm)/day Cellulose Lactose Xylose Starch Length (cm)/day Maltose Glucose Trehalose Sorbose 6 Advances in Pharmacological and Pharmaceutical Sciences 1.8 1.6 1.4 1.2 0.8 0.6 0.4 0.2 Sample concentration (mg/mL) P aeruginosa S aureus Bacillus sp K pneumonae S typhii K oxytosa (a) 250 500 750 1000 1500 2000 3000 0 500 1000 1500 2000 Sample concentration (µg/mL) Sample concentration (µg/mL) Mycelia Mycelial extract Fruit extract Mature fruit (b) (c) Figure 2: Different activities as shown by the G. lucidum extracts. (a) Bar graph showing inhibition zones with G. lucidum’s extracts on various opportunistic bacteria compared to standard antibiotics and control ethanol; (b) graph illustrating the reducing power of G. lucidum extracts compared to BHTas standard; BHT � butylated hydroxytoluene, Myc � mycelia, and FRT �fruit.; and (c) graph for total antioxidant potential assay by the phospho-molybdenum method; Myc � mycelia, MFRT �mature fruit, and ACRB � ascorbic acid. maximum only of 17.25 mg eqv BHT/g at 2 mg/mL were equivalent/g extract) (Figure 2(c)). However, there was no observed for the mycelial extract. However, the average significant difference in the activities between these extracts. reducing power value for the fruiting body was Higher results indicate that both the Ganoderma extracts 104.08± 7.59 mg eqv BHT/g with a maximum value of have potent radical scavenging effects for nitrous oxide and 111.67 mg eqv BHT/g at 1.5 mg/mL fruit extract. In superoxide anion radicals. (is property might be due to the G. lucidum, phenolic compounds are directly correlated to presence of carotenoids (terpenoids) in Ganoderma extracts. their antioxidant activities [14]. 4. Conclusions 3.5.3. Phospho-Molybdenum Method. Among both the ex- tracts of G. lucidium, fruit extract showed higher antioxidant In conclusion, G. lucidum can be optimally cultivated in sawdust substrate and PDA plates. (e optimum conditions potential (150.6± 56.92 mg ascorbic acid equivalent/g ex- tract) than mycelia extract (144.28± 81.72 mg ascorbic acid for growth were temperature 28–32 C, pH 4.5–5.5, carbon Antioxidant activity (mg ascorbic eq/g extract) Diameter of zone of inhibition (cm) 100 Fruit extract 200 Fruit extract 200 Mycelial extract 100 Mycelia Reducing activity (mg eqv BHT/g) extract ETH C Erythromycin Penicillin Advances in Pharmacological and Pharmaceutical Sciences 7 K. G. Ramawat, Eds., Springer International Publishing, source sorbose, and trehalose. 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Optimization of Growth Conditions and Biological Activities of Nepalese Ganoderma lucidum Strain Philippine

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Copyright © 2021 Krishna Subedi et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
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10.1155/2021/4888979
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Abstract

Hindawi Advances in Pharmacological and Pharmaceutical Sciences Volume 2021, Article ID 4888979, 7 pages https://doi.org/10.1155/2021/4888979 Research Article Optimization of Growth Conditions and Biological Activities of Nepalese Ganoderma lucidum Strain Philippine 1 1,2 1,3 4 Krishna Subedi, Buddha Bahadur Basnet , Raju Panday , Manisha Neupane , and Giri Raj Tripathi Central Department of Biotechnology, Tribhuvan University, Kirtipur, Kathmandu, Nepal Faculty of Sciences, Nepal Academy of Science and Technology, Khumaltar, Lalitpur, Nepal National Forensic Science Laboratory, Khumaltar, Lalitpur, Nepal Department of Biotechnology, National Institute of Science and Technology, Lainchaur, Kathmandu, Nepal Correspondence should be addressed to Buddha Bahadur Basnet; budbsn.btechnep@gmail.com Received 25 July 2021; Revised 17 September 2021; Accepted 21 September 2021; Published 4 October 2021 Academic Editor: Ghulam Hussain Copyright © 2021 Krishna Subedi et al. (is is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Ganoderma lucidum has been extensively studied for its valuable medicinal importance. In this study, the artificial cultivation of G. lucidum strain Philippine in different culture media, including sawdust substrate, was performed and optimized on the Potato Dextrose Agar (PDA) media. Phytochemical, antibacterial, and antioxidant analyses were performed and compared between the ethanol extracts prepared from two different cultures (fruit from synthetic log culture and mycelia from PDA media culture). Both the 200 mg/mL and 100 mg/mL concentrations of extracts inhibited all the tested bacteria, and the results were promising than the corresponding control using antibiotics. (e fruit extract showed higher antioxidant potential (150.6 ± 56.92 mg ascorbic acid equivalent/g extract) than mycelial extract (144.28± 81.72 mg ascorbic acid equivalent/g extract). (e results indicate that fruiting bodies of G. lucidum cultivated in a complex dust medium possess higher antioxidant properties than mycelia culture, which can be further explored for therapeutic applications. species include polysaccharides, including triterpenoids, 1. Introduction nucleotides, sterols, steroids, fatty acids, and proteins/pep- Ganoderma (Basidiomycota, Polyporales) is one of the well- tides with the most pharmacologically active compounds known medicinal polypore fungi in the family Gano- triterpenoids and polysaccharides [4]. It has been reported dermataceae and is widely used in East Asia, America, and that G. lucidum contains over 400 bioactive compounds, other countries [1]. Among the known, more than 400 which have several medicinal effects, remarkably such as species, only red, white, black, yellow, blue, and purple antitumor and anti-inflammatory, antimicrobial, antifungal, Reishi have been explored for their potential health-bene- antiviral (specifically against herpes and HIV) effects, ficial properties [2]. Of them, red Reishi (G. lucidum) and antioxidative, and radical scavenging effects [5–9]. black Reishi (G. sinensis) have shown the most significant Several reports revealed the successful artificial culti- health-strengthening effects [3]. East Asian countries con- vation on solid substrates utilizing sawdust and agricultural sumed Reishi for their medicinal value rather than nutri- wastes (rice bran, wheat bran, sugarcane bagasse, rice husks, peanut hulls, coconut fiber, banana leaves, etc.) as well as in tional value to extend the life span and increase youthful vigour and vitality [1]. tea waste [3]. Generally, G lucidum is cultivated on solid- G. lucidum, a cosmopolitan mushroom species, is a state fermentation and takes about six months to form the polypore rack mushroom that changes color during the fruiting body [10]. However, the process is time consuming morphogenesis process from orange-white to bright red. (e and difficult to control, dictating other alternatives for major chemical constituents of G. lucidum and related growing the fungi. One such method of choice is the 2 Advances in Pharmacological and Pharmaceutical Sciences submerged cultivation of mycelia to produce bioactive 2.3. Growth Parameter Optimization in Plate Culture compounds, which can be rapid, economically feasible, and 2.3.1. pH Optimization. PDB (100 mL) was prepared and controllable [11]. adjusted to varied pH from 3.5 to 6.75 (0.25 interval). (en, Most human diseases result from the uncontrolled agar powder (1.5 g) was added to each bottle, shaken, and production of reactive oxygen species (ROS), including free autoclaved before pouring into sterile Petri plates in trip- radicals. When unchecked, the endogenous mechanism for licates. A very minute mat with agar was plugged by using a free radical scavenging in living cells can lead to insufficient borer and inoculated into plates, incubated at 28 C for four neutralization of free radicals, resulting in major deterio- days, and their margin was marked. An increase in mycelial ration conditions such as cardiovascular diseases and cancer length was reported every day, and the growth rate was [12]. Molecules derived from natural sources such as fungi calculated. play a significant role in developing and discovering novel drugs for treatment. Currently, many active compounds from G. lucidum are being studied as treatment options [13]. 2.3.2. Temperature Optimization. Laboratory-prepared PBD (is study cultivates the G. lucidum in various media and was adjusted to pH 5.0. After adding agar, the plates were determines the optimum growth parameters (pH, temper- ° ° incubated at different temperatures (20 C to 36 C) in trip- ature, and carbon sources). In addition, the determination of licates and corresponding control for four days. An increase phenolic content, flavonoid content, antioxidant activity, in mycelia length was measured on the subsequent days, and antibacterial activity of extracts from the mycelia and which indicated lengthwise growth per day. fruiting body was performed. 2. Materials and Methods 2.3.3. Carbon Source Optimization. Liquid broth (LB) (100 mL) media were prepared without a carbohydrate 2.1. Strain Used. G. lucidum strain Philippine was purchased source, and pH was adjusted to 5.0. (en, 15 g different from the Centre for Agricultural and Technological Train- carbohydrate sources (sucrose, trehalose, glucose, lactose, ing, Lalitpur, and cultured in Potato Dextrose Agar (PDA) cellulose, maltose, sorbose, and xylose) as labeled in the flask (Hi-Media, Mumbai, India) plates and slants. (e cultured were added into respective flasks and autoclaved. Each plates were kept at 4 C for research purposes. medium was poured into plates in triplicates, inoculated with small mycelium spawn, and incubated for four days at 28 C, and their margin (apex) was marked. Everyday, the 2.2. Mass Culture increase in mycelia length was measured, which indicated lengthwise growth per day. 2.2.1. Culture on Synthetic Logs. A synthetic log was pre- pared by mixing sawdust powder, raw wheat bran, and raw rice bran. Polypropylene bags (12 ×12 cm ) were filled with 200 g of substrate, sealed with a cotton plug, autoclaved, and 2.4. Sample Preparation and Extraction. (e fruiting bodies maintained to 65% moisture. (e prepared synthetic logs from solid culture and upper mycelia culture from lawn were inoculated with fresh spawn and incubated for 60 days culture were harvested and then dried at 60 C until a at 28 C in diffused light with a relative humidity of 90–95%, constant weight was obtained. (e harvested culture was which was maintained with sprinkling sterile water (0.1% powdered using liquid nitrogen (−196 C) and crushing by calcium) twice daily. Few 1 mm pores were punched, and using a mortar and pestle. 10 gm of each powdered sample sterile cotton was loosely plugged for air circulation. was extracted by 400 mL ethanol using Soxhlet’s apparatus at 60 C, and the extract was dried using a rotary vacuum evaporator. Finally, a 100 mg/mL solution was prepared as a 2.2.2. Culture in Potato Dextrose Broth (PDB). PDB was working solution. prepared in the laboratory by boiling potatoes (300 g) in 1 L water and filtering to obtain the potato broth. Glucose (25 g) was added, and the final pH was adjusted to 5.0 before 2.5. Phytochemical Estimation autoclaving. (e sterile media were then inoculated with 2-3 pieces of fresh mycelia taken from potato dextrose agar 2.5.1. Total Phenolic Content Estimation. Total phenolic (PDA) plate and cultured in a shaking incubator (120 rpm) content was determined with the Folin–Ciocalteu (FC) re- at 28 C for 18 days. agent as in [14]. Briefly, 100 μL of each sample was dissolved in 500 μL (1 :10 v/v dilution) FC reagent and 1.5 mL (20%) sodium carbonate. (e mixture was vortexed for 30 s and 2.2.3. Culture on PDA. PDA plates inoculated with fresh incubated for 2 h in the dark at room temperature for color and actively growing mycelia and incubated at 28 C for one development. Absorbance was then measured at 765 nm week to obtain fresh inoculums as well as for one month for using the Genesys UV/Vis spectrophotometer (CA, USA). extraction. PDA plates were maintained at 4 C for long-term Total phenolic content (TPC) was measured as milligrams of storage. (e growth optimization of the culture was carried gallic acid equivalent/g (mgGAE/g) of sample dry weight out for optimal growth and further experiments. using the standard curve and the equation. Advances in Pharmacological and Pharmaceutical Sciences 3 2.5.2. Total Flavonoid Content (TFC) Estimation. Total fla- adding sample extract to 2.5 mL phosphate buffer (0.2 M, pH vonoid content in the extracts was estimated using the alu- 6.6) and 2.5 mL potassium ferricyanide (1%, w/v) and in- minium chloride colorimetric method [14]. Briefly, 0.5 mL of cubated for 30 min at 50 C. (en, 2.5 mL of 10% tri- plant extract (20 mg/mL) was mixed with 0.1 mL of 10% chloroacetic acid was added to the mixture and centrifuged aluminum chloride, 0.1 mL of 1.0 M potassium acetate, and for 10 min at 3000g. 2.5 mL of the supernatant was then 1.3 mL 90% ethanol. Absorbance was measured at 415 nm in diluted with 2.5 mL distilled water and mixed with 0.5 mL the Genesys UV/Vis spectrophotometer after incubating the freshly prepared ferric chloride (0.1%, w/v). (e absorbance mixture for 40 min at room temperature. TFC was then was measured at 700 nm using the Genesys UV Vis spec- calculated using quercetin as a standard and expressed as mg trophotometer after incubating for 30 min. Increased ab- quercetin equivalent/g (mgQE/g) of sample dry weight. sorbance of the reaction mixture indicated increased reducing power. All experiments were performed in trip- licate using butylated hydroxytoluene as a positive control. 2.6. Antibacterial Activity. Six clinical strains of bacteria (Staphylococcus aureus, Pseudomonas aeroginosa, Bacillus 2.7.3. Phospho-Molybdenum Method. (e total antioxidant subtilis, Salmonella typhii, Klebsiella pneumoniae, and Kleb- estimation of Ganoderma extracts was carried out according siella oxytoca) were obtained from Manmohan Memorial to the work in [6]. Briefly, 0.1 mL aqueous extract (100 μg/ Community Hospital, (amel, Kathmandu. (e antibacterial mL) was mixed with 1 mL of the reagent solution (0.6 M activity was determined by the agar well diffusion method H SO , 28 mM sodium phosphate, and 4 mM ammonium 2 4 according to the work in [15]. Briefly, MHA agar plates were molybdate). Capped tubes were then incubated in a water inoculated with bacterial strain under aseptic conditions, and bath at 95 C for 90 min and cooled to room temperature. 6 mm wells were filled with 20 μL of the test samples (mature ° Absorbance was measured at 695 nm in the Genesys UV Vis fruit extract, lawn culture mycelia) and incubated at 37 C for spectrophotometer. Total antioxidant activity was expressed 24 hours. (e diameter of the zone of inhibition was mea- as ascorbic acid equivalent in mg/g of the extract. sured in millimeters. Likewise, 18–24 h-old single bacterial colonies on agar plates were used to prepare the bacterial suspension with the turbidity of 0.5 McFarland standard 2.8. Statistical Analysis. All the assays were carried out in (equivalent to 1.5 ×10 colony-forming units (CFU)/mL). (e triplicates. One-way analysis of variance (ANOVA) followed turbidity of the bacterial suspension was measured at 600 nm. by Tukey’s test (p< 0.05) was performed to test any sig- Ethanol was used as a negative standard, while penicillin and nificant difference among means using Statistical Package erythromycin were used as positive standards. for the Social Sciences (SPSS) v. 20.0 (IBM Corp., NY, USA). 3. Results and Discussion 2.7. In Vitro Antioxidant Assay 3.1. Influence of Culture Media. (e mycelia growth pattern 2.7.1. Free Radical Scavenging Assay (RSA). Free RSA was of G. lucidum on three different culture media was com- measured as described in [14]. Briefly, ethanol (1 mL) pletely different from one another. containing G. lucidum extract in different concentrations was added to 0.4 mL of 0.2 mL DPPH (2,2-diphenyl-1- picrylhydrazyl) solution. Also, ascorbic acid (3.125 μg/ 3.1.1. Growth Pattern in PDA Media. (e mycelium on PDA mL–100 μg/mL) as standard was taken in different test tubes. media initially produced straight, velvety, whitish cottony (e sample volume was adjusted to 1 mL, adding ethanol. intermingled fabric mat-like colonies, which turned into (en, 0.4 mL DPPH was added to these tubes, shaken gently, yellowish-brown patches with time (45 days’ incubation). and allowed to stand for 45 min at room temperature in the Fresh growing apex had straight silken microfibers heading dark. (en, absorbance was measured at 517 nm in the at the top with an interfiber distance of nearly 0.5 mm. Genesys UV Vis spectrophotometer. RSA was expressed as the inhibition percentage and calculated as follows. 3.1.2. Growth Pattern in Synthetic Logs. Saw dust-powered Abs is the absorbance of DPPH radical in meth- control supplement with raw wheat bran and raw rice bran was used anol, and Abs is the absorbance of DPPH radical in sample as the previous investigation on the cultivation of G. lucidum sample extract. IC value, which represents the minimum using different biomasses which showed saw dust is a good inhibitory concentration of extract required to scavenge 50% substrate among different substrates [17]. Polypropylene of the DPPH free radicals, was calculated as bags (200 g substrate) were covered with spawn in 22 days. pi> 50% − 50 conc> 50% IC � EXP􏼠LN(conc> 50%) − 􏼠 ∗ LN􏼒 􏼓􏼡􏼡. However, bags were opened before browning. (e yellowish- pi> 50% − pi< 50% conc< 50% brown color appeared at the top and sides in still closed bags, (1) indicating more oxygen is necessary for sporophore for- mation as lignin degradation takes place only in aerobic conditions. After six days, the crown changed into a thick 2.7.2. Ferric Reducing Antioxidant Assay (FRAP). FRAP was cap with a white margin. Concentric striations were seen on performed similarly to the protocol adopted by Subhashini a flattened cap and deep-brown coloration at the center with et al. [16]. Briefly, reducing power ability was measured by its thickening. According to Sudheer et al., the shape and 4 Advances in Pharmacological and Pharmaceutical Sciences color of fruiting bodies are dependent on the light provided study, Jayasinghe et al. reported the most suitable temper- during the cultivation [9]. Once harvested, second and third ature for mycelia growth at 30 C [16]. fruiting bodies also developed from the synthetic log having enough substrates. To facilitate irrigation and nutrient ab- 3.2.3. Carbon Source. (e carbon source was optimized by sorption from soil for further fruiting, logs were buried in using different carbohydrates in basal minimum (10 g/L). humid soil rich in humus before primordial formation. Usually, fungi prefer glucose over other carbon compounds due to fast metabolization for cellular energy production 3.1.3. Growth Pattern in PDB Media. In shaking culture ° [16]. At the optimized temperature (30 C) and pH (5.0), after 18 days, the biomass remained submerged and round sorbose was found to be the best carbon source for mycelial wet cotton plug like bulk. In contrast, stationary culture growth of G. lucidum, which was closely followed by tre- remained floating and spread fully, forming a disc 8 mm in halose. Other substrates, i.e., glucose, maltose, and starch, thickness outwardly and 4 mm inwardly with a hydrophobic followed them in descending order. Both sorbose and tre- upper surface and jellied underneath. Biomass was filtered, halose yielded 1.08 cm of mycelial growth per day. On the dried, and weighed about 1.08 g/100 mL from shaking cul- contrary, G. lucidum was almost reluctant to utilize cellulose ture. However, Yang et al. reported, on long incubation (45 and lactose as carbon sources (Figure 1(c)). days), the upper surface in stationary culture changed to yellowish-brown. Mycelia from the disc started to grow upward, adhering to the bottle glass wall as the substratum 3.3. Extraction, Phenolic, and Flavonoid Content. with almost no mycelia remaining free in the bulk liquid Extraction yields were the highest for extracts of G. lucidum similar to the immobilized culture with foam sheet [16]. MCE (11.000%) and then FCE (10.625%). (e mycelia ex- Moreover, the medium was shaken for four days and kept tracts differed significantly in their flavonoid and phenolic still for having the most biomass and ganoderic acid ac- contents. (us, total phenol concentrations in mature fruit cumulation. Maturation takes about 90 days for carpo- and lawn mycelia were 5.6 μg GAE/mg and 0.9448 μg GAE/ phores, whereas suitable biomass and specific metabolites mg, while flavonoid content varied from 45.875 μg QE/mg to can be enriched in less than 20 days [18]. 34.375 μg QE/mg (Table 1). Some researchers have reported and quantified flavonoids in G. lucidum [8]. 3.2. Influence of Physical Factors in Lawn Culture. A semi- solid medium was used to optimize variables for the max- 3.4. Bioassay imum mycelial growth. PDA plates were incubated at room temperature in diffused or no light and 90–95% relative 3.4.1. Antibacterial Activity. (e zone of inhibition in the humidity. Ventilation was facilitated to increase fresh air to agar well diffusion method for FCE and MCE at concen- exchange CO gas. trations 200 and 100 μg/mL with standard drug erythromycin (ERM) and penicillin (PNC) is presented in Figure 2. It is observed that both 200 mg/mL and 100 mg/mL inhibited all 3.2.1. pH. Many fungi can grow in acidic to slightly alkaline the bacteria, and the results were more promising than the media, adjusting to various pH adversities within pH 2.0 and corresponding control antibiotics. Antimicrobial compounds 9.0 [8]. (e optimal pH range for promoting mycelia growth in mushrooms, such as terpenes, lectins, and polysaccharides, of Ganoderma species has been reported to be 5.5–6.0 [10]. act on the bacterial cytoplasmic membrane [7]. For However, in this research, higher growth was observed G. lucidum, various extracts are equally effective when between pH 4.5 and 5.5 (Figure 1(a)). About 1.08 cm in- compared with gentamycin sulphate. Quereshi et al. reported crease in mycelial length per day was observed over a pH that the most active antimicrobial components are generally range of 4.5 to 5.5. Higher and sustained mycelial growth water insoluble; it is expected that low-polarity organic sol- was obtained in the alkaline medium than in the acidic vents would yield more active antimicrobial extracts, and so, medium, whereas growth tended to halt at or below pH 3.0. ethanol is suitable to some extent [19]. According to Mishra Similarly, Lee et al. showed that G. lucidum had a broad pH et al., G. lucidum and other species extract combined with range of 5.0–9.0 for growth, and mostly favorable growth chemotherapeutic agents have been used to treat various was found at pH 5.0 [10]. bacterial infections [15]. Triterpenoids and polysaccharides play a key role in the antibacterial activity. However, terpe- 3.2.2. Temperature. Generally, the minimum and maximum noids, isoflavonoids, and tannins have stronger antimicrobial principal temperatures for the mycelial growth and biomass effects than other compounds singly. Smania et al. observed ° ° of G. lucidum were 9 C and 32 C, respectively [16]. Fungi can methyl australate, a derivative from G. lucidum, showing naturally adjust to various fluctuations in temperature as maximum antibacterial activity against E. coli, P. aeruginosa, they can adapt to induce sporulation on adverse conditions and S. aureus while the least inhibition zone was recorded for to protect the germplasm. In the present study, the highest Bacillus species [20]. Moreover, some components such as ° ° growth was observed between 28 C and 32 C at a previously ganomycin, triterpenoids, and aqueous extracts from optimized pH of 5.0 (Figure 1(b)). Lengthwise mycelial G. lucidum species have a broad-spectrum in vitro antibac- growth increased with increasing incubation temperature terial activity against Gram-positive bacteria, Gram-negative and peaked at 30 C, plunging downwards. Like the current bacteria, and Helicobacter pylori. Advances in Pharmacological and Pharmaceutical Sciences 5 1 1.2 0.9 0.8 0.7 0.8 0.6 0.5 0.6 0.4 0.4 0.3 0.2 0.2 0.1 0 0 3 3.5 4 4.5 5 5.5 6 6.5 7 15 20 25 30 35 40 pH Temperature (°C) (a) (b) 0.9 0.8 0.7 0.6 0.5 0.4 0.3 0.2 0.1 Carbon source (c) Figure 1: Growth optimization of G. lucidum using different parameters: (a) pH; (b) temperature; and (c) carbon source in PDA plates. Table 1: Comparative yield and phytochemical composition of the mature fruit and mycelia of Ganoderma lucidum strain Philippine. S. Raw Crude weight Extract yield Yield Phenolic content (mg GAE/gm of Flavonoid content (mg QE/gm of no. material (g) (g) (%) extract)± SD extract)± SD Mature 1 8 0.85 10.625 5.6± 0.080 45.875± 0.060 fruit Lawn 2 5 0.55 11 0.9448± 0.010 34.375± 0.020 mycelia 3.5. In Vitro Antioxidant Assay potential and vice versa [14]. IC values for ascorbic acid, FCE, and MCE were found to be 30.60134 μg/ml, 3.5.1. DPPH Scavenging Assay. (e scavenging activity 176.7767 μg/mL, and 157.4901 μg/mL, respectively. Ascorbic profiles of the FCE and MCE are shown in Figure 2. (e RSA acid is a far more potent antioxidant than G. lucidum extract, increased with the concentrations of mature fruit extract; having far high IC values. FCE showed a little higher IC 50 50 however, the mycelia extract was more consistent with the value than MCE. Ascorbic acid is a pure compound having increasing concentration. (e mature fruit extract scavenging high antioxidant activity. Despite its lower value, G. lucidum effect on DPPH at concentrations ranging from 100 to extracts showing a positive trend with ascorbic acid signified 300 mg/mL was thrice. In particular, there was an increase in potent antioxidant ability. the scavenging effect of mature fruit extracts up to a 300 mg/ mL concentration (60%), beyond which there was little sig- nificant increase, even up to 1000 mg/mL. On the contrary, 3.5.2. Ferrous Reducing Assay (FRA). (e FRA assay is mycelia extract exhibited a little progressive increase in the widely used to estimate the total antioxidant power of 3+ scavenging effect from 10–1000 mg/mL (about 55%). mushrooms by their capacity to convert ferricyanide (Fe ) 2+ Antioxidant capacity is expressed as their IC is cal- to ferrocyanide (Fe ) [14]. In the present study, the fruiting culated and compared with standards such as ascorbic acid. body had higher reducing power than the mycelia. (e (e lower the IC , the higher will be its antioxidant average reducing power of 13.58± 3.18 mg eqv BHT/g and Length (cm) Length (cm)/day Cellulose Lactose Xylose Starch Length (cm)/day Maltose Glucose Trehalose Sorbose 6 Advances in Pharmacological and Pharmaceutical Sciences 1.8 1.6 1.4 1.2 0.8 0.6 0.4 0.2 Sample concentration (mg/mL) P aeruginosa S aureus Bacillus sp K pneumonae S typhii K oxytosa (a) 250 500 750 1000 1500 2000 3000 0 500 1000 1500 2000 Sample concentration (µg/mL) Sample concentration (µg/mL) Mycelia Mycelial extract Fruit extract Mature fruit (b) (c) Figure 2: Different activities as shown by the G. lucidum extracts. (a) Bar graph showing inhibition zones with G. lucidum’s extracts on various opportunistic bacteria compared to standard antibiotics and control ethanol; (b) graph illustrating the reducing power of G. lucidum extracts compared to BHTas standard; BHT � butylated hydroxytoluene, Myc � mycelia, and FRT �fruit.; and (c) graph for total antioxidant potential assay by the phospho-molybdenum method; Myc � mycelia, MFRT �mature fruit, and ACRB � ascorbic acid. maximum only of 17.25 mg eqv BHT/g at 2 mg/mL were equivalent/g extract) (Figure 2(c)). However, there was no observed for the mycelial extract. However, the average significant difference in the activities between these extracts. reducing power value for the fruiting body was Higher results indicate that both the Ganoderma extracts 104.08± 7.59 mg eqv BHT/g with a maximum value of have potent radical scavenging effects for nitrous oxide and 111.67 mg eqv BHT/g at 1.5 mg/mL fruit extract. In superoxide anion radicals. (is property might be due to the G. lucidum, phenolic compounds are directly correlated to presence of carotenoids (terpenoids) in Ganoderma extracts. their antioxidant activities [14]. 4. Conclusions 3.5.3. Phospho-Molybdenum Method. Among both the ex- tracts of G. lucidium, fruit extract showed higher antioxidant In conclusion, G. lucidum can be optimally cultivated in sawdust substrate and PDA plates. (e optimum conditions potential (150.6± 56.92 mg ascorbic acid equivalent/g ex- tract) than mycelia extract (144.28± 81.72 mg ascorbic acid for growth were temperature 28–32 C, pH 4.5–5.5, carbon Antioxidant activity (mg ascorbic eq/g extract) Diameter of zone of inhibition (cm) 100 Fruit extract 200 Fruit extract 200 Mycelial extract 100 Mycelia Reducing activity (mg eqv BHT/g) extract ETH C Erythromycin Penicillin Advances in Pharmacological and Pharmaceutical Sciences 7 K. G. Ramawat, Eds., Springer International Publishing, source sorbose, and trehalose. A similar yield percentage was Cham, Switzerland, pp. 1863–1893, 2019. obtained from both mycelia and fruiting bodies as 10.62% [10] J. Magday, M. Bungihan, and R. Dulay, “Optimization of and 11%, respectively. (e phytochemical properties, anti- mycelial growth and cultivation of fruiting body of Philippine bacterial properties, and antioxidants were comparable in wild strain of Ganoderma lucidum,” Current Research in both G. lucidum mycelia grown in the lab and sawdust Environmental & Applied Mycology, vol. 4, no. 2, pp. 162–172, substrate logs. (us, further evaluation and identification of specific compounds are required to fully understand the [11] C Jasmina, M. Staji, and J. Vukojevi, “Potential of sub- mechanism in the biological system and drug discovery. mergedly cultivated mycelia of Ganoderma spp. as antioxi- dant and antimicrobial agents,” Current Pharmaceutical Data Availability Biotechnology, vol. 17, pp. 275–282, 2015. [12] D. M. Fernando, R. L. Wijesundera, P. 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Published: Oct 4, 2021

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