Iranian Journal of Medical Microbiology

Collection of Mushroom Samples
The F. fomentarius was isolated from Mazandaran forests in 2017 with the cooperation of Sari Agricultural Sciences and Natural Resources University. In each case, the fungal specimen was harvested in a healthy, complete and appropriate manner based on the color, shape, size and surface decoration of the cap, base, blades and many other characteristics. After morphological confirmation by mycologists, it is kept at 4°C.

Cultivation of F. fomentarius and Polysaccharides Extraction
The F. fomentarius mycelium was cultured in potato dextrose agar (PDA) (manufactured by Merck, Germany) in a petri dish and incubated for 5 days at 28°C (BINDER, USA). For seed culture, 10 mm² pieces of fungus grown in PDA medium were transferred to 100 mL of Potato dextrose broth (PDB) medium (manufactured by Merck, Germany) for 7 days at 28°C and 150 rpm in shaker incubator (JAL TAJHIZ, Iran). Suitable culture medium of this fungus includes glucose (5%), peptone (0.2%), malt extract (1%), yeast extract (0.2%), MgSO₄.7H₂O (0.25%) and KH₂PO₄ (0.5%). The initial pH of this medium was adjusted to 6 by adding NaOH (1M) and HCl (1M). After autoclaving, 5% v/v inoculum was added and incubated at 28°C and 150 rpm for 4 days (11). To extract the F. fomentarius polysaccharide, after separating the biomass using Whatman paper, absolute ethanol with a ratio of 4:1 v/v was added to the supernatant. After overnight refrigeration, the polysaccharide precipitates were centrifuged for 10 min and 10,000 rpm (AWEL/MF20-R/France). The biomass and polysaccharide were lyophilized (OPERON) and the final product was stored at room temperature.

Optimization of Culture Medium Compositions by Taguchi Method
Taguchi method was used to investigate the effect of independent variables of MgSO₄.7H₂O, pH, yeast extract, and inoculum percentage on biomass and polysaccharide production. The Taguchi L9 array was used to examine four variables at three levels (Table 1), the experiments were performed according to this array, and the results were analyzed using Design Expert 11 software (State-Ease, USA).

Biological Assessments
Antibacterial Activity
The colony-forming unit (CFU) method was used to evaluate the antibacterial activity of polysaccharides. Staphylococcus aureus (UTMC 1429) and Escherichia coli (PTCC 1269) were obtained from the the Research Center for Technology and Microbial Products of the University of Tehran and Persian Type Culture Collection. Bacteria were cultured in Müller-Hinton Broth (manufactured by Merck, Germany) and after 24 hours of incubation (BINDER, USA), 0.5 McFarland solution was prepared from them. A 5% solution of each polysaccharide with 0.5 McFarland solution was prepared from each bacterium in a volume of 500 μL and kept for 24 hours in a shaker incubator (JAL TAJHIZ, Iran) at 37°C and 140 rpm. The mixture was diluted with phosphate buffer saline (1:10) and 10 μL of it cultured in nutrient agar medium (manufactured by Merck, Germany). In the control state, 10 μL of McFarland 0.5 solution of each bacteria was cultured. Then, all culture media were incubated and after 24 hours, bacterial colony count was performed with a gel dock (Quantum, France) (14).

Antioxidant Activity
DPPH (2,2-diphenyl-1-picryl-hydrazyl-hydrate) test was used to evaluate the antioxidant activity of polysaccharides. After preparing 2 mg/mL polysaccharide solution in water, the polysaccharide solution and DPPH solution were mixed in a ratio of 1:4 v/v and placed in a shaker incubator for 30 minutes (10). Then the absorbance of the samples was measured at 517 nm with ELISA reader (Carry 100 Bio, Australia) and antioxidant activity was calculated according to the following formula:
:(eq.1)
DPPH• scavenging effect (%) = [(A0 – sample A)/A0] × 100
 
A0: Adsorption of DPPH solution at 517 nm
sample A: Absorption of the test sample at 517 nm

Cytotoxicity Assay
MKN45, AGS, A549, KYSE-30, and 5637 cancer cell lines were purchased from the Iranian Genetic Resources Center. Cancer cells were cultured in RPMI 1640 medium with 2 mM L-glutamine and 15% FBS and stored in a CO₂ incubator at 37°C. To evaluate the effect of polysaccharide, its solution was prepared in sterile water (1 mg/mL) and used at specific concentrations (50, 100, 200 µg/mL) to treat cells for 24 and 72 hours. In this experiment, positive control included culture medium and negative control included untreated cell culture. MTS test was used to evaluate cell viability. A total of 2000 cancer cells were cultured in each well of 96 well plates (24 hours). After treatment with specified concentrations of polysaccharide for 24 and 72 hours, 10 µM MTS solution (1:10 diluted in RPMI 1640) was added to each well and incubated for 3 hours (15). Then, adsorption of each sample was read at 495 nm using ELISA reader (Carry 100 Bio, Australia) and the cell viability percentage was calculated using the following formula:
:(eq.2)
Cell viability: Sample absorbance/ Control absorbance × 100
 
Statistical Analysis
Data were analyzed statistically by ANOVA method using Design Expert, version 11 (State Ease, USA). P-values below 0.05 (P<0.05) were considered to be statistically significant.

 

Source	Sum of squares	df	Mean square	F-value	P-value
Model	41.69	4	10.42	9.57	0.0251
MgSO4.7H2O (A)	5.55	1	5.55	5.55	0.0868
pH (B)	1.06	1	1.06	0.9722	0.3800
Yeast extract (C)	6.41	1	6.41	5.88	0.0723
Inoculum (D)	28.67	1	28.67	26.34	0.0068
Residual	4.35	4	1.09
Cor Total	46.05	8

 

 
Figure 3. Antibacterial (A) and antioxidant (B) activity of polysaccharide obtained from the Taguchi L9 array.
 
 
Figure 4. Cell viability diagram of 5 cancer cell lines treated with F. fomentarius polysaccharide after 24 (A) and 72 hours (B)

In this study, the optimization of biomass and polysaccharide production of the medicinal fungus F. fomentarius and its polysaccharide biological properties were investigated. As observed in previous studies (4) and this study, the suitable culture medium for this fungus includes glucose, peptone, malt extract, yeast extract, MgSO₄.7H₂O, and KH₂PO₄. Studies show that peptone in this environment plays an important role in increasing the growth and production of fungal enzymes (16). Optimization of culture medium using the Taguchi method showed that MgSO₄.7H₂O concentration and initial pH have a significant effect (P<0.05) on polysaccharide production of this fungus. With the increase of MgSO₄.7H₂O and the initial pH, the production of polysaccharide of F. fomentarius increases. pH is involved in permeability, cell membrane function, and production of secondary metabolites (11). Magnesium is an important element in the metabolism of fungi and the stability of cell membranes. It is also involved in DNA replication, cell division, and many enzymatic reactions (11, 17, 18). Chen et al. )2008(, increased the polysaccharide production of this fungus (3.64 g/L) by optimizing the culture medium. In this study, under optimal conditions (4 g/L MgSO₄.7H₂O, initial pH=8), the production of polysaccharide of the F. fomentarius reached 5.410 g/L. This amount is 1.5 times the production of polysaccharides in common fungal culture medium (PDB) (11).
Studies show that physicochemical conditions of the culture medium such as carbon source, nitrogen source, mineral sources, pH and culture temperature play an important role in the biological activity of fungal metabolites (19, 20). F. fomentarius polysaccharide inhibits the growth of S. aureus and E. coli by 50% and 25%, respectively. Polysaccharide of this fungus contains terpenoids and polyphenols with antibacterial activity and with their increase this activity increases (21). Studies show that the type and volume of solvent used to extract polysaccharides play a role in the content of polyphenols. The use of polar solvents such as ethanol increases polyphenols (10, 22). Kalyoncu et al. (2010), found that the antioxidant activity of the polysaccharide of the F. fomentarius was 5.97%. By optimizing the culture conditions in this study, the antioxidant activity of polysaccharide increased to 16.11%. Most biological properties such as antioxidant, antibacterial, and antiproliferative activity are observed when the initial pH of the fungal culture medium is in the range of 5-7 (23). The difference between the initial pH and the composition of the culture medium causes a difference in the phenolic content of the polysaccharide. These compounds are associated with antioxidant activity and reduction of OH^•, O₂^•, and NO^• radicals (24). Investigations in this study and previous studies (4, 5, 11) show that this polysaccharide has an antiproliferative effect on cancer cells, but this effect varies in cell lines and increases with increasing of the concentration.  The antiproliferative effect of F. fomentarius polysaccharide was observed (KYSE-30> A549 563749> AGS> MKN-45) after 72 hours. In the treatment of KYSE-30 cells with 200 µg/mL polysaccharide, cell viability reaches 40% after 72 hours. Also, after 72 hours, the life expectancy of A549 and 5637 cancer cells was 45% and that of MKN-45 and AGS cancer cells was 66% and 62%, respectively. Today, the polysaccharides of the fungi like Ganoderma   lucidum and Tinea versicolor are used as supplements in the treatment of various cancers. The anti-cancer activity of these polysaccharides includes inhibiting the growth of cancer cells and stimulating the immune system (25). The antiproliferative activity of fungal polysaccharides is related to the chemical structure and composition of their monosaccharides. The polysaccharide of F. fomentarius is rich in beta-glucan, which has anti-cancer properties. This compound destroys transcription proteins in cancer cells and arrests the cell cycle in the G1 phase. It also induces apoptosis in cells by damaging organelles, the cell nucleus, and fragmenting DNA (10, 26, 27, 28). The results of this study showed that the polysaccharide of the medicinal F. fomentarius, due to its antibacterial, antioxidant and antiproliferative properties, can be a suitable option for the treatment of many diseases and can be used as a dietary supplement.

In this study, the production of biomass and polysaccharide of the Iranian medicinal F. fomentarius was optimized using the Taguchi method. Inoculum percentage had significant effects on biomass production and MgSO₄.7H₂O concentration and initial pH had significant effects (P<0.05) on polysaccharide production of this fungus and under optimal conditions (4 g/L MgSO₄.7H₂O, initial pH=8) polysaccharide production increases 1.5 times and reaches 5.410 g/L. Biological assessments showed that this polysaccharide inhibits the growth of S. aureus and E. coli 50% and 25%, respectively (P<0.05). Also, its antioxidant activity increases 2 times and reaches 16.11%. The antiproliferative effect of F. fomentarius polysaccharide is different on different cancer cells and increases with increasing concentration. The cell viability of KYSE-30 treatment with 200 µg/mL polysaccharide, reaches 40% after 72 hours. Also, the cell viability of A549 and 5637 cancer cells is 45% and the cell viability of MKN-45 and AGS cancer cells is 66% and 62%, respectively.
 

This article is taken from the master's thesis (19/06/28690), which would like to thank all the professors who helped us in the Faculty of New Sciences and Technologies, University of Tehran.
 

Funding for this study was provided by the authors in collaboration with the University of Tehran.

 

Authors declared no conflict of interests.