Shiitake mushrooms are one of the most popular mushrooms worldwide. Among shitake mushroom, Lentinula edodes is the most famous and has been most commonly used food and traditional medicine.¹ Shiitake mushroom has shown to present medicinal compounds such as polysaccharides, sterols, terpenoids, and lipoids, by which has anti-inflammatory, antimicrobial, anti-tumor effects.^2,3 Also, the extract from this mushroom showed antimicrobial and antibiofilm activity against oral bacteria and biofilm.^4,5

Candida species are detected up to 90% in healthy persons and present in the oral cavity of up to 75% of the population.^6,7 Furthermore, Candida albicans was identified about 80% of the isolated Candida species.⁸ The characteristic related to the virulence of C. albicans is the change of morphology which grow either budding yeast and hyphal form by growth condition.⁹ The morphology of this fungus is changed by environmental pH, physiological temperature, serum and CO₂.⁶ The yeast forms are commonly found on the mucosal surface, and the hyphal forms are detected in epithelial layer.¹⁰ Furthermore, hyphal form but not yeast form are found in epithelial layer at sites of infection.¹¹ Therefore, the transition between yeast and hyphal forms is termed dimorphism, that is an important pathogenicity for oral candidiasis. Also, when the virulence of C. albicans is analyzed by global analysis using 177 mutant strains tested, attenuation of the virulence was significantly correlated with reduced hyphal morphogenesis.¹² Another characteristic of C. albicans is possible to form biofilm of hyphal type on denture. The biofilm formed on denture induces denture related stomatitis.

The elderly population is gradually increasing due to the development of medical technology and equipment. This phenomenon can be expected to increase the use of dentures. Therefore, the aim of this study was to investigated antifungal activity of shiitake mushroom against yeast and hyphal type of C. albicans.

Shiitake mushroom was used Lentinula edodes. Slide shiitake mushroom (300 g) was soaked in 500 ml of distilled water or ethyl alcohol with magnetic bar. The prepared mushroom was incubated at room temperature for 24 h on magnetic stirrer. The extract from the mushroom was collected by filtering with Whatman filter paper (GE healthcare, Chicago, USA), and the solvent was evaporated with vacuum evaporator (IKA, Staufen, Germany). The weight of the dried extract was measured and solved at 1 g/ml with distilled water and ethyl alcohol. The solution was filtrated with polyvinylidene fluoride (PVDF) filter (0.25 μm of pore size) (Millipore, Billerica, USA).

Candida albicans ATCC 10231 was used in this study. The fungus was cultivated in trypticase soy broth (TSB; BD biosciences, San jose, USA) at 37°C in shaking incubator. Furthermore, to form C. albicans biofilm with hyphal type, C. albicans was cultivated with Ham’s F-12 medium (Hyclone, Logan, USA) at 37°C in an incubator under 5% CO₂ condition.

The antifungal activity of shiitake extract was processed by the methods by protocol of Clinical Laboratory Standard.¹³ 180 μl of TSB was dispensed into the well of a 96-well plate (SPL Lifescience, Pocheon, Korea), and the solution of the shiitake extracts were added into the 12^th row of well containing TSB. The extract was performed 2-fold serial dilutions from 11th column to 2^nd column with micropipette. The fungus was counted with a hemacytometer (Marienfeld, Lauda-Konigshöfen, Germany) and adjusted the concentration to 1 × 10⁶ cells/ml in TSB. 20 μl of the suspension was inoculated into the prepared well. The plate was incubated 37°C under aerobic condition for 36 h. The fungal growth was measured with optical density at a 600 nm wavelength by a microplate reader (Biotek, Winooski, USA). Hyphal C. albicans was formed on 12-well plate using Ham’s F-12. After observe the hyphal type using a phase contrast microscope, the antifungal activity of the shiitake extract was investigated. The ethanol was diluted with phosphate buffered saline (PBS, pH 7.2) at 12.5, 25, and 50 mg/ml of concentration. The extract was treated on hyphal C. albicans for 1 min, and hyphal C. albicans on the well was disrupted with a scraper (SPL bioscience). The fungal suspension was transferred into 1.5 ml tube and serially diluted from 10 to 105 fold. 50 μl of the diluted suspension was inoculated on trypticase soy agar (TSA) plate. The plate was incubated 37°C for 36 h, and the fungal colonies were counted. To evaluate effect of the extract on denture-related stomatitis, A specimen was fabricated using a 3D printer (Form 2; formlabs Co., Somerville, USA) and acrylic resin (digital Denture resins; formlabs Co.). The specimens were printed with a diameter of 12 mm with a thickness of 2 mm parallel to the bottom of the LASER. Next, the surface of the specimens was washed with isopropyl alcohol and cured using ultra violet and heating. Enzyme based denture cleanser (Dongahwa, Seoul, Korea) solved with 100 ml of sterile tap water, and the extract (25 mg/ml) was solution was added into the solution. Candidal biofilm formed disk were placed into the solution. After incubating for 5 min, the disks were washed with sterile tap water and placed into each well of 12-well polystyrene plate (SPL Lifescience) containing 1 ml of TSB. Candidal biofilm on the disk was physically disrupted with a scraper (SPL bioscience). The fungal suspension was transferred into 1.5 ml tube and serially diluted from 10 to 105-fold. 50 μl of the diluted suspension was inoculated on trypticase soy agar plate. The plate was incubated 37°C for 36 h, and the fungal colonies were counted.

IBM SPSS Statistics Ver. 23 (IBM, Armonk, USA) was used for statistical analysis. In order to analyze the statistical difference in the data, data distribution was determined using the Kolmogorov-Smirnov test. The values among groups were analyzed by a non-parametric Kruskal-Wallis test and Mann-Whitney U-test, and statistical significance was defined by a P value of less than 0.05.

First, Ethanol and water extract from shiitake mushroom was investigated against yeast type of C. albicans. The ethanol extract significantly inhibited the growth of C. albicans above 6.25 mg/ml of concentration (P < 0.05), and the water extract inhibited the candidal growth above 25 mg/ml of concentration (P < 0.05, Fig. 1). The ethanol extract showed more antifungal activity compared to the water extract.

As the concentration of the ethanol extract increased, it showed strong fungicidal effect on hyphal biofilm (Fig. 2). However, the water extract did not show antifungal activity against hyphal biofilm (data not shown). The ethanol extract significantly showed antifungal activity against candidal biofilm above 25 mg/ml of concentration (P < 0.05).

Generally, most elderly people use denture cleansers to remove fungus and debris on denture. Therefore, the antifungal effect of a mixture of denture cleanser and the shiitake extract was investigated. Denture cleanser has strong antifungal activity against candidal biofilm on denture. However, the cleanser did not completely remove C. albicans (Fig. 3). The denture cleanser solution containing the shiitake extract showed more antifungal activity against candidal biofilm on denture compared to single condition of the cleanser.

C. albicans causes oral candidiasis, which is especially common in immune compromised patients or the elderly.^14,15 Also, denture stomatitis occurs in people who wear dentures and is associated with C. albicans.¹⁶ The elderly population is gradually increasing due to the development of medical technology and equipment. This phenomenon can be expected to increase the use of dentures. Therefore, the importance of denture hygiene is emphasized, and the need for a more effective denture cleanser is emerging. This study was investigated antifungal activity of extract from shiitake mushroom to prevent oral candidiasis.

Shiitake mushroom is used general food and is recognized medical value due to its nutritional components. This mushroom has bio-active polysaccharide (lentinan, heteroglucan, xylomannan, and β-glucan), free sugar (arabinose, arabitol, glycerol, mannitol, mannose, and trehalose), vitamins (B2, B12, C, D and E), organic acid (cinnamic acid, phenolic acid, and benzoic acid), and fiber.^17,18 These components of shiitake mushroom show antitumor, antiinflammation, antioxidant and antimicrobial activity.^1-3,17 When extract from shiitake mushroom using water and ethanol was investigated antifungal activity against C. albicans, the ethanol extract showed stronger antifungal activity compared to the water extract. These results indicates that hydrophobic components of shiitake mushroom may be suitable to prevent oral candidiasis. Also, comparing other studies. Mushroom extracts with antifungal activity were reported as organic acid such as benzoic acid, cinnamic acid, and phenolic acid, which is consistent with the results of this study.¹⁸ Basis of these results, the ethanol extract was investigated antifungal activity against hyphal type of C. albicans. Since the hyphal type forms biofilm, C. albicans was formed biofilm on 12-well plate to investigated antifungal activity against hyphal type. The ethanol extract showed antifungal activity against hyphal C. albicans. In this study, hyphal C. albicans was evaluated for two reasons. First, the hyphal type forms a biofilm, which forms a protective barrier on the outside of the biofilm with exopolysaccharide.¹⁹ The protective barrier makes it resistant to antifungal agents, and the resistance to antifungal agents is greater than that of the yeast type. Another reason is that C. albicans mainly exists as a hyphal type in oral cavity. Therefore, in order to investigate the antifungal effects in clinical area, it is necessary to use the hyphal type of C. albicans. Recently, resistance to antifungal agents of human disease-related fungi is increasing, and thus a treatment is being sought using antifungal agents in crop.^20,21 Shiitake mushroom is also a crop and may be a candidate to be an agricultural fungicide.

Next, to investigate the effect on denture-related stomatitis, a candidal biofilm was formed on denture resin and its removal ability was tested. Since the antifungal effects of the ethanol extract on candidal biofilm was investigated, the effect when mixed with a denture cleanser was investigated next. The ethanol extract showed a synergistic effect with the denture cleanser on candidal biofilm. This result indicates that the denture cleanser containing enzymes and other constituents had no effect on the ethanol extract. Denture cleanser has been reported to induce oral mucosal injury, chemical burn, and gastric perforation.^22-24 Therefore, it may be safer denture users by reducing or pulling out the strong toxic substances of denture cleanser and using the extract of shiitake mushroom. In addition, a further study is necessary to investigate antifungal activity against candidal biofilm on denture base resin by using the extract of shiitake mushroom and changing the composition of denture cleanser.

The present study showed that the ethanol extract from shiitake mushroom has strong antifungal activity against yeast and hyphal type of C. albicans. Also, the ethanol extract showed a synergistic effect with the denture cleanser on candidal biofilm on denture. The extract from shiitake mushroom may be a candidate to prevent oral candidiasis.