Candida spp. is a commensal member of the oral cavity of most of the population. However, local or systemic factors may disrupt this balance, causing superficial and disseminated infections in humans (Tong and Tang, 2017). Besides host factors, micro-organisms require several mechanisms to colonize epithelial cells, invade deep organs, and evade the human immune system. Extracellular enzyme production, such as proteinase and phospholipase, stands out among these mechanisms (Staniszewska, 2020).

Secreted aspartyl proteinase is an enzyme family that can degrade several physiologically important substrates such as albumin, immunoglobulin, and collagen, and immune components such as antibodies, elements of the complement system, and cytokines (Dabiri et al., 2018). Phospholipases are hydrolytic enzymes that attack the phospholipids of any cell membrane and have been associated with the ability of fungus adherence to the host cell (Zhao et al., 2020; Sriphannam et al., 2019).

Using natural products aims to replace conventional chemical drugs, mushroom properties should be examined because they may show active compounds produced by these organisms. Some mushrooms have been studied for their ability to use micro-organisms as a food resource and promote their activity (Bach et al., 2017; Abidin et al., 2017). Several substances have been isolated from basidiomycetes, especially Lentinula, which can assist host cell response to biologically active substances stimulating the maturation, differentiation, or proliferation of cells involved in defense mechanisms, increasing host resistance against various cancers and infectious diseases (Chaturvedi et al., 2018).

Recent studies have reported an increased interest in alternative medicine and natural therapies, especially using substances with antimicrobial properties, with mushrooms representing a good possibility (Fisher et al., 2018). Among these basidiomycetes, Lentinula edodes and Pleurotus pulmonarius have shown promising results as antimicrobial agents (Sakamoto et al., 2017; Válková et al., 2017). The L. edodes mushroom, known as ‘Shiitake’, has important pharmacological properties such as antimicrobial and antioxidant activities. Experiments with compounds produced during mycelial growth and substances found in basidiocarp have demonstrated this ability (Ruilova et al., 2019; Vetter, 2019). Regarding Pleurotus spp., investigations have shown that mushrooms of this genus can also produce antimicrobial agents (Govindaraj and Renganathan, 2017).

Therefore, this study aims to evaluate the activity of mushroom extracts from L. edodes, P. pulmonarius, and Pleurotus sajor-caju species against eight yeast species and evaluate proteinase and phospholipase productions in Candida spp. samples, verifying the effect on phospholipase production through the exposition of these samples to two mushrooms filtrates (SHI and PUL).

Mushroom culture conditions

The mushrooms were grown in the Mycology Laboratory of the Department of Microbiology and Parasitology of the Institute of Biology, Federal University of Pelotas, RS, Brazil. After the collection for extraction, the basidiomata were washed under running water, drained on a paper towel, sliced, ??and put in a forced-air oven, where they remained for 15 days at 35°C for drying.

Preparation of the filtered mushroom extracts

The dried mushrooms were packed in paper bags, placed in desiccators at room temperature, and protected from light until used. Dried mushrooms were ground in a blender for 1 min (50 g mushroom/L of distilled water). The mixture remained in the refrigerator at 4°C for 24 h, then filtered through cotton, and centrifuged at 5000 g and at 4°C for 1 h. The supernatant was filtered through a Whatman No. 1 filter and cellulose acetate membranes of 0.45 and 0.25 µm in aseptic conditions in a laminar flow.

Antifungal susceptibility assay

This study applied the methodology of the CLSI according to the document M27-A3 (CLSI, 2008a), which allowed testing pure and diluted extracts (50%) against eight yeasts with broth microdilution. The antifungigram assay was conducted adapted to plant protection, using the mushroom extracts diluted at 1:2 and 1: 4 against Candida sake, C. albicans, Candida parapsilosis, Candida globose, Rhodotorula species, Kloeckera japonica, and Cryptococcus laurentii yeasts. The results were released after 24 and 48 h.

Sample and culture conditions

Eighty-one C. albicans samples were collected from individuals with denture stomatitis to evaluate the extracellular activities of phospholipase and proteinase. The samples were collected from the palate of individuals with sterile swabs. The specimens were seeded in the SDA medium with 0.2 mg/mL of chloramphenicol and incubated at 37°C for 24 to 48 h. C. albicans strains were identified with (1) microculture assay in corn meal agar, (2) growth in CHROMagar^TM Candida, (3) growth in the hypertonic medium, and (4) ID 32 C assimilation assay (bioMérieux). The ATCC 62342^TM strain was used as the control for phospholipase and proteinase productions.

Exposure of Candida samples to filtered mushroom extracts

The effect of both mushroom filtrates on phospholipase activity was evaluated with the Kadir  methodology  with some alterations (Kadir et al., 2007). Ten C. albicans samples with positive phospholipase production were randomly selected for the assay. A 0.5 McFarland turbidity suspension was prepared from each of the 10 isolated samples. Of these suspensions, 0.5 mL was added to tubes containing 2 mL of phosphate-buffered saline (PBS; control) and others containing 1.9 mL of PBS and 0.1 mL of filtered mushroom extract. The tubes were incubated for 30 min at 37°C. Then, the agent was removed with two rounds of washing with sterile PBS and centrifuged for 10 min at 3000 turns. The supernatant was completely removed, and the pellet was re-suspended in 2 mL of sterile PBS.

Evaluation of the extracellular activities of phospholipase and proteinase enzymes

Eighty-one C. albicans samples were tested in triplicate to verify phospholipase and proteinase activities. The assay medium for phospholipase was Sabouraud Dextrose Agar (SDA) with 57.3 g of sodium chloride, 0.55 g of calcium chloride, and 100 mL of sterile egg yolk (enriched egg yolk) per liter of distilled water. The assay medium for proteinase was bovine serum albumin (BSA) agar containing 5 g of BSA, 1.45 g of yeast nitrogen base (YNB), 20 g of glucose, and 20 g of Agar-Agar per liter of distilled water. All samples were cultivated in 4% SDA for 24 h. Then, they were diluted in distilled water on the scale of 0.5 McFarland, and 20 µl of each sample was applied to the assay medium. The plates were incubated at 37°C for 72 h for proteinase and phospholipase. The enzymatic activity was determined with the formation of a halo around a fungal colony and measured by the division of diameter of the colony (DC) / DC + zone of precipitation (Zp),  according  to  the method described by Price et al. (1982). and illustrated in Figure 1. The values obtained allowed determining whether C. albicans presents high, low, or no enzymatic activity. High activity had values lower than 0.63, low activity had values between 0.99 and 0.64, and no activity had a value of 1.00. These values work for the activities of both enzymes analyzed in this study.

Statistical analysis

The results were analyzed with Student t-test and ANOVA, using the SigmaStat 3.5 software for both tests to determine whether the statistical difference was calculated, considering p < 0.05.

Antifungal susceptibility assay

After reading the results at 24 and 48 h, C. parapsilosis was sensitive to SHI, PUL, and PSC filtered extracts diluted at 1:2 and SHI at 1:4. A C. globose sample was sensitive to SHI and PUL filtered extracts diluted at 1:2 and SHI at 1:4. Other specimens of C. globose, K. japonica, and C. laurentii were only sensitive to the PSC extract diluted at 1:2. Rhodotorula spp. was sensitive to SHI and PSC extracts diluted at 1:2 and PSC at 1:4. C. sake and C. albicans did not show sensitivity to any of the three filtered mushroom extracts at any dilution.

Most of the 81 isolated strains identified as C. albicans (n ??= 70). C. parapsilosis (n = 5), Candida guilliermondii (n = 2), Candida lipolytic (n = 2), Candida glabrata (n = 1), and Candida tropicalis (n = 1) samples were also isolated. These C. albicans samples presented a high proteinase production rate, with Zp values between 0.167 and 0.581. Phospholipase was produced in 55 strains of C. albicans, with Zp values from 0.422 to 0.905. The 11 non-albicans samples also showed a high proteinase production rate, with Zp from 0.217 to 0.391. Phospholipase was produced in only four non-albicans strains (two C. parapsilosis, one C. lipolytic, and one C. tropicalis). Table 1 shows the mean phospholipase and proteinase production values according to species.

Neither SHI nor PUL mushroom filtrates showed any effect on fungal growth at the concentration used for the anti-phospholipase assay. Table 2 shows the mean phospholipase activity values for the 10 strains of C. albicans exposed to the two mushroom filtrates.

Evaluation of the extracellular activities of phospholipase and proteinase enzymes

The mean value of phospholipase produced by C. albicans isolates not exposed to mushroom filtrates was 0.574.

The brief exposure to SHI and PUL filtrates reduced extracellular phospholipase with Zp values of 0.652 and 0.636, respectively. The difference in Zp values between the two  groups  exposed  to  mushroom  filtrates was not significant (p = 0.991), while there was a statistically significant difference between the two test groups and the control (p < 0.05).

The interest in alternative non-chemical antimicrobial agents has grown considerably in recent years worldwide. The filtered extract of the L. edodes culture has significant antimicrobial activity against different species of phytopathogenic bacteria such as Ralstonia solanacearum, and the main bioactive components responsible for this antimicrobial effect are its acids, mainly oxalic acid (Kwak et al., 2016). Moreover, another study showed that exposing C. albicans and Candida neoformans yeasts to Lentinus subnidus and Lenzites species mushroom extracts at concentrations from 12.5 g/mL to 100 mg/mL made them sensitive to the lowest concentrations (Chakraborty et al., 2019).

The   antimicrobial   ability   of   mushrooms,  especially Pleurotus spp., also seems effective against Bacillus subtilis bacteria. Studies have demonstrated the sensitivity of bacterial species from the fermentation broth and mycelium of Pleurotus ostreatus (Muszy?ska et al., 2018; Al-Bahrani et al., 2017).

Pleurotus spp. is known for its high nutrient and medicinal content. The antimicrobial action of this genus occurs with the production of antibiotics such as pleurotin, produced by some species (Purnomo et al., 2017). Another study confirmed the presence of compounds such as lecithin, which also has therapeutic properties (El-Enshasy et al., 2019; Masri et al., 2017). P. ostreatus also showed antimicrobial activity, decreasing the bacterial adhesion of Listeria innocua by up to 96% (Klan?nik et al., 2017). Additionally, the Pisaster giganteus extract associated with Lignosus rhinocerus, Hericium erinaceus, and Schizophyllum commune extracts was effective against the DENV-2 serotype of the dengue virus, presenting low toxicity to Vero cells when infected by the virus (Ellan et al., 2019).

L. edodes also has antitumor and antioxidant properties, and has hypocholesterolemic activity, which was demonstrated in experiments with compounds produced during mycelial growth and substances found in the basidiocarp (Yoo et al., 2019).

Using mushrooms as antimicrobial, particularly antifungal agents is possible, as several of them can produce toxic metabolites. The results obtained in this study showed new perspectives on the use of macroscopic fungi pathogenic to humans and animals.

Regarding the Candida genus, several studies have been concerned with attenuating or inhibiting virulence factors instead of causing the death of this yeast. Kadir tested two subtherapeutic doses of chlorhexidine in ten C. albicans samples from patients with denture stomatitis and showed a significant decrease in phospholipase activity (Kadir et al., 2007).

Studies aiming to attenuate or inhibit the virulence factor of the Candida genus generally use antifungal agents that inhibit the ergosterol synthesis of fungal cells, such as fluconazole, itraconazole, amphotericin B, flucytosine, and nystatin. Lyon and Resende (2006) verified decreased adhesion ability and phospholipase and proteinase productions in C. albicans samples after exposure to fluconazole. This inhibition of virulence factors by antifungal agents seems similar among Candida spp.

The present study also evaluated the phospholipase activity of C. albicans isolates (phospholipase producers) exposed to two mushroom filtrates. Exposing the samples to the two mushroom filtrates for 30 min significantly reduced the mean value of phospholipase production (Meza-Menchaca et al., 2019). In a similar study, the anti-enzymatic activity in phospholipase is attributed to ergosterol peroxides, characterized as an ergosterol derivate, a compound present in the cell membrane of fungi. Additionally, the aqueous chloroform extract found in the fruiting bodies of mushrooms with Lactarius matsutake has also inhibited the enzymatic activity of phospholipase (Gao et al., 2007).

Thus, after verifying the filtered mushroom extracts tested, the results promise an inhibitory effect on phospholipase production. Further studies are required to evaluate the inhibitory effect and its mechanism, as well as complementary studies with a higher number of samples tested.

The authors have not declared any conflict of interests.

The authors thank the Brazilian Council of  Scientific  and Technological Development (CNPq).