Introduction
Edible mushrooms are recognized as functional foods due to their remarkable nutritional characteristics (Cohen et al., 2014). These organisms can have valuable resources for maintaining good health, and have numerous scientifically proven properties, including antiviral, immunomodulatory, hyperlipidemic, antioxidant (Brugnari et al., 2016; Acharya, Khatua, & Ray, 2017; Dulay et al., 2017; Finimundy et al., 2018), anti-inflammatory, antitumor (Cao, Liu, Hou, & Li, 2015), hyperglycemic, antithrombotic and antimicrobial properties (Schillaci, Arizza, Gargano, & Venturella, 2013; Owaid, Al-Saeedi, & Al-Assaffii, 2015; 2017; Castillo, Pereira, Alves, & Teixeira, 2017; Das, Saha, Joshi, & Das, 2017; Suresh, Ambika, Noorjahan, & Kalaiselvam, 2017). Such biological properties are conferred to macromolecules and molecules of lower molecular weight, such as polysaccharides, proteins, peptides and cerebrosides, isoflavones, triacylglycerols, steroids, amines, sesquiterpenes, among others (Fu, Liu, & Zhang, 2016).
Among the metabolites produced by these macrofungi, some of these which are derived from their secondary metabolism have antimicrobial activity, and are classified as terpenoids, polyacetylenes and phenolics (Shen, Shao, Chen, & Zhou, 2017). Recent research has indicated that several strains of the genus Pleurotus possess antimicrobial properties, such as Pleurotus japonicus, which inhibits the pathogen Bacillus subtilis via the antibiotic 6-deoxyilludin M (Hara, Yoshida, Morimoto, & Nakano, 1987).
Another species, P. eryngii, has attracted the attention of several researchers since it presents a basidiome with desirable organoleptic characteristics that are superior in relation to other mushrooms of the same genus (Iqbal et al., 2018; Ma et al., 2018). These have been reported to include medicinal properties, such as antitumoral, antioxidant, antimicrobial, hypoglycemic, immunomodulatory, antihemolytic properties, among others (Mariga et al., 2014; Xu et al., 2016; Zhang et al., 2016; Kim et al., 2017; Madhanraj et al., 2019). Such attributes may come from the basidiomes themselves or from the liquid filtrates from the cultivation of this organism (Owaid et al., 2015). Fu et al. (2016) and Zhang et al. (2019) present a brief review of the research in the literature and reaffirm that this mushroom species is interesting for the development of therapeutic remedies, due to the bioactive compounds which are presented. In this context, our study contributes to the continuity of studies regarding this mushroom species, given the continued interest in the search for natural antimicrobial compounds as an alternative to existing antimicrobial agents. Thus, this research evaluated the antimicrobial activity of P. eryngii from its filtrates obtained after submerged cultivation.
Material and methods
Microorganism
The P. eryngii DPUA 1816 culture used in the assay was provided by the DPUA Culture Collection at the Universidade Federal do Amazonas and reactivated in potato dextrose agar (PDA) culture medium with yeast extract (YE) 0.5% (p v-1). The cultures were maintained in the dark at 25°C, for 10 days (Kirsch, Pinto, Porto, Porto, & Teixeira, 2011).
Submerged cultivation
The culture was carried out in a culture medium based on an infusion of 200 g L-1 of purple-skinned sweet potato (PSSP), with different concentrations of glucose, in the presence and absence of yeast extract – YE (2 g L-1). The medium containing only sweet potato infusion was used as a control. From the pure cultures, three mycelium fragments (Ø = 1cm) were removed, added to the culture medium and incubated at 150 rpm, for 15 days, at 25°C. At the end of cultivation, samples of the fermented broth were separated from the mycelial biomass by vacuum filtration and placed under refrigeration at 4°C (Rufino et al., 2011).
A second cultivation was carried out to evaluate the growth of macrofungi during 18 days, in medium 4 (M4), under the same conditions as the previous cultivation. Samples, in triplicate, were randomly taken every 2 days, for evaluation of antimicrobial activity. Fungal growth was measured from dehydrated biomass at 60°C until constant weight (Kirsch, Macedo, & Teixeira, 2016).
Preparation of test microorganisms
The antimicrobial activity was evaluated against test microorganisms of the Amazon bacteria collection (CBAM) of the Leonidas and Maria Deane Institute (Fiocruz Amazonas): Escherichia coli CBAM 0007, Staphylococcus aureus CBAM 0026 and the Cefar diagnostic culture collection (CCCD): Escherichia coli CCCD-E005, Staphylococcus aureus CCCD-S009, Pseudomonas aeruginosa CCCD-P004, Candida albicans CCCD-CC001, Candida parapsilosis CCCD-CC004 and Candida tropicalis CCCD-CC002. The reactivation of the bacteria was performed in Müeller-Hinton agar medium (MH), incubated at 37ºC for 24 hours and the yeast in Sabouraud agar medium (SDA), incubated for 48 hours at 25ºC (Clinical and Laboratory Standards Institute [CLSI], 2009).
Antimicrobial activity
The determination of antimicrobial activity was made using the method of diffusion in agar (CLSI, 2009) with cups cut out of the agar (cup-plates) - according to the recommendation of the Clinical and Laboratory Standards Institute (CLSI, 2009). Aliquots of 100 µL of the microbial suspensions, prepared in sterile, distilled water and standardized using the McFarland scale (1.0), were sown uniformly in the culture mediums using swabs. Then, 100 µL of the samples obtained from the submerged cultivation were added. As a control, itraconazole (30 mg mL-1) and tetracycline hydrochloride (0.05 mg mL-1) were used as positive controls for yeast and bacteria, respectively.
Statistical analyses
The results were evaluated using analysis of variance (ANOVA) to determine significant differences (Tukey test p < 0.05), using the Minitab software, version 17 (Minitab Statistical Software, 2014).
Results and discussion
The results indicated that the metabolite broths of P. eryngii in all tested mediums were unable to inhibit the growth of the bacterias E. coli (CBAM 0007 and CCCD-E005), S. aureus (CBAM 0026 and CCCD-S009) and P. aeruginosa CCCD-P004, as can be seen in Table 1. This result is similar to the study of Yilmaz, Yildiz, Tabbouche, Kiliç, & Can, (2016), who tested P. ostreatus against the bacteria S. aureus ATCC 25923, E. coli ATCC 25922, P. aeruginosa ATCC 27853. Similarly, Costa, Silva, Araújo, and Carvalho (2018) did not observe the inhibition halo (IH) of organic extracts (hexane, dichloromethane, ethyl acetate and methanol) from P. ostreatus against E. coli, P. aeruginosa and S. aureus, although they did achieve IH in tests with β-glucan, isolated from this species.
Table 1. Antimicrobial activity of metabolites from submerged cultivation of Pleurotus eryngii in different culture medium.
Key: Control (200 g L-1 of purple-skinned sweet potato ); M1, M2, M3 or M4 (200 g L-1 of purple-skinned sweet potato PSSP, 10, 20, 30 and 40 g L-1 of glucose and 2 g L-1 of YE); M5, M6, M7 and M8 (200 g L-1 of purple-skinned sweet potato , 10, 20, 30 or 40 g L-1 of glucose and 0 g L-1 of YE). (-) no inhibition halo; (±) standard deviation. In each column, averages that share the same letter do not differ from each other by the Tukey test (p < 0.05).
Although the bacteria tested were resistant in this study, the inhibition of P. aeruginosa using biomass extracted from P. eryngii, P. sajor-caju, P citrinopileatus, P. ostreatus and P. florida in hot water has been mentioned in the literature, and presented an IH which was equivalent to 8.8, 9.0, 8.1, 9.4 and 9.7 mm, respectively (Özdal, Gülmez, Gür-Özdal, & Algur, 2019). Similarly, Fasoranti, Ogidi, and Oyetayo (2018) verified the sensitivity of P. aeruginosa (IH= 14 and 15 mm), S. aureus (IH= 13.7 and 15.7 mm) and E. coli (IH= 15.7 and 14 mm), in relation to the mushrooms P. pulmonarius and P. ostreatus. Sathyan, Majeed, Majitha, and Rajeswary (2017) evaluated the extracts Dimethyl Ether, chloroform and methanol from P. ostreatus, P. eryngii and P. djamor, respectively, and found that the first extract had an IH of 12.20 mm against E. coli, chloroform had an of 17.5 mm and methanol an HI of 12.2 mm against P. aeruginosa. Despite this, E. coli was in fact resistant to P. eryngii and P. djamor in our study.
However, antifungal assays revealed more promising data (Table 1), since, with the exception of the sample M1 against C. albicans CCCD-CC001 and the sample M2 against C. tropicalis CCCD-CC002, the others were able to form inhibition halos against all the evaluated yeasts. Of these, we can highlight: C. albicans CCCD-CC001 from samples M4, M7 and M8, without statistical difference between them, with an IH of 13.5, 12.5 and 12.17 mm respectively; C. tropicalis CCCD-CC002, in samples M1, M4 and M7, presented an IH greater than 11 mm; and finally, samples M1 (27.67 mm), M4 (26.33 mm) and M5 (25.83 mm) against C. parapsilosis CCCD-CC004, the latter being the most sensitive yeast in the samples tested.
These data are superior to those of Nwachukwu and Uzoeto (2010), who tested ethanolic extracts from P. squarrosolus against C. albicans and obtained a maximum IH of 7.10 mm. Similarly, in the study by Akyuz, Onganer, Erecevit, and Kirbag (2010), extracts of P. eryngii var. eryngii, P. eryngii var. ferulae, P. ostreatus and P. sajor-caju were evaluated and obtained IH results of 7.5 and 8.5 mm against C. albicans for only the first two fungi mentioned. Fasoranti et al. (2018) were not successful with organic extracts of P. pulmonarius and P. ostreatus against the yeasts C. albicans and C. parapsilosis, since both presented resistance to the tested species. This research is similar to that of Popa, Voaides, Cornea, and Zagrean (2016), who also indicated resistance of C. albicans ATCC 10321 and C. parapsilosis CBS 604 to P. eryngii.
After observing that the metabolic broths of P. eryngii, obtained from the culture medium M4, produced larger inhibition halos against two of the three tested yeasts, despite there being no statistical difference, this medium was selected for mushroom cultivation during 18 days. However, even after a longer cultivation period, none of the samples inhibited the growth of bacteria (Table 2).
Table 2. Antimicrobial activity of metabolites derived from the cultivation of Pleurotus eryngii in M4 medium during a cultivation period of 18 days.
Key: (-) no inhibition halo presented; (±) standard deviation. In each column, averages that share the same letter do not differ from each other by the Tukey test (p < 0.05).
However, there was antifungal activity which appears to be favorable for all samples from the 12th day of culture onwards. This activity presented greater IH against C. albicans CCCD-CC001 and C. tropicalis CCCD-CC002 in 12 days and against C. parapsilosis CCCD-CC004 after 16 days of cultivation (Figure 1).
Comparatively, the IH obtained from the M4 sample during 18 days of cultivation (Table 2) were lower against all yeasts tested than those of 15 days (Table 1). This result may be related to the growth phases of the fungus, since, according to Figure 1, it is possible to associate the growth phases of P. eryngii in 18 days of cultivation with the antifungal activity presented. This indicates antifungal potential from the end of the exponential phase and beginning of the stationary phase, and as the fungal growth decreases, the antifungal action also decreases.
Figure 1. Antifungal activity of metabolites obtained from Pleurotus eryngii culture in M4 medium for 18 days
The results of this research are in accordance with those found by Subrata, Gunjan, Prakash, Mandal, and Krishnendu (2012), who verified antifungal activity against C. albicans using methanolic extracts of P. djamor, although no activity was noted for S. aureus, E. coli and P. aeruginosa. Extracts obtained using methanol, ethanol and water may also be favorable in antimicrobial tests, and may present satisfactory results against E. coli, S. aureus, P. aeruginosa, C. albicans and C. glabrata (Kalu & Kenneth, 2017). The use of extracts makes it possible to perform antimicrobial tests, such as minimum inhibitory concentration, in addition to the isolation and purification of substances capable of inhibiting pathogenic microorganisms (Schillaci et al., 2013; Yehia & Al-Sheikh, 2014; Li & Shah, 2014; Finimundy et al., 2018; Musa et al., 2018).
Cleudiane Pereira de Andrade cleudiane.andrade@hotmail.com
