1. INTRODUCTION
Mushrooms are eukaryotic heterotrophic organisms that absorb nutrients and play an essential role in decomposing organic materials to make them available for use by plants and other organisms. Most mushrooms are saprophytes. Out of the 1.5 million species of macrofungi or mushrooms, only 6.7% have been described and are primarily documented in temperate zones; those in tropical areas with high diversity remain largely unexploited and underexplored [1,2]. In tropical countries, the estimated number of fungal species is approximately 80,000 of which 16,000 are macrofungi or mushroom species [3].
The macrofungus Lentinus belongs to the family Polyporaceae and is prevalent worldwide due to its diverse species and cultural requirements, particularly in tropical countries where the rich rainforests provide optimal conditions for its growth [4]. Well-known Lentinus spp. exhibit unique dietary and therapeutic advantages and have been utilized by native populations across the world for their functional edible uses. Some species, such as Lentinus edodes in China [5] and Lentinus squarrosulus (LSQ) in the Philippines [6], are widely cultivated as food. Lentinus spp. are also rich in mycochemicals, including alkaloids, terpenoids, and flavonoids, which exhibit antibacterial activity against various bacteria, such as Staphylococcus aureus, Bacillus cereus, Escherichia coli, and Pseudomonas aeruginosa [7]. Further analysis using GC-MS identified several secondary metabolites from L. squarrosulus, including methyl linoleate, hexadecanoic acid methyl ester, methyl 2-oxo-1-pyrrolidine acetate and 9,12-octadecadienoic acid ethyl esters (linoleic acid ethyl ester), all of which possess antibacterial, anti-oxidant, anti-hypercholesterolemic and pesticide activities [8].
Despite their widespread distribution and potential for biological activity, only a few studies have focused on the cultivation of Lentinus spp. mycelium outside their natural habitat. In the present work, Lentinus spp. was collected from tropical areas in Indonesia were cultivated through solid-state fermentation (SSF) by employing organic agro-industrial waste such as fruit peel to produce many bioactive compounds [9]. A previous study used papaya peel as a medium for cultivating Panus tigrinus (Polyporaceae) and was reported greater mycelial biomass production than using banana or pineapple peel medium [10]. Papaya peel was also applied as growing medium for Russula and Pleurotus mushrooms, and all the mushroom cultures exhibited good biomass production [11]. Papaya peel contains 54.48% total carbohydrate, 10.56% protein, 54.48% water, 0.23% fat, and 5.25% ash, rendering it a potential substrate for mushroom growth and a good source of bioactive compounds [12].
The cultivation and antimicrobial activity of Lentinus spp. in papaya peel medium has never been reported and must be documented. Therefore, this study aimed to identify and examine the mycochemical content and bioactivity of Lentinus spp. cultivated by SSF using papaya peel as the medium.
2. MATERIALS AND METHODS
2.1. Chemicals and Materials
Potato dextrose broth (PDB) and Mueller–Hinton Agar (MHA) media were obtained from HiMedia (India). Sabouraud dextrose agar (SDA) was purchased from Merck (USA). Nutrient agar (NA) and nutrient broth (NB) were acquired from Oxoid (UK). DNA extraction and purification kits were purchased from FavorGen (Taiwan), PCR mix and agarose gel were obtained from ABclonal (USA), and the ladder for electrophoresis was bought from PCRBIOSYSTEMS (UK). Loading dye was sourced from GeneDirectX (USA). As a medium for the SSF of mushroom, California (Callina) variety papaya was purchased from a local supermarket.
2.2. Mushrooms Sampling
Mushrooms were collected from two provinces: the forest surrounding Suak Gual Village in Belitung, Indonesia (2°54’49 “S 107°23’13 “E) and the area under dead wood around rice fields in Sleman District, Yogyakarta, Indonesia (7°44’18 “S 110°30’06 “E). The samples were stored in zip-lock plastic bags, sun-dried, and kept at 4°C in the laboratory for further analysis.
2.3. Morphological Identification of Mushrooms
The morphological identification of the mushrooms involved macroscopic observations and species identification based on their basidiomes (fruiting body) [13,14].
2.4. Mycelium Preparation
The mushroom basidiomes were cleaned under running water and then cut into pieces. The samples were treated with a 10% sodium hypochlorite solution and rinsed with sterile distilled water. The partially dried basidiomes were then inoculated on PDA medium and incubated for 3–7 days at room temperature (25°C ± 2°C) to observe mycelial growth [15].
2.5. Molecular Identification of Mushrooms
DNA was extracted from the basidiomes using the FavorPrepTM Plant Genomic DNA Extraction Mini Kit from FAVORGEN (Favorgen, Taiwan) following the manufacturer’s guidelines. The isolated DNA was then amplified using PowerPol 2x PCR Mix with Dye from ABclonal (Massachusetts, USA). The PCR reaction mixture comprised 25 µl of PowerPol 2x PCR Mix with dye, 1 µl of ITS-1 primer (10 pmol/µM), 1 µl of ITS-4 primer (10 pmol/µM), 50 µl of nuclease-free water, and 8 µl of DNA template. The PCR amplification process consisted of the following steps: pre-denaturation at 94°C for 5 minutes, template DNA denaturation at 94°C for 30 seconds, annealing at 55°C for 45 seconds, DNA extension at 45°C for 2 minutes, DNA polymerisation at 72°C for 1 minute and a final cooling stage at 4°C. The amplification cycle was repeated 30 times. The PCR products were then run in electrophoresis using 1% agarose gel in TAE buffer. Purification was performed with FavorPrep GEL/PCR Purification Mini Kit (Favorgen, Taiwan), followed by sequencing (Genetika Science, Jakarta, Indonesia). The sequence results were then analyzed and compared with existing data in GenBank using the BLASTn tool from the National Center for Biotechnology Information (NCBI). Phylogenetic construction was performed using MEGA 11.0 (Pennsylvania State University, Pennsylvania, USA).
2.6. Mushroom Cultivation on Papaya Peel Medium by SSF
The papaya peel waste was thoroughly washed, cut, placed in an Erlenmeyer flask at a total weight of 20 g, and added with 2 ml of sterile distilled water. The medium was autoclaved and then allowed to cool for 24 hours before being aseptically inoculated with mycelium using a cork borer. Five plugs of mycelium (5 mm2) were evenly distributed within the papaya peel medium. The inoculated medium was then incubated for 20 days at room temperature (25°C ± 2°C) until it was completely colonized by the mycelium [11].
2.7. Sample Extraction
The mushroom mycelia grown in papaya peel medium were harvested, soaked in 96% ethanol 1:2 (w/v), macerated for 72 hours, filtered with Whatman paper, concentrated with a rotary evaporator, and finally dried. The extract was then kept at 4°C and used for mycochemical and bioactivity analysis [15].
2.8. Antimicrobial Assays
The extracts were prepared by dissolving them in 0.5% DMSO (1 g/ml). Six bacterial strains, Staphylococcus aureus ATCC 25923, Staphylococcus hominis InaCC B226, Staphylococcus epidermidis, Propionibacterium acnes ATCC 6919, P. aeruginosa ATCC 27853, and E. coli 25922, were used as the tested bacteria, and Candida albicans was used as the tested fungus. The tested bacteria were sub-cultured in NA medium and grown in NB medium 24 hours prior to testing. The bacterial cultures in NB were adjusted to the 0.5 McFarland standard and then swabbed onto sterile Petri dishes containing MHA medium. Meanwhile, C. albicans was grown on PDB medium for 48 hours prior to testing and then swabbed onto sterile Petri dishes containing SBA medium. Subsequently, 10 µl of each extract was applied to a disc and placed in a circle on the MHA medium containing the tested bacteria and the SBA containing the tested fungus. The plates were then incubated at 37°C for 20–24 hours for bacterial growth and at 30°C for 48–56 hours for fungal growth. The diameter of the clear zone around each disc was measured to assess each extract’s ability to inhibit microbial growth [16].
2.9. Mycochemical Analysis
Mycochemical testing included triterpenoids, tannins, alkaloids, saponins, and flavonoids.
2.9.1. Alkaloid test
For alkaloid testing, 5 ml of the extract was combined with 10 ml of MeOH and 10 ml of 2N HCL and then treated with Wagner reagent. Positive results are indicated by turbidity in the sample [17].
2.9.2. Terpenoid test
Terpenoid testing was performed using the Salkowski test. Initially, 5 ml of the extract was mixed with 2 ml of chloroform and then carefully layered with 3 ml of sulphuric acid. Positive results are characterized by a red–brown coloration [18].
2.9.3. Tannin test
Tannin was detected using a FeCl3 (ferric chloride) reagent. Briefly, 10 ml of the ethanol extract was combined with 5 ml of 5% ferric chloride solution. Color changes in the solution to grey or black indicate the presence of tannin compounds in the extract [19].
2.9.4. Saponin test
Saponin testing involved diluting 1 ml of the extract with 20 ml of distilled water and shaking the mixture in a cylindrical measuring cup for 15 minutes. The formation of a foam layer (approximately 1 cm thick) indicates the presence of saponin [20].
2.9.5. Flavonoid test
Flavonoid testing utilized a cyanidin reagent. Extracts containing flavonoids will produce a purplish color [21].
2.10. GC-MS
The extract was dissolved in a 1:1 (g/v) solution with 96% ethanol. The GC-MS instrument used in this research is the Agilent 7693A Autosampler equipped with the following specifications: a capillary column type HP-5MS UI (Agilent) (30 m × 250 µm × 0.25 µm). Helium was used as the carrier gas, the injector temperature was set at 250°C and the injection volume was 1 µl. The split ratio was 01:10 with a split speed of 1 ml/minute, and the initial temperature was maintained at 50°C for 1 minute. The temperature ramp began at 50°C for 1 minute, followed by an increase of 5°C/minute to 100°C for 3 minutes, then 5°C/minute to 150°C for 3 minutes, 5°C/minute to 250°C for 3 minutes and finally 10°C/minute to 280°C for 5 minutes. The detector employed was the Agilent 5977B Mass Spectrometry with a mass range of 30–550. The obtained results include a table of peak chromatograms and compounds [22].
2.11. Data Analysis
The results of the triple experiments are shown as the mean and standard deviation (SD). Antimicrobial data were analyzed with SPSS software (SPSS Inc., Chicago, IL, USA). One-way ANOVA was conducted, followed by a DMRT post-hoc test for significant differences (p < 0.5). Phylogenetic analysis was performed using MEGA 11.0 software with the maximum likelihood (ML) method and 100 repetitions [23].
3. RESULTS AND DISCUSSION
3.1. Mushroom Identification
The two Lentinus samples were isolated from dead trees in the tropical forests of Belitung Islands and from dead wood near rice fields full of litter in the Special Region of Yogyakarta Province. They were labeled B1 and Y1 and identified based on their morphological characteristics.
Both mushrooms belong to the order Polyporales because they have distinctive pores. This order consists of various types of basidiocarps, such as bracket-shaped basidiocarps (genus Fomitopsis), flat-shaped (genus Lentinus), smooth-shaped (genus Podoscypha), hymenophore with a poroid (genus Perenniporia) and resuspended to effusion shaped-reflection (genus Ceriporiopsis, Phlebia). The mushroom samples exhibit a distinct morphology, featuring light brown to brown scales, serrated edges, and white to yellow underside lamellae. They grow individually or in groups on wet wood, logs, and tropical tree stumps, are extensively found in tropical regions, and can withstand a broad range of temperatures. These characteristics are typical of the genus Lentinus [4,24].
Fungi can be identified using morphological and molecular methods. Morphological differences in color, surface texture of their caps, attachment of the cap to the stalk, spacing of gills, and stalk shape of the basidiomes between the mushroom samples were observed to determine their species (Fig. 1).
 | Figure 1. Front and back views of (a) Lentinus sajor-caju (LSJ) from Yogyakarta and (b) LSQ isolated from Belitung Province, Indonesia. [Click here to view] |
According to morphological observations, the B1 sample was suspected to be L. squarrosulus due to the presence of scabs, scales, or crusts, which are critical features of this species [24]. L. squarrosulus basidiocarp has a slightly creamy white color, a rough hood surface, and a slightly brown center. It has a pileus diameter of 2.7–5.5 cm and lamellae with a size of 1.2–4.7 cm attached to the stalk with shortly decurrent (shortened lamella length) and moderate inter-row lamella characters. The stipe is 1.8–3.5 cm long with a cylindrical shape (uniform stalk size from tip to stalk) as described in their identification key [13,14]. Meanwhile, the Y1 sample was suspected to be L. sajor-caju. L. sajor-caju basidiocarp is yellow-brown with a smooth, fibrous, and fleshy hood surface, a pileus diameter of 4.5–5.8 cm. The lamellae have a size of 3.9–4.5 cm and are attached with a vast distance (adnate); the spacing between lamella rows is tight. The stipe is 1.5 cm long with a root-like shape. The primary identification characteristic of L. sajor-caju is the presence of an annulus. However, no annulus was found in the Y1 sample possibly due to genetic variations, suggesting that L. sajor-caju typically develops pseudoannula upon maturation [25]. Additionally, the sample underwent color changes or was hygrophanous, transitioning from white to yellow-brown, further supporting its classification as L. sajor-caju [26].
Molecular identification was also carried out to ensure accurate species identification based on DNA sequences. The ITS region was utilized for molecular identification to identify the fungal species [27]. The morphological identification was corroborated by the molecular data obtained using the ITS-1 and ITS-4 primers for Basidiomycota fungi, which included the 5.8S rRNA gene, ITS-1 and ITS-2 regions [28]. The results showed a band with a length of approximately 600 bp for both mushroom species. BLAST analysis revealed that the mushroom isolated from Yogyakarta (Y1) shared 97% similarity and 100% query coverage with the L. sajor-caju species and was submitted to NCBI as L. sajor-caju PP236948.1. Meanwhile, the mushroom isolated from Belitung Islands (B1) exhibited 93% similarity and 94% query coverage with the L. squarrosulus species and was submitted to NCBI as L. squarrosulus PP211943. 1. A phylogenetic tree was constructed using the ML method as shown in Figure 2.
 | Figure 2. Phylogeny tree of fungi L. squarrosulus PP211943.1 (B1 sample) and L. sajor-caju PP236948.1 (Y1 sample). [Click here to view] |
Lentinus spp. are macrofungi distributed in tropical and subtropical regions and predominantly growing in tropical areas [29]. Despite their various benefits, efforts for their cultivation are still limited. Mushrooms are cultivated by SSF, which involves fermentation under sufficient humidity to support fungal growth and metabolic activities. This method aims to replicate the natural growth environment of isolated fungi as closely as possible [11]. SSF offers several advantages over submerged fermentation, including water and energy efficiency, ease of extraction, production of high-value products, and utilization of industrial waste substrates, making the former more economically viable [30]. This study selected papaya peel substrate due to its economic importance among the Caricaceae family and its widespread cultivation in tropical countries such as Indonesia [10]. Using papaya peel as a growth medium allows for the conversion of agro-industrial waste into value-added products [12]. L. squarrosulus and L. sajor-caju demonstrated robust growth on papaya peel medium, with distinct differences in mycelial colony growth.
3.2. ANTIMICROBIAL ACTIVITY
Papaya peel medium in SSF can provide a favorable environment for fungal mycelium growth because it has high contents of water, protein, and carbohydrates [31]. The extracts of LSQ and LSJ mycelia cultivated on papaya peel medium were tested for antimicrobial activity. The inhibition zone of the extracts against the tested microorganisms can be used to detect the strength of the compounds present in the sample in inhibiting microorganism growth [32].
LSQ exhibited potential antibacterial activity against S. aureus, S. epidermidis, and P. acnes with inhibition zone diameters of 3.37 ±1 0.15, 0.87 ± 0.80, and 5.33 ± 1.24 mm, respectively. However, it could not inhibit S. hominis, P. aeruginosa, or E. coli. This research is the first to report the antibacterial activity of LSQ against P. acnes, which has been overlooked so far. Our study found that LSJ did not inhibit all the tested bacteria. Meanwhile, the papaya peel extract (EP) inhibited the growth of S. aureus ATCC25923 and S. epidermidis with inhibition diameters of 3.00 ± 0.52 and 0.33 ± 0.57 mm, respectively. LSQ and LSJ did not have antifungal activity against Candida albicans (Table 1). A previous report showed that EP could not inhibit the growth of E. coli, P. acnes, S. aeruginosa, and P. hominis [33,34].
 | Table 1. Inhibition zone of papaya peel medium and extracts of L. squarrosulus and L. sajor-caju mycelia cultivated on papaya peel medium against six pathogenic bacteria. [Click here to view] |
In this study, the extracts had higher antimicrobial activities against Gram-positive bacteria; Gram-negative bacteria possess an external membrane coated with lipopolysaccharides which can block the entry of toxic compounds into cells [35,36]. This finding is in accordance with a previous report, which stated the ethanol extract of L. squarrosulus exhibited higher antibacterial activity against Gram-positive bacteria (S. aureus and B. cereus) compared with Gram-negative bacteria (P. aeruginosa and E. coli) [7]. Our study showed that all the tested extracts did not have potential antifungal activity against C. albicans. However, Mossebo et al. [37] reported that the extracts of Lentinus spp. basidiomes have antifungal activity against C. albicans. This variation in bioactivity may be attributed to the use of mushroom samples in different stages that may produce different profiles of antifungal compounds [38,37].
3.3. Bioactive Compounds of Mushroom Extracts
The bioactive compounds of the mushroom extracts were identified using a two-pronged approach: qualitative mycochemical testing and quantitative GC-MS. This comprehensive method revealed that EP contained a diverse range of bioactive compounds, including alkaloids, saponins, tannins, and terpenoids. Meanwhile, L. squarrosulus consisted of alkaloids, and L. sajor-caju contained only terpenoids (Table 2).
 | Table 2. Qualitative mycochemical test on papaya peel medium, extract of L. squarrosulus and L. sajor-caju mycelia cultivated on papaya peel medium. [Click here to view] |
Alkaloids have health benefits such as antimicrobial and anti-inflammatory properties, and terpenoids are used as anti-inflammatory and anticancer agents. Tannins exhibit anti-tumour and antiviral activities [20]. Papaya peel comprises active compounds such as terpenoids, tannins, saponins, alkaloids, and steroids [39]. A previous study reported that the ethanol extract of L. squarrosulus basidiomes consists of alkaloids, steroids, terpenoids, saponins, and anthraquinones, and the hot water with submerged L. sajor-caju mycelium contains terpenoids, alkaloids, and saponins [4]. The differences in mycochemical contents can be due to several factors, including mushroom growth stages and extraction solvents, with the secondary metabolites commonly found in the basidiome samples [40]. According to the antibacterial results, the LSQ extract containing alkaloids can inhibit the growth of S. aureus, S. epidermidis, and P. acnes. Alkaloids can inhibit bacterial growth by disrupting bacterial cell membranes, affecting DNA function, and inhibiting protein synthesis [41]. We found that EP contained alkaloids, saponins, tannins, and terpenoids, all of which may contribute to its antibacterial activity. Saponins could break down the structure of the cell membrane, allowing cell contents to flow out, and terpenoids cause cytoplasm coagulation [42]. Tannins could also deactivate enzymes, cell envelope protein carriers, and other proteins [43].
Table 3 lists more than 11 group compounds, including epoxides, organooxygen compounds, organonitrogen compounds, pyrans, furans, benzene, acids and their derivatives, lactones, oxazinanes, unsaturated hydrocarbons, fatty acids, and terpenoids, identified from the extracts. GC-MS results indicated that EP, LSQ, and LSJ contained 5-hydroxymethylfurfural, n-hexadecanoic acid, and 2,4-dihydroxy-2,5-dimethy-3(2H)-furan-3-one, with EP showing a higher peak area than the mushroom extracts (Fig. 3). Previous GC-MS testing did not detect these compounds, particularly in LSJ [44]. This finding suggested that the papaya peel medium used in the SSF might stimulate the production of these compounds in the cultivated mushrooms. These compounds may still be present in impure or crude form, so the resulting antibacterial activity is still low. This result is similar to the observations for Aspergillus niger, whose compound contents increased following SSF in rose flowers [45]. The 5-Hydroxymethylfurfural and n-hexadecanoic acid exhibit antibacterial activity against S. aureus, B. substilis, and E. coli [46]. The mode of action of 5-hydroxymethylfurfural might be related with its ability to bind with bacterial single-stranded DNA-binding protein. n-Hexadecanoic acid, which includes fatty acids and terpenoids, prevents the synthesis of important compounds, such as proteins, nucleic acids, and cell-wall components, disrupts cell membrane and bacterial DNA replication, and suppresses metabolic processes [47,48]. Additionally, these compounds possess anti-oxidant, anti-hypercholesterolemic, nematocidal, and pesticide activities [49]
 | Figure 3. Chromatogram of GCMS analysis results (a) papaya peel extract. (b) L. squarrosulus (LSQ) extract. (c) L. sajor-caju (LSJ) extract. [Click here to view] |
 | Table 3. GC-MS results of papaya peel medium and extracts of L. squarrosulus and L. sajor-caju mycelia cultivated on papaya peel medium. [Click here to view] |
GC-MS analysis of LSQ revealed its content of hexadecanoic acid methyl ester (10,85%), which was previously found in L. squarrosulus basidiome, 9,12-octadecadienoyl chloride (Z) (10,82%), and maltol (8,73%). Hexadecanoic acid methyl esters show promising antibacterial properties by modifying the structure of the cytoplasmic membrane and altering cell metabolism; as an antimicrobial agent, 9,12-octadecadienoyl chloride (Z,Z) could interact with water molecules, causing cell lysis and water loss due to osmotic disturbance [50,51]. Maltol may cause the shrinkage of the plasma membrane, leading to the degradation and lysis of bacterial cells [52]. These compounds from mushrooms were found using GC-MS, shedding light on their possible roles as active ingredients and their potential synergistic effects on the mushrooms’ bioactivities, especially as antibacterial agents.
4. CONCLUSION
Two Lentinus species, L. squarrosulus and L. sajor-caju, were isolated from the Belitung Islands and Yogyakarta (Indonesia). LSQ exhibited antibacterial activity against S. aureus, S. epidermidis, and P. acnes, with moderate to weak impedance. Meanwhile, LSJ did not inhibit the tested bacteria. Qualitative mycochemical detection showed that LSQ contained alkaloids. GC-MS revealed hexadecanoic acid, methyl ester, 9,12-octadecadienoyl chloride, and maltol as the major bioactive compounds of LSQ.
5. ACKNOWLEDGMENT
The authors thank the Faculty of Animal Husbandry, Universitas Gadjah Mada, Indonesia for their assistance in GC-MS analysis.
6. CONFLICTS OF INTEREST
The authors report no financial or any other conflicts of interest in this work.
7. FUNDING
The authors are grateful to the Doctoral Competency Improvement Program Universitas Gadjah Mada, Indonesia (No. 7743/UN1.P.II/Dit-lIT/pt.01.03/2023), for providing financial support for this research.
8. AUTHOR CONTRIBUTIONS
All authors made substantial contributions to conception and design, acquisition of data, or analysis and interpretation of data; took part in drafting the article or revising it critically for important intellectual content; agreed to submit to the current journal; gave final approval of the version to be published; and agree to be accountable for all aspects of the work. All the authors are eligible to be an author as per the international committee of medical journal editors (ICMJE) requirements/guidelines.
9. ETHICAL APPROVALS
This study does not involve experiments on animals or human subjects.
10. DATA AVAILABILITY
The data are available from the corresponding author upon appropriate request.
11. USE OF ARTIFICIAL INTELLIGENCE (AI)-ASSISTED TECHNOLOGY
The authors confirm that no artificial intelligence (AI)-assisted technology was used in the writing or editing of the manuscript, and no images were manipulated using AI.
12. PUBLISHER’S NOTE
All claims expressed in this article are solely those of the authors and do not necessarily represent those of the publisher, the editors and the reviewers. This journal remains neutral with regard to jurisdictional claims in published institutional affiliation.
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