DOES THE SOURCE MATTER? PHENOLIC COMPOUNDS AND ANTIOXIDANT ACTIVITY FROM MYCELIUM IN LIQUID MEDIUM, WILD AND CULTIVATED FRUITING BODIES OF THE NEOTROPICAL SPECIES GANODERMA TUBERCULOSUM

and an herbarium reference specimen is kept at FACEN Herbarium N°4887. The species was identified by its macro and As a continuation of the study of the biologically active compounds of native neotropical Ganoderma species in Paraguay, the content of total phenolic compounds, total antioxidants and percentage of antioxidant activity of the fractions extracted with ethanol, hexane, diethyl ether, ethyl acetate and water of mycelium in liquid medium, wild and cultivated fruiting bodies of G. tuberculosum , were analyzed. Biologically active compounds were highest in the diethyl ether fractions of wild fruiting bodies, 126.49 ± 4.19 mg. (GAE) g -1 (milligrams of Gallic acid equivalent per grams of crude extract), antioxidant compounds content of 130.94 ± 5.302 mg .g -1 AAE (milligrams of Ascorbic acid equivalent per grams of crude extract) and activity percentage of 70.93% compared to 67.39 ± 1.5 mg. (GAE) g -1 , 48.37 ± 0.73.g -1 AAE and 45.63% antioxidant activity. According to the values obtained in the present study, the fruiting body of Ganoderma tuberculosum is a promising source of antioxidant compounds for the food industry.


INTRODUCTION
Species of the genus Ganoderma P. Karst., known as the 'mushrooms of immortality' are perhaps the most studied taxa in terms of their biological and chemical properties (Ferreira et al., 2015;Saltarelli et al., 2015;Gong et al., 2019;Zhang et al., 2019).They have long been recognized to promote health and longevity in Asian countries (Boh et al., 2007).So far, more than 20 species of Ganoderma have been studied (Gong et al., 2019), most of them from Asia and Europe.Molecular phylogenetic analyses have shown that these species differ from the neotropical ones (Moncalvo & Buchanan, 2008;Costa-Rezende et al., 2017;Costa-Rezende et al., 2020).Few studies describe the biological and chemical properties of native neotropical Ganoderma species, and preliminary studies have shown great potential and the need for research to continue (Campi et al., 2021).Basidiome, hereafter fruiting body, has been the traditional source of fungal metabolites and nutraceuticals, moreover, it has been shown that the production of those compounds depends on the stage of growth, i.e. mycelium, primordial stages and mature fruiting body (Chen et al., 2012;Li et al., 2013).For example, in Ganoderma lucidum (Curtis) P. Karst., the triterpenoid content was shown to be variable: extremely low in cultured mycelia, markedly higher in the fruiting body primordial formation, but with a decrease in the mature fruiting body (Chen et al., 2012).These variations in concentrations could be related to the expression of the protein-coding genes as it has been shown that their expression in G. lucidum is higher in mycelia or primordial stages compared with those in the fruiting bodies (Li et al., 2013).Regarding phenolic and antioxidant compounds, there is inconsistency on which stage of growth has more content of phenolic compounds and/or antioxidant activity.On one hand, a higher concentration in fruiting body than in mycelium was reported (Reis et al., 2012;Muna et al., 2015;Prasad et al., 2015;Sulistiany et al., 2016) on the other, it has been shown that mycelium presents a higher concentration of those compounds (Carvajal et al., 2012;Tešanović et al., 2017).
In addition to the stage of growth/development of the source, i.e. mycelium primordia or fully developed fruiting bodies, and the kind/nature of the compound under study, the extraction method of the compounds plays as the third variable to take into account.The study of the biologically active fungal compounds requires analysis and fine-tuning of the extraction and purification methods for the different types of compounds (Chen et

Identification of species
The wild sample was collected in Central Department, Paraguay, growing on Tipuana tipu (Benth.)Kuntze and an herbarium reference specimen is kept at FACEN Herbarium N°4887.The species was identified by its macro and As a continuation of the study of the biologically active compounds of native neotropical Ganoderma species in Paraguay, the content of total phenolic compounds, total antioxidants and percentage of antioxidant activity of the fractions extracted with ethanol, hexane, diethyl ether, ethyl acetate and water of mycelium in liquid medium, wild and cultivated fruiting bodies of G. tuberculosum, were analyzed.Biologically active compounds were highest in the diethyl ether fractions of wild fruiting bodies, 126.49± 4.19 mg.(GAE) g -1 (milligrams of Gallic acid equivalent per grams of crude extract), antioxidant compounds content of 130.94 ± 5.302 mg .g - AAE (milligrams of Ascorbic acid equivalent per grams of crude extract) and activity percentage of 70.93% compared to 67.39 ± 1.5 mg.(GAE) g -1 , 48.37 ± 0.73.g -1 AAE and 45.63% antioxidant activity.According to the values obtained in the present study, the fruiting body of Ganoderma tuberculosum is a promising source of antioxidant compounds for the food industry.micromorphological features and confirmed by molecular analyses.For microscopic analysis, free-hand sections of fruiting body were mounted in 3-5% (w/v) aqueous potassium hydroxide (KOH) and 1% (w/v) aqueous phloxine or Melzer's reagent following Robledo et al. (2020).Basidiospores were measured in KOH and phloxine mounts under oil immersion at 100X magnification.Sequence of the ITS marker was obtained from culture.The extraction, amplification and sequencing followed Robledo et al. (2020).The new DNA sequence generated in the present work was combined with sequences retrieved from GenBank (NCBI) to construct a dataset.Scientific names and GenBank Accession Numbers of sequences are listed in Table 1.Dataset was composed of 84 ITS sequences of 18 Ganoderma species (Table 1).Foraminispora rugosa (Berk.)Costa-Rezende, Drechsler-Santos & Robledo was selected as outgroup (Costa-Rezende et al. 2017, 2020;Morera et al., 2021).ITS region was aligned using ProbCons 1.12 (Do et al., 2005) and alignments were manually inspected and adjusted using MEGA 6 (Tamura et al., 2013).ModelFinder (Kalyaanamoorthy et al., 2017) as implemented in the IQ-Tree software (Nguyen et al., 2015) was used to estimate the best-fit partitioning strategy and the best-fit model of nucleotide evolution for the dataset using 3 data blocks (ITS1, 5.8S and ITS2).Models were restricted for those implemented in MrBayes 3.2 (Ronquist et al., 2012).Bayesian inference (BI) and maximum likelihood (ML) phylogenetic analyses were applied to the concatenated datasets using the partition scheme and evolutionary models defined by ModelFinder.BI was performed following Robledo et al. (2020) in the CIPRES science gateway (Miller et al., 2010) (http://www.phylo.org/).Maximum likelihood searches were conducted with IQ-Tree, initially involving 100 ML searches, each one starting from one randomized stepwise addition parsimony tree.Branch supports were calculated using the UFBoot (ultrafast bootstrap approximation) (Hoang et al., 2018) implemented in IQ-TREE with 1000 replications.A node was considered strongly supported with BPP ≥ 0.95 or BS ≥95% (Hyde et al., 2013;Minh et al., 2020).

Preparation of samples
From the context of a fresh basidiome, hereafter fruiting body, mycelium (M) was isolated on Potato Dextrose Agar (PDA).The inoculum consisted of a 25-mm 2 surface agar plug from a 10-day-old culture that was transferred to flasks (500 mL) with 200 ml of a liquid medium.The growing medium consisted of (g.L -1 ): sucrose 35, peptone 5, yeast extract 2.5, KH2PO4 x H2O 1, MgSO4•7H2O 0.5, and vitamin B1 0.05 (Tang & Zhong, 2002).The inoculated flasks were then incubated at 28 °C in an incubator resting for 30 days, the mycelium was filtered through Whatman No. 4 paper.

Cultivation substrates
Spawns were prepared from the PDA isolated strain into heat resistant polypropylene bags with oatmeal (Avena sativa L.) that were previously sterilized for 1 hour at 121°C, then inoculated.The mycelium was allowed to grow for two weeks in the dark at 28°C.The formulation for the bag cultivation was sawdust of Eucalyptus sp. and Handroanthus sp. in a 1:1 ratio.This mixture was hydrated to reach a moisture content of approximately 50% of its total weight, 2% Ca(OH)₂ was added to maintain pH.Three kilograms of the mixed substrate were filled into heatresistant polypropylene bags and sterilized at 121°C for 2 hours.The substrate was inoculated with the spawns and incubated at 28°C for 30 days until the fruiting body were obtained (Figure 3).

Preparation and characterization of phenolic extracts
Solvent extractions with a polarity gradient were carried out following  (2023).The results were expressed as the mean of the triplicates of the absorbance in milligrams of Gallic acid equivalent per grams of crude extract (mgg -1 ) ± standard deviation (SD).

Concentration and antioxidant activity
The concentration and oxidant activity were determined with the DPPH• radical (2,2-diphenyl-2-picrylhydrazyl hydrate) absorbance method according to Campi et al. (2021).The results were expressed as the mean of the analysis performed in triplicate in milligrams of ascorbic acid equivalent per g of crude extract (mgg -1 ) ± standard deviation (SD).The percentage of activity (A) was calculated as: where λDPPH is the absorbance of DPPH, λSolution is the absorbance of the solution.

Statistical analyses
The data obtained in the trials were analyzed using the two-way ANOVA test (95% confidence interval; with previous verification of assumptions), together with the Tukey post-hoc test (95% confidence interval).One-way ANOVA analysis and Tukey's test were also used to compare the variables evaluated (i.e.source, extraction method, polarity of solvent) between the different fractions studied.These analyses and statistical graphs were performed using the statistical programs InfoStat v. 2019e (Di Rienzo et al., 2019) and Past v. 4.06b (Hammer et al., 2001).

Morphological and molecular analyses
Main macro and microscopical characters observed in the species under study agree with the reference descriptions (Welti et al., 2010).Macroscopically G. tuberculosum is characterized by large, demediate to commonly substipitate to stipitate basidiomata; pilear surface concentrically sulcate and zonate, dark reddish at the base, yellowish to white at the margin, a homogeneous context with melanoid deposits and 4-5 rounded pores per mm.Microscopically by the ovoid to ellipsoid basidiospores, 9-12 × 5.5-7.8µm and the tuberculated cutis cells with a weak amyloid reaction (Loyd et al., 2018).The morphological identification is confirmed with molecular analyses.Dataset included 84 terminals and 600 characters, of which 126 were parsimony informative, 132 variable and 466 constants.The partitions and evolutionary models selected were: HKY+F+G4 (ITS1 and ITS2) and K2P (5.8S).Bayesian and ML analyses resulted in similar topologies; the ML tree is presented in the Figure 1.The topology recovered is congruent with previous works (Loyd et al., 2018;Morera et al., 2021).The specimen under study groups with G. tuberculosum conforming a lineage recovered with maximum support (1/100).

Chemical profile
Our results showed that, in general, there are significant differences in phenolic compounds (TPC), antioxidant concentration and antioxidant activity between the development stages and growth conditions, as well as the extraction technique used (Figure 2A-C, Table 2).The highest concentrations of phenolic compounds were obtained with diethyl ether and ethyl acetate, being significantly higher in wild fruiting bodies than cultivated and mycelium (Figure 2A, Table 2).The fruiting bodies have a higher concentration of TPC than the cultured mycelia.
The difference between the concentrations of TPC in the fruiting bodies and mycelium, have been reported several studies (  2012) reported significant differences in TPC of G. lucidum between the fruiting body (FB) and the submerged mycelium (M) (28.64±0.28mg GAE g - 1 and 5.95±0.09mg GAE g -1 , respectively).Prasad et al. (2015) reported values of 21.60 ± 2.60 mg GAE g -1 (FB) and 10.63 ± 1.00 mg GAE g -1 (M) for G. applanatum (Pers.)Pat., while for G. lucidum values of 16.78 ± 1.46 (FB) and 10.63 ± 1.00 mg GAE g -1 (M) were found.On the other hand, Tan et al. (2015) reported values of 101.01 ± 0.59 (FB) and 20.55 ± 0.53 (M) mg GAE g -1 for G. neojaponicum Imazeki.These results show similarities to those obtained in our work (Figure 2A-C) where it is seen that in all organic fractions the concentration of phenolic and antioxidant compounds in the fruiting body is higher than in the mycelium.This shows that in the genus Ganoderma, phenolic compounds are mainly accumulated in the fruiting body.Analyzing the fractions in relation to the concentration of antioxidant compounds and antioxidant capacity, they showed a similar pattern than TPC, the highest concentrations were found in the fruiting body (Figure 2).Regarding the origin, in general, the wild fruiting body had higher concentration of TPC, antioxidant concentration and antioxidant activity, in accordance with previously observed patterns (Grangeia et al., 2011;Tan et al., 2015).
Another factor influencing the secondary metabolites levels was the extraction solvent, as its polarity has a direct influence on the phenolic content of the resulting extracts (Tan et al., 2015).A significant difference was found between crude extracts and solvent washes with increasing polarity.Diethyl ether and Ethyl acetate fractions were the ones with the highest concentration in TPC and antioxidant concentration, so a previous washing with non-polar solvents (e.g., petroleum ether, hexane) is recommended to clean the extract lipid components as much as possible.

CONCLUSION
In conclusion, wild fruiting bodies showed significantly higher concentrations of phenolic compounds than cultivated fruiting bodies and mycelium.Several factors can influence the differences in bioactive compound concentrations: the extraction procedure, the choice of solvents, the culture media for growing mushrooms and the liquid culture of the mycelium.It should also be noted that secondary metabolites are produced in response to environmental stress.Regarding the extraction solvents, it is concluded that the liquid-liquid extraction technique with polarity gradient favors the correct separation of the metabolites to be studied.
al., 2007; Heleno et al., 2012; Chen & Kang, 2019; Dong et al., 2019).Different extraction methods influence the results obtained, as well as the growth conditions of the fruiting body and mycelium (Heleno et al., 2012).Welti et al. (2010) have isolated ganoderic acid from the fruiting bodies from Ganoderma tuberculosum Murril.According to these researchers G. tuberculosum extracts might inhibit the growth of cancer cells.Espinosa-García et al. (2021) for their part, studied 14 extracts of mycelium from Ganoderma strains from Mexico and concluded that G. tuberculosum manifested activity in at least one of the six cancer cell lines tested (HBL-100 and T-47D [breast], HeLa [cervix], A-549 and SW 1 573 [lung], and WiDr [colon]), with a minimum concentration necessary to cause 50% growth inhibition of cancer cells (GI50) < 50 μg/mL−1.The present study continues with the exploration of the potential of native Ganoderma species from Paraguay (Campi et al., 2021) and the main objective was to evaluate the concentrations of phenolic compounds and antioxidants in different development stages and growth conditions, i.e. cultured mycelium, wild and cultivated fruiting bodies, of G. tuberculosum, considering different extraction protocols.

Figure 1
Figure 1 Phylogenetic tree based on ITS rDNA sequences generated by maximum likelihood showing the position and phylogenetic relationships of G. tuberculosum within the genus Ganoderma.Posterior probabilities (>0.95) and bootstrap values (>95%) are shown at the branches.Clade names follow Loyd et al. (2018) and Morera et al. (2021).Specimen studied is highlighted in boldface.

Figure 3
Figure 3 Ganoderma tuberculosum: A wild fruiting body, B-C: fresh and dried fruiting bodies cultivated indoor, D: Mycelium in liquid medium.

Table 1
Ganoderma species: specimens: origin and ITS Gen Bank accession numbers for sequences used in the phylogenetic analyses Legend: O = outrgoup.T= type specimen.* = Accession numbers for ITS1 and ITS2.New sequence is highlighted in boldface.

Table 2
Phenolic compounds, antioxidant concentration and antioxidant activity values for mycelium, wild and cultivated fruiting bodies of G. tuberculosum.
Espinosa-García et al.(2021)analyzed the mycelial extracts from G. tuberculosum obtained with chloroform-methanol (1:1) and they reported promising antioxidant activity, with values of 62.5 ± 3.9 μM TE/mg [micromolar Trolox equivalents (TE) per milligram of extract].Regarding phenolic compounds, they reported 26.9 ± 1.7 GAE/mg, for mycelia our results show values of 70.56 ± 1.51 GAE/mg for the ethyl acetate fraction from mycelium, this higher concentration could be due to the polarity of the solvent used.