Fomitopsis officinalis: Spatial (Pileus and Hymenophore) Metabolomic Variations Affect Functional Components and Biological Activities

Fomitopsis officinalis is a holartic polyporous mushroom that forms large fruiting bodies on old standing trees, fallen logs, or stumps. F. officinalis is a medicinal mushroom species that is most commonly used in traditional European medicine. In this study, we explore the spatial metabolic differences in F. officinalis’ mushroom parts, i.e., the cap (median and apical parts) and the hymenium. Additionally, chromatographic analysis was conducted in order to unravel the composition of specialized metabolites in the hydroalcoholic mushroom extracts. The potential antifungal and bacterial effects of extracts were tested against pathogen strains of Gram+ and Gram– bacteria, and yeast, dermatophytic, and fungal-pool species. Extracts from the apical part were the richest in terms of phenolic compounds; consistent with this finding, the extracts were also the most effective antiradical and antimicrobial agents with MIC values < 100 µg/mL for most of the tested bacterial and dermatophytic species. According to these findings, F. officinalis extracts are valuable sources of primary and secondary metabolites, thus suggesting potential applications in the formulation of food supplements with biological properties in terms of antioxidant and antimicrobial activities.


Introduction
Fomitopsis officinalis (Vill.) Bondartsev and Singer (syn. Laricifomes officinalis (Vill.) Kotl. and Pouzar) is a holartic polyporous mushroom belonging to the Fomitopsidaceae family (Polyporales order) that forms large fruiting bodies on old standing trees, fallen logs, or stumps. It is a slow-growing necrotrophic parasite causing intensive brown rot in wood. Basidiomata are perennial and sometimes reach a considerable size [1,2]. F. officinalis is easily distinguished from other Fomitopsis species due to its chalky texture, and distinctive smell and taste [1,2].

Mushroom Identification
The exact characterization and identification of medicinal mushrooms is fundamental for exploiting their full potential in the food and pharmaceutical industries [20]. The morphological characteristics of the fruiting body of F. officinalis (PeruMyc 3897) correspond to those reported by Bernicchia and Gorjón [21]. The taxonomic affiliation of the mushroom strains was performed via targeting the ITS region of the ribosomal DNA. Additionally, a BLAST search confirmed that our sample belonged to F. officinalis, as it showed a close match with the deposited sequences of these species (Table 1).

Untargeted LC-MS/MS-Based Metabolomics
In this study, the metabolomic profile of F. officinalis was evaluated through the mass spectrometry-ultraperformance liquid chromatography (UHPLC)-QTOF method. The raw data were processed with MS-DIAL in two separate sessions, one for the Pos and one for the Neg files. The obtained data were merged into a single data matrix that reports the mass and retention time of 8335 features, and their area in the respective 9 samples (from L1 to L9).

Statistical Data Analysis
RawData_Mz__RT_MetaboAnalyst.xlsx was uploaded to the MetaboAnalyst web platform to perform PCA, and heat-map, pathway, and functional analyses (Figures 1-3). To perform PCA, the data matrix was treated with autoscaling and normalized with the median.   heat map confirming that the samples were mainly divided into two clusters: hymenium and fruiting body. The latter was divided into the medium and apical clusters. Heat-map analysis shows that some characteristics were overexpressed only in the hymenophore, others only in the apical part of the fruiting body, and others only in the middle part of the fruiting body.  heat map confirming that the samples were mainly divided into two clusters: hymenium and fruiting body. The latter was divided into the medium and apical clusters. Heat-map analysis shows that some characteristics were overexpressed only in the hymenophore, others only in the apical part of the fruiting body, and others only in the middle part of the fruiting body.

Cluster Analysis
The data, treated as previously described, were used to create the dendrogram ( Figure 2A) and the heat maps ( Figure 2B). The distance between the features was calculated with the Euclidean algorithm.

Functional Analysis
Both PCA and cluster analysis showed a clear separation between the hymenium and the fruiting body. Therefore, the data were subjected to functional analysis to search for significantly altered metabolic pathways between these two groups.
The data were loaded into the MetaboAnalyst functional analysis module, which performs a putative annotation of the features on the basis of m/z and RT values obtained from spectrometric analysis. A putative list of 505 annotated metabolites (AnnotatiMetabo-Analyst.csv) was used to determine the active metabolic pathways using the Mummichog and GSEA algorithms. The result sof this analysis are shown in Figure 3, where the abscissa and ordinate show the −log10 of the p-value calculated with the GSEA and Mummichog algorithms, respectively. The size of the circles represents the enrichment factor of each pathway; the color fades from yellow into red in proportion to the −log10 of the probability that a given pathway was active. The pathways that were statistically significantly active in the hymenium with respect to the fruiting body fell into the upper and right quadrants of the figure. A total of 60 metabolic pathways were identified; the complete data are in the table "Results_mummichog_integ_pathway_enrichment". Table 2 shows the most significant pathways (combined p value < 0.2). The matrix showing the metabolites found with functional analysis was used to determine the metabolic differences between the apical and middle parts of the fruiting body, and statistical analyses show that they differed markedly from each other. The metabolic pathways that were overexpressed in the apical part compared to the middle part are listed in Table 3.

Extract Phenolic Composition and Antioxidant Activity
Phenolic compounds are important phytochemicals whose content in plant extracts is generally determined with the Folin-Ciocalteu spectrophotometric and HPLC methods. Table 4 shows the values of the total phenol content (TPC) and antioxidant activity of the extracts of the three parts of F. officinalis (hymenium, apical, and median parts). The values for total phenoliv content ranged from 89.61 mg GAE/100 g in the median part to 116.12 mg GAE/100 g in the apical part of the mushroom. The apical part (L4-L6) was richer in total phenols, which was confirmed with chromatographic analysis (Supplementary Material Tables S1 and S2, Figures S1-S4). Among the identified compounds, quercetin was the main phenolic compound.
The recent interest in phenolic compounds characterizing edible mushrooms has been due to their health-promoting properties, such as their antioxidant potential. In order to evaluate the antioxidant activity of extracts, three complementary spectrophotometric in vitro assays were carried out. ABTS and DPPH assays measure the ability of antioxidants to scavenge chromogen ABTS or chromogen DPPH free radicals, respectively, while the FRAP assay allows for evaluating the reducing capacity of the extracts. All these assays were compared with the Trolox standard, a water-soluble vitamin E analog.
In this paper, the highest values of ABTS (170.00 mg TE/g DW), DPPH (104.06 mg TE/g DW), and FRAP (198.00 mg TE/g DW) were found in apical part of F. officinalis. These results agree with the phenolic content; in fact, the lowest values of all spectrophotometric assays were found for the hymenium. A wide range of values of TPC and antioxidant activity was reported in a previous paper [22] that studied the optimization of the extraction of bioactives from Pleurotus ostreatus. TPC values ranged from 38.5 to 423.7 mg GAE/100 g and were all lower than those reported in this paper. Concerning antioxidant properties, Ianni et al. [22] reported a wide range for FRAP (6.0-70.0 mg TE/100 g value) and DPPH (8.7 to 172.0 mg TE/100 g) values, which are all lower than those obtained for F. officinalis, while ABTS values (110.7-1112.7 mg TE/100 g) were similar or lower, a comparison of the antioxidant assay results with those in the literature was sometimes not possible because the data of these assays were reported as EC 50 or radical scavenging activity (%) [11,19,[23][24][25]. A correlation study was also conducted considering all the extracts and all spectrophotometric parameters (Table 5). Good correlations were always obtained (R 2 higher than 0.6874), and the best correlation values were found for TPC vs. FRAP (R 2 = 0.9886) and DPPH vs. ABTS (R 2 = 0.9755).

Antimicrobial Activity
In the present study, the results of the antimicrobial effect of F. officinalis were evaluated. The antimicrobial activity of F. officinalis Extracts L1, L4, and L7 against the tested bacterial, yeast, and dermatophytic strains is shown in Tables 6-8. All F. officinalis extracts showed antimicrobial activity in the concentration range of 1.95-200 µg/mL, but with a wide variability in terms of potency and selectivity (Tables 6 and 7). The growth inhibition of the yeast strains (Table 7) showed no sensitivity to the L1, L4, and L7 F. officinalis extracts. Regarding the growth inhibition of dermatophytic isolates (    With reference to bacteria (Table 6), the highest antimicrobial activity of F. officinalis extracts was observed in Extract L1 (MIC 1.53-<1.53) against Bacillus subtilis (PeruMycA 6), and Extracts L4 and L7 (MIC 3.86 and 7.71 µg/mL) against Escherichia coli (ATCC 10,536) with an MIC range of 3.86-79.37 µg/mL; S. typhi (PeruMycA 7) was the most resistant strain to F. officinalis extracts with an MIC range of 158.74->200. There were only two cases in which there was the least sensibility for L1 against Pseudomonas aeruginosa (ATCC 15442) and Staphylococcus aureus (ATCC 6538). In this case, the extracts also showed a wide range of sensibility (MIC 7.71 (6.12-12.25)-158.74 (100-200) µg/mL). MIC values under 100 µg/mL were considered an indication of high antimicrobial activity [26]. The highest antimicrobial activity of L1 was observed against B. subtilis (MIC 1.53 µg/mL), L4 had a major affect towards E. coli (MIC 3.86 µg/mL), and the highest inhibition of L7 was again observed for B. subtilis (MIC 3.86 µg/mL). Collectively, Gram-negative bacterial strains (ATCC 10536 and 15442, PeruMycA 2, 3, and 6) were less sensitive to mushroom extracts than Gram-positive strains were.
Comparing antimicrobial activity results is not easy, as the used methodology to produce fungal extracts may vary widely, and susceptibility is not only species-specific, but even strain-specific [27].
Nevertheless, the reported MIC values in the present study could be compared to those reported for other Basidiomycota [27]. In a different study involving Pleurotus ostreatus [22], this was also true for the tested bacteria strains. P. ostreatus extracts showed major antibacterial activity towards Gram+ bacteria, and the highest MIC value (9.92 µg/mL) was observed against B. subtilis (PeruMyca 6), which was the same as F. officinalis, but the highest MIC concentration (<6.25 µg/mL) was towards E. coli (ATCC 10536).
Overall, regarding the growth-inhibitory effects exerted by the F. officinalis extracts towards the selected pathogen microbial strains, consistent with the intrinsic biological activity of the extract, namely, antiradical properties, Extracts L4-L6 from the apical part were antimicrobially the most effective. This could partly be related to the phenolic compound content [28]. Although the MIC values were higher than those of the reference antimicrobial drugs, apical extracts were effective at <200 µg/mL concentrations, which are generally well-tolerated by human and murine cells [29]. Furthermore, MIC values < 100 µg/mL were considered an indication of high antimicrobial activity [26].

Mushroom Strain
The fruiting bodies of the  Figure 4 shows an F. officinalis mushroom with the three investigated parts.

Molecular Identification
Total genomic DNA was extracted from the fruiting body according to Angelini et al. [19]. The internal transcribed spacer (ITS) region was amplified using primer combination ITS1F/ITS4 according to Angelini et al. [19]. The thermocycler was programmed as follows: 1 cycle of denaturation at 95 °C for 2.5 min; 35 cycles of denaturation at 95 °C for 20 s, annealing at 55 °C for 20 s and extension at 72 °C for 45 s; 1 final extension cycle at 72 °C for 7 min. The electrophoresis of the polymerase chain reaction (PCR) amplicons was performed on 1.2% agarose gel. PCR products were purified using the ExoSap-IT PCR Cleanup reagent (Affymetrix UK Ltd., High Wycombe, UK) and then submitted for sequencing to Macrogen Europe (Amsterdam, The Netherlands). The resulting chromatogram was proofread, and the generated sequence was deposited in GenBank with access number OQ809067 (F. officinalis PeruMyc 3897).

Mushroom Extract Preparation
The mushroom materials were separated into 3 samples: the hymenium, median, and apical parts of the F. officinalis fruiting body. Afterwards, they were dried in a ventilated stove at 40 °C. The dried mushroom materials separated into hymenium, median, and apical parts of the fruiting body were finely ground and extracted in 50 mL of EtOH:water 7:3 (v/v) for 30 min under ultrasonic agitation. Each extract was prepared in triplicate (Samples L1-L9).
The resulting extracts were then filtered through Whatman GF/C filters (Sigma, Germany), and the solvent was evaporated under reduced pressure (40 °C, 218 mbar) using a rotary vacuum evaporator (Rotavapor R-100, Büchi, Switzerland). The residue was kept at −20 °C until further use (Table 9).

Molecular Identification
Total genomic DNA was extracted from the fruiting body according to Angelini et al. [19]. The internal transcribed spacer (ITS) region was amplified using primer combination ITS1F/ITS4 according to Angelini et al. [19]. The thermocycler was programmed as follows: 1 cycle of denaturation at 95 • C for 2.5 min; 35 cycles of denaturation at 95 • C for 20 s, annealing at 55 • C for 20 s and extension at 72 • C for 45 s; 1 final extension cycle at 72 • C for 7 min. The electrophoresis of the polymerase chain reaction (PCR) amplicons was performed on 1.2% agarose gel. PCR products were purified using the ExoSap-IT PCR Cleanup reagent (Affymetrix UK Ltd., High Wycombe, UK) and then submitted for sequencing to Macrogen Europe (Amsterdam, The Netherlands). The resulting chromatogram was proofread, and the generated sequence was deposited in GenBank with access number OQ809067 (F. officinalis PeruMyc 3897).

Mushroom Extract Preparation
The mushroom materials were separated into 3 samples: the hymenium, median, and apical parts of the F. officinalis fruiting body. Afterwards, they were dried in a ventilated stove at 40 • C. The dried mushroom materials separated into hymenium, median, and apical parts of the fruiting body were finely ground and extracted in 50 mL of EtOH:water 7:3 (v/v) for 30 min under ultrasonic agitation. Each extract was prepared in triplicate (Samples L1-L9).
The resulting extracts were then filtered through Whatman GF/C filters (Sigma, Germany), and the solvent was evaporated under reduced pressure (40 • C, 218 mbar) using a rotary vacuum evaporator (Rotavapor R-100, Büchi, Switzerland). The residue was kept at −20 • C until further use (Table 9). The F. officinalis extract was mixed with 20% Na 2 CO 3 solution and the Folin-Ciocalteu's reagent, and the mixture was kept in the dark for 30 min before measuring the absorbance at 750 nm. The results, expressed as mg of gallic acid equivalents (GAE) per 100 g of dry weight (mg GAE/100g dw), were derived from the calibration curve of the gallic acid standard [22]. Table S3 shows the regression equation, linearity, and range of concentration of gallic acid.

Determination of Antioxidant Activity
For the ABTS assay, radical cation ABTS + ·was prepared via the reaction of ABTS with potassium persulfate solutions after being kept in the dark at room temperature for 12 h. The obtained reagent was diluted with ethanol until 0.70 (±0.02) absorbance had been obtained at 734 nm. An aliquot of an ABTS + /ethanol solution was added to the extract, and the mixture was left in the dark for 6 min [22].
For the DPPH assay, the DPPH reagent (0.06 mM in ethanol) was added to the extract, and the mixture had been kept in the dark for 30 min before the absorbance was measured at 517 nm [22].
For the FRAP assay, the reagent, prepared by mixing a TPTZ solution with a FeCl 3 solution and acetate buffer, was added to the sample extracts. The reaction mixture had been kept in the dark for 4 min before absorbance was measured at 593 nm [22].
The results of the antioxidant assays are expressed as mg Trolox equivalents (TE) per 100 g of dry weight (mg TE/100g dw) and were derived from a calibration curve of the Trolox standard (Table S3). Spectrometric data were acquired in the 40-1700 m/z range in both negative and positive polarity. The Agilent JetStream source was operated as follows: gas temperature (N 2 ) 200 • C, drying gas 10 L/min, nebulizer 50 psi, sheath-gas temp: 300 • C at 12 L/min. Raw data were processed using the MS-DIAL software (4.48) [30] to perform peakpicking, alignment, and peak integration. The MS signal threshold was set at 1000 counts.

Untargeted LC-MS/MS-Based Metabolomics and Statistical Analysis
A data matrix was obtained that accurately reported the mass and area of each revealed peak in each analyzed sample.
Metabolites were putatively annotated and metabolic pathways were predicted using the mummichog algorithm [31] implemented in the 'MS Peaks to Pathways' module of Metaboanalyst 5.0 [32]. This considers any possible adducts and different ionic polarities, and classifies annotated peaks on the basis of the t-test. In this case, the list of putative compounds was mapped onto the KEGG library of Saccaromices cerevisiae. ANOVA and functional meta-analysis were also performed with MetaboAnalyst. For the statistical analysis, samples were normalized via the median, followed by Pareto scaling.

HPLC Determination of Phenolic Compounds
The extracts were analyzed for quantitative phenolic determination using a re-versedphase HPLC-DAD in gradient elution mode [33]. The separation was conducted within 60 min of the chromatographic run, starting with the following separation conditions: 97% water with 0.1% formic acid, 3% methanol with 0.1% formic acid (Supplementary Table S1). The separation was performed on an Infinity lab Poroshell 120-SB reverse-phase column (C18, 150 × 4.6 mm i.d., 2.7 µm; Agilent, Santa Clara, CA, USA). Column temperature was set at 30 • C. The quantitative determination of phenolic compounds was performed via a DAD detector. The selected wavelengths are reported in Supplementary Table S2. The quantification was conducted through 7-point calibration curves with linearity coefficients (R 2 ) > 0.999 in the concentration range of 2-140 µg/mL. All standards were purchased from Merck Life Science (Milan, Italy), and had ≥95% purity. The limits of detection were lower than 1 µg/mL for all assayed analytes. The area under the curve from the HPLC chromatograms was used to quantify the analyte concentrations in the extract [33]. The Candida parapsilosis (ATCC 22019) and Candida krusei (ATCC 6258) strains were used as quality controls in the broth dilution antifungal test [34].

Antibacterial Activity
The MICs of the F. officinalis samples were determined in sterile 96-well microplates using the broth microdilution method of the Clinical and Laboratory Standards Institute, M07-A10 [34]. MICs were determined using extract concentrations in the range of 1.562-200 µg/mL, derived from serial twofold dilutions in Mueller-Hinton Broth (MHB).
For the preparation of the bacterial suspensions (inocula), three to five colonies of the bacterial strains used for the test were chosen from 24 h cultures on tryptic soy agar plates (TSA) and pregrown overnight in Mueller-Hinton broth (MHB) to reach a cell density of approximately 1-2 × 10 8 CFU/mL in each tube.
This was confirmed with the plating of serial dilutions of the inoculum suspensions on Mueller-Hinton Agar (MHA). The setup included bacterial growth controls in wells containing 10 µL of the test inoculum and negative controls without a bacterial inoculum. MIC end points were determined after 18-20 h incubation in ambient air at 35 • C.
The inoculum suspensions were prepared from 7-day-old cultures grown on Sabouraud Dextrose Agar (SDA; Difco) at 25 • C, and adjusted spectrophotometrically to optical densities that ranged from 0.09 to 0.11 (MacFarland standard).
Filamentous fungi (microconidia) and yeast inoculum suspensions were diluted to a ratio of 1:50 in RPMI 1640 to obtain twice the inoculum size, ranging from 0.2 to 0.4 × 10 4−5 CFU/mL. This was further confirmed by plating the serial dilutions of the inoculum suspensions on SDA.
MIC end points (µg/mL) were determined after 24 h (for yeasts) and 72 h (for dermatophytes) of incubation in ambient air at 30 • C (CLSI 2017, CLSI 2018). For the Fomitopsis extracts, ç MIC end points were defined as the lowest concentration that showed total growth inhibition.
ç MIC end points for fluconazole and griseofulvin were defined as the lowest concentration that inhibited 50% of the growth when compared with the growth control (CLSI 2017). Geometric means and MIC ranges were determined from the three biological replicates to allow for comparisons between the activities of the F. officinalis samples.

Statistical Analysis
The results of spectrophotometric assays (TPC, ABTS, DPPH, FRAP) are reported as the mean ± standard deviation (SD) of three replicates. Microsoft Excel 2016 (Microsoft Corporation, Redmond, WA, USA) was used for data analysis. Correlation analysis was performed with a linear regression model.

Conclusions
Due to technological developments, mass spectrometry matched with liquid chromatography could be largely employed in metabolomic studies with a broad perceived range, and advanced specificity and sensitivity. In the current study, this method was used to analyze the metabolic profiling of F. officinalis extracts (Samples L1, L4, and L7), showing satisfactory data quality. The present findings support more indepth investigations aimed at evaluating the influence of growth substrates on the antimicrobial and antioxidant properties of F. officinalis. Extracts from distinct parts of the fruiting body of F. officinalis revealed different concentrations of secondary metabolites, thus suggesting potential applications in the formulation of food supplements with biological properties, especially in terms of antioxidant and antimicrobial activities.

Supplementary Materials:
The following supporting information about quantitative determination of phenolic compounds and antioxidant activity can be downloaded at: https://www.mdpi.com/ article/10.3390/antibiotics12040766/s1; Table S1: Gradient elution condition of the HPLC-DAD-MS analyses; Table S2: Content in specialized metabolites; Table S3: Regression equation, linearity, range of concentration of standards (gallic acid for TPC, Trolox for ABTS, DPPH, and FRAP) used for the spectrophotometric assays; Chromatograms: Figures S1-S4.