Untargeted Metabolomics Used to Describe the Chemical Composition, Antioxidant and Antimicrobial Effects of Extracts from Pleurotus spp. Mycelium Grown in Different Culture Media

Pleurotus species isolated in vitro were studied to determine the effect of different media on their production of secondary metabolites, antimicrobial, and antioxidant activity. The different metabolites among Pleurotus samples covered a total of 58 pathways. Comparisons were made between the metabolic profiles of Pleurotus spp. mycelia grown in two substrates: Potato-dextrose-agar-PDA, used as control (S1), and PDA enriched with 0.5 % of wheat straw (S2). The main finding was that the metabolic pathways are strongly influenced by the chemical composition of the growth substrate. The antibacterial effects were particularly evident against Escherichia coli, whereas Arthroderma curreyi (CCF 5207) and Trichophyton rubrum (CCF 4933) were the dermatophytes more sensitive to the mushroom extracts. The present study supports more in-depth investigations, aimed at evaluating the influence of growth substrate on Pleurotus spp. antimicrobial and antioxidant properties.


Introduction
The genus Pleurotus (Fr.) P. Kumm. (Pleurotaceae, Basidiomycota) includes some of the main cultivated edible mushrooms in the world due to their gastronomic, nutritional, and medicinal properties, such as anti-inflammatory [1], antioxidant [2,3], antidiabetic [4], antitumor [5], and immunomodulating [6]. Due to their ability to improve protein content and quality, as well as the valuable health benefits of myco-chemicals or bioactive components present in these mushrooms [7][8][9], Pleurotus spp. can also be used to supplement different processed products such as bread and dairy foods. Pleurotus genus includes

Mushroom Material
The fruiting bodies of P. columbinus, P. ostreatus, P. nebrodensis, and P. eryngii species (P. eryngii var. thapsiae, P. eryngii var. ferulae, P. eryngii var. elaeoselini) were collected on different dates and in different locations (Table 1) and immediately transferred to the laboratory to obtain pure mycelial cultures.
Briefly, for the isolation of mycelia, context pieces (5 × 5 × 5 mm) were excised aseptically from the context of fresh Basidiomycota and transferred to Petri dishes containing Rose Bengal Chloramphenicol agar (Sigma-Aldrich, Milan, Italy) under sterile conditions. Petri dishes inoculated with 3-4 explants were then incubated at 25 • C in the dark for 7 days.
Basidiomata identification was carried out by morphological and molecular analysis [29,30] in consideration of the available data on the occurrence of Pleurotus species in Italy [31].

Molecular Identification
To test the morphological identification (Table 1), the ITS region of the nrDNA was used as a fingerprint marker, as suggested for the wide majority of fungi [32] and successfully applied in previous works by the authors themselves [33][34][35] The total genomic DNA was extracted using the ZR Fungal/Bacterial DNA Kit (Euroclone S.p.A., Milan, Italy). The genomic DNA quality and quantity were evaluated with BIORAD (Milan, Italy) model 200/2.0 Power Supply gel electrophoresis [0.8% agarose gel in 1× TBE buffer (89 mM Tris, 89 mM boric acid, 2 mM EDTA, pH 7.6)] in the presence of SafeView Nucleic Acid Stain (NBS Biologicals, Huntingdon, UK) and a MassRuler DNA Ladder Mix (Thermo Scientific, Vilnius, Lithuania), and visualized with Safe ImagerTM 2.0 Blue Light Trans illuminator Invitrogen (Parma, Italy). DNA samples were subsequently diluted with up to 10 µg/µL nuclease-free water before PCR amplification. The internal transcribed spacer (ITS) region of the nrDNA was amplified by ITS1F and ITS4 primers. SimpliAmp Thermal Cycler Applied Biosystems (Monza, Italy) was programmed as follows: one 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; one final extension cycle at 72 • C for 7 min. Electrophoresis of PCR amplicons was carried out on 1.2% agarose gel as described above. The PCR amplified ITS fragment was purified using the ExoSapIT PCR Cleanup reagent (Thermo Fisher, Monza, Italy) and then sequenced by Macrogen Europe (Netherlands) ( Table 2).

Preparation of Samples
The in vitro culture of Pleurotus spp. was performed in the following solid media: (1) Potato-dextrose-agar-PDA, used as control (S1), and (2) PDA enriched with 0.5% of wheat straw (S2). Each medium in flasks was autoclaved at 121 • C for 20 min and subsequently dispensed into 15 100 mm Petri dishes. The mycelium discs (1 cm diameter) of each Pleurotus mushroom were placed in Petri dishes containing each culture medium (20 mL) under aseptic condition and incubated at 25 • C in the darkness. After 15 days of growth (when mycelium reached maximum radial growth in the PDA medium) the mycelium was recovered from the medium. All samples, realized in duplicate, were lyophilized (FreeZone 4.5 model 7750031, Labconco, Kansas, MO, USA), quantified, and reduced to a fine-dried powder (Supplementary Material: Tables S1 and S2). Preparation of mycelia extract: the Antibiotics 2022, 11, 1468 4 of 21 lyophilized mycelia were extracted for 30 min with distilled and deionized water under ultrasonic agitation.

Untargeted LC-MS/MS-Based Metabolomics and Statistical Analysis
Untargeted LC/MS QTOF analysis was performed using a 1260 Infinity II LC System coupled with an Agilent 6530 Q-TOF spectrometer (Agilent Technologies, Santa Clara, CA USA). The LC consists of a quaternary pump, a thermostated column compartment, and an autosampler. Separation was carried out on an Agilent InfinityLab Poroshell 120 HILIC-Z, 2.1 × 150 mm, 2.7 µm at 25 • C, and 0.25 mL/min flow. The mobile phase consisted of a mixture of water (A) and water/ACN 15:85 (B) both containing a concentration of 10 mM ammonium acetate. Gradient was: time 0-3 min isocratic at A 2%, B 98%; time from 3 to 11 min: linear-gradient to A 30%, B 70; time 11-12 min linear gradient to A 60%, B 40%; time from 12 to 16 min: linear-gradient to A 95%, B 5%; time 16-18 min isocratic at A 95%, B 5%; time 18 min: stop run.
Spectrometric data were acquired in the 40-1700 m/z range both in negative and positive polarity. The Agilent JetStream source operated as follows: Gas Temp (N2) 200 • C, Drying Gas 10 L/min, Nebulizer 50 psi, Sheath Gas temp: 300 • C at 12 L/min. Raw data were processed using MS-DIAL software (4.48) [36] to perform peak-picking, alignment, and peak integration. The MS signal threshold was set at 1000 counts. In the end, a data matrix was obtained reporting the accurate mass and area of each peak revealed in each sample analyzed.
The putative annotation of metabolites and the prediction of metabolic pathways was performed using the mummichog algorithm [37], implemented in the 'MS Peaks to Pathways' module of Metaboanalyst 5.0 [38]. It considers any possible adducts and different ionic polarities and classifies the peaks annotated 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 statistical analysis, samples were normalized by the median, followed by pareto scaling.

HPLC-DAD-MS Determination of Phenolic Compounds
The HPLC apparatus consisted of two PU-2080 PLUS chromatographic pumps, a DG-2080-54 line degasser, a mix-2080-32 mixer, UV, diode array (DAD) and detectors, a mass spectrometer (MS) detector (expression compact mass spectrometer), Advion, Ithaca, NY 14850, USA), an AS-2057 PLUS autosampler, and a CO-2060 PLUS column thermostat (all from Jasco, Tokyo, Japan). Integration was performed by ChromNAV2 Chromatography software. Before the injection in the HPLC apparatus, the extracts were centrifuged at 3500× g for 15 min, and the supernatant was diluted to 20 mg/mL. The extracts were analyzed for phenol quantitative determination using a reversed-phase HPLC-DAD-MS in gradient elution mode ( Table 3). The separation was conducted within 60 mins of the chromatographic run, starting from the following separation conditions: 95% water with 0.1% formic acid, and 5% methanol with 0.1% formic acid ( Table 4). 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). The column temperature was set at 30 • C. Quantitative determination of phenolic compounds was performed via a DAD detector, at 254 nm. Quantification was done through 7-point calibration curves, with linearity coefficients (R 2 ) > 0.999, in the concentration range of 2-140 µg/mL. The limits of detection were lower than 1 µg/mL for all assayed analytes. The area under the curve from HPLC chromatograms was used to quantify the analyte concentrations in the extracts. The extracts were also qualitatively analyzed with an MS detector in negative ion mode. MS signal identification was realized through comparison with a standard solution and MS spectra present in the MassBank Europe database.

Scavenging Effects
The scavenging effect of mushroom extracts on DPPH and ABTS radicals was evaluated as previously reported [33].

Antimicrobial Effects
The in vitro antimicrobial activity of extracts was assessed against the following Gram-negative and Gram-positive bacterial strains: Escherichia coli (ATCC 10536), E. coli

Mushroom Identification
The exact characterization and identification of medicinal mushrooms were fundamental for exploiting their full potential in the food and pharmaceutical industries [18].
The morphological characteristics of Pleurotus spp. (Table S1) fruiting bodies corresponded to those reported in the literature [29].
The taxonomic affiliation of the mushroom strains was performed by targeting the ITS region of the ribosomal DNA. Additionally, a BLAST search confirmed that our samples belong to P. columbinus, P. eryngii var. thapsiae, P. ostreatus, P. nebrodensis, P. eryngii var. ferulae, and P. eryngii var. elaeoselini, as it showed a close match with deposited sequences of these species.

Untargeted LC-MS/MS-Based Metabolomics
In this study, the metabolomic profile of Pleurotus spp. was evaluated through mass spectrometry ultra-performance liquid chromatography-mass spectrometry (UHPLC)-QTOF method. The different metabolites in all Pleurotus samples covered a total of 58 pathways, including biotin metabolism, pantothenate and CoA biosynthesis, tryptophan metabolism, arginine biosynthesis, valine, leucine and isoleucine degradation, glutathione metabolism, one carbon pool by folate, vitamin B6 metabolism, sulfur metabolism, and riboflavin metabolism (Table 5).
Phenylalanine, tyrosine, and tryptophan biosynthesis - Arginine and proline metabolism + + + - Amino sugar and nucleotide sugar metabolism Amino sugar and nucleotide sugar metabolism - Nucleotide metabolism Purine metabolism Vitamin metabolism Nicotinate and nicotinamide metabolism - Pantothenate and CoA biosynthesis - Fatty acid metabolism Biosynthesis of unsaturated fatty acids - Terpenoid metabolism Terpenoid backbone biosynthesis - Other metabolic pathways Porphyrin and chlorophyll metabolism - * metabolic pathway absent (-), metabolic pathway present (+).
A comparative investigation for exploring the effect of different substrates on the metabolic profile was made between Pleurotus spp. mycelia were grown in substrate S2 with respect to substrate S1 taken as reference (Figures 1 and 2). The most evident thing was that the metabolic pathways were strongly influenced by the substrate. Some differences can be tentatively explained. For example, folate biosynthesis was greater for sample PE4 grown on substrate S2. Indeed, this substrate contained, among others, wheat straw which had a good content of vitamin B12 and folic acid [39]. Similar evidence was noted for the metabolic pathway of arginine and proline metabolism which was increased in sample PE2 grown on substrate S2. The same substrate was able to activate the arginine and proline pathways, compared to substrate S1.
In these figures, the pathways revealed by the functional analysis are represented by colored circles, whose abscissas correspond to the enrichment factor and ordinates to the -log of the p-Value. Also, the size of the circle represents the enrichment factor, the color from yellow to red is proportional to the -log of the p-value. The most regulated pathways are in the upper right-hand corner. One carbon pool by folate pathway was particularly expressed in samples PE2 and PO4, rather in the other Pleurotus samples. By contrast, the folate biosynthesis pathway was expressed at higher levels in PE1 and PE4 samples, whereas the pantothenate and CoA biosynthesis pathway were particularly high in the PO5 sample. Comparative investigation for exploring the effect of different substrates on the metabolic profile was made between Pleurotus spp. mycelia grown in substrates S2. Substrate S1 was taken as a reference and data were calculated as mean differences compared to S1 (calibrator of the relative quantification). In the figure, red indicates a higher probability of metabolic pathway activation, whereas blue suggests a minor one. Comparative investigation for exploring the effect of different substrates on the metabolic profile was made between Pleurotus spp. mycelia grown in substrates S2. Substrate S1 was taken as a reference and data were calculated as mean differences compared to S1 (calibrator of the relative quantification). In the figure, red indicates a higher probability of metabolic pathway activation, whereas blue suggests a minor one.

Phenolic Composition of The Extracts
The extracts were also investigated through HPLC-DAD-MS to determine the composition of phenolic compounds.
Among all tested extracts, 28 compounds have been identified (Supplementary Material:  Table S3 and chromatograms). Only caftaric acid was quantified in all extracts ( Figure 3).
Among these phytochemicals, the combination of caftaric acid and benzoic acid was recorded in 9 extracts, while PE1-S2, PN-S2, and PE4-S2 are characterized by the presence of only caftaric acid and catechin. Anyway, there was no effect of the substrate on the qualitative or quantitative composition.
Regarding P. ostreatus, caftaric acid and benzoic acid were the prominent compounds; by contrast in P. eryngii, with the only exception of the sample PE3, catechin is the main phytochemical. In P. nebrodensis (PN), the presence of caftaric acid was not influenced by the substrate composition, while S2 increased the catechin level. In P. columbinus (PC1) cultivated in substrate S2, there was a significant increase in total phenols, as also witnessed by the elevated level of the flavonoid hesperitin. We cannot exclude a possible influence of substrate S2 on the flavonoid biosynthesis pathway (KEGG map 00941), in P. columbinus.
As a final remark, the presence of phenolic compounds in these samples was also an index of potential scavenging/reducing and enzyme inhibition properties [40]. Additionally, phenolic compounds have also been demonstrated to exert antimicrobial effects [41,42]. In these figures, the pathways revealed by the functional analysis are represented by colored circles, whose abscissas correspond to the enrichment factor and ordinates to the -log of the p-Value. Also, the size of the circle represents the enrichment factor, the color from yellow to red is proportional to the -log of the p-value. The most regulated pathways are in the upper right-hand corner. One carbon pool by folate pathway was particularly expressed in samples PE2 and PO4, rather in the other Pleurotus samples. By contrast, the folate biosynthesis pathway was expressed at higher levels in PE1 and PE4 samples, whereas the pantothenate and CoA biosynthesis pathway were particularly high in the PO5 sample.

Phenolic Composition of The Extracts
The extracts were also investigated through HPLC-DAD-MS to determine the composition of phenolic compounds.

Antimicrobial Activity
The antimicrobial activity of the extracts is shown in Tables 6-11, also in comparison with reference antimicrobial drugs, namely ciprofloxacin, fluconazole, and griseofulvin. All extracts from Pleurotus mycelia displayed antimicrobial activity in the concentration range of 1.56 to 200 µg mL −1 . Regarding the yeasts, C. parapsilosis (YEPGA 6551) was the most sensitive strain to the PN-PO6 extracts (S2), with MIC ranges of 7.78->200 µg mL −1 , while C. albicans (YEPGA 6183) showed the least sensitivity to the mushroom extracts. The results of the growth inhibition of yeast strains highlighted, albeit partially, the major activity of the extract derived from the S2 growth substrate. With reference to bacteria, the strongest inhibition was observed for the Pleurotus extracts PC1 and PO5 (S1) [MIC <2.47->200 µg mL −1 against E. coli (ATCC 10536) and B. cereus PeryMycA 2]. Collectively, Gram-bacterial strains (E. coli PeruMyc 2 and 3, S. typhi 7, and P. aeruginosa ATCC 15442) were less sensitive to mushroom extracts than that of Gram+ ones, as already observed for F. torulosa [43]. All results from the tested extracts showed active inhibition of dermatophytes growth. Regarding A. curreyi (CCF 5207), A. insingulare, and T. rubrum (CCF 4933), they were the most sensitive fungal species to all mushroom extracts, with MIC range between 31.49 and 158.74 µg mL −1 . Values of MIC < 100 µg mL −1 were considered as an index of high antimicrobial activity [44].
Among these phytochemicals, the combination of caftaric acid and benzoic acid w recorded in 9 extracts, while PE1-S2, PN-S2, and PE4-S2 are characterized by the presen of only caftaric acid and catechin. Anyway, there was no effect of the substrate on t qualitative or quantitative composition.
Regarding P. ostreatus, caftaric acid and benzoic acid were the prominent co pounds; by contrast in P. eryngii, with the only exception of the sample PE3, catechin the main phytochemical. In P. nebrodensis (PN), the presence of caftaric acid was not inf enced by the substrate composition, while S2 increased the catechin level. In P. columbin (PC1) cultivated in substrate S2, there was a significant increase in total phenols, as a witnessed by the elevated level of the flavonoid hesperitin. We cannot exclude a possib influence of substrate S2 on the flavonoid biosynthesis pathway (KEGG map 00941), in columbinus.
As a final remark, the presence of phenolic compounds in these samples was also index of potential scavenging/reducing and enzyme inhibition properties [40]. Additio ally, phenolic compounds have also been demonstrated to exert antimicrobial effe [41,42]. In this context, the phenolic composition of the extracts was consistent with su sequent investigations on antioxidant, enzyme inhibition, and antimicrobial effects.

Antimicrobial Activity
The antimicrobial activity of the extracts is shown in Tables 6-11, also in comparis with reference antimicrobial drugs, namely ciprofloxacin, fluconazole, and griseofulv All extracts from Pleurotus mycelia displayed antimicrobial activity in the concentrati range of 1.56 to 200 μg mL −1 . Regarding the yeasts, C. parapsilosis (YEPGA 6551) was t most sensitive strain to the PN-PO6 extracts (S2), with MIC ranges of 7.78->200 μg mL while C. albicans (YEPGA 6183) showed the least sensitivity to the mushroom extracts. T results of the growth inhibition of yeast strains highlighted, albeit partially, the major tivity of the extract derived from the S2 growth substrate. With reference to bacteria, t strongest inhibition was observed for the Pleurotus extracts PC1 and PO5 (S1) [MIC <2.4 >200 μg mL −1 against E. coli (ATCC 10536) and B. cereus PeryMycA 2]. Collectively, Gra bacterial strains (E. coli PeruMyc 2 and 3, S. typhi 7, and P. aeruginosa ATCC 15442) we S2_PC1 S1_PE1 S2_PE1 S1_PO1 S2_PO1 S1_PN S2_PN S1_PE2 S2_PE2 S1_PE3 S2_PE3 S1_PO2 S2_PO2 S1_PO3 S2_PO3 S1_PO4 S2_PO4 S1_PE4 S2_PE4 S1_PO5 S2_PO5 S1_PO6 S2_PO6 µg/mL     The antimicrobial activity of Pleurotus samples can be hypothesized, albeit partially, as due to the presence of the so-called CP compounds (derivatives of phosphonate and phosphinate with substitution of alkyl group for hydrogen of phosphorus-hydrogen bonds), monobactams, that are beta-lactam antibiotics (containing a monocyclic beta-lactam nucleus) and sesquiterpenoid and triterpenoid, for which the following metabolic pathways have been highlighted: phosphonate and phosphinate metabolism (PC1 and PE2 samples), monobactam biosynthesis (PE2) and sesquiterpenoid and triterpenoid biosynthesis (PE3, PO4, PE4, and PO6), respectively. The presence of these metabolic pathways has never been indicated in the mycelia of Pleurotus spp. Many C-P compounds are known bioactive substances used in medicine (antibiotics) and agriculture (herbicide) such as fosfomycin, FR-33289, rhizocticin, and bialaphos, while monobactams are beta-lactam antibiotics containing a monocyclic beta-lactam nucleus. Sesquiterpenoid and triterpenoid (present in PE3, PO4, PE4, and PO6 samples) are a group of terpenoids consisting of three isoprene units known for their antimicrobial capacity [45].   * Mic values are reported as geometric means of three independent replicates (n = 3). MIC range concentrations are reported within brackets.

Antiradical Activity
Regarding the antiradical activity, experimental data were normalized and expressed as EC 50 values (µg mL −1 ) for each mushroom extract and Trolox, which was used as a reference antioxidant compound. The antiradical properties were investigated through both DPPH and ABTS, which are common assays used for measuring the intrinsic antioxidant properties of extracts. The results of these tests are shown in Table 12. Values for DPPH radical scavenging activity varied between 886 and 4871 (µg mL −1 ), with the higher potency demonstrated by sample PE3 cultivated in substrate S1. Values for ABTS radical scavenging activity varied between 87 and 377 (µg mL −1 ), and the best activity was shown by the samples PE2 in both substrates, with mean values in the range 87-94 µg mL −1 referred to the EC 50 ; thus, suggesting that the substrates can affect the sample properties in different, that cannot be generalized. For instance, in the DDPH test, samples PC1, PO2, PO3, PO4, and PO5 were not influenced by the change of growth substrate. Whilst in the ABTS test, the substrate always influenced the intrinsic activity, with both stimulating or inhibiting antiradical effects. We cannot exclude that the discrepancies observed between DPPH and ABTS tests can be regarded as the differences between ABTS and DPPH radicals. Indeed, ABTS has been described to be more accurate compared with DPPH, when applied to samples rich in hydrophilic, lipophilic, and highly pigmented antioxidant compounds [46]. Table 12. Antiradical properties of the tested Pleurotus extracts.

Conclusions
With the development of technology, liquid chromatography coupled to a massspectrometry approach can be widely applied in metabolomic studies currently, having a wide detected range and high specificity and sensitivity [47]. In the present study, this method was used to analyze the metabolic profiling of Pleurotus species mycelia, which showed satisfactory data quality. The literature about the characterization of Pleurotus metabolic pathways is unexpectedly poor, even for P. ostreatus and a few other species. It is almost missing as concerns P. columbinus, since it has been only recently accepted as an independent species. The present findings support further investigations aimed at evaluating the influence of growth substrate on Pleurotus spp. antimicrobial and antioxidant properties. The extracts from Pleurotus revealed valuable sources of primary and secondary metabolites, thus suggesting potential applications in the formulation of food supplements, above all in terms of antioxidant and antimicrobial properties.
Regarding the antimicrobial effects, the results from the present study did not point out the optimal substrate for the cultivation of fungi. However, the effect of the substrate was present and should be deeply considered in view of the production of antioxidant extracts from Pleurotus species.
As a concluding remark, in view of a modern concept of sustainability, waste products of agronomical chains can be considered promising substrates for fungal cultivation. Indeed, our results demonstrated that residual plant materials, still containing primary and secondary metabolites, can play pivotal roles in modulating selectively fungal metabolome, with a concomitant influence on the potential use as food and/or health-promoting agent.
Future studies still prove necessary to better define the interactions between plant phytocomplex and fungal response, to drive the cultivation of fungi towards both sustainable improvements of the chain production and search for innovative market products.