Bioactivity and Metabolomic Profile of Extracts Derived from Mycelial Solid Cultures of Hypsizygus marmoreus

The beech mushroom (Hypsizygus marmoreus) is a highly nutritious, edible medicinal mushroom native to East Asia. The present research investigated the impact of different substrates on the metabolite compositions of H. marmoreus mycelia cultivated in vitro. The substrates tested included malt extract agar, malt extract agar enriched with barley malt, and malt extract agar enriched with grape pomace. The study also assessed antimicrobial and antiradical activities of the extracts against gram-positive bacteria (Bacillus subtilis and Staphylococcus aureus), gram-negative bacteria (Escherichia coli, Salmonella typhi, and Pseudomonas aeruginosa), yeasts (Candida albicans, C. tropicalis, and C. parapsilosis), and dermatophytes (Trichophyton mentagrophytes, T. tonsurans, T. rubrum, Arthroderma quadrifidum, A. gypseum, A. curreyi, and A. insingulare). The results revealed that the H. marmoreus mycelia extracts demonstrated antibacterial and antifungal activities against the tested microorganisms. Extracts obtained from the cultivation in substrates enriched with either barley malt or grape pomace exhibited the highest antibacterial activity among all the tested bacterial strains except for P. aeruginosa. The same extracts showed the highest inhibitory effect against C. albicans and C. parapsilosis. Noteworthy, the extract from the mushroom cultivated in the substrate enriched with grape pomace also exhibited remarkable efficacy against T. mentagrophytes and T. tonsurans. Terpenoid and carbapenem compounds could be related to the antimicrobial properties of the extracts from mushrooms cultivated in substrates enriched with grape pomace. In comparison, the higher antiradical properties could be related to the content of indole compounds. In conclusion, growth substrate selection affects the nutritional and medicinal properties of H. marmoreus, making it a valuable contribution to the understanding of the cultivation of this mushroom.


Introduction
The nutritional and health-promoting properties of mushrooms have been known for thousands of years.Ancient civilizations in China, Japan, and other eastern countries have long recognized the various biological activities of mushroom extracts [1,2].Edible mushrooms are valued for their high protein levels, dietary fiber, vitamins, minerals, phenolic compounds, and low fat [1,3].Moreover, certain edible mushrooms contain numerous bioactive compounds that offer therapeutic benefits [4][5][6].
It can be found in different countries in North America, Asia, and Europe (among which Italy) [12].It thrives in beech stumps, withered maple, and other trees [13].Globally, there are less than 150 known records of this species, and it is not indexed as threatened in the IUCN Red List of Endangered Species.Even in Italy, H. marmoreus is considered a rare species.
In addition to their well-known antioxidant activity, phenolic compounds found in mushrooms can exhibit significant antimicrobial effects against a wide range of organisms [10].For instance, Oka et al. [14] investigated the antimicrobial activity of the volatile chemicals synthesized by H. marmoreus against eight phytopathogenic fungi, reporting significant inhibitory effects on Alternaria brassicicola.Shiono et al. [15] isolated and studied three polyacetylene compounds from the organic extract of H. marmoreus, finding that one exhibited antifungal activity against the pathogenic fungus Raffaelea quercivora.Wong et al. [16] evaluated different polarity extracts from H. marmoreus, observing their inhibition effects on Candida albicans.The medicinal properties of H. marmoreus are attributed to several bioactive molecules, including terpenoid compounds like hypsiziprenol A9, which have been shown to exert cytotoxic effects in human liver cancer HepG2 cells [17].The dietary effects of H. marmoreus are associated with various active components, mainly primary metabolites [8].As a result of its multiple uses, the production and consumption of this medicinal mushroom have been increasing worldwide.H. marmoreus, known as a white-rot fungus [18], possesses the ability to degrade lignocellulosic materials and can be cultivated on biological, agricultural, or agro-industrial wastes.Studies have identified various suitable substrates for mushroom cultivation, including rice straw, maize, oak wood, horse chestnut, sawdust, and cotton stalks [19].Edible mushroom cultivation is considered a biotechnological process that helps reduce and protect the environment from excessive solid waste, as stated by Sanchez [20].Previous studies have demonstrated that the production of cultivated mushrooms can be influenced by the fruiting body or mycelium production processes, with chemical changes affecting their pharmacological effects on health [21].Solid culture methods for fruiting body production typically require a lengthy period, thus making it important to optimize in vitro mycelium cultures with the final goal of developing innovative health-promoting products [22].Moreover, mycelium can be produced in a compact space within a shorter timeframe and with minimal risk of contamination [23].The interest in the pharmaceutical potential of mushrooms has surged in the past decade, as mushrooms are regarded as small pharmaceutical farm for the synthesis of compounds with potential health effects.
In this study, our main objectives were as follows: (a) selecting two different solid media for cultivating H. marmoreus mycelium based on preliminary experiments, (b) investigating and comparing the chemical profiles of H. marmoreus mycelium grown on these two different culture media, and (c) exploring the influence of medium ingredients on the antimicrobial and antiradical activities.To achieve these goals, we employed mass spectrometry (MS)-based metabolomics, which enables quantitative analyses with high selectivity and sensitivity, along with the potential to identify metabolites [24].Combining this approach with multivariate statistical analysis will facilitate the determination of the effects of different culture media on metabolomics and transcriptomic disparities in H. marmoreus mycelia.

Mushrooms
H. marmoreus (fruiting body) was collected in Perugia (via Roma) in December 2018.The voucher specimen (PeruMyc2422) was deposited in the herbarium of the Department of Chemistry, Biology, and Biotechnology (University of Perugia, Perugia, Italy).The isolation of the mycelia was carried out as previously described [3].

Molecular Identification
Genomic DNA was extracted from 15-day mycelia grown on malt extract agar (MEA) using the ZR Fungal/Bacterial DNA kit (Euroclone S.p.A., Milan, Italy).The amplification of the internal transcribed spacer (ITS) region was conducted with the fungal-specific forward primer, ITS1F 5 -CTTGGTCATTTAGAAGTAA-3 in combination with the reverse primer, ITS4 5 -TCCTCCGCTTATTGATATGC-3 [25].The conditions for PCR amplification are included in our previous study [3].

Phylogeny
The chromatogram was examined and edited using Chromas 2.6.6, while MEGA X software was employed for the subsequent sequence analysis.For the phylogenetic inference, the DNA sequences of H. marmoreus, Hypsizygus tessulatus (Bull.)Singer, and Hypsizygus ulmarius (Bull.)Redhead were retrieved from GenBank.Additionally, Ossicaulis lignatilis (Pers.)Redhead and Ginns was chosen as an outgroup for the ITS region analysis.Within the MEGA X package, the MUSCLE algorithm was utilized for pairwise and multiple sequence alignments, and the resulting alignments were further refined manually.To support the branches in the maximum likelihood (ML) analysis, a bootstrap (BS) value of 1000 pseudoreplicates was applied.

Total Phenolic Content
The total phenolic contents of the tested extracts were evaluated via the colorimetric method using the method described for phenolics by Acquaviva et al. [26].The Folin-Ciocalteu assay was utilized to determine the total phenolic, and the results were expressed as gallic acid equivalent (GAE).Details are reported as Supplementary Material.

Untargeted Ultra-Performance Liquid Chromatography-Mass Spectrometry (UHPLC)-Quadrupole Time of Flight (QTOF)-Based Metabolomics and Statistical Analysis
The untargeted 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 details are reported in our previous study [3] and available as a Supplementary Materials.

Antimicrobial Effects
The antimicrobial properties of the extracts were investigated as previously reported [27][28][29].Further details can be found in the supplementary, where the employed microbial species for the tests are also listed.

Molecular Identification of Hypsizygus marmoreus (PeruMyc2422)
The ITS-based phylogeny provides supporting evidence for placing the newly sequenced samples from our study within the context of H. marmoreus and H. tessulatus (Figure 1).However, the sequences of H. marmoreus and H. tessulatus retrieved from Gen-Bank in our study exhibited a high degree of genetic similarity, making it challenging to precisely classify H. marmoreus PeruMyc 2422 collected in Perugia.Furthermore, the alignment analysis uncovered a notable variation of four nucleotides unique to the sample we collected compared to the strains of H. marmoreus, H. tessulatus, and H. ulmarius obtained from GenBank.This observation highlights the importance of further investigation using additional molecular markers to gain deeper insights into the genetic differentiation within this group.Therefore, in order to enhance our understanding and achieve a more conclusive taxonomic classification, it may be necessary to conduct further investigations employing alternative molecular markers.
The antimicrobial properties of the extracts were investigated as previously repo [27][28][29].Further details can be found in the supplementary, where the employed micr species for the tests are also listed.

Molecular Identification of Hypsizygus marmoreus (PeruMyc2422)
The ITS-based phylogeny provides supporting evidence for placing the n sequenced samples from our study within the context of H. marmoreus and H. tessu (Figure 1).However, the sequences of H. marmoreus and H. tessulatus retrieved GenBank in our study exhibited a high degree of genetic similarity, making it challen to precisely classify H. marmoreus PeruMyc 2422 collected in Perugia.Furthermore alignment analysis uncovered a notable variation of four nucleotides unique to the sa we collected compared to the strains of H. marmoreus, H. tessulatus, and H. ulm obtained from GenBank.This observation highlights the importance of fu investigation using additional molecular markers to gain deeper insights into the ge differentiation within this group.Therefore, in order to enhance our understanding achieve a more conclusive taxonomic classification, it may be necessary to conduct fu investigations employing alternative molecular markers.

Statistical Data Analysis
The data produced in this experiment were explored using the statistical technique of unsupervised principal component analysis after applying autoscaling.The score plot graph (Figure 2) shows that the first two components together explain 44.1% of the variance of the samples.The ellipsoids, which comprise the 95% confidence interval, enclose the samples in homogeneous groups.The first component separates control (HC), H1, and H3 groups from H2 and H4.The second component separates HC and H4 from everything else.

Statistical Data Analysis
The data produced in this experiment were explored using the statist of unsupervised principal component analysis after applying autoscaling.graph (Figure 2) shows that the first two components together explain variance of the samples.The ellipsoids, which comprise the 95% confid enclose the samples in homogeneous groups.The first component separates H1, and H3 groups from H2 and H4.The second component separates HC everything else.

Cluster Analysis
The dendrogram (Figure 3) shows that the samples are mainly div clusters: HC (fungus grown in control medium) and all the other samples samples admixed with different concentrations of barley malt (H1 and pomace (H2 and H4).Further nodes of the dendrogram subdivide the sam respective clusters.A heatmap (Figure 4) was also made considering significant metabolites.The heatmap confirms that the samples are mainly two clusters.The first node divides the HC clusters, H1 and H3, from th clusters.This is consistent with the different substrates employed for th cultivation.Indeed, samples H1 and H3 were grown in a barley malt-enric while samples H2 and H4 were grown in a pomace-enriched medium.Malt extract agar 1%, as the control (HC); malt extract agar 1% enriched with 0.5% barley malt (H1); malt extract agar 1% enriched with 0.5% grape pomace (HC2); malt extract agar 1% enriched with 2% barley malt (HC3); malt extract agar 1% enriched with 2% grape pomace (HC4); principal component (PC).

Cluster Analysis
The dendrogram (Figure 3) shows that the samples are mainly divided into two clusters: HC (fungus grown in control medium) and all the other samples grown in the samples admixed with different concentrations of barley malt (H1 and H3) or grape pomace (H2 and H4).Further nodes of the dendrogram subdivide the samples into their respective clusters.A heatmap (Figure 4) was also made considering the 70 most significant metabolites.The heatmap confirms that the samples are mainly divided into two clusters.The first node divides the HC clusters, H1 and H3, from the H2 and H4 clusters.This is consistent with the different substrates employed for the mushroom cultivation.Indeed, samples H1 and H3 were grown in a barley malt-enriched medium, while samples H2 and H4 were grown in a pomace-enriched medium.Heatmap reports the metabolomics analysis results of the two main clusters into which the samples are divided.Malt extract agar 1%, as the control (HC); malt extract agar 1% enriched with 0.5% barley malt (H1); malt extract agar 1% enriched with 0.5% grape pomace (HC2); malt extract agar 1% enriched with 2% barley malt (HC3); malt extract agar 1% enriched with 2% grape pomace (HC4).

Functional Analysis
Figures 5-7 and Tables 1 and 2 summarize the MetaboAnalyst's functional meta-analysis result.The 10 pathways activated in a statistically significant way with respect to control (HC) are reported in relationships with the employed substrate.In many cases, it is noted that the H1 and H3 pairs and H2 activate certain pathways in a similar way.For example, zymosterol biosynthesis appears activated in H2 and H4 but not in H1 and H3, thus demonstrating the substrate's effect on mushroom metabolism [3].Heatmap reports the metabolomics analysis results of the two main clusters into which the samples are divided.Malt extract agar 1%, as the control (HC); malt extract agar 1% enriched with 0.5% barley malt (H1); malt extract agar 1% enriched with 0.5% grape pomace (HC2); malt extract agar 1% enriched with 2% barley malt (HC3); malt extract agar 1% enriched with 2% grape pomace (HC4).

Functional Analysis
Figures 5-7 and Tables 1 and 2 summarize the MetaboAnalyst's functional metaanalysis result.The 10 pathways activated in a statistically significant way with respect to control (HC) are reported in relationships with the employed substrate.In many cases, it is noted that the H1 and H3 pairs and H2 activate certain pathways in a similar way.For example, zymosterol biosynthesis appears activated in H2 and H4 but not in H1 and H3, thus demonstrating the substrate's effect on mushroom metabolism [3].6742 Hits: number of identified metabolites that are part of a specific pathway; Sig_Hits: number of identified metabolites that are part of a specific pathway, whose concentration has varied in a statistically significant manner; mummichog: American-Indian term for by-groups; GSEA: gene set enrichment analysis.Hits: number of identified metabolites that are part of a specific pathway; Sig_Hits: number of identified metabolites that are part of a specific pathway, whose concentration has varied in a statistically significant manner; mummichog: American-Indian term for by-groups; GSEA: gene set enrichment analysis.Hits: number of identified metabolites that are part of a specific pathway; Sig_Hits: number of identified metabolites that are part of a specific pathway, whose concentration has varied in a statistically significant manner; mummichog: American-Indian term for by-groups; GSEA: gene set enrichment analysis.

Total Phenol Content and Antioxidant Effects
The extracts from H. marmoreus were also tested for the amounts of phenolic compounds, calculated as gallic acid equivalents (Table 3).However, we have to consider that the determination of total phenols through the Folin-Ciocalteu assay is a rough approximation, thus not excluding the presence of other antioxidant compounds.The results showed that substrate did not influence the total phenol content in most of the samples, with the exception of the H4 extract, which displayed a 24.28% reduction in total phenols.This is consistent, albeit partially, with its lower activity in the DPPH assay (Table 4).On the other hand, in the ABTS assay, we observed a sensitive increase in the antiradical activity, especially in the extract H4 (Table 5) derived from mushrooms cultivated in substrates with the highest concentration in grape pomace (2%).The discrepancies observed between the antiradical activity, expressed as Trolox equivalents, with DPPH and ABTS assays may be related to a lower sensitivity of DPPH to the change of growth substrate [3].This is consistent with the higher accuracy of ABTS in measuring the antioxidant activity of extracts rich in lipophilic and highly pigmented compounds [30].Antioxidant properties of H. marmoreus have been reported in the literature [31].It is also sensitive to highlight the recent identification of indole compounds in extracts from H. marmoreus [32].Considering the antioxidant properties of such compounds, we cannot exclude that the highest antioxidant effect of extract H4, as shown by ABTS assay, could be related to the presence of indole compounds [33,34].Indeed, the H4 extract showed the highest content in indole compounds, as revealed by the metabolomics investigation (Figure 3).

Antimicrobial Activity
Table 6 presents the minimal inhibitory concentration (MIC) values of extracts from H. marmoreus mycelia against bacteria, yeasts, and dermatophytes.All extracts derived from H. marmoreus displayed antimicrobial activity in concentrations ranging from 1.56 to 200 µg mL −1 .Notably, Escherichia coli (PeruMycA 2) exhibited the highest sensitivity to the H4 extract, with an MIC range of 1.56-3.125µg mL −1 (GM, 2.48 µg mL −1 ).In contrast, Bacillus cereus (PeruMycA 4) and Pseudomonas aeruginosa (ATCC 15442) showed the least sensitivity to the mycelia extracts.Generally, gram-negative bacteria (E. coli PeruMyc 2 and 3, S. typhi 7, and P. aeruginosa ATCC 15442) displayed lower sensitivity to the extracts compared to gram-positive strains, similar to observations for F. torulosa and Pleurotus spp.[2,3,35].The growth inhibition results for yeast strains revealed significant, albeit partial, antimicrobial activity of the mycelia extracts derived from the growth substrate containing grape pomace.Notably, the mycelia extracts H1 and H4 exhibited strong inhibition (MIC 12.5-100 µg mL −1 ) against Candida albicans (YEPGA 6379) and C. parapsilosis (YEPGA 6551), respectively (Table 7).Additionally, all tested extracts effectively inhibited dermatophyte growth, with Trichophyton mantagrophytes (CCF 4823), T. tonsurans (CCF 4834), and T. rubrum (CCF 4933) being the most susceptible fungal species to all mycelia extracts, with MIC ranging from 6.26 to 200 µg mL −1 .MIC values below 100 µg mL −1 were considered indicative of high antimicrobial activity (Table 8).Regarding the mechanisms underlying the observed antimicrobial effects, according to the metabolomics analysis, we hypothesize that different secondary metabolites, including terpenoids and carbapenems, could be responsible for the antibacterial and antifungal effects of the extracts.In particular, the contemporary presence of terpenoids and carbapenems in the extracts prepared from mushrooms cultivated in the medium enriched with grape pomace further corroborates the overall higher efficacy of the H4 as an antimicrobial.However, it cannot exclude that other mushroom metabolites, including sterols and polyisoprenepolyols could be involved in the antimicrobial properties displayed by H. marmoreus [36].

Figure 1 .
Figure 1.Phylogenetic tree of Hypsizygus species inferred using maximum likelihood (ML) bas the internal transcribed spacer (ITS) region (bootstrap support = 1000).The sequences genera this study are represented by the Perumyc 2422 code, while the original names were maintain sequences obtained from GenBank.

Figure 1 .
Figure 1.Phylogenetic tree of Hypsizygus species inferred using maximum likelihood (ML) based on the internal transcribed spacer (ITS) region (bootstrap support = 1000).The sequences generated in this study are represented by the Perumyc 2422 code, while the original names were maintained for sequences obtained from GenBank.

s 16 Figure 5 .
Figure 5. Statistically significant pathways activated with respect to control (HC) and in relationships with the employed substrate.

Figure 5 . 16 Figure 5 .
Figure 5. Statistically significant pathways activated with respect to control (HC) and in relationships with the employed substrate.

Table 1 .
Statistically significantly pathways active in the barley malt samples vs. control (HC).

Table 2 .
Statistically significantly pathways active in the grape pomace samples vs. control (HC).

Table 1 .
Statistically significantly pathways active in the barley malt samples vs. control (HC).

Table 2 .
Statistically significantly pathways active in the grape pomace samples vs. control (HC).