Nutritional Composition and Bioactive Properties of Wild Edible Mushrooms from Native Nothofagus Patagonian Forests

Nothofagus forests of the Andean Patagonian region are home to numerous wild edible mushroom (WEM) species with interesting organoleptic characteristics, although many of them have unknown nutritional and nutraceutical profiles. The proximal composition, fatty and organic acids, soluble sugars, phenolic compounds, ergosterol, as well as antioxidant and antimicrobial activity of 17 WEMs were analyzed. Carbohydrates, the most abundant macronutrients, varied between 49.00 g/100 g dw (C. magellanicus) and 89.70 g/100 g dw (F. antarctica). Significantly higher values were found for total fat in G. gargal (5.90 g/100 g dw) followed by A. vitellinus (4.70 g/100 g dw); for crude protein in L. perlatum (36.60 g/100 g dw) followed by L. nuda (30.30 g/100 g dw); and for energy in G. gargal (398 Kcal/100g) and C. hariotii (392 Kcal/100g). The most effective extracts regarding the TBARS antioxidant capacity were those of Ramaria. This is the first time that a study was carried out on the chemical composition of G. sordulenta, C. xiphidipus, F. pumiliae, and L. perlatum. The promotion of sustainable use of WEMs, including their incorporation in functional diets that choose WEMs as nutritious, safe, and healthy foods, and their use in an identity mycogastronomy linked to tourism development, requires the detailed and precise nutritional and nutraceutical information of each species.


Introduction
Edible wild mushrooms are highly available functional foods. Its consumption has been developed and perpetuated in various countries from all over the world [1]. Their commercial and culinary importance is mainly due to their organoleptic properties, such as aroma and flavor, their nutritional qualities, and their medicinal characteristics [2][3][4][5], due to their high protein and fiber content, essential amino acids, bioactive compounds, and low lipids content [3,6].
Different mushrooms have been studied in search of new therapeutic alternatives, finding that they have bioactive properties [7][8][9] and that they constitute rich sources of nutraceuticals molecules [10,11], which are responsible for their antioxidant [12][13][14] and antitumor properties [15]. Antioxidants from edible natural products are currently widely studied for their ability to protect organisms and cells from damage caused by oxidative stress, which is one of the causes of aging and degenerative diseases [16].
Patagonian Andean forests comprise 3,240,996 h dominated by Nothofagus spp. (N. antarctica, N. dombeyi, and N. pumilio are the most representative species) [17]. The region harbors numerous species of wild fungi that are potentially edible, with high nutritional and

Nutritional Characterization
Samples of each species were analyzed for nutritional composition (protein, carbohydrates, fat, ash, and energy) using AOAC procedures [36]. The total carbohydrates were obtained by difference, and the energy values were calculated with the equation Energy (kcal) = 4 × (g protein + g carbohydrates) + 9 × (g fat). The results are expressed in kcal per 100 g of dry weight (dw).

Nutritional Characterization
Samples of each species were analyzed for nutritional composition (protein, carbohydrates, fat, ash, and energy) using AOAC procedures [36]. The total carbohydrates were obtained by difference, and the energy values were calculated with the equation Energy (kcal) = 4 × (g protein + g carbohydrates) + 9 × (g fat). The results are expressed in kcal per 100 g of dry weight (dw).

Chemical Composition 2.3.1. Free Sugars
Free sugars determination followed the methodology by Barros et al. [37]. Analysis was performed by liquid chromatography (HPLC, Knauer, Smartline 1000 systems, Berlin, Germany), coupled with a refraction index detector (Knauer Smartline 2300). The detected compounds were identified by comparison with the retention times of the standards. Trehalose was used as the internal standard. Results are expressed in g/100 g of dry weight (dw).

Fatty Acids
The fatty acids were identified by gas chromatography with flame ionization detection (GC-FID), as previously described by Pereira et al. [38]. The identification of fatty acids was made according to their relative retention times of the FAME peaks of the sample standards (mixture 37, 47885-U purchased from Sigma). To process the results, we used CSW 1.7 software (DataApex 1.7, Prague, Czech Republic); results are expressed as a relative percentage (%).

Ergosterol
The ergosterol was quantified after extraction following Vieira Junior et al. [39]. It was determined by high-performance liquid chromatography (HPLC) coupled to a UV detector (280 nm), as described by Cardoso et al. [40], and was identified and quantified by comparison with the pure chemical standard and expressed in mg/100g dw.

Organic Acids Composition
The organic acids were determined by high-performance liquid chromatography coupled to a photodiode detector (UFLC-PDA) following the methodology described by Barros et al. [37]. The detection of organic acids was achieved using a DAD system, applying a wavelength of 215 nm (and 245 nm for ascorbic acid). The quantification was carried out by comparing the area of their recorded peaks with the calibration curves obtained from the standards of the respective compound. The results are expressed in mg/100 g (fw).

Phenolic Composition
Samples of 0.5 g of freeze-dried specimens were used for the extract preparation. They were initially macerated at room temperature with the addition of a solution (30 mL) of ethanol/water (80:20, v/v), for 1 h (150 rpm). Ethanol was removed under reduced pressure. Afterwards, the aqueous phase of both extracts was frozen and lyophilized.
The identification and quantification of the phenolic compounds followed the previously optimized methodology [41], using a Dionex Ultimate 3000 UPLC system (Thermo Scientific, San Jose, CA, USA). The DAD and mass spectrometer (LTQ XL mass spectrometer, Thermo Finnigan, San Jose, CA, USA) were working in negative mode.

Evaluation of Antioxidant Activity
An in vitro assay based on the monitoring of malondialdehyde (MDA)-TBA complexes was carried out as previously reported [42] to measure the extract capacity to inhibit the formation of thiobarbituric acid reactive substances (TBARS). Porcine brain cells were used as biological substrates. The results are expressed as IC 50 values (mg/mL).

Evaluation of Antibacterial Activity
The extracts were tested against five Gram-negative bacteria, namely, Enterobacter cloacae (ATCC 49741), Escherichia coli (ATCC 25922), Pseudomonas aeruginosa (ATCC 9027), Salmonella enterica subsp. enterica serovar Enteritidis (ATCC 13076), and Yersinia enterocolitica (ATCC 8610), and three Gram-positive bacteria, namely, Bacillus cereus (ATCC 11778), Listeria monocytogenes (ATCC 19111), and Staphylococcus aureus (ATCC 25923). The minimum inhibitory (MIC) and minimum bactericidal concentrations were determined for all bacteria using colorimetric assays, following Pires et al. [44]. The MIC was defined as the lowest concentration inhibiting visible bacterial growth, determined by a change from yellow to pink coloration if the microorganisms are viable. The MBC was defined as the lowest concentration required to kill bacteria.
To evaluate the antifungal activity, the methodology described by Heleno et al. [45], using Aspergillus fumigatus (ATCC 204305) and Aspergillus brasiliensis (ATCC 16404), was used. The organisms were obtained from Frilabo, Porto, Portugal. The minimum inhibitory concentration (MIC) and minimum fungicidal concentration (MFC) were determined by a serial dilution technique using 96-well microplates. The lowest concentrations without visible growth (at the binocular microscope) were defined as the MICs. The lowest concentration with no visible growth was defined as the MFC, indicating 99.5% killing of the original inoculum. The commercial fungicide ketoconazole (Frilabo, Porto, Portugal) was used as positive control.

Statistical Analysis
Three independent samples per mushroom species were analyzed, and the data are expressed as the mean ± standard deviation. All statistical tests were performed at a 5% significance level in RStudio (version 1.1.485-© 2009-2022 RStudio, Inc.) [46]. The homogeneity of variance and normal distribution of the residuals were tested by means of the Shapiro-Wilk and Levene tests, respectively, to fulfill the one-way ANOVA requirements. All dependent variables were compared using Tukey's tests. When normality or heteroscedasticity could not be verified, the variables were Box-Cox transformed before performing the ANOVA. Kruskal-Wallis tests were carried out when a normal distribution and heteroscedasticity were not achieved after Box-Cox transformation.

Results and Discussion
The obtained chemical compositions and energetic values are shown in Table 1. Protein contents varied between 3.20 g/100 g dw in F. antarctica and 36.60 g/100 g dw in L. perlatum. The top-five values concerning of highest protein content was L. perlatum (36.60 g/100 g dw), L. nuda (30.30 g/100 g dw), H. dusenii (22.20 g/100 g dw), R. patagonica (18.10 g/100 g dw), and C. magellanicus (14.40 g/100 g dw). Comparing with previous studies, Ramaria patagonica and R. botrytis showed similar results than those reported by other authors with a value of 19.68 g/100 g dw [25] and 16.60 g/100 g dw [14]. However, in a Portuguese mushroom study [16], R. botrytis showed higher protein values (39.8 g/100 g dw) than our results. Flammulina velutipes (17.89 g/100 g dw) and C. aegerita (19.65 g/100 g dw) showed lower levels than those reported by Jacinto-Azevedo et al. [14]. Other studies on G. gargal showed similar results with values of 5.96 [25] and 5.00 g/100 g dw [38]. Previous studies on Cyttaria have reported higher values than those reported here; for example, C. espinosae had values of 17.46 g/100 g dw [14] and C. darwini values of 17.20 g/100 g dw [38,47]. However, C. hariotii showed similar results (3.35 g/100 g dw) than those reported by Toledo et al. [25]. The protein values for A. vittelinus, C. magellanicus, F. antarctica, and F. endoxantha are in concordance with other reports [25]. On a dry weight basis, mushrooms normally contain 19 to 35% protein. Therefore, regarding the amount of crude protein, mushrooms are positioned below most animal meats but well above most other foods, including milk, rice, and wheat [48]. Table 1. Proximate composition (g/100 g) and energetic value (kcal/100 g) of the studied wild mushrooms (mean ± SD). For each mushroom sample, means within a column with different letters differ significantly (p < 0.05).
Ash varied from 4.80 g/100 g in P. ostreatus to 32.00 g/100 g in C. magellanicus. Cyttaria hariotii yielded lower values (7.0 g/100 g dw) than what Schmeda-Hirschmann et al. [47] previously reported, and similar values than those reported by Jacinto-Azevedo et al. [14] for C. espinosae (4.90 g/100 g dw). However, lower values were observed in F. velutipes, C. aegerita, and R. botrytis [14], in G. gargal [47], and in F. antarctica and F. endoxantha [25]. Similar results to Toledo et al. [25] were found for L. nuda (8.58 g/100 g dw) and R. patagonica (8.47 g/100 g dw), but lower for L. nuda (18.5 g/100 g dw) compared to Barros et al. [11]. The ash content in edible mushrooms ranges from 1 to 29 g/100 g dry matter and comprise a source of essential minerals. Concentrations of P, K, Ca, Na, and Mg constitute more than 56% of the total ash content [48].
Concerning sugar composition (Table 2), mannitol and trehalose were the principal sugars, which is in agreement with the data presented in the literature; they are essential in energetic metabolism and necessary in the synthesis of storage or structural polysaccharides [38]. Mannitol content was significantly higher for A. vitellinus (8.83 g/100 g dw), R. botrytis (6.34 g/100 g dw), and R. patagonica (8.64 g/100 g dw), although absent in F. endoxantha, in concordance with Toledo et al. [25]. The support and expansion of the mushroom fruiting bodies is guaranteed by the presence of mannitol [49]. Trehalose predominated in C. xiphidipus (17.60 g/100 g dw), P. ostreatus (15.81 g/100 g dw), C. aegerita (13.54 g/100 g dw), and G. sordulenta (11.25 g/100 g dw), but was absent in R. botrytis and R. patagonica. The ingestion, hydrolysis, absorption, and metabolism of trehalose is highly similar to all the other digestible disaccharides [49]. On the other hand, fructose and one unidentified sugar were predominant in all three Fistulina species (F. endoxantha, F. antarctica, and F. pumiliae) in concordance with previous reports [25]. Fructose was also the predominant sugar in Flammulina velutipes (8.56 g/100 g dw), agreeing with Reis et al. [50], while it was absent in A. vitellinus, C. magellanicus, L. perlatum, and P. ostreatus, and present in lower abundance in the rest of the species. In terms of total sugar content, F. endoxantha revealed the highest value (33.88 g/100 g dw), while G. gargal the lowest (3.63 g/100 g dw). This is the first study that reports the composition in free sugars of endemic species C. xiphidipus, F. pumiliae, and G. sordulenta.
Organic acids comprise a group of mono-, di-, and tricarboxylic acids physiologically occurring as intermediates in a variety of intracellular metabolic pathways, such as catabolism of amino acids, the tricarboxylic acid cycle, and neurotransmitters, as well as in cholesterol biosynthesis [51]. The organic acid composition is presented in Table 2. Most of the analyzed specimens had oxalic, malic, and fumaric acids in their composition. On the other hand, quinic, shikimic, citric, and succinic acids were present just in a few species. Citric acid was detected in high amounts in C. magellanicus (57.40 mg/100 g dw), R. patagonica (57.31 mg/100 g dw), A. vitellinus (31.11 mg/100 g dw), and C. hariotii (23.41 mg/100 g dw).

Organic Acids Sugars
Within polyunsaturated fatty acids, ω-3 and ω-6 are the most abundant in mammals. Its precursors, α-linolenic acid (ALA) and linoleic acid (LA), are considered essential fatty acids, as the body requires them for normal operation but which cannot be synthesized endogenously [54]. Within the series of omega-3, the most important in the human diet are eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), both difficult to synthesize endogenously and with important functions in the human body. DHA is a structural fatty acid, since it forms part of cell membranes and is also important for visual (it makes up 20% of all fatty acids present in the retina) and neuronal development during gestation and early childhood [55,56]. In our study, C. hariotii showed high amounts of DHA (7.74%). Interestingly, some ethnomycological reports [57,58] showed that in the Selknam, Ahonikenk, and Kawesqar native Patagonian populations, after giving birth and while the quarantine lasted, the mothers lived exclusively on Cyttaria fungus (C. darwinii and C. hariotii). In the omega-6 series one has to pay special attention to γ-linolenic acid (GLA) and arachidonic acid (AA), important for prostaglandin production and anti-inflammatory activity [55]. From all the above, the importance of supplementing a diet with fatty acids, which are clearly present in edible mushrooms, can be deduced, since their current intake is insufficient.
The ergosterol content (Table 4) ranged from 0.40 mg/100 g in C. hariotii to 123.57 mg/100 g dw in G. gargal. In other studies of edible and medicinal mushrooms, similar differences have been reported, ranging from traces for Armilaria mellea, to values of 25.71 mg/100 g dw for Laetiporus sulphureus or 445.32 mg/100 g dw for Macrolepiota procera. The differences in ergosterol and other nutritional and bioactive compounds depend on the species, stage of development, tissues, nutrient substrate, and microclimate [26,59]. Vieira Junior et al. [39] showed that ergosterol biosynthesis and its bioconversion into ergocalciferol were also affected by the cultivation process in Agaricus subrufescens production, showing differences between field culture and controlled conditions. Ergosterol is metabolized into a prohormone-vitamin D. Its action is related with bone mineral metabolism and with the balance of phosphorus and calcium, related to various mechanisms such as secretion and effect of insulin, regulation of the renin-angiotensin-aldosterone system, endothelial function, cell cycle control and apoptosis, immunological self-tolerance, and immune response against infections, among other effects [60]. For these reason, edible mushrooms are promising sources of vitamin D, and thus able to improve food supplements for human consumption.

Species p-Coumaric
Acid Gallic Acid

p-Hidroxibenzoic Acid
Protocatechuic Acid  Table 6 shows the in vitro antioxidant activity of the studied species. Ramaria patagonica (156 µg/mL), R. botrytis (167 µg/mL), G. sordulenta (299 µg/mL), and A. vitellinus (551 µg/mL) presented the best results in the TBARS assays; meanwhile, L. perlatum (90 µg/mL), L. nuda (93 µg/mL), A. vitellinus (113 µg/mL), and G. sordulenta (155 µg/mL) presented the best results in OxHLIA, all with IC 50 values ≤ 1000 µg/mL. The result of the antioxidant activity in OxHLIA for L. perlatum is in concordance with its highest total levels of phenolic compounds. That the antioxidant activity of mushrooms correlates with the phenolic compounds content has already been reported [62]. Fistulina antarctica comparatively presented the lowest antioxidant activity (IC 50 of 2627 µg/mL for TBARS and of 1066 µg/mL for OxHLIA), in concordance with Toledo et al. [25]. Lepista nuda showed lower values (711 µg/mL) for the TBARS assay compared to those reported by other authors, with IC 50 values of 5800 µg/mL [11] and 6100 µg/mL [25]. The high antioxidant activity of Ramaria is in agreement with the literature; for example, for R. flava, R. botrytis, and R. subaurantiaca with DPPH assays by Jacinto-Azevedo et al. [14], and for R. patagonica with different assays by Toledo et al. [25]. The antioxidant activity by DPPH or reducing the power test was also evaluated applying different extracting methodologies in Grifola samples; for example, Brujin et al. [63,64], using different solvents or heat treatments in Grifola gargal, and Postemsky et al. [65], using wheat grain biotransformed with mycelium of G. gargal and G. sordulenta. Among the three edible Rusulla species, R. integra ethanolic extract showed the best antihemolytic activity, with an IC 50 value of 139 ± 3 µg/mL, also for a 60 min ∆t [25]. This is the first study on the anti-hemolytic capacity of wild edible species. Table 6. Antioxidant activity of the mushroom extracts measured by inhibition of lipid peroxidation (TBARS) and the oxidative hemolysis inhibition assay (OxHLIA). IC 50 values were expressed in µg/mL. na: no activity (∆t values less than 60 min were obtained). For each mushroom sample, means within a column with different letters differ significantly (p < 0.05). All extracts were tested against ten bacteria and fungi considered food contaminants (Table 7). Each mushroom species showed different intensities of positive antimicrobial activity against the tested microorganisms. The antibacterial effects were more effective against Salmonella enterocolitica, Yersinia enterocolitica (Gram-negative bacteria), and Staphylococcus aureus (Gram-positive bacteria). The antifungal effect was more effective in Aspergillus brasiliensis. Among these active extracts, those produced by A. vitellinus (MIC 1.25 mg/mL) F. velutipes (MIC 2.5 mg/mL), G. gargal (MIC 2.5 mg/mL), P. ostreatus (MIC 2.5 mg/mL), and R. botrytis (MIC 1.25 mg/mL) exhibited a good inhibitory activity against Yersinia enterocolitica; and the extracts from G. sordulenta and R. botrytis (MIC 0.3 mg/mL) against Staphylococcus aureus. The Gram-negative bacteria, Enterobacter cloacae, Escherichia coli, and Pseudomonas aeruginosa, and the Gram-positive bacteria Bacillus cereus and Listeria monocytogenes were less sensitive to the extracts used. C. hariotii showed no activity against the analyzed bacteria. None of the extracts presented bactericidal and fungicidal activity. All these results must be considered taking into account the already established fact that the chemical composition of mushrooms could vary with the genetic structure and strains within the same species. Our ranks also considered dehydrated, complete fruiting bodies, in the mature stage, with no stratification by site conditions nor post-harvest treatments. Maturation stage at harvest, a specific part of the mushroom analyzed (stem, cup, lamellae), and environmental variables, such as soil composition, as well as the postharvest preservation method (freeze dry, oven-dry, cooled, fresh) and cooking process may affect their chemical composition [3].

Conclusions
This study highlights the value of the native and endemic mushrooms of the Patagonian forest, regarding, for example, their antioxidant qualities, as in the case of Ramaria spp., or their energetic value, as in the case of G. gargal and C. hariotti. Species such as C. aegerita, F. velutipes, L. nuda, and P. ostreatus demonstrated the importance of edible mushroom with a cosmopolitan distribution growing in native forests, resulting in an invaluable source of food with high protein values, low contents of fat, along with other bioactive compounds with remarkable antioxidant and antimicrobial activity. The data provided by this study, along with previous ones, will strengthen and support the inclusion of new species of wild edible fungi in the Argentine food code. In this way, we expect to revalue these resources as non-timber forest products from Patagonia, promoting multiple and sustainable uses of native forests.

Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.

Data Availability Statement:
The data presented in this study are available upon request from the corresponding author.