Basidiomas of Cantharellus spp. were collected in different locations in the Mountain Region, Veracruz, Mexico, during June-October in the period 2019-2021 (Fig. 1). Fresh samples were dried using a freeze dry system (Labconco 4.5 Plus, Kansas, USA) and stored in vacuum sealed bags in complete darkness at 4 °C to preserve their bioactive components for further nutritional analysis. Specimens were deposited in the Herbarium CORU “Jerzy Rzedowski Rotter” of Faculty of Biological and Agricultural Sciences of the Universidad Veracruzana, Campus Córdoba, Mexico.

Figure 1:
Map of the Mountain Region, Veracruz, Mexico. Elaborated with QGIS v. 3.18.3 (QGIS Development Team, 2023).

DNA was isolated from dried specimens using a commercial DNA extraction kit (Plant/Fungi DNA Isolation Kit, Norgen Biotek Corp, Ontario, Canada). The internal transcribed spacer (ITS) region was amplified following the protocol of White et al. (1990), by using primers ITS3 and ITS4. The transcription elongation factor 1-alpha (tef-1α) was amplified using the primers tef1F and tef1R (Morehouse et al., 2003). The PCR products were purified and sequenced (Macrogen, 2023). Forward and reverse reads were assembled and edited with SeqMan Pro v. 7.1.0 (DNASTAR, 2023).

New sequences generated from this study were deposited in GenBank (Clark et al., 2016). Initial BLAST® (Altschul et al., 1990) searches of both ITS and tef-1α sequences were performed to estimate similarity with Cantharellus sequences in GenBank (GenBank, 2023). Species were identified molecularly based on the BLAST® results.

The reagents used in this study such as Folin-Ciocalteu phenol, gallic acid, aluminium chloride, sodium hydroxide, 1,4-dichlorobenzene, chloroform, ethyl acetate, acetic anhydride, sulfuric acid, acetone, petroleum ether, and methanol, Sodium Carbonate, Quercetin, were all purchased through Sigma-Aldrich (Sigma-Aldrich St. Louis, MO, USA).

A stock solution was prepared for nutraceutical analyses. One gram of lyophilized mushroom sample was extracted with 20 ml of 80:20 methanol-water. The extract was allowed to settle for 60 min and was subsequently subjected to three cycles of 5 min each, in an ultrasound (CIVEQ 8892, Mexico City, Mexico) and finally filtered through Whatman No. 4 paper. The extracts were stored at 4 °C for the analysis of bioactive compounds (Barros et al., 2008a; Ferreira et al., 2009).

The chemical composition (dry matter, moisture, crude protein, crude fat, crude fiber, carbohydrates, energetic value, and ashes) of the fungal samples were analyzed using the Association of Official Analytical Chemists procedures (AOAC, 1995).

The moisture content was determined by the loss of mass weight that occurs when the material is heated (AOAC, 1995). The crude protein content (N 4.38) of the samples was estimated by the Kjeldahl method (Micro Kjeldahl Digestor, Labconco, Kansas, USA). Crude fat was determined by extracting 2 g of pulverized mushrooms with petroleum ether, using a Soxhlet apparatus (PYREX, Mexico City, Mexico); the ash content was determined by incineration at 600±15 °C (Felisa® Zapopan, Jalisco, Mexico); the method for Crude fiber is determined gravimetrically after chemical digestion and solubilization of other materials in present.

Total energy was calculated according to the following equation (AOAC, 1995):

using the following formula:

Bioactive compounds were determined after 80% methanolic extraction. Phenols, flavonoids, ascorbic acid and carotenoids were determined according to the methodology described by Barros et al. (2008a). The antioxidant activity was carried out according to the protocol of Barros et al. (2007a), with some modifications.

For the determination of phenolic compounds in the fungal extracts, 1 ml sample was mixed with 1 ml of Folin and Ciocalteu phenol reagent. After 3 min, 1 ml of a saturated sodium carbonate solution was added to the mixture and adjusted to 10 ml with distilled water. The reaction was kept in the dark for 90 min, and then the absorbance at 725 nm was measured (Spectrophotometer, Thermo Scientific^TM Evolution^TM 260, Waltham, Massachusetts, USA). Gallic acid was used to prepare the standard curve (0.01-0.1 mg/ml) (Y = 0.091848 + 0.057127x; R2 = 0.9997). The results were expressed as mg of gallic acid equivalents (GAE) per g of extract.

Zero point five ml of the methanolic extract was mixed with 0.1 ml of 10% aluminum nitrate and 0.1 ml of 1 M potassium acetate. Subsequently, 4.3 ml of 80% methanol was added, leaving it to stand for 40 min, then absorbance was read at 415 nm. Quercetin (0-100 mg/l) was used to prepare the standard curve (Y = 0.010852 + 0.091128x; R2 = 0.9997). The results were expressed in mg of Quercetin Equivalent (QE) per g of extract.

Five ml of ascorbic acid standard solution was mixed with 5 ml of HPO₃. The mixture was titrated with 2.6 dichlorophenolindophenol until it presented a pink color that was maintained for 15 seconds. The ascorbic acid staining factor was determined using the following formula:

Subsequently, 2 ml of the sample was mixed to 10 ml with HPO₃, filtered through Whatman No. 4 filter paper. A 0.5 ml aliquot of the extract was taken and titrated with the dye (Barros et al., 2008a).

For β-carotene and lycopene determination, 100 mg of dry methanolic extract were mixed vigorously with 10 ml of acetone-hexane (4:6) for 1 min and filtered through Whatmann nº 4. The absorbance of the filtrate was read at 453, 505, and 663 nm. The β-carotene and lycopene content were calculated by applying the following equations:

The results were expressed as mg of carotenoid and lycopene per g of extract.

The antioxidant capacity was determined based on the method described by Brand-Williams et al. (1995). Zero point threeml of the previously prepared methanolic extract was taken (stored at 4 °C). A 0.1 ml sample was obtained from the supernatant, which was placed in amber flasks and mixed with 3.9 ml of DPPH (2,2-diphenyl-1-picryl-hydrazyl-hydrate) methanolic solution with a concentration of 6 × 10-5 mol/l. Then the mixture was left to settle protected from light at room temperature for 70 min. Afterwards, the absorbance of the samples was measured in the spectrophotometer (Thermo Scientific^TM Evolution^TM 260, Waltham, Massachusetts, USA) at 517 nm; the control sample consisted of 0.1 ml of methanol and 3.9 ml of DPPH solution. Results were expressed as mg of Trolox Equivalent (TE) per g extract solution, using a trolox calibration curve as standard (Y = 0.58509 - 0.0214x; R2 = 0.9992).

For each of the fungal species, all the assays were carried out in triplicate. Results were expressed as mean values and standard deviation (SD). Results were analyzed using one-way analysis of variance (ANOVA) followed by Tukey’s HSD test with α = 0.05, using the program R software v. 4.3.0 (R Core Team, 2020).

Seven sequences were newly generated in this study. The four Cantharellus specimens were molecularly identified based on the blast results of ITS and tef-1α sequence data. The results are shown in Table 1.

Table 1:
Results of GenBank BLAST® searches for ITS and tef-1α sequences of four Cantharellus Adans. ex Fr. species from Mexico. S = Similarity, QC = Query Cover, “-” = sequence data is not available. The sequences here generated are in bold.

In general, Cantharellus violaceovinosus M. Herrera, V.M. Bandala & L. Montoya showed the highest values of the parameters evaluated (expressed on dry weight basis) (Table 2). Crude protein values between 5.60 g/100 g (Cantharellus sp.) and 8.04 g/100 g (C. violaceovinosus) were found. Crude fat ranged from 4.55 g/100 g (Cantharellus sp.) to 9.67 g/100 g (C. violaceovinosus). Carbohydrates were an abundant macronutrient ranging from 4.28 g/100 g (C. veraecrucis Bandala, Montoya & M. Herrera) to 24.50 g/100 g (C. violaceovinosus). The ash content was between 3.93 g/100 g (C. roseocanus Redhead, Norvell & Danell) Redhead, Norvell & Moncalvo) and 9.22 g/100 g (C. violaceovinosus). Based on the proximal analysis, it was determined that a 100 g portion of edible ectomycorrhizal fungi can ensure an intake of 266.53 Kcal in the case of C. violaceovinosus, while the lowest caloric intake with 91.32 Kcal was recorded for C. veraecrucis.

Table 2:
Approximate chemical composition (g/100 g) and energetic value (Kj/100 g) of four species of the genus Cantharellus Adans. ex Fr. Results are expressed in a dry weight basis. Values represent average values (±standard deviation) n = 3. In each column different letters mean significant differences (p<0.05) according to Tukey’s comparison of means test (α = 0.05).

The main antioxidant component found (1.91-6.23 mg/g) was β-carotene extract (Table 3), followed by flavonoids 0.43-2.98 mg QE/g extract. Ascorbic acid was found in small amounts 0.16-0.17 mg/g extract. Lycopene was found in amounts ranging from 1.18 to 2.52 mg/g extract. Lower ranges were observed in total polyphenols 0.34-0.82 mg GAE/g extract. Regarding the total antioxidant activity, a range of 1.74-2.44 mg TE/g extract was detected. There were differences between the species studied in terms of the concentrations of bioactive compounds. The exception was registered for the case of lycopene where no species presented statistical differences. Cantharellus roseocanus presented the highest amount of β-carotenes and flavonoids, compared to the other three species evaluated. In contrast, C. roseocanus recorded the highest amount of polyphenols. Cantharellus sp. and C. violaceovinosus registered the highest total antioxidant activity.

Table 3:
Total bioactive compounds of four species of wild ectomycorrhizal fungi of the genus Cantharellus Adans. ex Fr. values represent average values (± standard deviation) n = 3. Different letters in the same row indicate significant differences (p<0.05) according to Tukey’s comparison of means test (α = 0.05).

⁶Author for the correspondence: rllarena@uv.mx