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Introduction
Edible mushrooms are an alternative that contributes to satisfy the nutritional needs of the population, mainly in developing countries, due to the low production costs, high protein content and large harvest volumes in little space and time, with a precious flavor and highly desirable aromas (Wang et al., 2014; Kalač, 2016; Guerin-Laguette et al., 2020). The nutritional value of edible mushrooms is due to their high content of crude proteins, crude fibers, vitamins, minerals, and low-fat content (Assemie and Abaya, 2022). Indeed, mushrooms are rich sources of nutraceuticals, for example, several species of Cantharellus Adans. ex Fr. In Mexico, Cantharellus cibarius Fr., collected in the state of Hidalgo, reported important components (López-Vázquez et al., 2017), whiles in other countries it has been reported to have antioxidant, antigenotoxic, anti-inflammatory and antimicrobial properties (Barros et al., 2008a; Kumari et al., 2011; Ebrahimzadeh et al., 2015; Kozarski et al., 2015). The trace elements, nutrients and bioactive components of the species, as well as their apricot aroma, make these species popular among consumers worldwide (Ayaz et al., 2011; Politowicz et al., 2017). However, most of the studies of this genus refer to the physiology, taxonomy and ecology of its species (Barros et al., 2008a; Kumari et al., 2011; Ebrahimzadeh et al., 2015; Kozarski et al., 2015).
In the Mountain Region, Veracruz, Mexico, the climatic conditions, and its enormous biodiversity originate unique characteristics that are conducive to the growth of a huge amount of edible wild ectomycorrhizal mushrooms. In Mexico an important mycological richness is measured, positioning itself worldwide as the second reservoir of edible species, only behind China (Wu et al., 2019; Pérez-Moreno et al., 2020). Some of these species have a high gastronomic value. In recent years, new Cantharellus species have been reported in this region and are locally appreciated as choice wild edible mushrooms (Herrera et al., 2018; Montoya et al., 2021). Despite the popularity of edible species of the genus Cantharellus in agricultural markets in the region, there are no data on their nutritional and nutraceutical values. Therefore, it is essential to realize such investigations, to improve the conservation and valuation of these natural resources and their habitats (Barros et al., 2008b).
The aim of this study was to analyze the nutritional and nutraceutical composition of four species of wild ectomycorrhizal fungi of the genus Cantharellus from the mountain region, Veracruz, Mexico.
Materials and Methods
Sampling
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 isolation, PCR amplification and sequencing
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).
DNA sequence assembly and species identification
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.
Standards and reagents
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).
Preparation of samples
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).
Nutritional value analysis
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:
Determination of total bioactive compounds
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.
Total phenol content
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 ScientificTM EvolutionTM 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.
Total flavonoid content
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.
Ascorbic acid content
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).
β-carotene and lycopene content
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.
Antioxidant activity
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 ScientificTM EvolutionTM 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).
Statistical analysis
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).
Results
Species identification
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.
| Species | GenBank accession no. | |||
|---|---|---|---|---|
| ITS | tef-1α | References | ||
| Cantharellus veraecrucis Bandala, Montoya & M. Herrera | Bandala 4505 Type | - | MT449712 | Montoya et al., 2021 |
| EJHM20220813 | - | OQ876854 S=100%, QC=79% | ||
| Cantharellus violaceovinosus M. Herrera, V.M. Bandala & L. Montoya | - | - | MF616521 | Herrera et al., 2018 |
| EJHM20220814 | OQ875961 | OQ876855 S=99.87%, QC=84% | ||
| Cantharellus roseocanus Redhead, Norvell & Danell) Redhead, Norvell & Moncalvo | UBC F23802 | KX592760 | - | Thorn et al., 2017 |
| EJHM20220922 | OQ875962 S=99.42%, QC=99% | OQ876856 S=99.77%, QC=95% | ||
| T. Volk CC29 | JX030415 | Foltz et al., 2013 | ||
| Cantharellus sp. | EJHM20220923 | OQ875963 S=98.46%, QC=100% | OQ876857 S=97.86%, QC=100% | |
| C-2 | LC085373 | LC085470, 1 | Ogawa et al., 2018 | |
Nutritional and nutraceutical value analysis
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).
| Parameter\Species | Cantharellus violaceovinosus M. Herrera, V.M. Bandala & L. Montoya | Cantharellus roseocanus Redhead, Norvell & Danell) Redhead, Norvell & Moncalvo | Cantharellus sp. | Cantharellus veraecrucis Bandala, Montoya & M. Herrera |
|---|---|---|---|---|
| Dry material | 7.81±1.15b | 6.55±0.68b | 8.37±0.54b | 17.05±2.49a |
| Crude protein | 8.04±0.78b | 7.74±1.06b | 5.60±0.83a | 7.86±0.85b |
| Crude fat | 9.67±0.95a | 9.62±0.88a | 4.55±0.63b | 5.8±0.42b |
| Crude fiber | 20.98±0.91a | 2.67±.05b | 1.64±0.8b | 2.50±.05b |
| Ash | 9.22±1.55a | 3.93±0.64b | 6.49±0.73b | 8.22±1.05a |
| Moisture | 92.69±1.19a | 94.45±1.32a | 92.66±1.01a | 84.56±1.07b |
| Carbohydrates | 24.50±5.78a | 15.25±4.34b | 12.81±1.90b | 4.28±1.61c |
| Energetic value | 266.53±14.64a | 222.73±15.43b | 166.78±6.69c | 91.32±6.46d |
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).
| Compounds | Cantharellus violaceovinosus M. Herrera, V.M. Bandala & L. Montoya | Cantharellus roseocanus Redhead, Norvell & Danell) Redhead, Norvell & Moncalvo | Cantharellus sp. | Cantharellus veraecrucis Bandala, Montoya & M. Herrera |
|---|---|---|---|---|
| Antioxidants (mg TE/g extract) | 2.28±0.04ab | 1.74±0.04c | 2.44±0.06a | 2.22±0.10b |
| Polyphenols (mg GAE/g extract) | 0.34±0.02c | 0.82±0.02c | 0.39±0.01b | 0.62±0.07a |
| Flavonoids (mg QE/g extract) | 0.43±0.12c | 2.98±0.26a | 0.43±0.01c | 0.51±0.03b |
| Ascorbic acid (mg/g extract) | 0.16±0.01ab | 0.17±0.01a | 0.18±0.01a | 0.12±0.01b |
| Lycopene (mg/g extract) | 2.52±0.38a | 2.55±1.23a | 2.14±0.38a | 1.18±0.11a |
| β-Carotene (mg/g extract) | 3.97±0.95b | 6.23±0.68a | 3.67±0.62b | 1.91±0.15c |
Discussion
To the best of our knowledge, this is the first nutritional and nutraceutical report of C. violaceovinosus, C. veraecrucis and C. roseocanus in Mexico. The analyzed mushrooms contain valuable nutraceuticals such as phenols, ascorbic acid and β-carotenoids that could be extracted to be used as functional ingredients, specifically against microbial infections, food safety through food diversification, and soil restoration using mycorrhizal plants (Pérez-Moreno et al., 2021).
Edible ectomycorrhizal mushrooms are rich sources of protein with low amounts of fat with almost 400 species of edible mushrooms in Mexico second only to China (Garibay-Orijel and Ruan-Soto, 2014; Aguilar-Romero et al., 2016). Their conservation is associated with its hosts in Mexico, the genera Pinus L. and Quercus L., stand out, as it is one of their diversification centers (Romero-Sánchez et al., 2018; Castillo-Mendoza et al., 2022). Therefore, understanding the sustainable management of edible ectomycorrhizal fungi is important, since they support the production of fruiting bodies (Gernandt and Pérez-de la Rosa, 2014; Aguilar-Romero et al., 2016).
In this study, six antioxidant activities are reported: total phenols, total flavonoids, ascorbic acid, lycopene and β-carotene, as well as its total antioxidant activity. A report of species of the genus Cantharellus shows similar levels of flavonoids and β-carotene (Kumari et al., 2011). On the other hand, the same authors report a higher level of polyphenol (7.67-12.46 mg GAE/g extract) than this study. Compared to other edible species, Vega et al. (2022) reported a total polyphenols level from 1.87 to 3.03 mg GAE/g extract in Pleurotus djamor (Rumph. ex Fr.) Boedijn. In Latin America, commercial Pleurotus (Fr.) P. Kumm. production is mainly generated in Brazil, Mexico, Colombia, Argentina and Guatemala. It is known that the characteristics of the fungi are affected by different factors such as the species, strain, host, harvest time, management techniques, edaphoclimatic conditions and ecosystem conditions, among others (Manzi et al., 2004; Agrahar-Murugkar and Subbulakshmi, 2005).
Total antioxidant values in edible mushrooms of regional importance are diverse, such as Pleurotus ostreatus (Jacq.) P. Kumm, which could present values of 4.3 to 9.0 mg of TE/g extract (Stastny et al., 2022), while in this study C. violaceovinosus showed 2.28 mg of TE/g extract. This characteristic is important since according to the health authorities, it is considered that prevention and treatment with nutraceuticals is a powerful instrument to maintain and promote health, longevity, and life quality of the population (Barros et al., 2008a).
It should be noted that the species Cantharellus cibarius is the one with the largest number of reports on its chemical and nutraceutical composition (Barros, et al., 2007b; Ferreira et al., 2009). The chemical composition of Cantharellus cibarius in other countries was previously described from India, and contains protein as the main macronutrient (Agrahar-Murugkar and Subbulakshmi, 2005; Kumari et al., 2011), while in the Mexican samples of the species investigated here, the main component is carbohydrates, representing a source of nutrients and nutraceuticals. Therefore, these fungal species represent a source of nutrients and nutraceuticals as an alternative in the diet of the inhabitants of the municipalities of the Mountain Region where there are indices of marginalization and, both, poverty and extreme poverty (CONEVAL, 2020).
These mushrooms represent a growing segment of the current food industry (Willis, 2018). In the species studied C. violaceovinosus showed the highest values of the parameters evaluated. For this reason, the Cantharellus species reported in this study can be used directly in the diet and to promote health, taking advantage of the antioxidant molecules, and additive and synergistic effects tthat is bioactive components could have (Barros et al., 2008a).
