10.6092/issn.2531-7342/15649
The chemical composition evaluation and the nutritional value of nine most widespread wild edible mushroom species (Agaricus arvensis, Cantharellus cibarius, C. lutescens, Craterellus cornucopioides, Hericium erinaceus, Hydnum repandum, Lactarius deliciosus, Pleurotus pulmonarius, and Ramaria flavescens) and much collected in northwestern Tunisia, have been analyzed and determined according to standard methodologies. The chemical composition of edible mushrooms was validated by statistical PCA analyzes. Overall results showed that most of the studied species had interesting values for almost all measured variables. In particular, the species H. erinaceus had the highest quantity of carbohydrates (89.70%), oleic (24.05%), and docosahexaenoic acid (3.19%), phenolic compounds (11.25 mg g-1 dw), flavonoids (57.5 mg g-1 dw), and minerals K, Mg and Ca but also the lowest content of proteins (4.80%) and carbohydrates (3.96%). On the other hand, the species P. pulmonarius had the lowest lipid content (7.30%) and the lowest caloric value (371.76 Kcal). These promising data can be exploited by taking advantage of the high-quality nutritional value of these interesting species.
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Chemical composition and nutritional value of nine wild edible mushrooms from Northwestern Tunisia
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University of Tunis El Manar, Laboratory of Bioresources, Environment and Biotechnology (LR22ES04), Higher Institute of Applied Biological Sciences of Tunisia (ISSBAT), University of Tunis El Manar, 9 Street Zouheir Essafi CP 1006, Tunis, Tunisia
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Chemical composition and nutritional value of nine wild edible mushrooms from Northwestern Tunisia
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                IJM - Italian Journal of Mycology
                ISSN 2531-7342 - Vol. 52 (2023): 32-49
                Journal homepage: https://italianmycology.unibo.it/

Research article

    Chemical composition and nutritional value of nine wild edible
               mushrooms from Northwestern Tunisia

     Zouhour Ouali1, Hatem Chaar2,3, Giuseppe Venturella4, Fortunato Cirlincione4,
                        Maria Letizia Gargano5, Atef Jaouani6
1
  National Gene Bank of Tunisia (NGBT), Boulevard du Leader Yasser Arafat, Charguia, CP 1080, Tunis, Tunisia
2
  National Institute of Agronomic Research of Tunisia (INRAT), University of Carthage, Tunisia, Rue Hédi Karray, CP
1004, Menzah 1, Tunisia
3
  National Institute of Agronomy of Tunisia (INAT), University of Carthage, Tunisia, 43, Avenue Charles Nicolle, CP
1043, Tunis-Mahrajène, Tunisia
4
  University of Palermo, Department of Agricultural, Food and Forest Sciences (SAAF), Viale delle Scienze, Bldg. 5,
I-90128 Palermo (Italy)
5
  University of Bari Aldo Moro, Department of Agricultural and Environmental Science, Via Amendola 165/A, I-70126
Bari (Italy)
6
  University of Tunis El Manar, Laboratory of Bioresources, Environment and Biotechnology (LR22ES04), Higher
Institute of Applied Biological Sciences of Tunisia (ISSBAT), University of Tunis El Manar, 9 Street Zouheir Essafi CP
1006, Tunis, Tunisia

Corresponding author e-mail: atef.jaouani@issbat.utm.tn


Article info
Received 19/10/2022; accepted 18/1/2023
https://doi.org/10.6092/issn.2531-7342/15649

Abstract
The chemical composition evaluation and the nutritional value of nine most widespread wild edible mushroom
species (Agaricus arvensis, Cantharellus cibarius, C. lutescens, Craterellus cornucopioides, Hericium
erinaceus, Hydnum repandum, Lactarius deliciosus, Pleurotus pulmonarius, and Ramaria flavescens) and much
collected in northwestern Tunisia, have been analyzed and determined according to standard methodologies.
The chemical composition of edible mushrooms was validated by statistical PCA analyzes. Overall results
showed that most of the studied species had interesting values for almost all measured variables. In particular, the
species H. erinaceus had the highest quantity of carbohydrates (89.70%), oleic (24.05%), and docosahexaenoic
acid (3.19%), phenolic compounds (11.25 mg g-1 dw), flavonoids (57.5 mg g-1 d.w), and minerals K, Mg and Ca
but also the lowest content of proteins (4.80%) and carbohydrates (3.96%). On the other hand, the species P.
pulmonarius had the lowest lipid content (7.30%) and the lowest caloric value (371.76 Kcal). These promising
data can be exploited by taking advantage of the high-quality nutritional value of these interesting species.

Keywords
Hericium erinaceus, Pleurotus pulmonarius, mineral composition, phenolics


Introduction

A wide diversity of wild mushrooms can be found in nature, of which about 2,300 are known as
useful species (FAO, 2009). However, 1,154 species are considered as edible. They were particularly
preferred for their culinary and organoleptic characteristics (source of aromatic substances with

Department of Agricultural and Food Science in partnership with the Italian Mycological Society (UMI).
The journal is hosted and maintained by ABIS-AlmaDL. © 2023 The Authors. This work is licensed under CC BY 4.0
Ouali et al.                                                     Italian Journal of Mycology 52 (2023): 32-49

https://doi.org/10.6092/issn.2531-7342/15649



unique flavors) (Reis et al., 2017). They were recognized much later for their good quantity of
nutritional elements (protein, carbohydrate total sugars, lipid, and composition of fatty acid) and their
nutraceutical properties (bioactive compounds, phenolics including flavonoids and tannins) essential
for human health (Gargano et al., 2017).
    They were becoming more important in the human diet for their crude dietary fiber content, mineral
elements (Falandysz and Borovička, 2013) and proteins that can serve as a good substitute for animal
proteins in vegetarian diet (Wang et al., 2014). These functional characteristics are mainly due to
their content of different types of biologically active compounds in fruit bodies, cultured mycelia,
and cultured broth (Chang and Wasser, 2017), essentially depending on species and substratum. In
addition, Vetvicka et al. (2019) had demonstrated, by clinical trials, the high medicinal value of
some edible mushrooms which contain physiological agents and metabolites having antitumour,
cardioprotective, antiviral, antibacterial, immunoregulatory properties. On the other hand, (i)
the first annotated checklist of macromycetes in northern Tunisia (Ouali et al., 2018) with 123
species recorded, (ii) the updated checklist with 145 new species recorded (Ouali et al., 2021) and
(iii) the record of new species in Tunisia (Jaouani et al., 2015; Ouali et al., 2020) show that this
country harbors a large reservoir of fungal diversity especially in northwestern areas. Therefore, it
is stimulating to better exploit this diversity, particularly species of important commercial interest
such as edible and medicinal mushrooms by determining their chemical composition and studying
their nutritional value to better valorize these resources. Despite the significant economic of wild
edible and medicinal mushrooms in the collecting areas (around 50 t/year) according to the National
Forest Exploitation Authority, Ministry of Agriculture, 2019 (personal communication), the study of
their chemical composition and their medicinal potential remains little studied, under-estimated and
limited to few mushrooms (Palazzolo et al., 2012; Kacem Jedidi et al., 2016). Thereby, other species
should be analyzed to exploit their nutritional, nutraceutical and medicinal potential. The aim of this
paper was to investigate the chemical composition of nine wild edible mushrooms species (eight
species are well known, the most collected and sold by villagers, and one rare species (Hericium
erinaceus) recently recorded (Ouali et al., 2020) in northwestern Tunisia. The study was focused on
identifying the mineral and biochemical constituents that contribute most to their nutritional value
and highlighting species that had the best correlation between these elements.

Material and Methods

Sample collection, identification and preparation
The sporomata of nine species of wild edible mushrooms were collected from two sites in northern
Tunisia (Fig. 1, Table 1): eight species were frequently harvested in the area, showing a great economic
interest as a source of income for local population, and a rare species known for its medicinal properties
(H. erinaceus). The macro-morphological and microscopic identification of collected mushrooms
was performed on fresh material according to the methodology adopted by Ouali et al. (2018). The
mushroom basidiomata (cap and stipe) were cleaned of forest debris and lyophilized (Unicryo MC4L,
UniEquip GmbH, Germany), reduced to fine dried powder, and mixed to obtain homogeneous samples
and stored, at room temperature, in opaque glass boxes, protected from light, until further analyses.




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                                  Fig. 1 - Map of Tunisia; study area: I = Jendouba:
                                  AinDraham - OuedEzzen forest; II = Beja: Nefza - Bellif
                                  1 forest.



Table 1 - Edible wild mushrooms studied with habitat characteristics (Em: Ectomycorrhizal; Pn: Parasite necrotrophic;
Sh: lignicolous saprotroph; St: terricolous saprotroph).
 Specimens                             Family          Ecological Governorates (Region -          Host vegetation
                                                       categories Site - GPS coordinates)
 Cantharellus lutescens Fr.            Cantharellaceae Em         Jendouba (Ain Draham            Conifer woods of
                                                                  – Oued Ezzen forest;            Pinus halepensis
                                                                  36°46’01,7 N; 8°47’39,3 E;
                                                                  554 m)
 Lactarius deliciosus (L.) Gray        Russulaceae     Em
 Agaricus arvensis Schaeff.            Agaricaceae     St                                         Mixed oak woods of
                                                                                                  Quercus canariensis
                                                                                                  and Q. suber
 Hericium erinaceus (Bull.) Pers.      Hericiaceae       Sh
 Hydnum repandumL.                     Hydnaceae         Em
 Ramaria flavescens (Schaeff.)         Gomphaceae        Em
 Cantharellus cibarius Fr.             Cantharellaceae   Em          Beja (Nefza - Bellif 1
                                                                     forest; 37°02’19,2 N;
                                                                     9°08’01,2 E; 183 m)
 Craterellus cornucopioides (L.)                         Em
 Pleurotus pulmonarius (Fr.) Quél. Pleurotaceae          Pn


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Analytical methods
Ash content was determined by sample calcination at 525 °C after 4 h. The assay of minerals (K, P,
Mg, Ca, Na, Fe, Cu, Zn, Mn, and Cr) was analyzed by atomic emission spectrometer with induction
coupled plasma (Perkin Elmer, Optima, 7300DV) according to ISO 11885 standards (2007). The
assay of total nitrogen (TN) was determined using modified Kjeldahl method, according to ISO
11261 standards (1995). The crude protein content was estimated by multiplying the nitrogen content
by the factor 4.38 (= 4.38 × N) (Barros et al., 2007). Total carbohydrates were extracted using the
method described by Barros et al. (2007) and their profiles were obtained by high performance liquid
chromatography HPLC (Agilent 1200 series, USA) equipped with a quaternary pump model coupled
to a refractometer index (RI) detector and with Aminex HPX-87 H ion-exchange columns (300 × 7.8
mm, Bio-Rad) as described by Heleno et al. (2009). The mobile phase was sulfuric acid 5 mmol 1 L-1
(in milliQ water) at a flow rate of 0.5 mL 1/min. This HPLC was connected to a computer running
WINILAB III software (Perichrom, France).
    Total lipids were extracted by the mixture chloroform/methanol (2:1, v:v) as the solvent containing
0.01% butylated hydroxyl toluene (BHT) as antioxidant. The organic solvent was evaporated under
a stream of nitrogen and the lipid content was determined gravimetrically. Then, extracted lipids
were suspended in chloroform/methanol (2:1 v/v) and stored at -30 °C prior to chromatographic
analysis. For fatty acid analysis, lipids extracts were esterified according to ISO 5509 (2000) standards
trans-esterification method. Fatty acids were determined by gas–liquid chromatography with flame
ionization detection (GLC–FID)/capillary column. The fatty acid profile was analyzed with a
chromatograph (HP 6890) equipped with a split–split less injector, a flame ionization detector (FID).
The temperatures of the injector and detector were maintained at 250 °C and 275 °C, respectively.
Separation was achieved on 30 m × 250 µm i.d fused Innowax capillary column (HP) coated with
a 0.25 µm film. Nitrogen was used as carrier gas at an internal pressure of 120 kPa. The column
temperature was programmed to rise from 50 to 180 °C at a rate of 4 °C/min, then programmed to
increase from 180 °C to 220 °C at 1.33 °C/min and then held for 7 min. Fatty acids were identified
through comparison of their retention times with those of a mixture of methyl esters (SUPELCO
PUFA-3). Methyl nonadecanoate C19:0 (Sigma) was used as internal standard, a nd then fatty acid
peaks were integrated and analyzed using HP chemstation software.
    Total carbohydrates were calculated by difference:
    Total carbohydrates = 100 – (g moisture + g protein + g fat + g ash).
    Total energy was calculated according to the following equations:
    Energy (kcal) = [4 × (g protein + g carbohydrate)] + [9 × (g lipid)]
    Extraction of phenolic compounds, flavonoids and tannins, from freeze-dried carpophores was
carried out according to the slightly modified protocol described by Palacios et al. (2011). The
total phenolic and flavonoids content in the mushroom methanolic extracts was estimated using the
colorimetric assays of the Folin-Ciocalteau and Aluminum Chloride (AlCl3), respectively. Gallic acid
was used to calculate the standard curve for the phenolic total content. The results were expressed as
milligrams of gallic acid equivalents per gram of dried mushroom (mg GAE g-1 dw). The total content
of the tannins in the mushroom methanolic extracts was evaluated by the colorimetric test with vanillin
4%. Catechin was employed to calculate the standard curve of the described method. For the total
contents of flavonoids and tannins, the results are expressed in milligrams of catechin equivalents
per gram of dried mushroom (mg CE g-1 dw). The absorbance was measured with spectrophotometer
(Perkin Elmer, Lambda 25, UV-VIS) at 760 nm, 510 nm and 500 nm for the phenolic compounds,
flavonoids, and tannins contents, respectively.




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Statistical analysis
For the ash, as well as mineral elements, phenolic compounds, flavonoids and tannins, the effect of
the fixed factor species was tested with an analysis of variance (ANOVA). The residual normality
as well as homogeneity of variances assumptions was assessed using Shapio test and Levene’s test,
respectively. LS-means were calculated and compared with Tukey test. These statistical analyses
were carried out using SAS 9.4 (SAS Institute Inc, Cary, North Carolina, USA. A principal component
analysis (PCA) was separately performed on the two groups of variables measured on mushroom
species: (1) mineral composition, and (2) biochemical composition (protein, carbohydrate, total
sugars, lipid, and composition of fatty acid) that were centered and reduced beforehand. Then, the
relationship between these two variable groups was examined using a co-inertia analysis (CoIA)
(Dolédec and Chessel, 1994). The PCA and CoIA were executed using respectively Vegan (Oksanen
et al., 2019), and ade4 (Dray et al., 2019) R-packages R-3.6.1.

Results and discussion

Mineral composition
All the measured mineral variables varied significant according to the species characteristics (Tab
2). Ash content ranged from 4.8 g kg-1 to17.6 g kg-1 dw, with an average (11.7 ± 3.9 g kg-1 dw). The
lowest mean values of ash were recorded in H. erinaceus, followed by L. delisiosus, and the highest
one was monitored in C. cibarius (Table 2). These data were higher than those found on similar
species in Tunisia (Kacem Jedidi et al., 2016) and in other countries such as Turkey (Onbaşili et al.,
2015) and Bangladesh (Colak et al., 2009). They indicate that mushrooms absorb major and trace
elements more than plants. Indeed, mushrooms have effective mechanisms of mineral absorbency
notably using low molecular weight organic acids (Mleczek et al., 2016).
    The K content varied considerably from 15.75 to 71.15 g kg-1 dw (mean 34.57 ± 15.00 g kg-1 dw),
whereas P content varied slightly from 1.0 to 12.9 g kg-1 dw (mean: 4.19 ± 3.30 g kg-1 dw). Both K
and P contents were more abundant than Mg (0.5–2.0 g kg-1; mean: 1.13 ± 0.47 g kg-1), Ca (0.1–1.7
g kg-1; mean: 0.72 ± 0.42 g kg-1 dw) and Na (0.02–2.15 g kg-1 dw; 0.52 ± 0.58 g kg-1 dw) (Table 2).
These results confirm those of Andres and Baumann (2012), reporting that the main constituents in
mushroom ashes were K and P (60% totally).
    Hericium erinaceus had the highest average contents of K, Mg and Ca elements, whereas A.
arvensis had the highest average of P content and the lowest mean contents of Ca and Na. Cantharellus
lutescens had the highest Na content while K, Mg and Ca contents in C. cibarius were higher, and
Na content was considerably lower, than those reported by Kacem Jedidi et al. (2016). However, H.
repandum had the same K content but a very low Na content compared to that reported by Kacem
Jedidi et al. (2017). The accumulation of these major elements in all species may be due to their
abundance in the soil or to the composition of substratum (Vaishaly et al., 2015).
    The trace elements, represented by Fe (mean: 280 ± 260 mg kg-1 dw) and Zn (39.2 ± 27.8 mg
kg dw), were more abundant than the other elements, Cu (18.5 ± 16.2 mg kg-1 dw), Mn (8.4 ±
   -1

11.8 mg kg-1 dw) and Cr (1.7 ± 3 mg kg-1 dw) which were lower in traces. This observation is in
agreement with Vaishaly et al., (2015), reporting that mushrooms are known as Zn accumulators.
All specimens possess lower Zn content and higher Fe content than reported by Colak et al. (2009),
except for lignicolous H. erinaceus which had the lowest Fe content and null contents of Cu, Zn, Mn
and Cr. This exception is probably due to the saprotrophic ecology of H. erinaceus and to the specific
bioaccumulation metabolism of metals by fungi. On the other hand, terricolous A. arvensis had the
highest content for both Cu and Zn.



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    In this study, Fe, Mn and Cr contents of C. cibarius were higher than those reported by Colak et
al. (2009) for the same species. These metal levels can be interesting for human health and assured
their daily recommended intake. In contrast, Fe and Zn contents of this species were higher, and
its Cu content was considerably lower than those reported by Kacem Jedidi et al. (2016), on the
same species, collected in the same areas in northwestern Tunisia in 2012-2013. This may be due
to yearly or microclimate variations, to the environmental conditions (site with ferralitic soil) of the
mushroom growth that have influence on the concentration of metal absorption by mushrooms. These
concentrations in Cu are higher than those in vegetables and should be considered as a nutritional
source of this element. Indeed, the Recommended Dietary Allowances (RDA) for adults is 0.9 mg Cu/
day (Andres and Baumann, 2012). Cu contents of the sampled mushrooms are not considered as a
health risk. This element acts as an antioxidant and contributes to the good functioning of the nervous
and immune system (EFSA NDA Panel 2009). In the present study, mushrooms were shown to be
safe in terms of heavy metal contents. Indeed, the studied fungi showed negligible and acceptable
levels of heavy metals (Fe, Zn, Cu, Mn and Cr) by reference to other data reported by Gałgowska and
Pietrzak-Fiećko (2020) and Širić et al. (2016). The mushrooms are characterized by high contents of
assimilated minerals that depend not only on the species, but also on the collecting site, the nature
of substratum (acidic and organic matter content of the soil) and even the distance from sources
of pollution (Andres and Baumann, 2012). However, the relationship between soil contamination
with trace elements and their content in sporomas is not tight enough to enable usage of mushroom
species as a reliable bio-indicator of local pollution (Andres and Baumann, 2012). This study shows
that H. erinaceus and P. pulmonarius are the species with the best mineral composition for human
consumption with average contents of major elements (K, P, Ca, and Mg) and low contents of heavy
metals (Cr, Mn, Zn).

Biochemical composition
Wild mushrooms were rich sources of protein and carbohydrates, and had low amounts of fat (Barros et
al., 2007). In this study, mushroom protein content was ranging from 4.8 g/100 g dw (for H. erinaceus)
to 33.94 g/100 g dw (for A. arvensis). Indeed, some mushrooms had protein content below meats, but
well above most other foods, including milk (25.2%), cereals (7.3% in rice, 12.7% in wheat and 9.4%
in corn) and most of wild plants. The protein content in A. arvensis (33.94 g/100 g of dw) was higher
not only than that reported by Kumar al. (2013), for the same species, but also higher than that of milk
and other wild and cultivated mushrooms. Several studies have proven mycoproteins’ nutritional,
health and environmental benefits and affirm their role in a healthful diet. Indeed, mycoproteins are
rich in essential amino acids and can help maintain healthy blood cholesterol levels, promote muscle
synthesis, control glucose and insulin levels, and increase satiety (Finnigan et al., 2019). Study on
human volunteers has revealed that the biological value of proteins in mycoproteins is similar to that
of milk proteins (Hashempour-Baltork et al., 2020). Hence, mycoproteins constitute a sustainable and
satisfying alternative protein source that can replace environmentally-damaging animal-based foods
(Xu et al., 2021). Several authors (Barros et al., 2008; Colak et al., 2009; Kumar et al., 2013; Kacem
Jedidi et al., 2016) reported higher protein contents in C. cibarius, C. cornucopioides, H. repandum
and P. pulmonarius. This variation in mushroom protein content could be depending on species,
development stage, level of available nitrogen in the soil or substrate and the location.




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Table 2 - Chemical composition/energetic value of Tunisian wild edible mushrooms: mean ± standard deviation (SD); n = 3.

                      A. arvensis      C. cibarius      C. lutescens      C. cornucopioides    H. erinaceus    H. repandum L. deliciosus        P. pulmonarius     R. flavescens
 Ash (%)              12.90 ± 0.35 d   17.50 ± 0.05 a   11.30 ± 0.45 ef   16.40 ± 0.01 b       5.50 ± 0.01 h   14.40 ± 0.01   6.40 ± 0.2 g      10.60 ± 0.53 f     12 ± 0.02 ed
                                                                                                               c
 K (g kg-1 dw)        43.04 b          37.69 c          26.64 f           35.78 d              70.91 a         28.51 e        16.99 g           26.82 f            26.87 f
 P (g kg-1 dw)        12.50 a          2.18 d           1.10 e            2.48 d               4.91 c          3.81 d         3.04 d            6.04 b             2.55 d
 Mg (g kg-1 dw)       1.66 ba          0.88 d           0.64 d            1d                   1.98 a          0.75 d         0.96 d            1.60 b             1.22 b
 Ca (g kg dw)
           -1
                      0.10 e           1.02 b           0.98 b            0.79 b               1.57 a          0.66 cb        0.33 e            0.53 c             0.70 c
 Na (g kg dw)
           -1
                      0.03 f           0.41 cbd         2.15 a            0.43 cd              0.86 b          0.25 ced       0.15 ed           0.52 cb            0.14 e
 Fe (g kg dw)
         -1
                      0.05 c           0.77 a           0.28 b            0.60 ba              0.02 c          0.57 a         0.08 c            0.05 c             0.26 b
 Cu (g kg dw)
           -1
                      0.05 a           0.05 a           0.02 a            0.02 a               0a              0.01 a         0.01 a            Trace a            0.02 a
 Zn (g kg dw)
           -1
                      0.12 a           0.03 ba          0.05 ba           0.08 ba              0b              0.03 ba        0.08 ba           0.04 ba            0.05 ba
 Mn (g kg-1 dw)       trace cd         0.04 a           trace cd          trace cd             0d              0.01 cb        trace d           0.02 b             0.02 b
 Cr (g kg-1 dw)       trace b          0.01 a           trace b           trace b              0b              trace b        trace b           trace b            trace b
 Total nitrogen (%)   7.75             2.31             1.95              2.69                 1.10            2.58           2.82              3.18               2.70


 Proteins (%)         33.94 ± 2.25     10.10 ± 1.50     8.55 ± 0.20       11.79 ± 1.08         4.80 ± 0.08     11.28 ± 1.12   12.35 ± 0.17      13.91 ± 0.08       11.80 ± 0.02
 Carbohydrates (%)    21.46 ± 0.42     35.94 ± 0.86     59.16 ± 2.25      6.20 ± 1.42          89.70 ± 1.25    51.64 ± 1.42   53.13 ± 0.65      62.63 ± 0.50       10.67 ± 0.72
 Mannitol (%)         6.06 ± 0.05      12.15 ± 0.04     15.46 ± 0.15      15.02 ± 0.20         2.58 ± 0.58     5.81 ± 0.88    13.43 ± 0.50      1.35 ± 0.50        10.69 ± 1.23
 Trehalose (%)        0.53 ± 0.00      9.63 ± 0.02      0.57 ± 0.05       0.20 ± 0.01          1.37 ± 0.36     9.11 ± 0.31    5.75 ± 0.05       7.63 ± 1.40        1.04 ± 0.75
 Total sugars (%)     6.60 ± 0.05      21.79 ± 0.55     16.03 ± 0.22      15.23 ± 0.18         3.96 ± 0.50     14.93 ± 0.50   19.18 ± 0.01      8.98 ± 1.66        11.73 ± 1.50
 Total Lipids (%)     26.80 ±1.51      32.50 ± 0.75     35.20 ± 0.62      61.65 ± 0.05         26.78 ± 0.75    18.28 ± 1.55   23.62 ± 0.75      7.30 ± 2.01        60.97 ± 0.05




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 Table 2 - Continued
 Fatty acids
 C14:0 (%)             0.30 ± 0.04     0.65 ± 0.02     0.33 ± 0.04     0.50 ± 0.05     0.24 ± 0.08    0.02 ± 0.00     0.28 ± 0.01     0.15 ± 0.01       0.04 ± 0.04
 C15:0 (%)             0.29 ± 0.04     0.60 ± 0.02     0.62 ± 0.04     0.68 ± 0.04     0.14 ± 0.00    0.89 ± 0.07     1.13 ± 0.02     0.13 ± 0.01       0.16 ± 0.00
 C16:0 (%)             12.60 ± 0.25    12.50 ± 0.10    13.96 ± 1.87    13.86 ± 1.15    24.29 ± 0.25   14.30 ± 0.50    14.74 ± 0.08    15.13 ± 0.56      17.06 ± 1.20
 C16:1 (%)             0.65 ± 0.05     0.32 ± 0.02     0.93 ± 0.75     0.50 ± 0.00     0.18 ± 0.00    0.43 ± 0.03     1.22 ± 0.02     3.44 ± 0.34       0.78 ± 0.79
 C17:0 (%)             17.30 ± 0.55    18.72 ± 1.15    4.18 ± 1.46     8.88 ± 2.00     14.83 ± 1.91   8.79 ± 0.75     12.23 ± 0.15    16.98 ± 0.82      6.43 ± 0.81
 C18:0 (%)             3.83 ± 0.03     3.77 ± 0.02     4.96 ± 0.83     5.67 ± 0.35     3.60 ± 0.78    2.09 ± 0.01     17.17 ± 0.05    3.73 ± 0.42       2.57 ± 0.22
 C18:1 (%)             2.05 ± 0.12     1.33 ± 0.01     14.76 ± 1.02    20.26 ± 0.22    24.05 ± 1.64   6.51 ± 0.10     9.18 ± 0.42     3.56 ± 0.15       20.43 ± 0.09
 C18:2 (%)             35.4 ± 1.12     1.88 ± 0.03     19.41 ± 1.75    21.02 ± 1.55    11.76 ± 0.91   26.11 ± 0.55    11.89 ± 0.15    15.45 ± 0.45      29.82 ± 0.90
 C18:3 (%)             0.07 ± 0.00     0.08 ± 0.00     0.07 ± 0.00     0.05 ± 0.00     0.00 ± 0.00    0.15 ± 0.00     0.17 ± 0.02     0.09 ± 0.05       0.07 ± 0.00
 C20:0 (%)             0.60 ± 0.01     1.08 ± 0.01     0.50 ± 0.02     0.24 ± 0.01     0.21 ± 0.01    0.38 ± 0.01     0.46 ± 0.03     0.80 ± 0.51       0.26 ± 0.02
 C20:1 (%)             0.53 ± 0.02     0.13 ± 0.00     0.03 ± 0.12     0.61 ± 0.20     0.46 ± 0.04    0.36 ± 0.01     0.77 ± 0.01     0.70 ± 0.22       0.50 ± 0.20
 C20:2n-6 (%)          0.14 ± 0.00     0.50 ± 0.01     0.13 ± 0.03     0.18 ± 0.02     0.04 ± 0.01    0.13 ± 0.00     0.25 ± 0.00     0.05 ± 0.01       0.08 ± 0.00
 C20:3n-6 (%)          0.40 ± 0.01     0.25 ± 0.00     23.14 ± 1.40    10.94 ± 1.50    0.02 ± 0.02    20.25 ± 1.50    1.59 ± 0.05     0.17 ± 0.02       2.28 ± 0.05
 C20:4n-6 (%)          0.37 ± 0.01     0.06 ± 0.00     0.21 ± 0.04     0.11 ± 0.00     0.01 ± 0.02    0.12 ± 0.00     0.15 ± 0.00     0.04 ± 0.00       0.01 ± 0.01
 C21:0 (%)             0.20 ± 0.05     0.18 ± 0.01     0.17 ± 0.02     0.16 ± 0.00     0.09 ± 0.00    0.17 ± 0.00     0.24 ± 0.01     0.10 ± 0.00       0.14 ± 0.02
 C22:0 (%)             0.26 ± 0.03     0.48 ± 0.02     0.12 ± 0.02     0.27 ± 0.04     0.10 ± 0.01    0.14 ± 0.00     0.30 ± 0.01     0.33 ± 0.00       0.36 ± 0.05
 C24:0 (%)             0.91 ± 0.05     0.10 ± 0.00     0.35 ± 0.01     1.13 ± 0.57     0.75 ± 0.03    0.25 ± 0.01     1.02 ± 0.07     2.32 ± 0.12       0.72 ± 0.04
 C22:6n-3 (%)          0.33 ± 0.02     0.42 ± 0.05     0.39 ± 0.05     0.69 ± 0.32     3.19 ± 0.57    1.23 ± 0.75     0.21 ± 0.01     0.22 ± 0.04       0.69 ± 0.70
 C24:1 (%)             0.03 ± 0.00     1.80 ± 1.12     0.33 ± 0.85     0.50 ± 0.02     1.07 ± 0.36    0.61 ± 0.10     0.32 ± 0.00     0.31 ± 0.05       0.16 ± 0.01

 Total SFA (%)         60 ± 1.51       78.93 ± 2.25    40.59 ± 1.50    45.15 ± 0.58    59.22 ± 1.20   44.09 ± 0.50    74.26 ± 0.05    75.87 ± 0.74      45.18 ± 1.35
 Total MUFA (%)        3.26 ± 0.85     3.60 ± 0.95     16.06 ± 0.36    21.86 ± 0.24    25.76 ±1.57    7.91 ± 0.40     11.49 ± 0.12    8.02 ± 0.65       21.87 ± 1.11
 Total PUFA (%)        36.70 ± 1.05    3.21 ± 0.90     43.35 ± 0.50    32.99 ± 0.33    15.02 ± 0.95   47.99 ± 0.50    14.26 ± 0.15    16.03 ± 0.21      32.95 ± 1.01
 Energy (Kcal) (%)     462.80 ± 0.03   476.66 ± 0.05   514.96 ± 0.70   626.83 ± 0.02   378 ± 0.01     416.20 ± 0.05   474.50 ± 0.05   371.86 ± 0.02     638.59 ± 0.02




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    The carbohydrates contents of the sampled mushrooms showed high variation from 6.2 to 89.70
g/100 g dw, in C. cornucopioides and H. erinaceus, respectively (Table 2), indicating that mushroom
carbohydrates are also an abundant macronutrient. Carbohydrates are the most important energy source
for human nutrition. They support the nervous and immune system, help control blood glucose and
insulin metabolism, participate in cholesterol and triglyceride metabolism, and help with fermentation
(Holesh et al., 2021). Nevertheless, all samples had lower carbohydrates contents than those found
before (Colak et al., 2009; Kumar et al., 2013; Kacem Jedidi et al., 2016), except for P. pulmonarius,
(62.63%). For total sugars contents, the highest (21.79 g/100 g dw) and the lowest (3.96 g/100 g dw)
were revealed in C. cibarius and H. erinaceus, respectively. To our knowledge this is the first report
of sugar profiles for A. arvensis, C. lutescens, C. cornucopioides, H. erinaceus, P. pulmonarius, and
R. flavescens. Mannitol and trehalose were detected in all analyzed samples as the most abundant
sugars in fungi (Andres and Baumann, 2012). In this study, mannitol was the main dominant sugar
in all the studied species, varying from 1.35 to 15.46 g/100 g dw in P. pulmonarius and C. lutescens,
respectively. Trehalose content was ranging from 0.2 to 9.63 g/100 g dw in C. cornucopioides and C.
cibarius respectively. Mannitol and trehalose contents obtained in C. cibarius and C. cornucopioides
were higher than those reported by Barros et al. (2008). H. repandum had twice as much trehalose
content and low mannitol content compared to that previously reported by Fernandes et al. (2013).
    The lipid content of the analyzed mushrooms was remarkable; it varied between 7.3 and 61.65
g/100 g of dw in P. pulmonarius and C. cornucopioides respectively. Based on the immediate analysis,
it can be calculated that an edible portion of 100 g of these dry mushrooms provides on average 484
kcal (2,022 kJ). The highest value was shown by R. flavescens (638.59 Kcal) while P. pulmonarius
had the lowest energy contribution (371.86 Kcal) due to its low fat content (7.3 g/100 g of dw). This
result confirmed those reported by Fernandes et al. (2013). Thereby, P. pulmonarius could rebalance
or supplement the menus too rich in lipids or integrate in hypo caloric diets (Gargano et al., 2017).
The chemical composition and energetic value of H. repandum agreed with the previous works of
Fernandes et al. (2013).
    The results of fatty acid composition, total saturated fatty acids (SFA), monounsaturated fatty
acids (MUFA), and polyunsaturated fatty acids (PUFA) of the studied mushrooms were also shown
in Table 2. Fatty acid composition analysis allowed the quantification of 19 fatty acids which varies
from one species to another. The main fatty acids detected at high contents were palmitic (C16: 0),
margaric (C17: 0), oleic (C18: 1) and linoleic acid (C18: 2). This finding agreed with the results
reported for mushrooms from Portugal (A. arvensis, C. cibarius, C. cornucopioides and L. deliciosus)
in which linoleic, oleic and palmitic acids were considered the most specific fatty acids (Barros et al.,
2007, 2008). SFA were the most abundant group of fatty acids in all the studied mushrooms, ranging
from 40.59% to 78.93%. The main SFA found in studies species is palmitic acid followed by margaric
acid. C. cibarius revealed the highest SFA content due to the contribution of palmitic, margaric acid,
and the lowest contents of MUFA and PUFA, respectively. As reported by Helano et al. (2015), SFA
in H. erinaceus predominate over PUFA and MUFA due to the high contents of palmitic and margaric
acid. Compared to Barros et al. (2007, 2008) studies, palmitic acid content was higher (the double)
in C. cornucopioides, while it was lower in A. arvensis and C. cibarius. For A. arvensis, C. cibarius,
C. cornucopioides and L. deliciosus, both linoleic and margaric acid contents were higher than that
found by Barros et al. (2007, 2008). The highest percentage of oleic acid and linoleic acid were
respectively observed in H. erinaceus and A. arvensis. The most predominant UFA was linoleic acid
followed by oleic acid, confirming what was found by Heleno et al. (2015). Linoleic acid (Omega
6) is the most abundant fatty acid in all studied species. It is the main aromatic compound of most
mushrooms, known as mushroom alcohol. Linoleic acid may contribute to the flavor of mushrooms
and is necessary for the human body (Ribeiro et al., 2009). Agaricus arvensis had the highest content

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of linoleic acid, the lowest content of MUFA and high contents of SFA and PUFA. This result agreed
with previous ones published by Barros et al. (2007). For A. arvensis, C. cibarius, C. cornucopioides
and L. deliciosus, linoleic acid contents were lower than that found by Barros et al. (2007, 2008).
However, H. repandum had higher contents of SFA and PUFA and lower content of MUFA than
those reported by Fernandes et al. (2013). Other fatty acids were also detected but in low amounts. H.
erinaceus had the highest content of docosahexaenoic acid (DHA or Omega-3). The highest content
of stearic acid (C18:0) was observed in L. deliciosus; it was the main fatty acid present in the genus
Lactarius as it has been showed by both Fernandes et al. (2013) and Vieira et al. (2014). Compared to
Barros et al. (2007) study, L. deliciosus had the highest content of palmitic acid and the lowest stearic
acid. A remarkable content of homo-γ-linolenic acid (C20:3- ω6; precursor of the Omega 3 family)
was found in C. lutescens, C. cornucopioides and H. repandum. The sample of H. repandum had
the highest content of PUFA due to the high contribution of linoleic acid and homo-γ-linolenic acid.
These two PUFA acids exerting a powerful regulatory role in the treatment of diabetic neuropathy
and in cardiovascular disease prevention (Coste et al., 2004). Further studies should be conducted
to evaluate other components such as vitamins (i.e., vitamin D2), and sterols (i.e., ergosterol) which
have been linked to antioxidant, anticancer, hepatoprotective, antiallergic, antimicrobial, and antiviral
activities (Ramos et al., 2019; Hu et al., 2021).
    Phenolic compounds, among the most well-known are flavonoids, were the most important groups
of secondary metabolites due to their powerful and essential antioxidant properties for human nutrition.
The results of phenolic (TP), flavonoids (TF) and tannins (TT) contents in methanolic extract of
the analyzed mushrooms were presented in Fig. 2. These contents varied significantly according to
Species. The TP content varied considerably from 2.49 to 11.25 mg GAE g-1 dw (overall mean: 5.41 ±
3.36 mg GAE g-1 dw), the TF content varied from 3.05 to 19.34 mg GAE g-1 dw (overall mean: 6.46
± 4.51 mg GAE g-1 dw), whereas the TT content varied slightly from 15.23 to 26.84 mg GAE g-1 dw
(overall mean: 21.29 ± 3.66 mg GAE g-1 dw). The analysis of (TP), (TF) and (TT) compound profiles
showed that H. erinaceus and R. flavescens were the richest species in these compounds.
    Hericium erinaceus had the highest average content of (TP) and the lowest average content of
(TT), while, the highest and the lowest average of (TF) content were recorded in R. flavescens and P.
pulmonarius, respectively. The (TP) average content of H. repandum was lower than that reported by
Keleş et al. (2011) (2.63 mg GAE g-1 dw versus 4020 g of GAE kg-1 dw). According to Charumathy
et al. (2017), the contents of (TP) and (TF) in hot water extract in H. erinaceus were higher than
that showed in methanolic extract of this study. Compared to Onbaşili et al. (2015), L. deliciosus
had higher (TT) content, lower (TP) content and almost double content of (TF). Mushroom samples
of C. cibarius, C. cornucopioides and L. deliciosus had higher (TP) compounds and (TF) contents
in comparison with data reported by Palacios et al. (2011). The present study confirms the results
reported by Charumathy et al. (2017) and Schillaci et al. (2013) showing that H. erinaceus and the
genus Pleurotus had excellent organoleptic qualities, high nutritional value and potential applications
in the pharmaceutical and medicinal sectors.




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     Fig. 2 - Content of total phenolic (TP), flavonoids (TF) and tannin (TT) compounds in Tunisian wild edible
     mushrooms (mg g-1 dw).

Principal Component Analyses (PCA)
The Principal Component Analyses (PCAs) were performed separately on both mineral and
biochemical variables. The PCA was performed on mineral variables and allowed to separate the
mushrooms species into four groups. The first group is composed of A. arvensis, H. erinaceus, R.
flavescens, P. pulmonarius, C. cornucopioides and C. cibarius. The three other groups are composed
each single species (L. deliciosus, C. lutesens or H. repandum) (Fig. 3). According to PCA, the four
groups are separate in relation to two origins (Jendouba and Beja). Just mushrooms from Beja were
represented only in the first group.
   Mushrooms of the first group showed heterogenous ecological categories (Em, St, Sh, Pn) whereas
the mushrooms of groups 2, 3 and 4 were all Em. According to mushroom species mineral composition,
the two Principal Components (Axes) explained 66.68% (Fig. 3).
   The scaling 1 (species scaling) biplot showed a gradient from left to right, starting with the first
single-species group (L. deliciosus) which displays the highest Fe, Cr, ash and Mn values, and the
lowest K value. The second group of species including H. erinaceus, P. pulmonarius, R. flavescens,
A. arvensis, C. cornucopioides and C. cibarius which had intermediate values for almost all mineral
measured variables. This group could be subdivided into three subgroups: the first subgroup formed by
H. erinaceus, P. pulmonarius and R. flavescens; they were not spread out by the variables contributing
to axes 1. Within this subgroup, the only two lignicolous saprotroph species (living on tree trunk), H.


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   Fig. 3 - Graphical projection of the nine wild edible mushrooms species based on PCA of 11 mineral variables
   (Ash, K, P, Mg, Ca, Na, Fe, Cu, Zn, Mn, Cr).


erinaceus and P. pulmonarius, had null or even trace values of Fe, Cu, Zn, Mn and Cr, and the highest
Mg values and an average values Na. Moreover, H. erinaceus had the highest K and Ca values and
an average value in P, while P. pulmonarius had average values of K, P, and Ca. The second subgroup
formed by C. cibarius and C. cornucopioides had almost similar values of all the measured chemical
minerals probably because they belong to the same taxonomic family. The last subgroup formed by
A. arvensis had the highest K value and the lowest Ca and Na values, whereas the third group formed
by C. lutesens had the highest Na and Ca values and the lowest P and Mg values. The fourth group
formed by H. repandum had high K value, and low Cr and Mn values.
    The scaling 2 biplot (variable scaling) showed that the variables were organized into groups.
The lower left part showed that the four ash variables, Cu, Mn and Cr were very highly, positively
correlated, and they were very highly, negatively correlated with K. The two variables Ca and Na had
a higher contribution than average in comparison to the other mineral variables; they were highly,
negatively correlated with both Zn and P. These latter variables as well as Ca and Na were positively
related to K and Mg. Fe and Zn content had nearly orthogonal arrows, indicating a correlation close
to 0.
    The PCA applied on biochemical composition profiles allowed to distinguish four mushroom
species groups: 1) L. deliciosus and C. cibarius; 2) R. flavescens, A. arvensis, H. erinaceus and P.
pulmonarius; 3) C. lutesens and C. cornucopioides and 4) H. repandum (Fig. 4). Concerning PCA
applied on mushroom species biochemical composition, the two first PCAs explained 53.44% (Fig.
4).
    The scaling 1 biplot showed also a gradient from left to right, starting with the first group formed by
L. deliciosus and C. cibarius having almost similar (C20:0), (C22:0) and (C16:1) fatty acids contents.
The second group of species formed by R. flavescens, A. arvensis, H. erinaceus and P. pulmonarius
having intermediate values of mannitol, (C20: 3n-6), lipids, PUFA, energy and the lowest values of
carbohydrates and (C20:1) fatty acid. Hericium erinaceus had average values in total lipids, while
P. pulmonarius had the highest SFA in particular docosanoic acid (C22:0) and lignoceric (C24:0).
Both samples H. erinaceus and P. pulmonarius have the lowest values in sugars, fatty acids C20:2n,
C20:3n, C20:4n, C21:0 and average values in energy, stearic and linoleic acid. A third group formed
by C. cornucopioides and C. lutesens having the highest trehalose, (C17:0) and SFA contents, and

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    Fig.4 - Graphical projection of the nine wild edible mushrooms species based on PCA of 30 biochemical variables
    (Total nitrogen, Proteins, Carbohydrates, Mannitol, Trehalose, Total sugar, Lipids and fatty acids).



the lowest (C18: 2), PUFA and lipids contents probably because they belong to the same taxonomic
family and same ecological category (ectomycorrhizal species). The fourth group formed by H.
repandum had high (C16:0) fatty acid content, and low (C20: 2n-6) content.
   The species A. arvensis, H. erinaceus, R. flavescens, C. lutesens, C. cibarius, and C. cornucopioides
are not spread out by the variables contributing to axes 1 and 2. The lower left part, of the scaling 2
biplot, shows that total sugars and mannitol were very highly, positively correlated, and that these
two variables were very highly, negatively correlated with carbohydrates. Furthermore, the group of
variables formed by energy, lipids and PUFA were very highly, positively correlated, and that these
variables were very highly, negatively correlated with SFA, trehalose and (C17:0). The group of
variables SFA and (C17:0) contributed the most to the explained variance by the two axes. These two
variables were highly correlated with lipids and PUFA contents group. The carbohydrates and MUFA
contents are positively correlated with these two groups.
   Total nitrogen, proteins and stearic acid (C18:0) displayed nearly orthogonal arrows to PCA1.
As far as that goes, lipids and PUFA displayed nearly orthogonal arrows to PCA2. These variables
indicating a correlation almost nil close to 0. While, other variables such as (C14:0), (C20:1), (C24:1)
and (C24:0), displayed a shorter arrow, and were less important for the ordination of mushroom
species.

Co-inertia analysis (CoIA)
Co-inertia analysis (CoIA) confirms the results of mineral and biochemical variables obtained by PCAs.
The mineral and biochemical profile study performed by the PCA and CoIA analysis by confirming
the results presented in Table 2. Figure 5 is a global view of the effect of all mineral and biochemical
variables based on the results of the principal component analysis (Fig. 3 and 4). The upper right-
hand plot (normed species scores) shows the positions of species on the co-inertia axes using mineral
composition (origins of the arrows) and biochemical composition (arrowheads) co-inertia weights.
The profile of the Co-inertia analysis shows the rapprochement of the chemical composition of the
nine species of mushrooms which can be grouped as follows: (i) H. erinaceus, P. pulmonarius, R.


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flavescens, L. deliciosus, and A. arvensis, (ii) C. cibarius and C. lutescens, (iii) C. cornucopioides
and (iv) H. repandum. The first group has the shortest arrows, showed the best concordance between
the two projections of the point of the two variable groups (mineral and biochemical). The lower
right-hand pair of plots showed the contributions of these groups of variables. Zn content correlates
positively with proteins, content Na and Ca negatively with proteins. P, Mg and K contents are all
negatively correlated with total sugars, mannitol, trehalose, lipids and energy since these variables
have higher values downstream, and positively correlated with fatty acids (C18: 0), (C18: 2), (C24:




   Fig. 5 - Graphic projection of the nine wild edible mushrooms species based on co-inertia analysis of 41 mineral
   and biochemical variables.




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0) and SFA, which increase downstream. According to the results of co-inertia analysis, P, Mg and
Ca contents on the one hand, and proteins, total sugars on the other hand, having the longest vectors
contributed more to the nutritional value of species.

Conclusion

This study is the first report on the chemical composition of nine wild edible mushrooms originated
from Tunisia. The analyzed mushrooms showed low amounts in fat and calories contents and
high levels of protein, carbohydrates, minerals and, phenolics flavonoids and omega-3 fatty acids.
Interestingly, H. erinaceus appeared to be the most nutritious mushroom; it had the highest amount of
carbohydrates, proteins, oleic and docosahexaenoic (DHA) acids, phenolic compounds and flavonoids
as well as minerals mainly K, Mg and Ca. Therefore, it could be recommended for the obese, diabetics,
and people with cardiovascular diseases. Despite the nutritional and therapeutic importance of H.
erinaceus, this species remains poorly known in Tunisia and is not protected. Therefore, special
awareness program of the importance of this species is needed for mycologists, foresters and general
public. Furthermore, P. pulmonarius had high carbohydrate content and the lowest lipid content with
the lowest caloric value which make them suitable for obesity diet plan. In conclusion our findings
support the development and the utilization of edible mushrooms as a complete food ingredient
or sustainable alternative material, because of their composition, their safety, and health benefits
(Chang and Wasser, 2017). Because of their availability and their low carbon footprint, they could
contribute to food security and rural sustainable development. Hence, there is urgent need to a holistic
approach for a sustainable management of such a valuable bio-resource. This would include: (i) the
conservation of these natural resources and their habitats, (ii) commercial exploitation by improving
culture techniques, and (iii) the determination of their biotechnological potential.

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