Mineral Composition of Three Popular Wild Mushrooms from Poland

The region of Warmia and Mazury is characterized by the special diversity and richness of its natural environment, including large forest complexes, where wild mushrooms are commonly collected and consumed. This study aimed to examine the differences in mineral content (calcium, magnesium, sodium, potassium, iron, zinc, copper, manganese) of three species of mushrooms collected in north-eastern Poland. The research material consisted of dried samples of king bolete (Boletus edulis), bay bolete (Boletus badius), and chanterelle (Cantharellus cibarius) collected in the region of Warmia and Mazury. The content of the above-mentioned elements in mushroom fruit bodies was determined using the flame atomic absorption spectrometry (acetylene-air flame) and the emission technique (acetylene-air flame) for sodium and potassium. For the majority of micro- and macroelements, the studies confirmed the presence of significant differences in their content, depending on the species of fungi. The studied mushrooms cover a significant percentage of daily demand for many of the minerals. This concerns mainly copper, zinc, and potassium, although none of the species was a good source of calcium and sodium. Among the analyzed mushrooms, chanterelle is the best source of most minerals.


Introduction
The province of Warmia and Mazury is one of the least degraded areas in Poland, taking into account the natural environment. The whole province is located within the so-called "green lungs", which covers the cleanest regions of the country. Forests are the natural wealth of the Warmia and Mazury region. The properties of woods (appropriate substrate, age of stands) favor the occurrence of undergrowth, e.g., mushrooms. According to the data from the Statistical Yearbook of Forestry, in 2018, the purchase of mushrooms amounted to 122 tons, which ranked the region 6th place in the country in terms of the amount of fungal raw material obtained [1,2]. The most valued mushrooms include Boletus edulis, Boletus badius, and Cantharellus cibarius.
Boletus edulis (King Bolete) is considered one of the most appreciated species of wild edible mushrooms and is a popular food throughout the world [3,4]. It is very popular mainly because of its aroma, texture, and the presence of nutrients as well as biologically active compounds, which determine its nutritional and medicinal properties [5][6][7]. The fruiting bodies of the bay boletus (Boletus badius) are tasty, which makes them widely used in gastronomy. Furthermore, they contain many valuable substances, such as antioxidants (anisole, BHT toluene, tocopherol) and rare metals (manganese, zinc, selenium). Chanterelles (Cantharellus cibarius) are a desirable trade item because of their attractive taste, durability during transport and storage, and the fact that the sporocarps are rarely affected by insect larvae. In addition, this mushroom is rich in natural vitamin C and contains high levels of potassium and vitamin D [8].
Currently, knowledge of the mineral content in edible mushrooms is relatively extensive. In many countries (South Africa, Turkey, Greece, Serbia, China, and others), numerous studies have been conducted on the mineral composition of various species of fungi to more accurately estimate their nutritional and health value and the mechanism of accumulation of individual components [22,[27][28][29][30][31][32]. In Poland, large-scale analyses have been conducted, among others, by Falandysz, whose numerous reports concern a wide spectrum of elements determined in many species of mushrooms collected in various sites in Poland and around the world [33][34][35][36]. Siwulski, Mleczek, and Rzymski have also carried out numerous studies on cultivated and wild mushrooms from non-contaminated and contaminated areas [26,[37][38][39][40][41].
Mushrooms owe their ability to accumulate micro-and macroelements to the specific structure of mycelium: the exposed surface of vegetative cells and large hyphae surfaces [42]. They are able to store minerals in large quantities even exceeding concentrations found in the medium in which they have grown [43,44]. The uptake of elements considered physiologically essential to mushrooms (K, P, Mg, Mn, Cu, Ca, Na, Zn) by mycelia and their deposition in fruiting bodies is species dependent [45]. Therefore, unlike vascular plants, mushrooms are able to accumulate high concentrations of minerals, even when growing in soils with low metal contents [35]. In this way, they become a particular rich source of minerals. In Kalač's opinion, the levels of elements in wild mushrooms significantly increase with the increasing age of mycelium and extended time between the fructifications [46]. Variability in the chemical composition of mushrooms within a species is greater than that of plants. Since each individual fruit body can result from the crossbreeding of different hyphae and so presents a distinct genotype, the mineral contents in a mushroom species vary widely. Except for the species, the essential factors affecting trace element level in fruit bodies are the level of substrate composition, soil, pH, enzyme activity, and local pollution with trace elements.
With this background, the aim of the study was to determine the accumulation level of minerals of three species of mushrooms collected from the Warmia and Mazury region as well as to evaluate mushroom contribution to the daily intake of the studied bioelements.

Material and Methods
The research material consisted of dried samples of king bolete (Boletus edulis) and bay bolete (Boletus badius), which were purchased from July to November from a company in the region of Warmia and Mazury. In the same period, samples of fresh chanterelles (Cantharellus cibarius) were obtained from mushroom selling stands, located in the province of Warmia and Mazury (Figure 1). Chanterelles were selected, cleaned (removal of impurities in the form of needles, pebbles, tree elements), and dried in a special drier according to PN-68/A-78508 [47] under laboratory conditions at 60 • C to obtain a constant weight. In total, 20 samples of each species of mushroom were collected (2 samples of each species per month), where each was divided into three-unit samples. The results in the table are presented as mean values of all samples (in total n = 60 per each species). In addition, each series of mineralized samples included a parallel reagent test.

Samples Preparation
Prepared unit samples of mushrooms were ground to a powder and weighed into glass tubes in an amount of about 1 g. The remainder was stored in closed polyethylene bags at 18 °C.

Samples Mineralization
Mineralization of samples was carried out according to the method described by Whiteside & Miner (1984) [48]. The weighed samples were mineralized using the "wet" method in a mixture of nitric and perchloric acids (3:1). The analysis was performed in an aluminum electric heating block with temperature programming (VELP DK 20-VELP Scientifica, Usmate, Italy), within 5-6 h, gradually increasing the temperature from 120 °C to 200 °C. The obtained colorless mineralizate was quantitatively transferred into 50 cm 3 volumetric flasks and filled up to the mark with deionized water. Reagent samples were prepared along with the samples.

Determination of Copper, Manganese, Iron, Zinc, Calcium and Magnesium
In previously prepared mineralizates, the contents of copper (Cu), manganese (Mn), iron (Fe), zinc (Zn), calcium (Ca), and magnesium (Mg) were determined by flame atomic absorption spectrometry (acetylene-air flame). The analyses were performed using an atomic absorption spectrometer-iCE 3000 SERIES-THERMO (Thermo-Scientific, Hemel Hempstead, Hertfordshire, UK), equipped with a GLITE data station, background correction (deuterium lamp) and appropriate cathode lamps. For the calcium determination, a 10% aqueous solution of lanthanum chloride was added to all measured solutions in a quantity ensuring a final La +3 concentration of 1%. The determination was carried out at wavelengths (in nm) for individual minerals: 324.8 copper; 279.5 manganese; 248.3 iron; 213.9 zinc; 285.2 magnesium; 422.7 calcium.

Determination of Sodium and Potassium
The sodium and potassium contents (Na and K) were determined by the emission technique (acetylene-air flame). The analyzes were performed using the atomic absorption spectrometer iCE 3000 SERIES-THERMO (Thermo-Scientific, Hemel Hempstead, Hertfordshire, UK), equipped with a GLITE data station, operating in an emission system. The determination was carried out at wavelengths (in nm) for individual elements: 589.0 sodium; 766.5 potassium.

Samples Preparation
Prepared unit samples of mushrooms were ground to a powder and weighed into glass tubes in an amount of about 1 g. The remainder was stored in closed polyethylene bags at 18 • C.

Samples Mineralization
Mineralization of samples was carried out according to the method described by Whiteside & Miner (1984) [48]. The weighed samples were mineralized using the "wet" method in a mixture of nitric and perchloric acids (3:1). The analysis was performed in an aluminum electric heating block with temperature programming (VELP DK 20-VELP Scientifica, Usmate, Italy), within 5-6 h, gradually increasing the temperature from 120 • C to 200 • C. The obtained colorless mineralizate was quantitatively transferred into 50 cm 3 volumetric flasks and filled up to the mark with deionized water. Reagent samples were prepared along with the samples.

Determination of Copper, Manganese, Iron, Zinc, Calcium and Magnesium
In previously prepared mineralizates, the contents of copper (Cu), manganese (Mn), iron (Fe), zinc (Zn), calcium (Ca), and magnesium (Mg) were determined by flame atomic absorption spectrometry (acetylene-air flame). The analyses were performed using an atomic absorption spectrometer-iCE 3000 SERIES-THERMO (Thermo-Scientific, Hemel Hempstead, Hertfordshire, UK), equipped with a GLITE data station, background correction (deuterium lamp) and appropriate cathode lamps. For the calcium determination, a 10% aqueous solution of lanthanum chloride was added to all measured solutions in a quantity ensuring a final La +3 concentration of 1%. The determination was carried out at wavelengths (in nm) for individual minerals: 324.8 copper; 279.5 manganese; 248.3 iron; 213.9 zinc; 285.2 magnesium; 422.7 calcium.

Determination of Sodium and Potassium
The sodium and potassium contents (Na and K) were determined by the emission technique (acetylene-air flame). The analyzes were performed using the atomic absorption spectrometer iCE 3000 SERIES-THERMO (Thermo-Scientific, Hemel Hempstead, Hertfordshire, UK), equipped with a GLITE data station, operating in an emission system. The determination was carried out at wavelengths (in nm) for individual elements: 589.0 sodium; 766.5 potassium.
The selected concentrations of the standard solutions of individual micro-and macronutrients formed the measuring range of the analytical method used in the experiment. To determine the equation of the relationship between the measure of the signal generated by the device and the content of the analyte in the sample, calibration curves for individual elements were prepared. For this purpose, three parallel absorbance measurements were made for each standard solution, starting with measurements for the blank. Calibration curves for copper, manganese, iron, zinc, magnesium, calcium, sodium, potassium, cadmium, and lead were created based on the average absorbance values. The equations of the straight lines describing the curves were determined by the method of least squares (linear regression) according to the formula: where a denotes slope coefficient (directional) of the straight line and b the straight line shift coefficient.
Linearity, i.e., the range of the content of the analyte, for which the output signal of the measuring device is proportional to this content, was determined based on the regression coefficient (R 2 ). The value of this parameter should meet the condition R 2 ≥ 0.999.
The accuracy of the method was checked based on an examination of the certified reference material INCT-TL-1 tea leaves, which were analyzed five times.

Statistical Methods
The obtained results of the content of macro-and micromineral elements in mushrooms were subjected to statistical analysis. The computer package Statistica 13.1 (StatSoft Inc., Tulsa, OK, USA) was used for calculations and MS Excel was used to present the results.
First, the measures of descriptive statistics were calculated, such as arithmetic mean, median, variance, standard deviation, and range, to characterize the distribution of values of the examined features. Then, the Kolmogorov-Smirnov test was performed to check the compliance of the distribution of the examined feature with the normal distribution and the median series test to check if the values have a random distribution.
Non-parametric tests were used to compare the average levels of feature values between samples due to a small number of samples, no normal distribution for most samples, or no random distribution of most samples.
To study the differences between the two independent groups, the U-Mann-Whitney test was used, which is equivalent to the parametric t-test for independent samples. This verifies the null hypothesis regarding "equality of means in two independent samples" against the alternative hypothesis saying that these means are not equal. When comparing average levels in several independent samples, the Kruskal-Wallis test was used to verify the null hypothesis assuming "equality of means in the tested samples" against the alternative hypothesis saying that these means are not equal. Using both tests, the significance level was p = 0.05. Fungi species were the assumed grouping factor. The results of the experiment and their statistical interpretation are presented in Table 1.

Results
The mean content of minerals in the studied species of mushrooms is presented in Table 1. The Cu content in the three species of mushrooms is varied. The highest amount of Cu at 48.4 mg/kg d.w.
was observed in C. cibarius. Much lower levels were determined for B. edulis (23.41 mg/kg d.w.) and B. badius (29.7 mg/kg d.w.). Fe content turned out to be varied between species. The highest Fe content was determined for C. cibarius (58.9 mg/kg d.w.), followed by B. edulis (48.9 mg/kg d.w.). The lowest content of this element was observed in the B. badius (38.8 mg/kg d.w.). The content of Mn in C. cibarius fruit bodies (23.7 mg/kg d.w.) was significantly higher than in B. edulis and B. badius (11.3 and 11.9 mg/kg d.w., respectively). In general, B. edulis, B. badius, and C. cibarius differed significantly in terms of Zn content.
The highest Ca content, almost three times higher than in B. edulis (75.3 mg/kg d.w.) and almost four-fold than in in B. badius (54.7 mg/kg d.w.), was determined in C. cibarius and equaled 211 mg/kg d.w. The highest content of this element was determined in B. badius (163 mg/kg d.w.), and the lowest was in C. cibarius (112.7 mg/kg d.w.), while B. edulis contained 158 mg/kg d.w. of Zn. Significant differences in K content were found between all the analyzed fungi. The highest content of this element was determined for C. cibarius (46,024 mg/kg d.w.), followed by B. badius (36,001 mg/kg d.w.) and B. edulis (29,136 mg/kg d.w.). The Mg contents for B. edulis and B. badius were 566 and 526 mg/kg d.w., respectively. The content of Mg in C. cibarius was significantly higher than in the other mushrooms (842 mg/kg d.w.). The sodium contents appear to be varied, where more than four times lower contents than in other species (142 mg/kg d.w.) were determined in C. cibarius. Significant differences between the results determined for B. edulis (653 mg/kg d.w.) and B. badius (568 mg/kg d.w.) were also indicated. Table 2 shows the calculated coverage of the daily demand for selected micro-and macroelements in the case of the consumption of 25 g of dried mushrooms, which can be equivalent to 250 g of fresh mushrooms. In relation to the recommended daily allowance (RDA) of Cu, the studied mushrooms can be a rich source of this element in the human diet. Daily demand is met to the highest degree after consumption of C. cibarius. It covers as much as 134-173% of the demand in children and adolescents and 173% in adults. The lowest percentage of daily demand for Cu is covered by B. edulis-children and adolescents: 65.0-83.6% and adults: 83.6%. The coverage of the daily requirement after consumption of B. badius by children and adolescents is 82.6-106% and by adults: 106%. The largest percentage of the demand for Fe in children, adolescents and adults is covered by C. cibarius The studied mushrooms are characterized by a high K content. By consuming C. cibarius, the adequate intake is met in 32.9-47.9% among children and adolescents and in 32.9% in adults. The lowest degree of coverage was provided by B. edulis (children and adolescents: 20.8-30.4%; adults: 20.8%), while for B. badius, 25.7-37.5% coverage (children and adolescents) and 25.7% coverage (adults) was obtained. The demand for Mg can be realized the most after consuming C. cibarius. The values obtained for children and adolescents were: 5.13-8.77% and for adults: 5.01-6.79%. Recommended daily intake of Mg, in the case of B. edulis was 3.45-5.89% (children and adolescents) and 3.37-4.56% (adults). The values obtained for the B. badius were 3.21-5.48% and 3.13-4.24%, respectively. The demand for Na does not exceed 1.10% for the consumption of each of the studied species of fungi. The highest value was obtained for B. edulis: 1.09-1.26, then for B. badius: 0.95-1.09%. Daily adequate intake (AI) of Na is realized in the case of C. cibarius only in 0.24-0.27%.   [28] in mushrooms from Turkey, while higher contents were determined by Kolundžić et al. (2017) [29] in C. cibarius from Serbia: 60 mg/kg d.w.  [50] observed 18.7 mg/kg d.w. Cu in mushroom species growing near heavily trafficked road in Poland. Cu content in the B. badius ranging from 9.70-13.6 mg/kg d.w. was observed by Mleczek et al. (2013) [37], who were examining fungi from five regions of Poland.   [43] analyzed species obtained from unpolluted areas of acidic sandy soils located in the Wielkopolska region and from areas where alkaline flotation tailings from copper production had been stored. They received results ranging from 14-17 mg/kg d.w. and 9-13 mg/kg d.w., respectively. These values differ from those obtained in the current study.   [42] [17] and Ayaz et al. (2011) [54] reported the value ranges of 100 to 180 mg/kg d.w. Brzezicha-Cirocka et al. (2016) [34] determined Fe at the level of 170-520 mg/kg d.w. in C. cibarius from Morąg. In this species analyzed in Turkey and Serbia, 588.5 and 234 mg/kg d.w., respectively, were recorded [28,29].  [43] determined a lower Fe content in the B. badius in Wielkopolska region: 24-29 mg/kg d.w. and 28-35 mg/kg d.w. from a polluted area of the province of Lower Silesia.   [33] determined the amount of this mineral at 82.5 mg/kg d.w. in the mushrooms from Kętrzyn. Mleczek et al. (2013) [37]  The results obtained in this study, regarding eight selected minerals contained in Boletus edulis, Boletus badius and Cantharellus cibarius from the Warmia and Mazury region do not differ much from the results presented by other authors [27][28][29][30][31][32][33][34]. However, undoubtedly, chemical composition and properties of the growing substrate, as well as the contamination of the environment determine element composition in mushrooms. The experiment confirmed the occurrence of significant differences in content of studies micro-and macroelements depending on the species of fungi. Also, the region of collecting mushrooms had an impact on the content of some minerals as there were observed differences in the values for mushrooms growing in different parts of the world (South Africa, China, Turkey, Serbia).

Brzezicha
To understand the role of soil geochemistry and soil pollution in accumulation of minerals in mushrooms fruiting bodies several studies have been conducted in Europe [56][57][58]. A few recent ones have shown that edible mushrooms growing in unpolluted areas can accumulate Cd and Hg at much higher levels as in soil substrate, while some species hyper accumulate As, Cd, Hg, and Pb in the mining and geo-anomalous areas [22,[59][60][61][62]. Research conducted by Pająk (2016) [53], who analyzed fungi collected from theŚwierklaniec Forest District, located near a metallurgical plant, which is (Huta Cynku "MiasteczkoŚląskie" (HCMŚ)) confirmed the high accumulation of metals, including Zn mushrooms growing in highly polluted areas, and thus the possibility of using it as a bioindicator of the degree of contamination of the natural environment with heavy metals. Mleczek et al. (2016) [43] showed the effect of substrate purity on the accumulation of individual minerals by mushroom fruiting bodies. The results revealed the existence of relationships between the content of elements and low-molecular-weight organic acids. The considerably higher content of the minerals in mushrooms growing on flotation tailings than in soil was related with higher acid contents.

Conclusions
Assuming a 100% bioavailability (in fact significantly lower as determined by many factors) of the studied minerals consumed with the analyzed mushrooms, the current study showed that these raw materials cover a significant percentage of the daily demand for many of the micro-and macroelements tested. This applies mainly to Cu, Zn, and K, although none of the species is a good source of Ca and Na. Among the mushrooms studied, Cantharellus cibarius is the best source of most minerals, including Cu, Fe, Mg, Ca, and K, although this requires further research to confirm the persistence of the observed trend.
The presence of minerals in human nutrition is very important. These elements are supplied to the body with food in the right proportions and determine the effectiveness of many life processes. The presence of a wide range of micro-and macroelements in edible mushrooms has prompted researchers to conduct numerous studies of the nutritional value of these raw materials. Moreover, the specificity of mushrooms and their bioindication abilities may constitute important criteria for determining their health quality and the degree of environmental pollution in their local area. The consumption of wild edible mushrooms is increasing worldwide. The knowledge and documentation of baseline mineral composition of wild growing mushrooms is essential to maintain nutritional needs, especially for many people who are vegetarian or maintain a vegan diet.
Author Contributions: M.G., participation in chemical analyses, interpretation and presentation and discussion of research results, statistical analysis; R.P.-F., participation in preparation of discussion of results, collection of references, corresponding author. M.G. and R.P.-F. contributed equally to this paper. All authors have read and agreed to the published version of the manuscript.
Funding: Project financially supported by Minister of Science and Higher Education in the range of the program entitled "Regional Initiative of Excellence" for the years 2019-2022, Project No. 010/RID/2018/19, amount of funding 12.000.000 PLN.

Conflicts of Interest:
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.