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Metallic elements (Ca, Hg, Fe, K, Mg, Mn, Na, Zn) in the fruiting bodies of Boletus badius
Food Chemistry 200 (2016) 206–214
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Metallic elements (Ca, Hg, Fe, K, Mg, Mn, Na, Zn) in the fruiting bodies of Boletus badius Anna K. Kojta ⇑, Jerzy Falandysz ´ sk, 63 Wita Stwosza Str., PL 80-952 Gdan ´ sk, Poland Laboratory of Environmental Chemistry & Ecotoxicology, University of Gdan
a r t i c l e
i n f o
Article history: Received 14 August 2014 Received in revised form 21 December 2015 Accepted 1 January 2016 Available online 2 January 2016 Keywords: Europe Food Foraging Forest Heavy metals Organic food
a b s t r a c t The aim of this study was to investigate and compare the levels of eight metallic elements in the fruiting bodies of Bay Bolete (Boletus badius; current name Imleria badia) collected from ten sites in Poland to understand better the value of this popular mushroom as an organic food. Bay Bolete fruiting bodies were collected from the forest area near the towns and villages of Ke˛trzyn, Poniatowa, Bydgoszcz, Pelplin, _ Włocławek, Zuromin, Chełmno, Ełk and Wilków communities, as well as in the Augustów Primeval Forest. Elements such as Ca, Fe, K, Mg, Mn, Na and Zn were analyzed by inductively coupled plasma atomic emission spectroscopy (ICP-OES), and mercury by cold vapor atomic absorption spectrometry (CV-AAS). This made it possible to assess the nutritional value of the mushroom, as well as possible toxicological risks associated with its consumption. The results were subjected to statistical analysis (Kruskal–Wallis test, cluster analysis, principal component analysis). Ó 2016 Elsevier Ltd. All rights reserved.
1. Introduction The fruiting bodies of edible wild mushrooms are popular in many countries (Kottke, Qian, Pritsch, Haug, & Oberwinkler, 1998) and demand for this kind of organic food is growing worldwide. It is assumed that the fruiting bodies of mushrooms are naturally rich in different compounds (Rudawska & Leski, 2005a; Kalacˇ, 2010). Consumption of wild and cultivated mushrooms is increasing from year to year (Dimitrijevic et al., 2015). In Poland, picking wild mushrooms for home use or resale is common. Export of the most common mushroom species from Poland is 3400 metric tons per year and include Common Chanterelle (Cantharellus cibarius), King Bolete (Boletus edulis), Orange Birch Bolete (Leccinum versipelle), Brown Birch Scaber Stalk (Leccinum scabrum), Bay Bolete (Boletus badius), Red Cracking Bolete (Xerocomus chryzenteron) and Yellow Cracking Bolete (Xerocomus subtomentosus) (GUS, 2013; Falandysz & Chwir, 1997; Falandysz, Kawano, S´wieczkowski, Brzostowski, & Dadej, 2003a). On the Polish territory, Bay Bolete (B. badius) (Fr.) Fr. is a commonly found in coniferous forests, but rarely in mixed forests. It belongs to the ectomycorrhizal species (Gumin´ska & Wojewoda, 1985).
⇑ Corresponding author. E-mail address: [email protected] (A.K. Kojta). http://dx.doi.org/10.1016/j.foodchem.2016.01.006 0308-8146/Ó 2016 Elsevier Ltd. All rights reserved.
This study is part of an ongoing study to examine the mineral composition of fruiting bodies of B. badius, demonstrate possible variations in mineral composition of morphological parts as well as similarities and differences in the content of fruiting bodies collected from different sites across Poland. 2. Material and methods Samples of Bay Bolete B. badius (Fr.) Fr. (current name Imleria badia (Fr.) Vizzini) (Species Fungorum, 2015) fruiting bodies were collected in 2000 from distant forests of Pelplin, Ełk, Ke˛trzyn, _ Augustów, Chełmno, Bydgoszcz, Włocławek, Zuromin, Poniatowa and Wilków in Poland (Fig. 1). The caps and stipes were separated and dried at room temperature for 1–3 days, and then at 85 °C in a laboratory dryer for 48 h to constant weight. The dried fruiting bodies were pulverized in a porcelain mortar and stored in sealed polyethylene bugs under dry and clean conditions. All the reagents used in this study were of analytical reagent grade, unless otherwise stated. Double distilled water was used for the preparation of the solutions. Prior to the analysis of the metallic elements, sub-samples (300–400 mg) were wet digested with 7 mL of concentrated nitric acid (SuprapureÒ, Merck, Darmstadt, Germany) in closed polytetrafluoroethylene (PTFE) vessels in a microwave oven (MARS 5 of CEM Corp., Matthews, NC, USA) under the following conditions: power – 1.2 kW; ramp 1–10 min; temp. 1–100 °C; hold 1–10 min, ramp 2–10 min; pressure – 800 psi; temp. 2–100 °C; hold 2–10 min; cool down: 5 min, and
A.K. Kojta, J. Falandysz / Food Chemistry 200 (2016) 206–214
207
_ Fig. 1. Location of sampling sites in Poland (1 – Pelplin, 2 – Ke˛trzyn, 3 – Chełmno, 4 – Ełk, 5 – Augustów Primeval Forest, 6 – Bydgoszcz, 7 – Zuromin, 8 – Włocławek, 9 – Poniatowa, 10 – Wilków).
then diluted to 25 mL with deionized water (Chudzyn´ski & Falandysz, 2008; Frankowska, Ziółkowska, Bielawski, & Falandysz, 2010). The analysis of metallic elements content in fruiting bodies other than mercury was performed using inductively coupled plasma atomic emission spectroscopy (IC P-OES; Optima 2000, Perkin Elmer, USA) (Brzostowski, Falandysz, Jarzyn´ska, & Zhang, 2011). Determination of total Hg was performed using coldvapour atomic absorption spectroscopy (CV-AAS) by direct sample thermal decomposition coupled with gold wool trap for Hg and, following desorption, quantitative measurement at 253.7 nm. The analytical instrument used was a mercury analyzer (MA-2000), Nippon Instruments Corporation, Tatsuki, Japan) equipped with an auto-sampler, and operated in low (3–20 ng Hg per sample) or high (25–150 mg Hg per sample) modes, as appropriate (Falandysz, Zhang, Wang, Krasin´ska, et al. 2015; Falandysz, Zhang, Wang, Saba, et al. 2015; Jarzyn´ska & Falandysz, 2011). A standard solution of 1.0 mg Hg mL 1 was obtained from the 10 mg mL 1 stock solution. Blank and 3, 5, 10, 15 and 20 lL (low mode) and 25, 50, 100, 150 and 200 lL (high mode) of 1.0 mg mL 1 Hg standard solutions were injected into the analyzer for the construction of calibration curves weekly. Procedures for the determination of mercury and the other metallic elements were validated through the analysis of certified references materials such as CTA-OTL-1 (Tobacco Leaves), INCTTL-1 (Tea Leaves) and CS-M 1 (dried fruiting bodies of mushroom Cow Bolete Suillus bovinus) produced by the Institute of Nuclear Chemistry and Technology in Warsaw, Poland. Analysis of certified reference materials showed recovery was as follows: Ca, K, Mg, Mn and Zn (81–101%, INCT-TL-1 and CTA-OTL-1), Fe and Na (106–109 %, INCT-TL-1) and Hg (94–98 %, CS-M 1). The limit of detection (LOD) was 0.0015 mg of Hg per kg dry weight (dw) and the quantification limit (LOQ) was 0.005 mg Hg kg 1 dw. In addition, for each set of 10 samples, one blank sample and one certified reference material sample were examined. There was no interference or contamination of blank samples (Brzostowski et al., 2011; Falandysz, Zhang, Wang, Saba, et al., 2015; Chudzyn´ski & Falandysz, 2008; Gucia et al., 2011).
3. Results and discussion The results are shown in Table 1. All metal concentrations are given in mg kg 1 dry weight (dw), except for potassium (given in mg g 1 dw). On the average, the contents of the elements tested were higher in the caps than in the stipes, with the exception of manganese, which was higher in the stipes. The data obtained were in good agreement with previously published data in both Polish and international research, but there were some differences related to factors affecting the accumulation of elements by the mycelium (e.g. by soil properties). In many cases, high contents of the elements determined in the fruiting bodies can be explained by soil content (Falandysz et al., 2003; Falandysz, Zhang, Wang, Krasin´ska, et al., 2015; Kojta, Jarzyn´ska, & Falandysz, 2012; Kojta et al., 2015; Tokalıog˘lu, Kartal, & Günes, 2001). The highest concentrations of the analyzed elements were determined in samples from Poniatowa (caps: 51 ± 6.3 g K kg 1, stipes: 516 ± 156 mg Ca kg 1, 118 ± 52 mg Mn kg 1 dw), Ke˛trzyn (caps: 923 ± 99 mg Mg kg 1, 294 ± 90 mg Zn kg 1 dw), Augustów Primeval Forest (caps: 0.61 ± 0.25 mg Hg kg 1, stipes: 1 _ 1205 ± 442 mg Na kg dw) and Zuromin (caps: 225 ± 162 mg Fe kg 1 dw). The lowest ratio of element concentrations in caps to stipes (Qc/s) was observed for Mn in fruiting bodies collected near Poniatowa (Qc/s 0.18 ± 0.13) and the highest for Fe in _ samples from Zuromin (Qc/s 5.1 ± 4.3).
3.1. Ca, K, Mg and Na in B. badius The concentrations of calcium in the caps varied from 70 ± 17 to 206 ± 85 mg kg 1 dw, and from 129 ± 11 to 516 ± 156 mg kg 1 dw for the stipes. Mleczek et al. (2013) analyzed whole fruiting bodies of B. badius collected from five areas within Poland and reported the highest concentrations (78 ± 25 mg Ca kg 1 dw) for samples from the Łódz´ region, and the lowest (60 ± 11 mg Ca kg 1 dw) for samples from Pomeranian Voivodeship. In comparison, Rudawska and Leski (2005a) reported a mean of 600 ± 200 mg Ca kg 1 dw and 900 ± 200 mg Ca kg 1 dw, for caps and stipes respectively.
208
Table 1 Mean (mg kg
1
), standard deviation, range and median values of elements concentrations in Bay Bolete fruiting bodies and cap to stipe concentration quotient values (QC/S).
Site, year and number of samples
Para-meter
Ca
Fe
Ka
Mg
Mn
Na
Hg
Zn
Pelplin, Pomeranian Voivodeship, 2000, n = 13
Caps
130 ± 86 (40–310) 115 150 ± 60 (70–250) 125 0.94 ± 0.66 (0.25–2.6) 0.70
67 ± 21 (40–100) 64 38 ± 15 (21–70) 34 1.9 ± 0.7 (1.1–4.1) 1.8
36 ± 5 (27–45) 36 29 ± 5 (22–38) 28 1.3 ± 0.2 (0.94–1.5) 1.3
750 ± 110 (450–915) 750 380 ± 94 (250–560) 400 2.1 ± 0.6 (1.0–3.7) 2.1
21 ± 10 (9–50) 20 29 ± 11 (12–59) 27 0.79 ± 0.31 (0.24–1.4) 0.72
570 ± 200 (240–940) 560 1100 ± 580 (179–2200) 870 0.67 ± 0.32 (0.28–1.3) 0.57
0.098 ± 0.047 (0.02–0.22) 0.091 0.064 ± 0.029 (0.02–0.12) 0.054 1.6 ± 0.4 (1.0–2.6) 1.4
NA
206 ± 85 (90–370) 170 189 ± 82 (80–360) 190 1.2 ± 0.54 (0.44–2.3) 1.1
85 ± 16 (57–110) 88 80 ± 19 (42–105) 81.5 1.1 ± 0.24 (0.72–1.4) 1.0
40 ± 3.8 (33–47) 40 35 ± 3.7 (29–42) 35 1.1 ± 0.14 (0.94–1.4) 1.2
923 ± 990 (782–1139) 920 700 ± 83 (570–945) 692 1.3 ± 0.18 (0.97–1.5) 1.3
26 ± 6.1 (16–41) 25 43 ± 14 (16–73) 45 0.66 ± 0.33 (0.41–1.8) 0.56
532 ± 92 (326–698) 557 506 ± 120 (304–741) 479 1.1 ± 0.21 (0.81–1.5) 1.2
0.069 ± 0.026 (0.041–0.15) 0.065 0.046 ± 0.015 (0.029–0.079) 0.046 1.5 ± 0.23 (1.3–2.1) 1.4
294 ± 90 (127–425) 305 211 ± 69 (119–394) 201.5 1.4 ± 0.32 (1.00–2.3) 1.4
78 ± 29 (40–130) 75 285 ± 112 (160–540) 265 0.31 ± 0.14 (0.074–0.50) 0.36
62 ± 26 (44–125) 58 63 ± 20 (36–96) 63 1.03 ± 0.38 (0.71–1.9) 0.91
38 ± 4.5 (31–45) 39 30 ± 4.7 (21–36) 31 1.3 ± 0.16 (1.00–1.5) 1.3
NA
NA
NA
NA
NA
NA
404 ± 115 (250–638) 408 412 ± 219 (174–827) 348 1.2 ± 0.60 (0.40–2.1) 1.4
0.12 ± 0.078 (0.021–0.31) 0.098 0.053 ± 0.033 (0.01–0.12) 0.047 2.4 ± 0.75 (1.4–3.8) 2.2
210 ± 36 (145–290) 212 139 ± 37 (89–227) 131 1.6 ± 0.34 (0.92–2.1) 1.5
101 ± 19 (80–150) 90 183 ± 431 (140–320) 170 0.58 ± 0.16 (0.28–0.94) 0.57
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
Cap
NA
NA
Stipe
NA
NA
QC/S
NA
NA
34 ± 1.1 (33–36) 34 27 ± 4.5 (19–35) 27 1.3 ± 0.23 (0.94–1.7) 1.3
706 ± 46 (654–846) 695 357 ± 35 (308–442) 355 2.0 ± 0.19 (1.6–2.3) 2.04
18 ± 2.5 (14–23) 17 35 ± 6.3 (22–46) 35 0.54 ± 0.13 (0.36–0.82) 0.53
529 ± 54 (420–602) 533 1205 ± 442 (924–2669) 1057 0.47 ± 0.10 (0.21–0.63) 0.46
0.61 ± 0.25 (0.29–0.96) 0.68 0.26 ± 0.13 (0.087–0.51) 0.23 3.0 ± 2.1 (1.04–9.6) 2.7
166 ± 12 (148–189) 165 111 ± 14 (85–130) 113 1.5 ± 0.23 (1.2–1.9) 1.5
Cap
70 ± 17 (40–90) 80 129 ± 11 (110–140) 130 0.55 ± 0.15 (0.29–0.73) 0.58
61 ± 22 (41–130) 55 34 ± 7.1 (25–50) 34 1.9 ± 0.69 (0.94–3.6) 1.9
34 ± 2.1 (29–37) 34 28 ± 1.6 (25–30) 28 1.2 ± 0.087 (1.07–1.4) 1.2
748 ± 45 (651–808) 766 422 ± 52 (304–520) 427 1.8 ± 0.29 (1.4–2.6) 1.8
13 ± 1.5 (11–15) 14 19 ± 2.8 (15–24) 20 0.71 ± 0.13 (0.5–1.0) 0.68
549 ± 88 (460–765) 531 679 ± 218 (430–1179) 596 0.85 ± 0.20 (0.54–1.2) 0.82
0.10 ± 0.034 (0.061–0.16) 0.095 0.050 ± 0.018 (0.029–0.081) 0.045 2.2 ± 1.06 (0.97–4.9) 2.02
189 ± 16 (161–209) 190 133 ± 12 (113–147) 139 1.4 ± 0.19 (1.1–1.8) 1.4
Stipes
QC/S
Ke˛trzyn, Warmian-Masurian Voivodeship, 2000, n = 16
Cap
Stipe
Chełmno, Kuyavian-Pomeranian Voivodeship, 2000, n = 9
Cap
Stipe
QC/S
Ełk, Warmian-Masurian Voivodeship, 2000, n = 15
Cap
Stipe
QC/S
Augustów Primeval Forest, Podlaskie Voivodeship, 2000, n = 15
Bydgoszcz, Kuyavian-Pomeranian Voivodeship, 2000, n = 15
Stipe
QC/S
NA
A.K. Kojta, J. Falandysz / Food Chemistry 200 (2016) 206–214
QC/S
NA
_ Zuromin, Masovian Voivodeship, 2000, n = 15
225 ± 162 (69–530) 160 48 ± 30 (29–140) 35 5.1 ± 4.3 (2.2–18) 3.7
32 ± 2.7 (29–39) 32 29 ± 3.5 (23–35) 29 1.1 ± 0.18 (0.86–1.5) 1.04
561 ± 40 (471–613) 567 385 ± 79 (215–559) 394 1.5 ± 0.41 (1.02–2.5) 1.4
6.1 ± 1.2 (5–8) 6 6.2 ± 2.7 (3–11) 5 1.1 ± 0.47 (0.45–2.3) 1.2
389 ± 139 (179–678) 390.5 525 ± 294 (144–1134) 470 0.86 ± 0.31 (0.29–1.6) 0.83
0.059 ± 0.028 (0.016–0.10) 0.066 0.024 ± 0.011 (0.001–0.041) 0.021 3.5 ± 3.6 (1.1–16) 2.6
199 ± 43 (135–273) 195 143 ± 27 (109–198) 143 1.4 ± 0.19 (1.1–1.9) 1.3
131 ± 48 (70–250) 135 239 ± 71 (120–370) 230 0.57 ± 0.18 (0.25–0.92) 0.56
105 ± 30 (71–180) 94 81 ± 23 (58–150) 75 1.3 ± 0.35 (0.71–1.9) 1.3
35 ± 2.3 (32–40) 35 24 ± 1.6 (22–28) 24 1.4 ± 0.12 (1.3–1.7) 1.4
725 ± 54 (656–873) 714 395 ± 37 (339–448) 385 1.8 ± 0.26 (1.5–2.5) 1.8
25 ± 7.4 (14–38) 28 29 ± 7.8 (11–42) 30 0.89 ± 0.22 (0.57–1.3) 0.84
486 ± 66 (373–581) 488 561 ± 238 (233–1139) 546 0.98 ± 0.33 (0.48–1.6) 0.84
0.12 ± 0.059 (0.049–0.23) 0.1 0.041 ± 0.026 (0.010–0.10) 0.033 3.6 ± 2.0 (1.5–7.9) 2.8
234 ± 29 (181–293) 235 145 ± 11 (120–160) 145 1.6 ± 0.20 (1.3–2.1) 1.6
96 ± 50 (40–210) 85 516 ± 156 (300–760) 530 0.20 ± 0.10 (0.053–0.37) 0.16
65 ± 21 (36–120) 63 67 ± 32 (33–150) 60 1.08 ± 0.49 (0.30–1.9) 1.03
51 ± 6.3 (33–59) 52 49 ± 8.04 (38–67) 50 1.04 ± 0.18 (0.73–1.4) 1.04
852 ± 88 (607–957) 853 590 ± 105 (425–773) 596 1.5 ± 0.32 (1.02–2.07) 1.5
16 ± 6.1 (9–31) 15 118 ± 52 (16–209) 109 0.18 ± 0.13 (0.077–0.56) 0.14
10 ± 5.0 (2–17) 9 26 ± 15 (9–68) 22 0.46 ± 0.29 (0.074–1.00) 0.42
NA
NA
NA
NA
NA
NA
Cap
NA
NA
NA
NA
NA
NA
NA
Stipe
NA
NA
NA
NA
NA
NA
QC/S
NA
NA
NA
NA
NA
NA
0.14 ± 0.10 (0.068–0.47) 0.11 0.078 ± 0.029 (0.025–0.12) 0.080 1.9 ± 1.0 (1.1–4.1) 1.5
Stipe
QC/S
Włocławek, Kuyavian-Pomeranian Voivodeship, 2000, n = 16
Cap
Stipe
QC/S
Poniatowa, Lublin Voivodeship, 2000, n = 12
Cap
Stipe
QC/S
Wilków, Lublin Voivodeship, 2000, n = 15
a
g kg
1
.
NA
NA
A.K. Kojta, J. Falandysz / Food Chemistry 200 (2016) 206–214
121 ± 50 (50–250) 120 131 ± 91 (40–310) 100 1.3 ± 0.94 (0.29–3.2) 1.1
Cap
209
210
A.K. Kojta, J. Falandysz / Food Chemistry 200 (2016) 206–214
The results obtained in this study are within the range reported in previous work by Kalacˇ (2009). Potassium concentrations in the samples were high, varying from 32 ± 1 to 51 ± 6 g kg 1 dw in caps and from 24 ± 1.6 to 49 ± 8 g kg 1 dw in stipes. Rudawska and Leski (2005a) and Mleczek et al. (2013) obtained similar results for K (caps: 36.5 ± 4.6 g kg 1 dw, stipes: 33.3 ± 2.8 g kg 1 dw), which could be attributed to essentiality and requirement of this element by fungus while less to the soil type (sandy) in the sites studied. Average concentrations of potassium in the fruiting bodies of B. badius ranged from 20.3 ± 2.5 g kg 1 dw (for the Pomeranian Voivodeship) to 23.9 ± 2.1 g kg 1 dw (for the Lower Silesian Voivodeship) (Mleczek et al., 2013). Magnesium concentrations were found to vary from 561 ± 40 to 923 ± 99 mg kg 1 dw in caps and from 357 ± 35 to 700 ± 83 mg kg 1 dw in the stipes. Mleczek et al. (2013) obtained similar results (319 ± 54–372 ± 91 mg kg 1 dw). Data published by Rudawska and Leski (2005a) showed higher Mg contents (caps: 1.2 ± 0.3, stipes: 0.7 ± 0.1 g Mg kg 1 dw). The mean sodium concentrations in B. badius were between 10 ± 5.0 and 574 ± 200 mg kg 1 dw in the caps and from 26 ± 15 to 1205 ± 442 mg kg 1 dw in the stipes. Stipes of Bay Bolete accumulated more sodium than the caps. Mleczek et al. (2013) reported sodium concentrations in the whole fruiting bodies that varied from 57 ± 15 (for specimens from Greater Poland Voivodeship) to 773 ± 20 mg kg kg 1 dw (Pomeranian Voivodeship). Recent publications provide data on levels of calcium, magnesium, mercury, potassium and phosphorus in the fruiting bodies of B. badius and the soil substratum (Falandysz & Bielawski, 2001; Falandysz et al., 2012; Malinowska, Szefer, & Falandysz, 2004; Mleczek et al. 2013). Elements in the samples were found at different levels in caps and stipes. The contents of K, P and Mg were higher in caps while the stipes contained more Ca (Kottke et al., 1998). Differences in the beneficial elements content of caps and stipes of fruiting bodies collected from different areas have been presented by Malinowska et al. (2004). 3.2. Fe, Mn, Zn and Hg in B. badius The concentrations of iron in the Bay Bolete fruiting bodies ranged from 33.6 ± 7.09 mg kg 1 dw in stipes harvested from Byd_ goszcz to 214 ± 162 mg kg 1 dw in caps from Zuromin. The highest level of this element found in B. badius stipes was 81 ± 23 mg kg 1 dw observed in specimens from Włocławek while the lowest concentration of 61 ± 22 mg kg 1 dw was found in caps obtained from Bydgoszcz. The iron content of this study are in good agreement with previously published data. The analyses of samples taken from Polish territory showed iron concentrations in whole fruiting bodies that varied from 38 to 129 mg kg 1 dw, while the contents of caps and stipes analyzed separately were 122 ± 12 and 50 ± 5.6 mg kg 1 dw, respectively (Rudawska & Leski, 2005a,b). Malinowska et al. (2004) published among others, data on the content of iron, manganese and zinc in the fruiting bodies of B. badius collected from 12 sites in the northern and north-eastern Poland. These researchers reported iron concentrations range from 34 ± 15 to 207 ± 58 mg kg 1 dw in caps and 32 ± 11 to 150 ± 66 mg kg 1 dw in the stipes. The results obtained by Ouzouni, Veltsistas, Paleologos, & Riganakos (2007) for the whole fruiting bodies of B. badius collected near the motorway in Kastamonu, Turkey, showed a higher mean iron concentrations of 377 ± 21 mg kg 1 dw. Manganese concentrations in the mushroom samples _ varied from 6.1 ± 1.2 (for specimens from Zuromin) to 26 ± 6.1 mg kg
1
stipes
6.2 ± 2.7
from
dw in caps (for specimens from Ke˛trzyn), and in _ (for specimens from Zuromin) to
118 ± 52 mg kg 1 dw (for specimens from Poniatowa). Bay Bolete caps contained more manganese than the stipes. Similar results were obtained by Malinowska et al. (2004) who reported Mn concentrations of between 9.8 ± 3.2 and 22 ± 5.1 mg kg 1 dw in the caps, and between 10 ± 3.5 and 53 ± 52 mg kg 1 dw in stipes. Rudawska and Leski (2005a) reported Mn concentrations in caps of 14 ± 1.2 mg kg 1 dw and 15 ± 3.2 mg kg 1 dw in the stipes. The same authors also published data for the whole fruiting bodies showing manganese range from 11 to 19.1 mg kg 1 dw (Rudawska & Leski, 2005b). The Mn results for the whole fruiting bodies collected near the motorway in Kastamonu (Turkey) was 25 ± 1.3 mg kg 1 dw. The zinc concentrations varied between 166 ± 1.6 and 294 ± 90 mg kg 1 dw in caps, and 111 ± 14 and 211 ± 69 mg kg 1 dw in the stipes. Studies of specimens from Poland reported a range from 109 ± 18 to 230 ± 82 mg kg 1 dw in stipes and 121 ± 24 to 230 ± 55 mg kg 1 dw in caps, while the data for the whole fruiting bodies ranged from 124 to 162 mg kg 1 dw (Falandysz et al., 2001; Rudawska & Leski, 2005a,b). Turkish researchers have published a mean of 64 ± 5.3 mg kg 1 dw in the whole fruiting bodies (Mendil, Uluo¯zlü, Hasdemir, & Cag˘lar, 2004). The Hg concentrations varied from 0.059 ± 0.028 (for samples _ from Zuromin) to 0.61 ± 0.25 mg kg 1 dw (for samples from Augustów Primeval Forest) in caps. In the stipes, the Hg concentra_ tions were from 0.024 ± 0.011 (for samples from Zuromin) to 0.26 ± 0.13 mg kg 1 dw (for samples from Augustów Primeval Forest). The results are consistent with previously published data (caps: 0.18 ± 0.050, 0.20 ± 0.070, 0.49 ± 0.30 mg kg 1 dw; stipes: 0.094 ± 0.045, 0.084 ± 0.028, 0.29 ± 0.20 mg kg 1 dw) (Falandysz et al., 2001; Chudzyn´ski & Falandysz, 2008). The obtained data on the total Hg content are also consistent with the results presented by Melgar, Alonso, and García (2009). 3.3. Metals’ cap to stalk quotients The capacity of this species to accumulate elements in parts of the fruiting bodies was evaluated by calculating the ratio of the concentrations in caps and stipes (Table 1). Concentrations in caps and stipes were higher than 1 in the case of Fe (Qc/s from _ 1.03 ± 0.38 – Chełmno to 5.1 ± 4.3 – Zuromin), K (1.04 ± 0.18 – Poniatowa to 1.4 ± 0.12 – Włocławek), Mg (1.3 ± 0.18 – Ke˛trzyn to 2.1 ± 0.64 – Pelplin), Hg (1.5 ± 0.23 – Ke˛trzyn to 3.6 ± 2.0 – _ Włocławek) and Zn (1.4 ± 0.19 – Zuromin to 1.6 ± 0.20 – Włocławek). The values shown above demonstrate an enhanced capacity for this species to accumulate mercury in the cap. In the case of Ca, Mn and Na, the ratios for most of the samples were less than one. _ Only in the case of samples from Ke˛trzyn (for Ca and Na), Zuromin (Ca, Mn) and Chełmno (Na) were values of Qc/s higher than 1. The lowest Qc/s values for Mn were observed for samples from Poniatowa (Qc/s 0.18 ± 0.13). Taking into account these results, it can be concluded that iron, potassium, magnesium, mercury, and zinc are readily accumulated in larger amounts in the caps, while calcium, manganese and sodium accumulate more in the stipes. 4. Statistical evaluation of the data Data obtained in this study were subjected to statistical procedures including multifunctional analysis. In order to demonstrate the possible spatial variability of mineral composition of B. badius, cluster analysis (CA) and principal component analysis (PCA) were performed. The obtained data were not normally distributed (Shapiro–Wilk test, p < 0.005) and the variances were not equal (Levene’s test, p < 0.005), so the Kruskal–Wallis test was used for comparing the contents of elements in the caps and stipes. Performing these nonparametric tests showed that there is a
A.K. Kojta, J. Falandysz / Food Chemistry 200 (2016) 206–214
significant difference in the content of all elements examined in caps, as well as the stipes (p < 0.0001). Cluster analysis was performed for the sampling sites. Hierarchical cluster analysis (Ward method, p < 0.05) allows to identify the most similar regions in terms of accumulation of tested elements in the fruiting bodies. Cluster analysis was performed separately for caps and stipes. The tree diagram for caps consisted of two main clusters. The first cluster consisted of samples from Augustów Primeval Forest while the second group consists of sam_ ples from Ke˛trzyn, Pelplin, Zuromin, Włocławek, Poniatowa, Chełmno, Bydgoszcz and several individual samples from Augustów Primeval Forest. The most similar regions were separated by clusters of the second degree of the second main cluster. Cluster analysis performed for stipes showed two main groups; the first consisted of samples from Augustów Primeval Forest, while the second group consisted of samples from Poniatowa, Chełmno,
211
_ Pelplin, Bydgoszcz, Zuromin, Ke˛trzyn, Włocławek, and several samples from Augustów Primeval Forest. As with the caps, the cluster of the second degree of the second main cluster are the most similar regions. Cluster analysis was also conducted for the elements determined (Ward method, p < 0.05). Tree diagram separated the elements in the caps and stipes into two main fractions, reflecting the interdependent relations between them. In the case of caps, the first fraction was composed of elements such as Ca, Mn, K, Mg and Zn, while the second fraction consisted of Fe, Na and Hg (Fig. 2). Cluster analysis performed for the stipes showed two clusters consisting of elements such as Ca, Fe, Na, Hg and K, Mg, Mn and Zn (Fig. 3). In summary, the most similar regions in terms of accumulation of elements in caps were Ke˛trzyn, Włocławek, Pelplin and Ponia_ towa, Chełmno, Bydgoszcz, and Zuromin. However, for the stipes,
Fig. 2. Tree diagram for the elements in the caps as objects (Ward’s Method, 1-Pearson r).
Fig. 3. Tree diagram for the elements in the stipes as objects (Ward’s Method, 1-Pearson r).
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areas with similar accumulation of elements were Pelplin, _ Zuromin, Bydgoszcz, Chełmno and Ke˛trzyn, Włocławek. Elements that accumulated in the caps in a similar manner were Ca, Mn, K, Mg and Zn, while the second group consisted of Fe, Na and Hg. On the other hand, for stipes, the first group was Ca, Fe, Na, Hg while the second consisted of K, Mg, Mn, Zn. Principal component analysis (PCA) allows the graphical presentation of the relationships that exist between the elements accumulated in the fruiting bodies. PCA was also performed to identify the principal components that represented the largest part of the variability in the data set. PCA carried out on the data showed three principal components (PCs), which together explained 71.9% of the observed variability. The first principal
component (PC1) explained 36.8% of the observed variability, PC2 explained 21.7%, while PC3 explained 13.4% of the observed variability. The first principal component was defined by elements such as K, Mg and Zn with a negative charge factor coordinates, second by Ca and Mn (positive charge factor coordinate) and the third principal component was represented by Fe (negative charge factor coordinate). Projection of these cases on the factor plane created in the PCA analysis was intended to illustrate which of the elements determine the PCs that are accumulated in similar amounts, and whether the sampling site affect their content in the fruiting bodies (Figs. 4 and 5). The results of the analysis indicated that the greatest differences in the concentrations were observed for elements such as K, Mg and Zn (in fruiting bodies) collected from
Fig. 4. Projection of the elements concentrations in the fruiting bodies set on the first and second factor-plane.
Fig. 5. Projection of the elements concentrations in the fruiting bodies set on the first and third factor-plane.
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seven sampling sites (Fig. 4). The results showed a statistically significant difference in accumulation of the elements in the fruiting bodies collected from different areas. Caps of Bay Bolete harvested _ in Bydgoszcz, Włocławek and Zuromin differed slightly in terms of K, Mg and Zn contents from caps and stipes collected from Ke˛trzyn. Samples from these sites differed in terms of the elements described by PC2 (Fig. 4). The elements in the third principal component showed a significantly reduced variability in the contents described by PC3 (Fig. 5). 5. Intake rates and toxicological risks The European Union has not established limits for Hg in edible mushrooms. There are currently no Polish standards for the content of this element in wild edible mushrooms. Poland is in the European Union so the former limit established in Poland is without force. Possible health risks due to the intake of mercury accumulated in fruiting bodies can be assessed by comparing the estimated exposure to the recommended doses. Joint Expert Committee on Food Additives of the Food and Agriculture Organization of the United Nations and the World Health Organization (WHO, 1989; JECFA, 1978) issued a Provisional Tolerable Weekly Intake (PTWI; 0.005 mg Hg kg 1 body mass), an acceptable level of toxic metals that can be ingested within a week. Another indicator is the reference dose (RfD; 0.0004 mg Hg kg 1 body mass daily) set by the U.S. Environmental Protection Agency (US EPA, 2005). Reference dose is an estimate of daily human exposure to a substance without risk. To assess the possible risk due to the consumption of mercury accumulated in fruiting bodies, reference dose, and the value of the Provisional Tolerable Weekly Intake were used. The results obtained in this study indicate that mushrooms collected from the area of Augustów Primeval Forest slightly exceeded the reference dose for mercury calculated on a meal containing 300 g of fruiting bodies (caps: 0.0031 mg Hg kg 1 dw daily; stipes: 0.0013 mg Hg kg 1 dw daily). In the case of the PTWI, even eating large amounts of B. badius fresh caps (up to 4 kg per week) would not cause consumers to exceed this limit. 6. Conclusions The analysis of 126 samples of B. badius fruiting bodies was performed using validated methods to determine the nutritional value of the analyzed mushroom and toxicological risks associated with their consumption. Caps of Bay Bolete accumulated Ca, Mn, K, Mg, Zn and Fe, Na, Hg (CA, p < 0.005) in a similar manner. Principal component analysis showed the greatest variations in the concentrations of elements such as K, Mg and Zn in the fruiting bodies (p < 0.005). Mushroom B. badius consumed in a typical amount per week is safe with respect to the mercury content and intake dose. Acknowledgement The project was funded by the National Science Centre of Poland under of Decision No. UMO-2012/N/NZ7/00935. References Brzostowski, A., Falandysz, J., Jarzyn´ska, G., & Zhang, D. (2011). Bioconcentration potential of metallic elements by Poison Pax (Paxillus involutus) mushroom. Journal of Environmental Science and Health, Part A, 46, 378–393. Chudzyn´ski, K., & Falandysz, J. (2008). Multivariate analysis of elements content of Larch Bolete (Suillus grevillei). Chemosphere, 73, 1230–1239. Dimitrijevic, M. D., Mitic, J. J., Cvetkovic, J. S., Vesna, P., Jovanovic, S., Mutic, J. J., & Nikolic Mandic, S. D. (2015). Update on element content profiles in eleven wild
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Food Chemistry journal homepage: www.elsevier.com/locate/foodchem
Metallic elements (Ca, Hg, Fe, K, Mg, Mn, Na, Zn) in the fruiting bodies of Boletus badius Anna K. Kojta ⇑, Jerzy Falandysz ´ sk, 63 Wita Stwosza Str., PL 80-952 Gdan ´ sk, Poland Laboratory of Environmental Chemistry & Ecotoxicology, University of Gdan
a r t i c l e
i n f o
Article history: Received 14 August 2014 Received in revised form 21 December 2015 Accepted 1 January 2016 Available online 2 January 2016 Keywords: Europe Food Foraging Forest Heavy metals Organic food
a b s t r a c t The aim of this study was to investigate and compare the levels of eight metallic elements in the fruiting bodies of Bay Bolete (Boletus badius; current name Imleria badia) collected from ten sites in Poland to understand better the value of this popular mushroom as an organic food. Bay Bolete fruiting bodies were collected from the forest area near the towns and villages of Ke˛trzyn, Poniatowa, Bydgoszcz, Pelplin, _ Włocławek, Zuromin, Chełmno, Ełk and Wilków communities, as well as in the Augustów Primeval Forest. Elements such as Ca, Fe, K, Mg, Mn, Na and Zn were analyzed by inductively coupled plasma atomic emission spectroscopy (ICP-OES), and mercury by cold vapor atomic absorption spectrometry (CV-AAS). This made it possible to assess the nutritional value of the mushroom, as well as possible toxicological risks associated with its consumption. The results were subjected to statistical analysis (Kruskal–Wallis test, cluster analysis, principal component analysis). Ó 2016 Elsevier Ltd. All rights reserved.
1. Introduction The fruiting bodies of edible wild mushrooms are popular in many countries (Kottke, Qian, Pritsch, Haug, & Oberwinkler, 1998) and demand for this kind of organic food is growing worldwide. It is assumed that the fruiting bodies of mushrooms are naturally rich in different compounds (Rudawska & Leski, 2005a; Kalacˇ, 2010). Consumption of wild and cultivated mushrooms is increasing from year to year (Dimitrijevic et al., 2015). In Poland, picking wild mushrooms for home use or resale is common. Export of the most common mushroom species from Poland is 3400 metric tons per year and include Common Chanterelle (Cantharellus cibarius), King Bolete (Boletus edulis), Orange Birch Bolete (Leccinum versipelle), Brown Birch Scaber Stalk (Leccinum scabrum), Bay Bolete (Boletus badius), Red Cracking Bolete (Xerocomus chryzenteron) and Yellow Cracking Bolete (Xerocomus subtomentosus) (GUS, 2013; Falandysz & Chwir, 1997; Falandysz, Kawano, S´wieczkowski, Brzostowski, & Dadej, 2003a). On the Polish territory, Bay Bolete (B. badius) (Fr.) Fr. is a commonly found in coniferous forests, but rarely in mixed forests. It belongs to the ectomycorrhizal species (Gumin´ska & Wojewoda, 1985).
⇑ Corresponding author. E-mail address: [email protected] (A.K. Kojta). http://dx.doi.org/10.1016/j.foodchem.2016.01.006 0308-8146/Ó 2016 Elsevier Ltd. All rights reserved.
This study is part of an ongoing study to examine the mineral composition of fruiting bodies of B. badius, demonstrate possible variations in mineral composition of morphological parts as well as similarities and differences in the content of fruiting bodies collected from different sites across Poland. 2. Material and methods Samples of Bay Bolete B. badius (Fr.) Fr. (current name Imleria badia (Fr.) Vizzini) (Species Fungorum, 2015) fruiting bodies were collected in 2000 from distant forests of Pelplin, Ełk, Ke˛trzyn, _ Augustów, Chełmno, Bydgoszcz, Włocławek, Zuromin, Poniatowa and Wilków in Poland (Fig. 1). The caps and stipes were separated and dried at room temperature for 1–3 days, and then at 85 °C in a laboratory dryer for 48 h to constant weight. The dried fruiting bodies were pulverized in a porcelain mortar and stored in sealed polyethylene bugs under dry and clean conditions. All the reagents used in this study were of analytical reagent grade, unless otherwise stated. Double distilled water was used for the preparation of the solutions. Prior to the analysis of the metallic elements, sub-samples (300–400 mg) were wet digested with 7 mL of concentrated nitric acid (SuprapureÒ, Merck, Darmstadt, Germany) in closed polytetrafluoroethylene (PTFE) vessels in a microwave oven (MARS 5 of CEM Corp., Matthews, NC, USA) under the following conditions: power – 1.2 kW; ramp 1–10 min; temp. 1–100 °C; hold 1–10 min, ramp 2–10 min; pressure – 800 psi; temp. 2–100 °C; hold 2–10 min; cool down: 5 min, and
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_ Fig. 1. Location of sampling sites in Poland (1 – Pelplin, 2 – Ke˛trzyn, 3 – Chełmno, 4 – Ełk, 5 – Augustów Primeval Forest, 6 – Bydgoszcz, 7 – Zuromin, 8 – Włocławek, 9 – Poniatowa, 10 – Wilków).
then diluted to 25 mL with deionized water (Chudzyn´ski & Falandysz, 2008; Frankowska, Ziółkowska, Bielawski, & Falandysz, 2010). The analysis of metallic elements content in fruiting bodies other than mercury was performed using inductively coupled plasma atomic emission spectroscopy (IC P-OES; Optima 2000, Perkin Elmer, USA) (Brzostowski, Falandysz, Jarzyn´ska, & Zhang, 2011). Determination of total Hg was performed using coldvapour atomic absorption spectroscopy (CV-AAS) by direct sample thermal decomposition coupled with gold wool trap for Hg and, following desorption, quantitative measurement at 253.7 nm. The analytical instrument used was a mercury analyzer (MA-2000), Nippon Instruments Corporation, Tatsuki, Japan) equipped with an auto-sampler, and operated in low (3–20 ng Hg per sample) or high (25–150 mg Hg per sample) modes, as appropriate (Falandysz, Zhang, Wang, Krasin´ska, et al. 2015; Falandysz, Zhang, Wang, Saba, et al. 2015; Jarzyn´ska & Falandysz, 2011). A standard solution of 1.0 mg Hg mL 1 was obtained from the 10 mg mL 1 stock solution. Blank and 3, 5, 10, 15 and 20 lL (low mode) and 25, 50, 100, 150 and 200 lL (high mode) of 1.0 mg mL 1 Hg standard solutions were injected into the analyzer for the construction of calibration curves weekly. Procedures for the determination of mercury and the other metallic elements were validated through the analysis of certified references materials such as CTA-OTL-1 (Tobacco Leaves), INCTTL-1 (Tea Leaves) and CS-M 1 (dried fruiting bodies of mushroom Cow Bolete Suillus bovinus) produced by the Institute of Nuclear Chemistry and Technology in Warsaw, Poland. Analysis of certified reference materials showed recovery was as follows: Ca, K, Mg, Mn and Zn (81–101%, INCT-TL-1 and CTA-OTL-1), Fe and Na (106–109 %, INCT-TL-1) and Hg (94–98 %, CS-M 1). The limit of detection (LOD) was 0.0015 mg of Hg per kg dry weight (dw) and the quantification limit (LOQ) was 0.005 mg Hg kg 1 dw. In addition, for each set of 10 samples, one blank sample and one certified reference material sample were examined. There was no interference or contamination of blank samples (Brzostowski et al., 2011; Falandysz, Zhang, Wang, Saba, et al., 2015; Chudzyn´ski & Falandysz, 2008; Gucia et al., 2011).
3. Results and discussion The results are shown in Table 1. All metal concentrations are given in mg kg 1 dry weight (dw), except for potassium (given in mg g 1 dw). On the average, the contents of the elements tested were higher in the caps than in the stipes, with the exception of manganese, which was higher in the stipes. The data obtained were in good agreement with previously published data in both Polish and international research, but there were some differences related to factors affecting the accumulation of elements by the mycelium (e.g. by soil properties). In many cases, high contents of the elements determined in the fruiting bodies can be explained by soil content (Falandysz et al., 2003; Falandysz, Zhang, Wang, Krasin´ska, et al., 2015; Kojta, Jarzyn´ska, & Falandysz, 2012; Kojta et al., 2015; Tokalıog˘lu, Kartal, & Günes, 2001). The highest concentrations of the analyzed elements were determined in samples from Poniatowa (caps: 51 ± 6.3 g K kg 1, stipes: 516 ± 156 mg Ca kg 1, 118 ± 52 mg Mn kg 1 dw), Ke˛trzyn (caps: 923 ± 99 mg Mg kg 1, 294 ± 90 mg Zn kg 1 dw), Augustów Primeval Forest (caps: 0.61 ± 0.25 mg Hg kg 1, stipes: 1 _ 1205 ± 442 mg Na kg dw) and Zuromin (caps: 225 ± 162 mg Fe kg 1 dw). The lowest ratio of element concentrations in caps to stipes (Qc/s) was observed for Mn in fruiting bodies collected near Poniatowa (Qc/s 0.18 ± 0.13) and the highest for Fe in _ samples from Zuromin (Qc/s 5.1 ± 4.3).
3.1. Ca, K, Mg and Na in B. badius The concentrations of calcium in the caps varied from 70 ± 17 to 206 ± 85 mg kg 1 dw, and from 129 ± 11 to 516 ± 156 mg kg 1 dw for the stipes. Mleczek et al. (2013) analyzed whole fruiting bodies of B. badius collected from five areas within Poland and reported the highest concentrations (78 ± 25 mg Ca kg 1 dw) for samples from the Łódz´ region, and the lowest (60 ± 11 mg Ca kg 1 dw) for samples from Pomeranian Voivodeship. In comparison, Rudawska and Leski (2005a) reported a mean of 600 ± 200 mg Ca kg 1 dw and 900 ± 200 mg Ca kg 1 dw, for caps and stipes respectively.
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Table 1 Mean (mg kg
1
), standard deviation, range and median values of elements concentrations in Bay Bolete fruiting bodies and cap to stipe concentration quotient values (QC/S).
Site, year and number of samples
Para-meter
Ca
Fe
Ka
Mg
Mn
Na
Hg
Zn
Pelplin, Pomeranian Voivodeship, 2000, n = 13
Caps
130 ± 86 (40–310) 115 150 ± 60 (70–250) 125 0.94 ± 0.66 (0.25–2.6) 0.70
67 ± 21 (40–100) 64 38 ± 15 (21–70) 34 1.9 ± 0.7 (1.1–4.1) 1.8
36 ± 5 (27–45) 36 29 ± 5 (22–38) 28 1.3 ± 0.2 (0.94–1.5) 1.3
750 ± 110 (450–915) 750 380 ± 94 (250–560) 400 2.1 ± 0.6 (1.0–3.7) 2.1
21 ± 10 (9–50) 20 29 ± 11 (12–59) 27 0.79 ± 0.31 (0.24–1.4) 0.72
570 ± 200 (240–940) 560 1100 ± 580 (179–2200) 870 0.67 ± 0.32 (0.28–1.3) 0.57
0.098 ± 0.047 (0.02–0.22) 0.091 0.064 ± 0.029 (0.02–0.12) 0.054 1.6 ± 0.4 (1.0–2.6) 1.4
NA
206 ± 85 (90–370) 170 189 ± 82 (80–360) 190 1.2 ± 0.54 (0.44–2.3) 1.1
85 ± 16 (57–110) 88 80 ± 19 (42–105) 81.5 1.1 ± 0.24 (0.72–1.4) 1.0
40 ± 3.8 (33–47) 40 35 ± 3.7 (29–42) 35 1.1 ± 0.14 (0.94–1.4) 1.2
923 ± 990 (782–1139) 920 700 ± 83 (570–945) 692 1.3 ± 0.18 (0.97–1.5) 1.3
26 ± 6.1 (16–41) 25 43 ± 14 (16–73) 45 0.66 ± 0.33 (0.41–1.8) 0.56
532 ± 92 (326–698) 557 506 ± 120 (304–741) 479 1.1 ± 0.21 (0.81–1.5) 1.2
0.069 ± 0.026 (0.041–0.15) 0.065 0.046 ± 0.015 (0.029–0.079) 0.046 1.5 ± 0.23 (1.3–2.1) 1.4
294 ± 90 (127–425) 305 211 ± 69 (119–394) 201.5 1.4 ± 0.32 (1.00–2.3) 1.4
78 ± 29 (40–130) 75 285 ± 112 (160–540) 265 0.31 ± 0.14 (0.074–0.50) 0.36
62 ± 26 (44–125) 58 63 ± 20 (36–96) 63 1.03 ± 0.38 (0.71–1.9) 0.91
38 ± 4.5 (31–45) 39 30 ± 4.7 (21–36) 31 1.3 ± 0.16 (1.00–1.5) 1.3
NA
NA
NA
NA
NA
NA
404 ± 115 (250–638) 408 412 ± 219 (174–827) 348 1.2 ± 0.60 (0.40–2.1) 1.4
0.12 ± 0.078 (0.021–0.31) 0.098 0.053 ± 0.033 (0.01–0.12) 0.047 2.4 ± 0.75 (1.4–3.8) 2.2
210 ± 36 (145–290) 212 139 ± 37 (89–227) 131 1.6 ± 0.34 (0.92–2.1) 1.5
101 ± 19 (80–150) 90 183 ± 431 (140–320) 170 0.58 ± 0.16 (0.28–0.94) 0.57
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
Cap
NA
NA
Stipe
NA
NA
QC/S
NA
NA
34 ± 1.1 (33–36) 34 27 ± 4.5 (19–35) 27 1.3 ± 0.23 (0.94–1.7) 1.3
706 ± 46 (654–846) 695 357 ± 35 (308–442) 355 2.0 ± 0.19 (1.6–2.3) 2.04
18 ± 2.5 (14–23) 17 35 ± 6.3 (22–46) 35 0.54 ± 0.13 (0.36–0.82) 0.53
529 ± 54 (420–602) 533 1205 ± 442 (924–2669) 1057 0.47 ± 0.10 (0.21–0.63) 0.46
0.61 ± 0.25 (0.29–0.96) 0.68 0.26 ± 0.13 (0.087–0.51) 0.23 3.0 ± 2.1 (1.04–9.6) 2.7
166 ± 12 (148–189) 165 111 ± 14 (85–130) 113 1.5 ± 0.23 (1.2–1.9) 1.5
Cap
70 ± 17 (40–90) 80 129 ± 11 (110–140) 130 0.55 ± 0.15 (0.29–0.73) 0.58
61 ± 22 (41–130) 55 34 ± 7.1 (25–50) 34 1.9 ± 0.69 (0.94–3.6) 1.9
34 ± 2.1 (29–37) 34 28 ± 1.6 (25–30) 28 1.2 ± 0.087 (1.07–1.4) 1.2
748 ± 45 (651–808) 766 422 ± 52 (304–520) 427 1.8 ± 0.29 (1.4–2.6) 1.8
13 ± 1.5 (11–15) 14 19 ± 2.8 (15–24) 20 0.71 ± 0.13 (0.5–1.0) 0.68
549 ± 88 (460–765) 531 679 ± 218 (430–1179) 596 0.85 ± 0.20 (0.54–1.2) 0.82
0.10 ± 0.034 (0.061–0.16) 0.095 0.050 ± 0.018 (0.029–0.081) 0.045 2.2 ± 1.06 (0.97–4.9) 2.02
189 ± 16 (161–209) 190 133 ± 12 (113–147) 139 1.4 ± 0.19 (1.1–1.8) 1.4
Stipes
QC/S
Ke˛trzyn, Warmian-Masurian Voivodeship, 2000, n = 16
Cap
Stipe
Chełmno, Kuyavian-Pomeranian Voivodeship, 2000, n = 9
Cap
Stipe
QC/S
Ełk, Warmian-Masurian Voivodeship, 2000, n = 15
Cap
Stipe
QC/S
Augustów Primeval Forest, Podlaskie Voivodeship, 2000, n = 15
Bydgoszcz, Kuyavian-Pomeranian Voivodeship, 2000, n = 15
Stipe
QC/S
NA
A.K. Kojta, J. Falandysz / Food Chemistry 200 (2016) 206–214
QC/S
NA
_ Zuromin, Masovian Voivodeship, 2000, n = 15
225 ± 162 (69–530) 160 48 ± 30 (29–140) 35 5.1 ± 4.3 (2.2–18) 3.7
32 ± 2.7 (29–39) 32 29 ± 3.5 (23–35) 29 1.1 ± 0.18 (0.86–1.5) 1.04
561 ± 40 (471–613) 567 385 ± 79 (215–559) 394 1.5 ± 0.41 (1.02–2.5) 1.4
6.1 ± 1.2 (5–8) 6 6.2 ± 2.7 (3–11) 5 1.1 ± 0.47 (0.45–2.3) 1.2
389 ± 139 (179–678) 390.5 525 ± 294 (144–1134) 470 0.86 ± 0.31 (0.29–1.6) 0.83
0.059 ± 0.028 (0.016–0.10) 0.066 0.024 ± 0.011 (0.001–0.041) 0.021 3.5 ± 3.6 (1.1–16) 2.6
199 ± 43 (135–273) 195 143 ± 27 (109–198) 143 1.4 ± 0.19 (1.1–1.9) 1.3
131 ± 48 (70–250) 135 239 ± 71 (120–370) 230 0.57 ± 0.18 (0.25–0.92) 0.56
105 ± 30 (71–180) 94 81 ± 23 (58–150) 75 1.3 ± 0.35 (0.71–1.9) 1.3
35 ± 2.3 (32–40) 35 24 ± 1.6 (22–28) 24 1.4 ± 0.12 (1.3–1.7) 1.4
725 ± 54 (656–873) 714 395 ± 37 (339–448) 385 1.8 ± 0.26 (1.5–2.5) 1.8
25 ± 7.4 (14–38) 28 29 ± 7.8 (11–42) 30 0.89 ± 0.22 (0.57–1.3) 0.84
486 ± 66 (373–581) 488 561 ± 238 (233–1139) 546 0.98 ± 0.33 (0.48–1.6) 0.84
0.12 ± 0.059 (0.049–0.23) 0.1 0.041 ± 0.026 (0.010–0.10) 0.033 3.6 ± 2.0 (1.5–7.9) 2.8
234 ± 29 (181–293) 235 145 ± 11 (120–160) 145 1.6 ± 0.20 (1.3–2.1) 1.6
96 ± 50 (40–210) 85 516 ± 156 (300–760) 530 0.20 ± 0.10 (0.053–0.37) 0.16
65 ± 21 (36–120) 63 67 ± 32 (33–150) 60 1.08 ± 0.49 (0.30–1.9) 1.03
51 ± 6.3 (33–59) 52 49 ± 8.04 (38–67) 50 1.04 ± 0.18 (0.73–1.4) 1.04
852 ± 88 (607–957) 853 590 ± 105 (425–773) 596 1.5 ± 0.32 (1.02–2.07) 1.5
16 ± 6.1 (9–31) 15 118 ± 52 (16–209) 109 0.18 ± 0.13 (0.077–0.56) 0.14
10 ± 5.0 (2–17) 9 26 ± 15 (9–68) 22 0.46 ± 0.29 (0.074–1.00) 0.42
NA
NA
NA
NA
NA
NA
Cap
NA
NA
NA
NA
NA
NA
NA
Stipe
NA
NA
NA
NA
NA
NA
QC/S
NA
NA
NA
NA
NA
NA
0.14 ± 0.10 (0.068–0.47) 0.11 0.078 ± 0.029 (0.025–0.12) 0.080 1.9 ± 1.0 (1.1–4.1) 1.5
Stipe
QC/S
Włocławek, Kuyavian-Pomeranian Voivodeship, 2000, n = 16
Cap
Stipe
QC/S
Poniatowa, Lublin Voivodeship, 2000, n = 12
Cap
Stipe
QC/S
Wilków, Lublin Voivodeship, 2000, n = 15
a
g kg
1
.
NA
NA
A.K. Kojta, J. Falandysz / Food Chemistry 200 (2016) 206–214
121 ± 50 (50–250) 120 131 ± 91 (40–310) 100 1.3 ± 0.94 (0.29–3.2) 1.1
Cap
209
210
A.K. Kojta, J. Falandysz / Food Chemistry 200 (2016) 206–214
The results obtained in this study are within the range reported in previous work by Kalacˇ (2009). Potassium concentrations in the samples were high, varying from 32 ± 1 to 51 ± 6 g kg 1 dw in caps and from 24 ± 1.6 to 49 ± 8 g kg 1 dw in stipes. Rudawska and Leski (2005a) and Mleczek et al. (2013) obtained similar results for K (caps: 36.5 ± 4.6 g kg 1 dw, stipes: 33.3 ± 2.8 g kg 1 dw), which could be attributed to essentiality and requirement of this element by fungus while less to the soil type (sandy) in the sites studied. Average concentrations of potassium in the fruiting bodies of B. badius ranged from 20.3 ± 2.5 g kg 1 dw (for the Pomeranian Voivodeship) to 23.9 ± 2.1 g kg 1 dw (for the Lower Silesian Voivodeship) (Mleczek et al., 2013). Magnesium concentrations were found to vary from 561 ± 40 to 923 ± 99 mg kg 1 dw in caps and from 357 ± 35 to 700 ± 83 mg kg 1 dw in the stipes. Mleczek et al. (2013) obtained similar results (319 ± 54–372 ± 91 mg kg 1 dw). Data published by Rudawska and Leski (2005a) showed higher Mg contents (caps: 1.2 ± 0.3, stipes: 0.7 ± 0.1 g Mg kg 1 dw). The mean sodium concentrations in B. badius were between 10 ± 5.0 and 574 ± 200 mg kg 1 dw in the caps and from 26 ± 15 to 1205 ± 442 mg kg 1 dw in the stipes. Stipes of Bay Bolete accumulated more sodium than the caps. Mleczek et al. (2013) reported sodium concentrations in the whole fruiting bodies that varied from 57 ± 15 (for specimens from Greater Poland Voivodeship) to 773 ± 20 mg kg kg 1 dw (Pomeranian Voivodeship). Recent publications provide data on levels of calcium, magnesium, mercury, potassium and phosphorus in the fruiting bodies of B. badius and the soil substratum (Falandysz & Bielawski, 2001; Falandysz et al., 2012; Malinowska, Szefer, & Falandysz, 2004; Mleczek et al. 2013). Elements in the samples were found at different levels in caps and stipes. The contents of K, P and Mg were higher in caps while the stipes contained more Ca (Kottke et al., 1998). Differences in the beneficial elements content of caps and stipes of fruiting bodies collected from different areas have been presented by Malinowska et al. (2004). 3.2. Fe, Mn, Zn and Hg in B. badius The concentrations of iron in the Bay Bolete fruiting bodies ranged from 33.6 ± 7.09 mg kg 1 dw in stipes harvested from Byd_ goszcz to 214 ± 162 mg kg 1 dw in caps from Zuromin. The highest level of this element found in B. badius stipes was 81 ± 23 mg kg 1 dw observed in specimens from Włocławek while the lowest concentration of 61 ± 22 mg kg 1 dw was found in caps obtained from Bydgoszcz. The iron content of this study are in good agreement with previously published data. The analyses of samples taken from Polish territory showed iron concentrations in whole fruiting bodies that varied from 38 to 129 mg kg 1 dw, while the contents of caps and stipes analyzed separately were 122 ± 12 and 50 ± 5.6 mg kg 1 dw, respectively (Rudawska & Leski, 2005a,b). Malinowska et al. (2004) published among others, data on the content of iron, manganese and zinc in the fruiting bodies of B. badius collected from 12 sites in the northern and north-eastern Poland. These researchers reported iron concentrations range from 34 ± 15 to 207 ± 58 mg kg 1 dw in caps and 32 ± 11 to 150 ± 66 mg kg 1 dw in the stipes. The results obtained by Ouzouni, Veltsistas, Paleologos, & Riganakos (2007) for the whole fruiting bodies of B. badius collected near the motorway in Kastamonu, Turkey, showed a higher mean iron concentrations of 377 ± 21 mg kg 1 dw. Manganese concentrations in the mushroom samples _ varied from 6.1 ± 1.2 (for specimens from Zuromin) to 26 ± 6.1 mg kg
1
stipes
6.2 ± 2.7
from
dw in caps (for specimens from Ke˛trzyn), and in _ (for specimens from Zuromin) to
118 ± 52 mg kg 1 dw (for specimens from Poniatowa). Bay Bolete caps contained more manganese than the stipes. Similar results were obtained by Malinowska et al. (2004) who reported Mn concentrations of between 9.8 ± 3.2 and 22 ± 5.1 mg kg 1 dw in the caps, and between 10 ± 3.5 and 53 ± 52 mg kg 1 dw in stipes. Rudawska and Leski (2005a) reported Mn concentrations in caps of 14 ± 1.2 mg kg 1 dw and 15 ± 3.2 mg kg 1 dw in the stipes. The same authors also published data for the whole fruiting bodies showing manganese range from 11 to 19.1 mg kg 1 dw (Rudawska & Leski, 2005b). The Mn results for the whole fruiting bodies collected near the motorway in Kastamonu (Turkey) was 25 ± 1.3 mg kg 1 dw. The zinc concentrations varied between 166 ± 1.6 and 294 ± 90 mg kg 1 dw in caps, and 111 ± 14 and 211 ± 69 mg kg 1 dw in the stipes. Studies of specimens from Poland reported a range from 109 ± 18 to 230 ± 82 mg kg 1 dw in stipes and 121 ± 24 to 230 ± 55 mg kg 1 dw in caps, while the data for the whole fruiting bodies ranged from 124 to 162 mg kg 1 dw (Falandysz et al., 2001; Rudawska & Leski, 2005a,b). Turkish researchers have published a mean of 64 ± 5.3 mg kg 1 dw in the whole fruiting bodies (Mendil, Uluo¯zlü, Hasdemir, & Cag˘lar, 2004). The Hg concentrations varied from 0.059 ± 0.028 (for samples _ from Zuromin) to 0.61 ± 0.25 mg kg 1 dw (for samples from Augustów Primeval Forest) in caps. In the stipes, the Hg concentra_ tions were from 0.024 ± 0.011 (for samples from Zuromin) to 0.26 ± 0.13 mg kg 1 dw (for samples from Augustów Primeval Forest). The results are consistent with previously published data (caps: 0.18 ± 0.050, 0.20 ± 0.070, 0.49 ± 0.30 mg kg 1 dw; stipes: 0.094 ± 0.045, 0.084 ± 0.028, 0.29 ± 0.20 mg kg 1 dw) (Falandysz et al., 2001; Chudzyn´ski & Falandysz, 2008). The obtained data on the total Hg content are also consistent with the results presented by Melgar, Alonso, and García (2009). 3.3. Metals’ cap to stalk quotients The capacity of this species to accumulate elements in parts of the fruiting bodies was evaluated by calculating the ratio of the concentrations in caps and stipes (Table 1). Concentrations in caps and stipes were higher than 1 in the case of Fe (Qc/s from _ 1.03 ± 0.38 – Chełmno to 5.1 ± 4.3 – Zuromin), K (1.04 ± 0.18 – Poniatowa to 1.4 ± 0.12 – Włocławek), Mg (1.3 ± 0.18 – Ke˛trzyn to 2.1 ± 0.64 – Pelplin), Hg (1.5 ± 0.23 – Ke˛trzyn to 3.6 ± 2.0 – _ Włocławek) and Zn (1.4 ± 0.19 – Zuromin to 1.6 ± 0.20 – Włocławek). The values shown above demonstrate an enhanced capacity for this species to accumulate mercury in the cap. In the case of Ca, Mn and Na, the ratios for most of the samples were less than one. _ Only in the case of samples from Ke˛trzyn (for Ca and Na), Zuromin (Ca, Mn) and Chełmno (Na) were values of Qc/s higher than 1. The lowest Qc/s values for Mn were observed for samples from Poniatowa (Qc/s 0.18 ± 0.13). Taking into account these results, it can be concluded that iron, potassium, magnesium, mercury, and zinc are readily accumulated in larger amounts in the caps, while calcium, manganese and sodium accumulate more in the stipes. 4. Statistical evaluation of the data Data obtained in this study were subjected to statistical procedures including multifunctional analysis. In order to demonstrate the possible spatial variability of mineral composition of B. badius, cluster analysis (CA) and principal component analysis (PCA) were performed. The obtained data were not normally distributed (Shapiro–Wilk test, p < 0.005) and the variances were not equal (Levene’s test, p < 0.005), so the Kruskal–Wallis test was used for comparing the contents of elements in the caps and stipes. Performing these nonparametric tests showed that there is a
A.K. Kojta, J. Falandysz / Food Chemistry 200 (2016) 206–214
significant difference in the content of all elements examined in caps, as well as the stipes (p < 0.0001). Cluster analysis was performed for the sampling sites. Hierarchical cluster analysis (Ward method, p < 0.05) allows to identify the most similar regions in terms of accumulation of tested elements in the fruiting bodies. Cluster analysis was performed separately for caps and stipes. The tree diagram for caps consisted of two main clusters. The first cluster consisted of samples from Augustów Primeval Forest while the second group consists of sam_ ples from Ke˛trzyn, Pelplin, Zuromin, Włocławek, Poniatowa, Chełmno, Bydgoszcz and several individual samples from Augustów Primeval Forest. The most similar regions were separated by clusters of the second degree of the second main cluster. Cluster analysis performed for stipes showed two main groups; the first consisted of samples from Augustów Primeval Forest, while the second group consisted of samples from Poniatowa, Chełmno,
211
_ Pelplin, Bydgoszcz, Zuromin, Ke˛trzyn, Włocławek, and several samples from Augustów Primeval Forest. As with the caps, the cluster of the second degree of the second main cluster are the most similar regions. Cluster analysis was also conducted for the elements determined (Ward method, p < 0.05). Tree diagram separated the elements in the caps and stipes into two main fractions, reflecting the interdependent relations between them. In the case of caps, the first fraction was composed of elements such as Ca, Mn, K, Mg and Zn, while the second fraction consisted of Fe, Na and Hg (Fig. 2). Cluster analysis performed for the stipes showed two clusters consisting of elements such as Ca, Fe, Na, Hg and K, Mg, Mn and Zn (Fig. 3). In summary, the most similar regions in terms of accumulation of elements in caps were Ke˛trzyn, Włocławek, Pelplin and Ponia_ towa, Chełmno, Bydgoszcz, and Zuromin. However, for the stipes,
Fig. 2. Tree diagram for the elements in the caps as objects (Ward’s Method, 1-Pearson r).
Fig. 3. Tree diagram for the elements in the stipes as objects (Ward’s Method, 1-Pearson r).
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areas with similar accumulation of elements were Pelplin, _ Zuromin, Bydgoszcz, Chełmno and Ke˛trzyn, Włocławek. Elements that accumulated in the caps in a similar manner were Ca, Mn, K, Mg and Zn, while the second group consisted of Fe, Na and Hg. On the other hand, for stipes, the first group was Ca, Fe, Na, Hg while the second consisted of K, Mg, Mn, Zn. Principal component analysis (PCA) allows the graphical presentation of the relationships that exist between the elements accumulated in the fruiting bodies. PCA was also performed to identify the principal components that represented the largest part of the variability in the data set. PCA carried out on the data showed three principal components (PCs), which together explained 71.9% of the observed variability. The first principal
component (PC1) explained 36.8% of the observed variability, PC2 explained 21.7%, while PC3 explained 13.4% of the observed variability. The first principal component was defined by elements such as K, Mg and Zn with a negative charge factor coordinates, second by Ca and Mn (positive charge factor coordinate) and the third principal component was represented by Fe (negative charge factor coordinate). Projection of these cases on the factor plane created in the PCA analysis was intended to illustrate which of the elements determine the PCs that are accumulated in similar amounts, and whether the sampling site affect their content in the fruiting bodies (Figs. 4 and 5). The results of the analysis indicated that the greatest differences in the concentrations were observed for elements such as K, Mg and Zn (in fruiting bodies) collected from
Fig. 4. Projection of the elements concentrations in the fruiting bodies set on the first and second factor-plane.
Fig. 5. Projection of the elements concentrations in the fruiting bodies set on the first and third factor-plane.
A.K. Kojta, J. Falandysz / Food Chemistry 200 (2016) 206–214
seven sampling sites (Fig. 4). The results showed a statistically significant difference in accumulation of the elements in the fruiting bodies collected from different areas. Caps of Bay Bolete harvested _ in Bydgoszcz, Włocławek and Zuromin differed slightly in terms of K, Mg and Zn contents from caps and stipes collected from Ke˛trzyn. Samples from these sites differed in terms of the elements described by PC2 (Fig. 4). The elements in the third principal component showed a significantly reduced variability in the contents described by PC3 (Fig. 5). 5. Intake rates and toxicological risks The European Union has not established limits for Hg in edible mushrooms. There are currently no Polish standards for the content of this element in wild edible mushrooms. Poland is in the European Union so the former limit established in Poland is without force. Possible health risks due to the intake of mercury accumulated in fruiting bodies can be assessed by comparing the estimated exposure to the recommended doses. Joint Expert Committee on Food Additives of the Food and Agriculture Organization of the United Nations and the World Health Organization (WHO, 1989; JECFA, 1978) issued a Provisional Tolerable Weekly Intake (PTWI; 0.005 mg Hg kg 1 body mass), an acceptable level of toxic metals that can be ingested within a week. Another indicator is the reference dose (RfD; 0.0004 mg Hg kg 1 body mass daily) set by the U.S. Environmental Protection Agency (US EPA, 2005). Reference dose is an estimate of daily human exposure to a substance without risk. To assess the possible risk due to the consumption of mercury accumulated in fruiting bodies, reference dose, and the value of the Provisional Tolerable Weekly Intake were used. The results obtained in this study indicate that mushrooms collected from the area of Augustów Primeval Forest slightly exceeded the reference dose for mercury calculated on a meal containing 300 g of fruiting bodies (caps: 0.0031 mg Hg kg 1 dw daily; stipes: 0.0013 mg Hg kg 1 dw daily). In the case of the PTWI, even eating large amounts of B. badius fresh caps (up to 4 kg per week) would not cause consumers to exceed this limit. 6. Conclusions The analysis of 126 samples of B. badius fruiting bodies was performed using validated methods to determine the nutritional value of the analyzed mushroom and toxicological risks associated with their consumption. Caps of Bay Bolete accumulated Ca, Mn, K, Mg, Zn and Fe, Na, Hg (CA, p < 0.005) in a similar manner. Principal component analysis showed the greatest variations in the concentrations of elements such as K, Mg and Zn in the fruiting bodies (p < 0.005). Mushroom B. badius consumed in a typical amount per week is safe with respect to the mercury content and intake dose. Acknowledgement The project was funded by the National Science Centre of Poland under of Decision No. UMO-2012/N/NZ7/00935. References Brzostowski, A., Falandysz, J., Jarzyn´ska, G., & Zhang, D. (2011). Bioconcentration potential of metallic elements by Poison Pax (Paxillus involutus) mushroom. Journal of Environmental Science and Health, Part A, 46, 378–393. Chudzyn´ski, K., & Falandysz, J. (2008). Multivariate analysis of elements content of Larch Bolete (Suillus grevillei). Chemosphere, 73, 1230–1239. Dimitrijevic, M. D., Mitic, J. J., Cvetkovic, J. S., Vesna, P., Jovanovic, S., Mutic, J. J., & Nikolic Mandic, S. D. (2015). Update on element content profiles in eleven wild
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