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Journal of Environmental Science and Health, Part B: Pesticides, Food Contaminants, and Agricultural Wastes Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/lesb20
Efficacy of supplementation of selected medicinal mushrooms with inorganic selenium salts a
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Przemysław Niedzielski , Mirosław Mleczek , Marek Siwulski , Monika Gąsecka , Lidia ad
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Kozak , Iwona Rissmann & Patrycja Mikołajczak a
Department of Analytical Chemistry, Faculty of Chemistry, Adam Mickiewicz University in Poznań, Poznań, Poland b
Department of Chemistry, Poznan University of Life Sciences, Poznań, Poland
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Department of Vegetable Crops, Poznan University of Life Sciences, Poznań, Poland
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Department of Food, Nutrition and Food Contact Materials, Poviat Sanitary and Epidemiological Station in Poznań, Poznań, Poland Published online: 13 Oct 2014.
To cite this article: Przemysław Niedzielski, Mirosław Mleczek, Marek Siwulski, Monika Gąsecka, Lidia Kozak, Iwona Rissmann & Patrycja Mikołajczak (2014) Efficacy of supplementation of selected medicinal mushrooms with inorganic selenium salts, Journal of Environmental Science and Health, Part B: Pesticides, Food Contaminants, and Agricultural Wastes, 49:12, 929-937, DOI: 10.1080/03601234.2014.951576 To link to this article: http://dx.doi.org/10.1080/03601234.2014.951576
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Journal of Environmental Science and Health, Part B (2014) 49, 929–937 Copyright © Taylor & Francis Group, LLC ISSN: 0360-1234 (Print); 1532-4109 (Online) DOI: 10.1080/03601234.2014.951576
Efficacy of supplementation of selected medicinal mushrooms with inorganic selenium salts 2 PRZEMYSºAW NIEDZIELSKI1, MIROSºAW MLECZEK2, MAREK SIWULSKI3, MONIKA GASECKA ˛ , 1,4 2 3 LIDIA KOZAK , IWONA RISSMANN and PATRYCJA MIKOºAJCZAK 1
Department of Analytical Chemistry, Faculty of Chemistry, Adam Mickiewicz University in Pozna n, Pozna n, Poland Department of Chemistry, Poznan University of Life Sciences, Pozna n, Poland 3 Department of Vegetable Crops, Poznan University of Life Sciences, Pozna n, Poland 4 Department of Food, Nutrition and Food Contact Materials, Poviat Sanitary and Epidemiological Station in Pozna n, Pozna n, Poland
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The aim of the study was to evaluate the possibility of supplementation with inorganic forms of selenium (Na2SeO4 and Na2SeO3) in concentrations of 0, 0.1, 0.2, 0.4, 0.6, 0.8, 1.0 and 1.5 mM of three medicinal mushroom species: Agrocybe aegerita, Hericium erinaceus and Ganoderma lucidum. Tested mushroom species grew in Se additions of 0–0.6 mM (A. aegerita and H. erinaceus), while growth of G. lucidum bodies was observed for 0–0.8 mM. For the latter mushroom species, the total Se content was the highest. Content of Seorg was diverse; for control bodies it was the highest for G. lucidum (only organic forms were present), lower for A. aegerita (84% organic forms) and the lowest for H. erinaceus (56% organic forms). Accumulation of Se(IV) was generally significantly higher than Se(VI) for all tested mushroom species. There was no significant decrease of A. aegerita or G. lucidum biomass with the exception of G. lucidum bodies growing under 0.8 mM of Se species addition (15.51 § 6.53 g). Biomass of H. erinaceus bodies was the highest under 0.2 (197.04 § 8.73 g), control (191.80 § 6.06 g) and 0.1 mM (185.04 § 8.73 g) of both inorganic salts. The addition to the medium of Se salts brought about macroscopic changes in the fruiting bodies of the examined mushrooms. Concentrations exceeding 0.4 mM caused diminution of carpophores or even their total absence. In addition, colour changes of fruiting bodies were also recorded. At Se concentrations of 0.4 and 0.6 mM, A. aegerita fruiting bodies were distinctly lighter and those of H. erinaceus changed colour from purely white to white-pink. Keywords: Selenium, Agrocybe aegerita, Ganoderma lucidum, Hericium erinaceus, medicinal mushroom.
Introduction Selenium (Se) is a non-metal and is considered important for both humans and animals. This essential micronutrient may be protective for a few types of cancer by various mechanisms[1] and has antioxidative action.[2] Brozmanova et al.[3] pointed out the significant role of appropriate intake of total Se and particular species of this non-metal in correct human health. Se has a positive role in diet supplementation, cancer therapy and together with zinc ions has – in oral supplement form – metabolic effects in patients with liver cirrhosis/cancer.[4] On the other hand, Se may cause the dissemination and increased severity of disease,[5] and negative effects such as toxicity and DNA damage in the case of food collected from Se-polluted areas.[6] Generally, the most common situation is Se deficiency rather than its
Address correspondence to Miros»aw Mleczek, Department of Chemistry, Pozna n University of Life Sciences, Pozna n, Poland; E-mail: [email protected] Received May 21, 2014.
excess;[7] therefore, supplementation of Se is important regardless of the way of addition of this element.[8] Se in free form performs almost no physiological role, but linked with a chemical group or biomolecules (especially with amino acids) is a significant element responsible for correct functioning of living organisms. The most important are organic forms of Se, e.g., selenocysteine (Sec) and selenomethionine (Se-Met), with absorbency about 95% and 1.5 to 2-fold higher than the inorganic form of Se;[9] therefore, Se supplementation should be related mainly to accumulation of organic forms in mushroom bodies. According to the Scientific Committee on Food (SCF), the mean intake of Se by the European population is in the range 24–89 mg day¡1. Moreover, clinical studies on supplementation using Se-enriched yeast with presence of selenomethionine species in higher amounts showed no toxicity when Se intake was up to 343 mg day¡1.[9] Mushrooms are commonly used[10] and may contain diverse amounts of macro- and micronutrients, depending on their bioavailability. Cocchi et al.[11] tested the Se content in 60 species of edible mushrooms and described a
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wide range from 0.20 (Amanita rubescens) to 107 mg kg¡1 fresh matter (f.m.) (Boletus pinophilus) with relatively low content in Agrocybe aegerita (1.14–1.75 mg kg¡1 f.m.). Additionally, Kalac[12] presented the results of selected trace elements and selected European edible mushroom species, showing Se content from 20 mg kg¡1 DW (Boletus edulis). Additionally, Stijve et al.[13] mentioned Arbatrellus pescaprae, which is the most effective Se-accumulating mushroom (about 200 mg kg¡1 DW). Probably, the most interesting as regards the practical use of Se-enriched mushroom are the medicinal mushroom species, considering the many precious components such as free amino acids, soluble sugars,[14] and numerous other substances including bioactive proteins[15,16] and carbohydrates[17]. The most popular of them are Agaricus blazei, Agrocybe aegerita, Cordyceps sinensis,[18] Coriolus versicolor,[19] Ganoderma lucidum,[20] Hericium erinaceus[21,22] and Lentinula edodes.[23] The consequences of chemical characteristics of these mushrooms are specific traits, thanks to which they are able to protect DNA from hydroxyl radical oxidative damage,[24] exhibit anticancer and immunostimulating activity,[25] and prevent hypertension, high cholesterol levels, overweight or insomnia. Some medicinal mushrooms, e.g. Lentinula edodes, have the ability to biotransform an organic selenium compound (diacetophenonyl selenide) into elemental selenium, as described by Tsivileva et al.[26] The aim of this work was to assess the possibility of Se supplementation with inorganic Se salts (sodium selenite Na2SeO3 and sodium selenate Na2SeO4) of three medicinal mushroom species, Agrocybe aegerita, Hericium erinaceus and Ganoderma lucidum, with analysis of their biomass. Additionally, observation of macroscopic changes in tested mushroom fruiting bodies was performed.
concentrations of both selenium salts in the substrate were applied: 0.1, 0.2, 0.4, 0.6, 0.8, 1.0 and 1.5 mM of Na2SeO3 and Na2SeO4 (VI) (from Sigma-Aldrich). The substrates with selenium addition were mixed with grain spawn (on wheat grain) of the examined mushroom species (5% of substrate weight) and placed in polypropylene bottles of 1 dm3 volume. Each bottle was filled with 350 g of the substrate and closed with a cover with a filter. The bottles were used for H. erinaceus and A. aegerita. In the case of G. lucidum, 15 £ 30 cm polypropylene foil bags with a filter were used. Each bag contained the same quantity of the substrate as the above-described bottles. The incubation was conducted at the temperature of 25 C and 80–85% air relative humidity until the substrate became completely covered with mycelium. Next, the bottles with removed covers and bags with the top part of the foil cut off were placed in the cultivation chamber. For fructification, air relative humidity was maintained at 85–90% and temperature at 16 § 1 C for H. erinaceus, 18 § 1 C for A. aegerita and 25 § 1 C for G. lucidum. The cultivation was additionally illuminated with fluorescent light of 500 l£ intensity 12 h a day. The growth facility was aerated in such a way as to maintain CO2 concentration below 1000 ppm. Carpophores were harvested successively as they matured. Yield included whole fruiting bodies.
Materials and methods
Extraction procedure
Experiment design The substrate was prepared from a mixture of beech sawdust and flax shives (3:1 vol.) which was additionally supplemented with wheat bran in the amount of 20%, corn flour 5% and gypsum 1% in relation to the substrate dry matter (d.m.). The mixture was wetted with distilled water to the moisture content of 45%. The substrate prepared as described above was placed in polypropylene bags and sterilized at the temperature of 121 C for 1 h and next was cooled down to the temperature of 25 C. Selenium salts – sodium selenite Na2SeO3 (IV) and sodium selenate Na2SeO4 (VI)[27] – were dissolved in sterile water and the solution was added to the substrate (stirring intensely) in the amount allowing their appropriate concentration in the substrate and, at the same time, making sure that the substrate water content reached 60%. The following
Mushroom sample collection Collected mushroom fruiting bodies (caps and stipes together) were weighed, dried in an electric drier (SLW 53 STD, Pol-Eko) at 50 § 2 C for 48 h, weighed again for dry matter analysis and ground for 0.5 min in Cutting Boll Mill 200 by RETSCH. Three representative powdered samples for mushrooms growing in each experimental system were treated in the extraction procedure.
The extraction procedure was based on the work of Gonzalvez et al.;[28] it was modified to conditions needed for hyphenated system determination and has been described previously.[29] 1.0 g of the mushroom sample (homogenised by rubbing and sieving through a 0.02 mm sieve) was put into a glass flask containing 10 mL of 1.0 M phosphoric acid and extracted in an ultrasonic bath (30 min at ambient temperature). Next the solution was filtered by filter paper, and washed with 200 mL of water and 20 mL of phosphoric buffer or centrifuged. The pH of the solution was adjusted to 6.0–6.5 by addition of 10 mol/L solution of NaOH, and finally the solution was diluted to 20 mL by phosphate buffer. The Se species in the acid extracts were determined by hyphenated high performance liquid chromatography with hydride generation atomic absorption spectrometry detection (HPLC-HGAAS), immediately after the extraction procedure.
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Instrumentation HPLC-HG-AAS systems were constructed as described in previous papers.[29,30] In the present work, two independent analytical systems were used for Se species determination. The results are the mean of duplicate determination. The use of two analytical systems was not accidental but intended to ensure the quality of the results.[31,32] The first hyphenated analytical system consisted of a Shimadzu liquid chromatograph (LC-10A) equipped with a HPLC pump (LC-10AT), vacuum degasser unit (GT104), Rheodyne PEEK valve (IDEX, USA) and Supelco LC-SAX1 anion-exchange column (250 mm, 4.6 mm i.d., resin particle size 5mm) thermostatted by a column oven (CTO-10ASvp). The chromatographic run was isocratic at 3 mL min¡1 with an injection volume of 200 mL. The measurements were performed with a SpectrAA 220FS spectrometer (Varian, Australia) with an UltrAA Se hollow cathode lamp. The second analytical system was set up using the HPLC pump ProStar 240 Ternary Solvent Delivery Module (Varian, Australia), the Rheodyne PEEK (IDEX) chromatographic valve, the Zorbax SAX (250 mm length, 4.6 mm i.d.) HPLC column (Agilent, USA) and SpectrAA 280FS atomic absorption spectrometer (Agilent, USA) as a chromatographic detector. The chromatographic run was isocratic at 2.5 mL min¡1 with an injection volume of 200 mL. The Se species Se(VI) does not form a volatile hydride, so in order to obtain an analytical signal in the HG-AAS system it was necessary to perform preliminary reduction of Se(VI) to Se(IV). The reduction was carried out on-line by heating the sample (90–100 C) with a reducing agent: 0.5 mol/L thiourea solution in 10 mol/L hydrochloric acid.[29] The eluate from the chromatographic column was joined with the stream of the reducing agent (flow rate 1 mL min¡1 from the peristaltic pump) through a T-shape coupling and directed to the Tygon capillary loop (inner diameter 0.82 mm) heated on a water bath. The capillary loop outlet was connected to the hydride generation system. For both analytical systems the PEEK transfer tubing of the eluent from the LC column to the hydride generation unit was inserted into a Tygon sleeve. The continuous hydride generation system (VGA-77, Varian) consisted of a manually controlled, four-channel peristaltic pump with Tygon tubing (0.6 mm i.d.), one reaction coil (PTFE tubing 0.8 mm i.d., 75 cm length) and three-way connectors. The gas-liquid separator was made from glass and the interior dead volume was 3 mL. For atomization of the Se hydrides (detected at 196.0 nm), a heating controller, electrothermally heating mantle and a quartz tube (ETC-60, Varian) heated to 900 C were used. The chromatograms were displayed on the screen of a personal computer by using the signal graphics option of the AAS software (Varian), and as a separate print-out of the screen. Peak areas and height were calculated by the AAS software.
A number of validation parameters characterising the analytical method were determined. The limits of detection 0.1 mg kg¡1 for Se(IV) and Se(VI) respectively and the uncertainty of results (measured as RSD) at the level of 10% for both Se forms were obtained. As it was impossible to estimate the measurement traceability because of the lack of any certified reference materials for determination of inorganic Se species, the recovery of each Se species was measured upon addition of a standard to the sample. The recovery of 96–105% of each species was satisfactory. Total Se concentration was measured using electrothermal atomic absorption spectrometry with Zeeman background correction. The SpectrAA 280Z (Agilent) instrument with pyrolytic graphite tubes and Se hollow cathode lamp (wavelength 196.0 nm, slit 1.0 nm, current 10 mA) was used. The temperature programme was optimized: drying at 85–120 C for 55 s; ashing at 1000 C for 8 s; atomization at 2600 C. The palladium solution (10 mL of 500 mg/L for 20 mL of sample) was used as a chemical modifier. The limit of detection 0.01 mg kg¡1 and the uncertainty of results (measured as RSD) at the level of 5.0% were obtained. The traceability was measured by a standard addition procedure.
Gases and reagents Compressed argon gas of N-50 purity (99.999%) obtained from Linde (Poland) was employed as the carrier gas for Se vapour to the quartz cell without further purification. Water was redistilled and further purified with a Milli-Q water purification system (Millipore, USA). Standard solutions (1000 mg/L of Se) of selenite and selenate were prepared by dissolving appropriate amounts of sodium selenite (Na2SeO3), sodium selenate (Na2SeO4) and selenomethionine respectively, obtained from Sigma-Aldrich (USA). For total Se determination a commercial Se standard (Merck, Germany) was used. The standard stock solutions were stored in glass bottles at 4 C in darkness. Low concentration standards obtained by dilution of the stock solutions were prepared daily. Sodium tetrahydroborate (III), used as reducing solution (3% w/w), was prepared daily, by dissolving NaBH4 (Merck) in high-purity water and stabilizing with 1% (w/w) NaOH (Merck) solution to reduce its rate of decomposition. The solution was used without filtration. The HCl solution was of the highest quality grade (Suprapure, Merck). Thiourea of analytical quality grade was obtained from Merck. The buffered mobile phase was prepared by mixing together disodium hydrophosphate (Na2HPO4) 100 mmol/L and potassium dihydrophosphate (KH2PO4. 2H2O) 10 mmol/L obtained from Merck. Palladium solution (500 mg/L of palladium as nitrate) was prepared from commercial palladium chemical modifiers (Merck).
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Statistical analysis and calculations Results recorded in the course of the performed analyses were subjected to statistical analysis with STATISTICA v. 8.0 software. In order to compare content of Se in mushrooms of the same species, growing in particular systems, Tukey’s multiple comparison procedure was used, with the results marked with identical letters in rows or columns exhibiting no differences at the significance level a D 0.05. One-way analysis of variance was performed. The relationships between content of organic and inorganic Se forms in relation to addition of both Se species to the substrate were estimated by the correlation coefficient and nonlinear regression analyses together with the analysis of the single curvilinear regression effects and with the coefficient of determination (R2), as a measure of the good fit of regression.[33] Efficiency of Se species accumulation was characterized by the bioconcentration factor (BCF), calculated as the ratio of Se species content in the fruiting bodies to the content of this element in the substrate where the mushroom was grown. BCF values >1 indicate Se species accumulation, while BCF 1) for all tested mushroom species growing under all Se additions with the exception of A. aegerita growing
Table 1. Bioconcentration factor (BCF) values for particular Se species. Se addition [mM]
Se(IV)
Se(VI)
H. erinaceus 0.14 1.33 0.16 0.50 0.32 1.00 0.24 0.30 A. aegerita 0.02 2.30 10.87 2.00 0.09 0.23 0.14 0.01 G. lucidum 0.84 0.01 0.81 0.05 0.80 2.05 0.57 1.95 0.50 0.02
0.1 0.2 0.4 0.6 0.1 0.2 0.4 0.6 0.1 0.2 0.4 0.6 0.8
Seinorg
Seorg
Setotal
0.15 0.16 0.32 0.24
7.11 5.60 6.71 4.08
0.56 0.59 0.59 0.50
0.03 12.54 0.09 0.11
3.07 0.22 1.25 6.86
0.36 0.28 0.52 0.55
0.76 0.75 0.86 0.68 0.45
1.32 1.97 2.41 1.27 4.05
0.97 1.02 1.04 0.91 1.14
under 0.2 mM (BCF D 0.22). It is worth underlining the high BCF calculated for Se(IV) in A. aegerita bodies growing under 0.2 mM (BCF D 10.87). Mushroom biomass Each tested mushroom species was able to grow on substrates with Se salt supplementation 0.6 mM for each of them. Only G. lucidum fruiting bodies grew on substrates with 0.8 mM of both Se salts, but the biomass of them was significantly lower than mushrooms growing in other systems (Table 2). In the case of A. aegerita and G. lucidum bodies, there was not a significant decrease in biomass of these mushroom species with the exception of G. lucidum bodies growing under 0.8 mM of Se species addition (15.51 § 6.53 g). Biomass of H. erinaceus bodies was the highest under 0.2 (197.04 § 8.73 g), control (191.80 § 6.06 g) and
Table 2. Biomass [g] and biological efficiency (BE) of tested mushroom fruiting bodies. A. aegerita System
Biomass [g]
Control 0.1 0.2 0.4 0.6 0.8 1.0 1.5
178.25 § 6.51 190.76 § 12.84a 170.74 § 29.66a 177.22 § 11.87a 111.44 § 45.76a nb nb nb a
H. erinaceus BE [%]
Biomass [g]
50.93 54.50 48.78 50.63 31.84 nb nb nb
191.80 § 6.06 185.04 § 8.73ab 197.90 § 19.08a 141.58 § 10.72b 38.32 § 27.81c nb nb nb ab
G. lucidum BE [%]
Biomass [g]
BE [%]
54.80 52.87 56.54 40.45 10.95 nb nb nb
96.49 § 3.80 85.58 § 5.68a 121.92 § 7.73a 90.24 § 25.48a 117.36 § 12.84a 15.51 § 6.53b nb nb
27.57 24.45 34.83 25.78 33.53 4.43 nb nb
a
nb – no fruiting bodies. a,b,c Mean values (n D 3) § standard deviations; identical superscripts denote no significant (p < 0.05) difference between mean values in column according to Tukey’s HDS test (MANOVA).
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Fig. 2. Macroscopic changes in A. aegerita fruiting bodies.
0.1 mM (185.04 § 8.73 g) of both inorganic salts. For the other Se additions (0.4 and 0.6 mM), the biomass of H. erinaceus was significantly lower. BE calculated for each tested mushroom was higher for A. aegerita and H. erinaceus than for G. lucidum. The highest BE values for A. aegerita, H. erinaceus and G. lucidum bodies were 54.50, 54.80 and 34.83%, respectively, for bodies growing on substrate with 0.1 and 0.2 mM of Se (IV) and Se(VI) additions. The higher BE values calculated for bodies with Se addition than control bodies indicates the positive influence of Se supplementation on mushroom biomass stimulation. It is worth underlining that dry matter of A. aegerita and H. erinaceus was similar and significantly lower than dry matter of G. lucidum (Table 3). Presented data are important as regards the crop in practical use of tested mushroom species. The knowledge of Table 3. Dry matter [%] of tested fruiting bodies. System
A. aegerita
H. erinaceus
G. lucidum
Control 0.1 0.2 0.4 0.6 0.8 1.0 1.5
8.20 § 0.22b 9.10 § 0.16ab 8.80 § 0.08ab 8.93 § 0.29ab 9.60 § 0.51a nb nb nb
8.60 § 0.22a 9.17 § 0.26a 8.67 § 1.04a 10.33 § 0.31a 11.60 § 2.01a nb nb nb
41.80 § 1.00a 44.37 § 7.93a 34.10 § 5.05abc 39.47 § 10.73ab 21.43 § 0.93bc 17.43 § 3.15 nb nb
nb – no fruiting bodies. a,b,c Mean values (n D 3) § standard deviations; identical superscripts denote no significant (p < 0.05) difference between mean values in column according to Tukey’s HDS test (MANOVA).
biomass and BE level is essential in the planning of commercial mushroom cultivation. For graphical presentation of changes in biomass of tested mushroom species, in Figures 2–4, the macroscopic changes of mushroom fruiting bodies are presented. The first macroscopic changes (white parts of the cap border) of A. aegerita mushroom bodies under 0.4 mM of both Se salts, observed during supplementation with 0.6 mM, were in the case of mushroom fruiting bodies’ loss of colour. Additionally, fruiting bodies of this mushroom species growing on substrates with 0.4 and 0.6 mM addition were smaller than other collected bodies. In the case of white H. erinaceus, mushrooms were completely white. The slightly pink colour of fruiting bodies growing under 0.1 and 0.2 mM of Se salts was observed only, which suggests the possible start of a mechanism of inorganic Se detoxification and reduction of some Se into elementary Se nanoparticles with pink colour, as was described by Falcone and Nickerson[34] and Gharieb et al.[35] The number of carpophores formed by G. lucidum declined together with the increase of Se concentrations from 0.6 mM to complete absence of their formation when the concentration exceeded 0.8 mM. Moreover, the size of carpophores decreased when the concentration exceeded 0.4 mM.
Discussion About 25 proteins present in the human body need Se for their correct functioning; therefore, supplementation
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Fig. 3. Macroscopic changes in H. erinaceus fruiting bodies.
with inorganic Se species with significantly higher Seorg species content, e.g., for tested A. aegerita, pointed to the possibility to prepare mushroom supplements with higher Se or other elements (Cu, Zn) content. According to Navarro-Alarcon and Cabrera-Vique,[36] the daily requirement of Se is in the range 150–200 mg day¡1, while according to Dietary Reference Intakes, the recommended daily intake/adequate intake (RDA/AI)[37] of
Fig. 4. Macroscopic changes in G. lucidum fruiting bodies.
Se is in the range from 15 (infants) to 70 (lactation) mg day¡1 for different life stage groups. Taking into consideration the results presented in this work, especially the content of Seorg indicated that intake of tested medicinal mushroom species is related to too great exposure of the human organism to Se. Hartikainen[38] stated that an optimal Se addition level is important (for human health) as regards transport of this non-metal in the food chain.
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936 Accordingly, we suggest preparing a powder from the mushroom species tested in the experiment and developing a supplement for direct use with specifically determined Seorg and Seinorg contents. In cases where the content of inorganic species of Se is higher than organic Se, e.g., G. lucidum, there could be biotransformation of inorganic selenite present in the substrate into organic forms.[39] Dan[40] pointed out the possibility to use Se-rich Shiitake and Se-rich oyster mushrooms for preparation of new natural health products. The potential of Se-enriched edible mushrooms was also summarised by Ling et al.[40] Mao et al.[41] described the ability of fungus As-1 to accumulate Se growing on substrate supplemented with sodium selenite. The authors reported bodies’ growth inhibition when the Na2SeO4 concentration was 5 mg/L and mycelia growth inhibition when the Na2SeO4 concentration was 50 mg/L. Mushroom species supplemented with Se tested in this work can be used in practice, as previously reported by Guoyuan.[42] Stajic et al.[43] analysed the influence of Se addition on Pleurotus ostreatus (Jacq.: Fr.) growth, finding no significant influence of this non-metal on mycelial growth, as was also found in this work for other mushroom species. An interesting comparison of the present results can be made with the studies described by Savic et al.,[44] who tested accumulation of Se and growth of Pleurotus ostreatus (Hk-35 and P70) supplemented with the same inorganic salts. The authors reported stimulation effects on growth of mushroom bodies growing under 1–50 mg/L and toxic effects for mushrooms under 75, 100 and 150 mg/L. Results presented in this work indicated no growth of mycelium for three tested mushroom species in systems with addition of Se salts
Journal of Environmental Science and Health, Part B: Pesticides, Food Contaminants, and Agricultural Wastes Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/lesb20
Efficacy of supplementation of selected medicinal mushrooms with inorganic selenium salts a
b
c
b
Przemysław Niedzielski , Mirosław Mleczek , Marek Siwulski , Monika Gąsecka , Lidia ad
b
c
Kozak , Iwona Rissmann & Patrycja Mikołajczak a
Department of Analytical Chemistry, Faculty of Chemistry, Adam Mickiewicz University in Poznań, Poznań, Poland b
Department of Chemistry, Poznan University of Life Sciences, Poznań, Poland
c
Department of Vegetable Crops, Poznan University of Life Sciences, Poznań, Poland
d
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Department of Food, Nutrition and Food Contact Materials, Poviat Sanitary and Epidemiological Station in Poznań, Poznań, Poland Published online: 13 Oct 2014.
To cite this article: Przemysław Niedzielski, Mirosław Mleczek, Marek Siwulski, Monika Gąsecka, Lidia Kozak, Iwona Rissmann & Patrycja Mikołajczak (2014) Efficacy of supplementation of selected medicinal mushrooms with inorganic selenium salts, Journal of Environmental Science and Health, Part B: Pesticides, Food Contaminants, and Agricultural Wastes, 49:12, 929-937, DOI: 10.1080/03601234.2014.951576 To link to this article: http://dx.doi.org/10.1080/03601234.2014.951576
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Journal of Environmental Science and Health, Part B (2014) 49, 929–937 Copyright © Taylor & Francis Group, LLC ISSN: 0360-1234 (Print); 1532-4109 (Online) DOI: 10.1080/03601234.2014.951576
Efficacy of supplementation of selected medicinal mushrooms with inorganic selenium salts 2 PRZEMYSºAW NIEDZIELSKI1, MIROSºAW MLECZEK2, MAREK SIWULSKI3, MONIKA GASECKA ˛ , 1,4 2 3 LIDIA KOZAK , IWONA RISSMANN and PATRYCJA MIKOºAJCZAK 1
Department of Analytical Chemistry, Faculty of Chemistry, Adam Mickiewicz University in Pozna n, Pozna n, Poland Department of Chemistry, Poznan University of Life Sciences, Pozna n, Poland 3 Department of Vegetable Crops, Poznan University of Life Sciences, Pozna n, Poland 4 Department of Food, Nutrition and Food Contact Materials, Poviat Sanitary and Epidemiological Station in Pozna n, Pozna n, Poland
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The aim of the study was to evaluate the possibility of supplementation with inorganic forms of selenium (Na2SeO4 and Na2SeO3) in concentrations of 0, 0.1, 0.2, 0.4, 0.6, 0.8, 1.0 and 1.5 mM of three medicinal mushroom species: Agrocybe aegerita, Hericium erinaceus and Ganoderma lucidum. Tested mushroom species grew in Se additions of 0–0.6 mM (A. aegerita and H. erinaceus), while growth of G. lucidum bodies was observed for 0–0.8 mM. For the latter mushroom species, the total Se content was the highest. Content of Seorg was diverse; for control bodies it was the highest for G. lucidum (only organic forms were present), lower for A. aegerita (84% organic forms) and the lowest for H. erinaceus (56% organic forms). Accumulation of Se(IV) was generally significantly higher than Se(VI) for all tested mushroom species. There was no significant decrease of A. aegerita or G. lucidum biomass with the exception of G. lucidum bodies growing under 0.8 mM of Se species addition (15.51 § 6.53 g). Biomass of H. erinaceus bodies was the highest under 0.2 (197.04 § 8.73 g), control (191.80 § 6.06 g) and 0.1 mM (185.04 § 8.73 g) of both inorganic salts. The addition to the medium of Se salts brought about macroscopic changes in the fruiting bodies of the examined mushrooms. Concentrations exceeding 0.4 mM caused diminution of carpophores or even their total absence. In addition, colour changes of fruiting bodies were also recorded. At Se concentrations of 0.4 and 0.6 mM, A. aegerita fruiting bodies were distinctly lighter and those of H. erinaceus changed colour from purely white to white-pink. Keywords: Selenium, Agrocybe aegerita, Ganoderma lucidum, Hericium erinaceus, medicinal mushroom.
Introduction Selenium (Se) is a non-metal and is considered important for both humans and animals. This essential micronutrient may be protective for a few types of cancer by various mechanisms[1] and has antioxidative action.[2] Brozmanova et al.[3] pointed out the significant role of appropriate intake of total Se and particular species of this non-metal in correct human health. Se has a positive role in diet supplementation, cancer therapy and together with zinc ions has – in oral supplement form – metabolic effects in patients with liver cirrhosis/cancer.[4] On the other hand, Se may cause the dissemination and increased severity of disease,[5] and negative effects such as toxicity and DNA damage in the case of food collected from Se-polluted areas.[6] Generally, the most common situation is Se deficiency rather than its
Address correspondence to Miros»aw Mleczek, Department of Chemistry, Pozna n University of Life Sciences, Pozna n, Poland; E-mail: [email protected] Received May 21, 2014.
excess;[7] therefore, supplementation of Se is important regardless of the way of addition of this element.[8] Se in free form performs almost no physiological role, but linked with a chemical group or biomolecules (especially with amino acids) is a significant element responsible for correct functioning of living organisms. The most important are organic forms of Se, e.g., selenocysteine (Sec) and selenomethionine (Se-Met), with absorbency about 95% and 1.5 to 2-fold higher than the inorganic form of Se;[9] therefore, Se supplementation should be related mainly to accumulation of organic forms in mushroom bodies. According to the Scientific Committee on Food (SCF), the mean intake of Se by the European population is in the range 24–89 mg day¡1. Moreover, clinical studies on supplementation using Se-enriched yeast with presence of selenomethionine species in higher amounts showed no toxicity when Se intake was up to 343 mg day¡1.[9] Mushrooms are commonly used[10] and may contain diverse amounts of macro- and micronutrients, depending on their bioavailability. Cocchi et al.[11] tested the Se content in 60 species of edible mushrooms and described a
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wide range from 0.20 (Amanita rubescens) to 107 mg kg¡1 fresh matter (f.m.) (Boletus pinophilus) with relatively low content in Agrocybe aegerita (1.14–1.75 mg kg¡1 f.m.). Additionally, Kalac[12] presented the results of selected trace elements and selected European edible mushroom species, showing Se content from 20 mg kg¡1 DW (Boletus edulis). Additionally, Stijve et al.[13] mentioned Arbatrellus pescaprae, which is the most effective Se-accumulating mushroom (about 200 mg kg¡1 DW). Probably, the most interesting as regards the practical use of Se-enriched mushroom are the medicinal mushroom species, considering the many precious components such as free amino acids, soluble sugars,[14] and numerous other substances including bioactive proteins[15,16] and carbohydrates[17]. The most popular of them are Agaricus blazei, Agrocybe aegerita, Cordyceps sinensis,[18] Coriolus versicolor,[19] Ganoderma lucidum,[20] Hericium erinaceus[21,22] and Lentinula edodes.[23] The consequences of chemical characteristics of these mushrooms are specific traits, thanks to which they are able to protect DNA from hydroxyl radical oxidative damage,[24] exhibit anticancer and immunostimulating activity,[25] and prevent hypertension, high cholesterol levels, overweight or insomnia. Some medicinal mushrooms, e.g. Lentinula edodes, have the ability to biotransform an organic selenium compound (diacetophenonyl selenide) into elemental selenium, as described by Tsivileva et al.[26] The aim of this work was to assess the possibility of Se supplementation with inorganic Se salts (sodium selenite Na2SeO3 and sodium selenate Na2SeO4) of three medicinal mushroom species, Agrocybe aegerita, Hericium erinaceus and Ganoderma lucidum, with analysis of their biomass. Additionally, observation of macroscopic changes in tested mushroom fruiting bodies was performed.
concentrations of both selenium salts in the substrate were applied: 0.1, 0.2, 0.4, 0.6, 0.8, 1.0 and 1.5 mM of Na2SeO3 and Na2SeO4 (VI) (from Sigma-Aldrich). The substrates with selenium addition were mixed with grain spawn (on wheat grain) of the examined mushroom species (5% of substrate weight) and placed in polypropylene bottles of 1 dm3 volume. Each bottle was filled with 350 g of the substrate and closed with a cover with a filter. The bottles were used for H. erinaceus and A. aegerita. In the case of G. lucidum, 15 £ 30 cm polypropylene foil bags with a filter were used. Each bag contained the same quantity of the substrate as the above-described bottles. The incubation was conducted at the temperature of 25 C and 80–85% air relative humidity until the substrate became completely covered with mycelium. Next, the bottles with removed covers and bags with the top part of the foil cut off were placed in the cultivation chamber. For fructification, air relative humidity was maintained at 85–90% and temperature at 16 § 1 C for H. erinaceus, 18 § 1 C for A. aegerita and 25 § 1 C for G. lucidum. The cultivation was additionally illuminated with fluorescent light of 500 l£ intensity 12 h a day. The growth facility was aerated in such a way as to maintain CO2 concentration below 1000 ppm. Carpophores were harvested successively as they matured. Yield included whole fruiting bodies.
Materials and methods
Extraction procedure
Experiment design The substrate was prepared from a mixture of beech sawdust and flax shives (3:1 vol.) which was additionally supplemented with wheat bran in the amount of 20%, corn flour 5% and gypsum 1% in relation to the substrate dry matter (d.m.). The mixture was wetted with distilled water to the moisture content of 45%. The substrate prepared as described above was placed in polypropylene bags and sterilized at the temperature of 121 C for 1 h and next was cooled down to the temperature of 25 C. Selenium salts – sodium selenite Na2SeO3 (IV) and sodium selenate Na2SeO4 (VI)[27] – were dissolved in sterile water and the solution was added to the substrate (stirring intensely) in the amount allowing their appropriate concentration in the substrate and, at the same time, making sure that the substrate water content reached 60%. The following
Mushroom sample collection Collected mushroom fruiting bodies (caps and stipes together) were weighed, dried in an electric drier (SLW 53 STD, Pol-Eko) at 50 § 2 C for 48 h, weighed again for dry matter analysis and ground for 0.5 min in Cutting Boll Mill 200 by RETSCH. Three representative powdered samples for mushrooms growing in each experimental system were treated in the extraction procedure.
The extraction procedure was based on the work of Gonzalvez et al.;[28] it was modified to conditions needed for hyphenated system determination and has been described previously.[29] 1.0 g of the mushroom sample (homogenised by rubbing and sieving through a 0.02 mm sieve) was put into a glass flask containing 10 mL of 1.0 M phosphoric acid and extracted in an ultrasonic bath (30 min at ambient temperature). Next the solution was filtered by filter paper, and washed with 200 mL of water and 20 mL of phosphoric buffer or centrifuged. The pH of the solution was adjusted to 6.0–6.5 by addition of 10 mol/L solution of NaOH, and finally the solution was diluted to 20 mL by phosphate buffer. The Se species in the acid extracts were determined by hyphenated high performance liquid chromatography with hydride generation atomic absorption spectrometry detection (HPLC-HGAAS), immediately after the extraction procedure.
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Instrumentation HPLC-HG-AAS systems were constructed as described in previous papers.[29,30] In the present work, two independent analytical systems were used for Se species determination. The results are the mean of duplicate determination. The use of two analytical systems was not accidental but intended to ensure the quality of the results.[31,32] The first hyphenated analytical system consisted of a Shimadzu liquid chromatograph (LC-10A) equipped with a HPLC pump (LC-10AT), vacuum degasser unit (GT104), Rheodyne PEEK valve (IDEX, USA) and Supelco LC-SAX1 anion-exchange column (250 mm, 4.6 mm i.d., resin particle size 5mm) thermostatted by a column oven (CTO-10ASvp). The chromatographic run was isocratic at 3 mL min¡1 with an injection volume of 200 mL. The measurements were performed with a SpectrAA 220FS spectrometer (Varian, Australia) with an UltrAA Se hollow cathode lamp. The second analytical system was set up using the HPLC pump ProStar 240 Ternary Solvent Delivery Module (Varian, Australia), the Rheodyne PEEK (IDEX) chromatographic valve, the Zorbax SAX (250 mm length, 4.6 mm i.d.) HPLC column (Agilent, USA) and SpectrAA 280FS atomic absorption spectrometer (Agilent, USA) as a chromatographic detector. The chromatographic run was isocratic at 2.5 mL min¡1 with an injection volume of 200 mL. The Se species Se(VI) does not form a volatile hydride, so in order to obtain an analytical signal in the HG-AAS system it was necessary to perform preliminary reduction of Se(VI) to Se(IV). The reduction was carried out on-line by heating the sample (90–100 C) with a reducing agent: 0.5 mol/L thiourea solution in 10 mol/L hydrochloric acid.[29] The eluate from the chromatographic column was joined with the stream of the reducing agent (flow rate 1 mL min¡1 from the peristaltic pump) through a T-shape coupling and directed to the Tygon capillary loop (inner diameter 0.82 mm) heated on a water bath. The capillary loop outlet was connected to the hydride generation system. For both analytical systems the PEEK transfer tubing of the eluent from the LC column to the hydride generation unit was inserted into a Tygon sleeve. The continuous hydride generation system (VGA-77, Varian) consisted of a manually controlled, four-channel peristaltic pump with Tygon tubing (0.6 mm i.d.), one reaction coil (PTFE tubing 0.8 mm i.d., 75 cm length) and three-way connectors. The gas-liquid separator was made from glass and the interior dead volume was 3 mL. For atomization of the Se hydrides (detected at 196.0 nm), a heating controller, electrothermally heating mantle and a quartz tube (ETC-60, Varian) heated to 900 C were used. The chromatograms were displayed on the screen of a personal computer by using the signal graphics option of the AAS software (Varian), and as a separate print-out of the screen. Peak areas and height were calculated by the AAS software.
A number of validation parameters characterising the analytical method were determined. The limits of detection 0.1 mg kg¡1 for Se(IV) and Se(VI) respectively and the uncertainty of results (measured as RSD) at the level of 10% for both Se forms were obtained. As it was impossible to estimate the measurement traceability because of the lack of any certified reference materials for determination of inorganic Se species, the recovery of each Se species was measured upon addition of a standard to the sample. The recovery of 96–105% of each species was satisfactory. Total Se concentration was measured using electrothermal atomic absorption spectrometry with Zeeman background correction. The SpectrAA 280Z (Agilent) instrument with pyrolytic graphite tubes and Se hollow cathode lamp (wavelength 196.0 nm, slit 1.0 nm, current 10 mA) was used. The temperature programme was optimized: drying at 85–120 C for 55 s; ashing at 1000 C for 8 s; atomization at 2600 C. The palladium solution (10 mL of 500 mg/L for 20 mL of sample) was used as a chemical modifier. The limit of detection 0.01 mg kg¡1 and the uncertainty of results (measured as RSD) at the level of 5.0% were obtained. The traceability was measured by a standard addition procedure.
Gases and reagents Compressed argon gas of N-50 purity (99.999%) obtained from Linde (Poland) was employed as the carrier gas for Se vapour to the quartz cell without further purification. Water was redistilled and further purified with a Milli-Q water purification system (Millipore, USA). Standard solutions (1000 mg/L of Se) of selenite and selenate were prepared by dissolving appropriate amounts of sodium selenite (Na2SeO3), sodium selenate (Na2SeO4) and selenomethionine respectively, obtained from Sigma-Aldrich (USA). For total Se determination a commercial Se standard (Merck, Germany) was used. The standard stock solutions were stored in glass bottles at 4 C in darkness. Low concentration standards obtained by dilution of the stock solutions were prepared daily. Sodium tetrahydroborate (III), used as reducing solution (3% w/w), was prepared daily, by dissolving NaBH4 (Merck) in high-purity water and stabilizing with 1% (w/w) NaOH (Merck) solution to reduce its rate of decomposition. The solution was used without filtration. The HCl solution was of the highest quality grade (Suprapure, Merck). Thiourea of analytical quality grade was obtained from Merck. The buffered mobile phase was prepared by mixing together disodium hydrophosphate (Na2HPO4) 100 mmol/L and potassium dihydrophosphate (KH2PO4. 2H2O) 10 mmol/L obtained from Merck. Palladium solution (500 mg/L of palladium as nitrate) was prepared from commercial palladium chemical modifiers (Merck).
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Statistical analysis and calculations Results recorded in the course of the performed analyses were subjected to statistical analysis with STATISTICA v. 8.0 software. In order to compare content of Se in mushrooms of the same species, growing in particular systems, Tukey’s multiple comparison procedure was used, with the results marked with identical letters in rows or columns exhibiting no differences at the significance level a D 0.05. One-way analysis of variance was performed. The relationships between content of organic and inorganic Se forms in relation to addition of both Se species to the substrate were estimated by the correlation coefficient and nonlinear regression analyses together with the analysis of the single curvilinear regression effects and with the coefficient of determination (R2), as a measure of the good fit of regression.[33] Efficiency of Se species accumulation was characterized by the bioconcentration factor (BCF), calculated as the ratio of Se species content in the fruiting bodies to the content of this element in the substrate where the mushroom was grown. BCF values >1 indicate Se species accumulation, while BCF 1) for all tested mushroom species growing under all Se additions with the exception of A. aegerita growing
Table 1. Bioconcentration factor (BCF) values for particular Se species. Se addition [mM]
Se(IV)
Se(VI)
H. erinaceus 0.14 1.33 0.16 0.50 0.32 1.00 0.24 0.30 A. aegerita 0.02 2.30 10.87 2.00 0.09 0.23 0.14 0.01 G. lucidum 0.84 0.01 0.81 0.05 0.80 2.05 0.57 1.95 0.50 0.02
0.1 0.2 0.4 0.6 0.1 0.2 0.4 0.6 0.1 0.2 0.4 0.6 0.8
Seinorg
Seorg
Setotal
0.15 0.16 0.32 0.24
7.11 5.60 6.71 4.08
0.56 0.59 0.59 0.50
0.03 12.54 0.09 0.11
3.07 0.22 1.25 6.86
0.36 0.28 0.52 0.55
0.76 0.75 0.86 0.68 0.45
1.32 1.97 2.41 1.27 4.05
0.97 1.02 1.04 0.91 1.14
under 0.2 mM (BCF D 0.22). It is worth underlining the high BCF calculated for Se(IV) in A. aegerita bodies growing under 0.2 mM (BCF D 10.87). Mushroom biomass Each tested mushroom species was able to grow on substrates with Se salt supplementation 0.6 mM for each of them. Only G. lucidum fruiting bodies grew on substrates with 0.8 mM of both Se salts, but the biomass of them was significantly lower than mushrooms growing in other systems (Table 2). In the case of A. aegerita and G. lucidum bodies, there was not a significant decrease in biomass of these mushroom species with the exception of G. lucidum bodies growing under 0.8 mM of Se species addition (15.51 § 6.53 g). Biomass of H. erinaceus bodies was the highest under 0.2 (197.04 § 8.73 g), control (191.80 § 6.06 g) and
Table 2. Biomass [g] and biological efficiency (BE) of tested mushroom fruiting bodies. A. aegerita System
Biomass [g]
Control 0.1 0.2 0.4 0.6 0.8 1.0 1.5
178.25 § 6.51 190.76 § 12.84a 170.74 § 29.66a 177.22 § 11.87a 111.44 § 45.76a nb nb nb a
H. erinaceus BE [%]
Biomass [g]
50.93 54.50 48.78 50.63 31.84 nb nb nb
191.80 § 6.06 185.04 § 8.73ab 197.90 § 19.08a 141.58 § 10.72b 38.32 § 27.81c nb nb nb ab
G. lucidum BE [%]
Biomass [g]
BE [%]
54.80 52.87 56.54 40.45 10.95 nb nb nb
96.49 § 3.80 85.58 § 5.68a 121.92 § 7.73a 90.24 § 25.48a 117.36 § 12.84a 15.51 § 6.53b nb nb
27.57 24.45 34.83 25.78 33.53 4.43 nb nb
a
nb – no fruiting bodies. a,b,c Mean values (n D 3) § standard deviations; identical superscripts denote no significant (p < 0.05) difference between mean values in column according to Tukey’s HDS test (MANOVA).
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Fig. 2. Macroscopic changes in A. aegerita fruiting bodies.
0.1 mM (185.04 § 8.73 g) of both inorganic salts. For the other Se additions (0.4 and 0.6 mM), the biomass of H. erinaceus was significantly lower. BE calculated for each tested mushroom was higher for A. aegerita and H. erinaceus than for G. lucidum. The highest BE values for A. aegerita, H. erinaceus and G. lucidum bodies were 54.50, 54.80 and 34.83%, respectively, for bodies growing on substrate with 0.1 and 0.2 mM of Se (IV) and Se(VI) additions. The higher BE values calculated for bodies with Se addition than control bodies indicates the positive influence of Se supplementation on mushroom biomass stimulation. It is worth underlining that dry matter of A. aegerita and H. erinaceus was similar and significantly lower than dry matter of G. lucidum (Table 3). Presented data are important as regards the crop in practical use of tested mushroom species. The knowledge of Table 3. Dry matter [%] of tested fruiting bodies. System
A. aegerita
H. erinaceus
G. lucidum
Control 0.1 0.2 0.4 0.6 0.8 1.0 1.5
8.20 § 0.22b 9.10 § 0.16ab 8.80 § 0.08ab 8.93 § 0.29ab 9.60 § 0.51a nb nb nb
8.60 § 0.22a 9.17 § 0.26a 8.67 § 1.04a 10.33 § 0.31a 11.60 § 2.01a nb nb nb
41.80 § 1.00a 44.37 § 7.93a 34.10 § 5.05abc 39.47 § 10.73ab 21.43 § 0.93bc 17.43 § 3.15 nb nb
nb – no fruiting bodies. a,b,c Mean values (n D 3) § standard deviations; identical superscripts denote no significant (p < 0.05) difference between mean values in column according to Tukey’s HDS test (MANOVA).
biomass and BE level is essential in the planning of commercial mushroom cultivation. For graphical presentation of changes in biomass of tested mushroom species, in Figures 2–4, the macroscopic changes of mushroom fruiting bodies are presented. The first macroscopic changes (white parts of the cap border) of A. aegerita mushroom bodies under 0.4 mM of both Se salts, observed during supplementation with 0.6 mM, were in the case of mushroom fruiting bodies’ loss of colour. Additionally, fruiting bodies of this mushroom species growing on substrates with 0.4 and 0.6 mM addition were smaller than other collected bodies. In the case of white H. erinaceus, mushrooms were completely white. The slightly pink colour of fruiting bodies growing under 0.1 and 0.2 mM of Se salts was observed only, which suggests the possible start of a mechanism of inorganic Se detoxification and reduction of some Se into elementary Se nanoparticles with pink colour, as was described by Falcone and Nickerson[34] and Gharieb et al.[35] The number of carpophores formed by G. lucidum declined together with the increase of Se concentrations from 0.6 mM to complete absence of their formation when the concentration exceeded 0.8 mM. Moreover, the size of carpophores decreased when the concentration exceeded 0.4 mM.
Discussion About 25 proteins present in the human body need Se for their correct functioning; therefore, supplementation
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Fig. 3. Macroscopic changes in H. erinaceus fruiting bodies.
with inorganic Se species with significantly higher Seorg species content, e.g., for tested A. aegerita, pointed to the possibility to prepare mushroom supplements with higher Se or other elements (Cu, Zn) content. According to Navarro-Alarcon and Cabrera-Vique,[36] the daily requirement of Se is in the range 150–200 mg day¡1, while according to Dietary Reference Intakes, the recommended daily intake/adequate intake (RDA/AI)[37] of
Fig. 4. Macroscopic changes in G. lucidum fruiting bodies.
Se is in the range from 15 (infants) to 70 (lactation) mg day¡1 for different life stage groups. Taking into consideration the results presented in this work, especially the content of Seorg indicated that intake of tested medicinal mushroom species is related to too great exposure of the human organism to Se. Hartikainen[38] stated that an optimal Se addition level is important (for human health) as regards transport of this non-metal in the food chain.
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936 Accordingly, we suggest preparing a powder from the mushroom species tested in the experiment and developing a supplement for direct use with specifically determined Seorg and Seinorg contents. In cases where the content of inorganic species of Se is higher than organic Se, e.g., G. lucidum, there could be biotransformation of inorganic selenite present in the substrate into organic forms.[39] Dan[40] pointed out the possibility to use Se-rich Shiitake and Se-rich oyster mushrooms for preparation of new natural health products. The potential of Se-enriched edible mushrooms was also summarised by Ling et al.[40] Mao et al.[41] described the ability of fungus As-1 to accumulate Se growing on substrate supplemented with sodium selenite. The authors reported bodies’ growth inhibition when the Na2SeO4 concentration was 5 mg/L and mycelia growth inhibition when the Na2SeO4 concentration was 50 mg/L. Mushroom species supplemented with Se tested in this work can be used in practice, as previously reported by Guoyuan.[42] Stajic et al.[43] analysed the influence of Se addition on Pleurotus ostreatus (Jacq.: Fr.) growth, finding no significant influence of this non-metal on mycelial growth, as was also found in this work for other mushroom species. An interesting comparison of the present results can be made with the studies described by Savic et al.,[44] who tested accumulation of Se and growth of Pleurotus ostreatus (Hk-35 and P70) supplemented with the same inorganic salts. The authors reported stimulation effects on growth of mushroom bodies growing under 1–50 mg/L and toxic effects for mushrooms under 75, 100 and 150 mg/L. Results presented in this work indicated no growth of mycelium for three tested mushroom species in systems with addition of Se salts
