# Identification of new selenium compounds produced by edible mushrooms

Katarzyna Bierla, Marek Siwulski, Laurent Ouerdane, Ryszard Lobinski, Patrycja Mleczek, Mirosław Mleczek

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Katarzyna Bierla, Marek Siwulski, Laurent Ouerdane, Ryszard Lobinski, Patrycja Mleczek, et al. Identification of new selenium compounds produced by edible mushrooms. Food Chemistry, 2025, 496 (2), pp.146763. 10.1016/j.foodchem.2025.146763. hal-05365176

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# Identification of new selenium compounds produced by edible mushrooms

Katarzyna Bierla<sup>a,*</sup>, Marek Siwulski<sup>b</sup>, Laurent Ouerdane<sup>a</sup>, Ryszard Lobinski<sup>a,c</sup>, Patrycja Mleczek<sup>d</sup>, Mirosław Mleczek<sup>d</sup>

## ARTICLE INFO

Keywords:

Se-fortified mushroom
selenoneine derivatives
selenomethionine
ESI-MS
ICP-MS

## ABSTRACT

The ability of two common edible mushroom species, *Pleurotus ostreatus* and *Hericium coralloides*, to metabolize selenium when grown in the presence of selenite (Se(IV)) and selenate (Se(VI)) was investigated. The efficiency of selenium absorption was 4–8 times higher when Se(IV) was used as selenium source. Anion-exchange HPLC-ICP-MS showed complete (>98.5 %) metabolization of Se(IV) to organic forms, while non-metabolized Se(VI) reached 8–18 %, depending on the species. Metabolized selenium was principally (>40 %) recovered as water-soluble species, followed by ~20 % in water-insoluble polysaccharides and ~ 10 % in water-insoluble proteins. Selenomethionine was the most abundant species in *P. ostreatus*, accounting for over 45 % of absorbed selenium and much less abundant (15–18 %) in *H. coralloides*.

The water-soluble selenometabolome, accounting for 40–60 % of selenium in the samples, was characterized by HILIC with ESI-MS, revealing the presence of ten additional metabolites. These included several selenoneine derivatives and vinyl-selenocysteine-containing dipeptides, suggesting previously unreported pathways for selenium metabolism in mushrooms.

## 1. Introduction

Mushroom consumption is rising globally, driven by their nutritional value (de Brito et al., 2023). Low in fat but rich in protein, carbohydrates, dietary fiber, vitamins (e.g., niacin, folates, riboflavin), and minerals, mushrooms also contain bioactive compounds that boost immunity and health (Cheung, 2010; Ritota et al., 2023). The white button mushroom (*Agaricus bisporus*) is the most widely cultivated, with its mature form, the Portobello, prized for its texture, followed by the versatile oyster mushroom (*Pleurotus ostreatus*). The shiitake (*Lentinula edodes*) is an Asian cuisine staple, while enoki (*Flammulina velutipes*) is popular in soups. The lion's mane (*Hericium erinaceus*), known for its shaggy look, is valued for both culinary and medicinal uses (Khan et al., 2013).

Selenium is an essential trace element with significant health benefits, including antioxidant properties and immune system support. Supplementation with selenium increasingly favors its organic forms, in particular selenomethionine and selenocysteine, which are reported to have higher bioaccessibility than the inorganic forms (Ghaniem et al.,

2022; Rayman et al., 2008). Organic selenium compounds are commonly produced through advanced biotechnological processes involving yeast fermentation (Ouerdane & Mester, 2008; Wu et al., 2021) and bacterial metabolism (Jin et al., 2018; Zhu et al., 2023) in the presence of Se(IV) or Se(VI).

Mushrooms are known for their ability to accumulate selenium (Bhatia et al., 2013; da Silva et al., 2012; Kora, 2020), transforming it into various organic species (Yoshida et al., 2005). Among them, the most abundant compound- selenomethionine (SeMet) - was identified in *Ganoderma lucidum* (Xu et al., 2021), *H. erinaceus* (Hu et al., 2020), *L. edodes* (Ogra et al., 2004), *P. eryngii* (Ji et al., 2022) and *F. velutipes* (Dong et al., 2021) using HPLC-ICP-MS. Mushrooms were reported to produce selenocysteine (SeCys) and methylselenocysteine (MeSeCys) (Gergely et al., 2006; Maseko et al., 2013). Se-adenosyl compounds were identified in the water-soluble fraction of *H. erinaceus* (Egressy-Molnár et al., 2016). More recently, selenoneine was detected in *Boletus edulis* (Peer & Kuehnelt, 2024). The unique dietary properties of mushrooms and their ability to accumulate and metabolize selenium make them a promising dietary source of organic selenium.

<footnote><sup>a</sup> Université de Pau, E2S, CNRS, Institute of Analytical and Physical Chemistry for the Environment and Materials (UMR 5254), Hélioparc, 64053 Pau, France</footnote>

<footnote><sup>b</sup> Poznań University of Life Sciences, Faculty of Forestry and Wood Technology, Department of Vegetable Crops, Dąbrowskiego 159, 60-594 Poznań, Poland</footnote>

<footnote><sup>c</sup> Chair of Analytical Chemistry, Warsaw University of Technology, Noakowskiego 3, 00-664 Warszawa, Poland</footnote>

<footnote><sup>d</sup> Poznań University of Life Sciences, Faculty of Forestry and Wood Technology, Department of Chemistry, Wojska Polskiego 75, 60-625 Poznań, Poland</footnote>

<footnote>* Corresponding author.
E-mail address: katarzyna.bierla@univ-pau.fr (K. Bierla).</footnote>
<figure><img src="image_4.png" /><figcaption>Fig. 1. The mushrooms used in the study.</figcaption></figure>

The objective of this research was to evaluate the potential of two
popular mushroom species—the widely cultivated oyster mushroom
(*P. ostreatus*) and the lion's mane mushroom (*H. corraloides*) (Fig. 1) to
accumulate and metabolize selenium when grown in media supple-
mented with Se(IV) and Se(VI). The comparison included their uptake
capacity, the fractionation of the absorbed selenium among different
classes of biomolecules (such as sugars and proteins), and the charac-
terization of the water-soluble selenium metabolome using advanced
high-resolution accurate mass spectrometry (HRAM-MS).

**2. Material and methods**

**2.1. Reagent and samples**

**2.1.1. Preparation of substrate for mushroom cultivation**

The cultivation substrate was prepared from a mixture of alder and beech sawdust (1,1 vol) supplemented with 20 % of wheat bran, 5 % of corn flour, 3 % of soybean meal, 1 % of chalk, and 1 % of gypsum relative to the sawdust dry matter. The mixture was dampened with water to a moisture content of 45 %. Next, the substrate was placed in bags (polypropylene foil) and sterilized at a temperature of 120 °C for 1 h. As soon as the temperature fell below 25 °C, Se salts were dissolved. The Se solutions were added to the substrate, which was mixed intensely in an automatic stirrer for 15 min (POLYMIX PX-SR 90 D, Kinematica Littau-Luzern, Switzerland). After the addition of the solutions, the substrate moisture was 60 %. Selenium was added either as sodium hydrogen selenite for Se(IV) or sodium selenate for Se(VI), both from Acros Organics (Fair Lawn, New Jersey, NJ). The mushrooms studied came from the collection of cultivated and medicinal mushrooms at the Department of Vegetable Crops, Poznań University of Life Sciences.

**2.1.2. Mushroom (fruiting body) cultivation**

In preliminary studies, various doses of Se(IV) and Se(VI) were used. A dose of 0.5 mM was found to be the limiting dose for the Se(VI). Above this value, mycelial growth was partially inhibited, manifested by a prolonged incubation period. Changes in yield and fruiting body morphology also occurred. It can be assumed that above this limit, this form began to have a toxic effect on the tested species. Therefore Se was applied to each kg substrate in an amount of 0.5 mM for each Se species. The treated substrates were mixed using a POLYMIX PX-SR 90 D stirrer (Kinematica, Littau-Luzern, Switzerland) with *P. ostreatus* or *H. corraloides* spawn (mycelium on wheat grain) at 10 % of the substrate weight. The mixture was placed in bags (polypropylene foil, bag size 17 × 28 cm), each containing 400 g of substrate. Incubation was carried out

at 25 °C and 80–85 % air relative humidity until the substrate was fully colonized with mycelium.

The bags were placed in a cultivation chamber with the top part of the foil cut off. The relative humidity was maintained at 85–90 % and the temperature at 16 ± 1 °C. The cultivation was additionally illuminated with fluorescent light of 500 lx intensity for 10 h a day and aerated to maintain CO₂ concentration below 800 ppm. The experiment was conducted in triplicate. Fruiting bodies were harvested successively as they matured. Fresh fruiting bodies were dried in an electric Thermo-center oven (Salvislab, Rotkreuz, Switzerland) at 40 °C to a constant weight and ground for 0.5 min in a Cutting Boll Mill 200 (Retsch GmbH, Haan, Germany). Samples for further testing were taken from a pooled sample consisting of material from three replicates. Samples were pooled to obtain a more homogeneous material, as the study focused on identifying selenium-containing compounds and general differences between species and treatments rather than inter-individual variation.

**2.1.3. Solvent, reagents and solutions**

Milli-Q® Type 1 Ultrapure Water Systems (Millipore, Bedford, MA) deionized water was used throughout. Concentrated nitric acid (Fisher, Loughborough, UK) and 30 % hydrogen peroxide (Fluka, Steinheim, Germany) were used for sample digestions. Ammonium acetate (≥ 98 % for molecular biology), acetonitrile (≥99.9 %), formic acid (LC-MS grade) and hydrochloric acid (37 %) used for the preparation of mobile phases were from Fluka. A Se ICP MS standard stock solution (1003 µg mL⁻¹) SCP Science, Quebec, Canada) was diluted to prepare calibration curves. CRM SELM-1 (NRC, Ottawa, Canada), a selenium enriched yeast, was analyzed in parallel with the samples to validate the accuracy of total element determinations. SELM-1 was chosen as yeast as mushrooms they have cell walls made of chitin and glucans, therefore it was considered that the digestion will be similar for both of them.

**2.2. Instrumentation**

A DigiPrep MS system (SCP Science, Quebec, Canada) was used for sample digestion. The Bandelin Sonorex RK255H (Bandelin, Berlin, Germany) ultrasonic bath and Stuart SBS40 water bath were used for extraction procedures. Samples were centrifuged using an Eppendorf 5804R centrifuge (Eppendorf, Hamburg, Germany). Two chromatographic systems were employed for the separation of analytes. An Agilent 1200 Series (Agilent, Tokyo, Japan) HPLC system was used for anion exchange chromatography. A Dionex Ultimate 3000 RS (Thermo Scientific, Bremen, Germany) was used for RP and HILIC separations. The ICP MS spectrometers were ICP-MS 7700 (Agilent, Tokyo, Japan)
<table><caption>**Table 1**
Distribution of selenium among the different extractable fractions obtained from 3 individual determinations.</caption><thead><tr><th rowspan="2"></th><th colspan="4">Pleurotus ostreatus</th><th colspan="4">Hericium coralloides</th></tr><tr><th colspan="2">cultivated with Se(IV)</th><th colspan="2">cultivated with Se(VI)</th><th colspan="2">cultivated with Se(IV)</th><th colspan="2">cultivated with Se(VI)</th></tr><tr><th></th><th>µg g<sup>-1</sup></th><th>% of total</th><th>µg g<sup>-1</sup></th><th>% of total</th><th>µg g<sup>-1</sup></th><th>% of total</th><th>µg g<sup>-1</sup></th><th>% of total</th></tr></thead><tbody><tr><td>Se<sub>total</sub>*</td><td><strong>385 ± 7</strong></td><td></td><td><strong>55.9 ± 0.4</strong></td><td></td><td><strong>370 ± 4</strong></td><td></td><td><strong>88.2 ± 1.0</strong></td><td></td></tr><tr><td>water soluble</td><td>220 ± 8</td><td>56.9 ± 0.3</td><td>33.4 ± 0.9</td><td>59.7 ± 0.2</td><td>151 ± 4</td><td>40.7 ± 0.2</td><td>49.2 ± 2.2</td><td>55.7 ± 0.3</td></tr><tr><td>polysaccharide bound</td><td>70.8 ± 0.2</td><td>18.4 ± 0.1</td><td>8.9 ± 1.4</td><td>15.9 ± 0.6</td><td>64.8 ± 0.4</td><td>17.52 ± 0.05</td><td>12.2 ± 0.5</td><td>13.8 ± 0.1</td></tr><tr><td>water-insoluble protein bound</td><td>38.6 ± 0.3</td><td>10.0 ± 0.1</td><td>4.0 ± 0.3</td><td>7.1 ± 0.2</td><td>60.8 ± 0.4</td><td>16.45 ± 0.05</td><td>8.2 ± 0.7</td><td>9.3 ± 0.3</td></tr><tr><td>SDS-soluble</td><td>10.6 ± 0.1</td><td>2.75 ± 0.03</td><td>1.19 ± 0.11</td><td>2.14 ± 0.14</td><td>35.2 ± 0.2</td><td>9.53 ± 0.03</td><td>4.0 ± 0.2</td><td>4.5 ± 0.1</td></tr><tr><td>residue</td><td>10.0 ± 1.4</td><td>2.6 ± 0.2</td><td>1.0 ± 0.2</td><td>1.8 ± 0.3</td><td>20.1 ± 0.2</td><td>5.42 ± 0.03</td><td>2.4 ± 0.4</td><td>2.7 ± 0.3</td></tr><tr><td>Recovery</td><td></td><td>90.6</td><td></td><td>86.7</td><td></td><td>89.7</td><td></td><td>86.1</td></tr></tbody></table>

equipped with an integrated autosampler (I-AS) used for the total analysis and Agilent 7900× (Agilent, Tokyo, Japan) used for the LC couplings. A UPLC Acquity Ultimate 3000 system was coupled to an electrospray Orbitrap Lumos mass spectrometer (Thermo Fisher Scientific, Waltham, MA).

## 2.3. Procedures

### 2.3.1. Total selenium analysis

The total selenium content in mushrooms and extracts from sequential extraction was determined by ICP-MS following acid digestion. For this, 0.1 g of the sample was digested with 2 mL of HNO₃ and 0.5 mL of H₂O₂ at 65 °C for 4 h. After digestion, tubes were filled up to 50 mL with water. Blanks were processed in parallel with the samples. The calibration curves for quantification were prepared with PlasmaCAL Se standard (SCP Science, Canada).

### 2.3.2. Sequential extraction

The sequential extraction procedure was adapted from Jimenez et al. (Jiménez-Lamana et al., 2018). The modifications included the replacement of driselase (showing considerable proteolytic activity) by β-gluconase from *Trichoderma longibrachiatum* (Sigma-Aldrich, Mexico) and the concentrations of enzymes. In brief, the procedure included four steps: (1) 100 mg of a Se-rich mushroom sample were suspended in 5 mL of water, bath sonicated for 1 h and centrifuged at 4500 g for 10 min; (2) the pellet was resuspended with a solution of 5 mL of 6.7 mg L⁻¹ gluconase solution in 100 mM ammonium acetate at pH 5.1, incubated at 37 °C for 2 h and centrifuged at 4500 g for 10 min; (3) the pellet was resuspended with a solution of 6 mL of 6.7 mg L⁻¹ protease Type XIV (≥3.5 units mg⁻¹) (Sigma-Aldrich, Japan) in 30 mM Tris at pH 7.5, incubated at 37 °C for 17 h and centrifuged at 4500 g for 10 min; (4) the pellet was resuspended in a solution of 3 mL of 4 % (w/v) sodium dodecyl sulfate (SDS) (for molecular biology) (Sigma-Aldrich, St. Louis, MO, USA) in a water bath sonicated for 1 h and centrifuged at 4500 g for 10 min. The supernatant was collected and stored at 4 °C until analysis. The carry over from one fraction to the next was found not to exceed 1–2 % so it was decided not to wash the pellets in-between.

### 2.3.3. Total selenium determination in extracts from sequential extraction

250 µL of H₂O₂ (VWR International, Fontenay-sous-Bois, France) and 1 mL of conc. HNO₃ (Baker, Deventer, Netherlands) were added to 100, 250 or 500 µL of sample. The digestion was performed at 65 °C for 4 h. After digestion, the volume was made up to 15 mL so that the final HNO₃ concentration was 2 % (v/v). Digestions were made in duplicate. Blanks were analyzed in parallel with the samples.

### 2.3.4. HPLC-ICP MS analysis

Anion exchange – HPLC – ICP MS analyses were carried out using a PRP-X100 column (4.6 × 250 mm × 10 µm, Hamilton Bonaduz AG, Bonaduz, Switzerland) at the flow rate of 1 mL min⁻¹ (Bierla et al., 2018). The mobile phases were: A: 20 mM CH₃COOH - 10 mM triethylamine (≥ 99.5 %, GC) (Sigma-Aldrich, Steinheim, Germany) (pH

4.7), B: 200 mM CH₃COOH - 100 mM triethylamine (pH 4.7). The elution gradient was: 0–5 min: 0 % B, 5–25 min: 0–100 % B, 25–34,100 % B, 34–34.1 min: 100–0 % B. The injection volume was 50 µL. The calibration curves for quantification were prepared with solutions of sodium selenite, sodium selenate and selenomethionine (Sigma-Aldrich, Steinheim, Germany).

Hydrophilic interaction chromatography (HILIC) – ICP-MS analyses were carried out on a ZIC®-cHILIC column (2.1 × 150 mm × 3 µm, 100 Å; Merck-Millipore, Darmstadt, Germany) at the flow rate of 0.35 mL min⁻¹ at 30 °C. The mobile phases were: A: 10 mmol L⁻¹ ammonium formate (for mass spectrometry) (Sigma-Aldrich, India) in water, and B: as 10 mmol L⁻¹ ammonium formate in 9:1 v/v acetonitrile-water. The elution gradient was: 0–1 min, 95 % B; 1–19.5 min, down to 30 % “B”; 19.5–21.5 min, 30 % B; 21.5–22 min up to 95 % B", 22–30 min, 90 % B. The injection volume was 7 µL. The parameters for ICP MS settings were given in Table SI 1.

### 2.3.5. LC orbitrap ESI MS analysis and data processing

The HILIC eluate was screened using LC Orbitrap ESI-MS in untargeted mode to detect the presence of selenium compounds. The ion source was operated in both, positive and negative modes. The typical values of operational parameters are given in Table SI 2. A full scan mass spectrum in the m/z 150–1000 mass range was acquired at a resolution of 240,000. An AGC target of 4 × 10⁵ was used and a maximum injection time of 502 ms was set. Data dependent MS/MS scans were performed using high-energy collisional dissociation (HCD) fragmentation mode. The resolution for MS/MS scans was set to 60,000 and the isolation window for precursor isolation was 2.2 m/z with an offset of −1. An AGC target of 5 × 10⁴ was used and a maximum injection time of 300 ms was set.

Data processing was carried out using Compound Discoverer 3.3™ (ThermoFisher Scientific, Waltham, MA). This software was applied to mine for Se compounds based on their isotopic profile and identify all Se-compounds present in the analyzed samples detected using an untargeted approach (Bierla et al., 2023). The mass tolerance was set at 5 ppm. Fragments were assigned after data-dependent MS/MS.

## 3. Results and discussion

### 3.1. Selenium uptake and accumulation in mushrooms

#### 3.1.1. Total selenium uptake

The obtained data reveal a striking similarity in the pattern of selenium accumulation in *P. ostreatus* and *H. coralloides* as a function of their form. When grown under selenite-enriched conditions, both mushrooms accumulated high Se levels, reaching 370 and 385 µg g⁻¹ dry mass, respectively. However, under selenate-enriched conditions, Se accumulation was much lower, with concentrations of only 56 and 88 µg g⁻¹ (dry mass) for *P. ostreatus* and *H. coralloides*, respectively.

These results are consistent with the amounts of selenium previously reported to accumulate in cultivated mushrooms, including *Albatrellus pes-caprae* (400 µg g⁻¹ dm) (Falandysz, 2008), *A. bisporus* (J.E. Lange)

<footnote>*LOD and LOQ for the procedure were 0.0142 and 0.0224 µg g⁻¹ in the dry sample.</footnote>
Imbach (770 µg g⁻¹ dm) (Gergely et al., 2006) and 1300 µg g⁻¹ dm (Werner & Beelman, 2002). Our findings also corroborate earlier studies showing that Se(IV) is more effectively assimilated by mushrooms than Se(VI) (de Oliveira et al., 2023; Zhou et al., 2021). This differential uptake of Se species has implications for the cultivation of Se-enriched mushrooms for dietary supplementation. Selenate is more stable in oxidizing environments, highly soluble, and behaves similarly to sulfate (SO₄²⁻). Selenite is more reactive, less stable under oxidative conditions, and can bind readily to thiol groups (–SH) in proteins and glutathione, forming complexes.

### 3.1.2. The distribution of the accumulated selenium

The distribution of the accumulated selenium among different bio-molecular classes was investigated. For this purpose, mushrooms were fractionated into a water-soluble fraction, a water insoluble polysaccharide fraction and a water-insoluble protein fraction. Glucanase, an enzyme that catalyzes the hydrolysis of glycosidic bonds in polysaccharides, releasing smaller molecules such as glucose and potentially Se, was used to degrade the polysaccharide fraction. Protease was used to degrade the water insoluble proteins. The distribution of the accumulated selenium as a function of the mushroom species and selenium species is shown in Table 1.

Most of the selenium (>40 %, metabolized or residual) was recovered as a water-soluble fraction, regardless of the Se form or mushroom species. These results are consistent with values previously reported for various species of wild mushrooms (Huerta et al., 2005) as well as for cultivated mushrooms (Ogra et al., 2004). The percentage of selenium associated with the polysaccharide fraction was also relatively similar between the samples. Approximately 14–18 % of selenium was released after glucanase digestion, suggesting that selenium may be nonspecifically associated with polysaccharides or other glucan-containing substances that are part of mushroom cell walls. On the other hand, the selenium in the protein fraction, indicating its conversion into selenomethionine, was considerably higher when Se(IV) was used as the selenium source. This could also explain the higher accumulation efficiency observed when Se(IV) was used.

A relatively significant portion of selenium (5–10 %) could not be solubilized, indicating the presence of non-degradable polysaccharides or proteins, or reduced selenium. This is corroborated by the extraction of selenium from the residue using SDS, an ionic detergent designed to dissolve remaining membranes, disrupt protein-nucleic acid interactions, and break protein-protein, protein-lipid, and lipid-lipid connections, which were resistant to the enzymes used in previous steps. During this step, 2–10 % of selenium was recovered. The final step involved acidic digestion of the residual pellet, which allowed for the calculation of selenium recoveries for the entire procedure. Approximately 10 % of the selenium was unaccounted for, which is unlikely to be due to incompletely digested carbon, even if such matrix effects are known to enhance the Se signal. Such an enhancements were not observed for the yeast certified material with a comparable cell wall composition (chitin and glycans) analyzed together with the studied samples. Instead, volatilization provides a more plausible explanation. It was reported that vegetables and mushrooms release volatile sulfur compounds (VSCs) during preparation, contributing to their characteristic aromas and potential health effects. A recent study identified 15 different VSCs emitted from various vegetables and mushrooms (Park et al., 2024). Shiitake mushrooms were found to release 21 VSCs during drying (Xu et al., 2024). Additionally, up to 10 VSCs have been detected in shiitake mushrooms, with their concentrations varying depending on the extraction temperature (Dermiki et al., 2013; Wu & Wang, 2000). Selenium-containing volatile compounds were also identified in G. lucidum, with their presence strongly influenced by the total selenium content (Xu et al., 2021). Consistently, our SPME-GC-MS analysis revealed several selenium-containing species (m/z 93–270 with characteristic Se isotopic patterns), supporting the hypothesis that part of the missing selenium fraction may have been lost through volatilization,

<figure><img src="image_5.png" /><figcaption>**Fig. 2.** AE ICP MS chromatograms of SeMet, Se(IV) and Se(VI) at 10, 1 and 1 ng g⁻¹ respectively with gradient marked(a), water extract from mushroom *P. ostreatus* grown with Se(IV) (b) and Se(VI) (c) and *H. coralloides* grown with Se(IV) (d) and Se(VI) (e). The zones indicated as spotted areas show peaks from unidentified compounds.</figcaption></figure>
<caption>**Table 2**</caption>

Total content of the residual Se(IV) and Se(VI) and the biosynthesized SeMet and unknown Se-compounds in the different fractions obtained from 2 individual determinations.

<table><thead><tr><th rowspan="2"></th><th rowspan="2"></th><th rowspan="2"></th><th colspan="2">SeMet*</th><th colspan="2">Se(IV)**</th><th colspan="2">Se(VI)***</th><th colspan="2">Unknown****</th></tr><tr><th>µg g<sup>-1</sup></th><th>% of total</th><th>µg g<sup>-1</sup></th><th>% of total</th><th>µg g<sup>-1</sup></th><th>% of total</th><th>µg g<sup>-1</sup></th><th>% of total</th></tr></thead><tbody><tr><td rowspan="6">Pleurotus ostreatus</td><td rowspan="3">Se<br>(IV)</td><td>water extracted</td><td>104 ± 4</td><td>27.0 ± 0.2</td><td>3.9 ± 0.2</td><td>1.01 ± 0.06</td><td>ND</td><td>–</td><td>139 ± 8</td><td>35.9 ± 0.3</td></tr><tr><td>polysaccharide bound</td><td>39.0 ± 2.4</td><td>10.1 ± 0.2</td><td>0.39 ± 0.02</td><td>0.10 ± 0.02</td><td>ND</td><td>–</td><td>27.1 ± 2.2</td><td>7.0 ± 0.2</td></tr><tr><td>protein bound</td><td>29.2 ± 1.5</td><td>7.6 ± 0.2</td><td>0.73 ± 0.09</td><td>0.18 ± 0.06</td><td>ND</td><td>–</td><td>9.4 ± 1.3</td><td>2.4 ± 0.2</td></tr><tr><td rowspan="3">Se<br>(VI)</td><td>water extracted</td><td>19.1 ± 0.7</td><td>34.2 ± 0.2</td><td>ND</td><td>–</td><td>3.89 ± 0.09</td><td>6.97 ± 0.06</td><td>14.9 ± 2.3</td><td>26.8 ± 0.8</td></tr><tr><td>polysaccharide bound</td><td>6.2 ± 0.6</td><td>11.0 ± 0.3</td><td>ND</td><td>–</td><td>0.42 ± 0.05</td><td>0.75 ± 0.09</td><td>4.3 ± 0.6</td><td>7.7 ± 0.4</td></tr><tr><td>protein bound</td><td>2.9 ± 0.2</td><td>5.2 ± 0.1</td><td>ND</td><td>–</td><td>ND</td><td>–</td><td>1.1 ± 0.2</td><td>1.9 ± 0.03</td></tr><tr><td rowspan="6">Hericium coralloides</td><td rowspan="3">Se<br>(IV)</td><td>water extracted</td><td>13.4 ± 1.0</td><td>3.6 ± 0.2</td><td>ND</td><td>–</td><td>ND</td><td>–</td><td>88.9 ± 7.4</td><td>24.0 ± 0.4</td></tr><tr><td>polysaccharide bound</td><td>3.7 ± 0.2</td><td>0.99 ± 0.06</td><td>0.52 ± 0.06</td><td>0.14 ± 0.04</td><td>ND</td><td>–</td><td>45.3 ± 2.7</td><td>12.3 ± 0.2</td></tr><tr><td>protein bound</td><td>37.7 ± 1.2</td><td>10.2 ± 0.1</td><td>2.0 ± 0.3</td><td>0.55 ± 0.12</td><td>ND</td><td>–</td><td>18.6 ± 1.1</td><td>5.0 ± 0.1</td></tr><tr><td rowspan="3">Se<br>(VI)</td><td>water extracted</td><td>7.6 ± 0.5</td><td>8.6 ± 0.2</td><td>ND</td><td>–</td><td>14.2 ± 0.8</td><td>16.1 ± 0.2</td><td>23.0 ± 3.5</td><td>26.1 ± 0.8</td></tr><tr><td>polysaccharide bound</td><td>2.0 ± 0.2</td><td>2.3 ± 0.1</td><td>ND</td><td>–</td><td>1.1 ± 0.1</td><td>1.2 ± 0.1</td><td>8.5 ± 0.8</td><td>9.6 ± 0.3</td></tr><tr><td>protein bound</td><td>6.3 ± 0.4</td><td>7.2 ± 0.2</td><td>0.23 ± 0.05</td><td>0.3 ± 0.1</td><td>0.16 ± 0.03</td><td>0.18 ± 0.08</td><td>1.9 ± 0.7</td><td>2.2 ± 0.5</td></tr><tr><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr></tbody></table>

* LOD and LOQ for SeMet for the procedure were 0.0134 and 0.0224 µg g⁻¹ in the dry sample.

** LOD and LOQ for Se(IV) for the procedure were 0.0119 and 0.0199 µg g⁻¹ in the dry sample.

*** LOD and LOQ for Se(VI) for the procedure were 0.00715 and 0.0119 µg g⁻¹ in the dry sample.

**** content of unknown compound is an estimation achieved by manual integration on unknown peak and use of SeMet calibration curve for the calculations.

even though we were unable to quantify the volatilized compounds. These findings may explain the 10 % of the selenium unaccounted for in our procedure.

## 3.2. Conversion of inorganic selenium into organic forms

The presence of residual Se(IV) and Se(VI) was verified by anion-exchange HPLC-ICP MS (Ayouni et al., 2006; Bird et al., 1997; Dernovics, 2024). The chromatograms of the water extracts (Fig. 2) indicate virtually no residue of Se(IV) but a significant Se(VI) peak. The chromatograms of the water extracts also indicate the presence of SeMet and a prominent unidentified peak close to the void of the column. Despite the fact that the majority of published works claimed identification and quantification of SeCys₂ in the samples the doubts concerning its presence may arise as the analysis were performed using SAX (PRP-X100) columns. This separation does not appear to be the most suitable option as it is most commonly performed with mobile phases at pH 6, where SeCys₂ forms a zwitterion (Goessler et al., 1997) and therefore is not retained. In another work (Dong et al., 2021b), the pH was set to 7.5 but it seems that even when set to 8.5, it is not high enough to retain SeCys₂ and separate it from other non-retained organic Se-species (Dernovics, 2024; Larsen et al., 2001). The speciation and accumulation patterns varied between *P. ostreatus* and *H. coralloides* mushrooms clearly what is illustrated in Fig. 2b-d.

SeMet and residues (≤ 1.2 % of total Se content) of Se(IV) and Se(VI) were also determined by anion-exchange HPLC-ICP MS in the dissolved polysaccharide and protein fractions (Table 2). The residual amount of SeIV and SeVI may result from carry over from previous extraction steps. The data show that SeMet accounted for a significant proportion of the total selenium content. Specifically, the sum of SeMet in all three fractions accounted for 45–50 % in *Pleurotus* samples, which was notably higher compared to 15–18 % in *Hericium* samples. The SeMet contribution values obtained for *Pleurotus* samples are slightly lower than those reported in the literature for mushrooms from the same genus: *P. energi* 78 % (Ji et al., 2022), *P. florida* 65 % (Bhatia et al., 2013), *P. ostreatus* 53–78 % (Milovanovic et al., 2024), *P. pulmonarius* 84 % (Milovanovic et al., 2019), and considerably lower for *H. coralloides*: 64–68 % reported by Hu (Hu et al., 2020) and 49 % reported by Molnar (Egressy-Molnár et al., 2016) for a closely related species (*H. erinaceus*).

The contribution of unidentified peaks in anion-exchange ICP-MS chromatograms was estimated using the SeMet calibration curve, as ICP-MS sensitivity remains stable during elution. Notably, values for

unknown compounds in the water fractions were high, ranging from 34 to 87 % of the total selenium in the sample extracts and corresponding to 24–36 % of total selenium in the samples (Table 2). Subsequent experiments were conducted to characterize these unknown compounds and provide a detailed profile of Se compounds in all samples.

## 3.3. Characterization of the selenium metabolome using HILIC-ICP MS

The use of reversed-phase separation for Se-compounds in mushroom extracts resulted in weak or nearly no retention and no effective separation (Fig. SI 1), suggesting the hydrophilic nature of the compounds. The Se species were previously reported to be successfully retained and separated by ion-pairing reversed-phase high-performance liquid chromatography (IP-RP-HPLC) with ICP-MS detection. However, identification was limited to Se species for which commercial standards were available. The ion pairing agents used, para-hydroxybenzoic acid (Ayouni et al., 2006) and tetrabutylammonium phosphate (Milovanovic et al., 2019), were found to suppress electrospray ionization (ESI), precluding the use of ESI-MS for identification purposes. Consequently, hydrophilic interaction chromatography (HILIC), a technique previously employed for the separation of Se-compounds in yeast (Dernovics & Lobinski, 2008) and plants (Bierla et al., 2023; Sentkowska & Pyrzynska, 2018), was implemented here for enhanced separation and the identification of Se species in the mushroom extracts.

The HILIC-ICP-MS chromatograms (Fig. 3 a-d) show distinct variations between *P. ostreatus* and *H. coralloides* when grown with Se(IV) and Se(VI). Such differentiation in the profiles indicates the influence of the selenium valence state on its metabolism into organic compounds by the mushrooms.

While the HILIC-ICP-MS chromatograms corroborate the presence of SeMet, no peaks were observed at retention times of MeSeCys (RT 6.77 min) and SeCys₂ (RT 10.95 min) (Fig. SI 2), which are reported to be the most frequently detected compounds in previous works (Dong et al., 2021; Hu et al., 2020; Zhou et al., 2018). It should be remembered that the identity of compound can be only confirmed by spiking experiment when at least 2 different chromatographic modes are used and give a positive result. When using a single chromatographic technique, a spiking experiment can provide definitive conclusions only in the case of a negative result (Francesconi & Sperling, 2005).

The ESI-MS analysis carried out in parallel to ICP-MS analysis yielded significant insight into the identity of selenium species. The search for Se-species in the mass spectrum was performed according to the pattern
<figure><img src="image_6.png" /><figcaption>**Fig. 3.** HILIC-ICP-MS chromatograms of water extract from mushroom *P. ostreatus* grown with Se(IV) (a) and Se(VI) (b) and *H. coralloides* grown with Se(IV) (c) and Se(VI) (d), XIC (extracted ion chromatogram) HILIC-ESI-MS chromatogram of sample grown with Se(IV) (e). Compounds has been numbered according to Table 3</figcaption></figure>

picking protocol outlined by Bierla et al. (Bierla et al., 2023), along with a search within a laboratory curated database of Se-compounds. The list of detected Se-compounds is given in Table 3.

### 3.3.1. Selenomethionine and dehydroselenomethionine

The applied workflow allowed the identification of selenomethionine and its dehydrated homologue, dehydroselenomethionine. These compounds were detected based on their unique mass and isotopic signatures and their identity was revealed through MS² spectral analysis. Their different retention times during chromatographic separation ruled out the possibility of in-source fragmentation, suggesting their formation occurred prior to analytical assessment. SeMet (compound 3) (m/z 198.0028) was detected at RT 5.89 min. Note that there was another Se-compound (compound 2) (m/z 292.0559) present with a similar RT (5.72 min). The superposed XICs (Fig. 3 e) show that the compounds are coeluting and cannot be distinguished with ICP MS detection, potentially leading to an overestimation of SeMet concentration in the samples analyzed.

### 3.3.2. Selenoneine derivatives

In a recent study, selenoneine was reported as the most abundant Se-species in *Boletus edulis* (Peer & Kuehnelt, 2024). Selenoneine has garnered significant attention due to its remarkable radical-scavenging abilities, which are nearly 1000 times stronger than those of ergothioneine (Chen et al., 2024), a well-known antioxidant with notable chemopreventive properties (Alhasan et al., 2019). Beyond its exceptional antioxidant potential, selenoneine also plays a critical role in protecting iron-containing proteins, such as myoglobin and hemoglobin, from auto-oxidation. Furthermore, selenoneine's ability to bind metal ions is vital for its role in detoxifying harmful mercury ions, particularly by facilitating the removal of methylmercury (CH₃Hg⁻¹) from the bloodstream, thereby contributing to the regulation of heavy metal toxicity (Yamashita & Yamashita, 2010).

No intact selenoneine was detected in any of the mushrooms investigated, although ergothioneine was present. However, compounds at m/z 244.1115 and 292.0559 corresponding to methyl-ergothioneine and methyl-selenoneine (compound 2), respectively, were found in all analyzed samples. Their fragmentation patterns (Fig. SI 3), matching those found in spectral libraries, characterized by the sequential loss of CO₂ (mass 43.9899) and C₃H₉N (mass 59.0737), served as references for the identification of other compounds. The analytical approach enabled the identification of molecular structures corresponding to compounds detected at m/z 366.0563 (compound 7), 394.0512 (compound 9), and 397.0411 (compound 6), with the same loses of CO₂ (mass 43.9899) and C₃H₉N (mass 59.0737) (Fig. SI 4), which were hypothesized to be conjugates of selenoneine with lactic acid, succinic acid, and niacin, respectively. The proposed selenoneine-lactic acid conjugate (compound 7) likely results from the reaction of 2-hydroxyacrylic acid, a tautomer of pyruvic acid, with thiol groups, similar to its interaction with CoA in the Krebs cycle. The selenoneine-succinic acid conjugate is consistent with the known reactivity of fumarate-derived thiols, as observed in *Bacillus subtilis* (Niehaus et al., 2018). Notably, the conjugate with niacin was exclusively detected in *H. coralloides* samples, suggesting species-specific metabolic pathways that differentiate it from the other analyzed species. Overall, four selenoneine derivatives were identified in *H. coralloides* and three in *P. ostreatus*. This finding highlights potential variations in selenoneine metabolism and its role in fungal biochemical adaptations. However, the lack of detectable selenoneine could suggest susceptibility to oxidative changes, and therefor emphasize the need for optimized handling of raw material and processing protocols to preserve the integrity of selenium species in biological samples.

### 3.3.3. Other selen compounds

The pattern-picking approach used for data treatment facilitated the identification of additional masses, including m/z 293.0400, 323.0506,
<table><caption>**Table 3**
Selenium compounds detected in the water extracts of the investigated mushrooms.</caption><thead><tr><th rowspan="2"></th><th rowspan="2">RT [min]</th><th rowspan="2">Experimental m/z Se</th><th rowspan="2">Delta Mass [ppm]</th><th rowspan="2">Formula</th><th rowspan="2">Proposed structure</th><th colspan="2">P. ostreatus</th><th colspan="2">H. coralloides</th></tr><tr><th>Se(IV)</th><th>Se(VI)</th><th>Se(IV)</th><th>Se(VI)</th></tr></thead><tbody><tr><td>1</td><td>1.55</td><td>425.1801</td><td>0.02</td><td>C<sub>19</sub>H<sub>37</sub>O<sub>5</sub>Se<sup>+</sup></td><td></td><td>✓</td><td>✓</td><td>✓</td><td>✓</td></tr><tr><td>2</td><td>5.72</td><td>292.0559</td><td>0.12</td><td>C<sub>10</sub>H<sub>18</sub>O<sub>2</sub>N<sub>3</sub>Se<sup>+</sup></td><td></td><td>✓</td><td>x</td><td>✓</td><td>x</td></tr><tr><td>3</td><td>5.89</td><td>198.0028</td><td>0.07</td><td>C<sub>5</sub>H<sub>12</sub>O<sub>2</sub>N Se<sup>+</sup></td><td></td><td>✓</td><td>✓</td><td>✓</td><td>✓</td></tr><tr><td>4</td><td>7.88</td><td>195.9871</td><td>-0.19</td><td>C<sub>5</sub>H<sub>10</sub>O<sub>2</sub>N Se<sup>+</sup></td><td></td><td>✓</td><td>✓</td><td>✓</td><td>✓</td></tr><tr><td>5</td><td>8.32</td><td>293.0399</td><td>0.31</td><td>C<sub>10</sub>H<sub>17</sub>O<sub>3</sub>N<sub>2</sub>Se<sup>+</sup></td><td></td><td>✓</td><td>✓</td><td>✓</td><td>✓</td></tr><tr><td>6</td><td>8.47</td><td>397.0411</td><td>0.24</td><td>C<sub>15</sub>H<sub>17</sub>O<sub>4</sub>N<sub>4</sub>Se<sup>+</sup></td><td></td><td>x</td><td>x</td><td>✓</td><td>✓</td></tr><tr><td>7</td><td>9.08</td><td>366.0563</td><td>0.28</td><td>C<sub>12</sub>H<sub>20</sub>O<sub>5</sub>N<sub>3</sub>Se<sup>+</sup></td><td></td><td>✓</td><td>x</td><td>✓</td><td>x</td></tr><tr><td>8</td><td>10.47</td><td>325.0299</td><td>0.49</td><td>C<sub>10</sub>H<sub>17</sub>O<sub>5</sub>N<sub>2</sub>Se<sup>+</sup></td><td></td><td>✓</td><td>✓</td><td>✓</td><td>✓</td></tr><tr><td>9</td><td>10.58</td><td>394.0512</td><td>0.14</td><td>C<sub>13</sub>H<sub>20</sub>O<sub>6</sub>N<sub>3</sub>Se<sup>+</sup></td><td></td><td>✓</td><td>x</td><td>✓</td><td>✓</td></tr><tr><td>10</td><td>11.04</td><td>323.0506</td><td>0.26</td><td>C<sub>11</sub>H<sub>19</sub>O<sub>4</sub>N<sub>2</sub>Se<sup>+</sup></td><td></td><td>✓</td><td>x</td><td>✓</td><td>x</td></tr></tbody></table>

325.0299 and 425.1801, all exhibiting well-defined Se isotopic profiles. The *m/z* 325.0299 signal was attributed to gamma-glutamyl-vinyl-selenocysteine (compound 8), a selenium-containing analog of the sulfur compound previously isolated from *Vicia narbonensis*, known for its characteristic odor during seed germination (Enneking et al., 1998). The *m/z* 323.0506 peak corresponded to a β-methylene-norvaline conjugate with selenomethionine (SeMet) (compound 10), where β-methylene-L-norvaline itself is an atypical amino acid originally isolated from *Lactarius helvus* (Levenberg, 1968). Additionally, the *m/z* 293.0400 mass (compound 5) was identified as proline-vinyl-selenocysteine, further expanding the understanding of selenium-containing amino acid derivatives. The MS/MS spectra of these 3 compounds are given in Fig. SI 5. The *m/z* 425.1801 signal (compound 1) with assigned formula C<sub>19</sub>H<sub>37</sub>O<sub>5</sub>Se<sup>+</sup> could correspond to a selenized polyol which may be a fragment coming from extracellular fungal glycolipid biosurfactant. Four classes of fungal glycolipid biosurfactant synthesized by different fungi were described till now (Garay et al., 2018). Two additional compounds at *m/z* 346.0415 (RT 1.6 min) and 362.0364 (RT 5.75 min)

corresponding to Se-methyl-5 selenoadenosine and Se-methyl-5-selenoadenosine Se-oxide respectively, previously identified in *H. erinaceus* sample by Egressy-Molnar (Egressy-Molnar et al., 2016), were found in both mushroom species, but their low intensity and coelution with other compounds didn't allow for their fragmentation and formal identification.

These findings highlight the potential role of Se metabolism in various biological systems and emphasize the relevance of untargeted analytical approaches in characterizing novel selenium species.

The contrasting selenium species profiles observed between *P. ostreatus* and *H. coralloides* may reflect differences in selenium metabolism, as the two fungi belong to different families and genera. Previous studies have reported interspecific variability in the accumulation of various metals in fungi, supporting the notion of distinct metabolic pathways (Uzun et al., 2023). Although no detailed mechanisms are available in the literature, the observed differences may arise from species-specific uptake of Se(IV) and Se(VI), variations in enzymatic reduction and incorporation into organic forms such as SeMet, or
divergent binding of selenium to proteins and polysaccharides.

## 4. Conclusion

The study demonstrates that *P. ostreatus* and *H. coralloides* are highly efficient at metabolizing Se(IV), as opposed to Se(VI). Se(IV) is metabolized to SeMet and several derivatives of selenoneine, along with other organic species making it the first report of these derivatives in mushrooms and highlighting the complex nature of selenium metabolism in these mushrooms. The findings underscore that the selenium metabolism is strongly species-dependent, with distinct metabolic pathways governing the transformation and accumulation of selenium in different mushroom species. These insights contribute to a better understanding of how mushrooms process Se and its potential health benefits, emphasizing the importance of species-specific variations in Se biotransformation.

Supplementary data to this article can be found online at https://doi.org/10.1016/j.foodchem.2025.146763.

### CRediT authorship contribution statement

**Katarzyna Bierla:** Conceptualization, Data curation, Formal analysis, Investigation, Methodology, Software, Validation, Visualization, Writing – original draft, Writing – review & editing. **Marek Siwulski:** Conceptualization, Investigation, Methodology, Writing – original draft, Writing – review & editing. **Laurent Ouerdane:** Investigation, Writing – original draft. **Ryszard Lobinski:** Funding acquisition, Writing – original draft, Writing – review & editing. **Patrycja Mleczek:** Visualization. **Miroslaw Mleczek:** Conceptualization, Investigation, Methodology, Writing – original draft, Writing – review & editing.

### Declaration of generative AI and AI-assisted technologies in the writing process

During the preparation of this work the authors used https://quillbot.com/grammar-check in order to correct grammatical errors. After using this tool, the authors reviewed and edited the content as needed and take full responsibility for the content of the publication.

### Declaration of competing interest

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

### Data availability

No data was used for the research described in the article.

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