Comparative bioaccumulation and biotransformation of selenate, selenite, and selenomethionine in Cordyceps militaris

Abstract

Selenium (Se) is an essential trace element for humans. Cordyceps militaris is an edible mushroom with nutritional value. To develop a selenium biofortification strategy for C. militaris, the absorption and transformation of selenium from selenate, selenite, and selenomethionine in mycelia were investigated. The half maximal inhibitory concentrations (IC₅₀) of selenate, selenite and selenomethionine were 1028 mg L^-1, 68.2 mg L^-1, and 1.5 mg L^-1, respectively. The kinetic fitting results indicated that the absorption rate of selenomethionine is higher than that of selenite and selenate. The absorption of selenium was inhibited by carbonyl cyanide m-chlorophenylhydrazone (CCCP) and 2,4-dinitrophenol (2,4-DNP), while the addition of sulfur, phosphorus, and methionine reduced the uptake of selenate, selenite, and selenomethionine by 80.6%, 81.9%, and 14.2%, respectively. The mycelium can convert selenite into elemental selenium at 40 mg L^-1. The major selenium species in selenium-enriched mycelium are selenomethionine, selenocysteine, and Se-methylselenocysteine. Our results indicate that selenium biofortification of C. militaris mycelium using selenite is the optimal strategy. The selenium-enriched C. militaris mycelium could serve as a novel selenium source in human diets.

Graphical abstract

Highlights

• •The C. militaris mycelium are more sensitive to SeMet than Se(VI) and Se(IV).
• •The C. militaris mycelium shows a high ability to bioaccumulate selenium.
• •Energy metabolism and S, P, and Met pathways are involved in selenium absorption.
• •SeCys, SeMet, and MeSeCys are the main selenium species in Se-enriched mycelium.
• •Se-enriched C. militaris mycelium may serve as a novel dietary selenium source.

1. Introduction

Selenium is an essential trace element for normal human activity, with approximately 80% of it being present in selenoproteins [1,2]. Selenium is incorporated in the form of selenocysteine as the active site of 25 selenoproteins, including glutathione peroxidases, thioredoxin reductases, and iodothyronine deiodinases [3]. These selenoproteins catalyze the decomposition of lipid peroxides and hydrogen peroxide in human cells, thereby protecting the cells and cell membranes from oxidative damage [4]. The recommended daily allowances (RDA) and the tolerable upper intake levels (UL) of selenium for adults are 55 and 400 μg per day, respectively [1,4]. It is estimated that about 15% of the global population is at risk of selenium deficiency, which is mainly distributed in Brazil, Australia, China, Eastern Europe, and the Middle East [5,6]. Insufficient selenium supplementation leads to selenium-related diseases, including Keshan disease, Kashin-Beck disease, diabetes, coronary heart disease, and cerebrovascular diseases [7]. Currently, nutritional biofortification of foods has emerged as an effective strategy for preventing selenium deficiency.

Selenium generally exists in both organic species (selenomethionine, selenocysteine, etc.) and inorganic species (elemental selenium, selenide, selenite, selenate, etc.) in natural environments [8]. Organic selenium compounds are more available and safer for human health than that of inorganic selenium species [9]. Both selenite and selenate can be absorbed and converted to organic species by plants, animals, and microorganisms [10]. Among these organisms, mushrooms have the advantages of rapid growth cycle, high protein content, lower calories, and higher selenium biotransformation ability compared to plants and animals [11,12]. Lentinula edodes [13], Flammulina velutipes [14], Hypsizygus marmoreus [15], and Cordyceps cicadae [16] are excellent bioresources for selenium biofortification, which showed the ability to convert Se(IV) and Se(VI) into SeMet, MeSeCys and SeCys₂. Current studies on selenium biofortification in mushrooms primarily focus on the accumulation in fruiting bodies [17]. Nevertheless, the rule of selenium absorption and transformation in mycelium remains uncertain and requires further investigation.

Mycelium-based food (MBF) possesses rich nutritional value and eco-friendly characteristics, derived from mushroom mycelium [18,19]. Compared to the fruiting body, the mycelium showed rapid growth and stronger selenium biotransformation capacity. The cultivation of fruiting bodies of mushrooms typically requires a period of 1-3 months [20,21], while mycelium cultivation takes only 5-14 days [22,23]. Furthermore, the maximum bioconcentration factor (BCF) of selenium in mycelium (>40) was significantly superior to that of the fruiting bodies (<15) in edible fungi including Ganoderma lingzhi, Stropharia rugoso-annulata, Auricularia auricular, and Pleurotus eryngii. etc [20,[23], [24], [25]]. Cordyceps militaris, which belongs to the phylum Ascomycota and family Cordycipitaceae, is widely distributed in the world and is cultivated in China, Japan, South Korea, Europe, and North America [26,27]. In previous studies, the fruiting body of C. militaris exhibited a higher ability of selenium biotransformation than that of Auricularia auricula and Hericium erinaceus [24,28,29]. Although numerous studies have confirmed the selenium enrichment capability of C. militaris fruiting body, detailed investigations into the selenium enrichment and transformation properties of its mycelium remain limited.

The main objectives of this paper were: (1) to compare the effects of selenate, selenite and selenomethionine on the growth and selenium composition of C. militaris mycelium. (2) to explore the absorption and transformation abilities of selenium in C. militaris mycelium. (3) to evaluate the potential of selenium-enriched C. militaris mycelium as a selenium supplement.

2. Materials and methods

2.1. Materials and reagents

The C. militaris CP1 strain is preserved at the China Center for Type Culture Collection (CCTCC) under the accession number M2019671. All solvents and chemicals used were of analytical reagent grade. All solutions were prepared with deionized water (18.2 MΩ/cm), which was obtained using a Milli-Q system (Bedford, MA, USA). The reagent grade powder (purity >99%) of sodium selenate [Se(VI)], sodium selenite [Se(IV)] and selenomethionine (SeMet) was purchased from Sigma (St. Louis, MO, USA). The standard compounds (1 mg L^-1) including Se(VI), Se(IV), SeMet, selenocysteine (SeCys₂), and Se-methylselenocysteine (MeSeCys), were purchased from the National Reference Material Research Center (Beijing, China).

2.2. Growth of C. militaris mycelia in exogenous selenium

The C. militaris mycelia were cultured according to a previous method with modifications [30]. The C. militaris CP1 mycelia were cultured at 21 °C for 5 days in modified potato dextrose medium (MPD, 200 g L^-1 potato, 20 g L^-1 glucose, 3 g L^-1 peptone, 2 g L^-1 KH₂PO₄, 1 g L^-1 MgSO₄·7H₂O, and 25 mg L^-1 complex V_B) supplied with Se(VI), Se(IV), and SeMet at selenium concentrations of 40.0 to 5620.0 mg L^-1, 0.5 to 320.0 mg L^-1, and 0.1 to 10.0 mg L^-1, respectively. The cultured CP1 mycelia were centrifuged at 4000 × g for 5 min, washed three times with sterile water, lyophilized, and weighed. The dose-response curve was calculated using a logistic model [31,32]:

$$\mathrm{y}=\mathrm{c}/\left\{1+\exp \left[\mathrm{b}\left(\mathrm{x}-\mathrm{a}\right)\right]\right\}$$

y represents the tested parameter, x denotes the selenium concentration, c is the uninhibited value of y, b stands for the slope of mycelial inhibition, and a indicates the inhibitory concentration. The values of parameters a, b, and c of the model were estimated using the statistical program origin 2019b (Origin Lab, Northhampton, MA, USA). The 50% inhibition (IC₅₀) of mycelial growth was defined as the Se concentration that induced a 50% reduction in the biomass of mycelia compared with the control.

2.3. Morphological observation and energy spectrum analysis of C. militaris mycelia

The C. militaris CP1 mycelia were cultured for 5 days in the MPD medium supplied with 40.0 mg L^-1 Se(VI), 40.0 mg L^-1 Se(IV), or 2.5 mg L^-1 SeMet. Then the morphology of CP1 mycelia was observed by light microscope (Eclipse E200, Nikon, Japan), and transmission electron microscope (TEM) (Hitachi H7500, Tokyo, Japan) according to the previous methods [33,34]. The selenium in the mycelium was detected using an Energy Dispersive X-ray spectroscopy (EDS) detector (HT7700, Hitachi, Japan).

2.4. Selenium uptake by C. militaris mycelia

The mycelia of C. militaris were cultured in MPD medium for 4 days, collected by centrifugation, and inoculated in a new MPD medium supplied with Se(VI), Se(IV), or SeMet with at selenium concentrations of 0, 0.1, 0.5, 1, 2.5, 5, 10, 20, 40 and 80 mg L^-1, respectively. The mycelia were cultured in a shaker at 180 rpm for 24 h and then were collected by centrifuge at 4000 × g for 5 min, washed with sterile water three times, and lyophilized for total selenium analysis. For the time curve of selenium uptake by mycelia, Se(VI), Se(IV), and SeMet were added to MPD medium at a concentration of 1.0 mg L^-1, respectively. The mycelia were cultured in MPD medium with selenium at 180 rpm for 1 min, 5 min, 15 min, 30 min, 60 min, 120 min, and 240 min. The cultivated mycelium was centrifuged, washed with sterile water three times, and lyophilized for total selenium analysis as previously described. Each treatment was replicated four times. The absorption kinetic curves of selenium were fitted using the Michaelis-Menten equation [35]:

$$V=\frac{V_{\mathit{max}}*x}{K_m+x}$$

V (mg kg^-1 h^-1) represents the rate of selenium absorption by mycelium; K_m (mg L^-1) represents the affinity of mycelium for selenium; V_max (mg kg^-1 h^-1) represents the maximum absorption rate; x represents the independent variable in the experiment. The parameters of this model were estimated using the statistical program SigmaPlot (Systat Software, San Jose, CA, USA).

2.5. Selenium uptake pathways in the mycelia of C. militaris

The selenium absorption assays were performed according to the method with some modifications [36]. The mycelia of C. militaris were prepared as previous methods, and 2,4-dinitrophenol (DNP) and carbonyl cyanide m-chlorophenyl hydrazone (CCCP) were supplied in the MPD medium to a final concentration of 100 μM, respectively. After the mycelia were cultured for 10 min, the final concentrations of 1 mg L^-1 of Se(VI), Se(IV) and SeMet were added to the culture medium, respectively. For the sulfur and phosphorus uptake channel analysis, MgSO₄ and KH₂PO₄ in the MPD medium were replaced by MgCl₂ and KCl to prepare phosphorus-deficient and sulfur-deficient medium, respectively. Double-phosphorus and double-sulfur medium were prepared by adding twice the amount of MgSO₄ and KH₂PO₄ compared to the control. For the Met uptake channel analysis, the Met-supplemented medium was also prepared by adding Met to 10 mg mL^-1 in the medium. The MPD medium was used as the control. When the mediums described above were prepared, Se(VI), Se(IV), and SeMet were added at a concentration of 1.0 mg L^-1, respectively. The CP1 mycelia were incubated in the medium at 180 rpm for 2 h, collected by centrifugation, washed with sterile water for three times, and lyophilized. The total selenium was analyzed in the mycelia. Each treatment was repeated four times.

2.6. Determination of total selenium content

The total selenium was detected according to the methods described with some modifications [26]. Briefly, 0.5 mL of supernatant or 100 mg of mycelial powder was digested with 5 mL of 15.3 mol L^-1 HNO₃ in a far-infrared digestion furnace (Gelaimo Testing Equipment Co., Wuhan, China). The digested solutions were then reduced with 6 mol L^-1 HCl in a water bath at 95 °C for 3 h. The selenium content was then determined using a Hydride Generation Atomic Fluorescence Spectrometer (HG-AFS, Jitian Instruments Co., Beijing, China).

2.7. Determination of selenium speciation

Selenium species were determined using High Performance Liquid Chromatography coupled with Hydride Generation - Atomic Fluorescence Spectroscopy (HPLC-HG-AFS) (Beijing Jitian Instrument Co., Ltd., Beijing) [26]. Briefly, the mycelial solutions were centrifuged at 12,000 × g to collect the culture solutions and the mycelia. The culture solutions were filtered through a 0.22-μm filter unit (Millipore, Billerica, MA, USA) and mycelia were lyophilized and powdered before analysis. The lyophilized mycelial powder (200 mg) was mixed with 10 mL of 4 mg mL^-1 Proteinase K (Sigma Chemical Co., St. Louis, MO, USA) and incubated in a shaker at 150 rpm with 37 °C for 12 h. Then the supernatants were collected by centrifugation and filtered by a 0.22-μm filter unit. The selenium species were isolated by HPLC and detected by HG-AFS.

2.8. Statistical analysis

The data is presented as mean ± standard deviation (SD) in this study. Statistical analysis was conducted using SAS 9.0 (version 9.0, SAS Inc., Cary, NC, USA). The analysis of variance (ANOVA) was performed by Duncan's multiple range test and the pairwise analysis was conducted by two-tailed Student's t-test. Statistical significance was set at P < 0.05, with the following asterisk conventions: *P < 0.05, **P < 0.01.

3. Results

3.1. Effect of selenate, selenite, and SeMet on the growth of C. militaris mycelia

The biomasses of mycelia were significantly decreased for 48.4-85.0%, 27.38-53.53%, 62.17-82.11% in 1280.0-5620.0 mg L^-1 Se (Ⅵ), 80.0-320.0 mg L^-1 Se (Ⅳ), and 2.5-10 mg L^-1 SeMet treatments, respectively (Fig. 1A–D). In 40.0-320.0 mg/L Se (Ⅵ) treatments, the biomasses of mycelia significantly increased by 17.2%-46.8% (Fig. 1A and B). The biomasses of mycelia showed no significant difference between the control and the treatments of 640.0 mg L^-1 Se (Ⅵ), 5.0-40.0 mg L^-1 Se (Ⅳ), and 0.1-1.0 mg L^-1 SeMet (Fig. 1B–D). The median effect concentration (EC50) of Se (Ⅵ), Se (Ⅳ) and SeMet on mycelial growth were 1028.1 ± 222.1 mg L^-1, 68.2 ± 13.4 mg L^-1 and 1.5 ± 0.5 mg L^-1, respectively (Fig. 1E–G; Table S1). Meanwhile, there was obvious red coloration under 20-80 mg L^-1 Se (Ⅳ) treatments (Fig. 1A).

Fig. 1.

Effects of Se (Ⅵ), Se (Ⅳ), and SeMet on C. militaris mycelial growth. A, the mycelial growth in medium supplemented with a series of concentrations of Se (Ⅵ), Se (Ⅳ), and SeMet, respectively; B-D, biomass of C. militaris mycelia after 5 days incubation with Se (Ⅵ) (B), Se (Ⅳ) (C) and SeMet (D) treatments; E-F, the logistic fitting curves of C. militaris mycelia with Se (Ⅵ) (E), Se (Ⅳ) (F) and SeMet(G) treatments; The data are presented as means ± SD (n = 4). Different letters denote significant differences (p < 0.05) between mean values according to Duncan's test (ANOVA).

The mycelial pellet of C. militaris was white and spherical in the control treatment. The mycelial pellet in 2.5 mg L^-1 SeMet treatments was smaller than that in the control. However, in 40 mg L^-1Se(VI) and Se(IV) treatments, the mycelial pellets were not different compared to the control (Fig. 2A). In the SeMet treatment, the mycelial margin was sparse, whereas no difference was observed between the Se(VI), Se(IV), and control groups (Fig. 2B). Notably, the mycelial pellet exhibited a red coloration at the center in 40 mg L^-1 Se (Ⅳ) treatment (Fig. 2A). Meanwhile, transmission electron microscopy (TEM) demonstrated the existence of particles within and on the surface of the mycelium which were identified as selenium by Energy-dispersive X-ray spectroscopy (Fig. 2C and D).

Fig. 2.

Morphology of mycelia under different selenium treatments. A, Morphology of C. militaris mycelia treated with 40 mg L^-1 Se (Ⅳ), 40 mg L^-1 Se (Ⅵ), and 2.5 mg L^-1 SeMet; B, Morphology of mycelia under 40 mg L^-1 Se (Ⅳ), 40 mg L^-1 Se (Ⅵ), and 2.5 mg L^-1 SeMet observed by a light microscope; C, TEM image of the mycelium of C. militaris after 40 mg L^-1 Se (Ⅳ) treatment; D, Energy spectrum image of mycelium under 40 mg L^-1 Se (Ⅳ) treatment.

3.2. Time-dependent selenium accumulation in C. militaris mycelia

The absorptions of Se (Ⅳ), Se (Ⅵ), and SeMet in C. militaris mycelia were time dependent, which were fitted by the Michaelis-Menten equation (R² value of 0.8053-0.9795) (Fig. 3A; Table S1). The K_m of selenium absorption was 0.52 mg L^-1, 4.40 mg L^-1, and 44.41 mg L^-1 in treatments of Se (Ⅵ), Se (Ⅳ), and SeMet, respectively (Table S1). The maximum absorption rate of selenium in C. militaris mycelia was 155.00 ± 0.2 mg kg^-1 h^-1, 18.04 ± 0.5 mg kg^-1 h^-1, and 4.88 ± 0.2 mg kg^-1 h^-1 in treatments of SeMet, Se (Ⅳ), and Se (Ⅵ), respectively (Table S1). During mycelial growth, the selenium content in the supernatant decreased under Se (Ⅳ) and SeMet treatments. However, the selenium content in the supernatant increased after 60 min of growth under Se (Ⅵ) treatment (Fig. S1).

Fig. 3.

Absorption kinetic curves of selenium and bioconcentration factor in C. militaris mycelia. A, Time-dependent kinetics for Se(VI), Se (Ⅳ), and SeMet absorption within 1 mg L^-1. B, Concentration-dependent kinetics for Se(VI), Se (Ⅳ), and SeMet absorption. C, Bioconcentration factor (BCF) calculated for selenium; BCF = selenium content in the mycelium/selenium content in the substrate. The curves represent the fitted Michaelis-Menten kinetics. The data are presented as means ± SD (n = 4). Different letters denote significant differences (p < 0.05) between mean values according to Duncan's test (ANOVA).

3.3. Concentration-dependent selenium accumulation in C. militaris mycelia

The selenium contents in C. militaris mycelia increased and reached a plateau with selenium concentration in the culture medium (Fig. 3B). The absorption of Se (Ⅵ), Se (Ⅳ), and SeMet in mycelia exhibited a significant correlation with the concentration by fitting curve of absorption kinetics which R² value is 0.9084, 0.9805, and 0.9052, respectively (Table S2). The maximum absorption rates of selenium in mycelia were 1718.2 ± 265.7 mg kg^-1 h^-1, 1122.4 ± 59.2 mg kg^-1 h^-1, and 12.5 ± 0.8 mg kg^-1 h^-1 in treatment of SeMet, Se (Ⅳ) and Se (Ⅵ), respectively. Meanwhile, the K_m of selenium absorption were 32.8 ± 11.2 mg L^-1, 32.8 ± 11.2 mg L^-1, and 3.6 ± 0.9 mg L^-1 under SeMet, Se (Ⅳ) and Se (Ⅵ) treatments, respectively (Table S2). The BCF of selenium in mycelia were 6.22-0.14 and 588.23-16.2, which were decreasing with the increasing of Se(VI) and SeMet concentrations. However, in Se (Ⅳ) treatment, the BCF of selenium was increasing with selenium concentration from 0.1 mg L^-1 to 2.5 mg L^-1 and decreasing with concentration from 2.5 mg L^-1 to 80 mg L^-1. The BCF of SeMet in mycelium was significantly higher than that of Se (Ⅳ), and then than that of Se(VI) at the same selenium concentration (Fig. 3C).

3.4. The influence on selenium absorption of C. militaris mycelia

The absorptions of Se(VI), Se(IV), and SeMet in the C. militaris mycelia were significantly inhibited by CCCP and 2,4-DNP by 37.3%, 51.0%, and 62.9%, and 46.0%, 58.8%, and 62.1%, respectively (Fig. 4A and B). Compared to the control group, the absorption of Se(VI) by mycelium in sulfur-deficient medium was significantly increased by 80.6%, while phosphorus-deficiency showed no significant influence (Fig. 4C). In phosphorus-deficient medium, the absorption of Se(IV) by mycelium was significantly increased by 81.9%, while sulfur-deficiency showed no significant influence (Fig. 4D). The addition of methionine significantly reduces for 14.2% the absorption of SeMet in the mycelium compared to that of the control (Fig. 4E).

Fig. 4.

Selenium concentration in C. militaris mycelium with CCCP (A), 2,4-DNP (B), sulfur (C), phosphorus (D), methionine (E) treatments. *p < 0.05, **p < 0.01, assessed by two-tailed Student's t-tests.

3.5. Determination of selenium species in C. militaris mycelia

In Se(VI) treated mycelia, two selenium species, Se(VI) (0.022 mg kg^-1) and SeMet (0.142 mg kg^-1), were detected (Fig. 5B; Table 1). In Se(IV) treated mycelia, SeCys₂ (0.171 mg kg^-1), MeSeCys (0.224 mg kg^-1), and SeMet (0.193 mg kg^-1), were identified (Fig. 5C; Table 1). For mycelia treated with SeMet, SeCys₂ (0.178 mg kg^-1), MeSeCys (0.312 mg kg^-1), and SeMet (0.162 mg kg^-1), were detected (Fig. 5D; Table 1). In the culture supernatant, Se(VI) was detected in the Se(VI) treatment, while SeMet was detected in both the Se(IV) and SeMet treatments (Fig. 5A–D).

Fig. 5.

Chromatographic profile of selenium species in C. militaris mycelia. A, Standard curve of selenium species. B-D, Analysis of selenium species in C. militaris mycelium treated with 1 mg L^-1 Se(VI) (B), Se (Ⅳ) (C), and SeMet (D). The red curve denotes the selenium species in the supernatant, whereas the blue curve indicates the selenium species in the mycelium.

Table 1.

Selenium species in mycelia of C. militaris under selenate, selenite, and SeMet treatments.

Treat	Selenium species (mg kg-1)					Extraction rate (%)a
	SeCys2	MeSeCys	Se(IV)	SeMet	Se(VI)
Se (Ⅵ)	ND	ND	ND	0.142 ± 0.022	0.022 ± 0.005	75.2 ± 1.2
Se (Ⅳ)	0.171 ± 0.013	0.224 ± 0.024	ND	0.193 ± 0.031	ND	76.3 ± 0.8
SeMet	0.178 ± 0.021	0.312 ± 0.032	ND	0.162 ± 0.012	ND	78.6 ± 0.6

Note: SeCys₂, selenocystine; MeSeCys, Se-methylselenocysteine; Se(IV), selenite; SeMet, selenomethionine; Se(VI), selenate. ND: below the detection limit.

^aExtraction rate was calculated by the concentration of Se in enzymatic hydrolysate/the concentration of Se in mycelium. The data are presented as means ± SD (n = 3).

4. Discussion

4.1. Effect of Se (Ⅵ), Se (Ⅳ), and SeMet on the growth of C. militaris mycelia

The growth of C. militaris mycelia displayed significantly higher tolerance toward Se(VI) and Se(IV) compared to SeMet. The IC₅₀ of Se (Ⅵ) for C. militaris mycelia was approximately 15- fold and 685-fold higher than those of Se (Ⅳ) and SeMet, respectively (Fig. 1; Table S1). It was speculated that SeMet could replace methionine during protein synthesis, thus change the structure and function of proteins and affect the normal growth and metabolism of C. militaris mycelium [37,38]. Furthermore, the results showed that the K_m value of SeMet was 9 times that of Se (Ⅵ) (Fig. 3B). Similar researches indicated that the growth of Flammulina velutipes and Hericium erinaceus is more affected by SeMet than by Se(IV) and Se(VI), and the reason could be that SeMet is more efficient uptake from the substrate and bind to proteins quickly [28,34]. The biomass of the C. militaris mycelia increased under treatments of 40-320 mg L^-1 Se(VI) (Fig. 1B). The studies on Auricularia auricular [24] and Hypsizygus marmoreus [15] revealed similar results. Selenium has been demonstrated to act as an antioxidant and enhance the antioxidant capacity of the organism directly or by increasing the activity of antioxidant enzymes (e.g., superoxide dismutase (SOD)) at low concentrations [39]. However, the growth of C. militaris mycelia was significantly inhibited under treatments of high selenium concentration (Fig. 1B). Excess selenium can cause oxidative stress, leading to mycelial death or growth stagnation by harmful selenides or reactive oxygen species (ROS) generation, membrane phospholipid peroxidation and DNA damage [7,40].

4.2. The bioaccumulation of Se (Ⅵ), Se (Ⅳ), and SeMet in C. militaris mycelia

The C. militaris mycelium showed a high ability to bioaccumulate selenium. The utilization of mushroom fruiting bodies for selenium biofortification is currently a common method [41]. As another form of mushroom, the capability of mycelium to enrich and transform selenium remain unclear. The fruiting bodies of mushrooms possess strong selenium enrichment ability (BCF), such as C. militaris (1.21-5.68) [29], A. auricular (1.51-2.89) [24], and H. erinaceus (2.51- 4.24) [28]. In this study, the BCF values of C. militaris mycelia under Se(IV) treatment ranged from 11.06 to 47.69 (Fig. 3C), which were significantly higher than those in fruiting bodies of C. militaris, A. auricular, and H. erinaceus [24,28,29].

The mycelium of C. militaris can achieve bioaccumulation by synthesizing elemental selenium. The mycelial pellet of C. militaris was observed to turn red under 40 mg L^-1 Se(IV) treatment (Fig. 2A). The TEM- EDX analysis confirmed the presence of elemental selenium in mycelium (Fig. 2C and D). It is known that excessive uptake of selenite can disrupt the normal biological transformation mechanism of organisms [42]. Elemental selenium, as an inert and insoluble substance, exhibits lower toxicity toward humans and animals compared to other selenium species [43]. Additionally, the mycelia of F. velutipes, Pleurotus ostreatus, and Phycomyces blakesleeanus also showed the ability to convert Se (Ⅳ) into elemental selenium, which the reduction of Se (Ⅳ) depended on the concentration of and treatment time [34,44,45]. The impact of these conditions on the synthesis of elemental selenium in the C. militaris mycelium requires further investigation.

4.3. The absorption of Se (Ⅵ), Se (Ⅳ), and SeMet C. militaris mycelia

The absorptions of Se (Ⅵ), Se (Ⅳ), and SeMet in C. militaris mycelium were dependent on energy metabolism, and Se (Ⅵ), Se (Ⅳ), and SeMet uptake were related to sulfur, phosphorus, and methionine transporter, respectively (Fig. 4). Proton carriers CCCP and 2,4-DNP are typical oxidative phosphorylation uncouplers that disrupt the proton gradient (Δψ and ΔpH) in mitochondria, leading to the inhibition of ATP synthesis [46,47]. The addition of CCCP and 2,4-DNP significantly inhibited the absorption of Se(VI), Se (Ⅳ), and SeMet by C. militaris mycelium in this study (Fig. 4A and B). Similarly, 2,4-DNP significantly inhibited the selenite uptake by 11% in F. velutipes [34]. Previous studies have confirmed that the absorption of Se(VI) is related to sulfate transporters in Nannochloropsis oceanica, Triticum aestivum, and Saccharomyces cerevisiae [36,48,49], while phosphate transporter is the main contributor to Se (Ⅳ) absorption in Nicotiana tabacum, Oryza sativa, and Escherichia coli [[50], [51], [52]]. This is similar to our results that sulfur deficiency promoted the absorption of Se(VI), while phosphorus deficiency promoted the absorption of Se(IV) in C. militaris mycelium (Fig. 4C and D). Additionally, the addition of methionine significantly reduced the accumulation of SeMet in mycelium (Fig. 4E). SeMet is highly similar in chemical structure to methionine (differing only by the substitution of sulfur with selenium), thus it is recognized by mycelium as a methionine analog and is absorbed through the same transport system [[53], [54], [55]]. The C. militaris mycelium had a higher uptake capacity for SeMet compared to Se(IV) and Se(VI) (Fig. 3; Table S1). Similarly, the absorption efficiency of SeMet was 20-100 times higher than that of Se (Ⅵ) and Se (Ⅳ) by Triticum turgidum and Brassica napus, mainly due to the difference in transporter activity [56,57].

4.4. The biotransformation of Se (Ⅵ), Se (Ⅳ), and SeMet in C. militaris mycelia

The reduction of Se(VI) to Se(IV) appears to be the rate-limiting step for the transformation of Se(VI) into organic selenium in C. militaris mycelium, which is consistent with the result in fruiting body [29]. A portion was converted into SeMet after the absorption of Se(VI), while Se (Ⅳ) was undetected in mycelium. The Se(IV) was transformed into SeMet, SeCys₂, and MeSeCys in the mycelium. Notably, SeMet was detected in the supernatant in Se (Ⅳ) treatment. This result indicated that SeMet was accumulated in the mycelium and was subsequently partially released into the external environment. The SeMet was converted into SeCys₂ and MeSeCys (Fig. 5; Table 1). These results indicated that Se(VI) was initially converted to SeMet, followed by SeCys₂ and MeSeCys in C. militaris mycelium. In Saccharomyces cerevisiae, Se(VI) is initially reduced to Se(IV), which is subsequently converted into SeMet or selenocysteine. SeMet can be converted into SeCys₂, which undergoes methylation to form MeSeCys [[58], [59], [60]]. The biotransformation of selenium in C. militaris mycelium is similar to that in yeast. Notably, Se(IV) could be converted into SeMet and SeCys₂ by the fruiting bodies of C. militaris, and MeSeCys was undetected in the mycelium [29]. MeSeCys is a precursor of methylselenol and has strong anticancer activity [61]. It had been reported that MeSeCys was less toxic and has higher biological activity than inorganic or other organic selenium compounds [62,63]. Furthermore, MeSeCys was more easily released than SeMet and SeCys₂ from gastric digestion, whereas it was not released by cooking process of boiling water [28].

4.5. The application of Se-enriched mycelium of C. militaris in food products

Mycelium-based food (MBF), as a significant component of alternative meat and functional food products, has undergone rapid development in recent years [18,19,64]. The selenium-enriched C. militaris mycelium obtained in this study exhibited a total selenium content of up to 800 mg kg^-1 under high selenium exposure conditions, which primarily served to evaluate its selenium biotransformation capacity. In practical applications, such highly enriched mycelium would be applied as a functional ingredient or dietary supplement to achieve nutritionally appropriate selenium intake levels. For example, the daily consumption of approximately 70 mg of Se-enriched C. militaris mycelium supplemented with other food materials could satisfy the recommended dietary allowance (RDA) of 55 μg day^-1 [1]. The predominant selenium species were selenomethionine, selenocysteine, and Se-methylselenocysteine in Se enriched C. militaris mycelium.

5. Conclusions

The mycelia of C. militaris exhibited high efficiency in accumulating Se(VI), Se(IV) and SeMet. The absorption of selenium was related to sulfur, phosphorus, methionine channels or energy metabolism. The mycelium of C. militaris exhibited the highest tolerance toward Se(VI), followed by Se(IV) and SeMet. Furthermore, Se(VI) is converted into SeMet, whereas Se(IV) and SeMet can be transformed into SeCys₂, and MeSeCys. The selenium-enriched mycelium contains a high proportion of organic selenium, with MeSeCys being one of the major selenium species. This study focuses on the absorption, transformation, and composition of selenium in C. militaris mycelia, providing a viable approach for obtaining Se-enriched C. militaris mycelia. However, the impact of selenium on other nutrient components in the mycelia still needs to be further explored.

CRediT authorship contribution statement

Yong Zhang: Data curation, Formal analysis, Investigation, Methodology, Writing – original draft, Writing – review & editing. Ting Hu: Data curation, Investigation, Methodology, Writing – original draft. Bingjie Zhao: Investigation, Methodology. Yanbin Guo: Investigation, Methodology, Writing – original draft, Writing – review & editing.

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.

Appendix A. Supplementary data

The following is the Supplementary data to this article:

Data availability

Data will be made available on request.