Selenium Uptake by Hericium erinaceus Basidiocarps on Various Substrates and Their Effect on Growth and Yield

Abstract

This study investigates selenium (Se) biofortification in four strains of the medicinal mushroom Hericium erinaceus using Se-enriched substrates to assess Se uptake, distribution, and its impact on yield. Experimental substrates included lignocellulosic materials fortified with Se (0, 2, 6, and 18 µg/g) in the form of sodium selenite. Se accumulation varied by strain, with M9521 showing the highest bioaccumulation efficiency, particularly for an unknown Se compound, suggesting unique metabolic pathways. Other strains predominantly accumulated selenomethionine. The bioconcentration factor was highest at 6 µg/g Se, indicating optimal efficiency at this level. Moderate Se supplementation (2–6 µg/g) improved yield, though time to harvest was affected by Se levels in some strains. Substrate composition influenced Se uptake, with wheat bran yielding the highest Se content and soybean hulls maximizing biological efficiency. A strong correlation between C/N and Se content was observed. Se distribution was higher in outer basidiocarp layers, and light intensity during cultivation enhanced Se accumulation. Se uptake decreased with subsequent flushes at medium and high supplementation levels, while substrates maintained the same bioavailable Se. The results highlight the potential of Se-enriched Hericium erinaceus as a functional food source and the role of substrate and cultivation conditions in optimizing Se content.

1. Introduction

Hericium erinaceus is an edible and medicinal mushroom with a long history of use in traditional Chinese medicine [1]. It is distributed across the Northern Hemisphere in Europe, Asia, and North America. Due to its unique appearance, this mushroom has acquired several unusual names, such as lion’s mane, monkey head, bearded tooth mushroom, Yamabushitake in Japanese, and Houtou in Chinese [1,2]. It grows rarely parasitically on living hardwood trees, especially beech and oak, and saprophytically on their fallen logs and stumps [3]. The mycelium and basidiocarps contain many nutrients and bioactive compounds with broad therapeutic applications [4]. Hericium erinaceus has positive effects on the central nervous system, including the suppression of certain cancerous lines, depression, lipedema, and cognitive decline caused by aging [5]. It is also used to treat Alzheimer’s disease, Parkinson’s disease, ischemic stroke, or for the long-term prevention of stroke and dementia [2,6]. Mushrooms are cultivated and consumed for their high content of proteins, polysaccharides, and bioactive compounds, and they possess numerous nutritional and medicinal properties. Their ability to absorb and accumulate beneficial elements enhances their nutritional value, making them an interesting functional food supplement [7].

In some parts of the world, such as Europe or China, selenium (Se) intake is relatively low. The deficiency of Se in food can be addressed by manipulating Se concentrations in plants that accumulate this element. Biofortification is a strategy to improve the nutritional value of plants and deliver essential nutrients to the human diet [8]. According to the European Food Safety Authority (EFSA), adults can achieve sufficient Se intake by consuming 70 μg of Se daily. This dose ensures sufficient plasma selenoprotein P (SePP), reflecting functional Se saturation in the body [9]. A safe daily dose for an adult is up to 200 μg; however, a tolerable dose can reach up to 400 μg per day [10]. To prevent diseases caused by Se deficiency, Se intake should be at least 19 μg daily [11]. The most common inorganic Se compounds include selenites (SeO₃⁻²), selenates (SeO₄⁻²), and selenide (H₂Se), while organic Se compounds include selenopolysaccharides, selenoproteins, selenonucleic acids, selenoamino acids, and some volatile Se compounds [7]. Se is an essential element for humans, but can also be toxic, depending on its concentration and chemical form. Organic forms, such as selenomethionine (SeMet) and selenocysteine (SeCys), are safer and more bioavailable than inorganic forms [12]. Se is a part of selenoproteins and selenoenzymes in the human body, such as thioredoxin reductases, glutathione peroxidases, and iodothyronine deiodinases, which play important roles in physiological processes [13,14]. These processes include maintaining redox homeostasis, immunomodulation, detoxification, cancer prevention, supporting normal thyroid function, and aiding sperm maturation and motility [15]. Se also plays a role in physiological processes in the brain, protects against thyroid and heart diseases, has anti-inflammatory and antiviral functions, and its deficiency can lead to Keshan and Kashin–Beck diseases [16,17]. On the other hand, excessive Se supplementation can be dangerous, as high intake may cause serious health complications. Excess Se can lead to acute toxic effects, such as nausea, vomiting, abdominal pain, garlic breath, and cardiac symptoms [18].

Mushrooms are cultivated on by-products and waste from agriculture and the food industry [19]. These wastes include, for example, wood sawdust, straw, husks, oilseed cakes, bran, seed hulls, etc., and are referred to as lignocellulosic wastes [20,21]. This approach to waste processing contributes to sustainable agriculture, which is currently a major focus [19]. The composition of the substrate and the inclusion of alternative additives significantly influence the growth, yield, and nutritional profile of mushrooms. Factors such as temperature and pH further affect the production of biologically active compounds, emphasizing the importance of carefully controlled cultivation conditions. Proper substrate preparation and treatment also play a crucial role, directly impacting both the yield and overall success of mushroom cultivation [22,23,24,25].

Most cultivated edible mushrooms have low Se content and various substrate Se- additives are used to increase it [8,26,27,28,29]. The consumption of Se-enriched mushrooms may help prevent or support the treatment of oncological, neurodegenerative, cardiovascular, and immunological diseases, as well as HIV infection [15,30,31]. Due to the biological functions and beneficial effects of mushrooms, their metabolism, nutritional compound value, and bioavailability, research on Se-enriched mushrooms is becoming increasingly popular. Moreover, the ability of mushrooms to biotransform inorganic Se forms into organic forms creates more effective and safer Se sources. During metabolism, the mycelium and basidiocarps of mushrooms incorporate Se into macromolecules, forming Se-proteins, Se-polysaccharides, and other organic substances that exhibit lower toxicity and greater bioavailability compared to their inorganic counterparts [30,32]. In cultivating Se-enriched mushrooms, inorganic salts (Na₂SeO₃, Na₂SeO₄) are predominantly used, which are metabolically converted into organic substances such as SeMet, SeCys, selenocystine (SeCys₂), and methylselenocysteine (MeSeCys) [7,15]. The distribution of these compounds depends on the species and strain of the cultivated mushroom and on the cultivation conditions [7]. Besides Se-proteins, Se-polysaccharides, and Se-amino acids, Se-enriched mushroom cultivation also affects other biologically active metabolites that do not contain Se in their structure, such as basic amino acids, flavonoids, and polyphenols. Thus, Se-enriched mushroom cultivation positively impacts overall nutritional value. Small doses of Se promote mushroom growth and can increase yields; however, high Se concentrations inhibit fungal biomass growth and cause cell degradation [7,31]. Basidiocarps enriched with Se exhibit Se bioavailability of more than 50% in gastrointestinal digestion tests, suggesting that Se-enriched basidiocarps may serve as a promising natural source of daily Se intake for humans [8].

The aim of this study is to determine the effect of Se supplementation and fungal strains on harvest time, yields, Se content and Se species, the influence of substrate composition, and the optimal harvest time for Se accumulation. Current research primarily focuses on yields and Se content in the first flush, often neglecting subsequent flushes and variability of composition of primary substrate. However, from an economic perspective, it is crucial to address this issue, as most growers harvest mushrooms in multiple flushes.

2. Materials and Methods

2.1. Hericium Erinaceus Strains

Experiments were conducted in the 2023–2024, evaluating four strains of lion’s mane mushroom (Hericium erinaceous (Bull.) Pers.), strains are shown in Table 1. The cultures were maintained on MEA (malt extract agar), i.e., Agar-Agar, Kobe I (Carl Roth GmbH + Co. KG, Karlsruhe, Germany) with the addition of 2% malt extract (Carl Roth GmbH + Co. KG, Karlsruhe, Germany). Morphology of the basidiocarps displayed in Supplementary Figure S1.

2.2. Substrate Preparation and Growing Conditions

For substrate preparation a mix of oak and beech sawdust were used, obtained from company Albaflor s.r.o. (Čelákovice-Sedlčánky, Czech Republic). Additional materials included rapeseed press cake, wheat bran, spent brewers’ grain acquired from the Demonstration and Experimental Stable of Czech University of Life Sciences Prague, soybean hulls from CHOCOLAND a.s. (Kolín, Czech Republic) and gypsum from Kittfort Praha s.r.o. (Neratovice, Czech Republic). Polypropylene (PP) cultivation bags (model PP50/SEU4/V40-51) were purchased from SAC O2 nv (Deinze, Belgium). The composition of the substrates is in Table 2.

2.2.1. Spawn Preparation

The wheat grain was rinsed with water to remove coarse impurities and then hydrated to an optimal moisture level of 40–45%. Subsequently, gypsum 2.5% dry weight (d.w.) was added, and the mixture was poured into 1 L glass bottles with a cotton filter and sterilized for 3 h at 121 °C in an autoclave MLS-3781L (Sanyo Electric Co., Ltd., Moriguchi City, Japan).

After cooling, the bottles were inoculated with the respective Hericium strains from Petri dish in a sterile environment inside a flow box Flow FAST H (Faster s.r.l., Cornaredo, Italy) and then incubated at 24 °C for 20 days.

2.2.2. Substrate Preparation

For the first experiment, the substrate consisted of 79% sawdust, 20% wheat bran and 1% gypsum d.w., with four fortification levels, 0; 2; 6 and 18 μg/g, of Se wet weight (w.w.), using Na₂SeO₃ (Carl Roth GmbH + Co. KG, Karlsruhe, Germany) solution and one of four fungal strains. For experiments with different compositions of primary substrates, wheat bran was used alongside various alternatives., as shown in Table 3, and only one fortification level (6 μg/g) was used with one strain, MFTCCB134. The third experiment used the same substrate as in the first experiment and the strain M9521.

According to the methodology of individual experiments, a predetermined substrate composition was selected and adjusted to 64% moisture content. After homogenization, the substrate (2 kg) was filled into polypropylene bags and treated with steam for 24 h at 90 °C in a pasteurization chamber. Once cooled, the substrate was inoculated with 4% grain spawn inside a flow box Flow FAST H (Faster s.r.l., Cornaredo, Italy) and incubated at 24 °C without direct sunlight for 21–30 days. After 14 days of incubation, the bags were vented and turned upside down to minimize premature fructification. Each variant was cultivated in five replications (bags).

2.2.3. Growing Conditions

The cultivation itself took place in a growing box located in the Department of Horticulture, Czech University of Life Sciences. The conditions were maintained at a temperature range of 17–18 °C, with a relative humidity of 90–95%, and illumination of 500 lux for 24 h (except for one experiment with illumination of 35 lux for strain M9521). Regular ventilation ensured that the CO₂ concentration remained below 800 ppm. To initiate basidiocarps, an “X” incision measuring 3 × 3 cm was made in the bag using a sterile knife, through which the basidiocarps subsequently grew. The fully developed basidiocarps (in 2 to 3 flushes) were weighed and freeze-dried using a lyophilizer L10-55 (GREGOR Instruments s.r.o., Bělokozly, Czech Republic). The next flushes grew from the same opening in the bag. For selected strains, basidiocarps were harvested at two additional developmental stages: the beginning stage of basidiocarps development (two days after the appearance of primordia), and medium-developed basidiocarps (five days after the appearance of primordia). For strain M9521 at a concentration of 6 μg/g, the outer layer and inside of the basidiocarp was separated and analyzed.

The reflectance at individual wavelengths of the cultivation light was measured using a FieldSpec 4 Hi-Res instrument (ASD Inc., Boulder, CO, USA) at a distance of 15 cm from the light source, as shown in Supplementary Figure S2.

2.3. Determination of Total Selenium Content

Mushroom and substrate samples were lyophilized using a lyophilizer L10-55 (GREGOR Instruments s.r.o., Bělokozly, Czech Republic) and finely ground using a grinder (ETA a.s., Prague, Czech Republic). A homogeneous mushroom/substrate powder sample (0.2 g) was weighed (0.2 g), with an accuracy of 0.0001 g, into quartz tubes (35 mL). The samples were mineralized using a mixture of 4 mL HNO₃ Analpure^® (Analytika s.r.o., Prague, Czech Republic) and 2 mL H₂O₂ Rotipuran^® (Carl Roth GmbH + Co. KG, Karlsruhe, Germany) in a closed microwave digestion system Discover SP-D (CEM Corp., Matthews, NC, USA). The selected temperature program consisted of heating the samples to 180 °C over 10 min, followed by a 6 min hold at this temperature. The resulting digest was quantitatively transferred to 50 mL plastic tubes and diluted with ultrapure water (≥18.2 MΩ·cm⁻¹) using Milli-Q system (Millipore S.A.S., Molsheim, France) to a final volume of 40 mL. Se concentrations in the diluted digests were analyzed using inductively coupled plasma mass spectrometry (ICP-MS) using the Agilent 8900 (Agilent Technologies Inc., Santa Clara, CA, USA). The isotope ⁷⁸Se was measured in hydrogen mode to minimize spectral polyatomic interferences. External calibration was performed using a commercial certified Se solution ASTASOL-MIX (Analytika s.r.o., Prague, Czech Republic). The analyte signal was corrected by referencing the signal from the internal standards ⁷²Ge and ¹²⁵Te, which were continuously introduced into the nebulizer. The results were validated through parallel determination of Se content in a certified reference material, specifically bovine liver (BCR-185R, IRMM). The overall contamination introduced during the analytical procedure was evaluated using blank procedural blanks.

2.4. Determination of Bioavailable Se Content in Substrates

The homogenized substrates (2.5 g) were extracted with 25 mL of 0.1 M K₂HPO₄/KH₂PO₄ buffer (pH 7.0) to monitor temporal changes in the bioavailable fraction of Se in the substrates. This method was a modification of the procedure described by [33], which was originally developed to estimate soluble and ligand-exchangeable Se. The suspended samples were agitated for 4 h in a horizontal shaker set at 135 rpm. Subsequently, the samples were centrifuged at 3000× g for 10 min and filtered through a KA-5 filter with particle retention ≥ 8 μm (Papírna Perštejn s.r.o., Perštejn, Czech Republic). The resulting filtrates were diluted 10-fold with 2% HNO₃ and analyzed using ICP-MS. A solution of 0.01 M phosphate in 2% HNO₃ was used as the matrix for external calibration. Substrates samples were taken after important grow phases in four random samples: after heat treatment, after colonization, after the 1st flush, after the 2nd flush, and after the 3rd flush.

2.5. Determination of Se Species

Mushroom powder samples (200 mg) were weighed into 15 mL PP tubes and pre-incubated with 5 mL of 30 mM Tris-HCl buffer (pH 7.25) in an ultrasonic bath at 38 ± 2 °C for 30 min. The samples were then mixed with 1 mL of protease XIV (10 mg/mL, from Streptomyces griseus) and 1 mL of protease XXIII (10 mg/mL, from Aspergillus melleus), both purchased from Sigma Aldrich Chemie GmbH (Steinheim am Albuch, Germany), and dissolved in 30 mM Tris-HCl buffer. After brief homogenization on a vortex mixer (5 s), the samples were incubated in the ultrasonic bath for 120 min. Subsequently, the tubes were shaken on a rotator (30 rpm) for 30 min, centrifuged at 2690× g for 5 min, and filtered through 0.22 µm cellulose acetate syringe filters. The filtrate was diluted with Milli-Q water or mobile phase as needed. Two aliquots were prepared: one for determining total Se extraction efficiency, and the other for speciation analysis of Se by a chromatography–mass spectrometry technique (HPLC-ICP-MS). A high-performance liquid chromatography (HPLC) method, more specifically a reversed-phase ion-pair chromatography, was performed on an Agilent 1260 (Agilent Technologies Inc., CA, USA) equipped with a C18 Pyramid column (250 × 4.6 mm, 5 µm; Macherey-Nagel GmbH & Co. KG, Düren, Germany). An isocratic elution system was used to separate five individual species [SeCys₂, MeSeCys (MeSeCys), SeMet, selenite, and selenate]. The measurement conditions and instrumental parameters followed [34]. The mobile phase contained 20 mM ammonium acetate, 12 mM (NH₄)2SO₄, 1 mM tetrabutyl ammonium hydroxide (TBAH), and 2.5% (v/v) methanol, pH 5.2. The coupled instruments were calibrated in the range of 0.2–40 µg/L Se for each Se species considered.

2.6. Calculation of Biological Efficiency (BE)

Biological efficiency was calculated as the ratio of the fresh weight of basidiocarps (from all flushes) to the dry weight of the substrate, multiplied by 100. The result is expressed as a percentage, calculated using Equation (1) [24].

BE % = (weight of fresh mushrooms harvested (g)/(weight of dry substrate (g)) × 100(1)

2.7. Bioconcentration Factor (BCF)

BCF was calculated as the ratio of the selenium content in basidiocarps to the content of selenium in the substrate added by supplementation.

BCF = selenium in basidiocarps (μg/g)/content of selenium in the substrate (μg/g)(2)

2.8. Statistics

Statistical analysis was performed using TIBCO^® Statistica software, version 13.3 (TIBCO Software Ltd., Santa Clara, CA, USA). One-way and two-way analysis of variance (ANOVA) was used, followed by Fisher’s LSD test. Correlation matrices and regression and correlation analyses were used to examine dependencies. The closer the regression coefficient is to 1, the stronger the relationship: 0.0–0.2: very weak, 0.2–0.4: weak, 0.4–0.6: moderate, 0.6–0.8: strong, 0.8–1.0: very strong. The significance level was set at p = 0.05.

3. Results

3.1. The First Experiment with Selenium Fortification of Four Hericium erinaceus Strains

3.1.1. Content of Selenium in Basidiocarp in the First Flush

Figure 1 shows the influence of varying doses of Se added to the substrate (0; 2; 6 and 18 μg/g w.w.) on the Se concentration in the basidiocarps in first flush of four strains (VURV-F 5243, VURV-F 5242, M9521, and MFTCCB134).

Figure 1 summarizes Se accumulation in the basidiocarps of four H. erinaceus strains (VURV-F 5243, VURV-F 5242, M9521, MFTCCB134) under varying Se supplementation levels in the substrate (0, 2, 6, and 18 µg/g). Se concentrations in the basidiocarps increased progressively with higher supplementation levels for all strains, demonstrating a dose-dependent relationship. Among the strains, M9521 exhibited the highest Se accumulation, reaching 217.45 ± 7.9 µg/g d.w. at 18 µg/g supplementation, while MFTCCB134 had the lowest values across all supplementation levels, peaking at 129.52 ± 6.41 µg/g d.w. at 18 µg/g. VURV-F 5242 and VURV-F 5243 displayed intermediate Se concentrations, with maximum values of 159.76 ± 3.52 µg/g d.w. and 167.81 ± 4.25 µg/g d.w., respectively, at 18 µg/g. At 6 µg/g supplementation, Se concentrations ranged from 50.51 ± 2.76 µg/g (MFTCCB134) to 86.41 ± 2.61 µg/g (M9521). The control (0 µg/g supplementation) exhibited minimal Se levels across all strains, confirming the supplementation’s role in enhancing Se uptake. These findings highlight significant strain-specific differences in Se bioaccumulation and the strong influence of substrate Se concentration on uptake efficiency

Figure 2 demonstrates that all strains exhibited the highest bioconcentration factor (BCF) at 6 µg/g Se supplementation, suggesting this is the most effective level for Se bioaccumulation. Among the strains, M9521 consistently showed the highest BCF values, peaking at approximately 15, while MFTCCB134 exhibited the lowest BCF across all treatments. VURV-F 5242 and VURV-F 5243 displayed moderate responses, following a similar pattern. At the highest supplementation level (18 µg/g), all strains showed a decline in BCF, likely due to saturation or toxicity effects reducing Se uptake efficiency. This graph highlights both the strain-specific variability and the optimal supplementation level for maximizing Se uptake.

3.1.2. Different Developmental Stages of Strain VURV-F 5243 on Accumulation of Se

Se accumulation in the basidiocarps of Hericium erinaceus strain VURV-F 5243 increased significantly with higher Se supplementation levels in the substrate, with distinct trends observed across developmental stages (beginning, medium, and full). The medium developmental stages consistently exhibited the highest Se concentration, peaking at 217.95 ± 7.21 µg/g d.w. at 18 µg/g supplementation with significant difference, followed by the fully developed basidiocarps (167.81 ± 4.25 µg/g d.w.) and the beginning stage (163.05 ± 5.23 µg/g d.w.). The remaining fortification levels did not show a statistically significant difference. The Se concentration displayed a consistent increase from 2 µg/g to 6 µg/g supplementation across all stages, but the rate of accumulation was highest between 6 µg/g and 18 µg/g in the medium stage, indicating a greater capacity for Se uptake during this developmental stage. These results are shown in Figure 3 and suggest a dynamic interaction between the developmental stage and Se metabolism, with the medium stage serving as the most efficient period for Se enrichment in basidiocarps.

3.1.3. Growth and Yield Parameters

The influence of different Se concentrations on the yield and biological efficiency of various fungal strains was evaluated, as summarized in Table 4.

For strain M9521, the addition of Se had an impact on yield and biological efficiency. At 6 µg/g of Se, the highest total yield was observed (239.1 ± 6.2 g), corresponding to a biological efficiency (BE) of 33.2 ± 0.9% where significant difference against control was found. The control group had a lower total yield (193.0 ± 6.8 g) and BE (26.8 ± 0.9%).

Strain MFTCCB134 demonstrated the highest total yield (323.9 ± 25.4 g) and BE (45.0 ± 3.5%) at 2 µg/g Se. At the concentration of 2 µg/g Se, a statistically significant difference was observed compared to the control group. Furthermore, a statistically significant difference was found in all Se-supplemented variants compared to the other strains.

For strain VURV-F 5243, Se addition generally improved the total yield and BE. At 2 µg/g Se, the highest total yield was recorded (231.6 ± 17.8 g), with a BE of 32.2 ± 2.5% and a statistically significant difference compared to control. Higher Se levels (6 and 18 µg/g) did not further improve the yield compared to 2 µg/g, with total yields of 204.8 ± 2.3 g and 204.8 ± 12.4 g, respectively.

Strain VURV-F 5242 showed a similar trend, with the best performance at 2 µg/g Se, yielding 204.2 ± 9.7 g and achieving a BE of 28.4 ± 1.4%. Higher Se concentrations, such as 6 µg/g, reduced the total yield to 152.2 ± 18.5 g.

The results indicate that Se supplementation has a strain-specific impact on fungal growth and productivity. Moderate Se levels (2–6 µg/g) generally improved the total yield and biological efficiency with significant difference.

The cultivated strains of Hericium also differed in color. Strain VURV-F 5242 was always pure white, whereas the other strains exhibited a pinkish color during their early developmental stages. With increasing Se concentrations, the intensity of the pink color became more pronounced. However, as the basidiocarps matured, the pink color slightly faded. A similar pattern was observed in subsequent experiments. The strains also differed in their density, with strains M9521 and MFTCCB134 being the most compact and dense.

The influence of Se supplementation in the substrate on the time required for fructification and harvest was assessed for four Hericium erinaceus strains. Two key parameters were measured: the days from the initiation of fructification to the first harvest, and the days between the first and second harvest. The results are summarized in Table 5.

Strain M9521 exhibited the longest period from initiation of fructification to the first harvest, averaging 16.8 ± 0.2 days in the control, which was statistically significant. This value increased slightly with Se supplementation, reaching 17.2 ± 0.2 days at both 6 and 18 µg/g Se. The interval between the first and second harvest ranged from 9.2 ± 0.5 days at 2 µg/g Se to 10.6 ± 0.9 days in the control, showing moderate variability across treatments.

Strain MFTCCB134 exhibited a moderately fast time to first harvest among all strains, with 14.6 ± 0.6 days in the control. Se supplementation at 2 µg/g reduced the time further to 13.6 ± 0.4 days, while higher Se levels (6 and 18 µg/g) extended this period slightly to 15.8 ± 0.7 days. The interval between the first and second harvest was longest for this strain at 2 µg/g Se, reaching 15.6 ± 1.1 days, compared to 11.8 ± 0.2 days in the control.

VURV-F 5243 exhibited the fastest time to first harvest across all strains, averaging 12.2 ± 0.20 days in the control. This timing remained consistent across all Se supplementation levels, with a maximum of 13.2 ± 1.0 days at 2 µg/g Se. The interval between the first and second harvest was also the shortest among the strains, ranging from 7.6 ± 0.4 days at 6 µg/g Se to 8.2 ± 0.7 days at 18 µg/g Se.

Strain VURV-F 5242 showed intermediate timing for first harvest, with 15.2 ± 0.7 days in the control, increasing to 17.0 ± 0.3 days at 6 µg/g Se supplementation which was statistically different. The interval between the first and second harvest ranged from 9.2 ± 0.2 days at 2 µg/g Se to 11.00 ± 0.6 days in the control.

Se supplementation influenced the timing of fructification and harvest in Hericium erinaceus strains, with strain-specific and dose-dependent variations observed. Strain VURV-F 5243 consistently exhibited the shortest time to the first harvest, making it the fastest among the tested strains. Strain M9521 required the longest time to the first harvest across all treatments, while strain MFTCCB134 showed a balanced performance with reduced time to first harvest at lower Se supplementation, but extended intervals between harvests. These findings suggest that both Se supplementation and strain selection significantly impact the cultivation timeline of Hericium erinaceus.

3.1.4. Experiments Focused on Different Developmental Stages, Se Distribution Within the Basidiocarp, and Light Intensity of Strain M9521 at 6 µg/g Se Concentration

Because strain M9521 performed best at 6 µg/g Se concentration, yielding better compared to 18 µg/g, the later experiments focused on aspects such as the basidiocarp developmental stages, Se distribution within the basidiocarp, and the effect of lighting conditions.

At 6 ppm Se supplementation, Se accumulation in the basidiocarps varied significantly based light intensity during cultivation, as shown in Table 6. Cultivation under 500 lux resulted in a significantly higher Se concentration (96.85 ± 9.51 µg/g d.w.) compared to 35 lux (65.89 ± 3.24 µg/g d.w.), suggesting that higher light intensity promotes Se uptake. Furthermore, it was found that at lower intensity, the basidiocarps did not turn pink, and at an intensity of 35 lux, they were completely white.

Tissue location was not significantly different in total Se concentration, as shown in Supplementary Table S1. Nevertheless, the amount of Se was higher in the outer layer (103.69 ± 10.81 µg/g d.w.) compared to the inside of the basidiocarps (83.48 ± 9.22 µg/g d.w.), indicating a greater Se accumulation in surface tissues.

These results demonstrate that both tissue location and environmental conditions, such as light intensity, play a crucial role in Se distribution within basidiocarps at this supplementation level.

At 6 ppm Se supplementation, Se concentration in the basidiocarps of this strain remained consistent across developmental stages (beginning, medium, and full), with no statistically significant differences observed, as shown in Supplementary Table S2. These results suggest that Se uptake and distribution are uniform across the different growth stages at this supplementation level.

3.2. Different Substrate Compositions for Strain MFTCCB134

3.2.1. Content of Selenium in Basidiocarp in First Flush for Strain MFTCCB134

Se concentration in the basidiocarps and the BCF of H. erinaceus varied significantly across the tested substrate variants, as shown in Table 7. The WB substrate resulted in the significantly highest Se concentration (69.89 ± 2.43 µg/g d.w.) and the highest BCF (11.65 ± 0.40), indicating its superior efficiency in Se uptake. The BSG substrate also performed well, with Se concentration reaching 60.03 ± 3.07 µg/g d.w. and a BCF of 10.01 ± 0.51, ranking second among the variants. SBP and SH exhibited comparable Se concentrations (48.38 ± 1.08 µg/g d.w. and 43.71 ± 1.16 µg/g d.w., respectively) and BCF values (8.06 ± 0.18 and 7.29 ± 0.19, respectively), placing them in the mid-range. RPC displayed the lowest Se concentration (33.38 ± 2.54 µg/g d.w.) and BCF (5.56 ± 0.42), making it the least efficient substrate for Se bioaccumulation. The analysis of the C/N ratio revealed a very strong positive correlation (r = 0.97) with Se concentration in basidiocarps.

3.2.2. Different Developmental Stages of Strain MFTCCB134 on Accumulation of Se in Substrate WB

Best substrate in terms of accumulation was selected to more tests. At the 6 µg/g Se supplementation level on substrate WB, Se accumulation in the basidiocarps of H. erinaceus remained consistent across all stages (beginning, medium, and full), with no statistically significant differences observed (Supplementary Table S3). Se concentrations were slightly higher in the fully developed basidiocarps (69.89 ± 2.43 µg/g d.w.) compared to the medium (69.05 ± 3.66 µg/g d.w.) and beginning (68.91 ± 0.98 µg/g d.w.) stages. These results indicate that Se uptake and accumulation are stable throughout the development of basidiocarps at this supplementation level.

3.2.3. Growth and Yield Parameters for Strain MFTCCB134

The yield and biological efficiency of H. erinaceus varied across substrate variants (RPC, SH, WB, BSG), as shown in Table 8. The SH substrate produced a statistically significant higher overall yield, with a total of 580.6 ± 26.7 g per bag and a biological efficiency (BE) of 80.6 ± 3.7%, driven by its exceptional performance in the first (295.7 ± 27.4 g) and third flushes (139.3 ± 9.4 g) compared to the other substrates. In contrast, the RPC substrate resulted in the lowest total yield (377.9 ± 12.3 g) and BE (52.5 ± 1.7%), with the lowest first-flush yield (162.8 ± 13.5 g). WB and BSG substrates produced intermediate total yields (403.9 ± 22.3 g and 388.2 ± 14.4 g, respectively), with WB showing strong performance in the first flush (205.7 ± 23.5 g) but reduced yields in subsequent flushes. These results indicate that substrate selection significantly impacts productivity and BE, with SH emerging as the most effective substrate due to its consistently high yields across all flushes and superior efficiency. Weak negative correlation (r = −0.29) between the C/N ratio of the substrate and the biological efficiency (BE) across all flushes was found.

The time intervals for harvesting H. erinaceus across different substrate variants (RPC, SH, WB, and BSG) exhibited minor variability in the days required from initiation to the first harvest and between subsequent flushes.

3.3. Third Experiment: Strain M9521 and Different Flushes

Because strain M9521 was the best in terms of Se uptake from the substrate, further experiments were conducted on it to determine the effect of different flushes on accumulation of Se, as shown in Figure 4. Se concentration in the basidiocarps of H. erinaceus varied significantly across different substrate Se supplementation levels (control; 2 µg/g; 6 µg/g; and 18 µg/g) and flushes (first; second; and third). The control group exhibited minimal Se concentrations in all flushes, ranging from 0.16 ± 0.02 to 0.27 ± 0.01 µg/g d.w., with no significant differences among flushes. At 2 µg/g Se supplementation, Se concentrations were consistent across flushes, with values of 21.35 ± 1.58, 19.25 ± 0.6, and 19.56 ± 1.76 µg/g d.w. for the first, second, and third flushes, respectively. For 6 µg/g Se supplementation, the Se concentration peaked in the first flush (96.85 ± 9.51 µg/g d.w.) but decreased significantly in subsequent flushes (68.61 ± 7.98 µg/g in the second flush and 63.74 ± 2.94 µg/g in the third flush). Similarly, at 18 µg/g Se supplementation, the highest concentration was observed in the first flush (252.19 ± 5.99 µg/g d.w.), followed by significant reductions in the second (202.38 ± 8.59 µg/g) and third flushes (182.79 ± 11.28 µg/g). These findings indicate a decreasing trend in Se concentration across flushes at higher supplementation levels, reflecting reduced Se uptake efficiency in later flushes, while supplementation levels significantly influenced the overall Se content in basidiocarps.

Supplementary Table S4 presents the Se concentrations (Se_PO4) obtained from substrates at various stages of the cultivation process (6 µg/g Se treatment), following extraction with a buffered phosphate solution. The selenium concentration released after heat treatment remained stable at 4.27 ± 0.01 µg/g (d.w.), indicating that roughly half of the supplemented Se was not recovered prior to Hericium colonization. After colonization, Se_PO4 showed a slight increase to 4.78 ± 0.30 µg/g. Following the first flush, Se_PO4 decreased to 4.08 ± 0.29 µg/g, while after the second flush, it rose slightly to 4.34 ± 0.05 µg/g. Given the variability in Se_PO4 data, temporal changes in soluble and ligand-exchangeable Se were not statistically significant, and did not appear to accurately represent Se uptake by Hericium.

3.4. Speciation Analysis of Se in Hericium Samples

The dataset in Table 9 presents the results of speciation analysis of Se in hydrolysates of four strains (VURV-F 5243, MFTCCB134, VURV-F 5242, and M9521). The hyphenated technique was capable of detecting the following Se species (Supplementary Figure S3), along with their HPLC retention times (t_R): SeCys2 (2.47 min), MeSeCys (3.02 min), selenite (3.86 min), SeMet (4.35 min), and selenate (7.29 min). At t_R = 3.47 min, a major unknown compound was detected in specific strains and semi-quantified using calibration data from the nearest known peak (SeMet). The efficiency of the extraction procedure was 90 ± 6%, and column recovery reached 92 ± 8%.

The accumulation of Se species exhibited distinct patterns across the tested strains, potentially reflecting unique metabolic profiles. Selenite, selenate, and MeSeCys were below the detection limits (LODs) in all analyzed samples. The LODs for these species were estimated to range from 0.1 to 1.0 µg/g Se, depending on the dilution factor applied. The predominant Se species was SeMet in three of the four strains. In these strains, the SeMet content increased in a dose-dependent manner with the Se concentration in the substrate, peaking at 138 ± 6 µg/g Se in VURV-F 5243 grown in the 18 µg/g Se substrate. In contrast, the highest SeCys2 content was observed in VURV-F 5242, reaching 11 ± 1 µg/g Se when harvested from the 18 µg/g Se substrate. Interestingly, the M9521 strain displayed a distinctly different Se species profile, with the major Se species eluting at a t_R that did not match any of the calibration standards (Supplementary Figure S4). The content of this unknown Se species was estimated at up to 184 ± 13 µg/g Se, showing a strong linear response to Se supplementation, while SeMet levels remained low (≤2 µg/g Se) regardless of substrate Se concentration. The unknown species was not detected in VURV-F 5242, but was only present in MFTCCB134 (7 ± 2 µg/g Se) when grown in 18 µg/g Se substrate. In VURV-F 5243, the content of the unknown species showed irregular trends and was absent during the early developmental stages of the mushroom.

Overall, the results highlight substantial strain-specific differences in Se metabolism, particularly in the accumulation of SeCys2, SeMet, and the unidentified compound.

4. Discussion

The first experiment investigated the positive influence of different doses of Se in the form of Na₂SeO₃ added to the substrate on the resulting Se concentration in basidiocarps and growth parameters. This finding is supported by various authors in previous studies on various fungal species [8,26,27,28,29,35]. Hu et al. [8] recorded the highest Se concentrations in basidiocarps of Hericium erinaceus at 111.8 μg/g (with 40 μg/g Se addition), compared to the highest concentration of 217.45 ± 7.9 μg/g reported in this study, likely due to the specific ability of the strain to absorb Se.

The results suggest that the tested Hericium strains could be used for Se biofortification in the future, and potentially marketed as dietary supplements. According to the European Food Safety Authority (EFSA), adults require a daily Se intake of 70 μg [9]. For capsules weighing 300 mg of dried Hericium basidiocarp with Se-enriched substrate at 18 μg/g (with an average Se content of 161.1 μg Se/g across all strains), a capsule would contain 48.32 μg Se, covering 69% of the recommended daily intake.

The effect of basidiocarp maturity was also examined, revealing no significant differences in Se content among beginning, medium, and fully developed basidiocarps at Se enrichment levels up to 6 μg/g. However, at the highest fortification level, the highest Se content was observed in the medium-developed basidiocarps, potentially due to a dilution effect as the biomass increased while the Se uptake remained constant.

Biological efficiency varied across strains and Se concentrations. No statistically significant reduction in BE between controls was observed with increasing Se concentrations, consistent with other authors [27,28]. Lower Se supplementation (2–6 μg/g) in our experiments stimulated BE the most. The BE increase was biggest for strain MFTCCB134, highlighting variability among strains. Niedzielski et al. [29] enriched substrates with inorganic Se salts (Na₂SeO₃ and Na₂SeO₄), testing three mushroom species, including H. erinaceus. A slight, statistically insignificant BE increase was observed for Hericium at Se concentrations up to 0.2 mM, while concentrations above 0.4 mM showed negative effects and toxicity, with Hericium being the most sensitive among the tested species. On the other hand, Hu et al. [8] noted a decrease in BE for H. erinaceus at Se concentrations as low as 2.5 μg/g. A decrease with higher Se supplementations (50 mg/L) was also found in another study [36]. Despite the commercial strain MFTCCB134 achieving the highest yields, it had the lowest Se accumulation in basidiocarps. These results confirm significant differences, not only among mushroom species, but also between individual strains.

An interesting finding was the pink discoloration of basidiocarps in strains VURV-F 5243, M9521, and MFTCCB134 during earlier developmental stages, which intensified with higher Se concentrations in the substrate. Strain VURV-F 5242 remained white in all trials. A similar phenomenon was noted by Niedzielski et al. [29] in Hericium, where Se application caused slight pink discoloration, even in strains without this characteristic. These authors attributed the discoloration to potential detoxification mechanisms, reducing inorganic Se salts to elemental Se nanoparticles with a pink hue. However, in our study, slight pink coloration was also observed in control variants, possibly influenced by low temperatures, high light intensity, and continuous 24 h lighting at approximately 500 lux. Additional tests showed that reducing light intensity to 200 lux or lower and a 12 h light cycle resulted in white basidiocarps. The same was observed for a light intensity of 35 lux and 24 h lighting. This phenomenon appears strain-dependent, as other authors [37] cultivated Hericium under 1000 lux lighting without observing pink discoloration. The pink color could just indicate a stress reaction. In our study, we found that an increased intensity of light not only led to a pink coloration, but also resulted in a higher Se content in the basidiocarps. Further studies with various wavelengths and intensities should be conducted to confirm our findings. In our case, this was only an initial pilot study. Furthermore, it was observed that more Se is concentrated in the outer layers of basidiocarps. The increased Se content may be due to the fact that light is shining directly on the surface. All strains exhibited the highest BCF at 6 µg/g Se supplementation (around 13 for strain M9521), suggesting that this is the most effective level for Se bioaccumulation, which is higher than described by other authors [8,29].

In our study, it was demonstrated that the composition of substrate significantly affects the Se content in basidiocarps. The highest Se content was observed in the control variant WB (wheat bran) at 69.9 μg/g, with a statistically significant difference compared to other additives, except for the variant. The highest yield was achieved by variant SH (soybean hulls) with 80.6% biological efficiency (BE), which also had the second-lowest Se content in basidiocarps at 43.7 μg/g. A strong correlation (r = 0.97) was observed between the C/N ratio of the substrate and Se content in basidiocarps, with Se levels increasing as the C/N ratio increased. However, this ratio had no significant positive impact on BE. For instance, the variant RP had the lowest yields and the highest nitrogen content, and achieved the lowest yields. This aligns with the findings of Atila [38], who found no significant role between the N content and BE, but observed a strong correlation with lignin content. Conversely, Fan et al. [39] reported a correlation between a narrow C/N ratio. The yield differences and content of Se may not solely depend on the C/N ratio, but could also be influenced by the additive’s properties, such as particle size, lignin, cellulose and hemicellulose content, antinutritional factors (e.g., rapeseed press cake), oils, and other components. Rasalanavho et al. [40] confirmed the importance of locality, redox status, pH, and microbial activities on element uptake, including Se, in wild mushrooms.

In this study, the effect of soybean hulls, a by-product of soybean processing for the food industry, was tested. This material has not been extensively studied so far in scientific literature for Hericium. The focus was primarily on testing various meals from soybean, as demonstrated by Krupodorova and Barshteyn [41], who studied the mycelium yield of Hericium and other fungi on various meals, finding rapeseed meal to be one of the least favorable substrates for Hericium, while soybean meal was the most effective. Similarly, Siwulski and Sobieralski [42] reported that soybean meal and wheat bran provided the highest yields for Hericium cultivation, which aligns with our study. However, there are mentions in the literature regarding the use of hulls in other mushrooms; Liu et al. [43] observed the positive effects of soybean hulls on P. sajor-caju, Atoji-Henrique et al. [44] on Ganoderma lucidum, and Ryu et al. [45] on Pleurotus pulmonarius. Soybean hull enrichment is popular in America, and its use as an alternative additive to standard wheat bran could be considered in regions with increasing soybean cultivation.

At lower Se supplementation, the Se content remained consistent across all flushes. However, at medium and higher Se supplementation levels, Se content peaked in the first flush and declined in subsequent flushes, indicating a decrease in Se uptake efficiency over time. Despite consistent Se availability in the substrate, the accumulation decreased with successive flushes, emphasizing the need for further studies on Se dynamics during later growth stages.

Fortification studies of edible mushrooms have established that inorganic Se, typically in the form of selenite, can be efficiently converted into organically bound Se [7]. In our study, the absence of the source Se compound (selenite) and its oxidized form (selenate) in the chromatograms (Supplementary Figure S4) confirms that complete biotransformation occurred within the Hericium strains under investigation. Unfortunately, the metabolic pathways involved in the uptake and transformation of Se anions by mushrooms are only partially understood, and are occasionally inferred by analogy with Saccharomyces cerevisiae [46]. Speciation analysis of Se, commonly performed on enzymatic mushroom hydrolysates using HPLC-ICP-MS, is widely recognized as a fundamental tool for distinguishing individual Se species. We aimed to harness the capacity of Hericium to produce organically bound Se, which proved successful, as evidenced by the high SeMet contents in strains MFTCCB134 (up to 105 µg/g Se; 85% of total Se), VURV-F 5242 (up to 124 µg/g Se; 77% of total Se), and VURV-F 5243 (up to 138 µg/g Se; 81% of total Se. The SeMet contents align with the overall trend of total Se content in these strains. Other authors have also identified SeMet as the major Se species in Hericium [8,46]; however, they generally reported lower proportions relative to the total Se content, with both studies indicating approximately 50%. Surprisingly, in strain M9521, SeMet was detected only as a minor species (<2.0 µg/g Se), while an unknown species reached up to 184 µg/g Se (87% of total Se). In contrast, the unknown species was consistently detected as a minor component in strain VURV-F 5243, yet remained below LOD in all treatments of strain VURV-F 5242. It should be noted that other Se species, such as SeCys2 and SeMeCys, have been reported to exhibit variable profiles, not only among different edible mushrooms [7], but also within the same genus [27]. Factors including genetic differences, Se extraction efficiency, degree of protein proteolysis, chromatographic column recovery, sample stability, and cultivation conditions may all influence the observed species distribution. To rule out the possibility of artifacts, strains VURV-F5242 and M9521 were cultivated again, and the Se species profile was re-evaluated, yielding essentially the same observations. We further excluded chromatographic artifacts by confirming the reproducibility of t_R for all species, which permitted the simultaneous detection of SeMet, the unknown species, and spiked SeMeCys and selenite. The occurrence of minor unknown chromatographic peaks is commonly reported for mushrooms [8,27]; however, the confirmation of a novel or unusual Se species is rare. A notable example is the recent work [47] which documented a high accumulation of selenoneine in Boletus edulis samples. Our forthcoming research will aim to characterize the unknown Se species further, as the high-efficiency bioproduction of unusual Se species holds promising potential for therapeutic applications and food supplementation.

5. Conclusions

The findings demonstrate that Se supplementation significantly influences the growth, yield, and Se bioaccumulation of Hericium erinaceus, with strain- and condition-specific variations. Se concentration in basidiocarps increased with higher substrate Se levels, peaking at 217.45 µg/g d.w. for strain M9521 at 18 µg/g. The bioconcentration factor was highest at 6 µg/g Se supplementation, indicating optimal efficiency at this level. Key findings include strain-specific differences in Se metabolism, with some strains excelling in SeCys2 and SeMet production, while others favored the accumulation of unique Se compounds. Substrate composition also emerged as a critical factor, influencing both yield and Se content, with wheat bran and soybean hulls standing out as favorable additives. Growth stage analysis revealed stable Se accumulation at medium supplementation level of Se across developmental phases of basidiocarps for commercial strains. Light intensity and tissue location further influenced Se distribution, with higher accumulation observed under 500 lux and in the outer layers of basidiocarps. Another key finding was that Se accumulation in the basidiocarps decreases with each successive flush, with the highest selenium content consistently observed in the first flush across all substrate supplementation levels. Se concentrations (SePO₄) in the substrate the 6 µg/g Se treatment remained relatively stable throughout the cultivation process, with minor fluctuations after colonization and flushes, but no statistically significant changes, suggesting that soluble and ligand-exchangeable Se levels do not accurately reflect Se uptake by Hericium.

This study contributes to the limited research on the influence of substrate additives on Se uptake and yield across multiple flushes, identifying substrate composition as a critical factor. These insights contribute to the growing field of Se-enriched mushrooms as functional foods and provide valuable guidance for commercial cultivation practices. Further research is warranted to explore the pathways of novel Se compounds and refine cultivation strategies for maximum economic and nutritional benefits. This research provides insights into improving Se-enriched mushroom production and offers a foundation for future studies on enhancing Se metabolism in edible fungi.

Author Contributions

Conceptualization, I.J. and M.J.; methodology, M.J. and I.J.; software, M.K. and M.J.; validation, M.J., M.K. and L.P.; formal analysis, M.K. and L.P.; investigation, M.J., L.P. and J.M.; resources, M.J. and I.J.; data curation, L.P., M.J. and M.K.; writing—original draft preparation, M.J. and J.M.; writing—review and editing, M.K. and L.P.; visualization, M.J. and L.P.; supervision, I.J. and M.K.; project administration, M.K.; funding acquisition, L.P. All authors have read and agreed to the published version of the manuscript.

Data Availability Statement

The data presented in this study are available in this article and its Supplementary Materials.

Acknowledgments

We acknowledge Jana Najmanová and Vladimír Plachý (both Czech University of Life Sciences Prague, Czech Republic), research station Červený Újezd (Czech University of Life Sciences Prague, Czech Republic), Martin Rajtar (Mykoforest, Slovakia).

Conflicts of Interest

The authors declare no conflicts of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript; or in the decision to publish the results.

Footnotes

Disclaimer/Publisher’s Note: The statements, opinions and data contained in all publications are solely those of the individual author(s) and contributor(s) and not of MDPI and/or the editor(s). MDPI and/or the editor(s) disclaim responsibility for any injury to people or property resulting from any ideas, methods, instructions or products referred to in the content.

Figures and Tables

Figure 1. Effect of Se supplementation in substrate on Se content in basidiocarps in the first flush of four Hericium erinaceus strains.

Figure 2. Effect of Se supplementation in substrate on BCF of the first flush of four Hericium erinaceus strains.

Figure 3. Effect of different developmental stages of the VURV-F 5243 strain on the accumulation of Se in basidiocarps.

Figure 4. Effect of different flushes of strain M9521 on the accumulation of Se in basidiocarps.

Table 1

Selected Hericium erinaceus strains for experiments.

Strain	Origin
MFTCCB134	Mykoforest (Velčice, Slovakia). Commercial strain
VURV-F 5243	Crop Research Institute (Prague-Ruzyně, Czech Republic). Wild strain
VURV-F 5242	Crop Research Institute (Prague-Ruzyně, Czech Republic). Wild strain
M9521	Mycelia (Deinze, Belgium). Commercial strain

Table 2

Composition of initial substrates in dry weight.

Substrate	N (%)	C (%)	H (%)	S (%)	C/N	Se (μg/kg)
Soybean hulls	4.67	45.94	6.901	0.084	9.84	0.07
Wheat bran	2.91	41.84	6.543	0.053	14.38	0.04
Rapeseed press cake	5.32	44.98	6.653	0.425	8.45	0.24
Brewer’s spent grain	3.22	40.93	6.147	0.049	12.71	0.09
Oak/beech sawdust	0.23	45.41	6.156	n.d.	197.43	0.02
Gypsum	n.d.	n.d.	1.046	16.74	-	0.02
Substrate BSG	0.83	44.06	6.103	0.177	53.36	0.034
Substrate SH	1.12	45.06	6.254	0.184	40.39	0.03
Substrate RPC	1.25	44.87	6.204	0.252	36.02	0.064
Substrate WB	0.76	44.24	6.182	0.178	57.93	0.024

Notes: Substrate BSG = 79% sawdust, 1% gypsum, and 20% brewer’s spent grain; substrate SH = 79% sawdust, 1% gypsum, and 20% soybean hulls; substrate RPC = 79% sawdust, 1% gypsum, and 20% rapeseed press cake; and substrate WB = 79% sawdust, 1% gypsum, and 20% wheat bran); n.d. = not detected.

Table 3

Composition of experimental substrates.

Substrate	Se (μg/g w.w.)	Sawdust (% d.w.)	Wheat Bran (% d.w.)	Gypsum (% d.w.)	Soybean Hulls (% d.w.)	Rapeseed Press Cake (% d.w.)	Brewer’s Spent Grain (% d.w.)
First Experiment	0	79	20	1	0	0	0
	2	79	20	1	0	0	0
	6	79	20	1	0	0	0
	18	79	20	1	0	0	0
Substrate BSG	6	79	0	1	0	0	20
Substrate SH	6	79	0	1	20	0	0
Substrate RPC	6	79	0	1	0	20	0
Substrate WB	6	79	20	1	0	0	0

Table 4

The influence of different Se supplementation in substrate on the yield and biological efficiency of four Hericium erinaceus strains.

Strain	Se Supplemented in Substrate (µg/g)	Yield First Flush (g)	Yield Second Flush (g)	Total Yield per Bag (g)	Biological Efficiency (%)
M9521	0	142.6 ± 7.1 def	50.4 ± 4.3 a	193.0 ± 6.8 bc	26.8 ± 0.9 bc
	2	158.2 ± 8.6 efgh	44.5 ± 4.9 a	202.7 ± 6.9 bcd	28.2 ± 1.0 bcd
	6	179.0 ± 7.8 gh	60.2 ± 4.5 a	239.1 ± 6.2 de	33.2 ± 0.9 de
	18	145.2 ± 14.7 efg	49.6 ± 4.7 a	194.9 ± 15.4 bc	27.1 ± 2.1 bc
MFTCCB134	0	165.3 ± 7 fgh	97.5 ± 15.1 cd	262.8 ± 12.0 ef	36.5 ± 1.7 ef
	2	191.4 ± 12.9 h	132.5 ± 23.0 e	323.9 ± 25.4 g	45.0 ± 3.5 g
	6	172.3 ± 15.0 gh	108.8 ± 8.5 de	281.2 ± 20.1 f	39.1 ± 2.8 f
	18	183.1 ± 22.3 h	105.7 ± 5.7 cde	288.8 ± 18.6 fg	40.1 ± 2.6 fg
VURV-F 5243	0	100.5 ± 4.6 bc	90.3 ± 1.0 bcd	190.8 ± 5.0 ab	26.5 ± 0.7 ab
	2	139.1 ± 13.5 def	92.5 ± 10.1 cd	231.6 ± 17.8 cde	32.2 ± 2.5 cde
	6	129.8 ± 5.3 cde	75.0 ± 5.3 abc	204.8 ± 2.3 bcd	28.5 ± 0.3 bcd
	18	108.6 ± 2.5 bcd	96.2 ± 10.7 cd	204.8 ± 12.4 bcd	28.4 ± 1.7 bcd
VURV-F 5242	0	85.1 ± 22.0 ab	92.6 ± 17.5 cd	177.7 ± 13.8 ab	24.7 ± 1.9 ab
	2	93.0 ± 10.5 b	111.2 ± 10.9 de	204.2 ± 9.7 bcd	28.4 ± 1.4 bcd
	6	51.8 ± 10.7 a	100.4 ± 13.1 cd	152.2 ± 18.5 a	21.1 ± 2.6 a
	18	84.5 ± 12.4 ab	100.4 ± 10.1 cd	184.9 ± 9.1 ab	25.7 ± 1.3 ab

Notes: Values are shown as mean ± standard deviation. There is no significant difference between means with the same letter in the same column.

Table 5

The influence of different Se supplementation in substrate on the days to harvest after putting four Hericium erinaceus strains into the fruiting chamber.

Strain	Se Supplemented in Substrate (µg/g)	Days from Initiation of Fructification to First Harvest (Days)	Days from First to Second Harvest (Days)
M9521	0	16.8 ± 0.2 e	10.6 ± 0.9 cde
	2	17.0 ± 0.3 e	9.2 ± 0.5 abc
	6	17.2 ± 0.2 e	10.0 ± 1.1 bcd
	18	17.2 ± 0.2 e	9.4 ± 0.2 abc
MFTCCB134	0	14.6 ± 0.6 bc	11.8 ± 0.2 def
	2	13.6 ± 0.4 ab	15.6 ± 1.1 g
	6	15.8 ± 0.7 cde	12.4 ± 1.0 ef
	18	15.8 ± 0.7 cde	13.6 ± 0.8 f
VURV-F 5243	0	12.2 ± 0.2 a	7.8 ± 0.5 a
	2	13.2 ± 1.0 ab	7.8 ± 0.2 a
	6	12.4 ± 0.2 a	7.6 ± 0.4 a
	18	12.4 ± 0.4 a	8.2 ± 0.7 ab
VURV-F 5242	0	15.2 ± 0.7 cd	11.0 ± 0.6 cde
	2	16.2 ± 0.6 de	9.2 ± 0.2 abc
	6	17.0 ± 0.3 e	9.4 ± 0.5 abc
	18	16.0 ± 1.0 cde	10.8 ± 1.1 cde

Notes: Values are shown as mean ± standard deviation. There is no significant difference between means with the same letter in the same column.

Table 6

Selenium concentration in basidiocarps of strain M9521 across different light conditions.

Variant	Se Amount in Basidiocarps (µg/g d.w.)
500 lux	96.85 ± 9.51 a
35 lux	65.89 ± 3.24 b

Notes: Values are shown as mean ± standard deviation. There is no significant difference between means with the same letter in the same column.

Table 7

The influence of different substrate composition on content of Se in basidiocarps and the BCF of strain MFTCCB134.

Variant	Se in Basidiocarp (µg/g d.w.)	BCF
RPC	33.38 ± 2.54 a	5.56 ± 0.42 a
SH	43.71 ± 1.16 b	7.29 ± 0.19 b
WB	69.89 ± 2.43 d	11.65 ± 0.40 d
BSG	60.03 ± 3.07 c	10.01 ± 0.51 c

Notes: Values are shown as mean ± standard deviation. There is no significant difference between means with the same letter in the same column.

Table 8

The influence of different substrate compositions on the yield and biological efficiency of strain MFTCCB134.

Variant	Yield First Flush (g)	Yield Second Flush (g)	Yield Third Flush (g)	Total Yield per Bag (g)	Biological Efficiency (%)
RPC	162.8 ± 13.5 a	121.2 ± 12.7 ab	94.0 ± 15.2 a	377.9 ± 12.3 a	52.5 ± 1.7 a
SH	295.7 ± 27.4 b	145.6 ± 17.1 b	139.3 ± 9.4 a	580.6 ± 26.7 b	80.6 ± 3.7 b
WB	205.7 ± 23.5 a	100.5 ± 3.7 a	97.6 ± 8.6 a	403.9 ± 22.3 a	56.1 ± 3.1 a
BSG	170.3 ± 10.0 a	115.6 ± 11.0 ab	102.4 ± 24.1 a	388.2 ± 14.4 a	53.9 ± 2.0 a

Notes: Values are shown as mean ± standard deviation. There is no significant difference between means with the same letter in the same column.

Table 9

Se species in four Hericium erinaceus strains with different supplementation levels of Se in the substrate.

Strain	Se in Substrate (µg/g, w.w.)	Selenocystine (µg/g Se, d.w.)	Selenomethionine(µg/g Se, d.w.)	Unknown(µg/g Se, d.w.)
VURV-F 5243	2	0.58 ± 0.05	12.65 ± 0.83	1.67 ± 0.39
	6	1.34 ± 0.04	28.95 ± 0.41	19.73 ± 0.93
	18	5.16 ± 0.13	137.81 ± 5.66	0.71 ± 0.09
MFTCCB134	2	0.25 ± 0.04	9.85 ± 0.56	n.d.
	6	0.66 ± 0.09	42.89 ± 2.45	n.d.
	18	2.10 ± 0.05	104.93 ± 3.47	7.4 ± 1.54
VURV-F 5242	2	1.55 ± 0.19	16.27 ± 0.23	n.d.
	6	5.26 ± 0.13	59.93 ± 1.14	n.d.
	18	10.77 ± 0.65	123.97 ± 4.34	n.d.
M9521	2	0.19 ± 0.04	0.53 ± 0.31	19.67 ± 0.89
	6	0.35 ± 0.01	1.02 ± 0.17	62.74 ± 4.53
	18	0.99 ± 0.06	1.76 ± 0.21	184.33 ± 12.97
VURV-F 5243—small	6	1.46 ± 0.12	55.22 ± 2.82	n.d.
VURV-F 5243—medium	6	3.77 ± 0.08	53.69 ± 2.45	n.d.

Notes: Values are shown as mean ± standard deviation.; n.d. = not detected.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/agriculture15050460/s1, Figure S1: Photo of basidiocarps tested Hericium erinaceus strains: (A) MFTCCB134; (B) VURV-F 5243; (C) VURV-F 5242; (D) M9521; Figure S2: The reflectance at individual wavelengths of the cultivation light; Table S1: Selenium concentration in basidiocarps of strain M9521 in different tissue location; Table S2: Effect of different developmental stages of strain M9521 at fortification level 6 µg/g on accumulation of Se in basidiocarp; Table S3: The influence of different growth phases on content of Se in basidiocarps in 6 µg/g Se of strain MFTCCB134 on substrate WB; Table S4: Temporal changes in extractable Se in a 0.1 M phosphate-buffered solution (pH 7.0) during the experimental cultivation of Hericium strain M9521 at a supplementation level of 6 µg/g (w.w.); Figure S3: HPLC-ICP-MS chromatogram showing the injection of a mixed standard of Se species, each at a concentration of 5 µg/L; Figure S4: HPLC-ICP-MS chromatograms of proteolytic hydrolysates from two Hericium strains, each exhibiting a distinct major Se species.

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