Materials for mushroom cultivation

A culture of G. lingzhi mycelium was provided by the Expert Center of Innovative Agriculture at the Thailand Institute of Scientific and Technological Research. Yeast malt agar culture medium was purchased from Gibco (Billings, MT, USA). Nutritious liquid medium was prepared from a mixture of yeast malt extract (3.0 g), proteose peptone (5.0 g), and sucrose (10.0 g) in 1 L deionised water (all were AR grade chemicals). Sodium selenite (> 99%) was purchased from Sigma-Aldrich (St. Louis, MO, USA). Sawdust compost (used as a substrate) was prepared from a mixture of para rubber sawdust (100 kg), rice bran (6 kg), calcium hydroxide (1 kg), gypsum (0.5 kg), and magnesium sulphate (0.1 kg); all raw materials were acquired from a local store in Pathum Thani, Thailand.

All instrumentation and liquid media for mycelium culturing, including a 1-L conical flask and 5-L bioreactor (BioFlo ® 310; New Brunswick Scientific GmbH, Nürtingen, Germany), were sterilized by using an autoclave (Astell Scientific Ltd., Sidcup, UK) at 121 °C and 15 PSI for 30 min.

One-kilogram aliquots of sawdust compost spiked with four different concentrations of selenite (to obtain 20, 25, 50, and 100 mg.kg−1, prepared by weight disodium selenite and dissolved in deionized water) was transferred into a sterilized grow spawn bag (dimension in cm: 6.5 × 12.5-in. bag) and autoclaved at 121 °C, 15 PSI for 30 min.

Mycelium cultivation

G. lingzhi cultures were dispersed evenly in 1-L conical flasks containing selenite (0, 20, 25, 50, or 100 mg.L−1) at a ratio 50 g to 500 mL of liquid media, inoculated for 4 days and transferred to a 5-L bioreactor at 23 ± 5 °C, and stirred at 150 RPM for 5 days; then mycelia were harvested. The mycelium solution was filtered (Whatman GF/C Glass Microfiber Filters 1.2 µm × 47 mm), and the mycelium residue was freeze-dried (Labconco Corporation, Kansas City, MO, USA).

Mushroom (fruiting body) cultivation

A solution of G. lingzhi (without selenite) was inoculated in a compost bag containing sawdust without added selenium for the control, or with various concentrations of selenite (20, 25, 50, and 100 mg.kg−1, as described above). The mushrooms were cultivated on shelves in a room 4 m × 6 m × 2.8 m (high) at 25 °C and 60 to 70% relative humidity in the dark for 3 months. After the mushrooms were fully grown, reverse osmosis water was sprayed onto the mushroom fruit twice per day and a fluorescent lamp was applied to the mushrooms for 12 h per day. The harvest was conducted after 20 days of this cycle. The mushrooms were washed with deionized water, chopped, blended, and freeze-dried.

Determination of total selenium content in mycelium and fruiting body

Approximately 250 mg of dried sample was weighed into a Teflon microwave digestion vessel with 7 mL HNO3 and 0.5 mL 30% H2O2 (w/w). The vessels were capped and the rotor was placed into an Anton Paar Multiwave Pro microwave (Anton Paar; Graz, Austria) and digested by ramping the power to 1400 W over 15 min then holding for 30 min. After a cooling cycle at 0 W, samples were removed from the microwave and gravimetrically diluted to approximately 50 mL with deionized water (DIW).

The digested samples were further diluted to a matrix of approximately 2% HNO3 (v/v) and analysed on an Agilent 8800 Triple Quadrupole ICP-MS (Agilent Technologies; Santa Clara, CA, USA) in triple quadrupole mode using O2 as cell gas, monitoring the M+  > MO+ transition for 74Se, 76Se, 77Se, 78Se, 80Se, and 82Se. Solutions of NIST SRM 3149 (selenium standard solution) prepared in 2% HNO3 (v/v) were used for external calibration.

Determination of selenomethionine (SeMet) and water-extractable Se species

For the analysis of SeMet, approximately 250 mg of dried sample and an appropriate volume of a 82Se-SeMet standard solution (prepared from NRC CRM SEES-1 [21]), such that the 80Se/82Se ratio for SeMet was approximately equal to 1 (based on preliminary screening of the samples), were weighted into an Erlenmeyer flask with a ground glass joint. Twenty-four millilitres of 25% methanesulphonic acid and pre-cleaned glass beads were added. The flask was connected to a water-cooled condenser and refluxed on a hotplate for 16 h. Once cooled, the sample was filtered (0.2 µm, PVDF) and stored in the refrigerator until analysis. A sample of NRC CRM SELM-1 (selenized yeast [22]) was also prepared and analysed for quality control. In addition to samples, three blends of SeMet primary standard (from NRC CRM SENS-1 [23]) and spike (82Se-SeMet from SEES-1 [21]), and three blanks, were subjected to the same reflux method. Just prior to analysis, samples were diluted 2- to 25-fold with DIW, depending on the expected SeMet concentration.

Samples were analysed for SeMet by HPLC-ICP-MS, where an Agilent 1200 Series HPLC was coupled to an Agilent 8800 Triple Quadrupole ICP-MS (Agilent Technologies, Santa Clara, CA, USA). All samples were analysed using an Agilent Zorbax XDB C18 column, with 100 µL injected for the (diluted) refluxed samples and 25 µL for the aqueous extractions. Mobile phases consisted of 10 mM ammonium formate in DIW adjusted to pH 5.6 and 0.1% formic acid in methanol and were employed in a temperature controlled (40 °C) gradient dilution at 0.4 mL/min: 0–5 min at 5% methanol, ramping to 100% methanol from 5 to 14 min, holding at 100% methanol for 3 min, then re-equilibrating for 6.5 min after a 0.5-min ramp back to 5% methanol. For detection, the ICP-MS was operated in triple quadrupole mode with H2 as cell gas, monitoring the M+  > M+ transition for 74Se, 76Se, 77Se, 78Se, 80Se, and 82Se. To account for the methanol in the mobile phase, the ICP-MS was operated in “organic mode”, using a 1-mm injector, platinum cones, and a brass skimmer base. Oxygen was added to the plasma through the addition of an option gas (20% O2 in Ar) at a flow rate set at 15%. The radio frequency (RF) matching was adjusted such that reflected power was minimized. Additionally, a low flow of internal standard (1 mg.kg−1 Rh and In) was added to the HPLC eluent, post-column, via a “T” connection.

For quantification of SeMet in the refluxed samples, isotope dilution was employed, following the quadrupole isotope dilution approach, initially described by Pagliano et al. [24] and outlined below:

$$_=-\frac_^\left(2\right)}_^\left(3\right)}_+_^\left(1\right)}_^\left(3\right)}_+_^\left(1\right)}_^\left(2\right)}_}_^\left(1\right)}_+_^\left(2\right)}_+_^\left(3\right)}_}\bullet \frac_}_}$$

where

$$_^\left(i\right)}=_^\left(i\right)}\bullet \frac_^\left(^B\left(i\right)\right)}}_^B\left(i\right)\right)}}$$

$$_=\left(_-_^B\left(1\right)}\right)\bullet \left(_^B\left(2\right)}-_^B\left(3\right)}\right)$$

$$_=\left(_-_^B\left(2\right)}\right)\bullet \left(_^B\left(3\right)}-_^B\left(1\right)}\right)$$

$$_=\left(_-_^B\left(3\right)}\right)\bullet \left(_^B\left(1\right)}-_^B\left(2\right)}\right)$$

A:

analyte in sample

A*:

analyte in primary standard (natural isotopic composition)

B:

analyte in isotopically enriched standard

AB:

mixture of sample and enriched standard

A*B:

mixture of primary standard and enriched standard

wX:

mass fraction of X (X = A, A*, or B)

mX(XY):

mass of X used to prepare the blend of X and Y (X,Y = A, A*, or B)

rX:

isotope ratio in X as measured by mass spectrometry (X = A, A*, or B)

For the analysis of aqueous extractable selenium species, approximately 250 mg of dried sample and 10 mL of DIW were weighed into a 20-mL glass vial. The vials were inverted to mix the contents until the entire sample appeared wet, then they were placed in a sonic bath at room temperature and sonicated for 30 min. After sonication, solids were allowed to settle naturally, then a small amount of supernatant was filtered (0.2 µm, PVDF) for direct analysis.

The aqueous extracts were analysed for inorganic selenium speciation—selenate (Se(IV)) and selenite (Se(VI))—following a second HPLC-ICP-MS procedure. There, a Hamilton PRP-X100 anion exchange column was used, and a gradient elution at 0.8 mL/min and 40 °C was employed using DIW and 200 mM ammonium acetate/200 mM acetic acid, as follows: 2 mM eluent from 0 to 5 min, ramping to 200 mM from 5 to 15 min then holding until 23 min, with a re-equilibration at 2 mM until 30 min. The injection volume was 25 µL and the ICP-MS was operated in triple quadrupole mode with H2 as described above, but in normal mode with nickel cones, a 2.5-mm injector, and without the addition of oxygen to the plasma.

For quantification of the unidentified species in the aqueous extracts, their peak areas were compared to that of a standard of known concentration (selenate or selenite) for anion exchange, or SeMet for C18, accounting for differences in sensitivity at various times in the elution profile for the C18 separation. ICP-MS sensitivity is affected by the concentration of methanol in the eluent, so an initial step was taken, which involved running the gradient elution but replacing the internal standard solution with one containing 10 mg.kg−1 selenium such that there was a continuous flow of selenium to the ICP-MS. Sensitivity factors were calculated by comparing the signal at all time points (where peaks occurred in the samples) to that during the elution window for SeMet. Based on these factors, concentrations of all unidentified species could be estimated based on a single SeMet standard. This quantification approach has been previously used in our laboratory [25] and has been described in detail by Amayo et al. [26].

Analysis of selenium nanoparticles (SeNPs)

For the extraction of the SeNPs from the freeze-dried material of G. lingzhi, a mechanical lysis protocol was adapted from the one described by R. Álvarez-Fernández et al. [27] for SeNP extraction from yeast cells. For this, approximately 15 mg of the dried samples (fruiting bodies and mycelia) was suspended in water (1 mL) and approximately 850 mg of 500-μm-diameter glass beads were added to the suspension. Sample suspensions were placed in an ultrasonic bath for 10 min (Ultrasons, J.P. Selecta S.A., Abrea, Spain), followed by 5 min at maximum speed in a Vortex mixer (Vortex ZX3 VELP Scientifica, Usmate, Italy). This process was carried out twice. After removal of the glass beads, fungi lysates were centrifuged at 300 × g for 5 min to remove any tissue debris. Supernatants were collected and diluted with water (dilution 1:2000), prior to the analysis by single-particle ICP-MS (SP-ICP-MS). Earlier experiments revealed that this process could maintain the sizes and size distribution of Se nanoparticles and did not induce any agglomeration [27, 28].

All the SP-ICP-MS measurements were performed on the iCAP™ TQ ICP-MS (Thermo Fisher Scientific, Bremen, Germany), fitted with the “Single-Cell Sample Introduction System” (SC-SIS) (Glass Expansion, Melbourne, Victoria, Australia). Samples were introduced into the equipment at a flow rate of 10 μL.min−1 using a syringe pump Chemyx F100X (Chemyx Inc., Stafford, TX, USA) together with a 1-mL Hamilton syringe (Hamilton, Reno, NV, USA). Data acquisition was performed in triple quadrupole mode employing oxygen as reaction gas, monitoring the signals of 80Se+  > 80Se16O+ and 31P+  > 31P16O+ for the measurement of 80Se+ and 31P+, respectively. The measurement of phosphorous served to prove the successful cell lysis [27]. A dwell time of 5 ms was used. Detailed instrumental conditions are given in Table S1 and Álvarez-Fernández et al. [27].

For further data treatment, an established iterative procedure described by F. Laborda et al. [29] was followed. Using this method, the whole data set is averaged and the points above a threshold established at 5σ of the mean are collected as events and extracted from the data set for subsequent iterations. The process is repeated in an iterative way until the number of the detected events remains constant. The limit for possible outliers (multiple-cell events) was set at 3σ above the mean of the resulting data.

For the determination of the selenium mass in individual nanoparticles, an external calibration curve was built analysing ionic selenium solutions in the range 0–50 µg.L−1 prepared from an initial 1 g.L−1 selenium standard solution (Sigma-Aldrich, St. Louis, MO, USA). Transport efficiency was daily calculated using the gold nanoparticles (AuNPs) quality control material LGCQC5050 (LGC Standards Ltd., Teddington, UK).

Detected SeNPs further underwent a selective reaction for checking the presence of elemental Se (Se0) using sodium sulphite. In this reaction, Se0 transforms into soluble selenosulphate as described in the literature [30]. For this, 200 µL of fungi lysate was mixed with 200 µL of sodium sulphite solution (0.5 mol.L−1, Sigma-Aldrich, St. Louis, MO, USA). The mixture was heated to 50 °C and left to react for 20 min. After cooling down to room temperature, the solution was diluted with water (dilution 1:1000) and analysed by SP-ICP-MS.

As complementary tool, electron transmission microscopy (TEM) (JEOL-2000 21000F, Tokyo, Japan) was employed and the diameters of the detected SeNPs were manually determined employing the open software ImageJ (National Institutes of Health, Bethesda, MD, USA).