*2.6. Ergosterol Analysis*

The replicates and treatments were oven-dried at 60 ◦C for 48 h, milled in a laboratory blender (Waring Commercial, Christian, UK), homogenised, and the ergosterol content analysed using an adaptation of the rapid ultrasonic method developed previously [23]. This involved adding 2 mL of deionised water to a 5 g of sample. After 15 min, 10 mL methanol:ethanol (*<sup>v</sup>*:*v*, 4:1) was added and samples were stored at 4 ◦C for 2 h. Then, 20 mL hexane:propan-2-ol (*<sup>v</sup>*:*v*, 98:2) was added to each sample and they were ultrasonicated at 150 W in an ice bath with water for 3 min 20 s. After 30 s, 2 mL of the supernatant was centrifuged at 7000× *g* for 10 min, from which 1.5 mL was used for HPLC analysis.

The HPLC system was a Shimadzu Class-VP, which consisted of SCL-10Avp controller, SPD-10Avp detector and LC-10ADvp pump (Shimadzu Corporation, Japan). An amount of 100 μL was injected into a Lichrosorb Phenomenex column (250 × 4.6 mm, 10 μm) at 35 ◦C. Run time for samples was 15 min with ergosterol being detected at about 9.20 min. The flow rate of the mobile phase (hexane:propan-2-ol; *v*:*v*, 98:2) was 1.4 mL·min−1.

Calibration standards of 5, 25, 50, 100 and 150 <sup>μ</sup>g·g<sup>−</sup><sup>1</sup> ergosterol in hexane:propan-2-ol (*<sup>v</sup>*:*v*, 98:2) were used as an external calibration. A general recovery test was conducted from spiked samples using 4 replicates. The procedure was the same as explained above, with two changes: (a) 1 mL of internal standard was added after the deionised water and samples were allowed to stand for 15 min, (b) 19 mL of hexane:propan-2-ol (*<sup>v</sup>*:*v*, 98:2) was added instead of 20 mL. Average recovery rates for 50, 100, 500 and 1000 <sup>μ</sup>g·g<sup>−</sup><sup>1</sup> ergosterol from wheat grain matrix were 89.18 ± 14.44%, 104.35 ± 12.37%, 106.37 ± 9.81% and 94.41 ± 5.97%, respectively.
