*2.2. Preparation of Inoculum and Fermentation Medium*

The inoculum was prepared by collecting spores from the surface of the PDA dishes under aseptic conditions with 10 mL of sterile distilled water. The spore suspension was used to prepare the inoculum culture containing 1.25 <sup>×</sup> <sup>10</sup><sup>6</sup> spores/mL. The spore concentration was estimated using a Neubauer chamber (Marienfeld-Superior, Lauda-Königshofen, Germany). The inoculation medium (50 mL in 250 mL Erlenmeyer flasks) was prepared by reconstituting whey powder in water to contain lactose at 50 g/L concentration and pH 6.0. Since *Monascus purpureus* can not utilize lactose as the carbon source, β-galactosidase enzyme (Saphera 2600L) was added to the fermentation medium at a rate of 0.1% (*v*/*v*) (simultaneous hydrolysis and fermentation, SHF). The fermentation for inoculum preparation was performed in a shaking incubator operated at 200 rpm, 30 ◦C for 4 days.

Fifty milliliters of demineralized whey medium (pH 6.0, unless otherwise stated) containing 50 g/L lactose was the substrate in SHF. The pH of the whey medium was adjusted to pH 6.0 by using 1 N potassium hydroxide and sterilized at 121 ◦C for 15 min. The inoculation ratio was 2% (*v*/*v*) and the SHF's were carried out in a shaking incubator at 200 rpm, 30 ◦C for 8 days. Inoculation and fermentation media were supplemented with 5 g/L of MSG (MSG; Sigma Aldrich, St Louis, MO, USA) as the nitrogen source. Samples were collected at equal time intervals for the determination of biomass growth rate and red pigment synthesis.

Prehydrolysis and a separate fermentation (PHSF) method was also used for the production of the red pigment. In this method, the prehydrolysis of lactose in demineralised whey medium was done at 50 ◦C (pH 6.0) for 8 h enzymatically before fermentation with *M. purpureus*.
