*2.5. Analytical Methods*

The biomass was measured gravimetrically at the end of fermentation by filtering the mycelia through Whatman No: 1 filter paper, washing three times with distilled water and drying at 65 ◦C to constant weight in an oven (Memmert, UN 55, Schwabach, Germany). The biomass concentration was expressed as mycelial dry weight per unit volume of culture medium [39].

The supernatant obtained by centrifugation of the fermentation medium in the centrifuge (Hettich Universal 320 R, Tuttlingen, Germany) at 5400 rpm/20 min was filtered with Whatman No. 1 filter paper. The filtrate obtained was used for the quantification of the synthesized red pigments using a spectrophotometer (Thermo Scientific, Genesys 10S UV-VIS, Paisley, UK) at 510 nm and the readings were given in absorbance units (UA510) [40]. Sterile fermentation medium was used as the blank sample since the medium itself may have some absorbance at 510 nm wavelength. The pH values were determined with the pH-meter (WTW Inolab 7110, Weilheim, Germany). Lactose concentration in the medium was determined spectrophotometrically at 540 nm by the phenol-sulfuric acid method [41]. Total nitrogen content was determined by the Kjeldahl method [42] and the protein content of demineralized whey was calculated by multiplying the nitrogen content by the factor 6.25 in order to convert the nitrogen content into protein content. Moisture content of demineralized whey was determined by drying the sample to constant weight at 80 ◦C. The ash content of demineralized whey was determined by burning the sample for 12–18 h at 550 ◦C in a muffle furnace followed by cooling to room temperature and weighing.

Each experiment was performed in triplicate and the results were expressed as the mean ± standard deviation. The experiments were carried out in two repetitions and the analyses were carried out in three parallel samples. The results were expressed as the mean ± standard deviation. All statistical analyses were performed using the software IBM SPSS (v. 22). All data obtained were analyzed by one way analysis of variance, and tests of significant differences were determined by using Duncan's test at *p* < 0.05. In all the figures, mean values for all the factors given in x-axis that are not followed by the same letter (a–g) are significantly different (*p* < 0.05).
