*3.1. The Effect of Whey Type as Fermentation Substrate on Pigment Production*

Raw whey powder (W), demineralized whey powder (DM), deproteinized whey powder (DP), treated deproteinized raw whey powder (DPW) and treated deproteinized demineralized whey powder (DPDM) were prepared to investigate the effect of whey type on pigment production by *Monascus purpureus.* The simultaneous hydrolysis and fermentation (SHF) method was used in the fermentation experiments. The chemical composition of whey type has an important role in red pigment production since different types of whey have different protein and mineral contents [34]. type on pigment production by *Monascus purpureus.* The simultaneous hydrolysis and fermentation (SHF) method was used in the fermentation experiments. The chemical composition of whey type has an important role in red pigment production since different types of whey have different protein and mineral contents [34].

electricity, and water were obtained from the suppliers for cost analysis.

*3.1. The Effect of Whey Type as Fermentation Substrate on Pigment Production* 

measured by a TT-Technic PM 001 plug power meter device. Prices of whey, chemicals,

Raw whey powder (W), demineralized whey powder (DM), deproteinized whey powder (DP), treated deproteinized raw whey powder (DPW) and treated deproteinized demineralized whey powder (DPDM) were prepared to investigate the effect of whey

*Fermentation* **2021**, *7*, x FOR PEER REVIEW 5 of 15

**3. Results and Discussion** 

As seen from Figure 1, the highest pigment concentration (20.8 UA510 nm) was produced from demineralized whey (DM) medium. Similar pigment yield value of 20.2 UA510 nm was obtained with deproteinized and demineralized whey powder medium (DPDM). No significant difference (*p* > 0.05) in pigment yields were observed with DM and DPDM media. The lowest pigment yield was obtained from the DP medium (5.3 UA510 nm). While DP and DPW have yielded very low red pigment production values, DM and DPDM produced higher values. Lower pigment synthesis values observed in DP and PDW samples might have originated from the inhibitory effect of high concentration of cations (Na<sup>+</sup> , NH<sup>4</sup> + , K<sup>+</sup> , Mg+2, and Ca+2) and anions (Cl−, SO<sup>4</sup> −2 , PO<sup>4</sup> −3 , and citrate) present in whey medium [43,44]. Since the demineralization process decreased monovalent, divalent cation and anion levels in whey, pigment production values obtained from DM and DPDM are satisfactory compared to the literature [45]. Raw whey powder (W) yielded 12.1 UA510 nm pigment production value which was slightly higher than the values of DP and DPW as a substrate of pigment synthesis. The biomass concentration values observed in all types of whey were very close to each other and no significant correlation was observed between pigment and biomass production values (Figure 1). This indicates that biomass can reproduce in all types of whey, but the conditions for pigment production can be achieved at low anion and cation concentrations. As seen from Figure 1, the highest pigment concentration (20.8 UA510 nm) was produced from demineralized whey (DM) medium. Similar pigment yield value of 20.2 UA510 nm was obtained with deproteinized and demineralized whey powder medium (DPDM). No significant difference (*p* > 0.05) in pigment yields were observed with DM and DPDM media. The lowest pigment yield was obtained from the DP medium (5.3 UA510 nm). While DP and DPW have yielded very low red pigment production values, DM and DPDM produced higher values. Lower pigment synthesis values observed in DP and PDW samples might have originated from the inhibitory effect of high concentration of cations (Na+, NH4+, K+, Mg+2, and Ca+2) and anions (Cl−, SO4−2, PO4−3, and citrate) present in whey medium [43,44]. Since the demineralization process decreased monovalent, divalent cation and anion levels in whey, pigment production values obtained from DM and DPDM are satisfactory compared to the literature [45]. Raw whey powder (W) yielded 12.1 UA510 nm pigment production value which was slightly higher than the values of DP and DPW as a substrate of pigment synthesis. The biomass concentration values observed in all types of whey were very close to each other and no significant correlation was observed between pigment and biomass production values (Figure 1). This indicates that biomass can reproduce in all types of whey, but the conditions for pigment production can be achieved at low anion and cation concentrations.

**Figure 1.** The effect of whey types on the red pigment and biomass production by *M. purpureus* CMU 001 (Fermentation conditions: 30 °C, 8 days, 200 rpm, pH 6.0, 5 g/L monosodium glutamate (MSG), 50 g/L lactose). **Figure 1.** The effect of whey types on the red pigment and biomass production by *M. purpureus* CMU 001 (Fermentation conditions: 30 ◦C, 8 days, 200 rpm, pH 6.0, 5 g/L monosodium glutamate (MSG), 50 g/L lactose).

Demineralised whey powder with the highest pigment yield had the following composition (%): total sugar 70.46 ± 0.24, protein 5.32 ± 0.05, ash 3.32 ± 0.36, total nitro-Demineralised whey powder with the highest pigment yield had the following composition (%): total sugar 70.46 ± 0.24, protein 5.32 ± 0.05, ash 3.32 ± 0.36, total nitrogen 0.86 ± 0.01, moisture 2.72 ± 0.3. As long as the lactose present in whey is enzymatically hydrolyzed, DM whey with its high lactose sugar content (70.5%) is a suitable substrate for *M. purpureus* in the synthesis of the red pigment.
