*3.5. Effect of Monosodium Glutamate (MSG) Concentration on Pigment Production*

The previous studies showed that *M. purpureus* had higher red pigment production efficiency in the presence of MSG than other nitrogen sources [9,15]. Pigments produced by *Monascus* species are usually intracellular and insoluble in water. These pigments turn into extracellular and water-soluble red pigments as a result of a non-enzymatic reactions in the presence of MSG at neutral pH values. MSG replaces the ammonia in the orange pigment to produce the red pigment derivatives [51,55–57]. To investigate the effect of MSG concentration on red pigment production, SHF was performed with DM whey containing 75 g/L of lactose at pH 7.0 and 30 ◦C temperature during 8 days. As seen in Figure 5, MSG concentration had a significant effect on red pigment synthesis

by *M. purpureus*. The pigment production increased steadily and linearly up to 25 g/L of MSG concentration resulting in a maximum pigment synthesis of 45.7 UA510 nm and then declined at higher MSG concentrations. Atalay et al. [15] tested different nitrogen sources (monosodium glutamate, malt sprouts, corn steep liquor, peptone, urea, ammonium sulfate and yeast extract) for *Monascus* pigment production using residual beer as the fermentation medium and found that fermentation medium containing 7.5 g/L of MSG yielded the highest red pigment production of 18.5 UA510 nm. Sharmila et al. [1] obtained the maximum pigment synthesis by using 6 g/L of MSG as the nitrogen source. Babitha et al. [26] stated that Jackfruit seeds could not produce water-soluble pigments without using additional nitrogen sources. Zhang et al. [57] determined that glutamate and glycine was the most suitable source for growth of *Monascus*. Silbir and Göksungur [9] and Lee et al. [51] also used a submerged culture technique to determine the effect of various nitrogen sources on red pigment synthesis by *M. purpureus* and obtained maximum pigment production when MSG was used. Lee et al. [51] stated that increased MSG concentrations decreased pigment production while increasing biomass concentrations. Hamano and Kilikian [25] stated that the highest pigment production (20.7 U) was obtained in a fermentation medium containing 7.6 g/L of MSG concentration. Our results showed that the optimal MSG concentration found for DM is different and higher than the concentration values found for other substrates in the literature for the production of red pigment by *M. purpureus*. Demineralized whey medium might contain low concentration of nitrogen as stated before. When higher MSG concentrations are used to overcome this problem, *Monascus purpureus* gave higher pigment production values. *Fermentation* **2021**, *7*, x FOR PEER REVIEW 8 of 15 tion as 80 g/L in terms of maximum efficiency, pigment yield and cost efficiency for industrial applications. Parallel to our findings, they stated that the initial substrate concentration has a negligible effect on biomass (dry cell weight). The high glucose concentration in the fermentation medium can be an advantage for mycelium growth, but as fermentation progresses, the fermentation medium becomes more acidic and this can lead to low pigment yields [54].

**Figure 4.** The effect of initial lactose concentration on red pigment and biomass production by *M. purpureus* CMU 001 (Fermentation conditions: 30 °C, 8 days, 200 rpm, pH 7.0, 5 g/L MSG). **Figure 4.** The effect of initial lactose concentration on red pigment and biomass production by *M. purpureus* CMU 001 (Fermentation conditions: 30 ◦C, 8 days, 200 rpm, pH 7.0, 5 g/L MSG).
