*3.6. Effect of Inoculation Ratio on Pigment Production*

*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 To investigate the effect of inoculation ratio on red pigment production, DM medium was inoculated with 1, 2, 3, 4, 5, and 6% (*v*/*v*) spore suspension solutions. SHF was performed at 30 ◦C for 8 days (pH 7.0) with initial lactose and MSG concentrations of 75 g/L and 5 g/L, respectively (Figure 6). Although maximum pigment synthesis was obtained at an MSG concentration of 25 g/L, an MSG concentration of 5 g/L was used in the fermentation experiments due to economical considerations and feasibility of the process. It was observed that the highest pigment production of 38.4 UA510 nm was obtained when DM fermentation medium was inoculated with 5% of spore suspension. The results of this study showed that different inoculum ratios influenced the synthesis of the red pigment by *M. purpureus*, while 2 to 5% inoculum ratios had very little effect on the biomass concentration. Babitha et al. [26] studied pigment production by *M. purpureus* from jackfruit seed using solid-state fermentation and reported that low inoculum concentration produced insufficient biomass while high inoculum concentrations led to excessive

fermentation medium and found that fermentation medium containing 7.5 g/L of MSG

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 ob-

biomass formation depleting the nutrients in the fermentation medium that are necessary for the product formation. Atalay et al. [15] studied pigment production from residual beer using *M. purpureus* and obtained the highest pigment production of 18.5 UA510 nm with the fermentation medium inoculated with 2% (*v*/*v*) of culture medium. Silbir and Goksungur [9] also studied pigment production from brewer's spent grain in submerged fermentation system and found that 2% (*v*/*v*) spore suspension yielded the highest pigment production of 22.3 UA500 nm. tained 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 9 of 15

**Figure 5.** The effect of monosodium glutamate (MSG) concentration on the red pigment and biomass production by *M. purpureus* CMU 001 (Fermentation conditions: 30 °C, 8 days, 200 rpm, pH 7.0, 75 g/L lactose). **Figure 5.** The effect of monosodium glutamate (MSG) concentration on the red pigment and biomass production by *M. purpureus* CMU 001 (Fermentation conditions: 30 ◦C, 8 days, 200 rpm, pH 7.0, 75 g/L lactose). *Fermentation* **2021**, *7*, x FOR PEER REVIEW 10 of 15

beer using *M. purpureus* and obtained the highest pigment production of 18.5 UA510 nm with the fermentation medium inoculated with 2% (*v*/*v*) of culture medium. Silbir and **Figure 6.** The effect of inoculation ratio on the 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, 75 g/L lactose. **Figure 6.** The effect of inoculation ratio on the 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, 75 g/L lactose.

for the product formation. Atalay et al. [15] studied pigment production from residual

first 3 days of fermentation and increased afterwards to 8.0 at the end of the fermentation.

producing ammonia as a result of the deamination of amino acids. Dry biomass weight

**pH and Biomass (g/l)**

Red Pigment Lactose Biomass pH

steadily increased during the 8 days of fermentation period and then decreased.

#### Goksungur [9] also studied pigment production from brewer's spent grain in submerged fermentation system and found that 2% (*v*/*v*) spore suspension yielded the highest pig-*3.7. Kinetics of Red Pigment Production by M. Purpureus 3.7. Kinetics of Red Pigment Production by M. purpureus*

ment production of 22.3 UA500 nm. The mycelial development and pigment synthesis kinetics of *M. purpureus* were investigated by SHF under the optimized conditions (demineralized whey diluted to contain 75 g/L lactose, 5 g/L MSG concentrations, pH 7.0, 30 °C, 200 rpm, and 8 days). As shown in Figure 7, red pigment production started at the beginning of the exponential growth phase and reached a maximum value of 37 UA510 nm on the 8th days of fermentation. The red pigment concentration decreased after the 8th day of fermentation most probably due to the substrate limitation, possible chemical decomposition of the pigment, conversion to other products or oxidation by the microorganisms. The decrease in total sugar concentration during fermentation proved that hydrolyzed lactose was used by *M. purpureus* for growth and pigment synthesis. The pH value dropped during the The mycelial development and pigment synthesis kinetics of *M. purpureus* were investigated by SHF under the optimized conditions (demineralized whey diluted to contain 75 g/L lactose, 5 g/L MSG concentrations, pH 7.0, 30 ◦C, 200 rpm, and 8 days). As shown in Figure 7, red pigment production started at the beginning of the exponential growth phase and reached a maximum value of 37 UA510 nm on the 8th days of fermentation. The red pigment concentration decreased after the 8th day of fermentation most probably due to the substrate limitation, possible chemical decomposition of the pigment, conversion to other products or oxidation by the microorganisms. The decrease in total sugar concentration during fermentation proved that hydrolyzed lactose was used by *M. purpureus* for growth

**Figure 7.** Simultaneous hydrolysis and fermentation (SHF) profiles of mycelial growth of *M. purpureus* CMU 001, red pigment synthesis, lactose consumption, and pH under the optimum conditions (Fermentation conditions: 30 °C, 8 days, 200 rpm, pH 7.0, 5 g/L MSG, 75 g/L lactose). The standard deviation of each experimental point ranged from 1.5 to 4.8.

0123456789

**Incubation time (d)**

**Lactose (g/l) ,**

**Red Pigment (UA510nm)** 

and pigment synthesis. The pH value dropped during the first 3 days of fermentation and increased afterwards to 8.0 at the end of the fermentation. The pH increase in the last stage of fermentation was probably due to *M. purpureus* producing ammonia as a result of the deamination of amino acids. Dry biomass weight steadily increased during the 8 days of fermentation period and then decreased. total sugar concentration during fermentation proved that hydrolyzed lactose was used by *M. purpureus* for growth and pigment synthesis. The pH value dropped during the first 3 days of fermentation and increased afterwards to 8.0 at the end of the fermentation. The pH increase in the last stage of fermentation was probably due to *M. purpureus* producing ammonia as a result of the deamination of amino acids. Dry biomass weight steadily increased during the 8 days of fermentation period and then decreased.

The mycelial development and pigment synthesis kinetics of *M. purpureus* were investigated by SHF under the optimized conditions (demineralized whey diluted to contain 75 g/L lactose, 5 g/L MSG concentrations, pH 7.0, 30 °C, 200 rpm, and 8 days). As shown in Figure 7, red pigment production started at the beginning of the exponential growth phase and reached a maximum value of 37 UA510 nm on the 8th days of fermentation. The red pigment concentration decreased after the 8th day of fermentation most probably due to the substrate limitation, possible chemical decomposition of the pigment, conversion to other products or oxidation by the microorganisms. The decrease in

*Fermentation* **2021**, *7*, x FOR PEER REVIEW 10 of 15

b

<sup>b</sup> <sup>b</sup> <sup>b</sup> <sup>a</sup>

a

c

c

Red Pigment

Biomass

**Figure 6.** The effect of inoculation ratio on the red pigment and biomass production by *M. purpureus* CMU 001 (Fermen-

*3.7. Kinetics of Red Pigment Production by M. Purpureus* 

1.0 2.0 3.0 4.0 5.0 6.0

**Inoculation ratio (% v/v)**

tation conditions: 30 °C, 8 days, 200 rpm, pH 7.0, 5 g/L MSG, 75 g/L lactose.

f

**Red pigment (UA510nm),**

**Biomass (g/L)**

d

<sup>e</sup> <sup>d</sup>

**Figure 7.** Simultaneous hydrolysis and fermentation (SHF) profiles of mycelial growth of *M. purpureus* CMU 001, red pigment synthesis, lactose consumption, and pH under the optimum conditions (Fermentation conditions: 30 °C, 8 days, 200 rpm, pH 7.0, 5 g/L MSG, 75 g/L lactose). The standard deviation of each experimental point ranged from 1.5 to 4.8. **Figure 7.** Simultaneous hydrolysis and fermentation (SHF) profiles of mycelial growth of *M. purpureus* CMU 001, red pigment synthesis, lactose consumption, and pH under the optimum conditions (Fermentation conditions: 30 ◦C, 8 days, 200 rpm, pH 7.0, 5 g/L MSG, 75 g/L lactose). The standard deviation of each experimental point ranged from 1.5 to 4.8.

> Maximum pigment productivity and specific growth rate of *M. purpureus* were determined as 4.55 UAh−<sup>1</sup> and 0.023 h−<sup>1</sup> , respectively for SHF of DM whey at the optimized conditions in this study. Da Costa and Vendrusculo [45] investigated the use of hydrolyzed lactose, glucose, and lactose as a substrate for pigment production. While productivity values were calculated as 0.059 AU<sup>510</sup> h −1 , 0.072 AU<sup>510</sup> h −1 , and 0.032 AU<sup>510</sup> h −1 , specific growth rate values were determined as 0.031 h−<sup>1</sup> , 0.042 h−<sup>1</sup> , and 0.017 h−<sup>1</sup> for hydrolyzed lactose, glucose, and lactose, respectively. Atalay et al. [15] produced 18.5 UA<sup>510</sup> red pigment from residual beer by *M. purpureus* after 192 h of fermentation. Productivity and specific growth rate values were found as 2.3 UAh−<sup>1</sup> and 0.03 h−<sup>1</sup> respectively. Even though the calculated specific growth rate value of *M. purpureus* grown on DM whey was in the range reported by the other authors, pigment productivity was found to be higher than the other substrates. These findings make DM whey a productive and sustainable substrate for the production of red pigment by SHF of *M. purpureus*.
