*3.2. Effects of Different Fermentation Methods on Pigment Production*

Raw demineralised whey powder (DM) containing lactose as the carbon source in unhydrolyzed form was used for pigment production to determine the effect of lactose on growth and pigment production by *M. purpureus*. As seen in Figure 2, lactose found in DM was not utilized efficiently by *M. purpureus* for growth and pigment production. Other researcher also pointed out the difficulties of using lactose as the carbon source for the production of biopigments [45–48]. Therefore, it is necessary to hydrolyze lactose with β-galactosidase enzyme to its monomers of glucose and galactose for its proper utilization by *M. purpureus* as an energy source. In this study, two fermentation methods were

compared for lactose hydrolysis; simultaneous lactose hydrolysis and fermentation (SHF) and enzymatic pre-hydrolysis and separate fermentation (PHSF) for pigment production by *M. purpureus*. Demineralised whey powder (DM) containing 50 g/L lactose was used as the substrate in the fermentation experiments. As shown in Figure 2, the highest pigment production value (21.3 UA510 nm) was obtained with the SHF application. When lactose was hydrolyzed by the enzyme prior to fermentation (PHSF), lower red pigment synthesis (16.8 UA510 nm) was obtained compared to SHF. Similar biomass yields of 16.3 and 14.9 g/L were obtained for PHSF and SHF, respectively. Hence, SHF yielded higher pigment production values. Da Costa and Vendruscolo [45], determined pigment production by *Monascus ruber* CCT 3802 in the presence of several carbon sources such as glucose (20 g/L), lactose (20 g/L), and hydrolyzed lactose (20 g/L). Parallel to the results of our study, they found that the average production of yellow and orange pigments were higher in the hydrolyzed lactose medium by *M. ruber* compared to lactose. (SHF) and enzymatic pre-hydrolysis and separate fermentation (PHSF) for pigment production by *M. purpureus*. Demineralised whey powder (DM) containing 50 g/L lactose was used as the substrate in the fermentation experiments. As shown in Figure 2, the highest pigment production value (21.3 UA510 nm) was obtained with the SHF application. When lactose was hydrolyzed by the enzyme prior to fermentation (PHSF), lower red pigment synthesis (16.8 UA510 nm) was obtained compared to SHF. Similar biomass yields of 16.3 and 14.9 g/L were obtained for PHSF and SHF, respectively. Hence, SHF yielded higher pigment production values. Da Costa and Vendruscolo [45], determined pigment production by *Monascus ruber* CCT 3802 in the presence of several carbon sources such as glucose (20 g/L), lactose (20 g/L), and hydrolyzed lactose (20 g/L). Parallel to the results of our study, they found that the average production of yellow and orange pigments were higher in the hydrolyzed lactose medium by *M. ruber* compared to lactose.

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strate for *M. purpureus* in the synthesis of the red pigment.

*3.2. Effects of Different Fermentation Methods on Pigment Production* 

gen 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 sub-

Raw demineralised whey powder (DM) containing lactose as the carbon source in unhydrolyzed form was used for pigment production to determine the effect of lactose on growth and pigment production by *M. purpureus*. As seen in Figure 2, lactose found in DM was not utilized efficiently by *M. purpureus* for growth and pigment production. Other researcher also pointed out the difficulties of using lactose as the carbon source for the production of biopigments [45–48]. Therefore, it is necessary to hydrolyze lactose with β-galactosidase enzyme to its monomers of glucose and galactose for its proper utilization by *M. purpureus* as an energy source. In this study, two fermentation methods were compared for lactose hydrolysis; simultaneous lactose hydrolysis and fermentation

**Figure 2.** The effect of fermentation mode 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 MSG, 50 g/L lactose). **Figure 2.** The effect of fermentation mode 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 MSG, 50 g/L lactose).

#### *3.3. Effect of Initial pH on Pigment Production 3.3. Effect of Initial pH on Pigment Production*

The pH of fermentation medium is an important factor in red pigment synthesis by *Monascus* species since high pH values and the existence of suitable nitrogen source leads to the chemical modification of orange pigments changing into extracellular and water-soluble red pigments [49]. The effect of initial pH of fermentation medium was stud-The pH of fermentation medium is an important factor in red pigment synthesis by *Monascus* species since high pH values and the existence of suitable nitrogen source leads to the chemical modification of orange pigments changing into extracellular and water-soluble red pigments [49]. The effect of initial pH of fermentation medium was studied for pH range 5.0–9.0 using demineralised whey powder containing 50 g/L of lactose as the substrate. SHF (30 ◦C/8 days) was performed in 250 mL Erlenmayer flasks containing 50 mL of fermentation medium. As seen in Figure 3, the highest pigment synthesis of 25.3 UA510 nm was observed at the initial pH value of 7.0. A low amount of pigment formation was observed at high and low pH values. In the fermentation medium with an initial pH of 9.0, growth and pigment synthesis were found to be the lowest (4.4 UA510 nm). Other researchers have also reported that pH of fermentation medium was highly important for red pigment synthesis by *M. purpureus* [48,50]. Orozco and Kilikian [50] investigated the synthesis of pigments by *Monascus purpureus* and they obtained 11.3 U of red pigments at pH 8.5. They stated that high pH medium facilitates the transfer of intracellular pigments to the fermentation medium. Parallel to the findings of our study, Mukherjee and Singh [39] stated that *M. purpureus* produced more red pigments at pH values of 6.0–8.0. Lee et al. [51] also indicated that the pH range of 5.5–8.5 was suitable for red pigment production, whereas pH values higher than 8.5 and lower than 5.5 led to a decrease in red pigment production. Prapajati et al. [48] achieved maximum *Monascus* pigment synthesis at pH 8.5. It has been reported that water-soluble extracellular

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red pigment production increased at high pH values and at high MSG concentrations and the transition of pigments from the cell to fermentation medium was restricted at low pH values [49]. pigment production increased at high pH values and at high MSG concentrations and the transition of pigments from the cell to fermentation medium was restricted at low pH values [49].

ied for pH range 5.0–9.0 using demineralised whey powder containing 50 g/L of lactose as the substrate. SHF (30 °C/8 days) was performed in 250 mL Erlenmayer flasks containing 50 mL of fermentation medium. As seen in Figure 3, the highest pigment synthesis of 25.3 UA510 nm was observed at the initial pH value of 7.0. A low amount of pigment formation was observed at high and low pH values. In the fermentation medium with an initial pH of 9.0, growth and pigment synthesis were found to be the lowest (4.4 UA510 nm). Other researchers have also reported that pH of fermentation medium was highly important for red pigment synthesis by *M. purpureus* [48,50]. Orozco and Kilikian [50] investigated the synthesis of pigments by *Monascus purpureus* and they obtained 11.3 U of red pigments at pH 8.5. They stated that high pH medium facilitates the transfer of intracellular pigments to the fermentation medium. Parallel to the findings of our study, Mukherjee and Singh [39] stated that *M. purpureus* produced more red pigments at pH values of 6.0–8.0. Lee et al. [51] also indicated that the pH range of 5.5–8.5 was suitable for red pigment production, whereas pH values higher than 8.5 and lower than 5.5 led to a decrease in red pigment production. Prapajati et al. [48] achieved maximum *Monascus* pigment synthesis at pH 8.5. It has been reported that water-soluble extracellular red

**Figure 3.** The effect of initial pH on the red pigment and biomass production by *M. purpureus* CMU 001 (Fermentation **Figure 3.** The effect of initial pH on the red pigment and biomass production by *M. purpureus* CMU 001 (Fermentation conditions: 30 ◦C, 8 days, 200 rpm, 5 g/L MSG, 50 g/L lactose).

conditions: 30 °C, 8 days, 200 rpm, 5 g/L MSG, 50 g/L lactose).
