The results of biomass production in the culture environment and optimal pH
The pH of the fermentation environment is an important factor in the synthesis of red pigment by Monascus because high pH values and the presence of a suitable nitrogen and carbon source lead to the chemical change of orange pigments to extracellular and water-soluble red pigments (22). For this purpose, the optimal treatment was investigated in three basic pH ranges of 5 (22.5 g/l), 6.5 (26.75 g/l) and 8 (24.3 g/l). According to the obtained results, the maximum amount of biomass was obtained at pH 6.5 (26.75 g/l) and the initial pH was fixed at 6.5. Many factors, including the type of substrate, pH, nitrogen source, and carbon can be effective in increasing or decreasing the amount of pigment production by Monascus (1). Usually, at low pH, more yellow pigment (ancaflavin) is produced, and at higher pH, red pigment gradually dominates (4, 23).
New strategies to improve the stability characteristics and increase the application of the pigment have been applied (24, 25). The pigment was produced in the medium containing potato pomace through submerged fermentation by M. purpureus CH01 and the results showed that high temperatures decrease the stability with increasing pH (26). The pH effect on pigment produced by M. purpureus in submerged fermentation was evaluated and pH can affect the stability of the pigment, which is lost at lower pH values. It was also found that the red pigment compared to the yellow pigment and orange is more sensitive to pH and salt (23). In different studies, it has been reported that M. purpureus has more red pigments at pH values of 6-8 (15), and range of 5.5-8.5 (8). According to the report, red pigment production is positively affected by high pH values and high concentrations of monosodium glutamate. This means that increasing the pH level and adding higher amounts of monosodium glutamate can lead to an increase in the production of red pigments. On the other hand, the transfer of water-soluble extracellular pigments from the cell to the fermentation medium is hindered when the pH values are low. This implies that at lower pH levels, the ability of the pigments to move out of the cells and into the surrounding medium is limited (27).
Dairy sludge, a byproduct of milk fat separation and microorganism removal, contains valuable nutrients such as lactose, minerals, fat, whey protein, and nitrogenous compounds. It can serve as a carbon source and a potential source of nitrogen and minerals in various applications. (11). Nitrate resources have positive effects on different aspects of the process. Firstly, they enhance the mycelial morphology, which refers to the physical structure and appearance of the fungal mycelium. This suggests that the addition of nitrate resources can improve the growth and development of the fungi and regulate pigment color changes (26). Glucose is a primary energy source for many organisms, and its utilization has been shown to enhance metabolite production (1). In the studies conducted for the production of pigment by M. purpureus, the compounds of sucrose esters (7) and corn starch with oils (28) evaluated in fermentation medium.
Pigment extraction and purification
The extraction and purification of two pigment samples obtained from the control sample and the culture medium containing dairy sludge (optimal sample) was done and the images of the samples are depicted in Fig. 1a and b. According to the pictures of the samples, it can be seen that there is more color in the sample obtained from the optimum culture medium containing dairy sludge. The absorbance measured in the control medium for yellow (400 nm), orange (470 nm), and red (510 nm) pigments were 0.596, 0.326, and 0.308, respectively, and those measured in the optimum medium containing dairy sludge were 0.803, 0.547, and 0.689, respectively. The amount of 37 and 19 absorbance units (AU500) for the red pigment of the optimum and control samples was obtained, and also the amount of 4.85 and 2.5 g of dry color was obtained for the optimum and control sample, respectively, after extraction.
The LC-MS analysis provides valuable insights into the metabolites present in the purified samples, allowing for a more comprehensive characterization of the pigments and evaluation of their purity. Quantitative data was obtained through selective ion monitoring (SIM) of the pigment's hydrogen adult compound, with a mass-to-charge ratio (m/z) of 381 and 250 for optimum and control samples, respectively. The purity of the pigment was determined to be 91.9% and 85.5% optimum and control samples, respectively. The LC-MS spectrum of the samples is depicted in Fig 1c and d. The peaks corresponding to the pigment composition in optimum and control samples are observed within the inhibition time range of 5.5-8 and 4.5-8 min (Fig 2). This peak was compared with a standard sample for identification. Weak peaks in this range indicate the presence of small impurities in the sample. The results indicate that the addition of certain compounds in the culture medium has a significant effect on pigment production. In the optimal culture medium containing dairy sludge, monosodium glutamate, and glucose, the amount of pigment production was found to be twice as much compared to the control medium. This suggests that these compounds play a crucial role in enhancing pigment production.
Similar high extracellular pigment production of 34.12 U/ml was observed in submerged fermentation using a glucose-based medium with M. purpureus (8). Other studies have also demonstrated the influence of different culture media on pigment production. For instance, the culture medium based on whey resulted in a red pigment production efficiency of 1.12 UA510 with M. purpureus (22). Similarly, a pigment amount of 22.25 UA500 was reported in a culture medium containing monosodium glutamate, nitrogen, and waste (Silbir and Goksungur, 2019).
The type of medium used in the fermentation process has been consistently found to be a crucial factor in pigment production and metabolic products, including pigments, are known to be dependent on the culture conditions (26). Studies have highlighted the significance of specific compounds, such as monosodium glutamate, in improving pigment production, particularly red pigments (29). For example, it was found that jackfruit seed powder alone does not produce water-soluble pigments, but the addition of sodium magnesium glutamate enables the production of red water-soluble pigment (2).
Evaluation of pigment structure by 1H NMR
1H-NMR was employed to assess the purity of the optimal sample in comparison to the standard sample. Monascus spp. are responsible for the production of the primary color pigments, which exhibit a planktonic structure in shades of red, orange, and yellow. The six major pigments consist of rubropunctamine (C21H26NO4) and monascorubramine (C23H27NO4) in red, rubropunctatin (C21H22O5) and monascorubrin (C23H26O5) in orange, and monacin (C21H26O5) and enkaflavin (C23H30O5) in yellow (22).
The 1H-NMR spectra of the standard and optimal samples displayed significant similarity. However, a distinct peak at 1.2 ppm was observed solely in the control sample, indicating a potential impurity in the optimal sample (Fig. 2a). Both spectra exhibited a prominent peak around 1 ppm, corresponding to RCH3 (alkyl) groups (Fig. 2). The subsequent peak in the 2-3 ppm range likely originated from RC=OCH3 functional groups. Multiple peaks in the 3-4 ppm range were observed, possibly attributable to RCH2OH, RCH2OR, and RC=OOCH3 groups. Furthermore, two doublets appeared at 4.3 ppm and 4.9 ppm, indicating the presence of RNH and protons of amine groups as well as alcohol (30).
1H NMR spectral patterns at 0.87, 1.26, 1.28, 1.63 and 2.03 ppm readily indicated the presence of long alkyl chain attached to a carbonyl. In 1H NMR a broad peak was observed at 9.96 ppm that implied presence of an aldehyde group and also confirmed that out of two carbonyl groups (16).
Citrinin measurement by HPLC
The obtained results indicate a significant reduction in the amount of citrinin in the optimal sample compared to the control sample. The optimal sample had a citrinin content of less than the standard limit of 0.05 ppm, while the control sample had a citrinin content of 2.5 ppm (Fig. 3).
In a study analyzing citrinin and pigment production in a culture medium containing tyrosol, it was observed that the addition of tyrosol led to a decrease in citrinin content by approximately 51.5% compared to the control medium without tyrosol (5). The use of millet in the fermentation culture was found to decrease citrinin production in the pigment produced by M. purpureus (31). Similar results were observed in studies involving flavonoids such as apigenin, genistein, rutin, alpha-glucosylrutin, or troxerutin, which strongly reduced citrinin synthesis while increasing pigmentation (32, 33). Zhen et al. (2019) reported that NaCl inhibits citrinin synthesis but stimulates the synthesis of Monascus pigments (34). It is worth noting the biosynthesis that pathways of pigments and citrinin are related, but the results of the experiments suggest that these pathways are independent among different Monascus species under the conditions of this study. Citrinin can be produced through the tetractide biosynthesis of Monascus as well as from pentactides found in Penicillum and Aspergillus species (35). Overall, these findings demonstrate the potential to manipulate culture conditions and add specific compounds to regulate citrinin production and enhance pigment production in Monascus species.
Comparative tests of pigment antimicrobial effect
The results of the WDA test, as shown in Table 1, indicate that both the control and optimal samples exhibited the highest resistance (lowest halo size) against E. coli, while they showed the highest sensitivity (largest halo size) against the S. aureus strain. The results of the MIC (minimum inhibitory concentration) and MBC (minimum bactericidal concentration) tests, presented in Table 1, reveal that E. coli was the most resistant strain, while S. aureus was the most sensitive strain to both samples. These findings align with the results of the WDA test.
Overall, the pigment obtained from the optimum sample of the dairy sludge culture medium exhibited a significantly stronger antimicrobial effect against pathogenic microorganisms compared to the control sample which is more because of the pigment content. Pigments have long been used as natural and safe colors in Asian countries due to their excellent coloring and antibacterial properties (6). Extracts of fungal colorants have also been shown to possess antibacterial activities against pathogenic bacteria such as S. aureus (36, 37). According to a study, the antibacterial activity of the Monascus pigment was found to be higher than that of the commercial red pigment against all tested bacteria. The MBC for Bacillus cereus ATCC11778 was determined to be 256 mg/ml for the commercial pigment, while it was lower at 128 mg/ml for the Monascus pigment (19). It is worth noting that Gram-negative bacteria, including E. coli, have a complex cell membrane structure consisting of lipopolysaccharides and two layers of phospholipids. This outer layer acts as a barrier, making it more challenging for compounds to penetrate the cell membrane and exert their antimicrobial effects, which may explain the higher resistance observed in E. coli compared to S. aureus (11).
Measurement of antioxidant activity through inhibition of DPPH radicals and FRAP test
The optimal sample exhibited 55.26% and 0.105 µmol/l of antioxidant activity to inhibit DPPH radicals and FRAP reducing power, respectively. In comparison, the control sample showed 32.4% and 0.061 µmol/l for DPPH radicals and FRAP reducing power, respectively. These findings indicate the antioxidant activity of the dye produced in this study. Additionally, the pigment sample obtained from the dairy sludge culture medium displayed approximately twice the antioxidant activity compared to the control sample, which can be attributed to the presence of more red pigment in the optimal culture medium that red pigment has more biological activity. Monascus red pigment has more therapeutic effects and stability against heat and different pH than yellow and orange pigments (38). Furthermore, the pigment demonstrated antioxidant activity against the 2,2′-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) radical, with an IC50 of 14.42 µg/mL (36). Another study involving the pigment monashin, produced by M. purpureus, reported DPPH (26%), H2O2 (77%), and reducing power (0.57 AU) activities at concentrations of 37.5, 40, and 46.15 μg/ml, respectively (16).
Pigments produced by Monascus sp. belong to the azaphilone family, which are cyclic compounds with at least one chiral center. These azaphilones exhibit a wide range of biological activities, including anti-obesity effects such as adipogenesis and lipolysis, as well as anti-cancer, anti-inflammatory, anti-depressant, anti-osteoporosis, and anti-diabetic effects (6, 39, 40). The components of the pigments, reduces endothelial adhesion induced by reactive oxygen species (ROS) formation, TNFα and NF-κB activation. It also reduces amyloid-β toxicity and oxidative stress in Caenorhabditis elegans by increasing the activities of SHSP-16, glutathione S-transferase, and superoxide dismutase (2).
Biomass and pigment production kinetics in fermenter
The results showed interesting trends in both biomass and pigment production over time. Fig 4a illustrates the increase in biomass until 96 h of fermentation, accompanied by a decrease in substrate. This indicates successful growth and utilization of nutrients. However, after 96 h, biomass production declined. This could be attributed to the accumulation of toxic metabolites in the environment, leading to the death phase. Fig 4b presents the pigment production kinetic that, in the first 48 h, there was no significant difference in the amount of the three pigment combinations. After 48 h, the red monascorubramine pigment showed a significant increase compared to the other two pigments. The study suggests that sufficient nutrients and pigment production contribute to the ongoing logarithmic growth phase even after 96 h of fermentation. Monascorubramine pigment exhibited the highest production, followed by rubropunctatin and monascin. Specifically, after 96 h, 250 AU/g of monascorubramine pigments were produced.
To enhance pigment production in Monascus species, optimizing cultivation conditions is crucial. Factors such as inoculum size, temperature, initial pH, warm planting time, oxygen concentration, and nutritional components (nitrogen, carbon, and minerals) can be adjusted. Previous studies have shown similar results in optimizing cultivation conditions (41, 42).
Glucose has been found to be a favorable carbon source for higher biomass production compared to other sources. Additionally, glucose concentration influences the production of yellow and red pigments. Higher glucose concentrations tend to shift the maximum absorption towards red pigments. Also, the carbon-to-nitrogen ratio also plays a role in stimulating pigment production in filamentous fungi (4, 15). Specific studies have determined optimal conditions for red pigment production by M. purpureus. These conditions include a 2% inoculation rate (v/v), carbon source of 75 g/L lactose, nitrogen source of 25 g/L monosodium glutamate, and a pH of 7. Under these conditions, the maximum red pigment production was reported as 38.4 AU at 510 nm (22). The utilization of dairy sludge as a culture medium has been shown to significantly impact pigment production. Another study focused on optimizing GABA production and investigated dairy sludge as substrate. In the culture media contains dairy sludge, GABA production reached 359.45 ppm (12).
Determination of kinetic parameters in fermenter
The maximum specific growth rate (µmax) for M. purpureus was determined to be 0.029 1/h. The efficiency of cell production compared to the substrate (Yx/s) was found to be 0.29 g/g, and the specific rate of biomass production (qs) was calculated as 3.1 g/g.day (Table 2). A total production efficiency of 65% was achieved, which is considered high despite the use of a non-specific culture medium for this strain. In a similar study, the specific growth rate of M. purpureus on whey was determined to be 0.023/1/h, with a maximum pigment production efficiency of 4.55 AU (22). In line with results of this study, the productivity values of 0.059, 0.072, and 0.032 AU/h, respectively, and the specific growth rate values as 0.03 0.04, and 0.017 1/h for lactose, glucose, and hydrolyzed lactose were reported, respectively (43). Atalay et al. (2020) produced red pigment with M. purpureus after 192 h of fermentation with productivity and specific growth rate as 2.3 UA/h and 0.03/1/h, respectively (24). In another study, the addition of glucose to a rice paddy-based culture medium resulted in an increase in biomass production from 9.72 g/L to 25.35 g/L, while the yield of mycelium dry weight from the substrate remained constant at 0.36 g/g and 0.32 g/g, and a production efficiency of 21.2 U/ml in 336 h (qs) and a calculated specific growth rate of M. purpureus were reported as 0.06 U/ml/h (8). These findings indicate that the chosen culture medium in this study is highly productive and provides a stable substrate for the production of red pigment by M. purpureus.