{"paper_id":"302738d6-d629-4aa2-b6f5-70cfb1cb0506","body_text":"Improvement of red pigment production and citrinin reduction from Monascus purpureus using dairy sludge: potential health benefits and fermentation strategies | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Improvement of red pigment production and citrinin reduction from Monascus purpureus using dairy sludge: potential health benefits and fermentation strategies Samira Moradi, Seyed Ali Mortazavi This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-3936278/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract This study aimed to explore the production of red pigment from Monascus purpureus and its potential health benefits. The research started with the cultivation of M. purpureus in an environment containing dairy sludge. Subsequently, the extracted pigment was purified and subjected to various analyses, including liquid chromatography mass spectrometry (LCMS) and nuclear magnetic resonance (NMR) to verify its purity, high-pressure liquid chromatography (HPLC) to measure the citrinin levels, microbial testing, and assessment of antioxidant activity. Finally, fermentation was conducted in a batch system using a fermenter. M. purpureus was grown in a medium composed of dairy sludge, monosodium glutamate, and glucose, resulting in a biomass yield of 26.15 g/l. After extraction and purification, the optimal sample yielded 4.85 g of dry color, while the control sample produced 2.5 g. Analysis using NMR revealed similarities between the samples, while HPLC indicated low citrinin levels of less than 0.05 ppm in the optimal sample and 2.5 ppm in the control sample. LCMS analysis demonstrated a purity of 91.9% for the optimal sample, which also exhibited antimicrobial and antioxidant activity. In the fermenter, the sample obtained from optimal culture conditions displayed the highest concentration of the pigment monascorubramine, maximum specific growth rate of 0.029/1/h (µ max ), a cell yield (Y x/s ) of 0.29 g/g, and a production efficiency of 65% for M. purpureus . Overall, the produced pigment sample exhibited potential for use in the food industry due to its low citrinin content and high concentration of red pigment. Monascus purpureus Dairy sludge Pigment Fermenter Figures Figure 1 Figure 2 Figure 3 Figure 4 Introduction Color is a crucial characteristic of food in terms of its appearance, and consumers consider it as an indicator of quality. In recent years, there has been an increasing demand for natural colors due to concerns about the potential negative impact of synthetic colors on human and animal health ( 1 , 2 ). Microorganism colors are derived from certain species of bacteria, molds, and yeast. Notable examples include species from the genera Monascus, Paecilomyces, Serratia, Cordyceps, Streptomyces, and Penicillium ( 3 ). Monascus is a member of the Yomikota order and Monascus family, that identified over 20 species of Monascus, with Monascus pilosus , Monascus purpureus , and Monascus ruber being the most significant and widely utilized species in the food industry ( 4 , 5 ). Pigments produced by Monascus species are widely used in China and East Asian countries as natural food pigments in various products such as fish, Chinese cheese, ketchup, beverages like red wine, and meat products such as sausages and hamburgers as a substitute for nitrites ( 6 , 7 ). Monascus molds produce diverse metabolites, including pigments, monaculins, lovastatin, mucorvicin, a toxin called citrinin (monasidine A), and gamma-aminobutyric acid. M. purpureus is particularly important among the different species due to its high efficiency in pigment production and exhibits lower levels of citrinin, a type of mycotoxin, compared to other species of this mold ( 8 , 9 ). The presence of citrinin poses significant safety challenges in Monascus mold-based products, attracting global attention. Some Asian countries have also introduced their own limits, such as 0.05 µg and 0.2 µg citrinin/g food product in South Korea and Japan, respectively, while the American Food and Drug Administration has set it at 20 µg/kg for agricultural products ( 4 , 8 ). Various solutions have been proposed to reduce citrinin production, including optimizing culture conditions, incubation temperature, time, dissolved oxygen, and the use of additives such as specific amino acids, and ammonium sulfate ( 9 ). Most studies in this field have focused on the production of edible pigments during the fermentation process. However, little has been done to address citrinin production reduction during fermentation. Therefore, the main objective of this project is to minimize citrinin production. Another goal is to explore the use of agricultural wastes, such as dairy sludge, for red pigment production, a topic that has not been extensively researched. Dairy industries, being major producers of wastewater, generate dairy sludge, which contains high levels of organic compounds like carbohydrates and proteins. These can serve as carbon and nitrogen sources for microorganisms and reducing the overall production cost is also crucial for the industry ( 10 ). Pigment production using dairy sludge has not been done yet, and dairy sludge has been used in the production of lactic acid ( 11 ) and γ-aminobutyric acid (GABA) ( 12 ). The utilization of such diverse and readily available resources for pigment synthesis from M. purpureus highlights the possibility of sustainable and cost-effective production methods. Various substrates have been evaluated for pigment synthesis through M. purpureus fermentation include starch, Saba banana peel, residual beer, cheese whey, soybean meals, waste loquat kernels, date waste substrates, and whey ( 1 ). In this study, dairy sludge was used as a cheap and optimal culture medium and potato dextrose agar (PDA) medium was used as a control. After extracting and purifying the sample, the production of pigment was investigated through liquid chromatography mass spectrometry (LCMS) and citrinin by high pressure liquid chromatography (HPLC). Also, the antimicrobial and antioxidant activity of the produced pigment was studied. Finally, pigment production was evaluated in the fermenter. Materials and methods Microorganisms M. purpureus mold was obtained from Iran's Industrial Microorganisms Collection Center, grown on a solid culture medium containing starch yeast powder and agar solution and incubated at 30 ºC for 7 days. Then, it was kept in the refrigerator until use, and re-cultivation was prepared from it once every two weeks (13). Culture media and chemicals The PDA, potato dextrose broth (PDB) and mueller hinton agar (MHA) purchased from Sigma Aldrich Co. (Canada). Also, dairy sludge was sourced from dairy factory. All reagents of analytical grade used in the study were purchased from Merck Co. (Germany). Submerge fermentation The process of fermenting M. purpureus involves several steps as described in your previous statement were conducted: The spores produced from a 7-day culture of M. purpureus are washed from the surface of the culture medium using sterile phosphate buffered salt (PBS). A suspension is prepared with a concentration of 1.5 × 10 6 cells/ml (McFarland's 0.5 standard). 400 µl of the spore suspension are transferred to a 250 ml flask containing 40 ml of the original PDB culture medium, enriched with dairy sludge (10%), monosodium glutamate (1%), and glucose (10%). The flask is then kept in a dark environment for 14 days in a shaker incubator at a temperature of 30 °C and a rotation speed of 160 rpm. The initial pH of the culture medium is adjusted to 5, 6.5, and 8 to stimulate the microorganism to produce pigment (3). Biomass determination The fungal biomass was estimated by determining the amount of N-acetylglucosamine released by the acid hydrolysis of chitin, which is present in the mycelium cell wall. Chitin hydrolysis was performed using 10 M HCl in an autoclave at 130 °C for 2 h. The hydrolyzed mixture was neutralized to pH = 7, then mixed with acetylacetone reagent, followed by Ehrlich's reagent. Finally, the light absorption of the sample at 530 nm (compared to pure N-acetylglucosamine) was measured (14). Measurement of extracellular and intracellular pigments To separate the mycelium, all the contents of the culture medium were filtered using Whatman filter paper. Then, the strained culture medium was centrifuged for 15 min at 7511 × g and prepared solution was diluted with distilled water. The absorption of samples was measured using a visible ultraviolet spectrophotometer at 510 nm (red pigments), 470 nm (orange pigments) and 400 nm (yellow pigments). The remaining mycelium on the filter paper from the previous step was washed twice with distilled water, then cut into small pieces and added to 10 ml of 70% ethanol (v/v) (pH 2) and kept in a shaker at speed of 120 rpm for 2 h. The ethanolic solution containing intracellular pigment was centrifuged for 15 min at 7511 × g until the mycelium settled and the amount of pigment in the supernatant solution was measured with the mentioned method for intracellular pigment (14). Isolation and purification of pigment Fermented broth medium without cells was used for pigment purification. The filtered solution was concentrated using a rotary evaporator (Rotavapor R-210, Buchi, Switzerland) and then lyophilized and powdered. The powder was extracted using hexane (500 ml in total) for 1 h in a shaker (120 rpm) and concentrated using a rotary evaporator under vacuum. The extracted crude pigment was loaded into a silica gel column (60-120 mesh) and followed different ratios of hexane and ethyl acetate were used as detergents. The fractions eluted from the column that were read by spectrophotometer between 300 and 700 nm were combined. Finally, ethanol was used to wash the target compound (15). Analysis of pigment composition Nuclear magnetic resonance (NMR) The characteristics of the purified pigment were performed by NMR spectroscopy at room temperature using Bruker WM 500 spectrometer [500 MHz ( 1 H NMR)]. A small amount of dried purified pigment was dissolved in 500 ml of dimethyl sulfoxide (DMSO) and the solution was homogenized. Then, this mixture was analyzed by NMR to evaluate the number of protons ( 1 H) at 500.13 MHz to 125.77 MHz (16). LC-MS LC-MS (electrospray ionization, ESI) analysis was done on a Mightysil RP-18 GP column using an Agilent HPLC-MSD series 1100. The sample was first filtered through a 0.2 μm PTFE filter membrane and then placed on the automatic sampler. Next, the pigments were eluted on a Mightysil RP18 column (150 mm × 2 mm Kanto Chemical, Tokyo) using a linear gradient. The gradient ranged from acetonitrile-water containing 0.1% formic acid (60:40, v/v) to acetonitrile-water containing 0.1% formic acid (100:0, v/v). The flow rate was maintained at 0.2 mL/min, the oven temperature was set to 40 ºC, and running time for the analysis was 25 min. The pigment compounds were detected using electrospray ionization in positive ion mode MS/MS (17). Citrinin assay the measurement of citrinin was carried out using HPLC (Milford, USA). Specifically, 20 µl of the purified pigment sample was injected into an HPLC device equipped with a C18 reverse phase column (4.6 mm × 250 mm, 5 µm particle size). The mobile phase used was a mixture of acetonitrile and water (65:35 v/v), and the flow rate was set at 1 ml/min. To detect and quantify citrinin, a fluorescence detector was employed with an excitation wavelength of 331 nm and an emission wavelength of 500 nm (9). Measuring the antimicrobial activity of pigment Well Diffusion Agar (WDA) From the suspension of pathogenic bacteria Escherichia coli ATCC 25922, Pseudomonas aeruginosa PTCC 1707, Salmonella typhimurium PTCC 1609, and Staphylococcus aureus ATCC 25923 (obtained from the Center for Biological and Genetic Resources of Iran) with a concentration of half of McFarland, the amount of 10 µl was cultured on MHA medium and then wells with a diameter of 6-8 mm were created in the plates by the end of a sterile pipette and 100 µl of purified red pigment was added to the wells. After placing in an incubator for 48 h at a temperature of 37 ºC, the diameter of the growth halo around each well was measured (18). Minimum inhibitory concentration (MIC) and Minimum bactericidal concentration (MBC) determination In this method, the pigment along with the pathogenic bacteria is placed in a 96-well microplate and after incubation (24 h, 37 ºC), the absorbance of the sample was read using the ELISA method at 630 nm. In order to determine MBC, 10 µl were removed from the wells of the 96 microplates in which no color change was observed under sterile conditions and cultured on MHA culture medium. The plates were incubated in the temperature of 37 ºC, and after 24 h and were examined for growth. The first plate of concentrations cultured from the cell extract in which no colony was observed was considered as the MBC (19). Investigation of the pigment antioxidant properties Inhibition of DPPH radical The sample mixture (the concentration of 1 mg/ml) and DPPH radical solution of 0.2 mmol in 95% ethanol were combined and mixed. After keeping the sample for 30 min at room temperature and in a dark place, its absorbance was recorded at 517 nm and the radical scavenging activity of sample was calculated with following Eq. 1 (20). Eq. 1 radical scavenging activity % = (1- absorption of sample/ absorption of control) × 100 Ferric ion reducing antioxidant power measurement of the optimal sample (FRAP) 1 ml of the sample was mixed with 1 ml of distilled water and 1 ml of potassium ferricyanide (1%). After heating at 50 ºC for 20 min, 2.5 ml of 10% trichloroacetic acid was added to the solution. The resulting mixture was then centrifuged at 750 rpm for 5 min. Next, 2 ml of the supernatant was taken and mixed with 2 ml of distilled water and 1 ml of iron chloride (0.1%). The mixture was stirred and allowed to stand for 10 min at room temperature. Finally, the absorbance of the solution was measured at a wavelength of 700 nm. The percentage reducing activity of samples was calculated using the following Eq 2 (21). Eq 2 reducing activity % = [(1 - sample absorbance) /control absorbance] × 100 Fermentation in fermenter For the batch fermenter cultures, a glass vessel (New Brunswick Scientific) with a working volume of 3L was used and sealed with a stainless steel head plate. Agitation was achieved using a flat-bladed impeller rotating at 500 rpm, and sterile air was supplied at a rate of 0.003 L/min. Once cooled, sterile medium was added aseptically. After an initial inoculation of 1% (v/v) of the fermenter volume, feeding with a carbon source was initiated 36 h into fermentation. The feeding lasted for 8 h at a rate of 0.03 L/min. During the fermentation process, the temperature was automatically controlled at 30 °C, and pH was regulated using a steam-serializable pH electrode. Sterile solutions of 1 M sodium hydroxide or 1 M hydrochloric acid were used to adjust the pH as needed. To control foam formation, a foam controller added silicon antifoam as required. The fermentation lasted for a total of 96 h. Result and discussion 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 1 H NMR 1 H-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 1 H-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). 1 H 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 1 H 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 (µ max ) for M. purpureus was determined to be 0.029 1/h. The efficiency of cell production compared to the substrate (Y x/s ) was found to be 0.29 g/g, and the specific rate of biomass production (q s ) 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 (q s ) 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 . Conclusion The passage highlights the significance of pigments, particularly red pigments, in traditional foods as natural colors and preservatives. These pigments have diverse applications in the food industry, including coloring, preserving, flavoring, and functional food additives. In this study, the cultivation of M. purpureus in a medium containing dairy sludge, monosodium glutamate, glucose, and PDA resulted in increased bios and pigment production compared to the control medium. The study emphasizes the importance of selecting the appropriate culture medium, cultivation methods, extraction, and purification techniques to ensure the production of pigments with suitable purity, minimal citrinin content, and practical characteristics. Fermentation in the fermenter for 96 h maintained the strain in the logarithmic phase, leading to higher red pigment production and favorable kinetic characteristics. Based on these findings, the method described in the study holds great potential for industrial production and utilization of the produced pigment in various edible products. The resulting color exhibits a promising outlook for its application in the food industry. Declarations Acknowledgements This work is based upon research funded by Iran National Science Foundation (INSF) under project No. 4005146. Availability of data and materials The following supplementary materials can be downloaded at: https: Ethics approval and consent to participate Not applicable. Consent for publication Not applicable. Competing interests The authors declare that they have no competing interests. References Gong P, Shi R, Liu Y, Luo Q, Wang C, Chen W. Recent advances in Monascus pigments produced by Monascus purpureus: Biosynthesis, fermentation, function, and application. LWT. 2023:115162. Adin SN, Gupta I, Panda BP, Mujeeb M. Monascin and ankaflavin—Biosynthesis from Monascus purpureus, production methods, pharmacological properties: A review. Biotechnology and Applied Biochemistry. 2023;70(1):137-47. Silbir S, Goksungur Y. Natural red pigment production by Monascus purpureus in submerged fermentation systems using a food industry waste: Brewer’s spent grain. Foods. 2019;8(5):161. Agboyibor C, Kong W-B, Chen D, Zhang A-M, Niu S-Q. Monascus pigments production, composition, bioactivity and its application: A review. Biocatalysis and Agricultural Biotechnology. 2018;16:433-47. Erkaya S, Arslan NP, Orak T, Esim N, Taskin M. Evaluation of tyrosol and farnesol as inducer in pigment production by Monascus purpureus ATCC16365. Journal of basic microbiology. 2020;60(8):669-78. Choe D, Jang H, Jung HH, Shin CS, Johnston TV, Kim D, Ku S. In vivo anti-obesity effects of Monascus pigment threonine derivative with enhanced hydrophilicity. Journal of Functional Foods. 2020;67:103849. Qiao J, Zeng H, Ye W, Qiu R, Zeng X, Xin B, Xie T. Integrative addition of sucrose esters and immobilisation technology for enhancing yellow pigment yield of Monascus purpureus HBSD08 under submerged fermentation conditions and its molecular mechanism. LWT. 2023;186:115233. Liu J, Luo Y, Guo T, Tang C, Chai X, Zhao W, et al. Cost-effective pigment production by Monascus purpureus using rice straw hydrolysate as substrate in submerged fermentation. Journal of bioscience and bioengineering. 2020;129(2):229-36. Orozco SFB, Kilikian BV. Effect of pH on citrinin and red pigments production by Monascus purpureus CCT3802. World Journal of Microbiology and Biotechnology. 2008;24:263-8. Keivani H, Jahadi M, Ghasemisepero N. Optimizing submerged cultivation for the production of red pigments by Monascus purpureus on soybean meals using response surface methodology. Applied Food Biotechnology. 2020;7(3):143-52. Moradi S, Zeraatpisheh F, Tabatabaee-Yazdi F. Investigation of lactic acid production in optimized dairy wastewater culture medium. Biomass Conversion and Biorefinery. 2022:1-12. Falah F, Vasiee A, Tabatabaei-Yazdi F, Moradi S, Sabahi S. Optimization of γ-aminobutyric acid (GABA) production by Lactobacillus spp. from agro-food waste. Biomass Conversion and Biorefinery. 2022:1-13. Srianta I, Zubaidah E, Estiasih T, Yamada M. Comparison of Monascus purpureus growth, pigment production and composition on different cereal substrates with solid state fermentation. Biocatalysis and Agricultural Biotechnology. 2016;7:181-6. Xiong X, Zhen Z, Liu Y, Gao M, Wang S, Li L, Zhang J. Low‐frequency magnetic field of appropriate strengths changed secondary metabolite production and Na+ concentration of intracellular and extracellular Monascus purpureus. Bioelectromagnetics. 2020;41(4):289-97. Mukherjee G, Singh SK. Purification and characterization of a new red pigment from Monascus purpureus in submerged fermentation. Process Biochemistry. 2011;46(1):188-92. Mondal S, Pandit SG, Puttananjaiah MH, Harohally NV, Dhale MA. Structural and functional characterization of new pigment molecule Monashin from Monascus purpureus CFR410-11. Process Biochemistry. 2019;82:173-8. Matsuoka H, Honzawa S, Takahashi A, Yoshikawa H, Watanabe E, Watanabe T, et al. Photoisomerization of 2-[3-(2-thioxopyrrolidin-3-ylidene) methyl]-tryptophan, a yellow pigment in salted radish roots. Bioscience, biotechnology, and biochemistry. 2008;72(9):2262-8. Balouiri M, Bouhdid S, Harki E, Sadiki M, Ouedrhiri W, Ibnsouda SK. Antifungal activity of Bacillus spp. isolated from Calotropis procera AIT. Rhizosphere against Candida albicans. Asian J Pham Clin Res. 2015;8:213-7. Gökmen GG, Şılbır MS, Göksungur Y, Kışla D. Antimicrobial activity of red pigments derived from Monascus purpureus: A comparison to industrial red pigments. JSFA Reports. 2021;1(1):5-10. Pacifico S, Gallicchio M, Lorenz P, Duckstein SM, Potenza N, Galasso S, et al. Neuroprotective potential of Laurus nobilis antioxidant polyphenol-enriched leaf extracts. Chemical research in toxicology. 2014;27(4):611-26. Yudiarti T, Sugiharto S, Isroli I, Widiastuti E, Wahyuni HI, Sartono TA. Effect of fermentation using Chrysonillia crassa and Monascus purpureus on nutritional quality, antioxidant, and antimicrobial activities of used rice as a poultry feed ingredient. Journal of Advanced Veterinary and Animal Research. 2019;6(2):168. Mehri D, Perendeci NA, Goksungur Y. Utilization of whey for red pigment production by Monascus purpureus in submerged fermentation. Fermentation. 2021;7(2):75. Abdollahi F, Jahadi M, Ghavami M. Thermal stability of natural pigments produced by Monascus purpureus in submerged fermentation. Food Science & Nutrition. 2021;9(9):4855-62. Atalay P, Sargın S, Goksungur Y. Utilization of residual beer for red pigment production by Monascus purpureus in submerged fermantation. Fresenius Environ Bull. 2020;29:1025-34. de Almeida AB, Santos NH, de Lima TM, Santana RV, de Oliveira Filho JG, Peres DS, Egea MB. Pigment bioproduction by Monascus purpureus using corn bran, a byproduct of the corn industry. Biocatalysis and Agricultural Biotechnology. 2021;32:101931. Chen X, Chen M, Wu X, Li X. Cost‐effective process for the production of Monascus pigments using potato pomace as carbon source by fed‐batch submerged fermentation. Food Science & Nutrition. 2021;9(10):5415-27. Kang B, Zhang X, Wu Z, Qi H, Wang Z. Solubilization capacity of nonionic surfactant micelles exhibiting strong influence on export of intracellular pigments in M onascus fermentation. Microbial Biotechnology. 2013;6(5):540-50. Mahmoud GA-E, Soltan HA, Abdel-Aleem WM, Osman SA. Safe natural bio-pigment production by Monascus purpureus using mixed carbon sources with cytotoxicity evaluation on root tips of Allium cepa L. Journal of Food Science and Technology. 2021;58:2516-27. Babitha S, Soccol CR, Pandey A. Jackfruit Seed–a novel substrate for the production of Monascus pigments through solid-state fermentation. Food Technology and Biotechnology. 2006;44(4):465-71. Campoy S, Rumbero A, Martín JF, Liras P. Characterization of an hyperpigmenting mutant of Monascus purpureus IB1: identification of two novel pigment chemical structures. Applied microbiology and biotechnology. 2006;70:488-96. Marič A, Skočaj M, Likar M, Sepčić K, Cigić IK, Grundner M, Gregori A. Comparison of lovastatin, citrinin and pigment production of different Monascus purpureus strains grown on rice and millet. Journal of food science and technology. 2019;56:3364-73. Wang Y, Gao H, Xie J, Li X, Huang Z. Effects of some flavonoids on the mycotoxin citrinin reduction by Monascus aurantiacus Li AS3. 4384 during liquid-state fermentation. Amb Express. 2020;10(1):1-10. Huang Z, Zhang L, Wang Y, Gao H, Li X, Huang X, Huang T. Effects of rutin and its derivatives on citrinin production by Monascus aurantiacus Li AS3. 4384 in liquid fermentation using different types of media. Food chemistry. 2019;284:205-12. Zhen Z, Xiong X, Liu Y, Zhang J, Wang S, Li L, Gao M. NaCl inhibits citrinin and stimulates Monascus pigments and monacolin K production. Toxins. 2019;11(2):118. Jung H, Kim C, Kim K, Shin CS. Color characteristics of Monascus pigments derived by fermentation with various amino acids. Journal of agricultural and food chemistry. 2003;51(5):1302-6. Kaur M, Goel M, Mishra RC, Lahane V, Yadav AK, Barrow CJ. Characterization of the Red Biochromes Produced by the Endophytic Fungus Monascus purpureus CPEF02 with Antimicrobial and Antioxidant Activities. Fermentation. 2023;9(4):328. Feng L, Li Y, Sun G, Zhao X. Antibacterial effect of orange Monascus pigment against Staphylococcus aureus. Acta Alimentaria. 2019;48(2):169-76. Kim D, Ku S. Beneficial effects of Monascus sp. KCCM 10093 pigments and derivatives: A mini review. Molecules. 2018;23(1):98. Gao J-M, Yang S-X, Qin J-C. Azaphilones: chemistry and biology. Chemical reviews. 2013;113(7):4755-811. Nam K, Choe D, Shin CS. Antiobesity effect of a jelly food containing the L-tryptophan derivative of Monascus pigment in mice. Journal of Functional Foods. 2014;9:306-14. Hamdiyati Y, Kusnadi K, Yuliani LA, editors. Effect of Monascus purpureus inoculum concentration on pigment production in jackfruit seed flour substrate. AIP Conference Proceedings; 2016: AIP Publishing. Orak T, Caglar O, Ortucu S, Ozkan H, Taskin M. Chicken feather peptone: A new alternative nitrogen source for pigment production by Monascus purpureus. Journal of biotechnology. 2018;271:56-62. da Costa JPV, Vendruscolo F. Production of red pigments by Monascus ruber CCT 3802 using lactose as a substrate. Biocatalysis and agricultural biotechnology. 2017;11:50-5. Tables Table 1 Results related to the evaluation of pigment antimicrobial activity (WDA, MIC and MBC) of control and optimal samples. Strain WDA (mm) MIC (mg/ml) MBC (mg/ml) Optimal sample Control sample Optimal sample Control sample Optimal sample Control sample S. typhi 12.00 ± 0.48 cA 8.00 ± 0.23 dB 125±5 bB 500±10 aA 125±6 bB 500±12 aA E. coli 10.00 ± 0.50 dA 9.00 ± 0.21 cB 250±7 aB 550±10 bA 250±5 aB 550±5 bA L. innocua 14.00 ± 0.46 bA 10.00 ± 0.32 bB 125±5 bB 500±10 aA 125±7 bB 500±5 aA S. aureus 19.00 ± 0.45 aA 11.00 ± 0.24 aB 100±5 cB 250±8 cA 100±5 cB 250±10 cA Small letters a to b indicate significant differences between different strains in each column and capital letters A to B indicate significant differences between different samples in each row. Table 2 Kinetics parameters of M. purpureus fermentation in batch fermenter during 96 h in optimal culture environment. Kinetic parameters Responses Y x/s (g/g) 0.29±0.01 q s (g/g/day) 3.1±0.22 µ max (l/h) 0.029±0.003 Additional Declarations No competing interests reported. Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {\"props\":{\"pageProps\":{\"initialData\":{\"identity\":\"rs-3936278\",\"acceptedTermsAndConditions\":true,\"allowDirectSubmit\":true,\"archivedVersions\":[],\"articleType\":\"Research Article\",\"associatedPublications\":[],\"authors\":[{\"id\":272171737,\"identity\":\"276db95e-ac1b-4bc1-83f7-1895d638c1ce\",\"order_by\":0,\"name\":\"Samira Moradi\",\"email\":\"\",\"orcid\":\"\",\"institution\":\"Ferdowsi University of Mashhad\",\"correspondingAuthor\":false,\"prefix\":\"\",\"firstName\":\"Samira\",\"middleName\":\"\",\"lastName\":\"Moradi\",\"suffix\":\"\"},{\"id\":272171738,\"identity\":\"ea984189-8b18-43f3-916c-e6e8ed2063ab\",\"order_by\":1,\"name\":\"Seyed Ali Mortazavi\",\"email\":\"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAs0lEQVRIiWNgGAWjYBACgwMQWg7KlyCsxfIAA2MDkDYmXos9VEtiA9EOMzve+/zBhz926RuuHWD88IPBIp+wljPHDRtn8CTnbridwCzZwyBhSdA6sxtpjM08EswgLQzSQL8YELTFAKTlj0F9ugHQlt/Ea2FIOJwA1MJGpC1njjHO7Dlw3HDm7cQ2yx4DYrQcb2P48ONPtTzf7eTDN35U1BHWggRA8UOShlEwCkbBKBgFOAEAZNo7GYLo/7gAAAAASUVORK5CYII=\",\"orcid\":\"\",\"institution\":\"Ferdowsi University of Mashhad\",\"correspondingAuthor\":true,\"prefix\":\"\",\"firstName\":\"Seyed\",\"middleName\":\"Ali\",\"lastName\":\"Mortazavi\",\"suffix\":\"\"}],\"badges\":[],\"createdAt\":\"2024-02-07 08:59:16\",\"currentVersionCode\":1,\"declarations\":\"\",\"doi\":\"10.21203/rs.3.rs-3936278/v1\",\"doiUrl\":\"https://doi.org/10.21203/rs.3.rs-3936278/v1\",\"draftVersion\":[],\"editorialEvents\":[],\"editorialNote\":\"\",\"failedWorkflow\":false,\"files\":[{\"id\":51015757,\"identity\":\"d5d52624-64f2-483d-a49b-9bf67be4052d\",\"added_by\":\"auto\",\"created_at\":\"2024-02-12 18:47:07\",\"extension\":\"jpg\",\"order_by\":1,\"title\":\"Figure 1\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":321673,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003eImages related to (a) fermentation culture supernatant and (b) dry pigment produced by the strain in optimal medium (right) and control (left) and LCMS spectra of (c) optimal and (d) control pigment samples.\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"1.jpg\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-3936278/v1/ab18b3b15c2e43acf0bd4475.jpg\"},{\"id\":51015474,\"identity\":\"cdc186b3-d184-4576-a752-8baac93c1d9a\",\"added_by\":\"auto\",\"created_at\":\"2024-02-12 18:39:07\",\"extension\":\"jpg\",\"order_by\":2,\"title\":\"Figure 2\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":37373,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003e\\u003csup\\u003e1\\u003c/sup\\u003eHNMR spectra of (a) optimal and (b) control pigment samples.\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"2.jpg\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-3936278/v1/ae855426cb6cb99d88dabb34.jpg\"},{\"id\":51015475,\"identity\":\"87899f1b-e651-4189-a662-114148391f76\",\"added_by\":\"auto\",\"created_at\":\"2024-02-12 18:39:07\",\"extension\":\"jpg\",\"order_by\":3,\"title\":\"Figure 3\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":56963,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003eHPLC analysis of (a) optimal and (b) control pigment samples.\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"3.jpg\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-3936278/v1/21048196f232583e094c9a32.jpg\"},{\"id\":51015476,\"identity\":\"c8017980-b18c-4e64-9626-61ffb0afcf78\",\"added_by\":\"auto\",\"created_at\":\"2024-02-12 18:39:07\",\"extension\":\"jpg\",\"order_by\":4,\"title\":\"Figure 4\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":80754,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003eKinetics of (a) biomass production and substrate consumption and (b) pigment production by \\u003cem\\u003eM. purpureus\\u003c/em\\u003e in batch fermenter during 96 h in optimal culture environment.\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"4.jpg\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-3936278/v1/e5451cc2acc2c286ee54d071.jpg\"},{\"id\":51328198,\"identity\":\"f4ecf275-6c38-4208-9690-2c498c7f307d\",\"added_by\":\"auto\",\"created_at\":\"2024-02-19 17:01:14\",\"extension\":\"pdf\",\"order_by\":0,\"title\":\"\",\"display\":\"\",\"copyAsset\":false,\"role\":\"manuscript-pdf\",\"size\":739693,\"visible\":true,\"origin\":\"\",\"legend\":\"\",\"description\":\"\",\"filename\":\"manuscript.pdf\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-3936278/v1/dc388cf5-1ef1-44c1-9c15-d3981cabcd19.pdf\"}],\"financialInterests\":\"No competing interests reported.\",\"formattedTitle\":\"Improvement of red pigment production and citrinin reduction from Monascus purpureus using dairy sludge: potential health benefits and fermentation strategies\",\"fulltext\":[{\"header\":\"Introduction\",\"content\":\"\\u003cp\\u003eColor is a crucial characteristic of food in terms of its appearance, and consumers consider it as an indicator of quality. In recent years, there has been an increasing demand for natural colors due to concerns about the potential negative impact of synthetic colors on human and animal health (\\u003cspan citationid=\\\"CR1\\\" class=\\\"CitationRef\\\"\\u003e1\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR2\\\" class=\\\"CitationRef\\\"\\u003e2\\u003c/span\\u003e).\\u003c/p\\u003e \\u003cp\\u003eMicroorganism colors are derived from certain species of bacteria, molds, and yeast. Notable examples include species from the genera Monascus, Paecilomyces, Serratia, Cordyceps, Streptomyces, and Penicillium (\\u003cspan citationid=\\\"CR3\\\" class=\\\"CitationRef\\\"\\u003e3\\u003c/span\\u003e). Monascus is a member of the Yomikota order and Monascus family, that identified over 20 species of Monascus, with \\u003cem\\u003eMonascus pilosus\\u003c/em\\u003e, \\u003cem\\u003eMonascus purpureus\\u003c/em\\u003e, and \\u003cem\\u003eMonascus ruber\\u003c/em\\u003e being the most significant and widely utilized species in the food industry (\\u003cspan citationid=\\\"CR4\\\" class=\\\"CitationRef\\\"\\u003e4\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR5\\\" class=\\\"CitationRef\\\"\\u003e5\\u003c/span\\u003e). Pigments produced by Monascus species are widely used in China and East Asian countries as natural food pigments in various products such as fish, Chinese cheese, ketchup, beverages like red wine, and meat products such as sausages and hamburgers as a substitute for nitrites (\\u003cspan citationid=\\\"CR6\\\" class=\\\"CitationRef\\\"\\u003e6\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR7\\\" class=\\\"CitationRef\\\"\\u003e7\\u003c/span\\u003e).\\u003c/p\\u003e \\u003cp\\u003eMonascus molds produce diverse metabolites, including pigments, monaculins, lovastatin, mucorvicin, a toxin called citrinin (monasidine A), and gamma-aminobutyric acid. \\u003cem\\u003eM. purpureus\\u003c/em\\u003e is particularly important among the different species due to its high efficiency in pigment production and exhibits lower levels of citrinin, a type of mycotoxin, compared to other species of this mold (\\u003cspan citationid=\\\"CR8\\\" class=\\\"CitationRef\\\"\\u003e8\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR9\\\" class=\\\"CitationRef\\\"\\u003e9\\u003c/span\\u003e). The presence of citrinin poses significant safety challenges in Monascus mold-based products, attracting global attention. Some Asian countries have also introduced their own limits, such as 0.05 \\u0026micro;g and 0.2 \\u0026micro;g citrinin/g food product in South Korea and Japan, respectively, while the American Food and Drug Administration has set it at 20 \\u0026micro;g/kg for agricultural products (\\u003cspan citationid=\\\"CR4\\\" class=\\\"CitationRef\\\"\\u003e4\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR8\\\" class=\\\"CitationRef\\\"\\u003e8\\u003c/span\\u003e). Various solutions have been proposed to reduce citrinin production, including optimizing culture conditions, incubation temperature, time, dissolved oxygen, and the use of additives such as specific amino acids, and ammonium sulfate (\\u003cspan citationid=\\\"CR9\\\" class=\\\"CitationRef\\\"\\u003e9\\u003c/span\\u003e).\\u003c/p\\u003e \\u003cp\\u003eMost studies in this field have focused on the production of edible pigments during the fermentation process. However, little has been done to address citrinin production reduction during fermentation. Therefore, the main objective of this project is to minimize citrinin production. Another goal is to explore the use of agricultural wastes, such as dairy sludge, for red pigment production, a topic that has not been extensively researched. Dairy industries, being major producers of wastewater, generate dairy sludge, which contains high levels of organic compounds like carbohydrates and proteins. These can serve as carbon and nitrogen sources for microorganisms and reducing the overall production cost is also crucial for the industry (\\u003cspan citationid=\\\"CR10\\\" class=\\\"CitationRef\\\"\\u003e10\\u003c/span\\u003e). Pigment production using dairy sludge has not been done yet, and dairy sludge has been used in the production of lactic acid (\\u003cspan citationid=\\\"CR11\\\" class=\\\"CitationRef\\\"\\u003e11\\u003c/span\\u003e) and γ-aminobutyric acid (GABA) (\\u003cspan citationid=\\\"CR12\\\" class=\\\"CitationRef\\\"\\u003e12\\u003c/span\\u003e). The utilization of such diverse and readily available resources for pigment synthesis from \\u003cem\\u003eM. purpureus\\u003c/em\\u003e highlights the possibility of sustainable and cost-effective production methods. Various substrates have been evaluated for pigment synthesis through \\u003cem\\u003eM. purpureus\\u003c/em\\u003e fermentation include starch, Saba banana peel, residual beer, cheese whey, soybean meals, waste loquat kernels, date waste substrates, and whey (\\u003cspan citationid=\\\"CR1\\\" class=\\\"CitationRef\\\"\\u003e1\\u003c/span\\u003e).\\u003c/p\\u003e \\u003cp\\u003eIn this study, dairy sludge was used as a cheap and optimal culture medium and potato dextrose agar (PDA) medium was used as a control. After extracting and purifying the sample, the production of pigment was investigated through liquid chromatography mass spectrometry (LCMS) and citrinin by high pressure liquid chromatography (HPLC). Also, the antimicrobial and antioxidant activity of the produced pigment was studied. Finally, pigment production was evaluated in the fermenter.\\u003c/p\\u003e\"},{\"header\":\"Materials and methods\",\"content\":\"\\u003cp\\u003e\\u003cstrong\\u003eMicroorganisms\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cem\\u003eM. purpureus\\u003c/em\\u003e mold was obtained from Iran\\u0026apos;s Industrial Microorganisms Collection Center, grown on a solid culture medium containing starch yeast powder and agar solution and incubated at 30 \\u0026ordm;C for 7 days. Then, it was kept in the refrigerator until use, and re-cultivation was prepared from it once every two weeks\\u0026nbsp;(13).\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eCulture media and chemicals \\u0026nbsp;\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eThe PDA, potato dextrose broth (PDB) and mueller hinton agar (MHA) purchased from Sigma Aldrich Co. (Canada). Also, dairy sludge was sourced from dairy factory. All reagents of analytical grade used in the study were purchased from Merck Co. (Germany).\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eSubmerge fermentation\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eThe process of fermenting \\u003cem\\u003eM. purpureus\\u003c/em\\u003e involves several steps as described in your previous statement were conducted: The spores produced from a 7-day culture of \\u003cem\\u003eM. purpureus\\u003c/em\\u003e are washed from the surface of the culture medium using sterile phosphate buffered salt (PBS). A suspension is prepared with a concentration of 1.5 \\u0026times; 10\\u003csup\\u003e6\\u003c/sup\\u003e cells/ml (McFarland\\u0026apos;s 0.5 standard). 400 \\u0026micro;l of the spore suspension are transferred to a 250 ml flask containing 40 ml of the original PDB culture medium, enriched with dairy sludge (10%), monosodium glutamate (1%), and glucose (10%). The flask is then kept in a dark environment for 14 days in a shaker incubator at a temperature of 30 \\u0026deg;C and a rotation speed of 160 rpm. The initial pH of the culture medium is adjusted to 5, 6.5, and 8 to stimulate the microorganism to produce \\u0026nbsp;pigment\\u0026nbsp;(3).\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eBiomass determination\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eThe fungal biomass was estimated by determining the amount of N-acetylglucosamine released by the acid hydrolysis of chitin, which is present in the mycelium cell wall. Chitin hydrolysis was performed using 10 M HCl in an autoclave at 130 \\u0026deg;C for 2 h. The hydrolyzed mixture was neutralized to pH = 7, then mixed with acetylacetone reagent, followed by Ehrlich\\u0026apos;s reagent. Finally, the light absorption of the sample at 530 nm (compared to pure N-acetylglucosamine) was measured\\u0026nbsp;(14).\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eMeasurement of extracellular and intracellular pigments\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eTo separate the mycelium, all the contents of the culture medium were filtered using Whatman filter paper. Then, the strained culture medium was centrifuged for 15 min at 7511 \\u0026times; g and prepared solution was diluted with distilled water. The absorption of samples was measured using a visible ultraviolet spectrophotometer at 510 nm (red pigments), 470 nm (orange pigments) and 400 nm (yellow pigments).\\u0026nbsp;\\u003c/p\\u003e\\n\\u003cp\\u003eThe remaining mycelium on the filter paper from the previous step was washed twice with distilled water, then cut into small pieces and added to 10 ml of 70% ethanol (v/v) (pH 2) and kept in a shaker at speed of 120 rpm for 2 h. The ethanolic solution containing intracellular pigment was centrifuged for 15 min at 7511 \\u0026times; g until the mycelium settled and the amount of pigment in the supernatant solution was measured with the mentioned method for intracellular pigment\\u0026nbsp;(14).\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eIsolation and purification of pigment\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003e\\u0026nbsp;Fermented broth medium without cells was used for pigment purification. The filtered solution was concentrated using a rotary evaporator (Rotavapor R-210, Buchi, Switzerland) and then lyophilized and powdered. The powder was extracted using hexane (500 ml in total) for 1 h in a shaker (120 rpm) and concentrated using a rotary evaporator under vacuum. The extracted crude pigment was loaded into a silica gel column (60-120 mesh) and followed different ratios of hexane and ethyl acetate were used as detergents. The fractions eluted from the column that were read by spectrophotometer between 300 and 700 nm were combined. Finally, ethanol was used to wash the target compound\\u0026nbsp;(15).\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eAnalysis of pigment composition\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eNuclear magnetic resonance (NMR)\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eThe characteristics of the purified pigment were performed by NMR spectroscopy at room temperature using Bruker WM 500 spectrometer [500 MHz (\\u003csup\\u003e1\\u003c/sup\\u003eH NMR)]. A small amount of dried purified pigment was dissolved in 500 ml of dimethyl sulfoxide (DMSO) and the solution was homogenized. Then, this mixture was analyzed by NMR to evaluate the number of protons (\\u003csup\\u003e1\\u003c/sup\\u003eH) at 500.13 MHz to 125.77 MHz\\u0026nbsp;(16).\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cem\\u003e\\u0026nbsp;\\u003c/em\\u003e\\u003cstrong\\u003eLC-MS\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eLC-MS (electrospray ionization, ESI) analysis was done on a Mightysil RP-18 GP column using an Agilent HPLC-MSD series 1100. The sample was first filtered through a 0.2 \\u0026mu;m PTFE filter membrane and then placed on the automatic sampler. Next, the pigments were eluted on a Mightysil RP18 column (150 mm \\u0026times; 2 mm Kanto Chemical, Tokyo) using a linear gradient. The gradient ranged from acetonitrile-water containing 0.1% formic acid (60:40, v/v) to acetonitrile-water containing 0.1% formic acid (100:0, v/v). The flow rate was maintained at 0.2 mL/min, the oven temperature was set to 40 \\u0026ordm;C, and running time for the analysis was 25 min. The pigment compounds were detected using electrospray ionization in positive ion mode MS/MS\\u0026nbsp;(17).\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eCitrinin assay\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003ethe measurement of citrinin was carried out using HPLC (Milford, USA). Specifically, 20 \\u0026micro;l of the purified pigment sample was injected into an HPLC device equipped with a C18 reverse phase column (4.6 mm \\u0026times; 250 mm, 5 \\u0026micro;m particle size). The mobile phase used was a mixture of acetonitrile and water (65:35 v/v), and the flow rate was set at 1 ml/min. To detect and quantify citrinin, a fluorescence detector was employed with an excitation wavelength of 331 nm and an emission wavelength of 500 nm\\u0026nbsp;(9).\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eMeasuring the antimicrobial activity of pigment\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eWell Diffusion Agar (WDA)\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eFrom the suspension of pathogenic bacteria \\u003cem\\u003eEscherichia coli\\u003c/em\\u003e ATCC 25922, \\u003cem\\u003ePseudomonas aeruginosa\\u0026nbsp;\\u003c/em\\u003ePTCC 1707, \\u003cem\\u003eSalmonella typhimurium\\u003c/em\\u003e PTCC 1609, and \\u003cem\\u003eStaphylococcus aureus\\u0026nbsp;\\u003c/em\\u003eATCC 25923 (obtained from the Center for Biological and Genetic Resources of Iran) with a concentration of half of McFarland, the amount of 10 \\u0026micro;l was cultured on MHA medium and then wells with a diameter of 6-8 mm were created in the plates by the end of a sterile pipette and 100 \\u0026micro;l of purified red pigment was added to the wells. After placing in an incubator for 48 h at a temperature of 37 \\u0026ordm;C, the diameter of the growth halo around each well was measured\\u0026nbsp;(18).\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eMinimum inhibitory concentration (MIC) and Minimum bactericidal concentration (MBC) determination\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eIn this method, the pigment along with the pathogenic bacteria is placed in a 96-well microplate and after incubation (24 h, 37 \\u0026ordm;C), the absorbance of the sample was read using the ELISA method at 630 nm.\\u003c/p\\u003e\\n\\u003cp\\u003eIn order to determine MBC, 10 \\u0026micro;l were removed from the wells of the 96 microplates in which no color change was observed under sterile conditions and cultured on MHA culture medium. The plates were incubated in the temperature of 37 \\u0026ordm;C, and after 24 h and were examined for growth. The first plate of concentrations cultured from the cell extract in which no colony was observed was considered as the MBC\\u0026nbsp;(19).\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eInvestigation of the pigment antioxidant properties\\u0026nbsp;\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eInhibition of DPPH radical\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eThe sample mixture (the concentration of 1 mg/ml) and DPPH radical solution of 0.2 mmol in 95% ethanol were combined and mixed. After keeping the sample for 30 min at room temperature and in a dark place, its absorbance was recorded at 517 nm and\\u0026nbsp;the\\u0026nbsp;radical scavenging activity of sample\\u0026nbsp;was calculated with following Eq. 1\\u0026nbsp;(20).\\u003c/p\\u003e\\n\\u003cp\\u003eEq. 1\\u0026nbsp; \\u0026nbsp;\\u0026nbsp;radical scavenging activity % = (1- absorption of sample/ absorption of control) \\u0026times; 100\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eFerric ion reducing antioxidant power measurement of the optimal sample (FRAP)\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003e1 ml of the sample was mixed with 1 ml of distilled water and 1 ml of potassium ferricyanide (1%). After heating at 50 \\u0026ordm;C for 20 min, 2.5 ml of 10% trichloroacetic acid was added to the solution. The resulting mixture was then centrifuged at 750 rpm for 5 min. Next, 2 ml of the supernatant was taken and mixed with 2 ml of distilled water and 1 ml of iron chloride (0.1%). The mixture was stirred and allowed to stand for 10 min at room temperature. Finally, the absorbance of the solution was measured at a wavelength of 700 nm. The percentage reducing activity of samples was calculated using the following Eq 2 (21). \\u0026nbsp;\\u003c/p\\u003e\\n\\u003cp\\u003eEq 2 \\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp;reducing activity % = [(1 - sample absorbance) /control absorbance] \\u0026nbsp; \\u0026times; 100\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eFermentation in fermenter\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eFor the batch fermenter cultures, a glass vessel (New Brunswick Scientific) with a working volume of 3L was used and sealed with a stainless steel head plate. Agitation was achieved using a flat-bladed impeller rotating at 500 rpm, and sterile air was supplied at a rate of 0.003 L/min. Once cooled, sterile medium was added aseptically. After an initial inoculation of 1% (v/v) of the fermenter volume, feeding with a carbon source was initiated 36 h into fermentation. The feeding lasted for 8 h at a rate of 0.03 L/min. During the fermentation process, the temperature was automatically controlled at 30 \\u0026deg;C, and pH was regulated using a steam-serializable pH electrode. Sterile solutions of 1 M sodium hydroxide or 1 M hydrochloric acid were used to adjust the pH as needed. To control foam formation, a foam controller added silicon antifoam as required. The fermentation lasted for a total of 96 h.\\u0026nbsp;\\u003c/p\\u003e\"},{\"header\":\"Result and discussion\",\"content\":\"\\u003cp\\u003e\\u003cstrong\\u003eThe results of biomass production in the culture environment and optimal pH\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eThe 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\\u0026nbsp;(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\\u0026nbsp;(1). Usually, at low pH, more yellow pigment (ancaflavin) is produced, and at higher pH, red pigment gradually dominates\\u0026nbsp;(4, 23).\\u003c/p\\u003e\\n\\u003cp\\u003eNew strategies to improve the stability characteristics and increase the application of the pigment have been applied \\u0026nbsp;(24, 25). The pigment was produced in the medium containing potato pomace through submerged fermentation by \\u003cem\\u003eM. purpureus\\u003c/em\\u003e CH01 and the results showed that high temperatures decrease the stability with increasing pH (26). The pH effect on pigment produced by \\u003cem\\u003eM. purpureus\\u0026nbsp;\\u003c/em\\u003ein 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 \\u003cem\\u003eM. purpureus\\u003c/em\\u003e 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).\\u003c/p\\u003e\\n\\u003cp\\u003eDairy 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 \\u003cem\\u003eM. purpureus\\u003c/em\\u003e, the compounds of sucrose esters\\u0026nbsp;(7)\\u0026nbsp;and corn starch with oils\\u0026nbsp;(28)\\u0026nbsp;evaluated in fermentation medium.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003ePigment extraction and purification\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eThe 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.\\u0026nbsp;\\u003c/p\\u003e\\n\\u003cp\\u003eThe 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\\u0026apos;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.\\u003c/p\\u003e\\n\\u003cp\\u003eSimilar high extracellular pigment production of 34.12 U/ml was observed in submerged fermentation using a glucose-based medium with \\u003cem\\u003eM. purpureus\\u0026nbsp;\\u003c/em\\u003e(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 \\u003cem\\u003eM. purpureus\\u003c/em\\u003e (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).\\u003c/p\\u003e\\n\\u003cp\\u003eThe 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).\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eEvaluation of pigment structure by \\u003csup\\u003e1\\u003c/sup\\u003eH NMR\\u0026nbsp;\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003csup\\u003e1\\u003c/sup\\u003eH-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\\u0026nbsp;(22).\\u003c/p\\u003e\\n\\u003cp\\u003eThe \\u003csup\\u003e1\\u003c/sup\\u003eH-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\\u0026nbsp;(30).\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003csup\\u003e1\\u003c/sup\\u003eH 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 \\u003csup\\u003e1\\u003c/sup\\u003eH 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).\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eCitrinin measurement by HPLC\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eThe 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).\\u003c/p\\u003e\\n\\u003cp\\u003eIn 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 \\u003cem\\u003eM. purpureus\\u003c/em\\u003e (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.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eComparative tests of pigment antimicrobial effect\\u0026nbsp;\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eThe 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 \\u003cem\\u003eE. coli\\u003c/em\\u003e, while they showed the highest sensitivity (largest halo size) against the \\u003cem\\u003eS. aureus\\u003c/em\\u003e strain. The results of the MIC (minimum inhibitory concentration) and MBC (minimum bactericidal concentration) tests, presented in Table 1, reveal that \\u003cem\\u003eE. coli\\u003c/em\\u003e was the most resistant strain, while \\u003cem\\u003eS. aureus\\u003c/em\\u003e was the most sensitive strain to both samples. These findings align with the results of the WDA test.\\u003c/p\\u003e\\n\\u003cp\\u003eOverall, 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 \\u003cem\\u003eS. aureus\\u003c/em\\u003e (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 \\u0026nbsp;for \\u003cem\\u003eBacillus cereus\\u003c/em\\u003e 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 \\u003cem\\u003eE. coli\\u003c/em\\u003e, 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 \\u003cem\\u003eS. aureus\\u0026nbsp;\\u003c/em\\u003e(11).\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eMeasurement of antioxidant activity through inhibition of DPPH radicals and FRAP test\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eThe optimal sample exhibited 55.26% and 0.105 \\u0026micro;mol/l of antioxidant activity to inhibit DPPH radicals and FRAP reducing power, respectively. In comparison, the control sample showed 32.4% and 0.061 \\u0026micro;mol/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\\u0026prime;-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) radical, with an IC50 of 14.42 \\u0026micro;g/mL (36). Another study involving the pigment monashin, produced by \\u003cem\\u003eM. purpureus\\u003c/em\\u003e, reported DPPH (26%), H2O2 (77%), and reducing power (0.57\\u0026thinsp;AU) activities at concentrations of 37.5, 40, and 46.15\\u0026thinsp;\\u0026mu;g/ml, respectively\\u0026nbsp;(16).\\u003c/p\\u003e\\n\\u003cp\\u003ePigments 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\\u0026nbsp;(6, 39, 40). The components of the pigments, reduces endothelial adhesion induced by reactive oxygen species (ROS) formation, TNF\\u0026alpha; and NF-\\u0026kappa;B activation. It also reduces amyloid-\\u0026beta; toxicity and oxidative stress in Caenorhabditis elegans by increasing the activities of SHSP-16, glutathione S-transferase, and superoxide dismutase\\u0026nbsp;(2).\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eBiomass and pigment production kinetics in fermenter\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eThe 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.\\u003c/p\\u003e\\n\\u003cp\\u003eTo 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\\u0026nbsp;(41, 42).\\u003c/p\\u003e\\n\\u003cp\\u003eGlucose 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 \\u003cem\\u003eM. purpureus\\u003c/em\\u003e. 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).\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eDetermination of kinetic parameters in fermenter\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003e\\u0026nbsp;The maximum specific growth rate (\\u0026micro;\\u003csub\\u003emax\\u003c/sub\\u003e) for \\u003cem\\u003eM. purpureus\\u003c/em\\u003e was determined to be 0.029 1/h. The efficiency of cell production compared to the substrate (Y\\u003csub\\u003ex/s\\u003c/sub\\u003e) was found to be 0.29 g/g, and the specific rate of biomass production (q\\u003csub\\u003es\\u003c/sub\\u003e) 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 \\u003cem\\u003eM. purpureus\\u003c/em\\u003e 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 \\u003cem\\u003eM. purpureus\\u003c/em\\u003e 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 (q\\u003csub\\u003es\\u003c/sub\\u003e) and a calculated specific growth rate of \\u003cem\\u003eM. purpureus\\u003c/em\\u003e 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 \\u003cem\\u003eM. purpureus\\u003c/em\\u003e.\\u003c/p\\u003e\"},{\"header\":\"Conclusion\",\"content\":\"\\u003cp\\u003eThe passage highlights the significance of pigments, particularly red pigments, in traditional foods as natural colors and preservatives. These pigments have diverse applications in the food industry, including coloring, preserving, flavoring, and functional food additives. In this study, the cultivation of \\u003cem\\u003eM. purpureus\\u003c/em\\u003e in a medium containing dairy sludge, monosodium glutamate, glucose, and PDA resulted in increased bios and pigment production compared to the control medium. The study emphasizes the importance of selecting the appropriate culture medium, cultivation methods, extraction, and purification techniques to ensure the production of pigments with suitable purity, minimal citrinin content, and practical characteristics. Fermentation in the fermenter for 96 h maintained the strain in the logarithmic phase, leading to higher red pigment production and favorable kinetic characteristics. Based on these findings, the method described in the study holds great potential for industrial production and utilization of the produced pigment in various edible products. The resulting color exhibits a promising outlook for its application in the food industry.\\u003c/p\\u003e\"},{\"header\":\"Declarations\",\"content\":\"\\u003cp\\u003e\\u003cstrong\\u003eAcknowledgements\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eThis work is based upon research funded by Iran National Science Foundation (INSF) under project No. 4005146.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eAvailability of data and materials\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eThe following supplementary materials can be downloaded at: https:\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eEthics approval and consent to participate\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eNot applicable.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eConsent for publication\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eNot applicable.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eCompeting interests\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eThe authors declare that they have no competing interests.\\u003c/p\\u003e\"},{\"header\":\"References\",\"content\":\"\\u003col\\u003e\\n\\u003cli\\u003eGong P, Shi R, Liu Y, Luo Q, Wang C, Chen W. Recent advances in Monascus pigments produced by Monascus purpureus: Biosynthesis, fermentation, function, and application. LWT. 2023:115162.\\u003c/li\\u003e\\n\\u003cli\\u003eAdin SN, Gupta I, Panda BP, Mujeeb M. Monascin and ankaflavin\\u0026mdash;Biosynthesis from Monascus purpureus, production methods, pharmacological properties: A review. Biotechnology and Applied Biochemistry. 2023;70(1):137-47.\\u003c/li\\u003e\\n\\u003cli\\u003eSilbir S, Goksungur Y. Natural red pigment production by Monascus purpureus in submerged fermentation systems using a food industry waste: Brewer\\u0026rsquo;s spent grain. Foods. 2019;8(5):161.\\u003c/li\\u003e\\n\\u003cli\\u003eAgboyibor C, Kong W-B, Chen D, Zhang A-M, Niu S-Q. Monascus pigments production, composition, bioactivity and its application: A review. Biocatalysis and Agricultural Biotechnology. 2018;16:433-47.\\u003c/li\\u003e\\n\\u003cli\\u003eErkaya S, Arslan NP, Orak T, Esim N, Taskin M. Evaluation of tyrosol and farnesol as inducer in pigment production by Monascus purpureus ATCC16365. Journal of basic microbiology. 2020;60(8):669-78.\\u003c/li\\u003e\\n\\u003cli\\u003eChoe D, Jang H, Jung HH, Shin CS, Johnston TV, Kim D, Ku S. In vivo anti-obesity effects of Monascus pigment threonine derivative with enhanced hydrophilicity. Journal of Functional Foods. 2020;67:103849.\\u003c/li\\u003e\\n\\u003cli\\u003eQiao J, Zeng H, Ye W, Qiu R, Zeng X, Xin B, Xie T. Integrative addition of sucrose esters and immobilisation technology for enhancing yellow pigment yield of Monascus purpureus HBSD08 under submerged fermentation conditions and its molecular mechanism. LWT. 2023;186:115233.\\u003c/li\\u003e\\n\\u003cli\\u003eLiu J, Luo Y, Guo T, Tang C, Chai X, Zhao W, et al. Cost-effective pigment production by Monascus purpureus using rice straw hydrolysate as substrate in submerged fermentation. Journal of bioscience and bioengineering. 2020;129(2):229-36.\\u003c/li\\u003e\\n\\u003cli\\u003eOrozco SFB, Kilikian BV. Effect of pH on citrinin and red pigments production by Monascus purpureus CCT3802. World Journal of Microbiology and Biotechnology. 2008;24:263-8.\\u003c/li\\u003e\\n\\u003cli\\u003eKeivani H, Jahadi M, Ghasemisepero N. Optimizing submerged cultivation for the production of red pigments by Monascus purpureus on soybean meals using response surface methodology. Applied Food Biotechnology. 2020;7(3):143-52.\\u003c/li\\u003e\\n\\u003cli\\u003eMoradi S, Zeraatpisheh F, Tabatabaee-Yazdi F. Investigation of lactic acid production in optimized dairy wastewater culture medium. Biomass Conversion and Biorefinery. 2022:1-12.\\u003c/li\\u003e\\n\\u003cli\\u003eFalah F, Vasiee A, Tabatabaei-Yazdi F, Moradi S, Sabahi S. Optimization of \\u0026gamma;-aminobutyric acid (GABA) production by Lactobacillus spp. from agro-food waste. Biomass Conversion and Biorefinery. 2022:1-13.\\u003c/li\\u003e\\n\\u003cli\\u003eSrianta I, Zubaidah E, Estiasih T, Yamada M. Comparison of Monascus purpureus growth, pigment production and composition on different cereal substrates with solid state fermentation. Biocatalysis and Agricultural Biotechnology. 2016;7:181-6.\\u003c/li\\u003e\\n\\u003cli\\u003eXiong X, Zhen Z, Liu Y, Gao M, Wang S, Li L, Zhang J. Low‐frequency magnetic field of appropriate strengths changed secondary metabolite production and Na+ concentration of intracellular and extracellular Monascus purpureus. Bioelectromagnetics. 2020;41(4):289-97.\\u003c/li\\u003e\\n\\u003cli\\u003eMukherjee G, Singh SK. Purification and characterization of a new red pigment from Monascus purpureus in submerged fermentation. Process Biochemistry. 2011;46(1):188-92.\\u003c/li\\u003e\\n\\u003cli\\u003eMondal S, Pandit SG, Puttananjaiah MH, Harohally NV, Dhale MA. Structural and functional characterization of new pigment molecule Monashin from Monascus purpureus CFR410-11. Process Biochemistry. 2019;82:173-8.\\u003c/li\\u003e\\n\\u003cli\\u003eMatsuoka H, Honzawa S, Takahashi A, Yoshikawa H, Watanabe E, Watanabe T, et al. Photoisomerization of 2-[3-(2-thioxopyrrolidin-3-ylidene) methyl]-tryptophan, a yellow pigment in salted radish roots. Bioscience, biotechnology, and biochemistry. 2008;72(9):2262-8.\\u003c/li\\u003e\\n\\u003cli\\u003eBalouiri M, Bouhdid S, Harki E, Sadiki M, Ouedrhiri W, Ibnsouda SK. Antifungal activity of Bacillus spp. isolated from Calotropis procera AIT. Rhizosphere against Candida albicans. Asian J Pham Clin Res. 2015;8:213-7.\\u003c/li\\u003e\\n\\u003cli\\u003eG\\u0026ouml;kmen GG, Şılbır MS, G\\u0026ouml;ksungur Y, Kışla D. Antimicrobial activity of red pigments derived from Monascus purpureus: A comparison to industrial red pigments. JSFA Reports. 2021;1(1):5-10.\\u003c/li\\u003e\\n\\u003cli\\u003ePacifico S, Gallicchio M, Lorenz P, Duckstein SM, Potenza N, Galasso S, et al. Neuroprotective potential of Laurus nobilis antioxidant polyphenol-enriched leaf extracts. Chemical research in toxicology. 2014;27(4):611-26.\\u003c/li\\u003e\\n\\u003cli\\u003eYudiarti T, Sugiharto S, Isroli I, Widiastuti E, Wahyuni HI, Sartono TA. Effect of fermentation using Chrysonillia crassa and Monascus purpureus on nutritional quality, antioxidant, and antimicrobial activities of used rice as a poultry feed ingredient. Journal of Advanced Veterinary and Animal Research. 2019;6(2):168.\\u003c/li\\u003e\\n\\u003cli\\u003eMehri D, Perendeci NA, Goksungur Y. Utilization of whey for red pigment production by Monascus purpureus in submerged fermentation. Fermentation. 2021;7(2):75.\\u003c/li\\u003e\\n\\u003cli\\u003eAbdollahi F, Jahadi M, Ghavami M. Thermal stability of natural pigments produced by Monascus purpureus in submerged fermentation. Food Science \\u0026amp; Nutrition. 2021;9(9):4855-62.\\u003c/li\\u003e\\n\\u003cli\\u003eAtalay P, Sargın S, Goksungur Y. Utilization of residual beer for red pigment production by Monascus purpureus in submerged fermantation. Fresenius Environ Bull. 2020;29:1025-34.\\u003c/li\\u003e\\n\\u003cli\\u003ede Almeida AB, Santos NH, de Lima TM, Santana RV, de Oliveira Filho JG, Peres DS, Egea MB. Pigment bioproduction by Monascus purpureus using corn bran, a byproduct of the corn industry. Biocatalysis and Agricultural Biotechnology. 2021;32:101931.\\u003c/li\\u003e\\n\\u003cli\\u003eChen X, Chen M, Wu X, Li X. Cost‐effective process for the production of Monascus pigments using potato pomace as carbon source by fed‐batch submerged fermentation. Food Science \\u0026amp; Nutrition. 2021;9(10):5415-27.\\u003c/li\\u003e\\n\\u003cli\\u003eKang B, Zhang X, Wu Z, Qi H, Wang Z. Solubilization capacity of nonionic surfactant micelles exhibiting strong influence on export of intracellular pigments in M onascus fermentation. Microbial Biotechnology. 2013;6(5):540-50.\\u003c/li\\u003e\\n\\u003cli\\u003eMahmoud GA-E, Soltan HA, Abdel-Aleem WM, Osman SA. Safe natural bio-pigment production by Monascus purpureus using mixed carbon sources with cytotoxicity evaluation on root tips of Allium cepa L. Journal of Food Science and Technology. 2021;58:2516-27.\\u003c/li\\u003e\\n\\u003cli\\u003eBabitha S, Soccol CR, Pandey A. Jackfruit Seed\\u0026ndash;a novel substrate for the production of Monascus pigments through solid-state fermentation. Food Technology and Biotechnology. 2006;44(4):465-71.\\u003c/li\\u003e\\n\\u003cli\\u003eCampoy S, Rumbero A, Mart\\u0026iacute;n JF, Liras P. Characterization of an hyperpigmenting mutant of Monascus purpureus IB1: identification of two novel pigment chemical structures. Applied microbiology and biotechnology. 2006;70:488-96.\\u003c/li\\u003e\\n\\u003cli\\u003eMarič A, Skočaj M, Likar M, Sepčić K, Cigić IK, Grundner M, Gregori A. Comparison of lovastatin, citrinin and pigment production of different Monascus purpureus strains grown on rice and millet. Journal of food science and technology. 2019;56:3364-73.\\u003c/li\\u003e\\n\\u003cli\\u003eWang Y, Gao H, Xie J, Li X, Huang Z. Effects of some flavonoids on the mycotoxin citrinin reduction by Monascus aurantiacus Li AS3. 4384 during liquid-state fermentation. Amb Express. 2020;10(1):1-10.\\u003c/li\\u003e\\n\\u003cli\\u003eHuang Z, Zhang L, Wang Y, Gao H, Li X, Huang X, Huang T. Effects of rutin and its derivatives on citrinin production by Monascus aurantiacus Li AS3. 4384 in liquid fermentation using different types of media. Food chemistry. 2019;284:205-12.\\u003c/li\\u003e\\n\\u003cli\\u003eZhen Z, Xiong X, Liu Y, Zhang J, Wang S, Li L, Gao M. NaCl inhibits citrinin and stimulates Monascus pigments and monacolin K production. Toxins. 2019;11(2):118.\\u003c/li\\u003e\\n\\u003cli\\u003eJung H, Kim C, Kim K, Shin CS. Color characteristics of Monascus pigments derived by fermentation with various amino acids. Journal of agricultural and food chemistry. 2003;51(5):1302-6.\\u003c/li\\u003e\\n\\u003cli\\u003eKaur M, Goel M, Mishra RC, Lahane V, Yadav AK, Barrow CJ. Characterization of the Red Biochromes Produced by the Endophytic Fungus Monascus purpureus CPEF02 with Antimicrobial and Antioxidant Activities. Fermentation. 2023;9(4):328.\\u003c/li\\u003e\\n\\u003cli\\u003eFeng L, Li Y, Sun G, Zhao X. Antibacterial effect of orange Monascus pigment against Staphylococcus aureus. Acta Alimentaria. 2019;48(2):169-76.\\u003c/li\\u003e\\n\\u003cli\\u003eKim D, Ku S. Beneficial effects of Monascus sp. KCCM 10093 pigments and derivatives: A mini review. Molecules. 2018;23(1):98.\\u003c/li\\u003e\\n\\u003cli\\u003eGao J-M, Yang S-X, Qin J-C. Azaphilones: chemistry and biology. Chemical reviews. 2013;113(7):4755-811.\\u003c/li\\u003e\\n\\u003cli\\u003eNam K, Choe D, Shin CS. Antiobesity effect of a jelly food containing the L-tryptophan derivative of Monascus pigment in mice. Journal of Functional Foods. 2014;9:306-14.\\u003c/li\\u003e\\n\\u003cli\\u003eHamdiyati Y, Kusnadi K, Yuliani LA, editors. Effect of Monascus purpureus inoculum concentration on pigment production in jackfruit seed flour substrate. AIP Conference Proceedings; 2016: AIP Publishing.\\u003c/li\\u003e\\n\\u003cli\\u003eOrak T, Caglar O, Ortucu S, Ozkan H, Taskin M. Chicken feather peptone: A new alternative nitrogen source for pigment production by Monascus purpureus. Journal of biotechnology. 2018;271:56-62.\\u003c/li\\u003e\\n\\u003cli\\u003eda Costa JPV, Vendruscolo F. Production of red pigments by Monascus ruber CCT 3802 using lactose as a substrate. Biocatalysis and agricultural biotechnology. 2017;11:50-5.\\u003c/li\\u003e\\n\\u003c/ol\\u003e\"},{\"header\":\"Tables\",\"content\":\"\\u003cp\\u003eTable 1 Results related to the evaluation of pigment antimicrobial activity (WDA, MIC and MBC) of control and optimal samples.\\u003c/p\\u003e\\n\\u003ctable border=\\\"0\\\" cellspacing=\\\"0\\\" cellpadding=\\\"0\\\"\\u003e\\n \\u003ctbody\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd width=\\\"12.62135922330097%\\\" valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003eStrain\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd width=\\\"17.475728155339805%\\\" valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003eWDA (mm)\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd width=\\\"15.533980582524272%\\\" valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003e\\u0026nbsp;\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd width=\\\"11.650485436893204%\\\" valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003eMIC (mg/ml)\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd width=\\\"14.239482200647249%\\\" valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003e\\u0026nbsp;\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd width=\\\"14.239482200647249%\\\" valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003eMBC (mg/ml)\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd width=\\\"14.239482200647249%\\\" valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003e\\u0026nbsp;\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd width=\\\"12.62135922330097%\\\" valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003e\\u0026nbsp;\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd width=\\\"17.475728155339805%\\\" valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003eOptimal sample\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd width=\\\"15.533980582524272%\\\" valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003eControl sample\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd width=\\\"11.650485436893204%\\\" valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003eOptimal sample\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd width=\\\"14.239482200647249%\\\" valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003eControl sample\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd width=\\\"14.239482200647249%\\\" valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003eOptimal sample\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd width=\\\"14.239482200647249%\\\" valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003eControl sample\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd width=\\\"12.62135922330097%\\\" valign=\\\"bottom\\\"\\u003e\\n \\u003cp\\u003e\\u003cem\\u003eS. typhi\\u003c/em\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd width=\\\"17.475728155339805%\\\" valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003e12.00 \\u0026plusmn; 0.48\\u003csup\\u003ecA\\u003c/sup\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd width=\\\"15.533980582524272%\\\" valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003e8.00 \\u0026plusmn; 0.23\\u003csup\\u003edB\\u003c/sup\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd width=\\\"11.650485436893204%\\\" valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003e125\\u0026plusmn;5\\u003csup\\u003ebB\\u003c/sup\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd width=\\\"14.239482200647249%\\\" valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003e500\\u0026plusmn;10\\u003csup\\u003eaA\\u003c/sup\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd width=\\\"14.239482200647249%\\\" valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003e125\\u0026plusmn;6\\u003csup\\u003ebB\\u003c/sup\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd width=\\\"14.239482200647249%\\\" valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003e500\\u0026plusmn;12\\u003csup\\u003eaA\\u003c/sup\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd width=\\\"12.62135922330097%\\\" valign=\\\"bottom\\\"\\u003e\\n \\u003cp\\u003e\\u003cem\\u003eE. coli\\u003c/em\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd width=\\\"17.475728155339805%\\\" valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003e10.00 \\u0026plusmn; 0.50\\u003csup\\u003edA\\u003c/sup\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd width=\\\"15.533980582524272%\\\" valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003e9.00 \\u0026plusmn; 0.21\\u003csup\\u003ecB\\u003c/sup\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd width=\\\"11.650485436893204%\\\" valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003e250\\u0026plusmn;7\\u003csup\\u003eaB\\u003c/sup\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd width=\\\"14.239482200647249%\\\" valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003e550\\u0026plusmn;10\\u003csup\\u003ebA\\u003c/sup\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd width=\\\"14.239482200647249%\\\" valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003e250\\u0026plusmn;5\\u003csup\\u003eaB\\u003c/sup\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd width=\\\"14.239482200647249%\\\" valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003e550\\u0026plusmn;5\\u003csup\\u003ebA\\u003c/sup\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd width=\\\"12.62135922330097%\\\" valign=\\\"bottom\\\"\\u003e\\n \\u003cp\\u003e\\u003cem\\u003eL. innocua\\u003c/em\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd width=\\\"17.475728155339805%\\\" valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003e14.00 \\u0026plusmn; 0.46\\u003csup\\u003ebA\\u003c/sup\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd width=\\\"15.533980582524272%\\\" valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003e10.00 \\u0026plusmn; 0.32\\u003csup\\u003ebB\\u003c/sup\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd width=\\\"11.650485436893204%\\\" valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003e125\\u0026plusmn;5\\u003csup\\u003ebB\\u003c/sup\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd width=\\\"14.239482200647249%\\\" valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003e500\\u0026plusmn;10\\u003csup\\u003eaA\\u003c/sup\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd width=\\\"14.239482200647249%\\\" valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003e125\\u0026plusmn;7\\u003csup\\u003ebB\\u003c/sup\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd width=\\\"14.239482200647249%\\\" valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003e500\\u0026plusmn;5\\u003csup\\u003eaA\\u003c/sup\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd width=\\\"12.62135922330097%\\\" valign=\\\"bottom\\\"\\u003e\\n \\u003cp\\u003e\\u003cem\\u003eS. aureus\\u003c/em\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd width=\\\"17.475728155339805%\\\" valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003e19.00 \\u0026plusmn; 0.45\\u003csup\\u003eaA\\u003c/sup\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd width=\\\"15.533980582524272%\\\" valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003e11.00 \\u0026plusmn; 0.24\\u003csup\\u003eaB\\u003c/sup\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd width=\\\"11.650485436893204%\\\" valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003e100\\u0026plusmn;5\\u003csup\\u003ecB\\u003c/sup\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd width=\\\"14.239482200647249%\\\" valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003e250\\u0026plusmn;8\\u003csup\\u003ecA\\u003c/sup\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd width=\\\"14.239482200647249%\\\" valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003e100\\u0026plusmn;5\\u003csup\\u003ecB\\u003c/sup\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd width=\\\"14.239482200647249%\\\" valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003e250\\u0026plusmn;10\\u003csup\\u003ecA\\u003c/sup\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003c/tbody\\u003e\\n\\u003c/table\\u003e\\n\\u003cp\\u003eSmall letters a to b indicate significant differences between different strains in each column and capital letters A to B indicate significant differences between different samples in each row.\\u003c/p\\u003e\\n\\u003cp\\u003eTable 2 Kinetics parameters of \\u003cem\\u003eM. purpureus\\u003c/em\\u003e fermentation in batch fermenter during 96 h in optimal culture environment.\\u003c/p\\u003e\\n\\u003ctable border=\\\"1\\\" cellspacing=\\\"0\\\" cellpadding=\\\"0\\\"\\u003e\\n \\u003ctbody\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd width=\\\"50%\\\" valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003eKinetic parameters\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd width=\\\"50%\\\" valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003eResponses\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd width=\\\"50%\\\" valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003eY\\u003csub\\u003ex/s\\u0026nbsp;\\u003c/sub\\u003e(g/g)\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd width=\\\"50%\\\" valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003e0.29\\u0026plusmn;0.01\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd width=\\\"50%\\\" valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003eq\\u003csub\\u003es\\u0026nbsp;\\u003c/sub\\u003e(g/g/day)\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd width=\\\"50%\\\" valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003e3.1\\u0026plusmn;0.22\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd width=\\\"50%\\\" valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003e\\u0026micro;\\u003csub\\u003emax\\u003c/sub\\u003e (l/h)\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd width=\\\"50%\\\" valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003e0.029\\u0026plusmn;0.003\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003c/tbody\\u003e\\n\\u003c/table\\u003e\"}],\"fulltextSource\":\"\",\"fullText\":\"\",\"funders\":[],\"hasAdminPriorityOnWorkflow\":false,\"hasManuscriptDocX\":true,\"hasOptedInToPreprint\":true,\"hasPassedJournalQc\":\"\",\"hasAnyPriority\":false,\"hideJournal\":true,\"highlight\":\"\",\"institution\":\"\",\"isAcceptedByJournal\":false,\"isAuthorSuppliedPdf\":false,\"isDeskRejected\":\"\",\"isHiddenFromSearch\":false,\"isInQc\":false,\"isInWorkflow\":false,\"isPdf\":false,\"isPdfUpToDate\":true,\"isWithdrawnOrRetracted\":false,\"journal\":{\"display\":true,\"email\":\"info@researchsquare.com\",\"identity\":\"researchsquare\",\"isNatureJournal\":false,\"hasQc\":true,\"allowDirectSubmit\":true,\"externalIdentity\":\"\",\"sideBox\":\"\",\"snPcode\":\"\",\"submissionUrl\":\"/submission\",\"title\":\"Research Square\",\"twitterHandle\":\"researchsquare\",\"acdcEnabled\":true,\"dfaEnabled\":false,\"editorialSystem\":\"\",\"reportingPortfolio\":\"\",\"inReviewEnabled\":false,\"inReviewRevisionsEnabled\":true},\"keywords\":\"Monascus purpureus, Dairy sludge, Pigment, Fermenter\",\"lastPublishedDoi\":\"10.21203/rs.3.rs-3936278/v1\",\"lastPublishedDoiUrl\":\"https://doi.org/10.21203/rs.3.rs-3936278/v1\",\"license\":{\"name\":\"CC BY 4.0\",\"url\":\"https://creativecommons.org/licenses/by/4.0/\"},\"manuscriptAbstract\":\"\\u003cp\\u003eThis study aimed to explore the production of red pigment from \\u003cem\\u003eMonascus purpureus\\u003c/em\\u003e and its potential health benefits. The research started with the cultivation of \\u003cem\\u003eM. purpureus\\u003c/em\\u003e in an environment containing dairy sludge. Subsequently, the extracted pigment was purified and subjected to various analyses, including liquid chromatography mass spectrometry (LCMS) and nuclear magnetic resonance (NMR) to verify its purity, high-pressure liquid chromatography (HPLC) to measure the citrinin levels, microbial testing, and assessment of antioxidant activity. Finally, fermentation was conducted in a batch system using a fermenter. \\u003cem\\u003eM. purpureus\\u003c/em\\u003e was grown in a medium composed of dairy sludge, monosodium glutamate, and glucose, resulting in a biomass yield of 26.15 g/l. After extraction and purification, the optimal sample yielded 4.85 g of dry color, while the control sample produced 2.5 g. Analysis using NMR revealed similarities between the samples, while HPLC indicated low citrinin levels of less than 0.05 ppm in the optimal sample and 2.5 ppm in the control sample. LCMS analysis demonstrated a purity of 91.9% for the optimal sample, which also exhibited antimicrobial and antioxidant activity. In the fermenter, the sample obtained from optimal culture conditions displayed the highest concentration of the pigment monascorubramine, maximum specific growth rate of 0.029/1/h (\\u0026micro;\\u003csub\\u003emax\\u003c/sub\\u003e), a cell yield (Y\\u003csub\\u003ex/s\\u003c/sub\\u003e) of 0.29 g/g, and a production efficiency of 65% for \\u003cem\\u003eM. purpureus\\u003c/em\\u003e. Overall, the produced pigment sample exhibited potential for use in the food industry due to its low citrinin content and high concentration of red pigment.\\u003c/p\\u003e\",\"manuscriptTitle\":\"Improvement of red pigment production and citrinin reduction from Monascus purpureus using dairy sludge: potential health benefits and fermentation strategies\",\"msid\":\"\",\"msnumber\":\"\",\"nonDraftVersions\":[{\"code\":1,\"date\":\"2024-02-12 18:39:02\",\"doi\":\"10.21203/rs.3.rs-3936278/v1\",\"editorialEvents\":[{\"type\":\"communityComments\",\"content\":0}],\"status\":\"published\",\"journal\":{\"display\":true,\"email\":\"info@researchsquare.com\",\"identity\":\"researchsquare\",\"isNatureJournal\":false,\"hasQc\":true,\"allowDirectSubmit\":true,\"externalIdentity\":\"\",\"sideBox\":\"\",\"snPcode\":\"\",\"submissionUrl\":\"/submission\",\"title\":\"Research Square\",\"twitterHandle\":\"researchsquare\",\"acdcEnabled\":true,\"dfaEnabled\":false,\"editorialSystem\":\"\",\"reportingPortfolio\":\"\",\"inReviewEnabled\":false,\"inReviewRevisionsEnabled\":true}}],\"origin\":\"\",\"ownerIdentity\":\"18c8e7ce-ac7d-4304-b2f7-9d8dcc87da71\",\"owner\":[],\"postedDate\":\"February 12th, 2024\",\"published\":true,\"recentEditorialEvents\":[],\"rejectedJournal\":[],\"revision\":\"\",\"amendment\":\"\",\"status\":\"posted\",\"subjectAreas\":[],\"tags\":[],\"updatedAt\":\"2024-02-19T17:01:01+00:00\",\"versionOfRecord\":[],\"versionCreatedAt\":\"2024-02-12 18:39:02\",\"video\":\"\",\"vorDoi\":\"\",\"vorDoiUrl\":\"\",\"workflowStages\":[]},\"version\":\"v1\",\"identity\":\"rs-3936278\",\"journalConfig\":\"researchsquare\"},\"__N_SSP\":true},\"page\":\"/article/[identity]/[[...version]]\",\"query\":{\"redirect\":\"/article/rs-3936278\",\"identity\":\"rs-3936278\",\"version\":[\"v1\"]},\"buildId\":\"qtupq5eGEP_6zYnWcrvyt\",\"isFallback\":false,\"isExperimentalCompile\":false,\"dynamicIds\":[84888],\"gssp\":true,\"scriptLoader\":[]}","source_license":"CC-BY-4.0","license_restricted":false}