Monascus Pigments Research Articles

Methionine and S-Adenosylmethionine Regulate Monascus Pigments Biosynthesis in Monascus purpureus.

Amino acid metabolism could exert regulatory effects on Monascus pigments (MPs) biosynthesis. In this work, MPs biosynthesis regulated by methionine and S-adenosylmethionine (SAM) was investigated in Monascus purpureus RP2. The results indicated that the addition of methionine in fermentation significantly reduced MPs production by 60–70%, and it induced a higher expression of SAM synthetase Mon2A2272 and consequently led to SAM accumulation. However, the addition of SAM in fermentation promoted MPs production by a maximum of 35%, while over-expression of the gene Mon2A2272 led to a decrease in MPs yield, suggesting that SAM synthetase and SAM were likely to play different regulatory roles in MPs biosynthesis. Furthermore, the gene transcription profile indicated that SAM synthetase expression led to a higher expression of the transcriptional regulatory protein of the MPs biosynthesis gene cluster, while the addition of SAM gave rise to a higher expression of MPs biosynthesis activator and the global regulator LaeA, which probably accounted for changes in MPs production and the mycelium colony morphology of M. purpureus RP2 triggered by methionine and SAM. This work proposed a possible regulation mechanism of MPs biosynthesis by SAM metabolism from methionine. The findings provided a new perspective for a deep understanding of MPs biosynthesis regulation in M. purpureus.

Increased Water-Soluble Yellow Monascus Pigment Productivity via Dual Mutagenesis and Submerged Repeated-Batch Fermentation of Monascus purpureus.

Monascus pigments (MPs) have been used in the food industry for more than 2,000 years and are known for their safety, bold coloring, and physiological activity. MPs are mainly yellow (YMPs), orange (OMPs), and red (RMPs). In this study, a mutant strain Monascus purpureus H14 with high production of water-soluble YMPs (WSYMPs, λmax at 370 nm) was generated instead of primary YMPs (λmax at 420 nm), OMPs (λmax at 470 nm), and RMPs (λmax at 510 nm) produced by the parent strain M. purpureus LQ-6 through dual mutagenesis of atmospheric and room-temperature plasma and heavy ion beam irradiation (HIBI), producing 22.68 U/ml extracellular YMPs and 10.67 U/ml intracellular YMPs. WSYMP production was increased by 289.51% in optimal conditions after response surface methodology was applied in submerged fermentation. Application of combined immobilized fermentation and extractive fermentation improved productivity to 16.89 U/ml/day, 6.70 times greater than with conservative submerged fermentation. The produced WSYMPs exhibited good tone stability to environmental factors, but their pigment values were unstable to pH, light, and high concentrations of Ca2+, Zn2+, Fe2+, Cu2+, and Mg2+. Furtherly, the produced exYMPs were identified as two yellow monascus pigment components (monascusone B and C21H27NO7S) by UHPLC-ESI-MS. This strategy may be extended to industrial production of premium WSYMPs using Monascus.

Analysis of secondary metabolite gene clusters and chitin biosynthesis pathways of Monascus purpureus with high production of pigment and citrinin based on whole-genome sequencing.

Monascus is a filamentous fungus that is widely used for producing Monascus pigments in the food industry in Southeast Asia. While the development of bioinformatics has helped elucidate the molecular mechanism underlying metabolic engineering of secondary metabolite biosynthesis, the biological information on the metabolic engineering of the morphology of Monascus remains unclear. In this study, the whole genome of M. purpureus CSU-M183 strain was sequenced using combined single-molecule real-time DNA sequencing and next-generation sequencing platforms. The length of the genome assembly was 23.75 Mb in size with a GC content of 49.13%, 69 genomic contigs and encoded 7305 putative predicted genes. In addition, we identified the secondary metabolite biosynthetic gene clusters and the chitin synthesis pathway in the genome of the high pigment-producing M. purpureus CSU-M183 strain. Furthermore, it is shown that the expression levels of most Monascus pigment and citrinin clusters located genes were significantly enhanced via atmospheric room temperature plasma mutagenesis. The results provide a basis for understanding the secondary metabolite biosynthesis, and constructing the metabolic engineering of the morphology of Monascus.

Monascin and ankaflavin-Biosynthesis from Monascus purpureus, production methods, pharmacological properties: A review.

Monascus purpureus copiously yields beneficial secondary metabolites , including Monascus pigments, which are broadly used as food additives, as a nitrite substitute in meat products, and as a colorant in the food industry. Monascus yellow pigments (monascin and ankaflavin) have shown potential antidiabetic, antibacterial, anti-inflammatory, antidepressant, antibiotic, anticancer, and antiobesity activities. Cosmetic and textile industries are other areas where it has established its potential as a dye. This paper reviews the production methods of Monascus yellow pigments, biosynthesis of Monascus pigments from M. purpureus, factors affecting yellow pigment production during fermentation, and the pharmacological properties of monascin and ankaflavin.

Effects of mokF gene deletion and overexpression on the Monacolin K metabolism yields of Monascus purpureus

Monascus purpureus is a fungus known for producing various physiologically active secondary metabolites. Of these, Monacolin K, a compound with hypocholesterolemic effects, is controlled by the biosynthetic gene mokF. Here, mokF deletion and overexpression strains (F2 and C3, respectively) were constructed using genetic engineering and compared with the M. purpureus wild strain (M1). The results showed that Monacolin K production was reduced by 50.86% in F2 and increased by 74.19% in C3. Of the three strains, C3 showed the highest production of Monacolin K and the most abnormal morphology. In addition, mokF influenced the expression level of mokA-mokI and might play an important role in regulating the biosynthesis of secondary metabolites in M. purpureus. Overall, our study verified the function of mokF in M. purpureus using gene deletion and overexpression technology. KEY POINTS: • The deletion and overexpression strains of mokF gene were successfully constructed. • The deletion or overexpression of mokF gene directly affected Monacolin K production. •The mokF gene had little effect on Monascus pigments and cell biomass.

The stabilization of Monascus pigment by formation of Monascus pigment-sodium caseinate complex

This study aimed to evaluate the effect of sodium caseinate (SC) on the thermal stability of Monascus pigments (MPs) under acidic conditions (pH 3.0) during heating at 100 °C at different time (0, 30, 60, 90 and 120 min). The MPs solution was precipitated after heating in an acidic condition, while it was stable in the presence of SC. Particle size results indicated the formation of MP-SC complex nanoparticles. Their values (141) were much smaller than MP and significantly improved the solubility and stability of MP in acidic conditions. The formation of covalent interactions (CC at peak 1643–1654 and NO stretching at peak (1537–1539) and hydrogen bonds (CH binding at peak 1452–1454) between MPs and SC were clear. Overall, the formation of the MP-SC complex was attributed to the covalent interactions and hydrogen bonds between MPs and SC, leading to the inhibition of MPs aggregation and improved stability in acidic solution.

Effect of γ-Heptalactone on the Morphology and Production of Monascus Pigments and Monacolin K in Monascus purpureus.

Monascus is used widely in Asian countries and produces various biologically active metabolites, such as Monascus pigments (MPs) and monacolin K (MK). In this study, the effect of γ-heptalactone on secondary metabolites and mycelial growth during Monascus purpureus M1 fermentation was investigated. After the addition of 50 μM γ-heptalactone, the yields of MPs (yellow, orange, and red) reached maxima, increased by 115.70, 141.52, and 100.88%, respectively. The 25 μM γ-heptalactone groups showed the highest yield of MK was increased by 62.38% compared with that of the control. Gene expression analysis showed that the relative expression levels of MPs synthesis genes (MpPKS5, MpFasA2, mppB, mppC, mppD, mppG, mpp7, and mppR1/R2) were significantly upregulated after γ-heptalactone treatment. The relative expression levels of MK synthesis genes (mokA, mokC, mokE, mokH, and mokI) were significantly affected. The mycelium samples treated with γ-heptalactone exhibited more folds and swelling than that in the samples of the control group. This study confirmed that the addition of γ-heptalactone has the potential to induce yields of MPs and MK, and promote the expression of biosynthesis genes, which may be related to the transformation of mycelial morphology in M. purpureus.

Comparative transcriptomics discloses the regulatory impact of carbon/nitrogen fermentation on the biosynthesis of Monascus kaoliang pigments.

Carbon and nitrogen play a fundamental role in the production of Monascus pigments. However, their effects on pigment biosynthesis remain undetermined. In this study, we found that Monascus kaoliang produces pigments via liquid fermentation using glycerol and peptone as suitable carbon and nitrogen sources, respectively. Comparative transcriptomic profiling was performed using RNA sequencing. It indicated that the differentially expressed genes (DEGs) of carbon were enriched using amino acids and carbohydrates via the transport and metabolism pathways, respectively. DEGs of nitrogen were enriched only using general functional prediction pathways. These data provide a comprehensive interpretation of the linkage between primary and secondary metabolisms in M. kaoliang. Moreover, they provide insights into the effects of various substances involved in secondary metabolism.

Transcriptome analysis revealing molecular mechanisms of enhanced pigment yield by succinic acid and fluconazole

This study aimed to elucidate the molecular mechanisms through which succinic acid and fluconazole stimulate Monascus pigment biosynthesis under liquid fermentation conditions. The pigment yield was significantly improved by adding 0.35 g·L−1 succinic acid or 1.5 g·L−1 fluconazole. Transcriptome sequencing and RT-qPCR confirmation were performed to reveal transcriptome changes. The results indicated that the addition of succinic acid significantly decreased mRNA expression of genes involved in fatty acid biosynthesis while increasing expression of genes involved in pyruvate metabolism. Fluconazole significantly down-regulated transcripts involved in branched-chain amino acid metabolism, fatty acid metabolism, glycolysis/gluconeogenesis, and pyruvate metabolism, as well as the generation of acetyl-CoA for pigment biosynthesis. On the other hand, nitrogen metabolism and lysine degradation pathways were significantly enriched, which could stimulate the generation of acetyl-CoA. Therefore, the mechanism for enhancing pigment yield may be attributed to the competitive regulation of metabolic pathways toward acetyl-CoA biosynthesis. Additionally, up-regulation of some different key genes in the presence of fluconazole or succinic acid was involved in improving pigment production. This study deepens the theoretical understanding for enhancing pigment biosynthesis and provides a few potential approaches for improving pigment yield.

Inactivation of mrpigH Gene in Monascus ruber M7 Results in Increased Monascus Pigments and Decreased Citrinin with mrpyrG Selection Marker.

Monascus pigments (MPs) have been used as food colorants for several centuries in Asian countries and are currently used around the world via Asian catering. The MPs biosynthetic pathway has been well-illustrated; however, the functions of a few genes including mrpigH in the MPs gene cluster of M. ruber M7 are still unclear. In the current study, mrpigH was disrupted in Δmrlig4ΔmrpyrG, a highly efficient gene modification system, using mrpyrG as a selection marker, and ΔmrpigHΔmrlig4ΔmrpyrG::mrpyrG and ΔmrpigHΔmrlig4ΔmrpyrG have been obtained. Subsequently, their morphologies, biomasses, MPs and citrinin (CIT) production were analyzed, respectively. These results have revealed that the deletion of mrpigH has significant effects on the morphology and growth of M. ruber M7. Moreover, compared with M. ruber M7, the yields of MPs and CIT were drastically increased and decreased in mrpigH mutants, respectively.

Azaphilone alkaloids: prospective source of natural food pigments

Azaphilone, biosynthesized by polyketide synthase, is a class of fungal metabolites. In this review, after brief introduction of the natural azaphilone diversity, we in detail discussed azaphilic addition reaction involving conversion of natural azaphilone into the corresponding azaphilone alkaloid. Then, setting red Monascus pigments (a traditional food colorant in China) as example, we presented a new strategy, i.e., interfacing azaphilic addition reaction with living microbial metabolism in a one-pot process, to produce azaphilone alkaloid with a specified amine residue (red Monascus pigments) during submerged culture. Benefit from the red Monascus pigments with a specified amine residue, the influence of primary amine on characteristics of the food colorant was highlighted. Finally, the progress for screening of alternative azaphilone alkaloids (production from interfacing azaphilic addition reaction with submerged culture of Talaromyces sp. or Penicillium sp.) as natural food colorant was reviewed. KEY POINTS: • Azaphilic addition reaction of natural azaphilone is biocompatible • Red Monascus pigment is a classic example of azaphilone alkaloids • Azaphilone alkaloids are alterative natural food colorant.

An improved method for the purification and structural analysis of rubropunctatin from red yeast glutinous rice

An improved method for the separation and purification of rubropunctatin from red yeast glutinous rice was developed. In this method, silica gel column chromatography and thin layer chromatography were used to separate the Monascus pigments. Pure methanol and a mixture of ethyl acetate and methanol (13:7, v/v) were selected as the eluent and running solvent, respectively. When compared with previously reported methods, the developed method required fewer types of solvents, and could be suitable for large-scale laboratory production. The extracted pigment was analysed by ultra-high performance liquid chromatography quadrupole time of flight mass spectrometry (UPLC-Q-TOF-MS) and nuclear magnetic resonance (NMR) (1H NMR, 13C NMR). Structural analysis revealed a molecular mass of 353.2 m/z [M－H]－, and a structure including an azaphilone body with two side chains. This structure was consistent with that reported for rubropunctatin. Therefore, the improved purification method reported herein could efficiently be used for the extraction of rubropunctatin from red yeast glutinous rice.

Structurally diverse Monascus pigments with hypolipidemic and hepatoprotective activities from highland barley Monascus

Highland barley Monascus has historically been used in solid state fermentation and traditional fermented foods in Tibet. It is possessed of the characteristics of medicine and food. Three new 8,13-unsaturated benzocyclodiketone-conjugated Monascus pigments (1–3), three new benzofuran Monascus pigments (4–6), three new butylated malonyl Monascus pigments (7–9), and eleven known Monascus pigment derivatives (10−20) were isolated from highland barley Monascus for the first time. Their structures were determined by analyzing NMR, MS, UV, and IR spectral data and compared with relevant references. Among them, compounds 2, 4, 6 showed important inhibition of pancreatic lipase activity, and decreased significantly FFA-induced lipid accumulation in HepG2 liver cells. Additionally, compounds 1, 10, 14, 16, 18 exhibited certain hepatoprotective activities against the damage in acetaminophen-induced HepG2 cells. The plausible biogenetic pathway and preliminary structure activity relationship of the selected compounds were scientifically summarized and discussed in this work.

Effects of exogenous ascorbic acid on yields of citrinin and pigments, antioxidant capacities, and fatty acid composition of Monascus ruber

Monascus pigments considered as safe coloring agents had been widely used in food industries until citrinin, a nephrotoxic mycotoxin, was detected in Monascus products. Here, the effects of exogenous ascorbic acid (EAA) both on citrinin and pigments production in submerged cultures of Monascus ruber M2013082 were studied. With the addition of 16 mg/L EAA to the media, the yield of citrinin was reduced by 67.6 ± 0.02% and Monascus pigments was increased by 29.3 ± 0.21% at the culturing time of 132 h. The cultures with EAA showed excellent intracellular antioxidant enzyme activities and extracellular antioxidant activities including free radical scavenging, iron-chelating, and the reducing power, which suggested EAA protected cells from excess free radical damage and then inhibited pigments degradation and citrinin synthesis. Further fatty acid composition analysis showed the increased unsaturated/saturated fatty acid ratio from 1.25 ± 0.02 to 1.40 ± 0.03 and the IUFA value from 77.66 ± 0.32 to 80.67 ± 0.45 facilitated the secretion of intracellular pigments and therefore enhanced pigments production accordingly. Significant increase of fumaric and succinic acids indicated EAA activated Creb cycle resulted in less citrinin precursor of malonyl-CoA formation, which led to the reduction of citrinin synthesis.

Effect of arginine supplementation on Monacolin K yield of Monascus purpureus

Through a screening process, we previously revealed that the addition of arginine to Monascus fermentation broth significantly increased Monacolin K production, with 0.6 % arginine showing the best effects. Herein, we added 0.6 % arginine to further detect the effect of arginine on secondary metabolites and mycelium morphology of Monascus purpureus. When M. purpureus was grown in a medium supplemented with arginine, the Monacolin K yield increased from 94.6 to 228.4 mg/L, indicating an increase of approximately 2.4-fold. Further, there were significant increases in Monascus pigments. Scanning electron microscopy of Monascus mycelium morphology showed that arginine significantly altered the mycelial morphology and enhanced the extracellular secretion of Monacolin K. RT-qPCR showed that after the addition of arginine, expression of all the mokA-mokI genes were upregulated. These results indicate that arginine could increase the yield of Monacolin K, providing a basis for further analysis of Monacolin K biosynthesis.

Antimicrobial activity of red pigments derived from Monascus purpureus: A comparison to industrial red pigments

AbstractBackgroundIn recent years, the various advantages of fungal pigments over other natural or synthetic pigments have opened new avenues for wide range of applications in different industries. This study aims to determine the antimicrobial activity of fungal pigment produced by Monascus purpureus CMU001, against some Gr(+) and Gr(−) bacteria, yeasts and molds.MethodIn this study, the 96‐well microplate dilution and macro dilution methods were used to determine the minimum inhibitory concentration (MIC) values of pigments on tested microorganisms. Therefore, the microbial plating method was performed to observe the antifungal activity of pigment since the MIC values could not be determined on molds.ResultsThe antibacterial activity of Monascus pigment against all tested bacteria was higher than the commercial red pigment. The minimum bactericidal concentration on Bacillus cereus ATCC11778 was determined as 256 mg/ml for the commercial pigment and 128 mg/ml for Monascus pigment. The MIC values could not be determined for both pigments against yeast and molds even at the highest possible concentration.ConclusionThe Monascus pigments have a big potential to use in different industries such as foods, textile, and biomedical products, since the high inhibitory effects was confirmed on various food‐borne and pathogenic bacteria and fungi. Although the specific mechanism and properties of the antimicrobial activity of Monascus pigments are still unknown, the differences between lab‐produced and commercial pigments showed that the antimicrobial activity was based on the type of microorganism, growth conditions, and the composition of culture medium.

Utilization of low-cost agricultural by-product rice husk for Monascus pigments production via submerged batch-fermentation.

Monascus pigments (MPs) produced by the genus Monascus, have been utilized for more than 2000 years in the food industry. In the present study, by submerged batch-fermentation (SBF), we were able to obtain a mutant strain with a high tolerance of inhibitory compounds generated from rice husk hydrolysate, allowing the production of MPs. The mutant strain, M. Purpureus M523 with high rice husk hydrolysate tolerance was obtained using the atmospheric and room temperature plasma (ARTP) screening system, producing 39.48 U mL-1 extracellular total MPs (yellow and orange MPs), using non-detoxified rice husk diluted sulfuric acid hydrolysate (RHSAH) as the carbon source in SBF. Extracellular MPs (exMPs) production was enhanced to 72.1 and 80.7U mL-1 in supplemented SBF (SSBF) and immobilized fermentation (IF) using non-detoxified RHSAH, with productivities of 0.16 and 0.37U mL-1 h-1 , respectively. In addition, our findings revealed that despite having a high RHSAH tolerance, the mutant strain was unable to degrade phenolic compounds. Furthermore, we discovered that inhibitory compounds, including furfural (Fur) and 5'-hydroxymethyl furfural (5'-HMF), not only inhibit MP biosynthesis, but also regulate the conversion of pigment components. The low-cost agricultural by-product, rice husk, can serve as an efficient substitute for MP production with high productivity via IF by Monascus spp. © 2021 Society of Chemical Industry.

Triton X-100 supplementation regulates growth and secondary metabolite biosynthesis during in-depth extractive fermentation of Monascus purpureus

Extractive fermentation has been proven to be efficient in enhancing the secretion and production of secondary metabolites in submerged fermentation by Monascus spp., owing to increased cell membrane permeability and resolved product inhibition. In this study, we investigated the regulation effect of Triton X-100 on cell growth and secondary metabolite biosynthesis in submerged fermentation of M. purpureus DK. The results show that the maximum monascus pigments (MPs), citrinin (CIT) production, and specific growth rate are 136.86 U/mL, 4.57 mg/L, and 0.04 h−1, respectively, when 3 g/L of Triton X-100 is supplemented after fermentation for 10 d, and the extracellular MPs and CIT increased by 127.48% and 288.57%, respectively. RT-qPCR shows that the expression levels of MPs and CIT biosynthesis gene clusters are significantly upregulated, whereas those of glycolysis, tricarboxylic acid cycle, respiratory chains, and ATP synthase are downregulated. This study provides a vital strategy for extractive fermentation under extreme environmental conditions for further enhancing MP production.

Production of soluble dietary fibers and red pigments from potato pomace in submerged fermentation by Monascus purpureus

The goal of this study was to develop a submerged fermentation by Monascus purpureus for converting potato pomace, a low-cost byproduct generated in the potato starch industry, to a high value-added product containing soluble dietary fibers and Monascus pigments. Potato pomace was used directly without any pretreatments or hydrolysis in fermentation by M. purpureus to produce Monascus pigments, which was quantified by monitoring the absorbance of the fermentation broth at 492 nm wavelength and was found to reach a high level of 47.9 OD492/mL with a yield of 598.6 OD492/g pomace. Thermal stability analysis showed that the red pigments produced from potato pomace had a high activation energy of 71.6 kJ mol−1 at pH 5.0, indicating that the red pigment was thermally stable even at an acidic pH. Meanwhile, about 110.9 mg of soluble dietary fibers with porous microstructure, high solubility, and antioxidant activities were obtained from each gram of potato pomace after the fermentation. The results demonstrated the feasibility of converting potato pomace to a valuable product containing Monascus pigments and soluble dietary fibers by M. purpureus in submerged fermentation, providing an economical and environmentally friendly route for utilizing the agro-industrial waste byproduct.

Production of Natural Colorant by Monascus Purpureus FNCC 6008 using Rice and Cassava as Carbon Substrates

Consumer recognition of the adverse effects of synthetic colorant has increased awareness in utilizing natural colorants as an alternative. One of them is a microbial colorant and it is already studied for its safety and functional properties in the human body, i.e. hypercholesterolemia, antimicrobial, antioxidant, and anticancer. The functional properties of microbial colorant have driven many kinds of research about natural colorant produced by the microorganism. Monascus pigment is one of the popular red pigment synthesized by mold Monascus purpureus. This research was conducted to investigate the effects of particle size and incubation temperature on color value and water solubility of the natural colorant produced by M. purpureus FNCC 6008 using rice and cassava as carbon substrates. Every experiment was conducted in an erlenmeyer flask filled by 15 g of substrates, sterilized and incubated in an incubator chamber. Three particle sizes of 8, 10, and 16 mesh were employed on the incubation temperatures of 30, 32, and 34 oC for 14 days. Two parameters were measured from the sample to evaluate the results of the fermentation process, i.e. color intensity and water solubility of product. The highest color intensity of 59.6 CVU/gds was obtained from the rice substrate at the particle size of 10 mesh and incubation temperature of 32 oC. That particular fermentation condition resulted in a product with 71.4% product solubility.