Red Monascus Pigments Research Articles

Mutation Breeding of Monascus to Produce a High Yield of Orange Pigment and Low Citrinin Content Using the ARTP Method.

Monascus is a filamentous fungus with a long history of application in China, which can produce a variety of secondary metabolites, including Monascus red pigments, Monascus orange pigments, Monascus yellow pigments, and citrinin. There is widespread attention being paid to natural pigments because of their safety. Among the many natural pigments, orange pigment has a wide range of applications because of its unique color, but current production levels in the orange pigment industry are limited to a certain extent due to the insufficiently wide range of sources and low production. In this study, the ARTP mutation was used to obtain a strain with high-yield orange pigment and low citrinin. The strain RS7 was obtained through two-step mutagenesis, and all three pigments were improved to different degrees. The color value of orange pigment was elevated from the original 108 U/mL to 180 U/mL, an increase of 66.7% compared to the original strain, and the citrinin content was reduced by 69%. The result of microscopic morphology showed that RS7 has more wrinkles and is more convex than the R1 strain, but there was little change between the two strains. Therefore, the ARTP mutation influenced the growth and the biosynthesis of pigments in Monascus. In addition, the conditions of ultrasonic extraction of Monascus pigments were optimized using the response surface, and the separation of pigments was achieved with the method of thin-layer chromatography. Pigment stability results showed that the temperature had no significant effect on orange pigment, while tea polyphenol could improve its stability. This study generated a strain with high-yielding orange pigment and could lay a foundation for the future application of Monascus orange pigment in the food industry.

Exploring the Subcellular Localization of Monascus Pigments Biosynthases: Preliminary Unraveling of the Compartmentalization Mechanism.

Monascus pigments (MPs), a class of secondary metabolites produced by Monascus spp., can be classified into yellow, orange, and red MPs according to their differences in the wavelength of the maximum absorption. However, the biosynthetic sequence and cellular biosynthesis mechanism of different MPs components are still not yet completely clear in Monascus spp. In this study, the subcellular localization of five MPs synthases was investigated using fluorescent protein fusion expression. The results revealed that the proteins encoded by the MPs biosynthetic gene cluster were compartmentalized in various subcellular locations, including the mitochondrial polyketide synthase MrPigA, cytosolic enzymes consisting of the ketoreductase MrPigC, the oxidoreductase MrPigE, and the monooxygenase MrPigN, and the cell-wall-bound oxidoreductase MrPigF. Moreover, the correct localization of MrPigF to the cell wall was crucial for the synthesis of orange MPs. Lastly, we discussed the compartmentalized biosynthetic pathway of MPs. This study will not only be helpful in clarifying the biosynthetic sequence and biosynthesis mechanism of different MPs but also provides new insights into the cellular biosynthesis of secondary metabolites in filamentous fungi.

Monascus red pigments alleviate high-fat and high-sugar diet-induced NAFLD in mice by modulating the gut microbiota and metabolites.

Monascus red pigments (MRP) may have benefits against NAFLD with an unclear mechanism. This study aimed to explore the protective effect of MRP supplementation against NAFLD through regulation of gut microbiota and metabolites. The C57BL/6 mice animals were randomly allocated into the normal diet (NC), HFHS diet-induced NAFLD model, and MRP intervention group fed with HFHS diet. Serum lipid profiles and liver function parameters were measured. Liver and colon histopathology analysis was conducted to determine the injury in the liver and colon. 16S rRNA gene sequencing was employed to analyze gut microbial composition from fecal samples. Untargeted metabonomics was performed to analyze changes in metabolites in serum and fecal samples. MRP supplementation significantly improved the HFHS-induced alterations in body weight, lipid profiles, and liver function (p < .01). MRP supplementation decreased the abundance of Akkermansia, Candidatus saccharimonas, Dubosiella, and Oscillibacter, while increasing Lactobacillus, Lachnospiraceae NK4A136 group, and Rikenella in mice fed the HFHS diet. Furthermore, MRP supplementation improved the serum and fecal metabolic profiles induced by the HFHS diet, primarily involving the arachidonic acid metabolism, unsaturated fatty acid biosynthesis, and adipocyte lipolysis pathways. Liver function and lipid profiles were closely associated with the abundance of Lactobacillus, Streptococcus, Oscillibacter, Akkemansia, and Desulfovibrio (p < .01). These findings revealed that MRP supplementation may help restore gut microbiota composition and balance its metabolites, thereby improving NAFLD. This study presents a novel outlook on the potential benefits of MRP supplementation in ameliorating NAFLD and supports the application of MRP as a new functional food.

Insight into selenium biofortification and the selenite metabolic mechanism of Monascus ruber M7

Monascus species are functional fermentation fungi with great potential for selenium (Se) supplementation. This study investigated the effects of Se bio-fortification on the growth, morphology, and biosynthesis of Monascus ruber M7. The results demonstrated a significant increase in the yield of orange and red Monascus pigments (MPs) in red yeast rice (RYR) by 38.52% and 36.57%, respectively, under 20 μg/mL of selenite pressure. Meanwhile, the production of citrinin (CIT), a mycotoxin, decreased from 244.47 μg/g to 175.01 μg/g. Transcriptome analysis revealed significant upregulation of twelve genes involved in MPs biosynthesis, specifically MpigE, MpigF, and MpigN, and downregulation of four genes (mrr3, mrr4, mrr7, and mrr8) associated with CIT biosynthesis. Additionally, three genes encoding cysteine synthase cysK (Log2FC = 1.6), methionine synthase metH (Log2FC = 2.2), and methionyl-tRNA synthetase metG (Log2FC = 1.8) in selenocompound metabolism showed significantly upregulated. These findings provide insights into Se biotransformation and metabolism in filamentous fungi.

The effects of environmental factors on the synthesis of water-soluble Monascus red pigments via submerged fermentation: a review.

Monascus pigments (MPs) have been used as natural food pigments for many years. There is a high demand for Monascus red pigments (MRPs) to enhance color and for antibacterial and cancer prevention therapies in food and medicine. Most MRPs are not water soluble, and the yield of water-soluble MRPs is naturally low. On the other hand, water-soluble MRP is more cost effective for application in industrial mass production. Therefore, it is important to improve the yield of water-soluble MRPs. Environmental factors have a significant influence on the synthesis of water-soluble MRPs, which is crucial for the development of industrial production of water-soluble MRPs. This review introduces the biosynthetic pathways of water-soluble MRPs and summarizes the effects of environmental factors on the yield of water-soluble MRPs. Acetyl coenzyme A (acetyl-CoA) is a precursor for MPs synthesis. Carbon and nitrogen sources and the carbon/nitrogen ratio can impact MP production by regulating the metabolic pathway of acetyl-CoA. Optimization of fermentation conditions to change the morphology of Monascus can stimulate the synthesis of MPs. The appropriate choice of nitrogen sources and pH values can promote the synthesis of MRPs from MPs. Additives such as metal ions and non-ionic surfactants can affect the fluidity of Monascus cell membrane and promote the transformation of MRPs into water-soluble MRPs. This review will lay the foundation for the industrial production of water-soluble MRPs. © 2024 Society of Chemical Industry.

Precursor-directed production of water-soluble red Monascus pigments with high thermal stability via azaphilic addition reaction-based semi-synthesis

Red Monascus pigments (MPs) are a large group of polyketides from the fungus Monascus which have been widely used as food colorants. In this study, a variety of red MPs congeners were prepared to explore promising water-soluble candidates for application in liquid food formulations. The results showed that by combining the two-stage, low-pH fermentation strategy with a downstream purification step of fractional crystallization, precursors of red MPs, namely monascorubrin and rubropunctatin, were obtained with a purity of 91.9%. Then, via the azaphilic addition reaction, 18 types of red MPs congeners carrying different amino acid moieties (MPs-aa) were semi-synthesized. Compared to rubropunctamine and monascorubramine, the water solubility, pH and thermal stability of MPs-aa were improved greatly. MPs-His, MPs-Phe, MPs-Tyr and MPs-Trp were identified to be the most resistant to pasteurization. These findings provide water-soluble red MPs candidates with high thermal stability and an attractive approach for their large scale production.

Terminal carboxylation of branched carbon chain contributing to acidic stability of azaphilone pigments from a new isolate of Talaromyces amestolkiae.

Red Monascus pigments, a series of natural azaphilone alkaloids, have been utilized in China as a traditional food colorant for over 1000years. However, instability under an acidic condition is its drawback. A new strain of Talaromyces amestolkiae was isolated in the present work, which produced the azaphilone talaromycorubrin and the corresponding azaphilone alkaloid (N-MSG-talaromycorubramine) exhibiting good stability even at pH below 3. The azaphilone alkaloid with acidic stability, an alternative of Chinese traditional red Monascus pigments, is potential for application as natural food colorant in acidic foods. The acidic stability of azaphilone alkaloid also benefits for direct fermentation of N-MSG-talaromycorubramine under a low pH condition. More importantly, correlation relationship between the terminal carboxylation of branched carbon chain of azaphilone and the stability of azaphilone alkaloids under an acidic condition is set up for the first time, which makes designing other acidic stable azaphilone alkaloids via genetic engineering become possible.

Preparation, Multispectroscopic Characterization, and Stability Analysis of Monascus Red Pigments-Whey Protein Isolate Complex.

Monascus red pigments (MRPs) are mainly used as natural food colorants; however, their application is limited due to their poor stability. To expand their areas of application, we investigated the binding constants and capacity of MRPs to whey protein isolate (WPI) and whey protein hydrolysate (WPH) and calculated the surface hydrophobicities of WPI and WPH. MRPs were combined with WPI and WPH at a hydrolysis degree (DH) of 0.5% to form the complexes (DH = 0.0%) and (DH = 0.5%), respectively. Subsequently, the structural characteristics of complex (DH = 0.5%) and WPI were characterized and the color retention rates of both complexes and MRPs were investigated under different pretreatment conditions. The results showed that the maximum binding constant of WPI with MRPs was 0.670 ± 0.06 U-1 and the maximum binding capacity was 180 U/g. Furthermore, the thermal degradation of complex (DH = 0.0%), complex (DH = 0.5%), and MRPs in a water bath at 50-100 °C followed a first-order kinetic model. Thus, the interaction of WPI with MRPs could alter the protein conformation of WPI and effectively protect the stability of MRPs.

Monascus Red Pigment Liposomes: Microstructural Characteristics, Stability, and Anticancer Activity.

Monascus red pigments (MRPs), which are a kind of natural colorant produced by Monascus spp., are widely used in the food and health supplements industry but are not very stable during processing and storage. Thus, MRPs were embedded into liposome membranes using a thin-film ultrasonic method to improve stability in this study. Monascus red pigments liposomes (MRPL) exhibited spherical unilamellar vesicles (UV) with particle size, polydispersity indexes (PDI), and zeta potential of 20-200 nm, 0.362 ± 0.023, and -42.37 ± 0.21 mV, respectively. pH, thermal, light, metal ion, storage, and in vitro simulated gastrointestinal digestion stability revealed that, compared with free MRPs, liposomes embedding significantly enhanced the stability of MRPs when exposed to adverse environmental conditions. Furthermore, anticancer assay suggested that MRPL exhibited a stronger inhibitory effect on MKN-28 cells by damaging the integrity of cells, with the IC50 value at 0.57 mg/mL. Overall, MRPLs possess stronger stability in external environment and in vitro simulated digestion with greater anticancer activity, indicating that MRPLs have the potential for promising application in the functional foods and pharmaceutical industries.

Microencapsulation of Monascus red pigments by emulsification/internal gelation with freeze/spray-drying: Process optimization, morphological characteristics, and stability

Monascus red pigments (MRPs), natural food colorants from microorganisms, are widely used in the food industry but are not very stable during storage and processing. Therefore, this study aims to microencapsulate MRPs using an emulsification/internal gelation technique to improve the stability. The highest encapsulation efficiency (EE) of 70.32% ± 0.28% was achieved when the percentage of NaCl in phosphate buffered saline (PBS) was 0.88%, the mass ratio of CaCO3 to NaAlg was 3.13, and the molar ratio of calcium to acid was 4.15. Spray-dried MRPs microcapsules had a smaller average diameter (D [4,3] ≈ 3 μm) with a smooth surface and spherical shape. Moreover, the stability assays revealed that spray-dried MRPs microcapsules processed higher retention rate, especially under the treatments of heating, light and in vitro simulated gastrointestinal digestion. The spray-dried MRPs microcapsules exhibited the lowest degradation constant and longest half-life compared with free MRPs and freeze-dried MRPs microcapsules. These results demonstrated that combining emulsification/internal gelation with spray-drying is feasible for enhancing the stability of MRPs to broad its further application in the food industry.

Solid-state fermentation for the production of Monascus pigments from soybean meals

The aim of the present study was to investigate feasibility of soybean meal powder as a nitrogen substrate for the production of pigments by Monascus purpureus ATCC 16362 in solid-state fermentation using soybean meals as nitrogen sources to replace yeast extracts. Central composite design was used to assess the optimum level of soybean meal replacement (0–100%), ZnSO4.7H2O concentration (0–0.02 g/L) and thermal stress time of spore suspension at 70°C (50–90 s). Red Monascus pigment and biomass productions were assessed as dependent responses. A pigment yield of 7.06 AU/g dry fermented substrate was achieved using soybean meal powder with optimized process parameters such as a 50% initial moisture content and incubation at 30 °C for 21 days. Addition of Triton X-100 to solid-state fermentation media significantly increased red Monascus pigments (p < 0.05). Stability of the red Monascus pigments was assessed under storage time (90 days). At high temperatures of storage, degradation constant of the red pigments increased in the presence of light during the shelf-life. This study recommends use of Monascus purpureus for the efficient production of red pigments using low-cost substrates in solid-state fermentation by food industries.

Biosynthesis and polyketide oxidation of Monascus red pigments in an integrated fermentation system with microparticles and surfactants

Monascus red pigments are widely used in the food industry, mainly as intracellular red pigments. The low yields of extracellular red pigments (ERPs) make them unsuitable for large-scale industrial production. Herein, a novel integrated fermentation system (IFS) consisting of sodium starch octenyl succinate and Triton X-100 was explored for increasing yield, resulting in an ERP yield of 126.7 U/mL, 82.6% higher production than controls (69.4 U/mL). Major ERP components in control fermentations were monascopyridine A and monascopyridine B, but dehydro derivatives, rubropunctamine and monascorubramine, predominated in the test fermentations, presumably due to polyketide oxidation induced by Triton X-100. Improvement of hyphal morphology, membrane permeability, respiratory activity, and gene expression for red pigment biosynthesis is likely to be critical to increase yield and change the compositions. This study provides an effective strategy to accelerate the biosynthesis and secretion of Monascus pigments.

Combined analysis of inorganic elements and sugars for the identification of red pepper powder containing undeclared ingredients.

This study sought to identify adulterated red pepper powder (RPP) containing undeclared ingredients, such as salts, glucose, Monascus red pigments, and other plant ingredients (e.g., wheat flour, garlic powder, onion powder) using element and sugar analysis methods. Analytical data were obtained for 66 samples of authentic RPP and 12 samples of adulterated RPP. The variables selected to identify the authenticity of RPP include Na, Cl, K, maltohexaose, and maltoheptaose, which partly or totally originate from RPP, salts, or Monascus red pigments. All the 12 samples of commercial seasoned RPP used as models of adulterated RPP and all the 20 blind samples containing ≥10% (w/w) of commercial seasoned RPP were correctly identified by applying the range of the five variables found for the authentic RPP samples. Our findings suggest that combined analyses of the above five constituents could be used to identify adulterated RPP containing undeclared ingredients. PRACTICAL APPLICATION: Adulteration of high-priced spice products, including red pepper powder (RPP), has increasingly become a public concern worldwide as it endangers consumer health and represents economic fraud. This study provides analytical methods that can accurately determine the authenticity of RPP. They would become effective means for protecting producers and suppliers against unfair competition and consumers against health threats.

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.

Submerged culture of Penicillium sclerotiorum for production of rotiorin alkaloids by using biosynthetic and chemical catalytic cascade reactions

Red Monascus pigments, azaphilone alkaloids from Monascus sp., have been utilized as natural food colorant more than 1000 years in China. Other azaphilones are being developed as alternative sources of natural pigments. Rotiorin or rotiorin alkaloids are always only achieved by solid-state fermentation or liquid culture without stirring due to carbon/nitrogen metabolism affecting pH of the culture medium and the pH environment further affecting the rotiorin stability/reactivity. In the present work, based on the biocompatible reaction of rotiorin with primary amines using phosphate as catalyst, biosynthesis of rotiorin and chemical conversion of rotiorin into its alkaloids were designed as cascade reactions where rotiorin was prevented from degradation and rotiorin alkaloids were accumulated directly by submerged culture of P. sclerotiorum. This is a facile strategy for direct production of rotiorin alkaloids, which provides the precondition for exploiting rotiorin alkaloids as an alternative source of natural colorant.

Highly Efficient Production of Tailored Monascus Pigments by Using a Biocompatible Chemical Reaction Interfacing with Microbial Metabolism

A biocompatible reaction interfacing with microbial metabolism is an efficient approach for production of desired chemicals. Monascus pigments are a traditional natural food colorant from a microbial source. The major color components are red Monascus pigments (RMPs) with various primary amine resides as well as some minor components of yellow ones (YMPs) and orange ones (OMPs). Herein, we elucidate the pathway of OMP degradation involving nucleophilic attack of water on OMPs for the first time. On the basis of OMP degradation and the conversion of OMPs into RMPs sharing a similar reaction mechanism, i.e., nucleophilic attack of water or primary amine on the activated OMPs, we design cascade reactions including biosynthesis of OMPs and biocompatible chemical conversion of OMPs into the stable RMPs with a single glycine residue using a flexible combining chemocatalyst (phosphate ion) and micellar catalyst (Triton X-100). The cascade reactions ensure that OMPs maintain a very low concentration during microbial fermentation by rapid chemical conversion of OMPs into the stable RMPs. In this way, the metabolite profile of microbial fermentation is tailored to RMPs with a single glycine residue as the major color components while free of OMPs and YMPs. Meanwhile, the possible OMP degradation as well as feedback inhibition is eliminated, and a high concentration of RMPs 3.2 mM (1.4 g/L) is also achieved. Designing cascade reactions also has potential for the efficient microbial fermentation of other azaphilone alkaloids.

Monascorubin production by Penicillium minioluteum ED24 in a solid-state fermentation using sesame seed cake as substrate

Monascorubin is one of the red Monascus pigments produced by molds. It is a kind of alcohol-soluble pigment and has been widely used in food and medicine. Natural red pigments have been gaining numerous interests in the food industry as the synthetic red pigments such as azorubine were banned. In this study, the potential use of sesame seed cake as a low-cost substrate to produce monascorubin in a solid-state fermentation by Penicillium minioluteum ED24 was examined. Response surface methodology (RSM) was employed to study the effect of moisture content, dipotassium hydrophosphate (K2HPO4) concentration, concentration of trace ion solution, incubation temperature and inoculum size on the monascorubin production and P. minioluteum ED24 growth. The experiments were designed using central composite design (CCD). The results depicted that 20 g of sesame seed cake produced highest monascorubin at 46.17 µg/ml. While the optimum conditions for monascorubin production obtained from surface analysis are moisture content 65.45%, K2HPO4 concentration 10.43% v/w, concentration of trace ion solution 11.17% v/w, incubation temperature 29.13 °C and inoculum size 9.92% v/w. Meanwhile, the addition of 1% w/w of soluble starch and 1% w/w of mycological peptone enhanced the monascorubin production and fungal growth by 56.56% and 11.53% respectively. Finally, the potential use of sesame seed cake as a low-cost fermentation medium had been presented. This study also provides important insights on the production of monascus pigment by using non-Monascus fungi and lays a foundation for future utilizations of P. minioluteum ED24 for pigment production at industrial scale.

Optimization of production of Monascus ruber pigments on broth medium and their application in flavored yogurts

The aim of the present study was to optimize production conditions of yellow, orange and red pigments from the fungus Monascus ruber Went AUMC 5705 culture through submerged fermentation (SMF), then separation and application of the produced pigments individually in flavored yogurts. Maximum pigments productivity was achieved at incubation temperature of 30℃, inoculums size by 4% (V/V) with (36×104 spores /ml of inoculum), pH of 6.5 and incubation period of 11 days. The favorable carbon and nitrogen sources were Dextrin and monosodium glutamate at 4% (W/V) and 1.5% (W/V) concentrations respect. The present study has also revealed that the yogurt preparations supplemented with these pigments received highly acceptability. To the best of our knowledge, this is the first report on application of yellow, orange and red Monascus pigments individually in flavored yogurts. It is fervently hoped that in the future, these fungal pigments could receive greater attention in the field of food industries.

Monascorubrin and rubropunctatin: Preparation and reaction characteristics with amines

Monascorubrin and rubropunctatin are the two classical orange Monascus pigments, and serve as indispensable precursors in the formation of food colorants Monascus red pigments by amination with primary amines. However, this chemical transformation has been less investigated in vitro due to the hard preparation and preservation of monascorubrin and rubropunctatin. In this study, a way for the preparation of high-purity monascorubrin and rubropunctatin was developed. The in vitro reaction characteristics with various amino acids in different pH environments indicated that an alkaline environment could greatly promote the amination, while the amination was obviously inhibited in an acidic environment. The kinetic study of monascorubrin and rubropunctatin with ammonia suggested that their amination occurred via a second-order reaction. This study not only provides a better understanding of the chemical properties of monascorubrin and rubropunctatin, but also has significance in guiding the industrial applications of Monascus pigments.

Adulteration of beetroot red and paprika extract based food colorant with Monascus red pigments and their detection by HPLC-QTof MS analyses

An intense red food colorant found as a wholesale good for the European market and claimed as a mixture of beetroot red and paprika extract was investigated. The vibrant color intensity could not be readily explained because the measured amounts of dyes typical for beetroot red and paprika extract were too low. These facts led to the question of the source of the intense color, which is the focus of the present work. Efforts were made in the development of high performance liquid chromatography (HPLC) hyphenated with high resolution quadrupole/time-of-flight tandem mass spectrometry (QTof MS). Additional red color compounds could be detected and were identified as Monascus red pigments. N-leucyl-rubropunctamine and its isomer N-isoleucyl-rubropunctamine were found as the main compounds of Monascus red pigments in the food colorant. Starting from rubropunctatin, these compounds were synthesized in the lab. Chromatography and mass spectrometry confirmed unambiguously their presence in the colorant. Furthermore, an authentic sample of Monascus red pigments utilized as colorant in South East Asian countries showed excellent chromatographic and mass spectrometric agreement and confirmed the adulteration of the beetroot red and paprika extract based food colorant.