Rubropunctamine

Precursor-directed production of water-soluble red Monascus pigments with high thermal stability via azaphilic addition reaction-based semi-synthesis.[Pubmed:38144809]

Food Chem X. 2023 Oct 10;20:100940.

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.

Chemical characterization and toxicity evaluation of fungal pigments for potential application in food, phamarceutical and agricultural industries.[Pubmed:37113475]

Saudi J Biol Sci. 2023 May;30(5):103630.

Concerns associated with the use of synthetic colourants backs the demand for natural colourants. Thus, the current study aimed at characterizing crude fungal pigments produced by Penicillium multicolour, P. canescens, Talaromyces verruculosus, Fusarium solani and P. herquie. This included their antioxidant and antimicrobial properties together with acute toxicity evaluation on zebrafish embryos. The identification of pigment compounds was achieved through MS and IR data. The study demonstrated a substantial radical scavenging activity of extracts ranging from 65.49 to 74.46%, close to that of ascorbic acid (89.21%). Penicillium canescens and F. solani exhibited a strong antimicrobial activity against Escherichia coli and Enterococcus aerogenes and Salmonella typhi, Staphylococcus aureus and Bacillus cereus at MIC values ranging from 1.5 to 2.5 mg/mL. However, some levels of toxicity were observed for all extracts at a concentration range of 3-5 mg/mL. Pigment by P. multicolour, T. verruculosus and F. solani were tentatively identified through IR and MS data as sclerotiorin (yellow), Rubropunctamine (red) and bostrycoidin (red). In conclusion, the study demonstrates a market potential of filamentous fungi pigments due to their antioxidant, antimicrobial activities, and prominent colours. Although there are some toxicity concerns, further tests must be done using molecular docking, albino mice and cell linings.

A new endophyte Monascus ruber SRZ112 as an efficient production platform of natural pigments using agro-industrial wastes.[Pubmed:35871189]

Sci Rep. 2022 Jul 23;12(1):12611.

A number of biopigment applications in various industrial sectors are gaining importance due to the growing consumer interest in their natural origin. Thus, this work was conducted to valorize endophytic fungi as an efficient production platform for natural pigments. A promising strain isolated from leaves of Origanum majorana was identified as Monascus ruber SRZ112 produced several types of pigments. The nature of the pigments, mainly Rubropunctamine, monascin, ankaflavin, rubropunctatin, and monascorubrin in the fungal extract was studied by LC/ESI-MS/MS analyses. As a first step towards developing an efficient production of red pigments, the suitability of seven types of agro-industrial waste was evaluated. The highest yield of red pigments was obtained using potato peel moistened with mineral salt broth as a culture medium. To increase yield of red pigments, favourable culture conditions including incubation temperature, incubation period, pH of moistening agent, inoculum concentration, substrate weight and moisture level were evaluated. Additionally, yield of red pigments was intensified after the exposure of M. ruber SRZ112 spores to 1.00 KGy gamma rays. The final yield was improved by a 22.12-fold increase from 23.55 to 3351.87 AU g(-1). The anticancer and antioxidant properties of the pigment's extract from the fungal culture were also studied. The obtained data indicated activity of the extract against human breast cancer cell lines with no significant cytotoxicity against normal cell lines. The extract also showed a free radical scavenging potential. This is the first report, to our knowledge, on the isolation of the endophytic M. ruber SRZ112 strain with the successful production of natural pigments under solid-state fermentation using potato peel as a substrate.

Biosynthesis and polyketide oxidation of Monascus red pigments in an integrated fermentation system with microparticles and surfactants.[Pubmed:35759840]

Food Chem. 2022 Nov 15;394:133545.

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.

Effect of a Monascus sp. Red Yeast Rice Extract on Germination of Bacterial Spores.[Pubmed:34108955]

Front Microbiol. 2021 May 24;12:686100.

The pink-red color of traditional sausages (cured meat) is the result of nitrite addition and the formation of nitrosomyoglobin. However, the pleasant color of processed meat products is a side effect of nitrite addition while the main anticipated goal is to suppress the germination of clostridial spores. The fungus Monascus is known as a producer of oligoketide pigments, which are used in Asian countries, especially in China, for coloring foods, including meat products. Although, different biological activities of Monascus pigments have been tested and confirmed in many studies, their effect on germination of bacterial spores has never been investigated. This study is focused on testing the activity of red yeast rice (RYR) extract, containing monascin, rubropunctatin, Rubropunctamine complexes and monascuspiloin as the main pigments, on germination of Clostridium and Bacillus spores. It was found that addition of nitrite alone, at the permitted concentration, had no effect on spore germination. However, the combined effects of nitrite with NaCl, tested after addition of pickling salt, was efficient in inhibiting the germination of C. beijerinckii spores but had no effect on B. subtilis spores. In contrast, total suppression of C. beijerinckii spore germination was reached after addition of RYR extract to the medium at a concentration of 2% v/v. For B. subtilis, total inhibition of spore germination was observed only after addition of 4% v/v RYR extract to the medium containing 1.3% w/w NaCl.

Ammonium nitrate regulated the color characteristic changes of pigments in Monascus purpureus M9.[Pubmed:33398480]

AMB Express. 2021 Jan 4;11(1):3.

Monascus pigments (MPs) with different color characteristics, produced by submerged fermentation of Monascus purpureus M9, have potential application in food industry. In the present study, the effects and regulatory mechanisms of ammonium nitrate (AN) on the color characteristics of MPs were investigated. The concentration of intracellular pigments was significantly decreased when subjected to AN. The hue and lightness value indicated AN altered the total pigments appearance from original red to orange. The HPLC analysis for six major components of MPs showed that the production of rubropunctatin or monascorubrin, was significantly reduced to the undetectable level, whereas the yields of monascin, ankaflavin, Rubropunctamine and monascorubramine, were apparently increased with AN supplement. To be noted, via real-time quantitative PCR strategy, the expression level of mppG, closely relative to orange pigments biosynthesis, was significantly down-regulated. However, the expression of mppE, involved in yellow pigments pathway, was up-regulated. Moreover, the broth pH value was dropped to 2.5-3.5 in the fermentation process resulted from AN treatment, along with the increased extracellular polysaccharide biosynthesis. Taken together, the change of MPs categories and amounts by AN might be the driving force for the color characteristics variation in M. purpureus M9. The present study provided useful data for producing MPs with different compositions and modified color characteristics.

Effects of mrpigG on Development and Secondary Metabolism of Monascus ruber M7.[Pubmed:32872515]

J Fungi (Basel). 2020 Aug 29;6(3):156.

Monascus pigments (MPs) have been used as food colorants for several centuries in Asian countries and are now used throughout the world via Asian catering. The MP biosynthetic pathway has been well-illustrated, but the functions of a few genes, including mrpigG, in the MP gene cluster are still unclear. In the current study, in order to investigate the function of mrpigG in M. ruber M7, gene deletion (DeltamrpigG), complementation (DeltamrpigG::mrpigG) and overexpression (M7::PtrpC-mrpigG) mutants were successfully obtained. The morphologies and biomasses, as well as the MP and citrinin production, of these mutants were analyzed. The results revealed that the disruption, complementation and overexpression of mrpigG showed no apparent defects in morphology, biomass or citrinin production (except MP production) in DeltamrpigG compared with M. ruber M7. Although the MP profiles of DeltamrpigG and M. ruber M7 were almost the same-with both having four yellow pigments, two orange pigments (OPs) and two red pigments (RPs)-their yields were decreased in DeltamrpigG to a certain extent. Particularly, the content of rubropunctatin (an OP) and its derivative Rubropunctamine (an RP) in DeltamrpigG, both of which have a five-carbon side chain, accounted for 57.7%, and 22.3% of those in M. ruber M7. On the other hand, monascorubrin (an OP) and its derivative monascorubramine (an RP), both of which have a seven-carbon side chain, were increased by 1.15 and 2.55 times, respectively, in DeltamrpigG compared with M. ruber M7. These results suggest that the MrPigG protein may preferentially catalyze the biosynthesis of MPs with a five-carbon side chain.

Transfigured Morphology and Ameliorated Production of Six Monascus Pigments by Acetate Species Supplementation in Monascus ruber M7.[Pubmed:31936171]

Microorganisms. 2020 Jan 7;8(1):81.

Monascus species have been used for the production of many industrially and medically important metabolites, most of which are polyketides produced by the action of polyketide synthases that use acetyl-CoA and malonyl-CoA as precursors, and some of them are derived from acetate. In this study the effects of acetic acid, and two kinds of acetates, sodium acetate and ammonium acetate at different concentrations (0.1%, 0.25% and 0.5%) on the morphologies, biomasses, and six major Monascus pigments (MPs) of M. ruber M7 were investigated when M7 strain was cultured on potato dextrose agar (PDA) at 28 degrees C for 4, 8, 12 days. The results showed that all of the added acetate species significantly affected eight above-mentioned parameters. In regard to morphologies, generally the colonies transformed from a big orange fleecy ones to a small compact reddish ones, or a tightly-packed orange ones without dispersed mycelia with the increase of additives concentration. About the biomass, addition of ammonium acetate at 0.1% increased the biomass of M. ruber M7. With respect to six MPs, all acetate species can enhance pigment production, and ammonium acetate has the most significant impacts. Production of monascin and ankaflavin had the highest increase of 11.7-fold and 14.2-fold in extracellular contents at the 8th day when 0.1% ammonium acetate was supplemented into PDA. Intracellular rubropunctatin and monascorubrin contents gained 9.6 and 6.46-fold at the 8th day, when 0.1% ammonium acetate was added into PDA. And the extracellular contents of Rubropunctamine and monascorubramine were raised by 1865 and 4100-fold at the 4th day when M7 grew on PDA with 0.5% ammonium acetate.

Fluconazole treatment enhances extracellular release of red pigments in the fungus Monascus purpureus.[Pubmed:28333308]

FEMS Microbiol Lett. 2017 Apr 1;364(8).

Traditional methods for the production of food grade pigments from the fungus Monascus spp. mostly rely on submerged fermentation. However, the cell-bound nature and intracellular accumulation of pigments in Monascus spp. is a major hurdle in pigment production by submerged fermentation. The present study focused on the investigation of the effect of the antifungal agent fluconazole on red pigment production from Monascus purpureus (NMCC-PF01). At the optimized concentration of fluconazole (30 mug ml-1), pigment production was found to be enhanced by 88% after 96 h and it remained constant even after further incubation up to 168 h. Ergosterol, a sterol specific to fungi, was also extracted and estimated as a function of fungal growth. The concentration of ergosterol in fluconazole-treated fermentation broth was reduced by 49% as compared to control broth. Thus it could be responsible for facilitating the release of intracellular and cell-bound pigments. Nevertheless, the role of cell transporters in transporting out the red pigments cannot be ignored and deserves further attention. Qualitative analysis of red pigment by thin layer chromatography, UV spectroscopy and mass spectrometric analysis (ESIMS) has confirmed the presence of the well-known pigment Rubropunctamine. In addition, this fermentation process produces citrinin-free pigments. This novel approach will be useful to facilitate increased pigment production by the release of intracellular or cell-bound Monascus pigments.

Monascus secondary metabolites: production and biological activity.[Pubmed:23179468]

J Ind Microbiol Biotechnol. 2013 Feb;40(2):169-81.

The genus Monascus, comprising nine species, can reproduce either vegetatively with filaments and conidia or sexually by the formation of ascospores. The most well-known species of genus Monascus, namely, M. purpureus, M. ruber and M. pilosus, are often used for rice fermentation to produce red yeast rice, a special product used either for food coloring or as a food supplement with positive effects on human health. The colored appearance (red, orange or yellow) of Monascus-fermented substrates is produced by a mixture of oligoketide pigments that are synthesized by a combination of polyketide and fatty acid synthases. The major pigments consist of pairs of yellow (ankaflavin and monascin), orange (rubropunctatin and monascorubrin) and red (Rubropunctamine and monascorubramine) compounds; however, more than 20 other colored products have recently been isolated from fermented rice or culture media. In addition to pigments, a group of monacolin substances and the mycotoxin citrinin can be produced by Monascus. Various non-specific biological activities (antimicrobial, antitumor, immunomodulative and others) of these pigmented compounds are, at least partly, ascribed to their reaction with amino group-containing compounds, i.e. amino acids, proteins or nucleic acids. Monacolins, in the form of beta-hydroxy acids, inhibit hydroxymethylglutaryl-coenzyme A reductase, a key enzyme in cholesterol biosynthesis in animals and humans.

Computerized screening for novel producers of Monascus-like food pigments in Penicillium species.[Pubmed:18841978]

J Agric Food Chem. 2008 Nov 12;56(21):9981-9.

Monascus pigments have been used as natural food colorants in Asia for centuries. They are not authorized for use in the European Union and the United States mainly due to the risk of coproduction of the mycotoxin citrinin by Monascus spp. In the present study, we screened for novel producers of Monascus-like pigments from ascomycetous filamentous fungi belonging to Penicillium subgenus Biverticillium that are not reported to produce citrinin or any other known mycotoxins. The screening was carried out using the X-hitting algorithm as a tool to quickly screen through chromatographic sample data files of 22 different Penicillium extracts with 12 Monascus pigment extracts as controls. The algorithm searched for the most similar UV-vis spectra of the metabolites (cross hits) present in the pigment extracts to those of the selected reference metabolites viz. monascin, rubropunctatin, Rubropunctamine, and citrinin. The cross hits were then manually identified on the basis of their UV-vis and mass spectra. X-hitting was found to be a good tool in the rapid screening of crude pigment extracts. Monascus pigments were discovered in the extracts of two closely related species of Penicillium that were only distantly related to the genus Monascus. Monascorubrin, xanthomonasin A, and threonine derivatives of rubropunctatin were identified in the extract of Penicillium aculeatum IBT 14263, and monascorubrin was identified in the extract of Penicillium pinophilum IBT 13104. None of the tested Penicillium extracts showed the presence of citrinin. Thus, the present study brought out two novel promising sources of yellow, orange, and purple-red Monascus-like food pigments in the species of Penicillia that do not produce citrinin and opened the door to look for several more new promising sources of natural food colorants in the species of Penicillia.

Cytotoxic and antimitotic effects of N-containing Monascus metabolites studied using immortalized human kidney epithelial cells.[Pubmed:16598808]

Mol Nutr Food Res. 2006 Apr;50(4-5):406-12.

Recently the first Monascus metabolites with a pyridine ring were detected, the monascopyridines A and B. They are formally dehydrogenated derivatives of the red rice pigments Rubropunctamine and monascorubramine. Because of their structural similarity, the toxicological effects of these secondary metabolites were studied using immortalized human kidney epithelial cells. The cytotoxicity was determined with the following different endpoint detection methods: metabolic activity, trypan blue exclusion, and electronic cell counting. The compounds led to EC(50) values between 11 and 31 micromol/L but the pigments caused a stronger reduction of the cell viability. Also, the apoptotic potential was examined by measuring caspase 3 activity and detecting apoptotic bodies, but none of the tested compounds induced apoptosis. All four substances caused a rise of the mitotic index to about 9% (100 micromol/L monascopyridine A and B) and 20% (25 micromol/L Rubropunctamine and monascorubramine). The significant decrease of the ratio of cells in the ana- and telophase to cells in the prometa- and metaphase proved a stop of the mitosis at the meta- to anaphase control point. The compounds caused mitotic arrest and the formation of structural damages like c-mitosis through interaction with the mitotic spindle. These effects point to an aneuploidy inducing potential, which is linked to cancer formation.

Azaphilones, furanoisophthalides, and amino acids from the extracts of Monascus pilosus-fermented rice (red-mold rice) and their chemopreventive effects.[Pubmed:15686402]

J Agric Food Chem. 2005 Feb 9;53(3):562-5.

Six azaphilones, monascin (1), ankaflavin (2), rubropunctatin (3), monascorburin (4), Rubropunctamine (5), and monascorburamine (6), two furanoisophthalides, xanthomonasin A (7) and xanthomonasin B (8), and two amino acids, (+)-monascumic acid (9) and (-)-monascumic acid (10), isolated from the extracts of Monascus pilosus-fermented rice (red-mold rice) were evaluated for their inhibitory effects on 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced inflammation in mice, on the induction of Epstein-Barr virus early antigen (EBV-EA) by TPA in Raji cells, and on the activation of (+/-)-(E)-methyl-2[(E)-hydroxy-imino]-5-nitro-6-methoxy-3-hexemide (NOR 1), a nitric oxide (NO) donor. Among the compounds tested, seven compounds (1-6 and 10) on TPA-induced inflammation, and six compounds (1, 3-5, 9, and 10) on EBV-EA activation, exhibited potent inhibitory effects. All of the compounds tested showed moderate inhibitory effects on NOR 1 activation.