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Hypotriglyceridemic effect of Anka (a fermented rice product of monascus sp.) in rats.

Experimental rats with hypertriglyceridemia were prepared by feeding a high-fructose diet. Dried Anka powder (2%), a rice product fermented with Monascus sp., was mixed with basic high-fructose (30%) or basal-diet feed. Serum and liver lipids were measured after 6 months. The concentrations of serum triglycerides, total cholesterol, VLDL-C, and LDL-C had significantly decreased, whereas that of HDL-C had slightly increased in 30% fructose-Anka-fed rats as compared with the 30% fructose-fed rats, but hepatic lipase activity had increased in the Anka-fed groups. The ratio of lipoprotein lipase/hepatic lipase was not significantly different between 30% fructose-Anka-fed rats and 30% fructose-fed rats. The dietary intake and weight of these two groups were approximately the same. Similar results were obtained in noninduced hypertriglyceridemic rats. The concentrations of triglycerides and cholesterol did not significantly differ in the liver. Interestingly, Anka can suppress serum triglycerides in rats with induced hypertriglyceridemia. The antioxidant enzyme SOD activity was also measured in serum, and no significant change was observed. On the basis of these findings, we suggest that Anka may be used to suppress hypertriglyceridemia and hyperlipidemia in rats and possibly in man.

Animals↗

Growth, pigment production and protease activity of Monascus purpureus as affected by salt, sodium nitrite, polyphosphate and various sugars.

The effect of different levels of salt, sodium nitrite, polyphosphate and various sugars on growth, pigment production, protease activity and culture pH caused by Monascus purpureus was studied in broth medium and ground meat. The addition of sodium chloride (> 50.0 g l(-1)) and polyphosphate (> 3.0g l(-1)) to broth medium decreased mycelial growth, pigment production and protease activity of M. purpureus, whereas low concentrations of sodium nitrite (< 0.2 g l(-1)) promoted mycelial growth and pigment production. When the basal medium and ground meat contained salt, 150.0 g l(-1), the mould growth was stopped. The medium with fructose as carbon source proved to be the most suitable for mycelium growth and pigment production, with maltose and glucose being the second most productive. When sucrose and lactose were used as carbon sources, mycelium growth and pigment production were inhibited but the protease activity increased significantly. The mould showed more tolerance to salt and polyphosphate in ground meat than in broth medium and used sucrose as a carbon source as well as glucose for growth and pigment production in the meat mixture.

Animals↗

Metabolites of Monascus ruber in silages.

A total of 233 silages were examined and found that Monascus ruber was present in 43 samples with counts between 1 x 10(3) and 9 x 10(6) colony-forming units (CFU)/g (mean: 2 x 10(5) CFU/g). Monacolin K(L) and the hydroxy acid monacolin K(A) were detected by liquid chromatography-mass spectrometry in 45 and 50 of 233 samples at levels ranging from 25-15 600 and 28-65 400 microg/kg, respectively. Citrinin was found with high-performance liquid chromatography-fluorescence detection (FLD) in 14 (6%) samples, the concentrations varied between 2.4 and 64.2 microg/kg. The concentrations of citrinin were low and toxic effects are not anticipated. Monacolin K(A) and monacolin K(L) occur frequently and in considerable amounts in silages. These metabolites are believed to influence the metabolic activity of rumen anaerobic fungi resulting in a poorer digestion of crude fibre.

Animals↗

Biological activities of oligoketide pigments of Monascus purpureus.

Rubropunctatin (1), monascorubrin (2), monascin (3) and ankaflavin (4) were purified from the mycelium of Monascus purpureus by flash chromatography on silica gel or reversed phase. Their embryotoxicity towards chicken embryos decreased in the order 2 > 1 > 3 > 4. The lower homologues 1 and 3 exhibited teratogenic effects on these organisms. Significant antibiotic activities against Bacillus subtilis and Candida pseudotropicalis were found with compounds 1 and 2. Immunosuppressive activity on mouse T-splenocytes was most pronounced with compounds 3 and 4. None of the compounds showed significant cytotoxic activity towards rat hepatocytes in vitro. Incubation of resting cells of M. purpureus with glycine afforded the dark-red compounds 5 and 6 where the pyran moiety of 1 and 2 changed into the N-substituted dihydropyridine moiety by replacement of the O-atom by the amino group of glycine. Compounds 5 and 6 were less biologically active than the major pigments 1-4.

Abnormalities, Drug-Induced↗

Rhabdomyolysis due to red yeast rice (Monascus purpureus) in a renal transplant recipient.

Rhabdomyolysis is a known complication of hepatic 3-methylglutaryl coenzyme A reductase (HMG-CoA) inhibitor (statin) therapy for posttransplant hyperlipidemia, and thus monitoring for this effect is indicated. We report a case of an herbal preparation-induced rhabdomyolysis in a stable renal-transplant recipient, attributed to the presence of red yeast rice (Monascus purpureus) within the mixture. The condition resolved when consumption of the product ceased. Rice fermented with red yeast contains several types of mevinic acids, including monacolin K, which is identical to lovastatin. We postulate that the interaction of cyclosporine and these compounds through the cytochrome P450 system resulted in the adverse effect seen in this patient. Transplant recipients must be cautioned against using herbal preparations to lower their lipid levels to prevent such complications from occurring.

Adult↗

Pigmentation and Antibacterial Activity of Fast Neutron- and X-Ray-induced Strains of Monascus purpureus Went.

Seven new strains of Monascus purpureus Went were induced by neutron and x-ray irradiation. The quantity and quality of pigments produced by these strains differed. Strains N4S and N11S produced twice as much pigment as normal, while another strain, N14S, was albino. An unknown orange pigment was found in young colonies of the N11S strain. This orange pigment reacted with alcohols and malt extract medium to form red pigments. Strains N4S, N11S, X2P, and wild type inhibited the growth of certain bacteria, especially the Bacillus species. Strain N11S had more antibacterial activity than wild type. A major active compound was isolated with an ultraviolet absorption spectrum that was related to those of the red pigments found in this fungus. The active compound(s) was named monascidin.

Journal Article↗

Purification and characterization of an acid protease from Monascus kaoliang.

An acid protease from Monascus kaoliang was purified by consecutive applications of fractional acetone precipitation, batchwise CM-cellulose method and DEAE-cellulose column chromatography. The preparation was homogeneous on disc polyacrylamide gel electrophoresis at pH 4.5 and 7.5. The yield was about 30% with overall increase in specific activity of about 6-fold. The molecular weight as determined by SDS gel electrophoresis was about 34,000. The enzyme was a glycoprotease as indicated by specific carbohydrate staining on gels. It possessed the nature of an acid protease with a pH optimum at 3.0 toward heat-denatured casein and was stable over the range of pH 3.0 to 6.0. Reducing agents and thiol poisons had no effect on this enzyme, suggesting that free sulfhydryl groups were not required for enzyme activity. Diisopropyl fluorophosphate did not inactivate this protease, indicating the probable absence of serine residue in the active site. The enzyme was inactivated by reaction with the carboxy-group specific reagent, 1,2-epoxy-3-(p-nitrophenoxy) propane (EPNP). Pepstatin, a specific inhibitor for pepsin, was shown to inhibit this enzyme strongly. However, biacetyl (2,3-butadione) had little effect on this protease, although it inactivated pepsin to an 85% activity loss. Also, p-bromophenacyl bromide, another specific inhibitor of pepsin, failed to inactivate this acid protease.

Amino Acids↗

Production, Purification, and Characterization of alpha-Galactosidase from Monascus pilosus.

A Monascus pilosus strain was selected for production of intracellular alpha-galactosidase. Optimum conditions for mycelial growth and enzyme induction were determined. Galactose was one of the best enzyme inducers. The enzyme was purified by ammonium sulfate precipitation, gel filtration, and ion exchange chromatography and was demonstrated to be homogeneous by slab gel electrophoresis. The molecular weight of this enzyme, estimated by gel filtration, was about 150,000. The optimum conditions for the enzyme reaction was pH 4.5 to 5.0 at 55 degrees C. The purified enzyme was stable at 55 degrees C or below and in buffer at pH 3 to 8. The activity was inhibited by mercury, silver, and copper ions. The kinetics of this enzyme, with p-nitrophenyl-alpha-d-galactoside as substrate, was determined: K(m) was about 0.8 mM, and V(max) was 39 mumol/min per mg of protein. Enzymatic hydrolysis of melibiose, raffinose, and stachyose was analyzed by thin-layer chromatography.

Journal Article↗

Production and Identification of N-Glucosylrubropunctamine and N-Glucosylmonascorubramine from Monascus ruber and Occurrence of Electron Donor-Acceptor Complexes in These Red Pigments.

The filamentous fungus Monascus ruber produces water-soluble red pigments in a submerged culture when grown in a chemically defined medium containing glucose as a carbon source and monosodium glutamate as a nitrogen source. Two new molecules with polyketide structures, N-glucosylrubropunctamine and N-glucosylmonascorubramine, constituting under some conditions 10% of the total extracellular coloring matter when glucose as a carbon source was in excess (20 g/liter), were isolated and structurally characterized by high-pressure liquid chromatography, Dionex methods, (sup1)H and (sup13)C nuclear magnetic resonance spectroscopy, and mass spectrometry. The occurrence of the electron donor-acceptor complex effect was demonstrated by UV spectroscopy, polarography, and thin-layer voltammetry. The use of n-butanol as an extraction solvent stabilized the pigments against the effects of daylight for several months, promoting the stability of this type of complex.

Journal Article↗

Biosynthetic pathway of citrinin in the filamentous fungus monascus ruber as revealed by 13C nuclear magnetic resonance

Carbon isotope distribution of [13C]citrinin from Monascus ruber incubated with [13C]acetate revealed that the biosynthesis of the toxin originated from a tetraketide, instead of a pentaketide as has been shown for Penicillium and Aspergillus species. The production of polyketide red pigments and citrinin by M. ruber may therefore be regulated at the level of the tetraketide branch point.

Journal Article↗

Medium-chain fatty acids affect citrinin production in the filamentous fungus Monascus ruber.

During submerged culture in the presence of glucose and glutamate, the filamentous fungus Monascus ruber produces water-soluble red pigments together with citrinin, a mycotoxin with nephrotoxic and hepatoxic effects on animals. Analysis of the (13)C-pigment molecules from mycelia cultivated with [1-(13)C]-, [2-(13)C]-, or [1, 2-(13)C]acetate by (13)C nuclear magnetic resonance indicated that the biosynthesis of the red pigments used both the polyketide pathway, to generate the chromophore structure, and the fatty acid synthesis pathway, to produce a medium-chain fatty acid (octanoic acid) which was then bound to the chromophore by a trans-esterification reaction. Hence, to enhance pigment production, we tried to short-circuit the de novo synthesis of medium-chain fatty acids by adding them to the culture broth. Of fatty acids with carbon chains ranging from 6 to 18 carbon atoms, only octanoic acid showed a 30 to 50% stimulation of red pigment production, by a mechanism which, in contrast to expectation, did not involve its direct trans-esterification on the chromophore backbone. However, the medium- and long-chain fatty acids tested were readily assimilated by the fungus, and in the case of fatty acids ranging from 8 to 12 carbon atoms, 30 to 40% of their initial amount transiently accumulated in the growth medium in the form of the corresponding methylketone 1 carbon unit shorter. Very interestingly, these fatty acids or their corresponding methylketones caused a strong reduction in, or even a complete inhibition of, citrinin production by M. ruber when they were added to the medium. Several data indicated that this effect could be due to the degradation of the newly synthesized citrinin (or an intermediate in the citrinin pathway) by hydrogen peroxide resulting from peroxisome proliferation induced by medium-chain fatty acids or methylketones.

Acetates↗

The effect of different nitrogen sources on pigment production and sporulation of Monascus species in submerged, shaken culture.

Thirty-nine strains from the genus Monascus were cultivated aerobically to study the relation between nitrogen nutrition and sporulation and pigment production. The effects of yeast extract, nitrate, ammonium, and ammonium nitrate have been compared. During cultivation the pHs of the different media are not the same, resulting in the formation of different coloured pigments. When the source of nitrogen is yeast extract or nitrate the pH is around 6.5 and red pigments are formed, whereas with ammonium or ammonium nitrate the pH is around 2.5 and the pigments are orange. It is proposed that only the orange pigments, monascorubrin and rubropunctatin, are produced biosynthetically and that the other pigments are formed from these by chemical transformations depending on the cultural conditions. The presence of organic nitrogen is optimal for growth and unfavourable for pigment production. Reduced growth and best pigment formation occurs with the three other nitrogen sources. Nitrate stimulates conidiation and sexual reproduction, while ammonium is inhibitory. Pigment production is better when conidiation is reduced. A mechanism is proposed for the control of sporulation and pigment production.

Ascomycota↗

The effect of changes in pH on phosphate and potassium uptake by Monascus rubiginosus ATCC 16367 in submerged shaken culture.

Monascus rubiginosus ATCC 16367 was cultivated aerobically in media containing ammonium and nitrate as nitrogen source. The pH of the medium was adjusted at different times, the pH of the nitrate medium being lowered to the pH of the ammonium medium and the pH of the ammonium medium raised to that of the nitrate. More phosphate was taken up on the nitrate medium, but potassium uptake did not start until 24h. On the ammonium medium, both were taken up in parallel from the beginning, but the amount of phosphate taken up never reached the same level as on nitrate medium. When the pH was adjusted, the uptakes changed, especially on the ammonium medium where a great increase in phosphate uptake was observed. More conidia were formed on the nitrate medium and more pigment on the ammonium medium. When the pH of either media was adjusted, the development of conidia and pigment production changed to that of the other control medium where the pH evolved normally in the direction of the change, regardless of the source of nitrogen. The reasons for the development of conidia on nitrate medium or where the pH is high, and the production of pigment on ammonium medium or at low pH is discussed.

Ascomycota↗

A new series of coumarin derivatives having monoamine oxidase inhibitory activity from Monascus anka.

Monankarins A-F (1-6), a new series of pigments having conjugated pyrano-coumarin skeleton have been isolated from Monascus anka. Their structures have been elucidated by spectroscopic means and chemical modification. Monankarins A-D exhibited monoamine oxidase (MAO) inhibitory activity, while the activity was not observed in monankarins E and F or some other simple coumarins. Monankarin C (3) showed stronger inhibition of MAO-B than MAO-A in mice brain. However, the specificity was not found in mice liver MAO.

Animals↗

Protective effect of the mold Monascus anka against acetaminophen-induced liver toxicity in rats.

Antioxidant and hepatoprotective actions of the mold Monascus anka (also called Beni-Koji in Japan) against acetaminophen (AAP)-induced liver toxicity were investigated. Serum aspartate aminotransferase and glutathione S-transferase (GST) activities increased by AAP (180 mg/kg, i.p.) treatment were depressed when the Beni-Koji preparation (4 ml/kg, i.p.) was given 15 and 1 hr before AAP administration. The decrease in liver cytosolic GST activity by AAP, reflecting the release of the enzyme into serum, was also blocked by the mold. Cytochrome P450 activity was inhibited by the Beni-Koji preparation. These results suggest that M. anka prevents AAP-induced liver toxicity by both antioxidant action and the inhibition of AAP metabolism.

Acetaminophen↗

Antimicrobial activity of Monascus pilosus IFO 4520 against contaminant of Koji.

Antimicrobial activity of Monascus pilosus IFO 4520 was examined to prevent contamination during beni-koji making in the open air. The antibacterial effect of the beni-koji prepared with this strain occured with 30 mg/ml of beni-koji extract in combination with 0.5% lactic acid against two contaminants of koji, Micrococcus varians and Bacillus subtilis. There were two compounds, antibacterial and antiyeast substances, in the beni-koji extract. These results suggest a possibility of inhibiting the growths of contaminants during beni-koji making using beni-koji extract and lactic acid.

Anti-Bacterial Agents↗

Effect of yeast extract on growth kinetics of Monascus purpureus.

Growth kinetics and red pigment production of Monascus purpureus CCT 3802 was studied. A reproducible inoculum with extremely dispersed hyphae for bioreactor runs was obtained through a two-step cultivation in a shaker. First, the spores were cultivated in a complex medium rendering a suspension of vegetative cells. In the second step these cells were grown in a semisynthetic medium. Two types of media were employed in the bioreactor runs: a semisynthetic (glucose, salts, and yeast extract), and a synthetic, without yeast extract. The inclusion of yeast extract, caused an increase in cell yield on glucose (Yx/s) as high as 40%. Also, yeast extract probably yielded a higher proportion of red pigment associated with the cell, relative to the synthetic medium. On the other hand, cells grown on the synthetic medium were slightly higher producers of red soluble pigments.

Ascomycota↗

Biosynthesis of monacolins: conversion of monacolin L to monacolin J by a monooxygenase of Monascus ruber.

The microbial metabolites monacolins J and L are specific inhibitors of 3-hydroxy-3-methylglutaryl CoA reductase, the rate-limiting enzyme in cholesterol synthesis. The producing strain Monascus ruber M 4681 was found to convert exogenously added monacolin L to J. In this hydroxylation reaction 18O2 was incorporated into monacolin L, giving [18O]-monacolin J. The cell-free extracts of M. ruber quantitatively hydroxylated monacolin L to J, and molecular oxygen was required for the hydroxylation. The enzyme was located in the microsomal fraction and specific for NADPH. The enzyme activity was inhibited by metyrapone, carbon monoxide, sulfhydryl reagents and cytochrome c. The results indicate that monacolin L is the precursor of monacolin J, and that a monooxygenase is involved in this reaction.

Ascomycota↗