Monascus Spp Research Articles

Treatment of metabolic syndrome with ankaflavin, a secondary metabolite isolated from the edible fungus Monascus spp.

Edible fungi of the Monascus species have been used as traditional Chinese medicine in eastern Asia for several centuries. Monascus-fermented products possess a number of functional secondary metabolites, including anti-inflammatory pigments (such as monascin and ankaflavin [AK]), monacolins, and dimerumic acid. These secondary metabolites have anti-inflammatory, anti-oxidative, and anti-tumor activities. We found that AK positively regulates several transcription factors associated with the prevention of metabolic syndrome and other diseases, including peroxisome proliferator-activated receptor (PPAR)-gamma, PPAR-alpha, and nuclear factor (erythroid-derived 2)-like 2 (Nrf2). AK reduced hyperglycemia and enhanced pancreatic function via PPAR-gamma activation and increased lipid metabolism due to PPAR-alpha activation. The compound also exerted antioxidant effects via activation of Nrf2. These results suggest that AK belongs to the class of selective peroxisome proliferator-activated receptor modulators (SPPARMs), which are associated with a good safety profile when used in patients suffering from metabolic syndrome. Together with our studies to determine how AK production can be increased during Monascus fermentation, these data demonstrate the great potential of AK as a nutraceutical or therapeutic agent.

Monacolin K affects lipid metabolism through SIRT1/AMPK pathway in HepG2 cells

Monacolin K is the secondary metabolite isolated from Monascus spp. It is the natural form of lovastatin, which is clinically used to reduce the synthesis of cholesterol by inhibiting 3-hydroxy-3-methylglutaryl coenzyme A reductase. In the present study, monacolin K increased protein expression of SIRT1 and phosphorylation level of AMP-activated protein kinase (AMPK) in HepG2 cells. Through activation of SIRT1/AMPK pathway, monacolin K increased phosphorylation of acetyl CoA carboxylase and caused nuclear translocation of forkhead box O1. The western blotting results showed that monacolin K increased expression of adipose triglyceride lipase but decreased abundances of fatty acid synthase (FAS) and sterol regulatory element-binding protein 1 (SREBP1). Monacolin K also decreased the intracellular accumulation of lipids as demonstrated by Oil Red O staining. In addition, the immunostaining showed that monacolin K prevented the nuclear translocation of SREBP1, indicating the association with down-regulation of FAS. All the demonstrated effects of monacolin K were counteracted by nicotinamide or compound C, the inhibitors of SIRT1 or AMPK. In summary, monacolin K reduces the lipid content through SIRT1/AMPK pathway in HepG2 cells, which promotes catabolism and inhibits anabolism of lipid.

A preliminary survey on the occurrence of mycotoxigenic fungi and mycotoxins contaminating red rice at consumer level in Selangor, Malaysia

Red rice is a fermented product of Monascus spp. It is widely consumed by Malaysian Chinese who believe in its pharmacological properties. The traditional method of red rice preparation disregards safety regulation and renders red rice susceptible to fungal infestation and mycotoxin contamination. A preliminary study was undertaken aiming to determine the occurrence of mycotoxigenic fungi and mycotoxins contamination on red rice at consumer level in Selangor, Malaysia. Fifty red rice samples were obtained and subjected to fungal isolation, enumeration, and identification. Citrinin, aflatoxin, and ochratoxin-A were quantitated by ELISA based on the presence of predominant causal fungi. Fungal loads of 1.4 × 10(4) to 2.1 × 10(6) CFU/g exceeded Malaysian limits. Monascus spp. as starter fungi were present in 50 samples (100%), followed by Penicillium chrysogenum (62%), Aspergillus niger (54%), and Aspergillus flavus (44%). Citrinin was present in 100% samples (0.23-20.65 mg/kg), aflatoxin in 92% samples (0.61-77.33 μg/kg) and Ochratoxin-A in 100% samples (0.23-2.48 μg/kg); 100% citrinin and 76.09% aflatoxin exceeded Malaysian limits. The presence of mycotoxigenic fungi served as an indicator of mycotoxins contamination and might imply improper production, handling, transportation, and storage of red rice. Further confirmatory analysis (e.g., HPLC) is required to verify the mycotoxins level in red rice samples and to validate the safety status of red rice.

Studies on screening of higher γ-aminobutyric acid-producing Monascus and optimization of fermentative parameters

γ-Aminobutyric acid (GABA), a hypotensive agent, can be produced by Monascus spp. Two hundred and fifteen GABA-producing Monascus strains were isolated from fermented bean curd and red-mold rice. The strain M6 with the highest production level of GABA (3.657 g/L) was isolated from fermented bean curd by using PDB with 0.5% monosodium glutamate (MSG) and identified as Monascus ruber based on morphological and ITS region sequence comparisons. The strain M6 was irradiated by UV to raise GABA production. The mutant strain of M6-13 had the highest GABA yield of 5.527 g/L, which was 1.5 times higher than that of the original strain M6. In addition, the strain M6-13 still had stable yield of GABA and no reverse mutation after sub-cultured for 15 generations. GABA production level of the mutant strain M6-13 increased to 7.826 g/L in submerged flasks culture by use of the orthogonal experiment method. Based on this, process analysis and optimization of GABA production of the strain M6-13 were studied in 3.7-L vessel fermentor and the maximum GABA yield reached 13.470 g/L.

Characterization of a Multifunctional Monascus Isolate NTU 568 with High Azaphilone Pigments Production

Red mold rice is aerobic fungal grown rice on which Monascus sp. has been grown. NTU 568 strain was isolated from red mold rice by our laboratory. In our previous study, strain NTU 568 showed several bioactive functions of reducing blood serum lipid, relaying an anti-fatigue response, decreasing amyloid β peptide accumulation, and possibly preventing obesity. This research focused on the identification of strain NTU 568. The aim of this paper is to understand the taxonomic position of strain NTU 568 among other important Monascus spp. The morphological observations, which include colonies size, ascospores size, and the partial sequences of ribosomal RNA genes-the internal transcribed spacer 1/2 (ITS1/2), 5.8S/18S ribosomal RNA gene, polyketide biosynthesis gene and β-tubulin gene, were applied to identify the strain NTU 568. Comparisons of partial nucleotide sequences, morphology, and azaphilone pigment contents with the strains from different species (Monascus purpureus, Monascus ruber, and Monascus pilosus) were performed. The DNA sequences data indicated that strain NTU 568 was similar to BCRC 31615 (Monascus purpureus) obtained from the Food Industry Research and Development Institute (FIRDI). The contents of monascin (7060.8 mg/kg) and ankaflavin (6836.2 mg/kg) of red mold dioscorea by strain NTU 568 were higher than Monascus-grown products by different strains. The morphological observations, DNA sequences data, and secondary metabolite contents indicated that strain NTU 568 and BCRC 31615 were the same species, belonging to Monascus purpureus.

Exploring the Distribution of Citrinin Biosynthesis Related Genes among Monascus Species

Citrinin, a hepato-nephrotoxic compound to humans, can be produced by the food fermentation microorganisms Monascus spp. In this study, we investigated the distribution of mycotoxin citrinin biosynthesis genes in 18 Monascus strains. The results show that the acyl-transferase and keto-synthase domains of the pksCT gene encoding citrinin polyketide synthase were found in Monascus purpureus, Monascus kaoliang, and Monascus sanguineus. Furthermore, the ctnA gene, a major activator for citrinin biosynthesis, was found in M. purpureus and M. kaoliang, but was absent in M. sanguineus. The orf3 gene encoding oxygenase, located between pksCT and ctnA, was also present in M. purpureus and M. kaoliang. The pksCT gene was highly conserved in M. purpureus, M. kaoliang, and M. sanguineus, while the ctnA and orf3 genes were shown to be highly homologous in M. purpureus and M. kaoliang. In contrast, the PCR and Southern blot analyses suggest that pksCT, ctnA, and orf3 were absent or significantly different in Monascus pilosus, Monascus ruber, Monascus barkeri, Monascus floridanus, Monascus lunisporas, and Monascus pallens. A citrinin-producing phenotype was detected only in M. purpureus and M. kaoliang using high performance liquid chromatography (HPLC). These results clearly indicate that the highly conserved citrinin gene cluster in M. purpureus and M. kaoliang carry out citrinin biosynthesis. In addition, according to the phylogenetic subgroups established with the beta-tubulin gene, the citrinin gene cluster can group the species of Monascus.

Computerized Screening for Novel Producers of Monascus-like Food Pigments in Penicillium Species

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.

Production of carnation pigments in submerged culture of Monascus spp.

Production of carnation pigments in submerged culture of Monascus spp.

MOLECULAR PHYLOGENETIC ANALYSIS OF MONASCUS FUNGI BASED ON INTERNAL TRANSCRIBED SPACER REGION

A molecular analysis of internal transcribed spacer region has been carried out to reveal the relationship among 16 strains of Monascus spp. A primer set comprised primer ITS1 and ITS4 was used to amplify this region in which they were cloned and scqucnccd. We also compared the sequence result with M. purpureus AF458473, M.ruber AF458470, M. kaoliang AF451859, M. araneous AF458471 and M. pilosus AF451856 and one outgroup species Thermoascus crustaceus U18353. The result showed that 16 Monascus spp. were divided into two large clades while M. ruber AF458470 was basically separated from all those Monascus. One of the two large clades included the seven M. purpureus strains, M. purpureus AF458473, M. araneosus AF458471 and M. kaoliang AF451859. Another large cladc included the six Monascus sp. strains which typically have whitish colonies, the three M. ruber strains and M.pilosus AF451856. However, even outstanding morphological differences possessed by several white Monascus and one whitish M. purpureus strain, all Monascus strains were suggested to be very closely related with similarity >99% almost 100%. Although this ITS analysis could not discriminate cultural and morphological differentiation of Monascus strains studied, yet there is still little genetic variation within these strains. Key words : Molecular genetics/Monascus spp./fungi

Study on red fermented rice with high concentration of monacolin K and low concentration of citrinin

Red fermented rice (RFR), known as Asian traditional fermentation foodstuff, is now used as natural colorants and a dietary supplement all over the world. However, the discovery of citrinin in RFR has led to a controversy about the safety of RFR. In this paper, a mutant strain, Monascus spp. M12-69, was acquired by treatment with mutagenic agents from a wild strain M12 of Monascus screened from RFR samples gathered around China. According to the classification guide of Hawksworth and Pitt on Monascus genus, they belong to Monascus pilosus Sato. The conditions of solid state fermentation of M12-69 were optimized. At the optimum conditions, the concentrations of monacolin K and citrinin in RFR, which was dried at 50 °C to a constant weight, were 2.52 mg/g and 0.13 ng/g, respectively. These results reveal that Strain M12-69 is a potential strain, which can be used to produce RFR with high concentration of monacolin K and low concentration of citrinin.

Production of yellow pigments in submerged culture of a mutant of Monascus spp.

Abstract This paper communicates our new findings on a mutant of Monascus mold which is capable of producing a high concentration of yellow pigments (λ max 370 nm) instead of red pigments (λ max 420 and 500 nm) produced by its parents strains. Various factors affecting yellow pigment production have been examined. It was found that the mutant produced the maximal amount of pigment under the optimal conditions for pigment production of its parent strains. The mutation allowed a 10-fold increase in both yellow pigment production and amylolytic enzyme activity over those from a yellow pigment producer isolated previously by us.

Numerical taxonomy of Monascus species based on the electrophoretic analysis of intracellular enzymes

AbstractFour isozymes in fifteeen species of Monascus were analyzed using polyacrylamide gel electrophoresis. A numerical analysis was carried out on the basis of similarity values obtained from the electrophoretic mobilities of the enzymes. Non‐specific esterase and lactate dehydrogenase showed clearly distinguishable mobilities amont two clusters. From these results obtained, the strains used were divided into two clusters of Group‐I and Group‐II. Both groups were further subdivided into two subclusters. The type strain, M. ruber VAN TIEGHEM MR MO‐10 (IAM 8009) and M. kaoliang MR MO‐F2 were found to belong to the Group‐I. M. pilosus SATO MR MO‐4 (IAM8003) and M. pubigerus SATO MR MO‐8 (IAM 8004) were found to belong to the Group‐II. Nearly 100% similarity values were found between M. anka SATO MR MO‐6 (IAM 8001) and M. rubiginosus SATO IFO 4484, between M. anka var. rubellus SATO MR MO‐5 (IAM 8081) and M. purpureus WENT MR MO‐9 (IAM 8010), and, moreover, among M. vitreus SATO MR MO‐3 (IAM 8007), M. fuliginosus SATO IFO 4483 and M. serorubescens SATO 4487, respectively.Therefore, it was concluded that electrophoretic analysis of enzymes may be useful in presenting a new classification system of Monascus spp.

Comparative electrophoresis and some properties of alkaline proteinases produced by Monascus spp.

Some strains in the genus Monascus secreted extracellular alkaline proteinases on a wheat bran medium, along with propagation of cells. Crude enzymes prepared from six strains of five species and one variety of Monascus were separated by polyacrylamide slab gel electrophoresis and their activities were visualized with a printing technique on a casein-containing gel. We found that their proteinase activities were based on at least three different proteins which occurred commonly in all the strains examined. These enzymes were completely inhibited by phenylmethylsulfonyl fluoride and were insensitive to chelating and SH reagents. They were characterized as serine proteinases different from chymotrypsin- or trypsin-type enzymes by their insensitivity to the affinity-labeling reagents, N-α-tosyl-L-lysyl chloromethyl ketone and N-tosyl-L-phenylalanyl chloromethyl ketone.