Monascus Fungi Research Articles

Optimization of Color Fractionation of Monascus Pigment

Monascus fungi are unique among the various microorganisms that produce edible pigments, which consist of red, orange and yellow. These pigments are typically present in a mixture. Hence, pigment separation is needed. This study aims to optimize solvent systems that can effectively fractionate the pigments. The optimization was carried out using column chromatography and the data was analyzed using Central Composite Design (CCD). The interaction of the solvent systems ethyl acetate : formic acid : acetic acid : water was optimized. The optimal solvent system obtained was 98 : 14 : 12 : 28 (vol/vol.) for ethyl acetate : formic acid : acetic acid : water, respectively. The red and yellow pigment obtained were 2.42 mg and 0.93 mg, respectively. Based on the finding, 72.2% of the Monascus pigments was red while remaining was yellow.

Regulation of the pigment production by changing cellmorphology and gene expression of Monascus ruber in high-sugar synergistic high-salt stress fermentation.

Extreme environment of microbial fermentation is the focus of research, which provides new thinking for the production and application of Monascus pigment. In this work, the high-sugar synergistic high-salt stress fermentation (HSSF) of Monascus pigment was investigated. The Monascus fungus grew well under HSSF conditions with 35g L-1 NaCl and 150g L-1 glucose, and the extracellular yellow pigment and intracellular orange pigment yield in HSSF was 98% and 43% higher than that in conventional fermentation (CF), respectively. Moreover, the mycelial morphology was maintained in a better status with more branches and complete surface structure, indicating good biocatalytic activity for pigment synthesis. Four extracellular yellow pigments (Y1, Y2, Y3, Y4) were transformed into each other, and ratio of the relative content of intracellular orange pigments to yellow pigments (O/Y) significantly (p<0.05) changed. Moreover, the ratio of unsaturated fatty acids to saturated fatty acids (unsaturated/saturated) was significantly (p<0.05) increased, indicating that the metabolism and secretion of intracellular and extracellular pigment might be regulated in HSSF. The pigment biosynthesis genes mppB, mppC, mppD, MpPKS5, and MpFasB2 were up-regulated, whereas the genes mppR1, mppR2, and mppE were down-regulated, suggesting that the gene expression to regulate pigment biosynthesis might be a dynamic change process in HSSF. The HSSF system of MPs is successfully performed to improve the pigment yields. Mycelial morphology is varied to enhanced pigment secretion and gene expression is dynamically regulated to promote pigment accumulation in HSSF.

Metabolites analysis and new bioactive pigments from a Monascus purpureus strain with low citrinin

A Monascus purpureus strain with high mycelial biomass and no detected citrinin in liquid fermentation was selected from 20 strains. HPLC-DAD-HRMS analysis revealed that the EtOAc extract of the fermented mash contained 2 unknown compounds and 11 known metabolites of Monascus fungi. Isolations with preparative and semi-preparative HPLC resulted in four brownish red pigments. The two unknown pigments were identified with HRMS, CD, ECD, 1D and 2D NMR, and designated as monascuskaolin A (3) and B (8). The two known compounds were verified with 13C NMR as monascusazaphilone C and monasfluol B. Result of bioactivity tests showed that the two new pigments were able to significantly reduce the transcription levels of IL-1β, TNF-α, and TGF-β, and significantly promote the proliferation of 293 T cells, suggesting that they have anti-inflammatory function and may be good for human kidney. Metabolites studies suggested that M. purpureus RCEF7056 is a safe strain with functional food utilization potential.

Fungal Azaphilone Pigments as Promising Natural Colorants

Abstract—Microscopic fungi form and excrete numerous and diverse secondary metabolites, including pigments of various colors, which may be used as an alternative to chemical and plant colorants used in industry. Azaphilone compounds, first discovered in fungi of the genus Monascus, are among the promising classes of fungal pigments. The review analyzes the publications on formation of azaphilone-type pigments in Monascus fungi, as well as in Talaromyces and Aspergillus сavernicola. Brief information is provided concerning the antimicrobial, antitumor, anti-inflammatory, and hypolipidemic activities of azaphilone pigments. is given. Possible strategies for increasing the efficiency of the production process and directed synthesis of yellow, orange, and red pigments and their derivatives are discussed. In general, the review provides for assessment of the role of azaphilone pigments, as well as of the prospects and ways to expand their production for use as natural dyes in various fields.

A Review of Red Yeast Rice, a Traditional Fermented Food in Japan and East Asia: Its Characteristic Ingredients and Application in the Maintenance and Improvement of Health in Lipid Metabolism and the Circulatory System.

Red yeast rice has been used to produce alcoholic beverages and various fermented foods in China and Korea since ancient times; it has also been used to produce tofuyo (Okinawan-style fermented tofu) in Japan since the 18th century. Recently, monacolin K (lovastatin) which has cholesterol-lowering effects, was found in some strains of Monascus fungi. Since statins have been used world-wide as a cholesterol-lowering agent, processed foods containing natural statins are drawing attention as materials for primary prevention of life-style related diseases. In recent years, large-scale commercial production of red yeast rice using traditional solid-state fermentation has become possible, and various useful materials, including a variety of monascus pigments (polyketides) that spread as natural pigments, in addition to statins, are produced in the fermentation process. Red yeast rice has a lot of potential as a medicinal food. In this paper, we describe the history of red yeast rice as food, especially in Japan and East Asia, its production methods, use, and the ingredients with pharmacological activity. We then review evidence of the beneficial effects of red yeast rice in improving lipid metabolism and the circulatory system and its safety as a functional food.

Research of the physical and chemical properties and methods of Red rice (food colour) determination in sausage products

In Russia, in the production of meat and sausage products, the food colour, named as Red rice, is use. Red rice is obtain by the cultivation the strains of the Monascus fungus on various carbohydrate substrates, for example rice. That Red rice may contain the mycotoxin citrinin, but neither the purity of the food colour nor the safety profile are regulated. The aim of this work was to study the physical and chemical properties of Red rice and to develop method for its determination in sausage products. The experiments were carried out on model and commercial samples of sausages. The samples were analyzed using spectrophotometry and highefficiency planar and liquid chromatography. Spectrophotometric analysis revealed differences in solubility, coloring power, spectral characteristics, composition and ratio of pigments in commercial samples of Red rice. The extraction parameters of colouring substances from sausages model samples were determined. It has been established that Red rice is extracted with chloroform, acetone, ethanol and its aqueous solutions. Petroleum ether was proposed for degreasing sausages. Red rice from sausages was extracted with acetone because it did not extract synthetic colours and carminic acid. It was found, that 90% of Red rice is extracted from samples of sausages by double extraction with ultrasonic treatment with a power of 128 W. High performance thin layer chromatography method and high performance liquid chromatography method for the Red rice identification was proposed. The content of Red rice in the extracts was determined by spectrophotometric method. The developed method for the determination of Red rice was tested on commercial samples of sausage products.

Investigation of the mycelial morphology of Monascus and the expression of pigment biosynthetic genes in high-salt-stress fermentation.

Extreme environments, for example high-salt-stress condition, that can induce secondary metabolite biosynthesis in fungi are a promising and effective strategy for producing natural Monascus pigments used as food colourants and nutraceutical supplements. In this study, the relationship between the mycelial morphology and expression of pigment biosynthetic genes in high-salt-stress fermentation (HSF) with Monascus ruber CGMCC 10910 was investigated. The Monascus fungus grew well under HSF conditions with 35g/l NaCl, and the intracellular yellow pigment yield in HSF was 40% higher than that in conventional batch fermentation (CBF). Moreover, the mycelial morphology was maintained in a better state, with a hyphal diameter of 5-6μm in HSF, indicating good biocatalytic activity for pigment synthesis. The rate of the relative content of intracellular orange pigments to yellow pigments (O/Y) significantly (p < 0.05) changed, and the extracellular yellow pigments were transformed into each other, indicating that the pigment biosynthesis pathway was changed to promote yellow pigment accumulation in HSF. The pigment biosynthesis genes MpPKS5, MpFasB2, mppE, mppD and mppB were significantly (p < 0.05) up-regulated by approximately 58.4-106.1%, whereas the regulatory genes mppR1 and mppR2 were significantly (p < 0.05) down-regulated by approximately 23.2% and 59.0% in HSF. Notably, the mppE gene was highly correlated with (r > 0.95, p < 0.05) hyphal diameter. These findings indicated that the cultivation of the Monascus fungus under high-salt-stress conditions was beneficial for pigment biosynthesis by controlling the mycelial morphology to regulate gene expression. This study first described the relationship between the mycelial morphology and expression of pigment biosynthetic genes in Monascus during fermentation. KEY POINTS: • High-salt-stress fermentation (HSF) was first performed to improve Monascus pigment yield. • Pigment biosynthesis was enhanced by maintaining the mycelial morphology in an improved state in HSF. • Gene expression was up-/downregulated to promote yellow pigment accumulation in HSF. • The mycelial morphology was highly related to the expression of pigment biosynthetic genes in HSF.

The absolute configuration of anti-Vibrio citrinin dimeric derivative by VCD, ECD and NMR methods

Citrinin dimeric derivatives are bioactive polyketides previously reported from Penicillium, Aspergillus and Monascus fungi species. Due to the large distance between the stereogenic centers of the two monomer units, it was difficult to determine the absolute configuration of the whole molecule (1). In previous work, the absolute configuration of 1 was just proposed by biogenetic considerations. To address this problem, the experimental VCD of 1 was compared with the corresponding DFT calculations for two diastereomers (1a and 1b). Also, the experimental ECD and NMR spectra of 1 were combined for analysis with the corresponding theoretical predictions for different diastereomers. Additionally, compound 1 showed promising anti-Vibrio activity against pathogenic Vibrio spp. with MIC values ranging from 0.4 to 0.8 μM.

Orange, red, yellow: biosynthesis of azaphilone pigments in Monascus fungi.

Monascus azaphilone pigments (MonAzPs) are very widely used as food colorants, but their biosynthetic pathway has remained poorly characterized for more than half a century. In this study, the individual steps of MonAzPs biosynthesis in Monascus ruber M7 were elucidated by a combination of targeted gene knockouts, heterologous gene expression, and in vitro chemical and enzymatic reactions. This study describes the first rational engineering of MonAzPs biosynthesis and provides a roadmap for future pathway engineering efforts directed towards the selective production of the most valuable pigments and serves as a model for the biosynthesis of fungal azaphilones in general.

Production and biological activities of yellow pigments from Monascus fungi.

Monascus yellow pigments (MYPs), are azaphilone compounds and one of the three main components of total Monascus pigments (MPs). Thirty-five hydrophilic or hydrophobic MYPs have been identified, with the majority being hydrophobic. Apart from screening special Monascus strains, some advanced approaches, such as extractive and high-cell-density fermentations, have been applied for developing or producing new MYPs, especially extracellular hydrophilic MYPs. The outstanding performance of MYPs in terms of resistance to photodegradation, as well as tolerance for temperature and pH, give natural MYPs reasonable prospects, compared with the orange and red MPs, for practical use in the present and future. Meanwhile, MYPs have shown promising potential for applications in the food and pharmaceutical industries based on their described bioactivities. This review briefly summarizes the reports to date on chemical structures, biological activities, biosynthetic pathways, production technologies, and physicochemical performances of MYPs. The existing problems for MYPs are discussed and research prospects proposed.

Study of Viability of Solid-Phase Microextraction, in vivo, in the Extraction of Microbial Volatile Organic Compounds Associated to the Pigment Production Process by the Monascus Fungus, in Submerged Fermentation

Microbial volatile organic compounds (MVOCs) are present in several production processes in biotechnology. In this study it is evaluated the viability of use of a method involving solidphase microextraction (SPME), in vivo SPME, in the extraction of MVOCs, associated with the pigment production process by the Monascus fungus. PDMS/Car/DVB (50/30 μm) fiber in the headspace mode (HS-SPME) and gas chromatography-mass spectrometry (GC-MS) were used to fermentation broth study. Seventeen compounds were found associated with the presence of the fungus, being the main ones ethanol, 2-methyl-propanol, 3-methyl-butanol, 2-methyl-butanol and 2-phenyl-ethanol. These compounds have maximum concentration of 313.87, 5.51, 2.37, 0.95 and 0.70 mg L −1 . The values of biomass and red pigment found for fermentation for ten days were 4.03 ± 0.21 g L −1 and 3.96 ± 0.17 (UA480). Regular MVOC productions were observed in this study, which were useful in the prediction and control of fermentation by Monascus fungus.

Computational drug designing of fungal pigments as potential aromatase inhibitors

The existing aromatase inhibitors produced unwelcome effects impose the discovery of novel drugs with privileged selectivity, a reduced amount of toxicity and humanizing potency. In this study, we illuminate the binding mode of polyketide azaphilanoid pigments monascin, ankaflavin, monascorubrin and monascorubramine isolated from Monascus fungus to the aromatase by molecular docking. The 3-dimensional structure of aromatase enzyme (PDB: 4KQ8) was obtained from the Protein Data Bank. PatchDock docking software was used to analyze structural complexes of the aromatase with monascus pigments. Comparatively, the AutoGrid model presented the most briskly constructive binding mode of monascin to aromatase. Docked energies in kcal/mol are: monascin;-13.2; monascorubramine:-12.8, monascorubrin:-12.3; ankaflavin: -10.5. These outcomes exposed these ligands could be potential drugs to treat hormone dependent breast cancer.

Effects of endophytic and saprophytic fungi on in vitro methanogenesis

The predominance of pasture in the diet of ruminants in New Zealand presents specific challenges for mitigation of enteric methane emissions. However, there is preliminary evidence from both laboratory and animal-based studies that some fungi may suppress enteric methanogenesis. This study determined the antimethanogenic potential of a range of endophytic (Neotyphodium species) and saprophytic (Geotrichum, Monascus, Mortierella and Penicillium species) fungi. Candidate fungi were selected on the basis of their production of various classes of secondary compounds and screened in an in vitro batch-culture fermentation assay. No strains of endophyte in ryegrass or tall fescue suppressed methanogenesis compared with their endophyte-free controls. The supernatant fractions from three strains of Mortierella wolfii were the most promising fungi identified. They suppressed methanogenesis as effectively as an inhibitor of methanogenesis (bromoethane sulphonate at 30 µmoles/L), without also reducing overall fermentation. These strains should be investigated further by screening fresh cultures grown under contrasting conditions to establish some variation in putative bioactive compounds.

Biochemical aspects of red koji and tofuyo prepared using Monascus fungi

Red koji or red mold rice is prepared by growing a genus Monascus on steamed rice. For centuries, it has been used in Asia for the production of fermented foods including red rice wine and fermented tofu. Although red koji is an important source of various hydrolytic enzymes critical for food fermentation, information on the enzymatic properties in red koji has been limited. Hydrolytic enzymes produced by Monascus fungi may play important roles in ripening of tofuyo (Japanese fermented tofu) regarding the chemical and physical properties of the product. This review provides an introduction of red koji, its properties, and the application of hydrolytic enzymes, especially aspartic proteinases and carboxypeptidases from Monascus fungi. We also describe tofuyo and a novel fermented soybean protein food using a microbial action originating from red koji.

Influence of Carbon and Nitrogen Sources on Growth and Pigment Production by Monascus Pilosus C1 Strain

ABSTRACTPigments produced by Monascus fungi are widely used in food and biotechnology as natural colorants, flavors and preservatives. There are various factors influencing the pigments productions, among which are the nutrient media composition, pH, ambient temperature, mode of cultivation. In the present study the influence of different carbon and nitrogen sources on the pigments biosynthesis by Monascus pilosus C1 strain was investigated during batch cultivation at the following conditions: T = 30°C, pH 6.0, agitation 300 rpm and modified Chapec—Dox medium with different carbon (a range of sugars and alcohols) or nitrogen sources for 7 days. Some process kinetic parameters—YX/C, YP/C, Vav, were calculated and analized. It was found that the micelial growth, expressed as dry weight (DW) and the specific pigment production (SPP) were strongly stimulated by glucose as a carbon source. The DW and SPP reached values of 10.89 g DW l−1, and 1.97 OU mg DW−1 and 1.01 OU mg DW−1 for the total red and yellow pigments, respectively. Sodium glutamate combined with glucose increased effectively the pigments production up to 2.29 OU mg DW−1 and 1.67 OU mg DW−1 respectively for the total red and yellow pigments. The obtained yield coefficient of the studied processes indicated the feasibility of Monascus pilosus C1 strain for pigments production. None of the investigated carbon or nitrogen sources provoked citrinin biosynthesis.

Application of random amplified polymorphic DNA (RAPD) analysis coupled with microchip electrophoresis for high-resolution identification of Monascus strains

Monascus fungi are commonly used for a variety of food products in Asia, and are also known to produce some biologically active compounds. Since the use of Monascus is expected to increase in food industries, strain-level identification and management of Monascus will be needed in the near future. In the present study, random amplified polymorphic DNA (RAPD) analysis coupled with microchip electrophoresis was applied for this purpose. Evaluations of the analysis stability revealed that reproducible results could be obtained, although template DNA fragmentation could influence the resulting RAPD pattern. RAPD analysis using 15 Monascus strains consisting of four species, M. ruber, M. pilosus, M. purpureus, and M. kaoliang showed that each strain generated a unique RAPD pattern, which allows strain-level identification of Monascus. In addition, the phylogenetic tree constructed from RAPD patterns reflected M. ruber-M. pilosus and M. purpureus-M. kaoliang clusters inferred from both ITS and beta-tubulin gene sequences, which indicated that the RAPD pattern could reflect their phylogenetic traits to a certain extent. On the other hand, RAPD analysis did not support the monophyletic clustering of the four Monascus species used in this study, which suggests the necessity of reexamination of species boundaries in Monascus.

A Novel Fermented Food Product Prepared from Soybean Protein-Calcium-Gel with Monascus Fungus: Changes in Protein and Nitrogen Compounds during Fermentation

The aim of the present study was to investigate the changes in protein and nitrogen compounds in a novel fermented product generated from soybean protein-calcium-gel (SP-Cal-Gel) and Monascus fungus during fermentation. Slab SDS-polyacrylamide gel electrophoresis and measurement of nitrogen contents clearly revealed the occurrence of protein digestion in the form of degradation of α'-, α-, and β-subunits in β-conglycinin and of acidic polypeptide in glycinin; however, the band of basic polypeptide in glycinin still remained. The ratios of water-soluble nitrogen and TCA-soluble nitrogen to total nitrogen reached 52.4 or 46.6% after 60-days ripening, respectively. Moreover, involvement of a large amount of free amino acids, especially aspartic and glutamic acids, were shown in this novel fermented SP-Cal-Gel food product.

Mevinolin, citrinin and pigments of adlay angkak fermented by Monascus sp.

Adlay angkak a new developed product from an adlay substrate fermented by Monascus fungi can be used both as a natural coloring and a dietary supplement. However, not only useful secondary metabolites such as mevinolin and pigments are produced; the fungi also produce toxin substance called citrinin. This study conducted the cultivation of M. purpureus (ATCC 16365, BCC 6131, DMKU and FTCMU) and M. ruber TISTR 3006 on the adlay substrate for mevinolin, citrinin, pigments and glucosamine synthesis at room temperature (32–35 °C) for 28 days. The results elucidated that glucosamine levels expressed as the mold growth in solid-state fermentation corresponded as a relatively reliable indicator to the mevinolin, citrinin and pigments production. M. purpureus DMKU produced the lowest citrinin content of 0.26 ppm and the highest mevinolin content of 25.03 ppm with pigment concentrations expressed by absorbance at wavelengths of 400, 470 and 500 nm for yellow, orange and red pigments of 9.76, 3.03 and 3.43 units respectively and moisture content and pH of 83.51% and 6.54 respectively. This study suggested that M. purpureus DMKU has a potential for the production of adlay angkak within an authorized citrinin level.

Characterization of a non-pigment producing Monascus purpureus mutant strain.

A characterization of a non-pigment producing mutant Monascus purpureus M12 compared with its parental strain Monascus purpureus Went CBS 109.07 has been performed aiming to investigate the relation between pigment biosynthesis and other characteristics of these fungi. A comparison has been made of morphological features, some physiological properties and biochemical activities of both strains. The albino mutant exhibits an anamorph life cycle, high conidia forming capability, slower radial growth rate and temperature sensitivity. The assimilation capacity of both strains for mono-, disaccharides and some alcohols is in the same range (Yx/c 0.2 - 0.35), while the red strain has a higher fermentation capacity. In a selected albino mutant, the growth rate, metabolic activity and capacity for production of typical for Monascus fungi secondary metabolites were reduced considerably. Hydrolytic activity towards natural substrates expressed through glucoamylase and protease was approximately 10 fold lower in the non pigment producing strain (0.05 - 0.08 U/mg protein and 0.01 - 0.07 U/mg protein respectively) compared with the red one. Important qualitative differences between both strains was found in fatty acid composition and in the production of citrinin and monacolin. The mutant strain possessed C17, C20 and C22 fatty acids and did not produce citrinin.

Production, Purification and Properties of Acid Proteinase from Genus Monascus.

The screening was carried out to find strains in Monascus fungus that would produce a high activity of acid proteinase. Monascus sp. No. 3404 was found to have the highest activity of the enzyme, which was formed by addition of defatted soybean meal to the medium.The enzyme was purified to homogeneity from the culture broth of this organism grown on defatted soybean meal. The molecular weight of the purified enzyme was determined to be 43000 by gel filtration method and 46000 by SDS-polyacryl amide gel electrophoresis method. The optimum pH was 3.2 and the optimum temperature was 50°C. The enzyme was active on human hemoglobin, milk casein and bovine serum albumin, and Km values for them were calculated to be 0.80, 1.45 and 1.80mg/ml, respectively. The enzyme was markedly inhibited by pepstatin A and chymostatin, and Ki values for them were calculated to be 28nM and 0.13mM, respectively.