Monascus Spp Research Articles

Isolation and Characterization of β-Glucan Containing Polysaccharides from Monascus spp. Using Saccharina japonica as Submerged Fermented Substrate

Beta-glucan (β-glucan), a naturally occurring complex polysaccharide, has drawn attention for its diverse health benefits, including immune system modulation. β-glucan was extracted from two fungi, Monascus purpureus (Mp) and Monascus kaoliang (Mk), cultured in Saccharina japonica via submerged fermentation. The yield, solubility, total sugar, reducing sugar, protein content, Fourier Transform Infrared Spectroscopy (FTIR), X-ray Diffraction (XRD), Thermogravimetric analysis (TGA), Scanning Electron Microscopy (SEM), in vitro free radical scavenging activity, and cytotoxicity were analyzed. A significant yield of β-glucans, with the contents of 51.30 ± 1.54% in Mp and 44.24 ± 1.18% in Mk was observed on a dry weight basis. Water solubility slightly varied, measuring 36.25 ± 1.14% in Mp and 31.25 ± 0.94% in Mk. Total sugar and reducing sugar content in Mp and Mk derived β-glucans were 114.75 ± 2.54 mg/g and 100.25 ± 1.86 mg/g, 7.38 ± 0.78 mg/g, and 8.39 ± 0.46 mg/g, respectively. FTIR spectra resembled the standard, and TGA confirmed heat stability. XRD patterns indicated that the extracted β-glucans, including the standard one, showed the most prominent diffraction peaks in the lower 2θ range, suggesting similar crystalline phases; however, they differed in crystallinity and degree of amorphous content. SEM images displayed characteristic rough and fibrous shapes and surfaces for extracted β-glucans but it was uniform and of a regular shape in the standard sample. The isolated β-glucans exhibited in vitro free radical scavenging and no cytotoxicity was observed in the MTS assay. Therefore, utilizing S. japonica as a substrate in the fermentation process by Monascus spp. presents a unique opportunity in the production and utilization of β-glucans.

Nucleic acid demethylase MpAlkB1 regulates the growth, development, and secondary metabolite biosynthesis in Monascus purpureus.

Nucleic acid demethylases of α-ketoglutarate-dependent dioxygenase (AlkB) family can reversibly erase methyl adducts from nucleobases, thus dynamically regulating the methylation status of DNA/RNA and playing critical roles in multiple cellular processes. But little is known about AlkB demethylases in filamentous fungi so far. The present study reports that Monascus purpureus genomes contain a total of five MpAlkB genes. The MpAlkB1 gene was disrupted and complemented through homologous recombination strategy to analyze its biological functions in M. purpureus. MpAlkB1 knockout significantly accelerated the growth of strain, increased biomass, promoted sporulation and cleistothecia development, reduced the content of Monascus pigments (Mps), and strongly inhibited citrinin biosynthesis. The downregulated expression of the global regulator gene LaeA, and genes of Mps biosynthesis gene cluster (BGC) or citrinin BGC in MpAlkB1 disruption strain supported the pleiotropic trait changes caused by MpAlkB1 deletion. These results indicate that MpAlkB1-mediated demethylation of nucleic acid plays important roles in regulating the growth and development, and secondary metabolism in Monascus spp.

Transcriptomic and Metabolomic Analyses of Soybean Protein Isolate on Monascus Pigments and Monacolin K Production.

Monascus pigments (MPs) and monacolin K (MK) are important secondary metabolites produced by Monascus spp. This study aimed to investigate the effect of soybean protein isolate (SPI) on the biosynthesis of MPs and MK based on the analysis of physiological indicators, transcriptomes, and metabolomes. The results indicated that the growth, yellow MPs, and MK production of Monascus pilosus MS-1 were significantly enhanced by SPI, which were 8.20, 8.01, and 1.91 times higher than that of the control, respectively. The utilization of a nitrogen source, protease activity, the production and utilization of soluble protein, polypeptides, and free amino acids were also promoted by SPI. The transcriptomic analysis revealed that the genes mokA, mokB, mokC, mokD, mokE, mokI, and mokH which are involved in MK biosynthesis were significantly up-regulated by SPI. Moreover, the glycolysis/gluconeogenesis, pyruvate metabolism, fatty acid degradation, tricarboxylic acid (TCA) cycle, and amino acid metabolism were effectively up-regulated by SPI. The metabolomic analysis indicated that metabolisms of amino acid, lipid, pyruvate, TCA cycle, glycolysis/gluconeogenesis, starch and sucrose, and pentose phosphate pathway were significantly disturbed by SPI. Thus, MPs and MK production promoted by SPI were mainly attributed to the increased biomass, up-regulated gene expression level, and more precursors and energies.

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.

The serine/threonine protein kinase MpSTE1 directly governs hyphal branching in Monascus spp.

Monascus spp. are commercially important fungi due to their ability to produce beneficial secondary metabolites such as the cholesterol-lowering agent lovastatin and natural food colorants azaphilone pigments. Although hyphal branching intensively influenced the production of these secondary metabolites, the pivotal regulators of hyphal development in Monascus spp. remain unclear. To identify these important regulators, we developed an artificial intelligence (AI)–assisted image analysis tool for quantification of hyphae-branching and constructed a random T-DNA insertion library. High-throughput screening revealed that a STE kinase, MpSTE1, was considered as a key regulator of hyphal branching based on the hyphal phenotype. To further validate the role of MpSTE1, we generated an mpSTE1 gene knockout mutant, a complemented mutant, and an overexpression mutant (OE::mpSTE1). Microscopic observations revealed that overexpression of mpSTE1 led to a 63% increase in branch number while deletion of mpSTE1 reduced the hyphal branching by 68% compared to the wild-type strain. In flask cultures, the strain OE::mpSTE1 showed accelerated growth and glucose consumption. More importantly, the strain OE::mpSTE1 produced 9.2 mg/L lovastatin and 17.0 mg/L azaphilone pigments, respectively, 47.0% and 30.1% higher than those of the wild-type strain. Phosphoproteomic analysis revealed that MpSTE1 directly phosphorylated 7 downstream signal proteins involved in cell division, cytoskeletal organization, and signal transduction. To our best knowledge, MpSTE1 is reported as the first characterized regulator for tightly regulating the hyphal branching in Monascus spp. These findings significantly expanded current understanding of the signaling pathway governing the hyphal branching and development in Monascus spp. Furthermore, MpSTE1 and its analogs were demonstrated as promising targets for improving production of valuable secondary metabolites.Key points• MpSTE1 is the first characterized regulator for tightly regulating hyphal branching• Overexpression of mpSTE1 significantly improves secondary metabolite production• A high-throughput image analysis tool was developed for counting hyphal branching

Disruption of UDP-galactopyranose mutase expression: A novel strategy for regulation of galactomannan biosynthesis and monascus pigments secretion in Monascus purpureus M9

The impact of the cell wall structure of Monascus purpureus M9 on the secretion of extracellular monascus pigments (exMPs) was investigated. To modify the cell wall structure, UDP-galactopyranose mutase (GlfA) was knocked out using Agrobacterium-mediated transformation method, leading to a significant reduction in the Galf-based polysaccharide within the cell wall. Changes in mycelium morphology, sporogenesis, and the expression of relevant genes in M9 were also observed following the mutation. Regarding MPs secretion, a notable increase was observed in six types of exMPs (R1, R2, Y1, Y2, O1 and O2). Specifically, these exMPs exhibited enhancement of 1.33, 1.59, 0.8, 2.45, 2.89 and 4.03 times, respectively, compared to the wild-type strain. These findings suggest that the alteration of the cell wall structure could selectively influence the secretion of MPs in M9. The underlying mechanisms were also discussed. This research contributes new insights into the regulation of the synthesis and secretion of MPs in Monascus spp..

Effects of Main Nutrient Sources on Improving Monascus Pigments and Saccharifying Power of Monascus purpureus in Submerged Fermentation

Hong Qu (HQ), obtained through fermentation of various grains using Monascus spp., has been widely utilized as the main and characteristic initial saccharification and traditional fermentation starter in the food brewing industry. The quality, color, and flavor of HQ and HQ wine are closely related to the saccharifying power (SP) and Monascus pigments (MPs) of Monascus spp. In this study, to optimize the culture medium in submerged fermentation by M. purpureus G11 for improving SP and MPs, the effects of carbon source, nitrogen source, inorganic salts, and vitamins on SP activity and biosynthesis of MPs were explored through single-factor analysis and response surface Box–Behnken experiments. The results showed that the optimal medium composition was 6.008% rice powder, 1.021% peptone, 0.0049% CuSO4, and 0.052% vitamin B1. Validation experiments performed under the optimized fermentation conditions showed a significant increase in MPs and SP by 14.91% and 36.24%, with maximum MPs and SP reaching 112.61 and 365.12 u/mL, respectively. This study provides a theoretical basis for enhancing MPs and SP in M. purpureus for HQ production, to improve the production efficiency and shorten the production cycle of HQ-related fermentation products.

Overexpression of MrEsa1 accelerated growth, increased ascospores yield, and the polyketide production in Monascus ruber.

Esa1 has been proven to be an important histone acetyltransferase involved in the regulation of growth and metabolism. Monascus spp. with nearly 2000 years of edible history in East Asian countries can produce a variety of polyketides. It is unknown whether Esa1 plays a regulatory role in Monascus spp. In this study, we isolated the homology of histone acetyltransferase Esa1 (named MrEsa1) and constructed a mresa1-overexpressed strain. Western blot experiments showed that MrEsa1 hyperacetylated at K4 and K9 of the H3 subunit in Monascus ruber. Overexpression of mresa1 led to the larger colony diameter and increased dry cell mass; meanwhile, the conidia germination rate was significantly accelerated in the mresa1-overexpressed strain before 4 h, and the number of ascospores in the mresa1-overexpressed strain was significantly higher than that in WT. In addition, the Monascus azaphilone pigments (MonAzPs) and citrinin production of the mresa1-overexpressed strain were 1.7 and 2.4 times more than those of WT, respectively. Reverse transcription-quantitative polymerase chain reaction experiment suggested that mrpigB, mrpigH, mrpigJ, and mrpigK, involved in MonAzPs synthesis, and pksCT, mrl3, and mrl7, involved in citrinin synthesis, were upregulated in mresa1-overexpressed strain. This study provides important insights into the effect of MrEsa1 on the developmental process and the production of secondary metabolites in Monascus spp.

Versatile Strategy for the Construction of a Transcription Factor-Based Orthogonal Gene Expression Toolbox in Monascus spp.

Gene expression is needed to be conducted in an orthogonal manner and controllable independently from the host's native regulatory system. However, there is a shortage of gene expression regulatory toolboxes that function orthogonally from each other and toward the host. Herein, we developed a strategy based on the mutant library to generate orthogonal gene expression toolboxes. A transcription factor, MaR, located in the Monascus azaphilone biosynthetic gene cluster, was taken as a typical example. Nine DNA-binding residues of MaR were identified by molecular simulation and site-directed mutagenesis. We created five MaR multi-site saturation mutagenesis libraries consisting of 10743 MaR variants on the basis of five cognate promoters. A functional analysis revealed that all five tested promoters were orthogonally regulated by five different MaR variants, respectively. Furthermore, fine gene expression tunability and high signal sensitivity of this toolbox are demonstrated by introducing chemically inducible expression modules, designing synthetic promoter elements, and creating protein-protein interaction between MaRs. This study paves the way for a bottom-up approach to build orthogonal gene expression toolboxes.

Characterization of key upstream asexual developmental regulators in Monascus ruber M7

Monascus species are traditional edible and medicinal filamentous fungi with a very long history in East Asia, but their sparse production of asexual spores (conidia) limits their large-scale industrial application. Previous studies have revealed that the flbA ortholog plays a negative role in the conidiation of Monascus ruber M7. FlbA, together with FlbB, FlbC, FlbD, FlbE, and FluG, were shown to be upstream development activators (UDAs) in the asexual development pathway in the model fungus Aspergillus nidulans. In the present study, homologs of these UDAs in M. ruber M7 were identified and functionally characterized. Disruption of MrflbB, MrflbD, and MrflbE delayed and decreased conidiation in M7, whereas the deletion of MrflbC or MrfluG resulted in no detectable change in sporulation. Deletion of MrflbA not only increased conidial number but also postponed sexual development in M7. Furthermore, the deletion of some UDAs also affected the production of secondary metabolites, particularly citrinin, whose yields were dramatically decreased in the ΔMrflbC and ΔMrflbA mutants. These findings provide new insights and opportunities to further explore the asexual development mechanism and modify the conidial production in Monascus spp.

Construction of gene modification system with highly efficient and markerless for Monascus ruber M7.

Monascus spp. are traditional medicinal and edible filamentous fungi in China, and can produce various secondary metabolites, such as Monascus pigments (MPs) and citrinin (CIT). Genetic modification methods, such as gene knock-out, complementation, and overexpression, have been used extensively to investigate the function of related genes in Monascus spp.. However, the resistance selection genes that can have been used for genetic modification in Monascus spp. are limited, and the gene replacement frequency (GRF) is usually <5%. Therefore, we are committed to construct a highly efficient gene editing system without resistance selection marker gene. In this study, using M. ruber M7 as the starting strain, we successfully constructed a so-called markerlessly and highly genetic modification system including the mutants ΔmrpyrGΔmrlig4 and ΔmrpyrGΔmrlig4::mrpyrG, in which we used the endogenous gene mrpyrG from M. ruber M7 instead of the resistance marker gene as the screening marker, and simultaneously deleted mrlig4 related to non-homologous end joining in M. ruber M7. Then, the morphology, the growth rate, the production of MPs and CIT of the mutants were analyzed. And the results show that the mutant strains have normal mycelia, cleistothecia and conidia on PDA+Uridine(U) plate, the biomass of each mutant is also no different from M. ruber M7. However, the U addition also has a certain effect on the orange and red pigments yield of M. ruber M7, which needs our further study. Finally, we applied the system to delete multiple genes from M. ruber M7 separately or continuously without any resistance marker gene, and found that the average GRF of ΔmrpyrGΔmrlig4 was about 18 times of that of M. ruber M7. The markerlessly and highly genetic modification system constructed in current study not only will be used for multi-gene simultaneous modification in Monascus spp., and also lays a foundation for investigating the effects of multi-genes modification on Monascus spp..

Coconut Cream Agar as a simple and rapid semiquantitative method to screen citrinin-producing Monascus spp. isolates isolated from red fermented rice

Red fermented rice (RFR) is produced using Monascus spp. This product has some health benefits. However, RFR can also contain the mycotoxin, citrinin (CIT) and that has adverse effects on human health. The objective of the study was to develop a simple and rapid screening method for the detection of Monascus spp. isolates that can produce CIT by using Coconut Cream Agar (CCA). RFR was spread onto CCA and other media and incubated at 30 °C for 7 days. All the media were observed daily under ultraviolet (UV) light and any Monascus spp. colony that produced light blue fluorescence was recorded as a CIT-producer. Two different isolates (MF1 and MS1) isolated from CCA were selected for further analysis. All (100%; 10/10 plates) of CCA inoculated with MF1 produced light blue fluorescence after incubation for 4 days, meanwhile 30% (3/10 plates) of MS1 produced weak fluorescence on CCA after incubation for 7 days. Isolates MF1 and MS1 were identified as M. purpureus with the ability to produce CIT by having polyketide synthase (pksCT) and transcriptional regulator (ctnA) genes. CIT was quantified by high-performance liquid chromatography (HPLC). CCA is a simple and rapid method to detect CIT-producers of Monascus spp.

Optimization of the hongqu starter preparation process for the manufacturing of red mold rice with high gamma-aminobutyric acid production by solid-state fermentation.

Red mold rice (RMR) generally contains gamma-aminobutyric acid (GABA), which has several physiological functions. Monascus purpureus M162, with a high GABA production of 15.10 mg/g was generated by atmospheric and room temperature plasma mutation. Furthermore, we conducted a response surface methodology to produce a premium hongqu starter. The results revealed that under optimal conditions, that is, a substrate containing brown rice and bran in a brown rice: bran ratio of 9:1 (wt/wt), an inoculation size of 21.50 mL/100 g, a mixing frequency of one time/9 h, and a cultivation time of 7.20 days, the number of active spores, α-amylase activity, and saccharification power activity was 4.15 × 107 spores/g, 155 U/g, and 3260 U/g in the high-quality starter, respectively. These values were 224.32-fold, 139.64%, and 141.74% higher than those obtained with M. purpureus M162 inoculated into steamed indica rice, respectively, and 153.70-fold, 267.24%, and 151.63% higher than those obtained with the parent strain M. purpureus M1, respectively. The premium hongqu starter of M. purpureus M162 was inoculated into steamed indica rice to produce RMR with 15.93 mg/g of GABA. In conclusion, we proposed a novel strategy for functional RMR production with high GABA concentrations by solid-state fermentation with Monascus spp.

Enrichment of dimerumic acid in Monascus‐fermented rice and its in vivo antioxidant activity

AbstractMonascus‐fermented rice (MFR) has been used in traditional Chinese and Japanese medicine for the revitalization of blood, digestion problems, and cardiovascular disorders. Through a biotechnological approach, MFR is produced by the solid‐state fermentation of rice by Monascus spp. MFR has remarkable therapeutic properties as it contains several bioactive compounds, and dimerumic acid is one of the important siderophore‐based antioxidant fermentative metabolites found in it. This study aimed to screen out the medium composition for the production of dimerumic acid (DMA) and evaluate its antioxidant potential. Different strains of Monascus purpureus Microbial Type Culture Collection &amp; Gene Bank (MTCC) 410, 369, 1090 and Monascus ruber MTCC 1880, 2326 and Monascus pilosus CBS 286.34, CBS 290.34 with different solid substrate were screened for maximum yield of DMA. Solid‐state fermentation of long‐grain rice (Oryza sativa) by Monascus pilosus CBS 290.34 was the best for DMA production. The optimized medium parameters in rice for the highest yield of DMA were glucose (75.13 mg/g), soya extract (19.33 mg/g), malt extract (35.14 mg/g), and calcium carbonate (0.29 mg/g). The predicted values for medium parameters were in good agreement with the experimental data. MFR enriched with DMA showed potent antioxidant and lipid lowering capacity in both in vitro and in vivo. It can be explored further for the development of nutraceuticals as a natural antioxidant.

Effects of Chinese medicines on monacolin K production and related genes transcription of Monascus ruber in red mold rice fermentation.

Monacolin K (MK) is a secondary metabolite synthesized by polyketide synthases of Monascus spp. In this study, the combined supplementation of three medicines, including Citri Reticulatae Pericarpium (CRP), Fructus crataegi (FC), and Radix Angelicae Dahuricae (RAD), were mixed with nonglutinous rice and were optimized by response surface methodology to enhance the production of MK in fermented red mold rice (RMR). Under the optimum condition, MK production achieved 3.60 mg/g, which was 41.18% higher than RMR without medicines. The improved MK production was mainly caused by the up‐regulated transcription level of mokA, mokB, mokF, mokH, mokI, and mplaeA. Meanwhile, the inhibitory effect of Poria cocos (PC) on MK production (only 0.436 mg/g) was caused by significantly down‐regulated transcription of six tested genes. Therefore, this study is beneficial for better understanding of the possible mechanism of enhanced MK production by optimization of fermentation conditions.

Cost-effective pigment production by Monascus purpureus using rice straw hydrolysate as substrate in submerged fermentation

Monascus pigments (MPs), the secondary metabolites produced by the fungal strains of Monascus spp., hold commercial importance in not only the food and meat industries, but also therapeutic, cosmetic, and textile industries. To reduce the cost of MPs production, the utilization of rice straw hydrolysate as a substrate in submerged fermentation was investigated. The atmospheric and room temperature plasma (ARTP) mutation system was employed to develop a mutant strain Monascuspurpureus M630, with high total extracellular Monascus pigments (exMPs) production of 34.12U/mL in submerged fermentation with glucose-based medium. The results revealed that M. purpureus M630 produces 8.61U/mL and 20.86U/mL of exMPs in rice straw hydrolysate alone or in combination with glucose fermentation medium, respectively. Furfural (Fur) and 5'-hydroxymethyl furfural (5'-HMF), produced during pretreatment and hydrolysis of rice straw; are generally inhibitory for microbial growth and fermentation. Our findings revealed that M. purpureus M630 develops the tolerance and adaptation mechanisms in response to 5'-HMF and Fur during growth and MPs biosynthesis in rice straw hydrolysate. In conclusion, we report that rice straw hydrolysate can serve as an efficient and low-cost substitute for the MP production through submerged fermentation by Monascus spp.

High Expression Level of mok E Enhances the Production of Monacolin K in Monascus

ABSTRACTMonacolin K (MK) is a type of cholesterol-lowering agent produced by Monascus spp. The expression level of the mok E gene, a gene of the MK biosynthesis gene cluster, was previously speculated to be positively correlated with the production of MK. In this study, mok E was overexpressed in Monascus fuliginosusis CG-6. The expression levels were analyzed by RT-qPCR. Genome walking was used to analyze the insertion site of the exogenous gene. Results showed that MK production in transformants T-mok E1 (4083.2 μg/g), T-mok E2 (6186.3 μg/g), and T-mok E3 (4527.6 μg/g) was 1.7-fold, 2.5-fold, and 1.8-fold higher than that of the wild strain CG-6 (2469.8 μg/g), respectively. The expression level of mok E in T-mok E1, T-mok E2, and T-mok E3 was 1.83-fold, 4.82-fold, and 2.22-fold higher than that in the wild-type strain (WS CG-6), respectively. The AAA ATPase gene located upstream and acetyl-CoA synthetase gene located downstream of the insertion were obtained by genome walking. These results indicate that the expression of the mok E gene was positively correlated with the production of MK. The expression levels of the AAA ATPase gene and acetyl-CoA synthetase gene were enhanced by the insertion of an exogenous gene.

The Possible Mechanisms Involved in Citrinin Elimination by Cryptococcus podzolicus Y3 and the Effects of Extrinsic Factors on the Degradation of Citrinin.

Citrinin (CIT) is a toxic secondary metabolite produced by fungi belonging to the Penicillium, Aspergillus, and Monascus spp. This toxin has been detected in many agricultural products. In this study, a strain Y3 with the ability to eliminate CIT was screened and identified as Cryptococcus podzolicus, based on the sequence analysis of the internal transcribed spacer region. Neither uptake of CIT by cells nor adsorption by cell wall was involved in CIT elimination by Cryptococcus podzolicus Y3. The extracellular metabolites of Cryptococcus podzolicus Y3 stimulated by CIT or not showed no degradation for CIT. It indicated that CIT elimination was attributed to the degradation of intracellular enzyme(s). The degradation of CIT by C. podzolicus Y3 was dependent on the type of media, yeast concentration, temperature, pH, and initial concentration of CIT. Most of the CIT was degraded by C. podzolicus Y3 in NYDB medium at 42 h but not in PDB medium. The degradation rate of CIT was the highest (94%) when the concentration of C. podzolicus Y3 was 1 × 10⁸ cells/ml. The quantity of CIT degradation was highest at 28°C, and there was no degradation observed at 35°C. The study also showed that acidic condition (pH 4.0) was the most favorable for CIT degradation by C. podzolicus Y3. The degradation rate of CIT increased to 98% as the concentration of CIT was increased to 20 μg/ml. The toxicity of CIT degradation product(s) toward HEK293 was much lower than that of CIT.

Chemical profiling and quantification of monacolins and citrinin in red yeast rice commercial raw materials and dietary supplements using liquid chromatography-accurate QToF mass spectrometry: Chemometrics application

Red yeast rice (RYR) is prepared by fermenting rice with various strains of the yeast Monascus spp of the Aspergillaceae family. Depending on the Monascus strains and the fermentation conditions, the products may contain monacolins, pigments and citrinin as secondary metabolites. Authentic and commercial RYR samples were analyzed using UHPLC–DAD–QToF-MS for monacolins, pigments and citrinin. A separation by UHPLC was achieved using a reversed-phase column and a gradient of water/acetonitrile each containing formic acid as the mobile phase. Accurate mass QToF spectrometry was used to distinguish isobaric monacolins. Principle component analysis (PCA), a chemometric technique was used to discriminate between authentic RYR, commercial RYR raw materials and dietary supplements. Three authentic RYR samples, 31 commercial RYR raw materials and 14 RYR dietary supplements were analyzed. Monacolin K content in 600mg of authentic RYR samples ranged from 1.2mg to 1.38mg. Amounts of monacolin K in dietary supplements labeled as containing 600mg of RYR varied more than 40-fold from 0.03mg to 2.18mg. Monacolin K content of dietary supplements labeled as containing 1200mg RYR varied more than 20-fold from 0.22mg to 5.23mg. In addition to large variations in quantity of monacolin K found in dietary supplements, RYR dietary supplements contained ratios of monacolins that differed significantly from authentic samples. The results indicated that RYR commercial products are of variable quality and the analytical method is suitable for quality control testing of a variety of RYR products.

Chemical fingerprint analysis and characterization of monacolins, pigments, citrinin, sudan red G from red yeast rice samples using liquid chromatography-accurate QToF mass spectrometry

Red yeast rice (RYR) is prepared by fermentation of rice with various strains of the yeast Monascus spp belonging to family Aspergillaceae, the genus Monascus. Depending on the Monascus strains and the fermentation conditions, the products may contain polycetides called monacolins or polyketides (pigments), which are secondary metabolites. The preparations of red yeast rice have being used in food as a preservative, spice, coloring, rice wine or been sold as dietary supplements to lower blood levels of cholesterol and related lipids. The UHPLC-QToF-MS analysis of monacolins, pigments, citrinin and sudan red G from various RYR samples and dietary supplements was performed. A separation by LC was achieved using a reverse-phase column and a gradient of water/acetonitrile each containing formic acid as the mobile phase. MS-MS detection was used to provide structural information. Under typical ESI-MS conditions, monacolins produced abundant ions corresponding to the protonated molecules, sodium ion adducts and dimers [2 M+H]+. The elimination of the ester side chain followed by dehydration and dissociation of the lactone moiety were observed as the main fragmentation pathways for most of the monacolin-type of compounds.