Edible Filamentous Fungi from the Species Monascus: Early Traditional Fermentations,Modern Molecular Biology,and Future Genomics

Monascus spp. are filamentous fungi famous for their fermented products, especially red mold rice (RMR), a traditional fermented food in East Asian areas with a very long edible history documented back to the Han dynasty (BC 202‐AD 220) in China. Nowadays, RMR and its related products involve a very large industry from artisanal traditional fermentations to food companies to medicine manufacturers, which are distributed worldwide. Modern studies have shown that Monascus spp. are able to produce abundant beneficial secondary metabolites, such as monacolins (cholesterol‐lowering agents), γ‐amino butyric acid (an antihypertensive substance), dimerumic acid (an antioxidant), and pigments (food‐grade colorants), and some strains can also secrete citrinin, a nephrotoxic metabolite. Monascus‐related studies have received much attention because of their wide applications. However, to our knowledge, no systematic review on the progress of Monascus research has ever been published. In this review, the progress of research on Monascus is summarized into 3 stages: Monascus fermentation, Monascus molecular biology, and Monascus genomics. This review covers the past history, current status, and future direction of Monascus research, contributing to a comprehensive understanding of Monascus research progress.     

Red mold dioscorea: A potentially safe traditional function food for the treatment of hyperlipidemia

A study was undertaken to evaluate whether the interaction between Monascus-fermented products and lovastatin contributes to increased risk of rhabdomyolysis. Rhabdomyolysis is a potentially dangerous side effect of statin drugs. In this study with hyperlipidemic hamsters fed lovastatin only, lovastatin with 1-fold red mold dioscorea (RMD), and lovastatin, the functional components of red mold fermented products, HMG-CoA reductase inhibitors, did not exacerbate pre-existing diseases, and actually helped in improving existing disease conditions, respectively, as compared with the control. Administration of RMD, alone or in combination with lovastatin did not cause significant rhabdomyolysis as assessed by measuring the levels of creatinine phosphokinase. Further, we did not find any study that clearly implicates the involvement of RMD, which have long been considered a food product, in liver and kidney toxicity. RMD alone or in combination with lovastatin, does not increase the risk of rhabdomyolysis, even when administered at a high dosage (including HMG-CoA reductase inhibitors >75 mg/day/adult).     

Red mold dioscorea decreases blood pressure when administered alone or with amlodipine and is a potentially safe functional food in SHR and WKY rats

Hypertension is a risk factor for metabolic syndromes and cardiovascular diseases. Statins are antihypertensive, but can cause rhabdomyolysis as a side effect, which involves the breakdown of muscle fibres and release of myoglobin into the bloodstream. In this study, we used hypertensive rats to investigate whether the interaction between Monascus-fermented products (red mold dioscorea; RMD) and amlodipine increases the risk of rhabdomyolysis and toxicity. A single oral dose of RMD (176 mg/kg) significantly decreased systolic blood pressure (SBP) and diastolic blood pressure (DBP). Moreover, administration of RMD, alone or in combination with amlodipine, did not cause significant rhabdomyolysis and did not impair the metabolic or physiological functions of the liver or kidney. RMD treatment had hypocholesterolemic and hypotriacylglycerolemic effects, indicating that RMD may prevent the incidence of atherosclerosis. The findings of this study lend support to the potential use of RMD as a novel therapeutic and antihypertensive functional food.     

红曲霉合成莫纳克林K的研究进展

红曲霉是一种药食两用丝状真菌,广泛应用于食品、医药、生物催化等领域。莫纳克林K是红曲霉主要代谢功能活性产物,具有降脂、抗疲劳、抗菌、抗癌和保护神经等生物活性,是公认的降低胆固醇的理想药物。研究对比了红曲霉与土曲霉合成莫纳克林K基因簇的不同,阐述了红曲霉产莫纳克林K的合成途径;对莫纳克林K在医药和保健食品、饲料等领域中应用的情况和潜力进行了归纳和展望。由于天然红曲霉发酵产功能性物质过程中,伴随产生的真菌毒素桔霉素,对肾脏、肝脏、心脏和生殖系统有毒性影响,使红曲霉工业化发展受到限制。提高红曲霉的莫纳克林K产量,并降低桔霉素的产生,是解决功能红曲发展问题的重要途径。因此,从诱变育种、构建工程菌株、优化培养基、控制环境因子等方面总结了优选红曲霉高产莫纳克林K并避免桔霉素积累的技术方法。主要对红曲霉产莫纳克林K的合成途径、生物活性及应用领域、高产莫纳克林K的生物技术方法研究现状进行总结,在此基础上分析红曲霉产莫纳克林K中存在的问题,并提出建议与展望,以期为新型红曲保健食品的研究与开发提供理论依据和参考。     

Characteristics of Monascus sp. isolated from Monascus fermentation products

To screen for the strains suitable for industrial applications, 15 Monacus sp. were isolated from various Monascus fermentation products. The isolated strains were belonging to Monascus pilosus, Monascus purpureus, Monascus albidulus, and Monascus femeus based on the ITS sequence and morphological characteristics. They produced globular and hyaline conidia, and red, orange, or hyaline ascomata except for M. fumeus, in which ascomata was not observed in malt extract media. Each strain produced monacolin K and citrinin at different levels on different media: 15.79–20.05 mg/L of monacolin K and 0–255 μg/L of citrinin in submerged culture of monacolin K producing media, 1.27–2.69 mg/L and about 507.4 μg/L in YES medium, 0–0.78 g/kg and 0–9.7 mg/kg in Monascus red rice products, suggesting that it is necessary to adopt the similar conditions to a specific application for screening as well. These various isolated strains would be great resources for industrial application to develop Monascus fermented functional products.     

Optimization of Fermentation Parameters for Higher Lovastatin Production in Red Mold Rice through Co-culture of <Emphasis Type="Italic">Monascus purpureus</Emphasis> and <Emphasis Type="Italic">Monascus ruber</Emphasis>

Monascus, fermented rice (red mold rice), has been found to reduce the serum total cholesterol and triglyceride due to presence of lovastatin. Lovastatin acts as an inhibitor of 3-hydroxy-3-methyl glutaryl coenzyme A reductase. Coculture of Monascus purpureus MTCC 369 and Monascus ruber MTCC 1880 was used to produce red mold rice by solid-state fermentation. Optimization of different fermentation process parameters such as temperature, fermentation time, inoculum volume, and pH of the solid medium was carried out by Box–Behnken’s factorial design of response surface methodology to maximize lovastatin concentration in red mold rice. Maximum lovastatin production of 2.83 mg/g was predicted at 14th day in solid medium under optimized process condition.     

Antioxidant properties of methanolic extracts from monascal rice

Fungus Monascus (Monascaceae) possesses functional components effective in improving human health. The fungus was inoculated into rice and a traditional product (Monascus-fermented rice, anka, red koji) was produced after the colonization of fungal mycelia. Our objective was to evaluate the antioxidant properties of methanolic extracts from inoculated rice products [monascal polished rice (MPR) and monascal dehulled rice (MDR)] as compared to uninoculated rice products [polished rice (PR) and dehulled rice (DR)]. With regard to EC₅₀ values (mg extract/ml) of methanolic extracts, inhibitions of peroxidation were excellent and in the descending order of PR>DR>MDR>MPR. Effectiveness in reducing powers was in the descending order of MPR?MDR?DR>PR. Scavenging abilities on 1,1-diphenyl-2-picrylhydrazyl radicals were in the descending order of MPR>MDR>PR∼DR. Chelating abilities on ferrous ions were in the descending order of MPR∼MDR>PR>DR. Total phenols were the major naturally occurring antioxidant components found. Overall, monascal rice products were better in reducing power, scavenging and chelating abilities and higher in total phenol content than uninoculated rice products.     

Study on the fingerprint profile of Monascus products with HPLC–FD,PAD and MS

Monascus products have been widely used as food additives and pharmaceuticals. Citrinin is a toxic metabolite produced during the Monascus fermentation. In this work, a quick extraction method with ultrasonic treatment was studied for a complete extraction of citrinin from the fungi cells. Furthermore, the proposed HPLC method has the advantage of the simultaneous determination of Monascus pigments and citrinin without sample pretreatment. Under the optimized HPLC conditions, a baseline resolution of pigments, citrinin and other catabolites was achieved. The detection limit of citrinin reached 0.5 ng mL⁻¹ (S/N > 3/1) with a fluorescence detector (FD). Eight Monascus pigments were detected, with photodiode array detector (PAD), two of which not yet described. The HPLC fingerprint profile of the eight Monascus pigments and citrinin appears significant for the quality control of the Monascus product.     

Preparation and application of an immunoaffinity column based on an antibody with strong affinity and packing material with good stability

This article describes the production of an anti-citrinin antibody that showed a high affinity constant (K_a) of 6.28 × 10⁹ and good tolerance to organic solvent and low pH, the synthesis of a Cu (II)-embedded polymer that showed strong binding with this antibody and the preparation of packing material for an immunoaffinity column (IAC) that show good stability. Most of the IACs reported either use harsh elution conditions and are used only once or use gentle elution conditions and are reused many times. Here, through the combined use of a strong-affinity antibody and packing material with good stability, high recoveries during clean-up and yet simultaneously good stability of the IAC were successfully achieved. Under optimised conditions of 80% methanol (pH 3), the IACs were used to clean-up the extracts of Monascus colour and red yeast rice samples, followed by HPLC detection. The recoveries of citrinin from spiked samples at levels of 50–200 μg kg^?1 were in the range of 84–97%.     

Production and antioxidant activity of alcoholic beverages made from various cereal grains using Monascus purpureus NBRC 5965

Monascus sp., which produces red pigments, has been utilized for brewing red alcoholic beverages and fermented foods in China and Okinawa, Japan. Novel red alcoholic beverages with antioxidant activity were made from cereal grains of various colours utilizing the saccharifying activity of Monascus purpureus NBRC 5965. The alcoholic beverage made from black rice and beni koji prepared with M. purpureus NBRC 5965 showed a deep red colour, and the DPPH radical scavenging activity of the alcoholic beverage made from black rice and beni koji was around a 3300 µm Trolox equivalent/mL‐beverage. The antioxidant activity of this alcoholic beverage is thought to be a result of the combination of Monascus pigments and black rice anthocyanin. The DPPH radical scavenging activity and inhibitory activity of lipid peroxidation of an alcoholic beverage made with beni koji were higher than those of an alcoholic beverage made with ki koji prepared with Aspergillus oryzae. Copyright © 2016 The Institute of Brewing & Distilling     

Effects of addition of anka rice on the qualities of low-nitrite Chinese sausages

Anka rice (AR), previously inoculated with Monascus purpureus, was added during manufacturing of low-nitrite Chinese sausages. Chemical compositions and water activities of sausages were not affected. “L”, “a”, and “b” values of sausages with less nitrite (25 ppm) and 0.5% AR added were not significantly different from those with more nitrite (100 ppm) added. Colours of the sausages without AR were light red whereas those with AR added were darker red. Addition of AR did not inhibit lipid oxidation. Higher VBN (volatile basic nitrogen) values of the samples with AR added were observed. With addition of AR, the nitrite degrading rate was retarded. Microbial counts of the sausages with AR added were significantly higher than those of the controls (100 ppm nitrite). The low-nitrite Chinese sausage with addition up to 1.5% AR was acceptable when stored at 4 °C for 28 days.     

红曲色素高产菌的诱变选育与发酵优化

红曲红是一种由红曲霉生产的具有较高药用及营养价值的天然色素。为提高红曲红液态发酵产量,利用常压室温等离子体诱变技术(ARTP)对紫红曲霉（Monascus purpureus）LBBE进行诱变并优化了发酵条件。确定ARTP诱变条件为：红曲霉孢子浓度107个/mL、通气量10 SLM、功率80 W、诱变时间42 s。上述诱变条件下,经10轮诱变,红曲霉正向突变率从44.6 %降至3.8%,突变株LBBE-15的红曲红色价达到1 244 U/mL,较出发菌株提高1.26倍。红曲霉培养条件优化确定为：接种体积分数7%、硫酸锰1 g/L、初始pH 3.7。优化后的红曲红色价达到1 376 U/mL。50 L罐分批发酵显示,红曲红色价为642 U/mL。该研究结果为实现工业大规模生产提供了数据参考。     

Dimerumic acid,a novel antioxidant identified from Monascus-fermented products exerts chemoprotective effects: Mini review

Monascus has traditionally been used for the preparation of fermented products and as a food additive in Asia and is also a traditional Chinese medicine. The present review summarizes the underlying biochemical and molecular mechanisms that can explain anti-inflammation, antioxidation, hepatoprotection, inhibition of cancer cell invasion, and detoxification of dimerumic acid (DMA) isolated from Monascus-fermented products. DMA is an antioxidant that effectively inhibits liver damage and inflammation by attenuating oxidative stress in vitro and in vivo, which is stronger than silymarin, resveratrol, or curcumin. In addition, several lines of evidence have indicated that reactive oxygen species (ROS) promotes cancer cell invasion and suppress tumor development. DMA has been reported to attenuate ROS-induced invasion of SW620 (colorectal adenocarcinoma cells) greater than silymarin, resveratrol, and curcumin. These findings suggest that DMA has potential for development as a novel antioxidant for functional foods to act against several diseases-associated oxidative stress.     

Biochemical and morphological characterization of a new fungal contaminant in balsamic and cider vinegars

In this study we have determined the morphological, some physiological and biochemical characteristics of a fungal strain isolated from balsamic and cider vinegars. The examination of fungal contamination of 100 balsamic and cider vinegar samples showed that 90% contained more than 10⁴ colony forming units (CFU ml^?1), but in most samples (65%) values of more than 10⁵ CFU ml^?1 were obtained. The most frequent contamination was with Monascus sp. (80% of the samples examined showed 2 × 10³ to 6 × 10⁵ CFU ml^?1). This mould can produce secondary metabolites such as citrinin and monacolin K_L (lactone form), and monacolin K_A (acid form). Using high-performance liquid chromatography (HPLC) with fluorescence detection, citrinin was found in 68% of the samples. The concentration of these mycotoxins varied between 1.6 × 10^?2 µg ml^?1 and 7.2 × 10^?2 µg ml^?1. The concentrations of citrinin were very low, and we can anticipate that this compound at these concentrations has no toxic effects on renal cells. Monacolin was not detected in any sample studied.     

传统霉菌发酵食品中的霉菌DNA扩增

为了解市场上霉菌发酵食品能否扩增霉菌DNA,除了红曲霉外其他曲霉是否具有pksCT基因,以及加热灭菌条件对红曲黄酒中pksCT基因降解的影响。从市售的11种食品中提取DNA,选用ITS基因引物和pksCT基因引物进行聚合酶链式反应(polymerase chain reaction,PCR)扩增,并电泳分析。分别用麦曲、红曲发酵黄酒,对发酵前、发酵中、灭菌后、贮藏期样品扩增pksCT基因并测定橘霉素质量浓度;选择灭菌温度70、80、90、100℃,灭菌时间10、20、30 min,灭菌后样品扩增pksCT基因。结果表明有4种食品能用ITS引物扩增出条带,但只有红曲霉发酵的红腐乳能扩增出pksCT基因。红曲黄酒在发酵中、灭菌后能够扩增出pksCT基因,并且在80℃灭菌20 min和90℃灭菌10 min条件下,仍然可以扩增出pksCT基因,但90℃灭菌20 min或100℃灭菌10 min,DNA受到破坏。红曲霉发酵食品能够扩增出pksCT基因,曲霉发酵的食品未能用pksCT基因引物扩增出条带。通过PCR方法扩增出部分霉菌发酵食品的霉菌DNA并测序,有助于了解其加工中使用的菌种,继而为发酵加工食品的终端监测提供一种新方法。     

Optimization of Extraction Conditions of Citrinin from Red Yeast Rice by Orthogonal Design and Quantification of Citrinin by High-Performance Liquid Chromatography

An orthogonal experimental design was utilized to optimize extraction condition of mycotoxin citrinin from red yeast rice. The results showed that the combination of the following conditions was the optimum extraction condition: 50 % ethanol as extraction solvent, 1:5 as the solid-to-liquid ratio, and vortex for 5 min followed by 20 min ultrasonication as extraction manner. Under the optimum extraction condition, citrinin in 11 commercial red yeast rice products was extracted and quantified by optimized high-performance liquid chromatography (HPLC) conditions which consisted of acetonitrile and acidic water (pH 2.5) as mobile phase, flow rate of 1 mL/min, and fluorescence detector. The quantification results showed that seven products contained citrinin; the highest content of citrinin was 57.28 μg/g. The citrinin contamination rate in collected red yeast rice samples was 63.6 %. This investigation indicates that the optimized extraction method followed by HPLC fluorescence detection is a sensitive and effective method to quantify citrinin. The results also indicate that most red yeast rice products in China contain citrinin. However, there are no national criteria about citrinin limit in red yeast rice and the related products. Therefore, a national regulation standard in terms of quantification method should be set up to limit citrinin contamination and to control the circulation of red yeast rice products in China.     

Citrinin Determination in Red Fermented Rice Products by Optimized Extraction Method Coupled to Liquid Chromatography Tandem Mass Spectrometry (LC‐MS/MS)

A rapid and sensitive method was developed and validated for citrinin determination in red fermented rice products by liquid chromatography tandem mass spectrometry (LC‐MS/MS) under the selected reaction monitoring mode. Sample preparation was especially focused, and the quantitative methods of LC‐MS/MS and high‐performance liquid chromatography with fluorescence detection (HPLC‐FLD) were compared. In red fermented rice samples, the limit of detection was 1.0 μg/kg for LC‐MS/MS compared to 250 μg/kg for HPLC‐FLD, the limit of quantification was 3.0 μg/kg for LC‐MS/MS compared to 825 μg/kg for HPLC‐FLD. High correlation coefficient was obtained (R² = 0.999) within the linear range (0.1 to 100 μg/L) in the MS method. The recoveries ranging from 80.9% to 106.5% were obtained in different spiking concentrations. The average intra‐ and inter‐day accuracy ranged from 75.4% to 103.1%, and the intra‐ and inter‐day precisions were from 3.3% to 7.9%. The developed method was applied to 12 commercial red fermented rice products, and citrinin was found in 10 samples ranging from 0.14 to 44.24 mg/kg. Compared to traditional qualitative and quantitative methods, the newly developed LC‐MS/MS method for citrinin determination includes the merits of using a small amount of extraction solvent, simple preparation steps, and high sensitivity.     

Multi-commutated fluorometric optosensor for the determination of citrinin in rice and red yeast rice supplements

Citrinin is a toxic secondary metabolite first isolated from Penicillium citrinum, although is also produced by other species of Penicillium and Aspergillus. It has highly toxic, mutagenic, teratogenic and carcinogenic properties and is often found in crops, vegetables and fruit. To our knowledge there is no specific legislation on maximum levels permitted for citrinin, so no official analytical method is currently available for its determination. Our laboratory developed a fluorometric flow-through optosensor using Sephadex SPC-25 as solid support. Multi-commutated flow injection analysis was used for the construction of the manifold and for handling solutions. In this way, we minimised waste generation and human intervention, which are critical aspects when dealing with highly toxic compounds such as citrinin. The optimum excitation/emission wavelengths were set at 330/494 nm; the calibration curve was linear in the concentration range 35–900 ng ml^?1. A detection limit of 10.5 ng ml^?1 and relative standard deviations (RSDs) lower than 3% were obtained. The developed optosensor was applied to the determination of citrinin in rice and dietary supplements containing red yeast rice.     

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 degrees 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.