Insights into Monascus biology at the genetic level

The genus of Monascus was nominated by van Tieghem in 1884, but its fermented product—red mold rice (RMR), namely red yeast rice, has been used as folk medicines, food colorants, and fermentation starters for more than thousands of years in oriental countries. Nowadays, RMR is widely developed as food supplements around the world due to its functional compounds such as monacolin K (MK, also called lovastatin) and γ-aminobutyric acid. But the usage of RMR also incurs controversy resulting from contamination of citrinin (a kind of mycotoxin) produced by some Monascus strains. In the past decade, it has made great progress to Monascus spp. at the genetic level with the application of molecular biology techniques to restrain the citrinin production and increase the yields of MK and pigment in RMR, as well as aid Monascus classification and phylogenesis. Up to now, hundreds of papers about Monascus molecular biology (MMB) have been published in the international primary journals. However, to our knowledge, there is no MMB review issued until now. In this review, current understanding of Monascus spp. from the view of molecular biology will be covered and insights into research areas that need to be further investigated will also be discussed.     

Beneficial effects of <Emphasis Type="Italic">Monascus purpureus</Emphasis> NTU 568-fermented products: a review

Monascus-fermented products have been used in food, medicine, and industry dating back over a thousand years in Asian countries. Monascus-fermented products contained several bioactive metabolites such as pigments, polyketide monacolins, dimerumic acid, and γ-aminobutyric acid. Scientific reports showed that Monascus-fermented products proved to be effective for the management of blood cholesterol, diabetes, blood pressure, obesity, Alzheimer’s disease, and prevention of cancer development. This review article describes the beneficial effects about using Monascus-fermented products in human beings and animals.     

<Emphasis Type="Italic">Monascus purpureus</Emphasis>-fermented products and oral cancer: a review

Tobacco and alcohol consumption have been reported as major factors for the development of oral cancer. Edible fungi of the Monascus species have been used as traditional Chinese medicine in eastern Asia for several centuries. Monascus-fermented products have many functional secondary metabolites, including monacolin K, citrinin, ankaflavin, and monascin. In several recent studies performed in our laboratory, these secondary metabolites have shown anti-inflammatory, anti-oxidative, and anti-tumor activities. Many published studies have shown the efficacy of Monascus-fermented products in the prevention of numerous types of cancer. The current article discusses and provides evidence to support that Monascus-fermented metabolites may be developed as painting drugs for the mouth to prevent or cure oral carcinogenesis. This is a novel therapeutic approach focusing on tumor growth attenuation to improve patient survival and quality of life.     

Red mold,diabetes, and oxidative stress: a review

Type 2 diabetes is a major health concern and a rapidly growing disease with a modern etiology, which produces significant morbidity and mortality. The optimal management of type 2 diabetes aims to control hyperglycemia, hypertension, and dyslipidemia to reduce overall risks. Diabetes and its complications usually develop as oxidative stress increases. Monascus-fermented rice, also called red mold rice or red mold dioscorea are used in China to enhance food color and flavor. Red mold-fermented products are popular health foods that are considered to have antiobesity, antifatigue, antioxidation, and cancer prevention effects. This review article describes the antidiabetic and antioxidative stress effects on humans and animals of red mold-fermented products or their secondary metabolites.     

Effect of red mold rice supplements on serum and meat cholesterol levels of broilers chicken

Monacolin K is a secondary metabolite produced by Monascus species. It was found that it is able to decrease cholesterol levels. In this study, red mold rice (RMR) was added to the diet of Arbor Acres broiler chickens, and the cholesterol level in meat, as well as the concentration of triglyceride, the high-density lipoprotein cholesterol (HDL-C), and the low-density lipoprotein cholesterol (LDL-C) in the serum were evaluated. Four-week-old broilers are studied and divided into four groups in that each group contains 15 subjects. A 3-week experimental feeding trial was conducted in which three groups of broilers were fed 2.0, 5.0, and 8.0% of RMR (RMR groups) within their diet, respectively, and the result was compared to the control group. The results indicated that for each RMR group, the cholesterol content was significantly lower than that of the control group; in addition, their meat products contain higher level of unsaturated fatty acids. Triglyceride and cholesterol concentration in serum was also found to be considerably lower in RMR groups when compared to control group. Finally, in RMR groups, HDL-C/LDL-C and HDL-C/cholesterol ratios were all higher than those of the control group. In short, the results demonstrated that the cholesterol levels could be lowered by adding RMR to the diet of chickens.     

Simplified Resting Metabolic Rate—Predicting Formulas for Normal‐Sized and Obese Individuals

Objective: Resting metabolic rate (RMR) is known to be proportional to body weight and to follow allometric scaling principles. We hypothesized that RMR can be predicted from an allometric formula with weight alone as an independent variable. Research Methods and Procedures: An allometric, power‐law scaling model was fit to RMR measurements obtained from a cohort of patients being treated for weight loss. This, as well as many of the commonly used RMR‐predicting formulas, was tested for RMR prediction ability against a large publicly available RMR database. Bland‐Altman analysis was used to determine the efficacy of the various RMR‐predicting formulas in obese and non‐obese subjects. Results: Power law modeling of the RMR—body weight relationship yielded the following RMR‐predicting equations: RMR_Women = 248 × Weight^0.4356 ? (5.09 × Age) and RMR_Men = 293 × Weight^0.4330 ? (5.92 × Age). Partial correlation analysis revealed that age significantly contributed to RMR variance and was necessary to include in RMR prediction formulas. The James, allometric, and Harris‐Benedict formulas all yielded reasonable RMR predictions for normal sized and obese subjects. Discussion: A simple power formula relating RMR to body weight can be a reasonable RMR estimator for normal‐sized and obese individuals but still requires an age term and separate formulas for men and women for the best possible RMR estimates. The apparent performance of RMR‐predicting formulas is highly dependent on the methodology employed to compare the various formulas.     

Simultaneous Enrichment of Deglycosylated Ginsenosides and Monacolin K in Red Ginseng by Fermentation with Monascus pilosus

To improve its bioavailability and pharmacological effects in humans, red ginseng was fermented with a newly isolated fungus, Monascus pilosus KMU103. Most of the ginsenosides were converted to deglycosylated ginsenocides, such as Rh₁, Rh₂, and Rg₃. The total amount of ginsenosides Rh₁, Rh₂, and Rg₃ was 838.7 mg/kg in the red ginseng, and increased to 4,117 mg/kg after 50 L fermentation in 13% red ginseng and 2% glucose. In addition, the Monascus-fermented red ginseng contained 3,089 mg/kg of monacolin K, one of the metabolites produced by Monascus known to reduce cholesterol in the blood. This newly developed Monascus-fermented red ginseng should result in improved health effects, not only by biotransforming gisenosides to deglycosylated ones but also by creating additional bioactive compounds.     

Resting Metabolic Rate in Severely Obese Diabetic and Nondiabetic Subjects

Objectives : To compare the resting metabolic rate (RMR) between diabetic and nondiabetic obese subjects and to develop a predictive equation of RMR for these subjects. Research Methods and Procedures : Obese adults (1088; mean age = 44.9 ± 12.7 years) with BMI ≥ 35 kg/m² (mean BMI = 46.4 ± 8.4 kg/m²) were recruited. One hundred forty‐two subjects (61 men, 81 women) were diagnosed with type 2 diabetes (DM), giving the prevalence of DM in this clinic population as 13.7%. RMR was measured by indirect calorimetry, and several multivariate linear regression models were performed using age, gender, weight, height, BMI, fat mass, fat mass percentage, and fat‐free mass as independent variables. Results : The severely obese patients with DM had consistently higher RMR after adjustment for all other variables. The best predictive equation for the severely obese was RMR = 71.767 ? 2.337 × age + 257.293 × gender (women = 0 and men = 1) + 9.996 × weight (in kilograms) + 4.132 × height (in centimeters) + 145.959 × DM (nondiabetic = 0 and diabetic = 1). The age, weight, and height‐adjusted least square means of RMR between diabetic and nondiabetic groups were significantly different in both genders. Discussion : Severely obese patients with type 2 diabetes had higher RMR than those without diabetes. The RMR of severely obese subjects was best predicted by an equation using age, gender, weight, height, and DM as variables.     

Monascus fermentation of dioscorea for increasing the production of cholesterol-lowering agent—monacolin K and antiinflammation agent—monascin

Monacolin K, an inhibitor for cholesterol synthesis, is the secondary metabolite of Monascus species. The formation of the secondary metabolites of the Monascus species is affected by cultivation environment and method. This research uses sweet potato (Ipomoea batatas), potato (Solanum tuberosum), casava (Manihot esculenta), and dioscorea (Dioscorea batatas) as the substrates and discusses the best substrate to produce monacolin K. The results show that Monascus purpureus NTU 301, with dioscorea as the substrate, can produce monacolin K at 2,584 mg kg⁻¹, which is 5.37 times to that resulted when rice is used as the substrate. In addition, more amount of yellow pigment can be found in Monascus-fermented dioscorea than in Monascus-fermented rice. The certain composition of yellow pigment is identified as monascin, which has been shown as an antiinflammation agent exhibiting potent inhibitory effects on 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced inflammation in mice in previous studies. Therefore, dioscorea is concluded to be the best substrate for Monascus species to produce the cholesterol-lowering agent—monacolin K and antiinflammation agent—monascin.     

In vivo hypolipidemic effects and safety of low dosage Monascus powder in a hamster model of hyperlipidemia

Monascus or more commonly known as red mold rice is fermented rice on which Monascus purpureus has been grown. It has been a traditional Chinese food additive for thousands of years in China. Secondary metabolite product of Monascus, monacolin K, has been proven that it could be used as an antihypercholesterolemic agent. In this study, M. purpureus NTU568 mutated and selected from a monacolin K productivity strain—M. purpureus HM105 produced high quantities of monacolin K at a level of 9,500 mg kg⁻¹. This research focused on the effect of adding red mold rice powder of M. purpureus NTU568 to a hamster diet on total cholesterol (TC), triglyceride (TG), high-density lipoprotein cholesterol, and low-density lipoprotein cholesterol (LDL-C). In the results, the oral administration of Monascus powder in hyperlipidemia hamster was indeed proven to decrease TC, TG, and LDL-C levels. Plasma TC levels in hamster fed with Monascus powder at onefold dosage [10.78 mg (day 100 g bw)⁻¹] for 4 and 8 weeks were significantly lower (31.2 and 22.0%, respectively) than that in hyperlipidemia hamster. Plasma TG (30.1 and 17.9%) and LDL-C levels (36.0 and 20.7%) were also significantly lowered by feeding Monascus powder at onefold dosage for 4 and 8 weeks compared to hyperlipidemia hamster. In addition, examinations of liver TC and TG levels of hyperlipidemia hamster were also performed and showed similar effects on lipid-lowering action by oral administration of Monascus powder. Since citrinin is a mycotoxin that possesses nephrotoxic and hepatoxic effects, it has a negative impact on the safety of red mold rice for people. This study examined the liver somatic index [plasma glutamyl oxaloacetic transaminase (GOT) and glutamyl pyruvic transaminase (GPT) levels] and liver biopsy to investigate whether Monascus powder induced damage in liver. It was found that the plasma GOT and GPT levels were not significantly increased by feeding Monascus powder. There was no difference in the results of the liver biopsy between the Monascus powder-treated groups and the control group.     

Application of random amplified polymorphic DNA (RAPD) analysis coupled with microchip electrophoresis for high-resolution identification of <Emphasis Type="Italic">Monascus</Emphasis> 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 β-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.     

Isolation and characterization of a strain of <Emphasis Type="Italic">Klebsiella pneumoniae</Emphasis> with citrinin-degrading activity

Monascus-fermented products have been widely used in Taiwan and other Asian countries as health foods. Unfortunately, many Monascus strains concurrently produce trace amounts of toxic citrinin. This study isolated a strain NPUST-B11 with the ability to degrade citrinin as the only carbon source. The isolated strain NPUST-B11 was characterised and identified as Klebsiella pneumoniae by 16S rRNA gene analysis using UNI-F and UNI-R primers. The isolated strain was then incubated in the mineral broth containing 10 ppm of citrinin, 1.2% of glucose, 0.3% of peptone and 100 ppm of vitamin C under optimal conditions, including pH 7, 200 rpm and 37°C. Citrinin was rapidly degraded with incubation from 97.9% at 1 h to 8.67% at 5 h and completely depleted at 10 h. Overall, this strain could be useful for the degradation of citrinin in food products and other medical applications.     

Development and production of cholesterol-lowering <Emphasis Type="Italic">Monascus</Emphasis>-nata complex

This study develops a new foodstuff, the Monascus-nata complex, which combines the functions of cholesterol-lowering monacolin k and bacterial dietary-fibre. Two Monascus strains, M. ruber and M. pilosus were fermented within cubical bacterial cellulose, nata de coco, obtained from Acetobacter fermented coconut juice, in a conditioned medium. The production levels and stability of monacolin k in the cultured Monascus-nata complex were determined to develop optimal fermentation conditions. The results indicated that a medium that comprised 5% glucose and 1.5% ammonium phosphate at pH 6.0–7.0 produced the most monacolin k (157 mg/l) for Monascus pilosus NCHU M-35. However, monosodium glutamate (MSG) and 0.001% ZnSO₄ inhibited the intracellular accumulation of monacolin k. Monacolin k within the Monascus-nata complex was relatively resistant to washing and changes of pH, but thermal processing and freezing storage markedly reduced the amount present. This novel Monascus-nata complex is potentially a healthy foodstuff.     

Lower than predicted resting metabolic rate is associated with severely impaired cardiorespiratory fitness in obese individuals

Obese individuals have reduced cardiorespiratory fitness as compared with leaner counterparts. Regular exercise maintains or increases fitness and lean body mass. Lean body mass, in turn, has a direct impact on resting metabolic rate (RMR). Given these relationships, we sought to evaluate the association between RMR and cardiorespiratory fitness in obese individuals. We evaluated 64 obese individuals (78% female) with direct assessment of RMR and cardiorespiratory fitness via breath‐by‐breath measurement of oxygen consumption and carbon dioxide production at rest and during exercise. The mean age and BMI were 47.4 ± 12.2 years and 47.2 ± 9.2 kg/m², respectively. The majority of subjects, 69%, had a measured RMR above that predicted by the Harris‐Benedict equation. Compared with the higher RMR group, those with a lower than predicted RMR had increased BMI, with values of 52.9 vs. 44.7 kg/m², P = 0.001, respectively. Analysis of those demonstrating significant effort during cardiopulmonary exercise testing (peak respiratory exchange ratio ≥1.10) revealed a significantly higher peak oxygen uptake (VO₂ peak) in the higher RMR group (17.3 ± 3.5 ml/min/kg) compared with the lower RMR group (13.6 ± 1.9 ml/min/kg), P = 0.003. In summary, a lower than predicted RMR was associated with a severely reduced VO₂ peak and a higher BMI in this cohort. These data suggest that morbid obesity may be a vicious cycle of increasing BMI, reduced cardiorespiratory fitness, muscle deconditioning, and lower RMR. Collectively, these responses may, over time, exacerbate the imbalance between energy intake and expenditure, resulting in progressive increases in body weight and fat stores.     

Analyses of Monascus pigment secretion and cellular morphology in non‐ionic surfactant micelle aqueous solution

Monascus pigments produced by Monascus spp. are widely used as natural food colourants. Extractive fermentation technology can facilitate the secretion of intracellular Monascus pigments into extracellular non‐ionic surfactant micelle aqueous solution, so as to avoid the feedback inhibition and decomposition. In this study, behaviour of the trans‐membrane secretion of Monascus pigments was investigated using morphological and spectroscopic analyses. Laser scanning confocal microscopy (LSCM) traced that pigment secretion occurred through rapid trans‐membrane permeation in 4 min, with a simultaneous conversion in pigment characteristics. Approximately 50% of intracellular pigments (AU₄₇₀) extracted to extracellular broth with 40 g l^?1 Triton X‐100, indicating the capacity for pigment extraction was limited by the saturation concentrations of surfactant. Scanning electron microscope (SEM) and transmission electron microscope (TEM) imaging showed some damage in the cell wall but an intact cell membrane with a slightly increased mycelial diameter. However, the physiological properties of the cell membrane, including integrity, fluorescence intensity and permeability, were altered. A diagram was provided to demonstrate the behaviour of Monascus pigment secretion induced by Triton X‐100. This study lays a foundation for the further investigation of Monascus pigment metabolism and secretion in extractive fermentation.     

Improved Equations for Estimating the Resting Metabolic Rate

New models were developed for predicting the resting metabolic rate (RMR) with sufficient accuracy for use in epidemiologic studies and for weight control of individuals. For this purpose, the RMR of 213 women and 76 men was measured, and physical measurements were taken. The RMR was regressed on correlates of RMR, avoiding harmful degrees of collinearity by rejecting interregressor correlations exceeding r=0.5. For women, the best model (R₂=0.71) included the regressors age, race, weight, pulse rate, smoking, and body temperature. The best model for males (R2 = 0.81) included age, race, weight, blood pressure, smoking, time (of day the RMR was measured), and whether subjects had a meal prior to calorimetry. The models were cross validated internally and also validated using an external database. In both cases, the mean estimated RMR did not differ significantly from the measured RMR. The accuracy of the models was compared with four models reported in the literature, three of which overestimated the RMR by up to 17%. In conclusion, improved RMR prediction models have been developed, more accurate than existing models, rendering them suitable for application to epidemiological databases and for individual weight control programs.     

Production of the secondary metabolites γ-aminobutyric acid and monacolin K by <Emphasis Type="Italic">Monascus</Emphasis>

γ-Aminobutyric acid (GABA), a hypotensive agent, and monacolin K, a cholesterol-lowering drug, can be produced by Monascus spp. Under optimal culture conditions, the products of fermentation using Monascus spp. may serve as a multi-functional dietary supplement and can prevent heart disease. In this study, Monascus purpureus CCRC 31615, the strain with the highest amount of monacolin K, was identified from 16 strains using solid fermentation. Its GABA productivity was particularly high. Addition of sodium nitrate during solid-state fermentation of M. purpureus CCRC 31615 improved the productivity of monacolin K and GABA to 378 mg/kg and 1,267.6 mg/kg, respectively. GABA productivity increased further to 1,493.6 mg/kg when dipotassium hydrophosphate was added to the medium. Electronic Publication     

甘油影响红曲菌产色素的碳源选择性探究

红曲菌(Monascus spp.)是我国重要的药食同源微生物,红曲色素(Monascus pigments,MPs)是其主要次级代谢产物之一。有研究表明,甘油可促进红曲菌产MPs,但作用机制不明。以丛毛红曲菌(Monascus pilosus)MS-1为实验菌株,考察甘油与葡萄糖或蔗糖复合对红曲菌产MPs的影响。在不含碳源的合成培养基中,将甘油与葡萄糖或蔗糖复合,采用分光光度法和高效液相色谱法等分析MPs的产量和组分、生物量及发酵液pH。当甘油与葡萄糖复合,添加甘油后发酵液pH、生物量无显著变化(P0.05),总色价显著降低(P0.05)。当2 g/L或40 g/L甘油与蔗糖复合,发酵液pH显著降低而生物量及总色价显著增加(P0.05)。当40 g/L甘油与蔗糖复合时,总色价是仅以蔗糖为碳源时的16.5倍,且MPs同系物数量明显增多(P0.05)。在合成培养基条件下,甘油促进红曲菌产MPs具有碳源种类的选择性。该结果可为研究甘油影响红曲菌产MPs的作用机制提供参考,为甘油用于MPs生产提供依据。     

Differences in Resting Metabolic Rate between White and African‐American Young Adults

Objective: A reported lower resting metabolic rate (RMR) in African‐American women than in white women could explain the higher prevalence of obesity in the former group. Little information is available on RMR in African‐American men. Research Methods and Procedures: We assessed RMR by indirect calorimetry and body composition by DXA in 395 adults ages 28 to 40 years (100 African‐American men, 95 white men, 94 African‐American women, and 106 white women), recruited from participants in the Coronary Artery Risk Development in Young Adults (CARDIA), Birmingham, Alabama, and Oakland, California, field centers. Results: Using linear models, fat‐free mass, fat mass, visceral fat, and age were significantly related to RMR, but the usual level of physical activity was not. After adjustment for these variables, mean RMR was significantly higher in whites (1665.07 ± 10.78 kcal/d) than in African Americans (1585.05 ± 11.02 kcal/d) by 80 ± 16 kcal/d (p < 0.0001). The ethnic × gender interaction was not significant (p = 0.9512), indicating that the difference in RMR between African‐American and white subjects was similar for men and women. Discussion: RMR is ～5% higher in white than in African‐American participants in CARDIA. The difference was the same for men and women and for lean and obese individuals. The prevalence of obesity is not higher in African‐American men than in white men. Because of these reasons, we believe that RMR differences are unlikely to be a primary explanation for why African‐American women are more prone to obesity than white women.     

Environmental and genetic influences on body mass and resting metabolic rates (RMR) in a natural population of weasel Mustela nivalis

Body mass (BM) and resting metabolic rates (RMR) are two inexorably linked traits strongly related to mammalian life histories. Yet, there have been no studies attempting to estimate heritable variation and covariation of BM and RMR in natural populations. We used a marker‐based approach to construct a pedigree and then the ‘animal model’ to estimate narrow sense heritability (h²) of these traits in a free‐living population of weasels Mustela nivalis—a small carnivore characterised by a wide range of BM and extremely high RMR. The most important factors affecting BM of weasels were sex and habitat type, whereas RMR was significantly affected only by seasonal variation of this trait. All environmental factors had only small effect on estimates of additive genetic variance of both BM and RMR. The amount of additive genetic variance associated with BM and estimates of heritability were high and significant in males (h² = 0.61), but low and not significant in females (h² = 0.32), probably due to small sample size for the latter sex. The results from the two‐trait model revealed significant phenotypic (r_P = 0.62) and genetic correlation (r_A = 0.89) between BM and whole body RMR. The estimate of heritability of whole body RMR (0.54) and BM corrected RMR (0.45) were lower than estimates of heritability for BM. Both phenotypic and genetic correlations between BM corrected RMR and BM had negative signals (r_P = ?0.42 and r_A = ?0.58). Our results indicate that total energy expenditures of individuals can quickly evolve through concerted changes in BM and RMR.