Development of monacolin K-enriched ganghwayakssuk (Artemisia princeps Pamp.) by fermentation with Monascus pilosus

Monacolin K-enriched ganghwayakssuk (Artemisia princeps Pamp.) was developed by fermentation with Monascus sp. Among the 15 Monascus spp. isolated previously from Monascus fermentation products, Monascus pilosus KMU108 produced 2,219 mg/kg of monacolin K during ganghwayakssuk fermentation with no detectable citrinin. The optimum concentrations of ganghwayakssuk and glucose determined from the response surface methodology (RSM) design were 2.2% and 3.8%, respectively. By applying these conditions, the monacolin K productivity was increased to 3,007 mg/kg after 15 days of fermentation. On the other hand, other characteristics such as the total content of flavonoids and phenolic compounds, and the antioxidant activity were relatively unchanged. Therefore, Monascusfermented ganghwayakssuk is an excellent biomaterial for the development of functional foods because of its high level of monacolin K, known to lower cholesterol levels.     

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.     

不产桔霉素的红曲霉菌种深层发酵生产莫纳可林K

对三株在YES培养基中不产桔霉素的红曲霉菌种,在摇瓶中研究了它们液体发酵生产莫纳可林K的情况。在大米粉培养基中,红色红曲霉不产莫纳可林K,但是紫色红曲霉和烟灰色红曲霉均能产莫纳可林K,前者产量高于后者。在葡萄糖．甘油培养基中,后两者的产量均很低,但是如果在该培养基中添加酵母膏,紫色红曲霉能产生较为可观的莫纳可林K。在2L的发酵罐中,利用添加了酵母膏的葡萄糖-甘油培养基,紫色红曲霉在第13d的莫纳可林K产量可达104mg/L,培养过程中无桔霉素产生。     

Monacolin K production by citrinin‐free Monascus pilosus MS‐1 and fermentation process monitoring

Monacolin K (MK) is a naturally occurring hypocholesterolemic agent that specifically inhibits HMG‐CoA reductase. As a natural source of MK, Monascus‐fermented products are of special interest; however, some Monascus strains could produce citrinin, which is a nephrotoxin, as a contaminant in Monascus‐derived products. A Monascus pilosus strain (MS‐1) that produces high amounts of MK, but no citrinin, was screened in previous investigations. Herein, liquid‐state fermentation parameters of the MS‐1 strain were optimized using statistical methods to maximize the MK yield with potato juice as a basic medium. The maximum MK yield (326.74 μg/mL) was predicted with 50 mL of medium in a 250‐mL conical flask containing 30 g/L sucrose, 38.75 g/L soybean flour, 0.00105 mol/L Mg²⁺ at pH 5.48, and 8% v/v seed inoculum precultured for 42 h at 30°C, incubated at 30°C for 3 days, followed by further incubation for 11 days at 24.7°C. The verified MK yield was 390.68 μg/mL and the MK yield increased to 565.64 μg/mL after 21 days of fermentation. No citrinin was detected in MS‐1‐fermented products. The results suggest that citrinin‐free MK can be obtained from natural medium through liquid‐state fermentation in an economical way. This method will be of practical value to the industrial production of MK.     

固体发酵生产Monacolin K的动力学模型

对红曲霉菌株Monacus-3合成Monacolin K的发酵动力学进行了研究,建立了该菌株固体发酵合成Monacolin K的菌体生长、产物合成和底物消耗的动力学模型,并对模型参数进行了非线形回归。结果表明预测值与实验值有良好的拟和性,说明所建数学模型能较好地描述实际合成Monacolin K的固体发酵过程。     

Elevated yield of monacolin K in Monascus purpureus by fungal elicitor and mutagenesis of UV and LiCl

In China, Monascus spp., a traditional fungus used in fermentation, is used as a natural food additive. Monascus spp. can produce a secondary metabolite, monacolin K namely, which is proven to be a cholesterol-lowering and hypotensive agent. Hence, recently, many researchers have begun focusing on how to increase the production of monacolin K by Monascus purpureus. In the present study, we investigated the effect of the fungal elicitor and the mutagenesis of UV & LiCl on the amount of monacolin K produced by Monascus purpureus. The fugal elicitor, Sporobolomyces huaxiensis, was isolated from tea leaves and its filtrate was added into the culture filtrate of Monascus purpureus during growth to induct the production of monacolin K. The results showed that the highest amount of monacolin K produced by the liquid fermentation was 446.92 mg/mL, which was produced after the fungal elicitor was added to the culture filtrate of Monascus purpureus on the day 4; this amount was approximately 6 times greater than that of the control culture filtrate, whereas the highest amount of monacolin K produced by the mutated strain was 3 times greater than the control culture after the irradiation of UV light in the presence of 1.0 % LiCl in the medium.     

Changes of ginsenosides in Korean red ginseng (Panax ginseng) fermented by Lactobacillus plantarum M1

To obtain microorganisms for the microbial conversion of ginsenosides in red ginseng powder (RGP), Lactobacillus species (M1–M4 and P1–P4) were isolated from commercial ginseng products. Strain M1 was determined to be L. plantarum by 16S rRNA sequencing. Red ginseng powder (RGP) fermented by L. plantarum M1 had a high total content of ginsenosides (142.4 mg/g) as compared to the control (121.8 mg/g). In particular, the ginsenoside metabolites Rg3, Rg5, Rk1, Compound K (CK), Rh1, and Rg2 showed a high level in the fermented RGP (65.5 mg/g) compared to the control (32.7 mg/g). During fermentation for 7 days, total sugar content decreased from 8.55 mg/g to 4 mg/g, uronic acid content reached its maximum (53.43 μg/g) at 3 days, and total ginsenoside content increased to 176.8 mg/g at 4 days. In addition, ginsenoside metabolites increased from 38.0 mg/g to 83.4 mg/g at 4 days of fermentation. Using everted instestinal sacs of rats, the fermented red ginseng showed a high transport level (10.3 mg of polyphenols/g sac) compared to non-fermented red ginseng (6.67 mg of polyphenols/g sac) after 1 h. These results confirm that fermentation with L. plantarum M1 is very useful for preparing minor ginsenoside metabolites while being safe for foods.     

种间双亲灭活原生质体融合选育高产Monacolin K红曲菌

为获得高产MonacolinK的红曲菌菌株,将经农杆菌介导转化获得的携带潮霉素抗性基因且以甘油为原料液态发酵高产MonacolinK的发白红曲菌H2和以大米为原料固态发酵产Monaco-1inK的烟色红曲菌9908作为亲本,对其原生质体分别进行热灭活及紫外灭活,然后对灭活双亲用PEG作融合剂进行原生质体融合。从融合子中选出有潮霉素抗性的突变株,通过发酵与亲本对比,筛选得到一株以大米为原料固态发酵高产MonacolinK的融合株F12．11,其MonacolinK产量达到8．73mg／g;较发白红曲菌H2与烟色红曲菌9908分别提高了100．23％和48．98％;一株以甘油为原料液态发酵高产MonacolinK的融合株F13-2,其MonacolinK的产量达到1752．46mg／L,较发白红曲菌H2与烟色红曲菌9908分别提高了32．98％和1979．33％。     

Breeding of Monascus spp.with high monacolin K production by interspecific protoplast fusion of inactivated parental strains

为获得高产Monacolin K的红曲菌菌株,将经农杆菌介导转化获得的携带潮霉素抗性基因且以甘油为原料液态发酵高产Monacolin K的发白红曲菌H2和以大米为原料固态发酵产Monaco-lin K的烟色红曲菌9908作为亲本,对其原生质体分别进行热灭活及紫外灭活,然后对灭活双亲用PEG作融合剂进行原生质体融合.从融合子中选出有潮霉素抗性的突变株,通过发酵与亲本对比,筛选得到一株以大米为原料固态发酵高产Monacolin K的融合株F12-11,其Monacolin K产量达到8.73 mg/g;较发白红曲菌H2与烟色红曲菌9908分别提高了100.23％和48.98％;一株以甘油为原料液态发酵高产Monacolin K的融合株F13-2,其Monacolin K的产量达到1752.46 mg/L,较发白红曲菌H2与烟色红曲菌9908分别提高了32.98％和1979.33％.     

乙酸钠、烟酸对红曲菌白色变种产Monacolin K的影响

[背景]红曲是由红曲菌寄生在大米发酵而成的一种食用米曲,含有胆固醇抑制剂Monacolin K,但市售红曲中酸式Monacolin K含量低且普遍呈红色,其应用存在局限性.红曲菌白色变种3001-18具有不产色素和桔霉素而高产酸式Monacolin K的优点.[目的]研究微量营养物对红曲菌固态发酵Monacolin K...     

高产Monacolin K纯种红曲培养条件的研究

本实验对已有的十株红曲霉菌株进行纯种制曲,筛选出一株生长良好、Monacolin K含量高、不产桔霉素、产色素能力强及糖化酶活力相对较高的红曲霉菌株.以大米为原材料,采用液固联合制曲法,通过单因素实验对纯种红曲的培养条件进行优化.结果表明,固体培养基初始含水量为45％,接种量为8％,变温培养红曲时,红曲霉M-7所得纯种红曲最佳,其Monacolin K含量高达243 μg/g,色价450 U/g,糖化酶活力4 289 U/g.与优化之前比较,Monacolin K含量提高了37.2％.     

Effects of the principal nutrients on lovastatin production by Monascus pilosus

Lovastatin production is dependent on the substrates provided. We investigated how several carbon and nitrogen sources in the medium affect lovastatin production by Monascus pilosus. M. pilosus required a suitable concentration of organic nitrogen peptone for high lovastatin production. As sole carbon source with peptone, although glucose strongly repressed lovastatin production, maltose was responsible for high production. Interestingly, glycerol combined with maltose enhanced lovastatin production, up to 444 mg/l in the most effective case. Moreover, an isolated mutant, in which glucose repression might be relieved, easily produced the highest level of lovastatin, 725 mg/l on glucose-glycerol-peptone medium. These observations indicate that lovastatin production by M. pilosus is regulated by strict glucose repression and that an appropriate release from this repression by optimizing medium composition and/or by a mutation(s) is required for high lovastatin production.     

Secondary metabolites produced by the fungus Monascus pilosus and their anti-inflammatory activity

A detailed phytochemical study of the n-BuOH-soluble fraction of the 95% EtOH extract of red yeast rice fermented with the fungus Monascus pilosus BCRC 38072 (Eurotiaceae) revealed a new azaphilone derivative, namely monascusazaphilol (1) (rel-(3S,3aR,9aR)-3-(1-hydroxydecyl)-9a-methyl-6-((E)-propenyl)-3a,4,8,9a-tetrahydro-3H-furo[3,2-g]isochromene-2,9-dione), along with ten known compounds (2–11). Their structures were established by direct interpretation of their spectral data, mainly high resolution electrospray ionization mass spectrometry (HR-ESI-MS), 1D and 2D NMR (COSY, ROESY, HSQC and HMBC), and comparison of the spectroscopic data with those reported for structurally related compounds. We also evaluated the inhibitory effects of some isolates on the production of tumor necrosis factor (TNF-α) induced by lipopolysaccharide (LPS). Among the isolates, compounds 1–3 inhibited TNF-α production in U937 cells in vitro, and the IC₅₀ values were 1.24, 2.35 and 3.75 μg/ml, respectively.     

Simultaneous determination of ginsenosides in Panax ginseng with different growth ages using high-performance liquid chromatography-mass spectrometry

The contents of ginsenosides in Panax ginseng not only vary in different parts of the root, but also exhibit yearly variation. In this study, an HPLC-MS method was established in order to simultaneously analyse ginsenosides Rb1, Rb2, Rb3, Rc, Rd, Re, Rf, Rg1 and Rg2. The concentration of ginsenosides in the tap root and root fibre were compared and the yearly variations of nine ginsenosides elucidated. The results indicate that the total content of ginsenosides in the main root and the root fibre both attain a maximum level in the fourth year of growth, although the amount in the former is much higher than in the latter. The variation in the content of ginsenosides during a 2-6 year period suggests that cultivated P. Ginseng can be harvested after the fourth year. The current results will provide useful information for the quality control and good agricultural practice farming of ginseng.     

Selection of an effective red-pigment producing Monascus pilosus by efficient transformation with aurintricarboxylic acid

The filamentous fungus Monascus pilosus was genetically transformed with a reporter plasmid, pMS-1.5hp, by aurintricarboxylic acid (ATA) treatment to obtain an efficient red-pigment producing mutant. The transformation efficiency of Monascus pilosus was higher with the ATA-treatment than with either a non-restriction-enzyme-mediated integration (REMI) or a REMI method. This valid and convenient random mutagenesis method shows that ATA can be applied in fungi for efficient genetic transformation.     

Bifidus fermentation increases hypolipidemic and hypoglycemic effects of red ginseng

Antihyperlipidemic and antihyperglycemic effects of Red Ginseng (RG, steamed and dried root of Panax ginseng C. A. Meyer, family Araliaceae), major component of which is ginsenoside Rg3, and Bifidodoterium-fermented RG (FRG), major component of which is ginsenoside Rh2, were investigated. Orally administered RG and FRG potently reduced the serum triglyceride levels in corn-oil-induced hypertriglycemidemic mice as well as total cholesterol and triglyceride levels in Triton WR-1339-induced hyperlipidemic mice. Of the saponin and polysaccharide fractions of RG and FRG, the polysaccharide fraction inhibited postprandial blood glucose elevation of maltose- or starch-loaded mice and reduced the blood triglyceride levels in corn-oil-induced hypertriglycemidemic mice. The saponin fraction and its ginsenosides Rg3 and Rh2 reduced blood triglyceride and total cholesterol levels in Triton WR1339-induced hyperlipidemic mice. The inhibitory effect of FRG and its main constituents against hyperlipidemia and hyperglycemia in mice were more potent than those of RG. These findings suggest that hypolipidemic and hypoglycemic effects of RG can be enforced by Bifidus fermentation and FRG may improve hyperlipidemia and hyperglycemia.     

Accelerated solvent extraction of monacolin K from red yeast rice and purification by high-speed counter-current chromatography

Monacolin K from red yeast rice was extracted by accelerated solvent extraction (ASE). The effects of various extraction parameters including extraction temperature, static extraction time and cycle index on yield were investigated using a DIONEX ASE 300 system to select the optimal conditions by an orthogonal test design L₉ (3)³. The optimum extraction conditions were determined as follows: extraction temperature 120 °C, static extraction time 7 min, and cycle index 3. Under the optimal conditions, the yield of ASE extract and monacolin K was 5.35% and 9.26 mg/g of dry red yeast rice, respectively. A separation and purification method of monacolin K was then established using high-speed counter-current chromatography (HSCCC) with a two-phase solvent system composed of n-hexane–ethyl acetate–methanol–water (8:2:5:5, v/v/v/v). From 300 mg of crude extract, 51.2 mg of monacolin K was obtained with the purity of 98.7%. The chemical structure of isolated compound was identified by UV, ESI-MS and ¹H NMR.     

红曲霉液态发酵生产Monacolin K培养基的优化

利用正交试验设计对红曲霉TQ-57发酵生产Monacolin K的摇瓶发酵培养基进行了初步研究。结果表明:Monacolin K的最适发酵碳源为甘油,最佳添加质量分数为14%,最适氮源为蛋白胨,最佳氮源质量分数为3%,培养基初始pH为6.5,培养周期为8 d,在此条件下Monacolin K产量可达27.75 mg/L。     

Production, Purification, and Characterization of alpha-Galactosidase from Monascus pilosus

A Monascus pilosus strain was selected for production of intracellular alpha-galactosidase. Optimum conditions for mycelial growth and enzyme induction were determined. Galactose was one of the best enzyme inducers. The enzyme was purified by ammonium sulfate precipitation, gel filtration, and ion exchange chromatography and was demonstrated to be homogeneous by slab gel electrophoresis. The molecular weight of this enzyme, estimated by gel filtration, was about 150,000. The optimum conditions for the enzyme reaction was pH 4.5 to 5.0 at 55 degrees C. The purified enzyme was stable at 55 degrees C or below and in buffer at pH 3 to 8. The activity was inhibited by mercury, silver, and copper ions. The kinetics of this enzyme, with p-nitrophenyl-alpha-d-galactoside as substrate, was determined: K(m) was about 0.8 mM, and V(max) was 39 mumol/min per mg of protein. Enzymatic hydrolysis of melibiose, raffinose, and stachyose was analyzed by thin-layer chromatography.