Expression of pigmentation genes following electroporation of albino<Emphasis Type="Italic"> Monascus purpureus</Emphasis>

A UV-induced albino strain of Monascus purpureus was subjected to electroporation in the presence of genomic DNA from a wild-type red strain of the fungus. Eight colonies expressed color after several weeks of growth. The growth rates of all eight color variants were significantly greater than the recipient and donor strains under some culture conditions. Spectrophotometric analysis of the pigments extracted from the color variants revealed the pigments had absorbance spectra different from the DNA donor strain. These color variants may have resulted from transformation with wild-type DNA, mutation reversion, or activation of alternative pathway(s)—i.e., new mutations—that resulted in pigment production.     

Production of pigments byMonascus purpureus using sugar-cane bagasse in roller bottle cultures

Solid-state fermentation, using sugar-cane bagasse, and submerged fermentation, using a semi-synthetic medium, were performed for pigment production byMonascus purpureus in both stationary and rotary conditions. Rotary cultures gave higher yields of crude red and yellow pigments than stationary cultures whereas twice the amount was synthesized at an earlier time (day 8) in liquid medium (1,285U yellow pigment/bottle, 1,728U red pigment/bottle). Supplementing the liquid medium with 0.6% (v/v) corn oil doubled the extracellular pigment yield but halved fungal growth.     

Characterization of an hyperpigmenting mutant of Monascus purpureus IB1: identification of two novel pigment chemical structures

Monascus purpureus IB1 produces about 50-fold higher levels of azaphilone pigments than M. purpureus NRRL1596. Differently pigmented mutants were obtained from M. purpureus IB1 by nitrosoguanidine treatment. A highly pigmented strain, M. purpureus HP14, was found to lack the formation of the classical yellow and orange azaphilones and was found to produce only about 10% of the red azaphilone pigments. The intense color was associated with novel pigments as shown by high-performance liquid chromatography (HPLC). The addition of hexanoic acid to M. purpureus IB1 resulted in higher volumetric and specific red pigment productivity, but in a complete absence of the classical orange azaphilones, while the classical yellow and red azaphilone pigments were severely reduced; new peaks corresponding to less hydrophobic pigments were found in hexanoic-supplemented cultures by HPLC. Purification of pigments from hexanoic-supplemented cultures showed the presence of five new pigments as indicated by the absorption spectra and HPLC analysis. Two of them, R3 and Y3, were characterized by nuclear magnetic resonance as 9-hexanoyl-3-(2-hydroxypropyl)-6a-methyl-9,9a-dihydro-6H-furo[2,3-h]isochromene-6,8(6aH)-dione and 4-[2,4-dihydroxy-6-(3-hydroxybutanethioyloxy)-3-methylphenyl]-3,4-dihydroxy-3,6-dimethylheptanoic acid. These pigments were also found to be present in cultures of the high-producing mutant M. purpureus HP14. These new pigments are less hydrophobic than the classical azaphilones and may have better properties as natural colorants in the food industry.     

Effect of pH on citrinin and red pigments production by Monascus purpureus CCT3802

The production of red pigments and citrinin by Monascus purpureus CCT3802 was investigated in submerged batch cultures performed in two phases: in the first phase, cells were grown on glucose, at pH 4.5, 5.5 or 6.5; after glucose depletion, pH was adjusted, when necessary, to 4.5, 5.5, 6.5, 7.0, 8.0 or 8.5, for a production phase. The highest total red pigments absorbance of 11.3 U was 16 times greater than the lowest absorbance and was achieved with growth at pH 5.5, followed by production at pH 8.5, which causes an immediate reduction of the intra cellular red pigments from 75% to 17% of the total absorbance. The lowest citrinin concentration, 5.5 mg L⁻¹, was verified in the same culture while the highest concentration, 55 mg L⁻¹, was verified in cultures entirely carried out at pH 5.5. An alkaline medium, besides promoting intra cellular red pigments excretion, strongly represses citrinin synthesis.     

Effect of light on growth,pigment production and culture morphology of <Emphasis Type="Italic">Monascus purpureus</Emphasis> in solid-state fermentation

The capacity to sense and respond to light is widespread in animals, plants, fungi and bacteria. The effect of light quality on growth and pigment yield of Monascus purpureus was investigated. Incubation in total darkness increased red pigment production from 14. 5 OD/g dry substrate to 22 OD/g dry substrate. In contrast, growth of the fungus in direct illumination resulted in total suppression of pigment production. It was found that both red and blue light influenced pigment yield as well as culture morphology. The authors propose the existence of a light-perception system in Monascus purpureus.     

Response surface optimization of mixed substrate solid state fermentation for the production of lovastatin by Monascus purpureus

The objective of this work is to enhance the production of lovastatin using Monascus purpureus MTCC 369 in mixed substrate solid state fermentation using various solid substrates and to optimize the combination of the solid substrates by response surface methodology. Solid state fermentation was conducted in a 250 mL Erlenmeyer flask at 30°C for 14 days with initial moisture content of 40% and inoculum size of 10% active culture. Barley, long grain rice and sago starch were found to be the suitable substrates producing maximum lovastatin of 193.7 mg, 190.2 mg and 180.9 mg/g of dry solids. These substrates were further used in various combinations as designed by the central composite design for enhancing the lovastatin production using Monascus purpureus. To the best of our knowledge this is the first report on the production of lovastatin using a mixed substrate solid state fermentation using Monascus purpureus.     

Submerged fermentation in wheat substrates for production of Monascus pigments

Cereal grains are normally used as solid substrates for the production of Monascus metabolites. However, solid fermentation in these substrates requires complex control systems, whereas in liquid culture the control of the fermentation is simpler and consequently significant reductions in fermentation times can be achieved. In the same way, the use of submerged culture can benefit the production of many secondary metabolites and decrease production costs by reducing the labour involved in solid-state methods. A flour composed of a mixed variety of Canadian hard wheat was used as sole nutrient source to produce the pigments of Monascus purpureus Went (IMI 210765). Supplementation with NH₄Cl promoted biomass and orange dye formation, whereas the use of zinc sulphate favoured red dyes production. In submerged fermentations significant differences in final pigment yields were observed in the use of wheat-based broth at different concentrations in the presence of bran particles and/or gluten protein. It has been found that the viscosity of the broth had a significant effect on the growth morphology and production of pigments. Gluten-free wheat flour at concentrations of 3–5% was found to be the most suitable for liquid Monascus culture. The subsequent use of passive immobilization of Monascus served to enhance red pigment yields and to facilitate the downstream processing of the dyes.     

The influence of tapioca on the growth,the activity of glucoamylase and pigment production of <Emphasis Type="Italic">Monascus purpureus</Emphasis> UKSW 40 in soybean-soaking wastewater

The present study evaluates the usefulness of tapioca starch as additional carbon source for the growth of Monascus purpureus in soybean-soaking wastewater (SSW). The result revealed that M. purpureus grown on 2.0% (w/v) tapioca starch in SSW produced significantly (P < 0.05) higher amounts of biomass and production of the pigments (OD₄₀₀ and OD₅₀₀) when compared to those grown on glucose-or maltose-containing media. However, the glucoamylase activity of M. purpureus grown on the tapioca-SSW medium was not significantly increased when compared to those from the glucose-containing medium.     

Characterization of a non-pigment producing Monascus purpureus mutant strain

A characterization of a non-pigment producing mutant Monascus purpureus M₁₂ compared with its parental strain Monascus purpureus Went CBS 109.07 has been performed aiming to investigate the relation between pigment biosynthesis and other characteristics of these fungi. A comparison has been made of morphological features, some physiological properties and biochemical activities of both strains. The albino mutant exhibits an anamorph life cycle, high conidia forming capability, slower radial growth rate and temperature sensitivity. The assimilation capacity of both strains for mono-, disaccharides and some alcohols is in the same range (Y_X/C 0.2 – 0.35), while the red strain has a higher fermentation capacity. In a selected albino mutant, the growth rate, metabolic activity and capacity for production of typical for Monascus fungi secondary metabolites were reduced considerably. Hydrolytic activity towards natural substrates expressed through glucoamylase and protease was approximately 10 fold lower in the non pigment producing strain (0.05 – 0.08 U/mg protein and 0.01 – 0.07 U/mg protein respectively) compared with the red one. Important qualitative differences between both strains was found in fatty acid composition and in the production of citrinin and monacolin. The mutant strain possessed C₁₇, C₂₀ and C₂₂ fatty acids and did not produce citrinin. This revised version was published online in June 2006 with corrections to the Cover Date.     

Effect of pH and nitrogen source on pigment production by Monascus purpureus

The effect of pH and nitrogen source on pigment production by Monascus purpureus 192F using glucose as the carbon and energy source, was studied in pH-controlled, batch fermentor cultures using HPLC analysis to determine individual pigment concentrations. A maximum of four pigments were detected in fungal extracts. These were the yellow pigments monascin and ankaflavin, the orange rubropunctatin and the red pigment monascorubramine. Monascorubramine was present as the major product in all instances. Fungal growth and ankaflavin synthesis were favoured at low pH (pH 4.0), whereas production of the other pigments was relatively independent of pH. The nature of the nitrogen source affected fungal growth and pigment production, independent of pH. Ammonium and peptone as nitrogen sources gave superior growth and pigment concentrations compared to nitrate. Ankaflavin was not detected in nitrate cultures. The highest red pigment production was obtained using a glucose-peptone medium at pH 6.5, due to the secretion of red pigments into the medium under these conditions. Correspondence to: M. R. Johns     

Anka and anka pigment production

This study was conducted to determine the time-dependent changes of solid-state fermentation of rice with Monascus purpureus to produce anka and anka pigments. Growth of the fungus occurred prior to the synthesis of anka pigments. A steady increase in the yield of pigments occurred between the 5th and 15th days. After 15 days, growth of the fungus on rice substrate ceased and the yield of yellow anka pigments remained constant; however, orange anka pigments were reduced with a decreasing rate of 3.6 mg/g anka/day. Journal of Industrial Microbiology & Biotechnology (2001) 26, 280–282. Received 27 December 1999/ Accepted in revised form 27 December 2000     

Genetic transformation of Monascus purpureus DSM1379

Monascus purpureus was transformed into hygromycin B resistance with hygromycin B phosphotransferase (hph) fused to Aspergillus nidulans trpC or a putative Monascus purpureus gpd1 promoter by electroporation. Among five strains, only M. purpureus DSM1397 was a competent recipient. Normal growth and sporulation on media containing up to 500 mg hygromycin B l^–1 occurred up to five generations. Upon transformation of the strain with the green fluorescent protein gene (sgfp) as a model gene and hph as a selection marker, characteristic green fluorescence was observed under fluoromicroscopy indicating successful transformation.     

荧光物质(MFA、MFB)的分离、结构鉴定及金华地区高产菌株的筛选

采用硅胶柱层析及制备型液相色谱仪对红曲米中两种荧光物质进行分离纯化,使用高效液相色谱法(HPLC)检测荧光物质纯度,然后使用高分辨质谱(ESI-HRMS)对两种荧光物质进行分析,得到两种荧光物质的分子量分别为356和384,ESI-MS/MS二级质谱把两者鉴定为monasfluore A (MFA)和monasfluore B (MFB);从金华地区红曲米中分离得到10株红曲菌株,经固态发酵采用HPLC法分析,筛选获得1株高产MFA、MFB的菌株WZWZ,该菌株发酵制得红曲米中MFA含量为3.63 g/kg,MFB含量为7.29 g/kg,对WZWZ菌株进行外观形态学及显微观察、ITS基因序列测定与分析,最终将菌株WZWZ鉴定为紫色红曲霉(Monascus purpureus)。     

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     

Photoautotrophic Production of Lipids by Some <Emphasis Type="Italic">Chlorella</Emphasis> Strains

The microalgae Chlorella protothecoides UTEX 25, Chlorella sp. TISTR 8991, and Chlorella sp. TISTR 8990 were compared for use in the production of biomass and lipids under photoautotrophic conditions. Chlorella sp. TISTR 8990 was shown to be potentially suitable for lipid production at 30°C in a culture medium that contained only inorganic salts. For Chlorella sp. TISTR 8990 in optimal conditions in a stirred tank photobioreactor, the lipid productivity was 2.3 mg L⁻¹ h⁻¹ and after 14 days the biomass contained more than 30% lipids by dry weight. To attain this, the nitrogen was provided as KNO₃ at an initial concentration of 2.05 g L⁻¹ and chelated ferric iron was added at a concentration of 1.2 × 10⁻⁵ mol L⁻¹ on the ninth day. Under the same conditions in culture tubes (36 mm outer diameter), the biomass productivity was 2.8-fold greater than in the photobioreactor (0.125 m in diameter), but the lipid productivity was only 1.2-fold higher. Thus, the average low-light level in the photobioreactor actually increased the biomass specific lipid production compared to the culture tubes. A light-limited growth model closely agreed with the experimental profiles of biomass production, nitrogen consumption, and lipid production in the photobioreactor.     

Pigmentation and Antibacterial Activity of Fast Neutron- and X-Ray-induced Strains of Monascus purpureus Went

Seven new strains of Monascus purpureus Went were induced by neutron and x-ray irradiation. The quantity and quality of pigments produced by these strains differed. Strains N4S and N11S produced twice as much pigment as normal, while another strain, N14S, was albino. An unknown orange pigment was found in young colonies of the N11S strain. This orange pigment reacted with alcohols and malt extract medium to form red pigments. Strains N4S, N11S, X2P, and wild type inhibited the growth of certain bacteria, especially the Bacillus species. Strain N11S had more antibacterial activity than wild type. A major active compound was isolated with an ultraviolet absorption spectrum that was related to those of the red pigments found in this fungus. The active compound(s) was named monascidin.     

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.     

A new process for red pigment production by submerged culture of Monascus purpureus

Formation of red pigment by Monascus purpureus via diauxic growth on glucose and ethanol in submerged culture was optimized based on inoculum preparation and culture medium. A vegetative inoculum was prepared from spores grown on ethanol. The optimized culture medium was low in phosphates, and had an initial pH?of 5.5. The characteristics of Monascus purpureus grown on glucose and on ethanol were compared: the specific consumption rate of glucose (q_G) was higher than the specific consumption rate of ethanol (q_E), whereas the specific growth rate was greatest with ethanol. The specific production rate of red pigment (p_OD) and pigment yield (Y_OD/s) with glucose was twice that with ethanol. A novel fermentation process was developed with M. purpureus initially grown with controlled ethanol formation, and consumption of the latter during pigment formation.     

Suspended rice particles for cultivation of Monascus purpureus in a tower-type bioreactor

 Cultivation of Monascus purpureus (CCRC 31615) for the production of natural pigments was investigated. Traditionally, Monascus species were grown on rice by solid-state culture. For large-scale cultivation, solid-state cultures were associated with some problems such as contamination and scale-up. By using submerged cultures with rice particles, a stirred-tank fermentor was not suitable for submerged cultures as the impeller tended to break the particles into small pieces. A conventional bubble column was also unsuitable as its mixing capability was poor. In the present study, a modified bubble column with wire-mesh draft tubes was employed for the cultivation of M. purpureus. The proposed column had a shorter mixing time and a higher oxygen transfer rate relative to the conventional bubble column. The production of pigments using the proposed column was up to 80% higher than that achieved using the conventional bubble column. Received: 21 July 1999 / Received revision: 8 November 1999 / Accepted: 19 November 1999