Production of Monascus-Pigment in a Submerged Culture

The production of pigment by the molds belonging to the genus Monascus in a submerged culture was examined. The extracellular pigment was mainly studied. Monascus sp. No. 2 was found to be the most potent pigment producer. The optimum cultural conditions were: pH of the medium, 6.5; the temperature, 25°C; carbon sources, glucose or ethyl alcohol; nitrogen sources, polypeptone, yeast extract, monosodium glutamate or casamino acids. Glycine, l-threonine, l-arginine, l-alanine and l-tyrosine were found to be the most effective substances promoting pigment production.

Mycelial forms of this strain were correlated with pigment formation in submerged culture. As it grew into pellet type, the yield of pigment was at high level.

The Monascus-pigment in the fermentation liquid seemed to be firmly bound to the protein-like substances which made the pigment apparently soluble.     

Perstraction of intracellular pigments by submerged cultivation of <Emphasis Type="Italic">Monascus</Emphasis> in nonionic surfactant micelle aqueous solution

“Milking processing” describes the cultivation of microalgae in a water-organic solvent two-phase system that consists of simultaneous fermentation and secretion of intracellular product. It is usually limited by the conflict between the biocompatibility of the organic solvent to the microorganisms and the ability of the organic solvent to secret intracellular product into its extracellular broth. In the present work, submerged cultivation of Monascus in the nonionic surfactant Triton X-100 micelle aqueous solution for pigment production is exploited, in which the fungus Monascus remains actively growing. Permeabilization of intracellular pigments across the cell membrane and extraction of the pigments to the nonionic surfactant micelles of its fermentation broth occur simultaneously. “Milking” the intracellular pigments in the submerged cultivation of Monascus is a perstraction process. The perstractive fermentation of intracellular pigments has the advantage of submerged cultivation by secretion of the intracellular pigments to its extracellular broth and the benefit of extractive microbial fermentation by solubilizing the pigments into nonionic surfactant micelles. It is shown as the marked increase of the extracellular pigment concentration by the submerged cultivation of Monascus in the nonionic surfactant Triton X-100 micelle solution.     

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.     

Optimization of monascus red pigment fermentation by regulating chitinase activity level in fermentor

Enhancement of monascus pigment production in a co-culture of Monascus sp. J101 with Saccharomyces cerevisiae was accomplished using chitinase in the feeding of a S. cerevisiae culture filtrate. Batch fermentations were carried out at constant chitinase activity levels of 0.8?U and 0.5?U after 24?h of cultivation. Monascus red pigment concentrations obtained at the end of fermentation were 182.4 OD units and 147.3 OD units, respectively, for chitinase activity levels of 0.8?U and 0.5?U. The cell mass was higher at 0.8?U than at 0.5?U, whereas pigment production per unit cell mass was higher at 0.5?U than at 0.8?U. Kinetic equations involving cell growth rate, cell activity and chitinase activity levels were analyzed. Cell activity was expressed as a function of mean cell age. The optimum time for reduction of the chitinase activity level from 0.8?U to 0.5?U for maximization of monascus pigment production was numerically estimated to be 60?h. Based on this result, a batch fermentation was performed where the chitinase activity level was maintained at 0.8?U from 24?h to 60?h, then the level was lowered to 0.5?U after 60?h. This scheme resulted in a monascus pigment production of 198.3 OD units.     

Releasing intracellular product to prepare whole cell biocatalyst for biosynthesis of <Emphasis Type="Italic">Monascus</Emphasis> pigments in water–edible oil two-phase system

Selective releasing intracellular product in Triton X-100 micelle aqueous solution to prepare whole cell biocatalyst is a novel strategy for biosynthesis of Monascus pigments, in which cell suspension culture exhibits some advantages comparing with the corresponding growing cell submerged culture. In the present work, the nonionic surfactant Triton X-100 was successfully replaced by edible plant oils for releasing intracellular Monascus pigments. High concentration of Monascus pigments (with absorbance nearly 710 AU at 470 nm in the oil phase, normalized to the aqueous phase volume approximately 142 AU) was achieved by cell suspension culture in peanut oil–water two-phase system. Furthermore, the utilization of edible oil as extractant also fulfills the demand for application of Monascus pigments as natural food colorant.     

Negative effect of ammonium nitrate as nitrogen source on the production of water-soluble red pigments by Monascus sp.

Ammonium salts, especially ammonium nitrate, have been used as nitrogen sources for production of traditional water-insoluble Monascus pigments. However, we noted that defined media employing NH₄NO₃ as the sole nitrogen source in fermentations supported only poor pigment production by Monascus sp., and the pigments produced were mainly cell-bound. NH₄NO₃ was found not to (a) repress pigment synthase formation, (b) enhance synthase decay, or (c) serve as a nitrogen source for pigment production by resting cells; it had a weak inhibitory effect on the action of pigment synthase(s). The high level of cell-bound did not exert a feedback effect on the further synthesis of pigments. These observations indicate that the reason why NH₄NO₃ supports only low pigment production during fermentations is the poor ability of NH₄NO₃ to donate nitrogen in the Schiff-base reaction converting orange pigments to red ones.     

Fermentation and quality of yellow pigments from golden brown rice solid culture by a selected Monascus mutant

A single peak (λmax 370) yellow pigment-producing mutant derived from Monascus sp. TISTR 3179 was used for the pigment production in solid rice culture. Various factors affecting yellow tones were investigated. Hom-mali rice variety was the best amongst five Thai local varieties used for fungus culture. It was also better than corn, mungbean, soybean, potato, sweet potato, or cassava tubers. The moisture content and temperature were the key environmental factors affecting the color tones of creamy, tangerine, and golden brown rice solid cultures. The golden brown rice culture gave the highest yellow pigment concentration. Under an optimum room temperature of 28–32 °C, an initial moisture content of 42 %, and 7-day-old inoculum size of 2 % (v/w) the maximum yield at 2,224.63 A₃₇₀U/gdw of yellow pigment was produced. A mellow yellow powder at 550 A₃₇₀U/gdw could be obtained using spray-drying techniques. The powder had a moisture content of 5.15 %, a water activity value of 0.398, a hue angle of 73.70 ° (yellowish orange), high lightness (L*) of 74.63, color saturation (C*) of 28.97, a neutral pH of 7.42, 0.12 % acidity and solubility of 0.211 g/10 ml. It was noteworthy that the Chinese fresh noodle with spray-dried yellow powder showed no discoloration during 8-day storage.     

IM2, a new inducer of blue pigment production in Streptomyces sp. MAFF 10-06015

A new autoregulator designated as IM2, which induces blue pigment production in Streptomyces sp. MAFF 10-06015, was discovered. The culture conditions developed here for the production of the pigment by the strain did not require the addition of an artificial inducer such as γ-nonalactone or the autoregulator of S. virginiae MAFF 10-06014, IM, which induces the production of virginiamycin by this microorganism. The major improvements in the culture conditions for spontaneous pigment production included the inoculation conditions and the dilution of the medium. The method of IM2 assay was established and the time courses of IM2 production were followed in the cultures using flasks and a jar fermentor. It was confirmed that IM2 released once into the culture filtrate from the cells was taken up into the cells again. The concentration of IM required to induce pigment production in Streptomyces sp. MAFF 10-06015 was 50 u·ml⁻¹. However a concentration of 200 u·ml⁻¹ of IM2 was unable to induce the production of virginiamycin in S. virginiae MAFF 10-06014.     

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.     

STATISTICAL OPTIMIZATION OF PIGMENT PRODUCTION BY Monascus sanguineus UNDER STRESS CONDITION

Natural pigments are produced by the Monascus sp., which are used for coloring food substances. The intent of this study was to optimize the pigment yield and biomass produced from the unexplored Monascus sanguineus in submerged culture under stress conditions. For inducing thermal stress, the spores were incubated at various temperatures at higher ranges. For inducing osmotic stress, varied concentrations of NaCl, glycerol, and peptone were used. The medium components were optimized by response surface methodology (RSM). The combined effects of the four medium constituents mentioned were studied using a 2⁴ full factorial central composite design (CCD). The relationships between the predicted values and actual values, independent variable, and the response were calculated according to a second-order quadratic model. It was deduced that the variable with the leading effect was the linear effect of glycerol concentration. Furthermore, the quadratic effects of peptone and the interactive effects of temperature and glycerol were more noteworthy than other factors. The optimum values for the test variables in coded factors were found to be spores treated with 70°C for temperature, 0.25 M for glycerol, 0.51% (w/v) for peptone, and 1.25% (w/v) for NaCl, corresponding to a maximum red pigment yield of 55.67 color value units (CVU)/mL. With optimized conditions, the pigment yield was almost three times the yield observed with the control.     

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.     

Correlation of pigment production with mycelium morphology in extractive fermentation of Monascus anka GIM 3.592

Extractive fermentation with nonionic surfactants is a potential method for producing Monascus pigments. In this study, the correlation between mycelium morphology and pigment production was investigated in extractive fermentation of Monascus anka GIM 3.592. The results demonstrated that pigment biosynthesis was associated with mycelial morphology and the accumulation of granular inclusions in cells. The physiological status in terms of hyphal and pellet diameters exhibited an excellent correlation with pigment accumulation, especially the yield of extracellular pigment in extractive fermentation (r > 0.85, p < 0.05). Nonionic surfactants could reduce pigment yield by influencing the morphology of hyphae and mycelium pellets. High yields of both intracellular and extracellular pigments could be achieved by controlling variations in hyphal diameters in two-stage extractive fermentation. Moreover, continuous extractive fermentation led to stable pigment production, with a relatively high productivity of total pigments reaching 72.3 AU/day. This study proposed a simple method for monitoring pigment biosynthesis in extractive fermentation using mycelium morphology as an indicating factor.     

Production of citrinin by various species ofMonascus

Summary The production of citrinin by variousMonascus species was determinated using various culture mediums and conditions. The maximal production was obtained in fermentor usingM. ruber with concentrations of 380 mg/l. Since citrinin is a toxic product, it is essential that the production of red pigments as food additives fromMonascus sp. avoid the occurrence of citrinin; so, we argue that some nitrogen sources are unfavorable to the production of citrinin.     

Production of red pigments byMonascus purpureus in submerged culture

For the purpose of mass producingMonascus red pigments optimum medium composition and environmental conditions were investigated in submerged flask cultures. The optimum carbon and nitrogen sources were determined to be 30 g/L of glucose and 1.5 g/L of monosodium glutamate (MSG). Of the three metals examined, Fe²⁺ showed the stronges stimulatory effect on pigment production and some stimulatory effect was also found in Mn²⁺. Optimum pH and agitation speed were determined to be 6.5 and 700 rpm, respectively. Under the optimum culture conditions batch fermentation showed that the maximum biomass yield and specific productivity of red pigments were 0.20 g DCW/g glucose and, 32.5 OD₅₀₀ g DCW⁻¹ h⁻¹, respectively.     

Application of solid-phase extraction to agar-supported fermentation

Agar-supported fermentation (Ag-SF), a variant of solid-state fermentation, has recently been improved by the development of a dedicated 2 m² scale pilot facility, Platotex. We investigated the application of solid-phase extraction (SPE) to Ag-SF in order to increase yields and minimize the contamination of the extracts with agar constituents. The selection of the appropriate resin was conducted on liquid-state fermentation and Diaion HP-20 exhibited the highest recovery yield and selectivity for the metabolites of the model fungal strains Phomopsis sp. and Fusarium sp. SPE applied to Ag-SF resulted in a particular compartmentalization of the culture. The mycelium that requires oxygen to grow migrates to the top layer and formed a thick biofilm. The resin beads intercalate between the agar surface and the mycelium layer, and trap directly the compounds secreted by the mycelium through a “solid–solid extraction” (SSE) process. The resin/mycelium layer is easily recovered by scraping the surface and the target metabolites extracted by methanol. Ag-SF associated to SSE represents an ideal compromise for the production of bioactive secondary metabolites with limited economic and environmental impact.     

Simple Method for Shiga Toxin 2e Purification by Affinity Chromatography via Binding to the Divinyl Sulfone Group

Here we describe a simple affinity purification method for Shiga toxin 2e (Stx2e), a major causative factor of edema disease in swine. Escherichia coli strain MV1184 transformed with the expression plasmid pBSK-Stx2e produced Stx2e when cultivated in CAYE broth containing lincomycin. Stx2e bound to commercial D-galactose gel, containing α-D-galactose immobilized on agarose resin via a divinyl sulfone linker, and was eluted with phosphate-buffered saline containing 4.5 M MgCl₂. A small amount of Stx2e bound to another commercial α-galactose-immobilized agarose resin, but not to β-galactose-immobilized resin. In addition, Stx2e bound to thiophilic adsorbent resin containing β-mercaptoethanol immobilized on agarose resin via a divinyl sulfone, and was purified in the same manner as from D-galactose gel, but the Stx2e sample contained some contamination. These results indicate that Stx2e bound to D-galactose gel mainly through the divinyl sulfone group on the resin and to a lesser extent through α-D-galactose. With these methods, the yields of Stx2e and attenuated mutant Stx2e (mStx2e) from 1 L of culture were approximately 36 mg and 27.7 mg, respectively, and the binding capacity of the D-galactose gel and thiophilic adsorbent resin for Stx2e was at least 20 mg per 1 ml of resin. In addition, using chimeric toxins with prototype Stx2 which did not bind to thiophilic adsorbent resin and some types of mutant Stx2e and Stx2 which contained inserted mutations in the B subunits, we found that, at the least, asparagine (amino acid 17 of the B subunits) was associated with Stx2e binding to the divinyl sulfone group. The mStx2e that was isolated exhibited vaccine effects in ICR mice, indicating that these methods are beneficial for large-scale preparation of Stx2e toxoid, which protects swine from edema disease.     

Fermentative lactic acid production with a metabolically engineered yeast immobilized in photo-crosslinkable resins

In this study, the immobilization technique involving photo-crosslinkable resin gels was used for lactic acid production. Saccharomyces cerevisiae OC-2T T165R, a metabolically engineered yeast that produces optically pure l(+)-lactic acid, was immobilized in hydrophilic photo-crosslinked resin gels as a biocatalyst. Three resin gels, TEP 1, TEP 2 and TEP 3, were examined and all of them showed high performance as to lactic acid production. Resin gel TEP 1, which exhibited the highest productivity among the resin gels was used for 15 consecutive batch fermentations without decreases in productivity and mechanical deformation, indicating that it was a suitable carrier for long-term lactic acid fermentation. Moreover, the use of the immobilization technique can improve the productivity of the metabolically engineered yeast in the fermentation with or without extraction, showing promise for using the immobilized engineered yeast for lactic acid production.     

Production of Extracellular Pigment by a Mutant of Monascus kaoliang sp. nov

A hyperpigment-producing mutant, R-10847, was derived from Monascus kaoliang F-2 (ATCC 26264) through a series of mutagenesis steps. The mutant produced a large quantity of Monascus pigment when grown in mantou (steamed bread) by solid culture. The mutant produced pigments extracellularly by extruding the pigments outside the cell in a lump together with some viscous substances. The productivity of pigment was about 100-fold greater than that of the wild type. The mutant lost the capability of spore formation, the growth rate decreased, and both the size and quantity of conidia were reduced. The color of the pigment produced by the mutant changed from orange to deep red.     

Kinetic of orange pigment production from <Emphasis Type="Italic">Monascus ruber</Emphasis> on submerged fermentation

Pigments produced by species of Monascus have been used to coloring rice, meat, sauces, wines and beers in East Asian countries. Monascus can produce orange (precursor), yellow and red pigments. Orange pigments have low solubility in culture media and when react with amino groups they become red and largely soluble. The orange pigments are an alternative to industrial pigment production because the low solubility facilitates the downstream operations. The aim of this work was to study the kinetic on the production of orange pigments by Monascus ruber CCT 3802. The shaking frequency of 300 rpm was favorable to production, whereas higher shaking frequencies showed negative effect. Pigment production was partially associated with cell growth, the critical dissolved oxygen concentration was between 0.894 and 1.388 mgO₂ L^?1 at 30 °C, and limiting conditions of dissolved oxygen decreased the production of orange pigments. The maintenance coefficient (mo) and the conversion factor of oxygen in biomass (Yo) were 18.603 mgO₂ g _x ^?1  h^?1 and 3.133 g_x gO ₂ ^?1 and the consideration of these parameters in the oxygen balance to estimate the biomass concentration provided good fits to the experimental data.     

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.