Effects of cyclic AMP on development and secondary metabolites of Monascus ruber M-7

Aim: To study effects of cyclic adenosine monophosphate (cAMP) on development and secondary metabolites of Monascus ruber M‐7. Methods and Results: Plate culture, liquid‐state fermentation (LSF) and solid‐state fermentation (SSF) were used to evaluate effects of cAMP on colonial growth, spore formation and polyketide production of Strain M‐7. The results revealed that the variation trends of colonial sizes, numbers of sexual spores and red pigment contents of M‐7 were in a dose‐dependent manner. And generally they increased and decreased with cAMP concentrations in the ranges of low cAMP concentrations and high cAMP concentrations, respectively. But the variation trends of numbers of asexual spores and citrinin production in both LSF and SSF were opposite to those of colonial sizes, sexual sporulation and red pigment. Conclusions: The regulation of cAMP on development and secondary metabolites in Strain M‐7 was in a dose‐dependent pattern. And red pigment might convert to citrinin under changing cAMP concentrations. Significance and Impact of the Study: The effects of cAMP on Strain M‐7 in SSF give a new clue to enhance beneficial polyketides and reduce citrinin produced by M. ruber.     

Growth kinetics of biopigment production by Thai isolated <Emphasis Type="Italic">Monascus purpureus</Emphasis> in a stirred tank bioreactor

Monascus purpureus is a biopigment-producing fungi whose pigments can be used in many biotechnological and food industries. The growth kinetics of biopigment production were investigated in a liquid fermentation medium in a 5-l stirred tank bioreactor at 30°C, pH 7, for 8 days with 100 rpm agitation and 1.38 × 10⁵ N/m² aeration. Thai Monascus purpureus strains TISTR 3002, 3180, 3090 and 3385 were studied for color production, growth kinetics and productivity. Citrinin as a toxic metabolite was measured from the Monascus fermentation broth. The biopigment productions were detected from fermentation broth by scanning spectra of each strain produced. Results showed a mixture of yellow, orange and red pigments with absorption peaks of pigments occurring at different wavelengths for the four strains. It was found that for each pigment color, the color production from the strains increased in the order TISTR 3002, 3180, 3090, 3385 with 3385 production being approximately 10 times that of 3002. Similar results were found for growth kinetics and productivity. HPLC results showed that citrinin was not produced under the culture conditions of this study. The L*, a* and b* values of the CIELAB color system were also obtained for the yellow, orange and red pigments produced from the TISTR 3002, 3180, 3090 and 3385 strains. The colors of the pigments ranged from burnt umber to deep red.     

Kinetic analysis of red pigment and citrinin production by Monascus ruber as a function of organic acid accumulation

In submerged cultures performed in synthetic medium containing glucose and glutamate, the filamentous fungus Monascus ruber produced a red pigment and a mycotoxin, citrinin. In oxygen-limiting conditions, the production of these two metabolites was growth-associated, as was the production of primary metabolites. In oxygen-excess conditions, the profile of citrinin production was typical of a secondary metabolite, since it was produced mostly during the stationary phase. In contrast, the production of the pigment decreased rapidly throughout the culture, showing a profile characteristic of an inhibitory mechanism. The organic acids produced during the culture, L-malate and succinate, were shown to be slightly inhibitory against pigment production, while citrinin production was unaffected. However, this inhibition could not account for the observed profile of pigment production in batch cultures. Other dicarboxylic acids such as fumarate or tartrate showed a similar effect to that provoked by malate and succinate as regards pigment production. It was concluded that the decrease in red pigment production during the culture was due to the inhibitory effect of an unknown product whose accumulation was favored in aerobic conditions.     

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.     

Pigments and citrinin biosynthesis by fungi belonging to genus Monascus

Citrinin is a mycotoxin, which is produced by fungi belonging to the genus Monascus, known in biotechnology as producers of azaphilone pigments. The relation between biosynthesis of these secondary metabolites was investigated in different species of the genus Monascus in batch-culture at the following cultivation conditions: T = 28 degrees C, agitation 220 rpm, and a medium, which induce citrinin production, containing ethanol as a carbon source. The screening was carried out with 16 fungal strains and the biosynthesis of citrinin and pigments was monitored quantitatively at the standard conditions mentioned above. Some kinetic parameters of the process have been determined. The values of the growth yield coefficient Y(X/C) were between 0.32 and 0.57. The amount of the extracellular red and orange pigments at the end of cultivation varied for the different strains between 0.09 and 1.33 OU/ mg dry weight, and 0.15 and 0.96 OU/mg dry weight, respectively. The amount of the total pigments measured was between 0.16 and 3.6 OU/mg dry weight, and between 0.21 and 3.39 OU/mg dry weight. The determined ratio 500 nm/400 nm, characterizing the pigment production, ranged between 0.60 and 1.06. Twelve of the investigated strains produced citrinin and pigments, two of them produced only pigments. Two strains were not able to produce neither pigments nor citrinin. Thus, the biosynthesis of citrinin appeared to be strain-specific and does not correlate with the pigments' biosynthesis by the fungal strains belonging to the genus Monascus.     

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.     

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 citrinin by Monascus ruber submerged culture in chemically defined media

Following our investigations on citrinin production by Monascus ruber in a chemically defined medium, the kinetic behaviour of this toxin in the fermenter was studied with relation to production of pigments, biomass and nutrient consumption. Showing a secondary metabolite pattern, citrinin was produced when dμ/dt and dQ_s/dt were ≤ 0, while a high specific production rate of pigments occurred when dμ/dt and dQ_s/dt were > 0. No trophophase-idiophase transition was detected during the cuitivation, and the behaviour of pigment production was similar to that of a primary metabolite.     

Production of red mold rice using a modified Nagata type koji maker

In this research, a commercial koji maker with a rotary perforated bed of 5-m diameter was modified for red mold rice production. Monascus purpureus BCRC 31499 was selected for its high production capacities of monacolin K and red pigment. The selected strain was first cultivated in a 120-l submerged type fermentor at 34°C and 2 vvm aeration rate with 60 rpm agitation for 5 days using 20% liquefied rice porridge as carbon source. The high concentration red mold rice broth (>3.5 g/ml) was harvested for inocula and well mixed with cooked rice to an initial concentration of 2% v/w. The inoculated cooked rice then was directed into the modified koji maker, in which temperature and humidity profiles were kept at varied levels at different stages, respectively. Air was circulated to remove fermentation heat while the perforated bed rotated slowly for providing mild agitation. Lag phase of the Monascus sp. in the modified koji maker was determined to be 16 h by the time the koji temperature raised rapidly. Water was added into the koji bed by a water curtain at the 36th hour to keep the moisture content of the rice koji at 50% or above. At the final stage, temperature was adjusted to 34°C to direct red pigment production. After 7 days, 1,200-kg high quality red mold rice was harvested per batch. Labor costs, space, and fermentation time were reduced tremendously compared with those made by traditional methods.     

Improvement of monacolin K, γ-aminobutyric acid and citrinin production ratio as a function of environmental conditions of<Emphasis Type="Italic"> Monascus purpureus</Emphasis> NTU 601

Monascus, a traditional Chinese fermentation fungus, is used as a natural dietary supplement. Its metabolic products monacolin K and -aminobutyric acid (GABA) have each been proven to be a cholesterol-lowering drug and a hypotensive agent. Citrinin, another secondary metabolite, is toxic to humans, thus lowering the acceptability of red mold rice to the general public. In this study, the influence of different carbon and nitrogen sources, and fatty acid or oils, on the production of monacolin K, citrinin and GABA by Monascus purpureus NTU 601 was studied. When 0.5% ethanol was added to the culture medium, the production of citrinin decreased from 813 ppb to 561 ppb while monacolin K increased from 136 mg/kg to 383 mg/kg and GABA increased from 1,060 mg/kg to 7,453 mg/kg. In addition, response surface methodology was used to optimize culture conditions for monacolin K, citrinin and GABA production, and data were collected according to a three-factor (temperature, ethanol concentration and amount of water supplemented), three-level central composite design. When 500 g rice was used as a solid substrate with 120 ml water and 0.3% ethanol, the production of monacolin K at 30°C increased from 136 mg/kg to 530 mg/kg, GABA production increased from 1,060 mg/kg to 5,004 mg/kg and citrinin decreased from 813 ppb to 460 ppb.     

Influences of chemical fertilizers (in Vitro) on aflatoxin and citrinin synthesis by two strains of Aspergilli

The selective effect of various levels of phosphate and nitrate (as fertilizers) on biosynthesis of aflatoxin byAspergillus parasiticus var.globosus, and citrinin byA. terreus var.aureus was studied in defined culture medium. Phosphate at 35–175 mmol per 50 mL decreased aflatoxin production, but increased citrinin synthesis. Nitrate at 73–365 mmol per 50 mL stimulated the synthesis of aflatoxin but depressed that of citrinin. A rise in the levels of nitrate and phosphate led to a decrease in aflatoxin production, an increase in citrinin production and an accumulation of mycelial phosphate and nitrate contents.     

Production of carotenoids by Brevibacterium linens: variation among strains, kinetic aspects and HPLC profiles

This work describes carotenoid pigment production by the red bacterium Brevibacterium linens covering strain diversity, kinetic and analytical aspects. Pigment production of 23 B. linens strains ranged from 0.05 to 0.60 mg pigments L⁻¹ culture, with specific productivity from 0.2 to 0.6 mg pigments per g dry biomass. The pigment production time curve showed a sigmoid shape, that matched cell growth. HPLC analysis revealed three groups of peaks, possibly non-hydroxylated, mono- and di-hydroxylated carotenoids. Polar molecules were mainly represented. Journal of Industrial Microbiology & Biotechnology (2000) 24, 64–70. Received 19 April 1999/ Accepted in revised form 25 September 1999     

Medium-Chain Fatty Acids Affect Citrinin Production in the Filamentous Fungus Monascus ruber

During submerged culture in the presence of glucose and glutamate, the filamentous fungus Monascus ruber produces water-soluble red pigments together with citrinin, a mycotoxin with nephrotoxic and hepatoxic effects on animals. Analysis of the ¹³C-pigment molecules from mycelia cultivated with [1-¹³C]-, [2-¹³C]-, or [1,2-¹³C]acetate by ¹³C nuclear magnetic resonance indicated that the biosynthesis of the red pigments used both the polyketide pathway, to generate the chromophore structure, and the fatty acid synthesis pathway, to produce a medium-chain fatty acid (octanoic acid) which was then bound to the chromophore by a trans-esterification reaction. Hence, to enhance pigment production, we tried to short-circuit the de novo synthesis of medium-chain fatty acids by adding them to the culture broth. Of fatty acids with carbon chains ranging from 6 to 18 carbon atoms, only octanoic acid showed a 30 to 50% stimulation of red pigment production, by a mechanism which, in contrast to expectation, did not involve its direct trans-esterification on the chromophore backbone. However, the medium- and long-chain fatty acids tested were readily assimilated by the fungus, and in the case of fatty acids ranging from 8 to 12 carbon atoms, 30 to 40% of their initial amount transiently accumulated in the growth medium in the form of the corresponding methylketone 1 carbon unit shorter. Very interestingly, these fatty acids or their corresponding methylketones caused a strong reduction in, or even a complete inhibition of, citrinin production by M. ruber when they were added to the medium. Several data indicated that this effect could be due to the degradation of the newly synthesized citrinin (or an intermediate in the citrinin pathway) by hydrogen peroxide resulting from peroxisome proliferation induced by medium-chain fatty acids or methylketones.     

Optimization of culture conditions for the production of an extracellular ribonuclease by <Emphasis Type="Italic">Aspergillus niger</Emphasis> in a benchtop bioreactor

SummarySelf-directing optimization was successfully employed to determine the optimal combination of engineering parameters, viz., pH, aeration rate and agitation rate, for extracellular ribonuclease production by Aspergillus niger SA-13-20 in a batch bioreactor. Maximal RNase production of 5.38 IU ml^–1 was obtained at controlled pH of 2.33, aeration rate of 1.67 v/v/m and agitation rate of 850 rev/min. The effect of oxygen on the fermentation was also investigated. With increase in volumetric oxygen transfer coefficients (K_La), cell growth and RNase production first increased and then decreased. RNase production was further increased to 7.10 IU ml^–1 and the fermentation time was shortened from 96 to 72 h by controlling dissolved oxygen concentration at 10% saturation by aerating oxygen after about 28 h of fermentation under the above optimal condition. The kinetic model showed that RNase production by A. niger SA-13-20 was growth-associated.     

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.     

A factorial approach for a sugarcane juice-based low cost culture medium: increasing the astaxanthin production by the red yeast Phaffia rhodozyma

A sugarcane juice-based low cost culture medium was previously explored to produce the carotenoid pigment astaxanthin in liquid culture by the red yeast Phaffia rhodozyma (1300?μg astaxanthin/g of dry yeast and 6500?μg/l whole culture medium). Two peculiar limitations in Phaffia are growth temperature (<26?°C) and lack of sugar osmotolerance. Two advantages are the wide biochemical ability for the assimilation and metabolization of disaccharides and the prompt utilization of simple nitrogen sources. For instance, the sucrolytic/ureolytic enzymatic activities deserves exploration. In order to improve the culture medium composition and the conditions of fermentation for highly oxygenated carotenoids (e.g., astaxanthin) a study was carried out with a factorial design in two steps. As a first step, the production of astaxanthin was studied as a function of the nutrient concentration levels and their interactions. The production increase (μg/l) obtained was 23.0% but at the expense of 16.0% pigment content decrease (μg/g). In the second step, the variables pH and agitation level (OTR, oxygen transfer rate) were optimized and then, both goals were attained: the increase of pigment content (418?μg astaxanthin/g of yeast) as well as the absolute pigment production enhancement (1987?μg/l).     

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     

Citrinin-producing capacity of Monascus purpureus in response to low −frequency magnetic fields

Citrinin is a type of mycotoxin that negatively affects Monascus products. Application of a low-frequency magnetic field (LF-MF) decreased citrinin production by M. purpureus in liquid-state fermentation during shake flask culture. Under 30 °C incubation, six different magnetic field induction intensity (MF-II), four different exposure times and five exposure time periods were tested to discover optimal treatment conditions. The cultures were exposure to a MF-II of 1.6 mT from 0 to 2 d of incubation time. With LF-MF treatment, peak citrinin production decreased by 46.7% while yellow, red, orange pigments and monacolin K production increased by 31.3%, 40.3%, 41.7% and 29.3%, respectively, compared with control groups at 12 d of incubation. Moreover, the relative expression levels of the citrinin biosynthesis genes pksCT and ctnA were 0.46 and 0.43 times lower, respectively, than the control values relative to the GAPDH reference gene. This study provides evidence that LF-MF is a preferable way to alter M. purpureus metabolism to reduce citrinin production and to increase pigments and monacolin K production without affecting cell growth. Therefore, LF-MF may be used as a tool to process Monascus products to obtain important functional food additive while reducing the adverse effects of citrinin.     

Medium-chain fatty acids affect citrinin production in the filamentous fungus Monascus ruber

During submerged culture in the presence of glucose and glutamate, the filamentous fungus Monascus ruber produces water-soluble red pigments together with citrinin, a mycotoxin with nephrotoxic and hepatoxic effects on animals. Analysis of the (13)C-pigment molecules from mycelia cultivated with [1-(13)C]-, [2-(13)C]-, or [1, 2-(13)C]acetate by (13)C nuclear magnetic resonance indicated that the biosynthesis of the red pigments used both the polyketide pathway, to generate the chromophore structure, and the fatty acid synthesis pathway, to produce a medium-chain fatty acid (octanoic acid) which was then bound to the chromophore by a trans-esterification reaction. Hence, to enhance pigment production, we tried to short-circuit the de novo synthesis of medium-chain fatty acids by adding them to the culture broth. Of fatty acids with carbon chains ranging from 6 to 18 carbon atoms, only octanoic acid showed a 30 to 50% stimulation of red pigment production, by a mechanism which, in contrast to expectation, did not involve its direct trans-esterification on the chromophore backbone. However, the medium- and long-chain fatty acids tested were readily assimilated by the fungus, and in the case of fatty acids ranging from 8 to 12 carbon atoms, 30 to 40% of their initial amount transiently accumulated in the growth medium in the form of the corresponding methylketone 1 carbon unit shorter. Very interestingly, these fatty acids or their corresponding methylketones caused a strong reduction in, or even a complete inhibition of, citrinin production by M. ruber when they were added to the medium. Several data indicated that this effect could be due to the degradation of the newly synthesized citrinin (or an intermediate in the citrinin pathway) by hydrogen peroxide resulting from peroxisome proliferation induced by medium-chain fatty acids or methylketones.