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PubMed2003

Modelling the combined effect of temperature, pH and aw on the growth rate of Monascus ruber, a heat-resistant fungus isolated from green table olives.

AIMS: Growth modes predicting the effect of pH (3.5-5.0), NaCl (2-10%), i.e. aw (0.937-0.970) and temperature (20-40 degrees C) on the colony growth rate of Monascus ruber, a fungus isolated from thermally-processed olives of the Conservolea variety, were developed on a solid culture medium. METHODS AND RESULTS: Fungal growth was measured as colony diameter on a daily basis. The primary predictive model of Baranyi was used to fit the growth data and estimate the maximum specific growth rates. Combined secondary predictive models were developed and comparatively evaluated based on polynomial, Davey, gamma concept and Rosso equations. The data-set was fitted successfully in all models. However, models with biological interpretable parameters (gamma concept and Rosso equation) were highly rated compared with the polynomial equation and Davey model and gave realistic cardinal pHs, temperatures and aw. CONCLUSIONS: The combined effect of temperature, pH and aw on growth responses of M. ruber could be satisfactorily predicted under the current experimental conditions, and the models examined could serve as tools for this purpose. SIGNIFICANCE AND IMPACT OF THE STUDY: The results can be successfully employed by the industry to predict the extent of fungal growth on table olives.

Colony Count, Microbial↗
PubMed2005

Use of gradient plates to study combined effects of temperature, pH, and NaCl concentration on growth of Monascus ruber van Tieghem, an Ascomycetes fungus isolated from green table olives.

The effect of temperature, pH, and sodium chloride concentration on the growth of the Ascomycetes fungus Monascus ruber van Tieghem, the main spoilage microorganism during storage of table olives, was studied by using the gradient plate technique. Gradients of NaCl (3 to 9%, wt/vol) at right angles to gradients of pH (2 to 6.8) were prepared for the plates, which were incubated at 25, 30, and 35 degrees C. Visible fungal growth, expressed in optical density units, was recorded by image analysis and graphically presented in the form of three-dimensional grids. Results obtained from the plates indicated that the fungus was salt and acid tolerant, being able to grow at NaCl concentrations of up to 9% (wt/vol) and pH values of as low as 2.2, depending on the incubation temperature. The inhibitory effect of NaCl increased as the pH decreased progressively at 25 and 30 degrees C but not at 35 degrees C. Growth was better at 30 and 25 degrees C as judged by the larger extent of the plates covered by mycelium compared with that at 35 degrees C, where no growth was observed at pHs below 3.7. Differentiation between vegetative (imperfect-stage) and reproductive (perfect-stage) growth was evident on all plates, providing useful information about the effect of environmental conditions on the form of fungal growth. When the growth/no-growth surface model was obtained by applying linear logistic regression, it was found that all factors (pH, NaCl, and temperature) and their interactions were significant. Plots of growth/no-growth interfaces for P values of 0.1, 0.5, and 0.9 described the results satisfactorily at 25 and 35 degrees C, whereas at 35 degrees C the model predicted lower minimum pH values for growth in the range of 7 to 10% NaCl than those observed on the plates. Overall, it is suggested that the fungus cannot be inhibited by any combination of pH and NaCl within the limits of the brine environment, so further processing is required to ensure product stability in the market.

Culture Media↗
PubMed2006

Repression of secondary metabolite production by exogenous cAMP in Monascus.

The cAMP signal pathway controls various biological functions, including secondary metabolism of filamentous fungi. We found that exogenous cAMP represses the production of lovastatin, red pigments, and citrinin in Monascus. Interestingly, a mutant MK-1 with increased lovastatin and red pigments production was not influenced by cAMP on these productions, indicating that cAMP signaling might be lacking in MK-1.

Cyclic AMP↗
PubMed2006

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.

Carbon↗
PubMed2000

[Screening DPPH radical scavengers from Monascus sp].

A strain belongs to Monascus sp. was separated from hongqu(Chinese beni-coji) which was used to brew red wine in Changxin county, Zhejiang province, and its DPPH radical scavenging metabolites were studied. Results showed that the yield and radical scavenging activity of neutral extract with EtOAc were higher than that of acid and basic extract. After shaken at 30 degrees C, 100 r/min for 5 days, this strain produced largest amount of neutral extract. Further partitioning the neutral fraction with silica gel column chromatograph, LH-20 column chromatograph, MPLC and HPLC, we screened 15 free radical scavengers whose yield was more than 2 mg and purity was higher than 85% in HPLC (260 nm absorption). Several of them were analyzed with 1H-NMR and 13C-NMR spectrum from which we concluded that B1-3 and C3-1-7 were two kinds of substitute of benzene, and C3-1-7 may be 3-OH-4-OCH3-benzoic acid. The scavenging activity of 40 mumol/L B1-3 was about 65%, and that of C3-1-7 was lower than 56%, while as the control the scavenging activity of 40 mumol/L Vc and Ve was 80.4% and 78.4% respectively.

Benzene Derivatives↗
PubMed2003

[Effect of space flight on yield of Monascus purpureus].

OBJECTIVE: To select high Lovastatin-producing microbial breed by space flight. METHOD: Monascus purpureus species was carried into space by the recoverable spaceship, "Shenzhou 3". After flight, the strain was rejuvenized, segregated and selected. The content of Lovastatin produced in the solid fermentation was examined. RESULT: Mutants with high productivity of Lovastatin were obtained. A series of tests showed that the acquired character of the mutants was stable. CONCLUSION: Space flight is an effective method for the selection of fine strains.

Environmental Microbiology↗
PubMed2005

Serial analysis of gene expression in Monascus aurantiacus producing citrinin.

OBJECTIVE: To construct a tag expression library of Monascus aurantiacus that could produce citrinin maximally on the thirteenth (0.966 mg/mL) day in the submerged culture. METHODS: Total RNA was extracted from the mycelium, cDNA was synthesized using the SuperScript choice system, and then, a SAGE library was successfully constructed according to the MicroSAGE method. RESULTS: Five hundred and ninety eight clones were obtained in SAGE library, and 120 clones were picked out randomly for identification and sequencing purpose. Eighty nine clones had positive inserts, 26 clones had no inserts and the remaining 5 clones had no site of NlaIII enzyme in inserts. There were seven repeated clones. CONCLUSION: With the aid of SAGE2000 software, 901 tags were obtained from 89 clones, representing 686 unique transcripts. Six unique tags of them belong to highly expressed genes (Number of tags > or = 10) and 143 unique tags to moderately expressed genes (repeat tags > or = 2).

Anti-Bacterial Agents↗
PubMed2005

[Preparation and Regeneration of Protoplasts from Monascus purpureus and Genetic Transformation System.].

Generation of fungal protoplast is an essential tool for genetic transformation system. To establish protoplast-mediated genetic transformation system of Monascus purpureus, conditions for the protoplast isolation and regeneration of the mycelia of various enzymes and osmotic stabilizers were examined. To investigate suitable cell age for the protoplast preparation of mycelia of M. purpureus, the mycelia were cultured in different ways at 30oC. Mycelia obtained through cellophane - mediated culture for 30~40h were adequate to protoplast preparation. When lysing enzyme, cellulase and snailase were added to the mycelia in combination or alone, combination of lysing enzyme, cellulase and snailase accordingly at the concentration of 0.3%, 0.1% and 1% was most benefit for protoplast yield. When we applied various osmotic stabilizers at different concentrations to protoplast preparation, 1 mol/L MgSO4 was most effective for the protoplast release. The suitable incubation time with enzyme for the maximum release of protoplasts was 2.5-hr. When we investigate various osmotic stabilizers for the regeneration of the protoplasts of mycelia of strain M34 and N18, the complete medium containing 0.6 mol/L sucrose induced highest hyphal growth with regeneration frequency of 8.5% and 36.4%, respectively. PEG and CaCl2- mediated protoplast co-transformation of strain M34 with pBC-Hygro and pNL1, hygromycin B as selective marker, was fulfilled and 100 stable transformants per microgram DNA were obtained.

Hygromycin B↗
PubMed1999

Improvement of red pigment/citrinin production ratio as a function of environmental conditions by monascus ruber

The growth and metabolic behaviour of the filamentous fungus Monascus ruber were studied in submerged cultures under various aeration and agitation conditions. Improving the oxygen supply, by increasing either the air input or the agitation speed, resulted in modified metabolism: the biomass yield, the consumption of the nitrogen source (monosodium glutamate), and the production of secondary metabolites (red pigment and citrinin) all increased. However, the citrinin production increased more than that of the red pigment. In consequence, a low oxygen transfer coefficient was required to improve the red pigment/citrinin production ratio. Copyright 1999 John Wiley & Sons, Inc.

Journal Article↗
PubMed1980

Specificity of the acid protease from Monascus kaoliang towards the B-chain of oxidized insulin.

The proteolytic specificity of the acid protease from Monascus kaoliang has been investigated using the B-chain of performic acid-oxidized insulin as peptide substrate. Six splittings were detected after 1 h digestion and 12 splittings were found after 12 h incubation at 37 degrees C, pH 4.8. The bonds most susceptible to the acton of M. kaoliang acid protease were Phe(24)-Phe(25), Leu(15)-Tyr(16) and Tyr(16)-Leu(17). Among the acid proteases compared, the specificity of M. kaoliang acid protease on the B-chain of oxidized insulin is more closely related to that of penicillopepsin with which it has ten cleavage sites in common. N-Acetyl-L-phenylalanyl-L-3,5-diiodotyrosine, a synthetic substrate for pepsin, was resistant to the hydrolysis of M. kaoliang acid protease.

Ascomycota↗
PubMed2002

Dimerumic acid as an antioxidant from the mold, Monascus anka: the inhibition mechanisms against lipid peroxidation and hemeprotein-mediated oxidation.

This study aimed to investigate the antioxidant mechanism of dimerumic acid isolated as the active component with a radical scavenging action from the mold Monascus anka, traditionally used for the fermentation of foods. Dimerumic acid inhibited NADPH- and iron(II)-dependent lipid peroxidation (LPO) of rat liver microsomes at 20 and 200 microM, respectively. When ferrylmyoglobin was incubated with dimerumic acid, the myoglobin was scavenged and an electron spin resonance (ESR) signal with nine peaks was observed. The spin adduct was identified as a nitroxide radical by analysis of hyperfine structure. Similar ESR signal was also detected by incubation of dimerumic acid with peroxyl radicals. Thus, it was clarified that the antioxidant action of dimerumic acid is due to one electron donation of the hydroxamic acid group in the dimerumic acid molecule toward oxidants resulting in formation of nitroxide radical.

Animals↗
PubMed2000

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.

Journal Article↗
PubMed2002

Heat resistance of Monascus ruber ascospores isolated from thermally processed green olives of the Conservolea variety.

A heat-resistant fungus identified as Monascus ruber was isolated from thermally processed green olives of the Conservolea variety. Thermal death parameters of the ascospores were determined using Baranyi's primary predictive model in phosphate buffer (pH 7.0), citrate buffer (pH 4.0) and brine (pH 3.8) at two different NaCl levels (5.6% and 10.5%). The decimal reduction times (D values) at 70, 75 and 80 degrees C in phosphate buffer (pH 7.0) were 37.31, 7.14 and 1.66 min, respectively, and the calculated z value was 7.4 degrees C. In citrate buffer (pH 4.0), the relative D values were 38.61, 7.83 and 2.10 min and the z value was 7.9 degrees C. In brine, the D(70 degrees C), D75(75 degrees C) and D(80 degrees C) values increased from 46.08, 4.91 and 0.88 min to 72.99, 7.98 and 1.04 min, respectively, as the level of NaCl in brine increased from 5.6% to 10.5%, indicating a protective effect in ascospore inactivation. This effect was more pronounced at lower heating temperatures. It was concluded that a thermal process of F(80 degrees C) = 5 min could be considered as an effective thermal processing treatment. Results from this research could assist the table olive industry in designing an effective pasteurization plan to eliminate the fungus in thermally processed table olives.

Ascomycota↗
PubMed2000

Repeated-batch production of pigments by immobilised Monascus purpureus.

Extracellular pigment production by immobilised Monascus purpureus C322 has been studied in repeated-batch processes using different immobilising carriers such as Ca-alginate, polyurethane sponge, active carbon and pearlite. With Ca-alginate, pigment production was maximum (30.5 UA470 as process mean production, three batches) while the cell leakage was negligible (0.4 g 1(-1) free biomass) and the bead mechanical stability good; with this carrier, an extended repeated-batch fermentation (nine batches, 55 days) was carried out: the process pigment productivity was 3.87 UA470 day(-1).

Alginates↗
PubMed1999

Screening of antioxidant action of various molds and protection of Monascus anka against experimentally induced liver injuries of rats.

Antioxidant action of various molds, which are traditionally used for the production of foods or alcoholic beverages in Japan, was studied in vitro and in vivo. Antioxidant action was evaluated by scavenging stable free radical 1,1-diphenyl-2-picrylhydrazyl (DPPH) and lipid peroxidation of rat liver microsomes. Among 40 molds, 16 species showed the DPPH scavenging action, and the molds that can scavenge the DPPH radical inhibited lipid peroxidation. The mold with the strongest action, Monascus anka, was chosen for the investigation of a protective action against liver injury of rats. When galactosamine (GalN, 400 mg/kg) or GalN plus lipopolysaccharide (LPS, 0.5 microg/kg) was given intraperitoneally to rats (Sprague-Dawley), aspartate aminotransferase (AST) and glutathione (GSH) S-transferase (GST) activities in serum were significantly increased. However, such hepatotoxicities seen in the increase in serum enzyme levels were depressed when the extract prepared from M. anka was given 1 and 15 h before the toxic insultant. Liver microsomal GST activity, which is known to be activated by oxidative stress, was increased by GalN or GaIN plus LPS treatment and the increase was also inhibited by pretreatment with the extract. Pathomorphological changes in the liver caused by GalN treatment also were prevented by the mold extract. These results indicate that the extract of M. anka has radical scavenging action and ameliorates chemically induced hepatotoxicity.

Animals↗
PubMed2000

Dimerumic acid as an antioxidant of the mold, Monascus anka.

We previously reported that the mold Monascus anka, traditionally used for fermentation of food, showed antioxidant and hepatoprotective actions against chemically induced liver injuries. In the present study, the antioxidant component of M. anka was isolated and identified. The antioxidant was elucidated to be dimerumic acid. DPPH (1,1-diphenyl-2-picrylhydrazyl) radical was significantly scavenged by the antioxidant whereas hydroxyl radical and superoxide anion were moderately scavenged. When the antioxidant (12 mg/kg) was given to mice prior to carbon tetrachloride (CCl(4), 20 microl/kg, ip) treatment, the CCl(4)-induced liver toxicity in mice seen in an elevation of serum aspartate aminotransferase and alanine aminotransferase activities was depressed, suggesting the hepatoprotective action of the antioxidant. The liver microsomal glutathione S-transferase activity, which is known to be activated by oxidative stress or active metabolites, was increased by CCl(4) treatment and the increase was also depressed by pretreatment with the mold antioxidant. Thus these data confirmed that the dimerumic acid isolated from M. anka is the potential antioxidant and protective against CCl(4)-induced liver injury.

Alanine Transaminase↗
PubMed1999

A simple and rapid method for analyzing the Monascus pigment-mediated degradation of mutagenic 3-hydroxyamino-1-methyl-5H-pyrido[4,3-b]indole by in-capillary micellar electrokinetic chromatography.

A simple and rapid method is described for analyzing the Monascus pigment-mediated degradation of 3-hydroxyamino-1-methyl-5H-pyrido[4, 3-b]indole (Trp-P-2(NHOH)). We used the in-capillary micellar electrokinetic chromatography (MEKC). During the electrophoresis, the mutagen and the pigment, due to their different migration velocities, mix for a certain period of time to interact, and then they are separated and quantified. Using this technique, we were able to demonstrate that Trp-P-2(NHOH) is degraded by the pigment. The degradation was pigment-dose dependent, and because the pigment was recovered unchanged, it was deduced that the pigment acted catalytically for the degradation. The entire MEKC procedure takes 8 min.

Antimutagenic Agents↗
PubMed2000

Maximizing yellow pigment production in fed-batch culture of Monascus sp.

Yellow pigment production in exponential fed-batch cultivation of Monascus sp. was studied. Due to the difficulty of measuring the optical density for accurate determination of the cell concentration, a capacitance probe was employed on-line. The feed rate needed to keep the specific growth rate, mu, constant in fed-batch culture was determined on the basis of the cell concentration measured by the capacitance probe. Control of mu was improved by using updated information on the cell concentration compared with the simple feed-forward determination method using the initial cell concentration only. The highest specific pigment production rate was achieved with a mu of 0.02 h(-1) in the feeding phase. However, among several fermentation examined, the largest pigment production in the final step was obtained at a mu of 0.01 h(-1); in each case the same amount of substrates was used. An investigation of the optimal initial glucose concentration revealed that pigment production was maximum when the initial glucose concentration in the batch mode was 10 g/l and mu was 0.01 h(-1) in the fed-batch mode. It was also found that the pellet weight in the fermentation could be accurately estimated by image analysis. The ratio of the mycelium weight to the total cell weight estimated from information on the total cell weight and the estimated pellet weight was found to be more than 80%. However, no clear quantitative relationship could be discerned between the specific pigment production rate, rho, and the ratio of mycelium in the cell population.

Journal Article↗