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.     

Asymmetric Reduction of 2-Methyl-2-aryloxyacetic Acids by Glomerella cingulata

The mycotoxin citrinin had antifungal activity under acidic conditions. At the minimum inhibitory concentration, it completely inhibited cellular respiration and partially inhibited the incorporation of radioactive precursors into macromolecules in Saccharomyces cerevisiae. It had no effect on cell permeability. In mitochondrial preparations, it significantly inhibited succinate oxidase and NADH oxidase. Rhizopus chinensis was more sensitive than S. cerevisiae; its growth and mycelial respiration at acidic pH were completely inhibited by lower concentrations of citrinin. The pH-dependent antifungal activity of citrinin seems to be associated with its uptake by fungi. Approximately half of the citrinin taken up was found in mitochondria. The main site of the antifungal action of citrinin, therefore, appears to be the mitochondrial electron transport system.     

Effect of microelements on the biosynthesis of secondary metabolites by the fungusPenicillium citrinum thom VKM F-1079

Penicillium citrinum VKM F-1079 was found to produce clavine ergot alkaloids and citrinin, a secondaryO-heterocyclic metabolite. Citrinin was produced in the idiophase, whereas the production of ergot alkaloids paralleled fungal growth. The addition of manganese ions to the growth medium stimulated the biosynthesis of both citrinin and ergot alkaloids. Zinc ions stimulated only citrinin synthesis. The presence of these microelements in the growth medium influenced the proportion between the ergot alkaloids synthesized. Copper, manganese, and iron ions slightly affected fungal growth and alkaloid production. The effect of microelements on the main kinetic parameters of growth and alkaloid production was studied.     

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.     

Morphological changes induced in fungi by antibiotics

In tests of 31 antibiotics, 29 inhibited growth ofBotrytis cinerea and of these, 18 induced morphological changes. Terminal and lateral branching of the hyphae was induced by actinomycin D, aspergillic acid, citrinin, cyanein, cycloheximide, desertomycin and polyene antibiotics. Curling of the hyphae was induced by griseofulvin and narrowing of the hyphae by citrinin. Some antibiotics at different concentrations produced several types of morphological changes. For example, aspergillic acid, desertomycin and flavofungin also induced terminal bulging of the hyphae. Growth of the dimorphic fungusPaecilomyces viridis was inhibited by 24 antibioties, nine of which induced morphological changes. Branching of the hyphae was induced by azalomycin F, citrinin, eyanein, desertomycin, patulin, rugulosin and trichothecin. Griseofulvin had a curling effect. Except for rugulosin, the above antibiotics, in higher concentrations, induced yeast-like growth ofPaecilomyces viridis. Morphological changes were also induced by inhibitors of RNA and protein synthesis and by antibiotics injuring the cell membranes. Antibiotics with different mechanisms of action induced similar morphological changes.     

Isolation and characterization of a strain of <Emphasis Type="Italic">Klebsiella pneumoniae</Emphasis> with citrinin-degrading activity

Monascus-fermented products have been widely used in Taiwan and other Asian countries as health foods. Unfortunately, many Monascus strains concurrently produce trace amounts of toxic citrinin. This study isolated a strain NPUST-B11 with the ability to degrade citrinin as the only carbon source. The isolated strain NPUST-B11 was characterised and identified as Klebsiella pneumoniae by 16S rRNA gene analysis using UNI-F and UNI-R primers. The isolated strain was then incubated in the mineral broth containing 10 ppm of citrinin, 1.2% of glucose, 0.3% of peptone and 100 ppm of vitamin C under optimal conditions, including pH 7, 200 rpm and 37°C. Citrinin was rapidly degraded with incubation from 97.9% at 1 h to 8.67% at 5 h and completely depleted at 10 h. Overall, this strain could be useful for the degradation of citrinin in food products and other medical applications.     

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.     

Citrinin affects the oxidative metabolism of BHK-21 cells

The effects of citrinin on energy production along the respiratory chain and on glycolytic lactate production were examined in BHK-21 cultured cells. Citrinin inhibited the oxygen consumption rate by about 45 per cent. The respiratory rate of digitonin-treated cells energized with succinate, in the presence of ADP, was reduced by about 39 per cent. The mycotoxin inhibited the glucose utilization of BHK-21 cells by about 86 per cent. Cells treated with citrinin produced a small quantity of pyruvate, but were unable to produce lactate. It is concluded that BHK-21 cells cannot generate lactate when oxidative metabolism is inhibited by citrinin. The perturbations in BHK-21 cells caused by citrinin are due to alterations in mitochondrial function and in the glycolytic anaerobic pathway.     

红曲霉桔霉素的检测方法及红曲霉产桔霉素的判别方法*

建立了红曲霉真菌毒素桔霉素的HPLC检测方法。用谷氨酸和葡萄糖为唯一氮、碳源的培养基(MSG)液态摇瓶培养及红曲米固态培养,对30多株红曲霉产桔霉素的情况进行了普查,发现大多数红曲霉菌种可产生桔霉素。红曲霉的培养状态及条件对桔霉素的产生有重大影响。红曲霉菌种是否产桔霉素,可根据MSG培养基摇瓶发酵液中是否含有桔霉素来初步定性判断,为准确判断红曲霉菌是否产桔霉素,可采用多种培养法综合判断。     

Rapid semi-quantitative fluorimetric determination of citrinin in fungal cultures isolated from cheese and cheese factories

A new rapid semi-quantitative fluorimetric assay for citrinin production testing in mould cultures has been developed. The chemical structure of the citrinin makes it a weak native fluorophore. This fluorescence can be strongly enhanced in an acidic environment. A standard curve where the concentration of HCl needed to show the yellow fluorescence signal of different concentrations of citrinin was established, thus providing a semi-quantitative method to prove the capacity of toxin production of fungal cultures. Two Penicillium strains from the Spanish National Collection of Type Cultures, were studied for the toxin production on YES broth at 25°C for 21 d. The culture was assayed daily for the presence/absence and quantification of citrinin by adding the HCl concentration set, and also quantified by RP-HPLC as a confirmation procedure. Experiments demonstrate that 5 d are necessary to show the presence of citrinin. As an illustration, a total of 48 strains of Penicillium isolated from cheese and cheese factories were analysed with the proposed method.     

Production of lipase free of citrinin by Penicillium citrinum

Lipase (Glycerol ester hydrolase E.G. 3.1.1.3) from a Brazilian strain of Penicillium citrinum free of the mycotoxin citrinin has been investigated. Citrinin production was inhibited by using culture medium containing olive oil, soybean oil and corn oil as carbon sources. Potassium concentration and pH play an important role in citrinin production. Potassium concentration lower than 30 mM and pH below 4.5 inhibited the mycotoxin production. P. citrinum produced lipase free of extraneous proteins and citrinin when cultured using, as nitrogen source, ammonium sulphate (lipase activity of 7.88 U/mg) and yeast extract (lipase activity of 4.95 U/mg) with olive oil as carbon source. This data is relevant to the larger scale production of lipases for food technology applications, from Penicillium citrinum.     

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.     

The Influence of Glucose and Fructose on the Degradation of 2-Chlorophenol by Pseudomonas putida CP1

Summary Pseudomonas putida CP1 grew on 2-chlorophenol when supplied as the sole source of carbon. Chlorophenol degradation was stimulated in the presence of low concentrations of glucose (0.05–1%, w/v). Substrate removal was inhibited and there was a significant fall in pH with concentrations of glucose greater than 1.0% (w/v). When the pH was controlled at pH 7.0 inhibition of substrate removal was alleviated. The rate of removal of 2-chlorophenol was greater in the presence of fructose than in the presence of glucose. P. putida CP1 formed clumps of cells when grown on 2-chlorophenol and fructose but not on glucose. When the organism was grown on a combination of 2-chlorophenol and an additional carbon source clumping was present but to a lesser degree.     

Effect of acetone on production of aflatoxins and versicolorin pigments by resting cell cultures of aspergillus parasiticus

Resting cell cultures of Aspergillus parasiticus were grown in medium containing four different concentrations of glucose, with and without acetone. In addition, the effect of different equimolar concentrations of acetone, acetic acid, ethanol, and sodium acetate was compared at two glucose levels. Aflatoxin and versicolorin pigment production increased in resting cell medium containing increasing concentrations of glucose. In the presence of glucose high concentrations of acetone (1.0 and 0.25 M) inhibited secondary biosynthesis and low concentrations of acetone (0.1, 0.025 and 0.01 M) stimulated secondary biosynthesis of aflatoxins and versicolorin pigments.     

Regulation of endo-1,4-β-glucanase secretion fromTermitomyces clypeatus by carbon catabolic product(s)

Secretion of CMCase byTermitomyces clypeatus was only observed in the presence of a gluconeogenic amino acid, a citrate-cycle acid, maleate, subinhibitory concentrations of glucosamine, or fluoride in the medium. The enzyme was not secreted in the presence of caffeine or IBMX or theophylline, and these phosphodiesterase inhibitors lowered the secretion of CMCase by glutamate. The presence of both glucosamine and glutamate in a cellulose medium were, however, antagonistic to CMCase secretion. In a growth medium, xylose and glucose were equivalent carbon source for the fungus while succinate was a poor source and strongly repressed growth at higher concentrations. Growth ofT. clypeatus was highly favored in media containing xylose/glucose with succinate/glutamate. During growth ofT. clypeatus in a glucose medium, the intracellular glucose level was stabilized by the presence of succinate, glutamate or glucosamine in the medium. All these observation suggested that a negative cellular regulation, mediated by carbon catabolic product(s), existed inT. clypeatus which regulated the secretion of CMCase. A transient but significant increase of intracellular cAMP and cGMP levels was observed at the onset of mycelial growth in glucose and glucose/maleate media, respectively.     

Production of patulin and citrinin by <Emphasis Type="Italic">Penicillium expansum</Emphasis> from British Columbia (Canada) apples

Twenty-four isolates of Penicillium expansum Link from British Columbia (Canada) apples were cultured in yeast-extract sucrose (YES) at 25°C for 28 days to investigate production of patulin and citrinin. These isolates proved to be potent producers of citrinin, patulin, or in most cases, both mycotoxins. In every isolate, citrinin, patulin, or both compounds were produced at levels as high as 565 μg/mL (mean 269 μg/mL) and 100 μg/mL (mean 31 μg/mL), respectively. Of the 24 isolates, 4 produced citrinin only, and 2 produced patulin only. Overall, 83% of the isolates formed patulin and 91% formed citrinin. YES broth proved to be an effective medium for patulin and citrinin production. Other workers have noted that production of these mycotoxins in culture often presages production in fruits, so these results might help Canadian fruit processors evaluate and minimize mycotoxin levels in their products.     

Diphasic production of secondary metabolites byPenicillium notatum westling S-52

The levels of total lipids and their components, as well as of free and esterified fatty acids in the mycelium of growingPenicillium notatum vary during the diphasic production of citrinin. The changes in the ratio of C : N are reflected markedly in changes of free and esterified fatty acids as well as in the quantity of simple and complex lipids. Gas chromatography did not detect the presence of a C₁₀ fatty acid which would be an analogue of the postulated pentaketide from which citrinin is formed. Dedicated to Prof. P. Nemec on his 60th birthday.     

Production of water-soluble yellow pigments via high glucose stress fermentation of Monascus ruber CGMCC 10910

Monascus pigments are secondary metabolites of Monascus species and are mainly composed of yellow pigments, orange pigments and red pigments. In this study, a larger proportion of Monascus yellow pigments could be obtained through the selection of the carbon source. Hydrophilic yellow pigments can be largely produced extracellularly by Monascus ruber CGMCC 10910 under conditions of high glucose fermentation with low oxidoreduction potential (ORP). However, keeping high glucose levels later in the culture causes translation or a reduction of yellow pigment. We presume that the mechanism behind this phenomenon may be attributed to the redox level of the culture broth and the high glucose stress reaction of M. ruber CGMCC 10910 during high glucose fermentation. These yellow pigments were produced via high glucose bio-fermentation without citrinin. Therefore, these pigments can act as natural pigments for applications as food additives.

     

Conversion of the mycotoxin citrinin into dihydrocitrinone and ochratoxin A by Penicillium viridicatum

Summary The mycotoxin citrinin, produced by Penicillium viridicatum, was found to be unstable in broth culture. This instability was investigated using the techniques of ¹⁴C-labelling, mass spectrometry and thin layer chromatography. It was concluded that intracellular factors released by P. viridicatum were responsible for the instability of the mycotoxin. The major products resulting from the breakdown of citrinin were identified as 3,4-dihydro-6,8-dihydroxy-3,4,5-trimethyl-isocoumarin-7-carboxylic acid (dihydrocitrinone) and ochratoxin A.     

Activity of citrinin metabolized by rat and human microsome fractions in clastogenicity and SCE assays on Chinese hamster V79-E cells.

The mycotoxin citrinin is a potent inducer of chromosomal aberrations in the clastogenicity assay on V79-E cells when metabolized by rat and human liver microsomes. Rat and human liver microsomes, standardized on protein content, activate citrinin at equal levels. 5 X 10(-4) M citrinin induces complex translocations in a high frequency as well as defects of chromosomal coiling. Higher concentrations are cytotoxic, lower ones are almost inactive. After metabolization of mycotoxin by rat-kidney microsomes or an S9 mix fraction containing rat liver and kidney microsomes, toxic effects predominate and chromosomal aberrations are diminished. Clastogenic citrinin concentrations do not induce an increase of SCE frequency. Although the mode of action of this mycotoxin on chromosomal structure remains obscure, possible explanations are discussed.