Citrinin Production Research Articles

Natural occurrence of citrinin in Tunisian wheat grains

Citrinin (CIT) is a toxic metabolite produced by several filamentous fungi of the genera Penicillium, Aspergillus and Monascus, which has been known as a natural contaminant in cereal grains, foods and feedstuffs. CIT is a quinone methide with a powerful antibacterial effect, but toxic to humans and animals. This mycotoxin is mainly hepato-nephrotoxic, indeed it was implicated as a potential causative agent in human Balkan Endemic Nephropathy (BEN) associated with an increased frequency of urinary tract tumors in animals and humans. A total of 200 samples of wheat were collected during the harvest year of 2010 from the major cropping areas in Tunisia and they were analyzed for citrinin contamination. These samples were taken after the harvest which can last up to three months. During this period, Penicillium and Aspergillus can growth on the harvested crop and produce CIT. The aim of this study was to investigate for the first time the presence of CIT in widely-consumed cereals in Tunisia, especially in wheat, and to suggest some factors that can promote the production of CIT in Tunisia. To perform this study, we developed and validated in our laboratory conditions an HPLC method for quantitative analysis of CIT in solid cereal samples. Our results showed that the incidence of CIT contamination was 50%. The levels of contamination determined in the positive samples ranged between 01 and 170 μg/kg with an average of 28 μg/kg. These amounts of CIT in wheat are chiefly attributed to the geographic position of Tunisia as a Mediterranean country characterized by a climate that favors fungal proliferation and mycotoxins production.

Effect of Amino Acids on Red Pigments and Citrinin Production in Monascus ruber

Amino acids were used as sole nitrogen sources to examine their effects on the production of water-soluble red pigments and citrinin by Monascus ruber ATCC 96218 cultivated on chemically defined media. In general, when glycine, tyrosine, arginine, serine, or histidine were used as sole nitrogen sources, they favored the production of red pigments, and restricted the synthesis of the mycotoxin. In contrast, the production of citrinin was enhanced in media supplemented with either glutamate, alanine, or proline. Histidine was found to be the most valuable amino acid as it resulted in the highest production of red pigments and almost completely eliminated the formation of mycotoxin.

Effects of Blue Light on Mycelium Morphology, Citrinin Production and the Proportion of Sexual Spore of Monascus

To Monascus spp., light is not the required factor for its growth. But Monascus spp. has the capacity to sense and respond to light. This paper investigated the effects of blue light on growth and the changes of citrinin yield in Monascus 15. Our results demonstrated blue light was a stimulating signal for citrinin formation. Under the blue light illumination, the biomass of Monascus 15 was inhibited, but the citrinin yield increased when comparing with no light culture condition. Spores statistical results revealed that the blue light also influences the development of mycelium and spore formation.

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

To study effects of cyclic adenosine monophosphate (cAMP) on development and secondary metabolites of Monascus ruber M-7. 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. 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. 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.

Efficacy of aqueous medicinal plant extracts on growth and citrinin production by Penicillium citrinum isolated from rice grains.

In this study we investigated the efficacy of aqueous medicinal plant extracts obtained from Andrographis paniculata, Cymbopogon citratus, Eurycoma longifolia, Kaempferia galanga and Orthosiphon aristatus on growth and citrinin production by Penicillium citrinum isolated from rice grains under in vitro (liquid media) conditions. All medicinal plant extracts effectively reduced the growth of P. citrinum ranging from 30.1 to 88% and subsequent citrinin production ranging from 42.3 to 91.3% at 10 mg/ml concentration. Of the plant extracts tested, C. citratus effectively reduced the growth (88%) and citrinin production (91.3%) by P. citrinum at 10 mg/ml concentration followed by A. paniculata(76% and 83.69%). Our results showed that these plant extracts can be potentially used to reduce citrinin contamination in rice grains and probably in other food grains. Key words: Medicinal plants, P. citrinum, citrinin, TLC

Elimination of the mycotoxin citrinin production in the industrial important strain Monascus purpureus SM001

The application of the high-producing pigments industrial strain Monascus purpureus SM001 has been greatly limited by the synchronous production of mycotoxin citrinin. Here we have tried both traditional mutagenesis and metabolic engineering methods to eliminate the production of citrinin. Traditional chemical and physical mutagens were applied to induce mutagenesis, and a bio-screening method based on the antibacterial activity of citrinin against Bacillus subtilis was designed to select mutants. Among the resulting four citrinin-free mutants, only mutant MU2411 can maintain the similar pigments yield. A binary vector system was constructed and successfully disrupted the polyketide synthase gene pksCT in M. purpureus SM001 through the Agrobacterium tumefaciens-mediated transformation. The resulting citrinin-free Δ pksCT mutants maintained the same level of pigments yield. The established Monascus genetic system was comprehensively evaluated and showed high efficiency and specificity, which provides us a potential approach to manipulate and improve industrial Monascus strains.

Influence of temperature on citrinin accumulation by Penicillium citrinum and Peniccillium verrucosum in black table olives

Citrinin production characteristics of Penicillium citrinum and Penicillium verrucosum in black table olives stored at five different temperatures were investigated. Citrinin in both the media and olives were detected by thin layer chromatography (TLC). P. verrucosum produced citrinin at 20°C and 25°C on malt extract agar (MEA), czapek yeast extract agar (CYA), and yeast extract agar (YES), but not in olives. P. citrinum produced citrinin at 20°C, 25°C, and 30°C on both the media and in black olives. Citrinin was also detected in black olive samples stored in water at 30°C for 60 days of storage. NaCl in brine affected citrinin production in a positive way.

Production of patulin and citrinin by Penicillium expansum 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 28days 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 565µg/mL (mean 269µg/mL) and 100µg/mL (mean 31µg/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.

Red Yeast Rice Fermentation by Selected Monascus sp. with Deep-Red Color, Lovastatin Production but No Citrinin, and Effect of Temperature-Shift Cultivation on Lovastatin Production

Monascus pilosus NBRC4520 was selected for functional fermented food inoculation for its high lovastatin and low citrinin production with a deep-red color. For koji (mold rice) with high lovastatin production, separation of the growth phase and lovastatin production phase by shifting the temperature from 30 to 23 degrees C increased lovastatin production by nearly 20 times compared to temperature-constant cultivation. In addition, citrinin was not produced even in the lovastatin production phase, although the pigment was increased. With temperature-shift cultivation, 225 microg lovastatin/g dry koji was produced in 14 days without citrinin.

Construction of a Citrinin Gene Cluster Expression System in Heterologous Aspergillus oryzae

Filamentous fungi are considered an attractive resource for the discovery and production of bioactive compounds. To facilitate molecular breeding, biosynthetic genes must be rapidly identified. But, even after the chemical structure of a compound is identified, finding the corresponding biosynthetic genes in the fungal genome still remains a challenge. In an attempt to overcome this difficulty and to easily characterize each gene in a cluster, we constructed a heterologous expression system using Aspergillus oryzae. The approximate region covering the citrinin biosynthetic gene cluster from Monascus purpureus was introduced into A. oryzae via an Aspergillus-Escherichia coli shuttle cosmid vector without manipulating the gene structure. The transformants showed slight but reproducible citrinin production and definite transcription of the biosynthetic genes. Introducing additional copies of an activator gene (ctnA), controlled by the Aspergillus nidulans trpC promoter, into the citrinin-cluster-containing transformants enhanced the transcription of all the genes in the cluster and resulted in an almost 400-fold higher citrinin production compared to that of the parental transformant. This result suggested that CtnA controlled citrinin production in this system in the same way as in the native strain. This is the first report documenting the heterologous production of functional fungal secondary metabolites in A. oryzae following the introduction of an entire gene cluster.

Inhibition of citrinin production in Penicillium citrinum cultures by neem [Azadirachta indica A. Juss (Meliaceae)

The efficacy of different concentrations of aqueous neem leaf extract (3.12 to 50 mg/mL) on growth and citrinin production in three isolates of Penicillium citrinum was investigated under laboratory conditions. Mycotoxin production by the isolates was suppressed, depending on the concentration of the plant extract added to culture media at the time of spore inoculation. Citrinin production in fungal mycelia grown for 21 days in culture media containing 3.12 mg/mL of the aqueous extract of neem leaf was inhibited by approximately 80% in three isolates of P. citrinum. High-performance liquid chromatography was performed to confirm the spectrophotometric results. Vegetative growth was assessed, but neem extract failed to inhibit it. Neem leaf extract showed inhibition of toxin production without retardation in fungal mycelia growth.

Effect of dissolved oxygen concentration on red pigment and citrinin production by Monascus purpureus ATCC 36928

The present study investigated the effects of agitation speed, N (200, 500, 600 or 700 rpm), and dissolved oxygen concentration, C (120, >70, 70, 60, 10 or < 10%), on red pigment and citrinin production by Monascus purpureus ATCC 36928, cultivated in liquid medium by a batch process. The gas flow rate was the same for all runs with C controlled by means of the incoming gas composition control (air/N2 or air/O2). From the response surface plots it can be verified that the effect of C was greater than that of N on the production of both metabolites. The absorbance for red pigments varied from 1.6 U (C< 10%; N=200 rpm) up to 3.3 U (C=60%; N=600 rpm), an increase of 106%, while citrinin concentration increased 257%, from 14.2 to 50.7 mg.L-1. The most appropriate conditions were C=60% and N=600rpm, under which the highest red pigment absorbance (3.3U) and half of the highest citrinin concentration were obtained.

Ochratoxin A and citrinin producing species of the genus Penicillium from feedstuffs

In Spain, low ochratoxin A (OTA) levels have been detected in several pork products but there is no information published about the fungi involved in this OTA contamination. It is well known that P. verrucosum is much more frequently found on cereals in countries where they occasionally have OTA problems as in North European countries compared with South Europe where levels of OTA generally seem to be lower or not detected. Much less information is available about citrinin (CIT) and CIT producing species in cereals and their by products. The aim of this study was to determine, identify and characterize the occurrence of potential OTA and CIT producing Penicillium spp. from mixed feeds and raw materials purchased in the Spanish market and used as feedstuffs. A total of 155 Penicillium spp. isolates belonging to 34 species were analyzed in order to know if they are able to produce OTA and/or CIT. From these isolates, 11 P. verrucosum which were characterized by RAPD analyses, produced OTA. Fourteen isolates were CIT producers, 10 isolates of P. verrucosum and 4 of P. citrinum. Although the occurrence and abundance of OTA and CIT Penicillium producing species have been low in our study, our results confirm the potential risk of OTA and CIT production in feeds if stored improperly. Our results also confirm the occurrence of P. verrucosum in South European countries and that it is the only OTA producing Penicillium species in these substrates.

Occurrence of Penicillium verrucosum in retail wheat flours from the Spanish market

In Spain, low ochratoxin A (OTA) levels have been detected in wheat and different wheat products but no information has been published about the fungi involved in this OTA contamination. Some species of the genera Penicillium and Aspergillus are known to form OTA but few of them are known to contaminate foods with this mycotoxin. Penicillium verrucosum, an important OTA producer typical of temperate and cold climates, is much more frequently found on cereals in countries where they occasionally have OTA problems as in North European countries compared with South Europe, where levels of OTA generally seem to be lower or is not detected. The aim of this study was to determine, identify and characterize the occurrence of potential OTA-producing Aspergillus spp. and Penicillium spp. from retail wheat flours purchased in the Spanish market and used for human consumption. A total of 105 Aspergillus isolates were analyzed in order to know whether they are able to produce OTA and/or citrinin (CIT). None of these isolates were able to produce these mycotoxins. However, 17 suspected P. verrucosum isolates were recovered and confirmed by RAPD analyses. Eleven isolates were OTA producers and 14 isolates produced CIT. Our results confirm the potential risk of OTA and CIT production in wheat flours if stored improperly and the occurrence of P. verrucosum in South European countries. This was the only species able to produce these mycotoxins.

Improving the Ratio of Monacolin K to Citrinin Production of Monascus purpureus NTU 568 under Dioscorea Medium through the Mediation of pH Value and Ethanol Addition

Using dioscorea root as substrate of Monascus species was found to stimulate monacolin K (cholesterol-lowering agent) formation in our previous study, but the mycotoxin-citrinin has never been studied. This study used dioscorea root as the liquid medium to culture Monascus purpureus NTU 568 using a 6.6 L jar fermentor. Culture pH value, dioscorea concentration, and ethanol concentration were used as the factors of response surface methodology (RSM) to investigate the optimal culture condition for high monacolin K production and low citrinin formation. Monacolin K and citrinin formation of M. purpureus NTU 568 under submerged dioscorea medium were respectively found to be significantly formed by 148% and 147%, as compared to that under submerged rice medium. The reason is due to the pH value (3.5) of dioscorea medium involved in the formation of Monascus cell amount and secondary metabolite. RSM results further indicated that lowering the pH value to 2.5 would result in high monacolin K and citrinin concentrations as well as high biomass in fixed dioscorea amount, implying that pH value may stimulate the formation of monacolin K and citrinin through increasing Monascus cell amount. Lowering dioscorea and ethanol concentration was able to increase the ratio of monacolin K level to citrinin level. The optimal culture condition (pH 5.7, 1% dioscorea concentration, and 0.5% ethanol concentration) would increase monacolin K levels to 27.9 mg/g (by 47%) and decrease citrinin level to 2.15 microg/g (by 54%), as compared to control conditions (pH 3.5, 5% dioscorea, and ethanol free).

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.

Identification and In Vivo Functional Analysis by Gene Disruption of ctnA , an Activator Gene Involved in Citrinin Biosynthesis in Monascus purpureus

Citrinin, a secondary fungal metabolite of polyketide origin, is moderately nephrotoxic to vertebrates, including humans. From the red-pigment producer Monascus purpureus, a 21-kbp region flanking pksCT, which encodes citrinin polyketide synthase, was cloned. Four open reading frames (ORFs) (orf1, orf2, orf3, and orf4) in the 5'-flanking region and one ORF (orf5) in the 3'-flanking region were identified in the vicinity of pksCT. orf1 to orf5 encode a homolog of a dehydrogenase (similarity, 46%), a regulator (similarity, 38%), an oxygenase (similarity, 41%), an oxidoreductase (similarity, 26%), and a transporter (similarity, 58%), respectively. orf2 (2,006 bp with four introns) encodes a 576-amino-acid protein containing a typical Zn(II)2Cys6 DNA binding motif at the N terminus and was designated ctnA. Although reverse transcriptase PCR analysis revealed that all of these ORFs, except for orf1, were transcribed with pksCT under citrinin production conditions, the disruption of ctnA caused large decreases in the transcription of pksCT and orf5, together with reduction of citrinin production to barely detectable levels, suggesting that these two genes are under control of the ctnA product. Complementation of the ctnA disruptant with intact ctnA on an autonomously replicating plasmid restored both transcription and citrinin production, indicating that CtnA is a major activator of citrinin biosynthesis.

Suppression of ochratoxin biosynthesis by naturally occurring alkaloids

The effects of four alkaloids on the biosynthesis of ochratoxin A (OTA), ochratoxin B (OTB) and citrinin were examined on four OTA-producing aspergilli: Aspergillus auricomus, A. sclerotiorum and two isolates of A. alliaceus. Piperine and piperlongumine, natural alkaloids of Piper longum, significantly inhibited OTA production at 0.001% (w/v) for all aspergilli examined. Piperine and piperlongumine affected the polyketide synthesis step of OTA production and inhibited production of citrinin. Curcumin, a constituent of tumeric, completely inhibited mycelial growth of A. alliaceus isolate 791 at 0.1% (w/v) and decreased OTA production by ∼70% at 0.01% (w/v). Sesamin, a constituent of sesame oil, inhibited OTA and OTB production by 60 and 45%, respectively, at 0.1% (w/v), showing its effect was on chloroperoxidase and polyketide synthase activity. The potential advantage of these natural products to reduce ochratoxin contamination of agricultural commodities is discussed.

Regiones microsatélites (GTG) 5 en Penicillium productores de citrinina

Morphological and cultural characteristics, as well as biochemical properties, are the main criteria used in fungal taxonomy and in the standard description of fungi species. Sometimes, however, this criterion is difficult to apply due to fungal phenotypic variations. This is particularly true in the genus Penicillium. The aims of this work were to determine (GTG)5 microsatellite sequence in potentially citrinin-producing Penicillium strains and to investigate if this sequence could be useful to characterize such fungi. Penicillium citrinum Thom and Penicillium chrysogenum Thom were isolated from different foods. The identification of the isolates at species level was carried out according to classical taxonomy. The production of citrinin was determined by thin layer chromatography. This study proved that microsatellite regions exist as short repeated sequences in all tested strains. The patterns were very similar for all P. citrinum isolates and it was possible to group them in function of the quantity of citrinin produced. Yet, not similar clusters were obtained when P. chrysogenum isolates were analyzed.

NATURAL OCCURRENCE OF CITRININ AND BIOCONTROL OF ITS PRODUCING FUNGUS BY Trichoderma hamatum IN RICE GRAINS

Paddy rice was sampled from El-Sharkia, El-Gharbia, El-Dakahlia and Kafr El-Sheikh governorates, Egypt. Of the 29 samples taken, ten were contaminated with the mycotoxin citrinin. An average of 6.79 x 104 fungal spores per gram rice was found. The isolated fungi represented 47 species belong to 28 genera. The predomi-nant genera were Aspergillus, Cladosporium and Penicillium. Aspergilli were represented by 22 species; Aspergillus niger and A. flavus had the highest occur-rence. Penicillium viridicatum produced the highest amount of citrinin on glucose ammonium nitrate salts broth and rice grains and hence, this isolate was selected as a good producer of citrinin in this study. The presence of Trichoderma hamatum re-duced the amount of citrinin produced by P. viridicatum compared with its respec-tive control. The excessive growth of T. hamatum on P. viridicatum was increased with time. Viability of P. viridicatum conidia decreased by T. hamatum with an in-crease in the incubation period. Chitinases and 1,3-B-glucanase enzyme activity of T. hamatum increased with extending the incubation period on P. viridicatum cell walls up to maximum values at 72 and 84 h, respectively. T. hamatum led to a de-crease in the production of citrinin by P. viridicatum on rice grains compared with the respective control values.