The effect of Zataria multiflora Boiss Essential oil on the growth and citrinin production of Penicillium citrinum in culture media and cheese.

The effect of Zataria multiflora Boiss Essential oil (EO) on the growth, spore production, and citrinin production of Penicellium citrinum PTCC 5304 in the culture media as well as Iranian ultra-filtered white cheese in brine was investigated. Radial growth and spore production on the potato dextrose agar (PDA) were effectively inhibited by EO in a dose-dependent manner. At 200 ppm, the radial growth and sporulation declined by 92% and 100%, respectively. The growth was completely prevented at 400 ppm of EO on PDA and the minimum fungicidal concentration (MFC) of the oil was estimated at 400 ppm. Furthermore, the Zataria multiflora also significantly suppressed the mycelial growth and citrinin production in broth medium at all investigated concentrations (P < 0.05). At 150 ppm of EO, the citrinin accumulation and mycelial growth reduced by 88.6% and 89.6%, respectively. The EO was tested at all concentrations and the findings show an inhibitory effect of P. citrinum against the radial fungal growth and citrinin production in cheese. However, no concentration of EO could completely inhibit the growth and production of citrinin in cheese. We therefore concluded that Zataria multiflora has the potential to substitute the antifungal chemicals as a natural inhibitor to control the growth of molds in foods such as cheese.

Orf6 gene encoded glyoxalase involved in mycotoxin citrinin biosynthesis in Monascus purpureus YY-1.

As traditional edible fungi, Monascus spp. have been widely used as folk medicine, food colorants, and fermentation starters in East Asian countries for more than a thousand years. However, the presence of citrinin, which has nephrotoxic, hepatotoxic, and carcinogenic activities, raises suspicions about the safety of Monascus products. Citrinin biosynthesis in Monascus is known to occur via a polyketide pathway and a citrinin biosynthesis gene cluster, which include the characterized polyketide synthetase pksCT. A gene, orf6, encodes a protein that shows significant similarity to glyoxalase and is located between ctnE and orf1. This study analyzed orf6 function, and successfully obtained an orf6 disruption strain (Δorf6). Citrinin production was significantly greater (3.6-fold) in the Δorf6 strain than in the wild-type Monascus purpureus YY-1, and RT-PCR analysis further revealed increased expression of numerous genes of the citrinin biosynthesis gene cluster in Δorf6. Therefore, orf6 proved to be a major inhibitor, directly involved in citrinin biosynthesis. Moreover, pigment production in Δorf6 was reduced by approximately 30%, while the transcription levels of many genes involved in Monascus pigments (MPs) biosynthesis had increased. This dichotomy indicated that MPs and citrinin yields may be improved simultaneously; however, a portion of the pigments was consumed to protect the cells from oxidative damage in the Δorf6 strain. An Δorf6 revertant restored the citrinin and pigment yields to normal levels. This study makes a contribution to explore the citrinin biosynthesis pathway and provides some theoretical guidance to improving the safety of Monascus-related products.

Functional and Structural Analysis of Programmed C-Methylation in the Biosynthesis of the Fungal Polyketide Citrinin.

Fungal polyketide synthases (PKSs) are large, multidomain enzymes that biosynthesize a wide range of natural products. A hallmark of these megasynthases is the iterative use of catalytic domains to extend and modify a series of enzyme-bound intermediates. A subset of these iterative PKSs (iPKSs) contains a C-methyltransferase (CMeT) domain that adds one or more S-adenosylmethionine (SAM)-derived methyl groups to the carbon framework. Neither the basis by which only specific positions on the growing intermediate are methylated ("programming") nor the mechanism of methylation are well understood. Domain dissection and reconstitution of PksCT, the fungal non-reducing PKS (NR-PKS) responsible for the first isolable intermediate in citrinin biosynthesis, demonstrates the role of CMeT-catalyzed methylation in precursor elongation and pentaketide formation. The crystal structure of the S-adenosyl-homocysteine (SAH) coproduct-bound PksCT CMeT domain reveals a two-subdomain organization with a novel N-terminal subdomain characteristic of PKS CMeT domains and provides insights into co-factor and ligand recognition.

Deleting the citrinin biosynthesis-related gene, ctnE, to greatly reduce citrinin production in Monascus aurantiacus Li AS3.4384.

For thousands of years, fermentation products of the filamentous fungi Monascus spp. have been used extensively in the food and pharmaceutical industries. However, their development is limited because of the health threats from the mycotoxin citrinin, known to be produced by these fungi. Citrinin is recognized as a hepato-nephrotoxin which possesses potential genotoxicity, tumorigenicity, carcinogenicity, embryotoxicity, and teratogenicity. Studies have shown that citrinin biosynthesis is intimately related to pksCT, orf1, ctnA, orf3, ctnB and ctnG. The ctnE gene, which is located 3.3kb upstream of ctnA, encodes a protein that showed significant similarity to the dehydrogenase. In this study, the role of ctnE in citrinin biosynthesis was investigated by means of gene knockout technology. The ctnE disruptant significantly reduced citrinin production by 96%, which suggested that ctnE is important in citrinin biosynthesis. Moreover, the mutant produced 40% more pigments than the wild-type. This work contributes to the study of the citrinin biosynthesis pathway in Monascus, and the methodology described in this article can fundamentally lower the risk of citrinin contamination in Monascus aurantiacus Li AS3.4384 which has important significance for food safety.

Monascus secondary metabolites: production and biological activity.

The genus Monascus, comprising nine species, can reproduce either vegetatively with filaments and conidia or sexually by the formation of ascospores. The most well-known species of genus Monascus, namely, M. purpureus, M. ruber and M. pilosus, are often used for rice fermentation to produce red yeast rice, a special product used either for food coloring or as a food supplement with positive effects on human health. The colored appearance (red, orange or yellow) of Monascus-fermented substrates is produced by a mixture of oligoketide pigments that are synthesized by a combination of polyketide and fatty acid synthases. The major pigments consist of pairs of yellow (ankaflavin and monascin), orange (rubropunctatin and monascorubrin) and red (rubropunctamine and monascorubramine) compounds; however, more than 20 other colored products have recently been isolated from fermented rice or culture media. In addition to pigments, a group of monacolin substances and the mycotoxin citrinin can be produced by Monascus. Various non-specific biological activities (antimicrobial, antitumor, immunomodulative and others) of these pigmented compounds are, at least partly, ascribed to their reaction with amino group-containing compounds, i.e. amino acids, proteins or nucleic acids. Monacolins, in the form of ß-hydroxy acids, inhibit hydroxymethylglutaryl-coenzyme A reductase, a key enzyme in cholesterol biosynthesis in animals and humans.

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 approximately 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.

Nephrotoxigenic Penicillium species occurring on farm-stored cereal grains in western Canada.

The incidence of nephrotoxigenic Penicillium species on farm-stored cereals in western Canada was determined by morphological and metabolite profile examination. Of the 142 isolates examined 102 were toxin producers with 61 P. aurantiogriseum and 27 P. freii. Other nephrotoxigenic species included P. tricolor (6 isolates), P. verrucosum Chemotype II (4 isolates) and P. viridicatum Westling (4 isolates). The nephrotoxigenic Penicillium species profile for western Canada appears to differ from that of Denmark where P. verrucosum, P. cyclopium, P. freii and, to a lesser extent, P. aurantiogriseum, P. polonicum, and P. viridicatum predominate.

Citrinin.

Citrinin, a nephrotoxic mycotoxin, has been of growing importance also for the "International Agency for Research on Cancer", ever since its presumable role in the occurrence of Balcan endemic nephropathy (BEN) was discussed at the congress on "Mycotoxins, Endemic Nepthropathy and Urinary Tract Tumours" held in Lyon in June 1991 (12). In late 1991, citrinin was therefore also included in the list of toxins to be examined by the screening subcommittees on natural toxins of the International Live Science Institute, European Branch.