André El Khoury

Ochratoxin A: General Overview and Actual Molecular Status

Ochratoxin A (OTA) is a mycotoxin produced by several species of Aspergillus and Penicillium fungi that structurally consists of a para-chlorophenolic group containing a dihydroisocoumarin moiety that is amide-linked to L-phenylalanine. OTA is detected worldwide in various food and feed sources. Studies show that this molecule can have several toxicological effects such as nephrotoxic, hepatotoxic, neurotoxic, teratogenic and immunotoxic. A role in the etiology of Balkan endemic nephropathy and its association to urinary tract tumors has been also proved. In this review, we will explore the general aspect of OTA: physico-chemical properties, toxicological profile, OTA producing fungi, contaminated food, regulation, legislation and analytical methods. Due to lack of sufficient information related to the molecular background, this paper will discuss in detail the recent advances in molecular biology of OTA biosynthesis, based on information and on new data about identification and characterization of ochratoxin biosynthetic genes in both Penicillium and Aspergillus species. This review will also cover the development of the molecular methods for the detection and quantification of OTA producing fungi in various foodstuffs.

Fungal contamination and Aflatoxin B1 and Ochratoxin A in Lebanese wine-grapes and musts

Five hundred and ten strains of filamentous fungi were isolated from Lebanese grapes during 2005 at veraison and harvesting periods. Four hundred eighty-seven isolates belonged to the Aspergillus spp. (95.5%) and 23 belonged to the Penicillium spp. (4.5%). Black aspergilli constituted 56.9% (52.2% Aspergillus niger aggregates, 2.9% Aspergillus japonicus and 1.8% Aspergillus carbonarius) while the isolation rate of Aspergillus flavus the none habitual member of grape mycobiota was 43.1% of the total Aspergillus spp. isolated. All isolates were tested for the ability to produce the Ochratoxin A (OTA) and the Aflatoxin B1 (AFB1). A. carbonarius showed that it is the only species able to produce the OTA with a production ability of 100% and a maximum concentration reaching 8.38microg/g CYA. As for the aflatoxigenic ability, 43.4% of A. flavus isolates produced this mycotoxin with a maximum production reaching 22.6microg/g CYA while none of the other isolates showed a production capacity of this mycotoxin. Forty-seven samples of must produced from the collected grapes were also analyzed. None of these samples was contaminated by OTA at a detectable limit while 40% of these same samples were found to contain AFB1 with concentrations ranging from 0.01 to 0.46microgl(-1).

Sequencing, physical organization and kinetic expression of the patulin biosynthetic gene cluster from Penicillium expansum

Patulin is a polyketide-derived mycotoxin produced by numerous filamentous fungi. Among them, Penicillium expansum is by far the most problematic species. This fungus is a destructive phytopathogen capable of growing on fruit, provoking the blue mold decay of apples and producing significant amounts of patulin. The biosynthetic pathway of this mycotoxin is chemically well-characterized, but its genetic bases remain largely unknown with only few characterized genes in less economic relevant species. The present study consisted of the identification and positional organization of the patulin gene cluster in P. expansum strain NRRL 35695. Several amplification reactions were performed with degenerative primers that were designed based on sequences from the orthologous genes available in other species. An improved genome Walking approach was used in order to sequence the remaining adjacent genes of the cluster. RACE-PCR was also carried out from mRNAs to determine the start and stop codons of the coding sequences. The patulin gene cluster in P. expansum consists of 15 genes in the following order: patH, patG, patF, patE, patD, patC, patB, patA, patM, patN, patO, patL, patI, patJ, and patK. These genes share 60-70% of identity with orthologous genes grouped differently, within a putative patulin cluster described in a non-producing strain of Aspergillus clavatus. The kinetics of patulin cluster genes expression was studied under patulin-permissive conditions (natural apple-based medium) and patulin-restrictive conditions (Eagles minimal essential medium), and demonstrated a significant association between gene expression and patulin production. In conclusion, the sequence of the patulin cluster in P. expansum constitutes a key step for a better understanding of the mechanisms leading to patulin production in this fungus. It will allow the role of each gene to be elucidated, and help to define strategies to reduce patulin production in apple-based products.

Quantification of Fusarium graminearum and Fusarium culmorum by real-time PCR system and zearalenone assessment in maize

Zearalenone (ZEA) is a mycotoxin produced by some species of Fusarium, especially by Fusarium graminearum and F. culmorum. ZEA induces hyperoestrogenic responses in mammals and can result in reproductive disorders in farm animals. In the present study, a real-time PCR (qPCR) assay has been successfully developed for the detection and quantification of Fusarium graminearum based on primers targeting the gene PKS13 involved in ZEA biosynthesis. A standard curve was developed by plotting the logarithm of known concentrations of F. graminearum DNA against the cycle threshold (Ct) value. The developed real time PCR system was also used to analyze the occurrence of zearalenone producing F. graminearum strains on maize. In this context, DNA extractions were performed from thirty-two maize samples, and subjected to real time PCR. Maize samples also were analyzed for zearalenone content by HPLC. F. graminearum DNA content (pg DNA/ mg of maize) was then plotted against ZEA content (ppb) in maize samples. The regression curve showed a positive and good correlation (R²=0.760) allowing for the estimation of the potential risk from ZEA contamination. Consequently, this work offers a quick alternative to conventional methods of ZEA quantification and mycological detection and quantification of F. graminearum in maize.

Differentiation between Aspergillus flavus and Aspergillus parasiticus from pure culture and aflatoxin-contaminated grapes using PCR-RFLP analysis of aflR-aflJ intergenic spacer.

Aflatoxins (AFs) represent the most important single mycotoxin-related food safety problem in developed and developing countries as they have adverse effects on human and animal health. They are produced mainly by Aspergillus flavus and A. parasiticus. Both species have different aflatoxinogenic profile. In order to distinguish between A. flavus and A. parasiticus, gene-specific primers were designed to target the intergenic spacer (IGS) for the AF biosynthesis genes, aflJ and aflR. Polymerase chain reaction (PCR) products were subjected to restriction endonuclease analysis using BglII to look for restriction fragment length polymorphisms (RFLPs). Our result showed that both species displayed different PCR-based RFLP (PCR-RFLP) profile. PCR products from A. flavus cleaved into 3 fragments of 362, 210, and 102 bp. However, there is only one restriction site for this enzyme in the sequence of A. parasiticus that produced only 2 fragments of 363 and 311 bp. The method was successfully applied to contaminated grapes samples. This approach of differentiating these 2 species would be simpler, less costly, and quicker than conventional sequencing of PCR products and/or morphological identification.

A study on the physicochemical parameters for Penicillium expansum growth and patulin production: effect of temperature, pH, and water activity

Abstract Penicillium expansum is among the most ubiquitous fungi disseminated worldwide, that could threaten the fruit sector by secreting patulin, a toxic secondary metabolite. Nevertheless, we lack sufficient data regarding the growth and the toxigenesis conditions of this species. This work enables a clear differentiation between the favorable conditions to the P. expansum growth and those promising for patulin production. A mathematical model allowing the estimation of the P. expansum growth rate according to temperature, a W, and pH, was also developed. An optimal growth rate of 0.92 cm/day was predicted at 24°C with pH level of 5.1 and high a W level of 0.99. The models predictive capability was tested successfully on artificial contaminated apples. This model could be exploited by apple growers and the industrialists of fruit juices in order to predict the development of P. expansum during storage and apple processing.

Patulin transformation products and last intermediates in its biosynthetic pathway, E- and Z-ascladiol, are not toxic to human cells

Patulin is the main mycotoxin contaminating apples. During the brewing of alcoholic beverages, this mycotoxin is degraded to ascladiol, which is also the last precursor of patulin. The present study aims (1) to characterize the last step of the patulin biosynthetic pathway and (2) to describe the toxicity of ascladiol. A patE deletion mutant was generated in Penicillium expansum. In contrast to the wild strain, this mutant does not produce patulin but accumulates high levels of E-ascladiol with few traces of Z-ascladiol. This confirms that patE encodes the patulin synthase involved in the conversion of E-ascladiol to patulin. After purification, cytotoxicities of patulin and E- and Z-ascladiol were investigated on human cell lines from liver, kidney, intestine, and immune system. Patulin was cytotoxic for these four cell lines in a dose-dependent manner. By contrast, both E- and Z-ascladiol were devoid of cytotoxicity. Microarray analyses on human intestinal cells treated with patulin and E-ascladiol showed that the latter, unlike patulin, did not alter the whole human transcription. These results demonstrate that E- and Z-ascladiol are not toxic and therefore patulin detoxification strategies leading to the accumulation of ascladiol are good approaches to limit the patulin risk.

Secondary metabolism in Penicillium expansum: Emphasis on recent advances in patulin research

ABSTRACT The plant pathogenic fungus Penicillium expansum is a major concern of the global food industry due to its wide occurrence and ability to produce various mycotoxins, of which the most significant is patulin. Relatively less highlighted in the literature, in comparison with the other food-borne mycotoxins, patulin is one of the main factors in economic losses of vegetables and fruits. Otherwise, patulin is a health hazard which results in both short-term and long-term risks. This review includes knowledge on the biosynthetic mechanisms used for secondary metabolite production in P. expansum, with special emphasis on patulin biosynthesis. The abiotic factors triggering the production of patulin and the strategies developed to reduce or prevent the contamination by this mycotoxin are comprehensively discussed. The database presented in this review would be useful for the prioritization and development of future research.

Identification of the Anti-Aflatoxinogenic Activity of Micromeria graeca and Elucidation of Its Molecular Mechanism in Aspergillus flavus

Of all the food-contaminating mycotoxins, aflatoxins, and most notably aflatoxin B1 (AFB1), are found to be the most toxic and economically costly. Green farming is striving to replace fungicides and develop natural preventive strategies to minimize crop contamination by these toxic fungal metabolites. In this study, we demonstrated that an aqueous extract of the medicinal plant Micromeria graeca—known as hyssop—completely inhibits aflatoxin production by Aspergillus flavus without reducing fungal growth. The molecular inhibitory mechanism was explored by analyzing the expression of 61 genes, including 27 aflatoxin biosynthesis cluster genes and 34 secondary metabolism regulatory genes. This analysis revealed a three-fold down-regulation of aflR and aflS encoding the two internal cluster co-activators, resulting in a drastic repression of all aflatoxin biosynthesis genes. Hyssop also targeted fifteen regulatory genes, including veA and mtfA, two major global-regulating transcription factors. The effect of this extract is also linked to a transcriptomic variation of several genes required for the response to oxidative stress such as msnA, srrA, catA, cat2, sod1, mnsod, and stuA. In conclusion, hyssop inhibits AFB1 synthesis at the transcriptomic level. This aqueous extract is a promising natural-based solution to control AFB1 contamination.

A comparative study of procedures for binding of aflatoxin M1 to Lactobacillus rhamnosus GG

Several strains of lactic acid bacteria (LAB), frequently used in food fermentation and preservation, have been reported to bind different types of toxins in liquid media. This study was carried out to investigate the effect of different concentrations of Lactobacillus rhamnosus GG (ATCC 53103) to bind aflatoxin M1 (AFM1) in liquid media. AFM1 binding was tested following repetitive washes or filtration procedures in combination with additional treatments such as heating, pipetting, and centrifugation. The mixture of L. rhamnosus GG and AFM1 was incubated for 18 h at 37 °C and the binding efficiency was determined by quantifying the unbound AFM1 using HPLC. The stability of the complexes viable bacteria-AFM1 and heat treated bacteria-AFM1 was tested. Depending on the bacterial concentration and procedure used, the percentages of bound AFM1 by L. rhamnosus GG varied from as low as undetectable to as high as 63%. The highest reduction in the level of unbound AFM1 was recorded for the five washes procedure that involved heating and pipetting. Results also showed that binding was partially reversible and AFM1 was released after repeated washes. These findings highlight the effect of different treatments on the binding of AFM1 to L. rhamnosus GG in liquid matrix.