Thierry Liboz

Review of mycotoxin reduction in food and feed: from prevention in the field to detoxification by adsorption or transformation

Mycotoxins are secondary metabolites present worldwide in agricultural commodities and produced by filamentous fungi that cause a toxic response (mycotoxicosis) when ingested by animals. Prevention of mycotoxicoses includes pre- and post-harvest strategies. The best way to reduce the mycotoxin content in food and feed is the prevention of mycotoxin formation in the field, but this is often not sufficient, so other methods are needed. To decontaminate and/or detoxify mycotoxin-contaminated food and feed, the most prevalent approach in the feed industry is the inclusion of sorbent materials in the feed thus obtaining more or less selective removal of toxins by adsorption during passage through the gastrointestinal tract. Another reliable approach is to add enzymes or microorganisms capable of detoxifying some mycotoxins. Through a comprehensive review of published reports on the strategies for mycotoxin removal, this present work aims to update our understanding of mycotoxin removal. It provides an insight into the detoxification of mycotoxin present in food and feed. In the future, more emphasis needs to be placed on adsorption of mycotoxins in the gastrointestinal tract. Concerning the enzymatic transformation of mycotoxins, further efforts are required in understanding detoxification reactions, the toxicity of transformation products and in the characterization of enzymes responsible for transformations.

Comparative analysis of early embryonic sunflower cDNA libraries.

To gain information concerning cell functions and activities during sunflower embryogenesis, an expressed sequence tag (EST) approach was used to analyse gene expression in the early stages of sunflower embryos development. Confocal microscopy observations of whole-mounted embryos allowed us to identify precisely the major steps of the zygotic embryonic development. A time-course analysis was then employed to collect the embryonic material. Three cDNA libraries were constructed from microdissected embryos, and three other cDNA libraries were created using a classical day after pollination schedule. A total of 7106 ESTs were produced and assembled. The total number of putative different genes represents about 43.1 (3064 tentative contigs and singlets) of the analysed sequences. The unigenes that showed similarity to proteins with known or predicted functions (50.3) were classified into 15 different functional categories. The functional profiles were found to be quite similar for all studied embryo stages but statistical analysis revealed that successive and coordinate sets of genes are expressed at each embryonic stage. The analysis allowed us to identify abundant and differentially expressed genes at the early stages of embryos development as well as some putatively interesting genes, showing strong similarities with genes playing key roles in plant and animal embryogenesis. The data presented in this study not only provide a first global overview of the genes expression profile during sunflower embryogenesis but also represent an original and valuable tool for developmental genomics studies on exalbuminous dicots.

Sequence evaluation of four specific cDNA libraries for developmental genomics of sunflower.

Four different cDNA libraries were constructed from sunflower protoplasts growing under embryogenic and non-embryogenic conditions: one standard library from each condition and two subtractive libraries in opposite sense. A total of 22,876 cDNA clones were obtained and 4800 ESTs were sequenced, giving rise to 2479 high quality ESTs representing an unigene set of 1502 sequences. This set was compared with ESTs represented in public databases using the programs BLASTN and BLASTX, and its members were classified according to putative function using the catalog in the Kyoto Encyclopedia of Genes and Genomes (KEGG). Some 33% of sequences failed to align with existing plant ESTs and therefore represent putative novel genes. The libraries show a low level of redundancy and, on average, 50% of the present ESTs have not been previously reported for sunflower. Several potentially interesting genes were identified, based on their homology with genes involved in animal zygotic division or plant embryogenesis. We also identified two ESTs that show significantly different levels of expression under embryogenic and non-embryogenic conditions. The libraries described here represent an original and valuable resource for the discovery of yet unknown genes putatively involved in dicot embryogenesis and improving our knowledge of the mechanisms involved in polarity acquisition by plant embryos.

Microbial degradation of aflatoxin B1: Current status and future advances

Aflatoxin B1 (AFB1) is a natural toxin produced by many food-contaminant fungi and is a threat to human and animal health. This review summarizes current knowledge of the different ways to limit AFB1 in the food chain. We start by introducing current data and reviews available on the prevention of AFB1 occurrence, on AFB1 non-biological decontamination and biological adsorption. We then focus on microbial AFB1-degradation. The latter has already been well studied using living organisms, supernatants or purified enzymes. This review compiles information on the variety of protocols and the efficacy of the different sub-kingdoms or classes of microorganisms or their enzymes. We pay particular attention to publications closest to in vivo applications of microbial AFB1-degradation. In addition, this review also provides a summary of the currently known microbial degradation metabolites of AFB1 and their levels of toxicity, and provides recommendations on the most promising techniques to pursue the aim of minimizing ABF1 in the food supply.

Transformation of zearalenone to zearalenone-sulfate by Aspergillus spp.

The primary goal of this research was to assess the biotransformation of zearalenone (ZEA), an oestrogenic mycotoxin, into ZEA-sulfate by some Aspergillus section Nigri isolates. A. niger isolates were shown to be able to convert ZEA after 72 h of incubation at different ZEA concentrations (5 to 150 µg/ml). The product formula corresponding to ZEA-sulfate was determined by accurate mass measurement. This conjugate was shown to be less oestrogenic than ZEA using a MCF-7 proliferation assay. This study demonstrated that A. niger has the ability to transform ZEA over a broad range of ZEA concentrations (5 to 150 µg/ml) and that sulfonation could lead to a less toxic compound. These results are significant as little is known about the ability of fungi, and especially A. niger, to detoxify ZEA by sulfonation. This study could lead to a way of detoxifying feed naturally contaminated with mycotoxins.

In vitro interaction of actinomycetes isolates with Aspergillus flavus: impact on aflatoxins B1 and B2 production

This work aimed to study the interaction between Actinomycetal isolates and Aspergillus flavus to promote mutual antagonism in contact. Thirty‐seven soilborn Streptomyces spp. isolates were chosen as potential candidates. After a 10‐day in vitro co‐incubation period, 27 isolates respond to the criteria, that is, mutual antagonism in contact. Further aflatoxins B1 and B2 analysis revealed that those 27 isolates reduced aflatoxin B1 residual concentration from 38·6 to 4·4%, depending on the isolate. We selected 12 isolates and tested their capacity to reduce AFB1 in pure culture to start identifying the mechanisms involved in its reduction. AFB1 was reduced by eight isolates. The remaining AFB1 concentration varied between 82·2 and 15·6%. These findings led us to suggest that these eight isolates could be used as biocontrol agents against AFB1 and B2 with low risk of impacting the natural microbial equilibrium.

Reduction of aflatoxin production by Aspergillus flavus and Aspergillus parasiticus in interaction with Streptomyces

The aim of this study is to investigate aflatoxin gene expression during Streptomyces-Aspergillus interaction. Aflatoxins are carcinogenic compounds produced mainly by Aspergillus flavus and Aspergillus parasiticus. A previous study has shown that Streptomyces-A. flavus interaction can reduce aflatoxin content in vitro. Here, we first validated this same effect in the interaction with A. parasiticus. Moreover, we showed that growth reduction and aflatoxin content were correlated in A. parasiticus but not in A. flavus. Secondly, we investigated the mechanisms of action by reverse-transcriptase quantitative PCR. As microbial interaction can lead to variations in expression of household genes, the most stable [act1, βtub (and cox5 for A. parasiticus)] were chosen using geNorm software. To shed light on the mechanisms involved, we studied during the interaction the expression of five genes (aflD, aflM, aflP, aflR and aflS). Overall, the results of aflatoxin gene expression showed that Streptomyces repressed gene expression to a greater level in A. parasiticus than in A. flavus. Expression of aflR and aflS was generally repressed in both Aspergillus species. Expression of aflM was repressed and was correlated with aflatoxin B1 content. The results suggest that aflM expression could be a potential aflatoxin indicator in Streptomyces species interactions. Therefore, we demonstrate that Streptomyces can reduce aflatoxin production by both Aspergillus species and that this effect can be correlated with the repression of aflM expression.

Streptomyces–Aspergillus flavus interactions: impact on aflatoxin B accumulation

The aim of this work was to investigate the potential of Streptomyces sp. as biocontrol agents against aflatoxins in maize. As such, we assumed that Streptomyces sp. could provide a complementary approach to current biocontrol systems such as Afla-guard® and we focused on biocontrol that was able to have an antagonistic contact with A. flavus. A previous study showed that 27 (out of 38) Streptomyces sp. had mutual antagonism in contact with A. flavus. Among these, 16 Streptomyces sp. were able to reduce aflatoxin content to below 17% of the residual concentration. We selected six strains to understand the mechanisms involved in the prevention of aflatoxin accumulation. Thus, in interaction with A. flavus, we monitored by RT-qPCR the gene expression of aflD, aflM, aflP, aflR and aflS. All the Streptomyces sp. were able to reduce aflatoxin concentration (24.0–0.2% residual aflatoxin B1). They all impacted on gene expression, but only S35 and S38 were able to repress expression significantly. Indeed, S35 significantly repressed aflM expression and S38 significantly repressed aflR, aflM and aflP. S6 reduced aflatoxin concentrations (2.3% residual aflatoxin B1) and repressed aflS, aflM and enhanced aflR expression. In addition, the S6 strain (previously identified as the most reducing pure aflatoxin B1) was further tested to determine a potential adsorption mechanism. We did not observe any adsorption phenomenon. In conclusion, this study showed that Streptomyces sp. prevent the production of (aflatoxin gene expression) and decontamination of (aflatoxin B1 reduction) aflatoxins in vitro.

Mass cloning of differential and nondifferential transcript-derived fragments from cDNA-AFLP experiments in sunflower

We have developed a 1-step procedure for cloning multiple bands obtained through cDNA-amplified fragment length polymorphism (AFLP) experiments. This protocol enables us to clone differential and nondifferential transcript-derived fragments (TDFs) in the same experiment. It consists of a mass cloning of cDNA-AFLP amplification products, identification of clones of interest with electrophoresis, and characterization through sequencing. The entire experiment can be performed in 1 wk because of the reduced time and effort needed for cloning, and it can be applied to any PCR method that produces complex band patterns. This method, tested on a comparison of gene expression between globular and heart embryo stages in sunflower embryogenesis, enabled us to clone 10 differential TDFs in the same experiment.

Adsorption of zearalenone by Aspergillus japonicus conidia: new trends for biological decontamination in animal feed.

Zearalenone (ZEA) is an oestrogenic mycotoxin produced by Fusarium spp. on a wide variety of grains. The presence of ZEA impairs farm animal health. In this study, the ability to remove ZEA by conidia of Aspergilli was evaluated in vitro. This mycotoxin was successfully eliminated in vitro by living and heat-treated conidia just after conidial inoculation, especially by Aspergillus japonicus Mu541 (IMI389204) conidia. Consequently, components involved in adsorption are not affected by heat-treatment. The influence of pH on ZEA removal was also determined. Results have shown that ZEA removal was 53% at pH 2 while 2% removal was observed at pH 11. Then, the adsorption capacity was evaluated in vitro in porcine gastrointestinal tract conditions. We observed that there was better adsorption in acidic stomach conditions. Finally, the phenomenon has been modelled using Hills equation. This model was very suitable and confirmed better adsorption at acidic pH. Aspergilli section Nigri conidia were able to adsorb...