Nature and localisation of inhibitory biochemical elements.
In order to identify the biochemical elements encountered by Fusarium when it infects wheat or maize grains, scientists in the Mycology and Food Safety Research Unit (MycSA) have carried out field experiments on the kinetics of Fusarium infection. While monitoring the development kinetics of the fungus and its toxin production, they characterised changes affecting the biochemical composition of grains during filling.
In parallel, the localisation of "anti-toxin" entities was studied at the level of plant tissues following grain fractionation. Bran, meals and flours were inoculated with a panel of Fusarium. Fungal development and the production of toxins were quantified and compared.
Purification and identification of elements inhibiting toxin production in plant tissues.
Plant tissues in grains that may contain biochemical elements inhibiting the biosynthetic pathways of toxins were subjected to different fractionation procedures in order to obtain a fraction as pure as possible containing inhibitory activity. This fraction then underwent different analyses (involving mass spectrometry) to enable its identification.
Impact of candidate biochemical compounds on the biosynthesis of toxins by Fusarium.
Thanks to greater knowledge of pathways for the biosynthesis of toxins, some of the compounds present in grains at the time when Fusarium produces its toxins have shown, through their structure and chemical properties, that they might be able to interfere with toxin production. Thus any compounds endowed with pro- or anti-oxidant properties have been suspected. The efficacy of these compounds, and study of the mechanisms of inhibition, are analysed using approaches that couple biochemical and molecular techniques.
State of the art
The most significant results obtained to date have concerned the contamination of wheat by trichothecenes.
The outer coats of wheat grains: a reservoir of inhibitory compounds.
When seeded on wheat bran, fungi of the Fusarium species produced very few trichothecenes, but they developed significantly. The same wheat bran added to a culture medium providing optimum conditions for toxin production induced a marked inhibition of trichothecene synthesis. These clues have led scientists to suspect the presence of compounds inhibiting trichothecene biosynthesis in the outer coat of wheat grains.
Monomeric phenolic acids, potent effectors of trichothecene biosynthesis.
Concentrated in the outer coats of grains, phenolic acids in a monomeric form are also present at the initiation of toxin production by Fusarium, and some of them (particularly ferulic acid) at high concentrations. Studies performed by the MycSA Unit have demonstrated that ferulic, p-coumaric, caffeic, sinapic and chlorogenic acids very efficiently inhibited the production of trichothecenes at concentrations similar to those present in cereals. This inhibition appeared to be closely correlated to the antioxidant potential of these compounds. Phenolic acids thus blocked toxin synthesis by reducing the expression of the principal genes coding for enzymes in the biosynthetic pathways. Individually, phenolic acids act as "anti-toxin" compounds. The efficacy of mixtures of phenolic acids, as they are found in grains, is currently being assessed by the MycSA Unit.
What role can be attributed to phenolic acid oligomers?
A purification approach involving several chromatographic steps has made it possible to isolate a fraction of wheat bran exhibiting highly significant inhibitory activity. Analysis of this fraction by mass spectrometry showed that its inhibitory activity resulted from the presence of a biochemical entity, the structure of which (similar to that of phenolic acid dimmers) is currently being characterised. The "anti-toxin" efficacy of this biochemical element has proved to be markedly superior (nearly 10-fold) to that of phenolic acid monomers. To confirm the potential role of oligomeric forms of phenolic acids, researchers in the MycSA Unit have now initiated their chemical synthesis and will be testing the efficacy of the dimers thus produced.
Research carried out in the Unit has demonstrated the potential role for phenolic acids in modulating the levels of mycotoxins in cereals. These data are currently being validated in planta, in close collaboration with actors in the cereals sector, and particularly with seed producers.