Development of a biocontrol in order to reduce the contaminant toxins T2/HT2 in the brewing process. – TOXIFREE

Biological and biochemical methods are implemented. The microbiological cultures were realized on the solid and liquid media. The solid media allows a fast screening of the effective origins(stumps) of biocontrol, whereas the liquid environment(middle) allows to quantify better the phenomenon (a) of interaction whether it is regarding inhibition of the growth of the pathogenic, or its production of toxin T2. Sequential cultures are realized: preculture of the biocontrol G. candidum, then elimination of the strain, and the pathogenic Fusarium spp was added to the media pre-fermented by G. candidum. These sequential cultures are used to highlight certain mechanisms of interaction, to compare the efficiency on different Fusarium spp.The analytical chemistry allows to develop, to improve the dosages of the various métabolites microbial, whether it is the toxin T2 as the métabolites susceptible to intervene in the interaction.
Afterward, methods of follow-up of the expression of the genes will be used to verify what are the genes inhibited by the biocontrol.

A bibliographical report was realized. The analytical methods of dosage of different métabolites were organized or improved within the research team. The method of inoculation was standardized by using the spores of microorganisms. This allowed in particular to obtain first interesting results. The effect of a métabolite potentially inhibitive (literature) was validated by addition of the molecule in the environment of culture of various species of Fusarium (purchase of the pure molecule Sigma-Aldrich) .This metabolit seems to present an effect on the growth and on the production of the toxin by the tested species Fusarium. This métabolite seems produced in the first days of culture of the yeast (of 0 hours until 72 h) and disappears according to the fermentation.

Later experiences(experiments) must be led to be able to target / specify the moment and the peak of production of the metabolite by Geotrichum. This will be compared with the inhibitive effect of Geotrichum on Fusarium and to see if the effects are correlated. These results could help in the optimization of the implementation of the biocontrol in brewery.
experiments will then be realized to study the effect of this metabolit on the expression of the genes involved in the production of the toxin T2. we will use the data on the model strain Fusarium sporotrichoïdes for which the metabolic ways of production of toxin T2 are known. We shall look for if these ways metabolic can be found at Fusarium sporotrichoïdes (not known this day). Other mechanisms of interactions as the adsorption of the toxin will be studied (in particular via isotherms of sorption)

Poster at the french mycotoxine days (bordeaux-France)- january 2018

The goal of this project is to develop and optimize an innovating biocontrol to insure an efficient, safe and clean process in the brewing industry and particularly in the malting process. In Europe, the recent expansion of Fusarium spp. on cereals provokes a potential sanitary risk with the toxic molecules T2/HT2. The Barley-Malt-Beer chain is particularly affected and sensitive because of the environmental conditions prevailing during malting, which favour the development of Fusarium spp. brought by the barley at field. The French Barley-Malt-Beer network has considerable economic weight and the French malt industry is the fifth producer of malt in the world. In 2013, the European Union fixed recommended maximum concentrations of T2/HT2 in the cereals or derived products, depending on the cereals and their destination (animal or human feeds).
This project concerns the optimization of the biocontrol use, based on a preventive approach, using the yeast G. candidum. The experiments will consist in the knowledge’s improvement of the biocontrol action against Fusarium spp. in order to improve the use of the biocontrol mainly during the malting step.
The project is organised in two workpackages (WP). The first WP is focused on the indirect interaction between G. candidum and Fusarium spp. A preliminary work consists to repeat previous experiments from the Barsafe project to be sure having the same phenomenon of inhibition of G. candidum on F. langsethiae, and we will precise it : inhibition of the transcription, the translation or the excretion of mycotoxins. Then we will test the effect of G. candidum on another Fusarium spp.: F. sporotrichioïdes. The difference or not between the responses of these two Fusarium to G. candidum will contribute to the comprehension of the interaction mechanism, in order to improve the use of the biocontrol. Moreover, we will check if others phenomenon as degradation or/and adsorption of the mycotoxins by the biocontrol occur, as well as direct interaction by physical contact. Another task aims identifying the active agent produced by G. candidum. Literature cited some biological metabolites that seem able to inhibit the growth of others microorganisms. However, in our previous work, the Fusarium fungal growth was not really affected, but mainly toxin T2/HT2 concentration. We will research the presence of these molecules in our media thanks to purification and analytical methodologies available in the LGC.
The second Workpackage consists in optimizing the use of the biocontrol at several scales. In the first task, we preview to study and optimize the culture conditions of G. candidum in a laboratory bioreactor (medium composition, temperature, pH, batch/continuous process...) to increase the growth of G. candidum as well as the productivity and the yield of the active agent produced by G. candidum. As a final validation, the second task of WP3 will be to test the biocontrol in pilot experiments in an industrial environment. In this task, the IFBM will be solicited to provide us industrials who agree to test the biocontrol during the malting. In this step, we will follow the development of the G. candidum strain, we will control the presence of Fusarium spp., particularly F. langsethiae and F. sporotrichoïdes and the level of T2/HT2.
In this project, original and complementary approaches will combine molecular biology, fermentation, physiology and microbiology. The knowledge and the methodology developed in the previous Barsafe project, as well as the implication of two scientific experts will provide a solid “background” to the young researcher to quickly coordinate a project stopped 5 years ago. The results should allow developing and optimizing the use of an original and natural biocontrol in brewing industry, resulting in a better sanitary management of the process.