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Impact and identification of inhibitory peptides released by Saccharomyces cerevisiae on the malolactic fermentation

Rizk, Ziad. Impact and identification of inhibitory peptides released by Saccharomyces cerevisiae on the malolactic fermentation. PhD, Génie des Procédés et de l'Environnement, Institut National Polytechnique de Toulouse, 2016

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The production of most red wines and certain white and sparkling wine styles requires two consecutive fermentation steps. The first one is the alcoholic fermentation (AF) and is carried out mainly by Saccharomyces cerevisiae. At the end of the AF, the wines undergo malolactic fermentation (MLF) carried out mainly by Oenococcus oeni. However, the MLF is often difficult to trigger and accomplish because of the individual or synergistic antibacterial activity of several physical chemical wine parameters and yeast inhibitory metabolites. In this context, the study of the interactions that may occur between specific strains of yeasts and bacteria is important for choosing the adequate strain combination and inoculation strategy. In the present work, S. cerevisiae strain D strongly inhibited O. oeni strain X during sequential fermentations performed in synthetic grape juice (SGJ) media whereas S. cerevisiae strain A stimulated it. Protease and heat treatments of the SGJ media fermented by strain D showed the protein nature of the yeast inhibitory metabolites. Fractionation by ultrafiltration of the same media revealed that an extracellular peptidic fraction of 5-10 kDa was responsible for the inhibition. It was gradually released during AF and reached its highest concentration at late stages of the stationary phase. The MLF inhibition was maintained in natural grape juices and grape musts (Cabernet-Sauvignon and Syrah) presenting low and high phenolic contents. Therefore, the activity of the inhibitory peptides was not affected by grape phenolic compounds. The 5-10 kDa fraction was tested in vitro on cell-free bacterial cytosolic extracts containing the malolactic enzyme in a pH range between 3.5 and 6.7. Results showed that it was able to directly inhibit the malolactic enzyme activity with an increasing inhibitory kinetic correlated to the AF time at which it was collected. The 5-10 kDa peptidic fraction of the 60-80 % ammonium sulfate precipitate was submitted to analyses by both anionic and cationic exchange chromatography (AEXC and CEXC). Eluates recuperated with 0.5 M NaCl from both AEXC and CEXC contained inhibitory peptides and were further migrated by SDS-PAGE. The bands of interest were excised and sequenced by LC1D-nanoESI-LTQ-Orbitrap. Results gave 12 different peptidic fractions that may have worked synergistically. 2 GAPDH fragments of 0.9 and 1.373 kDa having a pI of 9.074 and a Wtm2p fragment of 2.42 kDa having a pI of 3.35 were involved in the MLF inhibition.

Item Type:PhD Thesis
Uncontrolled Keywords:
Institution:Université de Toulouse > Institut National Polytechnique de Toulouse - Toulouse INP (FRANCE)
Laboratory name:
Research Director:
Taillandier, Patricia and Mathieu, Florence
Deposited On:08 Jan 2018 15:05

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