New antibacterial peptides produced by Saccharomyces cerevisiae responsible for the inhibition of malolactic fermentation

Abstract

In winemaking, several antimicrobial peptides (AMPs) produced by different strains of Saccharomyces cerevisiae were found to be responsible for the inhibition of malolactic fermentation (MLF) carried out by some strains of Oenococcus oeni. However, only two AMPs produced by one of the yeast strains studied were totally identified and their mechanism of action was described. In an attempt to identify new AMPs, a 5-10 kDa peptidic fraction produced by an oenological strain of S. cerevisiae and previously shown to strongly inhibit MLF carried out by a strain of O. oeni was further purified. 

A synthetic grape juice medium fermented by the yeast strain was fractionated by ammonium sulfate precipitation combined with ultrafiltration. The 5-10 kDa peptidic fractions obtained at saturation degrees of 0 %-20 %, 20 %-40 % and 40 %-60 %, inhibited only the growth of O. oeni in vivo but not its ability to consume L-malic acid. The 5–10 kDa peptidic fraction recovered at a saturation degree of 60 %–80 % was the only one that inhibited both the bacterial growth and the malate consumption. It also inhibited the malolactic enzyme activity in vitro at a pH range between 3.5 and 6.7 in a cell-free enzymatic extract prepared from the same bacterial strain. Therefore, it was further purified by both anion and cation exchange chromatography. The eluates that inhibited the malolactic enzyme activity in vitro at the same pH range were migrated on Tricine SDS-PAGE and the protein bands were excised and sequenced by LC-MS/MS. 

The sequencing revealed nine peptides originating from eight proteins of S.cerevisiae that play diverse vital roles in yeast cells. Two GAPDH cationic fragments of 0.9 and 1.373 kDa having a pI of 10.5 and 11 respectively, Wtm2p and Utr2p anionic fragments of 2.42 kDa with a pI of 3.5 and 4 respectively were considered to contribute the most to the MLF inhibition. However, it is likely that one or more of the nine peptides have worked synergistically to inhibit MLF. In vivo, they are supposed to enter the bacterial cytoplasm and inhibit the malolactic enzyme by mechanisms yet to be identified. 

These results suggest that the 5-10 kDa fraction recovered at a saturation degree of 60 %-80 % contained at least two categories of peptides; the ones responsible for the bacterial growth inhibition and those responsible for the malate consumption inhibition. Whereas the fractions recovered between 0 % and 60 % contained only peptides that inhibited the bacterial growth.