OENO IVAS 2019 banner
IVES 9 IVES Conference Series 9 OENO IVAS 9 OENO IVAS 2019 9 Grape and wine microorganisms: diversity and adaptation 9 New antibacterial peptides produced by Saccharomyces cerevisiae responsible for the inhibition of malolactic fermentation

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.

DOI:

Publication date: June 23, 2020

Issue: OENO IVAS 2019

Type: Article

Authors

Nancy Nehme, Ziad Rizk, Youssef El Rayess, Chantal Ghanem, Florence Mathieu, Patricia Taillandier , Nancy Nehme

Lebanese Agricultural Research Institute (LARI)- Fanar Station- P.O. Box 90–1965, Jdeidet El-Metn, Fanar- Lebanon 
Université de Toulouse, Laboratoire de Génie Chimique, CNRS, INPT, UPS, Toulouse, France 

Contact the author

Keywords

antibacterial yeast peptides, Wtm2p, Utr2p, GAPDH 

Tags

IVES Conference Series | OENO IVAS 2019

Citation

Related articles…

The concept of terroir: what place for microbiota?

Microbes play key roles on crop nutrient availability via biogeochemical cycles, rhizosphere interactions with roots as well as on plant growth and health. Recent advances in technologies, such as High Throughput Sequencing Techniques, allowed to gain deeper insight on the structure of bacterial and fungal communities associated with soil, rhizosphere and plant phyllosphere. Over the past 10 years, numerous scientific studies have been carried out on the microbial component of the vineyard. Whether the soil or grape compartments have been taken into account, many studies agree on the evidence of regional delineations of microbial communities, that may contribute to regional wine characteristics and typicity. Some authors proposed the term “microbial terroir” including “yeast terroir” for grapes to describe the connection between microbial biogeography and regional wine characteristics. Many factors are involved in terroir including climate, soil, cultivar and human practices as well as their interactions. Studies considering “microbial terroir” greatly contributed to improve our knowledge on factors that shape the vineyard microbial structure and diversity. However, the potential impact of “microbial terroir” on wine composition has yet not received strong scientific evidence and many questions remain to be addressed, related to the functional characterization of the microbial community and its impact on plant physiology and grape composition, the origins and interannual stability of vineyard microbiota, as well as their impact on wine sensorial attributes. The presentation will give an overview on the role of microbiota as a terroir component and will highlight future perspectives and challenges on this key subject for the wine industry.

Evolution of the amino acids content through grape ripening: Effect of foliar application of methyl jasmonate with or without urea

The parameters that determine the grape quality, and therefore the optimal harvest time, suffer variations during berry ripening, related to climate change, with the widely known problem of the gap between technological and phenolic maturities. However, there are few studies about its incidence on grape nitrogen composition. For this reason, the use of an elicitor, methyl jasmonate (MeJ), alone or with urea, is proposed as a tool to reduce climatic decoupling, allowing to establish the harvest time in order to achieve the optimum grape quality. The aim was to study the effect of MeJ and MeJ+Urea foliar applications on the evolution of Tempranillo amino acids content throughout the grape maturation. Three treatments were foliarly applied, at veraison and 7 days later: control (water), MeJ (10 mM) and MeJ+Urea (10 mM+6 kg N/ha). Grape samples were taken at five stages of maturation: day before the first and second applications, 15 days after the second application (pre-harvest), harvest day, and 15 days after harvest (post-harvest). The amino acids analysis of the samples was carried out by HPLC. Results showed that the evolution of amino acids was similar regardless of the treatment; however, foliar applications influenced the nitrogen compounds content, i.e., there was no qualitative effect but quantitative one. Most of the amino acids reached their maximum concentration in pre-harvest, being higher in grapes from the treatments than in the control. In general, no differences in grape amino acids content were observed between MeJ and MeJ+Urea treatments. Foliar applications with MeJ and MeJ+Urea enhanced the grape amino acids content, without affecting their profile, helping to optimize their quality and allowing to establish a more complete grape ripening standard. Therefore, MeJ and MeJ+Urea foliar applications can be a simple agronomic practice, which has shown promising results in order to enhance the grape quality.

δ13C : A still underused indicator in precision viticulture  

The first demonstration of the interest of carbon isotope composition of sugars in grapevine, as an integrated indicator of vineyard water status, dates back to 2000 (Gaudillère et al., 1999; Van Leeuwen et al., 2001). Thanks to the isotopic discrimination of Carbon that takes place during plant photosynthesis, under hydric stress conditions, it is possible to accurately estimate the photosynthetic activity. Ever since, δ13C has been widely applied with success to zonation, terroir studies and vine physiology research, but is still not widely used by viticulturists. This is quite astonishing by considering the impact of global warming on viticulture and the need to improve water management, that would justify a widespread use of δ13C.
The lack of private laboratories proposing the analysis, the cost of the technology, as well as the long analytical delays, have been detrimental to its development. Some laboratories tried to overcome the analytical difficulties of isotopic analysis by using fourier transformed infrared spectroscopy, as a fast and cheap alternative to the official OIV method (IRMS). These claimed FTIR models have never been published or peer reviewed and cannot be considered robust. In this work, thanks to the recent acquisition of IRMS technology, new modern and robust applications of δ13C for viticulture are proposed. This includes the use of the analysis to make parcel separations at harvesting, the possibility to increase the precision of hydric stress cartography and the potential cost reduction when compared with Scholander pressure bomb analysis.

Teasing apart terroir: the influence of management style on native yeast communities within Oregon wineries and vineyards

Newer sequencing technologies have allowed for the addition of microbes to the story of terroir. The same environmental factors that influence the phenotypic expression of a crop also shape the composition of the microbial communities found on that crop. For fermented goods, such as wine, that microbial community ultimately influences the organoleptic properties of the final product that is delivered to customers. Recent studies have begun to study the biogeography of wine-associated microbes within different growing regions, finding that communities are distinct across landscapes. Despite this new knowledge, there are still many questions about what factors drive these differences. Our goal was to quantify differences in yeast communities due to management style between seven pairs of conventional and biodynamic vineyards (14 in total) throughout Oregon, USA. We wanted to answer the following questions: 1) are yeast communities distinct between biodynamic vineyards and conventional vineyards? 2) are these differences consistent across a large geographic region? 3) can differences in yeast communities be tied to differences in metabolite profiles of the bottled wine? To collect our data we took soil, bark, leaf, and grape samples from within each vineyard from five different vines of pinot noir. We also collected must and a 10º brix sample from each winery. Using these samples, we performed 18S amplicon sequencing to identify the yeast present. We then used metabolomics to characterize the organoleptic compounds present in the bottled wine from the blocks the year that we sampled. We are actively in the process of analysing our data from this study.

Terroir traceability in grapes, musts and wine: results of research on Gewürztraminer and Sauvignon Blanc grape varieties in northern Italy

In the study of terroir, a separate analysis of its many component factors can be of great help in accurately identifying a vineyard’s natural elements that impact wine quality and typicity. This research used a dedicated pluri-disciplinary approach to investigate the ecological characteristics, including geology and geographical features, of 14 vineyards that produce Gewürztraminer and Sauvignon Blanc cultivars in the alpine Alto Adige DOC wine region. Both the geopedological method using Vineyards Geological Identity (VGI) and the new Solar Radiaton Identity (SRI) topoclimatic classification method were used to provide analytical measurements and qualitative/quantitative characterisations. In addition, wide-ranging targeted and untargeted oenological and chemical analyses were carried out on grapes, musts and wines to correlate the soils’ geomineral and physical conditions with the biochemical properties of their fruits and wines. The research identified strong correlations between vineyard geo-identity and wine biofingerprint, confirming a mineral traceability of strontium rubidium ratio and some minerals distinctive to the local geology, such as K, Ca, Ag, Ba and Mn.  The study also discovered that particular geomineral and physical soil conditions of the studied vineyards are related to the different amount of amino acids, primary varietal aromas and polyphenols found in grapes, musts and wines. The research confirmed that winemaking technologies support oenological quality, although in some cases, human practices can overpower certain characteristic elements in wine, erasing the typical imprint left by the vineyards’ natural terroir, which becomes less traceable. Terroir abiotic ecological factors and vineyard identity can be classified in detail using the new VGI and SRI analysis methods to discover interrelationships between geo-pedological and topoclimatic conditions that impact wine quality. These methods are also helpful in identifying which ecological elements are exclusive to a particular vineyard or wine sub-region.