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…

Evaluation of climate change impacts at the Portuguese Dão terroir over the last decades: observed effects on bioclimatic indices and grapevine phenology

In the last decades the growers of the Portuguese Dão winegrowing region (center of Portugal) are experiencing changes in climate that are influencing either grape phenology berry health and ripening. Aiming to study the relationships between climate indices (CI), seasonal weather and grapevine phenology, in this work long-term climate and phenological data collected at the experimental vineyard of the Portuguese Dão research centre between 1958 and 2019 (61 years) for the red variety Touriga Nacional, was analyzed. The trends over time for the classical temperature-based indices (Growing Season Temperature – GST -, Growing Degree Days – GDD, Huglin Index – HI and Cool Night Index – CI) presented a significantly positive slope while the Dryness Index (DI) showed a negative trend over the last 61 years. Regarding grapevine phenology, an average advance of 4.5 days per decade in the harvest day was observed throughout the last 61 years. Consequently, the weather conditions during the ripening period have changed, showing an increasing trend over time in the average temperature (higher magnitude in the maximum than in the minimum temperature) and a decrease in the accumulated rainfall. A regression analysis showed that ~50% of harvest date variability over years was explained by the temperature-based indices variability. These observed effects of climate change on bioclimatic indices and corresponding anticipation of harvest date can still be considered advantageous for the Dão terroir as it allows to achieve an optimal berry ripening before the common equinox rains and, therefore, avoid the potential negative impacts of the rainfall on berry health and composition.

Impact on leaf morphology of Vitis vinifera L. cvs Riesling and Cabernet Sauvignon under Free Air Carbon dioxide Enrichment (FACE)

Atmospheric carbon dioxide (CO2) concentration has continuously increased since pre-industrial times from 280 ppm in 1750, and is predicted to exceed 700 ppm by the end of 21st century. For most of C3 plant species elevated CO2 (eCO2) improve photosynthetic apparatus results in an increased plant biomass production. To investigate the effects of eCO2 on morphological leaf characteristics the two Vitis vinifera L. cultivars, Riesling and Cabernet Sauvignon, grown in the Geisenheim VineyardFACE (Free Air Carbon dioxide Enrichment) system were used. The FACE site is located at Geisenheim University (49° 59′ N, 7° 57′ E, 94 m above sea level), Germany and was implemented in 2014 comparing future atmospheric CO2-concentrations (eCO2, predicted for the mid-21st century) with current ambient CO2-conditions (aCO2). Experiments were conducted under rain-fed conditions for two consecutive years (2015 and 2016). Six leaves per repetition of the CO2 treatment were sampled in the field and immediately fixed in a FAA solution (ethanol, H2O, formaldehyde and glacial acetic acid). After 24 h leaf samples were transferred and stored in an ethanol solution. Subsequently, leaf tissue was dehydrated using ethanol series and embedded in paraffin. By using a rotary microtomesections of 5 µm were prepared and fixed on microscopic slides. Subsequent the samples were stained using consecutive staining and washing solutions. Afterwards pictures of the leaf cross-sections were taken using a light microscope and consecutive measurements were conducted with an open source image software. Differences found in leaf cross-sections of the two CO2 treatments were detected for the palisade parenchyma. Leaf thickness, upper and lower epidermis and spongy parenchyma remained less affected under eCO2 conditions. The observed results within grapevine leaf tissues can provide first insights to seasonal adaptation strategies of grapevines under future elevated CO2 concentrations.

Soil, vine, climate change – what is observed – what is expected

To evaluate the current and future impact of climate change on Viticulture requires an integrated view on a complex interacting system within the soil-plant-atmospheric continuum under continuous change. Aside of the globally observed increase in temperature in basically all viticulture regions for at least four decades, we observe several clear trends at the regional level in the ratio of precipitation to potential evapotranspiration. Additionally the recently published 6th assessment report of the IPCC (The physical science basis) shows case-dependent further expected shifts in climate patterns which will have substantial impacts on the way we will conduct viticulture in the decades to come.
Looking beyond climate developments, we observe rising temperatures in the upper soil layers which will have an impact on the distribution of microbial populations, the decay rate of organic matter or the storage capacity for carbon, thus affecting the emission of greenhouse gases (GHGs) and the viscosity of water in the soil-plant pathway, altering the transport of water. If the upper soil layers dry out faster due to less rainfall and/or increased evapotranspiration driven by higher temperatures, the spectral reflection properties of bare soil change and the transport of latent heat into the fruiting zone is increased putting a higher temperature load on the fruit. Interactions between micro-organisms in the rhizosphere and the grapevine root system are poorly understood but respond to environmental factors (such as increased soil temperatures) and the plant material (rootstock for instance), respectively the cultivation system (for example bio-organic versus conventional). This adds to an extremely complex system to manage in terms of increased resilience, adaptation to and even mitigation of climate change. Nevertheless, taken as a whole, effects on the individual expressions of wines with a given origin, seem highly likely to become more apparent.

Variety and climatic effects on quality scores in the Western US winegrowing regions

Wine quality is strongly linked to climate. Quality scores are often driven by climate variation across different winegrowing regions and years, but also influenced by other aspects of terroir, including variety. While recent work has looked at the relationship between quality scores and climate across many European regions, less work has examined New World winegrowing regions. Here we used scores from three major rating systems (Wine Advocate, Wine Enthusiast and Wine Spectator) combined with daily climate and phenology data to understand what drives variation across wine quality scores in major regions of the Western US, including regions in California, Oregon and Washington. We examined effects of variety, region, and in what phenological period climate was most predictive of quality. As in other studies, we found climate, based mainly on growing degree day (GDD) models, was generally associated with quality—with higher GDD associated with higher scores—but variety and region also had strong effects. Effects of region were generally stronger than variety. Certain varieties received the highest scores in only some areas, while other varieties (e.g., Merlot) generally scored lower across regions. Across phenological stages, GDD during budbreak was often most strongly associated with quality. Our results support other studies that warmer periods generally drive high quality wines, but highlight how much region and variety drive variation in scores outside of climate.

Impact of changes in pruning practices on vine growth and yield

A gradual decline in vineyards has been observed over the past twenty years worldwide. This might be explained by the climate change, practices change or the increase of dieback diseases. To increase the longevity of vines, we studied the impact of different pruning strategies in four adult and four young vineyards located in France and Spain. In France, vineyards were planted with Cabernet franc on 3309C while Spanish trials were planted with Tempranillo grafted on 110R. Vegetative expression, yield, quality of berries and wood vessels conductivity were measured. The distribution of vegetative expression, yield and berry composition between primary and secondary vegetation were quantified. Finally, tomography was used to evaluate the implication of the treatments on sap flows.
First results show that i) the respectful pruning leads to an increase of 30 to 50% more secondary shoots than the aggressive pruning in France and between 15 and 20% in Spain, ii) there is no major effect on the yield over the first two years following the implementation of the new pruning practices, although the proportion of clusters from suckers is higher on the respectful pruning method. On young vines, the development of the trunk according to a respectful pruning leads to a loss of harvest 2 years after planting. This is due to the removal, on the future trunk, of the green suckers which carrying bunches. This operation carried out in spring rather than during winter pruning, would promote a better leaf / fruit balance when the plant comes into production, and could lead to better hydraulic conduction in the vessels of the trunk. Maintaining these trials for several years will provide more robust data to assess the impact of these practices on the vines over the long term.