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…

Geospatial trends of bioclimatic indexes in the topographically complex region of Barolo DOCG

Barolo DOCG is an economically important wine producing region in Northwest Italy. It is a small region of approximately 70 km2 gross area. The topography is very complex with steep sloped hills ranging in elevation from below 200 m to 550 m. Barolo DOCG wine is made exclusively from the Nebbiolo grape. Bioclimatic indexes are often used in viticulture to gain a better understanding of broader climate trends which can be compared temporally and geographically. These indexes are also used for identifying potential phenological timing, growing region suitability, and potential risks associated with expected climatic changes. Understanding how topography influences bioclimatic indexes can help with understanding of mesoscale climate behaviour leading to improved decision making and risk management strategies. The average monthly maximum and minimum temperatures, the Cool Night Index, the Huglin Index, and the monthly diurnal range (from July to October) were calculated using data from 45 weather stations within a 40 km radius of the Barolo DOCG growing area between the years 1996 and 2019. Linear and multiple regression models were developed using independent variables (elevation, aspect, slope) extracted from a digital elevation model to identify significant relationships. Bioclimatic indexes were then kriged with external drift using independent variables that showed significant relationships with the bioclimatic index using a 100 m resolution grid. The maximum monthly temperatures and the Huglin Index showed consistent significant negative relationships with elevation in all years. The minimum monthly temperatures showed no relationship with elevation but in some months a small but significant relationship was observed with aspect. Due to the lack of a relationship between minimum monthly temperatures and elevation compared to the significant relationship between maximum monthly temperatures and elevation, monthly diurnal range had a negative relationship with elevation.

The impact of sustainable management regimes on amino acid profiles in grape juice, grape skin flavonoids, and hydroxycinnamic acids

One of the biggest challenges of agriculture today is maintaining food safety and food quality while providing ecosystem services such as biodiversity conservation, pest and disease control, ensuring water quality and supply, and climate regulation. Organic farming was shown to promote biodiversity and carbon sequestration, and is therefore seen as one possibility of environmentally friendly production. Consumers expect organically grown crops to be free from chemical pesticides and mineral fertilizers and often presume that the quality of organically grown crops is different or higher compared to conventionally grown crops. Integrated, organic, and biodynamic viticulture were compared in a replicated field trial in Geisenheim, Germany (Vitis vinifera L. cv. Riesling). Amino acid profiles in juice, grape skin flavonoids, and hydroxycinnamic acids were monitored over three consecutive seasons beginning 7 years after conversion to organic and biodynamic viticulture, respectively. In addition, parameters such as soil nutrient status, yield, vigor, canopy temperature, and water stress were monitored to draw conclusions on reasons for the observed changes. Results revealed that the different sustainable management regimes highly differed in their amino acid profiles in juice and also in their skin flavonol content, whereas differences in the flavanol and hydroxycinnamic acid content were less pronounced. It is very likely that differences in nutrient status and yield determined amino acid profiles in juice, although all three systems showed similar amounts of mineralized nitrogen in the soil. Canopy structure and temperature in the bunch zone did not differ among treatments and therefore cannot account for the observed differences in favonols. A different light exposure of the bunches in the respective systems due to differences in vigor together with differences in berry size and a different water status of the vines might rather be responsible for the increase in flavonol content under organic and biodynamic viticulture.

Rootstock regulation of scion phenotypes: the relationship between rootstock parentage and petiole mineral concentration

Grapevine is grown as a graft since the end of the 19th century. Rootstocks not only provide tolerance to Phylloxera but also ensure the supply of water and mineral nutrients to the scion. Rootstocks are an important mean of adaptation to environmental conditions, because the scion controls the typical features of the grapes and wine. However, among the large diversity of rootstocks worldwide, few of them are commercially used in the vineyard. The aim of this study was to investigate the extent to which rootstocks modify the mineral composition of the petioles of the scion. Vitis vinifera cvs. Cabernet-Sauvignon, Pinot noir, Syrah and Ugni blanc were grafted onto 55 different rootstock genotypes and planted in a vineyard as three replicates of 5 vines. Petioles were collected in the cluster zone with 6 replicates per combination. Petiolar concentrations of 13 mineral elements (N, P, K, S, Mg, Ca, Na, B, Zn, Mn, Fe, Cu, Al) at veraison were determined. Scion, rootstock and the interaction explained the same proportion of the phenotypic variance for most mineral elements. Rootstock genotype showed a significant influence on the petiole mineral element composition. Rootstock effect explained from 7 % for Cu to 25 % for S of the variance. The difference of rootstock conferred mineral status is discussed in relation to vigor and fertility. Rootstocks were also genotyped with 23 microsatellite markers. Data were analysed according to genetic groups in order to determine whether the petiole mineral composition could be related to the genetic parentage of the rootstock. Thanks to a highly powerful design, it is the first time that such a large panel of rootstocks grafted with 4 scions has been studied. These results give the opportunity to better characterize the rootstocks and to enlarge the diversity used in the vineyard.

Assessing the climate change vulnerability of European winegrowing regions by combining exposure, sensitivity and adaptive capacity indicators

Winegrowing regions recognized as protected designations of origin (PDOs) are closely tied to well defined geographic locations with a specific set of pedoclimatic attributes and strictly regulated by legal specifications. However, climate change is increasingly threatening these regions by changing local conditions and altering winegrowing processes. The vulnerability to these changes is largely heterogenous across different winegrowing regions because it is determined by individual characteristics of each region, including the capacity to adapt to new climatic conditions and the sensitivity to climate change, which depend not only on natural, but also socioeconomic and legal factors. Accurate vulnerability assessments therefore need to combine information about adaptive capacity and climate change sensitivity with projected exposure to new climatic conditions. However, most existing studies focus on specific impacts neglecting important interactions between the different factors that determine climate change vulnerability. Here, we present the first comprehensive vulnerability assessment of European wine PDOs that spatially combines multiple indicators of adaptive capacity and climate change sensitivity with high-resolution climate projections. We found that the climate change vulnerability of PDO areas largely depends on the complex interactions between physical and socioeconomic factors. Homogenous topographic conditions and a narrow varietal spectrum increase climate change vulnerability, while the skills and education of farmers, together with a good economic situation, decrease their vulnerability. Assessments of climate change consequences therefore need to consider multiple variables as well as their interrelations to provide a comprehensive understanding of the expected impacts of climate change on European PDOs. Our results provide the first vulnerability assessment for European winegrowing regions at high spatiotemporal resolution that includes multiple factors related to climate exposure, sensitivity, and adaptive capacity on the level of single winegrowing regions. They will therefore help to identify hot spots of climate change vulnerability among European PDOs and efficiently direct adaptation strategies.

Impact of geographical location on the phenolic profile of minority varieties grown in Spain. II: red grapevines

Because terroir and cultivar are drivers of wine quality, is essential to investigate theirs effects on polyphenolic profile before promoting the implantation of a red minority variety in a specific area. This work, included in MINORVIN project, focuses in the polyphenolic profile of 7 red grapevines minority varieties of Vitis vinifera L. (Morate, Sanguina, Santafe, Terriza Tinta Jeromo Tortozona Tinta) and Tempranillo) from six typical viticulture Spanish areas: Aragón (A1), Cataluña (A2), Castilla la Mancha (A3), Castilla –León (A4), Madrid (A5) and Navarra (A6) of 2020 season. Polyphenolic substances were extracted from grapes. 35 compounds were identified and quantified (mg subtance/kg fresh berry) by HPLC and grouped in anthocyanins (ANT) flavanols (FLAVA), flavonols (FLAVO), hydroxycinnamic (AH), benzoic (BA) acids and stilbenes (ST). Antioxidant activity (AA, mmol TE /g fresh berry) was determined by DPPH method. The results were submitted to a two-way ANOVA to investigate the influence of variety, area and their interaction for each polyphenolic family and cluster analysis was used to construct hierarchical dendrograms, searching the natural groupings among the samples. Sanguina (A3) had the most of total polyphenols while Tempranillo (A5) those of ANT. Sanguina (A2) and (A3) reached the highest values of FLAVO, FLAVA and AA. These two last samples had also the maximum of AA. The effect cultivar and area were significant for all polyphenolic families analyzed. A high variability due to variety (>50%) was observed in FLAVA and the maximum value of variability due to growing area was detected in AA (86.41%), ANT and FLAVO (51%); the interaction variety*zone was significant only for ANT, FLAVO, EST and AA. Finally, dendrograms presented five cluster: i) Sanguina (A2); ii) Sanguina (A3); iii) Tempranillo (A5); iv) Tempranillo (A3); Terriza (A3,A5), Morate (A5,A6); v) Santafé (A1,A6); Tortozona tinta (A1,A3,A6); Tinta Jeromo (A3,A4).