Preliminary steps of a protocol to isolate transcription factors bound to a specific DNA locus in grapevine using CRISPR-dCas9 system

Non-Saccharomyces yeasts have been associated, for many years, with challenging alcoholic fermentation processes. However, during the last decade the use of non-Saccharomyces yeasts in wine production has become increasingly widespread due to the advantages they can offer in mixed inoculations with Saccharomyces cerevisiae (Sc). In this respect, Hanseniaspora vineae (Hv), in synergy with Saccharomyces spp, represents an interesting opportunity to impart a positive contribution to the aroma complexity of wines. In fact, it is a well-known producer of pleasant esters, such as 2-phenylethyl acetate. This study compares the performances of Hv (strain Hv-205) in sequential inoculation modality to Sc in three Chardonnay musts for base sparkling wine production. No significant differences were observed in basic chemical parameters between wines except for titratable acidity, with a significantly decrease (up to 1.5 g/L) in Hv processes due to malic acid degradation. The analysis of the aroma compounds revealed remarkable differences in concentration of volatile metabolites, among others up to 37-fold increase of 2-phenylethyl acetate. In contrast, lower concentration of its alcohol were detected, suggesting higher acetylation activity by Hv.

In the past decade, there has been an increasing amount of work dedicated to understanding micro-oxygenation in wine.

The use of toasted vine-shoots as an alternative enological tool to make differentiated wines has generated interest among researchers and wineries. However, the evolution of these wines in bottle and the effect on the sensory profile has not been studied so far.

The objective of the present work is to assess yeast effects on the development of wine varietal aroma throughout aging and on wine longevity.

Three independent experiments were carried out; two fermenting semi-synthetic musts fortified with polyphenols and aroma precursors extracted from Tempranillo (1) or Albariño (2) grapes and with synthetic precursors of polyfunctional mercaptans (PFMs), and a third in which a must, mixture of 6 different grape varieties was used. In all cases, fermentations were carried out by different Saccharomyces cerevisiae strains and one S. kudriavzevii, and the obtained wines were further submitted to anoxic accelerated aging to reproduce bottle aging. The volatile profile of the wines was analyzed using several chromatographic procedures, in order to provide a comprehensive evaluation of wine aroma. Aroma compounds analyzed included fermentation volatile metabolites, grape-derived aroma compounds including PFMs, and Strecker aldehydes (SA).

Results revealed that the effects of yeast on wine aroma throughout its self-life extend along three main axes:

1. A direct or indirect action on primary varietal aroma and on its evolution during wine
aging.

2. The direct production of SA during fermentation and/or their delayed formation by producing the required reagents (amino acids + dicarbonyls) for Strecker degradation
during anoxic aging.

3. Producing acids (leucidic, branched acids) precursors to fruity esters. More specifically, and leaving aside the infrequent de novo formation, the action of the different strains of yeast on primary varietal aroma takes four different forms:

1.- Speeding the hydrolysis of aroma precursors, which leads to early aroma formation without changing the amount of aroma formed. In the case of labile molecules, such as linalool, the enhancement of young wine aroma implies a short-living wine. 2.- Metabolizing the aroma precursor, reducing the amounts of aroma formed, which can be of advantage for negative aroma compounds, such as TDN or guaiacol; 3.- Transforming grape components into aroma precursors, increasing the amounts of aroma formed, as for ethyl cinnamate, leucidic acid or vinylphenols; 4.- Forming reactive species such as vinylphenols able to destroy varietal polyfunctional mercaptans.

Overall, it can be concluded that the yeast carrying alcoholic fermentation not only influences fermentative wine aroma but also affects to the wine varietal aroma, to its evolution during aging and to the development of oxidative off-odors

– Several practices can be applied to vineyards in order to ensure good healthy for grapevines, adequate yield and fruit quality. Among them, the use of cover crops is a relevant option for soil management. It increases the organic matter, improves water infiltration, reduces risks of soil erosion and greenhouse gas emissions, in addition improving biodiversity in the vineyard.