4.3. Fermentation Conditions

Two distinct fermentation batches were made. Simple and double deletion mutants of genes involved in linear ester biosynthesis were tested in a thermo-treated grape juice of Cabernet Sauvignon (CS) harvested in 2013 in the Bordeaux area and conserved at −20 °C. The assimilable nitrogen concentration of this grape juice was 200 mg N/L with mixed sources of 18 α-amino acids and ammonium nitrogen (60/40 ratio). The source of α-amino acids was previously described by [37]; the source of mineral nitrogen was a solution of (NH₄)₂SO₄. Fermentation took place in cylindric glass vessels of 300 mL with permanent stirring (200 rpm) according to the conditions described by [38].

A second fermentation batch was made in larger volumes in order to test the organoleptic impact of gene deletion. Two different grape varieties harvested in 2015 were used: a Merlot (Bordeaux area, France) and a Tempranillo (Rioja area, Spain). In order to mimic red-grape vinification, a specific protocol was developed. Full grapes were destemmed, crushed, pressed, and both the juice and the skins were conserved separately. Before freezing, potassium metabisulfite was added to the must to reach 50 mg/L of total SO₂. For both grape varieties, the sugar concentration was set at 230 g/L of reducing sugar by adding an equimolar amount of D-glucose and D-fructose. The assimilable nitrogen concentration was adjusted to 210 mg N/L, keeping a 66:34 balance ratio between amino acids and ammonium nitrogen source using the solution described above. Fermentations took place in 2.5 L cylindric glass flasks filled with 1.6 L of juice and with 400 mL of grape skin in order to keep a juice/solid ratio of 80:20, as is usual in oenology, and each flask was mixed twice a day to ensure a homogenous fermentation.

In all fermentation batches, the grape juice was inoculated with 1.10⁶ viable cell/mL obtained from 24 h precultures carried out in half-diluted grape must sterilized by membrane filtration (cellulose acetate 0.45 μm Sartorius Stedim Biotech, Aubagne, France). Yeast viability and concentration were estimated by flux cytometry “Cell Lab Quanta SC” (Beckman Coulter, USA, California) according to the procedure previously described [39]. Fermentation kinetics were monitored by CO₂ release [40]. At the end of the alcoholic fermentation, the wines were collected in glass bottles and stored at 10 °C for 1 week after the addition of SO₂ (50 mg/L).