*2.6. NMR Spectroscopy*

NMR spectra of a triplicate set of Albillo white wines fermented with *H. vineae* and *S. cerevisiae* yeas<sup>t</sup> strains, were carried out on a Bruker 600 Avance III HD spectrometer, equipped with a 5-mm 1H/D TXI probehead equipped with a z-gradient at 298 ± 0.1 K of temperature. The following set of NMR experiments were conducted:



**Table 1.** Targeted metabolites concentration (mg/L) of *Saccharomyces cerevisiae* and *Hanseniaspora vineae* wine samples obtained with the PULCON-NMR method [21].

> Means with the same letter are not significantly different (*p* < 0.05).

#### *2.7. Volatile Compounds from the Alcoholic Fermentation Analysis*

a

The volatile compounds of the wines obtained in fermentation assay were measured using an Agilent Technologies 6850 gas chromatograph, equipped with an integrated flame ionization detector (GC-FID) and DB-624 column (60 m × 250 μm × 1.40 μm). Analyses were performed according to the method described by [23]. The injector temperature was 250 ◦C, and the detector temperature was 300 ◦C. The column temperature was 40 ◦C for the first 5 min, rising linearly by a 10 ◦C/min until reaching 250 ◦C; this temperature was maintained for 5 min. Hydrogen was used as the carrier gas. The flow rate was 22.1 L/min. The injection split ratio was 1:10. The detection limit was 0.1 mg/L.

Calibration was performed using the following external standards: acetaldehyde, metanol, 1-propanol, diacetyl, ethyl acetate, 2-butanol, isobutanol, 1-butanol, acetoin, 2-methyl-1-butanol, 3-methyl-1-butanol, isobutyl acetate, ethyl butyrate, ethyl lactate, 2.3-butanediol, 3-ethoxy-1-propanol, isoamyl acetate, hexanol, 2-phenyl ethanol and 2-phenylethyl acetate.

#### *2.8. Proteins and Nucleic Acids Estimation by Absorbance at 260 and 280 nm*

The absorbance measurements were done through the ageing after centrifugation (1200 rcf for 3 min) using a 1-cm path-length quartz cuvette. All spectrometric measurements were obtained using an 8453 spectrophotometer from Agilent Technologies™ (Palo Alto, CA, USA).

#### *2.9. Polysaccharides Analysis (HPLC-RI)*

The polysaccharides content was measured after 156 days of ageing in the AOL assay, using an HPLC-RI technique. An 1100 HPLC chromatograph (Agilent Technologies, Palo Alto, CA, USA) equipped with a refractive index detector with Ultrahydrogel 250 molecular exclusion column (Waters) was used, according to the method described by [24]. The eluent was 0.1 M NaNO3 in deionized water (MilliQ). A calibration curve constructed from the following pullulan standards (polymaltotriose) (Shodex, Showa Denko K.K, Japan) were used to determine the concentration of polysaccharides in the samples: P-800 (788 kDa), P-400 (404 kDa), P-200 (212 kDa), P-100 (112 kDa), P-50 (47.3 kDa), P-20 (22.8 kDa), P-10 (11.8 kDa) and P-5 (5.9 kDa).
