**1. Introduction**

The inoculation of commercial *S. cerevisiae* yeas<sup>t</sup> strains is the most common practice in the industrial elaboration of commercial wines. However, nowadays, winemakers are trying to obtain quality wines with different organoleptic characteristics. In this regard, the use of different species of yeas<sup>t</sup> could be interesting. Many studies have been done with respect to obtaining differentiated quality products and the use of non-*Saccharomyces* yeasts for this purpose [1–3]. The use of *H. vineae* in wineries could be a good alternative to the traditional *Saccharomyces* fermentations. This yeas<sup>t</sup> and

others apiculate yeas<sup>t</sup> of the genus *Hanseniaspora*/*Kloeckera* are the main species present on mature grapes and play a significant role at the beginning of fermentation, producing enzymes and aroma compounds that expand the diversity of wine colour and flavor [4]. Normally, *H. vineae* appears in the first stages of the fermentation but it is quickly dominated by *S*. *cerevisiae* [5]. The main interest in this yeas<sup>t</sup> is due to the aromatic profile of the wines obtained [6]. This yeas<sup>t</sup> produces a fruity and floral aroma due to the increased amounts of acetate esters, primarily 2-phenylethyl acetate [7] and benzyl acetate. Other authors [8,9] investigated the potential of to the genus *Hanseniaspora* to produce acetate esters. In the same way, the modulation of the aeration during the growing stage of these yeasts can increase the aromatic diversity and quality of the wine obtained [10]. In addition, the *H*. *vineae* species can be used in pure culture because this yeas<sup>t</sup> might reach about 10% of the alcohol by volume of fermentative capacity under winemaking conditions [4]. In this respect, we conducted a semi-industrial assay in this study using *H*. *vineae* in pure culture compared to *S*. *cerevisiae* in the control.

Additionally, in this study, the use of *H. vineae* in aging on lees (AOL) has been assayed in comparison with other yeas<sup>t</sup> species. The AOL technique consists of a long contact of the yeas<sup>t</sup> lees with the wine. During this contact, the yeas<sup>t</sup> autolysis is produced with the breakdown of cell membranes, the release of intracellular constituents, the liberation of hydrolytic enzymes and the hydrolysis of intracellular biopolymers into low molecular weight products [11]. Among these compounds, the polysaccharides have an effect on the physico-chemical properties of the wine, as well as on the sensory properties [12]. The AOL improves the aromatic and gustatory complexity of wine, mainly by improving its body and reducing its astringency [13]. The main problem of this technique is that the AOL is a slow process, many studies have been done trying to accelerate the cell lysis like the use of emerging physical technologies such as high hydrostatic pressures and ultrasounds [14]. Another technique to reduce the ageing time is the use of yeas<sup>t</sup> species that have a high capacity to release polysaccharides into the wine. In previous studies, [15] certain wine spoilage yeasts like *Saccharomycodes ludwigii, Zygosaccharomyces bailii,* and *Brettanomyces bruxellensis* were shown to produce a greater quantity of polysaccharides compared to *S. cerevisiae* strains. In the same way, these authors classified the released polysaccharides according to their composition. Therefore, the AOL may depend on the yeas<sup>t</sup> used and its cell wall polysaccharide composition.

The main objective of this work is to obtain information about the use of *H. vineae* in alcoholic fermentation as well as in the AOL technique.

#### **2. Materials and Methods**

#### *2.1. Yeast Species Used in Alcoholic Fermentation*

The *H. vineae* yeas<sup>t</sup> strain used in this study was isolated by Professor Francisco Carrau (Facultad de Química, Universidad de la República, Montevideo, Uruguay) and it is currently under evaluation by "Oenobrands SAS, France".

The yeas<sup>t</sup> strain Fermivin 3C (*S. cerevisiae*) used as control in this study is a selected yeas<sup>t</sup> marketed by "Oenobrands SAS, France".

#### *2.2. Alcoholic Fermentation Conditions*

The Albillo grape variety (*Vitis vinifera L.*) was fermented at "Comenge Bodegas y Viñedos SA" (Curiel de Duero, Spain). The white must was fermented in triplicate in 120 L stainless steel barrels. The fermentation process was monitored following the daily variation of density and temperature. The samples were taken once at the end of the fermentation.

#### *2.3. Yeast Species Used in Ageing on Lees*

Two strains of *S. cerevisiae* were used as controls in the AOL assay, the strains 7VA and G37 (SC7VA, SCG37), both yeasts were isolated by the Chemistry and Food Technology Department of ETSI Agronómica, Alimentaria y de Biosistemas, Universidad Politécnica de Madrid.

Three species of non-*Saccharomyces* yeasts were used, the same *H. vineae* strain that had been previously used in the alcoholic fermentation trial, as well as *Lachancea thermotolerans* L31 strain (L31) isolated and selected by enotecUPM (Food Technology Department, ETSIAAB, Universidad Politécnica de Madrid) and *Schizosaccharomyces pombe* 938 (SP938, IFI, CSIC, Madrid, Spain).

The yeas<sup>t</sup> lees biomass used for the AOL assay was obtained by growing in 2 L of YEPD medium enriched with 100 g/<sup>L</sup> of glucose. The growth was carried out at 25 ◦C for three days. Then, the biomass was washed three times with deionized water, discarding the supernatant after each centrifugation, at 1200 rcf, for 3 min.

#### *2.4. Ageing on Lees Conditions*

The AOL was done in hydroalcoholic solution (13.5% *v*/*v*) sulphited to 60 mg/<sup>L</sup> with K2S2O5 and the pH was adjusted to 3.5 with phosphoric acid. The samples were prepared in triplicates, using ISO flasks of 0.5 L. The dosage of yeas<sup>t</sup> lees was 6 g/<sup>L</sup> and the ageing process was done at 16 ◦C in a dark room for 156 days. The samples were mixed once a week to simulate a *bâtonnage* process.

#### *2.5. Basic Oenological Parameters Analysis*

The values of ethanol (% *v*/*v*), pH, total acidity (g/L) expressed as tartaric acid, volatile acidity (g/L) expressed as acetic acid, malic acid (g/L), lactic acid (g/L) and glucose/fructose content (g/L) were obtained by Fourier transform infrared spectroscopy (FTIR), using an OenoFoss™ instrument (FOSS Iberia, Barcelona, Spain).
