Chemical characterization and enological potential of less frequent red grape Uruguayan varieties by study secondary metabolites

. Study of secondary grape metabolites, such as polyphenol compounds (anthocyanins, ﬂavonols, ﬂavan-3-ols), volatile compounds present in grapes in both free and glycoside forms (monoterpenols, norisoprenoids, benzenoids), allowed us to characterize grape varieties and to suggest the best winemaking practices to maximize their enological potential. The Vitis vin´ıferas studied in the present work are less frequent red grape varieties cultivated in small vineyards in southern Uruguay (Montevideo and Canelones Provinces): Ancellota, Aspiran Bouschet (syn. Lacryma Christi), Marselan (Grenache × Cabernet Sauvignon), Arinarnoa (Tannat × Cabernet Sauvignon), Egiodola (Abouriou × Tinta Negra Mole) and Caladoc (Malbec × Grenache).


Introduction
The grapevine (Vitis vinifera), belonging to the Vitaceae family, comprises species actually distributed in almost all the continents, usually under subtropical, Mediterranean and continental-temperate climatic conditions.The great majority of cultivars widely cultivated for wine, classified as Vitis vinifera L. spp vinifera, derive from wild forms (This et al., 2006).
However, and for different reasons (mainly pathogenic problems and climate conditions), the main cultivars used for grape production on a worldwide basis are changing rapidly.
Uruguay currently has almost 6,500 hectares (http:// www.inavi.com.uy/uploads/archivos/Estadisticas de Vinedos Nacionales 2017.pdf) of vineyards, being fundamentally a country of small producers where 100% of the establishments have been family properties for more than three generations.
Among the Uruguayan wines, those elaborated from Vitis vinifera cv Tannat are the most known, being this varietal representative of the country wines between international consumer.Nevertheless, work is being carried out to improve quality to obtain premium wines, through blends using less frequent grape varieties being introduced and employed in the wine production looking for sensory particularities.

Grape samples and polyphenolic compounds extraction
This study considered 6 red grape varieties (Vitis vinifera L.) cultivated in small vineyards in southern Uruguay in the 2016 vintage.The selected varieties were: 4 varieties obtained by crossing (Marselan, Arinarnoa, Egiodola and Caladoc), the Ancellota variety and a Tintorera cultivar (Aspiran Bouschet, in Uruguay syn.Lacryma Christi).Marselan is a crossing between Grenache and Cabernet Sauvignon; the Arinarnoa between Tannat and Cabernet Sauvignon; the Egiodola crossing between Abouriou and Tinta negra Mole; and Caladoc between Malbec and Grenache.
In order to extract anthocyanins and polyphenols compounds, forty grape berries were weighted, the seeds and skins were separated manually, and pulps were discarded (García-Marino et al., 2006; Boido et al., 2011).

Targeted analysis (HPLC-DAD)
The targeted analysis aimed at the identification and quantification of anthocyanins in skin extracts.It was

Non-targeted analysis (HPLC-MS)
The non-targeted analysis aimed at the tentative identification of free and bound phenols in both skin and seed extracts.The chromatographic separation was performed with a Thermo Ultimate TM 3000 HPLC was coupled with a tandem mass spectrometer (Q-Exactive TM ; Thermo Scientific, Bremen, Germany) furnished with a heated electrospray source (HESI-II).The mass analysis was performed in negative ion mode, adapting the method proposed by Barnaba et al. (2017).LC column (150 mm × 3 mm, 2.6 µm particle size; Thermo Fischer Scientific, Waltham, MA, USA) was used.

Volatile compounds analysis
The extraction of volatile compounds and aromatic precursors was carried out using an Isolute (IST Ldt., Mid Glamorgan, U.K.) ENV+ cartridge packed with 1 g of highly cross-linked-styrene-divinyl benzene (SDVB) polymer.The extractions of free and glycosylated volatile compounds was carried out according to the procedure described by Boido et al. (2013).GC-MS analysis was performed on a GC-MS QP 2010, using a Stabilwax capillary column (30 m, 0.25 mm, 0.25 µm film thickness; Restek Corporation, Bellefonte, PA.) according to Boido et al. (2013).

Volatile compounds
The aromatic precursors concentration in the varieties analyzed is presented in Table 1.All the varieties present low concentration of terpenes compounds, with the high concentration in Ancellota and Aspriran Bouschet.On the other hand, the total norisoprenoids compounds was higher in Arinarnoa with more than double the concentration of the other varieties.
The benzenoids and volatile phenols was the quantitative more important group of aromatic precursors compounds.Interesting, for the high anthocyanin content variety Aspirant Bouschet, the sum of benzenoids and volatile phenols compounds results in around 870 µg/kg, the highest value determined for the varieties analyzed.The Arinarnoa and Ancellota with 780 µg/kg and 760 µg/kg was the other two variety with highest values for these groups of compounds.
In conclusion, from these results, we infer that these less frequent red grape varieties cultivated have enological potential, which can easily be transferred to the wine by means of suitable winemaking practices.On the other hand, the data presented can be evaluated for the use of these varieties in blend wines with more planted varieties in the country.