Effects of altitude on berry flavor compounds in spine grapes

Abstract:【Obiective】Spine grape (Vitis davidii Foex) is a wild variety cultivated in southern China. It has strong tolerances to high-temperatures and drought and a strong disease-resistance. In recent years, because of its good adaptability in southern China, it has been widely cultivated in Hunan province and Jiangxi province, and not only brings high economic benefits to local growers but also offers high-quality grape berries for fresh consumption and for fermentation in Hunan province. Flavor compounds are important quality indexes for evaluating wine, and wine flavors are mainly derived from grape berries.The flavor of grape berry consists of sweetness, sourness and aroma. Soil, altitude, slope and cultivation management are important factors affecting the types and contents of metabolites in grape berry such as carbohydrates, phenols, amino acids and volatile substances. Altitude is one of the important factors effecting metabolism of the winemaking grape berry. In this study, we evaluated the flavor compounds of‘Ziqiu'and‘Seputao'berries growing in vineyards at 3 different altitudes (240 m, 620 m, 700 m) in Zhongfang county, Hunan province.【Methods】Samples each with 20 clusters were randomly collected from the vineyards when grape reached commercial maturity. Samples were stored at-40 ℃ for laboratory analysis. Reducing sugars in the berries were determined using direct titration method with Folin's solution; total acids of berries determined with alkali titration method. Extraction and analyses of individual anthocyanin were conducted on frozen grape berries with pedicel removed. Skin was taken from the frozen berries and grinded into powder. Grape powder (0.5 g) and 5 mL methanol solution containing 2% formic acid were added into a test tube, sonicated for 10 min (30 ℃, 40 Hz) , shaken in an orbit-al shaker at 25℃ for 30 min at a rate of 150 r· min^-1, and centrifuged at 8 000 r· min^-1 for 10 min. The supernatant was collected, and the above extraction operation was repeated 4 times. The supernatants were pooled in a 50 mL centrifuge tube and evaporated in a rotary evaporator at 30 ℃ until dry. The residues were re-dissolved in 10 m L of the solution of the mobile phase for high performance liquid chromatography (HPLC) . All the steps were operated in the dark. Detection of anthocyanins was carried out using an Agilent 1100 series HPLC-MSD trap VL instrument, equipped with a diode array detector and a reverse phase column (Zorbax Eclipse SB C-18 250 mm×4.6 mm, 5 μm) . The mobile phase A was Vwater∶Vformic acid∶Vacetonitrile=92∶2∶6; the mobile phase B was Vwater∶Vformic acid∶Vacetonitrile=44∶2∶54. Elution program was as follows: 0-18 min, 10%-25% B; 18-20 min, 25% B; 20-30 min, 25%-40% B; 30-35 min, 40%-70% B; 35-40 min, 70%-100% B. The flow rate was 1.0 mL· min-1; the column temperature was 50 ℃;the detection wavelength 525 nm; the wavelength range was 200-900 nm and injection volume was 30μL. MS conditions were as follows: Electrospray ionization (ESI) interface, positive ion model, 35 psi nebulizer pressure, 10 m L· min^-1 dry gas flow rate, 325 ℃ dry gas temperature, and scans between m/z100 and 1 000. Anthocyanins were quantified at 525 nm using malvidin-3-O-glucoside as standard and calibration curves obtained within a concentration range between 0.5 and 500 mg· L^-1, with a linear correlation coefficient greater than 0.999. For extraction and analyses of individual non-anthocyanin phenols, 2 g grape powder was macerated with 5 mL distilled water and 45 mL ethyl acetate in a conical flask, and shaken for 30 min in darkness. The supernatant was transferred in a 250 mL round bottom flask and the residue was extracted four times. The pooled supernatant was evaporated to dryness in a rotary evaporator at 33 ℃, and then re-dissolved in methanol (2 mL) . The non-anthocyanin phenols were measured by Agilent 1200 series HPLC-ESI-MS, equipped with a G1322 A degasser, a G1312 B bin pump, a G1367 C HiP-ALS autosampler, a G1316 B TCC, a G1314 C VWD and a reversed phase column (ZORBAX SB-C18, 3 mm × 50 mm, 5 μm) . The mobile phases were (A) aqueous 1% acetic acid and (B) acetonitrile containing 1% acetic acid. The wavelength was 280 nm and the injection volume was 2 μ L. The gradient was 0-15 min, l0%-26% B; 30-50 min, 40%-65% B; 50-60 min, 65%-95% B;60-63 min, 95%-10% B; 63-66 min, 10% B at a flow rate of 1.0 mL/min. The aroma components were detected by GC-MS.【Results】Nine anthocyanins were detected in‘Seputao'and‘Ziqiu'berry skins.The total amount of anthocyanins, non-acylated anthocyanins, caffeoyl anthocyanins and coumarin acylated in‘Ziqiu'grape decreased with increase of altitude (240 m>700 m>620 m) . Non-acylated anthocyanins were the dominate anthocyanins in‘Seputao'and highest in samples from the altitude of240 meters (66.93%) followed by those from 700 m (55.88%) and lowest in those from 620 m (50.22%) . Eighteen non-anthocyanin phenols were detected in‘Seputao'and‘Ziqiu'berry skins from different altitudes, including hydroxybenzoates, flavanols, hydroxycinnamic acids and flavonols. Myricetin was not detected in the skin of‘Ziqiu'grape, while rhamnosyl-3-O-galactoside and clovene-galactoside were not detected in the skin of‘Seputao'. The total non-anthocyanin phenols content in‘Ziqiu'grape at different altitudes were 240 m> 620 m> 700 m and 700 m > 240 m > 620 m in‘Se spine'.19, 21 and 21 volatile aroma compounds were detected in‘Ziqiu'grape at the altitude of 240 m, 620 m and 700 m, respectively, and the total amount of aroma was 3 016.30 μg· L^-1 at 240 m, 2 705.20 μg· L^-1 at 620 m and 1 681.35 μg· L^-1 at 700 m. But volatile components of aroma of‘Seputao'were 3 148.65 at 700 m, 2 967.19 μg· L^-1 at 240 m and 1 908.53 μg· L^-1 at 620 m. Overall, at the height of 700 meters above sea level, the contents of anthocyanins, non-anthocyanin phenolic compounds, and aroma compounds in‘Seputao'were significantly higher than those at the other two altitudes. At the altitude of 240 meters, the contents of reducing sugars, anthocyanins, non-anthocyanin phenolic compoundsand aroma compounds in‘Ziqiu'were the highest.【Conclusion】‘Ziqiu'grape at the altitude of 240 meters and‘Seputao'at the altitude of 700 meters have the highest flavor content, which is beneficial to wine making.