Received: 2 April 2019 y Accepted: 9 July 2019 y Published: 26 August 2019 DOI:10.20870/oeno-one.2019.53.3.2460 VINE AND WINE OPEN ACCESS JOURNAL Comparison of the anthocyanins composition of five wine-making grape cultivars cultivated in the Wujiaqu area of Xinjiang, China Ke Zhang 1#, Lin Yuan 2,3 #, Qian Li 1, Rui Wang 1 and Zhen-Zhen Zhang 1* 1College of Food Science and Pharmacy, Xinjiang Agricultural University, Urumqi, Xinjiang 830052, China 2College of Food Science and Engineering, Tianjin Agricultural University, Tianjin 300380, China 3College of Chemical Engineering, Tianjin University, Tianjin 300380, China *Corresponding author: [email protected] #These authors equally contributed to this work. ABSTRACT Anthocyanins biosynthesis are essentially determined by grape cultivar genotype. However, specific environmental and vine management conditions could alter the accumulation of anthocyanins. This study selected five wine- making grape cultivars cultivated in the Wujiaqu area of the Xinjiang region of China to investigate the profiles of anthocyanins. Results showed that a total of 15 anthocyanins were identified. Marselan contained 15 anthocyanins, whereas only 14 anthocyanins were present in Cabernet-Sauvignon and Cabernet Franc. Syrah and Petit Verdot were found to only contain 13 anthocyanins. Cabernet-Sauvignon and Cabernet Franc appeared to show the highest and lowest levels of the total anthocyanins, respectively. Cabernet-Sauvignon possessed the highest levels of the total monomeric and acylated anthocyanins, respectively. Malvidin-3-O-glucoside and its acylated derivatives (malvidin-3-O-(6-O-acetyl)-glucoside and malvidin-3-O-(6-O-coumaryl)-glucoside) were the dominant anthocyanins in these grape cultivars. Principal component analysis revealed that the acylated anthocyanins were the major anthocyanins that differentiated these grape cultivars. KEYWORDS anthocyanins, grapes, Xinjiang Region, principal component analysis, HPLC-MS OENO One 2019, 3, 549-559 © 2019 International Viticulture and Enology Society - IVES 549 Ke Zhang et al. INTRODUCTION Although some studies have reported on the performance of Cabernet Franc, Marselan and Grapes are one of the most popular fruits in the Petit Verdot , there is no related research in the world and have gained much interest in the field Wujiaqu area. The previous studies indicated that of food and nutritional sciences (Bakels, 2002). the anthocyanin content was affected by It has been confirmed that the consumption of environment conditions, soil status and grapes and wines could significantly lower the viticulture and the anthocyanin biosynthesis incidence of multiple chronic diseases, including pathway was altered by difference climate obesity, hypertension, diabetes and cancers conditions (Kliewer, 1970; Mori et al. , 2005; (Costa et al. , 2017; Iriti and Varoni, 2014). Such Xing et al. , 2015) Therefore, the anthocyanin health benefits in grapes and wines mainly result compounds and profiles of five red grapes from their high levels of antioxidants cultivated in the Wujiaqu area might be different (Frederiksen et al. , 2007). to grapes from other regions. The purpose of this study was to investigate the anthocyanin Polyphenols are the major antioxidants in grapes compounds of five wine grapes in the climactic and wines and these secondary metabolites play conditions of the Wujiaqu area to help improve a vital role in the determination of the sensory the quality of the grape berries using appropriate attributes of grapes (Zhao et al. , 2010). In cultivation techniques, to evaluate the addition, polyphenols, as the major antioxidants, winemaking potential of the different grape also determine the nutritional and bioactive cultivars and to enrich variety of grape cultivars. functions of grapes and wines (He and Giusti, 2010). Anthocyanins are a major polyphenol MATERIALS AND METHODS group and they have been confirmed to exert a significant role in the appearance of grapes and 1. Chemicals and reagents wines (Frederiksen et al. , 2007). During the The external standard malvidin-3-O-glucoside grape development period, anthocyanins are was purchased from ExtraSynthesis SA (Genay, mainly biosynthesized and accumulated in the France) with a purity of 95.0 %. The HPLC skin of grapes through the conjugation of grade methanol, acetonitrile, formic acid and anthocyanidins with sugar moiety (Filippetti et acetic acid were purchased from Fisher Scientific al. , 2015). The major anthocyanins in Vitis (Fairlawn, NJ, USA). The analytical grade ethyl vinifera L. grapes include cyanidin-3-O- acetate was from Beijing Chemical Reagent glucoside, petonidin-3-O-glucoside, dephinidin- Company (Beijing, China). Deionized water was 3-O-glucoside, peonidin-3-O-glucoside and purified using the Milli-Q purification system malvidin-3-O-glucoside. These monomeric (Millipore, Burlington, MA, USA). The other anthocyanins further experience acylation and/or reagents used in the study were of analytical polymerization to form anthocyanins derivatives grade unless specifically noted. in grapes (Hrazdina and Franzese, 1974). It has been accepted that the grape cultivar genotype 2. Experimental site and grape cultivars essentially determines the anthocyanins composition, whereas environmental and Fully ripened grapes of five cultivars – Cabernet cultivation conditions could play a role in Franc (No.327, Grafted 5BB), Cabernet- affecting the accumulation of anthocyanins in Sauvignon (No.338 Own-rooted), Marselan grapes through regulating the polyphenols (C980, Grafted 1130P), Petit Verdot (Grafted metabolisms (de Freitas and Mateus, 2011). 1130P) and Syrah (No.100, Grafted 1130P) – were hand harvested in a 2017 vintage at the The Wujiaqu area of northern Tianshan mountain experimental stage at the Tangting Xianlu winery region is an important wine production area in in the northern foothills of the Tianshan Xinjiang, with the same latitude as California Mountains in the Wujiaqu area (44 20’ north, 87 and Bordeaux. The area possesses a high day-to- 53’ east, altitude 440.5m). For the experiment, night temperature difference with a long we used V. vinifera L. CV . grapevines that were sunshine duration and a high sunshine intensity. root planted in 2013. Row and vine spacing were The major soil type in the area consists of sandy set to 3.0 m and 1.0 m, respectively, with rows and/or rocky soils. These conditions are oriented in the east –west direction. A modified extremely suitable for grape cultivation. In vertical shoot positioned system was employed, recent years, the major varieties produced in this with sloping trunks and with horizontal cordons area are Cabernet-Sauvignon and Merlot . positioned 0.5 m above ground. All vines were 550 © 2019 International Viticulture and Enology Society - IVES OENO One 2019, 3, 549-559 spur-pruned with 15 –18 nodes per linear meter (Agilent Technologies, Santa Clara, CA, USA) and were normalized to one or two bunches per was used to separate anthocyanins under a 1 shot. Nutrition and pest management was carried mL/min flow rate. The mobile phase consisted out according to industry standards for the of (A) 5.0 % v/v formic acid in water and (B) specified cultivar and region, as previously 5.0% v/v formic acid in acetonitrile. An elution described (Cheng et al. , 2014). The detailed gradient was programed as follows: 0–1 min, 3 harvest information of these cultivars is listed in % B isocratic; 1–12 min, 3 %–5 % B; 12–24 Table 1. The harvested grape samples were min, 15 %–25 % B; 24–28 min, 25 %–30 %B; placed in plastic bags with dry ice and and 28–32 min, 30 %B–40 %B. The column was immediately transported to our laboratory for maintained at 25°C during the elution program further analysis. and the wavelength on the diode array detector was set at 525 nm. The anthocyanins extract was 3. Physicochemical indexes measurements filtered through 0.45 µm filters and a 20 µL The berry weight of each grape cultivar was extract was injected into HPLC. A positive calculated by weighing 100 randomly picked electrospray ionization was used under a 30 psi berries. A PAL-2 digital Brix refractometer was nebulizer, a 12 mL/min drying gas and at a used to measure the total soluble solids ( °Brix ) 300°C drying temperature. A full mass scan with of the grape berries. The titratable acidity of each 100–1500 m/z was used to record the mass grape cultivar was analyzed by titrating the grape spectrum of anthocyanins. Anthocyanins were juice pH to 8.2 with NaOH and was expressed as tentatively identified by comparing their mass gram tartaric acid equivalents per liter of juice. spectrum with a published method (He et al. , 2010b). Each grape cultivar anthocyanin extract 4. Anthocyanins extraction was analyzed in triplicate. The external standard, malvidin-3-O-glucoside, was used to quantify The extraction of anthocyanins from these grape these anthocyanins and results were expressed as cultivars followed a published method with mg/g dry grape skin. minor modifications (He et al. , 2010b). In brief, the grape skins were hand -peeled and then 6. Statistical analysis freeze-dried using liquid nitrogen. The resultant skins were grounded into powder then the skin Data were expressed as the mean ± standard powder (0.1 g) was placed into a plastic deviation of triplicate tests. One-way analysis of centrifuge tube and then mixed with 1 mL 50 % variance (ANOVA) was applied to judge the formic acid solution. The mixture was sonicated means difference under a Turkey multiple range at room temperature for 20 min and then test with a significant level of 0.05, using SPSS centrifuged at 8,000 rpm for 5 min to collect the Statistical 19.0 (SPSS Inc., Chicago, IL, USA). supernatant. The sediment was mixed with 1 mL Principal component analysis was performed of 50 % formic acid solution and sonicated at using these detected anthocyanins as variables to room temperature for another 20 min. The unveil the similarity of these wine-making grape resultant mixture was then centrifuged at 8,000 cultivars using MetaboAnalyst 2.0 (http://www.