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agriculture Article Content of Polyphenolic Compounds and Antioxidant Potential of Some Bulgarian Red Grape Varieties and Red Wines, Determined by HPLC, UV, and NIR Spectroscopy Milena Tzanova * , Stefka Atanassova, Vasil Atanasov and Neli Grozeva Faculty of Agriculture, Trakia University, Studentski grad Str., 6000 Stara Zagora, Bulgaria; [email protected] (S.A.); [email protected] (V.A.); [email protected] (N.G.) * Correspondence: [email protected]; Tel.: +359-42-699-315 Received: 10 April 2020; Accepted: 26 May 2020; Published: 1 June 2020 Abstract: Today, good food criteria also include healthy capacity. So, the wine on our table should not only have good organoleptic qualities, but should be characterized by a high healthy potential. For the first time, extensive research was conducted on commercial red wine grape varieties cultivated in different Bulgarian regions in two consecutive years. Antioxidants, including trans-resveratrol, quercetin, and total phenolic content and antioxidant potential in wine grapes and wines were determined by HPLC, UV, and NIR methods. The results obtained showed similar concentration levels compared to the same varieties, produced in other countries. Trans-resveratrol showed the greatest contribution to the radical scavenging capacity. The factor with largest impact on the content of the tested substances was definitely the variety. Among agro-meteorological condition, temperature amplitude, rain fall, and UV irradiation before ripening had strong influences. Maintaining the balance between the level of synthesized and degraded and captured antioxidants during the wine making process was crucial to preserving the antioxidant properties of the final wine product. NIR spectroscopy showed very good accuracy of determination of trans-resveratrol, quercetin, total phenolic content, and the antioxidant activity of tested grape varieties and red wines. It could be a promising technique in the quantification of their antioxidant parameters. Keywords: trans-resveratrol; quercetin; radical scavenging capacity; red wine grape; red wine; HPLC; UV; NIR 1. Introduction The plants produce de novo compounds with small molecules called phytoalexins (from Greek phytos—“plant” and alekein—”fend off”). These secondary metabolites are responsible for the plant protection against diverse biotic and abiotic factors, like bacteria, fungi, and UV-irradiation [1]. Produced by vine substance called phenolic compounds, these known bio-antioxidants have a high potential [2]. During the process of vinification, they pass from the solid parts of the grapes into the must. Maceration is a very important stage in red wine production. Trans-resveratrol (t-RVT) belongs to the stilbene polyphenol subgroup and is potentially responsible for the “French paradox”—the French suffer relatively seldom from coronary heart disease, although their diet is rich in saturated fats [3]. A number of studies, reviewed by Novelle et al., on its influence on the human health as dietary supplement, demonstrated diverse bio-activities like antioxidant, cardio protective, anti-cancer, anti-diabetic, and anti-inflammatory agents [4]. Some selected natural and synthetic compounds are proven to work in synergy with trans-resveratrol. One of them is quercetin (QU, a flavonol, one of the subclasses of flavonoid Agriculture 2020, 10, 193; doi:10.3390/agriculture10060193 www.mdpi.com/journal/agriculture Agriculture 2020, 10, 193 2 of 14 compounds). A number of research teams studied and proved its antioxidant potential, as well as its anti-allergy, anticancer, blood pressure lowering, anti-inflammatory and antiviral activities [5]. In addition, quercetin increases the protective effect of trans-resveratrol as an antioxidant [6], an anti-inflammatory [7], a cardiovascular [8] and an anti-obesity agent [9]. The wine industry is currently requiring fast, low cost, and easy-to-operate techniques for routine grape and wine analysis. NIR spectroscopy is a non-destructive method, requiring minimal or no sample preparation, with good abilities in the analysis of different food products. Recently, several review papers can be found in the literature, demonstrating the abilities of NIR spectroscopy for determination of the content of flavanols, anthocyanins, and other phenolic compounds in wine and grapes [10,11]. Bulgarian wines have been on the international market for a long time, but data about the antioxidant properties, phenols, trans-resveratrol and quercetin are deficient. The aim of this study was the determination of their content in some ccommercial Bulgarian red wine grapes and wines by HPLC, UV, and NIR methods. Data about antioxidant constituents and antioxidant potential of a number of red wine grape varieties distributed on the Bulgarian and European market were collected. The impact of various agro-meteorological conditions on their content were established. 2. Materials and Methods 2.1. Samples The grapes grown in three different districts in Bulgarian viticulturally regions: Danubian plain in the North-Bulgarian town of Pleven (N: 43.407778◦ E: 24.620278◦, 116 m a.s.l.; moderate continental climate and average growing degree days in the last 3 years—3911 ◦C) and Thracian lowland in South-Bulgarian Region. Village of Mogilovo, Chirpan municipality (N: 42.333333◦ E: 25.4◦, 312 m a.s.l., moderate continental climate and average growing degree days in the last 3 years—3820 ◦C), and Village of Mezek, Svilengrad municipality (N: 41.73333◦ E: 26.08333◦, 168 m a.s.l., moderate continental climate and average growing degree days in the last 3 years—4346 ◦C). The regions provided good soil and climatic conditions for producing red wine grape varieties and wines of above average quality. From Pleven, eight varieties vintage 2017 were selected: three introduced—Merlot, Cabernet Sauvignon and Syrah, and five hybrids—Rubin, Kaylashki Rubin, Storgozia, Bouquet and Otel. During the vegetation seasons of 2017, no extreme climate events were observed. From Mogilovo, four introduced grape varieties were tested: Merlot, Cabernet Sauvignon, Syrah, and Malbec—vintage 2017 and 2018 wines, and wine grapes vintage 2018. From Mezek, three introduced grape varieties were tested: Merlot, Cabernet Sauvignon and Syrah—vintage 2018 wines, and wine grapes vintage 2018. Guyot training system was applied to all varieties and conventional methods of pest control were used during growing season. When grapes reached technological maturity, they were picked and used to produce red wines, in accordance with the generally accepted wine making procedures, which included the following steps: maceration in stainless tanks for 10–12 days at 7–8 ◦C; heating up to 23–24 ◦C, and yeast (Saccharomyces cerevisiae) adding; fermentation for 7–8 days at 25 ◦C–28 ◦C; post-enzymatic maceration for 10–12 days at 25 ◦C; wine draining in tanks; malolactic acid fermentation; and finally, wine decanting and maturing in wooden barrels for 10–12 months. Three samples of each batch were taken by the enologist of the corresponding winery at the moment of wine aging and were stored in amber glass bottles with cork stopper in a dark place, at 18 ◦C for one week prior to the analysis. Every sample was analyzed in triplicate. Grape samples were collected in the period from the middle of September to the beginning of October in 2018, at the moment of reaching of technological maturity. Small portions of the base, middle, and top of the grape cluster, located at the base, middle, and top of the vine were picked. Grapes free from visible blemish or disease were selected. Samples consisted of 3–5 kg of grape Agriculture 2020, 10, 193 3 of 14 Agricultureberries. 2020 After, 10, x sampling,FOR PEER REVIEW the grapes were peeled. The skins were immediately frozen, transported3 of 13 in a refrigerator, and stored for a maximum of 7 days at 12 C prior to the analysis. − ◦ 2.2. 2.2.HPLC HPLC Analysis Analysis For Forthe thequantification quantification of trans-re of trans-resveratrolsveratrol and and quercetin quercetin in ingrape grape berry berry skins, skins, the the method method validated validated by Tzanovaby Tzanova and andPeeva Peeva [12] [was12] wasapplied: applied: a sample a sample of 10 ofg thawed, 10 g thawed, at room at roomtemperature, temperature, berry skins berry were skins were weighed to the nearest 0.0001 g, grinded and homogenized with a mechanical homogenizer in weighed to the nearest ±0.0001 g,± grinded and homogenized with a mechanical homogenizer in 60 mL of a60 1% mL solution of a 1% of solution HCl in ofmethanol HCl in methanolfor 5 min for in 5a mindark in room. a dark The room. dispersed The dispersed samples samples were left were in the left darkin at the room dark temperature at room temperature overnight overnight and then andfiltered then through filtered a through 0.45 μm a membrane. 0.45 µm membrane. The solid Theresidue solid wasresidue washed was twice washed with twice10 mL with of 1% 10 mLHCl of in 1% methanol HCl in methanol.. The extracts The were extracts collected were collected and adjusted and adjusted to 100 to 100 mL with solvent, and stored at 12 C until initiation of the HPLC analysis. After filtration, mL with solvent, and stored at −12°C until− initiation◦ of the HPLC analysis. After filtration, through a 0.45through μm membrane, a 0.45 µm a membrane,sample of 1 amL sample of each of 1extract mL of eachwas placed extract into was the placed HPLC into auto the HPLCsampler, auto to sampler,assure the toinjection assure volume the injection of 20 volumeμl. of 20 µL. Five-pointFive-point calibration calibration graphs graphs (0.05; (0.05; 0.50; 0.50; 1.0; 1.0; 2.0 2.0 and and 5.0 5.0 mg/l) mg/ L)were were made made in in advance advance using using referencereference materials—trans-resveratrol materials—trans-resveratrol HPLC HPLC grade grade (not (not less less than than 99%) 99%) and and quercetin quercetin HPLC HPLC grade grade (not (not lessless than than 98%), 98%), purchased purchased from from Sigma-Aldrich Sigma-Aldrich (St.

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