Changes in phenolic composition and antioxidant activity of grape skins and seeds "Teran" (Vitis vinifera L.) during ripening

Fenolni spojevi prisutni u grožđu i vinu značajno doprinose organoleptičkim svojstvima vina te zahvaljujući antioksidacijskom djelovanju imaju blagotvoran učinak na ljudsko zdravlje. Grožđe i vino crnih kultivara vinove loze bogati su različitim fenolnim spojevima, dok su kožica i sjemenke bobice glavni izvori fenolnih spojeva, kako flavonoida tako i neflavonoida. Dosadašnja istraživanja pokazala su da sadržaj polifenola, ali i konačni kemijski sastav grožđa ovisi o kultivaru, klimatskim uvjetima, hranidbenom i vodnom statusu te agrotehničkim i ampelotehničkim zahvatima u vinogradu. S obzirom na ključnu ulogu koju polifenoli grožđa imaju u kvaliteti vina, provedena su mnogobrojna istraživanja kako bi se utvrdile promjene u sadržaju polifenola kožice i sjemenki grožđa tijekom dozrijevanja s ciljem odabira optimalnog trenutka berbe za željenu tipologiju vina. U relevantnim bazama podataka nisu pronađeni podaci o fenolnom sastavu i antioksidacijskoj aktivnosti kožice i sjemenki grožđa kultivara ´Teran´ tijekom dozrijevanja, već su takva istraživanja uglavnom provedena na introduciranim i drugim autohtonim kultivarima vinove loze. Cilj ovog istraživanja bio je utvrditi promjene fenolnog sastava i antioksidacijske aktivnosti kožica i sjemenki bobice grožđa tradicionalnog istarskog kultivara ´Teran´ (Vitis vinifera L.), tijekom dozrijevanja u dvije vegetacijske sezone. Određivanje udjela pojedinačnih fenolnih spojeva u ekstraktu kožica i sjemenki provedeno je metodom tekućinske kromatografije visoke djelotvornosti (HPLC), dok su ukupni fenolni spojevi u ekstraktu kožica i sjemenki određeni spektrofotometrijski. Antioksidacijska aktivnost kožica i sjemenki određena je spektrofotometrijski pomoću DPPH, FRAP i ABTS metode. Na temelju dvogodišnjeg istraživanja promjena fenolnog sastava i antioksidacijske aktivnosti kožica i sjemenki bobica grožđa cv. ´Teran´ tijekom dozrijevanja, najviša koncentracija svih pojedinačnih antocijana 2008. godine zabilježena je u posljednjem uzorkovanju, odnosno u trenutku branja grožđa, dok je 2009. godine ista zabilježena dva tjedna prije branja grožđa. Malvidin-3-glukozid, odnosno malvidin derivati, neacilirani i acilirani, bili su najzastupljeniji antocijani u grožđu u obje godine istraživanja. Koncentracije flavonola kožice konstantno su se povećavale do berbe u 2008. godini, dok su u 2009. godini najviše koncentracije flovonola zabilježene 7 dana prije berbe. Kvercetin je bio najzastupljeniji flavonol u svim terminima uzorkovanja. Koncentracije ukupnih flavanola kožice povećavale su se do berbe, dok su se pojedinačni flavanoli kožice različito kretali. Signifikantno najviše koncentracije flavanola u sjemenkama zabilježene su u trenutku berbe u obje godine istraživanja. Katehin je bio najzastupljeniji flavanol sjemenki u obje godine istraživanja. Postotni udio procijanidina B1 u obje godine istraživanja bio je veći u odnosu na udio procijanidina B2. Tijekom dozrijevanja grožđa zabilježeno je smanjenje postotnog udjela procijanidina B1 odnosno povećanje postotnog udjela procijanidina B2 u ukupnim oligomerima kožice i sjemenki u obje godine istraživanja. Vrijednosti antioksidativne aktivnosti sjemenki tijekom dozrijevanja mjerene FRAP i ABTS metodom u obje godine istraživanja bile su više od vrijednosti antioksidativne aktivnosti kožica, dok su vrijednosti mjerene DPPH metodom gotovo pa identične za sjemenke i kožice bobice. Nove spoznaje vezane uz promjene fenolnog sastava i antioksidacijske aktivnost kožice i sjemenki grožđa tijekom dozrijevanja, omogućit će stvaranje okvirnog fenolnog i antioksidacijskog profila kultivara 'Teran'. Ovo je posebice značajno sa stajališta definiranja cjelovitog koncepta tehnologije proizvodnje grožđa i vina, s ciljem dobivanja optimalnih koncentracija prirodnih antioksidansa grožđa i vina, o čemu uvelike ovisi i kakvoća, a pogotovo tržišna vrijednost grožđa i vina.Phenolic compounds present in grapes and wine significantly contribute to the organoleptic properties of wine, and due to their antioxidant activity, have beneficial effects on human health. Previous studies showed that the skin and seeds of grape berries are significant sources of phenolic compounds, such as flavonoids and nonflavonoids. Furthermore, previous research indicated that the content of polyphenols and the final chemical composition of grapes depends on the cultivar, climatic conditions, nutritional status, water availability and cultural practices in vineyards. Considering the key role that grape polyphenols have on wine quality, many studies have been conducted to determine changes in skin and seed during berry ripening. Only a part of grape flavonoids are selectively extracted into wine during vinification, with the final concentrations significantly depending on the grape cultivar but also on the conditions and techniques used during maceration. Using a selective extraction method for the extraction of polyphenols from grape skins and seeds it is possible to analyze quantitatively the polyphenolic potential of red grapes through quantitative information on the amount and localization in the grapes. Knowledge of the total polyphenolic composition and different concentrations of individual fractions and specific cultivar characteristics are important information for oenologists to select the optimal harvest time and the appropriate vinification technique for the desired wine typology. Data on the phenolic composition and antioxidant activity of the grape skin and seed on ´Teran´ cultivar during ripening were not found in the relevant databases, but such research was mainly conducted on introduced and other autochthonous grapevine cultivars. The aim of this study was to determine changes in the phenolic composition and antioxidant activity of the grape skins and seeds of the traditional Istrian cultivar ´Teran´ (Vitis vinifera L.) during ripening in two growing seasons. The research was conducted during 2008 and 2009 in two selected commercial vineyards of the wine-growing region Croatian Istria and Kvarner near Kosinožići and Motovun. The data on climatic conditions were collected from the nearest State Hydrometeorological Institute official stations which are about 3 km far from the experimental vineyards. The experimental treatments were conducted on five different harvesting dates during grape ripening starting from the sixth week to the tenth week after the verasion, when the technological maturity occurred. Each treatment had three replicates, ie 21 clusters were collected from each vineyard in each sampling period from 21 experimental vines included in the experiment in a random block arrangement from 3 marked rows (7 experimental vines in each row represented 1 replication). After separating the berries from the bunches, for further analytical procedures, a sample of 250 g of berries was selected per replicate. Extraction of polyphenols from grape skins and seeds was performed by a selective extraction method, optimized using the model of grape polyphenol extraction in the vinification process. For each replication, two extracts were prepared, one from the skins and the second one from grape seeds. The content of total phenols, proanthocyanidins, hydrolyzed tannins, and vanillin index in grape skin and seed extracts was determined spectrophotometrically. Total anthocyanins, absorbances at 420 nm, 520 nm and 620 nm, color intensity and hue were also spectrophotometrically determined in the skin extract. The identification and quantification of individual flavonoid phenols (flavan-3-ols, proanthocyanidins and flavonols) and non-flavonoid phenols (hydroxybenzoic acids) in the skin and seed extract were determined by high-performance liquid chromatography (HPLC). The antioxidant activity of the skin and seed was determined spectrophotometrically using the DPPH, FRAP and ABTS methods. Based on a two-year study of changes in the phenolic composition and antioxidant activity of the skins and seeds of grape berries cv. ´Teran´ during ripening the highest content of all individual anthocyanins in 2008 was recorded in the last sampling, at the time of grape harvest, while in 2009 it was recorded two weeks before grape harvest. Malvidin-3-glucoside and malvidin derivatives, unacylated and acylated, were the most abundant anthocyanins in grapes in both research years. During grape ripening, the concentrations of flavonols in the skins were constantly increasing until the time of harvest in 2008, while in 2009 the highest concentrations were recorded 7 days before harvest. Quercetin was the most abundant flavonol in all sampling terms. Concentrations of total skin flavanols increased to harvest, while the percentage of individual skin flavanols varied differently. Significantly the highest values of flavanol seeds concentration were recorded at the time of grape harvest in both studied years. Catechin was the most abundant seed flavanol in both research years. The percentage of procyanidin B1 in both research years was higher compared to the percentage of procyanidin B2. During grape ripening in both research years, a decrease in the percentage of procyanidin B1 and an increase in the percentage of procyanidin B2 in the total skins and seeds oligomers were recorded. The values of seeds antioxidant activity during ripening measured by FRAP and ABTS method in both research years were higher than the values of skins antioxidant activity, while the seeds and berry skins antioxidant activity values during ripening measured by the DPPH method were almost identical. New findings related to the changes in the phenolic composition and antioxidant activity of the skin and grape seeds during ripening, will enable the creation of a phenolic framework and antioxidant profile of the cultivar 'Teran'. This is especially important from the point of view of defining the overall concept of grape and wine production technology with the aim of obtaining optimal concentrations of natural antioxidants of grapes and wine, which greatly affects the quality and especially the market value of grapes and wine