Determination of phenolic profile of "Plavac mali" (Vitis vinifera L.) during ripening of the grapes and impact on wine quality

Senzorne karakteristike vina Plavca malog često uključuju negativna svojstva gorčine okusa i astrigentnosti nezrelih tanina što je dijelom posljedica pogrešnog roka berbe grožđa. S tehnološkog stajališta, sastav bobica u trenutku berbe od presudne je važnosti za proizvodnju visokokvalitetnih crnih vina. Tradicionalno odluka o berbi se donosi na temelju sadržaja šećera, ukupnih kiselina i pH-vrijednosti, a koji ukazuju samo na stupanj zrelosti pulpe (tehnološka zrelost), dok nam stupanj zrelosti kožice i sjemenki (fenolna zrelost) ostaje nepoznat. Grožđe ubrano temeljem osnovnih parametara ne garantira fenolnu dozrelost pa takva vina mogu imati loša senzorna svojstva. Cilj ovog istraživanja je bio procijeniti zrelost grožđa sorte Plavac mali kvantifikacijom osnovnih fizikalno-kemijskih i polifenolnih spojeva u moštu i vinu. Grožđe je ubrano u četiri različita roka na dvije različite lokacije pri sadržaju šećera od 17,6 do 21,40 °Brix, a što odgovara vinima sa 9,30 do 12,98 vol. % alkohola. Polifenolni spojevi su analizirani spektrofotometrijskim i kromatografskim analitičkim metodama, a intenzitet boje bobica je utvrđen koristeći nedestruktivni CIELab kolorimetrijski pristup. Identifikacija i kvantifikacija polifenolnih spojeva ekstrakata grožđa (kožice i sjemenki) i vina je provedena tekućinskom kromatografijom visoke djelotvornosti HPLC-DAD. Značajnost razlika u sastavu i koncentraciji pojedinih spojeva tijekom četiri roka berbe na dvije lokacije je testirana dvosmjernom analizom varijance. Rezultati su pokazali da dozrijevanjem grožđa raste udio ukupnih i pojedinačnih antocijana i flavonola, a smanjuje se udio monomernih i dimernih flavan-3-ola u kožici. Udio monomernih flavan-3-ola u sjemenkama se smanjuje, dok udio dimernih spojeva ostaje stabilan neovisno o roku berbe. Odgoda roka berbe ima značajan utjecaj na polifenolni sastav vina, grožđe ranijih rokova berbe daje vina siromašna tvarima boje, antocijanima i flavonolima, s visokim udjelom procijanidin monomernih i dimernih flavan-3-ola. Vina kasnijih rokova berbe su bolje obojena uslijed veće koncentracije antocijana i flavonola te sadrže veći udio prodelfinidin naspram procijanidin monomernih jedinica i nižu koncentraciju dimernih flavan-3-ola. Dobiveni rezultati pokazali su mogućnost procjene zrelosti grožđa preko vanjskih karakteristika, kao što su boja kožice bobica i sjemenki. Definirani su metaboliti koji najznačajnije utječu na razlike u sastavu grožđa i vina između četiri različita roka berbe. Obzirom na povezanost vanjskih svojstava kožice i sjemenke te kemijskih spojeva izrađena je vizualizacija tonske gradacije boje sjemenki i kožica bobice tijekom četiri različita roka berbe, a koja predstavlja kvalitetni indikator dozrelosti grožđa Plavca malog.Plavac Mali is major red grapevine of the Croatian wine industry. The sensory characteristics of Plavac Mali wine often include negative sensations of bitter taste and astringency of immature tannins, which is partly due to the wrong date of grape harvest. From a technological point of view, the composition of berries at the harvest is essential for production of high quality red wines. Traditionally the decision about harvest was taken based on sugar content, titratable acidity, and pH that only provide information about pulp ripeness, while the real degree of skin and seed maturity (phenolic maturity) remains unknown. The grape harvested based on conventional parameters does not guarantee phenolic maturation. Review of the similar researches on phenolic maturation, with emphasis on so far conducted researches on Plavac Mali, project hypothesis and aims are shown in the introduction chapter. Plavac Mali is late ripening variety and the race to produce high concentrated wines, as well as uneven nature of the grape berry ripening, and the wrong harvest timing indicators implies a delay in harvest with the aim of achieving better coloration and rounded, velvet tannins which often results in unbalanced wines with high alcohol, low acid content and high pH value. In this research project it is assumed that the specific ratios of metabolites (sugars, organic acids, phenolic compounds, nitrogen, and potassium) in grapes of the Plavac Mali of different harvest dates affect the chemical composition and the production of different types of wine. It is also assumed that the color of the skin and the seeds during maturation is correlated with the phenolic composition and can therefore be used as a good indicator of ripeness of Plavac Mali. The aims of this research project were to characterize grape and wine of Plavac Mali harvested at different technological ripeness, examine the most suitable, practical maturity parameters (general maturity indexes, total concentration of phenolic compounds, CIELab and RGB (HSV) variables of the color, HPLC profiles) and to determine the best time of harvest. Correlation between the concentration of the phenolic compounds and color development of the grapes was determined, and visualisation of the colour gradiation of the grape seeds during diferent harvest dates was made. The second chapter gives an overview of the latest research in the phenollic maturity of grapes and consequences on the quality of wines. Chapter is organized in five subdivisions and includes an overview of 198 literature references. The research carried out by different authors showed that phenolic compounds play an important role in the quality of red wine, particularly on sensory characteristics of the color, bitterness and astringency. The production of high quality wines is based on the optimum degree of phenol grape maturity. The concept of phenolic maturity at the level of the skin and the seed coat during maturation implies achieving the highest concentration of anthocyanins in the skin with the lowest extraction of the seeds proanthocyanidins in the total proantocyanidins. Red grape varieties easily reach the sugar content required for the production of high quality wines in the Mediterranean climates, but this is not the case with the phenolic compounds. In the last stage of the grape berry development, the phenolic compounds, especially the anthocyanins and the proanthocyanidins go through continuous quantitative and qualitative changes, and the intensity of the same depends on agro-ecological conditions and vineyard practice. Wines that are produced from unripe berries, whose skins and seeds did not achieve full phenolic maturity are poor in color, have bitter taste and astringent sensations. Precise research material and methods used in determination of grape maturity were described in third chapter. Grape samples were collected during four different harvest dates in two different collection vineyards, Duilovo and Baštica. Conventional oenological methods included 7 different analytical methods. Total polyphenolic capacity of the grape berries was determined through 3 spectrophotometric methods, and the separation of 5 different groups of polyphenolic compounds was performed with HPLC. Traditional measurements of sugar content, titratable acidity and pH are very important for the determination of grape ripeness. During recent years evaluation of the grape color development and the estimation of the skin and seed ripeness demands application of sophisticated analytical methods, primarily spectrophotometric, colorimetric, and the methods of the liquid chromatography. Unfortunately, these methods are not easily available to viticulturists and winemakers and require knowledge of analytical chemistry and expensive devices. As the wine market becomes more competitive, precise determination of the harvest date for production of the specific wine style will become normal in the future. An inexpensive way of determination of the best harvest date is the combination of traditional analyses and evaluation of the color of the grape which is possible with the new tool developed in this research project, visualization of the grape seed color. The fourth chapter consists of 25 tables, 24 graphs and 4 figures. The results show the significance of influence of four harvest dates and two locations on changes in the level of the conventional and polyphenolic parameters of grape and wine Plavac Mali by two-way analysis of variance. Techniques of the multivariate analysis PCA and Canonical correlation are used to determine the relationships between samples (PCA) and parameters (Canonical correlation). Significant difference in physiochemical and polyphenolic profile of grape berries of the four different harvest dates was determined. Wines produced from grapes of the different harvest dates showed a greater difference in basic oenological and polyphenolic profile. Comparison of the results of the polyphenolic profile of Plavac Mali grape and wine demonstrated the opportunity to evaluate maturity of the grapes by external parameters of color of grape skins and seeds. Due to the relationship between external properties of the skin and seeds and chemical compounds, visualization of the tonal gradation of the seed color during four different harvest dates was developed and the possibility of its use as a quality indicator of maturity of grapes Plavac Mali was determined. The research questions from hypothesis and raised by analysis were discussed in the fifth chapter, organized in three subdivisions. The results are compared to latest researches from this field. The proposed research provides a detailed insight into the potential of technological maturity of Plavac Mali. Conventional grape maturity indicators (° Brix, titratable acid and pH) as well as basic quality indicators, allowed a separation of grapes based on harvest date and/or growing location. However, it is not possible to correlate these indicators with the phenolic maturity of berries for the production of high quality wines. Berries of Plavac Mali reached optimal acidity content in the second harvest date, and optimal phenolic maturity in the third harvest date. The conclusions are stated based on main findings. A significant difference in the physiochemical profile of the Plavac Mali grape berries of four different harvest dates described by a series of variables (sugar, glucose, fructose, titratable acidity, pH; tartaric, malic and citric acid; potassium, phenolic constituents) was established. Wines produced from the grapes of different harvest dates differ in the basic enological physiochemical (content of real alcohol, total dry extract, reducing sugars, ashes, pH, titratable and volatile acidity) and polyphenolic composition (stilbene, phenolic acids, anthocyanins, flavonols, monomeric and dimeric components of flavan-3-ol). From the technological point of view a great difference was found between conventional and phenolic indicators used to determine the best time of harvest for production of high quality wines of Plavac Mali