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A new technology called solid phase microextraction (SPME) Arrow has recently been used in the analysis of food volatile organic compounds (VOCs). This technology is still not widely used for the analysis of wine VOCs, which play a crucial role in sensory properties. For optimisation of SPME Arrow extraction conditions, Box–Behnken experimental design and response surface methodology were used. The optimised conditions were extraction temperature, incubation time, and exposure time. The most significant factors affecting extraction efficiency were extraction temperature and exposure time. An increase in temperature and exposure time increased the analytical signal of most classes of VOCs extracted from both white and red wine samples. For VOCs in white wines, the optimal conditions are an extraction temperature of 50 °C, an incubation time of 10 min, and an exposure time of 60 min, while for VOCs in red wines, the optimal conditions are an extraction temperature of 60 °C, an incubation time of 17 min, exposure time of 53 min. The new optimised method allows more accurate and time-saving analysis of VOCs from wines, enabling the analysis of a large number of samples.

Prosušivanje grožđa jedan je od ključnih postupaka u proizvodnji prošeka, desertnog vina s dugom tradicijom proizvodnje u Hrvatskoj. Tradicionalne sorte koje su se koristile u proizvodnji prošeka su autohtone sorte Dalmacije. Jedna od sorata koja se zbog svoje rasprostranjenosti i karakteristika može koristiti u proizvodnji prošeka je autohtona sorta 'Plavina'. Tradicionalna proizvodnja prošeka uključuje prosušivanje grožđa na zraku, međutim razvojem tehnologije otvorile su se i mogućnosti sušenja u kontroliranim uvjetima. Stoga je cilj rada bio utvrditi promjene kakvoće grožđa sorte 'Plavina crna' u kontroliranim uvjetima sušenja. Rezultati su pokazali kako je sušenjem došlo do koncentriranja sadržaja bobice te se povećao sadržaj šećera i organskih kiselina. Prosušivanjem se smanjio sadržaj polifenolnih i hlapljivih spojeva. Kako bi se utvrdio točan utjecaj na konačni proizvod, potrebna su daljnja istraživanja

Global wine market is undergoing the changes in the consumer preferences, with the rising popularity of the sparkling wines. The similar trend is observed in the Republic of Croatia. Graševina, the most important white grapevine variety, showed great adaptability to climate conditions. The results of clonal selection pointed out the clone OB-435 as suitable for sparkling wine production. Thus, the aim of this study was to evaluate the quality of OB-435 sparkling wines. The sparkling wines were produced by traditional method and different concentrations of dosage were added- 1.5 mL L-1 (GR-1,5) and 2.5 mL L-1 (GR-2,5). The basic chemical parameters and sensory evaluation of sparkling wines, confirmed the suitability of this clone for sparkling wine production in the conditions of continental winegrowing hill Zagreb. Sparkling wine with lower dosage concentration was evaluated as better by different sensory methods, regardless the evaluators´ experiences.

Na globalnom tržištu vina dolazi do promjena u preferencijama potrošača te popularnost pjenušavih vina raste, a sličan trend događa se i u Republici Hrvatskoj. Graševina, kao najznačajnija bijela sorta vinove loze u Hrvatskoj, pokazala je veliku adaptabilnost na različite ekološke uvjete. Provedbom klonske selekcije, klon OB-435 izdvojio se svojim karakteristikama kao pogodan za proizvodnju pjenušavih vina. Stoga je cilj ovog rada bio ocijeniti kvalitetu pjenušavih vina proizvedenih od klona OB-435. Vina su proizvedena klasičnom metodom, te su dodane dvije koncentracije ekspedicijskog likera- 1,5 mL L-1 (GR-1,5) i 2,5 mL L-1 (GR-2,5). Osnovni kemijski parametri i senzorna analiza proizvedenih pjenušavih vina, potvrdila su pogodnost ovog klona za proizvodnju pjenušavih vina u uvjetima kontinentalnog Vinogorja Zagreb. Različitim senzornim metodama bolje je ocijenjeno pjenušavo vino s manjom dozom ekspedicijskog likera i voćno-cvjetnom aromom.

Polyphenols are secondary plant metabolites and important organic compounds present in grapes, must and wine. The importance of polyphenolic compounds present in grapes is mainly related to the quality of the wine. Those compounds affect the colour, taste, astringency, bitterness, and potential of wine aging. Therefore, the aim of this research was to determine and compare quantitative and qualitative changes in low molecular weight polyphenol content of 11 red grapevine varieties during ripening. Samples were collected periodically from the beginning of vérasion to the technological maturity. The polyphenolic compounds were analysed with high performance liquid chromatography (HPLC) with prior lyophilization and extraction. Based on the obtained results, changes in the composition and content of researched groups of polyphenolic compounds during maturation were determined.

Graševina' je jedan od osnovnih elemenata prepoznatljivosti hrvatskog vinogradarstva i vinarstva. Statistički gledano svaki četvrti trs u hrvatskim vinogradima je trs 'Graševine', a ako gledamo samo kontinentalnu Hrvatsku, gdje se ova sorta zapravo i jedino uzgaja 'Graševina' čini 50% svih vinograda. U ukupnoj proizvodnji vina koje se stavlja na tržište podrijetlom iz hrvatskih vinograda, ova sorta zauzima čak 40%. Proteklih nekoliko godina ponovno se aktualiziralo nekoliko pitanja oko ove sorte, posebno pitanje njezinog podrijetla. Dodatno, često se postavlja pitanje kako je ova sorta postala toliko popularna u Hrvatskoj i kakva je budućnost ove sorte. Da li će se zbog njenih karakteristika zbog kojih je često bila prvi izbor vinogradara u tijekom 20. stoljeća i dalje koristiti za podizanje novih nasada u sve toplijoj, ali neizvjesnoj i nepredvidivoj klimi budućnosti. U ovom radu su osim prikaza spoznaja vezanih uz navedene teme prikazane i karakteristike klonova 'Graševine' izdvojenih nakon provedene klonske selekcije ove sorte u kutjevačkom vinogorju koji su odnedavno registrirani i nalaze se na sortnoj listi.

'Graševina' is one of the basic elements of the recognizability of Croatian viticulture and winemaking. Statistically speaking, every fourth vine in Croatian vineyards is a 'Graševina', and if we look only at the continental part of Croatia, where this variety is actually grown, 'Graševina' makes up 50% of all vineyards. In the case of wine that is put on the market originating from Croatian vineyards, this variety occupies as much as 40% of the total volume. In the past few years, several questions regarding this variety have been brought up, especially the question of its origin. In addition, the question that often arises is how this variety became so popular in Croatia and what is the future of this variety and whether, due to its characteristics that often made it the first choice of winegrowers during the 20th century, it will continue to be used for planting new plantations in warmer, but an uncertain and unpredictable climate of the future. In this paper, in addition to the presentation of knowledge related to the mentioned topics, the characteristics of the 'Graševina' clones selected after the clonal selection of this variety in the Kutjevo wine growing area, which were recently registered, are presented.

Sortni tioli su izrazito mirisni i poželjni spojevi arome koji doprinose mirisu vina aromama tropskog voća. Njihova koncentracija ovisi o brojnim čimbenicima, od ampelotehničkih i enoloških postupaka do uvjeta skladištenja. Sorta grožđa je jedan od najvažnijih čimbenika, pri čemu su utvrđene razlike i između samih klonova pojedine sorte. Cilj ovog rada bio je istražiti utjecaj klonova sorte Pošip na koncentraciju sortnih tiola u vinima. Rezultati su pokazali značajne razlike između klonova pri čemu su najviše koncentracije utvrđene u vinu proizvedenom od klona POŠ-124. Dobiveni rezultati ističu klonsku selekciju kao važan korak u proizvodnji vina različitih stilova.

Varietal thiols are extremely volatile and desirable wine aroma compounds that contribute to tropical fruit notes. Their concentration depends on numerous factors, from agricultural practices to winemaking procedures and storage conditions. Among them, grape variety is one of the most important factors, and diversity among clones of a single variety has also been highlighted. The aim of this work was to determine the varietal thiols in wines produced from different clones of Pošip (Vitis vinifera L.) grape. The results showed significant differences between clones, with the highest concentrations of varietal thiols detected in wine produced from clone POŠ-124. The results suggest clone selection as an important step in diversifying the styles of produced wines.

The research of sanitary status and virus presence was conducted on three native Croatian grapevine varieties (Kujudžuša bijela, Zlatarica vrgorska and Trnjak crni) that are grown in Dalmatia region. The investigation was done on 492 vines from 31 vineyards. Vines were tested by ELISA (Enzyme-Linked Immunosorbent Assay) on four main economic viruses: arabis mosaic virus (ArMV), grapevine fanleaf virus (GFLV), grapevine leafroll-associated virus 1 and 3 (GLRaV–1, GLRaV-3) as a first step in clonal selection of these rare native grapevine varieties. Altogether, 40 vines were free of all tested viruses. The research confirms high rate of virus infection and eroded sanitary status of all investigated varieties. The above indicates necessity of implementation clonal and sanitary selection programme.

Grape drying is one of the key processes in prošek production, a sweet wine with long production history in Croatia. Traditional grape varieties use din prošek produsction are Dalmatian native varieties. One of the varieties that can be used due to its characteristics and cultivation area is 'Plavina'. Traditional prošek production includes sun-drying. However, with technology development the drying in contolled conditions was enabled. Thus, the aim of this work was to determine the changes in the grape quality of 'Plavina' during the drying in controlled conditions. Results showed that during the drying process the berry content was concentrated due to which sugar content and total acidity increased. On the other hand, the polyphenolic and volatile compounds decresed. To determine specific influences on final product, further research is necessary.

Graševina bijela is the most represented and economically most important grapevine variety in the Republic of Croatia. It’s mostly grown in viticultural regions of continental Croatia. Graševina adaptability to climatic conditions is one of many characteristics which makes this variety popular among wine growers. Intravarietal variability was detected among population of Graševina bijela which is the reason why is the project of clonal selection in Kutjevo initiated in 2004. From that period up to 2015, 12 clone candidates were selected, which were distinguished with positive characteristic from the rest of the population. Then, these 12 clone candidates were planted in the vineyard at the Experimental Station Jazbina for the purpose of the future study of their characteristics and for continuing process of the individual clonal selection. The study of the important economic characteristics was carried out when the vineyard came into full growth. There were significant differences in most important characteristic between clone candidates such as yield, number of clusters per vine and the basic quality indicators of the must.

U vinogradarskoj proizvodnji postupak cijepljenja predstavlja klasičan način dobivanja potomstva od matičnih biljaka vinove loze. To je najzastupljeniji način vegetativnog razmnožavanja, učinkovit je protiv filoksere, te daje dobre rezultate. Postupak se odvija u rasadniku, a nastali cjepovi predstavljaju klonove matičnih biljaka. In vitro razmnožavanje podrazumijeva brzo klonsko razmnožavanje biljnog materijala u sterilnim uvjetima. Prednosti in vitro razmnožavanja u odnosu na klasično razmnožavanje su kraći period dobivanja odraslih biljaka, te mogućnost ozdravljivanja biljnog materijala od virusa. Međutim, postupak in vitro razmnožavanja da bi bio uspješan zahtjeva testiranje pojedine sorte na kontrolirane uvjete. Cilj ovog rada je dati usporedbu prednosti i nedostataka klasičnog i in vitro razmnožavanja za vinovu lozu.

In viticulture, the classical procedure of obtaining the adult plants is the procedure of grafting. It is the most common procedure of vegetative propagation, highly efficient against phylloxera, with significantly good results. The procedure is performed in nursery and obtained grafts present clones of mother plants. In vitro multiplication implies the fast clonal propagation of plant material in a sterile conditions. Advantages of in vitro multiplication in comparison with classical one is short period for achieving mother plants with the possibility of virus eradication of plant material. However, the procedure of in vitro multiplication is more demanding considering the fact that each variety has different response to controlled culture conditions, which need to be tested. The aim of this study is to give a comparison of advantages and disadvantages of classical and in vitro multiplication of grapevine.

Graševina bijela najzastupljenija je i gospodarski najvažnija sorta u Republici Hrvatskoj. Uzgaja se u vinogradarskim regijama Zapadna i Istočna kontinentalna Hrvatska. Njena prilagodljivost klimatskim uvjetima jedna je od brojnih karakteristika koje ovu sortu čine popularnom među vinogradarima. Uočena je unutarsortna varijabilnost među populacijom Graševine bijele zbog čega je 2004. godine pokrenut projekt klonske selekcije na području vinogorja Kutjevo. U razdoblju od 2004. do 2015. godine, odabrano je dvanaest klonskih kandidata koji su se istaknuli od ostatka populacije po pozitivnim osobinama. Ovih dvanaest klonskih kandidata posađeno na Vinogradarsko-vinarsko pokušalište Jazbina s ciljem daljnjeg proučavanja njihovih karakteristika te nastavka postupka individualne klonske selekcije. Nakon ulaska nasada u puni rod, provedeno je proučavanje najbitnijih gospodarskih karakteristika. Utvrđene su značajne razlike između klonova za sljedeća svojstva: prirod, broj grozdova po trsu, osnovni pokazatelji kakvoće mošta.

Polifenolni sastav grožđa i vina predmet je brojnih istraživanja, ponajviše zbog utjecaja pojedinačnih polifenola na senzorna svojstva vina te njihovog blagotvornog učinka na ljudsko zdravlje. Zbog klimatskih promjena koje postaju učestalije sve više se susrećemo s problemom fenolne zrelosti grožđa. Stoga je cilj istraživanje bio utvrditi utjecaj inaktivnih kvasaca na dozrijevanje grožđa Plavine te postizanje optimalne fenolne zrelosti. Na početku fenofaze šare dio grožđa tretiran je inaktivnim kvascima (Lalvigne mature), a kao kontrolna varijanta poslužilo je grožđe sorte Plavina na kojoj nije proveden tretman. U uzorcima grožđa provedena je osnovna fizikalno-kemijska analiza te analiza aromatskog i polifenolonog sastava. Značajna razlika uočena je u koncentracijama petunidin-3-O-glukozida i peonidin-3-O-glukozida kao i kemferol-3-O-rutinozida te procijanidina B1 kod tretiranog grožđa dok je ukupna kiselost bila izraženija u kontrolnom tretmanu. U aromatskom profilu tretirano grožđe izdvojilo se većim sadržajem monoterpena dok su seskviterpeni bili zastupljeniji u kontrolnom tretmanu. S obzirom na proizvodna svojstva sorte Plavina kao što su tanka kožica, osjetljivost na bolesti, zbijen grozd i velike bobice s manjom mogućnosti nakupljanja suhe tvari, od kojih se najčešće proizvedu laganija, slabija obojena vina, manje bogata polifenolima, primijenjena tehnologija ukazuje na mogućnost unaprjeđenja kvalitativnih svojstava sorte Plavina a time i vina. Plavina, grožđe, polifenoli, inaktivni kvasci

Numerous studies have been dealing with the polyphenolic composition of grapes and wine, mostly due to the influence of individual polyphenols on the sensory properties of wine and their beneficial effect on human health. Due to climate change, which is becoming more frequent, we are increasingly facing the problem of phenolic ripeness of grapes. Therefore, the aim of the study was to determine the influence of inactive yeasts on the ripening of Plavina grapes and phenolic maturity. At the beginning of the veraison, a part of the grapes was treated with inactive yeasts (Lalvigne mature), and as a control variant, Plavina grapes were used, which were not treated. Basic physicochemical analysis and analysis of aromatic and polyphenolic composition were performed in grape samples. A significant difference was observed in the concentrations of petunidin-3-O-glucoside and peonidin-3-O-glucoside as well as kemferol-3-O-rutinoside and procyanidin B1 in the treated grapes while the total acidity was more pronounced in the control treatment. In the aromatic profile, treated grapes had higher content of monoterpenes, while sesquiterpenes were more present in the control treatment. Given the production characteristics of the Plavina variety such as thin skin, susceptibility to disease, compact grapes and large berries with less possibility of dry matter accumulation, which most often produce lighter, weaker colored wines, less rich in polyphenols, the applied technology indicates the possibility of improvement qualitative properties of the Plavina variety and thus of the wine.

Grape volatile organic compounds (VOCs) play an important role in the winemaking industry due to their contribution to wine sensory characteristics. Another important role in the winemaking industry have the grapevine varieties used in specific regions or countries for wine production. Due to the high variability of grapevine germplasm, grapevine varieties are as classified based on their genetic and geographical origin into genetic-geographic groups (GEN-GEO). The aim of this research was to investigate VOCs in 50 red grapevine varieties belonging to different GEN-GEO groups. The study included varieties from groups C2 (Italy and France), C7 (Croatia), and C8 (Spain and Portugal). The analysis of VOCs was performed by SPME-Arrow-GC/MS directly from grape skins. The analyzed VOCs included aldehydes, ketones, acids, alcohols, monoterpenes, and sesquiterpenes. The most abundant VOCs were aldehydes and alcohols, while the most numerous were sesquiterpenes. The most abundant compounds, aldehydes and alcohols, were found to be (E)-2-hexenal, hexenal, (E)-2-hexen-1-ol, and 1-hexanol. Using discriminant analysis, the GEN-GEO groups were separated based on their volatile profile. Some of the individual compounds contributing to the discrimination were found in relatively small amounts, such as benzoic acid, (E,E)-2,4-hexadienal, 4-pentenal, and nonanoic acid. The groups were also discriminated by their overall volatile profile: group C2 was characterized by a higher content of aldehydes and alcohols, and group C8 was characterized by a higher content of sesquiterpenes and acids. Group C7 was characterized by all low amount of all classes of VOCs.

The aim of this work was to determine the effect of different drying treatments: freeze-drying, room-drying, and oven-drying on the grape phenolic composition of the 'Regent' and 'Cabernet Sauvignon' varieties. After drying, the samples were grounded, submerged to ultrasound-assisted extraction, and analyzed with high-performance liquid chromatography (HPLC). This study demonstrated that the use of different drying methods significantly affects the content of polyphenols in grape skins extracts. The greatest content of anthocyanins-diglucoside was preserved using freeze-drying, i.e. 15706.86 mg/kg for 'Regent', while the content of anthocyanins-monoglucoside was best preserved by room-drying, i.e. 216778.68 and 9220.30 mg/kg for 'Regent' and 'Cabernet Sauvignon', respectively. The highest content of flavonol glycosides (2583.04 and 1429.64 mg/kg for 'Regent' and 'Cabernet Sauvignon', respectively), hydroxycinnamic acids (1303.31 and 544.88 mg/kg for 'Regent' and 'Cabernet Sauvignon', respectively), and stilbene (2321.52 and 79.36 mg/kg for 'Regent' and 'Cabernet Sauvignon', respectively) was observed in the oven-dried samples. By applying freeze-drying the most optimal content of flavan-3-ol was preserved. Contents of polyphenolic compounds in oven-dried samples after 6 months of storage were almost identical to those in the samples analyzed immediately after drying. The greatest rate of degradation was observed in the room-dried samples while it was moderate in the freeze-dried ones. The results of this experiment demonstrate that it is necessary to dry samples in different ways to obtain the highest content of a certain polyphenolic group of compounds. The application of a drying method is determined by the goal of the final dried product in terms of content and composition of different polyphenolic compounds; thus, the obtained results could have an application in scientific research and for commercial purposes as well.

The maceration process is one of the indispensable winemaking steps in red wine production that has a marked impact on phenolic compound extraction and can strongly influence the color and gustatory quality. Seed removal can have a substantial impact on the intensity of bitterness and astringency which are mainly influenced by the presence of flavanols. The main aim of this study was to investigate the influence of seed removal and extraction time on the phenolic composition, color parameters, and organoleptic properties of Plavac mali wines produced from the grapes from the Pelješac Peninsula. The achieved results showed that the control wines differ in terms of their highest content of total anthocyanins in both years and total flavonols in one year of investigation, while prolonged maceration without seeds decreases the concentration of phenolic acids as well as that of flavan-3-ols. A prolonged extraction time influenced the color intensity and tonality, moving it towards more yellow pigments. These changes could possibly be explained by the pronounced interaction between phenolic compounds and the formation of the new ones. Interestingly, seed removal probably diminished that change because the potential absorption of the anthocyanins by grape seeds and tannins were excluded. The highest quality was sensory defined in wines produced by prolonged maceration without seeds.

Since grapevine is highly susceptible to various pathogens, enormous amounts of pesticides are applied each season to achieve profitable production. One of the most destructive grapevine diseases is downy mildew, and their interaction has been in the spotlight for more than a decade. When it comes to a metabolome level, phenolic compounds are relevant to investigate due to their involvement in the plant immune system and known antifungal properties. Croatian grapevine germplasm is highly heterogeneous due to its long history of cultivation in diversified geographical regions. Since it has been found that native varieties react differently to the infection of Plasmopara viticola, the intention of this study is to define if the chemical background of the leaves, i.e., polyphenolic composition, is responsible for these dissimilarities. Therefore, the leaves of 17 genotypes, among which 14 were native and 3 were controls, were analyzed using high-performance liquid chromatography (HPLC) in four terms: before inoculation and 24, 48, and 96 h post inoculation (hpi). During this early phase, significant differences were found neither between the terms nor between the non-inoculated and inoculated samples, except for resveratrol-3-O-glucoside. By applying principal component analysis (PCA) using initial leaf polyphenolic composition, varieties of V. vinifera were clearly separated into three different groups corresponding to their International Organization of Vine and Wine (OIV) classes of susceptibility to P. viticola. Results obtained in this research suggest that the initial constitutive polyphenolic composition of the cultivar leaves has a crucial influence on their susceptibility to P. viticola, and this finding can be used to improve the success of grapevine breeding programs toward downy mildew resistance.

Varietal thiol concentration in wine is influenced by numerous factors, of which grape variety and winemaking practices are often highlighted as the most important. Therefore, the aim of this work was to study the effects of grape clone and yeast strain (Saccharomyces and non-Saccharomyces) on the varietal thiols concentrations and sensory characteristics of Graševina (Vitis vinifera L.) white wines. Two grape clones were evaluated (OB-412 and OB-445) along with three different commercial yeast strains (Saccharomyces cerevisiae Lalvin Sensy and Sauvy, and Metschnikowia pulcherrima Flavia). The results showed that the concentration of varietal thiols in Graševina wines amounted up to a total of 226 ng/L. The clone OB-412 was characterized by its significantly higher concentrations, especially of 3-sulfanylhexanol (3SH) and 3-sulfanylhexyl acetate (3SHA). Moreover, alcoholic fermentation with pure S. cerevisiae Sauvy yeasts generally resulted in higher thiol concentrations, while sequential fermentation involving M. pulcherrima positively affected only the 4-methyl-4-sulfanyl-pentan-2-one (4MSP) concentration. Finally, sensory analysis showed that fermentation with pure S. cerevisiae Sauvy yeast also produced more desirable wines. The results suggest that clonal, and especially yeast strain, selections are important modulators of the aroma and sensory properties of wine.

The Plasmopara viticola pathogen causes one of the most severe grapevine diseases, namely downy mildew. The response to P. viticola involves both visible symptoms and intricate metabolomic alterations, particularly in relation to volatile organic compounds, and depends on the degree of resistance of a particular variety. There are numerous native grapevine varieties in Croatia, and they vary in susceptibility to this oomycete. As previously reported, in vitro leaf disc bioassay and polyphenolic compound analysis are complementary methods that can be used to separate native varieties into various resistance classes. This research used the Solid Phase Microextraction-Arrow Gas Chromatography-Mass Spectrometry method to identify the early alterations in the VOCs in the leaves after P. viticola inoculation. Based on the absolute peak area of sesquiterpenes, some discrepancies between the sampling terms were noticed. The presence of certain chemical compounds such as humulene, ylangene, and α-farnesene helped distinguish the non-inoculated and inoculated samples. Although specific VOC responses to P. viticola infection of native varieties from various resistance classes could not be identified, the response of less susceptible native varieties and resistant controls was associated with an increase in the absolute peak area of several compounds, including geranylacetone, ß-ocimene, and (E)-2-hexen-1-ol.

The biological characteristics of grapevine viruses, such as their transmission and host range, are important for the adoption of successful prophylaxis strategies. The aim of this study was to investigate the traits of two newly described grapevine viruses widely distributed in Croatia, grapevine badnavirus 1 (GBV-1) and grapevine virus G (GVG). The vine mealybug (Planoccocus ficus) proved to be a vector of GBV-1 and GVG capable of vine-to-vine transmission with overall experimental transmission rates of 61% and 14.6%, respectively. Transmission was also demonstrated by grafting, with an overall transmission rate of 53.8% for GBV-1 and 100% for GVG, as well as by green grafting using the T-budding technique. Symptoms of GBV-1 and GVG were not observed on the woody cylinders of the indicators LN 33, Kober 5BB, 110 Richter and cvs. Chardonnay and Cabernet Sauvignon. Seed transmission and mechanical transmission were not confirmed. Electron microscopy revealed accumulation of GBV-1 particles and viroplasms in the cytoplasm, but no alternations of the cell structure. Infection with GVG revealed the proliferation of tonoplast-associated vesicles inside phloem cells and cell wall thickening.

Polyphenols are organic compounds that can be found in food, especially in fruits, vegetables, and their products. It was shown that their presence significantly affects the properties of food products and that the consumption of food rich in phenolic compounds has a beneficial effect on human health. The subjects of this research were polyphenols: anthocyanins, flavonols, and flavan-3-ols in the skin of grapevine variety Regent. Polyphenols from grape skins were extracted via microwave-assisted extraction (MAE) and matrix solid-phase dispersion (MSPD) as unconventional and green techniques. Therefore, the main aim of this work was to optimize the conditions for the extraction of polyphenolic compounds from grape skin using MAE and MSPD. The extracts were analyzed using high-performance liquid chromatography with a diode array detector and fluorescence detector. Analyses showed that MAE was a very effective method for extracting polyphenolic compounds from grape skin with 10 mL of 60% ethanol for 5 min at 40 °C. The best results for the MSPD extraction of polyphenolic compounds from grape skin were obtained with phenyl as an MSPD sorbent with 10 mL of acetonitrile:water 50:50 v/v as an elution solvent. This scientific research can be used for the better use of grapes as a basis for obtaining flavonoids for commercial purposes.

The investigation of virus incidence was conducted on 14 autochthonous Croatian grapevine cultivars on vines from 51 vineyard located in the Dalmatian region. A total of 1,116 vines were tested by ELISA on nine viruses: Arabis mosaic virus (ArMV), Grapevine fanleaf virus (GFLV), Grapevine fleck virus (GFkV), Grapevine leafroll-associated virus 1, 2, 3 and 7 (GLRaV-1, GLRaV-2, GLRaV-3 and GLRaV-7), Grapevine virus A and B (GVA and GVB). ELISA confirmed presence of eight viruses: GLRaV-3 in 888 vines (79.6%), GVA 685 (61.4%), GLRaV-1 455 (40.8%), GFkV 223 (19.9%), GFLV 219 (19.6%), GLRaV-2 46 (4.1%), ArMV 36 (3.2%) and GVB 35 (3.1%). Altogether 93 vines (8.3%) were free of all tested viruses, 168 (15.1%) were infected with a single virus, 368 (32.9%) with two, 314 (28.1%) with three, 127 (11.4%) with four, 43 (3.9%) with five, and 3 (0.3%) vines with six viruses. Symptoms of premature leaf color change and downward curling of leaf blades were common, while abnormal branching, short internodes, double nodes and irregular ripening were detected sporadically. The investigation confirms deteriorated sanitary status of all cultivars except Grk and Plavina and indicates the necessity of intensifying grapevine clonal and sanitary selection programs.

Dalmatian vineyards host many autochthonous varieties. The phenolic profile, defined by the relative proportions of different phenolic compounds, is specific for each grape variety. The aim of this study was to determine and analyze the flavonoid compounds of twenty rare red grape varieties. Nineteen phenolic compounds, represented by anthocyanins, flavanols, and flavonols, were detected and quantified using HPLC in three consecutive vintages. The content of grape skin anthocyanins (10414.06 (Plavac mali crni)-19.58 (Trišnjavac) mg kg−1 d.w. of grape skin), flavonols (1742.08 (Pošip crni)-215.56 (Crljenak viški) mg kg−1 d.w. of grape skin), and flavan-3-ols (448.04 (Pošip crni)-87.88 (Glavinuša) mg kg−1 d.w. of grape skin) showed significant differences in investigated varieties. According to the investigated phenolic compounds, Pošip crni, Ljutun, Zadarka, Dobričić, Plavac mali crni, and Trnjak differed from other investigated varieties. These local varieties can be perceived as an alternative to widespread varieties in Dalmatia. This was shown by one of the first studies on phenolic profiles of (mostly) rare autochthonous varieties.

Seed extracts are becoming more important due to their beneficial biological activities. The main constituents of seed extracts are flavan-3-ols, compounds important in winemaking. The coastal region in Croatia is rich in native grapevine varieties, which are used in wine production. The aim of the research was to analyze the flavan-3-ol profiles of 20 native varieties, and to evaluate the potential use of grape seeds as a source of flavan-3-ols. The flavan-3-ols from seeds were analyzed by HPLC. The predicted yield of flavan-3-ols was calculated using the analyzed profiles. In total, eight compounds were identified, with the most abundant compounds being catechin, epicatechin, and procyanidin B2. In general, the red grape varieties had higher content of flavan-3-ols than the white varieties, which was confirmed by PCA. The coastal region could potentially yield up to 73.97 kg/ha of flavan-3-ols, depending on the variety. The results show the diversity of flavan-3-ol profiles among Croatian varieties and their potential usage as a source of valuable nutraceuticals.

Grapevine badnavirus 1 (GBV-1) was recently discovered in grapevine using high throughput sequencing. In order to carry out large-scale testing that will allow for better insights into virus distribution, conventional and real-time PCR assays were developed using sequences both from previously known, and four newly characterized isolates. Throughout the growing season and dormancy, GBV-1 can be detected by real-time PCR using available tissue, with the possibility of false-negative results early in vegetation growth. GBV-1 real-time PCR analysis of 4302 grapevine samples from the Croatian continental and coastal wine-growing regions revealed 576 (~13.4%) positive vines. In the continental wine-growing region, virus incidence was confirmed in only two collection plantations, whereas in the coastal region, infection was confirmed in 30 commercial vineyards and one collection plantation. Infection rates ranged from 1.9 to 96% at the different sites, with predominantly autochthonous grapevine cultivars infected. Conventional PCR products obtained from 50 newly discovered GBV-1 isolates, containing the 375 nucleotides long portion of the reverse transcriptase gene, showed nucleotide and amino acid identities ranging from 94.1 to 100% and from 92.8 to 100%, respectively. The reconstructed phylogenetic tree positioned the GBV-1 isolates taken from the same vineyard close to each other indicating a possible local infection event, although the tree nodes were generally not well supported.

The aromatic and sensory profiles of monovarietal sparkling ciders made according to the modified Méthode Ancestrale or Pétillant Naturel (Pét-Nat) method were established. Three Croatian traditional apple varieties (‘Božićnica’, ‘Bobovac’, and ‘Crvenka’) were basic raw materials for Pét-Nat ciders in this study. The basic apple must and cider parameters were determined by applying OIV methods and nitrogenous compounds, total phenols, and color parameters were analyzed by spectrophotometer. Volatile compounds in final Pét-Nat ciders were determined by SPME-Arrow-GC/MS method and Odor Active Values (OAV) were calculated. The results show that variety significantly altered the pH value, color, aromatic and sensory profile of Pét-Nat ciders. The main contributors (OAV > 1) to the aroma of all Pét-Nat ciders were 1-hexanol, 1-propanol, (6Z)-nonen-1-ol, 1-dodecanol, hexanoic, octanoic and isovaleric acid, citronellol, ethyl hexanoate, ethyl butanoate, ethyl-9-decenoate and isoamyl acetate, eugenol and methionol. ‘Božićnica’ Pét-Nat was differentiated by a high concentration of 1-decanol and 4-ethylphenol, ‘Bobovac’ by 4-vinyl guaiacol and ‘Crvenka’ by 4-ethyl guaiacol. Sensory analysis showed that the highest rated overall quality was attributed to ‘Crvenka’ Pét-Nat cider, with the high-quality color, fruity odor (‘apple’,’apple juice/compote’, ‘pineapple’, and ‘buttery’) and well-balanced taste. This research demonstrates the possibilities in the production of natural sparkling cider from traditional Croatian apple varieties by analyzing the composition and quality of the final product for the first time.

Grapevine collections play an important role, especially in the study of viruses and virus-like pathogens. In 2009, after an initial ELISA screening for eight viruses (arabis mosaic virus, grapevine fanleaf virus, grapevine fleck virus, grapevine leafroll-associated viruses 1, 2, and 3, and grapevine viruses A and B), a collection of 368 grapevine accessions representing 14 different Croatian autochthonous cultivars and containing single or mixed infection of viruses was established to further characterize the viral pathogens. Subsequently, Western blot, RT-PCR, cloning, and sequencing revealed that grapevine rupestris stem pitting-associated virus was frequently found in accessions of the collection, with isolates showing substantial genetic diversity in the helicase and coat protein regions. High-throughput sequencing of 22 grapevine accessions provides additional insight into the viruses and viroids present in the collection and confirms the fact that Croatian autochthonous grapevine cultivars have high infection rates and high virome diversity. The recent spread of “flavescence dorée” phytoplasma in Europe has not spared the collection. After the first symptoms observed in 2020 and 2021, the presence of phytoplasma was confirmed by LAMP in six grapevine accessions and some of them were lost. Single or multiple viruses and viroids, as well as own rooted grapevines in the collection, make the plants susceptible to various abiotic factors, which, together with the recent occurrence of “flavescence dorée”, makes the maintenance of the collection a challenge. Future efforts will be directed towards renewing the collection, as 56% of the original collection has been lost in the last 13 years.

Grapevine leaves are photosynthetically the most active green organs providing carbohydrates that are of utmost importance for the regular vine’s metabolism and growth. Moreover, leaves are the pioneers of fungal infections caused by B. cinerea, E. necator, and P. viticola. Plant response to these microorganisms mostly depends on the content and composition of phenolic compounds abundantly found in the leaf’s outer tissues (epidermis, cuticle, trichomes). In order to obtain a satisfactory quantity of phenolic compounds, an experiment was conducted towards optimizing a solid–liquid extraction method. Variables were as follows: the type of organic solvent, the sample weight, the extraction temperature, and the extraction time. The optimal conditions were obtained by applying the response surface methodology. Therefore, by using acetonitrile as the organic solvent, conducting a single-step extraction at the temperature of 48 °C during the time period of 2 h and 25 min with a solid-to-solvent ratio of 1:56 g mL−1 (178 mg of leaves powder and 10 mL of extraction solvent) the optimal content of phenolic compounds was obtained. This protocol is reliable, fast, and relatively easy to perform for the determination of the abovementioned compounds.

(1) Background: Solid phase microextraction (SPME)-Arrow is a new extraction technology recently employed in the analysis of volatiles in food materials. Grape volatile organic compounds (VOC) have a crucial role in the winemaking industry due to their sensory characteristics of wine.; (2) Methods: Box–Behnken experimental design and response surface methodology were used to optimise SPME-Arrow conditions (extraction temperature, incubation time, exposure time, desorption time). Analyzed VOCs were free VOCs directly from grape skins and bound VOCs released from grape skins by acid hydrolysis.; (3) Results: The most significant factors were extraction temperature and exposure time for both free and bound VOCs. For both factors, an increase in their values positively affected the extraction efficiency for almost all classes of VOCs. For free VOCs, the optimum extraction conditions are: extraction temperature 60 °C, incubation time 20 min, exposure time 49 min, and desorption time 7 min, while for the bound VOCs are: extraction temperature 60 °C, incubation time 20 min, exposure time 60 min, desorption time 7 min.; (4) Conclusions: Application of the optimized method provides a powerful tool in the analysis of major classes of volatile organic compounds from grape skins, which can be applied to a large number of samples.

A survey of recently discovered vitiviruses was performed on 113 Croatian autochthonous grapevine cultivars from the national collection “Jazbina” using one-step RT-PCR. The presence of grapevine virus H (GVH) was confirmed in nine (7.9%) cultivars and grapevine virus G in eight (7.1%), while the presence of grapevine viruses I and J were not detected. GVH was transmitted by the vine mealybug (Planococcus ficus) from a source plant to grapevine seedlings with a 10.5% transmission rate using a combination of 10 first and second instars per plant with 48 and 72 h of acquisition and inoculation access period, respectively. Transmission correlated with the presence of grapevine leafroll-associated virus 3 (GLRaV-3) in the GVH-source plant and recipient seedlings. No alternative GVH host was identified. A comparison of 356 nt fragments of the RdRP and CP coding regions showed nucleotide identity between the Croatian GVH isolates in the range of 95.5–99.2% and 97.5–99.4% and amino acid identity between 95.8 and 100% and between 98.3 and 100%, respectively. Comparison with foreign isolates revealed nucleotide sequence similarity in the RdRP and CP between 94 and 100% and between 97.7–100%, respectively. To the best of our knowledge, this is the first report of GVH in Croatia and the first identification of the vine mealybug as a vector of GVH.

The population of Croatian autochthonous cultivars has a high degree of infection with economically important viruses, so it is necessary to carry out the elimination of the viruses in some cultivars to obtain healthy planting material. In this research, we tested in vitro meristem culture establishment on 18 autochthonous cultivars with different viral infections and the possibility of GLRaV-3 elimination through in vitro meristem culture. Plant material was sampled in a vineyard in two phenological stages, 10 days before flowering and 10 days after flowering of the grapevine. Apical meristem explants (1 mm) were placed into the MS culture medium supplemented with 0.5 mg/L benzyl adenine (BA) and 0.05 mg/L indol-3-acetic acid (IAA), and their survival, regeneration, and rooting were monitored. The results showed that the cultivar and the growth phase have a significant impact on the success of in vitro culture. In all cultivars studied higher success of in vitro culture establishment parameters (survival, regeneration, and rooting) was obtained in the case of explants sampled after flowering, with the exception of one cultivar for explants survival. Contrary to expectations, genotypes infected with three viruses (GLRaV-1, GLRaV-3, and GFLV) showed better results than genotypes infected with one or two viruses. The results showed successful in vitro establishment of Croatian autochthonous cultivar and GRLaV-3 elimination in one cultivar. However, due to the significant effect of cultivar, for routine application of this in vitro protocol on more than 100 autochthonous cultivars in need of sanitation, further studies should be conducted.

Wine quality is influenced by the presence of over 500 different chemical compounds, with polyphenols having a crucial role in color intensity and tonality, astringency, mouthfeel, and overall impression formation, especially in red wine production. Their concentrations in wine can vary notably depending on the grape variety, the temperature and the length of maceration process, aging duration, and yeast selection. Therefore, in this work, the main goal was to determine the influence of five commercially available Saccharomyces yeasts provided from Lallemand, France and AEB, Italy, on the phenolic compound composition and chromatic parameters of Plavac mali wines produced from the grapes from coastal Dalmatia, grown at two different micro-locations. The achieved results pointed out the marked difference in individual polyphenol compound adsorption between tested yeasts. Fermol Super 16 was the one with the lowest and Lalvin D21 the strongest adsorption ability, regardless of vine growing location. These differences can be explained by the content of some anthocyanins (delphinidin and petunidin-3-O-glucoside) and gallic acid, and some flavan-3-ols. Tested strains also influenced wine color intensity, pointing out the possibility of modulating the style of a Plavac mali by the use of commercial yeasts.

In the era of sustainable grapevine production, there is a growing demand to define differences between Vitis vinifera varieties in susceptibility to downy mildew. Croatia, as a country with a long tradition of grapevine cultivation, preserves a large number of native grapevine varieties. A leaf disc bioassay has been conducted on 25 of them to define their response to downy mildew, according to the International Organisation of Vine and Wine (OIV) descriptor 452-1, together with the stress response of the leaf discs using chlorophyll fluorescence and multispectral imaging with 11 parameters included. Time points of measurement were as follows: before treatment (T0), one day post-inoculation (dpi) (T1), two dpi (T2), three dpi (T3), four dpi (T4), six dpi (T5), and eight dpi (T6). Visible changes in form of developed Plasmopara viticola (P. viticola) sporulation were evaluated on the seventh day upon inoculation. Results show that methods applied here distinguish varieties of different responses to downy mildew. Based on the results obtained, a phenotyping model in the absence of the pathogen is proposed, which is required to confirm by conducting more extensive research.

Even though Saccharomyces cerevisiae starter cultures are still largely used nowadays, the non-Saccharomyces contribution is re-evaluated, showing positive enological characteristics. Among them, Lachancea thermotolerans is one of the key yeast species that are desired for their contribution to wine sensory characteristics. The main goal of this work was to explore the impact of L. thermotolerans commercial yeast strain used in sequential inoculation with S. cerevisiae commercial yeast on the main enological parameters and volatile aroma profile of Trnjak, Babić, Blatina, and Frankovka red wines and compare it with wines produced by the use of S. cerevisiae commercial yeast strain. In all sequential fermented wines, lactic acid concentrations were significantly higher, ranging from 0.20 mg/L in Trnjak up to 0.92 mg/L in Frankovka wines, while reducing alcohol levels from 0.1% v/v in Trnjak up to 0.9% v/v in Frankovka wines. Among volatile compounds, a significant increase of ethyl lactate and isobutyl acetate, geraniol, and geranyl acetate was detected in all wines made by use of L. thermotolerans. In Babić wines, the strongest influence of sequential fermentation was connected with higher total terpenes and total ester concentrations, while Trnjak sequentially fermented wines stood up with higher total aldehyde, volatile phenol, and total lactone concentrations. Control wines, regardless of variety, stood up with higher concentrations of total higher alcohols, especially isoamyl alcohol. The present work contributed to a better understanding of the fermentation possibilities of selected non-Saccharomyces strains in the overall red wine quality modeling.

For the characterization of grape cultivars, the profile and content of flavonoids are important because these compounds impact on grape and wine quality. A new extraction method for the recovery of flavonoids from grape skins was developed. The optimization of solid-liquid extraction of flavonoids was conducted, with respect to type of the organic solvent and their percentage in the extraction solvent as well as their extraction temperature and extraction time, using response surface methodology. Optimal conditions were obtained using 20 % acetonitrile as the organic modifier in extraction solvent, at an extraction temperature of 50 °C, an extraction time of 1 h in a single-step extraction and with a solid-to-solvent ratio of 1:80 g mL-1. The calculated relative standard deviations for the optimal extraction method were very low, measuring less than 5 %. The new optimal extraction method is inexpensive, simple, fast, accurate and selective for the recovery of simple flavonoids.

Grapes are rich in primary and secondary metabolites. Among the secondary metabolites, polyphenolic compounds are the most abundant in grape berries. Besides their important impacts on grape and wine quality, this class of compounds has beneficial effects on human health. Due to their antioxidant activity, polyphenols and phenolic acids can act as anti-inflammatory and anticancerogenic agents, and can modulate the immune system. In grape berries, polyphenols and phenolic acids can be located in the pericarp and seeds, but distribution differs considerably among these tissues. Although some classes of polyphenols and phenolic acids are under strict genetic control, the final content is highly influenced by environmental factors, such as climate, soil, vineyard, and management. This review aims to present the main classes of polyphenolic compounds and phenolic acids in different berry tissues and grape varieties and special emphasis on their beneficial effect on human health.

Commercial sparkling wine production represents a relatively low but important part of the Croatian wine production, especially in the Zagreb county. This study presents the results of volatile aroma compounds profile and organic acid composition of commercial sparkling wine samples from three vine-growing regions in Zagreb county. In total, 174 volatile aroma compounds were identified, separated between their chemical classes (aldehydes, higher alcohols, volatile phenols, terpenes, C13-norisoprenoids, lactones, esters, fatty acids, sulfur compounds, other compounds, other alcohols). Higher alcohols such as phenylethyl and isoamyl alcohol as well as 2-methyl-1-butanol, and esters such as diethyl succinate, ethyl hydrogensuccinate, and ethyl lactate had the strongest impact on the volatile compounds profile of Zagreb county sparkling wine. The presence of diethyl glutarate and diethyl malonate, compounds whose concentrations are influenced by yeast autolysis or caused by chemical esterification during the ageing process, was also noted. The influence of every single volatile aroma compound was evaluated by discriminant analysis using forward stepwise model. The volatile profiles of traditional sparkling wines from Croatia were presented for the first time. It is hoped the results will contribute to better understanding the quality potential and to evaluate possible differences on the bases of detected aroma concentrations and multivariate analysis.

Sulfur dioxide (SO2) is widely the most used enological additive with reductive, antiseptic and dissolving properties. According to increasing health concerns and the gradual decrease in total SO2 concentrations allowed in wine, alternative and supplementary agents for preservation are being investigated. For this reason, the current study was focused on the impact of different commercial reductive agents on white wine antioxidant activity and chemical composition. The effect of additives that combine sulfites, ascorbic acid and enological tannins were compared against standard 5% sulfurous acid (H2SO3) during the pre-fermentative treatments of Sauvignon Blanc must (Vitis vinifera L.). The basic parameters of quality, free amino-nitrogen and total polyphenoliccompounds in must were analyzed. Gas chromatography and spectrophotometric methods were used to investigate the overall volatile composition, antioxidant and chromatic parameters in wines. The obtained results undoubtedly pointed out the positive effect of sulfuric acid on the fermentation dynamics. Furthermore, application of combined reducing additives with potassium metabisulfite, L-ascorbic acid, gallotannins and ellagitannins, resulted in a higher antioxidant capacity and increased concentration of aromatic compounds and their odor activity values in Sauvignon Blanc wine.

Aim: The aim of this research was to evaluate if leaf removal and red geotextile reflective mulch “Vitexsol” could affect phenolic composition of wines from three white cultivars, Riesling italico, Traminer, and Manzoni bianco. Methods and results: A two-year study was conducted to evaluate the effects of leaf removal and reflective mulch on the phenolic composition of white wines from Zagreb vineyards in northwestern Croatia. Leaf removal (LR) and reflective mulch (RM) “Vitexsol”, made from weave of aluminum platelets protected by a transparent film and sewn together with red polypropylene threads, were tested separately and combined (LR+RM) on vines of Traminer, Riesling italico and Manzoni bianco in 2008 and 2009. LR and RM had no consistent effect on must sugar content and titratable acidity. All treatments resulted in higher total phenol and flavan-3-ol content in wines of all cultivars, but in different years. LR+RM generally resulted in the highest phenolic acid and individual flavan-3-ol content in all wines except Traminer. RM treatment had the least effect on phenolic composition of wines. Conclusion: LR+RM generally resulted in the highest content of most phenolic compounds, especially when compared to control wines. Significance and impact of the study: This work provides some useful informations for adjusting vineyard practices and thus optimizing phenolic quality of white wines.

Evaluation of the incidence of virus infections was conducted in two grapevine collection fields. The first was the National collection of Croatian native grapevine cultivars, situated at the “Jazbina” experimentation station in eastern Zagreb, where more than 120 different autochthonous grapevine cultivars are held, collected from different Croatian vine-growing regions. The second was a regional collection located in Risika, on the island of Krk (the North Adriatic region) containing 19 native cultivars from that region. During February 2009 from both collections, 95 plants were selected and tested for presence of eight viruses by ELISA, including: Arabis mosaic virus (ArMV), Grapevine fanleaf virus (GFLV), Grapevine fleck virus (GFkV), Grapevine leafroll-associated virus 1 (GLRaV-1), Grapevine leafroll-associated virus 2 (GLRaV-2), Grapevine leafroll-associated virus 3 (GLRaV-3), Grapevine virus A (GVA) and Grapevine virus B (GVB). The dominant virus in both collections was GLRaV-3, present in 75 vines (78.9%) in the National collection and in 73 vines (76.8%) in the Risika collection. The second most frequent virus in the National collection was GVA (60.0%), followed by GLRaV-1 (29.5%), GFkV (24.2%), GFLV (17.9%), ArMV (12.6%), GLRaV-2 and GVB (2.1%). In the Risika collection, GLRaV-3 was followed by GFLV (42.1%), GFkV (36.8%), GVA (32.6%), ArMV (23.2%), GLRaV-1 (11.6%), GLRaV-2 and GVB (1.1%). Mixed infections with two, three of four different viruses were also common in both collections. In the National collection the most common mixed infections were GLRaV-3 + GVA (15.8%) and GLRaV-1 + GLRaV-3 + GVA (14.7%), while in the Risika collection dominant were mixed infections with GLRaV-3 + GVA (10.5%) and GFLV + GLRaV-3 (8.4%). Free of all eight tested viruses were ten vines (10.5%) in the National collection and only seven vines (7.4%) in the Risika collection. This investigation has demonstrated that there has been deteriorated sanitary status of Croatian autochthonous cultivars, and indicates the need for the production of certified virus-free planting material.