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Botrytis cinerea Pers., the fungal plant pathogen and the causal agent of gray mould diseases in grapevine, is vastly responsible for substantial economic losses in table and wine grapes production worldwide by negatively affecting plant growth and causing the reduction of grape and wine quality. The conventional approach for pathogen control has been up to date based on synthetic fungicides with good effectiveness against pathogens but a negative impact on the environment. The growing level of harmful residues in the environment and some also detected in wines have led the European Union and many winemakers to limit the application of synthetic fungicides to earlier season. However, with a high risk of disease also late in the season, the need for other solutions is clear. Consequently, more and more research is focused on finding potential alternatives in the form of effective biological control agents. Although there are several reports of yeast’s biocontrol activity, they are up to date still poorly commercialized for such purposes. As the yeasts represent an important part of the grape microflora, competing with other microorganisms (including pathogens) for nutrients and space, we decided to examine the potential of autochthonous yeasts as "green" alternatives in fighting against phytopathogens such as B. cinerea in viticulture. With this aim, we tested the biocontrol activity of 119 different indigenous yeasts belonging to 30 different species of 17 genera against filamentous fungus B. cinerea, the causal agent of grey mould or botrytis bunch rot in grape. The yeasts were screened for putative multidimensional modes of action such as antifungal volatiles (VOC), in vitro inhibition of fungal mycelial growth, competition for nutrients, hydrolytic enzyme activities, and yeast tolerance to fungicides like copper, iprodione and cyprodinil/fludioxonil combination. With a qualitative detection of the hydrolysis activity by using screening methods based on solid medium with chitin or βD-glucosides as substrates, we found that many tested yeasts were capable of producing lytic enzymes with the ability to degrade the cell wall of phytopathogenic fungi and are potentially also able to produce VOCs via hydrolysis of grape glycosides as a result of β-glucosidase presence. Furthermore, we observed the capability of tested yeast to inhibit fungal mycelia growth on plate and assimilation of a wide variety of carbon sources however, no siderophore producers were detected. In general, the yeasts under observation were tolerant to the tested fungicides. Their fungicide resistance can indeed be regarded as a beneficial trait for potential biofungicide agent (PBA) candidates due to open possibilities of applications and combinations within low input pest management strategies in the vineyard. Finally, a field experiment in Pinot noir and Pinot gris vineyards was designed to study different combinations of optimized canopy microclimate manipulation (CMM) techniques and potential biocontrol agent (PBA) application. In experimental conditions, the ability of PBA’s to maintain appropriate population density for disease prevention was observed. In addition, the grape and wine quality parameters were analysed to observe the possible impact of implemented biocontrol yeast on final products. The biocontrol yeast Pichia guilliermondii ZIM 624 was selected and applied in experimental vineyards based on yeast testing results. We were able to detect and confirm PBA yeast’s suitable density on grapes until harvest. In the case of early defoliation for both varieties lower grape compactness was observed together with lower yield/ plant, regardless of PBA yeast/ no yeast application. Among grape basic quality parameters, the optimized techniques showed a positive effect on sugar content. Still, unexpectedly, in the treatments with biocontrol yeasts some trends toward higher acidity were noticed in Pinot gris.

V magistrskem delu smo obravnavali kakovostno in ekonomsko upravičenost vpeljave tehnološkega postopka hladne predfermentativne maceracije, s katerim smo želeli izboljšati predvsem barvne in aromatične lastnosti vina 'Refošk'. Najprej smo razložili pomen investicij in metode, s katerimi investicije ocenjujemo. V praktičnem poskusu smo pridelali vino po postopku hladne predfermentativne maceracije (HPM), za primerjavo pa smo vino pridelali še po postopku klasične fermentativne maceracije (FM) in fermentativne maceracije pri višji temperaturi (FMVT). V pridelanih vinih smo določali skupne polifenole, skupne in posamezne antocianine, barvo vina ter izvedli senzorično analizo. V ekonomskem delu smo ugotovili, kako bi investicijo financirali, in definirali lastno ceno. S pragom rentabilnosti smo predvideli smotrno prodajno ceno ter z metodami denarnih tokov in sedanje vrednosti ugotovili, da bi se nam investicija povrnila v devetem letu poslovanja. Rezultati kažejo, da je hladna predfermentativna maceracija vplivala na značilno večjo vsebnost skupnih polifenolov, medtem ko smo opazili značilno večjo vsebnost skupnih antocianinov pri FM in FMVT. Uporabljeni tehnološki postopki niso pokazali značilnega vpliva na povečanje vsebnosti posameznih antocianinov, malvidin-3-glukozida ter intenzitete in tona barve vina. Vino, pridelano po postopku HPM, je v primerjavi s FM in FMVT vini na senzorični analizi in testu všečnosti doseglo najmanj točk. ANALIZA KAKOVOSTNE IN EKONOMSKE UPRAVIČENOSTI VPELJAVE TEHNOLOŠKEGA POSTOPKA PREDFERMENTATIVNE MACERACIJE V PREDELAVI GROZDJA SORTE 'REFOŠK' MAGISTRSKO DELO Marko Benčina Mentorja: doc. dr. Melita Sternad Lemut, dr. Jan Reščič Nova Gorica, 2019 ZAHVALA Iskreno se zahvaljujem vsem, ki ste mi kakorkoli pomagali pri opravljanju teoretskega, eksperimentalnega in analitskega dela magistrske naloge, še posebej dr. Janu Reščiču in Jeleni Topić Božič, mag. ind. farm., za njuno potrpežljivost in pomoč pri laboratorijskem in analitskem delu. Posebna zahvala tudi mentorici doc. dr. Meliti Sternad Lemut, univ. dipl. inž. živ. tehnol. In somentorju Silvestru Vončini, univ dipl. ekon. za pomoč pri pisanju ekonomskega dela naloge. Brez Vas ne bi uspelo! NASLOV Analiza kakovostne in ekonomske upravičenosti vpeljave tehnološkega postopka predfermentativne maceracije v predelavi grozdja sorte 'Refošk' IZVLEČEK V magistrskem delu smo obravnavali kakovostno in ekonomsko upravičenost vpeljave tehnološkega postopka hladne predfermentativne maceracije, s katerim smo želeli izboljšati predvsem barvne in aromatične lastnosti vina 'Refošk'. Najprej smo razložili pomen investicij in metode, s katerimi investicije ocenjujemo. V praktičnem poskusu smo pridelali vino po postopku hladne predfermentativne maceracije (HPM), za primerjavo pa smo vino pridelali še po postopku klasične fermentativne maceracije (FM) in fermentativne maceracije pri višji temperaturi (FMVT). V pridelanih vinih smo določali skupne polifenole, skupne in posamezne antocianine, barvo vina ter izvedli senzorično analizo. V ekonomskem delu smo ugotovili, kako bi investicijo financirali, in definirali lastno ceno. S pragom rentabilnosti smo predvideli smotrno prodajno ceno ter z metodami denarnih tokov in sedanje vrednosti ugotovili, da bi se nam investicija povrnila v devetem letu poslovanja. Rezultati kažejo, da je hladna predfermentativna maceracija vplivala na značilno večjo vsebnost skupnih polifenolov, medtem ko smo opazili značilno večjo vsebnost skupnih antocianinov pri FM in FMVT. Uporabljeni tehnološki postopki niso pokazali značilnega vpliva na povečanje vsebnosti posameznih antocianinov, malvidin-3-glukozida ter intenzitete in tona barve vina. Vino, pridelano po postopku HPM, je v primerjavi s FM in FMVT vini na senzorični analizi in testu všečnosti doseglo najmanj točk. Na podlagi dosežene kakovosti in ekonomskih ocen smo sklenili, da bi bila investicija v postopek HPM v proučevanem letu ekonomsko manj upravičena. The master thesis studies the quality and economic viability of the introduction of the technological process of cold prefermentative maceration, with which we wanted to improve the color and aromatic characteristics of the Refošk wine. Firstly, we explained the importance of investments and the methods by which investments are evaluated. In a practical experiment, we made wine by the method of cold prefermentative maceration (HPM), and for comparison, the wine was produced by the method of classical fermentative maceration (FM) and fermentative maceration at a higher temperature (FMVT). In the produced wines we determined the total polyphenols, total and individual anthocyanins, wine colour and performed the sensory analysis. In the economic part we found out how to finance the investment and define its own price. With the profitability threshold, we predicted a reasonable selling price, and with the methods of cash flows and present value, we found out that the investment would be repaid in nineth year of economic period. The results show that cold prefermentative maceration significantly increased the content of total polyphenols, while significantly higher total anthocyanins were observed at FM and FMVT. The used technological processes did not show any significant effect on the increase in the content of individual anthocyanins, malvidin-3-glucoside, and the intensity and hue of the wine colour. Wine produced by the HPM procedure compared to the FM and FMVT wines, achieved least points on the sensory analysis and the preference test. We concluded that, considering the qualitative results and economic estimates, the investment in the HPM process in the studied year would be economically less justified.

Odstranjevanje listov v območju grozdov in uporaba mrež za zaščito pred ptiči sta pogosto uporabljeni vinogradniški strategiji, ki vplivata na mikroklimatske pogoje v grmu vinske trte ter posledično na količinske in kakovostne parametre pridelka. V okviru diplomskega dela smo v vinogradu sorte 'Sauvignonasse' v Pristavem (Brda), po načelu naključne razporeditve blokov trt, zastavili primerjavo med petimi ukrepi: UNT (brez tretiranja), ELR (razlistanje pred cvetenjem), LLR (razlistanje po cvetenju), REN (razlistanje po cvetenju in senčenje z rdečimi mrežami) ter BKN (razlistanje po cvetenju in senčenje s črnimi mrežami). Tekom dozorevanja smo v odvisnosti od ukrepa spremljali osnovne kakovostne, ob trgatvi pa tudi količinske parametre pridelka. Grozdje smo ločeno vinificirali in v vinih določili osnovne kakovostne parametre, vsebnost hlapnih tiolov ter opravili senzorično analizo. Z obravnavanimi ukrepi smo le v manjši meri vplivali na kakovostne parametre grozdja, medtem, ko smo, predvsem z ukrepom ELR, pomembneje vplivali na količino pridelka in težo grozdov. V vinih smo (v korist obravnavanjem ELR, LLR in REN) opazili razlike v koncentracijah sladkorja prostega ekstrakta, poleg različnih trendov vsebnosti skupnih titracijskih kislin pa smo z ukrepi povzročili tudi nekaj razlik v vsebnostih posameznih kislin (vinska, jabolčna) ter skupnih hlapnih kislin. V primerjavi s kontrolo smo opazili povečanje vsebnosti 4MMP pri obravnavnjih BKN in ELR ter povečanje 3MH pri LLR, BKN in REN. Natančnejši pregled povzročenih sprememb in rezultatov (enoletnih) primerjav je nakazal, da učinki na opazovane parametre niso tako preprosti in linearni, da bi omogočili sistematično optimizacijo oz. da so medsebojni vplivi različnih kombinacij vinogradniških strategij na parametre grozdja kompleksni ter jih moramo uvajati nadvse previdno. Cluster area leaf removal and the use of nets for protection against birds are widely used viticulture strategies that can influence canopy microclimate and consequently the crop quantitative and qualitative parameters. In our work, we set up a completely randomised experiment in the vineyard of ‘Sauvignonasse’ in Pristava (Goriška Brda) with the aim to compare five different strategies: UNT (without treatment), ELR (leaf removal before flowering), LLR (leaf removal after flowering), REN (leaf removal after flowering and shading with red nets) and BKN (leaf removal after flowering and shading with black nets). During the ripening period the grapes basic quality parameters of all the treatments were monitored, whereas at harvest also the crop quantity parameters were calculated. The grapes were separately vinificated and the basic wine qualitative parameters as well as the content of volatile thiols were analysed in addition to sensory evaluation of the experimental wines. The observed strategies affected the grape quality parameters only to a lesser extend, whereas, with ELR mainly, we considerably influenced yield and cluster weight. In wines (in favour of ELR, LLR and REN treatments) differences in sugar free extract were observed. In addition to different trends in total titratable acidity, the treatments caused also some differences in the content of individual acids (tartaric, malic) and total volatile acids. Compared to control, an increase in 4MMP content was observed in BKN and ELR and an increase of 3MH in LLR, BKN and REN treatments. A more detailed overview of the changes and results of (one year) comparisons indicated that the effects on the observed parameters are not as simple and linear as to allow the systematic optimization and the effect of interactions of different strategies (their combinations) on grape parameters are complex and must be introduced with great caution.

Kakovost vina je prvi vrsti odvisna od lastnosti uporabljenega grozdja, njegova kakovost in stil pa sta močno odvisna tudi od vinarjevih odločitev glede uporabljenih vinifikacijskih postopkov. Zahteve vinskega trga spodbujajo k izboljšanju klasičnih kot tudi k uvajanju alternativnih vinarskih strategij. V sklopu diplomskega dela smo preučevali vpliv postopka karbonske maceracije v predelavi grozdja sorte 'Merlot' na vsebnosti prostih aromatičnih spojin, skupnih antocianov ter skupnih polifenolov v vinih in sicer v primerjavi s kontrolnim, klasičnim postopkom pridelave mladega rdečega vina. Kemijske analize vzorcev smo opravili spektrofotometrično ter s pomočjo naprednih analitskih pristopov metabolnega profiliranja in tehnike GC-MS-MS. Preučili smo tudi senzorične lastnosti eksperimentalnih vin. Rezultati enoletnega poskusa so pokazali, da lahko s karbonsko maceracijo v vinu Merlot povečamo vsebnost skupnih prostih aromatičnih kislin, estrov in norizoprenoida damascenona. Nasprotno smo več aromatičnih alkoholov določili v kontrolnih vinih. Uporaba karbonske maceracije je povzročila slabši izplen skupnih antocianov, ne pa tudi skupnih polifenolov. Senzorične analize barve so potrdile več češnjevo rdečih odtenkov pri vinih, pridelanih s karbonsko maceracijo ter več vijoličnih in rubinastih pri kontrolnih vinih. Karbonska maceracija je povzročila boljšo intenzivnost ter kakovost vonja, vendar slabšo trajnost in intenzivnost okusa vina. Degustatorji so pri vinih, pridelanih s karbonsko maceracijo, zaznali več svežega sadja in mehkega rdečega sadja v vonju ter več svežine in nekoliko boljšo harmoničnost v okusu. Kontrolna vina so imela močnejše zaznave gozdnih sadežev v vonju, v okusu pa več grenkobe in astringence, a so degustatorjem delovala polnejše. Wine quality primarily depends on the quality of grapes, however, the quality and style of the wine largely depends also on winemaker's decisions regarding vinification procedures. The wine market promotes the improvements of classical as well as the introduction of alternative wine-making strategies. Within our thesis, we studied the impact of carbonic maceration in the processing of grapes of variety 'Merlot' on the content of free aromatic compounds, total anthocyanins and total polyphenols in the wines, all in comparison with control - classical method of young red wine production. Chemical analyses of the samples were carried out spectrophotometrically and with the help of advanced analytical approaches of metabolic profiling by GC-MS-MS tehnique. The sensory properties of experimental wines were also examined. The results of one year experiment showed that carbonic maceration in case of Merlot wines increased the amount of total free aromatic acids, esters and norizoprenoid damascenon. Contrary, more aromatic alcohols were detected in the control wines. The use of carbonic maceration resulted in lower yields of total anthocyanins, but not total polyphenols. Sensory analyses confirmed more cherry-red shades in wines produced with carbonic maceration and more purple and ruby shades in control wines. Carbonic maceration resulted in better quality and intensity of wine flavour, but poorer durability and intensity of wine taste. The tasters described the wines produced by carbonic maceration to have more fresh fruit and soft red fruit in the aroma and to be fresher with slightly better harmony of the taste. Control wines showed more forrest fruit notes in the flavour, whereas more bitterness and astringence in taste, but the tasters also found them richer in taste.

Poleg sorte, geo-klimatskih pogojev in vinogradniških strategij v vinogradu so tudi vinarjeve odločitve glede izbora vinifikacijskih postopkov tiste, ki lahko odločilno vplivajo na končno kakovost vina, vključno z njegovimi aromatskimi značilnostmi. Z različnimi prijemi ali postopki pred alkoholno fermentacijo in med njo lahko vplivamo predvsem na ekstrakcijo in ohranitev primarnih ter na tvorbo sekundarnih aromatskih spojin. V okviru diplomskega dela smo s pomočjo modernih analitskih pristopov metabolnega profiliranja in tehnike GC/MS/MS preučevali vpliv različnih tehnoloških postopkov predelave grozdja (krioekstrakcija s suhim ledom, stik s celimi jagodami med fermentacijo, klasični pristop-kontrola) na vsebnost prostih in vezanih aromatičnih spojin v mladih vinih Zelen, letnik 2014. Preučili smo tudi vpliv obravnavanih tehnologij na senzorične lastnosti, vezane na zaznave vonja v mladih vinih. Rezultati enoletnega poskusa so nakazali, da lahko s pomočjo krioekstrakcije v vinih Zelen povečamo izplene skupnih prostih aromatičnih kislin, estrov in terpenov, po drugi strani pa smo zaznali le trend povečanja vsebnosti skupnih višjih alkoholov pri vzorcih iz postopka dodajanja jagod med fermentacijo. V primeru vezanih aromatskih spojin smo s pomočjo postopka dodajanja jagod v primerjavi s kontrolo opazili trende povečanja vsebnosti nekaterih vezanih estrov, norizoprenoidov in višjih alkoholov, krioekstrakcija pa je večinoma povzročila trende k niţjim vsebnostim vezanih aromatskih spojin glede na kontrolne vzorce. Nazadnje so se tudi senzorična opazovanja z vonjem povezanih lastnosti vin zaključila preteţno v prid postopku z dodajanjem jagod med fermentacijo. Beside variety, geo-climatic conditions and viticulture strategies in the vineyard, also winemaker’s choices in the cellar play a significant role in final wine quality and aroma style. Different approaches or procedures, before and during the fermentation, can influence in particular the extraction and preservation of the primary and the formation of the secondary aromatic compounds. Within this study we were using modern analytical approach of metabolic profiling and techniques GC/MS/MS in order to study the influence of various winemaking approaches (crioextraction with dry ice, contact with the whole grape berries during fermentation, classic approach-control) on the content of free and bound aromatic compounds in young wines Zelen of vintage 2014. We also studied the influence of the observed technologies on the organoleptic characteristics related to the odor in young wines. The results of one-year experiment indicated that with the help of crioextraction we could improve the yielding of total free aromatic acids, esters and terpens in Zelen wines. On the other hand, we have noticed only a trend of an increase in the content of total higher alcohols in samples from the treatment with addition of whole grape berries during fermentation. In case of bound aromatic compounds we have observed the trends toward enhanced content of several bound esters, norisoprenoids and higher alcohols by adding berries in comparison with control. Crioextraction on the other hand mainly showed trends toward lower contents of bound aromatic compounds in relation to the control samples. Finally, also sensory observations of wine aromatic characteristics closed mainly in favour of the procedure with adding berries during the fermentation.