Jabučno-mliječna fermentacija (JMF) složen je biokemijski proces koji se obično nastavlja na završenu alkoholnu fermentaciju te ima važnu ulogu u proizvodnji većine crnih vina. Istraživanja na ovu temu navode da su glavni razlozi za njeno provođenje sniženje ukupne kiselosti koju prati porast pH vrijednosti, mikrobiološka stabilnost vina uslijed razgradnje jabučne kiseline te promjena fizikalno-kemijskih i senzorskih svojstava vina. Cilj ovog istraživanja bio je utvrditi utjecaj različitih tipova jabučno-mliječne fermentacije na kompatibilnost kvasca i bakterija, dužinu njenog trajanja, promjene koncentracija pojedinačnih organskih kiselina, spojeva arome te promjene u fizikalno-kemijskim i senzorskim svojstvima vina Teran (Vitis vinifera L.). Pokus je obuhvaćao sljedeće tretmane: kontrola (bez JMF), spontana jabučno-mliječna fermentacija, jabučno-mliječna fermentacija po završenoj alkoholnoj fermentaciji te koinokulacija kvasca i bakterija mliječne kiseline na početku alkoholne fermentracije. Korišten je komercijalni soj kvasca Uvaferm 299 (Saccharomyces cerevisiae) i dva komercijalna soja bakterija mliječne kiseline Uvaferm AlphaTM i LALVIN 31TM (Oenococcus oeni). Osnovna analiza vina provedena je prema metodama O.I.V.-a. Koncentracije pojedinačnih organskih kiselina određene su spektrofotometrijski i primjenom tehnike tekućinske kromatografije visoke djelotvornosti (HPLC). Za određivanje tvari arome korištena je instrumentalna metoda plinske kromatografije (GC). Sva vina senzorski su ocijenjena metodom 100 pozitivnih bodova, rangiranjem i opisno. Jabučno-mliječna fermentacija značajno je utjecala na smanjenje ukupne kiselosti i povećanje pH vrijednosti. Najviše koncentracije hlapive kiselosti utvrđene su u vinima spontane jabučno-mliječne fermentacije. U svim tretmanima kod kojih je primijenjena jabučno-mliječna fermentacija zabilježena je potpuna razgradnje jabučne kiseline, smanjenje koncentracije limunske kiseline, ukupnih viših alkohola te acetaldehida. Značajno više koncentracije etilnih estera, diacetila, acetoina i 2,3-butandiola zabilježene su u svim tretmanima s provedenom jabučno-mliječnom fermentacijom. Pravilnost u porastu koncentracije ukupnih acetatnih estera utvrđena je kod tretmana spontane jabučno-mliječne fermentacije. U istom tretmanu zabilježena je i značajno viša koncentracija etil acetata. U svim tretmanima s jabučno-mliječnom fermentacijom,alkoholna fermentacija je u potpunosti završila, bez zastoja ili usporavanja. Koinokulacija je utjecala na značajno kraće trajanje jabučno-mliječne fermentacije. Senzorskom analizom bolje su ocijenjena vina koinokulacije i naknadne inokulacije, bez značajnih razlika u kakvoći pojedinih svojstava. Karakterizirale su ih izraženije voćne arome, puniji okus, kremoznija struktura te blaža kiselost i manja astringentnost. Unatoč značajnim promjenama senzorskih svojstava, primjenomjabučno-mliječne fermentacije vina Teran nisu izgubila sortna obilježja, a koinokulacija kao tretman nije imala negativan utjecaj na protjecanjei ishod alkoholne fermentacije.
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Malolactic fermentation (MLF) is a complex biochemical process that usually continues, to complete alcoholic fermentation and plays an important role in the production of most red wines. Research on this topic states that the main reasons for its implementation are the reduction of total acidity accompanied by an increase in pH, microbiological stability of wine due to the decomposition of malic acid and changes in physicochemical and sensory properties of wine. Teran (Vitis vinifera L.) is an autochthonous grape variety, and the wine produced from it is characterized by high total acidity and as such, this research is extremely suitable. The aim of this study was to determine the influence of different types of MLFon the compatibility of yeast and bacteria, its duration, changes in concentrations of individual organic acids, aroma compounds and changes in physicochemical and sensory properties of Teran (Vitis vinifera L.). The chapter Review of previous research provides data on the influence of environmental factors on malolactic fermentation as well as those on strains of lactic acid bacteria (LAB) and provides an overview and comparison of different types of MLF (co-inoculation, inoculation during alcoholic fermentation, subsequent inoculation and spontaneous MLF). The subchapter on the time of LAB inoculation on physicochemical changes in wines deals with the influence on the decomposition dynamics of malic and citric acid. Numerous studies indicate the influence of LAB inoculation time on changes in aromatic components of wine such as esters, higher alcohols, volatile fatty acids, acetaldehyde and compounds formed by LAB metabolic activity (diacetyl, acetoin, 2,3-butanediol) as well as on the sensory profile of wine. The chapter Materials and Methods presents the course of research and data processing. A two-year study was conducted that included the influence of different malolactic fermentation techniques on changes in physicochemical, aromatic and sensory properties in wines of the red cultivar Teran (Vitis vinifera L.). The vineyard in which the manual harvest was carried out was 10 years old, planted on a lesivan red (terra rossa) cultivation form of two-limbed Guyot. It was planted on a Kober 5BB substrate, and the planting intervals were 2.20 x 0.80 m, which gives approximately 5600 vines per hectare. After mulching and crushing, the mash is uniform and then evenly distributed in stainless steel tanks. The experiment was set up in three replications (50 L x 3) for each treatment in the experiment: Treatment K (control - suppressed MLF), S (spontaneous MLF-MLF takes place by the action of epiphytic LAB's), KIA (co-inoculation of LAB Oenococcus oeni Uvaferm AlphaTM), KI31 (co-inoculation of LAB Oenococcus oeni LALVIN 31TM), NIA (subsequent inoculation of LAB Oenococcus oeni Uvaferm AlphaTM), NI31 (subsequent inoculation of LAB Oenococcus oeni LALVIN 31TM). Alcoholic fermentation was carried out with classical maceration for seven days with manual, immersion of the cap three times a day using a commercial strain of yeast Uvaferm 299 (Saccharomyces cerevisiae). Alcoholic and malolactic fermentation were conducted at a temperature of 24⁰C until their completion. In all treatments, samples for chemical analysis were taken at the following times: (I) in the must, after filling the vessels, and before sulfitation and the addition of oenological agents, (II) every 2 days during alcoholic fermentation until its completion, (III) every 2 days during malolactic fermentation until its completion, (IV) in young wine after completed alcoholic and malolactic fermentation, and before overflow and sulfitation. Chemical analysis of must include determination of sugar concentration, total acidity, pH value and the determination of
individual organic acids. Basic chemical analysis (% vol. Alcohol, reducing sugar, total extract, total acidity, pH, volatile acid, ash) was performed in wine samples. Concentrations of individual organic acids were determined spectrophotometrically and using high performance liquid chromatography (HPLC) techniques. The instrumental gas chromatography (GC) method was used to determine the flavor substance. All wines were sensory evaluated by the method of 100 points, ranking and descriptive.The Results section presents the main results obtained by the research. Malolactic fermentation significantly affected the reduction of total acidity with increasing pH. The highest concentrations of volatile acidity were found in wines of spontaneous malolactic fermentation. The time of LAB inoculation did not affect the concentration of volatile acidity, while lower concentrations were produced by wines obtained using the Uvaferm AlphaTM strain. The inoculation time and the LAB strain used had no effect on succinic acid concentrations, the reduction of which was observed only in spontaneous MLF treatments. Degradation of malic acid was in all MLF treatments regardless of inoculation time and strain LAB was complete while significantly the lowest lactic acid concentration was found in the control treatment. Citric acid concentrations recorded a statistically significant decrease in wines with MLF, with significantly higher degradation in the treatment of co-inoculation and the use of LALVIN 31TM strain. MLF significantly reduced the total concentration of higher alcohols and increased concentrations of ethyl esters in all treatments, while a significant increase in the concentration of acetate esters in both years was recorded only in the treatment of spontaneous MLF with a significant increase in ethyl acetate and hexyl acetate. Co-inoculation significantly reduced the concentrations of isoamyl alcohol, 2-phenylethanol, ethyl acetate and isoamyl acetate, and increased the concentrations of ethyl lactate and diethyl succinate. The UvafermAlphaTM strain had a significant effect on increasing the concentrations of isoamyl alcohol, 2-phenylethanol, ethyl lactate and diethyl succinate, and a decrease on ethyl acetate and isoamyl acetate. The most common volatile fatty acids were caproic and caprylic acid with concentrations above the sensory detection threshold regardless of treatment. Significantly the highest concentration of caproic acid was recorded in the treatment of subsequent inoculation with LALVIN 31TM strain, and caprylic in the treatment of co-inoculation with Uvaferm AlphaTM strain during 2005. MLF resulted in significantly lower concentrations of acetaldehyde and significantly higher concentrations of diacetyl, acetoin and 2,3-butanediol compared to control treatment. Significantly lower acetaldehyde concentrations were affected by inoculation time while LAB strains used for inoculation had no effect. An increase in 2,3-butanediol concentrations and a decrease in acetoin concentrations were found in the co-inoculation treatment using the LAB strain Uvaferm AlphaTM. The lengths of the LAG phase and the total duration of MLF showed regularity in both years of the study and lasted the shortest in treatments with co-inoculation, then with subsequent inoculation and the longest in spontaneous MLF. The duration of MLF was also affected by the LAB strain, whereas MLF was significantly shorter than the LALVIN 31TM strain using the Uvaferm AlphaTM strain. Furthermore, the presence of LAB did not negatively affect the viability of the yeast and in all treatments, alcoholic fermentation had the correct dynamics with a complete breakdown of sugar. The results of the sensory analysis of wine confirmed the positive impact of the application of selected LAB's on the quality of Teran wine. Co-inoculation and subsequent inoculation treatments were rated the best, and spontaneous MLF wines the worst in both years of the study. In relation to the control treatment, comparing the sensory properties of the aroma and taste of the wine, co-inoculations and subsequent inoculations had a more pronounced fruity character with an emphasis on plums with a lower intensity of cherry aroma and lower sensations of spicy
notes, while the taste of wines was fuller, creamier with less pronounced acidity and astringency. Despite the significant changes of sensory properties, wines produced by co-inoculation and subsequent inoculation did not lose the varietal characteristics of Teran wine, which proved that co-inoculation as a treatment did not have a negative impact.