Improvement of blackberry wines quality by pectolytic enzymes and selected yeast strains application

Iako u ukupnoj svjetskoj proizvodnji vina, voćna vina zauzimaju tek mali dio, u mnogim zemljama proizvodnja vina od različitih vrsta voća predstavlja značajan gospodarski čimbenik. U kontinentalnom dijelu naše zemlje postoji duga tradicija proizvodnje voćnih vina, ali je tek u posljednje vrijeme zabilježen povećani interes za njihovom proizvodnjom. Takav trend rezultat je pojačanog interesa za konzumiranjem namirnica koje imaju pozitivan utjecaj na zdravlje. Plodovi kupine (Rubus spp.) i proizvodi dobiveni njihovom preradom predstavljaju vrijedan izvor polifenola, posebice antocijana. Zbog visoke antioksidacijske aktivnosti, polifenolni spojevi se smatraju najznačajnijim dijetalnim antioksidansima. Mnoge biološke funkcije kao što su usporavanje starenja, anti-mutagena i anti-kancerogena svojstva, među ostalima, potječu i od antioksidacijskih aktivnosti. Voćna vina od kupina se proizvode alkoholnom fermentacijom šećera prisutnog u soku ili kaši od kupina. Kako ne postoji standardizirana tehnologija proizvodnje ovih vina, proizvodi koji su zastupljeni na tržištu uvelike se razlikuju u mirisnim i okusnim svojstvima, a time i sveobuhvatnom kakvoćom. Stoga je cilj ovog istraživanja bio utvrditi osnovni kemijski sastav, udio pojedinačnih organskih kiselina, ukupnih i pojedinačnih polifenolnih i aromatskih spojeva te antioksidacijsku aktivnost u vinima od kupina kultivara ʹThornfreeʹ. Uz određivanje prethodno navedenih kemijskih parametara, interes ovog istraživanja bio je usmjeren na definiranje utjecaja pektolitičkih enzima i soja kvasca na kemijski sastav i senzorna svojstva voćnih vina od kupina. Dvogodišnje istraživanje provedeno je u vinima dobivenim od kupina iz nasada smještenog u Garešnici, u sjeverozapadnoj Hrvatskoj. Istraživanje je bilo postavljeno u pet varijanti: varijanta A (BDX OE): vinifikacija korištenjem komercijalnog kvasca Uvaferm BDX Saccharomyces cerevisiae i enzima Lallzyme OE; varijanta B (BDX EX-V): vinifikacija korištenjem komercijalnog kvasca Uvaferm BDX Saccharomyces cerevisiae i enzima Lallzyme EX-V; varijanta C (71B OE): vinifikacija korištenjem komercijalnog kvasca Lalvin 71B Saccharomyces cerevisiae i enzima Lallzyme OE; varijanta D (71B EX-V): vinifikacija korištenjem komercijalnog kvasca Lalvin 71B Saccharomyces cerevisiae i enzima Lallzyme EX-V i varijanta E (KONTROLA): vinifikacija bez dodatka selekcioniranog kvasca i enzima tj.vinifikacija djelovanjem epifitne mikroflore. Kemijska analiza vina obuhvatila je provođenje osnovne fizikalno-kemijske analize vina prema metodama O.I.V., određivanje pojedinačnih polifenola primjenom tekućinske kromatografije visoke djelotvornosti (HPLC) te određivanje kvalitativnog i kvantitativnog udjela tvari arome primjenom instrumentalne metode plinske kromatografije (GC). Vina su senzorno ocijenjena metodom 100 bodova prema O.I.V.-u. Dobiveni rezultati istraživanja pokazali su značajnu različitost proizvedenih vina kako u kemijskom sastavu, tako i u senzornim svojstvima. Sva vina karakterizira relativno visoki sadržaj organskih kiselina, među kojima je najzastupljenija limunska kiselina. Nakon limunske prema zastupljenosti slijedi jabučna kiselina. Obzirom na sposobnost kvasca Lalvin 71B da djelomično metabolizira jabučnu kiselinu, vina berbe 2012. godine proizvedena pod njegovim utjecajem imala su, očekivano i nešto niži sadržaj ove kiseline. Koncentracija ukupnih fenola u vinima od kupina u skladu je sa njihovim sadržajem u crnim vinima. Antioksidacijska aktivnost vina je u pozitivnoj korelaciji sa sadržajem ukupnih fenola. Najzastupljenija fenolna kiselina je galna, iza koje prema zastupljenosti slijedi klorogenska kiselina. U vinima varijante 71B OE izmjeren je najviši sadržaj trans-resveratrola u obje godine proizvodnje. Iako ne postoji statistički opravdana razlika između varijanti u pokusu, vina kontrolne varijante odlikuju se visokim sadržajem procijanidina B2, u obje proizvodne godine. Glavni predstavnik antocijana je cijanidin-3-O-glukozid, koji je najzastupljeniji u vinima proizvedenim pod utjecajem kvasca Lalvin 71B. Monoterpeni su dominantna skupina aromatskih spojeva, unutar koje se prema najvišim vrijednostima nametnuo linalol. Zabilježena je velika razlika u koncentraciji ovog monoterpena između kontrolne varijante i ostalih varijanti u pokusu. Rezultati senzornog ocjenjivanja ukazuju na postojanje razlika između varijanti u pokusu. Najbolje su ocijenjena vina proizvedena pod utjecajem kvasca Lalvin 71Although in the world's total wine production fruit wines occupy only a small part, in many countries wine production of different types of fruit, represents a significant economic factor. In the continental part of our country there is a long tradition of producing fruit wines, but only recently there has been an increased interest in their production. Such trend is a result of increased interest in eating foods that have a positive impact on human health. The blackberry fruits (Rubus spp.) and the products obtained by their processing, represent a valuable source of polyphenols, particularly anthocyanins. Due to its high antioxidant activity, polyphenol compounds are considered to be the most important dietary antioxidants. Many biological functions such as delaying of aging, anti-mutagenic and anti-cancerous properties, among others, also come from antioxidant activities. Blackberry wines are produced by the fermentation of sugar present in the blackberry juice or mash. Most of them are marketed as dessert wines, which, due to the high content of alcohol and unprocessed sugars, are very often inharmonious in taste. In addition, the technology for the production of blackberry wines is not standardized, which often results in the emergence of products on the market that differs significantly in flavor and aroma and therefore in overall quality. One of the prerequisites for quality wine production is the implementation of controlled alcohol fermentation with the addition of pectolytic enzymes and use of selected yeast strains. Therefore, the aim of this study was to determine the basic chemical composition, the share of individual organic acids, total and individual polyphenols, aromatic compounds and antioxidant activity in blackberry wines made out of ʹThornfreeʹ cultivar. In addition to the determination of the previously mentioned chemical parameters, the interest of this study was focused on defining the influence of pectolytic enzymes and yeast strains on the chemical composition and sensory properties of blackberry wines. In the chapter Overview of previous research data about blackberry fruits and its general characteristics are presented, as well as previous research connected with the production and the composition of blackberry wine. Published data about composition of organic acids in wine as well as their concentrations, pointed out the influence of blackberry cultivar, wine technology and yeast selection. Subchapter about polyphenols is dealing with a large and diverse group of chemical compounds that can strongly influence wine quality. Previously published scientific research confirms the impact of wine technology on the content and composition of polyphenols. For that reason, it is important to determine the possible impact of different pectolytic enzymes and selected yeast strains on the concentration and composition of this group of compounds. Phenolic composition, antioxidant activity and color diversity of fruit wines was also investigated in Croatia, but without analizying influence of different winemaking technology, or application of different enzymes and yeast strains. The composition and synergistic effect of phenolic compounds represented in wines have an effect on antioxidant and other wine activities. Recent studies have shown that regular consumption of small amounts of red wine reduces the risk of coronary heart disease and atherosclerosis. This advantage is attributed to the antioxidant properties of polyphenolic compounds from wine. The aroma of wine is largely dependent on the aromatic compounds of fresh fruit, as well as the compounds formed during or after the process of alcohol fermentation. Published data about composition and concentrations of aromatic compounds in wine, pointed out the importance of the influence of the wine technology and yeast selection. Subchapter about production of fruit wines is dealing with a winemaking technology and with influence of enzymes and selected yeast strains on wine quality. In the chapter Material and methods the course of research and data processing is presented. Two years of research was carried out in wines obtained from blackberry plantations located in Garešnica, northwestern Croatia. The research was set up in five variants: variant A (BDX OE): vinification using commercial yeast Uvaferm BDX Saccharomyces cerevisiae and enzymes Lallzyme OE; variant B (BDX EX-V): vinification using commercial yeast Uvaferm BDX Saccharomyces cerevisiae and enzymes Lallzyme EX-V; variant C (71B OE): vinification using commercial yeast Lalvin 71B Saccharomyces cerevisiae and enzymes Lallzyme OE; variant D (71B EX-V): vinification using commercial yeast Lalvin 71B Saccharomyces cerevisiae and enzymes Lallzyme EX-V and variant E (CONTROL): vinification without the addition of selected yeast and enzymes i.e., epiphytic microflora was used. The chemical analysis of wine involved the basic physical-chemical analysis of wine according to O.I.V., determination of individual polyphenols using high performance liquid chromatography (HPLC) and determination of the qualitative and quantitative content of the aromatic compounds using the instrument gas chromatographic method (GC). Wines were evaluated by organoleptic method of 100 points. The obtained results of the research showed significant differences in the produced wines, in the chemical composition and in the sensory properties. All wines are characterized by a relatively high content of organic acids, among which the most common was the citric acid. According to its concentrations, citric acid was followed by malic acid. Given the ability of yeast Lalvin 71B to partially metabolize malic acid, wines from 2012. vintage which are produced under its influence had a slightly lower acidity content. Concentration of total phenols in blackberry wines is consistent with their content in red wines. The antioxidant activity of wines is in a positive correlation with the content of total phenols. The most common phenolic acid is gallic, which according to its content followed by the chlorogenic acid. In values of gallic acid were noticed the significant influence of the BDX yeast. In wines produced by its influence, the lowest level of gallic acid was found, in both years of study. The highest concentration of trans-resveratrol was determined in variant 71B OE, in both examined years. Although there were no statistically significant differences among some variants, we could notice that the CONTROL samples stood out with high values of procyanidin B2 in both production years. The predominant one among the studied anthocyanins is cyanidin-3-O-glucoside, whose values are the highest in wines produced under the influence of yeast Lalvin 71B. The fact that cyanidin-3-O-glucoside is predominant among anthocyanins in blackberry wines was confirmed in the previously published literature data. According to the obtained results, the yeast strain had a larger impact on the concentrations of individual anthocyanins in relation to the pectolytic enzymes used. However, the established differences can be explained by the fact that the anthocyanins are highly reactive and unstable compounds. Monoterpenes are the predominant group of aromatic compounds, among which linalool was the most prominent. There are large differences in the concentration of this monoterpene between the CONTROL variant and the other variants in this research. Higher values of total monoterpenes were detected in wines that were treated with pectolytic enzymes in relation to wines which were produced without the addition of enzymes. The result of sensory evaluation indicates that the variants in the experiment differs from each other. Best rated wines are those produced under the influence of yeast Lalvin 71B. With their quality, CONTROL wines did not fall behind other variants, on the contrary, they were better than some variants. At the end we can conclude that use of different pectolytic enzymes and selected yeast strains can significantly affect the chemical composition and quality of blackberry wines. In future, this data can help the wine producers in making decisions to which enzymes and yeast strains they can choose