Influence of enzymatic treatments on white wine composition

Most biochemical reactions involved in the wine-making process are catalyzed by enzymes. The use of enzymes of exogenous origin in wine production is due to the numerous technological and economical advantages demonstrated over time in the winemaking process. Understanding the important role played by enzymes in wine making technology contributes to the development of optimization strategies for the production process to improve the final quality of the wine. In order to accomplish this study, the influence of five oenological preparations with pectolytic and β-glucosidases enzymes types on the volatile compounds of white wines obtained from Fetească regală variety was analyzed by monitoring their evolution during the alcoholic fermentation to the final product. Wine samples have been physically and chemically analysed (pH, acidity, alcoholic strength, density, malic acid, lactic acid, sugar content, SO2, total dry extract and non-reducing extract) according to OIV Standards. Separation and identification of flavor compounds was performed using an Agilent 7890 gas chromatograph coupled to a 5975 C inert XL EI/CI MSD spectrophotometer. The organoleptic evaluation of wines was made according to a wide range of sensory descriptors. An important evolution of volatile compounds during fermentation was observed, depending on the type of enzyme administered, compared to the control sample. Enzymatic treatments did not significantly affect the physico-chemical composition of the wines obtained. The chromatic parameters of the wine samples varied according to the type of enzyme applied. The results of the study showed a significant influence of the enzymes on the organoleptic characteristics of the wines. Therefore, the aromatic quality of a wine is directly proportional to the chemical composition of the grapes and to the technology.


Introduction
Enzymes are biological catalysers used in winemaking technology for their numerous advantages on the product quality. These compounds may come from indigenous microflora of grapes, yeasts or other bacteria correlated with wine cellar, vinification conditions or vineyard [1].
The wine-maker can enrich the activity of these endogenous enzymes by using commercial products, usually obtained by fermenting pure culture of selected microorganism [2]. Pectinases, proteases and glycosidases are some of the enzymes produced by yeast, that present significance in oenology for their technological actions and their participation to aroma constitution. Pectinolytic enzymes have the ability to cleave long pectin chains into shorter and more soluble chain segments, that facilitate pressing operation of the grapes and clarification of musts, enhace the extraction of color and aroma compounds and facilitate the filtration of resulted wine. These enzymes can also facilitate the stabilisation of the wines, due to the enzymatic hydrolysis of the protein into smaller molecules, containing more nitrogen. The aroma compounds concentration of wines can be increased by glycosidases, which hydrolyse nonvolatile glycosidic precursors of the grapes. Yeasts are also important producers of enzymes [3]. Saccharomyces cerevisiae, considered the fundamental wine yeast, is not appreciated as a considerable producer of extracellular proteases, lipases or proteolytic enzymes [4]. The final quality of wines is influenced by a multitude of interactions between chemical compounds. During alcoholic fermentation, grapes derivates are converted by yeast into a range of sensorial relevant metabolites that gives wine its vinous character [5]. Commercial enzymes are utilized in wine-making technology for diverse biotransformation reactions from pre-fermentation stage pending alcoholic fermentation, post-fermentation and wine aging period [6]. Understanding the important role of enzymes in winemaking technology contributes to the development of strategies to optimize the production process, improving the wine's composition and its sensory characteristics [7].
The purpose of this experiment is to investigate the influence of enzymes pre-treatments on the physicochemical parameters, chromatic characteristics and aromatic profile.

Grapes samples and winemaking
The grapes representing Feteascȃ Regalȃ variety were harvested in autumn of 2018, from Iasi vineyard, with approximately 250 g/L sugar content. After destemming and crushing operations, the must was transferred into 50 L glass vessels. A dose of 20 g/hL Saccharomyces yeast (Levulia esperide, AEB) and 30 g/hL yeast nutrient (FERMO PLUS CH, AEB) (previously dissolved in warm must) was inoculated in each variant. For this experiment, five commercial enzymes based on pectolytic and β-glucosidases activity were added to musts before alcoholic fermentation, thus contributing to the release of aroma compounds (Endozym Thiol®, AEB -V1; Endozym β -Split®, AEB -V2; Zymovarietal aroma G®, SODINAL -V3; Endozym Ice®, AEB -V4; Zimarome®, BSG WINE -V5 and no enzyme-V6), at doses of 3 g/hL (all enzyme preparations were diluted with must 1:10) and 3 mL/hL respectively. 6 variants were obtained. The fermentation was carried out at [16][17][18] • C for about one week. For this experiment, samples were collected at two days differences during the alcoholic fermentation and stored in the freezer until the analysis. When the alcoholic fermentation was finished, each variant was filtered using sterile membrane filter followed by sulphitation (to preserve wine from microbiological damage) and bottled. The wine samples were stored under controlled condition and analysed after three months.

Chemicals
Standard reagents and pure commercial substances were purchased from Merck KGaA (Germany).
Standard physico-chemical parameters were reviewed according to International Organization of Vine and Wine Compendium methods of analysis [8]. Each variant was analysed for: total and volatile acidity, alcoholic strength, pH, density, total sugar, free and total sulphur dioxide, malic and lactic acid, total dry extract and non-reducing extract.
Sensory characteristics are important for the quality of wines. The wine samples were assessed for sensory characteristics by 10 tasters according to a wide range of sensory descriptors. The parameters were evaluated with ratings from 0 to 5, and the mean was registered in the tables. The sensory descriptors of samples were comparatively expressed in a chart.
Separation and identification of flavour compounds was performed using an Agilent 7890 gas chromatograph coupled to a 5975 C inert XL EI/CI MSD spectrophotometer. The column used for the analysis was HP-5MS fused silica capillary column (30 m · 0.25 mm I.D., 0.25 µm with a split ratio of 50:1 and inlet temperature was set at 250 • C. The MSD was used at 70 eV in the electron impact mode (EI), using the mass range from 50 to 800 Da at 150 • C. Peak identification of the aroma components was achieved by comparison of mass spectra with the mass spectral data collection from Wiley275 and NIST05a libraries.
Sample extraction: In a 15 mL glass tube, 5 mL of wine sample and 1 mL of dichloromethane were added, and the mixture was mechanically agitated for 45 minutes. The extract was separated from the aqueous phase by centrifugation at 7800 rpm for 10 minutes. This process was repeated twice, the extraction was performed after 20 minutes. The two organic phases were mixed and injected in the GC-MS.

Results and discussions
The present article highlights a comparative investigation on the action of enzymes on the quality of wines. Table 1 shows the physical-chemical parameters of wines. The alcoholic strength of a wine is dependent of the total sugar content of the origin grapes. The obtained samples were dry wines with over 12.7% vol. Alcoholic strength of wines was not influenced by the type of administrated enzymes.

Effects of enzymatic pre-treatment on basic parameters of resulted wine
Acidity and pH are relevant to the sensory perception of wine, essentially defining its structure and equilibrium. Excessive acidity increases the perception of sourness since low acidity decreases the flavour balance. Acid content is relevant for preservation and is responsible for sensory features of wines. Its content in wine may be influenced by grape variety, ripeness, climatic conditions, wine-making process, wine storage and pH [9]. No significant differences were recorded in total and volatile acidity of wines, which means that enzymes have no influence on this indicator. The total dry extract shows values between 19 and 19.6 g/L. The content of non-reducing extract of most Romanian wines varies between 13 and 35 g/L, according to grape variety, health conditions, applied technology and wine treatments [10]. The analysed samples registered values of non-reducing dry extract between 17.6 and 17.9 g/L.

Chromatic characteristics
Colour is a primary attribute by which the consumer assesses wine and food. Chromatic parameters of analysed wines is presented in Table 2.
Analysed samples registered a high clarity, with more yellow and red shades, except V1 that presented more green and yellow shades of colour. Parameter "a*"(green/red component) had the highest value on V6 (control sample) and the lowest on V1 sample (Endozym Thiol®). The highest values of "b*" (blue/yellow component) were registered in the case of V1 sample (Endozym Thiol®), and the lowest in V3 (Zymovarietal aroma G®). Some differences were recorded for "L*" that corresponded to brightness characteristics. It was expressed by a less green/red and more yellow colour of wine. The red pigment indicates the presence of low but still visible amounts of anthocyanins. Enzymatic treatment influenced chromatic parameters of analysed samples to varying degrees. The majority of wines treated with enzymatic preparations were characterized by the decrease in a* compared to the control sample, a less intense green colour and more red colour respectively. Also, all samples treated with enzymes were evaluated by an increase in b*, that means more yellow colour.

Volatile compounds
The aromatic profile of wines is influenced by numerous volatile compounds originating from the grapes or must, from yeast (fermentative aromas), or post-fermentative aromas (from aging processes) [11]. The strength and proportion of volatile substances set the category of wine odour. In Table 3, the most representative volatile compounds of analysed samples and their evolution during fermentation are represented. Enzymatic treatments determine an increase in the total amount of volatile compounds compared to control samples. The results denote that the content of volatile compounds significantly fluctuate during fermentation and storage.       These differences can be generated by enzymes reactions and yeast. Saccharomyces cerevisiae yeast can have a major contribution to wine aroma profile.
The aroma profile of analysed samples is expressed especially by higher alcohols, esters and acids. The highest concentration in alcohols was detected in V5 variant, followed by V6, V3, V2, and V1. On the other hand, the richest variant in esters was V1, followed by V6, V4, V3, V2 and V5. V1 variant registered the highest amount of acids, followed by V4, V3, V5, V2 and V6. Isoamylalcohol and benzenethanol were the highest among the total of identified alcohols, imprinting complexity to the aroma. Esters constitute one of the most important classes of aroma compounds, with an important effect on the fruity notes in wines (tropical fruits, banana, pineapple, pears). Butandienoic acid diethyl ester showed a high concentration in all variants. Organic acids enhance bitterness, freshness and astringency. Butanoic, hexanoic and decanoic acids presented the highest proportion. Phenylethylacetate and 1-hexyl acetate are specific for V1 sample (treated with Endozym Thiol®).
Significant differences can be observed referring to the quality of final wines, reported on the type of used enzyme. Enzymes released additional volatile compounds from the grapes, reducing the amount of alcohol, allowing a better perception and release of aroma compounds. The different distribution of volatile components in analysed wines is presented in Fig. 1.

Sensory characteristics
The final sensorial quality of wine is the result of a multitude of interaction between all the volatile components. The character of wine is influenced by the sweetness, acidity and astringency/bitterness, generated by non-volatile substances soluble in water or alcohol mixture. Together with volatile compounds, they are liable for the general aroma profile. Perceptible sweetness is significantly determined by numerous constituents, such as ethanol, acids or tannins. Detectable sweetness presents a diminishing effect on sourness and bitterness [18]. Sweet note was better remarked at V1 sample.
The principal organoleptic parameters of analysed wines are presented in Fig. 1. Following the sensory analysis, significant differences can be observed due to the type of enzyme preparation used as pre-treatment.
Enzymatic treatments generated an increase in flavor descriptors content compared to control sample. All wines were described as equilibrated, with a great acidity, that imprinted freshness and a good texture.
Control sample was characterized by hay notes, honey and vegetable flavour. V1 sample was remarked for its fruity taste (ripe fruits, exotic fruits, dry fruits), wild flowers and roses nose. Citric flavour was dominant in V2 sample and green fruits aroma is prevalent in V5.
Regarding the correlation between the chemical compounds and the results of sensorial analysis, the fruity notes (ripe fruits) of samples can be explained by the presence of 1-propanol, 1-hexyl acetate, 1-hexanol, ethyl octanoate, ethyl caproate. The presence of 1-hexanol explains the vegetal notes of samples. Ethyl caproate and isoamylacetate are the main responsible for the tropical aromas.

Conclusions
In this study, the pre-fermentative treatments did not appear to have a significant influence on the basic physicalchemical parameters. A perceptible colour difference between samples treated with enzyme preparations and control samples was observed, suggesting that enzymes preparation had a major effect on colour. Following the evolution of volatile compounds during fermentation, enzymes released additional volatile compounds from the grapes, reducing the amount of alcohol, thus allowing for a better perception and release of aroma compounds. Enzymatic treatments determine an increase in the total amount of volatile compounds compared to control sample. The most abundant compound in all samples is isoamylalcohol, followed by benzenethanol. Significant differences can be observed referring to the quality of final wines and on the type of used enzyme. The aroma profile of analysed samples is expressed especially by higher alcohols, esters, and acids. Following the sensory analysis, significant differences can be observed due to the type of enzyme preparation used as pre-treatment.
Enzymes are effective, specific and convenient to use and the need for a more in depth search is clear.