Study of Phenolic Compounds in Merlot Red Wines Obtained by Different Technologies

Study of Phenolic Compounds in Merlot Red Wines Obtained by Different Technologies GABRIELA IGNAT 1, GHEORGHE BALAN 2 *, ION SANDU 3,4, CARMEN LUIZA COSTULEANU 1, STEFAN TUDOSE SANDU VILLE 1 * 1 Ion Ionescu de la Brad University of Agricultural Sciences and Veterinary Medicine of Iasi, 3 M. Sadoveanu Alley, 700490, Iasi, Romania 2 Grigore T. Popa University of Medicine and Pharmacy, Faculty of Medicine, Institute of Gastroenterology and Hepatology of Iasi, 16 Universitatii Str., 700115, Iasi, Romania 3 Romanian Inventors Forum, 3 Sf. P. Movila Str., L11, III/3, 700089, Iasi, Romania 4 Alexandru Ioan Cuza University of Iasi, ARHEOINVEST Interdisciplinary Platform, 22 Carol I Blvd., 700506, Iasi, Romania The grape contains a high quantity of phenolic compounds responsible for color, which are transmitted into wines during the maceration-fermentation processes. The present paper s objectives are to monitor the extraction level, concentration evolution and analysis of these phenolic compounds in conjuncture with different maceration-fermentation procedures used. By approaching modern analytical methods, the correlation of studied phenolic compounds concentration variation with the maceration-fermentation technique will be quantified and statistically analyzed. Furthermore, by using a HPLC method of analysis (C18 column with 5 mm particle diameter and 25 cm in length) to determine the wine s anthocyan fingerprint, one can determine the grape variety used in winemaking, thus confirming its authenticity and typicity. The study concluded that the participation percentages of free and acylated anthocyans in the Merlot wine samples vary depending on the maceration technique used; the results show that the percentages of free anthocyans in wine vary between 77 and 91%, with lesser values for the acetylated and cumarilated anthocyans (8.8 22.7%). Key words: polyphenol, anthocyan profile, HPLC, authenticity, maceration Red wines, as the name shows, differ from white ones, mainly because of their high content of phenolic compounds, out of which some are specific to them (anthocyans). They are distinguishable by their aroma and taste which also are different from those in the white wines. Compared to aromatic wines, whose odor and taste are defined by many terpenic compounds, the red ones have a varietal aroma that is given by different compounds (for example, in the case of Cabernet Sauvignon, a great importance regarding aroma is represented by pyrazines [1] Ẇorld-wide, Romanian red wines enjoy a superior appreciation and solicitation to white wines, due to the four quality grape varieties predominant in our vineyards: Feteasca neagra, Merlot, Cabernet Sauvignon and Pinot Noir. Due to their phenolic compound content, red wines are less fragile, having considerably higher redox stability, in comparison to white wines [2-10]. Regarding the above information, it is understood that, in red wine technology, the main objective is extraction from skins and diffusion in must of these constituents that imprint the wine with their specific characteristics. The extraction of colored compounds and aromas from grape skins, its main deposit, is done in the pre-fermentative and even in the fermentation stages, though maceration [11, 12]. The present paper wants to underline the technological conditions which favor the phenolic compounds extraction from red grapes, in order to optimize the wine making methods and to obtain wines with superior chromatic characteristics. On the Romanian red wine market, the consumer is the decisive factor concerning the production, distribution and consumption circuit. The production level indicator is given by the consumption degree; thus, the consumer * email: balan.gheo@yahoo.com; stefan_ville@yahoo.com requirements, suggestions and dissatisfactions must dictate the production policy. A study conducted on a group of people living in the main 200 towns of Romania revealed the fact that 57% of 1500 participants acquired a certain type of wine on the influence of other people [13]. This is why the trend setters that influence the purchase of local red wines must be discovered. The quality red wines in Iasi region depends not only on the quality of the grapes used in the winemaking process, but also on the specific technology practiced. By optimizing the winemaking process, one can obtain high quality red wines with varietal tipicity, personality, positive olfactory and taste proprieties, with balanced astringency and beneficial implications on the consumer s health and, none the less, with a positive impact on enhancing overall red wine sales. Experimental part Materials and methods Research concerning the influence of different maceration-fermentation technological processes on the extraction degree for phenolic compounds from red grapes has been conducted in the Oenology Laboratory of the University of Agricultural Studies and Veterinary Medicine Iasi. Therefore, Merlot grapes have been harvested from Iasi-Copou vineyard, which were processed by using six maceration fermentation techniques: classical maceration, rotating-tank maceration, thermo-maceration, microwave maceration, ultrasound maceration and cryomaceration. Before applying the proposed experimental maceration procedures, the grapes (raw matter) were analyzed in regard to total anthocyan concentration and anthocyan profile. From the grape samples, several grapes were 1560 http://www.revistadechimie.ro REV.CHIM.(Bucharest) 67 No. 8 2016

randomly selected and were immediately frozen at under 35 C (to prevent polyphenolic extraction in the pulp) and the skins were removed; after a 48 hour drying period at max 36 C to prevent polyphenolic thermal degradation, 1g per sample was randomly separated and grinded with quartz stones. An acid-alcohol mixture (1% HCl in methanol) was used as solvent to extract the polyphenolic compounds. Thus, the dried skin samples were mixed with 100 ml solvent, freezed for 24 h at 35 C and filtered while cold; the extract was separated and the extraction process was repeated two more times (with 100mL and 50mL solvent), until the skins were pigment-free. The extract obtained was placed into a rotating evaporator (35 C and 0.2atm), where half of the methanol quantity was eliminated and replaced with double-distilled water. After separating through filtration the excess of alcohol-soluble compounds, the 250 ml hydro-alcoholic extracts were subjected to chromatographic and spectro-photometric analysis methods (total anthocyans and anthocyan profile by using a HPLC method) [3, 14-18]. The data was correlated with the climatic parameters registered in the maturation (pre-harvest) period (table 1). The characteristics of each maceration-fermentation technique used are: -Classical maceration (M-V1): selected yeasts were added to the must, which was in contact with the skins for 3 days at 20 C; when the alcoholic concentration reached 9%, the must was separated from the skins and the fermentation process continued until all sugars were depleted [19]. -Rotating-tank maceration (M-V2): selected yeasts were added to approximate 40L of marc, which was maintained in rotating tanks for three days, and the phases separation was conducted when the alcoholic separation reached 9%; the fermentation process ended in glass containers until all sugars were depleted [20]. -Microwave maceration (M-V3): 5 kg marc underwent microwave irradiation for 15 min at 650W power; the marc was then pressed; the fermentation process was conducted as stated above [21-23]. -Thermo-maceration (M-V4): the marc was subjected to thermal treatment at 60-75 C, for 30 min. A device for thermal treatment in must was used, with the following technical characteristics: tank capacity 20-40kg, maximum temperature 80 C, maximum productivity 40kg/ h, and 10 kw power. The minimal threshold for marc thermal treatment is 50 C. After the thermal treatment, the fermentation process took place as in the above cases [14, 17]. -Cryo-maceration (M-V5): fresh grapes have been slowly frozen at 30 C and then a fast destemming and crushing process took place; selected yeasts were added to the warmed must at 12 C. The fermentation process took place as in the above cases [25]. -Ultrasound maceration (M-V6): represents the easiest way to destroy the cellular wall and obtain the extract. Ultrasound cavitation builds powerful forces which mechanically destroy the cellular wall and improve the transfer. As the compound that must be solved is surrounded by an insoluble structure, in order to extract it, the cellular wall needs to be destroyed [26]. Therefore, the destemmed grapes were subjected to this treatment for 15 min (35 khz). This process is not widely used in red grape processing technology but it is successful in obtaining aromatic white wines. Many technological operations used were common to all variants: crushing and total destemming, SO 2 treatment of the marc, (doses of 0.05g/L) in order to insure antioxidant and antiseptic protection, proteolytic enzymes addition in order to increase fluid extraction, adding the same yeast to the must, Saccahromyces oviformis (from the collection bank of the Vine and Wine Research and Development Station Iasi), characterized by a high alcoholigenous capacity, SO 2 resistance and non-foaming effect, marc pressing using a low-capacity pneumatic press, alcoholic and malo-lactic fermentation (using endogenous lactic bacteria), oenological gelatin treatment (doses of 0.1g/L), racking, filtration with a sterile filter and bottling [27-31]. The phenolic compounds in the obtained wines were analyzed in regard to total anthocyan content and anthocyan fingerprint. An UV-Vis Analytik Jena Specord 200 spectrophotometer and a Hewlett-Packard HP-1100 HPLC with C18 column were used (Compendium of International Methods of Wine and Must Analysis). Results and discussions The anthocyans are visible phenolic compounds which accumulate in the grapes from their entry into the ripening period and continues for the duration of maturation. The total amount of anthocyans from grape skins determined at harvest and total anthocyans was calculated in mg per 1 gram dried skins (table 1). The anthocyanin profile analysis was done by HPLC, whereby the grape and wine anthocyanin compounds are individualized by means of a UV detector. For each chromatogram one identified and then calculated the participation percentage of the following anthocyans: delphinidin-3-monoglucoside (Dp-3-gl), cyanidin-3-monoglucoside (Ci-3-gl), petunidin-3- monoglucoside (Pt-3-gl), peonidin-3-monoglucoside (Po- 3-gl), malvidin-3-monoglucoside (Mv-3-gl), peonidin-3- acetyl-monoglucoside (Po-3-gl-a), malvidin-3-acetylmonoglucoside (Mv-3-gl-a), peonidin-3-coumarylmonoglucoside (Po-3-gl-c), malvidin-3-coumarylmonoglucoside (Mv-3-gl-c). Since the studied grape variety is obtained from Vitis vinifera, therefore contains only monoglucosidic anthocyans, five types of anthocyans were identified, namely delphinidin (Dp-3-gl), cyanidin (Ci-3-gl), petunidin Table 1 ANTHOCYAN PROFILE AND TOTAL ANTHOCYAN CONTENT IN MERLOT GRAPES AT HARVEST REV.CHIM.(Bucharest) 67 No. 8 2016 http://www.revistadechimie.ro 1561

Fig. 1. The chromatogram of the anthocyan profile of the extract obtained from Merlot grape skins at harvest with the retention lengths Table 2 TOTAL ANTHOCYAN CONTENT AND FREE ANTHOCYAN PROFILE OF MERLOT WINES (PARTICIPATION %) (Pt-3-gl) peonidin (Po-3-gl) and malvidin (Mv-3-gl); the but their color intensity during lengthy aging decreases, acylated derivatives for peonidin (Po-3-gl-A and Po-3-gl-c) and malvidin (Mv-3-gl-a and Mv-3-gl-c) have also been whereas a large part of free anthocyans oxidize, polycondensate or combine with other substances [3]. analyzed (acetylated and coumarylated). Since the diffusion of tannins is slower, to get more Figure 1 represents the chromatogram and the retention times of anthocyans in Merlot grapes at harvest. astringent wines, one requires a lengthier microwave maceration; in this case, the young wine have a more The ratio of acetylated and coumaryl derivatives is less expressed astringency that starts to diminish after aging than 1, fluctuating between 0 and 0.6 because the acetylated derivatives in Merlot grape skins are found in 2-3 years. In contrast, the color intensity decreases slightly as tannins, due to condensation, gain themselves color. very small quantities. Therefore, the microwave maceration procedure length In general, Merlot grapes contain small amounts acylated derivatives that vary according to the ripening should be chosen according to the type of wine and the period of maturation and aging that will be submitted. period (between 5 and 12%). Cryomaceration (M-V5) led to small quantities of The total anthocyan content (table 2) is different depending on the variety, vintage year and variant anthocyans being extracted, compared with the abovementioned processes (V1-V4) only166mg/l anthocyans conditions of maceration used. which is close to 55% of the quantity extracted by using Within the same variety and during the same harvest year, this parameter differs only due to the maceration the classical method (301 mg/l). Knowledge of wine anthocyan profile leads to the conditions. establishment of the variety from which the wine was Thus, the content of anthocyans in the experimental samples varies between 127.8 mg/l (M-V6) and 301.9 mg/ made and to confirm their authenticity. There is a limit for authentication based on wine anthocyan fingerprint; this L (M-V2). is because, after only 1-2 years of storage in bottles, only The variants where the fermentation process was conducted simultaneous with the maceration technique trace amounts of free anthocyans remain, which can be determined by a HPLC method. Anthocyanin profile of the (M-V1 and M-V2), the anthocyan extraction was favored wine expresses the participation percentage of free and by the presence of alcohol, by the higher temperature, by the marc homogenization and also by the changes in acylated anthocyans that make up the color of the wines. Because during maceration and wine storage period the ph,etc. acylated anthocyans are more stable and resistant to The first and second proposed variants, in terms of the concentration of anthocyans, contained normal values, condensation, for profiling anthocyans one analyzed the sum and the ratio of acetylated and coumarylated between 301.2 mg/l (M-V1) and 301.9 mg/l (M-V2) anthocyans contents. The results of Merlot wines that reflect (considering the fact that the climatic conditions were less than favorable for the grape maturation and for the the participation percentages of free and acylated anthocyans are presented in tables 2 and 3; figures 2 accumulation of large amounts of phenolic compounds in through 7 are the chromatograms that show the anthocyan their skins). By using microwaves to the maceration procedure, the amount of anthocyans found in Merlot wines profile.the analysis of Merlot wines anthocyan profile, in the color composition malvidin mostly participates (40.9 was 298 mg/l, and the wines obtained by thermomaceration to 80.2%), in varying percentages depending on the contained 290 mg/l. Although the exposure time to the microwave treatment is relatively short (15'), maceration variant. Wines obtained through cryomaceration have a different profile from other wines: the wine anthocyans reached normal concentrations. The malvidin 80%, peonidin 7.5%, petunidin 2.4% and diffusion of anthocyans from grape skins is much faster than the tannins and, because of this property, even after a delphinidin and cyanidin registered below par values. The results obtained in other experimental variants present limited exposure to microwaves, one can obtain intensely closer values among them. Except cryomaceration, one colored wines. These wines are less astringent when young, 1562 http://www.revistadechimie.ro REV.CHIM.(Bucharest) 67 No. 8 2016

Table 3 ACYLATED DERIVATES OF PEONIDIN AND MALVIDIN OF MERLOT WINES (PARTICIPATION %) Fig.2. The chromatogram of Merlot wine obtained by classical maceration (M-V1) with the retention lengths Fig. 3. The chromatogram of Merlot wine obtained by rotating tank maceration (M-V2). with the retention lengths can say that for Merlot wines, the anthocyan profile consists of: malvidin (40.9 to 63.5%), peonidin (6.3 to 12.3%), petunidin (9.7 to 11.5%), delphinidin (7.4 to 11.3%) and cyanidin (1.3 to 2.9%). The ultrasound maceration led to the extraction of a small amount of total anthocyans (127 mg/l), but the anthocyan profile is similar to other wines, keeping with the overall allure of Merlot. Thus, out of 89.2% of the total free anthocyans found, malvidin is 58.8%, peonidin 11.3%, petunidin 9.7%, delphinidin 8.1% and cyanidin 1.3%. The results analysis showed that wines contain a larger amount of acetylated anthocyans compared coumarylated anthocyans. which makes their ratio higher than one. between 1.1 and 2.89%. In this way. Merlot wines are distinguished from wines made from other varieties. for which this ratio has different values. Acylated anthocyan amount varies between 8.8 and 22.7% depending on the maceration variant used. Wines obtained by classical maceration (M.-V1). maceration in rotating tanks (M.-V2) and microwave maceration (M.-V3) contain the highest proportion of acylated anthocyans. representing 23% of the whole anthocyan profile; the other three variants (thermomaceration. cryomaceration and ultrasound maceration) have a smaller amount of the acylated anthocyans. which is 8.8 to 11.6% of the overall profile. Of the 4 acylated anthocyans which were determined. the malvidin acetylated anthocyan is characterized by higher participation percentages (all variants) (from 5.5 to 11.4%). followed by the malvidin coumarylated anthocyans (1.7 4.8%) and peonidin acetylated anthocyans (0.8 to 4.76%). The anthocyan fingerprint of Merlot analysis shows that wines have a high percentage of acylated anthocyans (8.8 to 22.7%). which gives them better resistance and color stability during maturation and aging. Oenological study phenomena is performed on highvolume collectivities. and in order to observe the entire population. only certain samples or samples of low volume are statistically tested. Thus. to assess statistical units of each sample a two-way analysis of variance (ANOVA - Two-Factor Without replication) was applied. in order to determine how the variants of the proposed maceration techniques affect the extraction levels of each of the anthocyans. After statistically testing the way the participation percentages of free anthocyans and acylated anthocyans of the wines obtained through the proposed experimental variants may or may not be influenced by the corresponding maceration method used. one obtained the following results (table 4). From the data presented it can be concluded that participation percentages of free anthocyans in Merlot wines profile produced in Iaºi vineyard are influenced, in statistical terms, by the maceration variations used (at least 95% probability coverage); thus, the participation percentages of delphinidin-3-monoglycoside, peonidin-3- monoglycoside and malvidin-3-monoglycoside are significantly influenced and the participation percentages REV.CHIM.(Bucharest) 67 No. 8 2016 http://www.revistadechimie.ro 1563

Fig. 4. The chromatogram of Merlot wine obtained by microwave maceration (M-V3) with the retention lengths Fig. 5. The chromatogram of Merlot wine obtained by thermomaceration (M-V4) with the retention lengths Fig. 6. The chromatogram of Merlot wine obtained by cryomaceration (M-V5) with the retention lengths Fig. 7. The chromatogram of Merlot wine obtained by ultrasound maceration (M-V6) with the retention lengths of cyanidin-3-monoglycoside is distinctly significantly influenced with 99% confidence level. On petunidin-3-monoglycoside, different technologies maceration exerted a very significant influence, in statistical terms, with a 99.9% coverage probability. Statistical analysis of the percentages of participation of acylated anthocyans reflects the fact that they are not significantly influenced by the macerating technology applied, except malvidin-3-coumaryl-monoglucoside, which was found to be statistically significantly influenced by the maceration technology used, in regard to its anthocyanin profile. Conclusions The total anthocyan content varies according to the maceration technique used; by using classical maceration fermentation and rotating tanks maceration fermentation techniques. the most balanced values were obtained, followed by thermomaceration. Ultrasound maceration was not favorable to the phenolic compound extraction 1564 http://www.revistadechimie.ro REV.CHIM.(Bucharest) 67 No. 8 2016

Notations: *- Very significant statistical influence (P-value < p = 0.001); ** - Distinctly significant statistical influence (P-value < p = 0.01);** *-Significant statistical influence (P-value < p = 0.05); ns - Insignificant statistical influence (P-value > p = 0.05) process through grape skins and the obtained wines had lesser quantities of anthocyans and tannins. In regard to the maceration method used, the free anthocyans are found in wines in different percentages. varying between 77 91%. In all analyzed wines, malvidin is found in the highest percentage, followed by peonidin, petunidin and delphinidin; cyanidin is found in the lowest proportions (0.2 2.9%). Although the sum of acetylated and cumarilated anthocyans vary with the maceration technology used, the obtained values are specific to every variety regardless of the technological technique used. Acknowledgements. The authors would like to thank the personnel of the Enology Research Center. 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