ANNALS OF AGRARIAN SCIENCE, vol. 12, no. 4, 2014 ИЗВЕСТИЯ АГРАРНОЙ НАУКИ, Том 12, Ном. 4, 2014 TECHNOLOGY OF PROCESSING OF AGRICULTURAL PRODUCTS ТЕХНОЛОГИЯ ПЕРЕРАБОТКИ СЕЛЬСКОХОЗЯЙСТВЕННЫХ ПРОДУКТОВ TRANSFORMATION OF BIOLOGICALLY ACTIVE STILBENOIDS FROM GRAPEVINE TO RED WINE, FOOD SUPPLEMENT AND BEVERAGE
M.G. Bezhuashvili, P.N. Vashakidze, N.G. Vepkhishvili, L.D. Elanidze Agricultural University of Georgia, Institute of Viticulture and Oenology 240, David Aghmashenebeli Alley, 0159, Tbilisi, Georgia; [email protected] Received: 22.06.2014; accepted: 02.09.2014
Cis and Trans piceid stilbenoids have been identified and determined in juice and skin of the red grape vine varieties growing in Georgia. The experiment used different grape varieties, such as Saperavi, Cabernet Sauvignon, Otskhanuri Sapere, Aleksandrouli, Mujuretuli, Shavkapito, Tavkveri, Aladasturi, Dzelshavi, Ojaleshi. The substance isolated by means of preparations was identified with acid hydrolysis, thin layer and high effective liquid chromatography and UV spectroscopy. It was found that the grape juice contains more trans piceid, while the grape skin contains more cis piceid. It was for the first time, trans resveratrol was identified in the shoots of Saperavi grape ( Vitis vinifera L.). Based on the gained data, the transformation of the grape stilbenoids in the red wine identified by us was fixed. We have developed the technologies to produce biologically active products containing stilbenoids by using the grapes.
INTRODUCTION For about 525 red and white vine varieties make Georgian vine gene bank. Saperavi is unique among the red vine varieties. It is used to produce high quality, competitive different type of wines carrying curative prophylactic value. Georgian vine varieties and wines made from them are characterized by wide spectrum of phenolic compounds; among them are proantocyanidins (olygomeric and polymeric), antocyanins, flavonols, flavanols, stilbenoids, phenolic carbonic acids, etc. As these phenolic compounds are localized in wines, they significantly influence the quality of the wine and its prophylactic curative value. [1 5]. Among vine phenolic compounds the group of stilbenoids take an important place. Stilbenoids are characterized by high biological activity and play phytoalexin role in the
1 plants. These are for example resveratrol, ε viniferin, etc. [6, 7]. Due to biological activity, stilbenoids are worth to be studied. In Georgia at first, the following stilbens were extracted and identified: trans resveratrol, its dimer ε viniferin and two tetrameric stilben [8 10]. The following red vine varieties spread in Georgia: Saperavi, Saperavi budeshurisebri, Cabernet Sauvignon, Otskhanuri Sapere, Aladasturi, Asuretuli Shavi, Chkhaveri, Aleksandrouli, Mujuretuli, Ojaleshi and wines made from these vine varieties contain trans resveratrol and its derivative ε viniferin and tetrameric stilben, from which dominates trans resveratrol. For instance its concentration in the wines made from Saperavi vine variety is as following: trans resveratrol – 2,56 mg/l, ε viniferin – 0,81 mg/l and tetrameric stilben – 1,22 mg/l [11 13]. In red vine varieties and wines of different countries among the stilbenoids the following substances are identified: resveratrol trans and cis isomers; ε ,δ viniferin, palidol, piceathanol, stilbenoid glucosides: trans, cis piceid, astringin, etc. [14 27]. Due to the studies of Georgian red vine varieties and grapes and also due to the fact that Saperavi juice is the main ingredient of biological active food supplement produced via our technology, we have conducted a research to identify stilbenoid glucoside in Saperavi grape juice.
OBJECTIVES AND METHODS Sound grape juice and skin of red grape varieties growing in Georgia were obtained in the technical ripeness period. Such varieties are: Saperavi, Cabernet Sauvignon, Otskhanuri Sapere, Aleksandrouli, Mujuretuli, Shavkapito, Tavkveri, Aladasturi, Dzelshavi and Ojaleshi. (1) We prepared the objects of study with grape juice and skin according to plan and used it for analysis; (2) we used grape juice of Saperavi to obtain acid hydrolysate, added concentrated HCL to it to reach a 10% acid concentration in the reaction area and accomplished hydrolysis at the temperature of 80°C for 3 hours. Under the given conditions, we carried acid hydrolysis of the individual study substances isolated by means of preparations. The hydrolysates were extracted with ethyl acetate, concentrated with rotational over flow and analyzed. UV spectrum of the compound under study was shot with spectrometer “Specord”. We used paper chromatography for qualitative analysis of sugars in acid hydrolysates. We used a mixture of solvents: N butanol ethyl acetate propanol acetic acid water (35:100:62:35:30) as a system. We developed a chromatogram with the mixture of water solutions of ammonium molybdate and ammonium chloride in acid area. Qualitative analysis was accomplished with thin layer chromatography (Sorbfil, ПТСХ П А 10х20; system – chloroform:methanol, 80:20). The chromatograms were developed with diazotized sulfanilic acid. HPLC analysis: chromatograph “Varian”, column Supelcosil TM LC18, 250X4,6 (mm); solvent A, 0.025%, trifluoroacetic acid; solvent B, acetonitrile (ACN)/A, 80/20, gradient regime 0 35 min. 20 50% B; 35 40 min. 50 100% B; 41 46 min. 100% B; 46 48 min. 100 20% B; 48 53 2 min. 20% B. Eluent transfer velocity 1ml/min, wavelength 306nm [9]. The samples for chromatography were filtered with a membrane filter (0.45 m) in advance.
RESULTS AND ANALISIS Unknown substances were found in Saperavi grape juice, which were not in evidence after the acid hydrolysis of the grape juice. Their chromatographic properties are shown in Table 1. One of the individual substances originated as a result of the acid hydrolysis of Saperavi grape juice, with its Rf (0.68), brownish orange coloration of lacquer, bluish violete illumination in the UV area and absorption maximum in the UV spectrum (300nm), is identified as trans resveratrol. Substance IV, through acid hydrolysis, forms trans resveratrol, and glucose is found in hydrolysate. This evidences the persence of trans resveratrol glucoside. Substance IV yields an absorption maximum of 300 350 nm in the UV area typical of stilbenoids of trans form. In particular, λ max (MeOH) – 308 nm; 335 nm. The study substance, after comparing the above mentioned properties with the present ones was identified as trans piceid (4 i,5 dihydroxystilbene 3 O β D glucopyranoside) (polydatine).
Table 1.1.1. Chromatographic properties of the study substance
Substance, Rf Lacquer Developer System N coloration
I 0.093 Brownish orange Diazotized Chloroform : sulfanilic acid methanol (80:20)
II 0.187 Brownish orange “ “ “ “
III 0.26 Yellow “ “ “ “
IV 0.39 Brownish orange “ “ “ “
V 0.49 Wine colored “ “ “ “
Grape
Juice Skin
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Air drying and crushing Self clearing Extraction with ethyl acetate Cleared juice Fraction with ethyl acetate Extraction with Concentration with a ethyl acetate rotational evaporator
Fraction with ethyl acetate Thickened extract
Concentration Mixing with adsorbent – “Sephadex – G25”, with a rotational Eluation with mixture MeOH:H 2O evaporator Stilbenoid -containing fraction – Plate preparation plate preparation
Thin -layer, liquid and preparation chromatograph Chromatograph analysis
Fig.1. Diagram of isolating stilbene containing fractions from grape juice and skin
Trans Piceid C20 H22 O8; Mr 390
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Cis Piceid C20 H22 O8
In ethyl acetate fractions of the grape juice of different varieties, a number of compounds were revealed by thin layer chromatography. Of them, Rf 0,26 corresponds to the study substance(III). As the diazotized sulfanilic acid appears, the substance yields a yellow spot. The study substance extracted individually and solved in methanol is characterized by maximum absorption at the wavelength of 285 nm. Glucose and aglycone were fixed in the hydrolyzate, which according to Rf 0, 65, spot painting and UV spectrum ( λ max(MeOH) 285 nm), are identified as cis resveratrol. Formation of cis resveratrol and glucose as a result of acid hydrolysis of the study substance, evidences the presence of the stilbenoid glucoside. The gained data, in particular, the typical area of maximal absorption (285 nm) and others, are the same as the literary data of the identification of cis piceid. Following the above mentioned, the study substance is identified as cis piceid. The red grape vine varieties were found to be different depending on the content of cis and trans piceid (Table 2).
Table 2. Content of cis and trans piceids in grape juice and skin
№ Variety Juice, mg/l Skin, mg/kg trans piceid cis piceid trans piceid cis piceid
1 Saperavi 12.40 5. 75 0.94 14. 21
2 Cabernet Sauvignon 6.96 2. 52 0.26 5. 32
3 Otskhanuri Sapere 5.11 3. 15 0.22 8. 76
4 Aleksandrouli 5.75 2. 27 0.28 4. 25
5 Mujuretuli 9.70 2. 11 0.30 2. 97
6 Shavkapito 4.35 1. 07 0.17 2. 53
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7 Tavkveri 3.82 0. 95 0.12 1. 63
8 Aladasturi 5.25 3. 57 0.46 12. 20
9 Dzelshavi 2.20 1. 72 0.18 2. 77
10 Ojaleshi 2.50 2.82 0.30 10.55
In addition, they were found to have a common feature the concentration of trans piceid in grape juice of each vine variety much exceeds that in the skin of varieties. Large amount of trans piceid is found in Saperavi grape juice (12.40 mg/l), while Saperavi grape skin contains an amount of trans piceid of all vine species (0.94 mg/kg). In the grape juice of red fruit varieties, the content of cis piceid varies within the range of 0, 95 5, 75 mg/l and it is 63 14, 21mg/kg in the grape skin. Vine varietal peculiarity was demonstrated in Georgian red wines while studying the trans resveratrol its derivative – dimer ε –––viniferin– and tetrameric stilben, also factors of Saperavi vine variety appellation region and winemaking technology was demonstrated, that is shown in the chart which compares the commercial type wines with the experiment wine that were clarified naturally (Table 3).
Table 3. Content of stilbenes (mg/l) in Georgian red wines
Experiment one year trans rezveratrol viniferin tetrameric stilben
Table dry
1.Sapearvi ( Kardenakhi) 2.56 0.81 1.22
2.Saperavi (Tsinandali) 2.13 0.88 1.92
3. Sapearvi (Napareuli) 2.35 0.78 1.59
4. Sapearvi budeshurisebri 2.01 0.63 0.84
5.Cabernet Sauvignon 1.26 0.52 0.96
6.Otskhanuri Sapere 2.23 0.65 1.12
7. Aladasturi 2.03 0.42 1.95
8.Asuretuli Shavi 0.9 0.51 0.75
9. Chakhveri 0.51 0.11 0.31
naturally s/s/
1.Saperavi (Akhasheni) 2.21 0.91 1.52
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2.Saperavi (Kindzmarauli) 1.87 0.66 1.32
3. Ojaleshi 2.05 0.53 1.87
4. Aleksandrouli 1.65 0.42 0.8
5. Mujuretuli 1.12 0.3 0.53
Commercial type wine :
1. Mukuzani dry 2.14 0.52 1.5
2. Kindzmarauli naturally s/s/ 1.5 0.41 1.01
3. Shumi dry 1.6 0.53 1.65
4 Shumi naturally s/s/ 1.21 0.38 0.93
5. Khvanchkara naturally s/s/ 0.9 0.32 0.62
6. Georgian Feast dry 1.7 0.55 1.37
7. Georgian Feast naturally s/s/ 1.4 0.49 1.11
8. Alazani Valley s/s/ 1.3 0.45 1.04
9. Biowine Saperavi
three years old naturally clarified 1.85 0.27 1.74
The stilben distribution was the same in Georgian red wine (experiment type and commercial type). Trans resveratrol is dominant, and then comes tetrameric stilben that is the dimeric product of the εεε ε viniferin and the last is εεε ε viniferin. According to the appellation region of Saperavi vine variety, Saperavi from Tsinandali region is characterized by low amount of trans resveratrol, but with the high concentration of its derivatives εεε ε viniefrin and tetrameric stilben. The intensive dimerisation of εεε ε viniferin of Saperai vine variety grown in Tsinandali region confirms this fact. Due to the technological processing the concentration of trans resveratrol and εεε ε viniferin are reduced. This fact is expressed by reduction of the stilbens in commercial type wines that is the result of technological processing. The intensive dimerisation of εεε ε viniferin and preservation of high concentration of tetrameric stilben in naturally clarified biological wines aged for three years also confirms this fact. We used stilbenoids from Saperavi, as one of the ingredients in the production technology of new products, such as biologically active food additive “Georgian Vitae rimas XXI” and alcoholic drink “Zigu+”. Their properties are given in the tables 4 6.
Table 4. Organoleptic characteristics of biologically active food supplement "Georgian Vitae rimas XXI"
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Name of characteristic Norm
External view Dense, syrup type liquid
Color Reddish brownish
Transparency Non transparent liquid
Taste and aroma Special
Table 5. Chemical characteristics of biologically active food supplement "Georgian Vitae rimas XXI"
Name of characteristics Number
Extracted substances gr/l 330 343
Total phenolic compounds gr/l 10 13
Proantocyanidins gr/l 8 11
Stilbenoids mg/ lTrans resveratrol 30 35 Trans piceid 16,0 17,5 ε viniferin 18 23 Tetrameric stilben 5 7 Catechins mg/l 700 900 among them: (+) catechins + ( ) epicatechin + (±)galocatechin + ( )epigalocatechin +
Color substances 1,995
Color intensivity (D 420 + D 520 +D 620 ) 29,85
K= D 420 /D 520 1,216
Phenolic acids and phenolic aldehyde among them are:
8 coniferyl aldehyde + 4 oxybenzo aldehyde + para cumaric acid + ferulic acid + vanillic acid + 4 oxybenzoic acid + lilac acid + caffeic acid + protocatechic acid + galic acid + other phenolic compounds acetovanillone (apocynin) mg/l 2 7 α conidedrin mg/l 5 8
Elements mg/l K 1008 Na 287,2 Ca 54,2 Mg 70,8 Fe 1,65
Ascorbic acid (Vita min C) not less than mg/l 50
Antiradical activity not less than % 130
The storage conditions should be considered in BASF technology. Particularly its should be stored in non transparent, dark, hermetically closed vessel at +5 0C for 6 months, after opening expiration time of BASF is one month.
Table 6. Physical chemical characteristics of alcoholic drink “Zigu+”
Name of indicator norm
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Physical evidence trasnparent liquid
Color red
Taste and Aroma Harmonical, typical for phenolic compounds
Alcohol volume % 18 20
Tartaric acidity g/l 6 – 7
Share of sugar mass % 16 18
Extract g/l, no less 165
Total phenolic compounds g/l no less 3.0
Total coloring substances mg/l, no less 500
trans resveratrol. mg/100ml, no less 3.0
ε viniferin mg/100ml, no less 1.0 antioxidant activity (%) 52
We identified the transformation of the stilbenoids of Saperavi identified by us in wine by studying the different parts of Saperavi. The identified transformation is shown in Fig. 2. Trans resveratrol was identified in Saperavi stem by us for the first time. Red wines are made though boiling the pomace without stem. However, if any new bulk wine is made with stem, then trans resveratrol is transported from it.
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Juice trans – Piceid
cis - Piceid
Seed Stem Red Wine Oligomeric trans - Stlbenes Resveratrol
ss Skin
trans- Resveratrol; cis- Resveratrol;ε- Viniferin;
Fig.2. Transformation of Stilbenoids from grapevine into red Wine CONCLUSION The content of the biologically active stilbenoids was identified in the red grape vine varieties (Vitis vinifera L.) growing in Georgia was identified through studies; transformation of stilbenoids from grape into wine was also fixed. Vine stilbenoids, in addition to wine, may be used to produce the biologically active products.
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ТРАНСФОРМАЦИЯ БИОЛОГИЧЕСКИ АКТИВНЫХ СТИЛЬБЕНОИДОВ ИЗ ВИНОГРАДА В КРАСНОМ ВИНЕ, ПИЩЕВОЙ ДОБАВКДОБАВКЕЕ И НАПИТКЕ
М.Г. Бежуашвили, П.Н. Вашакидзе, Н.Г. Вепхишвили, Л.Д. Эланидзе
В виноградном соке и кожице винограда красноягодных сортов, распространенных в Грузии, идентифицированы и определены стильбеноиды цис и транс пицеид. В эксперименте использованы сорта винограда Саперави, Каберне совиньон, Оцханури сапере, Александроули, Муджуретули, Шавкапито, Тавквери, Аладастури, Дзелшави, Оджалеши. Идентификация препаративно выделенных веществ проведена на основе их кислотного гидролиза, тонкослойной и высокоэффективной жидкостной хроматографии, УФ спектроскопии. Оказалось, что транс пицеид в большем количестве содержится в виноградном соке, а цис пицеид – в кожице винограда. Из гребней винограда сорта Саперави (Vitis vinifera L.) впервые идентифицирован транс резвератрол. На основе полученных данных определена трансформация идентифицированных нами стильбеноидов винограда в красном вине. Нами обработаны технологии приготовления стильбеноидсодержащих биологически активных продуктов виноградного происхождения.
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