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Color stabilisation properties of oenological tannins during wine ageing

A. Vignault, et al.; Université de Bordeaux, France | Universitat Rovira i Virgili, Spain

Vignault, A1,2., Gomez-Alonso, S3., Jourdes, M1., Canals, J.M2., Zamora, F2., Teissedre, P-L1

Université de Bordeaux, Unité de recherche Œnologie, EA 4577, USC 1366 INRAE, ISVV, 33882 Villenave d’Ornon cedex, France.
Departament de Bioquímica i Biotecnología, Facultat d’Enologia de Tarragona, Universitat Rovira i Virgili, C/Marcel.li Domingo 1, 43007 Tarragona, Spain.
Instituto Regional de Investigación Científica Aplicada, Universidad de Castilla-La Mancha, Ciudad Real, España

Email: adeline.vignault@gmail.com

Article extracted from the presentation held during Enoforum Web Conference (23-25 February 2021)

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Introduction

Anthocyanins are the main compounds responsible for the color of red wines. They are the major natural pigments in red wines, reaching typically 500 mg/L in young red wines (Burns et al., 2002), but their concentration depends on the grape variety and the growing conditions (Budić-Leto et al., 2009). 

In red wines from Vitis vinifera grapes, the main monomeric anthocyanins are the malvidin-3-O-glucoside, cyanidin-3-O-glucoside, delphinidin-3-O-glucoside, pelargodinin-3-O-glucoside, peonidin-3-O-glucoside and petunidin-3-O-glucoside (Castillo-Muñoz et al., 2009; Trouillas et al., 2016). These anthocyanins are also present in acylated forms with acetic, coumaric and caffeic acids (He et al., 2012). More derivatives of anthocyanins can be extracted or formed during the fermentation process (Busse-Valverde et al., 2011). The extraction and formation of new pigments or copigment complexes permit the color stabilization of red wine during the aging process since the monomeric anthocyanin content decreases constantly. 

Different phenomena can occur naturally to stabilize the color in red wines. Anthocyanins can form association structures with other colorless compounds (intermolecular copigmentation), with other anthocyanins (self-association), or in the case of coumaroylated and cafeoylated anthocyanins, the acylated esterified group can be associate with the pyrylium ring of the same anthocyanidin molecule (intramolecular copigmentation) (Boulton, 2001; Eiro and Heinonen, 2002).
The copigmentation phenomenon is characterized by the formation of a “sandwich complex,” meaning an hydrophobic interaction (π-π stacking) between the B ring of the anthocyanin and the copigment (phenolic compounds, for example) (Boulton, 2001). These complexes adopt a sandwich-like structure that protects the flavylium cation against nucleophilic attack  by water, avoiding the formation of the colorless hemiketal form (Santos-Buelga and De Freitas, 2009). Consequently, copigmentation increases wine color intensity (hyperchromic effect), but it can also change the color hue through a bathochromic shift (Clifford, 2000).
Finally, the formation of new pigments can occur, providing a large variety of pigments such as pyranoanthocyanins and polymeric anthocyanins (He et al., 2012). 
The mechanism of pigment formation or copigmentation differs with respect to the botanical origins of oenological tannins since they present different chemical structure. In this way, condensed tannins can combine directly or indirectly with anthocyanins while hydrolysable tannins cannot participate in condensation reactions with anthocyanins. However, hydrolysable tannins, can participate in copigmentation reactions, as well as protect wine anthocyanins from oxidation since they may regulate oxidation-reduction phenomena happening in wines (Zamora-Marín, 2003).

Among the functions attributed to oenological tannins, their enhancing effect on red wine color and stability is probably one of the main reasons for their potential use in winemaking. Red wine color plays a very important role for consumers in their acceptance and perception of the product (Wrolstad et al., 2005). For this reason, winemakers are interested in gaining a better understanding of the role played by polyphenols, and more particularly, the interactions between tannins/anthocyanins to produce deeply colored wines with great color stability during aging (Wrolstad et al., 2005). To achieve this goal, winemakers apply different oenological practices, for example, “reduced volume of juice” or thermovinification (Sacchi et al., 2005), addition of pectinolytic enzymes (Schwarz et al., 2005) and oak aging. Despite the various tools available to winemakers, none of these treatments have provided clear results without secondary problems or effects. The addition of oenological tannins has been proposed in the past, but due to the abundant diversity, their effects are not so well studied and clear. 

For these reasons, the aim of this work was to give model standards in terms of copigmentation proportion and level for the major anthocyanin of wine, malvidin-3-O-glucoside. Additionally, this publication aimed to verify and confirm the effectiveness of different botanical origin of oenological tannins on wine color stability to be applied as a new tool by winemakers.


Experimental methods

Five oenological tannins (quebracho, ellagitannin, gallotannin, grape-skin and grape-seed) were used to determine the influence of the time contact on the copigmentation effect. A model wine solution (12% vol. of ethanol, 4 g/L of tartaric acid and adjusted at pH 3.5) was prepared and supplemented with 0.1, 0.2 and 0.4 g/L of each commercial tannins and (-)-epicatechin (copigments). The (-)-epicatechin was used as reference standard.

Simultaneously, solutions containing 50 mg/L of malvidin-3-O-monoglucoside (pigment) and 0.1, 0.2 and 0.4 g/L of commercial tannins or (-)-epicatechin were prepared to reach copigment/pigment ratios of 2, 4 and 8 respectively. 

Finally, a solution containing only the malvidin-3-O-monoglucoside was prepared as positive control. Then, 1.5 mL of each solution was placed in Eppendorf tubes and maintained under airtight conditions. After 1, 7, 14 and 21 days, the full absorbance spectrum in the visible range (400-800 nm) was measured to determine the CIELAB coordinates.

In all cases, the spectrum of the solution containing only the copigment (oenological tannins or (-)-epicatechin) was subtracted from the spectrum of the corresponding mix copigment/pigment (oenological tannins/malvidin or (-)-epicatechin/malvidin). This subtraction was made to avoid the interferences due to the natural color of each copigment.

To determine the copigmentation index, the following equation (Equation 1) was proposed as an index to measure comparatively the effectiveness as copigments of the different commercial tannins.

Equation 1: Equation of the copigmentation index and the difference of color between two samples (ΔEab)

ΔEab x CS is the total color difference between the control solution malvidin-3-O-monoglucoside (CS) and a pure white color solution.
ΔEab x TS is the total color difference between the solution of malvidin-3-O-monoglucoside containing 0.4 g/L of commercial tannin (TS) and a pure white color solution. 
The CIELAB coordinates of a pure white color solution are L* = 100.00, a* = 0.00 and b* = 0.00.

The malvidin-3-O-monoglucoside and its degradation products were purchased and quantified using a 1260 Infinity high performance liquid chromatography system coupled to a diode array detector (DAD) and a 6545 quadrupole-time of flight (Q-TOF) mass spectrometer detector (Agilent, Waldbronn, Germany). The control software was MassHunter Workstation (version B.08.00). The Q-TOF used a Dual Jet Stream Electrospray Ionization (Dual AJS-ESI) source operated in the positive ionization mode and the following parameters were set: capillary voltage, 3500 V; fragmentor, 100; gas temperature, 300 °C; drying gas, 9 L/min; nebulizer, 40 psi; sheath gas temperature, 400 °C; sheath gas flow, 10 L/min; acquisition range, 100-1700 m/z; and fixed collision. Samples were analyzed by injection (20 μL) on a ZORBAX Eclipse XDB-C18 (2.1 × 150 mm; 3.5 μm particle, Agilent USA, Santa Clara-California), at 40 °C.

The solvent system, at a flow rate of 0.3 mL/min, was water acidified with 1% of formic acid (solvent A) and methanol with acetonitrile acidified with 1% of formic acid (30/70/1%; v/v) (solvent B). The elution gradient was (time, % of solvent A): 0 min, 99.0%; 2 min, 98.5%; 25 min, 97.0%; 45 min, 65.0%; 55 min, 20.0%; 60 min, 95.0% and then, 10 min equilibrium time was left between analysis. Quantification of malvidin-3-O-glucoside was realized using a calibration curve of malvidin-3-O-glucoside at 0, 0.25, 0.5, 0.75 and 1 g/L. Quantification of degradation products was realized using a calibration curve of syringic acid at 1, 2.5, 5, 10, 20 and 30 mg/L and malvidin-3-O-glucoside at 84 mg/L was used as external standard. All samples were analyzed in triplicates.


Discussion of the results 

The red-greenness (a*), yellow-blueness (b*) and lightness (L*) components of the solutions of malvidin-3-O-glucoside enriched with increasing concentrations of (-)-epicatechin and oenological tannins are displayed in Figure 1. Positive values of a* are in the direction of redness and negative values in the direction of the greenness. Positive values of b* are in direction of “yellowness”, and negative for “blueness”. Finally, low values in L* point towards black while high values point towards white (Wrolstad et al., 2005).

Figure 1: CIELAB color space of model wine solution of malvidin-3-O-glucoside (50 mg/L) supplemented by (-)-epicatechin and oenological tannins after 1, 7, 14 and 21 days of experimentation

In general, all tannins yielded a positive effect as copigments. Clear differences in effects were observed between oenological tannins. At day 1, gallotannins are clearly better copigments because their hyperchromic (decrease of L*) and bathochromic (decrease of b*) effects are considerably higher than the other ones. 
During the time, all the tannins except ellagitannins seems to present a quite stable copigmentation effect since the a* and b* parameters remains stable. Ellagitannins, presented a negative bathochromic effect with an important increase of b* during the time. 

The Copigmentation Index (Equation 1) proposed, allowed to measure the real effectiveness of commercial tannins and proposed a new tool for tannin manufacturers and potential consumers (winemakers) (Gombau et al., 2019). This index considers the total color difference (ΔEab) between an oenological tannin solution and a pure white color solution (L* = 100.00, a* = 0.00, b* = 0.00). Indeed, this is the best parameter to determine the real differences between the color of two solutions since it reflects the Euclidian distance between two point in the CIELAB space. This index considers also, the percentage of increase of ΔEab originated by the supplementation of the highest dose of tannins (0.4 g/L) to facilitate its quantification.

Figure 2 shows the results obtained for the copigmentation index of the different commercial tannins and (-)-epicatechin at 1, 7, 14 and 21 days. This copigmentation index indicate in a simple way the effectiveness of the different commercial tannins and (-)-epicatechin (reference) to improve wine color stabilization. This improve is mainly due to the copigmentation phenomenon, even if the formation of polymerized pigments can also take place.

Figure 2: Copigmentation index (%) of the oenological tannins and (-)-epicatechin at 1, 7, 14 and 21 days

At day 1, according to this index, the best copigment was the gallotannin (35.4 ± 1.2%) followed in decreasing order by ellagitannin (15.3 ± 1.3%), (-)-epicatechin (11.1 ± 0.6%) and then grape-skin (3.9 ± 1.2%), grape-seed (3.1 ± 0.8%) and quebracho tannins (3.9 ± 0.2%). 
Then on day 7, only the copigmentation index of (-)-epicatechin, gallotannin and quebracho tannin remain stable, since no significant differences were observed. On the contrary, in the case of ellagitannin, grape-skin and grape-seed tannin considerable differences were observed. Concerning ellagitannin, the copigmentation index decreases meanwhile for grape-skin and grape-seed tannins the copigmentation index increase. Nevertheless, the order of classification of the tannins as copigments was not changed by this decline or augmentation of the values. Indeed, gallotannin still remain the most efficient one followed by ellagitannin,   (-)-epicatechin and then grape-skin, grape-seed and quebracho tannins. 
On day 14, only gallotannin and quebracho tannin remain stable compared to day 1 and 7. Grape-seed and grape-skin tannins, stand constant compared to day 7 with an increase of the index compared to day 1. To the opposite copigmentation index of ellagitannin continue to decrease on day 14, becoming close to 0%. In the case of the (-)-epicatechin, used as reference, on day 14, a diminution of the copigmentation index start to be observed. According to this, on day 14, the effectiveness of the commercial tannins was changed, and the classification removed. Indeed, gallotannin remain the most efficient one, but this time followed by grape-skin and grape-seed tannins, quebracho tannin and finally (-)-epicatechin and ellagitannin. 
On day 21, for the first-time significant differences were observed for the gallotannin, with a slow decrease of the copigmentation index. Ellagitannin, grape-skin, grape-seed and quebracho tannin remain stable compared to day 14 meanwhile, (-)-epicatechin copigmentation index continue to decrease. At the end on day 21, gallotannin (27.4 ± 2.3%) stay the most efficient one, followed by grape-skin (7.1 ± 1.1%) and grape-seed tannins (6.5 ± 0.2%). Ellagitannin (0.0%), (-)-epicatechin (0.0%) and quebracho (0.0%) tannin were the lowest efficient ones.
These results are in accordance with the previous ones obtained for analysis of color component in which it has been shown that throughout the time gallotannin outstanding the most efficient one meanwhile ellagitannin presented a decrease of his hyperchromic and bathochromic effects.
The fact, that the copigmentation index of (-)-epicatechin and ellagitannin decrease significantly during the time from day 1 to day 21 can be explained by different phenomenon. Indeed, this decrease suggests that the copigmentation effectiveness of these tannins decline with the time or that the malvidin-3-O-glucoside has reacted with tannins to form new polymerized pigments with a lower contribution to color. In this regard, it has been reported that colorless pigments were formed in solutions containing malvidin-3-O-glucoside and oak extract and that the ellagitannin concentration decreases over time (Jordão et al., 2008). Even more so, malvidin-3-O-glucoside could be degraded with the time and formed degradation product colorless. 
In contrast, the copigmentation of grape-seed and grape-skin tannins increase significantly during the time. This could be explained by the fact that, the stacking structures responsible of copigmentation needs some time to be completely formed or, because of polymerized pigments with a higher contribution to the color have been formed (Dueñas et al., 2006; Malien-Aubert et al., 2001). Regarding quebracho tannin, it appears to be weak copigment since the copigmentation index was stable during the time but in any case, low. Concerning gallotannin, it is possible that the complex formed between them and the malvidin-3-O-glucoside is enough stable to be conserved during the time.

In order to confirm these hypotheses, the characterization and quantification of malvidin-3-O-glucoside and its degradation products was achieved, in order to understand and better explained the previous results obtained.
Indeed, the degradation of the anthocyanin could be a part of the explication and even more so explained the differences between the oenological tannins, regarding color stabilization. Two products corresponding to the degradation of the malvidin-3-O-glucoside were found. ​​​
These products were identified as the syringic acid and the formylphloroglucinol (also called 2,4,6-Trihydroxybenzaldehyde or phloroglucinol aldehyde). Various authors have also found these two compounds as degradation products of the malvidin-3-O-glucoside under different conditions (thermal degradation or enzymatic degradation mainly) (Keppler and Humpf, 2005; Piffaut et al., 1994; Zhao et al., 2013).
According to this, Figure 3 shows the concentration of malvidin-3-O-glucoside, formylphloroglucinol and syringic acid in the solution of malvidin added by the different copigments. In addition, to better visualize the results, the concentration of formylphloroglucinol and syringic acid were summed as the total concentration of degradation products. Only the results with the ratio Cp/p = 8 are presented, since it’s the most significant conditions to observe the differences between the different copigments. 
First, the malvidin-3-O-glucoside in model wine solution without any presence of copigments, present also a slight decrease of its concentration associated to the formation of degradation products. Additionally, the first day of the experiment was used as the control for determine the lost in malvidin-3-O-glucoside and the gain in the formation of degradation products compared to the other days of the experimentation.

Figure 3: Concentration in malvidin-3-O-glucoside loss and products of degradation gain in model wine solution with 50 mg/L added by different copigments (Cp/p =8) at different days of the experimentation

In all cases, as expected, the concentration in the anthocyanin decrease and the total concentration in products of degradation increase during the experimentation. Additionally, it should be highlighted that in general, syringic acid is preferentially formed than the formylphloroglucinol.  Nevertheless, in the case of the (-)-epicatechin, both products are formed in the same quantities and in the case of grape-seed tannin, the formylphloroglucinol was formed preferentially. 
Concerning grape-skin tannin, quebracho tannin and ellagitannin, difference between the loss of the anthocyanin and the gain in products of degradation was significant since the seventh day of the experimentation until the last day.
Regarding (-)-epicatechin the difference was significant only at the day 7 meanwhile for gallotannin the difference was significant only on the last day of the experimentation.
Finally, the concentration loss of the anthocyanin differs according to the copigment present in solution and the day of the experimentation. Nevertheless, the total concentration in the degradation products formed was similar for all the copigments tested. These results mean, that the differences noted in the loss of malvidin-3-O-glucoside in presence of the different copigment cannot be attributed to the degradation products. Indeed, the formation of these products explained a part of the loss of the anthocyanin but less than 33% and even more so, less than 70% in presence of ellagitannin. According to this, the rest of the anthocyanin loss can be involved in other reactions as the formation of polymerized pigments.
The only exception, is the gallotannin which present significant differences only the last day, meaning that probably no other reactions additionally to copigmentation come into play. 
Indeed, most of the malvidin-3-O-glucoside loss is explained by the formation of the degradation products meaning that malvidin-3-O-glucoside cannot be involved in other mechanisms. 
Finally, it should be highlighted that the maximum absorption of these two compounds (syringic acid and formylphloroglucinol) are 278 and 294 nm respectively, corresponding to colorless products (Zhao et al., 2013). This explained the changes noted in the color analyzes in which it has been observed in all cases a slight diminution of the color parameters. Indeed, the formation of this compounds, even if in few quantities, induce a quite diminution of the color of the solutions containing malvidin-3-O-glucoside and oenological tannins.  


Conclusions 

These results allowed us to conclude that botanical origin of oenological tannins influence their effectiveness regarding color stabilization. Indeed, hydrolysable tannins and more specifically gallotannins seem to be the most efficient compounds to stabilize the color of red wines during the aging by copigmentation. In contrast, condensed tannins seem to be weaker regarding the protection of the color by copigmentation even if grape-skin and grape-seed tannins appear to be more efficient than quebracho tannins. 
Nevertheless, the experimentation conducted at different time of contact between oenological tannins and malvidin-3-O-glucoside (1, 7, 14 and 21 days), enlighten us about the real effectiveness over time of the oenological tannins. 
Indeed, during all the time of the experiment, gallotannin still remain the most efficient one to improve color stabilization by having a great hyperchromic and bathochromic effect which is in accordance with the previous results obtained. On the contrary, ellagitannin appears at the beginning as a great candidate to improve the color stabilization but its effect during the time change conducting to a poor efficiency at the end. Grape-seed and grape-skin tannins, contrary to ellagitannins see their effects increase with the time of contact with the malvidin-3-O-glucoside. Finally, quebracho tannin, independently of the time contact remains as the lowest efficient one.
To conclude, oenological tannins appear as great candidate to stabilize the color of red wines and help to their improvement. More specifically, gallotannins are the candidate who should be used in priority, since its effect remain constant during the time and with the highest efficiency.
In the future, it should be great and helpful to determine, found and quantified the different polymerized pigments formed between oenological tannins and anthocyanins. 


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Published on 11/01/2022
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