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Ellagitannins

OENOLOGICAL TANNINS

Specific monograph for oenological tannins containing ellagitannins

OIV-OENO 675B-2022

Ellagitannins are a sub-class of hydrolysable tannins. Chestnut and oak tree tannins are included in this sub-class.

  1.                  Method for the determination of sub-class affiliations
    1.           Characterisation by high-performance liquid chromatography (HPLC)
      1.   Principle

This method is designed to verify the presence of ellagitannins in oenological tannins and measure their total concentration.

  1.   Reagents, materials and apparatus
    1.                    Reagents

Vescalagin (purity > 96%), CAS No. 36001-47-5

Ultrafiltered water (resistivity: 18.3 MΩ·cm)

Water (HPLC quality)

Methanol (HPLC quality)

Formic acid (HPLC quality)

  1.                    Materials

100-mL borosilicate-glass flask

Cellulose filters with 0.45 µm pore size diameter

Plastic 1-mL syringe

  1.                    Apparatus

Technical balance with precision of 0.01 g

Analytical balance with precision of 0.1 mg

Class-A volumetric glassware

Mass chromatographic system with spectrometry detection composed of:

-                      Gradient pump for binary or quaternary mix

-                      Injector with a loop of 10 µL

-                      Spectrophotometric detector at 280 nm fixe wavelength

-                      Column Phenomenex Kinetex (for example): 150 x 3.0 mm, 2.6 µm particle size

-                      ESI-SIM (Single Ion Monitoring mode via Electro Spray Ionisation) ionisation source

-                      Mass spectrometer detector: triple quadrupole time of flight (Q-Tof)

  1.   Preparation of samples and standards

Samples: weigh approximately 0.5 g of oenological tannin on the analytical balance and make a note of the weight. Dissolve the oenological tannin in 100 mL of ultrafiltered water in a 100-mL borosilicate-glass flask and mix well.

Preparation of standard solutions: put 10 mg of vescalagin in solution into 50 mL of ultrafiltered water, corresponding to a 200 mg/L concentration. Then carry out dilutions in ultrafiltered water to obtain 0.1, 0.2, 0.5, 1, 2, 5, 10, 20, 40, 50 and 100 mg/L concentrations for the calibration curve.

Solvent A: water (HPLC quality) containing 0.1% of formic acid (HPLC quality).

Solvent B: methanol containing 0.1% of formic acid (HPLC quality).

  1.   Procedure

Oenological tannin solution and standard solutions are filtered on 0.45 µm (pore size diameter) filters and analyse by chromatography under the following conditions given by way of example:

 Injected volume: 10 µL of oenological tannin solution or standard solution of vescalagin

 Detection at 280 nm

Composition of elution gradient: (time, % of solvent A)

0min, 99.0%; 2 min, 98.0%; 5 min, 97.0%; 6 min, 96.5%; 7 min, 96.0%; 8 min, 95.5%; 10 min, 95.0%; 14 min, 90.0%; 17 min, 85.0%; 23 min, 80%; 35 min, 1.0% and 10 min for equilibrium.             

Flow rate: 0.4 mL/min

Quantification and detection of the eight principal ellagitannins (vescalagin, castalagin, roburin A, B, C, D, E and grandinin) according to DAD (UV 280 nm) or the ESI-SIM scan with Q-ToF detection, which allows for more precise detection and quantification.

Figure 1: Example ellagitannin chromatogram at 280 nm

  1.           Conclusion

An oenological tannin is recognised as an ellagitannin (or ellagic tannin) when:

-                      its total polyphenol content is higher than 65% (gravimetric method in Annex 1 of the general monograph OIV-OENO 624-2022),

-                      its ellagitannin content is higher than 200 mg equivalent gallic acid per gram of oenological tannins (characterised by the HPLC method).

  1.                  Methods of measurement of properties and functionalities

 

The following compliance methods and criteria are only applicable when the property/functionality is claimed on the preparation of tannins.

 

 

  1.           Antioxidant ability
    1.   Principle

Determination of ellagitannins’ antioxidant ability to contribute to the protection of must and wine from oxidation.

  1.   Products
    1.                    Antioxidant capacity

DPPH (2,2-diphenyl-1-picrylhydrazyl): MM = 394.32

Trolox (6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid): MM = 250.29

Methanol at 99.9% volume

96-well microplate reader (FLUOstar Omega - BMG Labtech, for example)

  1.                    Direct oxygen consumption (OCR)

Ethanol at 96% volume, CAS No. 64-17-5

Tartaric acid: MM = 150.09, CAS No. 87-69-4

Iron (III) chloride hexahydrate: MM = 270.30, CAS No. 7705-08-0

Copper (II) sulfate pentahydrate: MM = 249.68, CAS No. 7758-98-7

Clear glass bottles with inserted pills of 0.75-L capacity

NomaSens oximeter (for example)

  1.   Protocols
    1.                    Antioxidant capacity (DPPH assay)

0.15 g/L oenological tannin solution: dissolve 37.5 mg of oenological tannins in 500  mL of model wine solution (distilled water, 12% vol. of ethanol, 4 g/L of tartaric acid and pH adjusted to 3.5). Dilution of oenological tannins solution could be needed if the measurement absorbance is higher than 1 unit (in this case the dilution should be included in the calculation).

1mM Trolox solution: dissolve 125 mg of Trolox in 500 mL of model wine solution (distilled water, 12% vol. of ethanol, 4 g/L of tartaric acid and pH adjusted to 3.5).

Calibration curve:  dissolve in 1, 0.8, 0.6, 0.4, 0.2 and 0.1 mL of 1mM Trolox solution into 0, 0.2, 0.4, 0.6, 0.8 and 0.9 mL of model wine solution. These quantities correspond to 1, 0.8, 0.6, 0.4, 0.2 and 0.1 mM final concentration of Trolox respectively.

6.10-5 M DPPH solution: dissolve 2.36 mg of DPPH in 100 mL of methanol. The solution should be freshly prepared.

  1.                    Direct oxygen consumption (OCR)

1 g/L oenological tannin solution: dissolve 0.75 g of oenological tannins in 750 mL of model wine solution.

Model wine solution: dissolve 4 g of tartaric acid, 2.25 mg of Iron (III) chloride hexahydrate and 0.225 mg of Copper (II) sulfate pentahydrate in 90 mL of ethanol and 660 mL of distilled water. The pH should be adjusted at 3.5.

  1.   Tests
    1.                    Antioxidant capacity

First a blank containing solely the DPPH reagent (RB) is measured at 515 nm by placing 190 µL of DPPH solution (1.3.1) in all the wells of the plate. Then, add 10 µL of oenological tannin solution (samples), distilled water (blank) or Trolox curve solution (standards) into the wells and measure (MS) at 515 nm after 30 min.

See Figure 2 for an example of how to fill the plate.

The formula to be applied for the calculation of the antioxidant capacity is as follows:

  1.                 
  2.                 

where “a” and “b” correspond respectively to the slope and the constant of the Trolox calibration curve: Absorbance = f ([Trolox])  Absorbance = ax + b

 

In all cases, ellagitannins (or ellagic tannins) should demonstrate an antioxidant capacity, and more specifically they should have more than 600 ± 50 mg equivalent Trolox per gram of tannins (commercial extract).

Figure 2: example 96-well plate

 

  1.                    Direct oxygen consumption (OCR)

First the model wine solution is saturated with oxygen at 8 mg/L by bubbling with air for 10 min at 20-25 °C. Then, add the oenological tannins to the model wine solution in the bottles filled to 0.75 cL. Sealed the bottles hermetically and shake to fully homogenise.

  1.               Measure the oxygen consumed every two days starting 1h after the filled of the bottles.
  2.               To determine the oxygen consumption rate, follow the pathway as shown in Figure 2: 
    •                   represent the oxygen consumption versus the time,
    •                   then represent the inverse of the oxygen consumed versus the inverse of the time,
    •                   the oxygen consumption rate corresponds to the inverse of the slope coefficient:

OCR t0 mg of O2 per L consumed per day and per g of tannins = 1/A, A being the slope coefficient

In all cases, ellagitannins (or ellagic tannins) should demonstrate an ability to consume the oxygen directly, and more specifically they should be able to consume at least 0.50 ± 0.05 mg of O2 per litre, per day and per gram of tannins (commercial extract).

 

 

Figure 3: Pathway to determine oxygen consumption rate

  1.           Antioxidasic ability
    1.   Principle

Determination of ellagitannins’ antioxidasic ability to contribute to antioxidasic protection in terms of the laccase activity of compounds in must and wine.

  1.   Products

Ethanol at 96% volume, CAS No. 64-17-5

Tartaric acid: MM = 150.09, CAS No. 87-69-4

Sodium acetate: MM = 82.03, CAS No. 6131-90-4

Syringaldazine (4-hydroxy-3,5-dimethoxybenzaldehyde azine): MM = 360.36, CAS No. 14414-32-5

Polyvinylpolypyrrolidone: PVPP, CAS No. 25249-54-1

Must botrytised with laccase activity

Distilled water (HPLC quality)

  1.   Protocols

2 g/L oenological tannin solution: dissolve 200 mg of oenological tannins in 100 mL of model wine solution (distilled water, 12% vol. of ethanol, 4 g/L of tartaric acid and pH adjusted to 3.5).

Buffer solution (8.2 g/L): dissolve 410 mg of sodium acetate in 50 mL of distilled water.

Syringaldazine solution (0.06 g/L): dissolve 30 mg of syringaldazine in 500 mL of ethanol.

  1.   Tests
  1.                  Add 4 mL of botrytized must to 1 mL of oenological tannin solution in tube, which will correspond to sample modality.
  2.                  Add 4 mL of botrytized must to 1 mL of model wine solution in tube, which will correspond to control modality.
  3.                  After 4 minutes (precisely), add 0.8 g of PVPP in both tube (sample and control modalities), stirred and centrifuged for 10 minutes at 8,500 rpm.
  4.                  Recover 1 mL of the supernatant (for both sample and control modalities), into 1.4 mL of buffer solution and 0.6 mL of syringaldazine solution. Put the mixture into a plastic spectrophotometer cuvette (10 mm path length).
  5.                  Measure the absorbance at 530 nm every minute for 5 minutes (including time measurements at 0 minutes).
  6.                  Then determine the laccase activity and the residual laccase activity by using the following equations and Figure 3:

𝐿𝑎𝑐𝑐𝑎𝑠𝑒 𝑎𝑐𝑡𝑖𝑣𝑖𝑡𝑦 = 46.15 𝑥 Δ𝐴 µ𝑚𝑜𝑙. 𝐿−1. 𝑚𝑖𝑛−1 = 46.15 𝑥 Δ𝐴 𝑈𝐿

% 𝑜𝑓 𝑟𝑒𝑠𝑖𝑑𝑢𝑎𝑙 𝑎𝑐𝑡𝑖𝑣𝑖𝑡𝑦 = (𝑙𝑎𝑐𝑐𝑎𝑠𝑒 𝑎𝑐𝑡𝑖𝑣𝑖𝑡𝑦𝑠𝑎𝑚𝑝𝑙𝑒/ 𝑙𝑎𝑐𝑐𝑎𝑠𝑒 𝑎𝑐𝑡𝑖𝑣𝑖𝑡𝑦𝑐𝑜𝑛𝑡𝑟𝑜𝑙) 𝑥 100

Figure 4: Example determination of .

In all cases, ellagitannins (or ellagic tannins) should demonstrate an antioxidasic ability, and more specifically they should be able to reduce the residual laccase activity by at least 40%. This value is valuable for must and wine containing less than 5 UL (units of laccases).

 

  1.           Colour stabilization
    1.   Principle

Determination of ellagitannins’ colour stabilisation properties to promote the expression, stabilisation and preservation of colour in red must and wine.

  1.   Products

Ethanol at 96% volume, CAS No. 64-17-5

Tartaric acid: MM = 150.09, CAS No. 87-69-4

Malvidin-3-O-glucoside: MM = 528.87, CAS No. 18470-06-9

  1.   Protocols

0.8 g/L oenological tannin solution: dissolve 80 mg of oenological tannins in 100 mL of model wine solution (distilled water, 12% vol. of ethanol, 4 g/L of tartaric acid and pH adjusted to 3.5).

0.1 g/L malvidin-3-O-glucoside solution: dissolve 10 mg of malvidin-3-O-glucoside in 100 mL of model wine solution (distilled water, 12% vol. of ethanol, 4 g/L of tartaric acid and pH adjusted to 3.5).

  1.   Tests
  1.                  Place 0.75 mL of oenological tannin solution and 0.75 mL of model wine solution in one  2-mL stoppered conical tube – hereinafter a “tube” – and keep it in the dark at room temperature. This tube will be called “T0”.
  2.                  Place 0.75 mL of malvidin-3-O-glucoside solution and 0.75 mL of model wine solution in one tube and keep it in the dark at room temperature. This tube will be called “M”.
  3.                  Place 0.75 mL of oenological tannin solution and 0.75 mL of malvidin solution in one tube and keep it in the dark at room temperature. This tube will be called “TM”.
  4.                  After 7 days, measure the absorbance at 450, 520, 570 and 630 nm of the three tubes (TM, T0 and M).
  5.                  Subtract the absorbance values of T0 to TM to obtain the absorbance avoiding the interferences due to the “natural” colour of the oenological tannin.

A(TM) – A(T0) = A(T)

  1.                  Then, determine the CIELAB coordinates (L*, a* and b*) corresponding to the tannin solution with malvidin-3-O-glucoside (T) and malvidin-3-O-glucoside solution (M) with the free MSCV software (https://www.unirioja.es/color/descargas.shtml) or equivalent.

The formulas to be applied for the calculation of the copigmentation index are as follows:

ΔEab.TS: total colour difference between the solution of malvidin-3-O-glucoside containing commercial tannins (T) and a pure white colour solution (W).

ΔEab.CS: total colour difference between the solution of malvidin-3-O-glucoside (M) and a pure white colour solution (W).

The CIELAB coordinates of a pure white colour solution are L* = 100.00, a* = 0.00 and b* = 0.00.

In all cases, ellagitannins (or ellagic tannins) should demonstrate an ability to stabilise the colour, and more specifically their copigmentation index should read as higher than 10.0 ± 2.0% after 7 days.

 

Note: Alternative methods of determination can be used in place of any of the methods described, on the condition that these have been internally validated.

 

  1.                  Bibliography
  •                   Sarneckis, C.J.; Dambergs, R.G.; Jones, P.; Mercurio, M.; Herderich, M.J.; Smith, P.A. Quantification of condensed tannins by precipitation with methyl cellulose: development and validation of an optimised tool for grape and wine analysis. Australian Journal of Grape Wine Research 2006, 12, 39–49.
  •                   Vignault, A.; González-Centeno, M.R.; Pascual, O.; Gombau, J.; Jourdes, M.; Moine, V.; Iturmendi, N.; Canals, J.M.; Zamora, F.; Teissedre, P.-L. Chemical characterization, antioxidant properties and oxygen consumption rate of 36 commercial oenological tannins in a model wine solution. Food Chemistry 2018, 268, 210–219.
  •                   Vignault, A.; Pascual, O.; Jourdes, M.; Moine, V.; Fermaud, M.; Roudet, J.; Canals, J.M.; Teissedre, P.-L.; Zamora, F. Impact of enological tannins on laccase activity. OENO One 2019, 53, 27–38.
  •                   Vignault, A.; Pascual, O.; Gombau, J.; Jourdes, M.; Moine, V.; Canals, J.M.; Teissedre, P.-L.; Zamora, F. Recent advances of the OIV working group on oenological tannins in the study of the functionalities of oenological. BIO Web of Conferences 2019, 15, 02015.
  •                   Vignault, A.; Gombau, J.; Pascual, O.; Jourdes, M.; Moine, V.; Canals, J.M.; Zamora, F.; Teissedre, P.-L. Copigmentation of Malvidin-3-O-Monoglucoside by Oenological Tannins: Incidence on Wine Model Color in Function of Botanical Origin, pH and Ethanol Content. Molecules 2019, 24, 1–15.
  •                   Vignault, A.; Gombau, J.; Jourdes, M.; Moine, V.; Canals, J.M.; Fermaud, M.; Roudet, J.; Zamora, F.; Teissedre, P.-L. Oenological tannins to prevent Botrytis cinerea damage in grapes and musts: kinetics and electrophoresis characterization of laccase. Food Chemistry 2020, 316, 126334.
  •                   Vignault, A. Tanins œnologiques : caractéristiques, propriétés et fonctionnalités. Impact sur la qualité des vins. Thèse de doctorat, Université de Bordeaux et Universitat Rovira i Virgili, 2019.