An international conference on grape quality specifications entitled "Grape Expectations" took place at U.C. Davis in May of 2000. This protocol derives from that conference and has resulted in part from the consensus of the expert speakers and from procedures offered there which, in our judgment, offered the best approach to the problem of maturity assessment. We offer this protocol as a standard method for red winegrape maturity assessment on the vine to determine harvest date.

We have based our protocol on the skin extraction method developed by C.A.S.V. (Chambre dAgriculture de la Gironde Service <Vigne>, realized through support of the Conseil Professionnel du Vin du Bordeaux) in preference to whole-berry methods offered from Australia and California, which are designed for fruit quality assessment at the winery delivery point. The AWRI method may well be the best choice for fruit quality assessment at the crusher because its NIR approach can assess spoilage and rot indicators in addition to phenolics.

We placed great importance on a method which could permit sensory analysis of fruit aromas extracted from skins. In our opinion the extractions obtained from the two whole berry methods seem unsuitable for sensory analysis. We were skeptical of the variability of seed extraction in whole berry methods, particularly the AWRI method, which uses a blender for quick preparation. Although probably necessary for a complete extraction, we were uncomfortable with the overnight heated digestion prescribed in the ETS method as a preparation method for sensory work. We also felt the presence of high sugar was likely to obscure determinations of fruit aromatic richness.

The Gironde method calls for a separation of skins from pulp and seeds. While potentially tedious, we feel this is essential for good sensory work, and results in a wine-like supernatant which may be examined visually, tasted, submitted to analytical quantifications.and preserved for future comparison with subsequent samples. We are in the process of testing an apparatus for counting, weighing and separating berries to facilitate and standardize the protocol.

Comparative (rather than quantitative) anthocyanin determination is a critical element of any acceptable protocol. The Gironde method has the longest track record in field use, and has earned acceptance in Europe for monitoring the peak and decline of pigment within a block during maturation. Since seed tannin is not extracted, co-pigmentation may be minimal for the method, and it should not be viewed as a total color or extractable color method for predicting wine composition.

Careful and consistent berry sampling is the key to a successful maturity assessment program. Since there is so much inherent compositional variation in a single cluster of grapes, and between clusters and vines in a given area, it is imperative that a minimum of 300 berries be used to represent the level of maturity in a given block. The following protocol for winegrape sensory and chemical analysis is suggested to optimize reliability:

Vineyard Sampling Protocol:

1. A person familiar with the vineyard block to be assessed should choose areas that most represent the entire block as sample sites.
2. The samples should be taken at approximately the same time each day to avoid environmental mitigating factors.
3. Approximately 2kg of fruit should be removed for each block sampled. This represents approximately 10 15 clusters, depending on cluster size. For ease of explanation, the author will refer to 12 clusters as a general number
4. Maturity sampling should begin approximately 15 20 days after mid-veraison for red varietals.
5. In a 10-20 acre site, choose 12 (or more) vines as sample sites and tag the vines with colored tape so each cluster is pulled from the same vine each time. These vines must be representative of the entire block to assure reliability of test results.
6. One entire cluster should be removed, using shears, from each of the vines chosen. This minimizes the damage to berries in the cluster and also minimizes sampling error due to vineyard variability.
7. A large plastic bin or box works well as the berries are not broken as easily as in a plastic bag.
8. Once all 12 clusters have been removed, the samples should be stored in a cooler on ice to avoid premature maceration and extraction of the berries. The optimal temperature for storage is between 50^oF and 60^oF, if possible. Care should be taken not to store them for any extended period of time over 62^oF.
9. The berries should be transported to the facility for testing as quickly as possible the same day, whenever possible.
10. At the lab, the clusters should be laid out, counted and weighed, with all data recorded.
11. After weighing, each berry should be removed from the cluster as carefully as possible to avoid premature maceration and oxidation. Count the number of berries per cluster, if that information is required.
12. After all the clusters have been treated as above, carefully mix all the berries together to achieve a representative sample of the entire lot. This large sample is as true a reflection of the block as is possible. The entire lot should contain at least 500 berries, depending on cluster size and weight.
13. Sort out a random sample of approximately 150 berries for grape sensory evaluation and weigh them. This number is used to determine sugar/berry to validate sampling compared to previous samples. Sort out another sample of approximately 300 berries for analytical examination of maturity. Weigh.
14. The sensory and analytical protocols are as follows:

1. Necessities for this assay:
• Citric acid solution (9.25g/L)
• Blender
• 96% alcohol
• balance to determine mass to 0.1g precision
• beakers and volumetric cylinders
• bottles for storage of the finished product
2. Sample 150 berries as described above.
3. Remove the skins by squeezing the pulp and pips out between the thumb and forefinger. An alternate method states that the berries can be dissected using a razor blade, making a slice around the equator of the berry and scooping the pulp out. Either may be utilized to separate the skins for sensory evaluation. Retain the skins and weigh them (approx. 60 120 gms). The juice separated can be used for Brix, pH and TA determinations. The seeds can be utilized to test their hardness as another indicator of berry maturity, described by Gordon Burns.
4. Put the skins in a blender and add 75ml of the citric acid solution. The final pH must be approximately 3.50. Blend this mixture using short pulsing strokes until well mixed.
5. Tare the balance with the beaker to be used. Pour the crushed skins/citric acid solution into the beaker. Wash the blender with a small aliquot of water and add the water back to the crushed skins mixture.
6. Add the equivalent of 8% alcohol, calculated on the original mass of the 150 berries as follows:

Wt of 96% alc to add:
= (dilution factor) x (0.08) x (wt of 150 berries)/ alcoholic strength

where dilution factor = 1.09 for 96% ethanol or 1.17 for 90% ethanol

EX: wt of alc to add = 1.09 x 0.08 x 75gms/.96 = 6.81gms of 96% alcohol

7. Add water back to the tared beaker to reach the original mass of the 150-berry sample in step 2 above.
8. Pour this turbid solution into a bottle that can be sealed. Store this sealed bottle overnight at room temperature. Alternately, the solution can be shaken every hour for 6 hours, then prepared.
9. Filter the solution through a very rough pad to remove the solids. Take care not to over-process, as aromatics can be easily affected.
10. Taste this clarified solution, taking care to note the intensity of the aromatics and the quality and concentration of the tannins present.
11. The points below can be used as a guide in evaluation of the grapeskins solution:
• COLOR: intensity from 1 to 5 (arbitrary, as extraction method will be the most important determinant of actual color concentration)
• FLAVOR:
1.Unripe grapes usually have cherry stem/vegetal/green type flavors and obvious high acid concentration

2. Slightly riper grapes give red fruit flavors such as strawberry/cherry or even green apple flavors, with marked tartness on the palate

3. Nearer maturity, grapes begin showing black fruit flavors but lack real intensity and concentration with slight green apple tartness still evident

4. Mature grapes show rich, deep black fruit/spice characters

• CONCENTRATION OF FLAVORS: from 1 to 5
• TANNIN QUALITY: are they green, bitter, hard, softening, etc.
• FINALLY, AND MOST IMPORTANTLY, IS THE MATURITY APPROPRIATE FOR THE STYLE OF WINE TO BE PRODUCED?
12. This protocol is entirely subjective and, therefore, dependent upon the style of wine desired. It is meant to be modified by each producer to meet their individual needs. It is suggested that careful records be maintained over time so a historical database can be built, using winegrape sensory data correlated to winegrape chemical maturity data and, finally, to finished wine data to develop a maturity profile best suited for each wine style.

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Polymeric Pigment and Tannin assay for use with Spectrophotometer

James F. Harbertson and Douglas O. Adams

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Tannin assay Adapted from A.E. Hagerman and L.G. Butler. Protein precipitation method for the quantitative determination of tannins. J. Agric. Food Chem. 26:809-812 (1978)

This method combines protein precipitation and traditional bleaching techniques to allow measurement of tannins and polymeric pigment using a Spectrophotometer.

Materials required

All materials required for colormetric assay are listed below.
All reagents and materials are available separately from Sigma or Fisher.

Name In Procedure	Description	Storage
Buffer A (Washing Buffer)	200 mM acetic acid 170 mM NaCI pH adjusted to 4.9 with NaOH	Stable at room temperature
Buffer B (Model B)	5 g/L potassium bitartrate (KHTa) pH adjusted to 3.3 w/HCl	Stable at room temperature
Buffer C (Resuspension Buffer)	5% triethanolamine (v/v) 5% SDS (w/v) pH adjusted to 9.4 w/HCI	Stable at room temperature
Protein Solution	1 mg/mL bovine serum albumin dissolved into Buffer A	Stored at 4 oC
Ferric Chloride Reagent	N HCI 10 mM FeCl3	Stable at room temperature
Bleaching Reagent	0.36 M Potassium Metabisulfite	Replace periodically
Catechin Standard	1 mg/mL (+)-catechin solution dissolved in a 10% EtOH	Make fresh each time

 

Hardware necessary for assay:
Spectrophotometer, 1.5 mL cuvets, a set of micropipets capable of measuring in the range of 1-1000 uL, 1.5 mL microfuge tubes, a microfuge test-tube rack, benchtop vortex and a Microcentrifuge capable of at least 14,000 RPM.

Procedure

Standard Curve

Perform all incubations at room temperature.

1. Prepare standard curve (+)-Catechin samples by taking from 50uL to 300uL of standard Catechin solution and adjusting the volume to 875uL with Buffer C (eg 100uL Catechin solution plus 775uL of Buffer C)
2. Add 125uL of the Ferric Chloride Reagent and vortex immediately (or invert). Make a zero tannin sample with 875uL Buffer C and 125uL of ferric chloride reagent.
3. Incubate the standard samples and the zero tannin for 10 minutes.
4. Read the absorbance at 510nm.
Wine Samples

This procedure describes how to measure tannins and polymeric pigment in wine samples.

1. Each wine sample requires two microfuge tubes. In the first tube (tube 1) 500uL of wine diluted from 1:1 up to 1:9 is added to 1 mL of Buffer A. A second tube (tube 2) with 500uL of the same dilution of wine added to 1mL of protein solution.
2. Tube 1 requires a 10 minute incubation and tube 2 requires a 15 minute incubation.
3. After 10 minutes take a 1 mL aliquot from tube 1 and read it in the spectrophotometer at 520 nm (Measurement A). (zero against 1 mL of Buffer A).
4. Add 80uL of bleaching reagent to that same aliquot and mix. After a 10 minute incubation read at 520 nm (Measurement B).
5. After the 15 minute incubation of tube 2 centrifuge for 5 minutes (14,000 RPM).
6. Remove 1 mL of the supernatant from tube 2 without disturbing the pellet. To that aliquot add 80uL of Bleaching Solution, and after a 10 minute incubation read at 520 nm (Measurement C).
7. Discard the remaining supernatant from tube 2 and wash the surface of the pellet and the walls of the tube with 250uL of Buffer A without disturbing the pellet.
8. Centrifuge for 1 minute (14,000 RPM).
9. Decant supernatant carefully (pipette if necessary). Add 875uL of Buffer C without disturbing the pellet and incubate for 10 minutes.
10. Vortex samples until pellet is completely dissolved, incubate for ten minutes and then read at 510 nm. This is the wine background A510.
11. Add 125uL of the Ferric Chloride Reagent and mix immediately. Incubate for 10 minutes and then read at 510 nm. This is Final A510.
Determination of polymeric pigments in wine

To all samples where the sample has been diluted with sulfur dioxide multiply the absorbance
by 1.08 (B and C).

Large polymeric pigment (LPP)
B-C

Small polymeric pigment (SPP)
C

Monomeric pigment
A-B

Total color at pH 4.9
A

Determination of the amount of tannin in a wine s ample
The amount of tannin in wine samples is calculated using the standard curve, after the background absorbance (the one obtained before the Ferric Chloride Reagent addition) is subtracted from the final absorbance (after adding the Ferric chloride Reagent).

The absorbance due to tannin is:
[(Final A510) - (A510 from zero tannin sample)]- (background A510 *0.875)

See Examples and Standard Curve on Page 4

The General Equation to determine the concentration of tannin in a wine is:

2 x (Abs-Intercept/Slope) (Dilution)

For example if you use this standard curve and your final absorbance is 0.500 and dillution is 5:

2 x (0.500-0.0075/0.0053)(5) = 929 mg/L CE (Catechin Equivalents)



Boulton - Modified Somers Color Analysis

We are basically following the method of Somers as modified by Roger Boulton to include copigmentation corrections. We have shortened the analysis time to 2 hours. We recommend incorporating this assay with Doug Adams astringency assay to obtain molar monomeric anthocyanin estimates as well as precipitable/non-precipitable polymeric pigment.

General Notes

• All samples should be obtained using a drop thief to get the most representative sample.
• All samples should be centrifuged and filtered through a minimum 1.0 filter. Be sure to thoroughly rinse the filter with the wine prior to collecting the filtrate used for analysis.
• Lack of clarification will cause staining and fouling of your expensive 1 mm quartz cuvettes. Periodically (about every 3-4 readings) clean the cuvette with 10% nitric acid to ensure accurate readings.
• All samples must be adjusted to pH 3.6 before analysis. We suggest this be done with your most reproducible pH meter and before clarification. Use the most concentrated base or acid practical to minimize the effect of dilution on the sample. If dilution caused by pH adjustment is greater than 1%, please record it.
Reagents
• 1 N HCl
• 10% Acetaldehyde. Available from Vinquiry (Shirley/ 838- 6312). Shelf life is 4 weeks.
• 5% Potassium Metabisulfite solution. Make fresh daily.
• pH 3.6 Buffer. 24 mL of pure ethanol is added to 176 mL of distilled H₂O. Dissolve 0.5 g of Potassium bitartrate into the solution. If pH does not = 3.6, adjust with concentrated HCl or NaOH as needed.
Necessary Equipment
• Spectrophotometer capable of reading at 280, 365, 420 and 520 nm
• Matched set of 1mm quartz cuvettes
• Glass 1cm cuvettes
• Micropipetters, lab centrifuge, test tubes etc.
1. Fill one 1 mm cuvette with water and the other with filtered wine. Be sure to zero the spectrophotometer with the water sample. Then, take the wine readings at 280, 365, 420 and 520 nm. Multiply all values by 10. These are the values for A₂₈₀, A₄₂₀, A₃₆₅ and A₅₂₀.
2. In a test tube (T1), pipet 100 L of wine and 10 mL of 1 N HCl. Cover, mix (or vortex) and let stand for 60 minutes. Read at 520 in a 10 mm 1 cm cuvette. Multiply this reading by 101. This is A^HCl..
3. In another test tube (T2), pipet 100L of 10% Acetaldehyde into 5.0 mL of w ine. Cover, mix and let stand for 45 minutes. Read at 520 nm in a 1mm cuvette. Multiply the reading by 10.2. This is A^Acet.
4. In a third test tube (T3), add 100 L of 3% Potassium metabisulfite to 5.0 mL of wine. Cover, mix and let stand for 15 minutes. Read at 520 nm in a 1 mm cuvette. Multiply reading by 10.2. This is A^SO2.
5. To a 1 cm cuvette containing 1900 L of buffer, add 100 L of wine. Let stand 10 minutes. Read at 520 nm. Multiply reading by 20. This is A²⁰.

Reading	What to add	Minutes	Cuvette	Multiplier
A280	Nothing	0	1mm	10
A420	Nothing	0	1mm	10
A365	Nothing	0	1mm	10
A520	Nothing	0	1 mm	10
AHCl	100 L wine 10 mL HCl	60	1 cm	101
AAcet	100 L Acetaldehyde 5.0 mL wine	45	1mm	10.2
ASO2	100 L 3% SO2 5.0 mL wine	15	1mm	10.2
A20	1900L buffer 100 L wine	10	1cm	20

Calculations:

Total Phenols = A₂₈₀

Copigmentation Co-factors = A₃₆₅

Visible Color = A₅₂₀

Color Intensity = A₄₂₀ +A₅₂₀

Hue = A₄₂₀/A₅₂₀

Color due to polymeric anthocyanins = A₅₂₀^SO2

& Yet others are called by different names or use a different equation for the same name

Fraction of color due to Copigmented Anthocyanin ( A^Acet- A₂₀)/ A^Acet

Fraction of color due to Free Anthocyanins (A₂₀ A_SO2) / A^Acet

Fraction of color due to Polymeric Pigment A_SO2/ A^Acet (Boulton)

% ionized anthocyanins or % anthocyanins in flavylium form:

also called anthocyanidins, these are monomeric anthocyanins such as malvidin, cyanidin, peonidin, petunidin, delphinidin

Total anthocyanins (Boulton)

This title is a little confusing. Boulton defines "anthocyanin" as referring only to monomeric pigment compounds. Therefore, he does not consider polymeric pigment as part of "total anthocyanins". Buried in this definition is the presumption that bleachable pigment is only monomeric, and also that there are no significant unbleachable monomers which can properly be called anthocyanins. While this value is good for attributing the contribution of these species to visible color, the measure at pH 3.6 is on the shoulder of the ionization curve, which will vary within the anthocyanin group and with alcoholic strength. This makes the application of a molar extinction coefficient (in order to estimate the molar ratio against flavonoid monomer) likely to be inaccurate by as much as a factor of two.

We believe that the Adams "bleachable pigment" may offer a better way to estimate molar monomeric anthocyanin.

 

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