Measuring Phenolics in Grapes
We measure Anthocyanins in grapes to anticipate the subsequent concentrattion of phenolics in wine. The expectation is that we can measure the effect of different viticultural practices on the development of phenolics in grapes and their maturity before harvest.
The process has been developed by Enotrex LLC (copyright 2010-2014) and provided by Gianni Colantuoni for research purposes. The central idea is that by pressing unripe grapes and exposing the pomace/skins to a warm alcohol solution we can simulates the phenolic extraction process that the ripe grapes will undergo later, yet estimate their Anthocyanin levels now. This page has the following sections:
- Setup: describes the laboratory equipment required to prepare the simulated wine samples
- Preparing the phenolics extraction buffer: describes how to prepare the alcohol solution which will extract the phenolics and create a simulated wine
- Process: describes the measurement process in detail
- Calculations: shows how the measurements can be presented for interpretation
The setup consist of:
- A Phenolics Extraction Buffer, an alcohol solution used create a model wine from grape samples.
- A Press; we use a hand operated juice press (an automated electric laboratory press is under development for more consistent results.
- A temperature controlled water bath, we use a Sous-Vide controller in a plastic container.
- A standard Spectral Analysis Setup as used for phenolics measurements with WineXray
Here is an annotated picture:
Preparation of Phenolics Extraction Buffer
The materials needed are:
- Potassium Hydrogen Tartrate (KHTa; 99%; manufactured by SIGMA-ALDRICH; Catalog Number 243531)
- Ethanol (CH3CH2OH, i.e., EtOH; Alcohol, Anhydrous, Reagent; manufactured by J. T. Baker; Catalog Number 9401-22)
- De-ionized water (DI H2O)
- Hydrochloric Acid (HCl; 1.0N and/or 0.1N Volumetric Solution; manufactured by J. T. Baker; Catalog Numbers 5620-02, 5611-02)
The composition of the buffer is: 5 g/l Potassium Hydrogen Tartrate; 12% Ethanol v/v; pH 3.3. Follow these steps to prepare 500mL of the Phenolics Extraction Buffer:
- Place 400 ml of de-ionized water in a 1000 ml graduated beaker.
- Weigh and add 2.5 g of Potassium Hydrogen Tartrate.
- Measure and add 80 ml of 100% Ethanol.
- With a magnetic heater/stirrer, stir for five (5) minutes; do not worry if all the Potassium Hydrogen Tartrate does not dissolve.
- Use a pH meter while stirring the solution to monitor its pH.
- Allow the pH to stabilize, and then adjust the pH of the solution to 3.3 using the Hydrochloric Acid.
- Bring the volume to 500 ml with de-ionized water.
Store the buffer at room temperature; it is to be used within 30 days
The measurement process has 9 steps for each sample:
- Collect 200 berries in a plastic bag. Count the Berries ( NB = 200) and weigh them (WB in grams)
- Crush berries in the bag. Assure all berries are broken and the pulp and seeds are visibly separated from the skins
- Empty the full content of the crushed sample bag into the press
- Apply pressure, allow the juice to flow into a holding container labelled “Fresh Juice”. Wait 30 seconds and collect any remaining juice in the container. Apply maximum pressure, wait another 30 seconds and collect all remaining juice. Measure the volume and weight of extracted juice: (VJ in mL, WJ in grams) and measure its approximate Sugar content per 100 g of solution (BX in Brix), pH (pH) and Titratable Acidity (TA in mg/L).
- Open the press and remove the compressed skins and seeds cake into a holding jar.labelled “Extraction Jar” Then poor an amount of “Extraction Buffer” which equals the amount of Fresh Juice extracted above (VJ) into the jar and mix well.
- Incubate the Extraction Jar for 3 hours in a water bath temperature controlled at 55 dC, then shake the Extraction Jar and let it cool down for 15 minutes.
- Empty the full contents of the Extraction Jar into the press
- Apply pressure, allow the juice to flow into a holding container labelled “Exposed Juice”. Wait 30 seconds and collect any remaining juice in the container. Apply maximum pressure, wait another 30 seconds and collect all remaining juice. Measure the volume and weight of the Exposed Juice (VE in mL and WE in grams). Discard the compressed seeds and skins cake.
- Measure the phenolic compounds in the Fresh Juice and the Exposed Juice Record Total Anthocyanins (tANT & EtANT in ppm Malvadin Equivalent) using the WineXray platform.
Enter the measurements into a spreadsheet (yellow fields) which calculates the following results (red fields) in weight per average berry [mg/Berry] or [% of Berry]