THE EFFECT OF HEAT ON THE ANTIOXIDANT ACTIVITY OF MERLOT AND CABERNET SAUVIGNON RED WINES.

Jui Kothari, Alexandra R. Eckburg, Disha Nataraj, Zachary J. Guritz ABSTRACT This experiment explored the effects of heat on the antioxidant activity of red wine. Two wines, a Merlot and a Cabernet Sauvignon, were heated to 25C, 50C, 75C and the boiling point of the solvent. A TEAC assay and a total polyphenolics measurement were used to analyze the antioxidant activity. Although there were slight variations in the radical scavenging activity of both wines over the temperature range, a direct linear relationship was not evident. The total polyphenolic content for the merlot increased until 75C and then significantly decreased. This suggests that heat treatment to 75C increases the amount of polyphenols in Merlot, but after this temperature, heat destroys the polyphenols. There was no evident correlation between the polyphenols and temperature in the Cabernet Sauvignon. These results indicate that there is no relationship between temperature and radical scavenging antioxidant activity in red wine, but temperature does affect the total polyphenolics content for Merlot, but not Cabernet Sauvignon. Therefore, there would be little benefit to keeping red wines in a temperature-controlled environment; however, extreme heat should be avoided.

INTRODUCTION Oxidation reactions produce free radicals that are highly unstable and cause further oxidation and creation of new free radicals. This starts a chain reaction that disrupts living cells. Antioxidants, like polyphenolic compounds (eg. Flavonoids), are substances that prevent the oxidation of free radicals by being oxidized themselves and therefore reduce aging and the risk of chronic heart disease. Additionally, polyphenols protect the lining of blood vessels in the heart. Red wine contains the antioxidant proanthocyanins, a group of bioflavonoids, known to possess broad pharmacological activities and therapeutic potentials1. Another vital component of red wine is resveratrol, a stilbene polyphenol, with the ability to

inhibit tumor growth that leads to cancer2. As a result, antioxidants are crucial in maintaining optimum health. This experiment tested the antioxidant activity of two red wines: a Merlot and a Cabernet Sauvignon. The hypothesis stated that the antioxidant activity of both wines would vary inversely with temperature i.e. antioxidant activity would decrease with increase in temperature. This is based on the principle that certain compounds denature and lose their potency when subjected to higher temperatures. The Trolox Equivalent Antioxidant Capacity (TEAC) Assay and Total Polyphenolics Content (TPC) Measurement (currently used in the wine industry) were employed in this experiment to measure the activity of the antioxidants in red

wine. The results of the experiment could provide insight into the optimum storage temperature of red wines in order to maximize the antioxidant activity, and thus the overall health benefits. EXPERIMENTAL I. Wine Preparation: The Merlot and Cabernet Sauvignon wines used in this experiment were controlled as they were both obtained from the McManis Family Vineyard, 2012 (Figure 1).

Just before the samples for each assay were prepared, the wines were thawed in warm water and diluted to 1% with ethanol or water for the TEAC assay and total polyphenolics content (TPC) measurement respectively. Then, they were heated (Figure 3) in a 400 ml beaker on a hot plate. A thermometer, attached to a clamp was used to record the temperature. Aliquots of 1 ml were pipetted out of the beaker just as the thermometer registered the following temperatures: 25C, 50C, 75C, and the boiling temperature of the solvent (100C for water and 78C for ethanol). The aliquots were allowed to cool down to room temperature (18C).

Figure 1- Merlot and Cabernet Sauvignon Wines

The study was not limited to one type of wine but extended to two, so that the results could be extrapolated to wines in general. Immediately after the wine bottles were opened with a corkscrew, 30 ml of each wine was flash frozen in liquid nitrogen (Figure 2) to prevent oxidation by atmospheric oxygen that would alter the antioxidant activity3.

Figure 3- Wine-heating set-up

Figure 2- Flash freezing of wine to prevent oxidation

II. TEAC Assay: The Trolox equivalent antioxidant capacity (TEAC) assay compares the total antioxidant activity in a sample to a standard, Trolox4. The substrate used was a radical chemical species of the monocation 2,2 -azinobis-(3ethylbenzothiazoline-6-sulfonic acid), abbreviated ABTS.+ 4. It is relatively stable, is easily reduced by important antioxidants and exhibits a characteristic absorption spectrum in the visible range of light4. The ABTS.+ reagent was prepared by mixing 2 ml of 0.014M ABTS solution ( prepared by dissolving 0.1801g salt in deionized water) and 0.0049M potassium persulfate solution( prepared by dissolving 0.1325g salt in

deionized water). Being light and temperature sensitive, the reagent was stored in an amber vial, covered in parafilm and refrigerated. It required 12 hours to be ready for use. The 2.5mM Trolox stock solution was prepared by dissolving 0.01564g of salt in 25ml ethanol in a volumetric flask. It was stored in an amber vial and refrigerated to prevent exposure to light and temperature. The stock was diluted to standards of 50 M, 100 M and 200 M in ethanol. A blank was set on the spectrophotometer (Beckman Coulter) at a wavelength of 735 nm using water. Cuvettes were prepared with 2.9 ml of ABTS.+ reagent and 0.1ml of ethanol (control) or Trolox standard or wine sample (Figure 4). The mixtures were allowed to react for 6 minutes and were then tested.

Cuvettes were prepared with 225 L water (control) or epicatechin standard or wine sample. Next, 1.5 ml diluted Folin-Ciocalteau reagent was added (reaction time of 6 minutes), followed by 1.5 ml 7% (w/v) sodium carbonate solution (reaction time of 30 minutes) (Figure 5). The cuvettes were tested in the spectrophotometer at 750 nm.

Figure 5- TPC Assay cuvette set up 1

RESULTS I.TEAC Assay: The solutions in the cuvettes turned blue over time and this hue deepened with increasing concentration of Trolox but stayed the same shade for the wine samples. This indicates that the antioxidant concentration of wine at various temperatures were not visibly different. The raw data for the Trolox standards are as follows:
Table 1- Spectrophotometric results for TEAC assay

Figure 4- TEAC Assay cuvette set up

III. TPC Measurement: Epicatechin is a common flavonoid and a monomeric component of polymeric proanthocyanidins4 which makes it an appropriate standard for the measurement of total polyphenolic content of red wine. 10mM Epicatechin stock solution was prepared by dissolving 0.0726g of salt in ethanol. Further dilutions of 50 M, 100 M and 200 M were made to create a standard curve. Folin-Ciocalteau reagent was made fresh, 1 part in 10, in deionized water. This reagent reacts with polyphenolic antioxidants to form compounds that absorb light in the visible region. 7.0% solution (w/v) of sodium carbonate was made in deionized water.

Sample 50 M 100 M 200 M ABTS.+ 25 C 50 C 75 C 100 C ABTS.+ 25 C 50 C 75 C 100 C ABTS.+

Trolox

Merlot

CS

Absorbance at 735 nm 0.36067 0.25849 8.7213 e-2 0.49392 0.14223 0.13197 0.16842 0.13688 0.34822 -4.1141 e-2 1.3296 e-2 -3.3753 e-2 6.9323 e-2 0.18112

The percentage inhibition of was calculated from the raw data shown above using the following formula:

where,
Graph 2- Effect of heat on ABTS.+ Absorbance of Merlot

For Example, % inhibition for 50 M

= 26.98% The processed data is as follows:

Table 2- Percentage Inhibition of standard and wine samples

Sample 50 M Trolox 100 M 200 M 25 C 50 C Merlot 75 C 100 C 25 C 50 C CS 75 C 100 C

% Inhibition 26.98 47.67 82.34 59.16 62.10 51.63 60.69 77.28 92.66 118.64 61.73

Graph 3- Effect of heat on ABTS.+ Absorbance on CS

II. TPC Measurement: The TCP Measurements showed a similar degradation in color as the TEAC Assay. The blue color of Epicatechin samples deepened with increase in concentration (Figure 6) whereas the color of wine samples remained almost the same (Figure 7). The colors of the solutions tested are displayed below:

The TEAC Assay graphs are presented below:

Figure 6- Blue color degradation of epicatechin standards Graph 1- Effect of Trolox on ABTS.+ Absorbance

Using the best fit equation (y= 0.0105x) of this graph, the TCP measurements for the wine samples were calculated from their respective absorbance at 750 nm as follows: For Merlot at 25 C: TPC Measurement = 0.36067/ 0.0105 = 30.95 M Similarly, calculations were carried out for the remaining samples and displayed as a bar graph:
Figure 7- Color Degradation of wine samples juxtaposed with color of unreacted wine subject to various temperatures Table 4- TCP Measurement for wine samples

The spectrophotometric results are tabulated below:

Table 3- Spectrophotometric results for TPC Measurements

Sample Epicatec -hin 50 M 100 M 200 M 25 C 50 C 75 C 100 C 25 C 50 C 75 C 100 C

Merlot

CS

Absorbance at 750 nm 0.7689 0.8390 2.1470 0.32494 0.59557 0.60344 0.28759 1.37850 0.39471 1.79740 1.08520

Sample 25 C 50 C Merlot 75 C 100 C 25 C 50 C CS 75 C 100 C

TPC (M) 30.95 56.72 57.47 27.39 138.29 37.59 171.18 103.35

The Epicatechin standard Graph is as follows:

DISCUSSION The data from the TEAC assay demonstrates that there is no proportional linear relationship between % inhibition of the Merlot and Cabernet Sauvignon and increase in heating temperature. This is deduced from the Rsquared values, which are less than 0.9 (0.026 for Merlot; 0.518 for Cabernet Sauvignon). This provides evidence for the idea that heat treatment has little to no effect on the radical scavenging activity of the wine antioxidants. The results of the total polyphenolics measurement indicate that the total polyphenolics content of both wines increase slightly until 75C, after which the content decreases. Although these changes are visible, they are very slight. The initial increase in TPC is

Graph 4- Epicatechin Standard Graph

potentially caused by the conversion of insoluble phenolic compounds to soluble ones1. After 75C, however, the antioxidant activity decreases. The t-test is a statistical hypothesis test used to determine whether the means of two groups are statistically different from each other. The t-test is performed to assess if the % inhibition and the total polyphenolics are statistically different for Merlot and Cabernet Sauvignon. The calculations were done using Graphpad Software5. and the results are discussed below: % Inhibition:
Table 5- t-test calculations for % Inhibition

TPC Measurements:
Table 5- t-test calculations for TPC Measurements

Group Mean SD (Standard Deviation) SEM (Standard Error of the mean) N (Number of values)

Merlot 43.1325 16.1908

Cabernet Sauvignon 115.6867 57.1654

8.0954

28.5827

Group Mean SD (Standard Deviation) SEM (Standard Error of the mean) N (Number of values)

Merlot 58.3950 4.6671

Cabernet Sauvignon 87.5775 24.2545

t = 2.3385 df = 6 Standard error of difference = 29.707 The two-tailed P value equals 0.0580 By conventional criteria, this difference is considered to be not quite statistically significant. Therefore, the effect of heat on the antioxidant activity does not statistically vary between the two red wines. This implies that the results may be applied to all red wines in general since the trend would remain the same. ERROR AND SCOPE FOR IMPROVEMENT The main reason for the greater fluctuations in the absorbance values of Cabernet compared to those of Merlot was the extended exposure to atmospheric oxygen. The Cabernet was the second wine to be tested and was exposed to air for a longer period of time than the Merlot.

2.3335

12.1273

t = 2.3630 df = 6 Standard error of difference = 12.350 The two-tailed P value equals 0.0561 By conventional criteria, this difference is considered to be not quite statistically significant.

When heating the wines, aliquots were removed just as the wine reached the desired temperature, but the wine was not maintained at each temperature for a fixed time interval. Therefore, the results are not conclusive for extended heating treatments as the amount of exposure to heat could alter the antioxidant activity and therefore should be controlled. Major errors in the experiment could have resulted due to the following reasons. Certain reagents like ABTS.+, Epicatechin and Trolox are sensitive to light. Although precautions were taken to limit their exposure, such as storing in amber bottles and using foil-covered glass measuring instruments, some unavoidable tear in the foil could lead to destabilization of reagents which in turn would affect the results. These reagents were also not consistently refrigerated as they were taken out each week for 2 and half hours. This would affect the stability and potency of molecules and create error in results due to incomplete reaction of denatured molecules. Another significant source of error is the condensation of on the cuvette resulting in the presence of bubbles and foggy walls. (Figure 8). This would interfere with the solution's ability to transmit light effectively in the spectrophotometer and would compromise the accuracy and precision of absorbance values. Furthermore, at the boiling temperature of the solvents (100C for water and 78C for ethanol), evaporation occurred which could potentially changed the concentration of the solution. In this case, data would not be indicative of the prepared dilution but would represent a more concentrated solution.

Figure 8- Bubble formation on cuvettes

Minor errors include errors in measurement that may have occurred due to the loss of substance during transfers between the apparatus. The presence of bubbles (sometimes unnoticed) in the micropipettes tip would restrict the correct uptake of solution and affect the concentration of mixture. Scope for improvement includes performing the experiment with more trials to ensure greater precision. Additionally, all other variables such as time exposure to a particular temperature, cooling time interval and final temperature of wine samples should be controlled to study in isolated the effect of heat on the antioxidant activity. More types of red wines could be investigated to show a common trend. Further investigations can be carried out to compare the effect of heat on antioxidant activity in red wine to white wine. ACKNOWLEDGMENT We thank Dr. Shelby Hatch, Bram Carlson and the Department of Chemistry, Northwestern University in assisting and guiding us with our research and giving us insightful feedback.

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