“Phenolic Measurement of Wine”

W.C. PAETZ
Michigan State University, Enology II,
Wine Production Laboratory - March 3, 2004

Abstract:

Wine color is a critical characteristic which can imply quality of a wine, or lack there of. Being able to measure and understand the status of this attribute is significant. Knowledge gained by examination of factual measured information as opposed to pragmatic opinion will obviously lead to understanding from which can be derived the control of important wine making aspects.

During this lab exercise I completed the process of examining the glycosolated anthocyanins and other phenolic substances found in a sample of Pinot- noir clone 115. My measurements included color density, color hue, degree of red pigment coloration, estimate of concentration of SO2 resistant pigments, total red pigments and total phenolics.

This was done using a spectrophotometer to measure the absorbance and reciprocal transmittance of light waves flowing through diluted samples of wine that had been mixed independently with acetaldehyde, sulfur dioxide and hydrochloric acid as well as a second reading done on samples that had a pH adjusted to 3.5. In both cases a control sample was measured as well.

INTRODUCTION:

What is a spectrophotometer? And why might being able to use one improve your wine making ability? According to the Usborne (1.) “When all different wavelengths of visible light fall on the eye at the same time, white light is seen. However white light can also undergo dispersion, whereby it is split into the visible light spectrum, ie. It’s different wave lengths by refraction this may occur accidentally, chromatic aberration or may be produced on purpose which is done by a spectrophotometer.”

According to Thermo Electron Corporation, (2.) “Spectrophotmeters have been used as a basic tool in wine making for over forty years. Not only can you obtain a fast, accurate profile of your wine. You can maximize the quality of this years vintage, ensure high volume uniformity, dependable test results are your guide to success. Testing can not only assist in meeting legal requirements it can help satisfy the palate as well. Overall quality can be increased through a variety of tests for substances such as:

Phenols….impart flavor, color and taste.
Lactic acid…signals the level of malo-lactic fermentation
Glycerol…indicates rot and mold in grapes
Argine…indicates the vine’s growth conditions
Other substances can be measured include Copper, Iron, Brightness and Hue, Tartaric Acid, Fructose and Glucose.”

Steve Voysey- Montana- Gisborne Senior Winemaker makes use of a state of the art UV spectrophotometer for enzyme mapping and colors of red wine. He is particularly proud of his show successes.(4.)

To understand how a spectrophotometer works it would be advisable to understand the following:(5.) “Many compounds absorb ultraviolet light (UV) or visible (Vis.) light. A beam of monochromatic radiation of radiant power P 0 , directed at a sample solution. Absorption takes place and the beam of radiation leaving the sample has radiant power P. The amount of radiation absorbed may be measured in a number of ways:
Transmittance T= P/P0
% Transmittance %T= 100T
Absorbance A= log P0/P, 1/T, 100/%T or 2-log %T

If light passes through a solution without any absorption, then absorbance is zero and percent transmittance is 100%. If all the light is absorbed, then % transmittance is zero and absorption is infinite.
Anthocyanin Equilibrium is Dependent upon pH and S02.(6.) Anthocyanin molecules contain a flavylium nucleus with a positively charged oxygen oxonium. In view of the existence of conjugated double bonds, the charge is delocalized on the entire cycle, which is stabilized by resonance. The colors of anthocyanin solutions are directly linked to pH. In an acid medium they are red, losing their color as pH increases. Maximum color loss is observed at values of 3.2 to 3.5. Colors vary from mauve to blue at pH values above 4, then fade to yellow in neutral or alkaline medium.
Because anthocyanin breakdown reactions include thermal degradation, oxidative degradation and degradation by acetone, it is important to know the wine constituent and environment at all times.

According to Ribereau, (8.) “The following compounds are present in the grape skin at maturity: benzoic and cinnamic acid, flavonols and tannins. They are distributed in the cells of the epidermis and the first sub-epidermal layers in both white and red grapes. In addition, the red grape skin contains athocyanins, essentially located in the hypodermal cell layers. Further anthocyanin composition varies from cultivar to cultivar, depending on the anthocyanidin substitution and heterosidic nature of the cultivar. Diglucosides are characteristic of American species (Vitis rupestris, Vitis riparia, Vitis labrusca) where as Vitas vinifera contains only monoglucosides.

The ripe grape skin also contains considerable amounts of aromatic substances and aroma precursors. In certain muscat varieties, the skin can contain more than half of the free terpenols of the berry. Finally the skin is covered by epicuticular wax , essentially constituted of oleanolic acid. All of this information is very important from a technological point of view. All methods of increasing the solid-liquid contact for color extraction or aroma dissolution should be favored during wine making.”

To observe the phenomenon of color and flavor extraction with out being able to measure and control it would be of limited value.

According to Jackson (9.) “The visual characteristics of a wine depend upon how its chemical and particulate nature transmit, absorb, and reflect visible radiation. Although such characteristics can be accurately measured with a spectrophotometer, the relevance of the data to human color perception is far from simple. Spectrophotmetric measurements assess the intensity of individual wave lengths, whereas the eye responds to light by combining the responses from the different types of receptor neurons (cones and rods). The cones respond relatively selectively to light in the blue, green , or red ranges of the visible spectrum where as the rods respond to low light intensity comparatively uniformly across the visible spectrum. Consequently, there is no simple relationship between spectrophotometric measurements and human color perception.”

The rationale for this experiment was to raise our level of knowledge and understanding of phenolic measurements. This research and technological development and training is targeted at wine making and meets real industry needs. Wine information such as flavonoid content and impact on the quality and healthiness of wine products lends measurable credibility to health claims. In addition the impact on flavonoids and phenol compounds of wine production and raw material processing needs to be understood.
Understanding this study would facilitate the special emphasis, which could be placed upon intimate wine product familiarity.

Materials and Methods

1.) Equipment:
1.) Wine Sample Pinot noir 115 Clone
2.) Two Spectrophotometers
3.) Cuvettes
4.) Distilled H2o
5.) Acetaldehyde, CH3CHO
6.) Sulfur dioxide, SO2
7.) HCL, hydrochloric acid
8.) Eight Test Tubes
9.) Pipettes

Method:
1.) Measure and record pH of wine sample
2.) Prepare labels for four test tubes: as follows: 1.wine, 2.wine +CH3CHO, 3. Wine + SO2 and 4. Wine + HCL
3.) Measure 20 ml CH3CO Place in test tube 2
4.) Measure 30 ml Na2S2o5 Place in test tube 3
5.) Measure 10 ml HCL Place in test tube 4
6.) Measure absorbance of each of the solutions in test tubes 1,2, and 3 @ 520, 420 and 280 dilute equally with distilled water if no reading on spectrophotometer
7.) Measure the absorbance of solution in test tube 4 @ 520 and 280
8.) Calculate the various spectral measures.
9.) Adjust wine to pH 3.5 and repeat process steps one through seven above.

Results

Please see appendix figures 1 and 2 for tables of direct measurement data.

Please see appendix figure 3 for calculated numerical data and dilution correction.

Discussion

An analysis that measures objective data obviously can be helpful to the knowledgeable wine maker. According to Serra Giuseppe, (3.) “The main advantages of using a spectrophotometer are:
Possibility of objective instrumental measurement of wine colour and it’s expression with numerical world-wide fixed parameters;

Repeatability and comparability of measured data in space and time, having the possibility to build an historical data base about the color of different products.

Possibility to fix objectively chromatic features typical of each product;
Possibility to correlate between them visual and instrumental valuations of the colour.
Possibility to correlate the color with other physic and chemical-analytical features of the product;
Better control of wine production process considering the standardisation of final product chromatic features.”

According to Patrick Iland, (7.) “Some authors have shown a positive correlation between wine colour density and quality ratings. This may be an indirect effect in that deeply coloured wines may also have higher levels of desirable aroma and flavor compounds. Spectral measures of wines can thus act as indicators of wine style (and in some cases quality) and are thus a useful analytical tool. They should be used in conjunction with other chemical and sensory quality indicators.”
In my opinion a wine may meet or even exceed all technologically measurable aspects and yet in spite of this, it conceivably may not meet the highest organoleptic scrutiny. The true test of a wine is made at the point in which it passes through a discerning customer’s lips and is judged worthy of repeat consideration.

Conclusion
This study was not only worth while but extremely important. However in order to truly understand the ramifications of this data and it’s relevance to making wine a much deeper understanding of chemistry and physics than I presently possess is required. I humbly assert that although we can go through the motions and even tabulate results, unless we truly know what the causative factors are we are merely collecting statistics and not adding to wine quality.

However for those gifted individuals who truly know and understand the complexities of this measurement, the value of it is quite incredible. Consider for a moment the medical health benefits of the phenols found in wine. (10.)

“Resveratrol (3, 4', 5 trihydroxystilbene) is a naturally occuring phytoalexin produced by some spermatophytes, such as grapevines, in response to injury. Given that it is present in grape berry skins but not in flesh, white wine contains very small amounts of resveratrol, compared to red wine. The concentrations in the form of trans- and cis- isomers of aglycone and glucosides are subjected to numerous variables. In red wine, the concentrations of the trans-isomer, which is the major form, generally ranges between 0.1 and 15 mg/L. As phenolic compound, resveratrol contributes to the antioxidant potential of red wine and thereby may play a role in the prevention of human cardiovascular diseases. Resveratrol has been shown to modulate the metabolism of lipids, and to inhibit the oxidation of low-density lipoproteins and the aggregation of platelets. Moreover, as phytoestrogen, resveratrol may provide cardiovascular protection. This compound also possesses anti-inflammatory and anticancer properties”

Other evidence is presented.(11.) “Wine has been part of human culture for 6,000 years, serving dietary and socio-religious functions. Its production takes place on every continent, and its chemical composition is profoundly influenced by enological techniques, the grape cultivar from which it originates, and climatic factors. In addition to ethanol, which in moderate consumption can reduce mortality from coronary heart disease by increasing high-density lipoprotein cholesterol and inhibiting platelet aggregation, wine (especially red wine) contains a range of polyphenols that have desirable biological properties. These include the phenolic acids (p-coumaric, cinnamic, caffeic, gentisic, ferulic, and vanillic acids), trihydroxy stilbenes (resveratrol and polydatin), and flavonoids (catechin, epicatechin, and quercetin). They are synthesized by a common pathway from phenylalanine involving polyketide condensation reactions. Metabolic regulation is provided by competition between resveratrol synthase and chalcone synthase for a common precursor pool of acyl-CoA derivatives. Polymeric aggregation gives rise, in turn to the viniferins (potent antifungal agents) and procyanidins (strong antioxidants that also inhibit platelet aggregation). The antioxidant effects of red wine and of its major polyphenols have been demonstrated in many experimental systems spanning the range from in vitro studies (human low-density lipoprotein, liposomes, macrophages, cultured cells) to investigations in healthy human subjects.

Several of these compounds (notably catechin, quercetin, and resveratrol) promote nitric oxide production by vascular endothelium; inhibit the synthesis of thromboxane in platelets and leukotriene in neutrophils, modulate the synthesis and secretion of lipoproteins in whole animals and human cell lines, and arrest tumour growth as well as inhibit carcinogenesis in different experimental models. Target mechanisms to account for these effects include inhibition of phospholipase A2 and cyclo-oxygenase, inhibition of phosphodiesterase with increase in cyclic nucleotide concentrations, and inhibition of several protein kinases involved in cell signalling. Although their bioavailability remains to be fully established, red wine provides a more favourable milieu than fruits and vegetables, their other dietary source in humans”

Further health claims regarding resveratrol. (12) “
“Resveratrol, a phytoalexin found in grapes and other food products, was purified and shown to have cancer chemopreventive activity in assays representing three major stages of carcinogenesis. Resveratrol was found to act as an antioxidant and antimutagen and to induce phase II drug-metabolizing enzymes (anti-initiation activity); it mediated anti-inflammatory effects and inhibited cyclooxygenase and hydroperoxidase functions (antipromotion activity); and it induced human promyelocytic leukemia cell differentiation (antiprogression activity).

The obvious implications and value of being able to isolate and measure these important wine constituents is quite amazing. I would conservatively assert that health conscious baby boomers have only begun to embrace wine for its salutary benefits to health and life expectancy. We in the wine industry have not begun to cultivate the potential of this information. Part of the reason for this is attributable to bureaucracy and conservative paradigms held by various government sanctioning organizations. Someday they will be compelled to succumb to the massive wave of evidence being collected in study after study. Some of which follow the exact process that we did in our lab as their foundation.

Appendix

Table I
Sample 1 Spectophotometer Results
Test Tube 520 Reading 420 Reading 280 Reading
1. .323 .268 3.459
2. .424 .306 3.604
3 .249 .211 3.610
4 .081 .029 .748

Table II
Sample 2 pH 3.5 Spectophotometer Results
Test Tube 520 Reading 420 Reading 280 Reading
1. .396 .286 3.62
2. .370 .294 3.53
3 .269 .233 3.28
4 .090 .027 4.87

Table 3 Final Numerical Data
Dilution
Description of Value Formula for Value Value correction

Wine Color Density =A520 +A420 0.591 11.82
Wine Color Hue =A420 / A520 0.8297214 16.59442724
Degree of Red Pigment colouruation % =(A520 / HCL520) X 100 3.9876543 79.75308642
Estimate of SO2 resistant pigments(a.u.) =A SO2 520 0.249 4.98
Total Red Pigments (a.u.) = =A HCL 520 0.081 1.62
Total Phenolics (a.u.) = =A HCL 280 - ASO2 520 VAL -0.168 3.36
Modified wine colour density =A CH3CHO 520 + CH3CHO 420 0.664 13.28
Modified wine colour Hue =A CH3CHO 420 / A CH3CHO 520 0.7945946 15.89189189
Modified Degree of Red Pigment colouruation % =(A CH3CHO 520 / A HCL 520) X 100 4.1111111 82.22222222
Modified Estimate of SO2 resistant pigments(a.u.) =A SO2 520 0.396 7.92
Modified Total Red Pigments (a.u.) = =A HCL 520 0.09 1.8
Modified Total Phenolics (a.u.) = =A HCL 280 - ASO2 520 VAL 4.601 92.02

References:

1.) The Usborne Illustrated Dictionary of Science, A Complete Reference Guide to Physics, Chemistry and Biology. Usborne Publishing Ltd. London England 2001
2.) Thermo Electron Corporation-“Squeeze Higher Quality From Your Next Vintage With Spectronic Spectrophotmeters.” 2003 http://www.thermo.com/com/cda/products/
3.) S. Giuseppe, “Procedure for the colour measure of wine and grapes in situ.” Consorzio Per L Assistenza Alle Piccole E Medie Imprese. April 2002.
4.) “Wine of the Week” a Webzine for Wine and Food Lovers. Editor S.Courtney November 2002 http://www.wineofthe week.com/persarch
5.) “Beer’s Law - Theoretical Principles” Sheffield Hallam University School of Science and Mathematics. 2004 http://www.shu.ac.uk/schools/sci/chem/tutorials/mols
6.) P. Ribereau-Gayon, Handbook of Enology Volume 2 The Chemistry of Wine Stabilization and Treatments. John Wiley and Sons Ltd. W. Sussex, England June 2003
7.) P. Iland , Techniques for chemical analysis and quality monitoring during wine making. Patrick Iland Wine Promotions, South Australia, Australia 2000
8.) P. Ribereau-Gayon, Handbook of Enology Volume 1 The Microbiology of Wine and Vinifications John Wiley and Sons Ltd. W. Sussex, England June 2001
9.) R. Jackson, Wine Science Principles, Practice, Perception Second Edition, Academic Press Harcourt Science and Technology Company, Sand Diego, Cal. 2000
10.) Frémont, L. (2000) Minireview - Biological effects of resveratrol. Life Sci. 66, 663-673
11.) Soleas, G.J., Diamandis, E.P. and Goldberg, D.M. (1997) Resveratrol: a molecule whose time has come? And gone? Clin. Biochem. 30, 91-113.
12.) Jang, M., Cai, L., Udeani, G.O., Slowing, K.V., Thomas, C.F., Beecher, C.W.W., Fong, H.H.S., Farnsworth, N.R., Kinghorn, A.D., Mehta, R.G., Moon, R.C. and Pezzuto, J.M. (1997) Cancer chemopreventive activity of resveratrol, a natural product derived from grapes. Science 275, 218-220.

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