We just had another update about what Sandy Paetz is doing at the
MSU Viticulture & Winemaking School - “Phenolic
Measurement of Wine”
This program was brought to our attention a few years ago when
a MSU student, Bernard Call, who took this course, purchased some grapes
at our farm and was carefully measuring the brix of the grapes with
a refractometer. The following year he returned to our farm after being
in the program for one year and he was very enthusiastic, interesting
and knowledgeable about the course.
Sandy Paetz, one of our past Sales Stand Associates, had also
enrolled in the MSU program. When the two of them found out
that they both were taking the courses, the conversations became very
stimulating and they agreed to forward some of their program notes to
us for our newsletter.
We have some inside stories of what Bernard and Sandy are doing
at MSU.
“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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