Field Test to Rapidly Screen Grape Juice for
Phenolic Content
Michael B. Sady 1*
1 Professor
of Chemistry,
* [Email: mbsady@wncc.edu FAX (775) 782-2415]
Acknowledgments: The author wishes to thank the owners of Lava Cap Winery in El Dorado Co. California USA for the grape and juice samples and providing the site for most of this study. In particular the author thanks David Jones, Charlie Jones, Tom Jones, and Eric Hays for their helpful comments during the study and on this manuscript.
The author also wishes to thank Thomas Hunter for the sample of Chardonnay must, and the owners of the Gardotti vineyard in Douglas Co. Nevada for the White Riesling grapes. The author’s class of Chem 220 are also acknowledged for testing the procedures and the gallic acid spectroassay of white grape juice. Anonymous reviewers of the American Journal of Enology and Viticulture are also acknowledged for their suggestions.
The author acknowledges the UCCSN Board of Regents for granting him a sabbatical leave the autumn 2003 term during which time this study was conducted.
Revised
Abstract
A field test, based on the modification of a documented colorimetric technique, was developed and used during the 2003 harvest season as a rapid screening method to visually estimate the phenolic content in fresh grape juice derived from varieties of Vitis vinifera. Two table and eleven wine grape varieties were evaluated with the field test to see if it could detect differences in phenolic content based on cultivar variety or provenance, or pressing method employed for extraction. The results showed that the field test can detect variations based on different varieties of grapes, but cannot detect differences between samples of the same variety nor at different times during the harvest season unless the provenance of the grape is different. However, grapes of the same variety growing in the same vineyard that were processed differently by either hand pressing or machine pressing, or grown on different sun-exposed aspects, did show differences in their phenolic content with the field test.
Keywords: phenolic content, grape juice, field test, pressing method, Vitis vinifera, provenance.
Introduction
Grapes, and their products such as juice and wine, contain considerable amounts of chemicals generally referred to as phenolic compounds. These phenolic compounds are primarily characterized structurally as flavonoids, and the non-flavonoid stilbenes, hydroxycinnamate and hydroxybenzoate derivatives, having at least one moiety of phenol that they each share in common. Phenolic compounds have been identified as being an important source of nutrition in the human diet, reported in the scientific literature to have antioxidant attributes that delay or inhibit cardiovascular deterioration (Waterhouse and Walzem 1998).
In wine the phenolic compounds play an important sensory role constituting a large source of naturally occurring astringent principles. Therefore, oenologists are increasingly committed to the study not only of the vine, harvesting and fermentation, but also to the principal ingredient that shapes the characteristics of the wines, namely the grape itself and the variability of concentration and composition of phenolic compounds that shape an individual wine type and style (Haslam 1998).
The composition of phenolic compounds in fresh grapes varies according to the cultivar, viticulture practices and growing conditions, and tissue. Among the tissues of the grape the juice and pulp account for the lowest proportion of phenolic compounds and are generally composed of only trace amounts of flavonoids, and primarily composed of hydroxycinnamate derivatives (Waterhouse and Walzem 1998; Singleton and Esau 1969). The major hydroxycinnamate component in grape juice, trans-caftaric acid, has a mean concentration of 127 mg/L in white varieties and 163 mg/L in red varieties of Vitis vinifera cultivars tested (Singleton et al. 1986).
Measuring phenolic compounds quantitatively and qualitatively in natural matrices can be achieved in the laboratory with a high level of selectivity and sensitivity (Floridi et al. 2003), and in grape juice and wines with quite sophisticated, yet simple and portable, biosensors (Jewell and Ebeler 2001). The study presented here utilizes a field test adapted from a procedure for the colorimetric visual estimation of phenolic content in grape juice and wines (Zoecklein et al. 1999, Schanderl 1962). One objective of this study was to see if the field test can be used in the vineyard, winery, laboratory, classroom, or home to screen samples for phenolic content rapidly, safely, and at negligible expense. Another objective of this study was to evaluate the field test during the harvest season for its ability to screen fresh grape juice samples for differences in phenolic content as a result of cultivar variety or provenance, or pressing methods used to produce the juice.
Materials and Methods
Grapes. The wine grapes tested in this study were harvested in summer/autumn (September-October) 2003 in El Dorado Co. California (USA) from "Lava Cap" (LC) Winery estate vineyard or from other "El Dorado" (ED) Co. vineyards delivered for processing to Lava Cap Winery, and in Douglas Co. Nevada (USA) from "Gardotti vineyard" (GV): Sauvignon Blanc (LC, September 16, 17, 18), Cabernet Sauvignon (LC, September 16, 19, 22), Cabernet Franc (ED, September 18), Syrah (ED, September 18; LC, September 25), Merlot (LC, September 19, 22; ED, September 23), Brunello-Sangiovese (ED, September 22), Grenache (LC, September 23), Zinfandel (LC, September 25), Chardonnay (ED, September 25), Viognier (ED, September 29), White Riesling (GV, October 2). The table grapes tested in this study were purchased fresh at a retailer in summer (August) 2003 of "unknown provenance" (UP): Red Flame Seedless (UP, August 21), and Thompson Seedless (UP, August 21).
Processing. All wine grapes in this study were pressed into juice and evaluated for phenolic content with the field test immediately on the same day as their harvest. All table grapes were pressed into juice and evaluated for phenolic content immediately on the same day as their purchase. Three clusters of a wine grape variety were selected randomly from "delivery" (D) bins on the day of harvest at the winery, or from the "vineyard" (V) three vines of a wine grape variety were chosen randomly and one cluster of grapes picked from each vine for evaluation, or three clusters of the table grapes were randomly selected when "purchased" (P).
From the three clusters 100 grapes were randomly selected and immediately deskinned by using the thumb and forefinger to squeeze out the pulp (with pips) and juice directly into a hand held garlic press (Zyliss susi, Lyss, Switzerland) and subjected to "hand pressing" (HP) at ambient air temperatures of 25-30 oC for the juice (discarding the flesh, pips, and skins). HP juice samples were unfiltered and free of any additives. Juice samples derived from "machine pressing" (MP) were harvested and delivered on the same day from the estate vineyard, or from a local vineyard, in bins to the winery and immediately destemmed in a Delta destemmer and pressed in a Howard presser at ambient air temperatures of 25-30 oC. MP juice samples were taken directly from the presser basin unfiltered and free of any additives.
Field test for estimating phenolic content in grape juice. Juice samples were evaluated using a modification of a procedure adapted from the Schanderl 1962 test that visually screens for phenolic content in wines and juices (Zoecklein et al. 1999, p. 459). Duplicate 10 mL samples were placed in clear plastic cups (250 mL capacity) and brought to pH 7 with a saturated solution of aqueous sodium bicarbonate (as opposed to the caustic sodium hydroxide suggested in Zoecklein) while swirling the cup until evolution of carbon dioxide gas ceased. Afterwards, 1-2 drops of the reagent 1% (w/v) aqueous ammonium iron (II) sulfate hexahydrate (Mallinckrodt Baker Inc., Phillipsburg, NJ) was added with swirling to each sample and the color of the resulting mixture and presence or absence of precipitate noted.
To preserve a record of the color and precipitate of each test approximately 0.5 mL of the test solution was blotted onto an absorbent filter paper circle (Fisherbrand P8 qualitative 12.5 cm diam, Fisher Scientific, Chicago, IL) and allowed to air dry and stored in the laboratory notebook. The dried filter papers were examined (using UV-absorbing protective glasses) with a portable ultraviolet lamp (Spectroline UV-4BNF, Westbury, NY) at short wavelengths (254 nm) and long wavelengths (365 nm) for any detectable absorbance or florescence that might be characteristic of a specific category of phenolic compounds (Harborne 1989).
Standards of salicylic acid (Fisher Scientific, Chicago, IL), gallic acid (Sigma-Aldrich, St. Louis, MO.), and quercitin (Twin Labs Inc., Ronkonkoma, NY), were prepared at concentrations of 1mg/mL in 70% (v/v) isopropanol:water and used to establish a visual estimation for comparison to the juice samples. The same field test evaluation for phenolic content as indicated above for juice samples was performed on 1 mL (corresponds to 1 mg) of each standard using distilled water to make up a total test volume of 10 mL.
Salicylic acid, a hydroxybenzoate derivative, was chosen as a relatively safe and inexpensive standard owing to its ubiquity in educational and research laboratories for comparing color intensities. For this study 1 mg of salicylic acid in a 10 mL volume is expressed as 100 mg/L of "Salicylic Acid Equivalents" (SAE) when comparing color intensities of samples of grape juice tested. Dilutions of the salicylic acid standard, using this field test, showed that the lower limits of visual detection of phenolic content ranges from 25 to 50 mg/L SAE. This lower limit is close to the lower end of the range of 16 to 430 mg/L of trans-caftaric acidin grape juice reported for cultivars of V. vinifera tested(Singleton et al. 1986). Gallic acid was employed as a phenolic standard in a spectrophotometric assay of store purchased bottled white grape juice. This sample of store purchased juice was comprised mainly of Thompson Seedless grape juice concentrate, vitamin C, citric acid, and potassium metabisulfite. The gallic acid standard gave a violet-black color with the field test and was used to develop a standard curve to quantify total phenolic content in the store purchased grape juice. The store purchased grape juice had an average total phenolic content of 45.3 mg/L Gallic Acid Equivalents (GAE), standard deviation +/- 1.75 mg/L, from 4 determinations tested.
Disposal after testing. The resulting cups of tested juice described above can be diluted with tap water and discarded in a solid waste disposal container.
Results and Discussion
The results in Table 1 illustrate that the field test is most effective at distinguishing between different grape varieties, and grapes of the same variety that are pressed differently, or of different provenance. Sauvignon Blanc, and Cabernet Sauvignon, grape juice samples of the same provenance showed no variation in their respective phenolic content profiles on three consecutive harvest dates. However samples of Sauvignon Blanc did show a visible difference in the color quality formed in the field test between HP and MP juice. Chardonnay and Grenache juice also appeared to reflect the differences of phenolic content as a result of different pressing methods. These observations appear to be confirmed by evidence that different pressing methods can influence the phenolic content of grape juice (Spanos and Wrolstad 1990; Talcott and Lee 2002).
Merlot, and Syrah, juice samples showed distinct differences in their respective field test profiles between grapes of different provenance. Sauvignon Blanc grapes grown on a north-facing aspect showed a marked reduction in the phenolic content when compared to other samples of Sauvignon Blanc growing in the same vineyard. The variations in phenolic content between samples of like cultivar but different provenance may be the result of different growing conditions that influence the amount of sunlight exposure. It has been demonstrated that Merlot berries with higher sun exposure have almost a ten times greater concentration of total flavonols than shaded berries (Spayd et al. 2002).
Among white cultivars tested the HP juice of Sauvignon Blanc had a higher visual intensity than Thompson Seedless, and among red cultivars the HP juice of Cabernet
Sauvignon had a higher visual intensity than Merlot. These results appear to correlate with earlier studies where Sauvignon Blanc and Thompson Seedless juice had respective concentrations of 97 and 56 mg/L of trans-caftaric acid, and Cabernet Sauvignon and Merlot juice had respective concentrations of 110 and 79 mg/L of trans-caftaric acid (Singleton et al. 1986).
Cabernet Franc, Brunello, and White Riesling grape juice gave the lowest visual estimation for phenolic content showing no precipitate or UV florescence. Viognier and Red Flame Seedless juice gave the highest visual estimation for phenolic content with large amounts of red-brown precipitate. A recent study suggests that the Red Flame Seedless variety has one of the highest phenolic contents of table grape cultivars tested (Cantos et al. 2002).
Standard salicylic acid tested positive for phenolic content with the field test and gave an intense purple colored solution, however precipitate and UV florescence were absent on the blotted filter paper. Quercitin also tested positive and gave a brownish-green colored solution with the field test, but likewise as with salicylic acid no precipitate or UV florescence. For those varieties in Table 1 testing UV florescent positive the observed green florescence was most intense at short wavelengths (254nm) and less so at long wavelengths (365nm). The absence of any UV green florescence at either of these wavelengths with the hydroxybenzoate derivative, salicylic acid, and flavonol, quercitin, suggests the florescent spots could result from a host of phenolic compounds yet to be determined (Harborne 1989).
The Zoecklein et al. 1999 procedure has a code for the resulting colors of test solutions suggesting the category of phenolic compound that may be present, e.g., blackish-violet implies the presence of gallic acid, brownish-red indicates presence of catechins, and red-brown precipitate the presence of ellagic acid. However one must be cautious in relying too much on color quality to actually identify specific categories of phenolic compounds. Colorimetric assays tend to overestimate phenolic content when compared to HPLC or other laboratory techniques due to specificity differences between the methods (Spanos and Wrolstad 1990). However, our spectroassay using the gallic acid standard to quantify the concentration of total phenolic compounds in white grape juice from concentrate compared well with the HPLC data of other studies (see Table V in Spanos and Wrolstad 1990). Thus it is important to consider the variations inherent with each analytical technique employed from study to study.
Conclusions
The study presented here is limited to screening samples of grape juice for a visual estimation of phenolic content, which, more than likely, overestimates the actual value. The field test is not intended to replace or compete with more sophisticated and analytical techniques employed in the laboratory to identify and quantify specific phenolic components. But this limitation does not detract from its utility in the field, vineyard, winery, classroom, or home as a safe, inexpensive, and rapid means to evaluate fresh grape juice samples for their phenolic content. As a visual estimation of relative phenolic content it appears the field test can distinguish differences between fresh grape juice samples of different cultivar varieties. The field test also appears to show differences in phenolic content resulting from different pressing methods. These observations are expected considering the evidence that the concentration of the chief phenolic component, trans-caftaric acid, found in grape juice is highly dependent upon the cultivar of grape and the pressing method employed to extract juice from berries (Vrhovsek 1998). The field test may also be able to distinguish between samples of grapes of the same variety but different provenance since differences in viticulture practices and growing conditions could influence variables, such as sun exposure, that can affect phenolic composition. Further sampling and use should confirm the application and utility of the field test.
Literature Cited
Cantos, E., J. C. Espin, and F. A. Tomas-Barberan. 2002. Varietal differences among the polyphenol profiles of seven table grape cultivars studied by LC-DAD-MS-MS. J. Agric. Food Chem. 50:5691-5696.
Floridi, S., L. Montanari, O. Marconi, and P. Fantozzi. 2003. Determination of free phenolic acids in wort and beer by coulometric array detection. J. Agric. Food Chem. 57:1548-1554.
Harborne J. B. 1989. General procedures and measurement of total phenolics. In Methods in Plant Biochemistry, Volume 1 Plant Phenolics. Dey, P. M. and J. B. Harborne (Eds., pp. 1-28. Academic Press, London.
Haslam, E. 1998. Practical Polyphenolics From Structure to Molecular Recognition and Physiological Action. Cambridge University Press, Cambridge, UK.
Jewell, W. T. and S. E. Ebeler. 2001. Tyrosinase biosensor for the measurement of wine polyphenolics. Am. J. Enol. Vitic. 52:219-222.
Schanderl, H.1962. Der einfluss von polyphenolen un gerbstoffen auf die physiologie der weinhefe und der wert des pH 7 test fur des auswahl von sektgrundweinen. Mitt.Rebe u.Wein, Serie A (Klosterneuburg) 12:265-274.
Singleton, V. L. and P. Esau. 1969. Phenolic Substances in Grapes and Wines and their Significance. Academic Press, New York.
Singleton, V.L. J. Zaya, and E. K. Trousdale. 1986. Caftaric and coutaric acids in fruits of Vitis. Phytochemistry 25:2127-2133.
Spanos, G. A. and R. E. Wrolstad. 1990. Influence of processing and storage on the phenolic composition of Thompson Seedless grape juice. J. Agric. Food Chem. 38:1565-1571.
Spayd, S. E., J. M. Tarara, D. L. Mee, and J. C. Ferguson. 2002. Separation of sunlight and temperature effects on the composition of Vitis vinifera cv. Merlot berries. Am. J. Enol. Vitic. 53:171-182.
Talcott, S. T. and J. H. Lee. 2002. Ellagic acid and flavonoid antioxidant content of Muscadine wine and juice. J. Agric. Food Chem. 50:3186-3192.
Vrhovsek, U. 1998. Extraction of hydroxycinnamoyltartaric acids from berries of different grape varieties. J. Agric. Food Chem. 46:4203-4208.
Waterhouse, A. L. and Walzem, R.
L. 1998. Nutrition of Grape Phenolics.
In Flavonoids in Health and
Disease. Rice-Evans, C. A. and L. Packer (Eds.), pp. 359-385. Marcel
Zoecklein, B. W., K. C. Fugelsang, B. H. Gump, and
F. S. Nury. 1999. Wine Analysis and
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Table 1 Results of field test screening of grape juice for phenolic content
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Variety of grape, Provenance, Date, Pressing,Visual intensity & color of test solution,
presence of precipitate, UV florescence a
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Sauvignon Blanc LC V 09/16 HP ++ brownish-red soln, violet ppt.,+ UV
Sauvignon Blanc LC B 09/16 MP ++ black soln, violet ppt.,+ UV
Sauvignon Blanc LC V 09/17 HP ++ brownish-red soln, violet ppt.,+ UV
Sauvignon Blanc LC B 09/18 MP ++ black soln, violet ppt.,+ UV
Sauvignon Blanc b LC B 09/18 MP + black soln, - ppt.,+ UV
Sauvignon Blanc LC V 09/18 HP ++ brownish-red soln, violet ppt.,+ UV
Cabernet Sauvignon LC V 09/16 HP ++ blackish-purple soln, violet ppt.,+ UV
Cabernet Sauvignon LC V 09/19 HP ++ blackish-purple soln, violet ppt.,+ UV
Cabernet Sauvignon LC V 09/22 HP ++ blackish-purple soln, violet ppt.,+ UV
Cabernet Franc ED B 09/18 HP + blackish-purple soln, - ppt.,- UV
Syrah ED B 09/18 HP + blackish-purple soln, - ppt.,+ UV
Syrah LC B 09/25 HP ++ blackish-purple soln, violet ppt.,+ UV
Merlot LC V 09/19 HP + brownish-red soln, - ppt.,+ UV
Merlot LC B 09/22 HP + brownish-red soln, - ppt.,+ UV
Merlot ED B 09/23 HP ++ brownish-red soln, violet ppt.,+ UV
Brunello ED B 09/22 MP + black soln, - ppt., - UV
Grenache LC B 09/23 HP ++ brownish-red soln, violet ppt., + UV
Grenache LC B 09/23 HP ++ brownish-red soln, violet ppt., + UV
Grenache LC B 09/23 MP + brownish-red soln, violet ppt., + UV
Zinfandel LC V 09/25 HP ++ blackish-purple soln, violet ppt.,+ UV
Viognier ED B 09/29 HP +++ brownish-red soln, red-brown ppt., +UV
White Riesling GV V 10/02 HP + black soln, - ppt., - UV
Chardonnay ED B 09/25 HP + blackish-purple soln, - ppt., +UV
Chardonnay ED B 09/25 MP ++ blackish-purple soln, violet ppt., +UV
Chardonnay c UP P 09/14 MP + black soln, - ppt., +UV
Red Flame Seedless UP P 08/21 HP +++ brownish-red soln, red-brown ppt., +UV
Thompson Seedless UP P 08/21 HP + brownish-red soln, red-brown ppt., +UV
a Visual intensity is based on a comparative scale expressed as mg/L "Salicylic Acid Equivalents" (SAE) corresponding to a phenolic content of +++ > 100 mg/L, ++ ~ 100 mg/L, + < 100 mg/L, and colors of the resulting test solution (soln), and presence or absence (-) of precipitate (ppt.) collected on filter paper. +/- UV indicates the presence or absence of green florescence of the filter paper spots when exposed to UV light at 254 nm. See Materials and Methods for all details, and abbreviations regarding provenance, and pressing method of grapes.
bThis batch of Sauvignon Blanc grapes are from a lot grown on a north-facing aspect in the LC vineyard.
c Must from a
wholesaler in the
Table 2. Analysis of processed fruit juices
and green tea (mg phenolics/L juice)*
*using method described in Sady, Michael B. J. Chem. Educ. 2005 82 1808A, and gallic acid standard (GAE)
Bottled White “Langers” grape juice 28.37
Bottled White “Fine Foods” grape juice 180.9
Green-White “Stash” tea 780.2
Frozen “Welches” White grape juice 174.6
Frozen “Apple Time” apple juice 299.8
Credit Chem
220 lab students Spring ‘07
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