Red Wine Tannin StructureâActivity Relationships during

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Red Wine Tannin Structure-Activity Relationships during Fermentation and Maceration Ralph S. Yacco, Aude Annie Watrelot, and James A. Kennedy J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.5b05058 • Publication Date (Web): 14 Jan 2016 Downloaded from http://pubs.acs.org on January 16, 2016

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Journal of Agricultural and Food Chemistry

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Red Wine Tannin Structure-Activity Relationships

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during Fermentation and Maceration

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Ralph S. Yacco, Aude A. Watrelot and James A. Kennedy†,*

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Department of Viticulture and Enology, California State University, 2360 E. Barstow Avenue

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MS VR89, Fresno, California, USA, 93740-8003

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†

Current Address: Constellation Brands, Inc., 12667 Road 24, Madera, California, USA 93637

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*Corresponding Author

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Tel: +1 559-661-5563

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Email: [email protected]

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Running Head: Tannin Structure-Activity Relationships

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KEYWORDS:

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proanthocyanidin, winemaking, thermodynamics, enthalpy of interaction, composition,

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molecular size, pigmented tannin

1 ACS Paragon Plus Environment


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ABSTRACT

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The correlation between tannin structure and corresponding activity was investigated by

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measuring the thermodynamics of interaction between tannins isolated from commercial red

21

wine fermentations and a polystyrene divinylbenzene HPLC column. Must and/or wine samples

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were collected throughout fermentation/maceration from five Napa Valley wineries. By varying

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winery, fruit source, maceration time and cap management practice, it was considered that a

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reasonably large variation in commercially-relevant tannin structure would result. Tannins were

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isolated from samples collected using low pressure chromatography and were then characterized

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by gel permeation chromatography and acid-catalyzed cleavage in the presence of excess

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phloroglucinol (phloroglucinolysis). Corresponding tannin activity was determined using HPLC

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by measuring the thermodynamics of interaction between isolated tannin and a polystyrene

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divinylbenzene HPLC column. This measurement approach was designed to determine the

30

ability of tannins to hydrophobically interact with a hydrophobic surface. The results of this

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study indicate that tannin activity is primarily driven by molecular size. Compositionally, tannin

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activity was positively associated with seed tannins and negatively associated with skin and

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pigmented tannins. Although measured indirectly, the extent of tannin oxidation as determined

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by phloroglucinolysis conversion yield suggests that tannin oxidation at this stage of production

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reduces tannin activity. Based upon maceration time, this study indicates that observed increases

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in perceived astringency quality, if related to tannin chemistry, are driven by tannin molecular

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mass as opposed to pigmented tannin formation or oxidation. Overall, the results of this study

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give new insight into tannin structure-activity relationships which dominate during extraction.


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INTRODUCTION Proanthocyanidins (PAs) are plant-derived phenolic compounds that are extracted from

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grapes during red wine production (1-9) and are responsible for astringency (10-12).

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Astringency is an important quality attribute of red wine and is known to vary with tannin

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structure. Because of the importance of tannin structure in overall wine quality, there is ongoing

45

interest in understanding and managing tannin structure during production operations.

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Rationalizing the role that tannin structure plays in perception has been studied (13-16).

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From these investigations, it is generally recognized that tannins interact with salivary protein

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and oral mucosa through a combination of hydrophobic interactions and hydrogen bonding.

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Specific studies have pointed out the importance of hydrophobic interaction (hereafter activity)

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as a driver in protein-tannin interaction (13) and the role that tannin structure has on

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corresponding activity (15, 16). Given this, developing objective analytical tools for managing

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tannin perception would presumably rely on being able to routinely measure variation in tannin

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activity.

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Given the importance of tannin concentration on perceived astringency, tannin analytical

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methodology has historically focused solely on red wine concentration determination. In

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contrast to this, analytical methodology that routinely measures tannin activity was lacking until

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recently. Based upon studies that have shown that considerable variation in red wine tannin

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structure exists (17-19), astringency variation with a basis in tannin structure is evident. For

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example, an increase in tannin galloylation has been shown to enhance flavan-3-ol activity with

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proteins suggesting that esterification of flavan-3-ols with gallic acid enhances astringency (10).



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The incorporation of color into the tannin polymer is also thought to modify tannin

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activity. Previous work has shown that anthocyanin and tannins react to form pigmented

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polymers. The formation of these molecules under wine conditions has been shown to increase

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the physical chemical stability of tannins and anthocyanin in the resulting wine (18). From a

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sensory standpoint, the formation pigmented polymers would be expected play an essential role

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in the modulation of astringency. Specifically, the formation of pigmented polymers would be

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expected to increase tannin glycosylation and charge (via the incorporation of anthocyanin into

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the tannin structure) and from this, it would be expected that pigmented tannin would have

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reduced ability to hydrophobically interact and by extension would be less astringent. This

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explanation is consistent with previous work showing that a proportional increase in pigmented

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polymer leads to a decrease in astringency when compared to unpigmented tannins (11).

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It is of interest to develop an understanding of how wine production operations influence

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tannin structure-activity relationships. Sensory studies have highlighted the importance of tannin

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structure on perception and it is of interest to develop a non-human analytical approach that

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draws the same conclusions as sensory studies. The focus of this study was to develop a tannin

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structure-activity understanding during the initial stage of wine production: fermentation and

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maceration, using a recently developed analytical method designed to measure tannin activity.

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This study included the isolation of tannins, subsequent structure and activity determination and

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finally, comparisons with previously published sensory studies.

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EXPERIMENTAL SECTION


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Chemicals and Samples. All solvents were HPLC grade. Acetonitrile, acetone,

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methanol, acetic acid, L-(+) ascorbic acid, hydrochloric acid, lithium chloride, o-phosphoric

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acid, N,N-dimethylformamide, and anhydrous sodium acetate were purchased from VWR

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International (Radnor, PA). Phloroglucinol, (-)-epicatechin and trifluoroacetic acid (99%

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spectrophotometric grade) were purchased from Sigma-Aldrich (St. Louis, MO).

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Samples from the 2014 harvest season were obtained from five participating Napa

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Valley, CA wineries and were collected throughout fermentation/maceration. A total of 91

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samples were collected, with collection intent being to develop a reasonably large set of samples

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with each sample having unique tannin composition. To achieve sample variation each winery

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was allowed to develop a controlled experiment of interest. Experiments varied according to

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maceration time (10-62 days), variety (Cabernet Sauvignon or Petit Verdot) and experiment

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(maceration variable).

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Samples were collected by participating winery personnel and were collected following

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cap management operations, from crush through pressing. At each sampling, flint glass bottles

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(375 mL) were filled to capacity and were kept at 4 °C until transported to the laboratory for

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tannin isolation. Samples were collected from participating wineries once or twice per week and

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were kept in an insulated container over dry ice (a styrofoam screen separated dry ice from

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samples) during transport. Once in the lab samples were stored at 4° C until tannin isolation and

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purification operations.. Tannin Isolation, Purification and Gravimetric Recovery. Tannin isolation and

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purification was conducted according to Aron and Kennedy (19). Briefly, samples

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(approximately 206 mL) were filtered through Whatman No. 1 filters (90mm, 11µm pore size),



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and then applied (~18mL/min) to a glass column (Kimble Kontes ChromaflexTM, 4.8x15 cm,

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Vineland, NJ) containing Toyopearl® chromatography resin (approximate bed volume of 272

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mL, HW 40C, Supelco Bellefonte, PA) equipped with a peristaltic pump assembly (Cole Parmer,

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drive Mflex, Pump head easy-load II SS, Chicago, IL). The column had previously been

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equilibrated with water containing 0.05% v/v trifluoroacetic acid (TFA). The applied must/wine

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was rinsed with 1.0 L water containing 0.05% v/v TFA to remove sugars and organic acids, 1.0

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L of 1:1 methanol:water containing 0.05% v/v TFA to remove anthocyanins and other low

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molecular weight phenolics, and finally, 300 mL of 2:1 acetone:water containing 0.05% v/v TFA

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to elute the tannin fraction of interest. The column was then washed with 500 mL water,

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followed by 200 mL 2:1 acetone:water, again with 500 mL water, and finally re-equilibrated

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with 500 mL water containing 0.05% v/v TFA. The tannin fraction was concentrated under

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reduced pressure at 38 °C to remove acetone and then lyophilized to a dry powder. Isolates were

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weighed to determine gravimetric recovery.

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Instrumentation. An Agilent 1260 (Santa Clara, CA) HPLC system was used to conduct

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all analyses. It consisted of a model G1331B pump and degasser, G1329B autosampler, G1315C

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DAD/UV-Vis Detector, G1316A column heater and a system controller.

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Tannin Activity, Pigmented Tannin and Concentration. The activity, pigmented

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tannin and concentration of tannin isolates were determined using the previously described

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HPLC method of Revelette et al. (20). This method is based upon the thermodynamics of

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interaction between injected tannin isolate and polystyrene divinylbenzene HPLC column as

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discussed by Barak and Kennedy (21). To prepare tannin isolates for activity measurement, 5

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mg of tannin was dissolved in 1 ml of an aqueous solution containing methanol (15% v/v), 40

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mM sodium acetate, and 20 mM HCl model wine. 6 ACS Paragon Plus Environment

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Tannin activities were determined by HPLC according to the method of Revelette et al.

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(20). Briefly, the HPLC method utilized a polystyrene divinylbenzene reversed-phase column

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(PLRP-S, 2.1 x 50 mm, 100Å, 3µm, Agilent Technologies, Santa Clara, CA) protected with a

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guard column (PRP-1, 3 x 8 mm, Hamilton Company, Reno, NV).

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The mobile phases consisted of 1.5% (w/w) 85% H3PO4 in water (mobile phase A) and 20%

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(v/v) mobile phase A in acetonitrile (mobile phase B) with a flow rate of 0.30 mL/min. The

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linear gradient was as follows: time in min (%B), 0 (14%), 10.0 (34%), 10.0-13.5 (34%), 15.3

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(70%), 15.3-17.0 (70%) and 17.0-20.0 (14%).

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In order to determine tannin activity, samples were run at four column temperatures (25-40

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°C, 5 °C increments). A blank sample (water) was run at all temperatures and was subtracted

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from sample signals to eliminate background absorbance. All temperatures were converted into

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Kelvin in order to calculate thermodynamic parameters in SI units. Tannin elution was

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monitored at 280 and 520 nm. Grape skin or wine tannin standard (mDP=39.0 and 15.4 (by

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phloroglucinolysis), respectively) served as an activity standard. Activity standards were

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prepared by dissolving tannin in an aqueous solution (5 g/L) containing methanol (15% v/v), 40

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mM sodium acetate, and 20 mM HCl. Activity standards did not vary by more than 10% across

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all experiments.

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Following baseline subtraction of a water blank, chromatograms were integrated by

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establishing a baseline at 0 mAU across the entire sample peak (TanninT, Figure 1). This peak

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area was then split just after the elution of (-)-epicatechin and at 16.8 minutes with the peak

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eluting after 16.8 min corresponding to TanninP.

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From the integration results, the alternative retention factor was calculated as follows:



=

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150

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(1)

where:

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= Total tannin peak area

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= Partial tannin peak area

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The alternative retention factor is related to thermodynamics of interaction according to the

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following equation:

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ln = −

∆°

+

∆°

+ ln∅

(2)

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where ∆H° and ∆S° are the specific enthalpy and entropy of interaction, respectively, R is the

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gas constant, T is temperature and ∅ is the mobile phase ratio. The specific enthalpy was

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calculated by plotting the natural logarithm (ln) of kalt versus the reciprocal of the column

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temperature in Kelvin at each of the four temperatures (i.e.: van′t Hoff plot). The specific

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enthalpy of interaction (tannin activity) was calculated from the slope of the best fit line (slope

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equivalent to −

162

∆°

).

The concentration of tannin (based upon ) was reported in (-)–epicatechin

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equivalents using an (-)-epicatechin standard, and was determined at 303 K (30 °C). In addition

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to measuring activity and concentration, percent pigmented tannin was determined by dividing

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the tannin peak (TanninT) area at 520 nm (in mAU) by that at 280 nm. Determining pigmented

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tannin by this HPLC approach has been discussed elsewhere (22).

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Gel Permeation Chromatography (GPC). With minor variations, the size distribution of tannin isolates was determined using the previously described method of Kennedy and Taylor


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(23). Briefly, the high-performance GPC method used to analyze tannins consisted of 2 PLgel

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(300 x 7.5 mm, 5µm, 1000 by 500Å) columns connected in series and protected by a guard

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column containing the same material (50 x 7.5 mm, 5 µm), all purchased from Agilent (Santa

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Clara, CA). The targeted sample injection amount was 40 µg. The isocratic method utilized a

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mobile phase consisting of N,N-dimethylformamide containing 1% v/v glacial acetic acid, 5%

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v/v water and 0.15 M lithium chloride. The flow rate was maintained at 1 ml/min with a column

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temperature of 60 °C and elution was monitored at 280 nm.

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Calibration curves were constructed using fractionated pre-veraison grape skin

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proanthocyanidins (PAs), by correlating their average molecular mass (determined by

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phloroglucinolysis) with their retention time at 50% elution. Initial PA (Vitis vinifera L. cv.

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Cabernet Sauvignon) isolation was accomplished according to the method described by Kennedy

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and Jones (24). For preparation of standards, 1.0 g of PA was dissolved in 50 mL 60% (v/v)

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HPLC grade methanol containing 0.05% v/v TFA and then applied to (~18mL/min) a glass

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column (Kimble Kontes ChromaflexTM, 4.8x15 cm, Vineland, NJ) containing Sephadex® LH20

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chromatography resin (Amersham, Uppsala, Sweden) to an approximate bed volume of 200 mL.

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The column was previously equilibrated with 60% v/v methanol containing 0.05% v/v TFA. The

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applied PA was fractionated using the solvent system described by Kennedy and Taylor (23) but

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formic acid was replaced with 0.05% v/v TFA. Eluted fractions were concentrated under reduced

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pressure at 38 °C to remove organic solvents and then lyophilized to a dry powder. Individual

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isolates were then used to construct a calibration curve.

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Acid-Catalysis in the Presence of Excess Phloroglucinol (Phloroglucinolysis). Tannin

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subunit composition and conversion yield of tannin isolates were determined using the

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previously described method of Kennedy and Jones (24) but with modified HPLC conditions as 9 ACS Paragon Plus Environment


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described by Kennedy and Taylor (23). Briefly, the reversed-phase HPLC method consisted of

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two Chromolith RP-18e (100 x 4.6 mm) columns connected in series and protected by a guard

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column containing the same material (4x4 mm), all purchased from EM Science (Gibbstown,

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NJ). The method utilized a binary gradient with water containing 1% v/v aqueous acetic acid

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(mobile phase A) and acetonitrile containing 1% v/v acetic acid (mobile phase B). Pump flow

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rate was kept at 3.0 mL/min with a column temperature of 30 °C and with elution monitored at

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280 nm. The linear gradient was as follows: time in min (%B); 0 (3%), 4.0 (3%), 14.0 (18%),

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14.01 (80%), 16.0 (80%), 16.01 (3%), 18.0 (3%). (-)-epicatechin was used as a quantitative

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standard and calculations for subunit composition and conversion yield were as described by

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Kennedy and Jones (24).

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reported as a proportion of the gravimetric recovery and was reported as a percentage.

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For conversion yield calculations, calculated subunit mass was

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Data Analysis. Box car plots of compositional data were constructed by determining the

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mean for each fermentation and across all maceration times for any given chemistry (e.g.: tannin

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activity). Following this, and within each fermentation, individual chemistry values were

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compared to the mean value and expressed as a percentage. Finally, across all fermentations and

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within specific maceration times (e.g.: crush, end of fermentation) box car plots were constructed

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using SigmaPlot (ver. 11.0, Systat Software, Inc., San Jose, CA).

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Principal component analysis of tannin structure variation including: extension subunit

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composition, terminal subunit composition, red color incorporation, molar mass at 50% elution,

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conversion yield, and activity was constructed using SAS (ver. 9.2, SAS Institute, Cary, NC) to

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determine the nature and extent of correlation between structure related variables and activity.

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RESULTS AND DISCUSSION

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This study was conducted in order to better understand the relationship between tannin

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structure and corresponding activity during red wine production operations. Given that tannin

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structure is expected to vary significantly during fermentation/maceration, this period of wine

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production was the focus of this investigation. Further, and in order to develop a commercially-

219

relevant dataset, tannins were isolated from samples collected from commercial fermentations of

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Napa Valley grown fruit and predominantly Cabernet Sauvignon. Although each winery

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conducted a controlled experiment, individual experiments were not replicated. Importantly, and

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for the purposes of this experiment, the intention was to develop upwards of 100 unique tannin

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samples so that tannin structure-activity relationships could be explored. Of less importance,

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given the lack of replication, was the interpretation of individual experiments. Nevertheless,

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some understanding of the effect of maceration on tannin structure and activity was possible

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given the experimental approach.

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Tannin Isolation. Tannins were isolated from samples collected throughout maceration

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and were purified according to the procedure developed by Aron and Kennedy (19). This

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procedure has been successfully used to isolate tannins from musts as well as wine and with

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good recovery. In order to determine the effectiveness of tannin isolation in the current study,

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the gravimetric recovery of tannin was compared to the in situ tannin concentration

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determination by HPLC. Given that the HPLC method being used for tannin activity assessment

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is based upon a previously developed HPLC method (21, 22), the tannins in the sample were

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quantified similarly yet under modified HPLC conditions (20).



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The results of the tannin isolation suggest that the method was effective (Figure 2).

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Specifically, there was a linear relationship between sample tannin concentration in must/wine

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by HPLC and corresponding gravimetric recovery (r2=0.84) and with a line slope approaching

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one (slope = 1.07). There was an increase in variation between the two analytical methods at

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higher concentrations. Based upon the y-intercept (0.22 g/L), the absolute value for

240

gravimetrically-determined tannin concentration was greater than the corresponding must/wine

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tannin concentration determined by HPLC. Two potential reasons for this variation in absolute

242

value include: 1) there were tannins eluting prior to the designated tannin peak by HPLC and 2)

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the presence of (-)-epigallocatechin subunits in the tannin samples leads to an error in HPLC

244

quantitation when using (-)-epicatechin as a quantitative standard because of the lower molar

245

absorptivity of (-)-epigallocatechin subunits.

246

For all individual fermentations, tannin concentration using the gravimetrically-

247

determined tannin amount increased with maceration time although there were some exceptions

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particularly at longer maceration times (Supporting Information S1). Specifically and for some

249

fermentations, the gravimetric recovery declined. This may have been a result of an increase in

250

the exposed surface area available for tannin adsorption (e.g.: yeast and grape cell wall area) and

251

subsequent fining.

252

Across all wineries, and consistent with results shown by Cerpa-Calderón and Kennedy

253

(17), the extraction of tannin during the initial stages of fermentation followed a Boltzmann

254

sigmoid extraction profile (r2 generally >0.92 for individual fermentations). Specifically, tannin

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extraction increased rapidly, generally reaching an apparent plateau within 10 days. The

256

consistency of this extraction profile with that of Cerpa-Calderón and Kennedy suggests that

257

initial extraction was dominated by skin tannin extraction. Based upon gravimetric recovery, the 12 ACS Paragon Plus Environment

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average plateau concentration was 1.42 g/L (SEM = 0.06 g/L) and the time to 50% of plateau

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concentration was 4.71 days (SEM = 0.88 day). While plateau tannin concentrations were

260

generally achieved within 10 days, total maceration time lasted for up to 62 days.

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Activity of Tannin Isolates. The major purpose of this study was to determine tannin

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activity variation as a function of maceration and to investigate tannin structural features that

263

influence corresponding activity. Tannin activity in this case is defined as the negative of the

264

enthalpy of interaction between tannin and a hydrophobic surface (polystyrene divinylbenzene

265

reversed phase HPLC column) (20-22).

266

Tannin activity values were at their lowest value at the beginning of maceration and

267

generally followed a similar pattern to that of concentration (Supporting Information S2).

268

Specifically, tannin activity increased and reached a maximal value at a time that was similar to

269

tannin concentration. From that point, tannin activity was more variable than concentration. For

270

several of the extended maceration experiments, tannin activity values declined after reaching a

271

maximum whereas for other experiments, tannin activity maintained its plateau value.

272

It was interesting to note that when comparing tannin activity with corresponding tannin

273

recovery across all fermentations (Figure 3), the activity appeared to increase exponentially to a

274

maximum value of 6828 J/mol (r2=0.77). This observation suggests that there was a theoretical

275

maximum value for tannin activity for these wines. This observation could be explained by the

276

role that adsorption sites (e.g.: yeast and grape cell walls) play relative to extraction and is

277

wholly consistent with observations made by others (25-29). With that said, there was

278

considerable variation in the concentration of tannin as the apparent tannin activity reached a

279

plateau. It is not surprising that variation in concentration-activity exists given that variables



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such as maceration time, temperature, grape berry cell integrity, grape cell wall area exposure to

281

extracted tannin, yeast population and surface area, anthocyanin concentration have all been

282

shown to influence tannin concentration and composition (1-9, 25-31).

283

Tannin Composition. In order to gain an insight into the factors that lead to variation in

284

tannin activity, a number of tannin structure elements were measured in isolates including

285

pigmented tannin, conversion yield and PA subunit composition (Figure 4, Supporting

286

Information S2). For Figure 4, the specific chemistry data for four time points (after crush,

287

following alcoholic fermentation, midway through maceration, and free run when pressed) were

288

averaged across all times and experiments. Individual experiments and time points were then

289

compared to the average value for a given time point to develop box car plots for comparison.

290

Given that individual fermentations were unreplicated and there were multiple wineries and fruit

291

sources, tendencies in chemistry are discussed. Conclusions drawn across all fermentations were

292

generally similar to individual fermentations in the case of tannin gravimetric recovery and

293

activity (comparison of Supporting Information Table S2 with Figures 4A and 4B).

294

Pigmented tannins are generally considered to be PAs that are covalently associated with

295

anthocyanins. In this study, the pigmented tannin contribution to tannin structure was

296

determined by monitoring the proportion of tannin that absorbed light at 520 nm, as described

297

elsewhere (22). Using this approach (Figure 4C) percentage of pigmented tannin tended to

298

increase during the early stages of maceration, reaching an apparent plateau in the region where

299

overall tannin extraction plateaued. During longer maceration times, the contribution of

300

pigmented tannin to the overall isolate declined. In absolute amounts, pigmented tannin was at

301

its highest before the end of fermentation (data not shown) suggesting that pigmented tannin

302

formation is occurring during fruit ripening, consistent with previous observations (32). 14 ACS Paragon Plus Environment

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One of the primary reasons for isolating tannins from musts and wines was so that the

304

phloroglucinolysis conversion yield could be determined in order to provide an impression of

305

tannin modification. Because of the difficulty in determining the extent to which tannins have

306

become modified structurally, conversion yield served as a means for determining how closely

307

tannin structure resembled PA structure initially present in fruit. PA conversion yield has been

308

shown to decline during fruit ripening, wine age and deliberate oxidation (12,33-37). There are

309

multiple reaction mechanisms that would be expected to lead to a reduction in conversion yield

310

including oxidation and acid-catalysis. Of specific interest for the current study is that a decline

311

in conversion yield has been associated with a reduction in tannin activity (21, 36).

312

The results of this study indicated that isolate conversion yield tended to increase with

313

maceration time and was highest at the end of maceration (Figure 4D, Supporting Information

314

Table S2), consistent with previous work (19). Based upon pigmented tannin and conversion

315

yield, the data presented in this study suggest that initially extracted tannins, while low in

316

concentration are more extensively modified than tannins extracted later. To the extent that

317

conversion yield can provide information on tannin modification and for the very longest

318

maceration times, conversion yield information suggests that tannin structure increasingly

319

resembles plant-based PAs with maceration time. Moreover, this result suggests that the

320

oxidative modification of tannin structure does not occur on this scale and method of production.

321

The size distribution of tannin isolates was determined by gel permeation

322

chromatography (23). Based upon the molecular mass at 50% elution time (Figure 5, Supporting

323

Information S2) tannin size increased with maceration time reaching a maximum in the same

324

region where isolate concentration plateaued; thereafter, the tannin molecular mass at 50%

325

elution declined. Previous studies have shown that seed tannins require a longer period of 15 ACS Paragon Plus Environment


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326

maceration to be extracted (8, 17). In addition, it has been shown that seed tannins are smaller

327

than skin tannin by mean degree of polymerization measurements (24, 38, 39). Given these

328

observations, the reduction in tannin size observed during the later stages of maceration suggests

329

that either seed tannin extraction is increasing (17) and/or fining by yeast and grape biomass is

330

occurring at this time (27, 40-42).

331

The PA composition of isolates was determined by phloroglucinolysis in order to provide

332

information on the role that skin and seed tannins are contributing to isolate composition.

333

Compositionally, subunits that are found in both skins and seeds ((+)-catechin, (-)-epicatechin)

334

or proportionally higher in seeds ((-)-epicatechin-3-O-gallate) (43) increased with maceration

335

time, while subunits that are specific to skin ((-)-epigallocatechin) (44) declined (Figures 4E and

336

4F, Supporting Information Table S2).

337

The proportion of (-)-epigallocatechin subunits in isolates was generally highest early in

338

maceration and remained high until tannin isolate concentration plateaued. Thereafter and for

339

most experiments, the proportion of (-)-epigallocatechin subunits declined. These results are

340

consistent with skin tannin extraction dominating extraction early and help to explain the

341

Boltzmann sigmoid extraction profile observed in this study.

342

Interestingly and at the earliest stages of extraction, some of the isolates had a low

343

proportion of (-)-epigallocatechin (Supporting Information Table S2, Figure 4E). Lower

344

proportions of (-)-epigallocatechin are consistent with PAs that are localized in the mesocarp of

345

the berry (28), and given the very low overall tannin extraction at this time, could be an

346

explanation for the lower than expected (-)-epigallocatechin extension subunit proportion.


Page 17 of 40

347


The observed increase in the proportion of (-)-epicatechin-3-O-gallate with maceration

348

time (Figure 4F) was correlated with a reduction in tannin molecular size and taken together

349

provide evidence that seed tannin extraction is dominating extraction during the latter stages of

350

maceration. This is also supported by the concomitant reduction in skin tannin specific (-)-

351

epigallocatechin subunits (8, 9, 45, 46).

352

Compositional data gathered in this study highlights the complexity of commercial

353

fermentations. Although these commercial fermentations all took place within the Napa Valley

354

and with grapes sourced from the Napa Valley, considerable variation in tannin structure

355

resulted. One of the conclusions drawn from Figure 4 was that tannin structure changes

356

significantly through the course of fermentation and maceration. In order to understand how

357

specific structure change influenced tannin activity, it was necessary to conduct principal

358

component analysis.

359

Tannin Structure-Activity Correlations (Principal Component Analysis). The

360

aforementioned HPLC method for measuring tannin activity is intended to be useful in the

361

understanding of how concentration-independent perceptual differences in red wine astringency

362

are a result of tannin-related chemistry. In order to provide this level of utility, relationships

363

between structure and corresponding activity need to be determined over a commercially-

364

relevant dataset and the results need to be compared with sensory studies that compare tannin

365

structure with perceived astringency.

366

It is proposed that the tannin structure influences corresponding activity with salivary

367

proteins and/or oral mucosa (15, 16, 47-52). In this study, principal component analysis was

368

used in order to determine if there are tannin structural elements that influence tannin activity.



369

Based upon this statistical approach (Figure 6), there were clear relationships between tannin

370

structure and corresponding activity.

371

Page 18 of 40

According to the PCA (Figure 6), tannin activity during fermentation/maceration is

372

primarily driven by tannin molecular mass followed by conversion yield and then compositional

373

elements (i.e.: pigmented tannin and subunit composition). To the extent that tannin activity

374

effects are analogous to tannin-protein interaction, tannin activity is predicted to be positively

375

associated with astringency. Given these results and based upon the current study, perceived

376

astringency would be maximal near day 10 in most fermentations, with isolate concentration and

377

activity both being maximal. With regard to activity specifically, tannin molecular mass in this

378

study was most strongly related to tannin activity. In previous studies, an increase in tannin

379

molecular mass was associated with an increase in perceived astringency (10, 48).

380

With regard to isolate composition, the PCA suggests a positive correlation between (-)-

381

epicatechin-3-O-gallate extension subunits and activity, while (-)-epigallocatechin extension

382

subunits had a negative correlation with activity. These results are consistent with seed versus

383

skin tannin associations with red wine astringency (14). From an activity standpoint, this

384

difference can be explained by flavan-3-ol esterification with gallic acid, which increases the

385

hydrophobicity of (-)-epicatechin-3-O-gallate over (-)-epigallocatechin (10, 47). Furthermore,

386

the higher more positive correlation between tannin activity and (+)-catechin over (-)-epicatechin

387

is consistent with previous studies that have found that (+)-catechin induced higher turbidity

388

upon reaction with proline rich peptides in comparison to (-)-epicatechin (49).

389 390

With respect to red color incorporation, the PCA shows a negative correlation between pigmented tannin and tannin activity. This correlation is consistent with sensory studies that have


Page 19 of 40


391

shown that tannin polymers bearing anthocyanin moiety within their structure are less astringent

392

in comparison to similar sized tannins with no anthocyanin (11). This can be explained by the

393

increased polarity that pigmented tannin polymers possess due to the glycoside portion of the

394

anthocyanin and the presence of the flavylium equilibrium form, when compared to tannin

395

polymers without anthocyanin.

396

Overall, while sensory studies were not conducted in the present study, the results shown

397

in Figure 6 are consistent with predicted effects of tannin structure on perceived astringency and

398

the proposed relationship between tannin activity and corresponding astringency.

399

Tannin Molecular Mass Distribution and Maceration. Given the role that tannin

400

molecular mass has on tannin activity, it was of interest to explore the effect of maceration on

401

tannin activity further (Figures 5 and 7). Across all experiments, the molecular mass distribution

402

of isolates was determined by gel permeation chromatography. From these results and for each

403

experiment, the cumulative mass distribution of the four time points mentioned previously, was

404

plotted corresponding to samples collected initially after crush, following alcoholic fermentation,

405

midway through maceration, and finally, free run when pressed (Figure 5 inset). Interestingly,

406

for all experiments, the size distribution was lowest for the earliest samples and highest for the

407

samples immediately following alcoholic fermentation. When averaged, the molecular mass

408

distribution declined with subsequent maceration time.

409

These results are consistent with earlier studies and suggest the predicted “tension”

410

between extraction and subsequent adsorption/fining at this time (25-28). Specifically, as

411

ethanol concentration increases so does the molecular mass of the extracted tannin. This would

412

be due to the solvent properties of ethanol relative to water (ethanol is more hydrophobic) and



Page 20 of 40

413

corresponding effect of tannin cell wall interaction. Following ethanol production, the

414

subsequent reduction in cumulative mass distribution could be explained by an increase in the

415

extraction of lower molecular mass seed tannins (which have been shown to be extracted more

416

slowly than skin tannins) and/or the grape berry tissues (or yeast biomass) in the fermenter may

417

be exposing adsorption sites which subsequently fine extracted tannin (27, 40-42). Based upon

418

tannin concentration and subunit composition (Supporting Information S2), the results of this

419

study suggest that both processes are occurring. A specific goal of this study was to develop an improved understanding of the extended

420 421

maceration technique used in wine production. One of the perceived benefits of extended

422

maceration is that astringency quality can be improved with longer maceration times. With

423

regard to tannin structure and corresponding activity, multiple explanations can be posited for

424

this observation including a reduction in concentration or a modification in structure leading to a

425

reduction in activity (e.g.: pigmented tannin formation, tannin oxidation, reduction in size). In

426

addition, non-tannin explanations could also explain this observation (e.g.: mannoprotein

427

interaction with tannin). Based upon the 62 day maceration experiment which was the most

428

comprehensively investigated fermentation (Figure 7, Supporting Information S2), and

429

considering tannin activity, molecular mass seems to be the major explanation for the observed

430

reduction in activity. Moreover, and considering the other chemistries monitored, tannin

431

oxidation, pigment incorporation and subunit composition are inconsistent with the observed

432

reduction in tannin activity with extended maceration (Figure 7 and inset comparison with Figure

433

6).

434 435

In light of the apparent role of tannin molecular mass on corresponding activity observed in these commercial experiments, this study highlights the importance of monitoring tannin 20 ACS Paragon Plus Environment

Page 21 of 40


436

molecular mass distribution in red wine production. Monitoring the molecular mass at 50%

437

elution provided information that was most strongly associated with tannin activity during this

438

stage of production. Moreover, the tannin molecular mass at 90% elution exhibited even more

439

significant change than that at 50% mass elution. Given the recent developments in

440

understanding the significance of adsorption on the selection of tannins in red wine (25-28) in

441

addition to the results presented here, there is some rationale for routinely monitoring tannin

442

molecular mass.

443

Relevance to Wine Production. The purpose of this study was to improve our

444

understanding of tannin structure-activity relationships, with an emphasis on commercial

445

production operations. This study focused on the earliest stage of production: fermentation and

446

maceration. The results indicate that tannin activity as measured by HPLC, provides structure-

447

activity information that is consistent with sensory studies. To date the cause and effect

448

approach to understanding tannin structure-astringency variation has been accomplished by

449

conducting sensory studies on precisely controlled variations in tannin structure. The utility of

450

measuring tannin activity by HPLC is highlighted given that tannin activity can be measured

451

more rapidly and with results that are consistent with sensory studies.

452

Understanding the utility of tannin activity determination by HPLC is shown in the

453

investigation of extended maceration studies. In this study we were able to show that tannin size

454

is the major tannin structure-related explanation for the perceived benefit of maceration time on

455

astringency quality. Specifically, at longer maceration times, tannin molecular mass declines

456

markedly. Other explanations including tannin oxidation, pigmented tannin formation were not

457

evident given the data presented here. Knowing that tannin activity reductions during extended

458

macerations are a result of a reduction in tannin molecular mass provides an opportunity to 21 ACS Paragon Plus Environment


459

reduce maceration time and associated risks. For example, if a reduction in tannin activity is

460

desired, the lowering of wine temperature just prior to pressing maximizes the opportunity for

461

adsorptive fining of large tannins. Alternatively, the addition of cell wall material (e.g.: yeast

462

hulls) while still on the skins would provide an opportunity to fine out larger tannins while still

463

extracting smaller-sized and less active seed tannins.

464

Page 22 of 40

This study highlights how tannin structure variation can lead to variations in the ability of

465

tannin to hydrophobically interact with a hydrophobic surface. Additionally, the ability to

466

measure activity information compliments concentration information that is common. The

467

results also highlight how tannin activity can help to explain astringency quality improvement

468

with extended maceration and provides a structural explanation for this.

469


Page 23 of 40


470

471

472 473 474 475 476

REFERENCES 1. Berg, H. W.; Akiyoshi, M. The effect of contact time of juice with pomace on the color and tannin content of red wines. Am. J. Enol. Vitic. 1956, 7, 84-90. 2. Berg, H. W.; Akiyoshi, M. Further studies of the factors effecting the extraction of color and tannin from red grapes. Food Res. 1958, 23, 511-517. 3. Zimman, A.; Joslin, W.S.; Lyon, M. L.; Meier, J.; Waterhouse, A. L. Maceration

477

variables affecting phenolic composition in commercial-scale cabernet Sauvignon

478

winemaking trials. Am. J. Enol. Vitic. 2002, 53, 93-98.

479

4. Cortell, J. M.; Halbleib, M.; Gallagher, A. V.; Righetti, T.; Kennedy, J. A. Influence of

480

vine vigor on grape (Vitis vinifera L. Cv. Pinot noir) and wine proanthocyanidins. J.

481

Agric. Food Chem. 2005, 53, 5798-5808.

482

5. Puertas, B.; Guerrero, R. F.; Jurado, M. S.; Jimenez, M. J.; Cantos-Villar, E. Evaluation

483

of alternative winemaking processes for red wine color enhancement. Food Sci. 2008, 14,

484

21-28.

485

6. Busse-Valverde, N.; Gomez-Plaza, E.; Lopez-Roca, J. M.; Gil-Munoz, R.; Fernandez-

486

Fernandez, J. I.; Bautista-Ortin, A. B. Effect of enological practices on skin and seed

487

proanthocyanidins in three varietal wines. J. Agric. Food Chem. 2010, 58, 11333-11339.

488

7. Ducasse, M. A.; Canal-Llauberes, R.-M.; de Lumley, M.; Williams, P.; Souquet, J. M.;

489

Fulcrand, H.; Doco, T.; Cheynier, V. Effect of macerating enzyme treatment on the

490

polyphenol and polysaccharide composition of red wines. Food Chem. 2010, 118, 369-

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376.



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8. Casassa, L. F.; Beaver, C. W.; Mireles, M. S.; Harbertson, J. F. Effect of extended

493

maceration and ethanol concentration on the extraction and evolution of phenolics, color

494

components and sensory attributes of Merlot wines. Aust. J. Grape Wine Res. 2013, 19,

495

25-39.

496

9. Casassa, L. F.; Larsen, R. C.; Beaver, C. W.; Mireles, M. S.; Keller, M.; Riley, W. R.;

497

Smithyman, R.; Harbertson, J. F.Impact of extended maceration and regulated deficit

498

irrigation (RDI) in Cabernet Sauvignon wines: characterization of proanthocyanidin

499

distribution, anthocyanin extraction, and chromatic properties. J. Agric. Food Chem.

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2013, 61, 6446-6457.

501

10. Vidal, S.; Francis, L.; Guyot, S.; Marnet, N.; Kwiatkowski, M. The mouth-feel properties

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of grape and apple proanthocyanins in a wine-like medium. J. Sci. Food Agric. 2003, 83,

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564-573.

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11. Vidal, S.; Francis, L.; Nobel, A. C.; Kwiatkowski, M.; Cheynier, V.; Waters, E. L. Taste

505

and mouth-feel properties of different types of tannin-like polyphenolic compounds and

506

anthocyanins in wine. Anal. Chim. Acta. 2004, 513, 57-65.

507

12. McRae, J. M.; Schulkin, A.; Kassara, E.; Holt, E. H.; Smith, P. A. Sensory properties of

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wine tannin fractions: implications for in-mouth sensory properties. J. Agric. Food Chem.

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2013, 61, 719-727.

510 511 512 513

13. Haslam, E. Natural polyphenols (vegetable tannins) as drugs: Possible modes of action. J. Nat. Prod. 1996, 59, 205-215. 14. Bennick, A. Interaction of plant polyphenol with salivary proteins. Crit. Rev. Oral Biol. 2002, 13, 184-196.


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15. Poncet-Legrand, C.; Gautier, C.; Cheynier, V.; Imberty, A. Interactions between flavan-

515

3-ols and poly (L-proline) studied by isothermal titration calorimetry: Effect of the tannin

516

structure. J. Agric. Food Chem. 2007, 55, 9235-9240.

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16. Le Bourvellec, C.; Renard, C. M. G. C. Interactions between polyphenols and

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macromolecules: Quantification methods and mechanisms. Crit. Rev. Food Sci. Nutr.

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2012, 52, 213-248.

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17. Cerpa-Calderón, F. K.; Kennedy, J. A. Berry integrity and extraction of skin and seed

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proanthocyanidins during red wine fermentation. J. Agric. Food Chem. 2008, 56, 9006-

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9014.

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18. Singleton, V. L.; Trousdale, E. K. Anthocyanin-tannin interactions explaining differences

524

in polymeric phenols between white and red wine. Am. J. Enol. Vitic. 1992, 43, 63-70.

525

19. Aron, P. M.; Kennedy, J. A. Compositional Investigation of Phenolic Polymers Isolated

526

from Vitis vinifera L. Cv. Pinot Noir during Fermentation. J. Agric. Food Chem. 2007,

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55, 5670-6580.

528

20. Revelette, M. R.; Barak, J. A.; Kennedy, J.A. High-Performance Liquid Chromatography

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Determination of Red Wine Tannin Stickiness. J. Agric. Food Chem. 2014, 62, 6626-

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6631.

531 532 533

21. Barak, J. A.; Kennedy, J.A.; HPLC Retention Thermodynamics of Grape and Wine Tannins. J. Agric. Food Chem. 2013, 61, 4270-4277. 22. Peng, Z.; Iland, P. G.; Oberholster, A.; Sefton, M. A.; Waters, E. J. Analysis of

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pigmented polymers in red wine by reverse phase HPLC. Austr. J. Grape Wine Res.

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2002, 8, 70-75.



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23. Kennedy, J. A.; Taylor, W. A. Analysis of Proanthocyanidins by High-Performance Gel Permeation Chromatography. J. Chromatogr. A. 2003, 995, 99-107.

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24. Kennedy, J. A.; Jones, G. P. Analysis of Proanthocyanidin Cleavage Products Following

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Acid-Catalysis in the Presence of Excess Phloroglucinol. J. Agric. Food Chem. 2001, 49,

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1740-1746.

541

25. Bindon, K. A.; Kennedy, J. A. Tissue-specific and developmental modification of grape

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cell walls influences the adsorption of proanthocyanidins. J. Agric. Food Chem. 2012, 60,

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9249-9260.

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26. Bindon, K. A.; Kennedy, J. A. Ripening-induced changes in grape skin

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proanthocyanidins modify their interaction with cell walls. J. Agric. Food Chem. 2011,

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59, 2696-2707

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27. Bindon, K. A.; Smith, P. A.; Kennedy, J. A. Interaction between grape-derived

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proanthocyanidins and cell wall material. 1. Effect on proanthocyanidin composition and

549

molecular mass. J. Agric. Food Chem. 2010, 58, 2520-2528.

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28. Bindon, K. A.; Smith, P. A.; Holt, H.; Kennedy, J. A. Interaction between grape-derived

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proanthocyanidins and cell wall material. 2. Implications for vinification. J. Agric. Food

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Chem. 2010, 58, 10736-10746.

553

29. Hanlin, R. L.; Hrmova, M.; Harbertson, J. F.; Downey, M. O. Review: Condensed tannin

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and grape cell wall interactions and their impact on tannin extractability into wine. Aust.

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J. Grape Wine Res. 2010, 16, 173-188.

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30. Bautista-Ortin, A. B.; Rodriguez-Rodriguez, P.; Gil-Munoz, R. Influence of berry

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ripeness on concentration, qualitative composition and extractability of grape seed

558

tannins. Aust. J. Grape Wine Res. 2012, 18, 123-130


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31. Bautista-Ortin, A. B.; Molero, N.; Marin, F. Reactivity of pure and commercial grape skin tannins with cell wall material. 2015, 240, 645-654.

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32. Kennedy, J. A.; Hayasaka, Y.; Vidal, S.; Waters, E. J.; Jones, G. P. Composition of grape

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skin proanthocyanidins at different stages of berry development. J. Agric. Food Chem.

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2001, 49, 5348-5355.

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33. McRae, J. M.; Day, M. P.; Bindon, K. A.; Kassara, S.; Schmidt, S. A.; Schulkin, A.;

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Kolouchova, R.; Smith, P. A. Effect of early oxygen exposure on red wine colour and

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tannins. Tetrahedron. 2015, 71, 3131-3137.

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34. Kennedy, J. A.; Matthews, M. A.; Waterhouse, A. L. Changes in grape seed polyphenols during fruit ripening. Phytochemistry 2000, 55, 77-85.

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35. Kennedy, J. A.; Troup, G. J.; Pilbrous, J. R.; Hutton, D. R.; Hewitt, D.; Hunter, C. R.;

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Ristic, R.; Iland, P. G.; Jones, G. P. Development of seed polyphenols in berries from

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Vitis vinifera L.cv Shiraz. Aust. J. Grape Wine Res. 2000, 6, 5348-5355.

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36. McRae, J. M.; Falconer, R. J.; Kennedy, J. A. Thermodynamics of grape and wine tannin

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interaction with polyproline: Implications for red wine astringency. J. Agr. Food Chem.

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2010, 58, 12510–12518.

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37. Jorgensen, E. M.; Marin, A. B.; Kennedy, J. A. Analysis of the oxidative degradation of

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proanthocyanidins under basic conditions. J. Agric. Food Chem. 2004, 52, 2292-2296.

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38. Cortell, J. M.; Kennedy, J. A. Effect of shading on accumulation of flavonoid compounds

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in (Vitis vinifera L.) Pinot noir and extraction on a model system. J. Agric. Food Chem.

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2006, 54, 8510-8520.



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39. Hanlin, R. L.; Kelm, M. A.; Wilkinson, K. L.; Downey, M. O. Detailed characterization

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of proanthocyanidins in skin, seeds and wine of Shiraz and Cabernet Sauvignon wine

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grapes (Vitis vinifera). J. Agric. Food Chem. 2011, 59, 13265-13276.

583

40. Nguela, J. M.; Vernhet, A.; Sieczkowski, N.; Brillouet, J.-M.; Interactions of condensed

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tannins with Saccharomyces cerevisiae yeast cells and cell walls: tannin location by

585

microscopy. J. Agric. Food Chem. 2015, 63, 7539-7545.

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41. Carew, A. L.; Smith, P.; Close, D. C.; Curtin, C.; Dambergs, R. G. Yeast effects on Pinot

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noir wine phenolics, color and tannin composition. J. Agric. Food Chem. 2013, 61, 9892-

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9898.

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42. Rodrigues, A.; Ricardo-da-Silva, J. M.; Lucas, C.; Laureano, O. Effect of winery yeast

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lees on Touriga Nacional red wine color and tannin evolution. Am. J. Enol. Vitic. 2013,

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64, 98-109.

592 593 594 595 596

43. Prieur, C.; Rigaud, J.; Cheynier, V.; Moutounet, M. Oligomeric and polymeric procyanidins from grape seeds. Phytochemistry. 1994, 36, 781-784. 44. Souquet, J. M.; Cheynier, V.; Brossaud, F.; Moutounet, M. Polymeric proanthocyanidins from grape skins. Phytochemistry. 1996, 43, 509-512. 45. Escribano-Bailon, M. T.; Guerra, M. T.; Rivas-Gonzalo, J. C.; Santos-Buelga, C.

597

Proanthocyanidins in skins from different grape varieties. Z. Lebensm. Unters. Forsch.

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1995, 200, 221-224.

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46. Gil, M.; Kontoudakis, N.; Gonzalez, E.; Esteruelas, M.; Fort, F. Influence of grape

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maturity and maceration length on color, polyphenolic composition, and polysaccharide

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content of Cabernet Sauvignon and Tempranillo wines. J. Agric. Food Chem. 2012, 60,

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7988-8001.


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47. Bajec, M. R.; Pickering, G. Astringency: mechanism and perception. Crit. Rev. Food Sci. Nutr. 2008, 48, 858-875.

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48. Ferrer-Gallego, R.; Quijada-Morin, N.; Bras, N. F.; Gomes, P.; de Freitas, V.; Rivas-

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Gonzalo, J. C.; Escribano-Bailon, M. T. Characterization of sensory properties of

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flavanols- A molecular dynamic approach. Chem. Senses. 2015, 40, 381-390.

608

49. Charlton, A. J.; Baxter, N. J.; Kahn, M. L.; Moir, A. J. G.; Haslam, E.; Davies, A. P.;

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Williamson, M. P. Polyphenol/peptide binding and precipitation. J. Agric. Food Chem.

610

2002, 50, 1593-1601.

611

50. Plumb, G. W.; de Pascual-Teresa, S.; Santos-Buelga, C.; Cheynier, V.; Williamson, G.

612

Antioxidant properties of catechins and proanthocyanidins: effect of polymerization,

613

galloylation and glycosylation. Free Radic. Res. 1998, 29, 351-358.

614 615 616

51. De Freitas, V. A.; Mateus, N. Structural features of procyanidin interactions with salivary proteins. J. Agric. Food Chem. 2001, 49, 940-945. 52. Rinaldi, A.; Iturmendi, N.; Jourdes, M.; Teissedre, P. L.; Moio, L. Transfer of tannin

617

characteristics from grape skins or seeds to wine-like solutions and their impact on

618

potential astringency. LWT-Food Sci. Technol. 2015, 63, 667-676.

619

620



Page 30 of 40

621

ACKNOWLEDGEMENT

622

The authors thank the following: American Vineyard Foundation and the Agricultural Research

623

Institute at California State University, Fresno for project funding. Sally Johnson (Pride

624

Mountain Vineyards), Nicole Marchese (Far Niente Winery), Megan Melief (Bella Union

625

Winery), Elizabeth Vianna (Chimney Rock) and Graham Wehmeier (Cornell Vineyard) are

626

thanked for juice and wine samples. Undergraduate research assistants Sasha Hazel, Melanie

627

Sherman and Rebecca Wolff for experimental support.

628

629

Supporting Information

630

S1, concentration of tannins in samples collected; S2, compositional and activity information for

631

tannin isolates. This material is available free of charge via the Internet at http://pubs.acs.org.

632

633

CONFLICT OF INTEREST DISCLOSURE

634

The authors declare no competing financial interest

635

636

637


Page 31 of 40

638


LIST OF FIGURES

639

640

Figure 1. Integrated chromatogram (280 nm, 30 °C) of isolated tannin from a Cabernet

641

Sauvignon fermentation following 9 days of maceration.

642

643

Figure 2. Relationship between tannin gravimetric recovery and corresponding in situ tannin

644

concentration determined by reversed-phase HPLC, and with best fit line shown.

645

646

Figure 3. Relationship between tannin activity and corresponding tannin concentration

647

determined by gravimetric recovery, and with best fit line shown.

648

649

Figure 4. Box car plots indicating median values (horizontal lines within each box), the upper

650

and lower quartile (upper and lower edges of the box), the upper and lower range (bars above

651

and below the box), and closed circles if data within each box fell outside of the 90% percentile.

652

Data includes tannin gravimetric recovery (A), activity (B), pigmented tannin (C), conversion

653

yield (D), percentage of (-)-epigallocatechin extension subunits (E), and percentage of (-)-

654

epicatechin-3-O-gallate extension subunits (F). Box car plots were constructed by determining

655

the mean for each fermentation and across all maceration times for any given chemistry (e.g.:

656

tannin activity, n=37). Following this, and within each fermentation, individual chemistry values

657

were compared to the mean value and expressed as a percentage. Finally, across all



Page 32 of 40

658

fermentations and within specific maceration times (e.g.: crush, end of fermentation; n=10, 9, or

659

8) box car plots were constructed.

660

661

Figure 5. Tannin molecular mass distribution for average and individual (inset) samples

662

collected at crush (red), after fermentation (blue), midway through maceration (pink) and free

663

run at pressing (aquamarine).

664

665

Figure 6. Principal component analysis vector plot for tannin activity (red vector) and associated

666

tannin structural features.

667

668

Figure 7. Tannin molecular mass distribution for 62 day extended maceration experiment with

669

samples collected at crush (red), after fermentation (blue), midway through maceration (pink)

670

and free run at pressing (aquamarine).


Page 33 of 40


671

672

Figure 1.

673

674 675

676

677

678



679

Page 34 of 40

Figure 2.

680 681


Page 35 of 40

682


Figure 3

683 684

685



686

Page 36 of 40

Figure 4.

687

689

690

691

% of Entire Set Mean Value (n=37)

180

688

160

n=9

A

B

n=8 n=10

140 120 n=10

100 80 60

40 3 Figure

Tannin Activity

TanninGravimetric Recovery

20

692

694

695

% of Entire Set Mean Value (n=37)

693

180 160

C

D

140 120 100 80 60 40 Conversion Yield

PAU520/PAU280 20

696 180

697

699

700

% 0f Entire Set Mean Value (n=37)

698

160

F

E

140 120 100 80 60 40 %ECG

%EGC 20 Crush

End of Ferm

Post Ferm

Free Run

Crush

End of Ferm

Post Ferm

Free Run

701

702

703 36 ACS Paragon Plus Environment

Page 37 of 40

704


Figure 5.

705

706 707

708



709

Page 38 of 40

Figure 6.

710

711 712


Page 39 of 40

713


Figure 7.

714

715 716

717

718



719

Page 40 of 40

TOC Graphic

720

721

722 723


Red Wine Tannin StructureâActivity Relationships during

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