Article pubs.acs.org/JAFC Factors Affecting Skin Tannin Extractability in Ripening Grapes Keren A. Bindon,*,† S. Hadi Madani,§ Phillip Pendleton,§,# Paul A. Smith,† and James A. Kennedy†,⊥ † The Australian Wine Research Institute, P.O. Box 197, Glen Osmond, SA 5064, Australia § Ian Wark Research Institute, University of South Australia, Mawson Lakes, SA 5095, Australia # School of Pharmacy and Medical Science, University of South Australia, G.P.O. Box 2471, Adelaide, SA 5001, Australia ⊥ Department of Viticulture and Enology, California State UniversityFresno, 2360 East Barstow Avenue, MS VR89, Fresno, California 93740-8003, United States *S Supporting Information ABSTRACT: The acetone-extractable (70% v/v) skin tannin content of Vitis vinifera L. cv. Cabernet Sauvignon grapes was found to increase during late-stage ripening. Conversely, skin tannin content determined following ethanol extraction (10, 20, and 50% v/v) did not consistently reflect this trend. The results indicated that a fraction of tannin became less extractable in aqueous ethanol during ripening. Skin cell walls were observed to become more porous during ripening, which may facilitate the sequestering of tannin as an adsorbed fraction within cell walls. For ethanol extracts, tannin molecular mass increased with advancing ripeness, even when extractable tannin content was constant, but this effect was negligible in acetone extracts. Reconstitution experiments with isolated skin tannin and cell wall material indicated that the selectivity of tannin adsorption by cell walls changed as tannin concentration increased. Tannin concentration, tannin molecular mass, and cell wall porosity are discussed as factors that may influence skin tannin extractability. KEYWORDS: tannin, anthocyanin, adsorption, ripening, skin, cell wall, porosity, BET, microscopy, molecular mass, gel permeation chromatography ■ INTRODUCTION Due to these conflicting results, it is evident that additional Generally, skin tannins from red grapes are more readily research is required, in particular as new analytical methods become available. Distinguishing extractable tannin from total extracted during vinification than seed tannins, regardless of tannin content is also an important consideration in studies that maceration time.1,2 In wines, later harvest dates are associated have implications for winemaking. Because existing research with an increase in total wine tannin concentration, as well as into the changes in grape skin tannin during ripening has an increase in the molar proportion of prodelphinidin 3,4 primarily employed exhaustive extraction techniques such as (epigallocatechin), which is derived from grape skins. As aqueous acetone or methanol, a pertinent question is the extent such, skin tannin concentration may serve as a useful marker for to which these methods relate to vinification conditions. phenolic maturity in ripening grapes. Reports on changes in Extraction conditions that exist during vinification reach only grape tannin quantity during ripening have used a diverse range between 10 and 16% v/v ethanol concentration in commercial fi of extraction, puri cation, and analytical methodologies. practice. In a study comparing the effectiveness of extraction Furthermore, to account for changes in berry weight during solvents for skin tannin,17 it was found that acetone was more ripening, it is necessary to report grape phenolic data effective than ethanol and that acetone-extracted tannin had a normalized as “content per berry” rather than simply as higher average molecular mass. It was also found that varying concentration (per gram berry or skin fresh weight). A review the ratio of water to solvent significantly affected the amount of studies that report tannin content or include berry mass and composition of the tannin extracted. The point was raised information revealed that skin tannin has been found to that aqueous ethanol may more accurately mimic the increase with the progression of ripening when analyzed using conditions of extraction during vinification. − adaptations of Porter’s assay5 9 and by the monitoring of Various studies have attempted to observe differences in tannin absorbance properties in the UV range via normal phase tannin extraction in ripening grapes using aqueous solutions, HPLC10 or methyl cellulose precipitation.3 Nevertheless, some either buffered18,19 or using dilute, acidified ethanol.5,7,20 It is authors10 have suggested that these increases in UV absorbance evident that differences exist in the partitioning of grape tannins may not originate directly from flavan-3-ol incorporation to the between soluble and cell wall-bound fractions, that the nature tannin polymer but could result from other structural of these fractions is significantly different, and that they change modifications. Furthermore, a study using selective precip- during ripening. A single study investigating the subcellular itation of skin tannin with protein11 found no change in content during ripening. Variable results have been reported using the Received: November 10, 2013 12 13 phloroglucinolysis method and showed either no change, Revised: January 15, 2014 variable,14 decreased,15 or increased16 skin tannin content per Accepted: January 17, 2014 berry in the late stages of ripening. Published: January 17, 2014 © 2014 American Chemical Society 1130 dx.doi.org/10.1021/jf4050606 | J. Agric. Food Chem. 2014, 62, 1130−1141 Journal of Agricultural and Food Chemistry Article partitioning of skin tannin19 compared extraction in buffered molecular mass distribution. Tannin and anthocyanin solutions solution (pH 7.5) to tannin remaining bound to the residue were combined with isolated cell walls to determine the effect following extraction, presumed to be cell wall fragments. Both of concentration on their respective adsorption affinities. In the buffer-soluble extract, designated the vacuolar fraction, and addition, scanning electron microscopy (SEM) and nitrogen the cell wall-bound fraction were further extracted using adsorption isotherms were used to characterize changes in cell acidified methanol.19 The experiment showed that the mean wall surface area (porosity) during ripening. degree of polymerization (mDP) of tannin within the vacuole did not change during grape development, yet the mDP of ■ MATERIALS AND METHODS tannins associated with the cell wall was higher and tended to Instrumentation. An Agilent model 1100 HPLC (Agilent 19 increase at the end of maturation. However, in said study, the Technologies Australia Pty Ltd., Melbourne, Australia) was used mDP of tannins isolated from cell walls was markedly lower with Chemstation software for chromatographic analyses. For SEM, a than observations from studies on the same cultivar using 70% Philips XL30 field emission scanning electron microscope (Philips, v/v acetone extraction.10,14,16 This may indicate that a greater Eindhoven, The Netherlands) was used. fraction of tannin is desorbed from the grape cell wall using Grape Sample Preparation and Extraction. Vitis vinifera L. cv. acetone. This phenomenon has been shown definitively for Cabernet Sauvignon grape samples were obtained from a commercial vineyard in the Langhorne Creek growing region of South Australia tannin extracted from apple using methanol or aqueous (Pernod Ricard Australia, Orlando Wines) at different commercial acetone, in which acetone extraction yielded tannin of higher 22 ripeness stages in the 2010 season. Berry samples were collected from mDP. Furthermore, in that work, aqueous acetone was shown three rows distributed within the vineyard block to obtain a to completely desorb high mDP tannin bound to cell wall representative sample, pooled, and processed fresh. A 200 berry material, in a fashion similar to the application of urea. It is subsample was processed fresh, and the juice centrifuged at 1730g for therefore evident that further research is needed to better 5 min and analyzed for total soluble solids (°Brix) using a digital 3 understand the factors which influence partitioning of tannin to refractometer. This data has been published previously. Triplicate extractable and nonextractable fractions. subsamples of 100 berries were prepared for cell wall isolation, and a Recent work using model studies has demonstrated the high further 36 subsamples of 10 berries each were prepared for phenolic ffi fi extraction experiments. a nity of puri ed grape cell wall material for tannin and The 100-berry samples were weighed, and skins were then highlighted that these interactions may limit tannin extraction separated from flesh and seed while kept on ice. Recovered skin during vinification. This may explain some of the significant material was weighed to determine the proportion of fresh skin variability that is observed in wine-extractable tannin between material to total berry mass, frozen in liquid nitrogen, and stored at − grape cultivars and grape ripeness stages.16,23 26 For grape skin −80 °C until used for the isolation of cell wall material. Cell wall tannin from red grapes, highly polymerized tannins had a low material was isolated from frozen grape skins as described previously.25 affinity for skin cell walls compared with tannin of intermediate For the purpose of comparing ripening-related changes in cell walls, molecular mass.16 However, it was found that with the two early-stage (preveraison and veraison) cell wall samples were retained from a previous study.25 progression of ripening, changes in skin cell wall composition The 10-berry samples were peeled, and the skins were rinsed