Phenolics and Ripening in Grape Berries – 249 From the ASEV 2005 Phenolics Symposium Phenolics and Ripening in Grape Berries Douglas O. Adams* Abstract: A key objective of this review is to describe the tissue types in the grape berry from a phenolics point of view, that is, an account of cell types based on the phenolic components they contain. To complement this, the major soluble phenolics of the grape are described with an emphasis on the relative abundance of some of the components in each class. The intent of this approach is that the juxtaposition of tissue types and phenolic com- position will help identify features of both that influence extraction during winemaking. Key words: phenolics, tannins, anthocyanins, flavonols, ripening, grape, Vitis vinifera Berry Anatomy and Phenolic Composition in the beak. Near veraison, when the seed coat still ap- pears yellow, the innermost cells of the outer integument The skin of a grape berry contains tannins and pig- undergo lignification. The cells outside the lignified layers ments, the pulp contains juice but no pigments, and the retain their thin primary cell walls except for the outermost seeds contain tannins, as shown in Figure 1. While this ones. The outer walls of these cells thicken to form a cu- figure is a serviceable representation of the grape, it lacks ticle on the surface of the seed. The thin-walled cells be- sufficient resolution to provide any insight into how phe- tween the cuticle and the inner lignified layers are perhaps nolics are extracted from fruit during winemaking. the most important in terms of phenolics because they Considine and Knox (1979) published an excellent ac- contain nearly all of the soluble seed phenolics. The famil- count of grape berry pericarp development and distin- iar seed browning that accompanies fruit ripening after guished two cell types in the skin: the single layer of veraison is thought to be the result of tannins and flavan- clear epidermal cells on the outside of the fruit and six 3-ols in this layer undergoing oxidation. Thus the brown- hypodermal layers beneath the epidermis. The exact num- ing of seeds during ripening is not associated with the ber of hypodermal layers depends on the variety (Hardie process of lignification, but instead is attributed to oxida- et al. 1996). The pulp was described as having three easily tion of the polyphenols in the thin-walled cells that lie distinguished tissues: the outer mesocarp, the inner meso- outside the lignified cells of the hard seed coat. During carp, and the vascular tissue separating the inner and initial growth of the seed, the polyphenols accumulate outer mesocarp. The vascular tissue in turn is comprised along with fresh weight and both reach a maximum around of xylem, phloem, and vascular parenchyma. veraison. The seed undergoes water loss even though it is The structure denoted as the seed coat (testa) has two encased in a fleshy pericarp, thus “drying” in a fleshy very distinct layers, both of which are derived from the fruit. Seed drying and the accompanying browning occurs outer integument of the ovule during seed development. after veraison, during the period of ripening when sugars From a phenolics standpoint, these two layers could not accumulate and malic acid is metabolized in the skin and be more different. The inner cell layers of the outer in- mesocarp. It has been shown that the appearance and tegument undergo a series of periclinal divisions (parallel color of the seed coat might be a useful indicator of berry to the surface of the seed) that results in a layer of cells ripeness in Shiraz (Ristic and Iland 2005). that are thin in the fossetts of the seed but much thicker The study of pericarp development after anthesis re- ported by Hardie et al. (1996) is particularly valuable in un- derstanding the nature of cells making up the hypodermis Department of Viticulture and Enology, University of California, Davis, (skin) and mesocarp (flesh). Fourteen days after anthesis One Shields Ave., Davis, CA 95616. the hypodermal cells of Traminer had a mean radial cell *Corresponding author [email: [email protected]; fax: 530 752-0382] Acknowledgments: Work from the author’s laboratory (Figures 11 and 12) was supported by the American Vineyard Foundation, the Viticulture Con- sortium, and the California Competitive Grant Program for Research in Viticulture and Enology. I thank the ASEV for the opportunity to present this work in the phenolics symposium and the reviewers for making many helpful suggestions for revising the manuscript. This article was originally presented at the ASEV 56th Annual Meeting Phenolics Symposium, 20–21 June 2005, Seattle, WA. All phenolics sym- posium articles were peer reviewed by two fellow presenters, and James Harbertson, Mark Downey, and Sara Spayd served as technical editors of the articles. Copyright © 2006 by the American Society for Enology and Viticulture. Figure 1 A simple diagram of the grape berry used to describe the All rights reserved. tissues and phenolic composition in an introductory wine class. 249 Am. J. Enol. Vitic. 57:3 (2006) 250 – Adams width of 7.0 ± 0.5 m and a cell wall thickness of 0.2 ± <0.1 epicatechin make up most of the extension units in grape mm. At this time the cells of the inner mesocarp were al- tannins, with epicatechin usually being the more abundant ready larger than the hypodermal cells, having a mean ra- of the two. dial cell width of 17.8 ± 1.5 mm. However, the cell wall The tannins of grape are actually a very diverse set of thickness of the inner mesocarp cells was 0.17 ± <0.1 mm, biomolecules. They vary in size from dimers and trimers similar to that of the hypodermal cells 14 days post- up to oligomers with more than 30 subunits. Skin tannins anthesis. After the cell divisions and cell expansion asso- differ from seed tannins in several important ways. First, ciated with berry development (126 days postanthesis), the average size of skin tannins (expressed as their mean the radial cell width of the hypodermal cells declined from degree of polymerization: mDP) is much larger than seed 7.0 ± 0.5 mm to 5.4 ± 0.6 mm while the mesocarp cells ex- tannins, and skin tannins contain epigallocatechin sub- panded from 17.8 ± 1.5 mm to 68.0 ± 4.5 mm. Thus, by har- units whereas seed tannins generally lack epigallocat- vest the mesocarp cells had a radial width 9.7 times echin. However, the smaller seed tannins usually have a greater than the hypodermal cells of the skin. As the higher proportion of their subunits as epicatechin gallate width of the hypodermal cells declined and as the meso- whereas epicatechin gallate is usually not present in skin carp cells expanded, important changes occurred in the cell tannin (Cheynier 2005). This difference in signature sub- walls of these two tissues. From 14 to 126 days postan- units has been used to estimate the relative proportions thesis, the thickness of the hypodermal cell walls in- of skin tannins and seed tannins in Pinot noir wines (Pey- creased nearly 10-fold from 0.2 ± <0.1 mm to 1.9 ± 0.2 mm. rot des Gachons and Kennedy 2003). During this same time the cell walls of the mesocarp be- Anthocyanins. The anthocyanins are co-located with came 59% thinner, from 0.17 ± <0.1 mm to 0.1 ± <0.1 mm. tannins in the thick-walled hypodermal cells of the skin. Thus, in fruit at harvest hypodermal cells of the skin had Anthocyanidins found in grape are shown in Figure 3. A cell walls nearly 20 times thicker than the cell walls of the variety such as Pinot noir that lacks acylated pigments mesocarp cells. An estimate of relative cell volume based has only the basic five anthocyanins whereas varieties on values provided by Hardie et al. (1996) for cell dimen- that produce all three types of acylated derivatives could sions shows that the thin-walled cells of the mesocarp are have as many as 20. This diversity is multiplied further in about 75 times larger than the very thick-walled hypoder- vivo because at vacuolar pH there are two distinct chemi- mal cells. This representation of berry pericarp at harvest cal forms of each anthocyanin, the neutral quinonoidal is important for two reasons. First, these two tissues have anhydro base and the flavylium cation form (Figure 3). The remarkably different phenolic composition at harvest, and ratio of these two chemical forms of anthocyanin is highly second, the difference in cell size, and in particular the pH dependent with a pKa of ~4. difference in cell wall thickness, is crucial for understand- Hydroxycinnamates. The third most abundant class of ing the more practical question of how phenolic com- soluble phenolics in grape berries are the hydroxycinna- pounds in these tissues are extracted during winemaking. Most work on the polyphenolics of grape berries has focused on those that are soluble in aqueous or organic solvents. From a biological perspective, the insoluble cu- 1 Catechin tin of the epidermis and the insoluble lignin of the hard seed coat are just as phenolic and just as important as the tannins and pigments in the skin and the tannins in the 2 Epicatechin seed. Nevertheless, because of their importance in winemaking, this overview will consider only the soluble phenolics of the grape berry and will address them in the 3 Epigallocatechin order of their abundance. Phenolic Classes in the Grape Berry 4 Epicatechin gallate Tannins. The most abundant class of soluble poly- phenolics in grape berries are the polymeric flavan-3-ols (tannins) found in the hypodermal layers of the skin and the soft parenchyma of the seed between the cuticle and the hard seed coat.