A Selection of Grapes

This undistinguished, productive, drought resistant, vigorous white grape, Airén, from the La Mancha region of Spain, was said to be the most widely planted grape in the world. In part the justification for this claim relies upon the observation that it is planted at a very low density! Except for its use in blending to make other wines “lighter,” it has not found wide accep¬tance. In part, it appears that its lack of popularity is the result of what is reported to be a mild, neutral flavor, and advertising has not pushed wines produced from it to the fore. Although it is now common to attempt to analyze the headspace (or ullage) in bottled wine (as well as the wine itself ) by chromatographic and mass spectrometric techniques it is less common to find that the grapes (skin, must, and seeds) are also subjected to such analysis. Nonetheless, the phenolic composition of V. vinifera var Airén was subjected to just such analysis during ripening from véraison to “technological” maturity (i.e., maturity which might actually be earlier than harvest, the latter being the decision of the viticulturist and vintner). The analysis of the ethyl ether extract of macerated skins, seeds, and accumulated solids (the pomace) was undertaken. Procyanidins and anthocyanins which would (the authors claim) interfere with subsequent analysis would not move into the ether phase. It was also found (using controls) that other highly polar materials (e.g., carboxylic acids) were only poorly extracted from the macerated skins and seeds. The isolated compounds and some information about their sources are provided in Figures 14.1 and 14.2. The analysis of the seeds, skin, and must did lead to the conclusion that “the maximum concentrations of benzoic and cinnamic acids and aldehydes and flavonol aglycones and glycosides at the end of the ripening period did not coincide with the minimum concentrations of the flavan-3-ols and hydroxycinnamic tartaric esters.” Depending upon what was sought, this information might thus affect decisions concerning the harvest date.

The Leaf

Grape leaves are thin and flat. As is common among leaves in general, they are composed of different sets of specialized cells. Today, on average, sunlight reaching their surface is about 4% ultraviolet (UV) (<400 nm), 52% infrared (IR) (>750 nm) and 44% visible (VIS) radiation. Little of the UV and IR are used by plants. As with other leaves that are green, only the red and blue ends of the visible part of the electromagnetic spectrum are absorbed, thus leaving green available by reflection and transmission. On the surface of the leaf (Figure 8.1), the cells of the outermost layer (the epidermis) are designed to protect the inner cells where the workings needed for gathering the sunlight used for photosynthesis and other chemistry necessary to the life of the plant are found. That is, the more delicate cells, beneath the epidermis, are involved in production of carbohydrates as well as the movement of nutrients in and products out of the leaf. The epidermis, exposed to the atmosphere, has cells that are usually thicker and are covered by a waxy layer made up of long- chain carboxylic acids that have hydroxyl groups (–OH) at or near their termini. These so-called omega hydroxy acids can then form esters using the hydroxyl group of one and the carboxylic acid of the next. This yields long-chain polyester polymers called “cutin.” As indicated in the earlier discussion of cells and, in particular, regarding the fatty acids of cell walls, the fatty acids found in the epidermis generally consist of an even number of carbon atoms, and for cutin, the sixteen carbon (palmitic acid) family (Figure 8.2) and the eighteen carbon family (oleic acid bearing a double bond or the saturated analogue stearic acid) are common. While one terminal hydroxyl group is usual (e.g., 16-hydroxypalmitic acid, 18-hydroxyoleic acid, or its saturated analogue 18-hydroxystearic acid) more than one (allowing for cross-linking) is not uncommon (e.g., 10,16-dihydroxypalmitic and 9,10,18-trihydroxystearic acid).

Adding Sulfur Dioxide (SO2)

The judicious use of sulfur dioxide (SO2) will inhibit the growth of microorganisms (e.g., bacteria) present on the grape skins as the berries come from the vineyard. Its early use presumes the vintner has decided that the adventitious wild yeasts which might be destroyed or inhibited by sulfur dioxide will not contribute to the vintage. It appears that Saccharomyces cerevisiae might be less susceptible to the action of sulfur dioxide than other yeasts that may be present. So, if the particular strain of S. cerevisiae used can cope, it may be able to function unimpeded. Regardless, sulfur dioxide might still be used because, in addition to suppression of deleterious microorganisms, it appears to reduce oxidation of particularly fragile white wine components. In industrial settings, both gaseous sulfur dioxide and sulfur dioxide as a liquefied gas (boiling point – 10 °C [14 °F]) are used. In either form it is a dangerous tool. It is dangerous first because it is toxic and second because an excess of it will ruin the wine. In many cases, because its value is recognized as beneficial, sulfur dioxide is replaced by addition of either sodium metabisulfite (Na2S2O5) or potassium metabisulfite (K2S2O5) with the latter generally preferred. Indeed, while it is best to look at the MSDS. (Manufacturer’s Safety Data Sheet) before use, the solubility of the two salts is the same and given as 450 grams/ liter (g/ L) at 68 °F (20 °C) and the pH on dissolution as between 3.5 and 4.5. The potassium (K) salt appears, at this writing, to be more readily available in food quality (as opposed to chemical quality) grade. So, with regard to sulfur dioxide (SO2), and as shown in Figure 17.1, its structure is much more similar to water and to ozone than it is to carbon dioxide (CO2); sulfur lies beneath oxygen (O2) in the periodic table (silicon, Si, lies beneath carbon). Nonetheless, sulfur dioxide (SO2) reacts with water much the same way that carbon dioxide (CO2) does.

More Than Skin Deep

The grape berry is composed of skin, flesh (pulp) and seeds. After destemming (Chapter 13), the grapes are sent on for crushing. On crushing, the thick walls of the skin, including the waxy cuticle, are broken. Crushing the grapes (Figure 16.1) is a question of quantity. Small quantities are handled differently than large. The skins, including the contaminants thereon, as well as the majority of the materials discussed above for the individual grapes (i.e., phenols, anthocyanins, tanins, some acids, terpenes, pyrazines, and some carbohydrates including those attached to the anthocyanidins, forming anthocyanins) therein, are released. The cells of the pulp are also broken and released into the juice on crushing. This berry cell juice is mainly water (70–80% by weight) which contains the mixture of sugars (mostly glucose and fructose, but small concentrations of many other carbohydrates are also present), carboxylic acids (mostly tartaric and malic, but additional members of the tricarboxylic acid cycle, oxalic, glucuronic, etc. are also present), complex cross-linked polysaccharides from cell walls (pectins), some phenols and proteins (as well as the peptides and simple amino acids from which they are constructed), and minerals, including oxides of iron (Fe), phosphorus (P), and sulfur (S), as well as salts of potassium (K) and sodium (Na) brought up in the xylem to the growing berry. The seeds have their cellulose carbohydrate-based exterior coatings, which are also rich in complexed polyphenols (tannins). Additionally, amino acids, generally found as constituents of peptides, proteins, and enzymes, and their cofactors needed for all life, nucleic acids and their attached sugars needed for the next generation, are all present too. Thus, overall, the result of crushing the berries is a mixture consisting of skins, seeds, and fruit juice (the must = Latin vinum mustum = young wine). This mixture may, if the grapes were “white,” be cooled and the cap on the must—sometimes called the pomace (the solid portion of the must) removed early or late (usually between 12 and 24 hours) by the vintner. Most of the flavoring constituents are quickly extracted, and brightly colored phenols, tannins, anthocyanins, etc.

Roots, Shoots, Leaves, and Grapes

As noted earlier and as anticipated by Charles and Francis Darwin it has been argued that plants sense the direction of gravity (gravitropism) by movement of starch granules found in cells called statocytes that contain compartments (organelles) called statoliths. The synthesis of statoliths appears to occur in the plastid (plant organelle) compartments called amyloplasts (Figure 7.1, 1). It has been suggested that this gravitropic signal then leads to movement of plant hormones such as indole-3-acetic acid (auxin) (Figure 7.2), through the phloem opposite to the pull of gravity to promote stem growth. Chloroplasts (Figure 7.1, 2) are cell compartments (plastids or organelles) in which photosynthesis is carried out. The process of photosynthesis, discussed more fully later, is accompanied by the production of adenosine triphosphate (ATP) from adenosine diphosphate (ADP) and inorganic phosphate (Pi) (Figure 7.3). ATP is consumed and converted to ADP and Pi in living systems. The cycle of production and consumption allows ATP to serve as an “energy currency” to pay for the reactions in living systems. Beyond this generally recognized critical function of chloroplasts, it has recently been pointed out that light/ dark conditions affect alternative splicing of genes which may be necessary for proper plant responses to varying light conditions. The organelles or plastids which contain the pigments for photosynthesis and the amyloplasts that store starch are only two of many kinds of plastids. Other plastids, leucoplasts for example, hold the enzymes for the synthesis of terpenes, and elaioplasts store fatty acids. Apparently, all plastids are derived from proplastids which are present in the pluripotent apical and root meristem cells. The cell wall (Figure 7.1, 3) is the tough, rigid layer that surrounds cells. It is located on the outside of the flexible cell membrane, thus adding fixed structure. A representation of a portion of the cell wall (as made up of cellulose and peptide cross-linking) is shown below in Figure 7.7. The cells will have different sizes as a function of where they are found (e.g., leaf, stalk, root), but in every case, the cell wall limits the size of the membrane that lies within.

The Roots of the Grape Vine

Aside from grafting onto already established rootstock or the development of roots from a planted cane (vide supra), root systems develop from the radicle in the plant’s seed. Both as roots begin to form from the cane, and as the sprouting seed coat opens in response to soil temperature, moisture, and genetic programming left in place when the seed formed, the roots begin to grow and interact with the rhizosphere. Similarly, signals received by rootstock where grafting has been effected also occur. The roots begin to bring moisture and food to produce and support the stem and, eventually, the leaves, flowers, and fruit. Heavily fruited plants such as grapes require additional support for the stems. In the roots, epidermal (surface) cells elongate and develop into root hairs. Beneath the epidermal cells it appears that the phloem cells which bring the starch bodies (amyloplasts) to the root tips and help direct which way “down” is, develop first. Then xylem elements develop in order to move the minerals into the system. Most of the minerals are absorbed through channels developing in the walls of the growing undifferentiated cells (the meristems). Because of concentration gradients (i.e., there is less on one side of a cell membrane than on the other), some minerals appear to be actively transported into the cells of the xylem (presumably through similar channels) in response to signals emanating from the plant. From the xylem cells, the minerals and water move upward into the apical meristem and get distributed to other regions. Interestingly, although most of the cells are derived from the same group of meristems which thus might be considered true stem cells, it is genetic programming which permits that differentiation. Thus, the derivatives of the meristems undergo transformation and develop into various cell types that perform the different functions (Figure 6.1). Relatively recently there has been an increased interest in what has been the largely unexplored biology of roots.

General Comments

Viticulture, it will be recalled, is the art and science of vine-growing and grape-harvesting, and it was the subject of Sections I–IV (Chapters 1–14). Enology, the subject of this Section (Chapters 15– 20), is the art and science of winemaking. Much of what follows in this brief Chapter on General Comments is expanded upon in other Chapters in this Section. In making the wine, it appears to be generally agreed that where possible it is best to follow the traditional methods to produce the best results. However, it should be clearly understood that work is underway to engineer yeast to make it more alcohol tolerant and to use the yeast to produce specific compounds recognized as being particularly flavorful. Additionally, as the number of vintners has grown, finding the proper oak for casks is becoming ever harder. Therefore, the art of reworking old oak casks or even avoiding them altogether (e.g., by aging wine in the presence of oak chips) may be used. In the same vein, it is widely recognized that stoppers other than cork may be used, so that the day may come when the cork stopper will be a thing of the past. Traditionally, grapes are taken directly from the vineyard to be crushed, and it is still the case in many of the oldest and most respected vineyards that this practice will continue. However, as the use of pesticides and fungicides has increased, methods for rapid washing and then drying of grapes before crushing may be employed. The arguments against these extra steps are mainly two. First, lingering water would dilute the grape juice. Second, the adventitious yeasts that might be removed by washing or deactivated by drying are often desired for the production of the vintage. Indeed, it has been argued that unique fungi, which might be exclusive to the most prestigious vineyards, are important to the production of the best wines. The issue of washing versus not washing has been investigated, and it was concluded for the case examined that only minor changes are effected by washing.

The Grape Berry

Beginning with fruit set (generally the grape berry is now between 1.5 and 3.0 mm, i.e., less than 1/ 8 of an inch in diameter) the grape berry growth is divided into three stages. Stages I and III correspond to periods of rapid growth, and the intervening slow growth phase is called Stage II. Generally the slow growth stage (Stage II) corresponds to the slowing of Stage I and the acceleration of Stage III, but it is clear that different grape cultivars have stages of different lengths even under ostensibly identical conditions. In the first stage of fruit set (also called “nouaison”) the actual development of the flower ovary into the grape berry begins. The seeds in the two seed cavities (the locules) and the flesh (the pericarp) begin to take form. The pericarp separates into the exocarp (the skin with its cuticle—a thin wax coating) and the mesocarp. The mesocarp, as it grows and divides, will eventually (by the end of Stage III) account for more than 90% of the grape’s weight. The exocarp, significantly thinner than the mesocarp, may be only five or six cells thick, and the cuticle only several layers of lipids (waxy, fatty acid esters, and compounds similar to those of cell walls and the chloroplast envelope, see pages 30 and 31). It is in this stage that the as yet undeveloped berries are green and hard (it has been sug¬gested that this is because chlorophyll is present and photosynthesis in the berry—as well as in leaves—is occurring). The berries are low in sugar (sucrose) but high in carboxylic acids, predominately malic acid and tartaric acid along with, generally, a lesser amount of ascorbic acid (vitamin C), hydroxycinnamic acid, and some acidic tannins (Figures 13.1 and 13.2). The grape berry structure is generally divided into three types of tissue: skin, flesh, and seed (Figure 13.3). The first, skin, as already mentioned is also known as exocarp.

Working in the Dark

Three turns of the Calvin cycle (Figure 11.1), allow the conversion of three (3) equivalents of carbon dioxide (CO2) (i.e., 3 C1 units) along with three (3) equivalents of the five-carbon carbohydrate derivative, ribulose-1,5-bisphosphate (i.e., 3 C5 units) to yield three (3) not yet isolated six-carbon adducts, 2-carboxy-3-ketoribitol-1,5-bisphosphate (3 C1 + 3 C5 = 3 C6) to form. The three (3) C6 species then undergo fragmentation to yield six (6) equivalents of the three (3) carbon dihydroxy monocarboxylate, 3-phosphoglycerate (i.e., 3 C6 = 6 C3). A cartoon representation of this process is shown in Scheme 11.1 for one of the three CO2 units. Of the six (6) three-carbon unit equivalents, five (5) are used to regenerate three (3) equiv¬alents of ribulose-1,5-bisphosphate (i.e., 5 C3 = 3 C5), while the sixth three- carbon fragment is now available to combine with another to make a six (6) carbon sugar (2 C3 = 1 C6) such as glucose (C6H12O6) (Figure 11.2). Additionally, as shown in Figure 11.3, 3-phosphoglycerate can be used to make other small compound building blocks such as glyceric acid, lactic acid, pyruvic acid and even acetic acid (after decarboxylation). Ribulose- 1,5-bisphosphate (often abbreviated as RuBP), using the enzyme ribulose- 1,5- bisphosphate carboxylase (EC 4.1.1.39, carboxydismutase, rubisco), catalyzes the Mg2+- dependent conversion of the 1,5- bisphosphate ester of the carbohydrate ribulose with carbon dioxide (CO2) to produce two (2) equivalents of 3- phosphoglycerate (PGA). As shown in the Schemes 11.1 and 11.2. A hypothetical the six carbon intermediate, 2- carboxy- 3- ketoribitol- 1,5- bisphosphate, is often written. It is important to keep in mind that we want the 3- phosphoglycerate for purposes of construction of other important compounds. But, as noted above, three turns of the cycle are necessary to produce six (6) equivalents of 3- phosphoglycerate, and five (5) of them are reused in making the three (3) ribulose- 1,5- bisphosphates necessary to turn the cycle three (3) times.

The Light on the Leaves

As noted earlier in the general description of the plant cell, there is a site at which photosynthesis, the process which allows plants to capture sunlight and convert it into energy, occurs. It is this process which has produced oxygen on the planet, food for herbivores, and the cool green hills of Earth we enjoy today. The capture of sunlight allows the grape vine to grow and produce fruit. Of course, while the discussion of the “light reactions” (capture of sunlight) and the subsequent so-called “dark reactions” (producing carbohydrates) is necessarily brief here, it is, nonetheless, an exciting story. We are only now beginning to understand a little of it. The earlier picture (Figure 7.1) of the plant cell is repeated here (Figure 9.1) so that the position of the chloroplast is seen. Refer to page 24 for a discussion of the numbered items. As the leaves begin to develop alongside the apical meristem, proplastids, which are present in the meristematic regions of the plant, are formed. Proplastids grow into plas¬tids (such as amyloplasts and chloroplasts) as they mature in different ways dictated by the plant’s DNA. Some plastids (e.g., chloroplasts) carry pigments, discussed more fully below, that allow them to carry out photosynthesis. Others are used for storage of fat, starch (amyloplasts) or specialized proteins. Still other plastids are used to synthesize specialized compounds needed to form different tissues or to produce compounds for protection (e.g., tannins). Each plastid builds multiple copies of its DNA as it grows. If it is growing rapidly, it makes more genome copies than if it is growing slowly. The genes, ignoring epigenetic (literally “above the gene”) and postgenetic (literally “after the gene”) modifications, about which we still have much to learn, encode plastid proteins, the regulation of whose expression controls differentiation and thus which plastid is eventually formed. However, despite the differentiation of plastids, it appears that many plastids remain connected to each other by tubes called stromules through which proteins can be exchanged.