ABSTRACT
The study sets-out to investigate the amount of some selected class of polyphenols with cardiovascular benefits, present in red wine. The total phenolic content of the freshly opened red wine samples as determined by Folin-Ciocalteau Method (FCM) were in the range of 42-1625 mg/1 Gallic Acid Equivalents (GAE) (Mean= 1084.6). Samples J and N, which were of Spanish and Portuguese origins, respectively, contained the highest concentration of 1625 mg/1 GAE each, while the lowest concentration was recorded in sample C, a wine of Nigerian origin. Similarly, the total anthocyanin concentration of freshly opened wine samples was in the range of 20.5-166.7 mg/1 Malvidin-3-Glucoside Equivalents (Mean = 109.4), with sample R (an American wine) containing the highest concentration. The total tannin concentration was in the range of 225-2387 mg/l Epicatechin Equivalents (Mean= 1653.1). Wine Q, a South African wine, had the highest concentration of tannin. The effect of air exposure resulted in a time• dependent decline in the total phenolic concentration of the wines, with the decline being more pronounced after 60 mins of air exposure. Red wine samples with lower phenolic concentration were found to be less affected by the reduction in total phenol due to air exposure. The maximum loss in the total phenolic concentration on air exposure was recorded in sample S (a Spanish wine), whose phenol content decreased by as much as 87.4% after 120 mins. Maximum decrease in total anthocyanin content was observed in sample A (a Spanish wine) which decreased by as much as 58.5% after 120 mins. Total tannin was found to be more stable than other polyphenolic compounds present in the samples, as the total tannin concentration showed a less significant reduction over time. Tannin concentration of sample B (an Australian wine) and sample C, decreased by as much as 22% over the course of 120 mins. A similar trend in decline was observed during cold storage between 5-8C after 96 hours at 24-hour intervals. Sample K lost as much as 87.6% of its total phenolic content after 96 hours. A maximum loss of74.9% of the concentration of total anthocyanin was observed in wine C. For total tannin, a loss of 47% was observed in wine B after 120 mins. The result of the organoleptic (sensory) evaluation and the total phenolic concentration were non-significantly correlated, r = 0.29, p < 0.05. Likewise, there was a non-significant correlation between the result of the organoleptic (sensory) evaluation and the total tannin concentration, r = 0.39, p < 0.05. Total phenolic concentration and the retail price were significantly correlated, r = 0.55, p < 0.05. Similarly, Total tannin concentration and the retail price were also significantly correlated, r = 0.50, p < 0.05. Comparing the total phenolic content of the alcoholic (M = 1201.60, SD= 357.59) and non-alcoholic brands (M = 967.50, SD = 521.34), showed a non-significant difference t (18) = 0.26, p < 0.05 (p > 0.05). In conclusion, the various wine brands differed widely in their content of all classes of polyphenols tested. The polyphenols in the samples also differed in their susceptibility to oxidation during air exposure and cold storage after opening. These observations could be useful to consumers who are interested in wines because of their content of polyphenols, which are reported to offer some health benefits.
CHAPTER ONE
INTRODUCTION
Epidemiological studies have shown that a moderate wine consumption has beneficial effect on health. Wine consumption reduces the susceptibility of low density lipoprotein (LDL) to oxidation which is important for the prevention of arteriosclerosis development (Harbome and Williams, 2000; Heim et al., 2002). The practice of wine consumption has a favorable influence on the reduction of cancer incidence and on chronic inflammatory diseases, the development of both being associated with oxygen free radical (Scalbert et al., 2005). This has gained more awareness through the famous “French Paradox” which observed low death rates resulting from coronary heart disease (CHD) despite high intake of dietary cholesterol and saturated fat (Renaud and de Lorgeril, 1992). The more remarkable health promoting effect of wine in comparison to alcohol alone (in addition to other factors) is due to some biologically active compounds, present especially in red wine. Among alcoholic beverages, red wine has been reported to be more protective against coronary heart disease than other alcoholic beverages (Gronbaek et al., 1995). More than 500 different compounds, of which 160 are esters, have been
identified in different wine types. These include water (7487%, w/w), ethanol (10 14%),
saccharides (0.05-10%), organic acids (0.05-0.7%), phenols (0.01-0.2%), and glycerol (Soleas et al., 1997). Phenolic compounds have long been considered to be basic components of wines and over 200 compounds have been identified. The concentration of total phenolic compounds in commercially available red wines is rarely above 2.5 g/1 (Singleton et al., 1999). Two primary classes of phenolic compounds that occur in grapes and wine are flavonoids and non-flavonoids. Flavonoids commonly constitute > 85% of the phenolic content (2: 1 g/1) in red wines. In white wines, flavonoids typically comprise < 20% of the total phenolic content (< 50 mg/1). Their
dietary intake has been shown to be inversely related to coronary heart disease mortality
(Hoffmeister et al., 1999).
Phenolic compositions is an important aspect in high quality red wines, and are responsible for astringency and bitterness (Margaret et al., 2007), and play a role in colour stability. The phenolic profile of a wine has been shown to be influenced by different viticultural practices, as well as different enological techniques. The variety, vintage and region where the grapes are grown all affect the phenolic composition of the wine (Brossaud et al., 1999; Yokotsuka et al.,
1999). The amount of flavonoids extracted during vinification is influenced by many factors, including temperature, mixing, parameters of the fermentation vessel, duration of skin maceration, ethanol concentration, SO, yeast strain, pH, and pectolytic enzymes (Brossaud et al., 1999). The concentration of phenolic compounds in wine increases during skin fermentation and subsequently begins to decrease as phenols bind with proteins and yeast hulls (cell remnants), and precipitate. During fining and maturation, the concentration of phenolic compounds continues to decrease. Their concentration is further substantially decreased at aging and cold storage (Stratil et al., 2008). Research on non-enzymatic wine oxidation by exposure to atmospheric oxygen has been approached on two major scales; from a macroscopic point of view, and a step-by-step mechanism involved in oxidation. This advanced exploration of oxidation phenomena in wine has been largely undertaken since the beginning of the 1990s (Atanasova et al., 2002). However, while detrimental effects of excessive and prolong exposure are well established, little is known about the exact impact on wine quality due to low and short levels of oxygen exposure.
1.2 Wine
Wine is an alcoholic beverage (containing ethyl alcohol, which is commonly referred to as ethanol) made from fermented grapes or other fruits. Wine is a complex liquid. Although water, ethanol, glycerol, and various organic acids comprise the major portion of wine, the distinct identity comes from the organic compounds (such as terpenes, esters, and alcohols), polysaccharides and phenolics (such as anthocyanins and tannins) present in the wine (Eti~vant,
1991). Some aromatic compounds are present in the grapes from which the wine is made, and some are synthesized as by-products of fermentation by the yeast that turns the sugar in the grape must into ethanol. Still others are formed only after wine has been aged and are the result of oxidation and acid-catalyzed reactions (Peyrot des Gachons and Kennedy, 2003).
Polysaccharides are polymeric un-fermentable sugars that lend body and viscosity to a wine.
Without them, a wine might seem thin or watery. These compounds are formed during fruit ripening when the grape berry softens. The riper the grape, the more these components are found in the final wine (Goldberg, 1995). Polyphenolic compounds are widely distributed in vegetables and fruit trees. They are present in complex polymeric and glycosidic forms, that could not easily be degraded by digestive juices and so their absorption could be limited. In wine, during fermentation, these aggregates are broken down to monomeric forms (Peyrot des Gachons and Kennedy, 2003). The alcohol content of 10% or more contained in most table wines gives stability to phenols present in bottled wines, allowing their absorption (Goldberg, 1995).
1.2.1 Classification of Wine
Wines are grouped by employing a number of different methods. They could be grouped into different classes by grape variety, region of origin, colour, production technique, by the name of the wine maker or viticulturist (Liger-Belair, 2005). For the consumer, basically three (3) main
classes of wines are most easily discernible: table wines, sparkling wines, and fortified wines. Table wines (still or natural wines) make up the majority of wines available in the market.
1.2.1.1 Table Wines
Table wines contain between 10 and 14 percent alcohol and are traditionally consumed as part of a meal. They are further grouped by their colour, sugar content, the variety and origin of the grapes that were used (Don~che, 1993). Wines can be white, red, or pink in colour, depending on the grape variety and wine-making technique. Most of the table wines made fit for consumption are fermented until they are dry-this means that, the entire grape sugar moiety present in the grape must has been turned to alcohol by the yeast. Off-dry (slightly sweet) wines are made by stopping the fermentation before all the sugar is converted or by adding grape juice to the wine afterwards. In other wine-producing regions outside of Europe, California and Australia in particular, table wines are most times classified by the grape variety from which they are made (Cerdan et al., 2002). As a rule, at least 75 percent of the grapes used in the production of the particular wine must be of the named grape variety (Don~che, 1993). For example, Chardonnay is wine made from at least 75 percent chardonnay grapes. Wines classified this way are sometimes called varietals, and include wines such as Riesling, Cabernet sauvignon, and Merlot. The traditional European classification system puts more emphasis on the region or appellation; where the wine is from. The French system of Appellation d’Origine Control~e labels wines according to their geographical pedigree (Cerdan et al., 2002). The most famous wine-producing regions in France, and probably in the world, are Burgundy, in central France, and Bordeaux, a region on the southwestern coast of the country. Bordeaux ranks its best wineries, called chdteaux, and their vineyards-crus, into five classes called grand crus: Ch~teau Margaux,
Ch~teau Latour, Ch~teau Mouton-Rothschild and Ch~teau Lafite-Rothschild in Pauillac, plus Ch~teau Haut-Brion in Graves. Wines from these vineyards in France are considered to be among the highest-quality wines in the world (Prectorius and Hoj, 2005).
American wineries use a tag on their wine bottle labels called Appellation of Origin to indicate where the grapes were grown. An appellation can be a country, state, county, or geographically defined as American Viticultural Area (AVA). At least 85 percent of the grapes used to produce the wine must be from the viticultural area stated on the label (Don~che, 1993). The United States currently recognizes about 150 AVAs, distinguishable by geographical features. The largest growing region in the United States, California, has at least 85 AVAs, including the Napa and Sonoma valleys (Don~che, 1993).
1.2.1.2 Sparkling Wine
Sparkling wine is a modification of table wine by subjecting table wine to a second fermentation. The wine maker achieves this by adding a measured amount of sugar and fresh yeast to the dry wine (Liger-Belair, 2005). This can be carried out in a closed tank, or directly in the bottle. French champagne, the most famous sparkling wine, is produced in this way (Liger-Belair,
2005). The fresh yeast ferments the freshly added sugar, but this time the carbon dioxide gas remains in the sealed bottle, creating carbonation. The basic material is usually a dry white, rose, or red table wine. Sufficient sugar is added to the basic wine to produce a pressure of about five (5) or six (6) atmospheres. The gas bubbles to the surface when the sparkling wine is poured into a glass (Pernot and Valade, 1995). Under the Appellation d’Origine Control~e system, only sparkling wines produced in the Champagne region of northeastern France can officially use the name champagne. Sparkling wines produced in all other regions of the world, even those
produced using the traditional champagne method, are simply referred to as sparkling wines
(Pemot and Valade, 1995).
1.1.1.3 Fortified Wines
Fortified wines contain additional alcohol and are usually consumed in small amounts as aperitifs before meals or dessert wines after a meal (Cristovam and Paterson, 2003). Two (2) famous examples are port and sherry. In port (originated in Portugal) wine making, the grapes are crushed and the fermentation started but then stopped through the addition of more alcohol, which as a result kills the yeast. The resulting wine is sweet and has an alcohol content that is about 5-10% higher than table wine. Fortification has two purposes: (1) to raise the alcohol content sufficiently (usually 17-21 %) to prevent fermentation of all of the sugar and (2) to produce types with a special alcohol character. The alcohol used for fortification is usually (legally required in most countries) distilled from wine (Cristovam and Paterson, 2003; Liddle and Boero, 2003). The distillation of the fortifying spirits is made to a high percent alcohol, usually 95-96%. Industrial alcohol has also been employed in a few countries. Sherry (originally from Spain) is likewise produced by adding alcohol to a young dry wine in an oak barrel intentionally filled only halfway. Special yeasts called flor yeast grow on the surface of the wine and create the distinct nutty flavour characteristic of sherry (Martinez et al., 1998).
Brandy is produced from wine but is classified as distilled liquor, not as wine. Brandy is distilled from wine to concentrate the alcohol in the wine. After distillation the brandy is aged. Bottled brandy typically contains 40 percent alcohol and has been aged in oak barrels for several years (Singleton, 1995; Bertrand, 2003; Cantagrel, 2003)
1.2.2 The Wine Grapes and Vineyards
The main grapevine cultivated for wine production is the European wine grape is Vitis vinifera which probably originated in the Caucasus Mountains (Aguero et al., 2006). Today there are more than 5,000 varieties of Vitis vinifera grown in the world basically localized along the Black, Caspian, and Mediterranean seas.V. labrusca and V. rotundifolia have been domesticated in the eastern United States, the domestication of V. amurensis has been reported in Japan, and various interspecies hybrids have been used for wine production (Aradhya et al., 2003). The high sugar content of most V. vinifera varieties at maturity is the major factor in the selection of these varieties for use in much of the world’s wine production. Their natural sugar content, providing necessary material for fermentation, is sufficient to produce a wine with alcohol content of 10 percent or higher; wines containing less alcohol are unstable because of their sensitivity to bacterial spoilage (Bertsch et al., 2005).This grape plant prefers warm, dry summers and mild winters, and successful cultivation is limited only to temperate climates in both the northern and southern hemispheres. One of the major factors attracting wine makers to this grape is its tremendous range in composition (Bertsch et al., 2005). The pigment pattern of the skin varies from light greenish yellow to russet, to pink, red, reddish violet, or blue-black; the juice is generally colourless, although some varieties have a pink to red colour, and the flavour varies from quite neutral to strongly aromatic (examples include Gewiirztraminer, Cabernet Sauvignon, and Zinfandel). Some varieties, such as Pinot Noir, having rather neutral flavoured juice, develop a characteristic flavour when fermented on the skins and aged (Bouquet et al., 2006). The species V. labrusca and V. rotundifolia seldom contain sufficient natural sugar to produce a wine with alcohol content of 10 percent or higher and additional sugar is usually required. The most popular red varieties in the United States are zinfandel, cabernet sauvignon, grenache, merlot,
and pinot noir. The most popular white grapes are colombard, chardonnay, cheninblanc, and sauvignon blanc. Factors associated with a particular region or areas make the wine from that location unique (Bowers and Meredith, 1996). These factors (terroir) include local climate (rainfall, sunlight and temperature), location of grapevines (altitude and slope), and soil
(structure, composition, and water drainage). From studies, it has been observed that a grapevine
produces the best of its fruits when the moderate climate provides much sunshine and cool nights without frost, and the soil is well drained (Bowers and Meredith, 1997). Grapevines grow best in sandy, chalky, or rocky soils. Wine grapes are grown in vineyards, where individual vines are cultured and nurtured on a system of stakes and wires, called a trellis, to maximize exposure to the sun. The first harvest of grapes can be collected in the third year after the planting, and a full crop suitable for commercial use can be expected after five years (Bowers and Meredith, 1997). Grapevines may produce fruit for 20 to over 100 years. The grapevine growth cycle begins in early spring when new shoots appear on the buds of the grapevine. Grapes begin their growth cycle in the spring when average daily temperature is about 10° C (50° F). To reach maturity, they require a certain amount of heat above 10° C during the growing season. This amount of heat, called the heat summation, is calculated by totaling the number of degrees of average daily temperature over 10° C for each day of the growing season (Fanizza et al., 2005). A heat summation of about 1,800° is required for successful growth. If the heat summation is less than required, the grapes will not ripen; they will reach the end of the growing season with insufficient sugar and too much acidity (Franks et al., 2002). This condition, frequently occurring in the eastern United States, Switzerland, and other cool regions, can be corrected by adding sugar to the crushed grapes. Where the heat summation is much greater than required, as in Algeria and parts of California, the grapes mature earlier and with less acidity and colour than
those produced under cooler conditions (Maletic et al., 2004). The grapes begin to ripen in midsummer and are ready to be harvested in midfall, depending on the location, grape variety, weather, and the type of wine to be produced. By the end of fall, the vines lose their leaves and become dormant until the following spring. The particular character of wine is strongly affected by viticultural practices such as training, trellising, harvesting, and pruning (Prectorius and Hoj,
2005). Training and trellising enable the viticulturist to control the sun exposure to ensure an even grape ripening. Grapes harvested when they are not ripe may be low in sugar and may not ferment properly. On the other hand, overly ripe grapes are very high in sugar content and produce wine high in alcohol (Regner et al., 2000). Once the vines are dormant, the viticulturist prunes the vines to remove all the dead wood. Pruning enables the grower to control the size and shape of the vines, and also the number of buds that will develop the next year. Just as plants have pests that affect the quality and number of their products, grapevines in like manner have many natural enemies: insects, molds, bacteria, viruses, and animals such as deer and birds that eat the young shoots or the sweet grapes (Regner et al., 2000). These pests will affect the produce from the grapevine and can as well contaminate the wine due to by-products of metabolism as remnants from these pests. Certain soil-borne pests, such as the root louse Phylloxera, destroy the roots of European grapevines. Vines native to North and South America have a natural resistance to this insect, but they often produce grapes with an undesirable flavour (Bowers and Meredith, 1997). To circumvent this problem, American vineyards use grapevines grown from two different parts: the roots from resistant American vines and the part above the ground from European vines by a process known as grafting and works much like healing a “broken bone”. There are about 8 million hectares (20 million acres) of vineyards in the world producing 69 million tons of grapes each year. About 29 billion liters (8 billion gallons) of wine
are made from those grapes. Countries that produce large quantity of wine include France, Italy, Germany, the United States, Australia, and South Africa. Many other countries produce enormous quantities of table wines (Bowers and Meredith, 1997). In Europe there are, for example, Spain, Portugal, Switzerland, Hungary, Romania, Bulgaria, Greece, and Georgia. In North Africa and the Middle East there are Algeria, Tunisia, and Israel. In South America there are Brazil, Peru, Chile, and Argentina. The largest wine producers worldwide are France (19 percent), Italy (17 percent), and Spain (13 percent). The United States of America (USA) is the fourth largest producer in the world, producing about 9 percent of the world’s wine. California produces 90 percent of all the wine in the United States, with enologists in New York, Washington, Oregon, and at least 41 other states making the remainder of U.S. wine. In Asia the largest producer is Japan ((Bowers and Meredith, 1997; Fanizzaet al., 2005; Franks et al., 2002).
1.3 Red Wine
Red wine is a type of wine made from dark-coloured grape varieties. Its colour can be derived from a vast assortment of grape varietals ranging from grapes that are reddish, deep purple, and even a beautiful blue on the colour scale (Boulton, 1996). These grapes give rise to a wine that is colour classified with such descriptors as garnet, almost black, dark red, light red, ruby red, opaque purple, deep violet and maroon. The actual colour of the wine can range from intense violet, typical of young wines, through to brick red for mature wines and brown for older red wines. It is the grape skins that are responsible for the red wine’s distinct colour spectrum. The skins are in contact with the grape’s juice during the fermentation process, allowing the dispersion of both colour and tannins (Bird, 2005).The juice from most black grapes is greenish• white; the red colour comes from anthocyan pigments (also called anthocyanins) present in the skin of the grape; exceptions are the relatively uncommon teinturier varieties, which produce a xxiii
red coloured juice. Much of the red-wine production process therefore involves extraction of colour and flavour components from the grape skin (Jeff, 1999). The individual wine’s particular red hue depends on the grape type used in the process and the length of time the skin’s pigmentation is in contact with juice. There are right around 50 key red wine varietals that consistently manifest themselves in today’s worldwide wine market.
1.2.1 Red Wine Styles and Varieties
Red wines are made into a variety of styles. The stylistic differences are based on differences in wine characteristics such as grape variety, colour, flavour, body, mouth-feel, and aging potential. The styles range from simple, fruity, fresh, light coloured blushes and roses to complex, full bodied, rich and dark red, with long aging potential. Many factors such as a variety, soil, climate, growing conditions, and viticultural practices influence the fruit composition, and therefore, the style of wine that can be produced. In addition to fruit composition, winemaking techniques also play an important role in determining the wine style.
Many varieties are available for red wine production. The wines are usually produced as varietals, or as blends containing several varieties. A list of commonly used red wine varieties is given in Table 1.
Table 1: Commonly Used Red Wine Varieties
VINIFERA GROUP LABRUSCA FRENCH HYBRIDS OTHERS
Cabernet Sauvingnon Concord
Baco Noir
Northon/Cynthiana
Merlot
Pinot
Steuben
Chambourcin
Chancelor
St. Vincent
Vincent
Zinfandel Syrah (shiraz) Grenache Cabernet Franc Barbera
Gamay
Foch Rougeon Villard no ir colobel
Source: Bird, 2005
Varieties from the Vinifera group are most widely used for winemaking (Bird, 2005). In regions where Vinifera grapes are not grown, French hybrids, Labrusca, and other varieties are often used. Among the Vinifera group, Cabernet Sauvignon alone, or in combination with Merlot or Cabernet Franc is used in premium red wine production (Boulton, 1996). Pinot noir, the famous grape of Burgundy, makes excellent red wine. When grown in other parts of the world, the wine does not always attain the same level of quality as found in Burgundy. Zinfandel, though popular for blush wine, can also make dark, full-bodied, and flavourful red wine (Jeff, 1999). Syrah, the popular grape of Rhone and Australia makes fruity wines with softer tannins. Concord is the leading red wine variety among American grapes. Wines from these grapes have a strong flavour, which is often referred to as a “foxy” aroma. Another American red wine grape, Cynthiana/ Norton, does not have the foxy aroma and can make full-bodied, dark red wines (Bird, 2005). Among the varieties in the French hybrid category, Baco, Chambourcin, Foch, and Rougeon are commonly used for red wines. These varieties, with proper handling, make good red table wines. Fresh grapes make the best raw material for making red wine (Boulton, 1996). In a situation where fresh grapes are unavailable, frozen grapes or grape concentrate can be used, particularly for making smaller lots of wine (Jeff, 1999
1.4 Phenolics
Phenols are a large and complex group of compounds of pnmary importance to the characteristics and quality of red wine. They are also significant in white wines, but occur at much lower concentrations (Waterhouse and Edeler, 1999). Phenols and related compounds can affect the appearance, taste, mouth-feel, fragrance, and antimicrobial properties of wine. Although primarily of grape origin, smaller amounts may be extracted from wood cooperage. Only trace amounts are derived from yeast metabolism (Waterhouse and Edeler, 1999).
1.4.1 Polyphenols
Polyphenols also described as Polyhydroxyphenolsare a group of orgamc chemicals characterized by the presence of large multiples of phenol structural units. They are basically natural, but can also be synthetic. The unique physical, chemical, and biological (metabolic, toxic, therapeutic, etc.) properties of a particular class are characteristic of the number of these phenol structures bonding together to form the polyphenol (Quideau, 2011). The major polyphenolics found in wine are either members of the diphenylpropanoids (flavonoids) or phenylpropanoids (non-flavonoids). In addition, there are related (phenyl) compounds that do not possess one or more hydroxyl groups on the phenyl ring (and are correspondingly strictly not phenols).
This material content is developed to serve as a GUIDE for students to conduct academic research
QUANTIFICATION OF THE POLYPHENOLIC CONSTITUENTS OF RETAIL SAMPLES OF RED WINE>
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