Hybrid varieties from Podkarpacie: five for white wines (Aurora, Bianca, Muskat Odeski, Pearl of Zala, Prim), five for red ones (Alden, Frontenac, Leon Millot, Marechal Foch, Rondo) and Swenson Red for rose wine were also investigated [27]. Among the major discriminants tested, the extract was estimated from 146 (Prim) to 218.5 g/L (Leon Millot) and acidity from 7.3 (Prim) to 9.8 g malic acid/kg (Aurora) for white and from 8.1 (Leon Millot) to 17.9 g malic acid/kg (Frontenac) for red wines. Total polyphenols content expressed as mg catechin/kg d.w. was at the level from 14,380 (Frontenac) to 49,190 (Rondo). The level of polyphenols was also investigated in cultivars from the most famous Polish vineyard—Golesz, situated in Podkarpacie, next to Jasło [28]. Ten grapevine cultivars (five white—Muskat Odeski, Hibernal, Seyval Blanc, Jutrzenka, Bianca and five red-skinned—Marechal Foch, Frontenac, Heridan, Rondo, Swenson Red) harvested in 2006 were analysed. The level of polyphenols of white varieties was the lowest for Seyval Blanc and Bianca (40.5 mg/100 g f.w.) and the highest for Muskat Odeski (130 mg/100 g f.w.), for red varieties from 117 mg/100 g f.w. (Swenson Red) to 686.7 mg/ 100 g f.w. (Rondo). Both studies [27, 28] show that the richest in health-promoting polyphenols, among the varieties grown in Podkarpacie are white Muskat Odeski and red Rondo.

There are ongoing attempts to restore the vineyards in Szczecin Lowland. The climate of this area is significantly affected by the Baltic Sea and big water basins (Szczecin Lagoon, Dąbie Lake, the Odra River), providing additional moisture in the period of plants vegetation. The majority of the West Pomeranian Province belongs to the zone 7A on the Heinz and Schreiber's 'Map of zones of plant resistance to frost' [29, 30]. The quality of three *V. vinifera* varieties (Cabernet Sauvignon, Cabernet Franc and Pinot Noir) cultivated in Szczecin Lowland in Poland and in Bulgaria was compared [30]. Higher levels of extract, pH and lower organic acid content were observed in fruits grown in Bulgaria. Cabernet Sauvignon was characterized by the highest level of extract (av. 23.3%) and acidity irrespective of the harvesting area. The lowest extract level was observed in Cabernet Franc variety (17.2% grown in Poland and 22.9% in Bulgaria). Acidity of Pinot Noir was the lowest (0.65 and 0.42 grown in Poland and Bulgaria, respectively). NAI (anthocyanin index) was higher in fruits growing in Poland. Another studies [31] show that grapes from Regent cultivar had a higher content of organic acids, vitamin C and yielded more juice, compared to Cabernet Sauvignon fruits. Cabernet Sauvignon contained more substances giving the wine its red colour. Regent cultivar grapes expressed lower extract content (below 20%), which is in agreement with other authors research on the Regent variety grown in Poland [25, 32].

Cultivated in Poland, Cabernet Sauvignon grapes accumulate an average of 18% of sugar, while Regent 11–12.5% only. According to Polish regulations, grape musts from these fruits should be sweetened to reach the appropriate alcohol content in wines [31].

Also wines from Polish vineyards were under investigations. Seven white wines (Cuvee, Jutrzenka, Milia, Swenson Red, Bianca, Cuvee, Seyval Blanc) and three red ones (Cuvee and Leon Millot) produced in Golesz vineyard in 2003–2006 were analysed [33]. The acidity ranged from 4.0 (Milia) to 8.7 g malic acid/L (Seyval Blanc). The red wines were characterized by significantly higher antioxidant activity from 670 (Cuvee) to 745 mg Trolox/100 ml (Leon Millot) in comparison with white wines. Red wines were distinguished by high concentration of polyphenols, from 970 (Leon Millot) to 1350 mg of catechin/L (Cuvee). Wines from the same winery, from 2007 vintage obtained from red (Heridan, Frontenac, St. Croix, Sabbrevois, De Chaunac, Marechal Foch, Leon Millot, Cascade, Rondo and Regent) and white (St. Pepin, La Crescent, Adalmiina, Seyval Blanc, Swenson Red) were also scrutinized [34, 35]. The total phenolic content of the white wines varied from 180 (Seyval Blanc) to 242 mg/L (Swenson Red) and for the red ones from 970 (Leon Millot), 996 (Regent) to 1669 mg/L (Rondo). The total phenolic content of wines produced from *V. vinifera* fell between the ranges observed from the hybrid grapevines [35]. Rondo wine, produced from the multispecies hybrid grapevine, was the richest in total phenolics and phenolic acids.

in Central Poland: Auxerrois, Pinot Gris, Pinot Noir, Riesling and Chasselas Blanc, classified

Hybrid varieties from Podkarpacie: five for white wines (Aurora, Bianca, Muskat Odeski, Pearl of Zala, Prim), five for red ones (Alden, Frontenac, Leon Millot, Marechal Foch, Rondo) and Swenson Red for rose wine were also investigated [27]. Among the major discriminants tested, the extract was estimated from 146 (Prim) to 218.5 g/L (Leon Millot) and acidity from 7.3 (Prim) to 9.8 g malic acid/kg (Aurora) for white and from 8.1 (Leon Millot) to 17.9 g malic acid/kg (Frontenac) for red wines. Total polyphenols content expressed as mg catechin/kg d.w. was at the level from 14,380 (Frontenac) to 49,190 (Rondo). The level of polyphenols was also investigated in cultivars from the most famous Polish vineyard—Golesz, situated in Podkarpacie, next to Jasło [28]. Ten grapevine cultivars (five white—Muskat Odeski, Hibernal, Seyval Blanc, Jutrzenka, Bianca and five red-skinned—Marechal Foch, Frontenac, Heridan, Rondo, Swenson Red) harvested in 2006 were analysed. The level of polyphenols of white varieties was the lowest for Seyval Blanc and Bianca (40.5 mg/100 g f.w.) and the highest for Muskat Odeski (130 mg/100 g f.w.), for red varieties from 117 mg/100 g f.w. (Swenson Red) to 686.7 mg/ 100 g f.w. (Rondo). Both studies [27, 28] show that the richest in health-promoting polyphenols,

among the varieties grown in Podkarpacie are white Muskat Odeski and red Rondo.

research on the Regent variety grown in Poland [25, 32].

There are ongoing attempts to restore the vineyards in Szczecin Lowland. The climate of this area is significantly affected by the Baltic Sea and big water basins (Szczecin Lagoon, Dąbie Lake, the Odra River), providing additional moisture in the period of plants vegetation. The majority of the West Pomeranian Province belongs to the zone 7A on the Heinz and Schreiber's 'Map of zones of plant resistance to frost' [29, 30]. The quality of three *V. vinifera* varieties (Cabernet Sauvignon, Cabernet Franc and Pinot Noir) cultivated in Szczecin Lowland in Poland and in Bulgaria was compared [30]. Higher levels of extract, pH and lower organic acid content were observed in fruits grown in Bulgaria. Cabernet Sauvignon was characterized by the highest level of extract (av. 23.3%) and acidity irrespective of the harvesting area. The lowest extract level was observed in Cabernet Franc variety (17.2% grown in Poland and 22.9% in Bulgaria). Acidity of Pinot Noir was the lowest (0.65 and 0.42 grown in Poland and Bulgaria, respectively). NAI (anthocyanin index) was higher in fruits growing in Poland. Another studies [31] show that grapes from Regent cultivar had a higher content of organic acids, vitamin C and yielded more juice, compared to Cabernet Sauvignon fruits. Cabernet Sauvignon contained more substances giving the wine its red colour. Regent cultivar grapes expressed lower extract content (below 20%), which is in agreement with other authors

Cultivated in Poland, Cabernet Sauvignon grapes accumulate an average of 18% of sugar, while Regent 11–12.5% only. According to Polish regulations, grape musts from these fruits

Also wines from Polish vineyards were under investigations. Seven white wines (Cuvee, Jutrzenka, Milia, Swenson Red, Bianca, Cuvee, Seyval Blanc) and three red ones (Cuvee and Leon Millot) produced in Golesz vineyard in 2003–2006 were analysed [33]. The acidity ranged from 4.0 (Milia) to 8.7 g malic acid/L (Seyval Blanc). The red wines were characterized by significantly higher antioxidant activity from 670 (Cuvee) to 745 mg Trolox/100 ml (Leon

should be sweetened to reach the appropriate alcohol content in wines [31].

as both wine and table cultivars [26].

406 Grape and Wine Biotechnology

The chemical characterization of wine produced from 10 Polish grape cultivars planted near Krakow (Garlicki Lamus vineyard at the Experimental Research Station Garlica Murowana) was conducted [36]. Six white cultivars (Aurora, Bianca, Jutrzenka, Muskat Odeski, Seyval Blanc and Siberia) and four red ones (Marechal Foch, Leon Millot, Rondo, Regent) were investigated. The tartaric acid content varied from 224 (Leon Millot) to 705 mg/L (Marechal Foch) and from 458 (Aurora) to 1528 mg/L (Siberia) for the red and white wines, respectively. Among the red wines, Regent was the wine with the highest antioxidant activity (10.5 mM Fe) and polyphenol concentration (3.16 g GA/L), whereas the lowest values were noted for Leon Millot (5.75 mM Fe and 0.82 g GA/L, respectively). In the white wine group, Jutrzenka was observed to have the highest antioxidant potential (3.17 mM Fe) and polyphenols content (0.51 g GA/L), while Seyval Blanc had the lowest antioxidant activity (0.77. mM Fe) and Bianca total polyphenols content (0.28 g GA/L). The white wines were much richer in organic acids (succinic, malic and tartaric acids) in comparison with the red wines. The total acidity was lower, while the pH was higher in the red than in the white wines. Some of the wines, particularly red Regent and white Jutrzenka, displayed high antioxidant activity and polyphenols levels. It should be highlighted that wine production from Jutrzenka cultivar does not require any agro-chemical pretreatment, and thus it can be classified as ecological manufacturing [27].

Clarification is an important stage in wine production. Effectiveness of selected wine clarification methods (tannins, gelatine or bentonite) was also studied [37]. The results showed that the particular method does not determine the wine sensory quality but can affect the total extract. The application of the gelatin-tannic acid mixture was characterized by the higher efficiency compared to the use of bentonite.

## **4. Polish traditional fruit wines and meads**

Poland is one of the major fruit producers in Europe with approximately 3-million tons of fruit, mainly apples, which are also utilized for wine production [4]. In some Polish regions, fruit wines are recognized as traditional or regional products: e.g. wines from cherries (*Prunus avium*) in the region Świętokrzyskie; from sour cherries in the province Wielkopolska; from plum in the province Zachodniopomorskie [38].

The extensive studies on the apple wines conducted by, among others, Bonin [39], Satora et al. [40] and Kunicka-Styczyńska [41–43], aimed at the determination of the influence of fruit processing, pectinolytic enzyme application, biological deacidification on antioxidant and volatile compounds profile of wines. The trimeric fractions of proanthocyanidins were identified as the causes of bitterness of apples wines and eliminated by clarifying with selected gelatines with small amount of low molecular proteins [44, 45].

The adsorption of polyphenols in apple musts and wines clarification with some bentonite preparations was also investigated, finding CLARIT BW125 (dioxosilanes, China) as the most efficacious [19]. Later studies [46] focusing on the levels of nucleic acid degradation products of apple wines fermented with preparations made of sediment wine yeast autolysate, demonstrated an elevated concentration of purine and pyrimidine bases and uric acid. The inhibiting effect of tannin in chokeberry must on the winemaking process was also considered [47]. Much attention has been paid to assessing pulp processing conditions to maintain biologically active compounds, especially polyphenols [48]. The effect of pulp treatment on polyphenols changes in blackcurrants and sour cherries musts and wines was investigated resulting in the highest extraction of polyphenols obtained after pectinolysis with Rohapect MA Plus (AB Enzymes, Germany) and Pektopol PM (Pectowin, Poland), respectively [49, 50].

Tradition of mead-making in Poland has been cultivated for ages. The most popular traditional flavoured Polish meads are: (i) Bernardine mead—with the addition of hops, iris rhizomes and dried or fresh rose petals, rose jam or rose oil; (ii) Castellan mead—with the addition of hops, vanilla pods and roots or fresh celery leaves; (iii) Capuchin mead—with the addition of hops, ginger, vanilla pods and nutmeg; (iv) Hop mead—with the addition of hops and raisins; (v) Camp mead—with the addition of hops, cinnamon, cloves and dried juniper berries; (vi) Lithuanian mead—with dried juniper berries and elder flower; (vii) Spicy mead—with the addition of hops, cinnamon, ginger, nutmeg, cloves and peppercorns; (viii) Cracowian mead —with the addition of hops, cinnamon and lemon peel; (ix) Polish mead(also known as the Russian) —with the addition of hops, dried berries of blackcurrant and valerian root; (x) Mound mead—with the addition of orange and lemon; (xi) Old Polish mead—with the addition of ginger, cinnamon, pepper and elder flower; and (xii) Lord mead—with the addition of dried raspberry, lemon, orange and dried rose petals [51].

#### **5. Final remarks**

Polish winemaking relies both on frost tolerant grapevine cultivars and fruits grown in this climate zone. Fermented alcoholic beverages become increasingly important in a Polish export. According to Eurostat, sales to foreign markets reached 35.2-mln litres in 2013, which is about a quarter of the total output. Polish wines, both grape and fruit ones, are becoming more widely recognized not only in Europe but in the world, winning the markets of Germany, Slovenia, Slovakia, Denmark, Switzerland, Latvia as well as Australia, Japan, Hong Kong and the United States. To face the shrinking market of classic wines, some traditional manufacturers as well as newly established companies have attempted to premiumise the category. They also offered a wide range of quality fruit wines, priced at the level of grape wines, and frequently presented as organic (Aronica-Jantoń) or regional products (Vin-kon).

The modern history of Polish grape winery is built by enthusiasts, forming the vineyards in the regions to allow the grapevine cultivation. The legal conditions for the production of wine have also been changed. It is permissible to use sucrose in order to enrich the must before fermentation, or to raise the potential alcoholic strength of 3% in relation to the volume, which give the Polish winemakers greater opportunities to obtain the wine a pleasant reception. By 2015, the annual production of grape wine in Poland was limited to 50 thousand hectolitres, with no demand of any restrictions in planting vineyards. Starting from 2016, any restrictions in this regard are also ceased, which promoted the development of many small vineyards particularly in the southern regions of Poland. Mechanisms of support for wine producers have been replaced by a system of direct payments, as being in force for other agricultural crops [52]. Planting the vineyards and wine production become more popular.

In the light of the law, Poland is exempted from the obligation to the classification of vine varieties and any variety registered at least in one of the European countries can be grown in Polish vineyards. Moreover, amendments to the legal act simplify rules of wine labelling and allow for the name of the vine variety and vintage to be put on the label of all categories of wine. On the basis of the regulations, producers up to 1000 hectolitres of wine per year, only from the grapes originated from their own vineyards, are entitled to release Polish grape wines on the market freely and legally [53]. Apart from the legal regulations, return towards organic foods and oenological tourism fashion create additional opportunities for the Polish vineyards development and wider popularization of Polish grape wines.

## **Author details**

The extensive studies on the apple wines conducted by, among others, Bonin [39], Satora et al. [40] and Kunicka-Styczyńska [41–43], aimed at the determination of the influence of fruit processing, pectinolytic enzyme application, biological deacidification on antioxidant and volatile compounds profile of wines. The trimeric fractions of proanthocyanidins were identified as the causes of bitterness of apples wines and eliminated by clarifying with selected

The adsorption of polyphenols in apple musts and wines clarification with some bentonite preparations was also investigated, finding CLARIT BW125 (dioxosilanes, China) as the most efficacious [19]. Later studies [46] focusing on the levels of nucleic acid degradation products of apple wines fermented with preparations made of sediment wine yeast autolysate, demonstrated an elevated concentration of purine and pyrimidine bases and uric acid. The inhibiting effect of tannin in chokeberry must on the winemaking process was also considered [47]. Much attention has been paid to assessing pulp processing conditions to maintain biologically active compounds, especially polyphenols [48]. The effect of pulp treatment on polyphenols changes in blackcurrants and sour cherries musts and wines was investigated resulting in the highest extraction of polyphenols obtained after pectinolysis with Rohapect MA Plus (AB Enzymes, Germany) and Pektopol PM (Pectowin, Poland), respectively [49, 50].

Tradition of mead-making in Poland has been cultivated for ages. The most popular traditional flavoured Polish meads are: (i) Bernardine mead—with the addition of hops, iris rhizomes and dried or fresh rose petals, rose jam or rose oil; (ii) Castellan mead—with the addition of hops, vanilla pods and roots or fresh celery leaves; (iii) Capuchin mead—with the addition of hops, ginger, vanilla pods and nutmeg; (iv) Hop mead—with the addition of hops and raisins; (v) Camp mead—with the addition of hops, cinnamon, cloves and dried juniper berries; (vi) Lithuanian mead—with dried juniper berries and elder flower; (vii) Spicy mead—with the addition of hops, cinnamon, ginger, nutmeg, cloves and peppercorns; (viii) Cracowian mead —with the addition of hops, cinnamon and lemon peel; (ix) Polish mead(also known as the Russian) —with the addition of hops, dried berries of blackcurrant and valerian root; (x) Mound mead—with the addition of orange and lemon; (xi) Old Polish mead—with the addition of ginger, cinnamon, pepper and elder flower; and (xii) Lord mead—with the addition

Polish winemaking relies both on frost tolerant grapevine cultivars and fruits grown in this climate zone. Fermented alcoholic beverages become increasingly important in a Polish export. According to Eurostat, sales to foreign markets reached 35.2-mln litres in 2013, which is about a quarter of the total output. Polish wines, both grape and fruit ones, are becoming more widely recognized not only in Europe but in the world, winning the markets of Germany, Slovenia, Slovakia, Denmark, Switzerland, Latvia as well as Australia, Japan, Hong Kong and the United States. To face the shrinking market of classic wines, some traditional manufacturers as well as newly established companies have attempted to premiumise the category. They also offered

gelatines with small amount of low molecular proteins [44, 45].

of dried raspberry, lemon, orange and dried rose petals [51].

**5. Final remarks**

408 Grape and Wine Biotechnology

Alina Kunicka-Styczyńska\* , Agata Czyżowska, Katarzyna Rajkowska, Agnieszka Wilkowska and Piotr Dziugan

\*Address all correspondence to<EMAIL_ADDRESS>
Department of Biotechnology and Food Sciences, Institute of Fermentation Technology and Microbiology, Lodz University of Technology, Lodz, Poland

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**Analysis and Origin Authentication**

## **Determination of Trace Elements in Wine by Atomic Spectroscopy and Electroanalytical Methods Provisional chapterDetermination of Trace Elements in Wine by Atomic Spectroscopy and Electroanalytical Methods**

Niina J. Ronkainen Niina J. Ronkainen

Additional information is available at the end of the chapter

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/64932

#### **Abstract**

The chapter will outline the basic theory, advantages and disadvantages, experimental considerations and set up of various atomic spectroscopy, and electroanalytical quantification methods and their specific application to trace element determination in wines. The reader will gain an introduction to most popular elemental analysis methods used in beverage analysis. Copper, iron, manganese, and zinc will be used as examples of essential trace elements throughout the chapter that at high levels may affect the properties of wine as well as the sensory experience of the consumer. Furthermore, special considerations that should be given to wine as a sample matrix for quantitative analysis of inorganic elements and the use of standard addition methods will be described.

**Keywords:** trace element determination, atomic spectroscopy, electroanalytical meth‐ ods, wine, standard addition methods, stripping voltammetry

## **1. Introduction**

Wine is a widely consumed alcoholic beverage produced by yeast fermentation of natural sugars contained in grape juice. Wine has been produced and consumed for thousands of years and is thought to have originated from current Georgia where wine stained pottery from circa 6000 BC has been found [1]. Drinking wine daily is common in many cultures ranging from countries with old, well‐established wine cultures in Europe and America to rapidly growing wine markets such as China. In some countries, alcoholic beverages, including wine, account for >12% of the daily intake of beverages [2]. Although the per capita consumption of wine by Americans was only 9.42 L per person in 2010, the United States is the single largest wine

© 2016 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2016 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

market in the world due to the large number of American consumers [3]. However, Old World wine countries such as France, Italy, and Portugal had the highest per capita wine consumption in 2010 (45.7, 42.15, and 41.81 L, respectively) out of the selected wine producing countries (**Figure 1**) [3]. Algeria and Israel had the lowest wine consumption per person of the selected countries at 0.97 L in 2010 [3].

**Figure 1.** Per capita wine consumption by country (L per person) in 2007 and 2010 from selected wine‐producing coun‐ tries (Wine Institute, Trade Data and Analysis, 2012).

Although fewer studies have been published on trace metal content in alcoholic beverages compared to elemental analysis of foods such as fish, produce, peanuts, and tea, wines are one of the most studied alcoholic beverages. Most studies have focused on quantification of metals such as As, Ni, Cu, Cr, Cd, Pb, Zn, and others in wines from specific countries such as Spain [4, 5], Italy [6–10], Argentina [11–13], Australia [14], Turkey [15–17], Romania [18], and Croatia [19–21] and/or focused on studying regional variation in mineral content from wine‐producing regions and grape varieties within a single country.

Wine has significant economic impact and commercial value in addition to its social impor‐ tance. Wine production is one of the most important agricultural activities in many regions of the world. Wine has a complex sample matrix that contains various components such as macro‐ and microelements as well as lanthanides that contribute to its nutritional value. Wine composition also greatly affects its quality. The concentration of metals in wine is of great significance as it affects their conservation and color, in addition to impacting the organoleptic properties of wine, thereby affecting human consumption and the sensory experience [2, 22, 23]. Trace metals affect the organoleptic properties such as aroma, color, flavor, freshness, and taste. Cu, Fe, and Zn, for example, contribute to haze formation and taste effects in wine [24]. However, these minor metals are also favorable for yeast as they are an essential part required for prosthetic group of metalloenzymes that serve as biological catalysts. Other elements such as Ca, K, Mg, and Na are involved in regulating the cellular metabolism of yeasts by helping to maintain adequate ionic balance and pH [25]. Since metal species participate in oxidation‐ reduction reactions, physical properties of the wine such as color, turbidity, and astringency are largely dependent on metal composition of the wine [23]. Browning, the oxidative spoilage of wine which ultimately results in the loss of aromatic freshness and the appearance of precipitates of condensed phenolic material in the bottled wine, is activated and accelerated by the presence of Fe, Mn, and Cu [2, 22]. The presence and the levels of specific metals can also be used in quality control and the authentication of wines [5, 26]. In addition, metal profile from trace element analysis can act as a fingerprint that may be used to determine the origin, variety, and/or the type of wine and other beverages [27–31]. Therefore, trace element charac‐ terization of wine is powerful in fraud detection in commercially sold wines. The trace element composition of wine is influenced by factors such as grape varieties, soil at the vineyard, and viticultural practices [32, 33]. Knowledge of metal concentrations in wine is also of great importance in quality assurance (QA) of branded wines [34].

market in the world due to the large number of American consumers [3]. However, Old World wine countries such as France, Italy, and Portugal had the highest per capita wine consumption in 2010 (45.7, 42.15, and 41.81 L, respectively) out of the selected wine producing countries (**Figure 1**) [3]. Algeria and Israel had the lowest wine consumption per person of the selected

**Figure 1.** Per capita wine consumption by country (L per person) in 2007 and 2010 from selected wine‐producing coun‐

Although fewer studies have been published on trace metal content in alcoholic beverages compared to elemental analysis of foods such as fish, produce, peanuts, and tea, wines are one of the most studied alcoholic beverages. Most studies have focused on quantification of metals such as As, Ni, Cu, Cr, Cd, Pb, Zn, and others in wines from specific countries such as Spain [4, 5], Italy [6–10], Argentina [11–13], Australia [14], Turkey [15–17], Romania [18], and Croatia [19–21] and/or focused on studying regional variation in mineral content from wine‐producing

Wine has significant economic impact and commercial value in addition to its social impor‐ tance. Wine production is one of the most important agricultural activities in many regions of the world. Wine has a complex sample matrix that contains various components such as macro‐ and microelements as well as lanthanides that contribute to its nutritional value. Wine composition also greatly affects its quality. The concentration of metals in wine is of great

countries at 0.97 L in 2010 [3].

418 Grape and Wine Biotechnology

tries (Wine Institute, Trade Data and Analysis, 2012).

regions and grape varieties within a single country.

There are endogenous and exogenous sources of metals in commercial wines. Endogenous metals of natural origin mostly come from the soil on which vines are grown and reach the wine through the harvested grapes [34]. These primary metals make up for the largest part of total metal content in wine [35–37]. Concentrations of these metals are also characteristic of the type of soil at the vineyard, the climate conditions during the growth of the grapes, grape variety, and the maturity of the grapes [38]. The metals of secondary origin are introduced during growth of grapes or at different stages in winemaking starting with harvesting and finishing with storing bottled wine at a cellar. Environmental pollution, addition of fertilizers used in cultivation, as well as application of fungicides and pesticides during the growing season of the grapes increases the amounts of metals such as Cd, Cu, Mn, Pb, and Zn in the finished wine [11, 39–43]. Variation in levels of K, Ca, and Cu in wine may also results from fertilizers applied at the vineyard [44]. The environmental pollution introducing metals into the soil, grapes, and ultimately the wine could be due to nearby industry, waste management practices, traffic, and even mining operations. For example, wines produced at vineyards located near industrial areas or close to major highways often contain higher levels of Cd and Pb due to vehicle exhaust fumes and other emissions to air, water, and/or soil [11, 39, 41, 42]. During winemaking process, the long contact of acidic wine with materials such as aluminum, brass, glass, stainless steel, and wood used in wine‐making machinery and pipes, casks, and barrels used for handling and storing wine may also introduce metal contamination to the wine. The steps during the winemaking process often introduce elements such as Al, Cd, Cr, Cu, Fe, and Zn into the finished wine [11, 35]. Metal wine casks at a Sonoma Valley winery are shown in **Figure 2**.

**Figure 2.** Shows wine storage containers at a Sonoma Valley winery at California (USA).

Many studies have shown that moderate consumption of red wine may improve health and longevity when it is done in combination with a balanced diet [45]. Much attention has been given in the research and medical communities to the properties and activity of polyphenols. Polyphenols have been shown to provide some possible health benefits by acting as antioxi‐ dants that helps prevent cell damage. Many of these health benefits of wine are associated with polyphenol antioxidants such as resveratrol (3,5,4'‐trihydroxystilbene) that is common in grapes and many other plants [46]. Resveratrol obtained from moderate wine consumption was found to decrease the risk of cardiovascular events and decrease tumor growth in animal models [47, 48]. The powerful antioxidant properties of resveratrol are thought to reduce the oxidation of low‐density lipoproteins (LDL) and to inhibit platelet aggregation that is involved in the formation of atherosclerotic lesions [49–51]. However, due to the complex structure, properties, and activity, many of the possible positive effects of polyphenols on animal and human health are not yet fully understood. The structure of resveratrol (3,5,4'‐trihydroxystil‐ bene) is shown in **Figure 3** as an example of a potent, well‐studied organic antioxidant.

**Figure 3.** Structure of resveratrol (3,5,4'‐trihydroxystilbene), a potent and commonly found polyphenol in red wines with known antioxidant properties.

While wine when used in moderation may also be a good nutritional source for dietary intake of some essential minerals such as iron (Fe) and zinc (Zn) [34, 52], and other metals such as copper (Cu) are considered both an essential and a potentially toxic element. Copper in excess may cause serious health consequences or result in the long‐term bioaccumulation and toxicity in the body [53]. Excessive iron intake above the tolerable upper intake level (UL) causes gastrointestinal distress while excess copper intake is likely to result in liver damage. It has been established that even moderate daily consumption of wine contributes significantly to the human nutritional requirements for essential trace elements such as Cr, Co, Fe, Cu, Mn, Mo, Ni, Se, and Zn [54]. The estimated safe intake levels of Cu, Fe, Mn, and Zn established by the US and the European Union (EU) are summarized in **Table 1** [55–57].

a The estimated safe and adequate dietary intake for Mn was reported as 2–5 mg/day for adults [55].

**Figure 2.** Shows wine storage containers at a Sonoma Valley winery at California (USA).

Many studies have shown that moderate consumption of red wine may improve health and longevity when it is done in combination with a balanced diet [45]. Much attention has been given in the research and medical communities to the properties and activity of polyphenols. Polyphenols have been shown to provide some possible health benefits by acting as antioxi‐ dants that helps prevent cell damage. Many of these health benefits of wine are associated with polyphenol antioxidants such as resveratrol (3,5,4'‐trihydroxystilbene) that is common in grapes and many other plants [46]. Resveratrol obtained from moderate wine consumption was found to decrease the risk of cardiovascular events and decrease tumor growth in animal models [47, 48]. The powerful antioxidant properties of resveratrol are thought to reduce the oxidation of low‐density lipoproteins (LDL) and to inhibit platelet aggregation that is involved in the formation of atherosclerotic lesions [49–51]. However, due to the complex structure, properties, and activity, many of the possible positive effects of polyphenols on animal and human health are not yet fully understood. The structure of resveratrol (3,5,4'‐trihydroxystil‐ bene) is shown in **Figure 3** as an example of a potent, well‐studied organic antioxidant.

**Figure 3.** Structure of resveratrol (3,5,4'‐trihydroxystilbene), a potent and commonly found polyphenol in red wines

with known antioxidant properties.

420 Grape and Wine Biotechnology

<sup>b</sup> Scientific Committee on Food of the EU has reported 1–10 mg/day as an acceptable range of Mn intake [56]. The EU does not have a RDA for Mn.

**Table 1.** Recommended dietary allowance (RDA) and tolerable upper intake level (UL) of selected trace minerals by the Food and Nutrition Board (Washington DC) [57]. Population reference intake (PRI) recommendations by the European Union. The Scientific Committee on Food (SCF) guidelines by the European Union [56].

The World Health Organization and various regulatory agencies have established recom‐ mended maximum limits for the concentration of metals in beverages and water utilized in human consumption. In addition, individual countries have rules restricting maximum metal content in wines, which have to be met in order to export wine to these consumer markets. For example, Cu and Zn content must be <5 mg/L by law in wines sold in Australia and Germany [58]. The Office International de la Vigne et du Vin (OIV) sets the limits for Cu at 1 mg/L and Zn at 5 mg/L [58, 59]. Italy has established the following legal levels in wine: 1 mg/mL Br, 1 mg/L Cu, 0.2 mg/L Pb, and 5 mg/L Zn [60]. Wine quality is also greatly affected by their metal content. For example, concentrations of Fe >5 mg/L generally induces haze formation and oxidative spoilage of the wine [2]. Concentration of Cu(II) above 1 µg/mL and Fe(III) ions above 7 µg/mL can give unpleasant, astringent tastes in addition to producing cloudiness in wines that have high pH or high concentration of tannic substances [35, 61]. Typically, the limits on metal concentration imposed for alcoholic beverages are higher than those established for drinking water as the expected intake of alcoholic beverages such as wine is lower [62]. Most of the trace metals are thought to originate from the atmospheric deposition of airborne particulate matter on grapes, composition of the soil, residues of metal containing agrochem‐ ical products, transfer of metals from the soil via the roots to the grapes and the wine, vinification methods, and contamination from contact with the metal apparatus during winemaking, production, and packaging processes [2, 54]. For example, some of the copper in finished wine may be due to copper sulfate used for spraying the wines to prevent mildew growth. Most of the iron in wine is determined by the composition of the soil where the grapes for the wine are produced [39]. Elements such as As, Cd, Pb, and Br are considered to be potentially toxic [63].

The ability for discriminating wines regionally within or between countries based on their trace element profile suggests that the elements mainly originate from their movement from the rock to soil and from the soil to grapes used to produce the commercially sold wine [64, 65]. Ability to determine the region of production for a wine greatly benefits the distributors, producers, as well as the consumers. The denomination origin controlled (DOC) system is used in some wine‐producing countries to track the origin, to guarantee the wine quality, and to help eliminate fraud [66, 67]. Chemical characterization of finished wine is one of the key requirements to obtaining DOC certification. Wine authenticity may be examined by quanti‐ fying suitable geographic tracers in finished wine. For example, the origin may be tracked by examining the specific content of organic constituents that vary regionally such as anthocya‐ nins, flavanols, flavonols, and organic acids [68, 69], by studying the elemental composition profile of inorganic species and by analysis of variation in selected stable isotopes [70]. Chemometric approaches have been applied to large analytical data sets that are generated during wine composition analysis and authenticity determination [12, 21, 66, 67, 70, 71].

The multielemental composition profile in soils appears to be strongly affected by the solubility of inorganic species in the soil. This connection between soil composition and that of finished wine has been confirmed in studies where soil and wine samples were systematically charac‐ terized. Strong consensus has been demonstrated between soil and wine samples' element composition while clear differences were shown between the studied regions [70]. In addition to Argentinian wines, statistically significant correlation between vineyard soils and finished Czech wines has been reported [35]. Ultimately, this multielement profile creates a sort of geochemical "fingerprint" that is unique to the vineyard or at least the grape growing region. These regional trace element patterns have been well established in chemical literature using instruments such as inductively coupled plasma‐mass spectrometry, ICP‐MS [72–84]. Char‐ acterization of the geographic origin of wine is even more accurate when natural isotopic abundance ratio of a key element, such as strontium (87Sr/86Sr) that is independent of the grape variety, is combined with trace element profile analysis [70].

As described above, concentrations of metals in finished wine are characteristic of the type of soil at the vineyard but are also influenced by the climate conditions during the growth of the grapes, the variety of grapes, and their maturity [38]. An example of a vineyard located in the northern United States will now be described. **Figure 4** shows the scenery at Chateau Grand Traverse in northern Michigan. The soil at the vineyard consists of loamy sand/glacial till and has a pH of 7.2–8.0. Chateau Grand Traverse vineyards lie directly on the 45° North Parallel at the elevation of 640–850 feet (or 195–259 m). The climate on the Old Mission Peninsula is also moderated by the surrounding deep waters of the Traverse Bay, helping to prevent frost during the growing season. Growing season at the Old Mission Peninsula is usually 145–160 days and the heat units have a 5‐year average = 2260° days (+50°F or +10°C). Five‐year average rain fall is 24.69" (or 62.7 cm) with 80.00" (or 203.2 cm) average snowfall.

that have high pH or high concentration of tannic substances [35, 61]. Typically, the limits on metal concentration imposed for alcoholic beverages are higher than those established for drinking water as the expected intake of alcoholic beverages such as wine is lower [62]. Most of the trace metals are thought to originate from the atmospheric deposition of airborne particulate matter on grapes, composition of the soil, residues of metal containing agrochem‐ ical products, transfer of metals from the soil via the roots to the grapes and the wine, vinification methods, and contamination from contact with the metal apparatus during winemaking, production, and packaging processes [2, 54]. For example, some of the copper in finished wine may be due to copper sulfate used for spraying the wines to prevent mildew growth. Most of the iron in wine is determined by the composition of the soil where the grapes for the wine are produced [39]. Elements such as As, Cd, Pb, and Br are considered to be

The ability for discriminating wines regionally within or between countries based on their trace element profile suggests that the elements mainly originate from their movement from the rock to soil and from the soil to grapes used to produce the commercially sold wine [64, 65]. Ability to determine the region of production for a wine greatly benefits the distributors, producers, as well as the consumers. The denomination origin controlled (DOC) system is used in some wine‐producing countries to track the origin, to guarantee the wine quality, and to help eliminate fraud [66, 67]. Chemical characterization of finished wine is one of the key requirements to obtaining DOC certification. Wine authenticity may be examined by quanti‐ fying suitable geographic tracers in finished wine. For example, the origin may be tracked by examining the specific content of organic constituents that vary regionally such as anthocya‐ nins, flavanols, flavonols, and organic acids [68, 69], by studying the elemental composition profile of inorganic species and by analysis of variation in selected stable isotopes [70]. Chemometric approaches have been applied to large analytical data sets that are generated during wine composition analysis and authenticity determination [12, 21, 66, 67, 70, 71].

The multielemental composition profile in soils appears to be strongly affected by the solubility of inorganic species in the soil. This connection between soil composition and that of finished wine has been confirmed in studies where soil and wine samples were systematically charac‐ terized. Strong consensus has been demonstrated between soil and wine samples' element composition while clear differences were shown between the studied regions [70]. In addition to Argentinian wines, statistically significant correlation between vineyard soils and finished Czech wines has been reported [35]. Ultimately, this multielement profile creates a sort of geochemical "fingerprint" that is unique to the vineyard or at least the grape growing region. These regional trace element patterns have been well established in chemical literature using instruments such as inductively coupled plasma‐mass spectrometry, ICP‐MS [72–84]. Char‐ acterization of the geographic origin of wine is even more accurate when natural isotopic abundance ratio of a key element, such as strontium (87Sr/86Sr) that is independent of the grape

As described above, concentrations of metals in finished wine are characteristic of the type of soil at the vineyard but are also influenced by the climate conditions during the growth of the grapes, the variety of grapes, and their maturity [38]. An example of a vineyard located in the

variety, is combined with trace element profile analysis [70].

potentially toxic [63].

422 Grape and Wine Biotechnology

**Figure 4.** Shows the scenery at Chateau Grand Traverse, one of the oldest wineries in northern Michigan. The vineyard is located on a narrow peninsula, north of Traverse City on the shores of Lake Michigan.

The typical composition and properties of wine as a sample matrix for various quantitative analyses will be described in the next section. The sample composition and properties impact important decisions such as what type of sample preparation is needed (if any), should the use of standard addition methods be considered, what is the best instrumental analysis method for characterization of the sample and the quantification of target analytes, etc.

## **2. Wine as a sample matrix and winemaking treatments**

The sample matrix is, by definition, everything in the sample of interest other than the analyte being quantified. Wine is a relatively complex aqueous sample matrix for quantitative chemical analysis due to containing about 12–15% ethanol (by percent volume) and mixture of hundreds of different organic compounds (such as polyhydroxyalcohols, polyphenols, organic acids, polysaccharides, peptides, etc.) in addition to various inorganic species (such as selenium, iron, zinc, nickel, copper, etc.). Major metals in wine, Ca, K, Na, and Mg are typically at levels in the range of 10–103 µg/mL [36]. The concentration of K is usually the highest [85]. Al, Cu, Fe, Mn, Rb, Sr, and Zn are called minor metals that are usually found in the range of 0.1–10 µg/mL [34]. Trace metals include Ba, Cd, Co, Cr, Li, Ni, Pb, V, and others in the range of 0.1–100 ng/mL or less [34].

It is also important to note whether or not allowed additives, adjuvants, or fining agents were used in the winemaking process as these do affect the trace‐element levels in wine. Ca concentration in wines is often affected by the addition of CaCO3 or CaSO4 during winemaking to deacidify must and wine [11, 43]. Clarifying products such as bentonites are allowed in winemaking. It has been reported that some winemaking treatments such as betonies and yeast hulls can affect the final trace element composition of wine [6]. Bentonite, for example, is a natural highly absorbent clay containing sheet silicates that is widely used in winemaking. Bentonite contains exchangeable cations such as Ca, Mg, and Na. Bentonite, which mostly consists of montmorillonite‐type phyllosilicate, acts as a settling aid to clarify wine and to remove amino acids, minerals, polyphenols, and protein, thus minimizing the risk of haze formation in wine. Proteins are depleted in wines due to their adsorption on the surface of the silica layers. Yeast hulls, living and nonliving biomass of *Saccharomyces cerevisiae*, have also been reported to significantly lower heavy metal content of wines by biosorption [86–88].

Nicolini et al. quantified changes of several minerals in red and white wines resulting from the addition of bentonites and yeast hulls using inductively coupled plasma‐optical emission spectrometry (ICP‐OES) and inductively coupled plasma‐mass spectrometry [6]. They studied the changes in trace element composition resulting from Italian wines treated with 10 different types of bentonite at 1 g/L (the highest doses routinely used in winemaking) and two different yeast hulls at doses of 180 and 360 mg/L. The authors found that bentonite fining significantly lowered concentrations of K, Cu, Rb, and Zn [6]. Cu depletion of −43% was observed with bentonite fining. Meanwhile, certain elements such as Ce, Gd, La, Nd, Pr, and Y increased by about one order of magnitude due to bentonite fining that was used. Also, concentrations of Be, Tl, and U increased by about 4–6 times after bentonite fining [6]. Therefore, depending on the specific element, its concentration may increase or decrease in the wine upon treatment with bentonites.

Nonliving yeast cell walls have also been reported to reduce concentrations of certain cations due to biosorption‐involving protein‐polysaccharide complexes [89]. Treatment of wine by yeast hulls induced significant decreases of Ce, Cu, Fe, La, Sb, U, V, and Y content in red and white wines as reported in [6]. Higher depletion of the elements was seen at the higher doses of 360 mg/L yeast hulls exposed to wines for 3 h.

## **3. Matrix effect and the use of standard addition methods**

Quantification of certain inorganic trace and ultratrace elements such as arsenic, cadmium, and lead in complex sample matrix such as wine is often challenging due to their very low concentrations. Including a preconcentration step prior to quantitative analysis is often necessary for these types of dilute analytes as the analyte concentrations may be below the detection limit of the instrumental analysis technique. The preconcentration step may also help to isolate the analyte of interest from the complex sample matrix, thereby improving selectivity of the detection step and stability of the analyte. Evaporation of some solvent including ethanol and water is usually sufficient to preconcentrate wine samples to a high enough level for quantification of trace elements such as iron, copper, zinc, and manganese by spectroscopy. However, electrolytic deposition is a very efficient method for isolation and preconcentration of many inorganic trace elements prior to quantification using an electrochemical detection method such as stripping voltammetry (SV). Selected examples of spectroscopic and electro‐ analytical detection methods will be described later in this chapter.

zinc, nickel, copper, etc.). Major metals in wine, Ca, K, Na, and Mg are typically at levels in the

Rb, Sr, and Zn are called minor metals that are usually found in the range of 0.1–10 µg/mL [34]. Trace metals include Ba, Cd, Co, Cr, Li, Ni, Pb, V, and others in the range of 0.1–100 ng/mL or

It is also important to note whether or not allowed additives, adjuvants, or fining agents were used in the winemaking process as these do affect the trace‐element levels in wine. Ca concentration in wines is often affected by the addition of CaCO3 or CaSO4 during winemaking to deacidify must and wine [11, 43]. Clarifying products such as bentonites are allowed in winemaking. It has been reported that some winemaking treatments such as betonies and yeast hulls can affect the final trace element composition of wine [6]. Bentonite, for example, is a natural highly absorbent clay containing sheet silicates that is widely used in winemaking. Bentonite contains exchangeable cations such as Ca, Mg, and Na. Bentonite, which mostly consists of montmorillonite‐type phyllosilicate, acts as a settling aid to clarify wine and to remove amino acids, minerals, polyphenols, and protein, thus minimizing the risk of haze formation in wine. Proteins are depleted in wines due to their adsorption on the surface of the silica layers. Yeast hulls, living and nonliving biomass of *Saccharomyces cerevisiae*, have also been reported to significantly lower heavy metal content of wines by biosorption [86–88].

Nicolini et al. quantified changes of several minerals in red and white wines resulting from the addition of bentonites and yeast hulls using inductively coupled plasma‐optical emission spectrometry (ICP‐OES) and inductively coupled plasma‐mass spectrometry [6]. They studied the changes in trace element composition resulting from Italian wines treated with 10 different types of bentonite at 1 g/L (the highest doses routinely used in winemaking) and two different yeast hulls at doses of 180 and 360 mg/L. The authors found that bentonite fining significantly lowered concentrations of K, Cu, Rb, and Zn [6]. Cu depletion of −43% was observed with bentonite fining. Meanwhile, certain elements such as Ce, Gd, La, Nd, Pr, and Y increased by about one order of magnitude due to bentonite fining that was used. Also, concentrations of Be, Tl, and U increased by about 4–6 times after bentonite fining [6]. Therefore, depending on the specific element, its concentration may increase or decrease in the wine upon treatment

Nonliving yeast cell walls have also been reported to reduce concentrations of certain cations due to biosorption‐involving protein‐polysaccharide complexes [89]. Treatment of wine by yeast hulls induced significant decreases of Ce, Cu, Fe, La, Sb, U, V, and Y content in red and white wines as reported in [6]. Higher depletion of the elements was seen at the higher doses

Quantification of certain inorganic trace and ultratrace elements such as arsenic, cadmium, and lead in complex sample matrix such as wine is often challenging due to their very low concentrations. Including a preconcentration step prior to quantitative analysis is often

µg/mL [36]. The concentration of K is usually the highest [85]. Al, Cu, Fe, Mn,

range of 10–103

424 Grape and Wine Biotechnology

with bentonites.

of 360 mg/L yeast hulls exposed to wines for 3 h.

**3. Matrix effect and the use of standard addition methods**

less [34].

Matrix effect is a detectable change (i.e., an increase or a decrease) in the analytical signal response caused by sample components other than the analyte. Matrix interferences that are sometimes observed in atomic spectroscopy (AS) are often due to differences between surface tension and viscosity of test solutions that are often used to simulate the samples and real samples undergoing quantification. Standard addition is especially useful when the exact sample composition is not known, the composition varies between samples, or the sample is complex and the sample matrix affects the observed analytical signal. Therefore, standard addition methods are well suited for analyzing complex aqueous samples such as wine in which the likelihood of matrix effects is substantial. In standard addition, all samples under‐ going analysis are in the same matrix. The standard being added in standard addition procedures is the same substance as the analyte of interest. For example, a certified reference standard purchased from a chemical company for aqueous Zn at ppm levels will be added into aliquots of wines undergoing quantitative analysis for zinc based on standard addition approach. The additions result in an increased signal response such as an increase in absorb‐ ance, which is directly proportional to the amount of zinc standard added. The standard addition methods will now be described in more detail.

By following the standard addition approach, sample constituents including the possible matrix effect that may suppress or enhance the signal response should be identical in each case because the standards are prepared in aliquots of the same sample. Standard addition methods are also advantageous when the amount of sample is limited. For example, in clinical labora‐ tory setting, limited sample size could be encountered when blood or urine samples are obtained from premature babies or dehydrated elderly patients. In this scenario, standard additions can be carried out by successive introductions of increments of the standard to a single measured volume of the unknown sample. Signal measurements are then made on the original sample and on the sample plus the standard after each addition. In most cases, the sample matrix is nearly identical after each standard addition, the only difference being the concentration of the analyte that has increased leading to detectable increases in the signal response. For direct metal determination in wines without any pretreatments, the usage of ethanol‐containing standards or using the standard addition method is recommended in order to minimize chemical and physical interferences [90–93].

As described, standard addition method can take several forms. For example, the wine sample may be spiked by adding one or more increments of a standard solution containing known concentration of the trace element analyte, such as Zn2+, to sample aliquots containing identical volumes of wine. The standard being added should ideally be relatively concentrated so that the addition of small volumes is sufficient and the sample matrix is not significantly altered. Each solution is then diluted to a constant total volume using deionized water before meas‐ urement of the signal. **Figure 5** helps one to visualize stepwise a typical standard addition experiment with constant total volume. This procedure is sometimes called a graphic proce‐ dure for standard addition. This graphic procedure is necessary when the analysis method such as atomic spectroscopy consumes some of the prepared solution during the quantification step. Flame atomic absorption, for example, may consume 2–5 mL of the prepared solution when multiple absorbances are obtained in order to determine a more representative mean absorbance value. The sample introduction and atomization steps in flame‐atomic absorption spectroscopy (flame‐AAS) are very wasteful as the aerosol containing the analyte reaching the flame contains only about 5% of the initial sample. The excess liquid drawn from the sample solution through a capillary tube flows out to drain and ultimately a waste container.

**Figure 5.** Standard addition procedure shows the addition of equal volume aliquots of the unknown sample (wine) to be quantified to five identical containers, the addition of progressively larger volumes of standard of known concentra‐ tion and composition (middle layer), and finally, filling the flasks to the same total volume using deionized water prior to the quantitative analysis. Ultimately, all samples undergoing quantitative analysis are in the same sample matrix.

The signal response is determined and recorded for each resulting solution to help quantify the analyte or analytes of interest. It is critical in quantitative analysis, regardless of the detection method chosen, that the signal response to the added analyte is linear. Ideally, each aliquot of added standard in a graphic standard addition procedure should increase the signal by a factor of 1.5–3. The quantification by this procedure usually involves the following steps:

**4.** The equation for the line (*y* = *mx* + *b*) will be used to find the concentration of the analyte in flask 1 by setting *y*‐value equal to zero, and solving for *x* (*x* = −*b*/*m*). The magnitude (absolute value) of the *x*‐axis intercept is the original concentration of the unknown (in units matching the *x*‐axis).

concentration of the trace element analyte, such as Zn2+, to sample aliquots containing identical volumes of wine. The standard being added should ideally be relatively concentrated so that the addition of small volumes is sufficient and the sample matrix is not significantly altered. Each solution is then diluted to a constant total volume using deionized water before meas‐ urement of the signal. **Figure 5** helps one to visualize stepwise a typical standard addition experiment with constant total volume. This procedure is sometimes called a graphic proce‐ dure for standard addition. This graphic procedure is necessary when the analysis method such as atomic spectroscopy consumes some of the prepared solution during the quantification step. Flame atomic absorption, for example, may consume 2–5 mL of the prepared solution when multiple absorbances are obtained in order to determine a more representative mean absorbance value. The sample introduction and atomization steps in flame‐atomic absorption spectroscopy (flame‐AAS) are very wasteful as the aerosol containing the analyte reaching the flame contains only about 5% of the initial sample. The excess liquid drawn from the sample

426 Grape and Wine Biotechnology

solution through a capillary tube flows out to drain and ultimately a waste container.

**Figure 5.** Standard addition procedure shows the addition of equal volume aliquots of the unknown sample (wine) to be quantified to five identical containers, the addition of progressively larger volumes of standard of known concentra‐ tion and composition (middle layer), and finally, filling the flasks to the same total volume using deionized water prior to the quantitative analysis. Ultimately, all samples undergoing quantitative analysis are in the same sample matrix.

The signal response is determined and recorded for each resulting solution to help quantify the analyte or analytes of interest. It is critical in quantitative analysis, regardless of the detection method chosen, that the signal response to the added analyte is linear. Ideally, each aliquot of added standard in a graphic standard addition procedure should increase the signal by a factor of 1.5–3. The quantification by this procedure usually involves the following steps:

**1.** Calculate the concentration of added standard for each flask as measured after dilution.

**2.** Plot analytical signal versus concentration of added standard on a *xy* graph.

**3.** Add a linear regression line and the equation for the line.

An example of a standard addition plot obtained using graphical standard addition treatment is shown in **Figure 6** for the quantification of Zn in a white wine using flame‐atomic absorption spectroscopy. The increases is mean absorbance were proportional to the aliquots of Zn standard added, allowing the reliable determination of Zn in the original wine using linear regression. Each standard addition increased the absorbance signal by a factor of 1.8.

**Figure 6.** Graphical treatment of standard addition data in quantification of zinc. Concentration of Zn in a white wine from Golan Heights in Israel was determined using 0, 2.5, 5, 7.5, and 10 mL additions 10 ppm zinc standard (from PerkinElmer) to 50 mL volumetric flasks already containing the white wine samples. The first flask with mean absorb‐ ance of 0.017 only contained the wine sample and deionized water. Based on the standard addition experiment, this Vintage 2012 Galilee wine from Golan Heights (Israel) contained 1.16 mg/L or ppm of zinc.

## **4. Common analytical methods for trace element analysis**

Various analytical techniques have been used for metal analysis in water, foods, and beverages. The electroanalytical methods include stripping voltammetry and stripping polarography [4, 10, 94–97]. Other analysis methods include X‐ray fluorescence [98, 99] and near‐IR spectro‐ scopy [14, 29, 30]. Ion chromatography and capillary zone electrophoresis can also be applied to the analysis of metal cations in wines [5, 100, 101].

Atomic absorption and emission spectroscopy methods such as inductively coupled plasma‐ optical emission spectroscopy, inductively coupled plasma‐mass spectroscopy, graphite furnace‐atomic absorption spectroscopy (GF‐AAS), and flame‐atomic absorption spectroscopy are the most commonly used techniques in the determination of metal concentrations due to their high sensitivity and rapid results [7, 14, 30, 58, 102–107]. **Figure 7** shows an F‐AAS by PerkinElmer, which is very affordable and reliable for quantification of many trace elements using standard addition methods.

**Figure 7.** Flame‐atomic absorption spectrometer (F‐AAS), type AAnalyst 200 by PerkinElmer is shown.

Despite wine being a relatively complex aqueous sample matrix for chemical analysis due to containing ethanol and hundreds of different larger organic compounds (such as polyhydrox‐ yalcohols, organic acids, polyphenols, polysaccharides, peptides, etc.) in addition to various inorganic species, very few of these compounds actually interfere with atomic spectroscopy due to the high temperatures involved in the sample atomization steps [58]. Also, AAS, the official method of analysis for determination of elements such as Fe and Zn by the European Union Regulation [108], and recommended by the OIV and the American Society of Enologists, is affordable, selective, highly sensitive, often capable of direct measurements, and relatively easy to operate. Fe, Cu, Zn, and Mn, for example, are relatively simple to quantify using F‐ AAS. These four elements have significant impact on wine quality by contributing to haze formation and leading to undesirable changes in wine taste and aroma [23]. These elements may also impact human health via contributing to total dietary intake (in addition to other sources such as multivitamin supplements, food, and other beverages) and ultimately possible bioaccumulation and toxicity in the event of excessive intake [23]. The most common fuel/ oxidant combination for quantification of trace elements such as Cu, Fe, Mn, and Zn by F‐AAS in aqueous sample matrix is acetylene/air.

Quantitative analysis by atomic spectroscopy consists of an atomization step where the solid sample components are broken into atoms at high temperature (2000–8000 K) flame, furnace, or plasma after liquid portion of the sample has already evaporated. The inorganic analyte species is quantified based on absorption or emission of monochromatic ultraviolet or visible electromagnetic radiation of characteristic wavelength by the gas‐phase analyte. The analytes are typically at parts per million (µg/g) to parts per trillion (pg/g) levels for AS.

In ICP‐MS, the analyte trace elements are ionized by collisions with excited Ar+ or energetic electrons in plasma. ICP‐MS has detection limits that are 5–7 orders of magnitude lower than atomic spectroscopy based on F‐AAS. In addition, the linear range for ICP‐MS is 108 versus 102 for F‐AAS. The sample throughput for ICP‐MS is greater as most elements may be analyzed in one run and the sample volumes required for analysis are also less. ICP‐MS outperforms even other multielement techniques in element analysis due to its extremely low detection limits. However, the purchase cost and maintenance cost of an ICP‐MS system is substantially higher and the instrument requires a skilled operator. ICP‐MS has been utilized by many especially as an instrumental tool for characterizing wines according to their geographical origin [72–80, 109].

Stripping voltammetry is a very sensitive, low cost, and a popular electroanalytical chemistry method due to incorporating an electrolytic preconcentration step prior to the analysis of trace concentrations of electroactive species in solution. Multiple elements such as Cd and Cu may also sometimes be quantified from the same sample using SV during the same experiment. Extremely low detection limits for metal ions at sub‐ppb (or 10‐10–10−11 M) concentrations have been reported for SV. There are three parts in a stripping experiment: deposition, quiet time, and stripping. During the deposition step, the analyte of interest is accumulated onto the working electrode that may have been chemically modified previously. Deposition of the target species may be increased by forced convection such as stirring the solution, rotation of the working electrode, or creating flow conditions during the deposition step. The required deposition time (of typically 1–10 min) depends on the concentration of the analyte species in the sample. In anodic stripping voltammetry (ASV), the working electrode is held at a potential at least 0.4 V cathodic to the standard potential of the least easily reduced ion being quantified. The stirring is stopped and the system is allowed to reach equilibrium during a quiet time of about 10–15 s. Finally, the accumulated material is reduced or oxidized back into the solution during the stripping step. Change in electrochemical response (i.e., signal) during the stripping step is proportional to the concentration of the analyte that was in or on the working electrode. Anodic stripping voltammetry involves the reduction of a metal ion analyte from the sample during the preconcentration step. SV method for trace metal analysis in dry wines will be described briefly below as an example of sample preparation, solution conditions, and time required for electroanaytical quantification.

**Figure 7.** Flame‐atomic absorption spectrometer (F‐AAS), type AAnalyst 200 by PerkinElmer is shown.

in aqueous sample matrix is acetylene/air.

428 Grape and Wine Biotechnology

102

Despite wine being a relatively complex aqueous sample matrix for chemical analysis due to containing ethanol and hundreds of different larger organic compounds (such as polyhydrox‐ yalcohols, organic acids, polyphenols, polysaccharides, peptides, etc.) in addition to various inorganic species, very few of these compounds actually interfere with atomic spectroscopy due to the high temperatures involved in the sample atomization steps [58]. Also, AAS, the official method of analysis for determination of elements such as Fe and Zn by the European Union Regulation [108], and recommended by the OIV and the American Society of Enologists, is affordable, selective, highly sensitive, often capable of direct measurements, and relatively easy to operate. Fe, Cu, Zn, and Mn, for example, are relatively simple to quantify using F‐ AAS. These four elements have significant impact on wine quality by contributing to haze formation and leading to undesirable changes in wine taste and aroma [23]. These elements may also impact human health via contributing to total dietary intake (in addition to other sources such as multivitamin supplements, food, and other beverages) and ultimately possible bioaccumulation and toxicity in the event of excessive intake [23]. The most common fuel/ oxidant combination for quantification of trace elements such as Cu, Fe, Mn, and Zn by F‐AAS

Quantitative analysis by atomic spectroscopy consists of an atomization step where the solid sample components are broken into atoms at high temperature (2000–8000 K) flame, furnace, or plasma after liquid portion of the sample has already evaporated. The inorganic analyte species is quantified based on absorption or emission of monochromatic ultraviolet or visible electromagnetic radiation of characteristic wavelength by the gas‐phase analyte. The analytes

electrons in plasma. ICP‐MS has detection limits that are 5–7 orders of magnitude lower than atomic spectroscopy based on F‐AAS. In addition, the linear range for ICP‐MS is 108 versus

for F‐AAS. The sample throughput for ICP‐MS is greater as most elements may be analyzed

or energetic

are typically at parts per million (µg/g) to parts per trillion (pg/g) levels for AS.

In ICP‐MS, the analyte trace elements are ionized by collisions with excited Ar+

Brainina et al. [96] developed and reported a method for quantification of cadmium, copper, lead, and zinc in dry wines by stripping voltammetry with a thick‐film modified graphite‐ containing electrode (TFMGE). The reported method has an advantage of not requiring acid digestion of the wine samples or any other sample preparation approaches for the destruction of organic substances. Supporting electrolyte of 0.5 M HCl was for Cu, Pb, and Cd, and 0.1 M acetate buffer (pH 5.5) and 0.35 M NaCl for Zn. The deposition potentials were −0.8 V for Cu, −1.2 V for Pb and Cd, and −1.4 V for Zn. The wine samples were diluted with the supporting electrolyte prior to deposition step. The deposition times were 30–60 s for Cu, 120 s for Pb and Cd, and 10–30 s for Zn. Results obtained by the TFMGE analysis were compared with quantification of the selected elements in the same samples done using ICP‐MS. Of note, the fast and simple analysis method with TFMGE actually provided better reproducibility than methods involving wine decomposition prior to analysis. More recently, Burmakina et al. [110] developed a procedure for determining manganese(II) in wines by SV on a graphite electrode. The detection limit of manganese(II) was 50 µg/L with a linear range 0.1–3 mg/L [110].

## **5. Conclusions**

Wine is a common beverage that has immense economic impact on certain regions of the world. The theory, advantages and disadvantages, experimental considerations and set up of various sensitive atomic spectroscopy, and electroanalytical quantification methods were described in this chapter. Examples of quantification methods suitable for essential trace element as well as the determination of toxic elements generally found at considerably lower concentrations in wine were provided throughout the chapter. Many inorganic elements have been found to affect the properties of wine, how well it stores, as well as the sensory perception of the wine consumer. The unique trace element profile may also be used as a fingerprint to identify the authenticity of the wine if a fraud is suspected or provide information about the growing region of the grapes and the location of the vineyard. The complexity of wine as a sample matrix for quantification of inorganic elements and the use of standard addition methods to improve the reliability of the selected detection methods were described. Examples of common methods, the common origins of the inorganic elements in the finished wine, and reported levels of trace elements in wines were heavily referenced throughout the chapter.

## **Acknowledgements**

The author thanks Benedictine University for financial support of her work.

## **Author details**

Niina J. Ronkainen

Address all correspondence to<EMAIL_ADDRESS>
Department of Chemistry and Biochemistry, Benedictine University, Lisle, Illinois, USA

## **References**

[4] Arcos, M.T., Ancin, M.C., Echeverria, J.C., Gonzalez, A., & Garrido, J.J. (1993). Study of lability of heavy metals in wines with different degrees of aging through differential pulse anodic stripping voltammetry. Journal of Agricultural and Food Chemistry, 41(12), 2333–2339.

**5. Conclusions**

430 Grape and Wine Biotechnology

**Acknowledgements**

**Author details**

Niina J. Ronkainen

**References**

Wine is a common beverage that has immense economic impact on certain regions of the world. The theory, advantages and disadvantages, experimental considerations and set up of various sensitive atomic spectroscopy, and electroanalytical quantification methods were described in this chapter. Examples of quantification methods suitable for essential trace element as well as the determination of toxic elements generally found at considerably lower concentrations in wine were provided throughout the chapter. Many inorganic elements have been found to affect the properties of wine, how well it stores, as well as the sensory perception of the wine consumer. The unique trace element profile may also be used as a fingerprint to identify the authenticity of the wine if a fraud is suspected or provide information about the growing region of the grapes and the location of the vineyard. The complexity of wine as a sample matrix for quantification of inorganic elements and the use of standard addition methods to improve the reliability of the selected detection methods were described. Examples of common methods, the common origins of the inorganic elements in the finished wine, and reported levels of trace

elements in wines were heavily referenced throughout the chapter.

Address all correspondence to<EMAIL_ADDRESS>
The author thanks Benedictine University for financial support of her work.

Department of Chemistry and Biochemistry, Benedictine University, Lisle, Illinois, USA

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## **D-O-C Stable Isotopes, 14C Radiocarbon and Radiogenic Isotope Techniques Applied in Wine Products for Geographical Origin and Authentication D-O-C Stable Isotopes, 14C Radiocarbon and Radiogenic Isotope Techniques Applied in Wine Products for Geographical Origin and Authentication**

Paraskevi Chantzi, Anastasia-Elektra Poutouki and Elissavet Dotsika Elissavet Dotsika

Paraskevi Chantzi, Anastasia-Elektra Poutouki and

Additional information is available at the end of the chapter Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/64933

#### **Abstract**

Oxygen, deuterium and carbon isotopes were measured in wine products in two Greek vineyards, Amydaio (north) and Nemea (south). The enriched isotope values in Nemea reflects the higher evapo-transpiration rate and the more arid condition of Southern Greece. White wines were slightly more depleted than red wines of the same year and the same growing region probably due to different harvest periods. Further was pointed out the variety of isotope values with respect to vintage year indicating that the vintage year contributes to the development of isotopes in wine water. In both vineyards the trend lines intersect the oxygen and deuterium isotopes of irrigation water highlighting the source water and the initial isotopic composition of grape berries. δ13C values of ethanol confirmed the origin of C3 plants and the authentication of wine products without detecting adulteration with industrial alcohol. The results of 14C measurements in ethanol extracted from Greek wines follow the known pattern of 14C variations in atmospheric CO2. The homogeneity of 87Sr/86Sr and 144Nd/143Nd isotope values confirms that the territorial and geological signal is transferred through the vineyards in the final product, wine, certifying the exclusively provenance of the wine areas Amydaio and Nemea.

**Keywords:** oxygen isotope, carbon isotopes, radiocarbon, Greek wines, grape berries

© 2016 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2016 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

## **1. Introduction**

Wine is a product that has suffered serious damage from adulteration and mislabeling practices. The authenticity of a wine label, which confirms the alignment of the product with the relevant laws of alcohol production, is of great importance in many levels. First of all, the protection of the consumers against the globalization of food market, where the adulteration episodes are strongly existent, should be the dominant objective. On the other hand, the authentication in conjunction with the geographic origin maintains and defends traditional production techniques that influence the particular output features of wine and which are lost through the competition of mass production. In general, modern dietary habits governed by standardized products, as required by the food industry, resulting the lack of the link between products and their geographical origin influenced by the particular environment setting. There is also a great interest by wine producers and traders so that they can guarantee high quality wines offer a reliable label. However, fraud incentives persist and with upgraded methods. Therefore, there is a necessity for the upgrading of the authentication methods of the wine label. Toward this direction the use of stable isotopes 2 H, 13C, and 18O in diet samples started in the early 1970s and later enriched by the use of radiocarbon 14C and radiogenic isotopes Sr and Nd. The recognition of isotopes as food authenticity indicators was a point of reference in the food authentication approach. In fact, stable isotope ratio analysis of wine isotope ratio mass spectrometry is official methods in the European Union for the detection of chaptalization, addition of water, sweetening with sugar, and authentication of geographic origin and year of harvest.

## **2. Factors that influence carbon and oxygen isotopic values**

The nature ratio of stable isotopes of plant tissues is strongly influenced by raw materials of photosynthesis, water and carbon dioxide, as well as the fractionation process during the photosynthetic pathways. Regarding carbon dioxide, it should be noted that the atmospheric reservoir considered as constant where the δ13C composition barley varies from −7% [1]. Since then the atmospheric pool supplies fixed carbon stable isotope values, water ("source water") and fractionation processes are responsible for the differentiation in δ13C values in plants. According to the photosynthetic pathway that each plant follows three types are distinguished with characteristic range of δ13C values: C4 (Hatch-Slack, C4-dicarboxylic acid path-way) plants generally range from −9 to −19% [2, 3] while for C3 (Calvin cycle) plants typically range from −20 (open areas exposed to water stress) to −35% (closed canopy) [2, 3]. The third photosynthetic pathway called crassulacean acid metabolism (CAM) presents δ13C values between the end-members of C3 and C4 types. Generally, the CAM cycle corresponds to plants that store quantities of water in their tissues to use under demand conditions; therefore, the biggest distinction is limited to C3 and C4 photosynthetic pathways. The first represents the cycle that the first synthesized sugars are three carbon sugars while in C4 cycle first sugars fixed are four carbon sugars. Consequently, alcohol derived from grapes should reflect δ13C values of the corresponding photosynthetic pathway that the grapes belong, which is the C3 type. The addition of different origin alcohol from C4 type (synthetic or beet) could be traced as the δ13C values are more enriched than those of the C3 type. This intense discrimination is due to the fact that C3 cycle is "longer" enabling a greater isotopic segregation against the "short" C4 cycle which is closer related to the end of the products [4]. Moreover, since the plant interacts with atmospheric CO2 any observed fluctuations should be justified by the particular environmental characteristics of the region where the vineyards grow. In more detail, the intake water source for vineyards owns a crucial role in plant growth by determining the sugar content in grapes and therefore the alcohol formation. The "water deficit" causes stress to plant and affects its function process which in an attempt to maintain the needed humidity preventing the loss though evaporation closes its leave stomata [2, 5, 6]. As a direct consequence to this is the limited interaction with environmental CO2, which is the main factor in photosynthesis. The disruption of the system "leaf transpiration-carbon fixation per unit leaf area" results in a disruption in sugar formation in grape berries. Stress episodes lead to higher sugar level in fruits in contrast to those where water availability is stable and representative to the actual needs of the plant [7]. Consequently, 13C/12C isotope analysis was performed in wine samples. Ethanol samples which were extracted by number of wine samples were subjected in 18O/16O, 2 H/1 H, 13C/12C, and radiocarbon (14C) isotope analysis. As oxygen-18 and deuterium isotopes are affected by temperature, precipitation, humidity, altitude, latitude, and the distance from the sea [8, 9], the isotopic signature of vineyard's "source water" should be related to corresponding geographical region. The emphasis is given to oxygen isotopic values (18O) for the characterization of the plant organic matter as they present a more normal behavior. However, the interpretation of their δ18O values is more complicated than that of carbon as it does not present such a linear formation as CO2 due to the several oxygen sources (precipitation, atmospheric CO2, soil water) and the complexity of metabolic processes. The isotopic characteristics of feeding water, environmental conditions, photosynthetic fractionation, and the evapotranspiration effect on plant sap and leave stomata are considered the most dominant factors that gather the fixation of δ18O values. However, we should further consider the cellular CO2-H2O equilibrium as well as possible contribution of CO2 and/or H2O to the organic oxygen in the cell [4, 10, 11].

**1. Introduction**

442 Grape and Wine Biotechnology

year of harvest.

Wine is a product that has suffered serious damage from adulteration and mislabeling practices. The authenticity of a wine label, which confirms the alignment of the product with the relevant laws of alcohol production, is of great importance in many levels. First of all, the protection of the consumers against the globalization of food market, where the adulteration episodes are strongly existent, should be the dominant objective. On the other hand, the authentication in conjunction with the geographic origin maintains and defends traditional production techniques that influence the particular output features of wine and which are lost through the competition of mass production. In general, modern dietary habits governed by standardized products, as required by the food industry, resulting the lack of the link between products and their geographical origin influenced by the particular environment setting. There is also a great interest by wine producers and traders so that they can guarantee high quality wines offer a reliable label. However, fraud incentives persist and with upgraded methods. Therefore, there is a necessity for the upgrading of the authentication methods of the wine

in the early 1970s and later enriched by the use of radiocarbon 14C and radiogenic isotopes Sr and Nd. The recognition of isotopes as food authenticity indicators was a point of reference in the food authentication approach. In fact, stable isotope ratio analysis of wine isotope ratio mass spectrometry is official methods in the European Union for the detection of chaptalization, addition of water, sweetening with sugar, and authentication of geographic origin and

The nature ratio of stable isotopes of plant tissues is strongly influenced by raw materials of photosynthesis, water and carbon dioxide, as well as the fractionation process during the photosynthetic pathways. Regarding carbon dioxide, it should be noted that the atmospheric reservoir considered as constant where the δ13C composition barley varies from −7% [1]. Since then the atmospheric pool supplies fixed carbon stable isotope values, water ("source water") and fractionation processes are responsible for the differentiation in δ13C values in plants. According to the photosynthetic pathway that each plant follows three types are distinguished with characteristic range of δ13C values: C4 (Hatch-Slack, C4-dicarboxylic acid path-way) plants generally range from −9 to −19% [2, 3] while for C3 (Calvin cycle) plants typically range from −20 (open areas exposed to water stress) to −35% (closed canopy) [2, 3]. The third photosynthetic pathway called crassulacean acid metabolism (CAM) presents δ13C values between the end-members of C3 and C4 types. Generally, the CAM cycle corresponds to plants that store quantities of water in their tissues to use under demand conditions; therefore, the biggest distinction is limited to C3 and C4 photosynthetic pathways. The first represents the cycle that the first synthesized sugars are three carbon sugars while in C4 cycle first sugars fixed are four carbon sugars. Consequently, alcohol derived from grapes should reflect δ13C values of the corresponding photosynthetic pathway that the grapes belong, which is the C3

**2. Factors that influence carbon and oxygen isotopic values**

H, 13C, and 18O in diet samples started

label. Toward this direction the use of stable isotopes 2

## **3. Environmental and hydrogeological setting of study areas vineyards**

Tradition of Greece in winemaking is pronounced with the unequivocal uniqueness of Greek wines resulted by the combination of the Greek terroir and the high quality of local wine varieties. Therefore, the authenticity control and origin assignment of Greek wine products have obtained remarkable importance. In the context of this project, two characteristic wineproducing areas of continental Greece: Amydaio in west Macedonia, north Greece, and Nemea in Corinthia, south Greece are selected. The first constitutes a significant Greek vineyard between Vermion and Borra mountains where it is dominated by the cultivation of Xinomauro variety while the second concerns vineyards with the exclusive cultivation of Agiorgitiko variety. So an attempt was made to trace the correspondence of "VQPRD (*Vins de Qualite Produits dans une Region Determinee*) Amydaio and Nemea" labels with their geographical origin. Many techniques have been performed on geographical origin traceability; however, isotopes have proven to be a valuable tool with robust and reliable results [12–16]. The study model was designed according to the following plan: "source water (precipitation)-plant development (photosynthesis)-wine (final product)" as all the environmental effects on the vine development, reflect their isotope signature in the final product of wine.

The predominately climate type in Greece is the Mediterranean with warm to hot, dry summers and mild to cool, wet winters; however, the unique topography of Greece constitutes to a large variation of microclimates which enhance the diversity and variety of traditional products. Amydaio located in the northwestern part of Greece, 33 km from Florina town (West Macedonia) at an altitude of 650 m above sea level without any influence of the Aegean Sea. Its climate is characterized as purely continental but it becomes milder because of the presence of Vegoritida Lake. Specifically, the climate presents as temperate continental with heavy winders accompanied by snow and low temperatures. The plateaus exhibit a southeastern direction without interfering mountains along them and the prevailing northerly winds to keep the temperature low. Summers are mild with higher temperatures presented at lower altitudes. The minimum average temperature in the winter months and the average maximum temperature during the summer are about 2.5°C and 29.6°C, respectively. Although the region of Western Macedonia belongs in the eastern mainland characterized by less rainfall compared to the corresponding west, the climate displays wet character (up to 75% humidity in the winter months of December–February) due to the combination of mountain landscape and lakes (Vegoritida, Zazari, Petron, and Cheimaditida lakes). The main volume of precipitation is observed in the autumn months. The wider area of Amydaio belongs to a tectonic basin with NE-SW trend formed during the Tertiary, as a consequence of strong tensile stress in the region. The Amydaion basin is divided into two elongated sub(tectonic wells), characterized by different geological-stratigraphic evolution and surface morphology [17]. The basin was gradually sinking during the Medium-Upper Miocene, which led to the stratification of the basin and the creation of lignite deposit. Tectonic events that occurred during the Pleistocene and Holocene caused further subduction and sediment deposition, which reached 350 m. The repeatability deposition in lake-marshy environment allowed the creation of lignite in the region. The vineyards of VQPRD Amydaion developed in alluvial area of the basin, dominated by lacustrine sediments (clay, sand, and gravel).

Nemea is a town of Corinth Prefecture, located 42 km southwest of Corinth, near Mount Prophet Elias, at an altitude of 320 m and is presented as the greater wine-growing region of Greece which produced the renowned wines VQPRD (Vins de Qualite Produits dans une Region Determinee) Nemea. The climate is characterized as warm and temperate with little rainfall throughout the year. The rainfall distribution is uneven with respect to seasons (85% of precipitation falls on a wet season from October to April). In the western part (Feneou areas and Stymfalias) where the higher altitudes are observed, the precipitation episodes are often in contrast to the Eastern part which is characterized by lower altitudes. Temperature and sunlight follow the same trend related to altitude presenting the lower values in January and the highest in July. The inverse distribution is observed for moisture regime where the winder presents a peak (December) while July is the driest month. The prevailing winds that occur are those of NE and east direction without any strong episodes. The dominant geological formations that are encountered include both pre- and postalpine formations of terrestrial, lacustrine, and marine origin. Three large plane sections constitute the relief portrait of the wider study area: the coastal area between Corinth and Kiato known as the Vochas plane, the plane between Agios Basilios and Spathovouni villages, and the plane area around the historical city of Nemea. In contrast, at the SW and SE edge steeper scene is observed with carbonate formations of the pre-alpine basement to set up a mountainous terrain with steep slopes, deep ravines, and sharp mountain peaks. Intermediate areas are developed in a hilly or semimountainous setting with moderate slopes and rounded mountain peaks. The drainage network is developed due to the creation and activity of WNW-ESE faults and vertical to these transform faults (**Figure 1**) [18, 19].

**Figure 1.** Study area of Amydaio, north Greece, and Nemea, south Greece, Greek vineyards (Google earth modified picture).

## **4. Material and methods**

origin. Many techniques have been performed on geographical origin traceability; however, isotopes have proven to be a valuable tool with robust and reliable results [12–16]. The study model was designed according to the following plan: "source water (precipitation)-plant development (photosynthesis)-wine (final product)" as all the environmental effects on the

The predominately climate type in Greece is the Mediterranean with warm to hot, dry summers and mild to cool, wet winters; however, the unique topography of Greece constitutes to a large variation of microclimates which enhance the diversity and variety of traditional products. Amydaio located in the northwestern part of Greece, 33 km from Florina town (West Macedonia) at an altitude of 650 m above sea level without any influence of the Aegean Sea. Its climate is characterized as purely continental but it becomes milder because of the presence of Vegoritida Lake. Specifically, the climate presents as temperate continental with heavy winders accompanied by snow and low temperatures. The plateaus exhibit a southeastern direction without interfering mountains along them and the prevailing northerly winds to keep the temperature low. Summers are mild with higher temperatures presented at lower altitudes. The minimum average temperature in the winter months and the average maximum temperature during the summer are about 2.5°C and 29.6°C, respectively. Although the region of Western Macedonia belongs in the eastern mainland characterized by less rainfall compared to the corresponding west, the climate displays wet character (up to 75% humidity in the winter months of December–February) due to the combination of mountain landscape and lakes (Vegoritida, Zazari, Petron, and Cheimaditida lakes). The main volume of precipitation is observed in the autumn months. The wider area of Amydaio belongs to a tectonic basin with NE-SW trend formed during the Tertiary, as a consequence of strong tensile stress in the region. The Amydaion basin is divided into two elongated sub(tectonic wells), characterized by different geological-stratigraphic evolution and surface morphology [17]. The basin was gradually sinking during the Medium-Upper Miocene, which led to the stratification of the basin and the creation of lignite deposit. Tectonic events that occurred during the Pleistocene and Holocene caused further subduction and sediment deposition, which reached 350 m. The repeatability deposition in lake-marshy environment allowed the creation of lignite in the region. The vineyards of VQPRD Amydaion developed in alluvial area of the basin, dominated by lacustrine sediments (clay, sand,

Nemea is a town of Corinth Prefecture, located 42 km southwest of Corinth, near Mount Prophet Elias, at an altitude of 320 m and is presented as the greater wine-growing region of Greece which produced the renowned wines VQPRD (Vins de Qualite Produits dans une Region Determinee) Nemea. The climate is characterized as warm and temperate with little rainfall throughout the year. The rainfall distribution is uneven with respect to seasons (85% of precipitation falls on a wet season from October to April). In the western part (Feneou areas and Stymfalias) where the higher altitudes are observed, the precipitation episodes are often in contrast to the Eastern part which is characterized by lower altitudes. Temperature and sunlight follow the same trend related to altitude presenting the lower values in January and the highest in July. The inverse distribution is observed for moisture regime where the winder

vine development, reflect their isotope signature in the final product of wine.

and gravel).

444 Grape and Wine Biotechnology

In order to identify the isotopic link between the environmental factors of vineyards and wine products precipitation, ground water, wine water, as well as grape must samples during the wine-making process were collected for δ18O, δD δ13C, and 14C isotopic analysis. Sr and Nd radiogenic analysis was also performed in selected wine samples. Rain samples were collected (11 samples for Nemea and 12 samples for Amydaio) by using 125 mL glass bottles. Ground water samples (10 samples for Nemea and 17 samples for Amydaio) were taken from irrigation water boreholes related to vineyards by using 125 mL glass bottles. In total 70 (54 samples for Amydaio and 16 samples for Nemea) different bottled vintage wines were chosen and prepared for stable isotopic analysis by extraction (approximately 40 mL from each bottle) through the cork using a 10 mL Hamilton Gastight Syringe with Gauge 26 Point Style 5 (side hole) needles. Finally, unfermented grape must samples (16 samples for Nemea and 33 samples for Amydaio) were collected immediately after harvest. Then they were centrifuged for approximately 1 hour to extract grape solids and larger yeasts, syringe-filtered with a 0.45 mm and 0.22 mm Cameo IV filter and stored into 20 mL glass bottles (without the addition of sulfur dioxide). Moreover, 10 samples of wine ethanol were collected by using a Cadiot column distillation system to quantitatively separate the ethanol, according to the procedure described in OIV MA-AS-311-05.

All isotopic analyses were performed in Laboratory of Stable Isotope and Radiocarbon of Nanoscience & Nanotechnology, Institute in N.C.S.R. Demokritos (Athens, Greece). Stable isotope analysis of 18O/16O, D/H, and 13C/12C ratio in Greek wines, wine water, meteoric, and ground waters were carried out on a continuous flow Finnigan DELTA V plus (Thermo Electron Corporation, Bremen, Germany) stable isotope mass spectrometer [20, 21].

13C, 18O, and <sup>2</sup> H isotope analysis in ethanol with Thermo Scientific DELTA V Isotope Ratio Mass Spectrometers was performed according to Ref. [22]. Specifically, for δ13C determination 1 μL of purified ethanol was injected into a small tin container for liquids and closed securely to prevent evaporation. The Flash Elemental Analyzer (FlashEA) with a single reactor system combining combustion and reduction in one reaction was used. The Thermo Scientific ConFlo interface diluted the CO2 sample peak with 1 bar helium resulting in a split of about 1:12. For δ2 H and δ18O determination 0.1 μL of pure ethanol was injected with a 0.5 μL syringe into a glassy carbon reactor in a high temperature carbon reduction system of a FlashEA. The autosampler was used and samples were stored in 2 mL vials with standard caps and septa.

The results are expressed in standard delta notation (*δ*) as per mil (‰) deviation from the standard V-SMOW as: *δ* = ((*R*sample − *R*standard)/*R*standard) × 1000, where *R*sample and *R*standard = 2 H/1 H or 18O/16O or 13C/12C ratios of sample and standard, respectively.

Measurement precision, based on the repeated analysis of internal standard waters, was 1.5, 0.5, and 0.2% for δ2 H, δ18O, and δ13C, respectively.

All measurements were carried out according to laboratory standards that were periodically calibrated based on the international standards recommended by the IAEA.

14C activity was determined by using liquid scintillation counting (LSC) on a Packard Tri-Carb TR/SL. Radiogenic isotope of Sr and Nd were measured with thermal ionization mass spectrometry (TIMS, Nd) and inductively coupled plasma mass spectrometry (ICP-MS, Sr).

## **5. Results and discussion**

radiogenic analysis was also performed in selected wine samples. Rain samples were collected (11 samples for Nemea and 12 samples for Amydaio) by using 125 mL glass bottles. Ground water samples (10 samples for Nemea and 17 samples for Amydaio) were taken from irrigation water boreholes related to vineyards by using 125 mL glass bottles. In total 70 (54 samples for Amydaio and 16 samples for Nemea) different bottled vintage wines were chosen and prepared for stable isotopic analysis by extraction (approximately 40 mL from each bottle) through the cork using a 10 mL Hamilton Gastight Syringe with Gauge 26 Point Style 5 (side hole) needles. Finally, unfermented grape must samples (16 samples for Nemea and 33 samples for Amydaio) were collected immediately after harvest. Then they were centrifuged for approximately 1 hour to extract grape solids and larger yeasts, syringe-filtered with a 0.45 mm and 0.22 mm Cameo IV filter and stored into 20 mL glass bottles (without the addition of sulfur dioxide). Moreover, 10 samples of wine ethanol were collected by using a Cadiot column distillation system to quantitatively separate the ethanol, according to the procedure described

All isotopic analyses were performed in Laboratory of Stable Isotope and Radiocarbon of Nanoscience & Nanotechnology, Institute in N.C.S.R. Demokritos (Athens, Greece). Stable isotope analysis of 18O/16O, D/H, and 13C/12C ratio in Greek wines, wine water, meteoric, and ground waters were carried out on a continuous flow Finnigan DELTA V plus (Thermo

Mass Spectrometers was performed according to Ref. [22]. Specifically, for δ13C determination 1 μL of purified ethanol was injected into a small tin container for liquids and closed securely to prevent evaporation. The Flash Elemental Analyzer (FlashEA) with a single reactor system combining combustion and reduction in one reaction was used. The Thermo Scientific ConFlo interface diluted the CO2 sample peak with 1 bar helium resulting in a split of about 1:12. For

H and δ18O determination 0.1 μL of pure ethanol was injected with a 0.5 μL syringe into a glassy carbon reactor in a high temperature carbon reduction system of a FlashEA. The autosampler was used and samples were stored in 2 mL vials with standard caps and septa.

The results are expressed in standard delta notation (*δ*) as per mil (‰) deviation from the standard V-SMOW as: *δ* = ((*R*sample − *R*standard)/*R*standard) × 1000, where *R*sample and *R*standard = 2

Measurement precision, based on the repeated analysis of internal standard waters, was 1.5,

All measurements were carried out according to laboratory standards that were periodically

14C activity was determined by using liquid scintillation counting (LSC) on a Packard Tri-Carb TR/SL. Radiogenic isotope of Sr and Nd were measured with thermal ionization mass spectrometry (TIMS, Nd) and inductively coupled plasma mass spectrometry (ICP-MS, Sr).

or 18O/16O or 13C/12C ratios of sample and standard, respectively.

H, δ18O, and δ13C, respectively.

calibrated based on the international standards recommended by the IAEA.

H isotope analysis in ethanol with Thermo Scientific DELTA V Isotope Ratio

H/1 H

Electron Corporation, Bremen, Germany) stable isotope mass spectrometer [20, 21].

in OIV MA-AS-311-05.

446 Grape and Wine Biotechnology

13C, 18O, and <sup>2</sup>

0.5, and 0.2% for δ2

δ2

#### **5.1. Isotopic composition of meteoric water**

Twenty-nine water samples (ASt1 to ASt17 irrigation water samples and ASt18 to ASt29 precipitation water samples) were collected at the region of Amydaio in West Macedonia in North Greece, corresponding to the growing season of 2013. From Nemea, northeastern Peloponnese in southern Greece, a total of 21 water samples (NSt1 to NSt10 groundwater samples and NSt11 to NSt21 precipitation water samples) were collected, corresponding to the same growing season. The δ18O and δD values of Amydaio and Nemea are shown in **Figure 2**. In the same figure are also pictured the Global Meteoric Water Line with correlation equation: [23]

$$\text{SID} = 8 \times \delta^{18} \text{O} + 10 \tag{1}$$

and the Local Meteoric Line that correspond to Greece (LMWL) with correlation equation: [24]

$$\text{6D} = 8.7\text{\ $}^{18}\text{O} + 19.5\text{\$ } \tag{2}$$

**Figure 2.** Equation correlation δ18O–δ2 H both in wine water and grape berries of Greece and Europe generally.

Generally, an isotopic correlation between δ18O and δ2 H with a slope close to 8 indicates fresh precipitation of all types [23], as well as for surface water not subjected to excessive evaporation relative to input [24]. In contrast, slopes ranging from 6 to 4 reflect isotopic relations between δ18O and δ 2H for waters which are subjected to excessive evaporation relative to input [23].

Oxygen and deuterium isotope values for Amydaio precipitation water samples range from −9.9‰ to −9.6‰ and from −66.7‰ to −63.8‰, respectively, while for groundwater range from −8.8 to −6.4‰ and from −56.6‰ to −40.9‰, respectively. The correlation equation for Amydaio groundwater is δD = 6.86\*δ18O + 3.24 where the slope 7.59 suggests that the samples have not undergone evapotranspiration process. For Nemea precipitation water samples of δ18O and δD values range from −7.9‰ to −6.0‰ and from −48.1‰ to −35.2‰, respectively, while for groundwater samples range −6.1‰ to −4.8‰ and from −35.1‰ to −25.2‰, respectively. Evapotranspiration process seems not to have effected remarkably the irrigation water samples as their slope is 7.27 (δD = 7.27\*δ18O + 9.70). Groundwater from both study areas do not exhibit remarkable variations in δ18O and δ2 H values indicating the lack of seasonal and altitude impacts affecting the meteoric waters [25].

Similar equations with slightly greater slopes of 7 and 7.5were proposed for the northern part of Eastern Macedonia and for the Eastern Nemea, respectively [26]. A slope lower than 7 was reported for Central Macedonia [27], the southern part of Eastern Macedonia and Thrace [28], as well as the northern part of Epirus [29].

#### **5.2. Isotopic composition of wine water**

Amydaio vineyards are represented by 54 bottles of wine (AW1–AW54) consisting of 6 (AW1–AW6) Roditis (white wine), 24 (AW7–AW30) Xinomauro (red wine), and 24 ((AW31– AW54) Tannat (red wine). Nemea vineyards are represented by 16 bottles of wine (NW1– NW16) consisting of 11 (NW1–NW11) Agiorgitiko (red wine), 1 (NW12) Merlot-Agiorgitiko (red wine), 1 (NW13) Moschofilero-Rodidtis (white wine), and 3 (NW14–NW16) Roditis-Chardonnay (white wine).

The stable isotope values of Greek wine waters are presented in **Figure 2**. As Western and Southern Europe constitutes the most significant region, both in terms of vineyard area and quantity of production, French and Italian wines are also plotted in the same figure. Amydaio presents isotopic values ranging from −1.3‰ to 2.3‰ (mean value 1.1‰) for δ18O and from 3.9 to 22.3‰ (mean value 14.2‰) for δD, while Nemea samples range from 2.5‰ to 6.1‰ (mean value 3.8‰) and from 15.2‰ to 26.1‰ (mean value 21.4‰) for δ18O and δD, respectively. A first observation is the fact of isotope enrichment in the north-south direction. This trend has been also suggested by some authors for olive oils [30, 31]. δ13C values also related to the north-south enrichment observation with wine water values to range from −29.4‰ to −26.1‰ (mean value −27.7‰) for Amydaio wines and from −26.2‰ to −24.7‰ (mean value −25.6‰) for Nemea wines. Moreover, Nemea wines present slightly more positive values with larger variations verifying the warmer and drier conditions than those of Amydaio where the conditions are cooler and the precipitation episodes more frequent and constant. Indeed, ambient conditions affect intermediate steps in plant functions [32, 33] and proceeds for sugar formation, therefore carbon isotope could trace deferent environmental conditions. The enzymes favor the 12C isotope for the photosynthesis than 13C as it is found in greater concentration in atmospheric CO2 (98.9‰ for 12C and 1.1‰ for 13C). Moreover, this difference mass which cause a higher diffusion of 12CO2 than that of 13CO2 combined to the fact that 13C forms stronger chemical compounds as it is heavier than 12C induce in lower concentrations of 13C in plants than atmosphere [3]. Especially in water deficit conditions leaves stomata close resulting in a reduced atmospheric CO2 interchange, which leads to changes in δ13C values among C3-photosynthesis plants such as grapes [2, 5].

Wine water and groundwater samples correlated well enough in both vineyards implying that the "source water" represented by groundwater could be linked to wine products. The isotopic composition of groundwater and precipitation are mainly related to latitude, distance from the sea, and altitude [27]. Groundwater samples from Amydaio and Nemea reflect their meteoric origin without remarkable variations (**Figure 2**) so it is assumed that environmental setting of the vineyards control the isotopic composition of wine products. Schmidt et al. have already reported the δ18O correlation between carbohydrates and δ18O of leaf water which are linked to the isotopic composition of groundwater with the evapotranspiration ratio to be influenced by humidity and temperature [9].