doi: / Vol.14,2015 ComprehensiveReviewsinFoodScienceandFoodSafety 681 C 2015 Institute of Food Technologists

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1 Acetaldehyde as Key Compound for the Authenticity of Sherry Wines: A Study Covering 5 Decades Luis Zea, María P.Serratosa,JulietaMérida, and Lourdes Moyano Abstract: Sherry wines are consumed worldwide and are principally produced in the Jerez and Montilla-Moriles regions of southern Spain. Acetaldehyde is a relevant carbonyl compound in wines and one of the main responsible for the particular personality of Sherry wines with a ripe apple odor descriptor. Aldehyde dehydrogenase plays an important role in yeast acetaldehyde metabolism. Acetaldehyde contents in Sherry wines subjected to biological aging strongly depend on yeast populations, and the formation of velum depends on specific amino acids, oxygen availability, and the composition of the wine. Both biological and oxidative aging processes increase the acetaldehyde content in Sherry, although some of the acetaldehyde is oxidized to acetic acid and subsequently transformed into acetyl-coa. In sensory terms, 1,1-diethoxyethane and other acetals, acetoin, and sotolon are the main compounds formed from acetaldehyde in Sherry wines. The chemical browning pathway of wine by the condensation between phenols and acetaldehyde is especially important in Sherry wines. Acetaldehyde can inhibit the velum formation at higher concentration than its threshold tolerance; also, it could be responsible for the high mitochondrial DNA polymorphism observed in flor yeasts. Usually, acetaldehyde is used to control the biological aging of Fino Sherry. A faster production of acetaldehyde could be considered to shorten the aging process of Sherry. In recent years, the acetaldehyde formed during ethanol metabolism in alcoholic beverages has been associated with carcinogenic processes; however, no systematic data are available about this statement. Keywords: acetaldehyde, Sherry, wine, yeast Introduction The famous Sherry wines are principally produced in 2 southern areas of Spain, Jerez and Montilla-Moriles. The climate conditions strongly determine the particular composition of these wines and comprise moderately low average minimum temperatures (lows of 5 C in winter and highs of 17 C in summer) and high temperatures in summer, above 35 C or even above 40 C during the grape-ripening stage (Chaves and others 2007; Moreno and others 2007). The region is a dry zone, where rainfall is unevenly distributed throughout the year and also at times is about 400 mm or less. These favorable climatic conditions allow the white grape varieties used to obtain Sherry wines (Palomino Fino and Pedro Ximénez) to reach a high reducing sugar concentration, even above 250 g/l in some areas. Therefore, after alcoholic fermentation the base wines have a high ethanol content, about 15% vol. obtained naturally, so no alcohol addition is necessary. The musts have a low acidity (3 to 4.5 g/l expressed as tartaric MS Submitted 24/2/2015, Accepted 20/7/2015. Authors are with the Dept. of Agricultural Chemistry, Univ. of Córdoba, Campus de Rabanales, Edificio Marie Curie, Córdoba, Spain. Direct inquiries to author Zea ( qe1zecal@uco.es). acid) which is generally increased by the winemakers. Also, the musts are added to a S 2 concentration of around 100 mg/l. After the vintage, grapes are stemmed and pressed. The must obtained from the first pressing, called yema, and that obtained in subsequent ones, is centrifuged or racked in order to remove suspended solids. Then it is pumped to stainless steel tanks or concrete cones and allowed to ferment. During this process, special care is taken to keep the temperature below 25 to 26 C. Currently available refrigeration systems can efficiently produce even lower temperatures. Typical Fino, Amontillado, and loroso wines are 3 types of dry Sherry wines obtained under identical fermentation conditions (same wine base) but using different aging conditions and procedures as part of the criaderas and solera system (Figure 1). Basically, this industrial aging method involves storing the wine in 500-L American oak casks, which are stacked in rows called escalas. Fino wines result from biological aging, carried out by flor yeasts growing on the wine surface when the ethanol content is lower than 15.5% (v/v) (Figure 2). The aerobic metabolism developed by these yeasts causes some changes in the aroma of wine, mainly a high production of acetaldehyde, which endows the wine its typical flavor (García-Maiquez 1988; Martínez and others 1997a; Cortés and others 1998). Acetaldehyde is responsible for the C 2015 Institute of Food Technologists doi: / Vol.14,2015 ComprehensiveReviewsinFoodScienceandFoodSafety 681

2 Figure 1 Criaderasand solera Sherry aging system. YUNG WINE Biologic aging Alcoholization (18-19%) Alcoholization (18-19%) xidative aging xidative aging FIN AMNTILLAD LRS Figure 2 Flor yeasts growing on the wine surface. pungent character of Fino wine and it directly contributes the ripe apple notes to its aroma (Moyano and others 2002; Muñoz and others 2007; Zea and others 2007, 2008). loroso wines are obtained by oxidative aging, after addition of ethanol, up to about 18% (v/v), which prevents the growth of flor yeasts (Casas 1985; Domecq 1989; Zea and others 1996, 2008). Under these conditions, loroso wine exhibits a dark color as a result of the oxidation of phenolic compounds, thus distinguishing it clearly from Fino wine (Fabios and others 2000). ne of the most relevant changes during oxidative aging of wine is the increase of volatile acidity through chemical oxidation of ethanol via acetaldehyde (Tulyathan and others 1989; Bartowsky and Henschke 2004). Figure 3 Different aging processes for making the Sherry-type wines. Amontillado wines are obtained by aging in a two-step process including biological aging under similar conditions to those of Fino wines, followed by an increase in the ethanol content. The wines are then subjected to oxidative aging, as in loroso wines. Amontillado wines are thus the oldest and most valued of the 3 types, because they reach a more complex flavor than the other 2 with hazelnut notes (Medina and others 2003). Figure 3 shows the different aging processes involved in making the Sherry-type wines. A special Sherry is the famous Pedro Ximénez sweet wine which is elaborated from sun-dried grapes containing sugars of more than 400 g/l. The musts obtained are fortified to an ethanol content of 13.5% (v/v) and then subjected to oxidative aging in a criaderas and solera system. The increase in the concentration of acetaldehyde is one of the more singular features of biological aging; therefore, traditionally 682 Comprehensive Reviews in Food Science and Food Safety Vol. 14, 2015 C 2015 Institute of Food Technologists

3 - C 2 NAD C H Mg 2 NADH H TPP C C pyruvate alcohol decarboxylase dehydrogenase H 2 C Pyruvate Acetaldehyde Ethanol Figure 4 Acetaldehyde formation from ethanol by flor yeasts. the Sherry winemaking industry has used the acetaldehyde content as a marker for the progress and status of biological aging. In this way Zea and others (2001) conclude that acetaldehyde content allows the differentiation of Fino wines from other types of Sherry wines obtained by oxidative aging. However, a more precise measure of biological aging must considerer changes in an extensive number of aroma compounds, acetaldehyde included (Cortés and others 1998, 1999). Acetaldehyde is a precursor for the synthesis of other aroma products, thus indirectly contributing to numerous distinctive notes in the aroma profile of wine (Etiévant and Schreier 1995). Likewise, according to Moreno and others (2005), derivatives of acetaldehyde such as sotolon and 1,1- diethoxyethane could be considered markers in Sherries subjected to biological aging. The production of acetaldehyde has been shown to be influenced by aerobic metabolism, medium composition, the nature of insoluble materials used to clarify the musts, aging procedure, S 2 content, and aeration (Romano and others 1994; Berlanga and others 2001). Acetaldehyde values in Sherry wines subjected to biological aging strongly depend to yeast populations (Ibeas and others 1997; Martínez and others 1997b, 1998; Cortés and others 1998; Muñoz and others 2005), and the aging time (Moreno and others 2005). Usually, these wines (Fino type) show 230 to 550 mg/l of acetaldehyde although it is possible to find much higher concentrations (Martínez and others 1997b, 1998). In the other Sherry wine types the acetaldehyde exhibits lower contents than in Fino (Zea and others 2001, 2008; Medina and others 2003; Peinado and others 2004). Therefore, after a brief summary on the making of the typical Sherry wines, a review of the exceptional participation of acetaldehyde in the genuineness of Sherries is presented, with a focus on the production during the different aging processes, chemical evolution, and sensory features of the acetaldehyde. Formation of Acetaldehyde in Wine Formation during alcoholic fermentation During must fermentation acetaldehyde occurs from yeast metabolism of sugars via the action of pyruvate decarboxylase and alcohol dehydrogenase (Figure 4). Acetaldehyde is considered as a secondary product of the alcoholic fermentation. It is quantitatively the most important carbonyl compound produced during alcoholic fermentations, with final concentrations ranging from 10 to 200 mg/l. Furthermore, this aldehyde is the terminal electron acceptor in the alcoholic fermentation by Saccharomyces cerevisiae (Jackowetz and others 2011). There are great strains and strain differences in acetaldehyde production by yeasts; for example, 60 to 500 mg/l for S. cerevisiae (Table 1). When the acetaldehyde content was above 400 mg/l, the montuliensis strain exhibited higher population Table 1 Acetaldehyde concentration (mg/l) and percentage of the different strains isolated from velum in young Sherry wines (from Martínez and others 1997b). %ofs. cerevisiae strains Acetaldehyde Beticus Montuliensis Cheresiensis Rouxii thers * * Strains others than S. cerevisiae. numbers, whereas in the wine with lower contents the only strain present was beticus. While glucose and fructose are the primary substrates of acetaldehyde formation, metabolism of amino acids such as alanine also contributes to the formation of this compound (Henschke and Jiranek 1993; Boulton and others 1996). During the last 5 decades, a substantial number of studies have been performed on the formation of acetaldehyde by film yeasts in relation to Sherrie s production. Acetaldehyde is excreted mainly duringthegrowthperiod(martínez and others 1997b) and can be catabolized (Farris and others 1993). Factors such as temperature, oxygen, and S 2 define the synthesis of acetaldehyde by yeasts. While several authors reported that fermentation temperature did not influence the final acetaldehyde levels in wine (Amerine and ugh 1964), Romano and others (1994) note increased formation of this carbonyl compound at 30 C compared with that formed at 12 or 24 C. Nonetheless, Jackowetz and others (2011) studied the effect of different enological parameters on acetaldehyde kinetics during alcoholic fermentations. Results showed that S 2 addition, grape cultivar, and fermentation nutrition were important regulators of acetaldehyde production. Results calculate the acetaldehyde increases produced by S 2 addition and indicate that the must composition determines the metabolism of the yeasts on acetaldehyde. Likewise, the final acetaldehyde content was strongly reduced to final fermentation stage. S 2 is used as an antimicrobial, antioxidative, anti-enzymatic, and anti-acetaldehyde (taste improvement) additive in oenology procedures. The roles that S 2 carried out in wine fermentation and the binding of S 2 by acetaldehyde and other compounds such as pyruvic acid and α-ketoglutaric acid have been extensively studied by Romano and Suzzi (1996), Zoecklein and others (1995), Boulton and others (1996). So, only a concise summary of S 2 in regard to acetaldehyde is given below. C 2015 Institute of Food Technologists Vol. 14, 2015 Comprehensive Reviews in Food Science and Food Safety 683

4 Comparison of acetaldehyde and hexose kinetics showed a probable relationship between the time of occurrence of top acetaldehyde concentrations and the divergence of glucose and fructose degradation rates, which demands additional consideration. In any case, acetaldehyde kinetics was uniform during the fermentation processes with an initial increase to a highest value, followed by reutilization. Finally, is interesting to note that several parameters were found to affect acetaldehyde concentrations either individually or in combination. Formation during biological aging Acetaldehyde is synthesizedfrom ethanolbyflor yeasts by means of the enzyme alcohol dehydrogenase in the presence of NAD (see Figure 4). As discussed above, the increase in the concentration of this compound is one of the more important consequences of biological aging. As already mentioned, the acetaldehyde levels allow the differentiation of wines biologically aged from other Sherries subjected to oxidative aging; also, acetaldehyde is a precursor of other aroma compounds, thereby indirectly contributing to the flavor of wine (Etiévant 1995). therwise, flor yeasts increase the contents in other aroma compounds such as higher alcohols and acetates, ethyl esters, lactones, and terpenes (Williams 1989; Zea and others 1995). Industrially, the status of biological aging of Sherry has been evaluated by the acetaldehyde values in wine. Nevertheless, the relationship of its content to the aging time for Fino wines is quite complex because its formation is affected by several factors such as the population and metabolism of the flor yeasts used, the temperature, ethanol content, and the redox potential, among other (García-Maiquez 1988; Martínez and others 1997b). It is noteworthy that flor yeasts are very temperature-dependent microorganisms, mainly growing at about 20 C (Ibeas and others 1997). Consequently, their period of greatest activity lasts only a few months each year depending on the temperature conditions of each cellar, obtaining wines with different compositions although aged for a same number of years. This fact is increased by the effect of the periodic transfers performed during winemaking, which mix wines with different years of biological aging. n the other hand, it is usual that some commercial Fino wines have similar acetaldehyde contents but differ strongly in their sensory properties. Consequently, one must be careful in considering this compound as a marker for biological aging of these Sherry type wines. Flor yeasts have adapted to the ethanol produced by them, so they can develop in high ethanol concentrations. Therefore, it could be accepted that the formation of the velum can be a mechanism of adaptation of yeasts to wine, being basically influenced by a particular amino acid, oxygen availability, and the composition of wine. Consumption of glycerine was associated to the metabolism of yeasts; in contrast, acetaldehyde tended to be released (Berlanga and others 2006). In this situation the yeasts grow on the surface of wine by means of an oxidative metabolism in which oxygen is not a limiting factor. Under these conditions the yeasts change their shape, size, and hydrophobicity, and being less dense than the wine the cells rise to the surface (Martínez and others 1997a). The increase of hydrophobicity could be due to synthesis of hydrophobic proteins which facilitate cell aggregation and retention of gas bubbles, causing decreased density and increased buoyancy. However, Imura and others (1980) and Rose and Harrison (1991) suggest that this hydrophobicity may depend largely on the fatty acids of the cell wall. A direct correlation between velum formation by S. cerevisiae and level of unsaturation of long-chain fatty acids were showed by Farris and others (1993) and Van der Rest and others (1995). Similar results were obtained by Aguilera and others (1997), mainly for oleic and palmitic acids in Saccharomyces capensis. Formation during oxidative aging Wildenradt and Singleton (1974) researched the production of acetaldehyde from ethanol by the oxidation of phenolic compounds in model solutions. They observed that the production of this compound by direct oxidation of ethanol is very scarce, and that oxidation of ethanol to acetaldehyde take place by a coupled auto-oxidation of specific phenolic compounds (Figure 5). Hydrogen peroxide is a strong oxidant, resulting from phenolic oxidation, oxidizes ethanol to acetaldehyde. Ions of metals such as Fe, Cu, and Mn are somehow involved in the oxidation processes. Thus, Mn favors the formation of acetaldehyde, while Fe catalyzes the combination of the phenolic compounds. Some authors, such as Danilewicz (2003) and Waterhouse and Laurie (2006), explain in detail the origin of H 2 2 in wine and the role of metals in wine oxidation. Acetaldehyde production is influenced by free S 2 levels, because it can be added to quinones, thus decreasing the efficiency of the oxidation of ethanol. Formation of acetaldehyde by acetic acid bacteria Besides yeasts, acetic acid bacteria (AAB) originating from grapes and winery equipment (Joyeux and others 1984; Drysdale and Fleet 1988) can also produce acetaldehyde. The enological and practical implications of AAB have been studied by Drysdale and Fleet (1988) among others. This bacteria oxidizes ethanol to acetaldehyde and acetic acid, and contents of up to 250 mg/l acetaldehyde can be produced (Adams and Moss 1995). This value overcomes the perception threshold (100 to 125 mg/l) for acetaldehyde, and could negatively affect the organoleptic property. According to Zoecklein and others (1995) and Fugelsang (1997), this compound has a tendency to accumulate under low oxygen conditions and/or alcohol contents higher than 10% (v/v) as an alternative of being oxidized to acetic acid. Formation of acetaldehyde by lactic acid bacteria Currently, it is not clear whether wine lactic acid bacteria (LAB) can produce acetaldehyde. n the contrary, the synthesis of acetaldehyde by dairy LAB is well confirmed, and a great deal of documentation on this fact is available. The amount of acetaldehyde formed by dairy LAB ranges with species and strain, and is generally less than 30 mg/l (Kneifel and others 1992). Acetaldehyde is synthesized path the metabolism of glucose, 2-deoxy-d-ribose-5- phosphate, and threonine (Rysstad and others 1990; Grozeva and others 1994). It is well know that these precursors are also present in wine, and it will be of practical interest to establish if wine LAB can originate acetaldehyde from these substrates. In addition, other microorganisms, such as LAB and non- Saccharomyces yeasts, may coexist with flor yeasts, causing sensorial deviations and deterioration of the Fino wine. The activity muramidase of enzyme hydrolytic Lysozyme can lyse Gram-positive bacteria; the IV in 1997 (resolution EN 10/97) approved its use in winemaking industry. This enzyme has been mainly used to control the development of LAB and to decrease or prevent heterolactic fermentation during biological aging of Sherry wines (Lasanta and others 2010). Taking into account, a lysozyme contents near to 6 g/hl allow to effectively control LAB populations in wines with high gluconic acid levels. Furthermore, when lysozyme is used in combination with flor velum yeast, volatile acidity content is reduced. However, there have been no studies 684 Comprehensive Reviews in Food Science and Food Safety Vol. 14, 2015 C 2015 Institute of Food Technologists

5 R R 2 H 2 () H 2 () H 2 2 H 2 2 CH 2 CH 2 H 2 Figure 5 Acetaldehyde production by chemical oxidation of ethanol. on the effect of lysozyme on velum; in this way, Roldán and others (2012) argued that lysozyme does not affect the flor yeast during the fermentation or velum growth. However, if yeast inoculation is carried out under submerged culture conditions during biological aging, doses of lysozyme around 10 g/hl affect yeast cells metabolism and its membrane hydrophobicity, inhibiting their aggregation and flotation and, consequently, the development of velum. Therefore, the yeast inoculation process and the methods used for the addition of lysozyme influence velum progress, its metabolism, and the wine characteristics. Changes of Acetaldehyde in Wine Ethanol is the main source of energy for the yeast cell and it is transformed into acetaldehyde using alcohol dehydrogenase (Plata and others 1998). Some of the acetaldehyde is oxidized to acetic acid and subsequently transformed into acetyl-coa; this then enters either the glyoxylate cycle (to form succinic acid) or the Krebs cycle. 1,1-Diethoxyethane, or diethylacetal, is one of the principals acetals in Sherry wines. This compound is synthesized by the reaction between ethanol and acetaldehyde according to Misselhorn (1974) through chemical and biochemical (flor yeasts) pathways (via 3 in Figure 7). This compound is an active odorant contributing with fruity and balsamic notes to the sensory profile of wines (Muñoz and others 2007; Zea and others 2008). Numerous authors have pointed out that the reaction of acetaldehyde with the alcohols is a relevant process to develop strongly woody notes. However, according to Etiévant (1995) the only acetal that can actually contribute to wine aroma is 1,1-diethoxyethane. In Porto wines, similar to loroso Sherry, Da Silva Ferreira and others (2002) found that during the oxidative aging the isomers of acetals 5-hydroxy-2-methyl-1,3-dioxane, and 4-hydroxymethyl-2- methyl-1,3-dioxolane, linearly increased their concentrations with time (r > 0.95) up to above the perception threshold levels. Among the many compounds formed by the reaction of acetaldehyde we must emphasize acetoin. It is a major compound, mainly produced during the biological aging through reductive acetoinic condensation of 2 acetaldehyde molecules (Romano and Suzzi 1996). According to Berlanga and others (2001) this reaction is influenced by acetaldehyde content and re-oxidation of NADH. In sensory terms, acetoin provides to Fino wines the bitter almond flavor so characteristic of them. Sotolon (3-hydroxy-4,5-dimethyl-2(5H)-furanone) is a lactone resulting from the reaction between α-ketobutyric acid and acetaldehyde during the development of yeast film, which takes place through the mechanism proposed by Pham and others (1995). These authors concluded that, in a synthetic medium resembling wine, when adding different organic acids and acetaldehyde, under temperature, ph, and alcoholic content conditions similar to those of aging wine in barrels, sotolon is formed by a purely chemical mechanism from α-ketobutyric acid and acetaldehyde (Figure 6). The formation of sotolon increases by increasing temperature and decreasing ph and alcoholic content. A number of authors attribute a high odor impact to sotolon, with nut, curry, and cotton candy notes in both Sherry biologically and oxidatively aged wines and others produced under oxidative aging conditions (Escudero and Etiévant 1999; López and others 1999; Cutzach and others 2000; Kotseridis and Baumes 2000). As a practical application, based on the value of r-squared of the linear model applied to the dor activity values (AVs) of sotolon and 1,1-diethoxyethane, versus aging time in a Fino wine, the odorant activity of these compounds would be a good marker of the biological aging of Sherry (Moreno and others 2005). Figure 7 shows the classic scheme published by Zea and others (1996), including other principal reactions of acetaldehyde: Path 1, acetaldehyde with free S 2 to produce ethanol-sulfonic acid. Acetaldehyde favors the condensation between different phenolic compounds (path 2). The resulting products are of highmolecular mass so easy precipitation allows a decrease of color and astringency of wine. This reaction is largely responsible for the pale color and smooth taste of Fino wine. Path 4, acetoin is formed from 2 acetaldehyde molecules (as mentioned above) whose subsequent reduction produces 2,3- butanediol. Although the evolution of this compound during wine aging is complex, an increase during biological aging has been observed. Both acetoin and, particularly, 2,3-butanediol, are essential components of the aroma profile of Sherry wines. ther quantitatively less important reactions involving acetaldehyde are the disproportionation to ethanol and acetic acid (path 5), or directly the chemical oxidation to acetic acid (path 6). In any case, acetic acid is consumed by flor yeasts, thus is not affecting the bouquet of the wine. A chemical browning pathway of wine passes through the condensation between phenols and acetaldehyde produced by yeast in the alcoholic fermentation. This reaction should be especially important in Sherry wines biologically aged because they show high acetaldehyde contents (Barón and others 1997; López- Toledano and others 2004; Mérida and others 2005, 2006). The condensation reaction between ()-catechin and acetaldehyde (Figure 8) was studied in model solutions in the presence and absence of yeasts to estimate its contribution to color changes in C 2015 Institute of Food Technologists Vol. 14, 2015 Comprehensive Reviews in Food Science and Food Safety 685

6 Threonine deaminase C H 3 NH 2 L-Threonine C C H 3 C A-Ketobutyric acid Aldol condensation C H 3 C CH Acetaldehyde Cyclization Sotolon Figure 6 Formation of sotolon from α-ketobutyric acid and acetaldehyde. CH ACETALDEHYDE C H 3 S 2 H 2 1 HC Sulfonic acid ethanol Polyphenols Tannins 2 C H 3 HC C 2 H 5 S 3 C 2 H 5 Condensation compounds 3 Ethanol Acetaldehyde Acetaldehyde 2 1,1-Diethoxyethane H C 4 HC H 2 Acetoin 5 CH 2 Ethanol C Acetic acid 6 C Acetic acid CH HC 2,3-Butanediol Figure 7 Classic scheme showing acetaldehyde reactions in wine. white wines. Results show that the yeasts retain the oligomers produced in the reaction, their retention ability increasing higher polymerization degrees. Consequently, the color of model solutions, evaluated as the absorbance at 420 nm, decreased after the addition of yeasts. This reaction takes place very slowly in similar conditions to those in wine, including acetaldehyde in high concentration as it is present in Sherry wines. Wine model solutions were used to research the ability of dehydrated yeasts to retain the brown pigments formed from the reaction between acetaldehyde and ()-catechin. The 2 most important flor yeast strains implicated in the biological aging of Sherry wines, S. cerevisiae capensis and bayanus, showed a higher capacity to retain colored compounds than S. cerevisiae fermentative yeast. The capensis strain exhibited a higher color reduction capacity than bayanus. Such differences may explain the different degrees at which browning products are eliminated at different times of the year during the biological aging of wines (Mérida and others 2006). Influence of Acetaldehyde on the Yeast Velum Jones (1990) and Ristow and others (1995) stated that acetaldehyde can inhibit the velum formation at higher concentrations than its threshold tolerance. Flor formation and flor tolerance have been related to the ability by S. cerevisiae to resist hostile conditions such as oxidative stress and the presence of acetaldehyde and ethanol. The toxicity of these compounds can originate reactive oxygen species and a decrease of cell viability (Fierro-Risco and others 2013). S. cerevisiae isolated in the biologically aged wines develop an oxidative metabolism on the wine surface where the mitochondria could play a determinant role (Moreno-García and others 2014). A study on 8 strains of flor yeast was carried out to examine the enological behavior of velum during laboratory experiments on biological aging. Strains with identical chromosomal and mitochondrial DNA (mtdna) patterns and the same origin showed a more closely related enological behavior, although the kinetics of growth and acetaldehyde accumulation in the wine were found 686 Comprehensive Reviews in Food Science and Food Safety Vol. 14, 2015 C 2015 Institute of Food Technologists

7 H H H H d - d - H HC H H H H 2 () Catechin H d - () Catechin H H CH H H Dimer Figure 8 Condensation reaction between ()-catechin and acetaldehyde. to be strain-dependent. Moreover, some strains were marked by high acetaldehyde accumulation in their pure cultures during the various phases of velum development. These results provide valuable information for planning technical strategies to improve the biological aging process in the Sherry wine industry (Rodríguez and others 2013). Flor yeasts grow and survive in Fino Sherry wine where the frequency of respiratory-deficient mutants is very low. Mitochondria from flor yeasts are highly acetaldehyde- and ethanol-tolerant, and resistant to oxidative stress. Nevertheless, limitation fragment size polymorphism (LFSP) of mtdna from flor yeast populations is very high and reflects variability induced by the high concentrations of acetaldehyde and ethanol of Sherry wine on mtdna. Therefore, mtdna LFSP increases as the concentration of these compounds also increases, originating a total loss of mtdna in cells. Yeasts with functional mitochondria are subjected to constant changes, so that flor yeast mtdna can evolve extremely rapidly and may serve as a reservoir of genetic diversity, whereas minority mutants are eventually eliminated because metabolism in Sherry wine is oxidative (Castrejón and others 2002). There are studies on the consequence of acetaldehyde and ethanol on cell chromosomal DNA which showed that ethanol induces DNA breaks, but that the former has a stronger damaging effect on chromosomal DNA. In this respect, some authors suggest that the superior capability of flor yeast mitochondria to tolerate acetaldehyde and ethanol is partly due to their possession of highly efficient active superoxide dismutases. Some authors suggest that flor yeast possesses distinct genetic features that are different from those of other wine yeasts, for example, low chromosomal polymorphism (Ibeas and Jiménez 1996). High ethanol concentrations found in wines aged under flor yeast may be responsible for such genetic differences (Ibeas and others 1997; Martínez and others 1998; Mesa and others 2000). Acetaldehyde can cause double-strand DNA breaks and has been reported to be responsible for the high mitochondrial DNA polymorphism observed in flor yeast. Acetaldehyde could also cause large-scale chromosomal rearrangement which may result in amplification of chromosomal segments (Infante and others 2003). Berlanga and others (2001) investigated the result of periodic aeration on the metabolism of a S. cerevisiae strain on velum growth and during biological aging of Sherries. Aeration improved adenylate energy charge, develop, and viability of the yeast cells. Besides, the concentration of several compounds, such as acetaldehyde, ethanol, glycerine, acetoin, higher alcohols, and acetic acid related with the intracellular redox equilibrium. Acetaldehyde stretched to its highest contents after the second aeration, which corresponded with the depletion of the source of nitrogen in the medium. The activity of the enzymes alcohol dehydrogenases I and II reduced right away after aeration, increasing afterward once all of the dissolved oxygen in the wine had been consumed by yeast. The enzymatic activity of aldehyde dehydrogenase was observed only after the first aeration, and it may be a result of the changes of the contents of acetic acid in wine. In biological aging, unfavorable conditions develop such as high acetaldehyde and ethanol contents and oxidative stress due to C 2015 Institute of Food Technologists Vol. 14, 2015 Comprehensive Reviews in Food Science and Food Safety 687

8 respiratory metabolism can be shown. Ethanol is extremely lethal to metabolism and growth of yeast cells, while acetaldehyde can inhibit the metabolism in a large range and is more toxic than ethanol (Jones 1990). The capacity of yeast cells to withstand the stress situations might be associated with their population dynamics for the period of aging, likewise an analogous relationship has been observed during winemaking (Ivorra and others 1999). Reply to stress conditions need the initiation of signal transduction pathways, which requires the formation of protective molecules, including heat shock proteins (Parsell and others 1994; Hohmann and Mager 1997; Estruch 2000). In this regard, it is quite possible that the protein (Hsp104p) is responsible for stress tolerance in laboratory yeast strains performing respiratory metabolism. However, this fact is not observed for yeast strains growing under fermenting conditions in wine with high levels of glucose (Carrasco and others 2001) or for brewery yeast strains (Brosnan and others 2000). To investigate the strength of yeast cells to the immediate addition of acetaldehyde Aranda and others (2002) studied the effect on the velum at several concentrations. The viability of yeast cells did not decrease when acetaldehyde was added up to a final content of 2 g/l in tubes that were entirely filled with the yeast solution after incubation for some hours at 20 C. The authors incorporated acetaldehyde suddenly at this final content to cells that had been previously incubated at 20 C with ethanol at a level of 12% (v/v), similar to that currently taking place in the barrels, since in soleras no viable cells were found with concentrations higher than 1 g/l (Martínez and others 1997a). Under these conditions, differences in tolerance were observed between strains; as in the case of ethanol stress, flor yeasts isolated from soleras showed the lowest decrease in viability, with values close to or even greater than 50%. ther prevalent strains in some soleras (Esteve-Zarzoso and others 2001) exhibited a viability of greater than 80% after acetaldehyde addition. Fermentative yeast strains used in winemaking obtained the opposite result, with viability reduced to less than 40%. These results show that tolerance to ethanol and acetaldehyde determinates the dynamics in the populations of wine yeasts between the stages of alcoholic fermentation and biological aging, probably jointly with the differences in their ability to produce acetaldehyde or in the rate of velum developed. Shortening of Sherry Aging The main disadvantage of the solera system is the low efficiency of the process because every year only 20% of wine stored in the cellar can be marketed; also, the cost of the large amount of oak barrels needed and evaporation losses during the long time of biological aging leads to a profit situation less favorable than for other wines (Suárez and Íñigo 1990). Thus, acceleration methods have been tested aimed at decreasing the time of biological aging of Fino wines, considering mainly microbiological and physicchemical factors. Among them, acetaldehyde content is traditionally considered the best indicator of biological aging. Since flor yeasts grow under aerobic conditions it is logical that different methods of aeration have been tested to reduce the biological aging, and the formation of the velum is not strictly necessary, for example by using submerged yeast cultures or increasing the surface/volume ratio. This method is known as system try. Cortés and others (1999) studied the effect of an acceleration test, performed on various aroma compounds of Fino Sherry wines. The test was carried out with periodic aeration and an increased surface/volume ratio, and the wine was aged under a pure culture of S. cerevisiae (race capensis) flor yeast velum. The authors subjected the results to multifactor analysis of variance, and the compounds, simultaneously depending on acceleration conditions and aging time (P < 0.01), were subjected to principal component analysis. The first component (86.14% of the overall variance) was mainly defined by acetaldehyde and its derivatives 1,1-diethoxyethane and acetoin. These compounds reached higher concentrations in accelerated aging wines than in control wines in a shorter time, and they do not show browning problems. Results indicated that these compounds can be used as indicators for biological aging of Fino Sherry wines; in addition, the assay demonstrates that this condition can be applied to shorten the aging time. An experiment was performed by Peinado and others (2003) using submerged S. cerevisiae variety capensis cultures previously grown in glycerin. From results the authors concluded that the traditional biological aging process can be accelerated. The controlled agitation conditions used raised the contents of acetaldehyde, acetoin, and meso-2,3-butanediol in the wine, in addition, the flor yeast consumed more gluconic acid than in traditional biological aging. A possibility of shortening the biological aging of Fino Sherry was reported by Muñoz and others (2005) by separately inoculating with the flor yeast strains S. cerevisiae variety capensis and Saccharomyces bayanus and subjecting to periodic microaeration to a dissolved oxygen concentration of 4 mg/l. Experiments were conducted after formation of the yeast velum of Sherry wines obtained by biological aging during 0, 2, and 4 y. The influence of enological variables such as acetaldehyde, ethanol, volatile acidity, and glycerol was studied by a multivariate analysis which showed that the biological aging of the wines could be substantially shortened. Thus, 42 d after flor velum formation by S. cerevisiae variety capensis, 0- and 2-y-old wines showed values of variables above those obtained for the wine aged for 4 y. The wines inoculated with S. bayanus exhibited high acetaldehyde contents and ethanol levels above 15% (v/v), and wines with alcohol concentration below 14.5% were undesirable. According to this work, it is possible to enhance the biological aging process by the consecutive or simultaneous inoculation of S. bayanus together with S. cerevisiae variety capensis and periodically oxygenating the wine. Also, it is possible to obtain wines with different organoleptic properties by inoculating yeast strains in different proportions. Recently, Fierro-Risco and others (2013) performed experiments with microorganisms genetically modified (MGM), although it is not yet allowed in Europe at present. The use of some MGM may be permitted in Europe in the near future. However, a genetically modified strain of S. cerevisiae has already been commercialized in the USA for the production of wine (Husnik Volschenk and others 2006). The transformant strains obtained in this work do not carry any bacterial resistance markers; this fact could make it possible for these transformant strains to be given generally regarded as safe status in the future. If these transformant strains were transferred to the industry to carry out biological aging, they might be quite beneficial, since an increase in viability and active metabolism should accelerate wine aging, thus reducing the time needed to consider the wine mature. It would also guarantee velum stability, thus preventing wine from being spoiled due to velum loss and wine oxidation. Sensory Contribution to Wine The standardized wine aroma terminology, given by the American Society for Enology and Viticulture (Noble and others 1984, 1987), includes the term acetaldehyde with the general descriptor oxidized. Subsequently, a modified version of the wine aroma 688 Comprehensive Reviews in Food Science and Food Safety Vol. 14, 2015 C 2015 Institute of Food Technologists

9 Tree fruit Apple Grass S Pungent Varnish Nailpolish Alcohol Glue Coffee Figure 9 Aroma wheel proposed for Sherry wines. wheel was performed for the purpose to clarify and enhance the proposed list of standardized terminology (Figure 9), because several of the descriptive terms for wines on the traditional wheel are not characteristic of Sherry wines. Therefore, it is necessary to define an aroma wheel for Sherry wines because of the characteristic flavor profile that they present (Zea and others 2012). In this wheel acetaldehyde is included in the fruity series with ripe apple descriptor. Acetaldehyde is considered the principal compound responsible for particular aroma of wine subjected to oxidative aging. In fact, acetaldehyde contents increase during aging. However, 1,1- diethoxyethane, an acetal derivate of acetaldehyde, is a more potent odorant than acetaldehyde according to the results of Zea and others (2001) who studied commercial Amontillado and loroso Sherry wines. AVs of 1,1-diethoxyethane for these wines of 20 and 50 were obtained, respectively, while for acetaldehyde the AVs were less than 2. A perception threshold from 100 to 125 mg/l was measured by Zoecklein and others (1995) in wine for free acetaldehyde. In hydroalcoholic solution, the acetaldehyde threshold was fixed at 10 mg/l by sensory analysis performed by Moreno and others (2005), Zea and others (2007, 2008), and Moyano and others (2009) to calculate AVs in Sherry wines. As reported by previous authors, high acetaldehyde concentrations report to Sherry aromatic notes of ripe apples (Gómez García-Carpintero and others 2014) and nutty, bruised apple (Lage and others 2014). These sensory descriptors are typical of special wines such as Sherry or Vin Jaune (Culleré and others 2007), which usually present higher levels of acetaldehyde, but are undesired in most other wines. The aromatic profile of Jura flor-sherry wines (also called "yellow wines") has been studied by taste panel and only acetaldehyde, 1,1-diethoxyethane, and sotolon were described as key odorants (Collin and others 2012). A gas chromatography-olfactometry (GC-) analysis on the aroma of loroso Sherry wines obtained by industrial oxidative aging for 0, 3, 6, 9, and 14 y were performed by Zea and others (2010). Results showed 18 compounds with AVs >1 and perceived by sniffing, which were grouped pursuant to their odor descriptors into 8 odorant series (fruity, chemical, balsamic, vegetable, fatty, empyreumatic, floral, and spicy). Using the calculated AVs and odor descriptors, the authors studied the odor profile of Sherry wines in view of identify differences in the typical aroma compounds attributable to the aging process used (whether biological or oxidative). However, it is difficult and speculative to obtain conclusions because of the great number of compound implied in the aroma fraction of wines and the differential odor impact of each one. Thereby, the odor activity values of the compounds exhibiting similar descriptions was grouped into odorant series (S). This method improved the study of aroma fraction of Sherry wines due to reduce the number of variables to be interpreted, maintaining their relative importance according to the AV of each compound. The S, as an analytical tool, allows a comparison of the aroma fractions with different aging times so as to study the changes in the aromatic fingerprints during the aging stages (Zea and others 2012). Aroma profile of wines was governed by the fruity series showing the highest AVs during aging; this series, in addition to the chemical and balsamic series, were the best to differentiate the aroma of wines of diverse ages. The ethyl esters, plus acetaldehyde and its derivatives (related to the chemical reactivity of ethanol), raised their participation in a higher amount than those not synthesized from ethanol (non-ethyl esters, alcohols, and C 2015 Institute of Food Technologists Vol. 14, 2015 Comprehensive Reviews in Food Science and Food Safety 689

10 Table 2 dor activity values (AVs) for the odorants in Sherry wines. The AV data are averages of 9 values (from Zea and others 2008). Wines Amontillado Biologically aged Chemically aged Compound AV AV SL AV SL Sotolon 92 ± 8 40 ± 4 *** 55 ± 4 *** Eugenol 86 ± ± 11 *** 19 ± 6 *** Ethyl isobutanoate 36 ± 8 79 ± 12 *** 30 ± 6 Ns Ethyl 3-hydroxybutanoate 27 ± 4 18 ± 3 *** 31 ± 8 Ns Ethyl octanoate 26 ± 6 32 ± 10 Ns Nd *** 1,1-Diethoxyethane 21 ± ± 13 *** 51 ± 15 *** Acetaldehyde 19 ± 6 56 ± 5 *** 12 ± 2 Ns Ethyl hexanoate 13 ± 4 20 ± 4 Ns 17 ± 2 Ns β-citronellol 9 ± 2 13 ± 4 Ns Nd *** Z-ak lactone 7 ± 3 4 ± 2 Ns 5.7 ± 0.8 Ns Ethyl acetate 6 ± ± 0.7 *** 25 ± 7 *** Isoamyl alcohols 6.0 ± ± 2 *** 10 ± 2 *** 4-Ethylguaiacol 3.4 ± ± 0.8 Ns 5 ± 1 *** 2-Phenylethanol 2.6 ± ± 1 Ns 3.6 ± 0.5 Ns Ethyl lactate 1.9 ± ± 0.06 *** 3.1 ± 0.8 Ns 3-Methylbutanoic acid 1.0 ± ± 0.8 *** Nd *** SL. significance level versus Amontillado wine; ***, >99%; ns,<99%; Nd, not detected. compounds from cask wood) during the aging process Acetaldehyde, and its derivatives 1,1-diethoxyethane and 2,3-butanedione, exhibited medium AVs (between 10 and 50) and powerful olfactometry perception. The importance of acetaldehyde regarding the different aging processes of Sherry was studied by Zea and others (2008) in reference to Amontillado wines. The aroma profiles of these wines, obtained by combined biological-oxidative aging, consecutively, were contrasted to those of the others 2 Sherries produced only by biological (Fino) and oxidative (loroso) aging, respectively (Table 2). A total of 16 odorant-active compounds (AV >1) for 3 types of wine were obtained where sotolon and eugenol were very potent odorants for the wines, with AVs >50. However, the results of the factor analysis performed on the 16 odorants point out that the acetaldehyde, ethyl acetate, and eugenol were the compounds most differentiating the wines. The spicy notes prevailed in the Amontillado and the fruity series (including acetaldehyde) did so in biologically and oxidatively aged wines. Because the biological aging step (high acetaldehyde production by flor yeasts) in this type of wine is shorter than the oxidative aging, the results suggest biological aging affects the aroma of Amontillado Sherry to a greater degree than does oxidative aging. Toxicity of Acetaldehyde Resveratrol is a bioactive compound protecting human cells against oxidative damage and contributes to cardiovascular fitness. The processes implicated in the elaboration of Sherry wine (fermentation, clarification, cold stabilization, and filtration) significantly affect resveratrol contents; however, the most important factor is the biological aging, decreasing the resveratrol contents by 80% (Roldán and others 2010). These results were supported in numerous laboratory tests carried out in which several factors that could affect the resveratrol content during aging were considered: oxidative phenomena and a combination of acetaldehyde and flor yeast velum growth (Roldán and others 2010). The toxicity of acetaldehyde can seriously affect the cardiac muscle and liver cells of the consumers; it is very reactive and, because of its pronounced electrophilic activity, binds easily to protein and peptide individual amino acids, and also to DNA. It has been stated that acetaldehyde has the capacity to cross-link to proteins in the rat nasal respiratory mucosa in vivo, proposing that mutagenesis and carcinogenesis phenomena may be caused by reaction of acetaldehyde DNA (Feron and others 1991; Takashi and Shibamoto 1993). Studies about acetaldehyde inhalation using experimental animals have demonstrated its ability to induce carcinomas; however, there are no data available on the chronic oral damage of acetaldehyde or its probable production of cancer. More such studies are needed because acetaldehyde occurs in a large number of foods and beverages, possibly even environmentally. Acetaldehyde, itself, has also been suspected to cause long-term adverse effects in consumers. A recent study found that acetaldehyde in alcoholic beverages could result in saliva contents above levels previously considered as possibly carcinogenic (Lachenmeier and Sohnius 2008) and may cause a considerable lifetime health risk (Lachenmeier and others 2009). An increasing number of evidences now involves acetaldehyde as a major subjacent factor for the carcinogenicity of alcoholic beverages and especially for esophageal and oral cancer. This compound together with alcohol consumption is carcinogenic to humans, there is now sufficient evidence available for the esophagus, head, and neck as sites of carcinogenicity (Yokoyama and mori 2003; Lewis and Smith 2005; Fang and others 2011; Lachenmeier and Monakhova 2011). These studies offer a plausible mechanism to explain the increased risk for oral cancer associated with high acetaldehyde levels in some beverages Acetaldehyde is a carbonyl compound naturally present in alcoholic beverages from ethanol metabolism of yeasts, being generally regarded as a source of carcinogenicity to consumers. Nonetheless, no definitive data are accessible about its presence in alcoholic beverages and the possible dangerous of human exposure to this directly consumed form of this compound outside alcohol metabolism. Lachenmeier and Sohnius (2008) have analyzed and evaluated many different products (n = 1555). Beer (0 to 63 mg/l) had considerably lower acetaldehyde concentrations than wine (0 to 211 mg/l), or spirits (0 to 1159 mg/l). The highest acetaldehyde levels were generally found in fortified wines (12 to 800 mg/l). Considering a similar distribution between the beverage and saliva, the remaining acetaldehyde contents in the saliva after ingestion could be, on average, 195 μm for beer, 734 μm for wine, 1387 μm for spirits, or 2417 μm forfortified wine; these levels are higher than those considered as theoretically carcinogenic. 690 Comprehensive Reviews in Food Science and Food Safety Vol. 14, 2015 C 2015 Institute of Food Technologists

11 In summary, to confirm the directly carcinogenic potential of acetaldehyde it would require a more thorough research. While some possible preliminary interventions could be used as reduction of acetaldehyde in beverages by improvements in production technology or the use of acetaldehyde-binding additives. A reestimation of the generally recognized as safe status of this compound is well necessary, which does not appear to be in agreement with its damage to human health. Conclusion and Future Trends During the past several decades, the role of acetaldehyde in Sherry wines has been extensively studied, mainly its microbial, chemical, and sensory relationship. In Sherry, acetaldehyde can be produced by different alternatives (fermentative yeasts, aging process, and acetic and lactic bacterial activities). Aging Sherry by using pure culture of flor yeasts wines could be done to obtain different sensory properties. Acetaldehyde is one of the more relevant compound in wines and one of the main responsible of the particular personality of Sherries with ripe apple odor descriptor. Acetaldehyde is considered as an indicator of aging degree because of biological, chemical, and other changes during this process and its sensory attributes. Based on consumer demand and the development of an innovative treatment of the musts destined for the production of Sherries, we believe that new studies about a functional Sherry should be considered. In our opinion, the future of this possible product depends on the explicit demonstration of efficacy in promoting health. Therefore, it is essential to work on the identification, quantification, and standardization of bioactive compounds; research on bioavailability and metabolism of functional compounds; and the investigation of safety facets associated to functional Sherry intake. In addition, it should be necessary to improve marketing efforts in regard to the functional properties of Sherry wine. Acknowledgments The authors acknowledge to Univ. of Córdoba (Spain) for material and financial support. Author Contributions Each author focused on a specific topic, performed an accurate search of papers, and wrote the relevant section. Conflict of Interest The authors declare no conflict of interest. References Adams MR, Moss M Food microbiology. Madison: Royal Society of Chemistry. Aguilera F, Valero E, Millán C, Mauricio JC, rtega JM Cellular fatty acid composition of two physiological races of Saccharomyces cerevisiae during fermentation and flor veil formation in biological aging of fine wines. Cerevisia 2: Amerine MA, ugh CS Studies with controlled fermentation. VIII. Factors affecting aldehyde accumulation. 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