Grape and Wine Metabolites: Biotechnological Approaches to Improve Wine Quality Grape and Wine Metabolites: Biotechnological Approaches to Improve Wine Quality

Grape metabolites can be affected by many extrinsic and intrinsic factors, such as grape variety, ripening stage, growing regions, vineyard management practices, and edaphoclimatic conditions. However, there is still much about the in vivo formation of grape metabolites that need to be investigated. The winemaking process also can create distinct wines. Nowadays, wine fermentations are driven mostly by single-strain inoculations, allowing greater control of fermentation. Pure cultures of selected yeast strains, mostly Saccharomyces cerevisiae , are added to grape must, leading to more predictable outcomes and decreasing the risk of spoilage. Besides yeasts, lactic acid bacteria also play an important role, in the final wine quality. Thus, this chapter attempts to present an overview of grape berry physiology and metabolome to provide a deep understanding of the primary and secondary metabolites accumulated in the grape berries and their potential impact in wine quality. In addition, biotechnological approaches for wine quality practiced during wine alcoholic and malolactic fermenta- tion will also be discussed.


Introduction
Grape berry chemical composition is complex, containing hundreds of compounds. Water (75-85%) is the main component followed by sugars and then organic acids. Other important compounds include amino acids, proteins, and phenolic compounds. Berry sugar composition has a key role in wine quality, since it determines alcohol content in wines [1]. Grape sugar, acidity, pH, and color are considered to mark harvest. Bouquet and flavor are related to the winemaker's expertise, stabilization, and storage processes, but primarily they are related to grape varietal character and its particular expression in a given terroir.
Nowadays, wine fermentations are driven mostly by single-strain inoculations, allowing greater fermentation control, leading to more predictable outcomes and decreasing the risk of spoilage by other yeasts [2]. During must fermentation, Saccharomyces cerevisiae produces a plethora of active-aroma secondary metabolites and releases many aroma compounds from inactive precursors present in grape juice, which significantly affect the sensory quality of the final wine [3,4]. Besides yeasts, lactic acid bacteria (LAB) are members of the normal microbiota that appears in all type of wines (white and red), and, therefore, they also play an important role in their final quality. Malolactic fermentation (MLF), a long-standing process of deacidification in winemaking carried by LAB, is a reaction of l-malic acid decarboxylation to l-lactic acid. Complex metabolic activities also occur, thus suggesting that MLF can positively or negatively affect the final wine quality [5,6].

Morphology and anatomy of grape berries
After successful pollination and fertilization of ovules within a flower berry development initiates [7]. The formation and growth of grape (Vitis vinifera) berries follows a double sigmoid pattern with three distinct phases [8]: I, rapid cell division and expansion in green berries; II or lag phase, in which cell expansion ceases; and III, in which growth is reinitiated and the fruit matures. The berry fruit comprises up to four seeds surrounded by the inner endocarp, the middle mesocarp, pulp or flesh, and the outer exocarp or skin [8,9] (Figure 1). The exocarp consisting of a cuticle-covered epidermis, which represents 5-18% of the fresh weight of the fruit [10] and several layers of underlying thick-walled cells of hypodermis, contains most of the skin flavonoids [11], notably anthocyanins in the outermost layers of the red grape varieties [8], interspersed with cells rich in needle-like crystals (raphides) [12]. Epidermis has non-photosynthetic cells with vacuoles containing large oil droplets [8]. Small berries have greater color, tannins, and flavor compounds than large berries because skin has a higher percentage of the total mass of small berries [7]. Scanning electron microscopy showed very few but functional stomata on young berries and wax-filled stomata on older berries [13], which accumulate polyphenolics and abnormally high concentrations of silicon and calcium in the peristomatal protuberances of up to 200 µm diameter [14].
At harvest, the cuticle of grape berry had an amorphous outer region and a mainly reticulate inner region [15]. During fruit development, the composition of the cuticular waxes changed, being oleanolic acid the main constituent, representing 50-80% of the total weight [16]. The soft wax was a mixture of long chain fatty acids (C 16 and C 18 fatty acid esters [17]), alcohols, aldehydes, esters, and hydrocarbons [18].
The mesocarp consists of thin-walled parenchyma [12]. The cells are round to ovoid and contain large vacuoles, which are the primary sites for the accumulation of sugars and phenolics [8], water, and organic acids [9] during grape berry ripening. According to Coombe [19], the translucent and hydrated mesocarp composes 85-87% of the berry's spherical volume. Altogether these make up 99.5% of the juice mass and hence are the major determinants of berry size and quality [9,20]. The remaining 0.5% of berry components are phenolics, terpenoids, lipids, cellulose, and pectin [20]. The endocarp consists of crystal-containing cells (druses) and an inner epidermis [12].
Grape seeds are contained in locules (Figure 1), and are composed of an outer seed coat, the endosperm, and the embryo [9]. As with most seeds, the endosperm comprises the bulk of the grape seed and serves to nourish the embryo during early growth. The normal or perfect number of seeds in the grape is four [9], but lack of ovule fertilization or ovule abortion reduces the number of developing seeds, generally resulting in smaller berry size [7]. Based upon recent molecular evidence, auxin is synthesized in the ovule and transported to the pericarp upon fertilization, where it induces gibberellin (GA) biosynthesis. The GA then degrades DELLA proteins that repress ovary growth and fruit initiation [21]. The size of mature berries at harvest is also a function of the number of cells divisions before and after flowering, extent of growth of these cells [22], and the extent of preharvest shrinkage [23].
High level of tannins is observed in the seed coat [9,11]. Similar to the tannins and phenols found in the flesh, these tannins also decline greatly on a per-berry basis after véraison [24].
Berry vascular tissue develops directly from that of the ovary. It consists primarily of a series of peripheral bundles that ramify throughout the outer circumference of the berry and axial bundles that extend directly up through the stem [8]. Grape berry is provided through the berry stem or pedicel by a vascular system composed of xylem and phloem vessels [25]. Water, minerals, hormones, and nutrients are transported from the root system throughout the vine by the xylem tissue [25]. Present evidence indicates that in the final stages of grape development, water movement through the xylem vessels decreases markedly [25]. But, it seems that the fruit is not hydraulically isolated from the parent grapevine by xylem occlusion then, rather, is "hydraulically buffered" by water delivered via the phloem [9]. Berry is also supplied by the phloem, which is the vasculature involved in photosynthate (sucrose) transport from the canopy to the vine [25].

Sugars
One of the main features of the grape-ripening process is the accumulation of sugars in the form of glucose and fructose within the cellular medium, specific in vacuole. In addition, sugar content is an important indicator often used to assess ripeness and to mark grape harvest. But, it is also possible to quantify small traces of sucrose in V. rotundifolia and hybrids between V. labrusca and V. vinifera grapevines [26]. Liu et al. [27] analyzed sugar concentration of 98 different grape cultivars and concluded that glucose (45.86-122.89 mg/mL) and fructose (47.64-131.04 mg/mL) were the predominant sugars in grape berries. During grape berry maturation, sucrose is produced in leaves by photosynthetic carbon assimilation and is transported to the berry in the phloem [24]. Sucrose is loaded into the phloem by either a symplastic or apoplastic mechanism [28]. However, it is at véraison that begins the sugar accumulation and the imported sucrose is converted into hexoses as a result of the activity of invertases [29].
Grape berries accumulate glucose and fructose in equal amounts at a relatively constant rate during ripening [29]. In addition, after véraison there is a considerable accumulation of glucose and fructose in the vacuoles of mesocarp cells, while 20 days after this period, the hexose content of the grape berry is close to 1 M, with a glucose/fructose ratio of 1 [19,30]. Grape sugar concentration and composition is mainly determined by several factors, such as genotype [26,31], vineyard management [32,33], and climatic conditions [34,35]. Moreover, in last years, as a result of climate change, there is a tendency for a sugar increase in grapes [36]. But, according to Mira de Orduña [35], the extremely high sugar levels reached at harvest today, especially in warm climates, may be rather associated with the desire to optimize technical or polyphenolic and/or aromatic maturity.

Organic acids and nitrogenous compounds
l-Tartaric and l-malic acids contribute to around 90% of the organic acid content in mature grapes [37,38]. Minor amounts of citric, succinic, lactic, and acetic acids are also present in ripened grapes [39]. Despite l-tartaric and l-malic acids having similar chemical structures, they are synthesized and degraded by evidently different metabolic pathways in the grape berries. l-Tartaric acid synthesis in grape berries occurs during the period of grape growth [19,40]. Tartaric acid pathway using l-ascorbic acid (vitamin C) is considered to be responsible for >95% of grape l-tartaric acid production [41]. l-Malic acid synthesis indicates that-carboxylation of pyruvate or of phosphoenol pyruvate is the most important pathway [42]. Accumulation of acids usually occurs at the beginning of berry development. The organic acid content increases up to véraison and then declines. The content of organic acids is determined by a balance between their synthesis and degradation. l-Tartaric acid was the most prominent acid from véraison until the fruits were fully mature. l-Malic acid content increased gradually until véraison, after which it decreased with fruit ripening [37]. Grape acid composition is influenced by many factors such as grape variety, environmental conditions, and cultural practices [43]. High malate-producing grape varieties have been identified, such as Carignane, Chardonnay, Grenache, Malbec, and Pinot Noir, as well as high tartrate-producing grape varieties such as Merlot, Semillon, Riesling, and Thompson Seedless [44]. Temperature is a key factor in the rate of l-malic acid degradation during the berries ripening; with low temperatures, higher concentration of l-malic acid was observed [43]. l-Tartaric acid is presumed to be more stable when exposed to higher temperature, being the slight decreases during ripening due to dilution from berry expansion [45,46].
Grapes nitrogenous compounds include ammonium cations and organic nitrogenous compounds such as amino acids, hexose amines, peptides, nucleic acids, and proteins. As maturation happens, organic nitrogen progressively increases while ammonia slightly declines. The synthesis of amino acids, peptides, and protein occurs during the last 6-8 weeks of berry ripening [47]. In grapes, the main free amino acids include proline (up to 2 g/L), arginine (up to 1.6 g/L), and to a lesser extent, alanine, aspartic acid, and glutamic acid [48]. However, compositional differences in amino acids were observed by Stines et al. [49] among grape varieties, proline and arginine always being the major grape amino acids. In all grape varieties, most of the proline accumulation happened late in ripening, nearby 4 weeks of post-véraison. In opposite, arginine accumulation started before véraison and continued to maturity, excluding grape varieties in which a great level of proline accumulated [49]. The variation of amino acid profile and their concentration in grapes depends on grape variety, but also on viticultural management and environmental conditions [43,50,51].
According to Hsu and Heatherbell [52], grapes contain naturally a wide range of different proteins, up to 41 protein fractions with molecular mass ranging from 11.2 to 190 kDa and isoelectric point from 2.5 to 8.7 [53,54]. Soluble proteins in grape are globular proteins, mainly albumins [55,56]. There is a significant increase in grape total protein content after véraison being a small content of proteins synthesized significantly during grape ripening [55,57]. The most abundant grape proteins synthesized during ripening are pathogenesis-related proteins, including chitinases (32 kDa) and thaumatin-like proteins (24 kDa) [29,57,58].

Aroma and flavor compounds
Free and bound terpene grape content has been used to measure berry flavorant development and potential. Numerous types of flavorants existed in the form of glycosidic precursors. Analysis of the total precursor content by assessment of the glycoside glucose (GG) content of the grapes may yield a more complete depiction of the grape flavorant potential [59]. During grape maturity, changes in the concentration and diversity of aroma precursors and volatile compounds occurred [60,61]. Lacey et al. [60] observed that grapes grown under cool temperatures showed higher grape methoxypyrazine concentration than grapes grown under hot temperatures. Grape methoxypyrazine levels were relatively high at véraison but decreased markedly with grape ripening. However, since grape maturation is genetically controlled, it is considerably influenced by environmental conditions [60].

Phenolic compounds
Phenolic compounds are very important for wine quality because they are responsible for most of the wine sensory characteristics, particularly color and astringency. These groups of compounds constitute a diverse group of secondary metabolites that exist in grapes, mainly in the grape berries' skins and seeds [62] and also in grape stems [63]. The phenolic compounds in V. vinifera grapes include two classes of phenolic compounds: non-flavonoids and flavonoids. The non-flavonoid compounds include phenolic acids divided into hydroxybenzoic acids and hydroxycinnamic acids, but also other phenol derivatives such as stilbenes ( Figure  2). Non-flavonoids incorporate C 6 -C 3 hydroxycinnmates acids, C 6 -C 1 hydroxybenzoic acids, and C 6 -C 3 -C 6 stilbenes trans-resveratrol, cis-resveratrol, and trans-resveratrol glucoside. For flavonoid compounds, there are a large number of subclasses, such as flavonols, flavanols, and anthocyanins [64]. Flavonols are the most abundant phenolic compounds in grape skins [65], while grape seeds are rich in flavan-3-ols [66]. Flavonoids are characterized by a basic structure of 15 carbon atoms comprising two aromatic rings bound through a three carbon chain (C 6 -C 3 -C 6 ). The major C 6 -C 3 -C 6 flavonoids in grapes include conjugates of flavonols quercetin, and myricetin; flavan-3-ols (+)-catechin and (−)-epicatechin; and malvidin-3-Oglucoside and other anthocyanins (Figure 3a-c).
The quantification of phenolic acids, stilbenes, monomeric anthocyanins, flavan-3-ols, and proanthocyanidins in red grape varieties is summarized in Tables 1-3 and the quantification of phenolic acids, stilbenes, flavan-3-ols, and proanthocyanidins in white grape varieties is summarized in Table 4

Biotechnological approaches for wine quality
More than 800 volatile compounds have been identified in wines, with a concentration range from hundreds of mg/L to the µg/L or ng/L [90]. The wine bouquet is formed by secondary metabolites synthesized by an extensive range of microbial species (yeasts and bacteria). Wine alcoholic fermentation (AF) is the key for innovation or creation of biotechnology that will change the expanding market [91] (Figure 4). In addition to yeasts, LAB also appears in all type of wines, being responsible for MLF that normally occurs after AF but may also occur simultaneously [92]. During the winemaking process, indigenous populations of LAB vary quantitatively and qualitatively [93], through a succession of species and strains before, during and after the AF [94]. After a phase of latency, the surviving cells begin to multiply and entering the exponential growth phase, reaching populations from 10 6 to 10 8 cfu/mL, almost exclusively, constituted by strains of Oenococcus oeni, species that dominate this stage and performs the MLF. Normally, a great diversity of strains of Oenococcus oeni at the beginning of the MLF is detected, while at the end only one or two predominate [95].

Yeasts metabolites: the imperceptible search of perfection
Wine yeasts contribute to wine aroma by a number of mechanisms: (i) they utilize grape juice constituents and transform them into flavor-impacting components, then (ii) they produce enzymes capable to transform neutral grape compounds into flavor-active compounds, and finally (iii) they can synthesize many flavor-active compounds such as primary and secondary metabolites [96].
Esters, in wine, are mainly originated from yeast metabolism during AF. But, some esters are also found in grape berry [97], where they occur in small amounts, contributing to the aroma of V. vinifera varieties [98]. Esters are formed via an intracellular process, catalyzed by an acyl transferase or ester synthase [99]. The concentration of esters usually found in wine is mostly well above their sensory threshold levels. Fruity and floral terms in Chardonnay wines were related to 2-phenylethyl acetate, as a rose-like/honey aroma [100] ( These esters also appear in higher levels in wines after bio-reduction (deacidification) of wine's volatile acidity [102]. A study of overexpression S. cerevisiae alcohol acetyltransferases genes, ATF1p, ATF2p, and Lg-ATF1p, was performed by Verstrepen et al. [103]. Analysis of the fermentation products confirmed that the expression levels of ATF1 and ATF2 greatly affected the production of ethyl acetate and isoamyl acetate. But, factors such as oxygen and temperature that allow ester and higher alcohol synthesis must be monitored during AF [104]. Hexanoic acid Sweaty, cheesenotes 420-3000 Guth [115]; Ferreira et al. [118] Octanoic acid Grass acid-like 500-8800 Etievant [116]; Ferreira et al. [118] Decanoic acid Soapy 1000-15,000 Guth [115]; Ferreira et al. [118]  Ethanol and glycerol are quantitatively the largest group of alcohols found in wine. Both contribute to the textural aspects of wines [1]. The search of yeast that can impart specific desirable characteristics to wines led to investigations such as the production of optimal levels of glycerol (the overexpression of GPD1, GPD2, and FPS1, together with the deletion of the ALD6 acetaldehyde dehydrogenase gene) [105].
Medium-chain fatty acids and their ethyl esters are natural components of alcoholic beverages. Fatty acids (butyric, isovaleric, hexanoic, octanoic, and decanoic acids, among others; Table  5) are produced by yeasts as intermediates in the biosynthesis of long-chain fatty acids, important components of yeast membrane [106]. Their aroma goes from vinegar to pungent, rancid, and soapy, sweetie, fruit and butter [106] ( Table 5). One of the major problematic volatile acids is acetic acid. It can be formed as a by-product of AF, MLF, or as a product of the metabolism of acetic bacteria. Acetic acid affects the quality of certain types of wine when it is present above a given concentration [107] due to its unpleasant vinegar aroma.
Terpenes are one of the major grape components that contribute to wine aroma. This is especially valid to wines of Gewürztraminer and Muscat varieties, but these flavor compounds are also present in other grape varieties, where they supplement other varietal flavors and aromas. They are present in two forms: a free volatile and a non-volatile sugar-conjugated [108]. Geraniol (geranium flowers aroma) and linalool (rose or lavender-like aroma) are considered to be the most important of the monoterpene alcohols as they are present in higher levels and have lower perception thresholds than other major wine monoterpenes [109]. Monoterpenes can be released from their glycosides either by acid or by enzymatic hydrolysis. Hydrolysis during winemaking is caused by grape [110] or microorganisms enzymes taking part in the process [111]. In the yeasts that were selected in the past years, glycosidase activities have been used for the hydrolysis of glycoconjugated aromatic precursors in order to enhance wine sensorial quality [112]. Fungi are considered a promising genetic source for commercial production of recombinant β-glucosidase [113]. In a work by Zietsman et al. [114], an yeast strain (S. cerevisiae VIN13) was built to express and secrete the Aspergillus awamori encoding a B-type α-l-arabinofuranosidase (AwAbfB) in combination with either the β-glucosidases BGL2 from Saccharomycopsis fibuligera or the BGLA from Aspergillus kawachii. Coexpression of AwAbfB and BGL2 in VIN13 increased free monoterpenes in wines. Panelists confirmed wine aroma profile improvement, mainly in floral character [114]. Recently, Pardo et al. [109] found that the expression of Ocimum basilicum (sweet basil) geraniol synthase (GES) gene in an S. cerevisiae wine strain greatly changed terpene profile of wine made from a non-aromatic grape variety.

Lactic acid bacteria metabolites: beyond malolactic fermentation
The complexity and diversity of LAB metabolic activities in wine illustrates that MLF is more than a mere decarboxylation of l-malic acid into l-lactic acid, and it may affect positively and/ or negatively the quality of wine [123] ( Table 6). Besides to the decrease in acidity, MLF also improves sensorial characteristics and increases wines microbiological stability that undergone this important second fermentation [124,125].
Aromatic modifications are due to l-lactic acid, less aggressive, and due to the increase of a number of other compounds such as diacetyl, acetoin, 2,3-butanediol, esters mainly ethyl lactate and diethyl succinate, and some higher alcohols and aromatic aglycones released by the action of β-glucosidases [126][127][128]. Sumby et al. [129] have verified the impact that different strains of O. oeni had on wine aroma and related that to their ester hydrolysis and synthesis abilities. For the aromatic complexity of wines, the production of volatile sulfur compounds, particularly 3-methylsulfanyl-propionic acid with chocolate and toasted odors [130], and the activity of taninoacil hydrolase enzyme, commonly termed tannase, reducing wine astringency and turbidity [131], also contribute. Concerning to negative effects on wine quality, LAB may be responsible for the formation of ethyl carbamate by the degradation of arginine [124] and for the formation of biogenic amines such as histamine, tyramine, and putrescine by the degradation of precursor amino acids [132,133]. Also, although less frequent nowadays, bitterness by acrolein formation from glycerol degradation [134], butter aroma due to excessive production of diacetyl [135], flocculent growth [136], mannitol taint [137], ropiness [138], tartaric acid degradation [137], mousy offodor by acetamide production of tetrahydropyridines [139], the geranium off-odor [140], and the formation of 4-ethilguaiacol and 4-ethylphenol volatile phenols [141,142] are spoilage phenomenons that may occur after malolactic fermentation. Nevertheless, it is thought that the time between the completion of alcoholic fermentation and the start of malolactic fermentation is the most likely time that Brettanomyces multiplies and produces "Brett character," 4ethylphenol of flavor, in wine [143].
As what happens to other food products, some researchers defend the use of autochthones LAB strains, more adapted and efficient to regional vinification conditions, for keeping the typicity of wines, instead of using universal ones that may impart similar characteristics and thus leading to final products that are too similar and also for preserving the local microbial biodiversity [144,145]. According to Marcobal and Mills [146], the knowledge of some wine LAB whole genome, including the PSU1 O. oeni strain, allows deeper phylogenetic analyses and their relation with key pathways involved in carbon and nitrogen metabolism, which will foster modeling of O. oeni growth and metabolism in order to predict optimum strategies for efficiently performing the MLF with a desired flavor outcome.

Composition of grapes and wines: new analytical techniques
Several different analytical approaches are increasingly used to profile the volatile, nonvolatile, and elemental composition of grapes and wines (see recent reviews, e.g., [158,160]).
According to a review made by Ebeler [159], we can group these analytical approaches in (i) targeted analysis of compounds, (ii) non-targeted analysis and profiling of metabolites, (iii) elemental analysis, and (iv) relating chemical composition and sensory attributes ( Table 7).
Therefore, wine composition and hence wine origin are possible by combining several analytical techniques ( Table 7) that offer significant advantages for trace quantification of important aroma-active volatiles [174], [175] and taint compounds [163]. It is also possible to comprehensively profile metals [178], including those that affect chemical stability and oxidative reactions, and to characterize aroma qualities of complex mixtures [182]. Each of these tools, alone and in combination, is providing significant new insights into variables influencing grape and wine composition and flavor. Moreover, concerning to specific grape compounds, in past years, several methodologies were also developed focused on the identification, quantification, and also in extraction techniques. For example for phenolic compounds, substantial developments for individual phenolic analysis, such as benzoic and cinnamic acid, coumarins, tannins, lignins, lignans, and flavonoids, have occurred over the last 25 years. Thus, several extraction techniques have been employed namely for grape phenolic compounds, such as ultrasounds and microwaves [183], supercritical fluid extraction [184], subcritical water extraction [185], high hydrostatic pressure extraction [186], pulsed electric fields [187], and enzymatic treatment [188].

Final remarks
The study of the grape berry physiology and metabolome will provide a deep understanding of the primary metabolites including sugars, organic acids and amino acids, and some secondary metabolites accumulated in the grape berries such as phenolic compounds. This issue is of particular importance for viticulturists and oenologists in order to know how grape composition could affect wine quality. In addition, biotechnological approaches for wine quality, practiced during wine AF and MLF, are also a promising tool available for oenologists that improve wine quality, namely, their sensorial value.