Open access peer-reviewed chapter

Grape and Wine Metabolites: Biotechnological Approaches to Improve Wine Quality

By Fernanda Cosme, Berta Gonçalves, António Inês, António M. Jordão and Alice Vilela

Submitted: November 2nd 2015Reviewed: July 6th 2016Published: October 19th 2016

DOI: 10.5772/64822

Downloaded: 1391

Abstract

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 fermentation will also be discussed.

Keywords

  • grape physiology
  • grape metabolites
  • wine biotechnology
  • alcoholic fermentation
  • malolactic fermentation
  • microbial metabolites

1. 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 ofl-malic acid decarboxylation tol-lactic acid. Complex metabolic activities also occur, thus suggesting that MLF can positively or negatively affect the final wine quality [5, 6].

2. Grape berry physiology and metabolome

2.1. 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).

Figure 1.

Structure of a ripe grape berry. Illustrated by Sílvia Afonso.

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 (C16 and C18 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].

2.2. Grape primary and secondary metabolites

2.2.1. 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.

2.2.2. Organic acids and nitrogenous compounds

l-Tartaric andl-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]. Despitel-tartaric andl-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 usingl-ascorbic acid (vitamin C) is considered to be responsible for >95% of grapel-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 ofl-malic acid degradation during the berries ripening; with low temperatures, higher concentration ofl-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].

2.2.3. 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].

2.2.4. 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 C6-C3 hydroxycinnmates acids, C6-C1 hydroxybenzoic acids, and C6-C3-C6 stilbenes trans-resveratrol, cis-resveratrol, and trans-resveratrol glucoside.

Figure 2.

Main non-flavonoid compounds found in V. vinifera grapes.

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 (C6-C3-C6). The major C6-C3-C6 flavonoids in grapes include conjugates of flavonols quercetin, and myricetin; flavan-3-ols (+)-catechin and (−)-epicatechin; and malvidin-3-O-glucoside and other anthocyanins (Figure 3ac).

According to Pastrana-Bonilla et al. [67], the average concentration of the total phenolic compounds in different grape fractions varied from 2178.8 mg/g gallic acid equivalent in seeds to 374.6 mg/g gallic acid equivalent in skins. In addition, it is also possible to found low concentrations of phenolic compounds in pulps (23.8 mg/g gallic acid equivalent).

Figure 3.

(a) Main flavonoid compounds (anthocyanidins) found in V. vinifera grape varieties. (b) Main flavonoid compounds (flavan-3-ols and procyanidins) found in V. vinifera grape varieties. (c) Main flavonoid compounds (flavonols) found in V. vinifera grape varieties.

In general, the phenolic composition of grapes is influenced by different factors, such as grape variety [68, 69], sunlight exposition [70], solar radiation [71] altitude [72], soil composition [73], climate [70, 7476], cultivation practices [43, 74], exposure to diseases [77], and the degree of grape ripeness [63, 69].

The quantification of phenolic acids, stilbenes, monomeric anthocyanins, flavan-3-ols, and proanthocyanidins in red grape varieties is summarized in Tables 13 and the quantification of phenolic acids, stilbenes, flavan-3-ols, and proanthocyanidins in white grape varieties is summarized in Table 4.

Phenolic compoundsGrape varietyConcentrationReferences
Phenolic acidsNegroamaro7.3a
Gallic acidSusumaniello45.0aNicoletti et al. [78]
Malvasia Nera77.3a
Aglianico151.9a
Merlot66.6a
Carménère2.8bObreque-Slier et al. [79]
Cabernet Sauvignon3.5b
Merlot9.8cMontealegre et al. [80]
Cencibel7.3c
Cabernet Sauvignon9.0c
Shiraz6.8c
Protocatechuic acidNegroamaro42.0aNicoletti et al. [78]
Susumaniello8.5a
Malvasia Nera46.0a
Aglianico37.4a
Cesanese31.1a
Merlot328.7a
Cencibel1.5bMontealegre et al. [80]
Cabernet Sauvignon2.4b
Merlot1.7b
Shiraz2.4b
Merlot8.7cMontealegre et al. [80]
Cencibel3.3c
Cabernet Sauvignon7.1c
Shiraz6.2c
Caftaric acidPrimitivo1.89aNicoletti et al. [78]
Negroamaro8.5a
Susumaniello171.7a
Malvasia Nera171.9a
Aglianico320.4a
Cesanese28.8a
Alphonse645.0a
Merlot746.3a
Carménère0.6bObreque-Slier et al. [79]
Cabernet Sauvignon0.7b
StilbenesPrimitivo30.7aNicoletti et al. [78]
Trans-piceidNegroamaro4.14a
Susumaniello150.3a
Uva di Troia15.3a
Malvasia Nera98.0a
Aglianico75.7a
Cesanese12.0a
Merlot26.3a
Alphonse Lavallée24.1a
Castelão67.24cSun et al. [81]
Syrah10.43c
Tinta Roriz11.57c
Trans-resveratrolPrimitivo13.9aNicoletti et al. [78]
Negroamaro3.6a
Susumaniello63.0a
Uva di Troia4.6a
Malvasia Nera48.5a
Aglianico61.1a
Cesanese8.1a
Merlot9.2a
Alphonse Lavallée40.0a
Blauer Burgunder0.5dMikeš et al. [82]
Lemberger0.3d
Saint Laurent1.0d
Saint Laurent2.3dBalík et al. [83]
Blauer Portugieser0.4d
Andre0.4d
Castelão6.8dSun et al. [81]

Table 1.

Quantification of phenolic acids and stilbenes in red grape varieties.

amg/kg of berry dry weight.

bmg/kg of fresh grape skin.

cmg/kg of fresh grape seed.

dmg/kg dry skin.

Monomeric anthocyaninsGrape varietyConcentrationReferences
Delphinidin 3-O-glucosideCabernet-Sauvignon431.6aOrtega-Regules et al. [84]
Merlot231.7a
Syrah258.0a
Cabernet Sauvignon4.67bRevilla et al. [85]
Garnacha2.26b
Graciano6.81b
Mencia5.13b
Merlot7.53b
Tempranillo10.9b
Castelão Francês6.2cJordão et al. [86]
Touriga Francesa0.9c
Cyanidin 3-O-glucosideCabernet-Sauvignon53.1aOrtega-Regules et al. [84]
Merlot48.2a
Syrah27.9a
Cabernet Sauvignon0.90bRevilla et al. [85]
Garnacha1.02b
Graciano1.28b
Mencia2.15b
Merlot5.52b
Tempranillo3.26b
Castelão Francês2.6cJordão et al. [86]
Touriga Francesa0.1c
Petunidin-3-O-glucosideCabernet-Sauvignon337.4cOrtega-Regules et al. [84]
Merlot270.9a
Syrah385.2a
Cabernet Sauvignon4.21bRevilla et al. [85]
Garnacha3.73b
Graciano7.21b
Mencia6.68b
Merlot7.0b
Tempranillo11.11b
Castelão Francês8.5cJordão et al. [86]
Touriga Francesa2.5c
Peonidin 3-O-glucosideCabernet-Sauvignon259.5aOrtega-Regules et al. [84]
Merlot381.9a
Syrah299.2a
Cabernet Sauvignon4.87bRevilla et al. [85]
Garnacha12.69b
Graciano12.79b
Mencia14.85b
Merlot14.27b
Tempranillo7.81b
Castelão Francês11.7cJordão et al. [86]
Touriga Francesa3.6c
Cabernet-Sauvignon2506.3aOrtega-Regules et al. [84]
Malvidin 3-O-glucosideMerlot1834.7a
Syrah2889.7a
Cabernet Sauvignon41.45bRevilla et al. [85]
Garnacha64.69b
Graciano53.69b
Mencia47.40b
Merlot35.54b
Tempranillo46.35b
Castelão Francês59.2cJordão et al. [86]
Touriga Francesa46.3c

Table 2.

Quantification of monomeric anthocyanins in red grape varieties.

aμg/g grape skin.

bRelative amount of anthocyanidins (%).

c% weight of anthocyanins/weight grape.

Phenolic compoundsGrape varietyConcentrationReferences
Flavan-3-olsBaboso Negro51.61a
(+)-CatechinListán Negro54.25aPérez-Trujillo et al. [87]
Negramoll51.31a
Tintilla50.10a
Vijariego Negro49.09a
Touriga Nacional0.012–0.021bMateus et al. [88]
Touriga Francesa0.012b
Merlot240.0cMontealegre et al. [80]
Cencibel82.0c
Cabernet Sauvignon270.0c
Shiraz120.0c
(−)-EpicatechinBaboso Negro16.50aPérez-Trujillo et al. [87]
Listán Negro13.77a
Negramoll15.07a
Tintilla20.55a
Vijariego Negro16.13a
Touriga Francesa0.010bMateus et al. [88]
Merlot210.0cMontealegre et al. [80]
Cencibel60.0c
Cabernet Sauvignon130.0c
Shiraz130.0c
ProanthocyanidinsTouriga Nacional0.013bMateus et al. [88]
Procyanidin B3Merlot64.0cMontealegre et al. [80]
Cencibel43.0c
Cabernet Sauvignon50.0c
Shiraz55.0c
Procyanidin B1Baboso Negro15.95aPérez-Trujillo et al. [87]
Listán Negro15.00a
Negramoll14.69a
Tintilla13.64a
Vijariego Negro13.39a
Touriga Nacional0.184–0.260bMateus et al. [88]
Touriga Francesa0.090–0.138b
Merlot170.0cMontealegre et al. [80]
Cencibel74.0c
Cabernet Sauvignon150.0c
Shiraz100.0c
Procyanidin B4Merlot80.0cMontealegre et al. [80]
Cencibel39.0c
Cabernet Sauvignon57.0c
Shiraz33.0c
Procyanidin B2Baboso Negro10.39aPérez-Trujillo et al. [87]
Listán Negro5.74a
Negramoll7.55a
Tintilla9.92a
Vijariego Negro7.44a
Touriga Nacional0.020bMateus et al. [88]
Touriga Francesa0.011–0.015b
Merlot37cMontealegre et al. [80]
Cencibel21.0c
Cabernet Sauvignon41.0c
Shiraz23.0c

Table 3.

Quantification of flavan-3-ols and proanthocyanidins in red grape varieties.

aMolar percentages.

bmg/g dry weight.

cmg/kg of fresh grape seed.

Phenolic compoundsGrape varietyConcentrationReferences
Phenolic acidsGrüner Veltliner3.9a
Gallic acidHibernal4.0aMikeš et al. [82]
Malverina3.5a
Müller Thurgau2.6a
Rheinriesling2.1a
Welschriesling1.8a
Neuburger3.9a
Protocatechuic acidChardonnay4.8bMontealegre et al. [80]
Sauvignon Blanc4.4b
Moscatel3.6b
Gewürztraminer6.0b
Caftaric acidMoscato48.4cNicoletti et al. [78]
StilbenesChardonnay1.1aBalík et al. [83]
Trans-piceidWelschriesling0.4a
Pinot Gris0.6a
Trans-resveratrolMoscato3.89cNicoletti et al. [78]
Grüner Veltliner0.1aMikeš et al. [82]
Hibernal0.3a
Malverina0.3a
Müller Thurgau0.3a
Rheinriesling0.2a
Welschriesling0.5a
Neuburger1.5a
Chardonnay0.3b
Welschriesling1.6bBalík et al. [83]
Pinot Gris1.1b
Flavan-3-olsChardonnay123a
(+)-CatechinWelschriesling61.0aBalík et al. [83]
Pinot Gris481a
Ugni blanc2.6–222.0dDe Freitas and Glories [89]
Sémillon12–35.2d
Chardonnay390.0cMontealegre et al. [80]
Sauvignon Blanc200.1c
Moscatel350.0c
Gewürztraminer500.0c
Riesling400.0c
Viogner120.0c
(−)-EpicatechinChardonnay144aBalík et al. [83]
Welschriesling84.3a
Pinot Gris251a
Ugni blanc0.04–3.0dDe Freitas and Glories [89]
Sémillon0.03–1.6d
Chardonnay310.0cMontealegre et al. [80]
Sauvignon Blanc130.0c
Moscatel120.0c
Gewürztraminer150.0c
Riesling160.0c
Viogner110.0c
ProanthocyanidinsUgni blanc0.2–0.3dDe Freitas and Glories [89]
Procyanidin B3Sémillon0.01–0.2d
Chardonnay52.0cMontealegre et al. [80]
Sauvignon Blanc52.0c
Moscatel39.0c
Gewürztraminer56.0c
Riesling43.0c
Viogner51.0c
Procyanidin B1Ugni blanc1.1–1.9dDe Freitas and Glories [89]
Sémillon0.02–0.4d
Chardonnay380.0cMontealegre et al. [80]
Sauvignon Blanc250.0c
Moscatel330.1c
Gewürztraminer460.0c
Riesling620.0c
Viogner200.0c
Procyanidin B4Ugni blanc0.04dDe Freitas and Glories [89]
Chardonnay71.5cMontealegre et al. [80]
Sauvignon Blanc54.0c
Moscatel40.0c
Gewürztraminer70.0c
Riesling95.0c
Viogner53.0c
Procyanidin B2Ugni blanc0.06–0.2dDe Freitas and Glories [89]
Chardonnay33.0cMontealegre et al. [80]
Sauvignon Blanc19.0c
Moscatel15.0c
Gewürztraminer22.0c
Riesling33.0c
Viogner19.0c

Table 4.

Quantification of phenolic acids, stilbenes, flavan-3-ols, and proanthocyanidins in white grape varieties.

amg/kg fresh grape weight.

bmg/kg of fresh grape seed.

cmg/kg of berry dry weight.

dmg/g dry weight.

3. 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).

Figure 4.

Grape juice is converted into wine by the action of wine yeast and bacteria during alcoholic and malolactic fermentations. Some wine components are wholly generated by these microorganisms as part of metabolism, while others are essentially synthesized by the grapevine. Wine quality and style is determined by the quality and quantity of compounds produced or modified by must/wine microflora.

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 106 to 108 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].

3.1. 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] (Table 5). In red wines, ethyl butyrate (pineapple aroma), ethyl 2-methylbutyrate (sweet, floral, fruity, and apple), ethyl 3-methylbutyrate (strawberry, ethereal, buttery, and ripe), isoamyl acetate (banana-like aroma), ethyl hexanoate (anise seed, apple, or pineapple aroma), and ethyl octanoate (sweet, cognac, and apricot aroma) made a main contribution to the fruity character of wines [101] (Table 5). 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].

Compounds Odor description Det. Threshold
(µg/L) 
References 
Isoamyl acetateBanana30Guth [115]
2-PhenylethylacetateRoses, honey250Guth [115]
EthylpropionateEthereal, fruity, rum-like1800Etievant [116]
EthylisobutyrateStrawberry, ethereal, buttery,
ripe
15Etievant [116]; Ong and Acree [117]
Ethyl butyratePineapple20Guth [115]
Ethyl 2-methylbutyrate Sweet, floral, fruity, apple1–18Guth [115]; Ferreira et al. [118]
EthylisovalerateFruity3Ferreira et al. [118]
Ethyl hexanoateAnise seed, apple, pineapple5–14Guth [115]; Ferreira et al. [118]
Ethyl octanoateSweet, cognac, apricot2–5Guth [115]; Ferreira et al. [118]
DiethylsuccinateFruity, melon1200Peinado et al. [119]
AcetaldehydeGrass, green, apple, sherry100,000Carlton et al. [120]
BenzaldehydeAlmond3500Delfini et al. [121]
LinaloolRose, lavender25Ferreira et al. [118]
α-TerpineolLily of the valley300Mateo and Jiménez [122]
CitronellolCitronella100Guth [115]
GeraniolRose-like; geranium flowers~75Pardo et al. [109]
2-phenylethanolRoses10,000Guth [115]
Isoamyl alcoholMarzipan, burnt, whisky
-like
30,000Guth [115]
Butyric acidRancid, cheese173Ferreira et al. [118]
Isovaleric acidRancid, sweaty33.4Ferreira et al. [118]
Hexanoic acidSweaty, cheesenotes420–3000Guth [115]; Ferreira et al. [118]
Octanoic acidGrass acid- like500–8800Etievant [116]; Ferreira et al. [118]
Decanoic acidSoapy1000–15,000Guth [115]; Ferreira et al. [118]

Table 5.

Major wine-yeast aromatic compounds, odor description, and detection thresholds in white and red wines.

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.

3.2. 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 ofl-malic acid intol-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 tol-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 [126128]. 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.

CompoundsOdor descriptionDet. threshold (µg/L)References
4-EthylguaiacolBacon, spice, clove, or smoky aromas33Dai et al. [26]; Bartowsky [123]
4-EthylphenolHorse and barnyard odor440Barthelmebs et al. [147], [148]
Tetrahydropyridines Mousy off-odor60Swiegers et al. [149];
Harrison and Dake [150]
3- Methylsulfanyl-
propionic acid
Chocolate and toasted odors244Pripis-Nicolau et al. [151]
Ethyl lactateLactic, raspberry154–636Ferreira et al. [118]; Bartowsky [152]
Diethyl succinateFruity, melon1200Peinado et al. [119]; Bartowsky [152]
DiacetylButter200–2800Martineau and Henick-Kling [153]; Bartowsky and Henschke [154]
AcetoinNo negative organoleptic influence. Unpleasant buttery flavor at concentrations higher than threshold150Swiegers et al. [155]; Ehsani et al. [156]
2,3-ButanediolNeutral sensory qualities150Swiegers et al. [155];
Romano and Suzzi [157]

Table 6.

Major LAB aromatic compounds, odor description, and detection thresholds in wine.

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 off-odor 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,” 4-ethylphenol 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.

4. Composition of grapes and wines: new analytical techniques

Several different analytical approaches are increasingly used to profile the volatile, non-volatile, 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].

Analytical approachesAnalytical techniquesExamples and references
Targeted analysis of compounds (i)Selected ion monitoring and tandem mass spectrometric, MS/MS or MSnAnalysis of trace analytes, with important sensory properties—Ebeler [160] and Robinson et al. [161, 162]—such as 2,4,6-trichloroanisole (TCA)—Hjelmeland et al. [163]
Combination of liquid chromatography, LC with mass spectrometry, MS. MS/MS is the combination of two mass analyzers
in one mass spectrometry instrument, LC-MS/MS/LC-MS/MS.
Supercritical fluid chromatography (SFC)
Smoke-derived volatile phenols—guaiacol and their glycoside precursors, and anthocyanins from grapes and wines—Kennison et al. [164166], Hayasaka et al. [167], and Pati et al. [168].
Polyphenols from grape seed extracts—Kamangerpour et al. [169]
Non-targeted
analysis and
profiling of metabolites (ii)
Ultra-high performance liquid chromatography, UHPLC wish operates in the 20,000 psi range, combined with quadrupole time-of-flight mass spectrometry, qTOF and UHPLC-qTOF-MSVarietal classification of wines—Vaclavik et al. [170] and Flamini [171]
Ion cyclotron resonance mass spectrometry, ICR-MSCharacterization of Pinot Noir grapes and wines and chemodiversity comparison of different appellations: Vintage vs terroir effects—Roullier-Gall et al. [172, 173]
Gas chromatography combined with time-of-flight mass spectrometry, GC GC-TOF-MSIdentification of over 350 volatile compounds in Australian Cabernet Sauvignon wines—Robinson et al. [174, 175]
Nuclear Magnetic Resonance, NMR1H NMR metabolite profiling to relate chemical composition to sensory perception of body and mouthfeel of white wines—Kogerson et al. [176]
Elemental analysis
(iii)
Inductively coupled plasma mass Spectrometry, ICP-MSRelating elemental composition of wines to the vineyard that the grapes were grown or in wish winery they were made—Hopfer et al. [177].
Leaching of metals from stainless steel containers and from closures—Hopfer et al. [178]
Relating chemical composition and sensory attributes
(iv)
Categorical principal components analysis, CATPCA; principal components analysis, PCA and
partial least squares analysis, PLS
One or more compounds that correlate with specific aroma or flavor attributes—Polaskova et al. [179] and development of a flavor lexicon using new statistical nonparametric approaches—Vilela et al. [180] and Monteiro et al. [181]
In-instrument gas chromatography recomposition-olfactometry, GC-ROPerceptual characterization and analysis of aroma mixtures—Johnson et al. [182]

Table 7.

Analytical approaches, analytical techniques used to profile the volatile, non-volatile and elemental composition of grapes and wines.

5. 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.

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

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Fernanda Cosme, Berta Gonçalves, António Inês, António M. Jordão and Alice Vilela (October 19th 2016). Grape and Wine Metabolites: Biotechnological Approaches to Improve Wine Quality, Grape and Wine Biotechnology, Antonio Morata and Iris Loira, IntechOpen, DOI: 10.5772/64822. Available from:

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