**10. Enzymes for grape juice and wine production**

Grape (*Vitis vinifera*) production can be found on every continent except Antarctica because the vine plant is able to adapt to a wide range of environments, and because of the increasing importance of fermentation-based industries in the world economy. The grape berry has three major types of structures (**Figure 9**): pulp (80–85% of total weight), skin (7–11%), and seed (2–6%) [60], with the sheer bulk of juice being derived from the pulp, and remarkably, aromas and red grape juice colour is derived from the skin. Enological enzymes can be used at several stages in juice and wine processing and their application in the wine industry started in the 70s [61]. The main stages for winemaking are: a) pre-fermentation processing, b)

**Figure 8.** *Effect of 10 U PGzyme /ml at 40°C on the Hard-to-cook beans (HTCB) during soaking and cooking time [59].*

**Figure 9.** *Grape berry diagram and tissue organisation.*

fermentation, c) post-fermentation processing, and ageing. Enzymes can be used in the first and third stages to improve: juice extraction, clarification, filtration, colloidal stability (by preventing haze from forming later in processing), aroma extraction, and in the case of red grapes, to improve colour extraction.

#### *Fungal Pectinases in Food Technology DOI: http://dx.doi.org/10.5772/intechopen.100910*

Commercial pectolytic enzyme preparations commercialised for grape juice and wine production are actually complex mixtures of several enzymes that degrade the cell walls and middle lamella of grape tissues, as well as polysaccharide colloids of the produced grape juices. The main enzyme activity of commercial preparations is polygalacturonase (*exo*- and *endo*-PG) accompanied with pectin lyase (PNL) [62]. In addition, these commercial preparations contain other enzymes that act in synergy with the former activities, among which, cellulases, hemicellulases, and other glycosidases (α-L-rhamnosidase, α-L-arabinofuranosidase, β-D-glucosidase) potentiate the lytic effect of these commercial enzyme preparations. When grape berries are infected by the mould *Botrytis cinerea* (grey rot or "pourriture grise") it is recommended to use specific pectolytic preparations that also contain *exo* β-(1 ! 3) glucanases and β-(1 ! 6) glucanases that degrade the β-D-glucan macromolecules produced by the mould and responsible for grape juice and wine spoilage [63]. Currently, commercial pectolytic enzyme preparations for enological use come from fungi belonging to the *Aspergillus* [37] and *Trichoderma* [61] genera that possess the status of GRAS organisms. Moreover, the International Organisation of Vine and Wine (OIV) requires manufacturers to inform their customers if the enzymes supplied have been produced from genetically modified organisms (GMOs). This information must be given either on the product label or on the technical documentation (resolution OIV-OENO 485–2012) and currently, both consumers and winemakers are not in favour of the GMO origin.

The endogenous pectolytic activities of grape cells and those of the wine fermenting yeasts [64] also participate in the whole lytic process of grape tissues. It is worth noting that only a low percentage (about 11.5%) of indigenous fungi strains associated with grapes and wine have been reported to be able to produce significant amounts of extracellular pectinases under enological conditions. Those strains belonged to *Aureobasidium pullulans, Filobasidium capsuligenum* [65] and to the yeast species included in **Table 2**. Under winemaking conditions, ethanol and sulphurous anhydride can be present at concentrations reaching 15% (vol/vol) and 120 mg L�<sup>1</sup> respectively, which are quite restrictive conditions for grape and yeast pectinase activities [66] and to a lower extent, also for pectinase activities produced by filamentous fungi. These organisms have evolved to infect plant cells, they are well adapted to overcome the plant cell barriers and to degrade vegetable cell walls and pectic substances, as demonstrated by the number of pectinase encoding genes identified in fungi.

#### **11. Grape processing and juice extraction**

Destemming and crushing of grape berries are the first mandatory procedures for grape juice extraction, and simultaneously maceration starts as grape skins are torn into smaller pieces. At this stage, commercial pectolytic enzyme preparations can be used to degrade the polysaccharides of cell walls and middle lamella, thus facilitate juice release, liberation of polyphenols (pigments, tannins) and aroma molecular precursors that are located at the skin cells [67]. Pectolytic enzymes used in enology are selected preparations that are active at the acidic pH of grape juices (pH 3.0–4.0) under enological conditions and within wide limits of temperature [68], although at lower temperatures (15–5°C) the pectolytic activity decreases [37]. Pectolytic enzymes allow better extraction of the juice, but they also allow better extraction of the components of skins and occasionally of seeds. As shown in **Figure 9**, the pulp intermediate zone is easily released and crushed, whereas the peripheral zone, which includes skin cells, is the most difficult to extract and needs extra pressure to release its components. If grapes are not ripe enough, extraction of vegetal and astringent compounds from skins and seeds will depreciate the juice

quality. In this case, the fruit should be processed without the addition of pectolytic enzymes and avoiding maceration procedures, and consequently, the yield will considerably decrease, but the obtained juice will be low in bitterness and astringency. Thus, we see that enzyme addition at this stage should be wisely used to improve grape varietal characteristics and give character to the juice and wine. Normally, the more the damage is done to the skin and internal berry cell walls, the greater the release of berry components. Consequently, both aromas and flavours increase.

In the case of red-wine making, red grape skins are allowed to be present during the alcoholic fermentation and the polyphenols responsible for the red colour and sensory properties such as astringency or mouth-filling, which include anthocyanins and tannins among others, are further extracted as the ethanol produced during the fermentation facilitates their release from skin cells [69]. In the case of white and rosé wines, grape musts are submitted to pressing avoiding further contact with skins and seeds.
