**5.1 Extraction of fruit and vegetable juices**

The main industrial application of pectinases is the extraction and clarification of fruit and vegetable juices. Most of the microbial pectinases produced by the industry are dedicated to this purpose. Pectins are responsible for the turbidity and consistency of the juice causing an

Fig. 2. A: Pectic enzymes produced by different microorganisms growing on minimal medium supplemented with polygalacturonic acid and detected by a clear halo around the colonies. 1, *Xanthomonas campestris* CECT 97; 2, *Kluyveromyces marxianus* CECT 1043; 3, *Aspergillus niger* CECT 2088; 4, *Botryotinia fuckeliana* CECT 20518. B: Cloudy (peach) and

The microbial world has shown to be very heterogeneous in its ability to synthesize different types of pectolytic enzymes with different mechanisms of action and biochemical properties (Favela-Torres et al., 2005; Gummadi & Panda, 2003). Pectic enzymes are produced by both prokaryotic microorganisms, which primarily synthesize alkaline pectinases, and by eukaryotic microorganisms, mostly fungi that synthesize acid pectinases (Hoondal et al., 2002; Jayani et al., 2005; Kashyap et al., 2001). Furthermore, the production of these enzymes has also been described in yeast (Alimardani-Theuil et al., 2011; Blanco et

There are many studies that have been conducted related to the characterization of different microbial pectic enzymes concerning their mechanisms of action and biochemical properties. The optimal pHs that these enzymes may act range between 3.5-11, while the optimal temperatures vary between 40-75 °C (Gummadi & Panda, 2003; Kashyap et al., 2001). Table 2 shows an example of the diversity of pectic enzymes produced by microorganisms. Given the features of the substrate on which they act and the effect that is required of them, acidic and depolymerizing pectinases are of great interest for the food industry although some applications such as the extraction of oils requires the alkaline ones

The main industrial application of pectinases is the extraction and clarification of fruit and vegetable juices. Most of the microbial pectinases produced by the industry are dedicated to this purpose. Pectins are responsible for the turbidity and consistency of the juice causing an

clear (apple) juices. CECT: Spanish Type Culture Collection

al., 1999).

(Hoondal et al., 2002).

**5. Pectinases in the food industry** 

**5.1 Extraction of fruit and vegetable juices** 

increase in their viscosity which hinders its clarification, filtration and concentration (Alkorta et al., 1998). The degradation of pectic substances in mashed fruit purees is achieved through the addition of pectolytic enzymes resulting in an increase in juice yield and its clarification as well as a decrease in viscosity. Treatment with pectinases also provides filtering of the product (de Gregorio et al., 2002; Fernandez-Gonzalez et al., 2004; Ribeiro et al., 2010; Sarioglu et al., 2001; Souza et al., 2003).


Table 2. Biochemical properties of some pectinases

The fruit and vegetable juice industry uses mainly acidic pectinases of fungal origin, principally from *Aspergillus* spp*.* Commercial preparations are mixtures of polygalacturonases, pectate lyases and pectin esterases. Pectate lyases can act on the esterified pectin while the polygalacturonases act on the desesterified pectin thus it might require previous action of the pectin esterases. Pectic enzymes treatments vary depending on the type of juice (Fig. 2B):

#### **5.1.1 Clear juices (i.e., apple, pear, grape)**

In this type of product, pectolytic enzymes are added to increase the yield in juice during the pressing and for removing matter in suspension. In the case of apple juice, the most commonly used enzymes are those that can depolymerize the highly esterified apple pectin. Apple juice can be obtained through a two-step process consisting of a first treatment of the crushed apple mush with pectinases to obtain the premium juice followed by pomace liquefaction treatment made with a mixture of different pectinases and cellulases for the complete extraction of the juice (Will et al., 2000).

After washing and crushing, the apples are pressed to obtain the juice. Pectic enzymes are used to facilitate the pressing and juice extraction and to assist in the separation of a flocculant precipitate by means of sedimentation, filtration or centrifugation. If a cloudy product is required, the juice is pasteurised after pressing to inactivate residual enzymes. Centrifugation removes the large-size remains leaving small particles in suspension.

pulp and are indispensable for the fermentation process and the good quality of fermented beans (Ouattara et al., 2010; Schwan & Wheals, 2004). Similarly, treatment of tea leaves with pectic enzymes of fungal origin (at a dose adjusted to avoid damaging the leaf), facilitates

201

Wine is the result of the fermentation of grape juice. Pectinases are the most important enzymes used by the winemaking industry although commercial preparations may contain other enzymatic activities such as hemicellulases, glucanases and glycosidases (Rombouts &

Pectic enzymes are synthesized naturally by the plant and are present in the grape. However, they have low activity during the wine producing process (Ducasse et al., 2011). Microbial pectolytic enzymes especially of fungal origin are resistant to the conditions of fermentation and can be used to facilitate processes, improve quality and diversify products. So far, commercial enzymes are produced all from fungi, mainly of the genus *Aspergillus*. Although all enzymes are produced by *Aspergillus*, pectinase preparations currently available for the wine market are very different. Both the type of activities as well as their concentration in the preparations depend on the strain of *Aspergillus* used, the fermentation conditions for production, the nature of the fermented substrate and the degree of partial

Various research studies have shown that the addition of pectolytic enzymes leads to increased levels of methanol in wine (Revilla & González-SanJosé, 1998; Servili et al., 1992) due to the activity of pectin methyl esterase. Methanol is toxic and its maximum concentration in wine is regulated. Therefore, pectin methyl esterase activity should be at

The functions of pectic enzymes in the winemaking process are to support the extraction process, maximize juice yield, facilitate filtration and intensify the flavour and colour.

The addition of pectinase to the must reduces its viscosity and causes the grouping of suspended particles in larger aggregates that can be removed by sedimentation. If the enzymes are added to the pulp before pressing, must yield increases, facilitating the pressing and enhancing the colour. Macerating enzyme preparations for this purpose contain pectinases as well as cellulases (Ribereau-Gayon et al., 2006). A high level of polygalacturonase is very effective for clarification but may require the prior action of pectin lyase activity. For this reason the enzyme preparations with a high content of pectin lyase are desirable when very fast racking is required to prevent any problems related to must oxidation, development of endogenous microbiota and nutrient loss. In addition, wines made with pectic-enzyme-treated grape must significantly reduces filtration times (Blanco et

In the case of musts obtained from red grapes, the degradation of cell walls in the skin of the grapes through pectolytic enzyme treatment results in an increased release of phenolic

and accelerates the fermentation (Carr, 1985; Kashyap et al., 2001).

**6. Pectinases in the wine industry** 

low concentrations in commercial mixtures.

**6.1 Extraction, clarification and filtration** 

**6.2 Intensification and stabilization of colour** 

Pilnik, 1980).

purification.

al., 1997; Blunt, 2000).

However, if a clear juice is required, these suspended particles have to be withdrawn. In order to do this a treatment with mixtures of commercial enzymes is carried out containing pectinases, cellulases and hemicellulases. Finally, the fluid is centrifuged to obtain the clear juice (Grassin & Fauquembergue, 1996; Kashyap et al., 2001). Although it has been noted that the proper clarification of apple juice requires mixtures of polygalacturonase and pectin methyl esterase (Yamaski et al., 1964), subsequent studies have shown that it can be clarified by only using a pure pectin lyase (Ishii & Vokotsuka, 1973).

### **5.1.2 Cloudy juices (i.e., citrus, tomato, nectars)**

In the case of orange juice, where natural pectin esterases are present, pectin is only partially methylated. Polygalacturonases are the pectic enzymes which are most commonly used and of great interest for this type of fruit juice.

In the process of orange juice extraction, pectinases can be added at the end of the pulp wash extraction to reduce viscosity or, preferably, at the end of the first finisher. This leads to higher yield in juice, a better extraction of soluble solids and to a lower viscosity. The action of these enzymes just reduces the viscosity without attacking the insoluble pectin that maintains the stability of the cloud. Enzyme preparations should lack or have the least possible amount of pectin methyl esterases to avoid the clarification of the product. It has been suggested that the best enzyme might be pure pectin lyase (Kashyap et al., 2001; Rebeck, 1990).

#### **5.2 Maceration products of plant tissues**

The enzymatic maceration of plant tissues allows the transformation of these organized tissues in suspensions of intact cells that constitute the pulpy products that are used as a basis for preparing juices, nectars, baby food and some dairy products such as yoghurts. Enzyme preparations for this purpose contain cellulases, hemicellulases and pectic enzymes which should only act on the middle lamella of the plant tissue (Kashyap et al., 2001).

#### **5.3 Extraction of vegetable oil**

Vegetable oils of olive, sunflower, coconut, palm or canola are obtained by extraction with organic solvents such as hexane. The use of pectolytic enzymes, in this case preferably alkaline, allows the extraction of vegetable oils in an aqueous process. Enzyme preparations based on cellulases, hemicellulases and pectinases have been used successfully in the extraction of olive oil. The enzyme treatment not only improves oil yield and stability but also increases polyphenols and vitamin E content enhanzing its organoleptic quality (Hoondal et al., 2002; Iconomou et al., 2010; Kashyap et al., 2001; West, 1996).

#### **5.4 Coffee, cocoa and tea fermentation**

Traditionally, fermentation of coffee is made with pectolytic microorganisms in order to remove the layer of mucilage from the coffee beans. With the same purpose, commercial enzyme preparations containing pectinase is sprayed onto the beans to ferment. A cheaper alternative is to use, with the same purpose, the filtrate of inoculated fermentations. The enzyme treatment significantly reduces the fermentation time (Amorim & Amorim, 1977; Kashyap et al., 2001; Serrat et al., 2002; Silva et al., 2000). Cocoa fermentation is essential to develop the chocolate flavour. This fermentation is carried out by a succession of different microorganisms, some of them pectolytic. Pectic enzymes allow the degradation of the cocoa pulp and are indispensable for the fermentation process and the good quality of fermented beans (Ouattara et al., 2010; Schwan & Wheals, 2004). Similarly, treatment of tea leaves with pectic enzymes of fungal origin (at a dose adjusted to avoid damaging the leaf), facilitates and accelerates the fermentation (Carr, 1985; Kashyap et al., 2001).
