Applications of Pectinases in Industrial Sector

Abstract

Pectinases are one of the upcoming enzymes of fruit and textile industries. These enzymes break down complex polysaccharides of plant tissues into simpler molecules like galacturonic acids. The role of acidic pectinases in bringing down the cloudiness and bitterness of fruit juices is well established. Recently, there have been a good number of reports on the application of alkaline pectinases in the textile industry for the retting and degumming of fiber crops, production of good quality paper, fermentation of coffee and tea, oil extractions and treatment of pectic waste water. This review discusses various types of Pectinases and their applications in the commercial sector. [PUBLICATION ABSTRACT]

Headnote

Abstract: Pectinases are one of the upcoming enzymes of fruit and textile industries. These enzymes break down complex polysaccharides of plant tissues into simpler molecules like galacturonic acids. The role of acidic pectinases in bringing down the cloudiness and bitterness of fruit juices is well established. Recently, there have been a good number of reports on the application of alkaline pectinases in the textile industry for the retting and degumming of fiber crops, production of good quality paper, fermentation of coffee and tea, oil extractions and treatment of pectic waste water. This review discusses various types of Pectinases and their applications in the commercial sector.

Keywords: Pectinases, Industry, Application, Polysaccharides, Pectin.

Introduction

Pectinase is an enzyme that breaks down pectin. Pectin is one of the compounds found in plant cell walls; it is in the plate (middle lamella) that is the first part of the wall to be formed during cytokinesis, following cell division. Pectinase therefore helps to break down the cell walls. This increases the volume of juice obtained (increases the yield), lowers the viscosity of the juice (makes it more runny), reduces the cloudiness of the juice, cause by suspended pieces of cell wall.

Pectinases were some of the first enzymes to be used in homes. Their commercial application was first observed in 1930 for the preparation of wines and fruit juices. Only in the 1960s did the chemical nature of plant tissues become apparent and with this knowledge, scientists began to use a greater range of enzymes more efficiently. As a result, pectinases are today one of the upcoming enzymes of the commercial sector. Primarily, these enzymes are responsible for the degradation of the long and complex molecules called pectin that occur as structural polysaccharides in the middle lamella and the primary call walls of young plant cells (Kashyap et al., 2001).

Structure of Pectin

Chemically, pectic substances are complex colloidal acid polysaccharides, with a backbone of galacturonic acid residues linked by (1±4) linkages. The side chains of the pectin molecule consist of L -rhamnose, arabinose, galactose and xylose. The carboxyl groups of galacturonic acid are partially esterified by methyl groups and partially or completely neutralized by Sodium, Potassium or Ammonium ions. Based on the type of modifications of the backbone chain, pectic substances are classified into protopectin, pectic acid, pectinic acid and pectin (Miller, 1986).

Protopectin

This is a parent pectic substance and upon restricted hydrolysis yields pectin or pectinic acid. Protopectin is occasionally a term used to describe the water-insoluble pectic substances found in plant tissues and from which soluble pectic substances are produced (Kilara, 1982).

Pectic Acids

These are the galacturonans that contain negligible amounts of methoxyl groups. Normal or acid salts of pectic acid are called pectates.

Pectinic Acids

These are the galacturonans with various amounts of methoxyl groups. Pectinates are normal or acid salts of pectinic acids (Kilara, 1982). Pectinic acid alone has the unique property of forming a gel with sugar and acid or if suitably low in methyl content, with certain other compounds such as calcium salts.

Biotechnological Applications of Microbial Pectinases

Over the years, pectinases have been used in several conventional industrial processes, such as textile, plant fiber processing, tea, coffee, oil extraction, treatment of industrial wastewater, containing pectinacious material, etc. They have also been reported to work on purification of viruses (Salazar and Jayasinghe, 1999) and in making of paper (Reid I. and Richard 2004; Viikari et al., 2001) but are yet to be commercialized.

Pectinases are among the most important industrial enzymes. The biotechnological potential of pectinolytic enzymes from microorganisms has drawn a great deal of attention from various researchers worldwide as likely biological catalysts in a variety of industrial processes. Alkaline significance in the current biotechnological arena with wide ranging applications in textile processing, degumming of plant bast fibers, treatment of pectic wastewaters, paper making, and coffee and tea fermentations. The present review features the potential applications and uses of microbial alkaline pectinases, the nature of pectin, and the vast range of pectinolytic enzymes that function to mineralize pectic substances present in the environment. It also emphasizes the environmentally friendly applications of microbial alkaline pectinases thereby revealing their underestimated potential.

Fruit Juice Extraction

The largest industrial application of pectinases is in fruit juice extraction and clarification. Pectins contribute to fruit juice viscosity and turbidity. A mixture of pectinases and amylases is used to clarify fruit juices. It decreases filtration time up to 50% (Blanco et al., 1999). Treatment of fruit pulps with pectinase also showed an increase in fruit juice volume from banana, grapes and apples (Kaur and Kumar, 2004). Pectinases in combination with other enzymes, viz., cellulases, arabinases and xylanases, have been used to increase the pressing efficiency of the fruits for juice extraction (Gailing et al., 2000). Vacuum infusion of pectinases has a commercial application to soften the peel of citrus fruits for removal. This technique may expand in future to replace hand cutting for the production of canned segments (Baker and Wicker, 1996). Infusion of free stone peaches with pectin methylesterase and calcium results in four times firmer fruits. This may be applied to pickle processing where excessive softening may occur during fermentation and storage (Baker and Wicker ,1996).

Apple

Apple juice is manufactured as natural, unfiltered and unclarified, juice containing a high percentage of pulp; as a hazy juice that has been centrifuged to remove coarse particles but not filtered; and finally as filtered clear and amber colored juice prepared by enzymatic treatment (Kilara, 1982). Although pectinases that can depolymerize highly esterified pectin are the major types of enzymes used in apple juice processing, they are by no means the only enzymes used for this purpose. A combination of pectinases and cellulases has been reported to give a juice yield up to 100% (Alkorta et al., 1998). Another potential contributor to the haziness is starch. Unripe apples may contain up to 15% starch. This can be broken down using an amylase which is active at the pH of apple juice and added at the same time as the pectinases.

Grape Juice and Wine

Grape juice is not consumed in large amounts because it is too sweet (about 200 g Γ1 sugars) or too acid (up to 10 g Γ1 tartaric acid). It is mixed with other fruit juices such as apple and the technology of grape juice processing has been reviewed (Pederson, 1980; Luh and Kean, 1975). With the high pectin content (5±10 g Γ1) grapes are difficult to crush and to press. They are de-stemmed, crushed and heated to 60°C or 80°C to release color (in case of black grapes) from the skins and destroy the endogenous polyphenoloxidase. Then enzymes such as Cytolase PCL5 (Gist -Brocades) or Ultrazyme (Novo Nordisk) are added at approximately 50 g ton"1 to macerate the berries and increase the yield. The free running juice is separated from the solids by different kinds of filters (rotary vacuum, earth) and/ or by centrifugation. The filtered juice is cooled to 0°C to prevent fermentation and then depectinized, at approximately 200 ppm over about two weeks. Pectinases, mainly PG and hemicellulases like arabino-galactanases, allow insoluble particles to flocculate. At the same time, cold storage of the juice encourages removal of tartarate and reduces the total acidity to acceptable levels. The juice is then filtered with diatomaceous earth and concentrated, pasteurized and bottled.

Strawberry, Raspberry and Blackberry Juices

The production of clear juices and concentrates from strawberries, raspberries or blackberries requires enzymatic depectinization. The juices from these fruits contain high amounts of pectin that remains as colloidally dissolved residue' making the juices viscous (Will and Dietrich, 1992). The clarification, filtration, and concentration of these juices therefore become difficult. These residual pectins and hemicelluloses also bind to phenolic substances and protein during the processing and storage and result in the formation of irreversible complexes that enzymes cannot breakdown. An additional problem is contamination of strawberries and raspberries by B. cinerea (Grassin and Fauquembergue, 1996). This fungus secretes a b-1, 3±l,6-linked glucan in the berries,which forms gum and hence reduces the filter ability and the clarity of the juice. These glucans can be hydrolyzed by b-glucanases.

Textile Processing and Bio-Scouring of Cotton Fibers

Pectinases have been used in conjunction with amylases, lipases, cellulases and hemi-cellulases to remove sizing agents from cotton in a safe and eco-friendly manner, replacing toxic caustic soda used for the purpose earlier (Hoondal et al., 2000). Bio-scouring is a novel process for removal of non- cellulosic impurities from the fiber with specific enzymes. Pectinases have been used for this purpose without any negative side effect on cellulose degradation (Hoondal et al., 2000).

Degumming of Plant Bast Fibers

Bast fibers are the soft fibers formed in groups outside the xylem, phloem or pericycle, e.g. Ramie and sun hemp. The fibers contain gum, which must be removed before its use for textile making. The chemical degumming treatment is polluting, toxic and non-biodegradable. Biotechnological degumming using pectinases in combination with xylanases presents an eco-friendly and economic alternative to the above problem (Kapoor et al., 2001).

Retting of Plant Fibers

Pectinases have been used in retting of flax to separate the fibers and eliminate pectins (Hoondal et al., 2000) .

Waste Water Treatment

The wastewater from the citrus-processing industry contains pectinaceous materials that are barely decomposed by microbes during the activated-sludge treatment (Tanabe et al., 1986; Tanabe et al., 1987) have tried to develop a new wastewater treatment process by using an alkalophillic microorganism. Their soil isolate of an alkalophilic Bacillus sp. (GIR 621), produces an extracellular endopectate lyase in alkaline media at pH 10.0. Treatment with this strain has proved to be useful in removing pectic substances from the wastewater. For treatment of wastewater from citrus processing industries various processes have been investigated, which include: physical dewatering, spray irrigation, chemical coagulation, direct activated sludge treatment and chemical hydrolysis followed by methane fermentation (Tanabe et al., 1986; Tanabe et al., 1987). These processes have some defects, such as low efficiency due to chemical resistance of the pectic substances, high treatment cost, long treatment periods and complexity of the process vegetable food processing industries release pectin, containing wastewaters as by product. Pretreatment of these wastewaters with pectinolytic enzymes facilitates removal of pectinaceous material and renders it suitable for decomposition by activated sludge treatment (Hoondal et al., 2000).

Paper Making

Pulp and paper mills are beginning to use enzymes to solve problems in their manufacturing processes. Papermaking is essentially a continuous filtration process in which a dilute suspension of fibers, fiber fragments (fines), and inorganic filler particles, such as clay or CaC03, is formed into sheets. The need for water drainage leads to use of a filter fabric with holes large enough to allow passage of the fines and filler particles. In modern papermaking, retention aids are added to pulp to keep fines and filler particles in paper sheets and to speed the drainage of water. Cationic polymers of various structures are commonly used as retention aids (Horn D. and Linhart, 1996). Alkaline peroxide bleaching of pulps solubilizes polysaccharides, which are troublesome interfering substances (Holbom etal., 1991). Prominent among these polysaccharides are pectins, or polygalacturonic acids. The ability of polygalacturonic acids to complex cationic polymers (cationic demand) depends strongly on their degree of polymerization, monomers, dimers, and trimers of galacturonic acid do not cause measurable cationic demand, but hexamers and long chains have high cationic demand (Thornton etal., 1994). Pectinase can depolymerize polymers of galacturonic acids, and subsequently lower the cationic demand of pectin solutions and the filtrate from peroxide bleaching (Reid I. and Ricard, 2000).

Coffee and Tea Fermentation

Pectinase treatment accelerates tea fermentation and also destroys the foam forming property of instant tea powders by destroying pectins (Carr, 1985). They are also used in coffee fermentation to remove mucilaginous coat from coffee beans.

Paper and Pulp Industry

During papermaking, pectinase can depolymerise pectins and subsequently lower the cationic demand of pectin solutions and the filtrate from peroxide bleaching (Reid I. and Richard, 2004; Viikari et al., 2001).

Animal Feed

Pectinases are used in the enzyme cocktail, used for the production of animal feeds. This reduces the feed viscosity, which increases absorption of nutrients, liberates nutrients, either by hydrolysis of non- biodegradable fibers or by liberating nutrients blocked by these fibers, and reduces the amount of faeces (Hoondal etal., 2000).

Purification of Plant Viruses

In cases where the virus particle is restricted to phloem, alkaline pectinases and cellulases can be used to liberate the virus from the tissues to give very pure preparations of the virus (Salazar and Jayasinghe, 1999).

Oil Extraction

Citrus oils such as lemon oil can be extracted with pectinases. They destroy the emulsifying properties of pectin, which interferes with the collection of oils from citrus peel extracts (Scott, 1978).

Improvement of Chromaticity and Stability of Red Wines

Pectinolytic enzymes added to macerated fruits before the addition of wine yeast in the process of producing red wine resulted in improved visual characteristics (color and turbidity) as compared to the untreated wines. Enzymatically treated red wines presented chromatic characteristics, which are considered better than the control wines. These wines also showed greater stability as compared to the control (Revilla and González-San José, 2003).

Conclusion

Most of the studies performed so far have been concentrated with the screening, isolation, production, purification, characterization and applications of pectinolytic enzymes in increasing the fruit juice yield and its clarification. Some reports are available on the applications of pectinases in other industries. Study of the molecular aspects of pectinases and engineering of enzymes that are more robust with respect to their pH and temperature kinetics by the techniques of protein engineering and site directed mutagenesis should receive increased attention in the coming times. Future studies on pectic enzymes should be devoted to the understanding of the regulatory mechanism of the enzyme secretion at the molecular level and the mechanism of action of different pectinolytic enzymes on pectin substances.

References

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