Bioresource Technology 77 (2001) 215227

Review paper

Applications of pectinases in the commercial sector: a review

D.R. Kashyap a, P.K. Vohra a, S. Chopra a, R. Tewari b,*
a b

Department of Microbiology, Punjab University, Chandigarh 160014, India Department of Biotechnology, Punjab University, Chandigarh 160014, India Accepted 10 August 2000

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 has been a good number of reports on the application of alkaline pectinases in the textile industry for the retting and degumming of ber crops, production of good quality paper, fermentation of coee and tea, oil extractions and treatment of pectic waste water. This review discusses various types of pectinases and their applications in the commercial sector. 2001 Elsevier Science Ltd. All rights reserved.
Keywords: Pectinase; Industrial applications

1. Introduction Pectinases were some of the rst enzymes to be used in homes. Their commercial application was rst 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 eciently. 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. Pectinases are now an integral part of fruit juice and textile industries as well as having various biotechnological applications. The estimated value of sales of all industrial enzymes in 1995 was $1 billion, of which some $75 million was assessed for pectinases. By 2005, the whole world market for industrial enzymes is expected to be $1.72 billion (Godfrey and West, 1996). The main emphasis of this article is on the types of pectinases and their applications in industries.

2. Structure of pectin Chemically, pectic substances are complex colloidal acid polysaccharides, with a backbone of galacturonic acid residues linked by a (14) linkages. The side chains of the pectin molecule consist of L -rhamnose, arabinose, galactose and xylose. The carboxyl groups of galacturonic acid are partially esteried by methyl groups and partially or completely neutralized by sodium, potassium or ammonium ions. Based on the type of modications of the backbone chain, pectic substances are classied into protopectin, pectic acid, pectinic acid and pectin (Be 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.

Corresponding author. Tel.: +172-541441 ext. 4085; fax: +172541409. E-mail address: shaktit@glide.net.in (R. Tewari).

0960-8524/01/$ - see front matter 2001 Elsevier Science Ltd. All rights reserved. PII: S 0 9 6 0 - 8 5 2 4 ( 0 0 ) 0 0 1 1 8 - 8

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Pectins. A generic name for the mixture of widely diering compositions containing pectinic acid as the major component. Pectin in native form is located in the cell wall and it may be interlined with other structural polysaccharides and proteins to form insoluble protopectin. In an unripe fruit, pectin is bound to cellulose microbrils in the cell wall. Such pectin is insoluble and hence confers rigidity on cell walls. However, during ripening the structure of pectin is altered by naturally occurring enzymes in the fruits. These alterations involve the breakdown of the pectin chain or of side chains attached to the units, which make up the main chain. In either case, the result is that the pectin becomes more soluble and its grip on the surrounding cell walls is loosened and the plant tissue softens. The pectic substances account for about 0.54% of the weight of fresh material. When the tissue is ground, the pectin is found in the liquid phase (soluble pectin) causing an increase in viscosity and the pulp particles, whereas other pectin molecules remain bound to cellulose brils by means of side chains of hemicellulose and thus facilitate water retention (Pieri et al., 1989). Mechanical crushing of pectin-rich fruit yields a fruit juice with high viscosity, which remains bound to the pulp in the form of a jellied mass. It is dicult to extract this juice by pressing or using other mechanical methods. With the addition of pectinases the viscosity of the fruit juice drops, the pressability of the pulp improves, the jelly structure disintegrates and the fruit juice is easily obtained and with higher yields. The raw press juice is rich in insoluble particles, which are mainly made up of pectic substances. These particles are known as `cloud particles'. In these particles, a protein nucleus with a positive surface charge is coated by negatively charged pectin molecules (Pilnik and Voragen, 1993). This negative charge causes the pectin molecules to repel one another. Pectinases degrade this pectin and expose part of the positively charged protein beneath, thus reducing electrostatic repulsion between cloud particles which causes these particles to aggregate to larger particles. These larger particles eventually settle out, but to improve the process, occulating agents (nings) such as gelatin, tannin or bentonite (a type of clay) can be added. Yeasts and other microbes, which may have contaminated the juice, are also precipitated by nings. What is left is a transparent but by no means clear juice. A second centrifugation and subsequent ltration are needed to give the clear juice that many consumers prefer. 3. Classication of pectic enzymes Pectinases are classied under three headings according to the following criteria: whether pectin, pectic

acid or oligo-D -galacturonate is the preferred substrate, whether pectinases act by trans-elimination or hydrolysis and whether the cleavage is random (endo-, liquefying of depolymerizing enzymes) or endwise (exo- or saccharifying enzymes). The three major types of pectinases are as follows. 3.1. Pectinesterases (PE) Pectinesterases also known as pectinmethyl hydrolase, catalyzes deesterication of the methoxyl group of pectin forming pectic acid. The enzyme acts preferentially on a methyl ester group of galacturonate unit next to a non-esteried galacturonate unit. 3.2. Depolymerizing enzymes These are the enzymes: 3.2.1. Hydrolyzing glycosidic linkages They include: 3.2.1.1. Polymethylgalacturonases (PMG). Catalyze the hydrolytic cleavage of a-1,4-glycosidic bonds. They may be: 1. Endo-PMG: causes random cleavage of a-1,4-glycosidic linkages of pectin, preferentially highly esteried pectin. 2. Exo-PMG: causes sequential cleavage of a-1,4-glycosidic linkage of pectin from the non-reducing end of the pectin chain. 3.2.1.2. Polygalacturonases (PG). Catalyze hydrolysis of a-1,4-glycosidic linkages in pectic acid (polygalacturonic acid). They are also of two types: 1. End-PG: also known as poly (1,4-a-D -galacturonide) glycanohydrolase, catalyzes random hydrolysis of a1,4-glycosidic linkages in pectic acid. 2. Exo-PG: also known as poly(1,4-a-D -galacturonide) galacturonohydrolase, catalyzes hydrolysis in a sequential fashion of a-1,4-glycosidic linkages on pectic acid. 3.2.2. Cleaving Cleaving a-1,4-glycosidic linkages by trans-elimination, which results in galacturonide with an unsaturated bond between C4 and C5 at the non-reducing end of the galacturonic acid formed. These include: 3.2.2.1. Polymethylegalacturonate lyases (PMGL). Catalyze breakdown of pectin by trans-eliminative cleavage. They are: 1. Endo-PMGL: also known as poly(methoxygalacturonide) lyase, catalyzes random cleavage of a-1,4-glycosidic linkages in pectin.

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2. Exo-PMGL: catalyzes stepwise breakdown of pectin by trans-eliminative cleavage. 3.2.2.2. Polygalacturonate lyases (PGL). Catalyze cleavage of a-1,4-glycosidic linkage in pectic acid by trans-elimination. They are also of two types: 1. Endo-PGL: also known as poly (1,4-a-D -galacturonide) lyase, catalyzes random cleavage of a-1,4-glycosidic linkages in pectic acid. 2. Exo-PGL: also known as poly(1,4-a-D -galacturonide) exolyase, catalyzes sequential cleavage of a-1,4-glycosidic linkages in pectic acid. 3.3. Protopectinase This enzyme solubilizes protopectin forming highly polymerized soluble pectin. On the bases of their applications, pectinases are mainly of two types: acidic pectinases and alkaline pectinases. The important producers of these pectinases as reported in the literature are given in Table 1.

pecially from Aspergillus niger. The juices produced by these industries commercially include: (A) Sparkling clear juices, (apple, pear and grape juices, (B) Juices with clouds (citrus juices, prune juices, tomato juice and nectars), and (C) Unicellular products where the intent is to preserve the integrity of the plant cells by selectively hydrolyzing the polysaccharides of the middle lamella. The objectives of adding enzymes dier in these three types of fruit and vegetable juices. 4.1.1. Sparkling clear juices In the case of sparkling clear juices enzymes are added in order to increase the juice yield during pressing and straining of the juice and to remove suspended matter to give sparkling clear juices (free of haze). Some examples of such juices and their further uses are described below. 4.1.1.1. Apple. Apple juice is manufactured as natural, unltered and unclaried, juice containing a high percentage of pulp; as a hazy juice that has been centrifuged to remove coarse particles but not ltered; and nally as ltered clear and amber-colored juice prepared by enzymatic treatment (Kilara, 1982). Although pectinases that can depolymerize highly esteried 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

4. Application of pectinases 4.1. Acidic pectinases Acidic pectic enzymes used in the fruit juice industries and wine making often come from fungal sources, esTable 1 Characterization of microbial pectinases Producer Acidic pectinases Aspergillus niger CH4 Penicillium frequentans Sclerotium rolfsii Rhizoctonia solani Mucor pusilus Cloctridium thermosaccharolyticum Alkaline pectinases Bacillus sp. RK9 Bacillus sp. NT-33 Bacillus polymyxa Bacillus pumilis Amucola sp. Xanthomonas compestris Bacillus No. P-4-N Bacillus stearothermophillus Penicillium italicum CECT 22941 Bacillus sp. DT 7 Bacillus subtilis Pseudomonas syringae pv. Glycinea Type of pectinase

Opti. pH for activity 4.56.0 3.55.0 4.54.7 3.5 4.8 5.0 5.57.0

Opti. Temp. for activity (C) Below 50 50 55 50 40 3040

Reference

Endo-pectinase, Exo-pectinase Endopolygalacturonase (Endo-PG) Endo-PG Endo-PG PG Polygalacturonate hydrolase PGL PG PG PATE Pectate lyase (PAL) PATE PG PATE Pectin lyase Pectin lyase PAL PAL

Acuna-Arguelles et al., 1995 Borin et al., 1996 Channe and Shewal, 1995 Marcus et al., 1986 Al-Obaidi et al., 1987 Rijssel et al., 1993

10.0 10.5 8.49.4 8.0-8.5 10.25 9.5 1010.5 9.0 8.0 8.0 8.5 8.0

75 45 60 70 2530 65 70 50 60 6065 3040

Fogarty and Kelly, 1983 Cao et al., 1992 Nagel and Vaughn, 1961 Dave and Vaughn, 1971 Bruhlmann et al., 1994 Nasumo and Starr, 1967 Horikoshi, 1990 Karbassi and Vaughn, 1980 Alana et al., 1990 Kashyap et al., 2000 Chesson and Codner, 1978 Magro et al., 1994

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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 (strictly speaking, an amyloglucosidase) active at the pH of apple juice, added at the same time as the pectinases. Juice extraction. The initial steps in the extraction of juice from apples include washing, sorting and crushing of apples in a mill. Although pectinases are often added at this stage, better results are achieved if the apple pulp is rst stirred in a holding tank for 1520 min so that enzyme inhibitors (polyphenols) are oxidized by naturally occurring poly-phenol oxidase present in the fruit. As these polyphenols in the pulp inhibit pectic enzymes, addition of microbial polyphenol oxidases to the fruit juices has also been suggested. An alternative way to remove polyphenols in the fruit juices is the addition of polyvinyl pyrolidone (PVP) which oxidizes the phenolic substances hence creating an environment for the action of pectic enzymes (De Vos and Pilnik, 1973). Pectic enzymes are used in apple juice preparation to facilitate pressing or juice extraction and to aid in the separation of a occulent precipitate by sedimentation, ltration or centrifugation. Schols et al. (1990) described a new pectinase called rhamnogalacturonase and its role in the maceration of apple tissue. This enzyme was found in Aspergillus aculeatus initially, but it seems also to be produced by other Aspergillus spp. Treatment with pectinase takes anything from 15 min to 2 h depending upon the exact nature of the enzyme and how much is used, the reaction temperature and the variety of apple chosen (Kilara, 1982). Some varieties, such as Golden Delicious, are notoriously dicult to breakdown. During incubation the pectinase degrades soluble pectin in the pulp, which would otherwise hamper juice extraction in two ways. First, these slimy pectin particles become saturated with juice, which is then dicult to extract from the pulp and second, they block drainage channels in the pulp through which the juice must pass. It is also important that the pulp is not broken down too much, as it is then becomes dicult to press. Enzyme treatment is particularly eective with mature apples and those from cold storage (Rombouts and Pilnik, 1986). Clarication. Clarication is aected by pH temperature, contact time and enzyme concentration. A juice with low pH will clarify more readily than one with a higher pH, and as the temperature increases the rate of clarication also increases as long as the temperature is below denaturation temperature for the enzyme 4060C (Kilara, 1982). In general, the time required to obtain clarication is inversely proportional to the concentration of enzyme used at constant temperature over the range of 550C and treatment time of 216 h. If a cloudy product is required, the juice is pasteurized immediately after pressing, to denature any residual

enzymes. Centrifugation then removes large pieces of debris, leaving most of the small particles in suspension. For a clear juice these suspended particles have to be removed. A dose of commercial enzyme e.g., `Rapidase pomaliq', which contains pectinases, hemicellulases, and cellulases is the accepted way of removing suspended particles (Grassin and Fauquembergue, 1996). This enzyme is supplied by GistBrocades and is used at 200 600 g per ton of apples while stirring the pomace at 40 60 rpm for approximately 3 h at 50C. The liquied pomace is centrifuged. Fig. 1 shows the signicant steps involved in production of apple juice. Yamaski et al. (1964) showed that clarication of apple juice could be obtained by a mixture of PG and PME alone without the presence of contaminating enzymes from the apples. Recent work has shown that puried pectin lyase (PL) used at pH 34 can clarify apple juice (Ishii and Vokotsuka, 1971, 1973). This research has also shown that apple juice containing 9192% esteried pectin can be easily claried by pure PL. Clear apple juice can sometimes develop a haze during storage, especially refrigerated storage. This defect is termed `After Haze' and results from juice processed at

Fig. 1. Production of apple juice (Grassin and Fauquembergue, 1996).

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temperature higher than storage temperature. The haze may result from polymerization of polyphenols and oxidation of proanthocyanidins during milling and pressing. Another appearance defect called `Starch Haze' may sometimes develop in apple juice prepared from early-season apples (contain up to 15% starch). Starch haze can be removed by fungal amylases (diastase) added to juices heated to 77C to gelatinize the starch and then cooled to 52C before diastase addition. Pectic enzyme is added at the same time as the diastase. In conclusion, advantages of the use of pectinases in apple juice are the following: their availability allows the producer to diversify the type of products, i.e., cloudy, clear juices, clear concentrates, etc., and to increase the value of this raw material. In practice, the total time for the juice extraction is shorter than the classical process. Enzymes also help in production of juices and concentrates that are very stable and have a good taste with a reduced pomace waste, and reduced production cost (higher yield, less equipment, less labor in a concentration process). Finally, they also permit the processing of dierent fruits by the same process. Cider. Use of pectinases assists in the production of French ciders of high and consistent quality from cider apple varieties that are bitter, sweet or sour (Drilleau, 1985). Traditionally, the fermentation of apple juice was very slow (a few weeks to 3 months ) and with some residual sugars at the end, depending on the type of cider produced (dry, semi-sweet and sour). The cider was aromatic but with low acidity and sometimes with a bitter taste because of lactic, acetic acids and other contaminations. In cider production, the rst step is the preparation of apple, followed by clarication using commercial fungal pectinases. Pectic enzymes enable the cider producer to control and accelerate the mechanism of coagulum (a gel of pectinates) formation. The coagulum entraps pectateprotein and tannins, which are removed from the juice. The high quality of the juice thus obtained encourages good fermentation and produces an aromatic cider. 4.1.1.2. Pear. Juice processing. The composition of pear pulp and the changes during ripening and post-harvest storage are similar to those described for apple. There is a decrease in protopectin content and viscosity, and increase in pectate, during pear ripening (Rahunard and Neukon, 1964). During maturation of pears phenolic compounds are oxidized and form insoluble compounds (lignin). The presence of scleroids or stone cells in the pulp results in a lower textural quality (Ranadive and Haard, 1973). These stone cells are lignocellulosic and contain approximately 18% lignin and 82% carbohydrate, which makes ltration or ultralteration dicult. Araban, hemicellulose and cellulose contents are higher than in apple. This is why it is important to use pec-

tinases with strong arabanase and arabinosidase activities. The process is similar to that for apples. Pears are crushed and enzymes are added to the pulp in maceration step at about 75 g ton1 and left for 3060 min at ambient temperature. The pectinases PL, PME, PG and arabanases improve the extraction and yield of juice. After pressing, the juice is depectinized with pectinases containing high arabanase activities for about 1 h at 4550C. The juice is then ned and ltered or ultraltered and concentrated. Pears can also be processed to pur ees or nectars. Fruit nectars are pulpy fruit juices blended with sugar syrup and citric acid to produce a ready-to-drink beverage. They come from diluted pur ee or pulpy juice processing. The most important quality factor of these nectars is that the cloud should be stable. After mixing with water, sugar and acid, the cloud particles tend to settle down and sometimes form a gel with a clear supernatant layer: this separation is a serious defect which reduces the attractiveness of the product (Askar et al., 1990). The use of exogenous enzymes can improve the cloud stability in nectars, and make pur ee concentration easier. Enzyme preparations like Rapidase LIQ (Gist Brocades), Pectinex Ultra SP (Novo Nordisk), or Rohapect TF (Rohm) which have high PG and PL activities combined with cellulases and hemicellulases have been found to decrease the viscosity, keep cloud stability, and improve the ability to concentrate the product. The resulting pur ee is concentrated, deaerated, heated to a temperature of 8595C, sweetened with sugar syrup and acidied with citric acid so as to maintain constant sugars: acid ratio at 1516 Brix. The resultant nectar is canned or bottled, seal processed at 100C for 23 min and marketed. 4.1.1.3. Grape juice and wine. Grape juice is not consumed in large amounts because it is too sweet (about 200 g l1 sugars) or too acid (up to 10 g l1 tartaric acid). It is mixed with other fruit juices such as apple. Pederson (1980) and Luh and Kean (1975) have reviewed the technology of grape juice processing. With the high pectin content (510 g l1 ) grapes are dicult to crush and to press. They are destemmed, crushed and heated to 60C or 80C to release colour (in case of black grapes) from the skins and destroy the endogenous polyphenoloxidase. Then enzymes such as Cytolase PCL5 (GistBrocades) or Ultrazyme (Novo Nordisk) are added at approximately 50 g ton1 to macerate the berries and increase the yield. The free running juice is separated from the solids by dierent kinds of lters (rotary vacuum, earth) and/ or by centrifugation. The ltered juice is cooled to 0C to prevent fermentation and then depectinized, at approximately 200 ppm over about 2 weeks. Pectinases, mainly PG,

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and hemicellulases like arabinogalactanases, allow insoluble particles to occulate. 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 ltered with diatomaceous earth and concentrated, pasteurized and bottled. Pectic enzymes from fungi, typically Aspergillus niger, Penicillium notatum, or Botrytis cinerea are useful in wine making. Although juices of berries, peaches, apples, pears, and other fruits are employed (Robertson, 1977; Pilnik, 1982; Fogarty and Kelly, 1983), grape wines are produced in greatest volume and hence will be considered here. Pectic enzymes are used to reduce haze or gelling of grape juice at any one of three stages: at the rst stage, while the grapes are being crushed; at the second stage, which involves the must (free-run juice) before its fermentation or after; or at the last stage, once the fermentation is complete, when the wine is ready for transfer or bottling. The addition of pectinases at the rst stage is preferred since it increases the volume of free-run juice and reduces pressing time. The better release of anthocyanins of the red grapes into the juice achieved by pectic enzymes is another advantage of these enzymes, which has received attention from redwine manufacturers also (Pilnik and Voragen, 1993). Enzyme treatment of the juice at the second stage before or during the fermentation, settles out many suspended particles and often some undesirable microorganisms. Finally, addition of pectic enzymes to the fermented wine at the last stage increases ltration rate and clarity, but the level of enzyme supplemented must be adjusted to allow for the inhibitory eect of alcohol on pectinases. 4.1.1.4. Strawberry, raspberry, blackberry. 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 clarication, ltration, and concentration of these juices therefore become dicult. 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-1,31,6-linked glucan in the berries, which forms gum and hence reduces the lterability and the clarity of the juice. These glucans can be hydrolyzed by b-glucanases. The addition of enzymes such as Rapidase BE (Gist Brocades) to the berries at 100150 g ton1 during the maceration step makes extraction, ltration, and con-

centration of these juices easier. After pasteurization the juices are cooled down to 3035C and if necessary, treated with pectinase to remove any residual pectin still remaining in these juices. The juices are then ltered and concentrated. They are stored at low temperature and in dark conditions. 4.1.2. Cloudy juices Pectin enzymes containing high levels of polygalacturonase activity are added to fruit juices to stabilize the cloud of citrus juices, pur ees and nectars. Some examples are described below. 4.1.2.1. Orange. In contrast to apple pectin, which is highly methylated, pectin from orange is only partly methylated. This is because orange juice naturally contains large amounts of pectin esterase: an enzyme which strips methoxyl groups from pectin molecules. In the presence of calcium ions, insoluble calcium pectate is formed in orange juice leading to the undesirable precipitation of haze particles. Two methods are widely employed to prevent this cloud loss: the rst is to denature the pectin esterase by heating the juice. Unfortunately this spoils the juice avor. An alternative is to freeze the juice concentrate, thereby holding the enzyme in an inactive state. Orange juice is often sold like this, but the drawback is that frozen concentrates are expensive to store and transport. In the juice extraction process from oranges pectinases can be used at two stages of the process. They can be added either at the end of the pulp wash extraction at 0.52.0 g per 100 kg pulp at room temperature, to reduce the viscosity or can be added after the rst nisher at the same dose at room temperature and left for about 30 min (Rebeck, 1990). This gives a better extraction of sugars and soluble solids, resulting in a higher yield and therefore a lower viscosity. Enzymatic treatment increases cloud stability. In this treatment, degradation of the pectin is limited to lower the viscosity without attacking the insoluble pectin that maintains the stability of cloud. It is very important to use pectinases with the lowest activity of PME as possible, to avoid clarication of the pulp wash. The ideal enzyme would be a pure PL. On the other hand, Rapidase Citrus 2000 (supplied by GistBrocades) contains side activities, which decrease the bitterness of the pulp wash, and improve its taste and avor level. 4.1.2.2. Lemon. The traditional method of clarifying lemon juice relies upon the natural pectin esterase content of the fruit. The major products e.g., cloudy peel products are obtained principally from citrus peel. Peels and pulp are ground to particle size of 35 mm and mixed with water (1:1), heated at 95C to destroy the citrus PME, and cooled down at 50C. The mixture so obtained contains a high content of pectin, polysac-

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charides, glycoproteins, waxes and essential oils. It is treated with pectinases or pectinases plus cellulases. After 1 h, the mixture goes through a nisher and the extract liquid is further depectinized with enzyme if necessary. It is centrifuged, pasteurized, concentrated, bottled and marketed. Recovery of peel oils. Essential oils are located in special cells in the avedo of citrus fruits. They contain hydrocarbons (terpenes and sesquiterpenes), oxygenated compounds (aldehydes, esters, alcohols, ketones, phenols) and non-volatile residues (waxes, avonoids, fatty acids). After juice extraction, the peel particles and the oilwater emulsion are separated. The nished emulsion is passed through a sand cyclone and is fed to a centrifuge to produce an oil-rich emulsion, which is sent to a polisher that recovers the clear oil (Sterger, 1979). The oil is then concentrated up to 6080%. In the second stage, the polisher removes the remaining water and the very ne particles. A cold storage allows the oil to be dewaxed for a better purication. Enzymes can improve the process time, yield of oil from the emulsion, and the quality of the nal product. Better separation of the oilwater emulsion needs pectinases and proteases. By hydrolyzing the pectinprotein complexes, oil is released more easily into the aqueous phase. Citrus salads. Citrus salads can be prepared by treating peeled citrus fruits with pectinases to digest residual albedo still bound to the segments. These materials are immersed in pectinase solution e.g., 1 2% Rapidase citrus 2000 (GistBrocades), for 3060 min at room temperature. The products are then pasteurized for storage or chilled to be used for citrus salads. Dried animal feed. Peels, seed residues and pulp ejected by the citrus fruit extractor can be used to make dried animal feed. The mixture is lime treated to increase the pH to 8.0 to take advantage of the citrus PME. As this endogenous enzyme has optimal enzyme activity at these pH values, it causes the demethylation of citrus pectin (about 7080%). Enzymes such as cytolase M 102 (GistBrocades) are used to improve the extraction of fermentable sugars, and formation of calcium pectate, increase the rmness of the mixture, and allow better yields of extracted liquor. The pomace is then dried to 810% moisture and this is used as an alternative or a supplement to corn as a source of concentrated feed nutrient for dairy cattle and sheep. 4.1.2.3. Mango. Mango is processed as a pur ee or nectar, juice and various juice drink blends. A typical mango nectar is one with 2030% pur ee, pH around 3.5 and titrable acidity 0.20.3%. Exogenous enzymes are used to increase the concentration of mango pur ee or to produce clear juice and concentrate. Pectinases, hemi-

cellulases and cellulases are important activities used to degrade mango pulp. Enzymatic treatment of mango mash provides a yield of around 80% of total mango juice present in the fruit. 4.1.2.4. Apricot. Apricot nectar exhibits a phenomenon of cloud loss, which can be avoided by grinding the pulp very nely (Siliha and Pilnik, 1985). These cloud particles of apricot nectar are composed of 86% individual apricot cells, 5% broken cells, 4% cell wall fragments and other aggregates. Cells in the fruit pulp contains only primary cell walls that consist of approximately 90% polysaccharide and 10% protein (Weiss and Samann, 1972). Addition of pectolytic enzymes separately, or in combination with homogenization, leads to a cloud-stable apricot nectar, whereas homogenization alone fails to achieve cloud stability. 4.1.2.5. Guava. The major producers of guava are South Africa, India and Hawaii. It is eaten fresh or made into juice, nectar, pur ee, jam and jelly. Guava pur ee is reprocessed into nectars, juice drink blends, syrups, jams and jellies. Fruit pulps are treated with exogenous enzymes to improve juice extraction (Chen Chin et al., 1984). Guava pericarp, like pear, has two main cellular types: parenchyma and stone cells. Stone cells are woody materials that are strongly lignied and resistant to degradation by pectinases. The mesocarp portion of the guava contains 90% of the total cell wall material of guava pulp 77% of which are stone cells. The dominant structural feature in parenchyma cell walls is cellulose with side chains of glucans and xyloglucans and highly branched arabinans. Therefore a combination of enzymes such as pectinesterase, arabanase, hemicellulase, tannase and cellulase are used to improve the clarication of the guava juice. 4.1.2.6. Papaya. Papaya pur ee is prepared by conversion of papaya esh into a semi-liquid product. As the pur ee is viscous even if the fruit contains endogenous PME and PG, it is necessary to depectinize it before concentration. Pectinases such as Rapidase (GistBrocades) can be added for this purpose. The depectinized pur ee is then concentrated from 2.4 to 3 fold. 4.1.2.7. Pineapple. The processing of pineapple mainly involves the fruit being sliced or cubed and then canned. A cloudy or clear juice concentrate can be processed and is used to cover the slices and cubes in the cans. Pineapple juice results from the surplus juice drained from the eradicator, juice drained during crushed-pineapple preparation, or from fruits which are too small for slicing (these are byproducts of the canning factory). This juice is pasteurized, cooled down to produce cloudy juice or depectinized at 50C for 2030 min or at

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20C for a few hours to produce clear concentrate. Enzymes such as Rapidase pineapple (GistBrocades), pectinex Ultra SP (Novo nordisk) or Rohapect BIL (Rohm) are used to process a clear concentrate (Grassin and Fauquembergue, 1996). 4.1.2.8. Banana. Banana is widely appreciated for its avor and aroma (isoamylacetate) either as banana juice or a mixture with other juices. However, banana juices are too pulpy and too viscous to yield beverage juices easily. Banana can be processed at the green stage, mainly for alcohol production, or allowed to mature. Dierent enzymatic treatments are required to produce pur ee or clear concentrate juice. Ripe bananas have high sugar content, which increases the temperature of starch gelatinization. This is why exogenous amylases like thermostable bacterial amylase (BATs) are used after heating the pulp to 100C for 10 min for starch gelatinization. After cooling to 45C and pH correction with organic acid to 4.5, Hazyme (GistBrocades) is added at 15 g per 100 kg over 23 h to produce fermentable sugars, which can be used for ethanol production. Yellow, fully ripe, bananas are blanched at 85C for 23 min after peeling and grinding (this is done to inhibit banana polyphenol oxidase and prevent browning), or treated with 100 ppm sulfur dioxide. Banana pH is 4.54.8 and optimal for exogenous pectinases. Blanched pulp is macerated using pectic enzymes, pressed and the juice is claried or centrifuged before concentration. Enzyme treatment gives reduced viscosity, high yield and concentration of the juices. Alcohol can also be produced from these juices. 4.1.3. Unicellular products Unicellular products are those products which are formed by the transformation of organized tissues into a suspension of intact cells, resulting in products that can be used as base material for pulpy juices and nectars, as baby foods, as ingredients for the dairy products such as puddings and yogurt and as protoplasts for various biotechnological applications. The enzymes used for this purpose are referred to as ``macerases'' and this process is referred to as maceration. It is likely that the best enzyme preparation for maceration contains cellulases and hemicellulases in addition to the pectic enzymes. Some examples are described below. 4.1.3.1. Maceration of plant tissue. Maceration is a process by which organized tissue is transformed into a suspension of intact cells, resulting in pulpy products used as base material for pulpy juices and nectar, as baby foods and an ingredient for diary products such as puddings and yogurts. Enzymatic degradation of pectin after a mild mechanical treatment often improves product properties if the process is carried out to leave

as many cells as possible intact, and the aim of enzymatic treatment is the transformation of tissue into a suspension of intact cells (Bock et al., 1983). Pectin degradation should aect only the middle lamella of the plant tissues. Before maceration, the endogenous PE must be inactivated, and this is important for the maceration of many products (Dongowski and Bock, 1980). Exogenous pectinases for fruit juice extraction and clarications are based on the destruction of pectin and other dietary compounds of the fruits. Enzymatic maceration leads to limited pectin degradation, therefore preventing starch leaking out of the cells and avoiding the quality defect of gluiness of the reconstituted product. The process is often used for carrots (Bock et al., 1984) and dried instant potato mash (Bock et al., 1979). 4.1.3.2. Liquefaction and sachharication of biomass. Agricultural resources of fermentable carbohydrates such as sugar beat, sugarcane, wheat, rice, and potatoes must be processed in order to obtain fermentable sugars. Large quantities of plant cell-wall materials become available as byproducts in the beet-sugar and potato starch industries. A large spectrum of enzymes (pectinases, cellulases, hemicellulases) is needed for the production of fermentable sugars from polysaccharides and for the disruption of the cell wall matrix, so giving liquefaction of the material and the release of intracellular carbohydrates (Beldman et al., 1984). 4.1.3.3. Isolation of protoplasts. In plant breeding programmes, many desirable combinations of characters cannot be transmitted through conventional methods of genetic manipulation. A process other than the sexual cycle has now become available for higher plants and can lead to genetic recombination. This non-conventional genetic procedure, involving fusion between isolated somatic protoplasts (walless, naked cells) under in vitro conditions and subsequent development of their product (heterokaryon) to a hybrid plant, may be a useful tool for the induction of genetic variability and combination of traits which do not exist in nature (Gleba, 1978). Methods of protoplast isolation from the plants are classied into three main groups: (a) mechanical (nonenzymatic), (b) sequential enzymatic (two steps), and (c) mixed enzymatic (simultaneous) procedures. Mechanical isolation, which includes cutting of plasmolysed tissue with a sharp-edged knife and release of protoplasts by deplasmolysis, is now only of historical importance, because of the low yield of the protoplasts obtained by this method. Therefore, enzymatic processes involving pectinases and cellulases mixture have been tried for this purpose. Takebe et al. (1968) employed a sequential or twostep procedure for isolating protoplasts using commer-

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cial preparations of enzymes. This approach involves initial incubation of macerated plant tissues with pectinases. The tissues are then converted into protoplasts by a cellulase treatment. However, Power and Cocking (1969) mixed two enzymes together (simulaneous procedure) and isolated protoplasts in one step. In this mixed enzyme approach, plant tissues are plasmolysed in the presence of a mixture of pectinases (0.5% (w/v) macerozyme, Kinki Yakult Manufacturers, Nishinomiya, Japan, or 0.5% (w/v) pectinase, Sigma Chemicals, St. Louis MO, USA; or 5% (w/v) pectinol R10, Rohm and Hass, Philadelphia PA, USA) and cellulase (5% (w/v), Onozuka P1500 from Trichoderma viride, Meiji Seika Kaisha, Tokyo, Japan) in 20% (w/v) sucrose adjusted to pH 5.6 with 2 N HCl. The use of commercially available enzymes of fungal as well as bacterial origin has enabled the isolation of protoplasts from practically every plant tissue in which cells have not acquired lignication. 4.2. Alkaline pectinases Alkaline pectinases are mainly used in the degumming and retting of ber crops and pretreatment of pectic wastewater from fruit juice industries. These enzymes come mostly from bacterial sources. In the industrial sector, alkaline pectinases, mainly from Bacillus spp. are applied for the following purposes. 4.2.1. Retting and degumming of ber crops Pectinolytic enzymes are involved in the retting and degumming of jute, ax, hemp, ramie, kena (Hibiscus sativa), and coir from coconut husks (Chesson, 1980; Bruhlmann et al., 1994). Retting is a fermentation process in which certain bacteria (e.g., Clostridium, Bacillus) and certain fungi (e.g., Aspergillus, Penicillium) decompose the pectin of the bark and release ber (Sharma and Robinson, 1983). Commercially, retting is done by one of the two basic forms. In dew retting (an aerobic process) plant straw is thinly spread on the ground and exposed to the action of the fungi and aerobic bacteria for 210 weeks. Species of Cladosporium, Penicillium, Aspergillus and Rhodotorula have been isolated from dew-retted plants (Fogarty and Ward, 1972). Dew retting may produce ber of a lower quality than the alternative anaerobic process. Anaerobic retting is achieved by submerging straw sheaves in water pits, concrete tanks or in running fresh water (Chesson, 1980). Tank and other stagnant retts rapidly become depleted of dissolved oxygen encouraging the development of an anaerobic ora (Chesson, 1978). Species of Clostridia, especially Clostridium butyricum and Cl. felsineum are considered as the major retting agents (Hellinger, 1953; Vonzyakovskaya et al., 1974); Bacillus, Pseudomonas and Micrococcus spp. have been isolated from retting ax (Rosemberg and de Franca, 1967), jute

(Ahmed, 1963), sisal and coir (Jayasankar et al., 1967) and have been shown to macerate straw ber in the laboratory. During water retting of ax, ber separation is brought about by a pectinase enzyme produced by bacteria (Chesson, 1978). In a series of experiments conducted by Sharma (1987), a mixture of pectinase and Ultrazyme and an enzyme extracted from culture of Ceraceomyces sublaevis has been found to be the most suitable enzyme preparation for deploymerizing noncellulosic material present on dew retted bers (at 45C, pH 5.06.0). When characterized, the commercial enzymes used in this investigation contained a wide range of polysaccharide degrading enzymes (polygalacturonase, PL, xylanase and traces of cellulase). Pectinol AC (pectinase preparation) contained PG, xylanase and traces of PL. Ultrazyme contained xylanase as the main component of the preparation along with traces of PG. Ramie bers are an excellent natural textile, but decorticated ramie bers contain 2035% ramie gum, which mainly consists of pectin and hemicellulose; hence it is necessary to degum bers for meeting the requirement for textiles. In a classical degumming process, this gum is removed by treatment of decorticated bers with hot alkaline solution (1220% NaOH solution) with or without application of pressure (Cao et al., 1992). In addition to the high consumption of energy, this process also results in serious environmental pollution. The degumming with microbes and their enzymes is a likely means to solve this problem. A combined microbial and enzymatic process has been proposed to reduce the consumption of chemicals and energy (Paul and Bhattacharya, 1979; Gurucharanam and Deshpande, 1986). Although degumming with neutrophilic bacteria has been reported (Cao et al. (1992), only a few reports of degumming with alkalophilic bacteria have been published. For degumming mostly neutrophillic microbes have been studied so far (Gurucharanam and Deshpande, 1986), but due to diculties in prevention of heterogenous contamination and some other disadvantages, these strains have not been applied on an industrial scale. Deshpande and Gurucharanam (1985) reported that pretreating the decorticated ramie bers with alkali could accelerate the degumming process. In a series of experiments carried out by Cao et al. (1992), they have reported the isolation of a Bacillus sp. (strain NT-33) which could grow well at pH 10.0, which is of great advantage to the productivity and activity of degumming enzymes. Moreover, fermentation at high pH values also has the advantage of preventing contamination and thus an open fermentation process can be developed. This strain has been reported to remove more than 70% ramie gun after 24 h fermentation under alkaline conditions. The quality of degummed bers produced by this technique sat-

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ised the textile industry standards. Pectinolytic enzymes from actinomycetes have also shown good correlation between the pectate lyase activity and the degumming eects, resulting in good separation of the bast ber. 4.2.2. Treatment of pectic wastewater 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). These processes have some defects, such as low eciency due to chemical resistance of the pectic substances, high treatment cost, long treatment periods and complexity of the process. A soft-rot pathogen, Erwinia carotovora (FERM P7576), which secrets endo-pectate lyase, has been reported to be useful in the pretreatment of pectinaceous wastewater (Tanabe et al., 1986). However, because of the danger from its phytopathogenecity, indirect pretreatment by the pectolytic enzyme produced from the bacterium has also been compared and reported to solubilize almost all the pectic substances contained in the wastewater. 4.2.3. Production of Japanese paper Alkaline pectinase produced by Bacillus sp. and Erwinia carotovora, due to its strong macerating activity, has been used for retting of Mitsumata bast (Tanabe and Kobayashi, 1987). These retted basts have been used for the preparation of Japanese paper (Horikoshi, 1990). The strength of the pulp from bacterial retting is as high as that obtained by the conventional soda-ash cooking method. The paper sheets prepared from this pulp are very uniform and soft to touch. 4.2.4. Paper making Pulp and paper mills are beginning to use enzymes to solve problems in their manufacturing processes. Papermaking is essentially a continuous ltration process in which a dilute suspension of bers, ber fragments (nes), and inorganic ller particles, such as clay or CaCO3 , is formed into sheets. The need for water drainage leads to use of a lter fabric with holes large

enough to allow passage of the nes and ller particles. In modern papermaking, retention aids are added to pulp to keep nes and ller particles in paper sheets and to speed the drainage of water. Cationic polymers of various structures are commonly used as retention aids (Horn and Linhart, 1996). Alkaline peroxide bleaching of pulps solubilizes polysaccharides, which are troublesome interfering substances (Holbom et al., 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 et al., 1994). Pectinase can depolymerize polymers of galacturonic acids, and subsequently lower the cationic demand of pectin solutions and the ltrate from peroxide bleaching (Reid and Ricard, 2000). 4.2.5. Oil extraction Oils from rape seed (Canola), coconut germ, sunower seed, palm, kernel and olives are traditionally produced by extraction with organic solvents. The most commonly used solvent is hexane, which is a potential carcinogen. Cell-wall-degrading enzymes, including pectinase, may be used to extract vegetable oil in an aqueous process by liquefying the structural cell wall components of the oil-containing crop. Recently, the plant cell-wall-degrading enzyme preparation has begun to be used in olive oil preparation. The enzymes are added during grinding of the olives, thereby the oil is released easily in the subsequent separation techniques (West, 1996). Enzyme treatment hence causes an increase in yield of olive oil. The increase in the yield depends upon pH, temperature, and dosage of the enzyme used. Olivex, an enzyme preparation derived from A. aculeatus, that contains pectinolytic activity besides hemicellulases and cellulolytic activity has been shown to give good oil extraction and better stability when stored. The oil also shows increased content of polyphenols and vitamin E, which stabilizes the oil against rancidity. 4.2.6. Coee and tea fermentation Pectinases play an important role in coee and tea fermentation. Fermentation of coee using pectinolytic microorganisms is done to remove the mucilage coat from the coee beans. Pectic enzymes are sometimes added to remove the pulpy layer of the bean, threefourths of which consists of pectic substances. Cellulases and hemicellulases present in the enzyme preparation aid the digestion of the mucilage. A diluted commercial enzyme preparation is sprayed on to the beans at a dose

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of 210 g per ton at 1520C. The fermentation stage of coee processing is accelerated and reduced from 40 to 80 h to about 20 h by enzyme treatment. Since largescale treatment of coee with commercial pectinases is costly and uneconomical, inoculated waste mucilage is used as a source of microbial pectic enzymes. The fermentation liquid is washed, ltered and then sprayed on to the beans (Amorim and Amorim, 1977; Carr, 1985; Godfrey, 1985). Fungal pectinases are also used in the manufacture of tea. Enzyme treatment accelerates tea fermentation, although the enzyme dose must be adjusted carefully to avoid damage to the tea leaf. The addition of pectinase also improves the foam-forming property of instant tea powders by destroying tea pectins (Carr, 1985).

5. Commercial pectinases

Supplier C.H. Boehringer Sohn Ciba-Geigy, A.G. Grinsteelvaeket Kikkoman Shoyu, Co. Schweizerische Ferment, A.G. Societe Rapidase, S.A. Wallerstein, Co. Rohm, GmbH

Location Ingelheim, West Germany Basel, Switzerland Aarthus, Denmark Tokyo, Japan Basel, Switzerland Seclin, France Des Plaines, USA Darmstadt, West Germany

Brand name Panzym Ultrazyme Pectolase Sclase Pectinex Rapidase, Clarizyme Klerzyme Pectinol, Rohament

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