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Paper No. 6 Paper Title: Technology of Fruits and Vegetables Module No. 30 Module Title: Application of enzymes in fruit and vegetables processing 1.

1 Paper No. 6 Paper Title: Technology of Fruits and Vegetables Module No. 30 Module Title: Application of enzymes in fruit and vegetables processing 1. Introduction Fruits and vegetables are consumed as fresh, or processed into different type of products. In order to extend the shelf life and maintain the quality of fruit and vegetables, inactivation of some of the naturally occurring enzymes in fruits and vegetables is needed. Enzymes also exhibit activity towards specific subtract under mild processing conditions, which can be used to rise the process efficiency and product quality, therefore need to boost up the commercial enzyme preparations as aids in fruits and vegetables processing. Hence, there has been a striking growth in the enzyme market for the fruit and vegetable industry. In any industry, it is important to know, how to get the most out of their process. Use of enzymes is one such solution for the fruit processor. For example wines, which mature earlier can, have enhanced aroma and colour stability by using desirable enzyme. Furthermore, production yields and purities of intermediate raw materials can be improved, and by products, previously thought of as waste, may now be utilized by using some desirable enzyme. 2. Enzymes

2 Enzymes are proteins and biological catalysts produced by all living organisms and promoters of vital chemical activity in plant and animal cells. They are also biodegradable and work efficiently and rapidly in increasing the reaction rate of biochemical processes that otherwise proceed very slowly, or in some cases, not at all. They play an important role in food processing. 3. Role of enzymes in Fruits and Vegetables Enzymes have not only beneficial, but also detrimental effects on fruit and vegetable products. The oxidizing enzymes are normally responsible for the rapid browning of cut fruits for examples the browning of wine and fruit juices, the reddening and bluing of potatoes etc. Due to their participation in a vast number of biochemical reactions in fruits and vegetable, in addition to browning, enzymes causes detrimental effect on the appearance of the product, changes in nutritive value, colour, texture and flavor owing to uncontrolled activity of these enzymes. The hydrolytic enzymes which normally act on anaerobic condition bring changes in wines. Optimal temperature for enzyme activity is around C, hence they get deactivated when heated beyond this optimal temperature. Enzyme should be destroyed otherwise the ripening process continues which can causes spoilage of fruit and vegetables. Thus enzymes and micro-organisms in fruit and vegetable are deactivated by heat treatments, thus improving the fruit and vegetables' storage stability.

3 4. Legislation Situation Most of the enzymes employed are not pure enzymes, they contain further secondary activities. For example, pectinase is often used with cellulases, hemicellulases, and proteases. That is why various international organizations use the word enzyme preparations instead. The enzyme preparation technology and their use in processing are rising fast during the past decade. In EU member states, enzymes are generally used and considered as processing aids since most are used during food processing. Currently, there are some specified legal regulations for use of a specific enzyme in defined application in fruit and vegetable processing. In most member states, there is no general statutory notification or approval system for enzymes used as processing aids. Instead, the use of processing aids is controlled by general regulations requiring the safety of all food ingredients. Only France and Denmark have in place a statutory procedure for notification of the use of enzyme products which is based on specific legislation for food enzymes. International organizations like the JECFA or the FAO/WHO have issued generally accepted guidelines for the toxicological assessment and purity criteria of enzymes. 5. Application of Enzymes in Fruit and Vegetable Processing The product quality and operations efficiency like peeling, juicing, clarification and extraction of value-added products is improved by using

4 suitable enzyme. The enzymes are used first on the bench-top and then in the processing plant. Usually enzyme selection is based on fruit; however, the composition of fruit varies from period to period due to effect of season and harvest condition which can mislead the results, so enzymes selection can be based on other factors like type of fruit, process, fruit s ph, processing temperature, ripeness stage or plant personnel s past experiences. 5.1 Fruit juice processing The most vital economic operation is the preparation of fruit and vegetable juices that provides the consumer valuable fruit and vegetable constituents. Basically it involves extracting the liquid portion of fruit juice and preserving it by pasteurization or concentration, either in clear (filtered) or cloudy form. The commercially produced juices includes: (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. Kiwi fruits processing emerged from 1970s but got some problem regarding its processing due to difficulty in processing, as compared to other fruits. Kiwifruit processing causes various kind of problems such as changes in flavour, loss of the green colour, development of brown pigments, formation of hazes and precipitates in liquid products, and the sensation of an irritation

5 ("catch") in the throat when eaten. Enzyme treatments have been used successfully to aid juice extraction from kiwifruit pulp. As with many forms of juice processing, commercial enzymes like a mixture of polygalacturonase and pectinase are added to assist the extraction of juice from pulp. The use of enzymes permits the production of juices and concentrates of sparkling clarity once the pectin are sufficiently degraded Juice clarification Filtration is a necessary step in fruit juice processing to obtain clear juice before being processed further. About 70% of the juice can be extracted from fruits such as a grape by pressing, but it faces some problem related to filtration and clarification. For example, in case of fruits such as apple, polymeric substances such as starch and arabans, may make filtration difficult and cause post process cloudiness. Mixtures of enzymes are necessary at this processing step. The enzyme such as cellulases, amylases, and pectinases assist the different process like maceration, liquefaction, and clarification during fruit juice processing with the benefit of reducing processing costs and improving yields, quality and stability of juices manufactured. Amylases may be required after pressing to degrade starch for clear apple juice production. In case of lemon juice, traditionally pectin esterase was used for clarification purpose, which resulted in precipitation of the suspended solids as calcium pectate. But it took several weeks (4-6 weeks) and microbial problems

6 were encountered, so use of fungal enzyme, which are available for very acidic juices (ph ) are now commonly used. It achieves clarification within 6 hr. without any microbial problem. Nowadays, pomegranate processing is also getting momentum. Gelatin treatment is given to pomegranate juice for improvement over the conventional process of clarification. Before concentration, there is a need to eliminate insoluble solids and destroy pectin substances from the pressed juice and its clarification. Also heating processes increases the haze formation, hence the need of enzyme treatments for depectinizzation and destartification. For example, in case of unripe apple 15% starch is normally present. Although, juice is centrifuged (second normally carried out in centrifugation tank) but still 5% starch remains in the apple juice, hence amylase (also called amyloglucosidase) is added along with pectinase which are active at ph of apple juice, for the purpose of breakdown of this remaining 5% starch Mode of action Pectinases assist the clarification process in following way: Pectin has a tendency to form a protective coat around protein in suspensions which carry negative charge. In acidic condition (apple juice ph 3.5), they repel from each other s due to their same charge. Addition of pectinase causes the breakdown of this coat of pectin and thus exposes the positively charged protein part

7 beneath. Hence, electrostatic repulsion between cloud particles gets reduced and they can clump to each others. These clumps then can be easily removed either by settling or centrifugation. The flocculation agents such as gelatin, tannin or bentonite causes enhanced settling of cloud particles. Some agents adsorb the enzyme to the surface, so they are desired to be added after the enzymes have done their job Increase juice yield Juice production from banana, guava, mango, etc is problematic as they are not easily extractable like other fruits such as oranges and lemon. Enzyme treatment significantly improves the yield of the juice by efficiently degrading the cell walls as compared to conventional methods and thereby minimizes the waste and energy use per unit of the juice produced. The application of pectinase and other enzymes facilitates liquefication of the entire fruit. In addition, it improves saccharification and thus sweetness of the end product. In case of grapes, enzyme treatments help in efficient release of juice which is responsible for increase in its yield up to 12 percent. It can also improve color extraction while processing of red grapes. The kind of enzyme usage is mainly dependent on variety and growing condition of grapes. In case of mango, enzyme treatments along with manual and mechanical treatments are necessary for getting efficient yield of some mango varieties such as totapuri. In case of pineapple, enzyme treatment aids

8 in release of juice from the tough fibre. The yield of juice from waste fibre is also considerably enhanced, resulting in profit of the processor. Pectinases with a high proportion of pectinesterase and liquefying polygalacturonases are suitable both for mash treatment (carried out at 20-30ºC for min) and pomace extraction. The hydrolysis of the protopectin that binds the cells weakens the fruit tissue, causing the protopectin to dissolve and increasing the juice viscosity. This leads to reduction in press performance and juice yield. Pectinase treatment breaks the dissolved pectin in the juice phase immediately which reduces the viscosity of the press and makes run off the press faster. This gives greater yield and press capacity Juice Filtration Ultrafiltration is used for production of clear juices by removal of suspended solids, large molecules like proteins, enzymes and to some degree limit dextrins. Ultrafiltration is a cross-flow process (the juice flows through the membranes horizontally) and filters sterile juice in one step without the addition of clarifiers. Nanofiltration is used for improving hydrolysate quality (e.g. increases the DX of glucose syrup or increases the maltose content in maltose syrups). With ultrafiltration membranes, the separation capacity is characterized not by pore size but by the so-called separation limit. The low molecular weight constituents of the juice pass through the membrane whereas the high

9 molecular weight substances are held back and concentrated. The liquid phase that passes through the membrane is usually termed permeate, and the concentrated product stream, the retentate. Membrane fouling is particularly important from enzyme technology perspective. This problem is caused by colloidal constituents of the juices, which build up and subsequently clog the membranes. Treatment with large quantities of enzymes helps to guarantee the complete hydrolysis of pectin substances and starch and minimizes the risk of irreversible blockage of the costly membranes. These results are improved by using combination of pectinase, hemicellulase and protease enzyme Other uses of enzymes treatment Debittering The bitter flavour associated with citrus fruits such as grape fruit or novel oranges is caused by two distinct groups of compounds: flavonoids and limonoids. There has been extensive research carried out regarding reduction of the bitterness of these compounds. Naringin is the main bitter-tasting flavonoids, found in all parts of the fruit but especially found beneath the skin. Thus during juice extraction grapefruit is normally selected on the basis of low naringin

10 content or juices are blended (different grape fruit varieties) to minimize the degree of bitterness. In USA and Japan studies were conducted regarding control of bitterness by enzymatic hydrolysis of bitter components (naringinase-obtained from fungi) so as to improve the quality attributes of citrus juice. Naringinase is an enzyme that removes the glycoside groups from compounds causing bitterness such as naringin and limonin. It breakdowns naringin into intermediated compound, prunin and then non-bitter naringenin compound. This process needs correct degree of breakdown which is achieved by controlling the flow rate of enzyme, hence this process was getting difficult. Further resin was also used for absorbtion of naringin but it also adsorbs other desirable compounds. Unlike naringin and limonin, bitter compounds are also formed after only few hours, by natural chemical reaction (called delayed bitterness). Bitterness in juice is promoted by pasteurization treatments. In late 1980s, the enzyme limonin glucosyltransferese (LGT) was used to convert limonin into tasteless compound. Unfortunately it is present in small quantities during the ripeneing of the citrus fruits. In 1998, Japanese with the help American co-workers produced genetically modified orange trees that bear fruits with more limonoid glucosides. These would not cause the bitterness of citrus juice. The use of small amounts of this enzyme in citrus juice, aids in improvement of flavour by degradation of bitter compounds and also increase the saccharification of the juice.

11 Flavour improvement For white wines, especially muscatel or Gewürztraminer, cleaving of aroma precursors by beta glucosidase action has been shown to release the desired terpenols that improve flavour Cloud stability The stability of suspended matter in naturally cloudy apple juices and apple juice concentrates is determined by the content of water-soluble pectin, the size of the suspended particles and the charge intensity of the hydrated pectin in the suspended matter. The native starch present in the juice get dissolved by the heating processes which increases the viscosity of the juice and thus also provides conditions for better cloud stability. However, during storage the dissolved starch retrogrades in the juice, particularly in the concentrate, and finally precipitates. The pectinase helps in removal of pectin, release of juice and enables the juice to concentrate without dangers of gelling and high viscosity. But still some insoluble pectin remains in suspension which gives cloudy appearance. Pectinase with high pectin lyase activity rapidly split the high molecular weight, soluble pectin into medium-sized pectin fragments, thus reducing viscosity within a very short time by 25-50%. The selective pectin lyase activity prevents the total degradation of the pectin, which would cause cloud destabilization.

12 The maintenance of stable cloud in citrus fruit is a challenge for the fruit industry. It can be prevented by using artificial clouding agent, but these agents are prohibited in many countries. So use of pectinase is an effective method used for prevention of cloud in fruit and vegetables juices. Pectinase with lyase activity is useful for producing cloud-stable juice since the presence of methyl esters is important to maintain the stability of the cloud. The enzyme polygalacturonase rapidly cut the pectin chain so prevent formation of calcium linked precipitate in orange juice. The apple juice contains enzyme pectin esterase which strip methoxyl group from the pectin molecules. In the presence of calcium ions, insoluble calcium pectate is formed in orange juice. This kind of cloud can be prevented by addition of pectin esterase inhibitors (short pectin chain), which compete with longer pectin molecule for attention of enzyme, slowing the rate at which it can attack them. A typical enzyme dosage before the press ranges from 40 to 80 ml of enzyme per ton, with more required for fruits that have been in cold storage Peeling Enzymes are able to soften the peel by disruption of the albedo, so nearly 100% peel removal can be achieved in citrus fruits, thereby facilitating a significant reduction in energy costs. Compounds such as pectin, cellulose, and hemicelluloses are responsible for the adherence of the skin to the fruit.

13 Therefore, both pectinases (probably needed for the release of the pectins in the albedo) and cellulases (contribute to the hydrolysis of the polysaccharides of the cell wall) are required for the enzymatic peeling. Fruit are required to be washed with chlorinated water or hot water before enzyme treatments and then vacuum infused with enzyme solution and incubated in water heated to 40 C for 30 minutes. The enzyme penetrate into the peel albedo where the peel is digested to the point where it can be easily removed. 5.2 Vegetable Juice Processing Carrot is the most commonly processed vegetable. The enzyme treatments to carrot juice give homogeneity, better yield and optimum color extraction. Two processes are employed for manufacturing carrot juice or concentrate or carrot puree. Pectinase with high pectin lyase activity along with cellulase (legislation allows to use) is suitable for manufacturing juice or concentrate with increased juice yield and ß-carotene. A combination of different enzymes can also be useful for preparation of vegetables purees and concentrate e.g. beetroot, celery, parsley etc. Enzyme treatment has been also used to obtain superior tomato paste quality due to reducing holding time, particularly at the higher concentration at low temperatures. In addition, the viscosity of the paste is also reduced thereby reducing the cost of energy and time required in conventional processes. 5.3 Herbal Extraction

14 Herb extraction involves some methods used for extraction of some compounds from the herb by using suitable extraction liquid such as water, alcohol etc. while some method of concentrating that liquid so there is not any liquid remain in final product. The quality of extract materials from herbs such as Gingko biloba and Valeriana officinalis is significantly (p<0.05) improved by using suitable enzyme. Use of enzymes can also possible to extraction of heat labile biological active compounds under the cold condition efficiently and within a shorter time period. 5.4 Oil Processing 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. In olive oil processing, enzyme processing is important operation which significantly improve both oil quality and yield in olive oil processing. The increase in the yield depends upon ph, temperature, and dosage of the enzyme used. The enzyme preparations involve various kinds of exogenous enzyme species such as pectolytic, cellulolytic and hemicellulolytic. These enzymes individually or together with minor one is incorporated to the olive paste at the beginning of the malaxation step during mechanical oil extraction. They represent an important innovation in the extraction process and markedly increase the efficiency of the current mechanical processing systems.

15 Enzyme has a tendency to degrade the vegetable colloids (pectins, hemicelluloses, proteins, etc.) emulsifying the minute oil droplets and later on remove the oil-water emulsions. In addition, the rheological characteristics of the olive paste are also improved. They are water-soluble and come out in the liquid effluent (wastewater) during the final step (oily must be centrifugated) of the extraction cycle. Their effect on the parenchyma and hypoderm tissues increase the release of the minor components (biophenols, tocopherols, chloroplast colourings, etc.), thus enhancing the nutritional and healthful effects of VOO (Virgin Olive oil). They also increase the oil outputs (up to 2%, olive weight basis) and furthermore decrease the environmental impact (-30%) of the liquid effluent by lowering the values concerning COD (Chemical Oxygen Demand), BOD (Biological Oxygen Demand), total solids, volatile solids, residual fat and suspended solids. 5.5 Deoxygenation Glucose oxidase is an enzyme used in wine and fruit juice processing so as to remove oxygen from environment. This is very useful as presence of oxygen promotes enzymatic browning and oxidative rancidity reactions which deteriorates the quality of foods. 6. Conclusion There are many ways in which commercial enzymes touch our lives. They are tools of nature that help provide everyday products in an environmentally

16 conscious manner. Current commercial use of enzymes, together with new applications, will continue to play an important role in maintaining and enhancing the quality of life we enjoy today while protecting the environment for generations to come. References Kashyap DR, Vohra PK, Chopra S, Tewari R (2001). Applications of pectinases in the commercial sector: a review. Bioresource Technology 77: Alev Bayindirli (2010). Enzymes in Fruit and Vegetable Processing. CRC press, Taylor and Fancies Group, LIC, Boca Raton, London New York. Food Agricultural Organization. (2009). Handbook of Fruit and Vegetable Processing, Viale delle Terme di Caracalla, Rome, Italy.

Pectin. Gelation properties of pectins. 2 Hypothetical Pectin Structure. 3 Pectin. Gelation of high methoxyl pectins. Gelation of low methoxyl pectins

Pectin. Gelation properties of pectins. 2 Hypothetical Pectin Structure. 3 Pectin. Gelation of high methoxyl pectins. Gelation of low methoxyl pectins 1 Pectin X is either H or CH 3 Subject to enzyme attacks Found in virtually all land-based plant as a structural material Commercial pectin extracted from citrus peel, apple pomace, sugar beet, or sunflower