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
 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 30-50°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.

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

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

("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.

5.1.1 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
were encountered, so use of fungal enzyme, which are available for very acidic

juices (pH 2.2-2.8) 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.

5.1.1.1 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
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.

5.1.2 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

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 30-120 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.

5.1.3 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
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.

5.1.4 Other uses of enzymes treatment

5.1.4.1 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

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.

5.1.4.2 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.

5.1.4.3 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.
 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

5.1.4.4 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.
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

 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.

 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

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: 215-227.
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.