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Chapter 7. Use of enzymes for non-citrus fruit juice production

Chapter 7. Use of enzymes for non-citrus fruit juice production

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Liliana N. Ceci and Jorge E. Lozano

fruit juice industry is a very important enzyme consumer. Commercial

sources of fungal pectic enzymes have been used in fruit juice processing since the 1930s for clarifying fruit juices and disintegrating plant

pulps to increase juice yields. Industrially used enzymes are similar to

the naturally occurring pectinases, cellulases, and hemicellulases found

in fruit during ripening. Most enzymes are marketed on the basis that

they are generally recognized as safe (GRAS) for their intended use in

the juice process.

In this chapter, after reviewing the non-citrus fruit juice processing,

focus is put on the use of enzymes in this particular industry. Maceration

and clarification of apple juice through mixture of enzymes (pectinases,

cellulases, etc.) and the application of other specific enzymes in juice production, as amylases, are extensively covered. Particular items as enzyme

activity determination and pH dependence are treated in some detail.

Finally, the use of immobilized enzymes and other miscellaneous applications are also summarized.

7.1.1â•…Non-citrus fruit juice production: World market

Fruit and vegetable juice world trade averaged near US$4,000 million

last decade (FAOSTAT, 2005). By far the most important non-citrus fruit

commodities are apples, which are mostly processed in the form of

juices. In 2006, apple world production was 1.2 million tons. There are,

however, many other products elaborated from fruits, as canned, dried,

and frozen fruit, pulps, purees, and marmalades. In addition, developments in aseptic processing have brought new dimension and markets

to the juice industry. In the period 1997–2006 the world production of

concentrated apple juice increased 50%, following a growing trend. This

increase is explained by the extraordinary progress of China, which

elaborates half of the world total and grows at an average annual rate

of 30%, although with a more moderate trend at present. On the other

hand, big consumers such as the United States present a decreasing production trend, with an average annual decrease of 2%. The lower price

of imported juice, among other factors, explains this trend. Global apple

juice trade was expected to have another record year in 2007–2008. In

2007, the value of juice imports grew to $1.5 billion, up from $1 billion

in 2006 (USDA, 2008).

It is well known that fungal pectic enzymes have been used in commercial applications for fruit juice processing since the 1930s, such as

clarification and disintegration of plant pulps to increase juice yields.

Commercial enzymes are similar to the naturally occurring pectinases,

cellulases, and hemicellulases found in fruit during ripening. Most

enzymes are marketed on the basis that they are GRAS for their intended

use in the juice process.

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Chapter seven:â•… Use of enzymes for non-citrus fruit juice production


Enzymes are used in the juice industry to aid in the separation of

juice from the fruit cells and to clarify the juice by the removal of pectin

and naturally occurring starches that contribute to undesired viscosity,

poor filtration, and a cloudy appearance. A considerable number of food

enzymes are on the market.

7.2â•…Non-citrus fruit juice processing

Fruits are mainly water (75–90%), most located in vacuole causing turgor

to the fruit tissue, and fruit juice is prepared by mechanically squeezing

or macerating fresh fruits without the application of heat or solvents. In

many countries, the term fruit juice can only legally be used to describe

a product which is 100% fruit juice (FDA, 2001; Food England, 2003).

Fruit cell wall consists of crystalline cellulose microfibrils embedded in

an amorphous matrix of pectin and hemicelluloses. The definition of a

mature fruit varies with each type. Typically, sugar and organic acid levels, and their ratio indicate maturity stage. Juice is composed of water;

soluble solids (sugars and organic acids); aroma and flavor compounds;

vitamins and minerals; pectic substances; pigments; and, to a very small

degree, proteins and fats. During ripening, fruits decrease in acidity and

starch and increase in sugars. Juices are products for direct consumption and are obtained by the extraction of cellular juice from fruit; this

operation can be done by pressing or by diffusion. Fruit juices were categorized in juices without pulp (clarified or cloudy); and juices with pulp

(pulps, purees, and nectars). Juices obtained by removal of a major part of

their water content by vacuum evaporation or fractional freezing will be

defined as concentrated juices.

Non-citrus fruit processing plants can vary from a simple facility for

single juice extraction and canning, to a complex manufacturing facility

including ultrafiltration (UF) and reverse osmosis equipments, cold storage and waste treatment plant. A simplified characteristic flow diagram of

a non-citrus juice processing line is shown in Figure€7.1. Processed products can be either as single-strength or bulk concentrate, both in clarified

or cloudy juice. Basic steps in the production of fruit juices can be divided

into four principal stages: Front-end operations; juice extraction; juice

clarification and fining; juice pasteurization and concentration.

7.2.1â•…Extraction methods

The way to extract juice depends on the fruit variety. In general there are

few problems in reducing the size of pome fruits, as apples or pears. As

Figure€7.1 shows, after washing pome fruits are milled. For the disintegration fixed positioned or rotating grinding knives may be used. Fruit mills

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Liliana N. Ceci and Jorge E. Lozano

Non-citrus Fruit


(Brushing, spraying, etc.)

Grape, berries

Destemming, destoning, peeling

Milling, chopping,


Seeds removing


Enzymatic treatment

(Maceration, liquefaction)


Turbid juice

Heat treatment

Enzymatic treatment






Cloudy juice

Clear juice

Concentration step

Final product

Figure 7.1╇ Typical non-citrus fruit juices (clear or cloudy) processing line steps.

(Lozano, J.E. 2006. Fruit manufacturing: Scientific basis, engineering properties

and deteriorative reactions of technological importance. In Food Engineering Series;

Springer, USA, pp. 1–72. With permission.)

generally used are rotating disc mills, rasp or grater mill, and fixed blade

hammer mills, in which the rotor with fixed blades rotates within a perforated screen. Milled particles should be about the same size and bigger

enough to facilitate pressing. Grater mills are found more efficient with

firm fruit while hammer mills are more efficient than graters for mature

or softer fruit, provided speed is properly adjusted (Lozano, 2006).

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Chapter seven:â•… Use of enzymes for non-citrus fruit juice production


Apple juice produced with enzymes typically follows one of two primary methods of extraction; traditional pressing or decanter extraction

(Nagy et al., 1993). In the traditional pressing operation, whole apples are

milled and treated with enzymes prior to pressing to loosen cell walls

and promote free run juice. In a number of pressing operations, pressing

aids such as rice hulls are mixed with the apple mash prior to pressing.

Most systems for extracting juices from apple and similar fruit pulps use

some method of pressing juice through cloths of various thicknesses, in

which pomace is retained. These filter presses include (Lozano, 2003) rack

and cloth presses; horizontal pack press; and continuous belt press. In a

rack and cloth press the milled fruit pulp is placed in a cloth (plastic fabric)

forming a “cheese” of pulp separated by racks made of hardwood or plastic, which are stacked up to 1 m or more in height, and pressed hydraulically. After pressing, the juice is transferred to clarification tanks where

additional enzyme is added to the juice to degrade pectin and hydrolyze

starch, if required, prior to filtration. The enzymatic process will be later

described in detail.

Other extraction techniques include centrifugation, diffusion extraction, and ultrafiltration. First method includes horizontal decanters, which

are nowadays especially used for juice clarification. Diffusion extraction

was adapted from the sugar industry. Extraction is a typical counter current type process. The diffusion extraction process is influenced by a

number of variables, including temperature, thickness, water, and fruit

variety. Slices from extractors pass through a conventional press system

and the very dilute juice may be returned to the extractors. It comes out

that the extra juice yield from diffusion extraction compensates for the

extra energy cost involved for concentration. Membrane clarification will

be specifically considered further on.

Using decanter extraction pectinase preparations, and sometimes cellulase and hemicellulase preparations, are used to further reduce viscosity. Because the mash is heated and stirred in the presence of enzyme

prior to centrifugation, some additional juice is recovered that may not be

extracted during traditional pressing operation. The decanter extraction

process is sometimes described as liquefaction, or whole fruit liquefaction. However, the whole fruit is not completely liquefied.

7.2.2â•…Clarification and fining

As previously described, the conventional route to concentrate is to strip

aroma, then depectinize juice with enzymes; centrifuge to remove heavy

sediment; and filter through pressure precoat filters and polish filters.

The juice is then usually concentrated through multi stage vacuum

evaporator. This process involves a slight concentration of juice during

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Liliana N. Ceci and Jorge E. Lozano

the stripping step in which up to 10% volume is removed to eliminate

methanol released during depectinization with pectin methyl esterase.

However, viscosity increased with concentration, which may slow flocculation and filtration. 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.

Fruit juices, both clarified and opalescent, may be concentrated to

4€folds (≅ 50°Brix) with little problem with respect to natural pectin gelling.

At this point in the concentration process there is also little detectable heat

damage. This concentrate may be canned and frozen. For a clear juice these

suspended particles have to be removed (McLellan, 1996). Soluble pectin

remains in the juice, making it too viscous to filter quickly and enzymatic

depectinization is required. Depectinization degrades the viscous soluble

pectins and promotes the aggregation of cloud particles. As soluble pectin

forms a protective coat, negatively charged, around proteins in suspension,

causing particles to repel one another. The effect of pectinolytic enzymes

is to degrade pectin and expose part of the positively charged protein

beneath. The electrostatic repulsion between cloud particles is thereby

reduced so that they clump together. These larger particles will eventually

settle out. However, to improve the process flocculating or fining agents can

be added.

Fining agents (Table€7.1) work either by sticking to particles making

them heavy enough to sink or by using charged ions to cause particles to

stick to each other making them settle to the bottom.

Although this conventional clarification was a widely used practice in

the clarified juice industry, this technology has been practically replaced

by mechanical processes such as ultrafiltration and centrifugal decanters.

Conventional clarification left a transparent but by no means clear juice,

and further centrifugation and/or filtration is required to obtain the clear

juice that many consumers prefer. Yeasts and other microorganisms may

also be precipitated also by fining.

Another potential contributor to the haziness of juice, when unripe

fruits are processed, is starch. In the case of apples, starch may account

as much as 15% in weight. While centrifugation can remove most of the

starch, about 5% usually remains. Common practice is to hydrolyze starch

with amylases (amyloglucosidases) active at the pH of apple juice, added

at the same time as the pectinases.

Most apple juice is concentrated before storage by evaporating up to

75% of the water making both depectinization and destarching essential to avoid gelling and turbidity haze formation during concentration.

Increased haze formation occurs when fining with gelatin and bentonite is omitted. Optimization of fining and ultrafiltration steps can help

retarding or preventing post-bottling haze development.

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Chapter seven:â•… Use of enzymes for non-citrus fruit juice production


Table€7.1╇ Fruit Juice Clarification Agents







Filters or Polishers

Pectic Enzymes


A natural albuminous protein extracted from kelp and

sold as a very fine powder.

Mixture of gelatins and silicon dioxide, with the active

ingredient being animal collagen.

A liquid in which small silica particles have been

suspended. It is usually used in tandem with gelatins.

The dosage is 1 milliliter per gram of gelatins. This

fining agent aids in pulling proteins out of suspension.

Sold as a powder and as coarse granules. It is refined

clay. A better way is to add the same amount to a liter

of hot water, stir well, and let stand for 36 to 48 hours.

In this time the clay will swell and become almost

gelatins like.

Produced from sturgeon swim bladders, isinglass is

sold either as a fine white powder or as dry hard

fragments. It is protein, extracted from the bladders of

these fishes. This product is also available as a

prepared liquid called “super-clear.”

With a fine porosity pad, filters are very effective at

removing particles (yeast cells, proteins, etc.).

Almost all fruits contain pectin, some more than others.

When pectin enzymes are added, it eliminates pectin

haze. There is no other way to prevent this condition,

and if it is in a juice, the haze will never clear on its own

Source: Lozano, J.E. 2006. Fruit manufacturing: Scientific basis, engineering properties and

deteriorative reactions of technological importance. In Food Engineering Series; Springer,

USA, pp. 1–72. With permission.

7.3â•…Enzymes in the non-citrus fruit industry

Commercial pectic enzymes (pectinases) and other enzymes are an important part of fruit juice technology practically from the beginning of the

industrial processing of this product. Technical enzyme products have

been used in the process of making fruit juices since the 1930s (Grampp,

1976). They are used to assist the extraction and clarification of juices from

many fruits, including berries, stone fruits, grapes, apples, and pears among

others. When clarification is not required, as in the case of cloudy juices,

enzymes are still applied to enhance extraction or perform other modifications. Commercial pectinase preparations used in fruit processing generally contains a mixture of pectinesterase (PE), polygalacturonase (PG), and

pectinlyase (PL) enzymes (Dietrich et al., 1991). The methods employed are

basically the same for many fruits (Rombouts and Pilnik, 1978). Table€7.2

lists the main application of pectolytic enzymes.

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Table€7.2╇ Application of Pectolytic Enzymes to Fruit Processing

Enzymatic process

Examples of application

Clarification of fruit juices

Apple juice; depectinized juices can

also be concentrated without gelling

and developing turbidity

Soft fruit, red grapes, citrus, and apples,

for better release of juice (and colored

material); enzyme treatment of pulp of

olives, palm fruit and coconut flesh to

increase oil yield

Used to obtain nectar bases and baby


Used to obtain products with increased

soluble solids content (pectinases and

cellulases combined)

Enzyme treatment of pulp

Maceration of fruits and

disintegration by cell separation

Liquefaction of fruit

7.3.1â•…Mash enzymatic treatment

Although pectinases were originally used for clarifying and depectinizing juices, at the start of the 1970s, they were used for mash enzymatic

treatment of apples. The pectolytic enzymes that are normally used feature polygalacturonases as their main activity. In addition, they can also

include amylase, cellulase, and protease activities. The combined use of

macerating enzymes (pectinases and cellulases) significantly increases the

sugar content in the juice and the yield due to the complete hydrolysis

of polysaccharide macromolecules. For this reason, the process of mash

liquefaction is legally permitted only in certain countries. Pectolytic mash

enzymes primarily hydrolyze the somewhat less esterified pectins of the

membranes. Mash enzymatic treatment is used for accelerating the juicing

process increasing yields. In particular, ripe apples, which are usually very

soft, are very difficult to press economically without the use of enzymes.

Pectinases are often added to fruit pulp, after crushing in a mill. Better

results are achieved, however, if the pulp is first stirred in a holding tank

for 15–20 minutes so that enzyme inhibitors (polyphenols) are oxidized by

the native polyphenol oxidase present in fruits. The pulp is then heated to

an appropriate temperature before enzymatic treatment. For apples 30°C

is the optimal temperature, whereas stone fruits and berries generally

require higher temperatures (approx. 50°C).

Equipment for mash enzymatic treatment includes a unit for controlling the temperature of the mash, an enzyme dispenser unit, and a tank

for the enzymatic reactions. This enzymatic maceration must be done from

15 minutes to 2 hours previous to juice extraction by pressing, depending

upon the exact nature of the enzyme and how much is used, the reaction

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Chapter seven:â•… Use of enzymes for non-citrus fruit juice production


temperature and the variety of fruit. Some varieties such as Golden

Delicious apples are notoriously difficult to break down. Pectinases also

degrade soluble pectin in the pulp, reducing juice viscosity, which facilitates juice extraction. Enzymatic treatment is particularly effective with

mature apples and those from cold storage. In the apple juice industry, the

juice yield is only 75% without mashing enzymes, rising to the range of

91–96% with first mash enzymes. Moreover, a second enzymatic mashing

can increase yields almost to a 99%.

7.3.2â•…Other enzymes in non-citrus juice production

During the last decades, cellulases, arabanase, and glucose oxidase became

commercially available. The addition of cellulases during extraction at

50°C improves the release of color compounds from the skins of fruit. This

cellulase effect results particularly useful during the processing of blackcurrants and red grapes. Increasingly cellulases are being used at the time

of the initial pectinase addition to totally liquefy fruit tissues.

The polysaccharide araban, a polymer of the pentose arabinose, was

found as a component of post-concentration haze in fruit juice. Although

commercial pectinase preparations often contain arabanase, fruits with

arabans usually require additional arabanase to avoid haze problems.

Glucose oxidase catalyses the breakdown of glucose to produce gluconic acid and hydrogen peroxide. Glucose oxidase, usually coupled with

catalase to remove the hydrogen peroxide, is therefore used to remove

the oxygen from the headspace above bottled fruit juice drinks, reducing

deteriorative oxidation reactions.

7.4â•…Commercial enzymes activity determination

In general, fruit juice processors are lacking of reliable methods for checking the activity of the different commercial enzymes in use. Complete pectin breakdown during clarification, can only be ensured if all the three

types of pectinolytic enzymes (PG, PE, PL) are present in the correct proportion. Moreover, the successful application of a pectinase product also

depends on the substrate where they act, and the standardization of a fruit

substrate complicates the evaluation of pectinolytic activities. For example, different varieties of apples will give substrates with different acidity,

pH, and content of inhibitors or promoters of the enzymatic activity.

7.4.1â•…Temperature dependence on the pectic enzyme activities

Figure€7.2 shows the residual polygalacturonase (PG) activity of two commercial enzymes after 30 min of heating at different temperatures. It was

clearly demonstrated that enzyme started to become inactivated at temperatures higher than 50°C, which is a very well-defined breaking point were the

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Figure 7.2╇ Enzymatic residual activities after thermal treatment 30 min at different temperatures of enzyme solutions in 0.1 M citrate/0.2 M phosphate

buffers at optimum pH). (Ceci, L. and J.E. Lozano. 1998. Determination of enzymatic activities of commercial pectinases. Food Chemistry 31(1/2), 237–241. With


enzyme rapidly lose its activity. Moreover, the thermal inactivation kinetics

for PG has shown a first period characterized as a thermo-labile fraction,

and a second period defined as the thermo-resistant fraction of the enzyme

(Ceci and Lozano, 1998). Sakai et al. (1993) and Liu and Luh (1978) reported

that the optimal temperature for PG activity was in the range 30°–50°C.

Both authors also indicated that inactivation was notable for temperature greater than 50°C after a short period of heating. Figure€ 7.2 shows

the inactivation curve of lyase activity (PL) which also shown a breaking

point at approximately 50°C. Inactivation kinetics was mono-phase for PL

unlike PG. Liu and Luh (1978) found that commercial enzymes were more

heat tolerant than purified fractions, and attributed this phenomenon to

the heat protective action of impurities.

7.4.2â•…pH dependence on the pectic enzymes activities

Ceci and Lozano (1998) studied the effect of pH on commercial pectinase enzymes. Figure€ 7.3 shows the behavior of PG and PE activities

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Chapter seven:â•… Use of enzymes for non-citrus fruit juice production











Figure 7.3╇ Effects of pH on the enzymatic activities of Röhapect D5S. (Ceci, L. and

J.E. Lozano. 1998. Determination of enzymatic activities of commercial pectinases.

Food Chemistry 31(1/2), 237–241. With permission.)

of Röhapect D5S enzyme vs. pH. While in the case of PG activity the

optimum pH resulted in approximately 4.6; it was difficult to identify a

single optimal value for lyase activity. Instead an optimal range of pH

5–6 was defined.

Pectinolytic enzymes (PG and PE) show a rapid decrease in activity

at about pH = 5 and become practically inactivated near neutrality. This

problem becomes irrelevant because pH values of fruit juices are in general lower than 4. Therefore, as much as 40% of PG and PE inactivation

can be expected during the enzymatic clarification of fruit juices. It was

found, however that a broadening of the optimal activities range (Ates

and Pekyardimci, 1995) can be obtained after enzyme immobilization on

appropriate supports. When the fruit juice clarification is performed at relatively high temperatures (45º–50ºC), careful must be taken to avoid excessive inactivation when lyase activity is considered important, because PL

is more sensible to thermal inactivation.

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Liliana N. Ceci and Jorge E. Lozano

7.4.3â•…Enzymatic hydrolysis of starch in fruit juices

Polymeric carbohydrates like starch and arabans may cause difficult filtration and post-process cloudiness. In the case of a positive starch test, the

following problems may occur: slow filtration, membrane fouling, gelling

after concentration and post concentration haze. Apple juice is one of the

juices that can contain considerable amounts of starch, particularly at the

beginning of the season. Starch content of apple juice may be high in years

when there were relatively low temperatures during the growing season.

As the apple mature on the tree, the starch hydrolyzes into sugars, but

unripe apples may contain as much as 15% starch (Reed, 1975).

Starch must be degraded by adding starch splitting enzymes, together

with the pectinase during depectinization of the juice. Starch is generally

insoluble in water at room temperature. Because of this, starch in nature

is stored in cells as small granules. Once in the juice, starch granules must

be necessarily gelatinized to allow enzymatic hydrolysis. When an aqueous suspension of starch is heated the hydrogen bonds weaken, water is

absorbed, and the starch granules swell, and form a gel (Zobel, 1984).

Besides the generalized application of commercial amylase enzymes

in the juice industry, there is a lack of information on characteristic and

extent of gelatinization of apple starch during juice pasteurization. Starch

granules (Figure€7.4) are quite resistant to penetration by both water and

20 àm

Figure 7.4õ Scanning electron photomicrograph of an isolated apple starch granule (5 kV × 4,400). (Carrín, M. E., L. Ceci, and J.E. Lozano. 2004. Characterization

of starch in apples and its degradation with amylases. Food Chemistry 87, 173–178.

With permission.)

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