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Chapter 8. Enzymes in citrus juice processing

Chapter 8. Enzymes in citrus juice processing

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Domenico Cautela et al.

Since citrus juice is mostly consumed and appreciated as a cloudy drink,

it is of great importance to preserve juice cloudiness over time. Therefore a

deeper description will be given of the procedures aimed at the inactivation

of endogenous enzymes whose activities are detrimental to cloud stability.

As a final point, processes involving the use of enzymes for the production of some citrus products and by-products will be described.

8.2â•…Overview of citrus processing technologies

The technology used in citrus juice processing is more or less identical

throughout the world. Differences among processing systems and equipment employed in juice production arise both from local traditions and

from morphological differences among citrus species, and also according

to the kind of final product to be obtained.

A schematic representation of the main process for citrus juice manufacturing is presented in Figure€8.1. Steps that are common to all manufacturing processes before juice extraction include receiving, washing,

sizing, and selection of fruits.

The juice extraction occurs through mechanical breakage of juice sacs

present in the fruit endocarp. Differences among the extracting machines

on the market regard the techniques used for the juice extraction and

essential oil recovery.

In the in-line process the extraction of essential oil and juice takes

place simultaneously, while in other processes, the juice extraction and

oil recovery from the peel are carried out in different steps by employing

different equipment.

The In-Line System (FMC Corporation, Fairway Avenue Lakeland,

FL-USA) is the most used citrus juice extraction equipment worldwide.

Before the extraction process, it is necessary to select fruits of uniform size.

The “In-Line” extractor simultaneously processes three to eight fruits per

cycle. The system consists of three to eight upper and lower cups: the upper

cups are movable and descend toward the lower fixed cups. At the base of

the bottom cups, there are circular knives mounted on top of the strainer

tubes that act as juice pre-finisher elements. Both cups are formed of “finger-shaped” knives that intersect when the upper cups move down to the

bottom cups. While the two cups still fit one against the other, the peel is

separated from the fruit and cut in strips. The fruit, without peel, is pushed

through a strainer tube, where the juice is separated from seeds. Pulp and

remaining fruit components are flowed into a collector for the finishing

operations. The essential oil is extracted by compression of the peel strips

during the descendent movement of the upper cups and is recovered by

sprayed water. The water−oil emulsion, bits of peel, and other solid parts

of the fruit are transferred to a finisher for the removal of insoluble solids

and then to centrifuges for the separation of essential oil.

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Chapter eight:â•… Enzymes in citrus juice processing

Juice Processing Line



























Cloudifier Processing Line








Clear Juice Processing Line

Figure 8.1╇ Schematic representation of citrus juice manufacturing operations:

juice extractor (1); finisher (2); tank (3); pump (4); heat exchanger (5); evaporator

(6); peel tank-hammermill-mixing tank (7); enzyme tank and dosing pump (8);

reaction tank (9); decanter (10); ultrafiltration unit (11); concentrate juice (CJ); clear

concentrate juice (CJC); cloudy peel concentrate (CPC); enzymatic preparation (E);

fruit uploading (F); single strength juice (J); peel (P); pulps/cells (PC); pasteurized

juice (PJ); cold pressed essential oil (O); water (W).

In equipment such as the “Brown Extractor” (Brown International

Corporation, Winter Haven, FL-USA) and the Birillatrice (Fratelli Indelicato

S.r.l., Catania, Italy), the juice extraction is carried out on half-fruit and

precedes that of essential oil that is successively recovered from the juiceexhausted half-fruit peels by various systems.

These extractors require fruit calibration to work properly and operate

on the fruits cut in two halves, which are placed in special cups. The juice

extraction is accomplished using special bulbs (“reamers”). The reamer

can be fixed or can more or less quickly rotate during the squeezing. It is

possible to set the distance between the reamer and the fruit wall depending on the type of extraction required. Generally for a soft extraction this

distance is about 1.1 cm but it can be 0.2 cm in the case of hard extractions.

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Domenico Cautela et al.

The extraction pressure affects not only the yield in juice but also its composition. High pressures, in fact, increase the content of pectin, pulp, and

pectin methylesterase enzyme (PME).

The extracted juice goes through a sieve or a vibrating rotary filter,

incorporated in the extractor for the removal of seeds, cells, and coarse

components of albedo.

The half-peels without juice fall into the oil extracting section. In the

Birillatrice-Sfumatrice equipment, a rotating drum catches the peels which

rolls and are pressed against a stationary profile. This causes repeated

deformations of the half-peels and breakage of the oil sacs. A water rain

provided by a set of sprayers collects oil. The mixture of water and essential oil falls into a cistern and from there it is conveyed to the next steps for

separation and finishing.

In other processes the oil extraction precedes the juice extraction. In

this case the essential oil is extracted from the whole fruit by rasping by

machines called “peelers.” The rasped fruits are then moved into a juice

extractor. Machines operating with this technique are manufactured by

such Italian companies as Fratelli Indelicato S.r.l. (Catania) and Speciale

F. & C. S.r.l. (Catania).

The juice extractor cuts the peeled fruits into two halves that successively are squeezed against a stainless steel screen. After passing through

the screen, the juice is collected in closed tanks, while seeds and pulp are


Detailed descriptions of these extractors are available from the manufacturers (FMC Corporation, Brown International Corporation, Fratelli

Indelicato S.r.l., and Speciale F. & C. S.r.l.).

The extracted juice (single strength juice) contains significant amounts

of pulp, seeds, rags, and other fibrous materials that are removed during the finishing operations. Orange juice is approximately 11°Brix (%

w/w soluble solids), lemon juice has a content of soluble solids of 8°Brix,

and grapefruit and tangerine juices have a soluble solid content of about

10°Brix. The pulp content is reduced to approximately 4–5% v/v in downstream finishers. Juices with pulp content of about 1% can be obtained by

the use of centrifuges. The discarded pulp can be further processed to

produce pulp wash or extracts of water-soluble solids.

The juice obtained must be de-aerated to avoid the ascorbic acid oxidation during pasteurization and storage. The single strength juice is usually pasteurized employing tubular or plate heat exchangers. Generally,

the citrus juice is pasteurized to stabilize the product against the microbial growth and to inactivate endogenous enzymes before concentration

or aseptic packaging. The pasteurization process is mainly designed to

inactivate the heat stable forms of the enzyme pectin methylesterase

(PME) as they require higher temperatures than those needed for thermal

destruction of microorganisms.

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Chapter eight:â•… Enzymes in citrus juice processing


The activity of endogenous pectin methylesterase (EC triggers

a sequence of events that lead to the destabilization of the juice cloud resulting in syneresis and/or gelation of concentrate juices. The citrus industry

employs different time-temperature combinations for the thermal inactivation of PME depending on the type of juice to be processed. Temperatures

and holding times required for the pasteurization of cloudy and pulpy

juices are generally higher than that required for the production of clear

juice or for juice reconstituted from concentrate before aseptic packaging.

Combinations of time and temperature used to inactivate the PME

will be extensively treated in a separate section of this chapter.

Usually the juice is concentrated to overcome the seasonality of citrus

production and to reduce the cost of packaging, storage, and shipping.

The majority of orange juice produced in Florida and Brazil is converted

into concentrated juice.

The evaporators most frequently used in the process of concentration

are the “falling film evaporators” in single or multiple steps. Among them,

T.A.S.T.E. (Thermally Accelerated Short Time Evaporator) is widely used

for concentrating clear and cloudy juice. The operating principle of the

T.A.S.T.E. entails use of high temperatures (92–112°C) which, besides fast

evaporation, also accomplishes juice pasteurization. Flavors lost during

evaporation can be largely restored by the addition to concentrated juice

of aqueous aromas recovered from the evaporator condensates and oilbased essences extracted from citrus peel.

Orange juice is marketed mostly as 60 or 65°Brix concentrate, while

cloudy lemon juice is sold on the basis of its citric acid content. Generally,

lemon juice is concentrated to 400 or to 530 g/L (grams of citric acid per

liter). Grapefruit concentrates can be prepared by evaporation to high Brix

levels (60–65°Brix), as for the orange and tangerine juices, although 55°Brix

grapefruit concentrate is also commonly produced.

Due to the high acidic nature of lemon and lime juices, greater care

must be taken in their heat treatment. If the temperature to inactivate pectinases is too high, acid hydrolysis of pectin and sugars can occur. As

in the case of pectinase activity, this results in cloud loss and gelation.

Furthermore, the excessive acid content accelerates the juice browning

due to the Maillard reaction.

8.3â•…Pectin methylesterase (PME) in citrus juice

Citrus juice is usually consumed as cloudy drinks. The citrus cloud is a

fine suspension of particles that gives citrus juice the characteristics of

turbidity, color, flavor, aroma, and texture (Mizrahi et al., 1970; Baker and

Cameron, 1999). The cloud is composed of high molecular weight polymeric materials including proteins, pectin, hemicellulose, and cellulose

(Sinclair 1984).

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Domenico Cautela et al.

Cloud particles are between 0.4 and 5 µm in diameter (Klavons et al.,

1994), stable cloud is made of particles of about 2 µm diameter (Mizrahi

et al., 1970). The cloud stability of citrus juice is related to the molecular

weight of pectin (Hotchkiss et al., 2002), its degree of methylesterification

(Hills et al., 1949), and the intra-molecular distribution of methylester

groups in the pectin molecules (Baker, 1979; Joye and Luzio, 2000; Willats

et al., 2001; Wicker et al., 2003).

The pectin methylesterase catalyses the hydrolysis of methylester

groups of pectin, causing the formation of negative carboxylate groups

and releasing H+ ions and methanol.

The action of PME modifies high methoxyl pectin into calcium sensitive low methoxyl pectin. Once a critical degree of de-esterification is

reached, divalent cations, such as calcium, can cross-link the free acid

units on adjacent pectin molecules, forming insoluble calcium pectates.

Cross-linking increases the pectin apparent molecular weight and

reduces its solubility, thus leading to flocculation. Cloud is considered

definitively broken or lost in orange juice when light transmittance reaches

36% (Redd et al., 1986).

The activity unit of PME (PMEu) is defined as the amount of enzyme

that release 1 µmole of carboxylic acid groups in 1 ml of solution for one

minute, and is generally measured using the method proposed by Rouse

and Atkins (1954), which is based on the titration of carboxyl groups generated by the PME activity during the hydrolysis of 1% pectin solution

containing 0.1 M NaCl.

Rouse and Atkins (1954) reported a value of 1.1 PMEu/mL in lemon


For orange juice cv. Navel containing 5% pulp, Ingallinera et al. (2005)

reported a pectin methylesterase activity of 1.3 PMEu/mL and found values 2–3 times higher for the Sicilian red orange juices cv. Moro, Tarocco,

and Sanguinello with the same pulp content. The juice from cv. Tarocco

showed the highest activity (2.85 PMEu/mL).

The technologies of citrus juice extraction affect the composition and

the “cloud stability” of the product. An increase of enzyme activity is correlated to a higher content of peel incorporated into the juice during the

extraction process (Amstalden and Montgomery, 1994).

Cameron et al. (1999) showed that the PME extracted from Valencia

orange peel destabilizes the cloud more rapidly than those extracted from

rag or hand-squeezed juice. In addition, PME activity in juice extracted

with hard procedures destabilizes the cloud faster than in a juice extracted

with a softer process.

The activity of PME in juice varies according to the fruit cultivar and the

stage of fruit ripeness. Amstalden and Montgomery (1994) found a higher

PME activity in orange juices from cv. Valencia than in those produced from

other Brazilian orange cultivars (cv. Natal, Pera, and Pera Rio Coroa).

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