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Enzymatic Browning in Fruits, Vegetables and Seafoods

Maurice R. Marshall1, Jeongmok Kim2 and Cheng-I Wei3

Food Science and Human Nutrition Department

University of Florida

Gainesville, Florida 32611-0370

Telephone: (352) 392-1978

Fax: (352) 392-1988

Food Engineering Department

College of Engineering

Mokpo National University

Muan-Gun, Chinnam, 534-729 Korea



Nutrition and Food Science Department

328 Spidle Hall, Auburn University

Auburn, Alabama 36849-5605

©FAO, 2000

1. Introduction

1.1 General overview of enzymatic browning

1.2 Economic benefits of browning in fruits and vegetables

1.3 Economic losses due to browning in fruits and vegetables

1.4 Economic benefits of browning in aquatic foods

1.5 Economic losses due to browning in aquatic foods

2. Physiological role of enzymatic browning

2.1 Plants

2.2 Animals

2.3 Characteristics of polyphenol oxidase

2.3.1 Monophenol oxidase

2.3.2 Diphenol oxidase

2.3.3 Laccase

2.4 Phenolic substrates

3. Control of browning

3.1 Processing

3.1.1 Heating

3.1.2 Refrigeration

3.1.3 Freezing

3.1.4 Dehydration

3.1.5 Irradiation

3.1.6 High pressure treatment

3.1.7 Treatment with supercritical carbon dioxide

3.1.8 Ultrafiltration

3.1.9 Ultrasonication
3.2 Inhibitors

3.2.1 Reducing agents/ Antioxidants

a. Sulphiting agents

b. L-Ascorbic acid

c. Erythorbic acid

d. Cysteine

e. Phenolic antioxidant

3.2.2 Acidulants

a. Citric acid

3.2.3 Chelators


b. Phosphates

c. Maltol

d. Kojic acid

e. Polysaccharides
f. Carbon monoxide

3.2.4 Complex agents

a. Cyclodextrins

b. Chitosan

3.2.5 Enzyme inhibitors

a. 4-Hexylresorcinol

b. Halide salts

c. Honey

d. Amino acids, peptides and proteins

e. Aromatic carboxylic acids

f. Aliphatic alcohols

3.3 Other inhibitors

3.3.1 Killer enzymes

a. Ring-cleaving oxygenases

b. Catechol transferase
c. Proteases

3.3.2 Edible coatings

4. Molecular biology of phenoloxidases

4.1 New approaches for the control of PP

4.2 Antisense RNA approach

5. Conclusion




1.1 General overview of enzymatic browning

Appearance, flavour, texture and nutritional value are four attributes considered by consumers
when making food choices. Appearance which is significantly impacted by colour is one of the first
attributes used by consumers in evaluating food quality. Colour may be influenced by naturally
occurring pigments such as chlorophylls, carotenoids and anthocyanins in food, or by pigments
resulting from both enzymatic and non-enzymatic reactions. Enzymatic browning is one of the
most important colour reactions that affects fruits, vegetables and seafoods. It is catalysed by the
enzyme polyphenol oxidase (1,2 benzenediol; oxygen oxidoreductase, EC1.10.3.1) which is also
referred to as phenoloxidase, phenolase, monophenol oxidase, diphenol oxidase and tyrosinase.

Enzymatic browning is one of the most studied reactions in fruits, vegetables and seafoods.
Researchers in the fields of food science, horticulture, plant and postharvest physiology,
microbiology, and even insect and crustacean physiology have studied this reaction because of the
diversity of its impact in these systems.

1.2 Economic benefits of browning in fruits and vegetables

When asked to discuss browning in foods, those involved from production to processing, usually
reflect on its detrimental influence. Some enzymatic browning reactions are however very
beneficial to the overall acceptability of foods. Tea, coffee, and cocoa are important commodities
for many developing countries. India, China, Sri Lanka, and Kenya, are major producers of tea
(Ensminger et al. 1995). Black, oolong, and green tea are reliant on enzymatic browning for colour
and flavour development. Coffee is primarily produced in Brazil and Columbia. Ethiopia, Indonesia,
Vietnam, and a number of other developing countries also produce coffee for world markets.
Approximately 4.2 million metric tons of coffee beans are produced on an annual basis by the top
ten major coffee producing countries. Some discussion occurs as to the contribution of enzymatic
browning to colour development during coffee processing.

Cocoa is generally produced by countries located just north and south of the equator with Brazil
and West Africa supplying much of the world market. Colour development in cocoa is facilitated by
polyphenol oxidase activity during fermentation and drying. Polyphenol oxidases are also
responsible for development of the characteristic golden brown colour in dried fruits such as
raisins, prunes, dates and figs. Blanching is generally required for inactivation of the enzyme after
colour development, in order to minimize discolouration.

Polyphenol oxidases are believed to play key physiological roles both in preventing insects and
microorganisms from attacking plants and as part of the wound response of plants and plant
products to insects, microorganisms and bruising. As fruits and vegetables ripen, their
susceptibility to disease and infestation is increased due to a decline in their phenolic content.
Phenoloxidase enzymes endogenous to fruits and vegetables, catalyse the production of quinones
from their phenolic constituents. Once formed, these quinones undergo polymerization reactions,
leading to the production of melanins, which exhibit both antibacterial and antifungal activity and
assist in keeping the fruit and/or vegetable physiologically wholesome.

Research describing the antibacterial, anticancer and antioxidant nature of melanins has triggered
considerable interest in enzymatic browning. Nutritional recommendations for increasing the per
capita consumption of fruits and vegetables, as well as new technologies of convenience such as
the production of precut or minimally processed fruits and vegetables, will provide economic
benefit to many countries which market exotic products.

1.3 Economic losses due to browning in fruits and vegetables

Increases in fruit and vegetable markets projected for the future will not occur if enzymatic
browning is not understood and controlled. Enzymatic browning is one of the most devastating
reactions for many exotic fruits and vegetables, in particular tropical and subtropical varieties. It is
estimated that over 50 percent losses in fruit occur as a result of enzymatic browning (Whitaker
and Lee, 1995). Such losses have prompted considerable interest in understanding and controlling
phenoloxidase enzymes in foods. Lettuce, other green leafy vegetables, potatoes and other
starchy staples, such as sweet potato, breadfruit, yam, mushrooms, apples, avocados, bananas,
grapes, peaches, and a variety of other tropical and subtropical fruits and vegetables, are
susceptible to browning and therefore cause economic losses for the agriculturist. These losses are
greater if browning occurs closer to the consumer in the processing scheme, due to storage and
handling costs prior to this point. The control of browning from harvest to consumer is therefore
very critical for minimizing losses and maintaining economic value to the agriculturist and food
processor. Browning can also adversely affect flavour and nutritional value. Figure 1 shows
examples of enzymatic browning in fruits and vegetables.
Figure 1. Examples of enzymatic browning in apple, banana and potato (A. Fresh cut, B.

1.4 Economic benefits of browning in aquatic foods

Aquatic organisms rely on polyphenol oxidases to impart important physiological functions for
their development. Polyphenol oxidases are important in hardening of the shell (sclerotization),
after moulting in insects and in crustaceans such as shrimp and lobsters. Polyphenol oxidase is also
responsible for wound healing. The mechanism of wound healing in aquatic organisms is similar to
that which occurs in plants in that the compounds produced as a result of the polymerization of
quinones exhibit both antibacterial and antifungal activities. Unfortunately, polyphenoloxidase-
catalysed browning of the shell postharvest, adversely affects both the quality and consumer
acceptability of these products.

1.5 Economic losses due to browning in aquatic foods

Browning or melanosis in aquatic foods postharvest occurs primarily in crustaceans. These highly
prized and economically valuable products are extremely vulnerable to enzymatic browning.
Melanosis is usually more severe in lobsters if the head is retained during storage postharvest. If
the head is removed, care should be taken to thoroughly wash the tail in order to eliminate
proteases that activate latent polyphenol oxidases and promote browning. Although the products
of melanosis are not harmful and do not influence flavour or aroma, consumers will not select
these products since their brown discolouration connotes spoilage. Severe melanosis on these
products can cause tremendous economic losses due to the high value commanded by these
aquatic products in the marketplace. There are many examples of imported aquatic products
entering the United States, worth millions of dollars, that are reduced markedly or lost completely
owing to the severity of melanosis. Unfortunately, a majority of these products originate in
developing countries, which lack both the scientific and technical resources, and the processing
infrastructure required in order to prevent the occurrence of these devastating losses. Limited
susceptibility of a number of crustacean species to melanosis on the other hand, presents the
processor with the problem of deciding how to treat the product in order to prevent melanosis.

On the basis of the foregoing discussions, it is clear that browning has both beneficial and
deteriorative effects. Control of the deteriorative effects of browning therefore poses a major
challenge to the food scientist.

The control of browning in fruits and vegetables hinges upon an understanding of the
mechanism(s) responsible for browning in fruits, vegetables and seafoods, the properties of
polyphenol oxidase enzyme(s), their substrates and inhibitors, and the chemical, biological and
physical factors which affect each of these parameters. Once understood these mechanisms may
be applied in either preventing the browning reaction, or slowing its rate, thus extending the shelf
life of the product.

In order for the food scientist to better understand how to prevent enzymatic browning, it is
important to understand why polyphenol oxidase is present in plant and animal tissues. Despite
knowing and hypothesizing some functions of polyphenol oxidase in these tissues, researchers are
still trying to piece together the functional puzzle of this enzyme in both plant and animal systems.

2.1 Plants

Polyphenol oxidases were first discovered in mushrooms and are widely distributed in nature.
They appear to reside in the plastids and chloroplasts of plants, although freely existing in the
cytoplasm of senescing or ripening plants (Vaughn and Duke, 1984). Cloning and sequencing
studies of the copper A binding region of these enzymes shows high conservation between
polyphenol oxidases from plants, microorganisms and animals. Polyphenol oxidase is thought to
play an important role in the resistance of plants to microbial and viral infections and to adverse
climatic conditions. Phenolics, such as chlorogenic acid, caffeic acid and scopolin, etc., which are
substrates of this enzyme have been shown to exhibit fungicidal properties.

Polyphenol oxidase catalyses the initial step in the polymerization of phenolics to produce
quinones, which undergo further polymerization to yield dark, insoluble polymers referred to as
melanins. These melanins form barriers and have antimicrobial properties which prevent the
spread of infection or bruising in plant tissues. Plants, which exhibit comparably high resistance to
climatic stress, have been shown to posses relatively higher polyphenol oxidase levels than
susceptible varieties. Other enzyme systems in plants, such as chitinase, peroxidase, lipoxygenase,
phenylalanine ammonia lyase, b-1,3-glucanase, etc., also show increased activity when subjected
to stress.

It should be pointed out that the responses of enzymes to stress and infection are dependent on a
number of factors, one of which is the host plant itself. Figure 2 for example, shows that two
different species of tobacco plants exhibit weak peroxidase and polyphenol oxidase responses
when infected with tobacco mosaic virus. A hybrid of the two species however possesses
significantly higher levels of both enzymes. Additionally, the two parent species exhibit different
isozymes after infection. The hybrid however possesses isozymes of both parent species, whether
inoculated or uninoculated with tobacco mosaic virus. The hybrid is also highly resistant to viral
and fungal attack when compared to both parent species. Recent evidence suggests that the
enzyme may play a role in the growth and metabolism of chloroplasts and in plant growth
regulation (Sherman et al. 1995).
Figure 2. Peroxidase and polyphenoloxidase activities in two species of tobacco plants infected
with tobacco mosaic virus and a cross between the two. (Adapted from Goy et al., 1992)

2.2 Animals

Polyphenol oxidase exists in both insects and crustaceans as a zymogen or propolyphenol oxidase
form, and is thought to confer disease resistance in animals, insects and crustaceans. The enzyme
is also believed to be involved in both immunity and self-recognition. Microbial products such as
laminarin (b-1,3-glucan) which initiates phagocytosis have been shown to activate propolyphenol
oxidase to the active polyphenol oxidase form. Proteases are also believed to be involved in the
activation of the propolyphenol oxidase form. Söderhäll and Unestam (1979) activated crayfish
propolyphenol oxidase usingb-1,3-glucans, fungal glycoproteins, laminarin pentaose and
lipopolysaccharides. Some of these compounds appeared to activate a protease which triggered
the activation of propolyphenol oxidase.

Researchers have shown in shrimp and lobster that a latent polyphenol oxidase form was
activated either by trypsin or an endogenous enzyme (Savagaon and Sreenivasan, 1978; Ferrer et
al. 1989a; Chen et al. 1991a). These researchers proposed that activation in these species was the
result of proteolysis, which produced numerous isoenzymes. Other proteases (chymotrypsin and
pepsin) did not appear to produce the same level of activation as trypsin, although chymotrypsin
was shown to activate polyphenol oxidase in insects.
Figure 3 depicts propolyphenol oxidase activation by host defense mechanisms. Proteases, which
activate polyphenol oxidases, are thought to be induced by microbial activity. Secondary
metabolites, such as glucans, glycoproteins, laminarins, lipopolysaccharides, etc., produced by
microorganisms may also induce the activation of propolyphenol oxidase by proteases. These
metabolites are also capable of activating the propolyphenol oxidase even in the absence of
proteolytic activity.
Figure 3. Schematic of the activation of polyphenol oxidase by microorganisms.

Although host-defense has been postulated for polyphenol oxidase activity in animal systems, its
major role in crustaceans and insects is most probably related to hardening or sclerotization of the
chitin shell during the growth cycle. The enzyme appears to be located both in the cuticle and
hemolymph of insects in the propolyphenol oxidase form, which requires an activation process
(Figure 3). Since sclerotization or hardening of the shell is associated with the moulting of insects
and crustaceans, the activation of propolyphenol oxidase serves as an excellent mechanism for the
control of the various stages of moulting. Although polyphenol oxidases have been more widely
studied in insects, it is apparent that both crustaceans and insects undergo the sclerotization
process by similar mechanisms.

During the sclerotisation process, diphenols are oxidized by polyphenol oxidase to their
corresponding quinones. Quinones thus formed react with side groups on adjacent protein
molecules thus linking them together for hardening. During the growth process, crustaceans must
shed the exoskeleton, which confines them, and replace it with a larger one. Every tissue in the
crustacean is affected in some manner by moulting. Each moult cycle is initiated by the shedding
of the old exoskeleton (ecdysis). Moulting includes all morphological and physiological changes in
preparation for and recovery from ecdysis (Aiken, 1980).

Four stages of moulting (postmoult, intermoult, premoult, moult) were described by Travis (1954).
Figure 4 describes the different developmental stages of lobster cuticle formation. Polyphenol
oxidase levels correlating to these stages are shown in Figure 5. At stage A, which is representative
of a lobster that has already moulted or is at the postmoult stage of its development, the cuticle is
absent and only the epidermis is present. The shell has already hardened and polyphenol oxidase
activity is very low. At stage B, which would represent an intermoult stage, development of the
new cuticle under the old shell begins to take place. At stages C (early premoult) and D (late
premoult), the cuticle is more defined, with greater definition at stage D. During the latter stage
(stage D), the old shell is ready to be discarded and hardening of the new cuticle is initiated. It is at
this stage that the lobster is the most vulnerable to infection, since the new cuticle will not harden
until after a few days.
Figure 4. Different developmental stages in the formation of Florida Spiny Lobster Cuticle. A. No
new cuticle, only epidermis from old cuticle; B. Beginning signs of a newly forming cuticle; C.
Advanced stages of a newly forming cuticle; and D. Newly formed cuticle almost completely
formed as in late premoult.
Figure 5. Polyphenol oxidase activity and trypsin activated polyphenol oxidase activity at various
stages of lobster moulting.

The highest level of polyphenol oxidase activity correlates to the moulting stage at which the
formation of new cuticle occurs (Stages C and D) (Figure 4). Propolyphenol oxidase is present
throughout the moult cycle. Apparently similar propolyphenol oxidase and polyphenol oxidase
activities during the late premoult stage, also seems to suggest that the activated enzyme is
present during formation of the new cuticle and is required for the initiation of sclerotization.
Increased polyphenol oxidase levels with a corresponding increase in quinones at that stage may
very well serve to prevent the penetration of microorganisms through the unfinished cuticle thus
functioning as part of a host-defense mechanism.

Figure 6 provides an example of a visual scale for the progression of melanosis, while Table 1
shows the scale used to describe the progression of melanosis (black spot) on pink shrimp
(Penaeus dourarum).

Table 1. Colour scale used to describe the progression of melanosis (black spot) on pink shrimp.
(Adapted from Otwell and Marshall, 1986)

Melanosis Scale Description

0 Absent

2 Slight, noticeable on some shrimp

4 Slight, noticeable on most shrimp

6 Moderate, noticeable on most shrimp

8 Heavy, noticeable on most shrimp

10 Heavy, totally unacceptable

Figure 6. Melanosis progression scale of shrimp. (Courtesy of Dr. W.S. Otwell, University of
2.3 Characteristics of polyphenol oxidase

Polyphenol oxidase catalyses two basic reactions: hydroxylation to the o-position adjacent to an
existing hydroxyl group of the phenolic substrate (monophenol oxidase activity), and oxidation of
diphenol to o-benzoquinones (dipehnol oxidase activity). Both reactions utilize molecular oxygen
as a co-substrate. Whether a single enzyme system exhibits both mono- and di- phenol oxidase
activities is still unclear. However, when both monophenol- and diphenol oxidases are present in
plants, the ratio of monophenol to diphenol oxidase activity is usually 1:10 or as low as 1:40.
(Nicolas et al. 1994).

Polyphenol oxidase isozymes were first isolated from mushroom. Subunits of the enzyme were
observed to differ with respect to chemical, physical and kinetic properties. These subunit
differences were believed to be responsible for relative affinities of the enzymes for both mono-
and diphenolic substrates. Polyphenol oxidases isolated from mango possess two isozymes, both
of which show specificity for o-diphenols. Gross Michel banana polyphenol oxidase was capable of
oxidizing o-diphenols but not monophenols, while other varieties were shown to exhibit both
mono- and diphenol activities (Palmer, 1963). In insects, polyphenol oxidase isolated from the
cuticle exhibits only diphenol oxidase activity, while polyphenol oxidase from insect hemolymph
exhibits monophenol oxidase activity. Polyphenol oxidases associated with shrimp cuticle exhibit
both activities.

2.3.1 Monophenol oxidase

Monophenol oxidase catalyses the hydroxylation of monophenols to o-diphenols (Figure 7). The
enzyme is referred to as tyrosinase in animals, since L-tyrosine is the major monophenolic
substrate. In plants, the enzyme is sometimes referred to as cresolase owing to the ability of the
enzyme to utilize the monophenolic substrate, cresol. Tyrosinase activity is also used to describe
monophenol and diphenol oxidases in plant systems, although L-tyrosine is probably not a major
substrate for the enzyme in plant systems, considering the rich abundance of phenolics in plant
systems. This rich abundance of phenolics in plants is also the probable reason for referring to the
enzyme as a polyphenol oxidase.

Figure 7. Monophenol oxidase pathway producing the diphenol.

Monophenol oxidase activity is generally overlooked in plants since the hydroxylation reaction is
dramatically slower than the oxidation reaction required for quinone production and initiation of
the browning reaction. Monophenol oxidase (tyrosinase) has been given somewhat more
attention in insect and crustacean systems, owing to its physiological significance in conjunction
with diphenolase activity, in hardening of the cuticle for sclerotization.

The enzyme is also capable of metabolizing aromatic amines and o-aminophenols, both of which
are structurally very similar to mono- and diphenols (Toussaint and Lerch, 1987) (Figure 8).

Figure 8. Polyphenol oxidase activity for the substrates aromatic amines and o-aminophenols.
(From Toussaint and Lerch, 1987).

2.3.2 Diphenol oxidase

The oxidation of diphenolic substrates to quinones in the presence of oxygen is catalysed by

diphenol oxidase activity (Figure 9). Diphenol oxidases have received much attention owing to
their high catalytic rate and their association with the formation of quinones, which lead, to
production of the brown pigment, melanin (Figure 10).

Figure 9. Diphenol oxidase pathway producing the quinones.

Figure 10. Formation of melanin from tyrosine. (From Lerner, 1953).

Whitaker (1972a) classified the diphenol oxidase catalysed reaction as having an ordered Bi Bi
mechanism, owing to the involvement of three substrates: oxygen, diphenol and diphenol.
Mechanisms of the diphenol oxidase reaction are still unknown since no free radical intermediates
are formed and oxygen binds to the enzyme prior to binding of the o-diphenolic substrate. Figure
11 shows a simplified mechanism for the hydroxylation and oxidation of phenols by polyphenol
oxidase. Both mechanisms involve the two copper moieties on the polyphenol oxidase.
Figure 11. Simplified mechanism for the hydroxylation and oxidation of diphenol by

2.3.3 Laccase

Laccase (p-diphenol oxidase, E.C. is a type of copper-containing polyphenol

oxidase. It has the unique ability of oxidizing p-diphenols, thus allowing it to be distinguished
from o-diphenol oxidases such as catechol oxidase (Figure 12). Laccases are deep blue in colour in
the pure state, and are remarkably non-specific as to their reducing substrates. They are
glycoproteins with molecular weights ranging between 60 and 80 kDa, and a carbohydrate content
varying between 15 and 41 percent. The copper content of purified laccases varies between two
and four atoms per enzyme molecule or subunit (Thurston, 1994). Several phenolic substrates,
including polyphenols, methoxy-substituted phenols, diamines and a considerable range of other
compounds serve as substrates for laccase. Substrate oxidation by laccase results in the
generation of one free radical (Reinhammar and Malmström, 1981) and is accompanied by the
reduction of oxygen to water.
Figure 12. Comparison of reactions catalysed by catecholase (o-DPO) and laccase (p-DPO). (From
Walker, 1995).

Laccases occur in many phytopathogenic fungi and in certain higher plants (Mayer and Harel,
1991). Laccases from higher plants, and fungal laccases are known to be cytoplasmic. With the
exception of peaches (Harel et al. 1970) and apricots (Dijkstra and Walker, 1991), laccases do not
occur in fruits and vegetables. Laccase (genus Rhus) was first discovered in lacquer trees by
Yoshida (1883). It was later detected in fungi by Laborde (1896). A number of fungi
including Lactarius, Polyporus, Aspergillus, Pleurotus, Polystictus, Psalliota, Glomerella, Podospora,
Botrytis, Neurospora and Russula possess laccase activity (Franke, 1960). The carbohydrate
content of laccase from Rhus varies between 32 and 45 percent, while that of laccase
from Podospora has a carbohydrate content of 23 percent and that from Neurospora has a
carbohydrate content of 11 percent.

Fungal laccase from Neurospora exists as both an intracellular and an extracellular enzyme. Joel et
al. (1978) reported the occurrence of laccase in the cavity of the secretory ducts of all the
members of the Anacardiaceae. The presence of a laccase-type phenol oxidase was demonstrated
in the bacterium,Azospirillum lipoferum (Givaudan et al. 1993).

Laccase is a component of the lignin synthesizing system in woody tissues (Bao et al. 1993). It is
however unrelated to ligninolysis in some fungi. Laccase from Aspergillus nidulans is involved in
pigment synthesis (Hermann et al. 1983). Laccase produced by the fungus Botrytis cinerea are also
implicated in pathogenic infections which lead to soft rot infections in cucumbers (Viterbo et al.

Catechol oxidase and laccase are distinguishable both on the basis of their phenolic substrates,
and their inhibitor specificities (Table 2). Differences in the reaction mechanisms and the oxidation
levels of copper at the active site of catecholase and laccase account for differences in their
responses to certain inhibitors. Laccase activity is unaffected by CO, phenylhydrazine or 2,3-
naphthalenediol, all of which are inhibitors of catechol oxidase (Keilin and Mann, 1940). The
copper chelator diethyldithiocarbamate, cyanide azide and EDTA all serve as inhibitors of laccase

Table 2. Differential tests for catecholase and laccases.

Test Catecholase (o-DPO) Laccase (p-DPO)

Substrate specificity:

o-Dihydroxyphenols oxidized -

p-Dihydroxyphenols nil or slow oxidized

p-Cresol oxidized

Guiacol - oxidized

1-Naphthol - oxidized

p-Phenylene-diamine - oxidized

Syringaldazine - oxidized

Inhibitor specificity:

Cinnamic acid, p-coumaric acid and inhibition nil

ferulic acid

Polyvinylpyrrolidone (PVP) inhibition nil

Salicylhydroxamic acid (SHAM) inhibition nil

4-Hexyl-resorcinol inhibition nil

Quaternary ammonium compounds nil inhibition


2.4 Phenolic substrates

Phenolic compounds are widely distributed in the plant kingdom and are considered to be
secondary metabolites. Structurally they contain an aromatic ring bearing one or more hydroxyl
groups, together with a number of other substituents. Plants provide nearly all the phenols found
in higher animals, since higher animals are incapable of synthesizing compounds with benzonoid
rings from aliphatic precursors. The polyphenolic composition of fruits varies in accordance with
species, cultivar, degree of ripening and environmental conditions of growth and storage.
Phenolics also contribute to colour, astringency, bitterness, and flavour in fruits.

Phenolic compounds occurring in food materials are mostly of the flavonoid type. Of the naturally
occurring flavonoid compounds, anthocyanidins, flavonols, and cinnamic acid derivatives occur
most frequently in foods. Catechins are also naturally occurring compounds, which are structurally
related to other flavonoids having the basic nucleus of 1,3-diphenylpropane (Figure 13). Flavonols,
together with flavones and flavanones are light yellow in colour, and are collectively termed
anthoxanthin pigments. Quercetin, myricetin, and kaempferol are the most commonly occurring
flavonols (Figure 13), and are generally glycosylated. The majority of naturally occurring flavonols
possess a B-ring hydroxylation pattern similar to catechol. Catechol can therefore be considered to
be the significant o-dihydroxyphenol. It becomes extremely important as a model substrate in
enzymatic oxidation studies. Tyrosine on the other hand which is a monohydroxy phenol, is an
important amino acid. Hydroxylation of tyrosine leads to the formation of dihydroxyphenylalanine
Figure 13. Structures of common phenolic compounds.

Benzoic acid derivatives and cinnamic acid derivatives are shown in Figure 13. Caffeic acid
derivatives, such as chlorogenic (caffeoylquinic) acid and caftaric (caffeoyltartaric) acid, are often
among the major o-diphenolic compounds in plants that serve as substrates for polyphenol
oxidases. Chlologenic acid (Fig. 13) is one of the key substrates for enzymatic browning in apples.
Enzymatic oxidation of caftaric acid takes place immediately upon crushing grapes (Cheynier and
Moutounet, 1992).

Relatively few of the phenolic compounds in fruits and vegetables serve as substrates for
polyphenol oxidase. Catechins, cinnamic acid esters, 3,4-dihydroxy phenylalanine (DOPA), and
tyrosine (Table 3) are the most important natural substrates of polyphenol oxidase in fruits and
vegetables. The main substrates of polyphenol oxidase in certain fruits and vegetables do not
however commonly occur as phenolic constituents of plant material. The principal phenolic
substrate in banana for example, was identified as dopamine (3,4-dihydroxy phenylethylamine),
while that in dates is 3-o-caffeoylshikimic acid (dactylifric acid), and in yam-tuber tissues is a
catechin-like substrate known as catecholamine.

Table 3. Phenolic substrates of PPO in fruits, vegetables, and seafoods.

Source Phenolic substrates

Apple chlorogenic acid (flesh), catechol, catechin (peel), caffeic acid, 3,4-dihydroxyphenylalanine
(DOPA), 3,4-dihydroxy benzoic acid, p-cresol, 4-methyl catechol, leucocyanidin, p-coumaric acid,
flavonol glycosides

Apricot isochlorogenic acid, caffeic acid, 4-methyl catechol, chlorogenic acid, catechin, epicatechin,
pyrogallol, catechol, flavonols, p-coumaric acid derivatives

Avocado 4-methyl catechol, dopamine, pyrogallol, catechol, chlorogenic acid, caffeic acid, DOPA

Banana 3,4-dihydroxyphenylethylamine (Dopamine), leucodelphinidin, leucocyanidin

Cacao catechins, leucoanthocyanidins, anthocyanins, complex tannins

Coffee beans chlorogenic acid, caffeic acid

Eggplant chlorogenic acid, caffeic acid, coumaric acid, cinnamic acid derivatives

Grape catechin, chlorogenic acid, catechol, caffeic acid, DOPA, tannins, flavonols, protocatechuic acid,
resorcinol, hydroquinone, phenol

Lettuce tyrosine, caffeic acid, chlorogenic acid derivatives

Lobster tyrosine

Mango dopamine-HCl, 4-methyl catechol, caffeic acid, catechol, catechin, chlorogenic acid, tyrosine,
DOPA, p-cresol

Mushroom tyrosine, catechol, DOPA, dopamine, adrenaline, noradrenaline

Peach chlorogenic acid, pyrogallol, 4-methyl catechol, catechol, caffeic acid, gallic acid, catechin,

Pear chlorogenic acid, catechol, catechin, caffeic acid, DOPA, 3,4-dihydroxy benzoic acid,p-cresol

Plum chlorogenic acid, catechin, caffeic acid, catechol, DOPA

Potato chlorogenic acid, caffeic acid, catechol, DOPA, p-cresol, p-hydroxyphenyl propionic acid, p-
hydroxyphenyl pyruvic acid, m-cresol

Shrimp tyrosine

Sweet potato chlorogenic acid, caffeic acid, caffeylamide

Tea flavanols, catechins, tannins, cinnamic acid derivatives

The substrate specificity of polyphenol oxidase varies in accordance with the source of the
enzyme. Phenolic compounds and polyphenol oxidase are, in general, directly responsible for
enzymatic browning reactions in damaged fruits during postharvest handling and processing. The
relationship of the rate of browning to phenolic content and polyphenol oxidase activity has been
reported for various fruits such as apples (CoSeteng and Lee, 1987), grapes (Lee and Jaworski,
1988), and peaches (Lee et al. 1990).

In addition to serving as polyphenol oxidase substrates, phenolic compounds serve as inhibitors of

polyphenol oxidases. Various cinnamic acids were observed to act as substrate analogues and
serve as good inhibitors of apple polyphenol oxidase (Walker, 1995). Their inhibitory action
decreased in the following order: cinnamic acid > p-coumaric acid > ferulic acid > benzoic acid.


Enzymatic browning does not occur in intact plant cells since phenolic compounds in cell vacuoles
are separated from the polyphenol oxidase which is present in the cytoplasm. Once tissue is
damaged by slicing, cutting or pulping, however, the formation of brown pigments occurs. Both
the organoleptic and biochemical characteristics of fruits and vegetables are altered by pigment
formation. The rate of enzymatic browning in fruit and vegetables is governed by the active
polyphenol oxidase content of the tissues, the phenolic content of the tissue, pH, temperature and
oxygen availability within the tissue.

As described in Chapter 1, polyphenol oxidase catalyses the oxidation of phenols to o-quinones,

which are highly reactive compounds. O-quinones thus formed undergo spontaneous
polymerization to produce high-molecular-weight compounds or brown pigments (melanins).
These melanins may in turn react with amino acids and proteins leading to enhancement of the
brown colour produced. Many studies have focused on either inhibiting or preventing polyphenol
oxidase activity in foods. Various techniques and mechanisms have been developed over the years
for the control of these undesirable enzyme activities. These techniques attempt to eliminate one
or more of the essential components (oxygen, enzyme, copper, or substrate) from the reaction.

i) The elimination of oxygen from the cut surface of fruits or vegetables greatly retards the
browning reaction. Browning however occurs rapidly upon exposure to oxygen. Exclusion of
oxygen is possible by immersion in water, syrup, brine, or by vacuum treatment.

ii) This copper prosthetic group of polyphenol oxidases must be present for the enzymatic
browning reaction to occur. Chelating agents are effective in removing copper.

iii) Inactivation of the polyphenol oxidases by heat treatments such as steam blanching is
effectively applied for the control of browning in fruits and vegetables to be canned or frozen.
Heat treatments are not however practically applicable in the storage of fresh produce.
iv) Polyphenol oxidase catalyses the oxidation of phenolic substrates such as caffeic acid,
protocatechuic acid, chlorogenic acid, and tyrosine. Chemical modification of these substrates can
however prevent oxidation.

v) Certain chemical compounds react with the products of polyphenol oxidase activity and inhibit
the formation of the coloured compounds produced in the secondary, non-enzymatic reaction
steps, which lead to the formation of melanin.

Many techniques are applied in the prevention of enzymatic browning. Relatively new techniques,
such as the use of killer enzymes, naturally occurring enzyme inhibitors and ionizing radiation,
have been explored and exploited as alternatives to heat treatment and the health risks associated
with certain chemical treatments. Processing technologies applied in the control of enzymatic
browning in fruits and vegetables are now reviewed.

3.1 Processing

3.1.1 Heating

Heat treatment is the most widely utilized method for stabilizing foods because of its capacity to
destroy microorganisms and to inactivate enzymes. Steam blanching is one of the most effectively
applied methods of heat treatment for controlling enzymatic browning in canned or frozen fruits
and vegetables. Steam blanching is not however practical for the prevention of browning in fresh
foods. Temperatures applied in steam blanching treatments vary in accordance with the
thermostability of the enzyme to be inactivated as well as with the nature of the food product.
Pasteurization is generally conducted at temperatures ranging between 60 oC and 85 oC, while
blanching techniques are often operated at temperatures ranging between 70 oC and 105 oC or
higher. In general, exposure of polyphenol oxidases to temperatures in the 70-90 oC range, results
in the destruction of their catalytic activity (Vámos-Vigyázó, 1981). Blanching of green beans in an
automatic rotary hot water blancher at temperatures of 82 oC and above for 3.5 minutes, almost
completely inactivated catalase, lipoxygenase, and polyphenol oxidase activities (Lee et al. 1988).
Thermal inactivation profiles of important enzymes such as peroxidase, polyphenol oxidase, and
lipoxygenase in fruit and vegetable processing, follow first-order reaction kinetics. The thermal
inactivation profile of the thermostable fraction of several potato enzymes is shown in Figure 14
(Svensson, 1977). From this Figure it is obvious that at lower temperatures much longer times are
required to accomplish a 90 percent reduction in enzyme activity.
Figure 14. Thermal inactivation of the thermostable fraction of potato lipolytic acyl hydrolase,
lipoxygenase, polyphenol oxidase, and peroxidase. (From Svensson, 1977).

Thermal process characteristics for enzymatic reactions are described in accordance with kinetic
parameters such as decimal reduction times (D), inactivation rate constant (k), z-values (z), and
activation energies (Ea). The D value, or decimal reduction value, is defined as the time required to
inactivate 90 percent of the original enzyme activity at a given temperature. An inactivation
reaction, which follows first-order kinetics, has a D value equivalent to 2.303/k. Temperature
dependence of the D-value is given by the z-value, which represents the temperature increase
required in order to obtain a ten-fold (1-log cycle) decrease in D-value. For a first-order decay
process, the D value is equivalent to ln (10)k. Similar to the z-value is the activation energy (Ea),
which expresses the temperature dependence of the k-value as indicated in the Arrhenius

ln k = -Ea/RT + ln A

k = A (e-Ea/RT)

The Q10 value is the change in the rate of a reaction that occurs with a 10 oC change in

Rate T

Q10 = -----------------------

Rate T+10oC

The Q10 value can be related to the Arrhenius equation as,

Q10 = e10Ea/RT

Where Ea is independent of temperature, provided that conditions are appropriate and Q10 is
dependent on temperature.

Steam and water blanching are widely applied in the inactivation of enzymes and for stabilizing
frozen vegetables against off-flavour development, nutritional loss and discolouration. Blanching
also prevents chlorophyll and carotenoid pigments from undergoing enzymatic degradation. Time
and temperature must both be controlled during blanching. The time taken for the complete heat
inactivation of polyphenol oxidases varies considerably with the food product. Water blanching
prevents darkening in frozen sweet potatoes by significantly decreasing the polyphenol oxidase
activity (Ma et al. 1992). A blanch treatment at 100 oC for 3 min or 94 oC for 5 min is required to
obtain products with minimal darkening.

Blanching is nutritionally disadvantageous in that it results in losses in vitamins, flavours, colours,

texture, carbohydrates and other water-soluble components. Requirements for large amounts of
water and energy, coupled with waste disposal problems make blanching technically
disadvantageous. Blanched products generally require a cooling step prior to freezing. Additional
leaching takes place if this cooling step is carried out by immersion of the product in cold water.

3.1.2 Refrigeration

The rate of enzyme-catalysed reactions is controlled to a great extent by temperature. For every
10 oC temperature increase (in biological important ranges), there is a two-fold increase in the rate
of an enzyme-catalysed reaction, referred to as the temperature coefficient (Q10). On the other
hand, for every 10 oC reduction in temperature a similar decrease in the rate of biological activity
occurs. At low temperatures, reduced kinetic energy of the reactant molecules results in a
decrease in both mobility and "effective collisions" necessary for the formation of enzyme-
substrate complexes and their products.

Chilling involves the temporary storage of foods at temperatures above freezing. Some vegetables
(broccoli, berries, spinach, peas, etc.) are either pre-cooled or stored at chilling temperatures.
Other commodities (bananas, mangoes, avocados, tomatoes, etc.) are susceptible to chill injury
and should therefore not be stored below their respective critical temperatures (Fennema,
1975a). The chilling process is generally accomplished either with the use of moving air, water
(hydrocooling), ice, or vacuum (vacuum cooling).
Cold preservation and storage during distribution and retailing are necessary for the prevention of
browning in fruit, vegetables, and seafood, since refrigerated temperatures are effective in
lowering polyphenol oxidase activity.

3.1.3 Freezing

Freezing temperatures of -18 oC or below are often used for the long-term preservation of food.
Mechanisms of enzyme inactivation at freezing temperatures can be explained by several
hypotheses. Tappel (1966) proposed that solutes, such as salt, sugars, and other carbohydrates,
are effective enzyme inhibitors at high concentrations. Increased solute concentrations attained in
the semi-frozen state would therefore enhance both substrate and product inhibition. Fennema
(1975b) attributed the effects of freezing to changes in pH. According to his theory, changes in
buffer concentration and composition could cause changes in pH and thus acidity, due to
enhanced mobility of the hydrogen ion in ice as compared to that in water. Another factor
involved in enzyme inactivation at freezing temperatures is the perturbation of sulphydryl groups
essential for the activity of some enzymes. Perturbation of sulphydryl groups is attributed to
increased propensity for SS-SH interchange as a result of 1) increased concentration of disulfide
groups, 2) conformational changes, and 3) accelerated oxidation of sulphydryl groups due to
increased oxygen concentrations within the ice (Fennema, 1975b). Recent discussions have
attributed enzyme inactivation on freezing to the influence of water removal during the freezing
process. Removal of water during freezing causes changes that eventually result in an alteration of
the microenvironment of the enzyme, in a manner similar to dehydration.

As with high temperature inactivation, low-temperature inactivation has its drawbacks. Freezing
causes changes in texture and other freshness characteristics. Freezing can also lead to the
decompartmentalization of certain enzymes, substrates, and/or activators as a result of cell
disruption thus facilitating enzyme activity in the frozen state, which is enhanced upon thawing of
the food (Ashie and Simpson, 1996).

3.1.4 Dehydration

Physical Methods

Water generally exerts an enormous influence on enzyme activity in that it behaves both as a
solvent and a reactant (Ashie and Simpson, 1996). Water activity (aw) is defined as the ratio of the
partial pressure of the water vapour above a sample to that of saturated water vapour at the same
temperature. Water activity can affect reactions in solid media either by restricting the mobility of
the reactants or by altering the active conformation of substrate and enzyme. In general,
enzymatic activities decrease with a decrease in aw. Water activity can be controlled either by
partial drying or through the addition of water binding agents, such as polyols, sugars and salts, to
increase stability and inhibit microbial growth. Dehydration has varied effects on enzymes because
of their differing responses to the concentrations of various solutes, inhibitors, or activators.
Solutes such as sugars and proteins often have a protective effect against enzyme inactivation.
Freeze-drying is another dehydration process, which involves the removal of moisture from foods
in the frozen state by sublimation under high conditions of high vacuum. Low temperatures
applied in the process inhibit undesirable chemical and biochemical reactions while minimizing the
loss of volatile aromatic compounds. Rapid freezing during the process minimizes enzymatic
browning and generally yields a product of excellent quality, while slow freezing is somewhat less
effective in minimizing browning. Freeze-dried mushrooms prepared by a slow-freezing process
were observed to have higher polyphenol oxidase activity than those prepared by the Freon-12
dipping process (Fang and Chiang, 1975). Other physical methods of dehydration include spray-
drying, radiative-, solar- and microwave drying.

Chemical Methods

Salts can also have a dehydrating effect in that high salt concentrations remove water from the
reaction medium (i.e. reduce aw), making it unavailable for water-protein interactions, thus
resulting in reduced enzyme activity. Sodium chloride, sucrose, and other sugars, glycerol,
propylene glycol and modified corn syrups are some of the solutes used for dehydration. Salts are
also capable of modifying the thermodynamics of enzyme catalysed reactions. Polyvalent anions,
such as SO32- and PO42-, in general tend to stabilize enzymes, while polyvalent cations, such as
Ca2+ and Mg2+, tend to have a destabilizing effect on enzymes (Adams, 1991).

3.1.5 Irradiation

Food irradiation is increasingly recognized as a method for reducing postharvest food losses,
ensuring hygienic quality, and facilitating wider trade in foodstuffs. Food irradiation is unique in
that it makes use of ionizing radiation for improving the shelf life or wholesomeness of the
product. Food irradiation is effective in securing the long-term preservation of foods through the
inactivation of microorganisms. Meats, seafoods, fruits, vegetables, and cereal grains can be
preserved by irradiation. Food irradiation can extend the shelf life of those foods which, after
harvest, continue to be physiologically active, by delaying their maturation or sprouting.

Gamma rays (from cobalt-60 or cesium-137), X-rays, and accelerated electrons (electron beams)
are the forms of ionizing radiation applied in food processing. Food irradiation has been approved
for both preservation and processing applications in the United States. The effects of varied doses
of ionizing radiation on fresh fruits and vegetables are summarized in Table 4 (Kader, 1986).

Table 4. Effects of ionizing radiation on fresh fruits and vegetables.

Dose (kGy) Observed effects

Sprout inhibition in tuber, bulb, and root vegetables;

inhibition of growth in asparagus and mushroom
0.15-0.75 Insect disinfestation

0.25-0.50 Delayed ripening of some tropical fruits such as banana,

mango, and papaya

>1.75 Control of postharvest disease

1.00-3.00 Accelerated softening; development of off-flavors in some


Excessive softening; abnormal ripening; incidence of some

physiological disorders; impaired flavor

1 kilogray (kGy) = 1000 gray (Gy), which is the SI unit of energy absorbed (1 joule/kg) from ionizing
radiation. 1 Gy = 100 rad (1 rad = 100 erg/g), and 1 kGy=100 krad

Low-dose gamma irradiation of fresh shrimp decreased melanosis and significantly increased the
shelf life of shrimp stored on ice (Novak et al. 1967). However low-dose irradiation of shrimp after
the onset of melanosis, resulted in acceleration of the rate of melanosis. Browning in potato
tubers and tropical fruits after gamma-irradiation was extensively reviewed by Thomas (1984;
1986). Gamma irradiation up to 1 kGy inhibited sprouting in potato tubers and caused a reduction
in both total phenolic and chlorogenic acid contents (Pendharkar and Nair, 1987). Mondy and
Gosselin (1989) reported that a radiation dose of 1 kGy caused less darkening in potatoes than
higher doses. Increased blackspot formation in potatoes at higher irradiation doses may occur due
to the rupture of lipid membranes and the liberation of polyphenol oxidase from mitochondria,
which in turn oxidize phenolic substrates in the vacuole, and cause browning. Browning might
therefore be minimized by controlling the dosage level of the ionizing radiation applied.

Ionizing radiation at doses exceeding 1 kGy can introduce various types of physiological disorders
in food products. Free radicals produced during the treatment of food with ionizing radiation, are
capable of reacting with various food constituents and inducing undesirable side effects, such as
tissue darkening, lipid oxidation and decreased vitamin content. Nonenzymatic browning reactions
of free amino acids and proteins with reducing sugars, such as glucose, may be responsible for this

The sensitivity of enzymes to ionizing radiation is measured by D37, which is defined, as the dose
required to inactivate 63 percent of the original activity of the enzyme. Where food systems are
concerned, sensitivity is often expressed as DE, which is the dose required for 90 percent
inactivation of the enzyme. D37 values of some enzymes in model systems measured in the dry
state are shown below (Roozen and Pilnik, 1971).

Enzyme D37 (kGy)

Alkaline phosphatase 40-50

Pectin esterase 60

Peroxidase 30-70

Enzymes exhibit a high degree of stability in the absence of oxygen, presumably due to formation
of the superoxide anion (O2-), which can interact to raise the energy level of other active molecules
and residues. Combined treatments using both irradiation and heat or other methods have
demonstrated a synergistic effect in preventing enzymatic browning.

3.1.6 High pressure treatment

Enzyme denaturation is caused by rearrangement and/or destruction of noncovalent bonds such

as hydrogen bonds, hydrophobic interactions, and ionic bonds of the tertiary protein structure.
Pressure can influence biochemical reactions by reducing molecular spacing and increasing
interchain reactions. High-pressure treatment is a potentially viable technique for preserving food
quality through the inactivation of endogenous food enzymes. Pressures exceeding 5 kbar
generally cause irreversible denaturation of enzymes due to the weakening of hydrophobic
interactions and the breaking of intramolecular salt bridges (Cheftel 1992; Masson 1992). High-
pressure treatments can result in either reversible or irreversible changes in protein structure.
Loss of catalytic activity under high-pressure conditions however varies in accordance with the
enzymes, the nature of the substrates, the temperature and the duration of high pressure
processing (Cheftel, 1992; Kunugi, 1992).

High-pressure treatment is known to preserve quality carriers, such as flavour, taste, and vitamins
in foods (Cheftel, 1992). Depending on the nature of the food, it may also induce quality changes
in the food. A proper combination of pressure and temperature might be used to enhance enzyme
inactivation in certain foods. This combination would prevent off-flavour development, and
changes in colour, nutritional value, and structure (Farkas, 1987).

Polyphenol oxidase is highly pressure resistant. Pressures of 5 and 7 kbar are required for the
inactivation of apple polyphenol oxidase at pH 4.5 and 5.4, respectively (Nicoli et al. 1994).
Polyphenol oxidases from different sources show different pressure-temperature behaviours
(Table 5). Weemaes et al. (1998) showed that inactivation of polyphenol oxidase from apple,
grape, avocado and pear at room temperature (25 oC) became noticeable at »600, 700, 800 and
900 Mpa, respectively. However, plum polyphenol oxidase was not inactivated at room
temperature by pressures of up to 900 Mpa.

Table 5. Pressure stability of PPO derived from various sources.

Origin of Medium Treatment Activation/Inactivation Reference

Apple crude extract (pH 4.5) #300 Mpa/25 oC/1min activation Anese et al.
crude extract (pH 5.4) >500 Mpa/25 oC/1 min inactivation

crude extract (pH 7) #300 Mpa/25 oC/1min activation

>700 Mpa/25 oC/1min inactivation

#100 Mpa/25 oC/1min inactivation

Avocado partially purified 800 Mpa/25 oC inactivation Weemaes et al.


Onion purified enzyme in Tris > 700 Mpa/25 oC/10 min activation, which is Butz et al.,
buffer (pH 6.5, 0.01M) maximal at 500 MPa (1994)

Pear in slices 400 Mpa/25 oC/10 min activation Asaka and

Hayashi (1991)
partially purified 900 Mpa/25 oC slight inactivation
Weemaes et al.

Plum partially purified 900 Mpa/25 oC no inactivation Weemaes et al.


Potato in cubes 400 Mpa/<50 oC/15 min no inactivation Knorr (1993)

White partially purified 700 Mpa/25 oC inactivation Weemaes et al.

grapes (1998)

Pressure inactivation of polyphenol oxidase in Tris buffer at 45 oC required treatments of 900 Mpa
for a period exceeding 30 min (Figure 15). The inactivation of pure enzymes by pressure is
dependent on the immersion medium, the pH, as well as the temperature and duration of the
treatment. Food constituents can however have a protective effect on enzymes during high
pressure treatment. Mushroom polyphenol oxidase shows very high pressure stability, although it
is a thermosensitive enzyme which is readily inactivated by temperatures exceeding 50 oC
(Weemaes et al. 1997).

Figure 15. Effect of high hydrostatic pressure on relative activity of polyphenoloxidase in Tris
buffer (pH 7) at 45 oC. (From Seyderhelm et al., 1996).

3.1.7 Treatment with supercritical carbon dioxide (SC-CO2)

A pure substance exists in a state that exhibits both gas- and liquid-like properties at temperatures
and pressures above its critical point (Kiran and Zhuang, 1997). A supercritical fluid (SC) on the
other hand exists in the fluid state above its critical temperature and pressure, i.e. it has a density
similar to that of a liquid, surface tension close to zero, and its diffusivity and viscosity range
between that of a liquid and a gas. Carbon dioxide has a low critical temperature and pressure
(Rizvi et al. 1986), which makes it ideal in use as a SC fluid. Its nontoxicity, nonflammability, low
cost and availability (Hardardottir and Kinsella, 1988) coupled with its environmentally acceptable
nature make it highly desirable for food processing applications. SC carbon dioxide treatments are
effective in destroying microorganisms, as well as in inactivating unwanted enzymes in foods.
Their effectiveness in food preservation is due to the fact that upon dissolution in water, high
pressure CO2 produces carbonic acid, which effects a temporary reduction in pH thus inactivating
enzymes and microorganisms. SC carbon dioxide has been reported to inactivate PPO (Zemel,
1989). Purified Florida spiny lobster, brown shrimp, and potato polyphenol oxidases exhibited a
time-related decline in activity following treatment at 43 oC with high pressure carbon dioxide at
58 atm (Chen et al. 1992). Kinetic studies showed that crustacean polyphenol oxidases were more
vulnerable to SC carbon dioxide treatment than potato polyphenol oxidase.
3.1.8 Ultrafiltration (UF)

UF is a widely used membrane separation technology which has been shown to be effective in
stabilizing the colour of white wines and other fruit juices (Flores et al. 1988; Sims et al. 1990). The
use of ultrafiltration as an alternative to sulphiting for the control of enzymatic browning has been
studied (Sims et al. 1989; Goodwin and Morris, 1991). UF is believed to remove polyphenol
oxidase, but not lower-molecular-weight polyphenols or Maillard-reaction precursors, which could
undergo nonenzymatic browning during storage of the wine.

Banana PPO fractions are reported to have molecular weights in excesses of 30 kDa (Galeazzi et al.
1981), which is well within the range of molecular weight cut-offs for UF membranes. UF therefore
offers potential for improving the colour stability of banana juice without the application of heat,
which is known to alter the flavour of banana juice.

3.1.9 Ultrasonication

Heat treatment is capable of altering the organoleptic properties of foods and reducing the
bioavailability of certain nutrients. The combined use of heat and ultrasonic waves can contribute
to enzyme inactivation. When applied to a liquid, ultrasonic waves promote acoustic cavitation
leading to bubble formation. Bubble-liquid interfaces continuously undergo changes in shape and
size. Acoustic streams occurring within the liquid in the vicinity of the bubble, often cause severe
shear stresses which can promote enzyme denaturation. Ultrasonication also promotes chemical
reactions involving H× and OH× free radicals formed by the decomposition of water within the
oscillating bubbles (El. piner et al. 1965). These free radicals could be scavenged by amino acid
residues of the enzymes participating in structure stability, substrate binding, or catalytic functions
(Gebicki and Gebicki, 1993).

3.2 Inhibitors

Enzymatic browning can be inhibited by targeting the enzyme, the substrates (oxygen and
polyphenols) or the products of the reaction.

i) Inhibition targeted toward the enzyme

Mayer and Harel (1979) classified the inhibitors which act directly on polyphenol oxidase into two
groups. The first group, which consists of metal ion chelators, such as azide, cyanide, carbon
monoxide, halide ions and tropolone, is well documented for the inhibition of polyphenol oxidase
from various sources. The chloride ion was shown to be noncompetitive for apple polyphenol
oxidase, while other halide ions were observed to have a competitive inhibitory effect (Janovitz-
Klapp et al. 1990). The second group of inhibitors, which consists of aromatic carboxylic acids of
the benzoic and cinnamic series, has been widely studied (Janovitz-Klapp et al. 1990). Compounds
of this group behave as competitive inhibitors of polyphenol oxidase, owing to their structural
similarity with phenolic substrates.

ii) Inhibition targeted toward the substrate

Enzymatic browning can be controlled by removal of either the oxygen or phenolic substrates,
from the reaction medium. Elimination of oxygen is perhaps the most satisfactory methodology
for preventing phenol oxidase catalysed phenolic oxidation. The removal of oxygen can however
result in metabolic deviations since excessive reduction of oxygen induces anaerobic metabolism,
leading to breakdown and off flavour development in foods (Ballantyne et al. 1988).

Vacuum packaging of pre-peeled potatoes to exclude oxygen, was observed to extend their shelf
life (Langdon, 1987). Vacuum packaged products however rapidly undergo browning upon
exposure to air. Anaerobic conditions created by vacuum packaging are a cause for safety concern
in that they are potentially capable of supporting the growth of Clostridium botulinum and the
production of its toxin (Tamminga et al. 1978).

Specific adsorbents, which undergo complexation with the phenolic substrate may be applied in
the physical elimination of phenolic compounds from food systems. The use of cyclodextrins for
the removal of phenolic compounds from raw fruit and vegetable juices has been patented in the
United States (Hicks et al. 1990). Cyclodextrins are thought to inhibit polyphenol oxidase activity
through the formation of inclusion complexes with polyphenols (Sapers et al. 1989). Sulphated
polysaccharides also have an inhibitory effect on browning (Tong and Hicks, 1991). Apart from
possible complexation, sulphate groups are believed to exert their inhibitory effect through
chelation of the copper prosthetic group of the polyphenol oxidase (Tong and Hicks, 1991).

Enzymatic modification of phenolic substrates may serve to inhibit polyphenol oxidase activity. O-
methyltransferase for example converts o-dihydroxy phenolics to the corresponding methoxy
derivatives, which do not serve as substrates for polyphenol oxidase (Finkle and Nelson, 1963).
Similarly, protocatechuate 3,4-dioxigenase purified from Pseudomonas aeruginosa prevents the
browning of Gravenstein apple juice (Kelly and Finkle, 1969) due to substrate modification. These
enzyme modification techniques are not however feasible in commercial use, owing to the high
cost of these enzymes.

iii) Inhibition targeted toward the products

O-quinones, which are the products of diphenol oxidation, are capable of reacting with each other,
resulting in the formation of dimers of the original phenol. These dimers, which possess an o-
diphenolic structure, undergo re-oxidation resulting in the formation of larger oligomers of varying
colour intensities. Ascorbic acid (Hsu et al. 1988), thiol compounds (Henze, 1956), sulphites
(Sayavedra-Soto and Montgomery, 1986), and amino acids (Kahn, 1985) are however capable of
inhibiting dimer formation and re-oxidation either by reducing o-quinones to o-diphenols, or
through the formation of colourless addition products.

Classification of Inhibitors

The use of browning inhibitors in food processing is restricted by considerations relevant to

toxicity, wholesomeness, and effect on taste, flavour, texture, and cost. Browning inhibitors may
be classified in accordance with their primary mode of action. Six categories of polyphenol oxidase
inhibitors are applicable in the prevention of enzymatic browning (Table 6). These include (1)
reducing agents; (2) acidulants; (3) chelating agents; (4) complexing agents; (5) enzyme inhibitors;
(6) enzyme treatments. Each category of inhibitor is now discussed:

Table 6. Representative inhibitors of enzymatic browning.(Adapted from McEvily et al. 1992)

Reducing agents sulphiting agents

ascorbic acid and analogs



Chelating agents phosphates


organic acids

Acidulants citric acid

phosphoric acid

Enzyme inhibitors aromatic carboxylic acids

aliphatic alcohol



substituted resorcinols

Enzyme treatments oxygenases

o-methyl transferase


Complexing agents cyclodextrins

3.2.1 Reducing agents/Antioxidants

Reducing agents play a role in the prevention of enzymatic browning either by reducing o-
quinones to colourless diphenols, or by reacting irreversibly with o-quinones to form stable
colourless products. Reducing compounds are very effective in the control of browning. Sulphiting
agents are the most widely applied reagents for the control of browning in the food industry.

a. Sulphiting agents

Sulphites are the most widely used inhibitors of enzymatic browning. Sulphiting agents include
sulphur dioxide (SO) and several forms of inorganic sulphite that liberate SO2 under the conditions
of their use.

SO2: sulphur dioxide

SO32-: sulphite

HSO3-: bisulphite

S2O52-: metabisulphite

SO2 and sulphite salts form sulphurous acid (H2SO3) and exist as a mixture of the ionic species,
bisulphite (HSO3-) and sulphite (SO32-) anions in aqueous solution. The predominant ionic species
varies in accordance with pH, ionic environment, water activity, presence of non-electrolytes, and
concentration of the medium in which they are dissolved. Maximum HOS3-concentrations exist at
pH 4, while at pH 7, both SO32- and HSO3- exist in approximately equivalent concentrations (Green,
1976). Increased concentrations of sulfite at pHs of less than 5 were observed to enhance the
inhibition of polyphenol oxidase-catalysed browning (Sayavedra-Soto and Montgomery, 1986).
The dibasic acid undergoes ionization according to the following reaction scheme:

SO2×H2O -> (H2SO3)- -> HSO3- + H+

HSO3- ->SO32- + H+

with pKa values of 1.89 and 7.18 (25 oC, zero ionic strength) for the first and second ionizations,
respectively (Figure 16).
Figure 16. The distribution of species of sulfurous acid at various pH values. (From Ough, 1984).

Sulphites serve a multifunctional role in foods. They possess antimicrobial activity and inhibit both
enzymatic and non-enzymatic browning reactions. Madero and Finne (1982) proposed that
bisulphite exerted a competitive inhibitory effect on polyphenol oxidase, by binding a sulphydryl
group at the active site of the enzyme. Ferrer et al. (1989b) on the other hand, proposed that
bisulphate inhibition was due to the reaction of sulphites with intermediate quinones, resulting in
the formation of sulphoquinones, which irreversibly inhibited polyphenol oxidase, causing
complete inactivation. Mechanisms involved in the control of enzymatic browning by sulphites are
shown in Figure 17.
Figure 17. The primary role of reducing agents such as sulphiting agents in the inhibition of
enzymatic browning is to reduce the pigment precursors (quinones) to colourless, less-reactive
diphenols. (Adapted from Walker, 1977).

Although sulphites are very effective in controlling browning, they are subject to regulatory
restrictions owing to their potentially adverse effects on health. Many reports have described
allergic reactions in humans, following the ingestion of sulphite-treated foods by hypersensitive
asthmatics. The use of sulphiting agents in food processing is based on sulfur dioxide equivalence
(Modderman, 1986). Table 7 gives a list of sulphiting agents and their theoretical yields of sulfur
dioxide. The Joint Expert Committee on Food Additives (JECFA) of the World Health Organization
(WHO) and the Food and Agriculture Organization (FAO) recommend an acceptable sulphite daily
intake of 0-0.7 mg sulphur dioxide per kg of body weight.

Table 7. Chemicals yielding sulfur dioxide which are currently allowed for use in food as
preservatives. (Adapted from Green, 1976)

Chemical Formula Theoretical Yield (%) Solubility (mg/100 mL)

Sulphur dioxide SO2 100.0 11 at 20 oC

Sodium sulphite anhydrous Na2SO3 50.8 28 at 40 oC

Sodium sulphite (heptahydrate) Na2SO3 @7H2O 25.4 24 at 25 oC

Sodium hydrogen sulphite NaHSO3 61.6 300 at 20 oC

Sodium metabisulphite Na2S2O5 67.4 54 at 20 oC

Potassium metabisulphite K2S2O5 57.6 25 at 0 oC

Sulphites are currently applied for the inhibition of melanosis (blackspot) in shrimp, potatoes,
mushrooms, apples, and other fruits and vegetables. Sulphites are also applied in stabilizing the
flavour and colour of wines. Sulphite concentrations necessary for controlling enzymatic browning
vary widely in accordance with the food material and the time required for inhibition of the
browning reaction (Taylor et al., 1986). Where only monophenolic substrates, such as tyrosine are
present, as in the case of potatoes, relatively low levels of sulphite are effective in inhibiting
browning. On the other hand, where diphenols are present, as is the case in avocados, much
higher sulfite concentrations are required for the control of browning.

Sulphites no longer have "Generally Required as Safe Status" (GRAS) status for use on fruits and
vegetables served raw, sold raw or presented to the consumer as raw in the United States.
According to the United States Federal Register (1988) foods containing detectable levels of a
sulphiting agent, at 10 ppm regardless of source, must declare the sulfite and its content on the
ingredient label. More regulatory restrictions are likely to be globally applied to the use of
sulphites in foods since sulphite allergies pose a health risk in many populations. Regulations
enacted by the United States Food and Drug Administration (FDA) in 1995 prohibit the use of
sulphites in salad bars. As a result, there has been a considerable focus on identifying appropriate
sulphite substitutes for use in foods. The FDA has proposed maximum residual sulphur dioxide
levels for certain foods. In accordance with these proposed limits, residual sulphur dioxide levels
for fruit juices, dehydrated potatoes, and dried fruit, are 300, 500, and 2000 ppm respectively
(Federal Register, 1988). Shrimp products having residual sulphite levels in excess of 100 ppm are
considered adulterated, since these levels are considered unsafe (Federal Register, 1985).

b. L-Ascorbic acid

Ascorbic acid is a moderately strong reducing compound, which is acidic in nature, forms neutral
salts with bases, and is highly water-soluble. L-ascorbic acid (vitamin C) and its various neutral salts
and other derivatives have been the leading GRAS antioxidants for use on fruits and vegetables
and in fruit juices, for the prevention of browning and other oxidative reactions (Bauernfeind and
Pinkert, 1970).

Ascorbic acid also acts as an oxygen scavenger for the removal of molecular oxygen in polyphenol
oxidase reactions. Polyphenol oxidase inhibition by ascorbic acid has been attributed to the
reduction of enzymatically formed o-quinones to their precursor diphenols (Walker, 1977).
Ascorbic acid is however irreversibly oxidized to dehydroascorbic acid during the reduction
process, thus allowing browning to occur upon its depletion (Figure 18). More stable forms of
ascorbic acid derivatives, such as erythrobic acid, 2- and 3-phosphate derivatives of ascorbic acid,
phosphinate esters of ascorbic acid, and ascorbyl-6-fatty acid esters of ascorbic acid, have
however been developed to overcome these problems (Seib, 1985; Sapers and Hicks, 1989).
Ascorbic acid esters release ascorbic acid upon hydrolysis by acid phosphatases (Liao and Seib,
1988). Their relative effectiveness as browning inhibitors varies in accordance with the food
product (Bauernfeind and Pinkert, 1970). Compounds containing reactive amino or thiol groups
can greatly affect the reactivity of o-quinones.

Figure 18. Mechanism of prevention of colour formation by ascorbic acid.

Ascorbic acid causes a distinct yellow off-colour, when used in the prevention of melanosis in
shrimp (Otwell and Marshall, 1986). It is usually applied in conjunction with citric acid in order to
maintain a more acidic pH level. In addition, it is also believed to have a chelating effect on the
copper prosthetic group of polyphenol oxidase (Whitaker, 1972b).

c. Erythorbic acid

Erythorbic acid and its salt, sodium erythorbate, are strong reducing agents with GRAS status. They
both act as oxygen scavengers, thus eliminating oxygen as a substrate for browning reactions.
Erythorbic acid is the D-isomer of ascorbic acid but does not have vitamin C activity. Its use in
conjunction with citric acid has often been suggested as a substitute for sulphites in the control of
enzymatic browning. Current research suggests that L-ascorbic acid and erythorbic acid both
possess equivalent antioxidant properties. A combination of both acids is applied at the retail level
for inhibiting both oxidative rancidity and discolouration in vegetables, salads, apples, and frozen
seafood. Erythobic acid or sodium erythorbate can suppress browning reactions in frozen fruits.

d. Cysteine

Cysteine is an effective inhibitor of enzymatic browning. It is reported to be more effective than

sodium bisulphite as an antibrowning agent (Kahn, 1985). Concentrations of cysteine and other
thiols required for the achievement of acceptable levels of browning inhibition have however been
shown to have negative effects on taste. The inhibition of melanosis by cysteine is thought to be
due to the formation of colourless thiol-conjugated o-quinones (Pierpoint, 1966). Cysteine has also
been shown to reduce o-quinones to their phenol precursors (Walker, 1977; Cilliers and Singleton,

A mode of action for cysteine and cysteinyl addition in the control of browning proposed by
Richard-Forget et al. (1992) is illustrated in Figure 19. Cysteine-quinone adducts serve as
competitive inhibitors of polyphenol oxidase. Sulphydryl (thiol) compounds N-acetyl-L-cysteine
(NAC) and reduced glutathion (GSH) are also excellent inhibitors of browning of potato powder
(Figure 20, Friedman et al. 1992).
Figure 19. Effect of cysteine and cyteinyl addition compounds with o-quinones on the enzymatic
oxidation of o-diphenols. (From Richard-Forget et al., 1992).

Figure 20. Inhibition of potato browning by N-acetyl-L-cysteine (5% potato in water; inhibitor 0.8
mM; 5 h; room temperature). (From Friedman et al., 1992).

e. Phenolic antioxidants

Antioxidants are defined by the United States Food and Drug Administration (FDA) as substances,
which may be applied in preserving food by retarding deterioration, rancidity or discolouration
due to oxidation. Antioxidants inhibit oxidative processes by reacting with free radicals, through
metal chelation, and by scavenging singlet oxygen.

Both synthetic and naturally occurring phenolic antioxidants are used in food applications. Several
synthetic antioxidants, such as butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT),
tertiarybutyl hydroxyquinone (TBHQ) and propyl gallate (PG), are permitted for use in food. Their
structures are shown in Figure 21. Plant phenolic compounds such as tocopherols, flavonoid
compounds, cinnamic acid derivatives, and coumarins are naturally occurring compounds, which
have an antioxidant effect, that renders them inhibitory to polyphenoloxidase, and thus browning.
Figure 21. Major antioxidants used in food.

3.2.2 Acidulants

Ionizable groups of the protein structure of enzymes are affected by the pH of the food medium.
These groups must be in the appropriate ionic form in order to maintain the conformation of the
active site, bind substrates, or catalyse the enzymatic reaction (Segel, 1976). Changes in the
ionization status of enzymes are generally reversible. Irreversible denaturation can however occur
under conditions of extreme pH. The stability of the substrate is also affected by changes in pH,
since substrates can undergo chemical breakdown under extreme conditions of pH. Degraded
substrates often behave as enzyme inhibitors, since they share the molecular features of the
substrate (Tipton and Dixon, 1983).

Acidulants are generally applied in order to maintain the pH well below that required for optimum
catalytic activity of an enzyme. Acidulants such as citric, malic, and phosphoric acids are capable of
lowering the pH of a system, thus rendering polyphenol oxidase polyphenol oxidase inactive
(Richardson and Hyslop, 1985). Acidulants are often used in combination with other antibrowning

a. Citric acid

Citric acid is the one of the most widely used acidulants in the food industry. It is typically applied
at levels ranging between 0.5 and 2 percent (w/v) for the prevention of browning in fruits and
vegetables. In addition, it is often used in combination with other antibrowning agents such as
ascorbic or erythorbic acids and their neutral salts, for the chelation of prooxidants and for the
inactivation of polyphenol oxidase. Recommended usage levels for citric acid typically vary
between 0.1 and 0.3 percent (w/v) with the appropriate antioxidant at levels ranging between 100
and 200 ppm (Dziezak, 1986). Citric acid exerts its inhibitory effect on polyphenol oxidase by
lowering the pH as well as by chelating the copper at the active site of the enzyme.

3.2.3 Chelators

Enzymes generally possess metal ions at their active sites. Removal of these ions by chelating
agents can therefore render enzymes inactive. Chelating agents complex with prooxidative agents,
such as copper and iron ions, through an unshared pair of electrons in their molecular structures.
Chelators have been applied in various food processing applications, for enzyme inactivation
(McEvily et al. 1992). Chelators used in the food industry include sorbic acid, polycarboxylic acids
(citric, malic, tartaric, oxalic, and succinic acids), polyphosphates (ATP and pyrophosphates),
macromolecules (porphyrins, proteins), and EDTA.

Other non-GRAS chelating agents which are capable of inhibiting polyphenol oxidase include
cyanide, diethyldithiocarbonate, sodium azide and 2-mercaptobenzothiazole, carbon monoxide,
mercaptobenzthiazol, dimercaptopropanol, and potassium methyl xanthate.

Ascorbic acid also has a chelating effect on the prosthetic group of polyphenol oxidase.


EDTA is a chelating agent permitted for use in the food industry as a chemical preservative.
Calcium disodium EDTA (21 CFR 172.120) and disodium EDTA (21 CFR 172.135) have been
approved for use as food additives by the United States Food and Drug Administration (Anon,
1992). Highly stable complexes are formed by the sequestering action of EDTA compounds on
iron, copper, and calcium. Maximum chelating efficiency occurs at the higher pH values where
carboxyl groups exist in a dissociated state (Dziezak, 1986). EDTA is generally used in combination
with other chemical treatments for the prevention of enzymatic browning in foods. It not very
effective as an inhibitor of peach polyphenol oxidase (Wong et al. 1971).

A typical combination of anti-browning agents might consist of a chemical reducing agent

(ascorbic acid), an acidulant (citric acid) and a chelating agent (EDTA).

b. Phosphates

Polyphosphates, sodium acid pyrophosphate, and metaphosphate are chelating agents of limited
cold water solubility. They have been used as antibrowning agents for fresh-peeled fruits and
vegetables at concentrations as low as 0.5 to 2 percent (final concentration in the dip solution)
(McEvily et al. 1992). SporixTM , an acidic polyphosphate mixture (sodium acid pyrophosphate,
citric acid, ascorbic acid, and calcium chloride), has been observed to delay the onset of oxidation
and enzymatic browning in fruits and vegetables (Gardner et al. 1991).

c. Maltol

Maltol (3-hydroxy-2-methyl-4H-pyran-4-one), a g-pyrone derivative, is a relatively weak inhibitor

of the formation of pigmented products. Structurally, it contains an a, b-unsaturated keto-enol
constituent, which makes it a good chelator. Maltol does not however chelate copper at the active
site of polyphenol oxidase. It prevents browning either through its ability to conjugate o-
benzoquinones back to o-dihydroxyphenols or through irreversible inactivation of polyphenol
oxidase (Kahn, 1995).

d. Kojic acid

Kojic acid (5-hydroxy-2-hydroxymethyl-4H-pyran-4-one), a g-pyrone derivative, is a fungal

metabolite produced by many species of Aspergillus and Penicillium. It is a good chelator of
transition metal ions such as Fe (III) and Cu (II) (Beélik, 1956; Wiley et al. 1942). Kojic acid occurs in
many fermented Oriental foods (Kinoshita et al. 1968), and possesses both antibacterial and
antifungal activities. A mixture of ascorbic acid and kojic acid has been patented for use as an anti-
browning agent in foods (Fukusawa et al. 1982). Kojic acid has potential applicability in the
prevention of melanosis in both plant and seafood products. Saruno et al. (1979) demonstrated
that kojic acid from Aspergillus albus inhibited mushroom PPO activity. Kojic acid was also shown
to inhibit melanosis in pink shrimp (Applewhite et al. 1990). Chen et al. (1991b) determined that
kojic acid was a competitive inhibitor of the oxidation of chlorogenic acid and catechol by apple
polyphenol oxidase.

Kojic acid inhibits the rate of formation of pigmented products, as well as the rate of oxygen
uptake, when various o-dihydroxy- and trihydroxy phenols are oxidized by tyrosinase (Kahn, 1995).
Tyrosinase inhibition by kojic acid was thought to be due to the ability of kojic acid to bind copper
at the active site of the enzyme. Although kojic acid is a good inhibitor of polyphenol oxidase, its
toxicity is of concern. Wei et al. (1991) reported weak mutagenic activity of kojic acid in
a Salmonella typhimurium assay.
e. Polysaccharides

Various sulfated polysaccharides, including carrageenans, amylose sulfate, and xylan sulfate, were
determined to be effective browning inhibitors in both apple juice and diced apples (Tong and
Hicks, 1991). Pectin, a naturally occurring anionic polysaccharide at a concentration of 0.5 percent,
gave between 5 and 10 percent inhibition of apple juice browning (Tong et al. 1995). Carboxyl
groups present in pectin are believed to be capable of chelating the copper moiety of polyphenol
oxidase, thus preventing browning.

f. Carbon monoxide (CO)

Carbon monoxide (CO) is a known inhibitor of many copper-containing oxidases and behaves as a
noncompetitive inhibitor of phenolic substrates. It has been studied in preventing the
discolouration of Shitake mushrooms (Fujimoto et al. 1972). Polyphenol oxidase activity extracted
from freeze-dried mushroom powder was inhibited by CO (Albisu et al.1989). This inhibition was
however reversible, and removal of CO led to restoration of the initial activity. A two-step gas
treatment of potato strips with SO2 followed by CO, resulted in 93.7 percent and 99.9 percent
inactivation of prophenol oxidase and polyphenol oxidase respectively, after 60 days at room
temperature (Kramer et al. 1980). A number of safety problems are however associated with the
use of carbon monoxide gas.

3.2.4 Complexing agents

a. Cyclodextrins

The cyclodextrins (CDs) are a class of cyclic oligosaccharides produced by the action of
cyclomaltodextrin glucanotransferase (CGTase) on liquified starches. Industrially produced CDs
contain 6-8 glucose units per macrocycle, linked by a-(1,4)-glycosidic bonds and include
cyclomaltohexose (a-CD, 6 units), cyclomaltoheptaose (b-CD, 7 units), and cyclomaltooctose (g-CD,
8 units). The chemical structure and a diagrammatic representation of the functional structure of
beta-CD are depicted in Figure 22. Structurally, all of the C-6 (primary) hydroxyl groups project
from one side of the torus of the CD, while the C-2, 3 (secondary) hydroxyl groups project from the
other. The central cavity of CD is hydrophobic while the outer region of the oligosaccharide is
hydrophilic due to the presence of primary and secondary hydroxyls at both the narrow and wide
bases. CD's are highly insoluble. Their solubility can however be increased by the action of de-
branching enzymes such as isoamylase and pullulanase (Okada et al., 1988).
Figure 22.Schematic and chemical structure of a ß-cyclodextrin molecule.

The most important functional property of cyclodextrins is their ability to behave as clathrate-like
compounds in the formation of inclusion complexes with a range of guest molecules. If the guest
molecule is of suitable size and conformation that allow it to bind within the hydrophobic core,
complex formation takes place. Larger guest molecules form relatively weak complexes due to
partial binding. Greater inclusion activity of these larger guest molecules can however be obtained
by suitable chemical modification of the CD. This application is of particular interest to the food
industry for the molecular encapsulation of insoluble or volatile food ingredients (Pagington,

The use of CD has been proposed for the control of enzymatic browning in apple products
(Billaud et al. 1995; Sapers et al. 1989). CD inhibits juice browning through the binding of
polyphenol oxidase substrates. Polyphenols can be removed by b-CD and by insoluble polyvinyl
polypyrrolidone or polyethylene glycol (Osuga et al. 1994). The thermodynamics of inclusion
complexes of a-CD, b-CD, g-CD, and polymerized b-CD with chlorogenic acid as a substrate of apple
polyphenol oxidase were studied in order to elucidate a mechanism for the inhibition of juice
browning. Alpha -CD and g-CD were less effective than the b-CD in the inhibition of browning in
apple juice (Irwin et al. 1994). The internal cavity of b-CD is slightly apolar, thus allowing it to
induce inclusion complex formation with guest molecules such as phenolic substrates of
polyphenol oxidases, thereby preventing their oxidation to quinones and subsequent
polymerization to brown pigments. The adsorption of flavour or colour compounds by
cyclodextrins poses a major drawback to their use in food systems. Although the applicability of
cyclodextrins in fruit and vegetable juices has been patented (Hicks et al. 1990), cyclodextrins have
not yet been approved for food use by the United States FDA.

b. Chitosan

Chitosan, a naturally abundant polymer of b-(1®4)-N-acetyl-D-glucosamine, is derived from the

chitin of shellfish. Chitosan has antimicrobial properties, is soluble in dilute organic acids and is
capable of forming films or membranes. Chitosan is non-toxic, biodegradable, and a naturally
occurring product in our food supply. It has been shown to inhibit enzymatic browning in apple
and pear juices (Sapers, 1992). The addition of 200 ppm chitosan to McIntosh apple juice, resulted
in the inhibition of browning. Although the mechanisms by which chitosan inhibits browning are
not known, its inhibitory effect is probably a consequence of the ability of the positively charged
polymer to adsorb suspended polyphenol oxidase, its substrates, or products. Treatment of shrimp
with 2 percent chitosan resulted in a consistently reduced incidence of melanosis during storage
(Simpson et al. 1997). Chitosan also exhibited strong antimicrobial properties inhibiting several
microorganisms at concentrations ranging between 0.0075 - 0.01 percent (Simpson et al. 1997).

A research study conducted by Zhang and Quantick (1997) indicated that chitosan coating had
potential inhibitory activity on polyphenol oxidase and peroxidase activity in lychee (Litchi
chinensis Sonn.) fruit. Chitosan has been shown to improve the storability of fruits. Its
effectiveness in this respect is therefore thought to be due to the formation of a protective barrier
on the surface of fruit, which reduces the supply of oxygen for the enzymatic oxidation of
phenolics. Chitosan is non-toxic and is biologically safe (Hirano et al. 1990). Thus, the application
of a chitosan coating for the control of browning and quality improvement in fruits and vegetables
might be accomplished in combination with other methods such as low temperature and suitable

3.2.5 Enzyme inhibitors

a. 4-Hexylresorcinol

Substituted resorcinols, which are m-diphenolic compounds that are structurally related to
phenolic substrates, have a competitive inhibitory effect on polyphenol oxidase activity (McEvily et
al. 1991, 1992). Hydrophobic substitution with hexyl, dodecyl, and cyclohexyl groups at the 4-
position of the aromatic resorcinol ring increases the effectiveness of their competitive inhibitory
effect on polyphenol oxidase (McEvily et al. 1992). Studies conducted by McEvily et al. (1992)
revealed cyclohexyl-substituted resorcinols to have the lowest I50,i.e. the inhibitor concentration
that resulted in 50 percent inhibition of polyphenoloxidase activity, at a concentration of 0.2 mM
of the substituted resorcinol. Both the monophenolase and diphenolase activities of tyrosinase are
inhibited by 4-hexylresorcinol (4-HR). Four-hexylresorcinol has a long history of use in
pharmaceuticals and is considered to be safe and effective in use as an anti-browning agent
(Frankos et al. 1991).

Four-hexylresorcinol has several advantages over sulphites when applied in the control of
browning in foods. These include its specific mode of inhibitory action, effectiveness at low
concentrations, inability to bleach preformed pigments, and chemical stability. It has a synergistic
effect with ascorbic acid in the prevention of browning. Ascorbic acid reduces quinones generated
by polyphenoloxidase while 4-HR specifically interacts with polyphenol oxidase, and renders it
incapable of catalysing the enzymatic reaction (Kahn and Andrawis, 1985) (Figure 23).
Figure 23.The inhibitory effect of 4-hexylresorcinol on PPO. (Adapted from Kahn and Andrawis,

Four-hexylresorcinol is applicable in the control of browning in fresh and hot-air dried apple slices
as well as in apple juice (McEvily et al. 1992). Several studies have shown the effectiveness of 4-HR
in controlling enzymatic browning in shrimp (Otwell et al. 1992; McEvily et al. 1991), mushroom
(Osuga et al. 1994) and apple slices (Monsalve-Gonzalez et al. 1993). EverFreshTM a patented
product (United States patent # 5,049,438) which consists of 4-HR as the active ingredient and
sodium chloride as the carrier agent has been studied as an alternative to sulphites in the control
of enzymatic browning, or blackspot, in crustaceans (Lambrecht, 1995). Raw headless brown
shrimp dipped in 4-HR for 1 min, exhibited greater stability to blackspot formation for a longer
period of time than shrimp dipped in fresh water (controls) or 1.25 percent sodium
metabisulphite. After 7 days of storage at 2 oC, raw headless brown shrimp treated with water
showed 54 percent blackspot; sulphite-treated shrimp showed 11 percent blackspot, while 4-HR-
treated shrimp had only 3.6 percent blackspot (Figure 24). At day 14, blackspot on control and
sulphite-treated shrimp increased to 75 percent and 25 percent, respectively. The 4-HR treated
shrimp did not however show an increase in blackspotting. This compound has been proposed for
use on various fruits and vegetables by McEvily et al. (1991). Figure 25 shows inhibition of
enzymatic browning by 4-HR in star fruit.
Figure 24. Blackspot (%) in raw head-off brown shrimp treated with 4-HR and 1.25% sulfite, and
stored at 2 oC. (From Lambrecht, 1995).


Figure 25. 4-HR treatment (0.02% for 2 min dip) of star fruit. A. Treated B. Non-treated.

Four-hexylresorcinol is a chemically stable, water-soluble compound. Toxicological, mutagenic,

carcinogenic, and allergenic studies have shown that there are no risks associated with the levels
of 4-HR used in the treatment of shrimp (Frankos et al. 1991). Four-hexylresorcinol has obtained
GRAS status from the United States Food and Drug Administration, for use on shrimp (Federal
Register, 1992). Its use in the inhibition of shrimp melanosis has no effect on taste, texture, or
colour at residual levels of less than 1 ppm (Iyengar et al. 1991; King et al. 1991).

b. Halide salts

Inorganic halides are well-known inhibitors of polyphenol oxidases (Vámos-Vigyázó, 1981).

Janovitz-Klapp et al. (1990) determined that NaF was the most potent inhibitor of apple
polyphenol oxidase, followed by NaCl, NaBr, and NaI. The inhibition of enzymatic browning by
halides decreases with increasing pH. Sodium chloride and calcium chloride at concentrations of
ranging between 2 and 4 percent (w/v) are most commonly used in the food industry for the
inhibition of browning (Steiner and Rieth, 1989). Poplyphenol oxidase activity was observed to
decrease with increasing concentrations of NaCl for peach (Luh and Phithakpol, 1972), eggplant
and avocado (Knapp, 1965). Sodium zinc chloride was shown to be a highly effective browning
inhibitor when used in combination with calcium chloride, ascorbic acid, and citric acid (Bolin and
Huxsoll, 1989).

c. Honey
Honey has been shown to inhibit enzymatic browning. The use of honey as a natural browning
inhibitor is therefore of great consumer interest. Honey was shown to inhibit browning in apple
slices, grape juice and in model systems (Oszmianski and Lee, 1990). The browning of apple slices
was inhibited to a greater extent by 10 percent honey, than by a sucrose solution containing an
equivalent sugar concentration. Purification of honey by Sephadex G-15 column chromatography
revealed the compound in honey, responsible for the inhibition of polyphenol oxidase, to be a
small peptide of approximately 600 Da molecular weight. Proteins, peptides, and amino acids
exert an inhibitory effect on polyphenol oxidase activity by chelating the essential copper at the
active site of polyphenol oxidase, thus forming stable complexes with Cu2+ (Kahn, 1985). The honey
peptide is thought to exert its inhibitory effect through a similar mechanism.

Honey has been shown to contain antioxidants: tocopherols, alkaloids, ascorbic acid, flavonoids,
and phenolics. The antioxidant content and the efficacy of honeys in inhibiting polyphenol oxidase
activity vary in accordance with the type of honey (Chen et al, 1998). The effect of commercial
browning inhibitors (ascorbate and sodium metasulphite) and various honeys were compared in a
study on polyphenol oxidase activity and browning. Antioxidant content showed a positive
correlation to honey colour. The addition of various honeys to fresh potato homogenates, resulted
in a 0-50 percent reduction in polyphenol oxidase activity and a decrease of 0-7 units in the
browning index.

d. Amino acids, peptides and proteins

Amino acids, peptides or proteins can affect polyphenol oxidase-catalysed browning either
through direct inhibition of the enzyme or by reacting with the quinone products of polyphenol
oxidase catalysis (McEvily et al. 1992). Proteins, peptides and a-amino acids are capable of forming
stable complexes with Cu2+ (O. Sullivan, 1969). In addition, they are also capable of chelating
copper at the active site of polyphenol oxidase. Histidine and cysteine have particularly high
affinities for Cu2+ since, apart from having NH2 and COOH groups, histidine possesses an imidazole
ring and cysteine, a thiol group, both of which have metal binding capacity (Bell, 1977).

Kahn (1985) studied the effects of proteins, protein hydrolysates, and amino acids on o-
dihydroxyphenolase activity in mushroom, avocado and banana. Mushroom PPO was weakly
inhibited by mM concentrations of L-lysine, glycine, L-histidine and L-phenylalanine. L-cysteine was
the most effective amino acid in inhibiting o-dihydroxyphenolase activity. Amino acids inhibit
polyphenol oxidase activity through the formation of stable complexes with copper. In addition,
thiol containing inhibitors form sulfur adducts with the o-quinone, thus blocking polymer
formation and preventing browning (Figure 26). Mason and Peterson (1965) showed that N-
terminal primary amino groups, aliphatic amino groups (secondary amines in amino acids) and
thiol-containing amino acids react with o-benzoquinones and 4-methyl-o-benzoquinone, while
only thiol-containing compounds and aromatic amines react with oxidation products of DOPA
Figure 26. Postulated mechanism for the inhibition of PPO induced enzymatic browning by
thiols. (From Friedman and Molnar-Perl, 1990.

e. Aromatic carboxylic acids

Aromatic carboxylic acids of the benzoic acid and cinnamic acid series are polyphenol oxidase
inhibitors, owing to their structural similarity to phenolic substrates (Krueger, 1955). Undissociated
forms of these acids are capable of inhibiting polyphenoloxidase, through complexation with
copper at the active site of the enzyme. The degree of polyphenol oxidase inhibition by carboxylic
acids is pH dependent, and increases with a decrease in pH.

Cinnamic acid and its analogues, p-coumaric, ferulic, and sinapic acids were found to be potent
inhibitors of potato (Macrae and Duggleby, 1968) and apple polyphenol oxidases (Pifferiet al.
1974; Walker and Wilson, 1975). Cinnamic acid at levels of 0.01 percent was observed to be
effective in providing long-term inhibition of polyphenol oxidase in apple juice (Walker, 1976).
Benzoic acid and its derivatives had an inhibitory effect on polyphenol oxidase activity in
mushrooms (Kermasha et al. 1993) and grapes (Gunata et al. 1987).

f. Aliphatic alcohols

Although the inhibition of polyphenol oxidase by ethanol has been reported (Kidron et al. 1978),
there are no extensive studies which describe the effect of aliphatic alcohols on polyphenol
oxidase. Valero et al. (1990) studied the effects of natural aliphatic alcohols on grape
polyphenoloxidase. Inhibition was observed to increase with increasing chain length of the
aliphatic alcohol.

3.3 Other inhibitors

3.3.1 Killer enzymes

As paradoxical as it may seem, enzyme action can be exploited for the control of undesirable
enzyme activities. This is achievable in three ways: (1) substrate and/or product modification by
enzymes other than the target enzymes; (2) direct inactivation of the target enzyme by other
enzymes and (3) inactivation by secondary reactions of highly reactive products. The activities of
"killer enzymes" or "anti-enzyme enzymes" which inactivate other enzymes via direct proteolytic
activity have been demonstrated. The use of enzymes to control other enzyme-related processes
has also has been reported in the control of enzymatic/nonenzymatic browning.

a. Ring-cleaving oxygenases

Kelly and Finkle (1969) proposed irreversible modification of phenolic substrates by enzymes as
one mechanism for the control of browning. Apple juice treated with the bacterial enzyme
protocatechuate-3, 4-dioxygenase in combination with ascorbic acid prevented browning due to
the oxidative ring-opening reaction and ortho-fission of catechols by the enzyme. The enzyme
deprived polyphenol oxidase enzymes of required substrates (Kelly and Frinkle, 1969).

b. Catechol transferase

Finkle and Nelson (1963) proposed the use of catechol transferase (EC for the prevention
of browning in apple juice. O-methyl transferase, an enzyme capable of methylating the 3-position
of 3,4-dihydroxy aromatic compounds was observed to cause irreversible modification of phenolic
substrates, thus preventing them from serving as substrates of the browning reaction. Treatment
of apple juice with o-methyl transferase and s-adenosyl methionine resulted in conversion of
chlorogenic and caffeic acids, to feruloylquinic and ferulic acids respectively, both of which are
polyphenol oxidase inhibitors (Finkle and Nelson, 1963).

c. Proteases

The plant proteases ficin, papain and bromelain are sulphydryl enzymes of broad specificity
(Labuza et al. 1992; Taoukis et al. 1990) which are very effective browning inhibitors. Ficin was
observed to be effective in preventing black spot formation in shrimp under refrigerated
conditions (Taoukis et al. 1990). This inhibitory effect is thought to be due to either binding or
hydrolysis at specific sites necessary for polyphenol oxidase activity.

Pineapple juice was found to be effective in inhibiting browning in apple rings (Lozano-De-
Gonzalez et al. 1993). Bromelain, organic acids, sulphydryl compounds and certain metallic
constituents of pineapple juice are thought to be responsible for this inhibitory effect. Polyphenol
oxidase activity in plum juice was significantly reduced when the juice was passed through a
column containing immobilized proteases (Arnold et al. 1992).

Commercial application of enzyme treatments in the control of enzymatic browning is precluded

by their high cost. Combinations of anti-browning agents of a chemical nature are however more
affordable and effective in commercial use.

3.3.2 Edible Coatings

The use of edible coatings to minimize undesirable changes due to minimal processing has been
reported for several commodities (Baldwin et al., 1995). The coating of fruits and vegetables with
semi-permeable films has been shown to retard ripening through modification of endogenous CO2,
O2 and ethylene levels. Coatings are also useful as carriers of antioxidants and preservatives
(Cuppett, 1994). Edible coatings have the potential to retard water loss, to form a barrier to
oxygen, and to retain antioxidants, as well as preservatives, on the surface of cut tissue in order to
control discolouration. A polysaccharide/lipid bilayer formulation was observed to reduce
respiration in cut apples (Wong et al. 1994) through modification of the gas exchange between the
processed tissue and the external environment. Sucrose fatty acid esters reduced browning of
shredded cabbage (Sakane et al. 1990) which was attributed to reduction of oxygen at the cut
surface. Zhang and Quantick (1997) determined that an edible coating based on sucrose esters of
fatty acids significantly delayed pericarp browning of lychee fruit. Carboxymethyl cellulose/soy
protein coating formulations containing 0.5 percent ascorbic acid applied to freshly cut apples
were more effective in antibrowning activity than aqueous solutions of 0.5 percent ascorbic acid
alone (Baldwin et al. 1996). The cellulose matrix may well have a protective effect in preventing
the degradation of ascorbic acid by oxygen.


Polyphenol oxidases occur in the chloroplasts of almost all higher plants. Cloning of the genes
which code for polyphenol oxidase offers the potentials for determining the physiological role of
polyphenol oxidase within the chloroplast and for manipulating polyphenol oxidase levels within
specific organs. Polyphenol oxidase genes are encoded within the nucleus and undergo translation
within the cytoplasm. Once formed, propolyphenol oxidase is transported to the chloroplast
where it undergoes proteolytic cleavage, to produce the active polyphenol oxidase form
(Vaughn et al., 1988). Predicted molecular weights for polyphenol oxidase in plants, range
between 57 and 62 kDa (Hunt et al. 1993; Newman et al. 1993).

Polyphenol oxidase is generally present in low concentrations in all organisms. The enzyme is
difficult to obtain in a pure form due to pigment contamination and the occurrence of multiple
forms. With the advent of recombinant DNA technology, numerous amino acid sequences of
polyphenol oxidases have become available. Primary structures of polyphenol oxidases
from Streptomyces glaucescens (Huber et al. 1985), Streptomyces antibioticus (Bernan et al. 1985)
and Neurospora crassa (Lerch, 1982), tomato (Shahar et al. 1992; Newman et al. 1993), broad
bean (Cary et al. 1992) potato (Hunt et al. 1993), mice (Shibahara et al. 1986) and humans
(Kwon et al. 1987; Giebel et al. 1991) have been determined using cDNA sequencing techniques.
Polyphenol oxidases of closely related plants, such as tomato and potato, show approximately 91
percent exact homology, while those of tomato and fava bean show only 40 percent exact
homology (Wong, 1995).

Polyphenol oxidases from different sources exhibit molecular weight differences. Molecular
weights predicted for mature polyphenol oxidases on the basis of cDNA sequences were 58 kDa
for mouse, ~63 kDa for human and 128 kDa for mushroom. Mushroom polyphenol oxidase is
thought to contain four subunits having a total molecular weight of 128 kDa. Monomeric through
octameric forms of mushroom polyphenol oxidases are known to exist (Whitaker and Lee, 1995).
Plant polyphenol oxidases are nuclear-encoded copper metalloproteins having a molecular mass
of approximately 59 kDa and are localized in the membranes of plastids. Plant genes encoding
polyphenol oxidase have recently been cloned and characterised. Although the sequences of plant
polyphenol oxidase genes are very similar, only the putative copper binding sites are conserved
when plant genes are compared to mammalian, bacterial, or fungal tyrosinases. One possible
approach to lowering polyphenol oxidase activity and resultant enzymatic browning reactions is to
characterize and inactivate the genes which code for polyphenol oxidase. Inactivation can be
accomplished by generating antisense RNAs specific for polyphenol oxidase.

4.1 New approaches for the control of PPO

The involvement of polyphenol oxidase in browning has been studied for a long time. Many
questions still remain about the enzyme itself, as well as the mechanism of browning. Chemical
and physical methods for controlling enzymatic browning have been reviewed in Chapter 3.
Current approaches to understanding and controlling enzymatic browning are however specifically
focused on the use of antisense RNA. Antisense RNA techniques have several applications in plant
research. They are applicable in studying the in vivo function of particular genes and their
biochemical modes of action. Antisense genes have been successfully used for the alteration of
plant processes, such as flower pigmentation, fruit ripening and photosynthesis, and to determine
the function of cryptic genes. They may also be put to practical use in crop improvement.
Antisense RNAs were recently observed to selectively block the gene expression of other plant
enzymes such as polygalacturonase and peroxidase in tomatoes.

4.2 Antisense RNA approach

One of the most successful methods developed in recent years for the inhibition of gene
expression in plants has been the expression of introduced antisense genes. Antisense technology
is based on blocking information flow from DNA via RNA to protein by the introduction of an RNA
strand complementary to the sequence of the target mRNA. It is generally assumed that the
antisense RNA basepairs to its target mRNA thereby forming double-stranded RNA. Duplex
formation may impair mRNA maturation and/or translation or alternatively may lead to rapid
mRNA degradation.

Technically, it involves the insertion of a gene or a significant part of it, into the cell in a reverse
orientation. Messenger RNA encoded by this antisense gene undergoes hybridization with that
encoded by the endogenous gene, precluding production of the protein product (Figure 27). Gene
silencing or the elimination of expected phenotypic characteristics, through antisense techniques
has received much attention in recent years. The expression of a transgene (i.e. a gene that has
been introduced into plant cells through molecular biology techniques) or an endogenous gene
appears to be affected by the presence of a homologous transgene. Antisense-mediated control
has been observed in bacteria, fungi, plants and mammalian systems. The biological function of
naturally occurring antisense RNAs, if any, remains to be determined. Complementary mRNA
levels can be reduced in the presence of antisense genes.

Figure 27. Simplified schematic showing how antisense RNA can be used to control gene
expression at the translational level (P represents the promoter). (From Martinez and Whitaker,

Polyphenol oxidase genes have been characterized in broad bean, tomato, potato, and grape.
Seven genomic polyphenol oxidase genes were identified in tomato (Newman et al. 1993).
Characterization of polyphenol oxidase genes in various plant species has shown that these genes
are present in the plant genome as gene families. Such transcriptional regulation offers potential
for the design of antisense constructs. Antisense constructs would be required for maintenance of
the enzyme if polyphenol oxidase is required for chloroplast metabolism or for resistance to
pathogens or predation.

In addition to improving a fundamental knowledge of biological processes, the antisense approach

has been applied, to increasing the shelf life of fruit (Fray and Grierson, 1993). Commercial
applications of antisense technology now include alterations of flower colour, virus resistance and
fruit ripening. The application of antisense technology also extends to improving food quality.

Bachem et al. (1994) determined that the expression of polyphenol oxidase in potatoes was
decreased through the use of vectors carrying antisense polyphenol oxidase cDNAs. Approximately
70 percent of the transformed plants had lower polyphenol oxidase activity than controls, and on
visual scoring, a significantly low level of discolouration. Insertion of polyphenol oxidase in the
sense orientation resulted in very high polyphenol oxidase activity in the lines expressing the

Breeders have been working to decrease polyphenol oxidase levels in apples, bananas,
mushrooms, peaches and other plants for many years. Lack of bruising sensitivity in transgenic
potatoes, and the absence of any apparent detrimental side effects, opens up the possibility for
preventing enzymatic browning in a wide variety of food crops, without resorting to chemical and
physical treatments. Quite possibly, browning-resistant varieties may be developed in the future
through the insertion of antisense genes that prevent the production of polyphenol oxidase.


New approaches to control enzymatic browning are under study at universities, government
research laboratories, and industry. These alternatives must be evaluated on the basis of
effectiveness, cost, and regulatory status. Inhibitors of enzymatic browning must not affect
product flavour, texture, and colour. The choice of a particular anti-browning agent will depend on
these factors, as well as the method of treatment.

A variety of methodologies can be applied in the control of enzymatic browning. In addition to

physical treatment, a wide range of chemicals inhibit polyphenol oxidase activity. Only a limited
number of browning inhibitors are considered acceptable with respect to consumer safety and/or
cost and act as potential alternatives to sulphites. Whether chemical treatments of food products
are achievable or not will depend on their effectiveness and cost relative to that of alternative
approaches and on the regulatory status of their use as food additives.