Caballero B. (ed.) Encyclopaedia of Food Science, Food Technology and Nutrition. Ten-Volume Set

Подождите немного. Документ загружается.

borohydride. Reactive lysine can also be determined

after conventional acid hydrolysis, by the furosine

method. This method is based on the property of

fructosyl-lysine to produce upon acid hydrolysis a

new amino acid, i.e., furosine. The measurement of

the furosine level allows quantification of the

Amadori compound of lysine and calculation of

the amount of reactive lysine.

The Advanced Maillard Reaction

0011 This stage of the reaction starts with the degradation of

the sugar moiety of the Amadori compound involving

dehydration, scission, oxidation, and Strecker degrad-

ation, leading to the generation of new molecules (pre-

melanoidins) contributing to aromas and flavors that

vary in their characteristics according to the conditions

employed. Some of these molecules are chemically

more reactive than the initial sugar, e.g., a-dicarbonyls,

reductones, and aldehydes. They react not only with

free amino groups still present in the food, but also

with a larger number of radical groups such as hy-

droxyl, amines, and other nitrogen-containing mol-

ecules, vitamins, and the side chains of amino acids.

0012 Polymerization of premelanoidins leads to the for-

mation of the high-molecular-weight melanoidins,

the reactions involved being responsible for the devel-

opments of pigments.

0013 During the advanced stage of the Maillard reac-

tion, loss of lysine and of B-vitamins in foods is accel-

erated. Other amino acids become less bioavailable

because they are either chemically modified or a de-

crease in the total food nitrogen digestibility occurs.

Lysine lost as new unavailable addition product(s)

can be calculated by difference as ‘lysine in advanced

Maillard compound(s)’ (Table 2). These addition

products do not regenerate lysine on acid hydrolysis

and their chemical structures are still unknown.

Indirect Effects

0014 Associated with the presence of Maillard reaction

products, a good example of an indirect negative

nutritional effect of nonenzymatic browning is the

reduction in protein digestibility. This is due to the

inability of proteases and peptidases to hydrolyze

the peptide bonds now containing modified amino

acids. Feeding such a protein results in an increase

in the amount of fecal nitrogen and a decrease in the

bioavailability of the chemically unmodified amino

acids. Another indirect effect is a modification of the

metabolism (intestinal absorption, utilization, and

urinary excretion) of some minerals and trace elem-

ents (calcium, zinc, iron, and copper). The increase

in the urinary excretion of zinc, iron, and copper

in patients fed intravenously a sterilized aqueous

solution of free amino acids and glucose, and the

hyperzincuria observed in rats fed heated casein–

glucose and casein–lactose mixtures, would be due

to the chelating effect of Maillard reaction products.

However, this increase in urinary zinc being a rela-

tively minor route of excretion compared to fecal

excretion, zinc retention, and zinc status is not

reduced in rats.

Physiological Effects

0015Many physiological changes in tissues of animals

have been attributed to feeding Maillard reaction

products. It has not always been easy, however, to

distinguish between the changes that are directly due

to the reduced nutritive value of the reacted protein

and those that are probably induced by the Maillard

components of the test protein in the diet. Further-

more, in many of these studies which corresponded to

model systems, the reaction products tested were in-

corporated in the diets at such high levels that inter-

pretation of observation has remained difficult.

0016Reduced growth rate and increased relative weight

of liver, kidney, and cecum have been noted in rats fed

a severely reacted (brown) compared to an unreacted

mixture of egg protein and glucose. Parallel increases

in values for blood glucose and urea nitrogen, and

serum transaminases and alkaline phosphatase were

also observed with the reacted mixture. The lower

growth rate, attributable to the reduced nutritive

value of the reacted egg protein, may partly explain

the above differences. Long-term rat feeding trials,

designed to eliminate the influence of the nutritional

factors through increased dietary protein level, im-

proved protein quality, and a lower, more realistic

level of incorporation of the reacted egg protein

under test showed less dramatic effects. Nevertheless,

an apparent effect of the reacted brown Maillard

compound was evident in the depressed growth rate,

enlarged liver, spleen, and cecum, lowered serum tri-

glycerides, increased total iron, and histopathologi-

cally observed fatty changes in the liver.

tbl0002 Table 2 Lysine blockage in processed milk

Heat process Percentage lysine

as Amadori compound

Freeze-drying 0

Pasteurization 0

UHT sterilization 0–2

Spray-drying 0–2

Spray-drying infant formula 5–10

HTST sterilization 5–10

Conventional sterilization 10–15

Roller drying 20–50

According to the furosine method.

UHT, ultra heat-treated; HTST, high-temperature, short-time.

BROWNING/Toxicology of Nonenzymatic Browning 675

0017 Activities of intestinal mucosal dissacharidases

(lactase, sucrase, and maltase) were also reduced in

rats fed the reacted, brown egg protein, compared to

those fed the unreacted material. Some improvement

occurred on fortification of the diet with amino acids

(apparently) destroyed during reaction, although a

persistent decrease in the activity of these enzymes

demonstrated a specific inhibitory effect of the Mail-

lard compounds.

0018 Diarrhea is often observed in animals fed high

levels of severely browned Maillard products. This

is likely to be a direct consequence of the increased

amount of indigestible protein available for fermen-

tation in the cecum, which is often found to be

enlarged.

0019 During the progress of the nonenzymatic browning

reaction, additional glycosyl residues become fixed to

specific milk proteins leading to an increase in their

allergenicity. Using a skin reactivity test, a good

correlation has been shown between the degree of

browning and allergenic response of a modified

bovine b-lactoglobulin. Whilst this may explain one

of the mechanisms responsible for the enhancement of

the allergenicity of a protein, it does not explain the

‘how and why’ of allergy to cows’ milk protein.

0020 Maillard reaction products of low molecular

weight which are partially absorbed can affect the

activity of detoxifying enzymes. Rats fed browned

egg albumin exhibited increased hepatic benzo[a]-

pyrene hydroxylase activity and decreased colonic

aminopyrine N-demethylase activity compared to

animals fed nonbrowned egg albumin. This suggests

that Maillard reaction products may modify the

metabolism of endogenous substrates, exogenous

drugs, and other xenobiotics.

0021 Dietary melanoidins appear to decrease plasma

cholesterol in rats and to modify the composition of

fecal, neutral steroids, suggesting that melanoidins

influence the intestinal metabolism of cholesterol.

0022 Maillard reaction products also interact with

the microorganisms of the digestive tract. Thus they

strongly inhibit the activity of glycosyl transferase of

Streptococcus mutans which has an active role in the

development of dental caries. This inhibition would

hence reduce the adherence of this microorganism to

the tooth surface. Also, in the gut, the microflora is

able to regenerate the amino acids from their Ama-

dori compound and oxidize the advanced Maillard

reaction products.

Toxicity

0023 Studies of the toxicity of Maillard reaction products,

both in vitro and in vivo, are a recent develop-

ment. This interest comes from the observation that

Maillard reactions involving creatinine (a typical

molecule of animal tissues) or protein pyrolysis rep-

resent a carcinogenic potential due to the formation

of heterocyclic amines. In addition to these amines,

there exist mutagenic products of the premelanoidin

and melanoidin types resulting from heat-processed

carbohydrate-rich foods such as bakery products,

caramel, coffee, and other milk and meat products.

Mutagenic/Carcinogenic Heterocyclic Amines

0024Using the short-term Ames microbiological test, it

has been possible to detect mutagenic activity in

broiled fish and meat. A series of heterocyclic

amines has been isolated from cooked foods and

from protein and amino acid pyrolysates. Some of

them, 2-amino-3-methylimidazo [4,5-f ]quinoline

(IQ), 2-amino-3,4-dimethylimidazo [4,5-f]quinoline

(MeIQ), 2-amino-3-methylimidazo [4,5-f ]quinoxa-

line (IQx), 2-amino-3,8-dimethyl-imidazo [4,5-f ]qui-

noxaline (MeIQx), 2-amino-3,4,8-

trimethylimidazo[4,5-f ]quinoxaline (4,8-DiMeIQx),

and 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyri-

dine (PhIP), possess an imidazole ring which is

formed from creatinine. The remaining parts of the

molecules (pyridines and pyrazines) are derived from

Maillard reaction products formed by amino acids

and sugars. Since creatinine is present only in animal

protein sources, this class of substances is found only

in these foods, albeit at low levels in the charred

portions of broiled fish and the surface of grilled

meat (PhIP 1 p.p.b; MeIQx 1 p.p.b.) and in beef ex-

tracts (IQ 70 p.p.b; MeIQx 8–90 p.p.b.).

0025The other heterocyclic amines detected in foods

are pyrolysis products formed from trypto-

phan: 3-amino-1,4-dimethyl-5H-pyrido[4,5-b]indole

(Trp-P-1), 3-amino-1,4-dimethyl-5H-pyrido[4,5-

b]indole (Trp-P-2), from glutamic acid: 2-amino-6-

methyldipyrido-[1,2-a:3

-d]imidazole (Glu-P-1),

2-aminodipyrido-[1,2-a:3

-d]imidazole (Glu-P-2),

from phenylalanine: 2-amino-5-phenylpyridine

(Phe-P-1), and from soya bean globulin: 2-amino-

9H-pyrido[2,3-b]indole (AaC), 2-amino-3-methyl-

9H-pyrido[2,3-b]indole (MeAaC). The heterocyclic

amines which are produced in very small amounts in

foods have been found to be carcinogenic in rats and

mice (Table 3). The major target organs are the small

and large intestines, liver, lung, and blood vessels.

Mutagenicity of Melanoidin-Type Products

0026Heated mixtures of free amino acid or food proteins

and sugars possess a weak mutagenic activity in the

Ames test. This activity varies with the composition

of the mixture and the conditions of the reaction.

Generally, the mutagenic activity observed in vitro

correlates quite well with the formation of brown

676 BROWNING/Toxicology of Nonenzymatic Browning

pigments and with the presence of molecules with

high antioxidant capacity.

0027 The demonstration that carbonyl compounds can

produce free radicals lends confirmation to the ob-

servation that the mutagenicity of the antioxidant

fraction of the Maillard products is deactivated by

catalases. This apparent contradiction suggests that,

when applied at high concentration to in vitro tests,

Maillard products can induce mutations due to an

‘overpowering’ of their prooxidant activity, a phe-

nomenon already observed with other antioxidants,

such as the antioxidant vitamins and phenolic

compounds.

0028 When considering model systems reflecting normal

cooking conditions a relatively weak mutagenic ac-

tivity was observed compared to that of heterocyclic

amines. The typical mutagenic effects of this class of

compounds reflect base pair, in contrast to frameshift

mutations, as caused by heterocyclic amines. Further-

more, their mutagenic effect has been found to be

effectively inactivated by the liver microsomal

enzymes (S9 liver fraction), in contrast to heterocyc-

lic amines which require S9 activation to mutagenic/

carcinogenic species. (See Mutagens.)

0029Examples of heated foods with such a weak

mutagenic activity that is metabolically deactivated

in vitro are some commercial caramel preparations

(ammonia and sulfite/ammonia processes), hydro-

lyzed plant proteins and roasted coffee. However, it

has been confirmed in animal feeding studies that

melanoidin-containing foods such as roasted coffee

and roasted cocoa are neither mutagenic nor carcino-

genic. Moreover, tested in vivo under well-controlled

physiological conditions, Maillard reaction products

with antioxidant activity are found to act as anti-

mutagens and/or anticarcinogens.

Antioxidant and Antimutagenic Activity of

Melanoidin-Type Products

0030Many Maillard reaction products exhibit antioxidant

properties which are utilized with success in different

types of food. Such reaction products include preme-

lanoidins which are rich in carbonyl compounds as

well as melanoidins. The observed antioxidant prop-

erty depends on the conditions of the reaction and on

the amino acids. Thus, reaction mixtures containing

histidine have been found to have the highest antioxi-

dant activity. The mechanism itself of the antioxidant

tbl0003 Table 3 Mutagenic and carcinogenic effects of heterocyclic amines

Compound Source Mutagenicity Carcinogenicity

Revertants

(mg) TA 98

Species Concentration (%) Experimental

period(weeks)

Tar g e t o r ga n s

Products formed

from creatinine

IQ Broiled sardines 433 000 Rats 0.03 72 Liver, intestine, skin

Mice 0.03 96 Liver, lung

MeIQ Broiled sardines 661 000 Rats 0.03 40 Large intestine,

mammary gland

Mice 0.04 91 Liver, forestomach

MeIQx Fried beef 145 000 Rats 0.04 61 Liver, skin

Mice 0.06 84 Liver, lung

Pyrolysis products

formed from

tryptophan

Trp-P-1 Tryptophan pyrolysate 39 000 Rats 0.02, 0.015 52 Liver

Mice 0.02 89 Liver

Trp-P-2 Tryptophan pyrolysate 104 200 Mice 0.02 89 Liver

Pyrolysis products

formed from

glutamic acid

Glu-P-1 Glutamic acid pyrolysate 49 000 Rats 0.05 67 Liver, small

and large intestines

Mice 0.05 68 Liver, blood

vessels

Glu-P-2 Glutamic acid pyrolysate 1 900 Rats 0.05 104 Liver, small

and large,

intestines

Mice 0.05 84 Liver, blood vessels

IQ, 2-amino-3-methylimidazo [4,5-f ]quinoline; MeIQ, 2-amino-3,4-dimethylimidazo-[4,5-f ]quindine; MeIQx, 2-amino-3,8-dimethyl-imidazo [4,5-

f ]quinoxaline; Trp-p-1, 3-amino-1,4, dimethyl-5H-pyrido [4,5-b] indole; Trp-P-2, 3-amino-1-methyl-5H-pyrido[4,5-b]indole; Glu-P-1, 2-amino-6-

methyldipyrido-[1,2-a:3

-d]imidazole; Glu-P-2, 2-aminodipyrido-[1,2-a:3

-d]imidazole.

BROWNING/Toxicology of Nonenzymatic Browning 677

activity in food systems involves both the reducing

capacity of the carbonyl compounds in the premela-

noidins and melanoidins, and the property of the

melanoidin brown pigments to chelate prooxidant

metals, copper, and iron.

0031 That Maillard reaction products possess antimuta-

genic and/or anticarcinogenic properties has been

evidenced. Thus, such reaction products inhibit

mutagenic activity of several foodborne heterocyclic

amines and of aflatoxin B

. Antimutagenic and pos-

sible anticarcinogenic activities have been attributed

to melanoidins and a-dicarbonyl compounds gener-

ated either during the aminocarbonyl or carameliza-

tion reactions. The scavenging of active oxygen by

melanoidins, demonstrated by means of electron spin

resonance, can explain their antimutagenic effects.

The involvement of antioxidant reactions in the anti-

mutagenicity of heat-reaction products suggests

that these compounds might have anticarcinogenic

properties which include inhibition of nitrosamine

formation, scavenging of active oxygen involved in

cancer initiation and promotion, and also change in

the chemical structure of carcinogens during the

browning process.

Nitrosamines of Amadori Compounds

0032 Amadori compounds have been shown to be nitro-

sated to mutagenic N-nitroso derivatives. This has led

to the postulation that such compounds, if present in

human food or if formed in the stomach, will consti-

tute a health hazard. However, heated foods rich in

Maillard reaction products with antioxidant proper-

ties can both inhibit nitrosamine formation and

reduce nitrosamine-induced carcinogenicity. (See

Nitrosamines.)

See also: Amines; Browning: Nonenzymatic;

Nitrosamines

Further Reading

Eriksson C (ed.) (1981) The Maillard Reaction in Food.

Progress in Food and Nutritional Science 5. Oxford:

Pergamon Press.

Finot PA, Aeschbacher HU, Hurrell RF and Liardon R (eds)

(1990) The Maillard Reaction in Food Processing,

Human Nutrition and Physiology. Advances in Life

Sciences. Basel: Birkhauser.

Friedman M (ed.) (1991) Nutritional and Toxicological

Consequences of Food Processing. Advances in Experi-

mental Medicine and Biology 289. New York: Plenum

Press.

Lee TC, Pintauro SJ and Chichester CO (1982) Nutritional

and toxicological effects of non-enzymic Maillard

browning. Diabetes 31 (suppl. 3): 37–46.

Enzymatic – Biochemical

Aspects

J Nicolas and C Billaud, Conservatoire National des

Arts et Me

tiers, Paris, France

J Philippon (Retired), Centre National du Machinisme

Agricole du Ge

nie Rural des Eaux et des Fore

ts,

Antony, France

M A Rouet-Mayer, Centre National de la Recherche

Scientifique, Gif sur Yvette, France

Introduction

0001The discoloration which occurs in plant material after

cell disruption and results in the formation of brown

or sometimes yellow, black, or pink pigments is

mainly due to the process of enzymatic browning.

Besides plant materials which are primarily con-

cerned, enzymatic browning also affects some animal

materials, e.g., melanosis (black spot) of shrimp.

0002Cell disruption can arise either from mechanical

injury or temperature changes which lead to physio-

logical disorders or even cell death (e.g., for deep-

frozen products). This loss of cell integrity results

in the decompartmentation of phenolic substrates

and enzymes and then, in the presence of molecular

oxygen, the oxidative production of colored pig-

ments.

0003Brown pigmentation following this enzymatic

reaction, and subsequent nonenzymatic reactions, is

generally considered to be detrimental to food quality

from both the organoleptic and nutritional points of

view. The prevention of enzymatic browning has

always been a challenge to food scientists owing to

the loss of quality that it causes in many food prod-

ucts, e.g., fruits and vegetables during either storage

or processing. It is only in a few exceptional cases that

the enzymatic browning is desirable (prunes, dates,

tea, tobacco, etc.).

0004With a better knowledge of the mechanism of

browning reactions, it will be possible to propose

processes which avoid or at least minimize this

discoloration and which can be adapted to each par-

ticular product. (See Colorants (Colourants): Proper-

ties and Determination of Natural Pigments).

Nomenclature

0005Two kinds of enzymes are able to act upon diphenols

in the presence of molecular oxygen according to

the reaction scheme shown in Figure 1. Both have

the trivial name polyphenol oxidases, but they are

somewhat different in nature. The first class of

enzymes, catechol oxidases (1,2-benzenediol: oxygen

678 BROWNING/Enzymatic – Biochemical Aspects

oxidoreductase, EC1.10.3.1), catalyze two distinct

reactions (reactions (1) and (2) of Figure 1), namely

hydroxylation of monophenols into o-diphenols (cre-

solase activity) and oxidation of o-diphenols into o-

quinones (catecholase activity). Both reactions con-

sume oxygen and the overall stoichiometry is 1 mol

of oxygen for 1 mol of monophenol, giving 1 mol of

o-quinone and 1 mol of water. The second class, lac-

cases (benzenediol: oxygen oxidoreductase,

EC1.10.3.2), oxidize o-diphenols as well as p-diphe-

nols, forming their corresponding quinones (reaction

(3) of Figure 1). The stoichiometry is one atom of

oxygen for 1 mol of diphenol giving 1 mol of quinone

and 1 mol of water. The unique ability to oxidize p-

diphenols can be used to distinguish the laccase activ-

ity from that of the first class of polyphenol oxidases.

In all cases, the quinones formed are very reactive

and, depending on the nature and the concentration

of the reactive species in the medium, they can enter

in numerous secondary enzymatic and nonenzymatic

reactions which will be described later.

0006 The nomenclature of these enzymes is some-

what confusing since, besides the two designated

as EC1.10.3.1 and EC1.10.3.2, a third one,

EC1.14.18.1 (monophenol, l-DOPA: oxygen oxido-

reductase), exists. It is referred to as monophenol

monooxygenase (tyrosinase) and corresponds to the

same enzymes as EC1.10.3.1 but which always cata-

lyze the hydroxylation of monophenols. For the sake

of simplicity, we will use the general terms of poly-

phenol oxidase (PPO) for the first class (EC1.10.3.1)

and laccase for the second class (EC1.10.3.2). (See

Enzymes: Functions and Characteristics.)

Enzymatic Browning Factors

0007The different factors of enzymatic browning are

examined, including some minor ones which may

occasionally play an important role, e.g., peroxidases.

Enzymes

0008Polyphenol oxidases (EC1.10.3.1) PPOs are copper

oxidoreductases exhibiting both cresolase and cate-

cholase activities. However, many enzymatic prepar-

ations have a very low cresolase activity or none at

all. It is difficult to insure that the latter activity was

originally absent in the plant or has not either been

extracted or has been destroyed during extraction

owing to its lability. Cresolase activity is often lost

during purification. Thus, in a preparation, the ratio

of catecholase to cresolase activity (when the latter is

present) can vary from 1 to 40.

0009PPO is present in a wide variety of plants. In a

particular plant, PPO activity varies from one organ

to another and varies inside an organ, depending on

the tissue considered. PPOs have been found in differ-

ent cell fractions, in organelles (chloroplasts and,

more precisely, in thylakoids, mitochondria, peroxi-

somes) where the enzymes are tightly bound to mem-

branes and in the soluble fraction of the cell. The

degree of binding to membranes varies with the tissue

and its ontogenic state. Thus, the overall PPO activity

is higher and mostly present in bound forms in

young green fruits, whereas it generally decreases

and the proportion of soluble forms increases in ripe

fruits.

0010Extraction of the PPO activity from plant sources is

complicated by the presence of endogenous phenolic

substrates which are oxidized and then interact with

proteins. Besides destroying activity, this may induce

‘new’ enzymatic forms corresponding to artifacts.

As a consequence, this oxidation has to be prevented

by adding a reducing agent (e.g., ascorbic acid or

a thiol) and/or a phenol-complexing compound

(e.g., polyamide, polyvinylpolypyrrolidone (PVPP),

or polyethylene glycol) to the extracting solution.

Solubilization after preparation of an acetone powder

is a frequently used procedure. Detergents (Triton

PPO

(cresolase)

PPO

(catecholase)

Laccase

PPO

(catecholase)

(1)

(2)

(3)

R' R'

fig0001 Figure 1 Reactions catalyzed by polyphenol oxidases. Repro-

duced from Browning: Enzymatic-Biochemical Aspects. Encyclo-

paedia of Food Science, Food Technology and Nutrition, Macrae R,

Robinson RK and Sadler MJ (eds), 1993, Academic Press.

BROWNING/Enzymatic – Biochemical Aspects 679

X100 or X114) are also used, but probably induce

modifications of the enzyme structure and properties.

Most of the purification procedures are based on

fractional precipitation by ammonium sulfate

followed by one or several chromatographic steps.

Until the mid-1980s, the molecular mass of PPOs

was believed to be around 40–45 kDa. However,

PPOs from a number of plant species have now been

characterized with molecular masses ranging from 20

to 180 kDa. This disparate estimation of molecular

mass, traducing the heterogeneity and multiplicity

of forms of the enzyme, can partly be attributed to

artifacts of protein isolation and electrophoresis, as

exposed above. Moreover, they can correspond to

small multigene families for PPO. In fact, PPO genes

characterized from different leaf and fruit plant

species (including faba bean, apple, tomato, potato,

spinach, Virginian pokeweed, and grape berry) are

present in the plant genome as gene families. To

date, cDNAs for PPOs cloned from these various

plants have been found to encode mature peptides

of 56–67 kDa with 8–12 kDa putative transit pep-

tides. The nuclear encoded-plastid protein is thus

translocated as a precursor with this transit peptide

that can direct its transport into the thylakoid mem-

brane or lumen. The copper content of various plant

PPOs has not yet been well established, but their

deduced amino acid sequences from the cloned

PPOs genes have revealed two highly conserved

copper-binding domains, showing significant hom-

ology with that of closely related bacterial, fungal,

or mammalian tyrosinases.

0011 Most of the PPOs studied show an optimum activ-

ity between pH 4 and 7. However, there are often

discrepancies in the published values for PPOs from

the same source. In addition, several reports indicate

differences in pH optimum depending on cultivars

and maturity. These differences have been attributed

to variations in the proportions of isoenzymes which

have distinct pH optima. Moreover, pH optima were

found to vary with the phenolic substrate used for

several enzymatic preparations. The effect of tem-

perature on PPO activity has been less investigated

than that of pH. The optimum temperature ranges

from 15 to 40



C, depending on the same factors as

pH.

0012 Laccases (EC1.10.3.2) The laccases catalyze the

oxidation of o- and p-diphenols into their correspond-

ing quinones according to reaction (3) in Figure 1.

They occur much less frequently in the plant kingdom

than PPOs. Their presence has been mainly reported

in many fungi and in species of the genus Rhus (e.g.,

the Japanese lacquer tree). They are almost absent

from fruits and vegetables, with the exception of

certain peach cultivars and apricots. Nevertheless,

the distinction between PPO and laccase activities is

not always clear-cut since the presence of endogenous

phenols can induce the coupled oxidation of

p-diphenols and therefore leads to a false conclusion

on the presence of a laccase type of activity. Selective

inhibitors can be used to ascertain the type of activity.

Thus, cinnamic acid, salicylhydroxamic acid, 4-hex-

ylresorcinol, phenylhydrazine, and carbon monoxide

inhibit PPO activity more specifically, whereas cetyl

trimethyl ammonium bromide, a cationic detergent,

is more specific for laccases.

0013Most fungi laccases are glycoproteins with a basic

subunit consisting of a single polypeptide chain of 50–

70 kDa containing a considerable amount of carbo-

hydrate (10–45%) and four atoms of copper. The

effect of pH on laccase activity is similar to that

observed for PPO activity, i.e., an optimum pH

which ranges between 4 and 7.5 and depends on the

substrate being used.

0014Peroxidases (EC1.11.1.7) The peroxidases are en-

zymes whose primary function is to oxidize hydrogen

donors at the expense of peroxides. They are highly

specific for hydrogen peroxide, but they accept a wide

range of hydrogen donors, including polyphenols.

The overall reactions catalyzed are shown in eqns

(1) and (2).

þ H

! A þ 2H

O ð1Þ

2AH þ H

! AA þ 2H

O ð2Þ

0015Peroxidases are glycoproteins with a hematin com-

pound as cofactor. Their molecular weights range

between 30 and 55 kDa. Depending on the enzyme

source, the isoenzyme considered, and the hydrogen

donor substrate, the optimum activity is between pH

4 and 7. In many, but not all cases, the peroxidase

isoenzymes are activated by calcium ions. The higher

thermostability of some isoenzymes is well known,

and the residual peroxidase activity after blanching

is often used as an index of thermal treatment.

0016Although peroxidases are widely distributed,

especially in plants, they generally appear to be little

involved in enzymatic browning of fruits and vege-

tables following a mechanical stress. The explanation

could be that the peroxidase activity is limited by

the internal level of hydrogen peroxide. However,

their involvement in slow processes such as internal

browning during cold storage of fruits is possible.

0017Nevertheless, the direct involvement of peroxidase

in browning still needs confirmation, just as does that

of laccase which may not be present in some fruits

and vegetables. Therefore, the following sections will

be mainly devoted to PPO activity.

680 BROWNING/Enzymatic – Biochemical Aspects

Substrates

0018 Susceptibility to browning varies widely from one

plant to another. This variation is linked to both

quantitative and qualitative aspects of their phenolic

content. Thus, browning after bruising of fruit culti-

vars with similar amounts of total phenolics can be

either more or less intense. Among the wide variety of

phenolic compounds found in fruits and vegetables,

only a small number serves as direct substrates for

PPO. Caffeic acid derivatives and monomeric flavan-

3-ols (mainly (þ)-(gallo)catechin and ()-(gallo)epi-

catechin) often appear to be the best substrates. Other

quantitatively important classes of phenols, namely

anthocyanins, flavonols, and condensed forms of

flavan-3-ols (tannins), are weakly if not directly

oxidized by PPO. The same holds for other less

important classes of phenols (flavones, flavanones,

flavononols, chalcones, and dihydrochalcones). This

restricted activity is probably related to the presence

of a sugar moiety in many of these molecules which

could cause steric hindrance since the aglycone forms

are often good substrates of PPO. Nevertheless,

phenolic compounds which are not direct substrates

can actively participate in browning through coupled

oxidation reactions. Thus, in model systems, it has

been shown that the degradation of anthocyanins,

procyanidins, and flavonols by PPO is greatly accel-

erated in the presence of caffeic acid derivatives or

catechins. The o-quinones enzymatically formed

from either of the latter compounds are able to pro-

mote cooxidation reactions leading to both the deg-

radation of the former compounds and the

regeneration of good substrates for the enzymatic

reaction. This degradation and the phenolic copoly-

merization resulting from nonenzymatic coupled oxi-

dations (reactions (2) and (3) of Figure 2) lead to

products which may be intensely brown.

0019Many studies have been devoted to the specificity

of PPO towards phenolic substrates. Usually, the ap-

parent Michaelis constant (K

) is higher than 1 mmol

1

, indicating a relatively low affinity. However, for a

particular substrate, the K

values can vary widely

depending on the PPO source, and the same holds

for the relative rate of oxidation among different

(1)

(2)

(3)

PPO

o-Quinone

Brown

polymers

R' R'

fig0002 Figure 2 Reactions of o-quinones with phenolic compounds. (All reactions are nonenzymatic except those with polyphenol oxidases

(PPOs). Reactions (2) and (3) are able to regenerate the original phenol.) Products with differing color intensities are indicated by

asterisks. Reproduced from Browning: Enzymatic-Biochemical Aspects. Encyclopaedia of Food Science, Food Technology and Nutrition,

Macrae R, Robinson RK and Sadler MJ (eds), 1993, Academic Press.

BROWNING/Enzymatic – Biochemical Aspects 681

phenolics (Table 1). It is often suggested that the

preferred substrate is the most abundant phenolic,

although this is not always true.

0020 By contrast, few studies have concerned oxygen,

the other substrate. Steady-state kinetics carried out

on PPO indicate that it probably follows an ordered

Bi-Bi mechanism in which the oxygen binds first. The

reported values for equilibrium constant (K

mapp

)

are in the range 0.1–0.5 mmol l

1

, corresponding to a

rather low affinity for the oxygen (compared to, for

example, cytochrome oxidase: 0.5–1 mmol l

1

). (See

Oxidation of Food Components; Phenolic Com-

pounds; Tannins and Polyphenols.)

Reaction Products

0021 The primary products of enzymatic oxidation are

o-quinones. These molecules have different spectral

properties and their color depends on the pH and the

phenol from which they originate. Some of the molar

extinction coefficients at maximum wavelengths are

given in Table 2, and show a wide range of variation.

0022 The colors are different from their precursors, since

after oxidation catechin is bright yellow, chlorogenic

acid is dull orange-yellow, whereas DOPA is pink.

Moreover, the o-quinones are reactive compounds,

as illustrated in Figures 2 and 3. Consider Figure 2

for the reactions with phenolic compounds. o-Quin-

ones can react with another phenol molecule,

resulting in a dimer of the original phenol (reaction

(1) of Figure 2). This dimer, having an o-diphenolic

structure, can be subject to reoxidation either enzy-

matically or by another o-quinone and gives larger

oligomers with different color intensities. The o-quin-

ones can also react with a different phenol molecule,

either leading to a copolymer (reaction (2) of Figure 2)

or regenerating the original phenol and giving a dif-

ferent o-quinone (reaction (3): coupled oxidation of

Figure 2). In Figure 3, corresponding to reactions

with nonphenolic compounds, another coupled oxi-

dation reaction is observed with ascorbic acid (reac-

tion (1) of Figure 3), since the phenol is regenerated

with the simultaneous formation of dehydroascorbic

acid.

0023With sulfites colorless addition compounds are

formed, together with regenerated diphenol (reaction

(2) of Figure 3). The o-quinones can form additional

compounds with thiol groups by nucleophilic substi-

tution (reactions (3) and (4) of Figure 3). Cysteine,

either free or bound in small peptides (e.g., glu-

tathione) or in large proteins, generates colorless

compounds. However, due to their o-diphenolic

structures, these compounds can be either oxidized

by laccase or react with excess o-quinones (by a

coupled oxidation mechanism) and form intensely

colored products. The same kind of addition reac-

tions occur with amino groups (primary or secondary

amines), although a little less readily ( (reactions (5–7)

of Figure 3). Furthermore, substitutions with other

thiol or amino groups of proteins may occur, leading

to intra- and intermolecular cross-links. Lastly, water

slowly adds to the o-quinones to form triphenols

which are readily oxidized by excess o-quinone

(by a coupled reaction mechanism), leading to the

p-quinones (reaction (8) of Figure 3). (See Amines;

Ascorbic Acid: Properties and Determination.)

tbl0001 Table 1 Michaelis constant (K

: mmol l

1

) andV

(maximal rate) values (expressed as a percentage of the V

of chlorogenic acid) of

polyphenol oxidases from different sources for three common natural substrates

Apple Grape Pear Peach Apricot Potato

Chlorogenic acid 100 4.2 100 2.5 100 16.1 100 1.2 1.4

(þ)-Catechin 58 6.2 64 1 60 2.1 373 0.74

Caffeic acid 8.1 0.14 69 5.5 43 61 0.5 2.4–2.9

Adapted from:

Janovitz-Klapp A, Richard F, Goupy P, Nicolas J (1990) Kinetic studies on apple polyphenol oxidase. Journal of Agricultural and Food Chemistry 38:

1437–1441.

Macheix JJ, Fleuriet A, Billot J (1990) Fruit Phenolics. Boca Raton: CRC Press.

Vamos-Vigyazo L (1981) Polyphenol oxidase and peroxidase in fruits and vegetables. CRC Critical Reviews in Food Science and Nutrition 15: 49–127.

tbl0002Table 2 Molar extinction coefficients of quinones from different

o-diphenolic substrates of polyphenol oxidases

Substrate Wavelength (nm) Extinction

coefficient

Pyrocatechol

390 1417

3,4-Dihydroxyphenylacetic acid

390 1311

4-t-Butylcatechol

400 1150

4-Methylcatechol

400 1400

L-DOPA

480 3388

Hydrocaffeic acid

412 1124

Chlorogenic acid

420 2000

(þ)-Catechin

380 1200

Adapted from:

Waite JH (1976) Calculating extinction coefficients for enzymically

produced o-quinones. Analytical Biochemistry 75: 211–218.

Rouet-Mayer MA, Ralambosoa J, Philippon J (1990) Roles of o-quinones

and their polymers in the enzymic browning of apples. Phytochemistry 29:

435–440.

682 BROWNING/Enzymatic – Biochemical Aspects

0024 The reactivity (or in other words, stability) of the

o-quinones in these different cases is very variable. It

depends strongly on the substituted nature of the

parent phenol, and on the medium (composition,

pH, temperature, etc.). Thus, in the same conditions,

the o-quinones derived from 4-methylcatechol are

more stable than those from chlorogenic acid which,

in turn, are more stable than those from catechins.

Obviously, the presence of reactive molecules, with

amino or thiol groups, in the medium can greatly

affect the stability of the o-quinones. Moreover, the

reactivity with reducing compounds, namely those

entering in coupled oxidation reactions, is under the

control of the redox potentials of the systems

involved. Thus, the o-quinones of chlorogenic acid

are able to cooxidize catechins into o-quinones of

catechin and regenerate chlorogenic acid, whereas

the reverse is not true.

Inhibition of Enzymatic Browning

0025 Enzymatic browning is often an undesirable reac-

tion and its prevention is a major concern for food

scientists. Therefore, a large part of the many studies

devoted to enzymatic browning concern its control.

This section gives only the physicochemical basis for

the control of the reaction. The more applied view of

the problem in food technology will be described in

the next article. The different ways of browning con-

trol can be divided into three classes, depending on

whether they affect enzymes, substrates, or reaction

products. However, some inhibitors are able to act

simultaneously on more than one of these factors.

Action on Enzymes

0026First, heat treatment is often employed, and usually

short exposure to temperatures between 70 and 90



is sufficient to inactivate PPOs. High-pressure treat-

ments have also been proposed for the denaturation of

PPO. Very high pressures (400–500 MPa) are needed

to inactivate the enzyme at 25



C. However, PPO

appears to be more vulnerable to high-pressure CO

treatment (close to 6 MPa) or to a combined treatment

of heat and ultrasonic waves under moderate pressure

(manothermosonication). Since the optimum pH of

most PPOs lies between pH 4 and 7 (see earlier) on

NHPr

SPr

NHAA

Pr NH

DHA

2−

−

Asc A

(8)

(7)

(3)

(4)

(5)

(6)

(1) (2)

Laccase

Brown

polymers

and

Pr SH

RSH

Pro NH

Pro N

fig0003 Figure 3 Reactions of o-quinones with nonphenolic compounds. (All reactions are nonenzymatic except those with laccase.

Reactions (1), (2), (3), (6), and (8) are able to regenerate the original phenol.) Products with differing color intensities are indicated

by asterisks. Ox, further oxidation reactions by oxygen or o-quinone; Pr-SH and Pr-NH

, proteins; Pro-NH, proline; AA-NH

, amino

acids; Asc A, ascorbic acid; DHA, dehydroascorbic acid; R

SH, small thiol compounds (e.g., cysteine and glutathione). Reproduced

from Browning: Enzymatic-Biochemical Aspects. Encyclopaedia of Food Science, Food Technology and Nutrition, Macrae R, Robinson RK

and Sadler MJ (eds), 1993, Academic Press.

BROWNING/Enzymatic – Biochemical Aspects 683

the one hand, and the phenols are more readily oxi-

dized when the pH increases on the other, acidifica-

tion below pH 4 is often proposed, although PPOs are

still active in some cases (grape, apple, etc.).

0027 Besides these general treatments, more specific

chemical inhibitors are known. PPOs having copper

as a prosthetic group are inhibited by many metal-

chelating agents such as cyanide, azide, diethyldithio-

carbamate, and ethylxanthate, although inhibition

depends on the source of the PPO. Inhibition by halide

ions observed in numerous cases is pH-dependent,

increasing when the pH decreases; it may be caused

by the formation of a complex between the halide ion

and copper which is enhanced by low pH values. Due

to their structural resemblance to phenolic substrates,

aromatic carboxylic compounds are usually found to

be competitive inhibitors. As has been shown for

apple PPOs, the inhibitory properties are highly de-

pendent on structure (Table 3). Thus, for a particular

substituent, inhibition decreases in the order cin-

namic, benzoic, phenylpropionic, and phenylacetic

acid. In each series, inhibition is slightly enhanced

by p-hydroxy substitution and greatly reduced by

o-methoxy substitution. Moreover, the presence of a

benzene nucleus is not an absolute requirement for

the inhibitory effect since sorbic acid, an aliphatic

carboxylic acid with two conjugated double bonds,

is almost as effective as benzoic acid. In all cases,

inhibition increases as pH decreases and it was

shown that the un-ionized form of the carboxylic

group is mainly responsible for inhibition.

0028 More recently, substituted resorcinols have been

recognized as PPO inhibitors. Although resorcinol is

a poor PPO inhibitor, the 4-substituted resorcinols

exhibit the highest inhibition properties. Among

them, the 4-hexylresorcinol has been proposed as a

valuable sulfite alternative for use in the food industry

in order to prevent enzymatic browning. It has been

shown that 4-hexylresorcinol inhibits PPO in a non-

classical mechanism involving the rapid formation

of an enzyme–inhibitor complex which undergoes a

slow reversible reaction (slow-binding competitive

inhibitor).

Action on Substrates

0029Inhibition of enzymatic browning can also be

obtained by removal of one of the two substrates

(oxygen and phenolic compounds) from the reaction

medium. Complete oxygen removal is the most

satisfactory way to control the phenolic oxidation

catalyzed by PPOs. However, although this method

may be applied to dead tissues, either by creating a

physical barrier to oxygen diffusion or by vacuum

infiltration (a more detailed description on this sub-

ject will be given in the next chapter), it is inapplic-

able to living tissues due to the risk of metabolism

deviations caused by anaerobic conditions. Concern-

ing the phenolic substrates, two methods are avail-

able. The first one is the physical elimination by

ultrafiltration or by specific adsorbents. In the latter

case, the most widely used, polyvinylpyrrolidone

(PVP) and its insoluble form (PVPP), are very effective

in controlling enzymatic browning. Thus, PVP has

also been shown to be a competitive inhibitor of

PPOs. Other complexing agents of phenols can be

used, such as polyethylene glycol or polyamide. In

the same way, borate acts by complexing the

o-dihydroxy groups of phenolic substrates. Lastly,

the inhibitory properties of b-cyclodextrins, observed

in apple juice browning, are related to their abilities

to form inclusion complexes with PPO substrates. A

similar mechanism of browning inhibition could be

ascribed to some polysaccharides such as carragenan,

amylose sulfate, xylan, and chitosan.

0030The second way of removing phenolic compounds

is by their modification. This can be performed by

two kinds of enzymes. The first modification is

methylation of the o-diphenolic substrates of PPOs

by an O-methyltransferase (e.g., caffeic acid is con-

verted to ferulic acid). Unfortunately, this elegant

tbl0003 Table 3 Inhibition constants of sodium halides and some carboxylic acids for apple polyphenol oxidases at pH 4.5. All are

competitive inhibitors, with the exception of sodium chloride, which is noncompetitive

Inhibitor K

(inhibition constant) (mmoll

1

)Inhibitor K

(mmoll

1

)

Sodium iodide 117 Sodium bromide 106

Sodium chloride 20 Sodium fluoride 0.07

Benzoic acid 0.64 p-Hydroxybenzoic acid 0.57

Vanillic acid 10 Syringic acid 34.5

Cinnamic acid 0.092 p-Coumaric acid 0.04

Ferulic acid 0.29 Sinapic acid 15

Phenylacetic acid 13 Phenylpropionic acid 1.4

p-Hydroxyphenylpropionic acid 1.1 Sorbic acid 0.51

Adapted from Janovitz-Klapp A, Richard F, Goupy P, Nicolas J (1990) Inhibition studies on apple polyphenol oxidase. Journal of Agricultural and Food

Chemistry 38: 926–931.

684 BROWNING/Enzymatic – Biochemical Aspects